Biomedical subjects

Melinda E Wilson

Publications and source records attributed to Melinda E Wilson.

11 recordsLinked to original sources

PubMed2007

Changes in estrogen receptor-alpha mRNA in the mouse cortex during development.

Estrogen plays a critical role in brain development and is responsible for generating sex differences in cognition and emotion. Studies in rodent models have shown high levels of estrogen binding in non-reproductive areas of the brain during development, including the cortex and hippocampus, yet binding is diminished in the same areas of the adult brain. These binding studies demonstrated that estrogen receptors decline in the cortex during development but did not identify which of the two estrogen receptors was present. In the current study, we examined the expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the mouse cortex during the first month of life. Messenger RNA was isolated from cortical tissue taken from C57BL/6 mice on postnatal day (PND) 1, 4, 10, 18 and 25 and expression levels were determined by real-time PCR. ERalpha mRNA expression in the mouse cortex at PND 25 was significantly reduced as compared to PND 1 (p<0.01). ERbeta mRNA expression at PND 25 was significantly increased as compared to PND 1 (p<0.05). Although the increase in ERbeta mRNA was statistically significant, the ERbeta levels were extremely low in the isocortex compared to ERalpha mRNA levels, suggesting that ERalpha may play a more critical role in the developmental decrease of estradiol binding than ERbeta. Additionally, we measured ERalpha mRNA expression in organotypic explant cultures of cortex taken from PND 3 mice. Explants were maintained in vitro for 3 weeks. mRNA was isolated at several time points and ERalpha and ERbeta mRNA was measured by real-time RT-PCR. ERalpha and ERbeta mRNA levels reflected a similar pattern in vitro and in vivo, suggesting that signals outside the cortex are not needed for this developmental change. This study lays the groundwork for an understanding of the mechanisms of the developmental regulation of ERalpha mRNA.

Aging↗

PubMed2006

Nucleoside reverse transcriptase inhibitors prevent HIV protease inhibitor-induced atherosclerosis by ubiquitination and degradation of protein kinase C.

HIV protease inhibitors are important pharmacological agents used in the treatment of HIV-infected patients. One of the major disadvantages of HIV protease inhibitors is that they increase several cardiovascular risk factors, including the expression of CD36 in macrophages. The expression of CD36 in macrophages promotes the accumulation of cholesterol, the development of foam cells, and ultimately atherosclerosis. Recent studies have suggested that alpha-tocopherol can prevent HIV protease inhibitor-induced increases in macrophage CD36 levels. Because of the potential clinical utility of using alpha-tocopherol to limit some of the side effects of HIV protease inhibitors, we tested the ability of alpha-tocopherol to prevent ritonavir, a common HIV protease inhibitor, from inducing atherosclerosis in the LDL receptor (LDLR) null mouse model. Surprisingly, alpha-tocopherol did not prevent ritonavir-induced atherosclerosis. However, cotreatment with the nucleoside reverse transcriptase inhibitors (NRTIs), didanosine or D4T, did prevent ritonavir-induced atherosclerosis. Using macrophages isolated from LDLR null mice, we demonstrated that the NRTIs prevented the upregulation of CD36 and cholesterol accumulation in macrophages. Treatment of LDLR null mice with NRTIs promoted the ubiquitination and downregulation of protein kinase Calpha (PKC). Previous studies demonstrated that HIV protease inhibitor activation of PKC was necessary for the upregulation of CD36. Importantly, the in vivo inhibition of PKC with chelerythrine prevented ritonavir-induced upregulation of CD36, accumulation of cholesterol, and the formation of atherosclerotic lesions. These novel mechanistic studies suggest that NRTIs may provide protection from one of the negative side effects associated with HIV protease inhibitors, namely the increase in CD36 levels and subsequent cholesterol accumulation and atherogenesis.

Animals↗

PubMed2006

Estradiol negatively regulates HIV-LTR promoter activity in glial cells.

HIV-associated dementia results from neuronal loss and an alteration of neuronal function due to a loss of synapses. While HIV infection in astrocytes is limited, astrocytes exhibit a chronic nonproductive infection that can lead to the release of neurotoxic proteins. Additionally, infection can disrupt the normal neurotrophic role of astrocytes that results in neuronal death. Gonadal steroid hormones are known to act as trophic and protective factors in the brain under a variety of normal and pathological conditions. In the present study, to determine if estrogen plays a role in the ability of Tat to function as a transcriptional activator within astrocytes, we examined the effect of estrogen on regulation of viral transcription. We utilized an immortalized human astrocyte cell line (SVGA) stably transfected with a reporter plasmid containing the HIV-1IIIB LTR driving the chloramphenicol acetyltransferase (CAT) gene. The amount of transcriptional activity was measured by quantifying the amount of CAT produced. We determined that 17beta-estradiol treatment (1 nM) had no effect on basal LTR activity. Following transfection with a Tat-expressing plasmid, there was a 100-fold increase in CAT production. This induction was reduced by 40% in cells pretreated with 17beta-estradiol. 17beta- Estradiol only suppressed transcription stimulated by Tat. Furthermore, we determined that this effect was specific to 17beta-estradiol and estrogen receptor agonists. This activity was limited to astrocytes as no effect was observed in a monocytic cell line. Finally, the mechanism of action did not involve an alteration in levels of Cdk9 or Cyclin T1 proteins necessary for Tat activation of the HIV-1 LTR. This study demonstrates a novel activity of 17beta-estradiol in glial cells that could play a role in the maintenance of neuronal health during HIV infection of the central nervous system.

Analysis of Variance↗

PubMed2006

Intravenous 6-hydroxydopamine attenuates vasopressin and oxytocin secretion stimulated by hemorrhage and hypotension but not hyperosmolality in rats.

The present study sought to determine whether chemical destruction of peripheral catecholaminergic fibers with 6-hydroxydopamine (6OHDA) attenuates vasopressin (VP) and oxytocin (OT) secretion stimulated by hemorrhage, hypotension, and hyperosmolality. Rats received 6OHDA (100 mg/kg iv) or vehicle (1 ml/kg iv) on days 1 and 7, and experiments were performed on day 8. Serial hemorrhage (4 samples of 2 ml per 300 g body wt at 10-min intervals) increased plasma VP and OT levels in both groups; however, the increase in plasma VP and OT levels was significantly attenuated in 6OHDA-treated vs. control rats despite a significantly lower mean arterial blood pressure. Similarly, the increase in plasma VP and OT levels in response to hypotension produced by the selective arteriolar vasodilator diazoxide was significantly attenuated in 6OHDA-treated rats. In marked contrast to hemorrhage and hypotension, hyperosmolality produced by an infusion of 1 M NaCl (2 ml/h iv) stimulated increases in plasma VP and OT levels that were not different between 6OHDA-treated and control rats. In a parallel set of experiments, intravenous 6OHDA treatment reduced dopamine--hydroxylase immunoreactivity in the posterior pituitary but had no substantial effect in the hypothalamic paraventricular and supraoptic nuclei. In each experiment, the pressor response to tyramine (250 microg/kg iv) was significantly attenuated in 6OHDA-treated rats, thereby confirming that 6OHDA treatment destroyed sympathetic catecholaminergic fibers. Collectively, these findings suggest that catecholaminergic fibers located outside the blood-brain barrier contribute to VP and OT secretion during hemorrhage and arterial hypotension.

Animals↗

PubMed2006

Immune modulation by estrogens: role in CNS HIV-1 infection.

Experimental and epidemiological data suggest that estrogen can be protective in both brain injury and infection. While estrogens can act directly on neurons to promote neuronal survival, estrogen also has antiinflammatory properties that may contribute to overall neuroprotection. Accordingly, estrogens may have particular relevance in chronic neuroimmune disorders such as HIV dementia. As AIDS is now a leading cause of death among women in their reproductive years, understanding the role that female sex hormones might play in the physiology of HIV-1 infection is especially critical. Indeed, there is accumulating evidence that many manifestations of HIV differ in women. For instance, it is now well established that women present with a lower viral titer at the time of seroconversion, have lower HIV viral loads compared to men at similar stages of disease, and may have altered disease progression during pregnancy. Conversely, while epidemiological studies suggest that women may be more vulnerable to certain late-stage AIDS-related illnesses including HIV dementia, there is accumulating data that strongly suggest an estrogen-deficient state is associated with long-term HIV infection in some women. Evaluated as a whole, existing evidence indicates that estrogen can directly protect neurons from damage, can modulate brain inflammation, and could act to maintain low titers of the HIV-1 virus. Accordingly, it can be hypothesized that maintenance of serum estradiol levels could decrease the incidence of HIV dementia and other AIDS-related neurological syndromes in HIV-1 positive women. In this article, we both summarize current understanding and present new data related to the potential mechanisms whereby estrogen could modulate the mechanics and the consequences of HIV-1 infection in the brain and thereby thwart the development of HIV dementia.

AIDS Dementia Complex↗

PubMed2005

Estrogen receptor-alpha mediates gender differences in atherosclerosis induced by HIV protease inhibitors.

As part of highly active antiretroviral therapy, protease inhibitor treatment has significantly increased the lifespan of human immunodeficiency virus (HIV)-infected individuals. Many patients, however, develop negative side effects, including premature atherosclerosis. We have previously demonstrated that in male low density lipoprotein receptor (LDL-R) null mice, HIV protease inhibitors induce atherosclerotic lesions and cholesterol accumulation in macrophages in the absence of changes in plasma lipid levels. We determined that these increases were due to an up-regulation of the scavenger receptor, CD36. In the present study, we examined the effects of HIV protease inhibitors in female LDL-R null mice. Female mice given ritonavir and amprenavir (23 and 10 microg/mouse/day, respectively) developed fewer atherosclerotic lesions than males. Furthermore, peritoneal macrophages isolated from ritonavir-treated females had reduced levels of cholesterol accumulation as compared with males, and CD36 protein levels were increased to a significantly lesser degree in females than in males. To investigate the molecular mechanisms of this gender difference, we examined the effect of genetically removing estrogen receptor-alpha (ERalpha). In female mice lacking both LDL-R and ERalpha, the protective effect of gender was lost. Additionally, the reduced levels of cholesterol accumulation in macrophages observed in females was reversed. Furthermore, the absence of ERalpha resulted in increased expression of CD36 protein in a macrophage-specific manner in mice treated with ritonavir. These data demonstrate that ERalpha is directly involved in the regulation of cholesterol metabolism in macrophages and plays an important role in the gender differences observed in HIV protease inhibitor-induced atherosclerosis.

Animals↗

PubMed2005

Estrogen and brain inflammation: effects on microglial expression of MHC, costimulatory molecules and cytokines.

To model the effects of estrogen on adaptive immunity in the brain, we examined the effects of 17beta-estradiol on microglial parameters related to antigen presentation and T cell activation. Specifically, the effects of 17beta-estradiol on basal and LPS-induced surface staining of Class I and II MHC, as well as CD40, CD80, CD86, CD152, CD28, CD8, CD11b, Fas, FasL, and also ERalpha and ERbeta, were examined in N9 microglial cells. Additionally, the effects of 17beta-estradiol on basal and LPS-induced release of cytokines (TNF-alpha, IFN-gamma, IL-2, IL-4, and IL-10) were determined. Data indicate that estrogen increases IL-10 while decreasing TNFalpha and IFNgamma release from resting and LPS-stimulated N9 cells. Additionally, LPS-induced surface staining of MHC Class I, CD40, and CD86 was significantly attenuated by estrogen pretreatment. The basal percentage of cells positive for MHC Class I and II, CD40, and CD152, Fas, and FasL was significantly decreased by estrogen exposure. However, CD8, CD86, CD11b, and CD28 were unaffected by estrogen, and CD80 cell surface staining significantly increased following estrogen exposure. Taken together, these data indicate that estrogen can significantly decrease components of adaptive immunity in microglial cells, and highlight the multi-faceted regulatory effects of estrogen on microglial parameters related to antigen presentation and T cell interaction.

Animals↗

PubMed2003

HIV protease inhibitors promote atherosclerotic lesion formation independent of dyslipidemia by increasing CD36-dependent cholesteryl ester accumulation in macrophages.

Protease inhibitors decrease the viral load in HIV patients, however the patients develop hypertriglyceridemia, hypercholesterolemia, and atherosclerosis. It has been assumed that protease inhibitor-dependent increases in atherosclerosis are secondary to the dyslipidemia. Incubation of THP-1 cells or human PBMCs with protease inhibitors caused upregulation of CD36 and the accumulation of cholesteryl esters. The use of CD36-blocking antibodies, a CD36 morpholino, and monocytes isolated from CD36 null mice demonstrated that protease inhibitor-induced increases in cholesteryl esters were dependent on CD36 upregulation. These data led to the hypothesis that protease inhibitors induce foam cell formation and consequently atherosclerosis by upregulating CD36 and cholesteryl ester accumulation independent of dyslipidemia. Studies with LDL receptor null mice demonstrated that low doses of protease inhibitors induce an increase in the level of CD36 and cholesteryl ester in peritoneal macrophages and the development of atherosclerosis without altering plasma lipids. Furthermore, the lack of CD36 protected the animals from protease inhibitor-induced atherosclerosis. Finally, ritonavir increased PPAR-gamma and CD36 mRNA levels in a PKC- and PPAR-gamma-dependent manner. We conclude that protease inhibitors contribute to the formation of atherosclerosis by promoting the upregulation of CD36 and the subsequent accumulation of sterol in macrophages.

Animals↗

PubMed2002

Estradiol enhances Akt activation in cortical explant cultures following neuronal injury.

We have previously demonstrated that estradiol reduces cell death in cortical explant cultures following injury induced by metabolic inhibition in a receptor-dependent fashion. In this study, we examined whether cell death involves apoptosis and assessed the potential mediators of estradiol's actions. Cortical explant cultures were generated from postnatal day 3 rat pups. On day 7 in vitro, explants were injured by exposure to 1 mM 2-DG/2 mM KCN for 2 h to model the metabolic inhibition observed during ischemia. Explants were fixed in 4% paraformaldehyde at 2, 6, 10 and 24 h following the injury period and 18-microm thick sections were cut on a cryostat and stained with cresyl violet to assess cell death. The same sections were also labeled by TUNEL to determine whether cell death occurred by apoptosis. Other sections were used for immunohistochemistry to determine whether cells that stained positive for activated caspase 3 were also immunopositive for NeuN, a neuronal marker, or GFAP, an astrocyte marker. Protein was extracted for Western blot analysis from a separate set of explants collected at 0, 0.5, 1, 2 and 4 h following the conclusion of the injury. Estradiol treatment significantly reduced the number of cells undergoing apoptotic cell death as indicated by nuclear condensation visualized by cresyl violet staining (P<0.05). TUNEL staining revealed that the majority of pyknotic and fragmented nuclei were also TUNEL positive. Furthermore, caspase 3 activation appeared to be restricted to neurons. To examine a possible mechanism by which estradiol prevents apoptosis, we examined the level of activation of Akt kinase, which mediates antiapoptotic signals. Potential activation was measured by phosphorylation of Akt at Ser473 by Western blot analysis. In the absence of estradiol, pAkt levels were significantly increased at 2 h following the termination of injury. Explants that were pretreated with estradiol exhibited elevated levels of pAkt at 1 h following injury. Treatment with ICI 182,780 prevented the effect of estradiol. These studies suggest that estradiol prevents injury-induced apoptosis and that Akt activation may mediate these protective effects.

Alzheimer Disease↗

PubMed2002

Age differentially influences estrogen receptor-alpha (ERalpha) and estrogen receptor-beta (ERbeta) gene expression in specific regions of the rat brain.

Estradiol's ability to influence neurochemical events that are critical to female reproductive cyclicity and behavior decreases with age. We tested the hypothesis that decreases in estrogen receptor-alpha (ERalpha) and/or ERbeta mRNA explain the brain's declining responsiveness to estradiol. We assessed ERalpha and ERbeta mRNA levels in intact and ovariectomized estradiol-treated rats. ERbeta mRNA was detected in several brain regions and decreased by middle-age in the cerebral cortex and supraoptic nucleus of estradiol-treated rats. ERbeta mRNA levels exhibited a diurnal rhythm in the suprachiasmatic nucleus of young and middle-aged rats and this rhythm was blunted in old rats. We examined ERalpha mRNA in the periventricular preoptic, medial preoptic, ventromedial and arcuate nuclei, and it was decreased only in the periventricular preoptic nucleus of the old rats. In summary, the expression of ERalpha and ERbeta mRNAs is differentially modulated in the aging brain and changes are region specific.

Aging↗

PubMed2002

Neuroendocrine modulation and repercussions of female reproductive aging.

The menopause marks the end of a woman's reproductive life. During the postmenopausal period, plasma estrogen concentrations decrease dramatically and remain low for the rest of her life, unless she chooses to take hormone replacement therapy. During the past 20 years, we have learned that changes in the central nervous system are associated with and may influence the timing of the menopause in women. Recently, it has become clear that estrogens act on more than just the hypothalamus, pituitary, ovary, and other reproductive organs. In fact, they play roles in a wide variety of nonreproductive functions. With the increasing life span of humans from approximately 50 to 80 years and the relatively fixed age of the menopause, a larger number of women will spend over one third of their lives in the postmenopausal state. It is not surprising that interest has increased in factors that govern the timing of the menopause and the repercussions of the lack of estrogen on multiple aspects of women's health. We have used animal models to better understand the complex interactions between the ovary and the brain that lead to the menopause and the repercussions of the hypoestrogenic state. Our results show that when rats reach middle age, the patterns and synchrony of multiple neurochemical events that are critical to the preovulatory gonadotropin-releasing hormone (GnRH) surge undergo subtle changes. The precision of rhythmic pattern of neurotransmitter dynamics depends on the presence of estradiol. Responsiveness to this hormone decreases in middle-aged rats. The lack of precision in the coordination in the output of neural signals leads to a delay and attenuation of the luteinizing hormone surge, which lead to irregular estrous cyclicity and, ultimately, to the cessation of reproductive cycles. We also have examined the impact of the lack of estrogen on the vulnerability of the brain to injury. Our work establishes that the absence of estradiol increases the extent of cell death after stroke-like injury and that treatment with low physiological levels of estradiol are profoundly neuroprotective. We have begun to explore the cellular and molecular mechanisms that underlie this novel nonreproductive action of estrogens. In summary, our studies show that age-related changes in the ability of estradiol to coordinate the neuroendocrine events that lead to regular preovulatory GnRH surges contribute to the onset of irregular estrous cycles and eventually to acyclicity. Furthermore, we have shown that the lack of estradiol increases the vulnerability of the brain to injury and neurodegeneration.

Aging↗