Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralardeficient (Aralar ؊/؊ ) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar ؊/؊ mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar ؊/؊ mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar ؊/؊ mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.

Aralar, the neuronal Ca2؉ -binding mitochondrial aspartate-glutamate carrier, has Ca 2؉ binding domains facing the extramitochondrial space and functions in the malate-aspartate NADH shuttle (MAS). Here we showed that MAS activity in brain mitochondria is stimulated by extramitochondrial Ca 2؉ with an S 0.5 of 324 nM. By employing primary neuronal cultures from control and aralar-deficient mice and NAD(P)H imaging with two-photon excitation microscopy, we showed that lactate utilization involves a substantial transfer of NAD(P)H to mitochondria in control but not aralardeficient neurons, in agreement with the lack of MAS activity associated with aralar deficiency. The AGCs are one of the transporters responsible for the malateaspartate NADH shuttle (MAS). Because of the electrogenic nature of Asp/Glu exchange (20, 21), the AGC reaction is irreversible under physiological conditions and thus a potential site for regulation. The first aim of this work was to explore the potential of aralar as a brain AGC isoform to regulate MAS activity in brain at low Ca 2ϩ concentrations, below those required for the function of the mitochondrial Ca 2ϩ uniporter. MAS activity in brain mitochondria was found to have Ca 2ϩ -activation properties adequate for this purpose.The second aim of this work was to study the role of the aralar-MAS pathway in the supply of reducing equivalents to neuronal mitochondria. Aralar is expressed postnatally in rat and mouse brain, and it is located in neurons. Both MAS activity and aralar expression are acquired in parallel during neuronal maturation (22,23). Aralar is important for neuronal function as underscored by the finding that alterations in aralar gene and protein are associated with central nervous system diseases such as Mohr-Tranebjaerg syndrome, in which there is an impaired targeting of aralar to mitochondria (24), and autism (25). Aralar null mice also exhibit prominent motor coordination defects along with deficient myelination (26).We have employed primary neuronal cultures from control and aralar-deficient mice (26) and two-photon excitation microscopy imaging of NAD(P)H to monitor the transfer of reducing equivalents from cytosol to mitochondria. We show that MAS is the main pathway to transfer reducing equivalents to neuronal mitochondria. High [Ca 2ϩ ] i signals activate the Ca 2ϩ uniporter-mitochondrial dehydrogenases signaling pathway, whereas small [Ca 2ϩ ] i signals selectively activate MAS activity in neurons. We conclude that the aralar-MAS pathway plays an * This work was supported in part by Direcció n General de Investigació n del Ministerio de Ciencia y Tecnología Grant BMC2002-02072, Comunidad de Madrid Grant 08.5/ 0024/2003, Fondo de Investigaciones Sanitarias del Ministerio de Sanidad y Consumo 01/0395 (to J. S.), an institutional grant from the Fundació n Ramó n Areces to the Centro de Biología Molecular 'Severo Ochoa,' and by a Grant-in-aid for Scientific Research 16390100 from the Japan Society for the Promotion of Science (to K. K.). The costs of publicatio...

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