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.

(Patho-)physiological relevance of PINK1-dependent ubiquitin phosphorylation.

Mutations in PINK1 and PARKIN cause recessive, early-onset Parkinson's disease (PD). Together, these two proteins orchestrate a protective mitophagic response that ensures the safe disposal of damaged mitochondria. The kinase PINK1 phosphorylates ubiquitin (Ub) at the conserved residue S65, in addition to modifying the E3 ubiquitin ligase Parkin. The structural and functional consequences of Ub phosphorylation (pS65-Ub) have already been suggested from in vitro experiments, but its (patho-)physiological significance remains unknown. We have generated novel antibodies and assessed pS65-Ub signals in vitro and in cells, including primary neurons, under endogenous conditions. pS65-Ub is dependent on PINK1 kinase activity as confirmed in patient fibroblasts and postmortem brain samples harboring pathogenic mutations. We show that pS65-Ub is reversible and barely detectable under basal conditions, but rapidly induced upon mitochondrial stress in cells and amplified in the presence of functional Parkin. pS65-Ub accumulates in human brain during aging and disease in the form of cytoplasmic granules that partially overlap with mitochondrial, lysosomal, and total Ub markers. Additional studies are now warranted to further elucidate pS65-Ub functions and fully explore its potential for biomarker or therapeutic development.

Dopaminergic neuronal imaging in genetic Parkinson's disease: insights into pathogenesis.

OBJECTIVES: To compare the dopaminergic neuronal imaging features of different subtypes of genetic Parkinson's Disease. METHODS: A retrospective study of genetic Parkinson's diseases cases in which DaTSCAN (123I-FP-CIT) had been performed. Specific non-displaceable binding was calculated for bilateral caudate and putamen for each case. The right:left asymmetry index and striatal asymmetry index was calculated. RESULTS: Scans were available from 37 cases of monogenetic Parkinson's disease (7 glucocerebrosidase (GBA) mutations, 8 alpha-synuclein, 3 LRRK2, 7 PINK1, 12 Parkin). The asymmetry of radioligand uptake for Parkinson's disease with GBA or LRRK2 mutations was greater than that for Parkinson's disease with alpha synuclein, PINK1 or Parkin mutations. CONCLUSIONS: The asymmetry of radioligand uptake in Parkinsons disease associated with GBA or LRRK2 mutations suggests that interactions with additional genetic or environmental factors may be associated with dopaminergic neuronal loss.

PINK1-linked parkinsonism is associated with Lewy body pathology.

Phosphatase and tensin homolog-induced putative kinase 1 gene mutations have been associated with autosomal recessive early-onset Parkinson's disease. To date, no neuropathological reports have been published from patients with Parkinson's disease with both phosphatase and tensin homolog-induced putative kinase 1 gene copies mutated. We analysed the coding region of phosphatase and tensin homolog-induced putative kinase 1 gene in a large Spanish family with six members with parkinsonism. The phenotype was characterized by an early-onset (mean: 31.6, standard deviation: 9.6 years, range: 14-45 years), slowly progressive levodopa-responsive parkinsonism, initial gait impairment and psychiatric symptoms. We identified two segregating pathogenic phosphatase and tensin homolog-induced putative kinase 1 mutations that were either in homozygous or heterozygous compound state in all affected family members. We found an exon 7 deletion (g.16089_16383del293; c.1252_1488del) and a novel+1U1-dependent 5' splice-site mutation in exon 7 (g.16378G>A; c.1488+1G>A). Leukocyte-derived messenger RNA analysis showed that both mutations caused exon 7 skipping and c.1488+1G>A also lead to an in-frame transcript with a 33 base-pair deletion (p.L485_R497del) resulting from activation of a 5' cryptic exon 7 splice site. Single photon emission computed tomography quantification of striatal dopamine transporter binding (123I-Ioflupane) revealed a posterior-anterior gradient similar to that of idiopathic Parkinson's disease, but there was no correlation between striatal reduced uptake and disease duration. Post-mortem neuropathological examination of an early-onset Parkinson's disease carrier of two heterozygous compound phosphatase and tensin homolog-induced putative kinase 1 mutations showed neuronal loss in the substantia nigra pars compacta, Lewy bodies and aberrant neurites in the reticular nuclei of the brainstem, substantia nigra pars compacta and Meynert nucleus, but the locus ceruleus and the amygdala were spared. This is the first neuropathological report of the brain from an early-onset phosphatase and tensin homolog-induced putative kinase 1-linked parkinsonism showing that mutated phosphatase and tensin homolog-induced putative kinase 1 protein induces Lewy body pathology. Unbalanced preservation of the locus ceruleus may well play a role in the slow evolution of motor symptoms and, probably, in the psychiatric symptoms often encountered in Parkinson's disease associated with phosphatase and tensin homolog-induced putative kinase 1 mutation.