Glutathione redox cycle dysregulation in Huntington’s disease knock-in striatal cells

Abstract

a b s t r a c tHuntington's disease (HD) is a CAG repeat disorder affecting the HD gene, which encodes for huntingtin (Htt) and is characterized by prominent cell death in the striatum. Oxidative stress was previously implicated in HD neurodegeneration, but the role of the major endogenous antioxidant system, the glutathione redox cycle, has been less studied following expression of full-length mutant Htt (FL-mHtt). Thus, in this work we analyzed the glutathione system in striatal cells derived from HD knock-in mice expressing mutant Htt versus wild-type cells. Mutant cells showed increased intracellular reactive oxygen species (ROS) and caspase-3 activity, which were significantly prevented following treatment with glutathione ethyl ester. Interestingly, mutant cells exhibited an increase in intracellular levels of both reduced and oxidized forms of glutathione, and enhanced activities of glutathione peroxidase (GPx) and glutathione reductase (GRed). Furthermore, glutathione-S-transferase (GST) and g-glutamyl transpeptidase (g-GT) activities were also increased in mutant cells. Nevertheless, glutamate-cysteine ligase (GCL) and glutathione synthetase (GS) activities and levels of GCL catalytic subunit were decreased in cells expressing FL-mHtt, highly suggesting decreased de novo synthesis of glutathione. Enhanced intracellular total glutathione, despite decreased synthesis, could be explained by decreased extracellular glutathione in mutant cells. This occurred concomitantly with decreased mRNA expression levels and activity of the multidrug resistance protein 1 (Mrp1), a transport protein that mediates cellular export of glutathione disulfide and glutathione conjugates. Additionally, inhibition of Mrp1 enhanced intracellular GSH in wild-type cells only. These data suggest that FL-mHtt affects the export of glutathione by decreasing the expression of Mrp1. Data further suggest that boosting of GSH-related antioxidant defense mechanisms induced by FL-mHtt is insufficient to counterbalance increased ROS formation and emergent apoptotic features in HD striatal cells.

Insulin and IGF-1 improve mitochondrial function in a PI-3K/Akt-dependent manner and reduce mitochondrial generation of reactive oxygen species in Huntington’s disease knock-in striatal cells

“…4A,B). A rise in glutathione levels in mutant cells, despite decreased GCL activity, was previously demonstrated [17]. Therefore, we tested whether insulin or IGF-1 could alter GSH and GSSG levels.…”

“…Reduced activity of SOD1 was also observed in cortex and cerebellum of post-mortem human brain tissues [7] and in erythrocytes [5] of HD patients. In a previous study we demonstrated increased ROS levels and decreased GCL activity, despite increased glutathione levels in HD striatal cells, which occurred as a result of a lower capacity of multidrug resistance-associated protein 1 (Mrp1) to export GSH to the extracellular space [17].…”

“…We previously showed that the activity of enzymes of the glutathione redox cycle were enhanced, whereas GCL and glutathione synthetase (GS) activities and protein levels of GCLc were decreased in HD mutant striatal cells [17]. Therefore, we determined the effect of insulin and IGF-1 on GCLc protein levels.…”

“…Because caspase-3 activation was previously observed in HD striatal cells [17,47,48], correlating with nuclear apoptotic features [49], we determined the effect of insulin and IGF-1 on caspase-3 activation and nuclear morphology, namely apoptotic and senescent nuclei, in mutant versus wild-type cells. Insulin (0.1 nM) significantly protected against caspase-3 activation in mutant cells ( Fig.…”

“…The levels of reduced and oxidized glutathione (GSH and GSSG, respectively) were measured as described in [17]. Briefly, striatal cells were cultured during 48h at 33ºC.…”