Genetic Inhibition of Solute-Linked Carrier 39 Family Transporter 1 Ameliorates Aβ Pathology in a Drosophila Model of Alzheimer's Disease

The aggregation or oligomerization of amyloid-β (Aβ) peptide is thought to be the primary causative event in the pathogenesis of Alzheimer's disease (AD). Considerable in vitro evidence indicates that the aggregation/oligomerization of Aβ is promoted in the presence of Zn; however, the functional role of Zn in AD pathogenesis is still not well clarified in vivo. Zn is imported into the brain mainly through the solute-linked carrier (Slc) 39 family transporters. Using a genetically tractable Drosophila model, we found that the expression of dZip1, the orthologue of human Slc39 family transporter hZip1 in Drosophila, was altered in the brains of Aβ42-expressing flies, and Zn homeostasis could be modulated by forcible dZip1 expression changes. An array of phenotypes associated with Aβ expression could be modified by altering dZip1 expression. Importantly, Aβ42 fibril deposits as well as its SDS-soluble form were dramatically reduced upon dZip1 inhibition, resulting in less neurodegeneration, significantly improved cognitive performance, and prolonged lifespan of the Aβ42-transgenic flies. These findings suggest that zinc contributes significantly to the Aβ pathology, and manipulation of zinc transporters in AD brains may provide a novel therapeutic strategy.


Introduction
Alzheimer's disease (AD) is a major neurodegenerative disease affecting the elderly. The accumulation of amyloid-b (Ab) peptides, which either form the major component of senile plaques (SP) or the oligomer state in patient brains, is hypothesized to be the primary causative event in AD pathogenesis [1][2][3]. However, what drives the Ab accumulation and how this accumulation links to progression of the disease is not well understood.
Increasing evidence indicates that the disruption of metal homeostasis, particularly in Zn and Cu concentrations, is strongly correlated with the pathophysiological process of AD [4][5][6]. Although copper and, to a lesser extent, iron can induce partial Ab aggregation, they need a mildly acidic condition (pH 6.6) [7]. Zn 2+ is the only metal ion available to aggregate Ab at pH7.4-the normal physiological pH [8][9][10]. Elevated Zn was found and copurified with Ab from AD brain tissues, associated with markedly high Zn level in cerebral spinal fluid (SP) [11]. Measurements from well characterized late stage AD (LAD) also showed a significant increase of Zn in brain sections of hippocampus, multiple neocortical areas and amygdala compared to age-matched normal control subjects [4,5,[12][13][14]. Although several reports indicate that Zn induces Ab aggregation at low physiological concentrations [8,15,16], later studies showed that higher Zn concentrations are required for significant fibril formation [17,18]. These pieces of evidence were obtained mostly from in vitro experiments. Therefore how Zn status influences Ab pathology in vivo throughout life course remains unclear. Dietary intervention of zinc intake with zinc chelators in animals show some encouraging results [19,20,21]. However, genetic evidence is still lacking. More importantly, zinc chelators are usually not zinc specific, and may associate with other nonspecific phenotypes [22], precluding accurate mechanism analysis. Transport of Zn into cells is mediated by a set of zinc transporters called Zrt-Irt like proteins (Zips). Zips are characterized as influx transporters that mediate Zn 2+ uptake into cytoplasm from extracellular or vesicular sources [23,24], and are encoded by the solute-linked carrier (Slc) gene family, Slc39 [23]. At least 14 Zips have been identified in the human genome [23] and 8 in Drosophila [25]. Most Zips are predicted to have eight transmembrane domains (TM) with a histidine-rich loop between TM3 and TM4, and to be located at the plasma membrane [26]. Although the uptake of Zn from the brain's extracellular environment to intracellular compartments in neurons and glia cells is not completely understood, the Zips are thought to be involved in this process [23,24]. To our knowledge, the relationship between Zips and AD has not been explored to date.
Previously we and others have shown that expression of human Ab42 in Drosophila brains recapitulates the main symptoms of AD including age-dependent memory loss, formation of amyloid deposits and neurodegeneration [27,28]. In the current study, we found that the time course expression change of dZip1, an orthologue of human Slc39 family transporter hZIP1 in Drosophila, was reversed in brains of Ab42 flies as compared with normal control flies. We hypothesize that modulating dZip1 expression level might affect Zn accumulation in the brain and modify the AD pathological process. By creating dZIP overexpression and RNAi transgenic flies, we demonstrated that dZip1 is critically involved in Ab-induced AD pathological process, and by lowering dZip1 expression Ab toxicity can be markedly ameliorated.

dZip1 is a putative Zn importer involved in zinc uptake
Using the amino acid sequence of hZip1, BLAST searches revealed 8 putative Zips in Drosophila, among which CG9428encoded putative protein shared the highest similarity with human Zip1 (29% identity). We designate it as dZip1. Topology analysis of dZip1 revealed the presence of eight putative transmembrane domains, a histidine-rich loop between domains 3 and 4 which was predicted to occur within the cytosol, and extracellular N-and C-terminals ( Figure 1A). All these features are typical of Slc39 family members [23,24].
We measured dZip1 transcript levels in organs of w 1118 flies by semi-quantitative RT-PCR (sqRT-PCR). dZip1 is expressed in the gut and other organs including the brain ( Figure 1B left). To further explore the physiological role of dZip1 in flies, we created dZip1 over-expression (OE) and RNAi transgenic flies. We confirmed elevated and decreased dZip1 transcript levels accordingly by dZip1 over-expression or RNAi in whole body of transgenic flies driven by actin-Gal4 ( Figure 1B Right). We then tested the zinc sensitivity of these flies. Figure 1C shows that dZip1 OE flies were more sensitive to zinc overdose (t-test, p,0.001), while dZip1-RNAi flies were more tolerant to zinc overdose (t-test, p,0.001) in comparison with controls. These results suggest that dZip1 is indeed involved in zinc uptake.
To test whether brain Zn levels could be changed accordingly by specifically modulating brain dZip1 level, the pan-neuronal elav-Gal4 driver was used to drive dZip1 OE and RNAi in fly brains. Figure 1D showed qRT-PCR results of dZip1 transcript level in different transgenic fly brains. Two dZip1-RNAi transgenic lines were used, in which the knockdown effect of dZip1-RNAi #2 transgenic line (,1/10 of the dZip1 transcript level in control elav-Gal4 flies) was much stronger than dZip1-RNAi #1 transgenic line (,3/5 of the dZip1 transcript level in control elav-Gal4 flies) ( Figure 1D). dZip1 OE transgenic line showed ,6-7 fold increase of dZip1 transcript level compared with control brains ( Figure 1D). Inductively coupled plasma optical emission spectrometry (ICP-OES) result showed that over-expression of dZip1 in the fly brain markedly increased brain Zn accumulation (t-test, P,0.01), while knocking down dZip1 via RNAi decreased brain Zn level compared with the control elav-Gal4 flies (t-test, p,0.05) ( Figure 1E). Therefore, specific manipulation of dZip1 expression level in fly brains could affect the brain Zn status.
Ab42 affects dZip1 expression pattern in the course of aging Ab42 expression in fly brains could induce an age-dependent formation of amyloid deposits and neurodegeneration which may correlate with disturbed metal homeostasis, especially for zinc and copper. We therefore checked native dZip1 mRNA levels in brains of elav-Gal4.UAS-Ab42 (Ab42) flies and normal elav-Gal4 flies at different ages. We found that the dZip1 mRNA level was developmentally altered in Ab42 fly brains compared to elav-Gal4 fly brains ( Figure 2A). The dZip1 mRNA level in brains of w 1118 flies showed similar results as elav-Gal4 flies (data not shown), indicating that this is not due to the effect of the introduced Gal4 gene. These results imply that Ab expression may indeed lead to a zinc dyshomeostasis in the fly brains. Of note is that the endogenous dZip1 expression was lower in young adult Ab flies as compared to the control, although the brain zinc level of the Ab flies at the young stage was not significantly different ( Figure 2B), suggesting a complex regulation of zinc metabolism (involving participants such as other zinc importers and exporters besides dZip1) is involved in the brain zinc control.
Using ICP-OES we directly measured the Zn content in brains of 7-and 30-day old flies. By 7 days of age, differences of Zn content among different groups were still not apparent ( Figure 2B). With ageing, the Zn content in all the brains significantly increased; however, in comparison with 7-day old elav-Gal4 flies without Ab expression, 30-day old Ab42 flies increased ,170% of their Zn level, significantly higher than that of 30-day old elav-Gal4 flies (average ,65% increase of the control Zn). Over-expression of dZip1 further increased Zn accumulation in Ab42 fly brains, while dZip1 knockdown slowed brain Zn accumulation during aging and significantly reduced Zn accumulation compared to 30day old Ab42 flies (t-test, p,0.01). By testing Zn content of these flies at 20-day old ( Figure S1), although it's not obvious as that of 30-day, the trend is already apparent and statistically significant. These results indicate an intimate connection among Ab42 expression, aging and brain zinc accumulation, and the latter can be strongly affected by dZip1 expression interference.
Modulation of dZip1 expression correspondingly alters the course of Ab42-induced neurodegeneration Next, we used Hematoxylin and Eosin (H&E) staining to examine whether the extent of brain neurodegeneration in aged Ab42 flies (visualized as vacuolization in the brain region, arrowhead in Figure 3) could be changed by modulation of dZip1 expression. Compared to the control, Ab42-expressing brains with dZip1 knockdown were to a large extent normal ( Figure 3C), but when dZip1 was overexpressed degenerative changes were significantly more apparent in both the cortex and the neuropil region, where there were more and bigger bubbles ( Figure 3B). Counting the number of vacuoles in the cortex and neuropil revealed that over-expression of dZip1 increased brain vacuoliza-

Author Summary
Alzheimer's disease (AD) is characterized by extracellular amyloid plaques and altered metal ion (including Zn, Cu, Fe) concentrations in the brain. Amyloid plaques are the result of increased aggregation of Ab, while the in vivo role of metal ions such as Zn remains poorly understood. We found that the expression of a zinc transporter (dZip1) is altered in the brains of AD flies. Genetic manipulation of dZip1 to modulate its expression was accompanied by altered Ab accumulation, resulting in changes in the neurodegeneration development, cognitive performance, and lifespan of the AD flies. These genetic findings support the zinc role in AD pathology and implicate a new therapeutic target for treating AD.  PLoS Genetics | www.plosgenetics.org tion more than 2-fold ( Figure 3D, t-test, p,0.001), whereas dZip1 knockdown dramatically decreased brain vacuolization in Ab42 flies (t-test, p,0.001). Meanwhile, no significant different of neurodegenerative bubbles were found between dZip1 OE alone and age-matched control elav-Gal4 flies in 20-day old fly brains ( Figure 3E and 3F). To exclude possible off-target effect of the RNAi action, we confirmed the results with a differently constructed RNAi line, V3986, from the VDRC stock center. V3986 exhibited a similar level of dZip1 reduction as our own dZip1-RNAi line #2 ( Figure S2), and could roughly to the same extent rescue Ab-associated brain vacuolization ( Figure S2C and S2D). These results indicated that modulating dZip1 expression could change the brain neurodegenerative process.

Modulation of dZip1 expression level affects the climbing ability and lifespan of Ab42 flies
It has been shown that Ab42 flies start to display locomotor dysfunction after three weeks of age and their lifespan is significantly reduced [27,28]. We therefore tried to examine whether dZip1 expression levels could affect Ab42 flies' locomotion and lifespan. Two dZip1-RNAi transgenic lines were used, in which the knocking down effect of the dZip1-RNAi #2 transgenic line was much more obvious than the dZip1-RNAi #1 transgenic line ( Figure 1D). Assay of climbing ability demonstrated that Ab42 flies with dZip1 overexpression started to have a locomotor defect at 15day old age comparable with that of the control elav-Gal4.UAS-Ab42 flies at between 20-25 days ( Figure 4A). In contrast, Ab42 flies with decreased dZip1 levels through RNAi (#1) had a delayed climbing deficit ( Figure 4A). This rescuing effect on climbing ability was even more pronounced when the stronger line dZip1-RNAi #2 was used. As a further control, we tested the climbing ability of the transgenic flies without Ab42 expression ( Figure 4B). Only flies with over-expression of dZip1 manifested mild locomotor defect 30 days after eclosion as compared with the age-matched control elav-Gal4 flies. Again we confirmed the climbing rescuing effect with a different RNAi line V3986 and found it compared similarly with our dZip1-RNAi flies ( Figure  S2E). We conclude the locomotion defect as a result of Ab42 toxicity can be modulated through the change of dZip1 expression level.
Consistent with the result obtained in the climbing assay, the lifespan of Ab42 flies was shortened by over-expression of dZip1 and prolonged by RNAi-based knockdown of dZip1 expression ( Figure 5A and 5B). The dZip1-RNAi #2 transgenic line exhibited the strongest rescue, with 33.3% and 88.8% increase respectively in the median lifespan of Ab42 flies reared at 25uC and 29uC ( Figure 5D). Similar to that in the climbing assay, the lifespans of flies without Ab42 expression were largely indistinguishable except for that of the dZip1 OE (elav-Gal4.UAS-dZip1) flies, which displayed a noticeable 7.2% reduction ( Figure 5C and 5D). Our results indicate that a reduction of dZip1 expression in Ab42 flies leads to improved locomotor ability and longer lifespan. Consistently, zinc chelation with clioquinol extended Ab42 survival ( Figure S3). Interestingly, clioquinol appeared rescuing male Ab42 more effectively than females.

Modulation of dZip1 expression ameliorates Ab42induced early memory loss
A cardinal defect in Alzheimer's disease is memory loss. With extensively characterized Pavlovian olfactory aversive conditioning [29], a memory defect in adult Ab42 flies started to appear as early as 5-day-old. dZip1 knockdown significantly rescued memory loss at this stage ( Figure 6A). Paradoxically, overexpression of dZip1 also resulted in obvious memory recovery. As a control, we examined how alteration of dZip1 expression alone (in the absence of Ab42) might impact memory scores. Overexpressing or knocking down dZip1 did not significantly influence memory of 5-day-old normal flies ( Figure 6B and 6C), although overexpression of dZip1 might have a marginal beneficial effect. These results suggest that modulating dZip1 could alleviate the Ab42 toxicity on memory ability at early stages.

dZip1 overexpression stimulates while dZip1 inhibition decreases Ab42 deposition
The aforementioned experiments demonstrated dZip1 expression modulation can markedly alter the course of Ab-associated neurodegeneration. Towards further analysis of the mechanism underlining dZip1 effect on Ab toxicity, we first tried to determine where Zn was concentrated in these Ab fly brains. We raised 10day old Ab42 flies on normal food supplemented with ZnCl 2 and then used Zinquin staining to detect Zn distribution in vivo. The purpose of applying extra Zn is to enhance the fluorescent signal. Without Zinquin treatment, little signal was detected ( Figure 7B). Over-expression of dZip1 ( Figure 7E and 7F) appeared to produce stronger signals in the neocortex and neuropile region of the fly brain compared with control Ab42 ( Figure 7C and 7D) flies. Quantitative analysis of these signal intensities showed a significant difference ( Figure 7K, t-test, p,0.01). Only a faint signal was detected in dZip1-RNAi fly brains ( Figure 7G, 7H and 7K, t-test, p,0.05 at 40 h and t-test, p,0.001 at 72 h). Staining of the brains of elav-Gal4 flies without Ab expression showed faint signals similar to dZip1-RNAi flies ( Figure 7I and 7J). These results demonstrate a positive correlation between dZip1 level and Zn accumulation in the neocortex and neuropile region of the fly brain where Ab deposits were revealed ( Figure 8).
cytosol. The N-and C-terminals were predicted to be located outside of the cytosol. (B) dZip1 expression analysis. On the left shows dZip1expression in the brain, gut and whole body minus gut & brain of w 1118 adult flies, and on the right dZip1 expression in the whole body of actin-Gal4, actin-Gal4.UAS-dZip1-RNAi 1# and actin-Gal4.UAS-dZip1 adult flies (right). Expression analysis was performed with sqRT-PCR, with rp49 as the loading control. (C) dZip1 expression change leads to Zn sensitivity change. Three-day-old flies were exposed to 2.5 mM ZnCl 2 or no drug, dZip1 ubiquitous expression flies (actin-Gal4.UAS-dZip1) were significantly more sensitive to 2.5 mM ZnCl 2 treatment than the control flies (actin-Gal4) (t-test, p,0.001). Inhibiting dZip1 expression (actin-Gal4.UAS-dZip1-RNAi 1#) significantly enhanced flies' tolerance against 2.5 mM ZnCl 2 treatment (t test, p,0.001). No significant differences were found among all the genotypes when raised on normal food. (D) dZip1 mRNA level can be changed in fly brains by dZip1 RNAi and over-expression when using a pan-neuronal elav-Gal4 driver. Compared with control elav-Gal4 flies, brain dZip1 transcript level was repressed nearly twofold in elav-Gal4.UAS-dZip1-RNAi 1# transgenic flies, and was repressed approximately ten fold in elav-Gal4.UAS-dZip1-RNAi 2# transgenic flies, while it was approximately sevenfold over-expressed in elav-Gal4.UAS-dZip1 transgenic flies. The relative dZip1 expression levels against rp49 were from three independent biological replicates of each genotype and plotted with SEM (error bars). (E) Inductivelycoupled-plasma-optical-emission-spectrometry (ICP-OES) was used to detect the Zn content in 15 day old fly brains. Pan-neuronal expression of dZip1 (elav-Gal4.UAS-dZip1) increased the Zn level (t-test, P,0.01) while dZip1 RNAi (elav-Gal4.UAS-dZip1-RNAi 1#) reduced Zn accumulation (t-test, P,0.05). doi:10.1371/journal.pgen.1002683.g001 dZip1 Affects Drosophila Amyloid Pathology To determine if Zinc is causally related to Ab42 deposition, we overexpressed or knocked down dZip1 under the control of elav-Gal4 in Ab42 flies and subjected the brains to histochemical analysis. Ab42 peptides could form diffused amyloid deposits in fly brains [27,28]. Thioflavin-S (TS) staining of the whole brain was used to specifically visualize the Ab42 fibril deposits (Figure 8) [23]. Ab42 deposits were observed in the Kenyon cell body region of Ab42-expressing brains. The number of TS-positive deposits in Ab42 flies was significantly increased with aging ( Figure 8A and 8A1, 8B and 8B1). Over-expression of dZip1 markedly increased the number of Ab42 deposits compared with age-matched Ab42 flies ( Figure 8A and A1, ,212% at 25-day old relative to Ab42 flies at 25-day old, t-test, p,0.001). Conversely, RNAi-based knockdown of dZip1 in Ab42 flies significantly decreased the Ab42 deposits compared with age-matched Ab42 flies ( Figure 8B and B1, ,48% at 30-day old relative to Ab42 flies at 25-day old, t-test, p,0.001). Taken together, our results demonstrate that dZip1 over-expression can increase Ab42 accumulation whereas inhibiting dZip1 can decrease Ab42 deposition.

Modulation of dZip1 expression also affects the low aggregation form of Ab
The above TS staining was used to specifically detect the Ab42 fibril deposits. Recently, an alternative model for the Ab toxicity is put forth hypothesizing that amyloid oligomers rather than plaques are responsible for the disease [30]. The oligomer form, together with the monomeric form of Ab, is soluble in SDS whereas the fibril aggregate is not. We next investigated how SDSsoluble Ab was affected by modulating dZip1 expression. Fly brain   lysates were used for a Western blotting analysis. The result showed that the SDS-soluble Ab42 (low level aggregation forms) was dramatically decreased in accordance to reduced dZip1 level and increased when dZip1 was over-expressed ( Figure 8C and 8C1). In a separate experiment, SDS-insoluble but formic acidsoluble Ab42 (high level aggregation forms) was also examined; much decreased formic acid-soluble Ab42 level was observed when dZip1 expression was inhibited ( Figure S4), consistent with the TS-staining result.
Whole-mount immunohistochemical staining with Ab42 antibody also revealed a reduction of Ab42 level in the dZip1-RNAi fly brains (Figure 9). Abundant amyloid deposits were observed in the Kenyon cell body region of 20-day old Ab42-expressing brains ( Figure 9A, arrow). Such deposits were markedly decreased when dZip1 was knocked down ( Figure 9C) and greatly increased when dZip1 was over-expressed ( Figure 9B). Similar results were found in 30-day old fly brains ( Figure 9E and 9F) compared with agematched Ab42-expressing control brains ( Figure 9D). In the case of co-overexpression of dZip1 and Ab42, vacuolization became much more pronounced with aging ( Figure 9E). As a control for the Ab42 antibody, we used it against elav-Gal4 fly brains and found no meaningful signal, indicating the antibody reacts specifically with Ab ( Figure S5).
Because dZip1 RNAi led to a general reduction of Ab42 level, we tried to ask whether this was due to Ab42 expression inhibition or an increase of Ab42 degradation. Ab42 gene was directly under the control of elav-Gal4, and indeed no changes of RNA expression under the various genetic manipulations were observed ( Figure 10A and 10B, Figure S2B). We then suspected that zinc might reduce the rate of Ab42 clearance. Several proteases (NEP1-3, IDE) were proposed to act in the Ab degradation [31,32] and we thus explored whether they were affected by dZip1 expression in the Ab flies. Ab expression brought some changes to the expression of these genes, but introduction of dZip1-RNAi transgene in the Ab flies resulted no significant expression increase of these genes ( Figure 10C-10F). We did however, observed some decrease of NEP2 expression in dZip1 OE/Ab flies. Therefore we saw little evidence that the observed rescuing effect of dZip1 RNAi on Ab42 flies is mediated by an increase of these degrading proteases.
Together, we conclude dZip1 reduction decreases levels of Ab42, in both the high (fibril aggregates) and low aggregation forms.

Discussion
Previous studies suggest that heavy metals, especially Zn, have a close relationship with the development of Alzheimer's disease [4][5][6][8][9][10]. However, little genetic evidence exists that demonstrates a functional link between Ab and proteins involved in zinc assimilation. In this study, we showed that manipulating the Slc39 family protein dZip1 greatly altered the Ab toxicity. In particular, knocking down dZip1 in brains of Ab42 flies markedly decreased both Ab42 deposits and zinc accumulation. dZip1 knockdown ameliorated early memory loss, decreased the number of neurodegenerative vacuoles, significantly enhanced locomotor ability and prolonged life-span in Ab42-expressing flies. Taken together, our results provide strong evidence to support our hypothesis that knocking down protein dZip1 may mitigate Ab pathology and Ab-dependent behavioral defects in a Drosophila model of Alzheimer's disease.
The accumulation and aggregation of Ab42 peptide in the neocortex has been suggested to be caused by its abnormal interactions with neocortical metal ions especially Zn, which is constitutively found at high levels in the neocortical regions where they play important roles in normal physiology [11,33,34]. Our qRT-PCR results showed that the dZip1 transcript level was higher in brains of young control elav-Gal4 and w 1118 flies and decreased with age, but lower in brains of young Ab42 flies and increased with age. Paradoxically, dZip1 transcript level was lower in young adult Ab42 flies than age matched normal flies. Since at this stage Zn level is not any lower ( Figure 2B) in Ab42 flies, we suspect other Zn homeostasis genes might also be affected. Indeed, besides dZip1 quite a few other Zip or ZnT (Slc30 family transporter) genes are also expressed in the brain (data not shown and the Flyatalas: http://flyatlas.org), likely contributing to Zn uptake or export. Supporting this notion is a recent report showing that the expression level of the Slc30 family protein ZnT3 decreased with age in AD brains [35,36]. Thus the general Zn status is the result from the combination effect of all these Zn homeostasis genes. It is possible that Ab42 expression alters the Zn homeostasis starting from an early stage, although we are not totally clear why dZip1 is reduced at early stages but increased at late stages. One thing worthy of consideration is that total zinc level does not reflect well available cellular zinc. In other words, two brains with the same level of total zinc may have very different levels of zinc for use. dZip1 expression regulation may reflect the cell's native response to its own physiological states-to bring more or less zinc into cells. Because Ab42 can likely bind to zinc, and monomer and oligomers may have different binding characteristics, we speculate that Ab42 can affect zinc homeostasis even in the absence of noticeable total zinc level alteration. This dyshomeostasis could result or be reflected by native dZip1 expression changes.
Not all pathogenic effects of Ab in Drosophila correlate directly with its aggregation. An artificial mutation (L17P) with decreased Ab42 aggregation tendency is associated with lower toxicities, in term of locomotor ability and lifespan, but induces even earlier onset of memory defects than its normal counterpart [37]. Furthermore, although both Ab40 and Ab42 affect learning, only Ab42 causes degeneration [27]; inhibition of PI3K activity ameliorated the Ab42-induced early memory loss, but did not rescue neurodegeneration [38]. These results lead to the Relative Zinquin signals against background in the neocotex and neuropile region were quantitated using ImagJ software (K). The relative intensity of Zinquin signal, however, is by no menas a quantitative reflection of zinc levels [42]. Data are expressed as means 6 SEM and analyzed by Student's t-test. n = 4 for each genotype. Scale bar, 100 mm. doi:10.1371/journal.pgen.1002683.g007 speculation that neuronal dysfunction and neurodegeneration may be mediated by different mechanisms. In our study, while knocking down dZip1 and overexpressing dZip1 were associated with opposite effects on all other aspects of Ab42-induced toxicity, it is interesting that both knocking down dZip1 or overexpressing dZip1 lessened Ab42-induced early memory loss. While inhibiting Zn accumulation in fly brains could promote memory in Ab42expressing flies by reducing aggregation of Ab42, the amelioration of memory loss with dZip1-overexpression is likely due to a different mechanism. At the moment, we are not certain how this memory gain with dZip1 over-expression was achieved.
One might ask whether the dramatic effect seen with dZip1 modulation in Ab42 flies could be reproduced with dietary zinc supplement or chelation. High levels of zinc supplement (such as 2.5 mM) do significantly worsen the viability of Ab42 flies, however at this level normal flies are also affected ( Figure S3C and S3D). Using a zinc chelator clioquiniol, dietary feeding can rescue to some extent Abeta flies, interestingly mostly in male flies ( Figure  S3A and S3B). Thus genetic intervention is a much more effective method. We interpret this as systemic Zn overloading may cause damages to other tissues before enabling a dramatic Zn increase in the target organ. Likewise, zinc depletion at the organismal level is generally harmful: zinc is an essential nutrient vital to many biological processes. Indeed, high levels of clioquinol greatly impact even the survival of normal flies. Thus, we believe low zinc level can affect fly development and survival so that overall beneficial effect of zinc reduction by dietary measures is significantly less effective than targeted neuronal zinc reduction through genetic interventions. dZip1 repression results in less Ab42 level. Although other possibility cannot be excluded, we favor the model that zinc induces oligomerization of Ab42, as supported by numerous in vitro evidences. More oligomers result more fibril deposits. Perhaps the oligomer and the aggregated Ab42 are more stable than the monomer, so that the Ab42 level is dramatically reduced in dZip1 RNAi flies, where a larger fraction of Ab42 adopt the monomeric form more susceptible to clearance (Figure 11).
In summary, we have demonstrated the modulating effect of dZip1 on Ab42 toxicity in a Drosophila model of Alzheimer's disease. We observed Ab42 expression could cause a change of dZip1 expression pattern during ageing. Through genetic manipulation of dZip1 expression, we can modify the pathological process of Ab42. These results raise the possibility that Zip1, or more broadly Zn transporter genes expressed in the brain, could be a new kind of promising therapeutic target in AD pathology.

RNA isolation, semi-quantitative and quantitative RT-PCR
Total RNA was extracted from the brain, gut and carcase (whole body minus brain and gut) of 10 adults for each sample using TRIzol Reagent (Invitrogen) according to the manufacturers' instructions and subjected to DNA digestion using DNAse I (Ambion) immediately. The concentration and quality of DNAsetreated total RNA were then tested, and 800 ng total RNA from each sample was used to synthesize cDNA by using Superscript TM II Reverse Transcriptase kit (Invitrogen) with oligo(dT) primers. Semi-quantitative RT-PCR (sqRT-PCR) was performed using primers for rp49 (forward: 59-TACAGGCCCAAGATCGTGAA-39; reverse: 59-TCTCCTTGCGCTTCTTGGA-39) and dZip1 (forward: 59-ATTATCCTCGCCCTTTCGC-39; reverse: 59-TCACCCTCCGCT TCGTCAG-39). rp49 was used as the loading control. For quantitative RT-PCR (qRT-PCR), 20 fly brains were used for each sample, RNA extraction and cDNA synthesis were the same as described for sqRT-PCR. Primers for amplifying dZip1, NEP1, NEP2, NEP3 and Ab42 were listed as Table S1. Real-time PCR reactions were monitored on an iCycler (Bio-Rad) by means of SYBR Green (Bio-Rad) dye. mRNA expression levels were determined relative to rp49 expression by relative quantification. Statistical analysis was performed using the Student's t-tests.

Histology and immunostaining
For immunostaining analysis on paraffin sections, antigen retrieval was achieved by boiling the samples in 10 mM sodium citrate (pH 6.6) for 15 min. Immunostaining was performed using an avidin-biotin-peroxidase complex (ABC) kit (Vector Laboratories). For Ab staining, the primary antibody used was anti-Ab42 (Promega; 1:500). Appropriate secondary antibodies were diluted 1:200, and histochemical detection was done with DAB (Sigma-Aldrich) color development.

Quantification of neurodegeneration
Adult fly heads were fixed in Carnoy solution (ethanol:chloroform:acetic acid = 6:3:1) overnight at 4 Cu, and then embedded in the paraffin and sectioned at 6 m thickness. H&E staining was performed following standard protocols. Neurodegeneration was assessed by quantification of vacuoles with diameter greater than staining was used to detect the Ab42 fibril deposits in fly brains (bright green dots). Few deposits were found in control brains (elav-Gal4, A, top panel) at 25-day old. TS-positive deposits were found after Ab42 expression in fly brains (elav-Gal4.UAS-Ab42) at both 25 dae (A) and 30 dae (B). Quantitative contents of Ab42 deposits based on TS signals were summarized and expressed after normalization to 25-day old Ab42 flies (A1 and B1). The increase of Ab42 deposits was age-dependent. Overexpressing dZip1 in Ab42-expressing brains (elav-Gal4.UAS-Ab42/UAS-dZip1) significantly increased fibril deposits at 25 dae (A and A1). However, inhibiting dZip1 (elav-Gal4.UAS-Ab42/UAS-dZip1-RNAi 1#) dramatically decreased deposits density at 30 dae (B and B1). t-test, **P,0.01, ***P,0.001. Data are expressed as means 6 SEM. n = 6 or 8 hemispheres for each genotype. Scale bar, 15 mm. (C) dZip1 knockdown also decreases the low aggregated form of Ab42. Protein lysates from 30-day old fly heads were prepared for western blotting assay. SDSsoluble Ab42 were detected in elav-Gal4.UAS-Ab42 flies. Less and more SDS-soluble Ab42 were found respectively when dZip1 expression was inhibited and increased. (C1) is a quantitative measurement of (C). t-test, *P,0.05. n = 3 independent experiments. Data are expressed as means 6 SEM. doi:10.1371/journal.pgen.1002683.g008  All the four degrading enzymes shows higher expression level in fly brains without Ab42 expression than that with Ab42 expression. dZip1 RNAi did not significantly influence NEP or IDE expression in Ab42 flies compared to the control Ab42 flies, while dZip1 OE shows a trend of reducing NEP or IDE expression level, but only statistically significant for NEP2. The relative Ab42, NEP1-3 and IDE expression levels against rp49 were from three independent biological replicates and plotted with SEM (error bars). doi:10.1371/journal.pgen.1002683.g010 3 mm in the fly brains. At least five fly brains were analyzed for each genotype.

Fibril Ab42 deposit detection
Thioflavin-S (TS, Sigma) staining was performed to detect fibril Ab42 deposits. Fly brains were fixed in 4% paraformaldehyde and permeabilized by 2% triton. Brains were then transferred to 0.25% TS in 50% ethanol overnight. After 16 wash for 10 min in 50% ethanol and 36 wash with PBS, they were mounted with focusclear (Pacgen Biopharmaceuticals Inc.) and covered by cover slips. Slides were inspected with a Zeiss LSM 510 confocal microscope, aided by LSM 510 analysis software. TS-positive deposits located in the mushroom body somatic region were counted for comparison analysis.

Pavlovian olfactory associative memory recording
The training and testing procedures were as previously described [40,41]. During one training session, a group of 100 flies was sequentially exposed for 60 s to two odors, octanol (OCT) or methylcyclohexanol (MCH), with 45 s of fresh air in between. Flies were subjected to foot-shock (1.5 s pulses with 3.5 s intervals, 60 V) during exposure to the first odor (CS+) but not to the second (CS2). To measure ''immediate memory (also referred to as ''learning'')'', flies were transferred immediately after training to the choice point of a T-maze and forced to choose between the two odors for 2 min, at which time they were trapped in their respective T-maze arms, anesthetized, and counted. A performance index (PI) was calculated from the distribution of flies in the T-maze. A reciprocal group of flies was trained and tested by using OCT as the CS+ and MCH as the CS+, respectively. PIs from these two groups finally were averaged for an n = 1 and multiplied by 100. A PI of 0 represented a 50:50 distribution, whereas a PI of 100 represented 100% avoidance of the shock-paired odor.

Western blot analysis
SDS-soluble and SDS-insoluble but formic acid-soluble Ab42 were prepared as previously reported [27]. Lysates from equal number of fly heads were diluted in SDS sample buffer and separated by 10-20% Tris-Tricine gels (Invitrogen), and transferred to nitrocellulose membranes (Invitrogen). Membranes were boiled in PBS for 3 min. Membranes were blocked with 3% BSA and blotted with primary antibody. Primary antibodies used in this study were mouse anti-Ab42 (6E10, Covance Research Products) and rabbit anti-Actin (Sigma). After washing in TBST for 3 times, membranes were incubated with secondary antibodies for 1 hr at RT. After 3 times wash in TBST, membranes were incubated with ECL working solution (GE healthcare) and developed with films (Kodak). Data were analyzed with ImageJ sofeware (NIH).

Zinquin staining
10-day old adult flies reared on normal condition were transferred to vials with normal food supplied with 4 mM ZnCl 2 . Fly brains were then dissected at 40 and 72 h after transferring, respectively. The dissected brains were then incubated with 25 mM Zinquin (Sigma) for 30 min at 37uC, and washed 3 times with 16PBS buffer for 5 min each time. After that, brains were examined using conventional epifluorescence microscope (Nikon, Diaphot 300) equipped with a Nikon 1006, 1.4 NA Plan Apo oilimmersion objective. Zinquin signals in the neocortex and neuropile region of fly brains were quantitated by using ImagJ software.

Metal stress and content assay
For the metal stress assay, Drosophila was fed on normal medium containing 2.5 mM ZnSO 4 . Control flies were fed on normal medium in the absence of drug. Mortality was recorded every 24 h or a longer intervals. Each vial contained 20-25 flies, and the experiments were repeated at least three times. Figure 11. A model to explain zinc's effect on Ab. Zinc stimulates the polymerization of Ab, resulting more oligomer formation, which in turn produces more fibril deposit. When zinc is low, less oliogomers are formed. The monomers may be more susceptible to degradation than the polymers/fibrils so that the observed overall Ab level is reduced when zinc is low. doi:10.1371/journal.pgen.1002683.g011 For the metal content analysis, flies were reared on normal food and fly heads were collected at day 7, 20 and 30 after eclosion. Fly heads were dissolved in 1 ml 65% HNO 3 , boiled in 100uC water bath for 10 min and diluted to 10 ml for metal content analysis with inductively coupled plasma optical emission spectrometry (ICP-OES, IRIS Intrepid II XSP, Thermo Electron Corporation, USA).

Climbing assay
The climbing assay was referenced to Iijima et al. (2004) [27]. Briefly, twenty flies were placed in a plastic vial and gently tapped to the bottom. The number of flies at the top of the vial was counted after 18 s of climbing under red light (Kodak, GBX-2, Safelight Filter). The data shown represent results from a cohort of flies with four repeats tested serially for 5-50 days. The experiment was repeated more than three times.

Longevity assay
Flies of two days after eclosion were used for the experiment. Twenty to 23 flies were placed in a food vial. Each vial was kept at 25 or 29uC, 70% humidity, under a 12-h light-dark cycle. Food vials were changed every 2-3 days, and dead flies were counted at that time. At least 150 flies were prepared for each genotype, and the experiments were carried out more than three times. Percent increases in life span are based on comparing the median survivals. Prism (GraphPad) was used for statistical analysis of lifespan data. Mantel-Cox log-rank statistical analysis was used for testing statistical significance of the differences between the survivorship curves.

Statistical analysis
All data were analyzed by Student's t-test. Statistical results were presented as means 6 SEM. Asterisks indicate critical levels of significance (*P,0.05, **P,0.01 and ***P,0.001). Figure S1 20-day old Drosophila brain zinc levels are affected by Ab42 and dZip1 expression. Zn content in brains of 20-day old flies was measured by ICP-OES. dZip1 OE (elav-Gal4.UAS-Ab42/ UAS-dZip1) facilitated Zn accumulation in fly brains compared with control Ab42 flies (elav-Gal4 vs. elav-Gal4.UAS-Ab42). dZip1 RNAi slowed down the brain Zn accumulation process (elav-Gal4.UAS-Ab42/UAS-dZip1-RNAi 2# and elav-Gal4.UAS-Ab42/ UAS-dZip1(v3986) RNAi). Data on top of the bars represent the relative increased percentage of Zn level to control elav-Gal4.+/+ flies. Data are expressed as means 6 SEM and analyzed by Student's t-test. n = 3 for each genotype. (TIF) Figure S2 V3986, an independent dZip1 RNAi line, could similarly rescue Ab-associated brain vacuolization and climbing defect. (A) shows a representive of sqRT-PCR analysis of dZip1 expression in brains of 5-day old flies.V3986 RNAi line shows similar knock down effect with dZip1 RNAi 2#, which is better than v3987 RNAi line. rp49 was used as the loading control. (B) shows a representive of sqRT-PCR analysis of Ab42 expression in brains of 30-day old flies.V3986 RNAi line shows similar Ab42 expression level with dZip1 RNAi 2# line and dZip1 OE line, and no reducing Ab42 expression level was found compared to Ab42 flies. rp49 was used as the loading control. (C) Paraffin sections of 30day old fly brains were stained with H&E. V3986 RNAi line produced similar rescuing effect as dZip1 RNAi 2# line on Ab42 expression induced neurodegeneration (arrowheads indicate the vacuoles). Scale bar, 50 mm. (D) is a statistical analysis of (C) Ab42induced neurodegeneration under dZip1 expression modulation. Number of vacuoles (diameter.3 mm) on each section was counted and summarized. V3986 RNAi line produce similar rescuing effect as dZip1 RNAi 2# line, which has significant less vacuole numbers than control elav-Gal4.UAS-Ab42 flies (p,0.001). Data are expressed as means 6 SEM and analyzed by the Student's t-test. n = 9 for each genotype. (E) V3986 RNAi line has similar rescuing effect as dZip1 RNAi 2# line on Ab42 expression induced climbing defect. t-test, **P,0.01, ***P,0.001 (in comparison with elav-Gal4.UAS-Ab42 flies). n = 6 independent experiments. (TIF) Figure S3 Zinc chelation with clioquinol extends while zinc addition shortens Ab42 survival. Male (A) and female (B) elav-Gal4.UAS-Ab42 flies were raised on normal food with DMSO (control) and normal food supplied with 0.5 mM clioquinol (CQ) at 29uC. (C) shows the survival curves of elav-Gal4 and elav-Gal4.UAS-Ab42 flies raised on normal food or supplied with 2.5 mM ZnSO 4 , at 25uC. (D) was a Mantel-Cox log-rank statistical analysis of (C) survival curves. The percentage of survivorship was plotted against the age (dae). Foods were changed every 2-3 days, and the survival numbers of flies were counted. At least three biological repeats were used for each genotype.  Figure S5 The Ab42 antibody is specific to Ab42 in the fly brain. Paraffin sections of 20-day old elav-Gal4 and elav-Gal4.UAS-Ab42 fly brains were stained with antibody against Ab42. Panneuronal expression of Ab42 in fly brains induced Ab42 accumulation which was primarily located in the neuronal somatic region (arrow). No Ab42 signals were detected in age-matched control brains without Ab42 expression. Scale bar, 50 mm. (TIF)