RésuméHereditary spastic paraplegias (SPGs) are characterized by lower (and sometimes upper) limb spasticity and weakness. Interestingly, a subset of the associated genes code for endolysosomal proteins that participate in the recycling of mannose 6-phosphate receptors (M6PRs) from endosomes to the trans-Golgi
network (TGN). These receptors transport acid hydrolases bearing M6P signals to lysosomes. Previous work conducted by our team highlighted that maspardin, the protein deficient in SPG21 cases, is also located on endosomes and lysosomes. However, the intracellular function of maspardin (coded by the
SPG21 gene) remains unknown. Preliminary results revealed that SPG21 HeLa KO cells hypersecrete procathepsin D (an acid hydrolase precursor), suggesting that maspardin could be involved in M6PRs trafficking. Our team also found an increase of the nuclear/cytosolic ratio of the transcription factor TFEB
in those cells. TFEB is known to promote transcription of lysosomal and autophagy genes upon lysosomal stress. The underlying cause and molecular pathways leading to TFEB delocalization in SPG21 KO cells remains however elusive. In the present study, we investigated directly whether the subcellular trafficking of cation independent (CI)-M6PRs depends on maspardin and analyzed several regulation pathways of TFEB in SPG21 KO HeLa cells.
The study of the expression level of CI-M6PRs and the analysis of their intracellular distribution by immunofluorescence did not revealed any difference between control and SPG21 KO cells. A dynamic assay showed that CI-M6PRs recycling from endosomes to the TGN is unaltered, and so was the amount of
CI-M6PRs detected in clathrin-coated vesicles. Hence, either maspardin is not involved in CI-M6PRs recycling or other recycling pathways compensate for the maspardin deficiency.
Next, we analyzed the co-localization (by immunofluorescence) and association (by immunoprecipitation) of TFEB with 14-3-3 proteins (i.e. proteins that trap phosphorylated TFEB in the cytosol under nutrient rich condition) in control and KO cells. We observed a decrease of interaction of TFEB with 14-3-3 in the
KO cells suggesting that there is either less phosphorylation of TFEB (possibly by the mTOR kinase) in these cells, that phospho-TFEB is rapidly degraded, or that a larger pool of phospho-TFEB is dephosphorylated when maspardin is deficient. Our team found no mTOR deregulation in the KO cells.
Here, we used MG132 or Bortezomib proteasome inhibitors to test a putative phospho-TFEB degradation in the KO cells. This was not conclusive as these treatments resulted in TFEB dephosphorylation. Next, we tested whether the cytosolic enzyme calcineurin is hyperactivated (or more abundant) in the KO cells. Overall calcineurin levels were unchanged but an overnight treatment with the calcineurin inhibitor (FK506) resulted in a modest increase of phospho-TFEB levels, suggesting that a putative calcineurin hyperactivity in the KO cells is a track worth pursuing.
|la date de réponse||18 janv. 2021|
|Superviseur||Marielle Boonen (Promoteur)|