Spectrins are cytoskeletal proteins that help maintain membrane organization and integrity and aid in signaling events. They create elongated, rod-shaped polypeptides composed of α and β subunits that cross-link actin to form intricate networks along cell membranes. βII-spectrin, encoded by the SPTBN1 gene, is the most abundant β-spectrin in the nervous system. We recently identified a novel neurodevelopmental syndrome caused by de novo variants in the SPTBN1 gene. This syndrome presents as global developmental delay (DD) and intellectual disability (ID), comorbid with autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), seizures, and motor and behavioral abnormalities. Despite these clinical observations, the mechanisms driving this syndrome are unknown. The identified mutations are predominantly missense mutations within the calponin homology (CH) domains of βII-spectrin, the regions responsible for promoting actin binding. Neuroimaging of patients reveal disrupted brain morphology, including a thinning of the corpus callosum (CC), a structure critical for the inter-hemispheric signaling integration and brain circuitry. Our lab previously found that loss of βII-spectrin causes a significant axonal shortening in vitro along with reduced circuitry and CC thinning in vivo. These findings indicate that βII-spectrin is essential for long range axonal growth, stabilization, and circuitry within neural networks.

To better understand the impact of these variants on corpus callosum morphology, we utilized novel knock-in mouse models (KI) harboring p.Thr268Ser (T268S) and p.Leu250Arg (L250R) variants. These variants are among the most clinically severe identified in patients with the syndrome and reside within the CH2 domain, exhibiting both overlapping and differing clinical presentation. Notably, we found that the L250R variant induces protein aggregation, while both L250R and T268S cause axon shortening compared to wild-type (WT) controls. Based on these findings, I hypothesized that mutant βII-spectrin would lead to reductions in axon length and thinning of the CC in these KI mouse models. 

I collected brains from T268S and L250R KI mice and WT controls (n=6 mice/sex/group) at post-natal day (PND) 25, after completion of synaptogenesis and synaptic pruning. I stained cryosections for neurofilament, a marker of the neuronal cytoskeleton. Using high resolution confocal microscopy, I captured images along the corpus callosum. I then used ImageJ to compute and compare the thickness of the corpus callosum in 50 µm intervals extending outward on either side of the brain midline. 

Significant thinning of the corpus callosum was found in L250R KI mice when compared to their WT littermates. Thinning of the corpus callosum was also found in T268S KI mice but there is not a significant difference when compared to the WT littermates. Alterations in CC morphology indicate that mutant βII-spectrin disrupts inter-hemispheric long-range axon projections and cortical circuitry, which may contribute to alterations in development, learning, sociability, and motor functions. CC thinning could be due to axon shortening, breakage, or misguidance during development.