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A High-content screen identifies compounds promoting the neuronal differentiation and the midbrain dopamine neuron specification of human neural progenitor cells.

ABSTRACT: Small molecule compounds promoting the neuronal differentiation of stem/progenitor cells are of pivotal importance to regenerative medicine. We carried out a high-content screen to systematically characterize known bioactive compounds, on their effects on the neuronal differentiation and the midbrain dopamine (mDA) neuron specification of neural progenitor cells (NPCs) derived from the ventral mesencephalon of human fetal brain. Among the promoting compounds three major pharmacological classes were identified including the statins, TGF-?RI inhibitors, and GSK-3 inhibitors. The function of each class was also shown to be distinct, either to promote both the neuronal differentiation and mDA neuron specification, or selectively the latter, or promote the former but suppress the latter. We then carried out initial investigation on the possible mechanisms underlying, and demonstrated their applications on NPCs derived from human pluripotent stem cells (PSCs). Our study revealed the potential of several small molecule compounds for use in the directed differentiation of human NPCs. The screening result also provided insight into the signaling network regulating the differentiation of human NPCs.

SUBMITTER: Rhim JH

PROVIDER: S-EPMC4635364 | biostudies-literature | 2015 Nov

REPOSITORIES: biostudies-literature

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A High-content screen identifies compounds promoting the neuronal differentiation and the midbrain dopamine neuron specification of human neural progenitor cells.

Rhim Ji Heon JH Luo Xiangjian X Xu Xiaoyun X Gao Dongbing D Zhou Tieling T Li Fuhai F Qin Lidong L Wang Ping P Xia Xiaofeng X Wong Stephen T C ST

Scientific reports 20151106

Small molecule compounds promoting the neuronal differentiation of stem/progenitor cells are of pivotal importance to regenerative medicine. We carried out a high-content screen to systematically characterize known bioactive compounds, on their effects on the neuronal differentiation and the midbrain dopamine (mDA) neuron specification of neural progenitor cells (NPCs) derived from the ventral mesencephalon of human fetal brain. Among the promoting compounds three major pharmacological classes w ...[more]

PMID: 26542303

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Project description:Parkinson's disease (PD) is the second most common neurodegenerative disorder with prominent dopamine (DA) neuron degeneration. PD affects millions of people worldwide, but currently available therapies are limited to temporary relief of symptoms. As an effort to discover disease-modifying therapeutics, we have conducted a screen of 1,403 bioactive small molecule compounds using an in vivo whole organism screening assay in transgenic larval zebrafish. The transgenic model expresses the bacterial enzyme nitroreductase (NTR) driven by the tyrosine hydroxylase (th) promotor. NTR converts the commonly used antibiotic pro-drug metronidazole (MTZ) to the toxic nitroso radical form to induce DA neuronal loss. 57 compounds were identified with a brain health score (BHS) that was significantly improved compared to the MTZ treatment alone after FDR adjustment (padj<0.05). Independently, we curated the high throughput screening (HTS) data by annotating each compound with pharmaceutical classification, known mechanism of action, indication, IC50, and target. Using the Reactome database, we performed pathway analysis, which uncovered previously unknown pathways in addition to validating previously known pathways associated with PD. Non-topology-based pathway analysis of the screening data further identified apoptosis, estrogen hormone, dipeptidyl-peptidase 4, and opioid receptor Mu1 to be potentially significant pathways and targets involved in neuroprotection. A total of 12 compounds were examined with a secondary assay that imaged DA neurons before and after compound treatment. The z'-factor of this secondary assay was determined to be 0.58, suggesting it is an excellent assay for screening. Etodolac, nepafenac, aloperine, protionamide, and olmesartan showed significant neuroprotection and was also validated by blinded manual DA neuronal counting. To determine whether these compounds are broadly relevant for neuroprotection, we tested them on a conduritol-b-epoxide (CBE)-induced Gaucher disease (GD) model, in which the activity of glucocerebrosidase (GBA), a commonly known genetic risk factor for PD, was inhibited. Aloperine, olmesartan, and nepafenac showed significant protection of DA neurons in this assay. Together, this work, which combines high content whole organism in vivo imaging-based screen and bioinformatic pathway analysis of the screening dataset, delineates a previously uncharted approach for identifying hit-to-lead candidates and for implicating previously unknown pathways and targets involved in DA neuron protection.

Project description:Reductions in the GABAergic neurotransmitter system exist across multiple brain regions in schizophrenia and encompass both pre- and postsynaptic components. While reduced midbrain GABAergic inhibitory neurotransmission may contribute to the hyperdopaminergia thought to underpin psychosis in schizophrenia, molecular changes consistent with this have not been reported. We hypothesised that reduced GABA-related molecular markers would be found in the midbrain of people with schizophrenia and that these would correlate with dopaminergic molecular changes. We hypothesised that downregulation of inhibitory neuron markers would be exacerbated in schizophrenia cases with high levels of neuroinflammation. Eight GABAergic-related transcripts were measured with quantitative PCR, and glutamate decarboxylase (GAD) 65/67 and GABAA alpha 3 (α3) (GABRA3) protein were measured with immunoblotting, in post-mortem midbrain (28/28 and 28/26 control/schizophrenia cases for mRNA and protein, respectively), and analysed by both diagnosis and inflammatory subgroups (as previously defined by higher levels of four pro-inflammatory cytokine transcripts). We found reductions (21 - 44%) in mRNA encoding both presynaptic and postsynaptic proteins, vesicular GABA transporter (VGAT), GAD1, and parvalbumin (PV) mRNAs and four alpha subunits (α1, α2, α3, α5) of the GABAA receptor in people with schizophrenia compared to controls (p < 0.05). Gene expression of somatostatin (SST) was unchanged (p = 0.485). We confirmed the reduction in GAD at the protein level (34%, p < 0.05). When stratifying by inflammation, only GABRA3 mRNA exhibited more pronounced changes in high compared to low inflammatory subgroups in schizophrenia. GABRA3 protein was expressed by 98% of tyrosine hydroxylase-positive neurons and was 23% lower in schizophrenia, though this did not reach statistical significance (p > 0.05). Expression of transcripts for GABAA receptor alpha subunits 2 and 3 (GABRA2, GABRA3) were positively correlated with tyrosine hydroxylase (TH) and dopamine transporter (DAT) transcripts in schizophrenia cases (GABRA2; r > 0.630, GABRA3; r > 0.762, all p < 0.001) but not controls (GABRA2; r < - 0.200, GABRA3; r < 0.310, all p > 0.05). Taken together, our results support a profound disruption to inhibitory neurotransmission in the substantia nigra regardless of inflammatory status, which provides a potential mechanism for disinhibition of nigrostriatal dopamine neurotransmission.

Dataset Information

A High-content screen identifies compounds promoting the neuronal differentiation and the midbrain dopamine neuron specification of human neural progenitor cells.

Publications

A High-content screen identifies compounds promoting the neuronal differentiation and the midbrain dopamine neuron specification of human neural progenitor cells.

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