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Autophagic lipid metabolism sustains mTORC1 activity in TSC-deficient neural stem cells

Although mTORC1 negatively regulates autophagy in cultured cells, how autophagy impacts mTORC1 signaling, in particular in vivo, is less clear. Here we show that autophagy supports mTORC1 hyperactivation in NSCs lacking Tsc1, thereby promoting defects in NSC maintenance, differentiation, tumourigenesis, and the formation of the neurodevelopmental lesion of Tuberous Sclerosis Complex (TSC). Analysing mice that lack Tsc1 and the essential autophagy gene Fip200 in NSCs we find that TSC-deficient cells require autophagy to maintain mTORC1 hyperactivation under energy stress conditions, likely to provide lipids via lipophagy to serve as an alternative energy source for OXPHOS. In vivo, inhibition of lipophagy or its downstream catabolic pathway reverses defective phenotypes caused by Tsc1-null NSCs and reduces tumorigenesis in mouse models. These results reveal a cooperative function of selective autophagy in coupling energy availability with TSC pathogenesis and suggest a potential new therapeutic strategy to treat TSC patients.


Supplementary Discussion
Here, by generating and analyzing compound cKO mice with deletion of both Fip200 and Tsc1 in NSCs, we made a surprising finding that autophagy deficiency restored NSC maintenance and differentiation and reduced tumorigenesis caused by mTORC1 hyper-activation.
Mechanistically, these results establish a new paradigm for the regulation of mTORC1 by selective lipophagy and lipid catabolism, which were required to fuel mTORC1 hyper-activation in Tscdeficient NSCs and other cell types, as outlined in a working model (Fig. 6Q). In vivo inhibition of tumorigenesis by targeting either lipophagy or lipid catabolism also suggests new therapeutic strategies for TSC and other diseases caused by mTORC1 hyper-activation.
Previous studies showed that metabolism is extensively rewired in Tsc-deficient cells as a consequence of mTORC1 hyper-activation, leading to increased aerobic glycolysis through HIF1a and FA synthesis via SREBP and S6K1 signaling to promote lipid storage 16,51 . Our results suggested that active autophagy is important for meeting increased bioenergetic demands due to unrestrained biosynthesis in TSC-deficient cells in vivo and under nutrient-limiting conditions in vitro. Under energy stress, Tsc-deficient cells access stored energy in LDs via lipophagy and lysosomal FFA release for mitochondrial OXPHOS fuel, allowing cells to maintain high ATP levels and avoid bioenergetic crisis. ULK1 showed significantly increased phosphorylation at Ser555 by AMPK in addition to sustained high phosphorylation at Ser757 by mTORC1 in Tsc2 KO MEF (Fig. ED8G) and AMPKα KO blocked autophagy flux in these cells (Figs. ED8H, ED8I), suggesting that AMPK activation can overcome the inhibitory effect of mTORC1 under energy stress conditions. Autophagic flux is sustained despite Tsc1-deficiency and mTORC1 activation in SVZ tissue and primary NSC neurospheres. In both Tsc1-deficient neurospheres and MEFs, FIP200 deletion reduced autophagy, mitochondrial respiration and ATP content, and mTORC1 signaling. In vivo the absence of FIP200 increased the number of LDs in the SVZ of Tsc1 GFAP cKO mice. We observed comparable effects of TSC1 and FIP200 on autophagy and metabolic function in NSC and MEF cells, strongly supporting that autophagy inhibition by genetic or pharmacological inhibition reduced the energy supply and suppressed mTORC1 signaling to reverse tumorigenesis in Tsc-deficient NSCs from Tsc1 GFAP cKO mice. It is well known that a major consequence of mTORC1 hyper-activation is increased protein synthesis 60 , which utilizes approximately 20% -25% of total cellular energy and must be tightly coordinated with cellular energy status 61 . Under nutrient-rich conditions, increased glycolytic activity and OXPHOS 62 provide needed energy, and in the absence of glycolysis, lipophagy mechanisms described here could serve to sustain elevated protein synthesis and energy expenditure in Tsc-deficient cells.
Previous studies showed that both de novo FA synthesis and glycolysis play important roles in NSC proliferation 11, 63-65 . Further, -oxidization is required for NSC quiescence and subsequent neurogenic activities in hippocampus 66 . Nevertheless, we recognize that the absence of tracing experiments for metabolites to provide direct evidence for each intermediate of the working model (see Fig. 6Q) is a limitation of the study. Despite this limitation, both genetic and pharmacological approaches in animal and cell models clearly established a role for lipophagy to meet energy demands in Tsc1-null NSC.
These results add FAs to a growing list of metabolic outputs from lysosomal processing of autophagosomes in the regulation of bioenergetics upstream of mTORC1 in neoplasia and other cellular and disease processes. Recent studies showed that autophagy could degrade cytoplasmic protein and release amino acids to reactivate mTORC1 during the process of autophagic lysosomal reformation after prolonged amino acids starvation 67 . However, such a mechanism is unlikely to account for our findings in Tsc deficient cells, because our data (see Figs It is interesting to contrast the effects of FIP200 single knockout with the effects of FIP200 knockout in Tsc1-null NSCs. FIP200 single knockout mice were depleted of NSCs 39 , while FIP200 knockout in the TSC1-deficient background rescued NSC maintenance (this study). Such contrasting scenarios further highlight the metabolic re-wiring caused by mTORC1 hyperactivation and FIP200 deletion, which support ATP production even under energy stress conditions through autophagy of LDs as shown in this study. We propose that the role of FIP200 in normal NSCs is distinct from canonical autophagy that can be blocked by deletion of Atg5, Atg7 or Atg16L1 38 , but in neoplastic cells responding to energy stress, the autophagy regulatory function of FIP200 becomes required for cell growth. Consistent with this model, autophagy inhibition by FIP200 deletion, Atg7 KO, or Atg5 knockdown, as well as pharmacological inhibitors compromised ATP production and OXPHOS in cells with hyper-activation of mTORC1 (Figs. 2G, 2I-2O, ED6C). Importantly, specific disruption of FIP200 autophagy function using the FIP200-4A mutant allele in the TSC1-deficient mouse (i.e. 2cKI mice) reversed defective NSC phenotypes and murine SEN-like lesion formation, similar to ablation of FIP200 gene. Autophagy is required to permit cells with elevated mTORC1 to avoid bioenergetic crisis and AMPK activation under energy stress conditions through maintaining high cellular ATP levels via -oxidation of FFAs derived from lipophagy of LDs.
It is also interesting to note that, although not the focus of the current study, our results showing normal SVZ morphology, cell number, proliferation and various lineage markers as well as neurosphere formation from SVZ cells in 2cKO mice (see Fig. 1) suggest that Tsc1 deletion rescued previously observed NSC defects in FIP200 cKO mice 38 . However, p62 levels remain high in the SVZ of these mice (see Figs. 1C, 1D, ED1C). Thus, while our previous studies suggest that p62 accumulation is necessary 38 , it may not be sufficient to cause NSC defects in FIP200 cKO mice. Another alternative, but not necessarily mutually exclusive, explanation is that mTORC1 hyper-activation rescued the molecular defects downstream of p62 accumulation (thus will not decrease high p62 level per se as observed in Fig. 1 here) which is crucial for the defective NSC phenotypes in FIP200 cKO mice as suggested previously 38 .
These studies reveal differences between the function of Tsc1-deficient cells in vivo and in vitro. While multiple neural phenotypes of Tsc1 GFAP cKO mice were ameliorated by the loss of FIP200 autophagy function in both 2cKO and 2cKI mice, glucose restriction or 2-DG were required in combination with autophagy inhibition to activate AMPK and reverse mTORC1 hyperactivation in primary and immortalized cultured cells. Thus suppression of autophagy alone is effective to rescue the TSC-null phenotype in vivo. It is possible that glucose removal or 2-DG block of glycolysis in cell culture mimics the relatively low glucose concentration in the cerebrospinal fluid and SVZ 47 , which is necessary for activation of AMPK 27, 68 to reverse mTORC1 hyper-activation and associated phenotypes in vivo. In this regard, it is important to note that consistent results were obtained in SVZ cells and MEFs under glucose free conditions in culture, which provide a crucial connection between mechanistic studies using SVZ cells and

MEFs and observations in a brain and SVZ in vivo.
Additionally, the specific approach used for autophagy inhibition may influence the ability to counteract mTORC1 activation phenotypes. Pharmacologic 2-DG administration in combination with CQ blockade of murine SEN-like lesion formation began at a later time point (i.e. from P7) vs genetic autophagy inhibition in 2cKO and 2cKI mice at ~ E12.5 mediated by hGFAP-Cre recombination of Tsc1 and FIP200 alleles. Although CQ might exert a similar effect to FIP200 KO (and FIP200-4A mutant knockin) if administered at earlier developmental stages, we have found that CQ is toxic in neonatal or embryonic mice. Further, we and others previously showed that autophagy gene deletion (FIP200 and Atg5) decreased glycolysis in oncogene transformed mammary gland cells and MEF 69, 70 . In contrast, a recent study from Chen et al., indicate that CQ increase ECAR in macrophage, suggesting that genetic and pharmacologic approaches to inhibit autophagy may diverge in their effects on glycolysis 71 . Future studies will be necessary to clarify these possibilities to understand the mechanisms underlying the different targeting strategies.
Besides SEN-like lesions and other NSC defects, Tsc1 GFAP cKO mice exhibit an overall increase in brain mass and size (see Fig. ED1A), as described in previous studies 7, 9 . These studies have attributed the larger brain to enlargement of the ventricles by hydrocephalus 7, 9 , which was also found in our model (Fig. ED8J). Interestingly, our analyses suggested that in addition to hydrocephalus, abnormal postnatal cortex development, including increased neuron size and the number of astrocytes could contribute to the larger brain phenotype in the mutant mice (Figs. ED8J-8R, ED9). Moreover, autophagy inhibition by Fip200 ablation rescued all these defects, and reversed hyper-activation of mTORC1 in the cortex. Increased brain mass is associated with learning deficit and epileptic seizures in TSC patients. Our results indicate that the larger brain phenotype driven by mTORC1 hyper-activation is also dependent on FIP200-mediated autophagy.
Whether the increased brain mass/size is a consequence of pre-natal or post-natal effects of TSCdeficiency in CNS development is unclear. While pharmacologic inhibition of autophagy and oxidation repressed SEN-like and NSC defects in post-natal mice (Fig. 6), further studies will be required to determine the pre-natal and post-natal contributions of autophagy-linked -oxidation in the expansion of TSC-deficient brain mass and size.
Autophagy inhibition by ablation of FIP200 or other autophagy genes has been shown to decrease tumorigenesis and progression in several mouse models of cancer, including breast, lung and pancreatic cancers 69,72,73 . Targeting the links between autophagy and metabolic pathways have been suggested to play a role in the inhibition of tumor growth, including decreased glucose transport and consequent reduction of glycolysis in breast cancer cells 69,70,74 . In pancreatic cancer, autophagy is elevated due to the increased nuclear translocation of MiT/TFE family transcription factors, which promotes cancer cell survival through maintaining the intracellular amino acid pool as well as high OXPHOS and mitochondria functions 50, 75-78 . In lung cancer models, autophagy ablation reduced -oxidation leading to lipid accumulation caused by defective mitochondrial function and morphology, which has been suggested to be responsible for the regression of malignant adenoma and carcinoma to benign oncocytoma 73,79,80 . Interestingly, we found that targeting lipophagy or its downstream catabolic pathway inhibited tumorigenesis of murine SENlike lesions driven by mTORC1 hyper-activation in Tsc1 GFAP cKO mice. While future studies will be necessary to delineate the relationships of various metabolic pathways affected by autophagy inhibition in tumors driven by different oncogenes, our studies advance the notion of employing autophagy inhibition for cancer therapy, and establish new links between autophagic processing, lipid catabolism, and cellular bioenergetics. They suggest potentially new targets to treat aggressive cancers and neurodevelopmental defects caused by mTORC1 hyper-activation by revealing the role of lipophagy and its downstream catabolic pathways in the diseases.