Browsing by Author "Cunningham, Rebecca L."
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Item Androgen Modulation of CNS During Chronic Intermittent Hypoxia(2018-05) Snyder, Brina D.; Cunningham, Rebecca L.; Barber, Robert C.; Cunningham, J. Thomas; Schreihofer, Derek A.; Planz, John V.The underlying causes of age-related neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease, are unknown. It is likely conditions which contribute to an abundance of oxidative stress throughout life renders an individual more susceptible to late-life neurodegenerative processes. Sex differences are observed in the onset and prevalence of these diseases, suggesting estrogens and androgens influence these processes. This study investigates the early role of androgens under a known oxidative stressor, sleep apnea, which frequently goes untreated in the clinical population but is but is associated with an increased risk of late-life neurodegeneration. The hypoxic events of sleep apnea can be modeled in rats by the use of chronic intermittent hypoxia (CIH). Male rats are more susceptible to hypertensive effects of CIH, a key characteristic of sleep apnea. After one week of CIH treatment, they also exhibit oxidative stress and inflammation in circulation and in brain nuclei associated with early stages of Parkinson's disease or Alzheimer's disease. This led to the hypothesis that oxidative stress and inflammation would be associated with behavior deficits and these effects are mediated by androgens. Results show that oxidative stress and inflammatory dysregulation can be prevented by testosterone, but are highly exacerbated by testosterone's non-aromatizable metabolite, dihydrotestosterone (DHT). Administration of DHT also resulted in significant memory impairments under CIH. In the central nervous system, DHT significantly altered oxidative stress and pro-inflammatory signals, which may underlie its detrimental actions in an oxidative stress environment. There was also evidence of hypothalamic-pituitary-adrenal axis dysregulation, which can influence testosterone and circadian rhythms. These findings have broad implications for clinical populations with conditions which chronically increase oxidative stress and inflammation, while at the same time alter endocrine function. Conditions, such as untreated sleep apnea, may pose a latent risk for neurodegeneration and should be addressed early to prevent later detrimental effects.Item Blood-Based Inflammation Biomarkers of Neurocognitive Impairment in People Living with HIV(2020-05) Swanta, Naomi K.; Borgmann, Kathleen; Berg, Rance E.; Cunningham, Rebecca L.; Johnson, Leigh A.; Yan, Liang-JunRace and sex minorities are disproportionately affected by HIV in the United States. Approximately 50% of people living with HIV (PLWH) experience HIV-associated neurocognitive disorders (HAND). ART has decreased incidence of HAD but the less severe forms of HAND has increased. Diagnosis of HAND is challenging as the often-subtle forms of impairment are not as overt as dementia. HIV infection promotes neurocognitive dysfunction through persistent inflammation, which correlates with the severity of impairment. The objective of this study was to identify blood-based cytokines that associate with, and could predict, neurocognitive functioning in a demographically balanced cohort of PLWH. Inflammatory biomarkers of HIV-associated neurocognitive impairment could improve current diagnosis methods and may be specific for populations disproportionately affected by the HIV. Seven neurocognitive domains were evaluated in 121 seropositive African American, Non-Hispanic White and White Hispanic men and women using computerized assessments. A panel of 26 inflammation-associated cytokines were measured in plasma and blood mononuclear cells. Significant associations among neurocognitive functioning and HIV-related parameters, relevant sociodemographic variables and cytokine panel were determined using multivariate and univariate regression analyses. Following corrections for education, CD4 T cell counts, viral load and eliminating outliers. Our results demonstrate that chemokine C-C motif ligand (CCL) 8 significantly correlated with memory, complex attention, cognitive flexibility, psychomotor speed, executive functioning and processing speed. Tissue inhibitor of metalloproteinases-1 (TIMP-1) significantly correlated with the aforementioned domains except memory and processing speed. In addition, interleukin (IL) - 23 significantly associated with executive functioning and processing speed. The biomarkers demonstrated a negative correlation to neurocognitive functioning. Race disparities were identified in memory and CCL8, furthermore, sex disparities were identified in executive functioning and TIMP-1. The plasma biomarkers were evaluated as predictive classifiers of neurocognitive functioning. Decision tree models for NCI and executive functioning predicted visit two at 67.2% accuracy. Collectively, these data identified blood-based inflammatory biomarkers of neurocognitive function with the potential to assist in the diagnosis of HIV-associated neurocognitive impairments in PLWH.Item Chronic Intermittent Hypoxia Increases Oxidative Stress and Impairs Spatial Memory in Male and Female Rats(2023) Gardner, Jennifer J.; Mabry, Steve; Bradshaw, Jessica L.; Wilson, E. Nicole; Little, Joel; Goulopoulou, Styliani; Cunningham, Rebecca L.Obstructive sleep apnea (OSA) is characterized by complex phenotypes and increased long-term risk of neurodegenerative disease. The impact of OSA in women is unknown due to sex differences in clinical presentation contributing to underdiagnosis. Using chronic intermittent hypoxia (CIH) to model OSA in rodents, our previous studies have shown CIH exposure increases oxidative stress and inflammation in male rats. However, the impact of CIH in female rats remains unclear. The objective of this study was to assess sex differences in CIH-mediated oxidative stress and rodent behaviors associated with neurodegenerative disease. Young adult male and female Long Evans and Sprague Dawley rats were exposed to CIH or normoxia for 14-15 days. Spatial memory and fine and gross motor skills were assessed. Plasma oxidative stress was measured and neuronal expression in the dorsal hippocampus was quantified. Female rats exhibited better spatial memory than males with increased neuronal expression in the CA1 region of the hippocampus. In both males and females, CIH impaired spatial memory and increased circulating oxidative stress. Yet, CIH increased CA1 neuronal expression in female rats only. CIH did not impact gross or fine motor skills, regardless of sex. Our preliminary findings indicate CIH increases oxidative stress and impairs spatial memory in males and females, but the impact of CIH on hippocampal neurons and region-specific contributions to spatial memory may be sexually dimorphic.Item Chronic testosterone deprivation sensitizes the middle-aged rat brain to damaging effects of testosterone(2020-05) Smith, Charity; Schreihofer, Derek A.; Cunningham, Rebecca L.; Singh, Meharvan; Yang, Shaohua; Jones, Harlan P.Levels of the testosterone (T) fall in aging men. Recently, the number of men obtaining testosterone replacement therapy (TRT) has increased dramatically. However, other consequences of aging, such as increased oxidative stress, may result in detrimental effects when combined with TRT, including an increased stroke risk. Whether such a delay would alter the effects of TRT on stroke is not known. We hypothesized that a delay TRT following castration in middle-aged male rats would result in increased oxidative stress and a reduction in the neuroprotective effects of testosterone following stroke. We evaluated the effects of testosterone treatment after short (2 week) and long-term testosterone deprivation (10 weeks) in middle-aged male rats on cerebral ischemia, oxidative stress and cognitive function. Our data suggest testosterone treatment after long-term hypogonadism can exacerbate functional recovery after focal cerebral ischemia, however in the absence of injury improves cognition. Both effects are regulated by oxidative stress.Item Effects of Amyloid β on Recollective Memory: Sex and Hormone Differences(2023) Vera, Edward; Mabry, Steve; Wilson, Nicole; Bradshaw, Jessica L.; Gardner, Jennifer J.; Little, Joel; Rybalchenko, Nataliya; Cunningham, Rebecca L.PURPOSE: Alzheimer’s disease (AD) is linked with increased memory loss and inability to learn new topics. One of the defining neuropathological features of AD is amyloid beta (Aβ) plaques in brain regions, such as the hippocampus. The hippocampus brain region is important for memory and learning. AD risk is elevated in individuals older than 65 years old, especially menopausal women. Menopause is an aging associated endocrine event in which the ovaries stop producing estradiol but continue producing testosterone. Testosterone can be aromatized to estradiol, but aromatase is not functional in women with AD. Therefore, post-menopausal women with AD have more androgens than estrogens than pre-menopausal women and aged men. Androgens can be neuroprotective or neurotoxic depending on the cellular environment. It is unknown what the impact of androgens and sex are on amyloid beta’s effects on the brain, (e.g., hippocampus) and behavior (e.g., memory). We hypothesize that females with the hormonal condition of androgens in the absence of estrogens will exhibit increased recollective memory in response to hippocampal injection of Aβ. METHODS: To investigate the role of androgens and sex on Aβ associated memory impairments, adult male and female Sprague-Dawley rats were gonadectomized to remove circulating sex hormones. A subset of these rats was given either cholesterol or dihydrotestosterone (DHT), which cannot be converted into estrogen. To model AD, rats were injected with 5ug/ul of Aβ oligomer fibrils 1-40 or vehicle shams in the CA1 region of the hippocampus. One week after Aβ hippocampal injections, the rats were assayed for short term and long-term recollective memory via a 1-hour and 24-hour Novel Object behavioral test. The Novel Objective behavioral tests examines recollective memory by quantifying the time spent with a novel object versus the time spent with a known object. Data was quantified with a three-way ANOVA with sex, hormone, and Aβ as independent variables. Tukey’s was used as a post-hoc test. RESULTS: Sex differences were observed between hormone-deficient rats exposed to Aβ. Specifically, males exhibited worse short term recollective memory (1 hour novel object) compared to females. DHT had no effect on recollective memory, regardless of Aβ exposure. No effects were observed in the long-term recollective memory (24-hour novelty test). CONCLUSIONS: Our results indicate that Aβ 's effects on short term recollective memory is influenced by sex chromosomes, as we observed sex differences in the hormone deficient (cholesterol) treated animals. However, DHT did not impact these recollective memory. These results indicate that recollective memory in AD is impacted by the sex chromosomes and not androgens.Item Hypoxia and oxidative stress reduce placental efficiency and impair the balance between autophagy and cell death mechanisms in trophoblasts(2024-03-21) Gardner, Jennifer; Bradshaw, Jessica L.; de Nazare Oliveria da, Renee; Hula, Nataliia; Mabry, Steve; Wilson, E. Nicole; Cunningham, Rebecca L.; Goulopoulou, StylianiIntroduction: Hypoxia and oxidative stress can activate autophagy, a lysosomal degradation pathway that maintains cellular homeostasis. Impairments in autophagy mechanisms have been observed in placentas from obstetric complications associated with placental hypoxia and oxidative stress, such as preeclampsia and intrauterine growth restriction. Purpose: The objective of this study was to investigate the effects of hypoxia and oxidative stress on placental autophagy. We hypothesized that exposure to oxidative stress and hypoxia would alter the balance between cytotoxic and cytoprotective mechanisms in human trophoblast cells and rat placentas and would adversely affect placental efficiency. Methods: We used an in vitro model incorporating human trophoblast cells (BeWo cells) exposed to an oxidative stressor, antimycin A (10, 100, 320 μM) or vehicle for 4 hours. Trophoblast cell death and autophagy mechanisms were assessed via flow cytometry and western blotting. Additionally, we used a rodent model of gestational sleep apnea, a pregnancy complication associated with placental hypoxia. Long Evans timed-pregnant dams were exposed to chronic intermittent hypoxia (CIH; n=6-8) or normoxia (NX; n=8-9) during their sleep cycle from gestational day (GD) 15 to 20 (late pregnancy, term=21-23 days). Results: In trophoblast cells (n=5-9 independent experiments), antimycin A increased necrosis and LC3 A/B II/I ratio (autophagy marker) at 100 μM compared to vehicle (p<0.015). Necrosis remained elevated at 320 μM, while BAX (pro-apoptotic marker) and p62 (autophagosomal flux marker) were reduced compared to vehicle (p<0.0001). LC3 A/B II/I ratio returned to vehicle levels at 320 μM (p>0.05 vs. vehicle). Placental weights from CIH exposed dams were greater (NX: 0.51±0.02 g vs. CIH: 0.60±0.03 g, p=0.015) and fetal to placental weight ratios (marker of placental efficiency) were reduced compared to control pregnancies (NX: 5.25±0.13 vs. CIH: 4.43±0.14, p=0.0006) on GD20. Gestational CIH did not affect (p>0.05) fetal weights (NX: 2.76±0.06 g vs. CIH: 2.61±0.06 g), crown to rump length (NX: 3.32±0.03 cm vs. CIH :3.18±0.12 cm), abdominal girth (NX: 3.22±0.06 cm vs. CIH: 3.32±0.12 cm), or litter size (NX: 11.9±0.90 vs. CIH: 10.5±0.82). Conclusion: Oxidative stress alters the balance between cytotoxic and cytoprotective mechanisms in trophoblast cells, promoting cell necrosis. Although assessment of autophagy machinery and cell death in placentas from hypoxic pregnancies is ongoing, our results indicate that maternal CIH during pregnancy adversely affects placental efficiency.Item INHIBITION OF LET-7I AS A MEANS TO ENHANCE THE NEUROPROTECTIVE EFFICACY OF PROGESTERONE IN THE ISCHEMIC BRAIN(2018-05) Nguyen, Trinh V.; Singh, Meharvan; Basu, Alakananda; Ghorpade, Anuja; Cunningham, Rebecca L.; Yang, ShaohuaThe occurrence of ischemic stroke is relatively rare among pre-menopausal women. Strikingly, this risk doubles every 10 years after the menopausal transition; and women are likely to experience worse outcomes and higher mortality post stroke than men. Since both estrogen (E2) and progesterone (P4) levels decline precipitously following the menopause, this hormonal reduction may, at least partially, contribute to the higher risk and worse outcomes. By inference, these hormones could play a critical role in protecting women against ischemic stroke. In this dissertation project, we focus on P4, the relatively understudied of the two hormones. And while P4 has been shown to be a potent neuroprotectant in various experimental models of stroke, the underlying mechanisms remain unclear. One known mediator of P4's protective function is brain-derived-neurotrophic-factor (BDNF), which has an established role in promoting neuronal differentiation, survival, and synaptogenesis. In addition, emerging literature and data from our laboratory strongly support the indispensable role of glia in P4's neuroprotective program and thus, may also play a significant role in post-stroke recovery. We recently reported that P4 induces a significant release of BDNF from primary astrocytes, through a putative membrane-associated progesterone receptor consisting of progesterone-receptor-membrane-component-1 (Pgrmc1). This receptor is abundantly expressed in various regions of brain and mediates such effects of P4 in the central nervous system (CNS) as anti-apoptotic effects, spinogenesis, and BDNF release. What is not known, however, is how the expression of this receptor is regulated. This dissertation was aimed to elucidate what regulates the expression of Pgrmc1 and BDNF in glia and how such regulation influences the neuroprotective function of P4 in the ischemic brain. Based on the observation that Let-7i regulates the expression of Pgrmc1 in a peripheral cell type, and our in silico analysis that revealed that both Pgrmc1 and BDNF are potential targets of let-7i, we hypothesized that let-7i represses P4's neuroprotective effects by down-regulating the expression of both Pgrmc1 and BDNF in glia, leading to: 1) suppression of P4-induced BDNF release from glia, and 2) attenuation of the beneficial effects of P4 on neuronal survival and markers of synaptogenesis in the ischemic brain. Using primary cortical astrocytes as an experimental model, we found that let-7i negatively regulated the expression of Pgrmc1 and BDNF. This was correlated with a reduction in P4-induced BDNF release from these cells. Under such conditions of reduced expression of both Pgrmc1 and BDNF, P4 was unable to protect primary neurons against oxygen-glucose-deprivation (OGD) or regulate markers of synaptogenesis. In our in vivo model of transient ischemic stroke, we found that protective effects of P4 were greatly enhanced in animals that were concomitantly treated with an inhibitor (antagomir) of let-7i. The combined treatment also enhanced synaptogenesis in the peri-infarct region. Collectively, the data presented here suggested that in the ischemic brain, let-7i negatively influences P4-induced neuroprotection via regulation of the Pgrmc1/BDNF axis. As such, inhibition of let-7i maybe an effective means to enhance the efficacy of P4 in treating ischemic stroke.Item Is there a Neurobiological Rationale for the Utility of the Iowa Gambling Task in Parkinson's Disease?(IOS Press, 2021-04-13) Salvatore, Michael F.; Soto, Isabel; Alphonso, Helene M.; Cunningham, Rebecca L.; James, Rachael; Nejtek, Vicki A.Up to 23% of newly diagnosed, non-demented, Parkinson's disease (PD) patients experience deficits in executive functioning (EF). In fact, EF deficits may occur up to 39-months prior to the onset of motor decline. Optimal EF requires working memory, attention, cognitive flexibility, and response inhibition underlying appropriate decision-making. The capacity for making strategic decisions requires inhibiting imprudent decisions and are associated with noradrenergic and dopaminergic signaling in prefrontal and orbitofrontal cortex. Catecholaminergic dysfunction and the loss of noradrenergic and dopaminergic cell bodies early in PD progression in the aforementioned cortical areas likely contribute to EF deficits resulting in non-strategic decision-making. Thus, detecting these deficits early in the disease process could help identify a significant portion of individuals with PD pathology (14-60%) before frank motor impairment. A task to evaluate EF in the domain of non-strategic decision-making might be useful to indicate the moderate loss of catecholamines that occurs early in PD pathology prior to motor decline and cognitive impairment. In this review, we focus on the potential utility of the Iowa Gambling Task (IGT) for this purpose, given significant overlap between in loss of dopaminergic and noradrenergic cells bodies in early PD and the deficits in catecholamine function associated with decreased EF. As such, given the loss of catecholamines already well-underway after PD diagnosis, we evaluate the potential utility of the IGT to identify the risk of therapeutic non-compliance and a potential companion approach to detect PD in premotor stages.Item Membrane androgen receptor-induced oxidative stress: mechanism involved in neurodegeneration(2019-05) Tenkorang, Mavis A. A.; Cunningham, Rebecca L.; O'Bryant, Sid; Schreihofer, Derek; Barber, Robert C.Oxidative stress-associated neurodegenerative diseases, such as Parkinson's disease (PD), affect millions of people worldwide. Although aging is the greatest risk factor for PD, other significant factors may be implicated, such as sex hormones that can mediate sex differences. Men have a higher incidence and prevalence of PD than women. Therefore, testosterone, a primary male sex hormone and a known oxidative stressor, is implicated in PD pathophysiology. Since androgens can have negative effects on dopaminergic cells, it is imperative to understand the underlying mechanisms in order to determine what mediates the observed sex differences in PD prevalence. NADPH Oxidase 1 and 2 are major oxidative stress generators in the brain, thus potential targets for testosterone-induced oxidative stress and cell death. This dissertation project therefore investigates the role of androgens and membrane androgen receptor activation on NOX1/2. We hypothesize that in dopaminergic cells, testosterone activates the membrane androgen receptor (AR45) that is complexed with NOX1/2 to increase oxidative stress. In an oxidative stress environment, androgen activation of this AR45-NOX complex leads to cell death. Results indicate that classical androgen receptor (AR) antagonists do not block testosterone's negative actions in an oxidative stress environment. The effects of AR45-NOX complex on cell viability can be blocked by either degrading AR45 protein or blocking NOX activation by apocynin. Further, these results show that testosterone's detrimental effect on cells is via a non-genomic mechanism, specifically via a novel membrane androgen receptor, AR45. The findings of this study help identify key players in testosterone-induced neurodegeneration, which could serve as potential therapeutic targets for PD. Ultimately, this project provides novel mechanisms to explain thought provoking questions on male sex bias in PD.Item Neuroprotective and neurotoxic outcomes of androgens and estrogens in an oxidative stress environment(BioMed Central Ltd., 2020-03-29) Duong, Phong; Tenkorang, Mavis A. A.; Trieu, Jenny; McCuiston, Clayton; Rybalchenko, Nataliya; Cunningham, Rebecca L.BACKGROUND: The role of sex hormones on cellular function is unclear. Studies show androgens and estrogens are protective in the CNS, whereas other studies found no effects or damaging effects. Furthermore, sex differences have been observed in multiple oxidative stress-associated CNS disorders, such as Alzheimer's disease, depression, and Parkinson's disease. The goal of this study is to examine the relationship between sex hormones (i.e., androgens and estrogens) and oxidative stress on cell viability. METHODS: N27 and PC12 neuronal and C6 glial phenotypic cell lines were used. N27 cells are female rat derived, whereas PC12 cells and C6 cells are male rat derived. These cells express estrogen receptors and the membrane-associated androgen receptor variant, AR45, but not the full-length androgen receptor. N27, PC12, and C6 cells were exposed to sex hormones either before or after an oxidative stressor to examine neuroprotective and neurotoxic properties, respectively. Estrogen receptor and androgen receptor inhibitors were used to determine the mechanisms mediating hormone-oxidative stress interactions on cell viability. Since the presence of AR45 in the human brain tissue was unknown, we examined the postmortem brain tissue from men and women for AR45 protein expression. RESULTS: Neither androgens nor estrogens were protective against subsequent oxidative stress insults in glial cells. However, these hormones exhibited neuroprotective properties in neuronal N27 and PC12 cells via the estrogen receptor. Interestingly, a window of opportunity exists for sex hormone neuroprotection, wherein temporary hormone deprivation blocked neuroprotection by sex hormones. However, if sex hormones are applied following an oxidative stressor, they exacerbated oxidative stress-induced cell loss in neuronal and glial cells. CONCLUSIONS: Sex hormone action on cell viability is dependent on the cellular environment. In healthy neuronal cells, sex hormones are protective against oxidative stress insults via the estrogen receptor, regardless of sex chromosome complement (XX, XY). However, in unhealthy (e.g., high oxidative stress) cells, sex hormones exacerbated oxidative stress-induced cell loss, regardless of cell type or sex chromosome complement. The non-genomic AR45 receptor, which is present in humans, mediated androgen's damaging effects, but it is unknown which receptor mediated estrogen's damaging effects. These differential effects of sex hormones that are dependent on the cellular environment, receptor profile, and cell type may mediate the observed sex differences in oxidative stress-associated CNS disorders.Item Novel androgen receptor splice variant in the substantia nigra(2017-08-01) Contreras, Jo Garza; Cunningham, Rebecca L.; Basha, Riyaz; Salvatore, MichaelTestosterone can increase calcium influx and cell death in dopamine neurons via a putative membrane androgen receptor (mAR). The mAR induced calcium increase may be due to activation of G-proteins involved in calcium mobilization. Previous studies using an N-terminal targeted androgen receptor (AR) antibody yielded low AR expression in dopamine neurons. Studies in our lab show high AR expression using a C-terminal targeted AR antibody. This difference in expression may be due to an AR variant. We hypothesize an AR variant is present in the membrane of dopaminergic neurons and associated with G proteins. To identify the presence of AR in dopaminergic neurons. We performed immunoblot, sucrose gradient, and immunohistochemistry studies. To determine the protein-protein interaction between mAR and G-proteins we performed co-immunoprecipitation studies. Our results show AR45 localizes in the membrane lipid rafts of dopaminergic neurons. Furthermore, AR45 interacts with Gαq and Gαo G-proteins, which can impact calcium signaling.Item Novel Androgen Receptor Variant 45 in Brain Tissue(2016-03-23) Contreras, Jo; Duong, Phong; Cunningham, Rebecca L.; Downs, LincolnPurpose: A membrane associated androgen receptor has been implicated in the damaging effects of testosterone in neurodegenerative disorders, such as Parkinson’s disease. An androgen receptor variant (AR45) that has a C-terminus and a unique N-terminus has been found in peripheral tissues, such as the heart, skeletal muscle, uterus, and prostate, but has not been found in the brain. Recent findings have shown that a AR45 is present in the membrane fraction of a dopaminergic neuronal cell line (N27). Therefore, we wanted to determine if AR45 was present in cortical, hippocampal, and substantia nigral neurons that are lost during Alzheimer’s and Parkinson’s disease. Methods: In this study we used immunohistochemistry to determine the presence of AR45 presence in rat brain tissue. Tissue sections of 40 um were prepared using a cryostat. The sections were stained with primary antibodies specific for different androgen receptor sequences. The antibodies AR C-19 and AR N-20 were used to identify the C terminus and the N terminus, respectively. Results: Since AR-45 is lacking an N-terminus, we used N-20 antibody that binds to the N-terminus as a negative control. However, AR-45 does have a c-terminus, and thus we used the C-19 antibody as a marker for AR-45. C-19 immunofluorescence was present in the hippocampus, cortex, and substantia nigra. Interestingly, N-20 positive cells were not identified in the substantia nigra, indicating that only AR45 is present in these neurons. Conclusion: This is first study to show the presence of AR45-immunoreactive positive neurons in the brain. Now the receptor has been identified, more research is needed to determine the role of AR45 in neurodegenerative diseases.Item Novel Correlation between TGF-beta1/-beta3 and Hormone Receptors in the Human Corneal Stroma(MDPI, 2023-09-09) Choi, Alexander J.; Hefley, Brenna S.; Nicholas, Sarah E.; Cunningham, Rebecca L.; Karamichos, DimitriosThis study investigated the interplay between transforming growth factor beta (TGF-beta1/T1 and TGF-beta3/T3), and sex hormone receptors using our 3D in vitro cornea stroma model. Primary human corneal fibroblasts (HCFs) from healthy donors were plated in transwells at 10(6) cells/well and cultured for four weeks. HCFs were supplemented with stable vitamin C (VitC) and stimulated with T1 or T3. 3D construct proteins were analyzed for the androgen receptor (AR), progesterone receptor (PR), estrogen receptor alpha (ERalpha) and beta (ERbeta), luteinizing hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), gonadotropin-releasing hormone receptor (GnRHR), KiSS1-derived peptide receptor (KiSS1R/GPR54), and follicle-stimulating hormone subunit beta (FSH-B). In female constructs, T1 significantly upregulated AR, PR, ERalpha, FSHR, GnRHR, and KiSS1R. In male constructs, T1 significantly downregulated FSHR and FSH-B and significantly upregulated ERalpha, ERbeta, and GnRHR. T3 caused significant upregulation in expressions PR, ERalpha, ERbeta, LHR, FSHR, and GNRHR in female constructs, and significant downregulation of AR, ERalpha, and FSHR in male constructs. Semi-quantitative Western blot findings present the interplay between sex hormone receptors and TGF-beta isoforms in the corneal stroma, which is influenced by sex as a biological variable (SABV). Additional studies are warranted to fully delineate their interactions and signaling mechanisms.Item Oxidative stress and androgen replacement as a model for neurodegeneration in the SN56 cell line(2016-03-23) Smith, Charity; Cunningham, Rebecca L.; Schreihofer, Derek; Warren, MarshallBackground: Sleep apnea has been linked to oxidative stress (OxS) in the form of reactive oxygen species. The presence of oxidative stress has been shown to determine either the protective or toxic property of androgens in the N27 dopaminergic cell line, which has been used as a model of Parkinson’s disease. Androgen treatment prior to OxS protects N27 cells from injury, whereas androgen treatment after OxS increases the toxicity of OxS. We sought to determine whether similar oxidative stress dependent androgen effects also occurred in the SN56 cholinergic cell line, which has been used to model Alzheimer’s disease using intermittent hypoxia as an oxidative stress. Hypothesis: We hypothesize that testosterone given prior to oxidative stress is neuroprotective and testosterone given after oxidative stress is neurotoxic. Materials and Methods: The pre-treatment group was exposed to 100 nM of testosterone followed by 17-40 cycles of intermittent hypoxia in a chamber with fluctuating levels of oxygen. One cycle of IH involved reducing the chamber from 20% to 1.5% oxygen for 30 seconds followed by re-oxygenation for 4 minutes. The post-treatment group was first exposed to 17 cycles of IH and then treated testosterone. The control group used SN56 cells that were not exposed to androgen or IH. Cells were lysed at 6 & 24-hour intervals. Western blots were performed for two markers of apoptosis, Poly ADP ribose polymerase (PARP) and alpha spectrin, and were normalized for beta actin levels. Additional experiments used a cell viability assay to determine cell survival 24 hours after the same treatments and 30 or 40 cycles of IH. Results: Cleavage of PARP and alpha-spectrin was significantly increased in the post-treatment group that was lysed at 6 hours, but not in the pretreatment groups. Conversely, both pre and post treatment with testosterone appeared to reduce cell death induced by IH. Conclusions: The results of the cells lysed at 6 hours confirm the original hypothesis that androgens given prior to oxidative stress are neuroprotective while androgens given after oxidative stress are neurotoxic in SN56 cells. However, at longer time points, testosterone appeared to only be protective to SN56 cells. These data suggest that IH, such as occurs with sleep apnea, may interact with androgens in both detrimental and beneficial ways in cholinergic cells, as they do in dopaminergic cells.Item Prenatal Hypoxic Insults Impact Brain Vulnerability(2021-05) Wilson, Elizabeth N.; Cunningham, Rebecca L.; Goulopoulou, Styliani; Jones, Harlan P.; Sumien, NathalieMaternal hypoxic insults during gestation may lead to an increased risk for neurodegenerative diseases, such as Parkinson's disease (PD), in progeny. Maternal hypoxic stress is a common consequence of many late-stage prenatal stressors (e.g., preeclampsia, eclampsia, inflammation, placental abruption). It is unknown whether maternal hypoxic insults during late gestation have long-term effects on brain regions associated with PD, such as the nigrostriatal pathway. We hypothesized that late gestational maternal hypoxia would result in sustained nigrostriatal impairment in male progeny. To determine whether late-stage gestational hypoxia exposure induced PD-associated behaviors and oxidative stress in progeny, timed pregnant Long-Evans rats were exposed to five days (gestational days: 15-19) of chronic intermittent hypoxia (CIH) or room air normoxia. Progeny were tested during two developmental stages (pubertal and young adult) as late-stage gestational insults can impair the neuronal organization of the brain, which can impact pubertal and young adult functions. To examine PD-associated behavioral phenotype of motor dysfunction, we quantified fine and gross motor behaviors in an open field arena. To examine the integrity of the nigrostriatal pathway, we quantified ultrasonic vocalizations. Our results showed that maternal CIH during late gestation did not impact gross or fine motor behaviors nor circulating oxidative stress. However, maternal CIH during late gestation did impair the nigrostriatal pathway integrity during puberty and young adulthood in both male and female progeny. Long-lasting consequences of maternal CIH during late gestation was most evident in young adult male progeny. Overall, we conclude that maternal hypoxia during late gestation induced sustained nigrostriatal pathway impairment in males more than females, which may underlie the increased risk for PD in men compared to women.Item Presence of Androgen Receptor Variant in Neuronal Lipid Rafts(Society for Neuroscience, 2017-08-29) Garza-Contreras, Jo; Duong, Phong; Snyder, Brina D.; Schreihofer, Derek A.; Cunningham, Rebecca L.Fast, nongenomic androgen actions have been described in various cell types, including neurons. However, the receptor mediating this cell membrane-initiated rapid signaling remains unknown. This study found a putative androgen receptor splice variant in a dopaminergic N27 cell line and in several brain regions (substantia nigra pars compacta, entorhinal cortex, and hippocampus) from gonadally intact and gonadectomized (young and middle-aged) male rats. This putative splice variant protein has a molecular weight of 45 kDa and lacks an N-terminal domain, indicating it is homologous to the human AR45 splice variant. Interestingly, AR45 was highly expressed in all brain regions examined. In dopaminergic neurons, AR45 is localized to plasma membrane lipid rafts, a microdomain involved in cellular signaling. Further, AR45 protein interacts with membrane-associated G proteins Galphaq and Galphao. Neither age nor hormone levels altered AR45 expression in dopaminergic neurons. These results provide the first evidence of AR45 protein expression in the brain, specifically plasma membrane lipid rafts. AR45 presence in lipid rafts indicates that it may function as a membrane androgen receptor to mediate fast, nongenomic androgen actions.Item Rat Strain and Housing Conditions Alter Oxidative Stress and Hormone Responses to Chronic Intermittent Hypoxia(Frontiers Media S.A., 2018-11-06) Snyder, Brina D.; Duong, Phong; Tenkorang, Mavis A. A.; Wilson, E. Nicole; Cunningham, Rebecca L.Sleep apnea has been associated with elevated risk for metabolic, cognitive, and cardiovascular disorders. Further, the role of hypothalamic-pituitary-adrenal (HPA) activation in sleep apnea has been controversial in human studies. Chronic intermittent hypoxia (CIH) is a rodent model, which mimics the hypoxemia experienced by patients with sleep apnea. Most studies of CIH in rats have been conducted in the Sprague Dawley rat strain. Previously published literature suggests different strains of rats exhibit various responses to disease models, and these effects can be further modulated by the housing conditions experienced by each strain. This variability in response is similar to what has been observed in clinical populations, especially with respect to the HPA system. To investigate if strain or housing (individual or pair-housed) can affect the results of CIH (AHI 8 or 10) treatment, we exposed individual and pair-housed Sprague Dawley and Long-Evans male rats to 7 days of CIH treatment. This was followed by biochemical analysis of circulating hormones, oxidative stress, and neurodegenerative markers. Both strain and housing conditions altered oxidative stress generation, hyperphosphorylated tau protein (tau tangles), circulating corticosterone and adrenocorticotropic hormone (ACTH), and weight metrics. Specifically, pair-housed Long-Evans rats were the most sensitive to CIH, which showed a significant association between oxidative stress generation and HPA activation under conditions of AHI of 8. These results suggest both strain and housing conditions can affect the outcomes of CIH.Item REGULATION OF NORMAL CEREBRAL DEVELOPMENT AND MELANOCYTIC ONCOPATHOLOGY BY N-GLYCANASE 1(2021-05) Lin, Victor J.T.; Wang, Yu-chieh; Cunningham, Rebecca L.; Singh, Meharvan; Smith, Michael L.; Dimitrijevich, Slobodan DanThe congenital disorders of glycosylation (CDG) are a group of diseases with inborn errors in metabolism that result from the improper glycosylation of necessary biological molecules. Though they are a collection of rare diseases, unique CDGs now number in the hundreds. 1 In 2012, N-glycanase deficiency became the world's first known congenital disorder of deglycosylation (CDDG). The clinical presentations of these individuals possess variable expressivity ranging from all patients having global developmental delay, movement disorder and hypotonia, to patients with a mosaic phenotype with the affliction of other neurological (e.g. abnormal brain imaging, electroencephalogram abnormalities, acquired microcephaly, diminished reflexes, seizures) and non-neurological (e.g. hypo- or alacrima, elevated liver transaminases, and hepatocellular storage and vacuolization dysfunction) symptoms. Premature death has also been recorded for several patients with NGLY1 deficiency. 2 As one would assume with the novelty of this disease, molecular mechanisms regarding how the loss of NGLY1 glycanase leads to the widely detrimental effects on human neurological development are mostly unknown. We chose to embark on this journey to elucidate the pathogenesis of this disease, but along the way also asked very pertinent questions revolving around the importance of the NGLY1 protein in other cell types. As an inherited disease, NGLY1 deficiency is a consequence of the mutant NGLY1 gene that has errors in translation within cells from the very beginning of life at the zygote, blastocyst, and other embryogenic stages. Evidenced by the developmental abnormalities observed in NGLY1-deficient patients, the normal function of NGLY1 plays a role in ensuring the normal development of human cells. Given that cancer is characterized as the cell's inability to regulate its proliferation and differentiation capacity, we also hypothesized that the dysregulation of NGLY1 could be involved also in the oncogenic process and cancerous development. Using melanoma as a proof-of-principle cancer type in our studies, the significance of abnormally expressed NGLY1 in human cancer cells has been revealed. In addition, we concluded that NGLY1 represents a novel and promising target that can be suppressed for anti-melanoma effects. Meanwhile, to elucidate the cellular and molecular mechanisms underlying the neurodevelopmental defects due to NGLY1 deficiency, we thought that a human cell-based platform that can closely model the early stage of cerebral development in humans would be necessary to properly and effectively investigate the role of NGLY1 in human brain development. The recent discoveries regarding human cerebral organoids and their ability to self-organize and give rise to cell layers similar to the structure of the human fetal brain made our intended research in the NGLY1-deficient development of the human cerebrum timely and feasible. 3 Organoids established using human induced pluripotent stem cells (hiPSCs) that are reprogrammed from somatic cells harvested from patients that carry pathogenic mutations provide even closer personalized disease modeling for pathogenesis that occurs in the human body. With the goal of building cerebral organoids using hiPSCs reprogrammed from noninvasively collected somatic cells that is suitable for studying neurodevelopment, we decided to test the production of cerebral organoids using hiPSCs reprogrammed from urine samplederived urinary epithelial cells (UECs). Although UEC-derived hiPSCs required specific protocol optimization to properly form cerebral organoids, the cellular and transcriptomic features of these organoids were comparable to those of cerebral organoids developed from embryonic stem cells. Our data offered direct evidence for the suitability, advantage, and possible limitations of using human urine sample-derived COs to model and study neurodevelopment. To tackle the question regarding how NGLY1 deficiency perturbs human brain development, we leverage a platform composed of NGLY1-functional and -deficient cerebral organoids to test our hypothesis that NGLY1 deficiency biases cell fates through compromising stress-adaptation capacity and secretory factor signaling in human cells undergoing neurogenesis. Our studies revealed that, compared with NGLY1-functional cerebral organoids, the NGLY1-deficient ones showed the premature differentiation of neural stem cells (NSCs), higher vulnerability to multiple stressors, and the reduction of secretory factors that are linked to neuroprotective and neurodevelopmental function. We concluded that NGLY1 may play critical roles in regulating stress responses and maintaining NSCs in cerebral development. NGLY1 deficiency-associated neurological abnormalities, including microcephaly, are likely due to the disruption of the NSC signaling modulated by NGLY1.Item Regulation of Supraoptic Vasopressin Neurons during Hypernatremia and Hyponatremia(2019-05) Balapattabi, Kirthikaa; Cunningham, J. Thomas; Mifflin, Steve W.; Schreihofer, Ann M.; Ma, Rong; Cunningham, Rebecca L.This dissertation addresses the regulatory mechanisms of arginine vasopressin (AVP) secretion during salt loading (SL) and liver failure. The experiments focused on AVP neurons located in the supraoptic nucleus (SON) of hypothalamus that, along with other hypothalamic neurons, secrete AVP. This dissertation has two sections. The first section, specific aims 1a and 1b, examines AVP secretion with SL. Salt loading is associated with increased AVP release and mean arterial pressure. The male SL (2% NaCl for 7 days) rats secrete AVP despite increase in mean arterial pressure and were used as an animal model for this aim to study altered AVP neuron regulation. Previous work has shown that SL impairs baroreceptor mediated inhibition of rat AVP neurons through brain derived neurotrophic factor (BDNF) dependent activation of tyrosine receptor kinase B (TrkB) and downregulation of K+/Cl- co-transporter (KCC2). This mechanism diminishes the GABAA mediated inhibition of SON AVP neurons by increasing intracellular chloride ([Cl]i). However, the source of BDNF leading to this ionic plasticity is unknown. In specific aim 1a, adeno-associated viral vectors with shRNA against BDNF were used to test if the SON is the source of BDNF contributing to increased AVP release and elevated mean arterial pressure in SL rats. In specific aim 1b, virally mediated chloride imaging with ClopHensorN was combined with single cell Western blot analysis by capillary based Simple Wes technology to verify the expression of KCC2 in the SON AVP neurons and to determine the role of TrkB-KCC2 mechanism in increased [Cl]i in SL male rats.In Aim 2, a more clinically relevant animal model was used to study regulatory mechanism leading to inappropriate increase in AVP secretion. Chronic bile duct ligated (BDL) rats were used as animal model of liver failure induced hyponatremia due to inappropriate AVP release. The findings and approaches from specific aim 1 were used to test the role of BDNF-TrkB-KCC2 signaling in increased AVP secretion and hyponatremia during liver failure. The experiments in this dissertation advance our understanding about the pathophysiology of AVP secretion. There are several novel findings from this work. First, SON was identified as the source of BDNF contributing to increase in [Cl]i of SON AVP neurons and AVP secretion in SL rats. Additionally, this is the first study to correlate the KCC2 protein expression in individual AVP neurons with its function using chloride imaging. Finally, these results are the first to demonstrate a mechanism that contributes to the increase in AVP secretion resulting in hyponatremia during liver failure.Item Sex and age differences in social and cognitive function in offspring exposed to late gestational hypoxia(BioMed Central Ltd., 2023-11-12) Mabry, Steve; Wilson, E. Nicole; Bradshaw, Jessica L.; Gardner, Jennifer J.; Fadeyibi, Oluwadarasimi; Vera, Edward, Jr.; Osikoya, Oluwatobiloba; Cushen, Spencer C.; Karamichos, Dimitrios; Goulopoulou, Styliani; Cunningham, Rebecca L.BACKGROUND: Gestational sleep apnea is a hypoxic sleep disorder that affects 8-26% of pregnancies and increases the risk for central nervous system dysfunction in offspring. Specifically, there are sex differences in the sensitivity of the fetal hippocampus to hypoxic insults, and hippocampal impairments are associated with social dysfunction, repetitive behaviors, anxiety, and cognitive impairment. Yet, it is unclear whether gestational sleep apnea impacts these hippocampal-associated functions and if sex and age modify these effects. To examine the relationship between gestational sleep apnea and hippocampal-associated behaviors, we used chronic intermittent hypoxia (CIH) to model late gestational sleep apnea in pregnant rats. We hypothesized that late gestational CIH would produce sex- and age-specific social, anxiety-like, repetitive, and cognitive impairments in offspring. METHODS: Timed pregnant Long-Evans rats were exposed to CIH or room air normoxia from GD 15-19. Behavioral testing of offspring occurred during either puberty or young adulthood. To examine gestational hypoxia-induced behavioral phenotypes, we quantified hippocampal-associated behaviors (social function, repetitive behaviors, anxiety-like behaviors, and spatial memory and learning), hippocampal neuronal activity (glutamatergic NMDA receptors, dopamine transporter, monoamine oxidase-A, early growth response protein 1, and doublecortin), and circulating hormones in offspring. RESULTS: Late gestational CIH induced sex- and age-specific differences in social, repetitive, and memory functions in offspring. In female pubertal offspring, CIH impaired social function, increased repetitive behaviors, and elevated circulating corticosterone levels but did not impact memory. In contrast, CIH transiently induced spatial memory dysfunction in pubertal male offspring but did not impact social or repetitive functions. Long-term effects of gestational CIH on social behaviors were only observed in female offspring, wherein CIH induced social disengagement and suppression of circulating corticosterone levels in young adulthood. No effects of gestational CIH were observed in anxiety-like behaviors, hippocampal neuronal activity, or circulating testosterone and estradiol levels, regardless of sex or age of offspring. CONCLUSIONS: Our results indicate that hypoxia-associated pregnancy complications during late gestation can increase the risk for behavioral and physiological outcomes in offspring, such as social dysfunction, repetitive behaviors, and cognitive impairment, that are dependent on sex and age. Sleep apnea during late pregnancy is a common pregnancy complication that can impact the brain development of children born to mothers with sleep apnea. Children with impaired brain development may present with decreased social skills, memory issues, anxiety, and compulsivity. It is unclear if there is a cause and effect relationship between sleep apnea during late pregnancy and behavioral changes in offspring. Additionally, it is unknown whether male or female sex or age of the offspring affects these relationships. In this study, we exposed pregnant rats to a model of sleep apnea called chronic intermittent hypoxia (CIH) within late gestation and examined the behavior of the offspring and brain activity during puberty and young adulthood. We found that CIH during late pregnancy had long-term effects in the offspring that were different in males and females. Notably, female offspring displayed social impairments in response to late gestation CIH, whereas male offspring displayed cognitive dysfunction.