A common feature of Alzheimer's disease (AD) and Down's syndrome (DS) is a cascading microglia/astrocyte activation, characterized by the abundant production of proinflammatory cytokines, such as interleukin-1beta (IL-1beta). IL-1beta stimulates astrocytes to synthesize and release neuroactive agents such as S100beta, a cytokine that fosters neuronal dysfunction and death by raising intracellular free Ca2+concentration ([Ca2+]cyt). IL-1beta also upregulates expression and processing of beta-amyloid precursor protein (beta-APP) leading to increased production of amyloid beta-protein (A-beta) that is thought to play a major role in the pathogenesis of AD. Both, IL-1beta and A-beta increase [Ca2+]cyt in astrocytes by augmenting Ca2+ influx. The mechanisms underlying Ca2+ dysregulation, although unknown, may be causally implicated in reactive changes of astrocytes leading to neurotoxicity. They may involve activation of store- and/or receptor-operated Ca2+ entry (SOCE and ROCE, respectively) through TRPC-encoded store- and receptor-operated Ca2+ channels (SOCs, ROCs). The goal of this program is to test the hypothesis that dysregulation of Ca 2+homeostasis with elevated [Ca2+]cyt and ER Ca2+concentration ([Ca2+]ER), in IL-1beta or A-beta-treated astrocytes and in astrocytes from trisomy 16 (Ts16) mouse, an animal model of DS and AD, is the result of upregulated TRPC expression and enhanced SOCE and/or ROCE.
The Specific Aims are: 1. To investigate how acute and chronic (24 hr) IL-1beta - and A-beta-treatment affects Ca2+ homeostasis in freshly dissociated and primary cultured mouse cortical astrocytes using Ca2+-sensitive fluorochromes. SOCE will be probed by measuring the rates and magnitudes of SOCE-evoked changes in [Ca2+]cyt, and [Ca2+]ER. 2. To identify the molecular mechanisms responsible for IL-1beta- and Abeta-induced global and local Ca2+ signaling in astrocytes. To determine whether IL-1beta and A-beta affect expression and distribution of TRPC channels and ER Ca2+ transporters and receptors (SERCA2b, IP3 and ryanodine receptors) by using PCR, immunoblotting and high spatial resolution immunocytochemistry. 3. To determine whether inhibition of TRPC-encoded SOCs and/or ROCs down-regulates S 100beta and beta-APP expression in IL-1beta - and A-beta-treated cells and in Ts16 astrocytes (Ts16 mice and DS humans overexpress betaAPP and S100beta). Antisense oligos for TRPC(1-7) genes and Ca2+-free media will be used to eliminate Ca2+ influx. Expression of S100beta and beta-APP will be identified by immunoblotting. Determining the mechanisms of IL-1beta - and A-beta-induced Ca2+influx in astrocytes could lead to novel therapeutic strategies for AD.
