Abstract
BackgroundMutations 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. MethodsA 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. ResultsOur 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. ConclusionsOur 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.
Highlights
Parkinson's disease (PD) is a common neurodegenerative disorder in which dopaminergic neurons of the substantia nigra pars compacta (SNpc) are lost, leading to the classic motor symptoms of the disease
We demonstrate that levels of the endocytic proteins clathrin and endophilin are reduced in induced-pluripotent stem cell (iPSC)-derived dopaminergic cultures from LRRK2-G2019S and LRRK2-R1441C mutation carriers, as well as in 22month-old LRRK2 BAC transgenic rats carrying these same mutations
Control and LRRK2 iPSC lines produce ventral midbrain dopaminergic neurons iPSCs were derived from four healthy controls, and eight patients with PD who carry either the LRRK2-G2019S or LRRK2-R1441C mutation
Summary
Parkinson's disease (PD) is a common neurodegenerative disorder in which dopaminergic neurons of the substantia nigra pars compacta (SNpc) are lost, leading to the classic motor symptoms of the disease. Previous work has shown that LRRK2 fractionates with important synaptic proteins such as synapsin, synaptophysin, NSF, dynamin-1, and VAMP2 (Arranz et al, 2014; Belluzzi et al, 2016; Carrion et al, 2017; Piccoli et al, 2014) Further to this LRRK2 has been shown to directly interact with some key endocytic components including endophilin, dynamin and auxilin (Matta et al, 2012; Nguyen and Krainc, 2018; Piccoli et al, 2014). 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 LRRK2G2019S or the LRRK2-R1441C mutation, and LRRK2 BAC transgenic rats, and post-mortem human brain tissue from LRRK2-G2019S patients. 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. We find evidence of disrupted clathrin-mediated endocytosis and suggest that LRRK2-mediated PD pathogenesis may arise through dysregulation of this process
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