Secreted amyloid ß-proteins in a cell culture model include N-terminally extended peptides that impair synaptic plasticity.

Evidence for a central role of amyloid ß-protein (Aß) in the genesis of Alzheimer's disease (AD) has led to advanced human trials of Aß-lowering agents. The "amyloid hypothesis" of AD postulates deleterious effects of small, soluble forms of Aß on synaptic form and function. Because selectively targeting synaptotoxic forms of soluble Aß could be therapeutically advantageous, it is important to understand the full range of soluble Aß derivatives. We previously described a Chinese hamster ovary (CHO) cell line (7PA2 cells) that stably expresses mutant human amyloid precursor protein (APP). Here, we extend this work by purifying an sodium dodecyl sulfate (SDS)-stable, ∼8 kDa Aß species from the 7PA2 medium. Mass spectrometry confirmed its identity as a noncovalently bonded Aß40 homodimer that impaired hippocampal long-term potentiation (LTP) in vivo. We further report the detection of Aß-containing fragments of APP in the 7PA2 medium that extend N-terminal from Asp1 of Aß. These N-terminally extended Aß-containing monomeric fragments are distinct from soluble Aß oligomers formed from Aß1-40/42 monomers and are bioactive synaptotoxins secreted by 7PA2 cells. Importantly, decreasing ß-secretase processing of APP elevated these alternative synaptotoxic APP fragments. We conclude that certain synaptotoxic Aß-containing species can arise from APP processing events N-terminal to the classical ß-secretase cleavage site.

UV light absorption parameters of the pathobiologically implicated bilirubin oxidation products, MVM, BOX A, and BOX B.

The formation of the bilirubin oxidation products (BOXes), BOX A ([4-methyl-5-oxo-3-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide]) and BOX B (3-methyl-5-oxo-4-vinyl-(1,5-dihydropyrrol-2-ylidene)acetamide), as well as MVM (4-methyl-3-vinylmaleimide) were synthesized by oxidation of bilirubin with H2O2 without and with FeCl3, respectively. Compound identity was confirmed with NMR and mass spectrometry (MS; less than 1 ppm, tandem MS up to MS4). UV absorption profiles, including λmax, and extinction coefficient (ε; estimated using NMR) for BOX A, BOX B, and MVM in H2O, 15% CH3CN plus 10 mM CF3CO2H, CH3CN, CHCl3, CH2Cl2, and 0.9% NaCl were determined. At longer wavelengths, λmax's for 1) BOX A were little affected by the solvent, ranging from 295-297 nm; 2) BOX B, less polar solvent yielded λmax's of lower wavelength, with values ranging from 308-313 nm, and 3) MVM, less polar solvent yielded λmax's of higher wavelength, with values ranging from 318-327 nm. Estimated ε's for BOX A and BOX B were approximately 5- to 10-fold greater than for MVM.

Phosphoproteome analysis of cardiomyocytes subjected to beta-adrenergic stimulation: identification and characterization of a cardiac heat shock protein p20.

Posttranslational modification of target substrates underlies biological processes through activation/inactivation of signaling cascades. To concurrently identify the phosphoprotein substrates associated with cardiac beta-adrenergic signaling, the mouse myocyte phosphoproteome was analyzed using 2-D gel electrophoresis in combination with 32P autoradiography. Phosphoprotein spots, detected by silver staining, were identified using MALDI-TOF mass spectrometry in conjunction with computer-assisted protein spot matching. Stimulation with isoproterenol (1 micromol/L for 5 minutes) was associated with maximal increases in myocyte contractile parameters, and significant stimulation of the phosphorylation of troponin I (190+/-23%) and succinyl CoA synthetase (160+/-16%), whereas the phosphorylation of pyruvate dehydrogenase (48+/-10%), NADH-ubiquinone oxidoreductase (46+/-6%), heat shock protein 27 (18+/-3%), alphaB-crystallin (20+/-3%), and an unidentified 26-kDa protein (29+/-7%) was significantly decreased, compared with unstimulated cells (100%). After sustained (30 minutes) stimulation with isoproterenol, only the alterations in the phosphorylation levels of troponin I and NADH-ubiquinone oxidoreductase were maintained and de novo phosphorylation of a phosphoprotein (approximately 20 kDa and pI 5.5) was observed. The tryptic peptide fragments of this phosphoprotein were sequenced using postsource decay mass spectrometry, and the protein was subsequently cloned and designated as p20, based on its high sequence homology with rat and human skeletal p20. The mouse cardiac p20 contains the conserved domain sequences for heat shock proteins, and the RRAS consensus sequence for cAMP-PKA substrates. LC-MS/MS phosphorylation mapping confirmed phosphorylation of Ser16 in p20 on beta-agonist stimulation. Adenoviral gene transfer of p20 was associated with significant increases in contractility and Ca transient peak in adult rat cardiomyocytes, suggesting an important role of p20 in cardiac function. These findings suggest that cardiomyocytes undergo significant posttranslational modification via phosphorylation in a multitude of proteins to dynamically fine-tune cardiac responses to beta-adrenergic signaling.

Proteomic analysis of hyperdynamic mouse hearts with enhanced sarcoplasmic reticulum calcium cycling.

Depressed sarcoplasmic reticulum (SR) Ca-cycling is a hallmark of human and experimental heart failure. Strategies to improve this impairment by either increasing SERCA2a levels or decreasing phospholamban (PLN) activity have been suggested as promising therapeutic targets. Indeed, ablation of PLN gene in mice was associated with greatly enhanced cardiac Ca-cycling and performance. Intriguingly, this hyperdynamic cardiac function was maintained throughout the lifetime of the mouse without observable pathological consequences. To determine the cellular alterations in the expression or modification of myocardial proteins, which are associated with the enhanced cardiac contractility, we performed a proteomics-based analysis of PLN knockout (PLN-KO) hearts in comparison to isogenic wild-types. By use of 2-dimensional gel electrophoresis (2-DE), approximately 3300 distinct protein spots were detected in either wild-type or PLN-KO ventricles. Protein spots observed to be altered between PLN-KO and wild-type hearts were subjected to tryptic peptide mass fingerprinting for identification by MALDI-TOF mass spectrometry in combination with LC/MS/MS analysis. In addition, two-dimensional 32P-autoradiography was performed to analyze the phosphorylation profiles of PLN-KO cardiomyocytes. We identified alterations in the expression level of more than 100 ventricular proteins, along with changes in phosphorylation status of important regulatory proteins in the PLN-KO. These protein changes were observed mainly in two subcellular compartments: the cardiac contractile apparatus, and metabolism/energetics. Our findings suggest that numerous alterations in protein expression and phosphorylation state occurred upon ablation of PLN and that a complex functional relationship among proteins involved in calcium handling, myofibrils, and energy production may exist to coordinately maintain the hyperdynamic cardiac contractile performance of the PLN-KO mouse in the long term.

Apolipoprotein E affects amyloid formation but not amyloid growth in vitro: mechanistic implications for apoE4 enhanced amyloid burden and risk for Alzheimer's disease.

The transition from the partially folded soluble Abeta monomer to insoluble Abeta amyloidfibrils is seminal to the formation and growth of amyloid plaques in Alzheimer's disease (AD). A detailed understanding of the role of AD risk factors in these processes is essential to understanding the physiochemical nature of this conformational rearrangement. The apolipoprotein E epsilon4 allele, a risk factor for AD, affects AD pathology by increasing amyloid burden relative to the much more common epsilon3 allele. In the present study, in vitro models were employed to probe the effect of these proteins on kinetically distinct steps in Abeta fibrillogenesis. Formation of Abeta amyloid was faster in the presence of apoE4 than apoE3, while growth of existing plaques was unaffected by either isoform. Further, experiments with Abeta stereoisomers establish that this effect of apoE3 is mediated through interaction with oligomeric fibrillogenic intermediates rather than through specific contacts with monomeric Abeta. Consistent with the altered pathology and enhanced risk for AD associated with inheritance of the epsilon4 allele, we conclude that APOE epsilon4 is a risk factor for AD not due to a pathological gain of function of apoE4 but to a loss of protective function of apoE3.

Endogenous opioids inhibit early-stage pancreatic pain in a mouse model of pancreatic cancer.

BACKGROUND & AIMS: The endogenous opioid system is involved in modulating the experience of pain, the response to stress, and the action of analgesic therapies. Recent human imaging studies have shown a significant tonic modulation of visceral pain, raising the question of whether endogenous opioids tonically modulate the pain of visceral cancer. METHODS: Transgenic mice expressing the first 127 amino acids of simian virus 40 large T antigen, under the control of the rat elastase-1 promoter, that spontaneously develop pancreatic cancer were used to investigate the role of endogenous opioids in the modulation of pancreatic cancer pain. Visceral pain behaviors were assessed as degree of hunching and vocalization. RESULTS: Although mice with late-stage pancreatic cancer displayed spontaneous, morphine-reversible, visceral pain-related behaviors such as hunching and vocalization, these behaviors were absent in mice with early-stage pancreatic cancer. After systemic administration of the central nervous system (CNS)-penetrant opioid receptor antagonists naloxone or naltrexone, mice with early-stage pancreatic cancer displayed significant visceral pain-related behaviors, whereas systemic administration of the CNS-nonpenetrant opioid antagonist naloxone-methiodide did not induce an increase in visceral pain behaviors. CONCLUSIONS: Our findings suggest that a CNS opioid-dependent mechanism tonically modulates early and late-stage pancreatic cancer pain. Understanding the mechanisms that mask this pain in early stage disease and drive this pain in late-stage disease may allow improved diagnosis, treatment, and care of patients with pancreatic cancer.

Noninvasive imaging of peripherally injected Alzheimer's disease type synthetic A beta amyloid in vivo.

The pathological hallmark of Alzheimer's disease (AD) is accumulation in the brain of amyloid composed of the 40-mer peptide A beta. Many fundamental questions about the biology of (AD) remain unanswered because there is currently no method of quantifying A beta amyloid in vivo. A noninvasive method of detecting and quantifying A beta amyloid in vivo would have wide application for the premortem diagnosis of AD and the efficient evaluation of candidate therapeutics aimed at inhibiting the formation and growth of A beta amyloid. Taking advantage of the extraordinarily high affinity of A beta for itself, we have synthesized an N'-terminal diethylenetriaminepentaacetic acid (DTPA) derivative of A beta possessing the kinetic activity and specificity for A beta amyloid desired of a probe to be used for noninvasive imaging. DTPA-A beta(3-40) is readily labeled with (111)InOAc(3) to yield a stable probe with exquisite specificity for naturally occurring and synthetic A beta amyloid in vitro. Moreover, (111)In-DTPA-A beta(3-40), administered intravascularly can specifically deposit onto and label previously injected synthetic A beta amyloid and be imaged in vivo with a gamma camera. The present results demonstrate the design, synthesis, and use of an A beta amyloid-specific probe and methods for its use as a noninvasive imaging agent. In vivo imaging of A beta amyloid represents an important step toward the development of biochemically based objective tools for the assessment of progression of AD and efficacy of potential therapeutics.

Proteomic analysis of the reactive phenotype of astrocytes following endothelin-1 exposure.

Reactive gliosis is an invariant feature of the pathology of central nervous system (CNS) injury and a major determinant of neuronal survival and regeneration. To begin to understand the alterations in astrocyte protein expression that drive glial changes that occur following injury, we used an established model system (endothelin-1 stimulation of hypertrophy) and proteomic analysis to define a discrete set of differentially expressed proteins and post-translational modifications that occur as the astrocytes change from a quiescent to a reactive state. This orchestrated set of changes included proteins involved in cytoskeletal reorganization (caldesmon, calponin, alpha B-crystallin, stathmin, collapsing response mediator protein-2), cell adhesion (vinculin, galectin-1), signal transduction (RACK-1) and astrocyte differentiation (glutamine synthetase). Using proteomic analysis to understand what drives astrocyte expression of these functionally divergent molecules may offer insight into the mechanisms by which astrocytes can exhibit both pro-regenerative and anti-regenerative activities following CNS injury.

Endothelin receptor expression in the normal and injured spinal cord: potential involvement in injury-induced ischemia and gliosis.

The endothelins (ETs) are a family of peptides that exert their biological effects via two distinct receptors, the endothelin A receptor (ET(A)R) and the endothelin B receptor (ET(B)R). To more clearly define the potential actions of ETs following spinal cord injury, we used immunohistochemistry and confocal microscopy to examine the protein expression of ET(A)R and ET(B)R in the normal and injured rat spinal cord. In the normal spinal cord, ET(A)R immunoreactivity (IR) is expressed by vascular smooth muscle cells and a subpopulation of primary afferent nerve fibers. ET(B)R-IR is expressed primarily by radial glia, a small population of gray and white matter astrocytes, ependymal cells, vascular endothelial cells, and to a lesser extent in smooth muscle cells. Fourteen days following compression injury to the spinal cord, there was a significant upregulation in both the immunoexpression and number of astrocytes expressing the ET(B)R in both gray and white matter and a near disappearance of ET(B)R-IR in ependymal cells and ET(A)R-IR in primary afferent fibers. Conversely, the vascular expression of ET(A)R and ET(B)R did not appear to change. As spinal cord injury has been shown to induce an immediate increase in plasma ET levels and a sustained increase in tissue ET levels, ETs would be expected to induce an initial marked vasoconstriction via activation of vascular ET(A)R/ET(B)R and then days later a glial hypertrophy via activation of the ET(B)R expressed by astrocytes. Strategies aimed at blocking vascular ET(A)R/ET(B)R and astrocyte ET(B)Rs following spinal cord injury may reduce the resulting ischemia and astrogliosis and in doing so increase neuronal survival, regeneration, and function.