Biology subjects

Denha, S. A.

Publications and source records attributed to Denha, S. A..

3 recordsLinked to original sources

bioRxiv2026

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

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.

neuroscience↗

bioRxiv2023

Increased actin binding is a shared molecular consequence of numerous spinocerebellar ataxia mutations in β-III-spectrin

Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the SPTBN2 gene encoding the cytoskeletal protein {beta}-III-spectrin. Previously, we demonstrated that a L253P missense mutation, localizing to the {beta}-III-spectrin actin-binding domain (ABD), causes increased actin-binding affinity. Here we investigate the molecular consequences of nine additional ABD-localized, SCA5 missense mutations: V58M, K61E, T62I, K65E, F160C, D255G, T271I, Y272H, and H278R. We show that all of the mutations, similar to L253P, are positioned at or near the interface of the two calponin homology subdomains (CH1 and CH2) comprising the ABD. Using biochemical and biophysical approaches, we demonstrate that the mutant ABD proteins can attain a well-folded state. However, thermal denaturation studies show that all nine mutations are destabilizing, suggesting a structural disruption at the CH1-CH2 interface. Importantly, all nine mutations cause increased actin binding. The mutant actin-binding affinities vary greatly, and none of the nine mutations increase actin-binding affinity as much as L253P. ABD mutations causing high-affinity actin binding, with the notable exception of L253P, appear to be associated with early age of symptom onset. Altogether, the data indicate increased actin-binding affinity is a shared molecular consequence of numerous SCA5 mutations, which has important therapeutic implications.

biochemistry↗

bioRxiv2022

Identification of β-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia

{beta}-III-spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells, and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the cytoskeletal protein {beta}-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small molecule actin-binding modulators of the L253P mutant {beta}-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P {beta}-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay, we screened a 2,684-compound library of FDA-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous co-sedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a SCA5-specific therapeutic. Furthermore, our validated FRET-based in vitro HTS platform is poised for screening large compound libraries for {beta}-III-spectrin ABD modulators.

biophysics↗