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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS048263-05
Application #
7364140
Study Section
Neurodegeneration and Biology of Glia Study Section (NDBG)
Program Officer
Sieber, Beth-Anne
Project Start
2004-04-01
Project End
2010-01-31
Budget Start
2008-02-01
Budget End
2010-01-31
Support Year
5
Fiscal Year
2008
Total Cost
$260,490
Indirect Cost
Name
University of Maryland Baltimore
Department
Physiology
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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