$446 Poster Session P3: Molecular Mechanisms of Neurodegeneration - Synpatic Disruption

~ SYNAPTIC TARGETING BY ALZHEIMER'S RELATED ABETA OLIGOMERS

Pascale N. Lacor*, Maria C. Buhiel, Pierce C. Cain, Lei Chang, Marie P. Lambert, William L. Klein. Northwestern University, Evanston, IL, USA. Contact e-mail: p-lacor@northwestern.edu

Background: It has been hypothesized that memory loss in Alzheimer's disease is a synapse failure (Selkoe, Science 2002) attributable to soluble Abeta oligomers (Klein et al, TINS, 2001), which we called "ADDLs" (Lambert et al, PNAS, 1998). In experimental models, ADDLs impact LTP and reversal of LTD (Walsh et al, Nature, 2002; Wang et al, Brain Res., 2002). We have also established that ADDLs are significantly elevated in AD brain (Gong et al, PNAS, 2003). Objective(s): We have tested two key predictions, asking (i) Are ADDLs specific ligands for memory-relevant synapses? (ii) If ADDLs bind specifically to synapses, do they cause molecular/structural changes that could disrupt memory function? Methods: Highly differentiated cultures of synapse-rich hippocampal neurons were exposed to low doses of ADDLs. ADDL binding and distribution relative to synaptic markers was assessed by confocal immunofluorescence imaging. Additional imaging experiments were carried out to test the effects ADDLs might have on different groups of memory-relevant synaptic proteins: (1) synaptic lEG proteins implicated in memory processes such as Arc, (2) other plasticity-related proteins such as CaMKII and (3) the expression of pre- and post-synaptic proteins involved in synaptic function/structure (synaptophysin, spinophilin). Results: Two key findings have emerged. First, synthetic and AD-brain extract-derived ADDLs bind specifically to synaptic terminals (>90% colocalized with synaptic marker PSD-95). Control brain extracts showed no binding, and use of various antibodies verified that synaptic ligands were Abeta oligomers (not monomer or dimer). Second, targeting of synapses by ADDLs rapidly induced synaptic expression of Arc and CaMKII. Arc expression remained elevated for over 6hrs throughout the targeted dendritic arbors. Significant biphasic changes were observed over time in fluorescence intensity of synaptophysin and spinophilin (respectively pre- and post-synaptic markers). Conclusions: These findings establish the specific targeting of synapses by neurologically active Abeta oligomers (ADDLs). Induced expression of Arc has the potential to underlie dysfunctional changes previously observed in LTP and reversal of LTD, especially given the observed impact of Arc overexpression on AMPA-R specific synaptic depression (Rial Verde et al, SFN, 2003). We would suggest that such memory-relevant synaptic alterations by ADDLs could represent first neuropathological event in AD.

~ - J ~ 1713-ESTRADIOL PROMOTES DENDRITIC RAMIFICATION AND SYNAPSIN I EXPRESSION IN THE CULTURED CEREBRAL AND HIPPOCAMPAL NEURONS

Jiang-Ning Zhou*, Xiang-You Hu. University of Science and Technology of China, Hefei, China. Contact e-mail: jnzhou@ustc.edu.cn

Background: Accumulating data indicate that estrogen can promote the neuronal outgrowth mad synapse formation in culture. Objective(s): To evaluate the effects of 17[~-estradiol on ramification of dendritic trees and on the synapsin I expression in the cultured cerebral and hippocampal neurons. Methods: Primary neuronal cultures of cerebral cortex and hippocampus were obtained from embryos (E) (E17-E19) of Sprague-Dawley rats. Cul- tures were prepared according to Harms et al [10] with some modifications. Dissociated cells were plated onto 20 x 20 mm 2 glass coverslips in a density of 2.5 x 104 cells/cm 2. Cells were incubated with the cultivating medium at 37°C with 5%CO2, fed twice a week by replacing half of the medium. An estradiol-treated group, a tamoxifen-treated group, an estradiol/tamoxifen- treated group, and a control group were designed. After 24 h in experimental solutions, cells were processed for topological analysis and immunocyto- chemical analysis. Results: 17[3-estradiol treatment for 24 h significantly increased dendritic ramification, which was blocked by estrogen receptor antagonist tamoxifen. While the estradiol significantly increased synapsin I clusters in both dendritic spines and shafts, this effect was only weakened by tamoxifen. Conclusions: These findings suggest that, beside classical

nuclear estrogen receptor pathway, other nongenomic mechanisms may also be involved in the effects of estradiol on synapse formation.

• ASSOCIATION OF PKMZETA, AN ATYPICAL PKC, WITH DENDRITIC CHANGES AND NEUROFIBRILLARY TANGLES IN LIMBIC STRUCTURES IN ALZHEIMER'S DISEASE

Charles Y. Shao*, John E Crary, Todd C. Sacktor, Suzanne S. Mirra. SUNY Downstate Med. Ctr, Brooklyn, NY,, USA. Contact e-mail: charles.shao @ downstate, edu

Background: Protein kinase Mzeta (PKMzeta), an atypical protein kinase C found in brain, is critical to the maintenance of long-term potentiation (LTP) in rodents and memory persistence in Drosophila (Ling et al. Nat Neurosci. 2002, 5:295; Drier et al, Nat Neurosci. 2002, 5:316-324). Immunoelectron microscopy of rat hippocampus revealed a prominent distribution of PKMzeta in dendritic spines. Objective: These findings implicating a role of PKMzeta in synaptic plasticity and memory prompted us to examine involvement of PKMzeta in Alzheimer's disease (AD). Methods: Immunohistochemistry and confocal microscopy was performed on autopsy brain tissue derived from 24 cases of neuropathologically-confirmed AD using antibodies to PKMzeta, MAP2, PHF1, and actin. Results: PKMzeta immunoreactivity was associated with a small subset of neurofibrillary tangles in hippocampus, amygdala, and entorhinal cortex. However, tangles in other regions, e.g., neocortex, nucleus basalis, and brainstem, were PKMzeta-negative. An interesting subset of PKMzeta-positive but PHF- negative dystrophic neurites also was identified in the limbic structures. Double-labeling of these neurites demonstrated colocalization of PKMzeta with MAP2 and actin. In addition, virtually all Hirano bodies in CA1 were PKMzeta-positive, colocalizing as expected with actin and MAP2. Conclusions: The role of PKMzeta in LTP and memory in animal studies are paralleled by our demonstration of an intriguing distribution of this molecule in a subset of dendrites and neurofibrillary tangles in limbic regions in AD. Together, these findings suggest a role of PKMzeta in altered synaptic plasticity in Alzheimer's disease. (Supported by NIH AG 00959 to SSM)

~ ' ~ ALTERED EXPRESSION OF SYNAPTIC AND IMMEDIATE EARLY GENES IN A NEURONAL CULTURE MODEL OF ALZHEIMER'S DISEASE

Davide Tampellini .1 , Claudia G. Almeida 1 , Eric M. Snyder 2, Reisnke H. Takahashi ~, Giovanni Manfredil, Gunnar K. Gouras 1.1Weill Cornell Medical College, New York, NY, USA; 2Rockefeller University, New York, NY,, USA. Contact e-mail: dat2OO6@med.cornell.edu

Background: There is growing evidence that the earliest Alzheimer's disease (AD) pathogenesis takes place at a synaptic level. Immediate Early Genes (lEGs) are the first group of genes to be expressed following synaptic activation and are activated even in the presence of protein synthesis inhibitors. Among important synaptic IEGs are Arc, Homer-la and Zif268. The expression of these genes is associated with long-term potentiation (LTP), long-term memory (LTM) and transcription and binding of synaptic proteins, such as synapsin and type-1 metabotropic glutamate receptor. Alterations in mRNA levels of Arc, Homer-la and Zif268 have been reported in plaque-containing areas of old PS1-APP Tg mouse brains. Objective(s): To evaluate for changes in the expression of IEGs and synaptic genes in primary neuronal cultures of Tg2576 compared to wild type littermate control mice. Alterations in synapse relevant gene expression in Tg2576 neurons in vitro, that demonstrate AD characteristic A[3 accumulation and both structural and functional alteration in synapses, will be extended to Tg2576 versus wild type brains in vivo. Methods: Primary neuronal cultures are obtained from dissection of cortices and hippocampi of Tg2576 mice embryos and littermate controls. Tg2576 mouse harbor the human APP Swedish 670/671-mutation gene and are a well-established model of AD-fike [~-amyloidosis. Tg 2576 primary neurons secrete elevated levels of [~-amyloid and accumulate AIM2 subcellularly