Chronic activation of microglia is a driving factor in the progression of neuroinflammatory diseases, and
mechanisms that regulate microglial inflammatory signaling are potential targets for novel therapeutics. RGS10
strongly suppresses inflammatory signaling in microglia, and loss of RGS10 enhances inflammatory
neurotoxicity. More specifically, RGS10 suppresses expression of inflammatory genes Tumor Necrosis Factor a
(TNFa) and cyclooxygenase 2 (COX-2), following stimulation of Toll-like Receptor 4 (TLR4) by
Lipopolysaccharide (LPS). However, the mechanism by which RGS10 affects inflammatory signaling is unknown
and is independent of its canonical G protein targeted mechanism. We have identified a robust, novel interaction
between RGS10 and the endoplasmic reticulum (ER) localized calcium sensor STIM2 in microglial cells. STIM2
is a critical component of the store-operated calcium entry (SOCE) machinery, responsible for coupling depletion
of ER calcium stores with Orai channel-mediated extracellular calcium entry. STIM2-Orai activity regulates
inflammatory signaling through activation of the phosphatase calcineurin and its substrate Nuclear Factor of
Activated T cells (NFAT), a transcription factor that directly regulates expression of TNFa and COX-2. This
pathway provides a novel, plausible mechanism for G protein independent regulation of inflammatory gene
expression by RGS10. The goal of this proposal is to define the specificity, mechanisms, and functional
consequence of RGS10 interaction with STIM2 in microglia. In preliminary experiments, we have validated
RGS10-STIM2 interaction in multiple cell types and demonstrated that inhibition of STIM2, Orai channels, or
calcineurin strongly blocks the effect of RGS10 on LPS-stimulated COX-2 expression. We have also
demonstrated a marked increase in SOCE in RGS10 knockout microglia. We hypothesize that RGS10 inhibits
LPS-stimulated expression of COX-2 and TNFa by interacting with STIM2 at the ER and inhibiting the ability of
STIM2 to activate plasma membrane Orai channels and subsequent calcium dependent activation of calcineurin
and NFAT. We will 1. Define the structural and scaffolding requirements for STIM2-RGS10 interactions in
microglia. This aim will provide essential molecular details governing the novel interaction between RGS10 and
STIM2. 2. Define the role of STIM2 and downstream signaling in RGS10 regulation of inflammatory gene
expression. This aim will establish to what extent regulation of the STIM2-ORAI-Calcinurin pathway accounts
for RGS10’s ability to regulate G protein-independent inflammatory gene expression in microglia. 3. Define the
ability of RGS10 to regulate store-operated calcium signaling. This aim will establish a mechanism for
RGS10 regulation of SOCE. 4. Define the ability of G¿i to regulate RGS10 regulation of STIM2 signaling.
This aim will define the mutual regulation of RGS10 interactions with Gi and STIM2 pathways. Completion of
these studies will fundamentally shape understanding of RGS10’s role in cell physiology and disease, and will
establish unexpected, novel interfaces and activities as therapeutic targets in neuroinflammatory disease.