Novel insights into SLC25A46-related pathologies in a genetic mouse model

<div><p>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 <i>Slc25a46</i> 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.</p></div

Mitochondrial localization of the SLC25A46 protein.

<p><b>(A)</b> 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>(B)</b> Fluorescence microscopy of N2A cells transfected with plasmids expressing either EGFP, SLC25A46-EGFP or mutSLC25A46-EGFP carrying the <i>atc</i> mutation. DAPI: nuclear staining, mtTracker: mitotracker for mitochondrial staining. Scale bar: 20μm. <b>(C)</b> 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 <b>(D)</b> qPCR for mouse <i>Slc25a46</i> and western blots in mitochondrial extracts from <b>(E)</b> WT mice and <b>(F)</b> WT and <i>atc/atc</i> 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). <b>(G)</b> 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 <i>atc/atc</i> mice indicative of cytoplasmic localization. The insets are shown at higher magnification in the lower panel. Scale bar 20 μm.</p

Compromised Purkinje cell dendritic arborization, reduction in glutamatergic synapses and ultrastructural alterations in the cerebellar cortex of <i>atc/atc</i> mice.

<p><b>(A)</b> Toluidine-blue staining of cerebellar mid-sagittal sections from 4 week-old mice shows reduced thickness of the molecular layer (ML) in <i>atc/atc</i> animals. <b>(B)</b> Graph showing quantification of average ML thickness (n = 4 per genotype, p = 0.018). <b>(C)</b> Calbindin immunostaining and <b>(D)</b> Golgi impregnation reveal a significant reduction in Purkinje cell dendritic arborization in <i>atc/atc</i> mice. <b>(E)</b> Quantification of Purkinje cell dendritic length as marked in red in (D). <b>(F)</b> 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). <b>(G)</b> Immunostaining of sagittal cerebellar sections from 4 week-old mice reveals a significant decrease in the expression of vGlut1 both in PC-parallel fiber synapses in the ML as well as in granule cell-mossy fiber synapses in the IGL of the cerebellar cortex of <i>atc/atc</i> mice. The insets are shown at higher magnification in (i-iv). <b>(H, I)</b> Quantification of vGlut1 fluorescence intensity in ML and IGL (n = 4 mice per genotype, p<0.00001). <b>(J)</b> Similarly, immunostaining for vGlut2 revealed a significant reduction PC-climbing fiber synapses in the ML and granule cell-mossy fiber synapses in the IGL. The insets are shown at higher magnification in (i-iv). <b>(K)</b> The reduction in vGlut2-positive synapses is illustrated at higher magnification in double immunofluorescence micrographs showing calbindin-positive PCs and vGlut2-positive puncta. <b>(L, M)</b> Quantification of vGlut2 fluorescence intensity in ML and IGL (n = 4 mice per genotype, p<0.00001). Statistical significance was determined by two-tailed unpaired Student’s <i>t</i>-test. Data represent mean values ± SEM Scale bar 40μm in <b>A, C, G, J</b>; 20μm in <b>D, K</b>. <b>(N)</b> Electron microscopy shows <b>(i)</b> a longitudinally sectioned dendrite (d) across the field, with normal mitochondria and neurofilament distribution in WT mice; <b>(ii, iii)</b> Longitudinal (d) and transverse (*) sections of <i>atc/atc</i> PC dendrites, containing disorganized neurofilaments and remnants of mitochondria and other membrane organelles [inset in <b>(iii’)</b>], indicating degeneration. Synapses (S) are marked; (iv) Part of WT PC soma close to the nucleus (N) containing mitochondria and other organelles. <b>(v)</b> Part of <i>atc/atc</i> PC soma close to the nucleus (N) and <b>(vi)</b> PC dendrite containing atypical mitochondria (m) with cytoplasmic inclusions (arrowheads). <b>(vii and viii)</b> Structures of concentrically arranged, flattened cisternae, surrounding clusters of mitochondria (arrows) in the <i>atc/atc</i>. Scale bars (i, ii), 1 μm; (iii, iv, v, vii), 500 nm; (vi, viii), 200 nm. 2 mice per genotype were observed with similar findings.</p

Label free proteomic analysis.

<p>Label free proteomic analysis.</p

Cellular alterations in the retina and optic nerve of <i>atc/atc</i> mice.

<p><b>(A)</b> and at higher magnification <b>(A’)</b> Confocal images of 4-week-old mouse retinas showing reduced MAP2 immunostaining in the internal plexiform layer (IPL) of <i>atc/atc</i> mice where the RGC dendrites lie. <b>(B)</b> Amacrine cells immunoreactive for Pax6 and GAD65 are significantly reduced in the <i>atc/atc</i> retina. Quantification of Pax6⁺ cells in the ganglion cell layer (GCL) <b>(C)</b> and the inner nuclear layer (INL) <b>(D)</b> (n = 3 mice per genotype, p = <0.0001 for the GCL and p = 0.03 for the INL). Confocal images of 4-week-old mouse retina <b>(E)</b> and optic nerve head <b>(F)</b>, showing reduced NF immunoreactivity in the innermost part of the retina where RGC axons lie and disorganization of RGC axons in the optic nerve of <i>atc/atc</i> mice. <b>(G)</b> Toluidine blue stained semi-thin optic nerve sections of 4-week-old WT and <i>atc/atc</i> mice. <b>(H)</b> Quantification of axonal numbers per optic nerve section (n = 4 nerves per genotype, p = 0.007). <b>(I)</b> Double immunofluorescence of a myelinated fiber in the optic nerve labeled for CASPR and Na_v. Scale bars (A), 10 μm; (B, E, F) 20 μm; (G) 5μm; (I) 1 μm. <b>(J)</b> Quantification of Na_v+ nodal length (n = 206 WT and 212 <i>atc/atc</i> nodes pooled from 3 mice/genotype, p<0.00001; two-tailed unpaired Student’s <i>t</i>-test; mean values ± SEM).</p

NMJ alterations in <i>atc/atc</i> mice.

<p><b>(A)</b> Schema of the abdominal view of the mouse diaphragm where the endplate band along each costal muscle is visualized in green and the innervating phrenic nerve is shown in red. (Orientation cross: V, ventral; D, dorsal; R, right; L; left). <b>(B</b> and higher magnification in <b>C)</b> Confocal images of whole mount diaphragm specimens (costal muscle; see drawing in A) from 4-week old WT and <i>atc/atc</i> littermates. The endplates are visualized by binding of FITC conjugated a-btx on AChR clusters (green) and show a narrower distribution along the endplate band in the <i>atc/atc</i> diaphragm. <b>(D)</b> Quantification of endplate density (n = 3, p = 0.0043). <b>(E)</b> Quantification of individual end plate area (n = 204 WT and 347 <i>atc/atc</i> endplates pooled from 3 mice/genotype, p<0.00001). <b>(F)</b> Quantification of individual endplate volume by Imaris surface module (n = 150 WT and 296 <i>atc/atc</i> endplates pooled from 3 mice/genotype, p<0.00001). <b>(G)</b> Classification of WT and <i>atc/atc</i> endplates based on maturation level (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006656#sec012" target="_blank">Materials and methods</a>). P: Plaque; SP: Plaque with small perforations; LP: Plaque with large perforations; IP: Immature pretzel (p = 0.0023); MP: Mature pretzel (p = 0.00005) [n = 4; 200 endplates were characterized per sample), ** p≤0.01; *** p≤0.001; **** p≤0.0001; two-tailed unpaired Student’s <i>t</i>-test; mean values ± SEM in (D, G); mean values ± SD in (E, F)]. <b>(H-I)</b> Representative images of WT and <i>atc/atc</i> diaphragm depicting the increased density and smaller size endplates in <i>atc/atc</i> visualized by FITC-a-btx (green), coupled with NF immunostaining [red in merged (J, K) and white in single-channel images (I)]. The arrow in (H) indicates abnormal bifurcation of one fiber to innervate two distinct endplates in <i>atc/atc</i>. <b>(I)</b> Representative images of abnormal NMJs observed in <i>atc/atc</i> mice: poly-innervation of an endplate (white arrow); increased axonal ramification within the endplate (empty arrowheads); axon terminal protrusions extending beyond endplate boundaries reminiscent of retraction bulbs (white arrowheads); an axon that bifurcates and the two fibers innervate two distinct endplates (empty arrows) as also shown in (H). Scale bars (B, C), 200 μm; (G), 10 μm, (H), 20 μm; (I), 10 μm.</p

Complete rescue of the <i>ataxic</i> phenotype through complementation with the human <i>SLC25A46</i> gene.

<p><b>(A)</b> Schematic representation of the NotI digested 202.7Kb genomic fragment used for the generation of <i>TghuSLC25A46</i> mice. <b>(B)</b> Transgene copy number determination by real-time PCR in four transgenic lines, Tg1255, Tg1332, Tg1351 and Tg1358 (n = 6 per group), compared to the two <i>Slc25a46</i> copies of the WT mice, using a primer pair common for both mouse and human <i>Slc25a46</i> genes. <b>(C)</b> Expression pattern of human SLC25A46 on mitochondrial extracts from cerebrum (B), cerebellum (C), spinal cord (SC), muscle (M), heart (H), thymus (T), spleen (S) and liver (L) tissues of <i>TghuSLC25A46/Slc25a46</i>^{<i>atc/atc</i>} mice (Tg1351-<i>atc/atc</i>) through immunoblotting with antibodies against SLC25A46 and the mitochondrial protein GRP75. Complete rescue of <b>(D)</b> premature lethality (n = 8 per group), <b>(E)</b> body weight gain (n = 6 per group) and <b>(F)</b> muscle weakness (n = 6 per group) in <i>atc/atc</i> mice expressing human SLC25A46 (Tg1351-<i>atc/atc</i>). All mice used were sex matched littermates. <b>(G)</b> Confocal images of cerebellar sagittal sections immunostained for calbindin illustrate the restored PC phenotype and ML thickness in transgenic “rescue” Tg1351-<i>atc/atc</i> one-year old mice, as compared to WT and <i>atc/atc</i> mice. <b>(H)</b> Immunofluorescence of vGlut1 and vGlut2 shows normal distribution, in the ML and IGL of the cerebellum of Tg1351-<i>atc/atc</i> mice. Scale bar 40 μm.</p