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A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila.

ABSTRACT: The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntington's disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compounds that inhibit aggregation in the PC12 cell-based screen were tested in a Drosophila model of polyglutamine-repeat disease. The disruption of aggregation in PC12 cells strongly correlates with suppression of neuronal degeneration in Drosophila. Thus, the engineered PC12 cells coupled with the Drosophila model provide a rapid and effective method to screen and validate compounds.

SUBMITTER: Apostol BL

PROVIDER: S-EPMC156307 | biostudies-literature | 2003 May

REPOSITORIES: biostudies-literature

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A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila.

Apostol Barbara L BL Kazantsev Alexsey A Raffioni Simona S Illes Katalin K Pallos Judit J Bodai Laszlo L Slepko Natalia N Bear James E JE Gertler Frank B FB Hersch Steven S Housman David E DE Marsh J Lawrence JL Thompson Leslie Michels LM

Proceedings of the National Academy of Sciences of the United States of America 20030501 10

The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntington's disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compound ...[more]

PMID: 12730384

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Project description:The polyglutamine (polyQ) diseases are a group of inherited neurodegenerative diseases caused by the abnormal expansion of a CAG trinucleotide repeat that are translated into an expanded polyQ stretch in the disease-causative proteins. The expanded polyQ stretch itself plays a critical disease-causative role in the pathomechanisms underlying polyQ diseases. Notably, the expanded polyQ stretch undergoes a conformational transition from the native monomer into the β-sheet-rich monomer, followed by the formation of soluble oligomers and then insoluble aggregates with amyloid fibrillar structures. The intermediate soluble species including the β-sheet-rich monomer and oligomers exhibit substantial neurotoxicity. Therefore, protein conformation stabilization and aggregation inhibition that target the upstream of the insoluble aggregate formation would be a promising approach toward the development of disease-modifying therapies for polyQ diseases. PolyQ aggregation inhibitors of different chemical categories, such as intrabodies, peptides, and small chemical compounds, have been identified through intensive screening methods. Among them, recent advances in the brain delivery methods of several peptides and the screening of small chemical compounds have brought them closer to clinical utility. Notably, the recent discovery of arginine as a potent conformation stabilizer and aggregation inhibitor of polyQ proteins both in vitro and in vivo have paved way to the clinical trial for the patients with polyQ diseases. Meanwhile, expression reduction of expanded polyQ proteins per se would be another promising approach toward disease modification of polyQ diseases. Gene silencing, especially by antisense oligonucleotides (ASOs), have succeeded in reducing the expression of polyQ proteins in the animal models of various polyQ diseases by targeting the aberrant mRNA with expanded CAG repeats. Of note, some of these ASOs have recently been translated into clinical trials. Here we overview and discuss these recent advances toward the development of disease modifying therapies for polyQ diseases. We envision that combination therapies using aggregation inhibitors and gene silencing would meet the needs of the patients with polyQ diseases and their caregivers in the near future to delay or prevent the onset and progression of these currently intractable diseases.

Project description:Polyglutamine expansion in the exon 1 domain of huntingtin leads to aggregation into beta-sheet-rich insoluble aggregates associated with Huntington's disease. We assessed eight polyglutamine peptides with different permutations of N-methylation of backbone and side chain amides as potential inhibitors of polyglutamine aggregation. Surprisingly, the most effective inhibitor, 5QMe(2) [Anth-K-Q-Q(Me(2))-Q-Q(Me(2))-Q-CONH(2), where Anth is N-methylanthranilic acid and Q(Me(2)) is side chain N-methyl Q], has only side chain methylations at alternate residues, highlighting the importance of side chain interactions in polyglutamine fibrillogenesis. Above a 1:1 stoichiometric ratio, 5QMe(2) can completely prevent fibrillation of a synthetic aggregating peptide, YAQ(12)A; it also shows significant inhibition at substoichiometric ratios. Surface plasmon resonance (SPR) measurements show a moderate K(d) with very fast k(on) and k(off) values. Sedimentation equilibrium analytical ultracentrifugation indicates that 5QMe(2) is predominantly or entirely monomeric at concentrations of <or=1 mM and that it forms a 1:1 stoichiometric complex with a fibril-forming target, YAQ(12)A. 5QMe(2) inhibits not only nucleation of YAQ(12)A but also fibril extension, as shown by the fact that it also inhibits seeded fibril growth where the nucleation steps are bypassed. 5QMe(2) acts on its targets only when they are in the PPII-like conformation, but not after they undergo a transition to beta-sheets. Thus, 5QMe(2) does not disassemble preformed YAQ(12)A; this contrasts with our previously described, backbone N-methylated inhibitors of beta-amyloid aggregation [Gordon, D. J., et al. (2001) Biochemistry 40, 8237-8245; Gordon, D. J., et al. (2002) J. Pept. Res. 60, 37-55]. The mode of action of 5QMe(2) is reminiscent of that of chaperones, because it binds and releases its targets very rapidly and maintains them in a nonaggregation-prone, monomeric state, in this case, the polyproline II (PPII)-like conformation, as shown by circular dichroism spectroscopy.

Dataset Information

A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila.

Publications

A cell-based assay for aggregation inhibitors as therapeutics of polyglutamine-repeat disease and validation in Drosophila.

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