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. 2019 Feb 19;116(8):2977-2986.
doi: 10.1073/pnas.1812943116. Epub 2019 Feb 6.

Abnormal glycogen storage in tuberous sclerosis complex caused by impairment of mTORC1-dependent and -independent signaling pathways

Rituraj Pal  1   2 , Yan Xiong  3   2 , Marco Sardiello  1   2
Affiliations

Abnormal glycogen storage in tuberous sclerosis complex caused by impairment of mTORC1-dependent and -independent signaling pathways

Rituraj Pal et al. Proc Natl Acad Sci U S A. .

Abstract

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome that causes tumor formation in multiple organs. TSC is caused by inactivating mutations in the genes encoding TSC1/2, negative regulators of the mammalian target of rapamycin complex 1 (mTORC1). Diminished TSC function is associated with excess glycogen storage, but the causative mechanism is unknown. By studying human and mouse cells with defective or absent TSC2, we show that complete loss of TSC2 causes an increase in glycogen synthesis through mTORC1 hyperactivation and subsequent inactivation of glycogen synthase kinase 3β (GSK3β), a negative regulator of glycogen synthesis. Specific TSC2 pathogenic mutations, however, result in elevated glycogen levels with no changes in mTORC1 or GSK3β activities. We identify mTORC1-independent lysosomal depletion and impairment of autophagy as the driving causes underlying abnormal glycogen storage in TSC irrespective of the underlying mutation. The defective autophagic degradation of glycogen is associated with abnormal ubiquitination and degradation of essential proteins of the autophagy-lysosome pathway, such as LC3 and lysosomal associated membrane protein 1 and 2 (LAMP1/2) and is restored by the combined use of mTORC1 and Akt pharmacological inhibitors. In complementation to current models that place mTORC1 as the central therapeutic target for TSC pathogenesis, our findings identify mTORC1-independent pathways that are dysregulated in TSC and that should therefore be taken into account in the development of a therapeutic treatment.

Keywords: Akt; TSC; autophagy; glycogen; mTOR.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Abnormal elevation of glycogen levels due to dysregulation of the mTORC1/GSK3β axis in TSC. (A) Immunoblot analyses with lysates from MEFs. (B) MEFs were starved of serum (16 h) and treated with insulin (1 µM) for 15 min before the immunoblot analyses. (C and D) MEFs were grown in nutrient-rich media or starved with AAs before the immunoblot in C and immunofluorescence analyses using LAMP1 (green) and mTOR (red) antibodies in D. Bar, 50 μm. Bar diagrams represent percent colocalization (Mander’s coefficient) of mTOR and LAMP1 in, at least, 30 cells/conditions. (E and F) MEFs were treated with either DMSO or 300 nM rapamycin for 24 h before immunoblot and in vitro kinase assay in E and immunoblot analyses in F. GS in F stands for GS. l.e., long exposure; s.e., short exposure. (G) MEFs were treated as in E before the periodic acid Schiff (PAS) staining analyses. Bar, 100 μm in G. (H) Glycogen assay in Tsc2+/+ and Tsc2−/− MEFs. (I) Glycogen assay in Tsc2+/+ and Tsc2−/− MEFs grown in either nutrient-rich media or starved for GFs and AAs for 4 h. (J) PAS staining showing glycogen levels in human fibroblasts. Bar, 100 μm. (K) Human fibroblasts were treated with either DMSO or 300 nM rapamycin for 24 h before the PAS staining analyses. Bar, 100 μm. Experiments were performed with two to four replicates. Data represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 2.
Fig. 2.
Decreased autophagy elevates glycogen levels in TSC. (A and B) Immunoblot analyses with lysates from MEFs. (C) MEFs were treated with either DMSO or 300 nM rapamycin for 24 h before the immunoblot analyses. (D) Live-cell imaging of MEFs transiently transfected with GFP-LC3 followed by treatment as in C. The box plot represents absolute numbers of lipidated-LC3 puncta from at least 20 cells. Bar, 60 μm. (E) MEFs transiently transfected with GFP-RFP-P62 followed by treatment as in C. The box plot shows the percentage of yellow (GFP-RFP-positive) puncta from at least 15 cells. Bar, 40 μm. (F) MEFs were either grown in nutrient-rich media or starved of serum (GF) for 16 h or starved with AAs (4 h) before the immunoblot analyses. The line plot indicates the rate of LC3II degradation (LC3II/tubulin) upon AA starvation for 4 h. The bar diagrams show the rate of LC3I-to-LC3II conversion. (G) MEFs were grown in nutrient-rich media or starved with AAs or starved and treated with bafilomycin (160 nM) for 4 h before the PAS staining analyses. Bar, 120 μm. (H) Human fibroblasts were treated with either DMSO or 160 nM bafilomycin for 4 h before the PAS staining analyses. Bar, 120 μm. Experiments were performed with two to four replicates. Data represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 3.
Fig. 3.
Depletion of lysosomes in TSC. (A) Live-cell imaging of MEFs transiently transfected with GFP-RFP-LC3. The box plot represents the percentage of yellow (GFP-RFP-positive) puncta from at least 20 cells. Bar, 60 μm. (B) Immunoblot analyses with lysates from MEFs. (C) Lysotracker-red staining in MEFs. The box plot represents mean lysotracker fluorescence per cell from, at least, 100 cells in each condition. Bar, 40 μm. (D) MEFs were cultured before immunofluorescence labeling of endogenous LAMP1 (green) and DAPI (nucleus, blue). The box plot represents the mean LAMP1 fluorescence per cell from at least 50 cells in each condition. Bar, 40 μm. (E) MEFs were treated with either DMSO or 300 nM rapamycin for 24 h before the immunoblot analyses. (F) Immunoblot analyses of Tsc2−/− MEFs stably expressing TSC2-Flag. (G) PAS staining analysis of Tsc2−/− MEFs stably expressing TSC2-Flag. Bar, 120 μm. Experiments were performed with two to four replicates. Data represent means ± SEM. ***P < 0.001; N.S., not significant.
Fig. 4.
Fig. 4.
Excessive proteasomal degradation of LC3 and LAMP1/2 in TSC. (A and B) qRT-PCR analysis showing total mRNA in A and polyribosome-bound mRNA in B expression levels of Lamp1 and Map1lc3b in Tsc2−/− MEFs. Gene expression was normalized relative to the housekeeping gene Tubb. The dashed line indicates relative gene expression in Tsc2+/+ MEFs. Data represent means ± SEM. (C) MEFs were treated with either DMSO or 300 nM rapamycin for 24 h or 10 µM MG132 for 4 h before the immunoblot analyses. (D) MEFs were transiently transfected with GFP-LC3 and treated with either DMSO or 10 µM MG132 for 4 h before immunofluorescence assay. (E) MEFs were treated as in D before immunofluorescence assay. Representative images from n = 15 cells are shown in D and E where yellow or orange pixels indicate colocalization in the merged images. Bars, 80 μm in D and 40 μm in E. (F) qRT-PCR analysis showing mRNA expression changes in autophagy-lysosome genes in Tsc2−/− MEFs. Gene expression was normalized relative to the housekeeping gene ACTB (actin). The dashed line indicates relative gene expression in Tsc2+/+ MEFs. (G) Immunoblot analysis showing protein expression levels of autophagy-lysosome proteins in DMSO- or MG132-treated MEFs. (H) MEFs were treated with DMSO, 600 nM rapamycin, or 10 µM MG132 for 4 h (Top) or 160 nM bafilomycin for 4 h (Bottom) before the immunoblot analyses. Experiments were performed with two to four replicates. Data represent means ± SEM. *P < 0.05 and ***P < 0.001.
Fig. 5.
Fig. 5.
mTORC1-dependent and -independent regulation of autophagy in TSC patients. (A) The diagram shows the deletion identified in one TSC patient (TSC2Δex1-14). Human fibroblasts were treated with either DMSO or 300 nM rapamycin for 24 h before the immunoblot analyses. (B) Human fibroblasts were treated with either DMSO or 300 nM rapamycin for 24 h or 10 µM MG132 for 4 h before the immunoblot analyses. (C) The diagram shows the mutations harbored by two TSC patients (TSC2-H522T and TSC2-R1743Q). Immunoblot analyses show the expression of the indicated proteins in these patient-derived fibroblasts. (D and E) Human fibroblasts were treated with either DMSO or 300 nM rapamycin for 24 h in D and either DMSO or 10 µM MG132 for 4 h in E before the immunoblot analyses. Experiments were performed with two to four replicates. Data represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001; N.S., not significant.
Fig. 6.
Fig. 6.
Akt regulates glycogen levels through GSK3β and autophagy in TSC. (A) MEFs were starved of serum (16 h) followed by treatment with DMSO, MK2206 (10 µM), or rapamycin (300 nM) for 2 h before insulin stimulation (1 µM) for 15 min. (B) MEFs were treated with DMSO, rapamycin (300 nM), MK2206 (10 µM), or rapamycin + MK2206 for 24 h before the immunoblot analyses. (C) MEFs transiently transfected with GFP-LC3 followed by treatment with DMSO, rapamycin (300 nM), MK2206 (10 µM), or rapamycin + MK2206 for 24 h before live-cell imaging. The box plots represent absolute numbers of lipidated-LC3 puncta from at least 15 cells/conditions. Bar, 25 μm. (D) MEFs were treated as in C before the PAS staining analyses. Bar, 120 μm. (E) Human fibroblasts were treated as in C before the PAS staining analyses. Bar, 80 μm. (F) The model shows mTOR-dependent and -independent regulation of glycogen levels in TSC. Experiments were performed with two to three replicates. Data represent means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001.
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