(Patho)Physiology of LRRK2: Focus on Melanoma cells

Parkinson's disease (PD) is a progressive movement disorder affecting about 1.8% of people over the age of 65. Although neurodegenerative diseases are generally associated with decreased cancer risk, PD patients have an increased risk of melanoma and the two conditions may share critical biological pathways. Dopaminergic cells in the substantia nigra produce a special form of melanin known as neuromelanin, whilst melanocytes have a neural crest origin. Melanoma cells also express high levels of alpha-synuclein and LRRK2, two proteins commonly mutated in familial and sporadic PD. Our goal will be to shed new light on the cell biological function of Parkinsons associated genes in neural crest derived cells.

Many PD genes are linked to maintenance of mitochondrial integrity and/or endolysosomal trafficking. We will focus on LRRK2, a large multi-domain protein that uniquely contains both kinase and GTPase domains. LRRK2 is in complex with two other proteins, linked to PD, that are associated with membrane trafficking: the small GTPase Rab7L and the retromer component Vps35. The related GTPase Rab7A, a known regulator of retromer, autophagy and late endosomal functions, is strongly linked to melanoma progression. Importantly, melanoma cells are particularly sensitive to disruption of the endo-lysosomal pathway. This encourages the view that one function of LRRK2, related to pathophysiology, is the control of flux through the endosomal/lysosomal pathway. Note also that late endosomal compartments have been shown to control MAPK and mTOR signaling pathways and are essential for the autophagic clearance of aggregated material, which accumulates in PD. Pigmented melanoma cells offer a unique opportunity to study the role of LRRK2 in melanogenesis, a specialised endosome related pathway.

We will leverage our combined expertise in membrane trafficking, autophagy, mitophagy and quantitative mass spectrometric analysis of cell signaling to explore (dis)functions of LRRK2 in melanoma cells.
Specific components of the study will include:
CRISPR/Cas9 to develop isogenic melanoma cell panels bearing deletions or activating mutations in the LRRK2 gene.
cell lines +/- inhibitors in quantitative phosphoproteomics studies to discover melanoma-specific substrates of LRRK2. Alongside these discovery-mode experiments we will directly probe for changes in p-Rab proteins that have been previously suggested as LRRK2 substrates.
Investigation of the role of LRRK2 in autophagic clearance of alpha-synuclein
Development of our foundational data suggesting a role for LRRK2 in melanogenesis (light and electron microscopy).
Study mitochondrial form and function (allied to ongoing studies of other PD genes and existing observations in LRRK2 mutant patient derived fibroblasts)
Development of preliminary data linking LRRK2 to invasive properties of melanoma cells.

The project will build on foundations laid by a Marie Slodowska-Curie fellowship (finishing July 2017) and dovetail with ongoing studies of other PD related genes (PINK1, Parkin) with respect to mitochondrial function (2016-2019) funded by MRC and Parkinsons UK.
The project has the potential to open up new areas of LRRK2 biology and synthesise others. Whilst many groups are studying LRRK2 we believe our choice of melanoma setting provides some unique opportunities to impact the field.