Keigan M. Park

B.S. Penn State University
M.S. and Ph.D. University of Rochester

Then	Now

Thesis Title

Regulation of the Inositol (1,4,5) Trisphosphate Receptor by Tumor Necrosis Factor-alpha

Defense Date

October 29, 2009

Abstract

Inflammatory events have long been implicated in initiating and/or propagating the pathophysiology associated with a number of neurological diseases. During neuroinflammation, the activation of brain-resident immune cells leads to the production of pro-inflammatory cytokines. These immunomodulators affect neuronal Ca2+ handling processes, which shape membrane potential, influence gene transcription, and affect neuronal spiking patterns. Similar alterations in Ca2+ signaling are also implicated in neurological disease progression and cognitive decline. The mechanisms underlying the purported interplay that exists between neuroinflammation and Ca2+ homeostasis have yet to be clearly defined. To that end, we performed a series cell culture-based studies to finely dissect the effects of the central proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) on neuronal Ca2+ signaling. Exposure of C57BL/6 primary neurons to TNF-α resulted in significant enhancement of Ca2+ signals downstream of muscarinic and purinergic stimulation. Subsequent experiments ruled out the possible effects of cytokine addition on Ca2+ influx and clearance, which further defined the event as an increase in inositol 1,4,5 trisphosphate receptor (IP3R)-mediated Ca2+ release. Enhanced steady-state mRNA and protein levels of the type-1 IP3R following cytokine exposure positively correlated with this alteration in Ca2+ homeostasis. Furthermore, utilizing pharmacological interventions, it was determined that the activation of cJun N-terminal kinase (JNK) was a key step in this process. To fully delineate the signaling pathway responsible for enhanced type-1 IP3R mRNA, the effects of TNF-α signaling on the human IP3R promoter were examined in the Neuro2A mouse neuroblastoma cell line. A novel site 59 base pairs downstream of the transcription start site was shown to be responsible for the JNK-induced regulation, while electrophoretic mobility shift experiments were used to further define factors binding to this promoter region,. Finally, the use of a dominant negative SP-1 construct demonstrated the key role of this protein in the pathway by eradicating the effects of TNF-α on IP3R-mediated Ca2+ release. After defining this novel pathway in normal neuronal cells, its signaling characteristics in primary neurons isolated from triple-transgenic Alzheimer’s disease (3xTg-AD) mouse embryos was examined. This model, which has been previously shown to harbor alterations in ER-mediated Ca2+ release, gives rise to both of the hallmarks of human AD pathology (amyloid plaques and neurofibrillary tangles) and expresses enhanced levels of TNF-α as a function of age. Despite observing basally elevated ER-derived Ca2+ release, there was no enhancement in release detected following 3xTg-AD neuron treatment with TNF-α. In contrast, prolonged incubation with the pro-inflammatory cytokine led to a significant diminution of Ca2+ release following muscarinic activation. Subsequent experiments demonstrated that the lack of a TNF-α effect on IP3R-mediated Ca2+ release was due to a marked suppression of TNF receptor expression. The presence of this novel pathway, and its marked alteration in neurons destined for AD-related demise, indicates a key role for TNF-α in the alteration of Ca2+ homeostasis within the central nervous system. Since the modulation of Ca2+ responses arising from the IP3R and its downstream effectors may exact significant consequences on neuronal function, this signaling cascade could underlie the compromise in neuronal activity observed in the setting of chronic neuroinflammation.