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Methylmalonic acidemia is a rare metabolic disorder caused by the deficient activity of L-methylmalonyl-CoA mutase or its cofactor 5-deoxyadenosylcobalamin and is characterized by accumulation of methylmalonic acid (MMA) and alternative metabolites. The brain is one of the most affected tissues and neurologic symptoms, characterized by seizures, mental retardation, psychomotor abnormalities, and coma, commonly appear in newborns. The molecular mechanisms of neuropathogenesis in methylmalonic acidemia are still poorly understood, specifically regarding the impairments in neuronal development, maturation, and differentiation. In this study, we investigated the effects of MMA in both undifferentiated and differentiated phenotypes of SH-SY5Y human neuroblastoma cells. We observed an increase in glucose consumption and reduction in respiratory parameters of both undifferentiated and differentiated cells after exposition to MMA, suggesting that differentiated cells are slightly more prone to perturbations in respiratory parameters by MMA than undifferentiated cells. Next, we performed qPCR of mature neuronal-specific gene markers and measured mitochondrial functioning to evaluate the role of MMA during differentiation. Our results showed that MMA impairs the respiratory parameters only at the late stage of differentiation and downregulates the transcriptional gene profile of mature neuronal markers neuron-specific enolase (ENO2) and synaptophysin (SYP). Altogether, our findings point out important changes 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.

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The data presented here is related to negative results obtained with the recombinant expression of chitinase from four species of Leishmania parasites in two expression systems, performed in order to investigate the molecular characteristics of the Leishmania chitinase and its possible application in leishmaniasis diagnosis. Thus, heterologous Leishmania sp chitinase proteins were expressed in bacteria using the prokaryotic expression vector pET28a and Escherichia coli Mach-T1, and in Spodoptera frugiperda (Sf9) insect cells, using the eukaryotic bac-to-bac expression system (Thermo Fisher Scientific) to produce recombinant baculoviruses to infect Sf9. Biochemical and cellular analysis of the various recombinant forms of the Leishmania sp chitinase produced in prokaryotic and eukaryotic expression systems were performed through SDS-PAGE and Western blotting. Chitinase produced and purified from bacteria presented low yield and formed inactive aggregates. Heterologous chitinase obtained after infection of Sf9 insect cells with all the four Leishmania species recombinant baculoviruses presented high yield of insoluble proteins. Dot-blot serological tests presented inconclusive results against the recombinant Leishmania sp chitinases produced in both expression systems. The experiments described in this paper can help researchers to avoid errors when choosing a recombinant expression systems to produce Leishmania parasites proteins for biotechnological purposes.

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