Mutations in familial Parkinson's disease (PD) have been associated with the failure of protein degradation through the ubiquitin-proteasome system (UPS). Impairment of proteasome function has also been suggested to play a role in the pathogenesis of sporadic PD. We examined the proteasome activity in PC12 cells treated with 6-hydroxydopamine (6-OHDA), the dopamine synthetic derivate used in models of PD. We found that 6-OHDA treatment increased protein oxidation, as indicated by carbonyl group accumulation, and increased caspase-3 activity. In addition, there was an increase in trypsin-, chymotrypsin-, and postacidic-like proteasome activities in cells treated with 10-100 microM 6-OHDA, whereas higher doses caused a marked decline. 6-OHDA exposure also increased mRNA expression of the 19S regulatory subunit in a dose-dependent manner, whereas the expression of 20S- and 11S-subunit mRNAs did not change. Administration of the antioxidant N-acetylcysteine to 6-OHDA-treated cells prevented the alteration in proteasome functions. Moreover, reduction in cell viability owing to administration of proteasome inhibitor MG132 or lactacystin was partially prevented by the endogenous antioxidant-reduced glutathione. In conclusion, our data indicate that mild oxidative stress elevates proteasome activity in response to increase in protein damage. Severe oxidative insult might cause UPS failure, which leads to protein aggregation and cell death. Moreover, in the case of UPS inhibition or failure, the blockade of physiological reactive oxygen species production during normal aerobic metabolism is enough to ameliorate cell viability. Control of protein clearance by potent, brain-penetrating antioxidants might act to slow down the progression of PD.
"However, 6- OHDA treatment does not result in the formation of cytoplasmic inclusions, apparent in the lactacystin model. Moreover, in contrast to lactacystin, 6-OHDA may increase protein degradation and UPS activity, presumably in response to oxidative stress, as has been shown in in vitro studies ,  and . Thus, the advantage of the lactacystin model over the conventional 6-OHDA model is that, besides the loss of DA-ergic neurons in the SNc, it expresses the cardinal feature of PD, namely abnormal protein degradation due to impairment in the UPS function. "
[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to determine whether the dopamine (DA) precursor L-DOPA attenuates parkinsonian-like symptoms produced by the ubiquitin-proteasome system inhibitor lactacystin. Wistar rats were injected unilaterally with lactacystin (2.5μg/2μl) or 6-OHDA (8μg/2μl) into the substantia nigra (SN) pars compacta. Four weeks after the lesion, the animals were treated chronically with L-DOPA (25 or 50mg/kg) for two weeks. During L-DOPA treatment, the lactacystin-treated rats were tested for catalepsy and forelimb asymmetry. Rotational behavior was evaluated after apomorphine (0.25mg/kg) and L-DOPA in both PD models. After completion of experiments, the animals were killed and the levels of DA and its metabolites in the striatum and SN were assayed. We found that acute L-DOPA administration effectively decreased catalepsy and increased the use of the compromised forelimb in the cylinder test. However, the lactacystin group did not respond to apomorphine or acute L-DOPA administration in the rotational test. Repeated L-DOPA treatment produced contralateral rotations in both PD models, but the number of rotations was much greater in the 6-OHDA-lesioned rats. Both toxins markedly (>90%) reduced the levels of DA and its metabolites in the striatum and SN, while L-DOPA diminished these decreases, especially in the SN. By demonstrating the efficacy of L-DOPA in several behavioral tests, our study confirms the usefulness of the lactacystin lesion as a model of PD. However, marked differences in the rotational response to apomorphine and L-DOPA suggest different mechanisms of neurodegeneration evoked by lactacystin and 6-OHDA.
Behavioural Brain Research 03/2014; 261:79-88. DOI:10.1016/j.bbr.2013.12.019 · 3.03 Impact Factor
"In vitro knockdown of RNF11 in dopaminergic cells imparts neuroprotection against 6-OHDA-induced toxicity PC12 cells were used for the in vitro system since these cells have a dopaminergic phenotype and are responsive to 6-OHDA (Elkon et al., 2004; Zhu et al., 2012). We transfected PC12 cells with the following pAAV plasmids: shRNF11 that directs the expression of small hairpin RNA (shRNA) targeted against RNF11, shScramble that directs the expression of a scrambled version of shRNF11, and V5-RNF11 that directs the over-expression of V5-tagged RNF11. "
[Show abstract][Hide abstract] ABSTRACT: Chronic activation of the NF-κB pathway is associated with progressive neurodegeneration in Parkinson's disease (PD). Given the role of neuronal RING finger protein 11 (RNF11) as a negative regulator of the NF-κB pathway, in this report we investigated the function of RNF11 in dopaminergic cells in PD-associated neurodegeneration. We found that RNF11 knock-down in an in vitro model of PD mediated protection against 6-OHDA-induced toxicity. In converse, over-expression of RNF11 enhanced 6-OHDA-induced dopaminergic cell death. Furthermore, by directly manipulating NF-κB signaling, we showed that the observed RNF11-enhanced 6-OHDA toxicity is mediated through inhibition of NF-κB-dependent transcription of TNF-α, antioxidants GSS and SOD1, and anti-apoptotic factor BCL2. Experiments in an in vivo 6-OHDA rat model of PD recapitulated the in vitro results. In vivo targeted RNF11 over-expression in nigral neurons enhanced 6-OHDA toxicity, as evident by increased amphetamine-induced rotations and loss of nigral dopaminergic neurons as compared to controls. This enhanced toxicity was coupled with down-regulation of NF-κB transcribed GSS, SOD1, BCL2, and neurotrophic factor BDNF mRNA levels, in addition to decreased TNF-α mRNA levels in ventral mesenchephalon samples. In converse, knockdown of RNF11 was associated with protective phenotypes and increased expression of above-mentioned NF-κB transcribed genes. Collectively, our in vitro and in vivo data suggest that RNF11-mediated inhibition of NF-κB in dopaminergic cells exaggerates 6-OHDA toxicity by inhibiting neuroprotective responses while loss of RNF11 inhibition on NF-κB activity promotes neuronal survival. The decreased expression of RNF11 in surviving cortical and nigral tissue detected in PD patients, thus implies a compensatory response in the diseased brain to PD-associated insults. In summary, our findings demonstrate that RNF11 in neurons can modulate susceptibility to 6-OHDA toxicity through NF-κB mediated responses. This neuron-specific role of RNF11 in the brain has important implications for targeted therapeutics aimed at preventing neurodegeneration.
Neurobiology of Disease 01/2013; 54. DOI:10.1016/j.nbd.2012.12.018 · 5.08 Impact Factor
"These results are in agreement with those from recent studies by our lab and from others in which both caspase-9 and caspase-3 activation was observed in neuronal cultures after MPP + (1-methyl-4-phenylpyridinium) treatment (Kaul et al., 2003; Yang et al., 2004). Our results are also in agreement with previous studies, which have reported oxidative stress, alterations in mitochondrial function, cytochrome c release, and caspase-9 and caspase-3 activation during 6-OHDA-induced cell death in the PC12 pheochromocytoma cell line (Elkon et al., 2004), SH-SY5Y human neuroblastoma cells (Jordan et al., 2004), MN9D murine dopaminergic cells (Choi et al., 1999) and cerebellar granule cells (Dodel et al., 1999). "
[Show abstract][Hide abstract] ABSTRACT: The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 μM) for 24h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 μM) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKCδ) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 μM). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKCδ(D327A) and kinase dead PKCδ(K376R) or siRNA-mediated knockdown of PKCδ protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKCδ promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKCδ expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKCδ cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKCδ(D327A) protein protected against 6-OHDA-induced PKCδ activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKCδ is a key downstream event in dopaminergic degeneration, and these results may have important translational value for development of novel treatment strategies for PD.
Young-Eun Cho, Hyung-Suk Kim, Chen Lai, Ansley Stanfill, Ann Cashion
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