Suppression of Huntington's disease pathology in Drosophila by human single-chain Fv antibodies

Abstract

Misfolded neuronal proteins have been identified in a number of neurodegenerative disorders and have been implicated in the pathogenesis of diseases that include Alzheimer's disease, Parkinson's disease, prion-based dementia, Huntington's disease (HD), and other polyglutamine diseases. Although underlying mechanisms remain the subject of ongoing research, it is clear that aberrant processing, protein degradation, and aggregate formation or spurious protein association of the abnormal neuronal proteins may be critical factors in disease progression. Recent work in these diseases has demonstrated in vitro that specific engineered antibody species, peptides, or other general agents may suppress the formation of aggregates. We have modified an approach with intracellularly expressed single-chain Fv (sFv) antibodies (intrabodies) that bind with unique HD protein epitopes. In cell and tissue culture models of HD, anti-N-terminal huntingtin intrabodies (C4 sFv) reduce aggregation and cellular toxicity. Here, we present the crucial experiment of intrabody-mediated in vivo suppression of neuropathology, using a Drosophila model of HD. In the presence of the C4 sFv intrabody, the proportion of HD flies surviving to adulthood increases from 23% to 100%, and the mean and maximum lifespan of adult HD flies is significantly prolonged. Neurodegeneration and formation of visible huntingtin aggregates are slowed. We conclude from this investigation that engineered intrabodies are a potential new class of therapeutic agents for the treatment of neurodegenerative diseases. They may also serve as tools for drug discovery and validation of sites on mutant neuronal proteins that could be exploited for rational drug design.

Fig. 1.

C4 sFv increased the survival time of adult HD flies. Selected survival curves for cohort no. 1 are shown (see Table 2 for characterization of curves of all of the genotypes examined including cohort no. 2). HD (htt exon-1-Q93) flies without intrabody (open circle, dashed line); HD flies with C4^b sFv intrabody (closed circle, dashed line); HD flies with C4^c sFv intrabody (closed triangle, dash-dot line); and HD flies with control intrabody D10 sFv (open triangle, solid line) (a). Survival curves for control flies expressing C4^a sFv alone (solid triangle, solid line); expressing nonpathogenic htt exon-1-Q20 (solid circle, dashed line); and flies harboring but not expressing an htt exon-1-Q93 transgene (open circle, dotted line) (b). The number of flies that survived was determined for each cohort once per day. In a, asterisks indicate survival curves that were significantly different from flies expressing htt exon-1-Q93 alone (P < 0.0001). The different range and scale for the x axis in a and b are to accommodate the large difference in survival times for the two sets of data. Htt, htt exon-1-Q93 and htt Q20, htt exon-1-Q20. n = between 79 and 145 animals for each genotype.

Fig. 2.

C4 sFv increased the relative amount of soluble htt exon-1-Q93 present in fly head homogenates. Equal amounts of detergent soluble protein from heads were subjected to Western blot analysis. a probed for htt; b probed for HSP 70; and c probed for actin. Lane 1, undriven htt; lane 2, undriven htt and C4 sFv; lane 3, undriven htt and D10 sFv; lane 4, driven htt; lane 5, driven htt and C4 sFv; and lane 6, driven htt and D10 sFv. HSP70 and actin serve as loading controls. Note the increased relative abundance of htt exon-1-Q93 in the presence of C4 sFv (lane 5, Top). Molecular weight standards in kDa are shown at the right.

Fig. 3.

Neurodegeneration of photoreceptor cells is slowed in HD flies expressing C4 sFv. (a) Pseudopupil images from 1-day-old HD flies without C4 sFv (htt) or with C4 sFv (htt and C4 sFv). In the HD flies coexpressing C4 sFv, most ommatidia contain seven visible photoreceptors in contrast to flies expressing htt alone. We quantified these data as the distribution of the percent of ommatidia, containing the specified number of photoreceptors, in 1- (b) and 6-day-old (c) adult flies. For both days, the distribution is shifted significantly toward a greater numbers of photoreceptors in HD flies when C4 sFv was present (P < 0.0001). There was no statistical difference between the distribution in HD and D10 sFv HD flies. White bar, htt exon-1-Q93 expressing flies; gray bar, C4 sFv and htt exon-1-Q93; black bar, D10 sFv and htt exon-1-Q93. Error bars represent 95% confidence interval. Significance was calculated by using the nonparametric Mann–Whitney test. For each genotype, five animals were assayed with a total number of ommatidia scored for each animal between 137 and 272. Htt, htt exon-1-Q93.