Inflammatory responses to amyloidosis in a transgenic mouse model of Alzheimer's disease

Mutations in the amyloid precursor protein (APP) and presenilin-1 and -2 genes (PS-1, -2) cause Alzheimer's disease (AD). Mice carrying both mutant genes (PS/APP) develop AD-like deposits composed of beta-amyloid (Abeta) at an early age. In this study, we have examined how Abeta deposition is associated with immune responses. Both fibrillar and nonfibrillar Abeta (diffuse) deposits were visible in the frontal cortex by 3 months, and the amyloid load increased dramatically with age. The number of fibrillar Abeta deposits increased up to the oldest age studied (2.5 years old), whereas there were less marked changes in the number of diffuse deposits in mice over 1 year old. Activated microglia and astrocytes increased synchronously with amyloid burden and were, in general, closely associated with deposits. Cyclooxygenase-2, an inflammatory response molecule involved in the prostaglandin pathway, was up-regulated in astrocytes associated with some fibrillar deposits. Complement component 1q, an immune response component, strongly colocalized with fibrillar Abeta, but was also up-regulated in some plaque-associated microglia. These results show: i) an increasing proportion of amyloid is composed of fibrillar Abeta in the aging PS/APP mouse brain; ii) microglia and astrocytes are activated by both fibrillar and diffuse Abeta; and iii) cyclooxygenase-2 and complement component 1q levels increase in response to the formation of fibrillar Abeta in PS/APP mice.     

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.     

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.     

beta-site amyloid precursor protein cleaving enzyme 1 increases amyloid deposition in brain parenchyma but reduces cerebrovascular amyloid angiopathy in aging BACE x APP[V717I] double-transgenic mice

The generation of amyloid peptides (Abeta) from the amyloid precursor protein (APP) is initiated by beta-secretase (BACE), whereas subsequent gamma-secretase cleavage mediated by presenilin-1, produces Abeta peptides mainly of 40 or 42 amino acids long. In addition, alternative beta'-cleavage of APP at position 11 of the amyloid sequence results in N-truncated Abeta(11-40/42) peptides, but the functional significance or pathological impact is unknown. Here we demonstrate that in the brain of BACE x APP[V717I] double-transgenic mice, amyloidogenic processing at both Asp1 and Glu11 is increased resulting in more and different Abeta species and APP C-terminal fragments. Pathologically, BACE significantly increased the number of diffuse and senile amyloid plaques in old double-transgenic mice. Unexpectedly, vascular amyloid deposition was dramatically lower in the same BACE x APP[V717I] double-transgenic mice, relative to sex- and age-matched APP[V717I] single-transgenic mice in the same genetic background. The tight inverse relation of vascular amyloid to the levels of the less soluble N-terminally truncated Abeta peptides is consistent with the hypothesis that vascular amyloid deposition depends on drainage of excess tissue Abeta. This provides biochemical evidence in vivo for the preferential contribution of N-truncated Abeta to parenchymal amyloid deposition in contrast to vascular amyloid pathology.     

Reduction of amyloid angiopathy and Abeta plaque burden after enriched housing in TgCRND8 mice: involvement of multiple pathways

Diversity and intensity of intellectual and physical activities seem to have an inverse relationship with the extent of cognitive decline in Alzheimer's disease (AD). To study the interaction between an active lifestyle and AD pathology, female TgCRND8 mice carrying human APPswe+ind were transferred into enriched housing. Four months of continuous and diversified environmental stimulation resulted in a significant reduction of beta-amyloid (Abeta) plaques and in a lower extent of amyloid angiopathy. Neither human amyloid precursor protein (APP) mRNA/protein levels nor the level of carboxy-terminal fragments of APP nor soluble Abeta content differed between both groups, making alterations in APP expression or processing unlikely as a cause of reduced Abeta deposition. Moreover, DNA microarray analysis revealed simultaneous down-regulation of proinflammatory genes as well as up-regulation of molecules involved in anti-inflammatory processes, proteasomal degradation, and cholesterol binding, possibly explaining reduced Abeta burden by lower aggregation and enhanced clearance of Abeta. Additionally, immunoblotting against F4/80 antigen and morphometric analysis of microglia (Mac-3) revealed significantly elevated microgliosis in the enriched brains, which suggests increased amyloid phagocytosis. In summary, this study demonstrates that the environment interacts with AD pathology at dif-ferent levels.     

Minocycline does not affect amyloid beta phagocytosis by human microglial cells

Activated microglia accumulate in amyloid beta (Abeta) plaques containing amyloid associated factors SAP and C1q in Alzheimer's disease (AD) brain. Microglia are involved in AD pathogenesis by promoting Abeta plaque formation and production of pro-inflammatory cytokines. On the other hand, phagocytosis of Abeta by activated microglia may prevent Abeta-mediated neurotoxicity and Abeta plaque formation. Minocycline, a tetracycline derivative, is neuroprotective in various neurodegenerative models as well as human chronic neurological disorders. Minocycline attenuates the release of TNF-alpha by human microglia upon exposure to a mixture of Abeta, SAP and C1q. Here, we demonstrate that minocycline down-regulates the production of pro-inflammatory cytokines by human microglia without affecting their beneficial activity, phagocytosis of amyloid beta fibrils.     

Spatial and temporal control of age-related APP processing in genomic-based beta-secretase transgenic mice

Genetic mutations associated with Alzheimer's disease (AD) in the Amyloid Precursor Protein (APP) gene specifically alter the production of the APP processing product, amyloid-beta (Abeta) peptide, generated by beta- and gamma-secretases. The accumulation and deposition of Abeta is hypothesized to cause AD pathogenesis, leading to the debilitating neurological deficits observed in AD patients. However, it is unclear how processing of APP to generate Abeta corresponds with the age-dependent pattern of brain-regional neurodegeneration common in AD. We have previously shown that overexpression of BACE1, the primary beta-secretase gene, in mice expressing an AD mutant form of APP leads to significantly elevated regional Abeta levels, which coincide with the regional pattern of Abeta deposition. In the current study, we have used our genomic-based beta-secretase transgenic mice to determine how BACE1 regulates the spatial and temporal pattern of Abeta production throughout post-natal development. Specifically, we observed unique differences in the brain-regional expression pattern between neonatal and adult BACE1 transgenic mice. These alterations in the BACE1 expression profile directly corresponds with age-related differences in regional Abeta production and deposition. These studies indicate that modulation of BACE1 expression leads to dramatic alterations in APP processing and AD-like neuropathology. Furthermore, our studies provide further evidence that BACE1 plays a major role in the regulation of the APP processing pathway, influencing the age-dependent onset of AD pathogenesis.     

BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major components of plaque, beta-amyloid peptides (Abetas), are produced from amyloid precursor protein (APP) by the activity of beta- and gamma-secretases. beta-secretase activity cleaves APP to define the N-terminus of the Abeta1-x peptides and, therefore, has been a long- sought therapeutic target for treatment of AD. The gene encoding a beta-secretase for beta-site APP cleaving enzyme (BACE) was identified recently. However, it was not known whether BACE was the primary beta-secretase in mammalian brain nor whether inhibition of beta-secretase might have effects in mammals that would preclude its utility as a therapeutic target. In the work described herein, we generated two lines of BACE knockout mice and characterized them for pathology, beta-secretase activity and Abeta production. These mice appeared to develop normally and showed no consistent phenotypic differences from their wild-type littermates, including overall normal tissue morphology and brain histochemistry, normal blood and urine chemistries, normal blood-cell composition, and no overt behavioral and neuromuscular effects. Brain and primary cortical cultures from BACE knockout mice showed no detectable beta-secretase activity, and primary cortical cultures from BACE knockout mice produced much less Abeta from APP. The findings that BACE is the primary beta-secretase activity in brain and that loss of beta-secretase activity produces no profound phenotypic defects with a concomitant reduction in beta-amyloid peptide clearly indicate that BACE is an excellent therapeutic target for treatment of AD.     

Reactive astrocytes and alpha1-antichymotrypsin in Alzheimer's disease.

There is ample genetic, biochemical, cellular and molecular evidence to show that the amyloid beta peptide (Abeta), a proteolytic fragment of the amyloid precursor protein (APP), plays an important, if not causative role in Alzheimer's disease (AD). An additional hallmark of AD is the neuroinflammatory response that is associated with the amyloid deposition. We discovered that the acute phase protein alpha1-antichymotrypsin (ACT) is overexpressed by reactive astrocytes, and is tightly associated with virtually all amyloid plaques in the AD brain. It has also been shown that Abeta and ACT bind in vitro. Recently, we have reported that astrocytic expression of ACT in APP transgenic mice leads to an increased plaque deposition in ACT/APP doubly transgenic mice compared to the APP mice alone, suggesting that ACT interferes with Abeta clearance. The main objective of this review is to summarize the role of astrocytosis and ACT in the pathogenesis of AD.     

Specific uptake of Abeta1-40 in rat brain occurs in astrocyte,but not in microglia

In the brain of a patient with Alzheimer's disease, beta amyloid peptide (Abeta) is thought to be taken up by glial cells such as astrocyte and microglia to be degraded. However, it is unclear whether the Abeta is absorbed by astrocyte or microglia. The purpose of our study is to determine which type of glial cell, astrocyte or microglia, can take up Abeta. Beta amyloid 1-40 (Abeta1-40) was directly infused into the frontal cortex or hippocampus for 14 days. Dual-labeling immunohistochemistry for Abeta1-40 with an astrocytic (GFAP) or microglial (CD11b) marker was performed to examine co-localization of Abeta1-40 and glial markers. In the Abeta1-40 infused site, immunoreactivity of Abeta1-40 was observed only in astrocytes, not in microglia. In addition, Abeta40-1, a reverse peptide of Abeta1-40, was not taken up by astrocytes. These results suggested that the astrocyte-specific uptake of Abeta occurred in the rat brain.     

Cerebral microvascular amyloid beta protein deposition induces vascular degeneration and neuroinflammation in transgenic mice expressing human vasculotropic mutant amyloid beta precursor protein

Cerebral vascular amyloid beta-protein (Abeta) deposition, also known as cerebral amyloid angiopathy, is a common pathological feature of Alzheimer's disease. Additionally, several familial forms of cerebral amyloid angiopathy exist including the Dutch (E22Q) and Iowa (D23N) mutations of Abeta. Increasing evidence has associated cerebral microvascular amyloid deposition with neuroinflammation and dementia in these disorders. We recently established a transgenic mouse model (Tg-SwDI) that expresses human vasculotropic Dutch/Iowa mutant amyloid beta-protein precursor in brain. Tg-SwDI mice were shown to develop early-onset deposition of Abeta exhibiting high association with cerebral microvessels. Here we present quantitative temporal analysis showing robust and progressive accumulation of cerebral microvascular fibrillar Abeta accompanied by decreased cerebral vascular densities, the presence of apoptotic cerebral vascular cells, and cerebral vascular cell loss in Tg-SwDI mice. Abundant neuroinflammatory reactive astrocytes and activated microglia strongly associated with the cerebral microvascular fibrillar Abeta deposits. In addition, Tg-SwDI mouse brain exhibited elevated levels of the inflammatory cytokines interleukin-1beta and -6. Together, these studies identify the Tg-SwDI mouse as a unique model to investigate selective accumulation of cerebral microvascular amyloid and the associated neuroinflammation.     

Beta-site amyloid precursor protein-cleaving enzyme-1 (BACE1)-mediated changes of endogenous amyloid beta in wild-type and transgenic mice in vivo

Beta-site amyloid precursor protein-cleaving enzyme-1 (BACE1) initiates generation of amyloid beta (Abeta), a pathological hallmark of Alzheimer's disease. We investigated the impact of BACE1 protein level on endogenous Abeta. Endogenous Abeta and BACE1 protein levels were concurrently and significantly reduced during early life. However, Abeta levels were similar between BACE1 transgenic and wildtype mice. This suggests that BACE1 protein level has a minimal effect on the level of endogenous Abeta. Consequently, other factors must be involved in modulation of Abeta production in adult and ageing brain and investigation of such factors may yield therapeutic targets. Further, these results suggest that substantial inhibition of BACE1 in brain may be required for clinical benefit in Alzheimer's disease.     

Beta-site APP cleaving enzyme 1 (BACE1) is increased in remaining neurons in Alzheimer's disease brains

Alzheimer's disease (AD) is characterized by the extensive deposition of amyloid beta protein (Abeta) in the brain cortex. Abeta is produced from beta-amyloid precursor protein (APP) by beta-secretase and gamma-secretase. beta-Secretase has been identified as beta-site APP cleaving enzyme1 (BACE1). We produced rabbit polyclonal antibodies against the amino and the carboxyl terminals of BACE1. Using these antibodies, BACE1 was characterized in temporal lobe cortices by Western blotting and immunohistochemistry. Immunohistochemical studies employing anti-GFAP and anti-MAP2 antibodies as well as anti-BACE1 antibodies showed that BACE1 was expressed exclusively in neurons but not in glial cells. Brain samples were directly extracted by 0.5% SDS and analyzed by Western blotting and densitometer. Although the mean level of BACE1/mg protein in AD brains was not increased, the ratio of BACE1 to MAP2 or to NSE was significantly increased compared with that in control brains. Taken together, these findings suggest that those neurons that survive in AD brains might generate more BACE1 than normal neurons in control brains, indicating that increased BACE1 activity could be one of the causes of AD. This could justify the development of anti-BACE1 drugs for AD treatment.     

Age-dependent differential expression of BACE splice variants in brain regions of tg2576 mice

Plaques found in the brains of patients suffering from Alzheimer's disease (AD) mainly consist of beta-amyloid (Abeta), which is produced by sequential cleaving of amyloid precursor protein (APP) by two proteolytic enzymes, beta- and gamma-secretases. Any change in the fine balance between these enzymes and their substrate may contribute to the etio-pathogenesis of AD. Indeed, the protein level and enzymatic activity of beta-secretase (BACE), but not its mRNA level, were found elevated in brain areas of AD patients who suffer a high load of Abeta plaque formation. Similarly, increased BACE activity but no mRNA change was observed in a transgenic mouse model of AD, tg2576, in which over expression of the Swedish mutated human APP leads to Abeta plaque formation and learning deficits. Based on the recent demonstration of four BACE splice variants with different enzymatic activity, the discrepancy between BACE activity and mRNA expression may be explained by the altered BACE alternative splicing. To test this hypothesis, we studied the expression of all BACE splice variants in different brain areas of tg2576 mice at age of 4 months and 1 year old. We found developmental and regional differences between wild-type and tg2576 mice. Our results indicate that over expression of APP in tg2576 mice leads to the altered alternative splicing of BACE and the increase of its enzymatically more active splice variant (I-501).     

Mitochondria are a direct site of A beta accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression

Alzheimer's disease (AD) is a complex, neurodegenerative disease characterized by the impairment of cognitive function in elderly individuals. In a recent global gene expression study of APP transgenic mice, we found elevated expression of mitochondrial genes, which we hypothesize represents a compensatory response because of mitochondrial oxidative damage caused by the over-expression of mutant APP and/or amyloid beta (Abeta). We investigated this hypothesis in a series of experiments examining what forms of APP and Abeta localize to the mitochondria, and whether the presence of these species is associated with mitochondrial dysfunction and oxidative damage. Using immunoblotting, digitonin fractionation, immunofluorescence, and electron microscopy techniques, we found a relationship between mutant APP derivatives and mitochondria in brain slices from Tg2576 mice and in mouse neuroblastoma cells expressing mutant human APP. Further, to determine the functional relationship between mutant APP/Abeta and oxidative damage, we quantified Abeta levels, hydrogen peroxide production, cytochrome oxidase activity and carbonyl proteins in Tg2576 mice and age-matched wild-type (WT) littermates. Hydrogen peroxide levels were found to be significantly increased in Tg2576 mice when compared with age-matched WT littermates and directly correlated with levels of soluble Abeta in Tg2576 mice, suggesting that soluble Abeta may be responsible for the production of hydrogen peroxide in AD progression in Tg2576 mice. Cytochrome c oxidase activity was found to be decreased in Tg2576 mice when compared with age-matched WT littermates, suggesting that mutant APP and soluble Abeta impair mitochondrial metabolism in AD development and progression. An increase in hydrogen peroxide and a decrease in cytochrome oxidase activity were found in young Tg2576 mice, prior to the appearance of Abeta plaques. These findings suggest that early mitochondrially targeted therapeutic interventions may be effective in delaying AD progression in elderly individuals and in treating AD patients.     

Elimination of GD3 synthase improves memory and reduces amyloid-beta plaque load in transgenic mice

Gangliosides have been shown to be necessary for beta-amyloid (Abeta) binding and aggregation. GD3 synthase (GD3S) is responsible for biosynthesis of the b- and c-series gangliosides, including two of the four major brain gangliosides. We examined Abeta-ganglioside interactions in neural tissue from mice lacking the gene coding for GD3S (St8sia1), and in a double-transgenic (APP/PSEN1) mouse model of Alzheimer's disease cross-bred with GD3S-/- mice. In primary neurons and astrocytes lacking GD3S, Abeta-induced cell death and Abeta aggregation were inhibited. Like GD3S-/- and APP/PSEN1 double-transgenic mice, APP/PSEN1/GD3S-/- "triple-mutant" mice are indistinguishable from wild-type mice on casual examination. APP/PSEN1 double-transgenics exhibit robust impairments on a number of reference-memory tasks. In contrast, APP/PSEN1/GD3S-/- triple-mutant mice performed as well as wild-type control and GD3S-/- mice. Consistent with the behavioral improvements, both aggregated and unaggregated Abeta and associated neuropathology were almost completely eliminated in triple-mutant mice. These results suggest that GD3 synthase may be a novel therapeutic target to combat the cognitive deficits, amyloid plaque formation, and neurodegeneration that afflict Alzheimer's patients.     

Amyloid precursor protein-induced axonopathies are independent of amyloid-beta peptides

Overexpression of amyloid precursor protein (APP), as well as mutations in the APP and presenilin genes, causes rare forms of Alzheimer's disease (AD). These genetic changes have been proposed to cause AD by elevating levels of amyloid-beta peptides (Abeta), which are thought to be neurotoxic. Since overexpression of APP also causes defects in axonal transport, we tested whether defects in axonal transport were the result of Abeta poisoning of the axonal transport machinery. Because directly varying APP levels also alters APP domains in addition to Abeta, we perturbed Abeta generation selectively by combining APP transgenes in Drosophila and mice with presenilin-1 (PS1) transgenes harboring mutations that cause familial AD (FAD). We found that combining FAD mutant PS1 with FAD mutant APP increased Abeta42/Abeta40 ratios and enhanced amyloid deposition as previously reported. Surprisingly, however, this combination suppressed rather than increased APP-induced axonal transport defects in both Drosophila and mice. In addition, neuronal apoptosis induced by expression of FAD mutant human APP in Drosophila was suppressed by co-expressing FAD mutant PS1. We also observed that directly elevating Abeta with fusions to the Familial British and Danish Dementia-related BRI protein did not enhance axonal transport phenotypes in APP transgenic mice. Finally, we observed that perturbing Abeta ratios in the mouse by combining FAD mutant PS1 with FAD mutant APP did not enhance APP-induced behavioral defects. A potential mechanism to explain these findings was suggested by direct analysis of axonal transport in the mouse, which revealed that axonal transport or entry of APP into axons is reduced by FAD mutant PS1. Thus, we suggest that APP-induced axonal defects are not caused by Abeta.     

Hippocampal neuron loss exceeds amyloid plaque load in a transgenic mouse model of Alzheimer's disease

According to the "amyloid hypothesis of Alzheimer's disease," beta-amyloid is the primary driving force in Alzheimer's disease pathogenesis. Despite the development of many transgenic mouse lines developing abundant beta-amyloid-containing plaques in the brain, the actual link between amyloid plaques and neuron loss has not been clearly established, as reports on neuron loss in these models have remained controversial. We investigated transgenic mice expressing human mutant amyloid precursor protein APP751 (KM670/671NL and V717I) and human mutant presenilin-1 (PS-1 M146L). Stereologic and image analyses revealed substantial age-related neuron loss in the hippocampal pyramidal cell layer of APP/PS-1 double-transgenic mice. The loss of neurons was observed at sites of Abeta aggregation and surrounding astrocytes but, most importantly, was also clearly observed in areas of the parenchyma distant from plaques. These findings point to the potential involvement of more than one mechanism in hippocampal neuron loss in this APP/PS-1 double-transgenic mouse model of Alzheimer's disease.