Localization of the Inositol (1,4,5)-trisphosphate Receptor type I, via protein 4.1N, mediates neurite formulation through intracellular calcium(II) ion waves

Calcium (Ca2+) signaling is of crucial importance in neurons because it regulates neurotransmitter release, dendrite growth/guidance and has been implicated in neurodegenerative diseases such as Alzheimer's and Parkinson's. The Inositol (1,4,5)-trisphosphate Receptor type I (IP3R1) is a key component of neuronal Ca2+ signaling and functions to generate intracellular Ca2+ waves, which have been linked to several cellular processes including gene transcription, proliferation, and plasticity. The localization of IP3R1 is thought to play a significant role in this wave activity and protein 4.1N has been shown to bind and mediate IP3R1 positioning in neurons. However, despite the identification of this protein-protein interaction, no functional data regarding this relationship exists.;With the use of NGF differentiated PC12 cells as a model system for neuronal development, I investigated the IP3R1-protein 4.IN relationship to determine a functional role for this interaction. Experimentally, I utilized RNAi technology and over-expressed soluble binding regions of each protein which function as dominant negatives. Upon introduction of these genetic elements I assayed neurite formation in PC12 cells stimulated with NGF for 48 hr. Additionally, I investigated Ca2+ signaling dynamics during NGF induced differentiation with the IP3 producing agonist carbachol (CCID). Lastly, I introduced RNAi and DN constructs into PC12 cells differentiated with NGF for 24 hr to determine what affect the IP3R1/4.1N relationship has on Ca2+ wave formation in actively developing cells.;In knocking down IP3R1 and protein 4.1N expression, I observed robust attenuation of neurite formation. With respect to dominant negative experiments, soluble binding regions of IP3R1 and protein 4.1N co-localized with endogenous protein 4.IN and IP3R1, respectively, and strongly attenuated neurite formation. Concerning Ca2+ signaling during NGF differentiation, PC12 cells shifted their Ca2+ release pattern from homogeneous transients to spatially restricted waves that initiate at the terminal end of neurite extensions. When RNAi and dominant negative constructs were introduced in partially differentiated PC12 cells stimulated with CCH, both sets of molecules shifted signaling events from wave to homogeneous patterns of Ca2+ release. In generating these data, it is my conclusion that IP3R1 localization, via protein 4.1N, mediates neurite formation through intracellular Ca2+ waves.