Effect of ovarian hormones on genes promoting dendritic spines in laser-captured serotonin neurons from macaques

Abstract

Dendritic spines are the elementary structural units of neuronal plasticity and the cascades that promote dendritic spine remodeling center on Rho GTPases and downstream effectors of actin dynamics. In a model of hormone replacement therapy, we sought the effect of estradiol (E) and progesterone (P) on gene expression in these cascades in laser-captured serotonin neurons from rhesus macaques with complementary DNA array analysis. Ovariectomized rhesus macaques were treated with either placebo, E or E+P through Silastic implant for 1 month before euthanasia. The midbrain was obtained, sectioned and immunostained for tryptophan hydroxylase (TPH). TPH-positive neurons were laser captured using an Arcturus Laser Dissection Microscope (PixCell II). RNA from laser-captured serotonin neurons (n=2 animals/treatment) was hybridized to Rhesus Affymetrix GeneChips. With E±P treatment, there was a significant change in 744 probe sets (analysis of variance, P<0.05), but 10,493 probe sets exhibited a twofold or greater change. Pivotal changes in pathways leading to dendritic spine proliferation and transformation included twofold or greater increases in expression of the Rho GTPases called CDC42, Rac1 and RhoA. In addition, twofold or greater increases occurred in downstream effectors of actin dynamics, including p21-activated kinase (PAK1), Rho-associated coiled-coil-containing protein kinase (ROCK), PIP5K, IRSp53, Wiskott-Aldrich syndrome protein (WASP), WASP family Verprolin-homologous protein (WAVE), MLC, cofilin, gelsolin, profilin and three subunits of actin-related protein (ARP2/3). Finally, twofold or greater decreases occurred in CRIPAK, LIMK2 and myosin light chain kinase (MLCK). The regulation of RhoA, Rac1, CDC42, ROCK, PIP5k, IRSp53, WASP, WAVE, LIMK2, CRIPAK1, MLCK, ARP2/3 subunit 3, gelsolin, profilin and cofilin was confirmed with nested quantitative reverse transcriptase-PCR on laser-captured RNA (n=3 animals/treatment). The data indicate that ovarian steroids target gene expression of the Rho GTPases and pivotal downstream proteins, that in turn would promote dendritic spine proliferation and stabilization on serotonin neurons of the dorsal raphe nucleus.

Figure 1

Chart illustrating the key Rho GTPases and their downstream effectors for actin and myosin activation leading to dendritic spine protrusion and transformation. RNA extracted from laser captured serotonin neurons was labeled and hybridized to the Rhesus Affymetrix GeneChip (n=2 animals/treatment) and the results were analyzed with GeneSifter. The genes in red increased 2-fold or greater and the genes in blue decreased 2-fold or greater with E or E+P treatment compared to ovariectomized placebo treated controls.

Figure 2

Histograms illustrating changes in gene expression obtained with nested qRT-PCR on RNA extracted from laser captured serotonin neurons (n=3 animals/treatment). The Rho GTPases, RhoA, and Rac1 significantly increased with E alone (ANOVA p<0.0002 and 0.00115, respectively) whereas CDC42 increased with E and E+P (ANOVA p<0.05). Downstream effectors ROCK2 increased with E+P (ANOVA p<0.0047) whereas PIP5K and IRSp53 increased with E alone (ANOVA p<0.05 and #t-test p<0.0114, respectively). The variance in IRSp53 in the E+P treated group was significantly different from the ovx group (⁺F test p<0.0028). * p<0.05 different from ovx control with Student-Newman-Keuls posthoc pairwise comparison after ANOVA.

Figure 3

Histograms illustrating changes in gene expression obtained with nested qRT-PCR on RNA extracted from laser captured serotonin neurons (n=3 animals/treatment). Intermediaries WAVE and WASP were significantly increased with E treatment, respectively (ANOVA p< 0.0129 and #t-test p<0.0001). The variance in WASP in the E+P treated group was significantly different from the ovx group (⁺F test p<0.0027). The actin regulatory proteins ARP3 and cofilin significantly increased with E and E+P treatment (ANOVA p<0.0329 and p<0.0093, respectively). However, gelsolin and profilin increases only with E+P (ANOVA p<0.0029 and p<0.0001, respectively). * p<0.05 different from ovx control with Student-Newman-Keuls posthoc pairwise comparison after ANOVA.

Figure 4

Histograms illustrating changes in gene expression obtained with nested qRT-PCR on RNA extracted from laser captured serotonin neurons (n=3 animals/treatment). Downstream of Rac1, MLCK was significantly decreased by E and E+P treatment (ANOVA p<0.0166) whereas LIMK2 was significantly decreased by E+P treatment (#t-test p<0.05; F-test p<0.0001). The endogenous Pak1 inhibitor, CRIPAK, was significantly decreased by E and E+P treatment (ANOVA p<0.0001). * p<0.05 different from ovx control with Student-Newman-Keuls posthoc pairwise comparison after ANOVA.