Regulation of the inositol (1,4,5) trisphosphate receptor by tumor necrosis factor-alpha

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 cytokincs. These immunomodulators affect neuronal Can handling processes, which in turn shape the 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 of cell culture-based studies to finely dissect the effects of the central proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on neuronal Ca2+ signaling. Exposure of C57BL/6 primary neurons to TNF-alpha resulted in significant enhancement of Ca2+ signals following 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 Ca 2+ homeostasis. Furthermore, it was determined that the activation of dun 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-alpha 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-alpha on IP 3R-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 were 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-alpha as a function of age. Despite observing basally elevated ER-derived Ca 2+ release, there was no enhancement in release detected following 3xTg-AD neuron treatment with TNF-alpha. In contrast, prolonged incubation with the pro-inflammatory cytokine led to a significant diminution of Ca 2+ release following muscarinic activation. Subsequent experiments demonstrated that the lack of a TNF-alpha effect on IP3R-mediated Ca 2+ 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-alpha in the alteration of Ca2+ homeostasis within the central nervous system. Since the modulation of Ca2+ responses arising from the IP3 R 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.