Extracellular amyloid β peptides (Aβs) have long been thought to be a primary cause of Alzheimer's disease (AD). Now, detection of intracellular neuronal Aβ1–42 accumulation before extracellular Aβ deposits questions the relevance of intracellular peptides in AD. In the present study, we directly address whether intracellular Aβ is toxic to human neurons. Microinjections of Aβ1–42 peptide or a cDNA-expressing cytosolic Aβ1–42 rapidly induces cell death of primary human neurons. In contrast, Aβ1–40, Aβ40–1, or Aβ42–1 peptides, and cDNAs expressing cytosolic Aβ1–40 or secreted Aβ1–42 and Aβ1–40, are not toxic. As little as a 1-pM concentration or 1500 molecules/cell of Aβ1–42 peptides is neurotoxic. The nonfibrillized and fibrillized Aβ1–42 peptides are equally toxic. In contrast, Aβ1–42 peptides are not toxic to human primary astrocytes, neuronal, and nonneuronal cell lines. Inhibition of de novo protein synthesis protects against Aβ1–42 toxicity, indicating that programmed cell death is involved. Bcl-2, Bax-neutralizing antibodies, cDNA expression of a p53R273H dominant negative mutant, and caspase inhibitors prevent Aβ1–42-mediated human neuronal cell death. Taken together, our data directly demonstrate that intracellular Aβ1–42 is selectively cytotoxic to human neurons through the p53–Bax cell death pathway.
Residues 16 -20 of the -amyloid peptide (A) function as a self-recognition element during A assembly into fibers. Peptides containing this motif retain the ability to interact with A and, in some cases, potently inhibit its assembly. Replacing L-with D-amino acids could stabilize such peptides and permit their evaluation as therapeutic agents for Alzheimer's disease. Here we have assessed the effect that such a chiral reversal has on inhibitory potency. D-enantiomers of five peptides, KLVFFA, KKLVFFA, KFVFFA, KIVFFA, and KVVFFA, were unexpectedly more active as inhibitors in an in vitro fibrillogenesis assay. Circular dichroism showed that D-KLVFFA more effectively prevented A adopting the -sheet secondary structure correlated with fibrillogenesis. Electron microscopy showed that fiber formation was also more strongly inhibited by D-KLVFFA. Heterochiral inhibition was confirmed using D-A, on the principle that enantiomeric proteins exhibit reciprocal chiral biochemical interactions. With D-A, L-KLVFFA was the more potent inhibitor, rather than D-KLVFFA. Most significantly, D-peptides were more potent at reducing the toxicity of both A 1-40 and A 1-42 toward neuronal cells in culture. This unforeseen heterochiral stereoselectivity of A for D-peptide inhibitors should be considered during future design of peptide-based inhibitors of A neurotoxicity and fibrillogenesis. -Amyloid peptide (A)1 is a 4-kDa peptide that when assembled into amyloid, progressively accumulates in Alzheimer's disease (AD) (1, 2). The neurotoxic properties specifically associated with aggregated forms of this peptide provide key causal evidence linking A to the pathology of AD (3, 4). This link is supported by A immunization experiments in transgenic mice expressing mutant alleles of human amyloid precursor protein. Such vaccination reduces both the A peptide levels and deposits in the brain (5) and also attenuates the associated cognitive impairment in these mice (6, 7). Subsequent studies showed that full assembly of A into mature fibers similar to those found in plaques is not necessary for toxicity. Smaller aggregates with molecular weights corresponding to dimers (8, 9), trimers, and tetramers (10) exhibit toxicity in cell culture as well. These findings support and highlight the necessity that A assembly intermediates be included as targets in AD drug development.While the delineation of which assembly intermediate(s) retain toxicity will aid in the rational design of specific inhibitors, screening assays have already identified a number of antifibrillogenic compounds (11-13). Studies show that the A 16 -20 region is important for A assembly (14, 15), possibly as an A self-recognition motif (16). These findings led to the development of a series of peptides incorporating this sequence, which bind to the homologous region of A and block its oligomerization (17). In another study an eleven residue peptide containing three prolines, which have a low propensity to form -sheets, inhibited A fibril formation (18). Pe...
Thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurine drugs. TPMT genetic polymorphisms represent a striking example of the potential clinical value of pharmacogenetics. Subjects homozygous for TPMT*3A, the most common variant allele for low activity, an allele that encodes a protein with two changes in amino acid sequence, are at greatly increased risk for life-threatening toxicity when treated with standard doses of thiopurines. These subjects have virtually undetectable levels of TPMT protein.In this study, we tested the hypothesis that TPMT*3A might result in protein misfolding and aggregation. We observed that TPMT*3A forms aggresomes in cultured cells and that it aggregates in vitro, functional mechanisms not previously described in pharmacogenetics. Furthermore, there was a correlation among TPMT half-life values in rabbit reticulocyte lysate, aggresome formation in COS-1 cells, and protein aggregation in vitro for the three variant allozymes encoded by alleles that include the two TPMT*3A single-nucleotide polymorphisms. These observations were compatible with a common structural explanation for all of these effects, a conclusion supported by size-exclusion chromatography and CD spectroscopy. The results of these experiments provide insight into a unique pharmacogenetic mechanism by which common polymorphisms affect TPMT protein function and, as a result, therapeutic response to thiopurine drugs. thiopurine toxicity ͉ protein aggregation ͉ pharmacogenomics ͉ protein degradation
Light-chain amyloidosis (AL) is characterized by immunoglobulin light-chain fragments aggregating into amyloid fibrils that deposit extracellularly in vital organs such as the kidney, the heart, and the liver, resulting in tissue degeneration and organ failure, leading to death. Cardiac involvement is found in 50% of AL patients and presents the most severe cases with a life expectancy of less than a year after diagnosis. In this study, we have characterized the variable domain of a cardiac AL patient light chain called AL-09. AL-09 folds as a b-sheet and is capable of forming amyloid fibrils both in the presence of sodium sulfate and in self-seeded reactions under physiological conditions. Glycosaminoglycans such as dermatan sulfate and heparin promote amyloid formation of self-seeded AL-09 reactions, while the glycosaminoglycan chondroitin sulfate A stabilized oligomeric intermediates and did not elongate the preformed fibrils (nucleus) present in the reaction. Finally, the histological dye Congo red, known to bind to the cross b-sheet structure of amyloid fibrils, inhibits AL-09 amyloid fibril formation in the presence of sodium sulfate and in self-seeded reactions. This paper provides insight into the impact of different reagents on light-chain stability, structure, amyloid fibril formation, and inhibition.
Dark-field microscopy of blood from healthy individuals revealed the existence of pleomorphic microorganisms. These bacteria exhibited limited growth and susceptibility to antibiotics and could be detected by fluorescent in situ hybridization and flow cytometry. They were further characterized by analysis of their 16S rRNA and gyrB genes.In our search for spirochetes involved in Alzheimer's disease (13), we observed pleomorphic bacteria in the blood of healthy human subjects by dark-field microscopy. This was a surprising finding since it is generally acknowledged that the bloodstream in healthy humans is a sterile environment (7) except when there is a breach in the integrity of the tissue membranes (6). However, the concept of the occurrence of bacteria in the blood of healthy humans is now more plausible because of cultivation-independent laboratory approaches. The main techniques employed in such studies include PCR amplification and sequencing of the16S ribosomal DNA (rDNA). These methods have revealed the presence of a wide diversity of microorganisms in the environment, and indeed within the human body (12). In this report we present evidence based on molecular phylogenetic techniques and light and electron microscopy, as well as other conventional microbiological methods, for the existence of a population of bacteria in healthy human blood. In view of the apparent controversial nature of our findings, it was encouraging to note the recent report of Nikkari et al. (14), who detected blood-associated bacterial rDNA sequences by using real-time PCR methods and a probe targeting conserved regions of bacterial 16S rDNA, and an earlier report by Tedeshi et al. (16) on the presence of pleomorphic bacteria as intraerythrocytic parasites in clinically healthy human subjects.For light microscopic examination, blood samples from 25 healthy volunteers were drawn in a Vacutainer tube with no anticoagulants (Becton Dickinson, Franklin Lakes, N.J.); blood was drawn in the conventional manner involving antisepsis of the skin and avoidance of any introduction of external microorganisms by contamination. (Since external contamination was always a possibility, particular care and precaution were exercised at all times to avoid this. The specific procedures, as well as appropriate controls, are specified throughout the text.) A wet mount of the serum from the clotted blood of each sample, fresh or incubated at 30°C for between 5 to 7 days, was examined by dark-field microscopy (Leitz Dialux 20) for pleomorphic bacteria.For PCR amplification, a 0.5-ml aliquot of incubated blood containing pleomorphic bacteria was used for conventional extraction of DNA by the method of Higuchi (11). Three microliters of the extract was used for DNA amplification by the method of Edwards et al. (8) using the forward primer BSF8/20 (5Ј-AGAGTTTGATCCTGGCTCAG-3Ј) and the reverse primer BSR1541/20 (5Ј-AAGGAGGTGATCCAGCCGCA-3Ј). Thirty cycles of PCR were performed, with 1 cycle consisting of the following steps: (i) denaturation (1 min at 94°C), (i...
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