Aberrant Rab11-Dependent Trafficking of the Neuronal Glutamate Transporter EAAC1 Causes Oxidative Stress and Cell Death in Huntington's Disease

Abstract

Oxidative stress contributes to neurodegeneration in Huntington's disease (HD). However, the origins of oxidative stress in HD remain unclear. Studies in HD transgenic models suggest involvement of mitochondrial dysfunction, which would lead to overproduction of reactive oxygen species (ROS). Impaired mitochondria complexes occur in late stages of HD but not in presymptomatic or early-stage HD patients. Thus, other mechanisms may account for the earliest source of oxidative stress caused by endogenous mutant huntingtin. Here, we report that decreased levels of a major intracellular antioxidant glutathione coincide with accumulation of ROS in primary HD neurons prepared from embryos of HD knock-in mice (HD 140Q/140Q ), which have human huntingtin exon 1 with 140 CAG repeats inserted into the endogenous mouse huntingtin gene. Uptake of extracellular cysteine through the glutamate/cysteine transporter EAAC1 is required for de novo synthesis of glutathione in neurons. We found that, compared with wild-type neurons, HD neurons had lower cell surface levels of EAAC1 and were deficient in taking up cysteine. Constitutive trafficking of EAAC1 from recycling endosomes relies on Rab11 activity, which is defective in the brain of HD 140Q/140Q mice. Enhancement of Rab11 activity by expression of a dominant-active Rab11 mutant in primary HD neurons ameliorated the deficit in cysteine uptake, increased levels of intracellular glutathione, normalized clearance of ROS, and improved neuronal survival. Our data support a novel mechanism for oxidative stress in HD: Rab11 dysfunction slows trafficking of EAAC1 to the cell surface and impairs cysteine uptake, thereby leading to deficient synthesis of glutathione.

Protein Interacting with C-Kinase 1 Deficiency Impairs Glutathione Synthesis and Increases Oxidative Stress via Reduction of Surface Excitatory Amino Acid Carrier 1

“…Neurons (DIV9) were used for measuring ROS as described previously (Valencia and Morán, 2001;Li et al, 2010). Neurons were incubated with 10 M DCFDA at 37°C for 30 min.…”

“…All parameters used in confocal microscopy, including the laser excitation power, detector and offset gains, and the pinhole diameter, were consistent in each experiment. The measurement of DCF imaging was according to Li et al (2010). In brief, each field with at least two neurons was taken for imaging.…”

“…L-[ 14 C]cysteine (20 Ci/ml; PerkinElmer Life and Analytical Sciences) uptake was performed at room temperature for 30 s in PBS containing 1% BSA according to previous work (Li et al, 2010). The final concentrations of L-[…”

“…Using DCFDA, a cell-permeable nonfluorescent dye that is oxidized into green fluorescent DCF in the presence of oxidants (Kirkland et al, 2007;Li et al, 2010), two experiments were undertaken to measure ROS level in PICK1-deficient neurons. First, cortical neurons from WT and PICK1 Ϫ/Ϫ mice were cultured and labeled with DCFDA at DIV9.…”

“…To detect directly the cysteine transport, the uptake of radiolabeled cysteine was undertaken according to previous work (Li et al, 2010). As shown in Figure 6E, cultured cortical neurons (DIV9) from WT and PICK1 Ϫ/Ϫ mice took up cysteine with similar rates in the presence of low concentrations of L-cysteine (0.1, , and ␤-actin (actin) in 4-month-old WT and PICK1 Ϫ/Ϫ (KO) mice.…”