Open Access

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

  • Jonathan R. Fisher1, 2, 3,
  • Clare E. Wallace1, 2, 3,
  • Danielle L. Tripoli1, 2, 3,
  • Yvette I. Sheline4 and
  • John R. Cirrito1, 2, 3, 5Email author
Molecular Neurodegeneration201611:45

https://doi.org/10.1186/s13024-016-0112-5

Received: 15 October 2015

Accepted: 14 June 2016

Published: 18 June 2016

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 beta-amyloid Serotonin receptor SSRI Microdialysis PKA α-secretase

Background

One primary pathology of Alzheimer’s disease (AD) is the accumulation of insoluble amyloid-β (Aβ) plaques in the brain. Aβ is produced by sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases [1, 2]. Alternatively, activity of another type of enzymes, α-secretases, can cleave within the Aβ sequence and prevent its formation [3]. Concentration is a key factor that determines whether soluble Aβ peptide aggregates into oligomers and plaques [4, 5], with higher concentrations increasing the likelihood that toxic multimers of Aβ will form. Consequently, reducing Aβ levels is one promising target for AD therapy.

Serotonin, Aβ, and AD have been connected in several studies in the scientific literature. The amount of both serotonin (5-HT) and its receptors (5-HTRs) are reduced in human post-mortem AD studies [6, 7]. Serotonergic activity reduces Aβ production rates in vitro and 5-HTR agonists alter APP processing to increase soluble fragments of the protein that are consistent with a suppression in Aβ generation [811]. Manipulating serotonin levels in vivo shows similar effects. In our previous work, treating APPswe/PS1∆E9 (APP/PS1) mice with a single dose of a selective serotonin re-uptake inhibitor (SSRI) reduced brain interstitial fluid (ISF) Aβ concentrations by 25 % [12]. Serotonin treatment did not alter the Aβ elimination rate which suggests this reduction was not mediated by Aβ clearance mechanisms. Instead, α-secretase enzymatic activity was increased by SSRI treatment, suggesting that Aβ generation was suppressed. Chronic dosing with a SSRI over 4 months reduced brain Aβ plaque load and cerebrospinal fluid (CSF) Aβ levels in mice by 50 % [12]. Similar reductions in Aβ were seen by SSRI treatment of 3xTg AD mouse model [6]. The reduction in Aβ by SSRI is not limited to mice; young adult, cognitively normal, non-depressed individuals given a single dose of the SSRI citalopram also showed a reduction in CSF Aβ levels in a matter of hours [13].

The cellular mechanism of SSRI-induced Aβ reduction is likely complex. There are 15 identified serotonin receptors expressed in the brain [14]. Most 5-HTRs are G-protein coupled receptors (GPCRs) while 5-HT3R is the only ionotropic cation channel [15]. 5-HT1R and 5-HT5R couple to Gi/o signal proteins and typically lead to Protein Kinase C (PKC) activation [1618] while 5-HT2R signals through Gq proteins to activate calcium-calmodulin dependent kinase II (CaMKII) [15, 19]. 5-HT4R, 5-HT6R, and 5-HT7R activate Gs proteins, which usually increase cyclic AMP levels, and induce Protein Kinase A (PKA) activation [10, 15, 20]. Interestingly, stimulating some serotonin receptors can activate the extracellular signal-regulated kinase (ERK) [18, 2022]. Inhibition of MAP kinase-ERK kinase (MEK), the kinase that activates ERK, reduces the production of the α-secretase cleavage product sAPPα in vitro [23]. Also, inhibiting either ERK or MEK increased ISF Aβ levels in mice and blocked the SSRI-dependent reduction in Aβ [12]. Our current study shows serotonin-induced reductions of Aβ, rely on the Gs-linked serotonin receptors and PKA signaling. We also provide evidence that serotonergic-dependent suppression of Aβ is mediated through an increase in α-secretase enzymatic activity.

Results

Serotonin-induced reduction in ISF Aβ is receptor specific

Aβ is largely produced by neurons during synaptic activity and is continually released into the ISF [2427]. We used in vivo brain microdialysis to measure dynamic changes in ISF Aβ levels [28]. This method allows for serial collection of ISF Aβ every hour over the course of several days from awake and freely mobile mice [24]. Small molecule compounds can be administered by intraperitoneal (i.p.) injection or by infusing the agents directly into the brain via the microdialysis probe, a method called reverse microdialysis when agents cross the membrane and act locally around the probe. Local infusion circumvents the blood brain barrier and allows for continual administration over time.

We previously showed that treating mice with several SSRIs caused a 25 % decrease in ISF Aβ [12]. SSRI compounds are not selective for a specific 5-HTR; they compete with 5-HT for binding to the serotonin reuptake transporter which increases 5-HT concentrations [29], thus activating all serotonin receptors present. The SSRI-dependent reduction in brain Aβ requires ERK activation, and stimulation of 5-HTRs can stimulate ERK under certain conditions [12, 18, 2022]. These results suggest that any or all of the 15 different 5-HTRs could be responsible for the reduction in ISF Aβ. To determine the role of individual receptors, we administered a selective 5-HTR agonist for each receptor class by reverse microdialysis. Two to three month old APP/PS1 mice [30] were implanted with unilateral microdialysis probes in the hippocampus. Mice at this age do not yet show Aβ plaques which enabled us to study normal Aβ metabolism without the added variable of Aβ pathology [28, 31, 32]. Basal levels of ISF Aβ were evaluated for 9 h before treatment with selective 5-HTR agonists by reverse microdialysis (see Table 1 for dosages). Treatment with a selective agonist for 5-HT4R, 5-HT6R, or 5-HT7R significantly reduced ISF Aβ40 by approximately 25 %. Each of these three receptors was equally effective at lowering Aβ. Interestingly, reductions by each agonist alone are equivalent to reduction induced by 5-HT or citalopram SSRI (Fig. 1a and b), suggesting that each receptor alone is sufficient to induce the maximal effect on ISF Aβ. However, treatment with agonists selective for 5-HT1AR or 5-HT2CR did not produce significant changes in ISF Aβ40 (Fig. 1c and d). These results suggest that only a subset of 5-HTRs, specifically those that activate Gs-coupled proteins, are likely responsible for the reduction in ISF Aβ by serotonin. To ensure that the serotonin-mediated suppression in ISF Aβ is via a normal metabolic pathway and not an artifact of APP transgenic over-expression, we administered citalopram to wildtype, C57Bl6 mice. The SSRI reduced ISF murine Aβ levels by over 30 % compared to vehicle, similar to the effect found in transgenic mice (Fig. 1e).
Table 1

Pharmacological agents used for reverse microdialysis

Compound

Target

IC50 or Ki

Rev MD Concentration

Dosage Across MD Membrane

Selectivity Limit

Reference

Agonists

      

Ipsapirone

5-HT1AR

10 nM

1 μM

100 nM

58 nMa

[59]

WAY161503

5-HT2CR

4 nM

400 nM

40 nM

233 nM

[60]

ML10302

5-HT4R

4 nM

400 nM

40 nM

700 nM

[61]

ST1936

5-HT6R

13 nM

1.3 μM

130 nM

168 nM

[62]

AS19

5-HT7R

0.83 nM

83 nM

8.3 nM

6.6 nMb

[63]

Antagonists

      

GR113808

5-HT4R

0.02 nM

100 nM

10 nM

10 μM

[64]

SB258719

5-HT7R

31.6 nM

3.16 μM

316 nM

316 nM

[68]

Inhibitors

      

GM6001

ADAM/MMP

0.5 nM-27 nM

2.5 μM

250 nM

NA

[66]

KT5720

PKA

60 nM

6 μM

600 nM

2 μM

[67]

PKI14-22

PKA

36 nM

3.6 μM

360 nM

15 μM

[65]

All agents were chosen for their selectivity for their targets as assayed in previous literature. We used the Ki and IC50 values to determine necessary concentrations of each compound (reverse microdialysis (Rev MD) concentration). We assumed only 10 % of the drug would cross the membrane (Dosage Across MD membrane), and this amount of the compound would be further diluted in the brain ISF. The selectivity limit is the concentration above which each compound begins stimulating off-target receptors

aIpsapirone has been shown to stimulate bovine α-adrenergic receptors at 58 nM in vitro [59]

bAS19 can stimulate 5-HT1DR at 6.6 nM concentrations in vitro [63]. However, expression of this receptor is absent in adult mouse hippocampus [14], so effects of its stimulation should be neglible

Abbreviations: 5-HTR serotonin receptor, ADAM a disintegrin and metallopeptidase domain, MMP matrix metallopeptidase, PKA protein kinase A

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

The dose of each agonist was 5-175 times more selective for their targets than other receptor types. We estimate that only 10 % of each compound crosses the microdialysis membrane; however, this varies based on drug properties so we cannot rule out some off-target effects of these compounds. The concentration of 5-HT1AR agonist we used in this experiment could possibly activate α-adrenergic receptors, though we saw no reduction in Aβ levels like those seen with the potent adrenergic receptor agonist caffeine [33]. To verify that the reductions in ISF Aβ were indeed to due to 5-HTR activation, we co-administered a 5-HT7R antagonist and agonist to APP/PS1 mice (Fig. 1f). Pretreatment with the antagonist completely blocked the reduction in ISF Aβ by the agonist, demonstrating the specificity of this suppression in ISF Aβ is due to those receptors.

Citalopram also significantly reduced soluble APPα (sAPPα) levels in the ISF of young APP/PS1 by 17.4 ± 4.0 % (p <0.05) as assessed with 1,000 kDa MWCO microdialysis probes and assessed by Western blotting (Additional file 1: Figure S1). However, the SSRI had no effect on ISF Aβ oligomers in these young mice (Additional file 1: Figure S2).

Selective 5-HTR antagonists increase ISF Aβ

If 5-HT4R, 5-HT6R, and 5-HT7R mediated the reduction in ISF Aβ, then we postulated that blocking their activity may have the opposite effect. To test this hypothesis, we administered GR113808 and SB258719, selective antagonists for 5-HT4R and 5-HT7R respectively, alone or in combination via reverse microdialysis (Fig. 2a). We chose to examine 5-HT4R and 5-HT7R as there is evidence their expression is reduced with increasing age [34]. Antagonizing a single receptor did not significantly change Aβ40 levels after 24 h of administration. However, blocking both receptors caused a significant increase of 32 % in ISF Aβ40 (Fig. 2b). Taken together with data from Fig. 1a, our results demonstrate that basal activity of 5-HT4R and 5-HT7R is normally suppressing Aβ levels, but stimulated activity of only one receptor at a time is sufficient to reduce Aβ levels in acute time frames. This suggests there is a redundancy to 5-HT signaling that is capable of lowering Aβ levels.
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

Inhibition of PKA activity increases ISF Aβ levels

The strong effects of 5-HT4R, 5-HT6R, and 5-HT7R led us to examine downstream signaling pathways. All three of these receptors typically activate Gs proteins, increase cAMP levels, and induce PKA activation [15, 20]. To determine the role of PKA in serotonin-mediated ISF Aβ reduction, we administered KT5720, a small molecule selective PKA inhibitor, via reverse microdialysis. Treatment with KT5720 alone for 24 h significantly increased ISF Aβ levels by 30 %, suggesting PKA has a normal role in regulating basal ISF Aβ levels (Fig. 3a and b). Our previous results suggest that 5-HT4R, 5-HT6R, and 5-HT7R work together to regulate basal ISF Aβ levels; therefore, we stimulated all receptors with a SSRI in order to observe the effects of PKA inhibition. The KT5720 inhibitor also blocked the citalopram-dependent decrease in ISF Aβ levels. To confirm the specificity of the results, we replicated the experiment with a structurally-distinct PKA peptide inhibitor, PKI14-22 amide myristoylate [35]. PKI14-22 was pretreated by reverse microdialysis for 8 h, followed by co-administration of citalopram by i.p. injection (10 mg/kg). Both inhibitors of PKA completely blocked the effect of the SSRI (Fig. 3b). These results strongly suggest PKA activity is required for ISF Aβ reduction by 5-HT signaling.
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

Acute SSRI treatment does not change expression of Aβ metabolism-related genes/proteins

Activation of ERK is one event that can occur downstream of PKA signaling [15]. We have previously shown that pERK levels were elevated after SSRI treatment, and inhibiting the ERK signaling cascade increased ISF Aβ [12]. Once phosphorylated, activated ERK can trans-locate to the nucleus to modify nuclear proteins and suppress gene expression. Interestingly, reductions in secretase gene expression have been observed with chronic SSRI therapy in other studies [12, 36]. To determine if ERK is modifying gene expression to reduce ISF Aβ in the acute phase, we measured expression of genes involved in Aβ metabolism following treatment with citalopram. 2.5 month old APP/PS1 mice were treated with citalopram by i.p. injection (10 mg/kg) or PBS and were sacrificed 16 h later. This dosage of SSRI was enough to reduce ISF Aβ levels (Fig. 1), and 16 h was the earliest time that reductions in ISF Aβ reached statistical significance after SSRI treatment [12]. We performed quantitative real-time PCR (qPCR) for 34 genes involved in Aβ metabolism using purified hippocampal mRNA. Expression of immediate early gene FBJ osteosarcoma oncogene (cFOS) was used as a positive control; its expression is reduced by citalopram within 24 h [37]. Expression of each gene was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and normalized to the mean expression level of PBS-treated controls. CaMKII was used as the control for statistical analysis since our previous studies showed that inhibition of its activity had no effect on ISF Aβ levels [38], and we observed no changes in gene expression by qPCR. There was no significant change in APP expression following SSRI treatment. α-secretase (a disintegrin and metallopeptidase domain 10 (ADAM10), ADAM17, matrix metallopeptidase 9 (MMP9)), β-secretase (beta-site APP-cleaving enzyme (BACE1)), and γ-secretase (anterior pharynx defective 1 (Aph1), basigin, nicastrin, presenilin 1, presenilin 2, presenilin enhancer 2 (PSEN2)) gene expression were not significantly affected by SSRI. There was also no altered expression of MMP9, neprilysin, or low density lipoprotein receptor-related protein 1 (LRP1), three proteins involved with Aβ clearance (Fig. 4a). There was no significant change in major signaling genes such as PKA (PKA catalytic unit α (PKA Cα), PKA catalytic unit β (PKA Cβ), PKC (PKCα), CaMKII, MEK, ERK, or c-Jun N-terminal kinase (JNK). Expression of β-arrestin 2, an important scaffolding protein for many signaling pathways, showed no change after SSRI treatment. Expression of serotonin receptors was also not significantly changed following SSRI treatment (Fig. 4b and c). These negative results suggest that the acute reductions in ISF Aβ following citalopram treatment are unlikely to be mediated by changes in expression of genes linked to Aβ metabolism.
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

We also performed Western blots for several putative α-secretase proteins (ADAM10, ADAM17, MMP2) in hippocampi from mice treated with citalopram for 16 h (Additional file 1: Figure S3). We did not find any change in expression levels for these proteins in whole tissue extracts; however, we cannot discount the possibility of altered sub-cellular localization of these proteins or that their enzymatic activity could be enhanced [12] despite no change in expression.

α-secretase mediates the serotonin-induced reduction in ISF Aβ

We previously showed that α-secretase enzymatic activity was increased by SSRI while β-secretase activity was unchanged [12]. Also there was no difference in Aβ half-life after SSRI treatment which suggests that Aβ clearance was unaffected [12]. These results suggest the reduction of ISF Aβ by serotonin is induced by reducing Aβ production. Similar to other neurotransmitter receptors, we hypothesized that increased α-secretase activity may mediate this suppression of Aβ [3840]. Several proteins demonstrate α-secretase enzymatic activity in vitro such as ADAM10, ADAM17, and MMP9 [3, 41, 42]. The presence of multiple α-secretase enzymes can generate redundancy; therefore, inhibiting a single candidate may not stop all enzyme activity [43, 44]. Consequently, we administered the broad spectrum ADAM/MMP inhibitor, GM6001, by reverse microdialysis for 8 h and then co-administered citalopram by i.p. injection. Treatment with 25 μM GM6001 induced a dramatic increase in ISF Aβ, but levels stabilized near 260 % of baseline after a few hours (Fig. 5a). However, ISF Aβ levels for GM6001-treated mice were not significantly reduced after 16 h of citalopram treatment (Fig. 5b), suggesting that serotonin-induced reduction in ISF Aβ is mediated by α-secretase enzymatic activity.
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

Discussion

We found that only a subset of 5-HTRs are required to suppress brain Aβ levels in vivo. Serotonin receptors that activate PKA subsequently activate ERK to increase α-secretase enzymatic activity. This increased activity cleaves APP within the Aβ sequence to suppress brain ISF Aβ levels.

ISF Aβ levels are reduced by specific 5-HTRs signaling through PKA

Stimulation of serotonin receptors with SSRI antidepressants reduces brain ISF Aβ by 25 % in APP/PS1 transgenic mice [12]. As SSRI compounds are not selective for a specific 5-HTR, therefore any or all of the receptors could reduce ISF Aβ. In this study we demonstrate that stimulation of 5-HT4R, 5-HT6R, and 5-HT7R with selective agonists significantly lowered ISF Aβ while agonists for other 5-HTR subtypes did not (Fig. 1). Interestingly, the magnitude of reduction by 5-HT4R, 5-HT6R, or 5-HT7R activity alone was equal to the reduction induced by serotonin or SSRI. Further, blocking activity of one receptor has no effect on Aβ; blocking two of these receptors together is necessary to increase ISF Aβ (Figs. 1 and 2). This suggests there is a redundancy and compensation in the serotonin signaling pathway that is capable of suppressing Aβ generation. These results suggest that activity of only three 5-HTRs are primarily responsible for the reduction in Aβ after SSRI treatment. Our data are consistent with previous findings in the literature. Tesseur et al. showed 1 month treatment with a 5-HT4R selective agonist reduced soluble Aβ40 and plaque load in the hippocampus [45]. Also, Gianonni et al. showed therapy with a weak 5-HT4R agonist increased concentrations of the α-secretase product and reduced plaque load in 5xFAD mice [46]. 5-HTR subtypes 4, 6 and 7 are Gs-linked receptors that typically activate PKA. Tesseur et al. reported that 5-HT4Rs activate phospholipase C (PLC) to reduce Aβ levels [45]; further studies are necessary to determine if PKA and PLC work in concert or are mutually-exclusive pathways to modulate Aβ levels. It is worth noting that we used agonists for 5-HT 1A, 2C, 4, 6, and 7 receptors as representatives for each class. However, we cannot draw absolute conclusions whether other 5-HTR subtypes may have similar or even contrary effects on Aβ levels. Additionally, activation of 5-HT2CR had a trend to increase ISF Aβ levels (p = 0.0625), suggesting that other receptor subtypes may have the opposite effect on ISF Aβ. Whether or not other 5-HT receptors have opposing effects on Aβ remains to be explored. It is important to note the limitation that few compounds tested had a full dose–response performed to determine their most effective dose in vivo. Most of these selective compounds begin stimulating off-target receptors above a specific concentration. For example, a higher dosage for the 5-HT 1A or 2C agonists may change ISF Aβ levels, but any effects would be confounded by these off-target interactions. Identifying the minimum concentration that reduces ISF Aβ for each compound would be valuable from a therapeutic standpoint. However, the primary focus of this study was 5-HT signaling pathways instead of AD therapy.

5-HTR subtypes 4, 6 and 7 activate Gs proteins, typically increase cAMP levels, and induce PKA activation [15, 20]. The strong reduction of ISF Aβ induced by stimulating these receptors suggests that PKA may be involved. We showed that inhibition of PKA activity with two structurally-distinct, selective inhibitors significantly increased ISF Aβ and abolished the effects of SSRI (Fig. 3). This result suggests that PKA activity is necessary for the reduction in ISF Aβ by serotonergic signaling. Interestingly, the increase in Aβ after PKA inhibition was of a similar magnitude to the increase observed after ERK inhibition [12, 38]. The interactions between these two kinases are complex. Stimulating PKA with serotonin has been shown to induce ERK activation, but there is evidence that ERK can activate PKA after exposure to brain derived neurotrophic factor (BDNF) [20, 47]. However, treating APP/PS1 mice with BDNF had no effect on ISF Aβ [38]. Further work will be necessary to understand the relationship between these two kinases following SSRI treatment.

Citalopram does not change expression of Aβ metabolism related genes after acute SSRI treatment

We have shown that ERK inhibition raises Aβ and abolishes the effects of citalopram [12]. Generally, ERK can act within two cellular compartments; ERK can translocate into the nucleus to modify gene expression or can phosphorylate proteins in the cytoplasm. We saw no significant changes in expression in the major genes related to Aβ metabolism, serotonin receptors, or signaling pathways 16 h after SSRI treatment (Fig. 4). However, these results do not necessarily exclude the role of gene expression in Aβ production after SSRI treatment; other studies have shown reductions in secretase-related genes after chronic SSRI administration [12, 45]. However, those reductions were seen after months of treatment whereas our data were obtained after only a few hours. More research is necessary to understand when expression of Aβ metabolism genes begins to decline after SSRI treatment and what impact this has on Aβ. The rapid reduction in ISF Aβ we observe in the acute timeframe suggests a post-translational response to alter enzymatic activity. Additionally, our qPCR data also suggest ERK is acting outside the nucleus to reduce Aβ.

α-secretase mediates the effects of serotonin-induced reduction of Aβ

ADAM10 is the constitutive α-secretase protein in neurons, however ADAM17 and MMP9 can act as α-secretase as well [3, 41, 42]. Using a broad spectrum inhibitor of ADAM and MMP enzymes, GM6001, we showed that blocking α-secretase enzymatic activity alone increases ISF Aβ levels rapidly and, more importantly for this study, completely blocks the SSRI-dependent suppression in ISF Aβ levels (Fig. 5). Admittedly, broadly inhibiting ADAM/MMP proteins with GM6001 is mechanistically complicated; ADAM/MMP proteases could affect both Aβ generation and Aβ clearance [3, 48]. However, previous studies [12] have found citalopram SSRI does not affect Aβ clearance, meaning the effect here is likely on Aβ production. Unfortunately, the broad range of targets for GM6001 prevents us from identifying which α-secretase protease is responsible for the reduction in ISF Aβ after SSRI. Future studies using a more targeted approach with selective inhibitor compounds or genetic manipulation of the putative α-secretases will be necessary to answer that question.

Interestingly, treatment with a SSRI reduces both ISF Aβ levels and ISF sAPP-α levels. This result is actually opposite of what one might expect if serotonin increases α-secretase activity; however, the sAPP fragments have their own metabolic pathways that are not fully understood. The idea that APP cleavage produces a 1:1 relationship between all the fragments is not necessarily true. We find this new data intriguing and believe future studies are important and necessary to uncover the mechanisms are work here.

Our results suggest the simplified model presented in Fig. 6. Serotonin activates 5-HT4R, 5-HT6R, and 5-HT7R which then activate PKA. PKA signaling activates MEK and ERK. We hypothesize that activated ERK phosphorylates an α-secretase in the cytoplasm to increase its activity and reduce ISF Aβ. There is evidence that phosphorylation by ERK can increase α-secretase enzymatic activity [49]. ERK activation increases cleavage activity of ADAM17 in vitro, and mutating the putative ERK phosphorylation site blocks this increase [50]. Interestingly, ADAM10 contains a similar ERK consensus site which suggests it may also be an ERK substrate [51, 52]. Future research will determine if ERK directly phosphorylates α-secretase after SSRI exposure.
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

Activation of 5-HTR subtypes 4, 6, and 7 all reduce ISF Aβ by ~25 % which is the same amount as either SSRI treatment or direct 5-HT infusion (Fig. 1a, b). This suggests that these receptors likely activate a common signaling pathway, and activity of any of these receptors is sufficient to fully activate this pathway to suppress Aβ. Supportive of this hypothesis is that antagonizing one receptor alone does not change ISF Aβ, but antagonizing two receptors is necessary to increase Aβ by 30 % (Fig. 2). This suggests there is functional redundancy to serotonergic suppression of Aβ generation.

Conclusions

The dramatic reductive effects of SSRIs on Aβ in mice and humans are encouraging for AD therapy. Millions of individuals world-wide use SSRIs on a daily basis for Depression and other disorders; however, these agents are not without side effects. 5-HTRs can be found throughout the digestive system [53, 54], and their stimulation by SSRI therapy can cause unpleasant digestive problems [55]. Also, stimulation of 5-HT2R in vascular tissue can lead to constriction of blood vessels and sexual side effects [55, 56]. Focusing AD therapy on a single or subset of 5-HTRs may reduce some of the side effects of SSRI treatment. In this report, we have shown activation of 5-HT4R, 5-HT6R, and 5-HT7R by selective agonists can significantly reduce ISF Aβ in mice by 25 %. In other mouse studies, a similar reduction of Aβ levels is sufficient to completely block plaque growth and significantly reduce new plaque formation [13, 57]. The safety and efficacy of selective agonists for these receptors need to be tested in humans; however, they offer a tantalizing path to treating AD.

Methods

Animals

All experiment protocols using animals were performed in accordance to the guidelines established by the Animal Studies Committee at Washington University. We bred APPswe/PS1∆E9 hemizygous mice (Jackson Laboratory) [58] to wild type C3H/B6 mice and aged the APP/PS1 +/- offspring to 2–3.5 months for experiments. Mice were screened for APPswe and PS1ΔE9 transgenes by PCR from toe DNA.

Compounds

All pharmaceutical compounds were ordered from Tocris Biosciences except for serotonin hydrochloride (Sigma-Aldrich). All compounds delivered by reverse microdialysis were diluted in microdialysis buffer consisting of artificial cerebrospinal fluid (aCSF) with 0.15-2 % BSA (Sigma-Aldrich). Specificity of each compound for its target was established by activity assays against libraries of receptors as described in previous literature [5968]. The selectivity limit is the concentration above which the compound begins to stimulate off-target receptors. Concentrations used took into account the IC50 or Ki for each compound and only 10 % delivery across the membrane at a 1 μl/min flow rate (Table 1). This small volume of each compound would be diluted even further in brain CSF. Agonists are 5-175 times more selective for their targets than other receptor types. Citalopram hydrobromide (Toronto Research Chemicals) was diluted in PBS and injected i.p. at 10 mg/kg of body weight.

In vivo Aβ microdialysis

In vivo microdialysis to measure brain ISF Aβ and sAPP-α in the hippocampus of freely moving APP/PS1 mice was performed similar to previously described [12, 24, 28]. This method captures soluble molecules in the extracellular fluid that are below the 30 kDa molecular weight cutoff of the probes. Under volatile isoflurane anesthetic, guide cannula (BR-style; Bioanalytical Systems or Atmos; EICOM) were cemented above the left hippocampus (3.1 mm behind Bregma, 2.5 mm lateral to midline, and 1.2 mm below dura at a 12° angle). Two millimeter microdialysis probes were inserted through the guides so their membranes were completely contained in the hippocampus (30 kDa BR-2; Bioanalytical Systems; 1,000 kDa AtmosLM; EICOM). Microdialysis buffer was aCSF (perfusion buffer in mM: 1.3 CaCl2, 1.2 MgSO4, 3 KCl, 0.4 KH2PO4, and 122 NaCl, pH 7.35) containing 0.15-2 % BSA (Sigma-Aldrich) that was filtered through a 0.1 μM membrane. The flow rate was 1.0 μL/min. Samples were collected every 60 or 90 min into a refrigerated fraction collector (Univentor Limited) in polypropylene tubes and assessed for Aβx-40 by sandwich ELISA. Basal ISF Aβ levels were defined as the mean concentration of Aβ over the 9 h preceding drug administration. All Aβ values were normalized to the basal Aβ concentration for each animal. After establishing baseline ISF Aβ, pharmaceutical agents (serotonin, 5-HTR agonists, 5-HTR antagonists, enzyme inhibitors) were diluted in microdialysis perfusion buffer and infused directly into the hippocampus by reverse microdialysis (Table 1).

Aβ sandwich ELISA

ISF Aβx-40 levels were measured using sandwich ELISAs as described [12]. This ELISA detects both human and murine Aβ. A mouse anti-Aβ40 antibody (mHJ2; 10 μg/ml) against the C-terminus of Aβ was used to capture peptides and a biotinylated central domain antibody (mHJ5.1; 75 ng/ml) was used to detect them. This was followed by a streptavidin poly-HRP-40 assay to measure Aβ concentration (Fitzgerald Industries). All steps included washes with PBS containing 0.05 % Tween-20. The standard curve for the ELISA was synthetic Aβ40 (American Peptide) taken from a stock in formic acid to remove preformed aggregates. ELISA sample buffers included sufficient Tris to neutralize pH of the formic acid. To detect Aβ oligomers, ELISA plates were coated a mouse anti-Aβ antibody (mHJ3.4; 10 μg/ml) to capture of N-terminus of the peptide then detected using the same monoclonal antibody that was biotinylated (500 ng/ml). None of the buffers for this assay contain detergents as such agents can artificially produce Aβ aggregates. The standard curve for this ELISA is a synthetic dimer of human Aβ40 that contains a Ser26Cys mutation that enables us to covalently crosslink the peptides together. The dimer is then specifically isolated by size exclusion chromatography. Aβ dimers were a generous gift from Dr. David Brody, Washington University. The oligomer assay is similar to one used in [69]. All ELISAs were developed using Super Slow ELISA TMB (Sigma-Aldrich) and absorbance read on a Bio-Tek Epoch plate reader at 650 nm.

Quantitative real-time PCR (qPCR)

2.5 month old APP/PS1 mice were given 10 mg/kg i.p. injections of citalopram or PBS. Mice were sacrificed 16 h later, and their hippocampi were microdissected. Total RNA were extracted using the RNeasy Mini kit (Qiagen) and reverse transcribed using the High Capacity cDNA Reverse Transcription kit (Life Technologies). Individual primers were designed using Harvard Medical School Primer Bank (http://pga.mgh.harvard.edu/primerbank/index.html) [7072]. See Additional file 1 for primer sequences and database identification numbers. qPCR was performed using the Fast SYBR Green Master Mix (Applied Biosystems) in ABI 7900HT (Applied Biosystems) with the default thermal cycling program. Dissociation curves were analyzed following qPCR assay to confirm primer efficacy. Endogenous mouse GAPDH was used as a normalization reference. Relative mRNA levels were calculated by comparative Ct method using the ABI 7900HT Sequence Detection Systems version 2.0.5 and GenEx version 5 software (MultiD analyses). Levels of each mRNA were statistically compared to CaMKII mRNA levels, which our previous studies have shown does not mediate suppression of brain Aβ levels [38].

SDS-PAGE/western blot

ISF samples collected from 1,000 kDa MWCO microdialysis probes (AtmosLM, Eicom) in APP/PS1 mice treated with citalopram for 16 h were utilized for Western blotting for soluble APP-α. Hippocampus from APP/PS1 mice treated with citalopram for 16 h was lysed in 1 % Triton X-100, 0.1 % SDS in PBS by sonication. 20 μg of total protein was loaded per lane on SDS-PAGE gels. SDS-PAGE was performed using 4–12 % Bis-Tris NuPAGE gels (Invitrogen, Carlsbad, CA) under reducing conditions with 24 μl of microdialysis sample loaded into each lane. Samples were loaded onto the gels in alternating basal and drug-treated samples. Nitrocellulose blots were probed with either mouse-anti-human APP antibody directed against the C-terminus of sAPP-α (m6E10; BioLegend), rabbit-anti-ADAM10 antibody (H-300; Santa Cruz Biotech), rabbit-anti-ADAM17 (Abcam), goat-anti-MMP2 (C-19; Santa Cruz Biotech), or mouse-anti-GAPDH (Sigma-Aldrich) followed by either a sheep-anti-mouse antibody (GE Life Sciences), donkey-anti-rabbit antibody (Santa Cruz Biotech), or a donkey-anti-goat antibody (Santa Cruz Biotech) conjugated to peroxidase. Bands were detected with Lumigen-TMA6 (Amersham, Piscataway, NJ) and captured digitally using the Kodak ImageStation 440CF. Densitometry was performed using the Kodak 1D Image Analysis software. Tissue protein levels were normalized to GAPDH for each lane, then normalized to % vehicle for each blot, followed by data from two blots being combined for analysis.

Statistical analysis

Data in figures are presented as mean ± SEM. All statistical analysis was performed using Prism version 6.0 for Windows (GraphPad). For analysis of microdialysis data at endpoints, the final 3 data points for a given treatment phase were averaged, and one-way ANOVA analyses were performed with Dunnett’s corrections for multiple comparisons. Comparison of only two groups was performed using a two-tailed, unpaired Student t-test method. For qPCR analysis, mean levels of gene expression were analyzed by one-way ANOVA with Dunnett’s corrections for multiple comparisons. Values were accepted as significant if p-value ≤ 0.05.

Declarations

Acknowledgements

Thanks to Dr. Jungsu Kim at Mayo Clinic in Jacksonville for advice on qPCR primer design, help with data analysis, and primer sequences for APP. Thank you to Drs. Qingli Xiao and Jin-Moo Lee at Washington University for the MMP9 primer sequences. This work was funded by National Institutes of Health (NIH)/NIA (R01 AG042513; JRC), NIH/NIA (R21 AG045691; JRC) NIH/NINDS (P01 NS074969; JRC), NIH/NIA (R01 AG041502; YIS), NIH/NIA (P50), BrightFocus Foundation, the Knight Family, and the Charles F. and Joanne Knight ADRC at Washington University (JRC). Funding was also provided by the NIH/NIGMS Cellular and Molecular Training Program (GM007067; JRF).

Authors’ contributions

JF was responsible for designing and performing the experiments described in this text. He also analyzed the data, wrote the manuscript, and edited the document. DT and CEW performed experiments and analyzed data. YS aided with experimental design, data interpretation, and manuscript revision. JC assisted with experimental design, performed experiments, aided with data interpretation, and revised the manuscript. All authors read and approved the manuscript.

Competing interests

The authors declare that they have no competing interests.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Neurology, Washington University School of Medicine
(2)
Knight Alzheimer’s Disease Research Center, Washington University School of Medicine
(3)
Hope Center for Neurological Disorders, Washington University School of Medicine
(4)
Departments of Psychiatry, Radiology, and Neurology, University of Pennsylvania
(5)
Present Address: Washington University, Neurology

References

  1. Seubert P, Oltersdorf T, Lee M, Barbour R, Blomquist C, Davis D, Bryant K, Fritz L, Galasko D, Thal L. Secretion of beta-amyloid precursor protein cleaved at the amino terminus of the beta-amyloid peptide. Nature. 1993;361:260–3.View ArticlePubMedGoogle Scholar
  2. Edbauer D, Winkler E, Regula JT, Pesold B, Steiner H, Haass C. Reconstitution of gamma-secretase activity. Nat Cell Biol. 2003;5:486–8.View ArticlePubMedGoogle Scholar
  3. Lammich S, Kojro E, Postina R, Gilbert S, Pfeiffer R, Jasionowski M, Laass C, Fahrenholz F. Constitutive and regulated alpha-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease. Proc Natl Acad Sci U S A. 1999;96(March):3922–7.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Lomakin A, Teplow D, Kirschner D, Benedek G. Kinetic theory of fibrillogenesis of amyloid beta-protein. Proc Natl Acad Sci U S A. 1997;94:7942–7.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Bero AW, Yan P, Roh JH, Cirrito JR, Stewart FR, Raichle ME, Lee J-M, Holtzman DM. Neuronal activity regulates the regional vulnerability to amyloid-β deposition. Nat Neurosci. 2011;14:750–6.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Nelson RL, Guo Z, Halagappa VM, Pearson M, Gray AJ, Matsuoka Y, Brown M, Martin B, Iyun T, Maudsley S, Clark RF, Mattson MP. Prophylactic treatment with paroxetine ameliorates behavioral deficits and retards the development of amyloid and tau pathologies in 3xTgAD mice. Exp Neurol. 2007;205:166–76.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Reynolds GP, Mason SL, Meldrum A, De KS, Parnes H, Eglen M, Wong HF. 5-Hydroxytryptamine (5-HT)4 receptors in post mortem human brain tissue: distribution, pharmacology and effects of neurodegenerative diseases. Br J Pharmacol. 1995;114:993–8.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Arjona A, Pooler AM, Lee RK, Wurtman RJ. Effect of a 5-HT(2C) serotonin agonist, dexnorfenfluramine, on amyloid precursor protein metabolism in guinea pigs. Brain Res. 2002;951:135–40.View ArticlePubMedGoogle Scholar
  9. Nitsch RM, Deng M, Growdon JH, Wurtman RJ. Serotonin 5-HT2a and 5-HT2c receptors stimulate amyloid precursor protein ectodomain secretion. J Biol Chem. 1996;271:4188–94.View ArticlePubMedGoogle Scholar
  10. Robert SJ, Zugaza JL, Fischmeister R, Gardier AM, Lezoualc’h F. The human serotonin 5-HT4 receptor regulates secretion of non-amyloidogenic precursor protein. J Biol Chem. 2001;276:44881–8.View ArticlePubMedGoogle Scholar
  11. Hashimoto G, Sakurai M, Teich AF, Saeed F, Aziz F, Arancio O. 5-HT4 receptor stimulation leads to soluble AβPPα production through MMP-9 upregulation. J Alzheimers Dis. 2012;32:437–45.PubMedGoogle Scholar
  12. Cirrito JR, Disabato BM, Restivo JL, Verges DK, Goebel WD, Sathyan A, Hayreh D, D’Angelo G, Benzinger T, Yoon H, Kim J, Morris JC, Mintun MA, Sheline YI. Serotonin signaling is associated with lower amyloid-β levels and plaques in transgenic mice and humans. Proc Natl Acad Sci U S A. 2011;108:14968–73.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Sheline Y, West T, Yarasheski K, Swarm R, Jasielec M, Fisher J, Ficker W, Yan P, Xiong C, Frederiksen C, Chott R, Bateman R, Morris J, Mintun M, Lee J, Cirrito J. An Antidepressant Decreases CSF Amyloid-beta Production in Healthy Individuals and in Transgenic Mice. Sci Transl Med. 2014;6:236re4.View ArticlePubMedPubMed CentralGoogle Scholar
  14. Tanaka KF, Samuels BA, Hen R. Serotonin receptor expression along the dorsal-ventral axis of mouse hippocampus. Philos Trans R Soc Lond B Biol Sci. 2012;367:2395–401.View ArticlePubMedPubMed CentralGoogle Scholar
  15. Hannon J, Hoyer D. Molecular biology of 5-HT receptors. Behav Brain Res. 2008;195:198–213.View ArticlePubMedGoogle Scholar
  16. Leone AM, Errico M, Lin SL, Cowen DS, Lione AM. Activation of extracellular signal-regulated kinase (ERK) and Akt by human serotonin 5-HT(1B) receptors in transfected BE(2)-C neuroblastoma cells is inhibited by RGS4. J Neurochem. 2000;75:934–8.View ArticlePubMedGoogle Scholar
  17. Francken BJ, Josson K, Lijnen P, Jurzak M, Luyten WH, Leysen JE. Human 5-hydroxytryptamine(5A) receptors activate coexpressed G(i) and G(o) proteins in Spodoptera frugiperda 9 cells. Mol Pharmacol. 2000;57:1034–44.PubMedGoogle Scholar
  18. Adayev T, Ray I, Sondhi R, Sobocki T, Banerjee P. The G protein-coupled 5-HT1A receptor causes suppression of caspase-3 through MAPK and protein kinase Cα. Biochim Biophys Acta - Mol Cell Res. 2003;1640:85–96.View ArticleGoogle Scholar
  19. Lairez O, Cognet T, Schaak S, Calise D, Guilbeau-Frugier C, Parini A, Mialet-Perez J. Role of serotonin 5-HT2A receptors in the development of cardiac hypertrophy in response to aortic constriction in mice. J Neural Transm. 2013;120:927–35.View ArticlePubMedGoogle Scholar
  20. Norum JH, Hart K, Levy FO. Ras-dependent ERK activation by the human G(s)-coupled serotonin receptors 5-HT4(b) and 5-HT7(a). J Biol Chem. 2003;278:3098–104.View ArticlePubMedGoogle Scholar
  21. Launay J, Birraux G, Bondoux D, Callebert J, Choi D, Loric S, Maroteaux L. Ras Involvement in Signal Transduction by the Serotonin 5-HT2B Receptor. J Biol Chem. 1996;271:3141–7.View ArticlePubMedGoogle Scholar
  22. Johnson-Farley NN, Kertesy SB, Dubyak GR, Cowen DS. Enhanced activation of Akt and extracellular-regulated kinase pathways by simultaneous occupancy of Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT7A receptors in PC12 cells. J Neurochem. 2005;92:72–82.View ArticlePubMedGoogle Scholar
  23. Desdouits-Magnen J, Desdouits F, Takeda S, Syu LJ, Saltiel AR, Buxbaum JD, Czernik AJ, Nairn AC, Greengard P. Regulation of secretion of Alzheimer amyloid precursor protein by the mitogen-activated protein kinase cascade. J Neurochem. 1998;70:524–30.View ArticlePubMedGoogle Scholar
  24. Cirrito JR, Kang J-E, Lee J, Stewart FR, Verges DK, Silverio LM, Bu G, Mennerick S, Holtzman DM. Endocytosis is required for synaptic activity-dependent release of amyloid-beta in vivo. Neuron. 2008;58:42–51.View ArticlePubMedPubMed CentralGoogle Scholar
  25. Zhao J, Paganini L, Mucke L, Gordon M, Refolo L, Carman M, Sinha S, Oltersdorf T, Lieberburg I, McConlogue L. Beta-secretase processing of the beta-amyloid precursor protein in transgenic mice is efficient in neurons but inefficient in astrocytes. J Biol Chem. 1996;271:31407–11.View ArticlePubMedGoogle Scholar
  26. Kamenetz F, Tomita T, Hsieh H, Seabrook G, Borchelt D, Iwatsubo T, Sisodia S, Malinow R, Point W. APP Processing and Synaptic Function. Neuron. 2003;37:925–37.View ArticlePubMedGoogle Scholar
  27. Cirrito JR, Yamada K, Finn MB, Sloviter RS, Bales KR, May PC, Schoepp DD, Paul SM, Mennerick S, Holtzman DM. Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo. Neuron. 2005;48:913–22.View ArticlePubMedGoogle Scholar
  28. Cirrito JR, May PC, O’Dell M, Taylor JW, Parsadanian M, Cramer JW, Audia JE, Nissen JS, Bales KR, Paul SM, DeMattos RB, Holtzman DM. In vivo assessment of brain interstitial fluid with microdialysis reveals plaque-associated changes in amyloid-beta metabolism and half-life. J Neurosci. 2003;23:8844–53.PubMedGoogle Scholar
  29. Apparsundaram S, Stockdale DJ, Henningsen RA, Milla ME, Martin RS. Antidepressants Targeting the Serotonin Reuptake Transporter Act via a Competitive Mechanism. J Pharmacol Exp Ther. 2008;327:982–90.View ArticlePubMedGoogle Scholar
  30. Borchelt DR, Ratovitski T, van Lare J, Lee MK, Gonzales V, Jenkins N, Copeland NG, Price DL, Sisodia SS. Accelerated amyloid deposition in the brains of transgenic mice coexpressing mutant presenilin 1 and amyloid precursor proteins. Neuron. 1997;19:939–45.View ArticlePubMedGoogle Scholar
  31. Jankowsky JL, Slunt HH, Ratovitski T, Jenkins NA, Copeland NG, Borchelt DR. Co-expression of multiple transgenes in mouse CNS: a comparison of strategies. Biomol Eng. 2001;17:157–65.View ArticlePubMedGoogle Scholar
  32. Jankowsky JL, Slunt HH, Gonzales V, Jenkins NA, Copeland NG, Borchelt DR. APP processing and amyloid deposition in mice haplo-insufficient for presenilin 1. Neurobiol Aging. 2004;25:885–92.View ArticlePubMedGoogle Scholar
  33. Cao C, Cirrito JR, Lin X, Wang L, Wang L, Verges DK, Dickson A, Mamcarz M, Zhang C, Mori T, Arendash GW, Holtzman DM, Potter H. Caffeine suppresses amyloid-beta levels in plasma and brain of Alzheimer’s disease transgenic mice. J Alzheimers Dis. 2009;17:681–97.PubMedPubMed CentralGoogle Scholar
  34. Rodríguez JJ, Noristani HN, Verkhratsky A. The serotonergic system in ageing and Alzheimer’s disease. Prog Neurobiol. 2012;99:15–41.View ArticlePubMedGoogle Scholar
  35. Dalton GD, Smith FL, Smith P, Dewey WL. Alterations in brain Protein Kinase A activity and reversal of morphine tolerance by two fragments of native Protein Kinase A inhibitor peptide (PKI). Neuropharmacology. 2005;48:648–57.View ArticlePubMedGoogle Scholar
  36. Wortzel I, Seger R. The ERK Cascade: Distinct Functions within Various Subcellular Organelles. Genes Cancer. 2011;2:195–209.View ArticlePubMedPubMed CentralGoogle Scholar
  37. Gąska M, Kuśmider M, Solich J, Faron-Górecka A, Krawczyk MJ, Kułakowski K, Dziedzicka-Wasylewska M. Analysis of region-specific changes in gene expression upon treatment with citalopram and desipramine reveals temporal dynamics in response to antidepressant drugs at the transcriptome level. Psychopharmacology (Berl). 2012;223:281–97.View ArticleGoogle Scholar
  38. Verges DK, Restivo JL, Goebel WD, Holtzman DM, Cirrito JR. Opposing synaptic regulation of amyloid-β metabolism by NMDA receptors in vivo. J Neurosci. 2011;31:11328–37.View ArticlePubMedPubMed CentralGoogle Scholar
  39. Haring R, Fisher A, Marciano D, Pittel Z, Kloog Y, Zuckerman A, Eshhar, N, Heldman E. Mitogen-activated protein kinase-dependent and protein kinase C-dependent pathways link the m1 muscarinic receptor to beta-amyloid precursor protein secretion. J Neurochem. 1998;71:2094–103.View ArticlePubMedGoogle Scholar
  40. Nitsch RM, Slack BE, Wurtman RJ, Growdon JH. Release of Alzheimer amyloid precursor derivatives stimulated by activation of muscarinic acetylcholine receptors. Science. 1992;258:304–7.View ArticlePubMedGoogle Scholar
  41. Buxbaum JD, Liu K, Slack JL, Stocking KL, Peschon JJ, Johnson RS, Castner BJ, Cerretti DP, Black RA, Luo Y. Evidence That Tumor Necrosis Factor Alpha Converting Enzyme is Involved in Regulated alpha-Secretase Cleavage of the Alzheimer Amyloid Protein Precursor. J Biol Chem. 1998;273:27765–7.View ArticlePubMedGoogle Scholar
  42. Fragkouli A, Papatheodoropoulos C, Georgopoulos S, Stamatakis A, Stylianopoulou F, Tsilibary EC, Tzinia AK. Enhanced neuronal plasticity and elevated endogenous sAPPα levels in mice over-expressing MMP9. J Neurochem. 2012;121:239–51.View ArticlePubMedGoogle Scholar
  43. Kuhn P-H, Wang H, Dislich B, Colombo A, Zeitschel U, Ellwart JW, Kremmer E, Rossner S, Lichtenthaler SF. ADAM10 is the physiologically relevant, constitutive alpha-secretase of the amyloid precursor protein in primary neurons. EMBO J. 2010;29:3020–32.View ArticlePubMedPubMed CentralGoogle Scholar
  44. Suh J, Choi SH, Romano DM, Gannon MA, Lesinski AN, Kim DY, Tanzi RE. ADAM10 missense mutations potentiate β-amyloid accumulation by impairing prodomain chaperone function. Neuron. 2013;80:385–401.View ArticlePubMedPubMed CentralGoogle Scholar
  45. Tesseur I, Pimenova AA, Lo AC, Ciesielska M, Lichtenthaler SF, De Maeyer JH, Schuurkes JAJ, D’Hooge R, De Strooper B. Chronic 5-HT4 receptor activation decreases Aβ production and deposition in hAPP/PS1 mice. Neurobiol Aging. 2013;34:1779–89.View ArticlePubMedGoogle Scholar
  46. Giannoni P, Gaven F, de Bundel D, Baranger K, Marchetti-Gauthier E, Roman FS, Valjent E, Marin P, Bockaert J, Rivera S, Claeysen S. Early administration of RS 67333, a specific 5-HT4 receptor agonist, prevents amyloidogenesis and behavioral deficits in the 5XFAD mouse model of Alzheimer’s disease. Front Aging Neurosci. 2013;5(December):96.PubMedPubMed CentralGoogle Scholar
  47. Gao Y, Nikulina E, Mellado W, Filbin MT. Neurotrophins elevate cAMP to reach a threshold required to overcome inhibition by MAG through extracellular signal-regulated kinase-dependent inhibition of phosphodiesterase. J Neurosci. 2003;23:11770–7.PubMedGoogle Scholar
  48. Yin K-J, Cirrito JR, Yan P, Hu X, Xiao Q, Pan X, Bateman R, Song H, Hsu F-F, Turk J, Xu J, Hsu CY, Mills JC, Holtzman DM, Lee J-M. Matrix metalloproteinases expressed by astrocytes mediate extracellular amyloid-beta peptide catabolism. J Neurosci. 2006;26:10939–48.View ArticlePubMedGoogle Scholar
  49. Kojro E, Postina R, Buro C, Meiringer C, Gehrig-Burger K, Fahrenholz F. The neuropeptide PACAP promotes the alpha-secretase pathway for processing the Alzheimer amyloid precursor protein. FASEB J. 2006;20:512–4.PubMedGoogle Scholar
  50. Cisse M, Braun U, Leitges M, Fisher A, Pages G, Checler F, Vincent B. ERK1-independent α-secretase cut of β-amyloid precursor protein via M1 muscarinic receptors and PKCα/ε. Mol Cell Neurosci. 2011;47:223–32.View ArticlePubMedGoogle Scholar
  51. Gonzalez FA, Raden DL, Davis RJ. Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases. J Biol Chem. 1991;266:22159–63.PubMedGoogle Scholar
  52. Yavari R, Adida C, Bray-Ward P, Brines M, Xu T. Human metalloprotease-disintegrin Kuzbanian regulates sympathoadrenal cell fate in development and neoplasia. Hum Mol Genet. 1998;7:1161–7.View ArticlePubMedGoogle Scholar
  53. Bard J, Zgombick J, Adham N, Vaysse P, Branchek T, Weinshank RL. Cloning of a novel human serotonin receptor (5-HT7) positively linked to adenylate cyclase. J Biol Chem. 1993;268:23422–6.PubMedGoogle Scholar
  54. Prins NH, Akkermans LM, Lefebvre RA, Schuurkes JA. 5-HT(4) receptors on cholinergic nerves involved in contractility of canine and human large intestine longitudinal muscle. Br J Pharmacol. 2000;131:927–32.View ArticlePubMedPubMed CentralGoogle Scholar
  55. Rosenzweig-Lipson S, Beyer CE, Hughes ZA, Khawaja X, Rajarao SJ, Malberg JE, Rahman Z, Ring RH, Schechter LE. Differentiating antidepressants of the future: efficacy and safety. Pharmacol Ther. 2007;113:134–53.View ArticlePubMedGoogle Scholar
  56. Watts SW, Yang P, Banes AK, Baez M. Activation of Erk mitogen-activated protein kinase proteins by vascular serotonin receptors. J Cardiovasc Pharmacol. 2001;38:539–51.View ArticlePubMedGoogle Scholar
  57. Yan P, Bero AW, Cirrito JR, Xiao Q, Hu X, Wang Y, Gonzales E, Holtzman DM, Lee J-M. Characterizing the appearance and growth of amyloid plaques in APP/PS1 mice. J Neurosci. 2009;29:10706–14.View ArticlePubMedPubMed CentralGoogle Scholar
  58. Savonenko AV, Xu GM, Price DL, Borchelt DR, Markowska AL. Normal cognitive behavior in two distinct congenic lines of transgenic mice hyperexpressing mutant APPSWE. Neurobiol Dis. 2003;12:194–211.View ArticlePubMedGoogle Scholar
  59. Peroutka SJ. Selective interaction of novel anxiolytics with 5-hydroxytryptamine1A receptors. Biol Psychiatry. 1985;20:971–9.View ArticlePubMedGoogle Scholar
  60. Rosenzweig-Lipson S, Zhang J, Mazandarani H, Harrison BL, Sabb A, Sabalski J, Stack G, Welmaker G, Barrett JE, Dunlop J. Antiobesity-like effects of the 5-HT 2C receptor agonist WAY-161503. Brain Res. 2006;1073–1074:240–51.View ArticlePubMedGoogle Scholar
  61. Yang D, Soulier JL, Sicsic S, Mathé-Allainmat M, Brémont B, Croci T, Cardamone R, Aureggi G, Langlois M. New esters of 4-amino-5-chloro-2-methoxybenzoic acid as potent agonists and antagonists for 5-HT4 receptors. J Med Chem. 1997;40:608–21.View ArticlePubMedGoogle Scholar
  62. Valentini V, Frau R, Bordi F, Borsini F, Di Chiara G. A microdialysis study of ST1936, a novel 5-HT6 receptor agonist. Neuropharmacology. 2011;60:602–8.View ArticlePubMedGoogle Scholar
  63. Brenchat A, Romero L, García M, Pujol M, Burgueño J, Torrens A, Hamon M, Baeyens JM, Buschmann H, Zamanillo D, Vela JM. 5-HT7 receptor activation inhibits mechanical hypersensitivity secondary to capsaicin sensitization in mice. Pain. 2009;141:239–47.View ArticlePubMedGoogle Scholar
  64. Gale JD, Grossman CJ, Whitehead JWF, Oxford AW, Bunce KT, Humphrey PPA. GR113808: a novel, selective antagonist with high affinity at the 5-HT4 receptor. Br J Pharmacol. 1994;111:332–8.View ArticlePubMedPubMed CentralGoogle Scholar
  65. Glass DB, Cheng HC, Kemp BE, Walsh DA. Differential and common recognition of the catalytic sites of the cGMP-dependent and cAMP-dependent protein kinases by inhibitory peptides derived from the heat-stable inhibitor protein. J Biol Chem. 1986;261:12166–71.PubMedGoogle Scholar
  66. Grobelny D, Poncz L, Galardy RE. Inhibition of human skin fibroblast collagenase, thermolysin, and Pseudomonas aeruginosa elastase by peptide hydroxamic acids. Biochemistry. 1992;31:7152–4.View ArticlePubMedGoogle Scholar
  67. Gadbois DM, Crissman HA, Tobey RA, Bradbury EM. Multiple kinase arrest points in the G1 phase of nontransformed mammalian cells are absent in transformed cells. Proc Natl Acad Sci U S A. 1992;89:8626–30.View ArticlePubMedPubMed CentralGoogle Scholar
  68. Linnanen T, Brisander M, Unelius L, Hacksell U, Johansson AM. Atropisomeric derivatives of 2’,6'-disubstituted (R) -11-phenylaporphine : selective serotonin 5-HT 7 receptor antagonists. J Med Chem. 2001;44:1337–40.View ArticlePubMedGoogle Scholar
  69. Esparza TJ, Zhao H, Cirrito JR, Cairns NJ, Bateman RJ, Holtzman DM, Brody DL. Amyloid-beta oligomerization in Alzheimer dementia versus high-pathology controls. Ann Neurol. 2013;73:104–19.View ArticlePubMedGoogle Scholar
  70. Spandidos A, Wang X, Wang H, Seed B. PrimerBank: a resource of human and mouse PCR primer pairs for gene expression detection and quantification. Nucleic Acids Res. 2010;38(Database issue):D792–9.View ArticlePubMedGoogle Scholar
  71. Spandidos A, Wang X, Wang H, Dragnev S, Thurber T, Seed B. A comprehensive collection of experimentally validated primers for Polymerase Chain Reaction quantitation of murine transcript abundance. BMC Genomics. 2008;9:633.View ArticlePubMedPubMed CentralGoogle Scholar
  72. Wang X, Seed B. A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res. 2003;31:154e–154.View ArticleGoogle Scholar

Copyright

© The Author(s). 2016