Leptin antagonism improves Rett syndrome phenotype in symptomatic Mecp2-deficient mice

Abstract

Rett syndrome (RTT) is a severe X-linked neurodevelopmental disorder caused by mutations in MECP2. Elevated circulating levels of the adipocyte hormone leptin are consistently observed in patients and in mouse models, yet their contribution to disease progression has remained unclear. Here, we show that reducing leptin signaling—either pharmacologically or genetically—significantly alleviates RTT-like phenotypes in Mecp2-deficient mice. In males, these interventions preserved general health, prevented weight loss, and improved breathing and locomotor functions. At the neuronal level, they restored excitatory/inhibitory balance in the hippocampus and somatosensory cortex and rescued hippocampal synaptic plasticity. In females, delaying the pathological rise of leptin levels postponed symptom progression. These findings uncover leptin as a key contributor to RTT pathophysiology and position leptin-targeted interventions as a promising therapeutic strategy for this currently untreatable disorder.

Introduction

Rett syndrome (RTT; OMIM identifier #312750) is a rare non-inherited genetic neurodevelopmental disorder resulting from de novo mutations in the X-linked gene MECP2 (methyl-CpG-binding protein 2) [1]. The MECP2 protein binds to methylated DNA regulating the expression of thousands of genes and plays a crucial role in brain development and functioning [2]. Affected individuals experience an apparently normal development until around 6–18 months of age. They subsequently undergo a rapid regression phase marked by the loss of purposeful hand movements and acquired speech, breathing irregularities, seizures and severe cognitive deficits [3]. Several clinical features observed in humans with RTT are recapitulated in Mecp2-deficient mice, making these mouse models an essential tool for unraveling the cellular mechanisms underlying the disease and evaluating potential therapeutic interventions [4]. Notably, studies have demonstrated that phenotypic rescue is achievable in Mecp2-deficient mice when the Mecp2 gene is expressed even at late symptomatic stages [5]. This finding indicates that RTT is not an irreversible condition, and it provides hope for therapeutic interventions.

Beyond MECP2, the expression of various molecular targets, including neurotrophic factors and neuromodulators, is dysregulated, some of which hold therapeutic potentials [6]. Among these deregulated factors, elevated circulating levels of leptin have been observed in RTT patients and rodent models of the disease [[7], [8], [9], [10]]. Leptin, the product of the obese (ob) gene, is a circulating hormone secreted mainly from the white adipocytes. It crosses the blood brain barrier to act on the hypothalamus, where its main function is to regulate food intake [11]. However, it is now clear that the actions of leptin extend beyond the hypothalamus, encompassing a wide range of biological roles. This pleiotropic hormone modulates motivation [12], cognitive functions [13], anxiety [14], the excitability of neuronal networks [15], epileptiform activities [16], breathing activity [17], locomotor activity [18] and more [19,20]. Leptin also acts as an important neurotrophic factor during perinatal periods, promoting neuritic growth and synaptogenesis in various brain structures, including the hypothalamus [21] and hippocampus [[22], [23], [24], [25], [26]]. Given these important physiological and developmental functions, dysregulation of leptin availability or signaling, either excess or deficiency, can have far reaching effects, including social and cognitive impairments, altered breathing activity, seizures susceptibility, locomotion and abnormal brain development [[16], [17], [18],24,27]. Consequently, the elevated levels of leptin observed in RTT patients [7,8] may contribute to the various disorders associated with Rett syndrome.

To address this hypothesis, we first confirmed the presence of elevated serum leptin levels in male and female Mecp^2tm1−1Bird mice, hereafter referred to as Mecp2^-/y and Mecp2^+/− mice respectively. We then evaluated the therapeutic potential of reducing leptin signaling using two complementary approaches: pharmacological antagonism of leptin and genetic reduction of leptin production. The effects of these interventions were systematically examined in this RTT mouse model at both neuronal circuit and behavioral levels. These analyses were performed independently to provide a broad assessment of the therapeutic impact of leptin-targeting strategies.

Our findings demonstrate that the pharmacological and genetic anti-leptin strategies prevent the degradation of health status, weight loss, and the progression of breathing and locomotor deficits in male Mecp2^-/y mice. At the neuronal level, these interventions rescue the excitatory/inhibitory balance in the hippocampus and somatosensory cortex and mitigate the impairment of synaptic plasticity in the hippocampus. Moreover, genetic reduction of leptin production delays the appearance of breathing and motor difficulties, and rescue the excitatory/inhibitory balance in the hippocampus of female Mecp2^+/− mice. These results indicate a role of leptin in the pathogenesis of RTT, providing valuable insights into potential therapeutic strategy for treating this syndrome.

Materials and methods

Animals

All animal procedures were carried out in accordance with the European Union Directive of 22 September (2010/63/EU) and have been approved by the Ethical Committee for Animal Experimentation (APAFIS-Numbers 17605-2018-1119-1115-7094 and 41156-2023-0220-1011-9732) delivered by the French Ministry of Education and Research.

Experiments were performed on male wild type and Mecp^2tm1−1Bird mice [28] C57BL/6J genetic background (Jackson Laboratory, stock number 003890). Hemizygous mutant males (hereafter referred as to Mepc2^-/y mice) were generated by crossing heterozygous females (Mecp2^+/−) with C57BL/6 wild type males. Mecp2^-/y and Mecp2^+/− mice haplo insufficient for leptin (hereafter referred as to Mecp2^-/y;ob ^± and Mecp2^+/−;ob^+/−) were generated by crossing heterozygous females Mecp2^+/− with C57BL/6 heterozygous leptin deficient males mice (Jackson Laboratory genotyping protocol, strains B6.Cg-Lepob/J, ID 000632). Animals were housed in a temperature-controlled environment with a 12h light/dark cycle and free access to food and water. Genotyping was performed by PCR techniques according to Jackson Laboratory protocols.

Leptin and anti-leptin injection

Recombinant murine leptin (Peprotech, New Jersey, USA) was reconstituted in sterile water and daily injected (5 μg/g) sub-cutaneous at 12–14 h a.m during 10 days starting at postnatal day (P) 40. The pegylated super active mouse leptin antagonist (Peg-SMLA, Protein Laboratories, Rehovot, Israel) was reconstituted in sterile water [29]. As the half-life of the Peg-SMLA was about 24 h, mice were injected (5 μg/g) sub-cutaneous at 12–14 h a.m every other day during 10 days starting at P40. Control mice received same volume injections of vehicle. All animals were randomly assigned to treatment groups in all experiments described in this study.

Leptin and BDNF immunoassay

Blood samples were collected by two routes: submandibular bleeding, performed every 10 days from postnatal day 20–90 in female mice or decapitation (trunk blood samples) at P15, 30 and 50 in male mice and at P100-200 in female mice. Blood samples were centrifuged (10.000 rpm, 10 min, 4 °C) immediately after collection at 10–11 h a.m. Quantification of endogenous leptin was performed with Mouse Leptin ELISA Kit (BioVendor R&D^R, Brno, Czech Republic) following the manufacturer's protocol. The measured concentration of samples was calculated from the standard curve and expressed as ng/ml.

Hippocampal tissues were homogenized in RIPA buffer (150 mM NaCl, 1% Triton X100, 0.1% SDS, 50 mM Tris HCl), pH 8, containing protease inhibitors (Complete Mini; Roche). Lysates were centrifuged (10.000 g for 30 min at 4 °C). Loading was 200 μg of proteins as determined using a modified Bradford reaction (BioRad Laboratories). Quantification of mature- and proBDNF was performed with both proBDNF and mature BDNF Rapid ELISA Kit (Biosensis Pty Ltd., Thebarton, SA, Australia) in the concentrated solutions following the manufacturer's protocol.

Real-time reverse transcription quantitative polymerase chain reaction

Visceral and inguinal fat pads and gastrocnemius muscles were dissected, rapidly frozen in liquid nitrogen and stored at −80 °C. Total RNAs from fat pads and gastrocnemius muscle were isolated using a RNeasy Mini kit (74106, Qiagen, Germany) and RNA Fibrous Tissue Mini kit (74704, Qiagen) respectively, following the manufacturer's instructions and quantified by reading the absorbance at 260 nm (NanoPhotometer, IMPLEN). RNAs were converted to cDNA using 1 μg RNA and a QuantiTect Reverse Transcription kit (Qiagen) according to manufacturer's instructions. Quantitative RT-PCR was performed with a Light Cycler 480 SYBR Green IMaster (Roche Applied Science) following the manufacturer's instructions, using the following oligonucleotides (QuantiTect Primer Assays, Qiagen): Leptin (Mm_Lep_1_SG QT00164360 (NM_008493)), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, QT001199633). Relative mRNA values were calculated using the LC480 software and GAPDH as the housekeeping gene. PCR was performed in triplicate.

Slice preparation and electrophysiological recordings

Brains were removed and immersed into ice-cold (2°–4 °C) artificial cerebrospinal fluid (ACSF) with the following composition: 126 mM NaCl, 3.5 mM KCl, 2 mM CaCl2, 1.3 mM MgCl2, 1.2 mM NaH2PO4, 25 mM NaHCO3, and 11 mM glucose (pH 7.4) equilibrated with 95% O2 and 5% CO2. Hippocampal slices (350 μm thick) were cut using a vibrating microtome (Leica VT1000S, Germany) in ice-cold oxygenated choline-replaced ACSF. After recovery at 25 °C for at least 90 min, slices were transferred to a submerged recording chamber perfused with oxygenated ACSF (3 ml/min) at 34 °C.

Whole cell recordings were performed from hippocampal CA3 and somatosensory layer V pyramidal neurons in the voltage-clamp mode using an axopatch 200B amplifier (Molecular Devices LLC, San Jose, USA). To assess the excitatory/inhibitory balance, the glass recording electrodes (4–7 MΩ) were filled with a solution containing 100 mM K-gluconate, 13 mM KCl, 10 mM Hepes, 1.1 mM EGTA, 0.1 mM CaCl2, 4 mM Mg–adenosine 5′-triphosphate, and 0.3 mM Na–guanosine 5′-triphosphate. The pH of the intracellular solution was adjusted to 7.2, and the osmolality was adjusted to 280 mOsmol liter−1. With this solution, GABA_A-receptor mediated postsynaptic currents (GABA_A-PSCs) reversed at −70 mV. GABA_A-PSCs and Glut-PSCs were recorded at a holding potential of −45 and −70 mV respectively.

Field recordings were performed in area CA1. A cut was made between CA1 and CA3. The Schaffer collaterals/commissural fibers were stimulated using a bipolar tungsten electrode (66 μm; A-M Systems, WA, USA) placed on the surface of the stratum radiatum of CA1 (10–50 μs, 5–15 V, 0.03 Hz). Extracellular tungsten electrodes (California Fine Wire,CA, USA) were used to record dendritic field excitatory postsynaptic potentials (fEPSP) from the stratum radiatum of the CA1 region. The signals were amplified using a DAM80 amplifier (WPI, UK). LTP was induced by three 100 Hz trains of 100 stimuli 30 s apart at 50% of the maximal intensity. The slope of the fEPSP was measured and expressed relative to the preconditioning baseline.

Spontaneous synaptic currents and fEPSP were recorded with Axoscope software version 8.1 (Molecular Devices LLC, San Jose, USA) and analyzed offline with Mini Analysis Program version 6.0 (Synaptosoft).

pSTAT3 immunohistochemistry

Mice were deeply anesthetized with tiletamine/zolazepam (Zoletil, 40 mg/kg) and medetomidine (Domitor, 0.6 mg/kg) and trans-cardially perfused with 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (pH 7.4). After perfusion, brains were removed and post-fixed through immersion in cold solution of 4% PFA for 24 h and then stored at 4 °C in Phosphate-Buffered Saline (PBS) with 0.01% sodium azide until processing. Serial coronal brain sections (40 μm thickness) at the hypothalamic level (from the bregma coordinates −0.58 to −2.18) were done using a vibratome (Leica VT1000S, Germany) and collected in PBS.

Three randomly chosen sections per animal of the same coordinates range for each hypothalamic nucleus examined were used for the immuhistochemistry processing. The free-floating sections were first rinsed in Tris buffer salin (TBS) and pre-treated in 0.3% Glycine for 30 min at room temperature (RT). They were then permeabilized for 30 min in 0.3% Triton ×100 and were blocked in 5% normal donkey serum and 5% bovine serum albumin (BSA) for 1 h at RT. Section were incubated overnight at 4 °C in pSTAT3 antibody (1/200 Cat 9131, cell Signaling) diluted in TBS with 1% BSA. On the next day, sections were washed four times in TBS and incubated with a donkey anti rabbit Alexa Fluor 568 labeled secondary antibody (1/500) for 2 h. The stained sections were mounted on coverslips with mounting reagent (FluorSave reagent, Calbiochem) and kept away from light until analysis. The immunostaining in the nuclei of interest was acquired at 20x magnification using a Zeiss AxioImager M2 Apo1.2 microscope. Each hypothalamic structure examined was delineated and the nuclear profiles of all pSTAT3 positive labeled cells were manually counted from 3 sections using Fiji Image J analysis software from the NIH, to exclude any bias that could result from changes in cell soma size or shape among experimental conditions. The average number of cells counted in the 3 sections per area in each mouse was taken for statistical comparisons.

Plethysmography

The breathing activity of non-anesthetized freely moving mice was recorded using a constant flow whole body plethysmograph (EMKA technologies, Paris, France) with 200 ml animal chambers maintained at 25 ± 0.5 °C and ventilated with air (600 ml/min). The breathing activity of pairs of WT and Mecp2^-/y mice littermates was simultaneously recorded. Mice were allowed to acclimate to the experimental room for 1 h and to the plethysmography chamber and airflow for approximatively 30 min before breathing measurement. Breathing activity was recorded during 1 h. A differential pressure transducer measured the changes in pressure in the body chamber resulting from the animal's respiration. Signals from the pressure transducer were analyzed offline via the Spike 2 interface and software (Cambridge Electronic Design, Cambridge, UK). Only periods of quiet breathing without body movements were analyzed, during which the number of apneas (> three respiratory cycles) per hour was quantified. The breathing irregularity score was calculated as 100∗ABS (Int_n- Int_n-1/Int_n-1) for each respiratory cycle, where Int_n = inter-breath interval and Int_n-1 = inter-breath interval of the previous breath.

General health scoring and lifespan

Weight was measured every day from the beginning of the treatment. The phenotypic score was evaluated the day before treatment and the last day of treatment. Severity score, typically used in RTT phenotypic assessments [28], was used to group animals into 4 severity classes: absence of phenotype [4] to severe phenotype (0). The parameters scored are: tremor, posture, limb grasp, spontaneous activity in the home cage and general aspect. Mice that rapidly lost weight were euthanized for ethical reasons. The day of the sacrifice was considered as the endpoint of lifespan assessment.

Accelerating rotarod

A rotarod apparatus (Biological Research Apparatus, Gemonio, Italy) was used to measure the motor coordination. After a 5 min habituation session (4 r.p.m), each mouse was given three trials with the rate of rotation increasing from 4 to 40 r.p.m. over 5 min. The trial ended when the mouse fell from the rod or remained on the rotarod for at least 5 min. The time spent on the rotarod was recorded by an automated unit, which stopped as the mouse fell. The mouse was placed back in its home cage for 10 min between each trial. The latency to fall was determined as the best of the 3 trials.

Behavior

All the behavioral tests were performed by Phenotype Expertise, Inc. (France). For all tests, animals were first acclimated to the behavioral room for 30 min. WT and Mecp2^-/y mice underwent elevated plus maze, open field, three chamber test and spontaneous social interaction test at P50. WT and Mecp2^-/y treated mice underwent elevated plus maze and open field at P40, before the beginning of the treatment, and at P50, at the end of the treatment. Animal movements were video-tracked using Ethovision software 11.5 (Noldus).

Elevated-Plus Maze. The EPM was used to assess anxiety state of animals. The device consists of a labyrinth of 4 arms 5 cm wide located 80 cm above the ground. Two opposite arms are open (without wall) while the other two arms are closed by side walls. The light intensity was adjusted to 20 Lux on the open arms. Mice were initially placed on the central platform and left free to explore the cross-shaped labyrinth for 5 min. Time spent in open and closed arms, the number of entries in open arms, as well as the distance covered, are directly measured by the software.

Open-field. Open field was used to evaluate both the locomotor activity of the animals. Mice were individually placed in a 40 × 40 cm square arena with an indirect illumination of 60 lux for 10 min. Total distance traveled and time in center (exclusion of a 5 cm border arena) are directly measured by the software. Grooming (time and events) and rearing were manually counted in live using manual functions of the software, by an experimented behaviorist.

Three-chamber social preference test. A square arena 40 × 40 cm was used, with the wired cages placed in opposite diagonal corners. The task was carried out in four trials. In the first trial (habituation), a test mouse was placed in the center of the arena and was allowed to freely explore each chamber for 5 min. At the end of the trial, the animal was briefly removed from the arena to allow for the preparation of the following trial. In the second trial (social exploration), a 8- weeks old C57BL/6J congener mouse (S1) was placed randomly in one of the two wire cages to avoid a place preference. The second wire cage remained empty (E). Then the test mouse was placed in the center of the arena and allowed to freely explore for 10 min. A second 8-weeks old C57BL/6J congener mouse (S2) was placed in the second wire cage for the third trial (social discrimination). Thus, the tested mouse had the choice between a familiar mouse (S1) and a new stranger mouse (S2) for 10 min. At the end of the trial, the mouse was returned to home-cage for 30 min. For the fourth trial (short-term social memory), S2 was replaced by a new stranger mouse (S3), the familiar mouse (S1) staying the same. Then tested mouse was allowed to freely explore the arena for 10 min. Time spent in each chamber and time of contact with each wire cage (with a mouse or empty) were calculated. The measure of the real social contact is represented by the time spent in nose-to-nose interactions with the unfamiliar or familiar mouse. This test was performed using grouped house mice of 4 months old.

Spontaneous social interaction test. The tested mouse was placed into the same OF arenas that were used for the OF test and allowed to explore this empty arena for 2.5 min. Immediately following this initial stage, the mouse was again allowed to explore the OF for an additional 10 min with the target (Swiss mouse) present. The time spent in nose-to-nose, nose-to-body, and nose-to-genitals interactions was measured.

Statistics

Statistical analyses were conducted with GraphPad Prism (GraphPad software 5.01). Shapiro-Wilk normality test was used to determine the normality of distributions. We used a Two-tailed Mann-Whitney U test or Two-tailed unpaired t-test for comparison between two independent groups, a Wilcoxon matched pairs signed rank test or Two-tailed paired t-test for comparison between two matched data, a Two-tailed Kruskal-Wallis test followed by a Dunn's multiple comparison or a two-way ANOVA followed by a Tukey's multiple comparison for comparison between three or more independent groups, and a Fisher exact test for nominal comparison between two independent groups. The effect of tetanic stimulation of the slope of field EPSPs was analyzed using repeated measure ANOVA. The survival analysis was performed using a Kaplan-Meier log-rank test. Possible outliers within an experimental group were identified with Grubb's test and discarded from the final analysis. To ensure the consistency and reproducibility of our results, we conducted repeated trials in different acute hippocampal slices prepared from at least three different animals from three different littermates. All data are expressed as mean ± standard error to the mean (S.E.M.). For results expressed as percent of WT (i.e. serum and mRNA leptin, Fig. 1, all values (WT and Mecp2^-/y mice) were normalized to the mean WT value. In the figures, box plots represent the 1^rst and 3rd quartiles; whiskers show data range; horizontal lines show the median. The statistical data corresponding to the various figures are presented in the supplementary data as supplementary Tables 1–7.

Fig. 1.

Circulating leptin levels, leptin mRNA expression and p-STAT3 immunostaining are elevated in symptomatic Mecp2^-/y mice. A) Box plots of serum leptin levels determined in blood samples taken at postnatal day (P) 15, 30 and 50, expressed as percentage of age matched wild type (WT) mice. B) Scatter plots of serum leptin levels as a function of mice body weight of P30–P50 WT and Mecp2^-/y mice. C) Box plots of normalized visceral (V) and inguinal (In) white adipose tissue (WAT) mass of P50 WT and Mecp2^-/y littermates. D-F) Mean ± SEM plots of normalized leptin mRNA expression in gastrocnemius muscle (D), visceral WAT (E) and inguinal WAT (F) of P30 and P50 WT and Mecp2^-/y littermates. (G) Representative microphotographs pSTAT3 immunoreactivity in the delineated arcuate hypothalamic nucleus (bregma level −1.70) of WT Sham [1], WT Leptin [2] and KO Sham [3] and in the lateral hypothalamic area (bregma level −1.34) of WT Sham [4], WT Leptin [5] and KO Sham [6]. V: third ventricle; Opt: optic tract; fr: fornix; delineated MGP: Medial globus Pallidus. Scale bars, 50 μm. Quantification (mean ± SEM) of pSTAT3 immunoreactivity in the hypothalamic nuclei. LH: lateral hypothalamus; DM: dorsomedial nucleus; VMH: ventromedial nucleus; Arc: arcuate nucleus; PVN: paraventricular nucleus. Numbers in parentheses indicate the number of mice used. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, two-tailed unpaired student t-test (C–F), One-way ANOVA followed by a Tukey's multiple comparison (A, G).

Results

Serum leptin levels and leptin mRNA expression are altered in male Mecp2^-/y mice

As a prerequisite for investigating the role of leptin in RTT, we assessed the circulating levels of leptin, and the Lep mRNA expression levels from two main sources of leptin, the white adipose tissues (WAT) and gastrocnemius muscle, in male Mecp2^-/y and WT littermates. Mecp2^-/y mice had significantly higher circulating leptin levels than their WT counterparts (Fig. 1A). Despite weighting less than their WT littermates (Fig. 1B), inguinal (Fig. 1C), gonadic (1.03 ± 0.06 vs 1.89 ± 0.26 g/100g body weight, t = 2.61, df = 18, p = 0.017, Unpaired t-test) and sub-cutaneous (0.46 ± 0.07 vs 1.42 ± 0.2 g/100g body weight, t = 3.83, df = 16, p = 0.001, Unpaired t-test) WAT weighted significantly more in P50 Mecp2^-/y mice. The weight of visceral WAT was similar between WT and Mecp2^-/y littermates (Fig. 1C). Mecp2^-/y mice also exhibited higher expression of leptin mRNA in gastrocnemius muscle (Fig. 1D), as well as in visceral and inguinal WAT (Fig. 1E and F).

We next examined whether elevated circulating leptin levels in Mecp2-deficient mice reflect enhanced central signaling or leptin resistance [30,31]. To address this, we used phosphorylated STAT3 (pSTAT3) as a readout of leptin receptor activation. Under basal conditions, Mecp2^-/y mice showed increased numbers of pSTAT3-positive neurons across several hypothalamic nuclei compared with WT littermates (Fig. 1G), consistent with previous reports [30]. However, a 10-day anti-leptin treatment did not reduce pSTAT3 levels (Fig. 1G), suggesting that this activation may arise from leptin-independent mechanisms such as alternative signaling pathway or intrinsic consequences of Mecp2 deficiency. Furthermore, acute leptin injection (5 μg/g) robustly increased pSTAT3 in WT mice but had little to no effect in Mecp2^-/y mice (Fig. 1G). Together, these findings indicate impaired central leptin signaling in Mecp2^-/y mice, consistent with leptin resistance despite elevated circulating leptin levels.

Leptin antagonism prevents the progression of breathing deficits in male Mecp2^-/y mice

Respiratory disorders have been well described in patients and rodent models of RTT [32]. Leptin regulates breathing by acting on neurons of the brainstem respiratory network and carotid body cells [17]. We therefore investigated the possible contribution of leptin to RTT-associated breathing disorders. We first characterized the respiratory activity of Mecp2^-/y mice from our breeding colony. Consistent with previous studies [33], when compared with their age-matched WT littermates, P40 Mecp2^-/y mice developed respiratory disorders characterized by increased number of apneas (Fig. 2A), respiratory cycle duration variability expressed as breathing irregularity score (Fig. 2D) and minute ventilation (Fig. 2F). These breathing distresses worsen with age in Mecp2^-/y mice (Fig. 2C and E), while the respiratory parameters remained constant in WT mice (Fig. 2B and E). Next, to test the possible role of leptin in RTT-associated breathing disorders, P40 WT mice received daily sub-cutaneous injection of leptin recombinant (5 μg/g) during 10 consecutive days. Sham mice received the same volume of vehicle. We used plethysmography to record breathing activity of each mouse at P40, before starting treatment, and at P50, the last day of treatment. Leptin treatment of WT mice led to a significant increase in the number of apneas (Fig. 2B) and breathing irregularity score (Fig. 2E), while in sham WT mice these parameters remained unchanged (Fig. 2B and E). The mean value of minute ventilation was not affected by the leptin-treatment (Fig. 2G).

Fig. 2.

Leptin antagonism prevents the progression of breathing deficits of symptomatic Mecp2^-/y mice. A) Box plots of apnea frequency in P40 untreated WT and Mecp2^-/y mice. B, C) Plots of apnea frequency in treated WT (B) and Mecp2^-/y (C) mice. Lines indicate individual mice recorded the day before the treatment (P40) and at the end of the treatment (P50). Connected scares indicate mean ± SEM. D) Box plots of breathing irregularity score in P40 untreated WT and Mecp2^-/y mice. E) Box plots of the percentage of change of breathing irregularity score in treated- WT and Mecp2^-/y mice. F) Box plots of minute ventilation in P40 untreated WT and Mecp2^-/y mice. G) Box plots of the percentage of change minute ventilation in treated- WT and Mecp2^-/y mice. Numbers in parenthesis indicate the number of mice used. ∗∗P < 0.01; ∗∗∗P < 0.001, Two-tailed paired t-test (B, C), Two-tailed unpaired t-test (A, D-G), One-way ANOVA followed by a Tukey's multiple comparison (E, F).

We next investigated the effect of anti-leptin treatment (5 μg/g, every other day during 10 days) on the breathing activity of the Mecp2^-/y mice. While the sham and anti-leptin treated Mecp2^-/y mice show similar phenotypic profile at the beginning of the treatment (Fig. 2C), we found that the breathing distress worsen with age in sham treated Mecp2^-/y mice, but remained constant in anti-leptin treated Mecp2^-/y mice (Fig. 2C). At the end of the treatment, P50 anti-leptin treated Mecp2^-/y mice showed improved breathing parameters (apneas frequency and irregularity score) compared to sham Mecp2^-/y mice (Fig. 2D and E). The mean value of minute ventilation (Fig. 2F) of P50 Mecp2^-/y mice was not affected by the anti-leptin-treatment. We also tested the effect of chronic leptin treatment on Mecp2^-/y mice and found no effect on their breathing parameters (Supplementary Fig. S1A–C). Altogether, these data suggest a contribution of leptin to the progression of breathing difficulties in symptomatic Mecp2^-/y mice.

Leptin antagonism prevents the worsening of health condition of male Mecp2^-/y mice

We next assessed the efficacy of the anti-leptin treatment on the overall health of the Mecp2^-/y mice. While the sham and anti-leptin treated Mecp2^-/y mice show similar weight at the beginning of the treatment (15.4 ± 0.5 vs 14.1 ± 0.6 g, p = 0.13, Mann Whitney test), the thoroughly described body weight loss of Mecp2^-/y mice (Fig. 3A) was abolished by the 10-day anti-leptin treatment (Fig. 3B and C). The 10-day leptin treatment decreased the body weight of the WT (Fig. 3C) but not Mecp2^-/y mice (Supplementary Fig. S1D). We also performed a scoring of the health condition of the mice. The parameters assessed include tremor, general aspect, spontaneous activity, limb grasp and posture. WT and Mecp2^-/y mice showed significant difference at P40 (Fig. 3D). Mecp2^-/y mice showed altered general aspect, decreased spontaneous activity, and increased grasp and tremor (Fig. 3E). The health scoring performed before starting treatment (P40) and on the last day of treatment (P50) revealed a deterioration in all parameters assessed in sham Mecp2^-/y mice (Fig. 3G). Worsening of posture, tremor and general aspect were prevented by the anti-leptin treatment while spontaneous activity and limb grasp showed no significant improvement (Fig. 3G). Although it does not reach significance, this improvement in overall health after the anti-leptin treatment was accompanied by a decrease of early lethality, with 50% of the sham- and anti-leptin treated Mecp2^-/y mice surviving after the postnatal day 48.5 and 59 respectively (Fig. 3F). No lethality nor change in the health scoring were observed in WT mice treated with either vehicle or leptin (n = 10 for both).

Fig. 3.

Leptin antagonism ameliorates the general health and lifespan of symptomatic Mecp2^-/y mice. A) Box plots of the body weight of untreated P40 WT and Mecp2^-/y mice. B) Body weight change (% of P40) as a function of age in treated WT and Mecp2^-/y mice. The treatment started at P40 and ended at P50 (Mean ± SEM). C) Box plot of the body weight change (% of P40) at P50 in treated WT and Mecp2^-/y mice. D) Severity score of untreated P40 WT and Mecp2^-/y mice. mean ± SEM. E) Radar plot of the symptoms scored in P40 WT and Mecp2^-/y mice. F) Plot of the percentage of surviving mice as a function of age. The treatment started at P40 and was maintained until the death of the animal. G) Radar plot of the symptoms scored in treated Mecp2^-/y mice. Numbers in parenthesis indicate the number of mice used. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001, Two-tailed unpaired t-test (A,C,D), One-way ANOVA followed by a Tukey's multiple (C), Mann Whitney U test (E, G), Kaplan-Meier log rank test (F).

We next assessed the effect of the anti-leptin treatment on the locomotor activity and motor coordination of Mecp2^-/y mice in the open field and the accelerating rotarod tests. In the open field test, the distance traveled by the Mecp2^-/y mice was reduced compared to their WT littermates (Fig. 4A). This locomotor deficit worsened with age in Mecp2^-/y mice (Fig. 4B). The 10-day anti-leptin treatment prevented the degradation of locomotor activity observed in the sham Mecp2^-/y mice (Fig. 4B and C). Moreover leptin-treated WT mice performed similarly to their sham littermates (Fig. 4C). In the accelerating rotarod test, Mecp2^-/y mice showed a significant decrease in the latency to fall compared to WT littermates (Fig. 4D). This phenotype did not worsen with age (Fig. 4E). Neither the leptin- nor the anti-leptin treatment affected the performances of respectively WT and Mecp2^-/y mice (Fig. 4F). Overall, these observations show that the anti-leptin treatment ameliorates the general health condition, (i.e., posture, tremor, general aspect) and locomotor activity of the symptomatic Mecp2^-/y mice.

Fig. 4.

Leptin antagonism prevents the worsening of locomotor deficit but does not improve motor coordination nor anxiety alterations of symptomatic Mecp2^-/y mice. A) Box plots of the distance traveled by untreated P40 WT and Mecp2^-/y mice in an open field. B) Plots of distance traveled by treated Mecp2^-/y mice in an open field. Connected points indicate individual mice recorded the day before the treatment (P40) and at the end of the treatment (P50). Connected squares indicate mean ± SEM. C) Box plots of the distance traveled in an open field by P50 treated WT and Mecp2^-/y mice. D) Mean ± SEM plots of the latency to fall of untreated P40 WT and Mecp2^-/y mice in the accelerating rotarod. E) Plots of latency to fall of treated Mecp2^-/y mice. Connected points indicate individual mice recorded the day before the treatment (P40) and at the end of the treatment (P50). Connected squares indicate mean ± SEM. F) Box plots of the latency to fall by P50 treated WT and Mecp2^-/y mice. G, H) Box plots of the time spent in open arms (G) and dipping (H) by untreated P40 WT and Mecp2^-/y mice, and P50 treated WT- and Mecp2^-/y mice in an elevated plus maze. Numbers in parenthesis indicate the number of mice used. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001, two-tailed unpaired t-test when comparing untreated mice (A,C,F,G,H), two-tailed paired t-test (B,E), Mann Whitney U test (D), Fisher exact test (J), Mann Whitney test (D), One-way ANOVA followed by a Tukey's multiple comparisons when comparing treated mice (C,F,G,H,I).

Leptin antagonism does not affect anxiety in male Mecp2^-/y mice

Leptin has been reported to exert anti-depressant and pro-cognitive like effects in rodents [13,14]. We therefore investigated the possible contribution of leptin to cognitive behavioral deficits repeatedly reported in RTT models. Our first set of behavioral tests examined anxiety, cognition and social interaction in P50 WT and Mecp2^-/y mice from our breeding colony. We first compared the spontaneous exploration in the elevated plus maze (EPM) as a measure of anxiety and found that Mecp2^-/y mice spent more time in the open arms than their WT littermate (Fig. 4G). The mean dipping time was also increased in Mecp2^-/y mice (Fig. 4H) a behavior also consistent with decreased anxiety. However, no genotype effects were found in tests used to evaluate object recognition and social behavior (Supplementary Fig. S2). We therefore limited the study of the role of leptin to the EPM. Leptin-treated WT and anti-leptin treated-Mecp2^-/y mice performed similarly to their vehicle-treated littermates in the EPM, i.e., they stayed for a similar amount of time in the open arms and exhibited similar mean dipping time (Fig. 4H and G). These observations therefore show that the anti-leptin treatment does not modify the anxiety-related behavior of symptomatic Mecp2^-/y mice.

Leptin antagonism restores the E/I balance in CA3 hippocampal neurons and layer 5 somatosensory cortical neurons, and mitigates the hippocampal synaptic plasticity in male Mecp2^-/y mice.

Alteration in the synaptic excitation/inhibition (E/I) balance is a widespread feature of neuronal networks in Mecp2-deficient mice [34]. Leptin has been reported to modulate the development and functioning of both glutamatergic and GABAergic synapses [13,[23], [24], [25], [26]], raising the possibility that elevated leptin levels might contribute to the altered E/I balance in Mecp2^-/y mice. We performed whole cell patch clamp recordings of CA3 pyramidal neurons on acute hippocampal slices obtained from P50 WT and Mecp2^-/y mice. We found that, as already reported [35], the E/I balance was increased in Mecp2^-/y compared to their WT littermates (Fig. 5A and B). The 10-day leptin treatment led to a significant increase in the E/I balance in P50 WT mice (Fig. 5B), while having no effect in Mecp2^-/y mice (Supplementary Fig. S1E). Conversely, the 10-day anti-leptin treatment restored the E/I balance in Mecp2^-/y mice (Fig. 5B). Previous studies have reported hypoactivity in cortical regions of Mecp2-null mice. To investigate whether anti-leptin treatment could restore the E/I balance in cortical neurons of Mecp2^-/y mice, we performed whole-cell patch-clamp recordings in layer 5 somatosensory cortical neurons. Interestingly, in contrast to earlier reports [[36], [37], [38]], we observed an increased E/I balance in Mecp2^-/y mice compared to their WT littermates (Fig. 5C). This unexpected discrepancy may be attributable to differences in genetic background, the specific Mecp2 mutation studied, or variations in experimental methodology. Importantly, a 10-day anti-leptin treatment effectively normalized the E/I ratio in layer 5 pyramidal neurons (Fig. 5C). We found no differences in the amplitude of sEPSCs and sIPSCs regardless of the genotype and/or treatment of the mice, in either hippocampal and cortical neurons (Supplementary Table 1). Altogether, these results show that leptin contributes to the E/I imbalance in symptomatic Mecp2^-/y mice.

Fig. 5.

Leptin contributes to hippocampal and cortical synaptic alterations of symptomatic Mecp2^-/y mice. A) Examples of whole-cell recordings performed on CA3 pyramidal neurons at a holding potential of −45 and −70 mV at P50. The glutamatergic synaptic currents are inwards and the GABAergic synaptic currents are outwards. Scale bars: 100 pA, 2 sec. B, C) Box plots of the ratio frequency of spontaneous glutamatergic and GABAergic postsynaptic currents recorded from P50 untreated and treated WT and Mecp2^-/y CA3 pyramidal neurons (B) and layer V somatosensory pyramidal neurons (C). D-F) LTP was recorded for 50 min following tetanic stimulation at the Schaffer collateral-CA1 synapses of acute hippocampal slices from symptomatic (P50) untreated (D) and treated (E, F) Mecp2^-/y and WT mice. Insets show examples of fEPSP. Scale bar: 100 μV, 3 ms. Numbers in parenthesis indicate the number of slices recorded and mice used. Numbers in parenthesis indicate the number of cells recorded and mice used. ∗∗P < 0.01; ∗∗∗P < 0.001, two-tailed unpaired t-test when comparing untreated mice (B,C), One-way ANOVA followed by a Tukey's multiple comparison when comparing treated mice (B,C). Repeated measure ANOVA (D–F).

Impairment of synaptic plasticity is another common feature of mice RTT models [39] and leptin has been reported to modulate the strength of glutamatergic synapses [27]. We therefore examined the effect of leptin and leptin antagonist treatment on long term potentiation (LTP) at the hippocampal Schaffer collaterals-CA1 synapses of P50 WT and Mecp2^-/y mice. As already reported [39], the magnitude of early (0–2 min post-tetanus) and late (45–50 min post-tetanus) potentiation were reduced in Mecp2^-/y mice compared to WT littermates (Fig. 5D). The 10-day leptin treatment of WT mice attenuated the magnitude of late and early potentiation compared to sham-treated mice but these differences did not reach significance (Fig. 5E). Conversely, the 10-day anti-leptin treatment increased the magnitude of early and late potentiation in Mecp2^-/y mice (Fig. 5F). Altogether, these data demonstrate the major contribution of leptin elevation to the hippocampal LTP impairment in symptomatic Mecp2^-/y mice.

Studies have shown that the brain derived neurotrophic factor (BDNF) levels are reduced in Mecp2-deficient mice and that their recovery would be a potential candidate for the treatment of RTT [40]. Leptin modulates BDNF expression in several brain structures including the hypothalamus [41] and hippocampus [42]. To determine whether the anti-leptin treatment modulates BDNF expression, we quantified the levels of mBDNF and proBDNF proteins in P50 hippocampi using ELISA. As already reported [40], the level of mBDNF was significantly lower in Mecp2^-/y mice compared to their WT littermates (Supplementary Fig. S3). The level of proBDNF in Mecp2^-/y mice was not different from that of WT mice. The 10-day anti-leptin treatment did not restore the level of mBDNF in Mecp2^-/y mice (Supplementary Fig. S3). Therefore, BDNF is unlikely to mediate the effects on the ant-leptin treatment in the hippocampus of Mecp2^-/y mice.

RTT-associated symptoms are improved in male and female leptin haplo insufficient Mecp2-deficient mice

The data presented above show that leptin antagonism effectively mitigates the deterioration of, or even rescues, certain symptoms observed in symptomatic Mecp2^-/y mice. However, prolonged administration of a leptin antagonist presents challenges, as it may potentially lead to metabolic and/or immune disturbances. To reduce this drawback and further evaluate the possible contribution of leptin in RTT phenotype, we generated Mecp2^-/y mice that are haplo insufficient for leptin with the aim of restoring the physiological levels of leptin in Mecp2-deficient mice. Leptin haplo insufficient Mecp2^-/y mice were generated by backcrossing Mecp2^+/− female mice with heterozygous leptin deficient male mice (ob^+/−). The resulting double transgenic Mecp2^-/y;ob ^± mice showed normalized leptin levels at P40-50 (Fig. 6A). We next examined whether Mecp2^-/y;ob ^± mice exhibited any improvements at advanced symptomatic stage. Notably, the progressive deterioration in breathing activity typically observed in Mecp2^-/y mice was prevented in Mecp2^-/y;ob ^± mice (Fig. 6B and C). In addition, Mecp2^-/y;ob ^± mice displayed significantly higher body weight compared to Mecp2^-/y littermates, with this difference becoming increasingly pronounced as the disease progressed (Fig. 6D). Whole cell recordings performed on hippocampal slices revealed that the E/I balance (Fig. 6E–Supplementary Table 1) was improved in CA3 hippocampal neurons of Mecp2^-/y;ob ^± mice. Field recordings on hippocampal slices further revealed that the magnitude of early and late potentiation at the Schaffer collateral-CA1 synapses was increased in Mecp2^-/y;ob ^± mice (Fig. 6F). Analysis of the health condition of the Mecp2^-/y;ob ^± mice revealed some improvement at P40 but not at P50 (Fig. 6G). At P40, Mecp2^-/y;ob ^± mice showed fewer tremor and limb grasping compared to the Mecp2^-/y mice (Fig. 6H). The life expectancy of Mecp2^-/y;ob ^± mice was however not different compared to their Mecp2^-/y littermates (Fig. 6I). Overall, these data show that reducing leptin production improves some RTT-associated symptoms in Mecp2^-/y mice.

Fig. 6.

RTT-associated symptoms are improved in male leptin haplo insufficient mice. A) Box plot of serum leptin levels in P50 WT, Mecp2^-/y and Mecp2^-/y;ob/+ littermates. B) Mean ± SEM plot of apnea frequency in P40 and P50 WT, Mecp2^-/y and Mecp2^-/y;ob/+ littermates. Scatter plots show individual data points. C) Mean ± SEM change of apnea frequency at P50 (% of P40). D) Box plot of body weight in P40 and P50 WT, Mecp2^-/y and Mecp2^-/y;ob/+ littermates. E) Box plots of the frequency ratio of spontaneous glutamatergic and GABAergic postsynaptic currents recorded from CA3 pyramidal neurons at P50. F) LTP at the Schaffer collateral-CA1 synapses of acute hippocampal slices from P50 Mecp2^-/y and Mecp2^-/y;ob/+ mice. Numbers in parenthesis indicate the number of mice used and number of slices recorded. G) Average severity score and H) Radar plot of the symptoms scored in P40 and P50 WT, Mecp2^-/y and Mecp2^-/y;ob/+ littermates. I) Plot of the percentage of surviving mice as a function of age. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. One-way ANOVA followed by a Tukey's multiple comparison (A, B, D, E, G, H), Mann-Whitney test (C), two-tailed Kruskal-Wallis followed by a Dunn's multiple comparison (H), repeated measure ANOVA (F).

Given that RTT primarily affects females, we wondered whether RTT symptoms were also alleviated in female Mecp2^+/− mice that are haplo-insufficient for leptin (hereafter referred to as Mecp2^+/−;ob ^± mice). A characteristic feature of the RTT mouse model on a C57BL/6J genetic background is that, unlike males, females gain weight as the disease progresses. The hyperleptinemia associated with this weight gain could therefore counterbalance haplo-insufficiency and prevent the normalization of leptin levels in Mecp2^+/−;ob ^± mice. To address this point and identify a window for reliable assessment of leptin involvement in RTT symptoms, we performed a longitudinal measurement of circulating leptin levels in Mecp2^+/−, Mecp2^+/−;ob ^± and WT mice from postnatal day (P) 20–200. Mecp2^+/− mice showed significantly elevated circulating leptin levels from P40 onwards compared to their WT littermates (Fig. 7A). Moreover, at P40 and P50, but not at older stages, Mecp2^+/−;ob/+ mice showed normalized leptin levels (Fig. 7A). It is worth noting that from P100 Mecp2^+/− and Mecp2^+/−;ob ^± mice weighted more than WT (Fig. 7B). This suggests that weight gain beyond P100, and the associated increase in leptin production, prevents normalization of leptin levels in Mecp2^+/−;ob ^± mice.

Fig. 7.

RTT-associated symptoms are improved in female leptin haplo insufficient mice. A) Mean ± SEM plot of serum leptin levels determined in blood samples taken at postnatal day (P) 20 every 10 days until P200, in female WT, Mecp2^+/− and Mecp2^+/−;ob/+ littermates. B-D) Box plots of body weight (B), frequency of apneas (C) and frequency ratio of spontaneous glutamatergic and GABAergic postsynaptic currents (D) in P40-50 and P100-200 WT, Mecp2^+/− and Mecp2^+/−;ob/+ littermates. E) Evolution of the severity score of WT, Mecp2^+/− and Mecp2^+/−;ob/+ littermates. mean ± SEM. F) Radar plot of the symptoms scored in P60 and P150 WT, Mecp2^+/− and Mecp2^+/−;ob/+ littermates. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. Two-tailed Kruskal-Wallis followed by a Dunn's multiple comparison (F), One-way ANOVA followed by a Tukey's multiple comparison (A, B, C, D), Wilcoxon matched-pairs signed rank test (E).

Based on these observations, we conducted a behavioral and neuronal characterization of the female mice at two different stages: at an early stage (P40-50), when the leptin levels are normalized in Mecp2^+/−;ob ^± mice and at a late stage (P100-200), when the leptin levels are comparable between Mecp2^+/− and Mecp2^+/−;ob ^± mice. We assessed several parameters including breathing, phenotypic score and E/I ratio, (Fig. 7C–F). Notably, the Mecp2^+/−;ob ^± mice showed an improvement in breathing activity at P40-50, but this improvement was not maintained at P100-200 (Fig. 7C). Similarly, the E/I ratio recorded on CA3 hippocampal neurons was restored in P40-50 Mecp2^+/−;ob ^± mice (Fig. 7D–Supplementary Table 1). There was no significant difference in the E/I balance between WT, Mecp2^-/y and Mecp2^+/−;ob ^± mice at P100-200 (Fig. 7D). The longitudinal analysis of the health condition of the mice revealed a progressive decline in Mecp^2−/y mice (Fig. 7E), characterized by worsening of tremor and limb grasping from P50, followed by a decline in spontaneous activity from P100 (Fig. 7F). In Mecp2^+/−;ob ^± mice, the onset of tremors and limb grasping was delayed, though spontaneous activity showed no significant improvement (Fig. 7F). Overall these data show that delaying the leptin rise delays the worsening of RTT-associated symptoms in Mecp2^+/−;ob ^± mice.

Discussion

In the present study, we investigated whether reducing leptin signaling, either through pharmacological antagonism or genetic reduction of leptin production, could alleviate deficits observed in a RTT mouse model. The impact of these interventions was systematically examined at both neuronal circuit and behavioral levels, with the aim of providing a broad evaluation of the therapeutic potential of leptin-targeting strategies rather than establishing direct causal relationships between neuronal and behavioral outcomes. Here, we show that Mecp2^-/y and Mecp2^+/− mice have elevated circulating leptin levels compared to their age-matched WT littermates. Given the diverse central and peripheral roles of leptin, this finding provides a foundation for investigating its potential contribution to RTT pathogenesis. Remarkably, a 10-day treatment of male Mecp2-null mice from P40 with a leptin antagonist prevented the worsening of several symptoms including breathing difficulty, locomotor deficit, weight loss and degradation of general health condition. At the neuronal level, the anti-leptin treatment restored hippocampal synaptic plasticity as well as hippocampal and cortical excitatory/inhibitory balance. However, the leptin antagonism strategy showed limited effectiveness against anxiety, motor coordination deficits and early lethality. Consistent with the role of leptin to RTT pathogenesis, we also showed that leptin treatment of WT mice impaired the hippocampal excitatory/inhibitory balance, reduced hippocampal synaptic plasticity and caused breathing abnormality in male WT mice, thus mimicking some RTT symptoms. We further showed that genetic manipulation aimed at normalizing leptin level in male Mecp2-null mice prevents weight loss, the progression of breathing deficits, and rescues the excitatory/inhibitory balance and glutamatergic synaptic plasticity in the hippocampus. Finally, we showed that delaying the leptin rise in female Mecp2-deficient mice delays the worsening of their RTT-associated symptoms. These findings strengthen the link between leptin and RTT, providing a valuable insight into potential therapeutic strategy for this syndrome. Interestingly, elevated leptin levels have also been observed in children with autism spectrum disorders [[43], [44], [45]]. Yet, the clinical features seen in ASD only partially overlap with those of Rett syndrome, suggesting that the functional and physiological actions of leptin may depend, among other factors, on the specific mutations associated with the etiology of the disease.

Elevated circulating leptin is consistently observed in patients with Rett syndrome [7,8] and in Mecp2-deficient mice [9,10,30]. Notably, individuals with RTT, hyperleptinemia occurs in the absence of significant alterations in body mass index (BMI) or fat mass [7,8,46,47]. Alternative mechanisms, including chronic inflammation [48], hormonal deregulations [49], or medications [50] may contribute to this phenotype. In mice, however, elevated leptin is often associated with higher body weight [30,31] or fat mass [9,10]. Notably, we found that symptomatic male Mecp2-null mice exhibit increased fat mass despite reduced body weight, together with elevated WAT leptin mRNA expression. The underlying mechanisms remain unclear, but they are likely to involve non-cell-autonomous processes. Supporting this hypothesis, previous studies showed that neuron-specific Mecp2 knockout mice (Mecp2^tm1.1Jae KO mice) display both increased circulating leptin levels and higher WAT leptin mRNA expression [9], whereas adipocyte-specific Mecp2 knockout mice (Mecp2^Adi KO mice) exhibit down regulated WAT leptin mRNA expression without changes in circulating leptin levels [51]. Together, these findings suggest that both increased leptin synthesis per unit of adipose tissue and increased adiposity contribute to elevated circulating leptin. Finally, intramuscular adipose tissue, known to expand in neuromuscular and metabolic diseases [52], may also contribute, consistent with the muscle hypotrophy and fibrosis observed in symptomatic Mecp2-null mice [53].

Given the known facilitatory role of leptin on breathing and synaptic function, our finding that elevated leptin levels contribute to RTT associated symptoms may initially appear counter-intuitive. However, chronic hyperleptinemia is known to induce a state of reduced leptin sensibility referred to as “leptin resistance”, a phenomenon extensively characterized in the context of obesity. In line with this, rodents fed a high-fat diet, that causes elevated leptin levels and leptin resistance, also exhibit impairments in hippocampal synaptic function as well as alterations in breathing, locomotor activity and anxiety level [17,54]. Andrade and colleagues demonstrated that the absence of Mecp2 leads to hypothalamic leptin resistance in female mice [30]. Consistent with this, we found that peripheral leptin administration increased pSTAT3 immunostaining, a surrogate marker of leptin receptor activation, in hypothalamic nuclei of wild-type mice, but not in Mecp2^-/y littermates. Furthermore, leptin injection had no effect on body weight in Mecp2^-/y mice, unlike in WT mice (Supplementary Fig. S1D). These findings indicate that Mecp2 deficiency disrupts leptin signaling, at least within hypothalamic circuits, supporting the notion that elevated leptin does not necessarily translate into increased physiological function. This raises the question of how anti-leptin treatment alleviates RTT-associated symptoms. We propose that reducing central leptin levels may relieve leptin resistance, thereby restoring leptin sensitivity. This hypothesis is supported by studies in obesity models, where lowering circulating leptin improves leptin responsiveness, leading to reduced food intake, increased energy expenditure, and improved glucose homeostasis [55,56]. Conversely, daily leptin administration in WT mice can induce leptin resistance, potentially contributing to the observed phenotype. Future experiments are required to directly test whether anti-leptin treatment restores leptin sensitivity in Mecp2-deficient brains and to define the brain regions involved.

Several observations support the hypothesis that the beneficial effects of leptin antagonism rely on central action. Firstly, functional leptin receptors are expressed throughout the brain [57]. Furthermore, in vivo experiments demonstrate that local/systemic injections of leptin or chemogenetic/optogenetic activation of leptin receptor expressing neurons, modulate physiological and behavioral responses, and that targeted deletion of central leptin receptors prevent these effects [[58], [59], [60], [61], [62], [63], [64]]. Finally, in vitro studies indicate that leptin directly regulates the excitability and synaptic function of neurons within different brain regions [11]. The respiratory network is complex and the effects of leptin on respiratory control are not yet fully understood. Nevertheless, alterations in the E/I balance have been reported in respiratory nuclei expressing leptin receptors, such as the nucleus tractus solitarius, the rostro ventrolateral medulla, the Kölliker-Fuse nucleus and the dorsomotor vagal nucleus [[65], [66], [67], [68]]. More research is needed to determine whether leptin antagonism also restore the E/I balance in these brain regions. Additionally, while our findings support the central effects of the treatments, we cannot completely rule out a peripheral effect, for example on carotid bodies.

We have shown that the anti-leptin treatment is well tolerated with no premature deaths or overt side effects, indicating it potential therapeutic application. Additionally, leptin antagonist therapy has been reported to be effective in preventing or treating diseases where elevated leptin levels are involved [69,70]. It is noteworthy that leptin antagonist treatment has also shown improvements in bone mineralization [71] and muscle function [72], two peripheral symptoms observed in RTT. Furthermore, hyperleptinemia has been suggested to contribute to sympathetic overactivity and cardiac abnormalities observed in RTT [8]. These observations collectively suggest that leptin may play a role in various central and peripheral RTT clinical manifestations underscoring the broad therapeutic potential of this approach.

Given the progressive nature of RTT symptoms and the high inter-individual variability, we assessed their evolution (i.e., the difference between the values at the beginning and at the end of treatment) to estimate the potential efficacy of the leptin antagonist treatment. Our data indicate that treatment with the leptin antagonist during the symptomatic period delays the progression of certain symptoms but does not lead to their complete recovery or to an increase in lifespan. Nevertheless, modulation of leptin signaling may represent a strategy to improve functional outcomes and quality-of-life, even if it does not extend longevity in this severe model. The notion of a pre-symptomatic period in RTT has recently been questioned, as studies have shown subtle symptoms in RTT girls and early alterations in animal models before the appearance of overt symptoms [73]. Therefore, while translational applications may pose challenges, it will be interesting in the future to determine whether “pre-symptomatic” treatments have more significant positive effects. Such interventions could shed light on potential therapeutic strategies for intervening at an earlier stage to possibly improve treatment outcomes in RTT. More broadly, our findings suggest that leptin-targeting approaches could be integrated into combination therapies aimed at addressing multiple pathogenic mechanisms simultaneously, which may ultimately be required to impact survival in RTT.

Data availability

All datasets generated for this study are available on request to the corresponding author.

Author contributions

J-LG, YB, CM and GAW conceived the experiments and wrote the manuscript. YB, J-LG, DD, MB-H, PS and VV performed the experiments. CS performed the behavioral analysis. J-CG bred the colony. All authors approved the final version of the manuscript.

Funding

This work was supported by the National Bar Institute of Health (Grant 1R01HD092396, GAW & J-LG), the Association Française du Syndrome de Rett (J-LG), INSERM Transfert (J-LG), Fondation Lejeune (J-LG), and the French government under the France 2030 investment plan, as part of the Initiative d'Excellence d'Aix-Marseille Université - A∗MIDEX (AMX-19-IET-007) through the Marseille Maladies Rares Institute (MarMaRa) (YB).

Declaration of competing interest

The authors declare no conflict of interest.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

figs1.

Leptin treatment has no effect on Mecp2^-/y mice.P40 Mecp2^-/y received daily sub-cutaneous injection of leptin recombinant (5µg/g) during 10 days. Sham mice received the same volume of vehicle. A-C) Box plots of the percentage of change (% of P40) of apnea frequency (A), breathing irregularity score (B) and minute ventilation (C) in sham and leptin treated-Mecp2^-/y mice. D) Body weight change (% of P40) as a function of age in sham and leptin treated Mecp2^-/y mice. E) Box plots of the frequency ratio of spontaneous glutamatergic and GABAergic postsynaptic currents recorded on CA3 pyramidal neurons of sham- and leptin treated- Mecp2^-/y mice at P50. Numbers in parenthesis indicate the number of mice used. Two-tailed unpaired t-test.