Thesis (Ph.D.)--University of Rochester. School of Medicine and Dentistry. Dept. of Pharmacology and Physiology, 2009.
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 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-α) on neuronal
Ca2+ signaling. Exposure of C57BL/6 primary neurons to TNF-α 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 Ca2+ homeostasis. Furthermore, 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 JNKinduced
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 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-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.
