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Tabatabaee, Mitrasadat

University of British Columbia

Growing evidence indicates that dysregulation of glutamate signaling between neurons and astrocytes leads to the onset and progression of brain tumors (glioma). The aberrant morphology of glioma cells from astrocytic origin (astrocytoma cells) with elongated processes is also emerging as an underlying cause of progression and therapy resistance of gliomas. Whether glutamate dysregulation conducts this atypical morphology of astrocytoma cells has not been explored. Since normal astrocytes respond to glutamate by filopodiagenesis and processes extension, observing aberrantly elongated processes in response to excessive glutamate in astrocytoma cells is conceivable; yet it requires detailed empirical support to make it translatable to therapies. In this dissertation, the role of glutamate receptors and contribution of L-type voltage-gated calcium channels (CaVs) in elongation and directional protrusion of the astrocytoma cell processes toward glutamate were investigated. Protrusion of cellular processes toward glutamate and an intracellular calcium rise were observed in U118-MG astrocytoma cells. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), whose role in neuro-gliomal interactions has been reported, did not trigger filopodiagenesis in these cells. In contrast, use of selective agonists (kainate and phenyl-kainate) and an antagonist (CNQX) indicated that kainate receptors (KARs) contribute to the glutamate-induced morphological response. However, precisely releasing a KAR caged-agonist (DECM-PhKA) to the 3D-cultured cells showed that despite their role in processes extension, KARs do not contribute to the pathfinding and the directional motility towards glutamate. CaVs were also found to play a role in the glutamate-induced morphological response in U118-MG cells by using selective antagonists (nifedipine, verapamil and diltiazem). Interestingly, a real-time imaging probe (FluoBar1) revealed that the membranar density of CaV1.2 rises immediately upon exposure to glutamate. Results from application of selective ligands (gabapentinoids and antibody) suggested that an auxiliary subunit of CaVs, α2δ1, links glutamate sensing to the intracellular calcium rise and filopodiagenesis. Strong evidence for the role of α2δ1 was found in heterologous expression systems that acquired the ability to respond to glutamate upon transfection. Altogether, determining the involvement of ligand-gated KARs and voltage-gated CaVs for the first time in the morphology of astrocytoma cells paves the way for therapeutical purposes to prevent progression and malignancy of gliomas.

Text

2021-11-03

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/80143

Biochemistry and Molecular Biology

University of British Columbia

UBCO

http://creativecommons.org/licenses/by-nc-nd/4.0/