Progressive Ataxia, Memory Impairments, and Seizure Episodes in Spna2 R1098Q Mouse Variant Affecting Alpha II Spectrin's Scaffold Stability.

SPTAN1 spectrinopathies refer to a group of rare, inherited diseases associated with damage to non-erythrocytic α-II spectrin (α-II). They are linked to a range of mild to severe neuropathologies of the central and peripheral nervous systems, such as early infantile epileptic encephalopathy type 5, cerebellar ataxia, inherited peripheral neuropathy, and spastic paraplegia. Modeling human SPTAN1 encephalopathies in laboratory animals has been challenging partially because no haploinsufficiency-related phenotypes unfold in heterozygous Spna2 deficient mice nor stable transgenic lines of mice mimicking missense human SPTAN1 mutations have been created to date. Here, we assess the motor and memory performance of a dominant-negative murine Spna2 (SPTAN1) variant carrying a spontaneous point mutation replacing an arginine 1098 in the repeat 10th of α-II with the glutamine (R1098Q). By comparing groups of heterozygous R1098Q mice at different ages, we find evidence for progressive ataxia, and age-related deterioration of motor performance and muscle strength. We also document stress-induced, long-lasting seizure episodes of R1098Q mice and their poor performance in novel object recognition memory tests. Overall, we propose that the complexity of neuropathology-related phenotypes presented by the R1098Q mice recapitulates a number of symptoms observed in human patients carrying SPTAN1 mutations affecting α-II scaffold stability. This makes the R1098Q mice a valuable animal model for preclinical research.

An alpha II spectrin mutant peptide with unstable scaffold structure and increased sensitivity to calpain cleavage.

A spontaneous missense mutation in the alpha II spectrin (αII) gene, replacing a highly conserved arginine 1098 with the glutamine (R1098Q), causes progressive neurodegeneration in heterozygous mutant mice. The molecular mechanism underlying this phenotype is unknown but the accumulation of 150kD αII breakdown products in brains of homozygous mutant embryos suggests an imbalance in the substrate level control of αII cleavage by calpains. This is further supported by in silico simulation predicting unmasked calpain target site and increased spectrin scaffold bending and flexibility of R1098Q mutant peptide. Here, using spectroscopic and in situ enzymatic techniques, we aimed at obtaining direct experimental support for the impact of R1098Q mutation on the αII stability and its propensity for calpain-mediated degradation. Thermal circular dichroism analyses performed on recombinant wildtype and R1098Q mutant αII peptides, composed of spectrin repeat 9-10 revealed that although both had very similar secondary structure contents, thermal stability curve profiles varied and the observed midpoint of the unfolding transition (Tm) was 5.5 °C lower for the R1098Q peptide. Yet, the dynamic light scattering profiles of both peptides closely overlapped, implying the same thermal propensity to aggregate. Calpain digestion of plate-bound αII peptides with and without added calmodulin revealed an enhancement of the R1098Q peptide digestion rate relative to WT control. In summary, these results support the unstable scaffold structure of the R1098Q peptide as contributing to its enhanced intrinsic sensitivity to calpain and suggest physiologic relevance of a proper calpain/spectrin balance in preventing neurodegeneration.

Age-dependent ataxia and neurodegeneration caused by an αII spectrin mutation with impaired regulation of its calpain sensitivity.

The neuronal membrane-associated periodic spectrin skeleton (MPS) contributes to neuronal development, remodeling, and organization. Post-translational modifications impinge on spectrin, the major component of the MPS, but their role remains poorly understood. One modification targeting spectrin is cleavage by calpains, a family of calcium-activated proteases. Spectrin cleavage is regulated by activated calpain, but also by the calcium-dependent binding of calmodulin (CaM) to spectrin. The physiologic significance of this balance between calpain activation and substrate-level regulation of spectrin cleavage is unknown. We report a strain of C57BL/6J mice harboring a single αII spectrin point mutation (Sptan1 c.3293G > A:p.R1098Q) with reduced CaM affinity and intrinsically enhanced sensitivity to calpain proteolysis. Homozygotes are embryonic lethal. Newborn heterozygotes of either gender appear normal, but soon develop a progressive ataxia characterized biochemically by accelerated calpain-mediated spectrin cleavage and morphologically by disruption of axonal and dendritic integrity and global neurodegeneration. Molecular modeling predicts unconstrained exposure of the mutant spectrin's calpain-cleavage site. These results reveal the critical importance of substrate-level regulation of spectrin cleavage for the maintenance of neuronal integrity. Given that excessive activation of calpain proteases is a common feature of neurodegenerative disease and traumatic encephalopathy, we propose that damage to the spectrin MPS may contribute to the neuropathology of many disorders.