The seminal work by Hoffmann and collea gues, in which simulation predictions were used in coordination with experimental studies of I B knockout cells to reveal functional differences among three I B iso forms, established mathematical modeling as a vital tool for studying NF B signaling at a systems level. Subse quently a number of researchers have full report used modeling to investigate various aspects of NF B activity, Here we develop a mathematical model to describe NF B signaling in microglia. Beginning with a recently published model structure shown to be capable of pre dicting NF B signaling in other cell types, we attempt to identify model parameters to match experi mental data sets of NF B and IKK activation obtained from a microglial cell line.

The inability of the original model to recapitulate NF B activation that is consistent with experimental data regardless of model parameter choice leads us to expand the model to incorporate previously unmodeled dynamics of the I Ba ubiquitin proteasome degradation pathway. We also find that IKK activation in microglia is highly nonlinear, which prompts refinement of the upstream signaling module. We use the new model to predict the levels of another network component, total I Ba, and are able to validate this prediction experimentally. The results offer a vali dated model that can be used as a new tool to study the dynamics of NF B activation in microglia. While we find that many key features of canonical NF B activa tion are shared in microglia, the model suggests a potentially more prominent role for the ubiquitin system in regulating the dynamics of NF B activation.

We use numerical analyses of this model to gain insight into how microglia regulate both IKK and NF B activity in response to inflammatory stimuli. Our sensitivity anlayses emphasizes the dynamic nature of how key sys tem responses are regulated, a feature that may not be apparent from similar analyses. The analysis further highlights the robust yet fragile nature of the NF B sig naling pathway due to the multiple layers of feedback regulation. Results TNFa stimulates dynamic NF B and IKK activation in BV2 microglia To characterize the dynamics of canonical NF B activa tion in microglia, cells from the microglial cell line BV2 were cultured and treated with 10 ng ml TNFa.

Whole cell extracts were collected in triplicate over a time course Brefeldin_A following stimulation in five identical experiments conducted on different days. ELISA measurements of NF B p65 DNA binding activity show that NF B acti vation in BV2 microglia is strongly induced by TNFa. Five minutes following TNFa treatment NF B activation remains near basal levels but increases rapidly thereafter, reaching maximal activity near 20 min. Following the initial peak, NF B activity declines until approximately 90 min when it returns to a second, smal ler amplitude peak.