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Published as doi: 10.1096/fj.07-099689.
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Combinational approach of intrabody with enhanced Hsp70 expression addresses multiple pathologies in a fly model of Huntington’s disease

J. A. McLear*, D. Lebrecht*,{dagger}, A. Messer*,{dagger},1 and W. J. Wolfgang*,{dagger}

* Division of Genetic Disorders, Wadsworth Center, New York State Department of Health, Albany, New York, USA; and

{dagger} Department of Biomedical Science, School of Public Health, University of Albany, Albany, New York, USA

1Correspondence: Wadsworth Center, New York State Department of Health, 120 New Scotland Ave., Albany, NY 12208, USA. E-mail: messer{at}wadsworth.org


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular antibodies (intrabodies) and the chaperone, heat shock protein 70 (Hsp70), have each shown potential as therapeutics for neurodegenerative diseases in vitro and in vivo. Investigating combinational therapy in an established Drosophila model of Huntington’s disease (HD), we show that Hsp70 and intrabody actually affect different aspects of the disease. Overexpression of human Hsp70 resulted in improved survival of HD flies to eclosion and prolonged adult life compared with intrabody treatment alone. An additive effect on adult survival was observed when the two therapies were combined. Intrabody was more successful at suppressing neurodegeneration in photoreceptors than was Hsp70. Furthermore, Hsp70 treatment alone did not block aggregation of mutant huntingtin, a process slowed by intrabody. Expression of each is restricted to the nervous system, which implies different neuronal populations respond distinctly to these treatments. Importantly, a role for endogenous Hsp70 in suppression of mutant huntingtin pathology was confirmed by a separate set of genetic studies in which HD flies deficient for Hsp70 showed significantly increased pathology. We conclude that a combinational approach of intrabody with enhanced Hsp70 expression is beneficial in addressing multiple pathologies associated with HD and has potential application for other neurodegenerative disorders.—McLear, J. A., Lebrecht, D., Messer, A., Wolfgang, W. J. Combinational approach of intrabody with enhanced Hsp70 expression addresses multiple pathologies in a fly model of Huntington’s disease.


Key Words: scFv • polyglutamine • Drosophila • neurodegenerative disorder • chaperone protein


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HUNTINGTON DISEASE (HD), A GENETIC, neurodegenerative disease, results in motor dysfunction, cognitive decline, emotional disturbances, and premature death (1) . Age of onset is inversely correlated with the pathological expansion of >35 repeats of polyglutamine (polyQ) (2 , 3) in the huntingtin (htt) protein (4) . HD, along with Alzheimer’s disease, Parkinson’s disease (PD), spinal cerebellar ataxia (SCA), and prion-based dementia, is classified as a misfolded protein disorder based on the characteristic perinuclear and intranuclear pathogenic aggregations of truncated and full-length htt protein observed in human patients and in mouse models of HD (5 , 6) . Although the protein is found ubiquitously in eukaryotic cells, the function of wild-type htt and the role of mutant htt in cellular dysfunction remain elusive. In the brain, striatal medium spiny GABAergic neurons and cortical neurons are preferentially susceptible to neurodegeneration, whereas other cell types seem more resistant to early cell death (7) .

In efforts to understand the mechanism of disease, features of HD have been recapitulated in vitro and in vivo. In vitro, cells expressing the htt protein that has a pathological expansion of polyQ show greater intranuclear aggregation and toxicity, with increased expansion of CAG repeats and with decreased length of the non-CAG repeat regions of the htt protein (8 9 10 11) . Drosophila models of the disease exhibit CAG repeat-dependent reduced survival to adulthood, shortened life span of those adults that eclose, neurodegeneration, aggregation of mutant htt, dysregulation of transcription, and behavioral abnormalities (12 13 14 15) . In other fly models, axonal transport is disrupted (16 , 17) . Transgenic mice harboring the htt exon 1 CAG expansion exhibit aggregation of mutant htt, neurodegeneration, behavioral symptoms, and early death (18 19 20 21) . Furthermore, several lines of HD mice recapitulate the gene expression changes found in the human disorder (22) .

In an attempt to neutralize these pathogenic properties of mutant htt, we are currently investigating an intervention through the use of intracellular single-chain fragment variable (Fv) antibodies (intrabodies). The binding specificity of the intrabody makes it a unique and powerful tool to target intracellular toxic molecules. Selected from libraries and expressed as transgenes, intrabodies display target specificity that make them ideal candidates for gene therapy in situations where neutralization of toxic protein could be beneficial.

We have recently reviewed the potential of intrabodies as therapeutic agents for HD and other neurodegenerative disorders characterized by protein misfolding (23) . Experiments with the anti-HD intrabody C4-single-chain (sc) Fv showed a reduction of htt aggregation in non-neuronal cell lines cotransfected with htt exon 1 harboring a pathogenic polyQ repeat (24) ; they also showed a reduced polyQ-specific toxicity in organotypic slice cultures from mouse brains (25) . C4-scFv was found to selectively bind diffuse, soluble htt but not aggregates, suggesting that the intrabody prevents aggregation (26) . It is hypothesized that once accumulation of misfolded protein is reduced, the cell can more successfully degrade insoluble htt and reverse the pathology (27) .

In vivo, dramatic phenotypic rescue was observed in a Drosophila HD model expressing C4-scFv. The proportion of HD flies surviving to adulthood increased, and adult life span was prolonged, whereas neurodegeneration and aggregate formation were slowed (15) . However, rescue was incomplete, even with high expression levels of intrabody.

Ubiquitin, proteasomal subunits, and chaperone proteins involved in the unfolded protein response (UPR) system, have been found to colocalize with nuclear inclusions of htt (28) and other polyglutamine-aggregated proteins (29 , 30) . The chaperone protein, heat shock protein (Hsp)70, which accumulates with htt aggregates, assists in the folding of newly synthesized, misfolded, and aggregated proteins (31) . It is hypothesized that misfolded protein substrates of Hsp70 cycle between a chaperone-bound and a free state until they attain a native folding configuration (32) . Hsp70 is overwhelmed when misfolded proteins accumulate, triggering apoptotic pathways. In addition, overproduction of Hsp70 has been shown to block activation of apoptotic pathways (33) . In humans with HD, Hsp70 was found to be up-regulated in cells resistant to survival, providing a possible mechanism for the selective cell death observed in postmortem tissues (34) . Therefore, augmentation of intracellular stores of Hsp70, reduction of the presence of abnormal htt, inhibition of its interactions with other proteins, and manipulation of its solubility are attractive strategies for therapy of HD.

Intervention with htt-interacting molecules, including chaperones, has yielded modest success. Overexpression of the ubiquitin ligases, parkin, and C-terminus Hsp70 interacting protein resulted in decreased polyQ aggregation and improved cell survival (35 , 36) . Neuronal protection by chaperone proteins has also been demonstrated in several Drosophila models of polyQ-mediated neurodegenerative disorders, including Machado-Joseph disease (MJD)/SCA3 (37) , spinal and bulbar muscular atrophy (SBMA) (38) , and HD (39 , 40) . In a fly model of PD, coexpression of human Hsp70 with {alpha}-synuclein prevented dopaminergic cell loss (41) . Experiments with Hsp70 overexpression in mouse disease models have yielded less compelling results. Hsp70 overexpression ameliorated phenotypes of SBMA in transgenic mice (42) and protected against neurodegeneration in SCA1 (30) but had only modest effects in the R6/2 model of HD (43) .

In these studies, we have achieved additive rescue of HD pathology in vivo by combining Hsp70 and C4-scFv treatments, two previously established and promising therapies in the intact nervous system of Drosophila. By comparing the efficacies of these therapies applied both independently and together, we find that neuronal populations and pathologies show distinct responses to each treatment, with a combined application eliciting the most complete rescue. Importantly, we demonstrate for the first time that Hsp70 has an endogenous role in protection against HD pathology.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Fly stocks
Flies were raised on standard media, and all experimental crosses were conducted at 25°C. The P{w[+mW.hs]=GawB} elav [C155] (elav-Gal4), P{UAS-Hsap≤SPA1L.W}41.1 (HSP70 elevation [Hsp70+]), and w[1118]; Df(3R)Hsp70A, Df(3R)Hsp70B (Hsp70-deficent [HSP70 def.]) flies were obtained from the Bloomington Stock Center, Indiana University (Bloomington, IN, USA). The human htt exon 1 with a 20 polyQ repeat (UAS-htt exon-1-Q20) and UAS-htt exon-1-Q93 flies have been described and contain the entire exon 1 from htt harboring the polyQ region (13) . The UAS-C4scFv flies were previously described (15) .

Survival to adult emergence
Crosses were set such that 50% of the progeny harbored the UAS-transgenes but did not express them, and 50% carried the elav-Gal4 driver, resulting in expression of the transgenes in the nervous system. To avoid the use of balancers, which can affect relative survival rates, males carrying the elav-Gal4 driver on the X chromosome were crossed to virgin females homozygous for the UAS-transgenes. In the progeny, the males carry but do not express the UAS-transgenes, whereas in the females, the UAS-transgenes are driven by the elav-Gal4 on one X chromosome. The percentage survival to adult emergence was calculated as (expressing/nonexpressing) x 100. Bottles were cleared each day, and the numbers of males and females were counted until all of the viable F1 progeny had eclosed.

Survival analysis
Virgin females carrying the UAS-transgene of interest were crossed to males carrying elav-Gal4 driver on the X chromosome. Female progeny were collected within 24 h of emergence. For each genotype, 3 vials of 20 female flies were maintained at 25°C and transferred every other day to fresh food; the number of dead flies was counted each day. Survival curves were generated and data analyzed by Kaplan-Meier survival analysis method; statistical significance was tested using log rank statistics software, Statview (Cary, NC, USA).

Pseudopupil assay
Neurodegeneration was followed by means of the pseudopupil assay (13 , 44 , 45) . The average number of photoreceptors per ommatidium was calculated from 25–35 ommatidia from 5 flies per genotype. P values were determined by nonparametric Mann-Whitney analysis in Statview.

Histology
Mouth parts and air sacs were removed, and the heads were fixed in 4% formaldehyde in PBS containing 0.01% Triton X-100. Heads were submerged in O.C.T. embedding medium and frozen on dry ice. A cryostat was used to collect 7 µm horizontal serial sections through the brain for each genotype. Sections were mounted and immunostained using sheep-anti-htt exon-1 antibody (S830 diluted 1/12,000) (46) . Nonadjacent sections were selected for analysis near the midpoint of the lamina at x10 so that aggregates were not visible. Visible aggregates were counted with the x63 objective in the lamina cell bodies and neuropil, then the sum was divided by the area of the lamina to measure aggregate density. Four sections from 3 brains were analyzed per genotype.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
We have initiated a combinational effort to alleviate htt pathology via genetic and small-drug intervention with the goal of identifying promising therapeutic approaches for preclinical evaluation. This study sought to determine whether altered levels of Hsp70 could augment C4-scFv suppression of HD pathology in our fly model. We hypothesized that because each of these two molecules showed potential singly, an additive effect could result from the combination of increased Hsp70 and intrabody therapy. In addition, if endogenous Hsp70 is an important factor in disease progression, deficiency of Hsp70 should exacerbate pathology and possibly reduce C4-scFv efficacy. The effects of elevating or reducing Hsp70 levels were tested in an established fly model of HD using the UAS/Gal4 system, which drives expression of UAS-htt-exon1-Q93. Mutant htt expression was driven from an early embryonic stage onward, specifically in the nervous system by elav-Gal4 driver (13) . Hsp70+ was achieved using a well-characterized transgene of human Hsp70 (UAS-HSPA1L) that is 72% identical and 85% similar to the Drosophila Hsp70 protein (37 , 47) . A 50% reduction of wild-type Hsp70 levels (Hsp70 def.), was achieved using a heterozygous deficiency that removes the entire Hsp70 A and B loci (48 , 49) . Flies that were homozygous for the deficiency, although viable, were not generated at this time because of the close proximity of the UAS-htt-exon1–93Q transgene insertion site and the Hsp70 loci.

Alterations in Hsp70 levels modulate survival of HD flies and C4-scFv rescue
Reduced survival to eclosion and decreased longevity of adults are characteristics of this HD fly model (13) . Treatment of HD flies with C4-scFv intrabody was previously shown to partially rescue survival (15) . To further suppress htt pathology, we first determined whether altered expression of Hsp70, either with or without intrabody treatment, would modify eclosion of HD flies. Elav-Gal4 driven flies of each genotype were compared with their undriven siblings in these experiments (Fig. 1 ). In each comparison, the rate of eclosion for the undriven controls is considered to be 100%. In this study, only 20% of elav-driven HD flies (htt; n=212) survived to eclosion. The percentage of viable HD flies increased to 60% when the flies were treated with intrabody (htt+C4; n=637). Flies with Hsp70 overexpression (htt+Hsp70+) showed an 80% eclosion rate (n=423), whereas flies harboring both the C4-scFv and the human Hsp70 overexpression transgenes (htt+C4+Hsp70+) showed 81% eclosion (n=394). HD flies with Hsp70 deficiency (htt+Hsp70 def.) showed an eclosion rate of 8.4% (n=40), whereas intrabody treatment of Hsp70 def. HD flies (htt+C4+Hsp70 def.) improved the eclosion rate to 56% (n=193) (Fig. 1) . All genotypes were significantly different from each other with the exception of htt + Hsp70+ compared with htt + C4 + Hsp70+. Statistical values were derived by a Z test for 2 proportions (Supplemental Table 1.)


Figure 1
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  		Figure 1. Hsp70 def. reduces survival to adult emergence, whereas overexpression of Hsp70 improves rates of emergence. Hsp70 def. reduced eclosion rates of HD flies. Addition of intrabody compensated for this effect. Hsp70+ increased eclosion more effectively than did intrabody. The combination of intrabody with Hsp70+ did not have an additive effect. This chart represents the summation of 2 experiments. n = 40–637 per genotype.

Next, we asked whether Hsp70+ could improve survival in adult HD flies. Survival curves were generated to assess the effect of Hsp70 expression alterations alone and in combination with intrabody treatment (Fig. 2 ). Application of Hsp70+ dramatically improved the mean survival of HD flies by 92%, from 11.5 to 20 days (P<0.0001). Importantly, HD flies with Hsp70+ lived 47% longer than did HD flies with C4-scFv, with a mean survival of 15 days (P<0.0001). The combination of Hsp70+ and C4-scFv increased the mean survival to 22.8 days, which was a modest, although significant, 10.5% relative to flies with Hsp70+ alone (P=0.0134) (Fig. 2) . Mean and median survival times and values of significance between samples are represented in Supplemental Table 2.


Figure 2
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  		Figure 2. Altered Hsp70 levels alone and in combination with intrabody affect survival. Hsp70 def. reduced survival of HD flies (P<0.0001). This deficiency was partially compensated by treatment with intrabody to a level similar to HD fly survival (P=0.4410). Overexpression of Hsp70 rescued survival more effectively than C4 alone (P<0.0001). Combinational treatment of intrabody with Hsp70 had a modest additive effect (P=0.0134). This graph is representative of 3 independent experiments. n = 60 per genotype.

We also assessed the effect of decreased expression of Hsp70 on HD adult fly survival. In the presence of the Hsp70 def., the mean survival of HD flies was decreased from 11.5 to 7.9 days, a 31% decrease relative to HD flies with normal, endogenous levels of Hsp70 (P<0.0001) (Fig. 2) . Addition of intrabody improved mean survival of Hsp70 def. HD flies from 7.9 to 11 days, a 28% increase (P<0.0001). Mean and median survival times and values of significance are represented in Supplemental Table 2. To verify that transgenes were not affecting the UAS/Gal4 system directly and thereby altering htt levels, we generated flies harboring both UAS-GFP and UAS-Hsp70 transgenes under the control of elav-Gal4. We confirmed that GFP levels in flies with or without UAS-Hsp70 were equivalent, indicating that the Gal4 system was not altered by overexpression of Hsp70. From the preceding experiments, we conclude that Hsp70 plays an integral role in promoting survival in HD. Not only did overexpression of Hsp70 promote survival, but reduction of endogenous levels exacerbated pathology. Furthermore, intrabody function can be augmented in the presence of Hsp70.

C4-scFv suppresses neurodegeneration more effectively than Hsp70
Seven of the 8 photoreceptor cells that comprise the ommatidium of the fly eye can be quantified using a light microscope (13 , 44 , 45) . Loss of photoreceptors has been demonstrated to be a good proxy for degeneration in the nervous system of this HD fly model (14) . C4-scFv was previously shown to significantly slow neurodegeneration in our fly model (15) . In this study, we quantified neurodegeneration by assessing the average number of visible photoreceptors in adult flies at days 0, 6, 10, and 18. At day 0, the average number of visible photoreceptors in the HD flies was 5.7 (Fig. 3 A). The average increased to 6.7 with intrabody treatment (P<0.0001) and to 6.5 with Hsp70+ treatment (P<0.0001). With combinational treatment we saw an average of 6.9 photoreceptors per ommatidium (P<0.0001). By day 6, the number of visible photoreceptors was reduced across all genotypes, indicating progressive degeneration. The number of visible photoreceptors was statistically unchanged between the htt and htt + Hsp70+ fly genotypes, averaging 5.33 and 5.26 photoreceptors, respectively (P=0.5388). Intrabody, singly and in combination with Hsp70+, increased the number of photoreceptors to 5.9 and 5.7, respectively (P<0.0001). At day 6, intrabody was more effective at photoreceptor preservation than was Hsp70+ (P<0.0001) or combinational treatment (P=0.0004). By day 10, both HD flies and HD flies treated with Hsp70+ had an average of 5.1 photoreceptors per ommatidium (P=0.4788), whereas both intrabody and the combinational treatment slowed degeneration to 5.9 (P<0.0001) and 5.5 respectively (P=0.0581). Therefore, at day 10, intrabody was again more effective than Hsp70+ (P<0.0001) or the combinational treatment (P<0.0001). At day 18 only Hsp70+ and C4-scFv + Hsp70+ flies were viable. Neurodegeneration progressed between days 10 and 18 in each of these genotypes, and the combinational treatment, with 4.6 photoreceptors, continued to protect photoreceptors better than did Hsp70+ alone with 3.8 photoreceptors (Fig. 3A ) (P<0.0001). All P values are given in Supplemental Tables 3–6.


Figure 3
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  		Figure 3. Intrabody protects photoreceptors from neurodegeneration better than Hsp70+ and compensates for reduction of endogenous Hsp70. Genotypes were compared with HD at each time point. A) All treatments suppressed photoreceptor degeneration in HD flies at day 0 (P<0.0001). Intrabody alone and in combination with Hsp70+ suppressed neurodegeneration at days 0, 6, and 10. C4 was more effective than were C4 + Hsp70+ at day 6 (P=0.0004) and day 10 (P=0.0001). Hsp70+ preserved photoreceptors in HD flies at day 0 (P<0.0001) but not at day 6 (P=0.5388) or day 10 (0.4788). At day 18, flies with both treatments had more visible photoreceptors than did Hsp70+ flies (P<0.0001). B) Hsp70 def. exacerbated HD neuropathology at day 0. There was no difference between htt and htt + Hsp70 def. at day 6 (P=0.0881). C4 was protective against degeneration at days 0 and 6 in flies with normal and reduced levels of Hsp70 (P<0.0001). The efficacy of C4 was not reduced in Hsp70 def. flies at day 0 (P=0.4871) or day 6 (P=0.1719). *P < 0.05; **P < 0.0001

The pseudopupil assay was repeated in HD flies heterozygous for the Hsp70 def. (Fig. 3B ). At day 0, the average number of photoreceptors in Hsp70 def. flies was 5.6, a significant reduction compared with the 5.8 photoreceptors found in HD flies (P=0.0025). Hsp70 def. flies treated with intrabody showed photoreceptor preservation of 6.6 (P<0.0001). By day 6, neurodegeneration had increased across all genotypes. HD Hsp70 def. flies, with 5.1 photoreceptors, were no longer statistically different from HD flies, with 5.3 photoreceptors (P=0.0881). Flies with intrabody alone and intrabody in combination with Hsp70 def. continued to show significant preservation of photoreceptors, with 6.0 and 5.8 photoreceptors, respectively (P<0.0001). Importantly, at day 6, intrabody rescue was independent of Hsp70 levels, as the average number of photoreceptors found in HD flies treated with intrabody was the same in flies with normal or reduced levels of Hsp70 (P=0.1719). Further time points could not be evaluated because of the reduced longevity of the HD Hsp70 def. flies. From these results, we conclude that exogenous Hsp70 does not significantly reduce photoreceptor degeneration, whereas intrabody maintains a robust effect over time that is independent of endogenous Hsp70 levels. All values of significance for pseudopupil assays are shown in Supplemental Tables 3–6.

Elevating levels of Hsp70 does not change aggregate density whereas reducing levels of Hsp70 increases aggregate formation
C4-scFv was previously shown to slow aggregation of mutant htt in the HD fly model (15) . To determine whether altering levels of Hsp70 also affected aggregation, we measured the density of aggregates in the optic lamina at days 0, 6, and 20. In the presence of C4-scFv, htt aggregate density was reduced by 50% from a mean of 7.22 to 3.25 aggregates/µm2 x 10–3 in day 0 adult HD flies (P<0.0001). However, increased levels of Hsp70 did not significantly change aggregate density in HD flies at day 0 (6.75 aggregates/µm2x10–3; P=0.5301) (Fig. 4 ). HD flies with C4-scFv and elevated Hsp70 levels showed reduced aggregate density at this time (2.84, P<0.0001). By day 6, the HD flies, with aggregate densities of 6.43, were not significantly different from C4-scFv-treated HD flies with aggregate densities of 5.98 (P=0.7321). Increased levels of Hsp70 again did not significantly change aggregate density in HD flies (7.15, P=0.2027) or C4-scFv-treated HD flies (5.79, P=0.1599) at day 6. At day 20, flies expressing both C4-scFv and Hsp70+ showed significantly reduced aggregate density of 7.12 compared with htt + Hsp70+ flies with a density of 9.06 (P=0.0030). Only flies expressing Hsp70+ survived to day 20, precluding further comparisons (Fig. 4) .


Figure 4
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  		Figure 4. Intrabody but not Hsp70+ slows aggregate formation. Hsp70 overexpression did not reduce aggregation, whereas Hsp70 def. increased aggregate density. The increased density was suppressed by C4, showing that C4 could function independently of Hsp70 to suppress aggregation. Combination of C4 and Hsp70+ resulted in aggregate densities similar to C4 alone, indicating no additive effect.

Reducing Hsp70 levels by 50% led to a significant increase in aggregate densities at 0 (7.22–9.22, P=0.0131) and 6 days (6.43–7.88, P=0.0073). In the presence of C4-scFv, aggregate density was reduced in Hsp70 def. flies at day 0 (4.81, P=0.0033), compared with without intrabody, but by day 6 the efficacy of C4-scFv was lost (7.02, P=0.1828). In Hsp70 def. HD flies there was a modest although significant decrease in aggregate density from 0 to 6 days (9.02–7.88, P=0.0396). In summary, increasing Hsp70 levels did not influence aggregation in this HD model. As reported in the previous study, intrabody showed an effect in young adults but had no effect in older adults (15) . Together, data from Hsp70 def. experiments confirm that there is a role for endogenous Hsp70 in suppression of htt aggregation in vivo. In addition, intrabody can partially compensate for loss of Hsp70. Representative images of htt aggregation in the flies are shown in Supplemental Fig. 1.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The C4-scFv intrabody exhibits a robust ability to block mutant htt aggregation and toxicity in vitro and in vivo. In our HD fly model, its therapeutic potential increases survival, reduces neurodegeneration, and inhibits aggregation. However, rescue of the HD phenotype was only partial. We therefore explored means of enhancing intrabody protection through increased activity of chaperone proteins. The rationale for testing Hsp70 as a neural-protective treatment in our HD/intrabody study was 3-fold. Hsps act as chaperones and aid in the folding of nascent htt proteins and thereby decrease toxic levels of misfolded protein. Second, Hsp70 has an anti-apoptotic effect, which can potentially ameliorate the selective striatal cell death found in HD patients (34 , 50) . Finally, Hsp70 has shown therapeutic benefits in other models of polyQ-mediated and protein misfolding disorders (37 , 39 , 40 , 49) .

We hypothesized that the combination of intrabody and enhanced expression of the Hsp70 chaperone would create an additive rescue in our HD fly model. Furthermore, if endogenous Hsp70 plays a role in combating HD pathology, as previously reported in an MJD model (49) , then reduced levels of Hsp70 could increase pathology and possibly antagonize the therapeutic benefits of C4-scFv. This hypothesis was tested in a combinational approach using C4-scFv and Hsp70, exploiting simple, yet elegant, Drosophila genetics.

Flies harboring UAS-C4-scFv and UAS-Hsp70 transgenes, driven exclusively in neuronal cells from early embryogenesis onward, were analyzed for overall survival, selective neurodegeneration, and htt-exon1-Q93 aggregation. Hsp70 dramatically improved survival to adult emergence and prolonged survival of HD flies relative to C4-scFv-treated flies. A statistically significant, but therapeutically modest, additive improvement in adult survival was observed when Hsp70 and intrabody were combined. Exacerbation of HD pathology, characterized by reduced eclosion and adult survival, was observed in flies deficient in endogenous Hsp70. Therefore, it is important to note that endogenous Hsp70 also has a protective role in this model of HD. Similar results were observed in an MJD model (49) . Hsp70 def. HD flies treated with intrabody showed alleviation of pathology compared with untreated Hsp70 deficient flies, suggesting that C4-scFv can partially compensate for reduced endogenous Hsp70 levels.

Neurodegeneration, assessed over time, was dramatically reduced in both Hsp70+ and C4-scFv-treated flies at day 0 and on combination; a significant additive effect was observed. At later times, days 6 and 10, Hsp70+ showed no benefit, whereas intrabody was still protective.

Reduction of Hsp70 levels significantly increased neurodegeneration of HD flies at day 0. Importantly, C4-scFv fully maintained its ability to suppress neurodegeneration even in the presence of reduced Hsp70 levels.

The lack of neural protection in photoreceptors by Hsp70+ over time was unexpected. Because Hsp70 greatly enhanced survival of our HD fly population, we may have introduced a bias, which allowed us to ascertain neurodegeneration in a subpopulation of HD flies that without Hsp70 would have died. In this subpopulation, neurodegeneration may have been more extensive because the flies survived longer. Another potential caveat of our analysis is that a limited neuronal population was studied to measure neurodegeneration in this assay. Although degeneration of photoreceptors correlates well with degeneration of some neurons, an as yet unidentified vulnerable population may be better preserved in Hsp70 than in intrabody-treated flies. Because Hsp70 overexpression is restricted to the nervous system in our study, this possibility is strongly supported by the greatly improved survival in Hsp70-treated flies. It would be useful to further differentiate sensitivities to Hsp70 and intrabody treatments in other neuronal populations as this would be particularly relevant to human therapies. This approach could be evaluated using different neural Gal4 drivers to express transgenes in specific neural cell types. Furthermore, increased levels of Gal4 can trigger apoptotic neuronal loss in Drosophila (51) . It is therefore plausible that driving multiple transgenes, as we have done in this fly model, may result in vulnerability of the photoreceptor cell population.

The last aspect of the disease model that was investigated was the occurrence of htt aggregation. In this study, treatment with C4-scFv slowed but did not block aggregation in the optic lamina, as previously reported. The reduction in intrabody efficacy was not due to decreased protein levels, as immunohistochemistry for C4 was robust at all time points. The combined treatment of intrabody and Hsp70+ at days 0 and 6 resulted in no additional improvements compared with intrabody treatment alone. Reducing endogenous Hsp70 levels promoted aggregation. C4-scFv treatment of Hsp70 def. flies counteracted this increase in aggregation density, but not to the extent that intrabody treatment of flies with normal Hsp70 levels did. There was a modest yet significant reduction of aggregation from day 0 to 6 in htt + Hsp70 def. flies. Since day 6 is close to the average survival time for this genotype, this result again may reflect a selection of a healthier population of flies.

The observation that Hsp70+ had no effect on aggregation is in agreement with other fly models of Hsp70 rescue of neurodegenerative disorders where aggregation remained unchanged (37 38 39) . Although htt aggregation is associated with HD pathology, whether visible aggregates are toxic or protective remains controversial (52 53 54) . Because Hsp70 does not seem to elicit its protective effect by preventing or slowing visible aggregation, alternative modes of action occur via its antiapoptotic capability or detoxification of small, putative toxic prefibrillar intermediate molecules. It was previously reported that Hsp70 and Hsp40 act on early, structurally undefined intermediates in the polyQ assembly process (55) . In a model of PD, in which Lewy bodies accumulate, it was suggested that Hsp70 affects and stabilizes an open conformation of {alpha}-synuclein without affecting aggregation (56) . These mechanisms may explain why toxicity but not visible aggregate formation is rescued. Alternatively, subcellular distribution, tissue localization, and/or size of aggregates, parameters that were not investigated here, may correlate better with toxicity in this model. In fact, data from in vitro studies suggest that application of another intrabody, VL12.3, or fusion of this intrabody with Hsp70, does reduce aggregate size (unpublished results). Future studies will more closely examine the size and location of aggregates. Although Hsp70 alone does not prevent aggregation, other cochaperones in the same pathway may have an effect. For instance, the J domain of Hsp40 interacts with Hsp70 (57) and has been shown to reduce aggregates in vitro (36) .

HD is a progressive disease that can be detected by genetic testing. Therefore, patients can potentially be treated in presymptomatic stages of the disease. Because intrabodies appear to work by inhibiting misfolded htt, they may serve as a preventive therapy. We have recently shown that intrabodies against {alpha}-synuclein can counteract pathological aggregation and toxicity in PD models (58 , 59) . Intrabody technology has already shown promise as a therapeutic agent for diseases such as HIV (60) and cancer (61 , 62) .

Manipulation of Hsp70 levels may also be a realistic goal for clinical trials. Hsp70 is up-regulated in cells resistant to mutant htt toxicity (34) . Geldanamycin specifically binds to and inhibits a major molecular chaperone, Hsp90, thereby increasing Hsp70 levels (63) . This negative regulator of Hsp70 has shown benefit in PD and HD models (64 65 66) . A modified version of geldanamycin, 17-allylaminogeldanamycin (17-AAG), which exhibits lower toxicity, has been examined in phase I clinical trials of cancer, with encouraging results (63) . In a mouse model of polyQ-mediated motor neuron degeneration, 17-AAG was able to cross the blood-to-brain barrier and induce Hsp70 and Hsp40 (67 68 69) . In addition, Hsp70 can be secreted from and taken up by mammalian cells, suggesting exogenous therapeutic potential (70) . If Hsp70 treatment can result in tolerance of mutant htt and maintenance of tissue homeostasis, this would be an excellent therapy to deliver to presymptomatic patients or in early stages of the disease.

Several novel conclusions can be drawn from the present study. Hsp70+ and intrabody treatment each improved survival but to different extents. Even by increasing the levels of intrabody by 2-fold (15) , we never observed the robust level of survival rescue that was observed with Hsp70 alone. We demonstrate that whereas Hsp70 has its strongest effect on survival, intrabody remains most effective in neuronal preservation and aggregation reduction. Although Hsp70 has been shown to be up-regulated and colocalized with htt aggregation, this study is the first to explore the effects of deficiency of endogenous Hsp70 activity on htt pathology. Reducing endogenous levels of Hsp70 exacerbated all pathologies examined in this HD fly model. However, intrabody rescue of pathology was not dependent on wild-type levels of endogenous Hsp70.

These observations support the notion that the two molecules exert their protective mechanisms by distinct modes. Because HD is a complex disease, we believe the most effective approach to therapy appears to be a combined treatment, such as gene therapy and use of small molecules, that addresses multiple aspects of the disease. These data have implications not only for future clinical use in HD but for treatments of other neurodegenerative disorders.


   ACKNOWLEDGMENTS
 
We thank the histology and David Axelrod Institute light microscopy core facilities at the Wadsworth Center for valuable assistance. This work was supported by U.S. National Institutes of Health/National Institute of Neurological Disorders and Stroke grants to W.J.W. and A.M.

Received for publication October 11, 2007. Accepted for publication December 13, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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