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. 2022 Aug 4:13:959360.
doi: 10.3389/fgene.2022.959360. eCollection 2022.

CaMKII is a modulator in neurodegenerative diseases and mediates the effect of androgen on synaptic protein PSD95

Shixiong Mi  1   2   3 , Huan Chen  1   2 , Peijing Lin  4 , Peiyuan Kang  5 , Dan Qiao  1   2 , Bohan Zhang  1   2 , Zhao Wang  1   2 , Jingbao Zhang  6 , Xiangting Hu  6 , Chang Wang  1   2   3 , Huixian Cui  1   2   3 , Sha Li  1   2   3   7
Affiliations

CaMKII is a modulator in neurodegenerative diseases and mediates the effect of androgen on synaptic protein PSD95

Shixiong Mi et al. Front Genet. .

Abstract

Androgens rapidly regulate synaptic plasticity in hippocampal neurones, but the underlying mechanisms remain unclear. In this study, we carried out a comprehensive bioinformatics analysis of functional similarities between androgen receptor (AR) and the synaptic protein postsynaptic density 95 (PSD95) to evaluate the effect. Using different measurements and thresholds, we obtained consistent results illustrating that the two proteins were significantly involved in similar pathways. We further identified CaMKII plays a critical role in mediating the rapid effect of androgen and promoting the expression of PSD95. We used mouse hippocampal neurone HT22 cells as a cell model to investigate the effect of testosterone (T) on intracellular Ca2+ levels and the mechanism. Calcium imaging experiments showed that intracellular Ca2+ increased to a peak due to calcium influx in the extracellular fluid through L-type and N-type voltage-gated calcium channels when HT22 cells were treated with 100 nM T for 20 min. Subsequently, we investigated whether the Ca2+/CaMKII signaling pathway mediates the rapid effect of T, promoting the expression of the synaptic protein PSD95. Immunofluorescence cytochemical staining and western blotting results showed that T promoted CaMKII phosphorylation by rapidly increasing extracellular Ca2+ influx, thus increasing PSD95 expression. This study demonstrated that CaMKII acts as a mediator assisting androgen which regulates the synaptic protein PSD95Also, it provides evidence for the neuroprotective mechanisms of androgens in synaptic plasticity and reveals the gated and pharmacological mechanisms of the voltage-gated Ca2+ channel family for androgen replacement therapy.

Keywords: Ca2+; CaMKII; PSD95; androgen; protein interaction; semantic similarity.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Genomic investigation of the association between AR and PSD95. (A) Venn diagram of the interactors between AR and PSD95. (B,C) Functional enrichment analysis of the PSD95 interactors (B) and AR interactors (C). (D,E) Heatmap showing the semantic similarity between AR interactors and PSD95 interactors. Two thresholds of interaction confidence score, 0.9 (D) and 0.95 (E), were used, respectively. (F,G) Distribution of the simulated SS scores calculated using wang. Three simulation methods were used and colored in red, green, and blue, respectively. Two thresholds of interaction confidence score, 0.9 (F) and 0.95 (G), were used, respectively.
FIGURE 2
FIGURE 2
Significant high semantic similarity between AR interactors and PSD95 interactors. (A) Distribution of the simulated SS scores calculated using four different methods. The simulation methods were colored the same as Figure 1. (B) Heatmap showing the semantic similarity between AR interactors and PSD95 interactors using the four methods. Interaction confidence score of 0.9 was used in (A,B). (C) Distribution of the simulated SS scores calculated using four different methods. (D) Heatmap showing the semantic similarity between AR interactors and PSD95 interactors using the four methods. Interaction confidence score of 0.95 was used in (C,D).
FIGURE 3
FIGURE 3
Identification of CaMKII as a mediator for androgen and PSD95. (A,B) SS scores were calculated by five methods based on the ICS threshold of 0.95 (A) and 0.9 (B), respectively. (C) Interaction network including AR, PSD95 and their mediators. (D) CAMK2B were differentially expressed in two datasets GSE173955 and GSE159699.
FIGURE 4
FIGURE 4
Rapid effects of T on calcium in HT22 cells. (A) Schematic diagram of calcium imaging in the T dose-response experiment in HT22 cells labeled with Fluo-4AM. (B) Fluorescence images of T dose-response experiment in Fluo-4AM-labelled HT22 cells. Scale bars = 50 μm. (C) Representative traces showing the changes in Ca2+ (F/F0) induced by different doses of T. Black arrows indicate when HT22 cells were treated. (D) Statistical graph showing changes in Ca2+ (F/F0) induced by different doses of T (ns: non-significant; *p < 0.05).
FIGURE 5
FIGURE 5
The source of increased intracellular Ca2+ induced by T in HT22 cells. (A) Schematic diagram of calcium imaging in the calcium source experiment in HT22 cells labeled with Fluo-4AM. (B) Fluorescence images of the calcium source in Fluo-4AM-labeled HT22 cells. Scale bars = 50 μm. (C) Representative traces showing changes in Ca2+ (F/F0) induced by T when HT22 cells were cultured in calcium-containing and calcium-free extracellular fluids. The black arrow indicates when the HT22 cells were treated. (D) Statistical graph showing the changes in Ca2+ (F/F0) induced by T when HT22 cells were cultured in calcium-containing and calcium-free extracellular fluids (ns: non-significant; *p < 0.05).
FIGURE 6
FIGURE 6
Voltage-gated calcium channels are involved in the T-induced increase of Ca2+ in HT22 cells. (A) Schematic diagram of calcium imaging in the voltage-gated calcium channel experiment in HT22 cells labeled with Fluo-4AM. (B) Fluorescence images of the voltage-gated calcium channel experiment in Fluo-4AM-labeled HT22 cells. Scale bars = 50 μm. (C) Representative traces showing changes in Ca2+ (F/F0) induced by T in HT22 cells treated with amlodipine and CgTx. The black arrow indicates when the HT22 cells were treated. (D) Statistical graph showing the T-induced changes in Ca2+ (F/F0) in HT22 cells treated with amlodipine and CgTx (ns: non-significant; *p < 0.05).
FIGURE 7
FIGURE 7
Voltage-gated calcium channels are involved in T-induced increase of synaptic protein PSD95 in HT22 cells. (A) Immunofluorescence cytochemistry for PSD95 expression in HT22 cells pretreated with T, amlodipine, or CgTx. (B) Statistical analysis of immunofluorescence cytochemistry for PSD95 expression in HT22 cells pre-treated with T, amlodipine, or CgTx. Scale bars = 20 μm. (C) Western blotting for PSD95 expression in HT22 cells pre-treated with T, amlodipine, or CgTx. (D) Statistical graph of western blot analysis of PSD95 expression in HT22 cells pre-treated with T, amlodipine, or CgTx (*p < 0.05).
FIGURE 8
FIGURE 8
Voltage-gated calcium channels are involved in T-induced increase of CaMKII protein in HT22 cells. (A) Immunofluorescence cytochemistry for p-CaMKII expression in HT22 cells pre-treated with T, amlodipine, or CgTx. (B) Statistical analysis of immunofluorescence cytochemistry for p-CaMKII expression in HT22 cells pre-treated with T, amlodipine, or CgTx. Scale bars = 20 μm. (C) Western blotting for CaMKII and p-CaMKII protein expression in HT22 cells pre-treated with T, amlodipine, or CgTx. (D,E) Statistical graph of western blot analysis of CaMKII and p-CaMKII protein expression in HT22 cells pre-treated with T, amlodipine, or CgTx (*p < 0.05).
FIGURE 9
FIGURE 9
CaMKII protein is involved in the T-induced increase of synaptic protein PSD95 in HT22 cells. (A) Immunofluorescence cytochemistry of PSD95 expression in HT22 cells pre-treated with T or KN-93. (B) Statistical graph of immunofluorescence cytochemistry for PSD95 expression in HT22 cells pre-treated with T or KN-93. Scale bars = 20 μm. (C) Western blotting for PSD95 expression in HT22 cells pre-treated with T or KN-93. (D) Statistical graph of western blot analysis of PSD95 expression in HT22 cells pre-treated with T or KN-93 (*p < 0.05).
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