Activation of Cell Death Mediated by the Crosstalk between Caspase‐3 and Apoptosis Inducing Factor in the Brainstem upon Exposure to Monosodium Glutamate and Corticosterone

Abstract

Cellular stress induced by excessive glutamate or corticosterone leads to apoptosis. Our previous studies showed that Monosodium Glutamate (MSG) and Corticosterone (CORT) potentially activated the neuroendocrine stress axis and induced microvascular disruption, neuronal and glial cell apoptosis. The present study tested the hypothesis that whether specific death programs were activated according to cell types and further delineated the intracellular mechanisms involved in the apoptotic signaling cascade in the brain stem. We used glutamate and CORT paradigms to test this hypothesis. Young adult (2 months old) male Wistar rats were injected subcutaneously with MSG or CORT for 7 days and sacrificed on 8th, 15th and 30th days of the treatment to monitor recovery responses after 1, 8 and 23 days. Brain stem (BS) was used for expression of Glucocorticoid receptor (Nr3c1) and Caspase-3 by qPCR protocols. MSG upregulated Nr3c1 expression on day 8 and 30, and downregulated it on day 15, though not significant. The CORT treatment showed downregulation of Nr3c1 expression across the 3 time points, significant only on day 8. In the MSG treated group, Caspase-3 expression displayed an insignificant upregulation on day 8 and 30, and an insignificant downregulation on day 15. The CORT treatment displayed downregulated Caspase-3 expression on day 8 and 30 and an upregulation on day 15 though not significant. Immunohistochemical staining of the brain stem showed an increase in Caspase-3 immunoreactivity in microvasculature, and neuron and glia-like cells in both MSG and CORT groups. Cells from control rats were with either no signal or less immunoreactivity. Since the Caspase-3 apoptotic marker gene was not activated in the BS, there is a possibility that cell death in the BS might be mediated by a caspase-independent apoptotic pathway involving apoptosis inducing factor (AIF) protein. The mitochondrial AIF protein is an important cell death effector upon cellular stress and is redistributed from the mitochondria to the nucleus to function as a proapoptotic factor mostly independent of caspase activity. Immunolabeling with anti-Caspase-3 and anti-AIF showed that AIF was colocalized with Caspase-3 in neuron and glia-like cells that underwent apoptosis, whereas the AIF labeling was not detected in microvasculature of the BS upon exposure to MSG and CORT. No signal was detected in control groups. The results suggest that microvessels may undergo disruption through a spontaneous active caspase-3 dependent mechanism whereas neurons or glial cells undergo AIF mediated caspase-3 apoptosis execution pathway. The protein levels of AIF and Caspase-3 in cytosol, mitochondria, nucleus and whole cell lysates were detected by Western blot analysis. Our study is the first to report that multiple and distinctive death programs involving only Caspase-3 dependent microvascular disruption and AIF - Caspase-3 mediated neuronal and glial cell death occur in the BS. Furthermore potential disruption in blood-brain barrier integrity and neural connectivity occurs upon exposure to MSG and CORT.