Epidemiological studies have long suggested that Parkinson’s disease (PD) and cutaneous malignant melanoma (CMM) share a mutual genetic background. While genetic studies have predominantly focused on examining the role of common variants in mediating this shared etiology, our recent study looking at rare variation showed that a heterozygous loss of function (LOF) tyrosinase (TYR) mutation substantially increases PD risk in carriers. However, the functional mechanism behind TYR-associated PD risk has yet to be elucidated. Most of what is known regarding the function of TYR within the brain stems from its well-characterized role in peripheral melanogenesis, however much remains to be confirmed in the context of the role of TYR in neuromelanin (NM) formation. TYR is believed to drive dopamine (DA) synthesis in the absence of tyrosine hydroxylase (TH), however the role of TYR and TH in DA biogenesis and oxidation during development is unknown. NM is undetectable in brains until around the age of 3 years, with age-dependent accumulation throughout life. NM is known to consist of two chemically distinct types of pigment, eumelanin (EM) and pheomelanin (PM). PM (pink/yellow) is located at the core of NM, and EM (brown/black) on the surface, the latter known to prevent oxidative stress by binding to metals, reactive oxygen species (ROS) and other toxic cellular byproducts. The EM:PM ratio is therefore critical to NM function. A role for TYR in EM formation has been demonstrated. Using induced pluripotent stem cell (iPSC)-derived substantia nigra (SN) DA neurons generated from biallelic TYR mutation, CrispR-Cas9 TYR knockout (KO), TYR overexpressing (OE) and heterozygous TYR LOF mutant PD lines, we will investigate TYR’s role in DA synthesis and oxidation as well as its function in regulating the EM:PM ratio, and therefore NM production. Disruption of this ratio may lead to a reduction in binding capacity and an increase in cellular toxicity. Based on our preliminary data, we hypothesize that TYR drives DA synthesis and oxidation, and NM synthesis during brain development, and that loss of TYR results in lower EM:PM ratios due to the reduction of EM production which ultimately leads to dysfunctional NM. We further hypothesize that the increased PD risk associated with heterozygous TYR mutations is therefore mediated through the accumulation of increased cellular ROS and iron accumulation, which predispose SN neurons to degeneration. Using microanalytical ultra-performance chromatography (UPLC), we will then test NM collected from all lines as well as TYR mutation positive PD patient-derived SN neurons to accurately evaluate the EM:PM ratio and its effect on the binding capacity of NM. We expect that, while demonstrating that TYR is involved in DA synthesis, DA oxidation and NM biogenesis in an age-dependent manner, our study will provide direct evidence to support our hypothesis that reduced TYR increases PD risk through the dysregulation of EM. The validation of our hypotheses will not only confirm that TYR is involved in pigmentation of DA neurons, but will also establish that NM dysfunction as a novel mechanism in PD etiology. To our knowledge, this study will be the first to investigate the role of TYR in DA production, DA oxidation and NM biogenesis in the human brain through the use of iPSC-derived SN neurons, and the first to use patient-derived SN neurons to evaluate the functional mechanism leading to significantly increased PD risk in TYR LOF mutation carriers.
|Effective start/end date||9/30/20 → 8/31/21|
- National Institute on Aging (1R21AG064470-01A1)