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Functional Screening Of Lysosomal Storage Disorder Genes Identifies Modifiers Of Alpha-synuclein Neurotoxicity

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. Shulma

Abstract

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 Methods

LSD gene set enrichment analysis from PD GWAS

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

Overall, our results are potentially consistent with a model in which partial loss of function for multiple LSD genes may enhance αSyn neuropathology, as with GBA-PD, but that more complete loss of gene function may compromise CNS function, as in neuronopathic Gaucher disease and many other LSDs. For several genes of interest, we confirmed that both RNAi and heterozygous loss-of-function alleles caused decreased gene expression; however, we were not able to establish a predictable relationship between degree of knockdown and severity of locomotor phenotype, at least based solely on mRNA levels.

Discussion

In prior work, experimental manipulations of GBA, SMPD1, and SCARB2—which similarly cause sphingolipid storage disorders—have been demonstrated to induce accumulation and toxicity of αSyn [12,26,31]. Beyond sphingolipid/ceramide metabolism, our screen also identifies many fly homologs of genes causing human mucopolysaccharidoses (e.g., GLB1, IDS, IDUA, MAN2B1, MANBA). These disorders are characterized by accumulation of glycosaminoglycans, which are long chains of repeating, negatively charged disaccharide units that can be linked to protein cores to form proteoglycans. Though perhaps less well studied than sphingolipids, recent studies suggest that glycosaminoglycan metabolites have the potential to similarly influence αSyn aggregation [32]. In addition, proteoglycans are abundant at the cell surface and are a major constituent of the extracellular matrix, having recently been implicated in the propagation and/or internalization of pathologic αSyn species [33].

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 exist.

https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010760#abstract0
 

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