Patricia A. Merz

Ultrastructure of Paired Helical Filaments of Alzheimer's Neurofibrillary Tangle

Isolated paired helical filaments (PHF) of Alzheimers disease and senile dementia of the Alzheimer type (AD/SDAT) were studied by negative stain techniques and electron microscopy (EM). The helical structure of the PHF was confirmed by tilt analysis of negatively stained isolated PHF. A substructure of four protofilaments, each 3–5 nm in diameter, was also observed. A comparison of PHF substructure, neurofilaments and microtubules, revealed no similarity among the three structures.

Biochemical Differences among Scrapie-associated Fibrils Support the Biological Diversity of Scrapie Agents

Scrapie-associated fibrils (SAF) were isolated and purified from animals infected with three different scrapie agents: ME7 and 139A in mice, and 263K in hamsters. Mouse ME7 and 139A SAF differed from hamster 263K SAF in morphology, sedimentation rate and protein composition. SAF from the three scrapie agents were distinguishable from each other by their sensitivity to proteinase K digestion. SAF copurified with infectivity in both the hamster and mouse systems. SAF appear to be a unique class of structures which are related but specific for each individual scrapie agent. These properties may correlate with the biological and pathological differences seen among these agents.

Detection of scrapie-associated fibril (SAF) proteins using anti-SAF antibody in non-purified tissue preparations

Antisera raised to scrapie-associated fibril (SAF) proteins were used to detect scrapie-specific polypeptides in three different non-purified brain preparations: a synaptosomal-mitochondrial fraction, 20% brain homogenate and 20% brain homogenate extracted with Sarkosyl. The concentration of SAF proteins in the preparations was greater than the quantity of SAF as detected by negative stain electron microscopy. This suggests that not all of the protein exists in the form of SAF. An immunologically reactive 33K to 35K protein was detected in both normal and scrapie brain preparations. This protein was susceptible to complete proteinase K (PK) digestion in normal brain preparations and it is suggested that scrapie infection is responsible for post-translational modifications which confer PK resistance in scrapie preparations. These modifications may also play a role in the antigenic differences seen in a variety of scrapie agents. SAF-specific proteins were also detected in the spinal cords and spleens from scrapie-affected animals. Detergent extraction of material followed by PK treatment and Western blot analysis is a highly specific and sensitive method for the detection of SAF proteins. This procedure could be applied to human neurological diseases of unknown aetiology.

Nature of the scrapie agent: current status of facts and hypotheses.

Scrapie is a slow infection affecting the central nervous system (CNS) of sheep and goats (Dickinson, 1976). Experimentally the agent has been passaged in mice, hamsters and a number of other laboratory animal species. Kuru and Creutzfeldt-Jakob disease (CJD) are related diseases occurring in humans (Gajdusek, 1977). These diseases are caused by agents that have unconventional characteristics compared to those of known animal viruses (Gajdusek, 1977). The unusual physical, chemical and biological properties of the scrapie agent have led to an abundance of theories concerning its nature. Each of the major classes of macromolecules has been suggested as an important component of the scrapie agent. Hypotheses have suggested that scrapie agent is composed exclusively of protein (Griffith, 1967), exclusively of polysaccharide (Gibbons & Hunter, 1967), or exclusively of nucleic acid (Diener, 1972). Other hypotheses have suggested that it is a replicating membrane component (Gibbons & Hunter, 1967) or a filterable virus (Eklund et al., 1963). This is but a partial list of the theoretical constructs that have been proposed.

The nature of the unconventional slow infection agents remains a puzzle

Unconventional slow infections are progressive transmissible degenerative disorders of the central nervous system. The human diseases belonging to this group are Creutz-feld-Jakob disease, kuru, and Gerstmann-Straussler syndrome. Scrapie, transmissible mink encephalopathy, chronic wasting disease of mule deer and elk, and the recently discovered bovine spongiform encephalopathy are similar diseases found in animals. Unusual characteristics of the unconventional slow infections clearly distinguish these disorders from conventional infections. These include: unusually long incubation periods (from months to years); progressive CNS degeneration with characteristic histopathological lesions; the lack of an immune or inflammatory response; unconventional biological and physical properties of the etiologic agents. There has been considerable controversy concerning the nature of the causative agent. The 3 main hypotheses, virus, virino, and modified host protein, are reviewed relative to their ability to explain the properties of the agent and the unusual characteristics of the disease process.The discovery of an abnormal structure, termed scrapie associated fibrils (SAF) and an abnormally modified 33–37 kDa host-encoded glycoprotein unique to unconventional slow infections opened new areas of intense interest and investigation. SAF are abnormal filamentous structures which copurify with infectivity and possess characteristics of “amyloids.” The major component of SAF is the host-encoded scrapie-specific protease resistant glycoprotein. Considerable data has accumulated on the biochemistry, immunology and molecular biology of this host coded scrapie protein. The relationship of SAF and the scrapie-specific protein to the infectious agent is discussed in the context of each of the “nature of the agent” hypotheses.

Paired helical filaments associated with Alzheimer disease are readily soluble structures

Considerable controversy exists concerning the origin and composition of Alzheimer neurofibrillary tangles (ANT) and of paired helical filaments (PHF), the abnormal cytoplasmic fibers which ultrastructurally are the major components of ANT. Thus far, the unusual solubility properties of PHF have hindered the analysis of ANT. A new procedure is presented for isolating purified PHF which are soluble in the presence of sodium dodecyl sulfate. The purification protocol involves differential and rate zonal centrifugation, treatment with the detergents sarcosyl and sulfobetain 3-14, and sonication. The isolated PHF from Alzheimer disease/senile dementia of the Alzheimer type (7 cases) and Downs syndrome (one case) have been characterized structurally by negative-stain electron microscopy, biochemically by PAGE, and immunologically by both the ELISA technique and Western blot analysis using a monoclonal antibody prepared against ANT. Distinct polypeptides were shown to be associated with this structure and not seen in preparations from young and age-matched normal brains.

Microtubular interactions of presenilin direct kinesis of Aβ peptide and its precursors

In our previous study we demonstrated that presenilin 1 (PS1) interacts with cytoplasmic linker protein 170/Restin (CLIP‐170/Restin). Herein we show that disruption of the interaction of these proteins within neuronal cell‐lines (SY5Y and N2a) can be accomplished by the transfection of vectors that drive the expression of peptide fragments corresponding to their binding domains (BDPs). Interestingly, the disruption of the PS1/CLIP‐170 complex is associated with both decreased secretion of endogenous Aβ and decreased uptake of exogenous Aβ from the medium. BDP‐expressing cells were also more resistant to surges of Aβ secretion induced by thapsigargin and ionomycin (that elevate intracellular calcium concentrations) and mutations in PS1 linked to familial Alzheimers disease. Uptake of Aβ by SY5Y cells was amplified when preincubated with ApoE and was mediated through lipoprotein receptor‐related protein (LRP). BDP‐expressing cells or cells treated with PS1 anti‐sense oligonucleotides were less capable of taking up Aβ from the medium compared with controls, indicating that the PS1/CLIP‐170 interaction is involved and that PS1 cannot be substituted. In this study, we also mapped the minimum binding domains (mBDPs) of PS1 and CLIP‐70 to regions corresponding to the N‐terminal end of the large cytoplasmic loop of PS1 and the metal binding motif‐containing C‐terminal end of CLIP‐170. Further, our data obtained from experiments involving in vitro taxol‐polymerization of tubulin and confocal immmunofluorescence suggest that PS1, via CLIP‐170, may serve as an anchor to the microtubules for specific subcellular fractions containing amyloidogenic fragments. Interestingly, Notch is absent from this population of microtubule binding subcellular fractions and its cleavage was unaffected in cells transfected with the PS1‐based BDP. This raises the possibility that the interaction of PS1 with CLIP‐170 could provide the conceptual basis for anti‐amyloidogenic therapeutic strategies with improved specificity. However, this approach may be hampered by a low efficiency, because it may also block Aβ clearance from the interstitial space of the CNS.

Partial Copurification of Scrapie-Associated Fibrils and Scrapie Infectivity

The association between scrapie infectivity and scrapie-associated fibrils (SAF) during a partial purification procedure for infectivity was investigated. Scrapie infectivity and SAF can be separated from most membrane components by subcellular fractionation of infected mouse brain to obtain a synaptosomal fraction, followed by detergent treatment and density gradient centrifugation. After different detergent treatments, with either octyl glucoside or sodium N-lauroyl sarcosinate, SAF showed differing sedimentation characteristics but nevertheless cosedimented with scrapie infectivity in both cases. Copurification under two different conditions provides more evidence that SAF may be a form of the infectious agent of scrapie.

The Effects of Detergents on the Composition of Postsynaptic Densities

Abstract: A method of purifying postsynaptic densities (PSD) of Cohen et al. (1977) has been modified, primarily by the substitution of octyl glucoside as the detergent used to solubilize synaptosomal fractions. Subsequent extraction with other detergents resulted in the selective removal of specific polypeptides. In particular sulphobetaine 3–14 removed most of the β‐tubulin but not α‐tu‐ bulin. Sodium N ‐ lauroyI sarcosinate completely destroyed the structural integrity of the PSD when the invitro formation of intermolecular disulphide bonds was minimized. These results suggest that the structure of PSDs is more labile than previously thought and demonstrate a technique for further examining their composition.

Unclassified: Spongiform Encephalopathies

Disease: Scrapie of sheep and goats, transmissible mink encephalopathy, chronic wasting disease of captive mule deer and elk, and the human diseases kuru, Creutzfeldt-Jakob, and Gerstmann-Straussler syndrome.