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Alzheimer's 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 APP sw PS2 N141I double-transgenic APP152 mice develop Aβ plaques. Cross-breeding generates triple transgenic ( triple AD) 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 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 Aβ dependent, both at the protein and activity levels. Synergistic effects of Aβ 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 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.

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) α7, 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 α7 nAChR activity. We have examined the role of phosphorylation by combining molecular biology, biochemistry, and electrophysiology in SH-SY5Y neuroblastoma cells,Xenopusoocytes, rat hippocampal interneurons, and neurons from the supraoptic nucleus, and we found tyrosine phosphorylation of α7 nAChRs. Tyrosine kinase inhibition by genistein decreased α7 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 α7 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 α7 nAChR-mediated responses, whereas expression of active Src reduced α7 nAChR activity. Mutant α7 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 α7 receptors was not affected by kinase or phosphatase inhibitors, these data show that functional properties of α7 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.

Apolipophorin III (apoLp-III) from the greater wax moth Galleria mellonella is an exchangeable insect apolipoprotein that consists of five amphipathic alpha-helices, sharing high sequence identity with apoLp-III from the sphinx moth Manduca sexta whose structure is available. To define the minimal requirement for apoLp-III structural stability and function, a C-terminal truncated apoLp-III encompassing residues 1-91 of this 163 amino acid protein was designed. Far-UV circular dichroism spectroscopy revealed apoLp-III(1-91) has 50% alpha-helix secondary structure content in buffer (wild-type apoLp-III 86%), increasing to essentially 100% upon interactions with dimyristoylphosphatidylcholine (DMPC). Guanidine hydrochloride denaturation studies revealed similar stability properties for wild-type apoLp-III and apoLp-III(1-91). Resistance to denaturation for both proteins increased substantially upon association with phospholipid. In the absence of lipid, wild-type apoLp-III was monomeric whereas apoLp-III(1-91) partly formed dimers and trimers. Discoidal apoLp-III(1-91)-DMPC complexes were smaller in diameter (13.5 nm) compared to wild-type apoLp-III (17.7 nm), and more molecules of apoLp-III(1-91) associated with the complexes. Lipid interaction revealed that apoLp-III(1-91) binds to modified spherical lipoprotein surfaces and efficiently transforms phospholipid vesicles into discoidal complexes. Thus, the first three helices of G. mellonella apoLp-III contain the basic features required for maintenance of the structural integrity of the entire protein.

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein. Its function, as currently understood, lies in the stabilization of low-density lipophorin particles (LDLp) crossing the hemocoel in phases of high energy consumption to deliver lipids from the fat body to the flight muscle cells. Recent studies with native Galleria mellonella-apoLp-III gave first indications of an unexpected role of that protein in insect immune activation. Here we report the immune activation by the recombinant protein, documenting a newly discovered correlation between lipid physiology and immune defense in insects. The complete cDNA sequence of G. mellonella-apoLp-III was identified by mixed oligonucleotide-primed amplification of cDNA (MOPAC), 3'-RACE-PCR, and cRACE-PCR. The sequence coding for the native protein was ligated into a pET-vector; this construct was transfected into Escherichia coli and overexpressed in the bacteria. Photometric turbidity assays with human low density lipoprotein (LDL) and transmission electron microscopy studies on apoLp-III-stabilized lipid discs revealed the full functionality of the isolated recombinant apoLp-III with regard to its lipid-association ability. For proving its immune-stimulating capacity, apoLp-III was injected into the hemocoel of last instar G. mellonella larvae and the antibacterial activity in cell-free hemolymph was determined 24 h later. As a result, the hemolymph samples of injected insects contained strongly increased antibacterial activities against E. coli as well as clearly enhanced lysozyme-like activities. From Northern blot analysis of total RNA from insects injected with apoLp-III or the bacterial immune provocator lipopolysaccharide, it could be concluded that the transcription rate of apoLp-III mRNA does not vary in comparison to untreated last instar larvae.

Apolipophorin III (apoLp-III) was isolated from the haemolymph of last instar larvae of Galleria mellonella. The ultraviolet (u.v.) spectrum and the N-terminal amino acid sequence reveal high similarities with the apoLp-III from Manduca sexta. The protein is heat-stable. The molecular mass of apoLp-III was determined to be 18 077 Da using mass spectrometry. The heat treatment (90 degrees C, 30 min) resulted in a pI shift from 6.6 for the non-heated to 6.1 for the heat-treated apoLp-III without change in the molecular mass, indicating that a conformational change might have been caused by the heat treatment, rather than covalent alterations. Intrahaemocoelic injection of pure apoLp-III into last instar G. mellonella larvae is followed by a dose-dependent increase of antibacterial activity in cell-free haemolymph of treated larvae 24 h after injection. Furthermore, pure apoLp-III enhances the phagocytic activity of isolated haemocytes in vitro. The newly discovered role of apoLp-III in inducing immune-related functions in insects is discussed in regard to the known features of this molecule in lipid metabolism. Arylphorin, another heat-stable protein in G. mellonella haemolymph, was likewise isolated in this study. The protein was identified by N-terminal protein sequencing, the sequence obtained exactly matches the known sequence data for this protein. Copyright 1997 Elsevier Science Ltd. All rights reserved

Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) represents the successful combination of retentate chromatography and mass spectrometry, and this technology is an integral part of Ciphergen's ProteinChip System, which was designed to answer biomedical questions by performing protein analyses on a single experimental platform. The quantification capability of the ProteinChip System is essential in all proteomic applications for which this technology is used. Here we describe methods and results for three short experiments mimicking realistic analytical challenges to provide practical examples of quantification.

We established an in vitro transfer system with monolayers of isolated plasmatocytes from Galleria mellonella. The plasmatocytes represent the main phagocytically active haemocyte type in this lepidopteran insect. Plasmatocytes to which hydrophilic silica beads were added as a phagocytosing agent served as ‘donor’ cells. Supernatants from these donor cultures were transferred to freshly prepared ‘recipient’ plasmatocyte monolayers. Subsequently, FITC (fluorescein-isothiocyanate) labelled yeast cells were added to the recipient monolayers and the phagocytic activity was determined using an FITC quenching assay with trypan blue. The phagocytic activity in plasmatocyte monolayers which received supernatants from phagocytically active donor cells was significantly higher than the activity of cells receiving supernatants from non-activated donor cells. Time course studies revealed that the inducing capacity of the donor cell supernatants was highest 2–4h after starting phagocytosis of the silica beads. Isolation of the responsible phagocytosis stimulating factor is still underway. From our investigations we can conclude that it must be a very small (

An in vitro microscopic fluorescence assay determining the phagocytic activity of isolated plasmatocytes of Galleria mellonella is described. It was developed to quantify insect cellular immune reactions. The assay, a modification of a method originally established for vertebrate blood cells, is based on the quenching effect of trypan blue on fluorescein-isothiocyanate-labelled yeast cells. Only ingested yeast cells retain their fluorescence after quenching and can be easily distinguished from adhering ones. The technique is highly reproducible and easy to perform. Using this method a phagocytosis-stimulating effect caused by a haemolymph fraction > 100 kDa isolated from G. mellonella is demonstrated.

Intrahaemocoelic injection of silica beads into Galleria mellonella (wax moth) larvae provoked strong cellular and humoral reactions similar to those normally occuring during an antibacterial defence. The cellular reactions of the haemocytes—investigated by light and scanning electron microscopy—comprised degranulation of granular cells and phagocytosis by plasmatocytes. The humoral responses—measured in cell free haemolymph as the increase of antibacterial activity against Echerichia coli K12 D31 and of lysozyme activity against Micrococcus luteus cell walls—were significantly enhanced in comparison to controls. Only hydrophylic but not hydrophobic silica beads provoked strong reactions. Plasmatocytes (PLs), the main phagocytic haemocytes of G. mellonella, were isolated by using a newly adapted nylon wool column technique. PL monolayers, prepared from isolated cells, exhibited a high purity by consisting of at least 90% PL. Scanning electron microscopy studies revealed that 64% of the isolated PLs showed phagocytic activity against the sterile silica beads in vitro. Intrahaemocoelic injection of supernatants from monolayers with phagocytosing plasmatocytes into naive larvae led to a clearly higher antibacterial activity against E. coli in haemolymph of recipients than the injection of supernatants from monolayers with non-phagocytosing cells. The earlier supposition that haemocyte-released factors induce the humoral immune response is further supported by these results.

Injection of ion exchange beads or sterile latex beads into the haemocoel of wax moth ( Galleria mellonella ) larvae provokes an humoral immune response measurable as antibacterial activity in cell-free haemolymph. The influence of provocator surface properties on the intensity of response was investigated. Comparing the antibacterial activities provoked by injection of anion and cation exchangers it can be concluded, that anion exchangers are better provocators than cation exchangers. The influence of physicochemical parameters was confirmed by the fact, that injection of latex beads pretreated with sulphuric acid lead to enhanced antibacterial activities in larvae as compared to the activities provoked by injection of untreated beads. In order to prove if it is possible to enhance the provocation capacity of beads by binding molecules to their surfaces, the following substances were covalently coupled onto the beads prior to injection: fibronectin, peptides with a cell adhesive signal of fibronectin (GLY-ARG-GLY-ASP-SER-PRO-LYS, GLY-ARG-GLY-ASP-SER, ARG-GLY-ASP-SER), poly-lysine, adjuvant peptide, lysozyme, bovine serum albumin, bovine plasma, cell-free haemolymph and haemolymph lysate supernatant (haemolymph preparations from G. mellonella ) Coupling of adjuvant peptide, fibronectin or haemolymph preparations enhanced the provocation capacity of beads very clearly. Likewise, but to a lower extent, an enhancement was noticed for nearly all the other substances tested in comparison to results obtained by injection of unloaded beads. The results will be discussed in relation to the known facts about the influence of provocator characteristics onto the onset of a cellular reaction by arthropod and vertebrate cells.

which express either the 4-repeat tau domain with the FTDP-17 mutation Delta-K280 (TauRD/DeltaK280 “pro-aggregation mutant”) or the 4-repeat tau domain with Delta-K280 deletion and two proline mutations in the hexapeptide motifs (TauRD/DeltaK280/I277P/I308 “antiaggregation mutant”). Results: The DeltaK280 mutation accelerates the aggregation of tau, but the inserted proline residues inhibit the tau aggregation in vitro and in cell models. Inducible transgene expression in mice was driven by a forebrain-specific CaMKII promoter in a Tet-Off system and can be suppressed by doxycycline. The pro-aggregation mutant showed aggregated tau in sarkosyl insoluble fractions and Gallyas silver stained neurofibrillary tangles from 3 months onwards, even though the level of the human tau protein was lower than endogenous mouse tau. Tau preparations from pro-aggregation mutant mice revealed PHFs by electron microscopy. Consistent with the tau pathology the neuronal loss was age-dependent and visible in the dentate gyrus as early as 5 months. The immunohistochemisty results showed phosphorylated tau at S262 missorted into the somatodendritic compartment of cortical and hippocampal neurons. The anti-aggregation mutant with a similar expression level as the pro-aggregation mutant did not show aggregated tau or neuronal loss, but missorting into the somatodendritic compartment. The level of synaptophysin, a presynaptic marker and the number of spine-synapses were reduced in the stratum radiatum of the pro-aggregation mutant, but not of the antiaggregation mutant. Six weeks of switching off the tau transgene in the pro-aggregation mutant lead to 90% reduction in the level of soluble human tau protein, to complete reversal of the pathological somatodendritic localization and of phosphorylation in the repeat domain. Remarkably, the aggregation was only partly reversed. The remaining aggregates did not contain the exogenous human tau, but the endogenous mouse tau. Conclusions: The results argue that a toxic species of tau, once introduced into neurons, can “poison” endogenous tau and propagate its aggregation for extended time periods. Supported by MPG and DFG.

Across all cultures and over all time periods, as early as the third millennium BC, indigo, a very intense blue vat dye, has been used to dye textiles. Even today, indigo is regarded as one of the most popular blue colorants in items such as blue jeans. While synthetic indigo is used to color textiles today, throughout history, a variety of plants have provided indigo. In this special feature article, René Csuk and co-authors propose to use atmospheric solids analysis probe mass spectrometry (ASAP-MS) to very rapidly and reliably identify indigo colorants tiny amounts in ancient historic fabrics without any sample preparation. The ionization in ASAP experiments is effected by Penning ionization or by the transfer of protons originating from protonated clusters of water. Dr. René Csuk is Professor of organic bioorganic chemistry at the Martin-Luther Universität Halle-Wittenberg (Halle, Germany). His main research interests are in organic and medicinal chemistry, chemical biology and phytochemistry. Graphical Abstract: Historical samples can be investigated conveniently using ASAP®-MS experiments. ASAP®-MS allowed for rapid direct sampling without any preparation of the sample prior to its measurements, and reliable results were generated in less than 1 minute. As demonstrated for indigoid-type dyes, the efficacy of ASAP®-MS analysis is independent of the type of fiber, the age of the sample, the workmanship, and the state of preservation.

Nicotinic acetylcholine receptors (nAChRs) exist in many subtypes and are found in the peripheral and central nervous system where they mediate or modulate synaptic transmission. We review how tyrosine phosphorylation and kinases regulate muscle and neuronal nAChRs. Interestingly, although some of the same kinase players interact with the various receptor subtypes, the functional consequences are different. While concerted action of MuSK, Abl- and Src-family kinases (SFKs) regulates the synaptic distribution of nAChRs at the neuromuscular junction, SFKs activate heteromeric neuronal nAChRs in adrenal chromaffin cells, thereby enhancing catecholamine secretion. In contrast, the activity of homomeric neuronal nAChRs, as found in the hippocampus, is negatively regulated by tyrosine phosphorylation and SFKs. It appears that tyrosine kinases provide the means to regulate all nAChRs; but the functional consequences, even those caused by the same kinase family, are specific for each receptor subtype and location

Apolipophorin III (apoLp-III) is an exchangeable insect apolipoprotein consisting of five amphipathic alpha-helices. The protein is able to open reversibly on associating with hydrophobic surfaces and plays a role both in lipid transport and induction of immune responses. Point mutations were introduced at positions 66 (N--D) and/or 68 (K--E) between helices 2 and 3, a region possibly serving as a hinge for the opening of the molecule when associating with lipids. The lipid-binding properties of the mutant proteins were analyzed and compared with their immune inducing activities. Structural properties of the proteins were studied by far UV circular dichroism spectroscopy and their abilities to form discoidal complexes of dimyristoyl phosphatidylcholine (DMPC) vesicles were investigated. In comparison to wild-type apoLp-III, apoLp-III(N66D/K68E), and apoLp-III(K68E) displayed significantly decreased lipid-binding abilities and immune stimulating activities, while these effects were less noticeable with apoLp-III(N66D). The secondary structure of the double mutant apoLp-III(N66D/K68E) was similar to that of wild-type apoLp-III. A noticeable reduction of alpha-helical content could be observed for the single mutants apoLp-III(N66D) and apoLp-III(K68E), which was accompanied by an increase in percentage amount of beta-turns. The stability of the secondary structure determined by heat denaturation was not affected by mutagenesis. Furthermore, the ability of all proteins to form discoidal complexes of equal size and shape in the presence of dimyristoyl phosphatidylcholine indicated that the mutagenesis did not affect the molecular architecture in the lipid-associated conformation. The relationship between reduced lipid association and reduced immune stimulating activity supports the hypothesis that apoLp-III-induced immune activation is triggered by the conformational change of the protein.

A few years ago, it was shown that intrahemocoelic injection of the insect apolipoprotein apolipophorin III (apoLp-III) stimulates an immune response in larvae of the greater wax moth, Galleria mellonella . Since the mode of action of this activation process is unknown, we followed apoLp-III's pathway in the early phase of the immune-stimulating process, using biotin as a probe. Biotinylated apoLp-III was injected and localized using avidin-coupled horseradish peroxidase. The labeled protein was fully functional; the added amount of biotin per apoLp-III molecule used in this study only slightly decreased its ability to associate with phospholipase C-treated human low-density lipoprotein, as well as the immune-stimulating capability of apoLp-III. Gel electrophoresis with subsequent staining of biotin moieties and lipids revealed that apoLp-III undergoes lipid association in vivo within the first few minutes after injection. After two hours, no biotinylated apoLp-III was detectable in cell-free hemolymph. At this time, a subpopulation of hemocytes showed a distinct peroxidase staining. Control injections of biotinylated bovine serum albumin did not lead to similar results, giving evidence for the specificity of the phenomena observed. The results indicate that lipid association of apoLp-III occurs prior to endocytosis by immune-competent hemocytes, which is followed by the induction of a humoral immune response.

In this study the authors give immunocytochemical evidence for the presence of interleukin (IL)-1alpha- and tumour necrosis factor (TNF) alpha-like molecules in the haemocytes of last instar larvae from the greater wax moth Galleria mellonella. Similar results are demonstrated in a continuous haemocyte line (BTI-EA-1174-A) from the salt marsh caterpillar Estigmene acraea. In Galleria mellonella larvae granular cells show a strong positive reaction with both primary antibodies, whereas plasmatocytes are stained to a lesser extent. Cell line haemocytes also react positively with both antibodies. After activating the cells with lipopolysaccharide (LPS) staining of Estigmene acraea cells is decreased, whereas Galleria mellonella haemocytes show no visible reaction in comparison to non-activated cells.

The hemocyte line BTI-EA-1174-A from the lepidopteran insect Estigmene acraea responds to bacterial lipopolysaccharide (LPS) by an enhanced phagocytic reaction and a dose-dependent increase of lysozyme release [Wittwer et al., Dev Comp Immunol 21:323 (1997)]. This paper provides evidence for a strong proteolytic activity in cell culture supernatants occuring after addition of LPS (1 mg/ml). The proteolysis is caused by cell-released proteases and seems to be necessary for cell activation. Its inhibition by α2-macroglobulin results in a dose-dependent reduction in cellular response strength. Phagocytic reactions, as well as lysozyme release, are lowered to about half in the presence of 0.0001 mg/ml α2-macroglobulin. A nearly complete abolishment of activation was achieved with final concentrations of 1.0 mg/ml α2-macroglobulin. The data presented allow us to conclude that the LPS-triggered proteolytic activity is an important part of the activation process; it occurs outside of the cells and delivers immune response activating factors. Arch. Insect Biochem. Physiol. 39:91–97, 1998. © 1998 Wiley-Liss, Inc.

The suitability of the hemocyte cell line BTI-EA-1174-A from Estigmene acraea (Lepidoptera) to serve as a tool for studying insect immune reactions in vitro was investigated. Addition of bacterial lipopolysaccharides to the cultures caused enhanced phagocytosis of silica beads, as well as increased lysozyme activity in the cell culture supernatants. Addition of fungal beta 1,3-glucans did not result in any activation. The LPS-influenced (1 mg/mL) increase of phagocytic reactions against the silica beads was at its highest within 24 h after LPS-addition. Activated cells exhibited drastic changes in their morphology in connection with reduced cell numbers in the cultures but without increased mortality rates. LPS-dosages higher than 10 micrograms/mL LPS provoked significantly enhanced lysozyme activities. A maximal induction took place with 1 mg/mL LPS. The lysozyme activity started to rise 2 days after LPS-addition, further increase was observed up to the seventh day. The responsible protein was isolated from cell culture supernatants and N-terminally sequenced. The exact molecular mass was determined by mass spectrometry as 14.080 kDa. The amino acid sequence of the analysed portion revealed high sequence-similarity to the lysozymes of other lepidopteran insects as well as to hen egg lysozyme. Further results presented in this paper give indications for the existence of soluble molecules which are released by the cells and which enhance the LPS-triggered activation.