Clinical and animal studies have documented that hearts of the elderly are more susceptible to ischemia/reperfusion damage compared to young adults. Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins. Besides primary localization in the cytosol, Grx1 also exists in the mitochondrial intermembrane space (IMS). In contrast, Grx2 is confined to the mitochondrial matrix. Here we report that Grx1 is decreased by 50–60% in the IMS, but Grx2 is increased by 1.4–2.6 fold in the matrix of heart mitochondria from elderly rats. Determination of in situ activities of the Grx isozymes from both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria revealed that Grx1 was fully active in the IMS. However, Grx2 was mostly in an inactive form in the matrix, consistent with reversible sequestration of the active-site cysteines of two Grx2 molecules in complex with an iron–sulfur cluster. Our quantitative evaluations of the active/inactive ratio for Grx2 suggest that levels of dimeric Grx2 complex with iron–sulfur clusters are increased in SSM and IFM in the hearts of elderly rats. We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase. However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation. These findings suggest that insufficient ROS accumulates in the vicinity of dimeric Grx2 to activate it in situ.

Oxidative stress is implicated in the etiology of Parkinson's disease (PD), the second most common neurodegenerative disease. PD is treated with chronic administration of L-3,4-dihydroxyphenylalanine (Levodopa, L-DOPA), and typically increasing doses are used during progression of the disease. Paradoxically L-DOPA is a pro-oxidant and induces cell death in cellular models of PD through disruption of sulfhydryl homeostasis involving loss of the thiol-disulfide oxidoreductase functions of the glutaredoxin (Grx1) and thioredoxin (Trx1) enzyme systems (Sabens et al. 2010). Considering this loss of both Grx1 and Trx1 activities upon L-DOPA treatment, we sought to elucidate the mechanism(s) of L-DOPA induced apoptosis. In other contexts both the NFκB (nuclear factor κB) pathway and the ASK1 (apoptosis signaling kinase 1) pathway have been shown to be regulated by both Grx1 and Trx1, and both pathways have been implicated in cell death signaling in model systems of PD. Moreover, mixed lineage kinase (MLK) has been considered as a potential therapeutic target for PD. Using SHSY5Y cells as model dopaminergic neurons we found that NFκB activity was not altered by L-DOPA treatment, and the selective MLK inhibitor (CEP-1347) did not protect the cells from L-DOPA. In contrast, ASK1 was activated with L-DOPA treatment as indicated by phosphorylation of its downstream mitogen activated protein kinases (MAPK), p38 and JNK. Chemical inhibition of either p38 or JNK provided protection from L-DOPA induced apoptosis. Moreover, direct knockdown of ASK1 protected from L-DOPA induced neuronal cell death. These results identify ASK1 as the main pro-apoptotic pathway activated in response to L-DOPA treatment, implicating it as a potential target for adjunct therapy in PD.