Antihypertensive drug Valsartan promotes dendritic spine density by altering AMPA receptor trafficking

Abstract

Recent studies demonstrated that the antihypertensive drug Valsartan improved spatial and episodic memory in mouse models of Alzheimer's Disease (AD) and human subjects with hypertension. However, the molecular mechanism by which Valsartan can regulate cognitive function is still unknown. Here, we investigated the effect of Valsartan on dendritic spine formation in primary hippocampal neurons, which is correlated with learning and memory. Interestingly, we found that Valsartan promotes spinogenesis in developing and mature neurons. In addition, we found that Valsartan increases the puncta number of PSD-95 and trends toward an increase in the puncta number of synaptophysin. Moreover, Valsartan increased the cell surface levels of AMPA receptors and selectively altered the levels of spinogenesis-related proteins, including CaMKIIα and phospho-CDK5. These data suggest that Valsartan may promote spinogenesis by enhancing AMPA receptor trafficking and synaptic plasticity signaling.

Keywords: AMPA receptor; CaMKIIα; Spinogenesis; Valsartan.

Fig. 1

Valsartan increases dendritic spine density in primary hippocampal neurons. (A) Neurons (DIV21) were transfected with GFP and were treated with different doses of Valsartan (1 µM, 5 µM, or 10 µM) or vehicle (1% DMSO) for 24 hours. (B) Dendritic spine densities from (A) are presented. (C) Primary hippocampal neurons (DIV14) were transfected with GFP, and were treated with Valsartan (10 µM) or vehicle (1% DMSO) for 24 hours. (D) Dendritic spine densities from (C) are shown. Error bars represent S.E.M. *p<0.05, **p<0.01, ***p<0.001.

Fig. 2

Valsartan increases the puncta number of synaptophysin and PSD-95. (A–C) Primary hippocampal neurons (DIV21) were transfected with GFP and treated with Valsartan (10 µM) or vehicle (1% DMSO) for 24 hours. After 24 hours, immunostaining was conducted with anti-synaptophysin, and protein levels of synaptophysin and puncta number were measured. (B) Protein levels of synaptophysin from (A). (C) Puncta number of synaptophysin from (A). (D–F) Primary hippocampal neurons (DIV21) were transfected with GFP and treated with Valsartan (10 µM) or vehicle (1% DMSO) for 24 hours. After 24 hours, cells were immunostained with anti-PSD-95, and protein levels of PSD-95 and puncta number were measured. (E) Protein expression level for PSD-95 from (D) is shown. (F) Puncta number of PSD-95 from (D). Error bars represent S.E.M. *p<0.05.

Fig. 3

Valsartan alters the cell surface levels of AMPA receptors. (A–F) Primary hippocampal neurons (DIV21) were transfected with GFP and treated with Valsartan (10 µM) or vehicle (1% DMSO) for 24 hours. Neurons were then immunostained with GluN1, GluN2A, or GluN2B antibodies respectively, and total protein levels were measured. (B, D, F) Quantification of protein levels of GluN1, GluN2A, and GluN2B. (G, H, K, L) Primary hippocampal neurons (DIV21) were transfected with GFP, and were treated with Valsartan (10 µM) or vehicle (1% DMSO) for 24 hours. Live cell surface staining was conducted with N-terminal anti-GluA1 or anti-GluA2 antibodies, and the cell surface levels were measured. (H, L) Quantification of cell surface levels of GluA1 and GluA2. (I, J, M, N) Primary hippocampal neurons (DIV21) were transfected with GFP and treated with Valsartan (10 µM) or vehicle (1% DMSO) for 24 hours. Immunostaining with GluA1 or GluA2 antibodies were conducted and their total protein levels were measured. (J, N) Total levels of GluA1 and GluA2 are shown. Error bars represent S.E.M. *p<0.05, ***p<0.001.

Fig. 4

Valsartan regulates the levels of CaMKIIα and p-CDK5. Primary hippocampal neurons (DIV21) were transfected with GFP and treated with Valsartan (10 µM) or vehicle (1% DMSO) for 24 hours. (A–F) Neurons were immunostained using RasGRF1, p-ERK, or p-CREB antibodies as shown. (B, D, F) Protein expression levels of RasGRF1, p-ERK, and p-CREB. (G–J) Neurons (DIV21) were transfected with GFP and treated with Valsartan (10µM) or vehicle (1% DMSO) for 24 hours. Neurons were immunostained with CaMKIIα or CaMKIIβ antibodies. (H, J) Protein expression levels of CaMKIIα and CaMKIIβ. (K–T) Neurons were immunostained with p-JNK, p-AKT, p-CDK5, CDK5, or PKC antibodies. (L, N, P, R, T) Levels of p-JNK, p-AKT, p-CDK5, CDK5, and PKC are shown. Error bars represent S.E.M. *p<0.05, **p<0.01, ***p<0.001.