Abstracts

Variants in TANC2 cause a rare neurodevelopmental disorder (TANC2-related disorder; TRD) associated with epilepsy, ASD, and intellectual disability. TRD is caused by haploinsufficiency of the TANC2 gene due to autosomal dominant loss-of-function variants. During brain development, TANC2 is most abundant in excitatory neural progenitor cells. The molecular function of TANC2 is poorly defined, however, it has been postulated that it has synaptic scaffolding, vesicle transport, or mTOR pathway regulatory roles. We sought to gain a better understanding of the function of TANC2 and the impact of patient-specific variants on disease phenotypes. We hypothesized that a loss of TANC2 would lead to dysregulation of cell motility due to an increase in mTOR pathway activity.

We used an in vitro approach by collecting patient-derived dermal fibroblast and generating TANC2 knockouts (KO) in mouse Neuro-2a (N2a) cells using CRISPR/Cas9. Changes in mTOR signaling were assayed by Western blots probing for phosphorylated S6 (Ser 240/244), phosphor 4E-BP1 (Thr 37/46), and phosphor-AKT (Ser 473). In N2a cells, neuronal process outgrowth and soma size was measured following retinoic acid-induced differentiation. In fibroblasts, we used live-cell confocal microscopy to track cell migration using a wound healing assay and leading-edge analysis. To define cell proliferation, we incubated, fixed, and stained fibroblasts for DAPI to visualize all nuclei and EdU to visualize proliferating cells. At least three representative images per well were acquired from each of the slides per condition. Image analysis and quantification of DAPI and EdU positive cells were conducted using Fiji/ImageJ software.

We observed no significant changes in mTOR signaling compared to corresponding controls in either N2a cells or fibroblasts. In addition, we observed no changes in soma size or process outgrowth- phenotypes typically associated with mTOR pathway hyperactivation. Fibroblast cell migration results showed that TANC2 variant cells migrate only in straight lines while control lines migrate along a circuitous route. EdU staining revealed decreased cell proliferation compared to control cells. Interestingly, there was considerable variability in cell proliferation rates among fibroblast variants within the same family cohort, suggesting potential influences on cell proliferation rate separate from the variant itself.

We observed no changes in mTOR pathway signaling but did observe changes in migration and cell proliferation in TANC2 variant cells. Interestingly, TANC2 variants are associated with microcephaly and therefore, changes in cell migration and cell division may be relevant to this phenotype.  Future studies will investigate the underlying mechanisms of cell migration and cell division changes and potential therapeutic avenues for TRD.