Effect of IL-17 receptor A blockade with brodalumab in inflammatory diseases.

IL-17 cytokines are expressed by a variety of cells and mediate host defence against extracellular pathogens. IL-17 is upregulated at sites of inflammation and can synergize with other cytokines, such as TNF-α, to amplify the inflammatory response. Activation of these signalling pathways has been hypothesized to contribute to the underlying pathogenesis of several inflammatory diseases, including psoriasis, RA, PsA and asthma. Thus the IL-17 signalling pathway is an attractive target for the development of therapeutic agents to modulate aberrant inflammatory responses. This review of the clinical development of therapeutic agents that target IL-17 signalling pathways in inflammatory diseases focuses on brodalumab, a human anti-IL-17 receptor A mAb. The cumulative findings of early clinical studies with anti-IL-17 agents, including brodalumab, secukinumab and ixekizumab, provide strong evidence for the role of IL-17 signalling in the pathophysiology of certain inflammatory diseases and support the potential use of these agents in treating these diseases.

Mitochondrial bioenergetic deficit precedes Alzheimer's pathology in female mouse model of Alzheimer's disease.

Mitochondrial dysfunction has been proposed to play a pivotal role in neurodegenerative diseases, including Alzheimer's disease (AD). To address whether mitochondrial dysfunction precedes the development of AD pathology, we conducted mitochondrial functional analyses in female triple transgenic Alzheimer's mice (3xTg-AD) and age-matched nontransgenic (nonTg). Mitochondrial dysfunction in the 3xTg-AD brain was evidenced by decreased mitochondrial respiration and decreased pyruvate dehydrogenase (PDH) protein level and activity as early as 3 months of age. 3xTg-AD mice also exhibited increased oxidative stress as manifested by increased hydrogen peroxide production and lipid peroxidation. Mitochondrial amyloid beta (Abeta) level in the 3xTg-AD mice was significantly increased at 9 months and temporally correlated with increased level of Abeta binding to alcohol dehydrogenase (ABAD). Embryonic neurons derived from 3xTg-AD mouse hippocampus exhibited significantly decreased mitochondrial respiration and increased glycolysis. Results of these analyses indicate that compromised mitochondrial function is evident in embryonic hippocampal neurons, continues unabated in females throughout the reproductive period, and is exacerbated during reproductive senescence. In nontransgenic control mice, oxidative stress was coincident with reproductive senescence and accompanied by a significant decline in mitochondrial function. Reproductive senescence in the 3xTg-AD mouse brain markedly exacerbated mitochondrial dysfunction. Collectively, the data indicate significant mitochondrial dysfunction occurs early in AD pathogenesis in a female AD mouse model. Mitochondrial dysfunction provides a plausible mechanistic rationale for the hypometabolism in brain that precedes AD diagnosis and suggests therapeutic targets for prevention of AD.

Estradiol and neurodegenerative oxidative stress.

Estradiol is a potent preventative against neurodegenerative disease, in part, by activating antioxidant defense systems scavenging reactive oxygen species, limiting mitochondrial protein damage, improving electron transport chain activity and reducing mitochondrial DNA damage. Estradiol also increases the activity of complex IV of the electron transport chain, improving mitochondrial respiration and ATP production under normal and stressful conditions. However, the high oxidative cellular environment present during neurodegeneration makes estradiol a poor agent for treatment of existing disease. Oxidative stress stimulates the production of the hydroperoxide-dependent hydroxylation of estradiol to the catecholestrogen metabolites, which can undergo reactive oxygen species producing redox cycling, setting up a self-generating toxic cascade offsetting any antioxidant/antiapoptotic effects generated by the parent estradiol. Additional disease-induced factors can further perpetuate this cycle. For example dysregulation of the catecholamine system could alter catechol-O-methyltransferase-catalyzed methylation, preventing removal of redox cycling catecholestrogens from the system enhancing pro-oxidant effects of estradiol.

Progesterone receptors: form and function in brain.

Emerging data indicate that progesterone has multiple non-reproductive functions in the central nervous system to regulate cognition, mood, inflammation, mitochondrial function, neurogenesis and regeneration, myelination and recovery from traumatic brain injury. Progesterone-regulated neural responses are mediated by an array of progesterone receptors (PR) that include the classic nuclear PRA and PRB receptors and splice variants of each, the seven transmembrane domain 7TMPRbeta and the membrane-associated 25-Dx PR (PGRMC1). These PRs induce classic regulation of gene expression while also transducing signaling cascades that originate at the cell membrane and ultimately activate transcription factors. Remarkably, PRs are broadly expressed throughout the brain and can be detected in every neural cell type. The distribution of PRs beyond hypothalamic borders, suggests a much broader role of progesterone in regulating neural function. Despite the large body of evidence regarding progesterone regulation of reproductive behaviors and estrogen-inducible responses as well as effects of progesterone metabolite neurosteroids, much remains to be discovered regarding the functional outcomes resulting from activation of the complex array of PRs in brain by gonadally and/or glial derived progesterone. Moreover, the impact of clinically used progestogens and developing selective PR modulators for targeted outcomes in brain is a critical avenue of investigation as the non-reproductive functions of PRs have far-reaching implications for hormone therapy to maintain neurological health and function throughout menopausal aging.

Erenumab in chronic migraine with medication overuse: Subgroup analysis of a randomized trial.

OBJECTIVE: To determine the effect of erenumab, a human anti-calcitonin gene-related peptide receptor monoclonal antibody, in patients with chronic migraine and medication overuse. METHODS: In this double-blind, placebo-controlled study, 667 adults with chronic migraine were randomized (3:2:2) to placebo or erenumab (70 or 140 mg), stratified by region and medication overuse status. Data from patients with baseline medication overuse at baseline were used to assess changes in monthly migraine days, acute migraine-specific medication treatment days, and proportion of patients achieving ≥50% reduction from baseline in monthly migraine days. RESULTS: Of 667 patients randomized, 41% (n = 274) met medication overuse criteria. In the medication overuse subgroup, erenumab 70 or 140 mg groups had greater reductions than the placebo group at month 3 in monthly migraine days (mean [95% confidence interval] -6.6 [-8.0 to -5.3] and -6.6 [-8.0 to -5.3] vs -3.5 [-4.6 to -2.4]) and acute migraine-specific medication treatment days (-5.4 [-6.5 to -4.4] and -4.9 [-6.0 to -3.8] vs -2.1 [-3.0 to -1.2]). In the placebo and 70 and 140 mg groups, ≥50% reductions in monthly migraine days were achieved by 18%, 36% (odds ratio [95% confidence interval] 2.67 [1.36-5.22]) and 35% (odds ratio 2.51 [1.28-4.94]). These clinical responses paralleled improvements in patient-reported outcomes with a consistent benefit of erenumab across multiple measures of impact, disability, and health-related quality of life. The observed treatment effects were similar in the non-medication overuse subgroup. CONCLUSIONS: Erenumab reduced migraine frequency and acute migraine-specific medication treatment days in patients with chronic migraine and medication overuse, improving disability and quality of life. CLINICALTRIALSGOV IDENTIFIER: NCT02066415. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that erenumab reduces monthly migraine days at 3 months in patients with chronic migraine and medication overuse.

Erenumab in chronic migraine: Patient-reported outcomes in a randomized double-blind study.

OBJECTIVE: To determine the effect of erenumab, a human monoclonal antibody targeting the calcitonin gene-related peptide receptor, on health-related quality of life (HRQoL), headache impact, and disability in patients with chronic migraine (CM). METHODS: In this double-blind, placebo-controlled study, 667 adults with CM were randomized (3:2:2) to placebo or erenumab (70 or 140 mg monthly). Exploratory endpoints included migraine-specific HRQoL (Migraine-Specific Quality-of-Life Questionnaire [MSQ]), headache impact (Headache Impact Test-6 [HIT-6]), migraine-related disability (Migraine Disability Assessment [MIDAS] test), and pain interference (Patient-Reported Outcomes Measurement Information System [PROMIS] Pain Interference Scale short form 6b). RESULTS: Improvements were observed for all endpoints in both erenumab groups at month 3, with greater changes relative to placebo observed at month 1 for many outcomes. All 3 MSQ domains were improved from baseline with treatment differences for both doses exceeding minimally important differences established for MSQ-role function-restrictive (≥3.2) and MSQ-emotional functioning (≥7.5) and for MSQ-role function-preventive (≥4.5) for erenumab 140 mg. Changes from baseline in HIT-6 scores at month 3 were -5.6 for both doses vs -3.1 for placebo. MIDAS scores at month 3 improved by -19.4 days for 70 mg and -19.8 days for 140 mg vs -7.5 days for placebo. Individual-level minimally important difference was achieved by larger proportions of erenumab-treated participants than placebo for all MSQ domains and HIT-6. Lower proportions of erenumab-treated participants had MIDAS scores of severe (≥21) or very severe (≥41) or PROMIS scores ≥60 at month 3. CONCLUSIONS: Erenumab-treated patients with CM experienced clinically relevant improvements across a broad range of patient-reported outcomes. CLINICALTRIALSGOV IDENTIFIER: NCT02066415. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that for patients with CM, erenumab treatment improves HRQoL, headache impact, and disability.

Estradiol in vivo regulation of brain mitochondrial proteome.

We used a combined proteomic and functional biochemical approach to determine the overall impact of 17beta-estradiol (E2) on mitochondrial protein expression and function. To elucidate mitochondrial pathways activated by E2 in brain, two-dimensional (2D) gel electrophoresis was conducted to screen the mitoproteome. Ovariectomized adult female rats were treated with a single injection of E2. After 24 h of E2 exposure, mitochondria were purified from brain and 2D analysis and liquid chromatography-tandem mass spectrometry protein identification were conducted. Results of proteomic analyses indicated that of the 499 protein spots detected by image analysis, a total of 66 protein spots had a twofold or greater change in expression. Of these, 28 proteins were increased in expression after E2 treatment whereas 38 proteins were decreased in expression relative to control. E2 regulated key metabolic enzymes including pyruvate dehydrogenase, aconitase, and ATP-synthase. To confirm that E2-inducible changes in protein expression translated into functional consequences, we determined the impact of E2 on the enzymatic activity of the mitochondrial electron transport chain. In vivo, E2 treatment enhanced brain mitochondrial efficiency as evidenced by increased respiratory control ratio, elevated cytochrome-c oxidase activity and expression while simultaneously reducing free radical generation in brain. Results of these analyses provide insights into E2 mechanisms of regulating brain mitochondria, which have the potential for sustaining neurological health and prevention of neurodegenerative diseases associated with mitochondrial dysfunction such as Alzheimer's disease.

Progesterone and estrogen regulate oxidative metabolism in brain mitochondria.

The ovarian hormones progesterone and estrogen have well-established neurotrophic and neuroprotective effects supporting both reproductive function and cognitive health. More recently, it has been recognized that these steroids also regulate metabolic functions sustaining the energetic demands of this neuronal activation. Underlying this metabolic control is an interpretation of signals from diverse environmental sources integrated by receptor-mediated responses converging upon mitochondrial function. In this study, to determine the effects of progesterone (P4) and 17beta-estradiol (E2) on metabolic control via mitochondrial function, ovariectomized rats were treated with P4, E2, or E2 plus P4, and whole-brain mitochondria were isolated for functional assessment. Brain mitochondria from hormone-treated rats displayed enhanced functional efficiency and increased metabolic rates. The hormone-treated mitochondria exhibited increased respiratory function coupled to increased expression and activity of the electron transport chain complex IV (cytochrome c oxidase). This increased respiratory activity was coupled with a decreased rate of reactive oxygen leak and reduced lipid peroxidation representing a systematic enhancement of brain mitochondrial efficiency. As such, ovarian hormone replacement induces mitochondrial alterations in the central nervous system supporting efficient and balanced bioenergetics reducing oxidative stress and attenuating endogenous oxidative damage.

LDL protein nitration: implication for LDL protein unfolding.

Oxidatively- or enzymatically-modified low-density lipoprotein (LDL) is intimately involved in the initiation and progression of atherosclerosis. The in vivo modified LDL is electro-negative (LDL(-)) and consists of peroxidized lipid and unfolded apoB-100 protein. This study was aimed at establishing specific protein modifications and conformational changes in LDL(-) assessed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and circular dichroism analyses, respectively. The functional significance of these chemical modifications and structural changes were validated with binding and uptake experiments to- and by bovine aortic endothelial cells (BAEC). The plasma LDL(-) fraction showed increased nitrotyrosine and lipid peroxide content as well as a greater cysteine oxidation as compared with native- and total-LDL. LC/MS/MS analyses of LDL(-) revealed specific modifications in the apoB-100 moiety, largely involving nitration of tyrosines in the alpha-helical structures and beta(2) sheet as well as cysteine oxidation to cysteic acid in beta(1) sheet. Circular dichroism analyses showed that the alpha-helical content of LDL(-) was substantially lower ( approximately 25%) than that of native LDL ( approximately 90%); conversely, LDL(-) showed greater content of beta-sheet and random coil structure, in agreement with unfolding of the protein. These results were mimicked by treatment of LDL subfractions with peroxynitrite (ONOO(-)) or SIN-1: similar amino acid modifications as well as conformational changes (loss of alpha-helical structure and gain in beta-sheet structure) were observed. Both LDL(-) and ONOO(-)-treated LDL showed a statistically significant increase in binding and uptake to- and by BAEC compared to native LDL. We further found that most binding and uptake in control-LDL was through LDL-R with minimal oxLDL-R-dependent uptake. ONOO(-)-treated LDL was significantly bound and endocytosed by LOX-1, CD36, and SR-A with minimal contribution from LDL-R. It is suggested that lipid peroxidation and protein nitration may account for the mechanisms leading to apoB-100 protein unfolding and consequential increase in modified LDL binding and uptake to and by endothelial cells that is dependent on oxLDL scavenger receptors.

Factors impacting time to acceptance and publication for peer-reviewed publications.

OBJECTIVE: Timely publication of data is important for the medical community and provides a valuable contribution to data disclosure. The objective of this study was to identify and evaluate times to acceptance and publication for peer-reviewed manuscripts, reviews, and letters to the editor. RESEARCH DESIGN AND METHODS: Key publication metrics for published manuscripts, reviews, and letters to the editor were identified by eight Amgen publications professionals. Data for publications submitted between 1 January 2013 and 1 November 2015 were extracted from a proprietary internal publication-tracking database. Variables included department initiating the study, publication type, number of submissions per publication, and the total number of weeks from first submission to acceptance, online publication, and final publication. RESULTS: A total of 337 publications were identified, of which 300 (89%) were manuscripts. Time from submission to acceptance and publication was generally similar between clinical and real-world evidence (e.g. observational and health economics studies) publications. Median (range) time from first submission to acceptance was 23.4 (0.2-226.2) weeks. Median (range) time from first submission to online (early-release) publication was 29.7 (2.4-162.6) weeks. Median (range) time from first submission to final (print) publication was 36.2 (2.8-230.8) weeks. Time from first submission to acceptance, online publication, and final publication increased accordingly with number of submissions required for acceptance, with similar times noted between each subsequent submission. CONCLUSIONS: Analysis of a single-company publication database showed that the median time for manuscripts to be fully published after initial submission was 36.2 weeks, and time to publication increased accordingly with the number of submissions. Causes for multiple submissions and time from clinical trial completion to first submission were not assessed; these were limitations of the study. Nonetheless, publication planners should consider these results when evaluating timelines and identifying potential journals early in the publication planning process.

Efficacy and safety of the glycine transporter type-1 inhibitor AMG 747 for the treatment of negative symptoms associated with schizophrenia.

OBJECTIVE: To determine the safety and efficacy of AMG 747, an oral inhibitor of glycine transporter type-1 (GlyT1), as an add-on to antipsychotic therapy in clinically stable people with schizophrenia with enduring negative symptoms. METHOD: Analysis of pooled data from two phase 2 studies. Adults diagnosed with schizophrenia stabilized on antipsychotic medication randomized (2:2:2:3) to orally receive daily AMG 747 (5mg, 15mg, or 40mg) or placebo. Primary endpoint was Negative Symptom Assessment (NSA)-16 total score change from baseline to week 12. RESULTS: Studies were terminated early after a report of Stevens-Johnson Syndrome/Toxic Epidermal Necrolysis (SJS/TEN) in one participant (40-mg AMG 747). At termination, 232 participants had enrolled and 153 completed 12weeks of treatment. At week 12, change from baseline NSA-16 total score showed no differences between groups. Mean decrease in Positive and Negative Syndrome Scale (PANSS) Negative Symptom Factor Score (NSFS) and NSA-16 global score were greater with 15-mg AMG 747 than placebo (p<0.05). Changes in PANSS-Positive Symptom Factor Scale were not significantly different for any group. Changes in patient-reported outcomes (Sheehan Disability Scale and Quality of Life Enjoyment and Satisfaction Questionnaire) showed trends consistent with greater efficacy of 15-mg AMG 747 compared with placebo (p≤0.1). Adverse event rates were similar among all groups, with no clear differences observed. CONCLUSIONS: Significant treatment effects of 15-mg AMG 747, but not higher or lower doses, were observed on secondary endpoints but not on the primary outcome. These results replicate previous reports of an inverted-U dose response curve and suggest further evaluation of GlyT1 inhibitors in schizophrenia negative symptoms is warranted. TRIAL REGISTRATION: Clinicaltrials.govNCT01568216 (https://clinicaltrials.gov/ct2/show/NCT01568216) and NCT01568229 (https://clinicaltrials.gov/ct2/show/NCT01568229?term=NCT01568229&rank=1); EudraCT number 2011-004844-23 and 2011-004845-42.

Dose and temporal pattern of estrogen exposure determines neuroprotective outcome in hippocampal neurons: therapeutic implications.

To address controversies of estrogen therapy, in vitro models of perimenopause and prevention vs. treatment modes of 17beta-estradiol (E(2)) exposure were developed and used to assess the neuroprotective efficacy of E(2) against beta-amyloid-1-42 (Abeta(1-42))-induced neurodegeneration in rat primary hippocampal neurons. Low E(2) (10 ng/ml) exposure exerted neuroprotection in each of the perimenopausal temporal patterns, acute, continuous, and intermittent. In contrast, high E(2) (200 ng/ml) was ineffective at inducing neuroprotection regardless of temporal pattern of exposure. Although high E(2) alone was not toxic, neurons treated with high-dose E(2) resulted in greater Abeta(1-42)-induced neurodegeneration. In prevention vs. treatment simulations, E(2) was most effective when present before and during Abeta(1-42) insult. In contrast, E(2) treatment after Abeta(1-42) exposure was ineffective in reversing Abeta-induced degeneration, and exacerbated Abeta(1-42)-induced cell death when administered after Abeta(1-42) insult. We sought to determine the mechanism by which high E(2) exacerbated Abeta(1-42)-induced neurodegeneration by investigating the impact of low vs. high E(2) on Abeta(1-42)-induced dysregulation of calcium homeostasis. Results of these analyses indicated that low E(2) significantly prevented Abeta(1-42)-induced rise in intracellular calcium, whereas high E(2) significantly increased intracellular calcium and did not prevent Abeta(1-42)-induced calcium dysregulation. Therapeutic benefit resulted only from low-dose E(2) exposure before, but not after, Abeta(1-42)-induced neurodegeneration. These data are relevant to impact of perimenopausal E(2) exposure on protection against neurodegenerative insults and the use of estrogen therapy to prevent vs. treat Alzheimer's disease. Furthermore, these data are consistent with a healthy cell bias of estrogen benefit.

Estrogen protects neuronal cells from amyloid beta-induced apoptosis via regulation of mitochondrial proteins and function.

BACKGROUND: Neurodegeneration in Alzheimer's disease is associated with increased apoptosis and parallels increased levels of amyloid beta, which can induce neuronal apoptosis. Estrogen exposure prior to neurotoxic insult of hippocampal neurons promotes neuronal defence and survival against neurodegenerative insults including amyloid beta. Although all underlying molecular mechanisms of amyloid beta neurotoxicity remain undetermined, mitochondrial dysfunction, including altered calcium homeostasis and Bcl-2 expression, are involved in neurodegenerative vulnerability. RESULTS: In this study, we investigated the mechanism of 17beta-estradiol-induced prevention of amyloid beta-induced apoptosis of rat hippocampal neuronal cultures. Estradiol treatment prior to amyloid beta exposure significantly reduced the number of apoptotic neurons and the associated rise in resting intracellular calcium levels. Amyloid beta exposure provoked down regulation of a key antiapoptotic protein, Bcl-2, and resulted in mitochondrial translocation of Bax, a protein known to promote cell death, and subsequent release of cytochrome c. E2 pretreatment inhibited the amyloid beta-induced decrease in Bcl-2 expression, translocation of Bax to the mitochondria and subsequent release of cytochrome c. Further implicating the mitochondria as a target of estradiol action, in vivo estradiol treatment enhanced the respiratory function of whole brain mitochondria. In addition, estradiol pretreatment protected isolated mitochondria against calcium-induced loss of respiratory function. CONCLUSION: Therefore, we propose that estradiol pretreatment protects against amyloid beta neurotoxicity by limiting mitochondrial dysfunction via activation of antiapoptotic mechanisms.

Systematic review of reports describing potential impact of the Sunshine Act on peer-reviewed medical publications.

OBJECTIVE: The Physician Payments Sunshine Act, enacted in 2010, is intended to increase the transparency of relationships between US physicians and teaching hospitals and manufacturers of drugs, biologics, and medical devices. We examined current opinion regarding the impact of the Sunshine Act on peer-reviewed medical publications. RESEARCH DESIGN AND METHODS: We searched indexed databases (NLM/PubMed, EMBASE, and Scopus) and nonindexed sources (lay and medical press, medical websites, congress abstracts) for articles published between January 2010 and June 2015 that contained terms indicative of content related to the Sunshine Act (e.g., 'Sunshine Act', 'open payment program'). Nine publication professionals then systematically reviewed identified articles for publications-related content. MAIN OUTCOME MEASURES: Quantification and characterization of publications that focused on the Sunshine Act and its implications for medical publishing. RESULTS: Among 1200 indexed publications, 113 had content on the Sunshine Act. Thirty-one discussed its implications for publications; nine distinguished between financial and nonfinancial transfers of value. Of the 117 nonindexed publications with content on the Sunshine Act, 16 discussed implications for publications, and seven distinguished between financial and nonfinancial transfers of value. Reporting of such transfers of value was viewed as a potential barrier to participation in publications with industry support. CONCLUSIONS: There is limited literature on the impact of the Sunshine Act on peer-reviewed publications and limited physician awareness that publication support may be reported as a transfer of value.

Estradiol prevents neural tau hyperphosphorylation characteristic of Alzheimer's disease.

Alzheimer's disease (AD) is three times more prevalent in women than men, and epidemiological studies have shown that estrogen replacement in aging women forestalls the onset of AD. Hyperphosphorylation of the tau protein that forms the neurofibrillary tangles found in AD brains might be responsible for the breakdown of microtubules in affected neurons. The mechanisms by which tau protein is phosphorylated in the AD brain are not fully understood. Using a human neuroblastoma cell line (SH-SY5Y) and primary cultures of newborn male or female rat cerebral cortical neurons, we investigated the effect of 17beta-estradiol on tau protein expression and phosphorylation. We found that estradiol increased total tau and induced dephosphorylation at the proline-directed site of the molecule. Further, estradiol prevented okadaic acid-induced hyperphosphorylation of tau in both proline- and non-proline-directed sites, and antiestrogens blocked this effect. To our knowledge, this is the first report of an effect of estradiol on naturally occurring and induced tau phosphorylation. This assumes special significance because the estrogen action was found to be sexually dimorphic in rat cortical neurons and differentiation-sensitive in human neuroblastoma cells.

Impact of clinically relevant progestins on the neural effects of estradiol and the signaling pathways involved.

The natural ovarian hormone progesterone functions as an effective neuroprotective agent. However, in its native state it is not an efficient therapeutic compound because of its poor bioavailability. Thus, for practical therapeutic usage it became necessary to develop orally active progestogens for use in hormone therapy. We have shown that not all progestogens are equal in their ability to modulate neuronal survival and markers of neuronal plasticity. Thus, one cannot assume that all hormone therapies will perform the same, and it is crucial to determine the neural effects and interactions with estradiol of synthetic progestins used in place of natural progesterone. We have analyzed a number of clinically relevant progestins for neuroprotective efficacy. These in vitro analyses of neuroprotective efficacy could serve as a predictive index of clinical efficacy for progestins to protect against degenerative insults that lead to Alzheimer's disease. To aid in such therapeutic development, we determined the chemical structural features that predict progestin efficacy in the brain and showed that binding affinity does not predict neuroprotective efficacy or the direction of effect and thus cannot be used as an indicator of neurological benefit. In contrast, there was a set of common features of ligand-receptor interactions that are correlative and hopefully predictive of neuroprotective efficacy. Elucidation of the sites and targets of progestogen action should have a clear impact on both the use of hormone therapy for the prevention of neurodegenerative disease and the future design of target-specific hormone therapy formulations.

Impact of progestins on estrogen-induced neuroprotection: synergy by progesterone and 19-norprogesterone and antagonism by medroxyprogesterone acetate.

Estrogen replacement therapy is associated with improvement of cognitive deficits and reduced incidence of Alzheimer's disease. To compare the impact of therapeutically relevant progestins on estrogen-induced neuroprotection, we treated primary hippocampal neuron cultures with 17beta-E2 and progestin, alone and in combination, 48 h before glutamate insult. Estrogen, progesterone, and 19-norprogesterone, alone or in combination, protected against glutamate toxicity. In contrast, medroxyprogesterone acetate (MPA) failed to protect against glutamate toxicity. Not only was MPA an ineffective neuroprotectant but it attenuated the estrogen- induced neuroprotection when coadministered. We addressed the role of MAPK activation in neuroprotection by ovarian steroids. Estrogen and all three progestins tested, alone or in combination, activated MAPK, indicating another mechanism of protection. Bcl-2 expression has been shown to prevent cell death and is up-regulated by 17beta-E2. Progesterone and 19-norprogesterone, alone or in combination with estrogen, increased Bcl-2 expression. In contrast, MPA blocked estrogen-induced Bcl-2 expression when coadministered. These results may have important implications for the effective use of hormone replacement therapy in the maintenance of neuronal function during menopause and aging and for protection against neurodegenerative diseases such as Alzheimer's disease.

Mitochondria as therapeutic targets of estrogen action in the central nervous system.

Neuron viability and defense against neurodegenerative disease can be achieved by targeting mitochondrial function to reduce oxidative stress, increase mitochondrial defense mechanisms, or promote energetic metabolism and Ca2+ homeostasis. Exposure to estrogen prior to contact with toxic agents can protect neurons against a wide range of degenerative insults. The proactive defense state induced by estrogen is mediated by complex mechanisms ranging from chemical to biochemical to genomic but which converge upon regulation of mitochondria function. Estrogen preserves ATP levels via increased/enhanced oxidative phosphorylation and reduced ATPase activity thereby increasing mitochondrial respiration efficiency, resulting in a lower oxidative load. In addition, estrogen increases antiapoptotic proteins, Bcl-2 and Bcl-xL, which prevents activation of the permeability transition pore protecting against estrogen-induced increase in mitochondrial Ca2+ sequestration. These effects are likely to be enhanced by antioxidant effects of estrogen, preventing the initiation of the deleterious "mitochondrial spiral". The extent to which each of these mechanisms contribute to the overall proactive defense state induced by estrogen remains to be determined. However, each aspect of the cascade appears to make a significant if not obligatory impact on the neuroprotective effects of estrogens. Moreover each component of the cascade is required for estrogen regulation of mitochondrial function. Mechanisms of estrogen action and results of the clinical efficacy of estrogen therapy for prevention or treatment of Alzheimer's disease are considered in the context of clinical use of estrogen therapy and the design of brain selective estrogens or NeuroSERMs.

Impact of progestins on estradiol potentiation of the glutamate calcium response.

One mechanism by which estrogen may modulate cognitive function is through potentiation of glutamate-mediated rises in intracellular calcium ([Ca2+]i) with resultant effects on neuronal morphology and signaling. Since progesterone is a component of hormone replacement therapy (HRT), we sought to determine whether therapeutically relevant progestins attenuated or blocked estrogen potentiation of glutamate-induced [Ca2+]i rises. 17beta-estradiol and progesterone, alone or in combination, significantly potentiated the rise in [Ca2+]i. When co-administered, progesterone attenuated the estrogen response to the level seen with progesterone alone. In contrast, medroxyprogesterone acetate (MPA) had no effect when administered alone and completely blocked the 17beta-estradiol-induced potentiation when co-administered. These results may have important implications for effective use of HRT to maintain cognitive function during menopause and aging.

Dual action of estrogen on glutamate-induced calcium signaling: mechanisms requiring interaction between estrogen receptors and src/mitogen activated protein kinase pathway.

Conjugated equine estrogens (CEE) is the most widely prescribed pharmaceutical estrogen replacement therapy (ERT) for postmenopausal women in the United States and is the ERT of the Women's Health Initiative. Previous studies from our laboratory have demonstrated that CEE exerts neurotrophic and neuroprotective effects in neurons involved in learning and memory, and which are affected in Alzheimer's disease. The present work demonstrates that CEE potentiated the rise in intracellular calcium ([Ca(2+)](i)) following exposure to physiological concentrations of glutamate. In contrast, the reverse effect occurred in the presence of excitotoxic levels of glutamate exposure, where CEE attenuated the rise in [Ca(2+)](i). Potentiation of the glutamate response was mediated by the NMDA receptor, as the NMDA receptor antagonist MK-801 blocked the CEE-induced potentiation, whereas the L-type calcium channel blocker nifedipine did not. Further, the CEE-potentiated glutamate response was mediated by a src tyrosine kinase, as the tyrosine kinase inhibitor PP2 blocked the potentiation induced by CEE and neurons treated with CEE displayed increased phosphorylated tyrosine. The inhibition by CEE of [Ca(2+)](i) rise in the presence of excitotoxic levels of glutamate was mediated by mitogen activated protein kinase (MAPK), as the protective effect of CEE was blocked by inhibiting MAPK activation with PD98059. These data provide potential mechanisms to explain the cognitive enhancing and neuroprotective effects exerted by ERT.