Person:

Michel, Thomas

Nitric oxide (NO) and hydrogen peroxide (H(_2)O(_2)) play key roles in physiological and pathological responses in cardiac myocytes. The mechanisms whereby H(_2)O(_2)–modulated phosphorylation pathways regulate the endothelial isoform of nitric oxide synthase (eNOS) in these cells are incompletely understood. We show here that H(_2)O(_2) treatment of adult mouse cardiac myocytes leads to increases in intracellular Ca(^{2+}) ([Ca(^{2+})](_i)), and document that activity of the L-type Ca(^{2+}) channel is necessary for the H(_2)O(_2)-promoted increase in sarcomere shortening and of [Ca(^{2+})](_i). Using the chemical NO sensor Cu(_2)(FL2E), we discovered that the H(_2)O(_2)-promoted increase in cardiac myocyte NO synthesis requires activation of the L-type Ca(^{2+}) channel, as well as phosphorylation of the AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase kinase 1/2 (MEK1/2). Moreover, H(_2)O(_2)-stimulated phosphorylations of eNOS, AMPK, MEK1/2, and ERK1/2 all depend on both an increase in [Ca(^{2+})](_i) as well as the activation of protein kinase C (PKC). We also found that H(_2)O(_2)-promoted cardiac myocyte eNOS translocation from peripheral membranes to internal sites is abrogated by the L-type Ca(^{2+}) channel blocker nifedipine. We have previously shown that kinase Akt is also involved in H(_2)O(_2)-promoted eNOS phosphorylation. Here we present evidence documenting that H(_2)O(_2)-promoted Akt phosphorylation is dependent on activation of the L-type Ca(^{2+})channel, but is independent of PKC. These studies establish key roles for Ca(^{2+})- and PKC-dependent signaling pathways in the modulation of cardiac myocyte eNOS activation by H(_2)O(_2).

Glia play critical roles in maintaining the structure and function of the nervous system; however, the specific contribution that astroglia make to neurodegeneration in human disease states remains largely undefined. Here we use Alexander disease, a serious degenerative neurological disorder caused by astrocyte dysfunction, to identify glial-derived NO as a signalling molecule triggering astrocyte-mediated neuronal degeneration. We further find that NO acts through cGMP signalling in neurons to promote cell death. Glial cells themselves also degenerate, via the DNA damage response and p53. Our findings thus define a specific mechanism for glial-induced non-cell autonomous neuronal cell death, and identify a potential therapeutic target for reducing cellular toxicity in Alexander disease, and possibly other neurodegenerative disorders with glial dysfunction.

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules. Caveolin-1null mice have marked metabolic abnormalities, yet the underlying molecular mechanisms are incompletely understood. We found the redox stress plasma biomarker plasma 8-isoprostane was elevated in caveolin-1null mice, and discovered that siRNA-mediated caveolin-1 knockdown in endothelial cells promoted significant increases in intracellular H2O2. Mitochondrial ROS production was increased in endothelial cells after caveolin-1 knockdown; 2-deoxy-D-glucose attenuated this increase, implicating caveolin-1 in control of glycolytic pathways. We performed unbiased metabolomic characterizations of endothelial cell lysates following caveolin-1 knockdown, and discovered strikingly increased levels (up to 30-fold) of cellular dipeptides, consistent with autophagy activation. Metabolomic analyses revealed that caveolin-1 knockdown led to a decrease in glycolytic intermediates, accompanied by an increase in fatty acids, suggesting a metabolic switch. Taken together, these results establish that caveolin-1 plays a central role in regulation of oxidative stress, metabolic switching, and autophagy in the endothelium, and may represent a critical target in cardiovascular diseases.