Evidence from human data and in experimental models of Parkinson's Disease (PD) supports a role for inflammation in the pathophysiology of PD in humans. Consistent with this idea, a prospective study of hospital workers found that daily use of certain non-steroidal anti-inflammatory drugs for a period greater than 2 yrs lowered the risk of developing PD by 46%. Therapeutically, these findings raise the possibility that anti-inflammatory therapy could delay or prevent onset of PD. Tumor Necrosis Factor (TNF) is a potent inflammatory mediator produced by microglial cells in the brain in response to various stimuli that is selectively toxic to dopamine (DA) neurons, the death of which results in Parkinson's Disease. The overall goal of this project is to investigate the cellular and signaling mechanisms by which TNF affects DA neuron function and survival. And to test the hypothesis that TNF is the critical inflammatory mediator responsible for degeneration of midbrain DA neurons. We propose a dual-site model by which TNF, independent of the trigger that elicits its production, directly promotes formation or reactive oxygen and nitrogen species within neurons, exerts toxic effects on mitochondria, and activates death pathways in DA neurons. In this model, TNF further enhances oxidative stress on DA neurons by potentiating activation of microglial-derived oxidant species. To test our hypothesis, we induce endogenous TNF production in culture and in whole animals with bacterial lipopolysaccharide (LPS) and with the oxidative neurotoxin 6-hydroxydopamine (OHDA).
In Aim 1 we will use our newly engineered dominant negative inhibitors (DN-TNFs) to identify TNF-dependent mechanisms required for degeneration of DA neurons in rodent primary neuronal cultures of embryonic ventral mesencephalon (EVMCs). If our hypothesis is correct, timely inhibition of TNF signaling should halt DA neuron loss and provide neuroprotection from LPS and 6-OHDA.
In Aim 2, in vivo neuroprotection studies will ascertain the contributions from soluble and membrane-tethered TNF in mediating degeneration of DA neurons in response to LPS or 6-OHDA, establish whether TNF mediates the acute and/or progressive death of DA neurons, and investigate if TNFR expression is dysregulated by chronic inflammation or oxidative stress.
In Aim 3, the function of each TNF receptor and its downstream signaling pathways in regulation of microglia activation and DA neuron survival will be investigated. Experiments to determine whether R1 and R2 have opposing or synergistic actions will be performed with agonistic antibodies and newly engineered receptor-selective TNF variants in embryonic mouse midbrain neuron/glia co-cultures. Completion of these specific aims will provide critical new information on the role of TNF in regulating neuroinflammation and DA neuron survival and may provide mechanism-based strategies that target the TNF pathway strategies to slow or reverse PD progression within our lifetime.
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