Faculty Bibliography

Absence of functional presenilin 1 (PS1) protein leads to loss of gamma-secretase cleavage of the amyloid precursor protein (betaAPP), resulting in a dramatic reduction in amyloid beta peptide (Abeta) production and accumulation of alpha- or beta-secretase-cleaved C-terminal fragments of betaAPP (alpha- or beta-CTFs). The major C-terminal fragment (CTF) in brain was identified as Abeta[11-98], which is consistent with the observation that cultured neurons generate primarily Abeta[11-40]. In PS1-/- murine neurons and fibroblasts expressing the loss-of-function PS1Asp385Ala mutant, CTFs accumulated in the endoplasmic reticulum (ER), Golgi, and lysosomes, but not late endosomes. There were some subtle differences in the subcellular distribution of CTFs in PS1-/- neurons as compared to PS1Asp385Ala mutant fibroblasts. However, there was no obvious redistribution of full-length betaAPP or of markers of other organelles in either mutant. Blockade of ER-to-Golgi trafficking indicated that in PS1-/- neurons (as in normal cells) trafficking of betaAPP to the Golgi compartment is necessary before alpha- and beta-secretase cleavages occur. Thus, while we cannot exclude a specific role for PS1 in trafficking of CTFs, these data argue against a major role in general protein trafficking. These results are more compatible with a role for PS1 either as the actual gamma-secretase catalytic activity or in other functions indirectly related to gamma-secretase catalysis (e.g., an activator of gamma-secretase, a substrate-adaptor for gamma-secretase, or delivery of gamma-secretase to betaAPP-containing compartments)

Deposition of amyloid-beta (Abeta) aggregates in the brain is a defining characteristic of Alzheimer's disease (AD). Fibrillar amyloid, found in the cores of senile plaques, is surrounded by dystrophic neurites. In contrast, the amorphous Abeta (also called preamyloid) in diffuse plaques is not associated with neurodegeneration. Depending on the conditions, Abeta will also form fibrillar or amorphous aggregates in vitro. In this present study, we sought to characterize the properties of the amorphous aggregate and determine whether we could establish an in vitro model for amorphous Abeta. CD data indicated that Abeta40 assembled to form either a beta-structured aggregate or an unfolded aggregate with the structured aggregate forming at high peptide concentrations and the unstructured aggregate forming at low Abeta40 levels. The critical concentration separating these two pathways was 10 microm. Fluorescence emission and polarization showed the structured aggregate was tightly packed containing peptides that were not accessible to water. Peptides in the unstructured aggregate were loosely packed, mobile, and accessible to water. When examined by electron microscopy, the structured aggregate appeared as protofibrillar structures and formed classic amyloid fibrils over a period of several weeks. The unstructured aggregate was not visible by electron microscopy and did not generate fibrils. These findings suggest that the unstructured aggregate shares many properties with the amorphous Abeta of AD and that conditions can be established to form amorphous Abeta in vitro. This would allow for investigations to better understand the relationship between fibrillar and amorphous Abeta and could have significant impact upon efforts to find therapies for AD

The amyloid beta-peptide (Abeta) is a principal component of insoluble amyloid plaques which are characteristic neuropathological features of Alzheimer's disease. Abeta also exists as a normal soluble protein that undergoes a pathogenic transition to an aggregated, fibrous form. This transition can be affected by extraneous proteinaceous and nonproteinaceous elements, such as zinc ions, which may promote aggregation and/or stabilization of the fibrils. Protein chelation of zinc is typically mediated by histidines, cysteines and carboxylates. Previous studies have demonstrated that the Abeta-Zn2+ binding site is localized within residues 6-28 and that histidines may serve as the principal sites of interaction. To localize key residues within this region, a series of Abeta peptides (residues 1-28) were synthesized that contained systematic His/Ala substitutions. Circular dichroism and electron microscopy were used to monitor the effects of Zn2+ on the peptide beta-sheet conformation and fibril aggregation. Our results indicate that substitution of either His13 or His14 but not His6 eliminates the zinc-mediated effects. These observations indicate a specific zinc binding site within Abeta that involves these central histidine residues

Nicastrin, a transmembrane glycoprotein, forms high molecular weight complexes with presenilin 1 and presenilin 2. Suppression of nicastrin expression in Caenorhabditis elegans embryos induces a subset of notch/glp-1 phenotypes similar to those induced by simultaneous null mutations in both presenilin homologues of C. elegans (sel-12 and hop-1). Nicastrin also binds carboxy-terminal derivatives of beta-amyloid precursor protein (betaAPP), and modulates the production of the amyloid beta-peptide (A beta) from these derivatives. Missense mutations in a conserved hydrophilic domain of nicastrin increase A beta42 and A beta40 peptide secretion. Deletions in this domain inhibit A beta production. Nicastrin and presenilins are therefore likely to be functional components of a multimeric complex necessary for the intramembranous proteolysis of proteins such as Notch/GLP-1 and betaAPP

Numerous missense mutations in the presenilins are associated with the autosomal dominant form of familial Alzheimer disease. Presenilin genes encode polytopic transmembrane proteins, which are processed by proteolytic cleavage and form high-molecular-weight complexes under physiological conditions. The presenilins have been suggested to be functionally involved in developmental morphogenesis, unfolded protein responses and processing of selected proteins including the beta-amyloid precursor protein. Although the underlying mechanism by which presenilin mutations lead to development of Alzheimer disease remains elusive, one consistent mutational effect is an overproduction of long-tailed amyloid beta-peptides. Furthermore, presenilins interact with beta-catenin to form presenilin complexes, and the physiological and mutational effects are also observed in the catenin signal transduction pathway

While PrP(C) rearranges in the area of codons 104-113 to form PrP(Sc) during prion infections, the events that initiate sporadic Creutzfeldt-Jakob disease are undefined. As Cu(II) is a putative ligand for PrP(C) and has been implicated in the pathogenesis of Creutzfeldt-Jakob disease and other neurodegenerative diseases, we investigated the structural effects of binding. Incubation of brain microsomes with Cu(II) generated approximately 30-kDa proteinase K-resistant PrP. Cu(II) had little effect on fresh recombinant PrP23-231, but aged protein characterized by conversion of Asn-107 to Asp decreased alpha-helical content by approximately 30%, increased beta-sheet content 100%, formed aggregates, and acquired proteinase K resistance in the presence of Cu(II). These transitions took place without need for acid pH, organic solvents, denaturants, or reducing agents. Since conversion of Asn to Asp proceeds by a spontaneous pathway involving deamidation, our data suggest that covalent variants of PrP(C) arising in this manner may, in concert with Cu(II), generate PrP(Sc)-like species capable of initiating sporadic prion disease

Amyloid formation is a key pathological feature of Alzheimer's disease and is considered to be a major contributing factor to neurodegeneration and clinical dementia. Amyloid is found as both diffuse and senile plaques in the parenchyma of the brain and is composed primarily of the 40- to 42-residue amyloid-beta (Abeta) peptides. The characteristic amyloid fiber exhibits a high beta-sheet content and may be generated in vitro by the nucleation-dependent self-association of the Abeta peptide and an associated conformational transition from random to beta-conformation. Growth of the fibrils occurs by assembly of the Abeta seeds into intermediate protofibrils, which in turn self-associate to form mature fibers. This multistep process may be influenced at various stages by factors that either promote or inhibit Abeta fiber formation and aggregation. Identification of these factors and understanding the driving forces behind these interactions as well as the structural motifs necessary for these interactions will help to elucidate potential sites that may be targeted to prevent amyloid formation and its associated toxicity. This review will discuss some of the modulating factors that have been identified to date and their role in fibrillogenesis

Recent studies have suggested that non-fibrillar soluble forms of Abeta peptides possess neurotoxic properties and may therefore play a role in the molecular pathogenesis of Alzheimer's disease. We have identified solution conditions under which two types of soluble oligomers of Abeta40 could be trapped and stabilized for an extended period of time. The first type of oligomers comprises a mixture of dimers/tetramers which are stable at neutral pH and low micromolar concentration, for a period of at least four weeks. The second type of oligomer comprises a narrow distribution of particles that are spherical when examined by electron microscopy and atomic force microscopy. The number average molecular mass of this distribution of particles is 0.94 MDa, and they are are stable at pH 3 for at least four weeks. Circular dichroism studies indicate that the dimers/tetramers possess irregular secondary structure that is not alpha-helix or beta-structure, while the 0.94 MDa particles contain beta-structure. Fluorescence resonance energy transfer experiments indicate that Abeta40 moieties in amyloid fibrils or protofibrils are more similar in structure to those in the 0.94 MDa particles than those in the dimers/tetramers. These findings indicate that soluble oligomeric forms of Abeta peptides can be trapped for extended periods of time, enabling their study by high resolution techniques that would not otherwise be possible

Amyloid-beta (Abeta) assembly into fibrillar structures is a defining characteristic of Alzheimer's disease that is initiated by a conformational transition from random coil to beta-sheet and a nucleation-dependent aggregation process. We have investigated the role of organic osmolytes as chemical chaperones in the amyloid pathway using glycerol to mimic the effects of naturally occurring molecules. Osmolytes such as the naturally occurring trimethylamine N-oxide and glycerol correct folding defects by preferentially hydrating partially denatured proteins and entropically stabilize native conformations and polymeric states. Trimethylamine N-oxide and glycerol were found to rapidly accelerate the Abeta random coil-to-beta-sheet conformational change necessary for fiber formation. This was accompanied by an immediate conversion of amorphous unstructured aggregates into uniform globular and possibly nucleating structures. Osmolyte-facilitated changes in Abeta hydration also affected the final stages of amyloid formation and mediated transition from the protofibrils to mature fibers that are observed in vivo. These findings suggest that hydration forces can be used to control fibril assembly and may have implications for the accumulation of Abeta within intracellular compartments such as the endoplasmic reticulum and in vitro modeling of the amyloid pathway

Amyloid enhancing factor (AEF) is an activity that appears naturally during the course of persistent inflammation and precedes, by 24-48 h, AA amyloid deposition in appropriate murine models. AEF is defined by its biological properties, namely, when administered intravenously or intraperitoneally to a mouse, it primes the recipient for the rapid induction of AA amyloid when they are given an inflammatory stimulus. Available evidence indicates that AEF is protein in nature, but a specific molecular species (if a singular species exits) has not been identified. Past work (Ganowiak et al., Biochem. Biophys. Res. Commun. 199:306-312, 1994) has shown that AEF activity may be imparted to two different proteins (IAPP and beta-protein) provided each is organized in the form of an amyloid fibril. Since a characteristic property of proteins in amyloid fibrils is their beta-sheet organization, one possibility is that AEF activity, in part, depends on such organization, and other proteins with such properties may also have AEF activity. To investigate this possibility, silk, a protein which contains substantial beta-sheet content, was denatured in LiSCN and allowed to renature slowly under reducing conditions to form a gel. The denatured silk preparation was then sonicated thoroughly to permit intravenous injection and assessed for AEF activity. The modified silk, presented as small fibrils in a beta-sheet conformation as assessed by electron microscopy and circular dichroism, respectively. This silk at 0-50 micrograms/animal was administered intravenously as 'AEF' followed immediately by subcutaneous AgNO3 as the inflammatory stimulus. Six days later the spleens were examined for the presence of AA amyloid and following Congo red staining, the amount of amyloid quantified by image analysis. Modified silk without an inflammatory stimulus, and non-sonicated modified silk, failed to induce AA amyloid. Sonicated modified silk followed by AgNO3 induced large quantities of splenic AA amyloid in a dose dependent fashion. Modified silk in quantities as small as 1-5 micrograms/animal can function as AEF. The AEF properties of the modified silk were stable at 4 degrees C for at least 4 weeks (the longest period tested). This procedure may provide a means of standardizing AEF preparations