Genome editing in induced pluripotent stem cells rescues TAF1 levels in X‐linked dystonia‐parkinsonism

Background: The most likely genetic cause of X‐linked dystonia‐parkinsonism, a neurodegenerative movement disorder endemic to the Philippines, is a 2672‐bp‐long retrotransposon insertion in intron 32 of the TAF1 gene. The objectives of this study were to investigate whether (1) TAF1 expression is altered in induced pluripotent stem cells and differentiated neuronal models and (2) excision of the retrotransposon insertion restores normal TAF1 expression. Methods: Expression of TAF1 and its neuronal isoform were determined in induced pluripotent stem cells and in induced pluripotent stem cell‐derived cortical neurons and spiny projection neurons using quantitative PCR. Genome editing‐based excision of the retrotransposon insertion was performed on induced pluripotent stem cells from 3 X‐linked dystonia‐parkinsonism patients. Edited and unedited induced pluripotent stem cells from X‐linked dystonia‐parkinsonism patients and controls were differentiated into cortical neurons and spiny projection neurons, and TAF1 expression was compared across groups. Results: TAF1 was reduced in patient‐derived induced pluripotent stem cells (P < 0.05) and spiny projection neurons (P < 0.01). After genome editing, we observed higher TAF1 expression in edited compared with unedited induced pluripotent stem cells (P < 0.0001). In edited spiny projection neurons, TAF1 expression was also increased, but did not reach statistical significance. No expression differences were observed in cortical neurons. Conclusions: (1) TAF1 reduction in X‐linked dystonia‐parkinsonism is likely due to the retrotransposon insertion and is recapitulated in induced pluripotent stem cells and differentiated spiny projection neurons. (2) TAF1 reduction is a tractable molecular phenotype of X‐linked dystonia‐parkinsonism that can be driven by excision of the retrotransposon insertion. (3) Successful rescue of the molecular phenotype in an endogenous, genome‐edited model serves as a proof of principle that may successfully be transferred to other inherited neurodegenerative diseases. © 2018 International Parkinson and Movement Disorder Society     

Application of induced pluripotent stem cells to understand neurobiological basis of bipolar disorder and schizophrenia

The etiology of neuropsychiatric disorders, such as schizophrenia and bipolar disorder, usually involves complex combinations of genetic defects/variations and environmental impacts, which hindered, for a long time, research efforts based on animal models and patients’ non-neuronal cells or post-mortem tissues. However, the development of human induced pluripotent stem cell (iPSC) technology by the Yamanaka group was immediately applied to establish cell research models for neuronal disorders. Since then, techniques to achieve highly efficient differentiation of different types of neural cells following iPSC modeling have made much progress. The fast-growing iPSC and neural differentiation techniques have brought valuable insights into the pathology and neurobiology of neuropsychiatric disorders. In this article, we first review the application of iPSC technology in modeling neuronal disorders and discuss the progress in the accompanying neural differentiation. Then, we summarize the progress in iPSC-based research that has been accomplished so far regarding schizophrenia and bipolar disorder.     

Idiopathic Parkinson's disease patient‐derived induced pluripotent stem cells function as midbrain dopaminergic neurons in rodent brains

Patient‐specific induced pluripotent stem cells (iPSCs) are a promising source for cell transplantation therapy. In Parkinson's disease (PD) patients, however, their vulnerability and the transmission of pathological α‐Synuclein are possible drawbacks that may prevent PD‐specific iPSCs (PDiPSCs) from being used in clinical settings. In this study, we generated iPSCs from idiopathic PD patients and found that there was no significant vulnerability between dopaminergic (DA) neurons generated from healthy individuals and idiopathic PD patients. PDiPSC‐derived DA neurons survived and functioned in the brains of PD model rats. In addition, in the brains of α‐Synuclein transgenic mice, PDiPSC‐derived DA neurons did not cause pathological α‐Synuclein accumulation in the host brain or in the grafts. These results suggested that iPSCs derived from idiopathic PD patients are feasible as donor cells for autologous transplantation to treat PD. © 2017 Wiley Periodicals, Inc.     

Heterogeneous population of dopaminergic neurons derived from mouse embryonic stem cells: preliminary phenotyping based on receptor expression and function

The possibility exists that directed differentiation of mouse embryonic stem (mES) cells is capable of yielding enriched populations of dopaminergic neurons, but at present there is little understanding of the pharmacological properties of these cells; or whether such cells represent a pharmacologically, phenotypically similar population. In this study we used a simple culture protocol to generate dopaminergic neurons and offer a preliminary pharmacological investigation of these cells using Ca2+ imaging and [3H]-dopamine release studies. In fluo-4 AM loaded cells, 13-17 days postplating, and after the addition of tetrodotoxin some of the population of mouse embryonic stem cell-derived neurons responded to adenosine triphosphate (ATP), noradrenaline (NA), acetylcholine (ACh) and L-glutamate (L-glut) with elevations of Ca2+ influx. Within the microtubule-associated protein and tyrosine hydroxylase (TH)-positive cell population adenosine triphosphate, noradrenaline, acetylcholine and L-glutamate elicited positive elevations of Ca2+ in 74, 66, 58 and 67% of the population; cells could be further subdivided into three major pharmacologically distinct populations based on the combinations of agonist they responded to. Acetylcholine (30 microM) and noradrenaline (30 microM) were the only agonists to elicit significant tritium overflow from [3H]-dopamine loaded cells. The acetylcholine effect was blocked by atropine (1 microM) and tetrodotoxin (1 microM) and elevated by haloperidol (100 nM). The noradrenaline effects were reduced by cocaine (10 microM), but not by tetrodotoxin (100 nM). These data indicate that the dopaminergic neurons derived from mouse embryonic stem cells represent a heterogeneous population possessing combinations of purinergic, adrenergic, cholinergic and glutamatergic receptors located on the cell soma.     

Silver nanoparticles exhibit coating and dose-dependent neurotoxicity in glutamatergic neurons derived from human embryonic stem cells

Silver nanoparticles (AgNPs) are used extensively as anti-microbial agents in various products, but little is known about their potential neurotoxic effects. In this study, we used glutamatergic neurons derived from human embryonic stem cells as a cellular model to study 20 nm citrate-coated AgNPs (AgSCs) and Polyvinylpyrrolidone-coated AgNPs (AgSPs) induced neurotoxicity. AgSCs significantly damaged neurite outgrowths; increased the production of reactive oxygen species and Ca²⁺ influxes; reduced the expression of MAP2, PSD95, vGlut1 and NMDA receptor proteins at concentrations as low as 0.1 μg/ml. In contrast, AgSPs exhibited neurotoxicity only at higher concentration. Furthermore, our results showed that AgSCs induced glutamate excitotoxicity by the activation of calmodulin and the induction of nitric oxide synthase; increased the phosphorylation of glycogen synthase kinase-3 α/β at Tyr²¹⁶ and Tau at Ser³⁹⁶ and reduced the expression of Tau46, which are typically observed in Alzheimer’s disease. This study indicated that stem cells can provide an excellent platform for studying nanoparticle induced neurotoxicity.