Serotonin 6 receptor controls Alzheimer's disease and depression

Abstract

Alzheimer's disease (AD) and depression in late life are one of the most severe health problems in the world disorders. Serotonin 6 receptor (5-HT6R) has caused much interest for potential roles in AD and depression. However, a causative role of perturbed 5-HT6R function between two diseases was poorly defined. In the present study, we found that a 5-HT6R antagonist, SB271036 rescued memory impairment by attenuating the generation of Aβ via the inhibition of γ-secretase activity and the inactivation of astrocytes and microglia in the AD mouse model. It was found that the reduction of serotonin level was significantly recovered by SB271036, which was mediated by an indirect regulation of serotonergic neurons via GABA. Selective serotonin reuptake inhibitor (SSRI), fluoxetine significantly improved cognitive impairment and behavioral changes. In human brain of depression patients, we then identified the potential genes, amyloid beta (A4) precursor protein-binding, family A, member 2 (APBA2), well known AD modulators by integrating datasets from neuropathology, microarray, and RNA seq. studies with correlation analysis tools. And also, it was demonstrated in mouse models and patients of AD. These data indicate functional network of 5-HT6R between AD and depression.

Keywords: 5-HT6R; APBA1/2; Alzheimer’s disease; depression; serotonin.

Figure 1. Inhibitory effects of SB271046 on memory impairment in the AD mouse model

A.–C. Training trial was performed three times a day for 5 days. Swimming time A. and swimming distance B. to arrive at platform were automatically recorded. 24 hr after training trials, a probe test was performed. The time spent in the target quadrant and target site crossing within 60 s was represented C.. Each value is presented as mean ± S.E.M from 10 mice. D. To perform passive avoidance test, the mice were given electric shock when entered into the dark compartment for training on learning day. After one day, the retention time in illuminated compartment was recorded. Each value is presented as mean ± S.E.M. from 10 mice. *,^#Significant difference (^*,#P < 0.05 and **P < 0.01).

Figure 2. Inhibitory effects of SB271046 on Aβ1-42 generation and neuroinflammation in the AD mouse model

A. Aβ_1-42 level was measured in mouse brains by ELISA as described in Materials and Methods. Value is mean ± S.E.M of 8 mice. B., C. The activities of β-secretase B. and γ-secretase C. were carried out by using each assay kit as described in Methods. Value is mean ± S.E.M of 8 mice. D., E. The effect of SB271046 on reactive astrocytes and activated microglia cells was measured by immunohistochemical analysis. The sections of mice brain incubated with anti-glial fibrillary acidic protein (GFAP) D. and anti-ionized calcium binding adaptor molecule 1 (Iba1) E. antibodies, followed by the biotinylated secondary antibody (n = 8). The stained tissues were viewed with a microscope (×50 or 200). F. The sections of mouse brain were incubated with anti-iNOS antibody, and then followed by the biotinylated secondary antibody (n = 8). The resulting tissue was viewed with a microscope. ^*,#Significant difference (^*,#P < 0.05). The experiments shown in Figure 2 were repeated in triplicate with similar results.

Figure 3. Distribution of 5-HT6R in the mouse brain, and effect of SB271046 on serotonergic neurons in the AD mouse model

A. The DRN, Cx, and Hp (CA1, CA3, and DG) region of mice brain were analyzed using 5-HT₆R antibodies. The stained tissues were viewed with a microscope (×50 or 200). B. Serotonin level was measured by ELISA as described in Materials and Methods. Value is mean ± S.E.M of 8 mice. C. After tissue sections were permeabilized, TPH (green) was immunostained with rabbit anti-TPH antibody followed by Alexa-Fluor 488-conjugated secondary antibodies, and neuronal cells (red) were immunostained with mouse anti-NeuN, followed by Alexa-Fluor 568-conjugated secondary antibodies. And then sections were stained with DAPI (blue). The right panels show the merged images of the first, second, and third panels. D. GABA level was measured by ELISA as described in Materials and Methods. Value is mean ± S.E.M of 8 mice. ^*,# Significant difference (^*,# P < 0.05). The experiments shown in Figure 3 were repeated in triplicate with similar results.

Figure 4. Inhibitory effects of Fluoxetine on memory impairment in Aβ1-42-infused mice model

A.–C. Swimming time A. and swimming distance B. to arrive at platform were automatically recorded. 24 hr after training trials, a probe test was performed. The time spent in the target quadrant and target site crossing within 60 s was represented C.. Each value is presented as mean ± S.E.M from 10 mice. D. One day after the mice were given electric shock when entered into the dark compartment for training on learning day, the retention time in illuminated compartment was recorded. Each value is presented as mean ± S.E.M. from 10 mice. ^*,# Significant difference (^*,#P < 0.05 and **P < 0.01).

Figure 5. Total number of probe sets and significantly associated biological processes

A. Screen shot captures the number of probe sets by p-values from genome-wide correlation analysis. B. Pathways were significantly enriched in the probe sets which were correlated with the 5-HT levels (p < 0.05). The functional annotation was done using an interface in the SNCID that links the SNCID (http://sncid.stanleyresearch.org) and DAVID (http://david.abcc.ncifcrf.gov/).

Figure 6. The correlation between APBA1, APBA2 and 5-HT6R in depression and AD

A Correlation between expression levels of 5-HT₆R and two APBA genes, APBA1 and APBA2. Gene expression levels were measured in the hippocampus of individuals with depression and unaffected controls using RNA-Seq method. (B., C.) The mRNA level of 5-HT₁R, 5-HT₂R, 5-HT₃R, 5-HT₄R, 5-HT₅R, 5-HT₆R, and 5-HT₇R (B), and APBA1/2 (C) was determined by qRT-PCR in APP mutant mice of Alzheimer’s disease.