Search

G protein–coupled receptors (GPCRs) promote the expression of immediate early genes required for learning and memory. Here, we showed that β 2 -adrenergic receptor (β 2 AR) stimulation induced the nuclear export of phosphodiesterase 4D5 (PDE4D5), an enzyme that degrades the second messenger cAMP, to enable memory consolidation. We demonstrated that the endocytosis of β 2 AR phosphorylated by GPCR kinases (GRKs) mediated arrestin3-dependent nuclear export of PDE4D5, which was critical for promoting nuclear cAMP signaling and gene expression in hippocampal neurons for memory consolidation. Inhibition of the arrestin3-PDE4D5 association prevented β 2 AR-induced nuclear cAMP signaling without affecting receptor endocytosis. Direct PDE4 inhibition rescued β 2 AR-induced nuclear cAMP signaling and ameliorated memory deficits in mice expressing a form of the β 2 AR that could not be phosphorylated by GRKs. These data reveal how β 2 AR phosphorylated by endosomal GRK promotes the nuclear export of PDE4D5, leading to nuclear cAMP signaling, changes in gene expression, and memory consolidation. This study also highlights the translocation of PDEs as a mechanism to promote cAMP signaling in specific subcellular locations downstream of GPCR activation.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease with grave physical, emotional and socioeconomic consequences. Despite advances in targeted biologic and pharmacologic interventions that have recently come to market, many patients with RA continue to have inadequate response to therapies, or intolerable side effects, with resultant progression of their disease. In this review, we detail multiple biomolecular pathways involved in RA disease pathogenesis to elucidate and highlight pathways that have been therapeutic targets in managing this systemic autoimmune disease. Here we present an up-to-date accounting of both emerging and approved pharmacological treatments for RA, detailing their discovery, mechanisms of action, efficacy, and limitations. Finally, we turn to the emerging fields of bioengineering and cell therapy to illuminate possible future targeted therapeutic options that combine material and biological sciences for localized therapeutic action with the potential to greatly reduce side effects seen in systemically applied treatment modalities.

G protein-coupled receptors (GPCRs) transduce pleiotropic intracellular signals in mammalian cells. Here, we report that some antagonists of β adrenergic receptors (βARs) such as β-blocker carvedilol and alprenolol activate β2AR at nanomolar concentrations, which promote G protein signaling and cAMP/PKA activity without action of G protein receptor kinases (GRKs). The cAMP/PKA signal is restricted within the local plasma membrane domain, leading to selectively enhance PKA-dependent augment of endogenous L-type calcium channel (LTCC) activity but not AMPA receptor in hippocampal neurons. Moreover, we have engineered a mutant β2AR that lacks serine 204 and 207 in the catecholamine binding pocket. This mutant can be preferentially activated by carvedilol but not the orthosteric agonist isoproterenol. Carvedilol activates the mutant β2AR in hippocampal neurons augmenting LTCC activity through cAMP/PKA signaling. Together, our study identifies a mechanism by which β-blocker-dependent activation of GPCRs at low ligand concentrations promotes local cAMP/PKA signaling to selectively target membrane downstream effectors such as LTCC in neurons.

Background New pharmacotherapies to treat alcohol use disorders (AUD) are needed. Given the complex nature of AUD, there likely exist multiple novel drug targets. We, and others, have shown that the tetracycline drugs, minocycline and doxycycline, reduced ethanol (EtOH) drinking in mice. To test the hypothesis that suppression of high EtOH consumption is a general property of tetracyclines, we screened several derivatives for antidrinking activity using the Drinking-In-the-Dark (DID) paradigm. Active drugs were studied further using the dose–response relationship. Methods Adult female and male C57BL/6J mice were singly housed and the DID paradigm was performed using 20% EtOH over a 4-day period. Mice were administered a tetracycline or its vehicle 20 hours prior to drinking. Water and EtOH consumption was measured daily. Body weight was measured at the start of drug injections and after the final day of the experiment. Blood was collected for EtOH content measurement immediately following the final bout of drinking. Results Seven tetracyclines were tested at a 50 mg/kg dose. Only minocycline and tigecycline significantly reduced EtOH drinking, and doxycycline showed a strong effect size trend toward reduced drinking. Subsequent studies with these 3 drugs revealed a dose-dependent decrease in EtOH consumption for both female and male mice, with sex differences in efficacy. Minocycline and doxycycline reduced water intake at higher doses, although to a lesser degree than their effects on EtOH drinking. Tigecycline did not negatively affect water intake. The rank order of potency for reduction in EtOH consumption was minocycline > doxycycline > tigecycline, indicating efficacy was not strictly related to their partition coefficients or distribution constants. Conclusions Due to its effectiveness in reducing high EtOH consumption coupled without an effect on water intake, tigecycline was found to be the most promising lead tetracycline compound for further study toward the development of a new pharmacotherapy for the treatment of AUD.

Polyadenylation is an essential mechanism for the processing of mRNA 3′ ends. CstF-64 (the 64,000 Mr subunit of the cleavage stimulation factor; gene symbol Cstf2) is an RNA-binding protein that regulates mRNA polyadenylation site usage. We discovered a paralogous form of CstF-64 called τCstF-64 (Cstf2t). The Cstf2t gene is conserved in all eutherian mammals including mice and humans, but the τCstF-64 protein is expressed only in a subset of mammalian tissues, mostly testis and brain. Male mice that lack Cstf2t (Cstf2t-/- mice) experience disruption of spermatogenesis and are infertile, although female fertility is unaffected. However, a role for τCstF-64 in the brain has not yet been determined. Given the importance of RNA polyadenylation and splicing in neuronal gene expression, we chose to test the hypothesis that τCstF-64 is important for brain function. Male and female 185-day old wild type and Cstf2t-/- mice were examined for motor function, general activity, learning, and memory using rotarod, open field activity, 8-arm radial arm maze, and Morris water maze tasks. Male wild type and Cstf2t-/- mice did not show differences in learning and memory. However, female Cstf2t-/- mice showed significantly better retention of learned maze tasks than did female wild type mice. These results suggest that τCstf-64 is important in memory function in female mice. Interestingly, male Cstf2t-/- mice displayed less thigmotactic behavior than did wild type mice, suggesting that Cstf2t may play a role in anxiety in males. Taken together, our studies highlight the importance of mRNA processing in cognition and behavior as well as their established functions in reproduction.

Background Physicians have long reported that patients with chronic pain show higher tendencies for alcohol use disorder (AUD), and AUD patients appear to have higher pain sensitivities. The goal of this study was to test 2 hypotheses: (i) binge alcohol consumption increases inflammatory pain and mechanical and cold sensitivities; and (ii) tigecycline is an effective treatment for alcohol-mediated-increased pain behaviors and sensitivities. Both female and male mice were used to test the additional hypothesis that important sex differences in the ethanol (EtOH)-related traits would be seen. Methods “Drinking in the Dark” (DID) alcohol consuming and nondrinking control, female and male, adult C57BL/6J mice were evaluated for inflammatory pain behaviors and for the presence of mechanical and cold sensitivities. Inflammatory pain was produced by intraplantar injection of formalin (10 μl, 2.5% in saline). For cold sensation, a 20 μl acetone drop was used. Mechanical withdrawal threshold was measured by an electronic von Frey anesthesiometer. Efficacy of tigecycline (80 mg/kg i.p.) to reduce DID-related pain responses and sensitivity was tested. Results DID EtOH consumption increased inflammatory pain behavior, while it also produced sustained mechanical and cold sensitivities in both females and males. Tigecycline produced antinociceptive effects in males; a pro-nociceptive effect was seen in females in the formalin test. Likewise, the drug reduced both mechanical and cold sensitivities in males, but females showed an increase in sensitivity in both tests. Conclusions Our results demonstrated that binge drinking increases pain, touch, and thermal sensations in both sexes. In addition, we have identified sex-specific effects of tigecycline on inflammatory pain, as well as mechanical and cold sensitivities. The development of tigecycline as an AUD pharmacotherapy may need consideration of its pro-nociceptive action in females. Further studies are needed to investigate the mechanism underlying the sex-specific differences in nociception.

Background Alcohol use disorder (AUD) is a spectrum disorder characterized by mild to severe symptoms, including potential withdrawal signs upon cessation of consumption. Approximately five hundred thousand patients with AUD undergo clinically relevant episodes of withdrawal annually (New Engl J Med, 2003, 348, 1786). Recent evidence indicates potential for drugs that alter neuroimmune pathways as new AUD therapies. We have previously shown the immunomodulatory drugs, minocycline and tigecycline, were effective in reducing ethanol (EtOH) consumption in both the 2-bottle choice and drinking-in-the-dark paradigms. Here, we test the hypothesis that tigecycline, a tetracycline derivative, will reduce the severity of EtOH withdrawal symptoms in a common acute model of alcohol withdrawal (AWD) using a single anesthetic dose of EtOH in seizure sensitive DBA/2J (DBA) mice. Methods Naive adult female and male DBA mice were given separate injections of 4 g/kg i.p. EtOH with vehicle or tigecycline (0, 20, 40, or 80 mg/kg i.p.). The 80 mg/kg dose was tested at 3 time points (0, 4, and 7 hours) post EtOH treatment. Handling-induced convulsions (HICs) were measured before and then over 12 hours following EtOH injection. HIC scores and areas under the curve were tabulated. In separate mice, blood EtOH concentrations (BECs) were measured at 2, 4, and 7 hours postinjection of 4 g/kg i.p. EtOH in mice treated with 0 and 80 mg/kg i.p. tigecycline. Results AWD symptom onset, peak magnitude, and overall HIC severity were reduced by tigecycline drug treatment compared to controls. Tigecycline treatment was effective regardless of timing throughout AWD, with earlier treatment showing greater efficacy. Tigecycline showed a dose-responsive reduction in acute AWD convulsions, with no sex differences in efficacy. Importantly, tigecycline did not affect BECs over a time course of elimination. Conclusions Tigecycline effectively reduced AWD symptoms in DBA mice at all times and dosages tested, making it a promising lead compound for development of a novel pharmacotherapy for AWD. Further studies are needed to determine the mechanism of tigecycline action.

Hypoxia is increasingly recognized as an important contributing factor to the development of brain diseases such as Alzheimer's disease (AD). In the periphery, hypoxia is a powerful regulator of angiogenesis. However, vascular endothelial cells are remarkably heterogeneous and little is known about how brain endothelial cells respond to hypoxic challenge. The objective of this study is to characterize the effect of hypoxic challenge on the angiogenic response of cultured brain-derived microvascular endothelial cells. Brain endothelial cell cultures were initiated from isolated rat brain microvessels and subjected to hypoxia (1% O(2)) for various time periods. The results showed that hypoxia induced rapid (≤ 0.5h) expression of hypoxia-inducible factor 1α (HIF-1α) and that cell viability, assessed by MTT assay, was unaffected within the first 8h. Examination of brain endothelial cell cultures for pro- and anti-angiogenic proteins by western blot, RT-PCR and ELISA revealed that within 0.5 to 2h of hypoxia levels of vascular endothelial growth factor and endothelin-1 mRNA and protein were elevated. The expression of heme oxygenase-1 also increased but only after 8h of hypoxia. In contrast, similar hypoxia exposure evoked a decrease in endothelial nitric oxide synthase and thrombospondin-2 levels. Exposure of brain endothelial cell cultures to hypoxia resulted in a significant (p

Mitigating oxidative stress-induced damage is critical to preserving neuronal function in diseased or injured brains. This study explores the mechanisms contributing to the neuroprotective effects of pigment epithelium-derived factor (PEDF) in cortical neurons. Cultured primary neurons are exposed to PEDF and H2O2 as well as inhibitors of phosphoinositide-3 kinase (PI3K) or extracellular signal-regulated kinase 1/2 (ERK1/2). Neuronal survival, cell death and levels of caspase 3, PEDF, phosphorylated ERK1/2, and Bcl-2 are measured. The data show cortical cultures release PEDF and that H2O2 treatment causes cell death, increases activated caspase 3 levels and decreases release of PEDF. Exogenous PEDF induces a dose-dependent increase in Bcl-2 expression and neuronal survival. Blocking Bcl-2 expression by siRNA reduced PEDF-induced increases in neuronal survival. Treating cortical cultures with PEDF 24 h before H2O2 exposure mitigates oxidant-induced decreases in neuronal survival, Bcl-2 expression, and phosphorylation of ERK1/2 and also reduces elevated caspase 3 level and activity. PEDF pretreatment’s effect on survival is blocked by inhibiting ERK or PI3K. However, only inhibition of ERK reduced the ability of PEDF to protect neurons from H2O2-induced Bcl-2 decrease and neuronal death. These data demonstrate PEDF- mediated neuroprotection against oxidant injury is largely mediated via ERK1/2 and Bcl-2 and suggest the utility of PEDF in preserving the viability of oxidatively challenged neurons.

The Alzheimer's disease (AD) epidemic proceeds unabated. Estimates suggest 5.4 million Americans and 36 million people worldwide have AD. No single mechanism or pathologic mediator can account for AD progression. Currently no disease modifying therapies are available. There is a large literature documenting an association among cardiovascular risk factors (CVRFs), especially diabetes and hypoxia, with increased AD incidence. CVRFs directly impair vascular function and could mediate cerebrovascular dysfunction in AD. This is important as cerebrovascular dysfunction precedes cognitive decline and onset of neurodegenerative changes in AD and AD animal models. In this review we present evidence that thrombin may be a heretofore unexplored target for AD therapy. This idea is based on the following observations. Thrombin is elevated in the brain and cerebral microvasculature in AD, is directly neurotoxic, and causes pro-inflammatory effects in endothelial cells, microglia, and astrocytes. Diabetes- and hypoxia-induced cerebrovascular effects are mediated by thrombin. Thrombin inhibitors block the effects of hypoxia on brain endothelial cells and reduce vascular inflammation in transgenic AD mice. Based on reports that reducing cerebrovascular expression of inflammatory proteins in AD mice is associated with improved cognition, we propose thrombin inhibitors could prove useful for improving cognition in AD patients. The next generation of AD therapeutics should not focus on single target drugs but rather employ a multi-component cocktail approach. We propose thrombin inhibitors be considered as potential contributors to the dementia therapy pharmacopeia. The urgent need for disease-modifying drugs in AD demands new thinking about disease pathogenesis and exploration of novel drug targets.

Endothelial cells make up a minor population of cells in a tissue, but play a major role in tissue homeostasis, as well as in diverse pathologies. To understand the biology of cerebral endothelium, purification and characterization of the cerebrocortical endothelial cell population is highly desirable. For this purpose, rat brains are mechanically minced and subsequently digested enzymatically with collagenase. In this protocol, the capillary fraction (microvessels) and the fraction enriched in small arterioles and arteries (resistance vessels) are separated. Each produces its own homogenous endothelial culture, namely, MV-EC and RV-EC. The endothelial origin of these cells is identified by positive immunofluorescent staining for the endothelial cell surface antigen Factor VIII. Unlike MV-EC, RV-EC cultures are capable of serial cultivation for up to 15 passages. Primary MV-ECs are able to retain their characteristic endothelial morphology for 6-8 weeks.

Background Angiogenesis is tightly linked to inflammation and cancer. Regulation of angiogenesis is mediated primarily through activation of receptor tyrosine kinases, thus kinase inhibitors represent a new paradigm in anti-cancer therapy. However, these inhibitors have broad effects on inflammatory processes and multiple cell types. Sunitinib is a multitarget receptor tyrosine kinase inhibitor, which has shown promise for the treatment of glioblastoma, a highly vascularized tumor. However, there is little information as to the direct effects of sunitinib on brain-derived neurons. The objective of this study is to explore the effects of sunitinib on neuronal survival as well as on the expression of inflammatory protein mediators in primary cerebral neuronal cultures. Methods Primary cortical neurons were exposed to various doses of sunitinib. The drug-treated cultures were assessed for survival by MTT assay and cell death by lactate dehydrogenase release. The ability of sunitinib to affect NF-κB, COX2 and NOS2 expression was determined by western blot. The NF-κB inhibitors dicoumarol, SN50 and BAY11-7085 were employed to assess the role of NF-κB in sunitinib-mediated effects on neuronal survival as well as COX2 and NOS2 expression. Results Treatment of neuronal cultures with sunitinib caused a dose-dependent increase in cell survival and decrease in neuronal cell death. Exposure of neurons to sunitinib also induced an increase in the expression of NF-κB, COX2 and NOS2. Inhibiting NF-κB blunted the increase in cell survival and decrease in cell death evoked by sunitinib. Treatment of cell cultures with both sunitinib and NF-κB inhibitors mitigated the increase in COX2 and NOS2 caused by sunitinib. Conclusions Sunitinib increases neuronal survival and this neurotrophic effect is mediated by NF-κB. Also, the inflammatory proteins COX2 and NOS2 are upregulated by sunitinib in an NF-κB-dependent manner. These data are in agreement with a growing literature suggesting beneficial effects for inflammatory mediators such as NF-κB, COX2 and NOS2 in neurons. Further work is needed to fully explore the effects of sunitinib in the brain and its possible use as a treatment for glioblastoma. Finally, sunitinib may be useful for the treatment of a range of central nervous system diseases where neuronal injury is prominent.

Considerable evidence implicates hypoxia and vascular inflammation in Alzheimer's disease (AD). Thrombin, a multifunctional inflammatory mediator, is demonstrable in the brains of AD patients both in the vessel walls and senile plaques. Hypoxia-inducible factor 1α (HIF-1α), a key regulator of the cellular response to hypoxia, is also upregulated in the vasculature of human AD brains. The objective of this study is to investigate inflammatory protein expression in the cerebrovasculature of transgenic AD mice and to explore the role of thrombin as a mediator of cerebrovascular inflammation and oxidative stress in AD and in hypoxia-induced changes in brain endothelial cells. Immunofluorescent analysis of the cerebrovasculature in AD mice demonstrates significant (p < 0.01–0.001) increases in thrombin, HIF-1α, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinases (MMPs), and reactive oxygen species (ROS) compared to controls. Administration of the thrombin inhibitor dabigatran (100 mg/kg) to AD mice for 34 weeks significantly decreases expression of inflammatory proteins and ROS. Exposure of cultured brain endothelial cells to hypoxia for 6 h causes an upregulation of thrombin, HIF-1α, MCP-1, IL-6, and MMP2 and ROS. Treatment of endothelial cells with the dabigatran (1 nM) reduces ROS generation and inflammatory protein expression (p < 0.01–0.001). The data demonstrate that inhibition of thrombin in culture blocks the increase in inflammatory protein expression and ROS generation evoked by hypoxia. Also, administration of dabigatran to transgenic AD mice diminishes ROS levels in brain and reduces cerebrovascular expression of inflammatory proteins. Taken together, these results suggest that inhibiting thrombin generation could have therapeutic value in AD and other disorders where hypoxia, inflammation, and oxidative stress are involved.

Bidirectional communication between neurons and vascular cells is important to the maintenance of the central nervous system (CNS) milieu. Vascular endothelial growth factor (VEGF), through its ability to affect both vascular and neuronal cells, is likely a key protein in this process. Despite considerable literature documenting a neuroprotective function for VEGF, overexpression of this protein has also been shown in a wide variety of CNS diseases, including Alzheimer's disease (AD). Increased oxidative stress and elevated thrombin levels have also been documented in AD, specifically in the microvasculature. The aim of the current study is to examine endothelial cells and neurons in vitro to determine the effects of oxidative stress and thrombin on VEGF release as well as the effects of low and high dose VEGF on neuronal viability. The data show that microvessels isolated from AD patients secrete significantly higher levels of VEGF compared to control-derived vessels. Exposure of brain endothelial cells to oxidative stress (sodium nitroprusside, menadione, or hydrogen peroxide) or thrombin significantly increases VEGF expression. Exposure of cultured neurons to oxidative stress increases expression of thrombin. Treating rat cortical neurons with high dose VEGF (≥500 ng/ml) decreases neuronal survival and expression of the anti-apoptotic protein Bcl-2 while increasing proapoptic proteins caspase 3 and phosphorylated p38 MAPK. High dose VEGF also negates the decrease in amyloid-β evoked by low dose VEGF. These results suggest that despite literature supporting neuroprotective effects of this protein, caution is warranted prior to implementation of VEGF as a therapeutic in the brain.

As the population ages, the need for effective methods to maintain brain function in older adults is increasingly pressing. Vascular disease and neurodegenerative disorders commonly co-occur in older persons. Cerebrovascular products contribute to the neuronal milieu and have important consequences for neuronal viability. In this regard vascular derived neuroprotective proteins, Such as vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and pituitary adenylate cyclase activating peptide (PACAP) are important for maintaining neuronal viability, especially in the face of injury and disease. The objective of this study is to measure and compare levels of VEGF, PEDF and PACAP released from isolated brain microvessels of Fischer 344 rats at 6, 12, 18, and 24 months of age. Addition of acetaminophen to isolated brain microvessels is employed to determine whether this drug affects vascular expression of these neuroprotective proteins. Experiments on cultured brain endothelial cells are performed to explore the mechanisms/mediators that regulate the effect of acetaminophen on endothelial cells. The data indicate cerebrovascular expression of VEGF, PEDF and PACAP significantly decreases with age. The age-associated decrease in VEGF and PEDF is ameliorated by addition of acetaminophen to isolated brain microvessels. Also, release of VEGF, PEDF, and PACAP from cultured brain endothelial cells decreases with exposure to the oxidant stressor menadione. Acetaminophen treatment upregulates VEGF, PEDF and PACAP in brain endothelial cells exposed to oxidative stress. The effect of acetaminophen on cultured endothelial cells is in part inhibited by the selective thrombin inhibitor hirudin. The results of this study suggest that acetaminophen may be a useful agent for preserving cerebrovascular function. If a low dose of acetaminophen can counteract the decrease in vascular-derived neurotrophic factors evoked by age and oxidative stress, this drug might be useful for improving brain function in the elderly.

Our laboratory has documented that brain microvessels derived from patients with Alzheimer disease (AD) express or release a myriad of factors that have been implicated in vascular activation and angiogenesis. In addition, we have documented that signaling cascades associated with vascular activation and angiogenesis are upregulated in AD-derived brain microvessels. These results are consistent with emerging data suggesting that factors and processes characteristic of vascular activation and angiogenesis are found in the AD brain. Despite increases in proangiogenic factors and signals in the AD brain, however, evidence for increased vascularity in AD is lacking. Cerebral hypoperfusion/hypoxia, a potent stimulus for vascular activation and angiogenesis, triggers hypometabolic, cognitive, and degenerative changes in the brain. In our working model, hypoxia stimulates the angiogenic process; yet, there is no new vessel growth. Therefore, there are no feedback signals to shut off vascular activation, and endothelial cells become irreversibly activated. This activation results in release of a large number of proteases, inflammatory proteins, and other gene products with biologic activity that can injure or kill neurons. Pathologic activation of brain vasculature may contribute noxious mediators that lead to neuronal injury and disease processes in AD brains. This concept is supported by preliminary experiments in our laboratory, which show that pharmacologic blockade of vascular activation improves cognitive function in an animal model of AD. Thus, "vascular activation" could be a novel, unexplored therapeutic target in AD.

Diseases of the central nervous system (CNS) pose a significant health challenge, but despite their diversity, they share many common features and mechanisms. For example, endothelial dysfunction has been implicated as a crucial event in the development of several CNS disorders, such as Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, multiple sclerosis, human immunodeficiency virus (HIV)-1-associated neurocognitive disorder and traumatic brain injury. Breakdown of the blood–brain barrier (BBB) as a result of disruption of tight junctions and transporters, leads to increased leukocyte transmigration and is an early event in the pathology of these disorders. The brain endothelium is highly reactive because it serves as both a source of, and a target for, inflammatory proteins and reactive oxygen species. BBB breakdown thus leads to neuroinflammation and oxidative stress, which are implicated in the pathogenesis of CNS disease. Furthermore, the physiology and pathophysiology of endothelial cells are closely linked to the functioning of their mitochondria, and mitochondrial dysfunction is another important mediator of disease pathology in the brain. The high concentration of mitochondria in cerebrovascular endothelial cells might account for the sensitivity of the BBB to oxidant stressors. Here, we discuss how greater understanding of the role of BBB function could lead to new therapeutic approaches for diseases of the CNS that target the dynamic properties of brain endothelial cells.

This study examined the effects on cognitive behaviors of giving normal adult gerbils three compounds, normally in the circulation, which interact to increase brain phosphatides, synaptic proteins, dendritic spines, and neurotransmitter release. Animals received supplemental uridine (as its monophosphate, UMP; 0.5%) and choline (0.1%) via the diet, and docosahexaenoic acid (DHA; 300 mg/kg/day) by gavage, for 4 wk, and then throughout the subsequent period of behavioral training and testing. As shown previously, giving all three compounds caused highly significant (P

Background Most neurodegenerative diseases are age-related disorders; however, how aging predisposes the brain to disease has not been adequately addressed. The objective of this study is to determine whether expression of proteins in the cerebromicrovasculature related to inflammation, oxidative stress and neurotoxicity is altered with aging. Methods Brain microvessels are isolated from Fischer 344 rats at 6, 12, 18 and 24 months of age. Levels of interleukin (IL)-1β and IL-6 RNA are determined by RT-PCR and release of cytokines into the media by ELISA. Vessel conditioned media are also screened by ELISA for IL-1α, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α, (TNFα), and interferon γ (IFNγ). Immunofluorescent analysis of brain sections for IL-1β and IL-6 is performed. Results Expression of IL-1β and IL-6, both at RNA and protein levels, significantly (p < 0.01) decreases with age. Levels of MCP-1, TNFα, IL-1α, and IFNγ are significantly (p < 0.05-0.01) lower in 24 month old rats compared to 6 month old animals. Immunofluorescent analysis of brain vessels also shows a decline in IL-1β and IL-6 in aged rats. An increase in oxidative stress, assessed by increased carbonyl formation, as well as a decrease in the antioxidant protein manganese superoxide dismutase (MnSOD) is evident in vessels of aged animals. Finally, addition of microvessel conditioned media from aged rats to neuronal cultures evokes significant (p < 0.001) neurotoxicity. Conclusions These data demonstrate that cerebrovascular expression of proteins related to inflammation, oxidative stress and neurotoxicity is altered with aging and suggest that the microvasculature may contribute to functional changes in the aging brain.