Neurodegeneration and Reduced Muscle Innervation in a Mouse Model Lacking the SPECC1L C‐terminal Actin‐Binding Domain

Abstract

SPECC1L cytoskeletal protein is known to associate with microtubules via its second coiled coil domain and with actin filaments via a C-terminal calponin homology domain (CHD). Autosomal dominant point mutations in SPECC1L cluster in these two domains and result in a range of defects, including craniofacial malformations, omphalocele, as well as intellectual disability and autism. To study SPECC1L function, we generated many mouse alleles, including one with truncation of the C-terminal 510 amino acids that also removes CHD (ΔC510). Homozygous ΔC510 mutants are perinatal lethal while heterozygotes (hets) are viable and fertile. We noticed that some ΔC510 hets showed hyperactive behavior in their cages. A systematic actimeter-based analysis showed that these hyperactive ΔC510 hets travelled more distance and showed more stereotypies than non-hyperactive hets and wildtype. Interestingly, all hyperactive ΔC510 hets were females. Additional analyses with ΔC510 hets identified significant differences in marble burying and aspects of social chamber interaction, as well as poor performance on grip strength and inverted screen tests. The latter two measurements assessing muscle strength were significantly reduced in male ΔC510 hets. While SPECC1L is broadly expressed, its expression was strongest in cerebellar Purkinje cells and in nerves innervating the muscle. A detailed analysis of ΔC510 het cerebellums showed a significant loss of Purkinje cells. However, female ΔC510 hets showed a significant decrease in posterior lobes, which are associated with motor function and hyperactivity, while male ΔC510 hets showed a greater loss in anterior lobes associated with muscle strength and hypotonia. We also identified a significant decrease in the number of fully innervated neuromuscular junctions (NMJs) distally in both ΔC510 het adults and in E18.5 embryos, indicating abnormal NMJ development. At the molecular level, we identified that both wildtype and ΔC510proteins strongly associated with neurofilaments. In ΔC510 hets, the truncated SPECC1L and neurofilaments are abnormally expressed in the cerebellar molecular layer at presynaptic terminals. We propose that SPECC1L is required for normal localization of neurofilaments, and that loss of actin binding leads to abnormal presynaptic localization resulting in both Purkinje cell degeneration and poor NMJ innervation. Our results present novel neuronal function of SPECC1L and provide mechanistic insights into the underpinnings of the neuronal deficits observed in patients with SPECC1L mutations.