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Ovarian steroids regulate gene expression related to DNA repair and neurodegenerative diseases in serotonin neurons of macaques.

ABSTRACT: Depression often accompanies the perimenopausal transition and it often precedes overt symptomology in common neurodegenerative diseases (NDDs, such as Alzheimer's, Parkinson's, Huntington, amyotrophic lateral sclerosis). Serotonin dysfunction is frequently found in the different etiologies of depression. We have shown that ovariectomized (Ovx) monkeys treated with estradiol (E) for 28 days supplemented with placebo or progesterone (P) on days 14-28 had reduced DNA fragmentation in serotonin neurons of the dorsal raphe nucleus, and long-term Ovx monkeys had fewer serotonin neurons than intact controls. We questioned the effect of E alone or E+P (estradiol supplemented with progesterone) on gene expression related to DNA repair, protein folding (chaperones), the ubiquitin-proteosome, axon transport and NDD-specific genes in serotonin neurons. Ovx macaques were treated with placebo, E or E+P (n=3 per group) for 1 month. Serotonin neurons were laser captured and subjected to microarray analysis and quantitative real-time PCR (qRT-PCR). Increases were confirmed with qRT-PCR in five genes that code for proteins involved in repair of strand breaks and nucleotide excision. NBN1, PCNA (proliferating nuclear antigen), GADD45A (DNA damage-inducible), RAD23A (DNA damage recognition) and GTF2H5 (gene transcription factor 2H5) significantly increased with E or E+P treatment (all analysis of variance (ANOVA), P<0.01). Chaperone genes HSP70 (heat-shock protein 70), HSP60 and HSP27 significantly increased with E or E+P treatment (all ANOVA, P<0.05). HSP90 showed a similar trend. Ubiquinase coding genes UBEA5, UBE2D3 and UBE3A (Parkin) increased with E or E+P (all ANOVA, P<0.003). Transport-related genes coding kinesin, dynein and dynactin increased with E or E+P treatment (all ANOVA, P<0.03). SCNA (?-synuclein) and ADAM10 (?-secretase) increased (both ANOVA, P<0.02) but PSEN1 (presenilin1) decreased (ANOVA, P<0.02) with treatment. APP decreased 10-fold with E or E+P administration. Newman-Keuls post hoc comparisons indicated variation in the response to E alone versus E+P across the different genes. In summary, E or E+P increased gene expression for DNA repair mechanisms in serotonin neurons, thereby rendering them less vulnerable to stress-induced DNA fragmentation. In addition, E or E+P regulated four genes encoding proteins that are often misfolded or malfunctioning in neuronal populations subserving overt NDD symptomology. The expression and regulation of these genes in serotonergic neurons invites speculation that they may mediate an underlying disease process in NDDs, which in turn may be ameliorated or delayed with timely hormone therapy in women.

SUBMITTER: Bethea CL

PROVIDER: S-EPMC4508249 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Ovarian steroids regulate gene expression related to DNA repair and neurodegenerative diseases in serotonin neurons of macaques.

Bethea C L CL Reddy A P AP

Molecular psychiatry 20150120 12

Depression often accompanies the perimenopausal transition and it often precedes overt symptomology in common neurodegenerative diseases (NDDs, such as Alzheimer's, Parkinson's, Huntington, amyotrophic lateral sclerosis). Serotonin dysfunction is frequently found in the different etiologies of depression. We have shown that ovariectomized (Ovx) monkeys treated with estradiol (E) for 28 days supplemented with placebo or progesterone (P) on days 14-28 had reduced DNA fragmentation in serotonin neu ...[more]

PMID: 25600110

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Project description:Dendritic spines are the elementary structural units of neuronal plasticity and their proliferation and stabilization involve components of glutamate neurotransmission. In a model of hormone replacement therapy (HT), we sought the effect of estradiol (E) and progesterone (P) on gene expression related to glutamate neurotransmission in a laser captured preparation enriched for serotonin neurons from rhesus macaques. Microarray analysis was conducted (n=2 animals/treatment) and then confirmed for pivotal genes with qRT-PCR on additional laser captured material (n=3 animals/treatment). Ovariectomized rhesus macaques were treated with either placebo, E or E+P via Silastic implants for 1month prior to euthanasia. The midbrain was obtained, sectioned and immunostained for TPH. TPH-positive neurons were laser captured using an Arcturus Laser Dissection Microscope (Pixel II). RNA from laser captured serotonin neurons (n=2 animals/treatment) was hybridized to Rhesus Affymetrix GeneChips for screening purposes. There was a 2-fold or greater change in the expression of 28 probe sets related to glutamate processes in E and E+P treated animals. Quantitative (q) RT-PCR was conducted for 11 genes with a custom Taqman PCR array containing monkey specific primers and analyzed with ANOVA followed by Bonferroni's test. The log of the relative expression values indicated that in general, the responses to E and E+P were similar. Comparison of the relative expression or log relative expression in Ovx-controls to combined E and E+P treated groups with t-tests showed a significant increase in AMPA1 (GRIA1), AMPA2 (GRIA2), AMPA4 (GRIA4), NMDA2a (GRIN2A), metabotrophic glutamate receptor (GRM1), glutamine synthetase (GLUL), glutamate dehydrogenase (GLUD), glutamate cysteine ligase modifier subunit (GCLM), the glutamate transporter 2 (SLC1A2) and the glutamate transporter 3 (SLC1A3) with steroid treatment. There was no effect of steroid treatment on gene expression of the glutamate cysteine ligase catalytic subunit (GCLC). These data suggest that ovarian steroids target gene expression of ionotrophic and metabotrophic glutamate receptors in serotonin neurons. These receptors are present on dendritic spines and are necessary for spine maturation. The mRNAs coding for glutamate-related enzymes and transporters are likely derived from astrocytes or glutamate-containing terminals. Their induction by ovarian steroids indicates a complex upregulation of multiple components in the glutamate cycle and antioxidation, in addition to spine proliferation.

Project description:CART (cocaine and amphetamine regulated transcript) is a neuropeptide involved in the control of several physiological processes, such as response to psychostimulants, food intake, depressive diseases and neuroprotection. It is robustly expressed in the brain, mainly in regions that control emotional and stress responses and it is regulated by estrogen in the hypothalamus. There is a distinct population of CART neurons located in the vicinity of the Edinger-Westphal nucleus of the midbrain that also colocalize urocortin-1. The aims of this study were 1) to determine the distribution of CART immunoreactive neurons in the monkey midbrain, 2) to examine the effects of estrogen (E) and progesterone (P) on midbrain CART mRNA and peptide expression and 3) to determine whether midbrain CART neurons contain steroid receptors. Adult female rhesus monkeys (Macaca mulatta) were spayed and either treated with placebo (OVX), estrogen alone (E), progesterone alone (P) or E+P. Animals were prepared (a) for RNA extraction followed by microarray analysis and quantitative (q) RT-PCR (n=3/group); (b) for immunohistochemical analysis of CART and CART+tryptophan hydroxylase (TPH), CART+estrogen receptors (ER) or CART+progesterone receptors (n=5/group) and (c) for Western blots (n=3/group). Both E- and E+P-administration decreased CART gene expression on the microarray and with qRT-PCR. Stereological analysis of CART immunostaining at five levels of the Edinger-Westphal nucleus indicated little effect of E or E+P administration on the area of CART immunostaining. However, P administration increased CART-immunopositive area in comparison to the OVX control group with Student's t-test, but not with ANOVA. CART 55-102 detection on Western blot was unchanged by hormone administration. ERbeta and PR were detected in CART neurons and CART fibers appeared to innervate TPH-positive serotonin neurons in the dorsal raphe. In summary, E decreased CART mRNA, but this effect did not translate to the protein level. Moreover, P administration alone had a variable effect on CART mRNA, but it caused an increase in CART immunostaining. Together, the data suggest that CART neurons in the midbrain have a unique steroid response, which may be mediated by nuclear receptors, neuroactive steroids or interneurons.

Project description: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.

Project description:ObjectiveNuclear factor kappa B (NFkappaB) is a transcription factor that activates gene expression in response to proinflammatory cytokines, and elevated cytokines are associated with depression, which has a serotonergic component. We questioned (1) whether serotonin neurons contain NFkappaB, (2) whether NFkappaB detection with immunocytochemistry is changed in the dorsal raphe nucleus (DRN) by ovarian hormone treatment and (3) whether ovarian hormones regulate midbrain NFkappaB gene or protein expression.MethodsMonkeys were spayed and treated with placebo, estrogen (E), progesterone (P) or E+P for 1 month (n = 4 animals/treatment group), and the midbrain was harvested for immunocytochemistry and stereology. An antibody that detects nuclear location-specific (NLS)-NFkappaB p65 was applied, and the numbers of NLS-NFkappaB-immunopositive cells were counted in 9 sections of the DRN. Additional monkeys were used for Western blot analysis and quantitative reverse transcription-polymerase chain reaction (RT-PCR) for NFkappaB p65.ResultsIn placebo-treated macaques, neurons were double-immunostained for serotonin and nuclear NFkappaB p65 throughout the DRN. The mean total number of NFkappaB-positive cells equalled 2178 (and standard error of the mean [SEM] 129) in the placebo group, 1631 (SEM 221) in the E-treated group, 2314 (SEM 186) in the P-treated group and 1162 (SEM 100) in the E+P-treated group (analysis of variance p = 0.003). The E-treated and E+P-treated groups had a significantly lower density of cells stained positive for NFkappaB than the placebo or P-treated groups (post hoc). Unmasking of NLS-NFkappaB immunostaining in the DRN revealed dense immunostaining in the cytoplasm of large dorsal raphe neurons. There was no difference between treatment groups in the amount of NFkappaB p65 detected by Western blot or in the relative expression of NFkappaB p65 mRNA with quantitative RT-PCR.ConclusionsThese observations are consistent with the notion that gene and protein expression of NFkappaB are constitutive but that ovarian hormones can decrease the nuclear location of NFkappaB in dorsal raphe neurons and, thereby, decrease the ability of NFkappaB to drive gene expression in response to cytokines.

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Ovarian steroids regulate gene expression related to DNA repair and neurodegenerative diseases in serotonin neurons of macaques.

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Ovarian steroids regulate gene expression related to DNA repair and neurodegenerative diseases in serotonin neurons of macaques.

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