LEPTIN RESISTANCE

FIELD OF THE INVENTION

The present invention relates to the field of preventing or treating disorders or diseases caused by the occurrence of a leptin resistance in an individual.

BACKGROUND OF THE INVENTION

Leptin is a 16 kDa hormone discovered in 1994, which is predominantly secreted by adipocytes and encoded by the obese (ob) gene. Leptin is able to interact with a variety of peripheral tissues as well as regions of the brain through specific receptors.

In particular, leptin is known to act on the brain to reduce food intake by regulating the activity of neurons in the mediobasal hypothalamus (Ahima, R.S. & Flier, 2000, J.S. Leptin. Annu Rev Physiol 62, 413-437).

Leptin is well-known for its appetite-suppressing effect. Indeed, obesity is associated with high circulating leptin levels that fail to trigger neuronal responses (Considine, R.V., et al, 1996, N.Engl.J.Med. 334, 292-295 ; Frederich, R.C., et al, 1995, Nat Med 1, 1311-1314).

What is more, many studies have shown that administering exogenous Leptin induces satiety (i.e. reduced appetite) and weight-loss in obese patients (see for instance Farooqi et al, 2002, J. Clin. Invest. 1 10, 1093-1103 ; Licinio et al, 2004, Proc. Natl. Acad. Sci. USA 101, 4531-4536).

In particular, deficient leptin signalling in humans or in murine models, for instance models bearing a mutation of leptin (ob/ob mice) or the leptin receptor (db/db mice), is associated with increased food intake and reduced energy expenditure. The main consequence is the development of imbalance between intake and expenditure, which may be associated with the development of various disorders.

Most notably, leptin is known to regulate insulin sensitivity and glucose homeostasis, and is thus involved in a wide variety of other disorders associated with energy imbalance, apart from obesity, such as diabetes or cardiovascular diseases (Roth, 1998, Diabetes Metab. Rev., 13, 1-2). Apart from its role in food intake and glucose homeostasis, leptin has also been implicated in reward behaviour, hippocampal dependent learning and memory, and is further known for its anti-depressant and anti-convulsivant effects. Many of those actions of leptin can be linked to its effect in the central nervous system (or CNS), and more particularly to its effect in the basomedial hypothalamus. For a more complete review of the effects of leptin as a regulator of neuronal functions, one may for instance refer to Harvey (Harvey, 2007, Current Opinion in Pharmacology, 7 :643-647).

Thus, leptin is a particularly interesting hormone due to its diverse effects in health and disease, as an active agent, and for promising new therapies in a wide spectrum of conditions.

However, it has been observed that high circulating levels of leptin fail to promote weight loss in obesity. What is more, most forms of obesity, such as high fat-diet induced obesity, are associated with a diminished responsiveness to leptin (Miinzberg & Myers, 2005, Nature neuroscience, Vol. 8 (5), pp. 568-570)

More generally, various mechanisms have been proposed to be responsible for this attenuation of leptin signaling ; said attenuation, also referred herein as « leptin resistance », negatively impacts the prospects of treating an individual suffering from a leptin resistance, and more particularly individuals suffering from obesity. The mechanisms underlying resistance to leptin remain, however, poorly understood, and different models have been proposed (El-Haschimi et al, 2000, J Clin Invest 105, 1827- 1832).

A first hypothesis states that circulating leptin fails to reach its targets in the brain. Indeed, leptin transport into the brain appears to be a crucial limiting step in the modulation of its central effects, and participates in leptin resistance both in humans and rodents (Caro et al, 1996, Lancet 348, 159-161; Schwartz et al, 1996, Nat Med, 2, 589- 593).

Remarkably, exogenous leptin administered intraperitoneally triggers LepR signalling in specialized neurons in the arcuate nucleus, a major hypothalamic leptin- sensing site adjacent to the median eminence (ME), within 15 minutes, and in more dorsal hypothalamic sites involved in energy homeostasis 1-2 h later. This delay is reduced to minutes when leptin is administered intracerebroventricularly, suggesting that its transport from the periphery to the cerebrospinal fluid (CSF) is a limiting step in the propagation of leptinergic signaling (Faouzi et a/., 2007, Endocrinology, 148, 5414-5423). In particular, it has been proposed that leptin may be transported across the blood-brain barrier by a saturable transport system (Banks, W.A., 2004, Peptides, 25, 331-338).

Thus, the identity of this transport system, its localization within the brain as well as its contribution to the overall development of leptin resistance remains elusive.

A second hypothesis suggests that leptin resistance occurs due to an inhibition of one or more intracellular signaling cascade associated with isoforms of the leptin receptor (LepR or LR), in particular the long isoform (LepRb or LRb). For a more general review on the leptin receptor, one may for instance refer to Tartaglia (Tartaglia L.A., 1997, J.Biol.Chem, 272, 6093-6096). This signaling involves intracellular pathways, including STAT3, ERK and PI3K→Akt, which, up to now, had not been precisely defined (Robertson et al, 2008, Physiol Behav, 94, 637-642).

Thus, reversing leptin resistance has become a major challenge in obesity research.

Thus there remains a need for the identification of compounds and/or compositions useful for treating or preventing leptin resistance.

There is also an urgent need for compounds and/or compositions against leptin resistance, which are able to cross the blood-brain barrier efficiently.

In particular, there remains a need for alternative methods for treating or preventing leptin-resistant individuals and leptin-related disorders.

More particularly, there remains a need for improved methods for treating or preventing obesity, diabetes and cognitive disorders which have been associated with leptin and/or leptin resistance.

There is also a need for methods of screening for new compounds for treating or preventing leptin resistance.

SUMMARY OF THE INVENTION

The invention relates to new strategies for treating and/or preventing the occurrence of leptin resistance in an individual. In particular, the invention relates to ERK- pathway activating compounds for use for treating and/or preventing leptin resistance in an individual. The development of a leptin resistance may be particularly detrimental for individuals suffering from diabetes, obesity and/or cognitive disorders, and/or any other leptin-related disorder, as it may reduce the efficacy of a treatment in which the administration of leptin is involved, and/or which is aimed at modulating deficient leptin signaling.

Thus, according to one of its aspects, the invention relates to ERK-pathway activating compounds for treating or preventing leptin resistance in an individual.

In particular the invention relates to pharmaceutical compositions comprising an ERK-pathway activating compound in combination with leptin.

The invention further relates to methods for the screening of compounds for treating and/or preventing leptin resistance.

The present invention relates ERK-pathway activating compounds for use for treating and/or preventing leptin resistance in an individual in need thereof.

According to the invention, « leptin resistance » refers to a lack of leptin activity in the brain, which occurs because of a reduction or failure of sensitivity to leptin. A reduction or failure of sensitivity to leptin may be due to a defective leptin transport to the cerebrospinal fluid, and/or defective signalling pathway, downstream of leptin receptor.

The present invention further relates to ERK-pathway activating compounds for treating or preventing diabetes, obesity and/or cognitive disorders associated with leptin resistance.

In other embodiments, the invention relates to pharmaceutical compositions comprising an ERK-pathway activating compound in combination with leptin.

In other embodiments, the invention relates to methods for the screening of compounds for treating and/or preventing leptin resistance in an individual in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Tanycytes of the ME express functional LepR

RT-PCR analysis of the expression of mRNAs for LepR isoforms (from up to bottom: LepRa; LepRb; LepRc; LepRe) in primary cultures of tanycytes (from left to right: tanycytes from the hypothalamus; tanycytes from the ME, tanycytes from the ME, as obtained from Prevot et al. , 2003.

Figure 2: Tanycytes of the ME internalize leptin through clathrin-coated vesicles.

(A) Representative western blots and quantitative comparison of phosphorylated and total STAT3, Akt and ERK 15 min after leptin or vehicle treatment of cultured tanycytes (n = 4 per group). Values indicate means ± SEM. * : p < 0.05; ***: p < 0.001 (two-tailed one-way ANOVA, followed by Newman-Keuls multiple comparison test).

The quantitative comparison of phosphorylated STAT3, Akt and ERK is assessed by determining (from left to right; y axis) the ratio pSTAT3/STAT3; pAkt/Akt; pERK/ERK, for three different leptin concentrations (x axis; from left to right: 0, 1 and 10 (B) Representative western blots and quantitative comparison of leptin and clathrin in immunoprecipitated (IPP) clathrin-coated vesicles from tanycytes (y axis; ratio leptin/clathrin) treated for 15 min with vehicle, LAN (^g/ml) or leptin (1 μg/ml) in the presence or absence of U0126 (10 μΜ), LY294002 (30 μΜ) and WP1066 (30 μΜ). ND: not detected; ns: not statistically significant (p = 0.503).

Figure 3: Tanycytes of the ME release captured leptin via an ERK- dependent signalling pathway.

(A) Representative live-cell imaging of fluorescent leptin distribution within tanycytes over an acquisition time of 30 min. Left panel, xy transmission image of the cultured tanycytes that were analyzed. Right panel, xz reconstruction of section 1 showing the dynamics of leptin transport from 15 to 45 min following leptin administration. Scale bar: 50 μπι.

(B) Representative fluorescence profile in a single cell 15, 30 and 45 min after treatment. The intensity of fluorescence is given in the y axis (rack units)

(C) Representative western blots and quantitative comparison of leptin in cell lysates from leptin-loaded tanycytes and in the medium (released leptin) over 15 min, in the presence or absence of the pharmacological inhibitors of LepR signalling pathways used in Figure 2B. STAT3 was used as a loading control for cell lysate samples. ***: p < 0.001; a vs. b. D: not detected (two-tailed one-way ANOVA, followed by Tukey's multiple comparison test). The level of leptin in the tanycyte-cultured medium (TCM) is compared to the level of leptin present in the cell lysate (CL). The corresponding TCM/CL leptin ratio is indicated in the y axis.

Figure 4: EGF-mediated activation of ERK signaling in the ME restores leptin transport into the hypothalamus.

(A) EGF (1 mg/kg, 15 min) promotes ERK activation in the ME as shown by the quantitative comparison of western blots (left panels; n = 5 per group). The pERK/ERK ratio in median eminence (ME) tanycytes is indicated in the y axis. The experiment is done on (from left to right) wild-type mice, diet-induced obesity (DIO) mice and db/db mutant mice.

(B) Representative western blots and quantitative comparison of leptin in ME and MBH explants from (left panel) DIO (n = 4) and (right panel) db/db mice (n = 3) mice after i.p. leptin administration (3 mg/kg), with or without EGF treatment 15 min before sacrifice.

(C) Quantitation (n = 4) of pSTAT3-IR immunofluorescence (number of pSTAT3-IR neurons; y axis) after i.p. administration of leptin (3 mg/kg, 45 min; right column) or vehicle (45 min; left column) along with EGF treatment 15 min before sacrifice in DIO mice. ARH: arcuate nucleus of the hypothalamus; VMH: ventromedial nucleus of the hypothalamus; DMH: dorsomedial nucleus of the hypothalamus.

Figure 5: EGF-mediated activation of ERK signaling accelerates the restoration of leptin sensitivity in obese mice once they are replaced on a normal diet.

(A) Body weight (g) change ( y axis) over 20 weeks (x axis) in standard-chow- fed mice, mice with diet-induced obesity (DIO) and DIO mice replaced on a standard diet after 1 week of EGF treatment (1 mg/kg/24h; DIO-R+EGF, arrowheads) or no EGF treatment (DIO-R) (n = 6-8 per group).

(B) top panels: representative western blots and quantitative comparison of leptin in ME and MBH explants from standard-chow-fed, DIO, DIO-R and DIO-R+EGF mice (n = 3-4 per group) after i.p. leptin administration (3 mg/kg) at the time points (week 8; week 12; week 18) indicated by arrows in A; the ratio leptin/STAT3 is indicated in the y axis; bottom panel: body weight change (g) in standard-chow-fed, DIO, DIO-R and DIO-R+EGF mice after daily i.p. leptin administration for 3 days (n = 6-8 per group). ***: p < 0.001; **: p < 0.01; *: p < 0.05; ns: p > 0.05. D: not detected. Differences between groups were analyzed using a one-tailed one-way ANOVA in A and a two-tailed one-way ANOVA followed by Tukey's multiple comparison test in B. Leptin-induced body weight change (g) is indicated in the y axis.

Figure 6: EGF treatment affects glucose metabolism and restores leptin sensitivity and leptin transport.

(A) EGF treatment impact on weight gain effect of high-fat diet (HFD). Two way ANOVA (treatment - time) Mann- Whitney post-hoc test. *** p<0.001, **** pO.0001.

Body weight (g) is indicated in the y axis, as a function of time (weeks).

(B) EGF treatment impact on glucose metabolism after long-term treatment (8 weeks) by a glucose tolerance test. Two way ANOVA (treatment - time) Mann- Whitney post-hoc test. ** p<0.01, $$$ or *** pO.001, $$$$ or **** pO.0001.

Blood glucose (mg/dl) is indicated in the y axis, as a function of time

(minutes).

(C) top panel: representative western blots and quantitative comparison of leptin in ME and MBH explants from standard-chow-fed, DIO and DIO mice receiving long-term EGF treatment (lmg/kg/day, n = 4 per group) after i.p. leptin administration (3 mg/kg) at the time points indicated by arrows in (A).

The molar ratio leptin/STAT3 is indicated in the y axis of the figure. bottom panel: sensitivity to leptin is assessed by measuring by measuring body weight loss after 3 days of peripheral leptin administration (3mg/kg) in control and DIO- mice, with and without long-term EGF treatment (8 weeks); BW decrease does not differ between control mice and DIO mice receiving long-term EGF treatment; T-test, * p<0.05

The leptin-induced body weight change is indicated in the y axis of the figure.

(D) Perigonadal white adipose tissue (WAT) content (y axis) is compared between control mice, DIO mice, and DIO mice with long-term EGF treatment (x axis). One-way ANOVA (two-tailed) Tukey post-hoc test. *** p<0.001. Figure 7: Characterization of the selectivity of the pharmacological agents used to block the different LepRb-coupled signaling pathways in primary cultures of tanycytes.

Representative western blots for phosphorylated and total forms of STAT3, Akt and ERK from tanycytes treated for 15 min with vehicle or leptin in the presence or absence of (A) U0126 (10 μΜ), (B) LY294002 (30 μΜ) and (C) WP1066 (30 μΜ). Figure 8: Semaphorin 7A, or Sema7a, activates ERK phosphorylation (P-

ERK) in tanycytes, which express its receptor, integrin βΐ, in vitro.

Representative western blots for phosphorylated and total forms of βΐ -integrin and ERK from tanycytes treated for 30 min with vehicle or Sema7a (250 ng/ml). From top to bottom: Plexin CI; βΐ integrin; phosphorylated βΐ integrin, phosphorylated Erkl/2, Erk 1/2.

Figure 9: Tanycytes of the median eminence express functional ghrelin- mediated signalling in vitro.

Representative western blots and quantitative comparison of phosphorylated and total ERK 15 min after ghrelin or vehicle treatment of rat cultured tanycytes.

The y axis indicates the corresponding pERK/ERK ratio in cultured tanycytes, and the x axis indicates the concentration of ghrelin which is present within the medium culture.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have found that the leptin receptor (LepR) is expressed at the surface of a particular set of glial-like cells called the tanycytes (see example 1 herein).

Tanycytes are specialized ependymal cells which line the surface of the third ventricle. They tend to be enriched, among other ependymal cells, at the ventral part of the third ventricle, and more specifically a region of the third ventricle called the median eminence (ME). So far, they have been involved in the control of feeding and energy balance. More generally, they are emerging as chemosensors in contact with the cerebrospinal fluid (Bolborea & Dale, 2013, Trends in Neurosciences, 36(2), pp 91-100).

In particular, the inventors have assessed the activation of LepR signalling in the median eminence of adult wild-type C57B1/6 mice by immunohistochemically detecting phosphorylated STAT3 (pSTAT3) at various times after peripheral leptin administration (3 mg/kg). Strikingly, five minutes after leptin injection, pSTAT3 immunoreactivity (IR) was detected both in tanycytic processes, which contact the plexus of permeable fenestrated vessels at the pial surface of the brain (Mullier et al, 2010, J Comp Neurol 518, 943-962), and in their nuclei, whereas pSTAT3-IR in neurons was both infrequent and weak. By 10-15 min after leptin injection, although some tanycytes were still labelled, pSTAT3-IR in neurons dramatically increased, while at longer intervals, pSTAT3-IR occurred almost exclusively in neurons.

Thus, the inventors have shown that circulating leptin sequentially activates LepR signalling in ME tanycytes followed by neurons.

It has also been found according to the invention that peripherally administering leptin promotes the sequential activation of leptin receptor (LepR) in tanycytes of the median eminence (ME) which form the blood-CSF barrier.

It has been further shown according to the invention that, in mice deficient in LepR or with diet-induced obesity (DIO), blood-borne leptin that is taken up by tanycytes accumulates in the median eminence and fails to gain access to the mediobasal hypothalamus.

The experimental results obtained by the inventors fully support that tanycytes play a major role in leptin uptake and release within the brain and suggest that the loss of tanycytic LepR signaling is a major cause of the pathophysiology of leptin resistance.

The results disclose herein that leptin resistance occurs due to the accumulation of leptin in the median eminence. Thus, they propose that leptin which has been taken up by tanycytes cannot reach the mediobasal hypothalamus.

The identification of a relationship between leptin resistance, leptin uptake and leptin transport by tanycytes, led the inventors to develop new therapeutic strategies and compositions against leptin resistance in an individual. Also, the inventors have designed new tools for the screening of compounds which are useful for treating and/or preventing leptin resistance. It is underlined that, according to the applicant's knowledge, evidence for molecular mechanisms of leptin transport within the brain are disclosed for the first time in the present patent application, in particular methods for maintaining and/or restoring leptin transport, and more particularly leptin transport associated with obesity.

According to a first embodiment, the invention relates to ERK-pathway activating compounds for use for treating and/or preventing leptin resistance in an individual in need thereof.

As disclosed herein, the inventors have shown that the activation of ERK signaling, in particular by means of a pharmacological treatment, is required for the release of the exogenous leptin captured by tanycytes, and for restoring leptin transport in the mediobasal hypothalamus (MBH) of obese mice (see example 4 herein).

In particular, it is disclosed herein that activation of ERK-signaling in median eminence tanycytes can functionally restore tanycyte-dependent leptin release in the MBH of diet-induced obese leptin-resistant individuals. ERK and/or LepR-ERK signaling are thus shown to be an efficient therapeutic target in leptin-related diseases such as obesity or diabetes (see examples 5 and 6 herein).

The novel concept of a tanycytic conduit for peripheral metabolic hormones into the brain on the one side, and the involvement of LepR-ERK signalling in central leptin resistance on the other side, holds therapeutic potential not only in obesity or diabetes but also in impaired cognitive processes, since adiposity hormones such as leptin play important roles in the regulation of higher brain functions.

Accordingly, the present invention relates to ERK-pathway activating compounds for treating and/or preventing leptin resistance in an individual affected by diabetes, obesity, or a cognitive disorder.

Thus, the invention further relates to ERK-pathway activating compounds for treating and/or preventing diabetes, obesity or a cognitive disorder.

This invention also pertains to ERK-pathway activating compounds for treating and/or preventing leptin resistance in an individual affected by obesity.

According to the invention, "obesity" comprises diet-induced obesity and especially diet-induced leptin-resistant obesity. This invention also concerns ERK-pathway activating compounds for treating and/or preventing leptin resistance in an individual affected by diabetes, more particularly type II diabetes.

According to the invention, "diabetes" comprises type II diabetes.

This invention further relates to ERK-pathway activating compounds for treating and/or preventing leptin resistance in an individual affected with cognitive disorders.

According to the invention, a "cognitive disorder" comprises a cognitive disorder associated with leptin resistance, and is selected in the group comprising or consisting in major depression, adjustment disorder, schizoaffective disorder, schizophrenia, bipolar disorder, seizure, Alzheimer's disease, autism, anorexia, Binge- eating, bulimia nervosa, Prader-Willy syndrome. In particular, the invention relates to individuals suffering from a cognitive disorder associated with eating disorders such as Binge-eating, bulimia nervosa, Prader-Willy and anorexia.

An « ERK-pathway activating compound » refers to any compound which is able to induce, at a given concentration, the phosphorylation of the intracellular protein ERK within tanycytes, or alternatively to increase the ratio of intracellular pERK/ERK.

ERK is the acronym for « Extracellular signal-regulated kinase ».

According to the invention, the protein ERK refers both to ERK-1 and ERK-2, which may be also referred in the literature as MAP kinases 1 and 2 (MAPKl and ΜΑΡΚ2

Advantageously, the phosphorylation of the intracellular protein ERK may be specifically detected by western blot and immunohistochemistry using the rabbit polyclonal anti-phospho-p44/42 ERK primary antibody (1 : 1000; #9101, Cell Signaling Technology), as it is disclosed in the examples herein.

In particular the inventors provide an experimental framework to assess the phosphorylation of ERK on tanycytes, such as the protocols followed for assessing ERK- pathway activating properties of EGF, ghrelin, and semaphorin-7a (also referred herein as Sema7a), especially in examples 4A, 7, 8 or 9.

For the purpose of the present invention, an "ERK-pathway activating compound" may be determined by culturing 90% confluent tanycytes from the median eminence in a suitable medium (tanycyte-defined medium or TDM) consisting of DMEM/F12 (devoid of phenol red; Invitrogen) supplemented with 1% L-glutamine, 2% penicillin/streptomycin, 5 μg/ml insulin (Sigma, Saint Quentin Fallavier, France), and 100 μΜ putrescine (Sigma)) for two days, by incubating the said tanycytes in the said medium comprising a candidate compound for 15 minutes at a given concentration, and then by assessing the phosphorylation of ERK by western blot, relatively to control untreated tanycytes, and then selecting the said candidate compound as consisting of an "ERK- pathway activating compound" if an increased phosphorylation of ERK is measured. The assessment of the phosphorylation of an intracellular protein by western blotting is well- known in the art.

In a non-limitative way, the ERK-pathway activating compound may be obtained by chemical synthesis, by genetic engineering, by purification form natural sources or by a combination of two or more of these methods.

In a non-limitative way, the ERK-pathway activating compound may be a protein, which comprises a protein obtained by chemical synthesis, a protein obtained by purification from a natural source and a recombinant protein.

An ERK-pathway activating compound, or a composition comprising or consisting in an ERK-pathway activating compound, as defined herein may be suitable for respiratory, enteral or parenteral administration.

Advantageously, an ERK-pathway activating compound, or a composition comprising or consisting in an ERK-pathway activating compound, as defined herein may be suitable for enteral or parenteral administration, and preferably suitable for parenteral administration.

An ERK-pathway activating compound, or a composition comprising or consisting in an ERK-pathway activating compound, as defined herein may be also suitable for administration by the respiratory route, in particular intranasal administration.

Thus, an ERK-pathway activating compound as defined herein may be suitable for intravenous, intranasal, intraperitoneal, intracerebral or intracerebroventicular administration.

For instance, the ERK-pathway activating compound as defined herein may be suitable for intravenous, intraperitoneal, intracerebral or intracerebroventicular administration. According to one particular embodiment, an ERK-pathway activating compound as defined herein is suitable for intraperitoneal and intravenous administration. According to another particular embodiment, an ERK-pathway activating compound as defined herein is suitable for enteral administration, and especially for oral administration.

An « ERK-pathway activating » compound as defined herein may be selected in a group comprising or consisting in VEGF, fluoxetin, B-Raf, integrin β-l ligands, GHS-R-la ligands, and EGF receptor ligands.

In certain embodiments, the « ERK-pathway activating compound is selected from a group comprising: EGF, VEGF, fluoxetin, Sema7a, B-Raf, ghrelin, TGFa, neuregulins, betacellulin, FIB-EGF, amphiregulin, epigen and epiregulin.

In certain embodiments, the GHS-R-la ligand may be ghrelin.

In certain embodiments, the integrin βΐ-ligand may be Sema7a.

In certain embodiments, the EGF receptor ligand may be selected in a group comprising EGF, TGFa, neuregulins, betacellulin, HB-EGF, amphiregulin, epigen and epiregulin.

In certain embodiments, the ERK-pathway activating compound is chosen in the group comprising EGF, ghrelin and Sema7a, and preferably EGF.

Among ERK-pathway activators, epidermal growth factor (EGF) is a preferred compound, because of the abundant expression of erbBl (an EGF receptor) in tanycytes.

According to the invention, "ERK-pathway activating compounds" and/or "leptin" compounds include derivatives of the compounds listed previously and having the same biological activity. In particular, the invention relates to any ERK-pathway activating compound or leptin derivative having the same biological activity regarding respectively the phosphorylation of ERK within tanycytes and the activation of leptin signaling within tanycytes.

According to the invention, derivatives of an "ERK-pathway activating compound" encompass compounds that are covalently linked to, or non-covalently linked to, a functional molecule such as a detectable molecule and/or a targeting-ligand, e.g. a cell penetrating peptide. A covalent or non-covalent linkage of an "ERK-pathway activating compound" with a detectable molecule is useful mainly for detection, screening or diagnosis purposes. A covalent or non-covalent linkage of an "ERK-pathway activating compound" with a targeting ligand is useful mainly for medical purposes.

Examples of detectable molecules are well known in the art. One may for instance refer to a detectable molecule such as: (i) a radioactive moiety, (ii) a fluorescent molecule, (iii) a luminescent molecule, (iv) a receptor molecule specifically recognized by another ligand and (v) a metallic tag.

Fluorophores which may be advantageously chosen include, in particular, lanthanide complexes, metallic tags such as Europium and Terbium, fluorescein, fluorescein isothiocyanate (FITC), dichlorotriazinylamine, rhodamine, eosine, coumarin, les methyl-coumarin, pyrene, Malachite green, Cy ^® or Alexa ^® (Invitrogen) fluorophores such as Cy ^® 3, Cy ^® 5, Alexa Fluor ^® 488, Lucifer Yellow, Cascade Blue, Texas Red, dansyle chloride, phycoerythrin, luciferin, GFP and its variants, bore-dipyromethene (BODIPY), and others which are known in the art such as the ones described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; The Synthegen catalog (Houston, Tex.), Lakowicz, Principles of Fluorescence Spectroscopy, 2nd Ed., Plenum Press New York (1999).

According to one particular embodiment, leptin and/or an « ERK-pathway activating compound », may be labeled with a molecule such as the ones described in Leyris et al. (2011, Anal Biochem., 408:253-262), Schaeffer et al. (2013, Proc Natl Acad Sci U S A., 110: 1512-1517) and Vauthier et al. (2013, Anal Biochem, 436: 1-9 http://dx.doi.Org/10.1016/j .ab.2012.12.013).

Thus, an ERK-pathway activating compound or leptin may be labelled with a d2 fluorescent probe (Cisbio), in particular labelled on lysines with a N- hydroxysuccinimiude-activated d2 dye in 100 mM P0 ₄ buffer at pH 8.0.

An ERK-pathway activating compound or leptin may be also labelled with a red fluorescent probe (Cisbio, Ref. LOO20RED).

According to another particular embodiment, the ERK-pathway activating compound may be linked covalently or non-covalently, and preferably covalently, to a cell-penetrating peptide.

A cell-penetrating peptide is a peptide which facilitates cellular uptake of molecular cargos. Such cell-penetrating peptides are well known in the art and are used to deliver a large variety of cargoes such as proteins, DNA, antibodies, contrast agents, toxins and nanoparticular drug carriers such as liposomes.

In particular, a "cell-penetrating peptide", or CPP, may be an arginine-rich cell-penetrating peptide. According to a more particular embodiment a cell-penetrating peptide may be a TAT-derived cell-penetrating peptide; the TAT peptide is an arginine-rich cell penetrating pepide derived from the transactivating protein TAT of HIV- 1 (Peitz et al., 2002, PNAS, 99:7, pp 4489-4494).

According to a preferred embodiment, the said cell-penetrating peptide comprises, and preferably consists in sequence SEQ ID NO 1 :

NH2-GRKKRRQRRR-COOH

wherein G is Glycine, Q is Glutamine, K is Lysine and R is Arginine.

In other embodiments, a cell-penetrating peptide may consist of a peptide derived from maurocalcine, and preferably a peptide derived from maurocalcine as described in the PCT application n° WO 2006/051224.

Methods of treatment and/or prevention of leptin resistance

The inventors have shown that a treatment of an individual with an ERK-pathway activating compound, such as EGF, has both the effect of accelerating weight loss and restoring leptin sensitivity in a durable manner (see examples 5 and 6 herein). In particular, the ERK-pathway activating compound may be used in complement with a diet, such as a low-fat diet.

Thus, the invention further relates to the medical use of an ERK-pathway activating compound, either used alone or used in combination with leptin. The ERK- pathway activating compound and leptin may be administered through identical or different administration modes, and preferably identical administration modes.

According to the invention, the ERK-pathway activating compound may be administered to an individual in need thereof for a long-term period. A « long-term period » relates to a daily administration of more than 6 days, and preferably at least 8 weeks.

In particular, when an ERK-pathway activating compound is administered in combination with leptin, it is preferably administered sequentially.

A "sequential administration" refers to the administration of two distinct compounds, wherein either (i) a first compound, such as an ERK-pathway activating compound is administered alone prior to the administration of a composition comprising the second compound, such as leptin or (ii) a first compound, such as an ERK-pathway activating compound is administered alone subsequently to the administration of a composition comprising the second compound, such as leptin.

In particular, a sequential administration refers to the administration of two distinct compounds, wherein a first compound, such as an ERK-pathway activating compound is administered alone prior to the administration of a composition comprising the second compound, such as leptin.

According to a preferred embodiment, the ERK-pathway activating compound is administered to an individual for a long-term period, followed by administration of leptin.

In particular, the sequential administration of leptin after a first administration of an ERK-pathway activating compound is also referred herein as a "leptin sensitivity test".

For instance, an ERK-pathway activating compound such as EGF may be administered to an individual for at least a period of time of 8 weeks, then followed by a leptin sensitivity test, comprising the administration of leptin to the said individual, alone or in combination with the said ERK-pathway activating compound.

In particular, when the ERK-pathway activating compound is EGF, EGF may be administered to an individual orally or parenterally, and is preferably administered intravenously or intraperitoneally, in an amount ranging from 0,001 to 3 mg/kg, more particularly from 0,001 to 1 mg/kg. (see Tsuda N. et al, 2008, Brain Dev: 533-543).

Alternatively, the ERK-pathway activating compound may be administered to an individual by the respiratory route, and in particular intranasally.

Leptin may be administered to an individual orally or parenterally, and preferably intravenously or intraperitoneally, in an amount ranging from 0.1 mg / kg to 10 mg/kg.

Alternatively, leptin may be administered to an individual by the respiratory route, and in particular intranasally.

According to certain embodiments, the administration of ERK-pathway activating compounds and leptin is an intraperitoneal administration. Pharmaceutical compositions

The invention further relates to pharmaceutical compositions comprising or consisting in an ERK-pathway activating compound, such as EGF, in combination with leptin.

In some embodiments, the ERK-pathway activating compound and leptin are comprised in the same dosage unit, i.e. in the same liquid or solid dosage unit.

In some embodiments, the ERK-pathway activating compound and leptin are comprised in separate dosage units in the pharmaceutical composition.

In some embodiments, the pharmaceutical composition comprises, or consists of, a kit comprising:

- a first container comprising or consisting in an ERK-pathway activating compound, and

- a second container comprising or consisting in leptin and optionally the said ERK-pathway activating compound.

As used herein, a « container » comprising a pharmaceutical composition according to the invention may be any pharmaceutical container known in the art, preferably a sterile container, which is either directly suitable for injection, or which may be suitable for preparing an injectable sample, alone or when mixed with a second container according to the invention. Examples of said containers may be selected in a group comprising pills, tablets, capsules, bottles, syringes, needles, catheters, infusion pumps, ampullas, and more generally any small sealed vial which may be used to contain and preserve a sample. Said containers may be made of glass or plastic, such as polypropylene.

Advantageously, said containers are subsequently mixed in order to achieve a specific pharmaceutical composition according to the invention.

According to this particular embodiment, the amount of ERK-pathway activating compound and leptin in each container can be easily adjusted by the man skilled in the art, in order to reach a specific ratio of ERK-pathway activating compound to leptin.

Often, pharmaceutical compositions will be administered by injection. For administration by injection, pharmaceutical compositions may be formulated as sterile aqueous or non-aqueous solutions or alternatively as sterile powders for the extemporaneous preparation of sterile injectable solutions. Such pharmaceutical compositions should be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

Pharmaceutically acceptable carriers for administration by injection are solvents or dispersion media such as aqueous solutions (e.g., Hank's solution, alcoholic/aqueous solutions, or saline solutions), and non-aqueous carriers (e.g., propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate). Injectable pharmaceutical compositions may also contain parenteral vehicles (such as sodium chloride and Ringer's dextrose), and/or intravenous vehicles (such as fluid and nutrient replenishers); as well as other conventional, pharmaceutically acceptable, nontoxic excipients and additives including salts, buffers, and preservatives such as antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thirmerosal, and the like). Prolonged absorption of the injectable compositions can be brought about by adding agents that can delay absorption (e.g., aluminum monostearate and gelatin). The pH and concentration of the compositions can readily be determined by those skilled in the art.

Sterile injectable solutions are prepared by incorporating the active compound(s) and other ingredients in the required amount of an appropriate solvent, and then by sterilizing the resulting mixture, for example, by filtration or irradiation. The methods of manufacture of sterile powders for the preparation of sterile injectable solutions are well known in the art and include vacuum drying and freeze-drying techniques.

Methods for the screening of ERK-pathway activating compounds Without wishing to be bound by any particular theory, the inventors believe that tanycytes may constitute the missing link in the loop that connects behaviour, hormonal changes, signal transduction, central neuronal activation and finally, behaviour again. In particular, they have shown that leptin resistance occurs because of deficient or insufficient leptin release by tanycytes.

Thus, the relationship between deficient or inefficient signalling and impaired hormone trafficking led the inventors to develop new strategies for screening compounds within tanycytes, which are able to modulate the uptake and/or the release of active agents such as adiposity hormones.

Herebelow are provided methods for the screening of compounds for treating and/or preventing leptin resistance which have the advantage to be predictive, suitable for high-throughput screening and adaptable to the measurement of a diverse set of variables and secondary controls.

Primary cultures of tanycytes are known in the art and may be followed according to any known protocol, such as the ones described in the examples.

According to a first embodiment, the invention relates to an in vitro method for the screening of compounds for treating and/or preventing leptin resistance in an individual in need thereof comprising the steps of:

a) providing cultured tanycytes,

b) incubating said tanycytes with leptin and a candidate compound, c) measuring the level of release of intracellular leptin,

d) selecting the said candidate compound, when the said candidate compound is determined to increase the level of release of intracellular leptin.

Of course, those values may be advantageously compared to any reference value, such as for instance a reference value that is measured in control cultures of tanycytes which have not been incubated with leptin and/or the said candidate compound. Thus a suitable reference value may be easily determined by the man skilled in the art on the basis of his general technical knowledge and of the content of the present specification, especially the examples herein.

In certain embodiments of the screening method above, the culture medium can be replaced by a tanycyte conditioned medium (TCM) after step b).

Standard medium used to culture tanycytes may be composed of DMEM (Invitrogen, San Diego, CA) supplemented with 2% (v/v) antibiotics (penicillin/streptomycin; Invitrogen), 1% L-glutamine (Invitrogen) and 10% donor bovine serum (Invitrogen).

In particular, tanycytes may be cultured in a "tanycyte defined medium" or

TDM.

TDM corresponds to the above-defined medium (without serum) applied 24h before treatment and during treatment and composed of DMEM/F12 (devoid of phenol red; Invitrogen) supplemented with 1% L-glutamine, 2% penicillin/streptomycin, 5 μg/ml insulin (Sigma, Saint Quentin Fallavier, France), and 100 μΜ putrescine (Sigma).

A "tanycyte conditioned medium", or TCM, can be defined as the fresh medium (for instance a tanycyte defined medium or TDM) in which tanycytes are incubated following treatment with leptin for 15 minutes. Advantageously, the TCM has the same composition as the original medium, namely TDM.

According to a particular embodiment of the screening method described above, the level of leptin release may be assessed using any method known in the art, such as for example western blotting, confocal imaging or ELISA methods.

When western blotting is used, the level of leptin release may be achieved by culturing tanycytes in a tanycyte conditioned medium (TCM), and then measuring the level of leptin which has been released by western blotting at distinct time points.

Advantageously, the level of leptin release can then be assessed over time, when compared to a control culture.

What is more, leptin release may be assessed in the presence of other modulating agents such as inhibitors of ERK and/or inhibitors of leptin signaling. Examples of such inhibitors are known in the art, and described for instance in example 1.

When confocal imaging is used, the leptin is preferably a leptin labeled with a detectable molecule, such as a fluorescent leptin. According to that particular embodiment, the level of leptin release may be assessed by measuring the decrease, over time, of the intensity of the fluorescent signal up to extinction, and by comparing it to a reference value.

Although ERK-pathway activating compounds have been proven efficient in promoting leptin release from tanycytes, those methods are also useful for providing information on the modulation of leptin uptake by a given compound, either on the same tanycyte or on a different tanycyte.

Thus, according to a particular embodiment, the invention relates to methods for the screening of compounds which further comprise a step of measuring the level of leptin uptake by the tanycytes.

In particular the steps of measuring the level of (i) leptin uptake and ii) leptin release may be performed with the same cultured tanycytes or with distinct cultured tanycytes. The level of leptin uptake may be measured according to any method known in the art, such as the ones described in example 2. In particular, leptin uptake may be assessed by clathrin immunoprecipitation, immunohistochemistry, western blotting, confocal imaging, or ELISA methods.

Advantageously, when leptin uptake is assessed by confocal-imaging, a time- lapse study can be performed on living cells and both leptin uptake and leptin release may be assessed on the same tanycyte (see figure 3A).

Thus, according to an alternative embodiment, the invention relates to an in vitro method for the screening of compounds for treating and/or preventing leptin resistance in an individual in need thereof comprising the steps of:

a) providing cultured tanycytes,

b) incubating said tanycytes with leptin and a candidate compound, c) optionally measuring the level of leptin uptake by said tanycytes, d) measuring the level of release of intracellular leptin by said tanycytes, e) selecting the said candidate compound, when the said candidate compound is determined to (i) optionally increase the level of leptin uptake at step c) and (ii) increase the level of release of intracellular leptin at step d),

wherein steps c) and d) are performed with the same cultured tanycytes or with distinct cultured tanycytes.

According to a more particular embodiment, the in vitro method may comprise a step of measuring the level of leptin uptake comprising the step of performing a Homogeneous Time-Resolved Fluorescence-Based Leptin Receptor binding assay, such as the one described in (Vauthier et al, 2013, Anal Biochem, 436: 1-9 http://dx.doi.Org/10.1016/j .ab.2012.12.013).

According to another alternative embodiment, the invention relates to a method for the screening of compounds for treating and/or preventing leptin resistance comprising the steps of:

a) administering leptin and a candidate compound to a non-human mammal, b) sacrificing the said non-human mammal,

c) collecting tanycytes from the said non-human mammal,

d) measuring the level of leptin in the said tanycytes, e) selecting the said compound, if the said compound is determined to increase the level of leptin measured at step d).

In particular, a "non-human mammal" may be a rat or a mouse, such as an adult wild-type C57B1/6 mouse, a diet-induced obese mouse, a LepR ^dmh or an ob/ob mutant mouse.

The non-human mammal may be sacrificed according to any method known in the art, such as transcardiac perfusion of a fixative solution.

Of course, it may be possible to collect tanycytes from tissue explants comprising said tanycytes, wherein the said tissue explants may be the median eminence or the mediobasal hypothalamus of a non-human mammal. Thus, according to said embodiment, the level of leptin release by tanycytes may be assessed on the whole tissue explant.

According to a preferred embodiment, tanycytes are collected from the median eminence or the mediobasal hypothalamus of a non-human mammal, and more preferably from the median eminence.

It may also be convenient to assess the level of ERK-phosphorylation in tanycytes in presence of a candidate compound. Advantageously, the assessment of the level of ERK-phosphorylation may thus be achieved before, during or after screening.

According to a particular embodiment, the level of ERK phosphorylation, or activation, in tanycytes may be measured by immunofluorescence.

Thus, according to a preferred embodiment, the invention relates to a method for the screening of compounds for treating and/or preventing leptin resistance comprising the steps of:

a) administering the candidate compound to a first non-human mammal, b) sacrificing the said non-human mammal by transcardiac perfusion of a fixative solution,

c) collecting the tanycytes from the said first non-human mammal, d) measuring the level of ERK phosphorylation in tanycytes by immunofluorescence,

e) selecting the said compound, if the said compound is determined to increase the level of ERK phosphorylation measured at step d), f) administering leptin and the said compound to a second non-human mammal,

g) sacrificing the said second non-human mammal,

h) collecting the median eminence and/or the mediobasal hypothalamus from the said second non-human mammal,

i) measuring the level of leptin in the said median eminence and/or the mediobasal hypothalamus,

j) selecting the said compound, if the said compound is determined to increase the level of leptin measured at step i).

According to a particular embodiment, leptin may be labeled with a detectable molecule as described previously, such as a fluorescently-labeled leptin.

According to another particular embodiment, methods for the screening of compounds may comprise an additional step of quantifying the phosphorylation of a protein in a sample or a tissue explant, in particular a protein within the said tanycytes, wherein the said protein is preferably selected in the group consisting in: ERK, STAT3 and Akt, and preferably STAT3 and ERK.

Advantageously, the phosphorylation of STAT3 can be assessed to confirm the occurrence of intracellular leptin signaling.

The quantification of the phosphorylation of a protein can be achieved by any method known in the art, such as western blotting, immunoprecipitation and immunohistochemistry, or as shown in the examples.

The present invention will be more fully described with the aid of the following examples and figures which should be considered as illustrative and non- limiting.

EXAMPLES

A. MATERIALS AND METHODS OF THE EXAMPLES

A.l. Statistics

Differences between several groups were analyzed using one-way ANOVA followed by the Student-Newman-Keuls or Tukey multiple-comparison post hoc test. Differences between two groups were analyzed using an unpaired Student's t-test. The threshold for significance was set at p < 0.05.

A.2. Animal procedures

Three- to four-month-old male C57B1/6 mice purchased from Charles River

(L'Arbresle, France) were housed under a 12h/12h light-dark cycle, and provided ad libitum access to water and standard laboratory chow (Special Diet Services, Commenailles, France) or a high-fat diet containing 60% fat (Research Diets, New Brunswick, NJ, USA). ). In one experiment, groups of diet-induced obese (DIO) mice that had been on a high- fat diet for 8 weeks were then switched to a regular diet for 4 to 10 weeks (until week 12 and 18, respectively; DIO-R). Mutant LepR ^dmh (db/db) mice were purchased from the Jackson Laboratories and housed under the same conditions and fed with standard chow. Sprague Dawley rats were purchased from Janvier (Saint Berthevin, France) and housed in a temperature-controlled room (21-23°C) with a 12h/12h light-dark cycle. Animals had ad libitum access to tap water and pelleted rat food. The protocols used here were approved by the Direction of Veterinary Departments of Nord (59-350134) and were in accordance its guidelines for the Care and Use of Laboratory Animals as well as the European Communities Council Directive of November 24th, 1986 (86/609/EEC) regarding mammalian research.

A.3. Leptin sensitivity test in mice

Mice were divided into two groups, which received daily intraperitoneal (i.p.) injections of recombinant murine leptin (3 mg/kg; Peprotech) or vehicle (10 mM Tris-HCl buffer) for 3 days. Body weight was measured before and 24h after the treatment period.

A.4. Protein extraction, western blotting and immunoprecipitation

Proteins were extracted and processed as described previously (De Seranno, S. et al, 2004, J.Neurosci. 24, 10353-1036; Prevot et al, 2003, J.Neurosci. 23, 10622- 10632). A.5. Clathrin Immunoprecipitation

Cell lysates were prepared in 750 μΐ _^ of Tris buffer (pH 7.4; 25 mM Tris) containing 50 mM β-glycerophosphate, 1.5 mM EGTA, 0.5 mM EDTA, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 μg/mL leupeptin and pepstatin, 10 μg/mL aprotinin, and 100 μg/mL phenylmethylsulfonyl fluoride by homogenization of the fragments through 26-gauge needle. The cell lysates were cleared by centrifugation at 12,000 x g for 15 min. Protein content was determined using the Bradford method (Bio- Rad, Hercules, CA). Cell lysates were incubated with gentle rocking at 4°C for 2 h with 6 μg of anti-clathrin antibody. Thereafter 50 μΐ _^ of protein A-Sepharose beads in lysis buffer (1 : 1 blend) were added to each sample and incubated for 1 additional hour with gentle rocking at 4°C. The beads were pelleted by brief centrifugation and then washed three times with ice-cold lysis buffer and boiled for 5 min in 50 μΐ _^ of 2X sample buffer. When necessary, the samples were stored at -80°C until use. A.6. Antibodies used for western blotting experiments

Rabbit polyclonal anti-phospho-STAT3 (#9131; 1 : 1000 for immunoblotting), mouse monoclonal anti-STAT3 (#9139), rabbit polyclonal anti-phospho-p44/42 ERK (#9101), rabbit polyclonal anti-p44/42 MAPK (#9102), rabbit polyclonal anti-phospho- Akt (#4060) and rabbit polyclonal anti-Akt (#4691) were obtained from Cell Signaling Technology (1 : 1000 for immunoblotting). Rabbit polyclonal anti-leptin (500-P68) was purchased from Peprotech (1 : 1000 for immunoblotting). Clathrin mouse monoclonal antibody (ab2731; 1 :500 for immunoblotting) was purchased from abeam and goat polyclonal antibody anti-actin (scl616; 1 :500 for immunoblotting) from Santa Cruz Biotechnology. Secondary antibodies used for western blotting [anti-mouse IgGs (1 :2000), anti-rabbit IgGs (1 :2000) horseradish-peroxidase-conjugated antibodies] were purchased from Sigma (Saint-Quentin Fallavier, France).

A.7. pSTAT3 Immunohistochemistry

Adult male mice were injected intraperitoneally with leptin (3 mg/kg; Peprotech) or vehicle alone (10 mM Tris-HCl buffer) and perfused 5, 15 or 45 min later with a solution of 2% paraformaldehyde. Brains were postfixed, cryoprotected overnight at 4°C in the same fixative solution containing 20% sucrose, and embedded in Tissue Tek (Miles, Elkhart, IN) before freezing. Frozen 30 μιη-thick coronal sections were cut and processed for immunofluorescence as previously described (Bouret et al, 2012, J Neurosci 32, 1244-1252) using rabbit anti-pSTAT3 (Tyr705) (1 : 1000; Cell Signaling Technology) and mouse monoclonal anti-HuC/D (1 :200 ; Molecular probes) primary antibodies. Double-immunofluorescent images were acquired using an Axio Imager.Zl ApoTome microscope (AxioCam MRm camera, AxioVision 4.6 software system; Zeiss). Slides were then coded to obscure the treatment group, and pSTAT3 immunoreactive (IR) cells counted in two sections per animal. The mean number of immunopositive cells per section was compared between groups using ANOVA and Tukey's post hoc test.

A.8. pERK Immunohistochemistry

Adult male mice were injected intraperitoneally with EGF (1 mg/kg; R&D Systems) or vehicle alone (10 mM acetic acid in PBS) and perfused 15 min later with a solution of 4% paraformaldehyde in pH 9.5 borate buffer. Brains were postfixed, cryoprotected overnight at 4°C in the same fixative solution containing 20% sucrose, and embedded in Tissue Tek (Miles, Elkhart, IN) before freezing. Frozen 25 μπι-thick coronal sections were cut and processed for immunofluorescence as previously described (Bouret et al, 2012, J Neurosci 32, 1244-1252) using a rabbit anti-phospho-p42-44 ERK primary antibody (1 : 1000; #9101, Cell Signaling Technology).

A.9. Primary cultures of tanycytes of the median eminence

Tanycytes were isolated from the median eminence of the hypothalamus of 10-day-old (P10) rats and cultured as described previously (De Seranno, S. et al, 2004, J.Neurosci. 24, 10353-1036; Prevot et a/., 2003, J. Neurosci. 23, 10622-10632).

The tanycyte culture medium is composed of DMEM (Invitrogen, San Diego, CA) supplemented with 2% (v/v) antibiotics (penicillin/streptomycin; Invitrogen), 1% L- glutamine (Invitrogen) and 10% donor bovine serum (Invitrogen).

Example 1:

Tanycytes of the ME express functional LepR and internalize leptin through clathrin-coated vesicles. Primary cultures of tanycytes were obtained as described previously in the material & methods.

Detection of leptin receptor mRNA in cultured tanycytes.

Total RNA was isolated from 10 cm dishes of cultured tanycytes using QIAzol lysis reagent (Qiagen). LepR mRNA levels were quantified using NanoDrop technology. RT-PCR was carried out after denaturation at 80°C for 5 min and 75°C for 2 min. Reverse transcriptase was then added and RT performed as follows: 25°C for 10 min; 42°C for lh; 94°C for 5 min. This was followed by amplification as follows: 40 cycles at 94°C for 4 min, 55°C for 1 min and 72°C for 2 min.

The primers used for LepR mRNA detection were as follows:

LepRa-d forward 5 ' - AC ACTGTT AATTTC AC ACC AGAG-3 ' (SEQ ID N° 2);

LepRa reverse, 5'-CTTCAAAGAGTGTCCGTTCT-3' (SEQ ID N° 3);

LepRb reverse, 5'-TGGATAAACCCTTGCTCTTCA-3 ' (SEQ ID N° 4);

LepRc reverse, 5 ' -TGAAC AC AAC AAC AT AAAGCCC-3 ' (SEQ ID N° 5);

LepRd reverse, 5 ' - AACTTC ATGT AAAGAT AT AC-3 ' (SEQ ID N° 6);

LepRe forward, 5 ' -TGTT AT ATCTGGTT ATTGAATGG-3 ' (SEQ ID N° 7);

LepRe reverse, 5 ' -C ATT AAATGATTT ATT ATC AGAATTGC-3 ' (SEQ ID N° 8). RESULTS

The role of tanycytic LepR signalling in the passage of leptin from the periphery to the hypothalamus was investigated using primary cultures of rat tanycytes, obtained from the hypothalamus or the median eminence. A second sample of tanycytes from the ME is provided from Prevot et al., 2003. RT-PCR and sequence analysis revealed that cultured tanycytes expressed mRNAs for LepRa, b, c and e (see figure 1).

Importantly, after 15 min of leptin treatment, tanycytes displayed STAT3, Akt and ERK phosphorylation (Fig. 2B), demonstrating functional LepRb signalling. The treatment of cultures with fluorescent bioactive leptin revealed that leptin was internalized by tanycytes through clathrin-coated vesicles (see figure 2A)

This was confirmed by western blotting experiments performed on immunoprecipitated clathrin-coated vesicles, as described in the material & methods for western blotting, clathrin immunoprecipitation and suitable antibodies (see figure 2B; top panel).

Using pharmacological agents to selectively block various LepRb-related signalling pathways in tanycytes cultures (U0126 to inhibit MEKK1, the upstream activator of ERK; LY294002 to inhibit PI3K; WP1066 to inhibit STAT3) (see figure 7), it is shown that none of these pathways impacts leptin internalization by tanycytes (see figure 2B; bottom panel). This is confirmed by unaltered leptin internalization in cells treated with AG490, an inhibitor of JAK2 autophosphorylation, the well-studied "first" event in the LepR signalling cascade (data not shown).

Next, standard-chow-fed wild-type mice were treated with a mutated recombinant leptin antagonist (LAN), devoid of biological activity but with unmodified LepR-binding properties (Niv-Spector et al, 2005, Biochem J, 391, 221-230).

While LAN was as readily detected by western blotting as bioactive leptin (not shown), it did not accumulate in the ME or MBH when administered intraperitoneally

Thus, in conjunction with the absence of internalization of LAN (see figure 2B; bottom panel), these results indicate that:

(i) leptin internalization is not altered by the above-mentioned pharmaceutical agents, and that

(ii) leptin internalization involves a LepR-dependant but JAK2-independent mechanism.

Example 2:

Methods in vitro for assessing leptin uptake and release by cultured tanycytes.

Primary cultures of tanycytes were obtained as described previously in the material & methods.

Cell treatments

To observe the ability of tanycytes to transport leptin, 90% confluent cells (see material & methods for the culture of tanycytes) cultured in 10 cm dishes in TDM for 2 days were exposed to leptin (Peprotech) for 15 min. Dose-response studies were performed with leptin (0.1 to 10 μ§/ιη1) to identify the optimal concentration for the activation of leptin receptor (LepR) signaling pathways. At 1 μg/ml, leptin induced an increase in the phosphorylation of STAT3 and ERK, and this dose was used in all subsequent experiments.

A. Assessment of leptin uptake by cultured tanycytes using clathrin immunoprecipitation and confocal analysis.

The internalization of leptin was assayed by clathrin immunoprecipitation (see material & methods for western blotting and suitable antibodies) in tanycytes after 15 min of leptin treatment, and by clathrin staining on tanycytes cultured on coverslips and treated for 15 min with fluorescent leptin (Vauthier et al, 2013, Anal Biochem, 436: 1-9 http://dx.doi.Org/10.1016/j .ab.2012.12.013). For confocal analyses, an inverted laser scanning Axio Observer microscope (LSM 710; Zeiss) with an EC Plan NeoFluor 63X/1.4 NA oil-immersion objective (Zeiss) was used (Imaging Core Facility, University of Lille 2, France).

B. Assessment of leptin release by cultured tanycytes using western blotting Leptin release was studied by western blotting in tanycyte conditioned medium (TCM). TCM was obtained by collecting the fresh medium in which tanycytes were incubated following treatment with leptin for 15 minutes. To determine the role of LepR signaling pathways, tanycytes were incubated for 30 min in the presence of 10 μΜ MAPK inhibitor (U0126; Calbiochem), 30μΜ PI3K inhibitor (LY294002 ; Calbiochem) or 30μΜ STAT3 inhibitor (WP1066; Calbiochem). A leptin antagonist (LAN) known to bind LepR with the same affinity as leptin (Niv-Spector et al, 2005, Biochem J, 391, 221- 230) was used as a negative control for leptin internalization (1 μg/ml; Protein Laboratories Rehovot Ltd).

C. Time-lapse study of leptin uptake and release by cultured tanycytes

The time course of leptin internalization and relocation in tanycytes was visualized by confocal imaging of fluorescent leptin. Tanycytes were seeded onto poly-D- ly sine-coated 35mm glass-bottom dishes (MatTek, Ashland MA) and incubated in TDM for 24h before the experiment. Fluorescent leptin was diluted in TDM and administered (1 μg/mL) 15 min before image acquisition. Images were acquired using an inverted confocal microscope (model TCS SP2 AOBS; Leica, Heidelberg, Germany), equipped with a thermostat-controlled chamber. ΧΥΖλ-stack sequences (step 2 μπι, depth 22 μπι, 30 frames at 1 min intervals) were realized with a Plan Apochromatic objective (20X, 0.7 NA) using 633 nm HeNe laser excitation for the fluorescent leptin signal and a non- descanned photon multiplier (PMT) for transmission. NIH ImageJ software was used for 3D reconstruction and measurement of fluorescence intensity.

Example 3:

in vivo methods for assessing leptin uptake and release in leptin-treated mice.

Animal procedures and leptin sensitivity tests were followed as described previously in the material & methods section, unless otherwise mentioned.

A. Assessment of leptin uptake in vivo in the median eminence and the mediobasal hypothalamus.

To determine whether leptin is transported across the median eminence, 15 or 45 min following an intraperitoneal (i.p.) injection of leptin (3 mg/kg; Peprotech) or vehicle (10 mM Tris-HCl buffer), adult male C57B1/6 mice were killed by decapitation. After the rapid removal of the brain, the median eminence and mediobasal hypothalamus were microdissected with Wecker scissors (Moria, France) under a binocular magnifying glass. After dissection, each fragment was placed in a microcentrifuge tube, snap frozen in liquid nitrogen, and stored at -80°C until use. In order to have sufficient protein for the detection of pSTAT3, the median eminence and mediobasal hypothalamus from 2 mice were pooled for western blotting experiments.

B. Assessment of leptin uptake in vivo in hypothalamic tanycytes

Bioactive fluorescent leptin (Cisbio Bioassays) or fluorescent leptin antagonist (LAN; Cisbio Bioassays) (Vauthier et al, 2013, Anal Biochem, 436: 1-9 http://dx.doi.Org/10.1016/j .ab.2012.12.013) were injected into the jugular vein of adult male mice (25 nmoles/animal) anesthetized with ketamine/xylazine, and animals sacrificed 3 min post-injection to assess leptin uptake by tanycytes by fluorescent microscopy. Immunofluorescent images were acquired using an Axio Imager.Zl ApoTome microscope (AxioCam MRm camera, AxioVision 4.6 software system; Zeiss).

C. Assessment of leptin transport rescue by EGF-induced ERK signaling in the median eminence.

In this experiment, 2 consecutive intraperitoneal injections were carried out: leptin (3 mg/kg; Peprotech) was injected 45 min before sacrifice by decapitation, while EGF (1 mg/kg in vehicle; R&D systems) or vehicle alone (lOmM acetic acid in PBS) was injected 15 min before death. The same protocol used to assess leptin uptake in the median eminence and the mediobasal hypothalamus (see above) was then followed. Variations in pERK levels were detected using extracts of individual median eminences and western blotting. The cellular localization of the pERK labeling in the brain was assessed using immunohistofluorecence (see material & methods for western blotting, suitable antibodies and pERK immunohistochemistry).

Example 4:

Tanycytes of the ME release captured leptin via an ERK-dependent signalling pathway.

Cell cultures and assessment of leptin uptake and release by cultured tanycytes were followed as shown in example 2 and in the material & methods.

RESULTS

To assess the fate of internalized leptin, the uptake of fluorescently-labelled bioactive leptin by individual tanycytes in vitro is followed using videomicroscopy. Cultured tanycytes are highly polarized, like their in vivo counterparts, with cell bodies attached to the coverslip and extending ~20^m-long processes into the TDM medium. Fluorescent leptin was taken up by tanycytic end-feet and gradually transported towards the cell body, where it accumulated (see figure 3A and 3B), confirming that, contrary to previous assumptions (Peruzzo et al, 2004, Cell Tissue Res, 317, 147-164), clathrin- mediated transport in tanycytes proceeds in a basal-to-apical direction. The intensity of the fluorescent signal decreased over time and reached extinction (see figure 3A), suggesting that the captured leptin was eventually released. When tanycytes were loaded with leptin (1 μg/ml, 15 min), washed with PBS, and leptin release monitored 5 and 15 min later, the leptin content of the medium gradually increased, confirming this hypothesis.

The mechanisms underlying this leptin release involve intracellular vesicular trafficking, as shown by its reversible suppression by colchicine (data not shown).

We next sought to determine whether LepR signalling was also required for this process, using the inhibitors mentioned above. Although neither LY294002 nor WP1066 affected leptin release, U0126 led to its accumulation in tanycytes instead (see figure 3C), indicating that leptin release requires ERK signalling but not PI3K or STAT3.

Example 5:

EGF-mediated activation of ERK signaling in the median eminence (ME) restores leptin transport into the hypothalamus and accelerates the restoration of leptin sensitivity in obese mice once they are replaced on a normal diet.

The following experiments verify that defective leptin translocation from the ME to the MBH in db/db and diet-induced obesity (DIO) mice can be rectified by activating the ERK pathway.

Animal protocols, assessment of leptin uptake and leptin transport in vivo were followed as shown in example 3 and the material & methods.

In particular, the restoration of leptin sensitivity in DIO mice after EGF administration is assessed using representative photomicrographs and quantitation (n = 4) of pSTAT3-IR immunofluorescence after intraperitoneal administration of leptin (3 mg/kg, 45 min) or vehicle (45 min) along with EGF treatment 15 min before sacrifice. RESULTS

Epidermal growth factor (EGF) treatment (1 mg/kg, 15 min) resulted in the marked activation of ERK in ME tanycytes (see figure 4A), which abundantly express the EGF receptor.

Strikingly, the sequential treatment of db/db and DIO animals with leptin and

EGF restored peripheral leptin uptake by the MBH (see figure 4B) as well as the subsequent activation of pSTAT3 in hypothalamic neurons within 15 min by western blots (see figure 4C) and quantification with photomicrographs (data not shown).

In contrast, replacing DIO mice on a normal diet, previously shown to normalize body weight and restore leptin sensitivity in the long term, only restored leptin transport into the hypothalamus after several weeks (see figure 5 A and 5B). Daily EGF treatment (1 mg/kg) in DIO mice during the last week of their high-fat diet before their return to a normal diet dramatically accelerated weight loss and the restoration of leptin sensitivity (see figure 5A and 5B).

Example 6:

EGF treatment affects glucose metabolism and restores leptin sensitivity and leptin transport. Leptin sensitivity tests were followed as shown in the material & methods.

Animal protocols

12 weeks old male mice were placed on HFD or standard diet for 10 weeks. Mice placed on HFD showed a rapid increase in BW. The differences in BW of HFD-fed mice became significantly different from standard chow fed mice after 4 weeks. HFD-fed mice are separated in 3 groups. DIO group did not receive any treatment. DIO-EGF8w group receive daily intraperitoneal injection of EGF (1 mg/kg) from week 2 until the glucose tolerance test at week 8. DIO-EGF6d group received daily intraperitoneal injections of EGF (lmg/kg) at week 7 during 6 days before glucose tolerance test. Glucose tolerance test (GTT)

GTT was performed as previously described (Fan et al, 2000, Endocrinology 141 :3072-3079; Howard et al, 2004, Nat Med 10:734-738).

RESULTS

There was no significant difference in body weight (BW) observed in the 3 groups of high-fat diet (HFD)-fed mice. While standard diet-fed mice maintain their BW over the time, all groups of HFD-fed mice display a constant increase in BW (see figures 6A and 6C)

Thus EGF treatment does not impact the weight gain effect of HFD, although it may affect glucose metabolism. Indeed, glucose tolerance test performed after 8 weeks of HFD or standard diet revealed that long term EGF treatment in DIO mice allows a partial recovery of glucose metabolism. 6 days of EGF treatment before the test does not change the response of DIO mice to GTT (see figure 6B).

After 3 days of daily intraperitoneal injection (3mg/kg), control mice respond to leptin by a decrease in BW. Sensitivity to leptin is maintained in DIO-EGF8w mice as their decrease in BW does not differ from control mice. After 8 weeks of HFD, DIO mice which did not receive EGF treatment lost their sensibility to leptin and did not show any change in BW after peripheral leptin administration.

More particularly, DIO-EGF8w display an intermediary phenotype between control and DIO mice, showing that long-term EGF treatment is efficient to reduce perigonadal white adipose tissue (WAT) content in DIO mice in spite of their increased BW compared to control mice (see figure 6D).

This result shows evidence that long-term EGF treatment (8 weeks) during high-fat diet prevents the development of leptin resistance and allows partial recovery of glucose metabolism.

Example 7:

Semaphorin-7a is an ERK-pathway activating compound.

Tanycyte culture and pERK western blotting were performed as described previously in the material & methods, and EGF was replaced by Semaphorin-7a. The antibodies used were mouse anti-Plexin CI (R&D Systems, AF5375; 1 :500), rabbit anti-Bl -Integrin (Santa Cruz, sc-8978; 1 :500), rabbit anti P-βΙ integrin (Abeam Technology, AB5189; 1 :500), rabbit polyclonal anti-phospho-p44/42 MAPK (p- Erkl/2) (Thr202/Tyr204; Cell Signaling Technology, 9101L; 1 : 1000), rabbit polyclonal anti-p44/42 MAPK (Erkl/2) (Cell Signaling Technology, 9102L; 1 : 1000).

Tanycytes express the receptor to Semaphorin-7a (integrin βΐ), and Semaphorin-7a activates p-ERK signaling (see figure 8). The phosphorylation of the β-1 integrin receptor indicates that the receptor is activated in the presence of Sema7A.

This result shows evidence that Semaphorin-7a is a potent ERK-pathway activating compound in tanycytes.

Example 8:

Ghrelin activates p-ERK signaling and is internalised in tanycytes in vitro.

Tanycyte culture, pERK immunohistochemistry and western blotting, and in vitro and in vivo ghrelin binding were performed as described previously in the material & methods, and bioactive fluorescent ghrelin was used, as described in Leyris et al (2011, Anal Biochem, 408:253-262).

Fluorescent bioactive ghrelin derivative (3.3 kDa) is administered intravenously to standard-chow-fed wild-type mice 3 min before sacrifice. Photomicrographs showing tanycytic processes and cell bodies were labelled by fluorescent ghrelin (25 nmoles/animal), but not fluorescent LAN (25 nmoles/animal), 3 min after intravenous injection. Sections were counterstained with Hoechst (1 : 10000; Molecular Probes, Invitrogen) and coverslipped using Mowiol® (Calbiochem, USA).

RESULTS

A. Immunohistochemistry on tanycyte cell culture.

To determine the role of tanycytes in hypothalamic ghrelin uptake in vivo, we intravenously administered a fluorescent bioactive ghreline derivative to standard-chow- fed wild-type mice 3 min before sacrifice. At this short interval, fluorescent ghrelin was localized exclusively in median eminence tanycytes in the brain parenchyma. Labelling occurred both in tanycytic cell bodies along the floor of the third ventricle and in the process of tanycytes, stretching to the pial surface, indicating that blood-borne ghrelin is likely taken up by tanycytic end-feet and transported towards the cell body in contact with the cerebrospinal fluid (CSF) (data not shown).

Bioactive fluorescent ghreline derivatives developed by Cisbio Bioassays (Leyris et al, 2011, Anal Biochem, 408:253-262) was shown to bind arcuate neurons when injected intravenously 5 to 10 min after death (Schaeffer et al, 2013, PNAS, 110: 1512- 1517), but its initial capture by tanycytes had never been suspected so far.

B. Assessment of pERK signalling within tanycytes in the presence of ghrelin To determine whether ghrelin can promote GHSRla-like signalling in tanycytes we used primary cultures of rat tanycytes. After 15 min of ghrelin treatment (0, 1 or 10 μg/ml), tanycytes displayed increased ERK phosphorylation (see figure 9), as evidenced by the increased pERK/ERK ratio, demonstrating functional ghrelin-mediated signalling. In addition, cultured tanycytes internalized fluorescent bioactive ghrelin.

Those results show evidence that GHSRla ligands such as ghrelin are potent ERK-pathway activating compounds in tanycytes.