Huntington disease arises from a combinatory toxicity of polyglutamine and copper binding

Significance

The etiology of Huntington disease (HD), a progressive neurodegenerative disorder caused by polyglutamine (polyQ) expansion in huntingtin, is not well clarified. Here using a Drosophila model of HD, we show that altered expression of genes involved in copper metabolism can significantly modulate the HD progression. Dietary copper intervention also modifies HD phenotypes in the fly. Copper reduction dramatically decreases the level of toxic huntingtin levels. Strikingly, substitution of two potential copper-binding residues of huntingtin completely dissociates the copper-intensifying toxicity of huntingtin. Our results therefore indicate huntingtin entails both the copper-facilitated toxicity as conferred by direct copper binding and the copper-independent polyQ toxicity and suggest that an ideal HD therapy would need to take both of these actions into consideration.

Abstract

Huntington disease (HD) is a progressive neurodegenerative disorder caused by dominant polyglutamine (polyQ) expansion within the N terminus of huntingtin (Htt) protein. Abnormal metal accumulation in the striatum of HD patients has been reported for many years, but a causative relationship has not yet been established. Furthermore, if metal is indeed involved in HD, the underlying mechanism needs to be explored. Here using a Drosophila model of HD, wherein Htt exon1 with expanded polyQ (Htt exon1-polyQ) is introduced, we show that altered expression of genes involved in copper metabolism significantly modulates the HD progression. Intervention of dietary copper levels also modifies HD phenotypes in the fly. Copper reduction to a large extent decreases the level of oligomerized and aggregated Htt. Strikingly, substitution of two potential copper-binding residues of Htt, Met8 and His82, completely dissociates the copper-intensifying toxicity of Htt exon1-polyQ. Our results therefore indicate HD entails two levels of toxicity: the copper-facilitated protein aggregation as conferred by a direct copper binding in the exon1 and the copper-independent polyQ toxicity. The existence of these two parallel pathways converging into Htt toxicity also suggests that an ideal HD therapy would be a multipronged approach that takes both these actions into consideration.

Huntington disease (HD) is a neurodegenerative disorder caused by expansion of polyglutamine (polyQ) repeats within the functionally enigmatic huntingtin (Htt) protein (1). The disease is characterized by movement disorder, psychiatric symptoms, and cognitive dysfunction. Previous research suggested that the N-terminal fragment of Htt mediates HD progression (2, 3). Human Htt exon1 with expanded polyQ repeats have been shown to form aggregates in vitro and in vivo (3), a hallmark of this disease. Transgenic flies of human Htt exon1 with expanded polyQ repeats have been shown to manifest phenotypes that mimic many features of HD (reviewed in ref. 4). For example, transgenic flies expressing human Htt exon1 with 93 glutamines (hereafter referred as P463) showed reduced survival rate and lifespan driven by the pan-neuronal driver Elav-Gal4 and a progressive loss of pigment cells and rhabdomeres when driven by GMR-Gal4 (2). Htt-polyQ aggregation in vivo was also observed in transgenic flies expressing EGFP-tagged human Htt exon1 with 103 glutamines (hereafter referred as HttQ103-EGFP) (5). These fly models have been used in various forward genetics studies, such as identifying genetic modifiers (5) or therapeutic targets of HD (2, 6). Nevertheless, etiology of HD remains incompletely understood, and effective treatments to slow down or to stop the disease are lacking.

Metal accumulation is often found in the pathologically affected regions of many neurodegenerative diseases (7, 8). However, to a large extent whether this is the causative event is not well established. Several disease-relevant proteins or polypeptides such as Aβ, tau, and prion have even been shown directly interacting with several kinds of metal ions in vitro (8, 9), although whether this happens in vivo or what the physiological significance is of this interaction is mostly a mystery. Several previous observations implicated disruption of metal homeostasis in the progression of HD, but again their precise relevance to disease pathogenesis remains unclear. For example, manganese dyshomeostasis in HD cell models and HD mouse models has been identified (10). Many studies offered evidence that iron may be an important contributor to HD progression (11). Besides iron, the copper level is also increased in the CNS of human HD brains, HD mouse models, and a rat HD model (12–14). It was reported that in vitro copper could significantly accelerate the fibrillation and aggregation of purified recombinant Htt exon1 with polyQ tract (15), and the fragment containing the first 171 amino acids of human wild-type Htt and its glutamine-expanded mutant form directly interact with copper (13). Clioquinol (CQ), an antibiotic with divalent metal ion-binding ability and whose action mechanism is still controversial (16, 17), improved cell survival and behavioral and pathologic phenotypes in human Htt transgenic mice (18). It is obvious that diverse metals have been reported associated or linked with HD; however, more conclusive and specific genetic evidence, particularly in the context of animal nervous systems, are largely absent.

In Drosophila melanogaster, copper uptake and efflux are mediated by Copper transporter 1 (Ctr1A/B/C) and Drosophila melanogaster ATP7 (DmATP7) (19), respectively. Ctr1A/B/C are the three Drosophila homologs of human Ctr1, which localizes to the cell membrane and plays major roles in cellular copper uptake (20). DmATP7 is the only homolog of human ATP7A/ATP7B in Drosophila, and DmATP7 localizes to the Golgi apparatus and plays a key role in cellular copper efflux, which is essential for dietary copper absorption (21) and copper uptake in the neurodevelopment of D. melanogaster (22).

In this study, we demonstrated that Copper transporter 1B (Ctr1B) and DmATP7 participate in copper homeostasis in the brains of D. melanogaster. Using Drosophila models of HD, we found that altered expression of Ctr1B or DmATP7 in the brain, or modulation of dietary copper availability, significantly modified the phenotypes caused by human Htt exon1-polyQ expression. We also showed that substitution of two potential copper-binding residues of Htt Met8 and His82 eliminated the copper-modulating effect on Htt exon1-polyQ toxicity. Our study thus provides unique insights into understanding how copper participates in HD progression and suggests HD is not only a polyQ disease but also affected by copper.

Results

Identification of Copper Transporter Ctr1B as a Genetically Interacting Gene in Drosophila HD.

Metal accumulation has been reported in a number of neurodegenerative diseases (7, 8). We initiated a genetic screen trying to identify metal (copper, iron, and zinc) genes likely linked to HD in a Drosophila model (Table S1). Facilitated by the binary Gal4-UAS (upstream activation sequence) system, the Drosophila HD model was generated with expression in the Central Nervous System (CNS) (directed by the pan-neuronal Elav-Gal4) of Htt exon1 with an expanded polyQ (Htt exon1 Q93 and referred to as the P463 model) (2). These flies display reduced survival (eclosion), which was used as our initial screening parameter for suppressors or enhancers. We isolated Ctr1B RNAi as a potent suppressor of the fly HD model (Table S1). Neuronal Ctr1B RNAi rescued the survival rate of HD flies from 37 to 77% (Fig. 1A).

Fig. 1.

Ctr1B and DmATP7 modulation affects brain copper homeostasis and the eclosion of HD flies. (A) The reduced survival rate of HD flies (Elav > P463) can be modified by altering the expression of copper transporters. (B) Ctr1B and DmATP7 expression modulation in Drosophila brains. Adult heads of Elav >Ctr1B-RNAi, DmATP7-OE, or -RNAi flies were analyzed for mRNA abundance using real-time q-PCR, with RP49 as the internal control. (C) Effects of Ctr1B and DmATP7 expression modulation on MtnA and MtnB transcript levels. (D) Copper content in Elav >Ctr1B-RNAi and DmATP7-OE or -RNAi heads. Pan-neural Ctr1B RNAi or DmATP7 OE showed a significant alteration in head copper content. All values in A–D are presented as mean ± SEM; n ≥ 3. *P < 0.05, **P < 0.01, ***P < 0.001, 2-tailed Student t test. OE, overexpression.

Because Ctr1B has been shown to be a copper transporter in the fly (19, 23), we reasoned that other copper transporters might also interact with the HD model. In addition to Ctr1B, the Drosophila genome encodes three other copper transporters including copper transporter1A (Ctr1A), Copper transporter 1C (Ctr1C), and DmATP7. Ctr1A and Ctr1C are two additional Ctr1 family members that mediate copper import in Drosophila, and DmATP7 plays a role in exporting copper to the Golgi and copper efflux (21, 23). The embryonic lethality of neuronal Ctr1A knockdown precluded analysis of the role of Ctr1A in HD, and neuronal Ctr1C RNAi showed no effects on the phenotypes of the HD model, including survival, lifespan, eye degeneration, and protein aggregation, likely due to its low/absent expression in the brain (FlyAtlas). Neuronal overexpression of DmATP7 [DmATP7 overexpression (OE)] significantly suppressed the toxicity of HD in Drosophila, whereas DmATP7 RNAi exacerbated the lethality (Fig. 1A). DmATP7 OE increased the eclosion rate of HD flies from 37 to 73%, whereas DmATP7 RNAi reduced it to 15%. These results from another copper transporter reassured a role of copper homeostasis in HD.

As a confirmation of the effects of Ctr1B and DmATP7 on copper levels in the fly brain, we examined changes in the levels of metallothioneins when modulating expressions of these copper transporters. Metallothioneins (Mtn), as metal detoxifiers in the cells (24), are transcriptionally induced by copper and are considered sensitive indicators of intracellular copper levels (19, 24). Ctr1B-RNAi and DmATP7-OE flies showed a significant reduction of both MtnA and MtnB, whereas DmATP7-RNAi flies displayed increased levels of MtnA expression (Fig. 1 B–C). We further performed ICP-MS (inductively coupled plasma–mass spectrometry) to quantitate copper content and, as expected, showed that the fly-head copper levels were altered by expression modulation of the copper transporters (Fig. 1D). Notably, forcible overexpression of Ctr1B by itself results in early lethality when driven by Elav-Gal4 and rough eye when driven by GMR-Gal4 (19), precluding this aspect of analysis in the HD model.

Consistent with mouse and clinical findings (12, 13), HD flies by themselves exhibited somewhat elevated copper levels (Fig.S1E). This was accompanied with increased native expression of both Ctr1B and DmATP7 (Fig. S1 A–D), indicating a copper dyshomeostasis occurred in HD flies.

Taken together, Ctr1B RNAi and DmATP7 OE reduce the copper level in the brain, and, as a result, confer beneficial effects on HD flies, whereas copper elevation augments the toxicity of HD.

Expression Alterations of Copper Transporters Ameliorate a Spectrum of Phenotypes Associated with the Drosophila HD Model.

We subsequently examined the effects of copper transporters on the other phenotypes associated with Drosophila HD, including reduced eclosion rate, shortened lifespan, impaired mobility, and progressive degeneration of eyes and brain tissues (2, 25). Young (9 d) P463 flies present mobility impairment, which serves as an exquisitely sensitive readout of neuronal dysfunction and death (26). When raised at 25 °C for 9 d, about 40% HD flies reached the subjectively designated height within 7 s (Fig. 2A). Copper reduction significantly rescued, whereas copper accumulation exacerbated, the mobility impairment in HD flies. In contrast to our expectations, DmATP7 OE failed to rescue the mobility impairment but instead enhanced it. It turned out that unlike Ctr1B RNAi and DmATP7 RNAi, DmATP7 OE on its own resulted in a serious mobility defect (Fig. 2B), which explains why DmATP7 OE augments the mobility defect of P463.

Fig. 2.

Modulation of copper transporters alters a spectrum of phenotypes associated with HD flies. (A) The mobility defect of HD flies (Elav > P463) can be modified by adjusting copper transporter expressions. Values are presented as mean ± SEM; n ≥ 3. ***P < 0.001, **P < 0.01. (B) Elav > DmATP7 OE control flies exhibited obvious mobility defect on its own, explaining its failure in rescue. Values are presented as mean ± SEM; n ≥ 3. ***P < 0.001. (C) Effect of copper transporters on HD lifespan. (D) Effect of copper transporters on the eyes of HD flies. Eyes of GMR > P463 flies displayed leukasmus due to neurodegeneration. Loss of pigmentation Tin GMR > P463 eyes was markedly rescued by Ctr1B RNAi or DmATP7 OE, while enhanced by DmATP7 RNAi. (E) Rescue of the progressive loss of rhabdomeres in HD flies by modulating copper transporter expression. P463 expression in the eye was driven by GMR-Gal4. (F) A quantitative measurement of E. OE, overexpression.

P463 flies exhibit early adult death and reduced lifespan at 25 °C (Fig. 2C). We analyzed the effects of Ctr1B and DmATP7 on P463 lifespan. Consistent with the results described above, Ctr1B RNAi and DmATP7 OE extended the HD longevity, whereas DmATP7 RNAi shortened it (Fig. 2C). Ctr1B RNAi exhibited a stronger rescuing effect, with 133 and 75% increase, respectively, for the medium and the maximum lifespans of P463 flies raised at 25 °C. DmATP7 OE also exhibited a dramatic rescuing effect. DmATP7 RNAi, on the other hand, drove a significant reduction for the medium and maximum lifespans of P463 flies (Fig. 2C).

Drosophila compound eyes consist of regular arrangements of lens-like ommatidia (2). Expression of Htt exon1 Q93 within the eye using the eye driver (GMR-Gal4) leads to the death of both pigment cells (27) and rhabdomeres (2). These phenotypes of HD were rescued by Ctr1B RNAi and DmATP7 OE but enhanced by DmATP7 RNAi (Fig. 2D). Semithin resin sections showed that rather than the normal seven visible rhabdomeres per ommatidium, the average number of visible rhabdomeres in HD flies declined to ∼4.8, which was markedly improved by Ctr1B RNAi or DmATP7 OE to ∼5.8 and ∼5.7, respectively (Fig. 2E). The numbers of rhabdomeres for each genotype were quantified and are shown in Fig. 2F. Notably, Ctr1B OE and DmATP7 RNAi so much aggravated the eye degeneration of P463 flies that it was difficult to quantify the number of rhabdomeres (Fig. 2E).

Taken together, these results indicate that modulating Ctr1B or DmATP7 expression in the Drosophila brain or the eye modified a whole range of symptoms of HD examined.

Dietary Copper Intervention Mimics the Genetic Modulation in Affecting HD Progression.

As an alternative to examine the role of copper homeostasis in HD progression, we tested whether dietary copper intervention with copper supplementation or depletion could simulate the rescue effect of genetic modulation of copper transporters on HD flies. As a control, the concentrations of copper/copper chelators we used had no effect on the survival rate of wild-type flies (Fig. 3A). Consistently, the copper chelator bathocuproine disulfonate (BCS) or CQ significantly rescued the survival rate of P463, whereas supplementation of additional CuCl₂ worsened the survival defect (Fig. 3B). This result indicates that dietary intervention of copper uptake with copper chelators alleviates the progression of HD flies and further strengthens the notion that copper is involved in the pathogenesis of HD in vivo.

Fig. 3.

Dietary copper intervention modifies HD progression in Drosophila. (A) Copper or copper chelators supplemented in the food had no effect on the survival rate of wild-type Drosophila. (B) The survival rate of HD flies (Elav > P463) was significantly increased by dietary copper chelation and reduced by excessive copper. (C) The genetic and dietary copper intervention had additive benefits on the eclosion rate of HD flies. Values in B and C are presented as mean ± SEM; n ≥ 3. *P < 0.05, ***P < 0.001, 2-tailed Student t test. OE, overexpression.

We wondered whether combining genetic manipulation and diet intervention could exert a stronger rescue effect on HD. P463 flies with altered copper transporter expression levels were raised on media with reduced or elevated copper levels. Significantly, dietary intervention of copper uptake with copper chelators further improved the rescue effect of the Ctr1B RNAi to a survival rate of about 100% (Fig. 3C).

Decreasing Copper Content in Brains Reduces Htt–polyQ Aggregation in Drosophila.

The aforementioned experiments showed that modulations of copper in the brain markedly altered the course of HD-associated phenotypes in Drosophila. To further identify the mechanism underlining copper’s effect on HD progress, the accumulation of Htt proteins was examined. Accumulation of abnormal structural forms of Htt with expanded polyglutamine repeats is a hallmark of Huntington disease progression (28). To facilitate the visualization of Htt protein aggregates, another Drosophila HD model, HttQ103-EGFP, was used. In this model, the Htt exon1 protein with polyQ was tagged with a C-terminal EGFP and therefore could be easily detected as fluorescent signals (5). When driven by Elav-Gal4, HttQ103-EGFP displayed an age-dependent pattern of aggregate formation. However, Ctr1B RNAi and DmATP7 OE suppressed the protein aggregation, whereas DmATP7 RNAi exerted an opposite effect (Fig. 4A). Compared with Elav-Gal4>HttQ103-EGFP flies, only 20 or 40% as many aggregates were formed in Elav-Gal4>HttQ103-EGFP/Ctr1B-RNAi or Elav-Gal4> HttQ103-EGFPT/DmATP7-OE flies, whereas aggregates formed in Elav-Gal4 > HttQ103-EGFP/DmATP7-RNAi were increased to 160% of the control (Fig. 4B). These results indicate that excess copper in brains increases, whereas reduced copper level decreases, HttQ103-EGFP accumulation. A filter retardation assay combined with antibody staining further confirmed this conclusion (Fig. 4E). Much reduced and enhanced pellet signals were respectively observed after Ctr1B and DmATP7 RNA interference. This copper effect is not achieved through affecting Htt exon1-polyQ mRNA levels, which remained largely unaltered by the expression modulation of copper transporters (Fig. 4F).

Fig. 4.

Copper reduction reduces Htt exon1-polyQ aggregation in the brain. (A) Ctr1B knockdown or DmATP7 OE decreased, while DmATP7 RNAi increased, the aggregation of Htt exon1-polyQ. b is a magnified view of the regions highlighted in a. Detection of aggregation was facilitated by fluorescence. (B) A quantitative measurement of A. Values are presented as mean ± SEM; n ≥ 3. *P < 0.05, **P < 0.01. (C) Ctr1B knockdown or DmATP7 OE decreased, while DmATP7 RNAi increased, the low aggregated form of Htt exon1-polyQ. (D) A quantitative measurement of C. Values are presented as mean ± SEM; n ≥ 3. *P < 0.05, **P < 0.01. (E) Ctr1B knockdown or DmATP7 OE decreased, while DmATP7 RNAi increased, the aggregation of Htt exon1-polyQ. The aggregation status was analyzed by filter retardation assay. A polyQ antibody was used for the hybridization. (F) Htt exon1-polyQ transcript levels were not affected by expression changes of copper transporters. OE, overexpression.

Several previous publications indicated that the N-terminal fragment of Htt-polyQ is an important factor that drives HD progression (2, 3). Meanwhile, there is evidence that soluble oligomers may be particularly important for the development of HD and other neurodegenerative diseases (29). We therefore investigated how SDS-soluble Htt exon1-polyQ was affected by copper in HttQ103-EGFP flies. Western blotting of whole fly brain lysates showed that the SDS-soluble Htt exon1-polyQ was dramatically decreased in Ctr1B-RNAi or DmATP7-OE flies and increased in DmATP7-RNAi flies (Fig. 4 C–D). We conclude that copper reduction in the Drosophila brain decreases the aggregated as well as the SDS-soluble form of Htt exon1-polyQ, and vice versa.

Copper Effect on HD is Mediated by its Direct Binding to Htt Exon 1.

In a parallel screen involving the polyQ Drosophila model expressing 99 glutamines (30) under the control of GMR-Gal4, no suppressors (including Ctr1B) were isolated (Table S1). Indeed, Ctr1B RNAi and DmATP7 OE failed to rescue polyQ flies (Fig. S2B). This prompted us to investigate the interaction between Htt exon1 and copper. In line with this thinking, it has been reported that copper directly binds the first 171 amino acids of Htt (N171) in vitro (13). Both His82 and His98 residues of rodent Htt appear essential for the interaction. Nevertheless, human exon-1 (17Q) Htt (N84), which lacks His98, interacts with copper (II) as well, suggesting it has a conformation and mode of interaction with copper different from N171 (13).

Met, His, and Cys are residues commonly found in proteins involved in copper interaction. Careful examination of human Htt exon-1 sequence identified two potential copper coordinating residues besides the initiation codon, Met8 and His82 (Fig. 5A). We thus generated a mutant Htt exon1 polyQ (127Q) transgenic fly (hereafter referred to as Htt exon1*-polyQ) with substitutions of M8V and H82A to verify the role of these two residues in vivo.

Fig. 5.

Disabling copper binding of Htt exon1-polyQ markedly mitigates its toxicity. (A) The Htt exon1 and potential copper-binding residues. Htt exon1 contains three potential copper (II) coordinating residues, Met1, Met8, and His82. The Htt exon1*-polyQ is with M8V and H82A substitutions. (B) Transcript levels of the Htt exon1*-polyQ transgenic line and that of the control P463 used for this study. (C) Removing copper binding by amino acid replacements of Htt exon1-polyQ increased the eclosion rate to almost 100%. Assayed at 25 °C. Values are presented as mean ± SEM; n ≥ 3. ***P < 0.001. (D) The lifespan of Htt exon1-polyQ was greatly extended by removing copper binding when raised normally. Elav > Htt exon1*-polyQ flies and the P463 control were assayed at 25 °C. (E) The mobility of Elav > Htt exon1*-polyQ flies was also improved over P463. Assayed at 29 °C. Values are presented as mean ± SEM; n ≥ 3. ***P < 0.001. (F) Removing copper binding rescued the pigment loss as observed in Htt exon1-polyQ. The pigment loss of GMR > Htt exon1*-polyQ flies is much less than that of P463. Assayed at 29 °C. (G) Removing copper binding increased the lifespan of Htt exon1-polyQ raised on an elevated temperature. Elav > Htt exon1*-polyQ flies and the P463 control were assayed at 29 °C. OE, overexpression.

Expression of Htt exon1*-polyQ in Drosophila CNS revealed much ameliorated symptoms in loss of pigment of eyes, mobility impairments, early adult death, and protein aggregation in the brain. Although severities correlate with expression levels, even when the mutant gene is expressed at a higher level than the normal Htt exon1-polyQ, its toxicity is still significantly milder. Fig. 5 shows a Htt exon1*-polyQ line, a typical example, presented no eclosion defect (Fig. 5C). For a comparison, the eclosion rate was only ∼40% (Fig. 5C) in even less-expressed (based on RT-PCR, Fig. 5B) normal HD flies. The Htt exon1*-polyQ flies exhibited little or very minor defect in eclosion, mobility, or lifespan when raised normally (25 °C), whereas normal HD flies displayed severe defects (Fig. 5 C–D). To enhance the potential abnormality of the Htt exon1*-polyQ flies we assayed the phenotypes of these flies on an elevated temperature (29 °C). Under this condition the Htt exon1*-polyQ flies manifested much more noticeable defects, although still significantly milder than the control Htt exon1-polyQ flies. At 29 °C, as compared with that of the control, besides the improvement of the mobility defect (Fig. 5E) and eye degeneration (Fig. 5F), the Htt exon1*-polyQ flies lived up to about 3 wk, whereas normal HD flies lived for only ∼2 wk (Fig. 5G).

We subsequently tested the effects of Ctr1B and DmATP7 RNAi or overexpression on the phenotypes of the Htt exon1*-polyQ flies. Remarkably, altered copper levels in brains no longer modified the phenotypes of the mutant HD at all, including pigment loss in the eye (Fig. 6A), mobility impairments (Fig. 6B), early adult death (Fig. 6C), and aggregation of proteins in the brain (Fig. 6 D–E), suggesting that copper interacts directly with Htt exon1 to modulate the accumulation of Htt exon1-polyQ in vivo.

Fig. 6.

Substitution of the two potential copper-binding residues in Htt exon1 dissipates the copper-intensifying toxicity of Htt. (A) The eye toxicity as a result of Htt exon1*-polyQ expression was little affected by expression changes of copper transporters. (B) The mobility defect in the Htt exon1*-polyQ flies was not affected by altering the brain copper homeostasis. Values are presented as mean ± SEM; n ≥ 3. ***P < 0.001. (C and D) The lifespan of C and extent of protein aggregation in (D) the Htt exon1*-polyQ flies was no longer copper-dependent. The aggregation status was measured by TS staining. (E) The extent of protein aggregation as measured by filter retardation assay. Not much difference was observed, compared with Fig. 4E. (F) A model to explain copper’s effect on HD. OE, overexpression.

It is known that both copper and iron are strong inducers of reactive oxygen species (ROS). Previously it was shown that oxidation of Htt through cysteine can promote its oligomerization (31). Our Htt exon1-polyQ, however, lacks cysteine. To analyze in general ROS involvement in our observed phenotypes, we measured the levels of ROS in HD flies and those with altered expression of copper transporters (Fig. S3). Indeed, HD flies had higher ROS, and modulating copper levels by Ctr1B or DmATP7 also led to ROS-level change. One question is then what role oxidative stress plays in HD pathogenesis. Because in flies carrying mutant Htt-polyQ, ROS increase was also observed after the copper level was elevated by Ctr1B or DmATP7 expression change, we argue that if ROS would mediate the copper effect on HD, mutant Htt-polyQ phenotype would also likely worsen when ROS was accumulated. Our data nevertheless show that although copper brought in more ROS, the copper effect on the mutant Htt-polyQ was totally dissipated. The ROS theory therefore could not well explain the copper effect as observed. We cannot, however, exclude an auxiliary role of ROS in our case.

Discussion

Copper is essential for normal CNS development and function. However, excess copper also possesses toxicity (32); e.g., it has been established that brain copper accumulation is associated with neurodegeneration (33). Previous studies suggested that there might be a close relationship between copper and the progress of Huntington disease (13–15). In this study, we provided in vivo genetic evidence that manipulating copper transporters such as Ctr1B or DmATP7 in brains can greatly modulate HD progress.

In our screening for HD interacting genes, some overexpression and RNAi lines had severe defects on their own when driven by Elav-Gal4, so it was difficult to evaluate accurately the HD rescuing effect under these circumstances. In the case of DmATP7, for example, some strong lines were lethal when directed by Elav-Gal4, and we had to resort to some weaker lines (such as the EY line Bloomington #16866, a p-insertional line) to obtain good rescue. Even these less robust lines had some deleterious effects such as mobility defect. One possibility is that in DmATP7 OE flies, copper may accumulate in the Golgi or the secretion pathway, affecting movement ability. This happened regardless of HD or normal fly background. This explains why DmATP7 OE in HD still manifested mobility defect. Additionally, this experience shows the subtlety of fine-tuning these genes without greatly affecting the normal functions of the body and suggests an optimal screening of this kind should include both stronger and weaker lines and assays for two or more aspects of rescue, when applicable.

Rescue data from dietary copper intervention largely coincided well with those through genetic interventions. Combining Ctr1B RNAi and dietary copper restriction, HD survival could be further rescued to near 100%. Some inconsistencies also arose. Copper addition did not worsen the survival of Ctr1B-RNAi/P463 flies. We explain this by that in Ctr1B-RNAi flies there might not be sufficient copper transporter to mediate the extra copper uptake. In other words, in Ctr1B-RNAi flies the limiting factor for intracellular copper is not external copper level, but the residual copper transporter that is still expressed after RNA interference. In DmATP7-RNAi/P463 flies, further copper chelation through diet control did not aid the survival. We are not sure what happened in this scenario. One possibility that cannot be excluded is that it is likely a secondary result from too much copper reduction in the secretion pathway when combining DmATP7 RNAi and copper chelation.

Besides Huntington disease, there are a few other polyQ-related disorders known to date such as spinal and bulbar muscular atrophy (SBMA), six types of spinocerebellar ataxia (SCA1, 2, 3, 6, 7, and 17), dentatorubral-pallidoluysian atrophy (DRPLA), and Machado Joseph disease (MJD or SCA3). Many aspects of the genetics and molecular biology are common to these diseases. Whether these disorders share the same pathogenic mechanism linking to polyQ has caused considerable controversy (reviewed in ref. 34). Although we found copper modulates the disease progression of HD flies, altering the expression of copper transporters fails to affect the phenotypes of polyQ flies, including the survival and eye phenotypes. This implies that the interaction of copper with huntingtin protein is relatively specific to HD but may not be so to other polyQ diseases. Although a direct comparison of metal effects on HD flies and polyQ flies would be better performed if both using the same Elav-Gal4 driver (instead of Elav-Gal4 for HD but GMR-Gal4 for polyQ as shown in Table S1), the polyQ line we have obtained happens to be a fairly strong one and ecloses less than 5% when driven by Elav-Gal4. Nevertheless, we have the opposite set of data with Htt-polyQ under GMR-Gal4. Data from GMR-gal4 > P463 (Fig. 2D) and GMR-gal4 > Htt exon1*-polyQ (Fig. 6A) demonstrated that copper transporters can modulate the severity of HD disease progression but not that of the Htt exon1*-polyQ, nor the normal polyQ (Table S1), indicating copper does not work through polyQ.

Intracellular copper reduction resulted in a lower overall Htt exon1-polyQ level. We suggest that copper’s binding to Htt exon1-polyQ induces the oligomerization of the protein, translating to more fibril deposits. It is possible that, compared with the monomer, the oligomer and the aggregated Htt are more stable or less vulnerable to clearance, leading to increased Htt exon1-polyQ level. An alternative to explain the decreased Htt exon1-polyQ level in copper removal is that copper binding may make the Htt exon1-polyQ itself more stable. A model to explain the copper effect on Htt is shown in Fig. 6F.

In summary, we have demonstrated the modulating effect of copper in a Drosophila HD model. Through either genetic manipulation of Ctr1B or DmATP7 expression in the brain or regulating the dietary copper intake, we can modify the pathological progress of HD. Copper affects HD pathology through binding directly with Htt exon1 to affect its fibrillation. Elimination of this binding removes the copper effect on HD. These results indicate HD pathology entails a copper-modulated effect on top of its polyQ toxicity and suggest that these two distinct pathways should both be considered when treating HD.

Materials and Methods

All flies were raised on standard cornmeal media at 25 °C unless stated otherwise. The details of these flies are described in SI Materials and Methods.

Details of the molecular biology, western blot, analysis of brain Htt aggregates, metal content assay, metal stress assays, eclosion, mobility and longevity assays, eye degeneration analysis, and ROS measurement procedures can be found in SI Materials and Methods.