Functional investigation of the role of TYR mutations and neuromelanin in Parkinson's disease

TYR 突变和神经黑色素在帕金森病中作用的功能研究

基本信息

批准号：
9976866
负责人：
DIMITRI KRAINC
金额：
$ 23.7万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-30 至 2021-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9976866
关键词：
20 year old 3 year old Affect Age Binding Biogenesis Birth Brain Brain Stem CRISPR/Cas technology Cell Death Characteristics Chemicals Chromatography Cutaneous Melanoma Cytoplasmic Granules Data Development Disease susceptibility Dopamine Drops Elderly Enzymes Epidemiology Etiology Functional disorder Genetic Genetic study Human Investigation Iron Knock-out Knowledge Lead Levodopa Life Link Mediating Melanins Melanogenesis Metals Mitochondria Monophenol Monooxygenase Mutation Neurons Newborn Infant Oxidation-Reduction Oxidative Stress Oxides Parkinson Disease Pathology Pathway interactions Patients Performance Peripheral Pigmentation physiologic function Pigments Play Predisposition Production Quinones Reactive Oxygen Species Risk Role Structure Substantia nigra structure Surface Testing Time Tissues Toxic effect Tyrosine Tyrosine 3-Monooxygenase Validation Variant Viral age related alpha synuclein autooxidation base density disorder risk dopaminergic neuron dopaquinone epidemiology study eumelanin in vivo induced pluripotent stem cell loss of function loss of function mutation mutant mutation carrier neuromelanin novel overexpression oxidation pheomelanin prevent stem

项目摘要

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.

流行病学研究长期以来表明帕金森病（PD）和皮肤恶性肿瘤黑色素瘤 (CMM) 具有共同的遗传背景，而遗传研究主要集中于黑色素瘤。我们最近的研究考察了常见变异在调节这种共同病因中的作用，我们最近的研究着眼于罕见变异变异表明，杂合功能丧失（LOF）酪氨酸酶（TYR）突变显着增加然而，TYR 相关 PD 风险背后的功能机制尚未明确。关于 TYR 在大脑中的功能，大部分已知的信息都源于其在外周黑素生成中的明确作用，但仍有许多问题有待在其背景下得到证实。 TYR 在神经黑色素 (NM) 形成中的作用被认为在缺乏时驱动多巴胺 (DA) 合成。酪氨酸羟化酶 (TH)，然而 TYR 和 TH 在 DA 生物合成和氧化过程中的作用 NM 的发育情况尚不清楚，直到 3 岁左右才能在大脑中检测到，并且具有年龄依赖性。众所周知，NM 由两种化学上不同类型的色素组成，即真黑素。 (EM) 和褐黑素 (PM)。PM（粉色/黄色）位于 NM 的核心，EM（棕色/黑色）位于 NM 的核心。表面，后者已知可通过与金属、活性氧 (ROS) 和因此，EM:PM 比例对于 TYR 在 EM 中的作用至关重要。已证实使用诱导多能干细胞 (iPSC) 衍生的黑质 (SN) 形成。由双等位基因 TYR 突变、CrispR-Cas9 TYR 敲除 (KO)、TYR 过表达产生的 DA 神经元 (OE) 和杂合 TYR LOF 突变体 PD 系，我们将研究 TYR 在 DA 合成和氧化中的作用及其调节 EM:PM 比率的功能，因此 NM 生产可能会受到干扰。导致结合能力降低和细胞毒性增加。根据我们的初步数据，我们发现。研究发现 TYR 在大脑发育过程中驱动 DA 合成和氧化以及 NM 合成，并且由于 EM 产量减少，TYR 损失导致 EM:PM 比率降低，最终导致我们进一步发现，PD 风险增加与 TYR 杂合相关。因此，突变是通过增加细胞活性氧的积累和铁的积累来介导的，使用微量分析超高效色谱法使 SN 神经元易于退化。 (UPLC)，然后我们将测试从所有品系收集的 NM 以及 TYR 突变阳性 PD 患者衍生的 SN 神经元准确评估 EM:PM 比率及其对 NM 结合能力的影响。在证明 TYR 以年龄依赖性方式参与 DA 合成、DA 氧化和 NM 生物发生的同时，我们的研究将提供直接证据来支持我们的假设，即 TYR 减少通过 EM 失调增加 PD 风险我们的假设的验证不仅会证实这一点。 TYR 参与 DA 神经元的色素沉着，但也将确定 NM 功能障碍是一种新的据我们所知，这项研究将是第一个探讨 TYR 在 DA 中的作用的研究。通过使用 iPSC 衍生的 SN 神经元在人脑中产生、DA 氧化和 NM 生物发生，并第一个使用患者来源的 SN 神经元来评估导致显着的功能机制 TYR LOF 突变携带者的 PD 风险增加。