Abstract:
The N-methyl-D-aspartate (NMDA) receptor is an ionotropic glutamate receptor that mediates intracellular entry of calcium ions (Ca2+). Previous data have demonstrated that NMDA receptor functionality influences megakaryocyte and platelet biology, but details remained poorly defined. Significantly, previously published data were limited to in vitro and ex vivo studies utilising pharmacological modulators.
The primary aim of this thesis was to generate genetic models of NMDA receptor function in megakaryocytes and platelets. The NMDA receptor is built as a heterotetramer containing the obligate GluN1 subunit, encoded by the GRIN1 gene; loss of GluN1 impairs the formation of the ion channel pore, leading to NMDA receptor hypofunction. The CRISPR-Cas9 system was utilised to delete GRIN1 in Meg-01 cells, a leukaemic megakaryocytic cell line, generating the Meg-01-GRIN1-/- cell line model. The Pf4-Grin1-/- mouse was also characterised, which utilised the Cre-loxP system to drive conditional deletion of Grin1 in megakaryocytes and platelets in vivo.
Meg-01-GRIN1-/- cells provided insights into the role of NMDA receptors in Ca2+ homeostasis in megakaryocytic cells. This model demonstrated that NMDA receptor hypofunction remodelled the Ca2+ toolkit and decreased store-operated Ca2+ entry, leading to depleted intracellular Ca2+ stores and elevated cytosolic Ca2+ levels. Biological effects of NMDA receptor hypofunction included decreased cell proliferation, increased differentiation, and increased cell death. Meg-01-GRIN1-/- cells accumulated lysosome-related organelles and had increased ploidy but showed a shift in their differentiation markers towards the erythroid lineage. Increased levels of erythroid transcription factors were also observed, accompanied by endoplasmic reticulum stress and autophagy induction.
