Modulation of transglutaminase 2 by pituitary adenylyl cyclase activating polypeptide and nerve growth factor in neuroblastoma cells: a role in cell survival and neurite outgrowth

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Algarni, AS, 2018. Modulation of transglutaminase 2 by pituitary adenylyl cyclase activating polypeptide and nerve growth factor in neuroblastoma cells: a role in cell survival and neurite outgrowth. PhD, Nottingham Trent University.

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Abstract

Several studies have reported that the multifunctional enzyme tissue transglutaminase (TG2) play important roles in neurite outgrowth and modulation of neuronal cell survival. Recently, TG2 enzymatic activity has been shown to be regulated by protein kinases such as PKA and PKC, modulating through G-protein coupled receptors (GPCRs). However, the regulation of TG2 following stimulation of GPCRs coupled to PKC and PKA activation are not fully understood. In neuronal cells, the neurotrophic factors; pituitary adenylate cyclase activating polypeptide (PACAP) and nerve growth factor (NGF) promote the differentiation, maturation, neurite outgrowth and survival of neurons via their GPCR's; the pituitary adenylate cyclase type 1 receptor (PAC1) and the tyrosine kinase receptor (TrKA) including multiple protein kinase signalling pathways. As some of these kinase pathways are associated with modulation of intracellular TG2 activity, it is conceivable to hypothesised that PACAP and NGF directly regulates TG2 activity. If this was found to be the case, it sought to determine whether the PACAP and/or NGF-induced neurite outgrowth and modulation of neuronal cell survival involved TG2. The main aims of this study were to investigate the molecular mechanisms underlying TG2 modulation by PACAP and NGF and its role in neurite outgrowth and neuroprotection in differentiating mouse N2a and human SHSY5Y neuroblastoma cells.

In the first part, the regulation of TG2 transamidase activity by PACAP-27 and NGF in differentiating mouse N2a and human SH-SY5Y neuroblastoma cells was assessed. TG2 transamidase activity was determined using an amine incorporation and a peptide cross linking assay. In situ TG2 activity was assessed by visualising the incorporation of iotin-X-cadaverine using confocal microscopy. TG2 phosphorylation was monitored via immunoprecipitation and Western blotting. In the second part of this study, the role of TG2 in PACAP-27 and NGF-induced cytoprotection was investigated by monitoring hypoxia-induced cell death and assessing MTT reduction, LDH release, and caspase-3 activity. The neurite outgrowth was assessed using high-throughput analysis of immunofluorescently stained cells.

In differentiating N2a and SH-SY5Y cells, stimulation of PAC1 and TrKA receptor with PACAP-27 and NGF respectively, resulted in a time- and concentration- dependent activation of TG2 amine incorporation and protein crosslinking activity, which was abolished by TG2 inhibitors Z-DON and R283. Responses to PACAP-27 were attenuated mainly by pharmacological inhibition of protein kinase A (KT 5720 and Rp-cAMPs), MEK1/2 (PD 98059), PKB (Akt inhibitor XI) and by removal of extracellular Ca2+. Responses to NGF were abolished by MEK1/2 (PD 98059), PKB (Akt inhibitor XI) and PKC (Ro 31-8220). Immunoprecipitation technique showed that both PACAP-27 and NGF triggered the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by inhibition of MEK1/2 and PKA for PACAP and MEK1/2, PKB, PKC and removal of extracellular Ca2+ for NGF. Fluorescence microscopy demonstrated that PACAP-27 and NGF induced in situ TG2 activity. TG2 inhibition blocked PACAP-27- and NGF-induced attenuation of hypoxia-induced cell death and neurite outgrowth. Taken together, this study highlights for the first time the importance of TG2 in the cellular functions of PACAP and NGF in neuronal cells and provides initial characterisation of the molecular mechanisms possibly involved.

Item Type:	Thesis
Creators:	Algarni, A.S.
Date:	June 2018
Rights:	This work is the intellectual property of the author. You may copy up to 5% of this work for private study, or personal, non-commercial research. Any re-use of the information contained within this document should be fully referenced, quoting the author, title, university, degree level and pagination. Queries or requests for any other use, or if a more substantial copy is required, should be directed to the owner(s) of the Intellectual Property Rights.
Divisions:	Schools > School of Science and Technology
Record created by:	Linda Sullivan
Date Added:	13 Aug 2018 11:43
Last Modified:	13 Aug 2018 11:43
URI:	https://irep.ntu.ac.uk/id/eprint/34324