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Rubin Lab
Rubin Lab Publications
Featured Publications
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2024. Organoid culture promotes dedifferentiation of mouse myoblasts into stem cells capable of complete muscle regeneration. Nature Biotechnology. DOI:https://doi.org/10.1038/s41587-024-02344-7 Price FD, Matyas MN, Gehrke AR, Chen W, Wolin EA, Chen W, Holton KM, Gibbs RM, Lee A, Singu P, Sakakeeny J,Poteracki JM, Goune K, Pfeiffer IT, Boswell SA, Sorger PK, Srivastava M, Pfaff KL, Gussoni E, Buchanan SM, Rubin LL. 2024. Organoid culture promotes dedifferentiation of mouse myoblasts into stem cells capable of complete muscle regeneration. Nature Biotechnology. DOI:https://doi.org/10.1038/s41587-024-02344-7 Experimental cell therapies for skeletal muscle conditions have shown little success, primarily because they use committed myogenic progenitors rather than true muscle stem cells, known as satellite cells. Here we present a method to generate in vitro-derived satellite cells (idSCs) from skeletal muscle tissue. When transplanted in small numbers into mouse muscle, mouse idSCs fuse into myofibers, repopulate the satellite cell niche, self-renew, support multiple rounds of muscle regeneration and improve force production on par with freshly isolated satellite cells in damaged skeletal muscle. We compared the epigenomic and transcriptional signatures between idSCs, myoblasts and satellite cells and used these signatures to identify core signaling pathways and genes that confer idSC functionality. Finally, from human muscle biopsies, we successfully generated satellite cell-like cells in vitro. After further development, idSCs may provide a scalable source of cells for the treatment of genetic muscle disorders, trauma-induced muscle damage and age-related muscle weakness. -
Watts ME, Giadone RM, Ordureau A, Holton KM, Harper JW, Rubin LL. 2023. Analyzing the ER stress response in ALS patient derived motor neurons identifies druggable neuroprotective targets. Frontiers in cellular neuroscience. 17:1327361. Pubmed: 38314348 DOI:10.3389/fncel.2023.1327361 Watts ME, Giadone RM, Ordureau A, Holton KM, Harper JW, Rubin LL. 2023. Analyzing the ER stress response in ALS patient derived motor neurons identifies druggable neuroprotective targets. Frontiers in cellular neuroscience. 17:1327361. Pubmed: 38314348 DOI:10.3389/fncel.2023.1327361 Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron (MN) disease with severely limited treatment options. Identification of effective treatments has been limited in part by the lack of predictive animal models for complex human disorders. Here, we utilized pharmacologic ER stressors to exacerbate underlying sensitivities conferred by ALS patient genetics in induced pluripotent stem cell (iPSC)-derived motor neurons (MNs). In doing so, we found that thapsigargin and tunicamycin exposure recapitulated ALS-associated degeneration, and that we could rescue this degeneration via MAP4K4 inhibition (MAP4K4i). We subsequently identified mechanisms underlying MAP4K4i-mediated protection by performing phosphoproteomics on iPSC-derived MNs treated with ER stressors ±MAP4K4i. Through these analyses, we found JNK, PKC, and BRAF to be differentially modulated in MAP4K4i-protected MNs, and that inhibitors to these proteins could also rescue MN toxicity. Collectively, this study highlights the value of utilizing ER stressors in ALS patient MNs to identify novel druggable targets.Copyright © 2024 Watts, Giadone, Ordureau, Holton, Harper and Rubin. -
Ximerakis M*, Holton KM*, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. (* these authors contributed equally). 2023. Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature Aging. 3. DOI:https://doi.org/10.1038/s43587-023-00373-6 Ximerakis M*, Holton KM*, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. (* these authors contributed equally). 2023. Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature Aging. 3. DOI:https://doi.org/10.1038/s43587-023-00373-6 Aging is a complex process involving transcriptomic changes associated with deterioration across multiple tissues and organs, including the brain. Recent studies using heterochronic parabiosis have shown that various aspects of aging-associated decline are modifiable or even reversible. To better understand how this occurs, we performed single-cell transcriptomic profiling of young and old mouse brains following parabiosis. For each cell type, we catalogued alterations in gene expression, molecular pathways, transcriptional networks, ligand-receptor interactions, and senescence status. Our analyses identified gene signatures demonstrating that heterochronic parabiosis regulates several hallmarks of aging in a cell-type-specific manner. Brain endothelial cells were found to be especially malleable to this intervention, exhibiting dynamic transcriptional changes that affect vascular structure and function. These findings suggest novel strategies for slowing deterioration and driving regeneration in the aging brain through approaches that do not rely on disease-specific mechanisms or actions of individual circulating factors. -
Rapino F, Natoli T, Limone F, O'Connor E, Blank J, Tegtmeyer M, Chen W, Norabuena E, Narula J, Hazelbaker D, Angelini G, Barrett L, O'Neil A, Beattie UK, Thanos JM, de Rivera H, Sheridan SD, Perlis RH, McCarroll SA, Stevens B, Subramanian A, Nehme R, Rubin LL. 2023. Small-molecule screen reveals pathways that regulate C4 secretion in stem cell-derived astrocytes. Stem cell reports. 18(1):237-253. Pubmed: 36563689 DOI:S2213-6711(22)00551-3 Rapino F, Natoli T, Limone F, O'Connor E, Blank J, Tegtmeyer M, Chen W, Norabuena E, Narula J, Hazelbaker D, Angelini G, Barrett L, O'Neil A, Beattie UK, Thanos JM, de Rivera H, Sheridan SD, Perlis RH, McCarroll SA, Stevens B, Subramanian A, Nehme R, Rubin LL. 2023. Small-molecule screen reveals pathways that regulate C4 secretion in stem cell-derived astrocytes. Stem cell reports. 18(1):237-253. Pubmed: 36563689 DOI:S2213-6711(22)00551-3 In the brain, the complement system plays a crucial role in the immune response and in synaptic elimination during normal development and disease. Here, we sought to identify pathways that modulate the production of complement component 4 (C4), recently associated with an increased risk of schizophrenia. To design a disease-relevant assay, we first developed a rapid and robust 3D protocol capable of producing large numbers of astrocytes from pluripotent cells. Transcriptional profiling of these astrocytes confirmed the homogeneity of this population of dorsal fetal-like astrocytes. Using a novel ELISA-based small-molecule screen, we identified epigenetic regulators, as well as inhibitors of intracellular signaling pathways, able to modulate C4 secretion from astrocytes. We then built a connectivity map to predict and validate additional key regulatory pathways, including one involving c-Jun-kinase. This work provides a foundation for developing therapies for CNS diseases involving the complement cascade.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved. -
Paul KC, Krolewski RC, Lucumi Moreno E, Blank J, Holton KM, Ahfeldt T, Furlong M, Yu Y, Cockburn M, Thompson LK, Kreymerman A, Ricci-Blair EM, Li YJ, Patel HB, Lee RT, Bronstein J, Rubin LL, Khurana V, Ritz B. 2023. A pesticide and iPSC dopaminergic neuron screen identifies and classifies Parkinson-relevant pesticides. Nature communications. 14(1):2803. Pubmed: 37193692 DOI:10.1038/s41467-023-38215-z Paul KC, Krolewski RC, Lucumi Moreno E, Blank J, Holton KM, Ahfeldt T, Furlong M, Yu Y, Cockburn M, Thompson LK, Kreymerman A, Ricci-Blair EM, Li YJ, Patel HB, Lee RT, Bronstein J, Rubin LL, Khurana V, Ritz B. 2023. A pesticide and iPSC dopaminergic neuron screen identifies and classifies Parkinson-relevant pesticides. Nature communications. 14(1):2803. Pubmed: 37193692 DOI:10.1038/s41467-023-38215-z Parkinson's disease (PD) is a complex neurodegenerative disease with etiology rooted in genetic vulnerability and environmental factors. Here we combine quantitative epidemiologic study of pesticide exposures and PD with toxicity screening in dopaminergic neurons derived from PD patient induced pluripotent stem cells (iPSCs) to identify Parkinson's-relevant pesticides. Agricultural records enable investigation of 288 specific pesticides and PD risk in a comprehensive, pesticide-wide association study. We associate long-term exposure to 53 pesticides with PD and identify co-exposure profiles. We then employ a live-cell imaging screening paradigm exposing dopaminergic neurons to 39 PD-associated pesticides. We find that 10 pesticides are directly toxic to these neurons. Further, we analyze pesticides typically used in combinations in cotton farming, demonstrating that co-exposures result in greater toxicity than any single pesticide. We find trifluralin is a driver of toxicity to dopaminergic neurons and leads to mitochondrial dysfunction. Our paradigm may prove useful to mechanistically dissect pesticide exposures implicated in PD risk and guide agricultural policy.© 2023. The Author(s). -
Mayweather BA, Buchanan SM, Rubin LL. 2021. GDF11 expressed in the adult brain negatively regulates hippocampal neurogenesis. Molecular brain. 14(1):134. Pubmed: 34488822 DOI:10.1186/s13041-021-00845-z Mayweather BA, Buchanan SM, Rubin LL. 2021. GDF11 expressed in the adult brain negatively regulates hippocampal neurogenesis. Molecular brain. 14(1):134. Pubmed: 34488822 DOI:10.1186/s13041-021-00845-z Growth differentiation factor 11 (GDF11) is a transforming factor-β superfamily member that functions as a negative regulator of neurogenesis during embryonic development. However, when recombinant GDF11 (rGDF11) is administered systemically in aged mice, it promotes neurogenesis, the opposite of its role during development. The goal of the present study was to reconcile this apparent discrepancy by performing the first detailed investigation into the expression of endogenous GDF11 in the adult brain and its effects on neurogenesis. Using quantitative histological analysis, we observed that Gdf11 is most highly expressed in adult neurogenic niches and non-neurogenic regions within the hippocampus, choroid plexus, thalamus, habenula, and cerebellum. To investigate the role of endogenous GDF11 during adult hippocampal neurogenesis, we generated a tamoxifen inducible mouse that allowed us to reduce GDF11 levels. Depletion of Gdf11 during adulthood increased proliferation of neural progenitors and decreased the number of newborn neurons in the hippocampus, suggesting that endogenous GDF11 remains a negative regulator of hippocampal neurogenesis in adult mice. These findings further support the idea that circulating systemic GDF11 and endogenously expressed GDF11 in the adult brain have different target cells or mechanisms of action. Our data describe a role for GDF11-dependent signaling in adult neurogenesis that has implications for how GDF11 may be used to treat CNS disease.© 2021. The Author(s). -
Buchanan SM, Price FD, Castiglioni A, Gee AW, Schneider J, Matyas MN, Hayhurst M, Tabebordbar M, Wagers AJ, Rubin LL. 2020. Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells. Skeletal muscle. 10(1):28. Pubmed: 33036659 DOI:10.1186/s13395-020-00248-z Buchanan SM, Price FD, Castiglioni A, Gee AW, Schneider J, Matyas MN, Hayhurst M, Tabebordbar M, Wagers AJ, Rubin LL. 2020. Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells. Skeletal muscle. 10(1):28. Pubmed: 33036659 DOI:10.1186/s13395-020-00248-z Satellite cells are the canonical muscle stem cells that regenerate damaged skeletal muscle. Loss of function of these cells has been linked to reduced muscle repair capacity and compromised muscle health in acute muscle injury and congenital neuromuscular diseases. To identify new pathways that can prevent loss of skeletal muscle function or enhance regenerative potential, we established an imaging-based screen capable of identifying small molecules that promote the expansion of freshly isolated satellite cells. We found several classes of receptor tyrosine kinase (RTK) inhibitors that increased freshly isolated satellite cell numbers in vitro. Further exploration of one of these compounds, the RTK inhibitor CEP-701 (also known as lestaurtinib), revealed potent activity on mouse satellite cells both in vitro and in vivo. This expansion potential was not seen upon exposure of proliferating committed myoblasts or non-myogenic fibroblasts to CEP-701. When delivered subcutaneously to acutely injured animals, CEP-701 increased both the total number of satellite cells and the rate of muscle repair, as revealed by an increased cross-sectional area of regenerating fibers. Moreover, freshly isolated satellite cells expanded ex vivo in the presence of CEP-701 displayed enhanced muscle engraftment potential upon in vivo transplantation. We provide compelling evidence that certain RTKs, and in particular RET, regulate satellite cell expansion during muscle regeneration. This study demonstrates the power of small molecule screens of even rare adult stem cell populations for identifying stem cell-targeting compounds with therapeutic potential.
All Publications
2025
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Ximerakis M, Holton KM, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. 2025. Author Correction: Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature aging. 5(2):333. Pubmed: 39881191 DOI:10.1038/s43587-025-00804-6 Ximerakis M, Holton KM, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. 2025. Author Correction: Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature aging. 5(2):333. Pubmed: 39881191 DOI:10.1038/s43587-025-00804-6 -
Price FD, Matyas MN, Gehrke AR, Chen W, Wolin EA, Holton KM, Gibbs RM, Lee A, Singu PS, Sakakeeny JS, Poteracki JM, Goune K, Pfeiffer IT, Boswell SA, Sorger PK, Srivastava M, Pfaff KL, Gussoni E, Buchanan SM, Rubin LL. 2025. Organoid culture promotes dedifferentiation of mouse myoblasts into stem cells capable of complete muscle regeneration. Nature biotechnology. 43(6):889-903. Pubmed: 39261590 DOI:10.1038/s41587-024-02344-7 Price FD, Matyas MN, Gehrke AR, Chen W, Wolin EA, Holton KM, Gibbs RM, Lee A, Singu PS, Sakakeeny JS, Poteracki JM, Goune K, Pfeiffer IT, Boswell SA, Sorger PK, Srivastava M, Pfaff KL, Gussoni E, Buchanan SM, Rubin LL. 2025. Organoid culture promotes dedifferentiation of mouse myoblasts into stem cells capable of complete muscle regeneration. Nature biotechnology. 43(6):889-903. Pubmed: 39261590 DOI:10.1038/s41587-024-02344-7 Experimental cell therapies for skeletal muscle conditions have shown little success, primarily because they use committed myogenic progenitors rather than true muscle stem cells, known as satellite cells. Here we present a method to generate in vitro-derived satellite cells (idSCs) from skeletal muscle tissue. When transplanted in small numbers into mouse muscle, mouse idSCs fuse into myofibers, repopulate the satellite cell niche, self-renew, support multiple rounds of muscle regeneration and improve force production on par with freshly isolated satellite cells in damaged skeletal muscle. We compared the epigenomic and transcriptional signatures between idSCs, myoblasts and satellite cells and used these signatures to identify core signaling pathways and genes that confer idSC functionality. Finally, from human muscle biopsies, we successfully generated satellite cell-like cells in vitro. After further development, idSCs may provide a scalable source of cells for the treatment of genetic muscle disorders, trauma-induced muscle damage and age-related muscle weakness.© 2024. The Author(s). 2024
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Day W, Jayaram RH, Rubin LE, Grauer JN. 2024. Total Hip Arthroplasty in Patients Who Have Marfan Syndrome: Adverse Events and 5-Year Revision Rates. The Journal of arthroplasty. 39(9S2):S275-S278. Pubmed: 37952742 DOI:S0883-5403(23)01098-7 Day W, Jayaram RH, Rubin LE, Grauer JN. 2024. Total Hip Arthroplasty in Patients Who Have Marfan Syndrome: Adverse Events and 5-Year Revision Rates. The Journal of arthroplasty. 39(9S2):S275-S278. Pubmed: 37952742 DOI:S0883-5403(23)01098-7 ArrayCopyright © 2023 Elsevier Inc. All rights reserved. -
Price FD, Matyas MN, Gehrke AR, Chen W, Wolin EA, Chen W, Holton KM, Gibbs RM, Lee A, Singu P, Sakakeeny J,Poteracki JM, Goune K, Pfeiffer IT, Boswell SA, Sorger PK, Srivastava M, Pfaff KL, Gussoni E, Buchanan SM, Rubin LL. 2024. Organoid culture promotes dedifferentiation of mouse myoblasts into stem cells capable of complete muscle regeneration. Nature Biotechnology. DOI:https://doi.org/10.1038/s41587-024-02344-7 Price FD, Matyas MN, Gehrke AR, Chen W, Wolin EA, Chen W, Holton KM, Gibbs RM, Lee A, Singu P, Sakakeeny J,Poteracki JM, Goune K, Pfeiffer IT, Boswell SA, Sorger PK, Srivastava M, Pfaff KL, Gussoni E, Buchanan SM, Rubin LL. 2024. Organoid culture promotes dedifferentiation of mouse myoblasts into stem cells capable of complete muscle regeneration. Nature Biotechnology. DOI:https://doi.org/10.1038/s41587-024-02344-7 Experimental cell therapies for skeletal muscle conditions have shown little success, primarily because they use committed myogenic progenitors rather than true muscle stem cells, known as satellite cells. Here we present a method to generate in vitro-derived satellite cells (idSCs) from skeletal muscle tissue. When transplanted in small numbers into mouse muscle, mouse idSCs fuse into myofibers, repopulate the satellite cell niche, self-renew, support multiple rounds of muscle regeneration and improve force production on par with freshly isolated satellite cells in damaged skeletal muscle. We compared the epigenomic and transcriptional signatures between idSCs, myoblasts and satellite cells and used these signatures to identify core signaling pathways and genes that confer idSC functionality. Finally, from human muscle biopsies, we successfully generated satellite cell-like cells in vitro. After further development, idSCs may provide a scalable source of cells for the treatment of genetic muscle disorders, trauma-induced muscle damage and age-related muscle weakness. -
Ottesen TD, Amick M, Kirwin DS, Mercier MR, Brand J, Frumberg DB, Grauer JN, Rubin LE. 2024. Increasing Value in Subspecialty Training: A Comparison of Variation in Surgical Complications for Pediatric Versus Other Fellowship-trained American Board of Orthopaedic Surgery Candidates in the Treatment of Supracondylar Fractures. Journal of the American Academy of Orthopaedic Surgeons. Global research & reviews. 8(1). Pubmed: 38252550 DOI:e22.00239 Ottesen TD, Amick M, Kirwin DS, Mercier MR, Brand J, Frumberg DB, Grauer JN, Rubin LE. 2024. Increasing Value in Subspecialty Training: A Comparison of Variation in Surgical Complications for Pediatric Versus Other Fellowship-trained American Board of Orthopaedic Surgery Candidates in the Treatment of Supracondylar Fractures. Journal of the American Academy of Orthopaedic Surgeons. Global research & reviews. 8(1). Pubmed: 38252550 DOI:e22.00239 ArrayCopyright © 2024 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Orthopaedic Surgeons. -
Leow DM, Ng YK, Wang LC, Koh HW, Zhao T, Khong ZJ, Tabaglio T, Narayanan G, Giadone RM, Sobota RM, Ng SY, Teo AKK, Parson SH, Rubin LL, Ong WY, Darras BT, Yeo CJ. 2024. Hepatocyte-intrinsic SMN deficiency drives metabolic dysfunction and liver steatosis in spinal muscular atrophy. The Journal of clinical investigation. 134(12). Pubmed: 38722695 DOI:10.1172/JCI173702 Leow DM, Ng YK, Wang LC, Koh HW, Zhao T, Khong ZJ, Tabaglio T, Narayanan G, Giadone RM, Sobota RM, Ng SY, Teo AKK, Parson SH, Rubin LL, Ong WY, Darras BT, Yeo CJ. 2024. Hepatocyte-intrinsic SMN deficiency drives metabolic dysfunction and liver steatosis in spinal muscular atrophy. The Journal of clinical investigation. 134(12). Pubmed: 38722695 DOI:10.1172/JCI173702 Spinal muscular atrophy (SMA) is typically characterized as a motor neuron disease, but extraneuronal phenotypes are present in almost every organ in severely affected patients and animal models. Extraneuronal phenotypes were previously underappreciated, as patients with severe SMA phenotypes usually died in infancy; however, with current treatments for motor neurons increasing patient lifespan, impaired function of peripheral organs may develop into significant future comorbidities and lead to new treatment-modified phenotypes. Fatty liver is seen in SMA animal models, but generalizability to patients and whether this is due to hepatocyte-intrinsic survival motor neuron (SMN) protein deficiency and/or subsequent to skeletal muscle denervation is unknown. If liver pathology in SMA is SMN dependent and hepatocyte intrinsic, this suggests SMN-repleting therapies must target extraneuronal tissues and motor neurons for optimal patient outcome. Here, we showed that fatty liver is present in SMA patients and that SMA patient-specific induced pluripotent stem cell-derived hepatocyte-like cells were susceptible to steatosis. Using proteomics, functional studies, and CRISPR/Cas9 gene editing, we confirmed that fatty liver in SMA is a primary SMN-dependent hepatocyte-intrinsic liver defect associated with mitochondrial and other hepatic metabolism implications. These pathologies require monitoring and indicate the need for systematic clinical surveillance and additional and/or combinatorial therapies to ensure continued SMA patient health. 2023
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Rapino F, Natoli T, Limone F, O'Connor E, Blank J, Tegtmeyer M, Chen W, Norabuena E, Narula J, Hazelbaker D, Angelini G, Barrett L, O'Neil A, Beattie UK, Thanos JM, de Rivera H, Sheridan SD, Perlis RH, McCarroll SA, Stevens B, Subramanian A, Nehme R, Rubin LL. 2023. Small-molecule screen reveals pathways that regulate C4 secretion in stem cell-derived astrocytes. Stem cell reports. 18(1):237-253. Pubmed: 36563689 DOI:S2213-6711(22)00551-3 Rapino F, Natoli T, Limone F, O'Connor E, Blank J, Tegtmeyer M, Chen W, Norabuena E, Narula J, Hazelbaker D, Angelini G, Barrett L, O'Neil A, Beattie UK, Thanos JM, de Rivera H, Sheridan SD, Perlis RH, McCarroll SA, Stevens B, Subramanian A, Nehme R, Rubin LL. 2023. Small-molecule screen reveals pathways that regulate C4 secretion in stem cell-derived astrocytes. Stem cell reports. 18(1):237-253. Pubmed: 36563689 DOI:S2213-6711(22)00551-3 In the brain, the complement system plays a crucial role in the immune response and in synaptic elimination during normal development and disease. Here, we sought to identify pathways that modulate the production of complement component 4 (C4), recently associated with an increased risk of schizophrenia. To design a disease-relevant assay, we first developed a rapid and robust 3D protocol capable of producing large numbers of astrocytes from pluripotent cells. Transcriptional profiling of these astrocytes confirmed the homogeneity of this population of dorsal fetal-like astrocytes. Using a novel ELISA-based small-molecule screen, we identified epigenetic regulators, as well as inhibitors of intracellular signaling pathways, able to modulate C4 secretion from astrocytes. We then built a connectivity map to predict and validate additional key regulatory pathways, including one involving c-Jun-kinase. This work provides a foundation for developing therapies for CNS diseases involving the complement cascade.Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved. -
Neel DV, Basu H, Gunner G, Bergstresser MD, Giadone RM, Chung H, Miao R, Chou V, Brody E, Jiang X, Lee E, Watts ME, Marques C, Held A, Wainger B, Lagier-Tourenne C, Zhang YJ, Petrucelli L, Young-Pearse TL, Chen-Plotkin AS, Rubin LL, Lieberman J, Chiu IM. 2023. Gasdermin-E mediates mitochondrial damage in axons and neurodegeneration. Neuron. 111(8):1222-1240.e9. Pubmed: 36917977 DOI:S0896-6273(23)00123-X Neel DV, Basu H, Gunner G, Bergstresser MD, Giadone RM, Chung H, Miao R, Chou V, Brody E, Jiang X, Lee E, Watts ME, Marques C, Held A, Wainger B, Lagier-Tourenne C, Zhang YJ, Petrucelli L, Young-Pearse TL, Chen-Plotkin AS, Rubin LL, Lieberman J, Chiu IM. 2023. Gasdermin-E mediates mitochondrial damage in axons and neurodegeneration. Neuron. 111(8):1222-1240.e9. Pubmed: 36917977 DOI:S0896-6273(23)00123-X Mitochondrial dysfunction and axon loss are hallmarks of neurologic diseases. Gasdermin (GSDM) proteins are executioner pore-forming molecules that mediate cell death, yet their roles in the central nervous system (CNS) are not well understood. Here, we find that one GSDM family member, GSDME, is expressed by both mouse and human neurons. GSDME plays a role in mitochondrial damage and axon loss. Mitochondrial neurotoxins induced caspase-dependent GSDME cleavage and rapid localization to mitochondria in axons, where GSDME promoted mitochondrial depolarization, trafficking defects, and neurite retraction. Frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS)-associated proteins TDP-43 and PR-50 induced GSDME-mediated damage to mitochondria and neurite loss. GSDME knockdown protected against neurite loss in ALS patient iPSC-derived motor neurons. Knockout of GSDME in SOD1 ALS mice prolonged survival, ameliorated motor dysfunction, rescued motor neuron loss, and reduced neuroinflammation. We identify GSDME as an executioner of neuronal mitochondrial dysfunction that may contribute to neurodegeneration.Copyright © 2023 Elsevier Inc. All rights reserved. -
Paul KC, Krolewski RC, Lucumi Moreno E, Blank J, Holton KM, Ahfeldt T, Furlong M, Yu Y, Cockburn M, Thompson LK, Kreymerman A, Ricci-Blair EM, Li YJ, Patel HB, Lee RT, Bronstein J, Rubin LL, Khurana V, Ritz B. 2023. A pesticide and iPSC dopaminergic neuron screen identifies and classifies Parkinson-relevant pesticides. Nat Commun. 14. DOI:10.1038/s41467-023-38215-z Paul KC, Krolewski RC, Lucumi Moreno E, Blank J, Holton KM, Ahfeldt T, Furlong M, Yu Y, Cockburn M, Thompson LK, Kreymerman A, Ricci-Blair EM, Li YJ, Patel HB, Lee RT, Bronstein J, Rubin LL, Khurana V, Ritz B. 2023. A pesticide and iPSC dopaminergic neuron screen identifies and classifies Parkinson-relevant pesticides. Nat Commun. 14. DOI:10.1038/s41467-023-38215-z Parkinson's disease (PD) is a complex neurodegenerative disease with etiology rooted in genetic vulnerability and environmental factors. Here we combine quantitative epidemiologic study of pesticide exposures and PD with toxicity screening in dopaminergic neurons derived from PD patient induced pluripotent stem cells (iPSCs) to identify Parkinson's-relevant pesticides. Agricultural records enable investigation of 288 specific pesticides and PD risk in a comprehensive, pesticide-wide association study. We associate long-term exposure to 53 pesticides with PD and identify co-exposure profiles. We then employ a live-cell imaging screening paradigm exposing dopaminergic neurons to 39 PD-associated pesticides. We find that 10 pesticides are directly toxic to these neurons. Further, we analyze pesticides typically used in combinations in cotton farming, demonstrating that co-exposures result in greater toxicity than any single pesticide. We find trifluralin is a driver of toxicity to dopaminergic neurons and leads to mitochondrial dysfunction. Our paradigm may prove useful to mechanistically dissect pesticide exposures implicated in PD risk and guide agricultural policy. -
Paul KC, Krolewski RC, Lucumi Moreno E, Blank J, Holton KM, Ahfeldt T, Furlong M, Yu Y, Cockburn M, Thompson LK, Kreymerman A, Ricci-Blair EM, Li YJ, Patel HB, Lee RT, Bronstein J, Rubin LL, Khurana V, Ritz B. 2023. A pesticide and iPSC dopaminergic neuron screen identifies and classifies Parkinson-relevant pesticides. Nature communications. 14(1):2803. Pubmed: 37193692 DOI:10.1038/s41467-023-38215-z Paul KC, Krolewski RC, Lucumi Moreno E, Blank J, Holton KM, Ahfeldt T, Furlong M, Yu Y, Cockburn M, Thompson LK, Kreymerman A, Ricci-Blair EM, Li YJ, Patel HB, Lee RT, Bronstein J, Rubin LL, Khurana V, Ritz B. 2023. A pesticide and iPSC dopaminergic neuron screen identifies and classifies Parkinson-relevant pesticides. Nature communications. 14(1):2803. Pubmed: 37193692 DOI:10.1038/s41467-023-38215-z Parkinson's disease (PD) is a complex neurodegenerative disease with etiology rooted in genetic vulnerability and environmental factors. Here we combine quantitative epidemiologic study of pesticide exposures and PD with toxicity screening in dopaminergic neurons derived from PD patient induced pluripotent stem cells (iPSCs) to identify Parkinson's-relevant pesticides. Agricultural records enable investigation of 288 specific pesticides and PD risk in a comprehensive, pesticide-wide association study. We associate long-term exposure to 53 pesticides with PD and identify co-exposure profiles. We then employ a live-cell imaging screening paradigm exposing dopaminergic neurons to 39 PD-associated pesticides. We find that 10 pesticides are directly toxic to these neurons. Further, we analyze pesticides typically used in combinations in cotton farming, demonstrating that co-exposures result in greater toxicity than any single pesticide. We find trifluralin is a driver of toxicity to dopaminergic neurons and leads to mitochondrial dysfunction. Our paradigm may prove useful to mechanistically dissect pesticide exposures implicated in PD risk and guide agricultural policy.© 2023. The Author(s). -
Paul KC, Krolewski RC, Lucumi Moreno E, Blank J, Holton KM, Ahfeldt T, Furlong M, Yu Y, Cockburn M, Thompson LK, Kreymerman A, Ricci-Blair EM, Li YJ, Patel HB, Lee RT, Bronstein J, Rubin LL, Khurana V, Ritz B. 2023. Publisher Correction: A pesticide and iPSC dopaminergic neuron screen identifies and classifies Parkinson-relevant pesticides. Nature communications. 14(1):3747. Pubmed: 37353544 DOI:10.1038/s41467-023-39001-7 Paul KC, Krolewski RC, Lucumi Moreno E, Blank J, Holton KM, Ahfeldt T, Furlong M, Yu Y, Cockburn M, Thompson LK, Kreymerman A, Ricci-Blair EM, Li YJ, Patel HB, Lee RT, Bronstein J, Rubin LL, Khurana V, Ritz B. 2023. Publisher Correction: A pesticide and iPSC dopaminergic neuron screen identifies and classifies Parkinson-relevant pesticides. Nature communications. 14(1):3747. Pubmed: 37353544 DOI:10.1038/s41467-023-39001-7 -
Cronin SJF, Davidow LS, Arvanites AC, Rubin LL, Penninger JM, Woolf CJ. 2023. Implementation of a Drug Screening Platform to Target Expression in Injured Mouse Dorsal Root Ganglion Neurons. Bio-protocol. 13(9):e4666. Pubmed: 37188109 DOI:10.21769/BioProtoc.4666 Cronin SJF, Davidow LS, Arvanites AC, Rubin LL, Penninger JM, Woolf CJ. 2023. Implementation of a Drug Screening Platform to Target Expression in Injured Mouse Dorsal Root Ganglion Neurons. Bio-protocol. 13(9):e4666. Pubmed: 37188109 DOI:10.21769/BioProtoc.4666 Management of neuropathic pain is notoriously difficult; current analgesics, including anti-inflammatory- and opioid-based medications, are generally ineffective and can pose serious side effects. There is a need to uncover non-addictive and safe analgesics to combat neuropathic pain. Here, we describe the setup of a phenotypic screen whereby the expression of an algesic gene,, is targeted. GCH1 is the rate-limiting enzyme in the de novo synthesis of tetrahydrobiopterin (BH4), a metabolite linked to neuropathic pain in both animal models and in human chronic pain sufferers.is induced in sensory neurons after nerve injury and its upregulation is responsible for increased BH4 levels. GCH1 protein has proven to be a difficult enzyme to pharmacologically target with small molecule inhibition. Thus, by establishing a platform to monitor and target induced expression in individual injured dorsal root ganglion (DRG) neurons in vitro, we can screen for compounds that regulate its expression levels. This approach also allows us to gain valuable biological insights into the pathways and signals regulating GCH1 and BH4 levels upon nerve injury. This protocol is compatible with any transgenic reporter system in which the expression of an algesic gene (or multiple genes) can be monitored fluorescently. Such an approach can be scaled up for high-throughput compound screening and is amenable to transgenic mice as well as human stem cell-derived sensory neurons. Graphical overview.Copyright © 2023 The Authors; exclusive licensee Bio-protocol LLC. -
Berryer MH, Rizki G, Nathanson A, Klein JA, Trendafilova D, Susco SG, Lam D, Messana A, Holton KM, Karhohs KW, Cimini BA, Pfaff K, Carpenter AE, Rubin LL, Barrett LE. 2023. High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly. eLife. 12. Pubmed: 37083703 DOI:10.7554/eLife.80168 Berryer MH, Rizki G, Nathanson A, Klein JA, Trendafilova D, Susco SG, Lam D, Messana A, Holton KM, Karhohs KW, Cimini BA, Pfaff K, Carpenter AE, Rubin LL, Barrett LE. 2023. High-content synaptic phenotyping in human cellular models reveals a role for BET proteins in synapse assembly. eLife. 12. Pubmed: 37083703 DOI:10.7554/eLife.80168 Resolving fundamental molecular and functional processes underlying human synaptic development is crucial for understanding normal brain function as well as dysfunction in disease. Based upon increasing evidence of species-divergent features of brain cell types, coupled with emerging studies of complex human disease genetics, we developed the first automated and quantitative high-content synaptic phenotyping platform using human neurons and astrocytes. To establish the robustness of our platform, we screened the effects of 376 small molecules on presynaptic density, neurite outgrowth, and cell viability, validating six small molecules that specifically enhanced human presynaptic density in vitro. Astrocytes were essential for mediating the effects of all six small molecules, underscoring the relevance of non-cell-autonomous factors in synapse assembly and their importance in synaptic screening applications. Bromodomain and extraterminal (BET) inhibitors emerged as the most prominent hit class and global transcriptional analyses using multiple BET inhibitors confirmed upregulation of synaptic gene expression. Through these analyses, we demonstrate the robustness of our automated screening platform for identifying potent synaptic modulators, which can be further leveraged for scaled analyses of human synaptic mechanisms and drug discovery efforts.© 2023, Berryer et al. -
Berryer MH, Tegtmeyer M, Binan L, Valakh V, Nathanson A, Trendafilova D, Crouse E, Klein JA, Meyer D, Pietiläinen O, Rapino F, Farhi SL, Rubin LL, McCarroll SA, Nehme R, Barrett LE. 2023. Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells. iScience. 26(7):106995. Pubmed: 37534135 DOI:10.1016/j.isci.2023.106995 Berryer MH, Tegtmeyer M, Binan L, Valakh V, Nathanson A, Trendafilova D, Crouse E, Klein JA, Meyer D, Pietiläinen O, Rapino F, Farhi SL, Rubin LL, McCarroll SA, Nehme R, Barrett LE. 2023. Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells. iScience. 26(7):106995. Pubmed: 37534135 DOI:10.1016/j.isci.2023.106995 Emerging evidence of species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy or require intermediate purification steps. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs). These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal network development. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, mostly resembling human fetal astrocytes. Finally, hiAs generated from a trisomy 21 disease model identify expected alterations in cell-cell adhesion and synaptic signaling, supporting their utility for disease modeling applications. Thus, hiAs provide a valuable and practical resource for the study of basic human astrocyte function and dysfunction in disease.© 2023 The Authors. -
Driss LB, Lian J, Walker RG, Howard JA, Thompson TB, Rubin LL, Wagers AJ, Lee RT. 2023. GDF11 and aging biology - controversies resolved and pending. The journal of cardiovascular aging. 3(4). Pubmed: 38235060 DOI:10.20517/jca.2023.23 Driss LB, Lian J, Walker RG, Howard JA, Thompson TB, Rubin LL, Wagers AJ, Lee RT. 2023. GDF11 and aging biology - controversies resolved and pending. The journal of cardiovascular aging. 3(4). Pubmed: 38235060 DOI:10.20517/jca.2023.23 Since the exogenous administration of GDF11, a TGF-ß superfamily member, was reported to have beneficial effects in some models of human disease, there have been many research studies in GDF11 biology. However, many studies have now confirmed that exogenous administration of GDF11 can improve physiology in disease models, including cardiac fibrosis, experimental stroke, and disordered metabolism. GDF11 is similar to GDF8 (also called Myostatin), differing only by 11 amino acids in their mature signaling domains. These two proteins are now known to be biochemically different both and . GDF11 is much more potent than GDF8 and induces more strongly SMAD2 phosphorylation in the myocardium compared to GDF8. GDF8 and GDF11 prodomain are only 52% identical and are cleaved by different Tolloid proteases to liberate the mature signaling domain from inhibition of the prodomain. Here, we review the state of GDF11 biology, highlighting both resolved and remaining controversies. -
Watts ME, Giadone RM, Ordureau A, Holton KM, Harper JW, Rubin LL. 2023. Analyzing the ER stress response in ALS patient derived motor neurons identifies druggable neuroprotective targets. Frontiers in cellular neuroscience. 17:1327361. Pubmed: 38314348 DOI:10.3389/fncel.2023.1327361 Watts ME, Giadone RM, Ordureau A, Holton KM, Harper JW, Rubin LL. 2023. Analyzing the ER stress response in ALS patient derived motor neurons identifies druggable neuroprotective targets. Frontiers in cellular neuroscience. 17:1327361. Pubmed: 38314348 DOI:10.3389/fncel.2023.1327361 Amyotrophic lateral sclerosis (ALS) is a degenerative motor neuron (MN) disease with severely limited treatment options. Identification of effective treatments has been limited in part by the lack of predictive animal models for complex human disorders. Here, we utilized pharmacologic ER stressors to exacerbate underlying sensitivities conferred by ALS patient genetics in induced pluripotent stem cell (iPSC)-derived motor neurons (MNs). In doing so, we found that thapsigargin and tunicamycin exposure recapitulated ALS-associated degeneration, and that we could rescue this degeneration via MAP4K4 inhibition (MAP4K4i). We subsequently identified mechanisms underlying MAP4K4i-mediated protection by performing phosphoproteomics on iPSC-derived MNs treated with ER stressors ±MAP4K4i. Through these analyses, we found JNK, PKC, and BRAF to be differentially modulated in MAP4K4i-protected MNs, and that inhibitors to these proteins could also rescue MN toxicity. Collectively, this study highlights the value of utilizing ER stressors in ALS patient MNs to identify novel druggable targets.Copyright © 2024 Watts, Giadone, Ordureau, Holton, Harper and Rubin. -
Ximerakis M, Holton KM, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. 2023. Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature aging. 3(3):327-345. Pubmed: 37118429 DOI:10.1038/s43587-023-00373-6 Ximerakis M, Holton KM, Giadone RM, Ozek C, Saxena M, Santiago S, Adiconis X, Dionne D, Nguyen L, Shah KM, Goldstein JM, Gasperini C, Gampierakis IA, Lipnick SL, Simmons SK, Buchanan SM, Wagers AJ, Regev A, Levin JZ, Rubin LL. 2023. Heterochronic parabiosis reprograms the mouse brain transcriptome by shifting aging signatures in multiple cell types. Nature aging. 3(3):327-345. Pubmed: 37118429 DOI:10.1038/s43587-023-00373-6 Aging is a complex process involving transcriptomic changes associated with deterioration across multiple tissues and organs, including the brain. Recent studies using heterochronic parabiosis have shown that various aspects of aging-associated decline are modifiable or even reversible. To better understand how this occurs, we performed single-cell transcriptomic profiling of young and old mouse brains after parabiosis. For each cell type, we cataloged alterations in gene expression, molecular pathways, transcriptional networks, ligand-receptor interactions and senescence status. Our analyses identified gene signatures, demonstrating that heterochronic parabiosis regulates several hallmarks of aging in a cell-type-specific manner. Brain endothelial cells were found to be especially malleable to this intervention, exhibiting dynamic transcriptional changes that affect vascular structure and function. These findings suggest new strategies for slowing deterioration and driving regeneration in the aging brain through approaches that do not rely on disease-specific mechanisms or actions of individual circulating factors.© 2023. The Author(s). 2022
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Cronin SJF, Rao S, Tejada MA, Turnes BL, Licht-Mayer S, Omura T, Brenneis C, Jacobs E, Barrett L, Latremoliere A, Andrews N, Channon KM, Latini A, Arvanites AC, Davidow LS, Costigan M, Rubin LL, Penninger JM, Woolf CJ. 2022. Phenotypic drug screen uncovers the metabolic GCH1/BH4 pathway as key regulator of EGFR/KRAS-mediated neuropathic pain and lung cancer. Science translational medicine. 14(660):eabj1531. Pubmed: 36044597 DOI:10.1126/scitranslmed.abj1531 Cronin SJF, Rao S, Tejada MA, Turnes BL, Licht-Mayer S, Omura T, Brenneis C, Jacobs E, Barrett L, Latremoliere A, Andrews N, Channon KM, Latini A, Arvanites AC, Davidow LS, Costigan M, Rubin LL, Penninger JM, Woolf CJ. 2022. Phenotypic drug screen uncovers the metabolic GCH1/BH4 pathway as key regulator of EGFR/KRAS-mediated neuropathic pain and lung cancer. Science translational medicine. 14(660):eabj1531. Pubmed: 36044597 DOI:10.1126/scitranslmed.abj1531 Increased tetrahydrobiopterin (BH4) generated in injured sensory neurons contributes to increased pain sensitivity and its persistence. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme in the de novo BH4 synthetic pathway, and human single-nucleotide polymorphism studies, together with mouse genetic modeling, have demonstrated that decreased GCH1 leads to both reduced BH4 and pain. However, little is known about the regulation of expression upon nerve injury and whether this could be modulated as an analgesic therapeutic intervention. We performed a phenotypic screen using about 1000 bioactive compounds, many of which are target-annotated FDA-approved drugs, for their effect on regulating expression in rodent injured dorsal root ganglion neurons. From this approach, we uncovered relevant pathways that regulate expression in sensory neurons. We report that EGFR/KRAS signaling triggers increased expression and contributes to neuropathic pain; conversely, inhibiting EGFR suppressed GCH1 and BH4 and exerted analgesic effects, suggesting a molecular link between EGFR/KRAS and pain perception. We also show that GCH1/BH4 acts downstream of KRAS to drive lung cancer, identifying a potentially druggable pathway. Our screen shows that pharmacologic modulation of GCH1 expression and BH4 could be used to develop pharmacological treatments to alleviate pain and identified a critical role for EGFR-regulated GCH1/BH4 expression in neuropathic pain and cancer in rodents. -
Spetz JKE, Florido MHC, Fraser CS, Qin X, Choiniere J, Yu SJ, Singh R, Friesen M, Rubin LL, Salem JE, Moslehi JJ, Sarosiek KA. 2022. Heightened apoptotic priming of vascular cells across tissues and life span predisposes them to cancer therapy-induced toxicities. Science advances. 8(45):eabn6579. Pubmed: 36351019 DOI:10.1126/sciadv.abn6579 Spetz JKE, Florido MHC, Fraser CS, Qin X, Choiniere J, Yu SJ, Singh R, Friesen M, Rubin LL, Salem JE, Moslehi JJ, Sarosiek KA. 2022. Heightened apoptotic priming of vascular cells across tissues and life span predisposes them to cancer therapy-induced toxicities. Science advances. 8(45):eabn6579. Pubmed: 36351019 DOI:10.1126/sciadv.abn6579 Although major organ toxicities frequently arise in patients treated with cytotoxic or targeted cancer therapies, the mechanisms that drive them are poorly understood. Here, we report that vascular endothelial cells (ECs) are more highly primed for apoptosis than parenchymal cells across many adult tissues. Consequently, ECs readily undergo apoptosis in response to many commonly used anticancer agents including cytotoxic and targeted drugs and are more sensitive to ionizing radiation and BH3 mimetics than parenchymal cells in vivo. Further, using differentiated isogenic human induced pluripotent stem cell models of ECs and vascular smooth muscle cells (VSMCs), we find that these vascular cells exhibit distinct drug toxicity patterns, which are linked to divergent therapy-induced vascular toxicities in patients. Collectively, our results demonstrate that vascular cells are highly sensitive to apoptosis-inducing stress across life span and may represent a "weakest link" vulnerability in multiple tissues for development of toxicities. -
Gardezi M, Moore HG, Rubin LE, Grauer JN. 2022. Predictors of Physical Abuse in Elder Patients With Fracture. Journal of the American Academy of Orthopaedic Surgeons. Global research & reviews. 6(7). Pubmed: 35819835 DOI:e22.00144 Gardezi M, Moore HG, Rubin LE, Grauer JN. 2022. Predictors of Physical Abuse in Elder Patients With Fracture. Journal of the American Academy of Orthopaedic Surgeons. Global research & reviews. 6(7). Pubmed: 35819835 DOI:e22.00144 ArrayCopyright © 2022 The Authors. Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Orthopaedic Surgeons. -
Moore HG, Burroughs PJ, Rubin LE, Frumberg DB, Sculco PK, Grauer JN. 2022. Patients With Ehlers-Danlos Syndromes Experience Higher Rates of Prosthetic Dislocation After Total Hip Arthroplasty and Worse Implant Survival at 5 Years. The Journal of the American Academy of Orthopaedic Surgeons. 30(4):177-183. Pubmed: 34967762 DOI:10.5435/JAAOS-D-21-00347 Moore HG, Burroughs PJ, Rubin LE, Frumberg DB, Sculco PK, Grauer JN. 2022. Patients With Ehlers-Danlos Syndromes Experience Higher Rates of Prosthetic Dislocation After Total Hip Arthroplasty and Worse Implant Survival at 5 Years. The Journal of the American Academy of Orthopaedic Surgeons. 30(4):177-183. Pubmed: 34967762 DOI:10.5435/JAAOS-D-21-00347 ArrayCopyright © 2021 by the American Academy of Orthopaedic Surgeons. -
Pediaditakis I, Kodella KR, Manatakis DV, Le CY, Barthakur S, Sorets A, Gravanis A, Ewart L, Rubin LL, Manolakos ES, Hinojosa CD, Karalis K. 2022. A microengineered Brain-Chip to model neuroinflammation in humans. iScience. 25(8):104813. Pubmed: 35982785 DOI:10.1016/j.isci.2022.104813 Pediaditakis I, Kodella KR, Manatakis DV, Le CY, Barthakur S, Sorets A, Gravanis A, Ewart L, Rubin LL, Manolakos ES, Hinojosa CD, Karalis K. 2022. A microengineered Brain-Chip to model neuroinflammation in humans. iScience. 25(8):104813. Pubmed: 35982785 DOI:10.1016/j.isci.2022.104813 Species differences in brain and blood-brain barrier (BBB) biology hamper the translation of findings from animal models to humans, impeding the development of therapeutics for brain diseases. Here, we present a human organotypic microphysiological system (MPS) that includes endothelial-like cells, pericytes, glia, and cortical neurons and maintains BBB permeability at relevant levels. This human Brain-Chip engineered to recapitulate critical aspects of the complex interactions that mediate neuroinflammation and demonstrates significant improvements in clinical mimicry compared to previously reported similar MPS. In comparison to Transwell culture, the transcriptomic profiling of the Brain-Chip displayed significantly advanced similarity to the human adult cortex and enrichment in key neurobiological pathways. Exposure to TNF-α recreated the anticipated inflammatory environment shown by glia activation, increased release of proinflammatory cytokines, and compromised barrier permeability. We report the development of a robust brain MPS for mechanistic understanding of cell-cell interactions and BBB function during neuroinflammation.© 2022 The Authors. 2021
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Mayweather BA, Buchanan SM, Rubin LL. 2021. GDF11 expressed in the adult brain negatively regulates hippocampal neurogenesis. Molecular brain. 14(1):134. Pubmed: 34488822 DOI:10.1186/s13041-021-00845-z Mayweather BA, Buchanan SM, Rubin LL. 2021. GDF11 expressed in the adult brain negatively regulates hippocampal neurogenesis. Molecular brain. 14(1):134. Pubmed: 34488822 DOI:10.1186/s13041-021-00845-z Growth differentiation factor 11 (GDF11) is a transforming factor-β superfamily member that functions as a negative regulator of neurogenesis during embryonic development. However, when recombinant GDF11 (rGDF11) is administered systemically in aged mice, it promotes neurogenesis, the opposite of its role during development. The goal of the present study was to reconcile this apparent discrepancy by performing the first detailed investigation into the expression of endogenous GDF11 in the adult brain and its effects on neurogenesis. Using quantitative histological analysis, we observed that Gdf11 is most highly expressed in adult neurogenic niches and non-neurogenic regions within the hippocampus, choroid plexus, thalamus, habenula, and cerebellum. To investigate the role of endogenous GDF11 during adult hippocampal neurogenesis, we generated a tamoxifen inducible mouse that allowed us to reduce GDF11 levels. Depletion of Gdf11 during adulthood increased proliferation of neural progenitors and decreased the number of newborn neurons in the hippocampus, suggesting that endogenous GDF11 remains a negative regulator of hippocampal neurogenesis in adult mice. These findings further support the idea that circulating systemic GDF11 and endogenously expressed GDF11 in the adult brain have different target cells or mechanisms of action. Our data describe a role for GDF11-dependent signaling in adult neurogenesis that has implications for how GDF11 may be used to treat CNS disease.© 2021. The Author(s). -
Sadegh C, Ebina W, Arvanites AC, Davidow LS, Rubin LL, Macklis JD. 2021. Synthetic modified Fezf2 mRNA (modRNA) with concurrent small molecule SIRT1 inhibition enhances refinement of cortical subcerebral/corticospinal neuron identity from mouse embryonic stem cells. PloS one. 16(9):e0254113. Pubmed: 34473715 DOI:10.1371/journal.pone.0254113 Sadegh C, Ebina W, Arvanites AC, Davidow LS, Rubin LL, Macklis JD. 2021. Synthetic modified Fezf2 mRNA (modRNA) with concurrent small molecule SIRT1 inhibition enhances refinement of cortical subcerebral/corticospinal neuron identity from mouse embryonic stem cells. PloS one. 16(9):e0254113. Pubmed: 34473715 DOI:10.1371/journal.pone.0254113 During late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed "cortical" here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and that its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro. -
Tomov ML, O'Neil A, Abbasi HS, Cimini BA, Carpenter AE, Rubin LL, Bathe M. 2021. Resolving cell state in iPSC-derived human neural samples with multiplexed fluorescence imaging. Communications biology. 4(1):786. Pubmed: 34168275 DOI:10.1038/s42003-021-02276-x Tomov ML, O'Neil A, Abbasi HS, Cimini BA, Carpenter AE, Rubin LL, Bathe M. 2021. Resolving cell state in iPSC-derived human neural samples with multiplexed fluorescence imaging. Communications biology. 4(1):786. Pubmed: 34168275 DOI:10.1038/s42003-021-02276-x Human induced pluripotent stem cell-derived (iPSC) neural cultures offer clinically relevant models of human diseases, including Amyotrophic Lateral Sclerosis, Alzheimer's, and Autism Spectrum Disorder. In situ characterization of the spatial-temporal evolution of cell state in 3D culture and subsequent 2D dissociated culture models based on protein expression levels and localizations is essential to understanding neural cell differentiation, disease state phenotypes, and sample-to-sample variability. Here, we apply PRobe-based Imaging for Sequential Multiplexing (PRISM) to facilitate multiplexed imaging with facile, rapid exchange of imaging probes to analyze iPSC-derived cortical and motor neuron cultures that are relevant to psychiatric and neurodegenerative disease models, using over ten protein targets. Our approach permits analysis of cell differentiation, cell composition, and functional marker expression in complex stem-cell derived neural cultures. Furthermore, our approach is amenable to automation, offering in principle the ability to scale-up to dozens of protein targets and samples. -
Kebaish KJ, Galivanche AR, Varthi AG, Ottesen TD, Rubin LE, Grauer JN. 2021. Long-term Corticosteroid Use Independently Correlates With Complications After Posterior Lumbar Spine Surgery. Orthopedics. 44(3):172-179. Pubmed: 34039214 DOI:10.3928/01477447-20210416-01 Kebaish KJ, Galivanche AR, Varthi AG, Ottesen TD, Rubin LE, Grauer JN. 2021. Long-term Corticosteroid Use Independently Correlates With Complications After Posterior Lumbar Spine Surgery. Orthopedics. 44(3):172-179. Pubmed: 34039214 DOI:10.3928/01477447-20210416-01 With the increasing medical complexity of patients undergoing posterior lumbar surgery, more patients are pharmacologically immunosuppressed to manage chronic conditions. The effects of immunosuppression have become of greater interest across multiple surgical specialties. The goal of the current study was to investigate whether long-term corticosteroid use is independently associated with perioperative adverse outcomes among patients undergoing posterior lumbar surgery. Patients who underwent elective posterior lumbar spine surgery (decompression and/or fusion) were identified in the 2005-2016 National Surgical Quality Improvement Program (NSQIP) database. Patient factors, surgical factors, and 30-day perioperative outcomes for patients taking long-term corticosteroids were compared with those for patients who were not taking these drugs. Propensity matching and multivariate analysis were used to evaluate comparable patients while controlling for potentially confounding variables. In total, 140,519 patients undergoing posterior lumbar spine surgery were identified. Of these, 5243 (3.73%) were taking corticosteroids. After propensity matching and controlling for age, sex, body mass index, functional status, American Society of Anesthesiologists class, and surgical procedure, those taking corticosteroids were at greater risk for any adverse event (odds ratio, 1.45), a serious adverse event (odds ratio, 1.57), a minor adverse event (odds ratio, 1.47), infection (odds ratio, 1.48), reoperation (odds ratio, 1.48), and readmission (odds ratio, 1.47) (≤.001 for each). The findings confirmed that long-term corticosteroid use is associated with significant increases in perioperative adverse outcomes for patients undergoing elective posterior lumbar surgery, even with matching and controlling for potentially confounding variables. These findings can guide patient counseling and preemptive interventions before surgery for this patient population. [. 2021;44(3):172-179.]. -
Mercier MR, Galivanche AR, Brand JP, Pathak N, Medvecky MJ, Varthi AG, Rubin LE, Grauer JN. 2021. COVID-positive ankle fracture patients are at increased odds of perioperative surgical complications following open reduction internal fixation surgery. PloS one. 16(12):e0262115. Pubmed: 34972190 DOI:10.1371/journal.pone.0262115 Mercier MR, Galivanche AR, Brand JP, Pathak N, Medvecky MJ, Varthi AG, Rubin LE, Grauer JN. 2021. COVID-positive ankle fracture patients are at increased odds of perioperative surgical complications following open reduction internal fixation surgery. PloS one. 16(12):e0262115. Pubmed: 34972190 DOI:10.1371/journal.pone.0262115 Array -
Yuan W, Beaulieu-Jones B, Krolewski R, Palmer N, Veyrat-Follet C, Frau F, Cohen C, Bozzi S, Cogswell M, Kumar D, Coulouvrat C, Leroy B, Fischer TZ, Sardi SP, Chandross KJ, Rubin LL, Wills AM, Kohane I, Lipnick SL. 2021. Accelerating diagnosis of Parkinson's disease through risk prediction. BMC neurology. 21(1):201. Pubmed: 34006233 DOI:10.1186/s12883-021-02226-4 Yuan W, Beaulieu-Jones B, Krolewski R, Palmer N, Veyrat-Follet C, Frau F, Cohen C, Bozzi S, Cogswell M, Kumar D, Coulouvrat C, Leroy B, Fischer TZ, Sardi SP, Chandross KJ, Rubin LL, Wills AM, Kohane I, Lipnick SL. 2021. Accelerating diagnosis of Parkinson's disease through risk prediction. BMC neurology. 21(1):201. Pubmed: 34006233 DOI:10.1186/s12883-021-02226-4 Array -
Almada AE, Horwitz N, Price FD, Gonzalez AE, Ko M, Bolukbasi OV, Messemer KA, Chen S, Sinha M, Rubin LL, Wagers AJ. 2021. FOS licenses early events in stem cell activation driving skeletal muscle regeneration. Cell reports. 34(4):108656. Pubmed: 33503437 DOI:S2211-1247(20)31645-4 Almada AE, Horwitz N, Price FD, Gonzalez AE, Ko M, Bolukbasi OV, Messemer KA, Chen S, Sinha M, Rubin LL, Wagers AJ. 2021. FOS licenses early events in stem cell activation driving skeletal muscle regeneration. Cell reports. 34(4):108656. Pubmed: 33503437 DOI:S2211-1247(20)31645-4 Muscle satellite cells (SCs) are a quiescent (non-proliferative) stem cell population in uninjured skeletal muscle. Although SCs have been investigated for nearly 60 years, the molecular drivers that transform quiescent SCs into the rapidly dividing (activated) stem/progenitor cells that mediate muscle repair after injury remain largely unknown. Here we identify a prominent FBJ osteosarcoma oncogene (Fos) mRNA and protein signature in recently activated SCs that is rapidly, heterogeneously, and transiently induced by muscle damage. We further reveal a requirement for FOS to efficiently initiate key stem cell functions, including cell cycle entry, proliferative expansion, and muscle regeneration, via induction of "pro-regenerative" target genes that stimulate cell migration, division, and differentiation. Disruption of one of these Fos/AP-1 targets, NAD(+)-consuming mono-ADP-ribosyl-transferase 1 (Art1), in SCs delays cell cycle entry and impedes progenitor cell expansion and muscle regeneration. This work uncovers an early-activated FOS/ART1/mono-ADP-ribosylation (MARylation) pathway that is essential for stem cell-regenerative responses.Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved. -
Martinez-Pastor B, Silveira GG, Clarke TL, Chung D, Gu Y, Cosentino C, Davidow LS, Mata G, Hassanieh S, Salsman J, Ciccia A, Bae N, Bedford MT, Megias D, Rubin LL, Efeyan A, Dellaire G, Mostoslavsky R. 2021. Assessing kinetics and recruitment of DNA repair factors using high content screens. Cell reports. 37(13):110176. Pubmed: 34965416 DOI:S2211-1247(21)01676-4 Martinez-Pastor B, Silveira GG, Clarke TL, Chung D, Gu Y, Cosentino C, Davidow LS, Mata G, Hassanieh S, Salsman J, Ciccia A, Bae N, Bedford MT, Megias D, Rubin LL, Efeyan A, Dellaire G, Mostoslavsky R. 2021. Assessing kinetics and recruitment of DNA repair factors using high content screens. Cell reports. 37(13):110176. Pubmed: 34965416 DOI:S2211-1247(21)01676-4 Repair of genetic damage is coordinated in the context of chromatin, so cells dynamically modulate accessibility at DNA breaks for the recruitment of DNA damage response (DDR) factors. The identification of chromatin factors with roles in DDR has mostly relied on loss-of-function screens while lacking robust high-throughput systems to study DNA repair. In this study, we have developed two high-throughput systems that allow the study of DNA repair kinetics and the recruitment of factors to double-strand breaks in a 384-well plate format. Using a customized gain-of-function open-reading frame library ("ChromORFeome" library), we identify chromatin factors with putative roles in the DDR. Among these, we find the PHF20 factor is excluded from DNA breaks, affecting DNA repair by competing with 53BP1 recruitment. Adaptable for genetic perturbations, small-molecule screens, and large-scale analysis of DNA repair, these resources can aid our understanding and manipulation of DNA repair.Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved. -
Cheng YC, Snavely A, Barrett LB, Zhang X, Herman C, Frost DJ, Riva P, Tochitsky I, Kawaguchi R, Singh B, Ivanis J, Huebner EA, Arvanites A, Oza V, Davidow L, Maeda R, Sakuma M, Grantham A, Wang Q, Chang AN, Pfaff K, Costigan M, Coppola G, Rubin LL, Schwer B, Alt FW, Woolf CJ. 2021. Topoisomerase I inhibition and peripheral nerve injury induce DNA breaks and ATF3-associated axon regeneration in sensory neurons. Cell reports. 36(10):109666. Pubmed: 34496254 DOI:S2211-1247(21)01110-4 Cheng YC, Snavely A, Barrett LB, Zhang X, Herman C, Frost DJ, Riva P, Tochitsky I, Kawaguchi R, Singh B, Ivanis J, Huebner EA, Arvanites A, Oza V, Davidow L, Maeda R, Sakuma M, Grantham A, Wang Q, Chang AN, Pfaff K, Costigan M, Coppola G, Rubin LL, Schwer B, Alt FW, Woolf CJ. 2021. Topoisomerase I inhibition and peripheral nerve injury induce DNA breaks and ATF3-associated axon regeneration in sensory neurons. Cell reports. 36(10):109666. Pubmed: 34496254 DOI:S2211-1247(21)01110-4 Although axonal damage induces rapid changes in gene expression in primary sensory neurons, it remains unclear how this process is initiated. The transcription factor ATF3, one of the earliest genes responding to nerve injury, regulates expression of downstream genes that enable axon regeneration. By exploiting ATF3 reporter systems, we identify topoisomerase inhibitors as ATF3 inducers, including camptothecin. Camptothecin increases ATF3 expression and promotes neurite outgrowth in sensory neurons in vitro and enhances axonal regeneration after sciatic nerve crush in vivo. Given the action of topoisomerases in producing DNA breaks, we determine that they do occur immediately after nerve damage at the ATF3 gene locus in injured sensory neurons and are further increased after camptothecin exposure. Formation of DNA breaks in injured sensory neurons and enhancement of it pharmacologically may contribute to the initiation of those transcriptional changes required for peripheral nerve regeneration.Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved. -
Pediaditakis I, Kodella KR, Manatakis DV, Le CY, Hinojosa CD, Tien-Street W, Manolakos ES, Vekrellis K, Hamilton GA, Ewart L, Rubin LL, Karalis K. 2021. Modeling alpha-synuclein pathology in a human brain-chip to assess blood-brain barrier disruption. Nature communications. 12(1):5907. Pubmed: 34625559 DOI:10.1038/s41467-021-26066-5 Pediaditakis I, Kodella KR, Manatakis DV, Le CY, Hinojosa CD, Tien-Street W, Manolakos ES, Vekrellis K, Hamilton GA, Ewart L, Rubin LL, Karalis K. 2021. Modeling alpha-synuclein pathology in a human brain-chip to assess blood-brain barrier disruption. Nature communications. 12(1):5907. Pubmed: 34625559 DOI:10.1038/s41467-021-26066-5 Parkinson's disease and related synucleinopathies are characterized by the abnormal accumulation of alpha-synuclein aggregates, loss of dopaminergic neurons, and gliosis of the substantia nigra. Although clinical evidence and in vitro studies indicate disruption of the Blood-Brain Barrier in Parkinson's disease, the mechanisms mediating the endothelial dysfunction is not well understood. Here we leveraged the Organs-on-Chips technology to develop a human Brain-Chip representative of the substantia nigra area of the brain containing dopaminergic neurons, astrocytes, microglia, pericytes, and microvascular brain endothelial cells, cultured under fluid flow. Our αSyn fibril-induced model was capable of reproducing several key aspects of Parkinson's disease, including accumulation of phosphorylated αSyn (pSer129-αSyn), mitochondrial impairment, neuroinflammation, and compromised barrier function. This model may enable research into the dynamics of cell-cell interactions in human synucleinopathies and serve as a testing platform for target identification and validation of novel therapeutics.© 2021. The Author(s). 2020
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Walsh MB, Janzen E, Wingrove E, Hosseinibarkooie S, Muela NR, Davidow L, Dimitriadi M, Norabuena EM, Rubin LL, Wirth B, Hart AC. 2020. Genetic modifiers ameliorate endocytic and neuromuscular defects in a model of spinal muscular atrophy. BMC biology. 18(1):127. Pubmed: 32938453 DOI:10.1186/s12915-020-00845-w Walsh MB, Janzen E, Wingrove E, Hosseinibarkooie S, Muela NR, Davidow L, Dimitriadi M, Norabuena EM, Rubin LL, Wirth B, Hart AC. 2020. Genetic modifiers ameliorate endocytic and neuromuscular defects in a model of spinal muscular atrophy. BMC biology. 18(1):127. Pubmed: 32938453 DOI:10.1186/s12915-020-00845-w Array -
Toyohara T, Roudnicky F, Florido MHC, Nakano T, Yu H, Katsuki S, Lee M, Meissner T, Friesen M, Davidow LS, Ptaszek L, Abe T, Rubin LL, Pereira AC, Aikawa M, Cowan CA. 2020. Patient hiPSCs Identify Vascular Smooth Muscle Arylacetamide Deacetylase as Protective against Atherosclerosis. Cell stem cell. 27(1):178-180. Pubmed: 32619513 DOI:S1934-5909(20)30211-3 Toyohara T, Roudnicky F, Florido MHC, Nakano T, Yu H, Katsuki S, Lee M, Meissner T, Friesen M, Davidow LS, Ptaszek L, Abe T, Rubin LL, Pereira AC, Aikawa M, Cowan CA. 2020. Patient hiPSCs Identify Vascular Smooth Muscle Arylacetamide Deacetylase as Protective against Atherosclerosis. Cell stem cell. 27(1):178-180. Pubmed: 32619513 DOI:S1934-5909(20)30211-3 -
Toyohara T, Roudnicky F, Florido MHC, Nakano T, Yu H, Katsuki S, Lee M, Meissner T, Friesen M, Davidow LS, Ptaszek L, Abe T, Rubin LL, Pereira AC, Aikawa M, Cowan CA. 2020. Patient hiPSCs Identify Vascular Smooth Muscle Arylacetamide Deacetylase as Protective against Atherosclerosis. Cell stem cell. 27(1):147-157.e7. Pubmed: 32413331 DOI:S1934-5909(20)30157-0 Toyohara T, Roudnicky F, Florido MHC, Nakano T, Yu H, Katsuki S, Lee M, Meissner T, Friesen M, Davidow LS, Ptaszek L, Abe T, Rubin LL, Pereira AC, Aikawa M, Cowan CA. 2020. Patient hiPSCs Identify Vascular Smooth Muscle Arylacetamide Deacetylase as Protective against Atherosclerosis. Cell stem cell. 27(1):147-157.e7. Pubmed: 32413331 DOI:S1934-5909(20)30157-0 Although susceptibility to cardiovascular disease (CVD) is different for every patient, why some patients with type 2 diabetes mellitus (T2DM) develop CVD while others are protected has not yet been clarified. Using T2DM-patient-derived human induced pluripotent stem cells (hiPSCs), we found that in patients protected from CVD, there was significantly elevated expression of an esterase, arylacetamide deacetylase (AADAC), in vascular smooth muscle cells (VSMCs). We overexpressed this esterase in human primary VSMCs and VSMCs differentiated from hiPSCs and observed that the number of lipid droplets was significantly diminished. Further metabolomic analyses revealed a marked reduction in storage lipids and an increase in membrane phospholipids, suggesting changes in the Kennedy pathway of lipid bioassembly. Cell migration and proliferation were also significantly decreased in AADAC-overexpressing VSMCs. Moreover, apolipoprotein E (Apoe)-knockout mice overexpressing VSMC-specific Aadac showed amelioration of atherosclerotic lesions. Our findings suggest that higher AADAC expression in VSMCs protects T2DM patients from CVD.Copyright © 2020. Published by Elsevier Inc. -
Ahfeldt T, Ordureau A, Bell C, Sarrafha L, Sun C, Piccinotti S, Grass T, Parfitt GM, Paulo JA, Yanagawa F, Uozumi T, Kiyota Y, Harper JW, Rubin LL. 2020. Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons. Stem cell reports. 14(1):75-90. Pubmed: 31902706 DOI:S2213-6711(19)30444-8 Ahfeldt T, Ordureau A, Bell C, Sarrafha L, Sun C, Piccinotti S, Grass T, Parfitt GM, Paulo JA, Yanagawa F, Uozumi T, Kiyota Y, Harper JW, Rubin LL. 2020. Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson's Disease Discovered Using Isogenic Human Dopaminergic Neurons. Stem cell reports. 14(1):75-90. Pubmed: 31902706 DOI:S2213-6711(19)30444-8 Parkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved. -
Malpani R, Mclynn RP, Bovonratwet P, Bagi PS, Yurter A, Mercier MR, Rubin LE, Grauer JN. 2020. Coagulopathies Are a Risk Factor for Adverse Events Following Total Hip and Total Knee Arthroplasty. Orthopedics. 43(4):233-238. Pubmed: 32674174 DOI:10.3928/01477447-20200624-02 Malpani R, Mclynn RP, Bovonratwet P, Bagi PS, Yurter A, Mercier MR, Rubin LE, Grauer JN. 2020. Coagulopathies Are a Risk Factor for Adverse Events Following Total Hip and Total Knee Arthroplasty. Orthopedics. 43(4):233-238. Pubmed: 32674174 DOI:10.3928/01477447-20200624-02 Current literature suggests a correlation between preoperative coagulopathies and postsurgical adverse events (AEs). However, this correlation has not been specifically assessed in the total hip arthroplasty (THA) and the total knee arthroplasty (TKA) populations. Patients who underwent primary THA and TKA with coagulopathy data were identified from the 2011-2015 American College of Surgeons National Surgical Quality Improvement Program database. Coagulopathies studied were low platelets, high partial thromboplastin time (PTT), high international normalized ratio (INR), and other hematological conditions. Univariate and multivariate analyses were conducted to explore the relationship between coagulopathies and 30-day AEs following surgery in these populations. In total, 39,605 THA patients and 67,685 TKA patients were identified. Of these, approximately 16% had a coagulopathy. These patients tended to be older and have a dependent functional status, American Society of Anesthesiologists score of 3 or greater, and diabetes mellitus. In the THA cohort, low platelets, high PTT, high INR, and other hematological conditions were associated with increased odds of any AE, major AEs, and minor AEs. High INR and other hematological conditions were associated with an increased odds of hospital readmission. In the TKA group, low platelets, high INR, and other hematological conditions were associated with increased odds of any AE, major AEs, and minor AEs. High PTT was associated with increased odds of major AEs and readmissions. Presence of a coagulopathy was associated with multiple AEs following both THA and TKA. This shows that special attention should be paid patients with any form of coagulopathy to minimize the potential risk of AEs. [Orthopedics. 2020;43(4):233-238.].Copyright 2020, SLACK Incorporated. -
Buchanan SM, Price FD, Castiglioni A, Gee AW, Schneider J, Matyas MN, Hayhurst M, Tabebordbar M, Wagers AJ, Rubin LL. 2020. Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells. Skeletal muscle. 10(1):28. Pubmed: 33036659 DOI:10.1186/s13395-020-00248-z Buchanan SM, Price FD, Castiglioni A, Gee AW, Schneider J, Matyas MN, Hayhurst M, Tabebordbar M, Wagers AJ, Rubin LL. 2020. Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells. Skeletal muscle. 10(1):28. Pubmed: 33036659 DOI:10.1186/s13395-020-00248-z Satellite cells are the canonical muscle stem cells that regenerate damaged skeletal muscle. Loss of function of these cells has been linked to reduced muscle repair capacity and compromised muscle health in acute muscle injury and congenital neuromuscular diseases. To identify new pathways that can prevent loss of skeletal muscle function or enhance regenerative potential, we established an imaging-based screen capable of identifying small molecules that promote the expansion of freshly isolated satellite cells. We found several classes of receptor tyrosine kinase (RTK) inhibitors that increased freshly isolated satellite cell numbers in vitro. Further exploration of one of these compounds, the RTK inhibitor CEP-701 (also known as lestaurtinib), revealed potent activity on mouse satellite cells both in vitro and in vivo. This expansion potential was not seen upon exposure of proliferating committed myoblasts or non-myogenic fibroblasts to CEP-701. When delivered subcutaneously to acutely injured animals, CEP-701 increased both the total number of satellite cells and the rate of muscle repair, as revealed by an increased cross-sectional area of regenerating fibers. Moreover, freshly isolated satellite cells expanded ex vivo in the presence of CEP-701 displayed enhanced muscle engraftment potential upon in vivo transplantation. We provide compelling evidence that certain RTKs, and in particular RET, regulate satellite cell expansion during muscle regeneration. This study demonstrates the power of small molecule screens of even rare adult stem cell populations for identifying stem cell-targeting compounds with therapeutic potential. 2019
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Howell K, Gibbs RM, Rubin LL. 2019. Spinal Muscular Atrophy: Huge Steps. Cerebrum : the Dana forum on brain science. 2019. Pubmed: 32206168 DOI:cer-03-19 Howell K, Gibbs RM, Rubin LL. 2019. Spinal Muscular Atrophy: Huge Steps. Cerebrum : the Dana forum on brain science. 2019. Pubmed: 32206168 DOI:cer-03-19 ArrayCopyright 2019 The Dana Foundation All Rights Reserved. -
Lipnick SL, Agniel DM, Aggarwal R, Makhortova NR, Finlayson SG, Brocato A, Palmer N, Darras BT, Kohane I, Rubin LL. 2019. Systemic nature of spinal muscular atrophy revealed by studying insurance claims. PloS one. 14(3):e0213680. Pubmed: 30870495 DOI:10.1371/journal.pone.0213680 Lipnick SL, Agniel DM, Aggarwal R, Makhortova NR, Finlayson SG, Brocato A, Palmer N, Darras BT, Kohane I, Rubin LL. 2019. Systemic nature of spinal muscular atrophy revealed by studying insurance claims. PloS one. 14(3):e0213680. Pubmed: 30870495 DOI:10.1371/journal.pone.0213680 Array -
Koutmani Y, Gampierakis IA, Polissidis A, Ximerakis M, Koutsoudaki PN, Polyzos A, Agrogiannis G, Karaliota S, Thomaidou D, Rubin LL, Politis PK, Karalis KP. 2019. CRH Promotes the Neurogenic Activity of Neural Stem Cells in the Adult Hippocampus. Cell reports. 29(4):932-945.e7. Pubmed: 31644914 DOI:S2211-1247(19)31225-2 Koutmani Y, Gampierakis IA, Polissidis A, Ximerakis M, Koutsoudaki PN, Polyzos A, Agrogiannis G, Karaliota S, Thomaidou D, Rubin LL, Politis PK, Karalis KP. 2019. CRH Promotes the Neurogenic Activity of Neural Stem Cells in the Adult Hippocampus. Cell reports. 29(4):932-945.e7. Pubmed: 31644914 DOI:S2211-1247(19)31225-2 Local cues in the adult neurogenic niches dynamically regulate homeostasis in neural stem cells, whereas their identity and associated molecular mechanisms remain poorly understood. Here, we show that corticotropin-releasing hormone (CRH), the major mediator of mammalian stress response and a key neuromodulator in the adult brain, is necessary for hippocampal neural stem cell (hiNSC) activity under physiological conditions. In particular, we demonstrate functionality of the CRH/CRH receptor (CRHR) system in mouse hiNSCs and conserved expression in humans. Most important, we show that genetic deficiency of CRH impairs hippocampal neurogenesis, affects spatial memory, and compromises hiNSCs' responsiveness to environmental stimuli. These deficits have been partially restored by virus-mediated CRH expression. Additionally, we provide evidence that local disruption of the CRH/CRHR system reduces neurogenesis, while exposure of adult hiNSCs to CRH promotes neurogenic activity via BMP4 suppression. Our findings suggest a critical role of CRH in adult neurogenesis, independently of its stress-related systemic function.Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved. -
Ximerakis M, Lipnick SL, Innes BT, Simmons SK, Adiconis X, Dionne D, Mayweather BA, Nguyen L, Niziolek Z, Ozek C, Butty VL, Isserlin R, Buchanan SM, Levine SS, Regev A, Bader GD, Levin JZ, Rubin LL. 2019. Single-cell transcriptomic profiling of the aging mouse brain. Nature neuroscience. 22(10):1696-1708. Pubmed: 31551601 DOI:10.1038/s41593-019-0491-3 Ximerakis M, Lipnick SL, Innes BT, Simmons SK, Adiconis X, Dionne D, Mayweather BA, Nguyen L, Niziolek Z, Ozek C, Butty VL, Isserlin R, Buchanan SM, Levine SS, Regev A, Bader GD, Levin JZ, Rubin LL. 2019. Single-cell transcriptomic profiling of the aging mouse brain. Nature neuroscience. 22(10):1696-1708. Pubmed: 31551601 DOI:10.1038/s41593-019-0491-3 The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand-receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain ) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process. -
Watts ME, Wu C, Rubin LL. 2019. Suppression of MAP4K4 Signaling Ameliorates Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis-Molecular Studies Toward New Therapeutics. Journal of experimental neuroscience. 13:1179069519862798. Pubmed: 31320806 DOI:10.1177/1179069519862798 Watts ME, Wu C, Rubin LL. 2019. Suppression of MAP4K4 Signaling Ameliorates Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis-Molecular Studies Toward New Therapeutics. Journal of experimental neuroscience. 13:1179069519862798. Pubmed: 31320806 DOI:10.1177/1179069519862798 Amyotrophic lateral sclerosis (ALS), the most common motor neuron (MN) disease of adults, is characterized by the degeneration of upper MNs in the motor cortex and lower MNs in the brain stem and spinal cord. Our recent work suggests that a MAP kinase family member, MAP4K4 (mitogen-activated protein kinase kinase kinase kinase 4), regulates MN degeneration in ALS. Activation of MAP4K4 occurs prior to MN death and inhibition of MAP4K4 improves neurite integrity and neuronal viability in a cell autonomous manner. The mechanism through which MAP4K4 reduction specifically modulates MN viability can be attributed to the attenuation of the c-Jun apoptotic pathway, as well as to the activation of FoxO1-mediated autophagy that reduces the accumulation of protein aggregates. We additionally show the feasibility of MAP4K4 as a drug target using a MAP4K4-specific inhibitor, which improves the survival of both primary and induced pluripotent stem cell (iPSC)-derived MNs. Our studies are thus far the first to highlight a MAP4K4-initiated signaling cascade that contributes to MN degeneration in ALS, providing a new mechanism underlying MN death in disease and a druggable target for new therapeutics. We propose exciting future directions and unexplored avenues based upon this work. -
Idkowiak-Baldys J, Santhanam U, Buchanan SM, Pfaff KL, Rubin LL, Lyga J. 2019. Growth differentiation factor 11 (GDF11) has pronounced effects on skin biology. PloS one. 14(6):e0218035. Pubmed: 31181098 DOI:10.1371/journal.pone.0218035 Idkowiak-Baldys J, Santhanam U, Buchanan SM, Pfaff KL, Rubin LL, Lyga J. 2019. Growth differentiation factor 11 (GDF11) has pronounced effects on skin biology. PloS one. 14(6):e0218035. Pubmed: 31181098 DOI:10.1371/journal.pone.0218035 Growth differentiation factor 11 (GDF11) belongs to the TGF-β superfamily of proteins and is closely related to myostatin. Recent findings show that GDF11 has rejuvenating properties with pronounced effects on the cardiovascular system, brain, skeletal muscle, and skeleton in mice. Several human studies were also conducted, some implicating decreasing levels of circulating GDF11 with age. To date, however, there have not been any reports on its role in human skin. This study examined the impact of GDF11 on human skin, specifically related to skin aging. The effect of recombinant GDF11 on the function of various skin cells was examined in human epidermal keratinocytes, dermal fibroblasts, melanocytes, dermal microvascular endothelial cells and 3D skin equivalents, as well as in ex vivo human skin explants. GDF11 had significant effects on the production of dermal matrix components in multiple skin models in vitro and ex vivo. In addition, it had a pronounced effect on expression of multiple skin related genes in full thickness 3D skin equivalents. This work, for the first time, demonstrates an important role for GDF11 in skin biology and a potential impact on skin health and aging. -
Pak E, MacKenzie EL, Zhao X, Pazyra-Murphy MF, Park PMC, Wu L, Shaw DL, Addleson EC, Cayer SS, Lopez BG, Agar NYR, Rubin LL, Qi J, Merk DJ, Segal RA. 2019. A large-scale drug screen identifies selective inhibitors of class I HDACs as a potential therapeutic option for SHH medulloblastoma. Neuro-oncology. 21(9):1150-1163. Pubmed: 31111916 DOI:10.1093/neuonc/noz089 Pak E, MacKenzie EL, Zhao X, Pazyra-Murphy MF, Park PMC, Wu L, Shaw DL, Addleson EC, Cayer SS, Lopez BG, Agar NYR, Rubin LL, Qi J, Merk DJ, Segal RA. 2019. A large-scale drug screen identifies selective inhibitors of class I HDACs as a potential therapeutic option for SHH medulloblastoma. Neuro-oncology. 21(9):1150-1163. Pubmed: 31111916 DOI:10.1093/neuonc/noz089 Array© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. -
Wu C, Watts ME, Rubin LL. 2019. MAP4K4 Activation Mediates Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis. Cell reports. 26(5):1143-1156.e5. Pubmed: 30699345 DOI:S2211-1247(19)30028-2 Wu C, Watts ME, Rubin LL. 2019. MAP4K4 Activation Mediates Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis. Cell reports. 26(5):1143-1156.e5. Pubmed: 30699345 DOI:S2211-1247(19)30028-2 Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons (MNs). To date, its underlying mechanisms have yet to be clarified completely, and there are no truly effective treatments. Here, we show that MAP4K4, a MAP kinase family member, regulates MN death, with its suppression not only promoting survival but preventing neurite degeneration and decreasing mutant SOD1 levels through autophagy activation. Moreover, we report that MAP4K4 signaling specifically modulates MN viability via phosphorylated JNK3 and activation of the canonical c-Jun apoptotic pathway. Finally, we show the feasibility of MAP4K4 as a drug target by using an available MAP4K4-specific inhibitor, which improves survival of ESC and/or iPSC-derived MNs and MNs cultured from mouse spinal cords. In summary, our studies highlight a MAP4K4-initiated signaling cascade that induces MN degeneration, shedding light on the mechanism underlying MN degeneration and providing a druggable target for ALS therapeutics.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved. -
Yang SJ, Lipnick SL, Makhortova NR, Venugopalan S, Fan M, Armstrong Z, Schlaeger TM, Deng L, Chung WK, O'Callaghan L, Geraschenko A, Whye D, Berndl M, Hazard J, Williams B, Narayanaswamy A, Ando DM, Nelson P, Rubin LL. 2019. Applying Deep Neural Network Analysis to High-Content Image-Based Assays. SLAS discovery : advancing life sciences R & D. 24(8):829-841. Pubmed: 31284814 DOI:10.1177/2472555219857715 Yang SJ, Lipnick SL, Makhortova NR, Venugopalan S, Fan M, Armstrong Z, Schlaeger TM, Deng L, Chung WK, O'Callaghan L, Geraschenko A, Whye D, Berndl M, Hazard J, Williams B, Narayanaswamy A, Ando DM, Nelson P, Rubin LL. 2019. Applying Deep Neural Network Analysis to High-Content Image-Based Assays. SLAS discovery : advancing life sciences R & D. 24(8):829-841. Pubmed: 31284814 DOI:10.1177/2472555219857715 The etiological underpinnings of many CNS disorders are not well understood. This is likely due to the fact that individual diseases aggregate numerous pathological subtypes, each associated with a complex landscape of genetic risk factors. To overcome these challenges, researchers are integrating novel data types from numerous patients, including imaging studies capturing broadly applicable features from patient-derived materials. These datasets, when combined with machine learning, potentially hold the power to elucidate the subtle patterns that stratify patients by shared pathology. In this study, we interrogated whether high-content imaging of primary skin fibroblasts, using the Cell Painting method, could reveal disease-relevant information among patients. First, we showed that technical features such as batch/plate type, plate, and location within a plate lead to detectable nuisance signals, as revealed by a pre-trained deep neural network and analysis with deep image embeddings. Using a plate design and image acquisition strategy that accounts for these variables, we performed a pilot study with 12 healthy controls and 12 subjects affected by the severe genetic neurological disorder spinal muscular atrophy (SMA), and evaluated whether a convolutional neural network (CNN) generated using a subset of the cells could distinguish disease states on cells from the remaining unseen control-SMA pair. Our results indicate that these two populations could effectively be differentiated from one another and that model selectivity is insensitive to batch/plate type. One caveat is that the samples were also largely separated by source. These findings lay a foundation for how to conduct future studies exploring diseases with more complex genetic contributions and unknown subtypes. 2018
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Paik EJ, O'Neil AL, Ng SY, Sun C, Rubin LL. 2018. Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture. Scientific reports. 8(1):804. Pubmed: 29339826 DOI:10.1038/s41598-018-19291-4 Paik EJ, O'Neil AL, Ng SY, Sun C, Rubin LL. 2018. Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture. Scientific reports. 8(1):804. Pubmed: 29339826 DOI:10.1038/s41598-018-19291-4 Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) can provide sources for midbrain dopaminergic (mDA) neural progenitors (NPCs) for cell therapy to treat Parkinson's disease (PD) patients. However, the well-known line-to-cell line variability in the differentiation capacity of individual cell lines needs to be improved for the success of this therapy. To address this issue, we sought to identify mDA NPC specific cell surface markers for fluorescence activated cell sorting (FACS). Through RNA isolation after sorting for NPCs based on staining for cell-specific transcription factors followed by microarray, we identified two positive cell surface markers (CORIN and CD166) and one negative cell surface marker (CXCR4) for mDA NPC sorting. These three markers can enrich floor plate NPCs to 90% purity, and the sorted NPCs more efficiently differentiate to mature dopaminergic neurons compared to unsorted or CORIN alone mDA NPCs. This surface marker identification strategy can be used broadly to facilitate isolation of cell subtypes of interest from heterogeneous cultures. -
Ozek C, Krolewski RC, Buchanan SM, Rubin LL. 2018. Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice. Scientific reports. 8(1):17293. Pubmed: 30470794 DOI:10.1038/s41598-018-35716-6 Ozek C, Krolewski RC, Buchanan SM, Rubin LL. 2018. Growth Differentiation Factor 11 treatment leads to neuronal and vascular improvements in the hippocampus of aged mice. Scientific reports. 8(1):17293. Pubmed: 30470794 DOI:10.1038/s41598-018-35716-6 Aging is the biggest risk factor for several neurodegenerative diseases. Parabiosis experiments have established that old mouse brains are improved by exposure to young mouse blood. Previously, our lab showed that delivery of Growth Differentiation Factor 11 (GDF11) to the bloodstream increases the number of neural stem cells and positively affects vasculature in the subventricular zone of old mice. Our new study demonstrates that GDF11 enhances hippocampal neurogenesis, improves vasculature and increases markers of neuronal activity and plasticity in the hippocampus and cortex of old mice. Our experiments also demonstrate that systemically delivered GDF11, rather than crossing the blood brain barrier, exerts at least some of its effects by acting on brain endothelial cells. Thus, by targeting the cerebral vasculature, GDF11 has a very different mechanism from that of previously studied circulating factors acting to improve central nervous system (CNS) function without entering the CNS. -
Darnell M, O'Neil A, Mao A, Gu L, Rubin LL, Mooney DJ. 2018. Material microenvironmental properties couple to induce distinct transcriptional programs in mammalian stem cells. Proceedings of the National Academy of Sciences of the United States of America. 115(36):E8368-E8377. Pubmed: 30120125 DOI:10.1073/pnas.1802568115 Darnell M, O'Neil A, Mao A, Gu L, Rubin LL, Mooney DJ. 2018. Material microenvironmental properties couple to induce distinct transcriptional programs in mammalian stem cells. Proceedings of the National Academy of Sciences of the United States of America. 115(36):E8368-E8377. Pubmed: 30120125 DOI:10.1073/pnas.1802568115 Variations in a multitude of material microenvironmental properties have been observed across tissues in vivo, and these have profound effects on cell phenotype. Phenomenological experiments have suggested that certain of these features of the physical microenvironment, such as stiffness, could sensitize cells to other features; meanwhile, mechanistic studies have detailed a number of biophysical mechanisms for this sensing. However, the broad molecular consequences of these potentially complex and nonlinear interactions bridging from biophysical sensing to phenotype have not been systematically characterized, limiting the overall understanding and rational deployment of these biophysical cues. Here, we explore these interactions by employing a 3D cell culture system that allows for the independent control of culture substrate stiffness, stress relaxation, and adhesion ligand density to systematically explore the transcriptional programs affected by distinct combinations of biophysical parameters using RNA-seq. In mouse mesenchymal stem cells and human cortical neuron progenitors, we find dramatic coupling among these substrate properties, and that the relative contribution of each property to changes in gene expression varies with cell type. Motivated by the bioinformatic analysis, the stiffness of hydrogels encapsulating mouse mesenchymal stem cells was found to regulate the secretion of a wide range of cytokines, and to accordingly influence hematopoietic stem cell differentiation in a Transwell coculture model. These results give insights into how biophysical features are integrated by cells across distinct tissues and offer strategies to synthetic biologists and bioengineers for designing responses to a cell's biophysical environment. -
Shin HY, Pfaff KL, Davidow LS, Sun C, Uozumi T, Yanagawa F, Yamazaki Y, Kiyota Y, Rubin LL. 2018. Using Automated Live Cell Imaging to Reveal Early Changes during Human Motor Neuron Degeneration. eNeuro. 5(3). Pubmed: 29971247 DOI:10.1523/ENEURO.0001-18.2018 Shin HY, Pfaff KL, Davidow LS, Sun C, Uozumi T, Yanagawa F, Yamazaki Y, Kiyota Y, Rubin LL. 2018. Using Automated Live Cell Imaging to Reveal Early Changes during Human Motor Neuron Degeneration. eNeuro. 5(3). Pubmed: 29971247 DOI:10.1523/ENEURO.0001-18.2018 Human neurons expressing mutations associated with neurodegenerative disease are becoming more widely available. Hence, developing assays capable of accurately detecting changes that occur early in the disease process and identifying therapeutics able to slow these changes should become ever more important. Using automated live-cell imaging, we studied human motor neurons in the process of dying following neurotrophic factor withdrawal. We tracked different neuronal features, including cell body size, neurite length, and number of nodes. In particular, measuring the number of nodes in individual neurons proved to be an accurate predictor of relative health. Importantly, intermediate phenotypes were defined and could be used to distinguish between agents that could fully restore neurons and neurites and those only capable of maintaining neuronal cell bodies. Application of live-cell imaging to disease modeling has the potential to uncover new classes of therapeutic molecules that intervene early in disease progression. -
Benkler C, O'Neil AL, Slepian S, Qian F, Weinreb PH, Rubin LL. 2018. Aggregated SOD1 causes selective death of cultured human motor neurons. Scientific reports. 8(1):16393. Pubmed: 30401824 DOI:10.1038/s41598-018-34759-z Benkler C, O'Neil AL, Slepian S, Qian F, Weinreb PH, Rubin LL. 2018. Aggregated SOD1 causes selective death of cultured human motor neurons. Scientific reports. 8(1):16393. Pubmed: 30401824 DOI:10.1038/s41598-018-34759-z Most human neurodegenerative diseases share a phenotype of neuronal protein aggregation. In Amyotrophic Lateral Sclerosis (ALS), the abundant protein superoxide dismutase (SOD1) or the TAR-DNA binding protein TDP-43 can aggregate in motor neurons. Recently, numerous studies have highlighted the ability of aggregates to spread from neuron to neuron in a prion-like fashion. These studies have typically focused on the use of neuron-like cell lines or neurons that are not normally affected by the specific aggregated protein being studied. Here, we have investigated the uptake of pre-formed SOD1 aggregates by cultures containing pluripotent stem cell-derived human motor neurons. We found that all cells take up aggregates by a process resembling fluid-phase endocytosis, just as found in earlier studies. However, motor neurons, despite taking up smaller amounts of SOD1, were much more vulnerable to the accumulating aggregates. Thus, the propagation of disease pathology depends less on selective uptake than on selective response to intracellular aggregates. We further demonstrate that anti-SOD1 antibodies, being considered as ALS therapeutics, can act by blocking the uptake of SOD1, but also by blocking the toxic effects of intracellular SOD1. This work demonstrates the importance of using disease relevant cells even in studying phenomena such as aggregate propagation. -
Gibbs RM, Lipnick S, Bateman JW, Chen L, Cousins HC, Hubbard EG, Jowett G, LaPointe DS, McGredy MJ, Odonkor MN, Repetti G, Thomas E, Rubin LL. 2018. Toward Precision Medicine for Neurological and Neuropsychiatric Disorders. Cell stem cell. 23(1):21-24. Pubmed: 29887317 DOI:S1934-5909(18)30237-6 Gibbs RM, Lipnick S, Bateman JW, Chen L, Cousins HC, Hubbard EG, Jowett G, LaPointe DS, McGredy MJ, Odonkor MN, Repetti G, Thomas E, Rubin LL. 2018. Toward Precision Medicine for Neurological and Neuropsychiatric Disorders. Cell stem cell. 23(1):21-24. Pubmed: 29887317 DOI:S1934-5909(18)30237-6 The genetic complexity, clinical variability, and inaccessibility of affected tissue in neurodegenerative and neuropsychiatric disorders have largely prevented the development of effective disease-modifying therapeutics. A precision medicine approach that integrates genomics, deep clinical phenotyping, and patient stem cell models may facilitate identification of underlying biological drivers and targeted drug development.Copyright © 2018 Elsevier Inc. All rights reserved. -
Christiansen EM, Yang SJ, Ando DM, Javaherian A, Skibinski G, Lipnick S, Mount E, O'Neil A, Shah K, Lee AK, Goyal P, Fedus W, Poplin R, Esteva A, Berndl M, Rubin LL, Nelson P, Finkbeiner S. 2018. In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images. Cell. 173(3):792-803.e19. Pubmed: 29656897 DOI:S0092-8674(18)30364-7 Christiansen EM, Yang SJ, Ando DM, Javaherian A, Skibinski G, Lipnick S, Mount E, O'Neil A, Shah K, Lee AK, Goyal P, Fedus W, Poplin R, Esteva A, Berndl M, Rubin LL, Nelson P, Finkbeiner S. 2018. In Silico Labeling: Predicting Fluorescent Labels in Unlabeled Images. Cell. 173(3):792-803.e19. Pubmed: 29656897 DOI:S0092-8674(18)30364-7 Microscopy is a central method in life sciences. Many popular methods, such as antibody labeling, are used to add physical fluorescent labels to specific cellular constituents. However, these approaches have significant drawbacks, including inconsistency; limitations in the number of simultaneous labels because of spectral overlap; and necessary perturbations of the experiment, such as fixing the cells, to generate the measurement. Here, we show that a computational machine-learning approach, which we call "in silico labeling" (ISL), reliably predicts some fluorescent labels from transmitted-light images of unlabeled fixed or live biological samples. ISL predicts a range of labels, such as those for nuclei, cell type (e.g., neural), and cell state (e.g., cell death). Because prediction happens in silico, the method is consistent, is not limited by spectral overlap, and does not disturb the experiment. ISL generates biological measurements that would otherwise be problematic or impossible to acquire.Copyright © 2018 Elsevier Inc. All rights reserved. -
Ordureau A, Paulo JA, Zhang W, Ahfeldt T, Zhang J, Cohn EF, Hou Z, Heo JM, Rubin LL, Sidhu SS, Gygi SP, Harper JW. 2018. Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics. Molecular cell. 70(2):211-227.e8. Pubmed: 29656925 DOI:S1097-2765(18)30215-6 Ordureau A, Paulo JA, Zhang W, Ahfeldt T, Zhang J, Cohn EF, Hou Z, Heo JM, Rubin LL, Sidhu SS, Gygi SP, Harper JW. 2018. Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics. Molecular cell. 70(2):211-227.e8. Pubmed: 29656925 DOI:S1097-2765(18)30215-6 Flux through kinase and ubiquitin-driven signaling systems depends on the modification kinetics, stoichiometry, primary site specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons, and we model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site specificity of PARKIN-dependent target ubiquitylation, and we demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is inhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains.Copyright © 2018 Elsevier Inc. All rights reserved. -
Rodriguez-Muela N, Parkhitko A, Grass T, Gibbs RM, Norabuena EM, Perrimon N, Singh R, Rubin LL. 2018. Blocking p62-dependent SMN degradation ameliorates spinal muscular atrophy disease phenotypes. The Journal of clinical investigation. 128(7):3008-3023. Pubmed: 29672276 DOI:95231 Rodriguez-Muela N, Parkhitko A, Grass T, Gibbs RM, Norabuena EM, Perrimon N, Singh R, Rubin LL. 2018. Blocking p62-dependent SMN degradation ameliorates spinal muscular atrophy disease phenotypes. The Journal of clinical investigation. 128(7):3008-3023. Pubmed: 29672276 DOI:95231 Spinal muscular atrophy (SMA), a degenerative motor neuron (MN) disease, caused by loss of functional survival of motor neuron (SMN) protein due to SMN1 gene mutations, is a leading cause of infant mortality. Increasing SMN levels ameliorates the disease phenotype and is unanimously accepted as a therapeutic approach for patients with SMA. The ubiquitin/proteasome system is known to regulate SMN protein levels; however, whether autophagy controls SMN levels remains poorly explored. Here, we show that SMN protein is degraded by autophagy. Pharmacological and genetic inhibition of autophagy increases SMN levels, while induction of autophagy decreases these levels. SMN degradation occurs via its interaction with the autophagy adapter p62 (also known as SQSTM1). We also show that SMA neurons display reduced autophagosome clearance, increased p62 and ubiquitinated proteins levels, and hyperactivated mTORC1 signaling. Importantly, reducing p62 levels markedly increases SMN and its binding partner gemin2, promotes MN survival, and extends lifespan in fly and mouse SMA models, revealing p62 as a potential new therapeutic target for the treatment of SMA. 2017
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d'Ydewalle C, Ramos DM, Pyles NJ, Ng SY, Gorz M, Pilato CM, Ling K, Kong L, Ward AJ, Rubin LL, Rigo F, Bennett CF, Sumner CJ. 2017. The Antisense Transcript SMN-AS1 Regulates SMN Expression and Is a Novel Therapeutic Target for Spinal Muscular Atrophy. Neuron. 93(1):66-79. Pubmed: 28017471 DOI:S0896-6273(16)30901-1 d'Ydewalle C, Ramos DM, Pyles NJ, Ng SY, Gorz M, Pilato CM, Ling K, Kong L, Ward AJ, Rubin LL, Rigo F, Bennett CF, Sumner CJ. 2017. The Antisense Transcript SMN-AS1 Regulates SMN Expression and Is a Novel Therapeutic Target for Spinal Muscular Atrophy. Neuron. 93(1):66-79. Pubmed: 28017471 DOI:S0896-6273(16)30901-1 The neuromuscular disorder spinal muscular atrophy (SMA), the most common inherited killer of infants, is caused by insufficient expression of survival motor neuron (SMN) protein. SMA therapeutics development efforts have focused on identifying strategies to increase SMN expression. We identified a long non-coding RNA (lncRNA) that arises from the antisense strand of SMN, SMN-AS1, which is enriched in neurons and transcriptionally represses SMN expression by recruiting the epigenetic Polycomb repressive complex-2. Targeted degradation of SMN-AS1 with antisense oligonucleotides (ASOs) increases SMN expression in patient-derived cells, cultured neurons, and the mouse central nervous system. SMN-AS1 ASOs delivered together with SMN2 splice-switching oligonucleotides additively increase SMN expression and improve survival of severe SMA mice. This study is the first proof of concept that targeting a lncRNA to transcriptionally activate SMN2 can be combined with SMN2 splicing modification to ameliorate SMA and demonstrates the promise of combinatorial ASOs for the treatment of neurogenetic disorders.Copyright © 2017 Elsevier Inc. All rights reserved. -
Ahfeldt T, Litterman NK, Rubin LL. 2017. Studying human disease using human neurons. Brain research. 1656:40-48. Pubmed: 27060768 DOI:S0006-8993(16)30189-5 Ahfeldt T, Litterman NK, Rubin LL. 2017. Studying human disease using human neurons. Brain research. 1656:40-48. Pubmed: 27060768 DOI:S0006-8993(16)30189-5 Utilizing patient derived cells has enormous promise for discovering new drugs for diseases of the nervous system, a goal that has been historically quite challenging. In this review, we will outline the potential of human stem cell derived neuron models for assessing therapeutics and high-throughput screening and compare to more traditional drug discovery strategies. We summarize recent successes of the approach and discuss special considerations for developing human stem cell based assays. New technologies, such as genome editing, offer improvements to help overcome the challenges that remain. Finally, human neurons derived from patient cells have advantages for translational research beyond drug screening as they can also be used to identify individual efficacy and safety prior to clinical testing and for dissecting disease mechanisms. This article is part of a Special Issue entitled SI: Exploiting human neurons.Copyright © 2016 Elsevier B.V. All rights reserved. -
Feinberg K, Kolaj A, Wu C, Grinshtein N, Krieger JR, Moran MF, Rubin LL, Miller FD, Kaplan DR. 2017. A neuroprotective agent that inactivates prodegenerative TrkA and preserves mitochondria. The Journal of cell biology. 216(11):3655-3675. Pubmed: 28877995 DOI:10.1083/jcb.201705085 Feinberg K, Kolaj A, Wu C, Grinshtein N, Krieger JR, Moran MF, Rubin LL, Miller FD, Kaplan DR. 2017. A neuroprotective agent that inactivates prodegenerative TrkA and preserves mitochondria. The Journal of cell biology. 216(11):3655-3675. Pubmed: 28877995 DOI:10.1083/jcb.201705085 Axon degeneration is an early event and pathological in neurodegenerative conditions and nerve injuries. To discover agents that suppress neuronal death and axonal degeneration, we performed drug screens on primary rodent neurons and identified the pan-kinase inhibitor foretinib, which potently rescued sympathetic, sensory, and motor and SOD1 mutant neurons from trophic factor withdrawal-induced degeneration. By using primary sympathetic neurons grown in mass cultures and Campenot chambers, we show that foretinib protected neurons by suppressing both known degenerative pathways and a new pathway involving unliganded TrkA and transcriptional regulation of the proapoptotic BH3 family members BimEL, Harakiri,and Puma, culminating in preservation of mitochondria in the degenerative setting. Foretinib delayed chemotherapy-induced and Wallerian axonal degeneration in culture by preventing axotomy-induced local energy deficit and preserving mitochondria, and peripheral Wallerian degeneration in vivo. These findings identify a new axon degeneration pathway and a potentially clinically useful therapeutic drug.© 2017 Feinberg et al. -
Tripathi P, Rodriguez-Muela N, Klim JR, de Boer AS, Agrawal S, Sandoe J, Lopes CS, Ogliari KS, Williams LA, Shear M, Rubin LL, Eggan K, Zhou Q. 2017. Reactive Astrocytes Promote ALS-like Degeneration and Intracellular Protein Aggregation in Human Motor Neurons by Disrupting Autophagy through TGF-β1. Stem cell reports. 9(2):667-680. Pubmed: 28712846 DOI:S2213-6711(17)30271-0 Tripathi P, Rodriguez-Muela N, Klim JR, de Boer AS, Agrawal S, Sandoe J, Lopes CS, Ogliari KS, Williams LA, Shear M, Rubin LL, Eggan K, Zhou Q. 2017. Reactive Astrocytes Promote ALS-like Degeneration and Intracellular Protein Aggregation in Human Motor Neurons by Disrupting Autophagy through TGF-β1. Stem cell reports. 9(2):667-680. Pubmed: 28712846 DOI:S2213-6711(17)30271-0 Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron disease. Astrocytic factors are known to contribute to motor neuron degeneration and death in ALS. However, the role of astrocyte in promoting motor neuron protein aggregation, a disease hallmark of ALS, remains largely unclear. Here, using culture models of human motor neurons and primary astrocytes of different genotypes (wild-type or SOD1 mutant) and reactive states (non-reactive or reactive), we show that reactive astrocytes, regardless of their genotypes, reduce motor neuron health and lead to moderate neuronal loss. After prolonged co-cultures of up to 2 months, motor neurons show increased axonal and cytoplasmic protein inclusions characteristic of ALS. Reactive astrocytes induce protein aggregation in part by releasing transforming growth factor β1 (TGF-β1), which disrupts motor neuron autophagy through the mTOR pathway. These results reveal the important contribution of reactive astrocytes in promoting aspects of ALS pathology independent of genetic influences.Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved. -
Palomo V, Perez DI, Roca C, Anderson C, Rodríguez-Muela N, Perez C, Morales-Garcia JA, Reyes JA, Campillo NE, Perez-Castillo AM, Rubin LL, Timchenko L, Gil C, Martinez A. 2017. Subtly Modulating Glycogen Synthase Kinase 3 β: Allosteric Inhibitor Development and Their Potential for the Treatment of Chronic Diseases. Journal of medicinal chemistry. 60(12):4983-5001. Pubmed: 28548834 DOI:10.1021/acs.jmedchem.7b00395 Palomo V, Perez DI, Roca C, Anderson C, Rodríguez-Muela N, Perez C, Morales-Garcia JA, Reyes JA, Campillo NE, Perez-Castillo AM, Rubin LL, Timchenko L, Gil C, Martinez A. 2017. Subtly Modulating Glycogen Synthase Kinase 3 β: Allosteric Inhibitor Development and Their Potential for the Treatment of Chronic Diseases. Journal of medicinal chemistry. 60(12):4983-5001. Pubmed: 28548834 DOI:10.1021/acs.jmedchem.7b00395 Glycogen synthase kinase 3 β (GSK-3β) is a central target in several unmet diseases. To increase the specificity of GSK-3β inhibitors in chronic treatments, we developed small molecules allowing subtle modulation of GSK-3β activity. Design synthesis, structure-activity relationships, and binding mode of quinoline-3-carbohydrazide derivatives as allosteric modulators of GSK-3β are presented here. Furthermore, we show how allosteric binders may overcome the β-catenin side effects associated with strong GSK-3β inhibition. The therapeutic potential of some of these modulators has been tested in human samples from patients with congenital myotonic dystrophy type 1 (CDM1) and spinal muscular atrophy (SMA) patients. We found that compound 53 improves delayed myogenesis in CDM1 myoblasts, while compounds 1 and 53 have neuroprotective properties in SMA-derived cells. These findings suggest that the allosteric modulators of GSK-3β may be used for future development of drugs for DM1, SMA, and other chronic diseases where GSK-3β inhibition exhibits therapeutic effects. -
Rodriguez-Muela N, Litterman NK, Norabuena EM, Mull JL, Galazo MJ, Sun C, Ng SY, Makhortova NR, White A, Lynes MM, Chung WK, Davidow LS, Macklis JD, Rubin LL. 2017. Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease. Cell reports. 18(6):1484-1498. Pubmed: 28178525 DOI:S2211-1247(17)30072-4 Rodriguez-Muela N, Litterman NK, Norabuena EM, Mull JL, Galazo MJ, Sun C, Ng SY, Makhortova NR, White A, Lynes MM, Chung WK, Davidow LS, Macklis JD, Rubin LL. 2017. Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease. Cell reports. 18(6):1484-1498. Pubmed: 28178525 DOI:S2211-1247(17)30072-4 The mechanism underlying selective motor neuron (MN) death remains an essential question in the MN disease field. The MN disease spinal muscular atrophy (SMA) is attributable to reduced levels of the ubiquitous protein SMN. Here, we report that SMN levels are widely variable in MNs within a single genetic background and that this heterogeneity is seen not only in SMA MNs but also in MNs derived from controls and amyotrophic lateral sclerosis (ALS) patients. Furthermore, cells with low SMN are more susceptible to cell death. These findings raise the important clinical implication that some SMN-elevating therapeutics might be effective in MN diseases besides SMA. Supporting this, we found that increasing SMN across all MN populations using an Nedd8-activating enzyme inhibitor promotes survival in both SMA and ALS-derived MNs. Altogether, our work demonstrates that examination of human neurons at the single-cell level can reveal alternative strategies to be explored in the treatment of degenerative diseases.Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved. 2016
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Walker RG, Poggioli T, Katsimpardi L, Buchanan SM, Oh J, Wattrus S, Heidecker B, Fong YW, Rubin LL, Ganz P, Thompson TB, Wagers AJ, Lee RT. 2016. Biochemistry and Biology of GDF11 and Myostatin: Similarities, Differences, and Questions for Future Investigation. Circulation research. 118(7):1125-41; discussion 1142. Pubmed: 27034275 DOI:10.1161/CIRCRESAHA.116.308391 Walker RG, Poggioli T, Katsimpardi L, Buchanan SM, Oh J, Wattrus S, Heidecker B, Fong YW, Rubin LL, Ganz P, Thompson TB, Wagers AJ, Lee RT. 2016. Biochemistry and Biology of GDF11 and Myostatin: Similarities, Differences, and Questions for Future Investigation. Circulation research. 118(7):1125-41; discussion 1142. Pubmed: 27034275 DOI:10.1161/CIRCRESAHA.116.308391 Growth differentiation factor 11 (GDF11) and myostatin (or GDF8) are closely related members of the transforming growth factor β superfamily and are often perceived to serve similar or overlapping roles. Yet, despite commonalities in protein sequence, receptor utilization and signaling, accumulating evidence suggests that these 2 ligands can have distinct functions in many situations. GDF11 is essential for mammalian development and has been suggested to regulate aging of multiple tissues, whereas myostatin is a well-described negative regulator of postnatal skeletal and cardiac muscle mass and modulates metabolic processes. In this review, we discuss the biochemical regulation of GDF11 and myostatin and their functions in the heart, skeletal muscle, and brain. We also highlight recent clinical findings with respect to a potential role for GDF11 and/or myostatin in humans with heart disease. Finally, we address key outstanding questions related to GDF11 and myostatin dynamics and signaling during development, growth, and aging.© 2016 American Heart Association, Inc. -
Rigamonti A, Repetti GG, Sun C, Price FD, Reny DC, Rapino F, Weisinger K, Benkler C, Peterson QP, Davidow LS, Hansson EM, Rubin LL. 2016. Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System. Stem cell reports. 6(6):993-1008. Pubmed: 27304920 DOI:S2213-6711(16)30065-0 Rigamonti A, Repetti GG, Sun C, Price FD, Reny DC, Rapino F, Weisinger K, Benkler C, Peterson QP, Davidow LS, Hansson EM, Rubin LL. 2016. Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System. Stem cell reports. 6(6):993-1008. Pubmed: 27304920 DOI:S2213-6711(16)30065-0 Human pluripotent stem cells (hPSCs) offer a renewable source of cells that can be expanded indefinitely and differentiated into virtually any type of cell in the human body, including neurons. This opens up unprecedented possibilities to study neuronal cell and developmental biology and cellular pathology of the nervous system, provides a platform for the screening of chemical libraries that affect these processes, and offers a potential source of transplantable cells for regenerative approaches to neurological disease. However, defining protocols that permit a large number and high yield of neurons has proved difficult. We present differentiation protocols for the generation of distinct subtypes of neurons in a highly reproducible manner, with minimal experiment-to-experiment variation. These neurons form synapses with neighboring cells, exhibit spontaneous electrical activity, and respond appropriately to depolarization. hPSC-derived neurons exhibit a high degree of maturation and survive in culture for up to 4-5 months, even without astrocyte feeder layers.Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved. 2015
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Ng SY, Soh BS, Rodriguez-Muela N, Hendrickson DG, Price F, Rinn JL, Rubin LL. 2015. Genome-wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy. Cell stem cell. 17(5):569-84. Pubmed: 26321202 DOI:S1934-5909(15)00358-6 Ng SY, Soh BS, Rodriguez-Muela N, Hendrickson DG, Price F, Rinn JL, Rubin LL. 2015. Genome-wide RNA-Seq of Human Motor Neurons Implicates Selective ER Stress Activation in Spinal Muscular Atrophy. Cell stem cell. 17(5):569-84. Pubmed: 26321202 DOI:S1934-5909(15)00358-6 Spinal muscular atrophy (SMA) is caused by mutations in the SMN1 gene. Because this gene is expressed ubiquitously, it remains poorly understood why motor neurons (MNs) are one of the most affected cell types. To address this question, we carried out RNA sequencing studies using fixed, antibody-labeled, and purified MNs produced from control and SMA patient-derived induced pluripotent stem cells (iPSCs). We found SMA-specific changes in MNs, including hyper-activation of the ER stress pathway. Functional studies demonstrated that inhibition of ER stress improves MN survival in vitro even in MNs expressing low SMN. In SMA mice, systemic delivery of an ER stress inhibitor that crosses the blood-brain barrier led to the preservation of spinal cord MNs. Therefore, our study implies that selective activation of ER stress underlies MN death in SMA. Moreover, the approach we have taken would be broadly applicable to the study of disease-prone human cells in heterogeneous cultures.Copyright © 2015 Elsevier Inc. All rights reserved. -
Brennand KJ, Marchetto MC, Benvenisty N, Brüstle O, Ebert A, Izpisua Belmonte JC, Kaykas A, Lancaster MA, Livesey FJ, McConnell MJ, McKay RD, Morrow EM, Muotri AR, Panchision DM, Rubin LL, Sawa A, Soldner F, Song H, Studer L, Temple S, Vaccarino FM, Wu J, Vanderhaeghen P, Gage FH, Jaenisch R. 2015. Creating Patient-Specific Neural Cells for the In Vitro Study of Brain Disorders. Stem cell reports. 5(6):933-945. Pubmed: 26610635 DOI:S2213-6711(15)00308-2 Brennand KJ, Marchetto MC, Benvenisty N, Brüstle O, Ebert A, Izpisua Belmonte JC, Kaykas A, Lancaster MA, Livesey FJ, McConnell MJ, McKay RD, Morrow EM, Muotri AR, Panchision DM, Rubin LL, Sawa A, Soldner F, Song H, Studer L, Temple S, Vaccarino FM, Wu J, Vanderhaeghen P, Gage FH, Jaenisch R. 2015. Creating Patient-Specific Neural Cells for the In Vitro Study of Brain Disorders. Stem cell reports. 5(6):933-945. Pubmed: 26610635 DOI:S2213-6711(15)00308-2 As a group, we met to discuss the current challenges for creating meaningful patient-specific in vitro models to study brain disorders. Although the convergence of findings between laboratories and patient cohorts provided us confidence and optimism that hiPSC-based platforms will inform future drug discovery efforts, a number of critical technical challenges remain. This opinion piece outlines our collective views on the current state of hiPSC-based disease modeling and discusses what we see to be the critical objectives that must be addressed collectively as a field.Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved. -
Xue R, Lynes MD, Dreyfuss JM, Shamsi F, Schulz TJ, Zhang H, Huang TL, Townsend KL, Li Y, Takahashi H, Weiner LS, White AP, Lynes MS, Rubin LL, Goodyear LJ, Cypess AM, Tseng YH. 2015. Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes. Nature medicine. 21(7):760-8. Pubmed: 26076036 DOI:10.1038/nm.3881 Xue R, Lynes MD, Dreyfuss JM, Shamsi F, Schulz TJ, Zhang H, Huang TL, Townsend KL, Li Y, Takahashi H, Weiner LS, White AP, Lynes MS, Rubin LL, Goodyear LJ, Cypess AM, Tseng YH. 2015. Clonal analyses and gene profiling identify genetic biomarkers of the thermogenic potential of human brown and white preadipocytes. Nature medicine. 21(7):760-8. Pubmed: 26076036 DOI:10.1038/nm.3881 Targeting brown adipose tissue (BAT) content or activity has therapeutic potential for treating obesity and the metabolic syndrome by increasing energy expenditure. However, both inter- and intra-individual differences contribute to heterogeneity in human BAT and potentially to differential thermogenic capacity in human populations. Here we generated clones of brown and white preadipocytes from human neck fat and characterized their adipogenic and thermogenic differentiation. We combined an uncoupling protein 1 (UCP1) reporter system and expression profiling to define novel sets of gene signatures in human preadipocytes that could predict the thermogenic potential of the cells once they were maturated. Knocking out the positive UCP1 regulators, PREX1 and EDNRB, in brown preadipocytes using CRISPR-Cas9 markedly abolished the high level of UCP1 in brown adipocytes differentiated from the preadipocytes. Finally, we were able to prospectively isolate adipose progenitors with great thermogenic potential using the cell surface marker CD29. These data provide new insights into the cellular heterogeneity in human fat and offer potential biomarkers for identifying thermogenically competent preadipocytes. -
Schlaeger TM, Daheron L, Brickler TR, Entwisle S, Chan K, Cianci A, DeVine A, Ettenger A, Fitzgerald K, Godfrey M, Gupta D, McPherson J, Malwadkar P, Gupta M, Bell B, Doi A, Jung N, Li X, Lynes MS, Brookes E, Cherry AB, Demirbas D, Tsankov AM, Zon LI, Rubin LL, Feinberg AP, Meissner A, Cowan CA, Daley GQ. 2015. A comparison of non-integrating reprogramming methods. Nature biotechnology. 33(1):58-63. Pubmed: 25437882 DOI:10.1038/nbt.3070 Schlaeger TM, Daheron L, Brickler TR, Entwisle S, Chan K, Cianci A, DeVine A, Ettenger A, Fitzgerald K, Godfrey M, Gupta D, McPherson J, Malwadkar P, Gupta M, Bell B, Doi A, Jung N, Li X, Lynes MS, Brookes E, Cherry AB, Demirbas D, Tsankov AM, Zon LI, Rubin LL, Feinberg AP, Meissner A, Cowan CA, Daley GQ. 2015. A comparison of non-integrating reprogramming methods. Nature biotechnology. 33(1):58-63. Pubmed: 25437882 DOI:10.1038/nbt.3070 Human induced pluripotent stem cells (hiPSCs) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV), episomal (Epi) and mRNA transfection mRNA methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation. -
Wainger BJ, Buttermore ED, Oliveira JT, Mellin C, Lee S, Saber WA, Wang AJ, Ichida JK, Chiu IM, Barrett L, Huebner EA, Bilgin C, Tsujimoto N, Brenneis C, Kapur K, Rubin LL, Eggan K, Woolf CJ. 2015. Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts. Nature neuroscience. 18(1):17-24. Pubmed: 25420066 DOI:10.1038/nn.3886 Wainger BJ, Buttermore ED, Oliveira JT, Mellin C, Lee S, Saber WA, Wang AJ, Ichida JK, Chiu IM, Barrett L, Huebner EA, Bilgin C, Tsujimoto N, Brenneis C, Kapur K, Rubin LL, Eggan K, Woolf CJ. 2015. Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts. Nature neuroscience. 18(1):17-24. Pubmed: 25420066 DOI:10.1038/nn.3886 Reprogramming somatic cells from one cell fate to another can generate specific neurons suitable for disease modeling. To maximize the utility of patient-derived neurons, they must model not only disease-relevant cell classes, but also the diversity of neuronal subtypes found in vivo and the pathophysiological changes that underlie specific clinical diseases. We identified five transcription factors that reprogram mouse and human fibroblasts into noxious stimulus-detecting (nociceptor) neurons. These recapitulated the expression of quintessential nociceptor-specific functional receptors and channels found in adult mouse nociceptor neurons, as well as native subtype diversity. Moreover, the derived nociceptor neurons exhibited TrpV1 sensitization to the inflammatory mediator prostaglandin E2 and the chemotherapeutic drug oxaliplatin, modeling the inherent mechanisms underlying inflammatory pain hypersensitivity and painful chemotherapy-induced neuropathy. Using fibroblasts from patients with familial dysautonomia (hereditary sensory and autonomic neuropathy type III), we found that the technique was able to reveal previously unknown aspects of human disease phenotypes in vitro. -
Katsimpardi L, Rubin LL. 2015. Young systemic factors as a medicine for age-related neurodegenerative diseases. Neurogenesis (Austin, Tex.). 2(1):e1004971. Pubmed: 27502604 DOI:10.1080/23262133.2015.1004971 Katsimpardi L, Rubin LL. 2015. Young systemic factors as a medicine for age-related neurodegenerative diseases. Neurogenesis (Austin, Tex.). 2(1):e1004971. Pubmed: 27502604 DOI:10.1080/23262133.2015.1004971 It is widely known that neurogenesis, brain function and cognition decline with aging. Increasing evidence suggests that cerebrovascular dysfunction is a major cause of cognitive impairment in the elderly but is also involved in age-related neurodegenerative diseases. Finding ways and molecules that reverse this process and ameliorate age- and disease-related cognitive impairment by targeting vascular and neurogenic deterioration would be of great therapeutic value. In Katsimpardi et al. we reported that young blood has a dual beneficial effect in the aged brain by restoring age-related decline in neurogenesis as well as inducing a striking remodeling of the aged vasculature and restoring blood flow to youthful levels. Additionally, we identified a youthful systemic factor, GDF11 that recapitulates these beneficial effects of young blood. We believe that the identification of young systemic factors that can rejuvenate the aged brain opens new roads to therapeutic intervention for neurodegenerative diseases by targeting both neural stem cells and neurogenesis as well as at the vasculature. 2014
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Blum B, Roose AN, Barrandon O, Maehr R, Arvanites AC, Davidow LS, Davis JC, Peterson QP, Rubin LL, Melton DA. 2014. Reversal of β cell de-differentiation by a small molecule inhibitor of the TGFβ pathway. eLife. 3:e02809. Pubmed: 25233132 DOI:10.7554/eLife.02809 Blum B, Roose AN, Barrandon O, Maehr R, Arvanites AC, Davidow LS, Davis JC, Peterson QP, Rubin LL, Melton DA. 2014. Reversal of β cell de-differentiation by a small molecule inhibitor of the TGFβ pathway. eLife. 3:e02809. Pubmed: 25233132 DOI:10.7554/eLife.02809 Dysfunction or death of pancreatic β cells underlies both types of diabetes. This functional decline begins with β cell stress and de-differentiation. Current drugs for type 2 diabetes (T2D) lower blood glucose levels but they do not directly alleviate β cell stress nor prevent, let alone reverse, β cell de-differentiation. We show here that Urocortin 3 (Ucn3), a marker for mature β cells, is down-regulated in the early stages of T2D in mice and when β cells are stressed in vitro. Using an insulin expression-coupled lineage tracer, with Ucn3 as a reporter for the mature β cell state, we screen for factors that reverse β cell de-differentiation. We find that a small molecule inhibitor of TGFβ receptor I (Alk5) protects cells from the loss of key β cell transcription factors and restores a mature β cell identity even after exposure to prolonged and severe diabetes. -
Naryshkin NA, Weetall M, Dakka A, Narasimhan J, Zhao X, Feng Z, Ling KK, Karp GM, Qi H, Woll MG, Chen G, Zhang N, Gabbeta V, Vazirani P, Bhattacharyya A, Furia B, Risher N, Sheedy J, Kong R, Ma J, Turpoff A, Lee CS, Zhang X, Moon YC, Trifillis P, Welch EM, Colacino JM, Babiak J, Almstead NG, Peltz SW, Eng LA, Chen KS, Mull JL, Lynes MS, Rubin LL, Fontoura P, Santarelli L, Haehnke D, McCarthy KD, Schmucki R, Ebeling M, Sivaramakrishnan M, Ko CP, Paushkin SV, Ratni H, Gerlach I, Ghosh A, Metzger F. 2014. Motor neuron disease. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy. Science (New York, N.Y.). 345(6197):688-93. Pubmed: 25104390 DOI:10.1126/science.1250127 Naryshkin NA, Weetall M, Dakka A, Narasimhan J, Zhao X, Feng Z, Ling KK, Karp GM, Qi H, Woll MG, Chen G, Zhang N, Gabbeta V, Vazirani P, Bhattacharyya A, Furia B, Risher N, Sheedy J, Kong R, Ma J, Turpoff A, Lee CS, Zhang X, Moon YC, Trifillis P, Welch EM, Colacino JM, Babiak J, Almstead NG, Peltz SW, Eng LA, Chen KS, Mull JL, Lynes MS, Rubin LL, Fontoura P, Santarelli L, Haehnke D, McCarthy KD, Schmucki R, Ebeling M, Sivaramakrishnan M, Ko CP, Paushkin SV, Ratni H, Gerlach I, Ghosh A, Metzger F. 2014. Motor neuron disease. SMN2 splicing modifiers improve motor function and longevity in mice with spinal muscular atrophy. Science (New York, N.Y.). 345(6197):688-93. Pubmed: 25104390 DOI:10.1126/science.1250127 Spinal muscular atrophy (SMA) is a genetic disease caused by mutation or deletion of the survival of motor neuron 1 (SMN1) gene. A paralogous gene in humans, SMN2, produces low, insufficient levels of functional SMN protein due to alternative splicing that truncates the transcript. The decreased levels of SMN protein lead to progressive neuromuscular degeneration and high rates of mortality. Through chemical screening and optimization, we identified orally available small molecules that shift the balance of SMN2 splicing toward the production of full-length SMN2 messenger RNA with high selectivity. Administration of these compounds to Δ7 mice, a model of severe SMA, led to an increase in SMN protein levels, improvement of motor function, and protection of the neuromuscular circuit. These compounds also extended the life span of the mice. Selective SMN2 splicing modifiers may have therapeutic potential for patients with SMA.Copyright © 2014, American Association for the Advancement of Science. -
Ichida JK, Tcw J, Williams LA, Carter AC, Shi Y, Moura MT, Ziller M, Singh S, Amabile G, Bock C, Umezawa A, Rubin LL, Bradner JE, Akutsu H, Meissner A, Eggan K. 2014. Notch inhibition allows oncogene-independent generation of iPS cells. Nature chemical biology. 10(8):632-639. Pubmed: 24952596 DOI:10.1038/nchembio.1552 Ichida JK, Tcw J, Williams LA, Carter AC, Shi Y, Moura MT, Ziller M, Singh S, Amabile G, Bock C, Umezawa A, Rubin LL, Bradner JE, Akutsu H, Meissner A, Eggan K. 2014. Notch inhibition allows oncogene-independent generation of iPS cells. Nature chemical biology. 10(8):632-639. Pubmed: 24952596 DOI:10.1038/nchembio.1552 The reprogramming of somatic cells to pluripotency using defined transcription factors holds great promise for biomedicine. However, human reprogramming remains inefficient and relies either on the use of the potentially dangerous oncogenes KLF4 and CMYC or the genetic inhibition of the tumor suppressor gene p53. We hypothesized that inhibition of signal transduction pathways that promote differentiation of the target somatic cells during development might relieve the requirement for non-core pluripotency factors during induced pluripotent stem cell (iPSC) reprogramming. Here, we show that inhibition of Notch greatly improves the efficiency of iPSC generation from mouse and human keratinocytes by suppressing p21 in a p53-independent manner and thereby enriching for undifferentiated cells capable of long-term self-renewal. Pharmacological inhibition of Notch enabled routine production of human iPSCs without KLF4 and CMYC while leaving p53 activity intact. Thus, restricting the development of somatic cells by altering intercellular communication enables the production of safer human iPSCs. -
Katsimpardi L, Litterman NK, Schein PA, Miller CM, Loffredo FS, Wojtkiewicz GR, Chen JW, Lee RT, Wagers AJ, Rubin LL. 2014. Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science (New York, N.Y.). 344(6184):630-4. Pubmed: 24797482 DOI:10.1126/science.1251141 Katsimpardi L, Litterman NK, Schein PA, Miller CM, Loffredo FS, Wojtkiewicz GR, Chen JW, Lee RT, Wagers AJ, Rubin LL. 2014. Vascular and neurogenic rejuvenation of the aging mouse brain by young systemic factors. Science (New York, N.Y.). 344(6184):630-4. Pubmed: 24797482 DOI:10.1126/science.1251141 In the adult central nervous system, the vasculature of the neurogenic niche regulates neural stem cell behavior by providing circulating and secreted factors. Age-related decline of neurogenesis and cognitive function is associated with reduced blood flow and decreased numbers of neural stem cells. Therefore, restoring the functionality of the niche should counteract some of the negative effects of aging. We show that factors found in young blood induce vascular remodeling, culminating in increased neurogenesis and improved olfactory discrimination in aging mice. Further, we show that GDF11 alone can improve the cerebral vasculature and enhance neurogenesis. The identification of factors that slow the age-dependent deterioration of the neurogenic niche in mice may constitute the basis for new methods of treating age-related neurodegenerative and neurovascular diseases. -
Gould SE, Low JA, Marsters JC, Robarge K, Rubin LL, de Sauvage FJ, Sutherlin DP, Wong H, Yauch RL. 2014. Discovery and preclinical development of vismodegib. Expert opinion on drug discovery. 9(8):969-84. Pubmed: 24857041 DOI:10.1517/17460441.2014.920816 Gould SE, Low JA, Marsters JC, Robarge K, Rubin LL, de Sauvage FJ, Sutherlin DP, Wong H, Yauch RL. 2014. Discovery and preclinical development of vismodegib. Expert opinion on drug discovery. 9(8):969-84. Pubmed: 24857041 DOI:10.1517/17460441.2014.920816 Array -
Woodard CM, Campos BA, Kuo SH, Nirenberg MJ, Nestor MW, Zimmer M, Mosharov EV, Sulzer D, Zhou H, Paull D, Clark L, Schadt EE, Sardi SP, Rubin L, Eggan K, Brock M, Lipnick S, Rao M, Chang S, Li A, Noggle SA. 2014. iPSC-derived dopamine neurons reveal differences between monozygotic twins discordant for Parkinson's disease. Cell reports. 9(4):1173-82. Pubmed: 25456120 DOI:S2211-1247(14)00877-8 Woodard CM, Campos BA, Kuo SH, Nirenberg MJ, Nestor MW, Zimmer M, Mosharov EV, Sulzer D, Zhou H, Paull D, Clark L, Schadt EE, Sardi SP, Rubin L, Eggan K, Brock M, Lipnick S, Rao M, Chang S, Li A, Noggle SA. 2014. iPSC-derived dopamine neurons reveal differences between monozygotic twins discordant for Parkinson's disease. Cell reports. 9(4):1173-82. Pubmed: 25456120 DOI:S2211-1247(14)00877-8 Parkinson's disease (PD) has been attributed to a combination of genetic and nongenetic factors. We studied a set of monozygotic twins harboring the heterozygous glucocerebrosidase mutation (GBA N370S) but clinically discordant for PD. We applied induced pluripotent stem cell (iPSC) technology for PD disease modeling using the twins' fibroblasts to evaluate and dissect the genetic and nongenetic contributions. Utilizing fluorescence-activated cell sorting, we obtained a homogenous population of "footprint-free" iPSC-derived midbrain dopaminergic (mDA) neurons. The mDA neurons from both twins had ∼50% GBA enzymatic activity, ∼3-fold elevated α-synuclein protein levels, and a reduced capacity to synthesize and release dopamine. Interestingly, the affected twin's neurons showed an even lower dopamine level, increased monoamine oxidase B (MAO-B) expression, and impaired intrinsic network activity. Overexpression of wild-type GBA and treatment with MAO-B inhibitors normalized α-synuclein and dopamine levels, suggesting a combination therapy for the affected twin.Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved. -
Cherry JJ, Kobayashi DT, Lynes MM, Naryshkin NN, Tiziano FD, Zaworski PG, Rubin LL, Jarecki J. 2014. Assays for the identification and prioritization of drug candidates for spinal muscular atrophy. Assay and drug development technologies. 12(6):315-41. Pubmed: 25147906 DOI:10.1089/adt.2014.587 Cherry JJ, Kobayashi DT, Lynes MM, Naryshkin NN, Tiziano FD, Zaworski PG, Rubin LL, Jarecki J. 2014. Assays for the identification and prioritization of drug candidates for spinal muscular atrophy. Assay and drug development technologies. 12(6):315-41. Pubmed: 25147906 DOI:10.1089/adt.2014.587 Spinal muscular atrophy (SMA) is an autosomal recessive genetic disorder resulting in degeneration of α-motor neurons of the anterior horn and proximal muscle weakness. It is the leading cause of genetic mortality in children younger than 2 years. It affects ∼1 in 11,000 live births. In 95% of cases, SMA is caused by homozygous deletion of the SMN1 gene. In addition, all patients possess at least one copy of an almost identical gene called SMN2. A single point mutation in exon 7 of the SMN2 gene results in the production of low levels of full-length survival of motor neuron (SMN) protein at amounts insufficient to compensate for the loss of the SMN1 gene. Although no drug treatments are available for SMA, a number of drug discovery and development programs are ongoing, with several currently in clinical trials. This review describes the assays used to identify candidate drugs for SMA that modulate SMN2 gene expression by various means. Specifically, it discusses the use of high-throughput screening to identify candidate molecules from primary screens, as well as the technical aspects of a number of widely used secondary assays to assess SMN messenger ribonucleic acid (mRNA) and protein expression, localization, and function. Finally, it describes the process of iterative drug optimization utilized during preclinical SMA drug development to identify clinical candidates for testing in human clinical trials. 2013
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Yang YM, Gupta SK, Kim KJ, Powers BE, Cerqueira A, Wainger BJ, Ngo HD, Rosowski KA, Schein PA, Ackeifi CA, Arvanites AC, Davidow LS, Woolf CJ, Rubin LL. 2013. A small molecule screen in stem-cell-derived motor neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. Cell stem cell. 12(6):713-26. Pubmed: 23602540 DOI:S1934-5909(13)00139-2 Yang YM, Gupta SK, Kim KJ, Powers BE, Cerqueira A, Wainger BJ, Ngo HD, Rosowski KA, Schein PA, Ackeifi CA, Arvanites AC, Davidow LS, Woolf CJ, Rubin LL. 2013. A small molecule screen in stem-cell-derived motor neurons identifies a kinase inhibitor as a candidate therapeutic for ALS. Cell stem cell. 12(6):713-26. Pubmed: 23602540 DOI:S1934-5909(13)00139-2 Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease, characterized by motor neuron (MN) death, for which there are no truly effective treatments. Here, we describe a new small molecule survival screen carried out using MNs from both wild-type and mutant SOD1 mouse embryonic stem cells. Among the hits we found, kenpaullone had a particularly impressive ability to prolong the healthy survival of both types of MNs that can be attributed to its dual inhibition of GSK-3 and HGK kinases. Furthermore, kenpaullone also strongly improved the survival of human MNs derived from ALS-patient-induced pluripotent stem cells and was more active than either of two compounds, olesoxime and dexpramipexole, that recently failed in ALS clinical trials. Our studies demonstrate the value of a stem cell approach to drug discovery and point to a new paradigm for identification and preclinical testing of future ALS therapeutics.Copyright © 2013 Elsevier Inc. All rights reserved. -
Sen A, Dimlich DN, Guruharsha KG, Kankel MW, Hori K, Yokokura T, Brachat S, Richardson D, Loureiro J, Sivasankaran R, Curtis D, Davidow LS, Rubin LL, Hart AC, Van Vactor D, Artavanis-Tsakonas S. 2013. Genetic circuitry of Survival motor neuron, the gene underlying spinal muscular atrophy. Proceedings of the National Academy of Sciences of the United States of America. 110(26):E2371-80. Pubmed: 23757500 DOI:10.1073/pnas.1301738110 Sen A, Dimlich DN, Guruharsha KG, Kankel MW, Hori K, Yokokura T, Brachat S, Richardson D, Loureiro J, Sivasankaran R, Curtis D, Davidow LS, Rubin LL, Hart AC, Van Vactor D, Artavanis-Tsakonas S. 2013. Genetic circuitry of Survival motor neuron, the gene underlying spinal muscular atrophy. Proceedings of the National Academy of Sciences of the United States of America. 110(26):E2371-80. Pubmed: 23757500 DOI:10.1073/pnas.1301738110 The clinical severity of the neurodegenerative disorder spinal muscular atrophy (SMA) is dependent on the levels of functional Survival Motor Neuron (SMN) protein. Consequently, current strategies for developing treatments for SMA generally focus on augmenting SMN levels. To identify additional potential therapeutic avenues and achieve a greater understanding of SMN, we applied in vivo, in vitro, and in silico approaches to identify genetic and biochemical interactors of the Drosophila SMN homolog. We identified more than 300 candidate genes that alter an Smn-dependent phenotype in vivo. Integrating the results from our genetic screens, large-scale protein interaction studies, and bioinformatic analysis, we define a unique interactome for SMN that provides a knowledge base for a better understanding of SMA. -
Ding Q, Lee YK, Schaefer EA, Peters DT, Veres A, Kim K, Kuperwasser N, Motola DL, Meissner TB, Hendriks WT, Trevisan M, Gupta RM, Moisan A, Banks E, Friesen M, Schinzel RT, Xia F, Tang A, Xia Y, Figueroa E, Wann A, Ahfeldt T, Daheron L, Zhang F, Rubin LL, Peng LF, Chung RT, Musunuru K, Cowan CA. 2013. A TALEN genome-editing system for generating human stem cell-based disease models. Cell stem cell. 12(2):238-51. Pubmed: 23246482 DOI:S1934-5909(12)00645-5 Ding Q, Lee YK, Schaefer EA, Peters DT, Veres A, Kim K, Kuperwasser N, Motola DL, Meissner TB, Hendriks WT, Trevisan M, Gupta RM, Moisan A, Banks E, Friesen M, Schinzel RT, Xia F, Tang A, Xia Y, Figueroa E, Wann A, Ahfeldt T, Daheron L, Zhang F, Rubin LL, Peng LF, Chung RT, Musunuru K, Cowan CA. 2013. A TALEN genome-editing system for generating human stem cell-based disease models. Cell stem cell. 12(2):238-51. Pubmed: 23246482 DOI:S1934-5909(12)00645-5 Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease-dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.Copyright © 2013 Elsevier Inc. All rights reserved. 2012
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Wang Y, Arvanites AC, Davidow L, Blanchard J, Lam K, Yoo JW, Coy S, Rubin LL, McMahon AP. 2012. Selective identification of hedgehog pathway antagonists by direct analysis of smoothened ciliary translocation. ACS chemical biology. 7(6):1040-8. Pubmed: 22554036 DOI:10.1021/cb300028a Wang Y, Arvanites AC, Davidow L, Blanchard J, Lam K, Yoo JW, Coy S, Rubin LL, McMahon AP. 2012. Selective identification of hedgehog pathway antagonists by direct analysis of smoothened ciliary translocation. ACS chemical biology. 7(6):1040-8. Pubmed: 22554036 DOI:10.1021/cb300028a Hedgehog (Hh) signaling promotes tumorigenesis. The accumulation of the membrane protein Smoothened (Smo) within the primary cilium (PC) is a key event in Hh signal transduction, and many pharmacological inhibitors identified to date target Smo's actions. Smo ciliary translocation is inhibited by some pathway antagonists, while others promote ciliary accumulation, an outcome that can lead to a hypersensitive state on renewal of Hh signaling. To identify novel inhibitory compounds acting on the critical mechanistic transition of Smo accumulation, we established a high content screen to directly analyze Smo ciliary translocation. Screening thousands of compounds from annotated libraries of approved drugs and other agents, we identified several new classes of compounds that block Sonic hedgehog-driven Smo localization within the PC. Selective analysis was conducted on two classes of Smo antagonists. One of these, DY131, appears to inhibit Smo signaling through a common binding site shared by previously reported Smo agonists and antagonists. Antagonism by this class of compound is competed by high doses of Smo-binding agonists such as SAG and impaired by a mutation that generates a ligand-independent, oncogenic form of Smo (SmoM2). In contrast, a second antagonist of Smo accumulation within the PC, SMANT, was less sensitive to SAG-mediated competition and inhibited SmoM2 at concentrations similar to those that inhibit wild-type Smo. Our observations identify important differences among Hh antagonists and the potential for development of novel therapeutic approaches against mutant forms of Smo that are resistant to current therapeutic strategies. -
Annes JP, Ryu JH, Lam K, Carolan PJ, Utz K, Hollister-Lock J, Arvanites AC, Rubin LL, Weir G, Melton DA. 2012. Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication. Proceedings of the National Academy of Sciences of the United States of America. 109(10):3915-20. Pubmed: 22345561 DOI:10.1073/pnas.1201149109 Annes JP, Ryu JH, Lam K, Carolan PJ, Utz K, Hollister-Lock J, Arvanites AC, Rubin LL, Weir G, Melton DA. 2012. Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication. Proceedings of the National Academy of Sciences of the United States of America. 109(10):3915-20. Pubmed: 22345561 DOI:10.1073/pnas.1201149109 Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing β cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase β-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary β cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of β cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases β-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes. -
Wang Y, Davidow L, Arvanites AC, Blanchard J, Lam K, Xu K, Oza V, Yoo JW, Ng JM, Curran T, Rubin LL, McMahon AP. 2012. Glucocorticoid compounds modify smoothened localization and hedgehog pathway activity. Chemistry & biology. 19(8):972-82. Pubmed: 22921064 DOI:10.1016/j.chembiol.2012.06.012 Wang Y, Davidow L, Arvanites AC, Blanchard J, Lam K, Xu K, Oza V, Yoo JW, Ng JM, Curran T, Rubin LL, McMahon AP. 2012. Glucocorticoid compounds modify smoothened localization and hedgehog pathway activity. Chemistry & biology. 19(8):972-82. Pubmed: 22921064 DOI:10.1016/j.chembiol.2012.06.012 The Hedgehog signaling pathway is linked to a variety of diseases, notably a range of cancers. The first generation of drug screens identified Smoothened (Smo), a membrane protein essential for signaling, as an attractive drug target. Smo localizes to the primary cilium upon pathway activation, and this transition is critical for the response to Hedgehog ligands. In a high content screen directly monitoring Smo distribution in Hedgehog-responsive cells, we identified different glucocorticoids as specific modulators of Smo ciliary accumulation. One class promoted Smo accumulation, conferring cellular hypersensitivity to Hedgehog stimulation. In contrast, a second class inhibited Smo ciliary localization and signaling activity by both wild-type Smo, and mutant forms of Smo, SmoM2, and SmoD473H, that are refractory to previously identified Smo antagonists. These findings point to the potential for developing glucocorticoid-based pharmacological modulation of Smo signaling to treat mutated drug-resistant forms of Smo, an emerging problem in long-term cancer therapy. They also raise a concern about potential crosstalk of glucocorticoid drugs in the Hedgehog pathway, if therapeutic administration exceeds levels associated with on-target transcriptional mechanisms of glucocorticoid action.Copyright © 2012 Elsevier Ltd. All rights reserved. -
Hayhurst M, Wagner AK, Cerletti M, Wagers AJ, Rubin LL. 2012. A cell-autonomous defect in skeletal muscle satellite cells expressing low levels of survival of motor neuron protein. Developmental biology. 368(2):323-34. Pubmed: 22705478 DOI:10.1016/j.ydbio.2012.05.037 Hayhurst M, Wagner AK, Cerletti M, Wagers AJ, Rubin LL. 2012. A cell-autonomous defect in skeletal muscle satellite cells expressing low levels of survival of motor neuron protein. Developmental biology. 368(2):323-34. Pubmed: 22705478 DOI:10.1016/j.ydbio.2012.05.037 Mutations in the Survival of Motor Neuron (SMN) gene underlie the development of spinal muscular atrophy (SMA), which currently represents the leading genetic cause of mortality in infants and toddlers. SMA is characterized by degeneration of spinal cord motor neurons and muscle atrophy. Although SMA is often considered to be a motor neuron disease, accumulating evidence suggests that muscle cells themselves may be affected by low levels of SMN. Here, we examine satellite cells, tissue-resident stem cells that play an essential role in the growth and repair of skeletal muscle, isolated from a severe SMA mouse model (Smn(-/-); SMN2(+/+)). We found similar numbers of satellite cells in the muscles of SMA and wild-type (Smn(+/+); SMN2(+/+)) mice at postnatal day 2 (P2), and, when isolated from skeletal muscle using cell surface marker expression, these cells showed comparable survival and proliferative potential. However, SMA satellite cells differentiate abnormally, revealed by the premature expression of muscle differentiation markers, and, especially, by a reduced efficiency in forming myotubes. These phenotypes suggest a critical role of SMN protein in the intrinsic regulation of muscle differentiation and suggest that abnormal muscle development contributes to the manifestation of SMA symptoms.Copyright © 2012 Elsevier Inc. All rights reserved. -
Chen PC, Gaisina IN, El-Khodor BF, Ramboz S, Makhortova NR, Rubin LL, Kozikowski AP. 2012. Identification of a Maleimide-Based Glycogen Synthase Kinase-3 (GSK-3) Inhibitor, BIP-135, that Prolongs the Median Survival Time of Δ7 SMA KO Mouse Model of Spinal Muscular Atrophy. ACS chemical neuroscience. 3(1):5-11. Pubmed: 22348181 Chen PC, Gaisina IN, El-Khodor BF, Ramboz S, Makhortova NR, Rubin LL, Kozikowski AP. 2012. Identification of a Maleimide-Based Glycogen Synthase Kinase-3 (GSK-3) Inhibitor, BIP-135, that Prolongs the Median Survival Time of Δ7 SMA KO Mouse Model of Spinal Muscular Atrophy. ACS chemical neuroscience. 3(1):5-11. Pubmed: 22348181 The discovery of upregulated glycogen synthase kinase-3 (GSK-3) in various pathological conditions has led to the development of a host of chemically diverse small molecule GSK-3 inhibitors, such as BIP-135. GSK-3 inhibition emerged as an alternative therapeutic target for treating spinal muscular atrophy (SMA) when a number of GSK-3 inhibitors were shown to elevate survival motor neuron (SMN) levels in vitro and to rescue motor neurons when their intrinsic SMN level was diminished by SMN-specific short hairpin RNA (shRNA). Despite their cellular potency, the in vivo efficacy of GSK-3 inhibitors has yet to be evaluated in an animal model of SMA. Herein, we disclose that a potent and reasonably selective GSK-3 inhibitor, namely BIP-135, was tested in a transgenic Δ7 SMA KO mouse model of SMA, and found to prolong the median survival of these animals. In addition, this compound was shown to elevate the SMN protein level in SMA patient-derived fibroblast cells as determined by western blot, and was neuroprotective in a cell-based, SMA-related model of oxidative stress-induced neurodegeneration. 2011
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Rubin LL, Haston KM. 2011. Stem cell biology and drug discovery. BMC biology. 9:42. Pubmed: 21649940 DOI:10.1186/1741-7007-9-42 Rubin LL, Haston KM. 2011. Stem cell biology and drug discovery. BMC biology. 9:42. Pubmed: 21649940 DOI:10.1186/1741-7007-9-42 There are many reasons to be interested in stem cells, one of the most prominent being their potential use in finding better drugs to treat human disease. This article focuses on how this may be implemented. Recent advances in the production of reprogrammed adult cells and their regulated differentiation to disease-relevant cells are presented, and diseases that have been modeled using these methods are discussed. Remaining difficulties are highlighted, as are new therapeutic insights that have emerged. -
Tang T, Tang JY, Li D, Reich M, Callahan CA, Fu L, Yauch RL, Wang F, Kotkow K, Chang KS, Shpall E, Wu A, Rubin LL, Marsters JC, Epstein EH, Caro I, de Sauvage FJ. 2011. Targeting superficial or nodular Basal cell carcinoma with topically formulated small molecule inhibitor of smoothened. Clinical cancer research : an official journal of the American Association for Cancer Research. 17(10):3378-87. Pubmed: 21558397 DOI:10.1158/1078-0432.CCR-10-3370 Tang T, Tang JY, Li D, Reich M, Callahan CA, Fu L, Yauch RL, Wang F, Kotkow K, Chang KS, Shpall E, Wu A, Rubin LL, Marsters JC, Epstein EH, Caro I, de Sauvage FJ. 2011. Targeting superficial or nodular Basal cell carcinoma with topically formulated small molecule inhibitor of smoothened. Clinical cancer research : an official journal of the American Association for Cancer Research. 17(10):3378-87. Pubmed: 21558397 DOI:10.1158/1078-0432.CCR-10-3370 Array©2011 AACR. -
Makhortova NR, Hayhurst M, Cerqueira A, Sinor-Anderson AD, Zhao WN, Heiser PW, Arvanites AC, Davidow LS, Waldon ZO, Steen JA, Lam K, Ngo HD, Rubin LL. 2011. A screen for regulators of survival of motor neuron protein levels. Nature chemical biology. 7(8):544-52. Pubmed: 21685895 DOI:10.1038/nchembio.595 Makhortova NR, Hayhurst M, Cerqueira A, Sinor-Anderson AD, Zhao WN, Heiser PW, Arvanites AC, Davidow LS, Waldon ZO, Steen JA, Lam K, Ngo HD, Rubin LL. 2011. A screen for regulators of survival of motor neuron protein levels. Nature chemical biology. 7(8):544-52. Pubmed: 21685895 DOI:10.1038/nchembio.595 The motor neuron disease spinal muscular atrophy (SMA) results from mutations that lead to low levels of the ubiquitously expressed protein survival of motor neuron (SMN). An ever-increasing collection of data suggests that therapeutics that elevate SMN may be effective in treating SMA. We executed an image-based screen of annotated chemical libraries and discovered several classes of compounds that were able to increase cellular SMN. Among the most important was the RTK-PI3K-AKT-GSK-3 signaling cascade. Chemical inhibitors of glycogen synthase kinase 3 (GSK-3) and short hairpin RNAs (shRNAs) directed against this target elevated SMN levels primarily by stabilizing the protein. It was particularly notable that GSK-3 chemical inhibitors were also effective in motor neurons, not only in elevating SMN levels, but also in blocking the death that was produced when SMN was acutely reduced by an SMN-specific shRNA. Thus, we have established a screen capable of detecting drug-like compounds that correct the main phenotypic change underlying SMA. 2010
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Haramati S, Chapnik E, Sztainberg Y, Eilam R, Zwang R, Gershoni N, McGlinn E, Heiser PW, Wills AM, Wirguin I, Rubin LL, Misawa H, Tabin CJ, Brown R, Chen A, Hornstein E. 2010. miRNA malfunction causes spinal motor neuron disease. Proceedings of the National Academy of Sciences of the United States of America. 107(29):13111-6. Pubmed: 20616011 DOI:10.1073/pnas.1006151107 Haramati S, Chapnik E, Sztainberg Y, Eilam R, Zwang R, Gershoni N, McGlinn E, Heiser PW, Wills AM, Wirguin I, Rubin LL, Misawa H, Tabin CJ, Brown R, Chen A, Hornstein E. 2010. miRNA malfunction causes spinal motor neuron disease. Proceedings of the National Academy of Sciences of the United States of America. 107(29):13111-6. Pubmed: 20616011 DOI:10.1073/pnas.1006151107 Defective RNA metabolism is an emerging mechanism involved in ALS pathogenesis and possibly in other neurodegenerative disorders. Here, we show that microRNA (miRNA) activity is essential for long-term survival of postmitotic spinal motor neurons (SMNs) in vivo. Thus, mice that do not process miRNA in SMNs exhibit hallmarks of spinal muscular atrophy (SMA), including sclerosis of the spinal cord ventral horns, aberrant end plate architecture, and myofiber atrophy with signs of denervation. Furthermore, a neurofilament heavy subunit previously implicated in motor neuron degeneration is specifically up-regulated in miRNA-deficient SMNs. We demonstrate that the heavy neurofilament subunit is a target of miR-9, a miRNA that is specifically down-regulated in a genetic model of SMA. These data provide evidence for miRNA function in SMN diseases and emphasize the potential role of miR-9-based regulatory mechanisms in adult neurons and neurodegenerative states. -
Rihel J, Prober DA, Arvanites A, Lam K, Zimmerman S, Jang S, Haggarty SJ, Kokel D, Rubin LL, Peterson RT, Schier AF. 2010. Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation. Science (New York, N.Y.). 327(5963):348-51. Pubmed: 20075256 DOI:10.1126/science.1183090 Rihel J, Prober DA, Arvanites A, Lam K, Zimmerman S, Jang S, Haggarty SJ, Kokel D, Rubin LL, Peterson RT, Schier AF. 2010. Zebrafish behavioral profiling links drugs to biological targets and rest/wake regulation. Science (New York, N.Y.). 327(5963):348-51. Pubmed: 20075256 DOI:10.1126/science.1183090 A major obstacle for the discovery of psychoactive drugs is the inability to predict how small molecules will alter complex behaviors. We report the development and application of a high-throughput, quantitative screen for drugs that alter the behavior of larval zebrafish. We found that the multidimensional nature of observed phenotypes enabled the hierarchical clustering of molecules according to shared behaviors. Behavioral profiling revealed conserved functions of psychotropic molecules and predicted the mechanisms of action of poorly characterized compounds. In addition, behavioral profiling implicated new factors such as ether-a-go-go-related gene (ERG) potassium channels and immunomodulators in the control of rest and locomotor activity. These results demonstrate the power of high-throughput behavioral profiling in zebrafish to discover and characterize psychotropic drugs and to dissect the pharmacology of complex behaviors. 2009
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Brunton SA, Stibbard JH, Rubin LL, Guicherit OM, Kruse LI, Price S, di Lucrezia R, MacKinnon CH, Avery A, Park Y, Buxton D, Boyd EA. 2009. Potent agonists of the Hedgehog signaling pathway. Bioorganic & medicinal chemistry letters. 19(15):4308-11. Pubmed: 19500978 DOI:10.1016/j.bmcl.2009.05.096 Brunton SA, Stibbard JH, Rubin LL, Guicherit OM, Kruse LI, Price S, di Lucrezia R, MacKinnon CH, Avery A, Park Y, Buxton D, Boyd EA. 2009. Potent agonists of the Hedgehog signaling pathway. Bioorganic & medicinal chemistry letters. 19(15):4308-11. Pubmed: 19500978 DOI:10.1016/j.bmcl.2009.05.096 A family of biaryl substituted 1,4-diaminocyclohexanamides of 3-chlorobenzothiophene-2-carboxylic acid is reported as picomolar modulators of Hedgehog protein function. SAR for the 1,4-diaminocyclohexane group is shown to be exquisitely sensitive to substitution on the 4-amino group, and SAR for the 3-chlorobenzothiophene group is highly specific. Preliminary SAR studies of the biaryl substituent led to a picomolar compound with in vivo activity. -
Robarge KD, Brunton SA, Castanedo GM, Cui Y, Dina MS, Goldsmith R, Gould SE, Guichert O, Gunzner JL, Halladay J, Jia W, Khojasteh C, Koehler MF, Kotkow K, La H, Lalonde RL, Lau K, Lee L, Marshall D, Marsters JC, Murray LJ, Qian C, Rubin LL, Salphati L, Stanley MS, Stibbard JH, Sutherlin DP, Ubhayaker S, Wang S, Wong S, Xie M. 2009. GDC-0449-a potent inhibitor of the hedgehog pathway. Bioorganic & medicinal chemistry letters. 19(19):5576-81. Pubmed: 19716296 DOI:10.1016/j.bmcl.2009.08.049 Robarge KD, Brunton SA, Castanedo GM, Cui Y, Dina MS, Goldsmith R, Gould SE, Guichert O, Gunzner JL, Halladay J, Jia W, Khojasteh C, Koehler MF, Kotkow K, La H, Lalonde RL, Lau K, Lee L, Marshall D, Marsters JC, Murray LJ, Qian C, Rubin LL, Salphati L, Stanley MS, Stibbard JH, Sutherlin DP, Ubhayaker S, Wang S, Wong S, Xie M. 2009. GDC-0449-a potent inhibitor of the hedgehog pathway. Bioorganic & medicinal chemistry letters. 19(19):5576-81. Pubmed: 19716296 DOI:10.1016/j.bmcl.2009.08.049 SAR for a wide variety of heterocyclic replacements for a benzimidazole led to the discovery of functionalized 2-pyridyl amides as novel inhibitors of the hedgehog pathway. The 2-pyridyl amides were optimized for potency, PK, and drug-like properties by modifications to the amide portion of the molecule resulting in 31 (GDC-0449). Amide 31 produced complete tumor regression at doses as low as 12.5mg/kg BID in a medulloblastoma allograft mouse model that is wholly dependent on the Hh pathway for growth and is currently in human clinical trials, where it is initially being evaluated for the treatment of BCC. -
Ichida JK, Blanchard J, Lam K, Son EY, Chung JE, Egli D, Loh KM, Carter AC, Di Giorgio FP, Koszka K, Huangfu D, Akutsu H, Liu DR, Rubin LL, Eggan K. 2009. A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog. Cell stem cell. 5(5):491-503. Pubmed: 19818703 DOI:10.1016/j.stem.2009.09.012 Ichida JK, Blanchard J, Lam K, Son EY, Chung JE, Egli D, Loh KM, Carter AC, Di Giorgio FP, Koszka K, Huangfu D, Akutsu H, Liu DR, Rubin LL, Eggan K. 2009. A small-molecule inhibitor of tgf-Beta signaling replaces sox2 in reprogramming by inducing nanog. Cell stem cell. 5(5):491-503. Pubmed: 19818703 DOI:10.1016/j.stem.2009.09.012 The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog. -
Chen S, Borowiak M, Fox JL, Maehr R, Osafune K, Davidow L, Lam K, Peng LF, Schreiber SL, Rubin LL, Melton D. 2009. A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nature chemical biology. 5(4):258-65. Pubmed: 19287398 DOI:10.1038/nchembio.154 Chen S, Borowiak M, Fox JL, Maehr R, Osafune K, Davidow L, Lam K, Peng LF, Schreiber SL, Rubin LL, Melton D. 2009. A small molecule that directs differentiation of human ESCs into the pancreatic lineage. Nature chemical biology. 5(4):258-65. Pubmed: 19287398 DOI:10.1038/nchembio.154 Stepwise differentiation from embryonic stem cells (ESCs) to functional insulin-secreting beta cells will identify key steps in beta-cell development and may yet prove useful for transplantation therapy for diabetics. An essential step in this schema is the generation of pancreatic progenitors--cells that express Pdx1 and produce all the cell types of the pancreas. High-content chemical screening identified a small molecule, (-)-indolactam V, that induces differentiation of a substantial number of Pdx1-expressing cells from human ESCs. The Pdx1-expressing cells express other pancreatic markers and contribute to endocrine, exocrine and duct cells, in vitro and in vivo. Further analyses showed that (-)-indolactam V works specifically at one stage of pancreatic development, inducing pancreatic progenitors from definitive endoderm. This study describes a chemical screening platform to investigate human ESC differentiation and demonstrates the generation of a cell population that is a key milepost on the path to making beta cells. 2008
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Brunton SA, Stibbard JH, Rubin LL, Kruse LI, Guicherit OM, Boyd EA, Price S. 2008. Potent inhibitors of the hedgehog signaling pathway. Journal of medicinal chemistry. 51(5):1108-10. Pubmed: 18275133 DOI:10.1021/jm070694n Brunton SA, Stibbard JH, Rubin LL, Kruse LI, Guicherit OM, Boyd EA, Price S. 2008. Potent inhibitors of the hedgehog signaling pathway. Journal of medicinal chemistry. 51(5):1108-10. Pubmed: 18275133 DOI:10.1021/jm070694n A small family of phenyl quinazolinone ureas is reported as potent modulators of Hedgehog protein function. Preliminary SAR studies of the urea substituent led to a nanomolar Hedgehog antagonist. -
Yauch RL, Gould SE, Scales SJ, Tang T, Tian H, Ahn CP, Marshall D, Fu L, Januario T, Kallop D, Nannini-Pepe M, Kotkow K, Marsters JC, Rubin LL, de Sauvage FJ. 2008. A paracrine requirement for hedgehog signalling in cancer. Nature. 455(7211):406-10. Pubmed: 18754008 DOI:10.1038/nature07275 Yauch RL, Gould SE, Scales SJ, Tang T, Tian H, Ahn CP, Marshall D, Fu L, Januario T, Kallop D, Nannini-Pepe M, Kotkow K, Marsters JC, Rubin LL, de Sauvage FJ. 2008. A paracrine requirement for hedgehog signalling in cancer. Nature. 455(7211):406-10. Pubmed: 18754008 DOI:10.1038/nature07275 Ligand-dependent activation of the hedgehog (Hh) signalling pathway has been associated with tumorigenesis in a number of human tissues. Here we show that, although previous reports have described a cell-autonomous role for Hh signalling in these tumours, Hh ligands fail to activate signalling in tumour epithelial cells. In contrast, our data support ligand-dependent activation of the Hh pathway in the stromal microenvironment. Specific inhibition of Hh signalling using small molecule inhibitors, a neutralizing anti-Hh antibody or genetic deletion of smoothened (Smo) in the mouse stroma results in growth inhibition in xenograft tumour models. Taken together, these studies demonstrate a paracrine requirement for Hh ligand signalling in the tumorigenesis of Hh-expressing cancers and have important implications for the development of Hh pathway antagonists in cancer. -
Rubin LL. 2008. Stem cells and drug discovery: the beginning of a new era?. Cell. 132(4):549-52. Pubmed: 18295572 DOI:10.1016/j.cell.2008.02.010 Rubin LL. 2008. Stem cells and drug discovery: the beginning of a new era?. Cell. 132(4):549-52. Pubmed: 18295572 DOI:10.1016/j.cell.2008.02.010 Much of the attention focused on stem cells relates to their use in cell replacement therapy; however, stem cells may also transform the way in which therapeutics are discovered and validated. 2006
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Dellovade T, Romer JT, Curran T, Rubin LL. 2006. The hedgehog pathway and neurological disorders. Annual review of neuroscience. 29:539-63. Pubmed: 16776596 Dellovade T, Romer JT, Curran T, Rubin LL. 2006. The hedgehog pathway and neurological disorders. Annual review of neuroscience. 29:539-63. Pubmed: 16776596 The hedgehog pathway is a major regulator of embryonic development, and mutations that decrease its activity are known to be associated with severe defects in nervous system development. Recent evidence suggests hedgehog continues to function in adult tissue, normal as well as diseased, by regulating both cell proliferation and the production of growth and angiogenic factors. In the adult nervous system, this dual ability is especially important in regulating the behavior of neural stem and progenitor cells. This review summarizes information connecting hedgehog signaling and neural diseases, including neurodegenerative disorders and brain tumors, particularly medulloblastoma. We also describe the discovery and utility of small molecule agonists and antagonists of this pathway and their potential as novel types of therapeutics. -
Soundararajan P, Miles GB, Rubin LL, Brownstone RM, Rafuse VF. 2006. Motoneurons derived from embryonic stem cells express transcription factors and develop phenotypes characteristic of medial motor column neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 26(12):3256-68. Pubmed: 16554476 Soundararajan P, Miles GB, Rubin LL, Brownstone RM, Rafuse VF. 2006. Motoneurons derived from embryonic stem cells express transcription factors and develop phenotypes characteristic of medial motor column neurons. The Journal of neuroscience : the official journal of the Society for Neuroscience. 26(12):3256-68. Pubmed: 16554476 Embryonic stem (ES) cells differentiate into functional motoneurons when treated with a sonic hedgehog (Shh) agonist and retinoic acid (RA). Whether ES cells can be directed to differentiate into specific subtypes of motoneurons is unknown. We treated embryoid bodies generated from HBG3 ES cells with a Shh agonist and RA for 5 d in culture to induce motoneuron differentiation. Enhanced green fluorescent protein (eGFP) expression was used to identify putative motoneurons, because eGFP is expressed under the control of the Hb9 promoter in HBG3 cells. We found that 96 +/- 0.7% of the differentiated eGFP+ motoneurons expressed Lhx3, a homeobox gene expressed by postmitotic motoneurons in the medial motor column (MMCm), when the treated cells were plated on a neurite-promoting substrate for 5 d. When the treated embryoid bodies were transplanted into stage 17 chick neural tubes, the eGFP+ motoneurons migrated to the MMCm, expressed Lhx3, projected axons to the appropriate target for MMCm motoneurons (i.e., epaxial muscles), and contained synaptic vesicles within intramuscular axonal branches. In ovo and in vitro studies indicated that chemotropic factors emanating from the epaxial muscle and/or surrounding mesenchyme likely guide Lhx3+ motoneurons to their correct target. Finally, whole-cell patch-clamp recordings of transplanted ES cell-derived motoneurons demonstrated that they received synaptic input, elicited repetitive trains of action potentials, and developed passive membrane properties that were similar to host MMCm motoneurons. These results indicate that ES cells can be directed to form subtypes of neurons with specific phenotypic properties. -
Deshpande DM, Kim YS, Martinez T, Carmen J, Dike S, Shats I, Rubin LL, Drummond J, Krishnan C, Hoke A, Maragakis N, Shefner J, Rothstein JD, Kerr DA. 2006. Recovery from paralysis in adult rats using embryonic stem cells. Annals of neurology. 60(1):32-44. Pubmed: 16802299 Deshpande DM, Kim YS, Martinez T, Carmen J, Dike S, Shats I, Rubin LL, Drummond J, Krishnan C, Hoke A, Maragakis N, Shefner J, Rothstein JD, Kerr DA. 2006. Recovery from paralysis in adult rats using embryonic stem cells. Annals of neurology. 60(1):32-44. Pubmed: 16802299 Array -
Rubin LL, de Sauvage FJ. 2006. Targeting the Hedgehog pathway in cancer. Nature reviews. Drug discovery. 5(12):1026-33. Pubmed: 17139287 Rubin LL, de Sauvage FJ. 2006. Targeting the Hedgehog pathway in cancer. Nature reviews. Drug discovery. 5(12):1026-33. Pubmed: 17139287 Several key signalling pathways, such as Hedgehog, Notch, Wnt and BMP-TGFbeta-Activin (bone morphogenetic protein-transforming growth factor-beta-Activin), are involved in most processes essential to the proper development of an embryo. It is also becoming increasingly clear that these pathways can have a crucial role in tumorigenesis when reactivated in adult tissues through sporadic mutations or other mechanisms. We will focus here on the Hedgehog pathway, which is abnormally activated in most basal cell carcinomas, and discuss potential therapeutic opportunities offered by the progress made in understanding this signalling pathway. 2005
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Schnapp E, Kragl M, Rubin L, Tanaka EM. 2005. Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration. Development (Cambridge, England). 132(14):3243-53. Pubmed: 15983402 Schnapp E, Kragl M, Rubin L, Tanaka EM. 2005. Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration. Development (Cambridge, England). 132(14):3243-53. Pubmed: 15983402 Tail regeneration in urodeles requires the coordinated growth and patterning of the regenerating tissues types, including the spinal cord, cartilage and muscle. The dorsoventral (DV) orientation of the spinal cord at the amputation plane determines the DV patterning of the regenerating spinal cord as well as the patterning of surrounding tissues such as cartilage. We investigated this phenomenon on a molecular level. Both the mature and regenerating axolotl spinal cord express molecular markers of DV progenitor cell domains found during embryonic neural tube development, including Pax6, Pax7 and Msx1. Furthermore, the expression of Sonic hedgehog (Shh) is localized to the ventral floor plate domain in both mature and regenerating spinal cord. Patched1 receptor expression indicated that hedgehog signaling occurs not only within the spinal cord but is also transmitted to the surrounding blastema. Cyclopamine treatment revealed that hedgehog signaling is not only required for DV patterning of the regenerating spinal cord but also had profound effects on the regeneration of surrounding, mesodermal tissues. Proliferation of tail blastema cells was severely impaired, resulting in an overall cessation of tail regeneration, and blastema cells no longer expressed the early cartilage marker Sox9. Spinal cord removal experiments revealed that hedgehog signaling, while required for blastema growth is not sufficient for tail regeneration in the absence of the spinal cord. By contrast to the cyclopamine effect on tail regeneration, cyclopamine-treated regenerating limbs achieve a normal length and contain cartilage. This study represents the first molecular localization of DV patterning information in mature tissue that controls regeneration. Interestingly, although tail regeneration does not occur through the formation of somites, the Shh-dependent pathways that control embryonic somite patterning and proliferation may be utilized within the blastema, albeit with a different topography to mediate growth and patterning of tail tissues during regeneration. -
Paladini RD, Saleh J, Qian C, Xu GX, Rubin LL. 2005. Modulation of hair growth with small molecule agonists of the hedgehog signaling pathway. The Journal of investigative dermatology. 125(4):638-46. Pubmed: 16185261 Paladini RD, Saleh J, Qian C, Xu GX, Rubin LL. 2005. Modulation of hair growth with small molecule agonists of the hedgehog signaling pathway. The Journal of investigative dermatology. 125(4):638-46. Pubmed: 16185261 The hedgehog (Hh) family of intercellular signaling proteins is intricately linked to the development and patterning of almost every major vertebrate organ system. In the skin, sonic hedgehog (Shh) is required for hair follicle morphogenesis during embryogenesis and for regulating follicular growth and cycling in the adult. We recently described the identification and characterization of synthetic, non-peptidyl small molecule agonists of the Hh pathway. In this study, we examined the ability of a topically applied Hh-agonist to modulate follicular cycling in adult mouse skin. We report that the Hh-agonist can stimulate the transition from the resting (telogen) to the growth (anagen) stage of the hair cycle in adult mouse skin. Hh-agonist-induced hair growth caused no detectable differences in epidermal proliferation, differentiation, or in the endogenous Hh-signaling pathway as measured by Gli1, Shh, Ptc1, and Gli2 gene expression when compared with a normal hair cycle. In addition, we demonstrate that Hh-agonist is active in human scalp in vitro as measured by Gli1 gene expression. These results suggest that the topical application of Hh-agonist could be effective in treating conditions of decreased proliferation and aberrant follicular cycling in the scalp including androgenetic alopecia (pattern hair loss). 2004
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Dvash T, Mayshar Y, Darr H, McElhaney M, Barker D, Yanuka O, Kotkow KJ, Rubin LL, Benvenisty N, Eiges R. 2004. Temporal gene expression during differentiation of human embryonic stem cells and embryoid bodies. Human reproduction (Oxford, England). 19(12):2875-83. Pubmed: 15375076 Dvash T, Mayshar Y, Darr H, McElhaney M, Barker D, Yanuka O, Kotkow KJ, Rubin LL, Benvenisty N, Eiges R. 2004. Temporal gene expression during differentiation of human embryonic stem cells and embryoid bodies. Human reproduction (Oxford, England). 19(12):2875-83. Pubmed: 15375076 Array -
Romer JT, Kimura H, Magdaleno S, Sasai K, Fuller C, Baines H, Connelly M, Stewart CF, Gould S, Rubin LL, Curran T. 2004. Suppression of the Shh pathway using a small molecule inhibitor eliminates medulloblastoma in Ptc1(+/-)p53(-/-) mice. Cancer cell. 6(3):229-40. Pubmed: 15380514 Romer JT, Kimura H, Magdaleno S, Sasai K, Fuller C, Baines H, Connelly M, Stewart CF, Gould S, Rubin LL, Curran T. 2004. Suppression of the Shh pathway using a small molecule inhibitor eliminates medulloblastoma in Ptc1(+/-)p53(-/-) mice. Cancer cell. 6(3):229-40. Pubmed: 15380514 Medulloblastoma is the most common malignant pediatric brain tumor. Current treatment is associated with major long-term side effects; therefore, new nontoxic therapies, targeting specific molecular defects in this cancer, need to be developed. We use a mouse model of medulloblastoma to show that inhibition of the Sonic Hedgehog (Shh) pathway provides a novel therapy for medulloblastoma. A small molecule inhibitor of the Shh pathway, HhAntag, blocked the function of Smoothened in mice with medulloblastoma. This resulted in suppression of several genes highly expressed in medulloblastoma, inhibition of cell proliferation, increase in cell death and, at the highest dose, complete eradication of tumors. Long-term treatment with HhAntag prolonged medulloblastoma-free survival. These findings support the development of Shh antagonists for the treatment of medulloblastoma. -
Lin LF, Rubin LL, Xu M. 2004. A non-peptidyl neurotrophic small molecule for midbrain dopaminergic neurons. Journal of neurochemistry. 89(6):1387-95. Pubmed: 15189341 Lin LF, Rubin LL, Xu M. 2004. A non-peptidyl neurotrophic small molecule for midbrain dopaminergic neurons. Journal of neurochemistry. 89(6):1387-95. Pubmed: 15189341 Abstract A small organic molecule (CUR-162590) that selectively enhances survival of midbrain dopaminergic neurons was identified by screening small molecule compound libraries. In embryonic midbrain cultures, CUR-162590 increased dopamine uptake and the number of dopaminergic neurons without altering the number of total neurons or astroglia or the uptake of GABA or serotonin. CUR-162590 reduced apoptosis of cultured dopaminergic neurons and protected against death induced by toxins such as MPP(+). Several synthetic analogs of CUR-162590 also had similar bioactivities. CUR-162590 thus represents a new class of neurotrophic small molecules that may have utility in the treatment of Parkinson's disease, which is marked by degeneration of midbrain dopaminergic neurons. -
Kessaris N, Jamen F, Rubin LL, Richardson WD. 2004. Cooperation between sonic hedgehog and fibroblast growth factor/MAPK signalling pathways in neocortical precursors. Development (Cambridge, England). 131(6):1289-98. Pubmed: 14960493 Kessaris N, Jamen F, Rubin LL, Richardson WD. 2004. Cooperation between sonic hedgehog and fibroblast growth factor/MAPK signalling pathways in neocortical precursors. Development (Cambridge, England). 131(6):1289-98. Pubmed: 14960493 Sonic hedgehog (SHH) and fibroblast growth factor 2 (FGF2) can both induce neocortical precursors to express the transcription factor OLIG2 and generate oligodendrocyte progenitors (OLPs) in culture. The activity of FGF2 is unaffected by cyclopamine, which blocks Hedgehog signalling, demonstrating that the FGF pathway to OLP production is Hedgehog independent. Unexpectedly, SHH-mediated OLP induction is blocked by PD173074, a selective inhibitor of FGF receptor (FGFR) tyrosine kinase. SHH activity also depends on mitogen-activated protein kinase (MAPK) but SHH does not itself activate MAPK. Instead, constitutive activity of FGFR maintains a basal level of phosphorylated MAPK that is absolutely required for the OLIG2- and OLP-inducing activities of SHH. Stimulating the MAPK pathway with a retrovirus encoding constitutively active RAS shows that the requirement for MAPK is cell-autonomous, i.e. MAPK is needed together with SHH signalling in the cells that become OLPs. 2003
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Calcutt NA, Allendoerfer KL, Mizisin AP, Middlemas A, Freshwater JD, Burgers M, Ranciato R, Delcroix JD, Taylor FR, Shapiro R, Strauch K, Dudek H, Engber TM, Galdes A, Rubin LL, Tomlinson DR. 2003. Therapeutic efficacy of sonic hedgehog protein in experimental diabetic neuropathy. The Journal of clinical investigation. 111(4):507-14. Pubmed: 12588889 Calcutt NA, Allendoerfer KL, Mizisin AP, Middlemas A, Freshwater JD, Burgers M, Ranciato R, Delcroix JD, Taylor FR, Shapiro R, Strauch K, Dudek H, Engber TM, Galdes A, Rubin LL, Tomlinson DR. 2003. Therapeutic efficacy of sonic hedgehog protein in experimental diabetic neuropathy. The Journal of clinical investigation. 111(4):507-14. Pubmed: 12588889 Hedgehog proteins modulate development and patterning of the embryonic nervous system. As expression of desert hedgehog and the hedgehog receptor, patched-1, persist in the postnatal and adult peripheral nerves, the hedgehog pathway may have a role in maturation and maintenance of the peripheral nervous system in normal and disease states. We measured desert hedgehog expression in the peripheral nerve of maturing diabetic rats and found that diabetes caused a significant reduction in desert hedgehog mRNA. Treating diabetic rats with a sonic hedgehog-IgG fusion protein fully restored motor- and sensory-nerve conduction velocities and maintained the axonal caliber of large myelinated fibers. Diabetes-induced deficits in retrograde transport of nerve growth factor and sciatic-nerve levels of calcitonin gene-related product and neuropeptide Y were also ameliorated by treatment with the sonic hedgehog-IgG fusion protein, as was thermal hypoalgesia in the paw. These studies implicate disruption of normal hedgehog function in the etiology of diabetes-induced peripheral-nerve dysfunction and indicate that delivery of exogenous hedgehog proteins may have therapeutic potential for the treatment of diabetic neuropathy. -
Rubin LE, Stein PB, DiScala C, Grottkau BE. 2003. Pediatric trauma caused by personal watercraft: a ten-year retrospective. Journal of pediatric surgery. 38(10):1525-9. Pubmed: 14577080 Rubin LE, Stein PB, DiScala C, Grottkau BE. 2003. Pediatric trauma caused by personal watercraft: a ten-year retrospective. Journal of pediatric surgery. 38(10):1525-9. Pubmed: 14577080 Array -
Gabay L, Lowell S, Rubin LL, Anderson DJ. 2003. Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron. 40(3):485-99. Pubmed: 14642274 Gabay L, Lowell S, Rubin LL, Anderson DJ. 2003. Deregulation of dorsoventral patterning by FGF confers trilineage differentiation capacity on CNS stem cells in vitro. Neuron. 40(3):485-99. Pubmed: 14642274 The CNS is thought to develop from self-renewing stem cells that generate neurons, astrocytes, and oligodendrocytes. Other data, however, have suggested that astrocytes and oligodendrocytes are generated from separate progenitor populations. To reconcile these observations, we have prospectively isolated progenitors that do or do not express Olig2, an oligodendrocyte bHLH determination factor. Both Olig2(-) and Olig2(+) progenitors can behave as tripotential CNS stem cells (CNS-SCs) in vitro. Growth in FGF-2 causes induction of Olig2 in the former population, permitting oligodendrocyte differentiation; extinction of Olig2 in the latter cells permits astrocyte differentiation. The induction of Olig2 by FGF-2 is mediated, in part, via endogenous Sonic Hedgehog. These data indicate that clonogenic competence to generate neurons, astrocytes, and oligodendrocytes reflects a deregulation of dorsoventral patterning during expansion in vitro, raising the question of whether such trifatent cells actually exist in vivo. -
Williams JA, Guicherit OM, Zaharian BI, Xu Y, Chai L, Wichterle H, Kon C, Gatchalian C, Porter JA, Rubin LL, Wang FY. 2003. Identification of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions. Proceedings of the National Academy of Sciences of the United States of America. 100(8):4616-21. Pubmed: 12679522 Williams JA, Guicherit OM, Zaharian BI, Xu Y, Chai L, Wichterle H, Kon C, Gatchalian C, Porter JA, Rubin LL, Wang FY. 2003. Identification of a small molecule inhibitor of the hedgehog signaling pathway: effects on basal cell carcinoma-like lesions. Proceedings of the National Academy of Sciences of the United States of America. 100(8):4616-21. Pubmed: 12679522 The link between basal cell carcinoma (BCC) and aberrant activation of the Hedgehog (Hh) signaling pathway has been well established in humans and in mouse models. Here we report the development of assays, including two novel in vitro BCC models, which allowed us to screen for Hh inhibitors and test their validity as potential treatments for BCC. We identified a novel small molecule Hh inhibitor (CUR61414) that can block elevated Hh signaling activity resulting from oncogenic mutations in Patched-1. Moreover, CUR61414 can suppress proliferation and induce apoptosis of basaloid nests in the BCC model systems, whereas having no effect on normal skin cells. These findings directly demonstrate that the use of Hh inhibitors could be a valid therapeutic approach for treating BCC. -
Machold R, Hayashi S, Rutlin M, Muzumdar MD, Nery S, Corbin JG, Gritli-Linde A, Dellovade T, Porter JA, Rubin LL, Dudek H, McMahon AP, Fishell G. 2003. Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron. 39(6):937-50. Pubmed: 12971894 Machold R, Hayashi S, Rutlin M, Muzumdar MD, Nery S, Corbin JG, Gritli-Linde A, Dellovade T, Porter JA, Rubin LL, Dudek H, McMahon AP, Fishell G. 2003. Sonic hedgehog is required for progenitor cell maintenance in telencephalic stem cell niches. Neuron. 39(6):937-50. Pubmed: 12971894 To directly test the requirement for hedgehog signaling in the telencephalon from early neurogenesis, we examined conditional null alleles of both the Sonic hedgehog and Smoothened genes. While the removal of Shh signaling in these animals resulted in only minor patterning abnormalities, the number of neural progenitors in both the postnatal subventricular zone and hippocampus was dramatically reduced. In the subventricular zone, this was partially attributable to a marked increase in programmed cell death. Consistent with Hedgehog signaling being required for the maintenance of stem cell niches in the adult brain, progenitors from the subventricular zone of floxed Smo animals formed significantly fewer neurospheres. The loss of hedgehog signaling also resulted in abnormalities in the dentate gyrus and olfactory bulb. Furthermore, stimulation of the hedgehog pathway in the mature brain resulted in elevated proliferation in telencephalic progenitors. These results suggest that hedgehog signaling is required to maintain progenitor cells in the postnatal telencephalon. 2002
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Frank-Kamenetsky M, Zhang XM, Bottega S, Guicherit O, Wichterle H, Dudek H, Bumcrot D, Wang FY, Jones S, Shulok J, Rubin LL, Porter JA. 2002. Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists. Journal of biology. 1(2):10. Pubmed: 12437772 Frank-Kamenetsky M, Zhang XM, Bottega S, Guicherit O, Wichterle H, Dudek H, Bumcrot D, Wang FY, Jones S, Shulok J, Rubin LL, Porter JA. 2002. Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists. Journal of biology. 1(2):10. Pubmed: 12437772 Array