2. SLC25 family and the discovery of SLC25A46

SLC25A46 belongs to the solute carrier family 25 (SLC25), a superfamily that contains 53 nuclear-encoded mitochondrial carrier proteins in humans [27]. SLC25 members are characterized by the presence of three tandem repeats of about 100 amino acids, each containing two transmembrane alpha helices linked by a large loop [28]. The mature carrier protein thus consists of six transmembrane helices that form an aqueous pore and have a highly conserved consensus sequence, P-X-[D/E]-X-X-[R/K], at the C-terminal ends of the three odd-numbered transmembrane alpha helices, whose charged residues form salt bridges that close the pore on the matrix side [29]. SLC25 proteins may shuttle a variety of solutes across the mitochondrial membrane to participate in various metabolic pathways [30]. Although common mechanisms of substrate translocation have been proposed, SLC25 members vary greatly in their size, the nature of substrates they transport, the modes of transport employed, and the driving forces they employ [30–32].

neurons through biogenesis. In addition to undergoing the dynamic processes of mitochondrial fission and fusion, mitochondria are transported bidirectionally within neurites, in which they are distributed purposefully, facilitating energy transmission over long distances to meet local demands and, when necessary, undergo controlled degradation by mitophagy [2, 3]. Thus,

Recent evidence suggests that abnormal mitochondrial dynamics may contribute to both familial and sporadic neurodegenerative diseases [4]. Most proteins related to mitochondrial dynamics are encoded by genes in the nucleus. Mutations in such nuclear-encoded genes can cause monogenic disorders in which mitochondrial dysfunction is unequivocally central to the pathogenesis of the disease. For example, mutations in MFN1/2 and OPA1 cause Charcot-Marie-Tooth neuropathy (CMT) type 2A [5–7] and autosomal dominant optic atrophy (ADOA) [8–10], respectively. A dominant negative allele of DRP1 was identified in a neonate with a lethal mitochondrial and peroxisomal fission defect associated with abnormal brain development, optic atrophy, and various other congenital anomalies [11]. Defects in proteins involved in axonal transport have also been identified in patients with CMT and related neuropathies [12–14]. In addition, there is increasing evidence linking mitochondrial dysfunction to neuronal loss in age-related neurodegenerative disorders, including Alzheimer's disease and

Recent studies have implicated the 46th isoform of subfamily A of the solute carrier (SLC) family 25, termed SLC25A46, in mitochondrial dysfunction pathology. SLC25A46 is a mitochondrial outer membrane protein that was shown recently to be involved in mitochondrial dynamics, either playing a role in mitochondrial fission or serving as a regulator of mitofusin (MFN)1/2 oligomerization [17, 18]. Disorders caused by recessive SLC25A46 mutations were defined recently as a new syndrome (introduced and elaborated in Section 3.1) that has a broad clinical spectrum of neurological phenotypes, including peripheral neuropathy, early-onset optic atrophy, cerebellar degeneration, and congenital pontocerebellar hypoplasia (PCH), with variable ages of onset and severities [17, 19–26]. In this chapter, we will focus on the phenotypic and genetic characteristics of SLC25A46-related neurological diseases and our current understanding of the pathophysiological mechanisms linking dysfunctional SLC25A46 to neurodegeneration.

SLC25A46 belongs to the solute carrier family 25 (SLC25), a superfamily that contains 53 nuclear-encoded mitochondrial carrier proteins in humans [27]. SLC25 members are characterized by the presence of three tandem repeats of about 100 amino acids, each containing two transmembrane alpha helices linked by a large loop [28]. The mature carrier protein thus consists of six transmembrane helices that form an aqueous pore and have a highly conserved consensus sequence, P-X-[D/E]-X-X-[R/K], at the C-terminal ends of the three odd-numbered transmembrane alpha helices, whose charged residues form salt bridges that close the pore on the matrix side [29]. SLC25 proteins may shuttle a variety of solutes across the mitochondrial

mitochondrial dynamics play critical roles in neuronal homeostasis and survival.

Parkinson's disease [15, 16].

70 Recent Advances in Neurodegeneration

2. SLC25 family and the discovery of SLC25A46

A number of genetic conditions associated with SLC25 mitochondrial transporters have been characterized biochemically and genetically [33]. SLC25 members mediate a variety of cellular functions, and mutations in SLC25 genes have been linked to various defects, such as carnitine/ acylcarnitine carrier deficiency (OMIM 212138), HHH syndrome (OMIM 238970), aspartate/ glutamate isoform 1 and 2 deficiencies (OMIM 612949, 603471, 605814), congenital Amish

Figure 1. Schematic diagram of SLC25A46 structure and its interactions. (A) SLC25A46 consists of six conserved transmembrane alpha helices. (B) 3D structure of SLC25A46. (C) Potential interactions of SLC25A46 with dynamic proteins.

microcephaly (OMIM 607196), neuropathy with bilateral striatal necrosis (OMIM 613710), congenital sideroblastic anemia (OMIM 205950), neonatal epileptic encephalopathy (OMIM 609304), and citrate carrier deficiency (OMIM 190315) [33]. These disorders are characterized by specific metabolic dysfunctions related to the role of the particular carrier that has been affected. Most disease-related SLC25 members have been characterized in terms of substrate identification and associated metabolic pathways, with the exception of two orphan SLC25 members, namely SLC25A38 and SLC25A46 [33].

SLC25A46 was first mapped to chromosome 5 by genomic sequence analysis in 2006 [27]. Its location was further refined to chromosome 5q22.1 based on sequence alignment with NCBI's standard reference human assembly sequence, that is, the Genome Reference Consortium Human genome build 38. The largest transcript isoform of SLC25A46 contains eight exons, which encode a 418-amino acid protein. Quantitative real-time polymerase chain reaction (PCR) experiments in rodents have demonstrated variable expression of SLC25A46 in all tissues examined, with the highest levels occurring in the hindbrain, spinal cord, and coronal brain sections containing the corpus callosum, fornix, optic chiasm, thalamus, hypothalamus, midbrain, pons, and cerebellum, with particularly high levels in mouse embryo cerebellum [27].

Given the typical SLC25 molecular structure, the primary sequence of the SLC25A46 protein has been predicted to form six conserved transmembrane alpha helices, TM1–TM6, spanning a region between amino acids 100–418 (Figure 1) [22]. However, the otherwise highly conserved P-X-(D/E)-X-X-(R/K) consensus sequence characteristic of SLC25 proteins is altered in SLC25A46. Moreover, the N-terminus of SLC25A46 is about five times longer than that of other members of the family (100 vs. <20 amino acids). These unusual characteristics suggest that SLC25A46 is unlikely to have a conventional metabolite carrier function. Recently, studies have proposed that unlike most SLC25 members that are located in the inner mitochondrial membrane, SLC25A46 may be anchored to the outer mitochondrial membrane where it may act as a regulator of mitochondrial dynamics rather than as a substrate transporter.
