EGb 761, a standardized extract of Ginkgo biloba leaves, has been shown to have antioxidative properties. We have previously demonstrated that EGb 761 increases stress resistance and mean life span in the model organism Caenorhabditis elegans. In this study, the molecular mechanism of EGb 761 on alleviating effects of oxidative stress is further investigated using transgenic C. elegans expressing a jellyfish green fluorescent protein (GFP)-tagged inducible small heat-shock protein gene (hsp-16-2). The expression of hsp-16-2 induced by the pro-oxidant juglone and by heat shock was significantly suppressed by 86% and 33%, respectively, in the transgenic nematode fed with EGb 761. These effects of EGb 761 correlate with its ability to increase mean survival rate of the nematode in response to acute oxidative and thermal stresses, as well as to attenuate the basal levels of hydrogen peroxide in the organism. Thus, we interpret the suppression of hsp-16-2/GFP expression as an indication that EGb 761 decreases cellular stress resulting from exogenous treatments, therefore leading to a decreased transcriptional induction of the reporter transgene. These results support the hypothesis that EGb 761 augments the natural antistress system of C. elegans, thus increasing stress resistance and life span.

Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures. The same concentrations of tyrosine-containing peptides required to prevent the nitration and apoptosis of motor neurons induced by trophic factor deprivation and of PC12 cells induced by peroxynitrite also prevented peroxynitrite-mediated nitration of motor neurons, brain homogenates, and PC12 cells. The heat shock protein 90 chaperone was nitrated in both trophic factor-deprived motor neurons and PC12 cells incubated with peroxynitrite. Tyrosine-containing peptides did not affect the induction of PC12 cell death by hydrogen peroxide. Tyrosine-containing peptides should protect by scavenging peroxynitrite-derived radicals and not by direct reactions with peroxynitrite as they neither increase the rate of peroxynitrite decomposition nor decrease the bimolecular peroxynitrite-mediated oxidation of thiols. These results reveal an important role for free radicalmediated nitration of tyrosine residues, in apoptosis induced by endogenously produced and exogenously added peroxynitrite; moreover, tyrosine-containing peptides may offer a novel strategy to neutralize the toxic effects of peroxynitrite.

Activation of the extracellular ATP ionotropic receptor P2X7 stimulates motor neuron apoptosis, whereas its inhibition in cell and animal models of amyotrophic lateral sclerosis can be protective. These observations suggest that P2X7 receptor activation is relevant to motor neuron disease and that it could be targeted for therapeutic development. Heat shock protein 90 (Hsp90) is an integral regulatory component of the P2X7 receptor complex, antagonizing ligand-induced receptor activation. Here, we show that the repressive activity of Hsp90 on P2X7 receptor activation in primary motor neurons is highly sensitive to inhibition. Primary motor neurons in culture are 100-fold more sensitive to Hsp90 inhibition by geldanamycin than other neuronal populations. Pharmacological inhibition and down-regulation of the P2X7 receptor prevented motor neuron apoptosis triggered by Hsp90 inhibition, which occurred in the absence of extracellular ATP. These observations suggest that inhibition of a seemingly motor neuron specific pool of Hsp90 leads to ligand independent activation of P2X7 receptor and motor neuron death. Downstream of Hsp90 inhibition, P2X7 receptor activated the phosphatase and tensin homolog (TPEN), which in turn suppressed the pro-survival phosphatidyl inositol 3 kinase (PI3K)/Akt pathway, leading to Fas-dependent motor neuron apoptosis. Conditions altering the interaction between P2X7 receptor and Hsp90, such as recruitment of Hsp90 to other subcellular compartments under stress conditions, or nitration following oxidative stress can induce motor neuron death. These findings may have broad implications in neurodegenerative disorders, including amyotrophic lateral sclerosis, in which activation of P2X7 receptor may be involved in both autonomous and non-autonomous motor neurons death. Impact statement Here we show that a motor neuron specific pool of Hsp90 that is highly sensitive to geldanamycin inhibition represses ligand-independent activation of P2X7 receptor and is critical to motor neuron survival. Activation of P2X7 receptor by Hsp90 inhibition triggers motor neuron apoptosis through the activation of PTEN, which in turn inhibits the PI3 kinase/Akt survival pathway. Thus, inhibition of Hsp90 for therapeutic applications may have the unexpected negative consequence of decreasing the activity of trophic pathways in motor neurons. The inhibition of Hsp90 as a therapeutic approach may require the identification of the Hsp90 complexes involved in pathogenic processes and the development of inhibitors selective for these complexes.