Rebecca L. Cunningham

Progesterone Increases the Release of Brain-Derived Neurotrophic Factor from Glia via Progesterone Receptor Membrane Component 1 (Pgrmc1)-Dependent ERK5 Signaling

Progesterone (P4) is cytoprotective in various experimental models, but our understanding of the mechanisms involved is still incomplete. Our laboratory has implicated brain-derived neurotrophic factor (BDNF) signaling as an important mediator of P4s protective actions. We have shown that P4 increases the expression of BDNF, an effect mediated by the classical P4 receptor (PR), and that the protective effects of P4 were abolished using inhibitors of Trk receptor signaling. In an effort to extend our understanding of the interrelationship between P4 and BDNF signaling, we determined whether P4 influenced BDNF release and examined the role of the classical PR and a putative membrane PR, progesterone receptor membrane component-1 (Pgrmc1), as mediators of this response. Given recent data from our laboratory that supported the role of ERK5 in BDNF release, we also tested whether P4-induced BDNF release was mediated by ERK5. In this study, we found that P4 and the membrane-impermeable P4 (P4-BSA) both induced BDNF release from cultured C6 glial cells and primary astrocytes. Both these cells lack the classical nuclear/intracellular PR but express high levels of membrane-associated PR, including Pgrmc1. Using RNA interference-mediated knockdown of Pgrmc1 expression, we determined that P4-induced BDNF release was dependent on the expression of Pgrmc1, although pharmacological inhibition of the PR failed to alter the effects of P4. Furthermore, the BDNF release elicited by P4 was mediated by ERK5, and not ERK1/2. Collectively, our data describe that P4 elicits an increase in BDNF release from glia via a Pgrmc1-induced ERK5 signaling mechanism and identify Pgrmc1 as a potential therapeutic target for future hormone-based drug development for the treatment of such degenerative diseases as Alzheimers disease as well as other diseases wherein neurotrophin dysregulation is noted.

Androgens induce dopaminergic neurotoxicity via caspase-3-dependent activation of protein kinase Cδ

Aged men have a greater incidence of Parkinsons disease (PD) than women. PD is a neurodegenerative condition associated with the loss of dopamine neurons in the nigrostriatal pathway. This study examined the neurotoxic effects of androgens in a dopaminergic cell line (N27 cells) and the downstream signaling pathways activated by androgens. Treatment of N27 cells with testosterone- and dihydrotestosterone-induced mitochondrial dysfunction, protein kinase C (PKC)-delta cleavage, and apoptosis in dopaminergic neuronal cells. Inhibition of caspase-3 prevented the cleavage of PKCdelta from the full-length element to the catalytic fragment and apoptosis in N27 cells, suggesting that androgen-induced apoptosis is mediated by caspase-3-dependent activation of PKCdelta. Androgen-induced apoptosis may be specific to dopamine neurons as evidenced by a lack of testosterone-induced apoptosis in GnRH neurons. These results support a neurotoxic consequence of testosterone on dopaminergic neurons and may provide insight into the gender bias found in PD.

Pubertal exposure to anabolic androgenic steroids increases spine densities on neurons in the limbic system of male rats.

Human studies show that the number of teenagers abusing anabolic androgenic steroids (AAS) is increasing. During adolescence, brain development is altered by androgen exposure, which suggests that AAS may potentially alter central nervous system (CNS) development. The goal of the present study was to determine whether pubertal AAS exposure increased dendritic spine densities on neurons within the medial amygdala and the dorsal hippocampus. Pubertal gonadally intact male rats received the AAS testosterone propionate (5 mg/kg) or vehicle for 5 days/week for 4 weeks. To determine the long-term implications of pubertal AAS use, another set of males received the same AAS treatment and was then withdrawn from AAS exposure for 4 weeks. Results showed that pubertal AAS exposure significantly increased spine densities on neurons in the anterior medial amygdala, posterodorsal medial amygdala, and the cornu ammonis region 1 (CA1) of the hippocampus compared with gonadally intact control males. Spine densities returned to control levels within the anterior medial amygdala and the posterodorsal medial amygdala 4 weeks after withdrawal. However, spine densities remained significantly elevated after AAS withdrawal in the CA1 region of the hippocampus, suggesting that pubertal AAS exposure may have a long-lasting impact on CA1 hippocampal neuroanatomy. Since pubertal AAS exposure increased spine densities and most excitatory synapses in the CNS occur on dendritic spines, AAS may increase neuronal excitation. It is proposed that this increase in excitation may underlie the behavioral responses seen in pubertal AAS-treated male rats.

Oxidative Stress, Testosterone, and Cognition among Caucasian and Mexican-American Men with and without Alzheimer's Disease

BACKGROUND The use of testosterone among aging men has been increasing, but results from studies addressing the effectiveness of testosterone replacement therapy have been equivocal. OBJECTIVE Given our prior pre-clinical studies that reported a major influence of oxidative stress on testosterones neuroprotective effects, we investigated whether the negative effects of testosterone on brain function were predicted by oxidative load. METHODS In order to test our hypothesis, we determined whether circulating total testosterone and luteinizing hormone correlated with cognition in a subset of the Texas Alzheimers Research & Care Consortium (TARCC) cohort, consisting of Caucasian (n = 116) and Mexican-American (n = 117) men. We also assessed whether oxidative stress (as indexed by homocysteine levels) modified this relationship between sex hormones and cognition, and whether the levels of two antioxidants, superoxide dismutase-1 and glutathione S-transferase (GST), varied as a function of circulating testosterone. RESULTS In a low oxidative stress environment, testosterone was positively associated with the level of the antioxidant, GST, while no deleterious effects on cognitive function were noted. In contrast, under conditions of high oxidative stress (homocysteine levels >12 μmol/L), testosterone and luteinizing hormone were associated with cognitive impairment, but only among Caucasians. The ethnic difference was attributed to significantly higher GST levels among Mexican-Americans. CONCLUSION While testosterone may be beneficial under conditions of low oxidative stress, testosterone appears to have negative consequences under conditions of elevated oxidative stress, but only in Caucasians. Mexican-Americans, however, were protected from any deleterious effects of testosterone, potentially due to higher levels of endogenous antioxidant defenses such as GST.

Androgens exacerbate motor asymmetry in male rats with unilateral 6-hydroxydopamine lesion.

Parkinsons disease (PD) is a progressive neurodegenerative disorder characterized by dopamine neuron loss in the nigrostriatal pathway that shows greater incidence in men than women. The mechanisms underlying this gender bias remain elusive, although one possibility is that androgens may increase dopamine neuronal vulnerability to oxidative stress. Motor impairment can be modeled in rats receiving a unilateral injection of 6-hydroxydopamine (6-OHDA), a neurotoxin producing nigrostriatal degeneration. To investigate the role of androgens in PD, we compared young (2 months) and aged (24 months) male rats receiving gonadectomy (GDX) and their corresponding intact controls. One month after GDX, rats were unilaterally injected with 6-OHDA, and their motor impairment and asymmetry were assessed 2 weeks later using the cylinder test and the amphetamine-induced rotation test. Plasma samples were also collected to assess the concentration of testosterone and advanced oxidation protein products, a product of oxidative stress. GDX decreased lesion-induced asymmetry along with oxidative stress and increased amphetamine-induced rotations. These results show that GDX improves motor behaviors by decreasing motor asymmetry in 6-OHDA-treated rats, an effect that may be ascribed to increased release of striatal dopamine and decreased oxidative stress. Collectively, the data support the hypothesis that androgens may underlie the gender bias observed in PD.

Prepubertal Social Subjugation and Anabolic Androgenic Steroid-Induced Aggression in Male Rats

Abused children are more prone to abuse drugs, such as anabolic androgenic steroids (AAS), as teenagers and display violence as adults. AAS use has been linked with elevated aggression. Thus, exposure to child abuse and AAS may potentiate aggression. A social subjugation paradigm was used as an animal model of childhood abuse to determine whether prior subjugation increases AAS‐induced aggression in male rats. Prepubertal gonadally intact male rats were exposed to social subjugation, a novel cage experience, or remained undisturbed in their home cages. Experimental males were socially subjugated by being placed in the home cage of an adult male. At puberty, both subjugated and nonsubjugated rats were injected with either the AAS testosterone or vehicle. AAS treatment continued for 5 weeks. Aggression was measured during the last week of AAS exposure. AAS was then discontinued. Aggression was again tested 12 weeks after AAS withdrawal. Aggression was tested under three conditions: (i) physical provocation of the experimental male; (ii) provocation of the intruder male; and (iii) without provocation. Both AAS‐treated males and socially subjugated males displayed significantly more aggression than did controls. Elevated aggression by subjugated males was still present 17 weeks after social subjugation. AAS males also showed increased aggression 12 weeks after AAS withdrawal. However, exposure to both social subjugation and AAS had no long‐term effects on aggression. The results of the present study indicate that social subjugation may have lasting consequences on the expression of adaptive social behaviours.

Androgen receptors, sex behavior, and aggression.

Androgens are intricately involved in reproductive and aggressive behaviors, but the role of the androgen receptor in mediating these behaviors is less defined. Further, activity of the hypothalamic-pituitary-gonadal axis and hypothalamic-pituitary-adrenal axis can influence each other at the level of the androgen receptor. Knowledge of the mechanisms for androgens’ effects on behaviors through the androgen receptor will guide future studies in elucidating male reproductive and aggressive behavior repertoires.

ERK5/KLF4 signaling as a common mediator of the neuroprotective effects of both nerve growth factor and hydrogen peroxide preconditioning

Oxidative stress has long been implicated in the pathogenesis of various neurodegenerative disorders such as Alzheimer’s disease and stroke. While high levels of oxidative stress are generally associated with cell death, a slight rise of reactive oxygen species (ROS) levels can be protective by “preconditioning” cells to develop a resistance against subsequent challenges. However, the mechanisms underlying such preconditioning (PC)-induced protection are still poorly understood. Previous studies have supported a role of ERK5 (mitogen-activated protein [MAP] kinase 5) in neuroprotection and ischemic tolerance in the hippocampus. In agreement with these findings, our data suggest that ERK5 mediates both hydrogen peroxide (H2O2)-induced PC as well as nerve growth factor (NGF)-induced neuroprotection. Activation of ERK5 partially rescued pheochromocytoma PC12 cells as well as primary hippocampal neurons from H2O2-caused death, while inhibition of ERK5 abolished NGF or PC-induced protection. These results implicate ERK5 signaling as a common downstream pathway for NGF and PC. Furthermore, both NGF and PC increased the expression of the transcription factor, KLF4, which can initiate an anti-apoptotic response in various cell types. Induction of KLF4 by NGF or PC was blocked by siERK5, suggesting that ERK5 is required in this process. siKLF4 can also attenuate NGF- or PC-induced neuroprotection. Overexpression of active MEK5 or KLF4 in H2O2-stressed cells increased Bcl-2/Bax ratio and the expression of NAIP (neuronal apoptosis inhibitory protein). Taken together, our data suggest that ERK5/KLF4 cascade is a common signaling pathway shared by at least two important mechanisms by which neurons can be protected from cell death.

Chronic intermittent hypoxia induces oxidative stress and inflammation in brain regions associated with early‐stage neurodegeneration

Sleep apnea is a common comorbidity of neurodegenerative diseases, such as Alzheimers disease (AD) and Parkinsons disease (PD). Previous studies have shown an association between elevated oxidative stress and inflammation with severe sleep apnea. Elevated oxidative stress and inflammation are also hallmarks of neurodegenerative diseases. We show increased oxidative stress and inflammation in a manner consistent with early stages of neurodegenerative disease in an animal model of mild sleep apnea. Male rats were exposed to 7 days chronic intermittent hypoxia (CIH) for 8 h/day during the light period. Following CIH, plasma was collected and tested for circulating oxidative stress and inflammatory markers associated with proinflammatory M1 or anti‐inflammatory M2 profiles. Tissue punches from brain regions associated with different stages of neurodegenerative diseases (early stage: substantia nigra and entorhinal cortex; intermediate: hippocampus; late stage: rostral ventrolateral medulla and solitary tract nucleus) were also assayed for inflammatory markers. A subset of the samples was examined for 8‐hydroxydeoxyguanosine (8‐OHdG) expression, a marker of oxidative stress‐induced DNA damage. Our results showed increased circulating oxidative stress and inflammation. Furthermore, brain regions associated with early‐stage (but not late‐stage) AD and PD expressed oxidative stress and inflammatory profiles consistent with reported observations in preclinical neurodegenerative disease populations. These results suggest mild CIH induces key features that are characteristic of early‐stage neurodegenerative diseases and may be an effective model to investigate mechanisms contributing to oxidative stress and inflammation in those brain regions.

Cell Models for the Study of Sex Steroid Hormone Neurobiology.

To date many aspects of neurons and glia biology remain elusive, due in part to the cellular and molecular complexity of the brain. In recent decades, cell models from different brain areas have been established and proven invaluable toward understanding this complexity. In the field of steroid hormone neurobiology, an important question is: what is the profile of steroid hormone receptor expression in these specific cell lines? Currently, a clear summary of such receptor profiling is lacking. For this reason, we summarized in this review the expression of estrogen, progesterone, and androgen receptors in several widely used cell lines (glial and neuronal) derived from the forebrain and midbrain, based on our own data and that from the literature. Such information will aid in the selection of specific cell lines used to test hypotheses related to the biology of estrogens, progestins, and/or androgens.