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Identification of anti-inflammatory targets for Huntington's disease using a brain slice-based screening assay.

ABSTRACT: Huntington's disease (HD) is a late-onset, neurodegenerative disease for which there are currently no cures nor disease-modifying treatments. Here we report the identification of several potential anti-inflammatory targets for HD using an ex vivo model of HD that involves the acute transfection of human mutant huntingtin-based constructs into rat brain slices. This model recapitulates key components of the human disease, including the formation of intracellular huntingtin protein (HTT)-containing inclusions and the progressive neurodegeneration of striatal neurons-both occurring within the native tissue context of these neurons. Using this "high-throughput biology" screening platform, we conducted a hypothesis-neutral screen of a collection of drug-like compounds which identified several anti-inflammatory targets that provided neuroprotection against HTT fragment-induced neurodegeneration. The nature of these targets provide further support for non-cell autonomous mechanisms mediating significant aspects of neuropathogenesis induced by mutant HTT fragment proteins.

SUBMITTER: Reinhart PH

PROVIDER: S-EPMC3104027 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Identification of anti-inflammatory targets for Huntington's disease using a brain slice-based screening assay.

Reinhart Peter H PH Kaltenbach Linda S LS Essrich Christian C Dunn Denise E DE Eudailey Joshua A JA DeMarco C Todd CT Turmel Gregory J GJ Whaley Jennifer C JC Wood Andrew A Cho Seongeun S Lo Donald C DC

Neurobiology of disease 20110331 1

Huntington's disease (HD) is a late-onset, neurodegenerative disease for which there are currently no cures nor disease-modifying treatments. Here we report the identification of several potential anti-inflammatory targets for HD using an ex vivo model of HD that involves the acute transfection of human mutant huntingtin-based constructs into rat brain slices. This model recapitulates key components of the human disease, including the formation of intracellular huntingtin protein (HTT)-containin ...[more]

PMID: 21458569

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Project description:Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D(0) = 1.3 ± 0.1 Gy and ñ = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85?) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D(0) = 1.3 ± 0.1 Gy and ñ = 2.3 ± 0.3 for the vehicle control treated cells compared to D(0) = 1.2 ± 0.1 Gy and ñ = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation-induced apoptosis in NCCIT cells. Treatment of mice with a single intraperitoneal LY294002 dose of 30 mg/kg at 10 min, 4, or 24 h after LD(50/30) whole-body dose of irradiation (9.25 Gy) enhanced survival. This study documents that an unbiased siRNA assay can identify new genes, signaling pathways, and chemotypes as radiation mitigators and implicate the PI3K pathway in the human radiation response.

Dataset Information

Identification of anti-inflammatory targets for Huntington's disease using a brain slice-based screening assay.

Publications

Identification of anti-inflammatory targets for Huntington's disease using a brain slice-based screening assay.

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