Ascorbate prevents cell death from prolonged exposure to glutamate in an in vitro model of human dopaminergic neurons

Abstract

Ascorbate (vitamin C) is a nonenzymatic antioxidant highly concentrated in the brain. In addition to mediating redox balance, ascorbate is linked to glutamate neurotransmission in the striatum, where it renders neuroprotection against excessive glutamate stimulation. Oxidative stress and glutamatergic overactivity are key biochemical features accompanying the loss of dopaminergic neurons in the substantia nigra that characterizes Parkinson's disease (PD). At present, it is not clear whether antiglutamate agents and ascorbate might be neuroprotective agents for PD. Thus, we tested whether ascorbate can prevent cell death from prolonged exposure to glutamate using dopaminergic neurons of human origin. To this purpose, dopamine-like neurons were obtained by differentiation of SH-SY5Y cells and then cultured for 4 days without antioxidant (antiaging) protection to evaluate glutamate toxicity and ascorbate protection as a model system of potential factors contributing to dopaminergic neuron death in PD. Glutamate dose dependently induced toxicity in dopaminergic cells largely by the stimulation of AMPA and metabotropic receptors and to a lesser extent by N-methyl-D-aspartate and kainate receptors. At relatively physiological levels of extracellular concentration, ascorbate protected cells against glutamate excitotoxicity. This neuroprotection apparently relies on the inhibition of oxidative stress, because ascorbate prevented the pro-oxidant action of the scavenging molecule quercetin, which occurred over the course of prolonged exposure, as is also seen with glutamate. Our findings show the relevance of ascorbate as a neuroprotective agent and emphasize an often underappreciated role of oxidative stress in glutamate excitotoxicity. Occurrence of a glutamate-ascorbate link in dopaminergic neurons may explain previous contradictions regarding their putative role in PD.

The SH-SY5Y cell line in Parkinson’s disease research: a systematic review

“…Additional protocols used for differentiation may involve combinations of the above-mentioned methods, or a combination of 10 μM RA and 0.3-5 mM dibutyryl cyclic adenosine monophosphate (dbcAMP) [39, 40], or of 10 μM RA for 3 days and 80nM tissue plasminogen activator [41] or the protocol was not specified. Differentiation may also be caused by 200 ng/mL growth/differentiation factor 5 (GDF5) [42], recombinant bone morphogenetic protein 2 (BMP2) [42], staurosporine [43, 44] or 50 ng/mL glial cell line-derived neurotrophic factor (GDNF) [45]. The pros and cons of the differentiation of the SH-SY5Y cell line to obtain a relevant model for PD have been reviewed more extensively elsewhere [46].…”

“…One complementary strategy to study PD in cells is to interfere directly with one of these processes by administering specific compounds, with agonistic or antagonistic activity, such as hydrogen peroxide (oxidative stress), lactacystin/MG-123 (proteasome inhibitors), tunicamycin (N-glycosylation inhibitor, triggers ER stress), bafilomycin (inhibitor of vacuolar H + ATPase, leading to autophagy dysfunction), thapsigargin (inhibitor of the sarco/endoplasmic reticulum Ca 2 + ATPase, resulting in ER stress and autophagy inhibition), carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (inhibitor of oxidative phosphorylation, leading to mitochondrial dysfunction), Conduritol B epoxide (CBE) (GBA inhibitor), or salsolinol/staurosporine (cell death). Intriguingly, staurosporine, a broad-spectrum kinase inhibitor, has been used in some PD-related publications to induce cell death [64–72], while other publications have used it to induce DAergic differentiation and study PD-related features [43, 44]. Early studies on SH-SY5Y cells showed differentiation towards a neuronal phenotype upon treatment with staurosporine [73, 74], which later has been characterized as catecholaminergic-like [75].…”

“…Up to now, 14 genes have been consistently associated with familial PD ( red circles ; [38]). The analysis of the functions of the corresponding (mutated) proteins and the resulting cellular abnormalities has allowed the identification of the depicted main pathways underlying PD: mitochondrial, proteasomal and autophagy dysfunction, protein aggregation, dopamine metabolism and oxidative stress, leading to DAergic cell death [39–44]. Green circles : compounds dysregulating multiple cellular processes linked to PD and used to mimic PD with dotted lines pointing towards their targets.…”

Mitochondrial dysfunction in the APP/PSEN1 mouse model of Alzheimer’s disease and a novel protective role for ascorbate

“…A meta-analysis evaluating oxidative stress biomarkers confirmed decreased antioxidant capacity in the plasma of patients with Alzheimer’s disease, suggesting that antioxidant defenses are consumed more rapidly in these individuals [32]. Animal and cell culture studies offer additional evidence that supplementation with ASC not only ameliorates oxidative stress, but also mitigates the production of Aβ [33–37]. Previously published data from our group show a significant increase in soluble Aβ 1–42 and Aβ 1–40 peptides in cortical tissue from SVCT2 +/− ; APP/PSEN1 mice at 6 months compared with APP/PSEN1 mice that have normal SVCT2 transporter expression, indicating that compromised ASC capacity increases the momentum of Alzheimer’s disease pathological processes [38].…”

Antioxidants as a preventive therapeutic option for age related neurodegenerative diseases

“…Clinical assessments for use of vitamin C are less in comparison to use of tocopherol. In vitro study has also suggested the protective effect of vitamin A in AD pathology as it could offer destabilizing effects to amyloid fibrils [145] . The use of carotenoid has also been suggested by Zhang et al (2002) [146] in PD pathology.…”