Gateways for Glutamate Neuroprotection in Parkinson’s Disease (PD): Essential Role of EAAT3 and NCX1 Revealed in an In Vitro Model of PD

Abstract

Increasing evidence suggests that metabolic alterations may be etiologically linked to neurodegenerative disorders such as Parkinson’s disease (PD) and in particular empathizes the possibility of targeting mitochondrial dysfunctions to improve PD progression. Under different pathological conditions (i.e., cardiac and neuronal ischemia/reperfusion injury), we showed that supplementation of energetic substrates like glutamate exerts a protective role by preserving mitochondrial functions and enhancing ATP synthesis through a mechanism involving the Na+-dependent excitatory amino acid transporters (EAATs) and the Na+/Ca2+ exchanger (NCX). In this study, we investigated whether a similar approach aimed at promoting glutamate metabolism would be also beneficial against cell damage in an in vitro PD-like model. In retinoic acid (RA)-differentiated SH-SY5Y cells challenged with α-synuclein (α-syn) plus rotenone (Rot), glutamate significantly improved cell viability by increasing ATP levels, reducing oxidative damage and cytosolic and mitochondrial Ca2+ overload. Glutamate benefits were strikingly lost when either EAAT3 or NCX1 expression was knocked down by RNA silencing. Overall, our results open the possibility of targeting EAAT3/NCX1 functions to limit PD pathology by simultaneously favoring glutamate uptake and metabolic use in dopaminergic neurons.

The Neuroprotective Effect of L-Carnitine against Glyceraldehyde-Induced Metabolic Impairment: Possible Implications in Alzheimer’s Disease

“…We recently reported that in different neuropathological settings, including AD, mitochondrial functions could be retained by supplying alternative substrates (e.g., glutamate) [15,40]. Glutamate could be used by neuronal cells to compensate for energy deficits and, at the same time, to scavenge for ROS, thus improving mitochondrial performance by acting at different sites [15,40].…”

“…Both SH-SY5Y and rat primary cortical neurons were plated on 12-well plates, and at the end of the experimental procedure, the cells were incubated with 0.5 mL of MTT solution (0.5 mg/mL in PBS) in the dark at 37 • C and a 5% CO 2 atmosphere in a humidified incubator. After 1 h, the cells were washed with PBS, and the produced formazan crystals were dissolved in 0.5 mL of DMSO [15,40]. A decrease in mitochondrial activity resulted in a reduction in the amount of formazan produced and therefore in the absorbance value.…”

“…The intracellular ATP content was evaluated by using a commercially available luciferase-luciferin system (ATPlite, Perkin Elmer) as previously described [15,40]. Briefly, SH-SY5Y cells were plated on a 96-well "View Plate" (Perkin Elmer), subjected to specific treatments in culture medium and then analyzed for the ATP content according to the manufacturer's protocol.…”

“…Specific evaluation of the mitochondrial ROS levels was performed by using Mito-Tracker CM-H2XRos (Invitrogen Life Technologies, Carlsbad, CA, USA) as previously described [15,40]. Briefly, cells were plated on coverslips (coated with poly-D-lysine for rat primary cortical neurons) and subjected to specific treatments.…”

“…In this experimental setting, we found that LC exposure induced a significant increase in the intracellular ATP content (Figure 2A,B). Interestingly, LC-induced ATP generation completely relied on the oxidative phosphorylation process, since, in the presence of the ATP synthase inhibitor oligomycin (3 µg/mL) [15,40,42], ATP production was completely abolished (Figure 2A,B). When cells were pretreated with LC for 1 h and then exposed to GA for 24 h (without removing LC), the decrease in the ATP levels induced by the GA challenge was significantly blunted.…”