Transthyretin gates GLP-1R-mediated affect by metabolic stat

Transthyretin gates GLP-1R-mediated affect by metabolic stat | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Transthyretin gates GLP-1R-mediated affect by metabolic stat Shuibing Liu, Beining Lu, Yongli Jiang, Jie Zhou, Nana Zhang, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8322552/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract GLP-1 receptor agonists are widely used to treat diabetes and obesity, yet their variable neuropsychiatric side effects including anxiety and depression in some individuals have remained mechanistically unknown. Here, we identify transthyretin (TTR) as a context-dependent mediator and biomarker for GLP-1R–driven affective behavior. We show that in normal-weight mice, hippocampal GLP-1R activation triggers a cAMP/PKA/Gli3 cascade that represses TTR expression, leading to impaired ERK/CREB-dependent synaptic plasticity and promotes anxiety- and depression-like behaviors. Conversely, in diet-induced obese models where hippocampal TTR is upregulated, and GLP-1R activation normalizes TTR levels, rescuing affective deficits. Translating these findings, we show that GLP-1R agonist reduces elevated serum TTR in obese humans. We therefore propose that baseline TTR levels serve as a critical biomarker: obese individuals with high TTR may achieve both metabolic and mental health benefits, whereas individuals with normal TTR levels are at risk for drug-induced affective disturbances. Our work establishes a molecular basis for the psychiatric side effects of GLP-1R-targeting therapies and provides a rationale for TTR-guided personalization of treatment. Biological sciences/Neuroscience/Emotion Health sciences/Endocrinology/Endocrine system and metabolic diseases GLP-1R depression TTR Gli3 CREB signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction The rising global prevalence of metabolic diseases such as type 2 diabetes mellitus (T2DM) and obesity coincides with an increasing burden of mood disorders, underscoring a profound connection between metabolic homeostasis and affective regulation 1,2 . Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) not only improve glycemic control and body weight but also exert central effects influencing mood and neuroinflammation 3,4 . While GLP-1 RAs may alleviate depressive symptoms in some patients 5,6 , post-marketing surveillance has also reported anxiety and suicidal ideation in certain individuals 7,8,9,10 , creating a clinical paradox that remains mechanistically unresolved. The hippocampus represents a critical interface between metabolic and affective processing 11 . This region not only shows high GLP-1 receptor expression but is also vulnerable to both metabolic stress and depression-related plasticity deficits 3 . Our previous work demonstrated particularly elevated hippocampal GLP-1R expression in diabetic mice, suggesting this region as a key site for investigating mood-related drug effects 12 . Preclinical studies show that GLP-1 RAs can improve hippocampal function in metabolic disease models yet paradoxically induce anxiety-like behaviors and HPA axis activation in healthy rodents 13,14,15 . This bidirectional effect implies the existence of an unknown molecular switch that directs GLP-1R signaling toward beneficial or detrimental affective outcomes based on metabolic context. Transthyretin (TTR), a transport protein synthesized in the liver and choroid plexus, may constitute this critical switch. Peripheral TTR is elevated in obese humans and diet-induced obese mice, correlating with insulin resistance (HOMA-IR) and adipose inflammation 16,17 . Central TTR exerts neuroprotection and synaptic function by activating the MAPK/ERK/CREB pathway, which is critical for hippocampal plasticity and neuronal energy metabolism 18 . Notably, in silico pharmacogenomic analyses reveal that GLP-1R signaling intersects with TTR-regulated pathways enriched in dopamine metabolism and insulin sensitivity, suggesting TTR could functionally bridge the metabolic and neuropsychiatric dimensions of GLP-1R activation 19,20 . Still, whether TTR serves as a metabolic-state-dependent mediator of GLP-1R-induced affective behaviors, and how its regulation is embedded within GLP-1R-driven signaling networks, remained unknown. In this study, we formally tested the hypothesis that TTR acts as a central molecular switch that integrates GLP-1R signaling with metabolic status to coordinate affective state. We aimed to (1) determine how metabolic status shapes the impact of GLP-1RA on hippocampal TTR expression and synaptic function; (2) delineate the upstream signaling cascade GLP-1R-cAMP-PKA-Gli3 controlling TTR transcription; (3) establish TTR as a dual biomarker capable of predicting metabolic and neuropsychiatric outcomes in a human; and (4) explore TTR-targeting strategies to optimize the therapeutic utility of GLP-1RA. Our findings reveal a TTR-mediated pathway that unifies the metabolic and neuropsychiatric effects of GLP-1R, providing a framework for personalized metabolic therapy that addresses both physiological and affective health. Results GLP-1R activation improves anxiety/depression-like behaviors in diabetic and over-weight mice Given the established efficacy of GLP-1RAs in type 2 diabetes and obesity management 21 , we first evaluated the impact of GLP-1R activation on affective behaviors in metabolically impaired mouse models. As our previously reported 12 , db/db mice, a model of type 2 diabetes 22 , exhibited significant anxiety and depression-like behaviors. These included reduced center distance and time in the open field test (OFT; S-Fig. 1A-1D), decreased open-arm time and increased closed-arm time in the elevated plus maze (EPM; S-Fig. 1E-H), and prolonged immobility in the tail suspension test (TST) and forced swimming test (FST; S-Fig. 1I and 1J). Treatment with GLP-1R agonist Exendin-4 (Ex4) significantly reversed these behavioral deficits, increasing center and open-arm exploration while reducing immobility (S-Fig. 1B, 1F, 1I and 1J), suggesting an anxiolytic and antidepressant-like effect of GLP-1R activation in the diabetic mice. We next examined high-fat diet (HFD)-induced overweight (OW) mice 23 , which showed a significantly elevated Lee's index (S-Fig.1L) without hyperglycemia (S-Fig.1M). These mice exhibited anxiety-like behaviors in OFT and EPM (S-Fig.1N-1S) but not depression-like behavior in TST or FST (S-Fig.1T and 1U). Ex4 administration ameliorated anxiety-like behaviors in OW mice without affecting normoglycemia (S-Fig.1M-1U). GLP-1R activation induces anxiety/depression-like behaviors in normal-weight mice We next asked whether GLP-1R activation influences affective behaviors in normal-weight mice. Using a time-gradient Ex4 regimen (5 μg/kg/day for 1, 3, 5, and 7 days) 24 , we found that 3-day treatment produced the most consistent anxiety/depression-like phenotype: reduced center activity in OFT (Fig. 1A-1D) , decreased open-arm and increased closed-arm time in EPM (Fig.1E-1H), and prolonged immobility in TST and FST (Fig.1I and 1J). A subsequent dose-response study identified 5 μg/kg/day for 3 days as the optimal regimen (Fig.1K-1T; S-Fig.2A-2H), with no effect on blood glucose (S-Fig.2I). To confirm that these effects were GLP-1R-specific and compound-generalizable, we treated normal-weight mice with semaglutide (SMG, 0.1 mg/kg/day) 25 , another GLP-1RA for 1, 3, 5 or 7 days. Semaglutide also induced anxiety/depression- like behaviors, particularly after 7 days of treatment (S-Fig.3A-3H), without affecting glycemia (S-Fig.3I). Furthermore, Ex4 failed to elicit such behaviors in GLP-1R knockout mice (GLP-1R⁻/⁻). Collectively, these data demonstrate that GLP-1R activation is sufficient to induce affective deficits in normal-weight mice, an effect that is receptor-dependent and not secondary to glycemic changes. TTR is downregulated in the hippocampus of over-weight mice after GLP-1R activation To explore the molecular mechanisms of GLP-1R activation-induced anxiety/depression-like behaviors in normal-weight mice, we performed RNA-sequencing (RNA-seq) on hippocampal tissue 12 , in which GLP-1R expression is highest in this region among affective brain regions. RNA-seq revealed that Ex4 treatment (5 μg/kg/day for 3 days) induced the downregulation of 32 genes and upregulation of 5 genes in the hippocampus (Fig.2A). Among these differentially expressed genes (DEGs), TTR stood out due to its substantial fold change and stable expression profile (Fig.2A and 2B). TTR was further prioritized as a core candidate based on existing evidence linking it to central nervous system (CNS) disorders and its established role in neuronal and synaptic growth 26,27,28,29 . Subsequent validation by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry (IHC) sonsistently confirmed that Ex4 remarkably reduced TTR mRNA and protein levels in the hippocampus (Fig.2C-F). Together, these results suggest that TTR may act as a key downstream mediator of GLP-1R-triggered affective deficits in normal-weight mice. GLP-1R activation transiently reduces serum TTR levels in mice and humans To investigate the relevance of TTR in GLP-1R -mediated affective behaviors, we measured circulating TTR levels in both mice and humans following GLP-1R activation. In normal-weight mice, Ex4 treatment (5 μg/kg/day) led to a transient reduction in serum TTR, with levels significantly decreased after 3 and 5 days of treatment but returning to baseline by day 7 (Fig.3A). We next asked whether TTR fluctuations were behaviorally relevant. Correlation analyses in Ex4-treated normal-weight mice revealed that lower serum TTR levels were positively correlated with affective deficit as shown by decreased time spent in central area of OFT and in open arms of EPM (Fig.3B), and increased immobility time in TST and FST (Fig.3C). We then extended these findings to humans by analyzing serum TTR in obese individuals before and after treatment with the GLP-1R agonist semaglutide (Fig.3D). ELISA results revealed a marked decrease in serum TTR levels 1 month after semaglutide administration (Fig.3E), whereas serum TTR levels returned to pre-treatment baseline after 2- and 3- months continuous treatment (Fig.3F and 3G). These cross-species data indicate that GLP-1R activation induces a transient reduction in circulating TTR, and that TTR fluctuations correlate with affective behaviors in normal-weight mice, supporting its potential role as a translatable biomarker in GLP-1RA-mediated affective regulation. TTR is required for GLP-1R-mediated affective deficits in normal-weight mice To determine whether TTR is required for GLP-1R-induced anxiety/depressive-like behaviors, we performed local hippocampal injections of eplontersen, a TTR-targeting antisense oligonucleotide, in normal-weight mice. Eplontersen treatment (10 μg/kg, 1 μl/side, 3 days) effectively reduced TTR mRNA and protein levels in the hippocampus (Fig.4A-4C). In OFT, eplontersen-treated mice showed obvious anxiety/depression-like phenotypes, including reduced center distance and time in OFT (Fig.4D-4F), decreased open-arm exploration and increased closed-arm time in EPM (Fig.4G-4I), and increased immobility in TST and FST (Fig.4J and 4K), recapitulating the effects of Ex4. We next asked whether restoring TTR could rescue Ex4-induced behavioral deficits. Co-administration of recombinant TTR (reTTR, 5 μg/kg/day, 3 days) via tail vein injection (Fig.4L) prevented the Ex4-induced downregulation of hippocampal TTR protein (Fig.4M). Accordingly, reTTR fully reversed the anxiety/depression- like behaviors triggered by Ex4 across all behavioral tests (Fig.4N-4U). Collectively, these loss- and gain-of-function experiments demonstrate that TTR is both necessary and sufficient for mediating the affective impairments induced by GLP-1R activation in normal-weight mice. Metabolic state dictates the bidirectional role of TTR in GLP-1R–mediated affective behaviors Next, we asked whether TTR contributes to the affective improvement observed after GLP-1R activation in diabetic and OW mice. The results of western blot showed that TTR levels of db/db mice had no difference in the hippocampus and serum compared to wild-type (WT) mice, and Ex4 treatment did not alter TTR levels in this model (Fig.5A and 5B). In contrast, OW mice exhibited markedly elevated TTR levels in both the hippocampus and serum, and Ex4 treatment effectively normalized TTR levels (Fig.5C and 5D). We further analyzed the behavioral relevance of TTR dynamics in OW mice before and after Ex4 treatment. Serum TTR levels were negatively correlated with the time spent in the center of OFT and in the open arms of EPM before Ex4 treatment (Fig.5E), but the relevance was not obvious after Ex4 treatment (Fig.5F), suggesting that Ex4 attenuates anxiety/depression-like behaviors in OW mice by reducing pathologically elevated TTR. To confirm the causal role of elevated TTR in promoting anxiety/depression-like behaviors, we administered reTTR (5 μg/kg/day, 3 days) to normal-weight mice. reTTR treatment significantly induced anxiety/depression-like phenotypes across behavioral tests (Fig.5G-5N). Together, these results establish TTR as a bidirectional modulator of affective behavior, wherein its levels must be maintained within a physiological range. In OW mice, GLP-1R activation normalizes pathologically elevated TTR to produce anxiolytic effects, whereas in normal-weight mice, the same intervention suppresses physiological TTR levels, thereby leading to affective deficits. GLP-1R activation represses TTR expression through the cAMP/PKA/Gli3 transcriptional pathway To elucidate the mechanism by which GLP-1R activation downregulates TTR expression, we first used the UCSC Genome Browser database to perform bioinformatic screening of transcription factors (TFs) predicted to bind the TTR promoter ( S-Fig. 5). From the top 20 candidate transcription factors based on prediction scores (Fig.6A), Gli3 emerged as the only TF meeting all relevant criteria: functional association with GLP-1R signaling, documented repressor activity, and expression in the hippocampus 30,31,32 . We then used the JASPAR database to predict potential Gli3-binding sites within the TTR promoter (Fig.6B), and chromatin immunoprecipitation (ChIP) confirmed specific Gli3 binding to all three predicted sites, with the strongest enrichment observed at site 2 (Fig.6C). Since Gli3 can be phosphorylated by PKA, a cAMP-dependent kinase, to become a transcriptional repressor 33 , and given that cAMP/PKA is a canonical downstream pathway of GLP-1R 34 , we hypothesized that GLP-1R activation suppresses TTR via cAMP/PKA/Gli3 signaling. Consistent with this, Ex4 treatment significantly increased cAMP levels (Fig.6D) and upregulated both phosphorylated PKA (P-PKA) and total PKA, along with transcriptionally inhibitory Gli3 fragments (Fig.6E and 6F). To functionally link this pathway to the observed behavioral outcomes, we co-administered PKA inhibitor H-89 (10 mg/kg, I.P.) with Ex4 in normal-weight mice. H-89 abolished Ex4-induced anxiety/depression-like behaviors (Fig.6G-6N) and blocked PKA/Gli3 activation and TTR downregulation (S-Fig.6). These data indicate that GLP-1R activation drives affective deficits in normal-weight mice through a cAMP/PKA/Gli3-dependent repression of TTR. TTR regulates affective behaviors through the ERK/CREB signaling pathway Subsequently, we sought to identify the downstream signaling mechanism by which TTR modulates anxiety and depression-like behaviors. Based on previous reports that TTR promotes neurite outgrowth and neuroprotection via ERK/AKT/CREB signaling 18 , we examined whether this pathway is involved in the behavioral effects of GLP-1R activation. Western blot analysis revealed that Ex4 administration significantly reduced both phosphorylation and total protein levels of ERK and CREB but not AKT in the hippocampus of normal-weight mice (Fig.7A-7G). IHC further demonstrated a marked decrease in P-ERK and P-CREB immunoreactivity after Ex4 injection (Fig.7H-J). To determine whether these signaling changes were TTR-dependent, we assessed the effects of TTR knockdown and restoration. Eplontersen-mediated TTR inhibition similarly reduced P-ERK/ERK and P-CREB/CREB levels without affecting P-AKT/AKT levels in normal-weight mice (S-Fig.7A-7G). Conversely, reTTR supplementation reversed the Ex4-induced suppression of ERK and CREB signaling (Fig.7K-7Q). Furthermore, PKA inhibition by H-89 prevented Ex4-induced decreases in P-ERK and P-CREB (S-Fig.7H-7M), placing ERK/CREB downstream of the cAMP/PKA/Gli3/TTR axis. Collectively, these results imply that GLP-1R activation impairs ERK/CREB signaling via TTR downregulation, establishing this pathway as a key effector mechanism in the development of affective deficits. CREB activation rescues synaptic and affective deficits induced by GLP-1R signaling Given the established role of CREB in anxiety/depression regulation 35,36,37 , we asked whether its activation could counteract the affective deficits induced by GLP-1R activation. We co-administered Ex4 with CREB agonist 3',6-disinapoylsucrose (DIS, 10 mg/kg, i.p.) in normal-weight mice. DIS treatment significantly ameliorated Ex4-induced anxiety/depression-like behaviors, as evidenced by increased center time and distance in OFT (Fig.8A-8D), increased open-arm exploration and reduced closed-arm time in EPM (Fig.8E-8H), and decreased immobility in TST and FST (Fig.8I and 8J). Since CREB regulates synaptic plasticity by modulating the expression of genes involved in synaptic structure and function 38 , we next examined two important synaptic plasticity markers, PSD95 (a core scaffolding protein in the postsynaptic density) and Drebrin (an actin-binding protein regulating synaptic morphological remodeling). DIS administration reversed the Ex4-induced downregulation of PSD95 and Drebrin (Fig. 8K-8M). We further assessed glutamate receptor expression and phosphorylation, given their importance in synaptic transmission. DIS treatment abolished Ex4-induced reductions in total protein levels of GluA1 and GluN2B, as well as their phosphorylation at key residues (Ser831 and Ser845 for GluA1; Ser1303 and Thr1472 for GluN2B; Fig. 8N-8T). These data further confirm that TTR mediates GLP-1R-induced affective deficits by suppressing CREB signaling, and demonstrate that CREB activation restores synaptic protein expression and affective behaviors, highlighting its key role of CREB pathway balance in affective regulation. Discussion The expanding clinical use of GLP-1RAs has uncovered a paradox in their affective effects, ranging from mood improvement to the induction of anxiety or depression 7,39,40,41,42 . Proposed mechanisms for these disturbances, such as neurotransmitter imbalance and HPA axis dysregulation 43,44,45 , along with variable risks of suicidal ideation noted in observational studies, have remained fragmented. A unifying framework explaining these bidirectional affective outcomes has been lacking. Our work resolves this gap by identifying TTR as the central, metabolic-state-dependent molecular switch, and delineating the dedicated signaling axis underlying this regulation. Our core finding is that TTR operates within a narrow physiological “functional window” essential for affective homeostasis. In normal-weight mice, endogenous TTR sustains hippocampal ERK/CREB signaling and preserves synaptic integrity. GLP-1R activation engages the cAMP/PKA/Gli3 cascade to transcriptionally suppress TTR, driving its level below this functional threshold. This suppression of TTR leads to impaired ERK/CREB activity, reduced expression of synaptic proteins such as PSD95, Drebrin, and key glutamate receptors, and ultimately precipitates affective deficits. Conversely, in diet-induced obese mice with pathologically elevated TTR, the same GLP-1RAs intervention normalizes TTR levels, thereby restoring downstream signaling and alleviating anxiety-like behavior. This establishes a unifying principle: the behavioral efficacy of GLP-1RAs correlates with the normalization of TTR levels toward its physiological set-point. The causal role of TTR within this axis is rigorously validated by multiple experimental approaches: the PKA inhibitor H-89 blocks GLP-1RA-induced affective disturbances; hippocampal TTR knockdown recapitulates the phenotype; and recombinant TTR supplementation fully rescues the synaptic and behavioral impairments. Notably, our study bridges two previously disconnected fields: the neuroprotective roles of TTR in processes like amyloid clearance 46,47,48 , and the multifaceted central nervous system effects of GLP-1R signaling via pathways such as cAMP/PKA and PI3K/Akt 49,50 . We fill this critical gap by delineating a complete and dedicated GLP-1R/cAMP/PKA/Gli3/TTR/ERK/CREB axis. This pathway is distinct from the canonical metabolic branches of GLP-1R signaling, such as PI3K/Akt/mTOR that primarily govern glycemic control and β-cell survival. A key mechanistic novelty is the identification of Gli3 as the critical transcription factor linking GLP-1R activation to TTR repression. GLP-1R activation elevates hippocampal cAMP levels, triggering PKA-dependent phosphorylation of Gli3, which then enhancing its binding to the TTR promoter and suppressing TTR transcription. This specific mode of transcriptional regulation, linking GLP-1R signaling to TTR repression via Gli3, represents a novel finding not previously reported in the contexts of either GLP-1R or TTR biology. The pathway is highly specific: pharmacological inhibition of PKA abolishes Gli3 activation, TTR downregulation, and the consequent affective disturbances, without impairing the glucoregulatory function of GLP-1R, as evidenced by unchanged blood glucose levels in treated normal-weight mice. Downstream, the pathway selectively targets the ERK/CREB module and synaptic protein expression without altering AKT signaling, distinguishing it from the broader neurotrophic effects often associated with TTR. Finally, direct pharmacological activation of CREB rescues both synaptic deficits and the affective impairments induced by GLP-1RA, confirming CREB as the key downstream effector of TTR in affective regulation. This aligns with CREB’s well-documented role as a master regulator of mood-related synaptic plasticity 51,52,53 . The most immediate translational implication is the identification of serum TTR as a tractable pretreatment biomarker for personalized therapy. We propose a stratified clinical strategy: patients with elevated baseline TTR should receive GLP-1RAs therapy to achieve dual metabolic and affective benefits via TTR normalization. In contrast, individuals with physiological TTR levels should either avoid GLP-1RAs or co-administer a TTR stabilizer to prevent TTR suppression and mood risk, with serum TTR monitoring to track dynamic changes. This paradigm shifts the clinical approach from reactive side-effect management toward proactive, precision neuro-metabolic care. Our study has limitations that guide future research. The supportive human correlation data require validation in large-scale prospective trials to confirm the predictive value of TTR. The precise molecular interface linking TTR to the modulation of ERK/CREB activity warrants further elucidation, as do the potential cell-type-specific functions of TTR within the hippocampus. Furthermore, our findings open several transformative questions: Can pharmacological modulators of TTR be used as adjuvants to optimize the therapeutic window of GLP-1RAs? Is the TTR functional window a generalizable mechanism for affective comorbidity in other systemic diseases? Exploring these avenues will deepen our understanding of the metabolic-brain axis. In conclusion, we resolve the clinical paradox of GLP-1RAs by establishing TTR as a metabolic state-dependent switch and defining the TTR functional window concept. We provide a comprehensive mechanistic pathway from receptor to behavioral manifestation and, most importantly, deliver a practical biomarker strategy to guide personalized therapy. This work reframes affective alterations from unpredictable liabilities into mechanistically grounded and potentially predictable outcomes, thereby laying a solid foundation for the practice of precision neuro-metabolic medicine. Materials and Methods Animals Eight-week-old male C57BL/6J mice were provided by the Experimental Animal Center of the Fourth Military Medical University. GLP-1R⁻/⁻ mice were obtained from Beijing Viewsolid Biotechnology Co., Ltd., with WT littermates serving as controls. Six-week-old male db/db mice (BKS-Leprem2cd479/Gpt) and non-diabetic control mice (same strain, 19–38 g) were purchased from Chengdu GemPharmatech Co., Ltd. All mice were housed in plastic cages with ad libitum access to standard chow and water, in a colony room maintained under controlled environmental conditions: temperature (22–26°C), relative humidity (55–60%), and a 12-h light/dark cycle. Prior to the initiation of experimental procedures, the animals were acclimated to the laboratory setting for a minimum of one week. All mice were randomly assigned to experimental groups, and investigators were blinded to the grouping information. The sample size of experimental animals was minimized as much as reasonably possible, while ensuring adequate statistical power and meeting the requirements of the experimental design. All experimental protocols were approved by the Animal Care and Use Committee of the Fourth Military Medical University (No. 20240015). Materials Exendin-4 (Ex4; No. A3408, purity ≥ 99%) was purchased from APExBIO Technology LLC (Houston, TX, USA). Semaglutide (SMG, purity ≥ 95%) was synthesized by Anwante Biotechnology Co.,Ltd (Shanghai, China). Eplontersen, recombinant TTR (reTTR), 3’,6-Disinapoylsucrose (DIS) and H-89 were obtained from MedChemExpress (Monmouth Junction, NJ, USA). TTR (ZC-35797, ZC-57833) ELISA kits were purchased from ZCIBIO Technology Co.,Ltd (shanghai, China). The SYBR Premix Ex TaqII and PrimeScript RT reagent kits were obtained from Yeasen (Shanghai, China). For western blotting and IHC, the following antibodies were used: β-Actin (No.66009-1-Ig, Proteintech, Wuhan, China), TTR (No.PA5-67636, AntiProtech, S.F, CA, USA) for western blotting, P-ERK (No.9101S, Cell Signaling Technology, Boston, MA, USA), ERK (No.9102S, Cell Signaling Technology), P-AKT (No.4060, Cell Signaling Technology), AKT (No.4691, Cell Signaling Technology), P-CREB (No.9198S, Cell Signaling Technology), CREB (No.9197S, Cell Signaling Technology), PSD95 (No.ab2723, Abcam, Cambridge, UK), Drebrin (No.ab178408, Abcam), GluA1 (No.13185S, Cell Signaling Technology), P-GluA1-S845 (No.8084S, Cell Signaling Technology), P-GluA1-S831 (No.75574S, Cell Signaling Technology), GluN2B (No. 4207, Cell Signaling Technology), P-GluN2B-S1303 (No. 71335, Cell Signaling Technology), P-GluN2B-T1472 (No. 90125, Cell Signaling Technology), P-PKA (No.CY9083, Abways, Shanghai, China), PKA (No.GB11598-100, Servicebio, Wuhan, China), Gli3 (No.Ab307714, Abcam), TTR (No.EPR20971, Abcam) for IHC. Secondary antibodies, including goat anti-mouse IgG-HRP (No. EK010) and goat anti-rabbit IgG-HRP (No. EK020), were purchased from Zhuangzhibio (Xi’an, USA) Collection of human blood samples Venous blood samples were collected from adult obese participants with a body mass index (BMI) ranging from 29 to 45 kg/m² before SMG administration, and separately at 1 month, 2 months, or 3 months after administration, with each time point assigned to different subgroups of participants. The blood samples were directly centrifuged at 2,000×g for 10 minutes at 4°C, and the separated serum was used for the detection of TTR levels by enzyme-linked immunosorbent assay (ELISA). The study protocol received ethical approval from the Medical Ethics Committee of the Xijing Hospital of Fourth Military Medical University (Approval No. KY20252243-F-1) and was prospectively registered with the Chinese Clinical Trial Registry (Registration No. ChiCTR 2500104257. https://www.chictr.org.cn/showproj.html?proj=275079). All participants provided written informed consent. The investigation was conducted in full accordance with the ethical principles outlined in the Declaration of Helsinki. Stereotaxic intracerebral delivery Mice were fixed in a stereotaxic frame (RWD68001, Shenzhen RWD Life Science, Shenzhen, China). A double cannula (guide cannula, length 7.5 mm, internal diameter 0.34 mm, external diameter 0.48 mm, center to center distance 2.6 mm) was aimed at 0.5 mm above the intended sites of injection, namely the hippocampus (AP: -2.06 mm; ML: ±1.3 mm; DV: -1.5 mm). The guide cannulas were affixed to the skull with machine screws and dental cement, and stylet was inserted into the cannula to keep them unimpeded. Mice were used for following experiments after one-week recovery. For the injection of Eplontersen (10 μg/kg), mice were anesthetized with isoflurane (3-4% for induced anesthesia and 1-1.5% for maintained anesthesia). Eplontersen was microinjected into the hippocampus using a Hamilton syringe (10 μl) connected to a glass capillary tip, with a total volume of 1μl per side, administered over three consecutive days. Tail vein injection Mice were secured in a restraint device with tails exposed. Bilateral lateral tail veins were visualized, and the more distinct vein was selected as the injection site. The injection area was disinfected using a 75% ethanol swab. reTTR (5 μg/kg) was administered via a 1-mL syringe equipped with a 26G needle. Following injection, the puncture site was compressed with a dry cotton ball for 10–15 seconds to prevent hemorrhage. The administration was performed over three consecutive days. Open field test (OFT) The OFT served to assess anxiety-like behavior in mice, as previously reported 54 . The apparatus was a transparent Plexiglas square arena (30×30×30 cm). Mice were gently tail-held and placed head-outward at the center of the arena (15×15 cm central zone, 25% of total area), with video recording initiated immediately. Animals were permitted free exploration for 10 min. Behavior was recorded by a ceiling-mounted camera, and trajectory data were analyzed using a video-tracking system (DigBehv-LR 4, Shanghai Jiliang, China). The arena was thoroughly cleaned between trials to eliminate odor interference. Elevated plus maze (EPM) The EPM test was performed to further assess anxiety-like behavior in mice 55 . The apparatus consisted of a black Plexiglas cross-shaped platform (50–60 cm in height) with two open arms (25 × 8 × 0.5 cm, unenclosed) and two closed arms (25 × 8 × 0.5 cm, enclosed by 15 cm walls), intersecting at a central zone (8 × 8 cm). Mice were placed at the junction of the central platform facing an open arm, and behavior was recorded for 5 minutes using a ceiling-mounted camera. A video-tracking system (DigBehv-LR 4, Shanghai Jiliang, Shanghai, China) was employed to analyze metrics. The maze was thoroughly cleaned with 75% ethanol after each trial to eliminate odor interference. Tail suspension test (TST) The TST apparatus typically consists of a stand and clamping device, with anti-climbing barriers surrounding the setup. Mice were suspended by their tails using adhesive tape from a horizontal bar for 6 minutes, and behavior was recorded via a ceiling-mounted camera. The cumulative duration of complete immobility (defined as no limb movement except minor forepaw adjustments) during the final 4 minutes of the test was quantified. Forced swimming test (FST) The FST apparatus consisted of a transparent cylindrical tank (15 cm in diameter) filled with water to a depth of 20 cm (23-25℃). The water surface was positioned 10 cm below the tank's top edge to prevent climbing or escape. Mice were gently placed into the water for 6 minutes, and behavior was recorded via a video-tracking system. The cumulative duration of immobility (defined as minimal limb movement beyond passive floating) during the final 4 minutes of the test was quantified. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted using the RNA Easy Fast Tissue Kit (DP451, TIANGEN) following the manufacturer’s protocol. cDNA was synthesized from purified RNA with the FastKing cDNA Synthesis Kit (KR116, TIANGEN). The qPCR was conducted using RealUniversal Color PreMix (FP201, TIANGEN) and SYBR Premix Ex Taq II (TaKaRa) following recommended thermal cycling parameters. The sequences of the primers are listed as follows (Sangon Biological Engineering Technology, Shanghai, China): TTR, forward 5′-TCACCAGGAGAAGCCGTCACAC-3′ and reverse 5′-AAATACCAGTCCAGCGAGGCAAAG-3′; β-Actin, forward 5′-ACTGTCGAGTCGCGTCC-3′ and reverse 5′-CTGACCCATTCCCACCATCA-3′. β-Actin served as the reference gene for normalization, and transcript expression levels were quantified via the 2− ΔΔCt method. Western blot analysis Western blot analysis was performed as our previous report 56 . The hippocampus tissue was dissociated via sonication in RIPA lysis buffer containing phosphatase and protease inhibitors. The protein content of the collected samples was quantified using the BCA Protein Assay Kit. Equal amounts of protein (30 μg) were separated on SDS-PAGE gels then electro-transferred to PVDF membranes (Invitrogen, Thermo Fisher Scientific Inc., Waltham, USA). The membranes were in turn probed with primary antibodies overnight at 4°C after incubation for 1.5 h in 5% non-fat milk.β-Actin (1:1,000), TTR (1:1,000), P-ERK (1:1,000), ERK (1:1,000), P-AKT (1:1,000), AKT (1:1,000), P-CREB (1:1,000), CREB (1:1,000), PSD95 (1:1,000), Drebrin (1:1,000), GluA1 (1:1,000), P-GluA1-S845 (1:1,000), P-GluA1-S831 (1:1,000), GluN2B (1:1,000), P-GluN2B-S1303 (1:1,000), P-GluN2B-T1472 (1:1,000), P-PKA (1:1,000), PKA (1:1,000), Gli3 (1:1,000). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies at room temperature (RT) for 1 hour. Densitometric analysis of Western blot bands was conducted using a ChemiDoc XRS imaging system (Bio-Rad, Hercules, CA, USA) and subsequently quantified by ImageJ software (NIH, Bethesda, MD, USA) in accordance with the manufacturers' protocols. Band intensities for each sample were calculated as relative ratios normalized to the housekeeping protein β-actin. The relative intensity ratio of the control group was set to 100%, while the ratios of other experimental groups were expressed as percentages relative to the control group. RNA sequencing RNA-Seq was conducted by Beijing Biomarker Technologies Co., Ltd. Briefly, poly(A) RNA was isolated from 1 μg of total RNA using Dynabeads Oligo (25-61005, Thermo Fisher Scientific). Subsequently, the poly(A) RNA was sheared into short fragments with a Magnesium RNA Fragmentation Module (m6150, NEB) at 94°C for 5–7 minutes. The sheared RNA fragments were reverse-transcribed into complementary DNA (cDNA) using SuperScript™ II Reverse Transcriptase (1896649, Invitrogen). The resulting cDNA was purified following the enzymatic reactions, and library size selection was conducted with AMPure XP beads. Following treatment of U-labeled second-stranded DNA with heat-labile UDG enzyme (m0280, NEB), the resulting ligated products were subjected to PCR amplification. The final cDNA library had an average insert size of 300 ± 50 bp. Paired-end sequencing (PE150, 2 × 150 bp) was carried out on an Illumina NovaSeq™ 6000 platform (LC-Bio) in accordance with the manufacturer’s recommended protocol. HISAT2 ( https://ccb.jhu.edu/software/hisat2 ) was employed for aligning sequencing reads to the Mus sapiens GRCm38 reference genome. Subsequent to transcriptome assembly, StringTie was utilized to quantify the expression levels of all transcripts via computation of the FPKM value (FPKM = [total exon fragments / (mapped reads in millions × exon length in kB)]). Differentially expressed mRNAs (DE-mRNAs) were defined as those with a fold change (FC) > 1.5 or FC < 0.05 and a P-value < 0.05.Post-sequencing, DEGs analysis was performed by the BMK Cloud bioinformatics pipeline ( www.biocloud.net ). ELISA and glucose test Serum TTR levels in mice were quantified using a species-specific ELISA kit (ZC-57833) in accordance with the manufacturer’s protocols. Briefly, mice were anesthetized with diethyl ether, and intraorbital blood collection was performed using ophthalmic forceps to puncture the retro-orbital venous plexus. Approximately 0.5 mL of blood per mouse was collected into 1.5 mL centrifuge tubes, with the entire collection process completed within 1 minute. Subsequently, the blood samples were centrifuged at 1000 ×g and 4°C for 20 minutes, and the serum supernatant was harvested for subsequent ELISA analysis. For the quantification of TTR in human serum samples, a human-specific ELISA kit (ZC-35797) was employed, with all experimental procedures strictly adhering to the manufacturer’s guidelines. Blood glucose concentrations were measured using glucose test strips (Roche Diagnostics, Shanghai, China) in compliance with the product instructions. Prediction of the Gli3 binding sites in the promoter of TTR Since the sequences of the TTR and Gli3 genes in mice and humans exhibit sufficient similarity and show high homology, we used human gene sequences for prediction 30,57 . The human TTR promoter sequences were obtained from the UCSC database ( http://genome.ucsc.edu/ ). The region from 2000 bp upstream and 100 bp downstream of the transcriptional start site was considered the promoter region. The transcript IDs of human TTR was Human hg19 chr18:20797307-20798306, respectively. The Gli3 binding motif was retrieved from the JASPAR database ( http://jaspar.genereg.net/ ). The TTR promoter region was input into the JASPAR database to predict potential Gli3-binding sites, which were then ranked based on their scores. The top 3 binding sites were selected for ChIP validation. ChIP ChIP was conducted by Beijing Tsingke Biotech Co., Ltd. Briefly, DNA-protein crosslinking was achieved by treating cells with 1% formaldehyde at room temperature for 10 minutes. Cross-linked chromatin was subsequently sonicated in the lysis buffer provided with the kit, and soluble DNA fragments were collected following centrifugation at 20,000 ×g for 10 minutes. The resulting supernatants were used for ChIP assays. Protein A/G agarose beads conjugated with anti-Gli3 antibody or normal rabbit IgG (as a negative control) were added to the supernatants and incubated at 4°C for 4 hours. After incubation, the beads were washed twice using the kit-supplied wash buffer. Bound DNA was then eluted with elution buffer, decrosslinked at 65°C overnight, purified through phenol-chloroform-isoamyl alcohol extraction followed by ethanol precipitation, and subjected to qPCR. Following amplification, the PCR amplicons were resolved on a 2.5% ethidium bromide-stained agarose gel. IHC IHC was conducted by Hubei Bios Biological Technology Co., Ltd. Paraffin sections were sequentially dewaxed in environmental dewaxing agents, hydrated through a graded ethanol series, and rinsed with distilled water. Antigen retrieval was then performed via high-pressure heat treatment using EDTA (pH 9.0), with timing set to 1.5 minutes after the onset of steam. After retrieval, endogenous peroxidase activity was blocked with 3% H₂O₂ for 30 minutes at room temperature in the dark, followed by section blocking with 10% species-matched serum (30 minutes at room temperature). Primary antibodies—P-ERK (1:100), P-CREB (1:100), and TTR (1:200)—diluted in 10% serum were added, and sections were incubated overnight at 4°C. After rewarming to room temperature and washing with TBST, TBST-diluted secondary antibody was added for incubation at 37 °C for 45 minutes, with additional TBST washes thereafter. Color development was conducted using either DAB or a red chromogenic system (including TY reaction solution and red chromogenic working solution); the reaction duration was monitored microscopically, and color development was terminated by tap water rinsing. Sections were counterstained with hematoxylin for 1 minute, differentiated in acid alcohol, and blued, then mounted with an environmental mounting medium. After air-drying, slides were examined under a microscope for image acquisition and analysis. Statistics All statistical analyses were performed by blinded operators with respect to experimental conditions, and data analysis was conducted using GraphPad Prism software (version 8.02). Results are presented as mean ± SEM. For comparisons between two groups, independent samples t-tests (two-tailed) were applied. For multi-group comparisons, statistical differences were evaluated using one-way analysis of variance (ANOVA) followed by Tukey's posthoc test for pairwise comparisons. Statistical significance was defined as P < 0.05. Declarations Acknowledgments This work was supported by Scientific and Technological Innovation Team of Shaanxi Province (2023-CX-TD-63), Technology Innovation Talent Engineering Project (2023RCZZ003), Science and Technology Research Projects (2024GJJH03-02), National Natural Science Foundation of China (82571721, 82571359, 82404591). Author information Authors and Affiliations State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture, Department of pharmacy, The Third Affiliated Hospital of the Fourth Military Medical University, 710032, Xi’an, China Beining Lu, Yongli Jiang, Qingjuan Guo, Yuxuan Yan, Liukun Yang, Dake Song, Cheng Zeng, Jiao Yue, Chen Zeng, Zixuan Liu, Xinshang Wang, Shuibing Liu. Department of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an, 710032, China Beining Lu, Yongli Jiang, Fan Yang, Yuxuan Yan, Dake Song, Zixuan Liu, Xue Ma, Xinshang Wang, Shuibing Liu. Military Medical Innovation Center, Fourth Military Medical University, Xi’an 710032, China. Xinshang Wang. Department of Endocrinology and Metabolism, Xijing Hospital of The Fourth Military Medical University Jie Zhou, Nana Zhang Corresponding author Correspondence to Xue Ma, Xinshang Wang, Shuibing Liu. Ethics declarations Competing interests The authors have declared that no conflict of interest exists. Study approval The animal experimental procedures were approved by the Animal Care and Use Committee of the Fourth Military Medical University (No. 20240015). And the human study protocol received ethical approval from the Medical Ethics Committee of the Xijing Hospital of Fourth Military Medical University (Approval No. KY20252243-F-1) and was prospectively registered with the Chinese Clinical Trial Registry (Registration No. ChiCTR 2500104257. https://www.chictr.org.cn/showproj.html?proj=275079). All participants provided written informed consent. The investigation was conducted in full accordance with the ethical principles outlined in the Declaration of Helsinki. References Collaborators, G. B. D. A. B. Global, regional, and national prevalence of adult overweight and obesity, 1990-2021, with forecasts to 2050: a forecasting study for the Global Burden of Disease Study 2021. Lancet 405 , 813-838, doi:10.1016/S0140-6736(25)00355-1 (2025). Fanelli, G. et al. The interface of depression and diabetes: treatment considerations. Transl Psychiatry 15 , 22, doi:10.1038/s41398-025-03234-5 (2025). Drucker, D. J. Author Correction: GLP-1-based therapies for diabetes, obesity and beyond. Nat Rev Drug Discov 24 , 570, doi:10.1038/s41573-025-01231-3 (2025). Drucker, D. J. The benefits of GLP-1 drugs beyond obesity. Science 385 , 258-260, doi:10.1126/science.adn4128 (2024). Li, S., Sabbah, S. G., Kwan, A. T. H. & McIntyre, R. S. Repurposing glucagon-like peptide-1 (GLP-1) receptor agonists for the treatment of depression: A systematic review of preclinical, observational and clinical investigations. Eur Neuropsychopharmacol 99 , 56-67, doi:10.1016/j.euroneuro.2025.08.002 (2025). Llach, C. D. et al. Glucagon-like Peptide-1 receptor agonists as emerging therapeutics in bipolar disorder: a narrative review of preclinical and clinical evidence. Mol Psychiatry , doi:10.1038/s41380-025-03261-0 (2025). Wang, M. et al. Exploring potential associations between GLP-1RAs and depressive disorders: a pharmacovigilance study based on FAERS and VigiBase data. EClinicalMedicine 86 , 103385, doi:10.1016/j.eclinm.2025.103385 (2025). Ueda, P. et al. GLP-1 Receptor Agonist Use and Risk of Suicide Death. JAMA Intern Med 184 , 1301-1312, doi:10.1001/jamainternmed.2024.4369 (2024). Pierret, A. C. S. et al. Glucagon-Like Peptide 1 Receptor Agonists and Mental Health: A Systematic Review and Meta-Analysis. JAMA Psychiatry 82 , 643-653, doi:10.1001/jamapsychiatry.2025.0679 (2025). Kornelius, E., Huang, J. Y., Lo, S. C., Huang, C. N. & Yang, Y. S. The risk of depression, anxiety, and suicidal behavior in patients with obesity on glucagon like peptide-1 receptor agonist therapy. Sci Rep 14 , 24433, doi:10.1038/s41598-024-75965-2 (2024). Liu, Y. N. et al. A pancreas-hippocampus feedback mechanism regulates circadian changes in depression-related behaviors. Nat Neurosci 28 , 2078-2091, doi:10.1038/s41593-025-02040-y (2025). Yang, F. et al. Glucagon-like Peptide 1 Receptor Activation Inhibits Microglial Pyroptosis via Promoting Mitophagy to Alleviate Depression-like Behaviors in Diabetic Mice. Nutrients 15 , doi:10.3390/nu15010038 (2022). Gil-Lozano, M. et al. Corticotropin-releasing hormone and the sympathoadrenal system are major mediators in the effects of peripherally administered exendin-4 on the hypothalamic-pituitary-adrenal axis of male rats. Endocrinology 155 , 2511-2523, doi:10.1210/en.2013-1718 (2014). Kinzig, K. P. et al. CNS glucagon-like peptide-1 receptors mediate endocrine and anxiety responses to interoceptive and psychogenic stressors. J Neurosci 23 , 6163-6170, doi:10.1523/JNEUROSCI.23-15-06163.2003 (2003). Anderberg, R. H. et al. GLP-1 is both anxiogenic and antidepressant; divergent effects of acute and chronic GLP-1 on emotionality. Psychoneuroendocrinology 65 , 54-66, doi:10.1016/j.psyneuen.2015.11.021 (2016). Zemany, L. et al. Transthyretin Antisense Oligonucleotides Lower Circulating RBP4 Levels and Improve Insulin Sensitivity in Obese Mice. Diabetes 64 , 1603-1614, doi:10.2337/db14-0970 (2015). Singhal, A., Chauhan, A., Goyal, P. & Taneja, A. Transthyretin - A Novel Biomarker for Insulin Resistance and Atherosclerosis Risk in Prediabetics. J Assoc Physicians India 69 , 11-12 (2021). Gomes, J. R. et al. Transthyretin provides trophic support via megalin by promoting neurite outgrowth and neuroprotection in cerebral ischemia. Cell Death Differ 23 , 1749-1764, doi:10.1038/cdd.2016.64 (2016). Zhang, M. et al. Cardiovascular and brain effects of liraglutide in transthyretin amyloidosis (ATTR) mice models. Int J Med Sci 22 , 3229-3241, doi:10.7150/ijms.112264 (2025). Lohia, S. et al. Exploratory Study Analyzing the Urinary Peptidome of T2DM Patients Suggests Changes in ECM but Also Inflammatory and Metabolic Pathways Following GLP-1R Agonist Treatment. Int J Mol Sci 24 , doi:10.3390/ijms241713540 (2023). Nauck, M. A., Quast, D. R., Wefers, J. & Meier, J. J. GLP-1 receptor agonists in the treatment of type 2 diabetes - state-of-the-art. Mol Metab 46 , 101102, doi:10.1016/j.molmet.2020.101102 (2021). Suriano, F. et al. Novel insights into the genetically obese (ob/ob) and diabetic (db/db) mice: two sides of the same coin. Microbiome 9 , 147, doi:10.1186/s40168-021-01097-8 (2021). Ji, A. et al. Whey protein and soy protein prevent obesity by upregulating uncoupling protein 1 to activate brown adipose tissue and promote white adipose tissue browning in high-fat diet-fed mice. Food Funct 13 , 12836-12851, doi:10.1039/d2fo01935c (2022). Chan, Y. L. et al. Short term exendin-4 treatment reduces markers of metabolic disorders in female offspring of obese rat dams. Int J Dev Neurosci 46 , 67-75, doi:10.1016/j.ijdevneu.2015.05.009 (2015). Piening, A. et al. Obesity-related T cell dysfunction impairs immunosurveillance and increases cancer risk. Nat Commun 15 , 2835, doi:10.1038/s41467-024-47359-5 (2024). Fleming, C. E., Saraiva, M. J. & Sousa, M. M. Transthyretin enhances nerve regeneration. J Neurochem 103 , 831-839, doi:10.1111/j.1471-4159.2007.04828.x (2007). Fleming, C. E., Mar, F. M., Franquinho, F., Saraiva, M. J. & Sousa, M. M. Transthyretin internalization by sensory neurons is megalin mediated and necessary for its neuritogenic activity. J Neurosci 29 , 3220-3232, doi:10.1523/JNEUROSCI.6012-08.2009 (2009). Nunes, A. F. et al. Transthyretin knockout mice display decreased susceptibility to AMPA-induced neurodegeneration. Neurochem Int 55 , 454-457, doi:10.1016/j.neuint.2009.07.001 (2009). Fleming, C. E., Nunes, A. F. & Sousa, M. M. Transthyretin: more than meets the eye. Prog Neurobiol 89 , 266-276, doi:10.1016/j.pneurobio.2009.07.007 (2009). Matissek, S. J. & Elsawa, S. F. GLI3: a mediator of genetic diseases, development and cancer. Cell Commun Signal 18 , 54, doi:10.1186/s12964-020-00540-x (2020). Tang, C. et al. Hippo signaling activates hedgehog signaling by Taz-driven Gli3 processing. Cell Regen 12 , 3, doi:10.1186/s13619-022-00151-6 (2023). Zaky, D. A., Sayed, R. H. & Mohamed, Y. S. Liraglutide limits the immunogenic cell death-mediated ROS propagation and PI3K/AKT inactivation after doxorubicin-induced gonadotoxicity in rats: Involvement of the canonical Hedgehog trajectory. Int Immunopharmacol 119 , 110212, doi:10.1016/j.intimp.2023.110212 (2023). Happ, J. T. et al. A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction. Nat Struct Mol Biol 29 , 990-999, doi:10.1038/s41594-022-00838-z (2022). Xia, T., He, W., Luo, Z., Wang, K. & Tan, X. Achyranthes bidentata polysaccharide ameliorates type 2 diabetes mellitus by gut microbiota-derived short-chain fatty acids-induced activation of the GLP-1/GLP-1R/cAMP/PKA/CREB/INS pathway. Int J Biol Macromol 270 , 132256, doi:10.1016/j.ijbiomac.2024.132256 (2024). Blendy, J. A. The role of CREB in depression and antidepressant treatment. Biol Psychiatry 59 , 1144-1150, doi:10.1016/j.biopsych.2005.11.003 (2006). Sulser, F. The role of CREB and other transcription factors in the pharmacotherapy and etiology of depression. Ann Med 34 , 348-356, doi:10.1080/078538902320772106 (2002). Tang, Y. et al. Jiao-tai-wan and its effective component-berberine improve diabetes and depressive disorder through the cAMP/PKA/CREB signaling pathway. J Ethnopharmacol 324 , 117829, doi:10.1016/j.jep.2024.117829 (2024). Dinevska, M. et al. CREB: A multifaceted transcriptional regulator of neural and immune function in CNS tumors. Brain Behav Immun 116 , 140-149, doi:10.1016/j.bbi.2023.12.002 (2024). Wang, W. et al. Association of semaglutide with risk of suicidal ideation in a real-world cohort. Nat Med 30 , 168-176, doi:10.1038/s41591-023-02672-2 (2024). McGowan, B. et al. A systematic review and meta-analysis of the efficacy and safety of pharmacological treatments for obesity in adults. Nat Med 31 , 3317-3329, doi:10.1038/s41591-025-03978-z (2025). Schoretsanitis, G., Weiler, S., Barbui, C., Raschi, E. & Gastaldon, C. Disproportionality Analysis From World Health Organization Data on Semaglutide, Liraglutide, and Suicidality. JAMA Netw Open 7 , e2423385, doi:10.1001/jamanetworkopen.2024.23385 (2024). Shapiro, S. B. et al. Glucagon-like peptide-1 receptor agonists and risk of suicidality among patients with type 2 diabetes: active comparator, new user cohort study. BMJ 388 , e080679, doi:10.1136/bmj-2024-080679 (2025). Kolling, L. J. et al. Interaction of serotonin/GLP-1 circuitry in a dual preclinical model for psychiatric disorders and metabolic dysfunction. Psychiatry Res 337 , 115951, doi:10.1016/j.psychres.2024.115951 (2024). Holt, M. K. et al. Modulation of stress-related behaviour by preproglucagon neurons and hypothalamic projections to the nucleus of the solitary tract. Mol Metab 91 , 102076, doi:10.1016/j.molmet.2024.102076 (2025). Tokgoz, S. et al. Activation of the HPA Axis Does Not Explain Nonresponsiveness to GLP-1R Agonist Treatment in Individuals With Type 2 Diabetes. Diabetes 74 , 212-222, doi:10.2337/db24-0463 (2025). Benson, M. D. et al. Inotersen Treatment for Patients with Hereditary Transthyretin Amyloidosis. N Engl J Med 379 , 22-31, doi:10.1056/NEJMoa1716793 (2018). Adams, D. et al. Hereditary transthyretin amyloid neuropathies: advances in pathophysiology, biomarkers, and treatment. Lancet Neurol 22 , 1061-1074, doi:10.1016/S1474-4422(23)00334-4 (2023). Chompoopong, P., Mauermann, M. L., Siddiqi, H. & Peltier, A. Amyloid Neuropathy: From Pathophysiology to Treatment in Light-Chain Amyloidosis and Hereditary Transthyretin Amyloidosis. Ann Neurol 96 , 423-440, doi:10.1002/ana.26965 (2024). Yang, X. et al. Neuroprotective Mechanisms of Glucagon-Like Peptide-1-Based Therapies in Ischemic Stroke: An Update Based on Preclinical Research. Front Neurol 13 , 844697, doi:10.3389/fneur.2022.844697 (2022). Holscher, C. Protective properties of GLP-1 and associated peptide hormones in neurodegenerative disorders. Br J Pharmacol 179 , 695-714, doi:10.1111/bph.15508 (2022). Li, S. et al. Interleukin-13 and its receptor are synaptic proteins involved in plasticity and neuroprotection. Nat Commun 14 , 200, doi:10.1038/s41467-023-35806-8 (2023). Sakamoto, K., Karelina, K. & Obrietan, K. CREB: a multifaceted regulator of neuronal plasticity and protection. J Neurochem 116 , 1-9, doi:10.1111/j.1471-4159.2010.07080.x (2011). Guan, W., Ni, M. X., Gu, H. J. & Yang, Y. CREB: A Promising Therapeutic Target for Treating Psychiatric Disorders. Curr Neuropharmacol 22 , 2384-2401, doi:10.2174/1570159X22666240206111838 (2024). Jiang, Y. L. et al. Tanshinone IIA improves contextual fear- and anxiety-like behaviors in mice via the CREB/BDNF/TrkB signaling pathway. Phytother Res 36 , 3932-3948, doi:10.1002/ptr.7540 (2022). Wang, X. S. et al. Anxiolytic effects of Formononetin in an inflammatory pain mouse model. Mol Brain 12 , 36, doi:10.1186/s13041-019-0453-4 (2019). Wang, X. S. et al. Activation of G protein-coupled receptor 30 protects neurons by regulating autophagy in astrocytes. Glia 68 , 27-43, doi:10.1002/glia.23697 (2020). Slamova, I. et al. Plasmin activity promotes amyloid deposition in a transgenic model of human transthyretin amyloidosis. Nat Commun 12 , 7112, doi:10.1038/s41467-021-27416-z (2021). Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalFigures.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8322552","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":573838271,"identity":"d9ae428f-1cdf-4a5b-a2a3-aed60d71094c","order_by":0,"name":"Shuibing Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYBACPmYGBmbGBgk5+wOHDzA8MCBCCxtEi4Uxw8FjCQwJRGlhAGupSGw4fMaAIYEYh7Gx8xh/LtwhkdjYdubjh4SCw4nbGZgfPrqB12E8ZtIzz0gYN/Oc3SyRYHA4cWcDm7FxDgEtzLxtErJtEmc3gLVsOMDDJk1Ai/FnoBbGHvk3j38Qq8VAGqhFcQbDGTZibWErk+YF+sWA4ZiZRYJBuvGGwwT8ws9/ePNn3h11cgYMhx/f+PDHWnbD8eaHj/FpQQfNwGgiQTkI1JGofhSMglEwCkYCAAAG8krbBHp7qwAAAABJRU5ErkJggg==","orcid":"","institution":"Fourth military medical university","correspondingAuthor":true,"prefix":"","firstName":"Shuibing","middleName":"","lastName":"Liu","suffix":""},{"id":573838272,"identity":"04472c77-73f5-41fb-9643-0db4239cd5a3","order_by":1,"name":"Beining Lu","email":"","orcid":"","institution":"Department of pharmacy, The Third Affiliated Hospital of the Fourth Military Medical University,","correspondingAuthor":false,"prefix":"","firstName":"Beining","middleName":"","lastName":"Lu","suffix":""},{"id":573838273,"identity":"6ea05112-8f07-4122-8f8e-c0b4dbb6375d","order_by":2,"name":"Yongli Jiang","email":"","orcid":"","institution":"Department of pharmacy, The Third Affiliated Hospital of the Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yongli","middleName":"","lastName":"Jiang","suffix":""},{"id":573838274,"identity":"737afdc0-5055-40fb-bdf5-0954e9198d27","order_by":3,"name":"Jie Zhou","email":"","orcid":"","institution":"Department of Endocrinology and Metabolism, Xijing Hospital of The Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhou","suffix":""},{"id":573838275,"identity":"7f274c1e-b8f3-446b-ab13-393e3a98e4f5","order_by":4,"name":"Nana Zhang","email":"","orcid":"","institution":"Department of Endocrinology and Metabolism, Xijing Hospital of The Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Nana","middleName":"","lastName":"Zhang","suffix":""},{"id":573838276,"identity":"8d15f782-f32b-46c2-b7e9-0201c4bb1242","order_by":5,"name":"Qingjuan Guo","email":"","orcid":"","institution":"Department of pharmacy, The Third Affiliated Hospital of the Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qingjuan","middleName":"","lastName":"Guo","suffix":""},{"id":573838277,"identity":"4e1d2359-8c7d-4cc4-9ea9-3fb665d28bfb","order_by":6,"name":"Fan Yang","email":"","orcid":"","institution":"Department of Pharmacology, School of Pharmacy, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fan","middleName":"","lastName":"Yang","suffix":""},{"id":573838278,"identity":"b1a7c201-6f03-48ea-a0bb-ac6c9f9676de","order_by":7,"name":"Yuxuan Yan","email":"","orcid":"","institution":"Department of pharmacy, The Third Affiliated Hospital of the Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuxuan","middleName":"","lastName":"Yan","suffix":""},{"id":573838279,"identity":"095543f7-d5e3-4fb7-8cff-11e284853942","order_by":8,"name":"Liukun Yang","email":"","orcid":"","institution":"Department of pharmacy, The Third Affiliated Hospital of the Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Liukun","middleName":"","lastName":"Yang","suffix":""},{"id":573838280,"identity":"2a7dd481-df94-4acc-b351-2430f24dab7a","order_by":9,"name":"Dake Song","email":"","orcid":"","institution":"Fourth military medical university","correspondingAuthor":false,"prefix":"","firstName":"Dake","middleName":"","lastName":"Song","suffix":""},{"id":573838281,"identity":"35b18b2c-1eff-4be0-a241-f9f66248ccf3","order_by":10,"name":"Cheng Zeng","email":"","orcid":"","institution":"Department of pharmacy, The Third Affiliated Hospital of the Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Zeng","suffix":""},{"id":573838282,"identity":"0896f271-ac21-40b8-b3d5-e68f37372cef","order_by":11,"name":"Jiao Yue","email":"","orcid":"","institution":"Department of pharmacy, The Third Affiliated Hospital of the Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Yue","suffix":""},{"id":573838283,"identity":"61afdcb9-e4bb-4566-acef-f6e7318712ff","order_by":12,"name":"Yue Chen","email":"","orcid":"","institution":"Department of pharmacy, The Third Affiliated Hospital of the Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Chen","suffix":""},{"id":573838284,"identity":"c83f6f6a-6433-4c90-b53f-e4aeb256b434","order_by":13,"name":"Zixuan Liu","email":"","orcid":"","institution":"Department of Pharmacology, School of Pharmacy, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zixuan","middleName":"","lastName":"Liu","suffix":""},{"id":573838285,"identity":"b6e3a212-a7c1-47ad-b688-c8eee7220e78","order_by":14,"name":"Xue Ma","email":"","orcid":"","institution":"Department of Pharmacology, School of Pharmacy, Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Ma","suffix":""},{"id":573838286,"identity":"c909670c-278e-4313-8ac5-d443c474a59f","order_by":15,"name":"Xinshang Wang","email":"","orcid":"","institution":"Department of pharmacy, The Third Affiliated Hospital of the Fourth Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xinshang","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-12-10 03:30:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8322552/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8322552/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100198210,"identity":"cc8f26d7-9ae3-4436-a3cd-44142ee4aa7b","added_by":"auto","created_at":"2026-01-14 03:57:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3599912,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1R activation by Ex4 promotes depression-like and anxiety-like behaviors in normal-weight mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-D)\u003c/strong\u003e The OFT following repeated Ex4 injection (5μg/kg, days 1/3/5/7). (A) Representative movement traces. (B, C) Ex4 decreased center distance (B) and duration (C), without affecting total locomotion (D). \u0026nbsp;\u003cstrong\u003e(E-H)\u003c/strong\u003e The EPM following repeated Ex4. (E) Representative movement traces. (F-H) Ex4 reduced open arm time (F), increased closed arm time (G), and decreased total entries (H). \u003cstrong\u003e(I, J) \u003c/strong\u003eRepeated Ex4 increased immobility time in the tail suspension test (TST) (I) and forced swim test (FST) (J). \u003cstrong\u003e(K-N)\u003c/strong\u003e Acute Ex4 injection (2.5/5/10μg/kg, 3 days) in the OFT. (K) Representative traces. (L-N) Ex4 reduced center distance (L), center time (M), and total distance (N). \u003cstrong\u003e(O-R)\u003c/strong\u003e Effects of acute Ex4 administration in the EPM. (O) Representative traces. (P-R) Treated mice spent less time in the open arms (P), more time in the closed arms (Q), and showed a reduction in total arm entries (R). \u003cstrong\u003e(S, T)\u003c/strong\u003e TST (S) and FST (T) showed that Ex4 treatment significantly increased immobility time in a dose-dependent manner. Ex4: Exendin-4 *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8322552/v1/d0d24cf1f3e60db733fe9347.png"},{"id":100370101,"identity":"62a5c13e-4d49-4677-af77-ddcbddfbfaa6","added_by":"auto","created_at":"2026-01-16 07:59:58","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2228077,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGLP-1R activation suppresses TTR expression in the hippocampus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B)\u003c/strong\u003eHippocampal transcriptome analysis after Ex4 treatment, presented as a volcano plot (A) and heatmap (B). \u003cstrong\u003e(C)\u003c/strong\u003e qRT-PCR validation of TTR mRNA downregulation by Ex4. n=6. \u003cstrong\u003e(D)\u003c/strong\u003e Western blot analysis confirming TTR protein reduction in Ex4-treated mice. n=6. \u003cstrong\u003e(E, F)\u003c/strong\u003e IHC confirmed the downregulation of TTR in the hippocampus following Ex4 treatment. n=3. Ex4: Exendin-4 *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8322552/v1/e7f6baab0b8f11002f3c4dea.png"},{"id":100198207,"identity":"cc94155c-8299-4966-a956-ba6964141c51","added_by":"auto","created_at":"2026-01-14 03:57:20","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1174937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eActivation of the GLP-1 receptor temporally decreases serum TTR levels in mice and humans.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Ex4 differentially reduced serum TTR in normal-weight mice. \u003cstrong\u003e(B, C)\u003c/strong\u003eIn normal-weight mice, serum TTR levels were positively correlated with active exploratory behaviors and negatively correlated with immobility time.\u003cstrong\u003e (D)\u003c/strong\u003eRepresentative figure of ELISA testing following blood collection in obese individuals after semaglutide treatment. \u003cstrong\u003e(E-G)\u003c/strong\u003e This effect was translatable to humans, where the GLP-1R agonist SMG caused a transient decrease in serum TTR in obese patients. n=6. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8322552/v1/9c1e3d29009a8238686a6291.png"},{"id":100369703,"identity":"1b510ded-b396-43ab-9ee4-f25c9ecc592f","added_by":"auto","created_at":"2026-01-16 07:59:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2175569,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTTR is essential for GLP-1R-mediated anxiety/depression-like behaviors in normal-weight mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic of intracerebroventricular (ICV) cannula implantation for drug delivery. \u003cstrong\u003e(B, C)\u003c/strong\u003e qRT-PCR and Western blot confirm that Eplontersen effectively knocked down hippocampal TTR. \u003cstrong\u003e(D-K)\u003c/strong\u003e Knockdown of hippocampal TTR with Eplontersen is sufficient to induce anxiety-like (D-I)and depression-like (J, K) behaviors, mimicking the effect of Ex4. \u003cstrong\u003e(L)\u003c/strong\u003eSchematic of tail vein injection for reTTR delivery and intraperitoneal injection of Ex4. \u003cstrong\u003e(M) \u003c/strong\u003eIntravenous reTTR restored hippocampal TTR levels under Ex4 challenge. \u003cstrong\u003e(N-U)\u003c/strong\u003e TTR supplementation fully reversed the anxiety-like (N-S) and depression-like (T, U) behaviors induced by Ex4. n=6. Ex4: Exendin-4, reTTR: recombinant TTR *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8322552/v1/fad7f7bb7807ba15ac80226a.png"},{"id":100369650,"identity":"cf74d3c3-2991-4e6f-a220-911b3c84c200","added_by":"auto","created_at":"2026-01-16 07:59:12","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2475345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMetabolic state regulates the bidirectional involvement of TTR in affective behaviors mediated by GLP-1R.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-D) \u003c/strong\u003eConserved TTR downregulation in overweight mice (C, D) but not in db/db mice (A, B). (For A, C, n=3; For B, D, n=6). \u003cstrong\u003e(E, F) \u003c/strong\u003eIn overweight mice, serum TTR levels are positively correlated with active exploratory behaviors(E), but this correlation disappeared following Ex4 administration(F). n=6. \u003cstrong\u003e(G-N)\u003c/strong\u003e Injection of recombinant TTR is sufficient to induce anxiety-like (G-L) and depression-like (M, N) behaviors.\u003cstrong\u003e \u003c/strong\u003en=6. Ex4: Exendin-4 *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8322552/v1/ef6b1d572b6ee2301764d87d.png"},{"id":100198214,"identity":"2cf596f9-eef9-4670-83b8-53f6372a2109","added_by":"auto","created_at":"2026-01-14 03:57:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1920939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTTR expression is repressed by GLP-1R activation through the cAMP/PKA/Gli3 transcriptional pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The UCSC and Jasper databases predict the top 20 transcription factors with the strongest binding affinity to the TTR promoter. \u003cstrong\u003e(B, C) \u003c/strong\u003eChIP confirms TTR binding to the Gli3 promoter.\u003cstrong\u003e (D-H)\u003c/strong\u003e Ex4 treatment activates the cAMP/PKA pathway and upregulates the TTR-target transcription factor Gli3. n=6.\u003cstrong\u003e (I-P)\u003c/strong\u003eBehavioral deficits induced by Ex4 were rescued by the PKA inhibitor H-89, as evidenced by normalized performance in the OFT (I-K), EPM (L-N), TST (O), and FST (P) Ex4: Exendin-4 *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8322552/v1/d29ef95f41b3a25be4b341d7.png"},{"id":100198215,"identity":"2b288400-bd8f-4f5d-aaad-309845ed9fe3","added_by":"auto","created_at":"2026-01-14 03:57:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5189150,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTTR modulates affective behaviors by the ERK/CREB signaling affective.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-G)\u003c/strong\u003e Ex4administration selectively suppressed the ERK/CREB pathway, as evidenced by decreased p-ERK/ERK and p-CREB/CREB ratios in western blot analysis, while AKT signaling remained unchanged. n=6.\u003cstrong\u003e (H-J) \u003c/strong\u003eIHC confirmed the co-downregulation of p-ERK and p-CREB in the hippocampus following Ex4 treatment. n=3.\u003cstrong\u003e (K-Q)\u003c/strong\u003e The suppressive effects of Ex4 on ERK and CREB phosphorylation were rescued by concomitant reTTR administration. n=6. Ex4: Exendin-4 *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8322552/v1/bfe12d6fbc45367ee5beb530.png"},{"id":100198212,"identity":"4f750538-6099-4f0b-b2de-a3683f24cf34","added_by":"auto","created_at":"2026-01-14 03:57:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3060448,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCREB activation ameliorates Ex4-induced behavioral and synaptic impairments.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-J) \u003c/strong\u003eBehavioral analysis demonstrates that the CREB agonist DIS counteracts Ex4-induced anxiety-like (A-H, OFT \u0026amp; EPM) and depression-like (I, J, TST \u0026amp; FST) behaviors.\u003cstrong\u003e (K-T) \u003c/strong\u003eAt the molecular level, DIS rescues the Ex4-mediated downregulation of key synaptic markers, including PSD95 and Drebrin (L, M), and restores the expression and phosphorylation of glutamate receptor subunits GluA1 (O-Q) and GluN2B (R-T) in the hippocampus. Representative traces (A, E, I, J) and immunoblots (K, N) are shown. n=6. Ex4: Exendin-4 *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8322552/v1/e7241ffd2a5563c3f399515a.png"},{"id":104781196,"identity":"7394c1d5-4073-43a9-8c97-bd24c89c98d6","added_by":"auto","created_at":"2026-03-17 07:55:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23856335,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8322552/v1/fc4f32e3-134e-483a-bfd3-fabbe9206124.pdf"},{"id":100369155,"identity":"1e7b3355-e93f-4633-8b44-726ef3db5c66","added_by":"auto","created_at":"2026-01-16 07:58:44","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3675958,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-8322552/v1/f49334eb851beb86957c5374.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Transthyretin gates GLP-1R-mediated affect by metabolic stat","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe rising global prevalence of metabolic diseases such as type 2 diabetes mellitus (T2DM) and obesity coincides with an increasing burden of mood disorders, underscoring a profound connection between metabolic homeostasis and affective regulation\u003csup\u003e1,2\u003c/sup\u003e. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) not only improve glycemic control and body weight but also exert central effects influencing mood and neuroinflammation\u003csup\u003e3,4\u003c/sup\u003e. While GLP-1 RAs may alleviate depressive symptoms in some patients\u003csup\u003e5,6\u003c/sup\u003e, post-marketing surveillance has also reported anxiety and suicidal ideation in certain individuals\u003csup\u003e7,8,9,10\u003c/sup\u003e, creating a clinical paradox that remains mechanistically unresolved.\u003c/p\u003e\n\u003cp\u003eThe hippocampus represents a critical interface between metabolic and affective processing\u003csup\u003e11\u003c/sup\u003e. This region not only shows high GLP-1 receptor expression but is also vulnerable to both metabolic stress and depression-related plasticity deficits\u003csup\u003e3\u003c/sup\u003e. Our previous work demonstrated particularly elevated hippocampal GLP-1R expression in diabetic mice, suggesting this region as a key site for investigating mood-related drug effects \u003csup\u003e12\u003c/sup\u003e. Preclinical studies show that GLP-1 RAs can improve hippocampal function in metabolic disease models yet paradoxically induce anxiety-like behaviors and HPA axis activation in healthy rodents\u003csup\u003e13,14,15\u003c/sup\u003e. This bidirectional effect implies the existence of an unknown molecular switch that directs GLP-1R signaling toward beneficial or detrimental affective outcomes based on metabolic context.\u003c/p\u003e\n\u003cp\u003eTransthyretin (TTR), a transport protein synthesized in the liver and choroid plexus, may constitute this critical switch. Peripheral TTR is elevated in obese humans and diet-induced obese mice, correlating with insulin resistance (HOMA-IR) and adipose inflammation\u003csup\u003e16,17\u003c/sup\u003e. Central TTR exerts neuroprotection and synaptic function by activating the MAPK/ERK/CREB pathway, which is critical for hippocampal plasticity and neuronal energy metabolism\u003csup\u003e18\u003c/sup\u003e. Notably, in silico pharmacogenomic analyses reveal that GLP-1R signaling intersects with TTR-regulated pathways enriched in dopamine metabolism and insulin sensitivity, suggesting TTR could functionally bridge the metabolic and neuropsychiatric dimensions of GLP-1R activation\u003csup\u003e19,20\u003c/sup\u003e. Still, whether TTR serves as a metabolic-state-dependent mediator of GLP-1R-induced affective behaviors, and how its regulation is embedded within GLP-1R-driven signaling networks, remained unknown.\u003c/p\u003e\n\u003cp\u003eIn this study, we formally tested the hypothesis that TTR acts as a central molecular switch that integrates GLP-1R signaling with metabolic status to coordinate affective state. We aimed to (1) determine how metabolic status shapes the impact of GLP-1RA on hippocampal TTR expression and synaptic function; (2) delineate the upstream signaling cascade GLP-1R-cAMP-PKA-Gli3 controlling TTR transcription; (3) establish TTR as a dual biomarker capable of predicting metabolic and neuropsychiatric outcomes in a human; and (4) explore TTR-targeting strategies to optimize the therapeutic utility of GLP-1RA. Our findings reveal a TTR-mediated pathway that unifies the metabolic and neuropsychiatric effects of GLP-1R, providing a framework for personalized metabolic therapy that addresses both physiological and affective health.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eGLP-1R activation improves anxiety/depression-like behaviors in diabetic and over-weight mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the established efficacy of GLP-1RAs in type 2 diabetes and obesity management\u003csup\u003e21\u003c/sup\u003e, we first evaluated the impact of GLP-1R activation on affective behaviors in metabolically impaired mouse models. As our previously reported\u003csup\u003e12\u003c/sup\u003e, \u003cem\u003edb/db\u003c/em\u003e mice, a model of type 2 diabetes\u003csup\u003e22\u003c/sup\u003e, exhibited significant anxiety and depression-like behaviors. These included reduced center distance and time in the open field test (OFT; S-Fig. 1A-1D), decreased open-arm time and increased closed-arm time in the elevated plus maze (EPM; S-Fig. 1E-H), and prolonged immobility in the tail suspension test (TST) and forced swimming test (FST; S-Fig. 1I and 1J). Treatment with GLP-1R agonist Exendin-4 (Ex4) significantly reversed these behavioral deficits, increasing center and open-arm exploration while reducing immobility (S-Fig. 1B, 1F, 1I and 1J), suggesting an anxiolytic and antidepressant-like effect of GLP-1R activation in the diabetic mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe next examined high-fat diet (HFD)-induced overweight (OW) mice\u003csup\u003e23\u003c/sup\u003e, which showed a significantly elevated Lee\u0026apos;s index (S-Fig.1L) without hyperglycemia (S-Fig.1M). These mice exhibited anxiety-like behaviors in OFT and EPM (S-Fig.1N-1S) but not depression-like behavior in TST or FST (S-Fig.1T and 1U). Ex4 administration ameliorated anxiety-like behaviors in OW mice without affecting normoglycemia (S-Fig.1M-1U).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGLP-1R activation induces anxiety/depression-like behaviors in normal-weight mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next asked whether GLP-1R activation influences affective behaviors in normal-weight mice. Using a time-gradient Ex4 regimen (5 \u0026mu;g/kg/day for 1, 3, 5, and 7 days)\u003csup\u003e24\u003c/sup\u003e, we found that 3-day treatment produced the most consistent anxiety/depression-like phenotype: reduced center activity in OFT (Fig. 1A-1D) , decreased open-arm and increased closed-arm time in EPM (Fig.1E-1H), and prolonged immobility in TST and FST (Fig.1I and 1J). A subsequent dose-response study identified 5 \u0026mu;g/kg/day for 3 days as the optimal regimen (Fig.1K-1T; S-Fig.2A-2H), with no effect on blood glucose (S-Fig.2I).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo confirm that these effects were GLP-1R-specific and compound-generalizable, we\u0026nbsp;treated normal-weight mice with\u0026nbsp;semaglutide (SMG, 0.1 mg/kg/day)\u003csup\u003e25\u003c/sup\u003e, another\u0026nbsp;GLP-1RA\u0026nbsp;for 1, 3, 5 or 7 days. Semaglutide also induced anxiety/depression- like behaviors, particularly after 7 days of treatment (S-Fig.3A-3H), without affecting glycemia (S-Fig.3I). Furthermore, Ex4 failed to elicit such behaviors in GLP-1R knockout mice (GLP-1R⁻/⁻). Collectively, these data demonstrate that GLP-1R activation is sufficient to induce affective deficits in normal-weight mice, an effect that is receptor-dependent and not secondary to glycemic changes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTTR is downregulated in the hippocampus of over-weight mice after GLP-1R activation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the molecular mechanisms of GLP-1R activation-induced anxiety/depression-like behaviors in normal-weight mice, we performed RNA-sequencing (RNA-seq) on hippocampal tissue \u003csup\u003e12\u003c/sup\u003e, in which GLP-1R expression is highest in this region among affective brain regions. RNA-seq revealed that Ex4 treatment (5 \u0026mu;g/kg/day for 3 days) induced the downregulation of 32 genes and upregulation of 5 genes in the hippocampus (Fig.2A). Among these differentially expressed genes (DEGs), TTR stood out due to its substantial fold change and stable expression profile (Fig.2A and 2B). TTR was further prioritized as a core candidate based on existing evidence linking it to central nervous system (CNS) disorders and its established role in neuronal and synaptic growth\u003csup\u003e26,27,28,29\u003c/sup\u003e. Subsequent validation by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry (IHC) sonsistently confirmed that Ex4 remarkably reduced TTR mRNA and protein levels in the hippocampus (Fig.2C-F). Together, these results suggest that TTR may act as a key downstream mediator of GLP-1R-triggered affective deficits in normal-weight mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGLP-1R activation transiently reduces serum TTR levels in mice and humans\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the relevance of TTR in GLP-1R -mediated affective behaviors, we measured circulating TTR levels in both mice and humans following GLP-1R activation. In normal-weight mice, Ex4 treatment (5 \u0026mu;g/kg/day) led to a transient reduction in serum TTR, with levels significantly decreased after 3 and 5 days of treatment but returning to baseline by day 7 (Fig.3A). We next asked whether TTR fluctuations were behaviorally relevant. Correlation analyses in Ex4-treated normal-weight mice revealed that lower serum TTR levels were positively correlated with affective deficit as shown by decreased time spent in central area of OFT and in open arms of EPM (Fig.3B), and increased immobility time in TST and FST (Fig.3C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe then extended these findings to humans by analyzing serum TTR in obese individuals before and after treatment with the GLP-1R agonist\u0026nbsp;semaglutide (Fig.3D). ELISA results revealed a marked decrease in serum TTR levels 1 month after semaglutide administration (Fig.3E), whereas serum TTR levels returned to pre-treatment baseline after 2- and 3- months continuous treatment (Fig.3F and 3G). These cross-species data indicate that GLP-1R activation induces a transient reduction in circulating TTR, and that TTR fluctuations correlate with affective behaviors in normal-weight mice, supporting its potential role as a translatable biomarker in GLP-1RA-mediated affective regulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTTR is required for\u0026nbsp;GLP-1R-mediated affective deficits in normal-weight mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether TTR is required for GLP-1R-induced anxiety/depressive-like behaviors, we performed local hippocampal injections of eplontersen, a TTR-targeting antisense oligonucleotide, in normal-weight mice. Eplontersen treatment (10 \u0026mu;g/kg, 1 \u0026mu;l/side, 3 days) effectively reduced TTR mRNA and protein levels in the hippocampus (Fig.4A-4C). In OFT, eplontersen-treated mice showed obvious anxiety/depression-like phenotypes, including reduced center distance and time in OFT (Fig.4D-4F), decreased open-arm exploration and increased closed-arm time in EPM (Fig.4G-4I), and increased immobility in TST and FST (Fig.4J and 4K), recapitulating the effects of Ex4.\u003c/p\u003e\n\u003cp\u003eWe next asked whether restoring TTR could rescue Ex4-induced behavioral deficits. Co-administration of recombinant TTR (reTTR, 5 \u0026mu;g/kg/day, 3 days) via tail vein injection (Fig.4L) prevented the Ex4-induced downregulation of hippocampal TTR protein (Fig.4M). Accordingly, reTTR fully reversed the anxiety/depression- like behaviors triggered by Ex4 across all behavioral tests (Fig.4N-4U). Collectively, these loss- and gain-of-function experiments demonstrate that TTR is both necessary and sufficient for mediating the affective impairments induced by GLP-1R activation in normal-weight mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMetabolic state dictates the bidirectional role of TTR in GLP-1R\u0026ndash;mediated affective behaviors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNext, we asked whether TTR contributes to the affective improvement observed after GLP-1R activation in diabetic and OW mice. The results of western blot showed that TTR levels of\u003cem\u003e\u0026nbsp;db/db\u003c/em\u003e mice had no difference in the hippocampus and serum compared to wild-type (WT) mice, and Ex4 treatment did not alter TTR levels in this model (Fig.5A and 5B). In contrast, OW mice exhibited markedly elevated TTR levels in both the hippocampus and serum, and Ex4 treatment effectively normalized TTR levels (Fig.5C and 5D). We further analyzed the behavioral relevance of TTR dynamics in OW mice before and after Ex4 treatment. Serum TTR levels were negatively correlated with the time spent in the center of OFT and in the open arms of EPM before Ex4 treatment (Fig.5E), but the relevance was not obvious after Ex4 treatment (Fig.5F), suggesting that Ex4 attenuates anxiety/depression-like behaviors in OW mice by reducing pathologically elevated TTR.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo confirm the causal role of elevated TTR in promoting anxiety/depression-like behaviors, we administered reTTR (5 \u0026mu;g/kg/day, 3 days) to normal-weight mice. reTTR treatment significantly induced anxiety/depression-like phenotypes across behavioral tests (Fig.5G-5N). Together, these results establish TTR as a bidirectional modulator of affective behavior, wherein its levels must be maintained within a physiological range. In OW mice, GLP-1R activation normalizes pathologically elevated TTR to produce anxiolytic effects, whereas in normal-weight mice, the same intervention suppresses physiological TTR levels, thereby leading to affective deficits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGLP-1R activation represses TTR expression through the cAMP/PKA/Gli3 transcriptional pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the mechanism by which GLP-1R activation downregulates TTR expression, we first used the UCSC Genome Browser database to perform\u0026nbsp;bioinformatic screening of transcription factors (TFs) predicted to bind the TTR promoter (\u003cstrong\u003eS-Fig.\u003c/strong\u003e5). From the top 20 candidate transcription factors based on prediction scores (Fig.6A), Gli3 emerged as the only TF meeting all relevant criteria: functional association with GLP-1R signaling, documented repressor activity, and expression in the hippocampus\u003csup\u003e30,31,32\u003c/sup\u003e. We then used the JASPAR database to predict potential Gli3-binding sites within the TTR promoter (Fig.6B), and chromatin immunoprecipitation (ChIP) confirmed specific Gli3 binding to all three predicted sites, with the strongest enrichment observed at site 2 (Fig.6C).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince Gli3 can be phosphorylated by PKA, a cAMP-dependent kinase, to become a transcriptional repressor\u003csup\u003e33\u003c/sup\u003e , and given that cAMP/PKA is a canonical downstream pathway of GLP-1R\u003csup\u003e34\u003c/sup\u003e, we hypothesized that GLP-1R activation suppresses TTR via cAMP/PKA/Gli3 signaling. Consistent with this, Ex4 treatment significantly increased cAMP levels (Fig.6D) and upregulated both phosphorylated PKA (P-PKA) and total PKA, along with transcriptionally inhibitory Gli3 fragments (Fig.6E and 6F). To functionally link this pathway to the observed behavioral outcomes, we co-administered PKA inhibitor H-89 (10 mg/kg, I.P.) with Ex4 in normal-weight mice. H-89 abolished Ex4-induced anxiety/depression-like behaviors (Fig.6G-6N) and blocked PKA/Gli3 activation and TTR downregulation (S-Fig.6). These data indicate that GLP-1R activation drives affective deficits in normal-weight mice through a cAMP/PKA/Gli3-dependent repression of TTR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTTR regulates affective behaviors through the ERK/CREB signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSubsequently, we sought to identify the downstream signaling mechanism by which TTR modulates anxiety and depression-like behaviors. Based on previous reports that TTR promotes neurite outgrowth and neuroprotection via ERK/AKT/CREB signaling\u003csup\u003e18\u003c/sup\u003e, we examined whether this pathway is involved in the behavioral effects of GLP-1R activation. Western blot analysis revealed that Ex4 administration significantly reduced both phosphorylation and total protein levels of ERK and CREB but not AKT in the hippocampus of normal-weight mice (Fig.7A-7G). IHC further demonstrated a marked decrease in P-ERK and P-CREB immunoreactivity after Ex4 injection (Fig.7H-J).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo determine whether these signaling changes were TTR-dependent, we assessed the effects of TTR knockdown and restoration. Eplontersen-mediated TTR inhibition similarly reduced P-ERK/ERK and P-CREB/CREB levels without affecting P-AKT/AKT levels in normal-weight mice (S-Fig.7A-7G). Conversely, reTTR supplementation reversed the Ex4-induced suppression of ERK and CREB signaling (Fig.7K-7Q). Furthermore,\u0026nbsp;PKA inhibition by H-89 prevented Ex4-induced decreases in P-ERK and P-CREB (S-Fig.7H-7M), placing ERK/CREB downstream of the cAMP/PKA/Gli3/TTR axis. Collectively, these results imply that GLP-1R activation impairs ERK/CREB signaling via TTR downregulation, establishing this pathway as a key effector mechanism in the development of affective deficits.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCREB activation\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;rescues synaptic and affective deficits induced by GLP-1R signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven the established role of CREB in anxiety/depression regulation\u003csup\u003e35,36,37\u003c/sup\u003e, we asked whether its activation could counteract the affective deficits induced by GLP-1R activation. We co-administered Ex4 with CREB agonist 3\u0026apos;,6-disinapoylsucrose (DIS, 10 mg/kg, i.p.) in normal-weight mice. DIS treatment significantly ameliorated Ex4-induced anxiety/depression-like behaviors, as evidenced by increased center time and distance in OFT (Fig.8A-8D), increased open-arm exploration and reduced closed-arm time in EPM (Fig.8E-8H), and decreased immobility in TST and FST (Fig.8I and 8J).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSince CREB regulates synaptic plasticity by modulating the expression of genes involved in synaptic structure and function\u003csup\u003e38\u003c/sup\u003e, we next examined two important synaptic plasticity markers, PSD95 (a core scaffolding protein in the postsynaptic density) and Drebrin (an actin-binding protein regulating synaptic morphological remodeling). DIS administration reversed the Ex4-induced downregulation of PSD95 and Drebrin (Fig. 8K-8M). We further assessed glutamate receptor expression and phosphorylation, given their importance in synaptic transmission. DIS treatment abolished Ex4-induced reductions in total protein levels of GluA1 and GluN2B, as well as their phosphorylation at key residues (Ser831 and Ser845 for GluA1; Ser1303 and Thr1472 for GluN2B; Fig. 8N-8T). These data further confirm that TTR mediates GLP-1R-induced affective deficits by suppressing CREB signaling, and demonstrate that CREB activation restores synaptic protein expression and affective behaviors, highlighting its key role of CREB pathway balance in affective regulation.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe expanding clinical use of GLP-1RAs has uncovered a paradox in their affective effects, ranging from mood improvement to the induction of anxiety or depression\u003csup\u003e7,39,40,41,42\u003c/sup\u003e. Proposed mechanisms for these disturbances, such as neurotransmitter imbalance and HPA axis dysregulation\u003csup\u003e43,44,45\u003c/sup\u003e, along with variable risks of suicidal ideation noted in observational studies, have remained fragmented. A unifying framework explaining these bidirectional affective outcomes has been lacking. Our work resolves this gap by identifying TTR as the central, metabolic-state-dependent molecular switch, and delineating the dedicated signaling axis underlying this regulation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur core finding is that TTR operates within a narrow physiological\u0026nbsp;“functional window”\u0026nbsp;essential for affective homeostasis. In normal-weight mice, endogenous TTR sustains hippocampal ERK/CREB signaling and preserves synaptic integrity. GLP-1R activation engages the cAMP/PKA/Gli3 cascade to transcriptionally suppress TTR, driving its level below this functional threshold. This suppression of TTR leads to impaired ERK/CREB activity, reduced expression of synaptic proteins such as PSD95, Drebrin, and key glutamate receptors, and ultimately precipitates affective deficits. Conversely, in diet-induced obese mice with pathologically elevated TTR, the same GLP-1RAs intervention normalizes TTR levels, thereby restoring downstream signaling and alleviating anxiety-like behavior. This establishes a unifying principle: the behavioral efficacy of GLP-1RAs correlates with the normalization of TTR levels toward its physiological set-point. The causal role of TTR within this axis is rigorously validated by multiple experimental approaches: the PKA inhibitor H-89 blocks GLP-1RA-induced affective disturbances; hippocampal TTR knockdown recapitulates the phenotype; and recombinant TTR supplementation fully rescues the synaptic and behavioral impairments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNotably, our study bridges two previously disconnected fields: the neuroprotective roles of TTR in processes like amyloid clearance\u003csup\u003e46,47,48\u003c/sup\u003e, and the multifaceted central nervous system effects of GLP-1R signaling via pathways such as cAMP/PKA and PI3K/Akt\u003csup\u003e49,50\u003c/sup\u003e. We fill this critical gap by delineating a complete and dedicated GLP-1R/cAMP/PKA/Gli3/TTR/ERK/CREB axis. This pathway is distinct from the canonical metabolic branches of GLP-1R signaling, such as PI3K/Akt/mTOR that primarily govern glycemic control and\u0026nbsp;β-cell survival. A key mechanistic novelty is the identification of Gli3 as the critical transcription factor linking GLP-1R activation to TTR repression. GLP-1R activation elevates hippocampal cAMP levels, triggering PKA-dependent phosphorylation of Gli3, which then enhancing its binding to the TTR promoter and suppressing TTR transcription. This specific mode of transcriptional regulation, linking GLP-1R signaling to TTR repression via Gli3, represents a novel finding not previously reported in the contexts of either GLP-1R or TTR biology. The pathway is highly specific: pharmacological inhibition of PKA abolishes Gli3 activation, TTR downregulation, and the consequent affective disturbances, without impairing the glucoregulatory function of GLP-1R, as evidenced by unchanged blood glucose levels in treated normal-weight mice. Downstream, the pathway selectively targets the ERK/CREB module and synaptic protein expression without altering AKT signaling, distinguishing it from the broader neurotrophic effects often associated with TTR. Finally, direct pharmacological activation of CREB rescues both synaptic deficits and the affective impairments induced by GLP-1RA, confirming CREB as the key downstream effector of TTR in affective regulation. This aligns with CREB’s well-documented role as a master regulator of mood-related synaptic plasticity\u003csup\u003e51,52,53\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe most immediate translational implication is the identification of serum TTR as a tractable pretreatment biomarker for personalized therapy. We propose a stratified clinical strategy: patients with elevated baseline TTR should receive GLP-1RAs therapy to achieve dual metabolic and affective benefits via TTR normalization. In contrast, individuals with physiological TTR levels should either avoid GLP-1RAs or co-administer a TTR stabilizer to prevent TTR suppression and mood risk, with serum TTR monitoring to track dynamic changes. This paradigm shifts the clinical approach from reactive side-effect management toward proactive, precision neuro-metabolic care.\u003c/p\u003e\n\u003cp\u003eOur study has limitations that guide future research. The supportive human correlation data require validation in large-scale prospective trials to confirm the predictive value of TTR. The precise molecular interface linking TTR to the modulation of ERK/CREB activity warrants further elucidation, as do the potential cell-type-specific functions of TTR within the hippocampus. Furthermore, our findings open several transformative questions: Can pharmacological modulators of TTR be used as adjuvants to optimize the therapeutic window of GLP-1RAs? Is the TTR functional window a generalizable mechanism for affective comorbidity in other systemic diseases? Exploring these avenues will deepen our understanding of the metabolic-brain axis.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we resolve the clinical paradox of GLP-1RAs by establishing TTR as a metabolic state-dependent switch and defining the TTR functional window concept. We provide a comprehensive mechanistic pathway from receptor to behavioral manifestation and, most importantly, deliver a practical biomarker strategy to guide personalized therapy. This work reframes affective alterations from unpredictable liabilities into mechanistically grounded and potentially predictable outcomes, thereby laying a solid foundation for the practice of precision neuro-metabolic medicine.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight-week-old male C57BL/6J mice were provided by the Experimental Animal Center of the Fourth Military Medical University. GLP-1R⁻/⁻ mice were obtained from Beijing Viewsolid Biotechnology Co., Ltd., with WT littermates serving as controls. Six-week-old male db/db mice (BKS-Leprem2cd479/Gpt) and non-diabetic control mice (same strain, 19–38 g) were purchased from Chengdu GemPharmatech Co., Ltd. All mice were housed in plastic cages with \u003cem\u003ead libitum\u003c/em\u003e access to standard chow and water, in a colony room maintained under controlled environmental conditions: temperature (22–26°C), relative humidity (55–60%), and a 12-h light/dark cycle. Prior to the initiation of experimental procedures, the animals were acclimated to the laboratory setting for a minimum of one week. All mice were randomly assigned to experimental groups, and investigators were blinded to the grouping information. The sample size of experimental animals was minimized as much as reasonably possible, while ensuring adequate statistical power and meeting the requirements of the experimental design. All experimental protocols were approved by the Animal Care and Use Committee of the Fourth Military Medical University (No. 20240015).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eExendin-4 (Ex4; No. A3408, purity ≥ 99%) was purchased from APExBIO Technology LLC (Houston, TX, USA). Semaglutide (SMG, purity ≥ 95%) was synthesized by Anwante Biotechnology Co.,Ltd \u0026nbsp;(Shanghai, China). Eplontersen, recombinant TTR (reTTR), 3’,6-Disinapoylsucrose (DIS) and H-89 were obtained from MedChemExpress (Monmouth Junction, NJ, USA). TTR (ZC-35797, ZC-57833) ELISA kits were purchased from ZCIBIO Technology Co.,Ltd (shanghai, China). The SYBR Premix Ex TaqII and PrimeScript RT reagent kits were obtained from Yeasen (Shanghai, China).\u003c/p\u003e\n\u003cp\u003eFor western blotting and IHC, the following antibodies were used: β-Actin (No.66009-1-Ig, Proteintech, Wuhan, China), TTR (No.PA5-67636, AntiProtech, S.F, CA, USA) for western blotting, P-ERK (No.9101S, Cell Signaling Technology, Boston, MA, USA), ERK (No.9102S, Cell Signaling Technology), P-AKT (No.4060, Cell Signaling Technology), AKT (No.4691, Cell Signaling Technology), P-CREB (No.9198S, Cell Signaling Technology), CREB (No.9197S, Cell Signaling Technology), PSD95 (No.ab2723, Abcam, Cambridge, UK), Drebrin (No.ab178408, Abcam), GluA1 (No.13185S, Cell Signaling Technology), P-GluA1-S845 (No.8084S, Cell Signaling Technology), P-GluA1-S831 (No.75574S, Cell Signaling Technology), GluN2B (No. 4207, Cell Signaling Technology), P-GluN2B-S1303 (No. 71335, Cell Signaling Technology), P-GluN2B-T1472 (No. 90125, Cell Signaling Technology), P-PKA (No.CY9083, Abways, Shanghai, China), PKA (No.GB11598-100, Servicebio, Wuhan, China), Gli3 (No.Ab307714, Abcam), TTR (No.EPR20971, Abcam) for IHC. Secondary antibodies, including goat anti-mouse IgG-HRP (No. EK010) and goat anti-rabbit IgG-HRP (No. EK020), were purchased from Zhuangzhibio (Xi’an, USA)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollection of human blood samples\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVenous blood samples were collected from adult obese participants with a body mass index (BMI) ranging from 29 to 45 kg/m²\u0026nbsp;before SMG administration, and separately at 1 month, 2 months, or 3 months after administration, with each time point assigned to different subgroups of participants. The blood samples were directly centrifuged at 2,000×g for 10 minutes at 4°C, and the separated serum was used for the detection of TTR levels by enzyme-linked immunosorbent assay (ELISA). The study protocol received ethical approval from the Medical Ethics Committee of the Xijing Hospital of Fourth Military Medical University (Approval No. KY20252243-F-1) and was prospectively registered with the Chinese Clinical Trial Registry (Registration No. ChiCTR 2500104257. https://www.chictr.org.cn/showproj.html?proj=275079). All participants provided written informed consent. The investigation was conducted in full accordance with the ethical principles outlined in the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStereotaxic intracerebral delivery\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were fixed in a stereotaxic frame (RWD68001, Shenzhen RWD Life Science, Shenzhen, China). A double cannula (guide cannula, length 7.5 mm, internal diameter 0.34 mm, external diameter 0.48 mm, center to center distance 2.6 mm) was aimed at 0.5 mm above the intended sites of injection, namely the hippocampus (AP: -2.06 mm; ML:\u0026nbsp;±1.3 mm; DV: -1.5 mm). The guide cannulas were affixed to the skull with machine screws and dental cement, and stylet was inserted into the cannula to keep them unimpeded. Mice were used for following experiments after one-week recovery. For the injection of Eplontersen (10 μg/kg), mice were anesthetized with isoflurane (3-4% for induced anesthesia and 1-1.5% for maintained anesthesia). Eplontersen was microinjected into the hippocampus using a Hamilton syringe (10 μl) connected to a glass capillary tip, with a total volume of 1μl per side, administered over three consecutive days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTail vein injection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were secured in a restraint device with tails exposed. Bilateral lateral tail veins were visualized, and the more distinct vein was selected as the injection site. The injection area was disinfected using a 75% ethanol swab. reTTR (5 μg/kg) was administered via a 1-mL syringe equipped with a 26G needle. Following injection, the puncture site was compressed with a dry cotton ball for 10–15 seconds to prevent hemorrhage. The administration was performed over three consecutive days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOpen field test (OFT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe OFT served to assess anxiety-like behavior in mice, as previously reported\u003csup\u003e54\u003c/sup\u003e. The apparatus was a transparent Plexiglas square arena (30×30×30 cm). Mice were gently tail-held and placed head-outward at the center of the arena (15×15 cm central zone, 25% of total area), with video recording initiated immediately. Animals were permitted free exploration for 10 min. Behavior was recorded by a ceiling-mounted camera, and trajectory data were analyzed using a video-tracking system (DigBehv-LR 4, Shanghai Jiliang, China). The arena was thoroughly cleaned between trials to eliminate odor interference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElevated plus maze (EPM)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe EPM test was performed to further assess anxiety-like behavior in mice\u003csup\u003e55\u003c/sup\u003e. The apparatus consisted of a black Plexiglas cross-shaped platform (50–60 cm in height) with two open arms (25 × 8 × 0.5 cm, unenclosed) and two closed arms (25 × 8 × 0.5 cm, enclosed by 15 cm walls), intersecting at a central zone (8 × 8 cm). Mice were placed at the junction of the central platform facing an open arm, and behavior was recorded for 5 minutes using a ceiling-mounted camera. A video-tracking system (DigBehv-LR 4, Shanghai Jiliang, Shanghai, China) was employed to analyze metrics. The maze was thoroughly cleaned with 75% ethanol after each trial to eliminate odor interference.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTail suspension test (TST)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe TST apparatus typically consists of a stand and clamping device, with anti-climbing barriers surrounding the setup. Mice were suspended by their tails using adhesive tape from a horizontal bar for 6 minutes, and behavior was recorded via a ceiling-mounted camera. The cumulative duration of complete immobility (defined as no limb movement except minor forepaw adjustments) during the final 4 minutes of the test was quantified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eForced swimming test (FST)\u003c/strong\u003e\u003c/p\u003e\n\u003ch4\u003eThe FST apparatus consisted of a transparent cylindrical tank (15 cm in diameter) filled with water to a depth of 20 cm (23-25℃). The water surface was positioned 10 cm below the tank's top edge to prevent climbing or escape. Mice were gently placed into the water for 6 minutes, and behavior was recorded via a video-tracking system. The cumulative duration of immobility (defined as minimal limb movement beyond passive floating) during the final 4 minutes of the test was quantified.\u0026nbsp;\u003c/h4\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(qRT-PCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using the RNA Easy Fast Tissue Kit (DP451, TIANGEN) following the manufacturer’s protocol. cDNA was synthesized from purified RNA with the FastKing cDNA Synthesis Kit (KR116, TIANGEN). The qPCR was conducted using RealUniversal Color PreMix (FP201, TIANGEN) and SYBR Premix Ex Taq II (TaKaRa) following recommended thermal cycling parameters. The sequences of the primers are listed as follows (Sangon Biological Engineering Technology, Shanghai, China): TTR, forward 5′-TCACCAGGAGAAGCCGTCACAC-3′ and reverse 5′-AAATACCAGTCCAGCGAGGCAAAG-3′; β-Actin, forward 5′-ACTGTCGAGTCGCGTCC-3′ and reverse 5′-CTGACCCATTCCCACCATCA-3′. \u0026nbsp;β-Actin served as the reference gene for normalization, and transcript expression levels were quantified via the 2−\u003csup\u003eΔΔCt\u003c/sup\u003e method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot analysis was performed as our previous report\u003csup\u003e56\u003c/sup\u003e. The hippocampus tissue was dissociated via sonication in RIPA lysis buffer containing phosphatase and protease inhibitors. The protein content of the collected samples was quantified using the BCA Protein Assay Kit. Equal amounts of protein (30 μg) were separated on SDS-PAGE gels then electro-transferred to PVDF membranes (Invitrogen, Thermo Fisher Scientific Inc., Waltham, USA). The membranes were in turn probed with primary antibodies overnight at 4°C after incubation for 1.5\u0026nbsp;h in 5% non-fat milk.β-Actin (1:1,000), TTR (1:1,000), P-ERK (1:1,000), ERK (1:1,000), P-AKT (1:1,000), AKT (1:1,000), P-CREB (1:1,000), CREB (1:1,000), PSD95 (1:1,000), Drebrin (1:1,000), GluA1 (1:1,000), P-GluA1-S845 (1:1,000), P-GluA1-S831 (1:1,000), GluN2B (1:1,000), P-GluN2B-S1303 (1:1,000), P-GluN2B-T1472 (1:1,000), P-PKA (1:1,000), PKA (1:1,000), Gli3 (1:1,000). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse secondary antibodies at room temperature (RT) for 1 hour. Densitometric analysis of Western blot bands was conducted using a ChemiDoc XRS imaging system (Bio-Rad, Hercules, CA, USA) and subsequently quantified by ImageJ software (NIH, Bethesda, MD, USA) in accordance with the manufacturers' protocols. Band intensities for each sample were calculated as relative ratios normalized to the housekeeping protein β-actin. The relative intensity ratio of the control group was set to 100%, while the ratios of other experimental groups were expressed as percentages relative to the control group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-Seq was conducted by Beijing Biomarker Technologies Co., Ltd.\u0026nbsp;Briefly, poly(A) RNA was isolated from 1 μg of total RNA using Dynabeads Oligo (25-61005, Thermo Fisher Scientific). Subsequently, the poly(A) RNA was sheared into short fragments with a Magnesium RNA Fragmentation Module (m6150, NEB) at 94°C for 5–7 minutes. The sheared RNA fragments were reverse-transcribed into complementary DNA (cDNA) using SuperScript™ II Reverse Transcriptase (1896649, Invitrogen). The resulting cDNA was purified following the enzymatic reactions, and library size selection was conducted with AMPure XP beads. Following treatment of U-labeled second-stranded DNA with heat-labile UDG enzyme (m0280, NEB), the resulting ligated products were subjected to PCR amplification. The final cDNA library had an average insert size of 300 ± 50 bp. Paired-end sequencing (PE150, 2 × 150 bp) was carried out on an Illumina NovaSeq™ 6000 platform (LC-Bio) in accordance with the manufacturer’s recommended protocol. HISAT2 (\u003ca href=\"https://ccb.jhu.edu/software/hisat2\" target=\"https://www.doubao.com/chat/_blank\"\u003ehttps://ccb.jhu.edu/software/hisat2\u003c/a\u003e) was employed for aligning sequencing reads to the \u003cem\u003eMus sapiens\u003c/em\u003e GRCm38 reference genome. Subsequent to transcriptome assembly, StringTie was utilized to quantify the expression levels of all transcripts via computation of the FPKM value (FPKM = [total exon fragments / (mapped reads in millions × exon length in kB)]). Differentially expressed mRNAs (DE-mRNAs) were defined as those with a fold change (FC) \u0026gt; 1.5 or FC \u0026lt; 0.05 and a P-value \u0026lt; 0.05.Post-sequencing, DEGs analysis was performed by the BMK Cloud bioinformatics pipeline (\u003ca href=\"https://www.biocloud.net/\" target=\"_blank\"\u003ewww.biocloud.net\u003c/a\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eELISA and glucose test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum TTR levels in mice were quantified using a species-specific ELISA kit (ZC-57833) in accordance with the manufacturer’s protocols. Briefly, mice were anesthetized with diethyl ether, and intraorbital blood collection was performed using ophthalmic forceps to puncture the retro-orbital venous plexus. Approximately 0.5 mL of blood per mouse was collected into 1.5 mL centrifuge tubes, with the entire collection process completed within 1 minute. Subsequently, the blood samples were centrifuged at 1000\u0026nbsp;×g and 4°C for 20 minutes, and the serum supernatant was harvested for subsequent ELISA analysis. For the quantification of TTR in human serum samples, a human-specific ELISA kit (ZC-35797) was employed, with all experimental procedures strictly adhering to the manufacturer’s guidelines. Blood glucose concentrations were measured using glucose test strips (Roche Diagnostics, Shanghai, China) in compliance with the product instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrediction of the Gli3 binding sites in the promoter of TTR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSince the sequences of the TTR and Gli3 genes in mice and humans exhibit sufficient similarity and show high homology, we used human gene sequences for prediction\u003csup\u003e30,57\u003c/sup\u003e. The human TTR promoter sequences were obtained from the UCSC database (\u003ca href=\"http://genome.ucsc.edu/\"\u003ehttp://genome.ucsc.edu/\u003c/a\u003e). The region from 2000 bp upstream and 100 bp downstream of the transcriptional start site was considered the promoter region. The transcript IDs of human TTR was Human hg19 chr18:20797307-20798306, respectively. The Gli3 binding motif was retrieved from the JASPAR database (\u003ca href=\"http://jaspar.genereg.net/\" target=\"https://yiyan.baidu.com/chat/_blank\"\u003ehttp://jaspar.genereg.net/\u003c/a\u003e). The TTR promoter region was input into the JASPAR database to predict potential Gli3-binding sites, which were then ranked based on their scores. The top 3 binding sites were selected for ChIP validation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChIP\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChIP was conducted by Beijing Tsingke Biotech Co., Ltd. Briefly, DNA-protein crosslinking was achieved by treating cells with 1% formaldehyde at room temperature for 10 minutes. Cross-linked chromatin was subsequently sonicated in the lysis buffer provided with the kit, and soluble DNA fragments were collected following centrifugation at 20,000 ×g for 10 minutes. The resulting supernatants were used for ChIP assays. Protein A/G agarose beads conjugated with anti-Gli3 antibody or normal rabbit IgG (as a negative control) were added to the supernatants and incubated at 4°C for 4 hours. After incubation, the beads were washed twice using the kit-supplied wash buffer. Bound DNA was then eluted with elution buffer, decrosslinked at 65°C overnight, purified through phenol-chloroform-isoamyl alcohol extraction followed by ethanol precipitation, and subjected to qPCR. Following amplification, the PCR amplicons were resolved on a 2.5% ethidium bromide-stained agarose gel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIHC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIHC was conducted by Hubei Bios Biological Technology Co., Ltd. Paraffin sections were sequentially dewaxed in environmental dewaxing agents, hydrated through a graded ethanol series, and rinsed with distilled water. Antigen retrieval was then performed via high-pressure heat treatment using EDTA (pH 9.0), with timing set to 1.5 minutes after the onset of steam. After retrieval, endogenous peroxidase activity was blocked with 3% H₂O₂ for 30 minutes at room temperature in the dark, followed by section blocking with 10% species-matched serum (30 minutes at room temperature). Primary antibodies—P-ERK (1:100), P-CREB (1:100), and TTR (1:200)—diluted in 10% serum were added, and sections were incubated overnight at 4°C. After rewarming to room temperature and washing with TBST, TBST-diluted secondary antibody was added for incubation at 37 °C for 45 minutes, with additional TBST washes thereafter. Color development was conducted using either DAB or a red chromogenic system (including TY reaction solution and red chromogenic working solution); the reaction duration was monitored microscopically, and color development was terminated by tap water rinsing. Sections were counterstained with hematoxylin for 1 minute, differentiated in acid alcohol, and blued, then mounted with an environmental mounting medium. After air-drying, slides were examined under a microscope for image acquisition and analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll statistical analyses were performed by blinded operators with respect to experimental conditions, and data analysis was conducted using GraphPad Prism software (version 8.02). Results are presented as mean ± SEM. For comparisons between two groups, independent samples t-tests (two-tailed) were applied. For multi-group comparisons, statistical differences were evaluated using one-way analysis of variance (ANOVA) followed by Tukey's posthoc test for pairwise comparisons. Statistical significance was defined as \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Scientific and Technological Innovation Team of Shaanxi Province (2023-CX-TD-63), Technology Innovation Talent Engineering Project (2023RCZZ003), Science and Technology Research Projects (2024GJJH03-02), National Natural Science Foundation of China (82571721, 82571359, 82404591).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors and Affiliations\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eState Key Laboratory of Oral \u0026amp; Maxillofacial Reconstruction and Regeneration, National Clinical Research Center for Oral Diseases, Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture, Department of pharmacy, The Third Affiliated Hospital of the Fourth Military Medical University, 710032, Xi’an, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBeining Lu, Yongli Jiang, Qingjuan Guo, Yuxuan Yan, Liukun Yang, Dake Song, Cheng Zeng, Jiao Yue, Chen Zeng, Zixuan Liu, Xinshang Wang, Shuibing Liu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Pharmacology, School of Pharmacy, Fourth Military Medical University, Xi’an, 710032, China\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBeining Lu, Yongli Jiang, Fan Yang, Yuxuan Yan, Dake Song, Zixuan Liu, Xue Ma, Xinshang Wang, Shuibing Liu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMilitary Medical Innovation Center, Fourth Military Medical University, Xi’an 710032, China.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXinshang Wang.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDepartment of Endocrinology and Metabolism, Xijing Hospital of The Fourth Military Medical University\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJie Zhou, Nana Zhang\u003c/p\u003e\n\u003cp\u003eCorresponding author\u003c/p\u003e\n\u003cp\u003eCorrespondence to Xue Ma, Xinshang Wang, Shuibing Liu.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no conflict of interest exists.\u003c/p\u003e\n\u003cp\u003eStudy approval\u003c/p\u003e\n\u003cp\u003eThe animal experimental procedures were approved by the Animal Care and Use Committee of the Fourth Military Medical University (No. 20240015). And the human study protocol received ethical approval from the Medical Ethics Committee of the Xijing Hospital of Fourth Military Medical University (Approval No. KY20252243-F-1) and was prospectively registered with the Chinese Clinical Trial Registry (Registration No. ChiCTR 2500104257. https://www.chictr.org.cn/showproj.html?proj=275079). All participants provided written informed consent. The investigation was conducted in full accordance with the ethical principles outlined in the Declaration of Helsinki.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCollaborators, G. B. D. A. B. Global, regional, and national prevalence of adult overweight and obesity, 1990-2021, with forecasts to 2050: a forecasting study for the Global Burden of Disease Study 2021. \u003cem\u003eLancet\u003c/em\u003e\u003cstrong\u003e405\u003c/strong\u003e, 813-838, doi:10.1016/S0140-6736(25)00355-1 (2025).\u003c/li\u003e\n\u003cli\u003eFanelli, G.\u003cem\u003e et al.\u003c/em\u003e The interface of depression and diabetes: treatment considerations. \u003cem\u003eTransl Psychiatry\u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, 22, doi:10.1038/s41398-025-03234-5 (2025).\u003c/li\u003e\n\u003cli\u003eDrucker, D. J. Author Correction: GLP-1-based therapies for diabetes, obesity and beyond. \u003cem\u003eNat Rev Drug Discov\u003c/em\u003e\u003cstrong\u003e24\u003c/strong\u003e, 570, doi:10.1038/s41573-025-01231-3 (2025).\u003c/li\u003e\n\u003cli\u003eDrucker, D. J. The benefits of GLP-1 drugs beyond obesity. \u003cem\u003eScience\u003c/em\u003e\u003cstrong\u003e385\u003c/strong\u003e, 258-260, doi:10.1126/science.adn4128 (2024).\u003c/li\u003e\n\u003cli\u003eLi, S., Sabbah, S. G., Kwan, A. T. H. \u0026amp; McIntyre, R. S. Repurposing glucagon-like peptide-1 (GLP-1) receptor agonists for the treatment of depression: A systematic review of preclinical, observational and clinical investigations. \u003cem\u003eEur Neuropsychopharmacol\u003c/em\u003e\u003cstrong\u003e99\u003c/strong\u003e, 56-67, doi:10.1016/j.euroneuro.2025.08.002 (2025).\u003c/li\u003e\n\u003cli\u003eLlach, C. D.\u003cem\u003e et al.\u003c/em\u003e Glucagon-like Peptide-1 receptor agonists as emerging therapeutics in bipolar disorder: a narrative review of preclinical and clinical evidence. \u003cem\u003eMol Psychiatry\u003c/em\u003e, doi:10.1038/s41380-025-03261-0 (2025).\u003c/li\u003e\n\u003cli\u003eWang, M.\u003cem\u003e et al.\u003c/em\u003e Exploring potential associations between GLP-1RAs and depressive disorders: a pharmacovigilance study based on FAERS and VigiBase data. \u003cem\u003eEClinicalMedicine\u003c/em\u003e\u003cstrong\u003e86\u003c/strong\u003e, 103385, doi:10.1016/j.eclinm.2025.103385 (2025).\u003c/li\u003e\n\u003cli\u003eUeda, P.\u003cem\u003e et al.\u003c/em\u003e GLP-1 Receptor Agonist Use and Risk of Suicide Death. \u003cem\u003eJAMA Intern Med\u003c/em\u003e\u003cstrong\u003e184\u003c/strong\u003e, 1301-1312, doi:10.1001/jamainternmed.2024.4369 (2024).\u003c/li\u003e\n\u003cli\u003ePierret, A. C. S.\u003cem\u003e et al.\u003c/em\u003e Glucagon-Like Peptide 1 Receptor Agonists and Mental Health: A Systematic Review and Meta-Analysis. \u003cem\u003eJAMA Psychiatry\u003c/em\u003e\u003cstrong\u003e82\u003c/strong\u003e, 643-653, doi:10.1001/jamapsychiatry.2025.0679 (2025).\u003c/li\u003e\n\u003cli\u003e Kornelius, E., Huang, J. Y., Lo, S. C., Huang, C. N. \u0026amp; Yang, Y. S. The risk of depression, anxiety, and suicidal behavior in patients with obesity on glucagon like peptide-1 receptor agonist therapy. \u003cem\u003eSci Rep\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, 24433, doi:10.1038/s41598-024-75965-2 (2024).\u003c/li\u003e\n\u003cli\u003e Liu, Y. N.\u003cem\u003e et al.\u003c/em\u003e A pancreas-hippocampus feedback mechanism regulates circadian changes in depression-related behaviors. \u003cem\u003eNat Neurosci\u003c/em\u003e\u003cstrong\u003e28\u003c/strong\u003e, 2078-2091, doi:10.1038/s41593-025-02040-y (2025).\u003c/li\u003e\n\u003cli\u003e Yang, F.\u003cem\u003e et al.\u003c/em\u003e Glucagon-like Peptide 1 Receptor Activation Inhibits Microglial Pyroptosis via Promoting Mitophagy to Alleviate Depression-like Behaviors in Diabetic Mice. \u003cem\u003eNutrients\u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, doi:10.3390/nu15010038 (2022).\u003c/li\u003e\n\u003cli\u003e Gil-Lozano, M.\u003cem\u003e et al.\u003c/em\u003e Corticotropin-releasing hormone and the sympathoadrenal system are major mediators in the effects of peripherally administered exendin-4 on the hypothalamic-pituitary-adrenal axis of male rats. \u003cem\u003eEndocrinology\u003c/em\u003e\u003cstrong\u003e155\u003c/strong\u003e, 2511-2523, doi:10.1210/en.2013-1718 (2014).\u003c/li\u003e\n\u003cli\u003e Kinzig, K. P.\u003cem\u003e et al.\u003c/em\u003e CNS glucagon-like peptide-1 receptors mediate endocrine and anxiety responses to interoceptive and psychogenic stressors. \u003cem\u003eJ Neurosci\u003c/em\u003e\u003cstrong\u003e23\u003c/strong\u003e, 6163-6170, doi:10.1523/JNEUROSCI.23-15-06163.2003 (2003).\u003c/li\u003e\n\u003cli\u003e Anderberg, R. H.\u003cem\u003e et al.\u003c/em\u003e GLP-1 is both anxiogenic and antidepressant; divergent effects of acute and chronic GLP-1 on emotionality. \u003cem\u003ePsychoneuroendocrinology\u003c/em\u003e\u003cstrong\u003e65\u003c/strong\u003e, 54-66, doi:10.1016/j.psyneuen.2015.11.021 (2016).\u003c/li\u003e\n\u003cli\u003e Zemany, L.\u003cem\u003e et al.\u003c/em\u003e Transthyretin Antisense Oligonucleotides Lower Circulating RBP4 Levels and Improve Insulin Sensitivity in Obese Mice. \u003cem\u003eDiabetes\u003c/em\u003e\u003cstrong\u003e64\u003c/strong\u003e, 1603-1614, doi:10.2337/db14-0970 (2015).\u003c/li\u003e\n\u003cli\u003e Singhal, A., Chauhan, A., Goyal, P. \u0026amp; Taneja, A. Transthyretin - A Novel Biomarker for Insulin Resistance and Atherosclerosis Risk in Prediabetics. \u003cem\u003eJ Assoc Physicians India\u003c/em\u003e\u003cstrong\u003e69\u003c/strong\u003e, 11-12 (2021).\u003c/li\u003e\n\u003cli\u003e Gomes, J. R.\u003cem\u003e et al.\u003c/em\u003e Transthyretin provides trophic support via megalin by promoting neurite outgrowth and neuroprotection in cerebral ischemia. \u003cem\u003eCell Death Differ\u003c/em\u003e\u003cstrong\u003e23\u003c/strong\u003e, 1749-1764, doi:10.1038/cdd.2016.64 (2016).\u003c/li\u003e\n\u003cli\u003e Zhang, M.\u003cem\u003e et al.\u003c/em\u003e Cardiovascular and brain effects of liraglutide in transthyretin amyloidosis (ATTR) mice models. \u003cem\u003eInt J Med Sci\u003c/em\u003e\u003cstrong\u003e22\u003c/strong\u003e, 3229-3241, doi:10.7150/ijms.112264 (2025).\u003c/li\u003e\n\u003cli\u003e Lohia, S.\u003cem\u003e et al.\u003c/em\u003e Exploratory Study Analyzing the Urinary Peptidome of T2DM Patients Suggests Changes in ECM but Also Inflammatory and Metabolic Pathways Following GLP-1R Agonist Treatment. \u003cem\u003eInt J Mol Sci\u003c/em\u003e\u003cstrong\u003e24\u003c/strong\u003e, doi:10.3390/ijms241713540 (2023).\u003c/li\u003e\n\u003cli\u003e Nauck, M. A., Quast, D. R., Wefers, J. \u0026amp; Meier, J. J. GLP-1 receptor agonists in the treatment of type 2 diabetes - state-of-the-art. \u003cem\u003eMol Metab\u003c/em\u003e\u003cstrong\u003e46\u003c/strong\u003e, 101102, doi:10.1016/j.molmet.2020.101102 (2021).\u003c/li\u003e\n\u003cli\u003e Suriano, F.\u003cem\u003e et al.\u003c/em\u003e Novel insights into the genetically obese (ob/ob) and diabetic (db/db) mice: two sides of the same coin. \u003cem\u003eMicrobiome\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 147, doi:10.1186/s40168-021-01097-8 (2021).\u003c/li\u003e\n\u003cli\u003e Ji, A.\u003cem\u003e et al.\u003c/em\u003e Whey protein and soy protein prevent obesity by upregulating uncoupling protein 1 to activate brown adipose tissue and promote white adipose tissue browning in high-fat diet-fed mice. \u003cem\u003eFood Funct\u003c/em\u003e\u003cstrong\u003e13\u003c/strong\u003e, 12836-12851, doi:10.1039/d2fo01935c (2022).\u003c/li\u003e\n\u003cli\u003e Chan, Y. L.\u003cem\u003e et al.\u003c/em\u003e Short term exendin-4 treatment reduces markers of metabolic disorders in female offspring of obese rat dams. \u003cem\u003eInt J Dev Neurosci\u003c/em\u003e\u003cstrong\u003e46\u003c/strong\u003e, 67-75, doi:10.1016/j.ijdevneu.2015.05.009 (2015).\u003c/li\u003e\n\u003cli\u003e Piening, A.\u003cem\u003e et al.\u003c/em\u003e Obesity-related T cell dysfunction impairs immunosurveillance and increases cancer risk. \u003cem\u003eNat Commun\u003c/em\u003e\u003cstrong\u003e15\u003c/strong\u003e, 2835, doi:10.1038/s41467-024-47359-5 (2024).\u003c/li\u003e\n\u003cli\u003e Fleming, C. E., Saraiva, M. J. \u0026amp; Sousa, M. M. Transthyretin enhances nerve regeneration. \u003cem\u003eJ Neurochem\u003c/em\u003e\u003cstrong\u003e103\u003c/strong\u003e, 831-839, doi:10.1111/j.1471-4159.2007.04828.x (2007).\u003c/li\u003e\n\u003cli\u003e Fleming, C. E., Mar, F. M., Franquinho, F., Saraiva, M. J. \u0026amp; Sousa, M. M. Transthyretin internalization by sensory neurons is megalin mediated and necessary for its neuritogenic activity. \u003cem\u003eJ Neurosci\u003c/em\u003e\u003cstrong\u003e29\u003c/strong\u003e, 3220-3232, doi:10.1523/JNEUROSCI.6012-08.2009 (2009).\u003c/li\u003e\n\u003cli\u003e Nunes, A. F.\u003cem\u003e et al.\u003c/em\u003e Transthyretin knockout mice display decreased susceptibility to AMPA-induced neurodegeneration. \u003cem\u003eNeurochem Int\u003c/em\u003e\u003cstrong\u003e55\u003c/strong\u003e, 454-457, doi:10.1016/j.neuint.2009.07.001 (2009).\u003c/li\u003e\n\u003cli\u003e Fleming, C. E., Nunes, A. F. \u0026amp; Sousa, M. M. Transthyretin: more than meets the eye. \u003cem\u003eProg Neurobiol\u003c/em\u003e\u003cstrong\u003e89\u003c/strong\u003e, 266-276, doi:10.1016/j.pneurobio.2009.07.007 (2009).\u003c/li\u003e\n\u003cli\u003e Matissek, S. J. \u0026amp; Elsawa, S. F. GLI3: a mediator of genetic diseases, development and cancer. \u003cem\u003eCell Commun Signal\u003c/em\u003e\u003cstrong\u003e18\u003c/strong\u003e, 54, doi:10.1186/s12964-020-00540-x (2020).\u003c/li\u003e\n\u003cli\u003e Tang, C.\u003cem\u003e et al.\u003c/em\u003e Hippo signaling activates hedgehog signaling by Taz-driven Gli3 processing. \u003cem\u003eCell Regen\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 3, doi:10.1186/s13619-022-00151-6 (2023).\u003c/li\u003e\n\u003cli\u003e Zaky, D. A., Sayed, R. H. \u0026amp; Mohamed, Y. S. Liraglutide limits the immunogenic cell death-mediated ROS propagation and PI3K/AKT inactivation after doxorubicin-induced gonadotoxicity in rats: Involvement of the canonical Hedgehog trajectory. \u003cem\u003eInt Immunopharmacol\u003c/em\u003e\u003cstrong\u003e119\u003c/strong\u003e, 110212, doi:10.1016/j.intimp.2023.110212 (2023).\u003c/li\u003e\n\u003cli\u003e Happ, J. T.\u003cem\u003e et al.\u003c/em\u003e A PKA inhibitor motif within SMOOTHENED controls Hedgehog signal transduction. \u003cem\u003eNat Struct Mol Biol\u003c/em\u003e\u003cstrong\u003e29\u003c/strong\u003e, 990-999, doi:10.1038/s41594-022-00838-z (2022).\u003c/li\u003e\n\u003cli\u003e Xia, T., He, W., Luo, Z., Wang, K. \u0026amp; Tan, X. Achyranthes bidentata polysaccharide ameliorates type 2 diabetes mellitus by gut microbiota-derived short-chain fatty acids-induced activation of the GLP-1/GLP-1R/cAMP/PKA/CREB/INS pathway. \u003cem\u003eInt J Biol Macromol\u003c/em\u003e\u003cstrong\u003e270\u003c/strong\u003e, 132256, doi:10.1016/j.ijbiomac.2024.132256 (2024).\u003c/li\u003e\n\u003cli\u003e Blendy, J. A. The role of CREB in depression and antidepressant treatment. \u003cem\u003eBiol Psychiatry\u003c/em\u003e\u003cstrong\u003e59\u003c/strong\u003e, 1144-1150, doi:10.1016/j.biopsych.2005.11.003 (2006).\u003c/li\u003e\n\u003cli\u003e Sulser, F. The role of CREB and other transcription factors in the pharmacotherapy and etiology of depression. \u003cem\u003eAnn Med\u003c/em\u003e\u003cstrong\u003e34\u003c/strong\u003e, 348-356, doi:10.1080/078538902320772106 (2002).\u003c/li\u003e\n\u003cli\u003e Tang, Y.\u003cem\u003e et al.\u003c/em\u003e Jiao-tai-wan and its effective component-berberine improve diabetes and depressive disorder through the cAMP/PKA/CREB signaling pathway. \u003cem\u003eJ Ethnopharmacol\u003c/em\u003e\u003cstrong\u003e324\u003c/strong\u003e, 117829, doi:10.1016/j.jep.2024.117829 (2024).\u003c/li\u003e\n\u003cli\u003e Dinevska, M.\u003cem\u003e et al.\u003c/em\u003e CREB: A multifaceted transcriptional regulator of neural and immune function in CNS tumors. \u003cem\u003eBrain Behav Immun\u003c/em\u003e\u003cstrong\u003e116\u003c/strong\u003e, 140-149, doi:10.1016/j.bbi.2023.12.002 (2024).\u003c/li\u003e\n\u003cli\u003e Wang, W.\u003cem\u003e et al.\u003c/em\u003e Association of semaglutide with risk of suicidal ideation in a real-world cohort. \u003cem\u003eNat Med\u003c/em\u003e\u003cstrong\u003e30\u003c/strong\u003e, 168-176, doi:10.1038/s41591-023-02672-2 (2024).\u003c/li\u003e\n\u003cli\u003e McGowan, B.\u003cem\u003e et al.\u003c/em\u003e A systematic review and meta-analysis of the efficacy and safety of pharmacological treatments for obesity in adults. \u003cem\u003eNat Med\u003c/em\u003e\u003cstrong\u003e31\u003c/strong\u003e, 3317-3329, doi:10.1038/s41591-025-03978-z (2025).\u003c/li\u003e\n\u003cli\u003e Schoretsanitis, G., Weiler, S., Barbui, C., Raschi, E. \u0026amp; Gastaldon, C. Disproportionality Analysis From World Health Organization Data on Semaglutide, Liraglutide, and Suicidality. \u003cem\u003eJAMA Netw Open\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, e2423385, doi:10.1001/jamanetworkopen.2024.23385 (2024).\u003c/li\u003e\n\u003cli\u003e Shapiro, S. B.\u003cem\u003e et al.\u003c/em\u003e Glucagon-like peptide-1 receptor agonists and risk of suicidality among patients with type 2 diabetes: active comparator, new user cohort study. \u003cem\u003eBMJ\u003c/em\u003e\u003cstrong\u003e388\u003c/strong\u003e, e080679, doi:10.1136/bmj-2024-080679 (2025).\u003c/li\u003e\n\u003cli\u003e Kolling, L. J.\u003cem\u003e et al.\u003c/em\u003e Interaction of serotonin/GLP-1 circuitry in a dual preclinical model for psychiatric disorders and metabolic dysfunction. \u003cem\u003ePsychiatry Res\u003c/em\u003e\u003cstrong\u003e337\u003c/strong\u003e, 115951, doi:10.1016/j.psychres.2024.115951 (2024).\u003c/li\u003e\n\u003cli\u003e Holt, M. K.\u003cem\u003e et al.\u003c/em\u003e Modulation of stress-related behaviour by preproglucagon neurons and hypothalamic projections to the nucleus of the solitary tract. \u003cem\u003eMol Metab\u003c/em\u003e\u003cstrong\u003e91\u003c/strong\u003e, 102076, doi:10.1016/j.molmet.2024.102076 (2025).\u003c/li\u003e\n\u003cli\u003e Tokgoz, S.\u003cem\u003e et al.\u003c/em\u003e Activation of the HPA Axis Does Not Explain Nonresponsiveness to GLP-1R Agonist Treatment in Individuals With Type 2 Diabetes. \u003cem\u003eDiabetes\u003c/em\u003e\u003cstrong\u003e74\u003c/strong\u003e, 212-222, doi:10.2337/db24-0463 (2025).\u003c/li\u003e\n\u003cli\u003e Benson, M. D.\u003cem\u003e et al.\u003c/em\u003e Inotersen Treatment for Patients with Hereditary Transthyretin Amyloidosis. \u003cem\u003eN Engl J Med\u003c/em\u003e\u003cstrong\u003e379\u003c/strong\u003e, 22-31, doi:10.1056/NEJMoa1716793 (2018).\u003c/li\u003e\n\u003cli\u003e Adams, D.\u003cem\u003e et al.\u003c/em\u003e Hereditary transthyretin amyloid neuropathies: advances in pathophysiology, biomarkers, and treatment. \u003cem\u003eLancet Neurol\u003c/em\u003e\u003cstrong\u003e22\u003c/strong\u003e, 1061-1074, doi:10.1016/S1474-4422(23)00334-4 (2023).\u003c/li\u003e\n\u003cli\u003e Chompoopong, P., Mauermann, M. L., Siddiqi, H. \u0026amp; Peltier, A. Amyloid Neuropathy: From Pathophysiology to Treatment in Light-Chain Amyloidosis and Hereditary Transthyretin Amyloidosis. \u003cem\u003eAnn Neurol\u003c/em\u003e\u003cstrong\u003e96\u003c/strong\u003e, 423-440, doi:10.1002/ana.26965 (2024).\u003c/li\u003e\n\u003cli\u003e Yang, X.\u003cem\u003e et al.\u003c/em\u003e Neuroprotective Mechanisms of Glucagon-Like Peptide-1-Based Therapies in Ischemic Stroke: An Update Based on Preclinical Research. \u003cem\u003eFront Neurol\u003c/em\u003e\u003cstrong\u003e13\u003c/strong\u003e, 844697, doi:10.3389/fneur.2022.844697 (2022).\u003c/li\u003e\n\u003cli\u003e Holscher, C. Protective properties of GLP-1 and associated peptide hormones in neurodegenerative disorders. \u003cem\u003eBr J Pharmacol\u003c/em\u003e\u003cstrong\u003e179\u003c/strong\u003e, 695-714, doi:10.1111/bph.15508 (2022).\u003c/li\u003e\n\u003cli\u003e Li, S.\u003cem\u003e et al.\u003c/em\u003e Interleukin-13 and its receptor are synaptic proteins involved in plasticity and neuroprotection. \u003cem\u003eNat Commun\u003c/em\u003e\u003cstrong\u003e14\u003c/strong\u003e, 200, doi:10.1038/s41467-023-35806-8 (2023).\u003c/li\u003e\n\u003cli\u003e Sakamoto, K., Karelina, K. \u0026amp; Obrietan, K. CREB: a multifaceted regulator of neuronal plasticity and protection. \u003cem\u003eJ Neurochem\u003c/em\u003e\u003cstrong\u003e116\u003c/strong\u003e, 1-9, doi:10.1111/j.1471-4159.2010.07080.x (2011).\u003c/li\u003e\n\u003cli\u003e Guan, W., Ni, M. X., Gu, H. J. \u0026amp; Yang, Y. CREB: A Promising Therapeutic Target for Treating Psychiatric Disorders. \u003cem\u003eCurr Neuropharmacol\u003c/em\u003e\u003cstrong\u003e22\u003c/strong\u003e, 2384-2401, doi:10.2174/1570159X22666240206111838 (2024).\u003c/li\u003e\n\u003cli\u003e Jiang, Y. L.\u003cem\u003e et al.\u003c/em\u003e Tanshinone IIA improves contextual fear- and anxiety-like behaviors in mice via the CREB/BDNF/TrkB signaling pathway. \u003cem\u003ePhytother Res\u003c/em\u003e\u003cstrong\u003e36\u003c/strong\u003e, 3932-3948, doi:10.1002/ptr.7540 (2022).\u003c/li\u003e\n\u003cli\u003e Wang, X. S.\u003cem\u003e et al.\u003c/em\u003e Anxiolytic effects of Formononetin in an inflammatory pain mouse model. \u003cem\u003eMol Brain\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 36, doi:10.1186/s13041-019-0453-4 (2019).\u003c/li\u003e\n\u003cli\u003e Wang, X. S.\u003cem\u003e et al.\u003c/em\u003e Activation of G protein-coupled receptor 30 protects neurons by regulating autophagy in astrocytes. \u003cem\u003eGlia\u003c/em\u003e\u003cstrong\u003e68\u003c/strong\u003e, 27-43, doi:10.1002/glia.23697 (2020).\u003c/li\u003e\n\u003cli\u003e Slamova, I.\u003cem\u003e et al.\u003c/em\u003e Plasmin activity promotes amyloid deposition in a transgenic model of human transthyretin amyloidosis. \u003cem\u003eNat Commun\u003c/em\u003e\u003cstrong\u003e12\u003c/strong\u003e, 7112, doi:10.1038/s41467-021-27416-z (2021).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"GLP-1R, depression, TTR, Gli3, CREB signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-8322552/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8322552/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"GLP-1 receptor agonists are widely used to treat diabetes and obesity, yet their variable neuropsychiatric side effects including anxiety and depression in some individuals have remained mechanistically unknown. Here, we identify transthyretin (TTR) as a context-dependent mediator and biomarker for GLP-1R–driven affective behavior. We show that in normal-weight mice, hippocampal GLP-1R activation triggers a cAMP/PKA/Gli3 cascade that represses TTR expression, leading to impaired ERK/CREB-dependent synaptic plasticity and promotes anxiety- and depression-like behaviors. Conversely, in diet-induced obese models where hippocampal TTR is upregulated, and GLP-1R activation normalizes TTR levels, rescuing affective deficits. Translating these findings, we show that GLP-1R agonist reduces elevated serum TTR in obese humans. We therefore propose that baseline TTR levels serve as a critical biomarker: obese individuals with high TTR may achieve both metabolic and mental health benefits, whereas individuals with normal TTR levels are at risk for drug-induced affective disturbances. Our work establishes a molecular basis for the psychiatric side effects of GLP-1R-targeting therapies and provides a rationale for TTR-guided personalization of treatment.","manuscriptTitle":"Transthyretin gates GLP-1R-mediated affect by metabolic stat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-14 03:57:15","doi":"10.21203/rs.3.rs-8322552/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6baa26da-758f-4467-bfa1-35da7de12e2c","owner":[],"postedDate":"January 14th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":61055791,"name":"Biological sciences/Neuroscience/Emotion"},{"id":61055792,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases"}],"tags":[],"updatedAt":"2026-03-12T09:57:17+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-14 03:57:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8322552","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8322552","identity":"rs-8322552","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}