Protection against APOE4 -associated aging phenotypes with the longevity-promoting intervention 17α-estradiol in male mice.

The apolipoprotein ε4 allele ( APOE4 ) is associated with decreased longevity, increased vulnerability to age-related declines, and disorders across multiple systems. Interventions that promote healthspan and lifespan represent a promising strategy to attenuate the development of APOE4 -associated aging phenotypes. Here we studied the ability of the longevity-promoting intervention 17α-estradiol (17αE2) to protect against age-related impairments in APOE4 versus the predominant APOE3 genotype using early middle-aged mice with knock-in of human APOE alleles. Beginning at age 10 months, male APOE3 or APOE4 mice were treated for 20 weeks with 17αE2 or vehicle then compared for indices of aging phenotypes body-wide. Across peripheral and neural measures, APOE4 was associated with poorer outcomes. Notably, 17αE2 treatment improved outcomes in a genotype-dependent manner favoring APOE4 mice. These data demonstrate a positive APOE4 bias in 17αE2-mediated healthspan actions, suggesting that longevity-promoting interventions may be useful in mitigating deleterious age-related risks associated with APOE4 genotype.

Effects of APOE Genotype and Western Diet on Metabolic Phenotypes in Female Mice.

Western diets high in sugars and saturated fats have been reported to induce metabolic and inflammatory impairments that are associated with several age-related disorders, including Alzheimer's disease (AD) and type 2 diabetes (T2D). The apolipoprotein E (APOE) genotype is associated with metabolic and inflammatory outcomes that contribute to risks for AD and T2D, with the APOE4 genotype increasing risks relative to the more common APOE3 allele. In this study, we investigated the impacts of the APOE genotype on systemic and neural effects of the Western diet. Female mice with knock-in of human APOE3 or APOE4 were exposed to control or Western diet for 13 weeks. In the control diet, we observed that APOE4 mice presented with impaired metabolic phenotypes, exhibiting greater adiposity, higher plasma leptin and insulin levels, and poorer glucose clearance than APOE3 mice. Behaviorally, APOE4 mice exhibited worse performance in a hippocampal-dependent learning task. In visceral adipose tissue, APOE4 mice exhibited generally higher expression levels of macrophage- and inflammation-related genes. The cerebral cortex showed a similar pattern, with higher expression of macrophage- and inflammation-related genes in APOE4 than APOE3 mice. Exposure to the Western diet yielded modest, statistically non-significant effects on most metabolic, behavioral, and gene expression measures in both APOE genotypes. Interestingly, the Western diet resulted in reduced gene expression of a few macrophage markers, specifically in APOE4 mice. The observed relative resistance to the Western diet suggests protective roles of both female sex and young adult age. Further, the data demonstrate that APOE4 is associated with deleterious systemic and neural phenotypes and an altered response to a metabolic stressor, findings relevant to the understanding of interactions between the APOE genotype and risks for metabolic disorders.

Role of estrogen in women's Alzheimer's disease risk as modified by APOE.

Alzheimer's disease (AD) is characterized by numerous sexual dimorphisms that impact the development, progression, and probably the strategies to prevent and treat the most common form of dementia. In this review, we consider this topic from a female perspective with a specific focus on how women's vulnerability to the disease is affected by the individual and interactive effects of estrogens and apolipoprotein E (APOE) genotype. Importantly, APOE appears to modulate systemic and neural outcomes of both menopause and estrogen-based hormone therapy. In the brain, dementia risk is greater in APOE4 carriers, and the impacts of hormone therapy on cognitive decline and dementia risk vary according to both outcome measure and APOE genotype. Beyond the CNS, estrogen and APOE genotype affect vulnerability to menopause-associated bone loss, dyslipidemia and cardiovascular disease risk. An emerging concept that may link these relationships is the possibility that the effects of APOE in women interact with estrogen status by mechanisms that may include modulation of estrogen responsiveness. This review highlights the need to consider the key AD risk factors of advancing age in a sex-specific manner to optimize development of therapeutic approaches for AD, a view aligned with the principle of personalized medicine.

A Qualitative Assessment of Patient Satisfaction with Radical Cystectomy for Bladder Cancer at a Single Institution: How Can We Improve?

PURPOSE: To evaluate patient satisfaction (with emphasis on preoperative education) with radical cystectomy for bladder cancer at our institution, the University of Missouri Hospital, qualitatively in order to identify specific areas where improvements can be made. MATERIALS AND METHODS: We developed a patient survey that used open-ended questions to identify positive and negative experiences that contributed to patient satisfaction. We administered the survey to radical cystectomy patients who met inclusion criteria and agreed to participate. We recorded, transcribed and qualitatively coded the responses. We identified four themes under which both positive and negative responses were placed, and constructed two diagrams to better illustrate contributors to patient experience and satisfaction. RESULTS: We identified 25 patients who met inclusion criteria. Of those, 13 participated in the survey. Regarding overall experience, 92.3% of patients rated their care as excellent or good. Regarding preoperative education, 76.9% of patients reported they definitely or somewhat received enough information on what to expect after surgery, and 76.9% definitely received enough guidance on how to care for themselves after surgery. From qualitative coding of patient responses to open-ended questions, we identified preoperative preparation, delivery of care, caregiver availability, and patient-centered care as themes that contributed positively and negatively to patient experience. CONCLUSION: Although the overall patient satisfaction could be perceived as high (92.3%), qualitative analysis revealed several areas where improvements can be made to improve patient experience with radical cystectomy at our institution. As previously expected, preoperative preparation was a contributor.

Aging Reduces Estradiol Protection Against Neural but Not Metabolic Effects of Obesity in Female 3xTg-AD Mice.

Vulnerability to Alzheimer's disease (AD) is increased by several risk factors, including midlife obesity, female sex, and the depletion of estrogens in women as a consequence of menopause. Conversely, estrogen-based hormone therapies have been linked with protection from age-related increases in adiposity and dementia risk, although treatment efficacy appears to be affected by the age of initiation. Potential interactions between obesity, AD, aging, and estrogen treatment are likely to have significant impact on optimizing the use of hormone therapies in postmenopausal women. In the current study, we compared how treatment with the primary estrogen, 17ß-estradiol (E2), affects levels of AD-like neuropathology, behavioral impairment, and other neural and systemic effects of preexisting diet-induced obesity in female 3xTg-AD mice. Importantly, experiments were conducted at chronological ages associated with both the early and late stages of reproductive senescence. We observed that E2 treatment was generally associated with significantly improved metabolic outcomes, including reductions in body weight, adiposity, and leptin, across both age groups. Conversely, neural benefits of E2 in obese mice, including decreased ß-amyloid burden, improved behavioral performance, and reduced microglial activation, were observed only in the early aging group. These results are consistent with the perspective that neural benefits of estrogen-based therapies require initiation of treatment during early rather than later phases of reproductive aging. Further, the discordance between E2 protection against systemic versus neural effects of obesity across age groups suggests that pathways other than general metabolic function, perhaps including reduced microglial activation, contribute to the mechanism(s) of the observed E2 actions. These findings reinforce the potential systemic and neural benefits of estrogen therapies against obesity, while also highlighting the critical role of aging as a mediator of estrogens' protective actions.

Staining and Quantification of ß-Amyloid Pathology in Transgenic Mouse Models of Alzheimer's Disease.

Studies of Alzheimer's disease (AD) using experimental systems most often involve transgenic mouse models that are characterized by neural accumulation of ß-amyloid protein (Aß), which is widely hypothesized to have a key role in AD pathogenesis. Quantification of Aß in transgenic mice typically is accomplished through both biochemical and histochemical approaches. In this chapter, we describe two techniques for the histological detection of Aß, immunostaining with Aß antibodies and staining with the amyloid dye thioflavin S, and its quantification using digital imaging.

Effects of aging, high-fat diet, and testosterone treatment on neural and metabolic outcomes in male brown Norway rats.

Risk for Alzheimer's disease (AD) is affected by multiple factors, including aging, obesity, and low testosterone. We previously showed that obesity and low testosterone independently and interactively exacerbate AD-related outcomes in young adult rodents. The goals of the present study are two-fold: to examine whether the effects of an obesogenic diet differ with increasing age and to determine if testosterone treatment in middle-aged and aged animals mitigates negative effects of the diet. Male brown Norway rats were maintained on control or high-fat diets for 12 weeks beginning in young adulthood, middle age, or advanced age. Separate cohorts of middle-aged and aged animals were treated with testosterone during dietary manipulations. Endpoints included metabolic indices, inflammation, cognitive performance, and neural health outcomes. Aging was associated with poorer outcomes that were generally exacerbated by high-fat diet, especially at middle age. Testosterone treatment was largely without benefit, exerting only subtle effects on a select number of measures. Understanding how the deleterious effects of obesity are affected by advancing age and the ability of protective strategies such as testosterone to reduce these effects may provide significant insight into both the development and prevention of age-related cognitive decline and AD.

APOE genotype affects metabolic and Alzheimer-related outcomes induced by Western diet in female EFAD mice.

Development of Alzheimer's disease (AD) is regulated by interactive effects of genetic and environmental risk factors. The most significant genetic risk factor for AD is the ε4 allele of apolipoprotein E ( APOE4), which has been shown to exert greater AD risk in women. An important modifiable AD risk factor is obesity and its associated metabolic dysfunctions. Whether APOE genotype might interact with obesity in females to regulate AD pathogenesis is unclear. To investigate this issue, we studied the effects of Western diet (WD) on female EFAD mice, a transgenic mouse model of AD that includes human APOE alleles ε3 (E3FAD) and ε4 (E4FAD). EFAD mice were fed either control (10% fat, 7% sugar) or WD (45% fat, 17% sugar), and both metabolic and neuropathologic outcomes were determined. Although E4FAD mice generally exhibited poorer metabolic status at baseline, E3FAD mice showed greater diet-induced metabolic impairments. Similarly, E4FAD mice exhibited higher levels of AD-related pathology overall, but only E3FAD showed significant increases on select measures of ß-amyloid pathology after exposure to WD. These data demonstrate a gene-environment interaction between APOE and obesogenic diets in females. Understanding how AD-promoting effects of obesity are modulated by genetic factors will foster the identification of at-risk populations and development of preventive interventions.-Christensen, A., Pike, C. J. APOE genotype affects metabolic and Alzheimer-related outcomes induced by Western diet in female EFAD mice.

Humanin Prevents Age-Related Cognitive Decline in Mice and is Associated with Improved Cognitive Age in Humans.

Advanced age is associated with a decline in cognitive function, likely caused by a combination of modifiable and non-modifiable factors such as genetics and lifestyle choices. Mounting evidence suggests that humanin and other mitochondrial derived peptides play a role in several age-related conditions including neurodegenerative disease. Here we demonstrate that humanin administration has neuroprotective effects in vitro in human cell culture models and is sufficient to improve cognition in vivo in aged mice. Furthermore, in a human cohort, using mitochondrial GWAS, we identified a specific SNP (rs2854128) in the humanin-coding region of the mitochondrial genome that is associated with a decrease in circulating humanin levels. In a large, independent cohort, consisting of a nationally-representative sample of older adults, we find that this SNP is associated with accelerated cognitive aging, supporting the concept that humanin is an important factor in cognitive aging.

TSPO ligand PK11195 improves Alzheimer-related outcomes in aged female 3xTg-AD mice.

Alzheimer's disease (AD) pathogenesis is a multifactorial process that involves numerous pathways within the central nervous system. Thus, interventions that interact with several disease-related pathways may offer an increased opportunity for successful prevention and treatment of AD. Translocator protein 18 kD (TSPO) is a mitochondrial protein that is associated with regulation of many cellular processes including inflammation, steroid synthesis, apoptosis, and mitochondrial respiration. Although TSPO ligands have been shown to be protective in several neurodegenerative paradigms, little work has been done to assess their potential as treatments for AD. Female 3xTg-AD mice were administered the TSPO ligand PK11195 once weekly for 5 weeks beginning at 16 months, an age characterized by extensive ß-amyloid pathology and behavioral impairments. Animals treated with PK11195 showed improvements in behavior and modest reductions of in both soluble and deposited ß-amyloid. The finding that short-term PK11195 treatment was effective in improving both behavioral and pathological outcomes in a model of late-stage AD supports further investigation of TSPO ligands as potential therapeutics for the treatment of AD.

Age-dependent regulation of obesity and Alzheimer-related outcomes by hormone therapy in female 3xTg-AD mice.

Depletion of ovarian hormones at menopause is associated with increased Alzheimer's disease (AD) risk. Hormone loss also increases central adiposity, which promotes AD development. One strategy to improve health outcomes in postmenopausal women is estrogen-based hormone therapy (HT), though its efficacy is controversial. The window of opportunity hypothesis posits that HT is beneficial only if initiated near the onset of menopause. Here, we tested this hypothesis by assessing the efficacy of HT against diet-induced obesity and AD-related pathology in female 3xTg-AD mice at early versus late middle-age. HT protected against obesity and reduced ß-amyloid burden only at early middle-age. One mechanism that contributes to AD pathogenesis is microglial activation, which is increased by obesity and reduced by estrogens. In parallel to its effects on ß-amyloid accumulation, we observed that HT reduced morphological evidence of microglial activation in early but not late middle-age. These findings suggest that HT may be effective during human perimenopause in reducing indices of obesity and AD-related pathology, a conclusion consistent with the window of opportunity hypothesis.

Studies of the Effectiveness of Bisphosphonate and Vanadium-Bisphosphonate Compounds In Vitro against Axenic Leishmania tarentolae.

Leishmaniasis is a disease that is a significant problem for people, especially in tropical regions of the world. Current drug therapies to treat the disease are expensive, not very effective, and/or of significant side effects. A series of alkyl bisphosphonate compounds and one amino bisphosphonate compound, as well as alendronate and zoledronate, were tested as potential agents against Leishmania tarentolae. Also, two polyoxometalates (POMs) with nitrogen-containing bisphosphonate ligands, vanadium/alendronate (V5(Ale)2) and vanadium/zoledronate (V3(Zol)3), were tested against L. tarentolae and compared to the results of the alendronate and zoledronate ligands alone. Of the compounds evaluated in this study, the V5(Ale)2 and V3(Zol)3 complexes were most effective in inhibiting the growth of L. tarentolae. The V5(Ale)2 complex had a larger impact on cell growth than either alendronate or orthovanadate alone, whereas zoledronate itself has a significant effect on cell growth, which may contribute to the activity of the V3(Zol)3 complex.

The APOE4 allele shows opposite sex bias in microbleeds and Alzheimer's disease of humans and mice.

The apolipoprotein APOE4 allele confers greater risk of Alzheimer's disease (AD) for women than men, in conjunction with greater clinical deficits per unit of AD neuropathology (plaques, tangles). Cerebral microbleeds, which contribute to cognitive dysfunctions during AD, also show APOE4 excess, but sex-APOE allele interactions are not described. We report that elderly men diagnosed for mild cognitive impairment and AD showed a higher risk of cerebral cortex microbleeds with APOE4 allele dose effect in 2 clinical cohorts (ADNI and KIDS). Sex-APOE interactions were further analyzed in EFAD mice carrying human APOE alleles and familial AD genes (5XFAD (+/-) /human APOE(+/+)). At 7 months, E4FAD mice had cerebral cortex microbleeds with female excess, in contrast to humans. Cerebral amyloid angiopathy, plaques, and soluble Aß also showed female excess. Both the cerebral microbleeds and cerebral amyloid angiopathy increased in proportion to individual Aß load. In humans, the opposite sex bias of APOE4 allele for microbleeds versus the plaques and tangles is the first example of organ-specific, sex-linked APOE allele effects, and further shows AD as a uniquely human condition.

Menopause, obesity and inflammation: interactive risk factors for Alzheimer's disease.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder, the development of which is regulated by several environmental and genetic risk factors. Two factors theorized to contribute to the initiation and/or progression of AD pathogenesis are age-related increases in inflammation and obesity. These factors may be particularly problematic in women. The onset of menopause in mid-life elevates the vulnerability of women to AD, an increased risk that is likely associated with the depletion of estrogens. Menopause is also linked with an abundance of additional changes, including increased central adiposity and inflammation. Here, we review the current literature to explore the interactions between obesity, inflammation, menopause and AD.

Immediate early gene activity-regulated cytoskeletal-associated protein regulates estradiol-induced lordosis behavior in female rats.

Sensory feedback is an important component of any behavior, with each instance influencing subsequent activity. Female sexual receptivity is mediated both by the steroid hormone milieu and interaction with the male. We tested the influence of repeated mating on the level of sexual receptivity in ovariectomized rats treated with estradiol benzoate (EB) once every fourth day to mimic the normal phasic changes of circulating estradiol. Females were divided into two groups: naïve, which were tested for lordosis behavior once, and experienced rats, which were tested for lordosis after each EB injection. To monitor the effect of mating, the number of neurons expressing the immediate early gene activity-regulated cytoskeleton-associated protein (Arc) were counted in the mediobasal hypothalamus. Females were unreceptive following the first EB treatment, but the mating induced Arc expression. In naïve rats, each subsequent EB injection increased the levels of sexual receptivity. This ramping was not observed in experienced rats, which achieved only a moderate level of sexual receptivity. However, experienced females treated with EB and progesterone were maximally receptive and did not have Arc expression. To test whether the expression of Arc attenuated lordosis, Arc antisense oligodeoxynucleotides (asODN) were microinjected into experienced females' arcuate nuclei. Arc expression was attenuated, and the experienced EB-treated females achieved maximal sexual receptivity. These results demonstrate that Arc expression in the hypothalamus might influence future sexual receptivity and provides evidence of learning in the arcuate nucleus. The loss of Arc results in unrestrained sexual receptivity.

Selective androgen receptor modulator RAD140 is neuroprotective in cultured neurons and kainate-lesioned male rats.

The decline in testosterone levels in men during normal aging increases risks of dysfunction and disease in androgen-responsive tissues, including brain. The use of testosterone therapy has the potential to increase the risks for developing prostate cancer and or accelerating its progression. To overcome this limitation, novel compounds termed "selective androgen receptor modulators" (SARMs) have been developed that lack significant androgen action in prostate but exert agonist effects in select androgen-responsive tissues. The efficacy of SARMs in brain is largely unknown. In this study, we investigate the SARM RAD140 in cultured rat neurons and male rat brain for its ability to provide neuroprotection, an important neural action of endogenous androgens that is relevant to neural health and resilience to neurodegenerative diseases. In cultured hippocampal neurons, RAD140 was as effective as testosterone in reducing cell death induced by apoptotic insults. Mechanistically, RAD140 neuroprotection was dependent upon MAPK signaling, as evidenced by elevation of ERK phosphorylation and inhibition of protection by the MAPK kinase inhibitor U0126. Importantly, RAD140 was also neuroprotective in vivo using the rat kainate lesion model. In experiments with gonadectomized, adult male rats, RAD140 was shown to exhibit peripheral tissue-specific androgen action that largely spared prostate, neural efficacy as demonstrated by activation of androgenic gene regulation effects, and neuroprotection of hippocampal neurons against cell death caused by systemic administration of the excitotoxin kainate. These novel findings demonstrate initial preclinical efficacy of a SARM in neuroprotective actions relevant to Alzheimer's disease and related neurodegenerative diseases.

Modulation of the arcuate nucleus-medial preoptic nucleus lordosis regulating circuit: a role for GABAB receptors.

Estradiol rapidly activates a microcircuit in the arcuate nucleus of the hypothalamus (ARH) that is needed for maximal female sexual receptivity. Membrane estrogen receptor-α complexes with and signals through the metabotropic glutamate receptor-1a stimulating NPY release within the ARH activating proopiomelanocortin (POMC) neurons. These POMC neurons project to the medial preoptic nucleus (MPN) and release ß-endorphin. Estradiol treatment induces activation/internalization of MPN µ-opioid receptors (MOR) to inhibit lordosis. Estradiol membrane action modulates ARH gamma-aminobutyric acid receptor-B (GABAB) activity. We tested the hypothesis that ARH GABAB receptors mediate estradiol-induced MOR activation and facilitation of sexual receptivity. Double-label immunohistochemistry revealed expression of GABAB receptors in NPY, ERα and POMC expressing ARH neurons. Approximately 70% of POMC neurons expressed GABAB receptors. Because estradiol initially activates an inhibitory circuit and maintains activation of this circuit, the effects of blocking GABAB receptors were evaluated before estradiol benzoate (EB) treatment and after at the time of lordosis testing. Bilateral infusions of the GABAB receptor antagonist, CGP52432, into the ARH prior to EB treatment of ovariectomized rats prevented estradiol-induced activation/internalization of MPN MOR, and the rats remained unreceptive. However, in EB-treated rats, bilateral CGP52432 infusions 30 min before behavior testing attenuated MOR internalization and facilitated lordosis. These results indicated that GABAB receptors were located within the lordosis-regulating ARH microcircuit and are necessary for activation and maintenance of the estradiol inhibition of lordosis behavior. Although GABAB receptors positively influence estradiol signaling, they negatively regulate lordosis behavior since GABAB activity maintains the estradiol-induced inhibition.

A Novel Membrane Estrogen Receptor Activated by STX Induces Female Sexual Receptivity through an Interaction with mGluR1a.

Membrane initiated estradiol signaling has been shown to be vital for multiple physiological processes. Several receptors have been proposed to mediate the actions of estradiol at the membrane. Here, we examined the ability of STX, an agonist of a novel putative membrane estrogen receptor, to activate sexually receptive behavior in the female rat. Infusions of STX into the arcuate nucleus of the hypothalamus resulted in the activation and internalization of µ-opioid receptors in the medial preoptic nucleus, an action that is required for lordosis behavior. Indeed, STX was able to augment sexual receptivity in female rats given a sub-behavioral dose of estradiol. However, if the mGluR1a antagonist, LY367,385, was administered prior to STX, its circuit-activating effects, the internalization of µ-opioid receptors, were lost. This suggests that the receptor stimulated by STX activates rapid membrane-initiated signaling through an interaction with mGluR1a - an effect previously described for estrogen receptor-α at the membrane.

Membrane-initiated estradiol actions mediate structural plasticity and reproduction.

Over the years, our ideas about estrogen signaling have greatly expanded. In addition to estradiol having direct nuclear actions that mediate transcription and translation, more recent experiments have demonstrated membrane-initiated signaling. Both direct nuclear and estradiol membrane signaling can be mediated by the classical estrogen receptors, ERα and ERß, which are two of the numerous putative membrane estrogen receptors. Thus far, however, only ERα has been shown to play a prominent role in regulating female reproduction and sexual behavior. Because ERα is a ligand-gated transcription factor and not a typical membrane receptor, trafficking to the cell membrane requires post-translational modifications. Two necessary modifications are palmitoylation and association with caveolins, a family of scaffolding proteins. In addition to their role in trafficking, caveolin proteins also serve to determine ERα interactions with metabotropic glutamate receptors (mGluRs). It is through these complexes that ERα, which cannot by itself activate G proteins, is able to initiate intracellular signaling. Various combinations of ERα-mGluR interactions have been demonstrated throughout the nervous system from hippocampus to striatum to hypothalamus to dorsal root ganglion (DRG) in both neurons and astrocytes. These combinations of ER and mGluR allow estradiol to have both facilitative and inhibitory actions in neurons. In hypothalamic astrocytes, the estradiol-mediated release of intracellular calcium stores regulating neurosteroid synthesis requires ERα-mGluR1a interaction. In terms of estradiol regulation of female sexual receptivity, activation of ERα-mGluR1a signaling complex leads to the release of neurotransmitters and alteration of neuronal morphology. This review will examine estradiol membrane signaling (EMS) activating a limbic-hypothalamic lordosis regulating circuit, which involves ERα trafficking, internalization, and modifications of neuronal morphology in a circuit that underlies female sexual receptivity.