alpha-chymotrypsin and Parkinson-Disease

The aggregation of misfolded proteins, such as α-synuclein in Parkinson's disease (PD), occurs intracellularly or extracellularly in the majority of neurodegenerative diseases. The immunoproteasome has more potent chymotrypsin-like activity than normal proteasome. Thus, degradation of α-synuclein aggregation via immunoproteasome is an attractive approach for PD drug development. Herein, we aimed to determine if novel compound, 11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime (named as J24335), is a promising candidate for disease-modifying therapy to prevent the pathological progression of neurodegenerative diseases, such as PD. The effects of J24335 on inducible PC12/A53T-α-syn cell viability and cytotoxicity were evaluated by MTT assay and LDH assay, respectively. Evaluation of various proteasome activities was done by measuring the luminescence of enzymatic activity after the addition of different amounts of aminoluciferin. Immunoblotting and real-time PCR were employed to detect the expression of various proteins and genes, respectively. We also used a transgenic mouse model for behavioral testing and immunochemical analysis, to assess the neuroprotective effects of J24335. J24335 inhibited wild-type and mutant α-synuclein aggregation without affecting the growth or death of neuronal cells. The inhibition of α-synuclein aggregation by J24335 was caused by activation of immunoproteasome, as mediated by upregulation of LMP7, and increased cellular chymotrypsin-like activity in 20S proteasome. J24335-enhanced immunoproteasome activity was mediated by PKA/Akt/mTOR pathway activation. Moreover, animal studies revealed that J24335 treatment markedly mitigated both the loss of tyrosine hydroxylase-positive (TH-) neurons and impaired motor skill development. This is the first report to use J24335 as an immunoproteasome enhancing agent to antagonize pathological α-synuclein-mediated neurodegeneration.

Topics: alpha-Synuclein; Animals; Chymotrypsin; Disease Models, Animal; Mice; Mice, Transgenic; Neurodegenerative Diseases; Parkinson Disease; Proteasome Endopeptidase Complex

A growing body of evidence suggests that α-synuclein accumulation may play an important role in the pathogenesis of Parkinson's disease. The aim of this study was to investigate the roles of the proteasome and autophagy pathways in the clearance of wild-type and mutant α-synuclein in PC12 cells.. PC12 cells overexpressing either wild-type or A30P mutant α-synuclein were treated with the proteasome inhibitor epoxomicin, the macroautophagy inhibitor 3-MA and the macroautophagy activator rapamycin alone or in combination. The cell viability was assessed using MTT assay. Immunofluorescence and Western blot analysis were used to detect the level of α-synuclein, LAMP-2A, E1 activase, and E2 ligase in the cells. Chymotrypsin-like proteasomal activity was measured using a commercial kit.. When the proteasome and macroautophagy in the wild-type and mutant cells were inhibited with epoxomicin and 3-MA, respectively, the cell viability was significantly decreased, and the α-synuclein level was increased. Both epoxomicin and 3-MA activated the chaperone-mediated autophagy (CMA) by increasing the level of the CMA-limiting enzyme LAMP-2A. Furthermore, 3-MA or epoxomicin significantly decreased chymotrypsin-like proteasomal activity. 3-MA or epoxomicin did not change E1 activase expression in either mutant or wild-type cells, but increased E2 ligase expression, especially when used together. Macroautophagy inducer rapamycin increased the cell viability and reduced epoxomicin-induced α-synuclein accumulation. Interestingly, CMA was also activated by rapamycin.. Our results demonstrate the existence of complex crosstalk between different forms of autophagy and between autophagy and the proteasome pathway in the clearance of α-synuclein in PC12 cells.

Topics: Adenine; alpha-Synuclein; Animals; Autophagy; Cell Survival; Chymotrypsin; Humans; Oligopeptides; Parkinson Disease; PC12 Cells; Point Mutation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats

The aim of this work was to evaluate the role of ubiquitin-proteasome system (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson's disease (PD) cellular models.. We used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patient population we evaluated the aSN levels in the plasma and the influence of several demographic characteristics in the above mentioned determinations.. We found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a downregulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC. We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomer levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients.. aSN oligomers are ubiquitinated and we identified a ubiquitin-dependent clearance insufficiency with the accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC.

Topics: Adult; Age Factors; Aged; Aged, 80 and over; Analysis of Variance; Case-Control Studies; Cell Line, Tumor; Cell Proliferation; Chymotrypsin; Citrate (si)-Synthase; Electron Transport Complex I; Female; Green Fluorescent Proteins; Humans; Immunoprecipitation; Lactic Acid; Leukocytes, Mononuclear; Male; Middle Aged; Mitochondria; Neuroblastoma; Parkinson Disease; Plasma; Proteasome Endopeptidase Complex; RNA, Small Interfering; Statistics as Topic; Tetrazolium Salts; Thiazoles; Transfection; Ubiquitin; Ubiquitination; Up-Regulation

Alzheimer's disease (AD) is a severe chronic neurodegenerative disease. During aging and neurodegeneration, misfolded proteins accumulate and activate the ubiquitin-proteasome system. The aim of the present study is to explore whether ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, ubiquitin or proteasome activity are affected in peripheral blood mononuclear cells (PBMC) of AD, mild cognitive impairment (MCI) and Parkinson's disease (PD) patients compared to healthy subjects. PBMCs were isolated from EDTA blood samples and extracts were analyzed by Western Blot. Proteasome activity was measured with fluorogenic substrates. When compared to healthy subjects, the concentration of enzyme E1 was increased in PBMCs of AD patients, whereas the concentration of the enzyme E2 was decreased in these same patients. Ubiquitin levels and proteasome activity were unchanged in AD patients. No changes in enzyme expression or proteasome activity was observed in MCI patients compared to healthy and AD subjects. In PD patients E2 levels and proteasomal activity were significantly reduced, while ubiquitin and E1 levels were unchanged. The present investigation demonstrates the differences in enzyme and proteasome activity patterns of AD and PD patients. These results suggest that different mechanisms are involved in regulating the ubiquitin-proteasomal system in different neurodegenerative diseases.

Topics: Aged; Alzheimer Disease; Animals; Blood Cells; Chymotrypsin; Cognition Disorders; Female; Humans; Male; Mental Status Schedule; Middle Aged; Neuropsychological Tests; Parkinson Disease; Proteasome Endopeptidase Complex; Rats; Statistics, Nonparametric; Ubiquitin; Ubiquitin-Activating Enzymes

This study demonstrated that DA and its oxidative metabolites: H2O2 and aminochrome (AM), cyclized DA quinones, could all directly inhibit proteasome activity. DA and AM, especially AM, could induce intensive and irreversible proteasome inhibition, whereas proteasome inhibition induced by H2O2 was weaker and GSH reversible. It was concluded that DA induced irreversible proteasome inhibition via DA-derived quinones, rather than through small molecular weight ROS. The AM was also more toxic than H2O2 to dopaminergic MN9D cells. Furthermore the cytotoxicity and proteasome inhibition induced by DA, AM and H2O2 could be abrogated by GSH, ascorbic acid (AA), Vitamin E, SOD (superoxidase dismutase) or CAT (catalase) with different profiles. Only GSH was potent to abrogate DA, AM or H2O2-induced cell toxicity and proteasome inhibition, as well as to reverse H2O2-induced proteosome inhibition. Therefore, therapeutic strategies to increase GSH level or to use GSH substitutes should function to control PD onset and development.

Topics: Animals; Arachidonic Acid; Catalase; Cell Line; Chymotrypsin; Dopamine; Glutathione; Humans; Hydrogen Peroxide; Indolequinones; Kinetics; Leupeptins; Mice; Oxidative Stress; Parkinson Disease; Protease Inhibitors; Proteasome Inhibitors; Superoxide Dismutase; Vitamin E

Alpha-synuclein (alpha-syn) is a major protein component of the neuropathological hallmarks of Parkinson's disease and related neurodegenerative disorders termed synucleinopathies. Neither the mechanism of alpha-syn fibrillization nor the degradative process for alpha-syn has been elucidated. Previously, we showed that wild-type, mutated, and fibrillar alpha-syn proteins are substrates of calpain I in vitro. In this study, we demonstrate that calpain-mediated cleavage near and within the middle region of soluble alpha-syn with/without tyrosine nitration and oxidation generates fragments that are unable to self-fibrillize. More importantly, these fragments prevent full-length alpha-syn from fibrillizing. Calpain-mediated cleavage of alpha-syn fibrils composed of wild-type or nitrated alpha-syn generate C-terminally truncated fragments that retain their fibrillar structure and induce soluble full-length alpha-syn to co-assemble. Therefore, calpain-cleaved soluble alpha-syn inhibits fibrillization, whereas calpain-cleaved fibrillar alpha-syn promotes further co-assembly. These results provide insight into possible disease mechanisms underlying synucleinopathies since the formation of alpha-syn fibrils could be causally linked to the onset/progression of these disorders.

Topics: alpha-Synuclein; Calpain; Chymotrypsin; Humans; Hydrolysis; Microscopy, Immunoelectron; Nerve Degeneration; Nerve Tissue Proteins; Nitrates; Parkinson Disease; Peptide Fragments; Peroxynitrous Acid; Recombinant Proteins; Solubility; Synucleins; Tyrosine

Recently, impairment of the ubiquitin-proteasome system is suggested to be responsible for the neuronal death in ageing and Parkinson's disease. The specific degeneration of dopamine neurons containing neuromelanin (NM) suggests that NM itself may be involved in the cellular dysfunction and death, even though the direct link has never been reported. We examined the effects of NM isolated from the human substantia nigra on the proteasome activity in human dopaminergic SH-SY5Y cells. NM reduced the activities of 26S proteasome, as shown in situ using a green fluorescent protein homologue targeted to 26S proteasome and also in vitro using ubiquitinated lysozyme as a substrate. However, NM did not affect 20S proteasome activity in vitro. NM reduced the amount of PA700 regulatory subunit of 26S proteasome, but did not affect that of alpha- and beta-subunits of 20S proteasome. These results suggest that NM may inhibit the ubiquitin-26S proteasome system, and determine the selective vulnerability of dopamine neurons in ageing and related disorders.

Topics: Adult; Aging; Cell Death; Chymotrypsin; Dopamine; Genetic Vectors; Green Fluorescent Proteins; Humans; In Vitro Techniques; Melanins; Microscopy, Phase-Contrast; Muramidase; Ornithine Decarboxylase; Parkinson Disease; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Spectrometry, Fluorescence; Ubiquitin

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.

Topics: Animals; Antioxidants; Caspase 3; Caspases; Cell Survival; Chymotrypsin; Dopamine; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Glutathione; Neurons; Oxidation-Reduction; Oxidative Stress; Oxidopamine; Parkinson Disease; PC12 Cells; Proteasome Endopeptidase Complex; Protein Subunits; Rats; RNA, Messenger; Trypsin