RESUMEN
Owing to the presence of multiple enzymatic domains, LRRK2 has been associated with a diverse set of cellular functions and signaling pathways. It also has several pathological mutant-variants, and their incidences show ethnicity biases and drug-response differences with expression in dopaminergic-neurons and astrocytes. Here, we aimed to assess the cell-intrinsic effect of the LRRK2-I1371V mutant variant, prevalent in East Asian populations, on astrocyte yield and biology, involving Nrf2-mediated glutathione machinery, glutamate uptake and metabolism, and ATP generation in astrocytes derived from LRRK2-I1371V PD patient iPSCs and independently confirmed in LRRK2-I1371V-overexpressed U87 cells. Astrocyte yield (GFAP-immunopositive) was comparable between LRRK2-I1371V and healthy control (HC) populations; however, the astrocytic capability to mitigate oxidative stress in terms of glutathione content was significantly reduced in the mutant astrocytes, along with a reduction in the gene expression of the enzymes involved in glutathione machinery and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Simultaneously, a significant decrease in glutamate uptake was observed in LRRK2-I1371V astrocytes, with lower gene expression of glutamate transporters SLC1A2 and SLC1A3. The reduction in the protein expression of SLC1A2 was also directly confirmed. Enzymes catalyzing the generation of γ glutamyl cysteine (precursor of glutathione) from glutamate and the metabolism of glutamate to enter the Krebs cycle (α-ketoglutaric acid) were impaired, with significantly lower ATP generation in LRRK2-I1371V astrocytes. De novo glutamine synthesis via the conversion of glutamate to glutamine was also affected, indicating glutamate metabolism disorder. Our data demonstrate for the first time that the mutation in the LRRK2-I1371V allele causes significant astrocytic dysfunction with respect to Nrf2-mediated antioxidant machinery, AT -generation, and glutamate metabolism, even with comparable astrocyte yields.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Humanos , Ácido Glutámico/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Glutamina/metabolismo , Astrocitos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Enfermedad de Parkinson/metabolismo , Glutatión/metabolismo , Adenosina Trifosfato/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismoRESUMEN
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 peptide-associated 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.