P3-352 BETA-SECRETASE MODULATION ANDAMYLOID-BETA PRODUCTION IN RAT BRAINPRIMARY CULTURES

Daniele Zacchetti, Barbara Bettegazzi, Alessandra Consonni,Romina Macco, Ilaria Pelizzoni, Franca Codazzi, Fabio Grohovaz, SanRaffaele Scientific Institute and University, Milano, Italy. Contact e-mail: zacchetti.daniele@hsr.it

Background: The processing of the amyloid-beta precursor protein by abeta-secretase is the rate-limiting step in the production of amyloid-betawithin the central nervous system. Two beta-secretases have been identi-fied, BACE1, mainly expressed in neurons, and BACE2, with a moreubiquitous expression pattern. Their expression is tightly regulated at boththe transcriptional and translational level. In primary cultures from ratbrain, we investigated: i) the regulation of BACE1 expression in neurons;ii) the regulation of beta-secretase activity, in resting and activated astro-cytes, and its contribution to amyloid-beta production. Methods: Primarycultures of neurons and astrocytes from rat brain were employed. Thetranslational control of BACE1 expression was evaluated by transfection ofreporter genes preceded by the BACE1 transcript leader. For the evaluationof endogenous beta-secretases, we tested their activity by an in vitro assay,and BACE1/2 expression by RNA analysis and Western blotting. Results:In a previous work we demonstrated that the translation driven by theBACE1 transcript leader (as evaluated by reporter genes) is higher inneurons than in astrocytes or other commonly used cell lines. In the presentstudy, we investigated the molecular determinants of this control by ana-lyzing the features of the BACE1 mRNA and monitoring changes inendogenous BACE1 upon various treatments. This analysis revealed thepresence of both inhibitory and stimulatory sequences in the transcriptleader. Furthermore, we demonstrated the presence of a translational blockthat is specifically bypassed in neurons. In parallel, we studied how beta-secretase activity is regulated in astrocytes. We did not detect BACE1protein, even under conditions of astrocyte activation induced by variousstimuli (including pro-inflammatory cytokines). Our data show that beta-secretase activity in astrocytes was due to the expression of BACE2. On theother hand, activated astrocytes displayed a reduced BACE2 expression aswell as a decrease in beta-secretase activity. We are currently investigatingthe role of astrocyte activation on the production of amyloid-beta. Con-clusions: BACE1 expression is regulated at the translational level by aneuronal-specific mechanism. Astrocytes do not express BACE1 and, uponactivation with pro-inflammatory cytokines, they down-regulate BACE2expression, thus decreasing beta-secretase activity and, likely, amyloid-beta production.

P3-353 REGIONAL SUSCEPTIBILITY TOINFLAMMATION: NEURON-GLIAINTERACTIONS IN THE HIPPOCAMPUS ANDSUBSTANTIA NIGRA AND RESCUE BY THENMDA ANTAGONIST MEMANTINE

Holly Brothers1, Francesca Cerbai2, Yannick Marchalant1, Gary Wenk1,1Ohio State University, Columbus, OH, USA; 2Universita degli Studi diFirenze, Columbus, OH, USA. Contact e-mail: brothers.23@osu.edu

Background: Neurodegenerative diseases are characterized by inflamma-tion, and areas with the highest concentrations of activated microglia tendto be the most vulnerable to loss of function. Chronic neuroinflammationmay contribute to degeneration by creating a microenvironment of elevatedglutamate availability in which intracellular calcium levels increase andcontribute to excitotoxicity. Lipopolysaccharide (LPS) infused intraven-tricularly for four weeks causes an increase in microglia activation that iscorrelated with hippocampal dysfunction and loss of tyrosine hydroxylaseimmunoreactive cells in the substantia nigra. The uncompetative NMDAchannel antagonist, memantine, has been shown to reduce microglia acti-vation and restore neuroplasticity. NMDA receptors are not located onmicroglia, indicating that memantine reduces activation of microglia indi-rectly through its effect upon neurons. To investigate the mechanism by

which neurons may signal microglia to remain inactive, we analyzedneuronal communication markers such as high-mobility-group-box-1(HMGB1) and CD200. Methods: Rats were infused continuously witheither LPS or aCSF into the fourth ventrical for four weeks though an Alzetminipump. Animals were treated with either saline or memantine duringthe last two weeks of the four week infusion period. Immunohistologicalmethods were used to visualize major-histocompatibility-complex II(MHC II) on activated microglia, HMGB-1, and CD200. Results: LPSincreased the number of activated microglia in the hippocampus andpreliminary data show that microglia activation is increased in the sub-stantia nigra. Memantine treatment decreased the number of activatedmicroglia in the hippocampus and appears to have done the same in thesubstantia nigra. Preliminary data also suggest that LPS caused a reductionin the number of tyrosine hydroxylase immunoreactive cells in the nigraand that memantine restored this number. Conclusions: Memantine re-duced established microglia activation in the hippocampus and preliminarydata indicate a role for memantine in the reduction of inflammation in thesubstantia nigra and the rescue of tyrosine hydroxylase positive neurons.Our data also indicate a role for HMGB1 and CD200 in neuronal-microgliacommunication. A better understanding the role of the NMDA receptor andthe biomolecules that control the activation states of microglia may lead tomore effective approaches for regulating chronic inflammatory diseases inhumans.

P3-354 ROLE OF IL-6–MEDIATED CHRONICNEUROINFLAMMATION ON AMYLOIDPATHOLOGY

Paramita Chakrabarty, Kasey Nimcheski, Todd E. Golde,Karen Jansen, Christophe Verbeeck, Pritam Das, Mayo Clinic,Jacksonville, FL, USA. Contact e-mail: chakrabarty.paramita@mayo.edu

Background: The pathological cascades leading to A�-mediated neuro-degeneration are still ambiguous. Secondary factors like chronic neuroin-flammation and reactive gliosis have been postulated to contribute to thisneurodegenerative process. Though an early upsurge in microglial activa-tion is associated with AD neuropathology, the exact role of neuroinflam-mation in development of A�-associated pathology is unknown. We planto examine whether any or all of the following scenarios are feasible invivo - 1) microglial activation may be beneficial in removing toxic A�

aggregates; 2) neuroinflammation may exacerbate A� pathology by caus-ing runaway neuronal toxicity and/or 3) microglial activation may bemostly a bystander phenomenon. Methods: Using rAAV mediated genedelivery technique, we have achieved high levels of expression of differentcytokines in APP (CRND8 and Tg2576) transgenic mice. This enables usto bypass the often confounding problems inherent in creating bitransgenicmice and allows us to dissect the role of individual cytokines and resultantglial involvement in development of A� pathology. Results: Here weexplore the role of IL-6 overexpression in modulating A� pathology inAPP transgenic mice. Since cytokine-mediated signaling pathways mayshow a differential effect on A� production, deposition or aggregationdepending on context and timing of expression, we have overexpressedIL-6, a pro-inflammatory cytokine, in both plaque-free neonate mice and inadult mice with pre-formed plaques. In spite of overwhelming microglialand astrocytic pathology, neonate expression of IL-6 does not lead to anysignificant alterations either in A� pathology in adult mice or in steadystate APP levels in young transgenic mice. However, focal hippocampalexpression of IL-6 in adult 5 month old CRND8 mice leads to a modest butsignificant decrease in A� levels. Conclusions: Thus, chronic expressionof IL-6 does not appear to have a direct role in APP production or A�

production and deposition. However, chronic neuroinflammatory condi-tions, when initiated in adult mice, appear to mitigate A� pathology. Wehypothesize that this decrease in A� levels may be attributed to increasedglial phagocytotic function. Functional assessment of IL-6 mediated glialactivation and resultant glial phenotypes vis-a-vis their potential role in A�

clearance mechanisms will be further discussed.

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