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

The spinocerebellar ataxia type 5 (SCA5) L253P mutation in β-III-spectrin causes high-affinity actin binding. Here we developed a CRISPR knock-in mouse to determine the i <i>n vivo</i> 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 β-III-spectrin in Purkinje neurons. This is marked by loss of β-III-spectrin in distal dendrites, accumulation of β-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 α-II-spectrin, accumulate around the nucleus, form at an early age, and are larger in homozygous <i>β-III-spectrin</i> ^{<i>L253P/L253P</i>} compared to heterozygous <i>β-III-spectrin</i> ^{<i>L253P/+</i>} mice. In contrast, neurons of the hippocampus and cerebral cortex, where β-III-spectrin is also known to be expressed, abnormally accumulate β-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 β-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 β-III-spectrin localization, including decreased levels in distal dendrites, is likely associated with a disruption of β-III-spectrin function in postsynaptic signaling. Consistent with this, and in agreement with prior findings in knockout mice, the L253P <i>β-III-spectrin</i> 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.