RESEARCH ARTICLE Novel insights into SLC25A46-related pathologies in a genetic mouse model Maria Eirini Terzenidou1,2☯, Aikaterini Segklia3☯, Toshimi Kano1,2, Florentia Papastefanaki3, Alexandros Karakostas1,2, Maria Charalambous3, Fotis Ioakeimidis1,2, Maria Papadaki1,2, Ismini Kloukina4, Margarita Chrysanthou-Piterou5, Martina Samiotaki2, George Panayotou2, Rebecca Matsas3³, Eleni Douni1,2³* 1 Laboratory of Genetics, Department of Biotechnology, Agricultural University of Athens, Athens, Greece, 2 Biomedical Sciences Research Center "Alexander Fleming", Vari, Greece, 3 Laboratory of Cellular and Molecular Neurobiology, Hellenic Pasteur Institute, Athens, Greece, 4 Center of Basic Research, Biomedical a1111111111 Research Foundation of the Academy of Athens, Athens, Greece, 5 Laboratory of Neurobiology, a1111111111 Histochemistry and Electron Microscopy, 1st Department of Psychiatry, University of Athens Medical School, a1111111111 Eginition Hospital, Athens, Greece a1111111111 a1111111111 ☯ These authors contributed equally to this work. ³ These authors are joint senior authors on this work. * [email protected] OPEN ACCESS Abstract Citation: Terzenidou ME, Segklia A, Kano T, The mitochondrial protein SLC25A46 has been recently identified as a novel pathogenic Papastefanaki F, Karakostas A, Charalambous M, et al. (2017) Novel insights into SLC25A46-related cause in a wide spectrum of neurological diseases, including inherited optic atrophy, Char- pathologies in a genetic mouse model. PLoS Genet cot-Marie-Tooth type 2, Leigh syndrome, progressive myoclonic ataxia and lethal congenital 13(4): e1006656. https://doi.org/10.1371/journal. pontocerebellar hypoplasia. SLC25A46 is an outer membrane protein, member of the Sol- pgen.1006656 ute Carrier 25 (SLC25) family of nuclear genes encoding mitochondrial carriers, with a role Editor: Edmund Kunji, Dunn Human Nutrition Unit, in mitochondrial dynamics and cristae maintenance. Here we identified a loss-of-function UNITED STATES mutation in the Slc25a46 gene that causes lethal neuropathology in mice. Mutant mice man- Received: July 26, 2016 ifest the main clinical features identified in patients, including ataxia, optic atrophy and cere- Accepted: February 26, 2017 bellar hypoplasia, which were completely rescued by expression of the human ortholog. Published: April 4, 2017 Histopathological analysis revealed previously unseen lesions, most notably disrupted cytoarchitecture in the cerebellum and retina and prominent abnormalities in the neuromus- Copyright: © 2017 Terzenidou et al. This is an open access article distributed under the terms of the cular junction. A distinct lymphoid phenotype was also evident. Our mutant mice provide a Creative Commons Attribution License, which valid model for understanding the mechanistic basis of the complex SLC25A46-mediated permits unrestricted use, distribution, and pathologies, as well as for screening potential therapeutic interventions. reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Author summary Funding: This research was partially financed by Neurodegenerative diseases encompass a wide range of clinical conditions remaining the European Union (European Social Fund-ESF) incurable while the genetic and molecular basis of most of these conditions remains and Greek national funds through the Operational unknown due to their heterogeneity and the lack of animal models. Mutations in nuclear Program "Education and Lifelong Learning" of the genes encoding mitochondrial proteins have recently emerged as novel causalities in dis- National Strategic Reference Framework (NSRF) Research Funding Programs THALIS-UOC eases affecting the nervous system. SLC25A46, a novel mitochondrial protein, has recently "Mitochondrial dysfunction in neurodegenerative been identified as a pathogenic target in a wide spectrum of rare genetic neurological diseases" (grant code 377226) to RM and ED, PLOS Genetics | https://doi.org/10.1371/journal.pgen.1006656 April 4, 2017 1 / 28 SLC25A46 role and pathology in a mouse model ARISTEIA II Grant "DnaJmito" (grant code 4676) to ED and the Hellenic General Secretariat for diseases, including optic atrophy, Charcot-Marie-Tooth type 2, Leigh syndrome, progres- Research and Technology Grant SYNERGASIA sive myoclonic ataxia and lethal congenital pontocerebellar hypoplasia. Following a Noiseplus 09SYN-21-969 to RM. The funders had genetic approach, we identified a novel neurological mouse model caused by a functional no role in study design, data collection and analysis, decision to publish, or preparation of the mutation in the Slc25a46 gene. Our SLC25A46 mutant mice constitute the first genetic manuscript. animal model for this disease and represent an ideal tool for elucidating the underlying molecular mechanisms. The aim of this study was to characterize the complex phenotype Competing interests: The authors have declared that no competing interests exist. displayed by the mutant mice in order to understand the mechanistic basis of the SLC25A46-mediated pathologies. Introduction Mitochondria are highly dynamic organelles with a central role in a plethora of cellular pro- cesses, including ATP synthesis through oxidative phosphorylation (OXPHOS), metabo- lism, apoptosis, and reactive oxygen species generation, with important implications for neurodegenerative diseases. Recent discoveries show that mitochondrial shape and bioen- ergetics are tightly linked. Mitochondria have a characteristic structure as they are sur- rounded by two membranes: the inner membrane (IM) and the outer membrane (OM). The IM is further subdivided into the inner boundary membrane (IBM), which is adjacent to the OM and the cristae that protrude into the matrix. IM proteins involved in mitochon- drial fusion and protein translocation are preferentially located in the IBM, whereas pro- teins involved in oxidative phosphorylation are enriched in the cristae membrane. A variety of proteins regulate cristae biogenesis, with optic atrophy 1 (OPA1) and the mito- chondrial contact site (MICOS) complex being master regulators in this process [1]. The OM separates the mitochondria from the cytosol, yet it allows the passage of metabolites through the voltage-dependent anion channel (VDAC) and nuclear-encoded proteins through the translocase of the outer membrane (TOM) complex [2,3]. Mitochondria fuse and divide dynamically in response to changes in the cellular environment through family members of dynamin-related GTPases. Dynamin-related protein 1 (Drp1) controls mito- chondrial fission while mitofusins (MFN) in the outer membrane and OPA1 in the inner membrane regulate mitochondrial fusion. Interestingly, mutations in MFN2 are associated with Charcot-Marie-Tooth type 2 (CMT2) [4] while mutations in OPA1 are the major cause of dominant optic atrophy [5,6]. Recent findings also demonstrate that OPA1 con- trols mitochondrial dynamics by sensing changes in nutrient availability through mito- chondrial solute carriers [7]. Solute Carrier 25 (SLC25) is a large family of more than 50 nuclear-encoded transporters embedded mainly in the inner mitochondrial membrane that shuttle a variety of substrates participating in numerous metabolic pathways [8]. SLC25 members vary greatly in their size, the nature of transported substrates and the modes of transport, whereas common mecha- nisms of substrate translocation have been proposed [9,10]. To date, numerous SLC25-related rare metabolic diseases have been identified, caused by mutations in 13 genes encoding SLC25 proteins with various symptomatology, depending on the affected metabolic pathways and their significance in specific tissues [11]. Even though the majority of these disease-related SLC25 members have been biochemically characterized through identification of their sub- strates and correlated metabolic pathways, there are yet two orphan SLC25 members, SLC25A38 and SLC25A46, causing disorders in humans whose substrates have not been iden- tified [11]. SLC25A46 has recently been identified as a cause in heterogeneous clinical condi- tions, including optic atrophy, axonal peripheral neuropathy Charcot-Marie-Tooth type 2 PLOS Genetics | https://doi.org/10.1371/journal.pgen.1006656 April 4, 2017 2 / 28 SLC25A46 role and pathology in a mouse model (CMT2) [12], an optic atrophy spectrum disorder [13], Leigh syndrome [14], progressive myo- clonic ataxia with optic atrophy and neuropathy [15] and lethal congenital pontocerebellar hypoplasia [16]. It has been proposed that human SLC25A46 is a mitochondrial outer mem- brane protein with a role in mitochondrial dynamics and cristae maintenance that interacts with MFN2, OPA1 and members of the MICOS complex [12,14]. However, the effects of its ablation in animal models in vivo have not been addressed yet. Thus, the unavailability of genetic animal models has compromised the in-depth dissection of the related pathology and its correlation with the human disorders. In the present study, following a forward genetics approach, we identified a loss-of-function mutation in the Slc25a46 gene that causes lethal neuropathology in mice. Mutant mice mani- fest the main clinical features described in patients, including ataxia, optic atrophy and cere- bellar hypoplasia as well as additional previously unidentified neuropathological alterations, while they were rescued by expression of the human ortholog. The aim of this study was to characterize the complex