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Published as doi: 10.1096/fj.07-103887.
 (The FASEB Journal. 2008;22:3348-3357.)
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Sodium dodecyl sulfate-insoluble oligomers are involved in polyglutamine degeneration

S. L. Alan Wong*,{dagger}, Wing Man Chan*,{dagger} and H. Y. Edwin Chan*,{dagger},{ddagger},1

* Laboratory of Drosophila Research,

{dagger} Molecular Biotechnology Programme, and

{ddagger} Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong, China

1Correspondence: Department of Biochemistry, The Chinese University of Hong Kong, Shatin N.T., Hong Kong, China. E-mail: hyechan{at}cuhk.edu.hk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In polyglutamine (polyQ) degeneration, disease protein that carries an expanded polyQ tract is neurotoxic. Expanded polyQ protein exists in different conformations that display distinct solubility properties. In this study, an inducible transgenic Drosophila model is established to define the pathogenic form of polyQ protein at an early stage of degeneration in vivo. We show that microscopic polyQ aggregates are neither pathogenic nor protective. Further, no toxic effect of sodium dodecyl sulfate (SDS) -soluble polyQ protein is observed in our model. By means of filtration, 2 forms of SDS-insoluble protein species are identified according to their size. Coexpression of an ATPase-defective form of the molecular chaperone Hsc70 (Hsc70-K71S) selectively reduces the abundance of the large SDS-insoluble polyQ species, but such modulation has no modifying effects on degeneration. Notably, we detect a distinct Hsc70-K71S-resistant, small, SDS-insoluble polyQ oligomeric species that is closely correlated with degeneration. Our data highlight the toxic role of SDS-insoluble oligomers in polyQ degeneration in vivo.—Wong, S. L. A., Chan, W. M., and Chan, H. Y. E. Sodium dodecyl sulfate-insoluble oligomers are involved in polyglutamine degeneration.


Key Words: aggregate • Drosophila • neurodegeneration • solubility


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
POLYGLUTAMINE (POLYQ) DISEASES are inherited neurodegenerative disorders that are caused by glutamine-coding CAG codon expansion in the affected genes (1) . CAG expansion results in the production of an expanded polyQ tract in the respective disease protein. To date, 9 polyQ diseases have been identified, including Huntington’s disease (HD), spinobulbar muscular atrophy (SBMA), dentatorubropallidoluysian atrophy, Machado-Joseph disease (MJD), and various types of spinocerebellar ataxia. Disease protein with an expanded polyQ tract triggers late-onset progressive neuronal degeneration and cell death (2) .

The toxicity of polyQ protein can be attributed to its interference with gene transcription (3 , 4) , protein folding (5 , 6) , and protein degradation mechanisms (7) in the affected neurons. Expanded polyQ protein exists in a number of biophysical conformations, including monomers, spherical oligomers, protofibrils, and aggregates (8 9 10 11 12 13 14) . It has been proposed that these polyQ conformers are associated with toxicity in different disease models. For example, expanded polyQ protein monomers in a β-sheet conformation initiates cytotoxicity when injected into cultured cells (8) . Treatment of cells with green tea polyphenol modulates spherical oligomer formation and suppresses polyQ-mediated toxicity (9) . Further, both castration (12) and reduction of polyQ transgene expression (14) in mice eliminate the accumulation of protofibrils and prevent neurodegeneration. Polyglutamine protein aggregates sequester cellular factors such as transcription factors and molecular chaperones, which compromise their cellular functions and eventually cause degeneration (15) . Conversely, polyQ aggregates have also been reported to attenuate toxicity in vitro (16) . To date, the pathogenic polyQ protein conformers are not well defined and still under investigation (15 , 17 18 19) .

Here we establish an inducible transgenic Drosophila model to investigate the pathogenic roles of polyQ conformers in vivo and find that microscopic polyQ aggregates neither promote nor alleviate degeneration. Expanded polyQ protein conformers display distinct biochemical solubility properties (8 , 11 12 13 , 20 21 22 23 24) . Our data show that neither sodium dodecyl sulfate (SDS) -soluble nor large (>0.22 µm) SDS-insoluble polyQ protein shows correlation with the degenerative phenotype. By means of filtration, we detect a small (<0.22 µm) SDS-insoluble polyQ protein species in our model, and the accumulation of this species is closely associated with degeneration. Our data also demonstrate that this small SDS-insoluble polyQ protein species occurs predominantly in the form of spherical oligomers.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Drosophila genetics
Flies were raised at 18 or 25°C on cornmeal medium supplemented with dry yeast. The fly strains used include UAS-HA-MJDtrQ27 (25) , UAS-HA-MJDtrQ78(s) (25) , UAS-EGFP (26) , gmr-GAL4, UAS-HSPA1L, UAS-GFP::lacZ.nls, UAS-HSC70–4.K71S, UAS-P35, and tubP-GAL80ts (Bloomington Drosophila Stock Center, Indiana University, Bloomington, IN, USA).

Generation of enhanced green fluorescent protein (EGFP) -polyQ76-FLAG transgenic fly lines
The CAG repeat was polymerase chain reaction-amplified from UAS-MJDtrQ78(s) fly genomic DNA and then cloned into pUAST-EGFP vector using EcoRI and NotI enzymes to generate pUAST-EGFP-polyQ76-FLAG plasmid. Transgenic flies were generated by microinjection. The UAS-EGFP-polyQ76-FLAG transgene encodes for an expanded polyQ polypeptide (Q76) that fuses with the N terminus of a FLAG tag and the C-terminus of EGFP. The following primers were used: MJDtrQFLAGF, 5' CGG AAT TCT ACT TTG AAA AAC AGC AG 3'; and MJDtrQFLAGR, 5' TGC GGC CGC TCA TCA CTT ATC GTC ATC GTC CTT GTA ATC TCC TGA TAG GTC CCG 3'.

Immunoprecipitation and Western blotting
For immunoprecipitation, 50 fly heads were homogenized in lysis buffer [50 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1 mM EDTA; 1 mM PMSF; 0.02% Nonidet P-40, protease inhibitor cocktail (Sigma-Aldrich Corp., St. Louis, MO, USA)]. Lysates were then incubated with anti-HA affinity gel beads HA-7 (Sigma) at 4°C overnight. After 3 rounds of washing (50 mM Tris-HCl, pH 7.6; 150 mM NaCl; 1 mM EDTA; 0.02% Nonidet P-40), the immunoprecipitated HA-tagged MJDtrQ78 protein samples were eluted in 6x SDS sample buffer. The samples were analyzed either unfiltered or after filtration through a 0.22 µm hydrophilic polyvinylidene difluoride (PVDF) membrane (Ultrafree-MC; Millipore, Bedford, MA, USA). For standard Western blotting, 15 fly heads were homogenized in 75 µl of 6x SDS sample buffer. Western blotting was performed as described previously (21 , 27) . Primary antibodies used were rabbit anti-HA (1:500; Zymed Laboratories, Burlingame, CA, USA), rat anti-heat shock protein 70 (Hsp70) 7FB (1:500) (28) , and mouse anti-β-tubulin E7 (1:2500; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, under the auspices of the National Institute for Child Health and Human Development), and the secondary antibodies used were affinity-purified goat anti-rabbit, goat anti-rat, and goat anti-mouse IgG (H+L) peroxidase conjugate (1:2000; Chemicon, Temecula, CA, USA).

Filter retardation assay
Ten fly heads were homogenized in sample buffer (2% SDS and 50 mM dithiothreitol). Lysates were heated at 99°C for 5 min and filtered through a 0.22 µm cellulose acetate membrane (Sartorius, Goettingen, Germany). Proteins retained on the membrane were detected using rabbit anti-HA antibody (1:500; Zymed) and affinity-purified goat anti-rabbit IgG (H+L) peroxidase conjugate (1:2000; Chemicon).

Immunofluorescence
Immunostaining of adult eye sections was performed as described previously (20 , 27) using rabbit anti-HA (1:150; Zymed) as primary antibody and goat anti-rabbit IgG (H+L) fluorescein isothiocyanate conjugate (1:150; Zymed) as secondary antibody; 4',6'-diamidino-2-phenyl indole (DAPI) was used to label the cell nuclei.

Pseudopupil assay
MJDtrQ78-expressing fly eyes were examined under a light microscope (Olympus CX31; Olympus, Tokyo, Japan) using an x60 oil objective as described previously (29) . At least 100 ommatidia from 5–10 fly eyes were used to calculate the average number of rhabdomeres per ommatidium in 3 independent experiments. For EGFP-polyQ76-FLAG flies, pseudopupil and EGFP signals were simultaneously detected under a fluorescence microscope (Olympus BX51). At least 80 ommatidia from 8 fly eyes were examined to calculate the average number of rhabdomeres per ommatidium in 2 independent counts.

Atomic force microscopy (AFM)
Proteins isolated from 100 fly heads were immunoprecipitated as described above and eluted in 2% SDS. The eluted protein samples were filtered through a 0.22 µm hydrophilic PVDF membrane (Ultrafree-MC; Millipore), spotted onto a silica wafer, and incubated for 4 min at room temperature. The samples were then rinsed with distilled water, dried with compressed air, and imaged in air with a digital multimode Nanoscope III scanning probe microscope (Digital Instruments/Veeco Instruments, Plainview, NY, USA) that was operated in tapping mode.

Statistical analyses
Difference in the mean measurements from immunofluorescence and Western blotting experiments were compared using unpaired Student’s t test (2 groups). The difference between the average numbers of rhabdomeres per ommatidium in pseudopupil assay was compared using a Mann-Whitney U test. Linear regression was used in the correlation studies, as shown in Figs. 1 and 4 , with P < 0.05 denoting statistical significance.


Figure 1
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  		Figure 1. Induced expression of the MJDtrQ78 protein in adult flies. A) Western blotting of MJDtrQ78 protein. SDS-insoluble (stacking gel, vertical bar), but not SDS-soluble monomeric (~35 kDa, arrow), MJDtrQ78 protein was detected at 4 dpi. β-Tubulin was used as the loading control. B) Detection of SDS-insoluble MJDtrQ78 protein by filter retardation assay. An increasing amount of SDS-insoluble MJDtrQ78 protein was retained on a 0.22 µm filter membrane from 1 to 4 dpi. C) The induction of MJDtrQ78 expression led to progressive accumulation of microscopic polyQ aggregates (green) from 2 dpi. DAPI was used to stain the cell nuclei (red). Double-headed arrows represent depth of the retina. The higher-magnification image (rightmost panel) illustrates the size of microscopic aggregates observed at 4 dpi. A total of 245 microscopic aggregates (detected from 2 to 4 dpi) were counted, and their average diameter was estimated to be ~0.91 ± 0.37 µm. Scale bars = 20 µm; 5 µm (rightmost panel). D) Quantification of C. Percentage of cells with aggregates was calculated by dividing the number of aggregates by the number of DAPI-stained cell nuclei. E) Plot of B and D. A linear correlation (R2=0.985; P<0.001) between the percentage of cells with microscopic aggregates and the amount of SDS-insoluble MJDtrQ78 protein was observed. Quantification was calculated from 3 independent experiments. Error bars = SEM. Genotype: w; gmr-GAL4 UAS-MJDtrQ78/+; tubP-GAL80ts/+.


Figure 2
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  		Figure 2. Induced expression of MJDtrQ78 protein causes progressive degeneration. A) Control flies showed a normal number of rhabdomeres per ommatidium up to 18 dpi. The induced expression of MJDtrQ78 caused a progressive reduction in the average number of rhabdomeres per ommatidium, starting from 12 dpi. Coexpression of the caspase inhibitor P35 protein, but not the control GFP-lacZ fusion protein, with MJDtrQ78 restored the rhabdomeres per ommatidium count at 12 dpi. B, C) Quantification of A, calculated from 3 independent experiments. *P < 0.05; Mann-Whitney U test. Error bars = SEM. Genotypes: w; gmr-GAL4/+; tubP-GAL80ts/+ (control), w; gmr-GAL4 UAS-MJDtrQ78/+; tubP-GAL80ts/+, w; gmr-GAL4 UAS-MJDtrQ78/UAS-GFP::lacZ.nls; tubP-GAL80ts/+, and w; gmr-GAL4 UAS-MJDtrQ78/+; tubP-GAL80ts/UAS-P35.


Figure 3
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  		Figure 3. Effects of the mutant Hsc70-K71S molecular chaperone on the microscopic MJDtrQ78 aggregates and MJDtrQ78-induced degeneration. A) The induced expression of MJDtrQ78 resulted in aggregate formation in adult fly eyes. The coexpression of Hsc70-K71S with MJDtrQ78 significantly reduced the percentage of cells with aggregates (defined as in Fig. 1D ) at 12 dpi. *P < 0.05; unpaired Student’s t test. B) The induced expression of MJDtrQ78 caused a reduction in the average number of rhabdomeres per ommatidium at 12 and 18 dpi, but not at 4 dpi. Coexpression of MJDtrQ78 with Hsc70-K71S resulted in a similar average number of rhabdomeres per ommatidium as in the MJDtrQ78 control at all time points examined. Quantification was calculated from 3 independent experiments. Error bars = SEM. Genotypes: w; gmr-GAL4 UAS-MJDtrQ78/+; tubP-GAL80ts/+ and w; gmr-GAL4 UAS-MJDtrQ78/UAS-Hsc70–4.K71S; tubP-GAL80ts/+.


Figure 4
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  		Figure 4. Accumulation of polyQ aggregates is not correlated with neuronal degeneration in the EGFP-polyQ76-FLAG model. Rhabdomeric integrity (top panel) and polyQ aggregates (bottom panel) were simultaneously monitored in EGFP-polyQ76-FLAG-expressing flies at 2–3 days post eclosion (dpe). No linear correlation was observed between the area occupied by polyQ aggregates (defined as the total area of green fluorescence signals over the area of a single ommatidium) and the average number of rhabdomeres per ommatidium (R2=0.001; P=0.752). Scale bar = 10 µm. Genotype: w; gmr-GAL4/+; UAS-EGFP-polyQ76-FLAG/+.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
An inducible transgenic polyQ disease model in Drosophila
Constitutive transgenic expression of a truncated expanded human MJD gene product, MJDtrQ78, in Drosophila caused polyQ protein aggregate formation in larval imaginal eye discs and degeneration in adult flies (Supplemental Fig. 1A) (21 , 25 , 27 , 30) . However, the occurrence of pupation in the flies between larval and adult stages interrupts the continual monitoring of protein aggregation and polyQ toxicity, which makes defining the moment at which degeneration begins difficult. In an attempt to carry out a continuous longitudinal study in vivo, we used the temperature-sensitive GAL80 (GAL80ts) expression system (31) to delay MJDtrQ78 transgene expression until adulthood (Fig. 1 A; Supplemental Fig. 2). This strategy allowed us to analyze the effects of polyQ protein expression in a single developmental stage and investigate the effects of early pathological events. The MJDtrQ78 transgene expression was initiated when adult flies that carried the gmr-GAL4, UAS-MJDtrQ78 and tubP-GAL80ts transgenes were transferred from 18°C (nonpermissive temperature) to 25°C (partially permissive temperature) (31) .

The induction of expanded polyQ protein expression causes accumulation of SDS-insoluble protein and microscopic aggregates
On induced expression of the MJDtrQ78 transgene, SDS-insoluble MJDtrQ78 protein was observed in the stacking gel at 4 days postinduction (dpi; Fig. 1A ). However, monomeric SDS-soluble MJDtrQ78 protein (~35 kDa) was not detected (Fig. 1A ), probably because of its rapid conversion to SDS-insoluble form, and resided in the stacking gel. We were able to detect such SDS-soluble MJDtrQ78 monomers only by means of immunoprecipitation from a large pool of protein samples (see Fig. 5A ). Through use of filter retardation assay, progressive accumulation of SDS-insoluble MJDtrQ78 protein was detected from 1 dpi (Fig. 1B ). These results indicate that the induction of MJDtrQ78 transgene expression in adult flies promotes a temporal buildup of SDS-insoluble polyQ protein. We next investigated the formation of polyQ aggregates in our inducible model using fluorescence microscopy. Polyglutamine protein aggregates were first detected in adult retina at 2 dpi, then increased in abundance over time (Fig. 1C, D ). We defined these polyQ proteins as "microscopic aggregates" (Fig. 1C ), and the average diameter of these aggregates was estimated to be ~1 µm. We also observed a close correlation (R2=0.985; Fig. 1E ) between the accumulation of SDS-insoluble MJDtrQ78 protein (Fig. 1B ) and microscopic aggregates (Fig. 1C, D ) in our model.


Figure 5
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  		Figure 5. Effect of the mutant Hsc70-K71S molecular chaperone on different biochemical forms of MJDtrQ78 protein. A) Immunoprecipitation of MJDtrQ78 protein. The induced expression of the MJDtrQ78 transgene resulted in the production of monomeric SDS-soluble polyQ protein (~35 kDa, arrow) at 4 dpi. The coexpression of MJDtrQ78 with Hsc70-K71S resulted in a similar level of SDS-soluble monomeric polyQ protein as in the MJDtrQ78 control. SDS-insoluble MJDtrQ78 protein was also immunoprecipitated at both 4 and 12 dpi (stacking gel, vertical bar). The MJDtrQ78 protein was detected in the eluent (Elu), but not in the flow-through (FT). B) A buildup of SDS-insoluble MJDtrQ78 protein was detected in the stacking gel from 4 to 12 dpi. C) Filter retardation assay on SDS-insoluble MJDtrQ78 protein. An increasing amount of large (>0.22 µm) SDS-insoluble MJDtrQ78 protein was detected from 4 to 12 dpi. D) A small (<0.22 µm) SDS-insoluble MJDtrQ78 protein species was detected after filtration and was found to accumulate in the stacking gel from 4 to 12 dpi. E, F) The coexpression of Hsc70-K71S with MJDtrQ78 decreased the total amount of SDS-insoluble MJDtrQ78 protein in the stacking gel at 12 dpi (E). Less large SDS-insoluble MJDtrQ78 protein was also detected by filter retardation assay (F). G) Coexpression of Hsc70-K71S with MJDtrQ78 resulted in a similar amount of small SDS-insoluble MJDtrQ78 protein in the stacking gel as in the MJDtrQ78 control at 12 dpi. β-Tubulin was used as the loading control (B, D, E, G). Quantification was calculated from 3 (A, B, D–G) or 4 (C) independent experiments. *P < 0.05; unpaired Student’s t test. Error bars = SEM. Genotypes: w; gmr-GAL4 UAS-MJDtrQ78/+; tubP-GAL80ts/+ and w; gmr-GAL4 UAS-MJDtrQ78/UAS-Hsc70–4.K71S; tubP-GAL80ts/+.

The induction of expanded polyQ protein expression causes degeneration
Pseudopupil assay is a commonly used method to measure polyQ-mediated degeneration (29 , 32 33 34 35 36 37 38) . We used this method to monitor polyQ degeneration in our inducible model. The continuous induction of MJDtrQ78 transgene expression caused progressive deterioration of rhabdomeric integrity from 12 dpi (Fig. 2 A, B). In addition, the coexpression of caspase inhibitor protein P35, but not GFP-lacZ, restored the MJDtrQ78-induced loss of rhabdomeres at 12 dpi (Fig. 2A, C ). However, the suppressive effect of P35 was lost at a later time point (18 dpi, Fig. 2C ). This result demonstrates that caspase activation is associated with the early stage of polyQ-induced degeneration.

The level of microscopic aggregates does not correlate with polyQ degeneration
To investigate the role of microscopic aggregates in polyQ toxicity, we attempted to manipulate their abundance in MJDtrQ78-expressing cells. Hsp70 is an ATPase that plays a crucial role in protein refolding and degradation in cells (39 , 40) . A substitution mutation (K71S) in the ATPase domain of Hsp70 has been found to impair its protein refolding activity but does not affect its protein degradation function (41) . When MJDtrQ78 and the heat shock cognate 70 (Hsc70; a constitutively expressed form of Hsp70) mutant protein Hsc70-K71S were coexpressed, the number of cells containing microscopic polyQ aggregates was significantly reduced (Fig. 3 A).

Pseudopupil assay was used to assess degeneration of flies that coexpressed MJDtrQ78 and Hsc70-K71S. No significant difference in the average number of rhabdomeres per ommatidium was observed between MJDtrQ78 and MJDtrQ78/Hsc70-K71S flies at either 4 or 12 dpi (Fig. 3B ). Similar results were also observed at a more advanced stage of degeneration (18 dpi; Fig. 3B ). To further validate our findings, we took advantage of an EGFP-polyQ76-FLAG fly model (Supplemental Fig. 3) that allows simultaneous monitoring of both polyQ aggregation (GFP fluorescence) and rhabdomere integrity in the same fly eye (Fig. 4 ). Consistent with that which we observed in the inducible MJDtrQ78 model (Fig. 3) , neither a positive nor a negative correlation was observed between polyQ aggregation and degeneration in EGFP-polyQ76-FLAG flies (R2=0.001; Fig. 4 ). All these data indicate that polyQ-induced degeneration is independent of the abundance of microscopic aggregates.

Detection of small SDS-insoluble polyQ oligomers that are associated with toxicity
Furthering the fluorescence microscopy, our biochemical data revealed the existence of SDS-soluble and -insoluble forms of MJDtrQ78 protein (Fig. 5 A). We then investigated the pathogenic polyQ conformers by means of biochemical and biophysical methodologies. We were able to isolate both SDS-soluble and -insoluble MJDtrQ78 proteins from a large pool of flies at 4 dpi by immunoprecipitation (Fig. 5A ). Nevertheless, we did not detect any SDS-soluble MJDtrQ78 protein at 12 dpi when degeneration was observed (Fig. 5A ). We detected a temporal accumulation of SDS-insoluble MJDtrQ78 protein in the stacking gel from 4 to 12 dpi (Fig. 5B ), which occurred concomitantly with degeneration (Fig. 3B ). We therefore argued that polyQ toxicity is associated with a buildup of SDS-insoluble protein species. By means of filtration, we further fractionated polyQ proteins according to their biophysical properties. Using a membrane filter with a molecular cutoff size of 0.22 µm, we were able to separate SDS-insoluble MJDtrQ78 with sizes larger and smaller than 0.22 µm. And we detected buildup of both >0.22 µm (Fig. 5C ) and <0.22 µm (Fig. 5D ; Supplemental Fig. 4) SDS-insoluble MJDtrQ78 protein species from 4 to 12 dpi.

We then investigated whether the abundance of the SDS-insoluble MJDtrQ78 protein would be altered by Hsc70-K71S coexpression. We found that the level of total SDS-insoluble MJDtrQ78 protein retained on the stacking gel was reduced significantly (Fig. 5E ). Meanwhile, Hsc70-K71S coexpression also reduced the level of the large (>0.22 µm) SDS-insoluble MJDtrQ78 protein species (Fig. 5F ). Notably, we found that the small (<0.22 µm) SDS-insoluble MJDtrQ78 protein species was resistant to Hsc70-K71S coexpression (Fig. 5G ). As the small SDS-insoluble MJDtrQ78 protein species accumulated with time (Fig. 5D ) when degeneration progressed (Fig. 3B ) and its level did not decrease in the presence Hsc70-K71S at 12 dpi (Fig. 5G ), we demonstrated a tight linkage between its abundance and degeneration (Fig. 3B ).

We previously reported (5 , 27) a biphasic expression profile of endogenous molecular chaperone genes in the constitutive MJDtrQ78 model. An induction of endogenous Hsp70 level, at both the mRNA and protein levels, was initially observed in MJDtrQ78-expressing flies. As degeneration progressed, a rapid decline of endogenous Hsp70 expression occurred. We observed a similar expression profile of endogenous Hsp70 protein in the inducible MJDtrQ78 model (Fig. 6 A; Supplemental Fig. 5). Because the endogenous Hsp70 level declined as the small (<0.22 µm) SDS-insoluble MJDtrQ78 protein species accumulated in MJDtrQ78 flies from 4 to 12 dpi (Fig. 5D ), we sought to investigate the effect of wild-type Hsp70 on the small SDS-insoluble MJDtrQ78 protein species. We first confirmed that overexpression of the human Hsp70 protein (HSPA1L) (30) was able to suppress neurodegeneration in our inducible model (Fig. 6B ). In contrast to the accumulation of the small SDS-insoluble MJDtrQ78 protein species observed in MJDtr78-expressing flies at 12 dpi (Fig. 5D ), we found that the level of this species was significantly reduced on Hsp70 coexpression (Fig. 6C ). As the suppression of neurodegeneration mediated by Hsp70 overexpression was accompanied by a marked reduction of the small SDS-insoluble MJDtrQ78 protein species, this observation further supports the notion that such species are substantially associated with polyQ toxicity.


Figure 6
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  		Figure 6. Expression of wild-type Hsp70 suppresses neurodegeneration and reduces small SDS-insoluble MJDtrQ78 protein species. A) Reduction of endogenous Hsp70 protein level in MJDtrQ78 flies. The level of endogenous Hsp70 protein level was initially induced on MJDtrQ78 expression at 4 dpi, but it was largely reduced at 12 dpi when neurodegeneration became prominent (Fig. 2) . B) Overexpression of human Hsp70 (HSPA1L) rescued neurodegeneration. Coexpression of HSPA1L with MJDtrQ78 significantly suppressed neurodegeneration at both 12 and 18 dpi. C) HSPA1L-mediated suppression of neurodegeneration was accompanied by a significant reduction of small (<0.22 µm) SDS-insoluble MJDtrQ78 protein at 12 dpi. Quantification was calculated from 3 independent experiments (B, C). *P < 0.05; Mann-Whitney U test (B) or unpaired Student’s t test (C). Error bars = SEM. Genotypes: w; gmr-GAL4 UAS-MJDtrQ78/+; tubP-GAL80ts/+ and w; gmr-GAL4 UAS-MJDtrQ78/UAS-HSPA1L; tubP-GAL80ts/+.

AFM has previously been used to investigate polyQ structures (8 9 10 11 12 13 14) . To determine the biophysical properties of the small (<0.22 µm) SDS-insoluble polyQ protein species, AFM was performed on the 0.22 µm membrane-filtered SDS-insoluble MJDtrQ78 protein samples (Fig. 7 ). We detected round particles within a size range of 5–60 nm (Fig. 7) , and the structure and size of these particles matched closely with those defined as polyQ spherical oligomers in previous reports (9 10 11) . Moreover, we observed a buildup of such spherical oligomers (5–20 nm) from 4 to 12 dpi (Fig. 7) , which is in good agreement with our filtration results (Fig. 5D ). As the accumulation of such small SDS-insoluble polyQ oligomers occurred in parallel with degeneration (Fig. 3B ), our data therefore indicate that they strongly associate with polyQ toxicity.


Figure 7
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  		Figure 7. Detection of small SDS-insoluble polyQ oligomers on induced expression of MJDtrQ78 protein. A) AFM images of the immunoprecipitated small SDS-insoluble MJDtrQ78 protein species. The higher magnification image (top right panel) highlights the round particles (arrows) that were observed. Arrowhead shows SDS-insoluble ordered array of round particles. Height, rather than diameter, was used to calculate particle size. A height scan across a round particle indicated its size to be ~12 nm. B) A significant increase in the number of round particles (5 to 20 nm) was detected from 4 to 12 dpi in 0.22 µm membrane-filtered SDS-insoluble MJDtrQ78 protein samples. Quantification was calculated from 3 independent experiments. *P < 0.05; unpaired Student’s t test. Error bars = SEM. Scale bars = 500 nm. Genotype: w; gmr-GAL4 UAS-MJDtrQ78/+; tubP-GAL80ts/+.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Proteins with an expanded polyQ tract trigger late-onset progressive neuronal degeneration (2) . The constitutive expression of polyQ protein in the Drosophila eye is known to cause degenerative phenotypes (Supplemental Fig. 1) (20 , 21 , 25 , 27 , 30 , 42 , 43) . Expanded polyQ protein exists in diverse biophysical conformations, including monomers and oligomers (17) . Here, we established an inducible transgenic Drosophila model to determine the polyQ conformers that underlie degeneration in vivo. This model recapitulates pathological features of polyQ disease, including formation of SDS-insoluble protein (Fig. 1A, B ), microscopic aggregates (Fig. 1C ), and progressive degeneration (Fig. 2A, B ). We also observed that the coexpression of caspase inhibitor P35 could mitigate only early- (up to 12 dpi) but not late-stage (from 18 dpi) degeneration (Fig. 2C ). The inability of P35 to suppress late-stage neurodegeneration in our model is also consistent with previous findings from a fly model of HD in which the mutant huntingtin transgene was expressed since third instar larval stage (35) . Taken together, our data therefore indicate that caspase activation (44) is involved during early stages (<12 dpi in our model) of polyQ pathogenesis.

Protein aggregation is a common pathological feature in most neurodegenerative disorders (45) , including polyQ diseases (2) . In this study, the induced expression of MJDtrQ78 protein resulted in the formation of microscopic polyQ protein aggregates (Fig. 1C, D ). It has been previously shown (46) that an ATPase domain deletion mutant of Hsc70 interacts with expanded polyQ protein and suppresses protein aggregation in vitro. When we reduced the number of cells containing microscopic polyQ aggregates by coexpressing the ATPase-defective Hsc70 mutant chaperone protein Hsc70-K71S with MJDtrQ78 (Fig. 3A ), we observed no modulation of the severity of degeneration (Fig. 3B ). K71S mutation reportedly (41) promotes protein degradation through a nonproteasome inhibitor-sensitive mechanism, but further investigation is needed to confirm whether the same pathway is involved in polyQ degeneration. Nonetheless, our data suggest that microscopic protein aggregates do not play a causal role in triggering polyQ-induced early degeneration. We also conducted an independent study using an EGFP-polyQ76-FLAG fly model (Supplemental Fig. 3) to further investigate the role of protein aggregation in polyQ degeneration. Because both EGFP-polyQ76-FLAG protein aggregates and toxicity (rhabdomeric deterioration) can be assessed simultaneously in EGFP-polyQ76-FLAG flies, this model allows us to evaluate the correlation of the 2 parameters in the same fly eye (Fig. 4) . Consistent with the results obtained from our inducible MJDtrQ78 model (Fig. 3) , we did not observe any correlation between polyQ aggregates and degeneration in the EGFP-polyQ76-FLAG flies (Fig. 4) . Our findings are also in line with previous studies that found no correlation between the occurrence of microscopic protein aggregates and neurodegeneration in polyQ patients, including those with MJD (47) .

Polyglutamine protein exists in several biophysical conformations that are not readily distinguished under the resolution of light microscopy. The polyQ conformers display distinct biochemical solubility properties (8 9 10 11 12 13 14 , 20 21 22 23 24 , 48 49 50 51) when extracted with solvents such as SDS. For example, monomeric polyQ protein (8) has been reported to be SDS soluble, whereas spherical oligomers (11) and fibrils (13 , 52) have been demonstrated to be SDS insoluble. We adopted a biochemical approach in our subsequent investigations. Monomeric soluble disease protein is one of the toxic species that is suspected of being involved in polyQ degeneration (8 , 16 , 53) . In this study, monomeric SDS-soluble MJDtrQ78 protein could be detected only by immunoprecipitation (when large numbers of flies were used) but not in standard Western blotting analysis, and its level diminished over time from 4 to 12 dpi (Fig. 5A ). These observations are consistent with the idea that expanded MJD protein has a tendency to accumulate in SDS-insoluble form (54) . As mentioned, SDS-insoluble but not SDS-soluble MJDtrQ78 protein was observed at 12 dpi (Fig. 5A ) when degeneration became evident (Fig. 2) , indicating that monomeric SDS-soluble polyQ protein does not pose immediate toxicity in our model. The detection of SDS-soluble ordered arrays of polyQ oligomers has been reported in an SBMA transgenic mouse model, and such polyQ species have been shown to be associated with degeneration (12) . However, we did not detect these species in our inducible model (Fig. 5A ), perhaps because of a difference in the time of detection, expression levels of the polyQ protein, or types of disease protein used.

A temporal accumulation of total SDS-insoluble MJDtrQ78 protein (Fig. 5B ) was detected alongside degeneration (from 4 to 12 dpi; Fig. 3B ) in our inducible model. Based on their size difference, 2 forms of SDS-insoluble protein species were identified. We found that the large (>0.22 µm) SDS-insoluble MJDtrQ78 protein accumulated from 4 to 12 dpi (Fig. 5C ). Although its abundance was greatly reduced on Hsc70-K71S coexpression at 12 dpi (Fig. 5F ), degeneration remained severe in these flies (Fig. 3B ). We further investigated the protein level of the large SDS-insoluble MJDtrQ78 in Hsc70-K71S-coexpressed flies at both 4 and 12 dpi and did not find any trend of accumulation (Supplemental Fig. 6). Our data indicate that large SDS-insoluble MJDtrQ78 conformers do not associate with polyQ toxicity in our model. Progressive accumulation of a small (<0.22 µm) SDS-insoluble MJDtrQ78 protein species was also demonstrated from 4 to 12 dpi in our model (Fig. 5D ). Notably, unlike large SDS-insoluble conformers (Fig. 5F ), the level of small SDS-insoluble MJDtrQ78 protein was unaffected by Hsc70-K71S coexpression (Fig. 5G ) when degeneration became evident at 12 dpi (Fig. 3B ). We also found that the amount of the small SDS-insoluble protein species was significantly reduced in MJDtrQ78 flies coexpressed with wild-type human Hsp70 HSPA1L (Fig. 6C ). Because HSPA1L overexpression has been shown to suppress polyQ toxicity (Fig. 6B ) (30) , our results highlight the toxic role of the small SDS-insoluble MJDtrQ78 protein species in our inducible polyQ model. AFM analysis was performed to further characterize the biophysical properties of this toxic form of MJDtrQ78 protein species. We detected an increase in the population of spherical polyQ oligomers (size range of 5 to 20 nm) from 4 to 12 dpi (Fig. 7) . This result shows that the toxic small SDS-insoluble MJDtrQ78 protein species occurs primarily in the form of spherical oligomers in vivo.

To conclude, we established an inducible model in Drosophila to study polyQ-induced degeneration. By biochemical separations, we detected 3 distinct types of polyQ protein species, namely, 1) monomeric SDS-soluble protein, 2) large (>0.22 µm) SDS-insoluble protein, and 3) small (<0.22 µm) oligomeric SDS-insoluble protein. Of these, only the small SDS-insoluble oligomers are closely associated with degeneration in our inducible model. This result underscores the toxic role of this species in polyQ pathogenesis. Because this study focused mainly on examining the effects of polyQ expression on early degeneration, our findings do not rule out the contribution of other polyQ species to long-term toxicity.


   ACKNOWLEDGMENTS
 
We thank E. Chen for making the pUAST-EGFP-polyQ76-FLAG construct, J. Lam for performing initial characterization on EGFP-polyQ76-FLAG transgenic lines, A. Li for the excellent technical assistance on the AFM analysis, M. Feder for antibodies, and C. O'Kane and members of the Laboratory of Drosophila Research for critical comments on the manuscript. This work was supported by the Hong Kong Research Grants Council (CUHK4314/03M).

Received for publication December 18, 2007. Accepted for publication April 24, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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