ABL inhibition activates TFEB and promotes cellular clearance in the lysosomal storage disorder Niemann-pick type C.
Type
Tesis DoctoradoAuthor
Contreras Soto, Pablo AndrésAbstract
Lysosomes are cellular organelles that play a fundamental role in cellular homeostasis because they are involved in different process such as: membrane repair, degradation and recycling of nutrients, macromolecules metabolism and autophagy. Deficiencies in lysosomal proteins promote the accumulation of different molecules in the organelle causing multiples lysosomal diseases, such as Niemann-Pick type C (NPC), which is characterized by lysosomal ...
Ver más
Lysosomes are cellular organelles that play a fundamental role in cellular homeostasis because they are involved in different process such as: membrane repair, degradation and recycling of nutrients, macromolecules metabolism and autophagy. Deficiencies in lysosomal proteins promote the accumulation of different molecules in the organelle causing multiples lysosomal diseases, such as Niemann-Pick type C (NPC), which is characterized by lysosomal cholesterol accumulation due to mutations in the genes encoding for the NPC1 and NPC2 proteins.
Interestingly, in the last time the transcription factor EB (TFEB) has been described as the master regulator of lysosomal biogenesis and gene expression related to autophagy. The activity and nuclear translocation of TFEB depends on its phosphorylation status in serine 211 and 142, which are regulated by the serine/threonine kinase mTORC1. At basal conditions, TFEB is phosphorylated in these serines and is retained in the cytoplasm due to its attachment to the cytosolic chaperone 14-3-3.
Previously, our lab described that the tyrosine kinase ABL is activated in NPC disease. The Inhibition of this kinase using Imatinib, an ABL specific inhibitor, prevents the neuronal death in the cerebellum and improves the locomotor function and survival of the NPC mice.
In addition, it has been described that the use of pharmacological ABL inhibitors induces the autophagy flux and an increase in the lysosomal levels. Nevertheless, the cellular mechanism by which ABL inhibition induces these changes are still unknown. Considering this data, the hypothesis of this thesis is the following: “ABL signaling promotes TFEB cytoplasmic localization, inhibiting cellular clearance and contributing to the pathogenesis of the lysosomal storage disease Niemann-Pick type C”.
To test our hypothesis, first we evaluated TFEB nuclear translocation by the high-content screening technique, using different ABL inhibitors. We found that ABL inhibition by these inhibitors promotes TFEB nuclear translocation. The same results were obtained analyzing nuclear-cytoplasmic fractions, as well as evaluating endogenous TFEB cellular localization. In addition, we observed the same results; TFEB nuclear translocation and an increase in the expression of TFEB target genes, using a siRNA against ABL1. These results indicate that ABL inhibition activates TFEB.
Then, we evaluated TFEB downstream cell biological process in conditions of ABL inhibition. We observed that ABL inhibition increases the autophagy flux in the stable H4 LC3-GFP-mRFP cell line. In addition, the results show that ABL inhibition, using Imatinib or a siRNA against ABL1 increases Lamp1 lysosomal protein levels and lysotracker staining. In addition, we found that under ABL inhibition lysosomes are attached to the plasma membrane,, suggesting an increase in lysosomal exocytosis. Thus, our results support the idea that the increase in autophagy flux, quantity of lysosomes and lysosomal exocytosis, all of them biological process that are regulated by TFEB, are downstream events of ABL inhibition.
On the other hand, we evaluated if TFEB activation induced by ABL inhibition is dependent or independent of mTORC1. To do that, we evaluated the phosphorylation levels in TFEB serine 142 and 211, using specific antibodies for them. We found that ABL inhibition does not change the levels of phosphorylated serine 142 and decreases the levels of phosphorylated serine 211 on TFEB. Due to these results, we then evaluated the activity of mTORC1 following the phosphorylation of an mTORC1 target protein. We found that the ABL inhibitors, Imatinib or Nilotinib, do not change the activity of mTORC1. These results demonstrate that ABL inhibition activates TFEB independent of mTORC1.
Then, we assessed whether the tyrosine kinase ABL phosphorylates TFEB on tyrosine. Using TFEB immunoprecipitation and an in vitro phosphorylation assay, we found that the activity of ABL was enough to promote an increase in the TFEB tyrosine phosphorylation levels. Concordantly, site directed mutagenesis showed that TFEB tyrosine 173 is relevant for maintaining TFEB cytoplasmic localization and impact on the S211 phosphorylation levels. These results demonstrate that ABL phosphorylates TFEB on tyrosine and these phosphorylations are important to retain TFEB in the cytoplasm.
Subsequently, because ABL phosphorylates TFEB on tyrosine and its inhibition promotes TFEB nuclear translocation, we asked if this signaling pathway could have a role in cellular clearance, a phenomena that is regulated by TFEB. The results show that in different models in which cholesterol accumulation was promoted, ABL inhibition promotes cellular clearance cholesterol accumulated in lysosomes. This clearance effect was not observed in TFEB knock out cells, demonstrating that ABL inhibition promotes cellular clearance in a TFEB dependent way.
The next step was to evaluate if the ABL/TFEB signaling pathway was relevant in the NPC lysosomal disease, which is characterized by lysosomal cholesterol accumulation. Our data show that in fibroblast from NPC patients, ABL kinase is active, and its inhibition promotes a reduction in cholesterol accumulation and TFEB nuclear translocation. These results correlates with an increase in Lamp1 and lysotracker levels and in TFEB target gene expression, measured in fibroblast from NPC patients treated with the ABL inhibitors, Imatinib and Nilotinib.
Finally, we corroborated using in vivo models of the disease, that the inhibition or absence of ABL promotes a reduction in cholesterol accumulation and TFEB translocation to the nucleus in the purkinje neurons.
In summary, we found a novel-signaling pathway, which involves the ABL kinase and TFEB. The inhibition of ABL promotes TFEB nuclear translocation, increasing lysosomal gene expression and causing an increase in cellular clearance in NPC models. Therefore, inhibition of ABL/TFEB signaling pathway arises as a therapeutic target not only for NPC disease, but also for different diseases that are characterized by lysosomal dysfunction.
Ver menos
Date de publicación
2019Academic guide
Alvarez Rojas, Alejandra
Zanlungo, Silvana
Metadata
Show full item recordThe following license files are associated with this item: