Nunzia Pastore

Wilson Disease Protein ATP7B Utilizes Lysosomal Exocytosis to Maintain Copper Homeostasis

Summary 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.

Gene transfer of master autophagy regulator TFEB results in clearance of toxic protein and correction of hepatic disease in alpha‐1‐anti‐trypsin deficiency

Alpha‐1‐anti‐trypsin deficiency is the most common genetic cause of liver disease in children and liver transplantation is currently the only available treatment. Enhancement of liver autophagy increases degradation of mutant, hepatotoxic alpha‐1‐anti‐trypsin (ATZ). We investigated the therapeutic potential of liver‐directed gene transfer of transcription factor EB (TFEB), a master gene that regulates lysosomal function and autophagy, in PiZ transgenic mice, recapitulating the human hepatic disease. Hepatocyte TFEB gene transfer resulted in dramatic reduction of hepatic ATZ, liver apoptosis and fibrosis, which are key features of alpha‐1‐anti‐trypsin deficiency. Correction of the liver phenotype resulted from increased ATZ polymer degradation mediated by enhancement of autophagy flux and reduced ATZ monomer by decreased hepatic NFκB activation and IL‐6 that drives ATZ gene expression. In conclusion, TFEB gene transfer is a novel strategy for treatment of liver disease of alpha‐1‐anti‐trypsin deficiency. This study may pave the way towards applications of TFEB gene transfer for treatment of a wide spectrum of human disorders due to intracellular accumulation of toxic proteins.

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

ABSTRACT 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.

Sustained Reduction of Hyperbilirubinemia in Gunn Rats After Adeno-Associated Virus-Mediated Gene Transfer of Bilirubin UDP-Glucuronosyltransferase Isozyme 1A1 to Skeletal Muscle

Crigler-Najjar syndrome is an autosomal recessive disorder with severe unconjugated hyperbilirubinemia due to deficiency of bilirubin UDP-glucuronosyltransferase isozyme 1A1 (UGT1A1) encoded by the UGT1A1 gene. Current therapy relies on phototherapy to prevent life-threatening elevations of serum bilirubin levels, but liver transplantation is the only permanent treatment. Muscle-directed gene therapy has several advantages, including easy and safe access through simple intramuscular injections, and has been investigated in human clinical trials. In this study, we have investigated the efficacy of adeno-associated viral (AAV) vector-mediated muscle-directed gene therapy in the preclinical animal model of Crigler-Najjar syndrome, that is the Gunn rat. Serotype 1 AAV vector expressing rat UGT1A1 under the control of muscle-specific creatine kinase promoter was injected at a dose of 3×10(12) genome copies/kg into the muscles of Gunn rats and resulted in expression of UGT1A1 protein and functionally active enzyme in injected muscles. AAV-injected Gunn rats showed an approximately 50% reduction in serum bilirubin levels as compared with saline-treated controls, and this reduction was sustained for at least 1 year postinjection. Increased excretion of alkali-labile metabolites of bilirubin in bile and urine was detected in AAV-injected animals. High-performance liquid chromatography analysis of bile from AAV-injected Gunn rats showed a metabolite with retention time close to that of bilirubin diglucuronide. Taken together, these data show that clinically relevant and sustained reduction of serum bilirubin levels can be achieved by simple and safe intramuscular injections in Gunn rats. AAV-mediated muscle directed gene therapy has potential for the treatment of patients with Crigler-Najjar syndrome type 1.

Transcriptional activation of RagD GTPase controls mTORC1 and promotes cancer growth

A transcriptional regulatory mechanism enables cellular adaptation to nutrient availability and supports cancer metabolism. Preparing for the feast during the fast A protein kinase complex known as mTORC1 plays a key role in cellular metabolism and nutrient sensing. Di Malta et al. elucidated a mechanism that regulates the metabolic changes that are necessary during the fast-to-feed transition. During starvation, a pair of transcription factors promotes the expression of a pair of guanosine triphosphatases that are required for mTORC1 activity and for its recruitment to the lysosome. However, mTORC1 activity also requires amino acids, which are lacking during starvation. Nevertheless, the cell becomes “primed” by this process so that on refeeding, it efficiently reactivates mTORC1, which is recruited to the lysosomal surface. This mechanism is particularly important in cancer cells. Science, this issue p. 1188 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.

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.

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

Background: Several rare lung proteinopathies are characterized by lung fibrosis due to accumulation of misfolded proteins in epithelial cells. Results: The PiZ mouse models the pathological characteristics of these lung proteinopathies, and the pathology is ameliorated by autophagy enhancing therapies. Conclusion: Autophagy represents a key proteostasis mechanism for lung proteinopathies and a potential therapeutic target. Significance: The PiZ mouse is an attractive animal model of lung proteinopathies. 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.

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.

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).

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.