Research ArticleAlzheimer’s Disease

Disrupted hippocampal growth hormone secretagogue receptor 1α interaction with dopamine receptor D1 plays a role in Alzheimer′s disease

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Science Translational Medicine  14 Aug 2019:
Vol. 11, Issue 505, eaav6278
DOI: 10.1126/scitranslmed.aav6278
  • Fig. 1 Aβ physically interacts with GHSR1α.

    (A) Measurement of PLA-positive dots for Aβ/GHSR1α complex in hippocampi from subjects with AD. ***P < 0.001, unpaired Student’s t test. n = 4 healthy donors or subjects with AD. (B) Representative images of quantification in (A). Arrows indicate Aβ/GHSR1α PLA-positive dots. (C) Analysis of Aβ/Ghsr1α PLA-positive dots in the hippocampal region from 4- and 9-month-old 5×FAD mice. **P < 0.01, unpaired Student’s t test. n = 4 mice per group. (D) Representative three-dimensional (3D) reconstructed images. The slices from 9-month-old Ghsr null mice were used as negative control. (E to G) Analysis of Ghsr1α/Aβ PLA-positive dots in HEK 293T cells expressing different forms of Ghsr1α treated with vehicle or 5 μM oligomeric Aβ42 for 24 hours. Anti-FLAG antibody was used to detect Ghsr1α and its mutants. †P < 0.001 versus cells expressing full-length Ghsr1α without oligomeric Aβ42 treatment and #P < 0.001 versus cells expressing Ghsr1α ∆aa42–116 with oligomeric Aβ42 treatment, unpaired Student’s t test. n = 4 to 7. (F) Representative 3D reconstructed images of Ghsr1α/Aβ PLA-positive dots in HEK 293T cells expressing different forms of Ghsr1α treated with vehicle or oligomeric Aβ42 (top panels) and (G) representative 3D reconstructed images of immunofluorescent staining of different forms of Ghsr1α (bottom panels) recognized by anti-FLAG antibody. (H) Densitometry of all immunoreactive bands generated from Co-IP on HEK 293T cells expressing different forms of Ghsr1α treated with vehicle or 5 μM oligomeric Aβ42 for 24 hours. ***P < 0.001, one-way ANOVA followed by Bonferroni post hoc analysis. Data were collected from three independent experiments. n (from left to right) = 3, 5, 2, 3, and 3. Nonimmune immunoglobulin G (IgG) to replace specific FLAG antibody was used for examining specificity of Co-IP. (I) Representative immunoblots showing the interaction of oligomeric Aβ42 with Ghsr1α and Ghsr1α ∆aa42–116. (J) Representative immunoblots showing the input of Ghsr1α and Ghsr1α ∆aa42–116. DAPI, 4′,6-diamidino-2-phenylindole; NS, not significant; IP, immunoprecipitation; WB, Western blot.

  • Fig. 2 Interaction with Aβ disrupts GHSR1α activity.

    (A to C) Impact of oligomeric Aβ42 (2 μM, 5-min pretreatment) on Ghsr1α FlAsH-FRET response in the presence or absence of MK0677 (50 μM). (A) FRET ratio quantified from data collected from a microplate reader. The effect of Ghsr1α antagonist JMV2959 (50 μM) against MK0677-induced Ghsr1α activation was used as positive control. **P < 0.01 compared with other groups, two-way ANOVA followed by Bonferroni post hoc analysis. n = 12 per group. (B) Representative confocal microscopy images for FRET pseudo-color ratio (FlAsHexECFP/emFIAsH/ECFPexECFP/emECFP) (top), FlAsHexECFP/emFIAsH (middle, green) and ECFPexECFP/emECFP (bottom, red). (C) Representative 3D reconstructed images for Ghsr1α and Drd1 expression in Ghsr1α/Drd1 coexpressing HEK 293T cells. (D) Analysis of Ghsr1α/Drd1 PLA-positive dot intensity in Ghsr1α/Drd1 coexpressing HEK 293T cells. ***P < 0.001, unpaired Student’s t test. Data were collected from three independent experiments. n = 78 cells for vehicle-treated group and n = 60 cells for the group with oligomeric Aβ42 treatment (5 μM, 24 hours). Anti-Ghsr1α and anti-Drd1 antibodies were used in this experiment. (E) Representative 3D reconstructed images for Ghsr1α/Drd1 PLA-positive dots in Ghsr1α/Drd1 coexpressing HEK 293T cells. (F) Analysis of GHSR1α/DRD1 PLA-positive dots in hippocampal sections from patients with AD and healthy controls. ***P < 0.001, unpaired Student’s t test. n = 5 per group. (G) Representative images of GHSR1α/DRD1 PLA dots. Arrows indicate GHSR1α/DRD1 PLA-positive dots. (H) Analysis of Ghsr1α/Drd1 PLA-positive dots in hippocampal CA1 region in 4- and 9-month-old 5×FAD mice. *P < 0.05 and **P < 0.01, unpaired Student’s t test. n = 3 for each group. (I) Representative 3D reconstructed images of Ghsr1α/Drd1 PLA-positive dots in the hippocampus of 4- and 9-month-old nonTg and 5×FAD mice. Ghsr null mice at 9 months old were used as critical negative control.

  • Fig. 3 Loss of Ghsr replicates AD-like phenotypes.

    (A) Analysis of synaptic density in CA1 regions from 4- and 9-month-old mice. **P < 0.001 and ***P < 0.001 nonTg versus other groups at the same age, one-way ANOVA followed by Bonferroni post hoc analysis. Four-month-old mice: nonTg, n = 4; 5×FAD, n = 7; Ghsr null mice, n = 5; and Ghsr null/5×FAD, n = 4. Nine-month-old mice: nonTg, n = 4, 5×FAD, n = 4, Ghsr null mice, n = 4, and Ghsr null/5×FAD, n = 3. (B) Representative 3D reconstructed images of synapse staining. Vesicular glutamate transporter 1 (vGLUT1, blue) and postsynaptic density 95 (PSD95, red) were used to visualize pre- and postsynaptic terminals, respectively. The overlapped staining of vGLUT1 and PSD95 indicates synapses. (C) Time course of LTP and representative fEPSP responses during the baseline period (black trace) and 30 s after theta burst simulation (red trace) in four groups of mice at 9 months old. *P < 0.05 and ***P < 0.001 nonTg versus other groups, one-way ANOVA followed by Bonferroni post hoc analysis. nonTg, n = 5; 5×FAD, n = 4; Ghsr null, n = 5; and Ghsr null/5×FAD, n = 5. (D to I) Spatial navigation of four groups of mice in the Morris water maze test. (D and G) Spatial learning of four groups of mice at 4 (D) and 9 (G) months old. *P < 0.05, **P < 0.01, and ***P < 0.001, nonTg versus other groups on the same day, one-way ANOVA followed by Bonferroni post hoc analysis. (E and H) Spatial reference memory of different groups of mice at 4 (E) and 9 (H) months of age. *P < 0.05, one-way ANOVA followed by Bonferroni post hoc analysis. (F and I) Swimming speed of four groups of mice at 4 (F) and 9 (I) months old. Four-month-old mice: nonTg, n = 7; 5×FAD, n = 9; Ghsr null mice, n = 10; and Ghsr null/5×FAD, n = 6. Nine-month-old mice: nonTg, n = 8; 5×FAD, n = 11; Ghsr null mice, n = 5; and Ghsr null/5×FAD, n = 8.

  • Fig. 4 Combined Ghsr1α/Drd1 activation rescues hippocampal synapse in vitro.

    (A) Effect of different treatments (1.5 μM MK0677, 2 μM SKF81297, or in combination) on synaptic density in hippocampal neurons in the presence or absence of oligomeric Aβ42 (1 μM, 24 hours). ***P < 0.001 vehicle-treated versus oligomeric Aβ42–treated groups, two-way ANOVA followed by Bonferroni post hoc analysis. Data were collected from three independent experiments. n = 30 to 48 neurites. (B) Representative 3D reconstructed images of synapse staining. vGLUT1 (blue) and PSD95 (red) were used to visualize pre- and postsynaptic terminals, respectively. The dendrites were stained with MAP2 (green). The overlaid staining of vGLUT1/PSD95 identifies synapses. (C) Effect of different treatments (1.5 μM MK0677, 2 μM SKF81297, or in combination) on Ghsr1α/Drd1 complex in hippocampal neurons in the presence or absence of oligomeric Aβ42 (1 μM, 24 hours). **P < 0.01 and ***P < 0.001 vehicle-treated versus oligomeric Aβ42-treated groups, two-way ANOVA followed by Bonferroni post hoc analysis. Data were collected from three independent experiments. n = 8 to 10 neurons. (D) Representative images of Ghsr1α/Drd1 PLA-positive dots. (E) Time course of LTP and representative fEPSP responses during the baseline period (black trace) and 30 s after theta burst simulation (red trace) in five treatment groups at 4 months of age. **P < 0.01 5×FAD MK0677/SKF81297 versus 5×FAD saline, one-way ANOVA followed by Bonferroni post hoc analysis. nonTg saline, n = 9; 5×FAD saline, n = 10; 5×FAD MK0677/SKF81297, n = 7; 5×FAD MK0677, n = 9; and 5×FAD SKF81297, n = 9. (F to H) mEPSC frequency (F) and amplitude (G) in the indicated groups of 4-month-old mice. *P < 0.05 and **P < 0.01, one-way ANOVA followed by Bonferroni post hoc analysis. n = 6. (H) Representative traces of mEPSC recordings.

  • Fig. 5 Coactivation of Ghsr1α and Drd1 preserves Ghsr1α activity from Aβ toxicity.

    (A) Effect of SKF81297 (100 μM) and MK0677 (50 μM) alone or in combination on Ghsr1α FIAsH-FRET response in the presence or absence of oligomeric Aβ42 (2 μM, 5-min pretreatment). Cells expressing Ghsr1αFIAsH/ECFP alone or coexpressed with Drd1 were used. Data were collected from a microplate reader. ***P < 0.001, two-way ANOVA followed by Bonferroni post hoc analysis. Data were collected from three independent experiments. n = 9 to 25 samples. (B and C) Effect of different treatments (1.5 μM MK0677, 2 μM SKF81297, or in combination) on Aβ/Ghsr1α complex in oligomeric Aβ42 (1 μM, 24 hours)-treated hippocampal neurons. *P < 0.05, one-way ANOVA followed by Bonferroni post hoc analysis. Data were collected from three independent experiments. n = 10 neurons per group.

  • Fig. 6 Combined Ghsr1α/Drd1 activation protects hippocampal synapse and cognition in vivo.

    (A to C) Spatial navigation analysis in four groups of mice treated with vehicle (saline) or MK0677/SKF81297 (MK0677, 1 mg/kg and SKF81297, 1.5 mg/kg) performing the Morris water maze test. (A) Spatial learning. **P < 0.01 and ***P < 0.001 5×FAD saline versus other groups, two-way ANOVA followed by Bonferroni post hoc analysis. (B) Spatial reference memory. ***P < 0.001 5×FAD saline versus other groups, two-way ANOVA followed by Bonferroni post hoc analysis. (C) Swimming speed. nonTg saline, n = 8; 5×FAD saline, n = 8; nonTg MK0677/SKF81297, n = 7; and 5×FAD MK0677/SKF81297, n = 5. (D and E) Analysis of synaptic density in the hippocampal CA1 region. **P < 0.01 and ***P < 0.001 5×FAD saline versus other groups, two-way ANOVA followed by Bonferroni post hoc analysis. nonTg saline, n = 3; 5×FAD saline, n = 4; nonTg MK0677/SKF81297, n = 4; and 5×FAD MK0677/SKF81297, n = 4. (E) Representative 3D reconstructed images of synapse staining in the CA1 region. vGLUT1 (blue) and PSD95 (red) were used to visualize pre- and postsynaptic components, respectively. The overlaid staining of vGLUT1 and PSD95 indicates synapses. (F and G) Analysis of Ghsr1α/Drd1 complex in the CA1 region. ***P < 0.001 5×FAD saline versus other groups, two-way ANOVA followed by Bonferroni post hoc analysis. n = 4 per group. (G) Representative 3D reconstructed images of Ghsr1α/Drd1 PLA-positive dots (red). Nuclei were stained with DAPI. (H and I) Analysis of Aβ/Ghsr1α PLA-positive dots in CA1 region. ***P < 0.001, unpaired Student’s t test. n = 4 per group. (I) Representative 3D reconstructed images of Aβ/Ghsr1α PLA dots (red). Nuclei were stained with DAPI.

  • Fig. 7 Hippocampal amyloidosis and tau phosphorylation remain unaltered in treated 5×FAD mice.

    (A) Analysis of APP expression level in the hippocampus by using immunoblotting. Unpaired Student’s t test. n = 4 per group. The right panel shows representative images of immunoblotting. β-Actin was used as the loading control. (B) Aβ deposition in the hippocampal region was measured and analyzed by immunostaining using Aβ antibody. Unpaired Student’s t test. 5×FAD saline mice, n = 6 and 5×FAD MK0677/SKF81297 mice, n = 5. The right panel shows representative images of Aβ staining (red). The neurons were identified by the staining of NeuN (green). (C) Soluble Aβ40 and Aβ42 amounts in hippocampal homogenate were detected by ELISA assay. Unpaired Student’s t test. 5×FAD saline mice, n = 6 and 5×FAD MK0677/SKF81297 mice, n = 5. (D) Analysis of intracellular Aβ in hippocampal CA1 neurons. Unpaired Student’s t test. 5×FAD saline mice, n = 8 and 5×FAD MK0677/SKF81297 mice, n = 5. The right panel shows representative images. Aβ was recognized by anti-Aβ antibody (red). Neurons were labeled by anti–β-III-tubulin (green). Nuclei were identified by the staining of DAPI (blue color). The overlaid staining of Aβ and β-III-tubulin indicates intraneuronal Aβ. Scale bar, 20 μm. (E) Congo red staining was used to label extracellular parenchymal Aβ plaques. Unpaired Student’s t test. 5×FAD saline mice, n = 8 and 5×FAD MK0677/SKF81297 mice, n = 5. The right panel shows representative images of Aβ plaque staining. (F) Immunoblotting analysis of tau phosphorylation at different motifs and total tau in mouse hippocampal tissues. Unpaired Student’s t test. n = 4 per group. The lower panel shows representative images of immunoblotting. β-Actin was used as the loading control. P-tau stands for phosphorylated tau, and T-tau stands for total tau.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/505/eaav6278/DC1

    Materials and Methods

    Fig. S1. Expression of GHSR1α increased in hippocampi from subjects with AD and 9-month-old 5×FAD mice.

    Fig. S2. Expressions of Ghsr1α and Drd1 were validated in transfected HEK 293T cells.

    Fig. S3. Schematic diagram shows the sequence of full-length Ghsr1α and its truncating mutants.

    Fig. S4. FLAG-tagged Ghsr1α and its truncating mutants were similarly expressed in HEK 293T cells.

    Fig. S5. The interaction between Ghsr1α mutants and Aβ42 was assessed by using Co-IP.

    Fig. S6. Schematic diagram represents FlAsH-FRET assay for Ghsr1α activity and the impact of oligomeric Aβ42 on Ghsr1α activation.

    Fig. S7. GHSR1α/DRD1 complex density was negatively correlated with hippocampal soluble Aβ40 or Aβ42 amounts in subjects with AD.

    Fig. S8. Expression of hippocampal DRD1 remained unaltered in hippocampi from subjects with AD and 5×FAD mice.

    Fig. S9. Oligomeric Aβ42 did not affect agonist-induced activation of Drd1 or form complex with Drd1.

    Fig. S10. Input/output curves of fEPSPs were similar in four types of transgenic mice.

    Fig. S11. Aβ deposition in the hippocampus remained unchanged in Ghsr null/5×FAD mice as compared with their 5×FAD littermates.

    Fig. S12. Serum ghrelin amounts were similar in four types of transgenic mice.

    Fig. S13. Loss of Ghsr1α suppressed postsynaptic CaMKII activation in the hippocampus.

    Fig. S14. The optimal doses of MK0677 and SKF81297 were determined by their augmenting effect on synaptogenesis in cultured hippocampal neurons.

    Fig. S15. Time course of LTP and fEPSP amplitudes was not changed by different treatments on hippocampal slices from nonTg mice.

    Fig. S16. The doses of MK0677/SKF81297 treatment were optimized on the basis of the influence on body weight, serum ghrelin, and behavioral performance.

    Fig. S17. MK0677/SKF81297 [MK0677 (1 mg/kg) and SKF81297 (1.5 mg/kg)] treatment on mice had no effect on hepatic, renal, and hippocampal cell density.

    Fig. S18. MK0677/SKF81297 [MK0677 (1 mg/kg) and SKF81297 (1.5 mg/kg)] treatment improved neurogenesis in the dentate gyrus of 5×FAD mice.

    Table S1. Human brain tissue information.

    Table S2. Raw data (provided as separate Excel file).

    References (6472)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Expression of GHSR1α increased in hippocampi from subjects with AD and 9-month-old 5×FAD mice.
    • Fig. S2. Expressions of Ghsr1α and Drd1 were validated in transfected HEK 293T cells.
    • Fig. S3. Schematic diagram shows the sequence of full-length Ghsr1α and its truncating mutants.
    • Fig. S4. FLAG-tagged Ghsr1α and its truncating mutants were similarly expressed in HEK 293T cells.
    • Fig. S5. The interaction between Ghsr1α mutants and Aβ42 was assessed by using Co-IP.
    • Fig. S6. Schematic diagram represents FlAsH-FRET assay for Ghsr1α activity and the impact of oligomeric Aβ42 on Ghsr1α activation.
    • Fig. S7. GHSR1α/DRD1 complex density was negatively correlated with hippocampal soluble Aβ40 or Aβ42 amounts in subjects with AD.
    • Fig. S8. Expression of hippocampal DRD1 remained unaltered in hippocampi from subjects with AD and 5×FAD mice.
    • Fig. S9. Oligomeric Aβ42 did not affect agonist-induced activation of Drd1 or form complex with Drd1.
    • Fig. S10. Input/output curves of fEPSPs were similar in four types of transgenic mice.
    • Fig. S11. Aβ deposition in the hippocampus remained unchanged in Ghsr null/5×FAD mice as compared with their 5×FAD littermates.
    • Fig. S12. Serum ghrelin amounts were similar in four types of transgenic mice.
    • Fig. S13. Loss of Ghsr1α suppressed postsynaptic CaMKII activation in the hippocampus.
    • Fig. S14. The optimal doses of MK0677 and SKF81297 were determined by their augmenting effect on synaptogenesis in cultured hippocampal neurons.
    • Fig. S15. Time course of LTP and fEPSP amplitudes was not changed by different treatments on hippocampal slices from nonTg mice.
    • Fig. S16. The doses of MK0677/SKF81297 treatment were optimized on the basis of the influence on body weight, serum ghrelin, and behavioral performance.
    • Fig. S17. MK0677/SKF81297 [MK0677 (1 mg/kg) and SKF81297 (1.5 mg/kg)] treatment on mice had no effect on hepatic, renal, and hippocampal cell density.
    • Fig. S18. MK0677/SKF81297 [MK0677 (1 mg/kg) and SKF81297 (1.5 mg/kg)] treatment improved neurogenesis in the dentate gyrus of 5×FAD mice.
    • Table S1. Human brain tissue information.
    • References (6472)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2. Raw data (provided as separate Excel file).

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