Methylmalonate Impairs Mitochondrial Respiration Supported by NADH-Linked Substrates: Involvement of Mitochondrial Glutamate Metabolism Daniela R. Melo, 1 Sandra R. Mirandola, 1 Nilson A. Assunc ¸a ˜o, 2 and Roger F. Castilho 1 * 1 Departamento de Patologia Clı ´nica, Faculdade de Cie ˆncias Me ´dicas, Universidade Estadual de Campinas, Campinas, Sa ˜o Paulo, Brazil 2 Instituto de Cie ˆncias Ambientais, Quı ´mica e Farmace ˆutica, Departamento de Cie ˆncias Exatas e da Terra, Universidade Federal de Sa ˜o Paulo, Diadema, Sa ˜o Paulo, Brazil The neurodegeneration that occurs in methylmalonic acidemia is proposed to be associated with impair- ment of mitochondrial oxidative metabolism resulting from methylmalonate (MMA) accumulation. The pres- ent study evaluated the effects of MMA on oxygen consumption by isolated rat brain mitochondria in the presence of NADH-linked substrates (a-ketoglutarate, citrate, isocitrate, glutamate, malate, and pyruvate). Respiration supported either by glutamate or gluta- mate plus malate was significantly inhibited by MMA (1–10 mM), whereas no inhibition was observed when a cocktail of NADH-linked substrates was used. Measurements of glutamate transport revealed that the inhibitory effect of MMA on respiration maintained by this substrate is not due to inhibition of its mito- chondrial uptake. In light of this result, the effect of MMA on the activity of relevant enzymes involved in mitochondrial glutamate metabolism was investigated. MMA had minor inhibitory effects on glutamate dehy- drogenase and aspartate aminotransferase, whereas a-ketoglutarate dehydrogenase was significantly inhibited by this metabolite (K i 5 3.65 mM). More- over, measurements of a-ketoglutarate transport and mitochondrial MMA accumulation indicated that MMA/a-ketoglutarate exchange depletes mitochon- dria from this substrate, which may further contribute to the inhibition of glutamate-sustained respiration. To study the effect of chronic in vivo MMA treatment on mitochondrial function, young rats were intraperitone- ally injected with MMA. No significant difference was observed in respiration between isolated brain mitochondria from control and MMA-treated rats, indicating that in vivo MMA treatment did not lead to permanent mitochondrial respiratory defects. Taken together, these findings indicate that the inhibitory effect of MMA on mitochondrial oxidative metabolism can be ascribed to concurrent inhibition of specific enzymes and lower availability of respiratory sub- strates. V V C 2012 Wiley Periodicals, Inc. Key words: brain mitochondria; glutamate; methylmalonic acidemia; neurodegeneration; oxidative metabolism Methylmalonic acidemia is a group of inherited disorders affecting catabolic pathways of the branched chain amino acids, thymine, odd carbon number fatty acids, and the side chain of cholesterol. Methylmalonic acidemia is usually caused by mutations in the MUT gene that lead to partial (mut – ) or complete (mut8) defi- ciency in the activity of methylmalonyl-CoA mutase (MCM), a mitochondrial enzyme that catalyses the con- version of L-methylmalonyl-CoA to succinyl-CoA (Fenton et al., 2001; Tanpaiboon, 2005; Venditti, 2005). Defects in the uptake of or steps involved in the process- ing of cobalamin, the precursor of the cofactor for MCM activity 5 0 -deoxyadenosyl cobalamin (AdoCbl), can also result in methylmalonic acidemia. Hypomyeli- nation, cerebral atrophy, and neurodegeneration with basal ganglia lesions affecting mainly the globus pallidus are common neurological features of the disease (Brismar and Ozand, 1994; Harting et al., 2008). Much evidence indicates that oxidative metabolism dysfunction is involved in the pathophysiology of methylmalonic acidemia (for reviews see Wajner and Coelho, 1997; Sandra R. Mirandola’s current address is German Center for Neurodege- nerative Diseases, Bonn, Germany *Correspondence to: Dr. Roger F. Castilho, Departamento de Patologia Clı ´nica, Faculdade de Cie ˆncias Me ´dicas, Universidade Estadual de Cam- pinas, Campinas, SP 13083-887, Brazil. E-mail: roger@fcm.unicamp.br Contract grant sponsor: Sa ˜o Paulo Research Foundation (FAPESP); Contract grant sponsor: Brazilian National Council for Scientific and Technological Development (CNPq). Received 9 September 2011; Revised 28 November 2011; Accepted 8 December 2011 Published online 20 February 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jnr.23020 Journal of Neuroscience Research 90:1190–1199 (2012) ' 2012 Wiley Periodicals, Inc.