Age-associated insolubility of parkin in human midbrain is linked to redox balance and sequestration of reactive dopamine metabolites

Abstract

The mechanisms by which parkin protects the adult human brain from Parkinson disease remain incompletely understood. We hypothesized that parkin cysteines participate in redox reactions and that these are reflected in its posttranslational modifications. We found that in post mortem human brain, including in the Substantia nigra, parkin is largely insoluble after age 40 years; this transition is linked to its oxidation, such as at residues Cys95 and Cys253. In mice, oxidative stress induces posttranslational modifications of parkin cysteines that lower its solubility in vivo. Similarly, oxidation of recombinant parkin by hydrogen peroxide (H2O2) promotes its insolubility and aggregate formation, and in exchange leads to the reduction of H2O2. This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. In prkn-null mice, H2O2 levels are increased under oxidative stress conditions, such as acutely by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxin exposure or chronically due to a second, genetic hit; H2O2 levels are also significantly increased in parkin-deficient human brain. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at the primate-specific residue Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation in vitro. By probing sections of adult, human midbrain from control individuals with epitope-mapped, monoclonal antibodies, we found specific and robust parkin reactivity that co-localizes with neuromelanin pigment, frequently within LAMP-3/CD63+ lysosomes. We conclude that oxidative modifications of parkin cysteines are associated with protective outcomes, which include the reduction of H2O2, conjugation of reactive dopamine metabolites, sequestration of radicals within insoluble aggregates, and increased melanin formation. The loss of these complementary redox effects may augment oxidative stress during ageing in dopamine-producing cells of mutant PRKN allele carriers, thereby enhancing the risk of Parkinson’s-linked neurodegeneration.

Discovery of small-molecule positive allosteric modulators of Parkin E3 ligase

“…When we started this drug discovery campaign, we reasoned that activating wildtype Parkin could be therapeutic for idiopathic Parkinson’s disease patients based on the following observations: (1) Parkin is found in an insoluble form in the aged human brain and thus likely enzymatically dead ( Tokarew et al., 2021 ). (2) Some studies suggest that insoluble Parkin is elevated in PD patients ( Lavoie et al., 2005 ; Wang et al., 2005 ).…”

Peiminine Reduces ARTS-Mediated Degradation of XIAP by Modulating the PINK1/Parkin Pathway to Ameliorate 6-Hydroxydopamine Toxicity and α-Synuclein Accumulation in Parkinson’s Disease Models In Vivo and In Vitro

“…This oxidative modification is associated with neuroprotection, including reducing H 2 O 2 , neutralizing reactive DA metabolites, chelating free radicals in insoluble aggregates, and increasing melanin formation. Therefore, parkin mutants may lose the ability to complement this redox effect, which increases oxidative stress and causes DA neurons to age, thereby increasing the risk of PD [47]. Therefore, PMN can prevent neuronal damage and degeneration by improving expression of parkin.…”

PINK1/Parkin Pathway Activation for Mitochondrial Quality Control – Which Is the Best Molecular Target for Therapy?

“…Two recent studies of activated dimeric PINK1 by crystallography ( Rasool et al, 2022 ) and Cryo electron microscopy ( Gan et al, 2022 ) have demonstrated a putative mechanism of kinase activation caused by oxidation and phosphorylation. PINK1 accumulates on the outer mitochondrial membrane in response to mitochondrial depolarization and its interaction with the translocase of the outer membrane (TOM complex) ( Tokarew et al, 2021 ) leads to autophosphorylation of Thr257 as well as Ser228 of the C-loop and Ser402 of the activation loop. The structures demonstrate that dimeric interface of PINK1 requires insert2 and that phosphorylation of the C-loop as well as ordering of insert 3 is necessary for conformational changes in PINK1 required to bind Ubiquitin.…”