Abnormal Glycogen Storage in Tuberous Sclerosis Complex Caused by Impairment of Mtorc1-Dependent and -Independent Signaling Pathways

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

Rituraj Pala,b,1, Yan Xionga,b, and Marco Sardielloa,b,1

aJan and Dan Duncan Neurological Research Institute, Texas Children’s Hospital, Houston, TX 77030; and bDepartment of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030

Edited by Kun-Liang Guan, University of California, San Diego, La Jolla, CA, and accepted by Editorial Board Member Christopher K. Glass December 31, 2018 (received for review July 26, 2018) Tuberous sclerosis complex (TSC) is an autosomal dominant glycogen synthesis and degradation and is disrupted in multiple syndrome that causes tumor formation in multiple organs. TSC is diseases (7, 8). Altered glycogen homeostasis in astrocytes is caused by inactivating mutations in the genes encoding TSC1/2, causally linked to mild and severe seizure disorders in the epi- negative regulators of the mammalian target of rapamycin lepsy family of brain disorders, such as Lafora disease (9, 10). complex 1 (mTORC1). Diminished TSC function is associated with Impaired glycogen metabolism is also a critical component of excess glycogen storage, but the causative mechanism is un- tumor formation (8). TSC is characterized by tumor formation in known. By studying human and mouse cells with defective or multiple organs and most often manifests with severe epileptic absent TSC2, we show that complete loss of TSC2 causes an seizures (1), indicating overlap with the outcomes of impaired increase in glycogen synthesis through mTORC1 hyperactivation glycogen homeostasis. Previous findings have associated hyper- and subsequent inactivation of glycogen synthase kinase 3β activation of mTORC1 and excess glycogen storage in cells from (GSK3β), a negative regulator of glycogen synthesis. Specific TSC patients and in a mouse model of TSC (11). Glycogen TSC2 pathogenic mutations, however, result in elevated glycogen synthesis requires several enzymatic reactions, including elon- levels with no changes in mTORC1 or GSK3β activities. We identify gation of nascent glycogen chains by the action of glycogen CELL BIOLOGY mTORC1-independent lysosomal depletion and impairment of synthase (GS) (12, 13). Insulin triggers inhibitory phosphoryla- autophagy as the driving causes underlying abnormal glycogen tion of glycogen synthase kinase-3 (GSK3α/β), leading to de- storage in TSC irrespective of the underlying mutation. The defec- phosphorylation and activation of GS (12, 14). In the absence of tive autophagic degradation of glycogen is associated with abnor- a functional TSC complex, GSK3β is phosphorylated and inac- mal ubiquitination and degradation of essential proteins of the tivated by the mTORC1 substrate S6K1, and various studies autophagy-lysosome pathway, such as LC3 and lysosomal associ- have shown evidence of aberrant phosphorylation of GSK3β ated membrane protein 1 and 2 (LAMP1/2) and is restored by the in human and animal tissues with deficient TSC (14, 15). combined use of mTORC1 and Akt pharmacological inhibitors. In mTORC1 also inhibits the autophagy initiator ULK1 through complementation to current models that place mTORC1 as the direct phosphorylation; mTORC1 hyperactivity therefore leads central therapeutic target for TSC pathogenesis, our findings iden- to decreased autophagosome formation and autophagic im- tify mTORC1-independent pathways that are dysregulated in TSC pairment (16), a mechanism that could contribute to glycogen and that should therefore be taken into account in the development of a therapeutic treatment. Significance TSC | mTOR | glycogen | autophagy | Akt Tuberous sclerosis complex (TSC) is a genetic disease characterized by tumor formation in multiple organs. The identification of uberous sclerosis complex (TSC), an autosomal dominant dysregulated cellular processes in TSC is necessary for the selec- disorder that is caused by loss-of-function mutations in either T tion of possible therapeutic interventions. The current models TSC1 (encoding TSC1, also known as hamartin) or TSC2 place impaired mTORC1 signaling at the core of TSC pathogene- (encoding TSC2 or tuberin), is characterized by a wide spec- sis. In this study, we identify an mTORC1-independent pathway trum of clinical manifestations in multiple organs including that drives excess glycogen storage in TSC via impaired the skin, brain, eyes, lungs, heart, and kidneys (1, 2). TSC1 and autophagic degradation of glycogen caused by defects in the TSC2 form the TSC complex, which functions as a GTPase- autophagy-lysosome system. Importantly, we find that the activating protein toward the small GTPase Rheb (3). Rheb is combined use of mTORC1 and Akt pharmacological inhibitors an essential positive regulator of mTORC1, a complex that co- restore glycogen homeostasis. Our findings uncouple mTORC1- ordinates several signaling pathways to regulate cell metabo- dependent from mTORC1-independent processes that are lism (4). In conditions of abundance of amino acids (AAs), dysregulated in TSC and pinpoint multiple pharmacologically mTORC1 translocates to the lysosomal surface where it interacts actionable entry points that could be leveraged to develop with Rheb, which stimulates the kinase activity of mTORC1 (4). a therapeutic treatment. In response to growth factors (GFs), such as insulin, Akt phosphorylates TSC2, leading to rapid dissociation of the TSC Author contributions: R.P. and M.S. designed research; R.P. and Y.X. performed research; complex from Rheb and resulting in Rheb conversion from an R.P. and M.S. analyzed data; and R.P. and M.S. wrote the paper. inactive (GDP-bound) to an active (GTP-bound) state, finally The authors declare no conflict of interest. activating mTORC1 (3). In the absence of TSC2, mTORC1 is This article is a PNAS Direct Submission. K.-L.G. is a guest editor invited by the hyperactive and insensitive to GF stimulation while remaining Editorial Board. responsive to changes in AA levels (3). Hyperactivation of Published under the PNAS license. mTORC1 is deemed as a major force driving TSC pathogenesis 1To whom correspondence may be addressed. Email: [email protected] or [email protected]. caused by inactivating mutations in TSC2 (5, 6). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Glycogen is a critical source of energy supply in cells. Glyco- 1073/pnas.1812943116/-/DCSupplemental. gen homeostasis is regulated by opposing pathways governing Published online February 6, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1812943116 PNAS | February 19, 2019 | vol. 116 | no. 8 | 2977–2986 Downloaded by guest on September 29, 2021 accumulation in TSC via impaired clearance of glycogen by the cells with the glycogen-degrading enzyme diastase (22) com- autophagy-lysosome pathway. How mTORC1 dysregulation leads pletely eliminated the PAS signal, confirming its specificity in to disruption of glycogen homeostasis and whether mTORC1- detecting glycogen (SI Appendix, Fig. S1F). As expected, trans- independent mechanisms also contribute to impaired glycogen fection of constitutively active GSK3β resulted in a significant metabolism are questions that still remain unanswered. decrease in glycogen levels compared with transfection of WT In the present study, we report that aberrant glycogen storage GSK3β (SI Appendix, Fig. S1G). Together, these results indicate in TSC is caused by impairment of mTORC1-GSK3β-dependent that the mTORC1/GSK3β axis plays a critical role in glycogen and -independent pathways, depending on the specific mutation synthesis in the absence of TSC2. We also checked whether in- in the TSC2-encoding gene. We show that key proteins of the creased glycogen levels could be due to decreased expression of autophagy-lysosome pathway are targeted to proteasomal deg- glycogen phosphorylase (GP), which breaks down cytosolic gly- radation in TSC cells and that this causes lysosomal depletion cogen and functions as the rate-limiting enzyme for glycogenol- and autophagic impairment. Finally, we show that stimulation of ysis (23). Real-time qPCR and immunoblotting showed that GP autophagy by modulation of mTOR-dependent and -independent mRNA and protein levels were significantly up-regulated in − − + + pathways synergistically promotes the clearance of excess glycogen Tsc2 / MEFs compared with Tsc2 / MEFs, therefore ruling in TSC cells. These results unveil the unanticipated involvement of out a role for GP in intracellular accumulation of glycogen in mTOR-independent pathways in impaired regulation of cell me- TSC (SI Appendix, Fig. S1 H and I). GF starvation was unable to − − tabolism in TSC and identify a possible strategy of pharmacological lower glycogen levels in Tsc2 / MEFs to levels comparable to + + manipulation to improve the aberrant storage of glycogen. Tsc2 / MEFs, consistent with mTORC1 insensitivity to GFs (SI Appendix, Fig. S1J). As an additional approach to measuring Results intracellular glycogen levels, we used a glycogen assay kit (24). Abnormal Elevation of Glycogen Levels Due to Dysregulation of Consistent with the PAS staining results, the glycogen assay − − Glycogen Synthesis via the mTORC1/GSK3β Axis in TSC. To examine showed a significant increase in glycogen levels in Tsc2 / MEFs + + mTORC1 regulation of GSK3β activity and glycogen metabolism compared with the Tsc2 / MEFs; in both cell lines, glycogen in TSC, we first analyzed the mTORC1 pathway in TSC2-null levels were significantly decreased upon GF and AA starvation − − mouse embryonic fibroblasts (Tsc2 / MEFs) under various (Fig. 1 H and I). We also observed elevated glycogen levels in conditions. We found a significant increase in basal mTORC1 three patient-derived TSC fibroblast lines (Fig. 1J and SI Ap- − − + + activity in Tsc2 / MEFs compared with wild-type (WT) (Tsc2 / ) pendix, Fig. S1K). Rapamycin treatment or GF and AA starva- MEFs as measured by phosphorylation levels of mTORC1 sub- tion decreased glycogen levels in all TSC fibroblast lines (Fig. 1L strates 4E-BP1 and S6K1 (and its substrate S6) (Fig. 1A). Insulin and SI Appendix, Fig. S1L). Together, these results demonstrate + + stimulation of GF-starved Tsc2 / MEFs activated the Akt- abnormal elevation of glycogen levels due to dysregulation of − − mTORC1 pathway, whereas GF-starved Tsc2 / MEFs showed glycogen synthesis via the mTORC1/GSK3β axis in TSC. insulin-mediated activation of Akt but resistance to activation of mTORC1 (Fig. 1B). Consistent with earlier studies (17), mTORC1 Impairment of Autophagy Leads to Excess Glycogen Storage in TSC. − − was still sensitive to AA availability in Tsc2 / MEFs even though to Owing to the role of mTORC1 as a major repressor of autophagy + + a lower degree than in Tsc2 / MEFs (Fig. 1C). Confocal micros- (25), we investigated the mTORC1-dependent autophagy regu- − − copy showed a significant increase in mTOR localization at lyso- latory pathway in TSC mutant cells. In Tsc2 / MEFs we ob- − − + + somes in Tsc2 / MEFs compared with Tsc2 / MEFs (Fig. 1D). served a significant increase in mTORC1-mediated inhibitory This was confirmed by a decreased dissociation of Raptor, an phosphorylation of the autophagy initiating kinase ULK1 and a mTORC1 subunit (18), from the heavy membrane-bound frac- reduction in LC3I-to-LC3II conversion (a readout of autopha- − − + + tion in Tsc2 / MEFs compared with Tsc2 / MEFs in response gosome formation) (Fig. 2A). Consistent with the autophagy to AA removal (SI Appendix, Fig. S1A), whereas no significant inhibition, we observed an accumulation of the autophagosome changes were observed in the levels of Raptor in the total cell substrate p62/SQSTM1, a marker of autophagic flux (26), in − − homogenate (SI Appendix, Fig. S1B). Thus, the partial resistance Tsc2 / MEFs (Fig. 2B). Treatment with rapamycin increased of mTORC1 to AA starvation due to loss of TSC2 could be the LC3II/LC3I ratio but not total LC3 levels (Fig. 2C). To explained by mTORC1 retention at lysosomes where it remains perturb mTORC1 activity genetically, we used RNA interference activated. to knockdown Raptor with two different shRNAs targeting the Next, we investigated the impact of hyperactive mTORC1 on Raptor gene as described in previous studies (27, 28). Knock- GSK3β activity. In vitro kinase assays showed that endogenous down of Raptor showed a partial but significant increase in − − − − S6K1 immunoprecipitated from GF-starved Tsc2 / MEFs but LC3II levels in Tsc2 / MEFs (SI Appendix, Fig. S2A). Trans- + + not Tsc2 / MEFs could phosphorylate recombinant GSK3β on fection of a GFP-LC3 vector to visualize autophagic vesicles − − S9, and this phosphorylation was significantly inhibited by cell showed decreased GFP-LC3-positive puncta in Tsc2 / MEFs + + pretreatment with the mTORC1 inhibitor rapamycin (19) (Fig. compared with Tsc2 / MEFs; rapamycin treatment resulted in − − 1E). In Tsc2 / MEFs, mTORC1-mediated inhibitory phos- an increased number of autophagic vesicles in both cell lines, but − − phorylation of GSK3β resulted in dephosphorylation of GS at the number of autophagic vesicles in Tsc2 / MEFs remained + + S641 and thus activation of GS, which was partially inhibited by lower compared with Tsc2 / MEFs (Fig. 2D), suggesting that a − − pretreatment with rapamycin (Fig. 1F). Tsc2 / MEFs stably mTORC1-independent pathway is also involved in the impair- − − transfected with a constitutively active form of GSK3β (HA- ment of autophagy in Tsc2 / MEFs. Next, we tested the effect of GSK3β-S9A) (20) showed a significant increase in the phos- rapamycin-induced autophagy on p62 accumulation by using a phorylation levels of GS at S641 compared with cells transfected tandem GFP-RFP-p62 construct (29), which emits red fluores- with WT GSK3β (HA-GSK3β-WT) (SI Appendix, Fig. S1C). cence from mature autophagolysosomes (GFP is quenched in Interestingly, pretreatment with rapamycin increased GS phos- the low pH environment of the lysosome) and yellow fluores- phorylation in cells transfected with WT GSK3β but did not cence when autophagosomes are unable to fuse with the lyso- − − further increase GS phosphorylation in cells transfected with somes. As expected, Tsc2 / MEFs showed an increase in yellow + + constitutively active GSK3β (SI Appendix, Fig. S1D). Consistent puncta compared with the Tsc2 / MEFs, which was significantly with the increased GS activity, PAS staining (21) showed in- decreased with rapamycin treatment (Fig. 2E). − − + + creased glycogen levels in Tsc2 / MEFs compared with Tsc2 / AA starvation resulted in a significant increase in the clearance + + − − MEFs, which was significantly diminished by rapamycin treat- of LC3I and LC3II in both Tsc2 / and Tsc2 / MEF lines, which ment (Fig. 1G and SI Appendix, Fig. S1C). Treatment of fixed was blocked by treatment with bafilomycin, a potent late-stage

2978 | www.pnas.org/cgi/doi/10.1073/pnas.1812943116 Pal et al. Downloaded by guest on September 29, 2021 autophagic flux inhibitor (SI Appendix, Fig. S2B) (26). Addi- levels (SI Appendix, Fig. S2D), suggesting that autophagic flux tionally, immunoblot analyses showed clearances of p62 in re- does not alter glycogen synthesis or cytosolic glycogen degrada- + + − − sponse to AA removal in both Tsc2 / and Tsc2 / MEF lines (SI tion. Together, these results confirm the AA starvation-mediated + + − − Appendix, Fig. S2C). Tsc2 / and Tsc2 / cells treated with increase in autophagic flux. We found that, although AA star- bafilomycin did not show any significant changes in GS or GP vation increased autophagic flux, GF starvation promoted

+/+ -/- A B Tsc2 Tsc2 C +/+ +/+ -/- -/- Tsc2 Tsc2 sc2 - + - + Insulin T Tsc2 + - + - Amino acids (AA) TSC2 TSC2 TSC2 P-S6K1(T389) P-Akt(S473) P-S6K1(T389) T-S6K1 T-Akt P-4E-BP1(S65) P-S6K1(T389) T-S6K1 T-4E-BP1 T-S6K1 P-4E-BP1(T37/46) T-4E-BP1 P-S6(S240/244) P-S6(S240/244) Tubulin T-S6 T-S6 P18 P-4E-BP1(S65) Tubulin T-4E-BP1 +/+ -/- Tubulin Tsc2 Tsc2 + + + + ATP +/+ -/- - + - + Rapa D E Tsc2 Tsc2 -AA Lysate TSC2 DAPI LAMP1 mTOR Merge - + - + Rapamycin (Rapa) P-GSK3 (s.e.) TSC2

+/+ P-GSK3 (l.e.)

P-GSK3 S9) CELL BIOLOGY sc2 GSK3 T-GSK3 T

T-GSK3 / Recombinent

Lysate P-S6K1(s.e.) -/- P-S6(S240/244) IgG

Tsc2 T-S6 P-S6K1(l.e.) IgG Tubulin

S6K1 IPs T-S6K1 IgG

F +/+ -/- G H I Tsc2 Tsc2 +/+ -/- Tsc2 Tsc2 - + - + Rapa TSC2 DMSO DMSO Rapa P-GS (S641) T-GS P-S6 T-S6 Tubulin

DMSO Rapa J K

TSC2-WT TSC2-H522T TSC2-H522T TSC2- R1743Q TSC2-R1743Q ∆ex1-14 TSC2 ∆ ex1-14 TSC2

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.GSinF 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 μminG.(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.

Pal et al. PNAS | February 19, 2019 | vol. 116 | no. 8 | 2979 Downloaded by guest on September 29, 2021 +/+ -/- DMSO Rapa A B C Tsc2 Tsc2 D +/+ -/- +/+ -/- +/+ sc2 - + - + Rapa sc2 T Tsc2

Tsc2 T sc2

TSC2 T TSC2 TSC2 p62 I LC3 P-S6K1(T389) II Tubulin P-S6(S240/244) -/-

T-S6K1 sc2 T-S6 T P-4E-BP1(S65) Tubulin T-4E-BP1 P-ULK1(S757) T-ULK1 I II LC3 Tubulin

GFP-p62 RFP-p62 Merge E +/+ -/- F Tsc2 Tsc2

+ - + + - + GF +/+ DMSO + + - + + - AA

Tsc2 TSC2 P-Akt(S473) Rapa T-Akt P-S6K1(T389) I II T-S6K1 -/- DMSO LC3 sc2 T Tubulin Rapa

TSC2- G -AA H ∆ex1-14 TSC2-H522T R1743Q TSC2 +AA -AA +Baf +/+ DMSO Tsc2 -/- Tsc2 +Baf

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.

+ + − − autophagosome formation in Tsc2 / and Tsc2 / MEFs as creased autophagy flux described above. Similar to the experi- shown by the increased conversion of LC3I to LC3II (Fig. 2F); ments in MEFs, treatment with bafilomycin also increased the however, the rate of autophagosome formation (the LC3II/LC3I accumulation of intracellular glycogen in TSC fibroblasts (Fig. ratio) and autophagic flux (clearance of LC3II) were significantly 2H). Together, these results demonstrate that decreased auto- − − + + lower in Tsc2 / MEFs compared with the Tsc2 / MEFs (Fig. phagy leads to excess glycogen storage in TSC. 2F), suggesting impaired autophagy initiation and autophagic flux in TSC. mTORC1-Independent Depletion of Lysosomes in TSC. An essential We next investigated the role of autophagy in glycogen step of autophagy is the fusion of autophagosomes with lyso- + + clearance in TSC. Glycogen clearance in both Tsc2 / and somes to form autophagolysosomes. We investigated whether − − Tsc2 / MEF lines was sensitive to AA starvation and blocked by the loss of TSC2 impairs the formation of autophagolysosomes. + + − − treatment with bafilomycin; however, the clearance levels were To this aim, we transfected Tsc2 / and Tsc2 / MEFs with a − − much lower in Tsc2 / MEFs (Fig. 2G), consistent with the de- tandem GFP-RFP-LC3 construct that emits red fluorescence

2980 | www.pnas.org/cgi/doi/10.1073/pnas.1812943116 Pal et al. Downloaded by guest on September 29, 2021 − − from mature autophagolysosomes and yellow fluorescence when Reexpression of TSC2 in Tsc2 / MEFs restored LC3II and autophagosomes are unable to fuse with lysosomes (30). Indeed, LAMP1 protein levels (Fig. 3F) and diminished the accumulation we found a significant increase in the number of yellow puncta in of glycogen (Fig. 3G). Collectively, these results reveal mTORC1- − − + + Tsc2 / MEFs compared with Tsc2 / MEFs (Fig. 3A), in- independentimpairmentoflysosomal biogenesis in TSC. dicating impaired fusion of autophagosomes with lysosomes − − upon deficiency of TSC2. We then tested whether lysosomal Excessive Proteasomal Degradation of LC3II and LAMP1 in Tsc2 / biogenesis is also impaired in TSC. Immunoblot experiments MEFs. Given that abnormal glycogen accumulation is associated − − revealed lysosomal depletion in Tsc2 / MEFs as detected by with lysosome depletion and impaired autophagy in TSC, we severely decreased levels of LAMP1 (Fig. 3B). Conversely, the next sought to determine the mechanism of reduction of − − levels of proteins that associate with the cytosolic side of the LC3 and LAMP1 in Tsc2 / MEFs. We first investigated the lysosomal membrane were unchanged (p18, HBXIP) or in- transcriptional status of the corresponding genes. Real-time creased (Rheb) (Fig. 3B). To confirm that the decrease in qRT-PCR analyses showed that transcription of both Map1lc3b − − LAMP1 levels reflects a reduced cellular pool of lysosomes, we (encoding LC3) and Lamp1 genes is increased in Tsc2 / MEFs + + performed microscopy analyses with two independent lysosomal compared with Tsc2 / MEFs (Fig. 4A), indicating that depletion markers lysotracker (31) and LAMP1 (SI Appendix, Fig. S3A). of LC3 and LAMP1 results from post-transcriptional events. To − − We found a significant decrease in both lysotracker and LAMP1 test whether translation of these proteins is impaired in Tsc2 / − − fluorescences in Tsc2 / MEFs (Fig. 3 C and D). mTORC1 acts MEFs, we performed a polyribosome fractionation analysis (33) as a negative regulator of the expression of lysosomal proteins to assess translation efficiency of Map1lc3b and Lamp1 mRNAs − − (32). The decrease in expression of LAMP1 protein in Tsc2 / (Fig. 4B and SI Appendix, Fig. S4A). The results showed that MEFs, however, could not be rescued by rapamycin treatment translation of Map1lc3b and Lamp1 mRNAs was not decreased − − + + (Fig. 3E). Similarly, shRNA-mediated knockdown of Raptor did in Tsc2 / MEFs compared with Tsc2 / MEFs thereby in- − − not show any significant increase in LAMP1 levels in Tsc2 / dicating that the observed depletion of LC3 and LAMP1 must MEFs (SI Appendix, Fig. S3B), suggesting that diminished result from post-translational events. We then investigated LAMP1 expression is independent of the activity of mTORC1. whether LC3 and LAMP1 are eliminated by ubiquitin proteasomal

Merge Lyso- DAPI LAMP1 CELL BIOLOGY A GFP-LC3 RFP-LC3 Merge B +/+ -/- C D DAPI tracker Merge sc2 T Tsc2 +/+ +/+ +/+ sc2 TSC2 sc2 T T sc2 T LAMP1 -/- -/- P18 -/- Tsc2 sc2 Tsc2 HBXIP T Rheb

Tubulin

-/- E F Tsc2 G +/+ -/- Tsc2 Tsc2 Flag-pcDNA Flag-TSC2-WT + + - - Flag-pcDNA - + - + Rapa - - + + Flag-TSC2-WT TSC2 -/- Flag-TSC2-WT Tsc2 LAMP1 LAMP1

I LC3 P-S6(S240/244) II T-S6 Tubulin Tubulin

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.

Pal et al. PNAS | February 19, 2019 | vol. 116 | no. 8 | 2981 Downloaded by guest on September 29, 2021 AB C +/+ -/- Tsc2 sc2 T - - - + Rapa - - + - MG132 TSC2 LAMP1 I II LC3 P-S6K1(T389) T-S6K1

Ubiquitin Merge Ubiquitin +/+ -/- D GFP-LC3 G Tsc2 Tsc2 Tubulin

+/+ - + - + MG132 F TSC2 Tsc2 +MG132 ATG3

-/- ATG7 proteins Beclin1 Tsc2

+MG132 VPS34 Autophagy-related GALNS LAMP1 Ubiquitin Merge E PPT1 TPP1 +/+ CathepsinD Lysosomal proteins

sc2 (mature form) T +MG132

Ubiquitin -/-

Tsc2 Tubulin +MG132

+/+ -/- +/+ -/- H Tsc2 Tsc2 Tsc2 Tsc2

- + - - + - MG132 - + - + Baf - - + - - + Rapa TSC2 TSC2 I LC3 II P-S6K1(T389) p62

T-S6K1 Tubulin I LC3 II p62 Tubulin

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 μminD and 40 μminE.(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.

− − system (UPS)-mediated degradation in Tsc2 / cells. Immuno- interaction of both LAMP1 and LC3 with ubiquitin protein (SI blot analyses showed that rapamycin treatment completely Appendix, Fig. S4B). Confocal microscopy showed increased inhibited mTORC1 activity but resulted in only a partial rescue numbers of ubiquitin-positive LC3 and LAMP1 puncta in − − + + of LC3I-to-LC3II conversion and no obvious effects on LAMP1 Tsc2 / MEFs compared with Tsc2 / MEFs (Fig. 4 D and E), − − and total LC3 protein levels in Tsc2 / cells (Fig. 4C). Treatment indicating that these proteins are eliminated by proteasomal with MG132, a potent inhibitor of proteasomal degradation (34), degradation. We also investigated the expression patterns of other however, resulted in a significant rescue in LAMP1 and LC3 components of the autophagy-lysosome pathway. qRT-PCR results levels (Fig. 4C). Coimmunoprecipitation analyses showed an showed that several other autophagy and lysosomal genes were

2982 | www.pnas.org/cgi/doi/10.1073/pnas.1812943116 Pal et al. Downloaded by guest on September 29, 2021 − − up-regulated in Tsc2 / MEFs (Fig. 4F). However, unlike LAMP1 MEFs (SI Appendix, Fig. S6A). Accordingly, either inhibition − − and LC3, their corresponding protein products were also up- decreased GS activity by phosphorylating GS at S641 in Tsc2 / regulated (Fig. 4G). In addition, treatment with MG132 did MEFs. As expected, Akt inhibition did not alter mTORC1 ac- − − not show any effects on these protein levels, suggesting that tivity in Tsc2 / MEFs, demonstrating that both mTORC1 and modulation of LAMP1 and LC3 levels is protein specific. Akt can independently regulate the GSK3β/GS signaling path- Transcription factor EB (TFEB) is the master regulator of way. mTORC1 activity is insensitive to GF availability and Akt − − autophagy and lysosomal genes (35, 36). A previous study has regulation in Tsc2 / cells (3, 20). Consistently, we observed that shown that both TFEB levels and nuclear localization are in- insulin stimulation induced a significant increase in mTORC1 + + creased in cells lacking either the TSC1 or the TSC2 protein activity in Tsc2 / MEFs, whereas mTORC1 was constitutively (37). This study has also shown that the expression of TFEB active and resistant to GF starvation or insulin stimulation in − − − − target genes is increased in Tsc2 / MEFs. Consistent with Tsc2 / MEFs (Fig. 6A). Insulin stimulation induced an increase + + these studies, we also observed a significant increase in TFEB in the phosphorylation of Akt and GSK3β in both Tsc2 / and − − + + − − expression levels in Tsc2 / MEFs compared with Tsc2 / Tsc2 / MEFs. Although insulin-mediated phosphorylation of MEFs (SI Appendix,Fig.S4C). In addition, subcellular frac- GSK3β at S9 was decreased by Akt inhibition in both MEF lines, tionation analyses in MEFs showed a significant increase in rapamycin was able to decrease GSK3β phosphorylation only in − − − − nuclear localization of TFEB in Tsc2 / MEFs (SI Appendix, Tsc2 / MEFs, suggesting that mTORC1 regulation of GSK3β − − Fig. S4D), a hallmark of TFEB activation (35, 36). These data activity is specific to Tsc2 / MEFs. In addition, inhibition of Akt suggest that the transcriptional up-regulation of genes of the decreased GSK3β phosphorylation independently of mTORC1 − − autophagy-lysosome pathway is associated with increased activity in Tsc2 / MEFs as evidenced by the absence of changes − − TFEB activity in Tsc2 / MEFs. Unlike rapamycin, the MG132- in S6K1 phosphorylation upon MK2206 treatment (Fig. 6A). mediated increase in LC3II levels did not show a concomitant Together, these results confirm that mTORC1 and Akt regulate − − decrease in p62 protein levels in either MEF line (Fig. 4H). On GSK3β activity in Tsc2 / MEFs independently. Consistent with the other hand, unlike bafilomycin, MG132 did not show a our previous study (39), we observed that inhibition of Akt in- significant increase in p62 protein levels (Fig. 4H). These re- creases the expression of LAMP1 independently of mTORC1 sults confirmed that MG132 does not induce autophagy nor activity (Fig. 6B). Immunoblot assays showed that treatments inhibit autophagic flux. Accordingly, MG132 treatment was un- with rapamycin or the Akt inhibitor MK2206 each resulted in an − − − − able to alter glycogen burden in Tsc2 / MEFs (SI Appendix, Fig. ∼twofold increase in LC3I-to-LC3II conversion in Tsc2 / cells, S4C), confirming that the MG132-mediated increase in LC3II whereas the combined treatment elicited an ∼fivefold increase, CELL BIOLOGY levels is caused by the accumulation of ubiquitinated LC3 proteins. indicating a synergistic effect of mTORC1 and Akt inhibition on − − Δex1-14 Similar to Tsc2 / MEFs, TSC2 fibroblasts showed in- autophagy induction (Fig. 6B). This was supported by a much − − creased mTORC1 activity and inhibitory phosphorylation of greater increase in GFP-LC3-positive puncta in Tsc2 / MEFs GSK3β along with a decrease in LAMP2 and LC3II protein when cells were treated with rapamycin and MK2206 in combi- levels (Fig. 5A). Interestingly, rapamycin treatment normalized nation, compared with the single treatments (Fig. 6C). The ad- − − both mTORC1 and GSK3β activities but resulted in only a dition of bafilomycin to Tsc2 / cells treated with MK2206 partial rescue of LC3I-to-LC3II conversion and no obvious ef- resulted in a significant increase in LC3II levels (SI Appendix, fects on LAMP2 and total LC3 protein levels (Fig. 5A). Treat- Fig. S6B), indicating that the observed increase in LC3II upon ment with MG132, however, resulted in a significant rescue in Akt inhibition is not due to autophagic blockage. The addition of − − − − LAMP2 and LC3 levels (Fig. 5B). Different from Tsc2 / MEFs MG132 to Tsc2 / cells treated with MK2206, however, did not Δex1-14 and TSC2 fibroblasts, patient-derived lines harboring the further increase LC3II levels compared with the treatment with missense mutations H522T and R1743Q had no changes in MK2206 alone (SI Appendix, Fig. S6C), indicating that Akt in- − − mTORC1 and GSK3β activities (Fig. 5C). Similar to Tsc2 / hibition prevents UPS-mediated degradation of LC3II thereby Δex1-14 MEFs, TSC2 cells showed an increase in GP levels and a promoting autophagy. − − decrease in phosphorylation of GS at S641, however, TSC2- Consistent with these results, PAS staining of Tsc2 / MEFs H522T and TSC2-R1743Q cells did not show any obvious showed that treatment with rapamycin or with MK2206 each led changes in either GP expression levels or phosphorylation of GS to a partial decrease in glycogen levels, whereas combined at S641 (SI Appendix, Fig. S5). Interestingly, both TSC2-H522T treatment resulted in a dramatic clearance of glycogen, which + + and TSC2-R1743Q cells showed a significant decrease in both was reduced to levels comparable to those of Tsc2 / MEFs (Fig. LAMP2 and LC3II protein levels (Fig. 5C). As expected, phos- 6D). Similar results were observed by using the glycogen assay kit phorylation of GSK3β at S9 in these two fibroblast lines was (SI Appendix, Fig. S6D). To corroborate these results, we in- insensitive to rapamycin treatment (Fig. 5D). Rapamycin treat- vestigated the combined effect of Akt inhibition and knockdown − − ment instead resulted in a small but significant increase in LC3I- of Raptor in Tsc2 / MEFs. We first confirmed that shRNA- to-LC3II conversion but no changes in total LC3 levels in both mediated knockdown of Raptor did not alter Akt activity (SI TSC2-H522T and TSC2-R1743Q cells (Fig. 5D). Immunoblot Appendix, Fig. S6E). Although Raptor knockdown showed a − − analyses of both cell lines revealed a significant increase in significant decrease in glycogen levels in Tsc2 / MEFs, in- LC3 protein levels upon MG132 treatment (Fig. 5E), similar to hibition of Akt showed a further decrease in glycogen levels in the other lines analyzed. Together, these results reveal excessive these cells (SI Appendix, Fig. S6F). Combined rapamycin and proteasomal degradation of proteins of the autophagy-lysosomal MK2206 treatment also resulted in a dramatic clearance of gly- pathway as a characteristic and common feature of TSC cellular cogen in patient-derived TSC fibroblasts (Fig. 6E). Collectively, pathology. these results establish that glycogen levels are independently regulated by Akt and mTORC1 via modulation of GSK3β ac- mTORC1-Dependent and -Independent Modulation of Intracellular tivity and autophagy and demonstrate that combined inhibition Glycogen Levels in TSC. Previous studies have shown that Akt of Akt and mTORC1 synergistically counteracts abnormal gly- − − regulates GSK3β activity and autophagy independently of cogen accumulation in Tsc2 / cells. mTORC1 (38, 39). Therefore, we investigated whether glycogen levels in TSC can be modulated by Akt independently of Discussion mTORC1. We found that inhibition of either mTORC1 (by The identification of the cellular pathways whose regulation is rapamycin) or Akt (by MK2206) caused an increase in GSK3β disrupted in TSC is the first necessary step toward the selection − − activity by inhibiting GSK3β phosphorylation at S9 in Tsc2 / of possible targets for therapeutic intervention. In this study, we

Pal et al. PNAS | February 19, 2019 | vol. 116 | no. 8 | 2983 Downloaded by guest on September 29, 2021 have identified two independent signaling cascades leading to brain, and the dysregulation of its metabolism is often found abnormal glycogen storage in TSC according to different associated with epileptic seizures (9). Accumulation of glycogen mechanisms. Impaired mTORC1-GSK3β signaling stimulates due to impairment of glycogen metabolism is also shown to be glycogen synthesis; on the other hand, ubiquitination and sub- associated with many cancers (8). It remains to be established sequent proteasomal degradation of LAMP1/2 and LC3II pro- whether aberrant glycogen storage plays a primary pathogenic mote intracellular glycogen accumulation through lysosomal role or contributes to disease progression. Owing to the role of depletion and impairment of the autophagy-lysosome pathway dysregulated glycogen metabolism in epilepsy and tumor for- (see the model in Fig. 6F). The characterization of the mecha- mation, our findings offer perspectives in the understanding of nisms that lead to aberrant UPS-mediated degradation of TSC disease pathogenesis that warrant the study of the role of autophagic proteins in TSC warrants further investigation. Im- aberrant glycogen storage in tumor formation and epilepsy in TSC. portantly, TSC is characterized by the formation of tumors in Whereas current therapeutic strategies for TSC are primarily multiple organs, and, most often, TSC patients develop epilepsy. focused on mTORC1 inhibition, mTORC1-independent pro- Brain glycogen is thought to be a putative energy store in the cesses that potentially contribute to TSC pathogenesis are poorly

A B

C D

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.

2984 | www.pnas.org/cgi/doi/10.1073/pnas.1812943116 Pal et al. Downloaded by guest on September 29, 2021 +/+ -/- ACTsc2 Tsc2 B +/+ -/- Tsc2 sc2 - + + + - + + + Insulin(Ins) T - - + - + Rapa - - + - - - + - MK2206 - - - + + MK2206 +/+ -/- - - - + - - - + Rapa Tsc2 +DMSO Tsc2 +DMSO TSC2 TSC2 P-Akt(S473) P-Akt(S473) T-Akt T-Akt P-GSK3 / (S21/9) -/- -/- Tsc2 +Rapa Tsc2 +MK2206 P-GSK3 (S9) (short exp.) P-GSK3 / (S21/9) T-GSK3 / (long exp.)

P-S6K1(T389) T-GSK3 / -/- Tsc2 +MK2206 T-S6K1 P-S6(S240/244) +Rapa

P-S6(S240/244) T-S6

T-S6 I LC3 II Tubulin LAMP1 D +/+ -/- -/- Tubulin Tsc2 +DMSO Tsc2 +DMSO Tsc2 +Rapa

-/- -/- CELL BIOLOGY Tsc2 +MK2206 Tsc2 +Rapa +MK2206

E +Rapa DMSO +Rapa +MK2206 TSC2-H522T TSC2- R1743Q F mTOR-dependent pathway mTOR-independent pathway ∆ex1-14

TSC2 mTORC1 mTORC1 Ub Ub Ub Ub basal Ub Ub Ub Ub active Ub Ub LAMP LC3 Akt LAMP LC3 Akt ˧ ˧ ˧ ˧

GSK3 GSK3

GS GS ˧ ˧

Glycogen Autophagy Glycogen Autophagy accumulation accumulation

Degraded LAMP Degraded LC3

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.

Pal et al. PNAS | February 19, 2019 | vol. 116 | no. 8 | 2985 Downloaded by guest on September 29, 2021 understood. One of the best-characterized hallmarks of TSC comprehensive therapeutic strategy aimed at rescuing disrupted pathology in multiple organs is defective autophagy. Our data autophagy and glycogen homeostasis in TSC. reveal an unexpected mTORC1-independent component that participates in the disruption of autophagy in TSC. In this study, Materials and Methods we observed that inhibition of Akt completely restores LC3 Patient fibroblasts were obtained from the Coriell Cell Repository following − − protein levels in Tsc2 / MEFs to normal levels. Our data in- the vendor’s guidelines. All patient fibroblast experimental procedures were dicate that inhibition of Akt not only increases synthesis of LC3, reviewed and approved by the IRB Committee at Baylor College of Medicine. but also provides stability to the LC3 protein. It will be inter- Coriell identification numbers for the patient fibroblasts: TSC2-H522T line, ID GM03958; TSC2-R1743Q line, ID GM06102; TSC2Δex1-14 line, ID GM04520. esting to explore in future studies the mechanism(s) by which For GF starvation, cells were grown in serum free media for 16 h. For AA modulation of Akt regulates ubiquitination of LC3. These starvation, cells were grown in dialyzed serum for 4 h. For insulin stimulation, studies could also help identify candidates that contribute to cells were starved of serum (GF, 16 h) and treated with insulin (1 μM) for autophagic impairment in TSC. Importantly, our data show that 15 min. Cells were treated with DMSO or drugs (where indicated) for 2 h stimulation of autophagy via mTORC1-independent Akt inhi- before the insulin stimulation. See SI Appendix, Materials and Methods for bition improves glycogen clearance in TSC cells and together details of the experimental procedures. with pharmacological mTORC1 inhibition synergistically activates autophagy and completely rescues glycogen clearance. Therefore, ACKNOWLEDGMENTS. We thank Dr. Hamed Jafar-Nejad and Dr. Kartik Venkatachalam for critically reading the manuscript. We thank Vera P. − − our study establishes that mTORC1-independent pathways should Krymskaya for the Tsc2 / MEFs and Ross Poche for the GFP-LC3 and GFP-RFP- be targeted along with mTORC1 for the development of a more LC3 constructs. This work was supported by NIH Grant NS079618 (to M.S.).

1. Henske EP, Józwiak S, Kingswood JC, Sampson JR, Thiele EA (2016) Tuberous sclerosis 21. Criado O, et al. (2012) Lafora bodies and neurological defects in malin-deficient mice complex. Nat Rev Dis Primers 2:16035. correlate with impaired autophagy. Hum Mol Genet 21:1521–1533. 2. Martin KR, et al. (2017) The genomic landscape of tuberous sclerosis complex. Nat 22. Skurat AV, Segvich DM, DePaoli-Roach AA, Roach PJ (2017) Novel method for de- Commun 8:15816. tection of glycogen in cells. Glycobiology 27:416–424. 3. Menon S, et al. (2014) Spatial control of the TSC complex integrates insulin and nu- 23. Baqué S, Guinovart JJ, Gómez-Foix AM (1996) Overexpression of muscle glycogen trient regulation of mTORC1 at the lysosome. Cell 156:771–785. phosphorylase in cultured human muscle fibers causes increased glucose consumption 4. Sancak Y, et al. (2008) The Rag GTPases bind raptor and mediate amino acid signaling and nonoxidative disposal. J Biol Chem 271:2594–2598. to mTORC1. Science 320:1496–1501. 24. Harriman G, et al. (2016) Acetyl-CoA carboxylase inhibition by ND-630 reduces hepatic 5. Cao J, et al. (2017) Tuberous sclerosis complex inactivation disrupts melanogenesis via steatosis, improves insulin sensitivity, and modulates dyslipidemia in rats. Proc Natl mTORC1 activation. J Clin Invest 127:349–364. Acad Sci USA 113:E1796–E1805. 6. Kwiatkowski DJ, et al. (2016) Mutations in TSC1, TSC2, and MTOR are associated with 25. Jung CH, Ro S-H, Cao J, Otto NM, Kim D-H (2010) mTOR regulation of autophagy. response to rapalogs in patients with metastatic renal cell carcinoma. Clin Cancer Res FEBS Lett 584:1287–1295. 22:2445–2452. 26. Rubinsztein DC, et al. (2009) In search of an “autophagomometer”. Autophagy 5: 7. Vilchez D, et al. (2007) Mechanism suppressing glycogen synthesis in neurons and its 585–589. demise in progressive myoclonus epilepsy. Nat Neurosci 10:1407–1413. 27. Peterson TR, et al. (2009) DEPTOR is an mTOR inhibitor frequently overexpressed in 8. Zois CE, Favaro E, Harris AL (2014) Glycogen metabolism in cancer. Biochem multiple myeloma cells and required for their survival. Cell 137:873–886. Pharmacol 92:3–11. 28. Thoreen CC, et al. (2009) An ATP-competitive mammalian target of rapamycin in- 9. DiNuzzo M, Mangia S, Maraviglia B, Giove F (2015) Does abnormal glycogen structure hibitor reveals rapamycin-resistant functions of mTORC1. J Biol Chem 284:8023–8032. contribute to increased susceptibility to seizures in epilepsy? Metab Brain Dis 30: 29. Lotfi P, et al. (2018) Trehalose reduces retinal degeneration, neuroinflammation and 307–316. storage burden caused by a lysosomal hydrolase deficiency. Autophagy 14:1419–1434. 10. Cloix J-F, Hévor T (2009) Epilepsy, regulation of brain energy metabolism and neu- 30. Zhou C, et al. (2012) Monitoring autophagic flux by an improved tandem fluorescent- rotransmission. Curr Med Chem 16:841–853. tagged LC3 (mTagRFP-mWasabi-LC3) reveals that high-dose rapamycin impairs au- 11. Meikle L, et al. (2005) A mouse model of cardiac rhabdomyoma generated by loss of tophagic flux in cancer cells. Autophagy 8:1215–1226. Tsc1 in ventricular myocytes. Hum Mol Genet 14:429–435. 31. Fan F, et al. (2012) Labeling lysosomes and tracking lysosome-dependent apoptosis 12. Bouskila M, et al. (2010) Allosteric regulation of glycogen synthase controls glycogen with a cell-permeable activity-based probe. Bioconjug Chem 23:1309–1317. synthesis in muscle. Cell Metab 12:456–466. 32. Martina JA, Chen Y, Gucek M, Puertollano R (2012) MTORC1 functions as a transcrip- 13. Roach PJ (2002) Glycogen and its metabolism. Curr Mol Med 2:101–120. tional regulator of autophagy by preventing nuclear transport of TFEB. Autophagy 8: 14. Zhang HH, Lipovsky AI, Dibble CC, Sahin M, Manning BD (2006) S6K1 regulates 903–914. GSK3 under conditions of mTOR-dependent feedback inhibition of Akt. Mol Cell 24: 33. Gennarino VA, et al. (2015) NUDT21-spanning CNVs lead to neuropsychiatric disease 185–197. and altered MeCP2 abundance via alternative polyadenylation. eLife 4:e10782. 15. Ali SM, Sabatini DM (2005) Structure of S6 kinase 1 determines whether raptor-mTOR 34. Dueber EC, et al. (2011) Antagonists induce a conformational change in cIAP1 that or rictor-mTOR phosphorylates its hydrophobic motif site. JBiolChem280:19445–19448. promotes autoubiquitination. Science 334:376–380. 16. Yu J, Parkhitko A, Henske EP (2011) Autophagy: An ‘Achilles’ heel of tumorigenesis in 35. Sardiello M, et al. (2009) A gene network regulating lysosomal biogenesis and TSC and LAM. Autophagy 7:1400–1401. function. Science 325:473–477. 17. Demetriades C, Doumpas N, Teleman AA (2014) Regulation of TORC1 in response to 36. Settembre C, et al. (2011) TFEB links autophagy to lysosomal biogenesis. Science 332: amino acid starvation via lysosomal recruitment of TSC2. Cell 156:786–799. 1429–1433. 18. Kim D-H, et al. (2002) mTOR interacts with raptor to form a nutrient-sensitive complex 37. Peña-Llopis S, et al. (2011) Regulation of TFEB and V-ATPases by mTORC1. EMBO J 30: that signals to the cell growth machinery. Cell 110:163–175. 3242–3258. 19. Manning BD (2017) Game of TOR–The target of rapamycin rules four kingdoms. N 38. Manning BD, Toker A (2017) AKT/PKB signaling: Navigating the network. Cell 169: Engl J Med 377:1297–1299. 381–405. 20. Pal R, Bondar VV, Adamski CJ, Rodney GG, Sardiello M (2017) Inhibition of ERK1/ 39. Palmieri M, et al. (2017) mTORC1-independent TFEB activation via Akt inhibition 2 restores GSK3β activity and protein synthesis levels in a model of tuberous sclerosis. promotes cellular clearance in neurodegenerative storage diseases. Nat Commun 8: Sci Rep 7:4174. 14338.

2986 | www.pnas.org/cgi/doi/10.1073/pnas.1812943116 Pal et al. Downloaded by guest on September 29, 2021