Expression of macrophage colony-stimulating factor receptor is increased in the AbetaPP(V717F) transgenic mouse model of Alzheimer's disease

Inflammation is an important neuropathological change in Alzheimer's disease (AD). However, the pathophysiological factors that initiate and maintain the inflammatory response in AD are unknown. We examined AbetaPP(V717F) transgenic mice, which show numerous brain amyloid-beta (Abeta) deposits, for expression of the macrophage colony-stimulating factor (M-CSF) and its receptor (M-CSFR). M-CSF is increased in the brain in AD and dramatically augments the effects of Abeta on cultured microglia. AbetaPP(V717F) animals 12 months of age showed large numbers of microglia strongly labeled with an M-CSFR antibody near Abeta deposits. M-CSFR mRNA and protein levels were also increased in brain homogenates from AbetaPP(V717F) animals. Dystrophic neurites and astroglia showed no M-CSFR labeling in the transgenic animals. A M-CSF antibody decorated neuritic structures near hippocampal Abeta deposits in transgenic animals. M-CSF mRNA was also increased in AbetaPP(V717F) animals in comparison with wild-type controls. Simultaneous overexpression of M-CSFR and its ligand in AbetaPP(V717F) animals could result in augmentation of Abeta-induced activation of microglia. Because chronic activation of microglia is thought to result in neuronal injury, the M-CSF system may be a potential target for therapeutic intervention in AD.     

Peripherally administered human umbilical cord blood cells reduce parenchymal and vascular beta-amyloid deposits in Alzheimer mice

Modulation of immune/inflammatory responses by diverse strategies including amyloid-beta (Abeta) immunization, nonsteroidal anti-inflammatory drugs, and manipulation of microglial activation states has been shown to reduce Alzheimer's disease (AD)-like pathology and cognitive deficits in AD transgenic mouse models. Human umbilical cord blood cells (HUCBCs) have unique immunomodulatory potential. We wished to test whether these cells might alter AD-like pathology after infusion into the PSAPP mouse model of AD. Here, we report a marked reduction in Abeta levels/beta-amyloid plaques and associated astrocytosis following multiple low-dose infusions of HUCBCs. HUCBC infusions also reduced cerebral vascular Abeta deposits in the Tg2576 AD mouse model. Interestingly, these effects were associated with suppression of the CD40-CD40L interaction, as evidenced by decreased circulating and brain soluble CD40L (sCD40L), elevated systemic immunoglobulin M (IgM) levels, attenuated CD40L-induced inflammatory responses, and reduced surface expression of CD40 on microglia. Importantly, deficiency in CD40 abolishes the effect of HUCBCs on elevated plasma Abeta levels. Moreover, microglia isolated from HUCBC-infused PSAPP mice demonstrated increased phagocytosis of Abeta. Furthermore, sera from HUCBC-infused PSAPP mice significantly increased microglial phagocytosis of the Abeta1-42 peptide while inhibiting interferon-gammainduced microglial CD40 expression. Increased microglial phagocytic activity in this scenario was inhibited by addition of recombinant CD40L protein. These data suggest that HUCBC infusion mitigates AD-like pathology by disrupting CD40L activity.     

Microglia lacking E Prostanoid Receptor subtype 2 have enhanced Abeta phagocytosis yet lack Abeta-activated neurotoxicity

Experimental therapies for Alzheimer's disease (AD) are focused on enhanced clearance of neurotoxic Abeta peptides from brain. Microglia can be neuroprotective by phagocytosing Abeta; however, this comes at the cost of activated innate immunity that causes paracrine damage to neurons. Here, we show that ablation of E prostanoid receptor subtype 2 (EP2) significantly increased microglial-mediated clearance of Abeta peptides from AD brain sections and enhanced microglial Abeta phagocytosis in cell culture. The enhanced phagocytosis was PKC-dependent and was associated with elevated microglial secretion of the chemoattractant chemokines, macrophage inflammatory protein-1alpha and macrophage chemoattractant protein-1. This suggested that microglial activation is negatively regulated by EP2 signaling through suppression of prophagocytic cytokine secretion. However, despite this enhancement of Abeta phagocytosis, lack of EP2 completely suppressed Abeta-activated microglia-mediated paracrine neurotoxicity. These data demonstrate that blockade of microglial EP2 is a highly desirable mechanism for AD therapy that can maximize neuroprotective actions while minimizing bystander damage to neurons.     

CD40 signaling regulates innate and adaptive activation of microglia in response to amyloid beta-peptide

Although deposition of amyloid beta-peptide (Abeta) as Abeta plaques involves activation of microglia-mediated inflammatory responses, activated microglia ultimately fail to clear Abeta plaques in the brains of either Alzheimer's disease (AD) patients or AD mouse models. Mounting evidence suggests that chronic microglia-mediated immune response during Abeta deposition etiologically contributes to AD pathogenesis by promoting Abeta plaque formation. However, the mechanisms that govern microglia response in the context of cerebral Abeta/beta-amyloid pathology are not well understood. We show that ligation of CD40 by CD40L modulates Abeta-induced innate immune responses in microglia, including decreased microglia phagocytosis of exogenous Abeta(1-42) and increased production of pro-inflammatory cytokines. CD40 ligation in the presence of Abeta(1-42) leads to adaptive activation of microglia, as evidenced by increased co-localization of MHC class II with Abeta. To assess their antigen-presenting cell (APC) function, cultured microglia were pulsed with Abeta(1-42) in the presence of CD40L and co-cultured with CD4(+) T cells. Under these conditions, microglia stimulate T cell-derived IFN-gamma and IL-2 production, suggesting that CD40 signaling promotes the APC phenotype. These data provide a mechanistic explanation for our previous work showing decreased microgliosis associated with diminished cerebral Abeta/beta-amyloid pathology when blocking CD40 signaling in transgenic Alzheimer's mice.     

Overexpression of monocyte chemotactic protein-1/CCL2 in beta-amyloid precursor protein transgenic mice show accelerated diffuse beta-amyloid deposition

Microglia accumulation at the site of amyloid plaques is a strong indication that microglia play a major role in Alzheimer's disease pathogenesis. However, how microglia affect amyloid-beta peptide (Abeta) deposition remains poorly understood. To address this question, we developed a novel bigenic mouse that overexpresses both amyloid precursor protein (APP) and monocyte chemotactic protein-1 (MCP-1; CCL2 in systematic nomenclature). CCL2 expression, driven by the glial fibrillary acidic protein promoter, induced mononuclear phagocyte (MP; monocyte-derived macrophage and microglial) accumulation in the brain. When APP/CCL2 transgenic mice were compared to APP mice, a fivefold increase in Abeta deposition was present despite increased MP accumulation around hippocampal and cortical amyloid plaques. Levels of full-length APP, its C-terminal fragment, and Abeta-degrading enzymes (insulin-degrading enzyme and neprilysin) in APP/CCL2 and APP mice were indistinguishable. Sodium dodecyl sulfate-insoluble Abeta (an indicator of fibrillar Abeta) was increased in APP/CCL2 mice at 5 months of age. Apolipoprotein E, which enhances Abeta deposition, was also increased (2.2-fold) in aged APP/CCL2 as compared to APP mice. We propose that although CCL2 stimulates MP accumulation, it increases Abeta deposition by reducing Abeta clearance through increased apolipoprotein E expression. Understanding the mechanisms underlying these events could be used to modulate microglial function in Alzheimer's disease and positively affect disease outcomes.     

Regulation of Fcγ receptors and immunoglobulin G-mediated phagocytosis in mouse microglia

As resident macrophages in the CNS, microglia can transform from a surveillance state to an activated phenotype in response to brain injury. During this transition microglia become highly capable phagocytic cells. Invading pathogens undergo opsonization with immunoglobulins and microglia recognize these opsonized pathogens through interaction with their cognate F_c receptors. In mice, both FcγRI and FcγRIIb receptors are involved in IgG-mediated phagocytosis of opsonzied pathogens. At sites of inflammation, microglial activity is regulated by T-cell derived cytokines. Here we first investigated the effects of IFN-γ, IL-4, IL-13 and GM-CSF on expression of FcγRI and FcγRIIb mRNA levels in both primary microglia and microglial cell line N9. Using quantitative real-time PCR we show that IFN-γ induced a 4-fold increase in the mRNA level of FcγRI but did not induce changes in FcγRIIb expression. IL-4 and IL-13 induced approximately 2-fold increases in expression of FcγRIIb mRNA, but had no effect on FcγRI expression. GM-CSF increased both FcγRI and FcγRIIb mRNA expression. We then characterized the ability of these same cytokines to regulate phagocytosis of immune complexes composed of IgG and the bacteria Staphylococcus aureus. IFN-γ and GM-CSF both induced approximately 2-fold increases in IgG-mediated phagocytosis whereas IL-4 and IL-13 both decreased IgG-mediated phagocytosis by about one-third. None of the cytokines influenced basal levels of phagocytosis. These findings demonstrate a highly selective cytokine-induced regulation of both phagocytosis-related Fcγ receptor subtypes and IgG-mediated phagocytosis itself in microglia. This selective regulation has implications for our understanding of the pathophysiology of CNS infection and autoimmune disease.     

Noradrenaline deficiency in brain increases beta-amyloid plaque burden in an animal model of Alzheimer's disease

Loss of Locus coeruleus (LC) noradrenergic (NA) neurons occurs in several neurodegenerative conditions including Alzheimer's disease (AD). In vitro and in vivo studies have shown that NA influences several features of AD disease including inflammation, neurodegeneration, and cognitive function. In the current study we tested if LC loss influenced beta amyloid (Abeta) plaque deposition. LC neuronal degeneration was induced in transgenic mice expressing mutant V717F human amyloid precursor protein (APP) by treatment with the selective neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine DSP4 (5mg/kg every 2 weeks beginning at age 3 months). At 9 months of age, when control mice show low amyloid load, DSP4-treated mice showed an approximately 5-fold increase in the average number of Abeta plaques. This was accompanied by an increase in the levels of APP C-terminal cleavage fragments. DSP4-treatment increased both microglial and astroglial activation. In vivo, DSP4-treatment decreased expression and activity of the Abeta degrading enzyme neprilysin, while in vitro NA increased phagocytosis of Abeta1-42 by microglia. These findings suggest that noradrenergic innervation from LC are needed to maintain adequate Abeta clearance, and therefore that LC degeneration could contribute to AD pathogenesis.     

Proinflammatory effects of M-CSF and A beta in hippocampal organotypic cultures

Macrophage colony stimulating factor (M-CSF) is a microglial activator expressed at increased levels in the brain in Alzheimer's disease. In monotypic microglial cultures, M-CSF strongly augments amyloid beta (Abeta) induced microglial production of proinflammatory cytokines and nitric oxide. However, this augmentation could be due to strong autocrine and paracrine effects in monotypic cultures. We used hippocampal organotypic cultures to test M-CSF/Abeta augmentation in a system modeling intact brain. Combined M-CSF/Abeta treatment increased interleukin-1 (IL-1) and macrophage inflammatory protein 1-alpha expression by microglia, whereas inducible nitric oxide synthase (iNOS) expression was localized primarily to astroglia. Induction of cytokines and iNOS was also observed after lipopolysaccharide treatment of organotypic hippocampal cultures, but iNOS expression was localized mainly to microglia rather than astrocytes. Treatment with M-CSF/Abeta did not result in neuronal death. These results demonstrate that combined M-CSF/Abeta treatment results in a strong inflammatory response in the organotypic environment without inducing neurotoxicity.     

Peripheral T cells derived from Alzheimer's disease patients overexpress CXCR2 contributing to its transendothelial migration, which is microglial TNF-alpha-dependent

The mechanism of circulating T cells entry into the brain in Alzheimer's diseases (AD) remains unclear. Here, we showed that peripheral T cells derived from AD patients overexpress CXCR2 to enhance its transendothelial migration. T cells migration through in vitro blood-brain barrier model was effectively blocked by anti-CXCR2 antibody or IL-8 (a CXCR2 ligand) RNAi in human brain microvascular endothelial cells (HBMECs). Amyloid beta (Abeta) injection in rat hippocampus upregulated CXCR2 expression accompanied with increased T cells occurrence in the brain, and this enhanced T cells entry was effectively blocked by CXCR2 antagonist. Furthermore, anti-TNF-alpha antibody blocked IL-8 production in HBMECs and T cells transendothelial migration caused by the culture supernatant of microglia treated with Abeta. Blockage of intracerebral TNF-alpha abolished the upregulation of CXCR2 in peripheral T cells and the increased T cells occurrence in the brain induced by Abeta injection in rat hippocampus. These data suggest that CXCR2 overexpression in peripheral T cells is intracerebral microglial TNF-alpha-dependent and TNF-alpha primes T cells transendothelial migration in Alzheimer's diseases.     

Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice

We have shown that interaction of CD40 with CD40L enables microglial activation in response to amyloid-beta peptide (Abeta), which is associated with Alzheimer's disease (AD)-like neuronal tau hyperphosphorylation in vivo. Here we report that transgenic mice overproducing Abeta, but deficient in CD40L, showed decreased astrocytosis and microgliosis associated with diminished Abeta levels and beta-amyloid plaque load. Furthermore, in the PSAPP transgenic mouse model of AD, a depleting antibody against CD40L caused marked attenuation of Abeta/beta-amyloid pathology, which was associated with decreased amyloidogenic processing of amyloid precursor protein (APP) and increased circulating levels of Abeta. Conversely, in neuroblastoma cells overexpressing wild-type human APP, the CD40-CD40L interaction resulted in amyloidogenic APP processing. These findings suggest several possible mechanisms underlying mitigation of AD pathology in response to CD40L depletion, and validate the CD40-CD40L interaction as a target for therapeutic intervention in AD.     

Abeta peptide conformation determines uptake and interleukin-1alpha expression by primary microglial cells

Microglia clear amyloid beta (Abeta) after immunization. The interaction of Abeta with the microglial cell surface also results in cytokine expression. Soluble oligomers and protofibrils of Abeta may be more neurotoxic than Abeta fibrils. We investigated the effects of oligomeric, protofibrillar and fibrillar Abeta40 and Abeta42 peptides on uptake and IL-1alpha expression by primary microglia. Abeta peptide assemblies were extensively characterized. Primary microglial cells were exposed to different Abeta40 and Abeta42 assemblies and IL-1alpha expression was quantified. To study uptake, microglial cells were exposed to different assemblies of Cy3-labeled Abeta. We found that Abeta42 and Abeta40 oligomers and fibrils induced IL-1alpha expression, but protofibrils did not. We also observed that all forms of Abeta42 (oligomer, protofibril and fibril) and Abeta40 fibrils were taken up by the microglial cells. These results demonstrate that microglial cells can take up non-fibrillar Abeta and that oligomeric peptide induces an inflammatory response. The uptake of oligomeric and protofibrillar Abeta by microglia merits further investigation as a potential means for removing these neurotoxic species from the brain.     

Evidence supporting a role for anti-Abeta antibodies in the treatment of Alzheimer's disease

Antibodies against Abeta have been suggested as potential therapeutic strategies for the treatment of Alzheimer disease (AD) for nearly 8 years. Animal studies have been very encouraging in that both active and passive immunization of transgenic mice can reduce amyloid load and reverse memory deficits found in these mice. Three mechanisms have been proposed to explain these results: (a). catalytic conversion of fibrillar Abeta to less toxic forms, (b). opsonization of Abeta deposits leading to microglial phagocytosis, or (c). promote the efflux of Abeta from the brain to the circulation. Evidence exists supporting all three mechanisms, which, it should be noted, are not mutually exclusive. Phase 2 clinical trials of active immunization with vaccines against human Abeta1-42 were halted due to an unacceptable incidence of meningoencephalitic reactions (6% of patients treated). However, a recent report from a fraction of the patients in this trial found that those patients developing antibodies which reacted with brain amyloid deposits had a significantly slower progression of cognitive loss over a period of 12 months. This supports the continued cautious testing of passive immunization and, possibly even active immunization against the Abeta peptide using preparations less likely to cause autoimmune reactions in the central nervous system.     

Toll-like receptor 4 promotes α-synuclein clearance and survival of nigral dopaminergic neurons

Toll-like receptors (TLRs) mediate innate immunity, and their dysregulation may play a role in α-synucleinopathies, such as Parkinson's disease or multiple system atrophy (MSA). The aim of this study was to define the role of TLR4 in α-synuclein-linked neurodegeneration. Ablation of TLR4 in a transgenic mouse model of MSA with oligodendroglial α-synuclein overexpression augmented motor disability and enhanced loss of nigrostriatal dopaminergic neurons. These changes were associated with increased brain levels of α-synuclein linked to disturbed TLR4-mediated microglial phagocytosis of α-synuclein. Furthermore, tumor necrosis factor-α levels were increased in the midbrain and associated with a proinflammatory astroglial response. Our data suggest that TLR4 ablation impairs the phagocytic response of microglia to α-synuclein and enhances neurodegeneration in a transgenic MSA mouse model. The study supports TLR4 signaling as innate neuroprotective mechanism acting through clearance of α-synuclein.     

Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model

We have previously shown that chronic treatment with the monoclonal antibody m266, which is specific for amyloid beta-peptide (Abeta), increases plasma concentrations of Abeta and reduces Abeta burden in the PDAPP transgenic mouse model of Alzheimer's disease (AD). We now report that administration of m266 to PDAPP mice can rapidly reverse memory deficits in both an object recognition task and a holeboard learning and memory task, but without altering brain Abeta burden. We also found that an Abeta/antibody complex was present in both the plasma and the cerebrospinal fluid of m266-treated mice. Our data indicate that passive immunization with this anti-Abeta monoclonal antibody can very rapidly reverse memory impairment in certain learning and memory tasks in the PDAPP mouse model of AD, owing perhaps to enhanced peripheral clearance and (or) sequestration of a soluble brain Abeta species.     

HIV-1 TAT inhibits microglial phagocytosis of Abeta peptide

Human immunodeficiency virus (HIV)-associated dementia (HAD) is a subcortical neuropsychiatric syndrome that has increased in prevalence in the era of highly active antiretroviral therapy (HAART). Several studies demonstrated increased amyloidosis in brains of HIV patients and suggested that there may be a significant number of long-term HIV survivors with co-morbid Alzheimer's disease (AD) in the future. We show HIV-1 Tat protein inhibits microglial uptake of Abeta1-42 peptide, a process that is enhanced by interferon-gamma (IFN-gamma) and rescued by the STAT1 inhibitor (-)-epigallocatechin-3-gallate (EGCG). It is hypothesized that reduced Abeta uptake occurs through IFN-gamma mediated STAT1 activation. This process promotes a switch from a phagocytic to an antigen presenting phenotype in microglia through activation of class II transactivator (CIITA). Additionally, we show that HIV-1 Tat significantly disrupts apolipoprotein-3 (Apo-E3) promoted microglial Abeta uptake. As Tat has been shown to directly interact with the low density lipoprotein (LRP) receptor and thus inhibit the uptake of its ligands including apolipoprotein E4 (Apo-E4) and Abeta peptide in neurons, we further hypothesize that a similar inhibition of LRP may occur in microglia. Future studies will be required to fully characterize the mechanisms underlying IFN-gamma enhancement of HIV-1 Tats disruption of microglial phagocytosis of Abeta and Apo-E3.     

Primary phagocytosis of viable neurons by microglia activated with LPS or Abeta is dependent on calreticulin/LRP phagocytic signalling

ABSTRACT: BACKGROUND: Microglia are resident brain macrophages that can phagocytose dead, dying or viable neurons, which may be beneficial or detrimental in inflammatory, ischaemic and neurodegenerative brain pathologies. Cell death caused by phagocytosis of an otherwise viable cell is called 'primary phagocytosis' or 'phagoptosis'. Calreticulin (CRT) exposure on the surface of cancer cells can promote their phagocytosis via LRP (low-density lipoprotein receptor-related protein) on macrophages, but it is not known whether this occurs with neurons and microglia. METHODS: We used primary cultures of cerebellar neurons, astrocytes and microglia to investigate the potential role of CRT/LRP phagocytic signalling in the phagocytosis of viable neurons by microglia stimulated with LPS (lipopolysaccharide) or nanomolar concentrations of amyloid-beta peptide1-42 (Abeta). Exposure of CRT on the neuronal surface was investigated using surface biotinylation and western blotting. A phagocytosis assay was also developed using BV2 and PC12 cell lines to investigate CRT/LRP signalling in microglial phagocytosis of apoptotic cells. RESULTS: We found that BV2 microglia readily phagocytosed apoptotic PC12 cells, but this was inhibited by a CRT blocking antibody or LRP-blocking protein (Receptor-Associated Protein: RAP). Activation of primary rat microglia with LPS or Abeta resulted in loss of co-cultured cerebellar granule neurons, and this was blocked by RAP or antibodies against CRT or against LRP, preventing all neuronal loss and death. CRT was present on the surface of viable neurons, and this exposure did not change in inflammatory conditions. CRT antibodies prevented microglia-induced neuronal loss when added to neurons, while LRP antibodies prevented neuronal loss when added to the microglia. Pre-binding of CRT to neurons promoted neuronal loss if activated microglia were added, but pre-binding of CRT to microglia or both cell types prevented microglia-induced neuronal loss. CONCLUSIONS: CRT exposure on the surface of viable or apoptotic neurons appears to be required for their phagocytosis via LRP receptors on activated microglia, but free CRT can block microglial phagocytosis of neurons by acting on microglia. Phagocytosis of CRT-exposing neurons by microglia can be a direct cause of neuronal death during inflammation, and might therefore contribute to neurodegeneration and be prevented by blocking the CRT/LRP pathway.     

The role of the immune system in clearance of Abeta from the brain.

In Alzheimer's disease (AD), there is abnormal accumulation of Abeta and tau proteins in the brain. There is an associated immunological response, but it is still unclear whether this is beneficial or harmful. Inflammation in AD, specifically in the form of microglial activation, has, for many years, been considered to contribute to disease progression. However, two types of evidence suggest that it may be appropriate to revise this view: first, the disappointing results of prospective clinical trials of anti-inflammatory agents and, second, the observation that microglia can clear plaques in AD following Abeta immunization. Although Abeta immunization alters AD pathology, there is limited evidence so far of benefit to cognitive function. Immunization against microorganisms is almost always used as a method of disease prevention rather than to treat a disease process that has already started. In animal models, immunotherapy at an early age can protect against Abeta accumulation and it will be interesting to see if this can usefully be applied to humans to prevent AD.     

Survival of immunoglobulin G-opsonized Toxoplasma gondii in nonadherent human monocytes.

Toxoplasma gondii is a protozoan parasite that is able to penetrate human monocytes by either passive uptake during phagocytosis or active penetration. It is expected that immunoglobulin G (IgG) opsonization will target the parasite to macrophage Fc gamma receptors for phagocytic processing and subsequent degradation. Antibody-opsonized T. gondii tachyzoites were used to infect nonadherent and adherent human monocytes obtained from the peripheral blood of seronegative individuals. The infected monocytes were evaluated for the presence of intracellular parasites and the degree of parasiticidal activity. A marked difference in both the numbers of infected macrophages and numbers of parasites per 100 macrophages was observed in the nonadherent cells when compared with those of the adherent cell population. When macrophage Fc gamma receptors were down-modulated, opsonized tachyzoites retained their ability to penetrate the host cell at a rate similar to that observed for unopsonized parasites. These results suggest that antibody opsonization of T. gondii does not prevent active penetration of human monocytes by the parasite and, furthermore, has little effect on intracellular replication of the parasite.