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

• 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
• 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

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）共享共同的遗传背景。虽然遗传学研究主要集中在 研究常见变异在介导这种共同病因中的作用，我们最近的研究着眼于罕见的 变异表明，杂合性酪氨酸酶（TYR）突变功能丧失（LOF）显著增加 携带者的PD风险。然而，TYR相关PD风险背后的功能机制尚未得到证实。 阐明。关于TYR在脑内的功能的大多数已知源于其在外周黑素生成中的充分表征的作用，然而在本发明的背景下仍有许多有待证实。 TYR在神经黑色素（NM）形成中的作用。TYR被认为在缺乏多巴胺（DA）的情况下驱动多巴胺（DA）的合成。 酪氨酸羟化酶（TH），然而，酪氨酸和TH的作用，DA的生物合成和氧化过程中， 发展未知。NM在大脑中直到3岁左右才被检测到，具有年龄依赖性 一生的积累。NM由两种化学性质不同的色素组成， (EM)和褐黑素（PM）。PM（粉红色/黄色）位于NM的核心，EM（棕色/黑色）位于 表面，后者已知通过结合金属、活性氧（ROS）和 其他有毒的细胞副产物因此，EM：PM比率对NM功能至关重要。TYR在EM中的作用 形成了示范。使用诱导多能干细胞（iPSC）衍生的黑质（SN） 由双等位基因TYR突变、CrispR-Cas9 TYR敲除（KO）、TYR过表达（K 0）产生的DA神经元 (OE)和杂合TYR LOF突变PD系，我们将研究TYR在DA合成和氧化中的作用 以及其在调节EM：PM比率中的功能，从而调节NM产生。这一比例的破坏可能 导致结合能力降低和细胞毒性增加。根据初步数据，我们 假设TYR驱动大脑发育过程中DA的合成和氧化以及NM的合成， TYR的损失导致EM：PM比率降低，这是由于EM产生的减少，最终导致 功能失调的NM。我们进一步假设与杂合子TYR相关的PD风险增加 因此突变是通过增加细胞ROS的积累和铁积累介导的， 使SN神经元易于变性。使用微量分析超高效色谱 （UPLC），然后我们将检测从所有细胞系收集的NM以及TYR突变阳性PD患者来源的SN 神经元，以准确地评估EM：PM比率及其对NM结合能力的影响。我们希望， 同时证明TYR以年龄依赖的方式参与DA合成、DA氧化和NM生物合成，我们的研究将为我们的假设提供直接证据， 通过EM失调增加PD风险。我们假设的验证不仅证实了这一点 TYR参与DA神经元的色素沉着，但也将确立NM功能障碍作为一种新的疾病。 PD病因学机制。据我们所知，这项研究将是第一个调查TYR在DA中的作用 通过使用iPSC衍生的SN神经元在人脑中的DA产生、DA氧化和NM生物发生， 并首次使用患者来源的SN神经元来评估导致显著 TYR LOF突变携带者的PD风险增加。