Meigen Yu, Hui Ye, Ruth B. De-Paula, Carl Grant Mangleburg, Timothy Wu, Tom V. Lee, Yarong Li, Duc Duong, Bridget Phillips, Carlos Cruchaga, Genevera I. Allen, Nicholas T. Seyfried, Ismael Al-Ramahi, Juan Botas, Joshua M. Shulman
Abstract
sHeterozygous variants in the glucocerebrosidase (GBA) gene are common and potent risk factors for Parkinson’s disease (PD). GBA also causes the autosomal recessive lysosomal storage disorder (LSD), Gaucher disease, and emerging evidence from human genetics implicates many other LSD genes in PD susceptibility. We have systemically tested 86 conserved fly homologs of 37 human LSD genes for requirements in the aging adult Drosophila brain and for potential genetic interactions with neurodegeneration caused by α-synuclein (αSyn), which forms Lewy body pathology in PD. Our screen identifies 15 genetic enhancers of αSyn-induced progressive locomotor dysfunction, including knockdown of fly homologs of GBA and other LSD genes with independent support as PD susceptibility factors from human genetics (SCARB2, SMPD1, CTSD, GNPTAB, SLC17A5). For several genes, results from multiple alleles suggest dose-sensitivity and context-dependent pleiotropy in the presence or absence of αSyn. Homologs of two genes causing cholesterol storage disorders, Npc1a / NPC1 and Lip4 / LIPA, were independently confirmed as loss-of-function enhancers of αSyn-induced retinal degeneration. The enzymes encoded by several modifier genes are upregulated in αSyn transgenic flies, based on unbiased proteomics, revealing a possible, albeit ineffective, compensatory response. Overall, our results reinforce the important role of lysosomal genes in brain health and PD pathogenesis, and implicate several metabolic pathways, including cholesterol homeostasis, in αSyn-mediated neurotoxicity.
Introduction
Parkinson’s disease (PD) is a common and incurable neurodegenerative disorder with strong evidence for genetic etiology [1]. Heterozygous carriers for variants in the glucocerebrosidase (GBA) gene have an approximately 5-fold increased risk of PD, and GBA variants also modify PD clinical manifestations, causing more rapid progression and susceptibility for dementia [1,2]. Whereas partial loss-of-function is associated with PD, complete or near-complete loss of GBA causes Gaucher disease, a recessive lysosomal storage disorder (LSD) [3,4]. GBA encodes the lysosomal enzyme glucocerebrosidase (GCase), which catalyzes the breakdown of glucosylceramide, a substrate that accumulates along with other, more complex sphingolipids in Gaucher disease and possibly PD [5].
Materials and method
PD GWAS summary statistics [10] were analyzed using MAGMA v1.10 [17]. Gene location and European reference files for the GRCh37 genome build were downloaded from MAGMA webpage (https://ctg.cncr.nl/software/magma), and the BEDTools v2.26.0 [57] intersect function was used to interrogate SNPs from the PD GWAS summary statistics. MAGMA annotation, gene analysis and gene-set analysis steps were performed using default parameters. The list of LSD genes [8] (S1 Table) was used under the—set-anot parameter for the gene-set analysis, and selected genes were excluded for the sensitivity analysis. The X-linked LSD genes, GLA, IDS, and LAMP2, were excluded from GWAS, so summary statistics were not available for aggregate variants tests.
Results
Previously, we and others have discovered evidence for an aggregate burden of rare genetic variants among LSD genes in association with PD risk [8,9]. Although several LSD genes have also been implicated at susceptibility loci from PD genome-wide association studies [10], to our knowledge, a systematic analysis for common variant associations across all LSD genes has not been performed. Leveraging publicly available summary statistics from 56,306 PD cases and 1.4 million control subjects [10], we used the multi-marker analysis of genomic annotation (MAGMA) tool [17] to examine LSD genes for enrichment of variants associated with PD. MAGMA computes an overall gene-set test statistic considering all variants falling within gene intervals, including adjustments for gene size and regional linkage disequilibrium. The full LSD gene set was significantly enriched for variants associated with PD risk (n = 51 loci, p = 0.0011) (S1 Table); the X-linked LSD genes (GLA, IDS, and LAMP2) were excluded from the available genome-wide association dataset. In order to identify possible drivers for the gene set association, we examined MAGMA output considering each of the LSD genes independently. These results identify GBA and 9 other LSD genes with aggregate evidence for common variant associations (p < 0.05): IDUA, SCARB2, CLN8, GNPTAB, ARSA, GALC, CLN5, NAGLU, and CTSD. We also performed a sensitivity analysis showing that the LSD gene set association remains significant after excluding either GBA (n = 50 loci, p = 0.014) or the top 3 genes (GBA plus SCARB2 and IDUA) (n = 47 loci, p = 0.03), which are similarly localized to regions with genome-wide significant associations in the dataset. These results reinforce the genetic connection between causes of LSDs and PD, revealing an important role for common variant associations.
Discussion
Mounting evidence supports an important connection between the genetic mechanisms of LSDs and PD. Using a cross-species, functional screening strategy, we discover 14 conserved human LSD genes with homologs that robustly enhance αSyn-mediated neurotoxicity when their activity is reduced in Drosophila models. The majority of these genes also show evidence for αSyn-independent requirements, and in several cases our data is suggestive of dose-sensitivity and context-dependent pleiotropy similar to GBA in PD and Gaucher disease. Two of the genes identified by our screen, GBA and SMPD1, are established PD risk genes [2,7,26], and our results confirm and extend data from other animal and cellular models (discussed below). In other cases, the evidence from human genetics may be more modest, but our discovery of genetic interactions with αSyn mechanisms increases the possibility that these genes may be bona fide PD risk factors. For example, in our prior analysis of exome sequencing data, CTSD and SLC17A5 showed suggestive associations with PD risk [8]; however, the rarity of variants and available sample sizes have limited statistical power to confirm such loci. Our results also provide experimental support for LSD genes, such as SCARB2 and GNPTAB, which are candidates at susceptibility loci from PD GWAS [10]. Indeed, GWAS rarely identify responsible genes definitively, but instead highlight regions that usually contain many viable candidates. As a group, we and others previously showed that genes causing LSDs harbor an aggregate burden of damaging rare variants among PD cases [8]. Here, using available GWAS data, we also demonstrate similar significant enrichment for more common variants associated with PD risk. Overall, the LSD genes prioritized by our functional screening strategy are outstanding candidates for further investigation as PD risk factors, including using both human genetics and experimental approaches.
Acknowledgments
We thank our colleagues, Drs. Chun Han, Michael Hoch, and Linda Partridge for generously providing Drosophila strains. We also thank the Bloomington Drosophila stock center, the Vienna Drosophila RNAi Center, the TRiP at Harvard Medical School, the National Institute of Genetics, Japan, and FlyBase. We are grateful to Dr. Laurie Robak for helpful discussions and feedback on the manuscript.
Citation: Yu M, Ye H, De-Paula RB, Mangleburg CG, Wu T, Lee TV, et al. (2023) Functional screening of lysosomal storage disorder genes identifies modifiers of alpha-synuclein neurotoxicity. PLoS Genet 19(5): e1010760. https://doi.org/10.1371/journal.pgen.1010760
Editor: Bingwei Lu, Stanford University School of Medicine, UNITED STATES
Received: July 25, 2022; Accepted: April 25, 2023; Published: May 18, 2023
Copyright: © 2023 Yu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: Drosophila proteomics data are included with supporting information (S1 and S2 Datasets). Human proteomics were obtained from the Parkinson’s Progression Markers Initiative (PPMI) database (www.ppmi-info.org/access-data-specimens/download-data). In order to ensure regulatory compliance for human subjects research and the informed consent process, PPMI data are available to qualified investigators following submission of a data use agreement and short online application. All numerical data contributing to graphical and statistical analysis is included in the S3 Dataset.
Funding: JMS, JB, and NTS were supported by grants from the National Institutes of Health (U01AG061357, R01AG057339). JMS was additionally supported by NIH (R21AG068961), Huffington Foundation, the Burroughs Wellcome Foundation (Career Award for Medical Scientists), the Effie Marie Cain Chair in Alzheimer’s Research, a gift from Terry and Bob Lindsay, and the Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital. MY was supported by a grant from the National Institutes of Health (F31NS115364). HY received support from the Parkinson’s Foundation (PF-PRF-830012) and Alzheimer’s Association (AARF-21-848017). The Pathology and Histology Core at Baylor College of Medicine is supported by NIH grant P30CA125123. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exis
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