A tricyclic antidepressant, amoxapine, reduces amyloid-β generation through multiple serotonin receptor 6-mediated targets

Abstract

Alzheimer's disease (AD) is a major and devastating neurodegenerative disease, and the amyloid-β (Aβ) hypothesis is still the central theory for AD pathogenesis. Meanwhile, another major mental illness, depression, is one of the risk factors for AD. From a high-throughput screening (HTS), amoxapine, a typical secondary amine tricyclic antidepressant (TCA), was identified to reduce Aβ production. A follow-up investigation on antidepressants showed that most of the TCAs harbour similar activity. Previous studies have indicated that TCAs improve cognitive function in AD mouse models as well as in preliminary clinical data; however, the underlying mechanism is controversial, and the effect on Aβ is elusive. Thus, we developed a secondary screening to determine the molecular target of amoxapine, and serotonin receptor 6 (HTR6) was identified. Knockdown of HTR6 reduced the amoxapine's effect, while the HTR6 antagonist SB258585 mimicked the activity of amoxapine. Further mechanistic study showed that amoxapine and SB258585 reduced Aβ generation through multiple HTR6-mediated targets, including β-arrestin2 and CDK5. Taken together, our study suggests that amoxapine, though no longer a first-line drug for the treatment of depression, may be beneficial for AD and further structural modification of TCAs may lead to desirable therapeutic agents to treat both AD and depression.

Figure 1

Amoxapine, a typical secondary amine TCA, reduces Aβ generation in a dose-dependent manner. (A) Representative results of the screening for chemicals that reduce Aβ generation. Two hours after cell seeding, HEK293-APPsw cells were treated with 10 μM of chemicals for 24 hours, and the Aβ concentration in the supernatant was measured by ELISA. Amoxapine is highlighted in blue. (B) The levels of Aβ produced by SK-N-SH cells in response to vehicle (0.1% DMSO), 10 μM BACE inhibitor IV (BSI IV), 10 μM L685,458, or the indicated compounds at 1 μM, 3 μM or 10 μM for 24 hours. (C,D) The levels of sAPPα (C) and sAPPβ (D) produced by SK-N-SH cells in response to vehicle (0.1% DMSO), 100 μM TAPI-1, 10 μM BSI IV, or the indicated compounds at 10 μM for 24 hours. (E,F) The measurements of α-secretase (E) and BACE1 (F) activity by fluorogenic substrate assay after treatment with vehicle (0.1% DMSO), 100 μM TAPI-1, 10 μM BSI IV, or 10 μM of the indicated chemicals. For the cellular treatment, SK-N-SH cells were treated with the indicated compounds for 24 hours before designated to the fluorogenic substrate assay. For the membrane treatment, the membrane fraction of SK-N-SH cells was prepared before compound treatment. (G) Representative image of a western blot showing the expression of α-secretase (ADAM10) and BACE1 after treatment with vehicle (0.1% DMSO), 100 μM TAPI-1, 10 μM BSI IV, 10 μM L685,458, or 10 μM amoxapine for 24 hours. Actin was used as loading control. (H) Representative image of a western blot showing the expression of APP after treatment with vehicle (0.1% DMSO), 100 μM TAPI-1, 10 μM BSI IV, 10 μM L685,458, or 10 μM amoxapine for 24 hours. Actin was used as a loading control. (I,K) The statistical analysis of G and H using ImageJ. Data are presented as the mean ± s.e.m. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the control of each group. One-way ANOVA with post hoc comparison test (B–F and I,K).

Figure 2

Serotonin receptor 6 mediates the effect of amoxapine. (A) The mRNA level of HTR6 and HTR2B in SK-N-SH cells with the infection of scrambled, HTR6 or HTR2B gene-specific shRNA. (B,C) The levels of Aβ produced by SK-N-SH cells after treatment with vehicle (0.1% DMSO) or amoxapine at 10 μM for 24 hours in the cells infected as described in (A). (D) The levels of Aβ produced by SK-N-SH cells in response to the indicated HTR6 antagonists at 1 μM, 3 μM or 10 μM for 24 hours. (E) The levels of Aβ produced by SK-N-SH cells in response to the indicated HTR2B antagonists at 1 μM, 3 μM or 10 μM for 24 hours. Data are presented as the mean ± s.e.m. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the control of each group or the control of the shNC group. ^$p < 0.05, ^$$p < 0.01 and ^$$$p < 0.001 compared to amoxapine of the shNC group. One-way ANOVA with post hoc comparison test (A,D and E) and two-way ANOVA with post hoc comparison test (B,C).

Figure 3

Amoxapine modulates Aβ generation in human neuronal cells differentiated from NSCs. (A) Representative image of neuronal differentiated human neural stem cells (NSCs) stained with Tuj1, Sox2, MAP2, GFAP or DAPI. Scale bar, 50 μm. (B) The mRNA level of HTR6 in human NSCs and NSC differentiated neuronal cells. (C) The levels of Aβ produced by neuronal differentiated NSCs in response to vehicle (0.1% DMSO), 10 μM BSI IV, 10 μM L685,458, or the indicated compounds at 3 μM or 10 μM for 24 hours. (D) The levels of Aβ produced by neuronal differentiated NSCs after treatment with vehicle (0.1% DMSO), amoxapine or SB258585 at 10 μM in the cells with the infection of scrambled or HTR6 gene-specific shRNA. Data are presented as the mean ± s.e.m. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the control of each group or the control of shNC group. ^$p < 0.05, ^$$p < 0.01 and ^$$$p < 0.001 compared to amoxapine of the shNC group. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 compared to SB258585 of the shNC group. Two-tailed t-test (B), one-way ANOVA with post hoc comparison test (C) and two-way ANOVA with post hoc comparison test (D).

Figure 4

Amoxapine reduces Aβ generation through multiple HTR6-mediated targets. (A) The cAMP responses after stimulation with the indicated compounds at the indicated concentrations in SK-N-SH cells infected with HTR6 lentivirus. (B) The mRNA level of Gα_s in SK-N-SH cells with the infection of scrambled or Gα_s gene-specific shRNA. (C) The levels of Aβ produced by SK-N-SH cells after treatment with vehicle (0.1% DMSO), amoxapine or SB258585 at 10 μM for 24 hours in the cells infected as described in (B). (D) Representative image of a western blot showing the expression of β-arrestin2 in SK-N-SH cells with the infection of scrambled or β-arrestin2 gene-specific shRNA. Actin was used as loading control. (E) The statistical analysis of D using ImageJ. (F) The levels of Aβ produced by SK-N-SH cells after treatment with vehicle (0.1% DMSO), amoxapine or SB258585 at 10 μM for 24 hours in the cells infected as described in (D). (G) Representative image of a western blot showing the expression of CDK5 in SK-N-SH cells with the infection of scrambled or CDK5 gene-specific shRNA. Actin was used as loading control. (H) The statistical analysis of (G) using ImageJ. (I) The levels of Aβ produced by SK-N-SH cells after treatment with vehicle (0.1% DMSO), amoxapine or SB258585 at 10 μM for 24 hours in the cells infected as described in (G). (J) The levels of Aβ produced by SK-N-SH cells after treatment with vehicle (0.1% DMSO), amoxapine or SB258585 at 10 μM for 24 hours in SK-N-SH cells with 45 min pretreatment with 50 μM of the CDK5 inhibitor roscovitine. Data are presented as the mean ± s.e.m. *p < 0.05, **p < 0.01 and ***p < 0.001 compared to the control of each group or the control of shNC group. ^$p < 0.05, ^$$p < 0.01 and ^$$$p < 0.001 compared to amoxapine of the shNC group. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 compared to SB258585 of the shNC group. Two-tailed t-test (B), one-way ANOVA with post hoc comparison test (E and H), and two-way ANOVA with post hoc comparison test (C,F,I and J).