NEUROBIOLOGY OF AGING, VOLUME 11, 1990 ABSTRACTS OF SECOND INTERNATIONAL CONFERENCE ON ALZHEIMER'S DISEASE MECHANISMS OF NEURONAL DEGENERATION

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MAb Alz-50 recognizes a neuronal antigen of 68kDa termed A68 which is spec i f i ca l l y expressed in Alzheimer brain tissue (Ann. Neurol., 22 (1987) 521). Alz-50 was also shown to cross-react with Tau proteins (J.Biol.Chem., 263 (1988) 7943) which are the major antigenic component of PHF. This f inding suggests that A68 might be an aberrant form of Tau. We have shown that in AD, two Tau proteins with molecular weight (MW) of 64 and 69kDa, named Tau 64 and 69, are spec i f i ca l l y detected in areas that are affec- ted by the neuro f ib r i l l a ry degeneration (J.Neurol.Sci. , 92 (1989) 133; Acta Neuropathol., in press). Tau 64 and 69 are due to an abnormal phosphorylation of Tau proteins as demonstrated by the decrease of the i r MW af ter a lkal ine phosphatase treatment. The cross-reaction of Alz-50 with Tau proteins and the close MW of Tau 69 le t us think that they might be the same protein.

In order to ver i fy this hypothesis, we compared the binding of Alz-50 (generous g i f t of Abbott Lab.) with those of anti-Tau and anti-PHF antisera on western blots of brain homogenates from patients with AD. Alz-50 was essent ia l ly directed against Tau 64 and 69, l ike the anti-PHF and i t never stained a protein around 68kDa other than Tau 69. When samples were treated with alkal ine phosphatase, Alz-50 stained dephosphorylated products of Tau 64 and 69 (MW of 50 to 60kDa). Besides, on immunoblots of two- dimensionally resolved proteins, Alz-50 never stained a 68kDa component other than Tau 69.

These results strongly suggest that A68 and Tau 69 are the same protein. The further study of Tau 69/A68, which is a re l ia - ble marker of the neuro f ib r i l l a ry degeneration, might complement those of the amyloid precursor to track the biochemical dysfunc- tions that lead to neuronal death and dementia.

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PAIRED HELICAL FILAMENTS (PHF) IN ALZHEIMER DISEASE ARE PHOSPHORYLATED AT MULTIPLE SITES. *LR. Murthy and K. Iqbal. Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA.

PHF are known to contain microtubule associated protein tau in an abnormally phosphorylated form/s (Proc. Natl. Acad. Sci. USA, 83:4913-17, 1986; ibid 86:5646-50, 1989). The present study was undertaken to determine the molecular nature of PHF phosphorylation. PHF were isolated from Alzheimer disease (AD) brains, with (PHF.C.SDS) or without (PHF.SDS) digestion with collagenase/dispase, followed by sodium dodecyl sulfate (SDS) treatment (Acta Neuropathol. 62:167-177, 1984). Aliquots of PHF.C.SDS and PHF.SDS were each treated with alkaline phosphatase for 18 hrs. at 37°C (protein: enzyme ratio of 1:10 in 100mm Tris, pH 7.8) to study the accessibility of the phosphate groups in PHF. Non- phosphoprotein phosphates were removed from the PHF samples by extensive extractions with trichloroacetic acid and ether (Methods in Enzymology 99, 7-14, 1983). Each PHF sample was then hydrolyzed in 6N HCI in vapor phase at 100°C for 4 hrs. The protein hydrolyzates and standard phosphoaminoacids were each derivatised with pheny- lisothiocyanate. The derivatised phosphoaminoacids were then analysed by High Performance Liquid Chromatography using a C18 silica based reverse phase column. Per ug protein, PHF.C.SDS contained 15.1+1 pmoles phosphoserine (PSer), 17.2+0.8 pmoles phosphothreonine (PThr) and 10.9-t-0.8 pmoles phosphotyrosine (PTyr). The phosphatase treatment decreased PThr to nondetectable levels, PTyr to 3.8 +0.5 pmoles (65% reduction) and PSer to 11.9 + 0.7 pmoles (21% reduction). PHF.SDS contained, per ug protein, 78.7+4.3 pmoles PSer, 8.1+0.8 pmoles PThr and 6.5+0.1 pmoles PTyr, which by the phosphatase treatment resulted to nondetectable levels for PThr and PTyr, and to 18.8+0.8 (76% reduction) pmoles for PSer. These studies suggest that PHF are phosphorylated at multiple sites and by more than one protein kinase. The large differences in the amounts of PSer in PHF.C.SDS and PHF.SDS are most likely due to the cleavage and the loss of tau fragments from the former by the collagenase/dispase treatment. (Supported in part by the Alzheimer's Disease Research Program of American Health Assistance Foundation, Rockville, MD., and by NIH grants AG05892, NS18105 and AG04220).

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A CASEIN KINASE I ACTIVITY ASSOCIATED WITH THE A68 PROTEINS FROM ALZHEIMER BRAIN. *I. Vincent & P. Davies. Albert Einstein College of Medicine, Bronx, NY, 10461.

A protein kinase activity has been found associated with purified preparations of the A68 proteins from Alzheimer brain. The kinase phosphorylates both serine and threonine residues in the A68 proteins and their degradation products. Second messengers such as Ca ~ , c-AMP and c-GMP have no effect on the phos~horylation acitivity, while 10~M hemin abollshes labelling of the proteins. In comparison with histone, casein is the preferred exogenous substrate. However, the kinase is not inhibited by heparin or activated by spermine and protamine, suggesting a resemblance to Casein kinase I rather than Casein kinase II. Immunoprecipitation with the monoclonal antibody 126 results in co-precipitation of the kinase activity with the A68 proteins. Furthermore, when the A68 preparatlon is UV irradiated in the presence of the photoaffinity @~P analog 8-azidoadenosine- 5'-triphosphate[~- P], labelled ATP-protein adducts are observed comigrating with the A68 proteins on Western blots. Since azido-ATP becomes specifically immobilized in the catalytically active ATP binding site, it is suggested that the kinase activity observed in these preparations is an intrinsic property of the A68 protein.

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THE DEVELOPMENTAL REGULATION OF PHF-1 REACTIVE TAU PROTEINS. "S.G. Greenberg and P. Davies, Albert Einstein College of Medicine, Bronx, New York, 10461 USA.

While immunohistochemical and sequence analysis has indicated that tau is a component of paired helical filaments (PHF), PHF-associated tau can be distinl~ished from normal tan proteins. For example, PHF-associafed tau tends to be slightly higher in MW, more acidic and contains apparently unique antigeftic sites. In aprevious report, Greenberg and Schein (1989) described the isolation ot a sensitive PHF-reactlve monoclonal antibody (PHF-1) which displayed a relatively weak cross reactivity, with normal human tau. In the current study, we have extended our analysis of PHF-1 immunoreactivi~ with tau protems that were isolated during different stages of human development and during Ahheimer's Disease (AD).

In AD, PHF-1 immunoreactivity appears to be primarily associated with PHF. In PHF-enriched preparations, PHF-1 recognizes the 57-68 kd tau proteins as well as proteins frith MW ranging from20 to greater than 400]~d. Re-eleetrophoresis experiments indicate that many of the high and low MW proteins result from the aggregation or degradation of PHF-associated tau.

In addition to PHF-assoeiated tau, PHF-1 recognizes a relatively minor population of soluble tau proteins in AD anffto a lesser extent in normaI human adults. However, unlike the majority of soluble tau proteins in human adults, the prominent PHF-1 reactive tau proteins display a MW and pI that is similar to PHF-associated tau. Thus, the soluble PHF-1 reactive tau proteins appear to represent the precursor pool of PHF-associated tan.

Examination of soluble tau isolated during different stal~es of human development revealed that PHF-1 distinctly recognizes a 57"kd fetal tau protein. Although PHF-1 i rr~munoreactivi_ty is dramatically reduced by 2 months after birth, distinct ctaanges in the MW to the adult tau isoforms was not detectable until 2 years of age. Quantitative analysis of western blots revealed that PHF-1 tau immunoreactivity is approximately 71 fold greater in the fetus as compared to 0-96 year old normal individuals. By cpntr~t , the levels of Alz 50 immunoreactivity were not significantly alterea m mese same preparations. Thus, the levels of PH-F-1 reactive tau appears to be selectively regulated during human development.

Preliminary e.xperiments using solubIe tau isolated from adult rats indicate that li~e humans, PHF-1 appears to react with a specific subset of tan proteins. However by contrast to human adults, PHF-1 reactive tau appears to be present at a concentration that is within the same range as Adz 50 reactive tau. This result suggests that the selective decrease m PHF-1 reactive tau proteins in human adults represents a recent evolutionary event. Consequently, events whicficause the accumulation of PHF-1 reactive tau in human adults may result in the abnormal formation of paired helical filaments.

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NEUROFILAMENT GENE EXPRESSION IN ALZHEIMER'S DISEASE

*Smita Kittur, John Hoh, Claudia Kawas, Wal laceTourtel lot te, Will iam Marksberry, Wil l iam Adler. NIA, Baltimore, MD, VA Med. Ctr. LosAngeles, CA. U.Ky. Lexington, Ky.