Abstract
The elevated level of the amino acid metabolite homocysteine (Hcy) is known as a risk factor for ischemic stroke. The molecular mechanisms responsible for neurotoxicity of Hcy remain largely unknown in ischemic brains. The previous studies have shown that Hcy decreases the proliferation and viability of neural stem cells (NSCs) in vivo and in vitro. Autophagy is required for the maintenance of NSCs homeostasis. In the current study, we hypothesized that the toxic effect of Hcy on NSCs may involve the changes in autophagy level following cerebral ischemia/reperfusion injury. The results showed that Hcy reduced cell viability, increased LDH release, and induced nonapoptotic cell death in primary NSCs exposed to oxygen–glucose deprivation)/reoxygenation (OGD/R). Treatment with autophagy inhibitor 3-methyladenine (3MA) partly reversed the decrease in the viability and prevented LDH release triggered by Hcy combined with OGD/R. Increased punctate LC3 dots co-localizing with Nestin-stained NSCs were also observed in the subventricular zone of Hcy-treated MCAO animals, which were partially blocked by 3MA. In vitro studies further revealed that Hcy induced the formation of autophagosomes, markedly increased the expression of the autophagic markers and decreased p-ERK, p-PI3K, p-AKT, and p-mTOR levels. In addition, MHY1485, an activator of mTOR, reduced Hcy-induced increase in LC3 and Beclin 1 protein levels, meanwhile ERK and PI3K activators (TPA, curcumin for ERK and IGF-1 for PI3K, respectively) enhanced Hcy-triggered mTOR inhibition in OGD/R NSCs. Our findings suggest that Hcy may cause excessive autophagy by downregulation of both PI3K-AKT- and ERK- dependent mTOR signaling, thereby facilitates the toxicity of Hcy on NSCs in ischemic brains.
Highlights
Ischemic stroke, as a major pathological stroke type, is one of the leading causes of mortality and disability worldwide
To verify the toxic effect of Hcy on the cultured rat neural stem cells (NSCs) subjected to oxygen–glucose deprivation (OGD)/R, the cell viability was determined by the MTT assay, and cell necrosis was evaluated by the LDH-release assay
To assess the role of autophagy in Hcytreated NSCs subjected to OGD/R, the cells were treated with three different concentrations (0.05, 0.1, and 0.5 mM) of 3MA for 72 h, subsequently, cell viability and injury were evaluated by the MTT assay and LDH release
Summary
As a major pathological stroke type, is one of the leading causes of mortality and disability worldwide. Ischemic insults have been reported to trigger neurogenesis from neural stem cells (NSCs)/progenitor cells located in the subventricular zone (SVZ) and the subgranular zone (SGZ) in the dentate gyrus of adult mammalian brains, and these cells migrate to the Homocysteine (Hcy), a sulfur-containing amino acid, was derived from the methionine metabolism. Wang et al Cell Death and Disease (2019)10:561 neural cells are sensitive to prolonged HHcy treatment, and it contributes to chronic progressive neurological disease[6,7]. The neurotoxicity of Hcy involves negative regulation of NSCs viability, proliferation, and differentiation capacities[8,9]. Rabaneda et al presented in vitro and in vivo evidence demonstrating that Hcy exerted an inhibitory effect on mice adult brain neurogenesis and antiproliferative effect on basic fibroblast growth factor (bFGF)-stimulated neural progenitor cells isolated from the postnatal SVZ10. The underlying mechanism for direct neurotoxic effects of Hcy on NSCs has remained obscure
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