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1.
Mol Psychiatry ; 23(6): 1453-1465, 2018 06.
Article in English | MEDLINE | ID: mdl-28242870

ABSTRACT

Bipolar disorder (BD) is a progressive psychiatric disorder with more than 3% prevalence worldwide. Affected individuals experience recurrent episodes of depression and mania, disrupting normal life and increasing the risk of suicide greatly. The complexity and genetic heterogeneity of psychiatric disorders have challenged the development of animal and cellular models. We recently reported that hippocampal dentate gyrus (DG) neurons differentiated from induced pluripotent stem cell (iPSC)-derived fibroblasts of BD patients are electrophysiologically hyperexcitable. Here we used iPSCs derived from Epstein-Barr virus-immortalized B-lymphocytes to verify that the hyperexcitability of DG-like neurons is reproduced in this different cohort of patients and cells. Lymphocytes are readily available for research with a large number of banked lines with associated patient clinical description. We used whole-cell patch-clamp recordings of over 460 neurons to characterize neurons derived from control individuals and BD patients. Extensive functional analysis showed that intrinsic cell parameters are very different between the two groups of BD neurons, those derived from lithium (Li)-responsive (LR) patients and those derived from Li-non-responsive (NR) patients, which led us to partition our BD neurons into two sub-populations of cells and suggested two different subdisorders. Training a Naïve Bayes classifier with the electrophysiological features of patients whose responses to Li are known allows for accurate classification with more than 92% success rate for a new patient whose response to Li is unknown. Despite their very different functional profiles, both populations of neurons share a large, fast after-hyperpolarization (AHP). We therefore suggest that the large, fast AHP is a key feature of BD and a main contributor to the fast, sustained spiking abilities of BD neurons. Confirming our previous report with fibroblast-derived DG neurons, chronic Li treatment reduced the hyperexcitability in the lymphoblast-derived LR group but not in the NR group, strengthening the validity and utility of this new human cellular model of BD.


Subject(s)
Bipolar Disorder/metabolism , Cell Differentiation/physiology , Neurons/drug effects , Adult , Antimanic Agents/therapeutic use , Antipsychotic Agents/therapeutic use , Biomarkers, Pharmacological/metabolism , Bipolar Disorder/genetics , Case-Control Studies , Dentate Gyrus/drug effects , Female , Hippocampus/drug effects , Humans , Induced Pluripotent Stem Cells/physiology , Lithium/therapeutic use , Lithium Compounds/therapeutic use , Male , Patch-Clamp Techniques
2.
Mol Psychiatry ; 21(1): 49-61, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26503761

ABSTRACT

The brain's serotonergic system centrally regulates several physiological processes and its dysfunction has been implicated in the pathophysiology of several neuropsychiatric disorders. While in the past our understanding of serotonergic neurotransmission has come mainly from mouse models, the development of pluripotent stem cell and induced fibroblast-to-neuron (iN) transdifferentiation technologies has revolutionized our ability to generate human neurons in vitro. Utilizing these techniques and a novel lentiviral reporter for serotonergic neurons, we identified and overexpressed key transcription factors to successfully generate human serotonergic neurons. We found that overexpressing the transcription factors NKX2.2, FEV, GATA2 and LMX1B in combination with ASCL1 and NGN2 directly and efficiently generated serotonergic neurons from human fibroblasts. Induced serotonergic neurons (iSNs) showed increased expression of specific serotonergic genes that are known to be expressed in raphe nuclei. iSNs displayed spontaneous action potentials, released serotonin in vitro and functionally responded to selective serotonin reuptake inhibitors (SSRIs). Here, we demonstrate the efficient generation of functional human serotonergic neurons from human fibroblasts as a novel tool for studying human serotonergic neurotransmission in health and disease.


Subject(s)
Cytological Techniques/methods , Fibroblasts/physiology , Serotonergic Neurons/physiology , Animals , Astrocytes/physiology , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Line , Cell Transdifferentiation/physiology , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , GATA2 Transcription Factor/genetics , GATA2 Transcription Factor/metabolism , Genetic Vectors , Homeobox Protein Nkx-2.2 , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Human Embryonic Stem Cells/physiology , Humans , LIM-Homeodomain Proteins/genetics , LIM-Homeodomain Proteins/metabolism , Lentivirus/genetics , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome , Zebrafish Proteins
4.
Mol Psychiatry ; 20(11): 1350-65, 2015 Nov.
Article in English | MEDLINE | ID: mdl-25385366

ABSTRACT

An increasing number of genetic variants have been implicated in autism spectrum disorders (ASDs), and the functional study of such variants will be critical for the elucidation of autism pathophysiology. Here, we report a de novo balanced translocation disruption of TRPC6, a cation channel, in a non-syndromic autistic individual. Using multiple models, such as dental pulp cells, induced pluripotent stem cell (iPSC)-derived neuronal cells and mouse models, we demonstrate that TRPC6 reduction or haploinsufficiency leads to altered neuronal development, morphology and function. The observed neuronal phenotypes could then be rescued by TRPC6 complementation and by treatment with insulin-like growth factor-1 or hyperforin, a TRPC6-specific agonist, suggesting that ASD individuals with alterations in this pathway may benefit from these drugs. We also demonstrate that methyl CpG binding protein-2 (MeCP2) levels affect TRPC6 expression. Mutations in MeCP2 cause Rett syndrome, revealing common pathways among ASDs. Genetic sequencing of TRPC6 in 1041 ASD individuals and 2872 controls revealed significantly more nonsynonymous mutations in the ASD population, and identified loss-of-function mutations with incomplete penetrance in two patients. Taken together, these findings suggest that TRPC6 is a novel predisposing gene for ASD that may act in a multiple-hit model. This is the first study to use iPSC-derived human neurons to model non-syndromic ASD and illustrate the potential of modeling genetically complex sporadic diseases using such cells.


Subject(s)
Autistic Disorder/pathology , Neurons/pathology , TRPC Cation Channels/metabolism , Animals , Antineoplastic Combined Chemotherapy Protocols/metabolism , Autistic Disorder/genetics , Autistic Disorder/physiopathology , Carboplatin/metabolism , Cell Differentiation/genetics , Cell Line , Cell Proliferation/genetics , Cells, Cultured , Child , Disease Models, Animal , Embryo, Mammalian , Etoposide/metabolism , Gene Expression Regulation/genetics , Humans , In Vitro Techniques , Induced Pluripotent Stem Cells/physiology , Inhibitory Postsynaptic Potentials/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitoxantrone/metabolism , Mutation/genetics , Neurons/metabolism , Prednisolone/metabolism , Signal Transduction/genetics , TRPC Cation Channels/genetics , TRPC6 Cation Channel
5.
Mem Inst Oswaldo Cruz ; 97(4): 547-52, 2002 Jun.
Article in English | MEDLINE | ID: mdl-12118289

ABSTRACT

Treatment of cancer using gene therapy is based on adding a property to the cell leading to its elimination. One possibility is the use of suicide genes that code for enzymes that transform a pro-drug into a cytotoxic product. The most extensively used is the herpes simplex virus thymidine kinase (TK) gene, followed by administration of the antiviral drug ganciclovir (GCV). The choice of the promoter to drive the transcription of a transgene is one of the determinants of a given transfer vector usefulness, as different promoters show different efficiencies depending on the target cell type. In the experiments presented here, we report the construction of a recombinant adenovirus carrying TK gene (Ad-TK) driven by three strong promoters (P CMV IE, SV40 and EN1) and its effectiveness in two cell types. Human HeLa and mouse CCR2 tumor cells were transduced with Ad-TK and efficiently killed after addition of GCV. We could detect two sizes of transcripts of TK gene, one derived from the close together P CMV IE/SV40 promoters and the other from the 1.5 Kb downstream EN1 promoter. The relative amounts of these transcripts were different in each cell type thus indicating a higher flexibility of this system.


Subject(s)
Adenoviridae/genetics , Antiviral Agents/pharmacology , Ganciclovir/pharmacology , Genetic Therapy/methods , Genetic Vectors/therapeutic use , Thymidine Kinase/genetics , Animals , Genes, Viral , Genetic Vectors/genetics , HeLa Cells , Humans , Mice , Promoter Regions, Genetic , Thymidine Kinase/therapeutic use , Tumor Cells, Cultured
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