International Journal of Molecular Sciences

Article Genetic Screen in Adult Drosophila Reveals That dCBP Depletion in Glial Cells Mitigates Huntington Disease Pathology through a Foxo-Dependent Pathway

Elodie Martin, Raheleh Heidari †,Véronique Monnier and Hervé Tricoire *

Unité de Biologie Fonctionnelle et Adaptative (BFA), Université de Paris-CNRS, UMR8251 4 rue Marie Andrée Lagroua Weill Halle, CEDEX 13, 75205 Paris, France; [email protected] (E.M.); [email protected] (R.H.); [email protected] (V.M.) * Correspondence: [email protected]; Tel.: +33-(1)-1-57-27-79-51 † Current Adress: F. Hoffmann-La Roche Ltd., Product Development Global Operations, CNS and Rare Diseases, Basel Headquarter, Building: 663, 4070 Basel, Switzerland.

Abstract: Huntington’s disease (HD) is a progressive and fatal autosomal dominant neurodegenera- tive disease caused by a CAG repeat expansion in the first exon of the huntingtin gene (HTT). In spite of considerable efforts, there is currently no treatment to stop or delay the disease. Although HTT is expressed ubiquitously, most of our knowledge has been obtained on neurons. More recently, the impact of mutant huntingtin (mHTT) on other cell types, including glial cells, has received growing interest. It is currently unclear whether new pathological pathways could be identified in these cells


compared to neurons. To address this question, we performed an in vivo screen for modifiers of
 mutant huntingtin (HTT-548-128Q) induced pathology in Drosophila adult glial cells and identified Citation: Martin, E.; Heidari, R.; several putative therapeutic targets. Among them, we discovered that partial nej/dCBP depletion in Monnier, V.; Tricoire, H. Genetic these cells was protective, as revealed by strongly increased lifespan and restored locomotor activity. Screen in Adult Drosophila Reveals Thus, dCBP promotes the HD pathology in glial cells, in contrast to previous opposite findings in That dCBP Depletion in Glial Cells neurons. Further investigations implicated the transcriptional activator Foxo as a critical downstream Mitigates Huntington Disease player in this glial protective pathway. Our data suggest that combinatorial approaches combined to Pathology through a Foxo-Dependent specific tissue targeting may be required to uncover efficient therapies in HD. Pathway. Int. J. Mol. Sci. 2021, 22, 3884. https://doi.org/10.3390/ Keywords: Huntington’s disease; Drosophila; CBP; Foxo; Wnt signaling ijms22083884

Academic Editor: Philippe Gasque

Received: 29 January 2021 1. Introduction Accepted: 6 April 2021 Huntington’s disease (HD) is a progressive and fatal autosomal dominant neurode- Published: 9 April 2021 generative disease characterized clinically by motor, cognitive, and psychiatric deficits. It is a rare disease with overall prevalence of 2–6 persons per 100,000, depending on the geo- Publisher’s Note: MDPI stays neutral graphic area [1]. HD is caused by a CAG repeat expansion in exon 1 of the gene encoding with regard to jurisdictional claims in the large (3144 a.a.) huntingtin (HTT) protein. When the number of trinucleotide repeats published maps and institutional affil- exceeds 40 in the mutated HTT gene (mHTT), the disease is fully penetrant with an age of iations. onset around 45 years, depending on the length of the repeat (reviewed in [2]). The more striking feature of the disease is prominent neurodegeneration in striatal medium spiny neurons and cortical pyramidal neurons, but, since HTT is expressed throughout the body, HD is also associated to peripheral dysfunctions notably in muscle, gut, and liver [3–5]. To Copyright: © 2021 by the authors. date, no clinical trial has demonstrated treatment efficacy to slow down the disease [6,7]. Licensee MDPI, Basel, Switzerland. Identification of the causative mutation in the HTT gene led to the development of This article is an open access article a plethora of HD models from yeast to mammals. Among mammalian models, a large distributed under the terms and number of mice models have been generated that allowed functional studies in differ- conditions of the Creative Commons ent genetic conditions. These include transgenic mHTT models, knock-in models, and Attribution (CC BY) license (https:// transgenic YAC and BAC mice (reviewed in [8,9]). A large number of Drosophila models creativecommons.org/licenses/by/ were also developed, taking advantage of the sophisticated genetic tools available in this 4.0/).

Int. J. Mol. Sci. 2021, 22, 3884. https://doi.org/10.3390/ijms22083884 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 3884 2 of 16

organism and its short lifespan [10]. Drosophila models reproduce many features of the human pathology, including CAG repeat length dependence of neurodegeneration, and have also been used to investigate HD in peripheral tissues [11–13]. Using mammalian and invertebrate models, some key physiopathological mechanisms of HD have been identified, including altered neurotransmitter release and receptor activity, Ca2+ signal- ing impairments, mitochondrial dysfunctions, transcription dysregulation, and axonal transport impairments (reviewed in [14,15]). Considering the prominent striatal neurodegeneration in HD, many of the initial investigations using these in vivo or cellular models expressing mHTT have been focused on neuronal impairments. In the past few years, more efforts have been dedicated to understand how other brain cells and notably astrocytes contribute to HD [16]. Indeed, astrocytes are critical for proper brain function since they associate with pre and post- synaptic compartments of the synapse, modulate synaptic neurotransmitter concentrations and provide the neuron with energy and substrates for neurotransmission. In addition, reactive astrocytes may participate to dysfunction in HD brain, although several studies suggest that this may represent a late event in the disease [17,18]. Interestingly, a recent study using a conditional mHTT-expressing BAC HD mouse model demonstrates that astrocyte-specific depletion of mHTT significantly rescues motor, psychiatric-like, neu- ropathological, and electrophysiological phenotypes in mice [19]. This strongly suggests that astrocytes are important contributors to the progression of the deleterious phenotypes observed in HD. Other types of glial cells, such as oligodendrocytes, are also believed to play an important role in the pathology [16]. However, it is currently not known whether similar pathways are affected in glial cells and neurons, and whether HD genetic modifiers identified in neurons could behave similarly in glial cells. It is thus important to improve our knowledge on these questions since discrepancies between these different brain cells may, in part, explain the poor success of clinical trials in HD. To address this issue, we used an in vivo inducible glial model of HD and screened a set of genes, some of which having been previously related to HD by genetic or genomic studies. We uncovered several potent modifiers of HD pathology in glial cells, expanding the number of potential therapeutic targets. Unexpectedly, while some modifiers act in the same direction in neuronal and glial tissues, we discovered that depletion of dCBP in glial cells strongly improved the phenotypes of mHTT expressing animals in these cells, in contrast to previous findings obtained in neuronal tissue [20]. Investigating several putative targets of the CBP acetyltransferase, we found that this phenotypic improvement was lost in Foxo null animals, suggesting that dCBP repress Foxo in mHTT expressing glial cells and that dCBP depletion relieves this repression and contributes to HD mitigation. In agreement with this hypothesis, we showed that genetic interventions in the insulin pathway leading to increased Foxo signaling in the glial cells were also protective.

2. Results 2.1. Genetic Modifiers Can Be Identified in an Adult Glial Inducible Model of HD A few previous studies performed in Drosophila looked at mechanisms of toxicity of mHTT in glial cells [21–26]. However, these studies were not designed to discriminate between developmental and adult-specific defects induced by mHTT. Here, we searched whether we could modify the course of HD in flies by genetic interventions exclusively in adult animals where mHTT is targeted specifically in glial cells. To this aim, we used a RU486-inducible GeneSwitch line under the control of the glial specific promoter repo (repoGS, [21]). The expression of mHTT in repoGS > UAS-HTT-548-128Q flies, induced by RU486 feeding from the first day of adulthood, led to a strong decrease of lifespan (Figure1a) . Induction of the repoGS alone did not impact lifespan in different genetic backgrounds (Figure S1). Compared to other studies using a classical repoGAL4 line, this indicates that mHTT expression in glial cells, specifically at the adult stage, is sufficient to induce toxicity, irrespective of developmental damages. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 16

(Figure 1a). Induction of the repoGS alone did not impact lifespan in different genetic backgrounds (Figure S1). Compared to other studies using a classical repoGAL4 line, this Int. J. Mol. Sci. 2021, 22, 3884 indicates that mHTT expression in glial cells, specifically at the adult stage, is sufficient3 of to 16 induce toxicity, irrespective of developmental damages.

FigureFigure 1. 1. IdentificationIdentification of of HD HD modifiers modifiers in in Drosophila Drosophila glial glial cells cells.. (a ()a )Progeny Progeny issued issued from from a across cross betweenbetween the the GeneSwitch GeneSwitch repoGS repoGS line line and and a UAS a UAS-HTT-548-128Q-HTT-548-128Q transgenic transgenic line lineexhibit exhibit strongly strongly re- ducedreduced lifespan lifespan (mean (mean lifespan lifespan (MLS (MLS)) = 10.7, = 10.7,n = 205)n = w 205)hen wheninduced induced with 100 with µg 100/mLµ ofg/mL RU486 of incor- RU486 poratedincorporated in the infood the at food the atemergence the emergence (RU100), (RU100), compared compared to uninduced to uninduced flies (RU0, flies (RU0, MLS MLS= 52.8, = 52.8,n = 80,n =pLogRan 80, pLogRankk < 10−10 <).10 (b−) 10A). repoG (b) AS repoGS > UAS- >HTT UAS-HTT-548-128Q-548-128Q line was line crossed was crossedwith lines with to linesbe tested to be ortested to various or to variouscontrol lines control (see lines Materials (see Materials and Methods and Methods and Table and S4 for Table detailed S4 for genotypes) detailed genotypes) and the MLSand of the the MLS progeny of the progenywas scored. was The scored. distribution The distribution of the MLS of the was MLS highly was highlyreproducible reproducible for the for con- the trols (MLS = 13.2, 12.0, 12.2; SD = 1.13, 0.66, 0.92; Nexp = 17, 9, 6, respectively, from left to right) controls (MLS = 13.2, 12.0, 12.2; SD = 1.13, 0.66, 0.92; Nexp = 17, 9, 6, respectively, from left to right) while a large dispersion of the MLS in tested lines illustrates the identification of HD modifiers. while a large dispersion of the MLS in tested lines illustrates the identification of HD modifiers. With With a threshold of >20% MLS fold change, 24 enhancers and 22 suppressors were identified. a threshold of >20% MLS fold change, 24 enhancers and 22 suppressors were identified. Next, we constructed a repoGS > UAS-HTT-548-128Q recombinant line and crossed Next, we constructed a repoGS > UAS-HTT-548-128Q recombinant line and crossed it with a set of RNAi lines targeting genes that were selected for their potential role in HD. it with a set of RNAi lines targeting genes that were selected for their potential role in SomeHD. gain Some of gain function of function (GOF) lines (GOF) were lines also were used also in the used course in the of coursethis study. of this A complete study. A listcomplete of the genes list of tested the genes with tested their with mammalian their mammalian orthologues orthologues is provided is provided in Table inS1, Table while S1, thewhile description the description of the genotypes of the genotypes of the tested of the flies tested can flies be found can be in found Table in S2. Table Most S2. of Most the linesof the used lines in usedthis study in this were study validated were validated in previous in previous studies, studies,as indicated as indicated in Table inS4. Table We comparedS4. We compared the lifespan the of lifespan the male of theflies male resulting flies resulting from these from crosses these to crosses the one to of the control one of malescontrol issued males from issued repoG fromS > repoGS UAS-HTT > UAS-HTT-548-128Q-548-128Q crossed crossedwith control with controllines adapted lines adapted to the testedto the genotypes tested genotypes (see M&M (see for M&M the choice for the of choicethese lines). of these In the lines). 27 independent In the 27 independent longevity experimentslongevity experiments performed in performed this study, in we this observed study, we a observedhighly reproducibility a highly reproducibility for the differ- for entthe control different lines control with linesa small with dispersion a small dispersionaround the around mean lifespan the mean (standard lifespan deviation (standard <9%,deviation Figure <9%, 1b). In Figure contrast,1b). Ina much contrast, larger amuch dispersion larger was dispersion observed was for observedthe tested for lines, the inditestedcating lines, that indicating HD glial modifiers that HD glialcan be modifiers identified can in this be identified way. Using in a this stringent way. thresh- Using a old,stringent we considered threshold, as wepositives considered the genetic as positives conditions the genetic where conditionsthe change whereof mean the lifespan change comparedof mean lifespanto the one compared of the control to the lines one of exceed the control 20% (corresponding lines exceed 20% to (correspondinga Z-value of 2.3) to. Witha Z-value this threshold, of 2.3). Withwe identified this threshold, 24 enhancers we identified and 22 24suppressors enhancers ( andTable 22 S3). suppressors The full results(Table of S3). theThe screen full resultsare available of the screenin Table are S2 available and we indiscuss Table below S2 and some we discussof the genes below identifiedsome of the as modifiers genes identified of the mHTT as modifiers longevity of the phenotype, mHTT longevity with a special phenotype, emphasis with aon special con- ditionsemphasis leading on conditions to extended leading lifespan. to extended lifespan.

22.2..2. Known Known M Modulatorsodulators of of N Neuronaleuronal polyQ polyQ-Induced-Induced T Toxicityoxicity E Exhibitxhibit V Variousarious E Effectsffects in HD Adult AGlialdult G Cellslial Cells WeWe first first assayed assayed in in our our adult adult glial glial HD HD model model the the potency potency of of a aset set of of chaperones chaperones to to rescuerescue the the pathology pathology when when overexpressed, overexpressed, as as it it is is the the case case in in several several models models of of polyQ polyQ diseases targeting neuronal cells [27]. Overexpression of two chaperones, Hsc70Cb and diseases targeting neuronal cells [27]. Overexpression of two chaperones, Hsc70Cb and HSP68, and of the DNAJ1 co-chaperone robustly increased the longevity of the HD flies HSP68, and of the DNAJ1 co-chaperone robustly increased the longevity of the HD flies while overexpression of the human HSPA4L chaperone was inefficient (Figure2a). Similarly, like in neurons [28], loss of function of Rab5, which interacts indirectly with mHTT through HAP40, exacerbates the glial pathology while two independent overexpressing lines led to a mild 10–13% increase in lifespan (Figure2a). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 16

while overexpression of the human HSPA4L chaperone was inefficient (Figure 2a). Simi- larly, like in neurons [28], loss of function of Rab5, which interacts indirectly with mHTT through HAP40, exacerbates the glial pathology while two independent overexpressing lines led to a mild 10–13% increase in lifespan (Figure 2a). Reduction of HDAC1 has been shown to protect neuronal cells in various polyQ models, including an HD Drosophila model [29–31]. We found that RNAi inhibition in adult glial cells did not lead to a significant increase in lifespan (Figure 2b). Similarly, we did not find a protective effect after RNAi depletion of genes involved in the kynurenine pathway like the tryptophan 2, 3-dioxygenase (v/TDO) or the kynurenine 3-monooxygen- ase (cn/KMO) (Table S2; Figure 2b), in spite of their involvement in neuronal toxicity in HD and SCA3 [32,33]. Similarly, several genes (faf, Gbeta13, Synd, Taf4, Pgi, and Gapdh1), encoding proteins that interact physically with mHTT and have been identified as modu- lators of toxicity in the fly eye neurons [34], were also unable to change the fly lifespan Int. J. Mol. Sci. 2021, 22, 3884 4 of 16 when inactivated or overexpressed in glial cells (Table S2). Altogether, these results point to the existence of cell-specific modifiers of HD pathology.

Figure 2. Analysis of known neuronal HD modifiers in glial cells. A repoGS > UAS-HTT-548-128Q Figure 2. Analysis of known neuronal HD modifiers in glial cells. A repoGS > UAS-HTT-548-128Q line was crossed with transgenic lines allowing modulation of expression of known neuronal HD line was crossed with transgenic lines allowing modulation of expression of known neuronal HD modifiers.modifiers. The The adult adult progeny progeny (see (see Tab Tablele S4 S4 for for detailed detailed genotypes) genotypes) was was transferred transferred at emergence at emergence on flyon food fly food containing containing RU486 RU486 (100 (100µg/mLµg/mL)) to induce to induce the transgenes the transgenes in glial in cells glial and cells scored and scored for their for lifespan.their lifespan. (a) Overexpression (a) Overexpression of chaperones of chaperones Hsc70Cb Hsc70Cb (UAS (UAS-Hsc70Cb),-Hsc70Cb), HSP68 HSP68 (UAS (UAS-HSP68),-HSP68), the DNAJ1the DNAJ1 co-chaperone co-chaperone (UAS (UAS-DNAJB1)-DNAJB1) and andof Rab5 of Rab5 (UAS (UAS-Rab5)-Rab5) are protective are protective against against mHTT mHTT in- ducedinduced glial glial pathology pathology as compared as compared to control to control (ML (MLSS = 18.7, = 18.7, 18.3, 18.3, 17.5, 17.5, 14.5; 14.5; pLogRan pLogRankk < 10−10 <, 1010−−1010, , −10 −6 1010−10, 10, 10;− n10 =, 10154,−6 ;148,n = 164, 154, 76 148,, respectively). 164, 76, respectively). In contrast, In contrast,RNAi mediated RNAi mediated depletion depletion of Rab5 of(Rab5 Rab5- −10 RNAi)(Rab5-RNAi) is strongly is strongly deleterious deleterious (MLS = 6 (MLS.3; pLogRan = 6.3; pLogRankk < 10 ; n = < 6 100).− Control10; n = 60).-RNAi1 Control-RNAi1 flies: MLS = 12.9, flies: n = 122. (b) RNAi mediated depletion of HDAC1 (HDAC1-RNAi), cinnabar/KMO (cn/cn-RNAi) and MLS = 12.9, n = 122. (b) RNAi mediated depletion of HDAC1 (HDAC1-RNAi), cinnabar/KMO vermilion/TDO (v/v-RNAi) have no significant effect or a slightly deleteriou-s effect on lifespan of (cn/cn-RNAi) and vermilion/TDO (v/v-RNAi) have no significant effect or a slightly deleteriou-s glial HD flies (MLS = 13.2, 13.8, 11.9, 12.8; pLogRank > 10−2, 10−2, <10−4; n = 140, 142, 201, respectively). −2 −2 −4 Controleffect on-RNAi1 lifespan flies: of MLS glial = HD 13.2, flies n = (MLS147. = 13.2, 13.8, 11.9, 12.8; pLogRank > 10 , 10 , <10 ; n = 140, 142, 201, respectively). Control-RNAi1 flies: MLS = 13.2, n = 147. 2.3. Impact of the Modulation of Energy Production Pathways in HD Adult Glial Cells Reduction of HDAC1 has been shown to protect neuronal cells in various polyQ mod- els, includingOne important an HD difference Drosophila between model neurons [29–31]. and We foundastrocytes that RNAiis their inhibition different inregula- adult tionglial of cells energy did metabolic not lead to pathways a significant that increaseresults in in improved lifespan (Figure resistance2b). to Similarly, oxidative we stress did inotn astrocytes. find a protective We addressed effect after the question RNAi depletion of the sensitivity of genes involvedof glial cells in theto energy kynurenine pathways path- perturbationsway like the tryptophan by targeting 2, several 3-dioxygenase enzymes (v /TDO)involved or either the kynurenine in glycolysis 3-monooxygenase (Pgi, Pfk, fbp, Gapdh1,(cn/KMO) and (Table Pyk) or S2; in Figure the Krebs2b), incycle spite (Mdh1, of their Nc73EF, involvement SdhA, and in neuronal SdhB). To toxicity our surprise, in HD RNAiand SCA3-induced [32,33 depletion]. Similarly, of many several of genes these (genesfaf, Gbeta13 did not, Synd impact, Taf4 ,thePgi longevity, and Gapdh1 of ),the encod- HD fliesing proteins(Table S2), that suggesting interact physically that glial cells with may mHTT develop and have some been compensatory identified as mechanisms. modulators Oneof toxicity exception in the was fly the eye pyruvate neurons kinase [34], weregene also(Pyk) unable for which to change expression the flyof one lifespan RNAi when line resultsinactivated in a orstrong overexpressed lifespan increase in glial (Pyk cells- (TableRNAi1, S2). +44%, Altogether, in 4 independent these results experiments, point to the Figureexistence 3a). of A cell-specific lower but modifierssignificant of effect HD pathology.was also observed with a stronger RNAi line (Pyk-RNAi2) which in addition resulted in decreased lifespan when expressed with a re- poGS2.3. Impact line alone, of the Modulationas expected of from Energy [3 Production5]. No such Pathways decrease in was HD Adultobserved Glial with Cells the Pyk- RNAi1One line important (Figure S2). difference The human between ortholog neurons of and Pyk, astrocytes PKM, has is theirbeen differentshown to regulation interact of energy metabolic pathways that results in improved resistance to oxidative stress in astrocytes. We addressed the question of the sensitivity of glial cells to energy pathways perturbations by targeting several enzymes involved either in glycolysis (Pgi, Pfk, fbp, Gapdh1, and Pyk) or in the Krebs cycle (Mdh1, Nc73EF, SdhA, and SdhB). To our surprise, RNAi-induced depletion of many of these genes did not impact the longevity of the HD flies (Table S2), suggesting that glial cells may develop some compensatory mechanisms. One exception was the pyruvate kinase gene (Pyk) for which expression of one RNAi line results in a strong lifespan increase (Pyk-RNAi1, +44%, in 4 independent experiments, Figure3a). A lower but significant effect was also observed with a stronger RNAi line (Pyk- RNAi2) which in addition resulted in decreased lifespan when expressed with a repoGS line alone, as expected from [35]. No such decrease was observed with the Pyk-RNAi1 line (Figure S2). The human ortholog of Pyk, PKM, has been shown to interact physically with mHTT in several studies [34,36] and it is possible that the outcome of Pyk depletion in HD glia is highly sensitive to the level of this protein. This will require further investigations in the future. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 16

physically with mHTT in several studies [34,36] and it is possible that the outcome of Pyk Int. J. Mol. Sci. 2021, 22, 3884 depletion in HD glia is highly sensitive to the level of this protein. This will require further5 of 16 investigations in the future.

Figure 3. Identification of modifiers of mHTT-induced glial pathology involved in energy pro- Figure 3. Identification of modifiers of mHTT-induced glial pathology involved in energy produc- duction. A repoGS > UAS-HTT-548-128Q line was crossed with transgenic lines modulating Pyk tion. A repoGS > UAS-HTT-548-128Q line was crossed with transgenic lines modulating Pyk (Pyk- RNAi1(Pyk-RNAi1 and Pyk and-RNAi2) Pyk-RNAi2) and srl (srl and-GOF)srl (srl-GOF) genes (see genes Table (see S4 for Table detailed S4 for genotypes) detailed genotypes) and the prog- and enythe were progeny scored were for scoredtheir lifespan for their after lifespan induction after with induction RU100. with (a) Adult RU100. glia (a depletion) Adult glia of Pyk depletion with 2 ofindependent Pyk with 2RNAi independent lines results RNAi in increased lines results lifespan in increased of mHTT lifespan expressing of mHTTflies (MLS expressing = 18.7, 14.4; flies pLogRan(MLS = 18.7,k < 10 14.4−10, 10; pLogRank−5; n = 106, <131 10, −respectively).10, 10−5; n = Control 106, 131, flies: respectively). MLS = 13.2, ControlN = 147. flies:(b) Overexpres-MLS = 13.2, sionN = of 147 srl. is(b also) Overexpression strongly protective,of srl is as also shown strongly by two protective, independent as shown experiments by two (MLS= independent 19.9, 18.2; ex- pLogRanperimentsk < (MLS=10−10, 10 19.9,−10; n 18.2;= 144, pLogRank 136, respectively). < 10−10 ,Control 10−10; nflies:= 144, MLS 136, = 13.3, respectively). n = 106. Control flies: MLS = 13.3, n = 106. In HD animals, depletion of succinate dehydrogenase has been observed by several groupsIn [3 HD7,38 animals,]. We found depletion that overexpression of succinate dehydrogenaseof the SdhB enzyme has been(but not observed SdhA), byusing sev- a eralUAS groups transposon [37,38 inserted]. We found upstream that overexpressionof the gene, conferred of the SdhBa weak enzyme protection (but (11% not SdhA),± 3%) againstusing glial a UAS HD transposon induced toxicity inserted (Table upstream S2), although of the this gene, was conferred below our a str weakingent protection thresh- old.(11% ± 3%) against glial HD induced toxicity (Table S2), although this was below our stringentFinally, threshold. we assayed the protection conferred by overexpression of spargel (srl), the DrosophilaFinally, orthologue we assayed of PGC1α, the protection a major conferredregulator of by mitochondrial overexpression biogenesis. of spargel A (srl) previ-, the ousDrosophila study has orthologue shown that of PGC1this treatmentα, a major was regulator efficient of mitochondrialagainst mHTT biogenesis.-induced dysfunc- A previ- tionsous studyin a fly has neuronal shown model that this [39 treatment]. Glial overexpression was efficient against of spargel mHTT-induced resulted here dysfunctions in a strong 50%in a increase fly neuronal of mean model lifespan [39]. Glial (Figure overexpression 3b). This confirms of spargel theresulted interest here of in pharmaceutical a strong 50% in- strategiescrease of aimed mean lifespanat boosting (Figure PGC1α3b). Thisin HD confirms which thecould interest be eff oficient pharmaceutical in various brain strategies cells [40aimed]. at boosting PGC1α in HD which could be efficient in various brain cells [40]. 2.4. HD Modifier Genes Involved in Calcium Signaling 2.4. HD Modifier Genes Involved in Calcium Signaling As perturbations in calcium regulation and signaling have been well documented As perturbations in calcium regulation and signaling have been well documented in in HD neurons, we assayed the importance of some genes in these pathways in HD glia. HD neurons, we assayed the importance of some genes in these pathways in HD glia. Cells use several ways to modulate cytoplasmic Ca2+ levels, notably by using the inositol Cells use several ways to modulate cytoplasmic Ca2+ levels, notably by using the inositol 1,4,5-trisphosphate (InsP3) to activate the inositol 1,4,5-trisphosphate receptor (IP3R) that 1,4,5-trisphosphate (InsP3) to activate the inositol 1,4,5-trisphosphate receptor (IP3R) that releases Ca2+ from the endoplasmic reticulum (ER). Importantly, mutant HTT, ataxin-2 and releases Ca2+ from the endoplasmic reticulum (ER). Importantly, mutant HTT, ataxin-2 ataxin-3 proteins specifically bind to the carboxy-terminal region of IP3R1 in rat neurons and ataxin-3 proteins specifically bind to the carboxy-terminal region of IP3R1 in rat neu- and increase its sensitivity to InsP3 [41,42]. In response to depletion of Ca2+ in the ER store, rons and increase its sensitivity to InsP3 [41,42]. In response to depletion of Ca2+ in the ER Orai and Stim activate a store-operated Ca2+ entry (SOCE) from the extracellular matrix store, Orai and Stim activate a store-operated Ca2+ entry (SOCE) from the extracellular across the plasma membrane, leading to a second rise in cytosolic Ca2+, which is then matrix across the plasma membrane, leading to a second rise in cytosolic Ca2+, which is pumped back into the ER by the SERCA pump [43]. then pumpedIn our Drosophila back into the adult ER gliaby the HD SERCA model, pump RNAi-induced [43]. depletion of Itpr (the single IP3RIn in our fly) Drosophila did not rescue adult the glia fly HD lifespan model, significantly RNAi-induced (Figure depletion4a). A RNAi of Itpr line (the targeting single IP3OraiR in did fly) not did improve not rescue either the thefly lifespan HD phenotype significantly while (Figure additional 4a). A crosses RNAi withline targeting two Orai Oraimutants did not resulted improve in a either 5% to 16%the HD increase phenotype in lifespan. while Moreover, additional RNAi-induced crosses with two depletion Orai mutantsof Stim, resulted the major in partnera 5% to of16% Orai, increase did not in lifespan. result in Moreover, phenotypic RNAi improvement-induced depl (Tableetion S2). ofTherefore, Stim, the wemajor consider partner that of ourOrai, data did do not not result provide in phenotypic evidence for improvement a protection ( ofTable HD S2). glial cells by inhibition of SOCE in Drosophila.

Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 16

Int. J. Mol. Sci. 2021, 22, 3884 6 of 16 Therefore, we consider that our data do not provide evidence for a protection of HD glial cells by inhibition of SOCE in Drosophila.

Figure 4. Figure 4. RepressionRepression of of SERCA SERCA is is protective protective in in HD HD glia glia.. A A repoG repoGSS > > U UAS-HTT-548-128QAS-HTT-548-128Q line line was was crossedcrossed with with transgenic transgenic lines lines modulating modulating Itpr Itpr (Itpr (Itpr-RNAi)-RNAi) or or SERCA SERCA (SERCA (SERCA-RNAi1-RNAi1 and and SERCA SERCA-- RNAi2)RNAi2) (see (see Tab Tablele S4 S4 for for detailed detailed genotypes) genotypes) and and the the lifespan lifespan of of the the progeny progeny after after induction induction with with RU100RU100 was was compared. compared. (a ()a )Adult Adult glia glia depletion depletion of of Itpr Itpr did did not not change change the the median median lifespan lifespan of of the the mHTTmHTT expressing expressing flies flies but but marginally marginally increased increased their their mean mean lifespan lifespan (MLS (MLS = = 13.1 13.1 vs. vs. 12.0 12.0 for for controls; controls; nn = =99, 99, 150 150,, respectively; respectively; pLogRan pLogRankk < 1 <0−4 10).− (4b).) Adult (b) Adult glia gliadepletion depletion of SERCA of SERCA strongly strongly increased increased the −10 meanthe mean lifespan lifespan of the of mHTT the mHTT expressing expressing flies (MLS flies (MLS= 16.4, = 18.3; 16.4, n 18.3;= 59, n150;= 59, pLogRan 150; pLogRankk < 10 , respec- < 10−10, tively). Control-RNAi2 flies: MLS = 11.5, n = 141. respectively). Control-RNAi2 flies: MLS = 11.5, n = 141.

InIn contrast contrast to to these these negative negative results, results, we we found found that that deplet depletionion of of SERCA SERCA in in glial glial cells cells conferredconferred a a strong strong protection protection to the HD fliesflies withwith increasesincreases inin lifespan lifespan ranging ranging from from 39% 39% to to61% 61% with with two two independent independent RNAi RNAi lines line (Figures (Figure4b). 4b). This This suggests suggests that that modulation modulation of theof thecytoplasmic cytoplasmic Ca Ca2+ levels2+ levels in in glial glial cells cells can can be be a therapeutica therapeutic strategy, strategy, like like in neurons.in neurons.

22.5..5. Depletion Depletion of of dCBP dCBP in in G Gliallial C Cellsells M Mitigatesitigates HD HD P Pathologyathology ChangesChanges in in cytoplasmic cytoplasmic calcium calcium concentrations concentrations may may activate activate downstream downstream pathways pathways byby activation activation of of various various kinases. kinases. This This may may result resultss in in the the activation activation of of the the CREB CREB transcrip- transcrip- tiontion factor factor through through interaction interaction with with co co-activators-activators like like CBP/p300 CBP/p300 and and CRTCs CRTCs (Reviewed (Reviewed in [44,45in [44]).,45 Calcium]). Calcium dependent dependent phosphatases phosphatases like likecalcineurins calcineurins act as act negative as negative regulators regulators of thisof thispathway. pathway. We depleted We depleted by RNAi by RNAi some some members members of this of pathway this pathway and assayed and assayed the con- the sequencesconsequences of such of suchdepletions depletions on HD on toxicity HD toxicity in glial in cells. glial cells.We found We found no effect no effecton fly on sur- fly vivalsurvival of depletion of depletion of calcineurin of calcineurin subunits, subunits, the the single single Drosophila Drosophila CRTC CRTC gene gene and and CrebA CrebA (Table(Table S2). S2). Depletion Depletion of of CrebB CrebB significantly significantly reduced reduced the the lifespan lifespan of of HD HD flies flies but but overex- overex- pressionpression of of this this gene gene did did not not impact impact significantly significantly this this phenotype phenotype (Figure (Figure 5a).5a). Strikingly, Strikingly, RNAiRNAi depletion depletion in in glial glial cells cells of of the the single single Drosophila Drosophila CBP/p300 CBP/p300 ortholog ortholog dCBP, dCBP, encoded encoded byby the the nejirenejire (nej(nej) )gene, gene, increased increased lifespan lifespan by by 80% 80% (Figure (Figure 5b).5b). A A similar similar treatment treatment in in adult adult neuronsneurons was was inefficientinefficient to to improve improve mHTT-dependent mHTT-dependent decrease decrease in lifespan in lifespan (Figure (Figure5c). Since 5c). Sincethis linethis hasline not has been not usedbeen inused previous in previous experiments, experiments, we checked we checked that it efficientlythat it efficiently reduced reducednej mRNA nej expressionmRNA expres bysion 60–80% by 60 (Figure–80% S3).(Figure Altogether, S3). Altogether, this suggests this suggests that depletion that de- of pletiondCBP inof DrosophiladCBP in Drosophila adult glia adult may glia specifically may specifically rescue HD rescue pathology HD pathology in glial cells in glial in a CREB independent manner. cells in a CREB independent manner. To understand how dCBP depletion improves the HD lifespan phenotype, we first asked, by qPCR and Western blot analysis, whether it reduced the level of mHTT pro- duced in our glial model. This was not the case neither for the mHTT mRNA nor the pro- tein level (Figure 5d; Figure S4a,b). Indeed, in both cases, we observed a trend towards increased levels when dCBP is depleted. This may result from a better survival of glial cells expressing mHTT, compared to cells containing wild-type levels of dCBP. Then, we assayed several available RNAi or shRNA dCBP lines to ensure that the protective effects cannot be ascribed to repression of an off-target gene. All the tested lines led to an increase

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Int. J. Mol. Sci. 2021, 22, 3884 7 of 16 of fly lifespan, ranging from 40% to 88% (Table S2; Figure 6a). Overexpressing dCBP in glial cells did not change significantly the lifespan of HD flies (Table S2; Figure 6a).

FigureFigure 5. 5. RepressionRepression of of dCBP dCBP is is protective inin HDHD glia.glia. ( a(a,b–)b A) A repoGS repoG >S UAS-HTT-548-128Q> UAS-HTT-548-128Q line line was wascrossed crossed with with transgenic transgenic lines lines modulating modulating genes genes involved involved in the in CREBthe CREB pathway pathway (see Table(see Tab S4 forle S4 de- fortailed detailed genotypes) genotypes) and the and lifespan the lifespan of the progenyof the progeny after induction after induction with RU100 with wasRU100 compared. was compared.(a) Adult (aglia) Adult depletion glia depletion of CrebA of (CrebA-RNAi)CrebA (CrebA- orRNAi) overexpression or overexpression of CrebB of (UAS-CrebB)CrebB (UAS-CrebB) did not did change not changethe median the median lifespan lifespan of the of mHTT the mHTT expressing expressing flies (MLSflies (MLS = 11.0, = 11.0, 13.1; 13.1; pLogRank pLogRan > 0.1;k > 0n.1;= n 74 =, 7 143,4, 143, respectively). In contrast, repression of CrebB (CrebB-RNAi) was deleterious for the flies (MLS respectively). In contrast, repression of CrebB (CrebB-RNAi) was deleterious for the flies (MLS = 8.7; = 8.7; pLogRank < 10−10; n = 80). Control flies: MLS = 11.5, 13.3; n = 121, 106 for control-RNAi2 and pLogRank < 10−10; n = 80). Control flies: MLS = 11.5, 13.3; n = 121, 106 for control-RNAi2 and UAS-control, respectively. (b) Adult glia depletion of nej/dCBP (nej-RNAi1) strongly increases the medianUAS-control, lifespan respectively. of mHTT expressing (b) Adult flies glia (MLS depletion = 25.0; of pLogRan nej/dCBPk < (nej-RNAi1) 10−10; n = 86). strongly Control increases flies: MLS the = median12.4; n = lifespan145. (c) Adult of mHTT neuronal expressing depletion flies of (MLS nej (nej = 25.0;-RNAi1) pLogRank does not < modify 10−10; then = reduced 86). Control median flies: lifespanMLS = of 12.4; neuronaln = 145. mHTT (c) Adult expressing neuronal flies depletion (MLS = 15.8; of nej pLogRan (nej-RNAi1)k > 0.2; does n = not72). modifyControl the flies: reduced MLS = median15.6; n = lifespan 110. (d)of The neuronal level of mHTT expression expressing of huntingtin flies (MLS in =repoG 15.8;S pLogRank > UAS-HTT >- 0.2;548n-128Q= 72). flies Control co- expressingflies: MLS RNAi = 15.6; constructsn = 110. (d targe) Theting level dCBP of expression or control of transgenes huntingtin was in repoGS analyzed > UAS-HTT-548-128Q by Western blot, withflies antibody co-expressing directed RNAi against constructs LaminC targeting as a loading dCBP control. or control The transgenes specificity was of the analyzed human by HTT Western an- 1118 tibodyblot, withwas checked antibody in directed a w strain. against Overall, LaminC the as data a loading do not control. indicate The a reduced specificity expression of the human of mHT HTTT after dCBP depletion. See Table S4 for detailed genotypes and Figure S4 for quantification. antibody was checked in a w1118 strain. Overall, the data do not indicate a reduced expression of mHTT after dCBP depletion. See Table S4 for detailed genotypes and Figure S4 for quantification.

To understand how dCBP depletion improves the HD lifespan phenotype, we first asked, by qPCR and Western blot analysis, whether it reduced the level of mHTT produced in our glial model. This was not the case neither for the mHTT mRNA nor the protein level (Figure5d; Figure S4a,b). Indeed, in both cases, we observed a trend towards increased levels when dCBP is depleted. This may result from a better survival of glial cells expressing mHTT, compared to cells containing wild-type levels of dCBP. Then, we assayed several available RNAi or shRNA dCBP lines to ensure that the protective effects cannot be ascribed to repression of an off-target gene. All the tested lines led to an increase of fly lifespan, ranging from 40% to 88% (Table S2; Figure6a). Overexpressing dCBP in glial cells did not change significantly the lifespan of HD flies (Table S2; Figure6a).

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Figure 6.6. ExtendedExtended longevity longevity and and improved improved locomotor locomotor activity activity of HD of HD flies flies depleted depleted for dCBP for dCBP in glia. in ( aglia.) Several (a) Several independent inde- RNAipendent transgenes RNAi transgenes targeting targeting dCBP and dCBP transgenes and transgenes leading to leading dCBP overexpression to dCBP overexpression (see Table S4 (see for Tab detailedle S4 for genotypes) detailed weregen- assayedotypes) were for their assayed ability for to their modulate ability the to lifespanmodulate of repoGSthe lifespan > UAS-HTT-548-128Q of repoGS > UAS- flies.HTT- All548 the-128Q RNAi flies. lines All tested the RNAi increased lines thetested fly in lifespancreased while the fly the lifespan overexpressing while the linesoverexpressing have no effect, lines irrespective have no effect, of the irrespective sex of the testedof the sex animals of the(F-: tested female animals flies, otherwise(F-: female males).flies, otherwise Error bars: males). standard Error bars: deviation, standard provided deviation, when provided several when independent several independent experiments experiments were performed. were (performed.b) The same (bflies) The were same submitted flies were to submitted a negative geotaxisto a negative assay geotaxis and their assay performance and their evaluated performance as the evaluated mean height as the at Tmean = 7s. height at T = 7s. All the flies performed similarly until day 8 but, from day 12, the repoGS > UAS-HTT-548-128Q flies show All the flies performed similarly until day 8 but, from day 12, the repoGS > UAS-HTT-548-128Q flies show a strong deficit of a strong deficit of performance compared to control flies (p value < 0.0002, two sided t-test) which is almost abolished performance compared to control flies (p value < 0.0002, two sided t-test) which is almost abolished when dCBP is depleted. when dCBP is depleted. At 12 days, all the scores of the RNAi lines were not significantly different from the control line At(p > 12 0. days,1, two all sided the scorest-test). of At the 15 RNAi days, linesonly werethe score not significantlyof the RNAi1 different line was from not thesignificantly control line different(p > 0.1 ,from two sidedthe controlt-test). line At 15(p < days, 0.05, only two thesided score t-test) of the while RNAi1 the linescore was of notthe significantlyRNAi2 and RNA3 different lines from were the significantly control line ( pdifferent< 0.05, two from sided the tcontrol-test) while line the(p < score0.005, of two the sided RNAi2 t- andtest), RNA3 reflecting lines inc wereomplete significantly rescue. different Error bars: from SE the for control N independent line (p < 0.005, experiments two sided (Nt-test), = 2, 2, reflecting 5, 6, 4 at incompleteday 4, 8, 12, rescue.15, 20, respectively). Error bars: SE for N independent experiments (N = 2, 2, 5, 6, 4 at day 4, 8, 12, 15, 20, respectively).

2.6. Depletion of dCBP in Glial Cells Improves Locomotor Defects in HD 2.6. Depletion of dCBP in Glial Cells Improves Locomotor Defects in HD Having established the robustness of the lifespan rescue effect, we asked whether Having established the robustness of the lifespan rescue effect, we asked whether dCBP depletion could improve other phenotypes. We measured the performance of flies dCBP depletion could improve other phenotypes. We measured the performance of flies in in longitudinal geotaxis assays which are widely used in fly models of neurodegeneration. longitudinal geotaxis assays which are widely used in fly models of neurodegeneration. Uninduced repoGS > UAS-HTT-548-128Q expressing a control RNAi construct ex- Uninduced repoGS > UAS-HTT-548-128Q expressing a control RNAi construct exhibit hibit a shallow decrease of performances by day 12 that is more pronounced by day 20. a shallow decrease of performances by day 12 that is more pronounced by day 20. When the When the same flies were submitted to RU486 feeding from the first day of adulthood, same flies were submitted to RU486 feeding from the first day of adulthood, they present they present strong locomotor defects by day 12 where their score is similar to the one of strong locomotor defects by day 12 where their score is similar to the one of 20-days old 20-days old uninduced flies. Four-days and eight-days old flies depleted for dCBP be- uninduced flies. Four-days and eight-days old flies depleted for dCBP behaved similarly to haved similarly to control flies expressing a control RNAi, or to uninduced flies (where control flies expressing a control RNAi, or to uninduced flies (where mHTT is not induced mHTT is not induced by RU486 feeding). However, at days 12 and 15, the performances by RU486 feeding). However, at days 12 and 15, the performances of the dCBP depleted of the dCBP depleted flies were much better than the one of control flies expressing a flies were much better than the one of control flies expressing a control RNAi (Figure6b). controlAt day RNAi 20, their (Figure performances 6b). At day remained 20, their closeperformances to those ofremained uninduced close flies. to those Thus, of dCBPunin- duceddepletion flies. in Thus, adult dCBP glial cells depletion is sufficient in adult to improveglial cells several is sufficie keynt features to improve of HD several pathology key featuresin fly. of HD pathology in fly.

22.7..7. Glia D Depletionepletion of dCBP R Rescuesescues HD in a Foxo Foxo-Dependent-Dependent M Manneranner In addition addition to to its its action action on on histone histone acetylation, acetylation, CBP CBP can can modulate modulate the the activity activity of of tran- tran- scription factorsfactors likelike p53p53 or or Foxo Foxo through through direct direct acetylation acetylation [46 [,46,4747]. It]. can It ca alson also modulate modulate the theWnt Wnt pathway pathway in a in complex a complex manner manner through through interactions interactions with with Beta-catenin Beta-catenin and TCFsand TCFs [48]. [Interestingly,48]. Interestingly, mHTT mHTT has beenhas been shown shown to interact to interact with with p53 p53 [35 ][35] and and to alterto alter the the binding binding of ofBeta-catenin Beta-catenin in thein the destruction destruction complex complex [49], [ while49], while daf16/Foxo daf16/Foxo has beenhas been implicated implicatedin vivo in vivoin protection in protection against against HD in HD worms in worms [50]. [50].

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ToTo assay assay the the involvement involvement of of these these diff differenterent pathways pathways in in the the protection protection conferred conferred by by dCBPdCBP in inglial glial cells cells expressing expressing mHTT, mHTT, we we took took advantage advantage of the of theexistence existence of viable of viable loss lossof functionof function mutants mutants of p53 of andp53 foxoand infoxo fly.in When fly. When we co we-expressed co-expressed mHTT mHTT and an and RNAi an RNAi di- recteddirected against against dCBP dCBP in glial in glial cells cells of flies offlies devoid devoid of p53, ofp53, we still we stillobserved observed an increase an increase of theirof their longevity longevity compared compared to control to control flies flies (Figure (Figure 7a).7a). Thus, Thus, p53 p53 is is dispensable dispensable for for the the dCBPdCBP-mediated-mediated protection protection in in flies. flies.

a. b. c. 120 repoGS> UAS-HTT-548-128Q 120 repoGS>UAS-HTT-548-128Q 120 repoGS>UAS-HTT-548-128Q Control UAS-control 100 100 Control-RNAi1 100 nej-RNAi1/+ nej-RNAi1/+ UAS-Foxo 80 p53-LOF 80 nej-RNAi1/+; Foxo-LOF/+ 80 UAS-Foxo-TM nej-RNAi1; p53-LOF Foxo-LOF UAS-InR-DN 60 60 60

nej-RNAi1/+; Foxo-LOF UAS-Pten

% survival % % survival% % % survival 40 40 40

20 20 20

0 0 0 0 10 20 30 40 0 10 20 30 40 0 10 20 30 40 T (days) T (days) T (days) FigureFigure 7. 7.ExtendedExtended longevity longevity provided provided by by glial glial depletion depletion of ofdCBP dCBP is Foxo is Foxo-dependent.-dependent. We We challenged challenged repoG repoGSS > U >AS UAS-HTT--HTT- 548548-128Q-128Q flies flies exposed exposed to toRU100 RU100 to todifferent different genetic genetic conditions conditions (see (see Tab Tablele S4 S4 for for detailed detailed genotypes) genotypes) and and analyzed analyzed their their lifespan.lifespan. (a ()a )Flies Flies devoid devoid of p53p53 (p53-LOF)(p53-LOF) are are still still strongly strongly responsive responsive to dCBP to dCBP depletion depletion (nej-RNAi1; (nej-RNAi1; p53-LOF) p53- withLOF) lifespan with lifespanincreased increased by 83% by compared 83% compared to controls to controls (MLS =(MLS 21.8 = vs. 21.8 11.9 vs. for 11.9 controls; for controls;n = 100, n = 205, 100, respectively; 205, respectively;pLogRank pLogRan < 10k− 10< ). −10 10(b)). In (b contrast,) In contrast, flies flies devoid devoid of Foxoof Foxo (Foxo-LOF) (Foxo-LOF) are are unable unable to to respond respond toto dCBPdCBP depletion (nej (nej-RNAi1/+;-RNAi1/+; Foxo Foxo-LOF)-LOF) with lifespan similar to controls (MLS = 11.1 vs. 12.9 for controls; n = 107, 180, respectively; pLogRank > 0.2). When only with lifespan similar to controls (MLS = 11.1 vs. 12.9 for controls; n = 107, 180, respectively; pLogRank > 0.2). When one allele of foxo is lost (nej-RNAi1/+; Foxo-LOF/+), flies are still responsive (MLS = 23.9 vs. 12.9 for controls; n = 104, 180, only one allele of foxo is lost (nej-RNAi1/+; Foxo-LOF/+), flies are still responsive (MLS = 23.9 vs. 12.9 for controls; respectively; pLogRank < 10−10). (c) Overexpression of wild type Foxo (UAS-Foxo), activated Foxo (UAS-Foxo-TM) or re- −10 pressionn = 104, of 180 insulin, respectively; pathway pLogRank with overexpression < 10 ). ( cof) Overexpression a dominant negative of wild insulin type Foxoreceptor (UAS-Foxo), (UAS-InR activated-DN) or the Foxo PTEN (UAS- phosphataseFoxo-TM) or(UAS repression-Pten) extend of insulin lifespan pathway in repoG withS overexpression> US-HTT-548-128Q of a dominantflies. (MLS negative = 20.1, 18.9, insulin 18.8, receptor 17.4; n = (UAS-InR-DN)202, 170, 210, 206;or thepLogRan PTENk phosphatase< 10−10, respectively). (UAS-Pten) Control extend flies: lifespan MLS = in11.9, repoGS n = 226. > US-HTT-548-128Q flies. (MLS = 20.1, 18.9, 18.8, 17.4; n = 202, 170, 210, 206; pLogRank < 10−10, respectively). Control flies: MLS = 11.9, n = 226. In contrast, when we performed the same experiment in flies devoid of Foxo, we ob- servedIn a complete contrast, suppression when we performed of the protective the same effect experiment conferred in by flies dCBP devoid depletion of Foxo, (Fig- we ureobserved 7b). Thus, a complete at least one suppression copy of the of foxo the protectivegene is required effect conferredin this HD by protective dCBP depletion dCBP pathway.(Figure7 Moreover,b). Thus, atwe least found one that copy direct of the overexpressionfoxo gene is of required foxo was in able this to HD rescue protective HD pathologydCBP pathway. in glial Moreover,cells, as shown we found in Figure that direct7c, suggesting overexpression that dCBP of foxo depletionwas able could to rescue act byHD increasing pathology Foxo in glialactivity. cells, Since as shown transcriptional in Figure 7activityc, suggesting of Foxo that is also dCBP und depletioner the control could ofact the by insulin increasing pathway, Foxo we activity. assayed Since this transcriptionalpathway and found activity that of attenuation Foxo is also of under insulin the signalingcontrol ofby theexpression insulin of pathway, a dominant we negative assayed thisinsulin pathway receptor and (InR found-DN) thator overexpres- attenuation of insulin signaling by expression of a dominant negative insulin receptor (InR-DN) or sion of the phosphatase PTEN also increased the lifespan of flies expressing mHTT in glial overexpression of the phosphatase PTEN also increased the lifespan of flies expressing cells (Figure 7c). mHTT in glial cells (Figure7c).

2.8.2.8. Identification Identification of ofN Newew HD HD M Modifiersodifiers in in the the Wnt Wnt Pathway Pathway AA potential potential involvement involvement of the of the Wnt Wnt pathway pathway in HD in HDhas hasbeen been previously previously observed observed by Dupontby Dupont and collaborators and collaborators [23], using [23], usinga fly model a fly model in which in which a short a human short human HTT fragment HTT frag- encompassingment encompassing Exon 1 with Exon 93 1CAG with repeats 93 CAG was repeats expressed was during expressed development during development and adult- hoodand in adulthood glial cells inwith glial the cells repoGAL4 with the driver. repoGAL4 RepoGAL driver.4 >RepoGAL4 UAS-Htt-Ex1 > UAS-Htt-Ex1-93Q-93Q flies heter- ozygousflies heterozygous for either arm/beta for either-cateninarm/beta-catenin or pan/dTCFor orpan/dTCF overexpressingor overexpressing the Axin gene the Axin exhibitgene longerexhibit lifespan longer and lifespan improved and improved locomotor locomotor activity compared activity compared to their repoGAL to their4 repoGAL4 > UAS- HTT> UAS-HTT-Ex1-93Q/+-Ex1-93Q/+ controls [23 controls]. This was [23]. also This the was case also when the the case longer when HTT the- longer548-128Q HTT-548- frag- ment128Q was fragment expressed was specifically expressed in specifically adult glial incells adult together glial cellswith togethertransgenes with expressing transgenes a RNAiexpressing targeting a RNAiarm or targeting expressing arm a dominant or expressing negative a dominant dTCF (dTCF negative-DN) dTCFor the (dTCF-DN)Axin gene (Figureor the 8a;Axin Tablegene S2). (Figure This8 indicatesa; TableS2). that This interventions indicates thatin this interventions pathway limited in this to pathway adult- hoodlimited are toalso adulthood efficient areto improve also efficient the topathology. improve theWe pathology.extended the We number extended of the putative number therapeuticof putative targets therapeutic by screening targets byforscreening genes located for genes upstream located or upstreamdownstream ordownstream of dTCF. We of founddTCF. that We RNAi found-mediate that RNAi-mediatedd depletion of depletion Gint3, the of ortholog Gint3, the of ortholog UBXN6, of a UBXN6,positive aregula- positive tor of this pathway [51], was also sufficient to improve the lifespan of HD flies in our

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regulator of this pathway [51], was also sufficient to improve the lifespan of HD flies in our modelmodel ( (FigureFigure 88b).b). This was also the case for depletion of pygopygo andand ktokto ((FigureFigure 88b)b) whichwhich areare members members of of the the Wnt Wnt enhanceosome enhanceosome and and the the associated associated transcription transcription complex complex [ [5252].]. In In contrast,contrast, targeting grouchogroucho(the (the orthologue orthologue of of the the transcriptional transcriptional repressor repressor TLE TLE in mammals) in mam- mals)resulted resulted in decreased in decreased lifespan. lifespan.

FigureFigure 8. 8. IdentificationIdentification of of protective protective genetic genetic interventions interventions in in the the Wnt Wnt pathway. pathway. A A repoG repoGSS > > U UAS-AS- HTTHTT-548-128Q-548-128Q line line was was crossed crossed with with transgenic transgenic lines lines allowing allowing modulation modulation of the of Wnt the Wntpathway pathway and theand progeny the progeny (see Tab (seele Table S4 for S4 detailed for detailed genotypes) genotypes) was scored was scored for their for lifespan their lifespan after induction after induction with RU100.with RU100. (a) Like (a )in Like neurons, in neurons, repression repression of the Wnt of the pathway Wnt pathway in glial in cells glial by cells depletion by depletion of arm of(Arm arm- RNAi)(Arm-RNAi) and overexpression and overexpression of a dominant of a dominant negative negative dTCF genedTCF (UASgene- (UAS-dTCF-DN)dTCF-DN) or of the or ofAxin the geneAxin (UASgene- (UAS-Axn)Axn) are protective are protective for mHTT for mHTT toxicity toxicity (MLS (MLS= 27.4, = 17.4; 27.4, 17.4;n = 142,n = 47; 142, pLogRan 47; pLogRankk < 10−2 <, 10 −10−2, , −6 1010−,10 respectively)., 10−6, respectively). Control Controlflies: MLS flies: = MLS13.3, =n 13.3, = 106.n = Note 106. Notethat repression that repression of the of Wnt the Wnt pathway pathway in glial cells by depletion of arm (Arm-RNAi) increased lifespan by only 11% (pLogRank < 10−2), below in glial cells by depletion of arm (Arm-RNAi) increased lifespan by only 11% (pLogRank < 10−2), our 20% threshold criterium. (b) Additional members of the pathway (Gint3, pygo, kto) also pro- below our 20% threshold criterium. (b) Additional members of the pathway (Gint3, pygo, kto) also tected the flies from glial mHTT toxicity when depleted (MLS = 17.5, 17.9, 27.4, 15.2; n = 202, 170, 210;protected pLogRan thek flies < 1 from0−10, 10 glial−10, mHTT10−10, respectively). toxicity when Note depleted that depletion (MLS = 17.5, of skd 17.9, (Skd 27.4,-RNAi) 15.2; n =increased 202, 170, −10 −10 −10 lifespan210; pLogRank by 17% <(pLogRan 10 , 10k < 1,0 10−8), below, respectively). our 20% threshold Note that criterium. depletion ofIn skdcontrast, (Skd-RNAi) depletion increased of the −8 groucho/TLElifespan by 17% repressor (pLogRank (Gro-RNAi) < 10 ),reduce belowd ourlifespan 20% (MLS threshold = 9.8; criterium. pLogRank In < contrast,10−10; n = depletion37). Control of flies:the groucho/TLE MLS = 13.0, n repressor= 126. (Gro-RNAi) reduced lifespan (MLS = 9.8; pLogRank < 10−10; n = 37). Control flies: MLS = 13.0, n = 126. 3. Materials and Methods 3. Materials and Methods 33.1..1. Drosophila Drosophila Lines Lines and and Culture Culture Methods Methods TheThe repoGS repoGS line line was was previously previously described described in in [ [5353].]. The The UAS UAS-HTT-548-128Q-HTT-548-128Q line line was was kindlykindly provided provided by by F. F. Maschat. Maschat. The The lines lines used used for for genetic genetic screening screening are are listed listed in in Table Table S2 S2 andand were were obtained obtained from from the the Bloomington Bloomington stock stock center center or or the the Vienna Vienna Drosophila Drosophila Research Research CenterCenter (VDRC), (VDRC), except except lines lines related related to to the the drldrl andand drl2drl2 genesgenes that that were were provided provided by by JM. JM. Dura.Dura. The The fly fly food food used used for all experiments contained 82.582.5 mg/mLmg/mL yeast, 34 mg/mL corn corn meal,meal, 50 50 mg/mL sucrose, sucrose, 11.5 11.5 mg/mL mg/mL agar, agar, and and 27.8 27.8 μL/mLµL/mL methyl methyl 4 4-hydroxybenzoate-hydroxybenzoate (stock(stock solution solution 200 200 g/L g/L in in ethanol). ethanol).

33.2..2. Lifespan Lifespan Experiments Experiments

FliesFlies were were collected collected within within 24 24 h h of of eclosion under brief CO 2 anesthesia, housed housed in in groupsgroups of of 30, 30, and and raised raised at at 26 26 °C◦C under under a a 12 12 h h–12–12 h h light light-dark-dark cycle. cycle. They They were were transferred transferred everyevery two two days days on on fresh fresh food, food, and and dead dead flies flies were counted. For For induction, induction, RU486 RU486 was was incorporatedincorporated in in the the fly fly food food from from a a 10 10 mg/mL stock stock solution solution in in ethanol. ethanol. All All the the longevity longevity experimentsexperiments were were performed performed on on male male flies, flies, except except othe otherwiserwise mentioned. mentioned. DuringDuring the the screen, screen, we we paid paid a a particular particular attention attention to to use use adequate adequate controls controls for for each each transgenetransgene (TG) (TG) analyzed. analyzed. Thus, Thus, longevity longevity of of the the flies flies with with different different genotypes genotypes (such (such as as UASUAS-HTT-548-128/+;-HTT-548-128/+; repoGS/TG) repoGS/TG) were were compared compared to to appropriate appropriate control control flies: flies: aa UAS UAS-- mcd8mcd8-GFP-GFP was was used used for for GOF GOF constructs; constructs; a a Sgs1[v21206] Sgs1[v21206] when when TG TG corresponds corresponds to to a a VDRC VDRC line;line; TRiP.HMS02542 TRiP.HMS02542 or or TRiP.JF03252 TRiP.JF03252 lines lines when when TG TG corresponds corresponds to to a TRIP line. These These controlcontrol lines lines target target genes genes that that are are not not expressed expressed in in the the glial glial tissue tissue and and th thusus do do not not interfere interfere withwith the the pathology. pathology. Their Their use use prevented prevented from from detection detection of of any any false false positive positive due due to to GAL4 GAL4

Int. J. Mol. Sci. 2021, 22, 3884 11 of 16

dosage (due to the presence of 2 UAS sequences), non-specific effects linked to activation of RNAi pathways or different genetic backgrounds. Statistical significance of differences in survival curves was evaluated with logrank tests.

3.3. Quantification of Transcripts by qRT-PCR RNA extractions were performed with the PicoPureTM RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) and treated with dsDNase (Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNAs were synthesized from isolated total RNA samples using SusperScript™ III Reverse Transcriptase (Thermo Fisher Scientific). qPCRs were performed with the qPCR Mix (Promega, Madison, WI, USA) on a Light- Cycler480 (Roche, Basel, Switzerland). The ribosomal gene rp49 was used as an internal reference for normalization. The primers used for amplifications were for the HTT gene: 50-TGCCAGCACTCAAGAAGGACAC-30 and 50-TGAGCAGCACGCCAAGAATCAG-30; for the nej/dCBP gene: primers 1: 50-AGGGCGATACGGTCACACT-30 and 50-TGAACTGAT CCTTTTTGATTTGG-30 and primers 2: 50-GTGGGCACTCAGATGGGTATG-30 and 50- CATGCCTGGTATGGCGTTCA-30. Quantifications were made on three to four indepen- dent biological samples for each biological sample. Statistical significance was assessed by unpaired t-test using Prism V6.01 software (GraphPad Software, San Diego, CA, USA).

3.4. Western Blot Analysis Duplicate aliquots of 30 fly heads were lysed at 4 ◦C in 70 µL of KCl Buffer (50 mM Tris–HCl pH8, 10% Glycerol, 5 mM EDTA, 150 mM KCl) supplemented with 1% cOmplete protease inhibitor (Roche). Proteins were separated by SDS-PAGE on 4–12% polyacry- lamide gradient gels and then electrotransferred onto nitrocellulose membranes (Schleicher & Schuell BioScience, Dassel, Germany). Standard immunochemistry protocols were used with primary mouse anti-huntingtin (MAB2166, 1/1000, Sigma-Aldrich, Saint-Louis, MO, USA) and mouse HRP secondary antibodies (1/20,000, OriGene Technologies, Rockville, MD, USA). The primary mouse anti-LaminC (LC28.26, 1/1000, DSHB, Iowa City, IA, USA) was used as a loading control. Immunoreactivity was imaged and quantified with ImmobilonTM Western Chemiluminescent HRP Substrate (Millipore) on an Amersham Imager 600 apparatus (Cytiva).

3.5. Negative Geotaxis Assays Locomotor activity was assessed by negative geotaxis assays. Flies were placed in 5 tubes per genotype by groups of 10 and made to fall to the bottom by tapping the tubes five consecutive times. Movies were acquired using a HF12.5HA-1B Fujinon Camera. Five movies were made on the same samples of flies, separated by a rest period of 2 min. The ability to climb, given as a mean distance for each group of flies, was assessed at 7 s using the Fiji Software. Five independent experiments with such configuration were performed. Statistical analysis was performed with GraphPrism software by comparing each genotype to the control flies or to uninduced flies with an unpaired two sided t-test.

4. Conclusions HD is a devastating neurodegenerative disorder and although the causal dominant mutation in the HTT gene was identified more than 20 years ago, there is still no efficient treatment. The difficulties to uncover such a treatment may come from different reasons: (1) the complexity of the HD pathology since the mHTT protein interacts with hundreds of proteins and disrupts many cellular processes [14]; (2) the ubiquitous expression of HTT, meaning that specific pathways may be differentially affected in different cells types; (3) the fact that HD is not only a degenerative disease but has also a neurodevelopmental component [54]. It is thus important to identify which processes are altered by mHTT in each of these temporal phases. In this study, we searched for genetic modifiers of HD pathology in an in vivo Drosophila model. This model presents two main characteristics—it is dedicated to uncover Int. J. Mol. Sci. 2021, 22, 3884 12 of 16

mHTT induced pathological pathways specifically in glial cells which are under studied as compared to neurons—it specifically addresses pathological mechanisms occurring during adulthood, bypassing any neurodevelopmental effects that could interfere in these mechanisms. To these aims, we took advantage of a newly generated GeneSwitch glial specific line to express mHTT simultaneously to transgenes expressing RNAi or cDNA constructs for a set of 137 candidate genes and scored for changes in fly lifespan. From this screen, we uncovered 24 enhancer and 22 suppressor lines that are listed in Table S3. It would have been desirable to complete this genetic analysis with HD transcriptome data in the context of the suppressor genes. However, such an analysis was prevented by the small proportion of the glial cells in the fly brain (10% of total), resulting in insufficient purification of glial specific mRNAs in our hands. We also did not address the question of the specificity of such suppressors in the different glial cell types, due to the lack of specific GeneSwitch drivers. These two aspects are important directions of research in the future. Overall, our screen identified some genetic interventions that appeared to be efficient in brain cells in all situations, in spite of cell specificities (see below). This includes the protection provided by chaperone overexpression or the downregulation of the canonical Wnt signaling, as shown in this study and previous work. However, in many cases, it did not extend to the glial HD pathology the potency of several genes previously identified in screens for modification of mHTT induced photoreceptors degeneration in Drosophila. For instance, we did not observe significant changes in lifespan when we modulated the expression of p53 or members of the kynurenine pathway. This may reflect the fact that these genes modulate the pathology in some but not all the type of cells. Alternatively, it could result from different susceptibility to mHTT in the developmental phase (which has a major influence on eye phenotypes) and in adulthood. In both cases, it means that therapeutic interventions have to be evaluated at different stages and, when required, with improved targeting of the drugs. The different behavior of modifier genes observed between neurons and glial cells in HD may result from their strikingly different energy metabolism [55]. Indeed, using standard repoGAL4 and elavGAL4 lines to express an HTT-Ex1-93Q toxic fragment, Besson et al. showed that the co-overexpression of the mitochondrial decoupling protein UCP2 in glial cells but not in fly neurons resulted in protection, as revealed by extended lifespan and improved behavior [22]. In this study, we investigated whether downregulation in glial cells of enzymes involved in glycolysis (including the rate limiting Pfk) may protect flies from mHTT toxicity, as it has been observed in neurons [56]. In most cases, this was not the case, emphasizing the difference between the two types of cells. However, surprisingly, partial depletion of pyruvate kinase (Pyk) in glia appeared to protect the HD flies. An interesting potent mechanism to be further explored would be that Pyk depletion contribute to downregulation of the Wnt pathway, a major toxic pathway upregulated in HD (see below), as observed for its mammalian orthologue PKM2 [57,58]. We also document in this study strikingly different responses to mHTT in neurons or in glial cells, as exemplified by modulation of genes controlling calcium homeostasis and the dCBP (nej) gene. In the first case, we did not find improvement in lifespan when SOCE related genes or Itpr, the IP3R1 orthologue, were downregulated in glial cells, in contrast to studies performed in neuronal cell models and mouse models [41,59,60]. Surprisingly, using two independent RNAi constructs, we found that inactivation of the SERCA pump, that is required to replenish the ER with Ca2+, resulted in a significant improvement of the HD glial pathology. This suggests that perturbations of Ca2+ homeostasis may be significantly different in HD neurons and glial cells, a hypothesis that should be further tested in mouse models. The most striking case of HD-induced toxicity discrepancy between neurons and glial cells is illustrated by the nejire gene. To our surprise, we found that depletion of dCBP in adult glia alleviated the toxicity of mHTT in these cells, leading to increased lifespan and improved locomotor activity. Previously, in the framework of a HD fly eye model where a 127Q transgene was expressed, overexpression of dCBP was found to rescue Int. J. Mol. Sci. 2021, 22, 3884 13 of 16

photoreceptors degeneration [20]. A reason for these opposite results could come from the use of the eye as a read-out of toxicity. Indeed, eye development is exquisitely sensitive to loss or gain of function of dCBP [61] which complicates the interpretation of the data. Using the same line than in [20] (nejEP1179) to overexpress dCBP specifically in adult neurons of elavGS > UAS-HTT-548-128Q flies, we found that this intervention did not increase the short lifespan of these HD flies (data not shown). Depletion of the dHDAC1 (rpd3) deacetylase that counteracts dCBP activity, was also unable to rescue reduced fly lifespan in a non-inducible neuronal HD model although it has a positive effect on photoreceptor neurodegeneration during development [30], emphasizing again the differences of toxicity during development and adulthood in polyQ diseases. To our knowledge, no studies have been performed in mouse to investigate stage-dependent effects of CBP modulation on HD pathology, but they would be highly instructive in view of the fly data. How does dCBP depletion in glial cells exert its protective effect in the HD fly model? CBP acetylates both histones and other client proteins and has a plethora of interactors. We focused on some well characterized CBP targets and assayed whether their modulation could improve the HD glial pathology in flies. We did not found evidence for a role of CREB proteins or p53 in the glial protection. In contrast, overexpression of the single foxo gene in flies, either directly or by playing with insulin pathway members, rescued significantly the decrease of fly lifespan. Foxo acetylation by CBP represses its transcriptional activity and this occurs in an oxidative stress (OS) dependent manner in the case of the mammalian Foxo4 [46]. Increased OS is associated to HD, which may result to sub-optimal Foxo activity. Interestingly, the rescue of HD phenotypes by dCBP depletion was fully suppressed in animals depleted for Foxo, supporting such a model. However, we cannot exclude that other targets could be involved in the protective effect of dCBP depletion in glial cells. Indeed, among the new suppressors of the pathology uncovered in this study, pygo, kto, and skd are good candidates to mediate CBP-dependent pathological effects. Pygopus is a member of the evolutionary conserved Wnt enhanceosome [62] that binds Legless (BCL9 in mammals), an interactor of arm/Beta-catenin, and both genes are required for Wingless target gene transcription. Importantly Legless can also bind CBP which may mediate this transcriptional activation [52], likely through the mediator complex subunits Med12 and Med13 (kto and skd) [63]. Thus, taking into account that WNT and FOXO pathways may also directly interact [64,65], additional work is still required to elucidate the mechanisms involved in CBP contribution to HD pathology in glial cells. Altogether, our study emphasizes the need to take into account the cellular hetero- geneity of HD pathological mechanisms that may counteract some therapeutic strategies based on single cell types. Combinatorial approaches combined to specific tissue targeting may thus help to uncovered efficient therapies in HD.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22083884/s1, Figure S1: Induction of repoGS is not toxic in Drosophila glial cells; Figure S2: Depletion of Pyk in glial cells may be deleterious; Figure S3: Validation of RNAi1 for nej depletion; Figure S4: No evidence of reduced expression of mHTT after dCBP depletion; Table S1: Genes analyzed; Table S2: Full results of the screen; Table S3: Modifiers identified in the screen; Table S4: Correspondence between figure legends and full genotypes. Author Contributions: E.M., V.M. and H.T. designed the study. E.M., V.M. and R.H. performed the experiments and performed the statistical analysis. H.T. was the head investigator of the study and wrote the first draft of the manuscript. All authors contributed to the manuscript revision. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by the UE 7th PCRD (TREATPOLYQ, Grant agreement n◦ 264508 PITNGA-2010-264508). Institutional Review Board Statement: Not applicable. Data Availability Statement: Not applicable. Int. J. Mol. Sci. 2021, 22, 3884 14 of 16

Conflicts of Interest: The authors have no conflict of interest to report. All views and opinions expressed in this article are solely those of the author. They do not purport or imply to reflect the opinions or views of Roche Product Development Global Operations, whatsoever. The desig- nations employed or assumptions made in this publication and the presentation of material and analysis therein have not been assessed by Roche Product Development Global Operations and do not imply the expression of any opinion whatsoever on the part of Roche Product Development Global Operations.

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