Abstract
Missense mutations in the leucine rich repeat kinase 2 (LRRK2) gene are the leading genetic cause of autosomal dominant familial Parkinson’s disease. We previously reported that two mutations within the ROC domain, namely R1441C and A1442P, exhibit increased protein degradation leading to lowered steady state LRRK2 protein levels in HEK293 cells. More recently, the common WD40 domain LRRK2 haplotype, Met2397, which is a risk factor for Crohn’s disease, has been shown to lower steady state protein levels in HEK293 cells. In view of recent evidence implicating LRRK2 and inflamemation in PD, we investigated the effects of Met2397 on LRRK2 expression, and compared them to the Thr2397 variant and other LRRK2 mutants. In this study, we transfected HEK293 cells with plasmid constructs encoding the different LRRK2 variants, and analyzed the resulting protein levels by Western blot and flow cytometry. Here we found that both the Met2397 and Thr2397 haplotypes yield similar levels of LRRK2 protein expression and do not appear to impact cell viability in HEK293 cells, compared to other LRRK mutants. Thus, we have concluded that the Met2397 haplotype is unlikely to play a role in LRRK2 mediated or idiopathic PD.
Highlights
Parkinson’s disease (PD) is a neurodegenerative condition characterised by Lewy Bodies and the loss of dopaminergic neurons within the substantian igrapars compacta [1]
We previously reported that two mutations within the ROC domain, namely R1441C and A1442P, exhibit increased protein degradation leading to lowered steady state leucine rich repeat kinase 2 (LRRK2) protein levels in Human embryonic kidney 293 (HEK293) cells
The LRRK2 expression constructs used in this study (Figure 1(a)) include the threonine wild type (Thr2397), methionine wild type (Met2397), threonine R1441C (R1441C) and threonine A1442P (A1442P)
Summary
Parkinson’s disease (PD) is a neurodegenerative condition characterised by Lewy Bodies and the loss of dopaminergic neurons within the substantian igrapars compacta [1]. Missense mutations within the leucine-rich repeat kinase 2 (LRRK2) gene appear to account for the majority of familial cases [3]. Pathogenic mutations within the LRRK2 ROC domain account for around 10% of all LRRK2 mutations, and most commonly affect codon 1441, for which three different amino acid substitutions (R1441C/G/H) have been identified [4]. We previously reported that the ROC domain missense mutation A1442P, identified in a Western Australian patient, caused reduced LRRK2 steady state protein levels, as did the common R1441C mutation [5]. Mutations within the ROC domain of LRRK2 reduce GTP hydrolysis and LRRK2 protein autophosphorylation, which leads to dysregulated organelle degradation, altered signalling and immune dysfunction [1] [6] [7]. Cell death is regulated by the WD40 domain, as neurotoxicity is reduced in mutants lacking the WD40 domain [10]
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