Abstracts

This is a Late Breaking abstract

Rationale: TANC2 is a gene that encodes the TANC2 (tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2) protein–a post-synaptic density interacting scaffold protein. Variants in TANC2 have been associated with neurodevelopmental disorders such as epilepsy, intellectual disability, and autism. Recently, TANC2 was found to be a negative regulator of mammalian target of rapamycin (mTOR) signaling and Tanc2 heterozygous mice had abnormal and temporally variable changes in mTOR signaling, impaired synaptic plasticity, anxiety, and depression. These phenotypes were normalized with mTOR inhibitor treatment. However, the extent of mTOR dysregulation and whether or not other signaling cascades are involved has not been fully defined. Further, the molecular mechanism underlying TANC2 variants leading to epilepsy and other neurodevelopmental disorders are poorly understood. Therefore, to investigate the role of Tanc2 in mTOR pathway signaling we created a Tanc2 knockout N2a cell line. We hypothesize that KO of Tanc2 will result in increased phosphorylation of multiple mTORC1 substrates as well as hallmarks of mTOR pathway hyperactivation including, increased dendritic outgrowth, and increased cell size. _x000D_
Methods: CRISPR/Cas9 knockout (KO) cell lines were created by targeting exon 26 of Tanc2 using Neuro2a cells (N2aC). Wildtype and scramble N2aC were used in these experiments as controls. Tanc2 knockout was validated through next-generation sequencing, qPCR, and Western blot. Changes in cell signaling pathways were assayed using Western assay and included antibodies targeting specific phosphotargets within the mTOR pathway (ribosomal S6 protein (240/244; PS6); 4E-binding protein 1 (37/46; 4E-BP1)). Changes in dendritic arborization was assessed by immunocytochemistry (ICC) probing for MAP2 and a fluorescent secondary antibody. Cells were imaged on a spinning disk confocal microscope. Changes in dendritic arborization were quantified using ImageJ FIJI. Cell size was assessed by direct measurement of the diameter of the soma in digital images. All statistical analysis was performed using an ANOVA._x000D_
Results: Tanc2 KO cells were validated using next-generation sequencing, qPCR, and Western assay which revealed sequence misalignments, significantly decreased mRNA expression, and loss of Tanc2 proteins, respectively. Tanc2 knockout cells displayed significant differences in the levels of phosphorylation of proteins downstream of mTORC1 compared to wildtype and scramble cells (n=50; p< 0.05). Tanc2 KO cells also displayed statistically significant differences in cell soma diameter and dendritic outgrowth compared to wildtype and scramble controls (n=50; p< 0.05)._x000D_