Impaired motor activity in a CRISPR SCA5 L253P knock-in mouse is associated with selective beta-III-spectrin subcellular redistribution in the cerebellum

Avery, A. W.; O'Callaghan, B. L.; Thiel, M. T.; Denha, S. A.; O'Callaghan, D. G.; Cismas, E. M.; Lamp, J.; Orr, H. T.; Hays, T. S.

doi:10.64898/2026.03.14.711824

bioRxiv · 10.64898/2026.03.14.711824

Impaired motor activity in a CRISPR SCA5 L253P knock-in mouse is associated with selective beta-III-spectrin subcellular redistribution in the cerebellum

Avery, A. W.·O'Callaghan, B. L.·Thiel, M. T.·Denha, S. A.·O'Callaghan, D. G.·Cismas, E. M.·Lamp, J.·Orr, H. T.·Hays, T. S.

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Abstract

The spinocerebellar ataxia type 5 (SCA5) L253P mutation in {beta}-III-spectrin causes high-affinity actin binding. Here we developed a CRISPR knock-in mouse to determine the in vivo impact of L253P on Purkinje neurons and motor activity, and to establish a model for future testing of SCA5 therapeutics. Significantly, the knock-in mouse shows impaired motor activity on elevated beam assays at 20 weeks. In the cerebellum, L253P causes a subcellular redistribution of {beta}-III-spectrin in Purkinje neurons. This is marked by loss of {beta}-III-spectrin in distal dendrites, accumulation of {beta}-III-spectrin at the plasma membrane of the soma and proximal dendrites, and formation of inclusions in the soma. The inclusions additionally contain F-actin and -II-spectrin, accumulate around the nucleus, form at an early age, and are larger in homozygous {beta}-III-spectrinL253P/L253P compared to heterozygous {beta}-III-spectrinL253P/+ mice. In contrast, neurons of the hippocampus and cerebral cortex, where {beta}-III-spectrin is also known to be expressed, abnormally accumulate {beta}-III-spectrin at the plasma membrane but do not form inclusions. To gain greater insight into disease mechanisms, unbiased proteomics identified over 150 cerebellar proteins that physically associate with {beta}-III-spectrin. Of these, cluster analysis revealed a group of 41 proteins, including glutamate receptors, SERCA2, and CaMKII, linked to synaptic transmission. Thus, the effect of the L253P to alter {beta}-III-spectrin localization, including decreased levels in distal dendrites, is likely associated with a disruption of {beta}-III-spectrin function in postsynaptic signaling. Consistent with this, and in agreement with prior findings in knockout mice, the L253P {beta}-III-spectrin knock-in mouse here shows that CaMKII, a calcium sensor and key mediator of glutamate signaling, is ~2-fold activated. Further, the abundance of EAAT4, a glutamate transporter, is significantly reduced. The L253P knock-in mouse primes future preclinical testing of SCA5 therapeutics, such as small molecule modulators of spectrin-actin binding, and glutamate and calcium signaling pathways.

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Avery, A. W., O'Callaghan, B. L., Thiel, M. T., Denha, S. A., O'Callaghan, D. G., Cismas, E. M., Lamp, J., Orr, H. T., Hays, T. S.. 2026-03-17. Impaired motor activity in a CRISPR SCA5 L253P knock-in mouse is associated with selective beta-III-spectrin subcellular redistribution in the cerebellum. https://doi.org/10.64898/2026.03.14.711824

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