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Background: Clinical studies of β-amyloid (Aβ) immunotherapy in Alzheimer's disease (AD) patients have demonstrated reduction of central Aβ plaque by positron emission tomography (PET) imaging and the appearance of amyloid-related imaging abnormalities (ARIA). To better understand the relationship between ARIA and the pathophysiology of AD, we undertook a series of studies in PDAPP mice evaluating vascular alterations in the context of central Aβ pathology and after anti-Aβ immunotherapy., Methods: We analyzed PDAPP mice treated with either 3 mg/kg/week of 3D6, the murine form of bapineuzumab, or isotype control antibodies for periods ranging from 1 to 36 weeks and evaluated the vascular alterations in the context of Aβ pathology and after anti-Aβ immunotherapy. The number of mice in each treatment group ranged from 26 to 39 and a total of 345 animals were analyzed., Results: The central vasculature displayed morphological abnormalities associated with vascular Aβ deposits. Treatment with 3D6 antibody induced clearance of vascular Aβ that was spatially and temporally associated with a transient increase in microhemorrhage and in capillary Aβ deposition. Microhemorrhage resolved over a time period that was associated with a recovery of vascular morphology and a decrease in capillary Aβ accumulation., Conclusions: These data suggest that vascular leakage events, such as microhemorrhage, may be related to the removal of vascular Aβ. With continued treatment, this initial susceptibility period is followed by restoration of vascular morphology and reduced vulnerability to further vascular leakage events. The data collectively suggested a vascular amyloid clearance model of ARIA, which accounts for the currently known risk factors for the incidence of ARIA in clinical studies., (Copyright © 2013. Published by Elsevier Inc.)

Several anti-amyloid β (Aβ) antibodies are under evaluation for the treatment of Alzheimer's disease (AD). Clinical studies using the N-terminal-directed anti-Aβ antibody bapineuzumab have demonstrated reduced brain PET-Pittsburg-B signals, suggesting the reduction of Aβ plaques, and reduced levels of total and phosphorylated tau protein in the CSF of treated AD patients. Preclinical studies using 3D6 (the murine form of bapineuzumab) have demonstrated resolution of Aβ plaque and vascular burdens, neuritic dystrophy, and preservation of synaptic density in the transgenic APP mouse models. In contrast, few studies have evaluated the direct interaction of this antibody with synaptotoxic soluble Aβ species. In the current report, we demonstrated that 3D6 binds to soluble, synaptotoxic assemblies of Aβ(1-42) and prevents multiple downstream functional consequences in rat hippocampal neurons including changes in glutamate AMPA receptor trafficking, AD-type tau phosphorylation, and loss of dendritic spines. In vivo, we further demonstrated that 3D6 prevents synaptic loss and acutely reverses the behavioral deficit in the contextual fear conditioning task in transgenic mouse models of AD, two endpoints thought to be linked to synaptotoxic soluble Aβ moieties. Importantly C-terminal anti-Aβ antibodies were ineffective on these endpoints. These results, taken with prior studies, suggest that N-terminal anti-Aβ antibodies effectively interact with both soluble and insoluble forms of Aβ and therefore appear particularly well suited for testing the Aβ hypothesis of AD.

Two experiments were conducted to investigate the possibility of faster forgetting by PDAPP mice (a well-established model of Alzheimer's disease as reported by Games and colleagues in an earlier paper). Experiment 1, using mice aged 13-16 mo, confirmed the presence of a deficit in a spatial reference memory task in the water maze by hemizygous PDAPP mice relative to littermate controls. However, after overtraining to a criterion of equivalent navigational performance, a series of memory retention tests revealed faster forgetting in the PDAPP group. Very limited retraining was sufficient to reinstate good memory in both groups, indicating that their faster forgetting may be due to retrieval failure rather than trace decay. In Experiment 2, 6-mo-old PDAPP and controls were required to learn each of a series of spatial locations to criterion with their memory assessed 10 min after learning each location. No memory deficit was apparent in the PDAPP mice initially, but a deficit built up through the series of locations suggestive of increased sensitivity to interference. Faster forgetting and increased interference may each reflect a difficulty in accessing memory traces. This interpretation of one aspect of the cognitive deficit in human mutant APP mice has parallels to deficits observed in patients with Alzheimer's disease, further supporting the validity of transgenic models of the disease.

The aspartyl protease beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) initiates processing of amyloid precursor protein (APP) into amyloid beta (Abeta) peptide, the major component of Alzheimer disease (AD) plaques. To determine the role that BACE1 plays in the development of Abeta-driven AD-like pathology, we have crossed PDAPP mice, a transgenic mouse model of AD overexpressing human mutated APP, onto mice with either a homozygous or heterozygous BACE1 gene knockout. Analysis of PDAPP/BACE(-/-) mice demonstrated that BACE1 is absolutely required for both Abeta generation and the development of age-associated plaque pathology. Furthermore, synaptic deficits, a neurodegenerative pathology characteristic of AD, were also reversed in the bigenic mice. To determine the extent of BACE1 reduction required to significantly inhibit pathology, PDAPP mice having a heterozygous BACE1 gene knock-out were evaluated for Abeta generation and for the development of pathology. Although the 50% reduction in BACE1 enzyme levels caused only a 12% decrease in Abeta levels in young mice, it nonetheless resulted in a dramatic reduction in Abeta plaques, neuritic burden, and synaptic deficits in older mice. Quantitative analyses indicate that brain Abeta levels in young APP transgenic mice are not the sole determinant for the changes in plaque pathology mediated by reduced BACE1. These observations demonstrate that partial reductions of BACE1 enzyme activity and concomitant Abeta levels lead to dramatic inhibition of Abeta-driven AD-like pathology, making BACE1 an excellent target for therapeutic intervention in AD.

Neuropathologic examination of 3 patients with Alzheimer disease in the Elan Pharmaceuticals trial using antibodies specific for different Abeta species showed in one case, 4 months after the immunization, evidence of a stage of active plaque clearance with "moth-eaten" plaques and abundant Abeta phagocytosis by microglia. At 1 to 2 years after immunization, 2 cases showed extensive areas cleared of plaques (69% and 86% of the temporal cortex was plaque-free). Cortex cleared of plaques in all 3 cases had a characteristic constellation of features, including a very low plaque burden, sparse residual dense plaque cores, and phagocytosed Abeta within microglia. There was resolution of tau-containing dystrophic neurites, although other features of tau pathology (tangles and neuropil threads) remained and cerebral amyloid angiopathy persisted. Although most antibodies generated by Abeta42 immunization in humans bind the intact N-terminus, immunohistochemistry with specific antibodies showed clearance of all major species of Abeta (Abeta40, Abeta42, and N-terminus truncated Abeta). Abeta immunotherapy can clear all Abeta species from the cortex. However, if it is to be used for treatment of established Alzheimer disease, then the residual tau pathology and cerebral amyloid angiopathy require further study.

Immunotherapy targeting the amyloid beta (Abeta) peptide is a novel therapy under investigation for the treatment of Alzheimer's disease (AD). A clinical trial using Abeta(1-42) (AN1792) as the immunogen was halted as a result of development of meningoencephalitis in a small number of patients. The cytokine TGF-beta1 is a key modulator of immune responses that is increased in the brain in AD. We show here that local overexpression of TGF-beta1 in the brain increases both meningeal and parenchymal T lymphocyte number. Furthermore, TGF-beta1 overexpression in a mouse model for AD [amyloid precursor protein (APP) mice] leads to development of additional T cell infiltrates when mice were immunized at a young but not old age with AN1792. Notably, only mice overproducing both Abeta (APP mice) and TGF-beta1 experienced a rise in T lymphocyte number after immunization. One-third of infiltrating T cells were CD4 positive. We did not observe significant differences in B lymphocyte numbers in any of the genotypes or treatment groups. These results demonstrate that TGF-beta1 overproduction in the brain can promote T cell infiltration, in particular after Abeta(1-42) immunization. Likewise, levels of TGF-beta1 or other immune factors in brains of AD patients may influence the response to Abeta(1-42) immunization.

Abnormal subgranular zone (SGZ) neurogenesis is proposed to contribute to Alzheimer's disease (AD)-related decreases in hippocampal function. Our goal was to examine hippocampal neurogenesis in the PDAPP mouse, a model of AD with age-dependent accumulation of amyloid-beta(42) (Abeta(42))-containing plaques that is well studied with regard to AD therapies. A secondary goal was to determine whether altered neurogenesis in the PDAPP mouse is associated with abnormal maturation or number of mature cells. A tertiary goal was to provide insight into why hippocampal neurogenesis appears to be increased in AD post-mortem tissue and decreased in most AD mouse models. We report an age-dependent decrease in SGZ proliferation in homozygous PDAPP mice. At 1 year of age, PDAPP mice also had new dentate gyrus granule neurons with abnormal maturation and fewer dying cells relative to control mice. In contrast to decreased SGZ cell birth, PDAPP mice had increased birth of immature neurons in the outer portion of the granule cell layer (oGCL), providing insight into why some studies link AD with increased neurogenesis. However, these ectopic oGCL cells were still rare compared with SGZ proliferating cells, emphasizing that the primary characteristic of PDAPP mice is decreased neurogenesis. The decrease in SGZ neurogenesis was not associated with an age-dependent loss of dentate granule neurons. The altered neurogenesis in the PDAPP mouse may contribute to the age-related cognitive deficits reported in this model of AD and may be a useful adjunct target for assessing the impact of AD therapies., (J. Comp. Neurol. 495:70-83, 2006. (c) 2006 Wiley-Liss, Inc.)

Progress in understanding and treating Alzheimer's disease (AD) has been tremendously bolstered by the era of transgenic models of AD. The identification of disease-causing mutations in proteins such as amyloid-beta precursor protein (betaAPP) and presenilin1 (PS1), together with the discovery of other high risk factors (e.g., Apolipoprotein E4), as well as pathogenic mutations in the tau protein has led to the creation of several transgenic mice, including those expressing bi- and tri-genic constructs. Each model has unique pathologies that provide insights into disease mechanisms and interactive features of neuropathologic cascades. More importantly, therapeutic hypotheses are now testable in a manner unheard of less than 15 years ago. The wealth of new approaches currently in clinical and preclinical evaluations can be directly attributed to the impact of these animals on our ability to model relevant aspects of the disease. As a result, we may see containment or even the elimination of AD in the near future as a direct consequence of these advances.

Alzheimer's disease is characterized by neuronal degeneration in the cerebral cortex and hippocampus and subcortical neuronal degeneration in such nuclei as the locus coeruleus (LC). Transgenic mice overexpressing mutant human amyloid precursor protein V717F, PDAPP mice, develop several Alzheimer's disease-like lesions. The present study sought to determine whether there is also loss of LC noradrenergic neurons or evidence of degenerative changes in these animals. PDAPP hemizygous and wild-type littermate control mice were examined at 23 months of age, at a time when there are numerous amyloid-beta (Abeta) plaques in the neocortex and hippocampus. Tissue sections were stained immunohistochemically with an antibody against tyrosine hydroxylase (TH) to identify LC neurons. Computer imaging procedures were used to count the TH-immunoreactive somata in sections through the rostral-caudal extent of the nucleus. There was no loss of LC neurons in the hemizygous mice. In a second experiment, homozygous PDAPP and wild-type mice were examined, at 2 months and 24 months of age. Again there was no age-related loss of neurons in the homozygous animals. In the portion of the LC where neurons reside that project to the cortex and hippocampus, however, the neurons were decreased in size selectively in the 24-month-old transgenic animals. These data indicate that overt LC cell loss does not occur following abundant overexpression of Abeta peptide. However, the selective size reduction of the LC neuronal population projecting to cortical and hippocampal regions containing Abeta-related neuropathology implies that these cells may be subjected to a retrograde-mediated stress., (Copyright 2005 Wiley-Liss, Inc.)

Alzheimer's disease neuropathology is characterized by key features that include the deposition of the amyloid beta peptide (Abeta) into plaques, the formation of neurofibrillary tangles, and the loss of neurons and synapses in specific brain regions. The loss of synapses, and particularly the associated presynaptic vesicle protein synaptophysin in the hippocampus and association cortices, has been widely reported to be one of the most robust correlates of Alzheimer's disease-associated cognitive decline. The beta-amyloid hypothesis supports the idea that Abeta is the cause of these pathologies. However, the hypothesis is still controversial, in part because the direct role of Abeta in synaptic degeneration awaits confirmation. In this study, we show that Abeta reduction by active or passive Abeta immunization protects against the progressive loss of synaptophysin in the hippocampal molecular layer and frontal neocortex of a transgenic mouse model of Alzheimer's disease. These results, substantiated by quantitative electron microscopic analysis of synaptic densities, strongly support a direct causative role of Abeta in the synaptic degeneration seen in Alzheimer's disease and strengthen the potential of Abeta immunotherapy as a treatment approach for this disease.

The authors report a patient with Alzheimer disease (AD) without encephalitis who was immunized with AN-1792 (an adjuvanted formulation of Abeta-42). There were no amyloid plaques in the frontal cortex and abundant Abeta-immunoreactive macrophages, but tangles and amyloid angiopathy were present. The white matter appeared normal and minimal lymphocytic infiltration in the leptomeninges was observed. This case illustrates the effects of an Abeta-based immunization on AD pathogenesis in the absence of overt meningoencephalitis and leukoencephalopathy.

Transgenic mice mimicking certain features of Alzheimer's disease (AD)-pathology, namely amyloid plaques and neurofibrillary tangles, have been developed in an effort to better understand the mechanism leading to the formation of these characteristic cerebral lesions. More recently, these animal models have been widely used to investigate emergent therapies aimed at the reduction of the cerebral amyloid load. Several studies have shown that immunotherapy targeting the amyloid peptide (Abeta) is efficacious at clearing the amyloid plaques or preventing their formation, and at reducing the memory/behavior impairment observed in these animals. In AD, different types of plaques likely have different pathogenic significance, and further characterization of plaque pathology in the PDAPP transgenic mice would enhance the evaluation of potential therapeutics. In the present study, a morphological classification of amyloid plaques present in the brains of PDAPP mice was established by using Thioflavin-S staining. Neuritic dystrophy associated with amyloid plaques was also investigated. Finally, the efficacy of passive immunization with anti-Abeta antibodies on the clearance of Thio-S positive amyloid plaques was studied. Our results show that distinct morphological types of plaques are differentially cleared depending upon the isotype of the antibody.

Transgenic mice overexpressing mutant human amyloid precursor protein (PDAPP mice) develop several Alzheimer's disease (AD)-like lesions including an age-related accumulation of amyloid-beta (Abeta)-containing neuritic plaques. Although aged, heterozygous PDAPP mice also exhibit synaptic and glial cell changes characteristic of AD pathology, no evidence of widespread neuronal loss has been observed. The present study sought to determine whether homozygous PDAPP mice, which express very high levels of Abeta peptide, exhibit AD-like cholinergic degenerative changes, and whether the changes parallel the deposition of Abeta plaques. Mice were examined at 2 and 4 months and at 1 and 2 years of age. There was an age-related increase in the density of Abeta plaques in the cortex and hippocampus of the PDAPP animals; at 4 months of age there were very few plaques, and at 2 years there was a very high density of plaques. There was an age-related reduction in the density of cholinergic nerve terminals in the cerebral cortex; at 2 months there was a normal density of nerve terminals, but as early as age 4 months there was an approximately 50% reduction. However, at age 2 years there was no difference in the number or size of basal forebrain cholinergic somata compared with 2-month-old PDAPP mice. These data indicated that the homozygous PDAPP mouse exhibits cholinergic nerve terminal degenerative pathology and that the cortical neurodegenerative changes occur before the deposition of Abeta-containing neuritic plaques., (Copyright 2003 Wiley-Liss, Inc.)

Various transgenic mouse models of Alzheimer's disease (AD) have been developed that overexpress mutant forms of amyloid precursor protein in an effort to elucidate more fully the potential role of beta-amyloid (A beta) in the etiopathogenesis of the disease. The present study represents the first complete 3D reconstruction of A beta in the hippocampus and entorhinal cortex of PDAPP transgenic mice. A beta deposits were detected by immunostaining and thioflavin fluorescence, and quantified by using high-throughput digital image acquisition and analysis. Quantitative analysis of amyloid load in hippocampal subfields showed a dramatic increase between 12 and 15 months of age, with little or no earlier detectable deposition. Three-dimensional reconstruction in the oldest brains visualized previously unrecognized sheets of A beta coursing through the hippocampus and cerebral cortex. In contrast with previous hypotheses, compact plaques form before significant deposition of diffuse A beta, suggesting that different mechanisms are involved in the deposition of diffuse amyloid and the aggregation into plaques. The dentate gyrus was the hippocampal subfield with the greatest amyloid burden. Sublaminar distribution of A beta in the dentate gyrus correlated most closely with the termination of afferent projections from the lateral entorhinal cortex, mirroring the selective vulnerability of this circuit in human AD. This detailed temporal and spatial analysis of A beta and compact amyloid deposition suggests that specific corticocortical circuits express selective, but late, vulnerability to the pathognomonic markers of amyloid deposition, and can provide a basis for detecting prior vulnerability factors.

Transgenic PDAPP mice, which express a disease-linked isoform of the human amyloid precursor protein, exhibit CNS pathology that is similar to Alzheimer's disease. In an age-dependent fashion, the mice develop plaques containing beta-amyloid peptide (Abeta) and exhibit neuronal dystrophy and synaptic loss. It has been shown in previous studies that pathology can be prevented and even reversed by immunization of the mice with the Abeta peptide. Similar protection could be achieved by passive administration of some but not all monoclonal antibodies against Abeta. In the current studies we sought to define the optimal antibody response for reducing neuropathology. Immune sera with reactivity against different Abeta epitopes and monoclonal antibodies with different isotypes were examined for efficacy both ex vivo and in vivo. The studies showed that: (i) of the purified or elicited antibodies tested, only antibodies against the N-terminal regions of Abeta were able to invoke plaque clearance; (ii) plaque binding correlated with a clearance response and neuronal protection, whereas the ability of antibodies to capture soluble Abeta was not necessarily correlated with efficacy; (iii) the isotype of the antibody dramatically influenced the degree of plaque clearance and neuronal protection; (iv) high affinity of the antibody for Fc receptors on microglial cells seemed more important than high affinity for Abeta itself; and (v) complement activation was not required for plaque clearance. These results indicate that antibody Fc-mediated plaque clearance is a highly efficient and effective process for protection against neuropathology in an animal model of Alzheimer's disease.

Transgenic (Tg) mouse models overexpressing amyloid precursor protein (APP) develop senile plaques similar to those found in Alzheimer's disease in an age-dependent manner. Recent reports demonstrated that immunotherapy is effective at preventing or removing amyloid-beta deposits in the mouse models. To characterize the mechanisms involved in clearance, we used antibodies of either IgG1 (10d5) or IgG2b (3d6) applied directly to the brains of 18-month-old Tg2576 or 20-month-old PDAPP mice. Both 10d5 and 3d6 led to clearance of 50% of diffuse amyloid deposits in both animal models within 3 d. Fc receptor-mediated clearance has been shown to be important in an ex vivo assay showing antibody-mediated clearance of plaques by microglia. We now show, using in vivo multiphoton microscopy, that FITC-labeled F(ab')2 fragments of 3d6 (which lack the Fc region of the antibody) also led to clearance of 45% of the deposits within 3 d, similar to the results obtained with full-length 3d6 antibody. This result suggests that direct disruption of plaques, in addition to Fc-dependent phagocytosis, is involved in the antibody-mediated clearance of amyloid-beta deposits in vivo. Dense-core deposits that were not cleared were reduced in size by approximately 30% with full-length antibodies and F(ab')2 fragments 3 d after a topical treatment. Together, these results indicate that clearance of amyloid deposits in vivo may involve, in addition to Fc-dependent clearance, a non-Fc-mediated disruption of plaque structure.

Fluorodeoxyglucosepositron emission tomography (PET) studies find that persons with Alzheimer's disease have preferential reductions in posterior cingulate activity. Using fluorodeoxyglucose autoradiography, we found that transgenic mice overexpressing a mutant form of the human amyloid precursor protein have preferentially reduced activity in the same region, providing a potential brain imaging indicator of Alzheimer's disease in these animals. In this study, we considered the feasibility of using in vivo imaging techniques, such as PET, to detect this reduction despite their limitations in spatial resolution. Autoradiographic measurements of posterior cingulate activity were remeasured in the previously studied PDAPP transgenic and littermate control mice after the images were filtered to lower spatial resolutions. We continued to detect significantly lower cingulate activity in the transgenic mice when the images were blurred to 0.50 mm, failed to detect significantly abnormal activity when the images were blurred to 0.75 mm, and, indeed, found significantly higher activity when the images were blurred to 1.0 mm. Reversal in direction of the abnormality appears attributable to a previously observed truncation in the corpus callosum in PDAPP mice. With the possible exception of future in vivo imaging techniques that have a spatial resolution greater than or equal to 0.50 mm and high sensitivity, noninvasive functional brain imaging techniques like PET may not be suitable for detecting declines in regional activity in PDAPP mice. It remains possible that these imaging techniques will prove useful in transgenic mouse lines that do not exhibit the same morphological abnormalities in neighboring white matter regions., (2002 Elsevier Science (USA).)

Recent studies demonstrated that mice overexpressing the human mutant beta-amyloid precursor protein (hbetaAPP; PDAPP mice) show age-independent and age-related deficits in spatial learning. We used behavioral and electrophysiological techniques to determine in young and aged PDAPP mice whether deficits in spatial learning also involve alterations in sleep-wake states, thermoregulation and motor activity. Consistent with earlier studies, young PDAPP mice exhibited selective age-independent deficits using spatial, but not random and serial strategies in the circular maze. Aged PDAPP mice exhibited deficits using all search strategies. The core body temperature (Tb) in young and aged PDAPP mice was significantly lower than in age-matched non-transgenic (non-Tg) littermates. During the dark period, the motor activity (LMA) was significantly increased in young PDAPP mice, but not in aged PDAPP mice. During the light period, young PDAPP mice showed a reduction in the generation of rapid-eye-movement (REM) sleep. In contrast, aged PDAPP mice exhibited a reduction in the amount of time spent in W and an increase in SWS during the light period. Aged PDAPP mice also showed an increase in the amount of time spent in W and a reduction in REM sleep during the dark period. Our findings support previous reports indicating deficits in spatial learning in young and aged PDAPP mice. These data also suggest that PDAPP mice exhibit age-independent and age-related deficits in neural mechanisms regulating visuospatial learning, the total amount and the circadian distribution of sleep-wake states, thermoregulation and motor activity.

Research over the past ten years on Alzheimer's disease has pursued many opportunities. Notable amongst the various approaches are efforts related to the "amyloid hypothesis." This hypothesis posits that the beta amyloid peptide causes the extensive neuropathology and clinical decline associated with the disease. Extensive research in this area has shown that the beta amyloid peptide is produced by proteases termed "secretases" and it has been shown that blockade of secretase functions reduce the amount of beta amyloid peptide produced. An additional approach to reduce beta amyloid, through an increase in clearance mechanisms, is to immunize with the peptide itself and induce an antibody response. The specifically elicited antibodies then bind to and stimulate clearance of the peptide from the brain. These findings have stimulated several approaches to develop novel therapeutic strategies to treat Alzheimer's disease that either are about or have entered the clinic.

Persons with Alzheimer's disease (AD) have progressive reductions in the relative sizes of the corpus callosum and hippocampus. Homozygotic PDAPP transgenic mice over-expressing a mutant form of the human amyloid precursor protein have more pronounced reductions in these regions, which are apparent prior to the deposition of amyloid plaques and do not progress with advancing age. The length of the corpus callosum was reduced by two-thirds, the fornix commissure was negligible, and the hippocampal volume was reduced by one-third, suggesting a massive disconnection between the cerebral hemispheres and the hippocampi in PDAPP mice. These findings, which might account for the early, nonprogressive behavioral abnormalities observed in these animals, have implications for the study of AD.

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.

Overexpression of mutated human amyloid precursor protein (hAPP717V-->F) under control of the platelet-derived growth factor promoter (PDAPP minigene) in transgenic (tg) mice results in plaque formation and astroglial activation similar to Alzheimer disease (AD). However, the extent of the neurofibrillary pathology in this model is less understood. In order to determine if these mice develop AD-like neurofibrillary pathology, vibratome sections from PDAPP tg mice (4- to 20-months-old) were immunolabeled with antibodies against phosphorylated tau (AT8) and phosphorylated neurofilaments (SMI 312, TA51), and analyzed by laser scanning confocal and electron microscopy. Phosphorylated neurofilament-immunoreactive dystrophic neurites in plaques were first seen in mice at 10 to 12 months of age, while phosphorylated tau-immunoreactive dystrophic neurites were observed after 14 months of age. Immunoelectron microscopic analysis revealed that phosphorylated neurofilament immunoreactivity was diffusely distributed along filamentous aggregates (12-15 nm in diameter) in the plaque dystrophic neurites, and occasionally in neuronal cell bodies. In contrast, phosphorylated tau immunoreactivity was observed as clusters distributed along filamentous structures accumulating in the dystrophic neurites and around neurotubules in the axons. However, no paired helical filaments were observed. Taken together, these studies indicate that the PDAPP tg model recapitulates early cytoskeletal pathology similar to that observed in AD.

Mice that overexpress the human mutant amyloid precursor protein (hAPP) show learning deficits, but the apparent lack of a relationship between these deficits and the progressive beta-amyloid plaque formation that the hAPP mice display is puzzling. In the water maze, hAPP mice are impaired before and after amyloid plaque deposition. Here we show, using a new water-maze training protocol, that PDAPP mice also exhibit a separate age-related deficit in learning a series of spatial locations. This impairment correlates with beta-amyloid plaque burden and is shown in both cross-sectional and longitudinal experimental designs. Cued navigation and object-recognition memory are normal. These findings indicate that A beta overexpression and/or A beta plaques are associated with disturbed cognitive function and, importantly, suggest that some but not all forms of learning and memory are suitable behavioural assays of the progressive cognitive deficits associated with Alzheimer's-disease-type pathologies.

One hallmark of Alzheimer disease is the accumulation of amyloid beta-peptide in the brain and its deposition as plaques. Mice transgenic for an amyloid beta precursor protein (APP) mini-gene driven by a platelet-derived (PD) growth factor promoter (PDAPP mice), which overexpress one of the disease-linked mutant forms of the human amyloid precursor protein, show many of the pathological features of Alzheimer disease, including extensive deposition of extracellular amyloid plaques, astrocytosis and neuritic dystrophy. Active immunization of PDAPP mice with human amyloid beta-peptide reduces plaque burden and its associated pathologies. Several hypotheses have been proposed regarding the mechanism of this response. Here we report that peripheral administration of antibodies against amyloid beta-peptide, was sufficient to reduce amyloid burden. Despite their relatively modest serum levels, the passively administered antibodies were able to enter the central nervous system, decorate plaques and induce clearance of preexisting amyloid. When examined in an ex vivo assay with sections of PDAPP or Alzheimer disease brain tissue, antibodies against amyloid beta-peptide triggered microglial cells to clear plaques through Fc receptor-mediated phagocytosis and subsequent peptide degradation. These results indicate that antibodies can cross the blood-brain barrier to act directly in the central nervous system and should be considered as a therapeutic approach for the treatment of Alzheimer disease and other neurological disorders.

While transgenic mice have great promise in the study of Alzheimer's disease (AD), uncertainties remain about the extent to which they provide a model of the disorder or the best way to characterize disease progression. Using fluorodeoxyglucose (FDG) autoradiography, we found that transgenic mice over-expressing a mutant form of the human amyloid precursor protein have preferentially and progressively reduced activity in the posterior cingulate cortex and relatively spared activity in visual cortex, sensorimotor cortex, cerebellum and brain stem, a pattern previously demonstrated in FDG PET studies of persons with Alzheimer's disease, Brain imaging of posterior cingulate activity could provide an indicator of AD in suitable animals, helping to clarify disease mechanisms and screen candidate treatments.

Immunohistochemistry was used to analyse 18- and 26-month-old transgenic mice overexpressing the human beta-amyloid precursor protein under the platelet-derived growth factor-beta promoter with regard to presence and distribution of neuropeptides. In addition, antisera/antibodies to tyrosine hydroxylase, acetylcholinesterase, amyloid peptide, glial fibrillary acidic protein and microglial marker OX42 were used. These mice have been reported to exhibit extensive amyloid plaques in the hippocampus and cortex [Masliah et al. (1996) J. Neurosci. 16, 5795-5811]. The most pronounced changes were related to neuropeptides, whereas differences between wild-type and transgenic mice were less prominent with regard to tyrosine hydroxylase and acetylcholinesterase. The main findings were of two types; (i) involvement of peptide-containing neurites in amyloid beta-peptide positive plaques, and (ii) more generalized changes in peptide levels in specific layers, neuron populations and/or subregions in the hippocampal formation and ventral cortices. In contrast, the parietal and auditory cortices were comparatively less affected. The peptide immunoreactivities most strongly involved, both in plaques and in the generalized changes, were galanin, neuropeptide Y, cholecystokinin and enkephalin. This study shows that there is considerable variation both with regard to plaque load and peptide expression even among homozygotes of the same age. The most pronounced changes, predominantly increased peptide levels, were observed in two 26-month-old homozygous mice, for example, galanin-, enkephalin- and cholecystokinin-like immunoreactivities in stratum lacunosum moleculare, and galanin, neuropeptide Y, enkephalin and dynorphin in mossy fibers. Many peptides also showed elevated levels in the ventral cortices. However, decreases were also observed. Thus, galanin-like immunoreactivity could not any longer be detected in the diffusely distributed (presumably noradrenergic) fiber network in all hippocampal and cortical layers, and dynorphin-like immunoreactivity was decreased in stratum moleculare, cholecystokinin-like immunoreactivity in mossy fibers and substance P-like immunoreactivity in fibers around granule cells. The significance of generalized peptide changes is at present unclear. For example, the increase in the mainly inhibitory peptides galanin, neuropeptide Y, enkephalin and dynorphin and the decrease in the mainly excitatory peptide cholecystokinin in mossy fibers (and of substance P fibers around granule cells) indicate a shift in balance towards inhibition of the input to the CA3 pyramidal cell layer. Moreover, it may be speculated that the increase in levels of some of the peptides represents a reaction to nerve injury with the aim to counteract, in different ways, the consequences of injury, for example by exerting trophic actions. Further studies will be needed to establish to what extent these changes are typical for Alzheimer mouse models in general or are associated with the V717F mutation and/or the platelet-derived growth factor-beta promoter.

In AD certain brain structures contain a pathological density of A beta protein deposited into plaques. The effect of genetic mutations found in early onset AD patients was an overproduction of A beta 42, strongly suggesting that overproduction of A beta 42 is associated with AD. We hypothesized that an immunological response to A beta 42 might alter its turnover and metabolism. Young PDAPP transgenic mice were immunized with A beta 1-42, which essentially prevented amyloid deposition; astrocytosis was dramatically reduced and there was reduction in A beta-induced inflammatory response as well. A beta 1-42 immunization also appeared to arrest the progression of amyloidosis in older PDAPP mice. A beta immunization appears to increase clearance of amyloid plaques, and may therefore be a novel and effective approach for the treatment of AD.

Amyloid-beta peptide (Abeta) seems to have a central role in the neuropathology of Alzheimer's disease (AD). Familial forms of the disease have been linked to mutations in the amyloid precursor protein (APP) and the presenilin genes. Disease-linked mutations in these genes result in increased production of the 42-amino-acid form of the peptide (Abeta42), which is the predominant form found in the amyloid plaques of Alzheimer's disease. The PDAPP transgenic mouse, which overexpresses mutant human APP (in which the amino acid at position 717 is phenylalanine instead of the normal valine), progressively develops many of the neuropathological hallmarks of Alzheimer's disease in an age- and brain-region-dependent manner. In the present study, transgenic animals were immunized with Abeta42, either before the onset of AD-type neuropathologies (at 6 weeks of age) or at an older age (11 months), when amyloid-beta deposition and several of the subsequent neuropathological changes were well established. We report that immunization of the young animals essentially prevented the development of beta-amyloid-plaque formation, neuritic dystrophy and astrogliosis. Treatment of the older animals also markedly reduced the extent and progression of these AD-like neuropathologies. Our results raise the possibility that immunization with amyloid-beta may be effective in preventing and treating Alzheimer's disease.

In summary, PDAPP mice overexpressing a mutation associated with some cases of familial early-onset AD express several of the major pathological hallmarks associated with AD. Amyloid plaques in PDAPP mice appear quite similar to A beta deposits in AD as shown by a variety of different antibodies and stains, and are of both the diffuse and compacted varieties. Additionally, a subset of these amyloid plaques appear to be neuritic plaques. Neurodegenerative changes, including the loss of synaptic and dendritic proteins, abnormal phosphorylation of cytoskeletal elements, subcellular degenerative changes, and the deposition of lysosomal and acute phase proteins has also been seen in PDAPP mouse brains. Reactive astrocytosis and microgliosis have also been observed in association with the amyloid plaques in the PDAPP mice. No neurofibrillary tangles or paired helical filaments have been found in the mice to date. It remains unknown whether mice are capable of generating these in a manner comparable to AD in less than two years. Extensive behavioral analyses are currently being performed in these mice, and preliminary results indicate that the PDAPP mice are significantly impaired on a variety of different learning and memory tests. In conclusion, the PDAPP mouse model doesn't display all the pathological hallmarks of AD, but it does display most of them in a robust manner that increases with age and gene dosage. Therefore, this transgenic model provides evidence that alterations in APP processing and A beta production can result in AD-like neuropathology, can contribute to a mechanistic understanding of AD (since examination of AD brains yields a static view, and we are unable to view the development of various pathological changes), as well as providing an useful animal model for the testing of various therapeutic interventions directed towards specific aspects of the neurodegenerative process.

The PDAPP transgenic mouse, which overexpresses human amyloid precursor protein (APP717V-->F), has been shown to develop much of the pathology associated with Alzheimer disease. In this report, levels of APP and its amyloidogenic metabolites were measured in brain regions of transgenic mice between 4 and 18 months of age. While absolute levels of APP expression likely contribute to the rate of amyloid beta-peptide (Abeta) deposition, regionally specific factors also seem important, as homozygotic mice express APP levels in pathologically unaffected regions in excess of that measured in certain amyloid plaque-prone regions of heterozygotic mice. Regional levels of APP and APP-beta were nearly constant at all ages, while A beta levels dramatically and predictably increased in brain regions undergoing histochemically confirmed amyloidosis, most notably in the cortex and hippocampus. In hippocampus, A beta concentrations increase 17-fold between the ages of 4 and 8 months, and by 18 months of age are over 500-fold that at 4 months, reaching an average level in excess of 20 nmol of A beta per g of tissue. A beta1-42 constitutes the vast majority of the depositing A beta species. The similarities observed between the PDAPP mouse and human Alzheimer disease with regard to A beta42 deposition occurring in a temporally and regionally specific fashion further validate the use of the model in understanding processes related to the disease.

Overexpression of mutated human amyloid precursor protein (hAPP717V-->F) under control of platelet-derived growth factor promoter (PDAPP minigene) in transgenic (tg) mice results in neurodegenerative changes similar to Alzheimer's disease (AD). To clarify the pathology of these mice, we studied images derived from laser scanning confocal and electron microscopy and performed comparisons between PDAPP tg mice and AD. Similar to AD, neuritic plaques in PDAPP tg mouse contained a dense amyloid core surrounded by anti-hAPP- and antineurofilament-immunoreactive dystrophic neurites and astroglial cells. Neurons were found in close proximity to plaques in PDAPP tg mice and, to a lesser extent, in AD. In PDAPP tg mice, and occasionally in AD, neuronal processes contained fine intracellular amyloid fibrils in close proximity to the rough endoplasmic reticulum, coated vesicles, and electron-dense material. Extracellular amyloid fibrils (9-11 nm in diameter) were abundant in PDAPP tg and were strikingly similar to those observed in AD. Dystrophic neurites in plaques of PDAPP tg mouse and AD formed synapses and contained many dense multilaminar bodies and neurofilaments (10 nm). Apoptotic-like figures were present in the tg mice. No paired helical filaments have yet been observed in the heterozygote PDAPP tg mice. In summary, this study shows that PDAPP tg mice develop massive neuritic plaque formation and neuronal degeneration similar to AD. These findings show that overproduction of hAPP717V-->F in tg mice is sufficient to cause not only amyloid deposition, but also many of the complex subcellular degenerative changes associated with AD.

While the epsilon 4 allele of apolipoprotein E (ApoE) has been identified as a risk factor in Alzheimer's disease (AD), the mechanism by which this risk is conveyed is not understood. Immunohistochemical studies demonstrating ApoE-A beta colocalization in senile plaques, neurofibrillary tangles, and blood vessels and in vitro studies of ApoE-A beta interactions suggest that ApoE plays a role in amyloid processing and/or fibrillogenesis. We examined the ApoE-A beta association in diffuse and neuritic plaques in the neocortex, striatum, and cerebellum, and determined the ApoE genotype in 100 brains derived from dementia patients with neuropathologically confirmed AD. As expected, the epsilon 4 allele was overrepresented in AD patients compared with patients without neurological disease (p < 0.001). ApoE-positive plaque counts in neocortex were higher in epsilon 4/4 individuals than in individuals with other genotypes (p < 0.0005). Overall, in the 100 cases, ApoE-positive plaques were less frequent than A beta-positive plaques in contiguous sections (p < 0.0001). In all cases, A beta-positive diffuse plaques in the striatum failed to label with ApoE antibody, whereas the majority of cerebellar diffuse plaques showed A beta-ApoE colocalization. Possible explanations for these discrepancies include regional variation in amyloid processing and fibrillogenesis, varying stages of plaque evolution, and technical considerations.

Alzheimer's disease (AD) is the most common cause of progressive intellectual failure in aged humans. AD brains contain numerous amyloid plaques surrounded by dystrophic neurites, and show profound synaptic loss, neurofibrillary tangle formation and gliosis. The amyloid plaques are composed of amyloid beta-peptide (A beta), a 40-42-amino-acid fragment of the beta-amyloid precursor protein (APP). A primary pathogenic role for APP/A beta is suggested by missense mutations in APP that are tightly linked to autosomal dominant forms of AD. A major obstacle to elucidating and treating AD has been the lack of an animal model. Animals transgenic for APP have previously failed to show extensive AD-type neuropathology, but we now report the production of transgenic mice that express high levels of human mutant APP (with valine at residue 717 substituted by phenylalanine) and which progressively develop many of the pathological hallmarks of AD, including numerous extracellular thioflavin S-positive A beta deposits, neuritic plaques, synaptic loss, astrocytosis and microgliosis. These mice support a primary role for APP/A beta in the genesis of AD and could provide a preclinical model for testing therapeutic drugs.

In order to establish a direct relationship between beta-amyloid protein (beta AP) and in vivo neurotoxicity, we made intraparenchymal injections and Alzet pump infusions of beta AP into the hippocampus and cortex of adult rats. We tested a number of synthetic beta AP peptides (beta AP 1-40, 1-38, and 25-35) and peptide controls (scrambled and reversed 1-40, and scrambled and reversed 25-35) over a wide range of concentrations and in a variety of vehicles. The rats were sacrificed from 2-35 days following the implant, and the brains examined by standard immunohistochemical and histological methods used to evaluate the pathologies associated with Alzheimer's disease. We report the lack of Alzheimer related pathology and no significant morphological differences between the beta AP peptide and the peptide and vehicle control injections. These observations indicate that the simple intraparenchymal injection of beta AP in the rat brain is not an appropriate model of Alzheimer-related neurotoxicity.

A central unresolved issue in Alzheimer's disease is the origin of the extracellular amyloid beta protein (A beta P) found in senile plaques and its relationship to the dystrophic neurites that intimately surround it. Here the presence and distribution within senile plaques of various epitopes of the beta-amyloid precursor protein (APP) are compared with the distribution of A beta P itself and markers for plaque neurites. Several principal findings emerge: 1) antibodies to regions of APP outside of A beta P ('APP antibodies') recognize only a subgroup of senile plaques; 2) within these plaques, APP antibodies label discrete globular and granular structures morphologically resembling neurites; 3) virtually all of the plaques labeled by APP antibodies also contain neurites reactive with antibodies to tau; 4) double labeling with anti-tau and an APP antibody shows that the neuritelike profiles stained by the APP antibody are always closely associated with tau-positive neurites within the same plaque and that a minority of profiles appear to be labeled by both antibodies; and 5) antibodies to different regions throughout APP label the same profiles within plaques, suggesting the presence of the full-length precursor. The authors conclude that only a subgroup of senile plaques contain APP epitopes and that the immunostained structures are neurites. Because many A beta P-containing plaques in neocortex, cerebellum, and striatum were found to be devoid of any APP labeling, as were vascular A beta P deposits, it is unlikely that the extracellular A beta P is principally derived from the APP found within dystrophic neurites. The immunodetection of apparently full-length APP, an axonally transported protein, in selected plaque neurites provides yet another protein marker of neuritic dystrophy, possibly indicative of an aberrant regenerative response.