Amyloid beta/A4 Protein Precursor Research Articles

Developmental alcohol exposure leads to a persistent change on astrocyte secretome.

Fetal alcohol spectrum disorder is the most common cause of mental disabilities in the western world. It has been quite established that acute alcohol exposure can dramatically affect astrocyte function. Because the effects of early alcohol exposure on cell physiology can persist into adulthood, we tested the hypothesis that ethanol exposure in ferrets during a period equivalent to the last months of human gestation leads to persistent changes in astrocyte secretome invitro. Animals were treated with ethanol (3.5g/kg) or saline between postnatal day (P)10-30. At P31, astrocyte cultures were made and cells were submitted to stable isotope labeling by amino acids. Twenty-four hour conditioned media of cells obtained from ethanol- or saline-treated animals (ET-CM or SAL-CM) were collected and analyzed by quantitative mass spectrometry in tandem with liquid chromatography. Here, we show that 65 out of 280 quantifiable proteins displayed significant differences comparing ET-CM to SAL-CM. Among the 59 proteins that were found to be reduced in ET-CM we observed components of the extracellular matrix such as laminin subunits α2, α4, β1, β2, and γ1 and the proteoglycans biglycan, heparan sulfate proteoglycan 2, and lumican. Proteins with trophic function such as insulin-like growth factor binding protein 4, pigment epithelium-derived factor, and clusterin as well as proteins involved on modulation of proteolysis such as metalloproteinase inhibitor 1 and plasminogen activator inhibitor-1 were also reduced. In contrast, pro-synaptogeneic proteins like thrombospondin-1, hevin as well as the modulator of extracelular matrix expression, angiotensinogen, were found increased in ET-CM. The analysis of interactome maps through ingenuity pathway analysis demonstrated that the amyloid beta A4 protein precursor, which was found reduced in ET-CM, was previously shown to interact with ten other proteins that exhibited significant changes in the ET-CM. Taken together our results strongly suggest that early exposure to teratogens such as alcohol may lead to an enduring change in astrocyte secretome. Despite efforts in prevention, fetal alcohol spectrum disorders are a major cause of mental disabilities. Here, we show that developmental exposure to alcohol lead to a persistent change in the pattern of proteins secreted (secretome) by astrocytes. This study is also the first mass spectrometry-based assessment of the astrocyte secretome in a gyrencephalic animal. Cover Image for this issue: doi: 10.1111/jnc.13320.

Proteome Profiling of Human Cerebrospinal Fluid: Exploring the Potential of Capillary Electrophoresis with Surface Modified Capillaries for Analysis of Complex Biological Samples

A bottom-up proteomic approach, based on capillary electrophoresis (CE) in combination with matrix- assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToF MS), was used to analyze immunoaffinity depleted human cerebrospinal fluid (CSF) and compare it with a non-depleted sample. After enzymatic digestion and desalting, the tryptic peptides were separated by CE using PolyE-323 modified capillaries and fractionated off-line onto MALDI target plates for further analysis by MALDI-MS and MS/MS. The protein profile of the depleted sample was compared with non depleted CSF. Overall, 85 proteins were identified with 95% significance in both samples. The significance scores for proposed biomarkers, such as amyloid-like protein 1 precursor, could be increased up to 12 times after the depletion. Other proteins, often suggested to be related to neurodegenerative diseases, like amyloid beta A4 protein precursor, superoxide dismutase and apolipoprotein E precursor could only be found in the depleted CSF samples. The effect of a derivatization of tryptic peptides with 2- methoxy-4,5-dihydro-1H-imidazole reagent for protein identification with MS was also employed to increase the number of identified proteins and the sequence coverages. The results presented in this study illustrate the benefit of combining a sample pre-fractionation step and a label's ability to enhance the ionization efficiency with the potential of CE using PolyE-323 modified capillaries in the analysis of complex samples. The straight-forward approach that provides speed and simplicity resulting in high-resolution separations and low sample consumption represents an easily applicable separation technique that can serve as a complement to other currently existing analytical approaches needed in modern proteomic analysis of clinically relevant samples.

Long term dietary supplementation of organic selenium enhances expression of immune related genes in adult pigs

Ninety six pigs at 34.4 kg weight were allotted to two dietary treatments with 6 replicates per treatment and 8 pigs per pen: the control (no added Se) and the OS (0.36 mg/kg added Se from Se enriched yeast). Pigs were fed for 110 days until 130.0 kg body weight based on 3 phase feeding programs with nutrient levels recommended by National Research Council only except for Se. The control diet contained 0.18 mg/kg indigenous Se whereas the OS diet contained 0.54 mg/kg Se. Blood samples were collected at 1000 h and further processed for the microarray analysis. Microarray chips were prepared with 800 genes related to immune function of pigs. Among those, 27 genes related to improved immune status and innate immunity were up-regulated (P < 0.05) and those include growth arrest homeobox transcription factor (43.9%), toll-like receptor 2 precursor (12.2%), vanin-1 (65.5%), lysozyme C-3 precursor (10.8%), amyloid beta A4 protein precursor (91.1%), integrin beta-1 subunit (20.0%), myelin and lymphocyte protein (51.2%), etc. This study indicates that long term dietary supplementation (0.3%) of Se as an organic form improves the expression of genes that are related to enhanced immunity of pigs.

A New Text Mining Approach for Finding Protein-to-Disease Associations

Discovering significant relationships between biological entities from text documents is an important task for biologists in order to develop biological models for research and discovery, especially with the existing gigantic amounts of biomedical documents and the rate at which they are increasing everyday. We propose a new text mining method to extract associations between biological entities from text documents; and we focus and apply the method in our experiments on discovering proteins-to-diseases associations. The proposed method uses two sets of documents on the topic of interest [a negative set and positive (or relevant) set] and utilizes the concepts of expectation (ex), evidence (ev) and Z-scores in combining positive and negative evidences in determining the significant associations. Moreover, the method offers an efficient way to handle protein names, aliases and abbreviations and to disambiguate them from common abbreviations, gene symbols and such. We evaluated the method in discovering protein-to-disease associations from Medline abstracts and the results are very encouraging. We confirmed the correctness of the results, in each experiment, through articles from Medline. Our method was able to discover associations between certain proteins and various diseases like Alzheimer, Creutzfeldt-Jakob, Crohn Disease, Dengue, Jaundice, Lung cancer and more. For example, in Alzheimer test, the method ran on 83,933 abstracts and discovered that Alzheimer has significant association with 6 proteins, among them, Amyloid beta A4 protein precursor, Apolipoprotein E precursor and Presenilin 1 [PMIDs: 8596911, 1465129, 8346443, 12614323, 8766720 and 8878479]. We further tested our method on some already discovered and published relationships between genes and diseases and the method was also successful in supporting those discoveries.

APP is required during an early phase of memory formation

The amyloid beta/A4 protein precursor (APP) has been shown to be implicated in age-associated plastic changes at synapses that might contribute to memory loss in Alzheimer's disease. As APP has previously been reported to have multiple functions during normal development, we have employed a one-trial passive avoidance task in day-old chicks to study its role in the process of memory formation. Administration of anti-APP antibodies, injected 30 min pretraining, prevented memory for a one-trial passive avoidance task in day-old chicks without effects on general behaviour or initial acquisition. Amnesia was apparent by 30 min post-training and lasted for at least 24 h. The same result was obtained by down-regulation of APP expression by APP-antisense, injected 8-12 h pretraining. However, injections of anti-APP antibodies or APP antisense at later post-training time did not cause amnesia for the task. Unlike antibodies and antisense, injection of the APP328-332 pentapeptide, in either orientation, 30 min pretraining, rescued the memory and prevented antisense-induced amnesia. The post-training time within which the antibody- and antisense-induced amnesia, and within which the APP peptides prevent amnesia, correspond to that during which memory formation is vulnerable to disruption of the putative signal transduction functions of APP. These results suggest that: (i) APP is required during an early phase of memory formation, and (ii) the memory enhancing effect of secretory APP is localized within a 5-mer sequence of growth-promoting domain.

Changes in protein kinases in brain aging and Alzheimer's disease. Implications for drug therapy.

There is ample evidence for the involvement of aberrant protein phosphorylation reactions in aging and age-associated neurological disorders. Alzheimer's disease (AD) in particular. The exact nature of this involvement, however, is not yet elucidated. In the brain tissue of AD patients, there are numerous examples of altered protein phosphorylation pathways. Individual protein kinases and phosphorylation by these kinases in AD brain tissues have been found to be altered. Protein kinases studied include protein kinase C (PKC), protein tyrosine kinase (PTK), casein kinase II (CKII), Ca++/calmodulin-dependent kinase II and mitogen-activated protein (MAP) kinases, all of which are thought to be necessary for cell survival. Interestingly, different protein kinases are involved in different aspects of AD pathology. It is postulated that the perturbation of amyloid beta/A4-protein precursor (APP) metabolism triggers abnormal protein phosphorylation reactions responsible for dysfunction and eventual death of neurons in the brain. The association of APP mutation with certain familial types of AD strongly suggests that there might be a link between aberrant APP metabolism, protein phosphorylation cascades and the eventual expression of AD pathology (plaques and tangles) and neurodegeneration. In summary, recent studies emphasise the prime importance of protein phosphorylation in aging and AD. This raises the possibility that future pharmacological interventions might be devised to interfere with this kinase cascade for the prevention or treatment of age-associated neurological disorders.

Peptides containing the RERMS sequence of amyloid beta/A4 protein precursor bind cell surface and promote neurite extension

Amyloid beta/A4 protein precursor (APP) is secreted into medium by most cultured cells and can function as an autocrine factor. To study the biological function of secreted forms of APP (sAPP) on neurons, we used a clonal CNS neuronal line, B103, which does not synthesize detectable levels of APP. B103 cells transfected with APP construct developed neurites faster than the parent B103 cells when plated in a serum-free defined medium. Neurite outgrowth of B103 cells was promoted by the conditioned medium of APP-695-over-producing cells or by the bacteria-produced sAPP-695 (named KB75). A series of peptides having sequences between Ala-319 and Met-335 of APP-695 also stimulated neurite outgrowth of B103 cells. The sequence of five amino acids, RERMS (APP 328-332), within this stretch of sequence, was the shortest active peptide, although the concentration required for the neuritotropic activity was higher than that of KB75. Binding assay using 125I-labeled APP 17-mer peptide corresponding to Ala-319 to Met-335 of APP-695 as a ligand demonstrated specific and saturable cell-surface binding sites. The predicted KD value was 20 +/- 5 nM and the Bmax value was 80 +/- 8 fmol/10(6) cells. The binding could be displaced with KB75. A 17-mer peptide with reverse sequence neither induced neurite outgrowth nor competed for the binding. A bacteria-produced sAPP fragment lacking the active 17-mer sequence (named KB75 delta) did not compete with 125I-labeled 17-mer for binding or stimulate neurite extension. A peptide of sequence RMSQ (APP 330-333), which partially overlaps the active sequence RERMS, could block the neuritotropic effects of both KB75 and the 17-mer at higher concentrations. APP 17-mer was also found to induce the accumulation of inositol polyphosphates, suggesting that the APP 17-mer effects involve activation of inositol phospholipid signal transduction systems. These data indicate that sAPP induces neurite extension through cell-surface binding and that the domain containing the RERMS sequence (APP 328-332) represents the active site responsible for this function.

Ultrastructural localization of amyloid beta/A4 protein precursor in the normal rat brain.

The ultrastructural localization of amyloid beta/A4 protein precursor (APP) was studied immunohistochemically in normal rat brains using antibodies against different portions of APP. In cerebral cortical neurons and Purkinje cells. APP reaction products were located in the cytoplasm and on cell surface membranes. Some Golgi apparatuses and rough endoplasmic reticulum also showed APP immunoreactivity on their membranes and some vesicles near the trans face of the Golgi apparatuses were stained. In the neuropil of the cerebral cortex and the cerebellar molecular layer, many cell processes, which surrounded synapses and were considered to be astrocytic, were APP-positive. Foot processes around capillaries and subpial astrocytic processes were also immuno-positive. At the ultrastructural level, APP-positive astrocytic processes were identified.

Biologically active domain of the secreted form of the amyloid beta/A4 protein precursor.

The amyloid beta/A4 protein precursor (APP), a large transmembrane protein, is expressed ubiquitously in many organisms, as well as in a variety of cultured cells. Studies of the synthesis and processing of APP have revealed several intricate metabolic pathways for this protein. One of these pathways involves the cleavage of APP in the middle of the beta/A4 domain and results in the secretion of the large amino-terminal portion of the protein. The biological function of this secreted form of APP has been the subject of intense investigation by several groups and various activities have been described for the different domains of APP studied. Our initial approach was to create a fibroblast cell line in which APP expression is dramatically reduced. These fibroblasts, called A-1, have a very slow growth rate. Addition of exogenous APP in the medium of A-1 cells restores their growth to the level of normal parent fibroblasts, demonstrating a growth factor-like activity for the secreted form of APP. Using APP fragments made in bacteria as well as synthetic peptides, we have been able to locate the active site of APP within a domain of 17 amino-acids (Ala319-Met335). This domain of APP can stimulate neurite extension of cultured neuroblastoma cells and it is proposed that APP mediates this effect through binding to a cell surface receptor, triggering intracellular transduction mechanisms. Thus, the secreted form of APP can function as a growth and/or differentiation factor and the site involved in these activities is within a 17-mer domain in the middle of the molecule. Our current lines of research seek to further characterize the mechanisms of APP function as well as its activity in vivo.

Amino acid sequence RERMS represents the active domain of amyloid beta/A4 protein precursor that promotes fibroblast growth.

The growth of A-1 fibroblasts depends on exogenous amyloid beta/A4 protein precursor (APP), providing a simple bioassay to study the function of APP. Our preliminary study, testing the activity of a series of fragments derived from the secreted form of APP-695 (sAPP-695) on this bioassay, has shown that at least one of the active sites of sAPP-695 was localized within a 40-mer sequence (APP296-335, Kang sequence; Roch, J.-M., I. P. Shapiro, M. P. Sundsmo, D. A. C. Otero, L. M. Refolo, N. K. Robakis, and T. Saitoh. 1992. J. Biol. Chem. 267:2214-2221). In the present study, to further characterize the growth-promoting activity of sAPP-695 on fibroblasts, we applied a battery of synthetic peptides on this bioassay and found that: (a) the sequence of five amino acids, RERMS (APP328-332), was uniquely required for the growth-promoting activity of sAPP-695; (b) the activity was sequence-specific because the reverse-sequence peptide of the active domain had no activity; and (c) the four-amino-acid peptide RMSQ (APP330-333), which partially overlaps the COOH-terminal side of the active sequence RERMS, could antagonize the activity of sAPP-695. Furthermore, a recombinant protein which lacks this active domain (APP20-591 without 306-335) did not promote fibroblast cell growth, suggesting that this domain is the only site of sAPP-695 involved in the growth stimulation. The availability of these biologically active, short peptides and their antagonists should prove to be an essential step for the elucidation of APP involvement in regulation of cellular homeostasis.

Ultrastructural localization of Alzheimer amyloid beta/A4 protein precursor in the cytoplasm of neurons and senile plaque-associated astrocytes.

The ultrastructural localization of amyloid beta/A4 protein precursor (APP) in the brains of control and Alzheimer's disease patients was examined immunohistochemically using antisera against the N and C termini of APP. In both control and Alzheimer brains, immunoreaction for APP was seen in the cytoplasm of most neurons, on plasma membranes, outer membrane of mitochondria, granular substance and neurofilaments. Cell bodies and foot processes of astrocytes, containing glial filaments, were also labeled. In primitive and classic type senile plaques, APP immunoreaction products were localized in the astroglial processes that surrounded the amyloid mass of the senile plaques. Swollen degenerating neurites in the senile plaques were also labeled. Amyloid fibrils were negative with APP antisera.