OBJECTIVE: To investigate the effect of curcumin on dopamine neurons in Parkinson's disease (PD) and its mechanism. METHODS: SH-SY5Y human neuroblastoma cells were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to establish the PD cell model. The model cells were treated with curcumin and/or autophagy inhibitor 3-MA. After 48 h of drug treatment, the number of surviving dopamine neurons was detected by tyrosine hydroxylase immunofluorescence method. Western blotting was used to detect protein expression of α-Synuclein (α-Syn), transcription factor EB (TFEB) and autophagy-related proteins lysosome-associated membrane protein 2A (LAMP2A) and microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ); RT-PCR was used to detect mRNA expression of α-Syn. RESULTS: Compared with MPTP model group, curcumin increased the number of surviving dopamine neurons(<0.01), decreased both protein expression and mRNA expression of α-Syn (all <0.01), and increased protein expression of TFEB, LAMP2A and LC3-Ⅱ (all <0.01). When curcumin and 3-MA were given concurrently, the number of surviving dopamine neurons, protein expression of TFEB, LAMP2A and LC3-Ⅱ increased (<0.05 or <0.01), and both protein expression and mRNA expression of α-Syn decreased (<0.05 or <0.01) compared with MPTP model group; but the number of surviving dopamine neurons and protein expression of LAMP2A and LC3-Ⅱ decreased compared with curcumin group (all <0.05). CONCLUSIONS Curcumin exerts protective effect on dopamine neurons in PD, which may be associated with enhancing autophagy and promoting the clearance of α-Syn.
Animals ; Cell Line ; Curcumin ; pharmacology ; Dopaminergic Neurons ; drug effects ; Humans ; Mice ; Mice, Inbred C57BL ; Parkinson Disease ; alpha-Synuclein ; metabolism

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopamine (DA)-producing neurons in the substantia nigra pars compacta (SNpc) and by abnormal aggregation of alpha-synuclein. Previous studies have suggested that DA can interact with alpha-synuclein, thus modulating the aggregation process of this protein; this interaction may account for the selective vulnerability of DA neurons in patients with PD. However, the relationship between DA and alpha-synuclein, and the role in progressive degeneration of DA neurons remains elusive. We have shown that in the presence of DA, recombinant human alpha-synuclein produces non-fibrillar, SDS-resistant oligomers, while beta-sheet-rich fibril formation is inhibited. Pharmacologic elevation of the cytoplasmic DA level increased the formation of SDS-resistant oligomers in DA-producing neuronal cells. DA promoted alpha-synuclein oligomerization in intracellular vesicles, but not in the cytosol. Furthermore, elevation of DA levels increased secretion of alpha-synuclein oligomers to the extracellular space, but the secretion of monomers was not changed. DA-induced secretion of alpha-synuclein oligomers may contribute to the progressive loss of the dopaminergic neuronal population and the pronounced neuroinflammation observed in the SNpc in patients with PD.
Blotting, Western ; Cell Line, Tumor ; Dopamine/*metabolism ; Humans ; Levodopa/pharmacology ; Neurons/*metabolism/pathology/*secretion ; Parkinson Disease/metabolism/pathology ; Substantia Nigra/metabolism/pathology ; alpha-Synuclein/*biosynthesis/*secretion

&alpha;-synuclein (&alpha;-SN) has been postulated to play a pivotal role in the pathogenesis of Parkinson's disease (PD). However, the physiological functions of &alpha;-SN and the molecular and cellular mechanisms underlying neuronal loss remain unclear. Recent studies suggest that &alpha;-SN plays dual roles of neuroprotection and neurotoxicity depending on its concentration or level of expression. In the present study, we explored the potential mechanisms for &alpha;-SN to regulate neuronal survival. &alpha;-SN at different concentrations (0.1 to 40 mumol/L) with or without 50 mumol/L 6-hydroxydopamine (6-OHDA) were added into the culture medium of the SH-SY5Y dopaminergic neural cells. The cell viability was measured on post-treatment day 1, 2 and 3. The activity of proteasome inhibited by &alpha;-SN was tested by a proteasome activity assay system after 2 h of &alpha;-SN treatment. According to the activity of proteasome inhibited by &alpha;-SN, the correlative dose of proteasome inhibitor--lactacystin (10 nmol/L to 5 mumol/L) with or without 50 mumol/L 6-OHDA were used and the cell viability was assayed on post-treatment day 1, 2 and 3. The results showed that &alpha;-SN played dual roles of neuroprotection and neurotoxicity depending on its concentration. At low concentration (0.1 to 5 mumol/L), &alpha;-SN promoted the proliferation and protected neurons against the neurotoxicity of 6-OHDA; in contrast, at high concentration (10 to 40 mumol/L), &alpha;-SN possessed cytotoxicity. The results of lactacystin treatment implied that the dual roles of &alpha;-SN were related to the moderate and strong inhibition of proteasome activity. The MEK1/2 specific inhibitor PD98059 completely blocked the protection of both &alpha;-SN and lactacystin, suggesting that MAPK pathway might be involved in the neuroprotection of &alpha;-SN.
Acetylcysteine ; analogs & derivatives ; Apoptosis ; Cell Survival ; Cells, Cultured ; Dose-Response Relationship, Drug ; Humans ; Neurons ; drug effects ; Neuroprotective Agents ; pharmacology ; Neurotoxins ; adverse effects ; Oxidopamine ; adverse effects ; Parkinson Disease ; alpha-Synuclein ; adverse effects ; pharmacology

OBJECTIVETo investigate the effect of curcumin on oligomer formation and mitochondrial ATP-sensitive potassium channels (mitoKATP) induced by overexpression or mutation of &alpha;-synuclein.

METHODSRecombinant plasmids &alpha;-synuclein-pEGFP-A53T and &alpha;-synuclein-pEGFP-WT were transfected into PC12 cells by lipofectamin method, and intervened by application of curcumin (20 μmol/L) and 5-hydroxydecanoate (5-HD). Oligomer formation in the cultured cells was identified by Western blotting and Dot blotting. Cytotoxicity and apoptosis of the PC12 cells were measured by lactate dehydrogenase (LDH) and JC-1 assays. mitoKATP were identified by Western blotting and whole cell patch clamp.

RESULTSCurcumin has significantly reduced the oligomer formation induced by overexpression or mutation of &alpha;-synuclein in the cultured cells. LDH has decreased by 36.3% and 23.5%, and red/green fluorescence ratio of JC-1 was increased respectively by 48.46% and 50.33% after application of curcumin (P<0.05). Protein expression of Kir6.2 has decreased and mitoKATP channel current has significantly increased (P<0.05).

CONCLUSIONCurcumin can inhibit &alpha;-synuclein gene overexpression or mutation induced &alpha;-synuclein oligomers formation. It may block apoptosis induced by wild-type overexpression or mutation of &alpha;-synuclein. By stabilizing mitochondrial membrane potential. Opening of mitoKATP channel may have been the initiating protective mechanism of apoptosis induced by wild-type overexpression or mutation of &alpha;-synuclein. Curcumin may antagonize above cytotoxicity through further opening the mitoKATP channel.

Animals ; Apoptosis ; drug effects ; Cell Line ; Curcumin ; pharmacology ; Humans ; KATP Channels ; chemistry ; genetics ; metabolism ; Mitochondria ; drug effects ; genetics ; metabolism ; Mutation ; drug effects ; PC12 Cells ; Parkinson Disease ; drug therapy ; genetics ; metabolism ; physiopathology ; Rats ; alpha-Synuclein ; genetics

Chronic neuroinflammation is an integral pathological feature of major neurodegenerative diseases. The recruitment of microglia to affected brain regions and the activation of these cells are the major events leading to disease-associated neuroinflammation. In a previous study, we showed that neuron-released alpha-synuclein can activate microglia through activating the Toll-like receptor 2 (TLR2) pathway, resulting in proinflammatory responses. However, it is not clear whether other signaling pathways are involved in the migration and activation of microglia in response to neuron-released alpha-synuclein. In the current study, we demonstrated that TLR2 activation is not sufficient for all of the changes manifested by microglia in response to neuron-released alpha-synuclein. Specifically, the migration of and morphological changes in microglia, triggered by neuron-released alpha-synuclein, did not require the activation of TLR2, whereas increased proliferation and production of cytokines were strictly under the control of TLR2. Construction of a hypothetical signaling network using computational tools and experimental validation with various peptide inhibitors showed that beta1-integrin was necessary for both the morphological changes and the migration. However, neither proliferation nor cytokine production by microglia was dependent on the activation of beta1-integrin. These results suggest that beta1-integrin signaling is specifically responsible for the recruitment of microglia to the disease-affected brain regions, where neurons most likely release relatively high levels of alpha-synuclein.
Animals ; Antigens, CD29/genetics/*metabolism ; Cell Line, Tumor ; *Cell Movement ; Cells, Cultured ; Culture Media, Conditioned/*pharmacology ; Gene Regulatory Networks ; Humans ; Mice ; Mice, Inbred C57BL ; Microglia/drug effects/metabolism/*physiology ; Neurons/*metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 2/metabolism ; alpha-Synuclein/*pharmacology

The accumulation of abnormal protein aggregates is a major characteristic of many neurodegenerative disorders, including Parkinson's disease (PD). The intracytoplasmic deposition of alpha-synuclein aggregates and Lewy bodies, often found in PD and other alpha-synucleinopathies, is thought to be linked to inefficient cellular clearance mechanisms, such as the proteasome and autophagy/lysosome pathways. The accumulation of alpha-synuclein aggregates in neuronal cytoplasm causes numerous autonomous changes in neurons. However, it can also affect the neighboring cells through transcellular transmission of the aggregates. Indeed, a progressive spreading of Lewy pathology among brain regions has been hypothesized from autopsy studies. We tested whether inhibition of the autophagy/lysosome pathway in alpha-synuclein-expressing cells would increase the secretion of alpha-synuclein, subsequently affecting the alpha-synuclein deposition in and viability of neighboring cells. Our results demonstrated that autophagic inhibition, via both pharmacological and genetic methods, led to increased exocytosis of alpha-synuclein. In a mixed culture of alpha-synuclein-expressing donor cells with recipient cells, autophagic inhibition resulted in elevated transcellular alpha-synuclein transmission. This increase in protein transmission coincided with elevated apoptotic cell death in the recipient cells. These results suggest that the inefficient clearance of alpha-synuclein aggregates, which can be caused by reduced autophagic activity, leads to elevated alpha-synuclein exocytosis, thereby promoting alpha-synuclein deposition and cell death in neighboring neurons. This finding provides a potential link between autophagic dysfunction and the progressive spread of Lewy pathology.
Adenine/analogs & derivatives/pharmacology ; Animals ; *Autophagy/drug effects ; Cell Line ; *Exocytosis/drug effects ; Extracellular Space/*metabolism ; Humans ; Mice ; Mice, Knockout ; Microtubule-Associated Proteins/deficiency/metabolism ; Phagosomes/drug effects/metabolism ; Protein Structure, Quaternary ; Protein Transport/drug effects ; alpha-Synuclein/chemistry/*metabolism/secretion/toxicity

OBJECTIVETo silence the expression of alpha-synuclein in MN9D dopaminergic cells using vector mediated RNA interference (RNAi) and examined its effects on cell proliferation and viability.

METHODSWe identified two 19-nucleotide stretches within the coding region of the alpha-synuclein gene and designed three sets of oligonucleotides to generate double-stranded (ds) oligos. The ds oligos were inserted into the pENTR/H1/TO vector and transfected into MN9D dopaminergic cells. alpha-Synuclein expression was detected by RT-PCR, real-time PCR, immunocytochemistry staining and Western blot. In addition, we measured cell proliferation using growth curves and cell viability by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide (MTT).

RESULTSThe mRNA and protein levels of alpha-synuclein gene were significantly down-regulated in pSH2/alpha-SYN-transfected cells compared with control MN9D and pSH/CON-transfected MN9D cells, while pSH1/alpha-SYN-transfected cells showed no significant difference. Silencing alpha-synuclein expression does not affect cell proliferation but may decrease cell viability.

CONCLUSIONOur results demonstrated pSH2/alpha-SYN is an effective small interfering RNA (siRNA) sequence and potent silencing of mouse alpha-synuclein expression in MN9D cells by vector-based RNAi, which provides the tools for studying the normal function of alpha-synuclein and examining its role in Parkinson's disease (PD) pathogenesis. alpha-Synuclein may be important for the viability of MN9D cells, and loss of alpha-synuclein may induce cell injury directly or indirectly.

Animals ; Cell Line ; Cell Proliferation ; Cell Survival ; drug effects ; genetics ; Down-Regulation ; drug effects ; genetics ; Gene Silencing ; Genetic Vectors ; genetics ; Hybridomas ; Mice ; Mice, Inbred C57BL ; Nerve Degeneration ; genetics ; metabolism ; Neurons ; drug effects ; metabolism ; pathology ; Oligonucleotides ; genetics ; Parkinson Disease ; genetics ; metabolism ; Plasmids ; genetics ; RNA Interference ; RNA, Double-Stranded ; genetics ; pharmacology ; RNA, Small Interfering ; genetics ; Transfection ; methods ; alpha-Synuclein ; genetics ; metabolism