alpha-synuclein and 5-5--6-6--tetrachloro-1-1--3-3--tetraethylbenzimidazolocarbocyanine

alpha-Synuclein is a pathological component of PD (Parkinson's disease) by participating in Lewy body formation. JC-1 (5,5',6,6'-tetrachloro-1,1,3,3'-tetraethylbenzimidazolyl carbocyanine iodide) has been shown to interact with alpha-synuclein at the acidic C-terminal region with a K(d) of 2.6 microM. JC-1 can discriminated between the fibrillation states of alpha-synuclein (monomeric, oligomeric intermediate and fibrillar forms) by emitting the enhanced binding fluorescence of different colours at 590, 560 and 538 nm respectively with the common excitation at 490 nm. The fibrillation-state-specific interaction of JC-1 allowed us to perform real-time analyses of the alpha-synuclein fibrillation in the presence of iron as a fibrillation inducer, rifampicin as a fibrillation inhibitor, baicalein as a defibrillation agent and dequalinium as a protofibril inducer. In addition, various alpha-synuclein fibrils with different morphologies prepared with specific ligands such as metal ions, glutathione, eosin and lipids were monitored with their characteristic JC-1-binding fluorescence spectra. FRET (fluorescence resonance energy transfer) between thioflavin-T and JC-1 was also employed to specifically identify the amyloid fibrils of alpha-synuclein. Taken together, we have introduced JC-1 as a powerful and versatile probe to explore the molecular mechanism of the fibrillation process of alpha-synuclein in vitro. It could be also useful in high-throughput drug screening. The specific alpha-synuclein interaction of JC-1 would therefore contribute to our complete understanding of the molecular aetiology of PD and eventual development of diagnostic/therapeutic strategies for various alpha-synucleinopathies.

Topics: alpha-Synuclein; Amyloid; Benzimidazoles; Benzothiazoles; Carbocyanines; Computer Systems; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Models, Biological; Protein Binding; Protein Multimerization; Substrate Specificity; Thiazoles

Expression of CCAAT/enhancer-binding protein beta (C/EBP beta) and growth-arrest DNA damage-inducible 153/C/EBP beta homology protein (GADD153/CHOP) increased after incubation of human neuroblastoma SH-SY5Y cells with a range of dopamine concentrations. Dopamine (100 microM) caused an increase in C/EBP beta expression between 2 and 12 h of treatment, with no evident intracellular morphological changes. Dopamine (500 microM) led to the appearance of autophagic-like vacuoles and a marked increase in GADD153/CHOP between 6 and 24 h of treatment. The expression of alpha-synuclein, the main protein of Lewy bodies in Parkinson's disease and other neurological disorders, increased with a profile similar to C/EBP beta. In addition, overexpression of C/EBP beta caused a concomitant increase in the expression of alpha-synuclein but not of GADD153. In contrast, the overexpression of GADD153 did not alter the expression of alpha-synuclein. Inhibition of JNK by SP600125 reduced increases in C/EBP beta and alpha-synuclein expression, whereas inhibition of both JNK and p38MAPK (with SB203580) blocked the increase in GADD153 expression. We conclude that dopamine, through a mechanism driven by stress-activated MAPKs, triggers C/EBP beta and GADD153 expression in a dose-dependent way. Given that the promoter region of the alpha-synuclein gene contains distinct zones that are susceptible to regulation by C/EBP beta, this factor could be involved in the increased expression of alpha-synuclein after dopamine-induced cell stress. GADD153 increase seems to be related with the endoplasmic reticulum stress, autophagy and cell death observed at high dopamine concentrations.

Topics: alpha-Synuclein; Amines; Benzimidazoles; Blotting, Western; Carbocyanines; CCAAT-Enhancer-Binding Protein-beta; CCAAT-Enhancer-Binding Proteins; Cell Count; Cell Death; Cell Line, Tumor; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Fluorescent Antibody Technique; Gene Expression Regulation, Neoplastic; Heat-Shock Proteins; Humans; Molecular Chaperones; Nerve Tissue Proteins; Neuroblastoma; Proteomics; Synucleins; Time Factors; Transcription Factor CHOP; Transcription Factors; Transfection