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Biomedical subjects

T Wisniewski

Publications and source records attributed to T Wisniewski.

At least 55 records · Page 3Linked to original sources
PubMed1996

Micropreparative gel electrophoresis of low-molecular-weight peptides: purification of highly insoluble amyloid peptide fragments.

We have used the continuous-elution micropreparative gel electrophoresis device described by Baumann and Lauraeus (Anal. Biochem. 214, 142-148, 1993) to purify low-molecular-weight peptide fragments from in-gel digested standard proteins as well as highly in-soluble amyloid peptides. Alzheimer's amyloid beta-peptide, gelsolin-derived amyloid peptide of the Finnish type, and a novel amyloid of the British type were purified from either homogenized brain or kidney tissue material to a high degree of purity in a single run. Using the high resolving capacity of the Tris-Tricine-SDS buffer system of Schaegger and von Jagow (Anal. Biochem. 166, 368-379, 1978) we were able to isolate two synthetic peptides with M(r)4329 and 3284, differing only by 1045 in mass. The total peptide recovery, as determined by amino acid sequence analysis and scanning densitometry, ranged between 60 and 80%. In order to demonstrate the utility of this technique we subjected some of the purified peptides to direct N-terminal amino acid sequence analysis, mass spectrometry, microbore high-performance liquid chromatography, and immunochemical studies. Our results show that micropreparative gel electrophoresis is an effective tool for the isolation of not only larger polypeptides but also small peptide fragments in a form suitable for further biological use.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Alzheimer's beta-amyloid peptide is conformationally modified by apolipoprotein E in vitro.

Amyloid beta-peptide (A beta) is a major component of neuritic plaques, a feature of Alzheimer's disease (AD) brains. Recently, we showed that A beta adopts two major conformational states in solution, which differ in their abilities to form amyloid. These are highly amyloidogenic conformer (A beta ac) with a high content of beta-sheet and a slowly amyloidogenic conformer (A beta nac) with a random coil conformation. Apolipoprotein E (apoE), particularly the E4 isoform, which is genetically associated with AD, binds to A beta and modulates fibrillogenesis in vitro. In the present work, the influence of apoE on the conformation of A beta peptides was studied. The results suggest that, under the conditions used, apoE enhances amyloid formation by inducing the conformational transition from A beta nac into A beta ac. We propose that an important step in A beta fibrillogenesis is the transformation induced by apoE of the soluble non-amyloidogenic into the pathological amyloidogenic conformer of A beta.

Alzheimer Disease↗
PubMed1996

Transthyretin gene in Alzheimer's disease patients.

Amyloid beta (Abeta) is known to be the main component of Alzheimer's disease (AD) senile plaques. A homologous peptide is a normal component of biological fluids and is called soluble Abeta (sAbeta). Synthetic peptides homologous to Abeta form amyloid-like fibrils in vitro. This fibril formation can be inhibited by normal human cerebrospinal fluid (CSF) [Wisniewski et al., Ann. Neurol. 34 (1993)]. Furthermore, it has been proposed that normal transthyretin (TTR), which is a component of CSF, can itself bind sAbeta, preventing amyloid fibril formation, and that variants of TTR could be associated with AD [Schwarzman et al., Proc. Natl. Acad. Sci. USA, 91 (1994)]. Because of this possible association, we screened for TTR mutations from 47 sporadic and 19 early-onset familial AD patients using single strand conformation polymorphism analysis. Our results show no correlation between TTR variants and Alzheimer's disease in this group of patients.

Age of Onset↗
PubMed1996

C-terminal fragments of alpha- and beta-tubulin form amyloid fibrils in vitro and associate with amyloid deposits of familial cerebral amyloid angiopathy, British type.

Familial amyloidosis, British type, is an autosomal dominant disease characterized by progressive dementia, spastic paralysis and ataxia. The identity of the accumulating amyloid is not known, thus preventing the definitive classification of the disease. Biochemical methods were used to characterize the nature of the amyloid deposits from the brain tissue of one individual who died with this disease. The purified tissue material was subjected to trypsin digestion and subsequent N-terminal sequence analysis. Major tryptic fragments yielded the sequences VGINYQPPTVVPGGDLAK, FDLMYAK, GLTVPEL and GYLTVAAVFR, which are all tryptic fragments of the C-termini of human tubulin subunits alpha and beta. Synthetic peptides based on the sequences of these fragments formed amyloid fibrils in vitro fitting the characteristic definition of amyloid. These findings suggest that the C-terminal fragments of both alpha- and beta-tubulin are closely associated to the amyloid deposits of familial amyloidosis, British type.

Amino Acid Sequence↗
PubMed1996

HB-GAM is a cytokine present in Alzheimer's and Down's syndrome lesions.

The distribution of heparin binding growth associated molecule (HB-GAM) in the cerebral amyloidoses of Alzheimer's disease (AD) and Down's syndrome (DS), conditions characterized by the deposition of amyloid beta (A beta), was investigated immunohistochemically. Antibodies to HB-GAM, a cytokine which plays an important role in brain development and maturation, showed strong immunoreactivity with senile plaques in both AD and DS. Anti-HB-GAM reacted with pre-amyloid lesions, but only when markers of dystrophic neurites were present. The presence of HB-GAM in AD brains, but not in control brains, was confirmed by Western blotting. We suggest that the presence of HB-GAM in A beta lesions is a marker of neuronal injury.

Adolescent↗
PubMed1996

Amyloid beta 1-42 deposits do not lead to Alzheimer's neuritic plaques in aged dogs.

In alzheimer's disease, amyloid beta (A beta) is deposited in senile plaques and amyloid angiopathy. Longer A beta peptides, which extend to residue 42 (A beta 42), have been suggested to be critical for the seeding of amyloid. Aged dogs develop cerebral vessel amyloid and parenchymal preamyloid lesions. Preamyloid in humans is related to senile plaques, whereas in dogs such progression is rare. We evaluated the composition of aged canine vessel amyloid and preamyloid both biochemically and immunohistochemically. The vessel amyloid extended mainly to residue 40 (A beta 40), while preamyloid contained a mixture of A beta 17-42 and A beta 42, with minimal A beta 40. Our results suggest other factors besides A beta 42 are important for neuritic plaque formation.

Aging↗
PubMed1996

Apolipoprotein E and amyloidogenesis.

Alzheimer's amyloid beta-protein (A beta) is a modified, pathogenic form of a constitutive host protein, soluble amyloid beta-protein (sA beta). Both are conformational isomers encoded by the gene for the beta-amyloid precursor protein (APP), located on chromosome 21. sA beta and A beta have identical sequence but are thought to differ in their secondary structure and physicochemical properties, hence they are conformational isomers. sA beta is easily degraded, while A beta is particularly resistant. A beta has a high beta-pleated sheet content, while sA beta is thought to be more random-coil and/or alpha-helical. A beta, unlike sA beta, adopts an amyloidogenic conformation, forms aggregates and gives rise to fibrils. Most early-onset forms of Alzheimer's disease (AD) have been linked to mutations of the presenilin 1, presenilin 2 or APP genes, located on chromosomes 14, 1 and 21, respectively. Their relationship to amyloidogenesis is being investigated. On the other hand, the major risk factor for the most common form, sporadic and familial late-onset AD, is the presence of the apoE epsilon 4 allele. Recent studies have shown that a 10 kDa C-terminal fragment of apoE is complexed to A beta in neuritic plaques and that apoE isoforms can modulate amyloid formation in vitro. Moreover, thrombin cleavage of apoE generates a similar C-terminal fragment that can form amyloid-like fibrils. Thus neuritic plaques may contain both A beta and apoE amyloid fibrils. AD can be neuropathologically defined by the presence of several interacting proteins that can adopt an amyloidogenic conformation. This has led us to hypothesize that in AD, amyloidosis may be reactive rather than causative.

Alzheimer Disease↗
PubMed1996

Sequence of deposition of heterogeneous amyloid beta-peptides and APO E in Down syndrome: implications for initial events in amyloid plaque formation.

Patients with trisomy 21 [Down syndrome (DS)] progressively develop amyloid beta-protein (A beta) deposits and then other features of Alzheimer's disease (AD), apparently due to increased gene dosage and thus expression of the beta-amyloid precursor protein. Because the neuropathological phenotype in older DS subjects closely resembles that of AD, the examination of DS brains of increasing age provides a unique model of the progression of AD. Here, we characterized the deposition of several A beta peptides and apolipoprotein E in formalin-fixed brain sections from 29 DS subjects between 3 and 73 years old. Amyloid plaque number and the percentage of cortical area they occupied were quantified by computerized image analysis. A beta ending at amino acid 42 (A beta 42) was the earliest form of A beta deposited in DS cortex. It was observed in 7 of 16 young (3-30 years) subjects, with the earliest deposition occurring at age 12. A beta ending at residue 40 (A beta 40) was not detected until approximately age 30, a time when degenerating neurites around A beta immunoreactive (IR) plaques were first observed, and the frequency of A beta 40 IR plaques then rose with age. Even in old (51-73 years) DS subjects, A beta 42 IR plaques were always more abundant than A beta 40 IR plaques. A beta peptides starting at aspartate 1 or pyroglutamate 3 were detected in small subsets of compacted, neuritic plaques beginning around age 30 and rose with age, the latter species always exceeding the former. Thus, the N-termini of the A beta 42 peptides abundantly deposited in very young DS subjects remain unknown. Apo E was detectable in a small subset of A beta 42 IR plaques beginning at age 12 and rose steadily with age; it clearly followed the deposition of A beta. Our analysis of very young DS brains suggests that amyloid plaque formation begins with A beta 42-ending peptides, and the number and percentage of cortical area of A beta 42 plaques increase very little with advancing age, while other heterogeneous A beta species and Apo E progressively accrue onto plaques containing A beta 42.

Adolescent↗
PubMed1996

Familial meningocerebrovascular amyloidosis, Hungarian type, with mutant transthyretin (TTR Asp18Gly)

Amyloid deposits in leptomeningeal vessels, subarachnoid, subpial, and subependymal cerebrospinal regions, spinal ganglia, peripheral nerves, and some internal organs (predominantly heart and kidney) characterize a dominantly inherited disease in a Hungarian family. We found four definitely and three probably affected members in this family of 56 persons in four generations. Clinical features in all definitely diseased patients include disturbance of memory, psychomotor deceleration, ataxia, and hearing loss. In most patients there was temporary disorientation, migraine-like headache with vomiting, and tremor. Some patients had nystagmus, pyramidal signs with spastic paraparesis, hallucinations, urinary retention, and obstipation. Single patients had facial tics and sleep disorders. Progressive visual disturbance and clinically manifest polyneuropathy were absent. CSF protein was markedly elevated in all patients. CT showed characteristic symmetric calcification along the sylvian fissure; MRI after contrast administration showed prominent enhancement at the surface of the sylvian fissures, brainstem, and cerebellum. Autopsy data was available in three definitely affected patients and in one unaffected family member. Immunohistochemistry identified the amyloid deposits as of the AF (transthyretin, TTR) type; DNA studies revealed a novel TTR missense mutation at codon 18 (TTR Asp18Gly). According to clinical features, pathologic alterations, and molecular studies, this disease is a novel type of systemic familial amyloidosis with disease manifestation clinically restricted to the CNS. It is similar to the oculoleptomeningeal amyloidoses but can be clinically diagnosed by characteristic CTs and the absence of progressive visual impairment.

Adult↗
PubMed1996

Molecular biology of brain aging and neurodegenerative disorders.

A significant component of the aging process is genetically determined. Numerous theories of aging exist, many of which postulate the existence of "longevity genes." Recent advances in molecular biological and other techniques have allowed a significantly greater understanding of aging and age-related disease. This will be illustrated by four genetic and sporadic diseases: Alzheimer's disease (AD) and related disorders, transthyretin dementia, cerebral amyloid angiopathy-Icelandic type and scrapie related diseases. Alzheimer's disease (AD), the most common of this group, is the leading cause of dementia in Western countries. Recent genetic and biochemical studies have shown the involvement of at least four genes in the pathogenesis of AD. In early-onset familial AD mutations in the beta PP, S182 (presenilin 1) and STM2 (presenilin 2 or E5-1) genes have been found, while in the more common late-onset AD the presence of the apolipoprotein E4 isotype is a major risk factor. Genetic studies have also helped to elucidate the etiology of rarer cerebral amyloidoses such as the recently described Hungarian amyloidosis that is characterized by meningocerebrovascular amyloid deposition, with resultant dementia. This disease is linked to a mutation in the transthyretin gene. It is hoped that in the near future this increase in knowledge will allow the development of therapeutic approaches to slow the aging process.

Aging↗
PubMed1996

Meningocerebrovascular amyloidosis associated with a novel transthyretin mis-sense mutation at codon 18 (TTRD 18G)

We describe a novel transthyretin mutation at codon 18 where Asp is replaced by Gly (D18G) in a Hungarian kindred. This mutation is associated with meningocerebrovascular amyloidosis, producing dementia, ataxia, and spasticity. Fifty different transthyretin mutations are related to amyloid deposition, typically producing a peripheral neuropathy or cardiac dysfunction. These symptoms are absent in this family. Up to now, amyloid-beta (A beta), cystatin C, and prion proteins have been known to be deposited as amyloid in the brain, leading to stroke or dementia. With this report we establish that transthyretin amyloid deposition can also produce central nervous system dysfunction as the major clinical symptom.

Amino Acid Sequence↗
PubMed1995

Amyloid beta binding proteins in vitro and in normal human cerebrospinal fluid.

A major neuropathological feature of Alzheimer's disease (AD) is the deposition of amyloid beta (A beta) in the form of senile plaques. The A beta peptide exists both in a beta-pleated sheet fibrillar form in amyloid deposits and as a normal soluble protein in biological fluids. Numerous proteins have been identified immunohistochemically to be associated with senile plaques, where A beta is the major constituent. Some of the latter have also been suggested to be carriers of the normal soluble A beta (sA beta) including apolipoprotein J (apoJ), apolipoprotein E (apoE) and transthyretin (TTR). We have found, using several different methods, that numerous proteins can bind synthetic A beta peptides when high concentrations are used; however, using an affinity anti-sA beta column we confirm that apoJ is the major binding protein in pooled human cerebrospinal fluid. On the other hand it is known that apoE co-purifies with A beta biochemically extracted from senile plaques. In AD tissue there may be a change in the major apolipoprotein binding A beta from apoJ to apoE.

Amino Acid Sequence↗
PubMed1995

Is Alzheimer's disease an apolipoprotein E amyloidosis?

The presence of the apolipoprotein E4 allele has been identified as a major risk factor for late-onset Alzheimer's disease. Apolipoprotein E has also been found immunohistochemically in Alzheimer's disease lesions. We biochemically isolated amyloid beta from senile plaques and found that a carboxyl-terminal fragment (residues 216-299) of apolipoprotein E co-purified. In vitro this fragment from recombinant apolipoprotein E could form amyloid-like fibrils, which were Congo-red positive. Thus senile plaques may contain both amyloid beta and apolipoprotein E amyloid fibrils.

Alzheimer Disease↗
PubMed1995

Fibrillogenesis in Alzheimer's disease of amyloid beta peptides and apolipoprotein E.

A central event in Alzheimer's disease is the conformational change from normally circulating soluble amyloid beta peptides (A beta) and tau proteins into amyloid fibrils, in the form of senile plaques and neurofibrillary tangles respectively. The apolipoprotein E (apoE) gene locus has recently been associated with late-onset Alzheimer's disease. It is not know whether apoE plays a direct role in the pathogenesis of the disease. In the present work we have investigated whether apoE can affect the known spontaneous in vitro formation of amyloid-like fibrils by synthetic A beta analogues using a thioflavine-T assay for fibril formation, electron microscopy and Congo Red staining. Our results show that, under the conditions used, apoE directly promotes amyloid fibril formation, increasing both the rate of fibrillogenesis and the total amount of amyloid formed. ApoE accelerated fibril formation of both wild-type A beta-(1-40) and A beta-(1-40A), an analogue created by the replacement of valine with alanine at residue 18, which alone produces few amyloid-like fibrils. However, apoE produced only a minimal effect on A beta-(1-40Q), found in the Dutch variant of Alzheimer's disease. When recombinant apoE isoforms were used, apoE4 was more efficient than apoE3 at enhancing amyloid formation. These in vitro observations support the hypothesis that apoE acts as a pathological chaperone, promoting the beta-pleated-sheet conformation of soluble A beta into amyloid fibres, and provide a possible explanation for the association of the apoE4 genetic isoform with Alzheimer's disease.

Alzheimer Disease↗