Microglia repetitively isolated from in vitro mixed glial cultures retain their initial phenotype.

In vitro culture of rodent microglia is a common system used to model proinflammatory changes in the brain. However, typical postnatal brain isolation protocols are time consuming and cell numbers acquired are often a rate-limiting factor for experimental progress. Large studies that rely on the use of primary microglia can, therefore, require excessive numbers of animals at considerable expense, additional technical support and culture incubator space. Although the addition of mitogens such as macrophage colony-stimulating factor, granulocyte macrophage-colony stimulating factor, and epidermal growth factor to the cultures can facilitate a higher yield, this adds additional expense and likely alters the microglial phenotype. We have defined a simple, inexpensive modification of our standard culture protocol that allows us to repetitively isolate microglia. In order to define a method for improving microglia yield, we utilized our standard mixed glial culture preparation derived from postnatal day 1-3 mouse brains. After isolating microglia from mixed cultures at 14 days in vitro, we added fresh media to the cultures for an additional 7 and 14 days to monitor microglial proliferation. We acquired a constant number of cells at each successive time point although the numbers were reduced from the first isolation. More importantly, in order to determine if our successive microglia isolates differed phenotypically we characterized several parameters of function. We compared their ability to secrete the proinflammatory cytokines interleukin-6 and tumor necrosis factor alpha after LPS stimulation. We also contrasted the phagocytic ability, morphology, and specific immunoreactivity (CD11b, CD68, CD45 and MHC II) of the culture ages. Our data demonstrate that microglia can be obtained from extended-time cultures provided periodic isolation is performed. Moreover, the cells retain a comparable in vitro phenotype. This demonstrates that cells from all ages can be combined for any given study. These findings are a viable and inexpensive way to increase and extend the microglial yield without increasing the number of animals used or adding costly mitogens. This method will be particularly useful for the preparation of microglia cultures from limited transgenic colonies.

Amyloid precursor protein cross-linking stimulates beta amyloid production and pro-inflammatory cytokine release in monocytic lineage cells.

Beta amyloid peptide-containing neuritic plaques are a defining feature of Alzheimer's disease pathology. Beta amyloid are 38-43 residue peptides derived by proteolytic cleavage of amyloid precursor protein. Although much attention has focused on the proteolytic events leading to beta amyloid generation, the function of amyloid precursor protein remains poorly described. Previously, we reported that amyloid precursor protein functions as a pro-inflammatory receptor on monocytic lineage cells and defined a role for amyloid precursor protein in adhesion by demonstrating that beta(1) integrin-mediated pro-inflammatory activation of monocytes is amyloid precursor protein dependent. We demonstrated that antibody-induced cross-linking of amyloid precursor protein in human THP-1 monocytes and primary mouse microglia stimulates a tyrosine kinase-based pro-inflammatory signaling response leading to acquisition of a reactive phenotype. Here, we have identified pro-inflammatory mediators released upon amyloid precursor protein-dependent activation of monocytes and microglia. We show that amyloid precursor protein cross-linking stimulated tyrosine kinase-dependent increases in pro-inflammatory cytokine release and a tyrosine kinase-independent increase in beta amyloid 1-42 generation. These data provide much needed insight into the function of amyloid precursor protein and provide potential therapeutic targets to limit inflammatory changes associated with the progression of Alzheimer's disease.

Regulation of beta-amyloid stimulated proinflammatory responses by peroxisome proliferator-activated receptor alpha.

Amyloid deposition within the brains of Alzheimer's Disease patients results in the activation of microglial cells and the induction of a local inflammatory response. The interaction of microglia or monocytes with beta-amyloid (A beta) fibrils elicits the activation a complex tyrosine kinase-based signal transduction cascade leading to stimulation of multiple independent signaling pathways and ultimately to changes in proinflammatory gene expression. The A beta-stimulated expression of proinflammatory genes in myeloid lineage cells is antagonized by the action of a family of ligand-activated nuclear hormone receptors, the peroxisome proliferator-activated receptors (PPARs). We report that THP-1 monocytes express predominantly PPAR gamma isoform and lower levels of PPAR alpha and PPAR delta isoforms. PPAR mRNA levels are not affected by differentiation of the cells into a macrophage phenotype, nor are they altered following exposure to the classical immune stimulus, lipopolysaccharide. Previous studies have found that PPAR gamma agonists act broadly to inhibit inflammatory responses. The present study explored the action of the PPAR alpha isoform and found that PPAR alpha agonists inhibited the A beta-stimulated expression of TNFalpha and IL-6 reporter genes in a dose-dependent manner. Moreover, the PPAR alpha agonist WY14643 inhibited macrophage differentiation and COX-2 gene expression. However, the PPAR alpha agonists failed to inhibit A beta-stimulated elaboration of neurotoxic factors by THP-1 cells. These findings demonstrate that PPAR alpha acts to suppress a diverse array of inflammatory responses in monocytes.

beta-Amyloid stimulation of microglia and monocytes results in TNFalpha-dependent expression of inducible nitric oxide synthase and neuronal apoptosis.

Reactive microglia associated with the beta-amyloid plaques in Alzheimer's disease (AD) brains initiate a sequence of inflammatory events integral to the disease process. We have observed that fibrillar beta-amyloid peptides activate a tyrosine kinase-based signaling response in primary mouse microglia and the human monocytic cell line, THP-1, resulting in production of neurotoxic secretory products, proinflammatory cytokines, and reactive oxygen species. We report that most of the amyloid-induced tyrosine kinase activity was stimulated after activation of Src family members such as Lyn. However, transduction of the signaling response required for increased production of the cytokines TNFalpha and IL1-beta was mediated by the nonreceptor tyrosine kinase, Syk. Additionally, beta-amyloid stimulated an NFkappaB-dependent pathway in parallel that was required for cytokine production. Importantly, TNFalpha generated by the monocytes and microglia was responsible for the majority of the neuorotoxic activity secreted by these cells after beta-amyloid stimulation but must act in concert with other factors elaborated by microglia to elicit neuronal death. Moreover, we observed that the neuronal loss was apoptotic in nature and involved increased neuronal expression of inducible nitric oxide synthase and subsequent peroxynitrite production. Selective inhibitors of inducible nitric oxide synthase effectively protected cells from toxicity associated with the microglial and monocytic secretory products. This study demonstrates a functional linkage between beta-amyloid-dependent activation of microglia and several characteristic markers of neuronal death occurring in Alzheimer's disease brains.

Inflammatory mechanisms in Alzheimer's disease: inhibition of beta-amyloid-stimulated proinflammatory responses and neurotoxicity by PPARgamma agonists.

Alzheimer's disease (AD) is characterized by the extracellular deposition of beta-amyloid fibrils within the brain and the subsequent association and phenotypic activation of microglial cells associated with the amyloid plaque. The activated microglia mount a complex local proinflammatory response with the secretion of a diverse range of inflammatory products. Nonsteroidal anti-inflammatory drugs (NSAIDs) are efficacious in reducing the incidence and risk of AD and significantly delaying disease progression. A recently appreciated target of NSAIDs is the ligand-activated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). PPARgamma is a DNA-binding transcription factor whose transcriptional regulatory actions are activated after agonist binding. We report that NSAIDs, drugs of the thiazolidinedione class, and the natural ligand prostaglandin J2 act as agonists for PPARgamma and inhibit the beta-amyloid-stimulated secretion of proinflammatory products by microglia and monocytes responsible for neurotoxicity and astrocyte activation. The activation of PPARgamma also arrested the differentiation of monocytes into activated macrophages. PPARgamma agonists were shown to inhibit the beta-amyloid-stimulated expression of the cytokine genes interleukin-6 and tumor necrosis factor alpha. Furthermore, PPARgamma agonists inhibited the expression of cyclooxygenase-2. These data provide direct evidence that PPARgamma plays a critical role in regulating the inflammatory responses of microglia and monocytes to beta-amyloid. We argue that the efficacy of NSAIDs in the treatment of AD may be a consequence of their actions on PPARgamma rather than on their canonical targets the cyclooxygenases. Importantly, the efficacy of these agents in inhibiting a broad range of inflammatory responses suggests PPARgamma agonists may provide a novel therapeutic approach to AD.

Cellular and molecular mechanisms of glial scarring and progressive cavitation: in vivo and in vitro analysis of inflammation-induced secondary injury after CNS trauma.

Post-traumatic cystic cavitation, in which the size and severity of a CNS injury progress from a small area of direct trauma to a greatly enlarged secondary injury surrounded by glial scar tissue, is a poorly understood complication of damage to the brain and spinal cord. Using minimally invasive techniques to avoid primary physical injury, this study demonstrates in vivo that inflammatory processes alone initiate a cascade of secondary tissue damage, progressive cavitation, and glial scarring in the CNS. An in vitro model allowed us to test the hypothesis that specific molecules that stimulate macrophage inflammatory activation are an important step in initiating secondary neuropathology. Time-lapse video analyses of inflammation-induced cavitation in our in vitro model revealed that this process occurs primarily via a previously undescribed cellular mechanism involving dramatic astrocyte morphological changes and rapid migration. The physical process of cavitation leads to astrocyte abandonment of neuronal processes, neurite stretching, and secondary injury. The macrophage mannose receptor and the complement receptor type 3 beta2-integrin are implicated in the cascade that induces cavity and scar formation. We also demonstrate that anti-inflammatory agents modulating transcription via the nuclear hormone receptor peroxisome proliferator-activated receptor-gamma may be therapeutic in preventing progressive cavitation by limiting inflammation and subsequent secondary damage after CNS injury.

Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of beta-amyloid and prion proteins.

Microglial interaction with amyloid fibrils in the brains of Alzheimer's and prion disease patients results in the inflammatory activation of these cells. We observed that primary microglial cultures and the THP-1 monocytic cell line are stimulated by fibrillar beta-amyloid and prion peptides to activate identical tyrosine kinase-dependent inflammatory signal transduction cascades. The tyrosine kinases Lyn and Syk are activated by the fibrillar peptides and initiate a signaling cascade resulting in a transient release of intracellular calcium that results in the activation of classical PKC and the recently described calcium-sensitive tyrosine kinase PYK2. Activation of the MAP kinases ERK1 and ERK2 follows as a subsequent downstream signaling event. We demonstrate that PYK2 is positioned downstream of Lyn, Syk, and PKC. PKC is a necessary intermediate required for ERK activation. Importantly, the signaling response elicited by beta-amyloid and prion fibrils leads to the production of neurotoxic products. We have demonstrated in a tissue culture model that conditioned media from beta-amyloid- and prion-stimulated microglia or from THP-1 monocytes are neurotoxic to mouse cortical neurons. This toxicity can be ameliorated by treating THP-1 cells with specific enzyme inhibitors that target various components of the signal transduction pathway linked to the inflammatory responses.

beta-Amyloid fibrils activate parallel mitogen-activated protein kinase pathways in microglia and THP1 monocytes.

The senile plaques of Alzheimer's disease are foci of local inflammatory responses, as evidenced by the presence of acute phase proteins and oxidative damage. Fibrillar forms of beta-amyloid (Abeta), which are the primary constituents of senile plaques, have been shown to activate tyrosine kinase-dependent signal transduction cascades, resulting in inflammatory responses in microglia. However, the downstream signaling pathways mediating Abeta-induced inflammatory events are not well characterized. We report that exposure of primary rat microglia and human THP1 monocytes to fibrillar Abeta results in the tyrosine kinase-dependent activation of two parallel signal transduction cascades involving members of the mitogen-activated protein kinase (MAPK) superfamily. Abeta stimulated the rapid, transient activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 in microglia and ERK2 in THP1 monocytes. A second superfamily member, p38 MAPK, was also activated with similar kinetics. Scavenger receptor and receptor for advanced glycated end products (RAGE) ligands failed to activate ERK and p38 MAPK in the absence of significant increases in protein tyrosine phosphorylation, demonstrating that scavenger receptors and RAGE are not linked to these pathways. Importantly, the stress-activated protein kinases (SAPKs) were not significantly activated in response to Abeta. Downstream effectors of the MAPK signal transduction cascades include MAPKAP kinases, such as RSK1 and RSK2, as well as transcription factors. Exposure of microglia and THP1 monocytes to Abeta resulted in the activation of RSK1 and RSK2 and phosphorylation of cAMP response element-binding protein at Ser133, providing a mechanism for Abeta-induced changes in gene expression.

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.

Low initial tau phosphorylation in human brain biopsy samples.

A rapid reversible tau phosphorylation at Ser 396/404 was observed in adult human cortical biopsy tissue and rat primary cortical cell cultures. Tau phosphorylation increased usually during the first 20-30 min in phosphate-buffered saline, followed by a decrease. The time course of tau phosphorylation and dephosphorylation in biopsy tissue could be lengthened by culturing in defined, oxygenated medium, instead of in phosphate-buffered saline. Phosphorylation of total protein in biopsy tissue occurred in two phases, with peaks at 30 and 90 min. The first peak of total protein phosphorylation coincided with the peak of tau phosphorylation, although both the first and second peaks of total protein phosphorylation coincided with the first and second peaks of neurofilament-H phosphorylation.