ALS/FTD mutant CHCHD10 mice reveal a tissue-specific toxic gain-of-function and mitochondrial stress response

Abstract

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10), a mitochondrial protein of unknown function, cause a disease spectrum with clinical features of motor neuron disease, dementia, myopathy and cardiomyopathy. To investigate the pathogenic mechanisms of CHCHD10, we generated mutant knock-in mice harboring the mouse-equivalent of a disease-associated human S59L mutation, S55L in the endogenous mouse gene. CHCHD10^S55L mice develop progressive motor deficits, myopathy, cardiomyopathy and accelerated mortality. Critically, CHCHD10 accumulates in aggregates with its paralog CHCHD2 specifically in affected tissues of CHCHD10^S55L mice, leading to aberrant organelle morphology and function. Aggregates induce a potent mitochondrial integrated stress response (mtISR) through mTORC1 activation, with elevation of stress-induced transcription factors, secretion of myokines, upregulated serine and one-carbon metabolism, and downregulation of respiratory chain enzymes. Conversely, CHCHD10 ablation does not induce disease pathology or activate the mtISR, indicating that CHCHD10^S55L-dependent disease pathology is not caused by loss-of-function. Overall, CHCHD10^S55L mice recapitulate crucial aspects of human disease and reveal a novel toxic gain-of-function mechanism through maladaptive mtISR and metabolic dysregulation.

Keywords: ALS; CHCHD10; CHCHD2; FTD; Knock-in mice; Mitochondrial integrated stress response; Mitochondrial myopathy; Neurodegeneration; Protein aggregation.

Figure 1.. Generation of CHCHD10^S55L mice

A. Alignment of human and mouse CHCHD10 protein sequences. Amino acid S55 in mice corresponds to the S59 in humans (highlighted in red). B. CRISPR guide RNA to create the TCA→TTA mutation in exon 2 of mouse CHCHD10 gene which changes the codon for serine to leucine. C. DNA sequence analyses of heterozygote and homozygote CHCHD10^S55L mice. The arrow and dashed box indicate the C→T mutation introduced by gene editing in the CHCHD10 gene. D. Kaplan-Meier survival analysis. Heterozygous CHCHD10^S55L mice (n=21) have shortened lifespan than WT mice (n=20). The average lifespan of male (n=8) and female (n=13) heterozygous CHCHD10^S55L mice is shown (inset). E. Heterozygous CHCHD10^S55L mice at 330 days of age show decreased body size and kyphosis. F. and G. Body weight measurements. Female and male CHCHD10^S55L heterozygote mice exhibit a progressive reduction in average body weight, which is more severe in homozygote CHCHD10^S55L mice. n=5 mice per group. Error bars indicate SD. *p<0.05, **p<0.01, ***p<0.001, by ANOVA with repeated measures and Holm-Sidak correction for multiple comparisons.

Figure 2.. CHCHD10^S55L mice manifest motor symptoms and skeletal muscle atrophy but no cognitive impairment.

A. Forepaw grip strength expressed as percent of WT average. n=10 mice per genotype (n=5 females, n=5 males). Error bars indicate SD. *p<0.05, **p<0.01, ***p<0.001 by ANOVA with repeated measures, Holm-Sidak correction for multiple comparisons. B. Exercise endurance measured by time on the treadmill expressed as percent of WT average. n=10 mice per genotype. Error bars indicate SD. *p<0.05, **p<0.01, ***p<0.001 by ANOVA with repeated measures, Holm-Sidak correction for multiple comparisons. C. Image of hind limb musculature of CHCHD10^S55L heterozygote, WT, and CHCHD10 KO mice at 330 days of age. D. Mice on the pole descent task. CHCHD10^S55L mice displayed hindlimb incoordination characterized by poor hindpaw pole grasping (magnified images, arrows). E. Average pole descent time in mice at 330 days of age. n=3 mice per group, 3 trials each mouse. Error bars indicate SEM. *p<0.05, by Student’s t-test. F. and G. Novel object recognition test. Average time spent with the novel object and total exploration time recorded during a 5-min testing period in mice at 250 days of age. n=13 WT (7 males and 6 females) and n=16 CHCHD10^S55L mice (8 males and 8 females) mice per group. Error bars indicate SEM. H. and I. Open field test. Average percent time spent in the center field relative to total time, and total distance travelled. n=13 WT (7 males, 6 females) and n=16 CHCHD10^S55L (8 males, 8 females) per group. Error bars indicate SEM. **p<0.01 by Student’s t-test.

Figure 3.. CHCHD10^S55L mice display cardiac hypertrophy exacerbated in breeding females.

A. Hearts at 330 days of age. Cardiac hypertrophy in CHCHD10^S55L mice is apparent relative to WT mice. B. Average heart weight relative to body weight (mg/g). n=7 WT (3 males, 4 females) and n=7 CHCHD10^S55L (3 males, 4 females) mice per group. Error bars indicate SEM. ***p<0.001 by Student’s t-test. C. H&E staining of heart sections of WT and CHCHD10^S55L mice. Left ventricular wall thickness is increased in CHCHD10^S55L heart. Scale bar = 1 mm. D. Kaplan-Meier survival analysis. CHCHD10^S55L breeding females (n=5) have shortened lifespan than CHCHD10^S55L naïve females (n=5). p=0.0018 by log-rank test. The orange bar indicates the time when females were breeding. E. Hearts of WT naïve and CHCHD10^S55L breeding females. F. Left ventricle ejection fraction measured by echocardiogram in WT naïve and CHCHD10^S55L breeding female mice at 80–90 days of age. n=5. Error bars indicate SEM. ***p<0.001 by Student’s t-test. G. Images captured from echocardiogram during ventricular diastole and systole. Scale bar = 3 mm.

Figure 4.. Histopathological and ultrastructural abnormalities in CHCHD10^S55L muscle and heart.

A. and B. H&E staining of myocardium in the apical region of hearts from mice at 330 days of age. The right panel of each image is a magnification of the area indicated by the square in the left panel. In B, arrows indicate cytoplasmic vacuoles. Scale bar = 50 μm. C. and D. Masson’s trichrome stain of myocardium of mice at 330 days of age. The right panel of is a magnification of the area indicated by the square in the left panel. In D, arrows indicate areas of cardiac interstitium with abundant collagen accumulation. Scale bar = 50 μm. E. Electron micrographs of mitochondria in WT, CHCHD10^S55L, and CHCHD10 KO cardiac tissue. Arrows highlight abnormal membranous structures in mitochondria. Scale bar = 500 nm. F. Quantification of abnormal heart mitochondria (% of total mitochondria). G and H. H&E staining of quadriceps femoris. The right panel of is a magnification of the area indicated by the square in the left panel. Scale bar = 100 μm. I. Electron micrographs of WT, CHCHD10^S55L, and CHCHD10 KO skeletal muscle. Scale bar = 1 μm. J. Quantification of abnormal skeletal muscle mitochondria (% of total mitochondria).

Figure 5.. CHCHD10^S55L mice have abnormal neuronal mitochondria and NMJ defects.

A. ChAT immunostaining of motor neurons in lumbar spinal cord. B. Average number of ChAT+ neurons per lumbar spinal cord section. Bars = 50 μm. n=4, 6 sections per mouse. Error bars indicate SEM. n.s., not significantly different by Student’s t-test. C. Electron micrographs of mitochondria in the soma of lumbar spinal cord motor neurons. Arrows indicate intra-mitochondrial vacuolization in CHCHD10^S55L motor neurons. Scale bar = 500 μm. D. Quantification of abnormal spinal cord motor neuron mitochondria (% of total mitochondria). E. Gastrocnemius NMJs labeled with TRITC-conjugated BTX (red) and NF-200 (green). Bars = 20 μm. F. Average numbers of innervated (BTX+ and NF-200+) NMJs in gastrocnemius expressed as a percent of total NMJs imaged. n=4, 10 NMJs per mouse. Error bars indicate SEM. *p<0.05 by Student’s t-test. G. Images for morphological analysis of NMJs labeled by BTX taken with fixed settings, and converted to binary images by applying identical thresholds. H. Average NMJ complexity assessed as the total length of branches in each NMJ. I. Average area of NMJs. In H and I, n=4 mice per group, 10 NMJs per mouse. Error bars indicate SEM. *p<0.05, **p<0.01 by Student’s t-test.

Figure 6.. CHCHD10^S55L mice have abnormal mitochondria in dopaminergic neurons.

A. TH immunostaining of substantia nigra pars compacta. Bars = 100 μm. B. Average TH+ neurons count in a 200 × 500 μm area encompassing the substantia nigra pars compacta expressed relative to WT. n=4 mice, 4 areas per mouse. n.s., not significantly different by Student’s t-test. C. Electron micrographs of mitochondria in immunogold labeled TH+ neurons of the substantia nigra. Abnormal cristae and vacuolization are visible in CHCHD10^S55L TH+ neurons. The right panel of each image is a magnification of the area indicated by the square in the left panel. Scale bar = 1 μm. D. Quantification of abnormal mitochondria in TH+ neurons (% of total mitochondria).

Figure 7.. Mutant CHCHD10 aggregates with CHCHD2 in mitochondria of affected tissues.

A. Western blot of heart cytosolic (C) and mitochondrial (M) fractions. D10, CHCHD10; D2, CHCHD2; Cyt c, cytochrome c, short (S) and long (L) exposures are shown for D10. The red color in L indicates overexposure. The inner membrane protein Tim23 was used as a loading control. B. CHCHD2 mRNA content measured by RNA-seq and expressed as average log2 fold change relative to WT control. Error bars indicate SEM. ***p<0.001 by Student’s t-test with Benjamini-Hochberg correction. C. Filter trap analysis of total homogenate (T), cytosolic (C) and mitochondrial (M) fractions from heart. Immunoreactive material was detected by immunoblot (IB) with either D10 or D2 antibodies. D. Cardiomyocytes immunolabeled with antibodies against CHCHD10 (red) and cytochrome oxidase subunit 1 (MTCOI). Bars = 25 μm. The lower panel in each image is a magnification of the area indicated by the square in the top panel. E. Filter trap analysis of specific brain regions for D10 and D2 aggregates. Immunoreactive material was detected by immunoblot (IB) with either D10 or D2 antibodies. Mid, midbrain; SC, spinal cord; CTX, cortex; Str, striatum, Cbl, cerebellum. F. Substantia nigra neurons immunostained for TH and CHCHD10. Bars = 10 μm. The right panel in each image is a magnification of the area indicated by the square in the middle panel. Hyper-intense CHCHD10 positive puncta are indicated by arrows. G. Average CHCHD10 immunolabeling intensity in TH+ neurons of the substantia nigra. n=20 WT and n=35 CHCHD10^S55L TH+ neurons, from 4 animals per group. Error bars indicate SEM. ***p<0.001 by Student’s t-test. H. Filter trap analysis of peripheral tissues for D10 and D2. SkM, skeletal muscle; Kdn, kidney; Spl, spleen Panc, pancreas. I. Kyte-Doolittle hydrophobicity plots of the α-helices of CHCHD10 WT and CHCHD10^S55L.

Figure 8.. TDP-43 pathology in mutant CHCHD10 mice.

A. Cardiomyocytes immunolabeled with antibodies against TDP-43 (green) and counterstained with the nuclear probe TOPRO3 (red). The right panels show the merged images. Arrowheads indicate TDP-43 inclusions. B. Cardiomyocytes immunolabeled with antibodies against TDP-43 (green) and TIA-1 (red). The right panels show the merged images. Arrowheads indicate inclusions positive for both TDP-43 and TIA-1. C. Midbrain neurons immunolabeled with antibodies against TDP-43 (green) and TH (red). Arrowheads indicate TDP-43 inclusions. D. Spinal cord motor neurons immunolabeled with antibodies against TDP-43 (green) and ChAT (red). Scale bars = 20 μm.

Figure 9.. CHCHD10^S55L hearts activate a transcriptional profile of mtISR.

A. and B. Volcano plots of transcriptomic changes in CHCHD10 KO and CHCHD10^S55L hearts relative to WT heart. C. Changes (log2 fold) in heart RNA content of genes involved in mtUPR and mtISR. Hif1α, hypoxia-induced factor 1α; Pck2, Phosphoenolpyruvate Carboxykinase 2; Hsp70, heat shock protein 70; Lonp1, Lon protease homolog. n.s. not significant. *p<0.05, ***p<0.001, by Student’s t-test with Benjamini-Hochberg correction. D. Schematic diagram of enzymes involved in serine biosynthesis and one-carbon metabolism under ATF4 transcriptional regulation. PHGDH, phosphoglycerate dehydrogenase; PSAT1, phosphoserine aminotransferase 1; PSPH, phosphoserine phosphatase; MTHFD2, methylenetetrahydrofolate dehydrogenase 2; MTHFD1L, methylenetetrahydrofolate dehydrogenase 1-like; CTH, Cystathionine gamma-lyase. E. Changes (log2 fold) in heart RNA content of genes involved in serine biosynthesis and one-carbon metabolism. ***p<0.001, by Student’s t-test with Benjamini-Hochberg correction. F. Changes (log2 fold) in heart RNA content of genes encoding subunits of the mitochondrial oxidative phosphorylation complex I (Ndufs7), II (Sdha), III (Uqcrfs1), IV (Cox7a1), and V (Atp5a1) and cytochrome c (cyc1). ***p<0.001, by Student’s t-test with Benjamini-Hochberg correction.

Figure 10.. mtISR leads to mitochondrial dysfunction and systemic metabolic remodeling in CHCHD10^S55L mice.

A. Western blots of total heart homogenates for MTHFD1L, MTHFD2, ATF4, p62, phospho-S6, total S6. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as a loading control. B. Cardiomyocyte immunolabeling for p62 (green) and Tom20 (red). The right panels show the merged images. Bars = 20 μm. C. Average oxygen consumption rates in freshly isolated heart mitochondria measured with complex I (glutamate/malate) and complex II (glutamate/succinate) substrates and expressed as a percentage of WT. n=6. Error bars indicate SEM. **p<0.01 by Students t-test. D. Average FGF-21 levels in mouse serum at 180 days (n = 10 WT, 6 CHCHD10^S55L, 4 KO), 280 days (n = 2 WT, 6 CHCHD10^S55L, 2 KO), and 380 days (n = 8 WT, 4 CHCHD10^S55L, 2 KO) expressed relative to WT. Error bars indicate SEM. ***p<0.001 by Student’s t-test. E. Visceral (top panel) and subcutaneous (lower panel) fat pads at necropsy. F. Schematic representation of the pathway of mTORC1/ATF4-mediated mtISR triggered by CHCHD10/2 aggregation and mitochondrial dysfunction in CHCHD10^S55L mice.