The role of the lysosome and transcription factor EB in tuberous sclerosis complex
Nidhiry, Anna S.
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Tuberous Sclerosis Complex (TSC) is a rare, autosomal dominant genetic disease that results from the loss-of-function mutations of either the TSC1 or TSC2 genes. It is a multisystemic disorder with manifestations in several organs including the lungs, kidneys, brain, skin, and heart. Loss of either TSC1 or TSC2 causes hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway resulting in cell proliferation and the continuous activation of multiple anabolic pathways that lead to tumor growth. Transcription factor EB (TFEB) is one of the many downstream targets of the mTORC1 pathway and is a master transcriptional regulator of lysosomal biogenesis. In the classical paradigm of TFEB-mTORC1 interaction, TFEB is negatively regulated by mTORC1. Interestingly, TFEB is upregulated and fully functional in Tsc1- and Tsc2- deficient, mTORC1 hyperactivated cells suggesting an alternate regulation involved in the context of TSC. The objective of this research was to investigate how TFEB upregulation and resulting lysosomal dysregulation in Tsc1- and Tsc2-deficient cells drives the pathogenesis of Tuberous Sclerosis Complex. By western blot analysis, elevated levels of TFEB were detected in Tsc1- and Tsc2-deficient cells compared to Tsc1- and Tsc2- expressing cells and multiple lysosomal proteins also showed increased expression in these cell lines. In Tsc1- and Tsc2-expressing MEFs, TFEB was phosphorylated and retained in the cytoplasm. Immunofluorescence imaging of TFEB in Tsc1- and Tsc2-deficient cells demonstrated nuclear localization. This was in contrast to previous studies, in which TFEB was retained in the cytoplasm upon mTORC1 activation. Immunohistochemistry staining of TFEB and the lysosomal protein, NPC1 (NPC Intracellular Cholesterol Transporter 1), in human TSC-associated renal cell carcinoma (RCC) also showed increased staining of TFEB and NPC1 in RCC compared to normal kidney tissue. This histological finding was confirmed in two mouse models of renal TSC, TSC2 +/- AJ mice and KSP-CreERT2 Tsc2fl/fl mice, which revealed strong expression of NPC1 in cyst-lining cells, specifically in the apical region, suggesting increased lysosome number and activity as a contributing factor to cystogenesis. These results suggest a novel mechanism in the development of Tuberous Sclerosis Complex through the dysregulation of lysosomal activity. Further investigation into the mechanism of nuclear localization of TFEB and its upregulation in Tsc1- and Tsc2- deficient cells may provide insight into the pathogenesis of the disease and could indicate a new therapeutic target for treating TSC and its associated disorders.