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Human Molecular Genetics, 2003, Vol. 12, No. 11 1253-1259
DOI: 10.1093/hmg/ddg144
© 2003 Oxford University Press

Prevention of polyglutamine oligomerization and neurodegeneration by the peptide inhibitor QBP1 in Drosophila

Yoshitaka Nagai1,*, Nobuhiro Fujikake2, Katsuhito Ohno3,4, Hiroyuki Higashiyama2, Helena A. Popiel1, Julia Rahadian1, Masamitsu Yamaguchi3, Warren J. Strittmatter5,6, James R. Burke5,6 and Tatsushi Toda1

1Division of Functional Genomics, Department of Post-Genomics and Diseases, Osaka University Graduate School of Medicine, 2-2-B9 Yamadaoka, Suita, Osaka 565-0871, Japan, 2The Fourth Department, Osaka Bioscience Institute, Suita, Osaka 565-0874, Japan, 3Chromosome Technology Group, Division of Biotechnology, Faculty of Textile Sciences, Kyoto Institute of Technology, Kyoto 606-8585, Japan, 4Division of Molecular Medicine, Aichi Cancer Center Research Institute, Chikusa-ku, Nagoya, Aichi 464-8681, Japan, 5Department of Medicine (Neurology) and 6Deane Laboratory, Duke University Medical Center, Durham, NC 27710-2900, USA

Received February 28, 2003; Accepted April 2, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Polyglutamine (polyQ) diseases are a growing class of inherited neurodegenerative diseases including Huntington's disease, which are caused by abnormal expansions of the polyQ stretch in each unrelated disease protein. The expanded polyQ stretch is thought to confer toxic properties on the disease proteins through alteration of their conformation leading to pathogenic protein–protein interactions including oligomerization and/or aggregation. Hypothesizing that molecules with selective binding affinity to the expanded polyQ stretch may interfere with the pathogenic properties, we previously identified Polyglutamine Binding Peptide 1 (QBP1) from combinatorial peptide phage display libraries. We show here that a tandem repeat of the inhibitor peptide QBP1, (QBP1)2, significantly suppresses polyQ aggregation and polyQ-induced neurodegeneration in the compound eye of Drosophila polyQ disease models, which express the expanded polyQ protein under the eye specific promoter. Most importantly, (QBP1)2 expression dramatically rescues premature death of flies expressing the expanded polyQ protein in the nervous system, resulting in the dramatic increase of the median life span from 5.5 to 52 days. These results suggest that QBP1 can prevent polyQ-induced neurodegeneration in vivo. We propose that QBP1 prevents polyQ oligomerization and/or aggregation either by altering the toxic conformation of the expanded polyQ stretch, or by simply competing with the expanded polyQ stretches for binding to other expanded polyQ proteins. The peptide inhibitor QBP1 is a promising candidate with great potential as a therapeutic molecule against the currently untreatable polyQ diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Polyglutamine (polyQ) diseases are a growing class of inherited neurodegenerative diseases, in which at least nine members have been discovered to date, including Huntington's disease, various types of spinocerebellar ataxia (type 1, 2, 3, 6, 7 and 17), dentatorubral-pallidoluysian atrophy and spinobulbar muscular atrophy (1–3). These diseases are caused by abnormal expansions of the polyQ stretch to greater than 40 repeats in each unrelated disease protein. In addition, the length of the polyQ repeat correlates with the age of disease onset and disease severity. In the pathogenesis of the polyQ diseases, expansion of the polyQ stretch is thought to confer toxic properties on the disease proteins, which are not present in the normal proteins, through alteration of their conformation leading to pathogenic protein–protein interactions including oligomerization and/or aggregation (1,2). Although many polyQ protein-interacting partners have been reported, such as Huntingtin-associated protein 1 (4), GAPDH (5) and CREB binding protein (CBP) (6,7), their roles in disease pathogenesis still remain controversial. Proteins with an expanded polyQ stretch have been shown to form aggregates in vitro, in cell culture models and in animal models, and the polyQ aggregate is a pathologic hallmark in the brains of polyQ disease patients (8–10). Furthermore, various cellular proteins have been reported to associate with the polyQ aggregates, including proteasome subunits (11,12), molecular chaperones (11,13), transcription factors (14,15), and cytoskeletal proteins (10,16), possibly leading to their dysfunction.

We hypothesized that molecules with selective binding affinity to the expanded polyQ stretch may interfere with the above pathogenic properties, and therefore be useful as a therapeutic tool. We previously identified the inhibitor peptide, Polyglutamine Binding Peptide 1 (QBP1; SNWKWWPGIFD) that preferentially binds expanded polyQ stretches, from combinatorial peptide libraries using the phage display technique (17). We showed that QBP1 inhibits polyQ protein aggregation in vitro and suppresses polyQ-induced cell death in cell culture. Our hypothesis was further supported by a subsequent report showing that an intrabody binding to the sequence adjacent to the polyQ stretch of the huntingtin (htt) protein inhibited its aggregation (18). Moreover, the monoclonal antibody 1C2 that preferentially recognizes the expanded, but not normal-length, polyQ stretches has also been shown to inhibit polyQ aggregation in vitro (19). However, QBP1 is the only inhibitor molecule that has been identified through combinatorial screening and is thought to recognize the altered conformation of the expanded polyQ stretch (17). Therefore, clarifying the ability of QBP1 to prevent polyQ-induced neurodegeneration in vivo is indispensable for developing a treatment for the polyQ diseases.

To elucidate the ability of QBP1 to inhibit polyQ aggregation and to prevent neurodegeneration in vivo, we employed Drosophila models to express both pathogenic polyQ proteins and (QBP1)2, a tandem repeat of QBP1 with greater inhibitory activity. Drosophila melanogaster is a useful in vivo model to study human neurodegenerative diseases, and several polyQ disease models have already been successfully established (20, 21). In this paper, we show the robust ability of (QBP1)2 to suppress polyQ aggregation and polyQ-induced neurodegeneration in Drosophila polyQ disease models.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
(QBP1)2 suppresses compound eye degeneration in Drosophila polyQ disease models
To examine the effect of QBP1 on polyQ-induced neurodegeneration in Drosophila, we first established transgenic fly lines designed to express a tandem repeat of QBP1 fused with cyan fluorescent protein (CFP) under the control of the GAL4-UAS system (22). This (QBP1)2–CFP fusion protein was previously demonstrated to inhibit polyQ aggregation and cell death in our cell culture model (17). We designed the genetic breeding experiments to initiate (QBP1)2–CFP expression before polyQ expression because QBP1 can prevent the progression of polyQ aggregation but cannot reverse the preformed polyQ aggregates in vitro (23). We used the Q92 fly lines that express a FLAG-tagged Q92 peptide directly under the control of the gmr promoter (21), and (QBP1)2–CFP expression was driven by the eyeless (ey) promoter that is activated earlier than the gmr promoter (24,25).

Earlier expression of (QBP1)2–CFP almost completely rescued the Q92W (weak phenotype) line from compound eye degeneration so that these flies could not be distinguished from wild-type flies under the light microscope (Fig. 1A–C). Expression of the control protein (SCR)2–CFP (a tandem repeat of a scrambled sequence of QBP1 fused with CFP) did not affect the polyQ-induced phenotype at all. Scanning electron microscopy of the rescued Q92W flies revealed no morphological abnormalities of the ommatidia such as fusion and disarrangement, which are observed in the Q92 flies, and only minimal defects of the bristles were observed (Fig. 1D–I). Expression of (QBP1)2–CFP showed significant, although not complete, suppression of eye degeneration even in the Q92S (strong phenotype) line (Fig. 1J–L). With this strong phenotype line, other suppressor molecules such as Hsp70 (26), also show low levels of suppression (data not shown).



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  		Figure 1. Suppression of compound eye degeneration by (QBP1)2–CFP in the Q92 flies. Light (A–C and J–L) and scanning electron (D–I) microscopic images of the compound eye of the Q92 flies expressing a FLAG-tagged Q92 peptide under the gmr promoter (Q92W, A–I; and Q92S, J–L). (D–F) Images at lower (x200) and (G–I) higher (x800) magnification. (A, D, G and J) Transgenic flies expressing only the Q92 protein. Fly genotypes are ey-GAL4/+;gmr-Q92W/+ (A, D and G), and ey-GAL4/+;gmr-Q92S/+ (J). (B, E, H and K) Transgenic flies co-expressing the Q92 protein and (QBP1)2–CFP. Fly genotypes are ey-GAL4/UAS–QBP1;gmr-Q92W/+ (B, E and H), and ey-GAL4/UAS–QBP1;gmr-Q92S/+ (K). (C, F, I and L) Transgenic flies co-expressing the Q92 protein and the control protein (SCR)2–CFP. Fly genotypes are ey-GAL4/UAS–SCR;gmr-Q92W/+ (C, F and I), and ey-GAL4/UAS–SCR;gmr-Q92S/+ (L). Note the dramatic suppression of compound eye degeneration by (QBP1)2–CFP, especially in the Q92W flies (B, E and H) showing an almost complete rescue in phenotype.

 
We also examined the effect of (QBP1)2–CFP on polyQ-induced eye degeneration when expressed at the same time as the polyQ proteins under the gmr promoter. We used the Q92 fly line and the MJDtr–Q78S fly line, the latter of which expresses the truncated form of the MJD–Q78 protein (20). The suppression of eye degeneration was not as dramatic as observed when (QBP1)2–CFP was expressed earlier, as expected (data not shown). All of these observations were confirmed using at least two independent QBP1 transgenic fly lines. (QBP1)2–CFP expression did not affect expression levels of the polyQ proteins (data not shown). In addition, expression of (QBP1)2–CFP alone in the compound eye by the ey-GAL4 or gmr-GAL4 driver did not cause any phenotypes (data not shown).

(QBP1)2 suppresses polyQ aggregate formation in a Drosophila polyQ disease model
It has remained unclear as to whether polyQ aggregation is cytotoxic or cytoprotective (27–31). To determine the ability of QBP1 to inhibit polyQ aggregation in vivo and the role of aggregation in polyQ-induced neurodegeneration, we examined polyQ aggregate formation in the eye imaginal discs of third instar larvae of the rescued Q92W flies expressing (QBP1)2–CFP earlier than the Q92 protein. The Q92 protein forms aggregates in the region posterior to the morphogenetic furrow in the eye discs of Q92 flies (21) (Fig. 2B). Expression of (QBP1)2–CFP dramatically suppressed Q92 aggregation so that almost no aggregates were detected in the Q92W flies, in which we observed an almost complete rescue of Q92-induced eye degeneration (compare Fig. 2A and E with B and F). The co-localization of (QBP1)2–CFP with the Q92 protein could not be observed because there were almost no Q92 aggregates present in the eye disc of these flies (Fig. 2C). Expression of (SCR)2–CFP did not have any effects on Q92 aggregation at all. This observation strongly suggests a tight correlation between polyQ aggregate formation and neurodegeneration.



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  		Figure 2. Suppression of Q92 aggregate formation by (QBP1)2–CFP in the Q92 flies. Confocal microscopic images are shown of eye-antennal imaginal discs of third instar larvae of the Q92W flies, stained with an anti-FLAG antibody to detect the Q92 protein (red) and an anti-V5 antibody to detect (QBP1)2–CFP or (SCR)2–CFP (green). (A and B) Merged images at lower magnification (x200). Eye portions are to the right and antennal portions to the left. The morphogenetic furrows are indicated by arrowheads. (C–F) Images at higher magnification (x630). Staining for (QBP1)2–CFP (C), (SCR)2–CFP (D), and the Q92 protein (E and F). (A, C and E) Transgenic flies co-expressing the Q92 protein and (QBP1)2–CFP. Fly genotype is ey-GAL4/UAS–QBP1;Q92W/+. (B, D and F) Transgenic flies co-expressing the Q92 protein and (SCR)2–CFP. Fly genotype is ey-GAL4/UAS–SCR;Q92W/+. Formation of the Q92 aggregates (arrows) is observed in the region posterior to the morphogenetic furrow (arrowhead) of the control flies (B, F). Q92 aggregates were almost completely undetectable upon (QBP1)2–CFP expression (compare A and E with B and F).

 
(QBP1)2 rescues premature death caused by polyQ expression in the nervous system in Drosophila
From the therapeutic point of view, it is crucial to determine whether QBP1 is effective against not only the neuronal cell death but also the death of the whole organism caused by the expanded polyQ protein. We therefore examined the effect of (QBP1)2–CFP on the life span of transgenic flies expressing the expanded polyQ protein in the nervous system. We used the transgenic fly line MJDtr–Q78W which has been reported to show premature death when the MJDtr–Q78 protein is expressed in neurons under the elav promoter (20). (QBP1)2–CFP expression under the elav promoter dramatically increased the survival rate of the MJDtr–Q78 expressing flies to a similar level to that of flies expressing MJDtr–Q27 alone, which do not show any signs of neurodegeneration (Fig. 3). Expression of the control protein (SCR)2–CFP, on the other hand, did not affect the survival rate of the MJDtr–Q78-expressing flies at all. Median life span remarkably increased from 5.5 days for flies co-expressing MJDtr–Q78 and (SCR)2–CFP, to 52 days for flies co-expressing MJDtr–Q78 and (QBP1)2–CFP. Expression of (QBP1)2–CFP alone by the elav-GAL4 driver did not cause any phenotypes (data not shown). This result suggests that QBP1 not only suppresses polyQ aggregation and neuronal cell death but is also capable of rescuing flies from the premature death caused by the deleterious expanded polyQ protein.



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  		Figure 3. Rescue of polyQ-induced premature death by (QBP1)2–CFP in the MJDtr–Q78 flies. Survival curves are shown of flies expressing the truncated form of the MJD protein (MJDtr) under the elav promoter. MJDtr–Q78+(QBP1)2 (open circles); flies co-expressing the MJDtr–Q78 protein and (QBP1)2–CFP. The elav-GAL4 transgene is on the X chromosome. Fly genotype is elav-GAL4/+;UAS–QBP1/+;UAS–MJDtr–Q78W/+. MJDtr–Q78+(SCR)2 (closed circles); flies co-expressing the MJDtr–Q78 protein and (SCR)2–CFP. Fly genotype is elav-GAL4/+;UAS–SCR/+;UAS–MJDtr–Q78W/+. MJDtr–Q27 (closed triangles); flies expressing the MJDtr–Q27 protein, used as a control. Fly genotype is elav-GAL4/+;+;UAS–MJDtr–Q27/+. Note that (QBP1)2–CFP expression dramatically increased the survival rate of the MJDtr–Q78 expressing flies.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this paper, we employed Drosophila models to elucidate the ability of QBP1 to inhibit polyQ aggregation and to prevent neurodegeneration in vivo, aiming to develop a treatment for the polyQ diseases. Drosophila melanogaster provides a powerful model to study human neurodegenerative diseases since molecules that mitigate polyQ-induced eye degeneration in the Drosophila polyQ disease models, such as Hsp70 and Hsp40, have also been shown to mitigate polyQ-induced neurodegeneration in transgenic mouse models (20,26,32).

We showed that (QBP1)2–CFP expression dramatically improves compound eye degeneration in the Drosophila polyQ disease models (Fig. 1), suggesting that QBP1 can prevent polyQ-induced neurodegeneration in vivo. Combined with our previous observation confirming the therapeutic effect of QBP1 in mammalian cells (17), QBP1 is a promising candidate for a therapeutic molecule to treat the human polyQ diseases. The suppression of polyQ-induced eye degeneration was dramatic upon earlier expression of (QBP1)2–CFP than the polyQ protein, while it was modest when (QBP1)2–CFP was expressed at the same time as the polyQ protein. This observation was consistent with our previous observation that QBP1 can prevent further progression of polyQ aggregation even after aggregate formation has started, but cannot reverse the preformed polyQ aggregates in vitro (23). In other words, QBP1 expression before the initiation of polyQ aggregate formation will optimize its inhibition of aggregation, suggesting that QBP1 treatment before onset of the disease should be the most effective therapeutic strategy.

It has been controversial as to whether the polyQ aggregation is the cause of neurodegeneration, unrelated by-products, or the consequence of the cell's attempt to eliminate the toxic proteins (27–31). In this study, we clearly showed an almost complete inhibition of polyQ aggregation by (QBP1)2 along with the robust suppression of polyQ-induced eye degeneration, suggesting a tight correlation between polyQ aggregation and neurodegeneration (Figs 1 and 2). Several reports have also indicated a correlation between the reduction in polyQ aggregate formation and the suppression of cell death in culture cells by expression of heat shock proteins (13,33,34) and by expression of an intrabody recognizing the htt protein (35). However, we cannot exclude the possibility that (QBP1)2 also acted on inhibiting the hypothetical pathogenic interactions of the polyQ protein with critical cellular proteins such as CBP and others (15).

In this study, we demonstrated that (QBP1)2 not only suppresses the polyQ-induced eye degeneration but also rescues polyQ-induced premature death in Drosophila disease models (Fig. 3). Although the mechanisms by which expression of the polyQ protein in neurons shortens the life span of the flies are unclear, the premature death is likely to result from the neuronal dysfunction caused by the polyQ protein as well as neuronal cell death itself (20,36). It has been argued that the initial neurological phenotypes of the polyQ diseases may be due to the neuronal dysfunction caused by the expanded polyQ protein occurring before cell death, and not by the neuronal cell death itself, based on studies of postmortem patient brains and transgenic mouse brains (37–40). Therefore, (QBP1)2 may rescue polyQ-induced premature death in flies by suppressing not only polyQ-induced cell death but also the neuronal dysfunction caused by the deleterious expanded polyQ protein.

The molecular mechanism by which the binding of QBP1 to the expanded polyQ stretch prevents aggregate formation has been unclear. One possibility is that QBP1 binding alters the hypothetical toxic conformation of the expanded polyQ stretch, which is thought to trigger polyQ protein oligomerization and/or aggregation. Another possibility is that QBP1 simply competes with the expanded polyQ stretches for binding to other expanded polyQ proteins, resulting in the inhibition of oligomerization. Congo red has also been shown to ameliorate polyQ aggregation and neurodegeneration (41). Congo red is likely to recognize the toxic conformation of the expanded polyQ protein, resulting in the inhibition of its oligomerization, since it is known to preferentially bind ß-sheet rich amyloid fibrils. QBP1 is also thought to recognize the altered conformation of the expanded polyQ protein because it preferentially binds the expanded polyQ protein, and not the normal protein (17). Therefore, molecules that bind the expanded polyQ stretch may be developed for therapeutic use to inhibit polyQ oligomerization and/or aggregation.

Our present study indicates the great potential of the inhibitor peptide QBP1 as a therapeutic molecule for the polyQ diseases, for which no effective therapy is currently available. Although QBP1, an 11 amino acid peptide, is too large to cross the cell membrane to reach the target polyQ protein efficiently, we have determined the minimal sequence of QBP1 required for inhibiting polyQ aggregation to be eight amino acids (23). In addition, protein transduction domains such as HIV-1 Tat and Antennapedia (42), which have been exploited for the intracellular delivery of various proteins including the 116 kDa ß-galactosidase into the mouse, may be applied for the efficient delivery of QBP1 into the suffering neurons of polyQ disease patients. We have already shown the intracellular delivery of QBP1 by the addition of a PTD and the inhibitory effect on polyQ aggregation and cytotoxicity in cell culture (23). Alternatively, structural analysis of the QBP1–polyQ complex will provide us with information on the mode of their interaction, which should facilitate the discovery of chemical compounds with a similar mode of interaction with the polyQ protein.

Attempts of peptide-based therapy against neurodegenerative diseases have been reported in the past. A peptide derived from the amyloid ß protein (Aß) sequence was demonstrated to bind Aß and to inhibit Aß fibrillogenesis in vitro and amyloid deposition in the rat brain (43). Recently, a bivalent peptide containing normal-length polyQ sequences connected by a spacer, which is thought to bind the expanded polyQ stretch, was reported to suppress polyQ aggregation and neurodegeneration in a Drosophila model (44). These past studies, together with the success of our approach, support the effectiveness of peptide-based treatment against neurodegenerative diseases. However, these past candidate approaches utilized peptides based on the sequence of the target proteins, and a combinatorial screening approach to obtain the optimal inhibitory peptide sequence has never been undertaken before. In this study, we utilized the inhibitor peptide QBP1, which shows the best binding affinity to the expanded polyQ stretch among several peptides derived from high-throughput screening using combinatorial peptide libraries by the phage display technique (17). We show the robust ability of QBP1 to inhibit polyQ aggregation, to suppress polyQ-induced eye degeneration, and most importantly to rescue premature death in Drosophila polyQ disease models. Our strategy of utilizing high-throughput screening to identify therapeutic molecules with selective binding affinity to the pathogenic proteins may also be applied for the other abnormal protein accumulating neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Drosophila genetics
Fly culture and crosses were performed under standard conditions at 25°C. Transgenic fly lines bearing gmr-Q92, UAS–MJDtr–Q27, and UAS–MJDtr–Q78 transgenes have been described previously (20,21). Fly lines bearing ey-GAL4, gmr-GAL4, and elav-GAL4 transgenes were obtained from the Drosophila Bloomington Stock Center (25,45,46). A DNA fragment coding for a tandem repeat of QBP1 fused with CFP was excised from the mammalian expression vector, (QBP1)2–CFP (17) and inserted into the BglII and NotI sites of the pUAST vector (22). The resultant DNA construct was injected into fly embryos to generate QBP1 fly lines bearing the UAS–QBP1 transgene by standard procedures. SCR fly lines bearing the UAS–SCR transgene were also generated as a control.

For the longevity assay, more than 50 flies of mixed sex (equal number of males and females) of each genotype were collected within 24 h after eclosion and maintained at 25°C. The number of dead flies was counted and fly medium vials were changed every 2–3 days.

Microscopy and immunohistochemistry
Light microscopic images were taken using a stereoscopic microscope model SZX9 (Olympus, Tokyo, Japan). Scanning electron microscopic images were taken using an electron microscope model S-3000N (Hitachi, Tokyo, Japan) in the low vacuum mode.

Eye discs from crawling third instar larvae were dissected by standard procedures and immunohistochemistry was performed. Tissue was blocked in PBS with 10% goat serum and 0.15% Triton X-100, and immunostained using a polyclonal anti-FLAG antibody at 1 : 100 dilution (Sigma, St Louis, MO, USA) and a monoclonal anti-V5 antibody at 1 : 250 dilution (Invitrogen Corp., Carlsbad, CA, USA) as primary antibodies, and secondary antibodies conjugated with Alexa 594 and Alexa 488 (Molecular Probes, Eugene, OR, USA), respectively. Images were taken using a confocal laser scanning microscope model LSM510 (Carl Zeiss, Oberkochen, Germany).


   ACKNOWLEDGEMENTS
 
We thank Dr A. Kakizuka for providing us with the Q92W and Q92S fly lines, and Dr N.M. Bonini for providing us with the MJDtr–Q27, Q78W and Q78S fly lines. We also thank Y. Nakabayashi for her technical assistance. This work was supported by grants from the Japan Society for the Promotion of Science, and the Ministry of Health and Welfare, Japan, and the Ministry of Education, Culture, Sports, Science and Technology, Japan.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +81 668793381; Fax: +81 668793389; Email: nagai{at}clgene.med.osaka-u.ac.jp Back


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 DISCUSSION
 MATERIALS AND METHODS
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