Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease

Abstract

This investigation was pursued to test the use of intracellular antibodies (intrabodies) as a means of blocking the pathogenesis of Huntington's disease (HD). HD is characterized by abnormally elongated polyglutamine near the N terminus of the huntingtin protein, which induces pathological protein-protein interactions and aggregate formation by huntingtin or its exon 1-containing fragments. Selection from a large human phage display library yielded a single-chain Fv (sFv) antibody specific for the 17 Nterminal residues of huntingtin, adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv intrabody was tested in a cellular model of the disease in which huntingtin exon 1 had been fused to green fluorescent protein (GFP). Expression of expanded repeat HD-polyQ-GFP in transfected cells shows perinuclear aggregation similar to human HD pathology, which worsens with increasing polyglutamine length; the number of aggregates in these transfected cells provided a quantifiable model of HD for this study. Coexpression of anti-huntingtin sFv intrabodies with the abnormal huntingtin-GFP fusion protein dramatically reduced the number of aggregates, compared with controls lacking the intrabody. Anti-huntingtin sFv fused with a nuclear localization signal retargeted huntingtin analogues to cell nuclei, providing further evidence of the anti-huntingtin sFv specificity and of its capacity to redirect the subcellular localization of exon 1. This study suggests that intrabody-mediated modulation of abnormal neuronal proteins may contribute to the treatment of neurodegenerative diseases such as HD, Alzheimer's, Parkinson's, prion disease, and the spinocerebellar ataxias. H untington's disease (HD) is a genetic disorder that derives from expanded CAG repeats in the huntingtin gene (1), which then encodes pathological huntingtin protein with abnormally long polyglutamine sequences (polyQ, or Qn). Huntingtin is expressed ubiquitously by human cells, with high levels in the brain, particularly the cortex and striatum. Murine model studies of HD have shown it occurs through dominant gain of function when a single abnormal allele is present (2-6). Polyglutamine lengths ranging up to 35 residues (Q35) are present in healthy individuals, whereas Q40 or longer are associated with HD pathogenesis (1, 7). Larger polyQ expansions are correlated with earlier onset and more severe symptoms. The polyglutamine length-dependent aggregation of huntingtin has been reported to involve interaction with other proteins as well as selfassociation. Intracellular protein aggregates are found in human HD brains at autopsy and in tissues of mice carrying transgenes with expanded-repeat huntingtin (8-11). Here we report on the use of intracellular antibodies (intrabodies) as a potential therapeutic strategy on the basis of their ability to inhibit aberrant protein aggregate formation in a cellular model for HD. This approach may be applicable to other diseases as well, because HD is a paradigm for several adult-onset neurodegenerative diseases tha...

Effects of intracellular expression of anti-huntingtin antibodies of various specificities on mutant huntingtin aggregation and toxicity

“…It is notable that true nuclear aggregates of htt are not usually seen in this cell system (Ϸ1% of the cells have clearly visible EGFP-labeled aggregates in the nucleus). A similar observation has been made for other tagged htt constructs expressed in 293 cells (18,26,34,35) and in PC12 cells (E. Schweitzer, personal communication). One explanation for this observation is that the rapid cell death caused by the expanded repeat construct does not allow time for aggregates to form in the nucleus, yielding instead the large aggregates seen in Fig.…”

“…Biochemical assays show that WM7 coexpression also strongly inhibits htt aggregation. A recent study of this type showed that intracellular expression of a mAb against the N terminus of htt also inhibits aggregate formation induced in cultured cells by mutant htt exon 1 (26). The latter paper did not, however, report the effects of this scFv on cell survival, and the connection between aggregates and toxicity remains controversial.…”

“…Intracellular expression of recombinant Abs is an approach to block the toxic effects of mutated proteins or other pathogenic agents with high selectivity (25). In fact, intracellular expression of an Ab against the N terminus of htt was shown to inhibit aggregate formation induced in cultured cells by mutant htt, although quantitative results on inhibiting htt toxicity were not reported (26). We have generated eight mAbs (MW1-8) that recognize polyQ, polyproline (polyP), or a unique epitope near the C terminus of htt exon 1 (27).…”

Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody

“…Intracellularly expressed antibodies with specificity for htt have been shown to reduce aggregation and toxicity in cellular and organotypic slice culture models of HD (1)(2)(3)(4). However, high intracellular antibody (intrabody) expression levels have been required to obtain moderate reductions in aggregation and toxicity.…”

Detection of Polyglutamine Protein Oligomers in Cells by Fluorescence Correlation Spectroscopy

“…These include small peptides binding to the poly(Q) stretch (17,25,41), intrabodies against the poly(Q) proteins (42,43), molecular chaperones targeting the misfolded proteins (32,44,45), and small chemical compounds inhibiting poly(Q) aggregation (29, 46 -48). However, none of these inhibitors have been shown to inhibit poly(Q) oligomer formation in cells except for QBP1, as shown in this study.…”