Recombinant Human GAD65 Protein [His]

Human embryonic stem cells (hESCs) may be applied to develop human-relevant sensitive in vitro test systems for monitoring developmental toxicants. The aim of this study was to identify potential developmental toxicity mechanisms of the histone deacetylase inhibitors (HDAC) valproic acid (VPA), suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA) relevant to the in vivo condition using a hESC model in combination with specific differentiation protocols and genome-wide gene expression and microRNA profiling. Analysis of the gene expression data showed that VPA repressed neural tube and dorsal forebrain (OTX2, ISL1, EMX2 and SOX10)-related transcripts. In addition, VPA upregulates axonogenesis and ventral forebrain-associated genes, such as SLIT1, SEMA3A, DLX2/4 and GAD2. HDACi-induced expression of miR-378 and knockdown of miR-378 increases the expression of OTX2 and EMX2, which supports our hypothesis that HDACi targets forebrain markers through miR-378. In conclusion, multilineage differentiation in vitro test system is very sensitive for monitoring molecular activities relevant to in vivo neuronal developmental toxicity. Moreover, miR-378 seems to repress the expression of the OTX2 and EMX2 and therefore could be a regulator of the development of neural tube and dorsal forebrain neurons.

Maternal infection during pregnancy increases the risk of neurodevelopmental disorders in the offspring. In addition to its influence on other neuronal systems, this early-life environmental adversity has been shown to negatively affect cortical -aminobutyric acid (GABA) functions in adult life, including impaired prefrontal expression of enzymes required for GABA synthesis. The underlying molecular processes, however, remain largely unknown. In the present study, we explored whether epigenetic modifications represent a mechanism whereby maternal infection during pregnancy can induce such GABAergic impairments in the offspring. We used an established mouse model of prenatal immune challenge that is based on maternal treatment with the viral mimetic poly(I:C). We found that prenatal immune activation increased prefrontal levels of 5-methylated cytosines (5mC) and 5-hydroxymethylated cytosines (5hmC) in the promoter region of GAD1, which encodes the 67-kDa isoform of the GABA-synthesising enzyme glutamic acid decarboxylase (GAD67). The early-life challenge also increased 5mC levels at the promoter region of GAD2, which encodes the 65-kDa GAD isoform (GAD65). These effects were accompanied by elevated GAD1 and GAD2 promoter binding of methyl CpG-binding protein 2 (MeCP2) and by reduced GAD67 and GAD65 mRNA expression. Moreover, the epigenetic modifications at the GAD1 promoter correlated with prenatal infection-induced impairments in working memory and social interaction. Our study thus highlights that hypermethylation of GAD1 and GAD2 promoters may be an important molecular mechanism linking prenatal infection to presynaptic GABAergic impairments and associated behavioral and cognitive abnormalities in the offspring.