Modulation of CNS Proinflammatory Cytokine Production

The abstract ot the current research plan, as originally submitted, is given here: The serine/threonine protein kinase p38a MAPK is an established therapeutic target for a number of peripheral inflammatory diseases where increased proinflammatory cytokine production contributes to pathology. In contrast, much less is known about the in vivo role of p38a MAPK in CNS dysfunction and its potential as a therapeutic target. Specifically, little information is available on the quantitative contribution of microglial p38a MAPK signaling in vivo to up-regulation of proinflammatory molecules that lead to disease-rel.evant neuropathology, the importance of p38a MAPK in the beneficial reparative and remodeling responses of activated microglia, or the role of neuronal p38a MAPK in CNS dysfunction responses. We hypothesize that p38a (and not the closely related p3813 MAPK isoform) is a key in vivo contributor to microglial inflammatory activation cascades that culminate in detrimental proinflammatory cytokine overproduction and subsequent neuronal/synaptic damage, and that suppression of p38 MAPK signaling in the microglia and/or neuron can lead to selective, beneficial outcomes. The field has been limited in its ability to pursue these questions because of the lack of CNS-penetrant, selective, small molecule p38a MAPK inhibitors. Our development of a novel, orally bioavailable, brain-penetrant, selective, small molecule p38a MAPK inhibitor (compound 069A) that attenuates hippocampal proinflammatory cytokine overproduction and leads to improved neurologic outcomes in a mouse CNS injury model now provides the opportunity to address these critical questions about p38 MAPK and CNS dysfunction in vivo as well as in vitro or in situ. We will use this unique chemical biology tool and novel knock-out (KO) and drug-resistant knock-in (KI) mice to pursue several important mechanistic investigations. First. we will test the importance of p38a MAPK and p3813 MAPK in vivo through the use of microglial p38a conditional KO and p3813 global KO mouse models subjected to stressor stimuli. The temporal onset and profile of microglial activation and synaptic dysfunction responses will be determined. Second, we will complement these in vivo studies by using cell culture models in order to explore in more detail the relative contributions of p38 MAPK isoforms in microglia and neurons. We will utilize microglial-neuronal co-cultures combined with pharmacological and genetic knock-down approaches to determine the importance of microglial and neuronal p38 MAPK isoforms to stressor-induced responses. Successful completion of this project will provide mechanistic insight intp the role of the key regulatory protein, p38a MAPK, in microglial activation and neuronal damage caused by stressors that induce CNS pathophysiology, and the contribution of microglial vs neuronal p38a MAPK to the CNS dysfunction responses. In addition, the studies will elucidate the potential role of the highly related p38 MAPK isoform, p3813, in the microglial and neuronal responses. Longer term, the knowledge generated by the proposed studies will provide a firmer foundation for future development of new classes of disease-modifying therapeutics and fuller interpretation of disease progression investigations. The current research plan includes one change in aim 1b of the R01 application, in order to eliminate any overlap with my Zenith award from the Alzheimer's Association. Specifically, we proposed in the Zenith award to test p38beta KO mice in an Abeta infusion study and measure selected endpoints of pathophysiology. In the R01 application, we proposed this same study, but also proposed additional experiments with using LPS as the stimulus instead of Abeta, and measuring anti-inflammatory and beneficial responses of microglia as well as pathophysiological endpoints. Therefore, in the current research plan, we will NOT do the Abeta infusion stUdy from aim 1b of the R01 application, and only do the LPS injection study instead (which is not part of the Zenith award). The rest of the current research plan remains the same as originally proposed.