CB1 agonism prolongs therapeutic window for hormone replacement in ovariectomized mice

Abstract

Hormone therapy (HT) is reported to be deficient in improving learning and memory in older postmenopausal women according to recent clinical studies; however, the reason for failure is unknown. A "window of opportunity" for estrogen treatment is proposed to explain this deficiency. Here, we found that facilitation of memory extinction and long-term depression by 17β-estradiol (E2) was normal in mice 1 week after ovariectomy (OVXST), but it was impaired in mice 3 months after ovariectomy (OVXLT). High-throughput sequencing revealed a decrease of miR-221-5p, which promoted cannabinoid receptor 1 (CB1) ubiquitination by upregulation of Neurl1a/b in E2-treated OVXLT mice. Blood samples from postmenopausal women aged 56-65 indicated decreases of miR-221-5p and 2-arachidonoylglycerol compared with samples from perimenopausal women aged 46-55. Replenishing of miR-221-5p or treatment with a CB1 agonist rescued the impairment of fear extinction in E2-treated OVXLT mice. The present study demonstrates that an HT time window in mice can be prolonged by cotreatment with a CB1 agonist, implying a potential strategy for HT in long-term menopausal women.

Keywords: Endocrinology; Neuroendocrine regulation; Neurological disorders; Neuroscience; Pharmacology.

Figure 1. Impaired memory extinction in OVX_LT mice treated with E2.

(A) Schematic of inhibitory avoidance. Latency to dark room was recorded in sham, OVX_ST, and OVX_LT mice. (B) Schematic of trace fear memory. Freezing time was recorded in sham, OVX_ST, and OVX_LT mice. CS, conditioned stimuli (tone); US, unconditioned stimuli (electric shock). (C) Schematic of novel object recognition. Interaction time with novel and familiar objects was recorded in sham, OVX_ST, and OVX_LT mice. Data are represented as mean ± SEM, n = 8 mice per group. *P < 0.05; **P < 0.01 vs. sham by 2-way ANOVA followed by Bonferroni’s post hoc test. Experimenters were blinded to the treatment.

Figure 2. E2 failed to facilitate LTD and increase spine density in OVX_LT mice.

(A) Sample images showing the location of MED64 probe at CA1. (B and C) LTP and LTD recording in CA1 of hippocampus. Insets show accumulative field excitatory postsynaptic potential (fEPSP) slope at CA1 from different groups and averaged fEPSP slope during the last 30 minutes. Arrows indicate the time point of TBS or LFS application. n = 8 slices from 4 mice per group. **P < 0.01 between the marked groups by 2-way ANOVA followed by Bonferroni’s post hoc test. (D) Sample images showing the location of MED64 probe at mPFC. (E and F) LTP and LTD recording in mPFC. Insets show accumulative fEPSP slope at mPFC from different groups and averaged fEPSP slope during the last 30 minutes. Arrows indicate the time point of TBS or LFS application. n = 8 slices from 4 mice per group. **P < 0.01 between the marked groups by 2-way ANOVA followed by Bonferroni’s post hoc test. (G) Samples of Golgi-Cox staining of mPFC pyramidal neurons for spine counting. (H) Top: Representative images of basilar dendrites. Bottom left: Summary of total spine counts from basilar dendrites per 10 μm. Bottom right: Number of spines including mushroom, stubby, thin, and filopodia types per 10 μm. n = 60 neurons from 6 mice per group. **P < 0.01 between the marked groups by 2-way ANOVA followed by Bonferroni’s post hoc test. (I) Top: Western blot samples showing the level of proteins in mPFC. Bottom: Summary of levels of GluN2A, GluN2B, GluN1, GluA1, and CB1 in mPFC. n = 8 mice per group. **P < 0.01 between the marked groups by 2-way ANOVA followed by Bonferroni’s post hoc test. Experimenters were blinded to the treatment. Data are represented as mean ± SEM.

Figure 3. Decreased CB1 in mPFC and rescue of impairment of memory extinction by activation of CB1 in E2-treated OVX_LT mice.

(A) Levels of CB1 in mPFCs of mice after OVX. n = 6 mice per group; 1-way ANOVA followed by Bonferroni’s post hoc test; *P < 0.05 and **P < 0.01 vs. sham group, ^##P < 0.01 vs. mice 1 week after OVX. (B) Synaptic fEPSP response to ACEA (CB1 agonist, 1 μM) perfusion in mPFC slices from different mice. Inset shows the averaged fEPSP slope from 90 to 120 minutes. n = 8 slices from 4 mice per group. **P < 0.01 between the marked groups by 1-way ANOVA followed by Bonferroni’s post hoc test. (C) Effects of ACEA (0.5 mg/kg, s.c.) combined with E2 (0.1 mg/kg, s.c.) on fear memory formation and extinction. (D) Effects of JZL184 (2-AG hydrolysis enzyme inhibitor, 8 mg/kg, s.c.) or WIN55,212-2 (nonselective cannabinoid receptor agonist, 1 mg/kg, s.c.) combined with E2 on fear memory formation and extinction, and blockage of AM251 (CB1-specific antagonist, 3 mg/kg, s.c.). (C and D) n = 8 mice per group. **P < 0.01 vs. sham or OVX_LT mice by univariate ANOVA after lower-bound correction as repeated-measurement data. (E) Images showing AAV-CMV-mCherry-Cnr1 injection in mPFC of OVX_LT mice. (F) Inhibitory avoidance test was carried out 4 weeks after AAV injection. n = 8 mice per group. **P < 0.01 vs. mCherry by univariate ANOVA after lower-bound correction as repeated-measurement data. (G) Representative images of basilar dendrites from mPFC neurons. Number of total and different spine subtypes. n = 60 neurons from 6 mice per group. **P < 0.01 between the marked groups by 2-way ANOVA followed by Bonferroni’s post hoc test. Experimenters were blinded to the treatment. Data are represented as mean ± SEM.

Figure 4. Decreased miR-221-5p after long-term estrogen deprivation.

(A) mRNA levels of CB1 were similar among different mice. n = 8 mice in each group. (B) Volcano plots of miRNA sequencing from the mPFC between OVX_LT + E2 and OVX_ST + E2 mice, OVX_LT + E2 and sham mice, and OVX_LT and OVX_ST mice. n = 3 mice per group. (C) Venn diagram shows the potential miRNAs among treatments. miR-221-5p and miR-541-3p were recognized as targets responsible for impaired fear memory extinction in OVX_LT + E2 mice. (D) Levels of miR-221-5p and miR-541-3p in mPFC of different groups were confirmed by quantitative PCR. n = 6 mice per group. *P < 0.05, **P < 0.01 between the marked groups by 2-way ANOVA followed by Bonferroni’s post hoc test. (E) Levels of miR-221-5p and miR-541-3p in plasma from 25- to 35-year-old, 36- to 45-year-old, 46- to 55-year-old, and 56- to 65-year-old women. Right panel: Levels of miR-221-5p in plasma after years of menopause. (F) 2-AG (left), total E2 (middle), and free E2 (right) in plasma from different ages. (E and F) *P < 0.05, **P < 0.01 between the marked groups by 1-way ANOVA followed by Bonferroni’s post hoc test. Experimenters were not blinded to the treatment. Data are represented as mean ± SEM.

Figure 5. miR-221-5p responsible for impairment of fear extinction.

(A) Sample images showing miR-221-5p agomir–Cy3 (red) and miR-541-3p agomir–Cy3 (red) injection and nucleus (DAPI, blue) in prefrontal cortex. (B) miR-221-5p agomir but not miR-541-3p agomir rescued memory extinction in E2-treated OVX_LT mice. n = 8 mice per group. **P < 0.01 vs. scramble by univariate ANOVA after lower-bound correction as repeated-measurement data. (C) miR-221-5p agomir rescued impairment of LTD induced by E2 in OVX_LT mice. Arrow indicates the point of LFS application. n = 8 slices from 4 mice. **P < 0.01 by unpaired Student’s t test. (D) Increase of CB1 by miR-221-5p mPFC injection in E2-treated OVX_LT mice. n = 5 mice per group. **P < 0.01 between the marked groups by 2-way ANOVA followed by Bonferroni’s post hoc test. (E) Combining miR-221-5p agomir with E2 + ACEA did not further increase memory extinction as compared with miR-221-5p + ACEA in OVX_LT mice. n = 8 mice per group. **P < 0.01 between the marked groups by univariate ANOVA after lower-bound correction as repeated-measurement data. (F) mPFC injection of miR-221-5p antagomir impaired fear memory extinction in E2-treated OVX_ST mice, similarly to what was seen in E2-treated OVX_LT mice. n = 8 mice per group. **P < 0.01 vs. scramble by univariate ANOVA after lower-bound correction as repeated-measurement data. Experimenters were blinded to the treatment except in C. Data are represented as mean ± SEM.

Figure 6. miR-221-5p regulating CB1 ubiquitination through Neurl1a/b-dependent pathway.

(A) Alignment of binding sequences for miR-221-5p in the 3′-UTRs of Neurl1a and neurl1b mRNAs. (B) Interaction between miR-221-5p and 3′-UTRs of Neurl1a or neurl1b mRNAs detected by luciferase reporter gene. n = 4 assays per group. *P < 0.05, **P < 0.01 vs. negative control (NC) by 1-way ANOVA followed by Bonferroni’s post hoc test. Rluc, renilla luciferase; Fluc, firefly luciferase. (C) mPFC injection of miR-221-5p agomir or antagomir regulated Neurl1a and Neurl1b expression in OVX_ST and OVX_LT mice. n = 6 mice per group. **P < 0.01 between the marked groups by 1-way ANOVA followed by Bonferroni’s post hoc test. (D) MG-132 (a 26S proteasomal inhibitor, 5 mg/kg, 1 week, once daily, i.p.) induced a larger increase of CB1 level in OVX_LT mice than that in OVX_ST mice. n = 6 mice per group. **P < 0.01 by unpaired Student’s t test. (E) E2-induced decrease of CB1 was blocked by MG-132 in OVX_LT mice. E2 increased Neurl1a and Neurl1b levels in OVX_LT mice, and higher Neurl1a and Neurl1 were found in OVX_LT mice compared with OVX_ST mice. n = 6 mice per group. **P < 0.01 between the marked groups by 2-way ANOVA followed by Bonferroni’s post hoc test. Experimenters were not blinded to the treatment. Data are represented as mean ± SEM.

Figure 7. Increase of CB1 ubiquitination through Neurl1a/b-dependent pathway in OVX_LT mice.

(A) Coimmunoprecipitation showing the interaction between CB1 and ubiquitin, Neurl1a, or Neurl1b in mPFC and the effects of ACEA (0.5 mg/kg, s.c.), E2 (0.1 mg/kg, s.c.), or miR-221-5p agomir treatment on it. Ubiquitinated CB1 was increased after E2 treatment in OVX_LT mice, which could be blocked by miR-221-5p agomir treatment. Phosphorylated GluA1 at Ser845 and Ser831 site was increased by E2 in OVX_ST mice but not in OVX_LT mice. n = 6 mice per group. **P < 0.01 between the marked groups by 2-way ANOVA followed by Bonferroni’s post hoc test. (B) Downregulation of Neurl1a and Neurl1b by mPFC injection of AAV-CMV-Neurl1a-shRNA-GFP and AAV-CMV-Neurl1b-shRNA-GFP in OVX_LT mice. n = 6 mice per group. (C) Levels of CB1 in mPFC of OVX_LT mice after injection of Neurl1a shRNA, Neurl1b shRNA, miR-221-5p agomir, and/or antagomir. n = 6 mice per group. **P < 0.01 between the marked groups by 1-way ANOVA followed by Bonferroni’s post hoc test. (D) Summary of latency in inhibitory avoidance test and freezing time in trace fear memory after extinction training in OVX_LT mice injected with Neurl1a/b shRNA (mixture of Neurl1a shRNA and Neurl1b shRNA) or ACEA. n = 8 mice per group. **P < 0.01 between the marked groups by univariate ANOVA after lower-bound correction as repeated-measurement data. Experimenters were not blinded to the treatment except in D. Data are represented as mean ± SEM.

Figure 8. Schematic of E2/CB1–mediated signaling in long-term estrogen deprivation.

Left: In long-term estrogen-deprived mice, E2 treatment activates downstream effector molecules. It leads to reduction of miR-221-5p, which targets the E3 ubiquitin protein ligases Neurl1a and Neurl1b. Higher E3 ubiquitin protein ligase activity, then, leads to excessive CB1 ubiquitination. Right: CB1 is a retrograde receptor and critical for induction of LTD. Reduction of CB1 leads to abnormal LTD induction and fear extinction in long-term estrogen deprivation. RISC, RNA-induced silencing complex.