| IntroductionEpidemiological evidence suggests that the incidence rate of Alzheimer's disease(AD) in post-menopausal women is twice of men, and the prevalence rate of depression is doubled compared to young women, indicating that the declined levels of sex hormones, especially estradiol(E2), caused by ovarian function failure are involved in the pathogenesis of AD and depression. Clinical data shows that women are more susceptible to depression when the level of E2 sharply declines. In recent years, although the eatrogen replacement therapy(ERT) has been applied in clinic as an adjunctive treatment of AD and depression, its effects are not clear. For example, women aged 50-63 years old received E2 treatment for more than six months had lower incidence of AD in the elderly(>78 years) compared to women of the same age without that treatment. However, for women aged 65 years old or older, E2 treatment had no effect on cognition. So far the mechanisms involved in the effects of E2 in the pathogenesis of AD and depression are still unclear. In addition, the mechanisms, targets and effective time window of E2 in the intervention treatment of AD also have no clear reports.The subventricular zone(SVZ) in lateral ventricles and the subgranular zone(SGZ) in hippocampal dentate gyrus(DG) in the adult mammalian retain the ability to produce and incorporate new neurons, which is refered to as adult neurogenesis. The newborn neurons in hippocampal DG, similar to the established ones, have morphological and functional characteristics, and can connect to hippocampal CA3 field and entorhinal cortex to integrate into the neural network. In this manner the newborn neurons can instead of degenerated neurons. The neuropathological hallmarks of AD include senile plaques, neuronal degeneration and neurofibrillary tangles. The main component of senile plaques is ?-amyloid peptide(A?). Our previous studies have reported that A? can stimulate the proliferation of hippocampal neural stem cells, but impair the survival of newborn neurons. The ability of hippocampal neurogenesis is thought to decrease in AD, indicating that the decline of neurogenesis is associated with impaired cognitive function. Little studies have reported the relevance of neurogenesis decline and cognition impairment. Especially to date there is no clear report about the involvement of E2 in the regulation of hippocampal neurogenesis in AD. The imaging data and autopsy report showed that the hippocampal volume in patients with depression is smaller than that in normal subjects. The antidepressant therapy can promote hippocampal neurogenesis and increase the hippocampal volume, and improve the symptoms of depression. Sigma-1 receptor(?1R) is a new antidepressant target, and can promote hippocampal neurogenesis. Chevallier et al. reported that the ?1R gene knockout(?1R-/-) induces sex-related depressive-like behaviors, that is male ?1R-/- mice appear depressive-like behaviors, but female ?1R-/- mice not.The present study employed APP/PS1 transgenic mice(AD mice) and ?1R-/-mice(mouse model of depression) as tools and focused on exploring the effects of E2 on cognitive-affective disorders and associated mechanisms of hippocampal neurogenesis, in order to provide new theoretical basis of the E2 treatment for postmenopausal women with AD and depression.Objective 1. Effects and effective time window of E2 on the A?-impaired cognition post-OVX and the role of hippocampal neurogenesis mechanisms.2. Effects of E2 on the depressive-like behaviors induced by ?1R knockout and the role of hippocampal neurogenesis mechanisms.The First Part Effects of E2 on the cognition-hippocampal neurogenesis impairment in OVX-AD mice and its molecular mechanismsMaterials and Methods 1. Animal models:(1) AD mice model: 5-11-month-old APPswe/PS1 d E9 transgenic(APP/PS1) mice, hereinafter referred to as the “AD mice”.(2) Menopausal mice model: mice were bilateral ovariectomy(OVX). 2. E2 treatment group:(1) Early stage post-OVX: 5-month-old mice were OVX?E2 treated for two consecutive months;(2) Middle stage post-OVX: 5-month-old mice were OVX?with an interval of two months?E2 treatment for two consecutive months;(3) Late stage post-OVX: 5-month-old mice were OVX?with an interval of four months?E2 treatment for two consecutive months. 3. Y maze and Morris water were used to test the hippocampus-dependent cognitive function in 11-month-old mice. 4. Hippocampal newborn neurons labelling: 5-bromodeoxyuridine(Brd U) was intraperitoneally injected three times within 24 h for mitotic labeling. Brd U immunostaining was performed at 24 h(1-d-old) after the last Brd U injection. 5. Ki67 immunostaining was used to evaluate the proliferation of hippocampal neural stem cells.6. Enzyme linked immunosorbent assay(ELISA) was used to quantify the levels of soluble and insoluble A?1-40 and A?1-42(A?1-40/42) in the hippocampus of AD mice. 7. A? immunostaining was used to observe the deposition of amyloid plaques. 8. Western Blot was used to analysis the level of p75 neurotrophin receptor(p75NTR) protein in hippocampal DG. 9. Real-time quantitative reverse transcription PCR(Q-RT-PCR) was used to check the levels of p75 NTR, Cyclin D1, Cylin E and Cylin A1 m RNA in hippocampal DG.Results and Summary 1. In comparison with 11-month-old WT mice, AD mice showed spatial cognitive deficits, e.g. a significant reduced alternation in Y maze and increased in escape-latency and decreased in spent time in platform quadrant in Morris water maze. OVX-WT mice did not show the changes in cognitive behavior compared to WT mice. However, OVX-AD mice showed worse cognitive function than that in AD mice—E2 deficiency post-OVX can aggravate the cognition impairment in AD mice. 2. In comparison with vehicle-treated 11-month-old OVX-AD mice, E2 treatment at early stage post-OVX could significantly improve the cognition function in AD mice; E2 treatment at middle stage post-OVX could partly improve the damaged cognitive function in AD mice; E2 treatment at late stage post-OVX had no effect on the cognition impairment in AD mice—The early stage is the effective time window of E2 to improve late AD cognitive impairment. 3. In comparison with OVX-AD mice, no significant changes were observed in the levels of soluble and insoluble A?1-42 or A?1-40 in the hippocampus of the three periods E2 treatment OVX-AD mice; furthermore, the shape and load of senile plaques in hippocampus also had no difference among these groups.4. In comparison with WT mice, the numbers of 1-d-old Brd U+ cells and Ki67+ cells were significantly increased in 7-month-old AD mice, but slightly increased in 9-month-old AD mice—The hippocampal neural stem cells show excessive proliferation in early AD; the numbers of 1-d-old Brd U+ cells and Ki67+ cells were reduced by approximately 50% compared to WT mice—The proliferation capacity of hippocampal neural stem cells decreases in late AD. 5. In comparison with WT mice, the proliferation of hippocampal stem cells had no changes in OVX-WT mice. However, compared with the same age of AD mice, the proliferation of hippocampal stem cells was significantly increased in 7-month-old OVX-AD mice, and was only slightly increased in 9-month-old OVX-AD mice, furthermore, the proliferation of hippocampal stem cells showed more decreased in 11-month-old OVX-AD mice—E2 deficiency can aggravate the excessive proliferation of in early AD, and eventually lead to proliferation exhaustion of neural stem cells in late AD. 6. In comparison with the same age OVX-AD mice, E2 treatment at early stage post-OVX could prevent the excessive proliferation of hippocampal stem cells in 7-month-old AD mice; E2 treatment at middle stage post-OVX could partly prevent the excessive proliferation of neural stem cells in 9-month-old AD mice; E2 treatment at late stage post-OVX had no effect on the proliferation of neural stem cells in 11-month-old AD mice—E2 treatment at the early stage post-OVX can prevent the excessive proliferation of hippocampal stem cells in AD mice. 7. In comparison with 11-month-old OVX-AD mice, E2 treatment at early stage post-OVX could protect the proliferation of hippocampal stem cells in OVX-AD mice; E2 treatment at middle stage post-OVX could partly protect the proliferation of neural stem cells in OVX-AD mice; E2 treatment at late stage post-OVX had no effect on the proliferation of neural stem cells in AD mice—E2treatment at the early stage post-OVX can alleviate the proliferative exhaustion of hippocampal stem cells in late AD. 8. In comparison with 7-month-old WT mice, the level of p75 NTR, but not the levels of Cyclin D1, Cyclin E or Cyclin A1, was increased in hippocampus of OVX-WT mice, the levels of Cyclin D1, Cyclin E and Cyclin A1, but not the level of p75 NTR were increased in hippocampus of AD mice; compared with AD mice, the level of p75 NTR was increased and the levels of Cyclin D1, Cyclin E, Cyclin A1 had a corresponding increase in OVX-AD mice. E2 treatment could inhibit the increased expression of p75 NTR in OVX-WT mice, and could inhibit the over-expression of p75 NTR and cell cycle factor in OVX-AD mice—A? stimulates the activation of p75NTR; E2 deficiency leads to the increased expression of p75 NTR. 9. Treatment with p75 NTR inhibitor TAPI-2 could decrease the over-expression of cell cycle factor, and significantly inhibit the excessive proliferation of hippocampal stem cells in OVX-AD mice—E2 through inhibiting the expression of p75 NTR prevents A?-stimulating hippocampal stem cells proliferation.Conclusion E2 treatment at early stage post-OVX through inhibiting the expression of p75 NTR can prevent over-activation of p75 NTR by A? stimulating, thus inhibits the excessive proliferation of neural stem cells in AD hippocampus, by which way E2 can prevent the proliferation exhaustion of hippocampal stem cells, and improve the cognition impairment in late AD.The Second Part Effects of E2 on the depressive-like behaviors in ?1R knockout mice and the role of hippocampal neurogenesis mechanismsMaterials and Methods 1. Animal models: 2-month-old Sigma-1 receptor(?1R) gene knockout mice(?1R-/-mice). 2. Drug and surgical treatment: female mice were bilaterally ovariectomized(OVX), male mice received subcutaneous E2 treatment for 14 consecutive days. Src inhibitor PP2 or Dasatinib, NMDA receptor NR2 B antagonist Ro25-6981 or NMDA receptor agonist NMDA was intraperitoneally injected for 14 consecutive days. 3. Emotional behaviors detection:(1) Tail suspension test(TST) was used to evaluate the depressive-like behaviors by the immobility time;(2) Forced swim test(FST) was used to evaluate the depressive-like behaviors by the immobility time. 4. Determination of serum hormone levels:(1) Enzyme linked immunosorbent assay(ELISA) was used to analysis the serum corticosterone levels;(2) Radioimmunoassay(RIA) was used to analysis the serum E2 levels in female mice and testosterone levels in male mice. 5. Detection of hippocampal neurogenesis:(1) Brd U immunostaining was performed on day 7, day 14, day 21 or day 28 after the last Brd U-injection to evaluate the survival of newborn neurons.(2) Brd U and Neu N(neuron specific nuclear protein) or GFAP(glial fibrillary acidicprotein) double labled immune-histofluorescence staining was used to evaluate the number of Brd U+/Neu N+ or Brd U+/GFAP+ cells. 6. Whole cell patch clamp recording was used to detecte the membrane properties of the granular cells and the density of NMDA receptor activated current(INMDA). 7. Western Blot was used to analysis the levels of NMDA receptor subunit NR2 B and NR2 A and Src phosphorylation in hippocampal DG.Results and Summary 1. In comparison with WT mice, male ?1R-/- mice appeared the depressive-like behaviors(the immobility times for TST and FST were significantly prolonged), but female ?1R-/- mice did not—?1R deficiency induced sex-related depressive-like behaviors. Compared with WT mice, the levels of serum corticosterone, serum E2 at proestrus or diestrus in female ?1R-/- mice or serum testosterone in male ?1R-/- mice did not show any changes. Female OVX-?1R-/-mice appeared the depressive-like behaviors, and E2 treatment could correct the depressive-like behaviors in male ?1R-/- mice—E2 can alleviate the depressive-like phenotype induced by ?1R deficiency. 2. In comparison with WT mice, male ?1R-/- mice showed a significant reduction in the numbers of 21-day-old and 28-day-old Brd U+ cells, and the number of Brd U+/Neu N+ cells reduced by approximately 30%, Brd U+/GFAP+ cells had no changes. However, female ?1R-/- mice did not show any changes of newborn cells—?1R deficiency induced Sex-related neurogenesis decrease. The number of 28-day-old Brd U+ cells reduced in female OVX-?1R-/- mice. E2 treatment could rescue the loss of 28-day-old Brd U+ cells in male ?1R-/- mice—E2 can reduce the loss of newborn neurons induced by ?1R deficiency.3. In comparison with WT mice, male ?1R-/- mice appeared the decreases in INMDA and phosphor-NR2 B, while female WT mice did not show the changes in INMDA and phosphor-NR2—?1R deficiency induced sex-related NMDA receptor dysfunction. However, female OVX-?1R-/- mice appeared the decreases in INMDA and phosphor-NR2 B, E2 treatment could correct the reduced INMDA and phosphor-NR2 B in male ?1R-/- mice. NMDA receptor agonist could improve the hippocampal neurogenesis and depressive-like behaviors in male ?1R-/- mice—E2 can correct the NMDA receptor dysfunction induced by ?1R deficiency, and protect the hippocampal neurogenesis and depressive-like behaviors. 4. In comparison with WT mice, the phosphor-Src was not altered in ?1R-/- mice. E2 treatment could elevate the level of phosphor-Src in male ?1R-/- mice. In addition, the Src inhibitor PP2 or dasatinib could abolish the E2-enhanced NMDA receptor function, and the E2-reduced loss of hippocampal newborn neurons and the E2-corrected depressive-like behaviors in male ?1R-/- mice—E2 through activating of Src can correct the NMDA receptor dysfunction induced by ?1R deficiency, thus alleviate depressive-like behaviors and the loss of newborn neurons.Conclusion E2 through enhancing phosphor-Src can improve the phosphor-NR2 B decline induced by ?1R deficiency, thus correct the downregulation of NMDA receptor, by which way E2 can alleviate the loss of newborn neurons and depressive-like behaviors in ?1R deficiency mice. |
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