P4-082 HEPARAN SULFATE PROTEOGLYCANACCUMULATION OCCURS CONCURRENT WITHBETA-AMYLOID PROTEIN PLAQUE DEPOSITIONIN APP TRANSGENIC MOUSE MODELS OFALZHEIMER’S DISEASE

Joel A. Cummings1, Eliezer Masliah2, Edward Rockenstein2,Alan D. Snow1, 1ProteoTech Inc., Kirkland, WA, USA; 2University ofCalifornia-San Diego, San Diego, CA, USA. Contact e-mail:snow@proteotech.com

Our previous studies over the last 15 years have demonstrated the impor-tance of heparan sulfate (HS) proteoglycans (PGs) and heparan sulfateglycosaminoglycans (GAGs) in the pathogenesis of beta-amyloid protein(A�) fibrillogenesis in Alzheimer’s disease (AD). In the present immuno-histochemical study, we used different commercially available HSPG andHS GAG antibodies to discern the temporal relationship between initialHSPG and A� accumulation in brains of two different APP transgenicmouse models during amyloid plaque development. Comparisons weremade in aging transgenic mice expressing 1) human APP-695 cDNAcontaining the Swedish (K670M/N671L) mutation (i.e. Tg2576), in whichinitial amyloid plaque development occurs late (at �7-10 months), and 2)human APP-751 cDNA containing the Swedish and London (V717I)mutations, in which initial plaque development occurs earlier (at �3-5months of age). Animals were sacrificed every 2-4 weeks at increasing agesfrom 5 months-24 months for the Tg2576 mice, and from 6-12 months forthe double mutation transgenic mice. The results demonstrated that in bothmouse models initial HSPG accumulation appeared to occur concurrentand co-localized with A� amyloid deposition in brain, as assessed byimmunostaining with antibodies that recognize HS GAGs (10E4), agrin,perlecan, and syndecan-2. More robust HSPG immunoreactivity in amy-loid plaque deposits was quite evident in the double mutation transgenicmice, compared to the single mutation transgenic mice, suggesting thatfactors that cause HSPG accumulation may be critical to the timing andinitial A� fibril and amyloid plaque development in AD and relateddisorders.

Supported by ProteoTech Inc and NIH SBIR Phase I Award AG022755.

P4-083 AMYLOID ION CHANNELS: A COMMONSTRUCTURAL LINK FOR PROTEIN-MISFOLDINGDISEASE PATHOGENESIS?

Jorge A. Ghiso1, Arjan Quist2, Ivo Doudevski2, Hai Lin3,Rushana Azimova4, Douglas Ng1, Blas Frangione1, Bruce Kagan4,Ratnesh Lal2, 1NYU School of Medicine, New York, NY, USA;2University of California, Santa Barbara, CA, USA; 3University ofPittsburgh, Pittsburgh, PA, USA; 4University of California, Los Angeles,CA, USA. Contact e-mail: ghisoj01@popmail.med.nyu.edu

Background: Cerebral amyloid diseases are considered to be part of anemerging complex group of chronic and progressive entities collectivelyknown as Disorders of Protein Misfolding that include, among manyothers, Alzheimer’s disease, polyglutamine-repeat disorders, cataracts,amyotrophic lateral sclerosis, Parkinson’s disease and other synucleinopa-thies, systemic and cerebral amyloidosis, tauopathies, prion diseases, andtype-II diabetes. In these disorders, soluble proteins normally found inbiological fluids change their conformation and form either insolublestructures that accumulate in the form of intra- and extra-cellular aggre-gates or fibrillar lesions usually associated with cell toxicity, complementactivation and the local release of inflammatory mediators and oxidativestress products. Recent studies have shown that pre-fibrillar conformationsof amyloid proteins are sufficient to induce cellular toxicity. However, the3D structural conformations of these globular structures, a key missing linkin designing effective prevention and treatment approaches, still remainundefined. Objective(s): To test the hypothesis that different amyloidmolecules form ion channel-like structures when incorporated in lipidbilayers. Methods: Recombinant or synthetic A�1-40, �-synuclein, ABri,

ADan, serum amyloid A, and amylin were inserted in reconstituted mem-branes. Structural and functional parameters were monitored by atomicforce microscopy (AFM), circular dichroism spectrometry, gel electro-phoresis, and electrophysiological recordings. Results: All these unrelatedproteins were initially monomeric or monomeric/dimeric, exhibited differ-ent secondary structures in solution and similar globular configurationsunder AFM. When reconstituted in lipid bilayers these small globularassemblies underwent supramolecular conformational changes. AFM im-ages of the resulting membrane structures were morphologically compat-ible with ion-channel-like structures. Electrophysiological recordings indi-cated that all these molecules, when inserted in lipid bilayers, elicit singleion-channel conductances. Proteins re-extracted from the membranesshowed higher degree of oligomerization when compared with the freshlysolubilized material (tetramers to octamers). Conclusions: In reconstitutedmembranes, amyloids form morphologically compatible ion channel-likestructures and elicit single ion-channel currents consistent with electro-physiological studies showing amyloid channel activity in cell membranes.Thus, by triggering destabilization of cellular ionic homeostasis, amyloidsmay directly induce cell dysfunction and degeneration in a variety ofprotein misfolding disorders.

Supported by NIH AG08721, AG05891, GM056290, the Alzheimer’sAssociation and the Alzheimer’s Disease Program, California Departmentof Health.

P4-084 AGE-RELATED ACCUMULATION OF AMYLOID-� PEPTIDE IN RHESUS HIPPOCAMPALNEURONS AND TERMINAL-LIKE STRUCTURES

Alexander H. Nicholas, Changiz Geula, Harvard Medical/ Beth IsraelDeaconess Medical Center, Boston, MA, USA. Contact e-mail:anichola@bidmc.harvard.edu

The amyloid-� (A�) peptide accumulates in plaques and is thought to bea major contributor to Alzheimer’s disease (AD) pathogenesis. Recentevidence indicates that production of small soluble A� oligomers, theiraccumulation in neurons and their secretion by neurons may be the earliestpathological process in AD, which is likely to lead to synaptic dysfunctionand neuronal degeneration. In this context, hippocampal pathology is ofparticular relevance given the central role of this structure in memoryprocessing and the severe memory deficit observed in AD. Age is theprimary risk factor in AD, suggesting the contribution of age-relatedchanges within the hippocampus to the disease process.

In the present study, age-dependent accumulation of A� in hippocampalneurons and terminals was examined in the rhesus monkey which, like thehuman, displays age-related accumulation of A� in plaques. Fixed sectionsfrom young (� 5 years) and old (�25 years) rhesus monkeys wereprocessed immunohistochemically using several A� antibodies.

We found an age-dependent accumulation of A� in hippocampal neuronsand terminal-like structures. No staining was observed in the hippocampiof young rhesus monkeys. In contrast, a subpopulation of neurons in thehippocampi of aged rhesus monkeys, displayed accumulation of A� withinthe cytoplasm. Labeling was seen in both pyramidal and non-pyramidalneurons in all CA fields. Significantly, intense terminal-like A� stainingwas observed in the outer molecular layer of the dentate gyrus in old rhesusmonkeys, corresponding with the termination of projection fibers from theentorhinal cortex which are exquisitely vulnerable to degeneration in AD.Control sections processed in the absence of primary antibody or in thepresence of non-specific IgG displayed no immunoreactivity.

Our findings suggest that age-related accumulation of A� in neurons andterminal-like structures within the primate hippocampus may be an earlyevent in the cascade of A� pathology. These observations are consistentwith previous reports that demonstrated elimination of A� immunoreactiveplaques in the molecular layer of the dentate gyrus in APP transgenic micefollowing unilateral lesions of the perforant pathway and suggest that

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