Noradrenergic signaling controls Alzheimer's disease pathology via activation of microglial ß2 adrenergic receptors.

In Alzheimer's disease (AD) pathophysiology, plaque and tangle accumulation trigger an inflammatory response that mounts positive feed-back loops between inflammation and protein aggregation, aggravating neurite damage and neuronal death. One of the earliest brain regions to undergo neurodegeneration is the locus coeruleus (LC), the predominant site of norepinephrine (NE) production in the central nervous system (CNS). In animal models of AD, dampening the impact of noradrenergic signaling pathways, either through administration of beta blockers or pharmacological ablation of the LC, heightened neuroinflammation through increased levels of pro-inflammatory mediators. Since microglia are the resident immune cells of the CNS, it is reasonable to postulate that they are responsible for translating the loss of NE tone into exacerbated disease pathology. Recent findings from our lab demonstrated that noradrenergic signaling inhibits microglia dynamics via ß2 adrenergic receptors (ß2ARs), suggesting a potential anti-inflammatory role for microglial ß2AR signaling. Thus, we hypothesize that microglial ß2 adrenergic signaling is progressively impaired during AD progression, which leads to the chronic immune vigilant state of microglia that worsens disease pathology. First, we characterized changes in microglial ß2AR signaling as a function of amyloid pathology. We found that LC neurons and their projections degenerate early and progressively in the 5xFAD mouse model of AD; accompanied by mild decrease in the levels of norepinephrine and its metabolite normetanephrine. Interestingly, while 5xFAD microglia, especially plaque-associated microglia, significant downregulated ß2AR gene expression early in amyloid pathology, they did not lose their responsiveness to ß2AR stimulation. Most importantly, we demonstrated that specific microglial ß2AR deletion worsened disease pathology while chronic ß2AR stimulation resulted in attenuation of amyloid pathology and associated neuritic damage, suggesting microglial ß2AR might be used as potential therapeutic target to modify AD pathology.

Soluble E-cadherin: a critical oncogene modulating receptor tyrosine kinases, MAPK and PI3K/Akt/mTOR signaling.

E-cadherin, a cell-cell adhesion glycoprotein, is frequently downregulated with tumorigenic progression. The extracellular domain of E-cadherin is cleaved by proteases to generate a soluble ectodomain fragment, termed sEcad, which is elevated in the urine or serum of cancer patients. In this study, we explored the functional role of sEcad in the progression of skin squamous cell carcinomas (SCCs). We found that full-length E-cadherin expression was decreased and sEcad increased in human clinical tumor samples as well as in ultraviolet (UV)-induced SCCs in mice. Interestingly, sEcad associated with members of the human epidermal growth factor receptor (HER) and insulin-like growth factor-1 (IGF-1R) family of receptors in human and UV-induced mouse tumors. Moreover, in both E-cadherin-positive (E-cadherin(+)) and -negative (E-cadherin(-)) cells in vitro, sEcad activated downstream mitogen-activated protein (MAP) kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling and enhanced tumor growth, motility and invasion, the latter via activation of matrix metalloproteinase-2 (MMP-2) and MMP-9. To this end, HER, PI3K or MEK inhibitors suppressed sEcad's tumorigenic effects, including proliferation, migration and invasion. Taken together, our data suggest that sEcad contributes to skin carcinogenesis via association with the HER/IGF-1R-family of receptors and subsequent activation of the MAPK and PI3K/Akt/mTOR pathways, thereby implicating sEcad as a putative therapeutic target in cutaneous SCCs.

Sustained expression of interleukin-1beta in mouse hippocampus impairs spatial memory.

Glial activation and neuroinflammation occur in neurodegenerative disease and brain injury, however their presence in normal brain aging suggests that chronic neuroinflammation may be a factor in age-related dementia. Few studies have investigated the impact of sustained elevation of hippocampal interleukin-1beta, a pro-inflammatory cytokine upregulated during aging and Alzheimer's disease, on cognition in mice. We utilized the IL-1beta(XAT) transgenic mouse to initiate bilateral hippocampal overexpression of interleukin-1beta to determine the influence of sustained neuroinflammation independent of disease pathology. Fourteen days following transgene induction, adult male and female IL-1beta(XAT) mice were tested on non-spatial and spatial versions of the Morris water maze. For the spatial component, one retention trial was conducted 48 h after completion of a 3 day acquisition protocol (eight trials per day). Induction of IL-1beta did not impact non-spatial learning, but was associated with delayed acquisition and decreased retention of the spatial task. These behavioral impairments were accompanied by robust reactive gliosis and elevated mRNA expression of inflammatory genes in the hippocampus. Our results suggest that prolonged neuroinflammation response per se may impact mnemonic processes and support the future application of IL-1beta(XAT) transgenic mice to investigate chronic neuroinflammation in age- and pathology-related cognitive dysfunction.

Enhanced cyclooxygenase-2 expression in olfactory-limbic forebrain following kainate-induced seizures.

Cyclooxygenase-2 is expressed at low levels in a subset of neurons in CNS and is rapidly induced by a multiplicity of factors including seizure activity. A putative relationship exists between cyclooxygenase-2 induction and glutamatergic neurotransmission. Cyclooxygenase-1 is constitutively expressed in glial cells and has been specifically linked to microglia. In this study we evaluated cyclooxygenase-2 protein immunocytochemically and found markedly enhanced immunostaining primarily in olfactory-limbic regions at 2, 6 and 24 h following kainate-induced status epilepticus. Impressive enhanced cyclooxygenase-2 immunoreactivity was localized in anterior olfactory nucleus, tenia tecta, nucleus of the lateral olfactory tract, piriform cortex, lateral and basolateral amygdala, orbital frontal cortex, nucleus accumbens (shell) and associated areas of ventral striatum, entorhinal cortex, dentate gyrus granule cells and hilar neurons, hippocampal CA subfields and subiculum. Alternate sections were processed for dual immunocytochemical analysis utilizing c-Fos and cyclooxygenase-2 antiserum to examine the possibility that the neuronal induction of cyclooxygenase-2 was associated with seizure activity. Neurons that showed a timeline of cyclooxygenase-2 upregulation were found to possess c-Fos immunopositive nuclei. Additional results from all seizure groups showed cyclooxygenase-1 induction in microglia, which was confirmed by Western blot analysis of hippocampus. Western blot and real-time quantitative RT-PCR analysis showed significant upregulation of cyclooxygenase-2 expression, confirming its induction in neurons. These data indicate that cyclooxygenase-2 induction in a neuronal network can be a useful marker for pathways associated with seizure activity.

Microglial response is poorly correlated with neurodegeneration following chronic, low-dose MPTP administration in monkeys.

Many investigators have reported extensive microglial activation in the mouse substantia nigra and striatum following acute, high-dose 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. Our previous work demonstrated tyrosine hydroxylase (TH)-positive fiber sprouting in the striatum in monkeys that had received a partial dopaminergic lesion using a low-dose, chronic MPTP administration paradigm. To characterize the microglial response, we utilized HLA-DR (LN3) to immunolabel the class II major histocompatibility complex (MHC II). In MPTP-treated monkeys, there was an intense microglial response in the substantia nigra, nigrostriatal tract, and in both segments of the globus pallidus. This response was morphologically heterogeneous, with commingled ramified, activated, and multicellular morphologies throughout the extent of these basal ganglia structures. Surprisingly, there was little evidence of microglial reactivity in the striatum despite evidence of neurodegeneration-by silver labeling and by loss of TH immunolabeling. Moreover, this pattern of microglial reactivity was the same in all animals that had received MPTP and seemed to be independent of the degree of neurotoxin-induced neurodegeneration. Thus, we conclude that microglial reactivity, per se, is not consistently associated with neurodegeneration, but depends on regional differences.

Neuroinflammation and anti-inflammatory therapy for Alzheimer's disease.

Neuroinflammation is now recognized as a prominent feature in Alzheimer's pathology and a potential target for therapy aimed at treatment and prevention of disease. This review provides a synopsis of current information about cellular and molecular mediators involved in Alzheimer's neuroinflammation as well as interactions between these mediators that influence pathology. Anti-inflammatory therapies, particularly nonsteroidal anti-inflammatory drugs, are considered from experimental and clinical perspectives and potential mechanisms underlying their apparent benefits are discussed. Finally, possible protective effects of the inflammatory response in Alzheimer's are described. Taken all together, evidence presented in this review suggests a scheme for Alzheimer's pathogenesis, with neuroinflammation playing a crucial role influencing and linking beta-amyloid deposition to neuronal damage and clinical disease.

Two-dimensional gel analysis of secreted proteins induced by interleukin-1 beta in rat astrocytes.

Interleukin-1 beta (IL-1 beta) is a pro-inflammatory cytokine produced in the brain by endogenous microglial cells responding to injury. Levels of IL-1 beta are elevated in several neurodegenerative disorders, including Alzheimer's disease. IL-1 beta, which can act as a mitogen for astrocytes, also elicits the expression and secretion of multiple factors and paracrine 'second messengers' such as other cytokines, nerve growth factor, prostaglandins and nitric oxide that may in turn modulate neuronal and glial responses to injury. Utilizing giant, high-resolution two-dimensional gel electrophoresis, we have sought to more fully define the potential range of protein mediators that are secreted by astrocytes treated with IL-1 beta. In cultured rat astrocytes, we observe dramatic increases in the secretion of eight different protein species after 24 h of treatment with human recombinant IL-1 beta (1 U/ml). Seven of the proteins are also induced by tumor necrosis factor-alpha or basic fibroblast growth factor. Based on immunoprecipitation with specific antisera, we have identified three of these proteins as plasminogen activator inhibitor type-1, ceruloplasmin, and complement component C3. The identities of the other proteins, including the IL-1 beta-specific induction, are currently unknown. Characterization of these downstream modulators of IL-1 beta action complements gene-based approaches and will provide a better understanding of astrocyte responses to injury as well as markers for astrocyte activation in neurodegenerative diseases.

Enhanced glial activation and expression of specific CNS inflammation-related molecules in aged versus young rats following cortical stab injury.

Aging is associated with increased glial responsiveness that may enhance the brain's susceptibility to injury and disease. To determine whether unique age-related molecular responses occur in brain injury, we assessed mRNA levels of representative central nervous system (CNS) inflammation-related molecules in young (3 months) and aged (36 months) Fisher 344/Brown Norwegian F1 hybrid rats following cortical stab. Enhanced glial activation in older animals was accompanied by increased expression of a subset of inflammation-related mRNAs, including IL-1beta, TNFalpha, IL-6, ICAM-1, inducible nitric oxide synthase (iNOS), metalloproteinase-9 (MMP-9), and complement 3alpha-chain 1 (C3alpha1). Recognition of these age-specific differences may guide development of novel treatment regimes for older individuals.

Inflammatory responses to amyloidosis in a transgenic mouse model of Alzheimer's disease.

Mutations in the amyloid precursor protein (APP) and presenilin-1 and -2 genes (PS-1, -2) cause Alzheimer's disease (AD). Mice carrying both mutant genes (PS/APP) develop AD-like deposits composed of beta-amyloid (Abeta) at an early age. In this study, we have examined how Abeta deposition is associated with immune responses. Both fibrillar and nonfibrillar Abeta (diffuse) deposits were visible in the frontal cortex by 3 months, and the amyloid load increased dramatically with age. The number of fibrillar Abeta deposits increased up to the oldest age studied (2.5 years old), whereas there were less marked changes in the number of diffuse deposits in mice over 1 year old. Activated microglia and astrocytes increased synchronously with amyloid burden and were, in general, closely associated with deposits. Cyclooxygenase-2, an inflammatory response molecule involved in the prostaglandin pathway, was up-regulated in astrocytes associated with some fibrillar deposits. Complement component 1q, an immune response component, strongly colocalized with fibrillar Abeta, but was also up-regulated in some plaque-associated microglia. These results show: i) an increasing proportion of amyloid is composed of fibrillar Abeta in the aging PS/APP mouse brain; ii) microglia and astrocytes are activated by both fibrillar and diffuse Abeta; and iii) cyclooxygenase-2 and complement component 1q levels increase in response to the formation of fibrillar Abeta in PS/APP mice.

Downregulation of neuronal cyclooxygenase-2 expression in end stage Alzheimer's disease.

Nonsteroidal anti-inflammatory drugs (NSAIDs) appear to delay the onset of Alzheimer's disease (AD). NSAIDs inhibit cyclooxygenase (COX), of which two isoforms exist. We report decreased neuronal COX-2 expression in AD subjects relative to nondemented controls using qualitative analysis of COX-2 immunoreactivity and quantification of COX-2 positive neurons in different hippocampal subfields. These changes also occurred in subjects with other dementia and thus may not be disease specific. The proportion of COX-2 positive neurons decreased in subjects with clinical dementia rating (CDR) 5 but not CDR 4, suggesting that this was a late event in the course of the disease. Furthermore, COX-2 was not preferentially associated with paired helical filament immunoreactivity, a marker of neuronal pathology. COX-2 immunoreactivity was also observed in astrocytes and cerebrovasculature. Indeed, the density of COX-2 immunopositive astrocytes was increased in AD temporal cortex. Based on our findings, it is unlikely that neuronal COX-2 contributes to pathology in end stage AD; however, COX-2 in other cell types may participate in the inflammation-related response associated with the disease.

Nonsteroidal anti-inflammatory drugs in orthodontic tooth movement: metalloproteinase activity and collagen synthesis by endothelial cells.

Orthodontic treatment is based on the biologic principle that prolonged pressure on teeth results in remodeling of periodontal structures, allowing for tooth movement. Periodontal remodeling is a complex process regulated in part by prostaglandins and adversely affected by the use of nonsteroidal anti-inflammatory drugs. We investigated the effects of indomethacin on collagenase activity and procollagen synthesis in rat endothelial cell cultures. Cyclooxygenase inhibition resulted in exacerbation of IL-1 beta-mediated collagenase B (MMP-9) production and activity, as well as attenuation of type IV procollagen synthesis levels by endothelial cells in vitro. Hence, the use of over-the-counter nonsteroidal anti-inflammatory drugs during tooth movement may result in aberrant remodeling of periodontal vasculature and other structures, ultimately affecting orthodontic treatment efficacy. Further studies are needed to establish novel pain relievers that do not interfere with orthodontic processes.

Inflammation and Alzheimer's disease.

Inflammation clearly occurs in pathologically vulnerable regions of the Alzheimer's disease (AD) brain, and it does so with the full complexity of local peripheral inflammatory responses. In the periphery, degenerating tissue and the deposition of highly insoluble abnormal materials are classical stimulants of inflammation. Likewise, in the AD brain damaged neurons and neurites and highly insoluble amyloid beta peptide deposits and neurofibrillary tangles provide obvious stimuli for inflammation. Because these stimuli are discrete, microlocalized, and present from early preclinical to terminal stages of AD, local upregulation of complement, cytokines, acute phase reactants, and other inflammatory mediators is also discrete, microlocalized, and chronic. Cumulated over many years, direct and bystander damage from AD inflammatory mechanisms is likely to significantly exacerbate the very pathogenic processes that gave rise to it. Thus, animal models and clinical studies, although still in their infancy, strongly suggest that AD inflammation significantly contributes to AD pathogenesis. By better understanding AD inflammatory and immunoregulatory processes, it should be possible to develop anti-inflammatory approaches that may not cure AD but will likely help slow the progression or delay the onset of this devastating disorder.

Cyclooxygenases in the central nervous system: implications for treatment of neurological disorders.

Recognition of two isoforms of cyclooxygenase and reports that nonsteroidal anti-inflammatory drugs may be beneficial in devastating neurological conditions such as Alzheimer's disease have led to increased interest in cyclooxygenase function in the nervous system. In the present paper we review current data on the multiplicity of cyclooxygenase and prostaglandin mediated effects in the central nervous system (CNS). We discuss CNS cells types, including neurons, glia, and cerebrovascular elements, where cyclooxygenases-1 and -2 are expressed under normal conditions or can be induced by physiological or pathological stimuli. We also address physiological processes such as pain sensitization, CNS inflammation and fever induction that are regulated or modified by cyclooxygenase activity. Finally, we describe potential roles of cyclooxygenase in neurological diseases and rationales for nonsteroidal anti-inflammatory drug use in the treatment of neurodegenerative disorders, stroke and CNS injury.

Cyclooxygenase-1 in human Alzheimer and control brain: quantitative analysis of expression by microglia and CA3 hippocampal neurons.

Epidemiological and clinical studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit cyclooxygenase (COX) slow the progression and delay the onset of Alzheimer disease (AD). Two isoforms of cyclooxygenase have been identified. Although much effort has recently been focused on the inducible COX-2 isoform, little is known about COX-1 expression in human brain. We report that COX-1 message and immunoreactivity are localized to human hippocampal CA3 and CA4 neurons, granular neurons in neocortical layer IV, and occasional cortical pyramidal neurons. Quantitative in situ hybridization showed no differences between COX-1 mRNA levels in control and AD CA3 hippocampal neurons. COX-1 immunoreactivity was also present in microglial cells in gray and white matter in all brain regions examined. COX-1 appeared to be expressed in microglial cells regardless of their activation state as determined by HLA-DR immunostaining. However, COX-1 immunopositive microglia were found in association with Abeta plaques, and the density of COX-1 immunopositive microglia in AD fusiform cortex was increased. This pattern suggests an overall increase of COX-1 expression in AD. Currently used NSAIDs inhibit both isoforms of cyclooxygenase. The present study shows that COX-1 is widely expressed in human brain, and raises the possibility that COX-1 may contribute to CNS pathology.

Adenovirus-mediated transgene expression in nonhuman primate brain.

Transgene expression in the brain of St. Kitts green monkey, Cercopithecus aethiops sabeus, was studied following injection of a serotype 5 adenoviral vector deleted in E1 and E3. The vector harbored the transgene for Escherichia coli beta-galactosidase (beta-Gal) with the simian virus 40 (SV40) nuclear localization signal under control of the Rous sarcoma viral (RSV) long terminal repeat. Several titers ranging from 5 x 10(7) to 2 x 10(9) plaque-forming units (PFU) in volumes ranging from 5 to 250 microl were injected into the caudate nuclei of 18 monkeys. Monkeys were treated with dexamethasone for 9 days, beginning the day prior to surgery, and were sacrificed at 1 week or at 1, 2, or 3 months. At 1 week, beta-Gal was expressed in thousands of cells, including both neurons and astrocytes. In addition, some dopaminergic neurons in the substantia nigra expressed transgene, suggesting retrograde transport of the vector. At 1 month 162,000+/-68,000 (SEM) or 65,000+/-29,000 beta-Gal-expressing cells persisted in striatum injected with 6 x 10(8) PFU in 30 microl or 5 x 10(7) PFU in 5 microl, respectively. Transgene expression was also observed in one of two monkeys sacrificed at 2 months and in a single monkey sacrificed at 3 months. No transgene expression was observed at 1 month in striatum injected with a higher titer (2 x 10(9) PFU in 100 microl) or more dilute vector (5 x 10(7) PFU in 30 microl). Staining for the major histocompatibility complex II (MHC II) subtype DR showed intense staining in sites injected with a higher vector titer, in which no transgene persisted at 1 month, whereas low to moderate staining was present in sites with high transgene expression. These observations suggest that there is an optimal range of vector titers for obtaining persistent transgene expression from E1E3-deleted adenovirus in primate brain, above which host responses limit transgene stability.

TNF alpha and IL-1beta mediate intercellular adhesion molecule-1 induction via microglia-astrocyte interaction in CNS radiation injury.

Radiation injury to the central nervous system (CNS) results in glial activation accompanied by expression of pro-inflammatory cytokines and adhesion molecules. In this study we demonstrate intercellular adhesion molecule-1 (ICAM-1) induction in the irradiated mouse brain at the mRNA and protein levels. Immunocytochemical analysis revealed that ICAM-1 protein was primarily expressed in endothelial cells and microglia. In vitro, ionizing radiation significantly induces TNF alpha, IL-1beta and ICAM-1 mRNA in primary microglia cultures. Interestingly, although ionizing radiation activated primary astrocyte cultures, it did not induce ICAM-1 expression. However, exposure of astrocytes to conditioned medium collected from irradiated microglia resulted in ICAM-1 induction, which was abrogated when the conditioned medium was pre-incubated with neutralizing antibodies raised against murine TNF alpha and IL-1beta. These results indicate that pro-inflammatory cytokines may be necessary for ICAM-1 expression in astrocytes in CNS radiation injury.

Cyclooxygenase-2: molecular biology, pharmacology, and neurobiology.

In the nervous system, prostanoids are well recognized as mediators in a variety of processes, including fever generation, modulation of the stress response, sleep/wake cycle, control of cerebral blood flow, and hyperalgesia. Two isoforms of cyclooxygenase (COX), the enzyme that catalyzes the conversion of arachidonic acid to prostanoids, are now recognized: a constitutively expressed COX-1 and a highly regulated COX-2. New molecular and pharmacologic tools have provided a better understanding of the roles of COX-generated prostanoids in the nervous system. Other studies reveal that COX may represent an important target for new therapeutic approaches to neurologic disorders. This review summarizes our current understanding of cyclooxygenase expression and prostanoid actions in the nervous system, with special reference to COX-2 and studies demonstrating its expression in different cell types responding to a variety of stimuli. A brief review of the molecular biology, pharmacology, and primary actions of COX-2 outside of the nervous system provides a context for understanding potential neurobiological roles for COX-2 and prostanoid production. Information about the role of COX in human neurological disorders, including cerebrovascular disease, Alzheimer' s disease, and hyperalgesia, is covered in the last section.

Interleukin-1 beta and tumor necrosis factor-alpha suppress dexamethasone induction of glutamine synthetase in primary mouse astrocytes.

Astrocytes play a key role in the protection of neurons from excitotoxicity by taking up excess glutamate and converting it to glutamine via the enzyme glutamine synthetase. In a number of cell types, glucocorticoid hormones induce glutamine synthetase. Glucocorticoids also down-regulate many genes induced by proinflammatory cytokines. As the glucocorticoid receptor has been shown to interact with transcription factors that may also be activated by the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), we hypothesized that IL-1beta or TNF-alpha might oppose the induction of glutamine synthetase by dexamethasone. Primary mouse cortical astrocytes were treated with 10(-7) M dexamethasone and doses of IL-1beta or TNF-alpha ranging from 0.02 to 5 ng/ml or 0.05 to 20 ng/ml, respectively. We found that both cytokines attenuated the dexamethasone induction of glutamine synthetase protein at 24 h and that the effect was dose-dependent. We also found that IL-1beta and TNF-alpha inhibited the induction of glutamine synthetase mRNA by dexamethasone, and that the induction of enzymatic activity was similarly prevented by IL-1beta. As glutamine synthetase can be induced by physiological levels of glucocorticoids, the release of proinflammatory cytokines following acute injury or in neurodegenerative disorders may hinder the ability of astrocytes to protect neurons from excitotoxicity.

Developmental regulation and PKC dependence of Alzheimer's-type tau phosphorylations in cultured fetal rat hippocampal neurons.

Attempts to describe a mechanism of neurofibrillary tangle formation often focus on site specific phosphorylations of tau protein. These have typically been described in both Alzheimer's disease and developing brains. Therefore, study of the developmental regulation of Alzheimer epitope tau phosphorylations may help explain their persistence or recurrence during Alzheimer's disease. Using fetal rat hippocampal cultures, we report a spatial and temporal expression of tau phosphorylation during neuronal differentiation. We have examined phosphorylation at the epitopes recognized by monoclonal antibodies, PHF-1 and Tau 1. Tau was highly phosphorylated at the PHF-1 epitope at all culture ages examined using both immunohistochemical staining and Western blots. Tau was heavily phosphorylated at the Tau 1 epitope only in older cultures. The populations of tau recognized by the two antibodies also exhibited different solubilities, suggesting different microtubule binding behaviors: tau phosphorylated at PHF-1 was retained in axons following solubilization whereas Tau 1 immunoreactive tau was not retained in any cell compartment. Finally, in this culture system, maintenance of phosphorylation at the PHF-1 epitope, but not the Tau 1 epitope, required protein kinase C activity. These results indicate unique regulatory mechanisms and roles for each of these phosphorylated tau epitopes.