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Huntington's disease (HD) is due to a mutation in the gene encoding for Huntingtin protein generating polyQ domain extension. Mutant Htt (mHtt) leads to important dysfunction of the BDNF/TrkB signaling. We previously described the 23aa Htt fragment P42, that attenuated the pathological phenotypes induced by mHtt. We reported that, in the R6/2 mouse model of HD, P42 rescued striatal TrkB level but marginally increased cortical BDNF. In the present study, our aim was to address P42 neuroprotection in presence of an external input of BDNF. We combined P42 administration with environmental enrichment (EE), induced by training in the Hamlet test. We examined the consequences of P42 + EE combination on different phenotypes in R6/2 HD mice: motor and cognitive performances, recorded at early and late pathological stages, and analyzed aggregated mHtt and BDNF levels in forebrain structures. Hamlet exploration (i.e., entries in Run, Hide, Eat, Drink and Interact houses) was gradually impaired in R6/2 mice, but maintained by P42 treatment until week 8. Topographic memory alteration measured at week 7 was attenuated by P42. Motor performances (rotarod) were significantly ameliorated by the P42 + EE combination until late stage (week 12). The P42 + EE combination also significantly decreased aggregated Htt levels in the hippocampus, striatum and cortex, and increased BDNF levels in the cortex and striatum. We concluded that combination between P42 treatment, known to increase TrkB striatal expression, and a BDNF-enhancing therapy such as EE efficiently delayed HD pathology in R6/2 mice. Use of dual therapies might be a pertinent strategy to fight neurodegeneration in HD.

Huntington's disease (HD) is caused by a mutation in the Huntingtin (HTT) protein. We previously reported that the 23aa peptide of HTT protein, P42, is preventing HD pathological phenotypes, such as aggregation, reduction of motor performances and neurodegeneration. A systemic treatment with P42 during the pre-symptomatic phase of the disease showed therapeutic potential in R6/2 mice. We here tested P42 effects when administered during the post-symptomatic phase. The P42 treatment alleviated deficits in motor performances, even when symptoms have already started. Because changes in the level and activity of brain-derived neurotrophic factor (BDNF) have been shown to play a central role in HD, we analysed the influence of P42 on BDNF deficit and associated phenotypes. Our data suggest that P42 is involved in the spatio-temporal control of bdnf and trkB mRNA and their protein levels. Related to this enhancement of BDNF-TrkB signalling, R6/2 mice treated with P42, exhibit reduced anxiety, better learning and memory performances, and better long-term potentiation (LTP) response. Finally we identified a direct influence of P42 peptide on neuronal plasticity and activity. These results suggest that P42 offers an efficient therapeutic potential not only by preventing aggregation of mutant HTT at early stages of the disease, but also by favouring some physiological functions of normal HTT, as P42 is naturally part of it, at the different stages of the disease. This makes P42 peptide potentially suitable not only to prevent, but also to treat HD.

One way to better understand the molecular mechanisms involved in the construction of a nervous system is to identify the downstream effectors of major regulatory proteins. We previously showed that Engrailed (EN) and Gooseberry-Neuro (GsbN) transcription factors act in partnership to drive the formation of posterior commissures in the central nervous system of Drosophila. In this report, we identified genes regulated by both EN and GsbN through chromatin immunoprecipitation ("ChIP on chip") and transcriptome experiments, combined to a genetic screen relied to the gene dose titration method. The genomic-scale approaches allowed us to define 175 potential targets of EN-GsbN regulation. We chose a subset of these genes to examine ventral nerve cord (VNC) defects and found that half of the mutated targets show clear VNC phenotypes when doubly heterozygous with en or gsbn mutations, or when homozygous. This strategy revealed new groups of genes never described for their implication in the construction of the nerve cord. Their identification suggests that, to construct the nerve cord, EN-GsbN may act at three levels, in: (i) sequential control of the attractive-repulsive signaling that ensures contralateral projection of the commissural axons, (ii) temporal control of the translation of some mRNAs, (iii) regulation of the capability of glial cells to act as commissural guideposts for developing axons. These results illustrate how an early, coordinated transcriptional control may orchestrate the various mechanisms involved in the formation of stereotyped neuronal networks. They also validate the overall strategy to identify genes that play crucial role in axonal pathfinding.

International audience; Huntington’s disease (HD) is a progressive neurodegenerative hereditary disease clinically characterised by the presence of involuntary movements, behavioural problems and cognitive decline. The disease-onset is usually between 30 and 50 years of age. HD is a rare disorder affecting approximately 1.3 in 10,000 people in the European Union. It is caused by an expanded CAG repeat in the first exon of the Huntingtin (HTT) gene, leading to an abnormal form of the Huntingtin protein (Htt) (polyQHtt), containing N-terminus, enlarged polyglutamine strands of variable length that stick together to form aggregates and nuclear inclusions in the damaged brain cells. Treatments currently used for Huntington’s disease are symptomatic and aimed at temporally relieving the symptoms of the disease; although some promising therapies are on study, there is no drug capable of stopping disease progression either in the form of delaying onset or slowing disability progression. The utilization of peptides interacting with polyQ stretches or with Htt protein to prevent misfolding and aggregation of the expanded polyQ protein is a fascinating idea, because of low potential toxicity and ability to target very initial steps in the pathophysiological cascade of the disease, such as aggregation or cleavage process. Indeed, several therapeutic peptides have been developed and were found to significantly slow down the progression of symptoms in experimental models of Huntington’s disease. This review is essentially focusing on the latest development concerning peptide strategy. In particular, we focused on a 23aa peptide P42, which is a part of the Htt protein. It is expected to work principally by preventing the abnormal Htt protein from sticking together, thereby preventing pathological consequences of aggregation and improving the symptoms of the disease. In the meantime, as P42 is part of the Htt protein, some therapeutic properties might be linked to the physiological actions of the peptide itself, considered as a functional domain of the Htt protein.

In all metazoans, the expression of group B HMG domain Sox transcription factors is associated with the earliest stages of CNS development. In Drosophila, SoxNeuro (SoxN) is involved in dorso-ventral patterning of the neuroectoderm, and in the formation and segregation of neuroblasts. In this report, we show that SoxN expression persists in a subset of neurons and glial cells of the ventral nerve cord at embryonic stages 15/16. In an attempt to address SoxN function in late stages of CNS development, we have used a chromatin immunoprecipitation approach to isolate genomic regions bound in vivo by SoxN. We identified several genes involved in the regulation of axon scaffolding as potential direct target genes of SoxN, including beat1a, semaphorin2a, fasciclin2, longitudinal lacking and tailup/islet. We present genetic evidence for a direct involvement of SoxN in axonal patterning. Indeed, overexpressing a transcriptionally hyperactive mutated SoxN protein in neurons results in specific defects in axon scaffolding, which are also observed in transheterozygous combinations of SoxN null mutation and mutations in its target genes.

In Drosophila, the subdivision into compartments requires the expression of engrailed (en) and hedgehog (hh) in the posterior cells and of cubitus-interruptus (ci) in the anterior cells. Whereas posterior cells express hh, only anterior cells are competent to respond to the hh signal, because of the presence of ci expression in these cells. We show here that engrailed and polyhomeotic (ph), a member of the Polycomb Group (PcG) genes, act concomitantly to maintain the repression of ci in posterior compartments during development. Using chromatin immunoprecipitation (ChIP), we identified a 1 kb genomic fragment located 4 kb upstream of the ci coding region that is responsible for the regulation of ci. This genomic fragment is bound in vivo by both Polyhomeotic and Engrailed. In particular, we show that Engrailed is responsible for the establishment of ci repression early during embryonic development and is also required, along with Polyhomeotic, to maintain the repression of ci throughout development.

Polycomb group (PcG) genes maintain cell identities during development in insects and mammals and their products are required in many developmental pathways. These include limb morphogenesis in Drosophila melanogaster, since PcG genes interact with identity and pattern specifying genes in imaginal discs and clones of polyhomeotic (ph) null cells induce abnormal limb patterning. Such clones are associated with ectopic expression of engrailed, hedgehog, patched, cubitus interruptus and decapentaplegic, in a compartment specific manner. Our results also reveal negative engrailed regulation by ph in both disc compartments: ph silences engrailed in anterior cells and maintains the level of engrailed expression in posterior ones. We suggest that PcG targets are not exclusively regulated by an on/off mechanism, but that the PcG also exerts negative transcriptional control on active genes.

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles during embryonic development. Although Engrailed is a transcription factor, little progress has been achieved in identifying its target genes. We report here the identification of an effector gene, the β3-tubulin gene, as a direct target of Engrailed. The cytological location of β3-tubulin, 60C, is a strong site of Engrailed binding on polytene chromosomes. Immunostaining analysis of a transgenic line containing a P[β3-tubulin-lacZ] construct shows an additional site of Engrailed binding at the location of the transgene. Molecular analysis allowed identification of several Engrailed binding sites, both in vitro and in vivo, within the first intron of the β3-tubulin locus. Engrailed binding sites identified in vitro are active in larvae. Furthermore, expression of β3-tubulin is de-repressed in the ectoderm of engrailed mutant embryos. Repression of β3-tubulin by Engrailed is also obtained when Engrailed is ectopically expressed in embryonic mesoderm. Finally, two different sets of Engrailed binding sites are shown to be involved in the early and late regulation of β3-tubulin by Engrailed during embryogenesis.

Background Huntington’s disease (HD) is caused by the abnormal expansion of the polyglutamine tract in the human Huntingtin protein (polyQ-hHtt). Although this mutation behaves dominantly, huntingtin loss of function also contributes to HD pathogenesis. Indeed, wild-type Huntingtin plays a protective role with respect to polyQ-hHtt induced defects. Methodology/Principal Findings The question that we addressed here is what part of the wild-type Huntingtin is responsible for these protective properties. We first screened peptides from the Huntingtin protein in HeLa cells and identified a 23 aa peptide (P42) that inhibits polyQ-hHtt aggregation. P42 is part of the endogenous Huntingtin protein and lies within a region rich in proteolytic sites that plays a critical role in the pathogenesis process. Using a Drosophila model of HD, we tested the protective properties of this peptide on aggregation, as well as on different polyQ-hHtt induced neuronal phenotypes: eye degeneration (an indicator of cell death), impairment of vesicular axonal trafficking, and physiological behaviors such as larval locomotion and adult survival. Together, our results demonstrate high protective properties for P42 in vivo, in whole animals. These data also demonstrate a specific role of P42 on Huntington’s disease model, since it has no effect on other models of polyQ-induced diseases, such as spinocerebellar ataxias. Conclusions/Significance Altogether our data show that P42, a 23 aa-long hHtt peptide, plays a protective role with respect to polyQ-hHtt aggregation as well as cellular and behavioral dysfunctions induced by polyQ-hHtt in vivo. Our study also confirms the correlation between polyQ-hHtt aggregation and neuronal defects. Finally, these results strongly suggest a therapeutic potential for P42, specific of Huntington’s disease.

Background In Huntington’s disease (HD), the ratio between normal and mutant Huntingtin (polyQ-hHtt) is crucial in the onset and progression of the disease. As a result, addition of normal Htt was shown to improve polyQ-hHtt-induced defects. Therefore, we recently identified, within human Htt, a 23aa peptide (P42) that prevents aggregation and polyQ-hHtt-induced phenotypes in HD Drosophila model. In this report, we evaluated the therapeutic potential of P42 in a mammalian model of the disease, R6/2 mice. Results To this end, we developed an original strategy for P42 delivery, combining the properties of the cell penetrating peptide TAT from HIV with a nanostructure-based drug delivery system (Aonys® technology), to form a water-in-oil microemulsion (referred to as NP42T) allowing non-invasive per mucosal buccal/rectal administration of P42. Using MALDI Imaging Mass Spectrometry, we verified the correct targeting of NP42T into the brain, after per mucosal administration. We then evaluated the effects of NP42T in R6/2 mice. We found that P42 (and/or derivatives) are delivered into the brain and target most of the cells, including the neurons of the striatum. Buccal/rectal daily administrations of NP42T microemulsion allowed a clear improvement of behavioural HD-associated defects (foot-clasping, rotarod and body weights), and of several histological markers (aggregation, astrogliosis or ventricular areas) recorded on brain sections. Conclusions These data demonstrate that NP42T presents an unprecedented protective effect, and highlight a new therapeutic strategy for HD, associating an efficient peptide with a powerful delivery technology. Electronic supplementary material The online version of this article (doi:10.1186/s40478-014-0086-x) contains supplementary material, which is available to authorized users.

The engrailed gene helps to direct Drosophila melanogaster development by encoding a homeodomain-containing DNA binding protein. To identify genes whose transcription engrailed regulates, we developed a method to isolate genomic sequences to which engrailed protein binds with high affinity. Fragments of genomic DNA were fractionated on an engrailed protein affinity column, and fragments that were retained in the presence of 0.4-1.0 M KCl were isolated and cloned. The isolated fragments include regions of the engrailed and cubitus interruptus gene promoters, both of which are candidate targets of engrailed, and most fragments contain regions that engrailed protein protects from DNaseI digestion. Chromosomal deletions that remove some of the engrailed binding sites (located either at 64D, 96B or 99D) interact genetically with engrailed. Characterization of a transcript encoded in region 64D revealed its dependence on engrailed protein.

Background Protective properties of P42 were studied in different models of Huntington’s disease (HD): HeLa cells, but also Drosophila and R6/2 models of HD. Our results identified that P42 prevents different phenotypes induced by the expression of the mutant protein, including aggregation process, defects in axonal trafficking and neurodegeneration. As a result P42 is able to rescue motor deficits but also anxiety and memory defects. We also showed that P42 is able to prevent but also to treat some of the phenotypes/symptoms of HD. Aims and results The study of the modes of action of P42 identified that P42 acts at different levels: i) it interacts with N17 domain, therefore preventing the first steps of the aggregation process; ii) as a part of the Htt protein, we analysed its role in vesicular trafficking along the axons, and found that P42 is one of the microtubule binding domain of the Htt protein and it is acting as an enhancer of the vesicular trafficking; iii) we also found that P42 is able to enhance the level of expression of cortical BDNF, defective in the context of the disease; iv) finally we identified a role of P42 in neuronal plasticity and activity. Conclusions These data showed that P42 allows different entry points in the disease, explaining its impact in both pre- and post-symptomatic windows. It is able to target the disease at different levels, not only by lowering the negative effect of the mutant polyQ-Htt, but also by enhancing the physiological functions of the normal Htt protein.

In Drosophila, Engrailed is a nuclear regulatory protein with essential roles in embryonic segmentation and in normal development of posterior compartments. One of its regulatory targets appears to be polyhomeotic (ph), a Polycomb group gene. We observed, by immunostaining, that Engrailed protein binds to the site of the polyhomeotic locus in region 2D of polytene chromosomes. The same analysis carried out on a transgenic line containing one copy of a P(ph-lacZ) construct shows an additional Engrailed-binding site at the location of the insert. In vivo, polyhomeotic depends on engrailed function in germ-band-elongated embryos, when engrailed and polyhomeotic genes are expressed in similar patterns. By in vitro immunoprecipitations and gel shift assays, we identified two classes of high affinity Engrailed-binding sites upstream of each of the two polyhomeotic transcription units. DNA fragments containing these sites were also immunoprecipitated from embryonic UV crosslinked chromatin in presence of anti-Engrailed antibody. These results suggest that polyhomeotic activation in germ-band-elongated embryos could be mediated by Engrailed-binding to these sites.

Background P42 is a 23aa peptide lying within hHtt that prevents aggregate formation in HeLa cells and ameliorates axonal transport and motor performances in Drosophila HD models (Mugat et al ., 2008; Arribat et al ., 2013). Additionally, it reduces aggregate formation, weight loss, and brain atrophy, and ameliorates motor performances in R6/2 mouse model (Arribat et al ., submitted). By mass spectroscopy we identified a shorter 14aa fragment, P42B, still present after three hours in P42TAT-containing brain extracts. Aims To test whether the fragment P42B still harbours active properties in HeLa cells and Drosophila HD models. Methods/techniques Cultured HeLa cells were co-transfected with GFP-hHtt171aa-138Q and cherry-P42 or cherry-P42B. P42 and P42B were tested alone, or combined to the transduction peptide TAT to increase cellular penetration. Transgenic flies expressing polyQ-hHtt in salivary glands (MS-1096-Gal4; UAS-HA-hHtt171aa-138Q/+) or eyes (GMR-Gal4; UAS-HA-hHtt67aa-93Q/+) were crossed with flies expressing UAS-P42GFP, UAS-P42TATGFP, UAS-P42BGFP, or UAS-GFP. MS-1096-Gal4; UAS-HA-hHtt171aa-138Q/+; +/+ flies were nourished since L1 with placebo, P42TAT or P42BTAT synthetic peptides containing yeast [0.2 mM] and sacrificed at L3. Aggregate formation was studied in L3 larval salivary glands by immunostaining and quantified with filter retardation assays. The eye degeneration was studied in adult Drosophila at day 0 and day 10. Results/outcome P42B prevents aggregate formation in HeLa cells. In the salivary glands from L3 larvae, immunostaining and filter retardation assays show that P42B, either genetically or orally-administered, is not able to significantly hamper aggregate formation. On the contrary, P42B was clearly able to prevent the eye degeneration in adult flies. Conclusions The efficacy of P42B is phenotype-dependent. This suggests either that the entire P42 peptide is required to fully hamper aggregate formation or that a threshold effects exists explaining the differences according to the phenotype.

Huntington's disease (HD) is caused by the expansion of the polyglutamine (polyQ) tract in the human Huntingtin (hHtt) protein (polyQ-hHtt). Although this mutation behaves dominantly, htt loss of function may also contribute to HD pathogenesis. Using a Drosophila model of HD, we found that Engrailed (EN), a transcriptional activator of endogenous Drosophila htt (dhtt), is able to prevent aggregation of polyQ-hHtt. To interpret these findings, we tested and identified a protective role of N-terminal fragments of both Drosophila and Human wild-type Htt onto polyQ-hHtt-induced cellular defects. In addition, N-terminal parts of normal hHtt were also able to rescue eye degeneration due to the loss of Drosophila endogenous dhtt function. Thus, our data indicate that Drosophila and Human Htt share biological properties, and confirm a model whereby EN activates endogenous dhtt, which in turn prevents polyQ-hHtt-induced phenotypes. The protective role of wild-type hHtt N-terminal parts, specifically onto polyQ-hHtt-induced cellular toxicity suggests that the HD may be considered as a dominant negative disease rather than solely dominant.

International audience; One challenging question in neurogenesis concerns the identification of cues that trigger axonal growth and pathfinding to form stereotypic neuronal networks during the construction of a nervous system. Here, we show that in Drosophila, Engrailed (EN) and Gooseberry-Neuro (GsbN) act together as cofactors to build the posterior commissures (PCs), which shapes the ventral nerve cord. Indeed, we show that these two proteins are acting together in axon growth and midline crossing, and that this concerted action occurs at early development, in neuroblasts. More precisely, we identified that their expressions in NB 6-4 are necessary and sufficient to trigger the formation of the PCs, demonstrating that segmentation genes such as EN and GsbN play a crucial role in the determination of NB 6-4 in a way that will later influence growth and guidance of all the axons that form the PCs. We also demonstrate a more specific function of GsbN in differentiated neurons, leading to fasciculations between axons, which might be required to obtain PC mature axon bundles.

In Drosophila, the ventral nerve cord (VNC) architecture is built from neuroblasts that are specified during embryonic development, mainly by transcription factors. Here we show that Engrailed, a homeodomain transcription factor known to be involved in the establishment of neuroblast identity, is also directly implicated in the regulation of axonal guidance cues. Posterior commissures (PC) are missing in engrailed mutant embryos, and axonal pathfinding defects are observed when Engrailed is ectopically expressed at early stages, prior to neuronal specification. We also show that frazzled, enabled, and trio, all of which are potential direct targets of Engrailed and are involved in axonal navigation, interact genetically with engrailed to form posterior commissures in the developing VNC. The regulation of frazzled expression in engrailed-expressing neuroblasts contributes significantly to the formation of the posterior commissures by acting on axon growth. Finally, we identified a small genomic fragment within intron 1 of frazzled that can mediate activation by Engrailed in vivo when fused to a GFP reporter. These results indicate that Engrailed's function during the segregation of the neuroblasts is crucial for regulating different actors that are later involved in axon guidance.

During embryogenesis and wing disc morphogenesis in Drosophila, different developmental mechanisms are used along the antero-posterior (A-P) axis. The establishment of antero-posterior polarity requires the secreted protein Hedgehog, which is only expressed in P compartments and which is a key effector of the Engrailed transcription factor. At the same time, it is essential that both engrailed and hedgehog (hh) remain in a repressed state in A compartments. In this article, we show that hh is maintained in a repressed state by the Polycomb group (PcG) chromatin proteins. We show that this process takes place during embryogenesis through two genomic elements that display genetic properties of a PRE. Interestingly, hh expression is not regulated by PcG genes in salivary glands, although at the same developmental stage PcG proteins repress hh in the A compartment of the wing disc. In addition, no PcG binding sites were found on polytene chromosomes, neither within hh transgenic constructs nor at the hh endogenous locus. Together, these results suggest that hh repression by the PcG acts in a tissue-specific manner.

Chromatin immunoprecipitation after UV crosslinking of DNA/protein interactions was used to construct a library enriched in genomic sequences that bind to the Engrailed transcription factor in Drosophilaembryos. Sequencing of the clones led to the identification of 203 Engrailed-binding fragments localized in intergenic or intronic regions. Genes lying near these fragments, which are considered as potential Engrailed target genes, are involved in different developmental pathways, such as anteroposterior patterning, muscle development, tracheal pathfinding or axon guidance. We validated this approach by in vitro and in vivo tests performed on a subset of Engrailed potential targets involved in these various pathways. Finally, we present strong evidence showing that an immunoprecipitated genomic DNA fragment corresponds to a promoter region involved in the direct regulation of frizzled2 expression by engrailed in vivo.

Background The starting point of our study was the observation that the ratio between wild-type and mutant polyQ-Htt is crucial in Huntington’s disease progression. Screening on polyQ-hHtt aggregation allowed the identification of a protective 23aa peptide (P42) lying within human Htt. Aims The aim was to test the protective properties of P42 on different HD models: HeLa cells, Drosophila, but also R6/2 mice. Methods/techniques The use of P42 at therapeutic ends in a mammalian model, required that this peptide was able to cross the blood-brain-barrier, and that we find a non-invasive route for chronic administration of P42. To this end, we associated two complementary strategies: i) we fused P42 to the protein transduction domain TAT to ensure its diffusion; ii) we adapted a nanostructure-based drug delivery system for brain targeting called Aonys®, where P42-TAT was formulated in a water-in-oil micro-emulsion (NP42T) and administered through buccal and/or rectal mucosa. In order to verify the pertinence of this way of administration, we also used an original imaging analysis of P42 derivatives, using MALDI Imaging Mass Spectrometry on brain sections. Results/outcome - We first verified that the fusion P42-TAT peptide did not change the functionality of P42, and identified a dose-response effect of P42-TAT action in HeLa cells. - To investigate the therapeutic potential of NP42T, buccal and rectal administrations were performed daily in R6/2 HD mouse model. The effects of NP42T treatment were analysed on a range of behavioural associated defects (foot-clasping, rotarod or body weights), and several markers (aggregation, astrogliosis or ventricle areas) recorded on brain sections. Conclusion Our data globally identify an efficient protective effect of the NP42T, leading to a clear recovery of all the phenotypes tested. This study not only shows that we identified an efficient peptide against HD, but it also describes a powerful delivery technology.

Engrailed is a nuclear regulatory protein with essential roles in embryonic segmentation and wing morphogenesis. One of its regulatory targets in embryos was shown to be the Polycomb group gene, polyhomeotic. We show here that transheterozygous adult flies, mutant for both engrailed and polyhomeotic, show a gap in the fourth vein. In the corresponding larval imaginal discs, a polyhomeotic-lacZ enhancer trap is not normally activated in anterior cells adjacent to the anterior-posterior boundary. This intermediary region corresponds to the domain of low engrailed expression that appears in the anterior compartment, during L3. Several arguments show that engrailed is responsible for the induction of polyhomeotic in these cells. The role of polyhomeotic in this intermediary region is apparently to maintain the repression of hedgehog in the anterior cells abutting the anterior-posterior boundary, since these cells ectopically express hedgehog when polyhomeotic is not activated. This leads to ectopic expressions first of patched, then of cubitus interruptus and decapentaplegic in the posterior compartment, except for the dorsoventral border cells that are not affected. Thus posterior cells express a new set of genes that are normally characteristic of anterior cells, suggesting a change in the cell identity. Altogether, our data indicate that engrailed and polyhomeotic interactions are required to maintain the anterior-posterior boundary and the posterior cell fate, just prior to the evagination of the wing.

The Drosophila Engrailed homeoprotein has been shown to activate directly a Polycomb-group gene, polyhomeotic, during embryogenesis. The molecular mechanism involved in this activation has been studied. Two different types of Engrailed-binding fragments have been detected within the polyhomeotic locus. The P1 and D1 fragments contain several 'TTAATTGCAT' motifs, whereas the D2 fragment contains a long 'TAAT' stretch to which multiple copies of Engrailed bind cooperatively. Another homeodomain-containing protein, Extradenticle, establishes protein-protein interactions with Engrailed on the D2 fragment. We have shown by CAT assays that both types of Engrailed-binding sites (P1 or D1 and D2), as well as Extradenticle, are necessary to obtain activation by Engrailed. In vivo, we have also shown that normal polyhomeotic expression depends on extradenticle expression. Moreover, in the absence of Extradenticle, overexpression of Engrailed protein represses polyhomeotic expression.

Background Huntington9s disease (HD) pathogenesis results from a dominant effect of polyQ-hHtt and a loss of function of Htt. Indeed, addition of wild-type Htt improves polyQ-hHtt-induced defects. Aims Screening on polyQ-hHtt aggregation in HeLa cells, we identified an inhibitory 23aa peptide (Pep42) lying within human Htt. Interestingly Pep42 acts specifically on HD model through a direct interaction with N17 domain, suggesting a role of Pep42 on nucleation and aggregation processes. The protective properties of Pep42 were confirmed on different polyQ-hHtt-induced phenotypes in HD Drosophila model. Therefore Pep42 presents a clear therapeutical potential that we further tested in R6(2) mice. Methods/techniques In order to attempt to cross the brain-blood barrier, Pep42 was fused to TAT-HIV. Then, we compared its protective effect after intraventricular injections, intravenous injections, vs buccal and rectal mucosa administrations in a water-in-oil microemulsion-based delivery vector (called Aonys, MedesisPharma). Results/outcome Pharmacokinetics analysed on brain sections at 6hrs and 24hrs after Pep42 treatment showed a nice spread of the peptide in the brain, still visible at 24 h. Finally, administration of Pep42/Aonys or empty vector was performed daily in 2 wks–11 wks old R6(2) mice for pre-symptomatic treatment and in 9–11 wks old R6(2) mice for post-symptomatic treatment. Several polyQ-hHtt behavioural associated defects were analysed (rotarod, foot-clasping or body weights), and several markers were followed on brain sections. These data globally show the protective effect of Pep42-TAT when delivered through Aonys vector, in both pre- and post-symptomatic treatments. Conclusions Altogether these data highlight a new therapeutic strategy for HD, associating an efficient peptide with a powerful delivery technology.

Six major transcripts encoding the α, β, and γ subunits of the larval serum protein LSP-1, the single subunit of the LSP-2, and the P1 and P6 polypeptides appear sequentially in the fat body tissue during the third larval stage of Drosophila melanogaster. Northern blot analysis shows that the transcription of the LSP-2 and P1 mRNAs is restricted temporally to the third instar and the early phase of pupal development and spatially to the fat body tissue. The synthesis and/or accumulation of the LSP-2 and P1 transcripts are differentially affected by mutations of the dor and occ loci of the 2B5 complex of genes involved in the trans-regulation of the activity of ecdysteroid-responsive genes. Application of the P element-mediated germ line transformation to the P1 gene shows that correct developmental expression of this gene requires no more than 1.5 kb of DNA sequences upstream from the mRNA start site.

P-element-mediated transformation was used to investigate 5' cis-acting regulatory sequences flanking the P1 gene of Drosophila melanogaster, which is selectively expressed in the fat body of late third instar larvae under the positive control of ecdysone. A hybrid gene was constructed by fusing a 1.5 kb DNA fragment directly adjacent to and including the first 25 transcribed bases of the P1 gene to the Escherichia coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene itself linked at its 3' end to the SV40 t antigen splicing and polyadenylation sequences. Five transformed lines of D. melanogaster containing only one copy of the hybrid gene were established. In each of these lines the gpt sequence is transcribed with the same spatial, temporal and hormonal specificities as those of the P1 gene. This provides evidence that control elements essential for the ecdysone and developmentally regulated expression of P1 are located within a 1.5 kb region 5' to this gene.

Publisher Summary This chapter discusses steroid-regulated vertebrate genes. The most extensively studied steroid-responsive gene is the mouse mammary tumor virus (MMTV) provirus. In a variety of cell types, chromosomally inserted proviruses give rise to a single transcript, initiating within the left long terminal repeat (LTR). This transcript is present at very low levels until the cells are exposed to a glucocorticoid hormone. Within minutes following hormone treatment, the titre of the MMTV transcript rises, reaching a level that is at least 50-fold above the basal level within 2 hours. Glucocorticoid receptor binding sites are located within the MMTV LTR, and the same region of the LTR is both necessary and sufficient to confer glucocorticoid inducibility. Receptor binding sites are clustered between 80 and 300 base pairs upstream of the transcriptional initiation sites, and additional sites are present at several positions internal to the transcript. The chapter discusses ecdysteroid-responsive genes from drosophila and glue genes.

Development in two wing-scalloping mutants ( scalloped and vestigial ) was analysed in mosaic flies. In strong alleles of these mutants the wings are reduced to vestiges. When a temperature-sensitive allele of scalloped was treated with heat pulses, particular wing areas were eliminated in a specific order. Homozygous mutant clones were made in heterozygous flies: Marginal clones in either the dorsal or ventral wing surface caused local scalloping, affecting both surfaces to the same extent. Internal Minute + clones survived well when surrounded by weaker Minute cells, but poorly when surrounded by normal Minute + cells. Large Minute + clones of either scalloped or vestigial could make distal parts that are never seen in the wings of mutant flies.

The Pl gene, together with the LSP-1 alpha, -1 beta, and -1 gamma, LSP-2, and P6 genes, is expressed exclusively in the larval fat body of D. melanogaster during the third instar. In vivo mapping of the cis-acting regulatory sequences of the P1 gene was carried out using hybrid constructs with three different reporter genes and a combination of transient and germline transformation assays. This revealed that regulatory elements involved in the setting up of the temporal and spatial specificities of transcription of the P1 gene are located in a short DNA region immediately upstream of the mRNA transcription start. This region includes an element that behaves as a fat-body transcriptional enhancer and element(s) required for ecdysone inducibility of transcription of the P1 gene.

Huntington’s disease (HD) is a rare genetic neurodegenerative disorder. Curative treatments are still actively sought. HD is induced by a mutation in the HTT gene inducing an abnormal expansion of the polyQ domain contained in the Huntingtin protein (Htt). Mechanisms and consequences of this mutation are now well described and allowed to identify an interaction of the Htt with the brain derived neurotrophic factor (BDNF) signaling pathway. BDNF is a neurotrophic factor, which plays important roles, through TrkB, one of its receptor, in neuronal development and plasticity. Mutant Htt (mHtt) down-regulates BDNF and TrkB transcription and transport along the axons.P42, a part of the Htt protein, is a 23aa peptide able to rescue HD pathological phenotypes, such as aggregation, axonal transport and neuronal viability.The aim of my PhD was to better understand the mechanisms of action of P42, in a purpose to optimize its therapeutic potential. To this end I developed different studies using different models.In a paper now accepted for publication in HMG, I first used a P42-based treatment on R6/2 HD mice, to analyze the effect of P42 on the BDNF/TrkB signaling pathway. To this end I analyzed pathologic phenotypes: behaviors or cell mechanisms developing in R6/2 mice and are related to the BDNF/TrkB pathway. I also measured BDNF and TrkB, mRNA or protein levels in both striatum and cortex. What I found is that P42 is acting on BDNF/TrkB pathway mainly by increasing the protein level of TrkB in the striatum.To observe the effect of P42 on vesicular transport, I rather used a Drosophila model, to perform live imaging based studies, in different transgenic conditions: with or without mHTT or P42.Also, in a way to better follow the progression of different pathological phenotypes and the effect of treatments on R6/2 mice, I benefited from a very recent and innovative tool, the HAMLET, which allows a multi-behavioral test.Finally, a bitherapy was used on R6/2 mice combining P42 and P3, a peptide raised against PolyQ mRNA that are also toxic.All those results contribute or will contribute to a better understanding of P42 mechanisms of action.; La maladie de Huntington (MH) est une maladie neurodégénérative progressive héréditaire. Aucun traitement curatif n’a encore été trouvé. La MH est provoquée par une mutation dans le gène HTT induisant l’augmentation anormale du domaine PolyQ (>36) contenue dans la Huntingtine (Htt), la protéine codée par le gène HTT. Les conséquences et les mécanismes de cette mutation sont maintenant bien décrits et ont permis d’identifier une interaction importante entre la Htt et la voie de signalisation du BDNF. Le BDNF est un facteur neurotrophique qui joue des rôles importants, à travers l’activation de son récepteur TrkB, dans le développement et le maintien des neurones et également dans la plasticité des réseaux synaptiques. La Htt mutante (mHtt) diminue l’expression et le transport du BDNF et de TrkB dans les neurones.P42, une partie de 23 acides aminés de la Htt, est capable de sauver de nombreux phénotypes pathologiques induits dans la MH.L’objectif de ma thèse était de mieux comprendre les mécanismes d’action de P42, dans le but d’optimiser son potentiel thérapeutique. Pour cela, j’ai développé plusieurs expériences sur plusieurs modèles. Dans un article, publié dans HMG, je montre les effets d’un traitement avec P42 sur la voie de signalisation BDNF/TrkB dans un modèle de MH : les souris R6/2. Pour cela, j’ai analysé plusieurs phénotypes pathologiques, comportements et mécanismes cellulaires développés par les souris R6/2 et connus pour être dépendants de la voie BDNF/TrkB. J’ai également mesuré les taux d’ARNm et de protéine, du BDNF et de TrkB, dans le cortex et le striatum. Ce que j’ai trouvé est que P42 agit sur la voie BNDF/TrkB principalement en augmentant l’expression de TrkB dans le striatum.Afin d’observer l’effet de P42 sur le transport vésiculaire, j’ai également utilisé la drosophile comme modèle. Grâce à ce modèle, j’ai pu observer in vivo le transport vésiculaire dans différentes conditions avec ou sans mHtt ou P42.Egalement, dans le but de mieux suivre l’évolution des différents phénotypes pathologiques induits par la MH et l’effet du traitement par P42, j’ai expérimenté le Hamlet test®, un test innovant multi-comportemental.Enfin, j’ai observé sur les souris R6/2, l’effet d’une bithérapie P42 avec P3, un peptide ciblant les effets toxiques induits par les ARN codants pour un PolyQ.Tous ces résultats permettent ou vont permettre de mieux comprendre les mécanismes d’action de P42.