The structural basis of tau filament formation

S132

BRAIN METABOLISM PREDICTORS OF 1594) REGIONAL MER’S DISEASE IN MILD COGNITIVE IMPAIRMENT.

Many

J de Lion.

Tarshish, Drwid

Antonio

.I Schlvrr.

Henv

Rusinek,

Alrxundra

Mudhu

Boppana.

Cormit, Brookhuvrn

National

Lab.

A Roche, NYU Upton,

Adam

J Scherrr.

Srh of Medicine.

New

1596) LEWY-LIKE TANT (A53T)

ALZHEI-

Chuim

Y

York, NY;

Bend AC,

Sommrr,

Samuel

NY

for

Mild cognitive impairment (MCI), defined by global wale, is a term used in clinical research to describe individuals wth predominant memory impairment that are considered to be at increased risk of developing Alzheimer’s Disease (AD). However, many MCI individual? do not develop AD over relatively short study intervals. The purpose of this 4 year longitudinal study was to identify the baseline regional cerebral glucose metabolic (CMRglu) and neuropsychologal predictors of two groups of MCI individuals (GDS=3); one that remains stable (n=lO) and one that declines to AD (n=h). Thiv way examined relative to a stable normal control group (n= 14) and a group with mild AD (“~8). The groups did not differ in age or education. Only the AD group showed at baseline significantly lower Mini-Mental Status Examination scores. Subjects received a baseline diagnostic examination that mcluded FDG-PET. MRJ and memory teats and at fc#oa-up the diagnostic exam and the memory teuts. FDG-PET scans were coregi\tered with MRIs and CMRglu was obtained from four whregions of the temporal lobe, the supramarginal gyrw, two subregions of the frontal lohe, the posterior cingulate gyms and the pow. Compared to the normal control group, the MCI group that declined to AD showed, after xan normalization. widespread metabolic reductions, most rtrikingly noted in the hippocampal formation and the temporo-par&al area (>20%). Significant reductiona in immediate and delayed memory performance were alao noted. There were no significant CMRglu or cognitive differences between the MCI group that remained stable at follow-up and the normal control group. However, there was a tendency for the stable MCI group to perform more poorly than the control group across all measures. Compared with declinmg MCI patients. the AD group showed significant temporo-panetal hypometaholic reductions. The results of this study suggest longitudinal decline verse stability can he predicted among MCI patients with indistinguishable baseline global severity ratings. Both CMRglu and memory performance predict the outcome. Such data highlight the need for improved clinical assewnent tools and for continued use of brain imaging in the early diagnosis of AD.

Herman

Base1 Switzerland; Bnrbieri.

PATHOLOGY

HUMAN

van der Putten, Alphonse

Novartis

PntholoRJ,

Bowl

IN MICE

Pmbst.

Pharmu

TRANSGENIC

FOR

MU-

a-SYNUCLEIN Karl-Heinz

Wiederhold,

Institute

Pathology,

for

AG, Base1 Switzerland;

Switzerland:

Graeme

Bilbe,

Novurti.s Basrl

Markus

Nwartis

Phannu

Switzerlund:

Tolnay,

Phrrrma

Institute

AG,

Bawl

Switxrland

Lewy pathology is a defining hallmark of degenerating neu~onb in postmorfem brain tissue of patients with neurodegenerative disorders including idiopathic Parkinson’s Disease (PD), dementia with Lewy bodies (DLB), and a Lewy Body variant of Alzheimer’s Disease (LBVAD). Lewy lesions appear to be central and may contribute mechamstically to dysfunction and degeneration of neurons in there diseases. A prime wapect for contributing to the pathophysiology of disease5 with Lewy pathology is the presynaptic protein ol-synuclein. It is a prominent constituent of Lewy structures in mo?t sporadic idiopathic as well as familial forms of PD, DLB and LBVAD. Furthermore, a-synuclein has the ability to self-aggregate forming insoluble proteinaceous Lewy body-like assemblies. Recently. two mutations (A53T or A30P) in the waynuclein gene were linked to early-onset familial PD with Lewy pathology. Alao, they are known to accelerate self aggregation of the cognate proteins and, in the AS3T mutant. to slow down degradation. We now have been able to recapitulate striking features of Lewy pathology in the mouse brain by neuronal expression of either the A53T mutant or wild-type human a-synuclein. Our animal models support a central role for a-synuclein in diseases with wsynucleinopathy and Lewy-pathology. They are the first rodent models with hallmarks of ruch diseases, and provide the means to address underlying fundamental aspects and to test therapeutic strategies.

1597) SOMAL SON’S

AND

NEURITIC

IN TRANSGENIC Philipp

J Kuhle,

Germany:

Mum&

Laurence

Veronikn

Mueller,

ACCUMULATION

DISEASE-ASSOCIATED

Ntwnann,

Ludwig

F HofJinunn

Mnvuyusu

Okochi.

THE

PARKIN-

[A30Pla-SYNUCLEIN

MICE

Ozmrn.

Jacobsen,

OF

MUTANT

F

Ludwig

Mamnilims La

Roche

UWP Leimer,

Maximilions

Univ qfMunich, Ltd.

Ludwig

Univ of Munich,

Lu Roche

Ho&wnn

Ltd.

Busel

Muenchen

Unrv

Germuny:

Helmut

Alicr Schindzielor:.

Base1 Swtserland; Maxrmiliuru

Munchen Swit:erland;

oj Munich.

Muenchpn

Germany

Oral Presentation:

Molecular

CELLULAR METABOLISM (5951 TIA-ASSOCIATED BRJ-L

Pathogenesis OF

FAMILIAL

I BRITISH

DEMEN-

Familial British Dementia (FBD), an autosomal dominant neurodegenerative dirorder, is clinically characterized by progrt?%ive spastic tetraparesis. cerehellar atexia and dementia. The genetic lesion underlying FBD is a T-A transversion at the termination codon of the BRI gene. The mutant gene encodes BRI-L, the precursor of ABri peptides that accumulate in amyloid deposits in FBD brain. We repofied that both BRI-L and its wild type counterpart. BRl, are constitutively processed by furin. rewlting in the secretion of ABri and Bri peptides, respectively. Notably, elevated levels of peptides were generated from the mutant BRI-L precurwr. We have extended these observations to examine the protease specificity and potential wuctural differences between the BRI and BRI-L substrates. First, inducible cxpreasion of al-antitrypsin Portland. a bioengineered furin inhibitor, inhibits the endoproteolysis of BRI and BRI-L. In addition, comparison of the actwities of several members of proprotein convertaw (PC) family reveal that furin is the most efficient in endoproteolysis of BRI and BRI-L. Other PCs show variable activities; PC6A and LPC has about -20% activity of furin while PACE% fails to show any activity. Interestingly, LPC, like furin, also exhibits enhanced cleavage of BRI-L hut PC6A and PC6B cleave wild type and mutant molecule with similar efficiency. Moreover. pulse-chase and MALDI-TOP analyses suggest that there is an additional cleavage downstream of furin cleavage site only in wild type BRI, not in mutant BRI-L. Finally, tryptic digestion of the peptides demonstrate that ABri has a intramolecular disulfide bond while Bri does not. Collectively, these results indicate that 1 I amino acids carhoxyl terminal extension in mutant BRI-L alters the conformation of the protein, by promoting intramolecular disulfide hond formation, that renders it more wsceptihle to furin cleavage.

Mutation< in the ol-hynuclein gene are nhsociated with fare cases of familial Parkinson‘s disease, and cu-synuclem is a major component of Lewy bodies and Lewy neuritea. Here we have investigated the localization of wild-type and mutant IA3OPlwqynuclein as well a\ P-cynuclein at the cellular and subcellular level. Our direct comparative study demonstrates extensive synaptic co-localization of u- and j3-synuclein m human and mouse brain. In a sucrose gradient equilibrium centrifw ration assay. a portton of Pmsynuclein floated into lower-density fractions, which also contained the \ynaptlc vesicle marker qynaptophysin. Likewise, wild-type and IA30P]a-synuclein were found in floating fractions. Subcellular fractionation of mouse brain revealed that both a- and P-synuclein were present in synaptosomes. In contrast to synaptophysin, P-synuclein and a-synuclein were recovered from the soluble fraction upon lysis of the synaptosomes. Thus, association of synucleins with synaptic vesicles is reversible. Synaptic co-localization of 01. and P-synuclem was directly visualized by confocal microscopy of double-stained human brain sectmns The ParkinTon’s disease-associated human mutant IA30P]a-synuclein was expressed in transgenic mouse brain under the control of a pan-neuronal promoter, Thy-l, ab well a? the dopamine neuron-specific promoter, TH. LA30Pla-Synuclein co-localized with P-
)5981 THE Jq‘j‘j Kuret, Norrhwestrm E King,

Lrtrr

STRUCTURAL

Ohm

St

Univ.

Univ, Chrago, 1 Binder,

BASlS OF TAU FILAMENT

Columbus,

OH;

IL: Lance Grau,

Norrhw~.~tem

Unrv.

Aida

Abruha,

FORMATION

Trurnon

Ohio St Umv, Columbus, Chicqo.

C

Gamblin,

OH; Michelle

IL

The polymerizatmn of the tau protein into filamentous inclusions is believed to play a key role in neurodegeneration. The resultant neurofibrillary pathology is a hallmark of Alzheimer’s disease (AD) and other neurodegenerative tauopathiea. In vitro studies of tau filament formation have shown that the addition of free fatty acids such as arachidonic acid under physiological redox conditions can induce the polymerization of pure recombinant tau protein into filaments. We have exploited this system to probe how tao protein structure and state of postranslational modification influences

Oral Preserttution:

Molecular

Pathogenesis

I

s133

the rate and extent of homopolymeriration. We found that tau isoforms arising from alternative splicing or from autaomal dominant miswwz mutations associated with

GENERATION OF AN ANIMAL MODEL OF AMYLOID DEPOSITION WITHOUT OVERPRODUCTION OF AMYLOID PRECURSOR PROTEIN

frontal temporal lobe dementia and parkinsonism linked to chromosome I7 (FTDP17) differ markedly in their efficiency of assembly. In addition, mimicry of tau phosphorylation by mutation of select wine residues to glutamic acid reridues greatly accelerated tau polymerization. Mutation of cyst&e residues showed that oxidation may contribute to final paired helical morphology, but is not a necessary prerequisite for efficient nucleation or elongation of tau filaments. Together these data suggest that tau polymerization ia modulated by multiple protein segments, borne of which mediate and others which inhibit polymerization. It is proposed that phosphorylation, truncation. alternative splicing, and misaenae mutation of residues within these begmat\ can greatly influence the ability of tau protein to form filaments.

LOSS OF PRESENHJN 1 LEADS TO EPIDERMAL SIA AND TUMORIGENESIS IN ADULT MICE

HYPERPLA-

Presenilin I (PSI) 1) a multi-pas\ tran.\membrane protein expressed in all tissues examined. PSI is essential for mammalian development and is required for extracellular production of A@ peptides. These activities are likely mediated by its absolute requirement in the proteolytic processing of two distinct molecules: Notch and amyloid precursor protein (APP). In addition, PSI associates with p-catenin. Here we report that this interaction plays a critical role in regulating @cat&n signaling pathway in viva Using transgenic mice deficient in moue PSI but rescued with human PSI whose pattern of expression is restricted to neuronal tissue, we show that lack of PS I expression in skin results in increased stability of p-catenin and activation of its downstream target cyclin Dl. Importantly, consistent with the established role\ of p-catenin and cyclin 01 in turmorigenesis, these mice develop epidermal hyperplasia and neoplaaia. Our results document that PSI ia a negative modulator of p-cat&n atability and downstream signaling and loaa of PSI ia linked to turmorigemc proce,a.

Dorothy G Flood. Muryv J Savage, Andrew G Reuume, Stephen P Trusko, Yin-Guo Lin. Karen S Dofman, Diane M Lang, Richard W Scott, Cephalon, Inc, West Chester, PA To date, animal models of amyloid (Ap) deposition have utilized the transgenic approach to overexpress amyloid precursor protein (APP) bearing familial Alzheima’\ disease (FAD) mutations. These mutations increase AP to the point that amyloid deposition occurs, but the tranagenes also increase APP productjon well beyond physiological level\. Recently A@ deposition has been further enhanced by crossing these APP transgenic mice with transgenics overproducing FAD mutant preaenilin-I (PSI). We have utilized a gene-targeting approach to generate AD models that do not involve protein overexpres\ion. Our first model introduced the Swedish K670N/M671L FAD mutations and “humanized” the mouse AD sequence in the APP gene. This mouse (APPNLh’N’~” ) produced normal levels of APP, overproduced human API -40 and l-42. but did not deposit AD as late ar 22 months of age. Next. the P264L mutation wa? introduced into the mouse PS-I gene. The P264L mutation is a non-conservative amino acid substitution in the cluster of mutations in exon 8, leading to an onset of FAD in the middle fortles to middle fifties. This mouse (PS-l’2”upL”L) also did not deposit A@ as late as I2 months of age. Crosses have produced APPNL1vNLhX PS- I PZM’-‘PZML d ou bl e gene-targeted mice. These mice have clevated levels of API-42, sufficient to cause AD deposition. A@ deposits were not present at 3 months of age but were seen at 6 months. Deposition was most prominent in telencephahc structures, consistent with the distribution of AP deposition in AD. The double gene-targeted mouse is a model of AP deposition that faithfully expresses normal levels of both APP and PS- 1 under the control of their endogenous promoters in a cell- and tissue-appropriate manner.

p%J

PRESENILIN 1 INTERACTS WITH A NEURONAL CELL ADHESION MOLECULE AND MEDIATES ITS INTRACELLULAR TRAFFICKING

Wim G Annarrt. Christine Roevc, Cay Esselens, Greet Snellings. Kathleen Craeswert.%. Hurt Dr Strooper, Gasthuisberg/K U LBUVEN. Lruvrn Belgium FAD ALLELES OF PSI AND PS2 HAVE DIVERGENT EFFECTS ON SKELETAL DEVELOPMENT, y-40- AND y-42SECRETASE David A Wevtuwu~. Ayhar M Chishti. FushenR Chen. Univ of Toronto, Toronto. ON (‘muda: Paul M Mathew’s. Nathan Kline Institute, Orangrhurg, NY; Gang Yu, Rohrrt Stromr, l/nil, of Toronto. Toronto. ON Canada; Richard Ro:mahel, Hop for Sick Children. Toronto. ON Cmudu; George A Carlson. McLaughlrn Resrtrrch Institute. Great Fcdls, MlPresenilin I (PSI) knockout (k/o) mice exhibit Ekeletal and CNS abnormalities. accumulation of APP-terminal fragments (CTFs) and absence of API-40 and API-42 peptides: theae defects are attributed to lack of secretase cleavage within the transmembrane domains of Notch and APP, respectively. We assessed biological activities of Familial Alzheimer Disease (FAD) alleler of PSl and PS2 by genetic complementation. Both PSI and PSZ transgenes expressed from the priori protein promoter rescued the skeletal abnormalities present m PSI k/o mice. Unlike the Ml46L FAD allele of PSI, however, Nl4lI and M239V PS2 tranagenes reduced APP CTFs in the brain by only 20-30s and failed to restore API-40 synthesis. Thu. whereas mutant PSl activates both y-42-\ecreta\e and y-40.secretase, mutant PS2 only activates the former endoprotease. These data make it unlikely that Notch in processed by y40.secretax. The ability to genetically diwoclate y-40. and y-42. secretase activity. a\ e\tahhrhed here, wdl be of use in awigning contributions of the two wcretaw pathways in developmental processe\ and m the pathogen&\ of AD.

Preaenilins are multitransmemhrane proteins that play important physiological roles in the proteolytic cleavage of APP and Notch and are key players in amyloid formation and development. It is unclear whether presenilin I (PSI) is the elusive y-wxretaae or a co-factor involved in efficient processing, targeting or sorting of APP. Notch and other candidate substrates. To explore further the exact function of PSI we used two-hybrid screening with the carboxy-terminus of PSI as a bait. We have ldentitied a PSI-interacting protein (PIP-l) which, like APP and Notch, is a type I tranmwnhrane protein. PIP-l is a cell adhesion molecule that is exclusively expressed in specific areas of the brain including hippocampus and dentate gyms where it localizes at the plasmamembrane of neurites. Both the topology and the neuronspecific expression make PIP-l a highly interwing protein to analyze the functlona of PSI m brain. In brain extracts, endogenous PIP-l interacts specifically with GST fused to the 39 carboxyterminal ammo acids of PSI and 2 m a salt-dependent way. Inversely, endogenous PSI-CTF could be precipitated using GST-PIP-l recombinant proteins. By in vitro coupled transcription/translation we were able to demonstrate that the intracellular domain of PIP-l is not required for the interaction with PSI as IS the case for APP. Instead, we narrowed the site of interaction with PSI to a sequence close to or wthin the tranamembrane region of PIP- I. Furthermore we found a vast accumulation of PIP-l at the plasmamembrane and in an as yet unidentified membrane compartment within the cell body of PSI :/hippocampal neurons, providing functional evidence for the interaction of PIP-l with PSI. Identifying the exact nature of the compartment where PIP-l accumulates is in progrech. ar well as studies aiming to elucidate the potential role of PSI in the proteolytic proccs\ing of PIP-I