Parkinson Disease-Linked Parkin Mediates Redox Reactions That Lower Oxidative Stress In Mammalian Brain

Abstract

We recently hypothesized that parkin plays a role in redox homeostasis and provided evidence that it directly reduces hydrogen peroxide (H 2 O 2 ) in vitro. Here, we examined this anti-oxidant activity in vivo. Informed by findings in human brain, we demonstrate that elevated oxidative stress promotes parkin insolubility in mice. In normal mouse brain parkin was partially oxidized, e.g., at cysteines 195 and 252, which was augmented by oxidative stress. Although under basal conditions H 2 O 2 levels were unchanged in adult prkn -/brain, a parkin-dependent reduction of cytosolic H 2 O 2 was observed when mitochondria were impaired, either due to neurotoxicant exposure (MPTP) or Sod2 haploinsufficiency. In accordance, markers of oxidative stress, e.g., protein carbonylation and nitrotyrosination, were elevated in the cytosol but not in mitochondria from prkn -/mice. This rise in oxidative stress was associated with altered glutathione homeostasis. In parkin's absence reduced glutathione concentrations were increased in cells, murine brain and human cortex. This compensation was not due to new glutathione synthesis but attributed to elevated oxidized glutathione (GSSG)-reductase activity. Moreover, we discovered that parkin also recycled GSSG to its reduced form. With this reaction, parkin became S-glutathionylated, e.g., at cysteines 59 and human-specific 95. This oxidative modification was reversed by glutaredoxin. Our results demonstrate that cytosolic parkin mediates anti-oxidant reactions including H 2 O 2 reduction and glutathione regeneration.These reducing activities lead to a range of oxidative modifications in parkin itself. In parkin-deficient brain oxidative stress rises despite changes to maintain redox balance. wordsIn oxidative stress-exposed, human embryonic kidney (HEK293) cells, which transiently over-expressed PRKN, we also observed HMW smear formation. Parkin became progressively gel excluded (aggregated) and insoluble following exposure to higher levels of H 2 O 2 (Fig. 1b). Since these HMW forms of parkin were reversed with dithiothreitol (DTT; Extended Data Fig. 1a, c), we postulated that these modifications occurred due to increasing oxidation of parkin's cysteines 17 . We tested this using Nethylmaleimide (NEM) and iodoacetamide (IAA), which irreversibly alkylate free thiols.There, we found that pre-incubation of cells with NEM or IAA blocked HMW smear formation of parkin and preserved its solubility during H 2 O 2 stress (Fig. 1b; Extended Data Fig. 1b). Complementing these findings, we mapped several cysteines in recombinant (r-) parkin, which became oxidized when the protein was subjected to rising H 2 O 2 levels in vitro, thereby promoting its aggregation and insolubility 11 .Intriguingly, exposure to DTT also lowered the solubility of cellular parkin (Extended Data Fig. 1c), which suggested that parkin's structure is altered under excessive oxidizing and excessive reducing conditions. These results demonstrated that human parkin acts as a redox-sensitive molecule, which undergoes thio...

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

“…2h,i ). Of note, in both models we confirmed the rise in H 2 O 2 levels (see below and El Kodsi et al [38]). In contrast to murine parkin, the solubility of endogenous Dj-1, encoded by a second, ARPD-linked gene, was not visibly affected on SDS/PAGE under these elevated oxidative stress conditions ( Fig.…”

“…Such changes could profoundly affect parkin function mediated by other domains, as has been shown in several studies involving its E3 ligase activity as a readout following modifications in the UbL domain [27, 29-31, 34, 55, 74-76] (and reviewed in Yi et al [77]). Our results do not exclude the possibility that other non-thiol-based, posttranslational modifications alter parkin solubility, such as phosphorylation at Ser65 [78], or at Ser77 [38], which we found in MPTP-treated murine brain (not shown).…”

“…Brains and hearts were collected from wild-type C57Bl/6J from Jackson laboratories, prkn -null from Dr. Brice’s laboratory [21], Sod2 +/− mice from Jackson laboratories; the bi-genic mouse ( prkn −/− // Sod2 +/− ) was created by crossing prkn -null mice with Sod2 haploinsufficient mice, and interbreeding heterozygous offspring. These bi-genic mice have been characterized elsewhere (El Kodsi et al, in preparation [38]). Mouse brains collected were homogenized on ice in a Dounce glass homogenizer by 20 passes in Tris salt buffer with or without the addition of 1% H 2 O 2 , transferred to ultracentrifuge tubes and spun at 55,000 and 4°C for 30 mins to extract the soluble fraction.…”

“…Eluted fractions (IP elute) along with controls (input, unbound, wash and recombinant parkin protein standards) were run on SDS/PAGE and blotted with anti-parkin. A sister gel was stained with Coomassie as described above and gel slices corresponding to band sizes 50-75 kDa were excised and analyzed by LC-MS/MS, as described in detail by Tokarew et al, 2020.…”

Autophagy and Redox Homeostasis in Parkinson’s: A Crucial Balancing Act

“…Lee et al recently reported that Parkin can also undergo methionine oxidation at M192, a residue mutated in early onset Parkinson's [ 301 ], and that this is reversed by methionine sulfoxide reductase B2 (MSRB2) released in response to damaged mitochondria, thereby promoting mitophagy [ 302 ]. Finally, El Kodsi et al have reported that Parkin can be glutathionylated in an antioxidant reaction (at preprint stage at the point of writing this review) [ 303 ].…”