Quantitative Immunoblotting Analyses Reveal that the Abundance of Actin, Tubulin, Synaptophysin and EEA1 Proteins is Altered in the Brains of Aged Mice.

Optimal synaptic activity is essential for cognitive function, including memory and learning. Evidence indicates that cognitive decline in elderly individuals is associated with altered synaptic function. However, the impact of aging on the expression of neurotransmitter receptors and accessory proteins in brain synapses remains unclear. To fill this knowledge gap, we investigated the effect of aging on the mouse brain by utilizing a subcellular brain tissue fractionation procedure to measure protein abundance using quantitative Western Blotting. Comparing 7-month- (control) and 22-month- (aged) old mouse tissue, no significant differences were identified in the levels of AMPA receptor subunits between the experimental groups. The abundance of GluN2B NMDA receptor subunits decreased in aged mice, whereas the levels of GluN2A did not change. The analysis of cytoskeletal proteins showed an altered level of actin and tubulin in aged mice while PSD-95 protein did not change. Vesicle protein analysis revealed that synaptophysin abundance is decreased in older brains whereas EEA1 was significantly increased. Thus, our results suggest that physiological aging profoundly impacts the abundance of molecules associated with neurotransmitter release and vesicle cycling, proteins implicated in cognitive function.

Inhibition of autophagy sensitises cells to hydrogen peroxide-induced apoptosis: Protective effect of mild thermotolerance acquired at 40°C.

Various toxic compounds produce reactive oxygen species, resulting in oxidative stress that threatens cellular homeostasis. Yet, lower doses of stress can stimulate defence systems allowing cell survival, whereas intense stress activates cell death pathways such as apoptosis. Mild thermal stress (40°C, 3h) induces thermotolerance, an adaptive survival response that renders cells less sensitive to subsequent toxic stress, by activating defence systems like heat shock proteins, antioxidants, anti-apoptotic and ER-stress factors. This study aims to understand how autophagy and apoptosis are regulated in response to different doses of H2O2, and whether mild thermotolerance can protect cervical carcinoma cells against apoptosis by stimulating autophagy. Autophagy was monitored through Beclin-1 and LC3 expression and acid compartment activity, whereas apoptosis was tracked by caspase activity and chromatin condensation. Exposure of HeLa and C33 A cells to H2O2 for shorter times (15-30min) transiently induced autophagy; apoptosis was activated after longer times (1-3h). Mild thermotolerance at 40°C enhanced activation of autophagy by H2O2. Disruption of autophagy using bafilomycin A1 and 3-methyladenine sensitised cells to apoptosis induced by H2O2, in non-thermotolerant cells and, to a lesser extent, in thermotolerant cells. Inhibition of autophagy enhanced apoptosis through the mitochondrial, death receptor and endoplasmic reticulum pathways. Autophagy was activated by lower doses of stress and protects cells against apoptosis induced by higher doses of H2O2. This work improves understanding of mechanisms that might be involved in toxicity of various compounds and could eventually lead to protective strategies against deleterious effects of toxic compounds.