Eduardo M. Castaño

Differences Between Vascular and Plaque Core Amyloid in Alzheimer's Disease

Abstract: The predominant protein of cerebrovascular and plaque core amyloid in Alzheimers disease, Downs syndrome, hereditary hemorrhage with amyloidosis—Dutch type, sporadic cerebral amyloid angiopathy, and age‐related amyloidosis is a unique polypeptide, called β protein. The length of the plaque amyloid protein was reported to be 42–43 residues, but the complete length of the cerebral vascular amyloid is not known. To clarify this issue, amyloid fibrils from the leptomeninges of an Alzheimers disease patient were isolated and the primary structure determined. The complete sequence of cerebrovascular β‐amyloid protein, although homologous to the plaque core amyloid protein previously reported, has 39 residues instead of 42. Amino terminal heterogeneity is present but minimal, and it is three residues shorter at the carboxy terminus. These differences are similar to those found in two cases of hereditary hemorrhage with amyloidosis—Dutch type. The differences between vascular and plaque β‐amyloid may reflect diverse processing of the β protein precursor in the vessel wall and brain parenchyma due to tissue‐specific endopeptidases.

Degradation of Soluble Amyloid β-Peptides 1–40, 1–42, and the Dutch Variant 1–40Q by Insulin Degrading Enzyme from Alzheimer Disease and Control Brains

Insulin degrading enzyme (IDE) is a metalloprotease that has been involved in amyloid β peptide (Aβ) degradation in the brain. We analyzed the ability of human brain soluble fraction to degrade Aβ analogs 1–40, 1–42 and the Dutch variant 1–40Q at physiological concentrations (1 nM). The rate of synthetic 125I-Aβ degradation was similar among the Aβ analogs, as demonstrated by trichloroacetic acid precipitation and SDS-PAGE. A 110 kDa protein, corresponding to the molecular mass of IDE, was affinity labeled with either 125I-insulin, 125I-Aβ 1–40 or 125I-Aβ 1–42 and both Aβ degradation and cross-linking were specifically inhibited by an excess of each peptide. Sensitivity to inhibitors was consistent with the reported inhibitor profile of IDE. Taken together, these results suggested that the degradation of Aβ analogs was due to IDE or a closely related protease. The apparent Km, as determined using partially purified IDE from rat liver, were 2.2 ± 0.4, 2.0 ± 0.1 and 2.3 ± 0.3 μM for Aβ 1–40, Aβ 1–42 and Aβ 1–40Q, respectively. Comparison of IDE activity from seven AD brain cytosolic fractions and six age-matched controls revealed a significant decrease in Aβ degrading activity in the first group, supporting the hypothesis that a reduced IDE activity may contribute to Aβ accumulation in the brain.

In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to alzheimer's disease β-protein

Two synthetic peptides corresponding to the reported 28-residue sequence of Alzheimers Disease beta-protein (SP28) and to residues 12-28 (SP17) were used to form fibrils in vitro. Synthetic fibrils bound Congo Red and closely resembled amyloid fibrils isolated from leptomeninges and senile plaques of Alzheimers brain by electron microscopy. A polyclonal antiserum to SP28 specifically decorated both synthetic and native amyloid by colloidal gold immunoelectron microscopy. Amyloid fibrils isolated from tissue were insoluble on SDS-Polyacrylamide gels, and tended to aggregate while synthetic amyloid fibrils were completely solubilized, releasing only monomers of SP28 and SP17. Anti-SP28 immunostained cerebrovascular and plaque core amyloid, but not neurofibrillary tangles, in tissue section. Western blot analysis showed that anti-SP28 reacted with a 4 kDa band released from amyloid core-enriched preparations and leptomeninges. By contrast, a 16 kDa band corresponding to the tetramer of beta-protein was not recognized. These data suggest that as little as a 17 residue sequence of beta-protein may be required to form fibrils and that the complete sequence of the 4 kDa beta-protein may be important in determining insolubility and the formation of intermediate size polymers.

Amyloid beta peptides in human plasma and tissues and their significance for Alzheimer's disease

We evaluated the amounts of amyloid beta (Aβ)) peptides in the central nervous system (CNS) and in reservoirs outside the CNS and their potential impact on Aβ plasma levels and Alzheimers disease (AD) pathology.

Comparative proteomics of cerebrospinal fluid in neuropathologically-confirmed Alzheimer's disease and non-demented elderly subjects

Abstract Objectives: Diagnostic tests able to reveal Alzheimers disease (AD) in living patients before cognitive ability is destroyed are urgently needed. Such tests must distinguish AD from other dementia causes, as well as differentiate subtle changes associated with normal aging from true pathology emergence. A single biomarker offering such diagnostic and prognostic capacities has eluded identification. Therefore, a valuable test for AD is likely to be based on a specific pattern of change in a set of proteins, rather than a single protein. Methods: We examined pooled cerebrospinal fluid (CSF) samples obtained from neuropathologically-confirmed AD (n=43) and non-demented control subjects (n=43) using 2-dimensional gel electrophoresis (2DE) proteomic methodology to detect differentially expressed proteins. Proteins exhibiting expression level differences between the pools were recovered and identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Results: Five differentially-expressed proteins with potential roles in amyloid-β metabolism and vascular and brain physiology [apolipoprotein A-1 (Apo A-1), cathepsin D (CatD), hemopexin (HPX), transthyretin (TTR), and two pigment epithelium-derived factor (PEDF) isoforms] were identified. Apo A-1, CatD and TTR were significantly reduced in the AD pool sample, while HPX and the PEDF isoforms were increased in AD CSF. Discussion: These results suggest that multi-factor proteomic pattern analysis of the CSF may provide a means to diagnose and assess AD.

Ten to fourteen residue peptides of Alzheimer's disease protein are sufficient for amyloid fibril formation and its characteristic xray diffraction pattern

The molecular basis of fibril formation in Alzheimers disease was explored by electron micrographic and x-ray diffraction analysis of a series of synthetic peptides corresponding to portions of the amino acid sequence of beta protein and that of its putative precursor. A minimum 14 residue peptide was identified that formed typical amyloid fibrils under physiological conditions. Of these 14 residues, 10 were sufficient to give an identical 4.76 A and 10.6 A diffraction pattern as that recently described for isolated neurofibrillary tangles, amyloid plaque cores and leptomeningeal amyloid fibrils.

Atherosclerosis, vascular amyloidosis and brain hypoperfusion in the pathogenesis of sporadic Alzheimer's disease

Abstract We postulate that severe atherosclerotic occlusion of the circle of Willis and leptomeningeal arteries is an important factor in the pathogenesis of some sporadic Alzheimers disease (AD) cases. These arterial stenoses are complicated by an overwhelming amyloid accumulation in the walls of leptomeningeal and cortical arteries resulting in a significant decrease in perfusion pressure and consequent ischemia/hypoxia of the brain tissue. We also propose that the distal areas of the white matter (WM) will be the first affected by a lack of oxygen and nutrients. Our hypotheses are supported by the following observations: (1) the number of stenoses is more frequent in AD than in the control population (p = 0.008); (2) the average index of occlusion is greater in AD than in the control group (p < 0.00001); (3) the index of stenosis and the total number of stenoses per case are positively correlated (R = 0.67); (4) the index of stenosis correlates with the neuropathological lesions of AD and with the MMSE psychometric test; (5) the number and degree of atherosclerosis of the anterior, middle and posterior cerebral arteries is more severe in cases of AD than in the control population; (6) atherosclerosis severity is apparently associated with the severity of the vascular amyloidosis; (7) the WM rarefaction correlates with the severity of the atherosclerosis and vascular amyloidosis; (8) the total cell count and microvessel count in the areas of WM rarefaction correlate with the neuropathological lesions of AD and with the MMSE score. Our data strongly suggest that severe hemodynamic disturbances contribute to sporadic AD and support the numerous observations indicating cardiovascular system participation in the pathogenesis of these dementias.

Fibrillogenesis of synthetic amyloid-β peptides is dependent on their initial secondary structure

Synthetic peptides containing the sequence of Alzheimers amyloid-beta peptide (A beta) spontaneously form amyloid-like fibrils in vitro, and have been extensively used to study the factors that modulate fibrillogenesis. Contradictory observations have been reported regarding the neurotoxicity of A beta and the influence of some A beta-binding proteins on in vitro A beta amyloid formation. In this study, we show that A beta 1-40 synthetic peptides obtained from different suppliers, have significantly distinct fibrillogenic properties. No differences were detected in the chemical structure or in the initial assembly state by mass spectroscopy, reverse-phase high performance liquid chromatography and denaturing or non-denaturing gel electrophoresis. However, there was a direct correlation between the ability of soluble peptides to form amyloid and their percentage of beta-sheet structure, as determined by electron microscopy, fluorescence associated to thioflavine T bound to amyloid, and circular dichroism. The data suggest that the determinant factor of A beta fibrillogenesis is the secondary structure adopted by the peptide in its soluble state.

Physicochemical characteristics of soluble oligomeric Aβ and their pathologic role in Alzheimer's disease

Abstract Extracellular fibrillar amyloid deposits are prominent and universal Alzheimers disease (AD) features, but senile plaque abundance does not always correlate directly with the degree of dementia exhibited by AD patients. The mechanism(s) and dynamics of Aβ fibril genesis and deposition remain obscure. Enhanced Aβ synthesis rates coupled with decreased degradative enzyme production and accumulating physical modifications that dampen proteolysis may all enhance amyloid deposit formation. Amyloid accumulation may indirectly exert the greatest pathologic effect on the brain vasculature by destroying smooth muscle cells and creating a cascade of negative impacts on cerebral blood flow. The most visible manifestation of amyloid dis-equilibrium could actually be a defense mechanism employed to avoid serious vascular wall degradation while the major toxic effects to the gray and white matter neurons are mediated by soluble oligomeric Aβ peptides with high β-sheet content. The recognition that dynamic soluble oligomeric Aβ pools exist in AD and are correlated to disease severity led to neurotoxicity and physical conformation studies. It is now recognized that the most basic soluble Aβ peptides are stable dimers with hydrophobic regions sequestered from the aqueous environment and are capable of higher order aggregations. Time course experiments employing a modified ELISA method able to detect Aβ oligomers revealed dynamic intermolecular interactions and additional experiments physically confirmed the presence of stable amyloid multimers. Amyloid peptides that are rich in β-sheet structure are capable of creating toxic membrane ion channels and a capacity to self-assemble as annular structures was confirmed in vitro using atomic force microscopy. Biochemical studies have established that soluble Aβ peptides perturb metabolic processes, provoke release of deleterious reactive compounds, reduce blood flow, induce mitochondrial apoptotic toxicity and inhibit angiogenesis. While there is no question that gross amyloid deposition does contribute to AD pathology, the destructive potential now associated with soluble Aβ suggests that treatment strategies that target these molecules may be efficacious in preventing some of the devastating effects of AD.

Different processing of Alzheimer's β-protein precursor in the vessel wall of patients with hereditary cerebral hemorrhage with amyloidosis-Dutch type

Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is an autosomal-dominant type of amyloidosis restricted to the small vasculature of the brain and clinically characterized by recurrent strokes. Amyloid fibrils from the leptomeninges of two patients were isolated and the primary structure determined. The complete sequence of the amyloid protein shows homology to the vascular (beta-protein) and plaque amyloid (A4-protein) obtained from Alzheimers Disease. However, it is three residues shorter (39 instead of 42) than that reported for the plaque amyloid. The difference at the carboxy terminal may reflect specific degradation that occurs in the vessel wall and not in the brain parenchyma.