Human Molecular Genetics, 2002, Vol. 11, No. 21 2657-2672
© 2002 Oxford University Press
Suppression of polyglutamine toxicity by a Drosophila homolog of myeloid leukemia factor 1
1Department of Physiology and Biophysics, Center for Neuroscience, School of Medicine and Biomedical Sciences, University at Buffalo, Buffalo, NY 14214, USA and 2California Institute of Technology, Division of Biology, Pasadena, CA 91125, USA
Received July 22, 2002; Accepted July 31, 2002
DDBJ/EMBL/GenBank accession no. AY037049
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The toxicity of an abnormally long polyglutamine [poly(Q)] tract within specific proteins is the molecular lesion shared by Huntington's disease (HD) and several other hereditary neurodegenerative disorders. By a genetic screen in Drosophila, devised to uncover genes that suppress poly(Q) toxicity, we discovered a Drosophila homolog of human myeloid leukemia factor 1 (MLF1). Expression of the Drosophila homolog (dMLF) ameliorates the toxicity of poly(Q) expressed in the eye and central nervous system. In the retina, whether endogenously or ectopically expressed, dMLF co-localized with aggregates, suggesting that dMLF alone, or through an intermediary molecular partner, may suppress toxicity by sequestering poly(Q) and/or its aggregates.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Excessive expansion of poly(CAG) tracts is associated with at least eight human hereditary neurodegenerative disorders (1). The expanded poly(CAG)s are translated to abnormally long polyglutamine [poly(Q)] tracts in the affected gene products, promoting their nuclear and/or cytoplasmic aggregation, and inducing cellular toxicity (2–4). In order to understand and possibly cure these diseases, the molecular components of the pathways involved in the associated neuronal degeneration have been investigated. These include components of protein folding (5–7), protein degradation (8,9), gene expression (10–17) and programmed cell death (18–20), as well as interacting proteins (21–23), neurotransmitters and their receptors (24–26).
The approach in this study is to search for novel genetic factors that suppress the cellular toxicity. Several Drosophila models have recapitulated the abnormal protein aggregation and neuronal toxicity associated with poly(Q) disorders (27–31). Independently of the context of the poly(Q) tract, be it in a full-length ataxin-1 in spinocerebellar ataxia type 1 or SCA1 (31), or in a truncated ataxin-3 in SCA3 (27), or in huntingtin in HD (28), or in a quasipure 108Q tract (29) or in a quasipure 127Q tract (30), expanded poly(Q) tracts in the disease size range consistently showed higher propensity to produce cellular inclusions and toxicity. However, perhaps based on differences in residues flanking the poly(Q) tract, these transgenic constructs vary in the manner in which they genetically interact with other, exogenously or endogenously expressed, genes. For instance, while the baculovirus anti-apoptotic gene P35 partially suppressed the eye phenotype in a SCA3 Drosophila model (27), it had no detectable effect in flies expressing a truncated huntingtin with an expanded poly(Q) (28). Similarly, either alone or in combination with the suppressor P-element reported here, P35 had no effect on the eye phenotype produced by 127Q (P. Kazemi-Esfarjani and S. Benzer, unpublished data).
In contrast to the latter observations, screens for dominant modifiers of poly(Q) toxicity, using either a full-length ataxin-1 82Q or plain 127Q polypeptide, in both cases identified dHDJ1, a Drosophila ortholog of Hsp40/HDJ1, a human co-chaperone protein (30,31). When overexpressed, dHDJ1 suppresses poly(Q) toxicity in the fly eye. Genetic and molecular analysis of this protein revealed that its N-terminal J domain mediates its suppressor activity via Hsp70, a chaperone involved in protein folding (32). Overexpression of Hsp70 was also able to suppress the toxicity of a truncated ataxin-3 with an expanded poly(Q) tract (33). Of considerable interest is the fact that overexpression of Hsp70 also suppressed the toxicity of 
-synuclein, associated with Parkinson's disease (34). Therefore, other modifier genes, such as the one reported here, might be relevant as therapeutic targets in other diseases with protein folding lesions.
As we have previously reported, the same genetic screen with 127Q identified another suppressor gene that encodes a protein with a C-terminal J domain and seven tetratricopeptide repeat (TPR) motifs (30). TPR motifs form suitable protein–protein interfaces (35). The human ortholog, hTPR2, was earlier discovered, in a two-hybrid screen, as a protein interactor of the guanine triphosphatase (GTPase)-activating protein-related domain (GRD) of neurofibromin, the neurofibromatosis type 1 (NF1) gene product (36). Later, independently, in another two-hybrid screen, hTPR2 was found to interact with hRad9, a 3' to 5' exonuclease and a proapoptotic and cell cycle checkpoint protein (37). Both in vivo and in vitro, this interaction was mediated through the TPR motifs and was greatly enhanced by the deletion or point mutation of the hTPR2 C-terminal J domain. Those observations open new avenues for research on the mechanism of suppression by dTPR2.
Identification of the genes and biochemical pathways that modify poly(Q) toxicity in Drosophila would potentially provide genetic targets for designing and testing therapeutic agents. This was demonstrated by suppression of poly(Q) toxicity in Drosophila by pharmacological inhibition of the deacetylation process of the histones involved in chromosomal structure and gene transcription (38). As an ongoing search for such modifier genes, we report here an entirely different gene that, similar to those mentioned above, suppresses poly(Q) toxicity without preventing the formation of intracellular inclusions. Interestingly, the homologous gene in humans is associated with a neoplastic disease.
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RESULTS |
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Cellular toxicity by expanded poly(Q)s
As before (30), P-element constructs were designed to express poly(Q) tracts of various lengths (20Q, 63Q and 127Q), carrying hemagglutinin (HA) protein tags on their C-terminal ends as an immunogenic marker. The constructs were microinjected into early embryos and transgenic lines were established. In vivo expression was regulated by the yeast transcription factor GAL4 acting on yeast upstream activating sequences (UAS) (30,39). To activate expression, these transgenic flies were crossed with other transgenic flies expressing GAL4 in the eye, using the promoter GMR (glass multimer reporter) (40–42), which is active in all retinal cells, from the time of their differentiation throughout adulthood (P. Kazemi-Esfarjani and S. Benzer, unpublished data). In a separate set of experiments, we used the neuron-specific driver Appl–GAL4 to express poly(Q) in the nervous system (43).
Control flies with a heterozygous insertion of GMR–GAL4 alone had fully developed eyes (Fig. 1A). When combined with a chromosome carrying UAS and a small poly(CAG) repeat (20Q), there was no evident change (Fig. 1B). Using an anti-HA antibody, immunohistological examination of head cryosections of 1-day-old flies carrying GAL4 alone, or GAL4 plus 20Q, revealed no poly(Q) aggregates, consistent with the experience of others (27–31). However, it should be noted that the 20Q peptide was also undetectable on western blots. In contrast, 63Q and 127Q flies had severely collapsed eyes, lacking pigmentation; in sections, anti-HA antibody revealed abundant poly(Q) clumps in the eye (Fig. 1C and D). As a control for non-specific suppression effects, the retina expressing lacZ in addition to 127Q was equally defective (Fig. 1E).
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Milder toxicity by longer sequences flanking the poly(Q) repeat
In mammalian cell culture systems, expanded poly(Q)s of a given length are less toxic in the context of a larger protein (44–46). To verify whether the same applies to flies, the eyes of flies expressing 20Q and 127Q were compared with eyes expressing the same repeat length as part of a truncated form of the Drosophila gene prospero, a neurogenic gene involved in neuronal fate specification in the central and peripheral nervous systems, which contains a segment coding for a 20Q poly(Q) tract ( pros20Qtr) [but does not share any other similarity with known poly(Q) disease genes] (47,48). Flies expressing either 20Q or pros20Qtr had apparently normal eye structure and pigment distribution (Fig. 2A and C). When flies expressing 127Q and pros127Qtr were compared, the latter showed milder toxicity, as judged by more pigmentation and less disturbed external structure (Fig. 2B and D). With truncated prospero constructs having a longer poly(Q) length ( pros223Qtr), a level of phenotypic severity similar to that of the expanded pure poly(Q) 127Q could be achieved (Fig. 2E). These results were confirmed with three independent transgenic lines for each construct. Therefore, in the fly, as in mammals, attenuation of poly(Q) toxicity increases with increasing size of the non-repeat portion of a protein.
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Identification of dmlf suppressor gene in a genetic screen
To screen for genes that suppress the toxicity of 127Q, the GMR–GAL4/UAS–127Q flies were crossed to 7000 de novo generated, random P-element insertions (30). To enhance expression, the P-elements contained 14 tandem UAS elements, which, in the presence of GAL4, drive the expression of downstream genomic sequences (49). Hence, if there is a gene downstream of a P-element insertion that codes for a modifier gene, it will be activated and cause a change in the eye phenotype.
Here, we describe a suppressor (EU2490, the 2490th newly generated P-element insertion tested), which dramatically counteracts the external eye and pigmentation defect caused by 127Q (Fig. 3C). Internal improvement was also seen in cryosections of the eye. Plasmid rescue of the P-element was performed and the DNA flanking the 3' end of the P-element was sequence (50). A BLAST search on the Berkeley Drosophila Genome Project (BDGP) server identified several expressed sequence tags (ESTs) with corresponding cDNA clones. A stretch of 
220 bp of the genomic DNA was 97% identical to the DNA sequence beginning 54 bp downstream of a predicted ATG start site of an open reading frame (ORF) in a cDNA clone, GH20101, from an adult head library (BDGP and Research Genetics). The ORF in GH20101 is 822 bp long and lies within a 1745 bp cDNA insert with 74 bp 5'-untranslated region (UTR) 849 bp 3'-UTR and an 18-base poly(A) tail (GenBank accession no. AY037049). The predicted translation product of the ORF is a 273-amino-acid protein with a molecular weight of 30 kDa, and it is homologous to the 268-amino-acid predicated protein, human myeloid leukemia factor 1 (hMLF1) (51), with 32% identity and 49% similarity. Therefore, it is referred to as Drosophila myeloid leukemia factor (dMLF) (52). Recently, in a yeast two-hybrid screen, dMLF protein was shown to interact with an N-terminal segment of Drosophila DNA replication-related element-binding factor (DREF) (52). However, the plasmid that expressed dMLF (pACT1-2) (52), isolated from a third instar larva cDNA library, contained a longer ORF predicting a 309-amino-acid protein. While otherwise identical, it is 36 residues longer at the C-terminal end than the 273-amino-acid protein predicted by the GH20101 plasmid in this study, and 41 residues longer than the 268-amino-acid protein predicted by hMLF1 cDNA.
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To investigate the possibility that the differences might be due to alternative splicing, a second, independent clone, LD47427, isolated from an embryo cDNA library, was sequenced, and all three cDNA sequences were matched against the genomic sequence of the dmlf gene (53). As with the pACT1-2 plasmid (52), LD47427 harbors an ORF that codes for a dMLF with 309 amino acids, and is generated by skipping a potential pre-mRNA splice junction in the genomic DNA. On the other hand, the ORF in GH20101 is generated using this early junction, resulting in splicing of an additional exon between the third and fourth exons in pLD47427. The stop codon that appears 10 bp from this alternative splice site results in a shorter ORF coding for 273 amino acids. Based on northern blot analysis, dmlf mRNA levels are high in unfertilized eggs, early embryos, pupae and adult males, and relatively low in adult females and larvae (52). Therefore, as dmlf mRNA is differentially expressed, it might also be alternatively spliced in embryo (LD47427) and third instar larva (pACT1-2) versus adult head (GH20101). A more comprehensive analysis is underway to clarify this aspect of dmlf expression.
Suppression of poly(Q) toxicity by transgenic expression of dmlf
To confirm whether the expression of dMLF is responsible for the suppression effect, the cDNA insert in GH20101 was placed in the same kind of P-element vector as UAS–127Q, and transgenic line were established. Three independently established lines, each carrying a heterozygous autosomal insertion of UAS–dmlf in the presence of GMR–GAL4/UAS–127Q, reproduced the improvement in external eye structure and pigmentation to an even greater extent than did the original P-element insertion (Fig. 3D and E). Similar suppression was obtained from the cDNA insert in LD47427 (not shown). Again, the internal eye structure was only slightly improved. We therefore tried higher doses of the suppressor gene. Three different transgenic lines were established, each carrying UAS–dmlf transgenic insertions on both the second and third chromosomes. These improved both the external and internal eye structures to almost normal (Fig. 3F). Nevertheless, as with the two suppressor genes described previously (30), numerous fluorescent aggregates were still present in the eye. Thus, despite the great reduction in toxicity, with none of these suppressors was the aggregation of poly(Q) prevented. Rather, they appeared to reduce the toxicity of the poly(Q), or enhance the ability of the cells to resist their toxic effect.
To investigate the overexpression effect of dmlf on the retinas expressing pros20Qtr or pros127Qtr, immunofluorescence experiments were performed as above. With respect to tissue morphology and the locations of DAPI-stained nuclei, retinas in which pros20Qtr was expressed appeared normal (Fig. 4A). As with 20Q alone, no aggregates were visible with anti-HA-dependent fluorescence staining. Nevertheless, the retinas of flies accumulated enough protein to produce a strong HA signal on western blots. Retinas that express pros20Qtr in the presence of two doses of dmlf transgenes also had normal morphology, except for some displacement of nuclei, perhaps as a result of dMLF interference with developmental processes (Fig. 4B). Compared with retinas expressing pros20Qtr,retinas that express pros127Qtr were thinner and had defective tissue morphology (Fig. 4C). As compared with pure 127Q, they also appeared to generate a smaller number of anti-HA-positive, fluorescent aggregates, with the caveat that some of the PROS127Qtr polypeptides might have lost their HA tags owing to degradation of a 360-amino-acid stretch separating the poly(Q) tract and the HA epitope tag. As negative controls for the suppression effect, neither one nor two doses of a modified form of lacZ, coding for a ß-galactosidase containing a nuclear localization signal, expressed together with pros127Qtr, had any apparent protective effect (Fig. 4D and E). On the other hand, when a single dose of dmlf transgene was co-expressed with pros127Qtr, the retinas were preserved (Fig. 4F and G). When two doses of dmlf transgenes were co-expressed with pros127Qtr, the retinas were similarly preserved as with a single dose (Fig. 4H).
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We examined whether dmlf overexpression has a detrimental effect on the cellular differentiation and development of the retina by assessing the number and location of the nuclei. The retinas were labeled with both anti-ELAV, an antibody raised against a neuron-specific nuclear protein, and DAPI to stain nuclei. In controls transgenic for GMR–GAL4 alone, or together with one or two doses of UAS–lacZ, the number of nuclei and their locations were essentially normal. The few misplaced nuclei were perhaps due to high levels of expression of GMR–GAL4, which causes some deterioration in the structure of the retina (Fig. 4I, J and K). In flies transgenic for GMR–GAL4 together with one or two doses of UAS–dmlf, similar numbers and localization of nuclei were observed (Fig. 4L, M and N). Therefore, even at a high expression level, dMLF may have little effect on retinal structure. Nonetheless, additional quantitative analyses at higher resolution are required to examine possible changes in the size and number of nuclei when dMLF is overexpressed.
The specificity of dMLF suppression effect with respect to poly(Q) protein type
As a first step towards verifying the application of dmlf suppressor activity in human poly(Q) disorders, dmlf suppression was further tested on a mutant form of huntingtin (Htt), the Huntington's disease gene product. dmlf transgenic flies were crossed to flies expressing a truncated form of huntingtin with 120 glutamines (HttQ120) (28). pros20Qtr and pros127Qtr flies were also tested in this experiment. Plastic sections of eyes of the resulting progeny were examined for the structure of their ommatidia, the units of the retina containing eight photoreceptors and their accessory cells. At 1 day of age, in the presence of active (with GMR–GAL4 driver) or inactive (without GMR–GAL4 driver; not shown) UAS transgenes for lacZ, or one or two doses of dmlf, the retinas expressing HttQ120 still maintained the arrangement of the facets or ommatidia, indicated by the presence of hexagonal borders containing the darkly stained circular forms of the photoreceptor rhabdomeres (Fig. 5A–D). However, with HttQ120 at 1, 2 or 5 weeks of age, the arrangement of ommatidia deteriorated, except in the presence of one or two doses of active (i.e. with GMR–GAL4 driver) UAS–dmlf transgenes (Fig. 5A–D). In the absence of GMR–GAL4 driver, UAS–dmlf transgenes were completely ineffective (not shown). The flies expressing pros20Q in the presence or absence of two doses of transgenic dmlf, had a preserved arrangement of ommatidia, even at 2 weeks of age (Fig. 5E and F). However, for pros127Qtr, not with lacZ (Fig. 5H and I), but with dmlf (Fig. 5J and K), the deterioration was stayed only in 1-day-old flies. dmlf transgenes were not effective in preserving the eye structure up to 2 weeks of age. (Note that the differences in the orientation of the ommatidia among different panels are due to the orientation of the photography and not to phenotypic effects.) Therefore, the dmlf suppression effect is not restricted to degeneration caused by pure poly(Q) polypeptides, but could also suppress the relatively milder toxicity of HttQ120 over a prolonged period, up to 5 weeks.
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dMLF is also effective in the central nervous system
To see whether dMLF protects other neuronal tissues against the deleterious effects of long poly(Q)s, we used the neuron-specific driver Appl–GAL4, derived from the promoter region of the gene for the Drosophila homolog of human amyloid precursor protein (APP) (43). Appl is expressed exclusively in postmitotic neurons of the central and peripheral nervous system, from mid to late stages of embryogenesis onward (54).
Flies carrying Appl–GAL4 alone developed normally, and survived for the full observation period of 20 days. The same was true for three independent UAS–20Q insertions in the presence of Appl–GAL4. UAS–63Q, however, had a strong toxic effect. Of four independent transgenic lines tested, three were pre-adult lethal, and one gave rise to only female adults. (Since the Appl–GAL4 transgene was on the X chromosome, dosage compensation may have produced higher expression in males, resulting in greater toxicity.) Three UAS–127Q lines were all pre-adult lethal in the presence of Appl–GAL4. Therefore, to test for suppression of toxicity by dmlf, 63Q females were chosen, using survival of adults versus age as a criterion.
In controls, with Appl–GAL4 alone (not shown) or expressing one or two doses of lacZ, the flies remained vital at 20 days (Fig. 6). In sections of the brain and thoracic ganglion, no anti-HA fluorescent staining was observed in these flies. Flies carrying Appl–GAL4 plus a heterozygous insertion of UAS–63Q, by day 12, became progressively lethargic, with almost all dead by day 20. Flies first became unable to climb the walls of the plastic vial, and died within a few hours. Anti-HA antibody on cryosections of 1-day-old adult Appl–GAL4/UAS–63Q flies revealed aggregates in the neuronal cell bodies of the cortices surrounding the neuropils of the brain and the thoracic ganglion. The fluorescent aggregates appeared to be almost exclusively localized to neuronal cell bodies, as evident by co-localization with staining by DAPI and the absence of anti-HA stain in synaptic neuropil regions. In plastic sections stained with toluidine blue, or in electron micrographs, no signs of neuronal degeneration were visible in either the brain or the thoracic ganglion, even in the last flies surviving up to 20 days. Death may be due to dysfunction of the neurons associated with poly(Q), rather than neurodegeneration, as has been in a reported mouse model for Huntington's disease and another for spinal and bulbar muscular atrophy (3,55).
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Expression of dmlf had a protective effect against 63Q on survival. At day 20, 55% of flies expressing 63Q along with dmlf were still active, while all of those expressing 63Q alone (or with lacZ) had also died (Fig. 6). Expression of dMLF alone from either one or two transgenes did not have any detectable effect on survival up to 20 days. Therefore, dmlf can protect against poly(Q) toxicity in neuronal tissues, as well as in the eye, supporting use of the eye as a convenient morphological index in screening for suppression of toxicity.
Examination of DREF in respect to poly(Q) toxicity and its suppression
In a recent publication, yeast two-hybrid screens uncovered dMLF as a protein binding to DREF, a transcription factor that regulates several genes involved in cell proliferation and DNA replication in Drosophila; the binding was verified by immunoprecipitation. Overexpression of dref in the eye caused a rough eye phenotype, with the loss of eye bristles. When dmlf was co-expressed with dref, the eyes were partially improved, indicated by partial restoration of the number and size of eye bristles, under scanning electron microscope (52). We therefore performed experiments to investigate a possible interaction in our system. In our hands, using light microscopy as in Figure 1, overexpression of dref caused severe eye degeneration, with loss of pigments. However, when flies with a UAS–dref transgene on the second chromosome and one or two UAS–dmlf transgenes on the third chromosome were crossed to GMR–GAL4 flies, in neither case was there any evident improvement in the pigmentation or the structure of the eyes in the progeny.
We also tested for possible modification of eye degeneration caused by 63Q by crossing the normal dref, or a dominant-negative form of dref (UAS–dref CR1) (56), or the zerknüllt gene (UAS–zen), a possible repressor of dref transcription and activity (57). UAS–dref, UAS–dref CR1 or UAS–zen were crossed to GMR–GAL4/UAS–63Q, and the progeny were examined as above. No detectable effect was observed (not shown).
Colocalization of dMLF with poly(Q) aggregates
To investigate the question of whether poly(Q) co-localizes with dMLF, through direct or indirect protein–protein interaction, we made transgenic flies to express dMLF with an N-terminal Flag epitope tag in the eye, using GMR–GAL4. Flies expressing dmlf–N-Flag alone, or together with 127Q, pros20Qtr or pros127Qtr, were sectioned and labeled with anti-Flag and anti-HA antibodies. In these experiments, 127Q was directly expressed from a GMR-like promoter with five GLASS response elements (5GR–127Q). The flies with this transgene had a slightly milder phenotype than did GMR–GAL4/UAS–127Q flies. In these flies, expressing dMLF–N-Flag produced a wider retina for better localization of poly(Q) aggregates and dmlf–N-Flag (Fig. 7A). As controls for subcellular localization and suppression effects, the cytoplasmic and nuclear forms of the lacZ gene product, ß-galactosidase, were expressed either alone or together with 127Q (Fig. 7B–E). Flag staining of flies carrying dmlf–N-Flag alone was relatively uniform throughout the retina (Fig. 7F, G and H). The same was true together with pros20Qtr (Fig. 7I, J and K). However, in the presence of 127Q or pros127Qtr transgenes, the staining was more punctuate, and many aggregates appeared positive for both anti-HA and anti-Flag antibodies (Fig. 7L–O). Using an anti-dMLF antibody (52), endogenously expressed dMLF was similarly localized to HA-positive aggregates in the retina (not shown).
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To better visualize the co-localization of dMLF and 127Q aggregates, confocal microscopy was performed using 5GR–127Q or another construct with one GLASS response element (1GR–127Q). In 1-day-old-adults, 1GR–127Q produces fewer 127Q aggregates with no morphologically detectable retinal degeneration. In flies co-expressing from UAS–lacZ (nuclear) and 1GR–127Q, confocal microscopy of the sections, double-stained with anti-ß-galactosidase and anti-HA antibodies, showed that all aggregates were contained within the nuclei, as indicated by the yellow signals and no green signals (Fig. 8A). The sections of the flies co-expressing from UAS–dmlf–N-Flag and 1GR–127Q, double-stained with anti-Flag and anti-HA antibodies, showed that, as in Figure 7, the anti-Flag antibody produces punctate staining, and most of them overlap with anti-HA signals, as indicated by the yellow signals (Fig. 8B). Furthermore, by scanning the entire diameter of these double-stained aggregates at 0.5 µm intervals and by examining each focal plane separately, it was clear that each punctuate yellow signal (overlapping Cy3 and FITC signals) was originating from a single aggregate. For comparison, confocal micrographs of retinas coexpressing from UAS–dmlf–N-Flag and 5GR–127Q were also included (Fig. 8C).
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These observations together indicate that dMLF is recruited into poly(Q) aggregates, either by directly interacting with poly(Q) fragments or through one or more intermediary proteins. This recruitment may play a part in the suppression mechanism.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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A dominant genetic screen resulted in the discovery that the dmlf gene suppresses the toxicity of abnormally expanded poly(Q)s. The suppression was effective for different poly(Q) lengths, different sizes of polypeptides containing the poly(Q) and different cell types in which they were expressed. These include 63Q and 127Q [the quasipure poly(Q)s], PROS127Qtr (composed of a 127Q tract within the 529 amino acids of the PROSPERO C terminus), and HttQ120 (composed of a 120Q tract within 262 amino acids of the N terminus of human huntingtin) (28). dmlf was effective in suppressing poly(Q) toxicity both in the eye and in neurons of the central (and peripheral) nervous system(s). When expressed alone, dmlf had little or no detectable detrimental effect.
Examination of the in vivo co-localization of dMLF and poly(Q) showed that endogenous and N-terminally Flag-tagged dMLF appear in the same aggregates with HA-labeled 127Q or PROS127Qtr. The transgenic dMLF might block various binding sites of aggregated poly(Q), preventing the recruitment of other cellular factors necessary for cell viability. This ‘sequestered poly(Q)’ hypothesis could also provide a possible mechanism for the suppression afforded by the chaperone Hsp70 (33) and the co-chaperone protein dHDJ1/Hsp40 (30,32). Overexpression of a single dose of dHDJ1 almost fully protected the eye morphology against 127Q toxicity, even though numerous aggregates were still present in the eye (30). Poly(Q) aggregates in transgenic flies expressing a truncated Machado–Joseph/SCA3 disease gene product, MJD/ataxin-3, with 78 glutamines, co-localized with a transgenic human Hsp70 (33), as well as a transgenic Flag-tagged dHDJ1 (32). Transgenic expression of Hsp70 or dHDJ1, or both together, increased the solubility of the aggregates, without having any detectable effect on their onset, size or number (32,33). Therefore, it is possible that suppression is not achieved merely through enhanced solubility of aggregates and with their removal by protein degradation machinery, but by sequestering them.
The sequestered poly(Q) hypothesis may also account for other observations. Ectopic expression of the transcriptional regulators CBP (10,11,13), TAF
130 (12) or Sp1 (58,59) could rescue the cells in culture, while other proteins are also affected by, interact with or are recruited by mutant poly(Q) proteins or their inclusions [e.g. the transcription factors TBP (60,61), p53 (13), CA150 (14), N-CoR (15), mSin3 (15) and CRX (62), as well as CBP, TAF
130 and Sp1]. These observations may be explained by the possibility that overexpression of any one of the factors would result in sequestration of soluble poly(Q) and/or its aggregates and saturation of its interacting sites, thus preventing the recruitment of other factors. In SBMA patients, some CBP-positive inclusions were negative for AR antibody, perhaps due to complete sequestration of the mutant AR aggregates by CBP (10). It is important to also consider the sequestration of soluble forms of poly(Q) peptides as the mediator of suppression versus that of their inclusions, because we were not able to detect the Drosophila homolog of CBP in the inclusions found in our fly model (not shown). Similarly, in a comprehensive set of experiments using three different HD mouse models, CBP could not be detected in the poly(Q)-dependent inclusions (63).
Based on this hypothesis, therapeutic peptides could be derived from such transcription factors that bind to and sequester poly(Q) peptides, thus suppressing their toxicity. Similarly, various segments of dMLF polypeptide could be examined for co-localization with poly(Q) aggregates and suppression of its toxicity and whether their interactions with poly(Q) are dependent on other factors. Such experiments could potentially provide biochemical targets or reagents for therapeutic intervention in poly(Q) disorders. Indeed, various peptides identified by phage display screen (64) or rational design (65) have been shown to be effective in reducing the toxicity of expanded poly(Q)s; though, in contrast to what has been observed with dMLF and dHDJ1 (30,32), reduction of poly(Q) toxicity by these peptides was associated with a delay or reduction in the formation of poly(Q) aggregates.
Presently, due to lack of any detectable genetic interaction between poly(Q) toxicity (and, for that matter, dmlf, as shown here) and the dref pathway, it is not possible to speculate on the role of the dref pathway in mediating poly(Q) toxicity or the suppression effect by dmlf. Nevertheless, it is intriguing that dMLF might be associated with a pathway involved in cell proliferation and DNA replication (52). The human MLF1 gene was originally identified as a portion of a chimeric product with the nucleolar transport protein nucleophosmin (NPM), in the chromosomal translocation t(3;5)(q25.1;q34), and is associated with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) (51). A murine homolog (HLS7) is associated with hematopoietic lineage switching from erythroid to myeloid (66). Remarkably, several other proteins associated with poly(Q) disease proteins are also involved in loss-of-function mutations or chromosomal translocations associated with various forms of cancer. For instance, inframe fusions of CBP and the leukemia-related transcription factors MORF (67), MLL (68,69) and MOZ (70), are associated with AML, and CBP associates physically with BRCA1 (71). Mutations of p53 are frequently associated with hematological malignancies (72). Huntingtin interacting protein 1 (HIP1) has been found as a chimeric gene with the platelet-derived growth factor ß receptor (PDGFßR) in a chromosomal translocation that causes chronic myelomonocytic leukemia (CMML) (73). These findings, and the lower incidence of cancer in individuals with HD (74), point to the involvement of common molecules and biochemical pathways in cancers and poly(Q) disorders.
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MATERIALS AND METHODS |
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Fly maintenance, transformation and handling
Flies were maintained on corn meal/agar/yeast at 25°C and 70% humidity. Microinjection solutions containing the transgenic constructs were composed of 13.5 µg transgenic vector, 4.5 µg p
25.1 transposase vector (75), 0.1 M sodium phosphate buffer (pH 7.8) and 5 mM potassium chloride, in 50 µl aqueous solution. Using Transjector 5246 and Femtotips II (Eppendorf), the transgenic constructs were microinjected into 5–30 min old w1118embryos reared at 18°C. Several transgenic lines were established for each construct. The UAS–lacZ trasgenic fly lines that were used as controls are as follows: (Fig. 1) cytoplasmic ß-galactosidase (ß-gal), genotype:w1118, P{w+mC=UAS–lacZ}; (Figs. 4–7) nuclear ß-gal, stock no. 3955, genotype w1118; P{w+mC=UAS–lacZ.NZ}20b; (Figs. 4–8) nuclear ß-gal, stock no. 3956, genotype w1118; P{w+mC=UAS–lacZ. NZ}J312 by Yash Hiromi; and (Fig. 7) cytoplasmic ß-gal, stock no. 1776, genotype w*; P{w+mC=UAS–lacZ.B}Bg4-1-2 by Norbert Perrimon. For survival test, the flies were housed at 20 per vial, using three to eight vials for each combination of transgenic inserts. Every 4 days, the flies were transferred to new vials and the dead flies were counted. These were defined by being completely motionless or unable to climb onto the vial wall.
Plasmid constructs
pUAS–63Q was constructed in the same manner as pUAS–20Q and pUAS–127Q as previously described (30). pUAS–pros20Qtr and pUAS–pros127Qtr produced by digesting full-length prospero constructs pUAS–pros20Q and pUAS–pros127Q (30) with PstI and BstEII, and replacing the PstI–BstEII fragment with an adapter fragment produced by annealing the oligonucleotides PstI–BstEII.adapterF 5'-G TAA TAA GCC GCC ACC ATG GGG-3' and PstI–BstEII.adapterR 5'-GTC ACC CCC ATG GTG GCG GCT TAT TAC TGC A-3' followed by its ligation into pUAS–pros20Q and pUAS–pros127Q plasmids. p5GR–127Q, containing five tandem GLASS response elements upstream of the 127Q sequence, was derived from pUAS–127Q and pGMR.1N (40) by removing the NheI–PstI fragment from pUAS–127Q, containing the five UAS and the minimal Hsp70 promoter, and replacing it with a NheI–PstI-digested PCR fragment amplified from pGMR.1N by the oligonucleotides GMR.PstIR 5'-AAA ACT GCA GTT AAA GGC ATT TCA AGG GTT TCC-3' and GMR.NheIF 5'-AAA AAA GCT AGC CCC AGT GGA AAC CCT TGA AAT G-3'. For p1GR-127Q, also derived from pUAS–127Q, containing a single GLASS response element upstream of the 127Q sequence, the NheI–PstI fragment from pUAS–127Q was replaced with an NheI–PstI fragment produced by annealing the oligonucleotides Glass1R.F 5'-CTA GCC CCA GTG GAA ACC CTT GAA ATG CCT TTA ACT GCA-3' and Glass1R.R 5'-GTT AAA GGC ATT TCA AGG GTT TCC ACT GGG G-3'. Subsequently, the intermediate p5GR and p1GR plasmids, containing five or one GLASS response elements, were digested with PstI and dephosphorylated by calf intestinal phosphatase (CIP; New England Biolabs, Inc.). Into this dephosphorylated PstI site was inserted a second NsiI–PstI-digested PCR-fragment, amplified from the Hsp70 minimal promoter region of pUAS–127Q (30), by the oligonucleotides Hsp70miniF 5'-CCA AAA TGC ATA GCG GAG ACT CTA GCG AGC GC-3' and pros11200R 5'-GGT AGT TTG TCC AAT TAT GTC ACA CCA-3'. This completed the p5GR and p1GR plasmid constructs. To produce p5GR–127Q or p1GR–127Q, the PstI–BamHI fragments of p5GR or p1GR were replaced by similar PstI-BamHI fragments from pUAS–127Q. PCR was performed in 200 µl thin-walled tube strips on a Stratagene Robocycler Gradient 96 Hot Top. The PCR reaction mixture contained 100 ng pGMR.1N or pUAS–127Q template, 50 pmol of each primer, 1x cloned Pfu buffer (Stratagene), 0.2 mM dNTP, 5% glycerol, 5% dimethylsulfoxide (DMSO) and 1.25 units cloned Pfu DNA polymerase (Stratagene) in a total volume of 62 µl aqueous solution. The thermal cycling parameters were an initial single cycle of denaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 65–80°C for 1 min, extension at 75°C for 1 min, and, finally, a single cycle of extension at 75°C for 10 min. For pUAS–dmlf, the PstI–XhoI fragments of GH20101 and LD47427 were cloned into the pINDY6–UAS transgenic vector after the removal of its PstI–XhoI fragment from its polyclonal site.
Immunofluorescence
Whole flies were submerged in Mirsky's fixative (National Diagnostics) for 1–2 min. They were then decapitated, and the heads were placed in OCT 4583 embedding medium (Sakura Finetek), frozen on dry ice and sectioned. Slides were dried on a 50°C hot plate for 30 s, then fixed in Mirsky's fixative for 30 min, followed by fixing in paraformaldehyde (4%) for 10 min at room temperature, and washed three times within 10 min, using PBS/Tween-20 (0.1%). The sections were blocked with 1% PBS/bovine serum albumin fraction V (Sigma), and then covered with 1 µg/ml of primary polyclonal Y-11 anti-HA (Santa Cruz Biotechnology, Inc.), primary monoclonal M5 anti-Flag (Sigma-Aldrich), primary monoclonal D19-2F3-2 anti-ß-gal (Roche Molecular), primary monoclonal anti-ELAV 7D4E6 (Dr Kalpana White), primary polyclonal anti-dCBP (Dr Sarah Smolik) or primary monoclonal anti-dMLF (Dr Masamitsu Yamaguchi) antibody in the block solution for 2 h at room temperature. They were washed 3x5 min with PBS/Tween-20 (0.1%), covered with 4 µg/ml of secondary FITC-labeled anti-rabbit or Cy3-labeled anti-mouse antibody (Jackson ImmunoResearch Laboratories) in the block solution for 1 h at room temperature, washed for 5 min with PBS/Tween-20 (0.1%), covered with 0.5 µg/ml 4', 6'-diamino-2-phenylindole (DAPI) for 1 min, and washed 3x15 min with the PBS/Tween-20. Finally, the sections were mounted in 0.1 mg/ml phenylenediamine (PDA)/0.5 µg/ml DAPI/90% glycerol and viewed on a Zeiss Axioplan fluorescent microscope and photographed by a SPOT-RT CCD digital camera using SPOT 3.0.6 AppleEvent software. Confocal micrographs were produced using an inverted Olympus IX70 microscope and Fluoview software. Photoshop 6.0.1 and Canvas 7.0 softwares were used to generate the figures.
Plastic tangential sections of the ommatidia of the retina
The proboscises were cut off and whole fly heads were fixed in paraformaldehyde (1%) and glutaraldehyde (1%) EM fixative overnight at 4°C. The heads were then rinsed with 0.1 M phosphate buffer for 0.5 h and post-fixed with 1% osmium tetroxide for 1–2 h. They were then rinsed with double-distilled water for 10 min and dehydrated once in 50%, 70%, 85%, and 95% ethanol, for 5 min each, twice in 100% ethanol for 10 min, and twice in 100% propylene oxide for 7 min. This was followed by infiltrating the heads with 1:1 ratio of propylene oxide: EM Bed-812 mixture (EM Bed-812 120 g, Epon 826 60 g, Epon 871 20 g, softener Dodecenylsuccinic anhydride 194 g, hardener Nadic methyl anhydride, mixed for 1 h and then in pure EM Bed-812 mixture with N,N-benzyldimethylamine (BDMA) for 3-4 hrs. The heads were embedded in pure EM Bed-812 mixture in EM embedding mold and the matrix was polymerized at 60°C overnight. The blocks containing the heads were sectioned at 1.2 µm on Sorval MT2-B Porter Blum ultra-microtome. The sections were stained with toluidine blue (1% toluidine blue and 1% borax in H2O) and mounted in Permount (Fisher Scientific). The slides were viewed on a Zeiss Axioplan microscope, using phase contrast filter and 100X oil-immersion objective, and photographed by SPOT-RT CCD digital camera using SPOT 3.0.6 AppleEvent software.
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ACKNOWLEDGEMENTS |
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We thank Dr Chris Q. Doe for the gift of prospero cDNA clone p139cAC1, Dr Kalpana White for Appl-GAL4 driver lines and anti-ELAV 7D4E6 monoclonal antibody, Drs Masamitsu Yamaguchi and Fumiko Hirose for anti-dMLF monoclonal antibody, UAS-dref, UAS-dref CR1 and UAS-zen, Dr Sarah Smolik for chicken anti-dCBP polyclonal antibody, Dr George Jackson for HttQ120 transgenic mutant HD fly line, Dr Laurent Seroude for the transgenic vector pINDY6, and Viveca Sapin, Rosalind Young, Amparo Gomez and Ekaterina Passetchnik for invaluable technical support. P.K.-E. was supported by fellowships from Cure HD Initiative/Hereditary Disease Foundation, and grants from The Wills Foundation, NINDS/NIH (NS42162), and Howard Hughes Medical Institute Biomedical Research Support Program (53000261), and grants to S.B. from the NSF, the NIH and the James G. Boswell Foundation.
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FOOTNOTES |
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* To whom correspondence should be addressed: Tel: +1 7168293290; Fax: +1 7168293050; Email: pkazemi{at}acsu.buffalo.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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