The Genetic Drivers of Juvenile, Young, and Early-Onset Parkinson's Disease in India.

BACKGROUND: Recent studies have advanced our understanding of the genetic drivers of Parkinson's disease (PD). Rare variants in more than 20 genes are considered causal for PD, and the latest PD genome-wide association study (GWAS) identified 90 independent risk loci. However, there remains a gap in our understanding of PD genetics outside of the European populations in which the vast majority of these studies were focused. OBJECTIVE: The aim was to identify genetic risk factors for PD in a South Asian population. METHODS: A total of 674 PD subjects predominantly with age of onset (AoO) ≤50 years (encompassing juvenile, young, or early-onset PD) were recruited from 10 specialty movement disorder centers across India over a 2-year period; 1376 control subjects were selected from the reference population GenomeAsia, Phase 2. We performed various case-only and case-control genetic analyses for PD diagnosis and AoO. RESULTS: A genome-wide significant signal for PD diagnosis was identified in the SNCA region, strongly colocalizing with SNCA region signal from European PD GWAS. PD cases with pathogenic mutations in PD genes exhibited, on average, lower PD polygenic risk scores than PD cases lacking any PD gene mutations. Gene burden studies of rare, predicted deleterious variants identified BSN, encoding the presynaptic protein Bassoon that has been previously associated with neurodegenerative disease. CONCLUSIONS: This study constitutes the largest genetic investigation of PD in a South Asian population to date. Future work should seek to expand sample numbers in this population to enable improved statistical power to detect PD genes in this understudied group. © 2023 Denali Therapeutics and The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Rab12 is a regulator of LRRK2 and its activation by damaged lysosomes.

Leucine-rich repeat kinase 2 (LRRK2) variants associated with Parkinson's disease (PD) and Crohn's disease lead to increased phosphorylation of its Rab substrates. While it has been recently shown that perturbations in cellular homeostasis including lysosomal damage can increase LRRK2 activity and localization to lysosomes, the molecular mechanisms by which LRRK2 activity is regulated have remained poorly defined. We performed a targeted siRNA screen to identify regulators of LRRK2 activity and identified Rab12 as a novel modulator of LRRK2-dependent phosphorylation of one of its substrates, Rab10. Using a combination of imaging and immunopurification methods to isolate lysosomes, we demonstrated that Rab12 is actively recruited to damaged lysosomes and leads to a local and LRRK2-dependent increase in Rab10 phosphorylation. PD-linked variants, including LRRK2 R1441G and VPS35 D620N, lead to increased recruitment of LRRK2 to the lysosome and a local elevation in lysosomal levels of pT73 Rab10. Together, these data suggest a conserved mechanism by which Rab12, in response to damage or expression of PD-associated variants, facilitates the recruitment of LRRK2 and phosphorylation of its Rab substrate(s) at the lysosome.

Lysosomes are cellular compartments tasked with breaking down large molecules such as lipids or proteins. They perform an essential role in helping cells dispose of obsolete or harmful components; in fact, defects in lysosome function are associated with a range of health conditions. For instance, many genes associated with an increased risk of developing Parkinson's disease code for proteins required for lysosomes to work properly, such as the kinase LRRK2. Previous work has shown that this enzyme gets recruited to the surface of damaged lysosomes, where it can modulate the function of another set of molecular actors by modifying them through a chemical process known as phosphorylation. Such activity is increased in harmful versions of LRRK2 linked to Parkinson's disease. However, the molecular mechanisms which control LRRK2 activity or its recruitment to lysosomes remain unclear. To examine this question, Wang, Bondar et al. first performed a targeted screen to identify proteins that can regulate LRRK2 activity. This revealed that Rab12, one of molecular actors that LRRK2 phosphorylates, can in turn modulate the activity of the enzyme. Further imaging and biochemical experiments then showed that Rab12 is recruited to damaged lysosomes and that this step was in fact necessary for LRRK2 to also relocate to these compartments. The data suggest that this Rab12-driven recruitment process increases the local concentration of LRRK2 near its Rab targets on the membrane of damaged lysosomes, and therefore leads to enhanced LRRK2 activity. Crucially, Wang, Bondar et al. showed that Rab12 also plays a role in the increased LRRK2 activity observed with two Parkinson's disease-linked mutations (one in LRRK2 itself and one in another lysosomal regulator, VPS35), suggesting that increased LRRK2 concentration on lysosomes may be a conserved mechanism that leads to increased LRRK2 activity in disease. Overall, these results highlight a new, Rab12-dependent mechanism that results in enhanced activity at the lysosomal membrane with variants associated with Parkinson's disease, and for LRRK2 in general when lysosomes are damaged. This knowledge will be helpful to develop therapeutic strategies that target LRRK2, and to better understand how increased LRRK2 activity and lysosomal injury may be linked to Parkinson's disease.

Parallel CRISPR-Cas9 screens identify mechanisms of PLIN2 and lipid droplet regulation.

Despite the key roles of perilipin-2 (PLIN2) in governing lipid droplet (LD) metabolism, the mechanisms that regulate PLIN2 levels remain incompletely understood. Here, we leverage a set of genome-edited human PLIN2 reporter cell lines in a series of CRISPR-Cas9 loss-of-function screens, identifying genetic modifiers that influence PLIN2 expression and post-translational stability under different metabolic conditions and in different cell types. These regulators include canonical genes that control lipid metabolism as well as genes involved in ubiquitination, transcription, and mitochondrial function. We further demonstrate a role for the E3 ligase MARCH6 in regulating triacylglycerol biosynthesis, thereby influencing LD abundance and PLIN2 stability. Finally, our CRISPR screens and several published screens provide the foundation for CRISPRlipid (http://crisprlipid.org), an online data commons for lipid-related functional genomics data. Our study identifies mechanisms of PLIN2 and LD regulation and provides an extensive resource for the exploration of LD biology and lipid metabolism.

Methods for genetic analysis of mammalian ER-associated degradation.

Identification and degradation of misfolded proteins by the ubiquitin-proteasome system (UPS) is crucial for maintaining proteostasis, but only a handful of UPS components have been linked to the recognition of specific substrates. Studies in Saccharomyces cerevisiae using systematic perturbation of nonessential genes have uncovered UPS components that recognize and ubiquitylate model substrates of the UPS; however, similar analyses in metazoans have been limited. In this chapter, we describe methods for using CRISPR/Cas9 technology combined with genome-wide high complexity single guide (sgRNA) libraries and a transcriptional shutoff strategy for phenotypic selection based on kinetic measurements of protein turnover to identify the genes required to degrade model clients of the mammalian ER-associated degradation system. We also discuss considerations for screen design, execution, and interpretation.

Ribosomal protein RPL26 is the principal target of UFMylation.

Ubiquitin fold modifier 1 (UFM1) is a small, metazoan-specific, ubiquitin-like protein modifier that is essential for embryonic development. Although loss-of-function mutations in UFM1 conjugation are linked to endoplasmic reticulum (ER) stress, neither the biological function nor the relevant cellular targets of this protein modifier are known. Here, we show that a largely uncharacterized ribosomal protein, RPL26, is the principal target of UFM1 conjugation. RPL26 UFMylation and de-UFMylation is catalyzed by enzyme complexes tethered to the cytoplasmic surface of the ER and UFMylated RPL26 is highly enriched on ER membrane-bound ribosomes and polysomes. Biochemical analysis and structural modeling establish that UFMylated RPL26 and the UFMylation machinery are in close proximity to the SEC61 translocon, suggesting that this modification plays a direct role in cotranslational protein translocation into the ER. These data suggest that UFMylation is a ribosomal modification specialized to facilitate metazoan-specific protein biogenesis at the ER.

Genome-wide CRISPR Analysis Identifies Substrate-Specific Conjugation Modules in ER-Associated Degradation.

The ubiquitin proteasome system (UPS) maintains the integrity of the proteome by selectively degrading misfolded or mis-assembled proteins, but the rules that govern how conformationally defective proteins in the secretory pathway are selected from the structurally and topologically diverse constellation of correctly folded membrane and secretory proteins for efficient degradation by cytosolic proteasomes is not well understood. Here, we combine parallel pooled genome-wide CRISPR-Cas9 forward genetic screening with a highly quantitative and sensitive protein turnover assay to discover a previously undescribed collaboration between membrane-embedded cytoplasmic ubiquitin E3 ligases to conjugate heterotypic branched or mixed ubiquitin (Ub) chains on substrates of endoplasmic-reticulum-associated degradation (ERAD). These findings demonstrate that parallel CRISPR analysis can be used to deconvolve highly complex cell biological processes and identify new biochemical pathways in protein quality control.

Redundant and Antagonistic Roles of XTP3B and OS9 in Decoding Glycan and Non-glycan Degrons in ER-Associated Degradation.

Glycoproteins engaged in unproductive folding in the ER are marked for degradation by a signal generated by progressive demannosylation of substrate N-glycans that is decoded by ER lectins, but how the two lectins, OS9 and XTP3B, contribute to non-glycosylated protein triage is unknown. We generated cell lines with homozygous deletions of both lectins individually and in combination. We found that OS9 and XTP3B redundantly promote glycoprotein degradation and stabilize the SEL1L/HRD1 dislocon complex, that XTP3B profoundly inhibits the degradation of non-glycosylated proteins, and that OS9 antagonizes this inhibition. The relative expression of OS9 and XTP3B and the distribution of glycan and non-glycan degrons within the same protein contribute to the fidelity and processivity of glycoprotein triage and, therefore, determine the fates of newly synthesized proteins in the early secretory pathway.