Reduced availability of high energy-content prey (nutritional stress) is a predominant hypothesis to explain the decline of Steller sea lion (Eumetopias jubatus) populations in western Alaska from the late 1970'€™s to the late 1990'™s. Animals may physiologically respond to consuming insufficient prey by increasing stress levels and decreasing metabolic rates. It may thus be possible to identify nutritional stress by measuring concentrations of glucocorticoids (stress) and thyroid hormones (metabolism) shed in feces. However, techniques to measure thyroid hormone concentrations from Steller sea lion feces have not been developed. We quantified variation of triiodothyronine (T3) and thyroxine (T4) concentrations in Steller sea lion feces following injections of thyrotropin (TSH) into four captive animals. Glucocorticoids (GC) were also assayed to examine any relationship to stimulated thyroid hormone secretion. We found that fecal T3 peaked 48 h post-injection and increased 25-57% in three sea lions (all animals, p=0.03). Pre-injection GC increases indicated stress from isolation for baseline fecal collections, but post-injection increases could not be confirmed as a response to TSH injections or as a product of the study design. The results demonstrated that pre- and post-injection changes in fecal GC and T3 concentrations were consistent with predictions of an increased stress response and metabolic rate within the animals. We then measured T3 and GC concentrations in 834 Steller sea lion fecal samples collected in 2005 and 2006 from 15 sites (haulouts and rookeries) between British Columbia and the Central Aleutian Islands. Overall, GC concentrations did not differ between haulout populations (western 2006 pre-pupping and eastern 2005 post-pupping). Fecal hard-part analyses revealed a lower energy-content diet in the western population, suggesting that diet quality is a relevant hypothesis to explain slightly higher GC concentrations found in the western population, specifically the Aleutian Islands region. However, nutritional stress could not be substantiated through T3 concentrations. The rookeries possessed the highest energy-content diets, but also exhibited a nutritional stress response with a significantly higher GC and lower T3 concentration than either haulout population (possibly related to lactation or decreased foraging opportunities), but T3 comparisons performed at scales of site and region were inconclusive.

Stress has long lasting effects on physiology, development, behavior, reproductive success and survival. These effects are mediated by glucocorticoids, such as corticosterone (Cort), via glucocorticoid receptors (GR), though the exact mechanisms underlying these effects are unknown. Early developmental stress affects the size of the avian song control nuclei (particularly HVC; proper name) and song quality in many songbirds, suggesting a direct link between brain and behavior. HVC is required for song learning and production. The complexity of the male zebra finch (Taeniopygia guttata) courtship song is important in female mate choice. Although the mechanisms behind the effects of developmental stress on song nuclei size and song quality are unknown, it is likely that elevated levels of Cort via GR within brain song nuclei play a significant role. We investigated the distribution, quantity, and subcellular-localization of GR- immunoreactive (GR-ir) neurons in the brains of male zebra finches 10 days post-hatch and in adulthood using immunohistochemistry. There was wide distribution of GR-ir neurons including two song nuclei HVC and robust nucleus of the arcopallium (RA). Distribution did not vary between the two ages but there were significant differences in the overall number of GR-ir neurons and their subcellular localization. We hypothesized that early Cort treatment would reduce song quality and HVC size in adult males. We inserted Cort implants in males at four days post-hatch and quantified the effects of early Cort treatment on adult song quality. Early Cort treatment decreased song similarity between the tutor and tutee’s songs and resulted in poorer copies of tutor song, but did not alter mean amplitude or song duration. Early Cort treatment reduced the HVC size in both juvenile and adult birds. This result suggests that the effect of developmental stress on the HVC size may be mediated through Cort via activation of GR within HVC as a mechanism by which HVC size and song quality are altered in developmentally stressed birds. These results suggest a potential role for Cort in mediating adverse effects of developmental stress in adult male zebra finches and highlight the developmental plasticity of the zebra finch brain.

The elderly are at increased risk for developing psychiatric diseases, which include Alzheimer's disease, depression, anxiety and suicide. The probability of multiple disease comorbidity is also increased in the elderly. At the cellular level, the loss of protein homeostasis is often at the root of disease emergence, and thus the scientific community is searching for ways to help maintain this balance. A vast group of proteins that are paramount to balancing and counterbalancing protein levels is the molecular chaperone protein group, which has evolved a tremendous variety of functions in the cell. They aid in protein trafficking, folding, receptor signaling, neurotransmission, vesicle forming and fusion, protein degradation, and apoptosis, among other activities. Despite their best efforts, disease still ensues, but because of their vast number and multiple abilities, it may be possible to modulate these proteins as a way to treat and prevent disease. Chaperones are of particular interest in diseases of aging, because chaperone induction and effectiveness is reduced with age. In addition, many diseases of the elderly are brought on by aberrant protein accumulation, like Alzheimer's disease. As a result, the hypothesis of this dissertation is whether the modulation of molecular chaperones changes disease pathology. A molecular chaperone family that is important to protein degradation is the Hsp70 chaperone complex. Hsp70 proteins have specialized function depending on cell type and cellular compartment, but Hsp70 proteins are very important for protein synthesis and degradation. As a result, they are in a position to contribute to the regulation of proteins that become aberrant. In recent years scientific literature has indicated that compounds that inhibit the enzymatic ATP hydrolysis of these proteins promote tau degradation, which accumulates in Alzheimer's disease. Alzheimer's disease is the sixth leading cause of death in the U.S., it is a progressive neurodegenerative disease, and is caused by the aberrant accumulation of the amyloid beta and tau proteins. Here, we show that treatment with the Hsp70 inhibitor methylene blue, reduces tau, saves neurons, and restores cognition, in a mouse model of tau accumulation (rTg4510). Cognitive rescue occurred despite a severe tangle load, equal to control treated tau transgenic mice. This study shows that reducing soluble tau can restore cognition, reducing tangles is not necessarily to ameliorate cognition, and saving neurons is not sufficient to increase cognition if they are burdened with soluble tau. This work shows that methylene blue does not affect the the number of tau tangles in this model, as suggested by in vitro data. It also suggests that further work into the development of Hsp70 ATPase inhibitors may find success in alleviating the soluble tau burden found in Alzheimer's disease. The co-chaperone FKBP5 is also of extreme importance, not because it is essential, but because research has implicated this protein with a host of psychiatric diseases. Single nucleotide polymorphisms in this gene, which increase the levels of FKBP5, interact with averse traumatic events to enhance the likelihood of developing mood and anxiety disorders, including major depressive disorder, post-traumatic stress disorder, bipolar disorder, and suicide. Moreover, we have found that FKBP5 protein levels increase with age in the human brain, increasing the risk for the elderly of developing disease if exposed to traumatic stress. Here, we tested the hypothesis that FKBP5 negatively regulates resilient behavior. We found that FKBP5 levels increase with age in the wild type mouse brain, and that wild type mice display reduced resiliency with age. FKBP5-/- mice, on the other hand, show enhanced resiliency to stress at all ages tested, and are protected from aging-induced despair. At the molecular level, FKBP5 is a robust inhibitor of the glucocorticoid receptor, which is responsible for the shut-off of the hypothalamic-pituitary-adrenal axis. In addition, excess glucocorticoid levels in the blood is a robust marker of psychiatric disease. Consequently, FKBP5 may be causing disease through enhanced levels of glucocorticoids. FKBP5-/- mice display reduced corticosterone after stress. Moreover, corticosterone production increases with age, and FKBP5-/- mice are protected from this increase. These studies are the first to show that reducing the levels of FKBP5 is a promising therapeutic option for the treatment of mood disorders in the elderly, resiliency naturally declines with age due to FKBP5, corticosterone levels after stress rise due to FKBP5, and that the ablation of this gene increases resiliency and prevents aging- induced despair. As a whole, these data show that the modulation of chaperone proteins has the potential for developing new therapies for the treatment of psychiatric diseases of the elderly.

Glucocorticoids (GCs) are endogenous steroid hormones that regulate a number of critical physiological processes. Psychological stress increases the level of GCs in the circulating system. The biological effect of elevated GCs on the heart is not well understood. We found that GCs induced Cyclooxygenase-1 (COX-1) and COX-2 gene expression in cardiomyocytes. COX-1 or COX-2 encodes the rate-limiting enzyme in the biosynthesis of prostanoids, which modulate crucial physiological and pathophysiological responses. The present studies aim to elucidate the signaling transduction pathway and the mechanism underlying GC induced COX expression.Our data demonstrate that GCs activate COX-1 gene expression through transcriptional regulation. COX-1 gene promoter studies support a role of Sp binding site in CT induced COX-1 gene expression. The nuclear protein binding to this site appears to be Sp3 transcription factor. Co-immunoprecipitation assays indicated a physical interaction between GR and Sp3 protein. Silencing of Sp3 transcription factor with small interfering RNA suppressed CT-induced COX-1 promoter activation. These data suggest that the activated GR interacts with Sp3 transcription factor that binds to COX-1 promoter to up-regulate COX-1 gene expression in cardiomyocytes.We also found that administration of GC in adult mice increased the level of COX-2 in the ventricles. With isolated neonatal cardiomyocytes, corticosterone (CT) induces the transcription of COX-2 gene. This response appears to be cardiomyocyte cell type specific and GC receptor (GR)-dependent. CT causes activation of p38 MAPK and subsequently CREB phosphorylation that mediates COX-2 gene expression. Mifepristone, a GR antagonist, failed to inhibit p38 and CREB activation and p38 inhibition failed to prevent activation of GR. These data suggest that two parallel signaling pathways, GR and p38 MAPK, act in concert to regulate the expression of COX-2 gene in cardiomyocytes.In addition to the investigation of mechanism and signaling transduction pathway, I have explored pharmacological agents that modulate COX expression. LY294002, a commonly used PI3K inhibitor, inhibited COX-2 gene expression via a PI3K-independent mechanism. Whereas GSK-3 inhibitors, such as lithium chloride, upregulated COX-2 gene expression, but suppressed GC-induced COX-1 expression. These data have paved the foundation for pharmacological manipulation of COX-1 and COX-2 gene expression in the heart.

Reduced availability of high energy-content prey (nutritional stress) is a predominant hypothesis to explain the decline of Steller sea lion (Eumetopias jubatus) populations in western Alaska from the late 1970'€™s to the late 1990'™s. Animals may physiologically respond to consuming insufficient prey by increasing stress levels and decreasing metabolic rates. It may thus be possible to identify nutritional stress by measuring concentrations of glucocorticoids (stress) and thyroid hormones (metabolism) shed in feces. However, techniques to measure thyroid hormone concentrations from Steller sea lion feces have not been developed. We quantified variation of triiodothyronine (T3) and thyroxine (T4) concentrations in Steller sea lion feces following injections of thyrotropin (TSH) into four captive animals. Glucocorticoids (GC) were also assayed to examine any relationship to stimulated thyroid hormone secretion. We found that fecal T3 peaked 48 h post-injection and increased 25-57% in three sea lions (all animals, p=0.03). Pre-injection GC increases indicated stress from isolation for baseline fecal collections, but post-injection increases could not be confirmed as a response to TSH injections or as a product of the study design. The results demonstrated that pre- and post-injection changes in fecal GC and T3 concentrations were consistent with predictions of an increased stress response and metabolic rate within the animals. We then measured T3 and GC concentrations in 834 Steller sea lion fecal samples collected in 2005 and 2006 from 15 sites (haulouts and rookeries) between British Columbia and the Central Aleutian Islands. Overall, GC concentrations did not differ between haulout populations (western 2006 pre-pupping and eastern 2005 post-pupping). Fecal hard-part analyses revealed a lower energy-content diet in the western population, suggesting that diet quality is a relevant hypothesis to explain slightly higher GC concentrations found in the western population, specifically the Aleutian Islands region. However, nutritional stress could not be substantiated through T3 concentrations. The rookeries possessed the highest energy-content diets, but also exhibited a nutritional stress response with a significantly higher GC and lower T3 concentration than either haulout population (possibly related to lactation or decreased foraging opportunities), but T3 comparisons performed at scales of site and region were inconclusive.

This thesis investigates the use of an enzyme immunoassay to measure keratin glucocorticoid concentrations in mammalian hair, bird feathers and reptilian shed skins. Keratin glucocorticoid concentrations were compared to fecal glucocorticoid concentrations produced during the period of keratin growth in the African Elephant (Loxodonta africana), the Western Lowland Gorilla (Gorilla gorilla gorilla), the Sumatran Orangutan (Pongo abelii), the Domestic Chicken (Gallus gallus domesticus), the Eastern Loggerhead Shrike (Lanius ludovicianus migrans), the African House Snake (Lamprophis fuliginosus) and the Eastern Massasauga Rattlesnake (Sistrurus catenatus catenatus). Complete biochemical validation was performed for the feather and shed skin corticosterone and fecal corticosterone enzyme immunoassays in the Domestic Chicken and the African House Snake. Biological validation was performed in the Domestic Chicken. Biological and physiological validation were attempted in the African House Snake. African Elephant, Western Lowland Gorilla and Sumatran Orangutan hair cortisol concentrations ranged from 2.10 - 312.70 ng/g. African Elephant hair corticosterone concentrations ranged from 2.68 - 20.70 ng/g. Domestic Chicken and Eastern Loggerhead Shrike feather corticosterone concentrations ranged from 1.31 - 8.09 pg/mm and from 1.09 - 6.59 pg/mm, respectively. African House Snake and Massasauga Rattlesnake shed skin corticosterone concentrations ranged from 4.42 - 124.35 ng/g and 3.82 - 22.85 ng/g, respectively. In the majority of cases, a statistically significant association was not found between summary statistical measures of fecal and keratin glucocorticoid concentrations. A statistically significant positive association was detected between hair cortisol and the coefficient of variation of fecal corticosterone in the African Elephants. A statistically significant negative association was detected between feather corticosterone and the 75th percentile and coefficient of variation of fecal corticosterone in the Eastern Loggerhead Shrikes. A statistically significant positive association was also detected between shed skin corticosterone and the mean fecal corticosterone from 3 weeks before to 1 week after the previous ecdysis in the African House Snake. Feather corticosterone concentrations were significantly higher in feathers from Domestic Chickens that were socially housed with roosters than in feathers from chickens housed individually in laying cages. A statistically significant difference was not detected between the shed skin corticosterone concentrations of the minimally handled control and the weekly handled experimental African House Snakes. Adrenocorticotropic hormone stimulation did not result in the physiological validation anticipated for shed skin corticosterone concentrations in the African House Snake. / Toronto Zoological Foundation and Ontario Ministry of Agriculture, Food and Rural Affairs

No description available.

Group X secretory phospholipase A2 (GX sPLA2) hydrolyzes membrane phospholipids producing free fatty acids and lysophospholipids. Previous studies from our lab suggest that mice with targeted deletion of GX sPLA2 (GX KO) have increased age-related weight gain due to an increase in overall adiposity. Paradoxically, this increased adiposity is associated with improved age-related glucose intolerance. GX KO mice also demonstrate a reduced inflammatory response to lipopolysaccharide injection. In vitro studies indicate this phenotype may be attributable to blunted macrophage mediated inflammatory responses. Given the role of macrophages in promoting adipose tissue (AT) inflammation and metabolic dysfunction in response to diet-induced obesity, we hypothesized that GX KO mice would be protected from the obesity related metabolic derangements associated with overfeeding. Unexpectedly, GX KO mice were only partially protected from high fat (HFD) diet-induced glucose intolerance and showed no improvement in HFD-induced insulin resistance. Moreover, GX KO mice were not protected against HFD-induced AT inflammation. GX sPLA2 is produced as a proenzyme (pro-GX sPLA2), and propeptide cleavage is required for enzymatic activity. Furin-like proprotein convertases (PCs) have recently been implicated in the proteolytic activation of pro-GX sPLA2; however the identity of individual PCs involved is unclear. Previous findings from our lab have shown that GX sPLA2 is expressed in the adrenals where it regulates glucocorticoid production. GX KO mice have increased plasma corticosterone levels under both basal and ACTH-induced stress conditions. However, how GX sPLA2 is regulated in the adrenals is still uncertain. We hypothesized that PCs may be involved in the proteolytic activation of pro-GX sPLA2 in the adrenals. Here we report the novel findings that the PCs, furin and PCSK6, proteolytically activate pro-GX sPLA2 in Y1 adrenal cells. Furthermore, we demonstrate that PC dependent processing of pro-GX sPLA2 is necessary for GX sPLA2 dependent suppression of steroidogenesis. Finally, we provide evidence that pro-GX sPLA2 processing by PCs is enhanced in response to adrenocorticotropic hormone (ACTH), suggesting a novel mechanism for negatively regulating adrenal steroidogenesis. Cumulatively, these studies provide valuable insight into the function and regulation of GX sPLA2.

Corticosteroid ligands activate the glucocorticoid receptor (GR). GR plays a role in glucose homeostasis, adipogenesis, inflammation, and mood and cognitive functions. Understanding the interplay of diverse forms of receptor regulation (including post-translational modification, cofactor interactions, ligand binding, and receptor localization) and their effects is important for understanding and developing more effective treatment for a variety of conditions. Prior to ligand binding, the naïve GR is primarily cytoplasmic, residing in a chaperone complex containing heat-shock proteins and immunophilins. Upon ligand-binding, alterations to the complex allow the receptor to dimerize and import into the nucleus. Nuclear GR interacts with transcriptional regulatory sequences and recruits cofactors to regulate specific gene expression. Upon hormone withdrawal, the original chaperone complex is reassembled and the receptor is exported to the cytoplasm. Interestingly, while the import of GR into the nucleus occurs very rapidly (t ½ = 5 min), the re-export is significantly slower (t ½ = 12-24h). Previous work by our lab and others has indicated the existence of a nuclear retention signal (NRS) within the GR. The NRS sequence of the GR, its interaction partners, and the role it might play in the activity of the receptor have not yet been fully defined. Work in the Hache lab indicates that mutation of the GR nuclear localization signal 1 (NL1) increases the export rate of nuclear GR to the cytoplasm, as well as compromising receptor import, suggesting that the NL1 overlapped an NRS sequence. In this work, I made a series of GR mutants, based on sequence from the SV40 large T antigen NLS, which lacks nuclear retention activity. Using these mutants, I found that GR nuclear retention is influenced by both specific residues within the hinge region and the location of the sequence within the receptor, as reintroduction of the NLS sequence at the N-terminus of the receptor retention mutant failed to reconstitute the retention activity. Agonist liganded and hormone-withdrawn receptor mutants showed a similar decrease in retention. By contrast, antagonist-withdrawn GR mutants were retained in the nucleus, possibly due to altered receptor configuration and interactions. Assays of GR-responsive promoter activation by receptor retention mutants showed that while no difference in the ability of retention mutants to activate transcription was seen at a simple promoter, activation of a complex promoter was compromised. This impaired transactivation for the SV506-523 mutant correlated with decreased histone H4 acetylation and PolII recruitment, while GR DNA-binding at the target promoter appeared to be unaffected. These results suggested that promoter-specific cofactor interactions might be implicated in GR nuclear retention. Loss of GR hinge interaction with Oct cofactors produced an incomplete loss of retention, suggesting overlapping signals, but not supporting Oct as a primary factor in GR retention. The overlap between important residues in GR nuclear retention and localization signals and the lack of retention shown by the SV40 NLS suggested that retention might be intrinsic to the sequence of particular NLS. Preliminary results suggest that the KT511-512 residues of GR may be of general importance in protein nuclear retention, while the role of proline is likely more variable. My research has focused on increasing our understanding of glucocorticoid receptor nuclear retention and its possible implications. I have determined that the KT511-512 residues of GR play an important role in its retention, and possibly also figure in nuclear retention of other proteins. These residues are involved in interactions which affect promoter-specific histone acetylation and transcriptional activation in GR, suggesting a reason for the existence of nuclear retention.

This study aimed to examine potential mechanisms for glucocorticoid (GC)-induced decreases in glucose clearance, and to determine if a reduction in oxidative stress load via dietary pre-treatment with an antioxidant-rich diet has a positive net effect on glucose tolerance following a sub-chronic treatment with the GC analogue dexamethasone (DEX). Rats fed a diet supplemented with 700IU of -tocopherol for two weeks had improved glucose clearance after five days of DEX-treatment relative to unsupplemented rats as well as decreased markers of oxidative stress. Following an intraperitoneal bolus of insulin, phosphorylation of AMP activated protein kinase (AMPK) was preserved in the supplemented groups despite no significant differences in upstream insulin signalling cascade intermediates between DEX-treated groups. This was corroborated by a similar increase (p<0.05) in phosphorylation of the downstream AMPK substrate acetyl CoA carboxylase. This study demonstrated that -tocopherol supplementation can attenuate GC-induced decreases in glucose clearance in an AMPK-dependent manner. / Nutrition and Metabolism