Low Levels of Citrin (SLC25A13) Expression in Adult Mouse Brain Restricted to Neuronal Clusters Laura Contreras, 1,2 Almudena Urbieta, 1 Keiko Kobayashi, 3 Takeyori Saheki, 4 and Jorgina Satru ´ stegui 1,2 * 1 Departamento de Biologı ´a Molecular, Centro de Biologı ´a Molecular Severo Ochoa CSIC-UAM, Universidad Auto ´noma de Madrid, Madrid, Spain 2 CIBER de Enfermedades Raras (CIBERER), Madrid, Spain 3 Department of Molecular Metabolism and Biochemical Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan 4 Institute for Health Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, Japan The mitochondrial aspartate-glutamate carriers (AGC) aralar (SLC25A12) and citrin (SLC25A13) are compo- nents of the malate aspartate shuttle (MAS), a major intracellular pathway to transfer reducing equivalents from NADH to the mitochondrial matrix. Aralar is the main AGC isoform present in the adult brain, and it is expressed mainly in neurons. To search for the other AGC isoform, citrin, in brain glial cells, we used a citrin knockout mouse in which the lacZ gene was inserted into the citrin locus as reporter gene. In agreement with the low citrin levels known to be present in the adult mouse brain, b-galactosidase expression was very low. Surprisingly, unlike the case with astroglial cultures that express citrin, no b-galactosidase was found in brain glial cells. It was confined to neuronal cells within dis- crete neuronal clusters. Double-immunolabelling experi- ments showed that b-galactosidase colocalized not with glial cell markers but with the pan-neuronal marker NeuN. The deep cerebellar nuclei and a few midbrain nuclei (reticular tegmental pontine nuclei; magnocellular red nuclei) were the regions where b-galactosidase expression was highest, and it was up-regulated in fasted mice, as was also the case for liver b-galactosi- dase. The results support the notion that glial cells have much lower AGC levels and MAS activity than neurons. V V C 2009 Wiley-Liss, Inc. Key words: mitochondrial aspartate-glutamate carrier; citrin; aralar; redox shuttle; neuron; cerebellum; fasting; OmniBank The malate aspartate shuttle (MAS) is, together with the glycerol-3-phosphate shuttle, a major intracellu- lar pathway to transfer glycolytic redox equivalents to the mitochondrial matrix (Dawson, 1979; McKenna et al., 2006). MAS is composed of two enzymes located in the cytosol and mitochondrial matrix compartments (aspartate transaminase and malate dehydrogenase) and of two transporters belonging to the mitochondrial carrier superfamily (Walker and Runswick, 1993). The oxoglu- tarate carrier (OGC) transports malate in exchange for a-ketoglutarate (electroneutral and reversible; Indiveri et al., 1991; Smith and Walker, 2003), whereas the aspartate-glutamate carrier (AGC) cotransports cytosolic glutamate (plus a proton) against mitochondrial aspartate (for review see Satrustegui et al., 2007). In mammals, two isoforms of the AGC exist: aralar, also named aralar1 (SLC25A12) and citrin (SLC25A13; del Arco and Satrustegui, 1998; Kobayashi et al., 1999; del Arco et al., 2000). Both have Ca 21 -binding motifs (EF-hands) in their N-terminal halves facing the intermembrane space (Palmieri et al., 2001). This allows the activation of AGCs and MAS in response to cytosolic calcium signals that do not reach the mitochondrial matrix (Pardo et al., 2006; Contreras et al., 2007; Marmol et al., 2009). Ca 21 activation of glutamate transport along the AGC is also responsible for calcium stimulation of respiration in brain mitochondria using glutamate and malate as respiratory substrates (Gellerich et al., 2008). The two AGC iso- forms differ mainly in terms of calcium sensitivity (Con- Laura Contreras’s current addresss is Department of Biomedical Sciences; University of Padua; Viale Giuseppe Colombo 3, 35121 Padua, Italy Contract grant sponsor: Ministerio de Educacio ´n y Ciencia; Contract grant number: BFU2005-C02-01; Contract grant number: BFU2008- 04084/BMC; Contract grant sponsor: Comunidad de Madrid; Contract grant number: S-GEN-0269-2006 MITOLAB-CM; Contract grant spon- sor: European Union; Contract grant number: LSHM-CT-2006-518153; Contract grant sponsor: Fundacio ´n Ramo ´n Areces (to the Centro de Biologı ´a Molecular Severo Ochoa). *Correspondence to: Jorgina Satru ´ stegui, Departamento de Biologı ´a Mo- lecular, Centro de Biologı ´a Molecular ‘‘Severo Ochoa’’ CSIC-UAM, c/ Nicola ´s Cabrera 1, Campus Cantoblanco, 28049-Madrid, Spain. E-mail: jsatrustegui@cbm.uam.es Received 8 July 2009; Revised 1 September 2009; Accepted 3 September 2009 Published online 11 November 2009 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.22283 Journal of Neuroscience Research 88:1009–1016 (2010) ' 2009 Wiley-Liss, Inc.