Christian Czech

Aβ42‐driven cerebral amyloidosis in transgenic mice reveals early and robust pathology

We have generated a novel transgenic mouse model on a C57BL/6J genetic background that coexpresses KM670/671NL mutated amyloid precursor protein and L166P mutated presenilin 1 under the control of a neuron‐specific Thy1 promoter element (APPPS1 mice). Cerebral amyloidosis starts at 6–8 weeks and the ratio of human amyloid (A)β42 to Aβ40 is 1.5 and 5 in pre‐depositing and amyloid‐depositing mice, respectively. Consistent with this ratio, extensive congophilic parenchymal amyloid but minimal amyloid angiopathy is observed. Amyloid‐associated pathologies include dystrophic synaptic boutons, hyperphosphorylated tau‐positive neuritic structures and robust gliosis, with neocortical microglia number increasing threefold from 1 to 8 months of age. Global neocortical neuron loss is not apparent up to 8 months of age, but local neuron loss in the dentate gyrus is observed. Because of the early onset of amyloid lesions, the defined genetic background of the model and the facile breeding characteristics, APPPS1 mice are well suited for studying therapeutic strategies and the pathomechanism of amyloidosis by cross‐breeding to other genetically engineered mouse models.

Amyloid-β and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer's disease mice

Alzheimers disease (AD) is characterized by amyloid-beta (Aβ)-containing plaques, neurofibrillary tangles, and neuron and synapse loss. Tangle formation has been reproduced in P301L tau transgenic pR5 mice, whereas APPswPS2N141I double-transgenic APP152 mice develop Aβ plaques. Cross-breeding generates triple transgenic (tripleAD) mice that combine both pathologies in one model. To determine functional consequences of the combined Aβ and tau pathologies, we performed a proteomic analysis followed by functional validation. Specifically, we obtained vesicular preparations from tripleAD mice, the parental strains, and nontransgenic mice, followed by the quantitative mass-tag labeling proteomic technique iTRAQ and mass spectrometry. Within 1,275 quantified proteins, we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Notably, deregulation of complex I was tau dependent, whereas deregulation of complex IV was Aβ dependent, both at the protein and activity levels. Synergistic effects of Aβ and tau were evident in 8-month-old tripleAD mice as only they showed a reduction of the mitochondrial membrane potential at this early age. At the age of 12 months, the strongest defects on OXPHOS, synthesis of ATP, and reactive oxygen species were exhibited in the tripleAD mice, again emphasizing synergistic, age-associated effects of Aβ and tau in perishing mitochondria. Our study establishes a molecular link between Aβ and tau protein in AD pathology in vivo, illustrating the potential of quantitative proteomics.

Intraneuronal Aβ accumulation precedes plaque formation in β-amyloid precursor protein and presenilin-1 double-transgenic mice

beta-Amyloid peptides are key molecules that are involved in the pathology of Alzheimers disease (AD). The source and place of the neurotoxic action of Abeta, however, is still a matter of controversial debates. In the present report, we studied the neuropathological events in a transgenic mouse model expressing human mutant beta-amyloid precursor protein and human mutant presenilin-1 in neurons. Western blot and immunohistochemical analysis revealed that intracellular Abeta staining preceded plaque deposition, which started in the hippocampal formation. At later stages, many neuritic Abeta positive plaques were found in all cortical, hippocampal and many other brain areas. Interestingly, intraneuronal Abeta staining was no longer detected in the brain of aged double-transgenic mice, which correlates with the typical neuropathology in the brain of chronic AD patients.

Time sequence of maturation of dystrophic neurites associated with Aβ deposits in APP/PS1 transgenic mice

Several novel transgenic mouse models expressing different mutant APPs in combination with mutant PS1 have been developed. These models have been analyzed to investigate the formation and progressive alterations of dystrophic neurites (DNs) in relation to Abeta deposits. In the most aggressive model, Abeta deposits appear as early as 2.5 months of age. Maturation of DNs was qualitatively quite similar among models and in some respect reminiscent of human AD pathology. From the onset of deposition, most if not all Abeta deposits were decorated with a high number of APP-, ubiquitin-, and MnSOD-immunoreactive DNs. Phosphorylated Tau DNs, however, appeared at a much slower rate and were more restricted. Mitochondrial dysfunction markers were observed in DNs: the frequency and the density per deposit of DNs accumulating cytochrome c, cytochrome oxidase 1, and Bax progressively increased with age. Later, the burden of reactive DNs was reduced around large compact/mature deposits. In addition, the previously described phenomenon of early intraneuronal Abeta accumulation in our models was associated with altered expression of APP protein as well as oxidative and mitochondrial stress markers occasionally in individual neurons. The present study demonstrates that oxidative and mitochondrial stress factors are present at several phases of Abeta pathology progression, confirming the neuronal dysfunction in APP transgenic mice.

Key factors in Alzheimer's disease: β-Amyloid precursor protein processing, metabolism and intraneuronal transport

During the last years it has become evident that the β‐amyloid (Aβ) component of senile plaques may be the key molecule in the pathology of Alzheimers disease (AD). The source and place of the neurotoxic action of Aβ, however, is still a matter of controversy. The precursor of the β‐amyloid peptide is the predominantly neuronal β‐amyloid precursor protein. We, and others, hypothesize that intraneuronal misregulation of APP leads to an accumulation of Aβ peptides in intracellular compartments. This accumulation impairs APP trafficking, which starts a cascade of pathological changes and causes the pyramidal neurons to degenerate. Enhanced Aβ secretion as a function of stressed neurons and remnants of degenerated neurons provide seeds for extracellular Aβ aggregates, which induce secondary degenerative events involving neighboring cells such as neurons, astroglia and macrophages/microglia.

Presenilins and Alzheimer’s disease: biological functions and pathogenic mechanisms

Alzheimers disease (AD) is the most common cause of dementia in the elderly population. Dementia is associated with massive accumulation of fibrillary aggregates in various cortical and subcortical regions of the brain. These aggregates appear intracellularly as neurofibrillary tangles, extracellularly as amyloid plaques and perivascular amyloid in cerebral blood vessels. The causative factors in AD etiology implicate both, genetic and environmental factors. The large majority of early-onset familial Alzheimers disease (FAD) cases are linked to mutations in the genes coding for presenilin 1 (PS1) and presenilin 2 (PS2). The corresponding proteins are 467 (PS1) and 448 (PS2) amino-acids long, respectively. Both are membrane proteins with multiple transmembrane regions. Presenilins show a high degree of conservation between species and a presenilin homologue with definite conservation of the hydrophobic structure has been identified even in the plant Arabidopsis thaliana. More than 50 missense mutations in PS1 and two missense mutations in PS2 were identified which are causative for FAD. PS mutations lead to the same functional consequence as mutations on amyloid precursor protein (APP), altering the processing of APP towards the release of the more amyloidogenic form 1-42 of Abeta (Abeta42). In this regard, the physical interaction between APP and presenilins in the endoplasmic reticulum has been demonstrated and might play a key role in Abeta42 production. It was hypothesized that PS1 might directly cleave APP. However, extracellular amyloidogenesis and Abeta production might not be the sole factor involved in AD pathology and several lines of evidence support a role of apoptosis in the massive neuronal loss observed. Presenilins were shown to modify the apoptotic response in several cellular systems including primary neuronal cultures. Some evidence is accumulating which points towards the beta-catenin signaling pathways to be causally involved in presenilin mediated cell death. Increased degradation of beta-catenin has been shown in brain of AD patients with PS1 mutations and reduced beta-catenin signaling increased neuronal vulnerability to apoptosis in cell culture models. The study of presenilin physiological functions and the pathological mechanisms underlying their role in pathogenesis clearly advanced our understanding of cellular mechanisms underlying the neuronal cell death and will contribute to the identification of novel drug targets for the treatment of AD.

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild‐type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild‐type APP and mutations implicated in Alzheimers disease and another brain beta‐amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane‐inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11–15 in the carbohydrate domain, the other encoded by exons 16–17 in the juxtamembraneous beta‐amyloid domain. Treatment of the cells with the N‐glycosylation inhibitor tunicamycin induced missorting of wild‐type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta‐amyloid‐dependent membrane proximal signal was the major contributor to axonal sorting, while N‐glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS‐insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta‐amyloid domain plays a major role in axonal delivery of APP.

Impaired Cu/Zn-SOD activity contributes to increased oxidative damage in APP transgenic mice.

Oxidative stress plays an important role in the pathogenesis of Alzheimers disease. To determine which mechanisms cause the origin of oxidative damage, we analyzed enzymatic antioxidant defense (Cu/Zn-superoxide dismutase Cu/Zn-SOD, glutathione peroxidase GPx and glutathione reductase GR) and lipid peroxidation products malondialdehyde MDA and 4-hydroxynonenal HNE in two different APP transgenic mouse models at 3-4 and 12-15 months of age. No changes in any parameter were observed in brains from PDGF-APP695(SDL) mice, which have low levels of Abeta and no plaque load. In contrast, Thy1-APP751(SL) mice show high Abeta accumulation with aging and plaques from an age of 6 months. In brains of these mice, HNE levels were increased at 3 months (female transgenic mice) and at 12 months (both gender), that is, before and after plaque deposition, and the activity of Cu/Zn-SOD was reduced. Interestingly, beta-amyloidogenic cleavage of APP was increased in female Thy1-APP751(SL) mice, which also showed increased HNE levels with simultaneously reduced Cu/Zn-SOD activity earlier than male Thy1-APP751(SL) mice. Our results demonstrate that impaired Cu/Zn-SOD activity contributes to oxidative damage in Thy1-APP751(SL) transgenic mice, and these findings are closely linked to increased beta-amyloidogenic cleavage of APP.

Intraneuronal APP/Aβ Trafficking and Plaque Formation in β-Amyloid Precursor Protein and Presenilin-1 Transgenic Mice

Neuropil deposition of β‐amyloid peptides Aβ40 and Aβ42 is believed to be the key event in the neurodegenerative processes of Alzheimers disease (AD). Since Aβ seems to carry a transport signal that is required for axonal sorting of its precursor β‐amyloid precursor protein (APP), we studied the intraneuronal staining profile of Aβ peptides in a transgenic mouse model expressing human mutant APP751 (KM670/671NL and V717I) and human mutant presenilin‐1 (PS‐1 M146L) in neurons. Using surface plasmon resonance we analyzed the Aβ antibodies and defined their binding profile to APP, Aβ40 and Aβ42. Immunohistochemical staining revealed that intraneuronal Aβ40 and Aβ42 staining preceded plaque deposition, which started at 3 months of age. Aβ was observed in the somatodendritic and axonal compartments of many neurons. Interestingly, the striatum, which lacks transgenic APP expression harbored many plaques at 10 months of age. This is most likely due to an APP/Aβ transport problem and may be a model region to study APP/Aβ trafficking as an early pathological event.

Metabolite Profiling of Alzheimer's Disease Cerebrospinal Fluid

Alzheimers disease (AD) is a neurodegenerative disorder characterized by progressive loss of cognitive functions. Today the diagnosis of AD relies on clinical evaluations and is only late in the disease. Biomarkers for early detection of the underlying neuropathological changes are still lacking and the biochemical pathways leading to the disease are still not completely understood. The aim of this study was to identify the metabolic changes resulting from the disease phenotype by a thorough and systematic metabolite profiling approach. For this purpose CSF samples from 79 AD patients and 51 healthy controls were analyzed by gas and liquid chromatography-tandem mass spectrometry (GC-MS and LC-MS/MS) in conjunction with univariate and multivariate statistical analyses. In total 343 different analytes have been identified. Significant changes in the metabolite profile of AD patients compared to healthy controls have been identified. Increased cortisol levels seemed to be related to the progression of AD and have been detected in more severe forms of AD. Increased cysteine associated with decreased uridine was the best paired combination to identify light AD (MMSE>22) with specificity and sensitivity above 75%. In this group of patients, sensitivity and specificity above 80% were obtained for several combinations of three to five metabolites, including cortisol and various amino acids, in addition to cysteine and uridine.