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 ? (TNF?) 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 TNF? 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 TNF? 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.

Public Health Relevance

Chronic inflammation drives progression of many neurodegenerative diseases including Parkinson?s disease and Alzheimer?s disease. The small protein RGS10 is a strong suppressor of neuroinflammation and holds great promise as a drug target in treating neurodegenerative disease, but its mechanism of action is unknown. This project aims to define a novel mechanism of RGS10 anti-inflammatory functions to pave the way for new drug development.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
High Priority, Short Term Project Award (R56)
Project #
1R56NS109303-01A1
Application #
9938890
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Morris, Jill A
Project Start
2019-07-15
Project End
2020-07-14
Budget Start
2019-07-15
Budget End
2020-07-14
Support Year
1
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Georgia
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602