TBC1D20 deficiency induces Sertoli cell apoptosis by triggering irreversible endoplasmic reticulum stress in mice.

Male 'blind sterile' mice with the causative TBC1 domain family member 20 (TBC1D20) deficiency are infertile with excessive germ cell apoptosis and spermatogenesis arrest at the spermatid stage. Sertoli cells are characterised as 'nurse cells' essential for normal spermatogenesis, but the role and corresponding molecular mechanisms of TBC1D20 deficiency in Sertoli cells of mice are not clear to date. In the present study, the histopathology of the testis and Sertoli cell proliferation and apoptosis were determined, and the corresponding molecular mechanisms were investigated by western blotting. Our data showed that TBC1D20 exhibits a testis-abundant expression pattern, and its expression level is positively associated with spermatogenesis. TBC1D20 is assembled in the Golgi and endoplasmic reticulum and is widely expressed by various germ cell subtypes and Sertoli cells. TBC1D20 deficiency in Sertoli cells led to an excessive apoptosis ratio and G1/S arrest. The increased apoptosis of TBC1D20-deficient Sertoli cells resulted from caspase-12 activation. TBC1D20-deficient Sertoli cells had an abnormal Golgi-endoplasmic reticulum structure, which led to endoplasmic reticulum stress, resulting in cell cycle arrest and excessive apoptosis. It suggested that TBC1D20 deficiency triggers irreversible endoplasmic reticulum stress resulting in G1/S arrest and excessive apoptosis in TBC1D20-deficient Sertoli cells, and TBC1D20 deficiency in Sertoli cells may also contribute to the infertility phenotype in 'blind sterile' male mice.

TBC1D20 mediates autophagy as a key regulator of autophagosome maturation.

In humans, loss of TBC1D20 (TBC1 domain family, member 20) protein function causes Warburg Micro syndrome 4 (WARBM4), an autosomal recessive disorder characterized by congenital eye, brain, and genital abnormalities. TBC1D20-deficient mice exhibit ocular abnormalities and male infertility. TBC1D20 is a ubiquitously expressed member of the family of GTPase-activating proteins (GAPs) that increase the intrinsically slow GTP-hydrolysis rate of small RAB-GTPases when bound to GTP. Biochemical studies have established TBC1D20 as a GAP for RAB1B and RAB2A. However, the cellular role of TBC1D20 still remains elusive, and there is little information about how the functional loss of TBC1D20 causes clinical manifestations in WARBM4-affected children. Here we evaluate the role of TBC1D20 in cells carrying a null mutant allele, as well as TBC1D20-deficient mice, which display eye and testicular abnormalities. We demonstrate that TBC1D20, via its RAB1B GAP function, is a key regulator of autophagosome maturation, a process required for maintenance of autophagic flux and degradation of autophagic cargo. Our results provide evidence that TBC1D20-mediated autophagosome maturation maintains lens transparency by mediating the removal of damaged proteins and organelles from lens fiber cells. Additionally, our results show that in the testes TBC1D20-mediated maturation of autophagosomes is required for autophagic flux, but is also required for the formation of acrosomes. Furthermore TBC1D20-deficient mice, while not mimicking severe developmental brain abnormalities identified in WARBM4 affected children, display disrupted neuronal autophagic flux resulting in adult-onset motor dysfunction. In summary, we show that TBC1D20 has an essential role in the maturation of autophagosomes and a defect in TBC1D20 function results in eye, testicular, and neuronal abnormalities in mice implicating disrupted autophagy as a mechanism that contributes to WARBM4 pathogenesis.