Faculty Bibliography

The accumulation of amyloid beta (Abeta) in the walls of small vessels in the cerebral cortex is associated with diseases characterized by dementia or stroke. These include Alzheimer's disease, Down syndrome, and sporadic and hereditary cerebral amyloid angiopathies (CAAs) related to mutations within the Abeta sequence. A higher tendency of Abeta to aggregate, a defective clearance to the systemic circulation, and insufficient proteolytic removal have been proposed as mechanisms that lead to Abeta accumulation in the brain. By using immunoprecipitation and mass spectrometry, we show that insulin-degrading enzyme (IDE) from isolated human brain microvessels was capable of degrading (125)I-insulin and cleaved Abeta-(1-40) wild type and the genetic variants Abeta A21G (Flemish), Abeta E22Q (Dutch), and Abeta E22K (Italian) at the predicted sites. In microvessels from Alzheimer's disease cases with CAA, IDE protein levels showed a 44% increase as determined by sandwich enzyme-linked immunosorbent assay and Western blot. However, the activity of IDE upon radiolabeled insulin was significantly reduced in CAA as compared with age-matched controls. These results support the notion that a defect in Abeta proteolysis by IDE contributes to the accumulation of this peptide in the cortical microvasculature. Moreover they raise the possibility that IDE inhibition or inactivation is a pathogenic mechanism that may open novel strategies for the treatment of cerebrovascular Abeta amyloidoses

To better understand the physiologic excretion and/or catabolism of circulating peripheral amyloid beta (Abeta), we labeled human Abeta40 (monomeric, with predominant unordered structure) and Abeta42 (mixture of monomers and oligomers in approximately 50:50 ratio, rich in beta-sheet conformation) with either Na(125)I or (125)I-tyramine cellobiose, also known as the cell-trapping ligand procedure, testing their blood clearance and organ uptake in B6SJLF1/J mice. Irrespective of the labeling protocol, the peptide conformation, and the degree of oligomerization, both Abeta40 and Abeta42 showed a short half-life of 2.5-3.0 min. The liver was the major organ responsible for plasma clearance, accounting for >60% of the peptide uptake, followed by the kidney. In vivo, hepatocytes captured >90% of the radiolabeled peptides which, after endocytosis, were preferentially catabolized and excreted into the bile. Biliary excretion of intact as well as partially degraded Abeta species became obviously relevant at doses above 10 microg. The use of biotin-labeled Abeta allowed the visualization of the interaction with HepG2 cells in culture, whereas competitive inhibition experiments with unlabeled Abeta demonstrated the specificity of the binding. The capability of the liver to uptake, catabolize, and excrete large doses of Abeta, several orders of magnitude above its physiologic concentration, may explain not only the femtomolar plasma levels of Abeta but the little fluctuation observed with age and disease stages

In prion diseases, the cellular prion protein (PrP(C)) is converted to an insoluble and protease-resistant abnormal isoform termed PrP(Sc). In different prion strains, PrP(Sc) shows distinct sites of endogenous or exogenous proteolysis generating a core fragment named PrP27-30. Sporadic Creutzfeldt-Jakob disease (sCJD), the most frequent human prion disease, clinically presents with a variety of neurological signs. As yet, the clinical variability observed in sCJD has not been fully explained by molecular studies relating two major types of PrP27-30 with unglycosylated peptides of 21 (type 1) and 19 kDa (type 2) and the amino acid methionine or valine at position 129. Recently, smaller C-terminal fragments migrating at 12 and 13 kDa have been detected in different sCJD phenotypes, but their significance remains unclear. By using two-dimensional immunoblot with anti-PrP antibodies, we identified two novel groups of protease-resistant PrP fragments in sCJD brain tissues. All sCJD cases with type 1 PrP27-30, in addition to MM subjects with type 2 PrP27-30, were characterized by the presence of unglycosylated PrP fragments of 16-17 kDa. Conversely, brain homogenates from patients VV and MV with type 2 PrP27-30 contained fully glycosylated PrP fragments, which after deglycosylation migrated at 17.5-18 kDa. Interestingly, PrP species of 17.5-18 kDa matched deglycosylated forms of the C1 PrP(C) fragment and were associated with tissue PrP deposition as plaque-like aggregates or amyloid plaques. These data show the presence of multiple PrP(Sc) conformations in sCJD and, in addition, shed new light on the correlation between sCJD phenotypes and disease-associated PrP molecules

The colocalization of cystatin C, an inhibitor of cysteine proteases, with amyloid beta (Abeta) in parenchymal and vascular amyloid deposits in brains of Alzheimer's disease (AD) patients may reflect cystatin C involvement in amyloidogenesis. We therefore sought to determine the association of cystatin C with Abeta. Immunofluorescence analysis of transfected cultured cells demonstrated colocalization of cystatin C and beta amyloid precursor protein (betaAPP) intracellularly and on the cell surface. Western blot analysis of immunoprecipitated cell lysate or medium proteins revealed binding of cystatin C to full-length betaAPP and to secreted betaAPP (sbetaAPP). Deletion mutants of betaAPP localized the cystatin C binding site within betaAPP to the Abeta region. Cystatin C association with betaAPP resulted in increased sbetaAPP but did not affect levels of secreted Abeta. Analysis of the association of cystatin C and Abeta demonstrated a specific, saturable and high affinity binding between cystatin C and both Abeta(1-42) and Abeta(1-40). Notably, cystatin C association with Abeta results in a concentration-dependent inhibition of Abeta fibril formation

In order to assess whether lipoproteins are physiologically able to balance and modulate the sAbeta homeostasis in vivo, soluble Abeta levels in lipoprotein-depleted plasma were measured as a function of age in normal controls, Alzheimer's disease (AD) patients, and Down's syndrome (DS) cases. The reshaping of sAbeta homeostasis, in particular the sAbeta42-lipoprotein interaction, takes place over normal-60's, whereas mild AD patients appear to have impaired this anti-amyloidogenic mechanism resulting in a significant increase of lipoprotein-free sAbeta42. Similar loss of function takes place in Down's syndrome patients. Lipoprotein-free sAbeta remains significantly elevated from the pre-symptomatic through the symptomatic stages of the disease, and declines with the progression of the AD-like pathology. The dissociation of sAbeta from lipoprotein-particles also occurs in brain parenchyma and the presence of soluble dimeric lipoprotein-free Abeta prior to its parenchymal deposition in AD brains would support the hypothesis that functionally declined lipoproteins may be major determinants in the production of metabolic conditions leading to higher levels of sAbeta species and cerebral amyloidosis

Immunization with amyloid-beta (Abeta) 1-42 has been shown to reduce amyloid burden and improve cognition in Alzheimer's disease (AD) model mice. In a human trial, possible cognitive benefit was found but in association with significant toxicity in a minority of patients. We proposed that immunization with nonfibrillogenic Abeta derivatives is much less likely to produce toxicity and have previously shown that one such derivative (K6Abeta1-30) can reduce amyloid burden in mice to a similar extent as Abeta1-42. Here, we immunized AD model mice (Tg2576) with Abeta1-30[E18E19] or with K6Abeta1-30[E18E19]. These peptides were designed to be nontoxic and to produce less T-cell response, which has been linked to toxicity. K6Abeta1-30[E18E19] induced primarily an IgM response, whereas Abeta1-30[E18E19] induced an IgG titer that was lower than previously seen with K6Abeta1-30 or Abeta1-42. However, both treated animal groups performed better than Tg controls in the radial arm maze. Amyloid burden was similar in Abeta1-30[E18E19]-vaccinated mice and their Tg controls, whereas the number of medium and small sized plaques was reduced (29-34%) in K6Abeta1-30[E18E19]-immunized mice compared with Tg controls. Amyloid burden in these mice correlated inversely with plasma IgM levels. The cognitive benefit and amyloid reduction in the K6Abeta1-30[E18E19]-vaccinated mice are likely to be related to peripheral clearance of Abeta, because IgM does not cross the blood-brain barrier because of its large size. Our results indicate that these nontoxic Abeta derivatives produce an attenuated antibody response, which is less likely to be associated with negative side effects while having cognitive benefits