Genetic ablation of the p66Shc adaptor protein reverses cognitive deficits and improves mitochondrial function in an APP transgenic mouse model of Alzheimer's disease.

The mammalian ShcA adaptor protein p66Shc is a key regulator of mitochondrial reactive oxygen species (ROS) production and has previously been shown to mediate amyloid ß (Aß)-peptide-induced cytotoxicity in vitro. Moreover, p66Shc is involved in mammalian longevity and lifespan determination as revealed in the p66Shc knockout mice, which are characterized by a 30% prolonged lifespan, lower ROS levels and protection from age-related impairment of physical and cognitive performance. In this study, we hypothesized a role for p66Shc in Aß-induced toxicity in vivo and investigated the effects of genetic p66Shc deletion in the PSAPP transgenic mice, an established Alzheimer's disease mouse model of ß-amyloidosis. p66Shc-ablated PSAPP mice were characterized by an improved survival and a complete rescue of Aß-induced cognitive deficits at the age of 15 months. Importantly, these beneficial effects on survival and cognitive performance were independent of Aß levels and amyloid plaque deposition, but were associated with improved brain mitochondrial respiration, a reversal of mitochondrial complex I dysfunction, restored adenosine triphosphate production and reduced ROS levels. The results of this study support a role for p66Shc in Aß-related mitochondrial dysfunction and oxidative damage in vivo, and suggest that p66Shc ablation may be a promising novel therapeutic strategy against Aß-induced toxicity and cognitive impairment.

T-cell brain infiltration and immature antigen-presenting cells in transgenic models of Alzheimer's disease-like cerebral amyloidosis.

Cerebral beta-amyloidosis, one of the pathological hallmarks of Alzheimer's disease (AD), elicits a well-characterised, microglia-mediated local innate immune response. In contrast, it is not clear whether cells of the adaptive immune system, in particular T-cells, react to cerebral amyloidosis in AD. Even though parenchymal T-cells have been described in post-mortem brains of AD patients, it is not known whether infiltrating T-cells are specifically recruited to the extracellular deposits of beta-amyloid, and whether they are locally activated into proliferating, effector cells upon interaction with antigen-presenting cells (APCs). To address these issues we have analysed by confocal microscopy and flow-cytometry the localisation and activation status of both T-cells and APCs in transgenic (tg) mice models of AD-like cerebral amyloidosis. Increased numbers of infiltrating T-cells were found in amyloid-burdened brain regions of tg mice, with concomitant up-regulation of endothelial adhesion molecules ICAM-1 and VCAM-1, compared to non-tg littermates. The infiltrating T-cells in tg brains did not co-localise with amyloid plaques, produced less interferon-gamma than those in controls and did not proliferate locally. Bona-fide dendritic cells were virtually absent from the brain parenchyma of both non-tg and tg mice, and APCs from tg brains showed an immature phenotype, with accumulation of MHC-II in intracellular compartments. These results indicate that cerebral amyloidosis promotes T-cell infiltration but interferes with local antigen presentation and T-cell activation. The inability of the brain immune surveillance to orchestrate a protective immune response to amyloid-beta peptide might contribute to the accumulation of amyloid in the progression of the disease.

Post-ischaemic silencing of p66Shc reduces ischaemia/reperfusion brain injury and its expression correlates to clinical outcome in stroke.

AIM: Constitutive genetic deletion of the adaptor protein p66(Shc) was shown to protect from ischaemia/reperfusion injury. Here, we aimed at understanding the molecular mechanisms underlying this effect in stroke and studied p66(Shc) gene regulation in human ischaemic stroke. METHODS AND RESULTS: Ischaemia/reperfusion brain injury was induced by performing a transient middle cerebral artery occlusion surgery on wild-type mice. After the ischaemic episode and upon reperfusion, small interfering RNA targeting p66(Shc) was injected intravenously. We observed that post-ischaemic p66(Shc) knockdown preserved blood-brain barrier integrity that resulted in improved stroke outcome, as identified by smaller lesion volumes, decreased neurological deficits, and increased survival. Experiments on primary human brain microvascular endothelial cells demonstrated that silencing of the adaptor protein p66(Shc) preserves claudin-5 protein levels during hypoxia/reoxygenation by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. Further, we found that in peripheral blood monocytes of acute ischaemic stroke patients p66(Shc) gene expression is transiently increased and that this increase correlates with short-term neurological outcome. CONCLUSION: Post-ischaemic silencing of p66(Shc) upon reperfusion improves stroke outcome in mice while the expression of p66(Shc) gene correlates with short-term outcome in patients with ischaemic stroke.

Active vaccination with ankyrin G reduces ß-amyloid pathology in APP transgenic mice.

Serum antibodies against amyloid-ß peptide (Aß) in humans with or without diagnosis of Alzheimer's disease (AD) indicate the possibility of immune responses against brain antigens. In an unbiased screening for antibodies directed against brain proteins, we found in AD patients high serum levels of antibodies against the neuronal cytoskeletal protein ankyrin G (ankG); these correlated with slower rates of cognitive decline. Neuronal expression of ankG was higher in AD brains than in nondemented age-matched healthy control subjects. AnkG was present in exosomal vesicles, and it accumulated in ß-amyloid plaques. Active immunization with ankG of arcAß transgenic mice reduced brain ß-amyloid pathology and increased brain levels of soluble Aß(42). AnkG immunization induced a reduction in ß-amyloid pathology, also in Swedish transgenic mice(.) Anti-ankG monoclonal antibodies reduced Aß-induced loss of dendritic spines in hippocampal ArcAß organotypic cultures. Together, these data established a role for ankG in the human adaptive immune response against resident brain proteins, and they show that ankG immunization reduces brain ß-amyloid and its related neuropathology.

Early accumulation of intracellular fibrillar oligomers and late congophilic amyloid angiopathy in mice expressing the Osaka intra-Aß APP mutation.

Pathogenic amyloid-ß peptide precursor (APP) mutations clustered around position 693 of APP-position 22 of the Aß sequence--are commonly associated with congophilic amyloid angiopathy (CAA) and intracerebral hemorrhages. In contrast, the Osaka (E693Δ) intra-Aß APP mutation shows a recessive pattern of inheritance that leads to AD-like dementia despite low brain amyloid on in vivo positron emission tomography imaging. Here, we investigated the effects of the Osaka APP mutation on Aß accumulation and deposition in vivo using a newly generated APP transgenic mouse model (E22ΔAß) expressing the Osaka mutation together with the Swedish (K670N/M671L) double mutation. E22ΔAß mice exhibited reduced α-processing of APP and early accumulation of intraneuronal fibrillar Aß oligomers associated with cognitive deficits. In line with our in vitro findings that recombinant E22Δ-mutated Aß peptides form amyloid fibrils, aged E22ΔAß mice showed extracellular CAA deposits in leptomeningeal cerebellar and cortical vessels. In vitro results from thioflavin T aggregation assays with recombinant Aß peptides revealed a yet unknown antiamyloidogenic property of the E693Δ mutation in the heterozygous state and an inhibitory effect of E22Δ Aß42 on E22Δ Aß40 fibrillogenesis. Moreover, E22Δ Aß42 showed a unique aggregation kinetics lacking exponential fibril growth and poor seeding effects on wild-type Aß aggregation. These results provide a possible explanation for the recessive trait of inheritance of the Osaka APP mutation and the apparent lack of amyloid deposition in E693Δ mutation carriers.

Separation of presenilin function in amyloid beta-peptide generation and endoproteolysis of Notch.

Most of the genetically inherited Alzheimer's disease cases are caused by mutations in the presenilin genes, PS1 and PS2. PS mutations result in the enhanced production of the highly amyloidogenic 42/43 amino acid variant of amyloid beta-peptide (Abeta). We have introduced arbitrary mutations at position 286 of PS1, where a naturally occurring PS1 mutation has been described (L286V). Introduction of charged amino acids (L286E or L286R) resulted in an increase of Abeta42/43 production, which reached almost twice the level of the naturally occurring PS1 mutation. Although pathological Abeta production was increased, endoproteolysis of Notch and nuclear transport of its cytoplasmic domain was significantly inhibited. These results demonstrate that the biological function of PS proteins in the endoproteolysis of beta-amyloid precursor protein and Notch can be separated.