Protective chaperones in alzheimer's disease: small heat shock proteins prevent in vitro AβM2 amyloid formation

Yogish C Kudva, C. S. Mueske, P. C. Butler, N. L. Eberhardt

Research output: Contribution to journalArticle

Abstract

Alzheimer's disease is a complex neurodegenerative disorder, involving two types of fibrils associated with neuronal death or abnormal fonction. The fibrils associated with neuritic plaques involve amyloid formation from APP-derived processed polypeptides, containing 39-43 amino acids. In studies of human islet amyloid polypeptide expression in COS cells, we demonstrated that intracelhitar amyloid formation is associated with cell death and hypothesized that protective mechanisms must exist to counteract the cytotoxic actions of intracellular amyloid (O'Brien TD et al., 1995, Am JPathol 147: 609-616). To begin to test this hypothesis, we examined the role of the ubiquitous heat shock proteins/chaperones on in vitro amyloid formation, using the Alzheimer's AβMZ as a model system. Amyloid formation was monitored by the increase in fluorescence of thioflavine T (ThT) bound to amyloid. Synthetic AβMI, verified for amino acid composition, sequence and purity, was dissolved in dimethylsulfoxide (100-3200 \iM) and amyloid formation was induced by dilution (1:20) into 10 mM Tris.HCl (pH 7.4), 100 mM NaCl. ThT fluorescence (XDl= 450 nm, >,= 482 nm) was measured at hourly intervals for 5-6 hours and the initial rate of fluorescence increase was analyzed by regression analysis. Confirming published observations, the time-dependent increase in ThT fluorescence was: (i) directly dependent on the concentration of AβMI and temperature, (ii) maximal at pH 6.5- 7.4, and (iii) enhanced by moderate concentrations of Na (50-100 mM) and inhibited by K (50-200 mM). The generation of the fluorescence signal was correlated with characteristic amyloid fibrils (8-10 nm width) by electron microscopy. Inclusion of small human heat shock proteins (sHsp), either human Hsp27 or E. coli Hsp25 (1.54- 6.2 pM) , inhibited amyloid formation by AβMI (20 uM) 70-100% as assessed by fluorescence assay (p < 0.05, ANOVA with Dunnett's multiple comparison tests) and electron microscopy. Specificity of the sHsp response was indicated by inability of the homologous ct-crystallin to inhibit amyloid formation. In conclusion, we have (i) validated a fluorescence assay that faithfully monitors AβM: amyloid formation and (ii) demonstrated that small heat shock proteins can inhibit amyloid formation. The data suggest that age-related changes in chaperone function could be a contributing factor toward the pathogenesis of Alzheimer's disease.

Original languageEnglish (US)
JournalJournal of Investigative Medicine
Volume44
Issue number3
StatePublished - 1996

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Small Heat-Shock Proteins
Amyloid
Alzheimer Disease
Fluorescence
Heat-Shock Proteins
Electron microscopy
In Vitro Techniques
Assays
Electron Microscopy
Islet Amyloid Polypeptide
Amino Acids
Heat-Shock Response
Crystallins
COS Cells
Amyloid Plaques
Cell death
Analysis of variance (ANOVA)
Dimethyl Sulfoxide
Regression analysis
Neurodegenerative Diseases

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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Protective chaperones in alzheimer's disease : small heat shock proteins prevent in vitro AβM2 amyloid formation. / Kudva, Yogish C; Mueske, C. S.; Butler, P. C.; Eberhardt, N. L.

In: Journal of Investigative Medicine, Vol. 44, No. 3, 1996.

Research output: Contribution to journalArticle

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abstract = "Alzheimer's disease is a complex neurodegenerative disorder, involving two types of fibrils associated with neuronal death or abnormal fonction. The fibrils associated with neuritic plaques involve amyloid formation from APP-derived processed polypeptides, containing 39-43 amino acids. In studies of human islet amyloid polypeptide expression in COS cells, we demonstrated that intracelhitar amyloid formation is associated with cell death and hypothesized that protective mechanisms must exist to counteract the cytotoxic actions of intracellular amyloid (O'Brien TD et al., 1995, Am JPathol 147: 609-616). To begin to test this hypothesis, we examined the role of the ubiquitous heat shock proteins/chaperones on in vitro amyloid formation, using the Alzheimer's AβMZ as a model system. Amyloid formation was monitored by the increase in fluorescence of thioflavine T (ThT) bound to amyloid. Synthetic AβMI, verified for amino acid composition, sequence and purity, was dissolved in dimethylsulfoxide (100-3200 \iM) and amyloid formation was induced by dilution (1:20) into 10 mM Tris.HCl (pH 7.4), 100 mM NaCl. ThT fluorescence (XDl= 450 nm, >,= 482 nm) was measured at hourly intervals for 5-6 hours and the initial rate of fluorescence increase was analyzed by regression analysis. Confirming published observations, the time-dependent increase in ThT fluorescence was: (i) directly dependent on the concentration of AβMI and temperature, (ii) maximal at pH 6.5- 7.4, and (iii) enhanced by moderate concentrations of Na (50-100 mM) and inhibited by K (50-200 mM). The generation of the fluorescence signal was correlated with characteristic amyloid fibrils (8-10 nm width) by electron microscopy. Inclusion of small human heat shock proteins (sHsp), either human Hsp27 or E. coli Hsp25 (1.54- 6.2 pM) , inhibited amyloid formation by AβMI (20 uM) 70-100{\%} as assessed by fluorescence assay (p < 0.05, ANOVA with Dunnett's multiple comparison tests) and electron microscopy. Specificity of the sHsp response was indicated by inability of the homologous ct-crystallin to inhibit amyloid formation. In conclusion, we have (i) validated a fluorescence assay that faithfully monitors AβM: amyloid formation and (ii) demonstrated that small heat shock proteins can inhibit amyloid formation. The data suggest that age-related changes in chaperone function could be a contributing factor toward the pathogenesis of Alzheimer's disease.",
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AU - Eberhardt, N. L.

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