Pathway-specific alteration of synaptic plasticity in Tg2576 mice

Various animal models of Alzheimer disease (AD) are characterized by deficits in spatial memory that are causally related to altered synaptic function and impairment of long-term potentiation (LTP) in the hippocampus. In Tg2576 AD mice, we compared LTP in 2 major hippocampal pathways, Schaffer collateral (SC) and mossy fiber (MF) pathways. Whereas LTP was completely abolished in the SC pathway of Tg2576 mice, we found no decrease in LTP induced by stimulation of the MF pathway. In fact, we found that in the MF pathway, LTP was slightly, but significantly, enhanced compared with that in the MF pathway of WT littermates. This pathway-specific impairment of LTP is not attributable to alterations in transmitter release, as indicated by an unaltered paired-pulse ratio. These results suggest that the spatial memory deficits normally seen in AD models arise primarily from LTP impairment at the SC pathway.     

Altered glycosylation of acetylcholinesterase in APP (SW) Tg2576 transgenic mice occurs prior to amyloid plaque deposition

Previous studies have shown that a minor glycoform of acetylcholinesterase (AChE) is increased in Alzheimer's disease brain and cerebrospinal fluid. This glycoform can be distinguished from other AChE species by its lack of binding to concanavalin A (Con A). In this study, the temporal relationship between AChE glycosylation and Abeta deposition was examined in Tg2576 mice. There was a significant (p < 0.05) difference in AChE glycosylation in Tg2576 mice compared with age-matched background strain control mice at 4 months of age. This difference in glycosylation was also observed in 8- and 12-month-old Tg2576 mice. In contrast, Abeta plaques were only seen in the Tg2576 mice at 12 months of age, and were not detected at 4 and 8 months of age. Soluble human-sequence Abeta was detected as early as 4 months of age in the transgenic mice. The altered AChE glycosylation was due to an increase in a minor AChE isoform, which did not bind Con A, similar to that previously observed to be increased in Alzheimer's disease brain and cerebrospinal fluid. The results demonstrate that in transgenic mice altered AChE glycosylation is associated with very early events in the development of AD-like pathology. The study supports the possibility that glycosylation may also be a useful biomarker of AD.     

Furin is an endogenous regulator of alpha-secretase associated APP processing

Constitutive and PKC-regulated alpha-secretase pathways have been reported to produce the secreted form of alpha-secretase-derived APP (sAPPalpha). Here, we examined putative role of furin in the regulation of alpha-secretase activity in vitro and in vivo. Overexpression of the prodomain of furin and infection with a furin-specific inhibitor significantly reduced the levels of sAPPalpha regardless of PKC activity, whereas total APP levels remained unchanged. Furin mRNA levels in the brains of AD patients and Tg2576 mice were significantly lower than those in controls, whereas ADAM10 and TACE mRNA levels were much alike between Tg2576 and littermate mice. Moreover, the injection of furin-adenovirus into Tg2576 mouse brains markedly increased alpha-secretase activity and reduced beta-amyloid protein (Abeta) production in infected brain regions. Our results suggest that furin enhances alpha-secretase activity via the cleavage of ADAM10 and TACE, and that attenuated furin activity is connected to the production of Abeta.     

Characterisation of the binding of amyloid imaging tracers to rodent Abeta fibrils and rodent-human Abeta co-polymers

Despite the application of amyloid imaging agents such as PIB, SB13, and FDDNP in Alzheimer's disease (AD) patients, the successful use of these agents in transgenic mice models of AD has not been reported to date. As a first step in understanding the behaviour of these ligands in transgenic models of AD, we have investigated in a series of in vitro ligand binding assays the interaction of selected agents, including PIB, FDDNP, SB13, and BSB, with amyloid fibrils produced from rodent Abeta(1-40) (roAbeta) peptide. The data indicate that the ligand binding affinities together with the pattern and number of binding sites on the roAbeta fibrils are broadly conserved with that reported previously for human Abeta(1-40) (huAbeta) fibrils. However, characterisation of huAbeta fibrils formed in the presence of increasing amounts of roAbeta (1, 5, 10% w/w) demonstrated a dose-dependent reduction in the number of high affinity [(3)H]Me-BTA-1 binding sites such that at the highest amount of roAbeta the specific signal was reduced by approximately 95%. These studies suggest that (i) the presence of small amounts of roAbeta in huAbeta fibrils has the potential to cause subtle ultrastructural alterations in the polymers and (ii) the weak binding signal observed in vivo in the transgenic mouse models of AD may in part be due to the decreased number of high affinity binding sites on the Abeta fibrils.     

Pin1 promotes production of Alzheimer's amyloid beta from beta-cleaved amyloid precursor protein

Here we show that prolyl isomerase Pin1 is involved in the Abeta production central to the pathogenesis of Alzheimer's disease. Enzyme immunoassay of brains of the Pin1-deficient mice revealed that production of Abeta40 and Abeta42 was lower than that of the wild-type mice, indicating that Pin1 promotes Abeta production in the brain. GST-Pin1 pull-down and immunoprecipitation assay revealed that Pin1 binds phosphorylated Thr668-Pro of C99. In the Pin1-/- MEF transfected with C99, Pin1 co-transfection enhanced the levels of Abeta40 and Abeta42 compared to that without Pin1 co-transfection. In COS7 cells transfected with C99, Pin1 co-transfection enhanced the generation of Abeta40 and Abeta42, and reduced the expression level of C99, facilitating the C99 turnover. Thus, Pin1 interacts with C99 and promotes its gamma-cleavage, generating Abeta40 and Abeta42. Further, GSK3 inhibitor lithium blocked Pin1 binding to C99 by decreasing Thr668 phosphorylation and attenuated Abeta generation, explaining the inhibitory effect of lithium on Abeta generation.     

Clathrin-mediated endocytic proteins are upregulated in the cortex of the Tg2576 mouse model of Alzheimer's disease-like amyloid pathology

Amyloid-β (Aβ) is cleaved from amyloid precursor protein (APP) predominantly after APP has trafficked through the secretory pathway and then become re-internalised by endocytosis. Clathrin-mediated and, more recently, clathrin-independent endocytosis have both been implicated in this process. Furthermore, endocytic abnormalities have been identified in cases of Alzheimer’s disease (AD), however, the relevance of these changes to the aetiology of the disease remains unclear. We therefore examined the expression of proteins related to these endocytic processes in the cortex of Tg2576 mice that overexpress the Swedish mutation in APP, and consequently overexpress Aβ, to determine if there were any changes in their associated pathways. We identified significant increases in the levels of clathrin, dynamin and PICALM, all proteins intimately involved with the clathrin-mediated endocytic pathway, in the transgenic animals. However, levels of proteins associated with flotillin or caveolin-mediated endocytic pathways remained unchanged. These results emphasise the importance of clathrin-mediated endocytosis in the aetiology of AD and reinforce the results of the recent GWAS studies that identified genes for clathrin-mediated endocytosis as susceptibility genes for AD. Such studies in transgenic mice will allow us to learn more about the role of clathrin-mediated endocytosis in AD.     

Advanced glycation endproducts and pro-inflammatory cytokines in transgenic Tg2576 mice with amyloid plaque pathology

Increased expression and altered processing of the amyloid precursor protein (APP) and generation of beta-amyloid peptides is important in the pathogenesis of amyloid plaques in Alzheimer's disease (AD). Transgenic Tg2576 mice overexpressing the Swedish mutation of human APP exhibit beta-amyloid deposition in the neocortex and limbic areas, accompanied by gliosis and dystrophic neurites. However, murine plaques appear to be less cross-linked and the mice show a lower degree of inflammation and neurodegeneration than AD patients. 'Advanced glycation endproducts (AGEs)', formed by reaction of proteins with reactive sugars or dicarbonyl compounds, are able to cross-link proteins and to activate glial cells, and are thus contributing to plaque stability and plaque-induced inflammation in AD. In this study, we analyze the tissue distribution of AGEs and the pro-inflammatory cytokines IL-1beta and TNF-alpha in 24-month-old Tg2576 mice, and compare the AGE distribution in these mice with a younger age group (13 months old) and a typical Alzheimer's disease patient. Around 70% of the amyloid plaque cores in the 24-month-old mice are devoid of AGEs, which might explain their solubility in physiological buffers. Plaque associated glia, which express IL-1beta and TNF-alpha, contain a significant amount of AGEs, suggesting that plaques, i.e. Abeta as its major component, can induce intracellular AGE formation and the expression of the cytokines on its own. In the 13-month-old transgenic mice, AGEs staining can neither be detected in plaques nor in glial cells. In contrast, AGEs are present in high amounts in both plaques and glia in the human AD patient. The data obtained in this show interesting differences between the transgenic mouse model and AD patients, which should be considered using the transgenic approach to test therapeutical strategies to eliminate plaques or to attenuate the inflammatory response in AD.     

Amyloid beta peptide (Abeta42) activates PLC-delta1 promoter through the NF-kappaB binding site

The abnormal deposition of amyloid beta peptide (Abeta) is a hallmark of Alzheimer's disease (AD). Phospholipase C-delta1 (PLC-delta1) is also known to abnormally accumulate in the brains of AD patients, but no report has addressed the relationship between these two events. This study investigated the effect of Abeta42 on the PLC-delta1 expression in human neuroblastoma cell lines. The PLC-delta1 mRNA level was increased by treatment with Abeta42 in a RT-PCR analysis. In the reporter assay, Abeta42 was found to activate the PLC-delta1 promoter activity in a dose-dependent manner. A novel NF-kappaB binding site in the PLC-delta1 promoter appeared to be responsible for the Abeta42 activity. First, the dominant negative forms of the NF-kappaB activating molecules, dominant negative TGF-beta activated kinase 1 (dnTAK1) and dnNIK (dominant negative NF-kappaB-inducing kinase), abolished the Abeta42 activity in the reporter assay. Second, the Abeta42 augmented a factor binding on the NF-kappaB site in the electrophoretic mobility shift assay (EMSA), which was abolished by a molar excess of the unlabeled consensus NF-kappaB oligonucleotide. These results suggest that the PLC-delta1 promoter is under the control of NF-kappaB, which mediates the expression of PLC-delta due to the Abeta42 treatment.     

Downregulation of myosin II-B by siRNA alters the subcellular localization of the amyloid precursor protein and increases amyloid-beta deposition in N2a cells

The Alzheimer's disease (AD) brain pathology is characterized by extracellular deposits of amyloid-beta (Abeta) peptides and intraneuronal fibrillar structures. These pathological features may be functionally linked, but the mechanism by which Abeta accumulation relates to neuronal degeneration is still poorly understood. Abeta peptides are fragments cleaved from the amyloid precursor protein (APP), a transmembrane protein ubiquitously expressed in the nervous system. Although the proteolytic processing of APP has been implicated in AD, the physiological function of APP and the subcellular site of APP cleavages remain unknown. The overall structure of the protein and its fast anterograde transport along the axon support the idea that APP functions as a vesicular receptor for cytoskeletal motor proteins. In the current study, we test the hypothesis that myosin II, important contributor to the cytoskeleton of neuronal cells, may influence the trafficking and/or the processing of APP. Our results demonstrate that downregulation of myosin II-B, the major myosin isoform in neurons, is able to increase Abeta deposition, concomitantly altering the subcellular localization of APP. These new insights might be important for the understanding of the function of APP and provide a novel conceptual framework in which to analyze its pathological role.     

Cellular localization of Nicastrin affects amyloid beta species production

The gamma-secretase complex, composed by presenilin, nicastrin, APH-1 and PEN-2, is involved in intramembranous proteolysis of membrane proteins, such as amyloid precursor protein or Notch. Cleavage occurs in multiple cellular compartments. Here, nicastrin mutants containing targeting signals to the endoplasmic reticulum, trans-Golgi network, lysosomes, or plasma membrane have been shown to yield active gamma-secretase complexes with different activities and specificities: wild-type and plasma membrane nicastrin complexes yielded the highest amounts of secreted amyloid-beta peptide (Abeta), predominantly Abeta40, whereas intracellular targeted mutants produced intracellular Abeta, with a comparatively higher amount of Abeta42. These results suggest that compartmental microenvironments play a role in gamma-secretase activity and specificity.     

Effect of a short- and long-term treatment with Ginkgo biloba extract on amyloid precursor protein levels in a transgenic mouse model relevant to Alzheimer's disease

Several clinical trials have reported beneficial effects of the Ginkgo biloba extract EGb761 in the prevention and therapy of cognitive disorders including Alzheimer’s disease (AD). The aim of the present long-term feeding trial was to study the impact of dietary EGb761 on Amyloid precursor protein (APP) metabolism in mice transgenic for human APP (Tg2576). Tg2576 mice were fed diets with and without EGb761 (300 mg/kg diet) for 1 and 16 months, respectively. Long-term treatment (16 months) with EGb761 significantly lowered human APP protein levels by ∼50% as compared to controls in the cortex but not in the hippocampus. However, APP levels were not affected by EGb761 in young mice. Current data indicate that APP seems to be an important molecular target of EGb761 in relation to the duration of the Ginkgo biloba treatment and/or the age of the animals. Potential neuroprotective properties of EGb761 may be, at least partly, related to its APP lowering activity.     

ACAT inhibition and amyloid beta reduction

Alzheimer's disease (AD) is a devastating neurodegenerative disorder. Accumulation and deposition of the beta-amyloid (Aβ) peptide generated from its larger amyloid precursor protein (APP) is one of the pathophysiological hallmarks of AD. Intracellular cholesterol was shown to regulate Aβ production. Recent genetic and biochemical studies indicate that not only the amount, but also the distribution of intracellular cholesterol is critical to regulate Aβ generation. Acyl-coenzyme A: cholesterol acyl-transferase (ACAT) is a family of enzymes that regulates the cellular distribution of cholesterol by converting membrane cholesterol into hydrophobic cholesteryl esters for cholesterol storage and transport. Using pharmacological inhibitors and transgenic animal models, we and others have identified ACAT1 as a potential therapeutic target to lower Aβ generation and accumulation. Here we discuss data focusing on ACAT inhibition as an effective strategy for the prevention and treatment of AD.     

Specific reaction of Met 35 in amyloid beta peptide with hypochlorous acid

Abstract

The reaction of the amyloid beta peptide (Aβ) with hypochlorous acid and hydroxyl radicals was analysed by spectrophotometry and mass spectrometry. N-acetylmethionine, Aβ25-35 and Aβ1-42 reacted rapidly with hypochlorous acid. The relative reaction rates of N-acetylmethionine and Aβ with hypochlorous acid was in the order N-acetylmethionine > Aβ25-35 > Aβ1-42. While the reaction of Aβ25-35 in the presence of a slight excess of hypochlorous acid resulted in complete conversion of Met35 to Met35 sulphoxide, Aβ1-42 required more than a 4-fold excess of hypochlorous acid for complete conversion of Met35. Identical products were obtained when Aβ25-35 and Aβ1-42 were treated with a hypochlorous acid generating system. Conversion of Met35 to Met35 sulphoxide in Aβ abolished the aggregation of Aβ25-35. Reaction of Aβ with hydroxyl radicals resulted in limited conversion of Met35 to Met35 sulphoxide. The specific reaction of Met35 in Aβ with hypochlorous acid to form Met35 sulphoxide has been analysed.     

Modulation of Alzheimer's pathology by cerebro-ventricular grafting of hybridoma cells expressing antibodies against Abeta in vivo

Accumulation in brain of the beta-amyloid peptide (Abeta) is considered as crucial pathogenic event causing Alzheimer's disease (AD). Anti-Abeta immune therapy is a powerful means for Abeta clearance from the brain. We recently showed that intravenous injections of anti-Abeta antibodies led to reduction, elevation or no change in brain Abeta42 concentrations of an AD mouse model. We report here, in a second passive immunization protocol, a different bioactivity of same antibodies to alter brain Abeta42 concentrations. Comparing the bioactivity of anti-Abeta antibodies in these two passive immunization paradigms underscores the potential of immune therapy for AD treatment and suggests that both the epitope recognized by the antibody and the mode of antibody administration are crucial for its biological activity.     

Aggregation and neurotoxicity of mutant amyloid beta (A beta) peptides with proline replacement: importance of turn formation at positions 22 and 23

Aggregation of the amyloid beta peptides (A beta 1-42 and A beta 1-40) plays a pivotal role in pathogenesis of Alzheimer's disease. Although it is widely accepted that the aggregates of A betas mainly consist of beta-sheet structure, the precise aggregation mechanism remains unclear. To identify amino acid residues that are important for the beta-sheet formation, a series of proline-substituted mutants of A beta 1-42 peptides at positions 19-26 was synthesized in a highly pure form and their aggregation ability and neurotoxicity on PC12 cells were investigated. All proline-substituted A beta 1-42 mutants except for 22P- and 23P-A beta 1-42 were hard to aggregate and showed weaker cytotoxicity than wild-type A beta 1-42, suggesting that the residues at positions 19-21 and 24-26 are important for the beta-sheet formation. In contrast, 22P-A beta 1-42 extensively aggregated with stronger cytotoxicity than wild-type A beta 1-42. Since proline has a propensity for beta-turn structure as a Pro-X corner, these data implicate that beta-turn formation at positions 22 and 23 plays a crucial role in the aggregation and neurotoxicity of A beta peptides.     

Role of high mobility group protein-1 (HMG1) in amyloid-beta homeostasis

In Alzheimer's disease (AD), fibrillar amyloid-beta (Abeta) peptides form senile plaques associated with activated microglia. Recent studies have indicated that microglial Abeta clearance is facilitated by several activators such as transforming growth factor-beta1 (TGF-beta1). The relationship between microglia and Abeta formation and deposition is still unclear. In the present study, high mobility group protein-1 (HMG1) inhibited the microglial uptake of Abeta (1-42) in the presence and absence of TGF-beta1. In addition, HMG1 bound to Abeta (1-42) and stabilized the oligomerization. In AD brains, protein levels of HMG1 were significantly increased in both the cytosolic and particulate fractions, and HMG1 and Abeta were colocalized in senile plaques associated with microglia. These results suggest that HMG1 may regulate the homeostasis of extracellular Abeta (1-42) and Abeta oligomerization.     

The influence of simvastatin, atorvastatin and high-cholesterol diet on acetylcholinesterase activity, amyloid beta and cholesterol synthesis in rat brain

OBJECTIVE: There is evidence to suppose that cholesterol-lowering medicine might confer protection against dementia, probably via modulation of cholesterol synthesis in the brain. The aim of the present study was to investigate the potential influence of statins and cholesterol diet on selected parameters relevant to Alzheimer's disease pathophysiology. METHODS: For 15 days, rats were orally administered simvastatin (10 or 20mg/kg b.wt.), atorvastatin (10 or 20mg/kg b.wt.), or aqua (control group); and one group was fed high-cholesterol (2%) diet. At the end of experiments brain (and plasma) cholesterol, lathosterol, hydroxymethylglutaryl-coenzyme A reductase protein, acetylcholinesterase activity, amyloid beta (40 and 42) and cholesterol synthesis rate (using the incorporation of deuterium from deuterated water) were determined and statistically compared to those of aqua. RESULTS: Both statins were able to lower cholesterol in the plasma, but none elicited an effect on total brain cholesterol. Significant reductions of brain lathosterol and cholesterol synthesis rate were observed after simvastatin and atorvastatin treatment. Acetylcholinesterase activity, amyloid beta and hydroxymethylglutaryl-coenzyme A reductase levels remained unaffected by the two drugs. CONCLUSIONS: This study brings additional evidence of a role for statins in cholesterol synthesis in the brain. Our data question the relationship between amyloid beta, acetylcholinesterase activity and cholesterol synthesis in the rat brain as well as the assumption about no exchange between peripheral and brain cholesterol pools.     

Assembly of hereditary amyloid beta-protein variants in the presence of favorable gangliosides

The mechanisms underlying regional amyloid beta-protein (Abeta) deposition in brain remain unclear. Here we show that assembly of hereditary variant Dutch- and Italian-type Abetas, and Flemish-type Abeta was accelerated by GM3 ganglioside, and GD3 ganglioside, respectively. Notably, cerebrovascular smooth muscle cells, which compose the cerebral vessel wall at which the Dutch- and Italian-type Abetas deposit, exclusively express GM3 whereas GD3 is upregulated in the co-culture of endothelial cells and astrocytes, which forms the cerebrovascular basement membrane, the site of Flemish-type Abeta deposition. Our results suggest that regional Abeta deposition is induced by the local gangliosides in the brain.     

Renin inhibitor aliskiren exerts neuroprotection against amyloid beta-peptide toxicity in rat cortical neurons

Accumulation of amyloid β-peptide (Aβ) in senile plaques, a pathological hallmark of Alzheimer's disease (AD), has been implicated in neuronal degeneration. Renin-angiotensin system (RAS) blockers, including the renin inhibitor aliskiren, are a group of clinically relevant anti-hypertensive agents. The present study was initiated to investigate whether aliskiren may modulate Aβ neurotoxicity as an additional function aside from its established property of lowering blood pressure. We found aliskiren conferred neuronal resistance to Aβ toxicity in primary rat cortical cultures. Moreover, both Aβ25-35 and Aβ1-42 induced renin expression in cortical neurons; in parallel, a heightened expression of renin was detected in the cerebral cortices of 9-month-old AD transgenic mice. Notably, aliskiren blocked Aβ-mediated neuronal induction of renin. We therefore concluded that aliskiren may carry neuroprotective action against Aβ toxicity. Furthermore, the aliskiren effects may involve downregulation of renin expression induced by Aβ.