Microglia are important in the inflammatory response in Alzheimer's disease (AD). We showed previously that macrophage colony-stimulating factor receptor (M-CSFR), encoded by the c-fms protooncogene, is overexpressed on microglia surrounding amyloid ␤ (A␤) deposits in the APP V717F mouse model for AD. The M-CSFR is also increased on microglia after experimental brain injury and in AD. To determine the relevance of these findings, we transiently expressed M-CSFR on murine BV-2 and human SV-A3 microglial cell lines using an SV40-promoted c-fms construct. M-CSFR overexpression resulted in microglial proliferation and increased expression of inducible nitric-oxide synthase, the proinflammatory cytokines interleukin-1␣, macrophage inflammatory protein 1-␣, and interleukin-6 and of macrophage colony-stimulating factor (M-CSF) itself. Antibody neutralization of M-CSF showed that the M-C-SFR-induced proinflammatory response was dependent on M-CSF in the culture media. By using a co-culture of c-fms-transfected murine microglia and rat organotypic hippocampal slices and a species-specific real time reverse transcriptase-polymerase chain reaction assay and enzyme-linked immunosorbent assay, we showed that M-CSFR overexpression on exogenous microglia induced expression of interleukin-1␣ by the organotypic culture. These results show that increased M-CSFR expression induces microglial proliferation, cytokine expression, and a paracrine inflammatory response, suggesting that in APP V717F mice increased M-CSFR on microglia could be an important factor in A␤-induced inflammatory response.

Microglia with increased expression of the macrophage colony-stimulating factor receptor (M-CSFR; c-fms) are found surrounding plaques in Alzheimer's disease (AD) and in mouse models for AD and after ischemic or traumatic brain injury. Increased expression of M-CSFR causes microglia to adopt an activated state that results in proliferation, release of cytokines, and enhanced phagocytosis. To determine whether M-CSFR-induced microglial activation affects neuronal survival, we assembled a coculture system consisting of BV-2 microglia transfected to overexpress the M-CSFR and hippocampal organotypic slices treated with NMDA. Twenty-four hours after assembly of the coculture, microglia overexpressing M-CSFR proliferated at a higher rate than nontransfected control cells and exhibited enhanced migration toward NMDA-injured hippocampal cultures. Surprisingly, coculture with c-fms-transfected microglia resulted in a dramatic reduction in NMDA-induced neurotoxicity. Similar results were observed when cocultures were treated with the teratogen cyclophosphamide. Biolistic overexpression of M-CSFR on microglia endogenous to the organotypic culture also rescued neurons from excitotoxicity. Furthermore, c-fms-transfected microglia increased neuronal expression of macrophage colony-stimulating factor (M-CSF), the M-CSFR, and neurotrophin receptors in the NMDA-treated slices, as determined with laser capture microdissection. In the coculture system, direct contact between the exogenous microglia and the slice was necessary for neuroprotection. Finally, blocking expression of the M-CSF ligand by exogenous c-fms-transfected microglia with a hammerhead ribozyme compromised their neuroprotective properties. These results demonstrate a protective role for microglia overexpressing M-CSFR in our coculture system and suggest under certain circumstances, activated microglia can help rather than harm neurons subjected to excitotoxic and teratogen-induced injury.

Alzheimer's disease (AD) 1 is characterized by amyloid-␤ peptide (A␤) plaques surrounded by microglia. A␤ is thought to be directly neurotoxic, and activated microglia are hypothesized to have negative effects on neurons through the release of effectors of inflammation (1). However, as brain macrophages microglia can clear A␤ by phagocytosis, primarily through macrophage scavenger receptors (MSR) (2-6). Immunization of transgenic mice modeling AD with A␤ results in clearance of plaques from the brain (7). Whereas some results suggest that microglial phagocytosis may be key in clearance of A␤ after immunization (8), other findings indicate that circulating antibodies may result in movement of A␤ out of the brain (9). This controversy has stimulated renewed interest in uptake of A␤ by microglia.A distinctive phenotypic feature of microglia surrounding A␤ plaques in APPV717F transgenic mice and in AD is enhanced expression of the macrophage colony-stimulating factor receptor (M-CSFR), translation product of the c-fms proto-oncogene (10, 11). Microglial M-CSFR expression is also increased after experimental ischemic and traumatic brain injury (12, 13). M-CSFR regulates proliferation, activation, and survival of cells in the monocyte-macrophage lineage through tyrosine kinase activation of diverse signal transduction pathways including: Src kinase, Ras-ERK, phosphoinositide 3-kinase, and p38 MAP kinase (14 -16). Deletion of M-CSFR expression results in decreased numbers of cells of the mononuclear phagocyte lineage (17). In AD macrophage colony-stimulating factor (M-CSF), the ligand for M-CSFR expressed by neurons and glia is also increased (18). Simultaneous increases in M-CSF and M-CSFR expression in the brain could result in significant changes in microglial function.We recently demonstrated that overexpression of M-CSFR by cultured microglia increases proliferation, stimulates release of pro-inflammatory and chemotactic cytokines, and induces a paracrine inflammatory response in a microglial-organotypic co-culture system (19). In the present study we sought to determine the effects of M-CSFR overexpression on A␤ phagocytosis by cultured mouse and human microglia. We hypothesized that M-CSFR-induced activation of microglia would increase their capacity to clear A␤ from culture medium. Although A␤ immunization clinical trials have been discontinued in humans for the present, identifying factors that enhance microglial clearance of A␤ may be of benefit in devising alternative means of decreasing A␤ burden in the brain in AD. EXPERIMENTAL PROCEDURES Microglial Cell Lines, Plasmid Transfections, and Tissue culture-The c-fms expression plasmid pTK1 was a gift from Dr. Rao Tekmal (Emory University, Atlanta, GA), and contains the complete mouse c-fms sequence under the control of an SV40 promoter (20). Transfections were carried out using mouse BV-2 and human SV-A3 microglial cells. The BV-2 immortalized microglial cell line has been characterized previously (21)(22)(23)(24). Because of phenotypic changes that occur at...

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