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- Materials and methods
We sought the effect of estradiol (E) and progesterone (P) on survival gene expression in laser captured serotonin neurons and in the dorsal raphe region of monkeys with cDNA array analysis. Spayed rhesus macaques were treated with either placebo, E or E + P via Silastic implant for 1 month prior to killing. First, RNA from a small block of midbrain containing the dorsal raphe was hybridized to Rhesus Gene Chips (n = 3/treatment). There was a significant change in 854 probe sets with E ± P treatment (anova, p < 0.05); however, only 151 probes sets exhibited a twofold or greater change. Twenty-five genes related to cell survival changed significantly. The expression of vascular endothelial growth factor, superoxide dismutase (SOD1), and the caspase inhibitor, BIRC4, was confirmed with quantitative RT-PCR. Then, RNA from laser captured serotonin neurons (n = 2/treatment) was hybridized to Rhesus Gene Chips. There was a significant change in 744 probe sets, but 10 493 probe sets exhibited a twofold or greater change. Pivotal changes in apoptosis and cell cycle pathways included twofold or greater increases in SOD1, IκBα, Fas apoptotic inhibitory molecule, fibroblast growth factor-receptor 2 (FGFR2), neurotrophic tyrosine kinase receptor 2 (NTRK2), phosphoinositide-3-kinase (p85 subunit), cyclic AMP dependent protein kinase (PKA) (catalytic subunit), calpain 2, and ataxia telangectasia mutated (ATM). Twofold or greater decreases occurred in TNF receptor interacting serine-threonine kinase 1 (RIP1), BH3 interacting domain death agonist (BID), apoptotic peptidase activating factor 1 (Apaf1), caspase recruitment domain 8 (CARD8), apoptosis inducing factor (AIF), Diablo and Cyclins A, B, D, and E. The regulation of SOD1, calpain 2, Diablo, and Cyclin D was confirmed with quantitative RT-PCR (n = 3/treatment). The data indicate that ovarian steroids target the cytokine-signaling pathway, caspase-dependent and -independent pathways and cell cycle proteins to promote serotonin neuron survival.
The serotonin system modulates a wide range of neural outcomes from emotion to intellect to metabolism and it is a target of pharmacotherapies, steroid hormones, cytokines, neuropeptides, and trophic factors, all of which impact the generation and efficacy of serotonin neurotransmission. Thus, any loss or degeneration of serotonin neurons could have profound ramification.
This laboratory has devoted effort toward understanding the actions of ovarian hormones in serotonin neurons and their terminal fields with a macaque model of surgical menopause. Serotonin neurons express estrogen receptor beta and progestin receptors (Bethea 1993; Gundlah et al. 2001). We found that estradiol (E) ± progesterone (P) supplementation, regulates the expression of pivotal serotonin-related genes and proteins in the monkey dorsal raphe in a pattern suggestive of increased serotonin production, increased serotonin turnover, increased neural firing, and decreased degradation (Bethea et al. 2002; Lu and Bethea 2002; Lu et al. 2003).
In another paradigm, we observed that stress-sensitive cynomolgus monkeys have fewer serotonin neurons than stress-resilient companions. Moreover, the lower serotonin cell number was strongly correlated to lower levels of E and P during their menstrual cycles. The stress-sensitive macaques also had overall lower expression of serotonin-related genes than stress-resilient counterparts reflecting the serotonin cell number (Bethea et al. 2005). These correlations raised the question of whether E and P could be neuroprotective for serotonin neurons.
This issue may be extremely important for menopausal women grappling with issues surrounding hormone therapy (HT). Women experience premature ovarian failure and loss of ovarian steroid production around 50 years of age. Thus, with extended lifespans, a woman may live 35–40 years without ovarian steroids. If serotonin neurons are gradually dying because of lack of steroid supported gene expression, then geriatric depression, anxiety, fretfulness, decreased coping skills, and increased vulnerability to stress can be predicted outcomes.
In a preliminary study, we probed the Human Affymetrix Gene Chip with RNA extracted from a small block of tissue containing the dorsal and median raphe nuclei (Reddy and Bethea 2005). This study indicated that several genes involved in neurotoxicity or programmed cell death (PDCD) (apoptosis) were regulated by E ± P. One very important pro-apoptosis gene, JNK1 (c-jun n-terminal kinase or MAPK8), was markedly decreased by HT. Subsequently, a rhesus macaque Affymetrix Gene Chip and better software for global analysis were marketed. In addition, the technology to microdissect individual neurons became available.
Therefore, in this study, we sought novel genes related to neuronal survival that are regulated by E and P in laser captured serotonin neurons of rhesus monkeys using the Rhesus Affymetrix cDNA array and quantitative (q) RT-PCR. We also probed the rhesus array with the RNA from the dorsal raphe block for comparative purposes. We found that E and P altered the expression of genes in the cytokine signaling pathway, in caspase-dependent and -independent pathways and in the cell cycle, which could have a significant impact on serotonin neuron survival. Moreover, seven pivotal gene changes predicted by the microarray were confirmed by qRT-PCR.
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- Materials and methods
In animal models, HT provides numerous beneficial effects on CNS function and viability. However, human trials have yielded mixed results which lately seem to be due to the age at which HT is initiated (Espeland et al. 2004; Rossouw et al. 2007). In our model of surgical menopause in adult monkeys, HT has positive effects on serotonin function (Bethea et al. 2002). In this model, HT is administered for 1 month so the effects may be mediated by genomic or non-genomic mechanisms. Now, we show that HT changes gene expression related to survival in serotonin neurons.
In this study, we included gene expression changes effected by E or E + P. Some genes showed similar inductions with the treatments while others exhibited a change with E + P and not E alone. In the laser-captured material, there was more variance in the E + P-treated group than in the E-treated group. It is of significant interest to re-examine the Affymetrix data sets and filter for differences between E and E + P to gain a better understanding of the potentially unique actions of P.
Expression increases in the DRN block
In the raphe block, HT induced a significant increase in SOD1. This critical enzyme scavages free oxygen radicals (reactive oxygen species) and thus, plays an important role in cell survival (Davis et al. 2007). HT induced a significant increase in several genes in the MAPK pathway such as VEGF, FGF-R2, NGF receptor-associated protein, RAS GRF, and MAP2K5. The proteins encoded by RAS GRF and MAP2K5 lead to activation of ERK and ERK5 which transcribe genes essential to cell survival (Almeida et al. 2005; Wu et al. 2005; Toborek et al. 2007) whereas VEGF has a newly recognized neuroprotective role (Gora-Kupilas and Josko 2005). The encoded FGF-R2 protein and the NGF receptor-associated protein are involved in signal transduction of their respective trophic factors and hence would be pro-survival. HT also increased the expression of three genes that code for endogenous caspase inhibitors called BIRC4 (also known as XIAP), MCL-1, and bifunctional apoptosis regulator (BFAR). This effect would decrease caspase activation and apoptosis along the caspase-dependent pathway (Nishihara et al. 2003). HT also increased the expression of two ubiquinases that shuttle α-synuclein and PARK 2 to the proteosome (Shimura et al. 2001), and this action could protect neurons from neurodegeneration associated with the accumulation of these proteins. E2F1 was previously found to increase (Reddy and Bethea 2005) and this action may block the activity of p53, which can initiate catastrophic entry into the cell cycle. Although E2F1 has been implicated in apoptosis (Hou et al. 2000), the integrity of the E2F1/Rb complex is neuroprotective in the presence of free oxygen radicals (Chong et al. 2006).
Expression decreases in the DRN block
Furthermore in the raphe block, HT decreased the expression of a number of pivotal genes in the stress-related, cytokine-signaling pathway. The expression of CXCL12, transforming growth factor (TGF)-β receptor 3 (TGFβ3), TNF α-induced protein (TNFAIP6), member 21 of the TNF superfamily (TNFRSF21), and the PTGFR were significantly decreased. Fas-associated factor 1 (FAF1) decreased significantly and the protein encoded by this gene binds to Fas antigen and can initiate apoptosis or enhance apoptosis initiated through Fas antigen. Intermediaries PAK and MAP2K4 which code for proteins that can activate JNK1 were also decreased. We previously found that HT decreased JNK1 expression as well (Reddy and Bethea 2005). JNK1 is a pivotal protein that can initiate a cascade leading to mitochondrial permeability and the release of either cytochrome c or AIF, leading to either caspase-dependent or -independent cell death. Thus, HT decreases gene expression in an intercellular signaling pathway that mediates various kinds of stresses.
The nibrin (NBS1) gene product is thought to be involved in DNA double-strand break repair and DNA damage-induced checkpoint activation. Thus, a decrease in nibrin may indicate inhibition of the cell cycle, which is neuroprotective for neurons (Marcelain et al. 2005). PDCD4 encodes a protein localized to the nucleus in proliferating cells and the expression of this gene is modulated by cytokines. The gene product is thought to play a role in apoptosis, but the specific role has not yet been determined (Yang et al. 2001).
It is likely that the gene changes observed in the block of tissue are global changes that occur in many cell types, which is an important data set. Although the changes were deemed significant by anova, we noticed that the absolute differences in signal intensity between the groups were not robust, suggesting that masking of differences may occur when multiple cell types are examined. When we compared the number of probe sets that changed twofold or greater in the raphe block with the laser captured neurons, there was a marked enrichment of probe sets that changed twofold or greater with HT in the laser-captured serotonin neurons. This highlights our perception that HT regulates gene expression in serotonin neurons, but detection of the changes may be obscured in a block of tissue that contains other cell types.
It is curious that TPH2 expression in the laser-captured neurons was similar with E and E + P whereas it was decreased with E + P relative to E alone in the DRN block although both treatment groups were significantly elevated compared with the OVX group. In an earlier analysis of TPH2 with in situ hybridization, there was no difference between the inductions by E or E + P which is consistent with the laser-capture data (Sanchez et al. 2005). Thus, the difference in TPH2 induction in the DRN block may be due to some unknown variance in the progestin responsiveness of this group of monkeys.
Expression changes in apoptosis pathways in laser-captured serotonin neurons
To further our hypothesis that HT protects serotonin neurons from cell death, we focused on twofold or greater expression changes in apoptosis and cell cycle pathways in the laser-captured neurons. As in the raphe block, SOD1 and FGF-R2 increased in the laser-captured serotonin neurons. This suggests that the effect of HT on SOD1 and FGF-R2 may be global effects. The likely decrease in reactive oxygen species that would ensue from elevated SOD1 protein and the increase in FGF-R2 signaling may be an important part of HT-induced neuroprotection. In the caspase-dependent pathway, HT decreased RIP1, BID, Apaf1, and CARD8. The BID gene encodes a death agonist that heterodimerizes with bcl-2. The encoded protein is a member of the bcl-2 family of cell death regulators. It is a mediator of mitochondrial damage induced by caspase 8; caspase 8 cleaves this encoded protein, and the COOH-terminal part translocates to mitochondria where it blocks bcl-2 and triggers cytochrome c release. Thus, decreasing expression of BID would promote cell survival. RIP1 encodes a TNF receptor (TNFRSF)-interacting serine–threonine kinase 1. It is an adaptor protein in the TNF signaling pathway that leads to apoptosis. Hence, a decrease in RIP1 would render the cell less susceptible to TNF signaling. Apaf1, or apoptotic peptidase activating factor 1, encodes a cytoplasmic protein that initiates apoptosis. Upon binding cytochrome c and dATP, this protein forms an oligomeric apoptosome. The apoptosome binds and cleaves procaspase 9, releasing its mature, activated form. Activated caspase 9 stimulates the subsequent caspase cascade that commits the cell to apoptosis. Therefore, the HT-induced decrease in Apaf1 in serotonin neurons is a pivotal means to decrease activity of the caspase-dependent apoptosis pathway. HT also decreased CARD8. CARD-containing proteins, such as CARD8, are involved in pathways leading to activation of caspases or nuclear factor kappa-B in the context of apoptosis or inflammation, respectively (Bouchier-Hayes et al. 2001). It is curious that we observed an increase in gene expression for procaspase 3. However, there appear to be mechanisms in place that would prevent its activation. Further work is underway to determine which of these changes in gene expression are manifested at the protein level and to determine active and inactive states of each protein.
Hormone therapy decreased an extremely pivotal gene in the caspase-independent pathway. Apoptosis inhibitory factor (AIF), also called PDCD8, was decreased fourfold with E + P treatment. The AIF gene encodes a flavoprotein essential for nuclear disassembly in apoptotic cells that is found in the mitochondrial intermembrane space in healthy cells. Induction of apoptosis results in the translocation of this protein to the nucleus where it effects chromosome condensation and fragmentation. In addition, this gene product induces mitochondria to release the apoptogenic proteins cytochrome c and caspase 9 so it also ties into the caspase-dependent pathway. Nonetheless, in certain cell lines, AIF is sufficient to induce apoptosis when the caspases are inhibited (Krantic et al. 2007). In preliminary western blots on subcellular fractions of raphe blocks, we find that the level of AIF in the mitochondria is decreased, and the amount of AIF translocated to the nucleus is significantly decreased in E + P-treated monkeys. This supports the notion that HT also acts through the caspase-independent pathway.
Expression changes in cytokine signaling in laser-captured serotonin neurons
Smac/Diablo is a second mitochondria-derived activator of caspases. Expression of Diablo was decreased threefold on the microarray and fivefold in the qRT-PCR assay with E + P treatment. Diablo encodes a protein that blocks the endogenous caspase inhibitor of apoptosis proteins (IAPs) (Verhagen et al. 2000) and it plays a pivotal role in the mediation of apoptosis through TRAIL and the TNF pathway (Deng et al. 2002). HT increased several IAP genes in the raphe blocks and Diablo would normally inhibit these IAPs, thereby removing a brake on the caspase-dependent pathway. A decrease in Diablo would allow activity of the IAPs. Moreover, we also observed a marked decrease in genes in the TNF/cytokine signaling pathway in the raphe block suggesting that HT may converge on multiple effectors of this pathway as a major mechanism of neuroprotection.
Hormone therapy induced a robust 23-fold increase in IκBα gene expression in serotonin neurons. The encoded protein binds to NFκB and prevents nuclear translocation and activation of stress-related genes, which can lead to apoptosis depending on the cell context (Post et al. 2000; Liang et al. 2007), although in some paradigms, NFkB has neuroprotective actions (Dhandapani et al. 2005). We previously demonstrated that NFκB colocalizes in serotonin neurons and that HT decreased the nuclear location of NFκB, but there was no change in the expression of NFκB at gene or protein levels (Bethea et al. 2006). The microarray data suggests an important mechanism by which HT reduced the translocation of NFκB to the nucleus. It is also worth noting that the 5HT1A autoreceptor gene contains NFκB response elements in the promoter and that HT decreases expression of the 5HT1A gene in the dorsal raphe. It follows that the HT-induced increase in IκBα and subsequent decrease in NFκB translocation may be mediating the effect of HT on 5HT1A gene expression.
Expression changes in intracellular signaling pathways in laser-captured serotonin neurons
Hormone therapy also increased several survival factors in serotonin neurons that are upstream of mitochondrial permeability. The expression of NTRK2 increased robustly with E treatment. This gene encodes a kinase that upon neurotrophin binding, phosphorylates itself and members of the MAPK pathway. Mutations in the gene have been associated with obesity and mood disorders (Adams et al. 2005; Gray et al. 2007). The expression of the gene coding the 85 kDa regulatory subunit of phosphoinositide-3-kinase increased twofold. This protein activates AKT, which in turn, phosphorylates and inactivates bcl-2 antagonist of cell death (BAD) (Datta et al. 1999). BAD is a member of the bcl-2 family that opens mitochondrial pores by binding to bcl-2, thereby releasing cytochrome c (Zhu et al. 2006; Koh 2007). HT also increased the gene expression of the catalytic subunit of PKA (cAMP-dependent protein kinase). PKA also inhibits the activity of BAD, which would decrease mitochondrial permeability and the release of pro-apoptotic proteins. Previous work showed that E prevented BAD phosphorylation in a model of ischemic neuronal death (Won et al. 2005). Of note, HT caused a robust threefold increase in calpain 2. This is interesting because until recently the calpains were considered pro-apoptotic. However, new data suggests that they may be neuroprotective and prevent signaling through the TNF pathway (Lu et al. 2002; Tan et al. 2006). Thus, the ability of HT to block TNF signaling by decreasing gene expression in the intracellular signaling cascade and by increasing calpain further suggests that this is a pivotal site of action for HT-induced neuroprotection.
Expression changes in cell cycle genes in laser-captured serotonin neurons
Hormone therapy decreased gene expression of four of the cyclins, which play essential roles in the progression of the cell cycle. The most pivotal of the cyclins, Cyclin D, decreased twofold on the microarray and nearly 10-fold in the qRT-PCR assay with E and E + P treatment. Cyclin D is essential for cells to re-enter the cell cycle and re-entry into the cell cycle is catastrophic for terminally differentiated cells, like serotonin neurons (Herrup et al. 2004). Therefore in neurons, cell cycle proteins are considered pro-apoptotic (Becker and Bonni 2004, 2005). In addition, the gene encoding the pivotal protein ATM was increased twofold in serotonin neurons. This protein is an important cell cycle checkpoint kinase that functions as a regulator of a wide variety of downstream proteins (Evan and Littlewood 1998). Activation of ATM plays a central role in shutting down cell cycle transitions through a series of effector molecules at each checkpoint (Lavin and Kozlov 2007). Altogether, these data suggest that another mechanism by which HT is neuroprotective is by preventing re-entry of serotonin neurons into the cell cycle.
Although the majority of expression changes support the hypothesis that ovarian hormones act to enhance survival, there were two minor inconsistencies. Cytochrome c was represented by three probe sets, which reported different changes so this was considered unreliable. The pro-apoptotic Bax gene was undetectable in the OVX and E + P-treated groups but it was detectable in the E-treated group, which was reported as an increase in expression. However, the marginal signal intensity did not engender confidence.
Upon review, it appears that HT is neuroprotective of serotonin neurons and probably other neurons in the midbrain by several major mechanisms. HT induces a decrease in gene expression in the TNF/cytokine signaling pathway and in Smac/Diablo, a mediator of apoptosis by this pathway, and it increases calpain which blocks TNF signaling; HT induces a decrease in expression of genes that encode pivotal proteins in the caspase-dependent and -independent pathways; HT increases gene expression of pivotal survival factors; and HT alters gene expression governing cell cycle initiation and progression. Thus, HT provides trophic support and inhibits the expression of genes that increase vulnerability to cytokines and stress in serotonin neurons and the surrounding neuropile. Further study of gene expression in other intracellular signaling pathways is underway.
It is important to recognize that our model does not include a gross insult to the CNS such as ischemia or neurotoxic lesion. The changes observed in gene expression occurred in 1 month in otherwise normal animals in paired housing with environmental enrichment. Extrapolating from this data is straightforward. In the long-term absence of ovarian steroids, serotonin neurons would be less resilient and may die with normal life stress. However, it is necessary to demonstrate that the changes observed in gene expression are manifested at the protein level, and that the proteins are active. Essential to validation of the hypothesis will be demonstration that ovariectomized primates have fewer serotonin neurons than ovarian intact controls. Juvenile macaques, with ovariectomy or fallopian tube ligation, are currently maturing in a semi-free ranging troop in an outdoor corral for the answer to this question.