Abstracts

Rationale: Variants in TANC2 can cause a range of neurodevelopmental disorders such as intellectual disability, autism spectrum disorder, and developmental delays. Further, many individuals with TANC2 variants develop early onset generalized seizures which are typically medically refractory. While the function of TANC2 remains unclear, it likely plays a critical role in brain development as expression is highest in developing and mature brain cells and Tanc2 KO in rodents is embryonically lethal. As previous work characterized Tanc2 as a potential mTOR inhibitor, we hypothesized that a reduction of TANC2 would result in mTOR pathway hyperactivation, a hallmark phenotype of mTORopathies. Previously, we found no correlation between Tanc2 and mTOR pathway activation in murine cells. Thus, to determine whether our findings were relevant to human disease, we aim to define the role of TANC2 on mTOR signaling in human cells. We hypothesize that reduced TANC2 in human cells does not impact mTOR signaling, mTOR functional localization, or cell morphology.

Methods: Patient-derived dermal fibroblasts were collected by skin biopsy and controls were obtained commercially. Human TANC2 knockout (KO) SK-N-SH cells were created via CRISPR/Cas9, with wildtype (WT) and scramble cells used as controls. TANC2 levels were analyzed via qPCR and immunocytochemistry (ICC). Amino acid starvation or rapamycin were used as mTOR inhibitor groups compared to baseline. SK-N-SH cells were treated for 7 days with retinoic acid to induce neuronal-like differentiation. The extent of mTOR pathway activity was quantified by Western Blot (WB) of phosphorylated Ser240/244 and Thr37/46 of ribosomal protein S6 and 4E-binding protein 1 (PS6 and P4E-BP1), respectively. Colocalization of mTOR and TANC2 and cell morphology were measured via ICC, confocal microscopy, and Image J analysis. Fibroblast cell size and SK-N-SK soma size, primary process number, and process length were measured with Vimentin and MAP2, respectively.

Results: Relative to controls, we observed reduced TANC2 in both patient fibroblasts and TANC2 KO SK-N-SH cells by qPCR and ICC (p< 0.05) yet no significant difference in mTOR pathway activity by WB of PS6 and P4E-BP1 (n= 3 blots per group). Additionally, ICC did not reveal colocalization between mTOR and TANC2 nor a significant difference in the measured cell morphological features.