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. 2016 Jun 18;11(1):45.
doi: 10.1186/s13024-016-0112-5.

Redundant Gs-coupled serotonin receptors regulate amyloid-β metabolism in vivo

Jonathan R Fisher  1   2   3 , Clare E Wallace  1   2   3 , Danielle L Tripoli  1   2   3 , Yvette I Sheline  4 , John R Cirrito  5   6   7   8
Affiliations

Redundant Gs-coupled serotonin receptors regulate amyloid-β metabolism in vivo

Jonathan R Fisher et al. Mol Neurodegener. .

Abstract

Background: The aggregation of amyloid-β (Aβ) into insoluble plaques is a hallmark pathology of Alzheimer's disease (AD). Previous work has shown increasing serotonin levels with selective serotonin re-uptake inhibitor (SSRI) compounds reduces Aβ in the brain interstitial fluid (ISF) in a mouse model of AD and in the cerebrospinal fluid of humans. We investigated which serotonin receptor (5-HTR) subtypes and downstream effectors were responsible for this reduction.

Results: Agonists of 5-HT4R, 5-HT6R, and 5-HT7R significantly reduced ISF Aβ, but agonists of other receptor subtypes did not. Additionally, inhibition of Protein Kinase A (PKA) blocked the effects of citalopram, an SSRI, on ISF Aβ levels. Serotonin signaling does not appear to change gene expression to reduce Aβ levels in acute timeframes, but likely acts within the cytoplasm to increase α-secretase enzymatic activity. Broad pharmacological inhibition of putative α-secretases increased ISF Aβ and blocked the effects of citalopram.

Conclusions: In total, these studies map the major signaling components linking serotonin receptors to suppression of brain ISF Aβ. These results suggest the reduction in ISF Aβ is mediated by a select group of 5-HTRs and open future avenues for targeted therapy of AD.

Keywords: Alzheimer’s Disease; Microdialysis; PKA; SSRI; Serotonin receptor; beta-amyloid; α-secretase.

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Figures

Fig. 1
Fig. 1
A small subset of 5-HT receptors reduce ISF Aβ levels in vivo. Selective agonists for individual 5-HT receptors or vehicle (DMSO) were infused via reverse microdialysis (rev md) in 2–3 month old APP/PS1 hemizygous mice. a As assessed by microdialysis, broad activation of 5-HTRs by serotonin (2 mM by rev md) directly or by citalopram (10 mg/kg i.p.), a SSRI, caused a decrease in ISF Aβ levels. Similarly, reverse microdialysis treatment with agonists for 5-HT4R (ML10302, 400 nM), 5-HT6R (ST1936, 1.3 μM), and 5-HT7R (AS19, 83 nM) induced a reduction in ISF Aβ in living mice. b After hours 22-24 of continuous treatment, ML10302, ST1936, and AS19 reduced ISF Aβx-40 by 25.2 ± 3.4 % (p < 0.01; n = 6), by 24.8 ± 6.1 % (p < 0.01; n = 6), and by 22.5 ± 5.5 % (p < 0.05; n = 6), respectively. Serotonin and citalopram also significantly reduced ISF Aβ by 26.5 ± 5.4 % (p < 0.01; n = 7) and 25.5 ± 3.4 % (p < 0.05; n = 5), respectively. c Agonist treatment for 5-HT1AR (Ipsapirone, 1 μM) or 5-HT2CR (WAY161503, 400 nM) showed no significant reduction of ISF Aβ. d After 24 h of continuous treatment, Ipsapirone reduced ISF Aβx-40 to 91.2 ± 9.4 % (n = 6) and WAY161503 increased ISF Aβx-40 to 119.9 ± 8.6 % (n = 8). e Citalopram (10 mg/kg, i.p.) administered to young C57Bl6 wildtype mice significantly reduced ISF Aβ levels by 32 ± 4.2 % at 21–24 h after treatment compared to vehicle-treated (PBS) mice (p < 0.001; n = 6). f Mice were treated with selective 5-HT7R antagonist SB258719 (3.16 μM) for 8 h followed by co-administration of 5-HT7R agonist, AS19, (83nM) for 24 h by reverse microdialysis (n = 4). The antagonist completely blocked the effect of the agonist. The antagonist alone had no significant effect on ISF Aβ levels. Data presented as mean ± SEM. Asterisks mark p-values < 0.05; double asterisks mark p-values < 0.01; triple asterisks mark p-values 0.001
Fig. 2
Fig. 2
Inhibiting 5-HT4R and 5-HT7R simultaneously increases ISF Aβ. Selective 5-HT4R antagonist GR113808 (100 nM), selective 5-HT7R antagonist SB258719 (3.16 μM), both antagonists together, or DMSO vehicle were infused via reverse microdialysis in 2.5 month old APP/PS1 hemizygous mice for 8 h. a As assayed by microdialysis, treatment with GR113808 or SB258719 alone had no detectable effect on ISF Aβx-40 (n = 4), but co-administration of both antagonists increased ISF Aβx-40. b 24 h of treatment with both antagonists significantly increased ISF Aβx-40 to 132 ± 11.2 % (p = 0.048, n = 10) by 21–24 h. Data presented as mean ± SEM. Asterisks mark p-values < 0.05
Fig. 3
Fig. 3
PKA activity modulates production of ISF Aβ. a Selective small molecule inhibitor KT5720 (6 μM), peptide inhibitor PKI14-22 amide (3.6 μM), or vehicle (DMSO or acetonitrile, respectively) were infused via reverse microdialysis (rev md) in 2.5 month old APP/PS1 hemizygous mice for 24 h. In two cohorts of mice after 8 h of inhibitor treatment, the SSRI citalopram was administered by i.p. injection at 10 mg/kg. b Inhibiting PKA with KT5720 alone for 24 h caused a significant increase in ISF Aβx-40 levels by 31.9 ± 6.0 % (p < 0.05; n = 4). By 16 h after co-administration of citalopram with either KT5720 or PKI 14-22, ISF Aβx-40 levels were significantly increased by 45.8 ± 10.5 % (p < 0.05, n = 6) and by 50.6 ± 19.6 % (p < 0.05, n = 6), respectively. Most importantly, each inhibitor completely blocked the effect of citalopram on ISF Aβ levels. Data represented as mean ± SEM. Asterisks mark p-values < 0.05
Fig. 4
Fig. 4
Acute SSRI treatment has no significant effect on expression of Aβ APP metabolism genes. 2.5 month old APP/PS1 hemizygous mice were treated with 10 mg/kg citalopram or PBS by i.p. injection. 16 h later hippocampi were microdissected for qPCR analysis. a qPCR analysis for genes involved in Aβ processing or clearance showed no significant changes in expression. In contrast, the positive control, cFos, was significantly reduced to 57.5 ± 0.04 % (p < 0.05). b qPCR analysis for genes encoding signaling proteins downstream of 5-HTR showed no significant changes in expression. c qPCR analysis for 5-HTR genes showed no significant changes in expression. Data are presented as mean ± SEM. Values are normalized to the mean expression level of the PBS controls (n = 6). Statistical significance was calculated by one-way ANOVA with comparison to CaMKII and corrected for multiple comparisons. Asterisks mark p-values < 0.05. See Additional file 1 for primer sequences and database numbers
Fig. 5
Fig. 5
Broad spectrum inhibition of α-secretase enzymes blocks the effects of SSRI. Broad spectrum ADAM/MMP inhibitor GM6001 (25 μM) or vehicle (DMSO) was infused via reverse microdialysis in 2.5 month old APP/PS1 hemizygous mice. 8 h later, the SSRI citalopram was administered by i.p. injection at 10 mg/kg. a As assessed by microdialysis, inhibiting ADAM/MMP enzymes dramatically increased ISF Aβx-40 levels, but there was no reduction in ISF Aβ after SSRI. b After 16 h, vehicle control with SSRI significantly reduced ISF Aβx-40 to 80.9 ± 2.4 % (p = 0.002, n = 6) of levels at hour 8. GM6001 treatment increased ISF Aβx-40 to 264.2 ± 21.1 % at time point 8. After 16 h of SSRI treatment, GM6001 ISF Aβx-40 levels were unchanged at 254.9 ± 16.3 % of baseline (p = 0.74, n = 6). Data represented as mean ± SEM. Asterisks mark p-values < 0.05
Fig. 6
Fig. 6
Model of Aβ reduction by serotonergic signaling. Serotonin binding to 5-HT4R, 5-HT6R, and 5-HT7R leads to the activation of PKA. PKA subsequently activates the MEK and ERK cascade. Activated ERK remains in the cytoplasm and phosphorylates α-secretase to increase its activity and reduce Aβ production
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