Peter J. Syapin

Regulation of haeme oxygenase-1 for treatment of neuroinflammation and brain disorders

Injury to the CNS elicits a host defense reaction that utilizes astrocytes, microglia, neurons and oligodendrocytes. Neuroinflammation is a major host defense mechanism designed to restore normal structure and function after CNS insult, but like other forms of inflammation, chronic neuroinflammation may contribute to pathogenesis. The inducible haeme oxygenase isoform, haeme oxygenase‐1 (HO‐1), is a phase 2 enzyme upregulated in response to electrophilic xenobiotics, oxidative stress, cellular injury and disease. There is emerging evidence that HO‐1 expression helps mediate the resolution of inflammation, including neuroinflammation. Whether this is solely because of the catabolism of haeme or includes additional mechanisms is unclear. This review provides a brief background on the molecular biology and biochemistry of haeme oxygenases and the actions of haeme, bilirubin, iron and carbon monoxide in the CNS. It then presents our current state of knowledge regarding HO‐1 expression in the CNS, regulation of HO‐1 induction in neural cells and discusses the prospect of pharmacological manipulation of HO‐1 as therapy for CNS disorders. Because of recognized species and cellular differences in HO‐1 regulation, a major objective of this review is to draw attention to areas where gaps exist in the experimental record regarding regulation of HO‐1 in neural cells. The results indicate the HO‐1 system to be an important therapeutic target in CNS disorders, but our understanding of HO‐1 expression in human neural cells is severely lacking.

Minocycline reduces ethanol drinking.

Alcoholism is a disease characterized by continued alcohol consumption despite recurring negative consequences. Thus, medications that reduce the drive to consume alcohol can be beneficial in treating alcoholism. The neurobiological systems that regulate alcohol consumption are complex and not fully understood. Currently, medications are available to treat alcoholism that act either by causing accumulation of a toxic metabolite of ethanol, or by targeting specific transmitter receptors. The purpose of our study was to investigate a new potential therapeutic pathway, neuroimmune interactions, for effects on ethanol consumption. We hypothesized that neuroimmune activity of brain glia may have a role in drinking. We utilized minocycline, a second generation tetracycline antibiotic that has immune modulatory actions, to test our hypothesis because it is known to suppress microglia, and to a lesser extent astroglia, activity following many types of insults to the brain. Treatment with 50mg/kg minocycline significantly reduced ethanol intake in male and female C57Bl/6J mice using a free choice voluntary drinking model. Saline injections did not alter ethanol intake. Minocycline had little effect on water intake or body weight change. The underlying mechanism whereby minocycline reduced ethanol intake requires further study. The results suggest that drugs that alter neuroimmune pathways may represent a new approach to developing additional therapies to treat alcoholism.

Alcohol and Nitric Oxide Production by Cells of the Brain

The L-arginine-nitric oxide pathway is important to both physiological and pathologic brain events. Brain tissue contains cells able to express all known isoforms of nitric oxide synthase, the rate-limiting enzyme in nitric oxide (NO) production and release. Effects of ethanol on NO production may be important to ethanol modification of brain function. Recent studies support this idea and demonstrate diverse interactions. For example, acute ethanol treatment decreases NMDA- and cytokine-stimulated NO synthesis by cortical neurons and glia, respectively, but enhances cytokine-stimulated NO synthesis in blood-brain barrier endothelial cells and does not affect norepinephrine-stimulated NO synthesis in medial basal hypothalamus. Furthermore, chronic ethanol enhances NMDA-stimulated NO synthesis in cortical neurons, but more potently decreases cytokine-induced NO synthesis in glial cells. The mechanisms underlying these effects are partially understood and include changes in NOS-2 gene expression. These observations illustrate that ethanol selectively affects NO production by brain cells, which may relate to reported behavioral interactions, but the extend and direction of change depends on cell type and length of exposure.

REGULATION OF STEROID HORMONE BIOSYNTHESIS IN R2C AND MA-10 LEYDIG TUMOR CELLS: ROLE OF THE CHOLESTEROL TRANSFER PROTEINS StAR AND PBR

The MA-10 mouse Leydig tumor cell line produces large amounts of steroids only in response to hormonal stimulation while the R2C rat Leydig tumor cell line is constitutively steroidogenic in nature. In an effort to uncover the potential reasons for constitutive steroidogenesis in R2C cells, we have recently shown that compared to MA-10 cells, R2C cells express much higher levels of the Scavenger Receptor Class B type 1 which results in a higher capacity for cholesteryl ester uptake through the selective uptake pathway. We also found an enhanced expression of Hormone Sensitive Lipase and the Steroidogenic Acute Regulatory protein in these cells and reasoned that they may further facilitate the conversion of cholesteryl esters to free cholesterol and its mobilization to the inner mitochondrial membrane, thus rendering them constitutively steroidogenic. Given the proposed role of the peripheral-type benzodiazepine receptor (PBR) in conferring a constitutively steroidogenic phenotype to the R2C cells, the current study was conducted to investigate the relationship between its expression in MA-10 and R2C cells and correlate it with the constitutive nature of R2C cell steroidogenesis. Our studies show that PBR expression as measured by PK 11195 ligand binding and Western analysis is much higher in MA-10 cells than R2C cells. We also determined that the affinity of ligand binding to the PBR is comparable in the two cell lines, suggesting that PBR is unlikely to be solely responsible for the constitutive nature of R2C cell steroidogenesis.

Effects of short chain alkanols on the inducible nitric oxide synthase in a glial cell line

Ethanol inhibits inducible nitric oxide synthase (iNOS) expression in C6 glioma cells by an unknown mechanism. Because relatively high concentrations are needed for inhibition in drug‐naïve cells (IC50∼150 mM), suppression due to cytotoxicity is one possible mechanism that has not been ruled out. Therefore, the present study examined the effects of ethanol and other alkanols on C6 glioma cell viability and iNOS activity to better understand the mechanism for inhibition. iNOS expression was induced in cell culture with lipopolysaccharide and phorbol ester treatment. Nitrite accumulation in culture medium, the in vitro conversion of [3H]‐L‐arginine to [3H]‐L‐citrulline, and immunoblotting were used to quantify iNOS induction and activity. Trypan blue exclusion, extracellular release of lactate dehydrogenase, and quantity of total cell protein were used as measures of viability. Short chain alkanols, methanol through 1‐heptanol, concentration‐dependently inhibited nitrite accumulation. Longer chain alkanols, 1‐octanol and 1‐decanol, did not except at cytotoxic concentrations. Experiments indicated short chain alkanol inhibition was not due to direct actions on iNOS catalytic activity, but that it transpires during iNOS induction. Immunoblots showed reduced iNOS protein levels. Correlation analysis ruled out iNOS inhibition as being due to decreased cell number, total cell protein, or cell viability. In contrast, there was significant correlation with physical measures of lipophilicity. In conclusion, inhibition of iNOS expression by ethanol and other short chain alkanols is not due to cytotoxicity. Instead, the strong correlation with lipophilicity suggests the inhibition derives from an interaction with unknown hydrophobic cellular sites.

Chronic ethanol inhibits CXC chemokine ligand 10 production in human A172 astroglia and astroglial-mediated leukocyte chemotaxis

Astroglia are the most prevalent cell type in the human central nervous system (CNS) and perform important roles in normal tissue homeostasis, during pathological events and following trauma. Astroglial-derived chemokines have important neurotrophic effects and are important to CNS immunocompetence and response to injury, in part, due to their direct role in leukocyte and microglial cell recruitment. However, while ethanol is known to induce CNS pathologies and to be peripherally immunosuppressive, ethanol effects on chemokine expression in human astroglia are essentially unknown. We have demonstrated that chemotaxis of human U937 leukocytic cells, across a 0.5 microm pore polycarbonate transmembrane insert, is induced in response to culture media collected from 10 microg/ml lipopolysaccharide (LPS) + 10 ng/ml interleukin (IL)-1beta-stimulated A172 human astroglia cells. The involvement of the chemokine CXCL10 (also known as interferon-gamma inducible protein or IP-10) in astroglial-induced chemotaxis of U937 cells has been indicated, as chemotaxis can be reduced by an anti-CXCL10 neutralizing antibody. Interestingly, chemotaxis of U937 cells, in response to astroglial-exposed media, is reduced when astroglia are chronically (9 days) exposed to 50 mM ethanol before stimulation with LPS + IL-1beta. Furthermore, we observed that LPS + IL-1beta-stimulated CXCL10 production is inhibited in human A172 astroglia exposed to chronic 50 mM ethanol. Thus, alterations in astroglial CXCL10 expression may disrupt CNS immunocompetence and play an important role in ethanol-induced CNS pathologies.

Alcohol Brain Damage and Neuroinflammation: Is There a Connection?

Many neurological disorders include a neuroinflammatory response in the central nervous system (CNS) that contributes to the pathogenesis and progression of the condition. However, the question of whether neuroinflammation plays a role in alcohol-induced brain damage has received little attention. This symposium at the 2004 World Congress on Biomedical Alcohol Research held in Mannheim, Germany considered for the first time the relationship between alcohol neuropathology and neuroinflammation. The organizers and co-chairs were Peter J. Syapin and Cynthia J. M. Kane. Neuroinflammatory responses including glial activation, demyelination, neuronal damage and neurodegeneration may contribute to the neurological deficits associated with alcoholism, alcohol abuse, fetal alcohol syndrome or alcohol-related neurodevelopmental disorder. A substantial body of literature indicates that alcohol directly affects astroglial cell function, including inflammation-related activity. In addition, emerging data indicate that alcohol affects microglial cell development and function in specific ways that interfere with microglial interactions with the immune system and with neurons. This symposium provided alcohol researchers with background information on CNS immunity and inflammatory processes and examined emerging experimental evidence that neuroinflammatory processes might be involved in alcohol-induced brain damage. A brief introduction was followed by four presentations, the first two describing the nature of immune reactions in the CNS and the role of astrocytes and microglia in neuroinflammatory processes. The presentations included: (1) Initiation of inflammation in the nervous system, by William Hickey; (2) Microglia – Interface between immune system and brain, by Helmut Kettenmann; (3) Ethanol and neuroinflammatory response in brain astroglial, by Peter Syapin; and (4) Microglia are targets of alcohol pathogenesis in the developing CNS, by Cynthia Kane.

Identification of cis-regulatory regions necessary for robust Nos2 promoter activity in glial cells: indirect role for NF-κB

Previous reports suggest the nitric‐oxide synthase 2 (Nos2) promoter contains negative and positive cis‐regulatory regions. This study identified such regions using rat C6 glial cells. Activity of the serially deleted rat Nos2 promoter fused to a luciferase reporter gene was found to vary with construct size independent of stimuli, decreasing markedly from 160 to 130 bp then increasing significantly from 110 to 94 bp. In contrast, time to peak activity was stimulus‐dependent but size‐independent; 4–8 h for a cytokine mixture or lipopolysaccharide + interferon‐γ, and 8–16 h for lipopolysaccharide + phorbol 12‐myristate 13‐acetate. Peak activity with heterologous promoters also varied; 4 h for 3.7 kb of the human Nos2A promoter, and 36 h for 1.8 kb of the murine promoter. Electrophoretic mobility shift assays and in vivo DNA footprinting data confirmed nuclear protein binding to promoter regions suspected of containing important regulatory sites based on reporter gene data. A binding site for NF‐κB was not required for Nos2 promoter activity. These findings provide significant new information on the relative importance of different regions of the rat Nos2 promoter for transcriptional activation and nitric oxide production by glial cells and support the existence of cell‐ and species‐specific mechanisms for transcriptional regulation of Nos2 activation.

Up-regulation of steroid biosynthesis by retinoid signaling: Implications for aging

Retinoids (vitamin A and its derivatives) are critical for a spectrum of developmental and physiological processes, in which steroid hormones also play indispensable roles. The StAR protein predominantly regulates steroid biosynthesis in steroidogenic tissues. We have reported that regulation of retinoid, especially atRA and 9-cis RA, responsive StAR transcription is largely mediated by an LXR-RXR/RAR heterodimeric motif in the mouse StAR promoter. Herein we demonstrate that retinoids are capable of enhancing StAR protein, P-StAR, and steroid production in granulosa, adrenocortical, glial, and epidermal cells. Whereas transient expression of RARα and RXRα enhanced 9-cis RA induced StAR gene transcription, silencing of RXRα with siRNA, decreased StAR and steroid levels. An oligonucleotide probe encompassing an LXR-RXR/RAR motif bound to adrenocortical and epidermal keratinocyte nuclear proteins in EMSAs. ChIP studies revealed association of RARα and RXRα with the StAR proximal promoter. Further studies demonstrated that StAR mRNA levels decreased in diseased and elderly men and women skin tissues and that atRA could restore steroidogenesis in epidermal keratinocytes of aged individuals. These findings provide novel insights into the relevance of retinoid signaling in the up-regulation of steroid biosynthesis in various target tissues, and indicate that retinoid therapy may have important implications in age-related complications and diseases.

Alcohol-induced Bone Loss and Quality during Adolescence is Improved by Green Tea Polyphenols

Our previous studies have shown significant osteo-protective effects of green tea polyphenols (GTP, green tea extract) in various bone loss models. To test the hypothesis that green tea supplementation would protect against binge alcohol-induced deterioration of bone quality in adolescent drinkers, we used a similar approach with green tea supplementation in drinking water. Using a six week, 2 × 3 factorial design of treatment × dose in male Sprague Dawley (SD) adolescent rats, bone parameters [femoral and lumbar vertebrae-4 (LV-4) area (BMA), bone mineral content (BMC), bone mineral density (BMD)], bone turnover biomarkers [serum osteocalcein (OC) and tartrateresistant acid phosphatase-5b (TRAP-5b)] and blood chemistry were measured. The blood chemistry results showed that alcohol administration significantly decreased albumin, glucose, alkaline phosphatase, and amylase levels; increased globulin, phosphorus, creatine kinase, cholesterol, and potassium levels; and had no effect on protein, calcium, blood urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase, sodium, and chloride. GTP supplementation significantly suppressed alkaline phosphatase levels and had no impact on other blood chemistry parameters. Alcohol administration lowered BMA, BMC, and BMD of the femur and LV-4, as well as serum TRAP-5b, but had no impact on serum OC. Supplementation of GTP into the drinking water increased BMD for the femur and BMC for the LV-4. GTP had no effect on scanned BMA or serum OC concentration. There was an interaction between the alcohol administration and GTP dosage in serum TRAP-5b and several parameters of bone strength, which reduced the negative effect on alcohol-induced bone modeling. In summary, our results show that a 6-week binge alcohol administration lowered bone mineral content, density, and strength in femura, reduced proximal tibial trabecular bone volume and lumbar vertebrae, and decreased cortical thickness at the tibial middiaphysis. Supplementation of GTP into the drinking water increased femoral bone mineral density and tibial cortical thickness at the mid-diaphysis. Importantly, GTP supplementation into drinking water improved overall bone quality in young binge-alcohol treated male rats through suppressing bone turnover rate.