Biology subjects

Hays, T. S.

Publications and source records attributed to Hays, T. S..

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↗

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↗

bioRxiv2021

Dynein acts to cluster glutamate receptors and traffic the PIP5 kinase, Skittles, to regulate postsynaptic membrane organization at the neuromuscular junction

Cytoplasmic dynein is essential in motoneurons for retrograde cargo transport that sustains neuronal connectivity. Little, however, is known about dyneins function on the postsynaptic side of the circuit. Here we report distinct postsynaptic roles for dynein at neuromuscular junctions (NMJs). Intriguingly, we show that dynein punctae accumulate postsynaptically at glutamatergic synaptic terminals. Moreover, Skittles, a phosphatidylinositol 4-phosphate 5-kinase that produces PI(4,5)P2 to organize the spectrin cytoskeleton, also localizes specifically to glutamatergic synaptic terminals. Depletion of postsynaptic dynein disrupts the accumulation of Skittles, PI(4,5)P2 phospholipid, and organization of the spectrin cytoskeleton at the postsynaptic membrane. Coincidental with dynein depletion, we observe an increase in the clusters size of ionotropic glutamate receptor (iGluR), and an increase in the amplitude and frequency of mEJPs. However, PI(4,5)P2 levels do not affect iGluR clustering and dynein does not affect the protein levels of iGluR subunits at the NMJ, suggesting a separate, transport independent function for dynein in iGluR cluster organization. As dynein punctae closely associate with iGluR clusters, we propose that dynein physically tethers iGluR clusters at the postsynaptic membrane to ensure proper synaptic transmission.

cell biology↗