An integrated transcriptomics and proteomics analysis reveals functional endocytic dysregulation caused by mutations in LRRK2

Abstract

Background Mutations in LRRK2 are the most common cause of autosomal dominant Parkinson's disease, and the relevance of LRRK2 to the sporadic form of the disease is becoming ever more apparent. It is therefore essential that studies are conducted to improve our understanding of the cellular role of this protein. Here we use multiple models and techniques to identify the pathways through which LRRK2 mutations may lead to the development of Parkinson's disease. Methods A novel integrated transcriptomics and proteomics approach was used to identify pathways that were significantly altered in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blotting, immunostaining and functional assays including FM1-43 analysis of synaptic vesicle endocytosis were performed to confirm these findings in iPSC-derived dopaminergic neuronal cultures carrying either the LRRK2-G2019S or the LRRK2-R1441C mutation, and LRRK2 BAC transgenic rats, and post-mortem human brain tissue from LRRK2-G2019S patients. Results Our integrated -omics analysis revealed highly significant dysregulation of the endocytic pathway in iPSC-derived dopaminergic neurons carrying the LRRK2-G2019S mutation. Western blot analysis confirmed that key endocytic proteins including endophilin I-III, dynamin-1, and various RAB proteins were downregulated in these cultures and in cultures carrying the LRRK2-R1441C mutation, compared with controls. We also found changes in expression of 25 RAB proteins. Changes in endocytic protein expression led to a functional impairment in clathrin-mediated synaptic vesicle endocytosis. Further to this, we found that the endocytic pathway was also perturbed in striatal tissue of aged LRRK2 BAC transgenic rats overexpressing either the LRRK2 wildtype, LRRK2-R1441C or LRRK2-G2019S transgenes. Finally, we found that clathrin heavy chain and endophilin I-III levels are increased in human post-mortem tissue from LRRK2-G2019S patients compared with controls. Conclusions Our study demonstrates extensive alterations across the endocytic pathway associated with LRRK2 mutations in iPSC-derived dopaminergic neurons and BAC transgenic rats, as well as in post-mortem brain tissue from PD patients carrying a LRRK2 mutation. In particular, we find evidence of disrupted clathrin-mediated endocytosis and suggest that LRRK2-mediated PD pathogenesis may arise through dysregulation of this process.

Sequential screening nominates the Parkinson’s disease associated kinase LRRK2 as a regulator of Clathrin-mediated endocytosis

“…This observation further supports prior studies that have demonstrated a reduction of CME in iPSC-derived LRRK2 R1441C and G2019S dopaminergic neurons as well as a significant decrease in several CME-associated proteins, including AP2. 28,60 Collectively, our data is most compatible with several previous studies suggesting that either too much or too little LRRK2 can deregulate cellular activity. [26][27][28][29]48 To date, GWAS have identified up to 90 independent PD-associated signals across 78 loci.…”

Vesicular Dysfunction and the Pathogenesis of Parkinson’s Disease: Clues From Genetic Studies

“…The large size of the protein and the number of distinct domain structure of the LRRK2 molecule is, however, consistent with the number of functions and the various protein domains is suspected to serve with functions including but not limited to, vesicular trafficking, autophagy and immune response (Rideout and Stefanis, 2014;Wallings et al, 2015). Crucially, LRRK2 associates with membranous structures and vesicles in mammalian brains and recently, integrated-omics analysis identified dysregulation of endocytic pathway in iPS derived DAergic neurons carrying G2019S mutation in the LRRK2 gene (Connor-Robson et al, 2019).…”

Modeling Parkinson’s Disease Using Induced Pluripotent Stem Cells

“…Another study also confirmed mitochondrial distribution and trafficking abnormalities in LRRK2 mutant neurons, accompanied by significantly low endogenous NAD+ levels and decreased protein lysine deacetylase activity, leading to bioenergy defects [71]. Stem Cells International confirmed that clathrin-mediated endocytosis was disrupted [73]. Recently, Nguyen and Krainc reported that LRRK2 interacted with auxilin to jointly damage clathrin-mediated endocytosis of synaptic vesicles.…”