Browsing by Author "Cunningham, Rebecca"
Now showing 1 - 20 of 35
- Results Per Page
- Sort Options
Item Androgen Receptors in the Middle Aged Male Rat Brain: Influence of Testosterone Deprivation on Expression(2017-03-14) Contreras, Jo; Cunningham, Rebecca; Fort*, Callie; Cuellar*, Elric; Lopez, Gladys; Metzger, Daniel; Oppong-Gyebi, Anthony; Schreihofer, Derek; Smith, Charity B.S.Purpose: 1) To determine whether long-term testosterone deprivation (LTTD) alters the levels and/or distribution of androgen receptors in the middle-aged male rat brain and 2) to determine whether testosterone replacement after LTTD influences androgen receptor levels. Methods: Twelve-month old male Fischer 344 rats were left intact or castrated for 2 weeks and replaced with subcutaneous implants containing testosterone (STTD). Additional groups were castrated for 10 weeks before being treated with testosterone (LTTD+T) or cholesterol (LTTD). Four weeks later, rats were euthanized and brains were collected for immunoblotting and immunohistochemistry (IHC) for androgen receptors (AR) using antibodies targeting the N-terminus or C-terminus of the protein. The cerebral cortex, hippocampus, thalamus, hypothalamus were examined, and testes were used as positive control tissue. Results: Contrary to expectations, the full-length AR (116 kDa) was barely detectable in the hippocampus and cerebral cortex by immunoblotting with antibodies directed to either end of the AR. Rather, smaller fragments were readily detected. Examination of the size of these fragments (~30, 37, 50, and 80 kDa) and consultation of the literature for the human AR, suggested that they represented calpain-dependent cleavage fragments. A series of control experiments was performed in an attempt to extract the full-length AR using rat testes as a positive control tissue. Protease inhibitors, EDTA, and the AR agonist dihydrotestosterone failed to reduce the appearance of fragments. Interestingly, the pattern of fragments from the hippocampus (80 [greater than] 37 kDa) differed from that from the cortex (37 [greater than] 80 kDa) suggesting differential processing. IHC of coronal brain sections though the forebrain revealed nuclear AR staining consistent with full-length AR in regions of high expression, including the hypothalamus. In agreement with immunoblotting AR staining in the cortex and hippocampus appeared to be cytoplasmic, rather than nuclear. No significant differences were observed between treatment groups. Conclusions: These data suggest that AR protein in some areas of the middle-aged male rat brain is rapidly degraded into fragments with altered localization and potential for transcriptional activity and/or signaling functions. Although no differences in expression were apparent between treatment groups, the differential processing of AR in the rat brain is a novel finding warranting further investigation.Item Chronic Intermittent Hypoxia Advances Hormonal Aging: Implications for Parkinson’s Related Sexual Dysfunction(2017-03-14) Schreihofer, Derek; Cunningham, Rebecca; Anderson, MarcPurpose: Chronic intermittent hypoxia (CIH) is an established model for sleep apnea and a common comorbidity in Parkinson’s disease (PD). Further, CIH is a known inducer of oxidative stress (OS), which is a key characteristic of PD and aging. Interestingly, in men both sleep apnea and PD are strongly linked with sexual dysfunction. However, it is unknown if CIH induces sexual dysfunction. Therefore, we examined the role of CIH on steroid hormones, sex behaviors, neuropeptides associated with social behaviors, and OS generation in young and old rats. Methods: Young (3-months) and old (12-months) male F344/BNF1 rats, were exposed to either mild CIH or normoxic conditions. CIH consisted of cycling oxygen levels from 21% to 10% over a span of 6 minutes during the rat’s sleep phase for a total of ten days. Sex behavioral tests were conducted to examine the influence of CIH. Specifically, the frequency and latencies of mounts, intromissions, and ejaculations were quantified. At the end of testing, plasma was collected and assayed for testosterone (T), corticosterone (C), vasopressin (AVP), oxytocin (OXY), and advanced oxidation protein products (AOPP). Results: Old rats had impaired sex behaviors compared to young rats. However, CIH induced sexual dysfunction in young rats, consistent with behaviors in old rats. Accordingly, in young rats CIH decreased T, increased C, and increased OS, as indicated by AOPP. CIH did not alter OXY and AVP in young rats. Interestingly, in old rats CIH had no effect on sexual behavior, T, C, OXY, or AVP, indicating that age may have a ceiling effect. Conclusions: Results show that mild CIH advances hormonal aging. Hormonal aging is an understudied phenomenon in PD and in sleep apnea. Therefore, PD progression may be halted by examining the influence of sleep apnea induced hormonal aging.Item Chronic Intermittent Hypoxia increases oxidative stress and inflammation(2015-03) Snyder, Brina; Shell, Brent; Cunningham, J. Thomas; Cunningham, RebeccaBackground: Inflammation has been linked with sleep apnea. Sleep apnea is a common comorbidity associated with neurodegenerative disorders, such as Parkinson’s disease and Alzheimer’s disease. Furthermore, neurodegenerative diseases have also been linked with inflammation. A possible mechanism underlying increased inflammation in these disorders is oxidative stress, a hallmark of neurodegeneration. To examine the role of oxidative stress on inflammation, we used chronic intermittent hypoxia (CIH), an established model for the hypoxemia associated with sleep apnea. CIH consists of recurring events of low oxygen followed by reoxygenation. Statement of Hypothesis: We hypothesize that CIH causes oxidative stress, which induces inflammation. Materials and methods: To test this hypothesis, plasma from adult male rats subjected to 7 days of CIH (3 minute periods of hypoxia (10% oxygen) and 3 minute periods of normoxia (21% oxygen) for 8 hours per day) or normoxia (room air) were tested for AOPP, an indicator of oxidative stress, and circulating inflammatory markers (such as IL-1b, IL-10, IL-4, IL-6). Additionally, a group of rats was administered a neurotropic AAV with shRNA for AT1a receptors in their forebrains and instrumented with telemetry for blood pressure recording prior to CIH treatment to determine the effects of angiotensin on CIH hypertension and oxidative stress. Significant results: Our results showed that CIH significantly increased circulating oxidative stress and inflammation. Interestingly, IL-1b, IL-2, and TNF-a inflammatory markers were associated with oxidative stress, unlike IL-10, IL-4, and IL-6 inflammatory markers. These markers were positively associated with IL-1b. Knockdown of angiotensin 1 receptors in the forebrain blocked the diurnal hypertension and CIH induced oxidative stress, indicating the involvement of CIH hypertension and central angiotensin receptors in CIH induced oxidative stress. Conclusions: These results indicate that both neurons and macrophages contribute to CIH induced oxidative stress and inflammation and that CIH oxidative stress and inflammation is dependent on central angiotensin receptors and CIH hypertension.Item Chronic intermittent hypoxia induces oxidative stress and inflammation in brain regions associated with neurodegeneration(2016-03-23) Shell, Brent; Cunningham, J. Thomas; Cunningham, Rebecca; Snyder, Brina D.Age is the highest risk factor for the development of neurodegenerative diseases (ND), such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). As life expectancy increases, the incidence of ND is projected to rise accordingly. Increased ND incidence will be associated with high healthcare costs. Currently, no cure exists for ND and diagnosis occurs at advanced stages, which foreshadows a financial healthcare crisis. Therefore, early identification of patients at risk for ND may provide opportunities for more effective therapies. Since ND is associated with increased oxidative stress (OS) and inflammation, these markers could be used to identify patients at risk for ND. Multiple environmental factors can be an oxidative stressor, and thus exacerbate inflammation induced ND risk. One such environmental factor that increases OS is sleep apnea (SA), a common ND comorbidity. However, it is unknown if SA induced oxidative stress activates neuroinflammation in areas associated with ND. To model SA in rats, chronic intermittent hypoxia was used. Male rats were exposed to six minute chronic intermittent hypoxia (CIH) cycles, during which oxygen levels were rapidly decreased from 21% to 10% then returned to normal room air levels, eight hours a day during the light phase for seven days. Plasma and tissue from hippocampus (HIPP), entorhinal cortex (ETC), substantia nigra (SN), rostral ventrolateral medulla (RVLM), and solitary tract nucleus (NTS) were collected and tested for levels of OS and inflammation, using Advanced Oxidative Protein Products (AOPP) and multiplex immunoassays, respectively. OS markers and inflammation were elevated in the plasma of rats exposed to CIH compared to control rats. Differences in neuroinflammatory markers within tissues were observed. Specifically, inflammatory markers in the RVLM were significantly decreased in animals exposed to CIH while TNF-a and IL-6 were elevated in the SN. TNF-a was positively associated with plasma OS and the cytokine associated with inflammatory cell recruitment, KC-Gro, exhibited the same pattern in the ETC. The ETC and SN are areas associated with initial neurodegenerative processes in AD and PD, respectively. CIH may contribute to processes involved in early ND pathology by elevating OS and inflammation in critical brain regions. These results indicate that SA can exacerbate ND by increasing OS-induced neuroinflammation. Therefore, treatment of SA could be one consideration in preventing ND.Item Chronic testosterone deprivation sensitizes the middle-aged rat brain to damaging effects of testosterone replacement(2020) Vann, Philip; Wong, Jessica; Sumien, Nathalie; Cunningham, Rebecca; Metzger, Daniel; Schreihofer, Derek; Oppong-Gyebi, Anthony; Contreras Garza, Jo; Kasanga, Ella; Smith, CharityHypothesis: We hypothesized that a delay in testosterone replacement therapy (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. Background: Levels of the hormone testosterone (T) fall in aging men. Recently, the number of men obtaining TRT has increased dramatically. However, other consequences of aging, such as oxidative stress, may result in detrimental effects when combined with TRT, including stroke risk. Methods: Twelve-month old male Fischer 344 rats were divided into 5 groups (n=9-14): 1) gonad Intact sham stroke (SH), 2) Intact stroke (IN), 3) short term castrate + T (ST), 4) long term castrate (LC), and 5) long term castrate + T (LCT). Rats were castrated 2 weeks (ST) or 10 weeks (LT, LCT) prior to T. Middle cerebral artery occlusion (Stroke) done via stereotaxic injection of endothelin 1 (ET1). Three, 7, and 14 days after stroke several behavior tests were done. Rats were humanely euthanized, and blood/brains were collected. Results: Plasma oxidative stress measured by Advanced Oxidative Protein Products (AOPP) was significantly negatively correlated with T levels. Long-term hypogonadism in middle-aged male Fischer 344 rats TRT exacerbated the detrimental behavioral effects of experimental focal cerebral ischemia. Conclusion: Data suggest that TRT after long-term hypogonadism can exacerbate functional recovery after focal cerebral ischemia.Item Determining the Viability of Biomarkers for Oxidative Stress Detection in Clinically Afflicted Cohorts(2015-03) Duong, Phong; Contreras, Jo G.; Snyder, Brina; O'Bryant, Sid; Cunningham, RebeccaIntro: In the US, there are more than 5 million Americans living with Alzheimer’s disease (AD) and approximately 500,000 more are suffering from Parkinson’s disease (PD). Oxidative stress (OS) and its deregulation of reactive oxygen species (ROS) have been implicated as a component of neurodegenerative diseases. Accumulation of ROS is accountable for increasing mitochondrial dysfunction that leads to neuronal apoptosis. In addition to ROS accumulation, OS can result in a number of cellular by-products such as Malondialdehyde (MDA), 8-Isoprostane, and Advanced Oxidation Protein Products (AOPP). Recent studies also suggest that high levels of testosterone can depress cellular resistance to oxidative stress. Purpose: The objective of this research is to determine the viability of laboratory assays as a detection tool for the evaluation of neurodegenerative diseases in aging males. Methods: 352 clinical plasma samples from the Texas Alzheimer’s Research and Care Consortium were used in assay evaluation. The samples were collected from either Caucasian or Hispanic males and evaluated based on 6 previously determined disease statuses. The AOPP Assay was used to detect chlorinated oxidation of plasma proteins. For AOPP, plasma samples were performed with a 1:7 dilution factor. Following preparation, the samples were then plated in duplicates and read at an absorbance of 340nm. To correlate with a previous testosterone study, a Testosterone ELISA was conducted with the use of Rabbit Anti-Free T-Antibody without sample dilution. Once plated, the samples were incubated for 60 minutes, washed, and read at an absorbance of 450nm. MDA can be accounted for in OS-resultant lipid peroxidation (LP). MDA levels in the clinical samples were examined using a Colorimetric TBARS Assay. The assay subjected the samples to boiling, centrifuging, and incubation in an ice bath. The supernatant was then removed, plated, and read at an absorbance of 532nm. Results: The absorbance reading from the AOPP assay and Colorimetric TBARS assay, yielded similar results across all six-disease statuses. Despite the differences in disease status, samples were determined to have triple the amount of MDA concentration compared the control. Conclusion: The similarity in values and discrepancy from the control indicates that AOPP and TBARS cannot accurately determine OS in banked plasma samples. T-ELISA is currently undergoing further scrutiny with the use of mass spectrometry.Item Effect of Acute Heat Exposure on the Pressor Response to a Voluntary Hypoxic Apnea: A Cross-tolerance Study.(2022) Saul, Benjamin; Hemingway, Holden; Richey, Rauchelle; Moore, Amy; Shokraeifard, Austin; Cope, Heidi; Yurvati, Albert; Cunningham, Rebecca; Smith, Michael; Romero, StevenThe pressor response induced by a voluntary hypoxic apnea is exaggerated in individuals with obstructive sleep apnea and is strongly correlated to sympathetic overactivity. Acute heat exposure alters neural control of blood pressure, but its effect on the pressor response to a voluntary hypoxic apnea has never been explored. Therefore, we tested the hypothesis that acute heat exposure would attenuate the pressor response to a voluntary hypoxic apnea, and thereby manifest as a form of physiological cross-tolerance. Eight adults (3 females, 26 +/- 2 yrs) were subjected to passive heat stress (water perfused suit) sufficient to increase body core temperature by 1.2°C. Voluntary hypoxic apneas were performed in duplicate before acute heat exposure (pre-heat) and in recovery when body core temperature returned to ≤ 0.3°C of baseline. Participants breathed gas mixtures varying FiO2 (21%, 16%, and 12%; randomized) for 1 min followed immediately by a 15 s end-expiratory apnea. Beat-by-beat arterial blood pressure (Finometer) and arterial oxygen saturation (finger pulse oximetry) were measured throughout. The pressor response was calculated as a difference between baseline mean arterial pressure and the peak response following each apnea. The change in arterial oxygen saturation during each apnea did not differ from pre-heat to recovery (FiO2 21%, pre-heat 0 +/- 1 % vs. recovery 0 +/- 2 %; FiO2 16%, pre-heat -4 +/- 1 % vs. recovery -4 +/- 2 %; FiO2 12%, pre-heat -8 +/- 3 % vs. recovery -10 +/- 4 %; P = 0.3 for interaction). The pressor response to a voluntary apnea was attenuated in recovery from acute heat exposure across all concentrations of FiO2 (FiO2 21%, pre-heat 19 +/- 8 mmHg vs. recovery 16 +/- 8 mmHg; FiO2 16%, pre-heat 27 +/- 8 mmHg vs. recovery 20 +/- 8 mmHg; FiO2 12%, pre-heat 33 +/- 11 mmHg vs. recovery 27 +/- 13 mmHg; P = 0.02 for main effect of time). These data suggest that acute heat exposure induces a cross-tolerance effect such that the pressor response to a voluntary hypoxic apnea is reduced. Acute heat exposure could improve hypertension in adults with obstructive sleep apnea, secondary to altered chemoreflex function and sympathetic neural control, and provide additional therapeutic options for this population to improve cardiovascular health.Item Impact of sex and hypoxia on brain region-specific expression of androgen receptor AR45 and G protein Gαq in young adult rats(2024-03-21) Wilson, Elizabeth; Bradshaw, Jessica; Mabry, Steve; Shrestha, Pawan; Gardner, Jennifer; Cunningham, RebeccaPurpose: Sex differences in oxidative stress-associated cognitive decline are influenced by sex hormone levels. However, little is known regarding the expression of hormone receptors in brain regions associated with cognitive function. Notably, oxidative stress-associated neuronal cell death is exacerbated through testosterone signaling via membrane-associated androgen receptor AR45 and G protein Gαq. The objective of this study was to elucidate the expression of AR45 and Gαq in brain regions associated with cognitive function. Additionally, we investigated whether chronic intermittent hypoxia (CIH), an oxidative stressor with sex-specific effects, would modulate AR45 and Gαq expression. Methods: Adult male and female Sprague-Dawley rats were exposed to CIH or normoxia for 14 days. We quantified AR45 and Gαq protein expression in various cognition-associated brain regions [dorsal hippocampal CA1, CA3, DG, and entorhinal cortex (ETC)] via western blotting. For comparisons, AR45 and Gαq protein expression were also assessed in brain regions outside the hippocampal-ETC circuit [thalamus (TH) and striatum (STR)]. Results: The highest AR45 levels were expressed in the CA1 while the lowest expression was observed in the STR. The highest Gαq levels were expressed in the DG and ETC while the lowest expression was observed in the TH. We observed no effect of sex on AR45 or Gαq expression regardless of brain region assessed. Similarly, there was no effect of CIH on AR45 expression in any of the brain regions examined. However, CIH exposure increased Gαq expression only in the CA3 regardless of sex. Conclusions: Our findings reveal enrichment of AR45 and Gαq protein expression within the hippocampal-ETC circuit, which is vulnerable to oxidative stress and neurodegeneration during cognitive decline. Moreover, our data suggest the CA3 is the most vulnerable region to CIH-mediated oxidative stress. Overall, these findings were observed in both sexes, indicating that there are no observed sex differences in AR45 and Gαq expression or their modulation by CIH.Item INDUCTION OF APOPTOSIS VIA TESTOSTERONE IN OXIDATIVELY DAMAGED DOPAMINERGIC CELLS(2014-03) Simmons, Olivia C.; Holmes, Shaletha S.; Cunningham, RebeccaFollowing an episode of stroke, reduction of blood supply to brain cells can lead to conditions of oxidative stress (OS) in brain cells, or neurons, specifically in the dopaminergic neurons. The loss of dopaminergic neurons manifests itself as Parkinson’s disease (PD). Classically, men have a higher incidence of developing PD post-stroke than females. This suggests a role of testosterone (T) in the development of PD after induction of OS in neurons. We postulate that T in OS-induced states will activate the pathway of apoptosis, or programmed cell death, via an enzyme called caspase to induce death of dopaminergic brain cells, and thus symptoms of PD. To test our hypothesis, we first pre-treated dopaminergic cell lines with hydrogen peroxide (H2O2) to simulate stroke-induced OS. The cells were then treated with differing concentrations of T (0, 1, 10, 100 nM), representing the physiologic ranges of T in humans. Expressions of pro-caspase-3 and pro-caspase-9, the uncleaved precursors to caspase-3 & caspase-9, respectively, in the cells were quantified using Western Blot analysis. Statistical significance of our findings was reported using ANOVA and Fisher’s post hoc analysis with SAS software. Our experiments showed a trend of decreased expression of pro-caspase-9, and a significant decrease in pro-caspase-3 expression in the H+T treatment conditions as compared to the control conditions. These results point to the apoptotic cell death pathway via caspase-3 and caspase-9 as the mechanism by which increased T levels lead to PD in stroke patients. Purpose (a): Following ischemic stroke, reduction of blood supply to brain cells can lead to conditions of oxidative stress (OS) in neurons, specifically in the dopaminergic neurons of the substantia nigra (SN). The loss of dopaminergic neurons manifests itself as Parkinson’s disease (PD). Classically, men have a higher incidence of developing PD post-stroke than females. This suggests a role of testosterone (T) in the development of PD after induction of OS in neurons. We postulate that T in OS-induced states will activate the caspase pathway of apoptosis to induce dopaminergic cell death, and thus symptoms of PD. Methods (b): To test our hypothesis, we first pre-treated N27 dopaminergic cell lines with hydrogen peroxide (H2O2) to simulate stroke-induced OS. The cells were then treated with differing concentrations of T (0, 1, 10, 100 nM), representing the physiologic ranges of T in humans. Expressions of pro-caspase-3 and pro-caspase-9, the uncleaved precursors to caspase-3 & caspase-9, respectively, in the cells were quantified using Western Blot analysis. Statistical significance of our findings was reported using ANOVA and Fisher’s post hoc analysis with SAS software and p <0.05 as significant. Results (c): Our experiments showed a trend of decreased expression of pro-caspase-9, and a significant decrease in pro-caspase-3 expression in the H+T treatment conditions as compared to the control conditions. Conclusions (d): These results point to the apoptotic pathway via caspase-3 and caspase-9 as the mechanism by which increased T levels lead to PD in stroke patients.Item Influence of Testosterone Deprivation and Replacement on Cognition and Oxidative Stress in Middle-Aged Male Rats(2017-03-14) Contreras, Jo; Metzger, Daniel; Oppong-Gyebi, Anthony; Kasanga, Ella; Vann, Philip; Sumien, Nathalie; Cunningham, Rebecca; Schreihofer, Derek; Smith, Charity B.S.Purpose: Data from aged men suggests a negative correlation between testosterone levels and cognitive function, including the development of mild cognitive impairment and Alzheimer’s disease. The purpose of this study was to 1) determine whether long-term testosterone deprivation (LTTD) impairs cognition and increases oxidative stress in the middle-aged male rat brain and 2) determine whether testosterone (T) replacement after LTTD can reverse these effects. Methods: Twelve-month old male Fischer 344 rats (13 per group) were left intact or castrated for 2 weeks and replaced with subcutaneous implants containing T (short-term T deprivation; STTD). Additional groups were castrated for 10 weeks before being treated with T (long-term T deprivation; LTTD+T) or cholesterol (LTTD). Rats underwent cognitive testing with the Morris water maze (MWM). A 4-day acquisition phase was used for rats to learn the location of a hidden platform. A retention day was used to determine whether rats remembered the platform location after it was removed. A 2-day reversal trial in which the platform was moved to a new location was used to examine mental flexibility. These tests require both hippocampal and cortical areas of the brain. Following MWM rats were euthanized and brains were collected for immunoblotting for markers of cell death (Spectrin) and oxidative stress responses (NFkB, COX2, NOX2) in the hippocampus and cerebral cortex. Plasma advanced oxidative protein products (AOPP) were used as a peripheral marker of oxidative stress. Total testosterone was measured by ELISA. Results: Castration reduced total testosterone to 40% of intact levels whereas testosterone implants increased levels back to those of intact males. Overall, intact rats performed significantly worse on the MWM than STTD and LTTD with or without T replacement. We saw no significant changes in blood AOPP among treatment groups. Similarly, there were no significant differences in the expression of oxidative stress regulated genes or Spectrin cleavage in the hippocampus. Cortical measurements are on-going. Conclusions: These data suggest that castration with or without T replacement improves cognitive function in middle-aged rats, but does not significantly alter oxidative stress in the brain or periphery. These data support the safety profile of testosterone replacement to physiological levels and do not recapitulate correlative data observed in men.Item Is Testosterone a Risk Factor for Ischemic Stroke and Neurodegeneration in Men?(2016-03-23) Cunningham, Rebecca; Holmes, Shaletha S.In the aging population, cardiovascular disease (i.e. stroke) is a common cause of mortality that affects 1 in 3 men. An ischemic event is characterized by oxidative stress (OS) and neuroinflammation. Testosterone is an oxidative stressor, which can be protective or detrimental depending on the environment. In aging males that have high levels of OS, testosterone can increase the risk for ischemic stroke. However, it is unknown if testosterone-induced OS can increase inflammation, such as COX2, a prominent mediator of neuroinflammation and oxidative stress, leading to neuronal death. Therefore, we hypothesize that testosterone, under OS conditions, will further increase OS, induce COX2 inflammation, and increase apoptosis. To test this hypothesis, we exposed a neuronal cell line (N27 cells) to a sublethal concentration of the pro-oxidant, tert-butyl hydrogen peroxide (H2O2) for 24 hours followed by the exposure to physiological levels of testosterone to assess oxidative stress and inflammatory signaling. In addition, N27 cells were exposed to ibuprofen prior to OS (H2O2) and hormone (testosterone) treatment. Under OS conditions, testosterone increased COX2 signaling and apoptosis in neurons. Further, ibuprofen attenuated the effects of testosterone in an OS environment. Our data shows testosterone promotes COX2 inflammation, which contributes to neurodegeneration in an OS environment. Notably, ibuprofen is a common and inexpensive over the counter anti-inflammatory therapeutic. Thus, ibuprofen may be a preventative intervention against ischemic events and neurodegeneration.Item Long-term effects of late gestational maternal hypoxic stress on mood disorders: Sex and age differences(2021) Mabry, Steve; Wilson, Elizabeth; Rybalchenko, Nataliya; Engelland, Rachel; Fadeyibi, Oluwadarasimi; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, RebeccaPURPOSE: In utero insults have been linked with increased fear and anxiety in progeny. In utero hypoxic stress is also associated with multiple gestational complications. We hypothesized that exposure to maternal hypoxia during late gestation will have a long-term impact on anxiety in progeny. METHODS: Pregnant female Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia: 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of 3 min hypoxia (10% O2) and 3 min normoxia for a total of 10 CIH cycles/hour. At weaning (PND 28), progeny was pair-housed with a conspecific of same sex and similar weight. To examine anxiety disorders, we quantified anxiety-related behaviors (time spent in center of open field arena, marble burying test, social and anti-social behaviors with conspecifics) along with quantifying food intake and circulating sex hormone levels during puberty (postnatal day, PND 40-45) and young adulthood (PND 60-65) in male and female progeny. RESULTS: Maternal CIH did not impact circulating sex hormones or food intake, regardless of sex or age of progeny. However, maternal CIH increased anxiety related behaviors in pubertal females but were not observed in young adulthood. Maternal CIH did not impact male progeny, regardless of age. CONCLUSIONS: Maternal CIH during gestation resulted in increased anxiety related behaviors in pubertal female progeny. Maternal hypoxia during late gestation may temporarily increase the risk for anxiety disorders in pubertal females.Item Long-term effects of late gestational maternal hypoxic stress on neurodegeneration: Sex and age differences(2021) Wilson, Elizabeth; Mabry, Steve; Rybalchenko, Nataliya; Engelland, Rachel; Fadeyibi, Oluwadarasimi; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, RebeccaIntroduction: In utero insults can lead to onset of neurodegenerative diseases, such as Parkinson's disease (PD). In utero hypoxic insults are associated with maternal sleep apnea or preeclampsia. It is unknown whether late gestational maternal hypoxic insults have long-term effects on brain regions associated with PD, such as the nigrostriatal pathway. We hypothesized that late gestational maternal hypoxia will result in sustained nigrostriatal impairment in male and female progeny. Methods: Timed pregnant Long-Evans rats were exposed to five days (gestational days: 15-20) of chronic intermittent hypoxia (CIH) or room air (normoxia 21% O2) for 8 hours during their sleep phase. Each CIH cycle was 6 min of alternating 3 min hypoxia (10% O2) and normoxia (21% O2) totaling 10 CIH cycles/hour. Gestational age and biometrics were recorded 12-16 hours after birth. At postnatal day, PND 28, progeny were pair-housed with a conspecific of the same sex and similar weight. We focused on PD associated oxidative stress and behavioral impairments in the nigrostriatal pathway. Gross motor (open field), fine motor (ultrasonic vocalizations), and cognition (spatial memory) were examined during puberty and young adulthood. Results: Maternal CIH had no effect on gestational age, progeny biometrics, or progeny circulating oxidative stress. Gross motor and cognitive functions were unaffected by maternal CIH. However, a sustained fine motor impairment was observed in both male and female progeny. Conclusion: Maternal hypoxia during late gestation induced sustained nigrostriatal pathway impairment, which may increase the risk for neurodegeneration.Item Long-term effects of prenatal chronic intermittent hypoxia insult on the substantia nigra(2021) Engelland, Rachel; Fadeyibi, Oluwadarasimi; Rybalchenko, Nataliya; Wilson, Elizabeth; Mabry, Steve; Osikoya, Oluwatobiloba; Cushen, Spencer; Goulopoulou, Styliani; Cunningham, RebeccaPurpose: Prenatal chronic intermittent hypoxia (CIH) was employed to evaluate the effects of hypoxic insults on the substantia nigra (SN), which is impacted by Parkinson's disease (PD). SN loss during PD is linked with oxidative stress (OS) and apoptosis. We hypothesized that exposure to late gestational maternal hypoxia would result in an increase in increased OS, but not apoptosis, in the SN of adult male and female progeny. Methods: During gestational days 15-20, pregnant Long-Evans rats were exposed to CIH or room air (normoxia) for 8 hours. CIH consisted of 3 min hypoxia (10% O2) and 3 min normoxia (21% O2). Animals were sacrificed at puberty (PND 44) or adulthood (PND 66). SN micropunches were obtained. OS was quantified by measuring calpain cleavage of spectrin. Results: OS (calpain cleavage of spectrin) was increased in the SN of adult male and female rats exposed to prenatal CIH compared to control (F1,17 = 3.606; p = 0.075). No effects on OS were observed in pubertal rats. Apoptosis (caspase-3 cleavage of spectrin) was not observed in any of the groups. Conclusions: These data suggest that prenatal CIH programming has a long-lasting impact on the SN of adult progeny, which may increase the susceptibility of SN to damage and PD risk. Although no sex differences were observed in this pilot study, we may see a sex effect upon increasing animal number, especially in male rats. This is consistent with the higher incidence of PD in men than in women.Item Mechanisms Underlying Membrane Androgen Receptor-Induced Neurodegeneration(2019-03-05) Duong, Phong; Wilson, Elizabeth; Cunningham, Rebecca; Tenkorang, MavisPurpose: A common characteristic of several neurodegenerative disorders is oxidative stress (OS). Many neurodegenerative disorders are more prevalent in men and postmenopausal women. Our lab has shown testosterone via a non-genomic mechanism exacerbates OS damage in neurons. Indeed, our lab was the first to discover the presence of the androgen receptor (AR) splice variant, AR45, in the brain. We found testosterone can initiate signaling cascades via this membrane associated AR (mAR), leading to increased OS. However, the mechanism for OS generation is unknown. NADPH Oxidase 1 and 2 (NOX 1/2) are major OS generators, and potential targets for androgen-induced OS and cell death. Based on our studies showing protein-protein interactions between NOX1/2, AR45 and Gαq, we hypothesize testosterone increases OS by activating mAR complexed with NOX 1/2, initiating IP3 signaling. Method: Using an immortalized neuronal cell line (N27 cells), we exposed cells to hydrogen peroxide (H2O2) prior to testosterone (100 nM) or DHT-BSA (500nM). Inhibitors were used to examine AR, IP3 and NOX1/2 signaling. Cell viability and OS were quantified. In addition to in vitro experiments, we examined the effects of NOX 1/2 on DHT exacerbation of chronic intermittent hypoxia, CIH (AHI=10) induced OS by treating adult male Long Evans rats with the NOX1/2 inhibitor, apocynin (4mg/kg). Results: Classical AR antagonists did not block testosterone’s negative effects, indicating classical AR does not mediate these effects. Since AR antagonists do not block mAR, we used an AR protein degrader, ASC-J9 (5uM). ASC-J9 blocked testosterone’s negative effects. Next, we examined signaling cascades associated with proteins complexed with mAR-AR45. To block NOX actions, we used apocynin (10 uM). Apocynin did not alter H2O2-induced cell loss, indicating H2O2 increases OS via a non-NOX mechanism. However, apocynin completely blocked testosterone induced cell loss and OS, suggesting the involvement of NOX1/2. Consistent with our in vitro data, apocynin also decreased OS generation in DHT-treated rats exposed to CIH, during sleep phase for 7 days. Inhibition of IP3 receptor blocked testosterone’s negative effects, indicating that testosterone may activate IP3 signaling via the mAR-NOX complex. Conclusion: NOX and IP3 play a crucial role in mAR-induced neurodegeneration. Future studies will examine the mAR-NOX complex as a therapeutic target for neurodegenerative diseases.Item Mild CIH Does Not Induce Cell Loss in the Substantia Nigra(2017-03-14) Snyder, Brina; Cunningham, Rebecca; Davis, WilliamPurpose: Sleep apnea severity has been associated with Parkinson’s disease (PD) severity in men. Chronic intermittent hypoxia (CIH) is an animal model for sleep apnea. Mild CIH increases oxidative stress (OS) and inflammation in substantia nigral dopamine neurons, a neuron type lost in PD. Currently there is no model for early stage PD, wherein cell loss is not evident. Clinical symptoms of PD are not observed until about 80% of the substantia nigra (SN) is lost. It is unknown what causes PD, nor is there a cure for PD. The purpose of this study was to determine if CIH impacted neuronal viability in the SN in order to establish an early stage PD model. Methods: Gonadally intact male Sprague Dawley rats were exposed to either room air (normoxia) or six-minute chronic intermittent hypoxia (CIH) cycles, during which oxygen levels were rapidly decreased from 21% to 10% then returned to normal room air levels, eight hours a day during the light phase for seven days. Animals were perfused and brain tissue containing the SN was prepared for 8-OhDg (OS damage marker) and DAPI (cell nuclear marker) immunohistochemical staining. Afterwards, tissue sections were mounted and imaged to analyze the specific effects of CIH on OS damage, cell nuclear size, and cell number. Specifically, 8-OhDg and DAPI expression within the SN were summed and averaged across multiple sections of the SN. Comparisons were made between normoxia and CIH groups. In addition to OS damage and cell number, cell nuclear sizes were quantified and averaged across sections. 8-OhDg and DAPI staining were visualized using a digital camera on fluorescent microscope. Results: CIH increased OS, as shown by increased 8-OhDg expression, in the SN compared to normoxia. No significant differences in cell number or cell nuclear size were found between CIH and normoxia. Conclusions: This is the first study to show that mild CIH does not alter SN cell number or nuclear size, even though CIH increases OS damage in cells. These results support the use of CIH as an early stage animal model for PD. Data generated from this model aid in the understanding of the PD and its pathophysiology.Item NADPH oxidase (NOX1) mediates testosterone-induced neurodegeneration(2018-03-14) Duong, Phong; Cunningham, Rebecca; Tenkorang, MavisBackground: Parkinson’s disease (PD) has been recorded as the second most common neurological disease. Oxidative stress (OS) plays a key role in the pathogenesis of PD. Several studies have established that Parkinson’s disease (PD) is sex biased, affecting more men than women. Testosterone, a primary male sex hormone and a known oxidative stressor, has been implicated in PD. Previous studies in our lab have shown that testosterone via a non-genomic mechanism exacerbates OS damage in dopaminergic neurons. However, the mechanism by which testosterone increases OS is unknown. We found that testosterone acts through a membrane associated androgen receptor (mAR) variant – AR45 leading to the activation of proinflammatory mediators; NF-κB and COX2. NADPH Oxidase 1 (NOX 1) is a major OS generator in cells, hence a potential contributor to the pathogenesis of neurological diseases. It is possible that NOX 1 complexes with the mAR to mediate this destructive process. Purpose: The primary objective of this study is to determine the underlying mechanism by which testosterone increases OS in dopaminergic neurons. We therefore hypothesize that in dopaminergic cells, testosterone increases oxidative stress by activating NOX 1. Ultimately, our goal is to identify pathways regulated by testosterone in dopaminergic neurons in order to provide effective pharmacological targets to enhance the treatment of PD. Methods: We used a dopaminergic cell line (N27 cells). For an oxidative stressor, we used tert-butyl-hydrogen peroxide (H2O2) to induce 20% cell loss prior to testosterone (100nm) administration. NOX 1 inhibitor, Apocynin was administered before H2O2 exposure. To examine membrane associated androgen receptor and not the classical androgen receptor, we used cell impermeable DHT-BSA (500 nM) to confirm that NOX 1’s effect is through a non-genomic mechanism. Cell viability and OS were quantified using the MTT and Reduced Thiols assays respectively. To determine if NOX 1 interacts with a mAR, we immunoprecipitated the mAR and probed for NOX 1. Results: Apocynin alone had no effect on cell viability and OS. Further, Apocynin alone, did not alter H2O2-induced cell loss, indicating that H2O2 increases OS via a non-NOX 1 mechanism. However, Apocynin blocked testosterone’s induced cell loss and OS generation suggesting that NOX 1 mediates testosterone’s damaging effects in an OS environment. Inhibition of NOX 1 also blocked DHT-BSA’s damaging effects on cell viability in an OS environment. NOX 1 protein also complexes with the mAR. Conclusion: Testosterone-induced cell loss is mediated by a NOX1/mAR complex, indicating that NOX 1 is involved in testosterone-induced OS generation. The findings of these experiments provide a better insight into testosterone’s role in neurodegeneration and its underlying mechanism.Item NADPH Oxidase (NOX1) Mediates Testosterone-Induced Neurodegeneration (2017)(2017-03-14) Cunningham, Rebecca; Tenkorang, MavisPurpose: One of the primary characteristics of Parkinson’s disease (PD) is oxidative stress (OS). Men have a higher risk for PD than women. Testosterone, a primary male sex hormone has been implicated in PD, and is a known oxidative stressor. Previous studies in our lab have shown that testosterone exacerbates OS damage in dopaminergic neurons. However, the mechanism by which testosterone increases OS is unknown. We hypothesize that in dopaminergic cells, testosterone increases OS by activating NOX 1, a major OS generator in cells. Methods: To test our hypothesis, we used a dopaminergic cell line (N27 cells). For an oxidative stressor, we used tert-butyl-hydrogen peroxide (H2O2) to induce 20% cell loss prior to testosterone (100nm) administration. NOX1 inhibitors (Apocynin, Diphenyleneiodonium-DPI) were administered before H2O2 exposure. Cell viability was quantified using the MTT assay. Results: Testosterone is only damaging in the presence of OS. DPI, alone, was damaging to N27 cells, hence this was no longer used as a NOX1 inhbitor. Unlike DPI, Apocynin had no effect on cell viability. Further, Apocynin did not alter H2O2-induced cell loss, indicating that H2O2 increases OS via a non-NOX1 mechanism. However, Apocynin blocked testosterone’s damaging effects in an oxidative stress environment. Conclusions: Testosterone-induced cell loss is mediated by NOX1, indicating that NOX1 is involved in testosterone induced OS generation. By understanding testosterone’s mechanism of action, potential therapeutic targets for Parkinson’s disease can be explored.Item Novel Androgen Receptor Protein in Brain: Implication for Parkinson's Disease(2017-03-14) Duong, Phong; Snyder, Brina; Cunningham, Rebecca; Garza-Contreras, JoObjective: Men have a two-fold increased risk for Parkinson’s disease (PD) than women. Testosterone, the major male sex hormone, 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 Gaq protein-coupled receptor (GPCR) that is involved in calcium mobilization. Currently, the mAR remains unidentified. Recent studies only found miniscule levels of androgen receptors (AR) in the substantia nigra (SN). This low AR expression in the SN may be due to absence of full length classical AR that contains an N terminus domain (NTD), especially as these studies used an antibody targeting the AR NTD. It is possible that ARs in the SN consist of a splice variant that does not possess a NTD, such as AR45. AR45 is not able to be assayed using an NTD antibody, and thus we used a C-terminus domain (CTD) antibody. Therefore, we hypothesize that the putative mAR is the AR45 splice variant that acts through a Gaq GPCR. Materials and Methods: We examined the expression of classical full length AR and AR45 in a dopaminergic N27 cell line and rat SN. Protein expression of AR and AR45 was quantified by Western blot analysis and immunohistochemistry (IHC). We used antibodies targeting either the NTD or CTD of the AR, along with antibodies targeting Gαq, Gαs and Gαo GPCRs. To determine the association between mAR and GPCR subunits we performed co-immunoprecipitation using AR-CTD and Gaq antibodies. Results: Our results showed that the SN and the N27 cells express very low AR-NTD positive cells, indicative of low full length classical AR expression. However, both N27 cells and SN showed very high levels of AR-CTD positive cells. Furthermore, protein expression of AR-CTD was observed at 45 kDa molecular weight, which is consistent with the AR splice variant, AR45. This AR45 splice variant was found to be associated with Gaq in both N27 cells and SN. Conclusions: Our data indicates that the mAR in dopaminergic neurons is the AR45 splice variant, which is associated with a Gaq subunit. These results provide a mechanism for our prior studies wherein testosterone increased intracellular calcium levels. This is the first observation of an AR splice variant in neuronal tissue. Further characterization of this protein may provide a novel therapeutic target to slow the progression of PD in men.Item Oxidative Stress and Release of Cell-free Mitochondrial DNA from Trophoblast Cells(2022) Gardner, Jennifer; Cushen, Spencer; Bradshaw, Jessica L.; Garlotte, Isabelle; Phillips, Nicole; Cunningham, Rebecca; Goulopoulou, StylianiCell free mitochondrial DNA (mtDNA) is an indicator of cellular stress and systemic inflammation. These properties are accentuated when mtDNA undergoes oxidative damage. In addition, toll-like receptor 9 (TLR9), a receptor of the innate immune system, is activated by mtDNA. Inflammation, oxidative stress, and cell death are characteristics of placental ischemia, a common feature of preeclampsia. Recent work from our lab has shown dysregulation of circulating cell-free mtDNA in pregnancies with preeclampsia and association of this dysregulation with preeclampsia diagnosis. However, mechanisms underlying the release of mtDNA remain unclear. We hypothesized that human trophoblast cells exposed to oxidative stress via antimycin A, an inhibitor of complex III of the electron transport chain, would induce release of mtDNA via cell death-dependent mechanisms, leading to increased TLR9 activation. BeWo cells (ATCC? CCL-98) were treated with increasing concentrations of antimycin A (10, 50, 100, 320 µM) and vehicle (ethanol, 0.16% v/v) for 4 hours. Supernatants were collected and snap frozen in liquid nitrogen. Absolute real-time qPCR quantification with TaqMan™ probes and chemistry was used to quantify cell-free mtDNA (amplification target: MT-ND5 gene) and nuclear DNA (nDNA). Flow cytometry was used to assess the activation of cell death mechanisms in response to oxidative stress. To determine TLR-9-associated immunostimulatory potency of cell culture supernatants, we used an engineered cell line of human embryonic kidney 293 cells transfected with a human TLR-9 gene (HEK-BlueTM hTLR9). Exposure of trophoblast cells to antimycin A did not induce the release of mtDNA (p>0.05) or nDNA (p>0.05). Similarly, there were no differences in TLR9 activation between groups (p>0.28). Antimycin A (320 µM) reduced cell viability (Vehicle: 64.44 ± 5.46% vs Antimycin A: 18.14 ± 5.78%, p< 0.05) and increased necrosis (Vehicle: 10.39 ± 3.11% vs Antimycin A (100, 320 µM): 30.51 ± 4.43%, 40.16 ± 5.08%, P< 0.05), while apoptosis levels remained unchanged (P>0.1). Activation of oxidative stress pathways, via inhibition of complex III of the electron transport chain, leads to cell death, but does not affect release of mtDNA. These data suggest other cellular mechanisms, such as mitophagy or activation of antioxidant pathways, may serve a cytoprotective role against oxidative stressors in trophoblast cells. This study extends our pre-clinical knowledge about the links between placental oxidative stress and immunogenic factors in trophoblast cells. These findings may contribute to development of novel therapeutic targets for treatment of maternal cardiovascular dysfunction in preeclampsia.