Abstract
Hypoxia is a severe stressful condition and induces cell death leading to neuronal loss both to the developing and adult nervous system. Central theme to cellular death is the activation of different classes of proteases such as caspases calpains and cathepsins. In the present study we investigated the involvement of these proteases, in the hypoxia-induced PC12 cell death. Rat PC12 is a model cell line for experimentation relevant to the nervous system and several protocols have been developed for either lethal hypoxia (oxygen and glucose deprivation OGD) or ischemic preconditioning (IPS). Nerve Growth Factor (NGF) treated PC12 differentiate to a sympathetic phenotype, expressing neurites and excitability. Lethal hypoxia was established by exposing undifferentiated and NGF-treated PC12 cells to a mixture of N2/CO2 (93:5%) in DMEM depleted of glucose and sodium pyruvate for 16 h. The involvement of caspases, calpains and lysosomal cathepsins D and E to the cell death induced by lethal OGD was investigated employing protease specific inhibitors such as z-VAD-fmk for the caspases, MDL28170 for the calpains and pepstatin A for the cathepsins D and E. Our findings show that pepstatin A provides statistically significant protection from cell death of both naive and NGF treated PC12 cells exposed to lethal OGD. We propose that apart from the established processes of apoptosis and necrosis that are integral components of lethal OGD, the activation of cathepsins D and E launches additional cell death pathways in which these proteases are key partners.
Highlights
Reduced oxygen supply to the brain causes severe adverse effects such as neurological handicaps due to neuronal cell death from energy shortage, free radicals damage, glutamate induced excitotoxicity, and launches numerous diverse cell survival and death mechanisms
Nerve Growth Factor (NGF) treated PC12 cells exposed to oxygen and glucose deprivation (OGD) showed a statistically significant reduction in the number of living cells down to 31.6%622.8, (p,0.001significance) compared to NGF differentiated PC12 cells growing in complete medium under normoxia
In this work we investigated the involvement of different classes of proteases in the cell death elicited by OGD on naive and NGF treated PC12 cells
Summary
Reduced oxygen supply to the brain causes severe adverse effects such as neurological handicaps due to neuronal cell death from energy shortage, free radicals damage, glutamate induced excitotoxicity, and launches numerous diverse cell survival and death mechanisms. OGD exposed PC12 cells reportedly suffer from mitochondrial dysfunction, oxidative stress and loss of energy that eventually lead to cell death [2,3,4]. This cell death in some cases is characterised as apoptotic [5,6] in others as necrotic [7] and in some recent reports, the process of autophagy is implicated in the cell death processes [8,9]. There are early reports indicating that in ischemia, the activation of the family of cysteine proteases, caspases, is upregulated [12]
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