Skip Navigation


Human Molecular Genetics Advance Access originally published online on December 8, 2007
Human Molecular Genetics 2008 17(6):882-894; doi:10.1093/hmg/ddm360
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
17/6/882    most recent
ddm360v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Liévens, J.-C.
Right arrow Articles by Birman, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liévens, J.-C.
Right arrow Articles by Birman, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

AKT-sensitive or insensitive pathways of toxicity in glial cells and neurons in Drosophila models of Huntington's disease

Jean-Charles Liévens1,*, Magali Iché2, Monique Laval1, Catherine Faivre-Sarrailh1 and Serge Birman2

1 Neurobiologie des Interactions Cellulaires et Neurophysiopathologie, CNRS UMR 6184, Université de la Méditerranée, Institut Jean-Roche, 13916 Marseille Cedex 20, France 2 Institut de Biologie du Développement de Marseille-Luminy, CNRS-Université de la Méditerranée, Campus de Luminy, Case 907, 13288 Marseille Cedex 9, France

* To whom correspondence should be addressed. Tel: +33 491698880; Fax: +33 491698977; Email: jean-charles.lievens{at}univmed.fr

Received September 26, 2007; Revised November 16, 2007; Accepted December 4, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOTE ADDED IN PROOF
 FUNDING
 REFERENCES
 
Huntington's disease (HD) is caused by an extended polyglutamine (polyQ) tract in the Huntingtin protein. Neuronal and glial dysfunction precedes the neurodegeneration and appears to be the primary cause for the early symptoms in HD. In recent years, development of Drosophila models of polyQ-related diseases facilitated research of candidate rescuer genes. In most cases, analysis in Drosophila was performed by assessing toxicity on retinal and/or brain neurons. However, none of the potential rescuers were evaluated on glial alterations. Here we used a genetic approach in Drosophila to characterize the phenotypic effects of mutant Huntingtin (mHtt) expressed in neurons or different glia subsets and we established a sensitive assay for evaluating modifiers of glial alterations. We determined the level of cell protection ensured by activation of the AKT and ERK anti-apoptotic kinases in the retina as well as in neurons and glia of the fly brain, compared with the rescuing effects of the HSP70 chaperone. We found that both AKT and HSP70 alleviated mHtt-induced toxicity in the retina. In contrast, their protective effects differed in the brain. HSP70 rescued neurodegeneration, locomotor defects and early lethality of flies expressing mHtt in neurons or glia. AKT failed to prevent brain neuronal death and lethality of flies, but significantly improved their locomotor performance when co-expressed with mHtt in glia. ERK had no beneficial effects in the retina or brain. These results indicate that mHtt activates distinct pathways of toxicity in Drosophila, either sensitive to AKT in retinal photoreceptors and glia, or independent in brain neurons.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOTE ADDED IN PROOF
 FUNDING
 REFERENCES
 
Huntington's disease (HD) is an inherited neurodegenerative disorder caused by the expansion of a CAG triplet repeat in the first exon of the IT15 gene, resulting in increased length of a polyglutamine (polyQ) stretch in the Huntingtin protein (Htt). Pathological symptoms appear with polyQ repeat >37 glutamine residues (1,2). At least eight additional neurodegenerative diseases including spinocerebellar ataxia (SCA) 1, 2, 3, 6, 7 and 17, dentatorubral pallidoluysian atrophy and bulbospinal muscular atrophy (SBMA) are also caused by expanded polyQ tract in specific proteins. Htt is ubiquitously expressed in brain, highly in neurons but yet significantly in glia. Moderate levels are also present in peripheral tissues such as testes, liver, heart and lungs (3–8). Despite such a widespread distribution, mutant Huntingtin (mHtt) induces late-onset neuronal loss preferentially in the striatum and cerebral cortex (9). There is evidence that cell brain function impairments precede neurodegeneration and appears to be the primary cause for the early phases of HD pathogenesis (10,11).

Over the last decade, neuronal dysfunction and death were the focus of many studies. Only recently, it was established that expression of mHtt in glia directly compromises the functions of these cells, such as glutamate removal from synaptic cleft (7,12). In post-mortem brains from HD patients, glial expression of glutamate and glucose transporters was found to be significantly reduced in the striatum (13,14), although astrocytes proliferate in this cerebral structure. Moreover, activated astrocytes express high levels of the apoptosis-related markers caspase-3 and poly(ADP-ribose) polymerase (15,16). Thus, disruption of glia functions and neuron–glia interaction might further challenge neurons already altered by intrinsic dysfunction. Testing therapeutic candidates on both neuronal and glial pathologies could prove invaluable to define novel reasoned strategies to treat HD.

The fruit fly Drosophila melanogaster is a powerful model organism to identify pathways involved in HD pathogenesis. Fly models replicate HD pathological features including axonal and glial dysfunctions, neuronal loss restricted to some cerebral structures and shortened adult lifespan (12,17–21). In recent years, a number of candidate rescuer genes were discovered from genetic analysis of polyQ diseases in Drosophila. The most effective modifiers were generally molecular chaperones and apoptosis- or prosurvival-related proteins. For instance, they include chaperone proteins such as HSP40, HSP70, HSC70 and Tetratricopeptide repeat protein 2 (TPR2) (22–28). Genetic manipulation of genes regulating apoptosis such as dIAP1, Apaf-1 and p53 (28–30) was also found to modulate polyQ toxicity. In most cases, rescuer gene analysis was performed by expressing expanded polyQ peptides in the fly retina and some of them were further tested on neuronal pathology. However, none of them were evaluated on glial pathology.

Epidermal growth factor receptor (EGFR) signalling is known to play major roles in cell development, differentiation and survival and is a key actor in the induction and maintenance of the astroglial reactivity in pathological situations (31). We and others reported that mHtt antagonizes the activation of AKT and ERK kinases by EGFR in cell-based and Drosophila HD models (12,32–34). Here we used a genetic approach in Drosophila to characterize the phenotypic effects of mHtt expression in neurons or different glia subtypes. We first developed a sensitive assay for evaluating modifiers of glial alterations. Then we evaluated the potential benefits of anti-apoptotic AKT and ERK kinases and HSP70 chaperone when co-expressed with mHtt in the eye photoreceptors and brain neurons or glia. Although both AKT and HSP70 alleviated the mHtt-induced cell toxicity in the retina, they did not share similar protective effects in the brain. HSP70 widely rescued neuronal degeneration in the brain, locomotor defects and early lethality of flies expressing mHtt in neurons or glia. AKT failed to prevent brain neuronal death and lethality of adult HD flies, but significantly improved their locomotor performance when co-expressed with mHtt in glia. Finally, active ERK expression did not improve HD phenotype in any cell type. Thus we provided in vivo evidence that increasing AKT delayed cell-specific HD phenotypes, whereas HSP70 suppressed mHtt-induced cell dysfunction and death.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 NOTE ADDED IN PROOF
 FUNDING
 REFERENCES
 
HSP70 and AKT similarly suppress cell toxicity in the HD Drosophila retina
The UAS-GAL4 system (35) was used to target expression of the first exon of human Htt with 93 CAG repeats (Httex1p Q93) to cell subtypes in the Drosophila nervous system. Identification of genetic modifiers in the Drosophila models of neurodegenerative disorders has been mostly performed in the retina to harness the power of rapid genetic testing. Previous studies showed that expression of various peptides containing an expanded polyQ tract in all cell types of the retina with the GMR-GAL4 driver resulted in progressive pigment cell degeneration (17,36,37). We similarly observed that Httex1p Q93 expression with GMR-GAL4 leads to late-onset eye depigmentation (Fig. 1A5). Numerous cells with pigment loss were observed beyond 1 month of age in adult flies. The Drosophila AKT gene was co-expressed with mHtt in the retina using two independent UAS-AKT1 insertion lines. In both cases, expression of AKT did not change the external appearance of the eye but strongly suppressed Httex1 Q93-mediated pigment cell degeneration (Fig. 1A8). In contrast, co-expression of the constitutively active Drosophila ERK (UAS-rolledSEM) did not prevent eye depigmentation (Fig. 1A7). ERK was by itself deleterious for eye formation inducing a rough phenotype (Fig. 1A3). As a control for non-specific suppression effects, green fluorescent protein (GFP) was co-expressed with Httex1p Q93 in the eye using UAS-GFP-65T with no detected change in eye mHtt-induced depigmentation (Fig. 1A6).


Figure 1
View larger version (57K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. AKT but not ERK rescues Httex1p Q93 toxicity in the fly retina. (A) External appearance of eyes from 42 day-old flies expressing either no transgene (panel 1), GFP (panel 2), ERK (panel 3) or AKT (panel 4) alone or the same plus Httex1p Q93 (panels 5–8) under the control of the eye driver GMR-GAL4. Co-expression of AKT prevented Httex1p Q93-induced progressive loss of eye pigmentation. (B) Representative deep pseudopupil photomicrographs of eyes from 9-day old flies expressing no transgene (control), Httex1p Q93 alone, or together with Drosophila AKT under control of the pan-neuronal driver elav-GAL4. Co-expression of AKT significantly alleviated photoreceptor loss in the retina. (C) Number of photoreceptor per ommatidium in 1- and 9-day old flies expressing Httex1p Q93 alone, or Httex1p Q93 with AKT. AKT significantly reduced Httex1p Q93-induced photoreceptor loss (P < 0.001) at 1 and 9 days of adult age (non-parametric Mann–Whitney U-test).

 
Expression of Httex1p Q93 with the neuronal-specific elav-GAL4 driver disrupted the regular arrangement of the retina decreasing the number of photoreceptors in each ommatidium as observed by pseudopupil analysis (Fig. 1B). Co-expression of AKT markedly slowed down photoreceptor degeneration. Figure 1C shows the distribution of photoreceptors per ommatidium 1 or 9 days after eclosion from pupae. The number of ommatidia that remained intact with seven photoreceptors was only 36% at day 1 and fell to 18% at day 9 in flies expressing Httex1p Q93 alone. AKT overexpression by itself had no effect on the photoreceptors (data not shown). Co-expression of AKT with Httex1p Q93 significantly rescued photoreceptor degeneration: the number of remaining intact ommatidia was doubled compared with flies expressing Httex1p Q93 alone at 1 and 9 days (Fig. 1C). ERK expression in photoreceptors had deleterious effects; as previously reported (38) eyes showed a rough phenotype with additional ectopic photoreceptors. Quantitative pseudopupil analysis could not be performed because of the strong eye malformation in the presence of ERK.

Since chaperone proteins are described as potent modifiers of expanded polyQ proteins in Drosophila models of SCA1 and SCA3 (22,28) or SBMA (39), we evaluated the effects of HSP70 in the eye of HD Drosophila model. We found that expression of HSP70 reduced retinal depigmentation (Fig. 2A) and photoreceptor loss (Fig. 2B). As observed with AKT, the number of ommatidia that remained intact was doubled at day 9 (Fig. 2C).


Figure 2
View larger version (37K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Co-expression of HSP70 rescues Httex1p Q93-induced toxicity in the fly retina. (A) External appearance of eyes from 47 day-old flies expressing either Httex1p Q93 alone or together with HSP70 under control of the eye driver GMR-GAL4. HSP70 reduced the Httex1p Q93-induced eye depigmentation. (B) Representative deep pseudopupil photomicrographs of eyes from 9-day old flies expressing Httex1p Q93 in the absence or presence of HSP70 under control of the neuron-specific driver elav-GAL4. (C) Number of photoreceptor per ommatidium in 9-day old flies. Overexpression of HSP70 significantly delayed photoreceptor loss in the retina. The P-value was <0.001 (non-parametric Mann–Whitney U-test).

 
Neuronal and glial expression of mHtt causes reduced lifespan and locomotor deficits
Previous studies described that the presence of Httex1p Q93 in fly neurons resulted in cell death, locomotor impairments and shortened lifespan (18,19). We further reported that expression of Httex1p Q93 in a subset of glial cells that contain the glutamate transporter, dEAAT1, was detrimental for adult Drosophila survival (12). Here, we more precisely compared the effects of mHtt selectively expressed in a large range of neurons or glia under the control of the elav-GAL4 or repo-GAL4 drivers, respectively. In both cases, adult flies died between days 8 and 28 of adult age with a similar lifespan curve, whereas control flies heterozygous for each driver lived >28 days at 25°C (Fig. 3A). Next, we used selective drivers to evaluate whether expression of mHtt in different glia subtypes has different effects on fly lifespan (Table 1). Based on morphological criteria and location in the nervous system, glial cells can be placed into three major subtypes in the Drosophila brain (40). Cortex glia extend cellular processes to the synapse and thus modulate synaptic neurotransmission, whereas neuropil glia ensheath and isolate axons. Surface glia cover the central nervous system and forms the blood-brain barrier. In the peripheral nervous system, peripheral glia enwrap and insulate axons, maintain the nerve-blood barrier and regulate transmission at the neuromuscular junction. Selective targeting of Httex1p Q93 into surface glia under control of moody-GAL4 driver (41) or into peripheral glia with gliotactin-GAL4 driver (42) did not affect fly behaviour nor the survival rate until 28 days after eclosion (Table 1). In adult Drosophila, glia that produce the unique glutamate transporter dEAAT1 correspond to cortex glia subsets in the brain and peripheral glia at the neuromuscular junction that cap the axon terminals and regulate glutamate transmission (43,44). Expression of Httex1p Q93 in dEAAT1-positive glia with dEAAT1-GAL4 driver induced lethality as early as the age of 12 days (Fig. 3A). These data indicate that the reduced lifespan we observed in the presence of mHtt in repo-GAL4-positive glia is not due to a breakdown of the blood-brain barrier but rather to alterations of perisynaptic glia positive for the glutamate transporter dEAAT1. However, since we have no specific driver to evaluate effects in neuropil glial cells, we cannot exclude a role for this glia subset.


Figure 3
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Expression of Httex1p Q93 in neurons or glia shortens Drosophila lifespan and induces locomotor deficits. (A) Survival rates of adult flies expressing either no transgene (control) or Httex1p Q93 either in neurons (elav-GAL4 driver), a large range of glia (repo-GAL4 driver) or glutamate transporter-expressing glia subsets (dEAAT1-GAL4 driver). Data were analyzed by the Kaplan–Meier method and statistical significance was assessed by logrank test (***P < 0.001 versus control). (B) Locomotor impairments evaluated by negative geotaxis test on 12-day old flies expressing either no transgene or Httex1p Q93 under control of the pan-neuronal driver elav-GAL4 and the glial drivers, repo-GAL4 and dEAAT1-GAL4. Bars indicate the proportion of flies that climbed to the top of the column or that remained at the bottom after 30 s. (C) Climbing scores of flies expressing Httex1p Q93 in neurons or glia at 4, 8 and 12 days. Climbing scores are defined as 1/2((ntotal + ntop – nbottom)/ntotal). Statistical significance was assessed by Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

 


View this table:
[in this window]
[in a new window]

  		Table 1. Comparison of mutant Huntingtin toxicity in Drosophila neurons and glia

 
Analysis of locomotor defects was assessed by measuring the climbing ability of flies using the negative geotaxis test as previously described (43). Figure 3B shows the percent of 12 day old flies that reached the top or remained at the bottom of the column 30 s after startle. Almost all control flies did not show behavioural defects and were able to reach the top of the column within 30 s (>70% of flies). In contrast, only 43 and 10% of flies expressing Httex1p Q93 under control of either elav-GAL4 and repo-GAL4 drivers, respectively, were able to reach the top of the column. Climbing scores (see section Materials and Methods) at 4, 8 and 12 days are presented in Figure 3C. Neuronal expression of Httex1p Q93 induced abnormal locomotor performance as early as 4 days and the defect remained stable at 8 and 12 days. In contrast, flies expressing Httex1p Q93 in Repo-positive glia showed a more progressive decline in locomotor ability between 4 and 12 days. More precisely, flies with either the elav or repo-driver displayed a similar reduced speed when they walk, whereas flies expressing Httex1p Q93 in Repo-positive glia also exhibited a temporary paralysis and/or seizure after being startled. This ‘bang-sensitive’ phenotype increased with the age and at 12 days, some flies remained paralyzed for up to 20 s after tapping. This explains why more flies remained at the bottom for the pan-glial driver repo-GAL4 (74%) when compared with the pan-neuronal driver elav-GAL4 (21%) at 12 days. Surprisingly, flies expressing Httex1p Q93 in dEAAT1-positive glia did not show any locomotor dysfunction, paralysis or seizure even at 12 days (Fig. 3B and C). This suggests that the locomotor deficits observed with the repo-GAL4 driver results from mHtt-induced defects in distinct glia subsets that do not express dEAAT1 such as the neuropil glia.

Effects of AKT and ERK on HD pathology in the fly nervous system
We then examined the protective effect of AKT and ERK on mHtt-induced brain alterations and the ensuing locomotor deficits and reduced survival. The effect of Httex1p Q93 was assessed by immunostaining of Fasciclin II to visualize the mushroom bodies, a central brain structure involved in locomotion, learning and memory (45). We observed that pan-neuronal expression of Httex1p Q93 with elav-GAL4 resulted in a decreased Fasciclin II immunostaining in the mushroom body {gamma}-lobe at 10 days (Fig. 4A). Quantitative analysis (Fig. 4B) showed that in the mHtt-expressing flies, the fluorescence intensity fell to 65% in the {gamma}-lobe, whereas Fasciclin II levels remained unchanged in the {alpha} and β lobes as compared to wild-type (WT) flies (fluorescence levels: 159 ± 7 versus 144 ± 5 in control and Httex1p Q93 flies, respectively). These results indicate that the decrease in fluorescence intensity did not result from a mHtt-induced down-regulation of Fasciclin II expression, but rather, was due to a selective degeneration of the Kenyon's cells that compose the {gamma}-lobe, as previously described in Drosophila models of polyQ disorders (18,28). We found that the co-expression of AKT or ERK did not modify the mHtt-induced decrease in the {gamma}-lobe fibre density. Therefore, AKT is able to prevent neuronal cell toxicity in the retina, but not in mushroom bodies.


Figure 4
View larger version (48K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Expression of HSP70 but not AKT and ERK rescues Httex1p Q93-induced mushroom body defects in the fly brain. (A and C) Mushroom bodies of 10-day old flies expressing no transgene (control), Httex1p Q93 alone and Httex1p Q93 with AKT, ERK or HSP70 under control of the pan-neuronal driver elav-GAL4. Mushroom bodies were visualized by Fasciclin II immunostaining. Magnification: x40. The {gamma}-lobe neuropil density was reduced in Httex1p Q93-expressing flies, whereas the {alpha} and β lobes remained intact. Overexpression of AKT or ERK did not prevent the Httex1p Q93-mediated reduction in Fasciclin II immunostaining in the {gamma} lobes. In contrast, HSP70 rescued the {gamma}-lobe degeneration. (B and D) Quantitative analysis of fluorescence intensity in the mushroom body {gamma} lobes of 10-day old flies. Statistical significance: **P < 0.01 as compared to Httex1p Q93 alone (Student's t-test).

 
Next, we observed that co-expression of AKT or ERK did not ameliorate the survival time of flies that expressed Httex1p Q93 either in neurons (Fig. 5A) or glia (Fig. 5B and C). As a control, we found that both protein kinases did not decrease lifespan when expressed alone, except for flies expressing ERK in dEAAT1-positive glia that died beyond 24 days. AKT was unable to prevent locomotor defects at 12 days caused by the neuronal expression of Httex1p Q93 (Fig. 6A). In contrast, AKT had a significant beneficial effect on the locomotor ability of the 12-day old Drosophila expressing mHtt in glia (Fig. 6A). In the presence of AKT, 62% of flies reached the top of the column, whereas only 22% of flies succeeded when Httex1p Q93 was expressed alone under regulation of repo-GAL4 driver. Thus, co-expression of AKT with mHtt in glia doubled the climbing score of flies (Fig. 6B). Actually, the flies remained paralyzed for a shortened period of time after tapping the column, indicating that AKT mostly ameliorated the bang-sensitive phenotype. Therefore, although AKT expression did not protect against mHtt-induced decrease in lifespan, it did partly restore neuron–glia interaction when co-expressed with mHtt in glia.


Figure 5
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. Lack of effect of AKT or ERK on Httex1p Q93-induced lifespan decrease. (A–C) Survival rates of flies expressing Httex1p Q93 alone, or together with AKT or ERK under control of neuronal (elav-GAL4) and glial (repo-GAL4 and dEAAT1-GAL4) drivers. Survival curves were generated by the Kaplan–Meier method and statistical significance was determined by logrank test (***P < 0.001). No significant change in survival rate was detected when AKT or ERK was co-expressed with Httex1p Q93. Expression of ERK alone (without Httex1p Q93) under control of dEAAT1-GAL4 driver shortened fly lifespan.

 


Figure 6
View larger version (27K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6. AKT rescues locomotor defects induced by expression of Httex1p Q93 in glia but not in neurons. (A) Locomotor performance and (B) climbing scores of 12 day-old flies expressing Httex1p Q93 alone or together with AKT under regulation of the neuronal or glial drivers, elav-GAL4 or repo-GAL4, respectively. AKT significantly ameliorated the locomotor ability of flies expressing Httex1p Q93 selectively in glia (*P < 0.05; **P < 0.01, Student's t-test).

 
Chaperone proteins suppress mHtt-induced toxicity on neuronal and glial pathologies in the fly nervous system
We investigated whether or not putative modifiers of HD phenotypes may also display a selective rescue of the neuronal and glial pathologies. We focused our screen on chaperone proteins that were proved to be the most effective modulators for neuronal dysfunction/death in various Drosophila models of polyQ disorders (22–28). Life expectancy of flies expressing mHtt in glia under control of the dEAAT1-GAL4 driver was used to assess the effect of modifiers (Table 2). We found that overexpression of human HSP70 chaperone or Drosophila homologs of HSP40 co-chaperones HDJ1 (dHDJ1) and MRJ (dMRJ) significantly delayed the early lethality of adult flies expressing mHtt in glia. Chaperones increased by >30% the life expectancy of flies expressing mHtt in dEAAT1-positive glia. TPR2 that regulates the multi-chaperone system (46) also improved survival rate of flies.


View this table:
[in this window]
[in a new window]

  		Table 2. Genetic modifiers for the lethality of flies expressing mutant Huntingtin in glia

 
Next, we selected one of these modifiers, HSP70, to analyze more precisely its effects on neuronal and glial pathologies in the fly nervous system. We found that HSP70 abolished the loss of Fasciclin II immunostaining in the {gamma}-lobe of mushroom bodies (Fig. 4C and D), indicating that HSP70 suppressed mHtt-induced neuronal toxicity in the eye as well as in the central brain.

We showed that co-expression of HSP70 significantly ameliorated lifespan of flies expressing Httex1p Q93 in neurons (Fig. 7A) or glia (Fig. 7B and C). Whereas almost all flies expressing Httex1p Q93 alone did not survive beyond 24 days, >57% of flies co-expressing HSP70 together with Httex1p Q93 remained alive at 24 days with any one of the drivers. The climbing ability of Httex1p Q93-expressing flies was also improved by HSP70 as late as 12 days of adult age (Fig. 7D). Accordingly, climbing scores were twice and 3-fold increased when HSP70 was co-expressed with Httex1p Q93 in neurons or glia, respectively.


Figure 7
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 7. HSP70 rescues both locomotor ability and survival in fly HD models. (A–C) Survival rates were determined on flies expressing Httex1p Q93 alone, or together with HSP70 under the control of the neuronal (elav-GAL4) and glial (repo-GAL4 and dEAAT1-GAL4) drivers. Cumulative survival curves were generated by the Kaplan–Meier method and statistical significance was determined by logrank test (***P < 0.001). HSP70 significantly increased the life expectancy of Httex1p Q93-expressing flies. (D) Climbing scores of 12 day-old flies expressing Httex1p Q93 alone or together with HSP70 under regulation of pan-neuronal or pan-glial drivers, elav-GAL4 or repo-GAL4, respectively. Statistical significance was assessed by using the Student's t-test (***P < 0.001). HSP70 significantly rescued the climbing ability of Httex1p Q93-expressing flies.

 
Finally, we further analyzed whether HSP70 may also rescue the mHtt-induced glial alterations. We previously showed that Httex1p Q93 expression in glia resulted in down-regulation of the glial glutamate transporter dEAAT1 (12). Protein levels of dEAAT1 were assessed in flies expressing GFP-tagged-dEAAT1 (UAS-dEAAT1-GFP) in the absence and presence of Httex1p Q93 alone or plus HSP70. Figure 8A and B shows representative views of adult brains at 10 days of age and quantitative analysis of the GFP fluorescence, respectively. The presence of Httex1p Q93 diminished the level of GFP-tagged dEAAT1 by 74% in both the optic lobes and central brain. Co-expression of HSP70 significantly prevented the decline in GFP-tagged dEAAT1. Fluorescence levels of GFP-tagged dEAAT1 in Httex1p Q93-expressing flies were doubled by the presence of HSP70. Therefore our data indicate that HSP70 strongly reduced the mHtt-induced pathology in neurons as well as in glia.


Figure 8
View larger version (36K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 8. Expression of HSP70 decreases Httex1p Q93-induced glial cell alterations. (A) dEAAT1-GFP fluorescence levels in whole-mount brains from 10 day-old adult flies. The dEAAT1-GFP fusion protein was expressed in glia subset under control of the dEAAT1-GAL4 driver either with no transgene (control), Httex1p Q93 alone, or Httex1p Q93 together with HSP70. Magnification: x20. HSP70 prevented the decline in GFP-tagged dEAAT1 that was observed in the presence of Httex1p Q93 in the optic lobe (OL) and the central brain (CB). (B) Quantitative analysis of fluorescence intensity in the optic lobe and the central brain. Data are expressed as the mean ± SEM of values. Statistical significance: ***P < 0.001 (Student's t-test).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 NOTE ADDED IN PROOF
 FUNDING
 REFERENCES
 
The overall aim of this study was to evaluate the rescuing potency of AKT and ERK anti-apoptotic kinases and HSP70 chaperone towards mHtt toxicity in different cell types of the Drosophila nervous system. We compared protection against retina degeneration with rescue of fly locomotor behaviour and early death when mHtt is expressed in brain neurons and glial cells, which is more the hallmark of HD than photoreceptor cell loss. Previously, genes identified in fly models as suppressors of expanded polyQ toxicity were generally found by screening for modifiers of the Drosophila eye phenotypes. Many of retinal rescuers were also neuroprotective in the fly brain (22–28). However, in a few cases, effects in the retina did not mirror protective effects in brain neurons (28). Among the modifiers, HSP70 chaperone protected against toxicity of polyQ proteins in both the retina and postmitotic brain neurons in Drosophila models (22,28,39) and thus it was used as a standard rescuer in the present study. We found that AKT and HSP70 had similar benefits on photoreceptor neuron loss in the fly eye. In contrast, their effects differed in the brain. Although HSP70 widely suppressed neuronal and glial phenotypes, we detected no beneficial effects of AKT on neuronal degeneration in mushroom bodies, locomotor deficits and lifespan when mHtt was expressed in neurons. Another level of complexity is that mHtt is also expressed in non-neuronal brain cells (7,47,48). Here we provided further evidence that the accumulation of mHtt in glial cell subsets may compromise neuron functioning and contribute to HD pathogenesis. Candidate modifiers should be tested on brain neurons but also on glia to enhance relevance to human diseases. Interestingly, increasing AKT signalling improved locomotor performance when mHtt was selectively expressed in glia but not in neurons, indicating that mHtt can induce AKT-sensitive or insensitive pathways of toxicity in distinct cell types.

Neuron and glia pathologies in HD Drosophila model
Drosophila HD models have been developed so far by expressing N-terminal fragments of human mHtt, whose total length is 3144 amino acids in its native form: the first 81 amino acids corresponding to exon 1 (19) as we used here, the first 548 amino acids (20) and recently the first 336 amino acids (49). In all cases, pan-neuronal expression replicates key HD features such as late onset and progressive neuronal dysfunction and degeneration, leading to decline in locomotor performance and premature death. Neuronal loss appears to be restricted to a few cerebral structures in Drosophila as it is the case in HD patient brains. Accordingly, when all neurons were immunostained with anti-Elav antibodies in HD flies, we did not observe massive neuronal loss in the brain (data not shown). As previously described by Agrawal et al. (18), we detected neuronal degeneration selectively in the mushroom body {gamma}-lobes, whereas the {alpha} and β lobes remained intact.

Neuron–glia interactions are crucial to ensure optimal brain function and accumulation of mHtt may compromise crosstalk between both cell types (7,12). Glial-specific expression of polyQ-expanded proteins was found to have adverse effects on nervous system function in Drosophila (12,50). Expression of polyQ-expanded human ataxin-3 in neurons or glia led to behavioural changes and reduced lifespan of flies (50). Here, we showed that pan-neuronal and pan-glial expressions of mHtt also triggered abnormal locomotor behaviour and shortened lifespan. Whereas in the both cases locomotor performance was compromised using the negative geotaxis test, only flies expressing mHtt in Repo-positive glia suffered from ‘bang-sensitivity’, i.e. temporary paralysis and/or seizure following a mechanical shock, such as after tapping a column to make the flies fall to the bottom. The bang-sensitive phenotype likely reflects the disruption of glial-specific functions by expanded polyQ proteins. The targeting of mHtt in surface and peripheral glia, which are implicated in maintenance of the blood-nervous system barrier, did not result in locomotor defects and shortened lifespan. Since disruption of the blood-brain barrier leads to paralytic or shaking phenotypes and reduced larval or adult survival time (41,51,52), our results suggest that mHtt does not strongly challenge this glial function. In contrast, when we expressed mHtt in dEAAT1-positive glia, flies exhibited early adult lethality, comparable to that observed when mHtt is expressed in Repo-positive glial cells, but surprisingly did not show reduced climbing ability. This suggests that locomotor defects are not related to alteration in perisynaptic glia that regulates glutamate transmission in the brain and at the neuromuscular junction in Drosophila. We previously reported that expression of mHtt does not produce a massive loss of glia (12). Similarly, overexpression of mHtt in cultured astrocytes, the mammalian counterpart of Drosophila cortex glia, did not cause obvious cell death. In HD mouse brain, degenerating glial cells have been observed but they were scarce (7). Therefore, the phenotype we observed is likely mediated by cell dysfunction rather than glia degeneration and strikingly differs according to the type of glial cells targeted.

Chaperone proteins modulate glia alterations in HD
Expression of chaperones was previously found to protect against toxicity of polyQ proteins in the retina and in brain neurons of Drosophila models (22–28). Here we provide additional evidence that several chaperones display glioprotective effects and thereby stand as worthwhile therapeutic targets in HD. Chaperones likely reduce toxicity at early stage of HD pathogenesis by directly acting on mHtt protein. However results from HD mouse model had disappointing outcome at this respect. HSP70 overexpression in R6/2 mouse model resulted in only modest effects on disease progression (53) or no improvement of the phenotype except for a delay in aggregate formation (54). Similarly, R6/2 mice transgenic for HSP27 chaperone did not display a phenotypic improvement (55). Overexpression of yeast HSP104 in another HD mouse model prolonged the lifespan, but did not rescue the motor deficits and weight loss of mice (56). One explanation is that molecular chaperones generally function in cooperation and thus the decreased levels of HSP70 and HSP40 in HD mouse models (54,56) might compromise the beneficial effects of one particular chaperone. Accordingly, co-expressed Drosophila Hdj1 (dHdj1) and HSP70 act synergistically to suppress toxicity in SCA3 fly model (57). Therefore, a more effective approach might be to induce stress response, thereby increasing expression of many chaperones. In support of this, expression of the active form of heat shock transcription factor 1, which regulates transcription of stress-inducible genes, reduces body weight loss and increases lifespan of R6/2 HD mice (58). The recent development of geldanamycin derivatives that cross the blood brain barrier and induce heat-shock response (59) could well lead to new therapeutic strategies.

AKT/ERK cell signalling and cell degeneration in HD pathology
There is strong support for an involvement of the protein kinases AKT or ERK in HD pathology. AKT is degraded in HD patient brain and its activity is decreased or increased, respectively, in the brain of mHtt-transfected rat or knock-in mouse models of HD (34,60). Altered levels of phosphorylated ERK are measured in mHtt-expressing cells and in neurons of R6/2 HD mice (61,62). Although ERK activation was found to be protective against mHtt-mediated cell toxicity in PC12 and cultured striatal cells (61), we observed no benefit of ERK activation on HD phenotype in Drosophila. Similarly, overexpression of MEK, the kinase upstream of ERK cascade, did not rescue polyQ-induced lethality in flies (25). One possible explanation is that ERK activation is sustained during pre-adult and adult life stages under our in vivo conditions, whereas ERK activation was transient in transfected cultured cells. Flies expressing active ERK displayed abnormal rough appearance and ectopic photoreceptors in the eye. Such deleterious effects on fly eye development were previously described after expression of gain of function alleles of components acting upstream of ERK such as EGFR, Ras or Raf (12,38,63,64). In contrast, expression of active ERK alone in the brain had no effects on mushroom body structure and on organismal lifespan, except for flies expressing ERK in dEAAT1-positive glia that died well beyond 24 days. Thus, the lack of protective effects of ERK on mHtt toxicity into the brain was likely not due to toxic effects of ERK.

In vitro studies previously showed that AKT abolished death of cultured striatal neurons expressing N-terminal fragments of expanded polyQ Htt (65,66). Here, we provide in vivo evidence that AKT rescued mHtt-dependent cell degeneration in the fly retina but not in brain neurons. Neuronal degeneration seems to be highly restricted in the brain since mHtt-mediated death was only detected in a specific subtype of Kenyon's cell giving rise to the axonal fibres of the {gamma}-lobes. Although AKT expression protected retinal cells from mHtt-mediated death, we did not detect beneficial effects on neuronal loss in mushroom bodies. Moreover, AKT failed to rescue early lethality when co-expressed with mHtt in all neurons or large subsets of glia. Therefore, the cellular pathways of mHtt-mediated cell death are likely different in the eye and brain cells, being respectively AKT-sensitive or insensitive.

There are a number of possible mechanisms by which AKT could lead to a prosurvival response. AKT was demonstrated to induce the phosphorylation of mHtt at Ser421, thereby reducing its toxicity (66). However, we expressed a shorter N-terminal fragment that does not contain this phosphorylation site. Prosurvival effect of AKT likely results from its anti-apoptotic function. For instance, AKT controls p53 levels via Mdm2 phosphorylation, the E3 ubiquitin ligase that triggers degradation of p53 (67,68) and could directly inactivate apoptosis pathway by phosphorylating the pro-apoptotic Bcl-2- related protein, BAD and Caspase 9/Apaf-1 complex (69). Accordingly, deletion of p53 was found to ameliorate HD pathologies in transgenic Drosophila and mouse models (30) and genetic inactivation of Apaf-1 suppressed mHtt-induced cell death in the fly retina (29). Alternatively, AKT could increase survival by inhibiting the stress-activated protein kinase pathways, JNK and p38 (69). Expression of mHtt causes increased activation of JNK in HD in vitro models (61,70,71). Pharmacological or genetic inhibition of JNK pathway protected against cultured cell apoptosis induced by mHtt and against neurodegeneration in a Drosophila HD model (33,61,71). Finally, AKT may also improve cell survival by direct effects on glycolysis and mitochondrial functions (reviewed in 72,73).

AKT signalling and behavioural deficits in HD models
Interestingly, we found that AKT ameliorated the locomotor performance of HD flies when selectively co-expressed with mHtt in glia but not in neurons. This was obviously due to a decrease in the duration of the bang-sensitive paralysis/seizure behaviour, suggesting that neuron–glia interactions were partly restored. Rescue was likely not caused by an improvement of glutamate transmission because (i) expression of mHtt in glia that contains the glutamate transporter dEAAT1 did not result in bang-sensitivity and (ii) dEAAT1 expression levels are not regulated by PI3K/AKT signalling pathway (12). Bang-sensitivity was previously described in mutants affecting sodium/potassium asymmetric distribution across the plasma membrane, ATP levels and mitochondrial energy production (74,75). Two particular functions of neuropil and cortex glia are to supply neurons with energy metabolites and to maintain energy-dependent ionic composition of the extracellular environment. Thus, abnormal energy metabolism in glia may explain the ensuing effects on neuron function. AKT is known to regulate glucose uptake, glycolysis and glycogen synthesis in a cell-intrinsic and cell type-specific manner (72,73). Therefore, we propose that AKT may rescue fly behaviour in HD model by stimulating glycolytic metabolism in glia. This view is supported by data from positron emission tomography studies in brain of early HD patients showing a selective impairment of cerebral glycolytic, not oxydative, metabolism that is predominantly a glial metabolic process (76). More recently, inhibitors of metabolism such as rotenone were found to rescue cell death in cellular and Drosophila eye models of HD (77). However, rescue by these inhibitors was not due to changes in ATP, reactive oxygen species or NADH levels but rather due to the activation of ERK or AKT (77).

In conclusion, we provided in vivo evidence that increasing AKT has beneficial effects on HD Drosophila phenotypes in a cell-type specific manner. AKT prevents some cell-intrinsic mechanisms leading to late neuronal death in the retina. Our data further suggest that increasing AKT signalling pathway may improve glial energy metabolism and dependent neuron functioning. Further works are required to understand the role and importance of energy metabolism in the alteration of neuron-glia interaction in HD pathogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
NOTE ADDED IN PROOF
 FUNDING
 REFERENCES
 
Drosophila strains and culture
Flies were grown at 25°C on a standard cornmeal/agar/yeast medium. The following fly stocks were used: dEAAT1-GAL4 and UAS-dEAAT1-GFP (43), moody-GAL4 (41), gliotactin-GAL4 (42), UAS-Httex1p Q93 (19), UAS-rolledSEM [ERK; (38)], UAS-dTpr2 (24), UAS-dHDJ1 (24) and UAS-dMRJ (26). All other strains: GMR-GAL4, repo-GAL4, elav-GAL4 (line c155), UAS-AKT (2 insertions) and UAS-HspA1L [HSP70; (22)] were obtained from the Bloomington Drosophila Stock Center.

Lifespan analysis
Female progeny were collected within 24 h of emergence. For each desired genotype, 10–20 flies per vial in 4–8 vials were incubated at 25°C. They were transferred into fresh vials every 2 days and the number of surviving flies was recorded. Cumulative survival curves were generated by the Kaplan–Meier method and statistical analysis was performed by logrank test (GraphPad Prism software).

Locomotion assay
Locomotor ability was assessed with a negative geotaxis assay as described previously (43). Female flies were anesthetized with CO2 and placed in a plastic column (length: 18 cm x diameter: 1.3 cm). After a recovery of 30 min, columns were disposed vertically and flies were gently tapped to the bottom of the column. WT flies quickly recovered and started climbing up the column. Flies that have reached the top or remained at the bottom of the column were counted after 30 s. Three trials were performed at 1 min of intervals. The data are presented as the number of flies at the top (ntop) or at the bottom (nbottom) and are expressed as percentage ± SEM of the total number of flies (ntotal). Climbing scores are defined as 1/2((ntotal + ntop – nbottom)/ntotal). Statistical significance was assessed by using the Student's t-test.

In situ fluorescence
Adult Drosophila brains expressing UAS-dEAAT1-GFP were dissected in phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, rinsed in PBS and mounted in Mowiol (Calbiochem, La Jolla, CA).

Immunocytochemistry
Reagents were prepared in PBS. Adult fly brains were dissected, fixed in 4% paraformaldehyde for 30 min, permeabilized with 0.3% Triton X-100 for 20 min, saturated in 0.1% Triton X-100 with 5 mg/ml bovine serum albumin for 2 h. Brains were incubated in the latter solution containing primary antibodies overnight, rinsed and incubated with fluorochrome-conjugated secondary antibodies. After rinsing, brains were mounted in Mowiol (Calbiochem).

The primary monoclonal antibodies against Fasciclin II (1/100 dilution, Developmental Studies Hybridoma Bank, Iowa) was used to visualize the fly mushroom bodies. Alexa-488-anti mouse antibodies (Molecular probes, Eugene, OR) were used as secondary antibodies at 1/400 dilution.

Fluorescence detection and quantification
For quantitative analysis of dEAAT1-GFP or Fasciclin II, microscopic examination was performed using a Nikon E800 fluorescent microscope (Champigny sur Marne, France) equipped with a Hamamatsu Photonics ORCA-ER digital camera (Massy, France). Quantification of the signal was done with the Image J software. Fluorescent intensities were expressed as percent of the mean of control flies. Data from at least six flies per condition were averaged and are presented as mean ± SEM. Statistical analysis was performed using a Student's t-test. A significance of P < 0.05 was required for rejection of the null hypothesis. Each experiment was performed twice.


   NOTE ADDED IN PROOF
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOTE ADDED IN PROOF
FUNDING
 REFERENCES
 
Since acceptance of this manuscript, Branco et al. have reported with another Drosophila model of HD that AKT overexpression reduced retinal phenotype, which is in agreement with our results. Conversely in a SCA1 fly model, AKT overexpression behaved as an enhancer of the phenotype in the retina. (Branco, J., Al-Ramahi, I., Ukani, L., Pérez, A.M., Fernandez-Funez, P., Rincón-Limas, D. and Botas, A.J. Comparative analysis of genetic modifiers in Drosophila points to common and distinct mechanisms of pathogenesis among polyglutamine diseases. Hum. Mol. Genet., in press).


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOTE ADDED IN PROOF
 FUNDING
REFERENCES
 
This work was supported by a fellowship to JCL and a grant both from the HighQ Foundation.


   ACKNOWLEDGEMENTS
 
We thank Leslie Thompson for UAS-Httex1p Q93 flies, Dirk Bohmann for the UAS-rolledSEM flies, Parsa Kazemi-Esfarjani for the UAS-dTpr2, UAS-dHDJ1 and UAS-dMRJ flies, Ulrike Gaul for the moody-GAL4 line and Vanessa Auld for gliotactin-GAL4 line, the Bloomington Drosophila Stock Center for transgenic flies and the Developmental Studies Hybridoma Bank for monoclonal antibodies.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 NOTE ADDED IN PROOF
 FUNDING
 REFERENCES
 

  1. The Huntington's Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell (1993) 72:971–983.[CrossRef][Web of Science][Medline]

  2. Stine O.C., Pleasant N., Franz M.L., Abbott M.H., Folstein S.E., Ross C.A. Correlation between the onset age of Huntington's disease and length of the trinucleotide repeat in IT-15. Hum. Mol. Genet. (1993) 2:1547–1549.[Abstract/Free Full Text]

  3. Gutekunst C.A., Levey A.I., Heilman C.J., Whaley W.L., Yi H., Nash N.R., Rees H.D., Madden J.J., Hersch S.M. Identification and localization of huntingtin in brain and human lymphoblastoid cell lines with anti-fusion protein antibodies. Proc. Natl Acad. Sci. USA (1995) 92:8710–8714.[Abstract/Free Full Text]

  4. Sharp A.H., Loev S.J., Schilling G., Li S.H., Li X.J., Bao J., Wagster M.V., Kotzuk J.A., Steiner J.P., Lo A., et al. Widespread expression of Huntington's disease gene (IT15) protein product. Neuron (1995) 14:1065–1074.[CrossRef][Web of Science][Medline]

  5. Landwehrmeyer G.B., McNeil S.M., Dure L.S.t., Ge P., Aizawa H., Huang Q., Ambrose C.M., Duyao M.P., Bird E.D., Bonilla E., et al. Huntington's disease gene: regional and cellular expression in brain of normal and affected individuals. Ann. Neurol. (1995) 37:218–230.[CrossRef][Web of Science][Medline]

  6. Li S.H., Schilling G., Young W.S.d., Li X.J., Margolis R.L., Stine O.C., Wagster M.V., Abbott M.H., Franz M.L., Ranen N.G., et al. Huntington's disease gene (IT15) is widely expressed in human and rat tissues. Neuron (1993) 11:985–993.[CrossRef][Web of Science][Medline]

  7. Shin J.Y., Fang Z.H., Yu Z.X., Wang C.E., Li S.H., Li X.J. Expression of mutant huntingtin in glial cells contributes to neuronal excitotoxicity. J. Cell Biol. (2005) 171:1001–1012.[Abstract/Free Full Text]

  8. Strong T.V., Tagle D.A., Valdes J.M., Elmer L.W., Boehm K., Swaroop M., Kaatz K.W., Collins F.S., Albin R.L. Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues. Nat. Genet. (1993) 5:259–265.[CrossRef][Web of Science][Medline]

  9. Vonsattel J.P., Myers R.H., Stevens T.J., Ferrante R.J., Bird E.D., Richardson E.P. Jr. Neuropathological classification of Huntington's disease. J. Neuropathol. Exp. Neurol. (1985) 44:559–577.[Web of Science][Medline]

  10. Rubinsztein D.C. Lessons from animal models of Huntington's disease. Trends Genet. (2002) 18:202–209.[CrossRef][Web of Science][Medline]

  11. Walker F.O. Huntington's disease. Lancet (2007) 369:218–228.[CrossRef][Web of Science][Medline]

  12. Liévens J.C., Rival T., Iche M., Chneiweiss H., Birman S. Expanded polyglutamine peptides disrupt EGF receptor signaling and glutamate transporter expression in Drosophila. Hum. Mol. Genet. (2005) 14:713–724.[Abstract/Free Full Text]

  13. Arzberger T., Krampfl K., Leimgruber S., Weindl A. Changes of NMDA receptor subunit (NR1, NR2B) and glutamate transporter (GLT1) mRNA expression in Huntington's disease—an in situ hybridization study. J. Neuropathol. Exp. Neurol. (1997) 56:440–454.[Web of Science][Medline]

  14. Gamberino W.C., Brennan W.A. Jr. Glucose transporter isoform expression in Huntington's disease brain. J. Neurochem. (1994) 63:1392–1397.[Medline]

  15. Vis J.C., Schipper E., de Boer-van Huizen R.T., Verbeek M.M., de Waal R.M., Wesseling P., ten Donkelaar H.J., Kremer B. Expression pattern of apoptosis-related markers in Huntington's disease. Acta Neuropathol (Berl). (2005) 109:321–328.[CrossRef][Medline]

  16. Hermel E., Gafni J., Propp S.S., Leavitt B.R., Wellington C.L., Young J.E., Hackam A.S., Logvinova A.V., Peel A.L., Chen S.F., et al. Specific caspase interactions and amplification are involved in selective neuronal vulnerability in Huntington's disease. Cell Death Differ. (2004) 11:424–438.[CrossRef][Web of Science][Medline]

  17. Jackson G.R., Salecker I., Dong X., Yao X., Arnheim N., Faber P.W., MacDonald M.E., Zipursky S.L. Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons. Neuron (1998) 21:633–642.[CrossRef][Web of Science][Medline]

  18. Agrawal N., Pallos J., Slepko N., Apostol B.L., Bodai L., Chang L.W., Chiang A.S., Thompson L.M., Marsh J.L. Identification of combinatorial drug regimens for treatment of Huntington's disease using Drosophila. Proc. Natl Acad. Sci. USA (2005) 102:3777–3781.[Abstract/Free Full Text]

  19. Steffan J.S., Bodai L., Pallos J., Poelman M., McCampbell A., Apostol B.L., Kazantsev A., Schmidt E., Zhu Y.Z., Greenwald M., et al. Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature (2001) 413:739–743.[CrossRef][Medline]

  20. Lee W.C., Yoshihara M., Littleton J.T. Cytoplasmic aggregates trap polyglutamine-containing proteins and block axonal transport in a Drosophila model of Huntington's disease. Proc. Natl Acad. Sci. USA (2004) 101:3224–3229.[Abstract/Free Full Text]

  21. Gunawardena S., Her L.S., Brusch R.G., Laymon R.A., Niesman I.R., Gordesky-Gold B., Sintasath L., Bonini N.M., Goldstein L.S. Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila. Neuron (2003) 40:25–40.[CrossRef][Web of Science][Medline]

  22. Warrick J.M., Chan H.Y., Gray-Board G.L., Chai Y., Paulson H.L., Bonini N.M. Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. Nat. Genet. (1999) 23:425–428.[CrossRef][Web of Science][Medline]

  23. Fernandez-Funez P., Nino-Rosales M.L., de Gouyon B., She W.C., Luchak J.M., Martinez P., Turiegano E., Benito J., Capovilla M., Skinner P.J., et al. Identification of genes that modify ataxin-1-induced neurodegeneration. Nature (2000) 408:101–106.[CrossRef][Medline]

  24. Kazemi-Esfarjani P., Benzer S. Genetic suppression of polyglutamine toxicity in Drosophila. Science (2000) 287:1837–1840.[Abstract/Free Full Text]

  25. Iijima-Ando K., Wu P., Drier E.A., Iijima K., Yin J.C. cAMP-response element-binding protein and heat-shock protein 70 additively suppress polyglutamine-mediated toxicity in Drosophila. Proc. Natl Acad. Sci. USA (2005) 102:10261–10266.[Abstract/Free Full Text]

  26. Fayazi Z., Ghosh S., Marion S., Bao X., Shero M., Kazemi-Esfarjani P. A Drosophila ortholog of the human MRJ modulates polyglutamine toxicity and aggregation. Neurobiol. Dis. (2006) 24:226–244.[CrossRef][Medline]

  27. Latouche M., Lasbleiz C., Martin E., Monnier V., Debeir T., Mouatt-Prigent A., Muriel M.P., Morel L., Ruberg M., Brice A., et al. A conditional pan-neuronal Drosophila model of spinocerebellar ataxia 7 with a reversible adult phenotype suitable for identifying modifier genes. J. Neurosci. (2007) 27:2483–2492.[Abstract/Free Full Text]

  28. Ghosh S., Feany M.B. Comparison of pathways controlling toxicity in the eye and brain in Drosophila models of human neurodegenerative diseases. Hum. Mol. Genet. (2004) 13:2011–2018.[Abstract/Free Full Text]

  29. Sang T.K., Li C., Liu W., Rodriguez A., Abrams J.M., Zipursky S.L., Jackson G.R. Inactivation of Drosophila Apaf-1 related killer suppresses formation of polyglutamine aggregates and blocks polyglutamine pathogenesis. Hum. Mol. Genet. (2005) 14:357–372.[Abstract/Free Full Text]

  30. Bae B.I., Xu H., Igarashi S., Fujimuro M., Agrawal N., Taya Y., Hayward S.D., Moran T.H., Montell C., Ross C.A., et al. p53 mediates cellular dysfunction and behavioral abnormalities in Huntington's disease. Neuron (2005) 47:29–41.[CrossRef][Web of Science][Medline]

  31. Junier M.P. What role(s) for TGFalpha in the central nervous system? Prog. Neurobiol. (2000) 62:443–473.[CrossRef][Medline]

  32. Song C., Perides G., Liu Y.F. Expression of full-length polyglutamine-expanded Huntingtin disrupts growth factor receptor signaling in rat pheochromocytoma (PC12) cells. J. Biol. Chem. (2002) 277:6703–6707.[Abstract/Free Full Text]

  33. Scappini E., Koh T.W., Martin N.P., O'Bryan J.P. Intersectin enhances huntingtin aggregation and neurodegeneration through activation of c-Jun-NH2-terminal kinase. Hum. Mol. Genet. (2007) 16:1862–1871.[Abstract/Free Full Text]

  34. Colin E., Regulier E., Perrin V., Durr A., Brice A., Aebischer P., Deglon N., Humbert S., Saudou F. Akt is altered in an animal model of Huntington's disease and in patients. Eur. J. Neurosci. (2005) 21:1478–1488.[Web of Science][Medline]

  35. Brand A.H., Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development (1993) 118:401–415.[Abstract]

  36. Warrick J.M., Paulson H.L., Gray-Board G.L., Bui Q.T., Fischbeck K.H., Pittman R.N., Bonini N.M. Expanded polyglutamine protein forms nuclear inclusions and causes neural degeneration in Drosophila. Cell (1998) 93:939–949.[CrossRef][Web of Science][Medline]

  37. Marsh J.L., Walker H., Theisen H., Zhu Y.Z., Fielder T., Purcell J., Thompson L.M. Expanded polyglutamine peptides alone are intrinsically cytotoxic and cause neurodegeneration in Drosophila. Hum. Mol. Genet. (2000) 9:13–25.[Abstract/Free Full Text]

  38. Peverali F.A., Isaksson A., Papavassiliou A.G., Plastina P., Staszewski L.M., Mlodzik M., Bohmann D. Phosphorylation of Drosophila Jun by the MAP kinase rolled regulates photoreceptor differentiation. Embo. J. (1996) 15:3943–3950.[Medline]

  39. Chan H.Y., Warrick J.M., Andriola I., Merry D., Bonini N.M. Genetic modulation of polyglutamine toxicity by protein conjugation pathways in Drosophila. Hum. Mol. Genet. (2002) 11:2895–2904.[Abstract/Free Full Text]

  40. Freeman M.R., Doherty J. Glial cell biology in Drosophila and vertebrates. Trends Neurosci. (2006) 29:82–90.[CrossRef][Web of Science][Medline]

  41. Bainton R.J., Tsai L.T., Schwabe T., DeSalvo M., Gaul U., Heberlein U. moody encodes two GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in Drosophila. Cell (2005) 123:145–156.[CrossRef][Web of Science][Medline]

  42. Schulte J., Tepass U., Auld V.J. Gliotactin, a novel marker of tricellular junctions, is necessary for septate junction development in Drosophila. J. Cell Biol. (2003) 161:991–1000.[Abstract/Free Full Text]

  43. Rival T., Soustelle L., Strambi C., Besson M.T., Iche M., Birman S. Decreasing glutamate buffering capacity triggers oxidative stress and neuropil degeneration in the Drosophila brain. Curr. Biol. (2004) 14:599–605.[CrossRef][Web of Science][Medline]

  44. Rival T., Soustelle L., Cattaert D., Strambi C., Iche M., Birman S. Physiological requirement for the glutamate transporter dEAAT1 at the adult Drosophila neuromuscular junction. J. Neurobiol. (2006) 66:1061–1074.[CrossRef][Web of Science][Medline]

  45. Fahrbach S.E. Structure of the mushroom bodies of the insect brain. Annu. Rev. Entomol. (2006) 51:209–232.[CrossRef][Web of Science][Medline]

  46. Brychzy A., Rein T., Winklhofer K.F., Hartl F.U., Young J.C., Obermann W.M. Cofactor Tpr2 combines two TPR domains and a J domain to regulate the Hsp70/Hsp90 chaperone system. Embo J. (2003) 22:3613–3623.[CrossRef][Medline]

  47. Singhrao S.K., Thomas P., Wood J.D., MacMillan J.C., Neal J.W., Harper P.S., Jones A.L. Huntingtin protein colocalizes with lesions of neurodegenerative diseases: An investigation in Huntington's, Alzheimer's, and Pick's diseases. Exp. Neurol. (1998) 150:213–222.[CrossRef][Web of Science][Medline]

  48. Hebb M.O., Denovan-Wright E.M., Robertson H.A. Expression of the Huntington's disease gene is regulated in astrocytes in the arcuate nucleus of the hypothalamus of postpartum rats. Faseb J. (1999) 13:1099–1106.[Abstract/Free Full Text]

  49. Kaltenbach L.S., Romero E., Becklin R.R., Chettier R., Bell R., Phansalkar A., Strand A., Torcassi C., Savage J., Hurlburt A., et al. Huntingtin interacting proteins are genetic modifiers of neurodegeneration. PLoS Genet. (2007) 3:e82.[CrossRef][Medline]

  50. Kretzschmar D., Tschape J., Bettencourt Da Cruz A., Asan E., Poeck B., Strauss R., Pflugfelder G.O. Glial and neuronal expression of polyglutamine proteins induce behavioral changes and aggregate formation in Drosophila. Glia (2005) 49:59–72.[CrossRef][Web of Science][Medline]

  51. Strigini M., Cantera R., Morin X., Bastiani M.J., Bate M., Karagogeos D. The IgLON protein Lachesin is required for the blood-brain barrier in Drosophila. Mol. Cell Neurosci. (2006) 32:91–101.[CrossRef][Medline]

  52. Auld V.J., Fetter R.D., Broadie K., Goodman C.S. Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila. Cell (1995) 81:757–767.[CrossRef][Web of Science][Medline]

  53. Hansson O., Nylandsted J., Castilho R.F., Leist M., Jaattela M., Brundin P. Overexpression of heat shock protein 70 in R6/2 Huntington's disease mice has only modest effects on disease progression. Brain Res. (2003) 970:47–57.[CrossRef][Web of Science][Medline]

  54. Hay D.G., Sathasivam K., Tobaben S., Stahl B., Marber M., Mestril R., Mahal A., Smith D.L., Woodman B., Bates G.P. Progressive decrease in chaperone protein levels in a mouse model of Huntington's disease and induction of stress proteins as a therapeutic approach. Hum. Mol. Genet. (2004) 13:1389–1405.[Abstract/Free Full Text]

  55. Zourlidou A., Gidalevitz T., Kristiansen M., Landles C., Woodman B., Wells D.J., Latchman D.S., de Belleroche J., Tabrizi S.J., Morimoto R.I., et al. Hsp27 overexpression in the R6/2 mouse model of Huntington's disease: chronic neurodegeneration does not induce Hsp27 activation. Hum. Mol. Genet. (2007) 16:1078–1090.[Abstract/Free Full Text]

  56. Vacher C., Garcia-Oroz L., Rubinsztein D.C. Overexpression of yeast hsp104 reduces polyglutamine aggregation and prolongs survival of a transgenic mouse model of Huntington's disease. Hum. Mol. Genet. (2005) 14:3425–3433.[Abstract/Free Full Text]

  57. Chan H.Y., Warrick J.M., Gray-Board G.L., Paulson H.L., Bonini N.M. Mechanisms of chaperone suppression of polyglutamine disease: selectivity, synergy and modulation of protein solubility in Drosophila. Hum. Mol. Genet. (2000) 9:2811–2820.[Abstract/Free Full Text]

  58. Fujimoto M., Takaki E., Hayashi T., Kitaura Y., Tanaka Y., Inouye S., Nakai A. Active HSF1 significantly suppresses polyglutamine aggregate formation in cellular and mouse models. J. Biol. Chem. (2005) 280:34908–34916.[Abstract/Free Full Text]

  59. Herbst M., Wanker E.E. Small molecule inducers of heat-shock response reduce polyQ-mediated Huntingtin aggregation. A possible therapeutic strategy. Neurodegener. Dis. (2007) 4:254–260.[CrossRef][Medline]

  60. Gines S., Ivanova E., Seong I.S., Saura C.A., MacDonald M.E. Enhanced Akt signaling is an early pro-survival response that reflects N-methyl-D-aspartate receptor activation in Huntington's disease knock-in striatal cells. J. Biol. Chem. (2003) 278:50514–50522.[Abstract/Free Full Text]

  61. Apostol B.L., Illes K., Pallos J., Bodai L., Wu J., Strand A., Schweitzer E.S., Olson J.M., Kazantsev A., Marsh J.L., et al. Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum. Mol. Genet. (2006) 15:273–285.[Abstract/Free Full Text]

  62. Liévens J.C., Woodman B., Mahal A., Bates G. Abnormal phosphorylation of Synapsin I predicts a neuronal transmission impairment in the R6/2 Huntington's disease transgenic mice. Mol. Cell Neurosci. (2002) 20:638.[CrossRef][Web of Science][Medline]

  63. Kumar J.P., Tio M., Hsiung F., Akopyan S., Gabay L., Seger R., Shilo B.Z., Moses K. Dissecting the roles of the Drosophila EGF receptor in eye development and MAP kinase activation. Development (1998) 125:3875–3885.[Abstract]

  64. Kim M., Cha G.H., Kim S., Lee J.H., Park J., Koh H., Choi K.Y., Chung J. MKP-3 has essential roles as a negative regulator of the Ras/mitogen-activated protein kinase pathway during Drosophila development. Mol. Cell Biol. (2004) 24:573–583.[Abstract/Free Full Text]

  65. Rangone H., Pardo R., Colin E., Girault J.A., Saudou F., Humbert S. Phosphorylation of arfaptin 2 at Ser260 by Akt Inhibits PolyQ-huntingtin-induced toxicity by rescuing proteasome impairment. J. Biol. Chem. (2005) 280:22021–22028.[Abstract/Free Full Text]

  66. Humbert S., Bryson E.A., Cordelieres F.P., Connors N.C., Datta S.R., Finkbeiner S., Greenberg M.E., Saudou F. The IGF-1/Akt pathway is neuroprotective in Huntington's disease and involves Huntingtin phosphorylation by Akt. Dev. Cell (2002) 2:831–837.[CrossRef][Web of Science][Medline]

  67. Mayo L.D., Donner D.B. A phosphatidylinositol 3-kinase/Akt pathway promotes translocation of Mdm2 from the cytoplasm to the nucleus. Proc. Natl Acad. Sci. USA (2001) 98:11598–11603.[Abstract/Free Full Text]

  68. Zhou B.P., Liao Y., Xia W., Zou Y., Spohn B., Hung M.C. HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat. Cell Biol. (2001) 3:973–982.[CrossRef][Web of Science][Medline]

  69. Song G., Ouyang G., Bao S. The activation of Akt/PKB signaling pathway and cell survival. J. Cell Mol. Med. (2005) 9:59–71.[Web of Science][Medline]

  70. Liu Y.F. Expression of polyglutamine-expanded Huntingtin activates the SEK1-JNK pathway and induces apoptosis in a hippocampal neuronal cell line. J. Biol. Chem. (1998) 273:28873–28877.[Abstract/Free Full Text]

  71. Garcia M., Charvin D., Caboche J. Expanded huntingtin activates the c-Jun terminal kinase/c-Jun pathway prior to aggregate formation in striatal neurons in culture. Neuroscience (2004) 127:859–870.[CrossRef][Web of Science][Medline]

  72. Plas D.R., Thompson C.B. Akt-dependent transformation: there is more to growth than just surviving. Oncogene (2005) 24:7435–7442.[CrossRef][Web of Science][Medline]

  73. Manning B.D., Cantley L.C. AKT/PKB signaling: navigating downstream. Cell (2007) 129:1261–1274.[CrossRef][Web of Science][Medline]

  74. Schubiger M., Feng Y., Fambrough D.M., Palka J. A mutation of the Drosophila sodium pump alpha subunit gene results in bang-sensitive paralysis. Neuron (1994) 12:373–381.[CrossRef][Web of Science][Medline]

  75. Fergestad T., Bostwick B., Ganetzky B. Metabolic disruption in Drosophila bang-sensitive seizure mutants. Genetics (2006) 173:1357–1364.[Abstract/Free Full Text]

  76. Powers W.J., Videen T.O., Markham J., McGee-Minnich L., Antenor-Dorsey J.V., Hershey T., Perlmutter J.S. Selective defect of in vivo glycolysis in early Huntington's disease striatum. Proc. Natl Acad. Sci. USA (2007) 104:2945–2949.[Abstract/Free Full Text]

  77. Varma H., Cheng R., Voisine C., Hart A.C., Stockwell B.R. Inhibitors of metabolism rescue cell death in Huntington's disease models. Proc. Natl Acad. Sci. USA (2007) 104:14525–14530.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
J. L. Marsh, T. Lukacsovich, and L. M. Thompson
Animal Models of Polyglutamine Diseases and Therapeutic Approaches
J. Biol. Chem., March 20, 2009; 284(12): 7431 - 7435.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
17/6/882    most recent
ddm360v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Liévens, J.-C.
Right arrow Articles by Birman, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Liévens, J.-C.
Right arrow Articles by Birman, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?