ALS-associated mutation SOD1G93A leads to abnormal mitochondrial dynamics in osteocytes

Abstract

While the death of motor neuron is a pathological hallmark of amyotrophic lateral sclerosis (ALS), defects in other cell types or organs may also actively contribute to ALS disease progression. ALS patients experience progressive skeletal muscle wasting that may not only exacerbate neuronal degeneration, but likely has a significant impact on bone function. In our previous published study, we have discovered severe bone loss in an ALS mouse model with overexpression of ALS-associated mutation SOD1 (G93A). Here we further provide a mechanistic understanding of the bone loss in ALS animal and cellular models. Combining mitochondrial fluorescent indicators and confocal live cell imaging, we discovered abnormalities in mitochondrial network and dynamics in primary osteocytes derived from the same ALS mouse model G93A. Those mitochondrial defects occur in ALS mice after the onset of neuromuscular symptoms, indicating that mitochondria in bone cells respond to muscle atrophy during ALS disease progression. To examine whether ALS mutation has a direct contribution to mitochondrial dysfunction independent of muscle atrophy, we evaluated mitochondrial morphology and motility in cultured osteocytes (MLO-Y4) with overexpression of mitochondrial targeted SOD1. Compared with osteocytes overexpressing the wild type SOD1 as a control, the SOD1 osteocytes showed similar defects in mitochondrial network and dynamic as that of the primary osteocytes derived from the ALS mouse model. In addition, we further discovered that overexpression of SOD1 enhanced the expression level of dynamin-related protein 1 (Drp1), a key protein promoting mitochondrial fission activity, and reduced the expression level of optic atrophy protein 1 (OPA1), a key protein related to mitochondrial fusion. A specific mitochondrial fission inhibitor (Mdivi-1) partially reversed the effect of SOD1 on mitochondrial network and dynamics, indicating that SOD1 likely promotes mitochondrial fission, but suppresses the fusion activity. Our data provide the first evidence that mitochondria show abnormality in osteocytes derived from an ALS mouse model. The accumulation of mutant SOD1 protein inside mitochondria directly causes dysfunction in mitochondrial dynamics in cultured MLO-Y4 osteocytes. In addition, the ALS mutation SOD1-mediated dysfunction in mitochondrial dynamics is associated with an enhanced apoptosis in osteocytes, which could be a potential mechanism underlying the bone loss during ALS progression.

β-aminoisobutyric Acid, l-BAIBA, Is a Muscle-Derived Osteocyte Survival Factor

“…Primary bone osteocytes from DMP1-GFP mice or MLO-Y4 cells were incubated with 200 nM MitoTracker Deep Red FM (Invitrogen, M22426) for 30 min at 37°C, for visualization of mitochondrial morphology and dynamics, or incubated with 50 nM TMRE (Invitrogen, T669) for 10 min at 37°C, for monitoring mitochondrial inner membrane potential as previously described (Wang et al, 2018; Yi et al, 2011; Zhou et al, 2010). MitoTracker Deep Red FM was excited at 633 nm and its emitted fluorescence was collected at 640–690 nm.…”

Species‐specific consequences of an E40K missense mutation in superoxide dismutase 1 (SOD1)

“…25 There were no differences in the morphology of the mitochondrial networks in cells expressing the WTH and Mut-H SOD1 variants and there were no statistically significant differences between the cells expressing the dog SOD1 variants evaluating the aspect ratio. Imbalances in their morphology can reflect impaired functionality and are reported in ALS-models.…”

“…Imbalances in their morphology can reflect impaired functionality and are reported in ALS-models. 25 There were no differences in the morphology of the mitochondrial networks in cells expressing the WTH and Mut-H SOD1 variants and there were no statistically significant differences between the cells expressing the dog SOD1 variants evaluating the aspect ratio. However, the form factor (branching) was significantly greater in cells expressing the Mut-D SOD1 protein compared to cells expressing the wild-type dog F I G U R E 2 Genomic sequencing from five different Perissodactyla species (Horse, Przewalski's horse, Onager, Zebra, Donkey.…”

“…1,3 A recent paper highlighted the importance of the residue at the 40 th codon (and its molecular bonds with 91 st codon) in human SOD1 in determining the correct structure of the enzyme, as substitution of the wild-type glutamic acid (E) residue to certain residues (aspartate (D), glycine (G), or valine (V)) introduced steric hindrance within the β-barrel plug, and increased protein accumulation. 3,22 Other common cellular perturbations seen in ALS-models include mitochondrial dysfunction and increased oxidative stress [23][24][25] but these (to date) have not been examined for the dog SOD1 mutation. These authors also reported normal enzymatic activity and dimerization of the mutant dog enzyme (SOD1 exists as a homodimer)likely because the 40 th codon, (in Greek Key loop 3), is remote from the active site, metalation sites, and structural dimerization bonds.…”

Structural and functional properties of bone are compromised in amyotrophic lateral sclerosis mice

“…In ALS SOD1 G93A mice, mitochondrial dysfunction was observed in cortical bone osteocytes due to accumulation of mutant SOD1 G93A protein (23). To determine if mitochondrial dysfunction continues despite absence of skeletal muscle atrophy, mutant SOD1 G93A protein was overexpressed in the osteocytic cell line MLO-Y4 cells.…”