Rapid appearance and local toxicity of amyloid-beta plaques in a mouse model of Alzheimer's disease.

Senile plaques accumulate over the course of decades in the brains of patients with Alzheimer's disease. A fundamental tenet of the amyloid hypothesis of Alzheimer's disease is that the deposition of amyloid-beta precedes and induces the neuronal abnormalities that underlie dementia. This idea has been challenged, however, by the suggestion that alterations in axonal trafficking and morphological abnormalities precede and lead to senile plaques. The role of microglia in accelerating or retarding these processes has been uncertain. To investigate the temporal relation between plaque formation and the changes in local neuritic architecture, we used longitudinal in vivo multiphoton microscopy to sequentially image young APPswe/PS1d9xYFP (B6C3-YFP) transgenic mice. Here we show that plaques form extraordinarily quickly, over 24 h. Within 1-2 days of a new plaque's appearance, microglia are activated and recruited to the site. Progressive neuritic changes ensue, leading to increasingly dysmorphic neurites over the next days to weeks. These data establish plaques as a critical mediator of neuritic pathology.

Microglia mediate the clearance of soluble Abeta through fluid phase macropinocytosis.

Alzheimer's disease is characterized by the progressive deposition of beta-amyloid (Abeta) within the brain parenchyma and its subsequent accumulation into senile plaques. Pathogenesis of the disease is associated with perturbations in Abeta homeostasis and the inefficient clearance of these soluble and insoluble peptides from the brain. Microglia have been reported to mediate the clearance of fibrillar Abeta (fAbeta) through receptor-mediated phagocytosis; however, their participation in clearance of soluble Abeta peptides (sAbeta) is largely unknown. We report that microglia internalize sAbeta from the extracellular milieu through a nonsaturable, fluid phase macropinocytic mechanism that is distinct from phagocytosis and receptor-mediated endocytosis both in vitro and in vivo. The uptake of sAbeta is dependent on both actin and tubulin dynamics and does not involve clathrin assembly, coated vesicles or membrane cholesterol. Upon internalization, fluorescently labeled sAbeta colocalizes to pinocytic vesicles. Microglia rapidly traffic these soluble peptides into late endolysosomal compartments where they are subject to degradation. Additionally, we demonstrate that the uptake of sAbeta and fAbeta occurs largely through distinct mechanisms and upon internalization are segregated into separate subcellular vesicular compartments. Significantly, we found that upon proteolytic degradation of fluorescently labeled sAbeta, the fluorescent chromophore is retained by the microglial cell. These studies identify an important mechanism through which microglial cells participate in the maintenance of Abeta homeostasis, through their capacity to constitutively clear sAbeta peptides from the brain.

Cerebrovascular dysfunction in amyloid precursor protein transgenic mice: contribution of soluble and insoluble amyloid-beta peptide, partial restoration via gamma-secretase inhibition.

The contributing effect of cerebrovascular pathology in Alzheimer's disease (AD) has become increasingly appreciated. Recent evidence suggests that amyloid-beta peptide (Abeta), the same peptide found in neuritic plaques of AD, may play a role via its vasoactive properties. Several studies have examined young Tg2576 mice expressing mutant amyloid precursor protein (APP) and having elevated levels of soluble Abeta but no cerebral amyloid angiopathy (CAA). These studies suggest but do not prove that soluble Abeta can significantly impair the cerebral circulation. Other studies examining older Tg2576 mice having extensive CAA found even greater cerebrovascular dysfunction, suggesting that CAA is likely to further impair vascular function. Herein, we examined vasodilatory responses in young and older Tg2576 mice to further assess the roles of soluble and insoluble Abeta on vessel function. We found that (1) vascular impairment was present in both young and older Tg2576 mice; (2) a strong correlation between CAA severity and vessel reactivity exists; (3) a surprisingly small amount of CAA led to marked reduction or complete loss of vessel function; 4) CAA-induced vasomotor impairment resulted from dysfunction rather than loss or disruption of vascular smooth muscle cells; and 5) acute depletion of Abeta improved vessel function in young and to a lesser degree older Tg2576 mice. These results strongly suggest that both soluble and insoluble Abeta cause cerebrovascular dysfunction, that mechanisms other than Abeta-induced alteration in vessel integrity are responsible, and that anti-Abeta therapy may have beneficial vascular effects in addition to positive effects on parenchymal amyloid.

Rapid microglial response around amyloid pathology after systemic anti-Abeta antibody administration in PDAPP mice.

Aggregation of amyloid-beta (Abeta) peptide in the brain in the form of neuritic plaques and cerebral amyloid angiopathy (CAA) is a key feature of Alzheimer's disease (AD). Microglial cells surround aggregated Abeta and are believed to play a role in AD pathogenesis. A therapy for AD that has entered clinical trials is the administration of anti-Abeta antibodies. One mechanism by which certain anti-Abeta antibodies have been proposed to exert their effects is via antibody-mediated microglial activation. Whether, when, or to what extent microglial activation occurs after systemic administration of anti-Abeta antibodies has not been fully assessed. We administered an anti-Abeta antibody (m3D6) that binds aggregated Abeta to PDAPP mice, an AD mouse model that was bred to contain fluorescent microglia. Three days after systemic administration of m3D6, there was a marked increase in both the number of microglial cells and processes per cell visualized in vivo by multiphoton microscopy. These changes required the Fc domain of m3D6 and were not observed with an antibody specific to soluble Abeta. These findings demonstrate that some effects of antibodies that recognize aggregated Abeta are rapid, involve microglia, and provide insight into the mechanism of action of a specific passive immunotherapy for AD.

Microglial phagocytosis induced by fibrillar beta-amyloid and IgGs are differentially regulated by proinflammatory cytokines.

Microglia undergo a phenotypic activation in response to fibrillar beta-amyloid (fAbeta) deposition in the brains of Alzheimer's disease (AD) patients, resulting in their elaboration of inflammatory molecules. Despite the presence of abundant plaque-associated microglia in the brains of AD patients and in animal models of the disease, microglia fail to efficiently clear fAbeta deposits. However, they can be induced to do so during Abeta vaccination therapy attributable to anti-Abeta antibody stimulation of IgG receptor (FcR)-mediated phagocytic clearance of Abeta plaques. We report that proinflammatory cytokines attenuate microglial phagocytosis stimulated by fAbeta or complement receptor 3 and argue that this may, in part, underlie the accumulation of fAbeta-containing plaques within the AD brain. The proinflammatory suppression of fAbeta-elicited phagocytosis is dependent on nuclear factor kappaB activation. Significantly, the proinflammatory cytokines do not inhibit phagocytosis elicited by antibody-mediated activation of FcR, which may contribute to the efficiency of Abeta vaccination-based therapy. Importantly, the proinflammatory suppression of fAbeta phagocytosis can be relieved by the coincubation with anti-inflammatory cytokines, cyclooxygenase inhibitors, ibuprofen, or an E prostanoid receptor antagonist, suggesting that proinflammatory cytokines induce the production of prostaglandins, leading to an E prostanoid receptor-dependent inhibition of phagocytosis. These findings support anti-inflammatory therapies for the treatment of AD.

Apolipoprotein E mediation of neuro-inflammation in a murine model of multiple sclerosis.

Apolipoprotein E (ApoE) functions as a ligand in receptor-mediated endocytosis of lipoprotein particles and has been demonstrated to play a role in antigen presentation. To explore the contribution of ApoE during autoimmune central nervous system (CNS) demyelination, we examined the clinical, cellular immune function, and pathologic consequences of experimental autoimmune encephalomyelitis (EAE) induction in ApoE knockout (ApoE(-/-)) mice. We observed reduced clinical severity of EAE in ApoE(-/-) mice in comparison to WT mice that was concomitant with an early reduction of dendritic cells (DCs) followed by a reduction of additional innate cells in the spinal cord at the peak of disease without any differences in axonal damage. While T cell priming was enhanced in ApoE(-/-) mice, reduced severity of EAE was also observed in ApoE(-/-) recipients of encephalitogenic wild type T cells. Expression of ApoE during EAE was elevated within the CNS of wild type mice, particularly by innate cells such as DCs. Overall, ApoE promotes clinical EAE, likely by mediation of inflammation localized within the CNS.

Regulation of microglial phagocytosis and inflammatory gene expression by Gas6 acting on the Axl/Mer family of tyrosine kinases.

Removal of apoptotic cells is an essential process for normal development and tissue maintenance. Importantly, apoptotic cells stimulate their phagocytosis by macrophages while actively suppressing inflammatory responses. Growth arrest specific gene 6 (Gas6) is involved in this process, bridging phosphatidylserine residues on the surface of apoptotic cells to the Axl/Mer family of tyrosine kinases which stimulate phagocytosis. Animals with mutations or loss of these receptors exhibit phenotypes reflective of impaired phagocytosis and a hyperactive immune response. We report that Gas6 induces phagocytosis in microglia through a novel non-classical phagocytic mechanism. Gas6 stimulates a type-II-related phagocytic response, but requires Vav phosphorylation and Rac activation, distinguishing it from the classical type II mechanism. Importantly, Gas6 suppressed lipopolysaccharide-induced expression of the inflammatory molecules IL-1beta and iNOS. Gas6 inhibited iNOS expression through suppression of promoter activity. The present data provide direct evidence for the role of Gas6 receptors in mediating an anti-inflammatory response to ligands found on apoptotic cells with the simultaneous stimulation of phagocytosis. These data provide a mechanistic explanation for the phenotype observed in animals lacking Axl/Mer receptors.

Fibrillar beta-amyloid-stimulated intracellular signaling cascades require Vav for induction of respiratory burst and phagocytosis in monocytes and microglia.

Microglial interaction with extracellular beta-amyloid fibrils (fAbeta) is mediated through an ensemble of cell surface receptors, including the B-class scavenger receptor CD36, the alpha(6)beta(1)-integrin, and the integrin-associated protein/CD47. The binding of fAbeta to this receptor complex has been shown to drive a tyrosine kinase-based signaling cascade leading to production of reactive oxygen species and stimulation of phagocytic activity; however, little is known about the intracellular signaling cascades governing the microglial response to fAbeta. This study reports a direct mechanistic link between the fAbeta cell surface receptor complex and downstream signaling events responsible for NADPH oxidase activation and phagosome formation. The Vav guanine nucleotide exchange factor is tyrosine-phosphorylated in response to fAbeta peptides as a result of the engagement of the microglia fAbeta cell surface receptor complex. Co-immunoprecipitation studies demonstrate an Abeta-dependent association between Vav and both Lyn and Syk kinases. The downstream target of Vav, the small GTPase Rac1, is GTP-loaded in an Abeta-dependent manner. Rac1 is both an essential component of the NADPH oxidase and a critical regulator of microglial phagocytosis. The direct role of Vav in fAbeta-stimulated intracellular signaling cascades was established using primary microglia obtained from Vav(-/-) mice. Stimulation of Vav(-/-) microglia with fAbeta failed to generate NADPH oxidase-derived reactive oxygen species and displayed a dramatically attenuated phagocytic response. These findings directly link Vav phosphorylation to the Abeta-receptor complex and demonstrate that Vav activity is required for fAbeta-stimulated intracellular signaling events upstream of reactive oxygen species production and phagosome formation.

Mechanisms of statin-mediated inhibition of small G-protein function.

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been reported to reduce the risk of Alzheimer disease. We have shown previously that statins inhibit a beta-amyloid (Abeta)-mediated inflammatory response through mechanisms independent of cholesterol reduction. Specifically, statins exert anti-inflammatory actions through their ability to prevent the isoprenylation of members of the Rho family of small G-proteins, resulting in the functional inactivation of these G-proteins. We report that statin treatment of microglia results in perturbation of the cytoskeleton and morphological changes due to alteration in Rho family function. Statins also block Abeta-stimulated phagocytosis through inhibition of Rac action. Paradoxically, the statin-mediated inactivation of G-protein function was associated with increased GTP loading of Rac and RhoA, and this effect was observed in myeloid lineage cells and other cell types. Statin treatment disrupted the interaction of Rac with its negative regulator the Rho guanine nucleotide dissociation inhibitor (RhoGDI), an interaction that is dependent on protein isoprenylation. We propose that lack of negative regulation accounts for the increased GTP loading. Isoprenylation of Rac is also required for efficient interaction with the plasma membrane, and we report that statin treatment dramatically reduces the capacity of Rac to interact with membranes. These results suggest a mechanism by which statins inhibit the actions of Rho GTPases and attenuate Abeta-stimulated inflammation.