Familial Alzheimer disease associated with A713T mutation in APP

Mutations in APP are associated with familial early-onset Alzheimer disease (FAD). Examination of the genomic sequence in one patient with FAD revealed a change located in the axon 17 of the APP gene at position 275329G>A (GenBank accession number: D87675; GI: 2429080); cDNA sequence 2137G>A (GenBank accession number: X06989; GI: 28720). This corresponds to the mutation A713T in APP. AD stage VI of neurofibrillary degeneration and stage C of Aβ-amyloid burden was found at the post-mortem neuropathological examination. Previous studies have suggested that the mutation A713T in APP is a silent mutation or polymorphism. However, we have not found this change in APP in a control population analyzed by the amplification-refractory mutation system (ARMS). It is concluded that A713T in APP is implicated in the pathogenesis of AD. Since the immunohistochemical study indicates that A713T mutation is not likely to relate with Aβ-amyloid processing, the causative role of this rare mutation remains to be warranted.     

The role of matrix metalloproteinase-9 in dementia

Neurofibrillary tangles and senile plaques, constituted of extracellular amyloid deposits (Aβ), are the two defining pathological hallmarks of Alzheimer's disease (AD). Inhibiting the synthesis or aggregation of Aβ or increasing its clearance may reduce the detrimental effects of this peptide and consequently improve cognitive functions in patients. Previous studies indicated that metalloproteinases are involved in Aβ degradation and the presence of matrix metalloproteinases (MMP) in AD plaques has been described. In this study, we examined the distribution of a functional polymorphism in the gene for MMP-9, −1562 C→T, in an independent population of 229 demented and 253 control individuals. We observed a weak protective effect of the high activity allele (T) in apolipoprotein E ε4 allele non-bearers (odds RATIO=0.5 (95% confidence interval, 0.3–0.9), P=0.04).     

BACE1 gene deletion: impact on behavioral function in a model of Alzheimer's disease

Accumulation of cerebral amyloid-β (Aβ) has been implicated as a putative causal factor in the development of Alzheimer's disease (AD). Transgenic mice like the PDAPP line overexpress human mutant Amyloid Precursor Protein (hAPP) and recapitulate many features of AD, including amyloid neuropathology and cognitive deficits. Inhibition of the β-site aspartyl cleaving enzyme (BACE1) enzyme responsible for the first proteolytic cleavage that ultimately generates Aβ has been proposed as a strategy for AD therapy. To assess the theoretical repercussions of β-secretase activity reduction in an in vivo model of AD, BACE1^−/− mice bred to the PDAPP line were examined in a series of behavioral tasks. Although BACE1 gene ablation abolished hAβ accumulation, BACE1^−/− mice had unexpected sensorimotor impairments, spatial memory deficits, and displayed seizures, phenotypes which were severe on the PDAPP background. These results suggest that while excess Aβ is functionally pathological, BACE1-mediated processing of APP and other substrates play a role in “normal” learning, memory and sensorimotor processes.     

Intranasal immunotherapy for the treatment of Alzheimer's disease: Escherichia coli LT and LT(R192G) as mucosal adjuvants

Alzheimer’s disease (AD) is the most common form of dementia worldwide, yet there is currently no effective treatment or cure. Extracellular deposition of amyloid-β protein (Aβ) in brain is a key neuropathological characteristic of AD. In 1999, Schenk et al. first reported that an injected Aβ vaccine given to PDAPP mice, an AD mouse model displaying Aβ deposition in brain, led to the lowering of Aβ levels in brain. In 2000, we demonstrated that intranasal (i.n.) immunization with human synthetic Aβ1–40 peptide for 7 months led to a 50–60% reduction in cerebral Aβ burden in PDAPP mice; serum Aβ antibody titers were low (26 μg/ml). More recently, we have optimized our i.n. Aβ immunization protocol in wild-type (WT) mice. When low doses Escherichia coli heat-labile enterotoxin (LT) were given as a mucosal adjuvant with Aβ i.n., there was a dramatic 12-fold increase in Aβ antibody titers in WT B6D2F1 mice treated two times per week for 8 weeks compared to those of mice receiving i.n. Aβ without adjuvant. A non-toxic form of LT, designated LT(R192G), showed even better adjuvanticity; anti-Aβ antibody titers were 16-fold higher than those seen in mice given i.n. Aβ without adjuvant. In both cases, the serum Aβ antibodies recognized epitopes within Aβ1–15 and were of the immunoglobulin (Ig) isotypes IgG2b, IgG1, IgG2a and low levels of IgA. This new and improved Aβ vaccine protocol is now being tested in AD mouse models with the expectation that higher Aβ antibody titers may be more effective in reducing cerebral Aβ levels.     

Amyloid β peptide levels and its effects on hippocampal acetylcholine release in aged, cognitively-impaired and -unimpaired rats

Excessive extracellular deposition of amyloid β (Aβ) peptide in neuritic plaques and degeneration of forebrain cholinergic neurones, which innervate the hippocampus and the neocortex, are the invariant characteristic features of Alzheimer's disease (AD). Studies of the pathological changes that characterize AD, together with several other lines of evidence, indicate that Aβ accumulation in vivo may initiate and/or contribute to the process of neurodegeneration observed in the AD brain. However, the underlying mechanisms by which Aβ peptide influences/causes degeneration of the basal forebrain cholinergic neurones in AD brains remain obscure. We reported earlier that nM concentrations of Aβ-related peptides, under acute conditions, can potently inhibit K⁺-evoked endogenous acetylcholine (ACh) release from the hippocampus and the cortex but not from striatum in young adult rats (J. Neurosci. 16 (1996) 1034). In the present study, to determine whether the effects of Aβ peptides alter with normal aging and/or cognitive state, we have measured Aβ_1–40 levels and the effects of exogenous Aβ_1–40 on hippocampal ACh release in young adult as well as aged cognitively-unimpaired (AU) and -impaired (AI) rats. Endogenous levels of Aβ_1–40 in the hippocampus are significantly increased in aged rats. Additionally, 10 nM Aβ_1–40 potently inhibited endogenous ACh release from the hippocampus of the three groups of rats, but the time-course of the effects clearly indicate that the cholinergic neurones of AI rats are more sensitive to Aβ peptides than either AU or young adult rats. These results, together with earlier reports, suggest that the processing of the precursor protein of Aβ peptide alters with normal aging and the response of the cholinergic neurones to the peptide possibly varies with the cognitive status of the animals.     

Callosal agenesis and absence of primary visual cortex induced by prenatal X rays impair navigation's strategy and learning in tasks involving visuo-spatial working but not reference memory in mice

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by cognitive and memory deterioration. Production and accumulation of β-amyloid peptide (Aβ) is central to the pathogenesis of AD. Recent studies have demonstrated that PKA/CREB-dependent signaling pathway and long-term potentiation are inhibited by sublethal concentrations of Aβ_1–42 in cultured hippocampus neurons. Here, we examined the effects of nobiletin on the Aβ-induced inhibition of CREB phosphorylation in cultured rat hippocampus neurons. A sublethal concentration of Aβ_1–42 or Aβ_1–40 decreased glutamate-induced CREB phosphorylation, whereas pretreatment with nobiletin reversed the Aβ-induced decrease in CREB phosphorylation. The effects of nobiletin on impairment of learning ability were also examined in chronically Aβ_1–40 infused AD model rats using the eight-arm radial maze. In the AD model rats, nobiletin showed protective effects on Aβ_1–40-induced impairment of learning ability. These results suggest that nobiletin has the potential for becoming a novel lead compound for drug development for AD.     

Ibuprofen effects on Alzheimer pathology and open field activity in APPsw transgenic mice

We previously showed the non-steroidal anti-inflammatory drug (NSAID) ibuprofen suppresses inflammation and amyloid in the APPsw (Tg2576) Tg2576 transgenic mouse. The mechanism for these effects and the impact on behavior are unknown. We now show ibuprofen’s effects were not mediated by alterations in amyloid precursor protein (APP) expression or oxidative damage (carbonyls). Six months ibuprofen treatment in Tg+ females caused a decrease in open field behavior (p < 0.05), restoring values similar to Tg− mice. Reduced caspase activation per plaque provided further evidence for a neuroprotective action of ibuprofen.

The impact of a shorter 3 month duration ibuprofen trial, beginning at a later age (from 14 to 17 months), was also investigated. Repeated measures ANOVA of Aβ levels (soluble and insoluble) demonstrated a significant ibuprofen treatment effect (p < 0.05). Post-hoc analysis showed that ibuprofen-dependent reductions of both soluble Aβ and Aβ42 were most marked in entorhinal cortex (p < 0.05). Although interleukin-1β and insoluble Aβ were more effectively reduced with longer treatment, the magnitude of the effect on soluble Aβ was not dependent on treatment duration.     

Aβ40 is a major form of β-amyloid in nonhuman primates

Because aged nonhuman primates show β-amyloid (Aβ) deposition in senile plaques and blood vessels similar to that seen in human aging and AD, we used C-terminal specific antibodies to Aβ₄₀ and Aβ₄₂ to investigate Aβ peptide length in the brains of 11 aged rhesus monkeys and a 59-year-old chimpanzee. In contrast to AD, where the earliest and most prominent form of Aβ in senile plaques is Aβ₄₂, in the monkey, Aβ₄₀-positive plaques predominated. The ratio of Aβ₄₎: Aβ₄₂-positive plaques averaged 2.08 in the monkey, as compared to a mean ratio of 0.37 in 68 human AD subjects (p < 0.001). Aβ₄₀ was also more prominent in the chimpanzee than in humans. Possible explanations for these findings include species differences in the cleavage of Aβ from the amyloid precursor protein or in the activity of a putative carboxy peptidase forming Aβ₄₀ from Aβ₄₂ in situ.     

A non-toxic ligand for voxel-based MRI analysis of plaques in AD transgenic mice

Amyloid plaques are a characteristic feature in Alzheimer's disease (AD). A novel non-toxic contrast agent is presented, Gd-DTPA-K6Aβ1–30, which is homologous to Aβ, and allows plaque detection in vivo. μMRI was performed on AD model mice and controls prior to and following intracarotid injection with Gd-DTPA-K6Aβ1–30 in mannitol solution, to transiently open the blood–brain barrier. A gradient echo T2^*-weighted sequence was used to provide 100 μm isotropic resolution with imaging times of 115 min. The scans were examined with voxel-based analysis (VBA) using statistical parametric mapping, for un-biased quantitative comparison of ligand-injected mice and controls. The results indicate that: (1) Gd-DTPA-K6Aβ1–30 is an effective, non-toxic, ligand for plaque detection when combined with VBA (p ≤ 0.01–0.001), comparing pre and post-ligand injection scans. (2) Large plaques can be detected without the use of a contrast agent and this detection co-localizes with iron deposition. (3) Smaller, earlier plaques require contrast ligand for MRI visualization. Our ligand when combined with VBA may be useful for following therapeutic approaches targeting amyloid in transgenic mouse models.     

Genotype-related changes of ganglioside composition in brain regions of transgenic mouse models of Alzheimer's disease

In this study, brain gangliosides of different transgenic mouse models of Alzheimer's disease (AD) were analyzed and compared with age-matched wild-type mice. Gangliosides were analyzed in cerebral cortex, a region with extensive Aβ plaques, and cerebellum, a non-vulnerable region with no Aβ containing plaques. There was a marked increase in simple gangliosides GM2 and GM3 only within the cortex of all mice expressing APP^SL. Additionally, loss of complex “a” gangliosides (GT1a, GD1a and GM1) was recorded in APP/PS1Ki model, whereas in APP^SL and APP/PS1 mice, the complex “b” gangliosides (GQ1b, GT1b and GD1b) moderately decreased. Surprisingly, expression of either mutant PS1^M146L or PS1 mutant FAD (Ki model) alone tended to lower the levels of both GM2 and GM3 within the cortex. Conversely, only slight changes of the ganglioside pattern were found in the cerebellum. Because ganglioside alterations occurring in APP transgenic mice were similar to those observed in human AD brain, these transgenic models would represent valuable tools to further investigate the role of altered ganglioside metabolism in the pathogenesis of AD.     

Bilateral Injections of Amyloid-β 25-35 into the Amygdala of Young Fischer Rats: Behavioral, Neurochemical, and Time Dependent Histopathological Effects

To examine the time course of the histopathological effects of bilateral injections of amyloid-β 25-35 (Aβ) and to determine if these effects are associated with a reduction in choline acetyltransferase activity and behavioral impairments, we injected Aβ (5.0 nmol) into the amygdala of young male Fischer rats. Control rats received vehicle infusions. For histological analysis, animals were sacrificed at 8, 32, 64, 96, and 128 days postoperatively (n = 21–33 per timepoint). Aβ induced neuronal tau-2 staining in the right, but not the left amygdala and hippocampus. Aβ also induced reactive astrocytosis and neuronal shrinkage within the right hippocampus and amygdala, respectively. As with tau-2, these same brain regions within the left hemisphere in the Aβ-treated rats were significantly less affected. In addition, Aβ appeared to induce microglial and neuronal interleukin-1β staining. The histopathological effects of Aβ peaked at 32 days postoperatively but were not associated with a reduction in amygdaloid choline acetyltransferase activity. In a separate experiment, behavioral effects of bilateral intra-amygdaloid injections of Aβ were analyzed at 34–52 days postoperatively. In an open field test, the treatment groups differed only in the numbers of rears emitted (p = 0.016). There was no effect of Aβ in the Morris water maze or in the acquisition and retention of a one-way conditioned avoidance response. These data suggest a laterality in the histopathological effects of Aβ and that the effects of single injections are in part transient. These findings also suggest a direct association between plaque and tangle formation in Alzheimer’s disease, and support the use of this rat model to screen drugs that may alter the initial pathological events associated with Alzheimer’s disease, that occur before the manifestations of extensive behavioral impairments become evident.     

A safer vaccine for Alzheimer's disease?

Recent reports indicate that amyloid-β (Aβ) vaccine-based therapy for Alzheimer’s disease (AD) may be on the horizon. There are, however, concerns about the safety of this approach. Immunization with Aβ1–42 may not be appropriate in humans because it crosses the blood–brain barrier, can seed fibril formation, and is highly fibrillogenic. Aβ1–42 fibrils can in turn cause inflammation and neurotoxicity. This issue is of a particular concern in the elderly who often do not mount an adequate immune response to vaccines. Our findings show that vaccination with nonamyloidogenic/nontoxic Aβ derivative may be a safer therapeutic approach to impede the progression of Aβ-related histopathology in AD. Although the site of action of the anti-Aβ antibodies has been suggested to be within the brain, peripheral clearance of Aβ may have a greater role in reducing cerebral amyloid plaques in these animals and eventually in AD patients. Antibodies in general are predominantly found outside the central nervous system (CNS) and will, therefore, primarily clear systemic Aβ compared to brain Aβ. This disruption of the equilibrium between central and peripheral Aβ should then result in efflux of Aβ out of the brain, and subsequent removal of plaques. Aβ therapy can be targeted to the periphery, which may result in fewer CNS side effects, such as inflammation. Future Aβ derived vaccines should include T_h epitopes, carriers and/or lipid moieties to enhance antibody production in the elderly, the population predominantly affected by AD.     

Suppression of p53-activated gene, PAG608, attenuates methamphetamine-induced neurotoxicity

Nitric oxide is involved in normal physiological functions and also in pathological processes leading to tissue damage due, in part, to its free radical nature (oxidative stress). Oxidative stress and vascular dysfunction have been recognized as contributing factors in the pathogenesis of Alzheimer disease (AD) and vascular dementia (VD). In order to study the possible links between these processes and dementia, we have analysed plasma amyloid-beta_(1–42) levels (Aβ) and total nitric oxide (NO_x), apolipoprotein E (ApoE), lipids, vitamin B12, and folate concentrations in the serum of 99 patients with dementia and 55 age-matched non-demented controls. Both nitrate and nitrite levels were measured by a colorimetric method using Griess Reagent and plasma Aβ levels were analysed by a hypersensitive ELISA method. Our data showed a significant decrease of serum NO_x levels in dementia, especially in probable AD and VD patients, as compared with controls. We observed a weak correlation between serum NO_x levels and cognitive deterioration in dementia; however, NO_x levels were not associated with ApoE and Aβ levels. In dementia and controls, a similar correlation pattern between HDL-cholesterol versus NO_x was found. No apparent association between NO_x, Aβ and AD-related genes [APOE (apolipoprotein E), PSEN1 (Presenilin 1)] was observed. Our data suggest that NO_x may contribute to the pathogenesis of dementia through a process mediated by HDL-cholesterol.     

Aβ as a bioflocculant: implications for the amyloid hypothesis of Alzheimer’s disease

Research into Alzheimer’s disease (AD) has been guided by the view that deposits of fibrillar amyloid-β peptide (Aβ) are neurotoxic and are largely responsible for the neurodegeneration that accompanies the disease. This ‘amyloid hypothesis’ has claimed support from a wide range of molecular, genetic and animal studies. We critically review these observations and highlight inconsistencies between the predictions of the amyloid hypothesis and the published data. We show that the data provide equal support for a ‘bioflocculant hypothesis’, which posits that Aβ is normally produced to bind neurotoxic solutes (such as metal ions), while the precipitation of Aβ into plaques may be an efficient means of presenting these toxins to phagocytes. We conclude that if the deposition of Aβ represents a physiological response to injury then therapeutic treatments aimed at reducing the availability of Aβ may hasten the disease process and associated cognitive decline in AD.     

Absence of association between Alzheimer disease and the −491 regulatory region polymorphism of APOE

A novel polymorphism (−491 A/T) within the regulatory region on the apolipoprotein E gene has recently been reported to be associated with risk for Alzheimer disease (AD). To test this association in an independent data set, we have examined this polymorphism in a sample of 88 well-characterized AD cases and compared the allele frequency and genotype frequencies for this polymorphism with those observed in 112 cognitively normal subjects drawn from the same ethnic group. These results suggest that in the current data set at least, the −491 A/T polymorphism is not associated with risk for AD, but may be in partial linkage disequilibrium with the APOE ε2/ε3/ε4 polymorphism.     

Increased amyloid beta-peptide (1-40) level in brain of streptozotocin-induced diabetic rats

The aims of the study were to investigate whether the level of amyloid β-peptide (Aβ) (1–40) was increased in brain of diabetic rats and whether the increase was associated with dysfunction of P-glycoprotein at the blood–brain barrier. A diabetes-like condition was induced by single administration of 65 mg/kg streptozotocin via i.p. injection. Aβ (1–40) levels in brain of the diabetic rats were measured using an enzyme linked immunosorbent assay (ELISA) kit. The in vivo brain-to-blood efflux and blood-to-brain influx transport of [¹²⁵I]-labeled human amyloid-β-peptide (hAβ) (1–40) were measured using the brain efflux index and brain permeability coefficient-surface area product, respectively. [¹⁴C]inulin served as a reference compound. The results showed that Aβ (1–40) levels significantly increased in temporal cortex and hippocampus of the diabetic rats. The brain remaining percentage of [¹²⁵I]hAβ (1–40) in diabetic rats significantly increased at 30 min after intracerebral microinjection, accompanied by decrease of the brain efflux index. Pretreatment of P-glycoprotein inhibitors verapamil or cyclosporin A significantly increased the brain remaining percentage of [¹²⁵I]hAβ (1–40). The brain permeability coefficient-surface area product of [¹²⁵I]hAβ (1–40) was increased in diabetic rats, accompanied by increased Aβ (1–40) levels in plasma. The present study demonstrated that a diabetic state could increase Aβ (1–40) levels in brain, which might be explained, at least in part, by the decline in brain-to-blood efflux of Aβ (1–40) due to deficient cerebral P-glycoprotein function in diabetic rats.     

Salidroside from Rhodiola sachalinensis Protects Neuronal PC12 Cells Against Cytotoxicity Induced by Amyloid-β

The amyloid β-peptide (Aβ)-induced oxidative stress is a well-established pathway of neuronal cell death in Alzheimer's disease (AD). Salidroside, one of the major compounds from the roots of Rhodiola species (Crassulaceae), was investigated in vitro for its cytoprotection against Aβ-induced toxicity on rat neuronal PC12 cells. Salidroside significantly reduced Aβ-induced cytotoxicity in a dose-dependent manner. Salidroside also reduced Aβ-mediated intracellular accumulation of reactive oxygen species and malondialdehyde (MDA), a product of lipid peroxides, by preventing Aβ-induced decline of antioxidant enzyme activities. These results suggest that salidroside protects neuronal PC12 cells from Aβ-induced cytotoxicity via its antioxidant pathway.     

Amyloid deposition precedes tangle formation in a triple transgenic model of Alzheimer's disease

Amyloid-β (Aβ) containing plaques and tau-laden neurofibrillary tangles are the defining neuropathological features of Alzheimer’s disease (AD). To better mimic this neuropathology, we generated a novel triple transgenic model of AD (3xTg-AD) harboring three mutant genes: β-amyloid precursor protein (βAPP_Swe), presenilin-1 (PS1_M146V), and tau_P301L. The 3xTg-AD mice progressively develop Aβ and tau pathology, with a temporal- and regional-specific profile that closely mimics their development in the human AD brain. We find that Aβ deposits initiate in the cortex and progress to the hippocampus with aging, whereas tau pathology is first apparent in the hippocampus and then progresses to the cortex. Despite equivalent overexpression of the human βAPP and human tau transgenes, Aβ deposition develops prior to the tangle pathology, consistent with the amyloid cascade hypothesis. As these 3xTg-AD mice phenocopy critical aspects of AD neuropathology, this model will be useful in pre-clinical intervention trials, particularly because the efficacy of anti-AD compounds in mitigating the neurodegenerative effects mediated by both signature lesions can be evaluated.     

Peripheral clearance of amyloid beta peptide by complement C3-dependent adherence to erythrocytes

Brain deposits of amyloid β peptide (Aβ) have been a diagnostic hallmark of Alzheimer's disease (AD) for nearly a century. Recent studies have demonstrated that Aβ is also present in peripheral blood. Here, we present evidence that circulating Aβ42 is subject to complement C3b-dependent adherence to complement receptor 1 (CR1) on erythrocytes, a classical set of mechanisms by which pathogens and proteins recognized as foreign are cleared from the bloodstream. Levels of Aβ42 targeted by this pathway differ significantly in AD compared to mild cognitive impairment and nondemented elderly controls.