@article{manole2023ngly1,
  title={NGLY1 mutations cause protein aggregation in human neurons},
  author={Manole, Andreea and Wong, Thomas and Rhee, Amanda and Novak, Sammy and Chin, Shao-Ming and Tsimring, Katya and Paucar, Andres and Williams, April and Newmeyer, Traci Fang and Schafer, Simon T and others},
  journal={Cell Reports},
  volume={42},
  number={12},
  year={2023},
  publisher={Elsevier}
}

@article{buswinka2023large,
  title={Large-scale annotated dataset for cochlear hair cell detection and classification},
  author={Buswinka, Christopher J and Rosenberg, David B and Simikyan, Rubina G and Osgood, Richard T and Fernandez, Katharine and Nitta, Hidetomi and Hayashi, Yushi and Liberman, Leslie W and Nguyen, Emily and Yildiz, Erdem and others},
  journal={bioRxiv},
  year={2023},
  publisher={Cold Spring Harbor Laboratory Preprints}
}

@article{okur2023long,
  title={Long-term NAD+ supplementation prevents the progression of age-related hearing loss in mice},
  author={Okur, Mustafa N. and Sahbaz, Burcin Duan and Kimura, Risako and Manor, Uri and Patel, Jaimin and Park, Jae-Hyeon and Andrade, Leo and Puligilla, Chandrakalap and Croteau, Deborah L. and Bohr, Vilhelm A.},
  journal={Aging Cell},
  pages={1474--9718},
  year={2023}
}

@article{boussaty2023altered,
  title={Altered Fhod3 Expression Involved in Progressive High-Frequency Hearing Loss via Dysregulation of Actin Polymerization Stoichiometry in The Cuticular Plate},
  author={Boussaty, Ely Cheikh and Ninoyu, Yuzuru and Andrade, Leo and Li, Qingzhong and Takeya, Ryu and Sumimoto, Hideki and Ohyama, Takahiro and Wahlin, Karl J and Manor, Uri and Friedman, Rick A},
  journal={bioRxiv},
  pages={2023--07},
  year={2023},
  publisher={Cold Spring Harbor Laboratory}
}

@article {Gatti2023.06.13.544768,
	author = {Priya Gatti and Cara Schiavon and Uri Manor and Marc Germain},
	title = {Mitochondria- and ER-associated actin are required for mitochondrial fusion},
	elocation-id = {2023.06.13.544768},
	year = {2023},
	doi = {10.1101/2023.06.13.544768},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Mitochondria play a crucial role in the regulation of cellular metabolism and signalling. Mitochondrial activity is modulated by the processes of mitochondrial fission and fusion, which are required to properly balance respiratory and metabolic functions, transfer material between mitochondria, and remove damaged or defective mitochondria. Mitochondrial fission occurs at sites of contact between the endoplasmic reticulum (ER) and mitochondria, and is dependent on the formation of mitochondria- and ER-associated actin filaments that drive the recruitment and activation of the fission GTPase DRP1. On the other hand, the role of mitochondria- and ER-associated actin filaments in mitochondrial fusion remains unknown. Here we show that preventing the formation of actin filaments on either mitochondria or the ER using organelle-targeted Disassembly-promoting, encodable Actin tools (DeActs) blocks both mitochondrial fission and fusion. We show that fusion but not fission is dependent on Arp2/3, and both fission and fusion are dependent on INF2 formin-dependent actin polymerization. Together, our work introduces a novel method for perturbing organelle-associated actin filaments, and demonstrates a previously unknown role for mitochondria- and ER-associated actin in mitochondrial fusion.Competing Interest StatementThe authors have declared no competing interest.},
	URL = {https://www.biorxiv.org/content/early/2023/06/13/2023.06.13.544768.1},
	eprint = {https://www.biorxiv.org/content/early/2023/06/13/2023.06.13.544768.1.full.pdf},
	journal = {bioRxiv}
}

Mitochondria play a crucial role in the regulation of cellular metabolism and signalling. Mitochondrial activity is modulated by the processes of mitochondrial fission and fusion, which are required to properly balance respiratory and metabolic functions, transfer material between mitochondria, and remove damaged or defective mitochondria. Mitochondrial fission occurs at sites of contact between the endoplasmic reticulum (ER) and mitochondria, and is dependent on the formation of mitochondria- and ER-associated actin filaments that drive the recruitment and activation of the fission GTPase DRP1. On the other hand, the role of mitochondria- and ER-associated actin filaments in mitochondrial fusion remains unknown. Here we show that preventing the formation of actin filaments on either mitochondria or the ER using organelle-targeted Disassembly-promoting, encodable Actin tools (DeActs) blocks both mitochondrial fission and fusion. We show that fusion but not fission is dependent on Arp2/3, and both fission and fusion are dependent on INF2 formin-dependent actin polymerization. Together, our work introduces a novel method for perturbing organelle-associated actin filaments, and demonstrates a previously unknown role for mitochondria- and ER-associated actin in mitochondrial fusion.Competing Interest StatementThe authors have declared no competing interest.

@Article{Pham2023,
	author={Pham, Tammy B. and Boussaty, Ely Cheikh and Currais, Antonio and Maher, Pamela and Schubert, David R. and Manor, Uri
	and Friedman, Rick A.},
	title={Attenuation of Age-Related Hearing Impairment in Senescence-Accelerated Mouse Prone 8 (SAMP8) Mice Treated with Fatty Acid Synthase Inhibitor CMS121},
	journal={Journal of Molecular Neuroscience},
	year={2023},
	month={May},
	day={01},
	volume={73},
	number={4},
	pages={307-315},
	abstract={In the senescence-accelerated mouse prone 8 (SAMP8) mouse model, oxidative stress leads to premature senescence and age-related hearing impairment (ARHI). CMS121 inhibits oxytosis/ferroptosis by targeting fatty acid synthase. The aim of our study was to determine whether CMS121 is protective against ARHI in SAMP8 mice. Auditory brainstem responses (ABRs) were used to assess baseline hearing in sixteen 4-week-old female SAMP8 mice, which were divided into two cohorts. The control group was fed a vehicle diet, while the experimental group was fed a diet containing CMS121. ABRs were measured until 13 weeks of age. Cochlear immunohistochemistry was performed to analyze the number of paired ribbon-receptor synapses per inner hair cell (IHC). Descriptive statistics are provided with mean{\thinspace}{\textpm}{\thinspace}SEM. Two-sample t-tests were performed to compare hearing thresholds and paired synapse count across the two groups, with alpha{\thinspace}={\thinspace}0.05. Baseline hearing thresholds in the control group were statistically similar to those of the CMS121 group. At 13 weeks of age, the control group had significantly worse hearing thresholds at 12 kHz (56.5 vs. 39.8, p{\thinspace}={\thinspace}0.044) and 16 kHz (64.8 vs. 43.8, p{\thinspace}={\thinspace}0.040) compared to the CMS121 group. Immunohistochemistry showed a significantly lower synapse count per IHC in the control group (15.7) compared to the CMS121 group (18.4), p{\thinspace}={\thinspace}0.014. Our study shows a significant reduction in ABR threshold shifts and increased preservation of IHC ribbon synapses in the mid-range frequencies among mice treated with CMS121 compared to untreated mice.},
	issn={1559-1166},
	doi={10.1007/s12031-023-02119-w},
	url={https://doi.org/10.1007/s12031-023-02119-w}
}

In the senescence-accelerated mouse prone 8 (SAMP8) mouse model, oxidative stress leads to premature senescence and age-related hearing impairment (ARHI). CMS121 inhibits oxytosis/ferroptosis by targeting fatty acid synthase. The aim of our study was to determine whether CMS121 is protective against ARHI in SAMP8 mice. Auditory brainstem responses (ABRs) were used to assess baseline hearing in sixteen 4-week-old female SAMP8 mice, which were divided into two cohorts. The control group was fed a vehicle diet, while the experimental group was fed a diet containing CMS121. ABRs were measured until 13 weeks of age. Cochlear immunohistochemistry was performed to analyze the number of paired ribbon-receptor synapses per inner hair cell (IHC). Descriptive statistics are provided with meanþinspace\textpmþinspaceSEM. Two-sample t-tests were performed to compare hearing thresholds and paired synapse count across the two groups, with alphaþinspace=þinspace0.05. Baseline hearing thresholds in the control group were statistically similar to those of the CMS121 group. At 13 weeks of age, the control group had significantly worse hearing thresholds at 12 kHz (56.5 vs. 39.8, pþinspace=þinspace0.044) and 16 kHz (64.8 vs. 43.8, pþinspace=þinspace0.040) compared to the CMS121 group. Immunohistochemistry showed a significantly lower synapse count per IHC in the control group (15.7) compared to the CMS121 group (18.4), pþinspace=þinspace0.014. Our study shows a significant reduction in ABR threshold shifts and increased preservation of IHC ribbon synapses in the mid-range frequencies among mice treated with CMS121 compared to untreated mice.

@article{doi:10.1126/science.abj5559,
  author = {Nazma Malik  and Bibiana I. Ferreira  and Pablo E. Hollstein  and Stephanie D. Curtis  and Elijah Trefts  and Sammy Weiser Novak  and Jingting Yu  and Rebecca Gilson  and Kristina Hellberg  and Lingjing Fang  and Arlo Sheridan  and Nasun Hah  and Gerald S. Shadel  and Uri Manor  and Reuben J. Shaw },
  title = {Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1},
  journal = {Science},
  volume = {380},
  number = {6642},
  pages = {eabj5559},
  year = {2023},
  doi = {10.1126/science.abj5559},
  URL = {https://www.science.org/doi/abs/10.1126/science.abj5559},
  eprint = {https://www.science.org/doi/pdf/10.1126/science.abj5559},
  abstract = {Cells respond to mitochondrial poisons with rapid activation of the adenosine monophosphate–activated protein kinase (AMPK), causing acute metabolic changes through phosphorylation and prolonged adaptation of metabolism through transcriptional effects. Transcription factor EB (TFEB) is a major effector of AMPK that increases expression of lysosome genes in response to energetic stress, but how AMPK activates TFEB remains unresolved. We demonstrate that AMPK directly phosphorylates five conserved serine residues in folliculin-interacting protein 1 (FNIP1), suppressing the function of the folliculin (FLCN)–FNIP1 complex. FNIP1 phosphorylation is required for AMPK to induce nuclear translocation of TFEB and TFEB-dependent increases of peroxisome proliferator–activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) messenger RNAs. Thus, mitochondrial damage triggers AMPK-FNIP1–dependent nuclear translocation of TFEB, inducing sequential waves of lysosomal and mitochondrial biogenesis. The kinase AMPK is a key sensor that helps to control energy homeostasis. Malik et al. reveal the mechanism by which AMPK controls the transcription factor TFEB to increase gene transcription and to support mitochondrial and lysosomal biogenesis. AMPK appears to act by direct phosphorylation of folliculin-interacting protein 1 (FNIP1). FNIP is part of a complex that acts as a GTP-activating protein for the GTPases RagC and RagD, which regulate the mechanistic target of rapamycin complex 1 protein kinase signaling complex on the lysosomal surface. This results in release of TFEB from the lysosome, allowing it to act at the nucleus. —LBR The energy-sensing protein kinase AMPK also regulates organelle biogenesis.}
  }

Cells respond to mitochondrial poisons with rapid activation of the adenosine monophosphate–activated protein kinase (AMPK), causing acute metabolic changes through phosphorylation and prolonged adaptation of metabolism through transcriptional effects. Transcription factor EB (TFEB) is a major effector of AMPK that increases expression of lysosome genes in response to energetic stress, but how AMPK activates TFEB remains unresolved. We demonstrate that AMPK directly phosphorylates five conserved serine residues in folliculin-interacting protein 1 (FNIP1), suppressing the function of the folliculin (FLCN)–FNIP1 complex. FNIP1 phosphorylation is required for AMPK to induce nuclear translocation of TFEB and TFEB-dependent increases of peroxisome proliferator–activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) messenger RNAs. Thus, mitochondrial damage triggers AMPK-FNIP1–dependent nuclear translocation of TFEB, inducing sequential waves of lysosomal and mitochondrial biogenesis. The kinase AMPK is a key sensor that helps to control energy homeostasis. Malik et al. reveal the mechanism by which AMPK controls the transcription factor TFEB to increase gene transcription and to support mitochondrial and lysosomal biogenesis. AMPK appears to act by direct phosphorylation of folliculin-interacting protein 1 (FNIP1). FNIP is part of a complex that acts as a GTP-activating protein for the GTPases RagC and RagD, which regulate the mechanistic target of rapamycin complex 1 protein kinase signaling complex on the lysosomal surface. This results in release of TFEB from the lysosome, allowing it to act at the nucleus. —LBR The energy-sensing protein kinase AMPK also regulates organelle biogenesis.

@ARTICLE{10.3389/fnagi.2023.1146245, 
  AUTHOR={Glavis-Bloom, Courtney and Vanderlip, Casey R. and Weiser Novak, Sammy and Kuwajima, Masaaki and Kirk, Lyndsey and Harris, Kristen M. and Manor, Uri and Reynolds, John H.},   
  TITLE={Violation of the ultrastructural size principle in the dorsolateral prefrontal cortex underlies working memory impairment in the aged common marmoset (Callithrix jacchus)},     
  JOURNAL={Frontiers in Aging Neuroscience},      
  VOLUME={15},           
  YEAR={2023},       
  URL={https://www.frontiersin.org/articles/10.3389/fnagi.2023.1146245},       
  DOI={10.3389/fnagi.2023.1146245},       
  ISSN={1663-4365},    
  ABSTRACT={Morphology and function of the dorsolateral prefrontal cortex (dlPFC), and corresponding working memory performance, are affected early in the aging process, but nearly half of aged individuals are spared of working memory deficits. Translationally relevant model systems are critical for determining the neurobiological drivers of this variability. The common marmoset (Callithrix jacchus) is advantageous as a model for these investigations because, as a non-human primate, marmosets have a clearly defined dlPFC that enables measurement of prefrontal-dependent cognitive functions, and their short (∼10 year) lifespan facilitates longitudinal studies of aging. Previously, we characterized working memory capacity in a cohort of marmosets that collectively covered the lifespan, and found age-related working memory impairment. We also found a remarkable degree of heterogeneity in performance, similar to that found in humans. Here, we tested the hypothesis that changes to synaptic ultrastructure that affect synaptic efficacy stratify marmosets that age with cognitive impairment from those that age without cognitive impairment. We utilized electron microscopy to visualize synapses in the marmoset dlPFC and measured the sizes of boutons, presynaptic mitochondria, and synapses. We found that coordinated scaling of the sizes of synapses and mitochondria with their associated boutons is essential for intact working memory performance in aged marmosets. Further, lack of synaptic scaling, due to a remarkable failure of synaptic mitochondria to scale with presynaptic boutons, selectively underlies age-related working memory impairment. We posit that this decoupling results in mismatched energy supply and demand, leading to impaired synaptic transmission. We also found that aged marmosets have fewer synapses in dlPFC than young, though the severity of synapse loss did not predict whether aging occurred with or without cognitive impairment. This work identifies a novel mechanism of synapse dysfunction that stratifies marmosets that age with cognitive impairment from those that age without cognitive impairment. The process by which synaptic scaling is regulated is yet unknown and warrants future investigation.}
}

Morphology and function of the dorsolateral prefrontal cortex (dlPFC), and corresponding working memory performance, are affected early in the aging process, but nearly half of aged individuals are spared of working memory deficits. Translationally relevant model systems are critical for determining the neurobiological drivers of this variability. The common marmoset (Callithrix jacchus) is advantageous as a model for these investigations because, as a non-human primate, marmosets have a clearly defined dlPFC that enables measurement of prefrontal-dependent cognitive functions, and their short (∼10 year) lifespan facilitates longitudinal studies of aging. Previously, we characterized working memory capacity in a cohort of marmosets that collectively covered the lifespan, and found age-related working memory impairment. We also found a remarkable degree of heterogeneity in performance, similar to that found in humans. Here, we tested the hypothesis that changes to synaptic ultrastructure that affect synaptic efficacy stratify marmosets that age with cognitive impairment from those that age without cognitive impairment. We utilized electron microscopy to visualize synapses in the marmoset dlPFC and measured the sizes of boutons, presynaptic mitochondria, and synapses. We found that coordinated scaling of the sizes of synapses and mitochondria with their associated boutons is essential for intact working memory performance in aged marmosets. Further, lack of synaptic scaling, due to a remarkable failure of synaptic mitochondria to scale with presynaptic boutons, selectively underlies age-related working memory impairment. We posit that this decoupling results in mismatched energy supply and demand, leading to impaired synaptic transmission. We also found that aged marmosets have fewer synapses in dlPFC than young, though the severity of synapse loss did not predict whether aging occurred with or without cognitive impairment. This work identifies a novel mechanism of synapse dysfunction that stratifies marmosets that age with cognitive impairment from those that age without cognitive impairment. The process by which synaptic scaling is regulated is yet unknown and warrants future investigation.

@article {Iyer2023.04.11.536299,
	author = {Manasi Iyer and Husniye Kantarci and Nicholas Ambiel and Sammy W. Novak and Leonardo R. Andrade and Mable Lam and Alexandra E. M{\"u}nch and Xinzhu Yu and Baljit S. Khakh and Uri Manor and J. Bradley Zuchero},
	title = {Oligodendrocyte calcium signaling sculpts myelin sheath morphology},
	elocation-id = {2023.04.11.536299},
	year = {2023},
	doi = {10.1101/2023.04.11.536299},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Myelin is essential for rapid nerve signaling and is increasingly found to play important roles in learning and in diverse diseases of the CNS. Morphological parameters of myelin such as sheath length and thickness are regulated by neuronal activity and can precisely tune conduction velocity, but the mechanisms controlling sheath morphology are poorly understood. Local calcium signaling has been observed in nascent myelin sheaths and can be modulated by neuronal activity. However, the role of calcium signaling in sheath formation and remodeling is unknown. Here, we used genetic tools to attenuate oligodendrocyte calcium signaling during active myelination in the developing mouse CNS. Surprisingly, we found that genetic calcium attenuation did not grossly affect the number of myelinated axons or myelin thickness. Instead, calcium attenuation caused striking myelination defects resulting in shorter, dysmorphic sheaths. Mechanistically, calcium attenuation reduced actin filaments in oligodendrocytes, and an intact actin cytoskeleton was necessary and sufficient to achieve accurate myelin morphology. Together, our work reveals a novel cellular mechanism required for accurate CNS myelin formation and provides mechanistic insight into how oligodendrocytes may respond to neuronal activity to sculpt myelin sheaths throughout the nervous system.Competing Interest StatementThe authors have declared no competing interest.},
	URL = {https://www.biorxiv.org/content/early/2023/04/12/2023.04.11.536299},
	eprint = {https://www.biorxiv.org/content/early/2023/04/12/2023.04.11.536299.full.pdf},
	journal = {bioRxiv}
}

Myelin is essential for rapid nerve signaling and is increasingly found to play important roles in learning and in diverse diseases of the CNS. Morphological parameters of myelin such as sheath length and thickness are regulated by neuronal activity and can precisely tune conduction velocity, but the mechanisms controlling sheath morphology are poorly understood. Local calcium signaling has been observed in nascent myelin sheaths and can be modulated by neuronal activity. However, the role of calcium signaling in sheath formation and remodeling is unknown. Here, we used genetic tools to attenuate oligodendrocyte calcium signaling during active myelination in the developing mouse CNS. Surprisingly, we found that genetic calcium attenuation did not grossly affect the number of myelinated axons or myelin thickness. Instead, calcium attenuation caused striking myelination defects resulting in shorter, dysmorphic sheaths. Mechanistically, calcium attenuation reduced actin filaments in oligodendrocytes, and an intact actin cytoskeleton was necessary and sufficient to achieve accurate myelin morphology. Together, our work reveals a novel cellular mechanism required for accurate CNS myelin formation and provides mechanistic insight into how oligodendrocytes may respond to neuronal activity to sculpt myelin sheaths throughout the nervous system.Competing Interest StatementThe authors have declared no competing interest.

@article{volpe2023roadmap,
      title={Roadmap on Deep Learning for Microscopy}, 
      author={Giovanni Volpe and Carolina Wählby and Lei Tian and Michael Hecht and Artur Yakimovich and Kristina Monakhova and Laura Waller and Ivo F. Sbalzarini and Christopher A. Metzler and Mingyang Xie and Kevin Zhang and Isaac C. D. Lenton and Halina Rubinsztein-Dunlop and Daniel Brunner and Bijie Bai and Aydogan Ozcan and Daniel Midtvedt and Hao Wang and Nataša Sladoje and Joakim Lindblad and Jason T. Smith and Marien Ochoa and Margarida Barroso and Xavier Intes and Tong Qiu and Li-Yu Yu and Sixian You and Yongtao Liu and Maxim A. Ziatdinov and Sergei V. Kalinin and Arlo Sheridan and Uri Manor and Elias Nehme and Ofri Goldenberg and Yoav Shechtman and Henrik K. Moberg and Christoph Langhammer and Barbora Špačková and Saga Helgadottir and Benjamin Midtvedt and Aykut Argun and Tobias Thalheim and Frank Cichos and Stefano Bo and Lars Hubatsch and Jesus Pineda and Carlo Manzo and Harshith Bachimanchi and Erik Selander and Antoni Homs-Corbera and Martin Fränzl and Kevin de Haan and Yair Rivenson and Zofia Korczak and Caroline Beck Adiels and Mite Mijalkov and Dániel Veréb and Yu-Wei Chang and Joana B. Pereira and Damian Matuszewski and Gustaf Kylberg and Ida-Maria Sintorn and Juan C. Caicedo and Beth A Cimini and Muyinatu A. Lediju Bell and Bruno M. Saraiva and Guillaume Jacquemet and Ricardo Henriques and Wei Ouyang and Trang Le and Estibaliz Gómez-de-Mariscal and Daniel Sage and Arrate Muñoz-Barrutia and Ebba Josefson Lindqvist and Johanna Bergman},
      year={2023},
      eprint={2303.03793},
      archivePrefix={arXiv},
      primaryClass={physics.optics}
}

@techreport{Sanders2023-nz,
  title    = "Biological research and self-driving labs in deep space supported
              by artificial intelligence",
  author   = "Sanders, Lauren M and Scott, Ryan T and Yang, Jason H and Qutub, Amina Ann and Garcia Martin, Hector and Berrios, Daniel C and Hastings, Jaden J A and Rask, Jon and Mackintosh, Graham and Hoarfrost, Adrienne L and Chalk, Stuart and Kalantari, John and Khezeli, Kia and Antonsen, Erik L and Babdor, Joel and Barker, Richard and Baranzini, Sergio E and Beheshti, Afshin and Delgado-Aparicio, Guillermo M and Glicksberg, Benjamin S and Greene, Casey S and Haendel, Melissa and Hamid, Arif A and Heller, Philip and Jamieson, Daniel and Jarvis, Katelyn J and Komarova, Svetlana V and Komorowski, Matthieu and Kothiyal,
              Prachi and Mahabal, Ashish and Manor, Uri and Mason, Christopher
              E and Matar, Mona and Mias, George I and Miller, Jack and Myers,
              Jerry G and Nelson, Charlotte and Oribello, Jonathan and Park,
              Seung-Min and Parsons-Wingerter, Patricia and Prabhu, R K and
              Reynolds, Robert J and Saravia-Butler, Amanda and Saria, Suchi
              and Sawyer, Aenor and Singh, Nitin Kumar and Snyder, Michael and
              Soboczenski, Frank and Soman, Karthik and Theriot, Corey A and
              Van Valen, David and Venkateswaran, Kasthuri and Warren, Liz and
              Worthey, Liz and Zitnik, Marinka and Costes, Sylvain V",
  abstract = "Space biology research aims to understand fundamental spaceflight
              effects on organisms, develop foundational knowledge to support
              deep space exploration and, ultimately, bioengineer spacecraft
              and habitats to stabilize the ecosystem of plants, crops,
              microbes, animals and humans for sustained multi-planetary life.
              To advance these aims, the field leverages experiments,
              platforms, data and model organisms from both spaceborne and
              ground-analogue studies. As research is extended beyond low Earth
              orbit, experiments and platforms must be maximally automated,
              light, agile and intelligent to accelerate knowledge discovery.
              Here we present a summary of decadal recommendations from a
              workshop organized by the National Aeronautics and Space
              Administration on artificial intelligence, machine learning and
              modelling applications that offer solutions to these space
              biology challenges. The integration of artificial intelligence
              into the field of space biology will deepen the biological
              understanding of spaceflight effects, facilitate predictive
              modelling and analytics, support maximally automated and
              reproducible experiments, and efficiently manage spaceborne data
              and metadata, ultimately to enable life to thrive in deep space.",
  journal  = "Nature Machine Intelligence",
  volume   =  5,
  number   =  3,
  pages    = "208--219",
  month    =  mar,
  year     =  2023
}

Space biology research aims to understand fundamental spaceflight effects on organisms, develop foundational knowledge to support deep space exploration and, ultimately, bioengineer spacecraft and habitats to stabilize the ecosystem of plants, crops, microbes, animals and humans for sustained multi-planetary life. To advance these aims, the field leverages experiments, platforms, data and model organisms from both spaceborne and ground-analogue studies. As research is extended beyond low Earth orbit, experiments and platforms must be maximally automated, light, agile and intelligent to accelerate knowledge discovery. Here we present a summary of decadal recommendations from a workshop organized by the National Aeronautics and Space Administration on artificial intelligence, machine learning and modelling applications that offer solutions to these space biology challenges. The integration of artificial intelligence into the field of space biology will deepen the biological understanding of spaceflight effects, facilitate predictive modelling and analytics, support maximally automated and reproducible experiments, and efficiently manage spaceborne data and metadata, ultimately to enable life to thrive in deep space.

@techreport{Scott2023-zn,
  title    = "Biomonitoring and precision health in deep space supported by
              artificial intelligence",
  author   = "Scott, Ryan T and Sanders, Lauren M and Antonsen, Erik L and
              Hastings, Jaden J A and Park, Seung-Min and Mackintosh, Graham
              and Reynolds, Robert J and Hoarfrost, Adrienne L and Sawyer,
              Aenor and Greene, Casey S and Glicksberg, Benjamin S and Theriot,
              Corey A and Berrios, Daniel C and Miller, Jack and Babdor, Joel
              and Barker, Richard and Baranzini, Sergio E and Beheshti, Afshin
              and Chalk, Stuart and Delgado-Aparicio, Guillermo M and Haendel,
              Melissa and Hamid, Arif A and Heller, Philip and Jamieson, Daniel
              and Jarvis, Katelyn J and Kalantari, John and Khezeli, Kia and
              Komarova, Svetlana V and Komorowski, Matthieu and Kothiyal,
              Prachi and Mahabal, Ashish and Manor, Uri and Garcia Martin,
              Hector and Mason, Christopher E and Matar, Mona and Mias, George
              I and Myers, Jerry G and Nelson, Charlotte and Oribello, Jonathan
              and Parsons-Wingerter, Patricia and Prabhu, R K and Qutub, Amina
              Ann and Rask, Jon and Saravia-Butler, Amanda and Saria, Suchi and
              Singh, Nitin Kumar and Snyder, Michael and Soboczenski, Frank and
              Soman, Karthik and Van Valen, David and Venkateswaran, Kasthuri
              and Warren, Liz and Worthey, Liz and Yang, Jason H and Zitnik,
              Marinka and Costes, Sylvain V",
  abstract = "Human exploration of deep space will involve missions of
              substantial distance and duration. To effectively mitigate health
              hazards, paradigm shifts in astronaut health systems are
              necessary to enable Earth-independent healthcare, rather than
              Earth-reliant. Here we present a summary of decadal
              recommendations from a workshop organized by NASA on artificial
              intelligence, machine learning and modelling applications that
              offer key solutions toward these space health challenges. The
              workshop recommended various biomonitoring approaches, biomarker
              science, spacecraft/habitat hardware, intelligent software and
              streamlined data management tools in need of development and
              integration to enable humanity to thrive in deep space.
              Participants recommended that these components culminate in a
              maximally automated, autonomous and intelligent Precision Space
              Health system, to monitor, aggregate and assess biomedical
              statuses.",
  journal  = "Nature Machine Intelligence",
  volume   =  5,
  number   =  3,
  pages    = "196--207",
  month    =  mar,
  year     =  2023
}

Human exploration of deep space will involve missions of substantial distance and duration. To effectively mitigate health hazards, paradigm shifts in astronaut health systems are necessary to enable Earth-independent healthcare, rather than Earth-reliant. Here we present a summary of decadal recommendations from a workshop organized by NASA on artificial intelligence, machine learning and modelling applications that offer key solutions toward these space health challenges. The workshop recommended various biomonitoring approaches, biomarker science, spacecraft/habitat hardware, intelligent software and streamlined data management tools in need of development and integration to enable humanity to thrive in deep space. Participants recommended that these components culminate in a maximally automated, autonomous and intelligent Precision Space Health system, to monitor, aggregate and assess biomedical statuses.

@article {Boussaty2023.02.15.528661,
	author = {Ely Cheikh Boussaty and Neil Tedeschi and Mark Novotny and Yuzuru Ninoyu and Eric Du and Clara Draf and Yun Zhang and Uri Manor and Richard H. Scheuermann and Rick Friedman},
	title = {Cochlear transcriptome analysis of an outbred mouse population (CFW)},
	elocation-id = {2023.02.15.528661},
	year = {2023},
	doi = {10.1101/2023.02.15.528661},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Age-related hearing loss (ARHL) is the most common cause of hearing loss and one of the most prevalent conditions affecting the elderly worldwide. Despite evidence from our lab and others about its polygenic nature, little is known about the specific genes, cell types and pathways involved in ARHL, impeding the development of therapeutic interventions. In this manuscript, we describe, for the first time, the complete cell-type specific transcriptome of the aging mouse cochlea using snRNA-seq in an outbred mouse model in relation to auditory threshold variation. Cochlear cell types were identified using unsupervised clustering and annotated via a three-tiered approach - first by linking to expression of known marker genes, then using the NS-Forest algorithm to select minimum cluster-specific marker genes and reduce dimensional feature space for statistical comparison of our clusters with existing publicly-available data sets on the gEAR website (https://umgear.org/), and finally, by validating and refining the annotations using Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH) and the cluster-specific marker genes as probes. We report on 60 unique cell-types expanding the number of defined cochlear cell types by more than two times. Importantly, we show significant specific cell type increases and decreases associated with loss of hearing acuity implicating specific subsets of hair cell subtypes, ganglion cell subtypes, and cell subtypes withing the stria vascularis in this model of ARHL. These results provide a view into the cellular and molecular mechanisms responsible for age-related hearing loss and pathways for therapeutic targeting.Competing Interest StatementThe authors have declared no competing interest.},
	URL = {https://www.biorxiv.org/content/early/2023/06/10/2023.02.15.528661},
	eprint = {https://www.biorxiv.org/content/early/2023/06/10/2023.02.15.528661.full.pdf},
	journal = {bioRxiv}
}

Age-related hearing loss (ARHL) is the most common cause of hearing loss and one of the most prevalent conditions affecting the elderly worldwide. Despite evidence from our lab and others about its polygenic nature, little is known about the specific genes, cell types and pathways involved in ARHL, impeding the development of therapeutic interventions. In this manuscript, we describe, for the first time, the complete cell-type specific transcriptome of the aging mouse cochlea using snRNA-seq in an outbred mouse model in relation to auditory threshold variation. Cochlear cell types were identified using unsupervised clustering and annotated via a three-tiered approach - first by linking to expression of known marker genes, then using the NS-Forest algorithm to select minimum cluster-specific marker genes and reduce dimensional feature space for statistical comparison of our clusters with existing publicly-available data sets on the gEAR website (https://umgear.org/), and finally, by validating and refining the annotations using Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH) and the cluster-specific marker genes as probes. We report on 60 unique cell-types expanding the number of defined cochlear cell types by more than two times. Importantly, we show significant specific cell type increases and decreases associated with loss of hearing acuity implicating specific subsets of hair cell subtypes, ganglion cell subtypes, and cell subtypes withing the stria vascularis in this model of ARHL. These results provide a view into the cellular and molecular mechanisms responsible for age-related hearing loss and pathways for therapeutic targeting.Competing Interest StatementThe authors have declared no competing interest.

@article{doi:10.1164/rccm.202202-0394OC,
  author = {Shang, Fenqing and Wang, Shen-Chih and Gongol, Brendoan and Han, So Yun and Cho, Yoshitake and Schiavon, Cara R. and Chen, Lili and Xing, Yuanming and Zhao, Yingshuai and Ning, Ming’an and Guo, Xuan and He, Fangzhou and Lei, Yuyang and Wang, Liuyi and Manor, Uri and Marin, Traci and Chou, Kun-Ta and He, Ming and Huang, Po-Hsun and Shyy, John Y.-J. and Malhotra, Atul},
  title = {Obstructive Sleep Apnea–induced Endothelial Dysfunction Is Mediated by miR-210},
  journal = {American Journal of Respiratory and Critical Care Medicine},
  volume = {207},
  number = {3},
  pages = {323-335},
  year = {2023},
  doi = {10.1164/rccm.202202-0394OC},
  note ={PMID: 36191258},
  URL = {https://doi.org/10.1164/rccm.202202-0394OC},
  eprint = {https://doi.org/10.1164/rccm.202202-0394OC},
  abstract = { Rationale: Obstructive sleep apnea (OSA)–induced endothelial cell (EC) dysfunction contributes to OSA-related cardiovascular sequelae. The mechanistic basis of endothelial impairment by OSA is unclear. Objectives: The goals of this study were to identify the mechanism of OSA-induced EC dysfunction and explore the potential therapies for OSA-accelerated cardiovascular disease. Methods: The experimental methods include data mining, bioinformatics, EC functional analyses, OSA mouse models, and assessment of OSA human subjects. Measurements and Main Results: Using mined microRNA sequencing data, we found that microRNA 210 (miR-210) conferred the greatest induction by intermittent hypoxia in ECs. Consistently, the serum concentration of miR-210 was higher in individuals with OSA from two independent cohorts. Importantly, miR-210 concentration was positively correlated with the apnea–hypopnea index. RNA sequencing data collected from ECs transfected with miR-210 or treated with OSA serum showed a set of genes commonly altered by miR-210 and OSA serum, which are largely involved in mitochondrion-related pathways. ECs transfected with miR-210 or treated with OSA serum showed reduced V˙o2 rate, mitochondrial membrane potential, and DNA abundance. Mechanistically, intermittent hypoxia-induced SREBP2 (sterol regulatory element–binding protein 2) bound to the promoter region of miR-210, which in turn inhibited the iron–sulfur cluster assembly enzyme and led to mitochondrial dysfunction. Moreover, the SREBP2 inhibitor betulin alleviated intermittent hypoxia–increased systolic blood pressure in the OSA mouse model. Conclusions: These results identify an axis involving SREBP2, miR-210, and mitochondrial dysfunction, representing a new mechanistic link between OSA and EC dysfunction that may have important implications for treating and preventing OSA-related cardiovascular sequelae. }
}

Rationale: Obstructive sleep apnea (OSA)–induced endothelial cell (EC) dysfunction contributes to OSA-related cardiovascular sequelae. The mechanistic basis of endothelial impairment by OSA is unclear. Objectives: The goals of this study were to identify the mechanism of OSA-induced EC dysfunction and explore the potential therapies for OSA-accelerated cardiovascular disease. Methods: The experimental methods include data mining, bioinformatics, EC functional analyses, OSA mouse models, and assessment of OSA human subjects. Measurements and Main Results: Using mined microRNA sequencing data, we found that microRNA 210 (miR-210) conferred the greatest induction by intermittent hypoxia in ECs. Consistently, the serum concentration of miR-210 was higher in individuals with OSA from two independent cohorts. Importantly, miR-210 concentration was positively correlated with the apnea–hypopnea index. RNA sequencing data collected from ECs transfected with miR-210 or treated with OSA serum showed a set of genes commonly altered by miR-210 and OSA serum, which are largely involved in mitochondrion-related pathways. ECs transfected with miR-210 or treated with OSA serum showed reduced V˙o2 rate, mitochondrial membrane potential, and DNA abundance. Mechanistically, intermittent hypoxia-induced SREBP2 (sterol regulatory element–binding protein 2) bound to the promoter region of miR-210, which in turn inhibited the iron–sulfur cluster assembly enzyme and led to mitochondrial dysfunction. Moreover, the SREBP2 inhibitor betulin alleviated intermittent hypoxia–increased systolic blood pressure in the OSA mouse model. Conclusions: These results identify an axis involving SREBP2, miR-210, and mitochondrial dysfunction, representing a new mechanistic link between OSA and EC dysfunction that may have important implications for treating and preventing OSA-related cardiovascular sequelae.

@article {Chaiamarit2023.03.19.533383,
	author = {Tai Chaiamarit and Adriaan Verhelle and Romain Chassefeyre and Nandini Shukla and Sammy Weiser Novak and Leonardo R. Andrade and Uri Manor and Sandra E. Encalada},
	title = {Mutant Prion Protein Endoggresomes are Hubs for Local Axonal Organelle-Cytoskeletal Remodeling},
	elocation-id = {2023.03.19.533383},
	year = {2023},
	doi = {10.1101/2023.03.19.533383},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Dystrophic axons comprising misfolded mutant prion protein (PrP) aggregates are a characteristic pathological feature in the prionopathies. These aggregates form inside endolysosomes -called endoggresomes-, within swellings that line up the length of axons of degenerating neurons. The pathways impaired by endoggresomes that result in failed axonal and consequently neuronal health, remain undefined. Here, we dissect the local subcellular impairments that occur within individual mutant PrP endoggresome swelling sites in axons. Quantitative high-resolution light and electron microscopy revealed the selective impairment of the acetylated vs tyrosinated microtubule cytoskeleton, while micro-domain image analysis of live organelle dynamics within swelling sites revealed deficits uniquely to the MT-based active transport system that translocates mitochondria and endosomes toward the synapse. Cytoskeletal and defective transport results in the retention of mitochondria, endosomes, and molecular motors at swelling sites, enhancing mitochondria-Rab7 late endosome contacts that induce mitochondrial fission via the activity of Rab7, and render mitochondria dysfunctional. Our findings point to mutant Pr Pendoggresome swelling sites as selective hubs of cytoskeletal deficits and organelle retention that drive the remodeling of organelles along axons. We propose that the dysfunction imparted locally within these axonal micro-domains spreads throughout the axon over time, leading to axonal dysfunction in prionopathies.Competing Interest StatementThe authors have declared no competing interest.},
	URL = {https://www.biorxiv.org/content/early/2023/03/21/2023.03.19.533383},
	eprint = {https://www.biorxiv.org/content/early/2023/03/21/2023.03.19.533383.full.pdf},
	journal = {bioRxiv}
}

Dystrophic axons comprising misfolded mutant prion protein (PrP) aggregates are a characteristic pathological feature in the prionopathies. These aggregates form inside endolysosomes -called endoggresomes-, within swellings that line up the length of axons of degenerating neurons. The pathways impaired by endoggresomes that result in failed axonal and consequently neuronal health, remain undefined. Here, we dissect the local subcellular impairments that occur within individual mutant PrP endoggresome swelling sites in axons. Quantitative high-resolution light and electron microscopy revealed the selective impairment of the acetylated vs tyrosinated microtubule cytoskeleton, while micro-domain image analysis of live organelle dynamics within swelling sites revealed deficits uniquely to the MT-based active transport system that translocates mitochondria and endosomes toward the synapse. Cytoskeletal and defective transport results in the retention of mitochondria, endosomes, and molecular motors at swelling sites, enhancing mitochondria-Rab7 late endosome contacts that induce mitochondrial fission via the activity of Rab7, and render mitochondria dysfunctional. Our findings point to mutant Pr Pendoggresome swelling sites as selective hubs of cytoskeletal deficits and organelle retention that drive the remodeling of organelles along axons. We propose that the dysfunction imparted locally within these axonal micro-domains spreads throughout the axon over time, leading to axonal dysfunction in prionopathies.Competing Interest StatementThe authors have declared no competing interest.

@article {Bosworth2023.03.02.529949,
	author = {AP Bosworth and M Contreras and S Weiser Novak and L Sancho and IH Salas and U Manor and NJ Allen},
	title = {Astrocyte glypican 5 regulates synapse maturation and stabilization},
	elocation-id = {2023.03.02.529949},
	year = {2023},
	doi = {10.1101/2023.03.02.529949},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {The maturation and stabilization of appropriate synaptic connections is a vital step in the development of neuronal circuits, however the molecular signals underlying these processes are not fully understood. We show that astrocytes, through production of glypican 5 (GPC5), are required for maturation and refinement of synapses in the developing mouse cortex. In the absence of astrocyte GPC5 thalamocortical synapses in the visual cortex show structural immaturity during the critical period, including smaller presynaptic terminals, decreased postsynaptic density area, and presence of more postsynaptic partners at multisynaptic connections. This structural immaturity is accompanied by a delay in developmental incorporation of GLUA2-containing calcium impermeable AMPARs at intracortical synapses. The functional impact of this is that mice lacking astrocyte GPC5 exhibit increased levels of ocular dominance plasticity in adulthood. This shows astrocyte GPC5 is necessary for maturation and stabilization of synaptic connections in typical development, with implications for understanding disorders with altered synaptic function, including Alzheimer{\textquoteright}s disease, where GPC5 levels are altered.Competing Interest StatementThe authors have declared no competing interest.},
	URL = {https://www.biorxiv.org/content/early/2023/03/02/2023.03.02.529949},
	eprint = {https://www.biorxiv.org/content/early/2023/03/02/2023.03.02.529949.full.pdf},
	journal = {bioRxiv}
}

The maturation and stabilization of appropriate synaptic connections is a vital step in the development of neuronal circuits, however the molecular signals underlying these processes are not fully understood. We show that astrocytes, through production of glypican 5 (GPC5), are required for maturation and refinement of synapses in the developing mouse cortex. In the absence of astrocyte GPC5 thalamocortical synapses in the visual cortex show structural immaturity during the critical period, including smaller presynaptic terminals, decreased postsynaptic density area, and presence of more postsynaptic partners at multisynaptic connections. This structural immaturity is accompanied by a delay in developmental incorporation of GLUA2-containing calcium impermeable AMPARs at intracortical synapses. The functional impact of this is that mice lacking astrocyte GPC5 exhibit increased levels of ocular dominance plasticity in adulthood. This shows astrocyte GPC5 is necessary for maturation and stabilization of synaptic connections in typical development, with implications for understanding disorders with altered synaptic function, including Alzheimer\textquoterights disease, where GPC5 levels are altered.Competing Interest StatementThe authors have declared no competing interest.

@article{sheridan_local_2022,
	title = {Local shape descriptors for neuron segmentation},
	issn = {1548-7091, 1548-7105},
	url = {https://www.nature.com/articles/s41592-022-01711-z},
	doi = {10.1038/s41592-022-01711-z},
	urldate = {2023-01-12},
	journal = {Nature Methods},
	author = {Sheridan, Arlo and Nguyen, Tri M. and Deb, Diptodip and Lee, Wei-Chung Allen and Saalfeld, Stephan and Turaga, Srinivas C. and Manor, Uri and Funke, Jan},
	month = dec,
	year = {2022},
}

@article{Calabrese2022,
abstract = {During early ischemic brain injury, glutamate receptor hyperactivation mediates neuronal death via osmotic cell swelling. Here we show that ischemia and excess NMDA receptor activation cause actin to rapidly and extensively reorganize within the somatodendritic compartment. Normally, F-actin is concentrated within dendritic spines. However, <5 min after bath-applied NMDA, F-actin depolymerizes within spines and polymerizes into stable filaments within the dendrite shaft and soma. A similar actinification occurs after experimental ischemia in culture, and photothrombotic stroke in mouse. Following transient NMDA incubation, actinification spontaneously reverses. Na+, Cl−, water, and Ca2+ influx, and spine F-actin depolymerization are all necessary, but not individually sufficient, for actinification, but combined they induce activation of the F-actin polymerization factor inverted formin-2 (INF2). Silencing of INF2 renders neurons vulnerable to cell death and INF2 overexpression is protective. Ischemia-induced dendritic actin reorganization is therefore an intrinsic pro-survival response that protects neurons from death induced by cell edema.},
author = {Calabrese, Barbara and Jones, Steven L and Shiraishi-Yamaguchi, Yoko and Lingelbach, Michael and Manor, Uri and Svitkina, Tatyana M and Higgs, Henry N and Shih, Andy Y and Halpain, Shelley},
doi = {10.1038/s41467-022-33268-y},
issn = {2041-1723},
journal = {Nature Communications},
number = {1},
pages = {6037},
title = {{INF2-mediated actin filament reorganization confers intrinsic resilience to neuronal ischemic injury}},
url = {https://doi.org/10.1038/s41467-022-33268-y},
volume = {13},
year = {2022}
}

During early ischemic brain injury, glutamate receptor hyperactivation mediates neuronal death via osmotic cell swelling. Here we show that ischemia and excess NMDA receptor activation cause actin to rapidly and extensively reorganize within the somatodendritic compartment. Normally, F-actin is concentrated within dendritic spines. However, <5 min after bath-applied NMDA, F-actin depolymerizes within spines and polymerizes into stable filaments within the dendrite shaft and soma. A similar actinification occurs after experimental ischemia in culture, and photothrombotic stroke in mouse. Following transient NMDA incubation, actinification spontaneously reverses. Na+, Cl−, water, and Ca2+ influx, and spine F-actin depolymerization are all necessary, but not individually sufficient, for actinification, but combined they induce activation of the F-actin polymerization factor inverted formin-2 (INF2). Silencing of INF2 renders neurons vulnerable to cell death and INF2 overexpression is protective. Ischemia-induced dendritic actin reorganization is therefore an intrinsic pro-survival response that protects neurons from death induced by cell edema.

@inproceedings{
prasad2022exploring,
title={Exploring the role of image domain in self-supervised {DNN} models of rodent brains},
author={Aaditya Prasad and Uri Manor and Talmo Pereira},
booktitle={SVRHM 2022 Workshop @ NeurIPS },
year={2022},
url={https://openreview.net/forum?id=KIlSyKTulXO}
}

@ARTICLE{10.3389/fcell.2022.974168,
  AUTHOR={Taiber, Shahar and Gozlan, Oren and Cohen, Roie and Andrade, Leonardo R. and Gregory, Ellen F. and Starr, Daniel A. and Moran, Yehu and Hipp, Rebecca and Kelley, Matthew W. and Manor, Uri and Sprinzak, David and Avraham, Karen B.},   
    
  TITLE={A Nesprin-4/kinesin-1 cargo model for nuclear positioning in cochlear outer hair cells},      
    
  JOURNAL={Frontiers in Cell and Developmental Biology},      
    
  VOLUME={10},           
    
  YEAR={2022},      
      
  URL={https://www.frontiersin.org/articles/10.3389/fcell.2022.974168},       
    
  DOI={10.3389/fcell.2022.974168},      
    
  ISSN={2296-634X},   
    
  ABSTRACT={Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, Nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how Nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of Nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of Nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between Nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.}
}

Nuclear positioning is important for the functionality of many cell types and is mediated by interactions of cytoskeletal elements and nucleoskeleton proteins. Nesprin proteins, part of the linker of nucleoskeleton and cytoskeleton (LINC) complex, have been shown to participate in nuclear positioning in multiple cell types. Outer hair cells (OHCs) in the inner ear are specialized sensory epithelial cells that utilize somatic electromotility to amplify auditory signals in the cochlea. Recently, Nesprin-4 (encoded by Syne4) was shown to play a crucial role in nuclear positioning in OHCs. Syne4 deficiency in humans and mice leads to mislocalization of the OHC nuclei and cell death resulting in deafness. However, it is unknown how Nesprin-4 mediates the position of the nucleus, and which other molecular components are involved in this process. Here, we show that the interaction of Nesprin-4 and the microtubule motor kinesin-1 is mediated by a conserved 4 amino-acid motif. Using in vivo AAV gene delivery, we show that this interaction is critical for nuclear positioning and hearing in mice. Nuclear mislocalization and cell death of OHCs coincide with the onset of hearing and electromotility and are solely restricted to outer, but not inner, hair cells. Likewise, the C. elegans functional homolog of Nesprin-4, UNC-83, uses a similar motif to mediate interactions between migrating nuclei and kinesin-1. Overall, our results suggest that OHCs require unique cellular machinery for proper nuclear positioning at the onset of electromotility. This machinery relies on the interaction between Nesprin-4 and kinesin-1 motors supporting a microtubule cargo model for nuclear positioning.

@article{Jeng2022,
  abstract = {The transduction of acoustic information by hair cells depends upon mechanosensitive stereociliary bundles that project from their apical surface. Mutations or absence of the stereociliary protein EPS8 cause deafness in humans and mice, respectively. Eps8 knockout mice (Eps8?/?) have hair cells with immature stereocilia and fail to become sensory receptors. Here, we show that exogenous delivery of Eps8 using Anc80L65 in P1?P2 Eps8?/? mice in vivo rescued the hair bundle structure of apical-coil hair cells. Rescued hair bundles correctly localize EPS8, WHIRLIN, MYO15, and BAIAP2L2, and generate normal mechanoelectrical transducer currents. Inner hair cells with normal-looking stereocilia re-expressed adult-like basolateral ion channels (BK and KCNQ4) and have normal exocytosis. The number of hair cells undergoing full recovery was not sufficient to rescue hearing in Eps8?/? mice. Adeno-associated virus (AAV)-transduction of P3 apical-coil and P1?P2 basal-coil hair cells does not rescue hair cells, nor does Anc80L65-Eps8 delivery in adult Eps8?/? mice. We propose that AAV-induced gene-base therapy is an efficient strategy to recover the complex hair-cell defects in Eps8?/? mice. However, this therapeutic approach may need to be performed in utero since, at postnatal ages, Eps8?/? hair cells appear to have matured or accumulated damage beyond the point of repair.},
  annote = {doi: 10.1016/j.omtm.2022.07.012},
  author = {Jeng, Jing-Yi and Carlton, Adam J and Goodyear, Richard J and Chinowsky, Colbie and Ceriani, Federico and Johnson, Stuart L and Sung, Tsung-Chang and Dayn, Yelena and Richardson, Guy P and Bowl, Michael R and Brown, Steve D M and Manor, Uri and Marcotti, Walter},
  doi = {10.1016/j.omtm.2022.07.012},
  issn = {2329-0501},
  journal = {Molecular Therapy - Methods & Clinical Development},
  month = {sep},
  pages = {355--370},
  publisher = {Elsevier},
  title = {{AAV-mediated rescue of <em>Eps8</em> expression <em>in&#xa0;vivo</em> restores hair-cell function in a mouse model of recessive deafness}},
  url = {https://doi.org/10.1016/j.omtm.2022.07.012},
  volume = {26},
  year = {2022}
}

The transduction of acoustic information by hair cells depends upon mechanosensitive stereociliary bundles that project from their apical surface. Mutations or absence of the stereociliary protein EPS8 cause deafness in humans and mice, respectively. Eps8 knockout mice (Eps8?/?) have hair cells with immature stereocilia and fail to become sensory receptors. Here, we show that exogenous delivery of Eps8 using Anc80L65 in P1?P2 Eps8?/? mice in vivo rescued the hair bundle structure of apical-coil hair cells. Rescued hair bundles correctly localize EPS8, WHIRLIN, MYO15, and BAIAP2L2, and generate normal mechanoelectrical transducer currents. Inner hair cells with normal-looking stereocilia re-expressed adult-like basolateral ion channels (BK and KCNQ4) and have normal exocytosis. The number of hair cells undergoing full recovery was not sufficient to rescue hearing in Eps8?/? mice. Adeno-associated virus (AAV)-transduction of P3 apical-coil and P1?P2 basal-coil hair cells does not rescue hair cells, nor does Anc80L65-Eps8 delivery in adult Eps8?/? mice. We propose that AAV-induced gene-base therapy is an efficient strategy to recover the complex hair-cell defects in Eps8?/? mice. However, this therapeutic approach may need to be performed in utero since, at postnatal ages, Eps8?/? hair cells appear to have matured or accumulated damage beyond the point of repair.

@article{RAMIREZ2022104803,
  title = {Cochlear ribbon synapse maturation requires Nlgn1 and Nlgn3},
  journal = {iScience},
  volume = {25},
  number = {8},
  pages = {104803},
  year = {2022},
  issn = {2589-0042},
  doi = {https://doi.org/10.1016/j.isci.2022.104803},
  url = {https://www.sciencedirect.com/science/article/pii/S2589004222010756},
  author = {Miguel A. Ramirez and Yuzuru Ninoyu and Cayla Miller and Leonardo R. Andrade and Seby Edassery and Ewa Bomba-Warczak and Briana Ortega and Uri Manor and Mark A. Rutherford and Rick A. Friedman and Jeffrey N. Savas},
  keywords = {genomics, neuroscience, cellular neuroscience, sensory neuroscience},
  abstract = {Summary
  Hearing depends on precise synaptic transmission between cochlear inner hair cells and spiral ganglion neurons through afferent ribbon synapses. Neuroligins (Nlgns) facilitate synapse maturation in the brain, but they have gone unstudied in the cochlea. We report Nlgn3 and Nlgn1 knockout (KO) cochleae have fewer ribbon synapses and have impaired hearing. Nlgn3 KO is more vulnerable to noise trauma with limited activity at high frequencies one day after noise. Furthermore, Nlgn3 KO cochleae have a 5-fold reduction in synapse number compared to wild type after two weeks of recovery. Double KO cochlear phenotypes are more prominent than the KOs, for example, 5-fold smaller synapses, 25% reduction in synapse density, and 30% less synaptic output. These observations indicate Nlgn3 and Nlgn1 are essential to cochlear ribbon synapse maturation and function.}
}

Summary Hearing depends on precise synaptic transmission between cochlear inner hair cells and spiral ganglion neurons through afferent ribbon synapses. Neuroligins (Nlgns) facilitate synapse maturation in the brain, but they have gone unstudied in the cochlea. We report Nlgn3 and Nlgn1 knockout (KO) cochleae have fewer ribbon synapses and have impaired hearing. Nlgn3 KO is more vulnerable to noise trauma with limited activity at high frequencies one day after noise. Furthermore, Nlgn3 KO cochleae have a 5-fold reduction in synapse number compared to wild type after two weeks of recovery. Double KO cochlear phenotypes are more prominent than the KOs, for example, 5-fold smaller synapses, 25% reduction in synapse density, and 30% less synaptic output. These observations indicate Nlgn3 and Nlgn1 are essential to cochlear ribbon synapse maturation and function.

@article{https://doi.org/10.1096/fasebj.2022.36.S1.0I661,
  author = {DelGiorno, Kathleen and Ma, Zhibo and Lytle, Nikki and Chen, Bob and Jyotsana, Nidhi and Weiser Novak, Sammy and Cho, Charles and Caplan, Leah and Ben-Levy, Olivia and Neininger, Abigail and Burnette, Dylan and Trinh, Vincent and Tan, Marcus and Manor, Uri and Mills, Jason and Goldenring, James and Lau, Ken and Wahl, Geoffrey},
  title = {Metaplasia-induced Epithelial Heterogeneity Directs Pancreatic Injury and Tumorigenesis},
  journal = {The FASEB Journal},
  volume = {36},
  number = {S1},
  pages = {},
  doi = {https://doi.org/10.1096/fasebj.2022.36.S1.0I661},
  url = {https://faseb.onlinelibrary.wiley.com/doi/abs/10.1096/fasebj.2022.36.S1.0I661},
  eprint = {https://faseb.onlinelibrary.wiley.com/doi/pdf/10.1096/fasebj.2022.36.S1.0I661},
  abstract = {Background and Aims Despite years of research, mechanisms of pancreatic injury and healing remain poorly understood. Acute pancreatitis is a painful and debilitating condition; chronic pancreatitis may be asymptomatic, but greatly enhances the risk of pancreatic ductal adenocarcinoma (PDAC). Acinar to ductal metaplasia (ADM), or the transdifferentiation of digestive enzyme producing acinar cells to ductal cells, is an early event in both conditions. While ADM is thought to function in healing and regeneration, it also represents a first step in tumorigenesis, demonstrating the duplicitous nature of this inherent plasticity. The goal of these studies was to define the populations arising in ADM, associated transcriptional changes, and their role in disease progression. Methods Acinar cells were lineage traced to follow their fate upon injury. Transcripts of more than 13,000 EYFP+ cells were determined using single cell RNA sequencing (scRNA-seq). Developmental trajectories were generated using several computational biology approaches that rely on non-overlapping assumptions. Data were compared to scRNA-seq studies of gastric metaplasia, oncogenic KrasG12D-induced ADM, and human pancreatitis. Results were confirmed using immunostaining and electron microscopy. Tuft and enteroendocrine cell (EEC) populations were quantified throughout tumorigenesis. KrasG12D was expressed in injury-induced ADM populations using several inducible Cre drivers. Results scRNA-seq of ADM from chronically injured pancreata revealed emergence of a mucin/ductal population that resembles gastric pyloric metaplasia. Developmental trajectories suggest that some pyloric metaplasia cells generate tuft or EEC populations as distinct lineages. Comparison to KrasG12D-induced ADM reveals populations associated with disease progression. Immunostaining demonstrates that tuft and EEC formation is an early event in tumorigenesis. Activation of KrasG12D in ADM populations results in neoplastic transformation and the formation of MUC5AC+ pit cells. Human pancreatitis samples reflect a pyloric metaplasia phenotype as well as the formation of tuft and EEC populations. Conclusions ADM under conditions of chronic injury results in the formation of a pyloric-type metaplasia which seeds disparate tuft and EEC lineages. This carefully orchestrated plasticity generates myriad epithelial cell types which likely mitigate injury, providing protection from the formation of pancreatitis and PDAC. KrasG12D expression is sufficient to drive neoplasia when targeted to injury-induced ADM populations offering an alternative origin for tumorigenesis. This program is conserved in human pancreatitis and provides insight into early events in pancreas diseases.},
  year = {2022}
}

Background and Aims Despite years of research, mechanisms of pancreatic injury and healing remain poorly understood. Acute pancreatitis is a painful and debilitating condition; chronic pancreatitis may be asymptomatic, but greatly enhances the risk of pancreatic ductal adenocarcinoma (PDAC). Acinar to ductal metaplasia (ADM), or the transdifferentiation of digestive enzyme producing acinar cells to ductal cells, is an early event in both conditions. While ADM is thought to function in healing and regeneration, it also represents a first step in tumorigenesis, demonstrating the duplicitous nature of this inherent plasticity. The goal of these studies was to define the populations arising in ADM, associated transcriptional changes, and their role in disease progression. Methods Acinar cells were lineage traced to follow their fate upon injury. Transcripts of more than 13,000 EYFP+ cells were determined using single cell RNA sequencing (scRNA-seq). Developmental trajectories were generated using several computational biology approaches that rely on non-overlapping assumptions. Data were compared to scRNA-seq studies of gastric metaplasia, oncogenic KrasG12D-induced ADM, and human pancreatitis. Results were confirmed using immunostaining and electron microscopy. Tuft and enteroendocrine cell (EEC) populations were quantified throughout tumorigenesis. KrasG12D was expressed in injury-induced ADM populations using several inducible Cre drivers. Results scRNA-seq of ADM from chronically injured pancreata revealed emergence of a mucin/ductal population that resembles gastric pyloric metaplasia. Developmental trajectories suggest that some pyloric metaplasia cells generate tuft or EEC populations as distinct lineages. Comparison to KrasG12D-induced ADM reveals populations associated with disease progression. Immunostaining demonstrates that tuft and EEC formation is an early event in tumorigenesis. Activation of KrasG12D in ADM populations results in neoplastic transformation and the formation of MUC5AC+ pit cells. Human pancreatitis samples reflect a pyloric metaplasia phenotype as well as the formation of tuft and EEC populations. Conclusions ADM under conditions of chronic injury results in the formation of a pyloric-type metaplasia which seeds disparate tuft and EEC lineages. This carefully orchestrated plasticity generates myriad epithelial cell types which likely mitigate injury, providing protection from the formation of pancreatitis and PDAC. KrasG12D expression is sufficient to drive neoplasia when targeted to injury-induced ADM populations offering an alternative origin for tumorigenesis. This program is conserved in human pancreatitis and provides insight into early events in pancreas diseases.

@article{MA2022604,
  title = {Single-Cell Transcriptomics Reveals a Conserved Metaplasia Program in Pancreatic Injury},
  journal = {Gastroenterology},
  volume = {162},
  number = {2},
  pages = {604-620.e20},
  year = {2022},
  issn = {0016-5085},
  doi = {https://doi.org/10.1053/j.gastro.2021.10.027},
  url = {https://www.sciencedirect.com/science/article/pii/S0016508521036659},
  author = {Zhibo Ma and Nikki K. Lytle and Bob Chen and Nidhi Jyotsana and Sammy Weiser Novak and Charles J. Cho and Leah Caplan and Olivia Ben-Levy and Abigail C. Neininger and Dylan T. Burnette and Vincent Q. Trinh and Marcus C.B. Tan and Emilee A. Patterson and Rafael {Arrojo e Drigo} and Rajshekhar R. Giraddi and Cynthia Ramos and Anna L. Means and Ichiro Matsumoto and Uri Manor and Jason C. Mills and James R. Goldenring and Ken S. Lau and Geoffrey M. Wahl and Kathleen E. DelGiorno},
  keywords = {ADM, Plasticity, Paligenosis, Tuft Cells, Enteroendocrine Cells},
  abstract = {Background & Aims
  Acinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression.
  Methods
  Acinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate post-injury. Transcripts of more than 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared with gastric metaplasia, KrasG12D-induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. KrasG12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining.
  Results
  scRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison with KrasG12D-induced ADM identifies populations associated with disease progression. Activation of KrasG12D expression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype.
  Conclusions
  Under conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse. KrasG12D expression is sufficient to drive neoplasia when targeted to injury-induced ADM populations and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases.}
}

Background & Aims Acinar to ductal metaplasia (ADM) occurs in the pancreas in response to tissue injury and is a potential precursor for adenocarcinoma. The goal of these studies was to define the populations arising from ADM, the associated transcriptional changes, and markers of disease progression. Methods Acinar cells were lineage-traced with enhanced yellow fluorescent protein (EYFP) to follow their fate post-injury. Transcripts of more than 13,000 EYFP+ cells were determined using single-cell RNA sequencing (scRNA-seq). Developmental trajectories were generated. Data were compared with gastric metaplasia, KrasG12D-induced neoplasia, and human pancreatitis. Results were confirmed by immunostaining and electron microscopy. KrasG12D was expressed in injury-induced ADM using several inducible Cre drivers. Surgical specimens of chronic pancreatitis from 15 patients were evaluated by immunostaining. Results scRNA-seq of ADM revealed emergence of a mucin/ductal population resembling gastric pyloric metaplasia. Lineage trajectories suggest that some pyloric metaplasia cells can generate tuft and enteroendocrine cells (EECs). Comparison with KrasG12D-induced ADM identifies populations associated with disease progression. Activation of KrasG12D expression in HNF1B+ or POU2F3+ ADM populations leads to neoplastic transformation and formation of MUC5AC+ gastric-pit-like cells. Human pancreatitis samples also harbor pyloric metaplasia with a similar transcriptional phenotype. Conclusions Under conditions of chronic injury, acinar cells undergo a pyloric-type metaplasia to mucinous progenitor-like populations, which seed disparate tuft cell and EEC lineages. ADM-derived EEC subtypes are diverse. KrasG12D expression is sufficient to drive neoplasia when targeted to injury-induced ADM populations and offers an alternative origin for tumorigenesis. This program is conserved in human pancreatitis, providing insight into early events in pancreas diseases.

@article {Newman2022.10.12.511955,
	author = {Laura E. Newman and Nimesha Tadepalle and Sammy Weiser Novak and Cara R. Schiavon and Gladys R. Rojas and Joshua A. Chevez and Ian Lemersal and Michaela Medina and Sienna Rocha and Christina G. Towers and Danielle A. Grotjahn and Uri Manor and Gerald S. Shadel},
	title = {Endosomal removal and disposal of dysfunctional, immunostimulatory mitochondrial DNA},
	elocation-id = {2022.10.12.511955},
	year = {2022},
	doi = {10.1101/2022.10.12.511955},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Maternally inherited mitochondrial DNA (mtDNA) encodes essential subunits of the mitochondrial oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation1. This function of mtDNA contributes to antiviral resistance, but unfortunately also causes pathogenic inflammation in many disease contexts2. Cells experiencing mtDNA stress due to depletion of the mtDNA-packaging protein, Transcription Factor A, Mitochondrial (TFAM), or HSV-1 infection exhibit elongated mitochondria, mtDNA depletion, enlargement of nucleoids (mtDNA-protein complexes), and activation of cGAS/STING innate immune signaling via mtDNA released into the cytoplasm3. However, the relationships between altered mitochondrial dynamics and mtDNA-mediated activation of the cGAS-STING pathway remain unclear. Here, we show that entire enlarged nucleoids are released from mitochondria that remain bound to TFAM and colocalize with cGAS. These nucleoids arise at sites of mtDNA replication due to a block in mitochondrial fission at a stage when endoplasmic reticulum (ER) actin polymerization would normally commence, which we propose is a fission checkpoint to ensure that mtDNA has completed replication and is competent for segregation into daughter mitochondria. Released nucleoids also colocalize with the early endosomal marker RAB5 as well as the late endosomal marker RAB7 in TFAM-deficient cells and in response to mtDNA stress caused by the HSV-1 UL12.5 protein. Loss of RAB7 increases interferon stimulated gene (ISG) expression. Thus, we propose that defects in mtDNA replication and/or segregation enact a late mitochondrial fission checkpoint that, if persistent, leads to selective removal of dysfunctional nucleoids by a mitochondrial-endosomal pathway. Early steps in this pathway are prone to mtDNA release and cGAS-STING activation, but the immunostimulatory mtDNA is ultimately disposed of through a mechanism involving RAB7-containing late endosomes to prevent excessive innate immune signaling. This mtDNA quality control pathway might represent a therapeutic target to prevent mtDNA-mediated inflammation and associated pathology.Competing Interest StatementThe authors have declared no competing interest.},
	URL = {https://www.biorxiv.org/content/early/2022/10/12/2022.10.12.511955},
	eprint = {https://www.biorxiv.org/content/early/2022/10/12/2022.10.12.511955.full.pdf},
	journal = {bioRxiv}
}

Maternally inherited mitochondrial DNA (mtDNA) encodes essential subunits of the mitochondrial oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation1. This function of mtDNA contributes to antiviral resistance, but unfortunately also causes pathogenic inflammation in many disease contexts2. Cells experiencing mtDNA stress due to depletion of the mtDNA-packaging protein, Transcription Factor A, Mitochondrial (TFAM), or HSV-1 infection exhibit elongated mitochondria, mtDNA depletion, enlargement of nucleoids (mtDNA-protein complexes), and activation of cGAS/STING innate immune signaling via mtDNA released into the cytoplasm3. However, the relationships between altered mitochondrial dynamics and mtDNA-mediated activation of the cGAS-STING pathway remain unclear. Here, we show that entire enlarged nucleoids are released from mitochondria that remain bound to TFAM and colocalize with cGAS. These nucleoids arise at sites of mtDNA replication due to a block in mitochondrial fission at a stage when endoplasmic reticulum (ER) actin polymerization would normally commence, which we propose is a fission checkpoint to ensure that mtDNA has completed replication and is competent for segregation into daughter mitochondria. Released nucleoids also colocalize with the early endosomal marker RAB5 as well as the late endosomal marker RAB7 in TFAM-deficient cells and in response to mtDNA stress caused by the HSV-1 UL12.5 protein. Loss of RAB7 increases interferon stimulated gene (ISG) expression. Thus, we propose that defects in mtDNA replication and/or segregation enact a late mitochondrial fission checkpoint that, if persistent, leads to selective removal of dysfunctional nucleoids by a mitochondrial-endosomal pathway. Early steps in this pathway are prone to mtDNA release and cGAS-STING activation, but the immunostimulatory mtDNA is ultimately disposed of through a mechanism involving RAB7-containing late endosomes to prevent excessive innate immune signaling. This mtDNA quality control pathway might represent a therapeutic target to prevent mtDNA-mediated inflammation and associated pathology.Competing Interest StatementThe authors have declared no competing interest.

@article {Okur2022.08.25.505332,
	author = {Mustafa N. Okur and Risako Kimura and Burcin Duan Sahbaz and Uri Manor and Jaimin Patel and Leo Andrade and Kala Puligilla and Deborah L. Croteau and Vilhelm A. Bohr},
	title = {Long-term NAD+ supplementation prevents the progression of age-related hearing loss in mice},
	elocation-id = {2022.08.25.505332},
	year = {2022},
	doi = {10.1101/2022.08.25.505332},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Age-related hearing loss (ARHL) is the most common sensory disability associated with human aging. Yet, there are no approved measures for preventing or treating this debilitating condition. With its slow progression, continuous and safe approaches are critical for ARHL treatment. Nicotinamide Riboside (NR), a NAD+ precursor, is well tolerated even for long-term use and is already shown effective in various disease models including Alzheimer{\textquoteright}s and Parkinson{\textquoteright}s Disease. It has also been beneficial against noise induced hearing loss and in hearing loss associated with premature aging. However, its beneficial impact on ARHL is not known. Using two different wild-type mouse strains, we show that long-term NR administration prevents the progression of ARHL. Through transcriptomic and biochemical analysis, we find that NR administration restores age-associated reduction in cochlear NAD+ levels, upregulates biological pathways associated with synaptic transmission and PPAR signaling, and reduces the number of orphan ribbon synapses between afferent auditory neurons and inner hair cells. We also find that NR targets a novel pathway of lipid droplets in the cochlea by inducing the expression of CIDEC and PLIN1 proteins that are downstream of PPAR signaling and are key for lipid droplet growth. Taken together, our results demonstrate the therapeutic potential of NR treatment for ARHL and provide novel insights into its mechanism of action.Competing Interest StatementV Bohr received Nicotinamide riboside from Chromadex Corp},
	URL = {https://www.biorxiv.org/content/early/2022/08/26/2022.08.25.505332},
	eprint = {https://www.biorxiv.org/content/early/2022/08/26/2022.08.25.505332.full.pdf},
	journal = {bioRxiv}
}

Age-related hearing loss (ARHL) is the most common sensory disability associated with human aging. Yet, there are no approved measures for preventing or treating this debilitating condition. With its slow progression, continuous and safe approaches are critical for ARHL treatment. Nicotinamide Riboside (NR), a NAD+ precursor, is well tolerated even for long-term use and is already shown effective in various disease models including Alzheimer\textquoterights and Parkinson\textquoterights Disease. It has also been beneficial against noise induced hearing loss and in hearing loss associated with premature aging. However, its beneficial impact on ARHL is not known. Using two different wild-type mouse strains, we show that long-term NR administration prevents the progression of ARHL. Through transcriptomic and biochemical analysis, we find that NR administration restores age-associated reduction in cochlear NAD+ levels, upregulates biological pathways associated with synaptic transmission and PPAR signaling, and reduces the number of orphan ribbon synapses between afferent auditory neurons and inner hair cells. We also find that NR targets a novel pathway of lipid droplets in the cochlea by inducing the expression of CIDEC and PLIN1 proteins that are downstream of PPAR signaling and are key for lipid droplet growth. Taken together, our results demonstrate the therapeutic potential of NR treatment for ARHL and provide novel insights into its mechanism of action.Competing Interest StatementV Bohr received Nicotinamide riboside from Chromadex Corp

@article{doi:10.1126/sciadv.abg3693,
  author = {Romain Chassefeyre  and Tai Chaiamarit  and Adriaan Verhelle  and Sammy Weiser Novak  and Leonardo R. Andrade  and André D. G. Leitão  and Uri Manor  and Sandra E. Encalada },
  title = {Endosomal sorting drives the formation of axonal prion protein endoggresomes},
  journal = {Science Advances},
  volume = {7},
  number = {52},
  pages = {eabg3693},
  year = {2021},
  doi = {10.1126/sciadv.abg3693},
  URL = {https://www.science.org/doi/abs/10.1126/sciadv.abg3693},
  eprint = {https://www.science.org/doi/pdf/10.1126/sciadv.abg3693},
  abstract = {Mutant prion protein particles form axonal aggregates inside endolysosomes via axonal transport and endosomal fusion. The pathogenic aggregation of misfolded prion protein (PrP) in axons underlies prion disease pathologies. The molecular mechanisms driving axonal misfolded PrP aggregate formation leading to neurotoxicity are unknown. We found that the small endolysosomal guanosine triphosphatase (GTPase) Arl8b recruits kinesin-1 and Vps41 (HOPS) onto endosomes carrying misfolded mutant PrP to promote their axonal entry and homotypic fusion toward aggregation inside enlarged endomembranes that we call endoggresomes. This axonal rapid endosomal sorting and transport-dependent aggregation (ARESTA) mechanism forms pathologic PrP endoggresomes that impair calcium dynamics and reduce neuronal viability. Inhibiting ARESTA diminishes endoggresome formation, rescues calcium influx, and prevents neuronal death. Our results identify ARESTA as a key pathway for the regulation of endoggresome formation and a new actionable antiaggregation target to ameliorate neuronal dysfunction in the prionopathies.}}

Mutant prion protein particles form axonal aggregates inside endolysosomes via axonal transport and endosomal fusion. The pathogenic aggregation of misfolded prion protein (PrP) in axons underlies prion disease pathologies. The molecular mechanisms driving axonal misfolded PrP aggregate formation leading to neurotoxicity are unknown. We found that the small endolysosomal guanosine triphosphatase (GTPase) Arl8b recruits kinesin-1 and Vps41 (HOPS) onto endosomes carrying misfolded mutant PrP to promote their axonal entry and homotypic fusion toward aggregation inside enlarged endomembranes that we call endoggresomes. This axonal rapid endosomal sorting and transport-dependent aggregation (ARESTA) mechanism forms pathologic PrP endoggresomes that impair calcium dynamics and reduce neuronal viability. Inhibiting ARESTA diminishes endoggresome formation, rescues calcium influx, and prevents neuronal death. Our results identify ARESTA as a key pathway for the regulation of endoggresome formation and a new actionable antiaggregation target to ameliorate neuronal dysfunction in the prionopathies.

@article {Priessner2021.11.02.466664,
	author = {Martin Priessner and David C.A. Gaboriau and Arlo Sheridan and Tchern Lenn and Jonathan R. Chubb and Uri Manor and Ramon Vilar and Romain F. Laine},
	title = {Content-aware frame interpolation (CAFI): Deep Learning-based temporal super-resolution for fast bioimaging},
	elocation-id = {2021.11.02.466664},
	year = {2021},
	doi = {10.1101/2021.11.02.466664},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {The development of high-resolution microscopes has made it possible to investigate cellular processes in 4D (3D over time). However, observing fast cellular dynamics remains challenging as a consequence of photobleaching and phototoxicity. These issues become increasingly problematic with the depth of the volume acquired and the speed of the biological events of interest. Here, we report the implementation of two content-aware frame interpolation (CAFI) deep learning networks, Zooming SlowMo (ZS) and Depth-Aware Video Frame Interpolation (DAIN), based on combinations of recurrent neural networks, that are highly suited for accurately predicting images in between image pairs, therefore improving the temporal resolution of image series as a post-acquisition analysis step. We show that CAFI predictions are capable of understanding the motion context of biological structures to perform better than standard interpolation methods. We benchmark CAFI{\textquoteright}s performance on six different datasets, obtained from three different microscopy modalities (point-scanning confocal, spinning-disk confocal and confocal brightfield microscopy). We demonstrate its capabilities for single-particle tracking methods applied to the study of lysosome trafficking. CAFI therefore allows for reduced light exposure and phototoxicity on the sample and extends the possibility of long-term live-cell imaging. Both DAIN and ZS as well as the training and testing data are made available for use by the wider community via the ZeroCostDL4Mic platform.Competing Interest StatementThe authors have declared no competing interest.},
	URL = {https://www.biorxiv.org/content/early/2021/11/03/2021.11.02.466664},
	eprint = {https://www.biorxiv.org/content/early/2021/11/03/2021.11.02.466664.full.pdf},
	journal = {bioRxiv}
}

The development of high-resolution microscopes has made it possible to investigate cellular processes in 4D (3D over time). However, observing fast cellular dynamics remains challenging as a consequence of photobleaching and phototoxicity. These issues become increasingly problematic with the depth of the volume acquired and the speed of the biological events of interest. Here, we report the implementation of two content-aware frame interpolation (CAFI) deep learning networks, Zooming SlowMo (ZS) and Depth-Aware Video Frame Interpolation (DAIN), based on combinations of recurrent neural networks, that are highly suited for accurately predicting images in between image pairs, therefore improving the temporal resolution of image series as a post-acquisition analysis step. We show that CAFI predictions are capable of understanding the motion context of biological structures to perform better than standard interpolation methods. We benchmark CAFI\textquoterights performance on six different datasets, obtained from three different microscopy modalities (point-scanning confocal, spinning-disk confocal and confocal brightfield microscopy). We demonstrate its capabilities for single-particle tracking methods applied to the study of lysosome trafficking. CAFI therefore allows for reduced light exposure and phototoxicity on the sample and extends the possibility of long-term live-cell imaging. Both DAIN and ZS as well as the training and testing data are made available for use by the wider community via the ZeroCostDL4Mic platform.Competing Interest StatementThe authors have declared no competing interest.

@article{Shadel2021,
  abstract = {Understanding basic mechanisms of aging holds great promise for developing interventions that prevent or delay many age-related declines and diseases simultaneously to increase human healthspan. However, a major confounding factor in aging research is the heterogeneity of the aging process itself. At the organismal level, it is clear that chronological age does not always predict biological age or susceptibility to frailty or pathology. While genetics and environment are major factors driving variable rates of aging, additional complexity arises because different organs, tissues, and cell types are intrinsically heterogeneous and exhibit different aging trajectories normally or in response to the stresses of the aging process (e.g., damage accumulation). Tackling the heterogeneity of aging requires new and specialized tools (e.g., single-cell analyses, mass spectrometry-based approaches, and advanced imaging) to identify novel signatures of aging across scales. Cutting-edge computational approaches are then needed to integrate these disparate datasets and elucidate network interactions between known aging hallmarks. There is also a need for improved, human cell-based models of aging to ensure that basic research findings are relevant to human aging and healthspan interventions. The San Diego Nathan Shock Center (SD-NSC) provides access to cutting-edge scientific resources to facilitate the study of the heterogeneity of aging in general and to promote the use of novel human cell models of aging. The center also has a robust Research Development Core that funds pilot projects on the heterogeneity of aging and organizes innovative training activities, including workshops and a personalized mentoring program, to help investigators new to the aging field succeed. Finally, the SD-NSC participates in outreach activities to educate the general community about the importance of aging research and promote the need for basic biology of aging research in particular.},
  author = {Shadel, Gerald S and Adams, Peter D and Berggren, W Travis and Diedrich, Jolene K and Diffenderfer, Kenneth E and Gage, Fred H and Hah, Nasun and Hansen, Malene and Hetzer, Martin W and Molina, Anthony J A and Manor, Uri and Marek, Kurt and O'Keefe, David D and Pinto, Antonio F M and Sacco, Alessandra and Sharpee, Tatyana O and Shokriev, Maxim N and Zambetti, Stefania},
  doi = {10.1007/s11357-021-00426-x},
  issn = {2509-2723},
  journal = {GeroScience},
  number = {5},
  pages = {2139--2148},
  title = {{The San Diego Nathan Shock Center: tackling the heterogeneity of aging}},
  url = {https://doi.org/10.1007/s11357-021-00426-x},
  volume = {43},
  year = {2021}
}

Understanding basic mechanisms of aging holds great promise for developing interventions that prevent or delay many age-related declines and diseases simultaneously to increase human healthspan. However, a major confounding factor in aging research is the heterogeneity of the aging process itself. At the organismal level, it is clear that chronological age does not always predict biological age or susceptibility to frailty or pathology. While genetics and environment are major factors driving variable rates of aging, additional complexity arises because different organs, tissues, and cell types are intrinsically heterogeneous and exhibit different aging trajectories normally or in response to the stresses of the aging process (e.g., damage accumulation). Tackling the heterogeneity of aging requires new and specialized tools (e.g., single-cell analyses, mass spectrometry-based approaches, and advanced imaging) to identify novel signatures of aging across scales. Cutting-edge computational approaches are then needed to integrate these disparate datasets and elucidate network interactions between known aging hallmarks. There is also a need for improved, human cell-based models of aging to ensure that basic research findings are relevant to human aging and healthspan interventions. The San Diego Nathan Shock Center (SD-NSC) provides access to cutting-edge scientific resources to facilitate the study of the heterogeneity of aging in general and to promote the use of novel human cell models of aging. The center also has a robust Research Development Core that funds pilot projects on the heterogeneity of aging and organizes innovative training activities, including workshops and a personalized mentoring program, to help investigators new to the aging field succeed. Finally, the SD-NSC participates in outreach activities to educate the general community about the importance of aging research and promote the need for basic biology of aging research in particular.

@misc{andreev2021biologists,
  title={Biologists need modern data infrastructure on campus}, 
  author={Andrey Andreev and Tom Morrell and Kristin Briney and Sandra Gesing and Uri Manor},
  year={2021},
  eprint={2108.07631},
  archivePrefix={arXiv},
  primaryClass={q-bio.OT}
}

@article{doi:10.1161/CIRCRESAHA.121.318902,
  author = {Yuyang Lei  and Jiao Zhang  and Cara R. Schiavon  and Ming He  and Lili Chen  and Hui Shen  and Yichi Zhang  and Qian Yin  and Yoshitake Cho  and Leonardo Andrade  and Gerald S. Shadel  and Mark Hepokoski  and Ting Lei  and Hongliang Wang  and Jin Zhang  and Jason X.-J. Yuan  and Atul Malhotra  and Uri Manor  and Shengpeng Wang  and Zu-Yi Yuan  and John Y-J. Shyy },
  title = {SARS-CoV-2 Spike Protein Impairs Endothelial Function via Downregulation of ACE 2},
  journal = {Circulation Research},
  volume = {128},
  number = {9},
  pages = {1323-1326},
  year = {2021},
  doi = {10.1161/CIRCRESAHA.121.318902},

  URL = {https://www.ahajournals.org/doi/abs/10.1161/CIRCRESAHA.121.318902},
  eprint = {https://www.ahajournals.org/doi/pdf/10.1161/CIRCRESAHA.121.318902}

}

@ARTICLE{10.3389/fcell.2021.624823,
  
  AUTHOR={Schiavon, Cara R. and Shadel, Gerald S. and Manor, Uri},   
    
  TITLE={Impaired Mitochondrial Mobility in Charcot-Marie-Tooth Disease},      
    
  JOURNAL={Frontiers in Cell and Developmental Biology},      
    
  VOLUME={9},           
    
  YEAR={2021},      
      
  URL={https://www.frontiersin.org/articles/10.3389/fcell.2021.624823},       
    
  DOI={10.3389/fcell.2021.624823},      
    
  ISSN={2296-634X},   
    
  ABSTRACT={Charcot-Marie-Tooth (CMT) disease is a progressive, peripheral neuropathy and the most commonly inherited neurological disorder. Clinical manifestations of CMT mutations are typically limited to peripheral neurons, the longest cells in the body. Currently, mutations in at least 80 different genes are associated with CMT and new mutations are regularly being discovered. A large portion of the proteins mutated in axonal CMT have documented roles in mitochondrial mobility, suggesting that organelle trafficking defects may be a common underlying disease mechanism. This review will focus on the potential role of altered mitochondrial mobility in the pathogenesis of axonal CMT, highlighting the conceptional challenges and potential experimental and therapeutic opportunities presented by this “impaired mobility” model of the disease.}
}

Charcot-Marie-Tooth (CMT) disease is a progressive, peripheral neuropathy and the most commonly inherited neurological disorder. Clinical manifestations of CMT mutations are typically limited to peripheral neurons, the longest cells in the body. Currently, mutations in at least 80 different genes are associated with CMT and new mutations are regularly being discovered. A large portion of the proteins mutated in axonal CMT have documented roles in mitochondrial mobility, suggesting that organelle trafficking defects may be a common underlying disease mechanism. This review will focus on the potential role of altered mitochondrial mobility in the pathogenesis of axonal CMT, highlighting the conceptional challenges and potential experimental and therapeutic opportunities presented by this “impaired mobility” model of the disease.

@techreport{Antonsen2021,
abstract = {We propose a ten-year research campaign to maximally adopt artificial intelligence (AI) and machine learning (ML) capabilities in space biology research and the design of spaceflight health systems. Fully leveraging AI/ML functionalities will enable generating and analyzing large amounts of data while requiring limited human time, and increasing knowledge gain of biological spaceflight effects. Further, integration of AI/ML into support systems for spacecraft, ecosystem, and astronaut health will increase the power of adverse event prediction and mitigation.},
author = {Antonsen, Erik and Babdor, Joel and Baranzini, Sergio E and Barker, Richard and Beheshti, Afshin and Berrios, Daniel C and Chalk, Stuart and Delgado-Aparicio, Guillermo M and Martin, Hector Garcia and Glicksberg, Benjamin S and Greene, Casey S and Haendel, Melissa and Hamid, Arif A and Hastings, Jaden JA and Heller, Philip and Hoarfrost, Adrienne and Jamieson, Daniel and Jarvis, Katelyn J and Kalantari, John and Khezeli, Kia and Komarova, Svetlana V and Komorowski, Matthieu and Kothiyal, Prachi and Mahabal, Ashish and Manor, Uri and Matar, Mona and Mias, George I and Miller, Jack and Jr, Jerry G Myers and Nelson, Charlotte and Oribello, Jonathan and Park, Seung-min and Parsons-Wingerter, Patricia and Prabhu, RK and Qutub, Amina Ann and Rask, Jon and Reynolds, Robert J and Saravia-Butler, Amanda and Saria, Suchi and Sawyer, Aenor J and Singh, Nitin Kumar and Soboczenski, Frank and Snyder, Michael and Soman, Karthik and Theriot, Corey A and Valen, David Van and Warren, Liz and Worthey, Liz and Yang, Jason H and Zitnik, Marinka},
institution = {NASA},
title = {{Machine Learning, Artificial Intelligence and Data Modeling for the Next Decade of Space Biology Research and Astronaut Health Support}},
year = {2021}
}

We propose a ten-year research campaign to maximally adopt artificial intelligence (AI) and machine learning (ML) capabilities in space biology research and the design of spaceflight health systems. Fully leveraging AI/ML functionalities will enable generating and analyzing large amounts of data while requiring limited human time, and increasing knowledge gain of biological spaceflight effects. Further, integration of AI/ML into support systems for spacecraft, ecosystem, and astronaut health will increase the power of adverse event prediction and mitigation.

@article{10.1371/journal.pgen.1009277,
  doi = {10.1371/journal.pgen.1009277},
  author = {Lehman, Bettina J. AND Lopez-Diaz, Fernando J. AND Santisakultarm, Thom P. AND Fang, Linjing AND Shokhirev, Maxim N. AND Diffenderfer, Kenneth E. AND Manor, Uri AND Emerson, Beverly M.},
  journal = {PLOS Genetics},
  publisher = {Public Library of Science},
  title = {Dynamic regulation of CTCF stability and sub-nuclear localization in response to stress},
  year = {2021},
  month = {01},
  volume = {17},
  url = {https://doi.org/10.1371/journal.pgen.1009277},
  pages = {1-34},
  abstract = {The nuclear protein CCCTC-binding factor (CTCF) has diverse roles in chromatin architecture and gene regulation. Functionally, CTCF associates with thousands of genomic sites and interacts with proteins, such as cohesin, or non-coding RNAs to facilitate specific transcriptional programming. In this study, we examined CTCF during the cellular stress response in human primary cells using immune-blotting, quantitative real time-PCR, chromatin immunoprecipitation-sequence (ChIP-seq) analysis, mass spectrometry, RNA immunoprecipitation-sequence analysis (RIP-seq), and Airyscan confocal microscopy. Unexpectedly, we found that CTCF is exquisitely sensitive to diverse forms of stress in normal patient-derived human mammary epithelial cells (HMECs). In HMECs, a subset of CTCF protein forms complexes that localize to Serine/arginine-rich splicing factor (SC-35)-containing nuclear speckles. Upon stress, this species of CTCF protein is rapidly downregulated by changes in protein stability, resulting in loss of CTCF from SC-35 nuclear speckles and changes in CTCF-RNA interactions. Our ChIP-seq analysis indicated that CTCF binding to genomic DNA is largely unchanged. Restoration of the stress-sensitive pool of CTCF protein abundance and re-localization to nuclear speckles can be achieved by inhibition of proteasome-mediated degradation. Surprisingly, we observed the same characteristics of the stress response during neuronal differentiation of human pluripotent stem cells (hPSCs). CTCF forms stress-sensitive complexes that localize to SC-35 nuclear speckles during a specific stage of neuronal commitment/development but not in differentiated neurons. We speculate that these particular CTCF complexes serve a role in RNA processing that may be intimately linked with specific genes in the vicinity of nuclear speckles, potentially to maintain cells in a certain differentiation state, that is dynamically regulated by environmental signals. The stress-regulated activity of CTCF is uncoupled in persistently stressed, epigenetically re-programmed “variant” HMECs and certain cancer cell lines. These results reveal new insights into CTCF function in cell differentiation and the stress-response with implications for oxidative damage-induced cancer initiation and neuro-degenerative diseases.},
  number = {1},

}

The nuclear protein CCCTC-binding factor (CTCF) has diverse roles in chromatin architecture and gene regulation. Functionally, CTCF associates with thousands of genomic sites and interacts with proteins, such as cohesin, or non-coding RNAs to facilitate specific transcriptional programming. In this study, we examined CTCF during the cellular stress response in human primary cells using immune-blotting, quantitative real time-PCR, chromatin immunoprecipitation-sequence (ChIP-seq) analysis, mass spectrometry, RNA immunoprecipitation-sequence analysis (RIP-seq), and Airyscan confocal microscopy. Unexpectedly, we found that CTCF is exquisitely sensitive to diverse forms of stress in normal patient-derived human mammary epithelial cells (HMECs). In HMECs, a subset of CTCF protein forms complexes that localize to Serine/arginine-rich splicing factor (SC-35)-containing nuclear speckles. Upon stress, this species of CTCF protein is rapidly downregulated by changes in protein stability, resulting in loss of CTCF from SC-35 nuclear speckles and changes in CTCF-RNA interactions. Our ChIP-seq analysis indicated that CTCF binding to genomic DNA is largely unchanged. Restoration of the stress-sensitive pool of CTCF protein abundance and re-localization to nuclear speckles can be achieved by inhibition of proteasome-mediated degradation. Surprisingly, we observed the same characteristics of the stress response during neuronal differentiation of human pluripotent stem cells (hPSCs). CTCF forms stress-sensitive complexes that localize to SC-35 nuclear speckles during a specific stage of neuronal commitment/development but not in differentiated neurons. We speculate that these particular CTCF complexes serve a role in RNA processing that may be intimately linked with specific genes in the vicinity of nuclear speckles, potentially to maintain cells in a certain differentiation state, that is dynamically regulated by environmental signals. The stress-regulated activity of CTCF is uncoupled in persistently stressed, epigenetically re-programmed “variant” HMECs and certain cancer cell lines. These results reveal new insights into CTCF function in cell differentiation and the stress-response with implications for oxidative damage-induced cancer initiation and neuro-degenerative diseases.

@article{DELGIORNO20201866,
  title = {Tuft Cells Inhibit Pancreatic Tumorigenesis in Mice by Producing Prostaglandin D2},
  journal = {Gastroenterology},
  volume = {159},
  number = {5},
  pages = {1866-1881.e8},
  year = {2020},
  issn = {0016-5085},
  doi = {https://doi.org/10.1053/j.gastro.2020.07.037},
  url = {https://www.sciencedirect.com/science/article/pii/S0016508520349994},
  author = {Kathleen E. DelGiorno and Chi-Yeh Chung and Vera Vavinskaya and H. Carlo Maurer and Sammy Weiser Novak and Nikki K. Lytle and Zhibo Ma and Rajshekhar R. Giraddi and Dezhen Wang and Linjing Fang and Razia F. Naeem and Leonardo R. Andrade and Wahida H. Ali and Hubert Tseng and Crystal Tsui and Vikas B. Gubbala and Maya Ridinger-Saison and Makoto Ohmoto and Galina A. Erikson and Carolyn O’Connor and Maxim Nikolaievich Shokhirev and Nasun Hah and Yoshihiro Urade and Ichiro Matsumoto and Susan M. Kaech and Pankaj K. Singh and Uri Manor and Kenneth P. Olive and Geoffrey M. Wahl},
  keywords = {COX1, COX2, Eicosanoids, Inflammation},
  abstract = {Background & Aims
  Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis.
  Methods
  We performed studies with LSL-KrasG12D/+;Ptf1aCre/+ mice (KC; develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice; do not develop tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice) and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA-sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasia (PanIN), 19 patients with intraductal papillary mucinous neoplasms (IPMNs), and 197 patients with PDA.
  Results
  Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D2 than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells after administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of prostaglandin D2, compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury after administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and intraductal papillary mucinous neoplasm had gene expression signatures associated with tuft cells and increased expression of Hpgds messenger RNA compared with PDA.
  Conclusions
  In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D2. These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type.}
}

Background & Aims Development of pancreatic ductal adenocarcinoma (PDA) involves acinar to ductal metaplasia and genesis of tuft cells. It has been a challenge to study these rare cells because of the lack of animal models. We investigated the role of tuft cells in pancreatic tumorigenesis. Methods We performed studies with LSL-KrasG12D/+;Ptf1aCre/+ mice (KC; develop pancreatic tumors), KC mice crossed with mice with pancreatic disruption of Pou2f3 (KPouC mice; do not develop tuft cells), or mice with pancreatic disruption of the hematopoietic prostaglandin D synthase gene (Hpgds, KHC mice) and wild-type mice. Mice were allowed to age or were given caerulein to induce pancreatitis; pancreata were collected and analyzed by histology, immunohistochemistry, RNA sequencing, ultrastructural microscopy, and metabolic profiling. We performed laser-capture dissection and RNA-sequencing analysis of pancreatic tissues from 26 patients with pancreatic intraepithelial neoplasia (PanIN), 19 patients with intraductal papillary mucinous neoplasms (IPMNs), and 197 patients with PDA. Results Pancreata from KC mice had increased formation of tuft cells and higher levels of prostaglandin D2 than wild-type mice. Pancreas-specific deletion of POU2F3 in KC mice (KPouC mice) resulted in a loss of tuft cells and accelerated tumorigenesis. KPouC mice had increased fibrosis and activation of immune cells after administration of caerulein. Pancreata from KPouC and KHC mice had significantly lower levels of prostaglandin D2, compared with KC mice, and significantly increased numbers of PanINs and PDAs. KPouC and KHC mice had increased pancreatic injury after administration of caerulein, significantly less normal tissue, more extracellular matrix deposition, and higher PanIN grade than KC mice. Human PanIN and intraductal papillary mucinous neoplasm had gene expression signatures associated with tuft cells and increased expression of Hpgds messenger RNA compared with PDA. Conclusions In mice with KRAS-induced pancreatic tumorigenesis, loss of tuft cells accelerates tumorigenesis and increases the severity of caerulein-induced pancreatic injury, via decreased production of prostaglandin D2. These data are consistent with the hypothesis that tuft cells are a metaplasia-induced tumor attenuating cell type.

@article{Schiavon2020,
  abstract = {The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are challenging to detect with fluorescence microscopy, making it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed fluorescent-protein-tagged actin nanobodies, termed ‘actin chromobodies' (ACs), targeted to organelle membranes to enable high-resolution imaging of sub-organellar actin dynamics.},
  author = {Schiavon, Cara R and Zhang, Tong and Zhao, Bing and Moore, Andrew S and Wales, Pauline and Andrade, Leonardo R and Wu, Melissa and Sung, Tsung-Chang and Dayn, Yelena and Feng, Jasmine W and Quintero, Omar A and Shadel, Gerald S and Grosse, Robert and Manor, Uri},
  doi = {10.1038/s41592-020-0926-5},
  issn = {1548-7105},
  journal = {Nature Methods},
  number = {9},
  pages = {917--921},
  title = {{Actin chromobody imaging reveals sub-organellar actin dynamics}},
  url = {https://doi.org/10.1038/s41592-020-0926-5},
  volume = {17},
  year = {2020}
}

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are challenging to detect with fluorescence microscopy, making it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed fluorescent-protein-tagged actin nanobodies, termed ‘actin chromobodies' (ACs), targeted to organelle membranes to enable high-resolution imaging of sub-organellar actin dynamics.

@article{Pan2020,
  abstract = {Cell polarity is fundamental to the development of both eukaryotes and prokaryotes, yet the mechanisms behind its formation are not well understood. Here we found that, phytohormone auxin-induced, sterol-dependent nanoclustering of cell surface transmembrane receptor kinase 1 (TMK1) is critical for the formation of polarized domains at the plasma membrane (PM) during the morphogenesis of cotyledon pavement cells (PC) in Arabidopsis. Auxin-induced TMK1 nanoclustering stabilizes flotillin1-associated ordered nanodomains, which in turn promote the nanoclustering of ROP6 GTPase that acts downstream of TMK1 to regulate cortical microtubule organization. In turn, cortical microtubules further stabilize TMK1- and flotillin1-containing nanoclusters at the PM. Hence, we propose a new paradigm for polarity formation: A diffusive signal triggers cell polarization by promoting cell surface receptor-mediated nanoclustering of signaling components and cytoskeleton-mediated positive feedback that reinforces these nanodomains into polarized domains.},
  author = {Pan, Xue and Fang, Linjing and Liu, Jianfeng and Senay-Aras, Betul and Lin, Wenwei and Zheng, Shuan and Zhang, Tong and Guo, Jingzhe and Manor, Uri and {Van Norman}, Jaimie and Chen, Weitao and Yang, Zhenbiao},
  doi = {10.1038/s41467-020-17602-w},
  issn = {2041-1723},
  journal = {Nature Communications},
  number = {1},
  pages = {3914},
  title = {{Auxin-induced signaling protein nanoclustering contributes to cell polarity formation}},
  url = {https://doi.org/10.1038/s41467-020-17602-w},
  volume = {11},
  year = {2020}
}

Cell polarity is fundamental to the development of both eukaryotes and prokaryotes, yet the mechanisms behind its formation are not well understood. Here we found that, phytohormone auxin-induced, sterol-dependent nanoclustering of cell surface transmembrane receptor kinase 1 (TMK1) is critical for the formation of polarized domains at the plasma membrane (PM) during the morphogenesis of cotyledon pavement cells (PC) in Arabidopsis. Auxin-induced TMK1 nanoclustering stabilizes flotillin1-associated ordered nanodomains, which in turn promote the nanoclustering of ROP6 GTPase that acts downstream of TMK1 to regulate cortical microtubule organization. In turn, cortical microtubules further stabilize TMK1- and flotillin1-containing nanoclusters at the PM. Hence, we propose a new paradigm for polarity formation: A diffusive signal triggers cell polarization by promoting cell surface receptor-mediated nanoclustering of signaling components and cytoskeleton-mediated positive feedback that reinforces these nanodomains into polarized domains.

@article{Boussaty2020,
  abstract = {This is the first genome-wide association study with the Hybrid Mouse Diversity Panel (HDMP) to define the genetic landscape of the variation in the suprathreshold wave 1 amplitude of the auditory brainstem response (ABR) both pre- and post-noise exposure. This measure is correlated with the density of the auditory neurons (AN) and/or the compliment of synaptic ribbons within the inner hair cells of the mouse cochlea. We analyzed suprathreshold ABR for 635 mice from 102 HMDP strains pre- and post-noise exposure (108 dB 10 kHz octave band noise exposure for 2 h) using auditory brainstem response (ABR) wave 1 suprathreshold amplitudes as part of a large survey (Myint et al., Hear Res 332:113–120, 2016). Genome-wide significance levels for pre- and post-exposure wave 1 amplitude across the HMDP were performed using FaST-LMM. Synaptic ribbon counts (Ctbp2 and mGluR2) were analyzed for the extreme strains within the HMDP. ABR wave 1 amplitude varied across all strains of the HMDP with differences ranging between 2.42 and 3.82-fold pre-exposure and between 2.43 and 7.5-fold post-exposure with several tone burst stimuli (4 kHz, 8 kHz, 12 kHz, 16 kHz, 24 kHz, and 32 kHz). Immunolabeling of paired synaptic ribbons and glutamate receptors of strains with the highest and lowest wave 1 values pre- and post-exposure revealed significant differences in functional synaptic ribbon counts. Genome-wide association analysis identified genome-wide significant threshold associations on chromosome 3 (24 kHz; JAX00105429; p < 1.12E-06) and chromosome 16 (16 kHz; JAX00424604; p < 9.02E-07) prior to noise exposure and significant associations on chromosomes 2 (32 kHz; JAX00497967; p < 3.68E-08) and 13 (8 kHz; JAX00049416; 1.07E-06) after noise exposure. In order to prioritize candidate genes, we generated cis-eQTLs from microarray profiling of RNA isolated from whole cochleae in 64 of the tested strains.},
  author = {Boussaty, Ely Cheikh and Gillard, Danielle and Lavinsky, Joel and Salehi, Pezhman and Wang, Juemei and Mendon{\c{c}}a, Aline and Allayee, Hooman and Manor, Uri and Friedman, Rick Adam},
  doi = {10.1007/s10162-020-00762-3},
  issn = {1438-7573},
  journal = {Journal of the Association for Research in Otolaryngology},
  number = {4},
  pages = {323--336},
  title = {{The Genetics of Variation of the Wave 1 Amplitude of the Mouse Auditory Brainstem Response}},
  url = {https://doi.org/10.1007/s10162-020-00762-3},
  volume = {21},
  year = {2020}
}

This is the first genome-wide association study with the Hybrid Mouse Diversity Panel (HDMP) to define the genetic landscape of the variation in the suprathreshold wave 1 amplitude of the auditory brainstem response (ABR) both pre- and post-noise exposure. This measure is correlated with the density of the auditory neurons (AN) and/or the compliment of synaptic ribbons within the inner hair cells of the mouse cochlea. We analyzed suprathreshold ABR for 635 mice from 102 HMDP strains pre- and post-noise exposure (108 dB 10 kHz octave band noise exposure for 2 h) using auditory brainstem response (ABR) wave 1 suprathreshold amplitudes as part of a large survey (Myint et al., Hear Res 332:113–120, 2016). Genome-wide significance levels for pre- and post-exposure wave 1 amplitude across the HMDP were performed using FaST-LMM. Synaptic ribbon counts (Ctbp2 and mGluR2) were analyzed for the extreme strains within the HMDP. ABR wave 1 amplitude varied across all strains of the HMDP with differences ranging between 2.42 and 3.82-fold pre-exposure and between 2.43 and 7.5-fold post-exposure with several tone burst stimuli (4 kHz, 8 kHz, 12 kHz, 16 kHz, 24 kHz, and 32 kHz). Immunolabeling of paired synaptic ribbons and glutamate receptors of strains with the highest and lowest wave 1 values pre- and post-exposure revealed significant differences in functional synaptic ribbon counts. Genome-wide association analysis identified genome-wide significant threshold associations on chromosome 3 (24 kHz; JAX00105429; p < 1.12E-06) and chromosome 16 (16 kHz; JAX00424604; p < 9.02E-07) prior to noise exposure and significant associations on chromosomes 2 (32 kHz; JAX00497967; p < 3.68E-08) and 13 (8 kHz; JAX00049416; 1.07E-06) after noise exposure. In order to prioritize candidate genes, we generated cis-eQTLs from microarray profiling of RNA isolated from whole cochleae in 64 of the tested strains.

@article{doi:10.1126/science.aaw9872,
  author = {Michael A. Badgley  and Daniel M. Kremer  and H. Carlo Maurer  and Kathleen E. DelGiorno  and Ho-Joon Lee  and Vinee Purohit  and Irina R. Sagalovskiy  and Alice Ma  and Jonathan Kapilian  and Christina E. M. Firl  and Amanda R. Decker  and Steve A. Sastra  and Carmine F. Palermo  and Leonardo R. Andrade  and Peter Sajjakulnukit  and Li Zhang  and Zachary P. Tolstyka  and Tal Hirschhorn  and Candice Lamb  and Tong Liu  and Wei Gu  and E. Scott Seeley  and Everett Stone  and George Georgiou  and Uri Manor  and Alina Iuga  and Geoffrey M. Wahl  and Brent R. Stockwell  and Costas A. Lyssiotis  and Kenneth P. Olive },
  title = {Cysteine depletion induces pancreatic tumor ferroptosis in mice},
  journal = {Science},
  volume = {368},
  number = {6486},
  pages = {85-89},
  year = {2020},
  doi = {10.1126/science.aaw9872},
  URL = {https://www.science.org/doi/abs/10.1126/science.aaw9872},
  eprint = {https://www.science.org/doi/pdf/10.1126/science.aaw9872},
  abstract = {Cell death can occur through different mechanisms, several of which are being explored as potential targets for cancer treatment. One form of cell death that has attracted recent interest is ferroptosis, which is triggered by high intracellular levels of lipid reactive oxygen species. Pancreatic cancer cells have high levels of reactive oxygen species but manage to avoid ferroptosis by importing extracellular cysteine. Studying mice bearing pancreatic tumors, Badgley et al. found that administration of a drug inhibiting cysteine import induced tumor-selective ferroptosis and inhibited tumor growth. Further work will be required to determine whether this therapeutic strategy will be effective in human pancreatic cancer, a tumor type for which new treatments are urgently needed. Science, this issue p. 85 A drug that lowers intracellular cysteine levels inhibits growth of pancreatic tumors in mice by inducing a specific form of cell death. Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC– is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC– subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.}
}

Cell death can occur through different mechanisms, several of which are being explored as potential targets for cancer treatment. One form of cell death that has attracted recent interest is ferroptosis, which is triggered by high intracellular levels of lipid reactive oxygen species. Pancreatic cancer cells have high levels of reactive oxygen species but manage to avoid ferroptosis by importing extracellular cysteine. Studying mice bearing pancreatic tumors, Badgley et al. found that administration of a drug inhibiting cysteine import induced tumor-selective ferroptosis and inhibited tumor growth. Further work will be required to determine whether this therapeutic strategy will be effective in human pancreatic cancer, a tumor type for which new treatments are urgently needed. Science, this issue p. 85 A drug that lowers intracellular cysteine levels inhibits growth of pancreatic tumors in mice by inducing a specific form of cell death. Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC– is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC– subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.

@ARTICLE{10.3389/fphys.2020.00088,
  
  AUTHOR={DelGiorno, Kathleen E. and Naeem, Razia F. and Fang, Linjing and Chung, Chi-Yeh and Ramos, Cynthia and Luhtala, Natalie and O’Connor, Carolyn and Hunter, Tony and Manor, Uri and Wahl, Geoffrey M.},   
    
  TITLE={Tuft Cell Formation Reflects Epithelial Plasticity in Pancreatic Injury: Implications for Modeling Human Pancreatitis},      
    
  JOURNAL={Frontiers in Physiology},      
    
  VOLUME={11},           
    
  YEAR={2020},      
      
  URL={https://www.frontiersin.org/articles/10.3389/fphys.2020.00088},       
    
  DOI={10.3389/fphys.2020.00088},      
    
  ISSN={1664-042X},   
    
  ABSTRACT={Chronic pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma (PDA), is a serious, widespread medical condition characterized by inflammation, fibrosis, and acinar to ductal metaplasia (ADM). ADM is a cell type transdifferentiation event where pancreatic acinar cells become ductal-like under conditions of injury or oncogenic mutation. Here, we show that chronic pancreatitis and ADM in genetically wild type mice results in the formation of a significant population of chemosensory tuft cells. Transcriptomic analyses of pancreatitis tuft cells identify expression of inflammatory mediators, consistent with a role for tuft cells in injury progression and/or resolution. Though similar to tuft cell populations in other organs and disease systems, we identified a number of key differences that suggest context-specific tuft cell functions. We evaluated seven different mouse strains for tuft cell formation in response to chronic injury and identified significant heterogeneity reflecting varying proclivity for epithelial plasticity between strains. These results have interesting implications in the role of epithelial plasticity and heterogeneity in pancreatitis and highlight the importance of mouse strain selection when modeling human disease.}
}

Chronic pancreatitis, a known risk factor for the development of pancreatic ductal adenocarcinoma (PDA), is a serious, widespread medical condition characterized by inflammation, fibrosis, and acinar to ductal metaplasia (ADM). ADM is a cell type transdifferentiation event where pancreatic acinar cells become ductal-like under conditions of injury or oncogenic mutation. Here, we show that chronic pancreatitis and ADM in genetically wild type mice results in the formation of a significant population of chemosensory tuft cells. Transcriptomic analyses of pancreatitis tuft cells identify expression of inflammatory mediators, consistent with a role for tuft cells in injury progression and/or resolution. Though similar to tuft cell populations in other organs and disease systems, we identified a number of key differences that suggest context-specific tuft cell functions. We evaluated seven different mouse strains for tuft cell formation in response to chronic injury and identified significant heterogeneity reflecting varying proclivity for epithelial plasticity between strains. These results have interesting implications in the role of epithelial plasticity and heterogeneity in pancreatitis and highlight the importance of mouse strain selection when modeling human disease.

@inbook{doi:https://doi.org/10.1002/9781119436812.ch13,
  author = {Manor, Uri},
  publisher = {John Wiley & Sons, Ltd},
  isbn = {9781119436812},
  title = {Organelle–Organelle Contacts},
  booktitle = {The Liver},
  chapter = {13},
  pages = {151-159},
  doi = {https://doi.org/10.1002/9781119436812.ch13},
  url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/9781119436812.ch13},
  eprint = {https://onlinelibrary.wiley.com/doi/pdf/10.1002/9781119436812.ch13},
  year = {2020},
  keywords = {calcium influx, cell biology, endoplasmic reticulum, ER-autophagosome contact, lipid droplet, lipid synthesis, mitochondrial fission, organelle-organelle contact, phospholipid synthesis},
  abstract = {Summary This chapter explores the implications of the evolutionary history and provides the mechanisms for organelle-organelle interactions in physiology and pathophysiology. The well-characterized organelle-organelle contact in cell biology is the endoplasmic reticulum (ER)-mitochondria junction. The vast majority of lipid synthesis enzymes are localized to the ER membrane, but several key enzymes reside within the mitochondrial outer membrane. Some phospholipid synthesis requires several key enzymes, some of which are on the ER, and others on mitochondria. Calcium influx from the ER to mitochondria appears to occur in subdomains, highlighting the existence of a highly conserved, highly organized molecular interaction that facilitates the process. Mitochondrial fission is mediated by calcium. VAPs, one of the key protein families mediating ER-autophagosome contacts and autophagosome biogenesis, mediate contacts with multiple other organelles, including the plasma membrane, mitochondria, lysosomes, and Golgi. Lipid droplets (LDs) were thought to be passive lipid-storage facilities until the discovery of multiple proteins and active functions for LDs.}
}

Summary This chapter explores the implications of the evolutionary history and provides the mechanisms for organelle-organelle interactions in physiology and pathophysiology. The well-characterized organelle-organelle contact in cell biology is the endoplasmic reticulum (ER)-mitochondria junction. The vast majority of lipid synthesis enzymes are localized to the ER membrane, but several key enzymes reside within the mitochondrial outer membrane. Some phospholipid synthesis requires several key enzymes, some of which are on the ER, and others on mitochondria. Calcium influx from the ER to mitochondria appears to occur in subdomains, highlighting the existence of a highly conserved, highly organized molecular interaction that facilitates the process. Mitochondrial fission is mediated by calcium. VAPs, one of the key protein families mediating ER-autophagosome contacts and autophagosome biogenesis, mediate contacts with multiple other organelles, including the plasma membrane, mitochondria, lysosomes, and Golgi. Lipid droplets (LDs) were thought to be passive lipid-storage facilities until the discovery of multiple proteins and active functions for LDs.

@article {Fang740548,
	author = {Linjing Fang and Fred Monroe and Sammy Weiser Novak and Lyndsey Kirk and Cara R. Schiavon and Seungyoon B. Yu and Tong Zhang and Melissa Wu and Kyle Kastner and Yoshiyuki Kubota and Zhao Zhang and Gulcin Pekkurnaz and John Mendenhall and Kristen Harris and Jeremy Howard and Uri Manor},
	title = {Deep Learning-Based Point-Scanning Super-Resolution Imaging},
	elocation-id = {740548},
	year = {2019},
	doi = {10.1101/740548},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Point scanning imaging systems (e.g. scanning electron or laser scanning confocal microscopes) are perhaps the most widely used tools for high resolution cellular and tissue imaging. Like all other imaging modalities, the resolution, speed, sample preservation, and signal-to-noise ratio (SNR) of point scanning systems are difficult to optimize simultaneously. In particular, point scanning systems are uniquely constrained by an inverse relationship between imaging speed and pixel resolution. Here we show these limitations can be mitigated via the use of deep learning-based super-sampling of undersampled images acquired on a point-scanning system, which we termed point-scanning super-resolution (PSSR) imaging. Oversampled, high SNR ground truth images acquired on scanning electron or Airyscan laser scanning confocal microscopes were {\textquoteleft}crappified{\textquoteright} to generate semi-synthetic training data for PSSR models that were then used to restore real-world undersampled images. Remarkably, our EM PSSR model could restore undersampled images acquired with different optics, detectors, samples, or sample preparation methods in other labs. PSSR enabled previously unattainable 2 nm resolution images with our serial block face scanning electron microscope system. For fluorescence, we show that undersampled confocal images combined with a multiframe PSSR model trained on Airyscan timelapses facilitates Airyscan-equivalent spatial resolution and SNR with \~{}100x lower laser dose and 16x higher frame rates than corresponding high-resolution acquisitions. In conclusion, PSSR facilitates point-scanning image acquisition with otherwise unattainable resolution, speed, and sensitivity.},
	URL = {https://www.biorxiv.org/content/early/2019/10/23/740548},
	eprint = {https://www.biorxiv.org/content/early/2019/10/23/740548.full.pdf},
	journal = {bioRxiv}
}

Point scanning imaging systems (e.g. scanning electron or laser scanning confocal microscopes) are perhaps the most widely used tools for high resolution cellular and tissue imaging. Like all other imaging modalities, the resolution, speed, sample preservation, and signal-to-noise ratio (SNR) of point scanning systems are difficult to optimize simultaneously. In particular, point scanning systems are uniquely constrained by an inverse relationship between imaging speed and pixel resolution. Here we show these limitations can be mitigated via the use of deep learning-based super-sampling of undersampled images acquired on a point-scanning system, which we termed point-scanning super-resolution (PSSR) imaging. Oversampled, high SNR ground truth images acquired on scanning electron or Airyscan laser scanning confocal microscopes were \textquoteleftcrappified\textquoteright to generate semi-synthetic training data for PSSR models that were then used to restore real-world undersampled images. Remarkably, our EM PSSR model could restore undersampled images acquired with different optics, detectors, samples, or sample preparation methods in other labs. PSSR enabled previously unattainable 2 nm resolution images with our serial block face scanning electron microscope system. For fluorescence, we show that undersampled confocal images combined with a multiframe PSSR model trained on Airyscan timelapses facilitates Airyscan-equivalent spatial resolution and SNR with \ 100x lower laser dose and 16x higher frame rates than corresponding high-resolution acquisitions. In conclusion, PSSR facilitates point-scanning image acquisition with otherwise unattainable resolution, speed, and sensitivity.

@article {DelGiorno2019.12.19.882985,
	author = {Kathleen E. DelGiorno and Chi-Yeh Chung and H. Carlo Mauer and Sammy Weiser Novak and Rajshekhar R. Giraddi and Dezhen Wang and Razia F. Naeem and Linjing Fang and Leonardo R. Andrade and Nikki K. Lytle and Wahida H. Ali and Crystal Tsui and Vikas B. Gubbala and Maya Ridinger-Saison and Makoto Ohmoto and Carolyn O{\textquoteright}Connor and Galina A. Erikson and Maxim Nikolaievich Shokhirev and Yoshihiro Urade and Ichiro Matsumoto and Vera Vavinskaya and Pankaj K. Singh and Uri Manor and Kenneth P. Olive and Geoffrey M. Wahl},
	title = {Tuft cells restrain pancreatic tumorigenesis through paracrine eicosanoid signaling},
	elocation-id = {2019.12.19.882985},
	year = {2019},
	doi = {10.1101/2019.12.19.882985},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Despite numerous advances in our understanding of pancreatic ductal adenocarcinoma (PDA) genetics and biology, this disease is expected to become the second leading cause of cancer-related U.S. deaths within the next few years. Incomplete understanding of how it arises precludes development of early detection and interception strategies to improve therapeutic outcomes. Acinar to ductal metaplasia involving genesis of tuft cells is one early step in PDA formation, but their functional significance has remained obscure due to their rarity and a lack of methods and relevant animal models for their molecular and functional analysis. Here, we show that deletion of tuft cell master regulator Pou2f3 eliminates pancreatic tuft cells and increases fibrosis, alters immune cell activation, and accelerates disease progression. We demonstrate that tuft cell expression of the prostaglandin D2 synthase Hpgds restrains pancreatic disease progression in early stages by inhibiting stromal activation. Analyses of human data sets are consistent with mouse studies. We propose that tuft cells and, by inference, the associated metaplastic lesions, play a protective role early in pancreatic tumorigenesis.Significance We find that tuft cell formation in response to oncogenic Kras is protective and restrains tumorigenesis through local production of anti-inflammatory substances, including paracrine prostaglandin D2 signaling to the stroma. Our findings establish tuft cells as a metaplasia-induced tumor suppressive cell type.},
	URL = {https://www.biorxiv.org/content/early/2019/12/20/2019.12.19.882985},
	eprint = {https://www.biorxiv.org/content/early/2019/12/20/2019.12.19.882985.full.pdf},
	journal = {bioRxiv}
}

Despite numerous advances in our understanding of pancreatic ductal adenocarcinoma (PDA) genetics and biology, this disease is expected to become the second leading cause of cancer-related U.S. deaths within the next few years. Incomplete understanding of how it arises precludes development of early detection and interception strategies to improve therapeutic outcomes. Acinar to ductal metaplasia involving genesis of tuft cells is one early step in PDA formation, but their functional significance has remained obscure due to their rarity and a lack of methods and relevant animal models for their molecular and functional analysis. Here, we show that deletion of tuft cell master regulator Pou2f3 eliminates pancreatic tuft cells and increases fibrosis, alters immune cell activation, and accelerates disease progression. We demonstrate that tuft cell expression of the prostaglandin D2 synthase Hpgds restrains pancreatic disease progression in early stages by inhibiting stromal activation. Analyses of human data sets are consistent with mouse studies. We propose that tuft cells and, by inference, the associated metaplastic lesions, play a protective role early in pancreatic tumorigenesis.Significance We find that tuft cell formation in response to oncogenic Kras is protective and restrains tumorigenesis through local production of anti-inflammatory substances, including paracrine prostaglandin D2 signaling to the stroma. Our findings establish tuft cells as a metaplasia-induced tumor suppressive cell type.

@article{Chu2019,
  abstract = {Cellular homeostasis relies on having dedicated and coordinated responses to a variety of stresses. The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a common stress that triggers a conserved pathway called the unfolded protein response (UPR) that mitigates damage, and dysregulation of UPR underlies several debilitating diseases. Here, we discover that a previously uncharacterized 54-amino acid microprotein PIGBOS regulates UPR. PIGBOS localizes to the mitochondrial outer membrane where it interacts with the ER protein CLCC1 at ER–mitochondria contact sites. Functional studies reveal that the loss of PIGBOS leads to heightened UPR and increased cell death. The characterization of PIGBOS reveals an undiscovered role for a mitochondrial protein, in this case a microprotein, in the regulation of UPR originating in the ER. This study demonstrates microproteins to be an unappreciated class of genes that are critical for inter-organelle communication, homeostasis, and cell survival.},
  author = {Chu, Qian and Martinez, Thomas F and Novak, Sammy Weiser and Donaldson, Cynthia J and Tan, Dan and Vaughan, Joan M and Chang, Tina and Diedrich, Jolene K and Andrade, Leo and Kim, Andrew and Zhang, Tong and Manor, Uri and Saghatelian, Alan},
  doi = {10.1038/s41467-019-12816-z},
  issn = {2041-1723},
  journal = {Nature Communications},
  number = {1},
  pages = {4883},
  title = {{Regulation of the ER stress response by a mitochondrial microprotein}},
  url = {https://doi.org/10.1038/s41467-019-12816-z},
  volume = {10},
  year = {2019}
}

Cellular homeostasis relies on having dedicated and coordinated responses to a variety of stresses. The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a common stress that triggers a conserved pathway called the unfolded protein response (UPR) that mitigates damage, and dysregulation of UPR underlies several debilitating diseases. Here, we discover that a previously uncharacterized 54-amino acid microprotein PIGBOS regulates UPR. PIGBOS localizes to the mitochondrial outer membrane where it interacts with the ER protein CLCC1 at ER–mitochondria contact sites. Functional studies reveal that the loss of PIGBOS leads to heightened UPR and increased cell death. The characterization of PIGBOS reveals an undiscovered role for a mitochondrial protein, in this case a microprotein, in the regulation of UPR originating in the ER. This study demonstrates microproteins to be an unappreciated class of genes that are critical for inter-organelle communication, homeostasis, and cell survival.

@article{10.1158/1538-7445.AM2019-5169,
    author = {DelGiorno, Kathleen E. and Chung, Chi-Yeh and Giraddi, Raj and Ke, Eugene and Maurer, H. Carlo and Ridinger-Saison, Maya and Ali, Wahida H. and Tsui, Crystal and Ramos, Cynthia and Naeem, Razia and Ohmoto, Makoto and Fang, Linjing and Luna, Gidsela and Fitzpatrick, Conor and O'Connor, Caz and Manor, Uri and Matsumoto, Ichiro and Olive, Kenneth P. and Wahl, Geoffrey M.},
    title = "{Abstract 5169: Pancreatic tumorigenesis evokes mechanisms of tissue injury and repair}",
    journal = {Cancer Research},
    volume = {79},
    number = {13_Supplement},
    pages = {5169-5169},
    year = {2019},
    month = {07},
    abstract = "{Despite numerous advances in our understanding of pancreatic ductal adenocarcinoma (PDA) genetics and biology, this disease is expected to become the second leading cause of cancer-related deaths in the U.S. by 2020. These statistics largely reflect the fact that by the time PDA is detected, it has already spread, making the study of early events in tumorigenesis invaluable. Harold Dvorak is credited with suggesting that tumors behave as wounds that do not heal, specifically that they are able to induce the stroma required for their maintenance and growth. Decades of research have provided an array of molecular mechanisms supporting this hypothesis. When injured, the pancreas undergoes acinar to ductal metaplasia (ADM) where digestive enzyme-producing acinar cells transdifferentiate to ductal cells; a process thought to allow for tissue healing and repair. Though a number of insightful studies have been conducted to determine the underlying mechanisms of this process, it is still incompletely understood. Using a number of high-resolution imaging techniques and lineage tracing models, we have found that chronic pancreatic injury is sufficient to induce formation of a number of differentiated cell types during ADM, including tuft cells, which are absent from the normal pancreas and may function in tissue repair.Tuft cells are solitary chemosensory cells found throughout the hollow organs of the respiratory and digestive tracts. Their expression of taste, neuronal, and inflammatory cell signaling factors is thought to enable monitoring of intraluminal homeostasis and local response via effectors. Previous studies demonstrate that, in mice, tuft cells are absent from the normal pancreas, but transdifferentiate from the acinar cell epithelium in response to oncogenic Kras expression. Interestingly, while they increase during the genesis of pancreatic intraepithelial neoplasia (PanIN), they are not detected in PDA. Tuft cell formation is also characteristic of human pancreatitis and PanIN. These data suggest a conserved, transient, but currently undefined role for tuft cells in early tumorigenesis. Here, we employ novel mouse models to elucidate this role and to identify consequences of tuft cell ablation. These studies suggest that an important function of tuft cells involves production of immune-modulatory factors in response to injury and oncogenesis. Consistent with this, we show that pancreas-specific Pou2f3 ablation eliminates tuft cell formation and enhances disease progression. Collectively, these data suggest that neoplastic lesions that form in response to oncogenic mutation evoke the cellular heterogeneity that occurs during ADM in response to tissue injury. We conclude that tuft cells and, by inference, the associated metaplastic and neoplastic lesions, play a protective role early in pancreatic injury and tumorigenesis.Citation Format: Kathleen E. DelGiorno, Chi-Yeh Chung, Raj Giraddi, Eugene Ke, H. Carlo Maurer, Maya Ridinger-Saison, Wahida H. Ali, Crystal Tsui, Cynthia Ramos, Razia Naeem, Makoto Ohmoto, Linjing Fang, Gidsela Luna, Conor Fitzpatrick, Caz O'Connor, Uri Manor, Ichiro Matsumoto, Kenneth P. Olive, Geoffrey M. Wahl. Pancreatic tumorigenesis evokes mechanisms of tissue injury and repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5169.}",
    issn = {0008-5472},
    doi = {10.1158/1538-7445.AM2019-5169},
    url = {https://doi.org/10.1158/1538-7445.AM2019-5169},
}

Despite numerous advances in our understanding of pancreatic ductal adenocarcinoma (PDA) genetics and biology, this disease is expected to become the second leading cause of cancer-related deaths in the U.S. by 2020. These statistics largely reflect the fact that by the time PDA is detected, it has already spread, making the study of early events in tumorigenesis invaluable. Harold Dvorak is credited with suggesting that tumors behave as wounds that do not heal, specifically that they are able to induce the stroma required for their maintenance and growth. Decades of research have provided an array of molecular mechanisms supporting this hypothesis. When injured, the pancreas undergoes acinar to ductal metaplasia (ADM) where digestive enzyme-producing acinar cells transdifferentiate to ductal cells; a process thought to allow for tissue healing and repair. Though a number of insightful studies have been conducted to determine the underlying mechanisms of this process, it is still incompletely understood. Using a number of high-resolution imaging techniques and lineage tracing models, we have found that chronic pancreatic injury is sufficient to induce formation of a number of differentiated cell types during ADM, including tuft cells, which are absent from the normal pancreas and may function in tissue repair.Tuft cells are solitary chemosensory cells found throughout the hollow organs of the respiratory and digestive tracts. Their expression of taste, neuronal, and inflammatory cell signaling factors is thought to enable monitoring of intraluminal homeostasis and local response via effectors. Previous studies demonstrate that, in mice, tuft cells are absent from the normal pancreas, but transdifferentiate from the acinar cell epithelium in response to oncogenic Kras expression. Interestingly, while they increase during the genesis of pancreatic intraepithelial neoplasia (PanIN), they are not detected in PDA. Tuft cell formation is also characteristic of human pancreatitis and PanIN. These data suggest a conserved, transient, but currently undefined role for tuft cells in early tumorigenesis. Here, we employ novel mouse models to elucidate this role and to identify consequences of tuft cell ablation. These studies suggest that an important function of tuft cells involves production of immune-modulatory factors in response to injury and oncogenesis. Consistent with this, we show that pancreas-specific Pou2f3 ablation eliminates tuft cell formation and enhances disease progression. Collectively, these data suggest that neoplastic lesions that form in response to oncogenic mutation evoke the cellular heterogeneity that occurs during ADM in response to tissue injury. We conclude that tuft cells and, by inference, the associated metaplastic and neoplastic lesions, play a protective role early in pancreatic injury and tumorigenesis.Citation Format: Kathleen E. DelGiorno, Chi-Yeh Chung, Raj Giraddi, Eugene Ke, H. Carlo Maurer, Maya Ridinger-Saison, Wahida H. Ali, Crystal Tsui, Cynthia Ramos, Razia Naeem, Makoto Ohmoto, Linjing Fang, Gidsela Luna, Conor Fitzpatrick, Caz O'Connor, Uri Manor, Ichiro Matsumoto, Kenneth P. Olive, Geoffrey M. Wahl. Pancreatic tumorigenesis evokes mechanisms of tissue injury and repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5169.

@article{Villanueva2019,
  abstract = {Pathological obesity can result from genetic predisposition, obesogenic diet, and circadian rhythm disruption. Obesity compromises function of muscle, which accounts for a majority of body mass. Behavioral intervention that can counteract obesity arising from genetic, diet or circadian disruption and can improve muscle function holds untapped potential to combat the obesity epidemic. Here we show that Drosophila melanogaster (fruit fly) subject to obesogenic challenges exhibits metabolic disease phenotypes in skeletal muscle; sarcomere disorganization, mitochondrial deformation, upregulation of Phospho-AKT level, aberrant intramuscular lipid infiltration, and insulin resistance. Imposing time-restricted feeding (TRF) paradigm in which flies were fed for 12 h during the day counteracts obesity-induced dysmetabolism and improves muscle performance by suppressing intramuscular fat deposits, Phospho-AKT level, mitochondrial aberrations, and markers of insulin resistance. Importantly, TRF was effective even in an irregular lighting schedule mimicking shiftwork. Hence, TRF is an effective dietary intervention for combating metabolic dysfunction arising from multiple causes.},
  author = {Villanueva, Jes{\'{u}}s E and Livelo, Christopher and Trujillo, Adriana S and Chandran, Sahaana and Woodworth, Brendon and Andrade, Leo and Le, Hiep D and Manor, Uri and Panda, Satchidananda and Melkani, Girish C},
  doi = {10.1038/s41467-019-10563-9},
  issn = {2041-1723},
  journal = {Nature Communications},
  number = {1},
  pages = {2700},
  title = {{Time-restricted feeding restores muscle function in Drosophila models of obesity and circadian-rhythm disruption}},
  url = {https://doi.org/10.1038/s41467-019-10563-9},
  volume = {10},
  year = {2019}
}

Pathological obesity can result from genetic predisposition, obesogenic diet, and circadian rhythm disruption. Obesity compromises function of muscle, which accounts for a majority of body mass. Behavioral intervention that can counteract obesity arising from genetic, diet or circadian disruption and can improve muscle function holds untapped potential to combat the obesity epidemic. Here we show that Drosophila melanogaster (fruit fly) subject to obesogenic challenges exhibits metabolic disease phenotypes in skeletal muscle; sarcomere disorganization, mitochondrial deformation, upregulation of Phospho-AKT level, aberrant intramuscular lipid infiltration, and insulin resistance. Imposing time-restricted feeding (TRF) paradigm in which flies were fed for 12 h during the day counteracts obesity-induced dysmetabolism and improves muscle performance by suppressing intramuscular fat deposits, Phospho-AKT level, mitochondrial aberrations, and markers of insulin resistance. Importantly, TRF was effective even in an irregular lighting schedule mimicking shiftwork. Hence, TRF is an effective dietary intervention for combating metabolic dysfunction arising from multiple causes.

@article {Araujo655803,
	author = {Jackeline S Araujo and Rui M P Silva-Junior and Tong Zhang and Cara R Schiavon and Qian Chu and Melissa Wu and Carmen L S Pontes and Anderson O Souza and Luciane C Alberici and Aline M Santos and Sadie Bartholomew Ingle and Alan Saghatelian and James Spudich and Uri Manor and Enilza M Espreafico},
	title = {A novel role for Myosin-Va in mitochondrial fission},
	elocation-id = {655803},
	year = {2019},
	doi = {10.1101/655803},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {In cancer cells metabolic changes and mitochondrial morphology are coupled. It is known that the cytoskeleton and molecular motors are directly involved in regulating mitochondrial morphology. Here we show that myosin-Va, an actin-based molecular motor, is required for the malignant properties of melanoma cells and localizes to mitochondria in these cells. Knockdown of myosin-Va increases cellular respiration rates and ROS production and decreases glucose uptake and lactate secretion. In addition, knockdown of myosin-Va results in reduced mitochondrial fission and correspondingly elongated mitochondria. We show that myosin-Va interacts with the mitochondrial outer membrane protein Spire1C, an actin-regulatory protein implicated in mitochondrial fission, and that Spire1C recruits myosin-Va to mitochondria. Finally, we show that during mitochondrial fission myosin-Va localization to mitochondria increases, and that myosin-Va localizes to mitochondrial fission sites immediately adjacent to Drp1 punctae. We conclude that myosin-Va facilitates mitochondrial fission. These data implicate myosin-Va as a target for the Warburg effect in melanoma cells.},
	URL = {https://www.biorxiv.org/content/early/2019/05/31/655803},
	eprint = {https://www.biorxiv.org/content/early/2019/05/31/655803.full.pdf},
	journal = {bioRxiv}
}

In cancer cells metabolic changes and mitochondrial morphology are coupled. It is known that the cytoskeleton and molecular motors are directly involved in regulating mitochondrial morphology. Here we show that myosin-Va, an actin-based molecular motor, is required for the malignant properties of melanoma cells and localizes to mitochondria in these cells. Knockdown of myosin-Va increases cellular respiration rates and ROS production and decreases glucose uptake and lactate secretion. In addition, knockdown of myosin-Va results in reduced mitochondrial fission and correspondingly elongated mitochondria. We show that myosin-Va interacts with the mitochondrial outer membrane protein Spire1C, an actin-regulatory protein implicated in mitochondrial fission, and that Spire1C recruits myosin-Va to mitochondria. Finally, we show that during mitochondrial fission myosin-Va localization to mitochondria increases, and that myosin-Va localizes to mitochondrial fission sites immediately adjacent to Drp1 punctae. We conclude that myosin-Va facilitates mitochondrial fission. These data implicate myosin-Va as a target for the Warburg effect in melanoma cells.

@article{Shi2019,
  abstract = {Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3–7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8–10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial–mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell–cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.},
  author = {Shi, Yu and Gao, Weina and Lytle, Nikki K and Huang, Peiwu and Yuan, Xiao and Dann, Amanda M and Ridinger-Saison, Maya and DelGiorno, Kathleen E and Antal, Corina E and Liang, Gaoyang and Atkins, Annette R and Erikson, Galina and Sun, Huaiyu and Meisenhelder, Jill and Terenziani, Elena and Woo, Gyunghwi and Fang, Linjing and Santisakultarm, Thom P and Manor, Uri and Xu, Ruilian and Becerra, Carlos R and Borazanci, Erkut and {Von Hoff}, Daniel D and Grandgenett, Paul M and Hollingsworth, Michael A and Leblanc, Mathias and Umetsu, Sarah E and Collisson, Eric A and Scadeng, Miriam and Lowy, Andrew M and Donahue, Timothy R and Reya, Tannishtha and Downes, Michael and Evans, Ronald M and Wahl, Geoffrey M and Pawson, Tony and Tian, Ruijun and Hunter, Tony},
  doi = {10.1038/s41586-019-1130-6},
  issn = {1476-4687},
  journal = {Nature},
  number = {7754},
  pages = {131--135},
  title = {{Targeting LIF-mediated paracrine interaction for pancreatic cancer therapy and monitoring}},
  url = {https://doi.org/10.1038/s41586-019-1130-6},
  volume = {569},
  year = {2019}
}

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis largely owing to inefficient diagnosis and tenacious drug resistance. Activation of pancreatic stellate cells (PSCs) and consequent development of dense stroma are prominent features accounting for this aggressive biology1,2. The reciprocal interplay between PSCs and pancreatic cancer cells (PCCs) not only enhances tumour progression and metastasis but also sustains their own activation, facilitating a vicious cycle to exacerbate tumorigenesis and drug resistance3–7. Furthermore, PSC activation occurs very early during PDAC tumorigenesis8–10, and activated PSCs comprise a substantial fraction of the tumour mass, providing a rich source of readily detectable factors. Therefore, we hypothesized that the communication between PSCs and PCCs could be an exploitable target to develop effective strategies for PDAC therapy and diagnosis. Here, starting with a systematic proteomic investigation of secreted disease mediators and underlying molecular mechanisms, we reveal that leukaemia inhibitory factor (LIF) is a key paracrine factor from activated PSCs acting on cancer cells. Both pharmacologic LIF blockade and genetic Lifr deletion markedly slow tumour progression and augment the efficacy of chemotherapy to prolong survival of PDAC mouse models, mainly by modulating cancer cell differentiation and epithelial–mesenchymal transition status. Moreover, in both mouse models and human PDAC, aberrant production of LIF in the pancreas is restricted to pathological conditions and correlates with PDAC pathogenesis, and changes in the levels of circulating LIF correlate well with tumour response to therapy. Collectively, these findings reveal a function of LIF in PDAC tumorigenesis, and suggest its translational potential as an attractive therapeutic target and circulating marker. Our studies underscore how a better understanding of cell–cell communication within the tumour microenvironment can suggest novel strategies for cancer therapy.

@inproceedings{antic2019decrappification,
  title={Decrappification, deoldification, and super resolution},
  author={Antic, Jason and Howard, Jeremy and Manor, Uri},
  booktitle={Facebook f8 conference},
  pages={1},
  year={2019}
}

@misc{zhang2018fanstore,
  title={FanStore: Enabling Efficient and Scalable I/O for Distributed Deep Learning}, 
  author={Zhao Zhang and Lei Huang and Uri Manor and Linjing Fang and Gabriele Merlo and Craig Michoski and John Cazes and Niall Gaffney},
  year={2018},
  eprint={1809.10799},
  archivePrefix={arXiv},
  primaryClass={cs.DC}
}

@article{doi:10.1021/acsbiomaterials.6b00802,
  author = {Curchoe, Carol Lynn and Manor, Uri},
  title = {Actin Cytoskeleton-Mediated Constriction of Membrane Organelles via Endoplasmic Reticulum Scaffolding},
  journal = {ACS Biomaterials Science \& Engineering},
  volume = {3},
  number = {11},
  pages = {2727-2732},
  year = {2017},
  doi = {10.1021/acsbiomaterials.6b00802},

  URL = { 
          https://doi.org/10.1021/acsbiomaterials.6b00802
      
  },
  eprint = { 
          https://doi.org/10.1021/acsbiomaterials.6b00802
      
  }
  ,
  abstract = { Intracellular organelles constantly undergo fission to facilitate turnover, transport, and functional changes. The cytoskeleton has long been understood to play a role in these events, and recent work strongly suggests that several conserved molecular players cooperate with the cytoskeleton to mediate the fission process. Membrane curvature-inducing, membrane scission proteins, and force-inducing cytoskeletal proteins all cooperate to drive the fission process. Recent work suggests that the endoplasmic reticulum serves as the linchpin that orchestrates and spatially organizes fission via these curvature-inducing, scission, and force-producing molecules. This all leads us to postulate a “universal theory” of organelle fission with distinct biophysical and biochemical features mediated by a finite number of physical and molecular constraints. This new physical paradigm deserves special attention from those who wish to model these processes, because previous theoretical and experimental attempts to elucidate these fission mechanisms have not included the organizing factor of the endoplasmic reticulum. Here we review the basic concepts of this new model for organelle fission, and explore the implications thereof. Previous studies that did not include this component can now be interpreted in light of these new data and serve as a useful guide for understanding how this process happens in vivo. Thus, this review provides direction for future modeling and experimental efforts to better understand how these complex systems and processes are regulated in both healthy and diseased biological systems. }
}

Intracellular organelles constantly undergo fission to facilitate turnover, transport, and functional changes. The cytoskeleton has long been understood to play a role in these events, and recent work strongly suggests that several conserved molecular players cooperate with the cytoskeleton to mediate the fission process. Membrane curvature-inducing, membrane scission proteins, and force-inducing cytoskeletal proteins all cooperate to drive the fission process. Recent work suggests that the endoplasmic reticulum serves as the linchpin that orchestrates and spatially organizes fission via these curvature-inducing, scission, and force-producing molecules. This all leads us to postulate a “universal theory” of organelle fission with distinct biophysical and biochemical features mediated by a finite number of physical and molecular constraints. This new physical paradigm deserves special attention from those who wish to model these processes, because previous theoretical and experimental attempts to elucidate these fission mechanisms have not included the organizing factor of the endoplasmic reticulum. Here we review the basic concepts of this new model for organelle fission, and explore the implications thereof. Previous studies that did not include this component can now be interpreted in light of these new data and serve as a useful guide for understanding how this process happens in vivo. Thus, this review provides direction for future modeling and experimental efforts to better understand how these complex systems and processes are regulated in both healthy and diseased biological systems.

@article{Graydon2017,
  abstract = {Ribbons are presynaptic structures that mediate synaptic vesicle release in some sensory cells of the auditory and visual systems. Although composed predominately of the protein Ribeye, very little is known about the structural dynamics of ribbons. Here we describe the in vivo mobility and turnover of Ribeye at hair cell ribbon synapses by monitoring fluorescence recovery after photobleaching (FRAP) in transgenic zebrafish with GFP-tagged Ribeye. We show that Ribeye can exchange between halves of a ribbon within $\sim$1 minute in a manner that is consistent with a simple diffusion mechanism. In contrast, exchange of Ribeye between other ribbons via the cell's cytoplasm takes several hours.},
  author = {Graydon, Cole W and Manor, Uri and Kindt, Katie S},
  doi = {10.1038/s41598-017-07940-z},
  issn = {2045-2322},
  journal = {Scientific Reports},
  number = {1},
  pages = {7467},
  title = {{In Vivo Ribbon Mobility and Turnover of Ribeye at Zebrafish Hair Cell Synapses}},
  url = {https://doi.org/10.1038/s41598-017-07940-z},
  volume = {7},
  year = {2017}
}

Ribbons are presynaptic structures that mediate synaptic vesicle release in some sensory cells of the auditory and visual systems. Although composed predominately of the protein Ribeye, very little is known about the structural dynamics of ribbons. Here we describe the in vivo mobility and turnover of Ribeye at hair cell ribbon synapses by monitoring fluorescence recovery after photobleaching (FRAP) in transgenic zebrafish with GFP-tagged Ribeye. We show that Ribeye can exchange between halves of a ribbon within $∼$1 minute in a manner that is consistent with a simple diffusion mechanism. In contrast, exchange of Ribeye between other ribbons via the cell's cytoplasm takes several hours.

@article{doi:10.1091/mbc.e16-07-0553,
  author = {Yao, Pamela J. and Manor, Uri and Petralia, Ronald S. and Brose, Rebecca D. and Wu, Ryan T. Y. and Ott, Carolyn and Wang, Ya-Xian and Charnoff, Ari and Lippincott-Schwartz, Jennifer and Mattson, Mark P.},
  title = {Sonic hedgehog pathway activation increases mitochondrial abundance and activity in hippocampal neurons},
  journal = {Molecular Biology of the Cell},
  volume = {28},
  number = {3},
  pages = {387-395},
  year = {2017},
  doi = {10.1091/mbc.e16-07-0553},
      note ={PMID: 27932496},

  URL = { 
      
          https://doi.org/10.1091/mbc.e16-07-0553
      
      

  },
  eprint = { 
      
          https://doi.org/10.1091/mbc.e16-07-0553
      
      

  }
  ,
  abstract = { Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. We present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway affects multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense, as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons. }
}

Mitochondria are essential organelles whose biogenesis, structure, and function are regulated by many signaling pathways. We present evidence that, in hippocampal neurons, activation of the Sonic hedgehog (Shh) signaling pathway affects multiple aspects of mitochondria. Mitochondrial mass was increased significantly in neurons treated with Shh. Using biochemical and fluorescence imaging analyses, we show that Shh signaling activity reduces mitochondrial fission and promotes mitochondrial elongation, at least in part, via suppression of the mitochondrial fission protein dynamin-like GTPase Drp1. Mitochondria from Shh-treated neurons were more electron-dense, as revealed by electron microscopy, and had higher membrane potential and respiratory activity. We further show that Shh protects neurons against a variety of stresses, including the mitochondrial poison rotenone, amyloid β-peptide, hydrogen peroxide, and high levels of glutamate. Collectively our data suggest a link between Shh pathway activity and the physiological properties of mitochondria in hippocampal neurons.

@article{ANDRADE2016139,
  title = {Tectorins crosslink type II collagen fibrils and connect the tectorial membrane to the spiral limbus},
  journal = {Journal of Structural Biology},
  volume = {194},
  number = {2},
  pages = {139-146},
  year = {2016},
  issn = {1047-8477},
  doi = {https://doi.org/10.1016/j.jsb.2016.01.006},
  url = {https://www.sciencedirect.com/science/article/pii/S1047847716300053},
  author = {Leonardo R. Andrade and Felipe T. Salles and M’hamed Grati and Uri Manor and Bechara Kachar},
  keywords = {Extracellular matrix, Electron microscopy, Immunogold-labeling, Tectorial membrane, Tectorins},
  abstract = {All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and β tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α- and β-tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4±1.5nm, width), resembling another collagen type protein, or chains of globular elements (15±3.2nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α- and β-tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti.}
}

All inner ear organs possess extracellular matrix appendices over the sensory epithelia that are crucial for their proper function. The tectorial membrane (TM) is a gelatinous acellular membrane located above the hearing sensory epithelium and is composed mostly of type II collagen, and α and β tectorins. TM molecules self-assemble in the endolymph fluid environment, interacting medially with the spiral limbus and distally with the outer hair cell stereocilia. Here, we used immunogold labeling in freeze-substituted mouse cochleae to assess the fine localization of both tectorins in distinct TM regions. We observed that the TM adheres to the spiral limbus through a dense thin matrix enriched in α- and β-tectorin, both likely bound to the membranes of interdental cells. Freeze-etching images revealed that type II collagen fibrils were crosslinked by short thin filaments (4±1.5nm, width), resembling another collagen type protein, or chains of globular elements (15±3.2nm, diameter). Gold-particles for both tectorins also localized adjacent to the type II collagen fibrils, suggesting that these globules might be composed essentially of α- and β-tectorins. Finally, the presence of gold-particles at the TM lower side suggests that the outer hair cell stereocilia membrane has a molecular partner to tectorins, probably stereocilin, allowing the physical connection between the TM and the organ of Corti.

@article{WEBSTER2016243,
  title = {Intravital Imaging Reveals Ghost Fibers as Architectural Units Guiding Myogenic Progenitors during Regeneration},
  journal = {Cell Stem Cell},
  volume = {18},
  number = {2},
  pages = {243-252},
  year = {2016},
  issn = {1934-5909},
  doi = {https://doi.org/10.1016/j.stem.2015.11.005},
  url = {https://www.sciencedirect.com/science/article/pii/S1934590915005032},
  author = {Micah T. Webster and Uri Manor and Jennifer Lippincott-Schwartz and Chen-Ming Fan},
  abstract = {Summary
  How resident stem cells and their immediate progenitors rebuild tissues of pre-injury organization and size for proportional regeneration is not well understood. Using 3D, time-lapse intravital imaging for direct visualization of the muscle regeneration process in live mice, we report that extracellular matrix remnants from injured skeletal muscle fibers, “ghost fibers,” govern muscle stem/progenitor cell behaviors during proportional regeneration. Stem cells were immobile and quiescent without injury whereas their activated progenitors migrated and divided after injury. Unexpectedly, divisions and migration were primarily bi-directionally oriented along the ghost fiber longitudinal axis, allowing for spreading of progenitors throughout ghost fibers. Re-orienting ghost fibers impacted myogenic progenitors’ migratory paths and division planes, causing disorganization of regenerated muscle fibers. We conclude that ghost fibers are autonomous, architectural units necessary for proportional regeneration after tissue injury. This finding reinforces the need to fabricate bioengineered matrices that mimic living tissue matrices for tissue regeneration therapy.}
}

Summary How resident stem cells and their immediate progenitors rebuild tissues of pre-injury organization and size for proportional regeneration is not well understood. Using 3D, time-lapse intravital imaging for direct visualization of the muscle regeneration process in live mice, we report that extracellular matrix remnants from injured skeletal muscle fibers, “ghost fibers,” govern muscle stem/progenitor cell behaviors during proportional regeneration. Stem cells were immobile and quiescent without injury whereas their activated progenitors migrated and divided after injury. Unexpectedly, divisions and migration were primarily bi-directionally oriented along the ghost fiber longitudinal axis, allowing for spreading of progenitors throughout ghost fibers. Re-orienting ghost fibers impacted myogenic progenitors’ migratory paths and division planes, causing disorganization of regenerated muscle fibers. We conclude that ghost fibers are autonomous, architectural units necessary for proportional regeneration after tissue injury. This finding reinforces the need to fabricate bioengineered matrices that mimic living tissue matrices for tissue regeneration therapy.

@article {10.7554/eLife.08828,
  article_type = {journal},
  title = {A mitochondria-anchored isoform of the actin-nucleating spire protein regulates mitochondrial division},
  author = {Manor, Uri and Bartholomew, Sadie and Golani, Gonen and Christenson, Eric and Kozlov, Michael and Higgs, Henry and Spudich, James and Lippincott-Schwartz, Jennifer},
  editor = {Lappalainen, Pekka},
  volume = 4,
  year = 2015,
  month = {aug},
  pub_date = {2015-08-25},
  pages = {e08828},
  citation = {eLife 2015;4:e08828},
  doi = {10.7554/eLife.08828},
  url = {https://doi.org/10.7554/eLife.08828},
  abstract = {Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.},
  keywords = {actin, mitochondria, endoplasmic reticulum, cytoskeleton, membrane, organelles},
  journal = {eLife},
  issn = {2050-084X},
  publisher = {eLife Sciences Publications, Ltd},
}

Mitochondrial division, essential for survival in mammals, is enhanced by an inter-organellar process involving ER tubules encircling and constricting mitochondria. The force for constriction is thought to involve actin polymerization by the ER-anchored isoform of the formin protein inverted formin 2 (INF2). Unknown is the mechanism triggering INF2-mediated actin polymerization at ER-mitochondria intersections. We show that a novel isoform of the formin-binding, actin-nucleating protein Spire, Spire1C, localizes to mitochondria and directly links mitochondria to the actin cytoskeleton and the ER. Spire1C binds INF2 and promotes actin assembly on mitochondrial surfaces. Disrupting either Spire1C actin- or formin-binding activities reduces mitochondrial constriction and division. We propose Spire1C cooperates with INF2 to regulate actin assembly at ER-mitochondrial contacts. Simulations support this model's feasibility and demonstrate polymerizing actin filaments can induce mitochondrial constriction. Thus, Spire1C is optimally positioned to serve as a molecular hub that links mitochondria to actin and the ER for regulation of mitochondrial division.

@article{10.1371/journal.pone.0127926,
    doi = {10.1371/journal.pone.0127926},
    author = {Orly, Gilad AND Manor, Uri AND Gov, Nir S.},
    journal = {PLOS ONE},
    publisher = {Public Library of Science},
    title = {A Biophysical Model for the Staircase Geometry of Stereocilia},
    year = {2015},
    month = {07},
    volume = {10},
    url = {https://doi.org/10.1371/journal.pone.0127926},
    pages = {1-13},
    abstract = {Cochlear hair cell bundles, made up of 10s to 100s of individual stereocilia, are essential for hearing, and even relatively minor structural changes, due to mutations or injuries, can result in total deafness. Consistent with its specialized role, the staircase geometry (SCG) of hair cell bundles presents one of the most striking, intricate, and precise organizations of actin-based cellular shapes. Composed of rows of actin-filled stereocilia with increasing lengths, the hair cell’s staircase-shaped bundle is formed from a progenitor field of smaller, thinner, and uniformly spaced microvilli with relatively invariant lengths. While recent genetic studies have provided a significant increase in information on the multitude of stereocilia protein components, there is currently no model that integrates the basic physical forces and biochemical processes necessary to explain the emergence of the SCG. We propose such a model derived from the biophysical and biochemical characteristics of actin-based protrusions. We demonstrate that polarization of the cell’s apical surface, due to the lateral polarization of the entire epithelial layer, plays a key role in promoting SCG formation. Furthermore, our model explains many distinct features of the manifestations of SCG in different species and in the presence of various deafness-associated mutations.},
    number = {7},

}

Cochlear hair cell bundles, made up of 10s to 100s of individual stereocilia, are essential for hearing, and even relatively minor structural changes, due to mutations or injuries, can result in total deafness. Consistent with its specialized role, the staircase geometry (SCG) of hair cell bundles presents one of the most striking, intricate, and precise organizations of actin-based cellular shapes. Composed of rows of actin-filled stereocilia with increasing lengths, the hair cell’s staircase-shaped bundle is formed from a progenitor field of smaller, thinner, and uniformly spaced microvilli with relatively invariant lengths. While recent genetic studies have provided a significant increase in information on the multitude of stereocilia protein components, there is currently no model that integrates the basic physical forces and biochemical processes necessary to explain the emergence of the SCG. We propose such a model derived from the biophysical and biochemical characteristics of actin-based protrusions. We demonstrate that polarization of the cell’s apical surface, due to the lateral polarization of the entire epithelial layer, plays a key role in promoting SCG formation. Furthermore, our model explains many distinct features of the manifestations of SCG in different species and in the presence of various deafness-associated mutations.

@article{Quintero2013,
  abstract = {Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.},
  annote = {doi: 10.1074/jbc.M113.511014},
  author = {Quintero, Omar A and Unrath, William C and {Stevens  Jr.}, Stanley M and Manor, Uri and Kachar, Bechara and Yengo, Christopher M},
  doi = {10.1074/jbc.M113.511014},
  issn = {0021-9258},
  journal = {Journal of Biological Chemistry},
  month = {dec},
  number = {52},
  pages = {37126--37137},
  publisher = {Elsevier},
  title = {{Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop *}},
  url = {https://doi.org/10.1074/jbc.M113.511014},
  volume = {288},
  year = {2013}
}

Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells.

@article{PhysRevE.88.022718,
  title = {Linking actin networks and cell membrane via a reaction-diffusion-elastic description of nonlinear filopodia initiation},
  author = {Ben Isaac, Eyal and Manor, Uri and Kachar, Bechara and Yochelis, Arik and Gov, Nir S.},
  journal = {Phys. Rev. E},
  volume = {88},
  issue = {2},
  pages = {022718},
  numpages = {10},
  year = {2013},
  month = {Aug},
  publisher = {American Physical Society},
  doi = {10.1103/PhysRevE.88.022718},
  url = {https://link.aps.org/doi/10.1103/PhysRevE.88.022718}
}

@article{doi:10.1073/pnas.1304644110,
  author = {David N. Furness  and Stuart L. Johnson  and Uri Manor  and Lukas Rüttiger  and Arianna Tocchetti  and Nina Offenhauser  and Jennifer Olt  and Richard J. Goodyear  and Sarath Vijayakumar  and Yuhai Dai  and Carole M. Hackney  and Christoph Franz  and Pier Paolo Di Fiore  and Sergio Masetto  and Sherri M. Jones  and Marlies Knipper  and Matthew C. Holley  and Guy P. Richardson  and Bechara Kachar  and Walter Marcotti },
  title = {Progressive hearing loss and gradual deterioration of sensory hair bundles in the ears of mice lacking the actin-binding protein Eps8L2},
  journal = {Proceedings of the National Academy of Sciences},
  volume = {110},
  number = {34},
  pages = {13898-13903},
  year = {2013},
  doi = {10.1073/pnas.1304644110},
  URL = {https://www.pnas.org/doi/abs/10.1073/pnas.1304644110},
  eprint = {https://www.pnas.org/doi/pdf/10.1073/pnas.1304644110},
  abstract = {Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.}}

Mechanotransduction in the mammalian auditory system depends on mechanosensitive channels in the hair bundles that project from the apical surface of the sensory hair cells. Individual stereocilia within each bundle contain a core of tightly packed actin filaments, whose length is dynamically regulated during development and in the adult. We show that the actin-binding protein epidermal growth factor receptor pathway substrate 8 (Eps8)L2, a member of the Eps8-like protein family, is a newly identified hair bundle protein that is localized at the tips of stereocilia of both cochlear and vestibular hair cells. It has a spatiotemporal expression pattern that complements that of Eps8. In the cochlea, whereas Eps8 is essential for the initial elongation of stereocilia, Eps8L2 is required for their maintenance in adult hair cells. In the absence of both proteins, the ordered staircase structure of the hair bundle in the cochlea decays. In contrast to the early profound hearing loss associated with an absence of Eps8, Eps8L2 null-mutant mice exhibit a late-onset, progressive hearing loss that is directly linked to a gradual deterioration in hair bundle morphology. We conclude that Eps8L2 is required for the long-term maintenance of the staircase structure and mechanosensory function of auditory hair bundles. It complements the developmental role of Eps8 and is a candidate gene for progressive age-related hearing loss.

@article{doi:10.4161/bioa.21733,
  author = {Uri Manor and M'hamed Grati and Christopher M. Yengo and Bechara Kachar and Nir S. Gov},
  title = {Competition and compensation},
  journal = {BioArchitecture},
  volume = {2},
  number = {5},
  pages = {171-174},
  year  = {2012},
  publisher = {Taylor & Francis},
  doi = {10.4161/bioa.21733},
      note ={PMID: 22954581},

  URL = { 
      
          https://doi.org/10.4161/bioa.21733
      
      

  },
  eprint = { 
      
          https://doi.org/10.4161/bioa.21733
      
      

  }
  ,
      abstract = { Stereocilia are actin protrusions with remarkably well-defined lengths and organization. A flurry of recent papers has reported multiple myosin motor proteins involved in regulating stereocilia structures by transporting actin-regulatory cargo to the tips of stereocilia.1-13 In our recent paper, we show that two paralogous class 3 myosins — Myo3a and Myo3b — both transport the actin-regulatory protein Espin 1 (Esp1) to stereocilia and filopodia tips in a remarkably similar, albeit non-identical fashion.1 Here we present experimental and computational data that suggests that subtle differences between these two proteins’ biophysical and biochemical properties can help us understand how these myosin species target and regulate the lengths of actin protrusions. }
}

Stereocilia are actin protrusions with remarkably well-defined lengths and organization. A flurry of recent papers has reported multiple myosin motor proteins involved in regulating stereocilia structures by transporting actin-regulatory cargo to the tips of stereocilia.1-13 In our recent paper, we show that two paralogous class 3 myosins — Myo3a and Myo3b — both transport the actin-regulatory protein Espin 1 (Esp1) to stereocilia and filopodia tips in a remarkably similar, albeit non-identical fashion.1 Here we present experimental and computational data that suggests that subtle differences between these two proteins’ biophysical and biochemical properties can help us understand how these myosin species target and regulate the lengths of actin protrusions.

@article{RaymondCMerrittUriManorFelipeTSallesMhamedGratiAndreaCDoseWilliamCUnrathOmarAQuinteroChristopherMYengo2012,
  abstract = {Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip-localization is lost when we remove the ESPN1 C-terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip-localization reveals a novel mechanism for the cell to regulate myosin tip-localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity, and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations. Go to:},
  author = {Merritt, Raymond C and Manor, Uri and Salles, Felipe T and Grati, M'hamed and Dose, Andrea C and Unrath, William C and Quintero, Omar A and Yengo, Christopher M and Kachar, Bechara},
  journal = {Current biology},
  number = {4},
  pages = {320--325},
  title = {{Myosin IIIB uses an actin-binding motif in its espin-1 cargo to reach the tips of actin protrusions}},
  volume = {22},
  year = {2012}
}

Myosin IIIA (MYO3A) targets actin protrusion tips using a motility mechanism dependent on both motor and tail actin-binding activity [1]. We show that myosin IIIB (MYO3B) lacks tail actin-binding activity and is unable to target COS7 cell filopodia tips, yet is somehow able to target stereocilia tips. Strikingly, when MYO3B is coexpressed with espin-1 (ESPN1), a MYO3A cargo protein endogenously expressed in stereocilia [2], MYO3B targets and carries ESPN1 to COS7 filopodia tips. We show that this tip-localization is lost when we remove the ESPN1 C-terminus actin-binding site. We also demonstrate that, like MYO3A [2], MYO3B can elongate filopodia by transporting ESPN1 to the polymerizing end of actin filaments. The mutual dependence of MYO3B and ESPN1 for tip-localization reveals a novel mechanism for the cell to regulate myosin tip-localization via a reciprocal relationship with cargo that directly participates in actin binding for motility. Our results are consistent with a novel form of motility for class III myosins that requires both motor and tail domain actin-binding activity, and show that the actin-binding tail can be replaced by actin-binding cargo. This study also provides a framework to better understand the late-onset hearing loss phenotype in patients with MYO3A mutations. Go to:

@article{Manor2011,
  abstract = {Myosin XVa (MyoXVa) and its cargo whirlin are implicated in deafness and vestibular dysfunction and have been shown to localize at stereocilia tips and to be essential for the elongation of these actin protrusions [1?4]. Given that whirlin has no known actin-regulatory activity, it remains unclear how these proteins work together to influence stereocilia length. Here we show that the actin-regulatory protein Eps8 [5] interacts with MyoXVa and that mice lacking Eps8 show short stereocilia compared to MyoXVa- and whirlin-deficient mice. We show that Eps8 fails to accumulate at the tips of stereocilia in the absence of MyoXVa, that overexpression of MyoXVa results in both elongation of stereocilia and increased accumulation of Eps8 at stereocilia tips, and that the exogenous expression of MyoXVa in MyoXVa-deficient hair cells rescues Eps8 tip localization. We find that Eps8 also interacts with whirlin and that the expression of both Eps8 and MyoXVa at stereocilia tips is reduced in whirlin-deficient mice. We conclude that MyoXVa, whirlin, and Eps8 are integral components of the stereocilia tip complex, where Eps8 is a central actin-regulatory element for elongation of the stereocilia actin core.},
  annote = {doi: 10.1016/j.cub.2010.12.046},
  author = {Manor, Uri and Disanza, Andrea and Grati, M'Hamed and Andrade, Leonardo and Lin, Harrison and {Di Fiore}, Pier Paolo and Scita, Giorgio and Kachar, Bechara},
  doi = {10.1016/j.cub.2010.12.046},
  issn = {0960-9822},
  journal = {Current Biology},
  month = {jan},
  number = {2},
  pages = {167--172},
  publisher = {Elsevier},
  title = {{Regulation of Stereocilia Length by Myosin XVa and Whirlin Depends on the Actin-Regulatory Protein Eps8}},
  url = {https://doi.org/10.1016/j.cub.2010.12.046},
  volume = {21},
  year = {2011}
}

Myosin XVa (MyoXVa) and its cargo whirlin are implicated in deafness and vestibular dysfunction and have been shown to localize at stereocilia tips and to be essential for the elongation of these actin protrusions [1?4]. Given that whirlin has no known actin-regulatory activity, it remains unclear how these proteins work together to influence stereocilia length. Here we show that the actin-regulatory protein Eps8 [5] interacts with MyoXVa and that mice lacking Eps8 show short stereocilia compared to MyoXVa- and whirlin-deficient mice. We show that Eps8 fails to accumulate at the tips of stereocilia in the absence of MyoXVa, that overexpression of MyoXVa results in both elongation of stereocilia and increased accumulation of Eps8 at stereocilia tips, and that the exogenous expression of MyoXVa in MyoXVa-deficient hair cells rescues Eps8 tip localization. We find that Eps8 also interacts with whirlin and that the expression of both Eps8 and MyoXVa at stereocilia tips is reduced in whirlin-deficient mice. We conclude that MyoXVa, whirlin, and Eps8 are integral components of the stereocilia tip complex, where Eps8 is a central actin-regulatory element for elongation of the stereocilia actin core.

@phdthesis{Manor2011,
  author = {Manor, Uri},
  school = {The Johns Hopkins University},
  title = {{Dynamic regulation of stereocilia length by unconventional myosins and their actin-regulatory cargo}},
  url = {https://www.proquest.com/openview/e12187bab2de2307454fd30851eef2cc/1?pq-origsite=gscholar&cbl=18750},
  year = {2011}
}

@article{Quintero2010,
  abstract = {Myosin IIIa (Myo3A) transports cargo to the distal end of actin protrusions and contains a kinase domain that is thought to autoregulate its activity. Because Myo3A tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate Myo3A biochemical activity, cellular localization, and cellular function. Inactivation of Myo3A 2IQ kinase domain with the point mutation K50R did not alter maximal ATPase activity, whereas phosphorylation of Myo3A 2IQ resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of Myo3A 2IQ autophosphorylation was unchanged by the presence of actin but was found to be dependent upon Myo3A 2IQ concentration within the range of 0.1 to 1.2 ?m, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of Myo3A lacking the kinase domain decreased when co-expressed with kinase-active, full-length Myo3A. The cellular consequence of reduced Myo3A tip localization was decreased filopodial density along the cell periphery, identifying a novel cellular function for Myo3A in mediating the formation and stability of actin-based protrusions. Our results suggest that Myo3A motor activity is regulated through a mechanism involving concentration-dependent autophosphorylation. We suggest that this regulatory mechanism plays an essential role in mediating the transport and actin bundle formation/stability functions of Myo3A.},
  annote = {doi: 10.1074/jbc.M110.144360},
  author = {Quintero, Omar A and Moore, Judy E and Unrath, William C and Manor, Uri and Salles, Felipe T and Grati, M'hamed and Kachar, Bechara and Yengo, Christopher M},
  doi = {10.1074/jbc.M110.144360},
  issn = {0021-9258},
  journal = {Journal of Biological Chemistry},
  month = {nov},
  number = {46},
  pages = {35770--35782},
  publisher = {Elsevier},
  title = {{Intermolecular Autophosphorylation Regulates Myosin IIIa Activity and Localization in Parallel Actin Bundles *<sup></sup>}},
  url = {https://doi.org/10.1074/jbc.M110.144360},
  volume = {285},
  year = {2010}
}

Myosin IIIa (Myo3A) transports cargo to the distal end of actin protrusions and contains a kinase domain that is thought to autoregulate its activity. Because Myo3A tends to cluster at the tips of actin protrusions, we investigated whether intermolecular phosphorylation could regulate Myo3A biochemical activity, cellular localization, and cellular function. Inactivation of Myo3A 2IQ kinase domain with the point mutation K50R did not alter maximal ATPase activity, whereas phosphorylation of Myo3A 2IQ resulted in reduced maximal ATPase activity and actin affinity. The rate and degree of Myo3A 2IQ autophosphorylation was unchanged by the presence of actin but was found to be dependent upon Myo3A 2IQ concentration within the range of 0.1 to 1.2 ?m, indicating intermolecular autophosphorylation. In cultured cells, we observed that the filopodial tip localization of Myo3A lacking the kinase domain decreased when co-expressed with kinase-active, full-length Myo3A. The cellular consequence of reduced Myo3A tip localization was decreased filopodial density along the cell periphery, identifying a novel cellular function for Myo3A in mediating the formation and stability of actin-based protrusions. Our results suggest that Myo3A motor activity is regulated through a mechanism involving concentration-dependent autophosphorylation. We suggest that this regulatory mechanism plays an essential role in mediating the transport and actin bundle formation/stability functions of Myo3A.

@article{Salles2009,
  abstract = {Actin filaments in stereocilia on the surface of inner ear sensory hair cells are continually renewed. Myosin IIIa transports the actin-binding/bundling protein espin to stereocilia tips and cooperates with espin in actin filament elongation.},
  author = {Salles, Felipe T and Merritt, Raymond C and Manor, Uri and Dougherty, Gerard W and Sousa, Aurea D and Moore, Judy E and Yengo, Christopher M and Dos{\'{e}}, Andr{\'{e}}a C and Kachar, Bechara},
  doi = {10.1038/ncb1851},
  issn = {1476-4679},
  journal = {Nature Cell Biology},
  number = {4},
  pages = {443--450},
  title = {{Myosin IIIa boosts elongation of stereocilia by transporting espin 1 to the plus ends of actin filaments}},
  url = {https://doi.org/10.1038/ncb1851},
  volume = {11},
  year = {2009}
}

Actin filaments in stereocilia on the surface of inner ear sensory hair cells are continually renewed. Myosin IIIa transports the actin-binding/bundling protein espin to stereocilia tips and cooperates with espin in actin filament elongation.

@article{Naoz2008,
  abstract = {We present a physical model that describes the active localization of actin-regulating proteins inside stereocilia during steady-state conditions. The mechanism of localization is through the interplay of free diffusion and directed motion, which is driven by coupling to the treadmilling actin filaments and to myosin motors that move along the actin filaments. The resulting localization of both the molecular motors and their cargo is calculated, and is found to have an exponential (or steeper) profile. This localization can be at the base (driven by actin retrograde flow and minus-end myosin motors), or at the stereocilia tip (driven by plus-end myosin motors). The localization of proteins that influence the actin depolymerization and polymerization rates allow us to describe the narrow shape of the stereocilia base, and the observed increase of the actin polymerization rate with the stereocilia height.},
  annote = {doi: 10.1529/biophysj.108.143453},
  author = {Naoz, Moshe and Manor, Uri and Sakaguchi, Hirofumi and Kachar, Bechara and Gov, Nir S},
  doi = {10.1529/biophysj.108.143453},
  issn = {0006-3495},
  journal = {Biophysical Journal},
  month = {dec},
  number = {12},
  pages = {5706--5718},
  publisher = {Elsevier},
  title = {{Protein Localization by Actin Treadmilling and Molecular Motors Regulates Stereocilia Shape and Treadmilling Rate}},
  url = {https://doi.org/10.1529/biophysj.108.143453},
  volume = {95},
  year = {2008}
}

We present a physical model that describes the active localization of actin-regulating proteins inside stereocilia during steady-state conditions. The mechanism of localization is through the interplay of free diffusion and directed motion, which is driven by coupling to the treadmilling actin filaments and to myosin motors that move along the actin filaments. The resulting localization of both the molecular motors and their cargo is calculated, and is found to have an exponential (or steeper) profile. This localization can be at the base (driven by actin retrograde flow and minus-end myosin motors), or at the stereocilia tip (driven by plus-end myosin motors). The localization of proteins that influence the actin depolymerization and polymerization rates allow us to describe the narrow shape of the stereocilia base, and the observed increase of the actin polymerization rate with the stereocilia height.

@article{MANOR2008502,
  title = {Dynamic length regulation of sensory stereocilia},
  journal = {Seminars in Cell & Developmental Biology},
  volume = {19},
  number = {6},
  pages = {502-510},
  year = {2008},
  note = {Controlling Size Within Cells Plant Development and Chromatin},
  issn = {1084-9521},
  doi = {https://doi.org/10.1016/j.semcdb.2008.07.006},
  url = {https://www.sciencedirect.com/science/article/pii/S1084952108000451},
  author = {Uri Manor and Bechara Kachar},
  keywords = {Length regulation, Stereocilia, Hair cells, Myosins, Actin protrusions, Hearing, Actin treadmilling},
  abstract = {Stereocilia, the mechanosensory organelles of hair cells, are a distinctive class of actin-based cellular protrusions with an unparalleled ability to regulate their lengths over time. Studies on actin turnover in stereocilia, as well as the identification of several deafness-related proteins essential for proper stereocilia structure and function, provide new insights into the mechanisms and molecules involved in stereocilia length regulation and long-term maintenance. Comparisons of ongoing investigations on stereocilia with studies on other actin protrusions offer new opportunities to further understand common principles for length regulation, the diversity of its mechanisms, and how the specific needs of each cell are met.}
}

Stereocilia, the mechanosensory organelles of hair cells, are a distinctive class of actin-based cellular protrusions with an unparalleled ability to regulate their lengths over time. Studies on actin turnover in stereocilia, as well as the identification of several deafness-related proteins essential for proper stereocilia structure and function, provide new insights into the mechanisms and molecules involved in stereocilia length regulation and long-term maintenance. Comparisons of ongoing investigations on stereocilia with studies on other actin protrusions offer new opportunities to further understand common principles for length regulation, the diversity of its mechanisms, and how the specific needs of each cell are met.

@article{WANG20083479,
title = {Quantification of co-occurring reaction rates in deep subseafloor sediments},
  journal = {Geochimica et Cosmochimica Acta},
  volume = {72},
  number = {14},
  pages = {3479-3488},
  year = {2008},
  issn = {0016-7037},
  doi = {https://doi.org/10.1016/j.gca.2008.04.024},
  url = {https://www.sciencedirect.com/science/article/pii/S0016703708002287},
  author = {Guizhi Wang and Arthur J. Spivack and Scott Rutherford and Uri Manor and Steven D’Hondt},
  abstract = {Net rates of biogeochemical reactions in subseafloor sediments can be quantified from concentration profiles of dissolved reactants or products and physical properties of the sediment. To study net rates of microbial activities in deep sediments, we developed a robust approach that is well suited to use over a broad range of sediment depths. Our approach is based on a finite-difference solution to a continuity equation that considers molecular diffusion, sediment burial, fluid advection, and reaction under the assumption of steady state. Numerical procedures are adopted to identify the maximum number of depth intervals with statistically different reaction rates. The approach explicitly considers downcore variation in physical properties and sample spacing. Uncertainties in the rate estimates are quantified using a Monte Carlo technique. We tested our approach using synthetic concentration profiles generated from analytical solutions to the continuity equation. We then applied the approach to concentration profiles of dissolved sulfate, sulfide, methane, and manganese in the 420-m thick sediment column of eastern equatorial Pacific Ocean Drilling Program Site 1226. Our results indicate that (i) sulfate reduction and iron reduction occur at most sediment depths, (ii) net methane production occurs in discrete depth intervals and (iii) manganese reduction occurs near the seafloor and deep in the sediments. These results provide quantitative evidence that multiple respiration pathways co-exist in the same depth intervals of these deep subseafloor sediments.}
}

Net rates of biogeochemical reactions in subseafloor sediments can be quantified from concentration profiles of dissolved reactants or products and physical properties of the sediment. To study net rates of microbial activities in deep sediments, we developed a robust approach that is well suited to use over a broad range of sediment depths. Our approach is based on a finite-difference solution to a continuity equation that considers molecular diffusion, sediment burial, fluid advection, and reaction under the assumption of steady state. Numerical procedures are adopted to identify the maximum number of depth intervals with statistically different reaction rates. The approach explicitly considers downcore variation in physical properties and sample spacing. Uncertainties in the rate estimates are quantified using a Monte Carlo technique. We tested our approach using synthetic concentration profiles generated from analytical solutions to the continuity equation. We then applied the approach to concentration profiles of dissolved sulfate, sulfide, methane, and manganese in the 420-m thick sediment column of eastern equatorial Pacific Ocean Drilling Program Site 1226. Our results indicate that (i) sulfate reduction and iron reduction occur at most sediment depths, (ii) net methane production occurs in discrete depth intervals and (iii) manganese reduction occurs near the seafloor and deep in the sediments. These results provide quantitative evidence that multiple respiration pathways co-exist in the same depth intervals of these deep subseafloor sediments.

@article{Manor2004,
  abstract = {A robust numerical procedure for biogeochemical interpretation and analysis of measured concentration profiles has been developed by Berg et al. 1 The model utilizes an approximation of Fick's Second Law to find constant reaction rates in equally spaced/sized ranges of depth (aka ‘zones'). This method works well for profiles several centimeters deep, where the resolution and complexity of behavior is uniform throughout the profile. However, it is limiting when attempting to analyze profiles several hundred meters in depth, in which case a model that can adjust accordingly to changes in sampling resolution and profile complexity would be more useful/accurate. Therefore, the concepts of the old model have been used and modified to make a new model that allows differently spaced and sized zones throughout the profile. It is evident that this approach is flexible enough to handle the complexity of profiles in marine sediments several hundred meters below the ocean floor. The program was written in MATLAB, which enables the user to analyze the profiles with a higher degree of accuracy in a fraction of the time (several hours vs. several minutes), due to MATLAB's matrix calculating abilities, providing a new useful tool for analyzing concentration profiles.},
  author = {Manor, Uri and Rutherford, Scott},
  journal = {SURFO Technical Report},
  pages = {52},
  title = {{An Improved Numerical Model For Determining Chemical Reaction Rates}},
  volume = {01},
  year = {2004}
}

A robust numerical procedure for biogeochemical interpretation and analysis of measured concentration profiles has been developed by Berg et al. 1 The model utilizes an approximation of Fick's Second Law to find constant reaction rates in equally spaced/sized ranges of depth (aka ‘zones'). This method works well for profiles several centimeters deep, where the resolution and complexity of behavior is uniform throughout the profile. However, it is limiting when attempting to analyze profiles several hundred meters in depth, in which case a model that can adjust accordingly to changes in sampling resolution and profile complexity would be more useful/accurate. Therefore, the concepts of the old model have been used and modified to make a new model that allows differently spaced and sized zones throughout the profile. It is evident that this approach is flexible enough to handle the complexity of profiles in marine sediments several hundred meters below the ocean floor. The program was written in MATLAB, which enables the user to analyze the profiles with a higher degree of accuracy in a fraction of the time (several hours vs. several minutes), due to MATLAB's matrix calculating abilities, providing a new useful tool for analyzing concentration profiles.