Characterization of the cellular function of TBC1D7, independent of- and as the third component of the TSC complex
Final Report Abstract
Tuberous sclerosis complex (TSC) is caused by mutations in TSC1 or TSC2, which form a functional complex (the TSC complex), which is a critical negative regulator of mTORC1. Together with the TBC1D7 protein, the TSC1 and TSC2 proteins form a negative regulatory complex upstream of mTORC1, a master regulator of anabolic cell growth and proliferation, metabolism and autophagy. TBC1D7 is a ubiquitous core component of the TSC complex, important for the stability and GAP activity of the TSC complex toward Rheb. Germline loss of function mutations in the TBC1D7 gene have not been identified in TSC patients, but loss of TBC1D7 has been found to give rise to a megalencephaly syndrome associated with elevated mTORC1 signaling. Here, I aimed to understand the functions of TBC1D7, dependent and independent of the TSC complex, which will provide important new insights into the molecular defects contributing to the pathological features of TSC. I used state-of-the-art CRISPR/Cas9 genome editing technique to generate whole body knock-out mice. We discovered a gait and grip strength in KO mice at weaning which resembled an “early ageing” phenotype. The loss of Tbc1d7 did not affect mice in their body weight or overall size. The specific abnormalities of the hind limbs of KO mice promoted us to look at the overall brain development in this mouse model to assess the possibility of a neuromuscular phenotype. Tbc1d7 loss seems to lead to enhanced apoptosis in the cerebellum thereby possibly affecting the motor movement. On a molecular basis the loss of Tbc1d7 was found to cause an increase in soma size of primary neurons. Furthermore primary neurons without Tbc1d7 were unable to polarize and mature normally and showed multiple axons per cell. All of the neuronal phenotype could be rescued by Rapamycin, a clinically used mTORC1 inhibitor. Therefore the data suggests that the phenotypes found are caused Tbc1d7 loss in the TSC complex and is due to aberrant mTORC1 activation. We are currently working on investigating the exact cortical development in the KO mice as well as the role of Tbc1d7 in the different brain regions. We further need a deeper understanding of the two pools of Tbc1d7 to differentiate between mTORC1-associated effects of Tbc1d7 and independent effects.
