Estrogen receptor α promotes Cav1.2 ubiquitination and degradation in neuronal cells and in APP/PS1 mice

Abstract

Cav1.2 is the pore-forming subunit of L-type voltage-gated calcium channel (LTCC) that plays an important role in calcium overload and cell death in Alzheimer's disease. LTCC activity can be regulated by estrogen, a sex steroid hormone that is neuroprotective. Here, we investigated the potential mechanisms in estrogen-mediated regulation of Cav1.2 protein. We found that in cultured primary neurons, 17β-estradiol (E2) reduced Cav1.2 protein through estrogen receptor α (ERα). This effect was offset by a proteasomal inhibitor MG132, indicating that ubiquitin-proteasome system was involved. Consistently, the ubiquitin (UB) mutant at lysine 29 (K29R) or the K29-deubiquitinating enzyme TRAF-binding protein domain (TRABID) attenuated the effect of ERα on Cav1.2. We further identified that the E3 ligase Mdm2 (double minute 2 protein) and the PEST sequence in Cav1.2 protein played a role, as Mdm2 overexpression and the membrane-permeable PEST peptides prevented ERα-mediated Cav1.2 reduction, and Mdm2 overexpression led to the reduced Cav1.2 protein and the increased colocalization of Cav1.2 with ubiquitin in cortical neurons in vivo. In ovariectomized (OVX) APP/PS1 mice, administration of ERα agonist PPT reduced cerebral Cav1.2 protein, increased Cav1.2 ubiquitination, and improved cognitive performances. Taken together, ERα-induced Cav1.2 degradation involved K29-linked UB chains and the E3 ligase Mdm2, which might play a role in cognitive improvement in OVX APP/PS1 mice.

Keywords: Alzheimer’s disease; Cav1.2; Estrogen receptor α; K29; Mdm2; ubiquitination.

Figure 1

Estrogen decreases Cav1.2 protein through ERα in primary cortical neurons. (a) Representative Western blots (top) and quantification (bottom) of Cav1.2 in primary cortical neurons in the absence (Ctl) and presence of estrogen at 10, 100 nM, and 1 μM, respectively; n = 4. (b) Representative Western blots (top) and quantification (bottom) of Cav1.2 in cortical neurons incubated with estrogen (100 nM) at different times; n = 3. (c) Representative Western blots (top) and quantification (bottom) of Cav1.2 in cortical neurons treated with DMSO (Ctl), the ER antagonist ICI182780 (1 μM, ICI), estrogen (100 nM, E2), and E2 with ICI, respectively n = 4. (d) Representative immunofluorescent images of Cav1.2 (green) relative to neuronal marker MAP2 (red), in cortical neurons treated with DMSO (Ctl), ICI, E2, and E2 with ICI for 24 hr (n = 3). Scale bar, 10 μm. (e) I‐V plots (top) and quantifications (bottom) of calcium mediated current (ICa²⁺) density (pA/pF) in primary cortical neurons treated with DMSO (Ctl, n = 15), ICI (n = 9), E2 (n = 9), and E2 with ICI (n = 11) for 24 hr. (f) Representative Western blots (top) and quantification (bottom) of Cav1.2 in primary cortical neurons treated with ERα agonist PPT (10 nM), ERβ agonist DPN (10 nM), and PPT with DPN for 24 hr; n = 6. (g) I‐V plots (top) and quantifications (bottom) of ICa²⁺ density (pA/pF) in cortical neurons treated with DMSO (Ctl, n = 15), PPT (n = 9), DPN (n = 9), and PPT with DPN (n = 11). (h) Representative Western blots (top) and quantification (bottom) of Cav1.2 in cortical neurons in the absence (Ctl) and presence of mock shRNA, mock shRNA with estrogen (E2, 100 nM), ERα shRNA2, and ERα shRNA2 with E2, respectively (n = 4). (i) Representative Western blots (top) and quantification (bottom) of Cav1.2 in cortical neurons in the absence (Ctl) and presence of mock shRNA, mock shRNA with estrogen (E2, 100 nM), ERβ shRNA3, and ERβ shRNA3 with E2, respectively (n = 3). *p＜0.05,**p＜0.01,***p＜0.001 (ANOVA)

Figure 2

K29‐linked UB chains are involved in ERα‐induced Cav1.2 ubiquitination and degradation. (a) Representative Western blots (top) and quantification (bottom) of Cav1.2 in primary neurons treated with PPT in the absence or presence of proteasomal inhibitor Mg132 (10 μM for 24 hr, n = 7). (b) Representative Western blots (top) and quantification (bottom) of Cav1.2 in cortical neurons treated with PPT in the absence or presence of lysosomal inhibitor chloroquine (CQ, 50 μM for 24 hr, n = 10). (c) Representative Western blots of the predicted molecular masses of full‐length and three fragments of Cav1.2 protein naturally occurred in primary cortical neurons, which shows that PPT decreases all fragments of Cav1.2 (n = 4). (d) Western blots of ubiquitin (UB, top) and Cav1.2 (bottom) in primary cortical neurons immunoprecipitated by Cav1.2 antibody under control conditions or after PPT treatment. (e) Representative Western blots (top) and quantification (bottom) of Cav1.2 in HT22 cells transiently expressing vector, or ubiquitin constructs WT, K0, K6, K11, K29, K33, K48, and K63, respectively (n = 4). In WT and K0, all 7 lysine (K) residues were included or mutated to arginine (R), respectively. In K6‐K63, only the numbered K residue was present, while the rest 6Ks were mutated to Rs. Overexpression of the ubiquitin mutant K6 or K29 significantly reduces Cav1.2 expression compared with control. (f) Top: Western blots of ubiquitin in HT22 cells without treatment (Ctl), or transiently expressing vector, K29 or K0 constructs, after cell extracts were immunoprecipitated by Cav1.2 antibody. Bottom: corresponding Western blots of Cav1.2 and HA tag under these conditions. (g) Representative Western blots (top) and quantification (bottom) of Cav1.2 in HT22 cells transiently expressing vector, mutant ubiquitin construct K6R and K29R alone or in combination, in the absence and presence of PPT, respectively (n = 3). (h) Representative Western blots (top) and quantification (bottom) of Cav1.2 in HT22 cells transiently expressing vector, mutant ubiquitin construct K6R or K29R, in the absence and presence of PPT, respectively (n = 5). (i) Representative Western blots (top) and quantification (bottom) of Cav1.2 in HT22 cells transiently expressing vector, wild‐type TRABID or mutated TRABID (C442A) construct, in the absence and presence of PPT, respectively (n = 4).*p＜0.05,**p＜0.01,***p＜0.001, ANOVA. TRABID: TRAF‐binding protein domain

Figure 3

E3 ligase Mdm2 associated with PEST sequence of Cav1.2 is involved in ERα‐induced Cav1.2 degradation. (a) Representative Western blots of Cav1.2 (top) and Mdm2 (bottom) in HT22 cell extracts immunoprecipitated by control antibody IgG or antibodies against Mdm2, Derlin‐1, or CHIP (top), or by Cav1.2 antibody (bottom), respectively. Extract (lane 1) represents 10% of total protein used for immunoprecipitation. Mdm2 shows a strong association with Cav1.2. (b) Representative Western blots of ERα (top) and Mdm2 (middle) in HT22 cell extracts immunoprecipitated by control antibody IgG or antibodies against Mdm2 or ERα, respectively. Proteins in input are shown on the bottom. PPT treatment increases ERα association with Mdm2. (c) Representative Western blots (top) and quantification (bottom) of Cav1.2 in HT22 cells transiently expressing vector, Mdm2 or Mdm2 inhibitor p14ARF, in the absence and presence of PPT, respectively (n = 3). (d) Top: Western blots of ubiquitin in SH‐SY5Y cells without treatment (Ctl), or transiently transfected with mock siRNA (Mock) or Mdm2 siRNA, after cell extracts were immunoprecipitated by Cav1.2 antibody. Bottom: corresponding Western blots of Cav1.2 under these conditions. (e) Schematic diagram depicting PEST1 and PEST3 sequences located in Cav1.2 protein. (f) Representative Western blots (top) and quantification (bottom) of Cav1.2 in primary neurons treated with 150 μM synthesized PEST peptides (PEST1 and PEST3) or synthesized scrambled peptides (ΔPEST1 and ΔPEST3), in the absence or presence of PPT (n = 4).*p＜0.05,**p＜0.01, ANOVA. Mdm2: double minute 2 protein; p14ARF: p14 alternate open reading frame

Figure 4

Mdm2 reduces Cav1.2 protein and enhances Cav1.2 ubiquitination in vivo. (a) Immunofluorescent images of cortical slices. Cells are successfully transfected with vector and Mdm2 as shown by green fluorescence (GFP). Cav1.2 and ubiquitin are shown as blue and red, respectively, whereas the merged images showing the colocalization of Cav1.2 and ubiquitin are shown as purple (Merge). The two columns under Vector or Mdm2 show general arrangement of cells (left) and the amplified view of immunofluorescence (right) from the rectangles marked in the left sides, respectively. The white arrows denote the purple dots indicating the colocalization of Cav1.2 and ubiquitin, whereas asterisk denotes the central area of cell body. (b) Quantitative analysis of Cav1.2 intensity between vector and Mdm2 (n = 36 cells/ 6 slices in each). (c) Quantitative analysis of ubiquitin intensity per Cav1.2 unit in cells transiently expressing vector or Mdm2 (n = 36 cells/ 6 slices in each). ***p＜0.001, unpaired t test

Figure 5

PPT treatment reduces Cav1.2 protein in ovariectomized APP/PS1 mice. (a and b) Representative Western blots (top) and quantification (bottom) of Cav1.2 in the hippocampus (a) and the cortex (b) of ovariectomized wild‐type mice without any treatment (WT, n = 8), or ovariectomized APP/PS mice treated with vehicle (AD, n = 7), 17β‐estradiol (E2, 30 μg/kg, n = 8), PPT (1 mg/kg, n = 8), and DPN (1 mg/kg, n = 7) for two weeks. E2 or PPT replacement attenuates Cav1.2 elevation in OVX APP/PS1 mice. (c) Representative immunofluorescent images from cortical slices probed with anti‐Cav1.2 (red) and anti‐ubiquitin (green) antibodies, in WT or OVX APP/PS1 mice. (d) Quantification of Cav1.2 immunofluorescent density using data related to C. (e) Western blots of ubiquitin (UB) in cortical extracts immunoprecipitated by Cav1.2 antibody, showing that Cav1.2 ubiquitination is significantly increased in APP/PS1 mice treated with E2 or PPT, but not with DPN. (f) Western blots of ERα (top) and Mdm2 (middle) in the hippocampal extracts of OVX APP/PS1 mice immunoprecipitated by Mdm2 antibody. Proteins in input are shown on the bottom. PPT treatment increases Mdm2 association with ERα and Cav1.2. *p＜0.05, **p＜0.01, ***p＜0.001, ANOVA

Figure 6

PPT treatment improves spatial and associative learning and memory performance in OVX APP/PS1 mice. (a) Compared with vehicle‐treated APP/PS1 mice (AD), E2 (AD + E2)‐ or PPT (AD + PPT)‐treated APP/PS1 mice show a significantly shorter latency on the third, fourth, and fifth day, respectively. No significant difference is found between DPN‐treated mice and AD mice. (b) In the hidden platform tests on the fifth day, the total distance spent on reaching the platform was recorded. Compared with vehicle‐treated APP/PS1 mice, PPT‐ or E2‐treated APP/PS1 mice show a significantly shorter distance. No significant difference is found between DPN‐treated mice and AD mice. (c) Representative road maps showing the movement trajectory of mice in hidden platform experiment on the fifth day. (d and e) In the probe trial on the sixth day, PPT‐ and E2‐treated mice show significantly more times traveling (annulus crossing, d) and significantly longer time staying (e) in the place where the hidden platform was previously placed, compared with AD. (f) Representative road maps showing the movement trajectory of mice in the probe trial on the sixth day. (g and h) Percentage freezing time as a function of training periods (g) and freezing times (H) are shown in wild‐type mice (WT), APP/PS1 mice treated with vehicle (AD), E2 (AD + E2), PPT (AD + PPT), and DPN (AD + DPN), respectively, on the first day. (i–l) On the second day (I and J) and third day (K AND L), percentage freezing time (i and k) and freezing times (j and l) are shown in the five groups of mice. Compared with AD + E2 or AD + PPT, mice in AD and AD + DPN exhibit significantly reduced freezing time and times of freezing at all points tested. No significance is shown between AD and AD + DPN. *p < 0.05, **p < 0.01 (ANOVA, n = 7–8 per group)