Faculty Bibliography

The prion protein (PrP) binds copper and under some conditions copper can facilitate its folding into a more protease resistant form. Hence, copper levels may influence the infectivity of the scrapie form of prion protein (PrPSc). To determine the feasibility of copper-targeted therapy for prion disease, we treated mice with a copper chelator, D-(-)-penicillamine (D-PEN), starting immediately following intraperitoneal scrapie inoculation. D-PEN delayed the onset of prion disease in the mice by about 11 days (p = 0.002), and reduced copper levels in brain by 29% (p < 0.01) and in blood by 22% (p = 0.03) compared with control animals. Levels of other metals were not significantly altered in the blood or brain. Modest correlation was observed between incubation period and levels of copper in brain (p = 0.08) or blood (p = 0.04), indicating that copper levels are only one of many factors that influence the rate of progression of prion disease. In vitro, copper dose-dependently enhanced the proteinase K resistance of the prion protein, and this effect was counteracted in a dose-dependent manner by co-incubation with D-PEN. Overall, these findings indicate that copper levels can influence the conformational state of PrP, thereby enhancing its infectivity, and this effect can be attenuated by chelator-based therapy

Amyloid deposition can take place in the walls of arteries, arterioles, and, less often, capillaries and veins of the central nervous system, a phenomenon known as cerebral amyloid angiopathy (CAA). The major clinicopathological manifestations of CAA include cerebral hemorrhage, ischemic lesions, and dementia. CAA may be classified according to the amyloid protein deposited. In the most common form, sporadic CAA, and in CAA related to sporadic Alzheimer disease (AD). A beta deposition is characteristic. CAA can also be severe in variants of familial AD caused by mutations of the amyloid-beta precursor protein or presenilin-1 genes in which deposition of A beta variants and/or wild-type A beta occurs. Other amyloid proteins involved in familial CAAs include 1) the mutant cystatin C (ACys) in hereditary cerebral hemorrhage with amyloidosis of Icelandic type, 2) variant transthyretins (ATTR) in meningo-vascular amyloidoses, 3) mutated gelsolin (AGel) in familial amyloidosis of Finnish type, 4) disease-associated prion protein (PrP(Sc)) in a variant of the Gerstmann-Straussler-Scheinker syndrome, and 5) ABri and ADan in CAAs observed in the recently described BRI2 gene-related dementias, familial British dementia and familial Danish dementia, respectively. This review addresses issues related to the correlation between morphology, biochemistry, and genetics, and briefly discusses both the pathogenesis and animal models of CAAs

Cruzipain, the major cysteine proteinase of Trypanosoma cruzi, might have other biological roles than its metabolic functions. In this report, we have explored the interaction of cruzipain with molecules of the immune system. The enzyme was used to digest all human IgG subclasses at different pH values and lengths of time. At pH 7.3, all subclasses were readily split at the hinge region. Immunoblot and amino acid sequence analysis showed fragments of IgG1 and IgG3 to be compatible with Fab and Fc, whereas IgG2 and IgG4 rendered Fab2 and Fc. In all cases the fragments produced might impair the binding capacities and the effector functions of specific IgG. At these cleavage sites cruzipain displays cathepsin L and/or cathepsin B activities and shows a clear preference for Pro at the P'2 position and polar residues at P1. Despite the activity of cruzipain within the hinge, the enzyme also cleaved all heavy chains between the CH2 and CH3 domains; producing Fc'-like-fragments of 14 kDa. These fragments are potential candidates to block or saturate Fc receptors on immunocompetent cells. At mild acidic pH cruzipain produced further degradation of the Fc of all subclasses, the Fd of IgG4 and partially the Fd of IgG1, with the consistent loss of any antibody activity. The L chains apparently were not affected. Thus, cruzipain should be able to modulate, depending on the subclass selected and the pH of the environment, the production and the length of different biologically active/inactive IgG fragments

Amyloid-beta peptide (Abeta) interacts with the vasculature to influence Abeta levels in the brain and cerebral blood flow, providing a means of amplifying the Abeta-induced cellular stress underlying neuronal dysfunction and dementia. Systemic Abeta infusion and studies in genetically manipulated mice show that Abeta interaction with receptor for advanced glycation end products (RAGE)-bearing cells in the vessel wall results in transport of Abeta across the blood-brain barrier (BBB) and expression of proinflammatory cytokines and endothelin-1 (ET-1), the latter mediating Abeta-induced vasoconstriction. Inhibition of RAGE-ligand interaction suppresses accumulation of Abeta in brain parenchyma in a mouse transgenic model. These findings suggest that vascular RAGE is a target for inhibiting pathogenic consequences of Abeta-vascular interactions, including development of cerebral amyloidosis

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid beta (Abeta) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Abeta wild type, yet whether IDE is capable of degrading Abeta bearing pathogenic substitutions is not known. We studied the degradation of all of the published Abeta genetic variants by recombinant rat IDE (rIDE). Monomeric Abeta wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Abeta Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Abeta wild type and the rest of the mutant peptides. In the case of Abeta Dutch, inefficient proteolysis was related to a high content of beta structure as assessed by circular dichroism. All of the Abeta variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13-15 and 18-21. SDS-stable Abeta dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Abeta. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Abeta in hereditary forms of Abeta diseases

Prion disease is characterized by a conformational change of the normal form of the prion protein (PrP(C)) to the scrapie-associated form (PrP(Sc)). Since the emergence of new variant Creutzfeldt-Jakob disease a potentially large human population is at risk for developing prion disease. Currently, no effective treatment or form of post-exposure prophylaxis is available for prion disease. We recently showed that active immunization with recombinant PrP prolongs the incubation period of scrapie. Here we show that anti-PrP antibodies following prion exposure are effective at increasing the incubation period of the infection. Stimulation of the immune system is an important therapeutic target for the prion diseases, as well as for other neurodegenerative illnesses characterized by abnormal protein conformation

We have reported that an amyloid-beta derivative, K6Abeta1-30-NH2 reduces amyloid burden in mice to a similar extent as previously shown for Abeta1-42 (Am J Pathol 159:439-47,2001). This derivative may be a safer alternative to Alzheimer's vaccination with Abeta1-42 because it has a low beta-sheet content while maintaining the main antigenic sites of Abeta. To determine the in vivo effect of other derivatives with similar in vitro properties, we immunized Tg2576 mice with Abeta1-30-NH2, in which amino acids 18 and 19 were substituted with glutamate (Abeta1-30E18E19). In a parallel study, mice were immunized with K6Abeta1-30E18E19. Freund's adjuvant was used to allow a comparison with our findings with K6Abeta1-30-NH2. Antibody titers were detectable, but much lower than we had observed for K6Abeta1-30-NH2 or Abeta1-42, indicating that the central hydrophobic region of Abeta may have an epitope important for modulating humoral response. Cognitive performance was assessed in a radial arm maze before sacrifice at 19-21 months. Control Tg mice had more errors than their wild-type littermates (p<0.01), and the Abeta1-30E18E19-treated mice (p<0.05). Mice receiving K6Abeta1-30E18E19 also performed better than their Tg controls (p<0.05). Histologically, no difference was observed in brain amyloid plaque burden in 6E10 stained brain sections from the Abeta1-30E18E19-vaccinated mice, compared to vehicle treated mice. Furthermore, amyloid burden did not correlate with cognitive performance. Analysis of plaque burden in the K6Abeta1-30E18E19-immunized mice is underway, as well as measurements of brain levels of Abeta to determine if these values will provide a better correlation with cognitive performance. A robust antibody response and a diminished plaque burden may not be necessary for a therapeutic effect of Abeta derived vaccines

P-component is a member of the pentraxin family of proteins that has been found associated with amyloid deposits in vivo in both systemic and localized forms of amyloidosis including AD. Recently, two novel familial forms of cerebrovascular amyloidosis have been described. These hereditary conditions, familial British dementia (FBD) and familial Danish dementia (FDD), both result from genetic alterations in the BRI2 gene and show striking clinical and neuropathological similarities with AD. Despite structural differences among the amyloid subunits (ABri in FBD, ADan in FDD, and Abeta in AD) all these disorders are characterized by the presence of neurofibrillary tangles and parenchymal and vascular amyloid deposits co-localizing with reactive microglia and activation products of the complement cascade. Immunohistochemical studies of FBD and FDD brain tissue identified P-component associated with the amyloid lesions. Using affinity chromatography, ELISA binding assays and electrophoretic techniques we were able to demonstrate a specific binding interaction between P-component and ABri/ADan peptides, study the biochemical parameters of the interaction, and compare them with that of Abeta amyloid species. Our studies revealed a high affinity, Calcium dependent, saturable binding between P-component and ABri/ADan peptides with Kd values in the low nanomolar range and in the same order of magnitude to those resulting from the interaction of P-component with Abeta1-40 and Abeta1-42. The high affinity interaction of P-component with ABri and ADan peptides in vitro suggests a likely mechanism for their in vivo co-localization in FBD and FDD lesions. Whether the presence of P-component protects the ABri/ADan amyloid deposits from enzymatic degradation as demonstrated for other amyloids is being investigated