Paul Mulvihill

Carbonyl-related posttranslational modification of neurofilament protein in the neurofibrillary pathology of Alzheimer's disease

Abstract: We present the first evidence for carbonyl‐related posttranslational modifications of neurofilaments in the neurofibrillary pathology of Alzheimers 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.

Extracellular neurofibrillary tangles reflect neuronal loss and provide further evidence of extensive protein cross-linking in Alzheimer disease

In this report we quantitatively assess the numbers of intracellular and extracellular neurofibrillary tangles (NFT) in the brains of a series of individuals with Alzheimers 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 Alzheimers disease that are directly and indirectly related to the presence of neurofibrillary pathology.

Immunochemical Properties of Ubiquitin Conjugates in the Paired Helical Filaments of Alzheimer Disease

Abstract: Immunocytochemical and peptide sequencing studies indicate that the regulatory protein ubiquitin (Ub) is incorporated into the paired helical filaments (PHF) of Alzheimer disease. In this study, we showed that some antibodies raised to PHF recognize epitopes of Ub. Analysis of the Ub sequences recognized by the antibodies raised to PHF, along with the known specificity of several monoclonal antibodies raised to artificial Ub conjugates, indicates the immunochemical representation of Ub residues 34–76 in PHF. The Ub epitopes recognized by antibodies raised to PHF are distinct from those recognized by antibodies raised to artificial Ub conjugates in two respects. First, antibodies that are raised to PHF and that recognize Ub react with PHF equally, whether denatured or not, whereas those raised to artificial Ub conjugates show greater reaction after denaturation. Second, mapping of the epitopes recognized by two monoclonal antibodies to PHF onto Ub indicates a distinction in the Ub residues recognized, compared with monoclonal antibodies raised to artificial Ub conjugates. The proximity of their epitopes to the site of conjugation, as well as their affinity for PHF polypeptides, suggests that the PHF antibodies that recognize Ub may be directed specifically to Ub epitopes defined by the protein conjugated to Ub.

Immunoaffinity demonstration that paired helical filaments of Alzheimer disease share epitopes with neurofilaments, MAP2 and tau.

Identification of the neuronal components incorporated into the neurofibrillary tangles of Alzheimer disease has primarily been derived from immunocytochemical procedures. Previous antibody studies have been able to directly determine the shared epitopes of known neuronal proteins with neurofibrillary tangles (NFT) only when the appropriate monoclonal antibodies were available. In this study, we use an immuno-affinity purification protocol to directly determine the properties of the epitopes recognized by two antisera which recognize NFT. Characterization of the purified antibodies demonstrates that NFT share epitopes with the two heavier neurofilament subunits. NFH and NFM, as well as MAP2 and tau. Further, this method indicates that the epitopes shared with neurofilaments and tau are distinct from each other.

Demonstration of a novel neurofilament associated antigen with the neurofibrillary pathology of Alzheimer and related diseases

A monoclonal antibody, termed NFT200, was raised after in vitro immunization with sonicated neurofibrillary tangle (NFT)-enriched fractions prepared from Alzheimer brain. The antigen to which NFT200 is directed was expressed in the paired helical filaments of NFT in sporadic and familial Alzheimer disease (AD), in the straight filaments of NFT in AD, progressive supranuclear palsy and of Pick bodies, and the NFT in several other conditions such as Parkinson-dementia complex of Guam and subacute sclerosing panencephalitis. Granulovacuolar degeneration of AD was also labeled with NFT200. Hirano bodies and amyloid deposits in AD, as well as Lewy bodies of idiopathic Parkinson disease lacked in the antigen. The NFT200-antigen was also expressed as a phosphatase-insensitive antigen in normal neurofilaments found in spinal cord and peripheral nerve axons but was absent from the perikaryal accumulation of neurofilaments induced by aluminum intoxication. Nevertheless, immunoblot studies failed to detect the NFT200 in isolated preparations of the neurofilament proteins, MAP-2, tau, ubiquitin or A4-amyloid peptide. The results indicate that the NFT200 monoclonal antibody is directed against a phosphatase-insensitive epitope of an axonal protein associated with neurofilaments but is labile to isolation and expressed as a stable epitope of a 200 kDa component of NFT.

Ubiquitin in Alzheimer and other Neurodegenerative Diseases

The majority of efforts in understanding the abnormal neuronal inclusions found in some neurodegenerative diseases are directed toward analyzing their chemical nature. As discussed in other chapters, this analysis has depended primarily on indirect techniques e.g. immunocytochemistry and structural studies, which lack the quantitation and rigor that direct biochemical analysis can provide. Although these studies have demonstrated the presence of cytoskeletal elements in several inclusions, they have not provided an answer regarding their major constituent. Another approach in the study of these inclusions is the elucidation of the mechanism underlying the formation of abnormal polymers; that is, the processes responsible for transformation of components, probably normally present in neurons, into structures unique to these diseases.

Alteration of the Neurofilament-Microtubule Network in Alzheimer Disease and Other Neurodegenerative Disorders

The major lesions consistently found in the brains of Alzheimer patients are neurofibrillary tangles (NFT) and senile plaques (SP). The prevalence of NFT and SP have been found to correlate with the severity of the dementia (1–3). NFT are primarily composed of paired helical filaments (PHF), although 15 nm straight filaments and amorphous components are also common constituents (4). SP, or neuritic plaques, are composed of a core of cerebral amyloid, 6 to 8 nm filaments (5), surrounded by altered neurites containing PHF (6). The chemical identity, mode of formation and physiological consequences of the PHF have been the focus of many studies on Alzheimer disease (AD).