Glutamate receptor dysregulation in the hippocampus of transgenic mice carrying mutated human amyloid precursor protein

Alzheimer's disease transgenic mice overexpressing human amyloid precursor protein (hAPP) with the Swedish double mutation (hAPP(Sw)) develop age-related amyloid deposition and behavioral and electrophysiologic changes by an unknown mechanism. Analysis of glutamatergic receptor subtypes in 4- and 15-month-old heterozygous hAPP(Sw) transgenic mice revealed a selective increase in AMPA receptor binding in the hippocampus of 15-month-old transgenic mice, which have established cortical and hippocampal amyloid deposits. There were no significant alterations of GluR1, GluR2, and GluR4 protein expression by semiquantitative confocal analysis or GluR1 mRNA by in situ hybridization. There was no significant alteration in NMDA, in group I and II metabotropic glutamate and in muscarinic receptor binding, or in striatal dopamine and adenosine receptor binding in 15-month-old mice. These data suggest that mutant APP overexpression or age-related amyloid deposition produce a subtle specific alteration in hippocampal glutamate receptors with aging.     

Neuroanatomical abnormalities in behaviorally characterized APP(V717F) transgenic mice

Histological analyses were performed on the brains of APP(V717F) transgenic (Tg)mice previously studied in a battery of behavioral tests. We describe here the regional and age-dependent deposition of amyloid in both heterozygous and homozygous Tg mice. We also report that Tg mice show significant and age-dependent changes in synaptic density measured by synaptophysin immunoreactivity. Surprisingly, a rather marked hippocampal atrophy is observed as early as 3 months of age in Tg mice (20-40%). Statistical analyses revealed that the deficits in object recognition memory are related to the number of amyloid deposits in specific brain regions, whereas deficits in spatial reference and working memory are related to the changes in synaptic density and hippocampal atrophy. Our study suggests that the behavioral deficits observed in Tg mice are only in part related to amyloid deposition, but are also related to neuroanatomical alterations secondary to overexpression of the APP(V717F) transgene and independent of amyloid deposition.     

Amyloid phenotype characterization of transgenic mice overexpressing both mutant amyloid precursor protein and mutant presenilin 1 transgenes.

Doubly transgenic mice (PSAPP) overexpressing mutant APP and PS1 transgenes were examined using antibodies to Abeta subtypes and glial fibrillary acidic protein (GFAP). Visible Abeta deposition began primarily in the cingulate cortex of PSAPP mice at approximately 10 weeks of age. By 6 months, the mice had extensive amyloid deposition throughout the hippocampus and cortex as well as other regions of the brain. Highly congophilic deposits consisting of N-terminal normal and modified forms of Abeta were identified, reminiscent of those found in human AD brain. Both immunohistochemistry and mass spectrometry showed that Abeta42 forms were underrepresented relative to Abeta40, and Abeta43 was undetectable. Deposits were associated with prominent gliosis which increased with age, but in 14-month-old PSAPP mice, GFAP immunoreactivity in the vicinity of amyloid deposits was substantially reduced compared to APP littermates. These mice have considerable utility in the study of the amyloid phenotype of AD.     

Aging increased amyloid peptide and caused amyloid plaques in brain of old APP/V717I transgenic mice by a different mechanism than mutant presenilin1.

Aging of transgenic mice that overexpress the London mutant of amyloid precursor protein (APP/V717I) (Moechars et al., 1999a) was now demonstrated not to affect the normalized levels of alpha- or beta-cleaved secreted APP nor of the beta-C-terminal stubs. This indicated that aging did not markedly disturb either alpha- or beta-secretase cleavage of APP and failed to explain the origin of the massive amounts of amyloid peptides Abeta40 and Abeta42, soluble and precipitated as amyloid plaques in the brain of old APP/V717I transgenic mice. We tested the hypothesis that aging acted on presenilin1 (PS1) to affect gamma-secretase-mediated production of amyloid peptides by comparing aged APP/V717I transgenic mice to double transgenic mice coexpressing human PS1 and APP/V717I. In double transgenic mice with mutant (A246E) but not wild-type human PS1, brain amyloid peptide levels increased and resulted in amyloid plaques when the mice were only 6-9 months old, much earlier than in APP/V717I transgenic mice (12-15 months old). Mutant PS1 increased mainly brain Abeta42 levels, whereas in aged APP/V717I transgenic mice, both Abeta42 and Abeta40 increased. This resulted in a dramatic difference in the Abeta42/Abeta40 ratio of precipitated or plaque-associated amyloid peptides, i.e., 3.11+/-0.22 in double APP/V717I x PS1/A246E transgenic mice compared with 0.43 +/- 0.07 in aged APP/V717I transgenic mice, and demonstrated a clear difference between the effect of aging and the effect of the insertion of a mutant PS1 transgene. In conclusion, we demonstrate that aging did not favor amyloidogenic over nonamyloidogenic processing of APP, nor did it exert a mutant PS1-like effect on gamma-secretase. Therefore, the data are interpreted to suggest that parenchymal and vascular accumulation of amyloid in aging brain resulted from failure to clear the amyloid peptides rather than from increased production.     

Progressive, age-related behavioral impairments in transgenic mice carrying both mutant amyloid precursor protein and presenilin-1 transgenes

This study provides a comprehensive behavioral characterization during aging of transgenic mice bearing both presenilin-1 (PS1) and amyloid precursor protein (APP(670,671)) mutations. Doubly transgenic mice and non-transgenic controls were evaluated at ages wherein beta-amyloid (Abeta) neuropathology in APP+PS1 mice is low (5-7 months) or very extensive (15-17 months). Progressive cognitive impairment was observed in transgenic mice for both water maze acquisition and radial arm water maze working memory. However, transgenicity did not affect Y-maze alternations, circular platform performance, standard water maze retention, or visible platform recognition at either age, nor did transgenicity affect anxiety levels in elevated plus-maze testing. In sensorimotor tasks, transgenic mice showed a progressive increase in open field activity, a progressive impairment in string agility, and an early-onset impairment in balance beam. None of these sensorimotor changes appeared to be contributory to any cognitive impairments observed, however. Non-transgenic mice showed no progressive behavioral change in any measure evaluated. Given the age-related cognitive impairments presently observed in APP+PS1 transgenic mice and their progressive Abeta deposition/neuroinflammation, Abeta neuropathology could be involved in these progressive cognitive impairments. As such, the APP+PS1 transgenic mouse offers unique opportunities to develop therapeutics to treat or prevent Alzheimer's Disease through modulation of Abeta deposition/neuroinflammation.     

Multi-metric behavioral comparison of APPsw and P301L models for Alzheimer's disease: linkage of poorer cognitive performance to tau pathology in forebrain

APPsw transgenic mice bearing the "Swedish" amyloid precursor protein (APP) mutation and JNPL3 transgenic mice bearing the P301L (Tau) mutation were compared to control non-transgenic (NT) mice in an extensive behavioral test battery administered between 5 and 8.5 months of age. APP mice were impaired in a variety of cognitive-based tasks prior to overt Abeta plaque development, making involvement of mutant APP overexpression and/or oligomeric Abeta assemblies most likely. Although Tau mice, as a group, were not impaired in any single behavioral measure, a collective assessment of behavioral measures through discriminant function analysis showed that Tau mice were impaired in overall behavioral (cognitive) performance. Moreover, correlation analyses involving Tau mice alone revealed linkage between poorer cognitive performance in all three water maze tasks and the number of neurofibrillary tangle (NFT)-containing neurons in neocortex and hippocampus. These findings indicate that: (1) APP mice show early and extensive cognitive impairment before evident Abeta deposition, and (2) the process or product of NFT formation in Tau mice is sufficient to deleteriously impact cognitive performance.     

Normal cognitive behavior in two distinct congenic lines of transgenic mice hyperexpressing mutant APP SWE

Amyloid deposition appears to be an early and crucial event in Alzheimer's disease (AD). To generate animal models of AD, mice expressing full-length amyloid precursor protein (APP), with mutations linked to FAD, have been created. These animals exhibit abnormalities characteristic of AD, including deposits of beta-amyloid (Abeta), neuritic plaques, and glial responses. In studies of cognition in these animals, there have been several reports of memory disturbances well before the appearance of amyloid deposits. We have developed two distinct lines of transgenic mice (C3-3 and E1-2) that express the "Swedish" variant of APP (APP(SWE)) at levels that are approximately three-fold higher than endogenous mouse APP. Both lines have been backcrossed to C57BL/6J mice for 10 generations. Here, we use longitudinal and cross-sectional studies to evaluate the cognitive performance of our animals, where the concentration of Abeta1-42 in brain increases with aging from low levels (2-10 pmol/g) at 6-14 months of age to relatively high levels (60-100 pmol/g) at 24-26 months, when deposits of Abeta were beginning to form. When 12-month-old mice were tested in tasks that assess reference and working memory, transgenic mice from both lines could not be distinguished from nontransgenic littermates. Further study of 24- to 26-month-old transgenic mice (C3-3 line) found no evidence of memory impairment despite the presence of high levels of human Abeta (60-100 pmol/g). Thus, the expression of APP(SWE) at approximately three-fold over endogenous levels, which is sufficient to induce amyloid deposition at advanced ages, does not significantly erode cognitive performance in aged mice.     

Neuroinflammation-induced acceleration of amyloid deposition in the APPV717F transgenic mouse

It has been postulated that neuroinflammation plays a critical role in the pathogenesis of Alzheimer's disease (AD). To directly test whether an inflammatory stimulus can accelerate amyloid deposition in vivo, we chronically administered the bacterial endotoxin, lipopolysaccharide (LPS), intracerebroventricularly (i.c.v.) to 2-month-old APPV717F+/+ transgenic (TG) mice, which overexpress a mutant human amyloid precursor protein (APP 717V-F) with or without apolipoprotein E (apoE) for 2 weeks. Two weeks following central LPS administration a striking global reactive astrocytosis with increased GFAP immunoreactivity was found throughout the brains of all LPS-treated wild-type and transgenic mice including the contralateral brain hemisphere. Localized microglial activation was also evident from lectin immunostaining adjacent to the cannula track of LPS-treated mice. Quantification of thioflavine-S-positive Abeta deposits revealed a marked acceleration of amyloid deposition in LPS-treated APPV717F+/+-apoE+/+ mice compared to nontreated or vehicle-treated APPV717F+/+-apoE+/+ mice (P = 0.005). By contrast, no amyloid deposits were detected by thioflavine-S staining in LPS or vehicle-treated apoE-deficient APPV717F TG mice. Our data suggest that neuroinflammation can accelerate amyloid deposition in the APPV717F+/+ mouse model of AD and that this process requires the expression of apoE.     

Behavioral deficits in APP(V717F) transgenic mice deficient for the apolipoprotein E gene

Both the beta-amyloid precursor protein (APP) and the apoliprotein E (apoE) genes are involved in the pathogenesis of Alzheimer's disease (AD). We previously showed that mice over-expressing a human mutated form of APP (APP(V717F)) display age-dependent recognition memory deficits associated with the progression of amyloid deposition. Here, we asked whether 10- to 12-month-old APP(V717F) mice lacking the apoE gene, which do not present obvious amyloid deposition, differ from APP(V717F) mice in the object recognition task. The recognition performance is decreased in both transgenic mouse groups compared to control groups. Moreover, some behavioral disturbances displayed by APP mice lacking apoE are even more pronounced than those of APP mice expressing apoE. Our results suggest that the recognition memory deficits are related to high levels of soluble Abeta rather than to amyloid deposits.     

Deposition of mouse amyloid beta in human APP/PS1 double and single AD model transgenic mice

The deposition of amyloid beta (Abeta) peptides and neurofibrillary tangles are the two characteristic pathological features of Alzheimer's disease (AD). To investigate the relation between amyloid precursor protein (APP) production, amyloid beta deposition and the type of Abeta in deposits, i.e., human and/or mouse, we performed a histopathological analysis, using mouse and human specific antibodies, of the neocortex and hippocampus in 6, 12 and 19 months old APP/PS1 double and APP and PS1 single transgenic mice. There was a significant correlation between the human amyloid beta deposits and the intrinsic rodent amyloid beta deposits, that is, all plaques contained both human and mouse Abeta, and the diffuse amyloid beta deposits also colocalized human and mouse Abeta. Furthermore, some blood vessels (mainly leptomeningeal vessels) show labeling with human Abeta, and most of these vessels also label with mouse Abeta. Our findings demonstrate that the human amyloid deposits in APP/PS1 transgenic mice are closely associated with mouse Abeta, however, they do not precisely overlap. For instance, the core of plaques consists of primarily human Abeta, whereas the rim of the plaque contains both human and mouse amyloid beta, similarly, human and mouse Abeta are differentially localized in the blood vessel wall. Finally, as early as amyloid beta deposits can be detected, they show the presence of both human and mouse Abeta. Together, these data indicate that mouse Abeta is formed and deposited in significant amounts in the AD mouse brain and that it is deposited together with the human Abeta.     

Inhibiting p38 mitogen-activated protein kinase attenuates cerebral ischemic injury in Swedish mutant amyloid precursor protein transgenic mice

Cerebral ischemia was induced using photothrombosis 1 hour after intraperitoneal injection of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB239063 into Swedish mutant amyloid precursor protein (APP/SWE) transgenic and non-transgenic mice. The number of surviving neurons in the penumbra was quantified using Nissl staining, and the activity of p38 MAPKs was measured by western blotting. The number of surviving neurons in the penumbra was significantly reduced in APP/SWE transgenic mice compared with non-transgenic controls 7 days after cerebral ischemia, but the activity of p38 MAPKs was significantly elevated compared with the non-ischemic hemisphere in the APP/SWE transgenic mice. SB239063 prevented these changes. The APP/SWE mutation exacerbated ischemic brain injury, and this could be alleviated by inhibiting p38 MAPK activity.     

Restricted diffusion in the brain of transgenic mice with cerebral amyloidosis

A prominent hallmark of Alzheimer's disease pathology is cerebral amyloidosis. However, it is not clear how extracellular amyloid-beta peptide (A beta) deposition and amyloid formation compromise brain function and lead to dementia. It has been argued that extracellular amyloid deposition is neurotoxic and/or that soluble A beta oligomers impair synaptic function. Amyloid deposits, by contrast, may affect diffusion properties of the brain interstitium with implications for the transport of endogenous signalling molecules during synaptic and/or extrasynaptic transmission. We have used diffusion-weighted magnetic resonance imaging to study diffusion properties in brains of young (6-month-old) and aged (25-month-old) APP23 transgenic mice and control littermates. Our results demonstrate that fibrillar amyloid deposits and associated gliosis in brains of aged APP23 transgenic mice are accompanied by a reduction in the apparent diffusion coefficient. This decrease was most pronounced in neocortical areas with a high percentage of congophilic amyloid and was not significant in the caudate putamen, an area with only modest and diffuse amyloid deposition. These findings suggest that extracellular deposition of fibrillar amyloid and/or associated glial proliferation and hypertrophy cause restrictions to interstitial fluid diffusion. Reduced diffusivity within the interstitial space may alter volume transmission and therefore contribute to the cognitive impairment in Alzheimer's disease.     

Acetylcholinesterase-positive fiber deafferentation and cell shrinkage in the septohippocampal pathway of aged amyloid precursor protein london mutant transgenic mice

Several lines of evidence implicate a cholinergic deficit in Alzheimer's disease (AD). Transgenic mice that overexpress clinical mutants of the human amyloid precursor protein (APP) have been generated that recapitulate many aspects of AD. We now analyzed the cholinergic system in aged APP/London transgenic mice. The major finding was the reorganization of acetylcholinesterase-positive fibers within the hippocampus and the reduced size of cholinergic cells in the medial septum. The reduction of acetylcholinesterase-positive fibers in the subiculum together with increased fiber density in the CA1 and in the dentate gyrus suggests a synaptic sprouting compensatory mechanism within the hippocampus. In the cortex, amyloid plaques were associated with intense acetylcholinesterase activity and surrounded by dystrophic acetylcholinesterase-positive fibers. Nevertheless, the overall pattern of cholinergic innervation was unchanged. These results demonstrate that overexpression of APP/London caused, besides amyloid plaques in aged mouse brain, also cholinergic deafferentation and cholinergic cell shrinkage.     

Lipopolysaccharide-induced-neuroinflammation increases intracellular accumulation of amyloid precursor protein and amyloid beta peptide in APPswe transgenic mice

The present study was designed to examine whether brain inflammation caused by systemic administration of lipopolysaccharides (LPS) alters the expression/processing of amyloid precursor protein (APP) and increases the generation of amyloid beta peptide (Abeta). APPswe transgenic (Tg) mice were treated with either LPS or phosphate-buffered saline (PBS). In LPS-treated APPswe mice, Abeta1-40/42 was 3-fold and APP was 1.8-fold higher than those in PBS-treated mice (P < 0.05) by ELISA, Western blots and immunoprecipitation-mass spectrometry (IP-MS) ProteinChip analysis. Numbers of Abeta- and APP-immunoreactive neurons (Abeta(+) and APP(+) neurons) increased significantly in LPS-treated APPswe mice; APP(+) and Abeta(+) neurons in neocortex were associated with an increased number of F4/80-immunoreactive microglia (F4/80(+) microglia) in their anatomical environment. Our findings demonstrate that experimental neuroinflammation increases APP expression/processing and causes intracellular accumulation of Abeta. It remains to be seen whether such events can cause neuronal dysfunction/degeneration and, with time, lead to extracellular Abeta deposits, as they occur in Alzheimer's disease.     

Alterations in cerebral blood flow and glucose utilization in mice overexpressing the amyloid precursor protein

We have used quantitative autoradiographic techniques to study the relationship between cerebral blood flow (CBF) and glucose utilization (CGU) in two lines of transgenic mice overexpressing Swedish mutant amyloid precursor protein (APP) and APP-derived Abeta peptides. Mice were studied at an age when there are no amyloid plaques. In the 2123 line, CBF was reduced only in telencephalic regions with no corresponding decrease in CGU. In 2576 transgenics, a line with higher levels of Abeta peptide, both CBF and CGU were reduced throughout the brain. The data indicate that Abeta induces alterations in resting CBF that are either associated with or independent of alterations in CGU and that occur in the absence of amyloid deposition in neuropil of blood vessels. These observations support the hypothesis that cerebrovascular and metabolic abnormalities are early events in the pathogenesis of Alzheimer's disease.     

A novel imaging probe for in vivo detection of neuritic and diffuse amyloid plaques in the brain

Extensive deposition of neuritic and diffuse amyloid plaques in the brain is a critical event for the pathogenesis of Alzheimer’s disease (AD) and considered to start before the appearance of clinical symptoms. In vivo detection of these brain β-amyloid (Aβ) deposits using positron emission tomography (PET), therefore, would be a useful marker for presymptomatic detection of AD. To develop a new agent for PET probe of imaging neuritic and diffuse amyloid deposits, novel fluorescent compounds, including styryl-fluorobenzoxazole derivatives, were examined. These compounds showed a high binding affinity for both synthetic Aβ1-40 and Aβ1-42 aggregates. Some of these compounds also displayed distinct staining of neuritic and diffuse amyloid plaques in AD brain sections. A biodistribution study of styryl-fluorobenzoxazole derivatives in normal mice exhibited excellent brain uptakes (4.5–5.5% injected dose/g at 2 min postinjection). Furthermore, iv administration of BF-145, a styryl-fluorobenzoxazole derivative, demonstrated specific in vivo labeling of compact and diffuse amyloid deposits in an APP23 transgenic mouse brain, in contrast to no accumulation in a wild-type mouse brain. These findings suggest that BF-145 is a potential candidate as a probe for imaging early brain pathology in AD patients.     

Neurodegenerative changes associated with beta-amyloid deposition in the brains of mice carrying mutant amyloid precursor protein and mutant presenilin-1 transgenes

Mutations of amyloid precursor protein (APP) and presenilin-1 (PS1) lead to an increase in beta-amyloid (Abeta) production. Despite the fact that a number of transgenic mice develop cerebral Abeta plaques, few have been subjected to ultrastructural investigation and the sequence of events leading to Abeta plaque formation is unclear. We therefore investigated the doubly transgenic (mutant APP(K670N,M671L)-mutant PS1(M146L)) mouse, which develops Abeta deposits much earlier than singly transgenic littermates. Widespread Abeta plaques with or without a distinct core were found in gray matter. Abeta plaques were also present in white matter. Astrocytosis was greater around gray matter plaques than around white matter plaques. In some plaques, Abeta cores were associated with cell profiles containing prominent endoplasmic reticulum and a homogeneous cytoplasm that appeared to be neuronal. The morphology and location of other profiles indicated them to be microglia or oligodendrocytes. Some Abeta fibrils appeared to lie within these profiles, but they may have been simply surrounded by the cell profile since the profile membrane was not always visible. Dark atrophic neurons, whose morphology suggested that they were apoptotic, were present around gray matter plaques. Cerebrovascular Abeta deposition was also observed in the brains of APP/PS1 transgenic mice. Thus, the amyloid deposition and neuropathology observed in APP/PS1 mouse brain are similar to those in Alzheimer's disease and they appear to develop earlier and become more severe than in the other transgenic models currently available.     

Age and damage induced changes in amyloid protein precursor immunohistochemistry in the rat brain

Alzheimer's disease (AD) is characterized by the extensive deposition of the 42-amino-acid β-amyloid or A4 protein in neuritic plaques and neurofibrillary tangles within the brain. This protein is liberated from the much larger amyloid protein precursor (APP). Multiple species of APP have been proposed, including several forms that contain a 56 amino acid insert sequence analogous to the Kunitz protease inhibitors. Although expression of APP mRNA is reportedly altered in AD brain and various roles for APP have been proposed, the pathogenesis of amyloid deposition and AD remains unclear. AD is also characterized by specific memory impairments associated with decreased cholinergic activity. While aging rats do not develop mature amyloid pathology, behaviorally impaired aged rats demonstrate an analogous cholinergic decline. In this study, we examined behaviorally characterized aged rats and normal young controls for changes in APP immunohistochemistry by using anti-APP antibodies, which detect N- or C-terminal regions and which distinguish APP species with or without the Kunitz protease inhibitor domain. The results show specific age- and behavior-related changes in cortical APP immunoreactivity as well as limited numbers of APP immunoreactive deposits in the aged rats. Additionally, we found that lesions of the fimbria-fornix pathway, which in part mimic the memory impairments and loss of cholinergic activity seen in AD, result in the marked accumulation of APP immunoreactive material in the region of cholinergic fiber degeneration in the hippocampus. These findings are discussed in relation to the pathogenesis of AD in humans. © 1994 Wiley-Liss, Inc.     

FBL2 regulates amyloid precursor protein (APP) metabolism by promoting ubiquitination-dependent APP degradation and inhibition of APP endocytosis

The ubiquitin-proteasome pathway is a major protein degradation pathway whose dysfunction is now widely accepted as a cause of neurodegenerative diseases, including Alzheimer's disease. Here we demonstrate that the F-box and leucine rich repeat protein2 (FBL2), a component of the E3 ubiquitin ligase complex, regulates amyloid precursor protein (APP) metabolism through APP ubiquitination. FBL2 overexpression decreased the amount of secreted amyloid β (Aβ) peptides and sAPPβ, whereas FBL2 mRNA knockdown by siRNA increased these levels. FBL2 overexpression also decreased the amount of intracellular Aβ in Neuro2a cells stably expressing APP with Swedish mutation. FBL2 bound with APP specifically at its C-terminal fragment (CTF), which promoted APP/CTF ubiquitination. FBL2 overexpression also accelerated APP proteasome-dependent degradation and decreased APP protein localization in lipid rafts by inhibiting endocytosis. These effects were not observed in an F-box-deleted FBL2 mutant that does not participate in the E3 ubiquitin ligase complex. Furthermore, a reduced insoluble Aβ and Aβ plaque burden was observed in the hippocampus of 7-month-old FBL2 transgenic mice crossed with double-transgenic mice harboring APPswe and PS1(M146V) transgenes. These findings indicate that FBL2 is a novel and dual regulator of APP metabolism through FBL2-dependent ubiquitination of APP.