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

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.

Age-Associated Insolubility of Parkin in Human Midbrain is Linked to Redox Balance and Sequestration of Reactive Dopamine Metabolites

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, which are reflected in its posttranslational modifications. We found that in human control brain, including the S. nigra, parkin is largely insoluble after age 40 years, which is linked to its oxidation, e.g., at Cys95 and Cys253. In mice, oxidative stress increases posttranslational modifications at parkin cysteines and reduces its solubility. Oxidation of recombinant parkin also promotes insolubility and aggregate formation, but in parallel, lowers hydrogen peroxide (H2O2). This thiol-based redox activity is diminished by parkin point mutants, e.g., p.C431F and p.G328E. Intriguingly, in parkin-deficient human brain H2O2 concentrations are elevated. In prkn-null mice, H2O2 levels are dysregulated under oxidative stress conditions, such as acutely by MPTP-toxin exposure or chronically due to a second genetic hit. In dopamine toxicity studies, wild-type parkin, but not disease-linked mutants, protects human dopaminergic M17 cells, in part through lowering H2O2. Parkin also neutralizes reactive, electrophilic dopamine metabolites via adduct formation, which occurs foremost at primate-specific Cys95. Further, wild-type but not p.C95A-mutant parkin augments melanin formation. In sections of normal, adult human midbrain, parkin specifically 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 redox effects may augment oxidative stress in dopamine producing neurons of mutant PRKN allele carriers, thereby contributing to neurodegeneration.

Nilotinib in Patients with Advanced Parkinson’s Disease: A Randomized Phase 2A Study (NILO-PD)

BackgroundNilotinib, a tyrosine kinase Abelson inhibitor, exhibits neuroprotective effects in preclinical Parkinson disease (PD) models.MethodsThis Phase 2A double-blind placebo-controlled study in moderate/advanced PD randomized participants 1:1:1 to placebo:150:300 mg nilotinib in matching capsules once daily for 6 months. The primary outcomes were safety and tolerability, the latter defined as ability to complete the study on assigned dose. Secondary outcomes included change in PD disability (Movement Disorder Society Unified Parkinson’s Disease Rating Scale (MDS-UPDRS), Part 3 OFF/ON). Additional exploratory outcomes included serum and cerebrospinal fluid (CSF) pharmacokinetic (PK) profile, and CSF dopamine metabolites.FindingsThe study screened 125 and enrolled 76 participants (39% screen failure) between November 2017 and December 2018 at 25 US sites. The last participant completed the study in September 2019. At baseline, mean (standard deviation) age was 64.6 years (7.5), disease duration 9.9 years (4.7), MDS-UPDRS Part 1-3 OFF score 66.4(19.3) and ON score 48.4(16.2), Montreal Cognitive Assessment (MoCA) score 27.1(2.2). Tolerability was 21(84%):19 (76%):20 (77%) in placebo:150:300 mg arm, respectively. Both active doses were safe. The most common reasons for drug suspension were elevations of amylase and/or lipase, which were dose-dependent. The 300 mg group had transitory worsening of MDS-UPDRS-3 ON at 1 month compared to placebo (p<0.01), which resolved by 6 months. There was no difference in the change of MDS-UPDRS-3 OFF from baseline to 6 months between the groups (p=0.17). CSF/serum PK ratio was 0.2-0.3%. There was no evidence of treatment-related elevation of any dopamine metabolites.InterpretationBoth doses of nilotinib were safe and tolerable in these participants, who were selected with strict inclusion/exclusion criteria. There was no evidence of any symptomatic benefit of nilotinib. The drug had low CSF exposure and failed to change dopamine metabolites. These findings do not warrant further testing of nilotinib in PD.FundingThe study was funded by Funded by Michael J Fox Foundation for Parkinson’s Research / The Cure Parkinson Trust / Van Andel Institute. Clinicaltrials.gov NCT03205488

Toxoplasma gondii Infection in Mice Impairs Long-Term Fear Memory Consolidation through Dysfunction of the Cortex and Amygdala

Chronic infection withToxoplasma gondiibecomes established in tissues of the central nervous system, where parasites may directly or indirectly modulate neuronal function. Epidemiological studies have revealed that chronic infection in humans is a risk factor for developing mental diseases. However, the mechanisms underlying parasite-induced neuronal dysfunction in the brain remain unclear. Here, we examined memory associated with conditioned fear in mice and found thatT. gondiiinfection impairs consolidation of conditioned fear memory. To examine the brain pathology induced byT. gondiiinfection, we analyzed the parasite load and histopathological changes.T. gondiiinfects all brain areas, yet the cortex exhibits more severe tissue damage than other regions. We measured neurotransmitter levels in the cortex and amygdala because these regions are involved in fear memory expression. The levels of dopamine metabolites but not those of dopamine were increased in the cortex of infected mice compared with those in the cortex of uninfected mice. In contrast, serotonin levels were decreased in the amygdala and norepinephrine levels were decreased in the cortex and amygdala of infected mice. The levels of cortical dopamine metabolites were associated with the time spent freezing in the fear-conditioning test. These results suggest thatT. gondiiinfection affects fear memory through dysfunction of the cortex and amygdala. Our findings provide insight into the mechanisms underlying the neurological changes seen duringT. gondiiinfection.

The Dipeptides Ile-Tyr and Ser-Tyr Exert Distinct Effects on Catecholamine Metabolism in the Mouse Brainstem

Catecholamine synthesis and transmission in the brain are influenced by the availability of Tyr in the body. In this study, we compared the effects of oral administration of Tyr-containing dipeptides Ile-Tyr, Ser-Tyr, and Tyr-Pro with Tyr alone on catecholamine metabolism in the mouse brainstem. Among these dipeptides, Ile-Tyr administration led to increases in dopamine, the dopamine metabolites homovanillic acid, and 3,4-dihydroxyphenylacetic acid, compared to administration of Ser-Tyr, Tyr-Pro, or Tyr alone. In comparison, administration of Ser-Tyr induced significantly increasing noradrenaline turnover, while Tyr-Pro administration suppressed dopamine turnover. Therefore, oral administration of Ile-Tyr, Ser-Tyr, and Tyr-Pro differentially affected metabolism of dopamine and noradrenaline. These observations strongly suggest that Tyr-containing dipeptides exert distinct effects on catecholamine metabolism in the brainstem when ingested orally.