PT  - JOURNAL ARTICLE
AU  - Laura. J. Smith
AU  - Magdalena. M. Bolsinger
AU  - Kai-Yin. Chau
AU  - Matthew. E. Gegg
AU  - Anthony. H. V. Schapira
TI  - The <em>GBA</em> variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines
AID  - 10.1101/2022.06.01.494130
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.06.01.494130
4099  - http://biorxiv.org/content/early/2022/06/01/2022.06.01.494130.short
4100  - http://biorxiv.org/content/early/2022/06/01/2022.06.01.494130.full
AB  - Sequence variants or mutations in the GBA gene are numerically the most important risk factor for Parkinson disease (PD). The GBA gene encodes for the lysosomal hydrolase enzyme, glucocerebrosidase (GCase). GBA mutations often reduce GCase activity and lead to impairment of the autophagy-lysosomal pathway, which is important in the turnover of alpha-synuclein, accumulation of which is a key pathological hallmark of PD. Although the E326K variant is one of the most common GBA variants associated with PD, there is limited understanding of its biochemical effects. We have characterised homozygous and heterozygous E326K variants in human fibroblasts. We found that E326K variants did not cause significant loss of GCase protein or activity, endoplasmic reticulum (ER) retention or ER stress, in contrast to the L444P GBA mutation. This was confirmed in human dopaminergic SH-SY5Y neuroblastoma cell lines over-expressing GCase with either E326K or L444P protein. Despite no loss of GCase activity, a significant increase of insoluble alpha-synuclein aggregates in E326K and L444P mutants was observed. Notably, SH-SY5Y over-expressing E326K demonstrated a significant increase in lipid droplet number under basal conditions, which was exacerbated following treatment with the fatty acid oleic acid. Similarly, a significant increase in lipid droplet formation following lipid loading was observed in heterozygous and homozygous E326K fibroblasts. In conclusion, the work presented here demonstrates that the E326K mutation behaves differently to common loss of function GBA mutations, however lipid dyshomeostasis and alpha-synuclein pathology is still evident.Competing Interest StatementThe authors have declared no competing interest.PDParkinson diseaseGCaseGlucocerebrosidaseEREndoplasmic reticulumGBA-PDGBA mutation-associated Parkinson diseaseGlcCerGlucosylceramideGDGlucosylsphingosine – GlcSph Gaucher diseaseALPAutophagic-lysosomal pathwayUPRUnfolded protein responseROSReactive oxygen speciesDMEMDulbecco’s modified eagle mediaFBSFoetal bovine serumNEAANon-essential amino acidsPBSPhosphate buffered salineECLElectro-chemi-luminescenceCBEConduritol-B-epoxideOAOleic acidSEMstandard error of the mean