High Glucose and Hypoxia-Mediated Damage to Human Brain Microvessel Endothelial Cells Induces an Altered, Pro-Inflammatory Phenotype in BV-2 Microglia In Vitro.

Diabetes is strongly linked to the development of Alzheimer's disease (AD), though the mechanisms for this enhanced risk are unclear. Because vascular inflammation is a consistent feature of both diabetes and AD, the cerebral microcirculation could be a key target for the effects of diabetes in the brain. The goal of this study is to explore whether brain endothelial cells, injured by diabetes-related insults, glucose and hypoxia, can affect inflammatory and activation processes in microglia in vitro. Human brain microvascular endothelial cells (HBMVECs) were either treated with 5 mM glucose (control), 30 mM glucose (high glucose), exposed to hypoxia, or exposed to hypoxia plus high glucose. HBMVEC-conditioned medium was then used to treat BV-2 microglia. Alterations in microglia phenotype were assessed through measurement of nitric oxide (NO), cytokine production, microglial activation state markers, and microglial phagocytosis. HBMVECs were injured by exposure to glucose and/or hypoxia, as assessed by release of LDH, interleukin (IL)-1ß, and reactive oxygen species (ROS). HBMVECs injured by glucose and hypoxia induced increases in microglial production of NO, tumor necrosis factor-α (TNFα) and matrix metalloproteinase (MMP)-9. Injured HBMVECs significantly increased microglial expression of CD11c and CLEC7A, and decreased expression of the homeostatic marker P2RY12. Finally, bead uptake by BV-2 cells, an index of phagocytic ability, was elevated by conditioned media from injured HBMVECs. The demonstration that injury to brain endothelial cells by diabetic-associated insults, glucose and hypoxia, promotes microglial inflammation supports the idea that the cerebral microcirculation is a critical locus for the deleterious effects of diabetes in the AD brain.

Sex Differences in Metabolic Indices and Chronic Neuroinflammation in Response to Prolonged High-Fat Diet in ApoE4 Knock-In Mice.

Late-onset Alzheimer's disease (LOAD) likely results from combinations of risk factors that include both genetic predisposition and modifiable lifestyle factors. The E4 allele of apolipoprotein E (ApoE) is the most significant genetic risk factor for LOAD. A Western-pattern diet (WD) has been shown to strongly increase the risk of cardiovascular disease and diabetes, conditions which have been strongly linked to an increased risk for developing AD. Little is known about how the WD may contribute to, or enhance, the increased risk presented by possession of the ApoE4 allele. To model this interaction over the course of a lifetime, we exposed male and female homozygote ApoE4 knock-in mice and wild-type controls to nine months of a high-fat WD or standard chow diet. At eleven months of age, the mice were tested for glucose tolerance and then for general activity and spatial learning and memory. Postmortem analysis of liver function and neuroinflammation in the brain was also assessed. Our results suggest that behavior impairments resulted from the convergence of interacting metabolic alterations, made worse in a male ApoE4 mice group who also showed liver dysfunction, leading to a higher level of inflammatory cytokines in the brain. Interestingly, female ApoE4 mice on a WD revealed impairments in spatial learning and memory without the observed liver dysfunction or increase in inflammatory markers in the brain. These results suggest multiple direct and indirect pathways through which ApoE and diet-related factors interact. The striking sex difference in markers of chronic neuroinflammation in male ApoE4 mice fed the high-fat WD suggests a specific mechanism of interaction conferring significant enhanced LOAD risk for humans with the ApoE4 allele, which may differ between sexes. Additionally, our results suggest researchers exercise caution when designing and interpreting results of experiments employing a WD, being careful not to assume a WD impacts both sexes by the same mechanisms.

Isoform-Specific Effects of Apolipoprotein E on Markers of Inflammation and Toxicity in Brain Glia and Neuronal Cells In Vitro.

Mutations to the cholesterol transport protein apolipoprotein E (ApoE) have been identified as a major risk factor for the development of sporadic or late-onset Alzheimer's disease (AD), with the e4 allele representing an increased risk and the rare e2 allele having a reduced risk compared to the primary e3 form. The reasons behind the change in risk are not entirely understood, though ApoE4 has been connected to inflammation and toxicity in both the brain and the periphery. The goal of this study was to better understand how the ApoE isoforms (ApoE2/3/4) confer differential AD-related risk by assessing cell-specific ApoE-related neuroinflammatory and neurotoxic effects. We compared the effects of ApoE isoforms in vitro on human astrocytes, a human immortalized microglia cell line (HMC3), and the human neuroblastoma cell line SH-SY5Y. Cells were treated for 24 h with or without recombinant ApoE2, ApoE3, or ApoE4 (20 nM) and inflammation and toxicity markers assessed. Our results indicated the expression of inflammatory cytokines IL-1ß, TNFα, and IL-6 in human astrocytes was increased in response to all ApoE isoforms, with ApoE4 evoking the highest level of cytokine expression. In response to ApoE2 or ApoE3, microglial cells showed reduced levels of microglial activation markers TREM2 and Clec7a, while ApoE4 induced increased levels of both markers. ApoE2 promoted neuron survival through increased BDNF release from astrocytes. In addition, ApoE2 promoted, while ApoE4 reduced, neuronal viability. Overall, these results suggest that ApoE4 acts on cells in the brain to promote inflammation and neuronal injury and that the deleterious effects of ApoE4 on these cells may, in part, contribute to its role as a risk factor for AD.

Inhibiting thrombin improves motor function and decreases oxidative stress in the LRRK2 transgenic Drosophila melanogaster model of Parkinson's disease.

Parkinson's disease (PD) is a complex neurodegenerative disease characterized by the presence of tremors, loss of dopaminergic neurons and accumulation of α-synuclein. While there is no single direct cause of PD, genetic mutations, exposure to pesticides, diet and traumatic brain injury have been identified as risk factors. Increasing evidence suggests that oxidative stress and neuroinflammation contribute to the pathogenesis of neuronal injury in neurodegenerative diseases such as PD and Alzheimer's disease (AD). We have previously documented that the multifunctional inflammatory mediator thrombin contributes to oxidative stress and neuroinflammation in AD. Here, for the first time, we explore the role of thrombin in a transgenic PD model, the LRRK2 mutant Drosophila melanogaster. Transgenic flies were treated with the direct thrombin inhibitor dabigatran for 7 days and locomotor activity and indices of oxidative stress evaluated. Our data show that dabigatran treatment significantly (p < 0.05) improved climbing activity, a measurement of locomotor ability, in male but had no effect on locomotor performance in female flies. Dabigatran treatment had no effect on tyrosine hydroxylase levels. Analysis of oxidative stress in male flies showed that dabigatran was able to significantly (p < 0.01) lower reactive oxygen species levels. Furthermore, Western blot analysis showed that the pro-oxidant proteins iNOS and NOX4 are elevated in LRRK2 male flies compared to wildtype and that treatment with dabigatran reduced expression of these proteins. Our results indicate that dabigatran treatment could improve motor function in PD by reducing oxidative stress. These data suggest that targeting thrombin may improve oxidative stress related pathologies in PD.

Equol, a Blood-Brain Barrier Permeable Gut Microbial Metabolite of Dietary Isoflavone Daidzein, Exhibits Neuroprotective Effects against Neurotoxins Induced Toxicity in Human Neuroblastoma SH-SY5Y Cells and Caenorhabditis elegans.

Emerging data support that plant food based isoflavones have ameliorating effects on a variety of neurodegenerative diseases including Parkinson's disease (PD). Our previous investigation revealed that dietary isoflavones including genistein (GEN), daidzein (DAI), and equol (EQL; a gut microbial metabolite of DAI) showed promising blood-brain barrier permeability and anti-neuroinflammatory activity in murine microglial BV2 cells. However, the neuroprotective effects of EQL against neurotoxins induced toxicity in PD related models remains unclear. Herein, EQL, along with GEN and DAI, were evaluated for their cytoprotective effect in a non-contact co-culture model with LPS-BV2-conditioned media and human neuroblastoma SH-SY5Y cells. In addition, their neuroprotective effects against PD related neurotoxins including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+) induced cytotoxicity were evaluated in SH-SY5Y cells. Furthermore, EQL was evaluated for its neuroprotective effects against MPP+ induced neurotoxicity using in vivo PD model including Caenorhabditis elegans lifespan assay. DAI (10 µM) and EQL (10 and 20 µM) showed cytoprotective effects by decreasing LPS-BV2-conditioned media induced cytotoxicity in SH-SY5Y cells by 29.2, 32.4 and 27.2%, respectively. EQL (10 and 20 µM) also showed neuroprotective effects by decreasing 6-OHDA and MPP+ induced cytotoxicity in SH-SY5Y cells by 30.6-34.5 and 17.9-18.9%, respectively. Additionally, data from the in vivo assay supported EQL's neuroprotective effect as it increases survival of C. elegans exposed to MPP+ from 72 to 108 h. Our findings support a growing body of evidence of the neuroprotective effects of dietary isoflavones and further studies are warranted to elucidate their mechanisms of action.

Blood-based biomarkers in Alzheimer disease: Current state of the science and a novel collaborative paradigm for advancing from discovery to clinic.

The last decade has seen a substantial increase in research focused on the identification of blood-based biomarkers that have utility in Alzheimer's disease (AD). Blood-based biomarkers have significant advantages of being time- and cost-efficient as well as reduced invasiveness and increased patient acceptance. Despite these advantages and increased research efforts, the field has been hampered by lack of reproducibility and an unclear path for moving basic discovery toward clinical utilization. Here we reviewed the recent literature on blood-based biomarkers in AD to provide a current state of the art. In addition, a collaborative model is proposed that leverages academic and industry strengths to facilitate the field in moving past discovery only work and toward clinical use. Key resources are provided. This new public-private partnership model is intended to circumvent the traditional handoff model and provide a clear and useful paradigm for the advancement of biomarker science in AD and other neurodegenerative diseases.

Guidelines for the standardization of preanalytic variables for blood-based biomarker studies in Alzheimer's disease research.

The lack of readily available biomarkers is a significant hindrance toward progressing to effective therapeutic and preventative strategies for Alzheimer's disease (AD). Blood-based biomarkers have potential to overcome access and cost barriers and greatly facilitate advanced neuroimaging and cerebrospinal fluid biomarker approaches. Despite the fact that preanalytical processing is the largest source of variability in laboratory testing, there are no currently available standardized preanalytical guidelines. The current international working group provides the initial starting point for such guidelines for standardized operating procedures (SOPs). It is anticipated that these guidelines will be updated as additional research findings become available. The statement provides (1) a synopsis of selected preanalytical methods utilized in many international AD cohort studies, (2) initial draft guidelines/SOPs for preanalytical methods, and (3) a list of required methodological information and protocols to be made available for publications in the field to foster cross-validation across cohorts and laboratories.

Molecular neuropsychology: creation of test-specific blood biomarker algorithms.

BACKGROUND: Prior work on the link between blood-based biomarkers and cognitive status has largely been based on dichotomous classifications rather than detailed neuropsychological functioning. The current project was designed to create serum-based biomarker algorithms that predict neuropsychological test performance. METHODS: A battery of neuropsychological measures was administered. Random forest analyses were utilized to create neuropsychological test-specific biomarker risk scores in a training set that were entered into linear regression models predicting the respective test scores in the test set. Serum multiplex biomarker data were analyzed on 108 proteins from 395 participants (197 Alzheimer patients and 198 controls) from the Texas Alzheimer's Research and Care Consortium. RESULTS: The biomarker risk scores were significant predictors (p < 0.05) of scores on all neuropsychological tests. With the exception of premorbid intellectual status (6.6%), the biomarker risk scores alone accounted for a minimum of 12.9% of the variance in neuropsychological scores. Biomarker algorithms (biomarker risk scores and demographics) accounted for substantially more variance in scores. Review of the variable importance plots indicated differential patterns of biomarker significance for each test, suggesting the possibility of domain-specific biomarker algorithms. CONCLUSIONS: Our findings provide proof of concept for a novel area of scientific discovery, which we term 'molecular neuropsychology'.

Sunitinib enhances neuronal survival in vitro via NF-κB-mediated signaling and expression of cyclooxygenase-2 and inducible nitric oxide synthase.

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.

Thrombin, a Key Driver of Pathological Inflammation in the Brain.

Neurodegenerative diseases, including Alzheimer's disease (AD), are major contributors to death and disability worldwide. A multitude of evidence suggests that neuroinflammation is critical in neurodegenerative disease processes. Exploring the key mediators of neuroinflammation in AD, a prototypical neurodegenerative disease, could help identify pathologic inflammatory mediators and mechanisms in other neurodegenerative diseases. Elevated levels of the multifunctional inflammatory protein thrombin are commonly found in conditions that increase AD risk, including diabetes, atherosclerosis, and traumatic brain injury. Thrombin, a main driver of the coagulation cascade, has been identified as important to pathological events in AD and other neurodegenerative diseases. Furthermore, recent evidence suggests that coagulation cascade-associated proteins act as drivers of inflammation in the AD brain, and studies in both human populations and animal models support the view that abnormalities in thrombin generation promote AD pathology. Thrombin drives neuroinflammation through its pro-inflammatory activation of microglia, astrocytes, and endothelial cells. Due to the wide-ranging pro-inflammatory effects of thrombin in the brain, inhibiting thrombin could be an effective strategy for interrupting the inflammatory cascade which contributes to neurodegenerative disease progression and, as such, may be a potential therapeutic target for AD and other neurodegenerative diseases.

Age-related decrease in cerebrovascular-derived neuroprotective proteins: effect of acetaminophen.

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.

Neurovascular dysfunction, inflammation and endothelial activation: implications for the pathogenesis of Alzheimer's disease.

Alzheimer's disease (AD) is an age-related disorder characterized by progressive cognitive decline and dementia. Alzheimer's disease is an increasingly prevalent disease with 5.3 million people in the United States currently affected. This number is a 10 percent increase from previous estimates and is projected to sharply increase to 8 million by 2030; it is the sixth-leading cause of death. In the United States the direct and indirect costs of Alzheimer's and other dementias to Medicare, Medicaid and businesses amount to more than $172 billion each year. Despite intense research efforts, effective disease-modifying therapies for this devastating disease remain elusive. At present, the few agents that are FDA-approved for the treatment of AD have demonstrated only modest effects in modifying clinical symptoms for relatively short periods and none has shown a clear effect on disease progression. New therapeutic approaches are desperately needed. Although the idea that vascular defects are present in AD and may be important in disease pathogenesis was suggested over 25 years ago, little work has focused on an active role for cerebrovascular mechanisms in the pathogenesis of AD. Nevertheless, increasing literature supports a vascular-neuronal axis in AD as shared risk factors for both AD and atherosclerotic cardiovascular disease implicate vascular mechanisms in the development and/or progression of AD. Also, chronic inflammation is closely associated with cardiovascular disease, as well as a broad spectrum of neurodegenerative diseases of aging including AD. In this review we summarize data regarding, cardiovascular risk factors and vascular abnormalities, neuro- and vascular-inflammation, and brain endothelial dysfunction in AD. We conclude that the endothelial interface, a highly synthetic bioreactor that produces a large number of soluble factors, is functionally altered in AD and contributes to a noxious CNS milieu by releasing inflammatory and neurotoxic species.

Cerebral microvascular endothelium and the pathogenesis of neurodegenerative diseases.

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.

Brain endothelial cells synthesize neurotoxic thrombin in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by neuronal death; thus, identifying neurotoxic proteins and their source is central to understanding and treating AD. The multifunctional protease thrombin is neurotoxic and found in AD senile plaques. The objective of this study was to determine whether brain endothelial cells can synthesize thrombin and thus be a source of this neurotoxin in AD brains. Microvessels were isolated from AD patient brains and from age-matched controls. Reverse transcription-PCR demonstrated that thrombin message was highly expressed in microvessels from AD brains but was not detectable in control vessels. Similarly, Western blot analysis of microvessels showed that the thrombin protein was highly expressed in AD- but not control-derived microvessels. In addition, high levels of thrombin were detected in cerebrospinal fluid obtained from AD but not control patients, and sections from AD brains showed reactivity to thrombin antibody in blood vessel walls but not in vessels from controls. Finally, we examined the ability of brain endothelial cells in culture to synthesize thrombin and showed that oxidative stress or cell signaling perturbations led to increased expression of thrombin mRNA in these cells. The results demonstrate, for the first time, that brain endothelial cells can synthesize thrombin, and suggest that novel therapeutics targeting vascular stabilization that prevent or decrease release of thrombin could prove useful in treating this neurodegenerative disease.

Dabigatran reduces thrombin-induced neuroinflammation and AD markers in vitro: Therapeutic relevance for Alzheimer's disease.

Background: Vascular risk factors such as atherosclerosis, diabetes, and elevated homocysteine levels are strongly correlated with onset of Alzheimer's disease (AD). Emerging evidence indicates that blood coagulation protein thrombin is associated with vascular and non-vascular risk factors of AD. Here, we examined the effect of thrombin and its direct inhibitor dabigatran on key mediators of neuro-inflammation and AD pathology in the retinoic acid (RA)-differentiated human neuroblastoma cell line SH-SY5Y. Methods: SH-SY5Y cells exposed to thrombin concentrations (10-100 nM) +/- 250 nM dabigatran for 24 h were analyzed for protein and gene expression. Electrophoretic mobility shift assay (EMSA) was used to determine DNA binding of NFkB. Western blotting, qRT-PCR and ELISA were used to measure the protein, mRNA, and activity levels of known AD hallmarks and signaling molecules. Results: Dabigatran treatment attenuated thrombin-induced increase in DNA binding of NFκB by 175% at 50 nM and by 77% at 100 nM thrombin concentration. Thrombin also augmented accumulation of Aß protein expression and phosphorylation of p38 MAPK, a downstream molecule in the signaling cascade, expression of pro-apoptotic mediator caspase 3, APP, tTau and pTau. Additionally, thrombin increased BACE1 activity, GSK3ß expression, and APP, BACE1, Tau and GSK3ß mRNA levels. Co-incubation with dabigatran attenuated thrombin-induced increases in the protein, mRNA, and activities of the aforesaid molecules to various extents (between -31% and -283%). Conclusion: Our data demonstrates that thrombin promotes AD-related pathological changes in neuronal cultures and suggests that use of direct oral anticoagulants may provide a therapeutic benefit against thrombin-driven neuroinflammation and downstream pathology in AD.

Thrombin Signaling Contributes to High Glucose-Induced Injury of Human Brain Microvascular Endothelial Cells.

BACKGROUND: Diabetes is one of the strongest disease-related risk factors for Alzheimer's disease (AD). In diabetics, hyperglycemia-induced microvascular complications are the major cause of end-organ injury, contributing to morbidity and mortality. Microvascular pathology is also an important and early feature of AD. The cerebral microvasculature may be a point of convergence of both diseases. Several lines of evidence also implicate thrombin in AD as well as in diabetes. OBJECTIVE: Our objective was to investigate the role of thrombin in glucose-induced brain microvascular endothelial injury. METHODS: Cultured Human brain microvascular endothelial cells (HBMVECs) were treated with 30 mM glucose±100 nM thrombin and±250 nM Dabigatran or inhibitors of PAR1, p38MAPK, MMP2, or MMP9. Cytotoxicity and thrombin activity assays on supernatants and western blotting for protein expression in lysates were performed. RESULTS: reatment of HBMVECs with 30 mM glucose increased thrombin activity and expression of inflammatory proteins TNFα, IL-6, and MMPs 2 and 9; this elevation was reduced by the thrombin inhibitor dabigatran. Direct treatment of brain endothelial cells with thrombin upregulated p38MAPK and CREB, and induced TNFα, IL6, MMP2, and MMP9 as well as oxidative stress proteins NOX4 and iNOS. Inhibition of thrombin, thrombin receptor PAR1 or p38MAPK decrease expression of inflammatory and oxidative stress proteins, implying that thrombin may play a central role in glucose-induced endothelial injury. CONCLUSION: Since preventing brain endothelial injury would preserve blood-brain barrier integrity, prevent neuroinflammation, and retain intact functioning of the neurovascular unit, inhibiting thrombin, or its downstream signaling effectors, could be a therapeutic strategy for mitigating diabetes-induced dementia.

Cerebrovascular expression of proteins related to inflammation, oxidative stress and neurotoxicity is altered with aging.

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)-1ß 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-1ß and IL-6 is performed. RESULTS: Expression of IL-1ß 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-1ß 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.

Thrombin, a Mediator of Coagulation, Inflammation, and Neurotoxicity at the Neurovascular Interface: Implications for Alzheimer's Disease.

The societal burden of Alzheimer's disease (AD) is staggering, with current estimates suggesting that 50 million people world-wide have AD. Identification of new therapeutic targets is a critical barrier to the development of disease-modifying therapies. A large body of data implicates vascular pathology and cardiovascular risk factors in the development of AD, indicating that there are likely shared pathological mediators. Inflammation plays a role in both cardiovascular disease and AD, and recent evidence has implicated elements of the coagulation system in the regulation of inflammation. In particular, the multifunctional serine protease thrombin has been found to act as a mediator of vascular dysfunction and inflammation in both the periphery and the central nervous system. In the periphery, thrombin contributes to the development of cardiovascular disease, including atherosclerosis and diabetes, by inducing endothelial dysfunction and related inflammation. In the brain, thrombin has been found to act on endothelial cells of the blood brain barrier, microglia, astrocytes, and neurons in a manner that promotes vascular dysfunction, inflammation, and neurodegeneration. Thrombin is elevated in the AD brain, and thrombin signaling has been linked to both tau and amyloid beta, pathological hallmarks of the disease. In AD mouse models, inhibiting thrombin preserves cognition and endothelial function and reduces neuroinflammation. Evidence linking atrial fibrillation with AD and dementia indicates that anticoagulant therapy may reduce the risk of dementia, with targeting thrombin shown to be particularly effective. It is time for "outside-the-box" thinking about how vascular risk factors, such as atherosclerosis and diabetes, as well as the coagulation and inflammatory pathways interact to promote increased AD risk. In this review, we present evidence that thrombin is a convergence point for AD risk factors and as such that thrombin-based therapeutics could target multiple points of AD pathology, including neurodegeneration, vascular activation, and neuroinflammation. The urgent need for disease-modifying drugs in AD demands new thinking about disease pathogenesis and an exploration of novel drug targets, we propose that thrombin inhibition is an innovative tactic in the therapeutic battle against this devastating disease.

Short-term treatment with dabigatran alters protein expression patterns in a late-stage tau-based Alzheimer's disease mouse model.

Proteins that regulate the coagulation cascade, including thrombin, are elevated in the brains of Alzheimer's disease (AD) patients. While studies using amyloid-based AD transgenic mouse models have implicated thrombin as a protein of interest, the role of thrombin in tau-based animal models has not been explored. The current study aims to determine how inhibiting thrombin could alter oxidative stress, inflammation, and AD-related proteins in a tau-based mouse model, the Tg4510. Aged Tg4510 mice were treated with the direct thrombin inhibitor dabigatran or vehicle for 7 days, brains collected, and western blot and data-independent proteomics using mass spectrometry with SWATH-MS acquisition performed to evaluate proteins related to oxidative stress, intracellular signaling, inflammation, and AD pathology. Dabigatran reduced iNOS, NOX4, and phosphorylation of tau (S396, S416). Additionally, dabigatran treatment increased expression of several signaling proteins related to cell survival and synaptic function. Increasing evidence supports a chronic procoagulant state in AD, highlighting a possible pathogenic role for thrombin. Our data demonstrate that inhibiting thrombin produces alterations in the expression of proteins involved in oxidative stress, inflammation, and AD-related pathology, suggesting that thrombin-mediated signaling affects multiple AD-related pathways providing a potential future therapeutic target.

Acetaminophen inhibits neuronal inflammation and protects neurons from oxidative stress.

BACKGROUND: Recent studies have demonstrated a link between the inflammatory response, increased cytokine formation, and neurodegeneration in the brain. The beneficial effects of anti-inflammatory drugs in neurodegenerative diseases, such as Alzheimer's disease (AD), have been documented. Increasing evidence suggests that acetaminophen has unappreciated anti-oxidant and anti-inflammatory properties. The objectives of this study are to determine the effects of acetaminophen on cultured brain neuronal survival and inflammatory factor expression when exposed to oxidative stress. METHODS: Cerebral cortical cultured neurons are pretreated with acetaminophen and then exposed to the superoxide-generating compound menadione (5 microM). Cell survival is assessed by MTT assay and inflammatory protein (tumor necrosis factor alpha, interleukin-1, macrophage inflammatory protein alpha, and RANTES) release quantitated by ELISA. Expression of pro- and anti-apoptotic proteins is assessed by western blots. RESULTS: Acetaminophen has pro-survival effects on neurons in culture. Menadione, a superoxide releasing oxidant stressor, causes a significant (p < 0.001) increase in neuronal cell death as well as in the release of tumor necrosis factor alpha, interleukin-1, macrophage inflammatory protein alpha, and RANTES from cultured neurons. Pretreatment of neuronal cultures with acetaminophen (50 microM) increases neuronal cell survival and inhibits the expression of these cytokines and chemokines. In addition, we document, for the first time, that acetaminophen increases expression of the anti-apoptotic protein Bcl2 in brain neurons and decreases the menadione-induced elevation of the proapoptotic protein, cleaved caspase 3. We show that blocking acetaminophen-induced expression of Bcl2 reduces the pro-survival effect of the drug. CONCLUSION: These data show that acetaminophen has anti-oxidant and anti-inflammatory effects on neurons and suggest a heretofore unappreciated therapeutic potential for this drug in neurodegenerative diseases such as AD that are characterized by oxidant and inflammatory stress.