Neuroprotective Mechanisms of Parkinsons's Disease Related Protein DJ-1
Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a loss of dopaminergic neurons in the substantia nigra region of the brain and the presence in surviving neurons of Lewy body inclusions enriched with fibrillar forms of the presynaptic protein, α-synuclein (aSyn). Dysfunction of the neuroprotective protein DJ-1 is thought to be involved in familial and sporadic cases of PD. Residue cysteine 106 (C106), which converts to cysteine sulfinic acid under oxidative conditions (yielding the more thermodynamically stable 2O form of DJ-1), is thought to play an important role in various neuroprotective effects of DJ-1. Previously we showed that human wild-type DJ-1 protects against neurotoxicity elicited by different PD stresses (rotenone, an inhibitor of mitochondrial complex I; MG132, a proteasome inhibitor; and aSyn over-expression) by inducing different pro-survival mechanisms, potentially because differences in the degree of C106 oxidation or in the subcellular localization of DJ-1 could result in the activation of different neuroprotective responses. Here, we describe the results of studies aimed at testing neuroprotective effects of DJ-1 against another PD-related insult, methamphetamine (METH), which acts via a different mechanism compared to the insults listed above. We also report on studies designed to explore DJ-1 mechanisms involved in neuroprotection against different PD-insults. Results from analyses of the binding of metal ions to wild-type DJ-1 (oxidized or unoxidized) and mutant forms of the protein are also presented. We found that METH exposure caused dopaminergic neurite retraction in primary midbrain cultures, and this METH-dependent neurite loss was less pronounced in cultures transduced with adenovirus encoding human WT DJ-1. In addition, we showed that DJ-1 levels were increased in SH-SY5Y neuronal cells exposed to METH, suggesting that DJ-1 up-regulation is a cellular defense mechanism against METH neurotoxicity. To test the effect of subcellular localization of DJ-1 on METH-induced neurite retraction, we overexpressed WT DJ-1 and DJ-1 variants localized to mitochondria (MLS-DJ-1) or the nucleus (NLS-DJ-1) in METH-treated midbrain cultures. We found that both targeted DJ-1 variants showed similar neuroprotection compared to WT DJ-1, suggesting that the subcellular localization of DJ-1 does not affect its ability to alleviate METH-induced neurite retraction. Further studies showed different degrees of neuroprotection by different DJ-1 variants. The familial mutant M26I (which becomes unstable under oxidative stress conditions) failed to protect against METH-induced neurite retraction, whereas two mutants with substitutions that prevent or modulate C106 oxidation, C106A and E18D, showed partial protection or no protection (respectively) compared to WT DJ-1. These results suggest that the neuroprotective activity of DJ-1 against METH neurotoxicity is disrupted by destabilizing mutations and substitutions preventing the conversion of C106 to its oxidized form. Additional experiments revealed that DJ-1 activates the Nrf2 transcription pathway, which plays a key role in regulating the cellular antioxidant response, in astrocytes. The ability to activate Nrf2 signaling varies among different DJ-1 mutants, though the effects of the mutations are different from those described above for DJ-1-mediated protection against METH-induced neurite retraction. Whereas over-expressed M26I and C106A induce an increase in Nrf2 signaling similar to WT DJ-1, E18D fails to do so. The ability of DJ-1 to activate Nrf2-mediated transcription is unaffected by targeting DJ-1 to mitochondria or the nucleus. Finally, we show that Nrf2 expression at the same levels as those observed in DJ-1-expressing astrocytes is sufficient to protect against METH-induced neurite retraction, suggesting that activation of the Nrf2 pathway could be one of the mechanisms by which DJ-1 carries out a protective response against METH neurotoxicity. Three neuroprotective pathways were characterized in terms of their involvement in DJ-1-mediated protection against neurotoxicity elicited by the PD-related insults rotenone, A53T aSyn-encoding virus, and MG132: rescue of mitochondrial dysfunction, activation of Nrf2-mediated transcription, and amelioration of UPS impairment. Our data suggest that DJ-1-mediated protection against rotenone neurotoxicity involves enhancement of mitochondrial function by the 2O form of DJ-1. In contrast, protection against A53Tmediated dopaminergic cell death may involve all three mechanisms. In particular, Nrf2-dependent transcription must be activated above a threshold level to alleviate A53T neurotoxicity. DJ-1 apparently interferes with MG132 neurotoxicity by modulating downstream targets of the UPS as opposed to one of the three pathways outlined above. The results of these studies provide insight into mechanisms by which DJ-1 alleviates neurodegeneration in the brains of PD patients, and they could stimulate the development of new strategies to treat PD or the neurotoxic effects of METH abuse. Our findings suggest that small molecules which activate Nrf2 signaling or stabilize the 2O form of DJ-1 could be therapeutically beneficial.
Rochet, Purdue University.
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