Impact of monomeric and aggregated wild‐type and A30P/A53T double‐mutant α‐synuclein on antioxidant mechanism and glutamate metabolic profile of cultured astrocytes

Abstract

Serving as a source of glutathione and up-taking and metabolizing glutamate are the primary supportive role of astrocytes for the adjacent neurons. Despite the clear physical association between astrocytes and α-synuclein, the effect of extracellular α-synuclein on these astrocytic functions has not yet been elucidated. Hence, we aim to assess the effect of various forms of α-synuclein on antioxidant mechanism and glutamate metabolism. Wild-type and A53T/A30P double-mutant α-synuclein, both in monomeric and aggregated forms, were added extracellularly to media of midbrain rat astrocyte culture, with their survival, oxidative, and nitrative stress, glutathione and glutamate content, expression of enzymes associated with oxidative stress and glutamate metabolism, glutamate and glutathione transporters being assessed along with the association/engulfment of these peptides by astrocytes. A30P/A53T peptide associated more with astrocytes, and low-extracellular K + concentration showed prominent reduction in the engulfment of the monomeric forms, suggesting that the association of the aggregated forms was greater with the membrane. The peptideassociated astrocytes showed lower survival and increased oxidative stress generation, owing to the decrease in nuclear localization of Nrf2 and increase in iNOS, and further aggravated by the decrease in glutathione content and related enzymes like glutathione synthetase, glutathione peroxidase, and glutathione reductase. Glutamate uptake increased in aggregate-treated cells due to the increase in GLAST1 expression, de novo synthesis of glutamate by pyruvate carboxylase, and/or glutamine synthase, bolstered by the differential glutamate dehydrogenase enzyme activity. We thus show for the first time that extracellular α-synuclein exposure leads to astrocytic dysfunction with respect to the antioxidant mechanism and glutamate metabolic profile. The impact was higher in the case of the aggregated and mutated peptide, with the highest dysfunction for the mutant aggregated α-synuclein treatment.

Astrocytes Differentiated from LRRK2-I1371V Parkinson’s-Disease-Induced Pluripotent Stem Cells Exhibit Similar Yield but Cell-Intrinsic Dysfunction in Glutamate Uptake and Metabolism, ATP Generation, and Nrf2-Mediated Glutathione Machinery

“…Light scatter for 10,000 gated events was used to identify cells. Secondary-antibody-stained astrocytes were used for gating (red line) out nonspecific staining and are represented by the shaded peaks in the flow cytometry histograms [ 23 ].…”

“…The protocol procedure was similar to that of earlier studies [ 23 , 49 ]. Briefly, astrocytes were treated with 100 mM glutamate for 2 h, and their glutamate contents were assessed using a Glutamic Acid Colorimetric Assay Kit per the manufacturer’s instructions.…”

“…The conversion of glutamate to α-ketoglutarate, with the reduction of NAD to NADH, emits fluorescence when excited with UV light (NADH). The GDH enzyme kinetics were studied using a previously reported procedure [ 23 , 50 ]. Astrocytes on a 22 mm coverslip were immersed in Hank’s balanced salt solution (HBSS, pH 7.4), with the addition of GDH (76 I.U./mL) and NAD (1 mM).…”

“…Autopsy data from PD patients have shown the presence of dystrophic reactive astrocytes. Reactive astrocytosis can lead to adverse effects on DA neurons through increased oxidative stress, decreased neurotrophic factor release, increased inflammation, or enhanced glutamate release, leading to excitotoxicity [ 21 , 22 , 23 , 24 , 25 ]. It has been shown that glutamate excitotoxicity leads to the loss of DA neurons and simultaneous motor dysfunction in PD [ 26 , 27 ], highlighting the function of healthy niche cells.…”

“…Unlike reports on diseases that are neurodegenerative in nature involving basal ganglia, such as multiple system atrophy and supranuclear palsy, postmortem studies of PD patients have reported lower levels of reduced glutathione in the substantia nigra (SN [ 32 ]). Researchers have now involved patient-derived cells to understand whether this impairment of glutamate uptake and metabolism or glutathione content is a cell-intrinsic factor in astrocytes derived from PD patients carrying familial mutations or whether this is due to oxidative stress [ 33 ] or extracellular α-synuclein aggregates [ 23 ] present during disease progression.…”