Novel insights into SLC25A46-related pathologies in a genetic ... - PLOS

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 Matsas3‡, Eleni Douni1,2‡*

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OPEN ACCESS Citation: Terzenidou ME, Segklia A, Kano T, Papastefanaki F, Karakostas A, Charalambous M, et al. (2017) Novel insights into SLC25A46-related pathologies in a genetic mouse model. PLoS Genet 13(4): e1006656. https://doi.org/10.1371/journal. pgen.1006656 Editor: Edmund Kunji, Dunn Human Nutrition Unit, UNITED STATES Received: July 26, 2016 Accepted: February 26, 2017 Published: April 4, 2017 Copyright: © 2017 Terzenidou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and 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. Funding: This research was partially financed by the European Union (European Social Fund-ESF) and Greek national funds through the Operational Program "Education and Lifelong Learning" of the National Strategic Reference Framework (NSRF) Research Funding Programs THALIS-UOC "Mitochondrial dysfunction in neurodegenerative diseases" (grant code 377226) to RM and ED,

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 Research Foundation of the Academy of Athens, Athens, Greece, 5 Laboratory of Neurobiology, Histochemistry and Electron Microscopy, 1st Department of Psychiatry, University of Athens Medical School, Eginition Hospital, Athens, Greece ☯ These authors contributed equally to this work. ‡ These authors are joint senior authors on this work. * [email protected]

Abstract The mitochondrial protein SLC25A46 has been recently identified as a novel pathogenic cause in a wide spectrum of neurological diseases, including inherited optic atrophy, Charcot-Marie-Tooth type 2, Leigh syndrome, progressive myoclonic ataxia and lethal congenital pontocerebellar hypoplasia. SLC25A46 is an outer membrane protein, member of the Solute Carrier 25 (SLC25) family of nuclear genes encoding mitochondrial carriers, with a role in mitochondrial dynamics and cristae maintenance. Here we identified a loss-of-function mutation in the Slc25a46 gene that causes lethal neuropathology in mice. Mutant mice manifest the main clinical features identified in patients, including ataxia, optic atrophy and cerebellar hypoplasia, which were completely rescued by expression of the human ortholog. Histopathological analysis revealed previously unseen lesions, most notably disrupted cytoarchitecture in the cerebellum and retina and prominent abnormalities in the neuromuscular junction. A distinct lymphoid phenotype was also evident. Our mutant mice provide a valid model for understanding the mechanistic basis of the complex SLC25A46-mediated pathologies, as well as for screening potential therapeutic interventions.

Author summary Neurodegenerative diseases encompass a wide range of clinical conditions remaining incurable while the genetic and molecular basis of most of these conditions remains unknown due to their heterogeneity and the lack of animal models. Mutations in nuclear genes encoding mitochondrial proteins have recently emerged as novel causalities in diseases affecting the nervous system. SLC25A46, a novel mitochondrial protein, has recently been identified as a pathogenic target in a wide spectrum of rare genetic neurological

PLOS Genetics | https://doi.org/10.1371/journal.pgen.1006656 April 4, 2017

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SLC25A46 role and pathology in a mouse model

ARISTEIA II Grant "DnaJmito" (grant code 4676) to ED and the Hellenic General Secretariat for Research and Technology Grant SYNERGASIA Noiseplus 09SYN-21-969 to RM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

diseases, including optic atrophy, Charcot-Marie-Tooth type 2, Leigh syndrome, progressive myoclonic ataxia and lethal congenital pontocerebellar hypoplasia. Following a genetic approach, we identified a novel neurological mouse model caused by a functional mutation in the Slc25a46 gene. Our SLC25A46 mutant mice constitute the first genetic 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 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 processes, including ATP synthesis through oxidative phosphorylation (OXPHOS), metabolism, apoptosis, and reactive oxygen species generation, with important implications for neurodegenerative diseases. Recent discoveries show that mitochondrial shape and bioenergetics are tightly linked. Mitochondria have a characteristic structure as they are surrounded 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 mitochondrial fusion and protein translocation are preferentially located in the IBM, whereas proteins involved in oxidative phosphorylation are enriched in the cristae membrane. A variety of proteins regulate cristae biogenesis, with optic atrophy 1 (OPA1) and the mitochondrial 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 mitochondrial 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 controls mitochondrial dynamics by sensing changes in nutrient availability through mitochondrial 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 mechanisms 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 substrates and correlated metabolic pathways, there are yet two orphan SLC25 members, SLC25A38 and SLC25A46, causing disorders in humans whose substrates have not been identified [11]. SLC25A46 has recently been identified as a cause in heterogeneous clinical conditions, including optic atrophy, axonal peripheral neuropathy Charcot-Marie-Tooth type 2


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(CMT2) [12], an optic atrophy spectrum disorder [13], Leigh syndrome [14], progressive myoclonic ataxia with optic atrophy and neuropathy [15] and lethal congenital pontocerebellar hypoplasia [16]. It has been proposed that human SLC25A46 is a mitochondrial outer membrane 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 manifest the main clinical features described in patients, including ataxia, optic atrophy and cerebellar 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 phenotype displayed by the mutant mice in order to understand the mechanistic basis of the heterogeneous SLC25A46-mediated pathologies.

Results Identification of a lethal recessive neurological phenotype with lymphoid abnormalities in mice through random mutagenesis Following a forward genetics approach through N-ethyl-N-nitrosourea (ENU) random mutagenesis [17,18], we have recently identified a novel mouse model of autosomal recessive neurological phenotype characterized by ataxia, unsteady locomotion, tonic-clonic seizures, limb-clasping during tail suspension, reduced muscle strength, and growth retardation with premature lethality by the age of 3 months (Fig 1A–1D, S1 Video). Because of the prominent neurological symptoms, this phenotype was designated as ataxic (atc). The first symptoms were noticeable at the second postnatal week with full penetrance in both sexes. From the third week, atc/atc mice stopped gaining weight (Fig 1A), their muscle strength began to deteriorate (Fig 1B) and by the fifth week of age the majority of atc/atc mice had died (Fig 1C). Heterozygous mice (atc/+) were indistinguishable from their wild-type (WT) littermates throughout life. In order to exclude the possibility that atc/atc mice succumb to starvation due to difficulties in accessing food caused by weakness and tremor, mashed wet food pellets were added inside the cage. No gross histopathological lesions such as hepatic steatosis were observed in atc/atc mice (S1 Fig). Apart from the neurological phenotype, atc/atc mice displayed severe thymic and splenic hypoplasia (Fig 1E–1N). Thymi of 4-week-old atc/ atc mice had approximately 30% of the WT littermate weight and flow cytometric analysis revealed significant reduced cellularity in all four thymic subpopulations, CD4+CD8-, CD4-CD8+, CD4+CD8+, and CD4-CD8- (Fig 1E–1I). Atc/atc mice also developed severe splenic hypoplasia (25% of WT weight) with a significant decrease in the absolute number of T lymphocytes (CD4+ and CD8+), B lymphocytes (B220+) and myeloid cells (CD11b+, Gr1+) (Fig 1J–1N). To examine whether the immune system is associated to the neurological phenotype we generated atc/atc mice lacking mature T and B lymphocytes upon genomic deletion of RAG-2 (recombination activating gene) gene that is necessary for immunoglobulin and T-cell receptor gene rearrangements [19]. Clinical evaluation of the double mutant mice showed that the neurological symptoms, survival, body weight, and muscle strength were not reversed (Fig 1O and 1P), implying that the lymphoid and neurological symptoms are distinct.


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Fig 1. Clinical characterization of the ataxic phenotype and identification of the causal mutation in Slc25a46. (A) Reduced body weight gain (n = 20 per group), (B) muscle weakness (n = 20 per group), (C) premature lethality (n = 40 per group) and (D) loss of hind limb extension reflex during tail suspension in ataxic (atc/atc) mice as compared to sex matched control (+/+ or atc/+) littermates. (E) Representative thymi dissected from WT and atc/atc mice. (F) Reduced thymus/body weight ratios in atc/atc mice as compared to WT littermates (n = 17 per group) accompanied by (G) lower total thymocyte numbers in atc/atc mice (n = 4–5 per group). (H) Percentages and (I) absolute numbers of thymic subpopulations (CD4+, CD8+, DP: Double Positive CD4+CD8+, DN: Double Negative CD4-CD8-) in WT (black columns, n = 5) and atc/atc (white columns, n = 4) littermates as determined by flow cytometry after staining with antibodies against CD4 and CD8. (J) Representative spleens dissected from WT and atc/atc mice. Reduction in spleen/body weight ratios in (K) atc/atc mice as compared to WT littermates (n = 17 per group) and (L) total splenocyte numbers (n = 5 per group). (M) Percentages and (N) absolute numbers of splenic subpopulations in WT and atc/atc littermates (n = 5 per group) through flow cytometry after staining with antibodies against CD4, CD8, B220 (B cells), CD11b and Gr1 (Myeloid). Atc/atc mice were crossed with Rag2 deficient mice (Rag-/-) that lack mature T and B lymphocytes and (O) body weight curves as well as (P) grip strength measurements demonstrate that the neurological phenotype is not reversed (WT and atc/atc/Rag-/-, n = 5 per group; atc/atc, n = 3). Data represent mean values ± SEM. Two-tailed unpaired Student’s t-test was performed for statistical analysis.


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* p0.05; ** p0.01; *** p0.001. (Q) Through genome-wide linkage analysis the causal mutation was mapped on chromosome 18. (R) Sequencing of genomic DNA samples from WT controls (+/+) and atc/atc littermates revealed an exonic C-to-T transition (red arrow) in the Slc25a46 gene that introduce a premature stop codon. https://doi.org/10.1371/journal.pgen.1006656.g001

Identification of the causal mutation in Slc25a46 gene To map the causal mutation we performed genetic linkage analysis using an initial set of 68 Simple Sequence Length Polymorphic markers (SSLPs) in 20 atc/atc and 20 not affected F2 mice, and identified clear linkage with chromosome 18 with a LOD score of 12 (Fig 1Q). Fine mapping by analyzing 37 additional F2 mice and screening with 2 Single Nucleotide Polymorphisms (SNPs) located in between the two extreme SSLPs, narrowed down the mutant locus to a genetic region of 8.84 Mbp between D18Mit22 and rs30151517 (S1 Table). Selected candidate gene sequencing, identified a C to T transition at the coding region of the Slc25a46 gene, introducing a premature termination codon and resulting in a truncated protein of 95 amino acids instead of the 418 amino acids composing WT SLC25A46 (Fig 1R). SLC25A46 is a member of the Solute Carrier 25 (SLC25) family of nuclear genes encoding mitochondrial carriers that shuttle a variety of substrates across the inner mitochondrial membrane [8]. 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. As the primary sequence of the SLC25A46 protein indicates the presence of six conserved transmembrane regions spanning a region between amino acids 100 to 418 [14], the identified nonsense mutation presumably leads to a loss-of-function allele. Functional point mutations in the SLC25A46 gene have recently been reported in heterogeneous neurological disorders [12,13,14,15,16]. Our mutant mice emerge as the first genetic mouse model for understanding the complex pathophysiological role of the SLC25A46 protein in vivo.

Mouse SLC25A46 is an outer mitochondrial membrane protein with prominent expression in the nervous system The majority of SLC25 members are embedded in the inner mitochondrial membrane. The mitochondrial localization of mouse SLC25A46 was confirmed by immunoblot analysis in WT central nervous system (CNS) tissues (Fig 2A) and by fluorescence microscopy upon expression of a chimeric SLC25A46-EGFP protein in the N2A mouse neuroblastoma cell line (Fig 2B). Similarly to human SLC25A46, the mouse ortholog localizes to the outer mitochondrial membrane, as shown upon swelling and proteinase K protection assays in mitochondria isolated from the cerebellum of WT mice (Fig 2C) or cell lines (S2 Fig). Analysis of SLC25A46 expression profile showed prominent expression in the cerebrum, cerebellum and spinal cord (Fig 2D and 2E), and complete absence of the full-length 46 kD protein in mitochondria from atc/atc mice (Fig 2F). However, the mRNA expression levels of Slc25a46 remained unaffected in atc/atc mice (S3 Fig), indicating that the truncating mutation does not lead to nonsensemediated mRNA decay. According to the "50 nucleotide rule" this premature termination codon is not predicted to subject to decay, as it is located 38 nucleotides apart from the next exon-exon junction [20]. Double immunofluorescence labelling for SLC25A46 and the alpha subunit of the mitochondrial enzyme pyruvate dehydrogenase E1 (PDH-E1a) revealed high expression of SLC25A46 in mitochondria of WT cerebellar Purkinje cells (PCs), while a diffuse cellular distribution was identified in atc/atc PC somata (Fig 2G), similarly to N2A cells expressing the mutant SLC25A46-EGFP protein (Fig 2B). These results demonstrate that the mutant SLC25A46 protein is not embedded in the mitochondrial membrane, thus being ensued with a compromised function.


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Fig 2. Mitochondrial localization of the SLC25A46 protein. (A) Cytosolic (Ct) and mitochondrial (Mt) fractions from cerebrum (B), cerebellum (C) and spinal cord (SC) tissues isolated from WT mice were analyzed by immunoblotting with antibodies for SLC25A46, the mitochondrial protein COX IV and the cytosolic protein GAPDH. (B) Fluorescence microscopy of N2A cells transfected with plasmids expressing either EGFP, SLC25A46-EGFP or mutSLC25A46-EGFP carrying the atc mutation. DAPI: nuclear staining, mtTracker: mitotracker for mitochondrial staining. Scale bar: 20μm. (C) Mitochondria and mitoplasts prepared by hypotonic swelling (Sw) of mitochondria from WT cerebellum, were treated with proteinase K (PK) and analyzed by immunoblotting with antibodies against SLC25A46, the outer membrane protein TOMM40, the intermembrane space protein Mitofilin, the integral inner membrane protein SLC25A5 and the matrix protein GRP75. Analysis of the SLC25A46 expression pattern in various tissues from WT mice at the mRNA level with (D) qPCR for mouse Slc25a46 and western blots in mitochondrial extracts from (E) WT mice and (F) WT and atc/atc mice with antibodies against the SLC25A46 and the mitochondrial protein prohibitin. Cerebrum (B), cerebellum (C), spinal cord (SC), muscle (M), heart (H), thymus (T), spleen (S) and liver (L). (G) Immunofluorescence labeling of 4-week-old mid-sagittal cerebellar sections for SLC25A46 shows a clear co-localization with the mitochondrial marker PDH-E1a in PCs of WT mice while a more diffuse distribution is apparent within the PC somata of atc/atc mice indicative of cytoplasmic localization. The insets are shown at higher magnification in the lower panel. Scale bar 20 μm. https://doi.org/10.1371/journal.pgen.1006656.g002


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Functional ablation of SLC25A46 results in cerebellar hypoplasia, compromised Purkinje cell dendritic arborization and reduced synaptic connectivity in atc/atc mice Because of the prominent SLC25A46 expression in the nervous system of WT mice and the ataxic phenotype of mutant animals, we examined the brain for possible histological alterations. Macroscopic examination showed no overt abnormalities in the gross anatomy of atc/ atc brain. However, histological staining revealed a selective 25% reduction in the size of the cerebellum (S4 Fig) with prominently reduced thickness of the molecular layer (ML) (Fig 3A and 3B). Although PC numbers and alignment were not affected, PC dendrites were severely stunted (Fig 3C–3F). PCs are the main output neurons of the cerebellar cortex receiving signals that modulate their activity through multiple synapses on their elaborate dendritic arbors [21]. Parallel fibers (the axons of granule neurons) extending from the internal granule layer (IGL) of the cerebellar cortex and climbing fibers originating from the inferior olivary nucleus form excitatory glutamatergic synapses on PC dendrites [22]. Additionally, mossy fibers coming from pre-cerebellar nuclei indirectly target PCs, via formation of synapses with granule cell dendrites within the IGL [23]. To elucidate the basis of cerebellar hypoplasia and PC impairment, we examined the distribution of glutamatergic synapses between PCs and parallel or climbing fibers in the ML, as well as the distribution of mossy fiber synapses in the IGL, which indirectly target PCs via granule cells, using antibodies against the vesicular glutamate transporters vGlut1 and vGlut2. Our analysis showed a significant reduction in vGlut1 and vGlut2 immunoreactivity both in the ML and the IGL, indicating a paucity of glutamatergic synapses in atc/atc mice (Fig 3G–3M). Interestingly, a reduction in vGlut1 and vGlut2 immunoreactivity was visible at postnatal day 9 when no obvious defects in PC morphology were discernible as yet (S5 Fig). This feature, which is compatible with the ponto-cerebellar hypoplasia observed in the human disease [16], suggests that the stunted PC dendritic growth observed later could be linked to or exacerbated by the compromised glutamatergic input. An alternative hypothesis is that the reduced glutamatergic input is a consequence rather than a cause of the underdeveloped PC dendritic arborization, possibly due to primary impaired mitochondrial dynamics. To examine the dendritic ultrastructure of PCs we employed electron microscopy (EM) in ultrathin sections of the cerebellum of 4-week-old atc/atc and WT mice. Unlike the well-preserved appearance of PC dendrites in the molecular layer of the cerebellum in WT mice (Fig 3Ni), several degenerated PC dendrites were identified in atc/atc mice as judged by their disorganized cytoskeleton, often containing remnants of mitochondria and other organelles (Fig 3Nii and 3Niii and inset Fig 3Niii’). Additionally, numerous atypical mitochondria with cytoplasmic inclusions were found both in the soma and dendrites of atc/atc PCs (Fig 3Nv and 3Nvi) that were not present in the WT (Fig 3Ni and 3Niv). Structural configurations containing concentric stacks of flattened cisternae that surrounded clusters of mitochondria were frequently observed in atc/atc (Fig 3Nvii and 3Nviii) but not in WT mice. Surprisingly, other alterations associated with mitochondrial ultrastructural organization or distribution, were not noted. Apart from aberrations in cerebellar cytoarchitecture, we observed significant astrogliosis and microgliosis in the spinal cord of mutant mice, indicating high levels of neuroinflammation, as estimated by the increase in GFAP+ astrocytes and resting (Iba1+/CD68-) or activated (Iba1-/CD68+) microglia by comparison with WT mice (S6 Fig). A reduction in the size of the spinal cord was also evident in atc/atc mice without any differences in neuronal density, motor neuron appearance or the profile of cholinergic nerve terminals (S7 Fig).


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Fig 3. Compromised Purkinje cell dendritic arborization, reduction in glutamatergic synapses and ultrastructural alterations in the cerebellar cortex of atc/atc mice. (A) Toluidine-blue staining of cerebellar mid-sagittal sections from 4 week-old mice shows reduced thickness of the molecular layer (ML) in atc/atc animals. (B) Graph showing quantification of average ML thickness (n = 4 per genotype, p = 0.018). (C) Calbindin immunostaining and (D) Golgi impregnation reveal a significant reduction in Purkinje cell dendritic arborization in atc/atc mice. (E) Quantification of Purkinje cell dendritic length as marked in red in (D). (F) Quantification of the branching distance of the thick primary dendrite into secondary and tertiary dendrites (n = 25 cells pooled from 3 mice per genotype, p = 0.0001). (G) Immunostaining of sagittal cerebellar sections from 4 week-old mice reveals a significant decrease in the expression of vGlut1 both in


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PC-parallel fiber synapses in the ML as well as in granule cell-mossy fiber synapses in the IGL of the cerebellar cortex of atc/atc mice. The insets are shown at higher magnification in (i-iv). (H, I) Quantification of vGlut1 fluorescence intensity in ML and IGL (n = 4 mice per genotype, p