A method of screening or assessing risk of GLP-1 receptor agonist-related mood disorders

Abstract

This invention discloses a method for screening or assessing the risk of GLP-1 receptor agonist-related mood disorders. It reveals for the first time that TTR is a key molecular switch for the bidirectional mood effects of GLP-1 receptor agonists, and its expression level needs to be maintained within a specific physiological window to ensure mood homeostasis. This invention assesses the risk of developing mood disorders after using GLP-1 receptor agonists by detecting the expression level or activity of TTR in subjects. This invention also provides kits for assessing the mood side effects of GLP-1 receptor agonists, methods for screening candidate drugs for the prevention or treatment of related mood disorders, and pharmaceutical compositions containing TTR or its agonists. This invention elucidates the molecular mechanism by which GLP-1 receptor agonists inhibit TTR expression through the cAMP / PKA / Gli3 signaling pathway, thereby affecting the ERK / CREB pathway and synaptic plasticity, providing novel targets and strategies for precise drug use, risk warning, and intervention for related mood disorders using GLP-1 receptor agonists.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical preparation technology, specifically to a method for screening or assessing the risk of GLP-1 receptor agonist-related mood disorders. Background Technology

[0002] Glucagon-like peptide-1 receptor agonists (GLP-1RAs), such as exenatide and smegglutide, have become star drugs for the treatment of type 2 diabetes and obesity. However, with their increasingly widespread clinical application, a significant clinical paradox has gradually emerged: although GLP-1RAs can improve depressive symptoms in some diabetic patients, post-marketing surveillance has also revealed that they can induce anxiety, depression, and even suicidal ideation in some individuals. The unclear mechanism of this bidirectional emotional effect poses significant uncertainty and safety risks to clinical use.

[0003] Currently, there is a lack of effective predictive tools and intervention targets for GLP-1RA-induced mood disorders. Existing research has proposed mechanisms such as neurotransmitter imbalance or hypothalamic-pituitary-adrenal axis dysfunction, but these are fragmented and cannot explain why the drugs produce drastically different mood effects under different metabolic states (e.g., normal weight versus obesity / diabetes). Therefore, there is an urgent clinical need for a biomarker that can predict an individual's risk of emotional responses to GLP-1RAs, as well as strategies to avoid or treat their mood side effects. Summary of the Invention

[0004] The present invention aims to address the following problems existing in the prior art: to provide a biomarker that can predict or assess the risk of mood disorders in an individual after receiving GLP-1RAs treatment, to elucidate its molecular mechanism, and to provide corresponding drug screening methods and treatment strategies to achieve precise and safe use of GLP-1RAs.

[0005] To achieve the above objectives, the present invention provides the following technical solution: Selecting C57BL / 6J normal-weight mice, GLP-1R knockout (GLP-1R...) - / -Mice, including db / db spontaneously diabetic mice and high-fat diet-induced obese mice, were randomly divided into a saline control group, a GLP-1R agonist treatment group (exenatide or smegglutinide), a TTR-targeted intervention group (TTR knockdown via intravenous injection of eplontersen or intravenous reTTR supplementation), and a signaling pathway regulation group (PKA inhibitor H-89 or CREB agonist DIS). Anxiety and depression-like behaviors in mice were assessed using open field, elevated cruciate maze, tail suspension, and forced swimming tests. At the molecular level, RNA sequencing was used to screen for differentially expressed genes in the hippocampus. qRT-PCR, Western blot, and immunohistochemistry were used to detect the expression and activation of key molecules such as PKA, Gli3, TTR, AKT, ERK, and CREB in hippocampal tissue. Chromatin immunoprecipitation was used to verify the binding of Gli3 to the TTR promoter, and ELISA was used to detect serum TTR levels and blood glucose levels. Inclusion criteria included mice with a BMI of 29-45 kg / m². 2 In obese subjects, venous blood was collected before treatment with smegglutide and 1, 2, and 3 months after treatment. Serum was separated and changes in TTR levels were detected.

[0006] The beneficial effects of this invention are:

[0007] 1. For the first time, TTR is revealed as a core molecular switch for the emotional effects of GLP-1RAs, resolving a clinical paradox.

[0008] This invention, through systematic molecular biology, behavioral science, and clinical sample validation, is the first to discover and confirm that TTR is a key molecular switch determining the bidirectional mood effects of GLP-1RAs. Research shows that TTR must be maintained within a narrow physiological "functional window" to ensure mood homeostasis. In overweight or diabetic states, TTR is pathologically elevated, and GLP-1RAs exert their beneficial anti-anxiety / depressive effects by downregulating it to normal levels; conversely, in normal-weight states, where TTR levels are already normal, intervention with GLP-1RAs suppresses it below the functional threshold, leading to mood deficits. This discovery perfectly explains the paradox of GLP-1RAs "improving mood while inducing anxiety / depression" that has plagued clinical practice for many years, providing a unified theoretical framework for the central nervous system side effects mechanism of this class of drugs and filling a theoretical gap in this field.

[0009] 2. Provide reliable predictive biomarkers to achieve accurate risk stratification.

[0010] This invention establishes TTR as a biomarker for predicting mood risk associated with GLP-1RAs. By detecting TTR levels (including TTR mRNA or protein levels in hippocampal tissue or serum) in subjects before or early in the course of medication, their risk of developing mood disorders can be effectively assessed.

[0011] If the baseline TTR level is normal or low, it suggests that the individual has a higher risk of developing anxiety and depression after taking the medication.

[0012] If the TTR level continues to decline after medication, it suggests that emotional side effects are occurring or about to occur.

[0013] Based on this, the present invention provides a kit containing TTR detection reagents, which can be used for risk screening before clinical medication and dynamic monitoring during medication, providing objective basis for clinicians to formulate individualized medication plans, fundamentally changing the current dilemma of "unpredictable and passive response" to the emotional side effects of GLP-1RAs.

[0014] 3. To open up entirely new drug intervention targets and provide effective strategies for treating mood side effects.

[0015] This invention clarifies the central role of TTR and its downstream ERK / CREB signaling pathway in emotion regulation, providing a novel drug target for the prevention and treatment of GLP-1RAs-induced mood disorders.

[0016] Targeted upregulation of TTR: By screening compounds that can upregulate TTR expression or enhance TTR activity, protective drugs specifically designed to alleviate the mood side effects of GLP-1RAs can be developed.

[0017] Direct TTR supplementation: This invention demonstrates that systemic supplementation of recombinant TTR (reTTR) can completely reverse GLP-1R agonist-induced anxiety / depression-like behavior and restore the normal expression of synaptic plasticity-related proteins (PSD95, Drebrin, GluA1, GluN2B).

[0018] Activation of downstream pathways: This invention also demonstrates that activating the CREB signaling pathway downstream of TTR can also salvage GLP-1RAs-induced mood deficits and synaptic damage.

[0019] Based on this, the present invention provides pharmaceutical compositions comprising TTR or its agonists, and combination therapy regimens of TTR and GLP-1RAs, providing a practical treatment option for patients experiencing the mood side effects of GLP-1RAs in clinical practice.

[0020] 4. Elucidate the complete molecular regulatory mechanism and provide a multi-level drug screening model.

[0021] This invention fully elucidates the molecular mechanism chain from "receptor activation" to "behavioral phenotype": GLP-1R activation, cAMP / PKA signaling activation, activation of the transcriptional repressor Gli3, TTR transcriptional repression, decreased TTR protein levels, impaired ERK / CREB pathway activity, downregulation of synaptic plasticity-related protein expression, and the occurrence of emotional defects.

[0022] Based on the revelation of this mechanism, this invention provides a multi-level drug screening model:

[0023] Screening models targeting TTR: can be used to screen compounds that regulate TTR expression;

[0024] Screening models targeting Gli3: can be used to screen compounds that intervene in downstream transcriptional regulation of GLP-1R;

[0025] Screening models targeting ERK / CREB can be used to screen protective compounds that repair synaptic function.

[0026] Cell-based signaling pathway reporting systems can be used for high-throughput screening of compounds that can block or reverse the GLP-1R signaling pathway.

[0027] These screening models provide a wide range of tools for new drug development, significantly reducing research and development costs and improving screening efficiency.

[0028] 5. Cross-species validation shows promising prospects for clinical translation.

[0029] The findings of this invention have not only been rigorously validated in various mouse models (normal-weight mice, db / db diabetic mice, and high-fat diet-induced overweight mice), but have also received preliminary confirmation in clinical human samples: obese patients treated with the GLP-1R agonist semaglutide exhibited a dynamic pattern of "transient decline followed by recovery" in serum TTR levels consistent with the mouse models. This cross-species consistency suggests that TTR has promising clinical translational potential as a biomarker and therapeutic target.

[0030] 6. It has a wide range of applications and significant social and economic benefits.

[0031] Application scenarios of this invention include, but are not limited to:

[0032] In the field of diagnostic products: developing TTR detection kits for pre-medication risk assessment and monitoring during medication use;

[0033] In the pharmaceutical field: developing novel drugs targeting TTR for the prevention and treatment of GLP-1RAs-related mood disorders;

[0034] In the clinical field: guiding individualized precision medicine of GLP-1RAs, improving treatment safety, and improving patients' quality of life;

[0035] In the field of scientific research: it provides new tools and ideas for studying the mechanisms of emotion regulation and the central nervous system side effects of drugs.

[0036] With the widespread use of GLP-1RAs (such as semaglutide and liraglutide) in the treatment of diabetes and obesity, the patient population is growing rapidly, and the problem of emotional side effects is becoming increasingly prominent. The implementation of this invention will significantly improve the safety of this type of drug, reduce discontinuation and treatment failure due to emotional side effects, and has significant social and economic benefits.

[0037] In summary, this invention redefines GLP-1RAs-related mood changes from "unpredictable adverse reactions" to "mechanism-based and potentially predictable outcomes," laying a solid scientific foundation for the practice of precision neurometabolic medicine and providing comprehensive technical solutions for the diagnosis, treatment, and drug development of related diseases. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0039] Figure 1 GLP-1R activation improves anxiety / depression-like behavior in diabetic and overweight mice.

[0040] Figure 2 GLP-1R activation induces anxiety / depression-like behavior in normal-weight mice.

[0041] Figure 3 GLP-1R activation downregulated TTR expression in the hippocampus of normal-weight mice.

[0042] Figure 4 GLP-1R activation can transiently reduce serum TTR levels in mice and humans, and TTR levels are associated with emotional behavior.

[0043] Figure 5 TTR is a core molecule essential for GLP-1R to mediate mood deficits in normal-weight mice.

[0044] Figure 6 Metabolic state determines the bidirectional role of TTR in GLP-1R-mediated emotional behavior.

[0045] Figure 7 GLP-1R activation inhibits TTR expression through the cAMP / PKA / Gli3 transcriptional pathway.

[0046] Figure 8 TTR regulates emotional behavior through the ERK / CREB signaling pathway.

[0047] Figure 9 CREB activation can salvage synaptic and emotional deficits induced by the GLP-1R signaling pathway. Detailed Implementation

[0048] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] TTR is a key molecule that integrates GLP-1R signaling with metabolic state to regulate anxiety / depression; its expression level needs to be maintained within a specific physiological window, and both excessively high and low levels can disrupt mood homeostasis. Furthermore, GLP-1R agonists can inhibit TTR expression by activating the cAMP / PKA / Gli3 signaling pathway, thereby downregulating ERK / CREB pathway activity, impairing hippocampal synaptic plasticity, and inducing anxiety / depression-like behaviors. Simultaneously, serum TTR levels may serve as a biomarker for the prognosis of both metabolic and mood disorders, providing a theoretical basis for the personalized application of GLP-1R agonists, and offering new targets and strategies for the intervention of metabolic-mood comorbidities, thus promoting the translational practice of precision medicine in the field of metabolic diseases.

[0050] To demonstrate this application, the following research was conducted:

[0051] The effects of GLP-1R activation on anxiety / depression are metabolically dependent.

[0052] In db / db diabetic and high-fat diet-induced obese mice, GLP-1R activation significantly improved their anxiety / depression-like behaviors, and this effect was independent of blood glucose changes. Conversely, in normal-weight mice, the same treatment induced significant anxiety and depression-like behaviors; this effect disappeared in GLP-1R knockout mice, confirming its receptor specificity and suggesting that metabolic state determines the direction of GLP-1R's emotional regulation.

[0053] Activation of GLP-1R downregulates TTR, and TTR levels are negatively correlated with anxiety / depression.

[0054] To elucidate the molecular mechanism by which GLP-1R activation induces anxiety / depression-like behavior in normal-weight mice, we used RNA sequencing, qRT-PCR, and Western blot analysis to find that exenatide significantly downregulated the mRNA and protein levels of TTR in the hippocampus of normal-weight mice. Further ELISA analysis of serum TTR levels revealed a significant decrease on days 3 and 5 after administration, with partial recovery by day 7, exhibiting dynamic fluctuations, which were negatively correlated with anxiety / depression-like behavior. In clinical validation, serum TTR in obese subjects also significantly decreased after one month of treatment with semaglutide, returning to baseline after 2–3 months, consistent with the trend in the mouse model, suggesting that TTR may be a potential biomarker in the mood regulation process of GLP-1R agonists.

[0055] Downregulation of TTR is a key step in GLP-1R activation-induced anxiety / depression in normal-weight mice.

[0056] To clarify the role of TTR in mediating GLP-1R-mediated mood regulation, this study conducted loss-of-function and recovery experiments. First, the TTR-targeting antisense oligonucleotide eplontersen was locally injected into the hippocampus of normal-weight mice, specifically knocking down its expression. Results showed that TTR mRNA and protein levels in the mouse hippocampus were significantly reduced, followed by exenatide-induced anxiety / depression-like behaviors, successfully mimicking the emotional phenotype induced by GLP-1R activation. Furthermore, supplementing with reTTR via tail vein injection revealed that exogenous reTTR not only corrected the exenatide-induced hippocampal TTR downregulation but also effectively improved the exenatide-induced anxiety / depression-like behaviors. These results suggest that TTR downregulation induced by GLP-1R activation in normal-weight mice is key to inducing anxiety / depression.

[0057] Metabolic state determines the dual role of TTR in the regulation of anxiety / depression-like behavior by GLP-1R.

[0058] To investigate the role of metabolic state in the regulation of emotional behavior by TTR, we examined the changes in TTR levels in the hippocampus and serum of diabetic and overweight mice after GLP-1R activation. Western blot results showed that, compared with wild-type mice, there were no significant differences in hippocampal and serum TTR levels in db / db mice, and the GLP-1R agonist exenatide did not affect TTR levels in db / db mice. TTR levels in the hippocampus and serum of overweight mice were significantly higher than in the control group, and exenatide could restore them to normal physiological levels. Correlation analysis showed that before exenatide treatment, baseline serum TTR levels in overweight mice were positively correlated with the severity of anxiety-like behaviors; after treatment, this correlation disappeared as TTR levels returned to normal, suggesting that TTR normalization is key to behavioral improvement. To verify that TTR upregulation can also induce anxiety / depression, we exogenously supplemented normal-weight mice with reTTR, and the results showed that the mice rapidly developed significant anxiety / depression-like behaviors. These results suggest that TTR function is bidirectional and needs to be maintained at a physiological level to ensure emotional homeostasis.

[0059] GLP-1R activation inhibits TTR expression by enhancing cAMP / PKA / Gli3 signaling transcription.

[0060] To elucidate the molecular mechanism by which GLP-1R activation downregulates TTR expression, we used the UCSC Genome Explorer database to predict and screen for candidate molecules of Gli3, a transcription factor associated with the GLP-1R signaling pathway, whose binding motif is highly enriched in the TTR promoter region. Chromatin immunoprecipitation experiments showed that exenatide enhanced the specific binding of Gli3 to specific regions of the TTR promoter, suggesting that Gli3 may directly regulate TTR transcription. ELISA and Western blot analysis showed that exenatide intervention significantly upregulated cAMP levels, PKA phosphorylation levels, and Gli3 mRNA and protein expression in the hippocampus of normal-weight mice, indicating that GLP-1R activation can effectively initiate the cAMP / PKA / Gli3 signaling cascade. Pretreatment with the PKA-specific inhibitor H-89 completely blocked exenatide-induced PKA / Gli3 pathway activation, TTR expression downregulation, and the resulting anxiety / depression-like behavioral phenotype. The results suggest that GLP-1R activation in normal-weight mice inhibits TTR expression by enhancing cAMP / PKA / Gli3 signaling transcription, thus inducing mood disorders.

[0061] TTR regulates synaptic plasticity and emotional behavior through the ERK / CREB pathway.

[0062] To further elucidate the downstream molecular mechanisms by which TTR regulates anxiety / depression, we examined the activity of the ERK / CREB pathway in the hippocampus, which regulates synaptic plasticity and neuronal energy metabolism. Western blot and immunohistochemical results showed that exenatide significantly reduced the phosphorylation levels of ERK and CREB in the hippocampus of normal-weight mice; Eplontersen-mediated TTR knockdown mimicked this effect, suggesting that TTR downregulation is sufficient to inhibit ERK / CREB pathway activation. Conversely, reTTR supplementation completely corrected the downregulation of ERK / CREB expression caused by GLP-1R activation, further confirming that TTR is an upstream molecule in this pathway. Furthermore, PKA inhibitors eliminated the inhibitory effect of GLP-1R activation on ERK / CREB phosphorylation, indicating that this pathway is regulated by the upstream cAMP / PKA signaling cascade.

[0063] To verify the role of the ERK / CREB pathway in TTR-mediated mood regulation, we intervened with the CREB-specific agonist 3',6-disinyl sucrose (DIS). DIS significantly ameliorated exenatide-induced anxiety / depression-like behaviors in normal-weight mice, while simultaneously inhibiting the expression and downregulation of synaptic markers (PSD95, Drebrin) and glutamate receptors (GluA1, GluN2B). These results indicate that TTR alleviates anxiety / depression by regulating the ERK / CREB signaling pathway and thus affecting hippocampal synaptic plasticity.

[0064] Experimental plan:

[0065] (1) The differential effects of GLP-1R activation on anxiety / depression-like behavior in mice under different metabolic states.

[0066] 1) Activation of GLP-1R improves anxiety / depression-like behavior in mice with metabolic disorders.

[0067] To investigate the regulatory role of GLP-1R agonists in metabolically impaired mood disorders, this study evaluated the effects of the GLP-1R agonist exenatide on anxiety / depression-like behaviors in two metabolically impaired mouse models. First, leptin receptor-deficient db / db mice were used as a type 2 diabetes model, and their mood-related phenotypes were assessed using a series of behavioral paradigms. The results showed that GLP-1R activation had a clear anti-anxiety and antidepressant effect in diabetic mice. Next, we validated this effect in a high-fat diet-induced overweight mouse model. These mice exhibited a significantly elevated Lee's index, but maintained normal blood glucose levels, suggesting a simple overweight state. The results indicate that exenatide intervention effectively improved anxiety-like behaviors in overweight mice, and this phenomenon may be independent of its glucose-lowering effect.

[0068] 2) Activation of GLP-1R induces anxiety / depression-like behavior in normal-weight mice.

[0069] To investigate the effects of GLP-1R activation on anxiety / depression-like behavior in normal-weight mice, this study used time- and concentration-gradient dosing to determine and validate the optimal conditions for exenatide (Ex4) to induce mood-related phenotypes. Time-gradient dosing experiments (5 μg / kg / d for 1, 3, 5, and 7 days) showed that 3 days of dosing stably induced anxiety / depression-like behavior in mice. Based on these dosing durations, concentration-gradient experiments (2.5, 5, and 10 μg / kg / d for 3 consecutive days) determined the optimal dose. Finally, the results were replicated under the optimal conditions (5 μg / kg / d for 3 consecutive days), and the results were consistent with the behavioral phenotypes described above. Furthermore, this dosing regimen did not affect blood glucose levels. To confirm the GLP-1R specificity of the above effects and their universality among different agonists, this study treated normal-weight mice with another GLP-1R agonist, smegglutide. The results showed that smegglutide also induced anxiety / depression-like behavior in mice without affecting blood glucose levels. Subsequently, to verify the receptor-dependent nature of this effect, the exenatide intervention experiment was repeated in GLP-1R knockout mice. Behavioral assessments showed that GLP-1R activation was sufficient to induce emotional and behavioral deficits in normal-weight mice, and that this effect was receptor-specific and not a secondary consequence of blood glucose changes.

[0070] (2) GLP-1R activation induces anxiety / depression-like behavior in mice by downregulating hippocampal TTR expression.

[0071] 1) Activation of GLP-1R downregulates TTR expression in the hippocampus of normal-weight mice.

[0072] ① To investigate the molecular mechanism by which GLP-1R activation induces anxiety / depression-like behavior in normal-weight mice, we performed RNA sequencing analysis on the hippocampus, a core brain region for emotion regulation. Combining volcano plots and heatmaps, we analyzed and screened each of the identified differentially expressed genes, and conducted a literature review based on disease relevance. We found that TTR expression was stably downregulated and significantly differentiated across all samples. Previous studies have clearly demonstrated that TTR participates in the regulation of central nervous system function, playing a crucial role in neuronal growth and synapse formation, while other differentially expressed genes have not been reported to be related to the central nervous system. Therefore, TTR was identified as a core candidate molecule for further validation.

[0073] ② The expression level of TTR was validated at multiple levels using qRT-PCR, Western blot, and immunohistochemistry. The results showed that, compared with the Saline group, exenatide treatment significantly reduced the mRNA and protein expression levels of TTR in the mouse hippocampus. These results indicate that GLP-1R activation specifically downregulates TTR expression in the hippocampus, suggesting that TTR may be a key downstream molecule mediating GLP-1R-related emotional and behavioral changes.

[0074] 2) Activation of GLP-1R transiently reduces serum TTR levels in mice and humans.

[0075] ① To clarify whether TTR is involved in GLP-1R-mediated emotion regulation, we systematically examined changes in circulating TTR levels in mice and humans after GLP-1R activation. In normal-weight mice, exenatide administration resulted in a transient decrease in serum TTR levels: compared with the Saline group, serum TTR levels were significantly lower on days 3 and 5 after exenatide treatment, returning to baseline levels on day 7. Further correlation analysis on mice on day 3 (the point of most significant TTR decrease) revealed a close correlation between serum TTR levels and anxiety / depression-like behavioral phenotypes: lower TTR levels were associated with shorter exploration time in the central region of the open field test, shorter entry time into the open arms of the elevated cruciate maze, and longer immobility time in tail suspension and forced swimming.

[0076] ② Next, we further validated the above findings in clinical samples. We analyzed serum TTR levels in obese subjects before and after semaglutide treatment. ELISA results showed that after one month of treatment, serum TTR levels in subjects were significantly lower than before treatment, while after two and three months of treatment, TTR levels returned to baseline levels. These animal and clinical studies are consistent with the findings that GLP-1R activation can induce a transient decrease in circulating TTR levels. Combined with the significant correlation between TTR levels and emotional behavior observed in normal-weight mice, this suggests that TTR may serve as a potential dynamic biomarker for the regulation of emotional function by GLP-1R agonists.

[0077] 3) TTR is a key molecule that activates GLP-1R to induce anxiety / depression in mice.

[0078] ① To clarify the functional necessity of TTR in GLP-1R activation-induced emotional and behavioral abnormalities, this study used local injection of TTR-targeting antisense oligonucleotides (Eplontersen) into the hippocampus to knock down TTR expression. qRT-PCR and Western blot results showed that Eplontersen effectively reduced the mRNA and protein levels of hippocampal TTR. Behavioral tests showed that mice in the Eplontersen group exhibited significant anxiety / depression-like phenotypes.

[0079] ② To investigate whether restoring TTR expression could reverse exenatide-induced behavioral abnormalities, reTTR was administered via tail vein in combination with exenatide. Western blot results showed that reTTR blocked exenatide-induced downregulation of hippocampal TTR protein. Behavioral testing showed that reTTR intervention completely reversed exenatide-induced anxiety / depression-like behaviors. In summary, knocking down hippocampal TTR can mimic the emotional and behavioral deficits caused by GLP-1R activation, while TTR supplementation can effectively block its effects. These experimental data suggest that TTR is a key molecule in the GLP-1R activation-induced emotional dysfunction in normal-weight mice.

[0080] (3) Metabolic state determines the bidirectional role of TTR in the regulation of anxiety / depression by GLP-1R.

[0081] 1) Activation of GLP-1R can reduce abnormally elevated TTR levels in obese mice and alleviate their anxiety / depression-like behavior:

[0082] To investigate whether TTR is involved in the mood-improving effect induced by GLP-1R activation in a metabolically abnormal mouse model, this study examined the changes in TTR expression in the hippocampus and serum of db / db diabetic mice and high-fat diet-induced obese mice. Western blot results showed that the TTR expression levels in the hippocampus and serum of db / db mice were not significantly different from those of WT mice, and exenatide treatment did not alter the TTR levels in this model mouse. In contrast, the TTR levels in the hippocampus and serum of obese mice were significantly higher than those in the control group, but exenatide treatment restored them to control levels. Further analysis of the dynamic changes in TTR in obese mice before and after exenatide treatment and their correlation with affective behavioral indicators revealed that before exenatide treatment, serum TTR levels were significantly negatively correlated with dwell time in the central region of the open field test and dwell time in the elevated cruciate open arm; however, these correlations disappeared after exenatide treatment.

[0083] 2) Supplementing with reTTR induced anxiety / depression-like behavior in normal-weight mice.

[0084] To verify the causal role of elevated TTR expression levels in inducing anxiety / depression-like behaviors, this study treated normal-weight mice with reTTR (5 μg / kg, for 3 consecutive days). Behavioral assessments showed that reTTR treatment induced significant anxiety and depression-like phenotypes in mice. In the open field test, compared with the Saline group, the reTTR group mice exhibited significantly shorter exploration distances, times, and total distances in the central region. In the elevated cruciate maze test, the reTTR group mice had shorter time to enter the open arms, longer time to remain in the closed arms, and a significantly reduced total number of arm entries. In the tail suspension test and forced swimming test, the reTTR group mice also showed significantly longer immobility times.

[0085] (4) TTR mediates the signaling pathway of GLP-1R regulating anxiety / depression.

[0086] 1) GLP-1R activation inhibits TTR expression through the cAMP / PKA / Gli3 pathway.

[0087] ① To elucidate the molecular mechanism by which GLP-1R activation downregulates TTR expression, this study first used the UCSC Genome Browser database to perform bioinformatics screening of transcription factors that could potentially bind to the TTR promoter region. Among the top 20 candidate transcription factors with the highest predicted scores, combined with a literature review, GLI family zinc finger protein 3 was the only factor that met the following screening criteria: functional association with the GLP-1R signaling pathway, transcriptional repressive activity, and expression in the hippocampus. JASPAR database prediction showed three potential Gli3 binding sites in the TTR promoter region; chromatin immunoprecipitation experiments confirmed that Gli3 can specifically bind to these sites, with site 2 showing the highest enrichment. Given that cAMP / PKA is a classic downstream pathway of GLP-1R, and that PKA can convert Gli3 into a molecule with transcriptional repressive activity through phosphorylation modification, this study hypothesizes that GLP-1R activation inhibits TTR expression through the cAMP / PKA / Gli3 signaling pathway. Experimental results showed that exenatide treatment significantly increased cAMP levels in the hippocampus of mice, upregulated the expression of phosphorylated PKA and total PKA, and increased the production of Gli3 fragments with transcriptional repression activity.

[0088] ② To verify the causal relationship between this pathway and behavioral phenotype, normal-weight mice were administered the PKA inhibitor H-89 in combination with exenatide. Behavioral assessment showed that H-89 completely reversed exenatide-induced anxiety and depression-like behaviors. Compared with the exenatide group, mice in the combined administration group showed increased exploration distance and time in the central open field area, with no change in total movement distance; increased time spent with arms open on the elevated cruciate prism, decreased time spent with arms closed, and increased total number of arm advances; and shortened immobility time in tail suspension and forced swimming. Western blot results further confirmed that H-89 could block exenatide-induced PKA / Gli3 pathway activation and TTR expression downregulation. These results indicate that GLP-1R activation induces anxiety / depression-like behaviors in normal-weight mice through TTR expression inhibition mediated by the cAMP / PKA / Gli3 pathway.

[0089] 2) ERK / CREB is a key downstream effector of TTR in regulating anxiety / depression.

[0090] ① To investigate the downstream signaling mechanism by which TTR regulates anxiety / depression-like behavior, we verified whether the ERK / AKT / CREB pathway is involved in GLP-1R activation-mediated behavioral effects, as studies have reported that TTR can promote neurite growth and exert neuroprotective effects through this pathway. Western blot results showed that exenatide treatment significantly inhibited the phosphorylation levels and total protein expression of ERK and CREB in the hippocampus of normal-weight mice, while having no significant effect on AKT expression. Immunohistochemical experiments further confirmed that the immunopositive signals of p-ERK and p-CREB in the hippocampus of mice were significantly reduced after exenatide injection.

[0091] ② To clarify whether the above signaling pathway changes depend on TTR, the effects of TTR knockdown and replenishment on this pathway were examined. Western blot showed that reTTR replenishment reversed the inhibitory effects of exenatide on ERK and CREB phosphorylation and total protein, but had no effect on AKT. Conversely, Eplontersen-mediated TTR knockdown mimicked the exenatide effect, significantly inhibiting ERK and CREB phosphorylation and total protein expression, as well as P-CREB / CREB, but had no significant effect on P-AKT / AKT. Furthermore, the PKA inhibitor H-89 blocked the exenatide-induced downregulation of ERK and CREB phosphorylation and total protein, suggesting that the ERK / CREB pathway is downstream of the cAMP / PKA / Gli3 / TTR signaling axis. These results indicate that GLP-1R activation inhibits ERK / CREB signaling pathway activity by downregulating TTR expression, and this pathway is a key effector mechanism by which GLP-1R activation induces abnormal emotional behavior in mice.

[0092] 3) Activating CREB reverses GLP-1R-induced synaptic plasticity damage and emotional and behavioral deficits.

[0093] ① Based on the well-established role of CREB in the regulation of anxiety and depression, this study further verified whether CREB activation could reverse GLP-1R agonist-induced affective and behavioral abnormalities. Normal-weight mice were administered a combination of intraperitoneal injections of exenatide and the CREB agonist 3',6-disinyl sucrose (DIS).

[0094] ② CREB participates in the regulation of synaptic plasticity by modulating the expression of genes related to synaptic structure and function. Western blot results showed that DIS treatment could inhibit the exenatide-induced downregulation of postsynaptic scaffold protein PSD95 and synaptic morphology regulator Drebrin. Further examination of glutamate receptor expression and phosphorylation levels revealed that DIS treatment could completely block the exenatide-induced downregulation of total GluA1 and GluN2B protein expression and restore the phosphorylation levels of key sites: GluA1 Ser831 and Ser845, and GluN2B Ser1303 and Thr1472. These results indicate that TTR mediates GLP-1R activation-induced emotional behavioral abnormalities by inhibiting the CREB signaling pathway; activating CREB can restore the expression of synaptic-related proteins and reverse abnormal behavioral phenotypes, suggesting that the balance of the CREB pathway plays a central role in emotion regulation.

[0095] We first assessed the effects of GLP-1R activation on emotional and behavioral changes in a metabolically impaired mouse model, such as... Figure 1 As shown, behavioral results suggest that GLP-1R activation has anxiolytic and antidepressant-like effects in diabetic mice, with *p<0.05, **p<0.01, and ***p<0.001. Next, we examined high-fat diet (HFD) induced overweight (OW) mice, a model of mice with significantly elevated Lee's index but no hyperglycemia. These mice exhibited anxiety-like behaviors in the open field test and elevated cruciate maze, but not depressive-like behaviors in the tail suspension test or forced swimming test. Exenatide (Ex4) intervention improved anxiety-like behaviors in overweight mice without affecting normal blood glucose levels.

[0096] like Figure 2 As shown, GLP-1R activation induced anxiety / depression-like behavior in normal-weight mice, with *p<0.05, **p<0.01, and ***p<0.001. Using a time-gradient exenatide (Ex4) dosing regimen (5 μg / kg / day, treated for 1, 3, 5, and 7 days), we found that the 3-day treatment induced the most stable anxiety / depression-like phenotype. Subsequent dose-response studies determined that 5 μg / kg / day for 3 consecutive days was the optimal dosing regimen. In summary, these data indicate that GLP-1R activation is sufficient to induce mood deficits in normal-weight mice.

[0097] like Figure 3As shown, GLP-1R activation downregulated TTR expression in the hippocampus of normal-weight mice, with *p<0.05, **p<0.01, and ***p<0.001. To investigate the molecular mechanism by which GLP-1R activation induces anxiety / depression-like behavior in normal-weight mice, we performed RNA sequencing (RNA-seq) on hippocampal tissue. The results suggest that TTR may be a key downstream mediator of GLP-1R-induced mood deficits in normal-weight mice.

[0098] like Figure 4 As shown, GLP-1R activation transiently reduced serum TTR levels in mice and humans, with *p<0.05, **p<0.01, and ***p<0.001. To investigate the relevance of TTR in GLP-1R-mediated emotional behavior, we examined circulating TTR levels in mice and humans after GLP-1R activation. Cross-species data indicated that GLP-1R activation induced a transient decrease in circulating TTR, and TTR fluctuations were associated with emotional behavior in normal-weight mice, supporting its potential role as a transformable biomarker for GLP-1RAs-mediated emotion regulation.

[0099] like Figure 5 As shown, TTR is a core molecule essential for GLP-1R-mediated mood deficits in normal-weight mice, with *p<0.05, **p<0.01, and ***p<0.001. To clarify whether TTR is necessary for GLP-1R-induced anxiety / depression-like behavior, we administered recombinant TTR via tail vein injection to normal-weight mice via local hippocampal injection of Epronsen. These loss-of-function and function recovery experiments confirmed that TTR is both a necessary and sufficient factor in mediating GLP-1R activation-induced mood impairment in normal-weight mice.

[0100] like Figure 6As shown, metabolic state determines the bidirectional role of TTR in GLP-1R-mediated emotional behavior, with *p<0.05, **p<0.01, and ***p<0.001. Next, we used Western blotting to investigate whether TTR is involved in regulating the mood improvement of diabetic and overweight (OW) mice by GLP-1R activation. To confirm the causal role of elevated TTR in promoting anxiety / depression-like behavior, we treated normal-weight mice with recombinant TTR (reTTR, 5 μg / kg / day for 3 consecutive days). The results established TTR as a bidirectional regulator of emotional behavior—its level must be maintained within physiological limits to ensure emotional homeostasis. In overweight mice, GLP-1R activation produced an anti-anxiety effect by restoring pathologically elevated TTR to normal; conversely, in normal-weight mice, the same intervention suppressed physiological levels of TTR, leading to mood deficits.

[0101] like Figure 7 As shown, GLP-1R activation inhibits TTR expression through the cAMP / PKA / Gli3 transcriptional pathway, where *p<0.05, **p<0.01, ***p<0.001. To elucidate the molecular mechanism by which GLP-1R activation downregulates TTR expression, we first used the UCSC Genome Browser database to perform bioinformatics screening on transcription factors (TFs) predicted to bind to TTR promoters. Subsequently, we predicted potential Gli3 binding sites within the TTR promoter region using the JASPAR database. Chromatin immunoprecipitation (ChIP) experiments confirmed that Gli3 specifically binds to all three predicted sites, with the highest enrichment at site 2. Since Gli3 can be phosphorylated by the cAMP-dependent kinase PKA and converted into a transcriptional repressor, and cAMP / PKA is known to be a classic downstream pathway of GLP-1R, we hypothesize that GLP-1R activation inhibits TTR expression through the cAMP / PKA / Gli3 signaling pathway. Consistent with this, Ex4 treatment significantly increased cAMP levels and upregulated phosphorylated PKA (P-PKA), total PKA, and the Gli3 fragment with transcriptional repressive activity.

[0102] like Figure 8As shown, TTR can regulate emotional behavior through the ERK / CREB signaling pathway, with *p<0.05, **p<0.01, and ***p<0.001. Based on previous studies reporting that TTR can promote neurite growth and exert neuroprotective effects through the ERK / AKT / CREB signaling pathway, we investigated whether this pathway is involved in the behavioral effects of GLP-1R activation. Western blotting and immunohistochemical (IHC) experiments confirmed that GLP-1R activation impairs the ERK / CREB signaling pathway by downregulating TTR expression, establishing this pathway as a key effector mechanism in the development of emotional deficits.

[0103] like Figure 9 As shown, CREB activation can rescue GLP-1R signaling pathway-induced synaptic and emotional deficits, with *p<0.05, **p<0.01, ***p<0.001. We administered Ex4 and the CREB agonist 3',6-disinyl sucrose (DIS, 10 mg / kg, intraperitoneal injection) to normal-weight mice. The results showed that DIS treatment significantly improved Ex4-induced anxiety / depression-like behavior. Since CREB regulates synaptic plasticity by modulating the expression of genes involved in synaptic structure and function, we then examined two important biomarkers of synaptic plasticity: PSD95 (a core scaffold protein in the postsynaptic compactum) and Drebrin (an actin-binding protein that regulates synaptic morphological remodeling). These data further confirm that TTR mediates GLP-1R-induced emotional deficits by inhibiting the CREB signaling pathway and demonstrate that CREB activation can restore synaptic protein expression and emotional behavior, highlighting the crucial role of CREB pathway balance in emotion regulation.

[0104] The sequences and companies of the key reagents used in this protocol are as follows:

[0105] Reagent Name sequence company Exenatide No. A3408 APExBIO, Inc. (USA) Transthyretin / TTR protein HEK293 MCE Corporation, USA Eplontersen HY-148089 MCE Corporation, USA H-89 HY-15979 MCE Corporation, USA 3′,6-Disinapoylsucrose (DIS) HY-N1414A MCE Corporation, USA

[0106] Preparation method:

[0107] 1) Exenatide

[0108] Accurately weigh exenatide powder and prepare a stock solution with a concentration of 1 mg / mL using physiological saline. Before administration, weigh the mice in each group, and then administer the corresponding volume of the drug solution via intraperitoneal injection at the required experimental concentration.

[0109] 2) Eplontersen

[0110] Accurately weigh Eplontersen powder, dissolve the drug in physiological saline and bring the volume to a final volume to prepare a 1 mg / mL stock solution. Before administration, weigh each group of mice, and then inject 1 μL of the drug solution locally into each hippocampus at a dose of 10 μg / kg using stereotaxic techniques.

[0111] 3) Transthyretin / TTR protein

[0112] Accurately weigh Transthyretin / TTR protein powder, dissolve the drug in physiological saline and bring the volume to a final concentration to prepare a 1 mg / mL stock solution. Before administration, weigh each group of mice, then administer the corresponding volume of the drug solution via tail vein injection at a dose of 5 μg / kg.

[0113] 4) H-89

[0114] Accurately weigh H-89 (PKA inhibitor) powder, dissolve the drug in physiological saline and make up to a final volume to prepare a 1 mg / mL stock solution. Before administration, weigh the mice in each group, and then administer the corresponding volume of the drug solution via intraperitoneal injection at a dose of 10 mg / kg.

[0115] 5) 3′,6-Disinapoylsucrose (DIS)

[0116] Accurately weigh DIS (CREB agonist) powder, dissolve the drug in physiological saline and make up to a final volume to prepare a 1 mg / mL stock solution. Before administration, weigh the mice in each group, and then administer the corresponding volume of the drug solution via intraperitoneal injection at a dose of 10 mg / kg.

[0117] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for screening or assessing the risk of GLP-1 receptor agonist-related mood disorders, characterized in that, Includes the following steps: The expression level or activity of TTR in the subjects was detected; Based on the expression level or activity of the TTR, assess the risk of mood disorders in the subject after using a GLP-1 receptor agonist; The mood disorders mentioned include anxiety, depression, or suicidal ideation.

2. The method according to claim 1, characterized in that, The expression level of TTR is the TTR mRNA or protein level in hippocampal tissue or serum.

3. A kit for assessing the risk of GLP-1 receptor agonist-related mood disorders, characterized in that, It includes reagents for detecting TTR expression levels or activity.

4. A method for screening candidate drugs for the prevention or treatment of GLP-1 receptor agonist-related mood disorders, characterized in that, Includes the following steps: Contact the candidate drug with a system expressing TTR; Detect changes in TTR expression levels or activity; If a candidate drug upregulates TTR expression or enhances its activity, it will be identified as a candidate drug for the prevention or treatment of GLP-1 receptor agonist-related mood disorders.

5. The method according to claim 4, characterized in that, The candidate drugs are screened using at least one of the following methods: (1) Inhibit the cAMP / PKA / Gli3 signaling pathway; (2) Activate the ERK / CREB signaling pathway.

6. The use of TTR or its agonists in the preparation of medicines for the prevention or treatment of GLP-1 receptor agonist-related mood disorders.

7. A pharmaceutical composition, characterized in that, It includes GLP-1 receptor agonists, as well as TTR or its agonists.

8. The pharmaceutical composition according to claim 7, characterized in that, The TTR or its agonist is used to prevent or alleviate mood disorders induced by GLP-1 receptor agonists.

9. A method for screening compounds that regulate TTR expression, characterized in that, Includes the following steps: The candidate compound was brought into contact with cells expressing the TTR gene; Detecting the transcriptional or translational level of the TTR gene; If a candidate compound upregulates or downregulates TTR expression, it is identified as a compound that regulates TTR expression.

10. A system for assessing the risk of GLP-1 receptor agonist-related mood disorders, characterized in that, include: The detection module is used to detect the expression level or activity of TTR in the subject and generate detection results; An assessment module, connected to the detection module, is used to receive the detection results and assess the risk of the subject developing mood disorders after using a GLP-1 receptor agonist based on the detection results.

Images