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Rubin, Lee

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Rubin, Lee
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    Blocking p62-dependent SMN degradation ameliorates spinal muscular atrophy disease phenotypes
    (American Society for Clinical Investigation, 2018-06-11) Rodriguez-Muela, Natalia; Parkhitko, Andrey; Grass, Tobias; Gibbs, Rebecca; Norabuena, Erika M.; Perrimon, Norbert; Singh, Rajat; Rubin, Lee
    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.
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    Using intracellular markers to identify a novel set of surface markers for live cell purification from a heterogeneous hIPSC culture
    (Nature Publishing Group UK, 2018) Paik, Elizabeth J.; O’Neil, Alison L.; Ng, Shi-Yan; Sun, Chicheng; Rubin, Lee
    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.
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    A neuroprotective agent that inactivates prodegenerative TrkA and preserves mitochondria
    (The Rockefeller University Press, 2017) Feinberg, Konstantin; Kolaj, Adelaida; Wu, Chen; Grinshtein, Natalie; Krieger, Jonathan R.; Moran, Michael F.; Rubin, Lee; Miller, Freda D.; Kaplan, David R.
    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 wt 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.
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    Reactive Astrocytes Promote ALS-like Degeneration and Intracellular Protein Aggregation in Human Motor Neurons by Disrupting Autophagy through TGF-β1
    (Elsevier, 2017) Tripathi, Pratibha; Rodriguez-Muela, Natalia; Klim, Joseph; de Boer, A. Sophie; Agrawal, Sahil; Sandoe, Jackson; Lopes, Claudia S.; Ogliari, Karolyn Sassi; Williams, Luis A.; Shear, Matthew; Rubin, Lee; Eggan, Kevin; Zhou, Qiao
    Summary 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.
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    Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication
    (Proceedings of the National Academy of Sciences, 2012) Annes, J. P.; Ryu, J. H.; Lam, K.; Carolan, Peter; Utz, K.; Hollister-Lock, J.; Arvanites, Anthony C.; Rubin, Lee; Weir, Gordon; Melton, Douglas
    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.
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    A small molecule that directs differentiation of human ESCs into the pancreatic lineage
    (Nature Publishing Group, 2009) Chen, Shuibing; Borowiak, Malgorzata; Maehr, René; Osafune, Kenji; Davidow, Lance; Lam, Kelvin; Peng, Lee F; Schreiber, Stuart; Rubin, Lee; Melton, Douglas
    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.
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    A TALEN Genome-Editing System for Generating Human Stem Cell-Based Disease Models
    (Elsevier BV, 2013) Ding, Qiurong; Lee, Youn-Kyoung; Schaefer, Esperance; Peters, Derek T.; Veres, Adrian; Kim, Kevin; Kuperwasser, Nicolas; Motola, Daniel L; Meissner, Torsten; Hendriks, William; Trevisan, Marta; Gupta, Rajat; Moisan, Annie; Banks, Eric; Friesen, Max; Schinzel, Robert T.; Xia, Fang; Tang, Alexander; Xia, Yulei; Figueroa, Emmanuel; Wann, Amy; Ahfeldt, Tim; Daheron, Laurence; Zhang, Feng; Rubin, Lee; Peng, Lee F; Chung, Raymond; Musunuru, Kiran; Cowan, Chad
    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.
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    Modeling pain in vitro using nociceptor neurons reprogrammed from fibroblasts
    (2015) Wainger, Brian; Buttermore, Elizabeth D.; Oliveira, Julia T.; Mellin, Cassidy; Lee, Seungkyu; Saber, Wardiya Afshar; Wang, Amy; Ichida, Justin K.; Chiu, Isaac; Barrett, Lee; Huebner, Eric A.; Bilgin, Canan; Tsujimoto, Naomi; Brenneis, Christian; Kapur, Kush; Rubin, Lee; Eggan, Kevin; Woolf, Clifford
    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. Here, we identify five transcription factors that reprogram mouse and human fibroblasts into noxious stimulus-detecting (nociceptor) neurons that recapitulate 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 exhibit 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 show that the technique can reveal novel aspects of human disease phenotypes in vitro.
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    Targeting Superficial or Nodular Basal Cell Carcinoma with Topically Formulated Small Molecule Inhibitor of Smoothened
    (American Association for Cancer Research (AACR), 2011) Tang, Tracy; Tang, Jean Y.; Li, Dongwei; Reich, Mike; Callahan, Christopher A.; Fu, Ling; Yauch, Robert L.; Wang, Frank; Kotkow, Karen; Chang, Kris S.; Shpall, Elana; Wu, Angela; Rubin, Lee; Marsters, James C., Jr.; Epstein, Ervin H., Jr.; Caro, Ivor; de Sauvage, Frederic J.
    Purpose: Inappropriate activation of the Hedgehog (Hh) signaling pathway in skin is critical for the development of basal cell carcinomas (BCC). We have investigated the anti-BCC efficacy of topically-applied CUR61414, an inhibitor of the Hh signal transduction molecule Smoothened. Experimental Design: In preclinical studies, we used a depilatory model to evaluate the ability of topical formulations of CUR61414 to repress Hh responsive cells found at the base of hair follicles in normal skin. We also tested the in vivo effects of topical CUR61414 on murine BCCs developed in \(Ptch1^{ +/−}\) K14-CreER2 p53 fl/fl mice. In a phase I clinical study, we evaluated the safety, tolerability, and efficacy of a multidose regimen of CUR61414 (0.09%, 0.35%, 1.1%, and 3.1%) applied topically to human superficial or nodular BCCs for up to 28 days. Results: In mice, topical CUR61414 significantly inhibited skin Hh signaling, blocked the induction of hair follicle anagen, and shrank existing BCCs. However, we observed no clinical activity of this formulation in human superficial or nodular BCCs in a phase I clinical study. Conclusions: Our data highlight some of the challenges of translating preclinical experience into successful human results for a topical anticancer agent.
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    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
    (American Chemical Society (ACS), 2013) Chen, Po C.; Gaisina, Irina N.; El-Khodor, Bassem F.; Ramboz, Sylvie; Makhortova, Nina; Rubin, Lee; Kozikowski, Alan P.
    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.