Age- and disease-dependent increase of the mitophagy marker phospho-ubiquitin in normal aging and Lewy body disease

Although exact causes of Parkinson disease (PD) remain enigmatic, mitochondrial dysfunction is increasingly appreciated as a key determinant of dopaminergic neuron susceptibility in both familial and sporadic PD. Two genes associated with recessive, early-onset PD encode the ubiquitin (Ub) kinase PINK1 and the E3 Ub ligase PRKN/PARK2/Parkin, which together orchestrate a protective mitochondrial quality control (mitoQC) pathway. Upon stress, both enzymes cooperatively identify and decorate damaged mitochondria with phosphorylated poly-Ub (p-S65-Ub) chains. This specific label is subsequently recognized by autophagy receptors that further facilitate mitochondrial degradation in lysosomes (mitophagy). Here, we analyzed human post-mortem brain specimens and identified distinct pools of p-S65-Ub-positive structures that partially colocalized with markers of mitochondria, autophagy, lysosomes and/or granulovacuolar degeneration bodies. We further quantified levels and distribution of the ‘mitophagy tag’ in 2 large cohorts of brain samples from normal aging and Lewy body disease (LBD) cases using unbiased digital pathology. Somatic p-S65-Ub structures independently increased with age and disease in distinct brain regions and enhanced levels in LBD brain were age- and Braak tangle stage-dependent. Additionally, we observed significant correlations of p-S65-Ub with LBs and neurofibrillary tangle levels in disease. The degree of co-existing p-S65-Ub signals and pathological PD hallmarks increased in the pre-mature stage, but decreased in the late stage of LB or tangle aggregation. Altogether, our study provides further evidence for a potential pathogenic overlap among different forms of PD and suggests that p-S65-Ub can serve as a biomarker for mitochondrial damage in aging and disease. Abbreviations: BLBD: brainstem predominant Lewy body disease; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DLB: dementia with Lewy bodies; DLBD: diffuse neocortical Lewy body disease; EOPD: early-onset Parkinson disease; GVB: granulovacuolar degeneration body; LB: Lewy body; LBD: Lewy body disease; mitoQC: mitochondrial quality control; nbM: nucleus basalis of Meynert; PD: Parkinson disease; PDD: Parkinson disease with dementia; p-S65-Ub: PINK1-phosphorylated serine 65 ubiquitin; SN: substantia nigra; TLBD: transitional Lewy body disease; Ub: ubiquitin尽管帕金森病（Parkinson disease, PD）的确切病因仍未明确，线粒体功能障碍愈发被认为是家族性与散发性帕金森病中多巴胺能神经元（dopaminergic neuron）易感性的关键决定因素。两个与隐性遗传性早发性帕金森病相关的基因，分别编码泛素（ubiquitin, Ub）激酶PINK1以及E3泛素连接酶（E3 Ub ligase）PRKN/PARK2/Parkin，二者协同调控一条保护性的线粒体质量控制（mitochondrial quality control, mitoQC）通路。 在应激状态下，这两种酶可协同识别受损线粒体，并以磷酸化多聚泛素（p-S65-Ub）链对其进行标记。这一特异性标记随后会被自噬受体识别，进而促进受损线粒体在溶酶体中的降解，即线粒体自噬（mitophagy）。 本研究对人类死后脑组织标本进行分析，鉴定出多群不同的p-S65-Ub阳性结构，这些结构可部分与线粒体、自噬体、溶酶体以及颗粒空泡变性小体（granulovacuolar degeneration body, GVB）的标志物共定位。我们进一步采用无偏倚数字病理技术，在两个大型脑组织样本队列中对该"线粒体自噬标记物"的水平与分布进行量化，这些样本来自正常衰老人群以及路易体病（Lewy body disease, LBD）患者。 体细胞p-S65-Ub结构的数量随年龄增长以及疾病进展在不同脑区中独立升高，而路易体病患者脑组织中p-S65-Ub水平的升高则与年龄以及Braak神经原纤维缠结分期（Braak tangle stage）相关。此外，我们观察到p-S65-Ub水平与路易体（Lewy body, LB）及神经原纤维缠结（neurofibrillary tangle）水平在疾病状态下存在显著相关性。 在路易体或神经原纤维缠结聚集的早期阶段，共存的p-S65-Ub信号与帕金森病病理标志物的共定位程度升高，但在晚期阶段则出现下降。 综上，本研究为不同类型帕金森病之间存在潜在的致病重叠提供了进一步证据，并提示p-S65-Ub可作为衰老与疾病状态下线粒体损伤的生物标志物。 缩写说明：BLBD：脑干为主型路易体病（brainstem predominant Lewy body disease）；CCCP：羰基氰化物间氯苯腙（carbonyl cyanide m-chlorophenyl hydrazone）；DLB：路易体痴呆（dementia with Lewy bodies）；DLBD：弥漫性新皮质型路易体病（diffuse neocortical Lewy body disease）；EOPD：早发性帕金森病（early-onset Parkinson disease）；GVB：颗粒空泡变性小体（granulovacuolar degeneration body）；LB：路易体（Lewy body）；LBD：路易体病（Lewy body disease）；mitoQC：线粒体质量控制（mitochondrial quality control）；nbM：梅恩特氏基底核（nucleus basalis of Meynert）；PD：帕金森病（Parkinson disease）；PDD：痴呆伴帕金森病（Parkinson disease with dementia）；p-S65-Ub：PINK1磷酸化丝氨酸65泛素（PINK1-phosphorylated serine 65 ubiquitin）；SN：黑质（substantia nigra）；TLBD：过渡型路易体病（transitional Lewy body disease）；Ub：泛素（ubiquitin）