Targeting postsynaptic glutamate receptor scaffolding proteins PSD-95 and PICK1 for obesity treatment

Abstract

Human genome-wide association studies (GWAS) suggest a functional role for central glutamate receptor signaling and plasticity in body weight regulation. Here, we use UK Biobank GWAS summary statistics of body mass index (BMI) and body fat percentage (BF%) to identify genes encoding proteins known to interact with postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartate (NMDA) receptors. Loci in/near discs large homolog 4 (DLG4) and protein interacting with C kinase 1 (PICK1) reached genome-wide significance (P < 5 × 10^-8) for BF% and/or BMI. To further evaluate the functional role of postsynaptic density protein-95 (PSD-95; gene name: DLG4) and PICK1 in energy homeostasis, we used dimeric PSD-95/disc large/ZO-1 (PDZ) domain-targeting peptides of PSD-95 and PICK1 to demonstrate that pharmacological inhibition of PSD-95 and PICK1 induces prolonged weight-lowering effects in obese mice. Collectively, these data demonstrate that the glutamate receptor scaffolding proteins, PICK1 and PSD-95, are genetically linked to obesity and that pharmacological targeting of their PDZ domains represents a promising therapeutic avenue for sustained weight loss.

Fig. 1.. Overview of PDZ domain–containing proteins and the associations of genetic variants in the corresponding gene regions with BF% and BMI.

(A) PDZ domain–containing proteins that interact with glutamatergic AMPA or NMDA receptors: GRIP1, GRIP2, PICK1, PSD-95 (DLG4), SAP97 (DLG1), and SAP102 (DLG3). (B) Genetic variants in the PICK1 (rs17752670) and DLG4 (rs2242449) loci reached genome-wide significance [shown as −log₁₀(P)] for association with BF% and BMI.

Fig. 2.. Human SNPs in the 1-Mb DLG4 region on chromosome 17 are associated with BF%.

(A) PheWAS for anthropometric traits in unrelated British individuals from the UK Biobank. The illustrated SNPs (x axis) are lead SNPs for the presented anthropometric traits (y axis). The highlighted SNPs rs2242449 and rs144129583 are lead SNPs for BF% in the locus. Icon colors are used to distinguish between the traits. (B) LD map of lead SNPs for anthropometric traits. Three LD blocks are identified in the locus chr17:6608115-7608115. LD blocks 1 and 3 show associations with BF% [rs2242449 (BF%): P = 7.22 × 10⁻⁹, β = 0.011, rs144129583 (BF%): P = 2.19 × 10⁻⁸, β = 0.022]. The strongest associations in LD blocks 2 and 3 are with height. (C) rs2242449 is associated with DLG4 transcript level in brain DLPFC tissue (P = 1.4 × 10⁻⁷) and located in an intron of DLG4. eQTL data were derived from Open Targets Genetics (https://genetics.opentargets.org/).

Fig. 3.. Human SNPs in the 1-Mb PICK1 region on chromosome 22 are associated with BF%.

(A) PheWAS for anthropometric traits in unrelated British individuals from the UK Biobank. The illustrated SNPs (x axis) are lead SNPs for the presented anthropometric traits (y axis). The highlighted SNPs rs17752670 and rs4821764 are lead SNPs for BF%. Icon colors are used to distinguish between the traits. (B) LD map of lead SNPs for anthropometric traits. Two LD blocks are identified in the locus chr22:37962013-38962013 that are independently associated with BF% (rs17752670: P = 9.59 × 10⁻⁹, β = −0.013, rs4821764: P = 9.42 × 10⁻¹⁸, β = −0.016). (C) PICK1 is located in between the lead SNPs, and eQTL analysis from Open Target Genetics (https://genetics.opentargets.org/) reveals significant association of both lead SNPs with PICK1 transcript level in blood.

Fig. 4.. Pharmacological inhibition of the PSD-95/nNOS/NMDA receptor complex reduces adiposity in DIO mice.

(A) Mechanism of action of PSD-95 inhibitor UCCB01-147 disrupting the PSD-95/nNOS/NMDA receptor complex. (B) Chemical structure of UCCB01-147. (C) Experimental design of the study in which DIO male C57BL/6J mice were treated with once-daily subcutaneous (s.c.) injections of UCCB01-147 (25 mg kg⁻¹; n = 10 mice and n = 7 cages) or vehicle (isotonic saline, n = 9 mice and n = 6 cages) over 14 days. (D) Change in body weight. (E) Total change in body weight. (F) Cumulative food intake. (G) Total food intake. (H) Change in body composition. (I) Compound tolerance test on day 0. (J) Area under the curve of (I). (K) Intraperitoneal glucose tolerance test on day 14. (L) Area under the curve of (K). (M) Plasma insulin levels. (N) Plasma cholesterol levels. (O) Plasma triglyceride levels. (P) Chemical structure of UCCB01-144AA, a double alanine mutant with abolished potency at PSD-95. DIO male C57BL/6J mice were treated with once-daily subcutaneous injections of UCCB01-147 (25 mg kg⁻¹; n = 6 mice and n = 6 cages), UCCB01-144AA (25 mg kg⁻¹; n = 6 mice and n = 6 cages), or vehicle (isotonic saline, n = 6 mice and n = 6 cages) over 5 days. (Q) Change in body weight. (R) Total change in body weight. (S) Cumulative food intake. (T) Total food intake. Data were analyzed by two-tailed unpaired t test (E, G, H, J, and L to O), one-way analysis of variance (ANOVA), multiple comparison, Bonferroni post hoc test (R and T), and two-way ANOVA, multiple comparisons, Bonferroni post hoc test (D, F, I, K, Q, and S). Data represent means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Asterisks denote comparison between UCCB01-147 and UCCB01-144AA (Q and S).

Fig. 5.. Pharmacological inhibition of PICK1 reduces adiposity in DOI mice.

(A) Mechanism of action of mPD5 inhibiting the PICK1/AMPA receptor complex. (B) Chemical structure of mPD5. (C) Study in which DIO male C57BL/6J mice were treated with once-daily subcutaneous injections of mPD5 (56 mg kg⁻¹; n = 9 mice and n = 5 cages) or vehicle (isotonic saline, n = 9 mice and n = 5 cages) over 14 days. (D) Change in body weight. (E) Total change in body weight. (F) Cumulative food intake. (G) Total food intake. (H) Change in body composition. (I) Compound tolerance test on day 0. (J) Area under the curve of (I). (K) Intraperitoneal glucose tolerance test on day 14. (L) Area under the curve of (K). (M) Plasma insulin levels. (N) Plasma cholesterol levels. (O) Plasma triglyceride levels. (P) Study in which DIO male wild type or global PICK1 knockout C57BL/6J mice were treated with once-daily subcutaneous injections of mPD5 (56 mg kg⁻¹; wild type: n = 4 mice and n = 4 cages; PICK1 knockout: n = 6 mice and n = 6 cages) or isotonic saline (wild type: n = 4 mice and n = 4 cages; PICK1 knockout: n = 6 mice and n = 6 cages) over 7 days. (Q) Change in body weight. (R) Total change in body weight. (S) Cumulative food intake. Data were analyzed by two-tailed unpaired t test (E, G, H, J, and L to O), one-way ANOVA, multiple comparisons, Bonferroni post hoc test (R), and two-way ANOVA, multiple comparisons, Bonferroni post hoc test (D, F, I, K, Q, and S). Data represent means ± SEM; *P < 0.05, **P < 0.01, and ****P < 0.0001. Asterisks denote the comparison between the mPD5-treated wild-type and PICK1 knockout groups (Q and S).

Fig. 6.. Metabolic phenotyping of UCCB01-147 and mPD5.

(A) Indirect calorimetry study in which DIO male C57BL/6J mice were treated with once-daily subcutaneous injections of UCCB01-147 (25 mg kg⁻¹; n = 10 mice and n = 10 cages), mPD5 (56 mg kg⁻¹; n = 9 mice and n = 9 cages), or vehicle (isotonic saline, n = 9 mice and n = 9 cages) over 10 days in metabolic cages. (B) Change in body weight. (C) Total change in body weight. (D) Cumulative food intake. (E) Total food intake. (F) Longitudinal curves of energy expenditure. (G) Average energy expenditure during dark and light periods. (H) Longitudinal curves of respiratory exchange ratio. (I) Average respiratory exchange ratio during the dark and light periods. (J) Longitudinal curves of locomotor activity. (K) Average locomotor activity during the dark and light periods. (L) Open field test in which lean chow-fed C57BL/6J mice were treated with a single subcutaneous injection of UCCB01-147 (25 mg kg⁻¹; n = 10 mice), mPD5 (56 mg kg⁻¹; n = 10 mice), or vehicle (isotonic saline, n = 10 mice). (M) Distance traveled. (N) Conditioned taste aversion study in which lean chow-fed C57BL/6J mice were dosed with a single subcutaneous injection of UCCB01-147 (25 mg kg⁻¹; n = 10 mice), mPD5 (56 mg kg⁻¹; n = 10 mice), semaglutide (30 nmol kg⁻¹; n = 10 mice), or vehicle (isotonic saline, n = 10 mice). (O) Total drinking volume. (P) Saccharin preference. Data were analyzed by one-way ANOVA, multiple comparisons, Bonferroni post hoc test (C, E, G, I, K, M, O, and P) and two-way ANOVA, multiple comparisons, Bonferroni post hoc test (B and D). Data represent means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Fig. 7.. Whole brain quantification of c-Fos expression in response to UCCB01-147 or mPD5 treatment.

(A) Experimental design of study with whole-brain 3D mapping and quantification of c-Fos induction in response to acute treatment with mPD5 (56 mg kg⁻¹; n = 8 mice), UCCB01-147 (25 mg kg⁻¹; n = 8 mice), or vehicle (isotonic saline, n = 8 mice) in lean C57BL/6J mice. (B) Heatmaps of c-Fos density using spherical averaging (350 μm diameter). (C) Total c-Fos–positive cells in response to treatments. (D) Volcano plot of brain regions regulated for mPD5 relative to vehicle-treated mice. Light red indicates a log₂ fold change >0 and Benjamini-Hochberg–adjusted P < 0.05. Light blue indicates a log₂ fold change <0 and Benjamini-Hochberg–adjusted P < 0.05. (E) Volcano plot of brain regions regulated for UCCB01-147 relative to vehicle-treated mice. Light red indicates a log₂ fold change >0 and Benjamini-Hochberg–adjusted P < 0.05. Light blue indicates a log₂ fold change <0 and Benjamini-Hochberg–adjusted P < 0.05. (F) Heatmap of c-Fos activity in brain regions involved in body weight and appetite regulation. Data represent log₂ fold change relative to the vehicle group average. (G) Top 20 regulated brain areas in response to acute mPD5 (56 mg kg⁻¹) treatment represented as log₂ fold change relative to the vehicle group average. (H) Top 20 regulated brain areas in response to acute UCCB01-147 (25 mg kg⁻¹) treatment represented as log₂ fold change relative to the vehicle group average. Data represent mean ± SEM; ****P < 0.0001. For brain region abbreviations, please refer to table S2.

Fig. 8.. Pharmacological PSD-95 and PICK1 inhibition differentially influence the brain proteome in a region-specific manner.

(A) Schematic anatomical location of dissected brain regions following 10 days of treatment. (B) PCA of all samples from different brain regions and treatment conditions. (C) UpSet plot for the number of unique and shared proteins that are differentially regulated with either UCCB01-147 (D) or mPD5 treatment compared to vehicle across brain regions. (E) Heatmap of log₂ fold change (logFC) for the 35 proteins that respond similarly across brain regions with mPD5 treatment compared to vehicle. (F) Venn diagrams comparing similar or unique proteins that are differentially regulated with mPD5 or UCCB01-147 treatment. (G) Volcano plots for proteins logFC with UCCB01-147 (H) or mPD5 treatment compared to vehicle in the brainstem. (I) Heatmap of row-wise z-scored logFCs clustered by row for all proteins with an adjusted P < 0.05 and an absolute LogFC > 1.5. (J) Overrepresentation analysis for enriched gene ontologies (CC, BP, and MF) within each cluster with the total proteome set as background. (K) LogFC to either mPD5 or UCCB01-147 compared to vehicle in each brain region for significantly regulated proteins that are also interactors with PICK1 and PSD-95.