Central aromatization: A dramatic and responsive defense against threat and trauma to the vertebrate brain.

Aromatase is the requisite and limiting enzyme in the production of estrogens from androgens. Estrogens synthesized centrally have more recently emerged as potent neuroprotectants in the vertebrate brain. Studies in rodents and songbirds have identified key mechanisms that underlie both; the injury-dependent induction of central aromatization, and the protective effects of centrally synthesized estrogens. Injury-induced aromatase expression in astrocytes occurs following a broad range of traumatic brain damage including excitotoxic, penetrating, and concussive injury. Responses to neural insult such as edema and inflammation involve signaling pathways the components of which are excellent candidates as inducers of this astrocytic response. Finally, estradiol from astrocytes exerts a paracrine neuroprotective influence via the potent inhibition of inflammatory pathways. Taken together, these data suggest a novel role for neural aromatization as a protective mechanism against the threat of inflammation and suggests that central estrogen provision is a wide-ranging neuroprotectant in the vertebrate brain.

Exogenous progesterone is neuroprotective following injury to the male zebra finch brain.

The use of progesterone following brain injury has a controversial history. On one hand, some lab-based models have showed progesterone as being neuroprotective, but on the other, clinical trials have showed quite the opposite. One of many complaints that arose from this discrepancy was the lack of a diverse pool of animal models and paradigms employed during the preclinical phase. However, over the past decade, the zebra finch has emerged as an optimal organism for the study of steroid-mediated neuroprotection. Following an injury, steroid hormones and receptors are upregulated, serving to decrease neuroinflammation and overall damage to the brain. As compared to other vertebrate models, zebra finches can upregulate expression of both estrogens and androgens at a faster and more robust response, suggesting that vertebrates differ in their neuroprotective mechanisms and timing following injury. Therefore, to expand the types organisms studied in pre-clinical trials, we chose to use zebra finches. While the majority of work in the zebra finch brain has focused on estrogens and androgens, we sought to clarify the role of progesterone following injury. Adult male zebra finches were given daily injections of progesterone following a penetrating injury and then were assessed for the size of injury and expression of various genes associated with neuroinflammation and cell survival. Treatment with progesterone decreased the injury size in zebra finches over controls and increased expression of various genes associated with cell survival and neuroinflammation. These data suggest that progesterone does mediate neuroprotection, most likely through the alteration of neuroinflammatory and cell survival pathways.

Crosstalk between Estrogen Withdrawal and NFκB Signaling following Penetrating Brain Injury.

OBJECTIVES: Characterized by neuroinflammation, traumatic brain injury (TBI) induces neuropathological changes and cognitive deficits. Estrogens are neuroprotective by increasing cell survival and this increase is mediated by a decrease in neuroinflammation. To further explore the relationship between estrogens, brain injury, and neuroinflammation, we examined the expression of the IKK/NFκB complex. The IKK/NFκB complex is a pleiotropic regulator of many cellular signaling pathways linked to inflammation, as well as three major cytokines (IL-1ß, IL-6, and TNF-α). We hypothesized that NFκB expression would be upregulated following injury and that this increase would be exacerbated when circulating estrogens were decreased with fadrozole (aromatase inhibitor). METHODS: Using adult zebra finches, we first determined the expression of major components of the NFκB complex (NFκB, IκB-α, and IκB-ß) following injury using qPCR. Next, male and female finches were collected at 2 time points (2 or 24 h after injury) and brain tissue was analyzed to determine whether NFκB expression was differentially expressed in males and females at either time point. Finally, we examined how the expression of NFκB changed when estrogen levels were decreased immediately after injury. RESULTS: Our study documented an increase in the expression of the major components of the NFκB complex (NFκB, IκB-α, and IκB-ß) following injury. Decreasing estrogen levels resulted in a surprising decrease in the NFκB complex studied here. DISCUSSION: These data further expand the model of how estrogens and other steroid hormones interact with the inflammatory pathways following injury and may prove beneficial when developing therapies for treatment of TBI.

Chondroitin sulfate proteoglycans mRNA expression and degradation in the zebra finch following traumatic brain injury.

Traumatic brain injury (TBI) is one of the leading causes of fatality and disability worldwide. From minutes to months following damage, injury can result in a complex pathophysiology that can lead to temporary or permanent deficits including an array of neurodegenerative symptoms. These changes can include behavioral dysregulation, memory dysfunctions, and mood changes including depression. The nature and severity of impairments resulting from TBIs vary widely given the range of injury type, location, and extent of brain tissue involved. In response to the injury, the brain induces structural and functional changes to promote repair and minimize injury size. Despite its high prevalence, effective treatment strategies for TBI are limited. PNNs are part of the neuronal extracellular matrix (ECM) that mediate synaptic stabilization in the adult brain and thus neuroplasticity. They are associated mostly with inhibitory GABAergic interneurons and are thought to be responsible for maintaining the excitatory/inhibitory balance of the brain. The major structural components of PNNs include multiple chondroitin sulfate proteoglycans (CSPGs) as well as other structural proteins. Here we examine the effects of injury on CSPG expression, specifically around the changes in the side change moieties. To investigate CSPG expression following injury, adult male and female zebra finches received either a bilateral penetrating, or no injury and qPCR analysis and immunohistochemistry for components of the CSPGs were examined at 1- or 7-days post-injury. Next, to determine if CSPGs and thus PNNs should be a target for therapeutic intervention, CSPG side chains were degraded at the time of injury with chondroitinase ABC (ChABC) CSPGs moieties were examined. Additionally, GABA receptor mRNA and aromatase mRNA expression was quantified following CSPG degradation as they have been implicated in neuronal survival and neurogenesis. Our data indicate the CSPG moieties change following injury, potentially allowing for a brief period of synaptic reorganization, and that treatments that target CSPG side chains are successful in further targeting this brief critical period by decreasing GABA mRNA receptor expression, but also decreasing aromatase expression.

Traumatized and inflamed--but resilient: glial aromatization and the avian brain.

Steroids like estrogens have potent effects on the vertebrate brain, and are provided to neural targets from peripheral and central sources. Estradiol synthesized within the vertebrate CNS modulates neural structure and function, including the pathways involved in neuroprotection, and perhaps, neural repair. Specifically, aromatase; the enzyme responsible for the conversion of testosterone to estradiol, is upregulated in the avian and mammalian brain following disruption of the neuropil by multiple forms of perturbation including mechanical injury, ischemia and excitotoxicity. This injury induced aromatase expression is somewhat unique in that it occurs in astroglia rather than neurons, and is stimulated in response to factors associated with brain damage. In this review, we focus on the induction, expression and consequences of glial aromatization in the songbird brain. We begin with a review of the anatomical consequences of glial estrogen provision followed by a discussion of the cellular mechanisms whereby glial aromatization may affect injury-induced neuroplasticity. We then present the current status of our understanding regarding the inductive role of inflammatory processes in the transcription and translation of astrocytic aromatase. We consider the functional aspects of glial aromatization before concluding with unanswered questions and suggestions for future studies. Birds have long informed us about fundamental questions in endocrinology, immunology, and neuroplasticity; and their unique anatomical and physiological characteristics continue to provide an excellent system in which to learn about brain trauma, inflammation, and neuroprotection.

The song remains the same: coactivators and sex differences in the songbird brain.

The majority of songbird species have sexually dimorphic neuronal circuits for song learning and production and these differences are paralleled by sex differences in behavior, with only males singing or singing at a higher rate than females. Therefore songbirds serve as an excellent model for studying the mechanisms that influence the sexually dimorphic development of the brain and behavior. Past research focused on the actions of steroid hormones or their receptors in the development of these sex differences. This review examines the distribution and action of steroid receptor coactivators in the songbird brain; more specifically the actions of RPL7, SRC-1, and CBP on the song control system. Coactivators enhance the transcriptional activity of the nuclear steroid receptors with which they associate, and therefore may play a role in modulating the development of sex differences in the brain and behavior. The actions of these proteins may help elucidate the hormonal mechanisms that underlie song nuclei development and steroid activated singing behavior in adulthood.

Steroid profiling in brain and plasma of adult zebra finches following traumatic brain injury.

Traumatic brain injury (TBI) is a serious health concern and a leading cause of death. Emerging evidence strongly suggests that steroid hormones (estrogens, androgens, and progesterone) modulate TBI outcomes by regulating inflammation, oxidative stress, free radical production, and extracellular calcium levels. Despite this growing body of evidence on steroid-mediated neuroprotection, very little is known about the local synthesis of these steroids following injury. Here, we examine the effect of TBI on local neurosteroid levels around the site of injury and in plasma in adult male and female zebra finches. Using ultrasensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS), we examined estrogens, androgens, and progesterone in the entopallium and plasma of injured and uninjured animals. Three days after injury, elevated levels of 17ß-estradiol (E2 ), estrone (E1 ), and testosterone (T) were detected near injured brain tissue with a corresponding increase in E2 also detected in plasma. Taken together, these results provide further evidence that TBI alters neurosteroid levels and are consistent with studies showing that neurosteroids provide neuroprotection following injury.

Neuroinflammation induces glial aromatase expression in the uninjured songbird brain.

BACKGROUND: Estrogens from peripheral sources as well as central aromatization are neuroprotective in the vertebrate brain. Under normal conditions, aromatase is only expressed in neurons, however following anoxic/ischemic or mechanical brain injury; aromatase is also found in astroglia. This increased glial aromatization and the consequent estrogen synthesis is neuroprotective and may promote neuronal survival and repair. While the effects of estradiol on neuroprotection are well studied, what induces glial aromatase expression remains unknown. METHODS: Adult male zebra finches (Taeniopygia guttata) were given a penetrating injury to the entopallium. At several timepoints later, expression of aromatase, IL-1ß-like, and IL-6-like were examined using immunohistochemistry. A second set of zebra birds were exposed to phytohemagglutinin (PHA), an inflammatory agent, directly on the dorsal surface of the telencephalon without creating a penetrating injury. Expression of aromatase, IL-1ß-like, and IL-6-like were examined using both quantitative real-time polymerase chain reaction to examine mRNA expression and immunohistochemistry to determine cellular expression. Statistical significance was determined using t-test or one-way analysis of variance followed by the Tukey Kramers post hoc test. RESULTS: Following injury in the zebra finch brain, cytokine expression occurs prior to aromatase expression. This temporal pattern suggests that cytokines may induce aromatase expression in the damaged zebra finch brain. Furthermore, evoking a neuroinflammatory response characterized by an increase in cytokine expression in the uninjured brain is sufficient to induce glial aromatase expression. CONCLUSIONS: These studies are among the first to examine a neuroinflammatory response in the songbird brain following mechanical brain injury and to describe a novel neuroimmune signal to initiate aromatase expression in glia.

Distribution and sexually dimorphic expression of steroid receptor coactivator-1 (SRC-1) in the zebra finch brain.

Coactivator proteins, such as steroid receptor coactivator-1 (SRC-1) greatly enhance gene expression by amplifying steroid-induced transcription regulated by receptors such as estrogen receptor. These proteins may also play a role in the development of sex differences in central nervous system as well the maintenance of the sexually dimorphic behaviors in adulthood. One well-studied sexually dimorphic behavior is singing in songbirds such as the Australian zebra finch (Taeniopygia guttata). Song learning and production is controlled by the song control system, a collection of sexually dimorphic nuclei found in the avian telencephalon. While the actions of steroid hormones on song nuclei development has been under debate, steroids, such as testosterone, influence singing behavior in adulthood. We hypothesize that the differential expression of coactivators in male and female brains aid in organizing the song nuclei during development and function in adulthood to aid in activating the song control nuclei to induce singing behavior. The distribution of SRC-1-immunoreactive neurons was localized in the brains of male and female zebra finches on the day of hatch (P1) and in adults. In adults SRC-1 immunoreactive cells are found in the four main song control nuclei as well as other steroid sensitive brain regions. We found that SRC-1 is sexually dimorphic in the adult zebra finch telencephalon, suggesting that coactivators may play a role in the maintenance of sexually dimorphic behaviors including singing.

Neuroprotective actions of brain aromatase.

The steroidal regulation of vertebrate neuroanatomy and neurophysiology includes a seemingly unending list of brain areas, cellular structures and behaviors modulated by these hormones. Estrogens, in particular have emerged as potent neuromodulators, exerting a range of effects including neuroprotection and perhaps neural repair. In songbirds and mammals, the brain itself appears to be the site of injury-induced estrogen synthesis via the rapid transcription and translation of aromatase (estrogen synthase) in astroglia. This induction seems to occur regardless of the nature and location of primary brain damage. The induced expression of aromatase apparently elevates local estrogen levels enough to interfere with apoptotic pathways, thereby decreasing secondary degeneration and ultimately lessening the extent of damage. There is even evidence suggesting that aromatization may affect injury-induced cytogenesis. Thus, aromatization in the brain appears to confer neuroprotection by an array of mechanisms that involve the deceleration and acceleration of degeneration and repair, respectively. We are only beginning to understand the factors responsible for the injury-induced transcription of aromatase in astroglia. In contrast, much of the manner in which local and circulating estrogens may achieve their neuroprotective effects has been elucidated. However, gaps in our knowledge include issues about the cell-specific regulation of aromatase expression, steroidal influences of aromatization distinct from estrogen formation, and questions about the role of constitutive aromatase in neuroprotection. Here we describe the considerable consensus and some interesting differences in knowledge gained from studies conducted on diverse animal models, experimental paradigms and preparations towards understanding the neuroprotective actions of brain aromatase.

Estrogen Formation and Inactivation Following TBI: What we Know and Where we Could go.

Traumatic brain injury (TBI) is responsible for various neuronal and cognitive deficits as well as psychosocial dysfunction. Characterized by damage inducing neuroinflammation, this response can cause an acute secondary injury that leads to widespread neurodegeneration and loss of neurological function. Estrogens decrease injury induced neuroinflammation and increase cell survival and neuroprotection and thus are a potential target for use following TBI. While much is known about the role of estrogens as a neuroprotective agent following TBI, less is known regarding their formation and inactivation following damage to the brain. Specifically, very little is known surrounding the majority of enzymes responsible for the production of estrogens. These estrogen metabolizing enzymes (EME) include aromatase, steroid sulfatase (STS), estrogen sulfotransferase (EST/SULT1E1), and some forms of 17ß-hydroxysteroid dehydrogenase (HSD17B) and are involved in both the initial conversion and interconversion of estrogens from precursors. This article will review and offer new prospective and ideas on the expression of EMEs following TBI.

Expression of glial CBP in steroid mediated neuroprotection in male and female zebra finches.

Under neurodegenerative conditions, reactive astrocytes upregulate both aromatase (estrogen synthase) as well as estrogen and androgen receptors. This increased steroidogenic signal promotes neuroprotection and repair by promoting neurogenesis and decreasing cell death, but also by modulating the release of inflammatory molecules. Thus, endocrine - immune cross-talk is an essential component of estrogen mediated neuroprotection following brain injury. However, the exact mechanisms underlying this cross-talk remains unknown. cAMP response element-binding protein-binding protein (CBP) may be involved in the modulation of both the endocrine and inflammatory response following injury. CBP acts as both an estrogen receptor (ER) coactivator and as a promotor for NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) target genes and previous data suggests that ER and NF-κB compete for CBP. When CBP is displaced, target genes for NF-κB are repressed and inflammation is decreased. To test the role of CBP following injury, we examined CBP expression following penetrating injury in adult male and female zebra finches. Using immunohistochemistry, we were able to specifically examine glial CBP expression, as glial cells are important mediators of the neuroendocrine response to damage. Male but not female zebra finches upregulated glial CBP following damage to the brain. To determine if this upregulation was estrogen dependent, we decreased local estrogen levels with fadrozole (aromatase inhibitor) and reexamined glial CBP expression following injury. Aromatase inhibition resulted in no change in overall glial CBP expression suggesting that circulating estrogens do not mediate the upregulation of glial CBP following injury. Thus CBP may play a role in the both the estrogen and immune response to injury.

Injury-induced expression of glial androgen receptor in the zebra finch brain.

Astrogliosis occurs following injury to the zebra finch brain. To date, only estrogen synthase (aromatase) has been identified in injury-induced astrocytes. The expression of other steroidogenic enzymes or their receptors remains unknown in the avian brain. However, in mammals, an upregulation of androgen receptors has been identified in glial cells. The aim of this study was to determine if the androgen receptor is upregulated following injury in adult zebra finches. Finches were given a single penetrating injury and brain tissue was collected 24 or 72 h later. Expression of androgen receptor was examined using immunohistochemistry and quantified using quantitative polymerase chain reaction (qPCR) analysis. Androgen receptors were localized to astrocytes versus neurons, further solidifying the role for astrocytes in neural recovery.

The selective estrogen receptor-alpha coactivator, RPL7, and sexual differentiation of the songbird brain.

The brain and behavior of the Australian zebra finch (Taeniopygia guttata) are sexually dimorphic. Only males sing courtship songs and the regions of the brain involved in the learning and production of song are significantly larger in males than females. Therefore the zebra finch serves as an excellent model for studying the mechanisms that influence brain sexual differentiation, and the majority of past research on this system has focused on the actions of steroid hormones in the development of these sex differences. Coregulators, such as coactivators and corepressors, are proteins and RNA activators that work by enhancing or depressing the transcriptional activity of the nuclear steroid receptor with which they associate, and thereby modulating the development of sex-specific brain morphologies and behaviors. The actions of these proteins may help elucidate the hormonal mechanisms that underlie song nuclei development. Research described in this review focus on the role of estrogen receptor coactivators in the avian brain; more specifically we will focus on the role of RPL7 (ribosomal protein L7; also known as L7/SPA) on sexual differentiation of the zebra finch song system. Collectively, these studies provide information about the role of steroid receptor coactivators on development of the zebra finch song system as well as on sexual differentiation of brain.

The sexually dimorphic expression of L7/SPA, an estrogen receptor coactivator, in zebra finch telencephalon.

Sex differences in the zebra finch (Taeniopygia guttata) brain are robust and include differences in morphology (song control nuclei in males are significantly larger) and behavior (only males sing courtship songs). In zebra finches, hormonal manipulations during development fail to reverse sex differences in song nuclei size and suggest that the classical model of sexual differentiation is incomplete for birds. Coactivators act to initiate transcriptional activity of steroid receptors, and may help explain why hormonal manipulations alone are not sufficient to demasculinize the male zebra finch brain. The present study investigated the expression and localization of L7/SPA (an estrogen receptor coactivator) mRNA and protein expression across the development of zebra finch song nuclei from males and females collected on P1 (song nuclei not yet formed), P10 (posthatch day 10, song nuclei formed), P30 (30 days posthatch, sexually immature but song nuclei formed and birds learning to sing), and adult birds (older than 65 days and sexually mature). Northern blot analysis showed a significant sex difference in P1 and adult L7/SPA mRNA expression while Western blot analysis also showed enhanced expression in the male brain at all age points. Both in situ hybridization and immunohistochemistry demonstrated that L7/SPA mRNA and protein were located in the song nuclei as well as expressed globally. Elevated coactivator expression may be a possible mechanism controlling the development of male song control nuclei, and coactivators such as L7/SPA may be important regulators of the masculinizing effects of estradiol on brain sexual differentiation.