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1.
Int J Exp Pathol ; 84(2): 91-9, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12801283

ABSTRACT

We recently described a transgenic mouse strain overexpressing hamster alphaA-crystallin, a small heat shock protein, under direction of the hamster vimentin promoter. As a result myelin was degraded and axonal dystrophy in both central nervous system (especially spinal cord) and peripheral nervous system occurred. Homozygous transgenic mice developed hind limb paralysis after 8 weeks of age and displayed progressive loss of myelin and axonal dystrophy in both the central and peripheral nervous system with ongoing age. Pathologically the phenotype resembled, to a certain extent, neuroaxonal dystrophy. The biochemical findings presented in this paper (activity of the enzymes superoxide dismutase, catalase and transglutamase, myelin protein zero expression levels and blood sugar levels) confirm this pathology and exclude other putative pathologies like Amyothrophic Lateral Sclerosis and Hereditary Motor and Sensory Neuropathy. Consequently, an excessive cytoplasmic accumulation of the transgenic protein or a disturbance of the normal metabolism are considered to cause the observed neuropathology. Therefore, extra-ocular alphaA-crystallin-expressing transgenic mice may serve as a useful animal model to study neuroaxonal dystrophy.


Subject(s)
Demyelinating Diseases/metabolism , Neuroaxonal Dystrophies/metabolism , alpha-Crystallin A Chain/metabolism , Animals , Blood Glucose/metabolism , Blotting, Southern , Blotting, Western , Catalase/metabolism , Demyelinating Diseases/etiology , Demyelinating Diseases/genetics , Disease Models, Animal , Mice , Mice, Transgenic , Myelin P0 Protein/metabolism , Neuroaxonal Dystrophies/etiology , Neuroaxonal Dystrophies/genetics , Superoxide Dismutase/metabolism , Transglutaminases/metabolism
2.
FEBS Lett ; 480(2-3): 79-83, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-11034304

ABSTRACT

alphaA-Crystallin, a small heat shock protein with chaperone-like activity, forms dynamic multimeric complexes. Recently we described the spontaneous generation of a mutant protein (super alphaA-crystallin) by exon duplication arisen via exon shuffling confirming a classic hypothesis by Gilbert [Nature 271 (1978) 501]. Comparison of super alphaA-crystallin, which is viable in a mouse skeletal muscle cell line, with normal alphaA-crystallin shows that it has diminished thermostability, increased exposure of hydrophobic patches, a larger complex size and lost its chaperone activity. However, super alphaA-crystallin subunits exchange as readily between complexes as does normal alphaA-crystallin. These data indicate that chaperone-like activity may vanish independent of subunit hydrophobicity and exchangeability.


Subject(s)
Crystallins/metabolism , Exons , Molecular Chaperones/metabolism , Anilino Naphthalenesulfonates , Animals , Cell Line , Chromatography, Gel/methods , Cricetinae , Crystallins/genetics , Crystallins/isolation & purification , Fluorescence , Fluorescent Dyes , Gene Expression , Heating , Mice , Microscopy, Electron/methods , Molecular Chaperones/genetics , Molecular Chaperones/isolation & purification , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism
3.
Invest Ophthalmol Vis Sci ; 41(12): 3893-7, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11053291

ABSTRACT

PURPOSE: To determine whether alpha-crystallin is capable of forming filament-like structures with other members of the crystallin family. METHODS: Water-soluble crystallins were isolated from calf lenses and fractionated into alpha-, betaH-, betaL-, and gamma-crystallins according to standard procedures. Chaperone-like activity of alpha-crystallin was determined in control and UV-A-irradiated lenses by the heat-induced aggregation assay of betaL-crystallin. Protein samples from this assay were analyzed by electron microscopy. In vitro filament formation was examined by transmission immunoelectron microscopy using specific antibodies directed against the crystallins. Involvement of intermediate filament constituents was excluded by the results of Western blot analysis, which were all negative. Moreover, the in vitro amyloid fibril interaction test using thioflavin T (ThT) was also performed. RESULTS: At the supramolecular level heating at 60 degrees C has no effect on the morphologic appearance of alpha-crystallin as observed by transmission electron microscopy. Moreover alpha-crystallin obtained from UV-A-irradiated lenses shows a virtually identical shape. However, heating in the presence of betaL-crystallin results in the formation of filament-like alphabeta-hybrids as demonstrated by immunoelectron microscopy using specific antibodies directed either against alpha- or betaL-crystallin. Parallel experiments with alpha-crystallin derived from UV-A-irradiated lenses showed even more pronounced filamentous structures, compared with the controls. Nonetheless, we were able to show that the UV-light treatment affected the chaperone-like capacity of alpha-crystallin, as revealed by a diminished ability to inhibit in vitro denaturation of betaL-crystallin. To exclude the presence of cytoskeletal contamination in the crystallin preparations, vimentin antibodies were also tested. These latter experiments were negative. The filamentous nature of the hybrids was further confirmed by the results obtained with the ThT assay earlier applied for the detection of amyloid fibrils. CONCLUSIONS: Crystallin hybrids have previously been detected in the water-soluble lens crystallin fraction. Our findings indicate that such endogenous hybrids, formerly called "rods," may result from stress-induced interaction between alpha-crystallin and other lens constituents such as betaL-crystallin. Because the hybrid formation is enhanced when alpha-crystallin from UV-A-irradiated lenses is used as one of the two components of the hybrid, one can only speculate that this formation may be one of the factors leading to UV-A cataract.


Subject(s)
Crystallins/metabolism , Cytoskeleton/metabolism , Heat Stress Disorders/metabolism , Animals , Cattle , Chromatography, Gel , Crystallins/ultrastructure , Cytoskeletal Proteins/metabolism , Cytoskeleton/ultrastructure , Heat Stress Disorders/pathology , Lens, Crystalline/metabolism , Lens, Crystalline/radiation effects , Microscopy, Immunoelectron , Molecular Chaperones/metabolism , Organ Culture Techniques , Protein Denaturation , Ultraviolet Rays
4.
Int J Exp Pathol ; 81(4): 271-82, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10971749

ABSTRACT

Homozygous mice transgenic for alphaA-crystallin, one of the structural eye lens proteins, developed hindlimb paralysis after 8 weeks of age. To unravel the pathogenesis of this unexpected finding and the possible role of alphaA-crystallin in this pathological process, mice were subjected to a histopathological and immunohistochemical investigation. Immunohistochemistry showed large deposits of alphaA-crystallin in the astrocytes of the spinal cord, and in the Schwann cells of dorsal roots and sciatic nerves. Additionally, microscopy showed dystrophic axons in the spinal cord and digestion chambers as a sign of ongoing demyelination in dorsal roots and sciatic nerves. Apart from a few areas with slight alphaA-crystallin-immunopositive structures, the brain was normal. Because the alphaA-crystallin protein expression appeared in specific cells of the nervous system (astrocytes and Schwann cells), the most plausible explanation for the paralysis is a disturbance of cell function caused by the excessive intracytoplasmic accumulation of the alphaA-crystallin protein. This is followed by a sequence of secondary changes (demyelination, axonal dystrophy) and finally arthrosis. In conclusion, alphaA-crystallin transgenic mice develop a peripheral and central neuropathy primarily affecting spinal cord areas at the dorsal side, dorsal root and sciatic nerve.


Subject(s)
Axons/ultrastructure , Crystallins/physiology , Demyelinating Diseases/etiology , Paralysis/etiology , Animals , Astrocytes/metabolism , Blotting, Western , Crystallins/genetics , Crystallins/metabolism , Demyelinating Diseases/metabolism , Demyelinating Diseases/pathology , Electrophoresis, Polyacrylamide Gel , Female , Male , Mice , Mice, Transgenic , Microscopy, Electron , Paralysis/metabolism , Paralysis/pathology , Schwann Cells , Spinal Cord/ultrastructure
5.
Ophthalmologica ; 214(1): 7-12, 2000.
Article in English | MEDLINE | ID: mdl-10657740

ABSTRACT

alphaB-Crystallin, which has homology with the small heat shock proteins, is the basic subunit of alpha-crystallin, a major component of the vertebrate eye lens. These crystallins have for a long time been thought to be absolutely lens specific. However, about a decade ago alphaB-crystallin has been detected extralenticularly in many tissues among which the central nervous system. Under pathological conditions the expression level of alphaB-crystallin frequently increases. For this reason it is considered to be a useful marker in a variety of neurodegenerative diseases. In this mini-review, a number of typical neurodegenerative disorders is dealt with in which alphaB-crystallin may play a role.


Subject(s)
Brain/metabolism , Crystallins/physiology , Neurodegenerative Diseases/metabolism , Neurons/metabolism , Spinal Cord/metabolism , Animals , Biomarkers , Humans , Prognosis
6.
Biochem Biophys Res Commun ; 262(1): 152-6, 1999 Aug 19.
Article in English | MEDLINE | ID: mdl-10448084

ABSTRACT

Amyloid beta (Abeta) is a 40- to 42-residue peptide that is implicated in the pathogenesis of Alzheimer's Disease (AD). As a result of conformational changes, Abeta assembles into neurotoxic fibrils deposited as 'plaques' in the diseased brain. In AD brains, the small heat shock proteins (sHsps) alphaB-crystallin and Hsp27 occur at increased levels and colocalize with these plaques. In vitro, sHsps act as molecular chaperones that recognize unfolding peptides and prevent their aggregation. The presence of sHsps in AD brains may thus reflect an attempt to prevent amyloid fibril formation and toxicity. Here we report that alphaB-crystallin does indeed prevent in vitro fibril formation of Abeta(1-40). However, rather than protecting cultured neurons against Abeta(1-40) toxicity, alphaB-crystallin actually increases the toxic effect. This indicates that the interaction of alphaB-crystallin with conformationally altering Abeta(1-40) may keep the latter in a nonfibrillar, yet highly toxic form.


Subject(s)
Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/toxicity , Crystallins/pharmacology , Molecular Chaperones/pharmacology , Neurons/pathology , Peptide Fragments/chemistry , Peptide Fragments/toxicity , Plaque, Amyloid/drug effects , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Animals , Animals, Newborn , Apoptosis/drug effects , Benzothiazoles , Cell Survival/drug effects , Cells, Cultured , Cerebral Cortex , Dose-Response Relationship, Drug , Hippocampus , Neurons/drug effects , Neurons/metabolism , Neurons/ultrastructure , Peptide Fragments/metabolism , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Plaque, Amyloid/ultrastructure , Protein Binding/drug effects , Protein Structure, Secondary/drug effects , Rats , Thiazoles
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