John E. Maggio

Aluminum, Iron, and Zinc Ions Promote Aggregation of Physiological Concentrations of β‐Amyloid Peptide

Abstract: A major pathological feature of Alzheimers disease (AD) is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human β‐amyloid peptide βA4, a 40‐mer whose neurotoxicity is related to its aggregation. Certain metals have been proposed as risk factors for AD, but the mechanism by which the metals may exert their effects is unclear. Radioiodinated human βA4 has been used to assess the effects of various metals on the aggregation of the peptide in dilute solution (10‐10M). In physiological buffers, 10‐3M calcium, cobalt, copper, manganese, magnesium, sodium, or potassium had no effect on the rate of βA4 aggregation. In sharp contrast, aluminum, iron, and zinc under the same conditions strongly promoted aggregation (rate enhancement of 100–1,000‐fold). The aggregation of βA4 induced by aluminum and iron is distinguishable from that induced by zinc in terms of rate, extent, pH and temperature dependence. These results suggest that high concentrations of certain metals may play a role in the pathogenesis of AD by promoting aggregation of βA4.

Amyloid beta-peptide is transported on lipoproteins and albumin in human plasma.

The amyloid β-peptide (Aβ) is the major constituent of neuritic plaques in Alzheimers disease and occurs as a soluble 40-42-residue peptide in cerebrospinal fluid and blood of both normal and AD subjects. It is unclear whether Aβ, once it is secreted by cells, remains free in biological fluids or is associated with other proteins and thus transported and metabolized with them. Such knowledge of the normal fate of Aβ is a prerequisite for understanding the changes that may lead to the pathological aggregation of soluble Aβ in vivo, the possible influence of certain extracellular proteins, particularly apolipoprotein E, on plaque formation, and the pharmacology of putative Aβ-lowering drugs. To address the question of Aβ distribution in human biological fluids, we incubated fresh human plasma from 38 subjects with physiological concentrations (0.5-0.7 nM) of radioiodinated Aβ1-40 and seven plasma samples with Aβ1-42. Lipoproteins and lipid-free proteins were separated and analyzed for bound iodinated Aβ1-40. We found that up to 5% of Aβ added to plasma is bound to selected lipoproteins: very low density, low density, and high density, but not lipoprotein(a). The large majority (≈89%), however, is bound to albumin, and very little Aβ is free. Aβ distribution in plasma was not significantly influenced by apolipoprotein E genotype. We conclude that Aβ is normally bound to and transported by albumin and specific lipoproteins in human plasma under physiological conditions.

Substance P receptors: localization by light microscopic autoradiography in rat brain using [3H]SP as the radioligand.

Substance P (SP) is a putative neurotransmitter in both the peripheral and central nervous systems. In the present report we have used a modification of the Young and Kuhar technique to investigate some of the SP receptors binding properties and the distribution of SP receptors in rat brain. Tritiated SP [( 3H]SP) absorbed extensively to glass but this adsorbtion was greatly reduced by preincubating the slide-mounted tissue sections in a solution containing the cationic polymer polyethylenimine. [3H]SP was found to bind to rat tissue in a saturable fashion with a Bmax of 14.7 fmol/mg tissue wet weight and a Kd of 1.1 nM. The rank order of potencies for displacing [3H]SP binding from rat tissue sections was SP greater than SP sulphoxide greater than DiMeC7 greater than Eledoisin greater than SP(5-11) greater than SP(COOH) greater than SP(1-9) amide. Using autoradiography coupled with LKB tritium-sensitive Ultrofilm or the dry emulsion-coated coverslip technique the distribution of [3H]SP binding sites was found to be very dense within olfactory bulb, amygdalo-hippocampal area and the nucleus of the solitary tract. Heavy concentrations of receptors were observed in the septum, diagonal band of Broca, striatum subiculum, hypothalamus, locus coeruleus, parabrachial nucleus and lobule 9 and 10 of the cerebellum. Moderate to low concentrations of receptors were observed in the cerebral cortex, globus pallidus, raphe nuclei and the trigeminal nucleus. Very low densities were observed in most aspects of the dorsal thalamus, substantia nigra and cerebellum (other than lobule 9 and 10). Comparisons of the present data with SP peptide levels indicate that in some areas of the brain there is a rough correlation between peptide and receptor levels. However, in other brain areas (olfactory bulb, globus pallidus and substantia nigra) there is little obvious correlation between the two.

Point Substitution in the Central Hydrophobic Cluster of a Human β-Amyloid Congener Disrupts Peptide Folding and Abolishes Plaque Competence†

Alzheimers disease (AD) is pathologically characterized by the presence of numerous insoluble amyloid plaques in the brain composed primarily of a 40-43 amino acid peptide, the human beta-amyloid peptide (A beta). The process of A beta deposition can be modeled in vitro by deposition of physiological concentrations of radiolabeled A beta onto preexisting amyloid in preparations of unfixed AD cerebral cortex. Using this model system, it has been shown that A beta deposition is biochemically distinct from A beta aggregation and occurs readily at physiological A beta concentrations, but which regions and conformations of A beta are essential to A beta deposition is poorly understood. We report here that an active congener, A beta (10-35)-NH2, displays time dependence, pH-activity profile, and kinetic order of deposition similar to A beta (1-40), and is sufficiently soluble for NMR spectroscopy in water under conditions where it actively deposits. To examine the importance of the central hydrophobic cluster of A beta (LVFFA, residues 17-21) for in vitro A beta deposition, an A beta (10-35)-NH2 analog with a single point substitution (F19T) in this region was synthesized and examined. Unlike A beta (10-35)-NH2, the F19T analog was plaque growth incompetent, and NMR analysis indicated that the mutant peptide was significantly less folded than wild-type A beta. These results support previous studies suggesting that the plaque competence of A beta correlates with peptide folding. Since compounds that alter A beta folding may reduce amyloid deposition, the central hydrophobic cluster of A beta will be a tempting target for structure-based drug design when high-resolution structural information becomes available.

Differential expression of substance P receptors in patients with Crohn's disease and ulcerative colitis

BACKGROUND & AIMS Although clinical and pathological differences exist between Crohns disease (CD) and ulcerative colitis (UC), distinguishing features are often absent, making diagnosis and treatment problematic. This study evaluated the differences in the expression of substance P (SP) receptors in patients with CD or UC. METHODS Tissue samples from patients with inflammatory bowel disease or control patients were obtained at surgery, processed for 125I-SP binding, and analyzed by quantitative autoradiography. RESULTS Patients with CD showed a massive increase in SP receptors in lymphoid aggregates, small blood vessels, and enteric neurons of the small and large bowel relative to controls. Six of 16 CD specimens had no pathological evidence of CD yet continued to express high concentrations of SP receptors. Pathologically positive patients with UC showed high concentrations of SP receptors on colonic lymphoid aggregates and small blood vessels but not enteric neurons. No increased SP binding was evident in clinically and pathologically quiescent UC colons and normal UC ileostomy samples. CONCLUSIONS The increased expression of SP receptors on the enteric neurons of patients with CD distinguishes CD from UC. The persistent increased SP binding in pathologically normal CD tissue may indicate a subclinical disease state. SP receptor expression may have important diagnostic, etiologic, and therapeutic usefulness in inflammatory bowel disease.

Receptors for sensory neuropeptides in human inflammatory diseases: implications for the effector role of sensory neurons.

Glutamate and several neuropeptides are synthesized and released by subpopulations of primary afferent neurons. These sensory neurons play a role in regulating the inflammatory and immune responses in peripheral tissues. Using quantitative receptor autoradiography we have explored what changes occur in the location and concentration of receptor binding sites for sensory neurotransmitters in the colon in two human inflammatory diseases, ulcerative colitis and Crohns disease. The sensory neurotransmitter receptors examined included bombesin, calcitonin gene related peptide-alpha, cholecystokinin, galanin, glutamate, somatostatin, neurokinin A (substance K), substance P, and vasoactive intestinal polypeptide. Of the nine receptor binding sites examined only substance P binding sites associated with arterioles, venules and lymph nodules were dramatically up-regulated in the inflamed tissue. These data suggest that substance P is involved in regulating the inflammatory and immune responses in human inflammatory diseases and indicate a specificity of efferent action for each sensory neurotransmitter in peripheral tissues.

Regional distribution of kassinin-like immunoreactivity in rat central and peripheral tissues and the effect of capsaicin

The regional distribution of kassinin-like immunoreactivity in rat central and peripheral tissues was investigated by radioimmunoassay and found to resemble closely that of substance P-like immunoreactivity. Neonatal capsaicin treatment caused a similar decrease to both kassinin-like and substance P-like immunoreactivity in primary sensory areas. These results suggest that more than one member of the tachykinin family of neuropeptides exist within the same neuron.

Zinc-Induced Aggregation of Human and Rat β-Amyloid Peptides In Vitro

Abstract: The major pathological feature of Alzheimers disease is the presence of a high density of amyloid plaques in the brain tissue of patients. The plaques are predominantly composed of human β‐amyloid peptide (Aβ), a 39–43‐mer peptide the neurotoxicity of which is related to its aggregation state. Previous work has demonstrated that certain metals that have been implicated as risk factors for Alzheimers disease (Al, Fe, and Zn) also cause substantial aggregation of Aβ. In particular, we reported that zinc cations at concentrations of >10−4M dramatically accelerate the rate of Aβ aggregation at physiological peptide concentrations at 37°C in vitro. In the present study, we investigate the effect of Zn2+ on aggregation of radiolabeled and unlabeled human and rat Aβ over a wide range of peptide concentrations in the presence and absence of salt and blocking protein. Aggregation was assayed by centrifugation and filtration using amino acid analysis, immunoassay, and γ‐counting for quantification over a wide range of concentrations of Zn2+ and Aβ above and below physiological values. The results of this study demonstrate the following: (a) Radio‐iodinated Aβ accurately tracked unlabeled Aβ, (b) zinc concentrations of at least 10−4M were required to induce significant aggregation of Aβ, and (c) rat and human Aβ species were cleared from aqueous solutions by similar concentrations of zinc. These results stand in significant quantitative disagreement (∼100‐fold in zinc concentration) with one previous study that reported significant aggregation of Aβ by <1 µM Zn2+. Differences between the present study and the latter study from another laboratory appear to result from inappropriate reliance on optical density to measure Aβ concentrations and nonspecific loss of Aβ to plastic in the absence of blocking protein.

The autoradiographic distribution of kassinin and substance K binding sites is different from the distribution of substance P binding sites in rat brain

In the present report we have used autoradiographic receptor binding techniques to compare the distribution of substance K (SK), kassinin (K) and substance P (SP) binding sites in the rat brain. Whereas the distribution of K and SK binding sites appeared to be identical, notable differences are apparent when comparing these binding sites to the distribution of SP binding sites. These results demonstrate that in many areas of the rat brain K and SK binding sites have a different distribution than SP binding sites. These results further suggest that different classes of mammalian tachykinins may each have their own set of receptors.

Receptor binding sites for substance P and substance K in the canine gastrointestinal tract and their possible role in inflammatory bowel disease.

The mammalian tachykinins, substance P, substance K (neurokinin A) and neuromedin K (neurokinin B), are putative peptide neurotransmitters in both the brain and peripheral tissues. We used quantitative receptor autoradiography to localize and quantify the distribution of binding sites for radiolabeled substance P, substance K and neuromedin K in the canine gastrointestinal tract. Substance P binding sites were localized to smooth muscle cells in the muscularis mucosa and muscularis externa, the smooth muscle and endothelium of arterioles and venules, neurons in the myenteric plexus, mucosal epithelial cells, exocrine cells and lymph nodules. Substance K binding sites were distributed in a pattern distinct from substance P binding sites and were localized to smooth muscle cells in the muscularis mucosa and muscularis externa, the smooth muscle and endothelium of arterioles and venules, and neurons of the myenteric plexus. Neuromedin K binding sites were not observed in any area of the canine gastrointestinal tract although they were localized with high specific/non-specific binding ratios in the canine spinal cord. These results indicate that there are at least two distinct types of tachykinin receptor binding sites in the canine gastrointestinal tract, one of which probably recognizes substance P and the other substance K as endogenous ligands. In correlation with previous physiological data, these substance P and substance K receptor binding sites appear to be involved in the regulation of a variety of gastrointestinal functions including gastric motility, mucosal ion transport, hemodynamics, digestive enzyme secretion and neuronal excitability. In addition these results demonstrate that receptor binding sites for substance P and substance K are expressed by cells involved in mediating inflammatory and immune responses. These data, together with our studies on surgical specimens from patients with inflammatory bowel disease, suggest that in a pathophysiological state tachykinins and their receptors may play a role in inflammatory bowel disease and should permit a rational approach to designing neuropeptide antagonists which may prove effective in treating inflammatory diseases.