THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE

puriliedPHFpreparaonshowed tha~thesepeptidesoorrespondedtoseq_cesfoundinIhe carboxyldomainofthetaumolecule.Recently,i~wasr~~thatapopulationofsoluble PHF is oompoxd of three major polypeptides (68.64 and 62 kDa) called PHF-tau or A68 polypeprides To establish to which extent these PHF and the associated PHF-tau polypeptides contam the whole tau molecule or only a fragment of it, we have studied in electron microscopy the immuooreactlvlty of isolated PHF with a panel of antibodies drreckd to specific ammo acid sequences spanning the tan molecule from the N-IO the C- termini. including sequences specific of the two types of inserts found in the amino doman of some tao isofonns. and with two anti-neurofilament antibodies (RT97 and 8D8) previously shown to label PHF and to cross-react with tau proteins. The PHF-tau polypeptides associakd with isolaled PHF were studied by Western blotting with rhe same ant&die0 The PHF present in A68 preparations were found lo be labeled by all anti-tau aotibodies. including the insert-specific anti-mu antibodies. IO addition. the antibody decoration of PHF suggeskd that the N-terminus of tau extended outwards the longihu3inal axis of the PHF The three mqor PHFtau polypeptides were labeled by all anti-tau antibodies recognizing sequences common to all tau isofonns from the N-to the C-termink The lower PHF-tau polypeptide was no, labeled by the msert-spenfic anti-tau a&bodies. Dephosphorylation of PHF-tau polypeptides led to a sluft on SDS-PAGE towards apparent lower molecular

labeled isolatedPHFandthePHF-ta~poiype~tides,althoughthelowerPHF-taupolypepttde was nol labeled by RT97. Both RT97 and 8D8 detected more efficiently the PHF-tau polypeptides than the normal tau proteins An epltope mapping study was performed by comparing the immunoreactivity of RT97.8DE and of the panel of anti-tau antibodies with the products of digestion of PHF-tau polypeptides by uypsin and a-chymouypsin. The 8D8 epitopewaslocalized ~nthecarboxyl-domainoftau,nearthe KSPV sequence,whereas the RT97 epitope was localized m the amino domain of tau. probably in the sequence correspondmg to Insert I These resulls in&care that isolated PHF and the associakd PHF-tau polypeptides contain epitopes from the whole sequences of different l.w isoforms,but show also mod&d sites recognized by Rl97 and 8IX anti-newofilament anobodxs.

Supportedbygrantsfrom Ihe "FondsdeRechercheDivry". and "FondsM.T detava"

215 COLOCALIZATION OF TAU AND B/A4 FRAGMENTS IN HIPPO- CAMPUS, W. Bondareff and C.H. Wischik. Dept of Psychiatry, University of So. California, Los Angeles, U.S.A. end H.R.C. Laboratory of Molecular Biology, Cambridge, U.K. Neurofibrillary tangles and senile plaques are

histological hallmerks of Alzheimer's disease. The submicroscopic structural unit of tangles, paired helical filaments (PiiP), are characterized by tau protein; senile plaques are characterized by B/A4. The formation and accumulation of these two proteins is central to the degeneration of neurons in AD. We report here results of a" immunohistochemical inquiry, using antibodies raised against fragments of tau and B/A4, which are overexpressed in AD. Polyclonal antitau antibodies included: BR133, raised

against N-terminal residues, 1-16; BR134, raised against C-terminal residues 339-352; BR135, raised against repeat region residues 323-335. Polyclonal anti-amyloid antibodies included: BR88 (anti-,3/A4:1-12; BR89 (anti- ,WA4:28-40). A monoclonal antibody, 18.8, was also raised against ,9/A4:1-12. Sections of hippocampus were treated, first with the primary antibody, then with a fluorescein or Texas Red labelled anti- gammaglobulin.

Confocal microscopy, by means of which 10-20 optical sections through a single pyramidal neuron were projected as a single 3-dimensional image, suggested that BR133, BR134 and mAh 18.8 exclusively immunolabelled intracellular tangles. These antibodies labelled, also, dystrophic neurites in the neuropil end in the periphery of the senile plaque. BR135 and BR88 labelled both intra and extracellular tangles. Labelling of dystrophic neurites was observed only after pronase pretreatment.

These findings suggest that the N-terminus of ,9/A4 co- localizes with the N- and C-tennini of tau in

intracellular tangles. The exclusively intracellular labelling suggests that epitopes recognized are localized in the superficial part of the PW and that some portion of the 100 C-terminal residues of APP appears to be associated with intracellular neurofibrillary pathology.

216 NucLFiorAR LOCALIZATION OF TAU AND A MAP4-LIKE POLYPEPTIDE IN IiUMAN NEUROBLASTOMA CELLS. Loomis,

*P.A. and L.I. Binder. Department of Cell Biology,

University of Alabama at Birmingham, Alabama, 35294 U.S.A.

Utilizing indirect immunofluorescent microscopy and SDS-PAGE we have detected both tau isoforms and a MAP4-like polypeptide in the nucleus of human and primate cells in culture. Nuclear tau localized to the fibrillar regions of the nucleolus in interphase cells and to the nucleolar organizer regions (NORs) on the acrocentric chromosomes in mitotic cells. The acrocentric chromosomes are 13, 14, 15, 21 and 22. Interestingly, nuclear tau was insoluble in SDS, requiring formic acid extraction prior to immunoblot analysis; a property shared with the paired helical filaments isolated from Alzheimers Disease brain. A monoclonal antibody, designated AHT-1, was generated from a fusion whose immunogen was a heat stable, perchloric acid soluble fraction from an Alzheimers diseased brain. which

AHT-1 recognizes a 200 kDa protein localized to the periphery of

nucleoli and co-localizes with tau interphase

on the NORs in mitotic cells. Polyclonal antibodies to NAP4 cross react with this heat stable 200 kDa polypeptide, suggesting that it may be a member of the NAP4 family of polypeptides. This data alludes to the possibility that more than one microtubule-associated protein (NAP) may be involved in nucleolar physiology. Additionally, the localization of tau and the MAP4- like polypeptide to the NORs of human acrocentric chromosomes may implicate these MAPS as part of the mechanism linking Downs Syndrome to Alzheimers Disease.

217 DYSTROPHIN AND ALzHEnlER’S DISEASE: Inl4”NOHISTOCHE”ICAL ANALYSIS OF CARSOXYL, ROD AND AMINO DOMAINS. J. Maguire, A. Lewis, J. Silbao, J. stevene, J. Trogadie, W. Oeanne, S. Young and S. Cohen. University of Toronto, Toronto, Ontario, Canada, and the Playfair Neurosciences Laboratory, Toronto, Ontario, Canada.

Recent studies indicate that alterations in membrsno- associated proteina may play an important role in the pathogeneaia of Alzheimer's disease (AD). The membrane protein dystrophin ia currently the focus of muscular dystrophy-related research, and has also been localized to mammalian neurons. The objectives of this imnunohistochemical study were to identify the distribution of the carboxyl (C), rod and amino (N) domains of the dystrophin protein in AD. Sections of archival frozen brain obtained from hiooocamas (SC\ end temwral cortex rTCI from 7 caeee of AD and 4 &-ma&h&l cbntrola &re studied ii& commercially available monoclonal antibodies to the C, N and rod domains of dystrophin. In AD cases, mature ~lasues with amvloid corea were outlined by N and rod immunoreactiv; cellular p;ocosses. The participation of the C-terminus in the mature amyloid plaques was minimal. These findings were not observed in the control cases. Analysis of diffuse ~laaues was orecluded bv frozen artifact and a prospective ‘stuby with snap-frozen -tissue is in progress. Dendritic processee were immunoreactive to all three domains (C, N and rod) and no distinctions between AD and controls were observed 6ith this material at the lioht microscooic level. Electron microscopic analysis with immunogold techniques is in proqress. Neurofibrillarv tanoles were occaeionallv identified ;ith anti-l and rod dom&s only. Fine glial-lik processes extending to and enveloping blood vessel walls in cortex and white matter were well demon&rated only with anti-C antibodies in both AD cases and controls.

These preliminary results indicate that the different dystrophin domains have distinct functional roles in the human brain, and that the rod and N components of dystrophin are associated with established plaques. The localization of the C- terminus to blood vessels implies a role in the blood brain barrier.

218 ABNORMALLY PHOSPHORYLATED TAU IS PRESENT BOTH IN PHF AND NON-PHF FORMS IN ALZHEIMER DISEASE BRAIN, E. Kijpke, K. Iqbal and I. Grundke-Iqbal. Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Rd., Staten Island, NY, 10314, USA. One of the most prominent brain lesions of Alzheimer disease (AD) is the intraneuronal accumulation of paired helical filaments (PHF). The major protein subunit of PHF is the microtubule- associated protein tau. Tau represents a family of phosphopolypeptides that are abnormally phosphorylated in AD brain. Previously, we described the isolation of abnormally phosphorylated tau (AD P-tau) from the 27,000 to 200,000 x g pellet of AD cerebral cortex and showed that unlike the sparingly soluble PHF, this pool of the protein had no ubiquitin immunoreactivity. We now report that the AD P-tau isolated from the 27,000 to 200,000 x

S56 THIRD INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE

g pellet is derived both from readily soluble PHF and from a non-PHF pool of the protein. To determine how much of this AD P-tau was derived from PHF, the 27,000 to 200,000 x g pellet was fractionated on a 1.50 to 2.50 M sucrose discontinuous gradient. The sample was distributed in seven distinct layers, which were examined by electron microscopy for the presence of PHF. The majority of PHF were present in layers 5 (57%) and 7 (25%). However, only 35% of the total AD P-mu was present in these layers as discerned by densitometric measurements on immunoblots of these fractions, developed with the mAb Tau-1, with or without prior dephosphorylation of the blots. In contrast, 54% of the AD P-tau was in sucrose layers 2-4, which contained mainly membranes, amorphous material, and only a small number of PHF (11%). This study demonstrates the presence of a pool of abnormally phosphorylated tau in addition to PHF in AD brain. (Supported in part by NIH fellowship F32 AGO5541 to E.K, NIH grants NS 18105, AGO4220, AG05892, AGO8076 and a grant from AHAF to LG.-I. and ICI.).

219

DEPHOSPHORYLATION OF MICROTUBULE ASSOCIATED PROTEIN TAU FROM ALZHEIMER DISEASE BRAIN CYTOSOL INCREASES ITS ABILITY TO PROMOTE IN VITRO ASSEMBLY OF MICROTUBULES, Alejandra Alonso, Inge Grundke-Iqbal and Khalid Iqbal. New York State Institute For Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA Previously we have shown that in brain of patients with Alzheimer disease (AD) tau is abnormally phosphorylated and that this abnormal phosphorylation appears to be one of the earliest events in AD. It is also known that the phosphorylation of tau decreases its ability to promote microtubule assembly. The present study was undertaken to examine the microtubule assembly-promoting activity of Alzheimer disease-tau and the effect of in vitro dephosphorylation on this biological activity. Two different pools of tau were isolated from AD brain under non-denaturing conditions: - One that sedimented between 27,000 x g to 200,000 x g (abnormally phosphorylated tau or AD-P tau) and - one that was soluble at 200,000 x g (AD-tau). Tau was further purified from both fractions by acidic precipitation followed by phosphocellulose chromatography. Dephosphorylation of tau was achieved by treatment with 100 uM alkaline phosphatase overnight at 37°C. Tubulin was isolated by phosphocellulose chomatography of 2-cycled assembled microtubules from rat brain. Assembly of microtubules was carried out by incubating tubulin and tau at 37C in 100 mM MES, 1 mM EGTA, 1 mM GTP an.d 1 mM MgCl,. The degree of tubulin polymerization, examined by electron microscopy, was markedly decreased when using the AD-P tau. This biological activity was recovered when AD-P tau was treated with alkaline phosphatase. The AD-tau also showed a decreased biological activity when compared to tau from normal brain. Like AD-P tau, the phosphatase treatment increased the microtubule assembly promoting- activity of AD-tau. These results suggest that phosphorylation of tau in AD brain decreases its ability to promote microtubule assembly.

220

ABNORMALLY PHOSPHORYLATED TAU IS ASSOCIATED WITH DEFECTIVE GTP BINDING TO THE B-SUBUNIT OF TUBULIN IN ALZHEIMER DISEASE BRAIN, Sabiha Khatoon, Inge Grundke-Iqbal, and Khalid Iqbal. New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA Binding of GTP to the exchangeable site on the #I subunit of tubulin promotes microtubule assembly. Previously we demonstrated defective microtubule assembly (Iqbal et al., 1986, Iancet 2421-426) and diminished GTP binding to the B-subunit of tubulin (Khatoon et al., 1989, Ann. Neurol. 16210-215) in Alzheimer disease (AD) brain. We had also shown that the microtubule-associated protein tau in AD brain is abnormally phosphorylated and is the major protein subunit of paired helical filaments (PHF) (Grundke-Iqbal, et al., 1986, Proc. Natl. Acad. Sci. USA 83:4913-4917; Grundke-Iqbal, et al., 1986, J. Biol. C&em. 261:6084-6089). Furthermore, we had proposed abnormal phosphorylation of tau to be a cause of the breakdown of the microtubule system in AD brain. Studies were undertaken to examine the role of the abnormally phosphotylated tau in the GTP binding defect. Tubulin was isolated from AD and control brain homogenates. Binding of [yPP]8N,GTP photoaffinity analogue to tubulin was lower in AD cases

than in normal aged cases. Using radioimmunoslot blot assays, employing monoclonal antibody Tau-1 and anti-mouse *zI-IgG as secondary antibody the levels of normal and abnormally phosphorylated tau were measured in the tubulin-enriched fractions. Tau-1 recognizes only normal and alkaline phosphatase-treated abnormally phosphorylated tau. Tubulin-enriched fractions from AD brain homogenates contained 1.3 + 0.5 ng abnormally phosphorylated tau per ug protein, whereas normal brain tubulin preparations contained none. Also, purified bovine tau added to bovine, normal human, and AD brain tubulin increased the GTP binding, as monitored by [yf2P]SN,GTP photolabeling to the /3 subunit of tubulin. Furthermore, the addition of an alkaline phosphtase- treated PHF-enriched fraction increased the binding of the GTP photoaffinity analogue to tubulin almost 2-fold. These studies suggest that abnormally phosphorylated tau might be responsible for the defective GTP binding to tubulin in AD brain. (Supported in part by NIH grants AG 05892, AG 08076, AG 04220, and NS 18105 and a grant from the American Health Assistance Foundation, Rockville, MD).

221 Abnormal phosphorylation in NFTs with and without Ubiquitination. Cruz-Sanchez F.F., Cardozo A., Rossi MI_, Tolosa E. Neurological Tissue Bank, University of Barcelona; Dept. of Neuropathology, Birmingham and Service of Neurology, Hospital Clinic0 y Provincial, Barcelona

Neurofibrillaty Tangles (NFTs) and other intracytoplasmic inclusions are the morphological manifestation of several degenerative processes affecting neurons from the central netvous system. These inclusions are usually composed of normal cytoskeletal components.

We present an immunohistological study of these inclusions, specially focusing on NITS from several neurological conditions including: Alzheimer Disease, Parkinson Disease, Pick Disease, Progressive Supranuclear Palsy, Motor Neuron Disease, Post Traumatic, and Post Encephalitic Parkinsonism. A panel of antibodies: MABs to NF and antisera to Ubiquitin, GFAP, two epitopes of TAU, and AC Z50 were used.

We found that NFTs from AD, Post Traumatic and MND have similar antigenic determinant which are different from NPTs in PSP, and Post. Enc. Park. other inclusion also showed different epitopes of NFTs.

Our results demonstrate that NITS in AD accumulate proteins related to abnormal phosphotylation and that an ubiquitination mechanism is operating. NFTs in other conditions showed accumulation of some of these proteins, but ubiquitination was not present. We conclude that a new concept of degeneration related to phosphorylation and ubiquitination in relation to neurodegenerative conditions can be put forward.

This highlight different pathophysiological mechanism in relation to aetiopathological factors in which time, type of neuronal population and pathogenic noxae could be involved.

222 BIOCHEMICAL QUANTIFICATION OF THE ALZHEIMER-TYPE

DEGENERATING PROCESS IN BRODMANN AREAS, USING TAU-PHF AS MARKERS: EVIDENCE FOR HETEROGENEITY

AS A FUNCTION OF TOPOGRAPHY AND AGE. A. Delacourte, P. Vermersch.

INSERM U 156, Place de Verdun, 59045 Line France.

Paired Helical Filaments are composed of a triplet of abnormally phosphorylated Tau proteins named Tau 55,64,69 or 7PHF. These pathological proteins are reliable markers of the Alzheimer-type neurofibrillary degeneration. We used these biochemical markers to map the degenerating process in Brcdmann areas of the brains from 5 sporadic, late onset AD patients with an almost identical duration of the disease. We also compared the intensity of the neurodegeneration as a function of age.

Material and methods: To quantify the degenerating process, we used an immunoblot method with antibodies specifically directed against rPHF [ Delacourte et al., Acta Neuropathol. 1990; 80: 11 l- 1171. For each