Ribeiro, C. A. J. | bioRxiv Science

bioRxiv2021

Methylmalonic acid impairs cell respiration and glutamate uptake in C6 rat glioma cells

Methylmalonic acidemia is an organic acidemia caused by deficient activity of L-methylmalonyl-CoA mutase or its cofactor cyanocobalamin and it is biochemically characterized by an accumulation of methylmalonic acid (MMA) in tissue and body fluids of patients. The main clinical manifestations of this disease are neurological and observable symptoms during metabolic decompensation are encephalopathy, cerebral atrophy, coma, and seizures, which commonly appear in newborns. This study aimed to investigate the toxic effects of MMA in a glial cell line presenting astrocytic features. Astroglial C6 cells were exposed to MMA (0.1-10mM) for 24 or 48 hours and cell viability, glucose consumption and oxygen consumption rate, as well as glutamate uptake and ATP content were analyzed. The possible preventive effects of bezafibrate were also evaluated. MMA significantly reduced cell viability after 48-hour period and increased glucose consumption during the same period of incubation. Regarding the energy homeostasis, MMA significantly reduced respiratory parameters of cells after 48-hour exposition, indicating that cell metabolism is compromised at resting and reserve capacity state, which might influence the cell capacity to meet energetic demands. Glutamate uptake and ATP content were also compromised after exposition to MMA, which can be influenced energy metabolism impairment, affecting the functionality of the astroglial cells. Our findings suggest that these effects could be involved in the pathophysiology of neurological dysfunction of this disease.

neuroscience↗

bioRxiv2020

Methylmalonic acid compromises mitochondrial respiration and reduces the expression of markers of differentiation in SH-SY5Y human neuroblastoma cells

Methylmalonic acidemia is a rare metabolic disorder characterized by the accumulation of methylmalonic acid (MMA) and alternatives metabolites which is caused by the deficient activity of L-methylmalonyl-CoA mutase or its cofactor 5-deoxyadenosylcobalamin (AdoCbl). The brain is one of the affected tissues by the accumulation of this metabolite in patients. The neurologic symptoms commonly appear in newborns and are clinically characterized by seizures, mental retardation, psychomotor abnormalities, and coma. The molecular mechanisms of neuropathogenesis in methylmalonic acidemia are still poorly understood, specifically regarding the impairments in neuronal development and maturation. In this study, we firstly investigated the neurotoxicity of MMA in both undifferentiated and 7-day RA-differentiated phenotypes of SH-SY5Y human neuroblastoma cells and found alterations in energetic homeostasis after the exposition to MMA. We observed an increase in glucose consumption and reduced respiratory parameters of both undifferentiated and differentiated SH-SY5Y cells after 48 hours of exposition to MMA. RA-differentiated cells slightly indicated to be more prone to perturbations in respiratory parameters by MMA than undifferentiated cells. In order to understand whether the presence of MMA during neuronal maturation could compromise this process in neuronal cells, we performed high-resolution respirometry to evaluate the mitochondria function and qPCR assay to evaluate mRNA levels of mature neuronal-specific genes in early-stage (day 3), and late-stage (day 7) of differentiation in cells co-treated with MMA 1mM during RA mediated differentiation. Our results showed that MMA compromises the respiratory parameters of routine, ATP-linked, and maximal respiration only at the late stage of differentiation as well as downregulates the transcriptional gene profile of mature neuronal markers ENO2 and SYP. Altogether, our finds point to important alterations observed during neuronal maturation and energetic stress vulnerability that can play a role in the neurological clinical symptoms at the newborn period and reveal important molecular mechanisms that could help the screening of targets to new approaches in the therapies of this disease.

biochemistry↗