MRC PPU Collaboration With Medical University of Vienna And Michael J Fox Foundation Identifies New Upstream LRRK2 Regulator

A possible mechanism by which the VPS35[D620N] mutation promotes Parkinson's Disease
A possible mechanism by which the VPS35[D620N] mutation promotes Parkinson's Disease

Missense mutations that induce hyper-activation of the LRRK2 protein kinase cause autosomal dominant Parkinson’s disease. LRRK2 phosphorylates a subgroup of Rab GTPases within their Switch-II effector binding motifs that impacts on their ability to associate with critical effectors. Little is known about the upstream pathways that control LRRK2 activity, other than Rab29 may activate LRRK2 by promoting its recruitment to the Golgi.

To identify further upstream components Rafeeq Mir, a postodoc in the Alessi lab, is exploring whether mutations in the ~20 genes that cause familial inherited Parkinson’s could also trigger the activation of the LRRK2 pathway and hence promote phosphorylation of Rab proteins at their Switch-II motif site.

One of the first candidates that Rafeeq screened was a mutation in the VPS35 gene (D620N). This encodes for the cargo binding component of the retromer complex that shuttles proteins from the endosomes to the golgi and plasma membrane.

Strikingly, Rafeeq found that in both heterozygous as well as homozygous VPS35[D620N] knock-in mouse embryonic fibroblasts, LRRK2 mediated phosphorylation of Rab10 was elevated around 5 to 6 fold, in a manner that is blocked by LRRK2 inhibitors. Rafeeq also obtained evidence that the VPS35[D620N] mutation enhances autophosphorylation of LRRK2 at Ser1292, suggesting that it is leading to a direct activation of LRRK2.

Pawel Lis, a postdoc in the Alessi lab working closely with Nicole Polinski and Terina Martinez at The Michael J Fox Foundation for Parkinson’s Research (MJFF) and Meng-Yun Chou at Abcam, characterised a series of new rabbit monoclonal antibodies that enable the detection of endogenous LRRK2 mediated phosphorylation of Rab8A and Rab12 to be readily measured. Using these reagents Pawel developed novel assays to show that the VPS35[D620N] mutation in mouse embryonic fibroblasts also markedly increased LRRK2 mediated phosphorylation of Rab8A and Rab12 over 4-fold, in a manner that is blocked by LRRK2 inhibitors.

Rafeeq also showed that the VPS35[D620N] mutation increased Rab10 phosphorylation in mouse tissues that express significant levels of LRRK2 (lung, kidney, spleen and brain). He also demonstrated that LRRK2 mediated Rab10 phosphorylation was significantly suppressed by knock-out or knock-down of VPS35 in wild type, LRRK2[R1441C] or VPS35[D620N] cells, further suggesting that VPS35 functions as an upstream regulator of the LRRK2 signalling pathway.

A key question was whether the VPS35[D620N] mutation activates the LRRK2 pathway in human. To address this Esther Sammler, a Clinical Programme leader in the MRC PPU, teamed up with Francesca Tonelli (a postdoc in the Alessi lab) and Alexander Zimprich and his colleagues at the Department of Neurology, Medical University of Vienna, as well as the Verein zur Förderung der wissenschaftlichen Forschung im in Vienna. In a wonderful series of experiments they were able to demonstrate that LRRK2 mediated Rab10 phosphorylation is indeed markedly increased in neutrophils as well as monocytes isolated from three Parkinson’s patients with a heterozygous VPS35[D620N] mutation, compared to both healthy donors and idiopathic Parkinson’s patients.

These studies establish the power of interrogating the impact that genetic mutations in human have on the LRRK2 signalling pathway, by studying Rab10 phosphorylation in patient derived-neutrophil and monocytes

Rafeeq also noted that the VPS35[D620N] mutation in mouse embryonic fibroblasts induced a significantly higher enhancement of Rab10 phosphorylation than was observed in cells with LRRK2 G2019S or R1441C knock-in pathogenic mutations. This prompted Esther Sammler to analyse available data relating to the age of onset of Parkinson’s disease with patients bearing VPS35[D620N], LRRK2[G2019S] and LRRK2[R1441G/C/H] mutations. Interestingly, data from 50 VPS35[D620N] patients revealed that the largest group of patients, namely 23, were diagnosed with PD in the 5th decade, with only 13 diagnosed in the 6th decade. In contrast, for LRRK2 G2019S carriers of which there are 277 in the MDS database, the largest groups of patients were diagnosed in 6th (73) and 7th decade (77). The age at onset of the LRRK2 R1441 mutations was similar to the G2019S. This data is therefore consistent with the notion that the VPS35[D620N] mutation promotes LRRK2 pathway activity more potently than LRRK2 pathogenic mutations, and that disease could be triggered through hyper-activation activation of LRRK2.

Our observations indicate that VPS35 controls LRRK2 activity and that the VPS35[D620N] mutation results in a gain of function, potentially causing Parkinson’s disease through hyperactivation of the LRRK2 kinase. This work is also consistent with previous elegant Drosophila genetic studies undertaken in the laboratories of Asa Abeliovich and others, that suggested VPS35, LRRK2 and perhaps Rab29 operate in a same signalling pathway.

In future work it will be essential to decipher the molecular mechanism by which the retromer complex controls LRRK2 and how the D620N mutation promotes LRRK2 mediated phosphorylation of Rab proteins. The image shown depicts one possible mechanism by which VPS35 could regulate the LRRK2 pathway through an as yet unknown intermediate.

Uncovering the mechanism by which VPS35 controls LRRK2 may enable pharmaceutical companies to elaborate compounds that target the retromer complex in a manner that specifically suppress LRRK2 activity without impacting on its other vital functions.

Another major implication of our findings is that Parkinson’s patients with VPS35[D620N] mutations might benefit from LRRK2 inhibitor treatment that have entered clinical trials in humans.

To read a copy of our paper that describes these findings please click here.