2025 Theses Doctoral

Molecluar mechanisms of motor unit dysfunction in an ALS mouse model

Park, JoonHyung

Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease affecting mostly spinal motor neurons. Its hallmarks include motor neuron loss, muscle atrophy, paralysis and death. Studies in ALS using genetic mutants in mouse models have identified numerous changes in motor neurons at early stages, including excitotoxicity, oxidative stress, denervation at neuromuscular junction (NMJ), mitochondrial abnormalities and motor neuron excitability. Such changes are accompanied by abnormal motor unit function and motor output. Despite attempts to identify early changes in ALS, the causal relationship between abnormal motor output – broadly defined as motor unit dysfunction – and the reported pathological changes are poorly understood.

Here, we used C57BL6/J-SOD1 G93A male mice as the animal model of ALS, together with behavioral, physiological and morphological assays to identify transcriptional changes in vulnerable motor neurons involved in motor dysfunction during ALS onset. We found that ALS mice travelled shorter distances as early as one month old, which progressively reduced to peak around ~P80 and dramatically decreased afterwards until death at ~P150. To identify changes responsible for the observed reduction in distance travelled in SOD1 mutants, we investigated mice at ages in which behavioral differences became apparent. We opted to evaluate the extent of motor unit pathology by focusing on a motor pool involved in running that is known to be vulnerable in ALS, the tibialis anterior (TA) muscle. Although TA motor units do not exhibit any detectable overt changes between SOD1 and control mice at P30, SOD1-G93A mice at P50 exhibited a significant ~40% loss of functional TA motor units, determined by in vivo experiments in which individual motor units were counted following incremental stimulation intensities and quantifying the elicited muscle force. This reduction was not accompanied by motor neuron loss. However, the loss in functional motor units was accompanied by significant decrease in the twitch force alongside ~40% NMJ denervation in the TA muscle. Taken together, the functional changes in motor units and NMJ denervation are deficits in motor unit function that can account for the reduction in distance travelled daily by SOD1 male mice.

We next wanted to identify transcriptional changes in the TA motor neurons of SOD1-G93A mice that could be involved in the observed motor unit pathology. To this end, large TA motor neurons (presumed to be motor neurons from the affected vulnerable motor units) from control and SOD1 males at P30 and P50 were subjected to RNAseq which were selectively collected via laser capture microdissection following labelling with a fluorescent tracer through intramuscular injection. The resultant differentially expressed genes included 6 upregulated and 10 downregulated transcripts. Among these, a mitochondrial gene, Chchd10, which mutations have recently been associated with ALS, revealed elevated levels in motor neurons. Reduction of Chchd10 protein levels within CNS, including motor neurons, via intracerebroventricular injection with an antisense oligonucleotide, we observed a significant protection against motor unit loss that was translated with an increase in distance travelled daily by ALS-treated mice. These results suggest that dysregulation of Chchd10 in SOD1 G93A is involved in the pathogenesis of disease. Furthermore, whilst the downregulation of Chchd10 did not rescue all characteristics of motor unit pathology it implies that multiple genes dysregulation may be involved and be responsible for the different characteristics of motor unit pathology early in the disease onset. It further highlights the downregulation of Chchd10 as a potential novel therapeutic target for ALS.