Adenosine signaling and its downstream target mod ( mdg 4 ) modify the pathogenic 1 effects of polyglutamine in a Drosophila model of Huntington ’ s disease 2 3

13 Dysregulation of adenosine (Ado) homeostasis has been observed in both rodent models 14 and human patients of Huntington’s disease (HD). However, the underlying mechanisms 15 of Ado signaling in HD pathogenesis are still unclear. In the present study, we used a 16 Drosophila HD model to examine the concentration of extracellular Ado (e-Ado) as well 17 as the transcription of genes involved in Ado homeostasis and found similar alterations. 18 Through candidate RNAi screening, we demonstrated that silencing the expression of 19 adenosine receptor (adoR) and equilibrative nucleoside transporter 2 (ent2) not only 20 significantly increases the survival of HD flies but also suppresses both retinal pigment cell 21 degeneration and the formation of mutant Huntingtin (mHTT) aggregates in the brain. We 22 compared the transcription profiles of adoR and ent2 mutants by microarray analysis and 23 identified a downstream target of AdoR signaling, mod(mdg4), which mediates the effects 24 of AdoR on HD pathology in Drosophila. Our findings have important implications for the 25 crosstalk between Ado signaling and the pathogenic effects of HD, as well as other human 26 diseases associated with polyglutamine aggregation. 27


Introduction 31
Adenosine (Ado) is one of the most common neuromodulators in the nervous 32 system of vertebrates as well as invertebrates and modulates synaptic transmission 1,2 . 33 Under normal conditions, the extracellular Ado (e-Ado) concentration is in the nanomolar 34 range, which is sufficient to modulate the appropriate adenosine receptors (AdoRs) in the 35 brain cells tonically 3 . However, under pathological circumstances the e-Ado level may 36 increase up to 100-fold. In these conditions, Ado functions as an imperfect neuroprotector;

59
Drosophila expressing human mHTT has previously been demonstrated as a 60 suitable model system for studying gene interactions in polyQ pathology, and has been 61 used to elicit a number of modifiers for symptoms of HD 16,17 . Drosophila e-Ado signaling 62 is a relatively simple system compared to mammals; it contains a single AdoR isoform 63 (cAMP simulation) and lacks P2X receptors 18,19 . Human homologs of the Drosophila 64 genes involved in the regulation of Ado homeostasis and AdoR are shown in Fig. S1. The upon immune challenges [23][24][25] . 72 In the present study, we performed a candidate RNAi screen examining the role of 73 Ado signaling in a Drosophila HD model. We co-expressed exon 1 with a polyglutamine 74 tract of normal human htt Q20 or pathogenic mhtt Q93 17 together with UAS-RNAi or UAS-75 overexpression constructs specific for adoR, Ado transporters, and Ado metabolic enzymes 76 in Drosophila. We demonstrated that the downregulation of adoR and ent2 expression 77 reduces cell death, mortality and the formation of mHTT aggregates. In addition, we 78 identified a number of differentially-expressed genes in response to Ado signaling and 79 showed that mod(mdg4) is a downstream target of AdoR that mediates its effect in HD 80 pathogenesis. Drosophila, we compared e-Ado levels in the hemolymph of last-instar larvae ubiquitously 95 expressing Q20 HTT and Q93 mHTT driven by the daughterless-Gal4 driver (da-GAL4). 96 The results showed that the e-Ado concentration in the hemolymph of Q93-expressing 97 larvae was significantly lower compared to larvae expressing Q20 or control da-GAL4 ( Fig.   98 1A).

99
Since e-Ado concentration may be associated with the level of extracellular ATP (e-ATP), 100 we also examined its titer in the hemolymph of larvae with the same genotypes as the above 101 experiment. As shown in Fig. 1B, there was no significant difference in e-ATP levels 102 between Q20, Q93, and control da-GAL4 larvae. We thus postulated that the lower level 103 of e-Ado in Q93 larvae might be affected by changes in proteins involved in Ado 104 metabolism or transportation.

106
Earlier reports have shown that the expression of several genes involved in Ado 107 homeostasis, including Ado receptor, transporters, and genes involved in Ado metabolism, 108 are abnormal in human HD patients as well as in HD mice 28-30 . Since homologous proteins 109 have also been shown to control Ado homeostasis in flies (Fig. S1), we compared the 110 expression of three Drosophila adgf genes (adgf-a, adgf-c, adgf-d), adenosine kinase 111 (adenoK), adenosine transporters (ent1, ent2, ent3, cnt2), and adenosine receptor (adoR) in 112 the brains of Q93-and Q20-expressing larvae. The results showed that the expression of 113 adgf-a and adgf-d, as well as transporters ent1, ent2, and ent3 in the brain of Q93 larvae 114 were significantly lower than in Q20 larvae (Fig. 1D). The expression of cnt2 and adoR 115 showed no difference between Q93 and Q20 larvae.

116
In order to assess progressive changes in transcription profiles associated with HD 117 pathogenesis, we further examined the expression of genes involved in Ado homeostasis 118 in the heads of 5-and 15-day-old adults, roughly corresponding to early-and late-stage HD 119 (Fig. S2C). Unlike in the larval stage, the expression of metabolic genes adgf-c, adgf-d, 120 and adenoK, and transporter ent1, in five-day-old adults was found to be higher in Q93 121 flies than Q20 flies (Fig. 1E). In addition, 15-day-old Q93 flies showed higher expression 122 of adgf-d and adenoK (Fig. 1F). Previous studies in Drosophila have shown that the   significantly reduced (to 50%) in adoR RNAi flies (Fig. 3C&D), and a similar suppression 155 of mHTT aggregate formation was also observed in 20-day-old HD flies (Fig. S4). An 156 examination of eye phenotypes in ent2 RNAi flies showed a significant reduction in retinal 157 pigment cell death (Fig. 3E), but surprisingly we did not observe a significant rescue of 158 cell death by silencing adoR (Fig S5). We therefore postulated that it might be due to 159 insufficient RNAi efficiency for suppressing AdoR signaling in the eye. To test this, we  although both still showed a significant difference to either Q93 control or Q93/gfp RNAi 176 control by weighted log-rank test (Fig. S6B). Hence, we concluded that overexpressing the 177 examined genes enhances the effect of mHTT, resulting in the increased mortality of HD 178 flies. Our results demonstrate that the overexpression and silencing of ent2 or adoR has a 179 stronger influence over HD pathology than genes involved in Ado metabolism.

181
In order to investigate whether there is a synergy between the effects of AdoR and 182 ENTs, we co-expressed adoR RNAi constructs with ent1 RNAi or ent2 RNAi in Q93- effects on lifespan which is longer than the knockdown of adoR alone. There seems to be 187 a synergy between ENT1 and AdoR suggesting that ENT1 may have its own effect, which 188 is partially independent from AdoR signaling.

189
Next, we investigated our hypothesis that the source of e-Ado, which contributes  To further confirm the genetic data related to AMPK activation or inhibition, we 214 pharmaceutically inhibited AMPK signaling by feeding the larvae with AMPK inhibitor,  To validate the microarray data, we knocked down adoR expression in the brain and   To further confirm that mod(mdg4) is downstream target of the AdoR pathway and 251 regulated by e-Ado signaling, we first checked the expression of mod(mdg4) in larval 252 brains and adult heads of HD flies using qPCR. In Q93 larvae, we found that both the 253 expression level of mod(mdg4) (Fig. 7A) and the e-Ado level was lower than in Q20-254 expressing controls (Fig. 1A). For the 15-day-old (roughly corresponding to late-stage HD) 255 Q93 adults, there was no difference in mod(mdg4) expression compared to Q20 control 256 adults (Fig. 7A). We next examined the epistasis relationship between ent2, adoR, and  ent2 and adoR (Fig. 8B). These results indicate that 260 mod(mdg4) serves as a downstream target of AdoR signaling involved in the process of 261 mHTT inclusion formation and other pathogenic effects (Fig. 7C).

262
The mod(mdg4) locus of Drosophila contains several transcription units encoded on both 263 DNA strands producing 31 splicing isoforms 45 . As shown in Fig. 5B, two of the 264 mod(mdg4)-specific microarray probes which target 11 mod(mdg4) splicing isoforms (Tab. 265 S2) were downregulated in all three datasets. We performed splice form-specific qPCR 266 analysis and found that adoR RNAi silencing leads to the downregulation of multiple 267 mod(mdg4) isoforms (Fig. 7D), suggesting that AdoR signaling regulates multiple isoforms. AdoR contributes to mHTT pathogenesis and aggregates formation. 294 We observed a non-additive interaction between AdoR and ENT2 characteristic for 295 epistasis relationship (Fig. 4B), indicating that ENT2 is required for the transportation of 296 Ado from the intra-to extracellular environment which activates AdoR and, in turn, 297 enhances the effects of mHTT. Our previous report showed that both ENT2 and AdoR  Interestingly, while the effects of ent2 and adoR RNAi in HD flies were found to 306 completely overlap, ent1 RNAi showed a synergistic effect, suggesting potential AdoR-307 independent mechanisms (Fig. 4A). These results correspond to our previous report 308 showing that Drosophila ENT1 has lower specificity for Ado transportation in comparison  (Fig. 6), we could not clarify which of the isoforms is 328 specifically involved in HD pathogenesis because AdoR signaling regulates multiple 329 isoforms (Fig. 7D). Interestingly, an earlier report on protein two-hybrid screening      Table S2.  Table   431 S1. The expression level was calculated using the 2 -∆∆Ct method. The ct values of target 432 genes were normalized to reference gene, ribosomal protein 49 (rp49).