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bioRxiv · 10.64898/2025.12.08.692907

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

Lescouzeres, L.·Butti, Z.·Chaineau, M.·Young, D.·You, Z.·Nicouleau, M.·Chen, C. X.-Q.·Haghi, G.·Aprahamian, N.·Zaouter, C.·Dufour, A.·Durcan, T.·Patten, S. A.
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Abstract

A 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. Using 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. We demonstrate that restoring calpastatin activity with a cell-permeable calpastatin-derived peptide or the small molecule, calpeptin, 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 revealed direct calpain-mediated cleavage of synaptic proteins in motor neurons derived from C9orf72 patients. Proteolysis of novel ALS-relevant synaptic and axonal proteins is prevented by calpeptin and calpastatin peptide treatments. Our findings establish the calpastatin 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.

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Lescouzeres, L., Butti, Z., Chaineau, M., Young, D., You, Z., Nicouleau, M., Chen, C. X.-Q., Haghi, G., Aprahamian, N., Zaouter, C., Dufour, A., Durcan, T., Patten, S. A.. 2025-12-11. Therapeutic modulation of the calpastatin/calpain pathway restores calpain-mediated synaptic proteolysis and preserves motor neurons survival and function in C9orf72 ALS. https://doi.org/10.64898/2025.12.08.692907

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