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| Author(s): |
Daniela Rodrigues de Melo
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Campinas, SP. |
| Institution: | Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas |
| Defense date: | 2012-02-17 |
| Examining board members: |
Roger Frigério Castilho;
Leonardo dos Reis Silveira;
Mario Henrique de Barros;
Marcondes Cavalcante Franca Junior;
Rodrigo Ramos Catharino
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| Advisor: | Roger Frigério Castilho |
| Abstract | |
Methylmalonic acidemia is an inherited metabolic disorder involving a deficiency in the activity of the enzyme methylmalonyl-CoA mutase or its cofactor 5'-deoxyadenosylcobalamin that results in an accumulation of methylmalonate (MMA) in the body. There is evidence that MMA impairs mitochondrial oxidative metabolism, leading to neurodegeneration. In this study we evaluated the in vitro effect of MMA on oxygen consumption by isolated rat brain mitochondria in the presence of different respiratory chain substrates. MMA (1-10 mM) strongly inhibited glutamate-supported and succinatesupported respiration. We confirmed that the inhibitory effect of MMA on succinatesupported respiration is due to inhibition of the mitochondrial dicarboxylate transporter by MMA, blocking succinate uptake by mitochondria. Glutamate transport measurements revealed that the MMA effect on glutamate-supported respiration is not due to inhibition of mitochondrial uptake of this substrate. While MMA showed a weak inhibitory effect on glutamate dehydrogenase and aspartate transaminase, _'alfa'-ketoglutarate dehydrogenase activity was significantly inhibited by MMA (Ki = 3.65 mM). 'alfa'-ketoglutarate transport measurements showed that an exchange can take place between extramitochondrial MMA and intramitochondrial 'alfa'-ketoglutarate, depleting this substrate and consequently causing inhibition of glutamate-supported respiration. We observed that isolated brain organelles can accumulate nearly three times the concentration of extramitochondrial MMA. In addition, MMA inhibition of respiration by diced rat brain tissue was partially prevented by malate. MMA effects in vivo were studied by measuring respiration and reactive oxygen species generation in isolated brain mitochondria from young rats chronically injected (ip, 15 d) with MMA. No differences were observed between control and MMA-treated samples, indicating that in vivo MMA treatment does not lead to permanent mitochondrial dysfunction. In addition, a study into the effect of MMA on the viability and metabolism of neuronal and glial cells was carried out. In the PC12 neuronal tumor cell line, MMA decreased cell viability after 24 hours of treatment. Mitochondrial membrane potential and respiration were evaluated after 7 hours of MMA treatment. MMA inhibited respiration in intact cells but did not alter respiration in permeabilized cells, where there is no substrate deprivation. Cell viability of U-87MG human glioblastoma cells was not affected by MMA treatment. However, in cells from a primary culture of rat cerebral astrocytes, viability and cell area were significantly reduced and morphological alterations were also noted. The most evident effects of MMA were observed in primary cells, suggesting that tumor cells are more resistant to the deleterious effects of MMA. Taken together, these results indicate that the inhibitory effect of MMA on mitochondrial oxidative metabolism can be ascribed to the concurrent inhibition of specific enzymes and transporters, limiting the availability of substrates for mitochondrial metabolic pathways (AU) | |