Olivera M. Mitrasinovic

Microglia Overexpressing the Macrophage Colony-Stimulating Factor Receptor Are Neuroprotective in a Microglial-Hippocampal Organotypic Coculture System

Microglia with increased expression of the macrophage colony-stimulating factor receptor (M-CSFR; c-fms) are found surrounding plaques in Alzheimers 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.

Accelerated Phagocytosis of Amyloid-β by Mouse and Human Microglia Overexpressing the Macrophage Colony-stimulating Factor Receptor

Microglia surrounding Aβ plaques in Alzheimers disease and in the APPV717F transgenic mouse model of Alzheimers disease have enhanced immunoreactivity for the macrophage colony-stimulating factor receptor (M-CSFR), encoded by the proto-oncogene c-fms. Increased expression of M-CSFR on cultured microglia results in proliferation and release of pro-inflammatory cytokines and expression of inducible nitric-oxide synthase. We transfected mouse BV-2 and human SV-A3 microglia to overexpress M-CSFR and examined microglial phagocytosis of fluorescein-conjugated Aβ. Flow cytometry and laser confocal microscopy showed accelerated phagocytosis of Aβ in mouse and human microglia because of M-CSFR overexpression that was time- and concentration-dependent. In contrast, microglial uptake of 1-μm diameter polystyrene microspheres was not enhanced by M-CSFR overexpression. Microglial uptake of Aβ was blocked by cytochalasin D, which inhibits phagocytosis. M-CSFR overexpression increased the mRNA for macrophage scavenger receptor A, and fucoidan blocking of macrophage scavenger receptors inhibited uptake of Aβ. M-CSFR antibody blocking experiments demonstrated that increased Aβ uptake depended on the interaction of the M-CSFR with its ligand. These results suggest that overexpression of M-CSFR in APPV717F mice may prime microglia for phagocytosis of Aβ after immunization.

Microglial overexpression of the M-CSF receptor augments phagocytosis of opsonized Aβ

The role of microglia in Alzheimers disease (AD) has come under intense scrutiny recently because microglia may clear amyloid beta (Abeta) by phagocytosis after immunization of transgenic mice. Increased expression of the macrophage colony-stimulating factor receptor (M-CSFR) is an important feature of microglia in AD and transgenic mouse models for AD. Increased expression of M-CSFR on mouse and human microglia accelerates phagocytosis of aggregated Abeta in part through macrophage scavenger receptors. We now show that Abeta phagocytosis by microglia overexpressing M-CSFR is further enhanced by antibody opsonization of Abeta. M-CSFR overexpression increased microglial phagocytosis of opsonized aggregated Abeta in culture medium, and accelerated ingestion of native Abeta from AD brain sections. M-CSFR overexpression also increased microglial expression of Fcgamma receptors, and blocking Fcgamma receptors attenuated the enhanced Abeta uptake observed after M-CSFR overexpression and antibody opsonization. Microglia in AD and in AD mouse models with increased expression of M-CSFR are likely to rapidly ingest opsonized Abeta after immunization, making high intracerebral antibody titers unnecessary.

Macrophage colony stimulating factor promotes phagocytosis by murine microglia

Macrophage colony stimulating factor (M-CSF) and its receptor are upregulated in the brain in Alzheimers disease. M-CSF induces activation and proliferation of microglial cells and expression of proinflammatory cytokines. Amyloid beta (Abeta) immunization experiments suggest that microglia have the capacity to aggressively clear Abeta from the brain under certain circumstances. We examined the role of M-CSF in phagocytosis of fluorescent microspheres and Abeta by cultured microglia. M-CSF treatment increased microglial cell phagocytosis of both microspheres and of Abeta. Antibody neutralization of M-CSF inhibited Abeta uptake induced by overexpression of the M-CSF receptor on microglia. These results suggest that M-CSF could be important in promoting microglial clearance of abnormal protein aggregates such as Abeta.

Biolistic expression of the macrophage colony stimulating factor receptor in organotypic cultures induces an inflammatory response.

The receptor for macrophage colony‐stimulating factor (M‐CSFR; c‐fms) is expressed at increased levels by microglia in Alzheimers disease (AD) and in mouse models for AD. Increased expression of M‐CSFR on cultured microglia results in a strong proinflammatory response, but the relevance of this cell culture finding to intact brain is unknown. To determine the effects of increased microglial expression of M‐CSFR in a complex organotypic environment, we developed a system for biolistic transfection of microglia in hippocampal slice cultures. The promoter for the Mac‐1 integrin α subunit CD11b is active in cells of myeloid origin. In the brain, CD11b expression is restricted to microglia. Constructs consisting of the promoter for CD11b and a c‐fms cDNA or an enhanced green fluorescent protein (EGFP) cDNA were introduced into monotypic cultures of microglia, neurons, and astrocytes. Strong CD11b promoter activity was observed in microglia, whereas little activity was observed in other cell types. Biolistic transfection of organotypic hippocampal cultures with the CD11b/c‐fms construct resulted in expression of the c‐fms mRNA and protein that was localized to microglia. Furthermore, biolistic overexpression of M‐CSFR on microglia resulted in significantly increased production by the hippocampal cultures of the proinflammatory cytokines interleukin (IL)‐1α macrophage inflammatory protein (MIP‐1α), and trends toward increased production of IL‐6 and M‐CSF. These findings demonstrate that microglial overexpression of M‐CSFR in an organotypic environment induces an inflammatory response, and suggest that increased microglial expression of M‐CSFR could contribute to the inflammatory response observed in AD brain.

P3-285 : Diagnostic and therapeutic assay development for A-beta using reactive oxygen species (ROS)-mediated signaling pathways: A systems biology perspective

were used. Inflammatory gene expression was assessed by RPA (in vitro) and RT-PCR (in vivo). Microglial migration and A phagocytosis was examined by Bowden chamber assay and FACS, respectively. A deposition and inflammatory molecules were detecetd by standard and confocal ICH. Results: NE stimulation (10 M) abolished amyloid beta (A ) stimulated microglia chemokine and cytokine expression, while the capacity of microglial cells to migrate and to phagocyte fibrillar amyloid beta peptides increased upon NE stimulation in a concentration dependent manner (10 nM-10 M). Similarly, DSP4 induced LC degeneration and NE depletion of APPV7171I transgenic mice resulted in a pronounced increase of mRNA and protein levels of cytokines, glial fibrillary acidic protein, inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Confocal immunohistochemistry revelead that iNOS was expressed by microglial cells in APPV717I control mice, while NE depleted APP717I mice showed a robust neuronal expression of iNOS. COX2 was predominantly expressed by IB4 positive microglial cells. NE depleted APPV717I transgenic mice showed more A 1-40 and 1-42 deposits within the hippocampus and frontal cortex compared to APP transgenic mice with an intact NE innervation. Confocal analysis of microglial cells and A revealed that the number of colabelled cells was significantly higher control APPV717I transgenics compared to NE depleted mice, suggesting that NE levels may modulate microglial phagocytosis also in vivo. Conclusions: Taken together this may indicate that LC degeneration and the subsequent decrease of NE concentrations facillitate the inflammatory reaction of microglial cells. At the same time, impaired microglial clearance may significantly contribute to A deposition in AD.