Neuronal Thread Protein Assay for Alzheimer's Disease

             Alzheimer’s disease is the most important cause of dementia in the United States, afflicting 1 o to 15 percent of elderly individuals . Although the diagnosis of Alzheimer’s disease is readily made by postmortem examination of the brain, the diagnosis in the living patient can b e difficult, particularly at early stages of the disease. Evaluation of new treatment protocols and investigations to further understand its natural history and epidemiology would be better effected if specific, sensitive, and readily assessed antemortem (prior to death} diagnostic markers for Alzheimer’s disease existed . As has been recognized for sometime, amyloid , an amorphous, fibrillar, in soluble protein, accumulates in brains with Alzheimer’s disease. Recently , a great deal of attention has been focused on the possibility of a mutation in the amyloid gene which could be responsible for amyloid accumulation in the brain. Indeed, this might be the case, but in several hereditary conditions in which amyloid does accumulated in the brain (mainly in blood vessel walls and the tissue between the neuronal cell bodies) because of specific mutations in the amyloid gene, there are no other associated lesions of Alzheimer’s disease and the affected individuals are not demented . These observations cast doubt upon the hypothesis that an abnormality in the amyloid  gene or processing of the protein is entirely responsible for or causes all of the clinical and neuropathological degeneration associated with Alzheimer’s disease .                 The major structural lesions in brains with Alzheimer’s disease include: loss of neurons with atrophy of the cerebral cortex; retraction of neuronal processes; abnormal (dystrophic} neuritic sprouts and dendritic processes suggesting failed attempts to reestablish connections with other neurons; accumulation of inclusions known as neurofibrillary tangles and paired helical filaments within neurons reflecting degeneration; and of course amyloid accumulation in the walls of blood vessels and in senile plaques. The major neuropathological changes correlated with dementia in Alzheimer’s disease are the density of neurofibrillary tangles and extent of dystrophic neuritic sprouts rather than senile plaques, per se. Therefore, any specific and reliable biological marker of Alzheimer’ s disease should be correlated with neurofibrillary tangles and dystrophic neurites, and not necessarily with senile plaques.                   Our research involves the study of a novel protein termed, “Neuronal thread protein”, which accumulates in brains with Alzheimer’s disease. We discovered neuronal thread protein, · or NTP, because we had been working with another similar but smaller molecule, pancreatic thread protein, which is a fibrillar protein that has structural features in common with Alzheimer’s paired helical filaments when viewed with the electron microscope. Using antibodies prepared against the pancreatic form of the protein, but which cross-react with NTP, we demonstrated that NTP accumulates in the same population of neurons that have Alzheimer’s neurofibrillary tangles, and in the same distribution as the dystrophic neuritic sprouts that are so prevalent in Alzheimer’s disease brains. Moreover, NTP was found to be accumulated in neurons predisposed to developing but lacking neurofibrillary tangles, perhaps suggesting that NTP immunoreactivity (antibody binding} marked neurons that have already degenerated but do not yet manifest neurofibrillary tangles. Using a more quantitative method of assessing immunoreactivity, we demonstrated significantly higher levels of NTP in Alzheimer’s disease compared with aged control brains. In additio n, we discovered that NTP was detectable in cerebrospinal fluid (CSF), and that the levels in Alzheimer’s disease were significantly higher than in aged controls. Analysis of the molecular weights by polyacrylamide gel electrophoresis disclosed that NTP extracted from brain is the same size as the NTP extracted from CSF , and both are larger than the pancreatic form of the protein. Correspondingly, cloning and molecular studies have demonstrated that the messenger RNA encoding NTP is larger than the one encoding the pancreatic form of the protein, confirming that these are two distinct molecules, despite similar antibody-binding properties.                 One of the most exciting preliminary observations was that accumulation of NTP in Alzheimer’s disease brains is associated with increased levels of NTP in CSF. To detect this abnormality, we utilized a highly sensitive radioimmunometric binding assay which we developed in our laboratory, and which will be used for further studies of NTP expression in Alzheimer’s disease brains and CSF. We expect that this assay will have broad utility as a diagnostic test for Alzheimer’s disease in the living patient. The focus of the present research proposal is to extend the original observations by assessing the sensitivity and specificity of this test with respect to diagnosing and perhaps re-defining Alzheimer’s disease on the basis of elevated levels of NTP in CSF. We intend to accomplish this specific aim by employing our already developed highly sensitive immunoradiometric assay to measure NTP levels in postmortem ventricular fluid from Alzheimer’s disease, aged normal controls, and individuals with neurodegenerative di seases other than Alzheimer’s disease, i.e. disease controls. In addition, we will initiate antemortem studies of CSF from patients with clinically suspected and postmortem docum ented Alzheimer’s disease, Parkinson’s disease (an important disease control group) , and normal aged controls.                   The second specific aim of this proposed research is to characterize NTP derived from Alzheimer’s disease CSF. This is necessary to develop new monoclonal antibodies which will disting ui sh the pancreatic and neuronal forms of the protein on the basis of an immunoassay, and without the need for more time consuming and complex assays such Western blots. NTP is approximately 20% larger than the pancreatic form of the protein. Therefore, in order to generate specific NTP monoclonal antibodies we will need to identify distinct antigenic regions (epitopes) in NTP that are not present in the pancreatic form of the protein. To do this, we will first purify large quantities of NTP from Alzheimer’s disease CSF by affinity chromatography using three different monoclonal antibodies ttiat we already have and which bind to NTP. We will determine a small portion of the protein sequence to confirm that the correct mol ecul e has be en extracted. The purifi ed NTP will be used to immunize mice to generat e monoclonal antibodies. We will screen the clones by ascertaining which antibodies bind to purified NTP and not to the purified pancreatic form of the protein, and which antibodies bind to Alzheimer’s disease brains and not to pancreatic acinar cells. Such antibodies will be critical to developing a highly specific immunoradiometric antemortem CSF-based diagnostic assay for Alzheimer’s disease.