Periodic Reporting for period 1 - MELANOPARK (Investigating the LRRK2-melanin connection through phosphoproteomics of isogenic melanoma cells)
Reporting period: 2015-07-01 to 2017-06-30
LRRK2 is a multi-domain protein, which uniquely possesses both protein kinase and GTPase catalytic activities. Autosomal dominant mutations of LRRK2 represent one of the principal genetic risk factors for Parkinson’s Disease (PD), which results in preferential loss of neuromelanin containing neurons from the substantia nigra. The (patho)physiological roles of LRRK2 are currently unclear. However, it has been linked to the regulation of endolysosmal membrane trafficking through complex formation with other Parkinson’s related genes (Rab7L and Vps35). LRRK2 is a large protein (2527 amino acids), mutations in which, are prominently associated with both familial and sporadic PD. Small molecule inhibitors of LRRK2 kinase activity have been developed and are commercially available. By interrogating the Broad Institute’s Cancer Cell line Encyclopedia, we noticed that LRRK2 and another PD-associated protein (α-synuclein) are particularly highly expressed in melanoma cells and furthermore that six genes associated with melanosome biogenesis/pigmentation such as tyrosinase (TYR) and melanin A are in the top 20 genes expressed across >1000 cell lines that are most correlated with LRRK2 expression.
The overall objective is to introduce melanocyte model systems to the host laboratory and to establish LRRK2 substrates or cellular consequences of activity which may be specific to melanin-containing cells.
For technical reasons we switched our cell model to a mouse model cell line (Melan-a) which displayed higher levels of LRRK2 whilst maintaining pigmentation. Conditions for siRNA-mediated depletion were optimised in this cell line. In these cells depletion of MITF had no effect on LRRK2 levels but depletion of LRRK2 led to a clear increase in MITF levels - the key transcription factor driving melanogenesis. Despite this effect, LRRK2 depletion did not influence primary parameters associated with melanogenesis, but did increase the velocity of melanosome transport along microtubules. In contrast, pharmacological inhibition using two structurally unrelated LRRK2 inhibitors increased pigmentation.
During the course of the project, the rab family of small GTPases, moved into focus as potential LRRK2 substrates, based on work published by others. Assays were established in the laboratory to monitor rab-phosphorylation using phos-Tag gels. A platform has been established for specific analyses of melanosome associated rab proteins.
The cell system and assays introduced to the host laboratory during this period will enable further pursuit of the connection between LRRK2 and MITF that this work has uncovered.