Serine 421 is a crucial mediator of pathology in a mouse model of Huntington's disease

Huntington's disease (HD) is a progressive, adult-onset neurodegenerative disease without cure or disease-modifying therapy caused by a polyglutamine (polyQ) expansion in the N-terminal region of the protein huntingtin (Htt). Pathological hallmarks of HD include selective neuron loss and protein aggregation, but the etiology remains unclear. Htt has a highly conserved phosphorylation site at serine 421 (S421-P), and basal levels of S421-P in the brain are inversely correlated to tissue vulnerability. Further, prior work found that S421-P diminishes the toxicity of mutant Htt fragments. However, whether S421-P affects the toxicity of mutant Htt in vivo remains unknown. To determine the role of S421-P in mutant Htt-induced neurodegeneration in vivo, we mutated the human Htt gene within a bacterial artificial chromosome (BAC), previously used to create the BACHD mouse model of HD, to express either an aspartic acid or alanine at position 421 to mimic tonic phosphorylation (S421D) or to prevent phosphorylation (S421A), respectively. We used the mutated BACs to generate new lines of transgenic mice and compared their behavior and neuropathology to the unmodified HD mice and wildtype controls. We found that S421D mice that expressed Htt in the cortex at levels similar to the original BACHD model had significantly less motor and psychiatric-like dysfunction than BACHD mice at multiple time points. Further, 12-month-old BACHD but not S421D mice showed evidence of striatal neurodegeneration when compared to wildtype controls despite a similar load of Htt inclusion bodies. S421A mutants with considerably lower mutant Htt expression demonstrated a mild behavioral phenotype similar to that reported in the BACHD-L (low expressing) line. Surprisingly, while S421D mice had similar levels as BACHD mice of both Htt mRNA and soluble protein in the cortex, they had relatively decreased steady-state levels of protein but not mRNA in the striatum. Biochemical analyses confirmed that S421D decreased the steady-state levels of soluble Htt in a striatal cell line and suggested that this occurs via increased Htt flux through the proteasome. In sum, our work suggests that S421 phosphorylation significantly ameliorates mutant Htt toxicity in mice and thereby validates phosphorylation at S421 as a therapeutic target in HD