Activator of primary cilia to treat cognitive dysfunctions or ciliopathies

Abstract

The present invention relates to the field of treat age-related cognitive declines. In this study, the inventors report that GPR158, which localizes at the PC, is required for OCN- dependent autophagy induction in hippocampal neurons. Consistently, the mobilization of key autophagy players by OCN in hippocampal neurons requires core PC-proteins. In aged hippocampi, neuronal PC present an abnormal morphology, correlated with a reduction of the major core ciliary proteins levels. Conversely, restoration of their levels in aged hippocampi is sufficient to improve autophagy and cognitive deficits. Lastly, they show that core PC-proteins are required to integrate the rejuvenating effects of OCN in the hippocampus, thereby ameliorating age-related cognitive deficits. Altogether, these findings demonstrate a new paradigm for the neuron / systemic milieu communication and significantly advance our understanding of the regulatory mechanism mediating the rejuvenating effects of the pro-youth blood factor, OCN, in hippocampal neurons. This study also provides the foundation for novel therapeutic strategies to treat age-related cognitive decline, as well as neurological deficits in ciliopathies. Thus, the present invention relates to an activator of the primary cilia (PC) for use in the restoration and / or improvement of cognitive functions in a subject in need thereof.

Description

[0001] ACTIVATOR OF PRIMARY CILIA TO TREAT COGNITIVE DYSFUNCTIONS OR CILIOPATHIES

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to an activator of the primary cilia (PC) for use in the restoration and / or improvement of cognitive functions in a subject in need thereof.

[0004] BACKGROUND OF THE INVENTION:

[0005] During aging, organisms undergo a decline in tissue function, leading to reduced systemic, cellular, and organ performance. One of the functional consequences of aging in the brain is the decrease cognitive capacity (1-3). Among the different brain regions affected by aging, the hippocampus is particularly susceptible (3-5). Central hallmarks of hippocampal aging include alterations in homeostasis and adaptive functional properties of the neuron, leading to deficits in synaptic plasticity and memory function (4,6-9). Many efforts are now focused on the identification of molecules and pathways involved in these processes to develop novel and broadly effective preventive or therapeutic strategies to treat age-related memory deficits and associated disorders.

[0006] Growing evidence demonstrates the functional importance of blood-factors-mediated inter-tissue communication in brain aging (9-11). Indeed, multiple reports have shown that administration of plasma derived from young animals is sufficient to improve hippocampal- dependent cognitive and neuronal impairments in aged mice (12-15). Moreover, it was shown that blood factors are essential to transfer the rejuvenating effects of lifestyle interventions, such as exercise on cognition to the aged brain (9,16). Leveraging technological advances in proteomics and transcriptomics analyses led to the identification of pro-youth blood-borne factors that can foster neuronal homeostasis and cognitive fitness. Among them, the inventors and others have identified the bone-derived hormone Osteocalcin (OCN), which crosses the blood-brain barrier and impacts neuronal homeostasis and cognitive fitness via activation of its receptor GPR158, a G-coupled receptor expressed in hippocampal neurons (17-19). Circulating OCN levels are dramatically reduced in aged mice, while their restoration is necessary and sufficient to improve age-related cognitive deficits (18,19,20).

[0007] The inventors recently demonstrated that autophagy machinery is required to mediate the effects of OCN in hippocampal neurons (20). Autophagy is a catabolic cellular process, whereby proteins and organelles are engulfed in autophagosomes (APs), formed by the activity of different autophagy (ATG) proteins, and then transported to lysosomes for degradation, contributing directly to cell metabolism and energy production (21,22,23). In terminally differentiated cells such as neurons, autophagy -mediated degradation of damaged intracellular material is key to maintain cellular homeostasis and promote cell survival (24).

[0008] Beyond its basal functions, autophagy pathway can also be induced by various physiological stimuli (stress, hormones, nutrients) to mediate adaptive cellular responses to changes in environmental conditions, influencing important physiological functions, such as energy metabolism, appetite and control of host defenses (25-27). In the brain, autophagy contributes to the adaptive neuronal response to novel stimuli and memory formation, by promoting activity-dependent synaptic plasticity in hippocampal neurons (20,28-31). During aging, neuronal autophagy levels are reduced in the hippocampus, which contributes to a progressive decline in hippocampal-dependent memory functions (20,31,32). Conversely, administration of factors from young blood is necessary and sufficient to restore the level of essential autophagy related proteins in hippocampal neurons of old mice. Moreover, these proteins are essential to mediate the antiaging effects of systemic factors, such as OCN or GDF11 (Growth differentiation factor 11) on age-related memory deficits (20,32,33). This is explained, in part, by the fact that maintenance of autophagy-related protein levels in hippocampal neurons is a prerequisite to foster cognitive fitness, influencing the capacity of neurons to conserve their ability to integrate novel stimuli (20). However, the regulatory mechanisms mediating the induction of neuronal autophagy by pro-youthful blood factors, remain largely elusive.

[0009] Recent work in the kidney has shown that autophagic machinery can be mobilized in response to systemic stimuli by direct or indirect connection with core primary cilia (PC)- proteins, namely intraflagellar transport IFT (IFT20 and IFT88) or Kinesin Family Member 3 A (KIF3A) (34-36). In neurons, the link between PC and autophagy machinery has not yet been characterized.

[0010] PC are non-motile single organelles with sensory functions, acting as cellular ‘antenna’ that sense and transmit changes in the systemic milieu (37,38). Importantly, PC are hubs for various G protein-coupled receptors (GPCRs), which represent one of the largest families of membrane protein sensors, involved in many vital physiological processes, including hormonal systems (39-41). This suggests that PC could be a dedicated platform for GPCRs to transmit changes in the systemic milieu via activation of specific signaling pathways (42,43). Interestingly, mutations in ciliary proteins are implicated in a wide variety of genetic disorders, known as ciliopathies, which, among other phenotypes, lead to neurological defects (44). Previous research indicates that depletion of core cilia proteins in different brain regions leads to altered cognitive function (45-47). Taken together, these observations led us to hypothesize that PC and / or core PC-proteins constitute a gateway in hippocampal neurons through which youthful systemic factors, such as OCN, modulate neuronal autophagy machinery and therefore cognitive fitness.

[0011] SUMMARY OF THE INVENTION:

[0012] In this study, the inventors report that GPR158, which localizes at the PC, is required for OCN-dependent autophagy induction in hippocampal neurons. Consistently, the mobilization of key autophagy players by OCN in hippocampal neurons requires core PC- proteins. In aged hippocampi, neuronal PC present an abnormal morphology, correlated with a reduction of the major core ciliary proteins levels. Conversely, restoration of their levels in aged hippocampi is sufficient to improve autophagy and cognitive deficits. Lastly, they show that core PC-proteins are required to integrate the rejuvenating effects of OCN in the hippocampus, thereby ameliorating age-related cognitive deficits. Altogether, these findings demonstrate a new paradigm for the neuron / systemic milieu communication and significantly advance our understanding of the regulatory mechanism mediating the rejuvenating effects of the pro-youth blood factor, OCN, in hippocampal neurons. This study also provides the foundation for novel therapeutic strategies to treat age-related cognitive decline, as well as neurological deficits in ciliopathies.

[0013] Thus, the present invention relates to an activator of the primary cilia (PC) for use in the restoration and / or improvement of cognitive functions in a subject in need thereof.

[0014] Particularly, the invention is defined by its claims.

[0015] DETAILED DESCRIPTION OF THE INVENTION:

[0016] A first aspect of the present invention relates to an activator of the primary cilia (PC) for use in the restoration and / or improvement of cognitive functions in a subject in need thereof.

[0017] In other word, the invention relates an activator of the primary cilia (PC) for use in the treatment of cognitive troubles in a subject in need thereof.

[0018] In one embodiment, the cognitive functions are knowledge, attention, memory and working memory, judgment and evaluation, reasoning and computation, problem solving and decision making, comprehension and production of language. In one embodiment, the cognitive troubles are troubles in knowledge, attention, memory and working memory, judgment and evaluation, processing speed, reasoning and computation, executive functioning, visuospatial abilities, problem solving and decision making, comprehension and production of language.

[0019] Thus, in one embodiment, the invention relates to an activator of an activator of the primary cilia (PC) for use in the restoration and / or improvement of memory in a subject in need thereof.

[0020] According to the invention, the activator of the primary cilia (PC) may be used in old people with age-related memory decline.

[0021] Thus, the invention also relates to an activator of the primary cilia (PC) for use in the restoration and / or improvement of the age-related memory decline or age-related memory loss in a subject in need thereof.

[0022] Thus, the invention also relates to an activator an activator of the primary cilia (PC) for use in the treatment of dementia in a subject in need thereof.

[0023] In others words, the invention relates to an activator of the primary cilia (PC) for use in the prevention and / or reversion of the deleterious effects of aging on cognitive function.

[0024] In one embodiment, the invention relates to an activator an activator of the primary cilia (PC) for use in the prevention and / or reversion of the deleterious effects of aging on memory.

[0025] According to the invention, the activator of the invention may be used in people with diseases which have an impact on the memory. Such diseases may be neurological disorders or diseases like Alzheimer’s disease, dementia, amnesia, Hyperthymestic syndrome, Huntington's disease, Parkinson's disease, Stress or Wernicke- Korsakoff s syndrome.

[0026] Thus, a second aspect of the invention relates to an activator of the primary cilia (PC) to restore and / or improve the memory in a subject in need thereof suffering from Alzheimer’s disease, dementia, amnesia, Hyperthymestic syndrome, Huntington's disease, Parkinson's disease, Stress or Wernicke- Korsakoff s syndrome.

[0027] According to the invention, the inventors showed that a downregulation of IFTs or Kinesin (IFT20, IFT25, IFT88, KiF3A, etc.) induces memory disorders associated with a reduction in autophagy. When they restored autophagy in these mice model of downregulation of IFTs or Kinesin, they recovered memory functions. Thus, activator of the primary cilia (PC) or autophagy activator like TAT-Beclin-1 or TETA can be used to treat ciliopathies and particularly ciliopathies with neurological disorders. Thus, a third aspect of the invention relates to an activator of the primary cilia (PC) or to an autophagy activator for use in the treatment of ciliopathies in a subject in need thereof.

[0028] According to the invention, the term “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. Particularly, the subject denotes an old human or a human with cognitive troubles and particularly memory troubles. Particularly, the subject denotes an human with a ciliopathy.

[0029] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting a disease of the invention or suspected to have contracted the disease as well as subjects who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). Activator of PC:

[0030] In a particular embodiment, the activator of PC can be the intraflagellar transport IFT (IFT20, IFT25 or IFT88), the Kinesin Family Member 3A (KIF3A) or TULP3 or fragment thereof and / or an agent for IFT20, IFT25, IFT88, KIF3 A or TULP3 protein expression.

[0031] As used herein, the term “the proteins of the invention” denotes the proteins IFT20, IFT25, IFT88, KIF3 A or TULP3

[0032] As used herein, the term “an agent for IFT20, IFT25, IFT88, KIF3 A or TULP3 protein expression” denotes an agent which can increase or restore the IFT20, IFT25, IFT88, KIF3A or TULP3 protein expression or increase the activity of the IFT20, IFT25, IFT88, KIF3A or TULP3. To increase or restore the IFT20, IFT88, KIF3A or TULP3 protein expression, the agent can also increase or restore the IFT20, IFT88, KIF3 A or TULP3 gene expression.

[0033] In order to test the functionality of a putative agent for IFT20, IFT25, IFT88, KIF3 A or TULP3 protein expression a test is necessary. For that purpose, to identify agent for IFT20, IFT25, IFT88, KIF3 A or TULP3 protein expression, it is possible to evaluate molecularly the autophagy levels and functionally to perform behavioral tests to assess memory functions.

[0034] As used herein, the term “IFT20” for “Intraflagellar transport protein 20 homolog” denotes a protein that in humans is encoded by the IFT20 gene. The gene is composed of 6 exons and is located on human chromosome 17pl 1.1. This gene is expressed in human brain, lung, kidney and pancreas, and lower expression were also detected in human placenta, liver, thymus, prostate and testis. IFT20 subunit of the particle is localized to the Golgi complex in addition to the basal body and cilia where all previous IFT particle proteins had been found. In living cells, fluorescently tagged IFT20 is highly dynamic and moves between the Golgi complex and the cilium as well as along ciliary microtubules. IFT20 has been shown to interact with SPEF2 in the testis, and plays a role in sperm motility. Its Entrez number accession is 90410 and its UniProt accession number is Q8IY31.

[0035] As used herein, the term “IFT25” for “Intraflagellar Transport 25” denotes a protein that in humans is encoded by the IFT25 gene. Component of the IFT complex B required for sonic hedgehog / SHH signaling. May mediate transport of SHH components: required for the export of SMO and PTCHI receptors out of the cilium and the accumulation of GLI2 at the ciliary tip in response to activation of the SHH pathway, suggesting it is involved in the dynamic transport of SHH signaling molecules within the cilium. Not required for ciliary assembly. Its role in intraflagellar transport is mainly seen in tissues rich in ciliated cells such as kidney and testis. Essential for male fertility, spermiogenesis and sperm flagella formation. Plays a role in the early development of the kidney. May be involved in the regulation of ureteric bud initiation (By similarity). Its Entrez number accession is 51668 and its UniProt accession number is Q9Y547.

[0036] As used herein, the term “IFT88” for “Intraflagellar transport protein 88 homolog” denotes a protein that is encoded by the IFT88 gene. This gene encodes a member of the tetratrico peptide repeat (TPR) family. Mutations of a similar gene in mouse can cause polycystic kidney disease. Two transcript variants encoding distinct isoforms have been identified for this gene. In 2012 a mutation was found to be responsible for a novel form of ciliopathy and anosmia in humans capable of remedy in mice by adenoviral mediated gene therapy. IFT88 has been shown to interact with BAT2 and WDR62. WDR62 is required for IFT88 localization to the cilia basal body and the cilia axoneme. Its Entrez number accession is 8100 and its UniProt accession number is Q13099.

[0037] As used herein, the term “KIF3A” for “Kinesin-like protein” denotes a protein that in humans is encoded by the KIF3A gene. KIF3A is one subunit of the heterotrimeric motor protein, kinesin-2, that was initially isolated from sea urchin egg / embryo cytosol using microtubule affinity purification. This motor consists of two kinesin-related subunits (called KIF3A and KIF3B or 3C in vertebrates) and an associated protein (KAP3), and it transports protein complexes, nucleic acids and organelles towards the "plus" ends of microtubule tracks within cells. Work done in a broad range of eukaryotic cells has revealed that heterotrimeric kinesin-2 is the primary motor protein driving the intraflagellar transport of tubulins and other axonemal building blocks from the base of the ciliary / flagellar axoneme to their site of assembly at the distal tips. This process is required for cilium assembly / maintenance and cilium-based signalling which play key roles in various cell and developmental processes. For example, in vertebrate embryos, kinesin-2 function is required for cilia-dependent nodal flow and the development of left-right asymmetry. KIF3A has been shown to interact with MAP3K10. Its Entrez number accession is 11127 and its UniProt accession number is Q9Y496.

[0038] As used herein, the term “TULP3” for “Tubby-like protein 3” denotes a protein member of the tubby gene family of bipartite transcription factors. Members of this family have been identified in plants, vertebrates, and invertebrates, and they share a conserved N-terminal transcription activation region and a conserved C-terminal DNA and phosphatidylinositolphosphate binding region. The encoded protein binds to phosphoinositides in the plasma membrane via its C-terminal region and probably functions as a membrane-bound transcription regulator that translocates to the nucleus in response to phosphoinositide hydrolysis, for instance, induced by G-protein-coupled-receptor signaling. It plays an important role in neuronal development and function. Two transcript variants encoding distinct isoforms have been identified for this gene. Its Entrez number accession is 7289 and its UniProt accession number is 075386.

[0039] In some embodiments, the IFT20, IFT25, IFT88, KIF3A or TULP3 protein of the invention is an isolated, synthetic or recombinant IFT20, IFT25, IFT88, KIF3A or TULP3 protein.

[0040] In some embodiments, the IFT20, IFT25, IFT88, KIF3A or TULP3 protein of the present invention comprises or consists of an amino acid sequence having at least 70% of identity with the sequence of IFT20, IFT88, KIF3A or TULP3 proteins.

[0041] According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; or 99, or 100% of identity with the second amino acid sequence. Amino acid sequence identity is preferably determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990). In particular the with the sequence of IFT20, IFT25, IFT88, KIF3 A or TULP3 proteins of the invention is a functional conservative variant of the with the sequence of IFT20, IFT25, IFT88, KIF3A or TULP3 proteins according to the invention. As used herein the term “function-conservative variant" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the with the sequence of IFT20, IFT25, IFT88, KIF3A or TULP3 proteins, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like). Accordingly, a "function-conservative variant" also includes an with the sequence of IFT20, IFT25, IFT88, KIF3 A or TULP3 proteins which has at least 70 % amino acid identity and which has the same or substantially similar properties or functions as the native or parent with the sequence of IFT20, IFT25, IFT88, KIF3A or TULP3 proteins to which it is compared.

[0042] A further aspect of the present invention relates to a fusion protein comprising the proteins or peptides according to the invention that is fused to at least one heterologous polypeptide. The term “fusion protein” refers to the protein or peptide according to the invention that is fused directly or via a spacer to at least one heterologous polypeptide.

[0043] According to the invention, the fusion protein comprises the protein or peptide according to the invention that is fused either directly or via a spacer at its C-terminal end to the N-terminal end of the heterologous polypeptide, or at its N-terminal end to the C-terminal end of the heterologous polypeptide.

[0044] As used herein, the term “directly” means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the protein or peptide is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous polypeptide.

[0045] In other words, in this embodiment, the last amino acid of the C-terminal end of said protein or peptide is directly linked by a covalent bond to the first amino acid of the N-terminal end of said heterologous polypeptide, or the first amino acid of the N-terminal end of said protein or peptide is directly linked by a covalent bond to the last amino acid of the C-terminal end of said heterologous polypeptide.

[0046] As used herein, the term “spacer” refers to a sequence of at least one amino acid that links the protein or peptide of the invention to the heterologous polypeptide. Such a spacer may be useful to prevent steric hindrances.

[0047] In some embodiments, the heterologous polypeptide is a cell-penetrating peptide, a Transactivator of Transcription (TAT) cell penetrating sequence, a cell permeable peptide or a membranous penetrating sequence.

[0048] The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).

[0049] The proteins, peptides or fusion proteins of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said proteins, peptides or fusion proteins, by standard techniques for production of amino acid sequences. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, California) and following the manufacturer’s instructions. Alternatively, the proteins, peptides or fusion proteins of the invention can be synthesized by recombinant DNA techniques as is now well-known in the art. For example, these fragments can be obtained as DNA expression products after incorporation of DNA sequences encoding the desired (poly) peptide into expression vectors and introduction of such vectors into suitable eukaryotic or prokaryotic hosts that will express the desired (poly) peptide, from which they can be later isolated using well- known techniques.

[0050] The proteins, peptides or fusion proteins of the invention can be used in an isolated (e.g., purified) form or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome).

[0051] In specific embodiments, it is contemplated that proteins, peptides or fusion proteins according to the invention may be modified in order to improve their therapeutic efficacy and their stability using well-known techniques. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution.

[0052] A strategy for improving drug stability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water- soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

[0053] For example, Pegylation is a well-established and validated approach for the modification of a range of polypeptides (Chapman, 2002). The benefits include among others: (a) markedly improved circulating half-lives in vivo due to either evasion of renal clearance as a result of the polymer increasing the apparent size of the molecule to above the glomerular filtration limit, and / or through evasion of cellular clearance mechanisms; (b) reduced antigenicity and immunogenicity of the molecule to which PEG is attached; (c) improved pharmacokinetics; (d) enhanced proteolytic resistance of the conjugated protein (Cunningham- Rundles et.al., 1992); and (e) improved thermal and mechanical stability of the PEGylated polypeptide.

[0054] Therefore, advantageously, the proteins, peptides or fusion proteins of the invention may be covalently linked with one or more polyethylene glycol (PEG) group(s). One skilled in the art can select a suitable molecular mass for PEG, based on how the pegylated polypeptide will be used therapeutically by considering different factors including desired dosage, circulation time, resistance to proteolysis, immunogenicity, etc.

[0055] In one embodiment, the PEG of the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CEE ("methoxy PEG"). In addition, such a PEG can consist of one or more PEG side-chains which are linked together. PEGs with more than one PEG chain are called branched PEGs. Branched PEGs can be prepared, for example, by the addition of polyethylene oxide to various polyols, including glycerol, pentaerythriol, and sorbitol. For example, a four-armed branched PEG can be prepared from pentaerythriol and ethylene oxide. One form of PEGs includes two PEG side-chains (PEG2) linked via the primary amino groups of a lysine (Monfardini et al., 1995).

[0056] To effect covalent attachment of PEG groups to the polypeptide, the hydroxyl end groups of the polymer molecule must be provided in activated form, i. e. with reactive functional groups (examples of which include primary amino groups, hydrazide (HZ), thiol, succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA), succinimidyl proprionate (SPA), succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC), N- hydroxysuccinimide (NHS), aldehyde, nitrophenyl carb onate (NPC), and tresylate (TRES)). Suitable activated polymer molecules are commercially available, e. g. from Shearwater Polymers, Inc., Huntsville, AL, USA, or from PolyMASC Pharmaceuticals pic, UK. Alternatively, the polymer molecules can be activated by conventional methods known in the art, e. g. as disclosed in WO 90 / 13540. Specific examples of activated linear or branched polymer molecules for use in the present invention are described in the Shearwater Polymers, Inc. 1997 and 2000 Catalogs (Functionalized Biocompatible Polymers for Research and pharmaceuticals, Polyethylene Glycol and Derivatives, incorporated herein by reference). Specific examples of activated PEG polymers include the following linear PEGs : NHS-PEG (e g. SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, and SCM- PEG), and NOR-PEG, BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDLPEG, ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs such as PEG2-NHS.

[0057] The conjugation of the proteins, peptides or fusion proteins and the activated polymer molecules is conducted by use of any conventional method. Conventional methods are known to the skilled artisan. The skilled person will be aware that the activation method and / or conjugation chemistry to be used depends on the attachment group(s) of the polypeptides as well as the functional groups of the PEG molecule (e.g., being amine, hydroxyl, carboxyl, aldehyde, ketone, sulfhydryl, succinimidyl, maleimide, vinylsulfone or haloacetate).

[0058] In one embodiment, the proteins, peptides or fusion proteins of the invention are conjugated with PEGs at amino acid D and E (for COOH), T, Y and S (for OH), K (for NH2), C (for SH if at least one cysteine is conserved) or / and Q and N (for the amide function).

[0059] In one embodiment, additional sites for PEGylation can be introduced by site-directed mutagenesis by introducing one or more lysine residues. For instance, one or more arginine residues may be mutated to a lysine residue. In another embodiment, additional PEGylation sites are chemically introduced by modifying amino acids on proteins, peptides or fusion proteins of the invention.

[0060] In one embodiment, PEGs are conjugated to the polypeptides or fusion proteins through a linker. Suitable linkers are well known to the skilled person. A preferred example is cyanuric chloride ((Abuchowski et al., 1977); US 4,179, 337).

[0061] Conventional separation and purification techniques known in the art can be used to purify pegylated polypeptides of the invention, such as size exclusion (e.g. gel filtration) and ion exchange chromatography. Products may also be separated using SDS-PAGE.

[0062] In one embodiment, the pegylated polypeptides provided by the invention have a serum half-life in vivo at least 50%, 75%, 100%, 150% or 200% greater than that of an unmodified polypeptide.

[0063] In some embodiments, the agent for with the sequence of IFT20, IFT25, IFT88, KIF3A or TULP3 proteins expression of the invention is selected from the group consisting of an isolated, synthetic or recombinant nucleic acid encoding for with the sequence of IFT20, IFT88, KIF3A or TULP3 proteins, a nucleic acid sequence encoding for the fusion protein, a nucleic acid encoding a fragment of with the sequence of IFT20, IFT25, IFT88, KIF3A or TULP3 proteins, a nucleic acid encoding a fragment of a peptide according to the invention, a cell expressing with the sequence of IFT20, IFT25, IFT88, KIF3 A or TULP3 proteins, and agent inducing with the sequence of IFT20, IFT25, IFT88, KIF3A or TULP3 proteins expression and their combinations.

[0064] In some embodiments, the nucleic acid encoding for with the sequence of IFT20, IFT25, IFT88, KIF3 A or TULP3 proteins for example comprises or consists of a sequence at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the nucleic acids sequence of the genes of the proteins of the invention.

[0065] As used herein, a sequence "encoding" an expression product, such as a RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide sequence encodes an amino acid sequence for that polypeptide, protein or enzyme. A coding sequence for a protein may include a start codon (usually ATG) and a stop codon.

[0066] These nucleic acid sequences can be obtained by conventional methods well known to those skilled in the art. Typically, said nucleic acid is a DNA or RNA molecule, which may be included in a suitable vector, such as a plasmid, cosmid, episome, artificial chromosome, phage or viral vector.

[0067] So, a further object of the present invention relates to a vector and an expression cassette in which a nucleic acid molecule encoding for a proteins, peptides or fusion proteins of the invention is associated with suitable elements for controlling transcription (in particular promoter, enhancer and, optionally, terminator) and, optionally translation, and also the recombinant vectors into which a nucleic acid molecule in accordance with the invention is inserted. These recombinant vectors may, for example, be cloning vectors, or expression vectors.

[0068] As used herein, the terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression (e.g. transcription and translation) of the introduced sequence.

[0069] Any expression vector for animal cell can be used. Examples of suitable vectors include pAGE107 (Miyaji et al., 1990), pAGE103 (Mizukami and Itoh, 1987), pHSG274 (Brady et al.,

[0070] 1984), pKCR (O'Hare et al., 1981), pSGl beta d2-4 (Miyaji et al., 1990) and the like.

[0071] Other examples of plasmids include replicating plasmids comprising an origin of replication, or integrative plasmids, such as for instance pUC, pcDNA, pBR, and the like.

[0072] Other examples of viral vectors include adenoviral, lentiviral, retroviral, herpes virus and AAV vectors. Such recombinant viruses may be produced by techniques known in the art, such as by transfecting packaging cells or by transient transfection with helper plasmids or viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv+ cells, 293 cells, etc. Detailed protocols for producing such replication-defective recombinant viruses may be found for instance in WO 95 / 14785, WO 96 / 22378, US 5,882,877, US 6,013,516, US 4,861,719, US 5,278,056 and WO 94 / 19478.

[0073] Examples of promoters and enhancers used in the expression vector for animal cell include early promoter and enhancer of SV40 (Mizukami and Itoh, 1987), LTR promoter and enhancer of Moloney mouse leukemia virus (Kuwana et al., 1987), promoter (Mason et al.,

[0074] 1985) and enhancer (Gillies et al., 1983) of immunoglobulin H chain and the like.

[0075] A further aspect of the invention relates to a host cell comprising a nucleic acid molecule encoding for a protein, peptide or a fusion protein according to the invention or a vector according to the invention. In particular, a subject of the present invention is a prokaryotic or eukaryotic host cell genetically transformed with at least one nucleic acid molecule or vector according to the invention. The term "transformation" means the introduction of a "foreign" (i.e. extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. A host cell that receives and expresses introduced DNA or RNA has been "transformed".

[0076] In a particular embodiment, for expressing and producing proteins, peptides or fusion proteins of the invention, prokaryotic cells, in particular E. coli cells, will be chosen. Actually, according to the invention, it is not mandatory to produce the proteins, peptides or fusion proteins of the invention in a eukaryotic context that will favour post-translational modifications (e.g. glycosylation). Furthermore, prokaryotic cells have the advantages to produce protein in large amounts. If a eukaryotic context is needed, yeasts (e.g. saccharomyces strains) may be particularly suitable since they allow production of large amounts of proteins. Otherwise, typical eukaryotic cell lines such as CHO, BHK-21, COS-7, C127, PER.C6, YB2 / 0, HEK293, mononuclear macrophage / monocyte-lineage hematopoietic precursors, Haematopoietic stem cells, Mononuclear precursor cells, osteoblast or inactive osteoclast could be used, for their ability to process to the right post-translational modifications of the fusion protein of the invention.

[0077] The construction of expression vectors in accordance with the invention, and the transformation of the host cells can be carried out using conventional molecular biology techniques. The protein, peptide or the fusion protein of the invention, can, for example, be obtained by culturing genetically transformed cells in accordance with the invention and recovering the proteins, peptides or fusion proteins expressed by said cell, from the culture. They may then, if necessary, be purified by conventional procedures, known in themselves to those skilled in the art, for example by fractional precipitation, in particular ammonium sulfate precipitation, electrophoresis, gel filtration, affinity chromatography, etc. In particular, conventional methods for preparing and purifying recombinant proteins may be used for producing the proteins in accordance with the invention.

[0078] A further aspect of the invention relates to a method for producing a protein, peptide or a fusion protein of the invention comprising the step consisting of: (i) culturing a transformed host cell according to the invention under conditions suitable to allow expression of said protein, peptide or fusion protein; and (ii) recovering the expressed protein, peptide or fusion protein. In a particular embodiment, the activator of the primary cilia (PC) is the osteocalcin (see for example the patent application WO2014152497) and modulator of IFTs proteins (see below).

[0079] Ciliopathies:

[0080] As used herein the term “primary cilia” refers to sensory organelles. Primary cilia and flagella project from the apical side of the cells and emerge from the basal body, a modified centriole structure anchored to the plasma membrane. Their structural core is a microtubulebased cytoskeleton called the axonema, which is surrounded by a membrane contiguous with the cell plasma membrane, but expressing specific signaling molecules. The axonema of primary cilia is composed of nine doublets of microtubules, whereas the axonema of motile cilia usually comprises two additional central microtubule singlets.

[0081] As used herein, the term “ciliopathy”, “ciliopathy disorder”, “ciliopathy disease”, “ciliopathic disease”, “ciliopathies” and ciliopathic disorders” are used interchangeably and refer to those genetic disorders of the cellular cilia, the cilia anchoring structures, the basal bodies, and / or ciliary function. Said ciliopathy may be characterized by ataxia, intellectual deficiency, visual dysfunction, male infertility, kidney dysfunction, liver dysfunction, skeletal dysplasia, olfactory dysfunction, encephalopathy or their combinations. Examples of such disorder include, but are not limited to, Alstrom Syndrome (ALMS), Bardet-Biedl Syndrome (BBS) (e g., BBS1, BBS2, BBS4, BBS5, BBS7, BBS9, BBS10, BBS12, ARL6, MKKS, TTC8, TRIM32), Joubert Syndrome (JBTS), Meckel-Gruber syndrome (MKS), Nephronophthisis (NPHP), Oral -facial-digital syndrome 1 (0FD1), Senior-Loken Syndrome, Polycystic kidney disease (PKD), Polycystic liver disease, primary ciliary dyskinsesia (PCD), Marden-Walker syndrome, situs inversus / Isomerism, retinal dystrophy, cerebello-oculo-renal syndrome, Ellis- van Creveld syndrome, Jeune asphyxiating thoracic dystrophy (JATD), Sensenbrenner syndrome, Leber congenital amaurosis, Retinitis pigmentosa (RP), Usher syndrome (USH) and FGFR3 -related disorders.

[0082] As cilia are a component of almost all vertebrate cells, cilia dysfunction can manifest as a constellation of features that include characteristically, retinal degeneration, renal disease (e.g. Polycystic kidney disease, Nephronophthisis (NPHP), Renal dysplasia...) and cerebral anomalies. Additional manifestations include congenital fibrocystic diseases of the liver, diabetes, obesity and skeletal dysplasias.

[0083] Autophagy activators;

[0084] As used herein, the term “autophagy” denotes a natural intracellular system that delivers cytoplasmic constituents (proteins and organelles) to the lysosome for degradation. Three forms of autophagy are commonly described: macroautophagy, microautophagy, and chaperone- mediated autophagy (CMA), along with mitophagy. The inventors focus on macroautophagy (which we will hereafter refer to as autophagy), the best-characterized autophagic mechanism in eukaryotic cells in which portions of the cytoplasm are sequestered within double- or multimembraned vesicles known as an autophagosome and then delivered to lysosomes for bulk degradation. Indeed, it is an essential proteostasis and stress response mechanism that maintains cellular health by regulating the quantity and quality of organelles and macromolecules through lysosomal degradation.

[0085] Activator of autophagy are well known in the art (see for example Ya-ping YANG et al 2013 and www.sigmaaldrich.com / life-science / cell-biology / cell-biology- products.html?TablePage=104899444). Activators of autophagy may be selected in the group consisting in Earle’s balanced salt solution (EBSS), Brefeldin A, Thapsigargin, Tunicamycin, Rapamycin, CCI-779, RAD001, AP23576, Small molecule enhancers rapamycin (SMER), Trehalose, Lithium chloride, L-690,330, Carbamazepine, Valproic acid sodium salt, N-Acetyl- D-sphingosine (C2-ceramide), Penitrem A, Calpastatin, Xestospongin B, Akebia saponin, Amiodarone hydrochloride, ATG13, GF 109203X synthetic, GF 109203X hydrochloride, N- Hexanoyl-D-sphingosine, MRT68921 dihydrochloride, Niclosamide, Qcl, Rottierin, STF- 62247, Tamoxifen, Temsirolimus, ULK Active, Z36 and Hydroxy citrate.

[0086] In a particularly embodiment, the activator of the autophagy is the beclin 1 of SEQ ID NO: 1 :

[0087] MEGSKTSNNS TMQVSFVCQR CSQPLKLDTS FKILDRVTIQ ELTAPLLTTA QAKPGETQEE ETNSGEEPFI ETPRQDGVSR RFIPPARMMS TESANSFTLI

[0088] GEASDGGTME NLSRRLKVTG DLFDIMSGQT DVDHPLCEEC TDTLLDQLDT QLNVTENECQ NYKRCLEILE QMNEDDSEQL QMELKELALE EERLIQELED

[0089] VEKNRKIVAE NLEKVQAEAE RLDQEEAQYQ REYSEFKRQQ LELDDELKSV

[0090] ENQMRYAQTQ LDKLKKTNVF NATFHIWHSG QFGTINNFRL GRLPSVPVEW

[0091] NEINAAWGQT VLLLHALANK MGLKFQRYRL VPYGNHSYLE SLTDKSKELP

[0092] LYCSGGLRFF WDNKFDHAMV AFLDCVQQFK EEVEKGETRF CLPYRMDVEK GKIEDTGGSG GSYSIKTQFN SEEQWTKALK FMLTNLKWGL AWVSSQFYNK In another embodiment, the activator of the autophagy is a peptide derived from the beclin 1 protein wherein the peptide has a sequence comprising residues 270 to 278 of the amino acid sequence SEQ ID NO: 1.

[0093] In another embodiment, the activator of the autophagy is a peptide derived from the beclin 1 protein wherein the peptide has a sequence comprising residues 270 to 283 of the amino acid sequence SEQ ID NO: 1.

[0094] A further aspect of the present invention relates to a fusion protein comprising a peptide according to the invention (which is an activator of the autophagy) that is fused to at least one heterologous polypeptide.

[0095] The term “fusion protein” refers to the polypeptide according to the invention that is fused directly or via a spacer to at least one heterologous polypeptide.

[0096] According to the invention, the fusion protein comprises the polypeptide according to the invention that is fused either directly or via a spacer at its C-terminal end to the N-terminal end of the heterologous polypeptide, or at its N-terminal end to the C-terminal end of the heterologous polypeptide.

[0097] As used herein, the term “directly” means that the (first or last) amino acid at the terminal end (N or C-terminal end) of the polypeptide is fused to the (first or last) amino acid at the terminal end (N or C-terminal end) of the heterologous polypeptide.

[0098] In other words, in this embodiment, the last amino acid of the C-terminal end of said peptide is directly linked by a covalent bond to the first amino acid of the N-terminal end of said heterologous polypeptide, or the first amino acid of the N-terminal end of said peptide is directly linked by a covalent bond to the last amino acid of the C-terminal end of said heterologous polypeptide.

[0099] As used herein, the term “spacer” refers to a sequence of at least one amino acid that links the peptide of the invention to the heterologous polypeptide. Such a spacer may be useful to prevent steric hindrances. According to the invention, the polypeptide may be coupled to the peptide through linkers or spacers known in the art, such as polyglycine, s-aminocaproic, etc.

[0100] In some embodiments, the heterologous polypeptide is a cell-penetrating peptide, a Transactivator of Transcription (TAT) cell penetrating sequence, a cell permeable peptide (see above for the definition) or a membranous penetrating sequence.

[0101] In a particular embodiment, the heterologous polypeptide is an internalization sequence derived either from the homeodomain of Drosophila Antennapedia / Penetratin (Antp) protein or the Transactivator of Transcription (TAT) cell penetrating sequence of SEQ ID NO 2: YGRKKRRQRRR. In another embodiment, the peptide of the invention is the TAT-Beclin 1 of SEQ ID NO:3 (a peptide derived form the beclin and fused to the cell-penetrating peptide TAT).

[0102] SEQ ID NO:3 of the TAT-beclin 1 :

[0103] YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT

[0104] In a particularly embodiment, the activator of the autophagy is TETA.

[0105] As used herein, the term “TETA” or “Triethylenetetramine” has its general meaning in the art and refers to the N,N'-Bis(2-aminoethyl)ethane-l,2-diamine. The term is also known as Trien; Trientine (INN); or Syprine®(brand name). The term “TETA” encompasses the trientine tetrahydrochloride (4HCL TETA) and the trientine dihydrochloride (2HCL TETA).

[0106] Therapeutic composition

[0107] Another object of the invention relates to a therapeutic composition comprising activator of the primary cilia (PC) for use in the restoration and / or improvement of cognitive functions in a subject in need thereof.

[0108] Another aspect of the invention relates to a therapeutic composition comprising an activator of the primary cilia (PC) or to an autophagy activator for use in the treatment of ciliopathies in a subject in need thereof.

[0109] Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

[0110] "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0111] The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

[0112] The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular, hippocampal stereotactic or subcutaneous administration and the like. In particular embodiment, the pharmaceutical compositions of the invention is formulated for systemic administration.

[0113] Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

[0114] The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

[0115] In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

[0116] Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent.

[0117] By a "therapeutically effective amount" of the activator of the primary cilia (PC) or the autophagy activator of the present invention as above described is meant a sufficient amount of the activator of the primary cilia (PC) or the autophagy activator for treating cognitive dysfunctions or ciliopathies. at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the activator of the primary cilia (PC) or the autophagy activator of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific activator of the activator of the primary cilia (PC) or the autophagy activator employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific activator of the activator of the primary cilia (PC) or the autophagy activator employed; the duration of the treatment; drugs used in combination or coincidental with the specific activator of the activator of the activator of the primary cilia (PC) or the autophagy activator or the autophagy activator employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the activator of the activator of the primary cilia (PC) or the autophagy activator at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the activator of the primary cilia (PC) of the present invention for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the activator of the primary cilia (PC) or the autophagy activator of the present invention, preferably from 1 mg to about 100 mg of the activator of the primary cilia (PC) of the present invention. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0118] In a particular embodiment, the activator of the primary cilia (PC) or the autophagy activator according to the invention may be used in a concentration between 0.01 pM and 20 pM, particularly, the activator of the primary cilia (PC) of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 pM.

[0119] Typically, the activator of the primary cilia (PC) or the autophagy activator of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0120] In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0121] Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising the activator of the primary cilia (PC) or the autophagy activator as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The activator of the primary cilia (PC) or the autophagy activator of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized agent of the present inventions into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze- drying techniques which yield a powder of the activator of the primary cilia (PC) or the autophagy activator of the present invention plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0122] In another aspect, the present invention relates to a method for restoring and / or improving cognitive functions in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of an activator of the primary cilia (PC) of the invention.

[0123] In another aspect, the present invention relates to a method for treating ciliopathies in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount an activator of the primary cilia (PC) or an autophagy activator.

[0124] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

[0125] Figure 1: OCN-GPR158 coupling system requires Core PC-proteins to induce autophagy machinery in hippocampal neurons.

[0126] A) Western blot analysis and quantification of LC3-II accumulation (LC3-II / p-actin ratio) in primary neurons previously infected with lentivirus expressing either shRNA-Gprl58 or shRNA-Scramble. These primary neuronal cultures (3 independent preparations) were treated 4 hrs with either vehicle (NaCl), osteocalcin (OCN) (10 ng / mL), bafilomycin Al (Baf) (100 mM), or OCN + Baf. P-actin was used as a loading control for each sample (n=3). B-C) Left panels are the representative fluorescent microscopy images of brain cross-sections collected 3 weeks after local stereotactic injections with either AAV-U6-shRNA-Scramble (B) or AAV-eSYN-shRNAmir-Scramble (C). The constructs express eGFP, which allows for verification of the site and efficacy of infection. Representation of western blot of IFT20 and GPR158 in 3 -month-old mice hippocampi 3 weeks after hippocampal stereotactic injections with either AAV-U6-shRNA-Ift20, AAV-eSYN-shRNAmir-Ift20 or their respective controls. P-actin was used as a loading control for WB analyses. D) Relative gene expression (qPCR performed in triplicate) of cFOS in the hippocampi of 3-month-old mice previously injected in the hippocampus with either AAV-eSYN-shRNA-Ift20mir or U6-shRNA-Scramble-mir. The mice were subjected to peripheral chronic infusion (peripheral osmotic mini pumps) of either vehicle (NaCl) or uncarb oxy lated OCN (30 ng / hr).

[0127] Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant by Student’s t test compared to control groups.

[0128] Figure 2. Downregulation of PC-core proteins induces autophagy machinery alterations in hippocampal neurons.

[0129] A) LC3 and SQSTMl / p62 puncta quantification performed on cross brain sections at the level of the hippocampal CA3 region performed in 3-month-old mice, 3 weeks after local stereotactic injections with either AAV-U6-shRNA-Ift20, AAV-U6-shRNA-Kif3a (n=4) or AAV-U6-shRNA-Scramble (n=5). B) Representative quantification of LC3-II accumulation (LC3-II / p-actin ratio) in 3 -Month-old mouse hippocampi, 3 weeks after local stereotactic injections with either eSYN-shRNA-Ift20mir (n = 9) or eSYN-shRNA-Scramble mir. P-actin was used as a loading control for each sample. Data are expressed as the mean ± SEM (2 independent cohorts). C) SQSTMl / p62 puncta quantification in CA3 region of the hippocampi of 3 -Month-old mice, 3 weeks after local stereotactic injections of AAVs expressing either eSYN-shRNA-Ift20-mir or eSYN-shRNA-Scramble-mir. NeuN staining was used to label neuronal nucleus (Scale Bar: 100 pm). Data were obtained from 2 independent cohorts. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant by Student’ s t test compared to control groups or by One-way ANOVA repeated-measure followed by a post hoc Bonferroni’s honestly significant difference (HSD) multiple comparisons test for pairwise differences between each group of mice.

[0130] Figure 3. Selective downregulation of PC-core proteins in hippocampal neurons leads to severe learning and memory impairments.

[0131] A-C) Behavioral analyses performed in 3 -month-old mice after hippocampal stereotactic injections with either AAV-U6-shRNA-Ift20, AAV-U6-shRNA-Kif3a or their respective AAV-U6-shRNA-Scramble. A) Novel Object Recognition test (NOR), B) Contextual fear conditioning (CFC) and C) Morris water maze test (MWM) were assessed 3 weeks after local AAVs injections. For the NOR, discrimination and preference indexes were measured 24 hrs after the training phase to assess memory performances. For the CFC, the percentage of freezing was measured for the training and testing phases. For the MWM, the graph shows the time to localize a submerged platform in the swimming area. All behavioral tests were performed in two independent cohorts of animals for each group and their respective control. D) Quantification of LC3-II accumulation (LC3-II / p-actin ratio) in mouse hippocampi stereotactically injected with either eSYN-shRNA-Ift20-mir or eSYN-shRNA-Scramble-mir; and following 5 consecutive days of daily infusion with either TAT-Scramble or TAT-Beclinl. P-actin was used as a loading control for each sample. The quantification is relative to eSYN- shRNA-Scramble-mir mice injected with TAT-Scramble. E) NOR performed in the same experimental conditions as Figure 3A (eSYN-shRNA-Scramble-mir + TAT-Scramble, eSYN- shRNA-Ift20-mir + TAT-Scramble, eSYN-shRNA-Ift20mir + TAT-Beclin-1). This analysis was performed in two independent cohorts of animals for each group. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant by Student’s t test compared to control groups by two-way ANOVA repeated-measure followed by a post hoc Bonferroni’s honestly significant difference (HSD) multiple comparisons test for pairwise differences between each group of mice.

[0132] Figure 4. PC-core proteins levels are reduced during brain aging and their restoration is sufficient to improve age-related autophagy and memory decline.

[0133] A-B) A) Quantification of IFT20 in 3 or 16-month-old mice 3 weeks after stereotactic injections with either AAV-mIft20 (expressing mouse Ift20 cDNA) or AAV-Scramble. (B) Quantification of IFT88, KIF3 A and IFT25 levels in the same experimental groups as D. P- actin was used as a loading control for each sample. These measurements were performed in two independent experiments. C) Puncta quantification performed on brain cross-sections, at the level of the hippocampal CA3 region of 3- or 16-month-old mice, 3 weeks after stereotactic injections with either AAV-mIft20 cDNA or AAV-Scramble (Scale Bar: 20 pm). D) NOR performed in 3 or 16-month-old mice, after hippocampal stereotactic injections with either AAV-mIft20 cDNA or AAV-Scramble. Discrimination and Preference indexes were measured 24 hrs after the training phase to assess memory performances. The NOR was performed in two independent experiments for each group of mice.

[0134] Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant by Student’s t test compared to control groups by One-way ANOVA repeated- measure followed by a post hoc Bonferroni’s honestly significant difference (HSD) multiple comparisons test for pairwise differences between each group of mice.

[0135] Figure 5. The PC-core protein IFT20 is required to mediate the rejuvenating effects of OCN on age-related memory deficits.

[0136] A) Novel Object Recognition test (NOR) performed in 3- and 16-month-old mice, after hippocampal stereotactic injections of either AAV-eSYN-Ift20-shRNA-mir or AAV-shRNA- Scramble-mir and infused peripherally and continuously through minipumps with uncarb oxy lated OCN (30 ng / hr) or vehicle (NaCl). Discrimination and preference indexes were measured for each group during the testing phase. The NOR was performed in two independent experiments for each group of mice. B) CREB protein levels in the hippocampus of 3-Mo, after peripheral chronic infusion (minipumps) with either vehicle (NaCl) or uncarb oxy lated OCN (30 ng / hr). C) Relative quantification (relative intensity) of pCREB levels performed at the level of the hippocampal CA3 region (Scale Bar: 20 pm). Brain cross-sections of 3- (n=4) and 16-month-old (n=4) collected 3 weeks after hippocampal stereotactic injections with either AAV-U6-Scramble or AAV-U6-mIft20 cDNA were used. Relative intensity quantification was measured compared to the 3-month-old locally injected with AAV-U6-Scramble. These measurements were obtained from two independent cohorts of mice for each group. Data are expressed as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant by Student’s t test compared to control groups by two-way ANOVA repeated-measure followed by a post hoc Bonferroni’s honestly significant difference (HSD) multiple comparisons test for pairwise differences between each group of mice.

[0137] Figure 6. novel physiological role of the autophagy-PC axis in brain.

[0138] EXAMPLE:

[0139] Material & Methods Animals

[0140] All experiments were performed on C57BL / 6J WT male mice (obtained from Janvier Laboratory, Le Genest St Isle, France). All mice were 3 or 16 months of age at the start of experiments. For all experiments, we used littermates as controls. Upon arrival, mice were housed at least 2 weeks before any behavioral or molecular testing. Male mice were housed 5 animals per cage in polycarbonate cages (35.5 * 18 x 12.5 cm), under a 12 hours light / dark cycle with ad libitum access to food and water prior to experimentation. In all experiments, animals were randomly assigned to treatment groups. Group sizes were determined after performing a power calculation to lead to an 80% chance of detecting a significant difference (P < 0.05). All behavioral experiments were performed in accordance with the European Communities for Experimental animal use (2010 / 63 / EU) and local ethical committee review procedures and protocols (APAFIS- 25139).

[0141] Western blot analysis

[0142] Mouse dorsal hippocampi were dissected, snap frozen and lysed in RIPA lysis buffer (25mM Tris HC1, pH 7.6, 150 mM NaCl, 1% NP40, 1% Na deoxycholate, 0.1% SDS and cOmplete protease and phosphatase inhibitors). The lysates were sonicated with BioRuptor Diagenode (5 cycles of 10" on combined with 30'off) and the protein concentration was analyzed in the supernatant by BCA assay. The lysates were loaded on a 12,5% or 10% SDS polyacrylamide gradient gel and transferred onto a PVDF membrane. The blots were blocked in Tris-buffered saline with Tween (TBST)-5% BSA and incubated with either mouse anti-P- actin (1 :5000, A-2228: Sigma), rabbit anti-IFT20 (1 : 1000, 13615: Proteintech), rabbit anti- IFT88 (1 : 1000, 13967: Proteintech), anti-KIF3A (1 : 1000, 13930: Proteintech), rabbit anti- IFT25 (1: 1000, 15732: Proteintech), rabbit anti-TULP3 (1: 1000, 13637: Proteintech), rabbit anti-GPR158 (1 : 1000, ABIN6258978: Antibodies online), mouse anti-Beclin 1 (1 : 1000, 612113: BD Transduction Laboratories, only the top band corresponding to non-cleaved Beclin 1 was quantified in this study), rabbit anti-LC3 (1 : 10000, L7543: Sigma), guinea pig anti-p62 (1 :2000, GP62-C: Progen), rabbit anti-phospho-CREB (1 : 1000, 06-519: Merck Millipore), rabbit anti-CREB (1 : 1000, 9197: Cell Signaling). Horseradish peroxidase-conjugated secondary antibodies (anti-mouse IgG, HRP -linked antibody (7076, Cell Signaling) and antirabbit IgG, HRP -linked antibody (7074: Cell Signaling) and anti-guinea pig IgG, HRP -linked antibody (A5545: sigma) revealed using an ECL kit Clarity Western ECL Substrate (BioRad) or Immobilon chemiluminescent HRP Substrate (Millipore) for protein detection. Selected films were scanned and quantified using BioRad Image Lab software (Version 5.2). P-actin bands were used for normalization. All Western-blot analyses were performed on freshly prepared hippocampal lysates.

[0143] N2a cell culture conditions

[0144] Murine neuroblastoma N2a cells were cultured in DMEM at 37°C in a humidified incubator with 5% CO2. At passage 11, N2a cells were treated with lOpM retinoic acid to induce differentiation four days prior to treatment.

[0145] Co-Immunoprecipitation

[0146] We followed the manufacturer’s protocol (Dynabeads Protein G - 10007D: Life Technologies-) for TULP3 Co-Immunoprecipitation performed in N2a cells. To that end, N2a cell lysates were prepared with RIPA and Protease and phosphatase inhibitors as described before, sonicated, centrifuged and analyzed the protein concentration by BCA assay. We linked the rabbit anti-TULP3 antibody (1 pg, 13637: Proteintech) or control rabbit: IgG (1 pg, 30000- 0A: Proteintech) to magnetic particles by incubation on a rotatory wheel for 30 min at room temperature, washed, and incubated with 400 pg of protein extract overnight at 4°C on a rotatory wheel. After 3 washes, we eluted the proteins, added loading buffer and DTT, denatured at 95°C, and resolved by western blot. The membranes were incubated with primary and secondary antibody as described above.

[0147] SQSTMl / p62 and LC3 puncta immunostaining and quantification

[0148] Mice were deeply anesthetized with a mixture Ketamine / Xylazine and transcardially perfused with cold PBS, followed by cold 4%PFA. Brains were post-fixed overnight in 4%PFA at 4°C. 30pm serial coronal floating sections were obtained using a vibratome. For the coimmunofluorescences, sections were blocked with 10% fetal bovine serum for 30 min at room temperature and then incubated with guinea pig anti-p62 (1 :200, GP62C: Progen) with rabbit anti-LC3 (1 :200, L7543: Sigma) overnight at 4°C. The sections were washed with PBS before and after being incubated with an Alexa Fluor - conjugated secondary antibodies (donkey anti - guinea pig IgG (1 :200, Alexa Fluor 555: Bioss inc.; and donkey anti - rabbit IgG (1 :200, Alexa Fluor 647: Bioss inc.) for 2 hrs at room temperature in blocking buffer. All sections were mounted onto gelatin-subbed slides and coverslipped using Mowiol with DAPI. Images were obtained using a Zeiss Apotome2 fluorescence microscope and analyzed using Zen light Zeiss LSM software. The number of cells with SQSTMl / p62 puncta was quantified on digital images with Icy software (http: / / icy.bioimageanalysis.org).

[0149] Phospho-CREB immunostaining and quantification Mice were deeply anesthetized with a mixture Ketamine / Xylazine and transcardially perfused with cold PBS, followed by cold 4%PFA. Brains were post-fixed overnight in 4%PFA at 4°C. 30pm serial coronal floating sections were obtained using a vibratome. For the coimmunofluorescences, sections were blocked with TBS-Triton 0.3% with 10% normal donkey serum for 1 hour at room temperature and then incubated with rabbit anti-pCREB (1:500, 06- 519: Merck Millipore) overnight at 4°C in 5% normal donkey serum in TBS-Triton 0.15%. The sections were washed with TBS-Tween 0.1% before being incubated with an Alexa Fluor - conjugated secondary antibodies (donkey anti - rabbit IgG (1 :200, Cy3 : Jackson Immuno) for 2h at room temperature in TBS-Triton 0.15%. All sections were mounted onto gelatin-subbed slides and coverslipped using Fluoromount G with DAPI. Images were obtained using a Zeiss Apotome2 fluorescence microscope and analyzed using Zen light Zeiss LSM software under the same setting. We acquired images from 5 hippocampal sections of the different groups, transformed them to a gray scale and analyzed immunoreactivity by calculating the integrated density of a region of interest after defining a threshold with ImageJ software (National Institutes of Health; available at http: / / rsb.info.nih.gov / ij / ).

[0150] Semi -quantitative RT-PCR

[0151] Brain tissues were immediately flash-frozen after dissection and total RNA was isolated with TRIzol Reagent using a homogenizer. Single-strand cDNA was synthesized from total RNA (2pg) by using SuperScript II Reverse Transcriptase. qRT-PCR was performed using iTAQ SYBR Green (BioRad) with specific primers.

[0152] Stereotaxic surgery

[0153] Mice were anesthetized by intraperitoneal injection of ketamine hydrochloride (20mg / ml BW) (1000 Virbac) and xylazine (lOOmg / ml BW) (Rompun 2%; Bayer) and placed in a stereotaxic frame (900SL-KOPF). Ophthalmic eye ointment was applied to the cornea to prevent desiccation during surgery. The area around the incision was trimmed and Vetoquinol was applied. All drugs were injected bilaterally into the dorsal hippocampi using the following coordinates (from Bregma, Paxinos and Franklin, 2008): X=+ / -1.4 mm, Y= 2.0 mm and Z=-1.34 mm. A 1 pl volume of either AAV or drugs was injected stereotactically over 4 min (injection rate: 0.25 pl / min). To limit reflux along the injection track, the needle was maintained in situ for 4 min between each 1 pl injection. The skin was stitched over the brain. At the end of the surgical procedure, the skin was stitched over the brain (Filapeau 5 / 0 3 / 8 T12) and an analgesic solution is administered subcutaneaously(Ketoprofen, 5mg / kg).

[0154] Surgery procedure for hippocampal cannula implantation Mice were anesthetized via the intraperitoneal injection of ketamine hydrochloride (20 mg / Kg bodyweight) (Virbac, Carros, France) and xylazine (100 mg / ml bodyweight) (Rompun 2%; Bayer, Leverkusen, Germany) and placed in a stereotaxic frame (900SL, -Kopf Instruments, Tujunga, CA, USA). To prevent desiccation during surgery, ophthalmic eye ointment was applied to the cornea. The area around the incision was trimmed and Vetoquinol was applied. After exposure of the dorsal skull, a hole was made at the following coordinates: anterior- posterior (AP) -2.0, medial lateral (ML) -1.4 (in mm, from the bregma). A guide cannula (C315GMN / 1.5, 26 gauge, Plastics One) and secured to the skull with dental cement (SuperBond C&B, Sun Medical, Shiga, Japan). Lastly, the scalp incision was sutured around the cannula and the mouse as observed in a recovery chamber until it was fully mobile. Lastly, the scalp incision was sutured around the cannula (Filapeau 5 / 0 3 / 8 T12) and an analgesic solution was administered subcutaneaously(Ketoprofen, 5mg / kg). The mouse as observed in a recovery chamber until it was fully mobile.

[0155] Drug preparation and hippocampal cannula infusion

[0156] TAT-Beclin 1 or TAT-Scramble was dissolved to a concentration of 1 pg / pl in PBS. For 7 days, the experimenter gently restrained the mouse and placed an injector cannula (C315IMN / 1.5 / 0.5; 33 gauge, extending 0.5 mm beyond the 1.5 mm guide cannula). The drug was infused (1.0 pl / side at a rate of 1 pl / min) using a syringe pump (Harvard Apparatus) and a 10 pl Hamilton syringe connected via a PE-50 polyethylene catheter to the injector cannula.

[0157] Osmotic pumps

[0158] Alzet micro-osmotic pumps (model 1002) were loaded 4 hrs before surgery with either vehicle saline solution (NaCl) or uncarboxylated OCN (30 ng / hr). Mice were anesthetized with isoflurane and osmotic pumps were surgically installed subcutaneously in the backs of the mice. Ophthalmic eye ointment was applied to the cornea to prevent desiccation during surgery. The incision in the back of the mice was sutured and the mouse was observed in a recovery chamber until it was fully mobile. Behavioral analyses started 1 week after Osmotic pumps installation.

[0159] Recombinant OCN

[0160] Mouse uncarboxylated OCN was purified from BL21 bacteria transformed with pGEX2TK-mOCN as previously describedl7,18. In brief, GST-OCN fusion protein was bacterially produced in BL21 pLyS transformed with pGEX- 2TK-mOCN after induction with IPTG. Cells were collected in lysis buffer (PBS IX 10 mM Tris, pH 7.2, 2 mM EDTA, 1% Triton, and IX protease and phosphatase inhibitor cocktail; 78443; Thermo Fisher Scientific). Following four freeze-thaw cycles and sonication, lysates were cleared by centrifugation. The supernatant was incubated with glutathione- Sepharose 4B (17075601; GE) for 4 hrs at 4°C. Following six washes with washing buffer (PBS IX and 1% Triton) and with PBS IX, OCN was then cleaved out from the GST moiety by using thrombin (27-0846-01; GE). Four fractions were collected, and each of them was incubated with benzamidine Sepharose (17-5123-10; GE) for 30 min at room temperature to remove thrombin. 10 ng (diluted in PBS IX) of OCN were injected per hemisphere in 16 month-old mouse hippocampi. Primary hippocampal neurons were treated with OCN at a concentration of 10 ng / ml of culture medium.

[0161] Pharmacological modulation of autophagy

[0162] For pharmacological hippocampal induction of autophagy, we performed intra- hippocampal injections of either Ipg TAT-Scramble (dissolved in PBS) or Ipg TAT-Beclin 1 (dissolved in PBS). The Tat-Beclin 1 (YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT, SEQ ID NO: 3) consisted of 11 amino acids of the TAT protein transduction domain (PTD) at the N terminus, a GG linker to increase flexibility, and 18 amino acids derived from Beclin 1, amino acids 267-284 containing 3 substitutions: H275E, S279D, Q281E. Control peptide, TAT-Scramble (YGRKKRRQRRRGGVGNDFFINHETTGFATEW, SEQ ID NO: 4), consisted of the TAT protein transduction domain, a GG linker, and a scrambled version of the C -terminal 18 amino acids from Tat-Beclin 1 All drugs were infused in a volume of 1 pl (bilaterally) in the dorsal hippocampus chronically using hippocampal cannulas for 5 consecutive days before the NOR or CFC behavioral tasks and maintained during the behavioral days (total 10 days). For brain collection hippocampal injections through cannulas were performed 12 hrs before sacrificed.

[0163] Adeno-associated viruses expressing shRNA

[0164] Adeno-associated viruses (AAV) serotype 9 expressing shRNA were purchased from Vector Biosystems Inc (Malvern PA). shRNAs specific to Ift20 (AAV9-GFP-U6-m-Ift20- shRNA) (shRNA sequence (VBLKO-248329), Kif3a (AAV9-GFP-U6-m-Kif3a-shRNA) (shRNA sequence (TRCN0000339511) or scrambled non-targeting negative control (AAV9- GFP-U6-scrmb-shRNA) were injected in a volume of I l (bilaterally), 3 weeks prior either behavioral tests or brain tissue collection. The AAV titers were between 1.0 and 4.3x1013 GC / ml. AAV expressing shRNAmir targeting IFT20 (AAV9-eSYN-GFP-Ift20-shRNAmir) (Use siRNA with sh-loop for shmiR. The shRNAmir is in 3'UTR of eGFP under the eSYN promoter) or IFT88 (AAV9-eSYN-GFP-mIft88-shRNAmir) specifically in neurons were purchased from Vector Biosystems Inc (Malvern PA). The shRNAmir are driven by the eSYN promoter, a hybrid promoter consisting of the 0,45Kb human Synapsin 1 promoter fragment (hSYNl). AAV9-eSYN-GFP-shRNA155mir was used as control. The AAV titers were between 1.3 and 6.2x1013 GC / ml. AAV overexpressing Ift20 (AAV9-CMV-mIft20-IRES- eGFP) was purchased from Vector Biosystems Inc (Mavern PA). AAV9-CMV-eGFP was used as control. The AAV titers were between 5.0 and 6.0x1012 GC / mL.

[0165] Behavioral tests

[0166] For all behavioral assessments, mice were handled for at least 20min over 3 consecutive days. Before the behavioral procedure, animals were transported a short distance from the holding mouse facility to the testing room in their home cages and left undisturbed for at least one hour prior to testing. Behavior was scored by 2 observers blind to the groups.

[0167] Novel object recognition paradigm (NOR)

[0168] The behavior sessions were recorded with a video camera. The testing arena consisted on four plastic boxes (60 * 40 * 32 cm). Mice could not contact or see each other during the exposures. The light intensity was equal in all parts of the arena. Two different objects were used, available in quadruplicate: (A) a blue ceramic pot (diameter 6.5 cm, maximal height 7.5 cm) and (B) a clear glass (diameter 6.5 cm, maximal height 6.5 cm). The objects elicited equal levels of exploration as determined in pilot experiments and training phase. Mice were always placed in the center of the arena at the start of each exposure.

[0169] The NOR paradigm consists of three phases over 4 days. On day 1 and 2 (habituation phases): mice were given 5 min to explore the arena, without any objects and were then taken back to their home. On day 3 (training phase): mice were allowed to explore, for 10 min, two identical objects arranged in a symmetric opposite position from the center of the arena and were then transported to their home cage. On day 4 (testing phase): mice were given 15 minutes to explore two objects: a familiar object and a novel one, in the same arena, keeping the same object localization. The object that serves as a novel object (A or B), as well as the left / right localization of the objects were counterbalanced within each group. Between exposures, arenas were cleaned with phagosphore, and the bedding replaced. The following behaviors were considered as exploration of the objects: sniffing or touching the object with the nose or with the front legs or directing the nose to the object at a distance < 1 cm. Investigation was not scored if the mouse was on top of the object or completely immobile. The preference index (time spent exploring the new object / the total time spent exploring both objects) and the discrimination index (time spent exploring the new object - time spent exploring the familiar object) / (total time spent exploring both objects) were calculated. As control, preference index for the (right / left) object location or for the object A versus B during the training phase of the NOR was measured in all groups of mice exposed to the test. Behavior was scored on videos by an observer blind to treatment and the total exploration time of the objects was quantified during the training and testing phases. 3-foot shock contextual fear conditioning (CFC)

[0170] The conditioning chambers were obtained from Bioseb (France). Each chamber was located inside a larger, insulated plastic cabinet that provided protection from outside light and noise (67 * 55 x 50 cm, Bioseb, France), and mice were tested individually in the conditioning boxes. Floors of the chamber consisted of 27 stainless steel bars wired to a shock generator with scrambler for the delivery of foot shock. Signal generated by the mice movements was recorded and analyzed through a high sensitivity weight transducer system. The analog signal was transmitted to the Freezing software module through the load cell unit for recording purposes and analysis of time active / time immobile (Freezing) was performed.

[0171] The CFC procedure took place over two consecutive days. On day 1 (training): mice were placed in the conditioning chamber, and received 3 foot-shocks (1 sec, 0.5 mA), which were administrated at 60, 120 and 180 sec after the animals were placed in the chamber. On day 2 (testing): contextual fear memory was assessed 24 hours after training by returning the mice to the conditioning chamber and measuring freezing behavior during a 4 min retention test. Freezing behavior was considered to occur if the animals froze for a period of at least two seconds. Freezing was scored and analyzed automatically using Packwin 2.0 software (Bioseb, France) and analyzed by one observer blind to mouse treatment or AAV-infections.

[0172] Morris Water Maze (MWM)

[0173] Morris water maze (MWM) with an automatic tracking system was employed for assessing spatial learning and memory. The apparatus was a white circular swimming pool (diameter: 200 cm, walls: 60 cm high), which was located in a room with various distal cues. The pool was filled with water (depth: 50 cm) maintained at 22°C ± 1°C, which was made opaque by the addition of a nontoxic white paint. A 12 cm round platform was hidden 1.0 cm below the water surface. The maze was virtually divided into four arbitrary, equally spaced quadrants delineated by the cardinal points north (N), east (E), south (S), and west (W). The pool was located in a brightly lit room. Extra maze geometric and high-contrast cues were mounted on the walls of the swimming pool with the ceiling providing illumination. Each daily trial consisted of four swimming trials, in which each mouse was placed in the pool facing the wall of the tank and allowing the animal to swim to the platform before 120 sec had elapsed. A trial terminated when the animal reached the platform, where it remained for 5 sec. Mice were removed and placed back in their home cages for a 5 min inter-trial interval. To prevent hypothermia, the animals were gently dried with a paper towel between and after the trials. The starting point differed at each trial, and different sequences of release points were used from day to day. Animal movements were recorded using ANY-maze (Stoelting Co.) to calculate parameters of the performance of mice.

[0174] Open Field Tests (OFT)

[0175] Each animal was placed in a 43 x 43 cm open field chamber and tested for 30 min. Mice were placed individually into the center of the open-field arena and allowed to explore freely. Mice were monitored throughout each test session by infrared light beam activity monitor using actiMot2 Software (PhenoMaster Software, TSE System, Germany). The overall motor activity was quantified as the total distance traveled. Anxiety was also quantified by measuring the percentage of the time and distance spent in the center versus periphery of the open-field chamber.

[0176] Light-Dark test (LPT)

[0177] Each animal was placed in a 43 x 43 cm open field also containing a dark compartment and tested for 10 min. Mice were placed individually into the dark chamber of the open-field arena and allowed to explore freely. Mice were monitored throughout each test session by infrared light beam activity monitor using actiMot2 Software (PhenoMaster Software, TSE System, Germany). Anxiety was quantified by measuring the number of entries in the light compartment, the latency to reach the light compartment and the percentage of distance spent in the light versus dark chamber of the open-field box.

[0178] Primary cultured hippocampal neurons

[0179] Hippocampal neurons were isolated from mouse embryos (embryonic day 16.5). After dissection, hippocampi were digested with trypsin 0.05% and EDTA 0.02% for 15 min at 37°C. After three washes with DMEM (61965059: Thermo Fisher Scientific) supplemented with 10% FBS, 100 U / ml penicillin-streptomycin and lx GlutaMAX (Thermo Fisher Scientific), cells were dissociated by pipetting up and down, and then plated. The dissociated cells were plated onto poly-L-lysine-coated plates or glass coverslips for microscopic examination. 24 hrs after plating, the media was replaced with Neurobasal medium (Thermo Fisher Scientific) containing B27 supplement (Thermo Fisher Scientific), GlutaMAX and Mycozap (Lonza) was changed two times per week and neurons were maintained in 5% CO2 and 37°C. To decrease the proportion of glial cells, 1 pM of Ara-C inhibitor (Cl 768, SIGMA) was added in the prewarmed complete Neurobasal, starting at DIV5. Experiments were performed on cells after 15 days of culture.

[0180] Neuronal stimulation treatment of primary hippocampal neurons

[0181] Prior to treatments, neurons were starved for 4 hours (Neurobasal without B27). For OCN, neurons were treated for 5 min or 4 hrs with neurobasal medium containing 10 ng / ml OCN. For the 4 hrs treatment, some neurons were treated with neurobasal medium containing either 10 ng / ml OCN, lOOmM Baf or both. For experiments using CREB inhibitor, neurons were treated with neurobasal medium containing either 10 ng / ml OCN, 73 nM CREB inhibitor 666-15 (SIGMA, 5383410001) or both for 4 hrs. After treatment, neurons were rinsed in PBS and proteins extracted in IX Laemmli buffer containing phosphatase and protease inhibitors or fixed in 4% PFA / 4% glucose for 20 min at room temperature. The coverslips were then washed 3 times in PBS and kept at 4°C.

[0182] Lentiviral infections of primary hippocampal neurons

[0183] Neurons were infected (MOI 10) at Day In Vitro (DIV) 1 with lentiviral shRNAs (titer l,80E+08 TU / ml). The shRNA sequence 5Z-GAG CCG CTC CAC TGA CGG CAC CAT CTT GG-3Ztargeting mouse Gprl58 was purchased from Origene (TL509693B) and cloned into pGFP-C-Lentivector VSV-G pseudotyped (Origene).

[0184] In vitro primary cilia staining

[0185] For the co-immunofluorescences, coverslips were permeabilized with 0.2% Triton X- 100 for 15 min; blocked with 3% BSA for 30 min at room temperature and then incubated with mouse anti-Arll3b (1 :200; N295B / 66 Neuromab) and rabbit anti-TULP3 (1 : 100; 13637-1-AP Proteintech) or rabbit anti-ACIII (1 :400; PA5-35382 ThermoFisher) and mouse anti-GPR158 (1 : 100; MAB 10286 Bio-techne) overnight at 4° C. The coverslips were washed with PBS before and after being incubated with an Alexa Fluor - conjugated secondary antibodies (donkey anti - mouse IgG (1:200, Alexa Fluor 488: Bioss inc.; and donkey anti - rabbit IgG (1 :200, Alexa Fluor 546: Bioss inc.) for 2 hrs at room temperature in blocking buffer. Hoechst fluorescent stain was used to stain nuclei (1 :5000; 62249 ThermoFisher) for 5 minutes at room temperature. Coverslips were mounted onto slides using Fluoromount Aqueous Mounting Medium (Sigma- Aldrich). Slides were imaged using fluorescent microscopy with a 63X objective on a ZEISS ApoTome fluorescence microscope.

[0186] In vivo primary cilia staining

[0187] Mice were deeply anesthetized with a mixture Ketamine / Xylazine and transcardially perfused with cold PBS, followed by cold 4%PFA. Brains were post-fixed overnight in 4% PFA at 4°C. 30pm serial coronal floating sections were obtained using a vibratome. Sections were permeabilized with 0.3% TritonX-100 for 10 min, blocked with 10% Normal donkey serum / 1% Triton X-100 for 1 hr at room temperature and then incubated with rabbit anti-ACIII (1 :400; PA5-35382 ThermoFisher) overnight at 4°C. The sections were washed with PBS before and after being incubated with a Cy™3 AffiniPure Donkey Anti-Rabbit IgG (H+L) secondary antibody (1 :200; 711-165-152 Jackson ImmunoResearch) for 2 hrs at room temperature in blocking buffer. After PBS washes, Hoechst fluorescent stain was used to stain nuclei (1 :5000; 62249 ThermoFisher) for 5 minutes at room temperature. All sections were mounted onto gelatin-subbed slides and coverslipped using Fluoromount Aqueous Mounting Medium (Sigma-Aldrich). Images were obtained using a Zeiss Apotome2 fluorescence microscope with a 40X objective.

[0188] Primary cilia counting and analysis

[0189] Cilia length analysis was performed on the microscopy analysis software program Imaris (version 9.2.0; Bitplane). Apotome images of the CA3 and DG hippocampal regions were imported and first exported to the Bitplane format. Primary cilia were counted using Imaris FilamentTracer. Bitplane images were analyzed by manually tracing over primary cilia using the Pencil tool on AutoPath mode. The start of the cilium was determined as the start of the red signal to the end of the same elongated signal. Cilia were only traced where Hoechst dye was apparent. If uncertainty regarding start and finish of cilium, the longest potential length value was always used. Imaris functionality provided automatic centering of traced cilia once a cilium was delineated manually. Cilia were traced in 3D. Imaris statistics of cilia length, number of cilia per image and cilia straightness were then exported to excel files for later data analysis. The number of cilia per image was normalized by dividing the number of cilia by the volume of the nuclei (pm3). The area was delimited by using the built-in Imaris surface tool. The area was manually drawn and the detection of voxels threshold was manually adjusted in order to either increase or decrease coverage of nuclei that needed to be included in the volume of calculation. Between 49 and 116 cilia were examined for each image in the totality of the CA3 region and between 85 and 368 cilia were examined for the upper layer of the dentate gyrus. Between 3 and 4 images were analyzed per mouse / region / age in the different experiments.

[0190] GPR158 quantification and analysis

[0191] GPR158 analysis within the PC was performed on the microscopy analysis software program Imaris (version 9.2.0; Bitplane). Apotome images of the primary hippocampal neurons were imported and first exported to the Bitplane format. Primary cilia were first outlined using Imaris FilamentTracer in 3D. Bitplane images were analyzed by manually tracing over primary cilia using the Pencil tool on AutoPath mode following the same method as described above. The localization of GPR158 was then measured in 3D using the Spot Detection tool. We performed an automatic segmentation of objects, based on GPR158 signal intensity, with an estimated diameter of the spots of 0.280 pm. The Model PSF -elongation along Z-axis and background subtraction options were selected. The Quality filter was manually adjusted to 57.6 on all images, so that the spots fit with GPR158 signal intensity. In the Spots menu, the Filter ‘Shortest distance to Cilia #’ was added and this process was repeated for all the cilia of the image. Then, the different distances to the cilia were set up (from 0 to 10 pm from the primary cilium) and the number of GPR158 spots were read for all the distance groups.

[0192] Quantification and statistical analysis

[0193] All values are expressed as mean ± s.e.m. Alpha was set to 0.05 for all analyses. Statistical parameters including the exact sample size (n), post hoc tests, and statistical significance are reported in every figure and figure legend. Number of mice was estimated to be sufficient on the basis of pilot experiments. Data were estimated to be statistically significant when P < 0.05 by nonparametric Mann-Whitney U test or Student’s t test, and one-way or two- way ANOVAs with repeated-measures when appropriate. Significant ANOVAs were followed by a two-way repeated-measures analysis of variance with a post-hoc Tukey’s HSD multiple comparisons test for pairwise differences between each mouse groups. In every figure, an asterisk denotes statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Data were analyzed using GraphPad Prism v5 software.

[0194] Results

[0195] OCN increases autophagy in hippocampal neurons through GPR158,

[0196] We previously showed that OCN can influence neuronal homeostasis and cognitive fitness by mobilizing autophagic machinery in hippocampal neurons (20). However, the mechanism by which this regulation occurs has not yet been elucidated. Recent work identified GPR158, an orphan GPCR expressed in neurons of the CA3 region of the hippocampus, to be a key regulator of OCN’s influence on memory (18,19). We first confirmed that Ocn treatment in primary hippocampal neurons significantly enhances autophagosome-like structure formation, as shown by measuring lipidation of the LC3 protein (LC3-II) levels (considered as a key readout in autophagosome-like structure biogenesis) in the presence and in the absence of lysosomal blockers (bafilomycin), respectively (Fig. 1A). Using primary hippocampal neurons infected with lentiviruses expressing shRNA against Gprl58, we next showed that this induction is dependent on GPR158 (Fig. 1A). This observation was further confirmed in vivo showing that local stereotaxic injections of OCN enhances hippocampal LC3 lipidation in WT, but not in Gprl58- / - mice (data not shown).

[0197] GPR158 is present at the PC of hippocampal neurons. Many studies recently proposed that the autophagic machinery can be triggered, in response to various stimuli such as serum deprivation and shear stress, by direct or indirect connection with core PC proteins, namely intra-flagellar transporters (IFT) (such as IFT20 and IFT88) and KIF3 A (Kinesin Family Member 3 A, a kinesin-2 family motor protein). These core proteins, allowing the transportation of ciliary cargoes, are essential for the formation, maintenance, and the signaling activity of the PC (48,49). The PC has been described as a signaling hub for various intracellular pathways dependent on GPCRs, that sense and transduce changes of the systemic milieu (40). Remarkably, we identified five ciliary-targeting VxPx motifs at the C-terminal domain of GPR158, suggesting its possible localization in the PC. Accordingly, co-staining of endogenous GPR158 with neuronal ciliary protein adenylate cyclase 3 (AC3), indicates its presence, at least partially, at the PC in hippocampal neurons (data not shown). Furthermore, OCN treatment increases the number of Gprl58 positive puncta associated with ACIII positive ciliary plasma membrane (data not shown). The presence of GPR158 was further illustrated via co-immunoprecipitation and coimmunofluorescence experiments between endogenous GRP 158 and the ciliary protein, Tubby- like protein 3 (TULP3), which is involved in the localization and trafficking of GPCRs to PC 50 (data not shown).

[0198] Core PC-proteins are required to mediate the induction of autophagy by OCN.

[0199] Next, we found that treatment of primary hippocampal neurons with OCN, or local stereotactic injections of OCN in the hippocampus, enhance the level of core PC-proteins, namely IFT20, IFT88, KIF3A and TULP3, together with increasing LC3 lipidation (LC3II levels) (data not shown). By contrast, core PC-proteins levels are lower in the hippocampus of either Ocn- / - or Gprl58- / - mice than in wild type (WT) littermates, which is correlated with reduced LC3II levels (data not shown). Therefore, using stereotactic injections of adeno- associated virus expressing small -hairpin-RNA (AAV), we generated mouse models in which the intra-flagellar Ift20 (AAV9-U6-shRNA-Ift20) or kinesin Kif3a (AAV9-U6-shRNA-Kif3a) proteins are selectively downregulated in the hippocampus. We found that these mice exhibit a significant reduction of hippocampal GPR158 levels in comparison to controls (AAV-U6- shRNA-Scramble injected mice) (Fig. IB). Similar results were obtained after Ift20 downregulation selectively in mature hippocampal neurons, generated by local stereotactic injections of AAVs expressing shRNAmir for Ift20 (AAV9-eSYN-Ift20-shRNAmir) under the control of the neuronal eSynapsin 1 (e-SYN) promoter (Fig. 1C). Moreover, we found that Ift20 downregulation in mature hippocampal neurons is sufficient to abolish the induction of the hippocampal autophagy machinery (data not shown) and neuronal activity (as shown by cFOS expression measurements) (Fig. ID) by OCN. Altogether, these data demonstrate that core PC- proteins are required to mediate the induction of autophagy machinery in hippocampal neurons, by the coupling of OCN and GPR158.

[0200] Core PC-proteins modulate autophagy levels in hippocampal neurons.

[0201] We sought to investigate a potential link between core PC-proteins and autophagy in the hippocampus. Accordingly, we found that downregulation of either Ift20 or Kif3a decreases autophagy in CA3, as shown by the reduction of LC3 puncta, together with the increase of SQSTMl / p62 positive puncta in comparison to controls (Fig. 2A). Of note this decrease was not observed in DG (data not shown). Similar results were obtained after selective Ift20 downregulation in mature hippocampal neurons (AAV9-eSYN-shRNA-Ift20-mir) (Fig. 2B-C). Indeed, we found an alteration of autophagic flux, as shown by a decreased in LC3II levels combined with an increase of SQSTMl / p62 positive puncta in comparison to controls (Fig. 2B- C). Interestingly, this reduction in autophagy observed after Ift20 postnatal downregulation is associated with longer PC axonemes (data not shown).

[0202] Core PC-proteins influence hippocampal-dependent memory.

[0203] Our previous findings indicate that neuronal autophagy in the hippocampus is essential to foster neuronal homeostasis and thereby memory function (20,33). Therefore, we investigated the functional impact of decreasing the levels of hippocampal core PC-proteins for the regulation of cognitive functions. To that end, we first subjected our mouse models of hippocampal Ift20 and Kif a downregulation to three independent behavioral tests assessing hippocampal-dependent learning and memory (51,52): The novel object recognition (NOR) test, the 3-foot-shocks cued fear conditioning (CFC) paradigm and the Morris water maze test (MWM) (Fig. 3A-C). NOR and CFC tests assess associative memory and rely on two phases: a training phase, in which mice are exposed to novel memory stimulations (NOR: exposure to two similar objects; CFC: 3 foot-shocks associated to a neutral environment) and a testing phase performed 24 hrs after the training phase, in which memory performances are evaluated (NOR: recognition of a novel exposed object; CFC: measurement of context-elicited freezing). In NOR, we found that the downregulation of both Ift20 or Kif3a severely impaired memory capacities, as demonstrated by a decrease in the time spent exploring a novel object compared to control mice (Fig. 3A). We next confirmed these results in CFC, by showing that mice downregulated for core PC-proteins exhibit a decrease in freezing during contextual memory testing in comparison to controls (Fig. 3B). Finally, spatial learning and memory was assessed using MWM, in which latencies to locate the platform were recorded for each trial. We found that downregulation of either Ift20 or Kifia in the hippocampus lead to a decrease in memory performances, as shown by the drastic increase in the latency to locate the escape platform in comparison to controls (Fig. 3C). By contrast, downregulated mice and their controls show comparable performances in the open field (OFT) and Light-Dark (LDT) tests, indicating that exploratory- and anxiety-like behaviors are unaffected (data not shown). Importantly, the downregulation of key core PC-proteins selectively in mature hippocampal neurons (after stereotactic injections of AAV9-eSYN-Ift20-shRNAmir or AAV9-eSYN-Ift88-shRNAmir), leads to similar memory deficits in NOR and CFC (data not shown), without affecting exploratory- and anxiety-like behaviors (data not shown). However, by contrast the selective downregulation of Ift20 in mature neurons did not induce any changes in MWM, suggesting potential implication of core PC-proteins in neuronal progenitors or maturing neurons in the hippocampus. Taken together, these results demonstrate that reducing the levels of key core PC-proteins in hippocampal neurons leads to severe impairments of memory without affecting their exploratory- and anxiety-like behaviors.

[0204] Autophagy induction counteracts cognitive deficits, despite Core PC-proteins downregulation.

[0205] To further examine the functional role of hippocampal core PC-protein-dependent autophagy in the regulation of cognition, we tested whether inducing hippocampal autophagy in mouse downregulated for neuronal Ift20 is sufficient to counteract the memory deficits observed in these mice. To that end, we performed hippocampal daily infusion of either TAT- Beclin 1 peptide, (an efficient pharmacological inducer of AP formation53) or TAT-scramble (control), after implantation of bilateral cannulas in animals downregulated for Ift20. We validated that TAT-Beclin-1 infusions improve the defects in autophagy observed after downregulation of neuronal Ift20 (Fig. 3D), as shown by the significative increase in LC3II accumulation upon TAT-Beclin-1 treatment. Then, mice were exposed to the NOR to assess memory function. As a result, we found that the improved level of autophagy is sufficient to restore the recognition and contextual memory deficits induced by neuronal Ift20 downregulation in the hippocampus (Fig. 3E).

[0206] Core PC-proteins are reduced in old hippocampi and restoring their levels is sufficient to improve age-related autophagy and memory deficits. Normal brain aging is characterized by a decline in autophagy machinery in hippocampal neurons leading to memory deficits (3,20). Importantly, we observed that this alteration is correlated to a marked decrease of core PC-constituents (such as IFT20, IFT25, IFT88 and KIF3A, both at the mRNA (data not shown) and protein levels (data not shown), in the hippocampus of aged (16-month-old) mice. This decrease is also associated with PC morphological abnormalities, characterized by a longer PC axoneme in hippocampal neurons in both CA3 (data not shown) and DG regions (data not shown). Interestingly, these morphological abnormalities were similar to the ones observed in PC after selective downregulation of Ift20 in hippocampal neurons of 3-month-old mice (data not shown). These results prompted us to examine whether restoring core PC-protein levels in old hippocampi could improve age-related neuronal autophagy decrease and memory deficits (Fig. 4A). To do so, we performed selective stereotactic injections of an AAV9 expressing Ift20 cDNA in aged mouse hippocampi. Three weeks after the injections, we confirmed that IFT20 level was restored in old hippocampi (Fig. 4A), which is accompanied by an improvement of the level of other core PC-proteins, such as IFT88, KIF3 A and IFT25 (Fig. 4B). Importantly, we found that restoration of Ift20 levels is sufficient to both increase the LC3II levels and reduce SQSTMl / p62 accumulation, suggesting an improvement of autophagy activity in old hippocampi (Fig. 4C). Exposing these mice to behavioral tests, we next observed that restoring neuronal IFT levels in old hippocampi, despite no change in PC size (data not shown), is sufficient to reverse age-related cognitive deficits (Fig. 4D). Together, these results indicate that the reduction of core PC-protein levels in the hippocampus during aging is associated with cognitive decline and that their restoration is sufficient to improve these deficits as well as neuronal autophagy decline in old hippocampi.

[0207] Neuronal PC-dependent autophagy mediates the rejuvenating effects of OCN on cognition.

[0208] These findings prompted us to conduct two independent experiments to test whether core PC-proteins are required to transduce the effects of OCN on age-related cognitive decline. First, we show that chronic infusion of recombinant OCN (via implantation of subcutaneous mini-osmotic pumps) in 16-month-old mice is sufficient to improve the enfeeblement of key core PC-proteins, namely IFT20 and TULP3 in the hippocampus (data not shown). Conversely, we show that the improvement of age-related autophagy decline and memory deficits after systemic OCN administration is abolished after Ift20 downregulation in hippocampal neurons (Fig. 5A). Altogether, these data strongly suggest a necessary contribution of core PC-proteins- dependent autophagy to foster cognitive fitness.

[0209] OCN regulates autophagy via activation of PC-dependent CREB signaling pathway in hippocampal neurons.

[0210] Activation of the transcription factor cAMP response element-binding protein (CREB) is decreased with age in the hippocampus (54-56). These alterations in CREB signaling contribute to cognitive deficits as observed in normal aging and various neuro-degenerative diseases (53-55). Importantly, multiple reports have shown that the rejuvenating effects of the young plasma administration on cognition are mediated, in part, through the activation of CREB (9,13). Accordingly, we first found that OCN enhances CREB and CREB phosphorylation levels in mouse hippocampi (Fig. 5B). More importantly, we show in primary hippocampal neurons that the increase in LC3II accumulation observed upon OCN is abolished following CREB inhibitor (666-15) treatment (data not shown). These observations suggest that CREB signaling pathway is required for the regulation of OCN-induced neuronal autophagy in hippocampal neurons. We next confirmed that CREB phosphorylation is lower in 16-month- old hippocampi, and we show that this decrease could be reversed following OCN administration (data not shown). Then, we found that hippocampal downregulation of either Ift20 or Kif3a drastically decreased neuronal pCREB / CREB ratio (data not shown) in the hippocampus. Conversely, the restoration of IFT20 levels (after local AAV9-U6-Ift20cDNA injections) is sufficient to improve CREB phosphorylation deficits in the hippocampi of 16- month-old mice (Fig. 5C). Lastly, we show that downregulation of neuronal Ift20 blocked the improvement of CREB phosphorylation by OCN in old mice (data not shown). Altogether, these data support the necessary contribution of PC-dependent cAMP response element-binding protein (CREB) signaling pathway to mediate the induction of autophagy machinery by OCN and thereby to the rejuvenating effects of OCN on age-related cognitive decline.

[0211] Conclusion

[0212] This study unravels a novel physiological role of the autophagy-PC axis in brain, where alteration of essential ciliary core PC-proteins during aging leads to autophagy impairments and thereby memory deficits (Fig. 6). This study proposes a novel paradigm in the mechanism controlling the communication between systemic milieu and neurons and its importance during brain aging for the maintenance of neuronal homeostasis and signal transduction in the hippocampus. PC could constitute a gateway in hippocampal neurons through which systemic factors modulate neuronal autophagy and thereby cognitive fitness. By shedding light on novel regulatory mechanism responsible for the decline of autophagy activity during aging, our findings also provide the foundation for the development of more potent therapeutic targets to prevent / treat age-related cognitive disorders, as well as neurological disorders in ciliopathies, which represent a major public health care challenge.

[0213] REFERENCES:

[0214] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. An activator of the primary cilia (PC) for use in the restoration and / or improvement of cognitive functions in a subject in need thereof.

2. An activator of the primary cilia (PC) for use in the treatment of cognitive troubles in a subject in need thereof.

3. The activator of the primary cilia (PC) for use wherein the cognitive troubles are troubles in knowledge, attention, memory and working memory, judgment and evaluation, processing speed, reasoning and computation, executive functioning, visuospatial abilities, problem solving and decision making, comprehension and production of language.

4. An activator of the primary cilia (PC) for use in the restoration and / or improvement of the age-related memory decline or age-related memory loss in a subject in need thereof.

5. An activator of the primary cilia (PC) for use to restore and / or improve the memory in a subject in need thereof suffering from Alzheimer’s disease, dementia, amnesia, Hyperthymestic syndrome, Huntington's disease, Parkinson's disease, Stress or Wernicke- Korsakoff s syndrome.

6. The activator of the primary cilia (PC) for use according to claims 1 to 5 wherein the activator is intraflagellar transport IFT (IFT20, IFT25 or IFT88), the Kinesin Family Member 3 A (KIF3 A) or TULP3 or fragment thereof and / or an agent for IFT20, IFT25, IFT88, KIF3 A or TULP3 protein expression.

7. A method for restoring and / or improving cognitive functions in a subject in need thereof comprising a step of administering to said subject a therapeutically effective amount of an activator of the primary cilia (PC) of the invention.

8. An activator of the primary cilia (PC) or to an autophagy activator for use in the treatment of ciliopathies in a subject in need thereof.

9. the autophagy activator for use according to the claim 8 wherein the activator of the TAT-Beclin-1 or TETA.

10. A therapeutic composition comprising activator of the primary cilia (PC) for use in the restoration and / or improvement of cognitive functions in a subject in need thereof.

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