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

Alzheimer's disease (AD) is characterized by amyloid-beta (Abeta)-containing plaques, neurofibrillary tangles, and neuron and synapse loss. Tangle formation has been reproduced in P301L tau transgenic pR5 mice, whereas APP(sw)PS2(N141I) double-transgenic APP152 mice develop Abeta plaques. Cross-breeding generates triple transgenic ((triple)AD) mice that combine both pathologies in one model. To determine functional consequences of the combined Abeta and tau pathologies, we performed a proteomic analysis followed by functional validation. Specifically, we obtained vesicular preparations from (triple)AD 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 Abeta dependent, both at the protein and activity levels. Synergistic effects of Abeta and tau were evident in 8-month-old (triple)AD 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 (triple)AD mice, again emphasizing synergistic, age-associated effects of Abeta and tau in perishing mitochondria. Our study establishes a molecular link between Abeta and tau protein in AD pathology in vivo, illustrating the potential of quantitative proteomics.

Functional genomics dissects pathomechanisms in tauopathies: mitosis failure and unfolded protein response.

BACKGROUND: Alzheimer's disease (AD) is characterized by beta-amyloid (Abeta) peptide-containing plaques and tau-containing neurofibrillary tangles. By intracerebral injection of Abeta(42), both pathologies have been combined in P301L tau mutant mice. Furthermore, in cell culture, Abeta(42) induces tau aggregation. While both Abeta(42) and mutant tau cause neuronal dysfunction, their modes of action are only vaguely understood. METHODS: To determine which processes are disrupted by Abeta(42) and/or P301L mutant tau, we used transcriptomic and proteomic techniques followed by functional validation and analysis of human AD tissue. RESULTS: Our transcriptomic study in the SH-SY5Y cell culture system revealed that Abeta(42) and P301L tau expression independently affect genes controlling the cell cycle and cell proliferation. Proteomics applied to Abeta(42)-treated P301L tau-expressing SH-SY5Y cells and the amygdala of Abeta(42)-injected P301L transgenic mice revealed that a significant fraction of proteins altered in both systems belonged to the same functional categories, i.e. stress response and metabolism. Among the proteins identified was valosin-containing protein (VCP), a component of the quality control system during endoplasmic reticulum stress. Mutations in VCP have recently been linked to frontotemporal dementia. CONCLUSION: Our data support the mitosis failure hypothesis that claims that aberrant cell cycle reentry of postmitotic neurons induces apoptosis. Furthermore, our data underline a role of Abeta(42) in the stress response associated with protein folding.

A decade of tau transgenic animal models and beyond.

The first tau transgenic mouse model was established more than a decade ago. Since then, much has been learned about the role of tau in Alzheimer's disease and related disorders. Animal models, both in vertebrates and invertebrates, were significantly improved and refined as a result of the identification of pathogenic mutations in Tau in human cases of frontotemporal dementia. They have been instrumental for dissecting the cross-talk between tau and the second hallmark lesion of Alzheimer's disease, the Abeta peptide-containing amyloid plaque. We discuss how the tau models have been used to unravel the pathophysiology of Alzheimer's disease, to search for disease modifiers and to develop novel treatment strategies. While tau has received less attention than Abeta, it is rapidly acquiring a more prominent position and the emerging view is one of a synergistic action of Abeta and tau in Alzheimer's disease. Moreover, the existence of a number of neurodegenerative diseases with tau pathology in the absence of extracellular deposits underscores the relevance of research on tau.

Alpha7 neuronal nicotinic acetylcholine receptors are negatively regulated by tyrosine phosphorylation and Src-family kinases.

Nicotine, a component of tobacco, is highly addictive but possesses beneficial properties such as cognitive improvements and memory maintenance. Involved in these processes is the neuronal nicotinic acetylcholine receptor (nAChR) alpha7, whose activation triggers depolarization, intracellular signaling cascades, and synaptic plasticity underlying addiction and cognition. It is therefore important to investigate intracellular mechanisms by which a cell regulates alpha7 nAChR activity. We have examined the role of phosphorylation by combining molecular biology, biochemistry, and electrophysiology in SH-SY5Y neuroblastoma cells, Xenopus oocytes, rat hippocampal interneurons, and neurons from the supraoptic nucleus, and we found tyrosine phosphorylation of alpha7 nAChRs. Tyrosine kinase inhibition by genistein decreased alpha7 nAChR phosphorylation but strongly increased acetylcholine-evoked currents, whereas tyrosine phosphatase inhibition by pervanadate produced opposite effects. Src-family kinases (SFKs) directly interacted with the cytoplasmic loop of alpha7 nAChRs and phosphorylated the receptors at the plasma membrane. SFK inhibition by PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656 (2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide) increased alpha7 nAChR-mediated responses, whereas expression of active Src reduced alpha7 nAChR activity. Mutant alpha7 nAChRs lacking cytoplasmic loop tyrosine residues because of alanine replacement of Tyr-386 and Tyr-442 were more active than wild-type receptors and insensitive to kinase or phosphatase inhibition. Because the amount of surface alpha7 receptors was not affected by kinase or phosphatase inhibitors, these data show that functional properties of alpha7 nAChRs depend on the tyrosine phosphorylation status of the receptor and are the result of a balance between SFKs and tyrosine phosphatases. These findings reveal novel regulatory mechanisms that may help to understand nicotinic receptor-dependent plasticity, addiction, and pathology.

A technical triade for proteomic identification and characterization of cancer biomarkers.

Biomarkers are needed to elucidate the biological background and to improve the detection of cancer. Therefore, we have analyzed laser-microdissected cryostat sections from head and neck tumors and adjacent mucosa on ProteinChip arrays. Two differentially expressed proteins (P = 3.34 x 10(-5) and 4.6 x 10(-5)) were isolated by two-dimensional gel electrophoresis and identified as S100A8 (calgranulin A) and S100A9 (calgranulin B) by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immunodepletion assay. The relevance of these single marker proteins was evaluated by immunohistochemistry. Positive tissue areas were reanalyzed on ProteinChip arrays to confirm the identity of these proteins. As a control, a peak with low P was identified as calgizzarin (S100A11) and characterized in the same way. This technical triade of tissue microdissection, ProteinChip technology, and immunohistochemistry opens up the possibility to find, identify, and characterize tumor relevant biomarkers, which will allow the movement toward the clonal heterogeneity of malignant tumors. Taking this approach, proteins were identified that might be responsible for invasion and metastasis.

A fast and reliable detection of indigo in historic and prehistoric textile samples.

We present a method requiring no sample preparation for the direct identification of indigoid colorants in tiny amounts in ancient historic fabrics using ASAP®-MS. Exact determinations were completed in less than 1 min. Copyright © 2015 John Wiley & Sons, Ltd.

Detection of tumor markers with ProteinChip technology.

The early diagnosis of cancer at a curable stage is crucial for the successful treatment of this disease. Most of the currently used tumor assays appear too late and rely on single biomarkers with high false-negative and/or false-positive rates. As an additional burden for the patient, the traditional assays often require biopsy material instead of less invasively taken samples like serum. With the hope for more reliable DNA- and RNA-based screening tools, the research activities of the past 20 years have focused on the genomic characteristics of cancer cells. But, up to now, the output from this strategy has been disappointingly low and the disillusionment is paired with a return to proteins as the real key players in all physiological and pathological processes. Meanwhile, comparative protein profiling is generally acknowledged as a promising way for the detection of specific and predictive protein patterns reflecting certain stages of cancer without dependency on single markers. To meet the new technological demands, the ProteinChip Biomarker System was developed for the Expression Difference Mapping analysis of several hundreds of samples per day on a single, uncomplicated platform; with software support for the construction of multi-marker predictive models. The Interaction Discovery Mapping platform is introduced as the next methodical step for investigations about protein binding partners of possible importance in diagnosis and therapy. This review summarizes the current state in cancer diagnosis, provides an introduction into the ProteinChip technology, and gives an update on publications and research collaborations in SELDI-based tumor marker discovery.

Surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI TOF-MS) and ProteinChip technology in proteomics research.

In this review article, we describe some of the studies that have been performed using the surface-enhanced laser desorption ionization (SELDI) time-of-flight mass spectrometry and ProteinChip technology over the past few years, and highlight both their findings as well as limitations. Proteomic applications, such as target or marker identification and target validation or toxicology, will be addressed. We will also provide an examination of SELDI technology and go into the question of where possible future research may lead us.

PICK1 interacts with alpha7 neuronal nicotinic acetylcholine receptors and controls their clustering.

Central to synaptic function are protein scaffolds associated with neurotransmitter receptors. Alpha7 neuronal nicotinic acetylcholine receptors (nAChRs) modulate network activity, neuronal survival and cognitive processes in the CNS, but protein scaffolds that interact with these receptors are unknown. Here we show that the PDZ-domain containing protein PICK1 binds to alpha7 nAChRs and plays a role in their clustering. PICK1 interacted with the alpha7 cytoplasmic loop in yeast in a PDZ-dependent way, and the interaction was confirmed in recombinant pull-down experiments and by co-precipitation of native proteins. Some alpha7 and PICK1 clusters were adjacent at the surface of SH-SY5Y cells and GABAergic interneurons in hippocampal cultures. Expression of PICK1 caused decreased alpha7 clustering on the surface of the interneurons in a PDZ-dependent way. These data show that PICK1 negatively regulates surface clustering of alpha7 nAChRs on hippocampal interneurons, which may be important in inhibitory functions of alpha7 in the hippocampus.

Differential lipid binding of truncation mutants of Galleria mellonella apolipophorin III.

Apolipophorin III (apoLp-III) is a prototype exchangeable apolipoprotein that is amenable to structure-function studies. The protein folds as a bundle of five amphipathic alpha-helices and undergoes a dramatic conformational change upon lipid binding. Recently, we have shown that a truncation mutant of Galleria mellonella apoLp-III comprising helices 1-3 is stable in solution and able to bind to lipid surfaces [Dettloff, M., Weers, P. M. M., Niere, M., Kay, C. M., Ryan, R. O., and Wiesner, A. (2001) Biochemistry 40, 3150-3157]. To investigate the role of the C-terminal helices in apoLp-III structure and function, two additional 3-helix mutants were designed: a core fragment comprising helix (H) 2-4, and a C-terminal fragment (H3-5). Each truncation mutant retained the ability to associate spontaneously with dimyristoylphosphatidylcholine (DMPC) vesicles, transforming them into discoidal complexes. The rate of apolipoprotein-dependent DMPC vesicle transformation decreased in the order H1-3 > H2-4 > H3-5. Truncation of two helices led to a significant decrease in alpha-helical content in buffer in each case, from 86% (wild-type) to 50% (H1-3), 28% (H2-4), and 24% alpha-helical content (H3-5). On the other hand, trifluoroethanol or complexation with DMPC induced the truncation mutants to adopt a high alpha-helical structure similar to that of wild-type protein (84-100% alpha-helical structure). ApoLp-III(H1-3) and apoLp-III(H2-4), but not apoLp-III(H3-5), were able to prevent phospholipase-C-induced low density lipoprotein aggregation, indicating that interaction of the C-terminal fragment with spherical lipoprotein surfaces was impaired. As lipoprotein binding is significantly affected and DMPC transformation rates are relatively slow upon removal of N-terminal helices, the data indicate that structural elements necessary for lipid interaction reside in the N-terminal part of the protein.

Biomarker discovery and identification in laser microdissected head and neck squamous cell carcinoma with ProteinChip technology, two-dimensional gel electrophoresis, tandem mass spectrometry, and immunohistochemistry.

Head and neck cancer is a frequent malignancy with a complex, and up to now not clear etiology. Therefore, despite of improvements in diagnosis and therapy, the survival rate with head and neck squamous-cell carcinomas is poor. For a better understanding of the molecular mechanisms behind the process of tumorigenesis and tumor progression, we have analyzed changes of protein expression between microdissected normal pharyngeal epithelium and tumor tissue by ProteinChip technology. For this, cryostat sections from head and neck tumors (n = 57) and adjacent mucosa (n = 44) were laser-microdissected and analyzed on ProteinChip arrays. The derived mass spectrometry profiles exhibited numerous statistical differences. One peak significantly higher expressed in the tumor (p = 0.000029) was isolated by two-dimensional gel electrophoresis and identified as annexin V by in-gel proteolytic digestion, peptide mapping, tandem mass spectrometry analysis, and immuno-deplete assay. The relevance of this single marker protein was further evaluated by immunohistochemistry. Annexin-positive tissue areas were re-analyzed on ProteinChip arrays to confirm the identity of this protein. In this study, we could show that biomarker in head and neck cancer can be found, identified, and assessed by combination of ProteinChip technology, two-dimensional gel electrophoresis, and immunohistochemistry. In our experience, however, such studies only make sense if a relatively pure microdissected tumor tissue is used. Only then minute changes in protein expression between normal pharyngeal epithelium and tumor tissue can be detected, and it will become possible to educe a tumor-associated protein pattern that might be used as a marker for tumorigenesis and progression.