ABSTRACT
Oxidative stress is well accepted as an important pathogenic factor in Parkinson disease, based largely on indirect evidence. Recently, we have developed antibodies that recognize specific advanced glycation end-products (anti-pentosidine and anti-pyrraline), protein modifications that are potentiated by oxidative stress in a process termed glycoxidation. We applied these antibodies immunocytochemically to affected regions in Parkinson disease and diffuse Lewy body disease brains. Additionally, we used antibodies to heme oxygenase-1, a putative marker of oxidative stress response. Immunoreactivity to pentosidine, pyrraline, and heme oxygenase-1 was seen in the substantia nigra of Parkinson disease and the neocortex of diffuse Lewy body disease. Heme oxygenase-1 was further demonstrated by immunoelectron microscopy in intimate association with filaments of cortical Lewy bodies. Immunolocalization of advanced glycation end-products and a marker of oxidative stress response induction provides evidence that glycoxidation and oxidative stress may be an important pathogenic factor in diseases characterized by Lewy body formation, and furthers the evidence that cytoskeletal proteins and their inclusions are susceptible to oxidative stress.
Subject(s)
Oxidative Stress/physiology , Parkinson Disease/metabolism , Aged , Aged, 80 and over , Arginine/analogs & derivatives , Arginine/analysis , Cerebral Cortex/chemistry , Cerebral Cortex/enzymology , Cross-Linking Reagents/analysis , Glycation End Products, Advanced/metabolism , Glycosylation , Heme Oxygenase (Decyclizing)/analysis , Humans , Immunohistochemistry , Locus Coeruleus/chemistry , Locus Coeruleus/enzymology , Lysine/analogs & derivatives , Lysine/analysis , Microscopy, Immunoelectron , Middle Aged , Neurons/chemistry , Neurons/enzymology , Neurons/ultrastructure , Norleucine/analogs & derivatives , Norleucine/analysis , Parkinson Disease/physiopathology , Pyrroles/analysis , Substantia Nigra/chemistry , Substantia Nigra/enzymologyABSTRACT
In this study, we evaluate the ability of several solvents to solubilize insoluble paired helical filaments (PHF) of Alzheimer disease. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitative assays to establish solvents of PHF. Using sequential categories of protein solvent to analyze insoluble PHF, only alkali or exhaustive proteolysis are effective in completely solubilizing PHF, while a variety of denaturants are ineffective. Alkali does not affect the phosphorylation state of PHF and complete dephosphorylation of PHF with hydrofluoric acid does not affect PHF solubility. These findings suggest that the 'hyperphosphorylation' of PHF proteins is not responsible for PHF insolubility. However the in vitro glycation of tau generates PHF that are insoluble in SDS and soluble in alkali. These findings suggest that protein crosslinks, including advanced glycation endproduct-derived crosslinks which were recently described in Alzheimer disease, play a major role in effecting PHF insolubility in vivo.
Subject(s)
Alzheimer Disease/metabolism , Neurofilament Proteins/analysis , Adolescent , Adult , Aged , Aged, 80 and over , Alkalies , Cell Fractionation , Cross-Linking Reagents/analysis , Cross-Linking Reagents/metabolism , Densitometry , Endopeptidases , Formates , Glycation End Products, Advanced/analysis , Guanidine , Guanidines , Hemostatics , Humans , Immunoblotting , Microscopy, Electron , Middle Aged , Neurofilament Proteins/metabolism , Neurofilament Proteins/ultrastructure , Phosphorylation , Protein Denaturation , Sodium Dodecyl Sulfate , Solubility , UreaABSTRACT
This study presents evidence for plasma membrane abnormalities of the dystrophic neurites in senile plaques of Alzheimer's disease. We found that the plasma membranes of dystrophic neurites are more labile to fixation than those membranes of other cells of the senile plaque or of normal neurites distant from senile plaques. Further, we found vesicles in the extracellular space adjacent to dystrophic neurites and similar to those within them, suggesting that the increased lability seen in our preparations may, in vivo, be associated with release of neuritic contents. Plasma membrane alterations may be critical to deposition of amyloid-beta in senile plaques from the abundant beta-protein precursor of dystrophic neurites. The consequences of altered membrane integrity, such as calcium influx, lipid peroxidation and free radical damage, could also be responsible for many of the pathological correlates of the disease.
Subject(s)
Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Neurites/metabolism , Neurites/pathology , Neurofibrillary Tangles/metabolism , Neurofibrillary Tangles/pathology , Oxidative Stress , Cell Membrane/metabolism , Cell Membrane/pathology , Cell Membrane/ultrastructure , Humans , Middle Aged , Neurites/ultrastructure , Neurofibrillary Tangles/ultrastructureABSTRACT
In this study, the direct comparison of biopsy and autopsy tissue by morphological and immunocytochemical techniques, respectively, was used to document cytoskeletal changes of dystrophic neurites (DN) of senile plaques in Alzheimer's disease. This dual approach demonstrated several unreported abnormalities which, together with analogous findings in several experimental models, suggest that DN are associated with deficiencies in fast axonal transport and replacement of the cytoskeleton by an array of related abnormal filaments.
Subject(s)
Actin Cytoskeleton/pathology , Alzheimer Disease/pathology , Axonal Transport , Neurites/pathology , Neurofibrillary Tangles/pathology , Actin Cytoskeleton/ultrastructure , Adult , Aged , Aged, 80 and over , Alzheimer Disease/metabolism , Alzheimer Disease/physiopathology , Amyloid beta-Protein Precursor/metabolism , Amyloid beta-Protein Precursor/ultrastructure , Humans , Immunohistochemistry , Microscopy, Immunoelectron , Middle Aged , Neurites/ultrastructure , Neurofibrillary Tangles/ultrastructureABSTRACT
This study demonstrates elastase immunoreactivity in the neurofibrillary pathology of Alzheimer disease. Using an antiserum against elastase, we show that elastase immunoreactivity is restricted to neurons and is markedly elevated in a proportion of neurofibrillary tangle-bearing neurons. Elastase is a proteolytic enzyme that might be a candidate protease for the generation of amyloid-ß from ß-protein precursor. These findings support the hypothesis that proteases play an important role in Alzheimer disease and furthers the notion that an imbalance in proteolytic regulation contributes towards the pathogenic presentation of the disease. Moreover, since α1-antitrypsin, the principal inhibitor of elastase, is highly susceptible to oxidative stress, our findings suggest a link between proteolytic imbalance and oxidative stress in the pathogenesis of Alzheimer disease.
ABSTRACT
Oxidative stress is increasingly implicated in a number of neurodegenerative disorders characterized by abnormal filament accumulation in affected neurons, including Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis. To further evaluate the role of oxidative stress in the neurodegenerative process and the accumulation of abnormal filaments, we examined the pathologic lesions in Pick disease and of corticobasal degeneration with immunocytochemistry by using antisera to heme oxygenase-1 (HO-1) - a putative marker of oxidative injury. Immunoreactivity to HO-1 was demonstrated in ballooned neurons, Pick bodies, neuropil threads, and glial inclusions (the latter two in a case of corticobasal degeneration). By immunoelectron microscopy, HO-1 immunolabelling of Pick bodies was closely associated with the abnormal filaments comprising the inclusion. Apparently unaffected neurons in all cases showed only background levels of HO-1 immunoreactivity. These data suggest that oxidative stress is important in the formation of the lesions characteristic of Pick disease and corticobasal degeneration. Moreover, taken together with our previous demonstration that HO-1 immunoreactivity is associated with the neurofibrillary pathology of Alzheimer disease, progressive supranuclear palsy, and subacute sclerosing panencephalitis, it appears that oxidative stress specifically targets the cytoskeleton in a variety of neurodegenerative disorders characterized by abnormal filament accumulation.
Subject(s)
Basal Ganglia/metabolism , Basal Ganglia/pathology , Cerebral Cortex/metabolism , Cerebral Cortex/pathology , Dementia/metabolism , Dementia/pathology , Oxidative Stress/physiology , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Brain/pathology , Brain/ultrastructure , Humans , Immunohistochemistry , Microscopy, Immunoelectron , Nerve Degeneration/physiology , Neurofibrillary Tangles/pathology , Neurons/ultrastructureABSTRACT
Previous studies demonstrated the specific association of heme oxygenase (HO)-1 protein to the neurofibrillary pathology of Alzheimer's disease (AD). In this study, we used reverse transcription-polymerase chain reaction methods to show the increased expression of HO-1 but not HO-2 mRNA transcripts in cerebral cortex and cerebral vessels from subjects with AD compared with age-matched non-AD controls. Neither the HO-1 nor the HO-2 mRNA levels was altered in the cerebellum, a brain region usually spared from the pathological alterations of AD. There was no clear evidence that the expression of HO-1 in these tissues was related to postmortem interval, cause of death, or the age of the subjects studied. Using immunoblotting methods, we further showed that HO-1 protein content was increased in neocortical and vascular samples from AD subjects compared with controls. Our findings suggest the specific induction of HO-1 mRNA and protein in the cerebral cortex and cerebral vessels but not HO-2 mRNA or protein in association with the pathological lesions of the disease.
Subject(s)
Alzheimer Disease/enzymology , Brain/blood supply , Cerebral Cortex/enzymology , Gene Expression , Heme Oxygenase (Decyclizing)/genetics , RNA, Messenger/biosynthesis , Aged , Base Sequence , Cerebellum/enzymology , Heme Oxygenase (Decyclizing)/biosynthesis , Humans , Molecular Sequence Data , Polymerase Chain Reaction , RNA-Directed DNA PolymeraseABSTRACT
We present the first evidence for carbonyl-related posttranslational modifications of neurofilaments in the neurofibrillary pathology of Alzheimer's disease (AD). Two distinct monoclonal antibodies that consistently labeled neurofibrillary tangles (NFTs), neuropil threads, and granulovacuolar degeneration in sections of AD tissue also labeled the neurofilaments within axons of the white matter following modification by reducing sugars, glutaraldehyde, formaldehyde, or malondialdehyde. The epitope recognized by these two antibodies shows a strict dependency for carbonyl modification of the neurofilament heavy subunit. The in vivo occurrence of this neurofilament modification in the neurofibrillary pathology of AD suggests that carbonyl modification is associated with a generalized cytoskeletal abnormality that may be critical in the pathogenesis of neurofibrillary pathology. Furthermore, the data presented here support the idea that extensive posttranslational modifications, including oxidative stress-type mechanisms, through the formation of cross-links, might account for the biochemical properties of NFTs and their resistance to degradation in vivo.
Subject(s)
Alzheimer Disease/metabolism , Glyceraldehyde/pharmacology , Malondialdehyde/pharmacology , Neurofibrillary Tangles/metabolism , Neurofilament Proteins/metabolism , Ribose/pharmacology , Alzheimer Disease/pathology , Epitopes , Humans , Immunoblotting , Microscopy, Electron , Neurofibrillary Tangles/immunology , Neurofibrillary Tangles/ultrastructure , Protein Processing, Post-TranslationalABSTRACT
The simultaneous presence of intracellular neurofibrillary tangles (NFT) and extracellular senile plaques in Alzheimer's disease (AD) suggests that the two lesions could be synergistically interrelated. However, although the main protein components of NFT and senile plaques, tau (tau) and amyloid beta-protein, respectively, are well characterized, the molecular mechanisms responsible for their deposition in lesions are unknown. We demonstrate, using four independent techniques, that tau directly interacts with a conformation-dependent domain of the amyloid beta-protein precursor (beta PP) encompassing residues beta PP714-723. The putative tau-binding domain includes beta PP717 mutation sites that are associated with familial forms of AD. Our findings strongly suggest that NFT and senile plaques, often thought of as independent structures, may play a role in each other's formation during the pathogenesis of AD.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Protein Precursor/metabolism , Brain/metabolism , tau Proteins/metabolism , Alzheimer Disease/etiology , Amino Acid Sequence , Amyloid beta-Protein Precursor/chemistry , Humans , Immunoblotting , Immunoenzyme Techniques , Immunohistochemistry , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolismABSTRACT
Apolipoprotein E (ApoE) genotype is a significant risk factor for the development of Alzheimer disease (AD) and the ApoE protein is associated with senile plaques (SP) and neurofibrillary tangles (NFT), the pathological lesions of AD. Despite this data, the relevance of ApoE to the disease pathogenesis is unknown. In this study we sought to understand the role that ApoE protein could play in the pathogenesis of AD. Using an in situ binding technique, we showed that ApoE bound avidly to SP and NFT in diseased brain. Molecular characterization of ApoE binding suggested that binding to NFT was mediated by tau, the main protein component of NFT, and that ApoE binding to SP was mediated by amyloid-beta, the main protein component of SP. There was no significant difference in binding or binding characteristics between the different ApoE isoforms, ApoE3 and ApoE4. These findings suggest that the interaction of ApoE with tau and amyloid-beta proteins in AD could play a important role in the formation of NFT and SP, respectively, contributing to the pathogenesis of AD.
Subject(s)
Alzheimer Disease/metabolism , Apolipoproteins E/metabolism , Neurofibrillary Tangles/metabolism , Alzheimer Disease/genetics , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Genotype , Hippocampus/metabolism , Hippocampus/pathology , Humans , Neurons/metabolism , tau Proteins/metabolismABSTRACT
In this report we quantitatively assess the numbers of intracellular and extracellular neurofibrillary tangles (NFT) in the brains of a series of individuals with Alzheimer's disease and of controls and correlate these with neuronal loss. Our data indicate that in some cases, NFT are not removed from the brain throughout the disease process. This finding, together with our previous demonstration of carbonyl-related modifications in NFT, provides additional evidence that the protein constituents of NFT are resistant to proteolytic removal, possibly as a result of extensive cross-links. Additionally, correlation between the number of NFT and neuronal loss indicates that there are at least two distinct mechanisms responsible for neuronal death in Alzheimer's disease that are directly and indirectly related to the presence of neurofibrillary pathology.
Subject(s)
Alzheimer Disease/pathology , Neurofibrillary Tangles/pathology , Aged , Cell Count , Cell Death , Entorhinal Cortex/pathology , Frontal Lobe/pathology , Humans , Microglia/pathology , Middle AgedSubject(s)
Alzheimer Disease/metabolism , Glycation End Products, Advanced/metabolism , Maillard Reaction , Nerve Tissue Proteins/metabolism , Alzheimer Disease/pathology , Amyloid beta-Peptides/metabolism , Animals , Free Radicals/metabolism , Glycation End Products, Advanced/analysis , Humans , Neurofibrillary Tangles/pathology , Oxidation-ReductionABSTRACT
Previously, we showed three differentially sulfated forms of chondroitin sulfate proteoglycans (CSPG) associated with senile plaques, astrocytes and neurofibrillary tangles in Alzheimer's disease. Here, monoclonal antibodies were used to demonstrate CSPGs in other neurodegenerative diseases. CSPGs were found associated with inclusions of Parkinson's, diffuse Lewy body, Pick's diseases, and progressive supranuclear palsy. Reacting astrocytes in each of these neurodegenerative diseases and Huntington's disease showed immunoreactivity for CSPG. CSPG distribution in a variety of neurodegenerative diseases suggests that similar mechanisms may be involved in the accumulation of proteoglycans in a number of filamentous inclusions.
Subject(s)
Astrocytes/chemistry , Chondroitin Sulfate Proteoglycans/analysis , Inclusion Bodies/chemistry , Nervous System Diseases/metabolism , Neurons/chemistry , Aged , Aged, 80 and over , Humans , Immunoenzyme Techniques , Middle Aged , Nerve Degeneration/physiology , Nervous System Diseases/pathologyABSTRACT
Heme oxygenase-1 is an important enzyme that degrades heme, a pro-oxidant, leading to the formation of antioxidant molecules. In this study we demonstrate by immunocytochemistry close association of heme oxygenase-1 with Alzheimer neurofibrillary pathology and with the neurofibrillary tangles found in progressive supranuclear palsy and subacute sclerosing panencephalitis. In Alzheimer's disease, using two different rabbit antisera against heme oxygenase-1 protein, we localized, using immunocytochemical methods, heme oxygenase-1 to neurofibrillary tangles, senile plaque neurites, granulovacuolar degeneration, and neuropil threads. Only light background staining was seen in young controls and sporadic lesion-related immunoreactivity in age-matched controls. The increase in heme oxygenase-1 protein in association with the neurofibrillary pathology of Alzheimer's disease and other diseases characterized by neurofibrillary tangles supports the notion that the generation of free radicals and oxidative stress plays a role in the pathogenesis of neurofibrillary pathology.
Subject(s)
Alzheimer Disease/enzymology , Heme Oxygenase (Decyclizing)/analysis , Neurofibrils/enzymology , Aged , Alzheimer Disease/pathology , Antibody Specificity , Humans , Neurofibrillary Tangles/enzymology , Neurofibrillary Tangles/pathology , Neurofibrils/pathologyABSTRACT
During aging long-lived proteins accumulate specific post-translational modifications. One family of modifications, termed Maillard reaction products, are initiated by the condensation between amino groups of proteins and reducing sugars. Protein modification by the Maillard reaction is associated with crosslink formation, decreased protein solubility, and increased protease resistance. Here, we present evidence that the characteristic pathological structures associated with Alzheimer disease contain modifications typical of advanced Maillard reaction end products. Specifically, antibodies against two Maillard end products, pyrraline and pentosidine, immunocytochemically label neurofibrillary tangles and senile plaques in brain tissue from patients with Alzheimer disease. In contrast, little or no staining is observed in apparently healthy neurons of the same brain. The Maillard-reaction-related modifications described herein could account for the biochemical and insolubility properties of the lesions of Alzheimer disease through the formation of protein crosslinks.
Subject(s)
Alzheimer Disease/metabolism , Arginine/analogs & derivatives , Glycation End Products, Advanced/metabolism , Hippocampus/metabolism , Lysine/analogs & derivatives , Norleucine/analogs & derivatives , Pyrroles/metabolism , Arginine/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Immunoenzyme Techniques , Lysine/metabolism , Maillard Reaction , Neurofibrillary Tangles/metabolism , Norleucine/metabolismABSTRACT
Amyloid beta (A beta) immunoreactivity has been demonstrated in all extracellular neurofibrillary tangles (E-NFT) and most intraneuronal neurofibrillary tangles (I-NFT). We undertook this immunocytochemical study to understand the relationship between A beta immunoreactivity localized in NFT and beta-protein precursor (beta PP). We found epitopes of amino-, mid-, and carboxyl-terminal domains of beta PP in I-NFT and the majority of E-NFT. NFT retained beta PP after ionic detergent extraction, demonstrating that beta PP is an integral component of NFT. Finding beta PP in regions of A beta immunoreactivity raises the possibility that beta PP or its fragments associate with amyloid, and that the stability of A beta is responsible for its dominance in amyloid deposits.