Tanycytic transcytosis inhibition disrupts energy balance, glucose homeostasis and cognitive function in male mice

Objectives In Western society, high-caloric diets rich in fats and sugars have fueled the obesity epidemic and its related disorders. Disruption of the body-brain communication, crucial for maintaining glucose and energy homeostasis, arises from both obesogenic and genetic factors, leading to metabolic disorders. Here, we investigate the role of hypothalamic tanycyte shuttles between the pituitary portal blood and the third ventricle cerebrospinal fluid in regulating energy balance. Methods We inhibited vesicle-associated membrane proteins (VAMP1-3)-mediated release in tanycytes by expressing the botulinum neurotoxin type B light chain (BoNT/B) in a Cre-dependent manner in tanycytes. This was achieved by injecting either TAT-Cre in the third ventricle or an AAV1/2 expressing Cre under the control of the tanycyte-specific promoter iodothyronine deiodinase 2 into the lateral ventricle of adult male mice. Results In male mice fed a standard diet, targeted expression of BoNT/B in adult tanycytes blocks leptin transport into the mediobasal hypothalamus and results in normal-weight central obesity, including increased food intake, abdominal fat deposition, and elevated leptin levels but no marked change in body weight. Furthermore, BoNT/B expression in adult tanycytes promotes fatty acid storage, leading to glucose intolerance and insulin resistance. Notably, these metabolic disturbances occur despite a compensatory increase in insulin secretion, observed both in response to exogenous glucose boluses in vivo and in isolated pancreatic islets. Intriguingly, these metabolic alterations are associated with impaired spatial memory in BoNT/B-expressing mice. Conclusions These findings underscore the central role of tanycytes in brain-periphery communication and highlight their potential implication in the age-related development of type 2 diabetes and cognitive decline. Our tanycytic BoNT/B mouse model provides a robust platform for studying how these conditions progress over time, from prediabetic states to full-blown metabolic and cognitive disorders, and the mechanistic contribution of tanycytes to their development. The recognition of the impact of tanycytic transcytosis on hormone transport opens new avenues for developing targeted therapies that could address both metabolic disorders and their associated cognitive comorbidities, which often emerge or worsen with advancing age.


INTRODUCTION
Since the mid-20th century, the development of the processed food industry in Western society has created a high-caloric eating culture, which, together with a sedentary lifestyle, resulted in an epidemy of obesity and a plethora of obesity-related disorders such as cardiovascular diseases, type 2 diabetes (T2D), steatohepatitis, cancer, and neurodegenerative disorders [1].Nutrient sensing mechanisms that maintain glucose and energy homeostasis involve body-brain communication by peripheral humoral and neuronal components, that will inform the brain about peripheral energy status and nutrient availability [2e4].Impairment of this communication due to both obesogenic and genetic factors can lead to the development of metabolic disorders.In recent decades, advances in neuroendocrinology have enabled the development of new strategies to treat metabolic diseases.These approaches aim to restore communication between the body and the brain that is often disrupted in conditions of energy excess [5e7].A deeper understanding of cells and molecules mediating communication between the periphery and the brain is essential for developing more effective and long-lasting therapies for metabolic diseases.Within the tuberal region of the hypothalamus, the median eminence serves as a key central hub for integrating signals related to energy balance by functioning as a circumventricular organ (CVO) [8].The median eminence is the structure closing the ventral aspect of the third ventricle (3V) adjacent to the arcuate nucleus of the hypothalamus (ARH) which is a major site of the metabolic brain containing the melanocortin system [6].The median eminence houses a dense capillary network with fenestrated endothelium, allowing blood-borne cues about the body's energy status to freely enter the median eminence and the ventromedial ARH [ [9,10]].However, the extent of passive molecule diffusion is limited, and the fenestration of capillary loops reaching the ARH is finely regulated according to energy status [11].Tanycytes are specialized ependymoglial cells that line the 3V floor.They form tight junctions that prevent blood-borne molecules from reaching deeper hypothalamic structures via the cerebrospinal fluid (CSF) [12e14].Fine processes of these cells establish direct contact with fenestrated vessels, controlling the bidirectional exchange of molecules between the bloodstream and the hypothalamus [14,15].This exchange delivers neurohormones into the bloodstream from neurons that regulate the anterior pituitary's activity and permits bloodborne molecules to reach their target neurons in the hypothalamus [16e19].Moreover, tanycytes themselves are central to intercellular communication processes within hypothalamic circuits that regulate energy homeostasis.They sense certain nutrients such as glucose and communicate their concentration to neurons [20].Selectively manipulating tanycytic activity using the DREADD technology or genetically altering their viability has profound consequences on energy homeostasis and circulating metabolic hormone levels [21e23].This is particularly relevant given that the conduit of blood-borne metabolic hormones into the hypothalamus by tanycytes is impaired in conditions of energy surplus [24,25].Our previous research indicates that the disruption of tanycyte shuttles may impair vesicular trafficking within these cells that is crucial for transcytosis processes [16].
To explore the importance of SNARE-dependent vesicle trafficking in tanycytes for metabolic regulation, we employed Cre recombinase-mediated expression of clostridial botulinum neurotoxin serotype B light chain (BoNT/B) in tanycytes in adult mice.Our findings demonstrate that the blockade of tanycytic transcytosis alone is sufficient to abrogate the access of blood-borne signals such as leptin into the mediobasal hypothalamus.This manipulation impairs glucose homeostasis in mice fed on a standard diet: it promotes intraabdominal fat deposition, glucose intolerance, hyperinsulinemia and impaired insulin sensitivity, particularly in the context of moderate overweight.Additionally, it heightens sensitivity to obesogenic diets.

Animals
All C57Bl/6J male mice were housed under specific pathogen-free conditions in a temperature-controlled room (21e22 C) with a 12h light/dark cycle, 40% humidity and ad libitum access to food and water.Tg(CAG-BoNT/B,EGFP)U75-56Fwp/J (BoNT/B-EGFP loxP-STOP-loxP ; JAX Stock No. 018056) mice were generated as previously described [26].Transgenic model mice were bred and genotyped in-house to generate experimental animals.All experiments were performed in 8e 20 weeks-old mice and with the approval of the Institutional Ethics Committees for the Care and Use of Experimental Animals of the University of Lille and the French Ministry of National Education, Higher Education and Research (APAFIS#2617e2015110517317420 v5), following the guidelines outlined by the European Union Council Directive of September 22, 2010 (2010/63/EU).

Indirect calorimetry study
Mice were individually housed and acclimatized to the cages for 48h before experimental measurements in a 12h light/dark cycle and an ambient temperature of 22 AE 1 C. Mice fed a chow diet were placed in metabolic cages 6 weeks after AAV1/2 Dio2Cre-induced recombination.Mice fed a high-fat diet (HFD) were placed in metabolic cages 10 weeks after AAV1/2 Dio2Cre injection, which was 4 weeks after they began consuming the HFD.Mice were analyzed for total energy expenditure, oxygen consumption, carbon dioxide production, and food intake using calorimetric cages (Labmaster, TSE Systems GmbH, Bad Homburg, Germany) [29].Fat oxidation was calculated as described before [30,31], using energy expenditure, oxygen consumption, and carbon dioxide production.Mice were monitored daily for body weight and body composition at the beginning and the end of the experiment.2.12.Mouse pancreatic islet isolation and culture Each of the ten mice used for islet isolation was fully anesthetized and sacrificed by cervical dislocation.The mouse was laid with the abdomen facing up and the skin was sterilized with 70% ethanol.An incision was performed around the upper abdomen to expose the pancreas and common bile duct.The pancreas was infused with type V collagenase (1.5 mg/ml) via the common bile duct.After perfusion, the pancreas was removed, collected in a 50 ml tube containing 2 ml of enzyme collagenase, and digested in a water bath at 37 C for 8e 10 min [33,34] 1.Expression levels were normalized to cyclophilin mRNA and expressed using formula 2eDCt.
Images were processed for morphometry using ImageJ software by an observer blind to experimental groups.

Immunohistochemistry and analysis
In fresh frozen tissue.Adult mice were sacrificed by decapitation at lights-on in fed state (for p-STAT3 and MECA32).Brains were harvested before being embedded directly in Tissue Tek (SakuraÒ) and frozen fresh.Coronal sections 20 mm thick were post-fixed with a 2% paraformaldehyde solution for 1 h and processed for immunofluorescence as previously described (Bouret et al., 2012) using primary antibodies directed against STAT3 phosphorylated at Tyr705 (p-STAT3, 1:1000; #9131, Cell Signaling Technology) and against plasmalemma vesicle-associated protein (clone MECA32; 1:500; 550563, BD Parmingen).Double immunofluorescence images were acquired using an Apotome Axio.Z2 microscope (AxioCam MRm camera, Zeiss).Slides were then coded to conceal treatment groups, and p-STAT3 immunoreactive (IR) cells were counted on eight representative sections per animal.MECA-32 immunoreactive vessels are visualized in the superficial plexus of the outer zone of the median eminence, but some MECA-32 immunoreactive vessels form intra-infundibular capillary loops.The total number of fenestrated loops and p-STAT3 immunopositive cells was assessed over the entire rostrocaudal surface of the median eminence and arcuate nucleus in each animal (8 representative median eminence sections per animal).The average number of immunopositive cells or vessels per area was then compared between groups.

Behavioral assessment
Visuospatial short-term memory and novelty preference were tested using the Y-maze [37].The Y-maze consists of three white wooden arms (24.0 cm Â 6.5 cm x 15 cm) on the floor and surrounded with visual cues on the walls.Mice were placed in the start arm, facing the end of this arm, and were allowed to explore the maze for 10 min while one arm was blocked (novel arm).Consequently, mice were placed in their home cage for 2 min before being allowed to explore all three arms for 5 min.The trajectories of the mice were recorded using EthoVision video tracking equipment and software (Noldus Bv, Wageningen, The Netherlands).The primary outcome measure was the percentage of time spent in the novel arm compared to the familiar arms, indicative of the subject's spatial memory and exploratory behaviour.The time spent in the novel arm and latency to enter the novel arm were compared between mice.The Y-maze test was performed in the morning (9:00).

Statistics
Results are given as means AE s.e.m.Samples or animals were excluded when their values were outside AE2 s d., or when an objective experimental failure was observed; studies were not formally randomized and investigators were not blind to the treatment group, except when mentioned.To test whether populations followed a Gaussian distribution, a normality test was performed (Kolgomorove Smirnov test for n ¼ 5e7, ShapiroeWilk test for n ! 7).For normal distributions, one or two-sided unpaired t-tests were used to compare two populations; for multiple-comparison tests, one or two-way ANOVA followed by Tukey's post hoc multiple-comparison test was used (unless otherwise indicated in the figure legends).For non-Gaussian distributions, ManneWhitney tests were used to compare two populations, and KruskaleWallis followed by Dunn's post hoc test for multiple comparisons.The ANCOVA analysis was performed using the NIDDK Mouse Metabolic Phenotyping Centers online tools (MMPC, www.mmpc.org/).Data analysis was performed using GraphPad Prism Software v.8.0 (version 8.4.2;GraphPad Software, La Jolla, California, USA).The threshold for significance was P < 0.05.

Botulinum neurotoxin type B expression in adult hypothalamic tanycytes promotes body weight gain while increasing carbohydrate consumption
To explore the specific function of tanycytes in transporting metabolic hormones from the bloodstream to the brain, we employed a mouse model where we inhibited vesicular transport within tanycytes using botulinum toxin light chain B (BoNT/B).BoNT/B hinders exocytosis in neurons and glial cells by cleaving vesicle-associated membrane proteins 1 and 2 (VAMPs), which facilitate vesicular fusion and transport [26,38].VAMP3, another target of BoNT/B, is involved both in endocytosis and exocytosis processes [39,40].For conditional cellspecific BoNT/B expression, we used males from a transgenic mouse line in which BoNT/B and EGFP can be expressed in a Credependent manner [26].To induce Cre expression and thus BoNT/B specifically in tanycytes of these mice, we injected either TAT-Cre into the 3rd ventricle [16] or of an AAV1/2 vector expressing the Cre recombinase under the control of the tanycytes-specific human deiodinase 2 (DIO2) promoter into the lateral ventricle [23].Control littermates were injected with an AAV1/2 vector expressing EGFP.Control-and Cre-injected animals are hereafter referred to as BoNT/ B Ctl and BoNT/B Tan mice, respectively (Figure 1A).The viral strategy selectively targeted tanycytes in the tuberal region of the hypothalamus (Figure 1B).Isolation of tanycytes using fluorescent-activated cell sorting (FACS) from median eminence-ARH microdissected explants (Figure 1C) showed strong induction of BoNT/B transcripts in BoNT/ B Tan mice in contrast to BoNT/B Ctl littermates, which did not show any signal (Figure 1D).Transcripts for VAMP1-3 were readily detected in FACS-sorted tomato-positive tanycytes isolated from tdTomato loxPÀ- STOP-loxP mice following TAT-Cre delivery into the third ventricle (Figure 1E) that express tanycytic markers, including Ppp1r1b and Dio2 (Figure 1F).Notably, as expected the presence of BoNT/B did not affect the expression of VAMP1 and VAMP2 genes remained unchanged in targeted tanycytes.However, there was a trend toward downregulation of VAMP3 transcripts compared to tanycytes from tdTomato mice (Figure 1E).Crucially, the expression of BoNT/B in tanycytes did not change their morphology (Figure 1B) nor did it induce cell death.No nuclear fragmentation, which would indicate apoptosis, was observed in the tanycytic cell body layer adjacent to the third ventricle (Supplementary Fig. 1A), and no cleaved caspase 3 signal was detected in the cell nuclei lining the wall and the floor of the third ventricle in mice expressing BoNT/B at 4 and 12 weeks after injection of the Cre-expressing viruses (Supplementary Figs.1B and C).Four weeks after Cre-mediated genetic recombination and intake of a chow diet, mice expressing BoNT/B in tanycytes exhibited increased food intake (Figure 1G), which was associated with a slight increase in body weight (Figure 1H, Supplementary Fig. 2A) compared to BoNT/B Ctl littermates.Surprisingly, indirect calorimetric analyses revealed elevated carbohydrate metabolism at the end of the resting period, as indicated by a higher respiratory exchange ratio (RER, Figure 1I), along with increased energy expenditure and ambulatory activity (Supplementary Figs.2BeC).ANCOVA analysis [41e43] revealed no significant interaction between total body mass (TBM) and energy expenditure (EE) across genotypes (p ¼ 0.51), and when adjusted for TBM, there was no significant difference in EE between control and BoNT/B-expressing mice (p ¼ 0.4).However, the observed difference in EE was significantly associated with the difference in TBM between groups (p ¼ 0.046), suggesting that the altered EE in BoNT/B-expressing mice is primarily due to changes in body mass rather than a direct effect of genotype on energy metabolism.At 12 weeks post recombination, BoNT/B Tan mice exhibited a more consistent net body weight gain compared to their control littermates (Figure 1H, Supplementary Fig. 2A).Interestingly, this occurred despite the absence of actual body weight differences between genotypes (Supplementary Fig. 2A).However, the BoNT/ B Tan mice showed two notable metabolic changes: increased accumulation of visceral fat (Figure 1J) and higher circulating levels of leptin (Figure 1K).Altogether the BoNT/B Tan mice appear to develop a "normal-weight obesity" phenotype, i.e, intra-abdominal accumulation of fat with slight overweight or no marked changes in body weight [44,45].When animals were placed on a high-fat diet (HFD) four weeks after the beginning of the experiment using AAV-mediated Cre recombination, body weight gain markedly accelerated in BoNT/B Tan mice, with significant changes occurring already within two weeks after initiating this diet (Figure 1L).As observed with the chow diet (Figure 1G, I), indirect calorimetry at four weeks on the HFD showed that BoNT/B Tan mice exhibited increased food intake (Figure 1M), accompanied by increased use of carbohydrates as the primary energy source during both resting and active periods (Figure 1N).However, no marked differences were observed in energy expenditure or ambulatory activity (Supplementary Figs.2DeE).Altogether these data show that impairing SNARE-dependent vesicle release from tanycytes enhances food intake and body weight.These changes were possibly caused by disruption of the feedback loops between peripheral tissues and the brain, leading to miscommunication regarding energy balance.

BoNT/B expression in tanycytes blocks blood-borne leptin transport into the hypothalamus
Increased food intake (Figure 1G) concomitant with elevated adiposity and circulating levels of leptin in BoNT/B Tan mice (Figure 1J,K) raises the possibility that these animals may be developing hypothalamic resistance to circulating leptin.This could be caused by defective leptin transport across the bloodebrain barrier into the CSF via tanycytes, as seen in various animal models at early stages of diet-induced obesity [16,24,46].First, to test whether this phenomenon occurs under physiological conditions, we assessed endogenous STAT3 activation in the ARH at the specific time of the day when lights are switched on, after overnight feeding, when circulating leptin levels are at their highest [47].This paradigm has previously been shown to be linked to endogenous LepR activation [11,48].Four weeks after Cre-induced recombination, the number of P-STAT-3 immunoreactive cells in the ARH was diminished by about 30% in BoNT/B Tan mice when compared to BoNT/B Ctl littermates (Figure 2A,B).In contrast, a trend towards an increased number of p-STAT3-immunoreactive cells was observed in the median eminence of BoNT/B Tan mice compared to control littermates, although this difference did not reach statistical significance (Figure 2C).This suggests that in BoNT/B Tan mice, circulating leptin extravasates from the fenestrated capillaries of the median eminence but appears to remain trapped within the median eminence parenchyma.There, it may activate local LepR-expressing cells without being transported into the hypothalamus.In support of this,  examination of the fenestral diaphragms of median eminence endothelial cells using antibodies to MECA-32 showed that the number of fenestrated capillary loops in the median eminence and the vmARH were comparable in BoNT/B Tan and control littermates (Figure 2A,D  and 2E).Finally, twelve weeks after Cre-induced recombination, we subjected BoNT/B Tan mice as well as their control littermates to intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) injection of exogenous leptin and measured food intake 24h and 12h later, respectively (Figure 2FeH).While exogenous leptin injected i.c.v.directly into the lateral ventricle reduced feeding in both groups of mice efficiently (Figure 2H), exogenous leptin injected intraperitoneally showed this effect only in BoNT/B Ctl mice, but not in BoNT/B Tan mice (Figure 2G).This confirmed that BoNT/B Tan mice are sensitive to central leptin but resistant to peripheral leptin suggesting a block of the tanycytic transport of blood-borne leptin into the hypothalamus.To further explore the impact of BoNT/B expression in tanycytes on bloodborne leptin transport into the ARH, we stereotaxically implanted microdialysis probes into the mediobasal hypothalamus of both BoNT/ B Ctl and BoNT/B Tan mice (Figure 2I).Leptin levels in the hypothalamus were monitored in dialysates over time.Forty minutes after administering an intraperitoneal bolus of leptin, we observed leptin transport into the mediobasal hypothalamus in BoNT/B Ctl mice, following a kinetic pattern that was not observed in BoNT/B Tan littermates.In the latter, leptin levels remained close to the detection limit of the approach (Figure 2J).Altogether, these results unequivocally demonstrate that tanycytic transcytosis is required for the transport of bloodborne leptin into the hypothalamus.

BoNT/B expression in hypothalamic tanycytes alters peripheral lipid homeostasis
Despite the higher visceral fat mass and leptinemia observed in BoNT/ B Tan mice compared to their BoNT/B Ctl littermates (Figure 1J-K), no significant differences were found in either fatty acid oxidation (Figure 3A) or serum levels of triglycerides (Figure 3B), cholesterol (Figure 3C), and non-esterified free fatty acids (NEFAS) between the two groups of mice (Figure 3D).To delve deeper into lipid metabolism, we examined the lipolysis and lipogenesis pathways in both the liver in visceral adipose tissue of BoNT/B Ctl and BoNT/B Tan littermates using Western blot analyses.In the liver, BoNT/B expression in tanycytes did not affect markers of de novo lipogenesis, including the total protein levels and phosphorylation of acetyl-CoA carboxylase (ACCa) and fatty acid synthase (FAS) (Figure 3E,H).Also, protein expression of carnitine palmitoyl transferase 1-A (CPT1-A), an indicator of fatty acid oxidation, remained unchanged (Figure 3F,H).However, there was a notable reduction in lipoprotein lipase protein (LPL) levels (Figure 3G, H), suggesting a decrease in hepatic lipid uptake.In the epididymal white adipose tissue, BoNT/B Tan mice showed higher ratio of phosphorylated ACCa (pACC)/ACCa, indicating a decrease in fatty acid synthesis (Figure 3I,L).No differences were observed in the protein levels of the fatty acid oxidation marker CPT1A (Figure 3J,L) or in the lipid uptake marker LPL (Figure 3K,L).However, BoNT/B Tan mice exhibited reduced phosphorylation of hormone-sensitive lipase (HSL) compared to control mice (Figure 3M, 3L), suggesting a decrease in triglyceride mobilization, possibly due to lower noradrenaline levels, an activator of HSL-mediated lipolysis (Figure 3N) [49,50].Overall, these results suggest that the blockade of tanycyte transcytosis in BoNT/B Tan mice may reduce lipid synthesis and mobilization in the white adipose tissue.This could be due to the incapacity of leptin to reach the ARH and promote peripheral lipid mobilization [51], as well as hindered signaling to the sympathetic nervous system via the melanocortin system [52], as suggested by the decreased noradrenalin levels in the BoNT/B Tan mouse model (Figure 3N).Nonetheless, it is also possible that other hormones are involved, their passage impeded, thereby affecting lipid metabolism in BoNT/B Tan mice.
3.4.Expression of BoNT/B in tanycytes promotes glucose intolerance and insulin resistance associated with pancreatic beta cell compensation At 12 weeks after BoNT/B induction in tanycytes, BoNT/B Tan mice exhibited impaired tolerance to exogenous glucose (Figure 4A).Monitoring of glucose-stimulated insulin release monitoring revealed heightened insulin secretion in BoNT/B Tan mice compared to control BoNT/B Ctl littermates, suggesting a beginning of insulin resistance (Figure 4B).An insulin tolerance test further confirmed this observation (Figure 4C).Additionally, insulin sensitivity and the homeostatic model assessment of insulin resistance (HOMA-IR) were found to be elevated in BoNT/B Tan mice compared to control mice (Figure 4D), indicating a prediabetic, insulin-resistant state in BoNT/B Tan overweight mice.To assess potential changes in pancreatic function in mice expressing BoNT/B in tanycytes, we examined glucose-stimulated insulin secretion (GSIS) in pancreatic islets isolated from both BoNT/B Tan and BoNT/ B Ctl littermates 12 weeks after Cre-mediated recombination.Consistent with the prediabetic, insulin-resistant phenotype observed in BoNT/B Tan mice, isolated islets demonstrated heightened insulin secretion in response to 20 mM glucose, but similar insulin content compared to those from control BoNT/B Ctl littermates (Figure 4E,F), suggesting the activation of compensatory mechanisms in b cells following insulin resistance in these animals [53].Gene expression profiling of b-cells from BoNT/B Ctl and BoNT/B Tan islets revealed notable transcriptional alterations in markers of b-cell function and endoplasmic reticulum (ER) stress.In b-cells from BoNT/B Tan mice compared to controls, the heightened GSIS was accompanied by upregulation of mRNA levels in genes implicated in glucose sensing and insulin secretion, including Slc2a2 and Glp-1r.Conversely, there was a decrease in expression of the proinsulin processing gene Pcsk1 (Figure 4G).Additionally, differences were noted in the expression of genes associated with pancreatic islet identity and function, such as reduced expression of Pdx1 involved in b-cell maturation and survival [54], and elevated levels of Hnf1a, MafA and NeuroD1 genes, all contributing to b-cell identity (Figure 4G).Notably, b cells from BoNT/ B Tan mice exhibited elevated expression of the early onset ER-stress marker Atf4 (Figure 4H), suggesting that the increased insulin secretion observed in these mice following insulin resistance may trigger an ER unfolded protein response and ER stress.This response is typically associated with impaired b-cell function under chronic high glucose [55].Despite this, no differences were observed yet in surface area (Figure 4I), size (Figure 4J), or morphology (Figure 4K) of pancreatic islets from BoNT/B Tan compared to those from BoNT/B Ctl mice.Overall, tanycytic transcytosis appears to be an essential part of the central regulation of pancreatic function, whose blockade results in glucose intolerance, insulin resistance and the disruption of b-cell function and identity independently of pancreatic islet morphology.

Blunting transcytosis in tanycytes alters spatial working memory
Next, we aimed to evaluate potential early cognitive effects of the metabolic changes observed in our mouse model, focusing on hippocampus-dependent behaviors that are often the first to show impairment in age-related cognitive decline [56,57].We employed the Ymaze test, which specifically assesses spatial working memory, a form of short-term memory, and exploratory behavior, both of which heavily rely on hippocampal function.Twelve weeks after viral injection, BoNT/ B Ctl mice subjected to this test showed normal behavior, spending more time exploring the newly accessible arm than the previously explored arms (Figure 5).In contrast, BoNT/B Tan mice spent an equal length of time in all arms, suggesting impaired spatial memory (Figure 5).These results suggest that disrupting tanycytic shuttling, and consequently body-brain communication, may progressively impair spatial working memory, contributing to the onset of cognitive decline in mice, particularly in the context of metabolic disturbances.

DISCUSSION
Over the last decade, tanycytes have been proposed as the conduit for peripheral information into the hypothalamus, aiding in the regulation of body homeostasis.To achieve this, they actively transport bloodborne signals across the blood-cerebrospinal fluid (CSF) barrier, which they themselves form [24]. Using the selective expression of BoNT/B in tanycytes to inhibit vesicular trafficking, we provide compelling evidence to show that tanycytic transcytosis, which shuttles not only circulating hormones and peripheral signals but also nutrients into the brain, is essential for maintaining energy homeostasis and cognitive processes.Previous studies have demonstrated that the conditional ablation or impaired function of median eminence tanycytes results in a phenotype similar to that observed in our model, characterized by increased adiposity, insulin resistance, elevated caloric intake, and leptin resistance [22,58].However, these studies also revealed compromised barrier properties in the median eminence, allowing peripheral cues to bypass the tanycytic barrier and tanycyte-mediated changes in barrier architecture induced by energy status [11] or circadian rhythms [59].Through a more targeted approach, we can now elucidate the specific role of tanycytic transport in modulating energy balance.By expressing BoNT/B in tanycytes, we impede the transport of blood-borne molecules transported via tanycytic transcytosis into the ARH, and deprive BBB-protected ARH neurons of peripheral cues.Our observations indicate that, in addition to promoting "normal-weight central obesity", a phenotype associated with adverse metabolic outcomes that is frequently observed in the human population [44,45,60,61], BoNT/B expression in hypothalamic tanycytes induces insulin resistance and glucose intolerance, underscoring the involvement of tanycytes and their transcytotic activity in the pathogenesis of type 2 diabetes.Our study highlights the crucial role of transcytotic vesicular trafficking in transporting peptide hormones, such as leptin, from the pituitary portal blood circulation to the hypothalamus for maintaining energy homeostasis.However, it is essential to recognize that blocking vesicular transcytotic activity with our current approach may disrupt the transport of various other blood-borne metabolic hormones released by peripheral tissues, which are known to exert an action in the hypothalamus.Therefore, the metabolic phenotype observed in BoNT/B Tan mice likely results from a global impairment of the access of multiple metabolic signals to the brain rather than the specific disruption of a single feedback loop.For instance, while our results demonstrate metabolic alterations in BoNT/B Tan mice, we cannot rule out the possibility that the insulin resistance observed is partially attributable to their increased intra-abdominal adiposity.In addition, BoNT/B may also impair the release of tanycyte-derived signals that play a role in these processes.For example, the impaired regulation of glucose homeostasis in BoNT/B Tan mice might result from the combined alteration of both leptin and insulin transport into the ARH.Notably, when insulin transport into the ARH was impaired by ablating insulin receptors in tanycytes, mice exhibited insulin resistance without changes in glucose tolerance [18].Conversely, mice with abrogated leptin transport due to the selective knockout of LepR in tanycytes showed increased insulin secretion at early stages despite maintaining normal glucose and insulin tolerance [16].Remarkably, the absence of both insulin and leptin receptors in ARH POMC neurons, one of the two key neuronal targets of the metabolic hormones transported by tanycytes, resulted in glucose intolerance, insulin resistance, and heightened glucose-stimulated insulin secretion from pancreatic islets [62], a phenotype similar to that observed in BoNT/B Tan mice.Environmental and age-related changes in tanycyte function can also significantly impact healthy aging.For instance, obese individuals appear to exhibit impaired transport of leptin from the bloodstream into the cerebrospinal fluid [63,64], a condition observed in diet-induced obesity in minipigs [46] and mice [24], as well as in mice with selective knockout of leptin receptors in tanycytes [16].This points to tanycytes as a prime target of aging processes.Whether this impairment extends to other peptide metabolic hormones remains to be explored.Nevertheless, it is well-established that midlife obesity and diabetes are risk factors for Alzheimer's disease and other forms of dementia later in life [65].The BoNT/B Tan mouse model, which exhibits alterations in spatial working memory, provides a unique opportunity to investigate the role of tanycytic dysfunction in multiple age-related conditions.This model mimics key features of age-related obesity, such as increased intra-abdominal fat accumulation without significant body weight gain and impaired glucose metabolism.It could therefore prove useful to explore not only whether tanycytic dysfunction contributes to these metabolic changes but also its role in cognitive decline, including dementia, and other hormone-regulated pathological processes associated with aging.Such a mechanism, is especially relevant considering that the bridge formed by tanycytes between the CSF and blood seems to be compromised in Alzheimer's patients [66], and that tanycytes also appear to mediate estrogenic effects on ARH neurons controlling metabolism [67], a finding that could be of particular significance in postmenopausal women who are susceptible to both metabolic dysfunctions and developing Alzheimer's disease.From a therapeutic point of view, in keeping with the central regulation of peripheral metabolic processes, recent studies have underscored the pivotal role of the brain in the mechanisms of action of emerging anti-obesity and anti-diabetic medications.For instance, our research has recently revealed that hindering tanycytic transcytosis using the same BoNT/B Tan mouse model used in the current study not only impedes the entry of the anti-obesity/anti-diabetic drug liraglutide into the brain but also mitigates its anti-obesity effects on parameters such as food intake, body weight, fat mass, and fatty acid oxidation [17].Novel drugs combining GLP-1R and glucosedependent insulinotropic polypeptide (GIP) coagonism are also known to exert their regulatory effects on body weight and food intake through central nervous system pathways [68,69], and it may be possible to enhance their efficacy or specificity by targeting their entry into the brain.To summarize, our study not only reveals that tanycytic transcytosis is an essential mechanism in the control of metabolic homeostasis by the brain, the blockade of which has far-ranging effects on both central and peripheral processes, providing clues to the pathophysiology of conditions such as type 2 diabetes and obesity as well as age-related cognitive decline, but also validates a tool e the BoNT/B Tan model e that can be used to elucidate these complex and interlinked regulatory processes and design effective therapeutic strategies for the pathologies that result from their breakdown.
[35,36]Anti-Chicken Alexa FluorÒ 488 ref: 703-545-155).All secondary antibodies were diluted in PBS and incubated with sections for 90 min at room temperature.Nuclei were counterstained by incubating sections with DAPI (1:5,000 D9542, Sigma Aldrich) for 5 min at RT.All labelled sections were mounted onto Superfrost glass slides and images acquired using an Apotome Axio.Z2 microscope (AxioCam MRm camera, Zeiss) or a STELLARIS Confocal microscope platform (Leica).2.16.Leptin sensitivity assayIndividually housed mice were subjected to fasting for 6h before the onset of the dark phase as previously described[35,36].Three hours before the refeeding, 2 groups of mice (both BoNT/B Ctl and BoNT/B Tan ) were challenged with leptin.A group received either murine leptin (3 mg/kg À1 ; Harbor-UCLA Medical Center) or vehicle (PBS pH 8.0) intraperitoneally.To check the ICV effect of leptin administration, a cannula (Plastic One) was stereotaxically placed in the lateral ventricle (anteroposterior, À0.3 mm; midline, AE1 mm; dorsoventral, À2.5 mm) and leptin (2 mg in 2 ml Harbor-UCLA Medical Center) or vehicle (2 ml of PBS pH 8.0) was administrated to a second group of mice.Body weight and food intake were monitored before,12h, and 24h after leptin administration.2.17.In vivo microdialysisA microdialysis cannula (CMA8 High Cut-off, 100 kDa, 1 mm membrane length; CMA microdialysis) was stereotaxically implanted in the mediobasal hypothalamus (anteroposterior, À1.3 mm; lateral, À0.3 mm; ventral, À6.1 mm) in BoNT/B Ctl and BoNT/B Tan isoflurane-anesthetized mice, maintained at 37 C core body temperature using a thermostatically controlled electrical blanket.The administered to mice and six dialysates of 20 min recovered.Dialysates were placed in a fraction collector (CMA 820) during the experiment and immediately stored at À80 C until analysis.At the end of the experiment, mice were decapitated and brains were stored immediately in fresh 4% paraformaldehyde.Microtome brain sections (40 mm) were counterstained with DAPI to verify probe location.Only mice in which the probe was positioned between anteroposterior À1.2 and À2.3 were included in analyses.