Therapeutic modulation of the calpastatin/calpain pathway restores calpain-mediated synaptic proteolysis and preserves motor neurons survival and function in C9orf72 ALS

BackgroundA hexanucleotide repeat expansion (GGGGCC) in the C9orf72 gene is the most prevalent genetic cause of ALS, with early neuromuscular junction (NMJ) dysfunction being a key pathological feature. Current therapies provide only limited symptomatic relief, underscoring the need for targeted, mechanism-based interventions. MethodUsing a C9orf72 ALS zebrafish model (C9-miR) and patient-derived induced pluripotent stem cell (iPSC) motor neurons, we identified significant downregulation of calpastatin, the endogenous inhibitor of calpains, a calcium-dependent protease family implicated in neurodegeneration. ResultsWe demonstrate that restoring calpastatin function through calpain inhibition with calpeptin or a novel calpastatin-derived peptide ameliorates locomotor deficits and NMJ dysfunction in the C9-miR zebrafish model. These interventions enhance synaptic vesicle turnover and quantal release at the NMJ while improving motor neuron excitability and synaptic integrity in iPSC-derived motor neurons. N-terminomic/TAILS mass spectrometry approaches revealed direct calpain-mediated cleavage of synaptic proteins in motor neurons derived from C9orf72 patients. Proteolysis of key synaptic proteins, such as spectrin, is prevented by calpeptin and calpastatin peptide treatments. ConclusionsOur findings establish the calpastatin/calpain axis as a pivotal regulator of synaptic function in C9orf72-associated ALS and identify it as a promising therapeutic target, offering a novel strategy to restore synaptic transmission and potentially halt disease progression.