Early death of ALS-linked CHCHD10-R15L transgenic mice with central nervous system, skeletal muscle, and cardiac pathology

Abstract

Mutations in coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) have been identified in patients suffering from various degenerative diseases including mitochondrial myopathy, spinal muscular atrophy Jokela type, frontotemporal dementia, and/or amyotrophic lateral sclerosis (ALS). The pathogenic mechanism underlying CHCHD10-linked divergent disorders remains largely unknown. Here we show that transgenic mice overexpressing an ALS-linked CHCHD10 p.R15L mutation leads to an abbreviated lifespan compared with CHCHD10-WT transgenic mice. The occurrence and severity of the phenotype correlates to transgene copy number. Central nervous system (CNS), skeletal muscle, and cardiac pathology is apparent in CHCHD10-R15L transgenic mice. Despite the pathology, CHCHD10-R15L transgenic mice perform comparably to control mice in motor behavioral tasks until very close to death. Although paralysis is not observed, these models provide insight into the pleiotropic nature of CHCHD10 and suggest a contribution of CNS, skeletal muscle, and cardiac pathology to CHCHD10 p.R15L-ALS pathogenesis.

Keywords: Molecular Biology; Molecular Physiology; Neuroscience.

Conflict of interest statement

The authors declare no competing interests.

© 2021 The Authors.

Figures

Graphical abstract

Figure 1

Development and survival analysis of CHCHD10 transgenic mice (A) 6.4kb human CHCHD10 transgene design indicating restriction enzyme cutting sites and exons 1–4. Gray boxes represent untranslated regions. Black boxes represent translated regions. (B) Electropherograms displaying the nucleotide sequences of CHCHD10-WT and CHCHD10-R15L transgenes identified in mouse tail DNA. The pathogenic G > T change leading to the R15L mutation is indicated in pink. A C > A change in the CHCHD10-R15L lines, indicated by the overlying asterisk, is a reported synonymous SNP in humans (rs179468) that was also introduced during site-directed mutagenesis. (C) Agarose gel displaying PCR amplification products of indicated mouse tail DNA using primers directed against human CHCHD10 and murine Actb. A non-transgenic mouse tail DNA sample and H2O sample are used as negative controls, demonstrating the specificity of the primers used. The relative transgene copy number is demonstrated qualitatively. (D) Western blot analysis of transgene expression at the protein level. Mouse forebrain lysate was prepared using RIPA buffer, and immunoblotting was carried out using the indicated antibodies. (E) Quantitation of transgenic human CHCHD10 protein expression in indicated mouse lines from n = 3 independent experiments. Data are represented as mean ± SEM. One-way ANOVA with Tukey's multiple comparison test, ∗p 

Figure 2

Behavioral testing of CHCHD10 transgenic mouse lines (A) Mouse rotarod performance. Mice were tested on an accelerating rotarod task every 60 days beginning at 60 days of age until all CHCHD10-R15L (M) mice in the cohort died. The overlying numbers indicate the number of mice from each line used to calculate mean latency to fall at each time point. Data are represented as mean ± SEM. Two-way ANOVA with Holm-Šidák post hoc statistical analysis summarized in Tables S1–S6. (B) Male mouse body weight measurements. Mouse body weight was measured every 60 days beginning at 60 days of age until all male CHCHD10-R15L (M) mice in the cohort died. The overlying numbers indicate the number of mice from each line used to calculate mean body weights at each time point. Data are represented as mean ± SEM. Two-way ANOVA with Holm-Šidák post hoc statistical analysis summarized in Tables S25–S30. (C) Female mouse body weight measurements. Mouse body weight was measured every 60 days beginning at 60 days of age until all female CHCHD10-R15L (M) mice in the cohort died. The overlying numbers indicate the number of mice from each line used to calculate mean body weights at each time point. Data are represented as mean ± SEM. Two-way ANOVA with Holm-Šidák post hoc statistical analysis summarized in Tables S19–S24. (D) The mean distance traveled by 4-month-old mice of the indicated lines in an open field test over five minutes. Data are represented as mean ± SEM. One-way ANOVA with Tukey's multiple comparison test, ns p > 0.05. (E) The mean distance traveled by 14- to 20-month-old mice of the indicated lines in an open field test over 5 minutes. Data are represented as mean ± SEM. One-way ANOVA with Tukey's multiple comparison test, ns p > 0.05. (F) The mean velocity of 4-month-old mice of the indicated lines in an open field test over 5 minutes. Data are represented as mean ± SEM. One-way ANOVA with Tukey's multiple comparison test, ns p > 0.05. (G) The mean velocity of 14- to 20-month-old mice of the indicated lines in an open field test over 5 minutes. Data are represented as mean ± SEM. One-way ANOVA with Tukey's multiple comparison test, ns p > 0.05. (H) The mean percentage time spent in the center of the open field by 4-month-old mice of the indicated lines. Data are represented as mean ± SEM. One-way ANOVA with Tukey's multiple comparison test, ns p > 0.05. (I) The mean percentage time spent in the periphery of the open field by 4-month-old mice of the indicated lines. Data are represented as mean ± SEM. One-way ANOVA with Tukey's multiple comparison test, ns p > 0.05. (J) The mean percentage time spent in the center of the open field by 14- to 20-month old mice of the indicated lines. Data are represented as mean ± SEM. One-way ANOVA with Tukey's multiple comparison test, ns p > 0.05. (K) The mean percentage time spent in the periphery of the open field by 14- to 20-month-old mice of the indicated lines. Data are represented as mean ± SEM. One-way ANOVA with Tukey's multiple comparison test, ns p > 0.05.

Figure 3

CNS pathology of CHCHD10-R15L transgenic mice (A) A 20-month-old male CHCHD10-WT (L) anterior horn from one side of a transverse spinal cord section stained using an antibody targeting CHCHD10. Region within black box outline is magnified in (B). Scale bar: 100 μm. (B) Higher magnification of region indicated in (A). Scale bar: 25 μm. (C) A 35-month-old male CHCHD10-WT (H) anterior horn from one side of a transverse spinal cord section stained using an antibody targeting CHCHD10. Region within black box outline is magnified in (D). Scale bar: 100 μm. (D) Higher magnification of region indicated in (C). Scale bar: 25 μm. (E) A 10-month-old male CHCHD10-R15L (L) anterior horn from one side of a transverse spinal cord section stained using an antibody targeting CHCHD10. Region within black box outline is magnified in (F). Scale bar, 100 μm. (F) Higher magnification of region indicated in (E) demonstrates the presence of neuritic processes with swellings along the length of the neuritic process. Example of a swelling is indicated by a black arrow. Scale bar, 25 μm. (G) A 13-month-old male CHCHD10-R15L (M) anterior horn from one side of a transverse spinal cord section stained using an antibody targeting CHCHD10. Region within black box outline is magnified in (H). Scale bar, 100 μm. (H) Higher magnification of region indicated in (G) demonstrates the presence many neuritic processes with swellings that appear either in isolation or in sequence along the length of the neuritic process. Examples of swellings are indicated by black arrows. Scale bar, 25 μm. (I) A 14-month-old female CHCHD10-R15L (H) anterior horn from one side of a transverse spinal cord section stained using an antibody targeting CHCHD10. Region within black box outline is magnified in (J). Scale bar, 100 μm. (J) Higher magnification of region indicated in (I) demonstrates the presence many neuritic processes with swellings along the length of the neuritic process. Examples of swellings are indicated by black arrows. Scale bar, 25 μm. (K) Quantitation of the number of swellings in the right and left lumbar spinal cord anterior horn gray matter per section of 8- to 14-month-old mice (n = 3, m = 3/genotype). Data are represented as mean ± SEM. One-way ANOVA with Tukey's multiple comparison test, ns p > 0.05, ∗p 

Figure 4

CNS axonal pathology evident in CHCHD10-R15L transgenic mice (A–C) Immunofluorescence of 10-month-old CHCHD10-R15L (M) transgenic mice spinal cord gray matter using antibodies targeting CHCHD10 and the dendritic protein, Map2. The staining profiles are mutually exclusive, indicating that neuronal dendrites do not harbor the pathological swellings (examples indicated by white arrows). Scale bars, 25 μm. (D–F) Immunofluorescence of 10-month-old CHCHD10-R15L (M) transgenic mice spinal cord gray matter using antibodies targeting CHCHD10 and SMI 32, non-phosphorylated neurofilament heavy chain protein. White arrows indicate SMI 32-positive neurites harbor pathological swellings. Scale bars, 25 μm. (G) Transmission electron microscopy image of 5-month-old CHCHD10-R15L (M) transgenic mouse spinal cord gray matter. A myelinated axon is apparent harboring a swelling resembling those observed under light microscopy (black asterisk). The white box outline is magnified in (H). (H) Membranous protrusions into the amorphous matrix of the swelling are apparent (black arrow).

Figure 5

Widespread myopathic abnormalities in CHCHD10-R15L transgenic mice (A–F) Hematoxylin and eosin staining of quadriceps of a 22-month-old female non-transgenic mouse (A), a 5-month-old male CHCHD10-WT (L) mouse (B), a 14-month-old female CHCHD10-WT (H) mouse (C), a 23-month-old female CHCHD10 R15L (L) mouse (D), a 15-month-old female CHCHD10-R15L (M) mouse (E), and a 12-month-old male CHCHD10-R15L (H) mouse (F). Abundant myopathic features are apparent in the CHCHD10-R15L transgenic mice including central nuclei, hematoxylin invasion, eosinophilic cores, and increased intermyofibrillary cellularity. Milder myopathic changes are apparent in CHCHD10-WT (H) skeletal muscle with occasional central nuclei. Scale bars, 50 μm. (G–N) Immunofluorescence of 14-month-old female CHCHD10-WT (H) and 15-month-old female CHCHD10-R15L (M) transgenic mouse quadriceps using antibodies targeting CHCHD10 and p62. Transgenic CHCHD10 protein expression is somewhat mosaic in appearance and often shares immunoreactivity with p62-positive inclusions. Scale bars, 25μm. (J, K) Muscle respirometry of permeabilized fibers from tibialis anterior (O) or soleus (P) from 7- to 8-month-old male mice (n = 3/group) demonstrating the oxygen consumption of the muscle fibers under sequential substrate, uncoupler, and inhibitor application conditions, as indicated. Data are represented as mean ± SEM. Unpaired t test, ns p > 0.05.

Figure 6

Echocardiography demonstrates transgene copy number and age-related decline in cardiac function (A–C) Representative short axis echocardiography views from a 6-month-old male CHCHD10-WT (L) transgenic mouse. (D–F) Representative short axis echocardiography views from a 7-month-old male CHCHD10-R15L (M) transgenic mouse. (G–I) Representative short axis echocardiography views from a 16-month-old male CHCHD10-R15L (H) transgenic mouse. (J) Echocardiography calculations derived from short axis M-Mode measurements of younger female mice. n = 3 6- to 7-month-old CHCHD10-WT (L) mice; n = 3 7- to 9-month-old CHCHD10-R15L (M) mice. Data are represented as mean ± SEM. Unpaired t test, ns p > 0.05. (K) Echocardiography calculations derived from short axis M-Mode measurements of younger male mice. n = 3 6- to 7-month-old CHCHD10-WT (L) mice; n = 3 6- to 7-month-old CHCHD10-R15L (M) mice. Data are represented as mean ± SEM. Unpaired t test, ns p > 0.05. (L) Echocardiography calculations derived from short axis M-Mode measurements of older female mice. n = 3 18-month-old CHCHD10-WT (H) mice; n = 3 18-month-old CHCHD10-R15L (M) mice; n = 1 15-month-old CHCHD10-R15L (H) mouse. Data are represented as mean ± SEM. Unpaired t test, ns p > 0.05. (M) Echocardiography calculations derived from short axis M-Mode measurements of older male mice. n = 7 12- to 18-month-old CHCHD10-WT (H) mice; n = 1 15-month-old CHCHD10-R15L (M) mouse; n = 5 14- to 16-month-old CHCHD10-R15L (H) mice. Data are represented as mean ± SEM. Unpaired t test, ns p > 0.05, ∗p  0.05.

Figure 7

Transgenic overexpression of CHCHD10 gives rise to CHCHD10-positive, p62-positive, ubiquitin-positive, protein accumulations in cardiac muscle (A–C) A 25-month-old male CHCHD10-WT (L) transverse heart section stained using the indicated antibodies. Scale bars, 100 μm. (D–F) A 20-month-old female CHCHD10-WT (H) transverse heart section stained using the indicated antibodies. Black arrows indicate examples of pathological protein accumulations. Scale bars, 100 μm. (G–I) A 23-month-old female CHCHD10-R15L (L) transverse heart section stained using the indicated antibodies. Black arrows indicate examples of pathological protein accumulations. Scale bars, 100 μm. (J–L) A 23-month-old male CHCHD10-R15L (M) transverse heart section stained using the indicated antibodies. Black arrows indicate examples of pathological protein accumulations. Scale bars, 100 μm. (M–O) A 12-month-old male CHCHD10-R15L (H) transverse heart section stained using the indicated antibodies. Black arrows indicate examples of pathological protein accumulations. Scale bars, 100 μm. (P–U) Immunofluorescence of a 23-month-old CHCHD10-R15L (M) transgenic mouse heart using antibodies targeting CHCHD10 and p62 demonstrating pathological protein inclusions (white arrows). Scale bars, 25 μm.

Figure 8

Chchd10-KO mouse behavioral testing (A) Male mouse body weight measurements. Mouse body weight was measured every 60 days beginning at 60 days of age. The overlying numbers indicate the number of mice from each line used to calculate mean body weights at each time point. Data are represented as mean ± SEM. Two-way ANOVA with Holm-Šidák post hoc statistical analysis summarized in Tables S35–S36. (B) Female mouse body weight measurements. Mouse body weight was measured every 60 days beginning at 60 days of age. The overlying numbers indicate the number of mice from each line used to calculate mean body weights at each time point. Data are represented as mean ± SEM. two-way ANOVA with Holm-Šidák post hoc statistical analysis summarized in Tables S37–S38. (C) Kaplan-Meier survival analysis of Chchd10-KO mice compared with C57BL/6J mice. Death of a mouse for unknown reasons is considered a death event, as indicated by a drop in the line. Mice still living or death of a mouse due to intervention by the investigators are considered censored events, as indicated by tick marks along a line. Median survival for Chchd10-KO mice is 851 days, n = 8 death events and n = 40 censored events. Median survival for C57BL/6J mice is undefined, n = 0 death events and n = 94 censored events. Gehan-Breslow-Wilcoxon test, p = 0.0107. (D) Mouse rotarod performance. Mice were tested on an accelerating rotarod task every 60 days beginning at 60 days of age. The overlying numbers indicate the number of mice from each line used to calculate mean latency to fall at each time point. Data are represented as mean ± SEM. Two-way ANOVA with Holm-Šidák post hoc statistical analysis summarized in Tables S39–S40. (E) Female mouse rotarod performance. Mice were tested on an accelerating rotarod task every 60 days beginning at 60 days of age. The overlying numbers indicate the number of mice from each line used to calculate mean latency to fall at each time point. Data are represented as mean ± SEM. Two-way ANOVA with Holm-Šidák post hoc statistical analysis summarized in Tables S41–S42. (F) Male mouse rotarod performance. Mice were tested on an accelerating rotarod task every 60 days beginning at 60 days of age. The overlying numbers indicate the number of mice from each line used to calculate mean latency to fall at each time point. Data are represented as mean ± SEM. Two-way ANOVA with Holm-Šidák post hoc statistical analysis summarized in Tables S43–S44.