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A model of SPRY3 - VGCC interactions relating to autism
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Date
2023
Authors
Bharatham Vijayaraghavan, Sashank
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
Published Version
Abstract
Autism Spectrum Disorder (ASD) is a neurobehavioral condition characterized by impaired social interaction and communication resulting from irregular brain development during infancy and adolescence. Globally, over 168 million cases of ASD have been diagnosed, with Ireland ranking sixth in prevalence (583.69 cases per 100,000 people). Heritability, estimated between 40% and 80% in twin and family studies, underscores the importance of identifying susceptibility genes in ASD research. The ASD susceptibility candidate gene SPRY3 encodes a receptor tyrosine kinase inhibitor and plays a regulatory role in branching morphogenesis. SPRY3 is highly expressed in cerebellar Purkinje cells. Spry3 and p75NTR have opposite expression patterns in the cerebellar vermis of the mouse and it has been hypothesized that reactivation of the epigenetically silenced Y-linked SPRY3 copy in the human might interact with the TrkB and p75NTR signaling pathways to cause Purkinje cell pathology.
The current work evaluates the potential that SPRY3-GFP colocalizes with neurotropic receptors in HEK293 cell line model and found that SPRY3-GFP colocalizes with TrkB-RFP and p75NTR-RFP. Significant colocalization of SPRY3 with EGFR-GFP and TrkA-RFP was also observed. This investigation detected no colocalization between SPRY3-RFP and CasR-GFP, which is a G-protein-coupled extracellular calcium-sensing receptor. The involvement of voltage-gated calcium channels (Cav) have been implicated in ASD and their role in the regulation of branching morphogenesis in the brain and lung, and the fact that calcium acts as a secondary messenger to modulate various signaling pathways involved in branching morphogenesis, we hypothesized a functional relationship between SPRY3 and calcium signaling mediated through Cav receptors. SPRY3 plays an important role in regulating axon branching of motor neurons. Similarly, growth cones rely on calcium signaling to respond to guidance cues and adjust their behavior accordingly and it is critical for proper axon extension and guidance. Using calcium imaging in SH-SY5Y cells, we found that adding KCl to SH-SY5Y cells expressing SPRY3-GFP significantly altered the function of Cav 1.2, Cav 1.3, and Cav 2.2. SPRY3-GFP did not alter the levels of calcium when the L-type inhibitor nifedipine was added to SH-SY5Y cells, shows that SPRY3 interacts with Cav 1.2 and Cav 1.3. Cav2.2 is a prominent VGCC in IMR-32 cells, where calcium levels are not altered. Also, we found that adding BDNF to SH-SY5Y cells transfected with SPRY3-GFP shown no changes in intracellular calcium levels. Overall, this study suggests an interaction between SPRY3 and L-type VGCCs, which may be relevant to ASD pathogenesis. In a pilot study, there was no significant difference in the mRNA expression of FMR1-, a known ASD gene in SPRY3-GFP transfected SH-SY5Y cells compared to GFP transfected SH-SY5Y cells.
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Keywords
Autism , SPRY3 , Voltage-gated calcium channels
Citation
Bharatham Vijayaraghavan, S. 2023. A model of SPRY3 - VGCC interactions relating to autism. MRes Thesis, University College Cork.