TFEB and TFE3 control glucose homeostasis by regulating insulin gene expression.

To fulfill their function, pancreatic beta cells require precise nutrient-sensing mechanisms that control insulin production. Transcription factor EB (TFEB) and its homolog TFE3 have emerged as crucial regulators of the adaptive response of cell metabolism to environmental cues. Here, we show that TFEB and TFE3 regulate beta-cell function and insulin gene expression in response to variations in nutrient availability. We found that nutrient deprivation in beta cells promoted TFEB/TFE3 activation, which resulted in suppression of insulin gene expression. TFEB overexpression was sufficient to inhibit insulin transcription, whereas beta cells depleted of both TFEB and TFE3 failed to suppress insulin gene expression in response to amino acid deprivation. Interestingly, ChIP-seq analysis showed binding of TFEB to super-enhancer regions that regulate insulin transcription. Conditional, beta-cell-specific, Tfeb-overexpressing, and Tfeb/Tfe3 double-KO mice showed severe alteration of insulin transcription, secretion, and glucose tolerance, indicating that TFEB and TFE3 are important physiological mediators of pancreatic function. Our findings reveal a nutrient-controlled transcriptional mechanism that regulates insulin production, thus playing a key role in glucose homeostasis at both cellular and organismal levels.

Increased expression or activation of TRPML1 reduces hepatic storage of toxic Z alpha-1 antitrypsin.

Mutant Z alpha-1 antitrypsin (ATZ) accumulates in globules in the liver and is the prototype of proteotoxic hepatic disease. Therapeutic strategies aiming at clearance of polymeric ATZ are needed. Transient receptor potential mucolipin-1 (TRPML1) is a lysosomal Ca2+ channel that maintains lysosomal homeostasis. In this study, we show that by increasing lysosomal exocytosis, TRPML1 gene transfer or small-molecule-mediated activation of TRPML1 reduces hepatic ATZ globules and fibrosis in PiZ transgenic mice that express the human ATZ. ATZ globule clearance induced by TRPML1 occurred without increase in autophagy or nuclear translocation of TFEB. Our results show that targeting TRPML1 and lysosomal exocytosis is a novel approach for treatment of the liver disease due to ATZ and potentially other diseases due to proteotoxic liver storage.

Nutrient-sensitive transcription factors TFEB and TFE3 couple autophagy and metabolism to the peripheral clock.

Autophagy and energy metabolism are known to follow a circadian pattern. However, it is unclear whether autophagy and the circadian clock are coordinated by common control mechanisms. Here, we show that the oscillation of autophagy genes is dependent on the nutrient-sensitive activation of TFEB and TFE3, key regulators of autophagy, lysosomal biogenesis, and cell homeostasis. TFEB and TFE3 display a circadian activation over the 24-h cycle and are responsible for the rhythmic induction of genes involved in autophagy during the light phase. Genetic ablation of TFEB and TFE3 in mice results in deregulated autophagy over the diurnal cycle and altered gene expression causing abnormal circadian wheel-running behavior. In addition, TFEB and TFE3 directly regulate the expression of Rev-erbα (Nr1d1), a transcriptional repressor component of the core clock machinery also involved in the regulation of whole-body metabolism and autophagy. Comparative analysis of the cistromes of TFEB/TFE3 and REV-ERBα showed an extensive overlap of their binding sites, particularly in genes involved in autophagy and metabolic functions. These data reveal a direct link between nutrient and clock-dependent regulation of gene expression shedding a new light on the crosstalk between autophagy, metabolism, and circadian cycles.

Potentiation of rifampin activity in a mouse model of tuberculosis by activation of host transcription factor EB.

Efforts at host-directed therapy of tuberculosis have produced little control of the disease in experimental animals to date. This is not surprising, given that few specific host targets have been validated, and reciprocally, many of the compounds tested potentially impact multiple targets with both beneficial and detrimental consequences. This puts a premium on identifying appropriate molecular targets and subjecting them to more selective modulation. We discovered an aminopyrimidine small molecule, 2062, that had no direct antimycobacterial activity, but synergized with rifampin to reduce bacterial burden in Mtb infected macrophages and mice and also dampened lung immunopathology. We used 2062 and its inactive congeners as tool compounds to identify host targets. By biochemical, pharmacologic, transcriptomic and genetic approaches, we found that 2062's beneficial effects on Mtb control and clearance in macrophages and in mice are associated with activation of transcription factor EB via an organellar stress response. 2062-dependent TFEB activation led to improved autophagy, lysosomal acidification and lysosomal degradation, promoting bacterial clearance in macrophages. Deletion of TFEB resulted in the loss of IFNγ-dependent control of Mtb replication in macrophages. 2062 also targeted multiple kinases, such as PIKfyve, VPS34, JAKs and Tyk2, whose inhibition likely limited 2062's efficacy in vivo. These findings support a search for selective activators of TFEB for HDT of TB.

Enhancement of hepatic autophagy increases ureagenesis and protects against hyperammonemia.

Ammonia is a potent neurotoxin that is detoxified mainly by the urea cycle in the liver. Hyperammonemia is a common complication of a wide variety of both inherited and acquired liver diseases. If not treated early and thoroughly, it results in encephalopathy and death. Here, we found that hepatic autophagy is critically involved in systemic ammonia homeostasis by providing key urea-cycle intermediates and ATP. Hepatic autophagy is triggered in vivo by hyperammonemia through an α-ketoglutarate-dependent inhibition of the mammalian target of rapamycin complex 1, and deficiency of autophagy impairs ammonia detoxification. In contrast, autophagy enhancement by means of hepatic gene transfer of the master regulator of autophagy transcription factor EB or treatments with the autophagy enhancers rapamycin and Tat-Beclin-1 increased ureagenesis and protected against hyperammonemia in a variety of acute and chronic hyperammonemia animal models, including acute liver failure and ornithine transcarbamylase deficiency, the most frequent urea-cycle disorder. In conclusion, hepatic autophagy is an important mechanism for ammonia detoxification because of its support of urea synthesis, and its enhancement has potential for therapy of both primary and secondary causes of hyperammonemia.

HDAC6-dependent ciliophagy is involved in ciliary loss and cholangiocarcinoma growth in human cells and murine models.

Reduced ciliary expression is reported in several tumors, including cholangiocarcinoma (CCA). We previously showed primary cilia have tumor suppressor characteristics, and HDAC6 is involved in ciliary loss. However, mechanisms of ciliary disassembly are unknown. Herein, we tested the hypothesis that HDAC6-dependent autophagy of primary cilia, i.e., ciliophagy, is the main mechanism driving ciliary disassembly in CCA. Using the cancer genome atlas database, human CCA cells, and a rat orthotopic CCA model, we assessed basal and HDAC6-regulated autophagy levels. The effects of RNA-silencing or pharmacological manipulations of ciliophagy on ciliary expression were assessed. Interactions of ciliary proteins with autophagy machinery was assessed by immunoprecipitations. Cell proliferation was assessed by MTS and IncuCyte. A CCA rat model was used to assess the effects of pharmacological inhibition of ciliophagy in vivo. Autophagy is increased in human CCA, as well as in a rat orthotopic CCA model and human CCA cell lines. Autophagic flux was decreased via inhibition of HDAC6, while it was increased by its overexpression. Inhibition of autophagy and HDAC6 restores cilia and decreases cell proliferation. LC3 interacts with HDAC6 and ciliary proteins, and the autophagy cargo receptor involved in targeting ciliary components to the autophagy machinery is primarily NBR1. Treatment with chloroquine, Ricolinostat (ACY-1215), or their combination decreased tumor growth in vivo. Mice that overexpress the autophagy transcription factor TFEB show a decrease of ciliary number. These results suggest that ciliary disassembly is mediated by HDAC6-regulated autophagy, i.e., ciliophagy. Inhibition of ciliophagy may decrease cholangiocarcinoma growth and warrant further investigations as a potential therapeutic approach.NEW & NOTEWORTHY This work identifies novel targets against primary ciliary disassembly that can lead to new cholangiocarcinoma therapeutic strategies. Furthermore, ciliary loss has been described in different tumors, increasing the significance of our research.

Activation of the c-Jun N-terminal kinase pathway aggravates proteotoxicity of hepatic mutant Z alpha1-antitrypsin.

Alpha1-antitrypsin deficiency is a genetic disease that can affect both the lung and the liver. The vast majority of patients harbor a mutation in the serine protease inhibitor 1A (SERPINA1) gene leading to a single amino acid substitution that results in an unfolded protein that is prone to polymerization. Alpha1-antitrypsin defciency-related liver disease is therefore caused by a gain-of-function mechanism due to accumulation of the mutant Z alpha1-antitrypsin (ATZ) and is a key example of an disease mechanism induced by protein toxicity. Intracellular retention of ATZ triggers a complex injury cascade including apoptosis and other mechanisms, although several aspects of the disease pathogenesis are still unclear. We show that ATZ induces activation of c-Jun N-terminal kinase (JNK) and c-Jun and that genetic ablation of JNK1 or JNK2 decreased ATZ levels in vivo by reducing c-Jun-mediated SERPINA1 gene expression. JNK activation was confirmed in livers of patients homozygous for the Z allele, with severe liver disease requiring hepatic transplantation. Treatment of patient-derived induced pluripotent stem cell-hepatic cells with a JNK inhibitor reduced accumulation of ATZ. CONCLUSION: These data reveal that JNK is a key pathway in the disease pathogenesis and add new therapeutic entry points for liver disease caused by ATZ. (Hepatology 2017;65:1865-1874).

Enhancing Autophagy with Drugs or Lung-directed Gene Therapy Reverses the Pathological Effects of Respiratory Epithelial Cell Proteinopathy.

Recent studies have shown that autophagy mitigates the pathological effects of proteinopathies in the liver, heart, and skeletal muscle but this has not been investigated for proteinopathies that affect the lung. This may be due at least in part to the lack of an animal model robust enough for spontaneous pathological effects from proteinopathies even though several rare proteinopathies, surfactant protein A and C deficiencies, cause severe pulmonary fibrosis. In this report we show that the PiZ mouse, transgenic for the common misfolded variant α1-antitrypsin Z, is a model of respiratory epithelial cell proteinopathy with spontaneous pulmonary fibrosis. Intracellular accumulation of misfolded α1-antitrypsin Z in respiratory epithelial cells of the PiZ model resulted in activation of autophagy, leukocyte infiltration, and spontaneous pulmonary fibrosis severe enough to elicit functional restrictive deficits. Treatment with autophagy enhancer drugs or lung-directed gene transfer of TFEB, a master transcriptional activator of the autophagolysosomal system, reversed these proteotoxic consequences. We conclude that this mouse is an excellent model of respiratory epithelial proteinopathy with spontaneous pulmonary fibrosis and that autophagy is an important endogenous proteostasis mechanism and an attractive target for therapy.

Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation.

Batten disease, one of the most devastating types of neurodegenerative lysosomal storage disorders, is caused by mutations in CLN3. Here, we show that CLN3 is a vesicular trafficking hub connecting the Golgi and lysosome compartments. Proteomic analysis reveals that CLN3 interacts with several endo-lysosomal trafficking proteins, including the cation-independent mannose 6 phosphate receptor (CI-M6PR), which coordinates the targeting of lysosomal enzymes to lysosomes. CLN3 depletion results in mis-trafficking of CI-M6PR, mis-sorting of lysosomal enzymes, and defective autophagic lysosomal reformation. Conversely, CLN3 overexpression promotes the formation of multiple lysosomal tubules, which are autophagy and CI-M6PR-dependent, generating newly formed proto-lysosomes. Together, our findings reveal that CLN3 functions as a link between the M6P-dependent trafficking of lysosomal enzymes and lysosomal reformation pathway, explaining the global impairment of lysosomal function in Batten disease.

TFEB regulates murine liver cell fate during development and regeneration.

It is well established that pluripotent stem cells in fetal and postnatal liver (LPCs) can differentiate into both hepatocytes and cholangiocytes. However, the signaling pathways implicated in the differentiation of LPCs are still incompletely understood. Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is known to be involved in osteoblast and myeloid differentiation, but its role in lineage commitment in the liver has not been investigated. Here we show that during development and upon regeneration TFEB drives the differentiation status of murine LPCs into the progenitor/cholangiocyte lineage while inhibiting hepatocyte differentiation. Genetic interaction studies show that Sox9, a marker of precursor and biliary cells, is a direct transcriptional target of TFEB and a primary mediator of its effects on liver cell fate. In summary, our findings identify an unexplored pathway that controls liver cell lineage commitment and whose dysregulation may play a role in biliary cancer.

Keeping the autophagy tempo.

Most essential physiological functions in mammals show a 24-h rhythmic pattern, which includes sleep-wake, feeding-non-feeding cycles and energy metabolism. Recent studies indicate that macroautophagy/autophagy also displays a robust circadian rhythmicity following the daily feeding pattern in adult mammals. We discovered that MiT-TFE transcription factors TFEB and TFE3, master regulators of autophagy and lysosomal biogenesis, are activated in a circadian manner and drive the expression of NR1D1/REV-ERBα, a key component of the core clockwork, thus revealing a molecular link between the nutrient-driven circadian cycle and the light-induced molecular clock. The dynamic balance between TFEB and TFE3 activation and NR1D1 expression is responsible for the modulation and oscillation of autophagy and metabolism genes.

Fasting Imparts a Switch to Alternative Daily Pathways in Liver and Muscle.

The circadian clock operates as intrinsic time-keeping machinery to preserve homeostasis in response to the changing environment. While food is a known zeitgeber for clocks in peripheral tissues, it remains unclear how lack of food influences clock function. We demonstrate that the transcriptional response to fasting operates through molecular mechanisms that are distinct from time-restricted feeding regimens. First, fasting affects core clock genes and proteins, resulting in blunted rhythmicity of BMAL1 and REV-ERBα both in liver and skeletal muscle. Second, fasting induces a switch in temporal gene expression through dedicated fasting-sensitive transcription factors such as GR, CREB, FOXO, TFEB, and PPARs. Third, the rhythmic genomic response to fasting is sustainable by prolonged fasting and reversible by refeeding. Thus, fasting imposes specialized dynamics of transcriptional coordination between the clock and nutrient-sensitive pathways, thereby achieving a switch to fasting-specific temporal gene regulation.

TFE3 regulates whole-body energy metabolism in cooperation with TFEB.

TFE3 and TFEB are members of the MiT family of HLH-leucine zipper transcription factors. Recent studies demonstrated that they bind overlapping sets of promoters and are post-transcriptionally regulated through a similar mechanism. However, while Tcfeb knockout (KO) mice die during early embryonic development, no apparent phenotype was reported in Tfe3 KO mice. Thus raising the need to characterize the physiological role of TFE3 and elucidate its relationship with TFEB TFE3 deficiency resulted in altered mitochondrial morphology and function both in vitro and in vivo due to compromised mitochondrial dynamics. In addition, Tfe3 KO mice showed significant abnormalities in energy balance and alterations in systemic glucose and lipid metabolism, resulting in enhanced diet-induced obesity and diabetes. Conversely, viral-mediated TFE3 overexpression improved the metabolic abnormalities induced by high-fat diet (HFD). Both TFEB overexpression in Tfe3 KO mice and TFE3 overexpression in Tcfeb liver-specific KO mice (Tcfeb LiKO) rescued HFD-induced obesity, indicating that TFEB can compensate for TFE3 deficiency and vice versa Analysis of Tcfeb LiKO/Tfe3 double KO mice demonstrated that depletion of both TFE3 and TFEB results in additive effects with an exacerbation of the hepatic phenotype. These data indicate that TFE3 and TFEB play a cooperative, rather than redundant, role in the control of the adaptive response of whole-body metabolism to environmental cues such as diet and physical exercise.

Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth.

The mechanistic target of rapamycin complex 1 (mTORC1) is recruited to the lysosome by Rag guanosine triphosphatases (GTPases) and regulates anabolic pathways in response to nutrients. We found that MiT/TFE transcription factors-master regulators of lysosomal and melanosomal biogenesis and autophagy-control mTORC1 lysosomal recruitment and activity by directly regulating the expression of RagD. In mice, this mechanism mediated adaptation to food availability after starvation and physical exercise and played an important role in cancer growth. Up-regulation of MiT/TFE genes in cells and tissues from patients and murine models of renal cell carcinoma, pancreatic ductal adenocarcinoma, and melanoma triggered RagD-mediated mTORC1 induction, resulting in cell hyperproliferation and cancer growth. Thus, this transcriptional regulatory mechanism enables cellular adaptation to nutrient availability and supports the energy-demanding metabolism of cancer cells.

TFEB and TFE3 cooperate in the regulation of the innate immune response in activated macrophages.

The activation of transcription factors is critical to ensure an effective defense against pathogens. In this study we identify a critical and complementary role of the transcription factors TFEB and TFE3 in innate immune response. By using a combination of chromatin immunoprecipitation, CRISPR-Cas9-mediated genome-editing technology, and in vivo models, we determined that TFEB and TFE3 collaborate with each other in activated macrophages and microglia to promote efficient autophagy induction, increased lysosomal biogenesis, and transcriptional upregulation of numerous proinflammatory cytokines. Furthermore, secretion of key mediators of the inflammatory response (CSF2, IL1B, IL2, and IL27), macrophage differentiation (CSF1), and macrophage infiltration and migration to sites of inflammation (CCL2) was significantly reduced in TFEB and TFE3 deficient cells. These new insights provide us with a deeper understanding of the transcriptional regulation of the innate immune response.

Correction of hyperbilirubinemia in gunn rats by surgical delivery of low doses of helper-dependent adenoviral vectors.

Helper-dependent adenoviral (HDAd) vectors are attractive for liver-directed gene therapy because they can drive sustained high levels of transgene expression without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic delivery because of a nonlinear dose response. Unfortunately, such high doses result in systemic vector dissemination and dose-dependent acute toxicity with potential lethal consequences. We have previously shown in nonhuman primates that delivery of HDAd in surgically isolated livers resulted in a significantly higher hepatic transduction with reduced systemic vector dissemination compared with intravenous delivery and multiyear transgene expression. Encouraged by these data, we have now employed a surgical vector delivery method in the Gunn rat, an animal model for Crigler-Najjar syndrome. After vector delivery into the surgically isolated liver, we show phenotypic correction at the low and clinically relevant vector dose of 1 × 10(11) vp/kg. Correction of hyperbilirubinemia and increased glucuronidation of bilirubin in bile was achieved for up to 1 year after vector administration. Surgical delivery of the vector was well tolerated without signs of acute or chronic toxicity. This method of delivery could thereby be a safer alternative to liver transplantation for long-term treatment of Crigler-Najjar syndrome type I.

Wilson disease protein ATP7B utilizes lysosomal exocytosis to maintain copper homeostasis.

Copper is an essential yet toxic metal and its overload causes Wilson disease, a disorder due to mutations in copper transporter ATP7B. To remove excess copper into the bile, ATP7B traffics toward canalicular area of hepatocytes. However, the trafficking mechanisms of ATP7B remain elusive. Here, we show that, in response to elevated copper, ATP7B moves from the Golgi to lysosomes and imports metal into their lumen. ATP7B enables lysosomes to undergo exocytosis through the interaction with p62 subunit of dynactin that allows lysosome translocation toward the canalicular pole of hepatocytes. Activation of lysosomal exocytosis stimulates copper clearance from the hepatocytes and rescues the most frequent Wilson-disease-causing ATP7B mutant to the appropriate functional site. Our findings indicate that lysosomes serve as an important intermediate in ATP7B trafficking, whereas lysosomal exocytosis operates as an integral process in copper excretion and hence can be targeted for therapeutic approaches to combat Wilson disease.

Autophagy master regulator TFEB induces clearance of toxic SERPINA1/α-1-antitrypsin polymers.

Deficiency of SERPINA1/AAT [serpin peptidase inhibitor, clade A (α-1 antiproteinase, antitrypsin), member 1/α 1-antitrypsin] results in polymerization and aggregation of mutant SERPINA1 molecules in the endoplasmic reticulum of hepatocytes, triggering liver injury. SERPINA1 deficiency is the most common genetic cause of hepatic disease in children and is frequently responsible for chronic liver disease in adults. Liver transplantation is currently the only available treatment for the severe form of the disease. We found that liver-directed gene transfer of transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, results in marked reduction of toxic mutant SERPINA1 polymer, apoptosis and fibrosis in the liver of a mouse model of SERPINA1 deficiency. TFEB-mediated correction of hepatic disease is dependent upon increased degradation of SERPINA1 polymer in autolysosomes and decreased expression of SERPINA1 monomer. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease in SERPINA1 deficiency. Moreover, this study suggests that TFEB-mediated cellular clearance may have broad applications for therapy of human disorders due to intracellular accumulation of toxic proteins.

Improved efficacy and reduced toxicity by ultrasound-guided intrahepatic injections of helper-dependent adenoviral vector in Gunn rats.

Crigler-Najjar syndrome type I is caused by mutations of the uridine diphospho-glucuronosyl transferase 1A1 (UGT1A1) gene resulting in life-threatening increase of serum bilirubin. Life-long correction of hyperbilirubinemia was previously shown with intravenous injection of high doses of a helper-dependent adenoviral (HDAd) vector expressing UGT1A1 in the Gunn rat, the animal model of Crigler-Najjar syndrome. However, such high vector doses can activate an acute and potentially lethal inflammatory response with elevated serum interleukin-6 (IL-6). To overcome this obstacle, we investigated safety and efficacy of direct injections of low HDAd doses delivered directly into the liver parenchyma of Gunn rats. Direct hepatic injections performed by either laparotomy or ultrasound-guided percutaneous injections were compared with the same doses given by intravenous injections. A greater reduction of hyperbilirubinemia and increased conjugated bilirubin in bile were achieved with 1 × 10(11) vp/kg by direct liver injections compared with intravenous injections. In sharp contrast to intravenous injections, direct hepatic injections neither raised serum IL-6 nor resulted in thrombocytopenia. In conclusion, ultrasound-guided percutaneous injection of HDAd vectors into liver parenchyma resulted in improved hepatocyte transduction and reduced toxicity compared with systemic injections and is clinically attractive for liver-directed gene therapy of Crigler-Najjar syndrome.