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-β precedes and induces the neuronal abnormalities that underlie dementia 1 . This idea has been challenged, however, by the suggestion that alterations in axonal trafficking and morphological abnormalities precede and lead to senile plaques 2 . 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 3 . 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.To explore the formation of amyloid plaques and to determine the effects of newly formed dense-cored plaques on the microarchitecture of the brain, we have developed a novel in vivo multiphoton imaging technique. This recognizes newly formed plaques and allows monitoring of their immediate vicinity thereafter to determine the rate of their formation and the temporal sequence of pathophysiological events. We imaged young (5-to 6-month-old) B6C3-YFP mice, an age when plaques begin to appear 4 (Fig. 1). We used three-colour imaging to establish fiduciary markers for repeated imaging: YFP positive neurons, dendrites and axons in the cortex, methoxy-XO4-labelled fibrillar amyloid-β deposits in the parenchyma and on vessel walls, and a fluorescent angiogram with Texas red dextran to image blood vessels. A volume of cortex (lamina I-III) that initially did not contain plaques was re-imaged until repeat imaging detected a new plaque, establishing its 'birthday'. To ensure that the appearance of a new plaque did not simply reflect a greater depth of imaging or a slightly different imaging volume, we went through each image stack and compared NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2012 January 23. We postulated that we would occasionally observe the appearance and growth of new plaques within an imaging volume if the time interval between imaging sessions was long enough. From one weekly imaging session to the next, most of the sites remained unchanged ( Supplementary Fig. 1a-c). However, we identified 14 new plaques: instances in which a plaque appeared in a second imaging session in a volume that had clearly been unoccupied in the first images one week earlier (Fig. 1a-c).We examined the spatial relation between newly identified plaques and blood vessels. Measurements of the distance between vessel wall and the edge of a plaque confirmed that dense-core plaques develop...

Microglia undergo a phenotypic activation in response to fibrillar ␤-amyloid (fA␤) 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 fA␤ deposits. However, they can be induced to do so during A␤ vaccination therapy attributable to anti-A␤ antibody stimulation of IgG receptor (FcR)-mediated phagocytic clearance of A␤ plaques.We report that proinflammatory cytokines attenuate microglial phagocytosis stimulated by fA␤ or complement receptor 3 and argue that this may, in part, underlie the accumulation of fA␤-containing plaques within the AD brain. The proinflammatory suppression of fA␤-elicited phagocytosis is dependent on nuclear factor B activation. Significantly, the proinflammatory cytokines do not inhibit phagocytosis elicited by antibody-mediated activation of FcR, which may contribute to the efficiency of A␤ vaccination-based therapy. Importantly, the proinflammatory suppression of fA␤ 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.

Alzheimer's disease is characterized by the progressive deposition of ␤-amyloid (A␤) within the brain parenchyma and its subsequent accumulation into senile plaques. Pathogenesis of the disease is associated with perturbations in A␤ homeostasis and the inefficient clearance of these soluble and insoluble peptides from the brain. Microglia have been reported to mediate the clearance of fibrillar A␤ (fA␤) through receptor-mediated phagocytosis; however, their participation in clearance of soluble A␤ peptides (sA␤) is largely unknown. We report that microglia internalize sA␤ 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 sA␤ is dependent on both actin and tubulin dynamics and does not involve clathrin assembly, coated vesicles or membrane cholesterol. Upon internalization, fluorescently labeled sA␤ 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 sA␤ and fA␤ 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 sA␤, the fluorescent chromophore is retained by the microglial cell. These studies identify an important mechanism through which microglial cells participate in the maintenance of A␤ homeostasis, through their capacity to constitutively clear sA␤ peptides from the brain.

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 ␤-amyloid (A␤)-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 A␤-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 A␤-stimulated inflammation.