Neurobiology of AgingProinflammatory effects of M-CSF and Aβ in hippocampal organotypic cultures
Introduction
In Alzheimer’s disease (AD) there is a local inflammatory process surrounding amyloid beta (Aβ) deposits in the brain [2], [53]. Numerous microglia are associated with Aβ plaques [8]. Activation of microglia by Aβ may contribute to neuronal injury in AD through the release of proinflammatory cytokines and cytotoxic factors [12], [22]. However, recent Aβ immunization experiments in transgenic mice indicate that in the presence of opsonizing antibody, activated microglia may have a beneficial effect by clearing Aβ from the brain [9], [54]. Identifying factors that promote microglial activation in AD could lead to therapies designed to augment Aβ immunization.
Stimuli that initiate and sustain microglial activation in AD are poorly defined. In monotypic microglial cultures a robust Aβ-induced pro-inflammatory response requires the presence of augmenting factors [29], [46]. However, many of the factors demonstrated to augment Aβ-induced microglial activation like interferon γ [36] are not present at increased levels in AD brain. The cytokine macrophage colony stimulating factor (M-CSF) strongly augments the pro-inflammatory effects of Aβ on cultured microglial cells [48]. M-CSF is increased in the central nervous system in AD patients [26] and the M-CSF receptor (M-CSFR) is increased on microglia in AD brain [3] and in the APPV717F transgenic mouse model for AD [49]. Further, microglia from AD patients express M-CSF when exposed to Aβ, and the magnitude of this expression is greater than that seen with microglia from non-demented elderly controls [43]. M-CSF is produced in the brain by neurons and glia [33], [40], [50], [60] and is essential for normal microglial maturation and function [61], [65]. These data suggest that in AD M-CSF may co-activate microglia with Aβ.
Although studies of M-CSF with monotypic microglia cultures have been informative, this system lacks the complexity of living brain. Further, strong autocrine and paracrine effects among microglia in monotypic culture may exaggerate biologic processes. Organotypic hippocampal cultures offer many advantages over monotypic cultures because interactions between neurons and glial cells are maintained [58]. It has been suggested that hippocampal cultures may be particularly useful for ex vivo modeling of neuroinflammatory processes [4], [16]. To test the hypothesis that the effects of Aβ plus M-CSF observed in monotypic microglial cultures would apply in an organotypic environment, we examined the effects of M-CSF and Aβ on the expression of cytokines and nitric oxide (NO) in organotypic hippocampal cultures using immunohistochemistry, ELISA, and TaqMan real-time RT-PCR. For comparison, we also examined the effects of the pro-inflammatory agent lipopolysaccharide (LPS). In addition, we tested for neurotoxicity due to M-CSF and Aβ by using lactate dehydrogenase (LDH) measurements, propidium iodide staining and the specific neuronal injury marker FluoroJade [51].
Section snippets
Hippocampal organotypic cultures
Hippocampal slice cultures were prepared according to the method of Stoppini et al. [58] with some modifications. Briefly, slice cultures were prepared from 7 day-old Sprague-Dawley rats (Simonsen, Gilroy, CA). Slices were cut at 400 μm on a McIlwain tissue chopper (Brinkman, Westbury, NY) and transferred to Millipore (Bedford, MA) MilliCell membrane inserts (0.4 μm). Slice culture media consisted of 50% D-Minimal Essential Medium (DMEM; Life Technologies, Gaithersburg, MD), 25% Hank’s Balanced
M-CSF augments Aβ induced mRNA expression for IL-1α, IL-6, MIP-1α, and iNOS
Combined treatment with M-CSF plus Aβ 1–40 induced expression of cytokines and iNOS in hippocampal organotypic cultures. Conventional RT-PCR using RNA from organotypic cultures showed no increase in expression of the mRNA encoding interleukin-6 (IL-6) in comparison with control values after 24 h of treatment with Aβ 1–40 alone, the control peptide Aβ 40–1 alone or Aβ 40–1 together with M-CSF. Weak expression of IL-6 mRNA was observed after treatment with M-CSF alone, but a marked increase in
Discussion
These results demonstrate a synergistic effect of Aβ and M-CSF on the production of the proinflammatory cytokines IL-1, IL-6, the chemokine MIP-1α, and on the expression of iNOS in hippocampal organotypic cultures. M-CSF is a microglial cell activator that is increased in AD brain, whereas the M-CSFR, encoded by the proto-oncogene c-fms, is strongly expressed on microglial cells in AD brain and in the APPV717F mouse model for AD [3], [26], [49]. Determining the effects of M-CSF on glia in an
Acknowledgments
Lan Yang, Feifei Zhao, Karen Schmidt, Dilek Simsek, Mark Rodgers, and Cheryl Luu provided assistance. Supported by National Institute of Mental Health grant MH57833 and by the Alzheimer’s Association.
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2014, Neuroscience and Biobehavioral ReviewsCitation Excerpt :β-Amyloid is pro-inflammatory (Ii et al., 1996) and its toxicity in neurons in co-culture with glia is prevented by iNOS inhibitors (Brown, 2007). Macrophage colony stimulating factor (M-CSF), a classical hematopoietic cytokine, robustly elevates iNOS expression primarily induced by β-amyloid in BV2 microglia (Murphy et al., 1998) and in astrocytes of hippocampal cultures (Vincent et al., 2002). Some cytokines, including interferon (IFN)-γ, IL-1β, and IL-12, individually induce iNOS expression/NO production in microglia and astrocytes; however, other cytokines such as TNF-α usually upregulate iNOS expression in association with IL-1β or IFN-γ (Jana et al., 2001; Saha and Pahan, 2006a; Trajkovic et al., 2001).
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These authors contributed equally to this work.