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  • A61K38/09—Luteinising hormone-releasing hormone [LHRH], i.e. Gonadotropin-releasing hormone [GnRH]; Related peptides
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• • A—HUMAN NECESSITIES
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Definitions

• GnRH neurons and olfactory neurons arise from the same group of progenitor cells in the olfactory placode and GnRH neurons migrate into the hypothalamus of the brain during further embryogenesis. This shared origin highlights a link between the GnRH system and the olfactory system. The present inventors thus further evaluated whether GnRH deficiency could be implied in the cognitive impairments found in cognitive disorders associated with olfactory dysfunction. Detailed description
• GnRH treatment allows reducing the expression of AD-associated proteins, AD being known to be associated with olfactory dysfunction (see e.g. Doty, 2012).
• GnRH is a neurohormone released in a pulsatile manner from GnRH neurons located in the hypothalamus. GnRH expression controls luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion from the anterior pituitary. Differential GnRH pulse frequencies and amplitudes alter the secretion patterns of FSH and LH.
• GnRH is a decapeptide.
• “GnRH” refers to the above GnRH decapeptide and to any water-soluble, ionizable form of GnRH, including free base, salts, or derivatives, homologs, or analogs thereof.
• GnRH refers to gonadorelin, and particularly to:
• Gonadorelin is a synthetic decapeptide that has the same amino acid sequence as endogenous GnRH synthesized in the human hypothalamus, and thus has the same pharmacological and toxicological profile as endogenous GnRH.
• the GnRH is administered in a“pulsatile” manner.
• GnRH is naturally secreted with a specific pulse frequency and amplitude. Said frequency and amplitude vary according to species, genders and age.
• the“pulsatile” administration reproduces the natural endogenous GnRH pulsatile peaks of a middle-age adult (i.e.
• men GnRH pulsatile peaks correspond to an administration of 10 to 40 ng/kg of GnRH every 60 to 180 minutes, particularly of 20 to 30 ng/kg of GnRH every 90 to 150 minutes.
• a typical GnRH pulsatile peak in men is 25ng/kg of GnRH every 120 minutes (see Boehm et al. 2015).
• GnRH- secreting neurons from Human Pluripotent Stem Cells (hPSCs), and in particular from Human Induced Pluripotent Stem Cells (hiPSCs), e.g. hiPSCs established from healthy donor fibroblasts.
• hPSCs Human Pluripotent Stem Cells
• hiPSCs Human Induced Pluripotent Stem Cells
• the production of such GnRH- secreting neurons does not involve the destruction of human embryos.
• the present invention aims at restoring GnRH pulsatile secretion for treating cognitive disorders, particularly associated with olfactory dysfunction.
• GnRH expression is regulated via the action of several miRNAs.
• members of the miRNA-200 family and miR-155 are known to regulate Zebl and Cebpb, respectively, two important repressors of GnRH promoter activators (see Messina et al (2016) as well as in the international patent application published under reference WO2017/182580).
• the present inventors have thus demonstrated that it is possible to restore pulsatile GnRH expression in a patient by overexpressing miRNA-200 family members (referred to as“miR-200”) and/or miR- 155.
• MicroRNAs are small, noncoding RNAs that are emerging as crucial regulators of biological processes.
• “MicroRNA”, “miRNA” or “miR” means a non-coding RNA of about 18 to about 25 nucleotides in length. These miRs could originate from multiple origins including: an individual gene encoding for a miRNA, from introns of protein coding gene, or from poly- cistronic transcript that often encode multiple, closely related microRNAs.
• MiR- 155 has the sequence shown under reference MI0000681 in the miR database and under reference ENSG00000283904 in the Ensembl database.
• the cognitive disorder according to the present invention is age-associated cognitive decline
• Parkinson disease progresses according to five stages known as the Hoehn and Yahr Scale.
• the early stages of Parkinson disease according to the present invention are stages I, II and earlier (i.e.“pre-stage Parkinson disease”). Accordingly, in another specific embodiment, the cognitive disorder according to the present invention is early-stage Parkinson disease.
• the cognitive disorder according to the present invention is associated with olfactory dysfunction (so as to specifically target cognitive impairments associated with GnRH deficiency).
• “Olfactory dysfunction” corresponds to an alteration in the sense of smell. Said alteration may be a total loss of the sense of smell, also called“anosmia”, or to a partial sense of smell which is referred to as“hyposmia”, or“microsmia”.
• Multiple olfactory tests are available to the skilled person who is used to use them for evaluating a patient’s olfactory function. Olfactory tests can be divided into psychophysical tests, electrophysiological tests, and psychophysiological tests (see e.g.
• test implies presenting familiar odorants to the patient who then has to choose the name of the odour from a list of options. Threshold tests can also be used. They aim at determining the lowest concentration of an odorant that can be discerned by a patient.
• FIG. 1 Ts65Dn mice show an age-dependent loss of the ability to recognize new odors and objects.
• A Schematic of experimental design performed to evaluate the ability of the mice to discriminate olfactory and visual cues at different stages during postnatal development.
• B Habituation/dishabituation test was used to assess the ability to differentiate between different odors. First, one odor is presented for four consecutive times, during habituation phase; and then, a new odor is presented during the dishabituation phase.
• C Novel object recognition test was used to evaluate the recognition memory. The object recognition score was calculated as the time that the animal spent exploring a new object during the trial 2, over the total exploration time.
• Ts65Dn mice were unable to differentiate between two distinct odors while they were equally able to recognize the introduction of new objects in their environment when compared to wild-type littermates (WT).
• E At adult ages, Ts65Dn mice showed a loss of the capacity to differentiate both different odors and objects when compared to WT littermates. * p
• Figure 2 Hippocampal and cortical APP, CTF and Tau-Cter expression levels in Ts65dn mice.
• A,B Quantification of protein levels of APP, CTF and Tau-Cter in hippocampus (A) and cortex (B) of 3-months and middle-age adult (8-12-months) Ts65dn and WT male mice, grafted with POA (WT-POA) or ungrafted (Sham).
• C,D Quantification of protein levels of APP, CTF and Tau-Cter in hippocampus (C) and cortex (D) of 3 -months and middle-age adult (8- 12- months) Ts65dn and WT female mice. GAPDH was used as a loading control.
• B-F Representative graphs for LH pulsatility assessment after 15 days of vehicle or LUTRELEF® subcutaneous administration.
• LUTRELEF® pulsatile infusion rescued the capacity to discriminate between different odors (H) and cognitive deficits (I) in Ts65Dn mice.
• FIG. 5 Acute chemogenetic inhibition of GnRH Receptor (GnRH-R) expressing neurons impairs cognitive and olfactory performance in adult control mice.
• A Schematic diagram illustrating the protocol to study the effect of the chemogenetic inhibition of GnRH-R expressing neurons on cognitive and olfactory performance.
• Six-month old Gnrhrr. Cre mice were tested before and after injection with an hM4D(Gi) DREADD viral vector. The two black dots indicate the injection sites of the virus.
• Ts65dn males were smaller and presented significant lower body- weight gain than wildtype littermates during the postnatal maturation and the pubertal transition. A marked delay on puberty onset was observed in male Ts65dn mice compared with wild-type littermates. Ts65dn males exhibited a delay in the balanopreputial separation, a smaller penis and their testicles did not descend into the scrotum, all of them are external signs used to follow postnatal sexual maturation. Body weight at balanopreputial separation was identical between Ts65Dn and wild- type littermates suggesting that retarded growth may be responsible for the delay in sexual maturation.
• Ts65Dn mice presented an irregular profile of the expression of major urinary proteins expression, which excretion in the urine is stimulated by testosterone, also used as a marker of sexual maturation in mice.
• Ts65dn males revealed severe hypogonadism, exhibiting lower testicular weight and smaller testes compared with wild-type mice.
• LH luteinizing hormone
• Ts65dn females Phenotypic characterization of sexual maturation in the females showed that, like in males, the body- weight gain was significant lower in Ts65dn than wild-type littermates during postnatal development and pubertal transition.
• Ts65dn females exhibited delayed vaginal opening, an indicator of the increase in circulating estradiol levels, but no difference was found in the day of the occurrence of the first estrus, which is strictly correlated with the acquisition of reproductive capacity, i.e., puberty.
• Body-weight at vaginal opening and at puberty onset was lower in Ts65Dn females that in wild-type littermates.
• Adult Ts65dn females also presented lower uterus weight in the diestrous phase.
• Ts65dn female mice exhibited regular estrous cyclicity, they showed attenuated fertility with fewer litters produced over a 120-day period and fewer pups per litter, when compared to wild-type littermates. However, no difference was detected in the pattern of LH secretion nor in circulating levels of FSH between Ts65dn and wild-type female mice in diestrus.
• Ts65dn mice exhibit an age-dependent loss of GnRH expression
• GnRH-immunoreactive fibers could indeed be readily traced to the medial habenula and the anteriordorsal amygdala (Ranee et al, 1994) and were often seen to be following or in close association with the wall of the lateral ventricles.
• the GnRH-immunoreactive expansive projection network seen in wildtypes was absent.
• the broad distribution of GnRH- immunoreactive fibers in extra-hypothalamic areas in wildtypes suggests that GnRH neurons, which control species survival, may also be involved in non-reproductive processes. Corollary, the absence of these extrahypothalamic GnRH fibers in Ts65Dn mice raise the intriguing hypothesis that this GnRH deficiency may contribute to the cognitive phenotype in this mouse model of DS.
• Ts65dn mice exhibit an age-dependent loss of olfactory and cognitive functions
• mice To evaluate whether the loss of GnRH-immunoreactivity seen in Ts65dn mice could be associated with olfactory and cognitive decline in these mice, we performed the habituation/dishabituation test to assess the ability of the mice to discriminate between different odors (Breton-Provencher et al, 2009) and the novel object recognition test to assess recognition memory (Leger et al, 2013) in prepubertal (P35, when GnRH immunoreactivity is comparable to control littermates) and adult (>P60, when Ts65Dn mice experience a loss in GnRH immunoreactivity) mice (Fig. la).
• amyloid precursor protein (App) gene triplication present in both DS patients and mice (Reeves et al, 1995) has been linked to the early-onset Alzheimer-disease (AD) phenotype observed in DS.
• AD Alzheimer-disease
• CTF C-terminal fragments
• Tau-Cter Tau C-terminal
• Ts65dn mice show an imbalance in the miRNA-gene network controlling Gnrh expression
• mice with acquired GnRH deficiency To determine whether these mice with acquired GnRH deficiency also recapitulate part of the behavioral phenotype of Ts65dn mice, we subjected adult Gnrh::Cre;DicerloxP/loxP mice to olfactory and cognitive tests.
• miR-99a, let-7c, miR-125b-2 and miR-155 are expressed by GnRH neurons and that their expression significantly increases between P7 and P12, i.e., at the onset of minipuberty (Messina et al., 2016).
• miR155 can also influence the expression of other miRNA species such as the expression of the members of the miR-200 family, which plays an essential role in controlling GnRH expression during postnatal development, including adulthood (Messina et al., 2016).
• miRNA-200 family and miR-155, are known to regulate Zebl and Cebpb, respectively, two important repressors of GnRH promoter activators (Messina et al, 2016).
• POA preoptic area
• GnRH neurons were isolated by fluorescent activated cell sorting (FACS) as was described previously (Messina et al, 2016), at P12, a developmental stage preceding the drastic drop in GnRH expression (data not shown).
• FACS fluorescent activated cell sorting
• miR-200b was selectively overexpressed in the hypothalamus of adult male Ts65dn mice using stereotaxic injections of adeno-associated viral vectors (AAV). Both olfactory and cognitive performances were assessed before and after the viral infection in each one of the mice subjected to this experimental protocol.
• AAV adeno-associated viral vectors
• GnRH replacement therapies reverse olfactory- and cognitive-associated impairments in Ts65Dn mice.
• Neonatal POA implantation did not restore the fertility in Ts65dn males (data not shown) neither estrous cyclicity in aged Ts65dn females (data not shown), showing that the restoration of olfactory capacity and cognition in these mice are uncoupled to the restoration of gonadal function.
• Western blot analysis revealed no change in APP and CTF expression in the cortex and the hippocampus of Ts65dn males after the implantation of neonatal POA compared with Ts65Dn control (Sham) (Fig. 2a-d), showing that the graft-mediated rescue is not associated to visible changes in these AD proteins.
• a decreased expression of the TauCter was seen in the cortex of Ts65Dn grafted with preoptic cells, when compared to sham-treated Ts65Dn mice (Fig. 2b).
• GnRH neurons release their neurohormone in a pulsatile manner; pulsatile GnRH release has been monitored in vivo both in the cerebrospinal fluid (Van Vugt et al., 1985) and in the pituitary portal blood (Clarke and Cummins, 1982). Because of its presence in the CSF, a deficiency in the secretion of GnRH, in addition to impinge on the reproductive function, could also alter the function of GnRH receptor-expressing neuronal populations (Granger et al., 2004; Wilson et al., 2006) in brain areas involved in cognition both in rodents and in humans via volume transmission.
• mice All mice were housed under specific pathogen-free conditions in a temperature controlled room (21-22°C) with a 12h light/dark cycle. The day the litters were bom was considered as day 0 of age (postnatal day 0; P0). Animals were weaned at P21 and were provided with ad libitum access to food and water.
• mice (B6EiC3 Sn.BLi A-T s( 1716)65Dn/DnJ; Stock no. 005252) carrying a partial trisomy of chromosome 16, the orthologous region of human chromosome 21, were purchased from Jackson Laboratories (New Harbor, ME, USA).
• Ts65Dn line has a genetic background wild-type (WT) for the Pde6b gene
• WT genetic background wild-type
• the line was maintained by crossing Ts65Dn trisomic females to Pde6b+ (C57BL/6JEiJ x C3 Sn.BLi A-Pde6b+/DnJ)Fl/J; Stock no 003647) males. This mating system results in WT and Ts65Dn animals.
• mice DicerLoxP/LoxP, Gnrh::Cre (Tg(Gnrhl ::Cre)lDlc), Gnrh::Gfp and Tg(CAG- BoNT/B,EGFP)U75-56wp/J (iBot) mice were a generous gift from Dr. Brian Harfe (University of Florida, FL) (Harfe et al., 2005), Dr. Catherine Dulac (Howard Hughes Medical Institute, Cambridge MA) (Yoon et al, 2005), Dr. Daniel J. Spergel (Section of Endocrinology, Department of Medicine, University of Chicago, IL) (Spergel et al., 1999) and Dr.
• mice were genotyped by PCR using the primers listed in supplementary table SI. Animal studies were approved by the Institutional Ethics Committees for the Care and Use of Experimental Animals of the University of Lille; all experiments were performed in accordance with the guidelines for animal use specified by the European Union Council Directive of September 22, 2010 (2010/63/EU). The sex of the animals used is specified in the text and/or figure legends. The genotype or/and treatment group of animals was blinded for the study except when the morphological or physiological differences were too obvious to be ignored.
• vaginal smears were performed daily and analyzed under an inverted microscope to identify the specific day of estrous cycle.
• Fertility index Female fertility indices were calculated from the number of litters per female during a 120-day long mating.
• Plasma testosterone levels were measured using a commercial ELISA (Demeditec Diagnostics, DEV9911) (Moore et al, 2015) according to the manufacturer’s instructions.
• membranes were washed three times with IX TBST before incubation with the secondary antibody (peroxidase anti-Rabbit IgG (H+L), 1 :2000 dilution, PI- 1000, Vector Laboratories) diluted in blocking buffer for 1 hour at RT. After incubation with secondary antibody, the membranes were washed three times with IX TBST. Immunoreactions were developed using the ECL detection kit (NELIOI; PerkinElmer, Boston, MA) and scanned using a desktop scanner (Epson Expression 1680 PRO).
• mice were sequentially presented with one odour for four consecutive trials for a duration of 1 min, and an inter-trial interval of 10 min was maintained to ensure the replacement of the odour. After four consecutive trials, a second odour was presented during a 1 min trial. Odours (20pl of 1 : 1000 dilution) were administered on a filter paper and placed in a perforated plastic box to avoid direct contact with the odour stimulus. The measurement consisted of recording the total amount of time the mouse spent sniffing the object during different trials.
• mice were euthanized by decapitation and trunk blood was collected for hormone level analyses.
• the preoptic area (POA) of the hypothalamus was dissected using Wecker scissors (Moria, France) under a binocular magnifying glass, placed in dry ice immediately and stored at - 80°C until further processing and assays.
• the sort decision was based on measurements of GFP fluorescence (excitation: 488nm, 50 mW; detection: GFP bandpass 530/30 nm, autofluorescence bandpass 695/40nm) by comparing cell suspensions from Gnrh::Gfp and Gnrh::Gfp;Ts65dn animals, as indicated in supplementary figure S5.
• GFP positive and negative cells were sorted directly into 10 pi extraction buffer [0.1% Triton® X-100 (Sigma- Aldrich) and 0.4 U/mI RNaseOUTTM (Life Technologies)].
• Tissues were cryosectioned (Leica cryostat) at 16 pm for P0 and at 35 pm (free-floating sections) for P12, P35 and adult brains, unless otherwise indicated.
• the animals received the one single intrap eritoneal (ip) injection of GnRH-1 or vehicle 2 hours before the habituation phase.
• ip eritoneal
• the animals received two ip injections of GnRH-1 peptide or vehicle. The first injection was given 2 hours before the start of the trial, and a second one 12 hours after the first injection to promote memory consolidation. On day 2, the animals received the ip injection 2 hours before the start of the first trial.
• the promoter of the rat gonadotropin releasing hormone receptor gene directs the expression of the human placental alkaline phosphatase reporter gene in gonadotrope cells in the anterior pituitary gland as well as in multiple extrapituitary tissues. Endocrinology 145, 983-993.
• AD2 a phosphorylation-dependent monoclonal antibody directed against tau proteins found in Alzheimer's disease. Brain Res Mol Brain Res.
• DynPeak an algorithm for pulse detection and frequency analysis in hormonal time series.

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