The role of microglial cell subsets in Alzheimer's disease.

Alzheimer disease (AD) is characterized by a progressive cognitive decline and accumulation of ß-amyloid (Aß forming senile plaques that are associated with inflammatory molecules and cells. Resident microglia and newly differentiated cells that are derived from the bone marrow are found in the vicinity of Aß plaques. Although these two types of microglia are not distinguishable by specific markers in the brain, they seem to possess different phenotype and functions. In mouse models of AD, bone marrow-derived microglia (BMDM) have been shown to delay or stop the progression of AD and preventing their recruitment exacerbates the pathology. Transplantation of competent hematopoietic stem cells or their genetic modifications ameliorate cognitive functions, reduce Aß accumulation and prevent synaptic dysfunctions. Improving the recruitment of genetically-modified BMDM may be considered as a powerful new therapeutic strategy to counteract AD. Here we review the role of microglia subsets in AD and how these cells have a great potential to fight against Aß accumulation and cognitive impairment.

Cognitive and non-cognitive behaviors in an APPswe/PS1 bigenic model of Alzheimer's disease.

Neuropsychiatric signs are critical in primary caregiving of Alzheimer patients and yet have been relatively ignored in murine models. In the present study, APPswe/PS1 bigenic mice had higher levels of irritability than non-transgenic controls as measured in the touch escape test. Moreover, APPswe/PS1 mice showed poorer nest building than controls and a higher duration of immobility in the forced swimming assay. These results are concordant with the hypothesis of increased apathy and depression-like behavior in an Alzheimer's disease model. In addition, APPswe/PS1 bigenic mice were deficient in retention of passive avoidance learning and left-right discrimination learning, concordant with previous findings in other Alzheimer-like models.

MyD88 signaling in brain endothelial cells is essential for the neuronal activity and glucocorticoid release during systemic inflammation.

Activation of neuronal circuits involved in the control of autonomic responses is critical for the host survival to immune threats. The brain vascular system plays a key role in such immune-CNS communication, but the signaling pathway and exact type of cells within the blood-brain barrier (BBB) mediating these functions have yet to be uncovered. To elucidate this issue we used myeloid differentiation factor 88 (MyD88)-deficient mice, because these animals do not show any responses to the cytokine interleukin-1beta (IL-1beta). We created chimeric mice with competent MyD88 signaling in either the BBB endothelium or perivascular microglia of bone marrow origin and challenged them with IL-1beta. Systemic treatment with the cytokine caused a robust transcriptional activation of genes involved in the prostaglandin E(2) (PGE(2)) production by vascular cells of the brain. Upregulation of these genes is dependent on a functional MyD88 signaling in the endothelium, because MyD88-deficient mice that received bone marrow stem cells from wild-type animals (for example, functional perivascular microglia) exhibited no response to systemic IL-1beta administration. MyD88 competent endothelial cells also mediate neuronal activation and plasma release of glucocorticoids, whereas chimeric mice with MyD88-competent perivascular microglia did not show a significant increase of these functions. Moreover, competent endothelial cells for the gene encoding Toll-like receptor 4 (TLR4) are essential for the release of plasma corticosterone in response to low and high doses of lipopolysaccharide. Therefore, BBB endothelial cells and not perivascular microglia are the main target of circulating inflammatory mediators to activate the brain circuits and key autonomic functions during systemic immune challenges.

Neuroprotective role of the innate immune system by microglia.

Innate immunity is a rapid series of reactions to pathogens, cell injuries and toxic proteins. A key component of this natural response is the production of inflammatory mediators by resident microglia and infiltrating macrophages. There is accumulating evidence that inflammation contributes to acute injuries and more chronic CNS diseases, though other studies have shown that inhibition of microglia is, in contrast, associated with more damages or less repair. The controversies regarding the neuroprotective and neurodegenerative properties of microglia may depend on the experimental approaches. Neurotoxic substances are frequently used to produce animal models of acute injuries or diseases and they may activate microglia either directly or indirectly by their ability to cause neuronal death and demyelination. Whether microglia and the immune response play a direct role in such processes still remains an open question. On the other hand, there are data supporting the role of resident microglia and those derived from the bone marrow in the stimulation of myelin repair, removal of toxic proteins from the CNS and the prevention of neurodegeneration in chronic brain diseases. The ability of glucocorticoids to provide a negative feedback on nuclear factor kappa B pathways in microglia may be a determinant mechanism underlying the ultimate fate of the inflammatory response in the CNS. This review presents new concepts regarding the neuroprotective role of the innate immune response in the brain and how microglia can be directed to improve recovery after injuries and prevent/delay neurodegeneration.

Innate immune receptor expression in normal brain aging.

Brain aging often results in cognitive impairment and is considered to be a major risk factor for neurodegenerative diseases. Earlier studies reported inflammatory responses in aged brain that could contribute to age-related neurodegeneration. Recently, innate immune receptors such as toll-like receptors (TLRs), so far implicated in defense against microorganisms, have been linked to pathogenesis of Alzheimer's disease. Therefore, we asked whether the transcription of TLRs (1-9) and CD14, could also be altered in physiological brain aging. Using real-time polymerase chain reaction (PCR), we indeed observed that TLR1, TLR2, TLR4, TLR5, TLR7 and CD14 expression was up-regulated in mouse brain in correlation with age. In contrast, transcriptions of TLR3, TLR6 and TLR8 were unchanged and the one of TLR9 was down-regulated. In situ hybridization further confirmed these results and identified the cellular source of TLR2 and TLR7 as mononuclear phagocytes. Together, this first systematic analysis demonstrates altered regulation of those innate immune receptors even in normal brain aging, which might be of relevance for understanding susceptibility to neurodegenerative processes associated with aging.

Neuroprotective properties of the innate immune system and bone marrow stem cells in Alzheimer's disease.

The role of innate immunity and microglia in the brain is currently a matter of great debate and controversy. While several studies have provided evidence that they contribute to neurodegeneration in various animal models of brain diseases and traumas, others have shown that their inhibition may in contrast be associated with more damages or less repair. We have recently reported the existence of two different types of microglia, the resident and the newly differentiated microglia that derive from the bone marrow stem cells. Of great interest is the fact that blood-derived microglial cells are associated with amyloid plaques and these cells are able to prevent the formation or eliminate the presence of amyloid deposits in mice that develop the major hallmark of Alzheimer's disease (AD). These newly recruited cells are specifically attracted to the beta-amyloid 40/42 isoforms in vivo and they participate in the elimination of these proteins by phagocytosis. This review presents the mechanisms involved in the control of the innate immune response by microglia and the beneficial properties of such a response in brain diseases, such as AD.

The LPS receptor (CD14) links innate immunity with Alzheimer's disease.

To rapidly respond to invading microorganisms, humans call on their innate immune system. This occurs by microbe-detecting receptors, such as CD14, that activate immune cells to eliminate the pathogens. Here, we link the lipopolysaccharide receptor CD14 with Alzheimer's disease, a severe neurodegenerative disease resulting in dementia. We demonstrate that this key innate immunity receptor interacts with fibrils of Alzheimer amyloid peptide. Neutralization with antibodies against CD14 and genetic deficiency for this receptor significantly reduced amyloid peptide induced microglial activation and microglial toxicity. The observation of strongly enhanced microglial expression of the LPS receptor in brains of animal models of Alzheimer's disease indicates a clinical relevance of these findings. These data suggest that CD14 may significantly contribute to the overall neuroinflammatory response to amyloid peptide, highlighting the possibility that the enormous progress currently being made in the field of innate immunity could be extended to research on Alzheimer's disease.

Cloning of murine TReP-132, a novel transcription factor expressed in brain regions involved in behavioral and psychiatric disorders.

In recent studies that addressed the transcriptional control of steroid synthesis, a transcriptional regulating protein of 132 kDa (TReP-132) was cloned and demonstrated to regulate expression of the human P450 side chain cleavage (P450scc) gene. In the present study, we describe the cloning and characterization of the mouse orthologue of the human factor, mouse transcriptional regulating protein (mTReP-132). mTReP-132 encodes a 1216-residue protein that is 85.5% homologous to the human protein. Both factors contain characteristic motifs, namely glutamine-, proline- and acidic-rich regions. The primary structure also exhibits two zinc fingers of the C(2)H(2) subtype, suggesting that this protein has the ability to act as a DNA binding transcription factor. mTReP-132 may also be a co-regulator of nuclear receptors because of two nuclear box motifs in this protein. Northern blot analysis demonstrated the expression of two transcripts of 4.4 and 7.5 kb in several tissues, but expression was clearly highest in the brain, thymus and testis of mice. In the brain, the hybridization signal was quite localized and strong in the basal ganglia, hippocampus, piriform cortex, cerebral cortex, ventromedial nucleus of the hypothalamus, and the dorsal and superior central nuclei of the raphe. Although classical steroidogenesis pathways have yet to be firmly established in the brain, expression of both mTReP-132 and P450scc provides anatomical evidence that mTReP-132 may regulate this key steroidogenic enzyme within specific regions involved in behavioral and psychiatric disorders. Moreover, the presence of both mTReP-132 and steroidogenic factor 1 (SF-1) transcripts in the ventromedial nucleus of the hypothalamus suggests a role for mTReP-132 in brain development and function. The molecular cloning and the highly specific expression of mTReP-132 across the brain further consolidate the hypothesis that this tissue is able to synthesize de novo steroids in a region-specific manner.

Analysis of the properties of Bacillus thuringiensis insecticidal toxins using a potential-sensitive fluorescent probe.

A potential-sensitive fluorescent probe, 3,3'-dipropylthiadicarbocyanine iodide, was used to analyze, at pH 7.5 and 10.5, the effects of Bacillus thuringiensis toxins on the membrane potential generated by the efflux of K(+) ions from brush border membrane vesicles purified from the midgut of the tobacco hornworm, Manduca sexta. Fluorescence levels were strongly influenced by the pH and ionic strength of the media. Therefore, characterization of the effects of the toxins was conducted at constant pH and ionic strength. Under these conditions, the toxins had little effect on the fluorescence levels measured in the presence or absence of ionic gradients, indicating that the ionic selectivity of their pores is similar to that of the intact membrane. Valinomycin greatly increased the potential generated by the diffusion of K(+) ions although membrane permeability to the other ions used to maintain the ionic strength constant also influenced fluorescence levels. In the presence of valinomycin, active toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C and Cry1E) efficiently depolarized the membrane at pH 7.5 and 10.5.

Role of host phosphotyrosine phosphatase SHP-1 in the development of murine leishmaniasis.

Activation of host phosphotyrosine phosphatase SHP-1 by Leishmania and its subsequent impact on tyrosine phosphorylation-based signaling cascades were shown to represent an important mechanism whereby this pathogen may alter host cell functions. Herein, we report that Leishmania-induced macrophage SHP-1 activity is necessary for its survival within phagocytes through the attenuation of nitric oxide-dependent and -independent microbicidal mechanisms. In vivo, Leishmania major infection, which footpad inflammation is mostly undetectable in SHP-1-deficient viable motheaten mice, was accompanied by increased inducible nitric oxide synthase and activation of neutrophils. These enhanced cellular activities were paralleled by a marked activation of signaling events usually negatively regulated by SHP-1. Overall, this study firmly establishes that modulation of the signaling terminator SHP-1 by Leishmania is essential for its installment and propagation.

Induction of proinflammatory molecules in mice with amyotrophic lateral sclerosis: no requirement for proapoptotic interleukin-1beta in neurodegeneration.

Recent studies have demonstrated the activation of caspase-1 and caspase-3 in mice expressing mutant superoxide dismutase 1 (SOD1), models of amyotrophic lateral sclerosis. Caspase-1 converts the prointerleukin-1beta into a potent proinflammatory molecule involved in the innate immune response and in neurodegenerative diseases. We report on the chronic expression of interleukin-1beta mRNA in the spinal cord of SOD1G37R mice, together with robust mRNA expression for the nuclear factor-kappaB (NF-kappaB) inhibitor IkappaBalpha, for other proinflammatory cytokines and chemokines (interleukin-6, tumor necrosis factor-alpha, monocyte chemoattractant protein-1) and for the toll-like receptor TLR2 involved in innate immunity. To further assess the interleukin-1beta contribution to neurodegeneration, we generated mice expressing SOD1G37R in a context of interleukin-1beta gene knockout. Surprisingly, the absence of interleukin-1beta had no effect on the life span of SOD1G37R mice, nor on the extent of motor axon degeneration at age 7 and 10 months. Whereas neither compensatory induction of the interleukin-1alpha mRNA nor increases in mRNA levels for IkappaBalpha, tumor necrosis factor-alpha and macrophage chemoattractant protein-1 occurred as a result of interleukin-1beta gene disruption, enhanced levels of TLR2 mRNA were detected in SOD1G37R mice lacking interleukin-1beta. We conclude that interleukin-1beta does not directly contribute to motor neuron degeneration in SOD1G37R mice, but it may act as a modulator of the innate immune response.

Circulating cell wall components derived from gram-negative, not gram-positive, bacteria cause a profound induction of the gene-encoding Toll-like receptor 2 in the CNS.

The recent characterization of human homologs of Toll may be the missing link for the transduction events leading to nuclear factor-kappaB (NF-kappaB) activity and proinflammatory gene transcription during innate immune response. Mammalian cells may express as many as 10 distinct Toll-like receptors (TLRs), although TLR2 is a key receptor for recognizing cell wall components of Gram-positive bacteria. The present study investigated the effects of circulating bacterial cell wall components on the expression of the gene-encoding TLR2 across the mouse brain. Surprisingly, while Gram-negative components caused a robust increase in TLR2 transcription within the cerebral tissue, peptidoglycan (PGN) and lipoteichoic acid (LTA), either alone or combined, failed to modulate the receptor transcript. Indeed, the mRNA levels for TLR2 in the choroid plexus and few other regions of the brain remained similar between vehicle-, LTA-, PGN-, and LTA/PGN-administered mice at all the times evaluated (i.e. 30 min to 24 h post-intraperitoneal injection). This contrasts with the profound de novo expression of TLR2 following a single systemic injection of the lipopolysaccharide (LPS). The signal was first detected in regions devoid of blood-brain barrier and few blood vessels and microcapillaries. A second wave of TLR2 expression was also detected from these structures to their surrounding parenchymal cells that stained for a microglial marker iba1. The rapid induction of IkappaBalpha (index of NF-kappaB activity) and up-regulation of the adaptor protein MyD88 suggest that LPS-induced TLR2 transcription may be dependent on the NF-kappaB pathway. These data provide the evidence that TLR2 is not only present in the brain, but its encoding gene is regulated by cell wall components derived from Gram-negative, not Gram-positive, bacteria. The robust wave of TLR2-expressing microglial cells may have a determinant impact on the innate immune response that occurs in the brain during systemic infection by Gram-negative, not Gram-positive, bacteria.

How circulating cytokines trigger the neural circuits that control the hypothalamic-pituitary-adrenal axis.

It is now no secret that the brain plays a crucial role in organizing, adapting and restraining the systemic inflammatory response via a complex cascade of mechanisms involving proteins of the innate immune system, molecules of the proinflammatory signal transduction pathways, prostaglandins (PGs) and specific populations of neurons. These neuronal circuits, in particular those controlling autonomic functions, are all together involved in engaging the physiological responses that may help eliminating the foreign material and adjust the inflammatory events to prevent detrimental consequences. For instance, elevation in plasma glucocorticoid levels is one of the most powerful endogenous and well-controlled feedback on the pro-inflammatory signal transduction machinery taking place across the organisms. The main Center that controls this neuroendocrine system is the paraventricular nucleus of the hypothalamus (PVN) that receives neuronal projections from numerous hypothalamic and extra-hypothalamic nuclei and areas. There is now compelling evidence that molecules produced by cells of the blood-brain barrier (BBB) may bind to their cognate receptors expressed at the surface of neurons that are responsible to trigger the hypothalamic-pituitary adrenal (HPA) axis. This review presents the new molecular insights regarding the pro-inflammatory signal transduction pathways that occur in these cells and how they are related to the neuroendocrine circuits mediating the increase in plasma glucocorticoid levels during systemic and localized immunogenic insults.

Inhibitory action of nitric oxide on circulating tumor necrosis factor-induced NF-kappaB activity and COX-2 transcription in the endothelium of the brain capillaries.

Circulating tumor necrosis factor alpha (TNF-alpha) has a profound stimulatory influence on mitogen-activated protein kinases that lead to nuclear factor kappa B (NF-kappaB) activity and transcription of the cyclooxygenase 2 (COX-2) gene in cells associated with the blood-brain barrier (BBB). This study investigated the hypothesis that nitric oxide (NO) acts as an endogenous modulator of TNF-induced NF-kappaB signaling and COX-2 transcription in the endothelium of the cerebral capillaries. To this end, rats were pretreated with the nonselective inhibitor of NO synthase (NOS) N(G)-nitro-L-arginine methyl ester (L-NAME) and killed 15, 45, and 90 minutes (min) after an i.v. injection of recombinant rat TNF-alpha. De novo expression of the inhibitory factor kappa B alpha (IkappaB alpha) was used as an index of NF-kappaB activity, whereas COX-2 mRNA induction was evaluated throughout the brain by in situ hybridization combined with immunohistochemistry. A single i.v. bolus of TNF caused a rapid expression of IkappaB alpha transcript first along large arterioles and small capillaries and thereafter within microglia across the brain parenchyma. The proinflammatory cytokine also provoked a strong transcriptional activation of the COX-2 gene that was quite specific to the cerebral endothelium as revealed by dual labeling using an antisera directed against the von Willebrand factor. Inhibition of NO synthesis did not by itself activate these proinflammatory molecules, but it enhanced the effects of circulating TNF-alpha in the BBB; the IkappaB alpha and COX-2 signal was significantly higher in microvascular-associated cells of animals that received both L-NAME and TNF-alpha treatments than those challenged with the proinflammatory cytokine alone. Rats treated with specific NOS inhibitors provided the evidence that these effects were mediated via the constitutive endothelial NOS (eNOS) and not the inducible form. These results indicate that eNOS-derived NO acts as an endogenous inhibitor of TNF-alpha-induced NF-kappaB activity and COX-2 transcription in the endothelium of the cerebral capillaries. This autoregulatory feedback of NO on these proinflammatory signal transduction events may be an essential element to prevent an exaggerated response that takes place in cells of the BBB during systemic immune challenges.

Role of interdomain salt bridges in the pore-forming ability of the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac.

The four salt bridges (Asp(222)-Arg(281), Arg(233)-Glu(288), Arg(234)-Glu(274), and Asp(242)-Arg(265)) linking domains I and II in Cry1Aa were abolished individually in alpha-helix 7 mutants D222A, R233A, R234A, and D242A. Two additional mutants targeting the fourth salt bridge (R265A) and the double mutant (D242A/R265A) were rapidly degraded during trypsin activation. Mutations were also introduced in the corresponding Cry1Ac salt bridge (D242E, D242K, D242N, and D242P), but only D242N and D242P could be produced. All toxins tested, except D242A, were shown by light-scattering experiments to permeabilize Manduca sexta larval midgut brush border membrane vesicles. The three active Cry1Aa mutants at pH 10.5, as well as D222A at pH 7.5, demonstrated a faster rate of pore formation than Cry1Aa, suggesting that increases in molecular flexibility due to the removal of a salt bridge facilitated toxin insertion into the membrane. However, all mutants were considerably less toxic to M. sexta larvae than to the respective parental toxins, suggesting that increased flexibility made the toxins more susceptible to proteolysis in the insect midgut. Interdomain salt bridges, especially the Asp(242)-Arg(265) bridge, therefore contribute greatly to the stability of the protein in the larval midgut, whereas their role in intrinsic pore-forming ability is relatively less important.

Regulation of the gene encoding the monocyte chemoattractant protein 1 (MCP-1) in the mouse and rat brain in response to circulating LPS and proinflammatory cytokines.

Accumulating evidence supports the existence of an innate immune response in the brain during systemic inflammation that is associated with a robust induction of proinflammatory cytokines and chemokines by specific cells of the central nervous system. The present study investigated the genetic regulation and fine cellular distribution of the monocyte chemoattractant protein-1 (MCP-1) in the brain of mice and rats in response to systemic immune insults. MCP-1 belongs to a superfamily of chemokines that have a leading role in the early chemotaxic events during inflammation. In situ hybridization histochemistry failed to detect constitutive expression of the chemokine transcript in the cerebral tissue except for the area postrema (AP) that exhibited a low signal under basal conditions. This contrasts with the strong and transient induction of the mRNA encoding MCP-1 following a single systemic bolus of lipopolysaccharide (LPS), recombinant interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha). These stimuli rapidly triggered (30 to 90 minutes) MCP-1 transcription in all the circumventricular organs (CVOs), the choroid plexus (chp), the leptomeninges, and along the cerebral blood vessels. The time-related induction and intensity of the signal differed among the challenges, route of administration and species, but MCP-1-expressing cells were always found in vascular-associated structures and those devoid of blood-brain barrier. At later times, few isolated microglia across the brain parenchyma depicted positive signal for MCP-1 mRNA. A dual-labeling procedure also provided convincing anatomical evidence that endothelial cells of the microvasculature and a few myeloid cells of the CVOs and chp were positive for the transcript during endotoxemia. This gene is under a sophisticated transcriptional regulation, as the hybridization signal returned to undetectable levels 12 to 24 hours after all the treatments in both species. Of interest are the data that only ligands that triggered nuclear factor kappa B (NF-kappa B) signaling had the ability to increase MCP-1 gene expression, because high doses of IL-6 remained without effects. These data provide the anatomical evidence that MCP-1 is expressed within specific populations of cells in response to systemic inflammatory molecules that use NF-kappa B as intracellular signaling system. This chemokine may therefore play a critical role in the cerebral innate immune response and contribute to the early chemotaxic events during chronic cerebral inflammation.

Corticotropin-releasing hormone and its receptors; an evaluation at the transcription level in vivo.

Activation of the hypothalamic-pituitary-adrenal (HPA) axis is the main defining feature of the stress response. The primary mediator of this response is corticotropin-releasing hormone (CRH), a 41-residue peptide acknowledged as the principal hypophysiotropic factor driving stress-induced adrenocorticotropic hormone (ACTH) secretion. Although CRH is widely distributed within the central nervous system (CNS), the paraventricular nucleus (PVN) of the hypothalamus is the principal site of the parvocellular neurosecretory neurons responsible for delivering CRH to the hypophyseal portal system, an event that initiates the activity of the pituitary-adrenal axis. Stress-induced transcriptional activation of CRH takes place quite uniquely in this hypothalamic nucleus, despite the robust constitutive hybridization signal for CRH mRNA across the brain. The fact that CRH itself is capable of mimicking these effects and that de novo but transient expression of its type one receptor occurs in the PVN are data that make this hypothalamic region of great interest to study the mechanisms that lead to such specific transcriptional activity. This review will present evidence of such phenomenon by stressors of different categories as well as the possible neuromediators involved.

Anti-inflammatory effects of prostaglandin E2 in the central nervous system in response to brain injury and circulating lipopolysaccharide.

Prostaglandin E2, a product of the cyclooxygenation of arachidonic acid released from membrane phospholipids, plays major roles in regulating brain injury and inflammation. Although prostaglandin E2 has frequently been considered as a possible inducer of brain damage and degeneration, it may exert beneficial effects in the CNS. Indeed, in spite of its classic role as a pro-inflammatory molecule, several recent in vitro observations indicate that prostaglandin E2 can inhibit microglial activation. This study investigated the effect of central prostaglandin E2 injection on circulating lipopolysaccharide-induced gene expression of different pro-inflammatory molecules in both vascular and parenchymal elements of the brain. Localized, but strong, expression of tumor necrosis factor-alpha and interleukin-1ss mRNA was found at the edge of the intracerebroventricular tract, which was largely prevented by the central prostaglandin E2 injection. Systemic lipopolysaccharide injection caused a profound transcriptional activation of cyclooxygenase-2 and the inhibitory factor kappaBalpha (IkappaBalpha, index of NF-kappaB activity) in the cerebral endothelium and tumor necrosis factor-alpha in microglial cells across the brain parenchyma. Although exogenous prostaglandin E2 increased lipopolysaccharide-induced NF-kappaB activity and cyclooxygenase-2 transcription in vascular-associated elements, it significantly reduced microglial activation and tumor necrosis factor-alpha expression in the brain parenchyma. These results indicate that prostaglandin E2 may play an important role in modulating the immune response occurring at the injured site and the pro-inflammatory signaling events taking place in both vascular- and microglial-associated elements of the CNS.

Toll-like receptor 4: the missing link of the cerebral innate immune response triggered by circulating gram-negative bacterial cell wall components.

The recent characterization of human homologues of Toll may be the missing link for the transduction events leading to NF-kappaB activity and proinflammatory gene transcription during innate immune response. Indeed, CD14 is not thought to participate directly in the cell signaling, but rather one or more of the mammalian Toll-like receptors (TLRs) acts in concert with the lipopolysaccharide (LPS) receptor to discriminate between microbial pathogens or their products and initiate transmembrane signaling. Mammalian cells may express as many as 10 distinct TLRs, although the importance of TLR4 in response to gram-negative bacteria and LPS is now supported by the fact that TLR4-mutated mice are LPS resistant. We investigated the expression of TLR4 across the rat brain under basal conditions and in response to systemic LPS and IL-1beta injection. We first cloned the rat TLR4 cDNA via RNA isolation and polymerase chain reaction (PCR) amplification with a proofreading polymerase. Total RNA was isolated from the rat liver tissue using Tri-Reagent and reverse transcribed into cDNA using Superscript II reverse transcriptase and an oligonucleotide primer with a degenerate 3' end of sequence 5'-T12(GAC)N-3'. Positive hybridization signal was found in the leptomeninges, choroid plexus (chp), subfornical organ, organum vasculosum of the lamina terminalis, median eminence, and area postrema. Scattered small cells also displayed a convincing hybridization signal within the brain parenchyma. Few well-defined nuclei exhibited positive TLR4 transcript: the supramamillary nucleus, cochlear nucleus, and the lateral reticular nucleus. The circumventricular organs, the leptomeninges, and chp also exhibited constitutive expression of the LPS receptor mCD14. In contrast to the strong up-regulation of the gene encoding mCD14 during endotoxemia, neither LPS nor IL-1beta caused a convincing increase in the TLR4 mRNA levels across the CNS. A down-regulation of the gene encoding TLR4 was found in the cerebral tissue of immune-challenged animals. The constitutive expression of both mCD14 and TLR4 may explain the innate immune response in the brain, which originates from the structures devoid of blood-brain barrier in presence of circulating LPS.