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

Yu‐Jie Lai,Bing‐Lin Zhu,Yuan‐Lin Ma,Ling He,Fei Sun,Jing Tang,Jian Yang,Zhen Yan,Ming‐Jian Xiong,Xiao‐Tong Hu,Lu Liu,Dong Luo,Bio Luo,Yan Long,John H Zhang,Xiao‐Juan Deng,Guo‐Jun Chen

doi:10.1111/acel.12961

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.

Highlights

Cav1.2 is the pore‐forming subunit of L‐type voltage‐gated calcium channels (LTCC) and accounts for approximately 70% of LTCC in the brain (Zamponi, Striessnig, Koschak, & Dolphin, 2015)
We found that E2 reduced Cav1.2 protein through estrogen receptor α (ERα)
C443A overexpression did not significantly affect PPT‐induced reduction of Cav1.2. These results indicated that K29‐linked UB chains were critical in mediating ERα‐induced ubiquitination and degrada‐ tion of Cav1.2 in neuronal cells

Summary

| INTRODUCTION

Cav1.2 is the pore‐forming subunit of L‐type voltage‐gated calcium channels (LTCC) and accounts for approximately 70% of LTCC in the brain (Zamponi, Striessnig, Koschak, & Dolphin, 2015). Further co‐immu‐ noprecipitation experiments showed that Cav1.2 ubiquitination was significantly increased by PPT (Figure 2d) These results suggested that UPS played a role in ERα regulation of Cav1.2. In cells transiently transfected with Mdm siRNA, the reduced Cav1.2 ubiquitination was concomitant with the dramatic increase in Cav1.2 protein (Figure 3d) These re‐ sults suggested that Mdm2‐associated ubiquitination was critical in ERα regulation of Cav1.2. Compared with control or scrambled peptides (ΔPEST1/ ΔPEST3), Cav1.2 was significantly increased in cells treated with PEST1/PEST3, in which PPT‐induced reduction of Cav1.2 was di‐ minished These results suggested that PEST motif in Cav1.2 played an important role in Mdm2‐mediated ubiquitination of Cav1.2. Mdm association with Cav1.2 or ERα was increased in mice treated with PPT, respectively (Figure 5f) These results indicated that ERα activation promoted the ubiquit‐ ination and degradation of Cav1.2 in OVX APP/PS1 mice. In the probe trial when the platform was removed, the passing times crossing over

| DISCUSSION

| EXPERIMENTAL PROCEDURES

Findings

CONFLICT OF INTEREST