Acid Sphingomyelinase Deficiency Normalizes Neuronal Function in GCase Deficiency - Unexpected Biological Rescue Effect of Combined Genetic Risk Factors for Parkinson’s Disease

Background: The additive mechanistic effect of genetic risk variants for Parkinson’s disease (PD) is a plausible but largely unproven hypothesis. We investigated the mechanistic interaction between the two lysosomal PD risk genes glucocerebrosidase 1 ( GBA1 ) and sphingomyelinase 1 ( SMPD1 ) in complementing model systems. Methods: Using CRISPR/Cas gene editing, smpd1 mutant ( smpd1 -/- ) zebrafish were generated and crossed to our previously characterised gba1 -/- zebrafish line, generating double knockouts ( gba1 -/- ; smpd1 -/- ). Spontaneous motor behaviour and survival were assessed in WT, single mutants and double mutants. HPLC-based sphingolipid quantification was combined with RNAseq based pathway analysis, assessment of the mitochondrial respiratory chain and quantification of lipid membrane oxidation for in-depth assessment of cellular health across all four genotypes. We also determined the effect of combined glucocerebrosidase (GCase) and acid sphingomyelinase (ASM) inactivation on autophagy and alpha-synuclein homeostasis in the human neuronal cell line SH-SY5Y. Results: Unexpectedly, ASM deficiency rescued the marked behavioural phenotype and prolonged survival in gba1 -/- ; smpd1 -/- double-mutant zebrafish compared to gba1 -/- . RNAseq-based pathway analysis confirmed a profound rescue of neuronal function and intracellular homeostasis. We identified complete reciprocal rescue of mitochondrial respiratory chain function and abolished lipid membrane oxidation in gba1 -/- ; smpd1 -/- compared to gba1 -/- or smpd1 -/- as the underlying rescue mechanism. The complementing in vitro experiments demonstrated an unexpected reduction of α-synuclein levels in human cell lines with combined GCase and ASM deficiency. Conclusions: Our study highlights the importance of functional validation for any putative mechanistic interactions between genetic risk factors and their overall effect on disease-relevant mechanisms rather than readily assuming an additive effect.