Dennis R. Sparkman

Transplants of mouse trisomy 16 hippocampus provide a model of Alzheimer's disease neuropathology.

Alzheimers disease, which is characterized by amyloid plaques and neurofibrillary tangles, may be attributed to the abnormal expression of gene(s) located on human chromosome 21. Genetic linkage studies have narrowed the region of candidate genes to 21q11.2–21q22 of the long arm of this chromosome. Several single copy sequences within this region, including the amyloid precursor protein (APP), have been mapped to mouse chromosome 16. Reliable strategies exist for breeding Trisomy 16 mice. However, the consequences of developmental overexpression of genes on chromosome 16 have not been previously investigated, because of the lethal effects of this aneuploidy during gestation. In the present report, we employ neural transplantation to study long‐term survival and pathogenesis in Trisomy 16 central nervous system tissues. Immunocytochemical staining with antiserum raised against the synthetic APP, beta‐A4 and alpha 1‐antichymotrypsin revealed numerous densely stained cells within hippocampal grafts of Trisomy 16 mice. Similarly, a population of grafted cells were positively stained following incubation with an antiserum raised against components of the pathological neurofibrillary tangle and with the monoclonal antibodies Tau 6.423 and ubiquitin.

Analysis of the core components of Alzheimer paired helical filaments A gas chromatography/mass spectrometry characterization of fatty acids, carbohydrates and long-chain bases

We have carried out a fatty acid and carbohydrate compositional analysis of the protease‐resistant core of paired helical filaments (prcPHF) isolated from six Alzheimers diseased brains. Fatty acids, long‐chain bases and monosaccharides were characterized by gas chromatography/mass spectrometry (GC/MS) of fatty acid methyl esters, trimethylsilylated long‐chain bases, peracetylated alditol acetates and trimethylsilyl methyl glycosides. Glucose and mannose were found to be the only carbohydrate components. Four of the six prcPHF samples contained only glucose while the remaining two samples contained between 30–40% mannose in addition to glucose. None of the samples were found to contain either hydroxylated fatty acids or long‐chain bases. The average fatty acid profile of prcPHF was highest in stearic (C18:0) and palmitic acids (C16:0) with less than 10% unsaturated fatty acids. By comparing the carbohydrate and lipid composition of prcPHF to similar data for other brain glycolipids, it was determined that prcPHF is a unique glycolipid, distinct from cerebrosides, gangliosides or brain phospholipids. The fatty acid and carbohydrate composition of a glycolipid isolated from a population of normal brains according to the prcPHF protocol was found be identical to that of prcPHF glycolipid. It is possible that subtle differences in structure or indigenous factors are responsible for the initiation of PHF formation in vivo.

Characterization of the Glycolipid Associated with Alzheimer Paired Helical Filaments

Abstract: In the present study, analytical techniques including gas chromatography/mass spectrometry (GC/MS)‐assisted carbohydrate linkage‐analysis, one‐ and two‐dimensional NMR, and matrix‐assisted laser desorption/ionization time of flight mass spectroscopy (MALDI‐MS) have been used to characterize the structure of the glycolipid associated with the paired helical filaments (PHF) isolated from the neurofibrillary tangles of Alzheimers diseased brain. The 1H NMR spectrum of acid‐hydrolyzed protein‐resistant core PHF (prcPHF) displays resonances that can be assigned to fatty acid and glucose. There are no resonances present that would indicate the presence of protein, amino acids, or a sphingosine base. Using two‐dimensional homonuclear correlated spectroscopy, homonuclear Hartmann‐Hahn, and heteronuclear multiple quantum coherence experiments, resonances in the 1H and 13C NMR spectrum of native PHF were assigned to a nonreducing terminal α‐1,6‐glycosidically linked glucose, an internal α‐1,6‐linked glucose, and an α‐1,2,6‐linked glucose. The narrow line‐widths observed for these residues suggest that they arise from glucose residues undergoing rapid segmental motion. The carbohydrate portion of the PHF‐associated glycolipid was analyzed using GC/MS linkage analysis and confirmed the presence of terminal and internal α‐1,6‐linked glucose and α‐1,2,6‐linked glucose in a molar ratio of 2:1:1. Three components of the PHF‐associated glycolipid fraction having masses 2,416, 2,325, and 2,237 Da were observed using MALDI‐MS. The least abundant, heavier mass component (2,416 Da) was best fit to a structure with a tridecamer of glucose having a single esterified C20 fatty acid (Glc13 + C20 or Glc13 + C20:1), whereas the more abundant, lower mass components were best fit to noncovalently associated glycolipid dimers, each with a glucose pentamer or hexamer having two C14, C16, or C18 esterified fatty acids {D[(Glc5 + C18) + (Glc6 + C16)] or D[(Glc5 + C14) + (Glc6 + C14)]}. The ratio of glucose to fatty acid calculated from these best‐fit structures of the more abundant mass components (5.5 ± 1.1:1.0) is in reasonable agreement with the same ratio calculated from peak integrations in the NMR spectra of acid‐hydrolyzed prcPHF (6.2 ± 1.6). Structural similarities between PHF‐associated glycolipid and other glycolipid amphiphiles known to form PHF‐like filaments indirectly suggest that this unique glycolipid may be an integral component of the PHF suprastructure.

Hippocampal grafts derived from embryonic trisomy 16 mice exhibit amyloid (A4) and neurofibrillary pathology.

Publisher Summary This chapter discusses the transplanted neocortical and hippocampal tissues derived from trisomic and control fetuses into the brains of normal recipient mice and monitored neuropathologcal changes occurring over 4–6 months within the grafts. In the experiments discussed in the chapter, embryos for transplantation were staged by vaginal plugs and the developmental age was confirmed by crown-rump length. Frontal cortex and hippocampal dissections were taken from 7 trisomic and 18 normal embryos assessed as E14-16. The trisomy 16 embryo was devoid of immunoreactivity to either antiserum. Neocortical and/or hippocampal sections derived from post mortem Alzheimer brain were included in each batch of immunocytochemical staining to confirm the activity and appropriate patterns of immunoreactivity of the antibodies. lmmunocytochemical staining with an anti-A4 antibody demonstrated the presence of deposits of A4 immunoreactivity around the cerebral vasculature within the trisomic grafts. The intracellular immunoreactivity of similar numbers of cells within trisomic grafts was observed with the A128 antiserum raised against purified paired helical filaments.

Alzheimer disease paired helical filament core structures contain glycolipid

The core structures of sodium dodecyl sulfate extracted, pronase digested paired helical filaments of Alzheimer disease were solubilized by heating in dimethyl sulfoxide. Electron microscopy revealed that after heating in dimethyl sulfoxide, intact paired helical filaments were no longer present in the dimethyl sulfoxide soluble fractions or in the insoluble lipofuscin-containing fractions. Enzyme-linked immunosorbent assays of the various fractions with the monospecific antibody A128 to paired helical filaments demonstrated 96% of the immunoreactivity to be in the dimethyl sulfoxide soluble fraction, and only 4% in the dimethyl sulfoxide insoluble fractions. Lyophilization of the dimethyl sulfoxide soluble supernatant and resuspension in water failed to reassociate the paired helical filaments, but did result in an insoluble precipitate. Analysis of the dimethyl sulfoxide solubilized paired helical filament fraction by nuclear magnetic resonance revealed it to be composed of glycolipid in a form that was distinct from similar fractions isolated from normal aged control brains. The aggregation of an altered glycolipid to form paired helical filaments in Alzheimer disease could explain their insolubility.

A Rapid One-Step Extraction Procedure for the Isolation of Ubiquitin from Human Erythrocytes for Antibody Production

A procedure is described that employs 5% perchloric acid extraction to isolate ubiquitin from human erythrocytes. The procedure is rapid and economical as it requires no specialized equipment. The extracted protein appeared to be highly purified as judged by electrophoresis and was identified as ubiquitin by immunoblotting and total amino acid analysis. The extraction yields about 78% of the ubiquitin in the hemolysate, which is a higher yield than is obtained with other procedures. The purified ubiquitin was used to make a polyclonal antiserum. As ubiquitin is a small and highly conserved protein, it is necessary to couple it to a larger immunogen to elicit an immune response. This ubiquitin antiserum was produced using an immunogen system that produces an immune response to the ubiquitin, but not to the carrier protein.

X-ray probe microanalysis of Alzheimer disease soluble and insoluble paired helical filaments

The paired helical filaments of Alzheimer disease, which have been shown to consist of both soluble and insoluble forms, were examined by X-ray probe microanalysis in order to determine if there existed differences in their elemental composition. The soluble paired helical filaments contained both sulfur and phosphorus, supporting their composition being enriched in a phosphorylated protein. The insoluble paired helical filament core structures, which retained their morphology after extensive protease digestion, contained only a small amount of sulfur over background, which suggests that they are not composed entirely of protein. This significant difference in sulfur and phosphorus indicates a difference in composition between the soluble and insoluble paired helical filaments, and that the paired helical filament core structures may attribute their insolubility to their being predominately non-proteinaceous.

Paired helical filaments are not the major binding sites for wheat germ and Dolichos biflorus agglutinins in the neurofibrillary tangles of Alzheimer's disease.

SummaryThe isolated paired helical filaments (PHF) that occur in the neurofibrillary tangles of Alzheimers disease were assayed to determine if they contained N-acetyl-glucosamine and N-acetyl-galactosamine residues. The enzyme-linked lectin assay was used to detect their total content in the PHF preparation. The assay employed biotinylated Dolichos biflorus and wheat germ agglutinins and was developed with avidin-horseradish peroxidase. The total PHF preparation was shown to contain very little of these glycosylated control proteins. Colloidal gold-labeled lectins were used to study the PHF by electron microscopy to assess whether the minor amount of lectin binding in the total preparation was directly associated with the PHF. These studies showed no significant association of the colloidal gold-labeled lectins with the isolated filaments. We conclude that the PHF themselves contain few or no N-acetyl-glucosamine or N-acetyl-galactosamine residues.

Production and characterization of a monospecific antiserum (A128) to disaggregated Alzheimer paired helical filaments.

Paired helical filaments (PHF), which constitute neurofibrillary tangles (NFT) and neuritic plaque (NP) neurites, serve as a useful marker for Alzheimer disease (AD). We have isolated AD PHF in a highly purified and disaggregated form for use as an immunogen to produce a heterologous polyclonal antiserum in rabbits. One rabbit was maintained long-term for the high quality of the antiserum it produced. Through absorptions with normal brain tissue, we were able to produce a monospecific antiserum which reacts only with NFT and NP neurites in AD brain tissue sections. We further demonstrated the specificity of this antiserum by electron microscopic immunohistochemistry, gel diffusion analysis, and immunoblotting. This antiserum also showed immunoreactivity to NFT of Down syndrome and progressive supranuclear palsy, and to the Pick bodies of Pick disease, but not to the Lewy bodies of idiopathic Parkinson disease. This well-characterized antiserum, all from one rabbit, offers several unique advantages to the study of the nature, origin, and interrelationships of filamentous protein abnormalities in AD and other neurodegenerative disorders.

A quantitative assessment of glycolipid and protein associated with paired helical filament preparations from Alzheimer's diseased brain

Protease resistant paired helical filaments (prcPHF) can be isolated from the brains of Alzheimers diseased patients. A second type of PHF, A68 PHF, may be extracted in soluble form from brain homogenate and induced to form filaments in vitro. Here we use a variety of analytical techniques to assess the protein, carbohydrate and fatty acid composition of prcPHF and A68 PHF. High-field ^1H NMR of both PHF preparations display similar fatty acid and carbohydrate proton resonances, consistent with the presence of a structurally similar glycolipid. Carbohydrate analysis showed that both preparations contained greater than 82% less than 12% C16:1 was significantly lower in A68 PHF than in prcPHF, both preparations contained otherwise similar fatty acid profiles with the most abundant lipid component being oleic acid (C18:1, 29.3 +/- 9.0%) followed by palmitic (C16:0, 28.5 +/- 5.6%) 17.6 +/- 7.5%) preparations revealed a profile reasonably consistent with that previously determined for PHF-tau but significantly higher in glycine and lower in lysine than would be predicted from the cDNA sequence. On a weight per cent basis, protein accounted for about 51% A68 PHF samples but only about 10% Carbohydrate and fatty acid accounted for about 39% A68 PHF samples but 74% preparations showed strong correlations between the total amount of tau protein and fatty acid. These results suggest that a glycolipid component forms an integral part of the PHF structure.