Mutant huntingtin and glycogen synthase kinase 3‐β accumulate in neuronal lipid rafts of a presymptomatic knock‐in mouse model of Huntington's disease

Abstract

Patients with Huntington's disease have an expanded polyglutamine tract in huntingtin and suffer severe brain atrophy and neurodegeneration. Because membrane dysfunction can occur in Huntington's disease, we addressed whether mutant huntingtin in brain and primary neurons is present in lipid rafts, which are cholesterol-enriched membrane domains that mediate growth and survival signals. Biochemical analysis of detergent-resistant membranes from brains and primary neurons of wild-type and presymptomatic Huntington's disease knock-in mice showed that wild-type and mutant huntingtin were recovered in lipid raft-enriched detergent-resistant membranes. The association with lipid rafts was stronger for mutant huntingtin than wild-type huntingtin. Lipid rafts extracted from Huntington's disease mice had normal levels of lipid raft markers (G(alphaq), Ras, and flotillin) but significantly more glycogen synthase kinase 3-beta. Increases in glycogen synthase kinase 3-beta have been associated with apoptotic cell death. Treating Huntington's disease primary neurons with inhibitors of glycogen synthase kinase 3-beta reduced neuronal death. We speculate that accumulation of mutant huntingtin and glycogen synthase kinase 3-beta in lipid rafts of presymptomatic Huntington's disease mouse neurons contributes to neurodegeneration in Huntington's disease.

Native Mutant Huntingtin in Human Brain

“…Preparation of Primary Neurons-Embryonic cortex of WT and HD 140Q/140Q mice were used as the source of primary neurons and prepared according to our previously published method (23). The protocol resulted in Ͼ99% neurons in the cultures.…”

“…Before electrophoresis, samples were diluted in 1ϫ Native PAGE TM sample buffer and incubated in 2 mM MgCl 2 and 1 unit/l benzonase (Sigma) for 15 min at room temperature. Parallel cultures prepared from the same embryos were used to detect viability by MTT assay as previously described (23,36).…”

“…Huntingtin is known to associate with membranes and phospholipids and inserts into the lipid bilayer (21)(22)(23). The association of htt with membranes and its potential structure as a protein enriched in heat repeats with a continuous hydrophobic core (noted above) suggested to us that htt in brain might be amenable to analysis by Blue Native PAGE.…”

“…Analysis of Native Mutant htt in HD 140Q/140Q Primary Neurons-In recent studies we showed that primary embryonic cortical neurons from homozygous HD 140Q/140Q mice develop elevated levels of reactive oxygen species at day 8 in vitro and increased cell death at day 10 in vitro compared with WT primary cortical neurons (23,36). The HD 140Q/140Q neurons in culture do not show visible signs of aggregates using anti-htt antibody.…”

Convergent pathogenic pathways in Alzheimer's and Huntington's diseases: shared targets for drug development

“…Lithium treatment has also shown benefits in mouse models of HD 57,110 , and levels of GSK3β are increased prior to symptom onset in an HD model 111 .The treatment of primary neurons derived from these mice with GSK3β inhibitors reduces neuronal death 111 (Suppl Table S3). …”