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Article ; Online: Implementation of a Drug Screening Platform to Target

Cronin, Shane J F / Davidow, Lance S / Arvanites, Anthony C / Rubin, Lee L / Penninger, Josef M / Woolf, Clifford J

2023 Volume 13, Issue 9, Page(s) e4666

Abstract: 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 ... ...

Abstract	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,
Language	English
Publishing date	2023-05-05
Publishing country	United States
Document type	Journal Article
ZDB-ID	2833269-6
ISSN	2331-8325 ; 2331-8325
ISSN (online)	2331-8325
ISSN	2331-8325
DOI	10.21769/BioProtoc.4666
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Article ; Online: Synthetic modified Fezf2 mRNA (modRNA) with concurrent small molecule SIRT1 inhibition enhances refinement of cortical subcerebral/corticospinal neuron identity from mouse embryonic stem cells.

Sadegh, Cameron / Ebina, Wataru / Arvanites, Anthony C / Davidow, Lance S / Rubin, Lee L / Macklis, Jeffrey D

PloS one

2021 Volume 16, Issue 9, Page(s) e0254113

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Animals ; Cell Differentiation ; Cells, Cultured ; DNA-Binding Proteins/genetics ; DNA-Binding Proteins/metabolism ; Mice ; Mice, Knockout ; Mouse Embryonic Stem Cells/cytology ; Mouse Embryonic Stem Cells/metabolism ; Neocortex/cytology ; Neocortex/metabolism ; Nerve Tissue Proteins/genetics ; Nerve Tissue Proteins/metabolism ; Neurons/cytology ; Neurons/metabolism ; RNA, Messenger/genetics ; Sirtuin 1/antagonists & inhibitors ; Transcription Factors/metabolism
Chemical Substances	DNA-Binding Proteins ; Nerve Tissue Proteins ; RNA, Messenger ; Transcription Factors ; Zfp312 protein, mouse ; Sirt1 protein, mouse (EC 3.5.1.-) ; Sirtuin 1 (EC 3.5.1.-)
Language	English
Publishing date	2021-09-02
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0254113
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Article ; Online: Using Automated Live Cell Imaging to Reveal Early Changes during Human Motor Neuron Degeneration.

Shin, Hye Young / Pfaff, Kathleen L / Davidow, Lance S / Sun, Chicheng / Uozumi, Takayuki / Yanagawa, Fumiki / Yamazaki, Yoichi / Kiyota, Yasujiro / Rubin, Lee L

eNeuro

2018 Volume 5, Issue 3

Abstract: 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 ...

Abstract	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.
MeSH term(s)	Benzazepines/administration & dosage ; Cell Death ; Cells, Cultured ; Embryonic Stem Cells/drug effects ; Embryonic Stem Cells/pathology ; Embryonic Stem Cells/physiology ; Humans ; Image Processing, Computer-Assisted/methods ; Indoles/administration & dosage ; Motor Neurons/drug effects ; Motor Neurons/pathology ; Motor Neurons/physiology ; Neurites/pathology ; Neurites/physiology ; Neurodegenerative Diseases/pathology ; Neurodegenerative Diseases/physiopathology ; Pattern Recognition, Automated
Chemical Substances	Benzazepines ; Indoles ; kenpaullone
Language	English
Publishing date	2018-06-29
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2800598-3
ISSN	2373-2822 ; 2373-2822
ISSN (online)	2373-2822
ISSN	2373-2822
DOI	10.1523/ENEURO.0001-18.2018
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Article ; Online: Assessing kinetics and recruitment of DNA repair factors using high content screens.

Martinez-Pastor, Barbara / Silveira, Giorgia G / Clarke, Thomas L / Chung, Dudley / Gu, Yuchao / Cosentino, Claudia / Davidow, Lance S / Mata, Gadea / Hassanieh, Sylvana / Salsman, Jayme / Ciccia, Alberto / Bae, Narkhyun / Bedford, Mark T / Megias, Diego / Rubin, Lee L / Efeyan, Alejo / Dellaire, Graham / Mostoslavsky, Raul

Cell reports

2021 Volume 37, Issue 13, Page(s) 110176

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Chromatin/genetics ; Chromatin/metabolism ; DNA Damage ; DNA Repair ; DNA Repair Enzymes/genetics ; DNA Repair Enzymes/metabolism ; High-Throughput Screening Assays ; Histones/genetics ; Histones/metabolism ; Humans ; Kinetics ; Open Reading Frames ; Tumor Suppressor p53-Binding Protein 1/genetics ; Tumor Suppressor p53-Binding Protein 1/metabolism
Chemical Substances	Chromatin ; Histones ; TP53BP1 protein, human ; Tumor Suppressor p53-Binding Protein 1 ; DNA Repair Enzymes (EC 6.5.1.-)
Language	English
Publishing date	2021-12-17
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2021.110176
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Article ; Online: Phenotypic drug screen uncovers the metabolic GCH1/BH4 pathway as key regulator of EGFR/KRAS-mediated neuropathic pain and lung cancer.

Cronin, Shane J F / Rao, Shuan / Tejada, Miguel A / Turnes, Bruna Lenfers / Licht-Mayer, Simon / Omura, Takao / Brenneis, Christian / Jacobs, Emily / Barrett, Lee / Latremoliere, Alban / Andrews, Nick / Channon, Keith M / Latini, Alexandra / Arvanites, Anthony C / Davidow, Lance S / Costigan, Michael / Rubin, Lee L / Penninger, Josef M / Woolf, Clifford J

Science translational medicine

2022 Volume 14, Issue 660, Page(s) eabj1531

Abstract: 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- ... ...

Abstract	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
MeSH term(s)	Analgesics/pharmacology ; Analgesics/therapeutic use ; Animals ; Biopterins/analogs & derivatives ; ErbB Receptors/genetics ; ErbB Receptors/metabolism ; GTP Cyclohydrolase/genetics ; GTP Cyclohydrolase/metabolism ; Humans ; Lung Neoplasms/drug therapy ; Lung Neoplasms/genetics ; Mice ; Neuralgia/drug therapy ; Neuralgia/metabolism ; Proto-Oncogene Proteins p21(ras)/genetics ; Proto-Oncogene Proteins p21(ras)/metabolism
Chemical Substances	Analgesics ; KRAS protein, human ; Biopterins ; EGFR protein, human (EC 2.7.10.1) ; ErbB Receptors (EC 2.7.10.1) ; GTP Cyclohydrolase (EC 3.5.4.16) ; Gch1 protein, mouse (EC 3.5.4.16) ; Proto-Oncogene Proteins p21(ras) (EC 3.6.5.2) ; sapropterin (EGX657432I)
Language	English
Publishing date	2022-08-31
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2518854-9
ISSN	1946-6242 ; 1946-6234
ISSN (online)	1946-6242
ISSN	1946-6234
DOI	10.1126/scitranslmed.abj1531
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Article ; Online: Patient hiPSCs Identify Vascular Smooth Muscle Arylacetamide Deacetylase as Protective against Atherosclerosis.

Toyohara, Takafumi / Roudnicky, Filip / Florido, Mary H C / Nakano, Toshiaki / Yu, Haojie / Katsuki, Shunsuke / Lee, Minjin / Meissner, Torsten / Friesen, Max / Davidow, Lance S / Ptaszek, Leon / Abe, Takaaki / Rubin, Lee L / Pereira, Alexandre C / Aikawa, Masanori / Cowan, Chad A

Cell stem cell

2020 Volume 27, Issue 1, Page(s) 178–180

Language	English
Publishing date	2020-06-03
Publishing country	United States
Document type	Published Erratum
ZDB-ID	2375354-7
ISSN	1875-9777 ; 1934-5909
ISSN (online)	1875-9777
ISSN	1934-5909
DOI	10.1016/j.stem.2020.05.013
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Article ; Online: Patient hiPSCs Identify Vascular Smooth Muscle Arylacetamide Deacetylase as Protective against Atherosclerosis.

Cell stem cell

2020 Volume 27, Issue 1, Page(s) 147–157.e7

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Animals ; Atherosclerosis ; Cell Proliferation ; Cells, Cultured ; Diabetes Mellitus, Type 2 ; Humans ; Induced Pluripotent Stem Cells ; Mice ; Mice, Inbred C57BL ; Muscle, Smooth, Vascular ; Myocytes, Smooth Muscle
Language	English
Publishing date	2020-05-14
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2375354-7
ISSN	1875-9777 ; 1934-5909
ISSN (online)	1875-9777
ISSN	1934-5909
DOI	10.1016/j.stem.2020.04.018
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Article ; Online: Large-Scale Production of Mature Neurons from Human Pluripotent Stem Cells in a Three-Dimensional Suspension Culture System.

Rigamonti, Alessandra / Repetti, Giuliana G / Sun, Chicheng / Price, Feodor D / Reny, Danielle C / Rapino, Francesca / Weisinger, Karen / Benkler, Chen / Peterson, Quinn P / Davidow, Lance S / Hansson, Emil M / Rubin, Lee L

Stem cell reports

2016 Volume 6, Issue 6, Page(s) 993–1008

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Biomarkers/metabolism ; Brain-Derived Neurotrophic Factor/pharmacology ; Cell Culture Techniques ; Cell Differentiation/drug effects ; Ciliary Neurotrophic Factor/pharmacology ; Glial Cell Line-Derived Neurotrophic Factor/pharmacology ; Humans ; Microtubule-Associated Proteins/genetics ; Microtubule-Associated Proteins/metabolism ; Nerve Net/cytology ; Nerve Net/physiology ; Neurogenesis/drug effects ; Neurogenesis/genetics ; Neurons/classification ; Neurons/cytology ; Neurons/drug effects ; Neurons/metabolism ; Observer Variation ; Pluripotent Stem Cells/cytology ; Pluripotent Stem Cells/drug effects ; Pluripotent Stem Cells/metabolism ; Repressor Proteins/genetics ; Repressor Proteins/metabolism ; Reproducibility of Results ; Smad Proteins/antagonists & inhibitors ; Smad Proteins/genetics ; Smad Proteins/metabolism ; Spheroids, Cellular/cytology ; Spheroids, Cellular/drug effects ; Spheroids, Cellular/metabolism ; T-Box Domain Proteins/genetics ; T-Box Domain Proteins/metabolism ; Tumor Suppressor Proteins/genetics ; Tumor Suppressor Proteins/metabolism
Chemical Substances	BCL11B protein, human ; Biomarkers ; Brain-Derived Neurotrophic Factor ; Ciliary Neurotrophic Factor ; Glial Cell Line-Derived Neurotrophic Factor ; MAP2 protein, human ; Microtubule-Associated Proteins ; Repressor Proteins ; Smad Proteins ; T-Box Domain Proteins ; TBR1 protein, human ; Tumor Suppressor Proteins ; BDNF protein, human (7171WSG8A2)
Language	English
Publishing date	2016-06-14
Publishing country	United States
Document type	Journal Article
ZDB-ID	2720528-9
ISSN	2213-6711 ; 2213-6711
ISSN (online)	2213-6711
ISSN	2213-6711
DOI	10.1016/j.stemcr.2016.05.010
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Article ; Online: Single-Cell Analysis of SMN Reveals Its Broader Role in Neuromuscular Disease.

Rodriguez-Muela, Natalia / Litterman, Nadia K / Norabuena, Erika M / Mull, Jesse L / Galazo, Maria José / Sun, Chicheng / Ng, Shi-Yan / Makhortova, Nina R / White, Andrew / Lynes, Maureen M / Chung, Wendy K / Davidow, Lance S / Macklis, Jeffrey D / Rubin, Lee L

Cell reports

2017 Volume 18, Issue 6, Page(s) 1484–1498

Abstract: 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 ... ...

Abstract	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.
Language	English
Publishing date	2017-02-07
Publishing country	United States
Document type	Journal Article
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2017.01.035
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Article: The DXPas34 repeat regulates random and imprinted X inactivation.

Cohen, Dena E / Davidow, Lance S / Erwin, Jennifer A / Xu, Na / Warshawsky, David / Lee, Jeannie T

Developmental cell

2007 Volume 12, Issue 1, Page(s) 57–71

Abstract: X chromosome inactivation (XCI) is initiated by expression of the noncoding Xist RNA in the female embryo. Tsix, the antisense noncoding partner of Xist, serves as its regulator during both imprinted and random XCI. Here, we show that Tsix in part acts ... ...

Abstract	X chromosome inactivation (XCI) is initiated by expression of the noncoding Xist RNA in the female embryo. Tsix, the antisense noncoding partner of Xist, serves as its regulator during both imprinted and random XCI. Here, we show that Tsix in part acts through a 34mer repeat, DXPas34. DXPas34 contains bidirectional promoter activity, producing overlapping forward and reverse transcripts. We generate three new Tsix alleles in mouse embryonic stem cells and show that, while the Tsix promoter is unexpectedly dispensable, DXPas34 plays dual positive-negative functions. At the onset of XCI, DXPas34 stimulates Tsix expression through its enhancer activity. Once XCI is established, DXPas34 becomes repressive and stably silences Tsix. Germline transmission of the DXPas34 mutation demonstrates its necessity for both random and imprinted XCI in mice. Intriguingly, sequence analysis suggests that DXPas34 could potentially have descended from an ancient retrotransposon. We hypothesize that DXPas34 was acquired by Tsix to regulate antisense function.
MeSH term(s)	Animals ; Base Sequence ; Consensus Sequence ; Down-Regulation ; Embryo, Mammalian/cytology ; Embryo, Mammalian/embryology ; Embryo, Mammalian/metabolism ; Embryonic Stem Cells/metabolism ; Female ; Gene Expression Regulation, Developmental ; Gene Targeting ; Genomic Imprinting ; In Situ Hybridization, Fluorescence ; Mice ; Models, Genetic ; Molecular Sequence Data ; Phylogeny ; Promoter Regions, Genetic/genetics ; RNA, Long Noncoding ; RNA, Untranslated/genetics ; Repetitive Sequences, Nucleic Acid/genetics ; Sequence Deletion ; Up-Regulation ; X Chromosome/genetics ; X Chromosome Inactivation/genetics
Chemical Substances	RNA, Long Noncoding ; RNA, Untranslated ; Tsix transcript, mouse
Language	English
Publishing date	2007-01
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2054967-2
ISSN	1878-1551 ; 1534-5807
ISSN (online)	1878-1551
ISSN	1534-5807
DOI	10.1016/j.devcel.2006.11.014
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