BET Bromodomain inhibition promotes neurogenesis while inhibiting gliogenesis in neural progenitor cells
ABSTRACT: Neural stem cells (NSCs) and progenitor cells (NPCs) are increasingly appreciated to hold great promise for regenerative medicine to treat CNS injuries and neurodegenerative diseases. However, evidence for effective stimulation of neuronal production from endogenous or transplanted NPCs for cell replacement with small molecules remains limited. To identify novel chemical entities/targets for neurogenesis, we had established a NPC phenotypic screen assay and validated it using known small-molecule neurogenesis inducers. Through screening small molecule libraries with annotated targets, we identified BET bromodomain inhibition as a novel mechanism for enhancing neurogenesis. BET bromodomain proteins, Brd2, Brd3, and Brd4 were found to be downregulated in NPCs upon differentiation, while their levels remain unaltered in proliferating NPCs. Consistent with the pharmacological study using bromodomain selective inhibitor (+)-JQ-1, knockdown of each BET protein resulted in an increase in the number of neurons with simultaneous reduction in both astrocytes and oligodendrocytes. Gene expression profiling analysis demonstrated that BET bromodomain inhibition induced a broad but specific transcription program enhancing directed differentiation of NPCs into neurons while suppressing cell cycle progression and gliogenesis. Together, these results highlight a crucial role of BET proteins as promising epigenetic regulators in NPC development and suggest a therapeutic potential of BET inhibitors in treating CNS injury and neurodegenerative diseases. NPCs were treated with (+)-JQ-1 or inactive enantiomer (-)-JQ-1 at 0.2 μM or 0.5 μM for 12 and 24 hrs in differentiation medium. The experiments were carried out in 3 independent preparations of NPCs (triplicates)
Project description:In this study, proteomic analysis on ZIKV-infected primary human fetal neural progenitor cells (NPCs) revealed that virus infection altered levels of cellular proteins involved in NPC proliferation, differentiation and migration.
Project description:To follow-up findings that miR-9 was abundantly expressed in control NPCs, significantly down-regulated in a subset of SZ NPCs, and that miR-9 levels/activity, neural migration and diagnosis were strongly correlated, we tested the effect of manipulating miR-9 at cellular, proteomic and transcriptomic levels. Unexpectedly, proteomic- and RNAseq-based analysis revealed that these effects were mediated primarily by small changes in expression of indirect miR-9 targets, rather than large changes in direct miR-9 targets; these indirect targets are enriched for migration-associated genes. Together these data indicate that aberrant levels and activity of miR-9 may be one of the many factors that contribute to SZ risk, at least in a subset of patients. Methods: We compared global transcription of forebrain NPCs from two control and two SZ patients with manipulated miR-9 levels by RNAseq. Results: Although RNAseq analysis revealed large inter-individual heterogeneity, we were able to resolve several functional consistencies in the effects of our miR-9 perturbations: i) the change in miR-9 activity was consistent with the inhibitory role of miR-9, ii) the gene expression fold-change of miR-9 target genes (between each perturbation and its corresponding control, summarized by the first principal component) was correlated (r=0.95, p=3.92e-04) with miR-9 fold change and iii) the differentially expressed (DE; p <0.01) gene list resulting from miR-9 perturbation (paired t-test) was enriched for miR-9 targets (1.53-fold, p=1.2e-5). Conclusions: We integrated the miR-9 perturbation RNAseq data with our existing RNAseq datasets contrasting control and SZ hiPSC NPC expression from our cohort 1 (six controls, four patients), to ask whether there was any relationship between the “SZ NPC signature” and “miR-9 perturbation” datasets; we observed that the DE (p-value <0.01) in “SZ NPC signature” is enriched for DE (fdr<0.01) in “miR-9 perturbation” (the overall enrichment is 2.31-fold (p=9.39e-09)); there is significant correlation between DE fold-change in these two datasets (overall genes r=0.188; p<10e-50). Effects were mediated primarily by small changes in expression of indirect miR-9 targets, rather than large changes in direct miR-9 targets; these indirect targets are enriched for migration-associated genes Biological duplicates of passage-matched NPCs from 1 control (female) and 1 SZ patient (female) were transduced with either RV-GFP or RV-miR-9-GFP; GFP-positive NPCs were purified by fluorescent activated cell sorting (FACS) and expanded for two passages. In parallel, passage-matched NPCs from 2 controls (1 male, 1 female) and 2 SZ patients (1 male, 1 female) were transiently transfected with either scrambled or miR-9 LNA probes. In both instances, miR-9 perturbation was confirmed by qPCR.
Project description:We analyzed the genome-wide binding of Sox2 and POU factor partner factors, Oct4 in ESCs (using published datasets PMID:18692474 and GSM307137, GSM307154, GSM307155) and Brn2 in NPCs. We found that Sox2 and Oct4 co-occupied a large subset of promoters and enhancers in ESCs, but that Sox2 and Brn2 co-occupy predominantly enhancers. Further, we overexpressed Brn2 in differentiating ESCs and showed that ectopic Brn2 recruited Sox2 to NPC-specific targets, resulting in skewed differentiation towards the neural lineage. Examination of transcription factor binding in ESCs, NPCs, and differentiating ESCs by ChIP-Seq.
Project description:Induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs) are a promising source of tailor-made cell therapy for neurological diseases. However, tumorigenicity and immunogenicity are major obstacles to translational use. Here we demonstrate epidural therapeutics of human iPSC-NPC grafts after experimental ischemic stroke to avoid surgical damage and intracerebral teratomas. We found that human iPSC-NPCs co-cultured trans-membranously with rat cortical cells subjected to oxygen-glucose deprivation, compared with human mesenchymal stem cells from bone marrow and umbilical cord Wharton's jelly, superiorly enhanced neural survival and growth as well as mitigated astrogliosis. Using comparative whole-genome microarrays and cytokine arrays, we identified a neurorestorative secretome from iPSC-NPCs and neutralization of the enriched cytokines potently abolished the neuroprotective effects in the iPSC-NPC co-cultures. Moreover, we implanted the human iPSC-NPCs epidurally using fibrin glue over the peri-infarct cortex at 7 days following permanent middle cerebral artery occlusion in adult rats. The cell-treated rats showed significant improvement in their paretic forelimb usage and grip strength from 10 days post-transplantation (dpt) onwards compared to the vehicle-treated rats, accompanied by ameliorated infarct/atrophy volumes, inflammatory infiltration and astrogliosis, as well as augmented angiogenesis, oligodendrocyte precursor cells and white matter integrity. Some iPSC-NPCs migrated into the peri-infarct cortex but poorly survived by 21 dpt. This proof-of-concept study demonstrates that a less invasive yet effective epidural delivery route of human iPSC-NPCs may promote functional remodeling of the peri-infarct brain predominantly through distinct paracrine effects. cDNA samples from iPSC-NPC were hybridized to the human genome U133 Plus 2.0 GeneChip arrays.
Project description:Down syndrome neurophenotypes are characterized by mental retardation and a decreased brain volume. In order to identify whether deficits in proliferation, differentiation or survival could be responsible for this phenotype, neural precursor cells (NPCs) were isolated from the developing E14 neocortex of Down syndrome partial trisomy Ts1Cje mice and euploid (WT) littermates. Proliferation, cell differentiation and cell death assays revealed that Ts1Cje NPCs proliferated at a slower rate, due to a longer cell cycle and that a greater number of cells were positive for glial fibrillary acidic protein. An increase in Ts1Cje NPC cell death was also noted. Gene expression profiling was conducted on RNA extracted from Ts1Cje and WT NPCs. Approximately 54% of triploid gene expression ratios were significantly greater than the expected diploid gene ratio of 1.0. A number of diploid genes associated with differentiation, glial function and proliferation were dysregulated. The evidence points to a delay in cellular cycling that could exert stress on the NPC population, which might result in cellular death and a mobilization of glial cell survival responses. Importantly, these phenotypic changes, which mimic those seen in Down syndrome individuals, do not require over-expression of amyloid precursor protein (App) or soluble superoxide dismutase 1 (Sod1). In conclusion, early developmental proliferation deficits in Down syndrome result in secondary morphological changes that can impact on cognitive development and function. Keywords: Down syndrome, Neocortical precursor cells, transcriptome, proliferation Neural precursor cells (NPCs) were isolated from mouse E14 neocortex of Down syndrome Ts1Cje mice and euploid littermates. We compared gene expression profiles from trisomic and wild-type cells using pangenomic microarrays.
Project description:Cellular senescence is a form of adaptive cellular physiology associated with aging. Cellular senescence causes a pro-inflammatory cellular phenotype that impairs tissue regeneration, has been linked to stress, and is implicated in several human neurodegenerative diseases. We had previously determined that neural progenitor cells (NPCs) derived from primary progressive multiple sclerosis (PPMS) patient induced pluripotent stem (iPS) cell lines failed to promote oligodendrocyte progenitor cell (OPC) maturation whereas NPCs from age-matched control cell lines did so efficiently. Herein, we report that expression of hallmarks of cellular senescence were identified in SOX2+ progenitor cells within white matter lesions of human progressive MS autopsy brain tissues and PPMS patient iPS-derived NPCs. Expression of cellular senescence genes in PPMS NPCs was found to be reversible by treatment with rapamycin which then enhanced PPMS NPC support for oligodendrocyte differentiation. A proteomic analysis of the PPMS NPC secretome identified high mobility group box-1 (HMGB1), which was found to be a senescence-associated inhibitor of oligodendrocyte differentiation. Transcriptome analysis of OPCs revealed that senescent NPCs induced expression of epigenetic regulators mediated by extracellular HMGB1. Lastly, we determined that progenitor cells are a source of elevated HMGB1 in human white matter lesions. Based on these data, we conclude that cellular senescence contributes to altered progenitor cell functions in demyelinated lesions in MS. Moreover, these data implicate cellular aging and senescence as a process that contributes to remyelination failure in progressive MS which may impact how this disease is modeled and inform development of future myelin regeneration strategies.
Project description:Expression profiles for isogenic (129SvJae x C57BL/6) murine embryonic stem (ES) cells, neural precursors (NPC) obtained through in vitro differentiation of the ES cells, and embryonic fibroblasts (MEF) obtained at day 13.5. Experiment Overall Design: 3 replicates of wt ES cells, 3 replicates of wt NPCs obtained by in vitro differentiation, 2 replicated of primary MEFs
Project description:Adult neurogenesis occurs in mammals and provides a mechanism for continuous neural plasticity in the brain.However, little is known about the molecular mechanisms regulating hippocampal neural progenitor cells (NPCs) and whether their fate can be pharmacologically modulated to improve neural plasticity and regeneration. Here, we report the characterization of a unique small molecule (KHS101) that selectively induces a neuronal differentiation phenotype. Mechanism of action studies revealed a link of KHS101 to cell cycle exit and specific binding to the TACC3 protein, whose knockdown in NPCs recapitulates the KHS101-induced phenotype. Upon systemic administration, KHS101 distributed to the brainandresulted in a significant increase in neuronal differentiation in vivo. Our findings indicate that KHS101 accelerates neuronal differentiation by interaction with TACC3 and may provide a basis for pharmacological intervention.directed at endogenous NPCs. Compare expression profile of KHS101-treated hippocampal progenitor cells (2 concentrations) vs. DMSO (negative control), retinoic acid (positive control)
Project description:Analysis of dopaminergic neuronal gene expression changes by Nurr1 and/or Foxa2 overexpression. Result provides that Foxa2 potentiates Nurr1-induced DA neuronal phenotype gene expression. To identify the syergism of Nurr1 and Foxa2 for developing DA neural precursors, neural precusor cells (NPCs) isolated from embryonic brain were treated control, Nurr1, Foxa2 and Nurr1-Foxa2 retrovirus. After treatment of retroviruses, NPCs were cultrued in N2 media withdrawn mitogen (bFGF, EGF) for differetiation of DA neuron. Total RNA was obtained from NPCs in differentiation day 2.