Identification of Targets from LRRK2 Rescue Phenotypes

Abstract

Parkinson’s disease (PD) is an age-dependent neurodegenerative condition. Leucine-rich repeat kinase 2 (LRRK2) mutations are the most frequent cause of sporadic and autosomal dominant PD. The exact role of LRRK2 protective variants (R1398H, N551K) together with a pathogenic mutant (G2019S) in aging and neurodegeneration is unknown. We generated the following myc-tagged UAS-LRRK2 transgenic Drosophila: LRRK2 (WT), N551K, R1398H, G2019S single allele, and double-mutants (N551K/G2019S or R1398H/G2019S). The protective variants alone were able to suppress the phenotypic effects caused by the pathogenic LRRK2 mutation. Next, we conducted RNA-sequencing using mRNA isolated from dopaminergic neurons of these different groups of transgenic Drosophila. Using pathway enrichment analysis, we identified the top 10 modules (p < 0.05), with “LRRK2 in neurons in Parkinson’s disease” among the candidates. Further dissection of this pathway identified the most significantly modulated gene nodes such as eEF1A2, ACTB, eEF1A, and actin cytoskeleton reorganization. The induction of the pathway was successfully restored by the R1398H protective variant and R1398H-G2019S or N551K-G2019S rescue experiments. The oxidoreductase family of genes was also active in the pathogenic mutant and restored in protective and rescue variants. In summary, we provide in vivo evidence supporting the neuroprotective effects of LRRK2 variants. RNA sequencing of dopaminergic neurons identified upregulation of specific gene pathways in the Drosophila carrying the pathogenic variant, and this was restored in the rescue phenotypes. Using protective gene variants, our study identifies potential new targets and provides proof of principle of a new therapeutic approach that will further our understanding of aging and neurodegeneration in PD.

Coding and Noncoding Variation in LRRK2 and Parkinson's Disease Risk

“… 8 , 41 , 42 , 43 A recent study has shown that both p.R1398H and p.N551K were able to counteract the putative pathogenic effects of p.G2019S in Drosophila models. 44 The literature therefore suggests that each of these variants has measurable effects on protein function in cells and in vivo that are consistent with the proposed direction of effects for risk versus protection. However, the relation between the functional impact and risk for PD needs to be further investigated for these coding variants.…”

“…In addition, LRRK2 p.R1398H has been shown to affect GTPase and Wnt signaling activity contrary to pathogenic LRRK2 mutations 8,41‐43 . A recent study has shown that both p.R1398H and p.N551K were able to counteract the putative pathogenic effects of p.G2019S in Drosophila models 44 . The literature therefore suggests that each of these variants has measurable effects on protein function in cells and in vivo that are consistent with the proposed direction of effects for risk versus protection.…”

Functional Analyses of Two Novel LRRK2 Pathogenic Variants in Familial Parkinson′s Disease

“…Only one transcriptomic study in Drosophila melanogaster has identified altered pathways associated with N551K, including alterations of the oxidoreductase pathway. 29 Structural analysis of full-length human LRRK2 has shown that the ANK and LRR domains interact with the kinase domain but not the ARM domain, which shows flexibility relative to the rest of the protein. 30 Rab proteins directly interact with LRRK2 via the ARM domain 31,32 but the H230R variant is not located in the potential Rab-interacting regions of this domain (residues 386-392).…”

Functional analyses of two novel LRRK2 pathogenic variants in familial Parkinson’s disease

“…Another rare variant, N551K, belonging to a protective haplotype (N551K-R1398H-K1423K) has been reported for PD patients (22,23) but the mechanisms explaining how this haplotype confers neuroprotection in PD is not clear and it has not been functionally assessed. Only one transcriptomic study in Drosophila melanogaster has identified altered pathways associated with N551K, including alterations of the oxidoreductase pathway (24). Structural analysis of full-length human LRRK2 has shown that the ANK and LRR domains interact with the kinase domain but not the ARM domain, which shows flexibility relative to the rest of the protein (25).…”