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A novel "molecular tweezer" inhibitor of ?-synuclein neurotoxicity in vitro and in vivo.

ABSTRACT: Aggregation of ?-synuclein (?-syn) is implicated as being causative in the pathogenesis of Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Despite several therapies that improve symptoms in these disorders, none slow disease progression. Recently, a novel "molecular tweezer" (MT) termed CLR01 has been described as a potent inhibitor of assembly and toxicity of multiple amyloidogenic proteins. Here we investigated the ability of CLR01 to inhibit assembly and toxicity of ?-syn. In vitro, CLR01 inhibited the assembly of ?-syn into ?-sheet-rich fibrils and caused disaggregation of pre-formed fibrils, as determined by thioflavin T fluorescence and electron microscopy. ?-Syn toxicity was studied in cell cultures and was completely mitigated by CLR01 when ?-syn was expressed endogenously or added exogenously. To determine if CLR01 was also protective in vivo, we used a novel zebrafish model of ?-syn toxicity (?-syn-ZF), which expresses human, wild-type ?-syn in neurons. ?-Syn-ZF embryos developed severe deformities due to neuronal apoptosis and most of them died within 48 to 72 h. CLR01 added to the water significantly improved zebrafish phenotype and survival, suppressed ?-syn aggregation in neurons, and reduced ?-syn-induced apoptosis. ?-Syn expression was found to inhibit the ubiquitin proteasome system in ?-syn-ZF neurons, resulting in further accumulation of ?-syn. Treatment with CLR01 almost completely mitigated the proteasome inhibition. The data suggest that CLR01 is a promising therapeutic agent for the treatment of Parkinson's disease and other synucleinopathies.

SUBMITTER: Prabhudesai S

PROVIDER: S-EPMC3337029 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature

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A novel "molecular tweezer" inhibitor of α-synuclein neurotoxicity in vitro and in vivo.

Prabhudesai Shubhangi S Sinha Sharmistha S Attar Aida A Kotagiri Aswani A Fitzmaurice Arthur G AG Lakshmanan Rajeswari R Ivanova Magdalena I MI Loo Joseph A JA Klärner Frank-Gerrit FG Schrader Thomas T Stahl Mark M Bitan Gal G Bronstein Jeff M JM

Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 20120401 2

Aggregation of α-synuclein (α-syn) is implicated as being causative in the pathogenesis of Parkinson's disease, multiple system atrophy, and dementia with Lewy bodies. Despite several therapies that improve symptoms in these disorders, none slow disease progression. Recently, a novel "molecular tweezer" (MT) termed CLR01 has been described as a potent inhibitor of assembly and toxicity of multiple amyloidogenic proteins. Here we investigated the ability of CLR01 to inhibit assembly and toxicity ...[more]

PMID: 22373667

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Project description:Methamphetamine is an amphetamine-type psychostimulant that can damage dopaminergic neurons and cause characteristic pathological changes similar to neurodegenerative diseases such as Parkinson's disease. However, its specific mechanism of action is still unclear. In the present study, we established a Parkinson's disease pathology model by exposing SH-SY5Y cells and C57BL/6J mice to methamphetamine. In vitro experiments were performed with 0, 0.5, 1.0, 1.5, 2.0 or 2.5 mM methamphetamine for 24 hours or 2.0 mM methamphetamine for 0-, 2-, 4-, 8-, 16-, and 24-hour culture of SH-SY5Y cells. Additional experimental groups of SH-SY5Y cells were administered a nitric oxide inhibitor, 0.1 mM N-nitro-L-arginine, 1 hour before exposure to 2.0 mM methamphetamine for 24 hours. In vivo experiments: C57BL/6J mice were intraperitoneally injected with N-nitro-L-arginine (8 mg/kg), eight times, at intervals of 12 hours. Methamphetamine 15 mg/kg was intraperitoneally injected eight times, at intervals of 12 hours, but 0.5-hour after each N-nitro-L-arginine injection in the combined group. Western blot assay was used to determine the expression of nitric oxide synthase, α-synuclein (α-Syn), 5G4, nitrated α-synuclein at the residue Tyr39 (nT39 α-Syn), cleaved caspase-3, and cleaved poly ADP-ribose polymerase (PARP) in cells and mouse brain tissue. Immunofluorescence staining was conducted to measure the positive reaction of NeuN, nT39 α-Syn and 5G4. Enzyme linked immunosorbent assay was performed to determine the dopamine levels in the mouse brain. After methamphetamine exposure, α-Syn expression increased; the aggregation of α-Syn 5G4 increased; nT39 α-Syn, nitric oxide synthase, cleaved caspase-3, and cleaved PARP expression increased in the cultures of SH-SY5Y cells and in the brains of C57BL/6J mice; and dopamine levels were reduced in the mouse brain. These changes were markedly reduced when N-nitro-L-arginine was administered with methamphetamine in both SH-SY5Y cells and C57BL/6J mice. These results suggest that nT39 α-Syn aggregation is involved in methamphetamine neurotoxicity.

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A novel "molecular tweezer" inhibitor of ?-synuclein neurotoxicity in vitro and in vivo.

Publications

A novel "molecular tweezer" inhibitor of α-synuclein neurotoxicity in vitro and in vivo.

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