A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB

Under basal conditions TFEB, a master regulator of lysosomal biogenesis, is sequestered in the cytosol due to mTORC1-dependent phosphorylation at the lysosomal membrane. Nutrient starvation or lysosomal dysfunction inhibit mTORC1 activity and induce nuclear translocation of TFEB inducing target gene expression.

The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. Here, we show that the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB activity. Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. In addition, the transcriptional response of lysosomal and autophagic genes to either lysosomal dysfunction or pharmacological inhibition of mTORC1 is suppressed in TFEB−/− cells. Interestingly, the Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to regulate starvation- and stress-induced nuclear translocation of TFEB. These data indicate that the lysosome senses its content and regulates its own biogenesis by a lysosome-to-nucleus signalling mechanism that involves TFEB and mTOR.

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.

Lysosomes are ubiquitous intracellular organelles that have an acidic internal pH, and play crucial roles in cellular clearance. Numerous functions depend on normal lysosomes, including the turnover of cellular constituents, cholesterol homeostasis, downregulation of surface receptors, inactivation of pathogenic organisms, repair of the plasma membrane and bone remodeling. Lysosomal storage disorders (LSDs) are characterized by progressive accumulation of undigested macromolecules within the cell due to lysosomal dysfunction. As a consequence, many tissues and organ systems are affected, including brain, viscera, bone and cartilage. The progressive nature of phenotype development is one of the hallmarks of LSDs. In recent years biochemical and cell biology studies of LSDs have revealed an ample spectrum of abnormalities in a variety of cellular functions. These include defects in signaling pathways, calcium homeostasis, lipid biosynthesis and degradation and intracellular trafficking. Lysosomes also play a fundamental role in the autophagic pathway by fusing with autophagosomes and digesting their content. Considering the highly integrated function of lysosomes and autophagosomes it was reasonable to expect that lysosomal storage in LSDs would have an impact upon autophagy. The goal of this review is to provide readers with an overview of recent findings that have been obtained through analysis of the autophagic pathway in several types of LSDs, supporting the idea that LSDs could be seen primarily as “autophagy disorders.”

Dysfunctional mitochondria are a well-known disease hallmark. The accumulation of aberrant mitochondria can alter cell homeostasis, thus resulting in tissue degeneration. Lysosomal storage disorders (LSDs) are a group of inherited diseases characterized by the buildup of undegraded material inside the lysosomes that leads to autophagic-lysosomal dysfunction. In LSDs, autophagic stress has been associated to mitochondrial accumulation and dysfunction. However, the mechanisms underlying mitochondrial aberrations and how these are involved in tissue pathogenesis remain largely unexplored. In normal conditions, mitochondrial clearance occurs by mitophagy, a selective form of autophagy, which relies on a parkin-mediated mitochondrial priming and subsequent sequestration by autophagosomes. Here, we performed a detailed analysis of key steps of mitophagy in a mouse model of multiple sulfatase deficiency (MSD), a severe type of LSD characterized by both neurological and systemic involvement. We demonstrated that in MSD liver reduced parkin levels resulted in inefficient mitochondrial priming, thus contributing to the accumulation of giant mitochondria that are located outside autophagic vesicles ultimately leading to cytochrome c release and apoptotic cell death. Morphological and functional changes were also observed in mitochondria from MSD brain but these were not directly associated with neuronal cell loss, suggesting a secondary contribution of mitochondria to neurodegeneration. Together, these data shed new light on the mechanisms underlying mitochondrial dysfunction in LSDs and on their tissue-specific differential contribution to the pathogenesis of this group of metabolic disorders.

Summary

Lysosomes are cellular organelles primarily involved in degradation and recycling processes. During lysosomal exocytosis, a Ca2+-regulated process, lysosomes are docked to the cell surface and fuse with the plasma membrane (PM), emptying their content outside the cell. This process has an important role in secretion and PM repair. Here we show that the transcription factor EB (TFEB) regulates lysosomal exocytosis. TFEB increases the pool of lysosomes in the proximity of the PM and promotes their fusion with PM by raising intracellular Ca2+ levels through the activation of the lysosomal Ca2+ channel MCOLN1. Induction of lysosomal exocytosis by TFEB overexpression rescued pathologic storage and restored normal cellular morphology both in vitro and in vivo in lysosomal storage diseases (LSDs). Our data indicate that lysosomal exocytosis may directly modulate cellular clearance and suggest an alternative therapeutic strategy for disorders associated with intracellular storage.

Graphical Abstract

Highlights

► TFEB-regulated transcription induces lysosomal docking to the plasma membrane (PM) ► TFEB promotes lysosomal fusion with the PM by raising Ca2+ levels through MCOLN1 ► TFEB can thus rescue pathological storage in lysosomal storage disease (LSD) cells ► In vivo TFEB gene delivery rescues storage, inflammation, and apoptosis in LSD mice

We propose an innovative, integrated, cost-effective health system to combat major non-communicable diseases (NCDs), including cardiovascular, chronic respiratory, metabolic, rheumatologic and neurologic disorders and cancers, which together are the predominant health problem of the 21st century. This proposed holistic strategy involves comprehensive patient-centered integrated care and multi-scale, multi-modal and multi-level systems approaches to tackle NCDs as a common group of diseases. Rather than studying each disease individually, it will take into account their intertwined gene-environment, socio-economic interactions and co-morbidities that lead to individual-specific complex phenotypes. It will implement a road map for predictive, preventive, personalized and participatory (P4) medicine based on a robust and extensive knowledge management infrastructure that contains individual patient information. It will be supported by strategic partnerships involving all stakeholders, including general practitioners associated with patient-centered care. This systems medicine strategy, which will take a holistic approach to disease, is designed to allow the results to be used globally, taking into account the needs and specificities of local economies and health systems.

Macroautophagy (a.k.a. autophagy) is a cellular process aimed at the recycling of proteins and organelles that is achieved when autophagosomes fuse with lysosomes. Accordingly, lysosomal dysfunctions are often associated with impaired autophagy. We demonstrated that inactivation of the sulfatase modifying factor 1 gene (Sumf1), a gene mutated in multiple sulfatase deficiency (MSD), causes glycosaminoglycans (GAGs) to accumulate in lysosomes, which in turn disrupts autophagy. We utilized a murine model of MSD to study how impairment of this process affects chondrocyte viability and thus skeletal development.

Background

Dosage imbalance is responsible for several genetic diseases, among which Down syndrome is caused by the trisomy of human chromosome 21.

Results

To elucidate the extent to which the dosage imbalance of specific human chromosome 21 genes perturb distinct molecular pathways, we developed the first mouse embryonic stem (ES) cell bank of human chromosome 21 genes. The human chromosome 21-mouse ES cell bank includes, in triplicate clones, 32 human chromosome 21 genes, which can be overexpressed in an inducible manner. Each clone was transcriptionally profiled in inducing versus non-inducing conditions. Analysis of the transcriptional response yielded results that were consistent with the perturbed gene's known function. Comparison between mouse ES cells containing the whole human chromosome 21 (trisomic mouse ES cells) and mouse ES cells overexpressing single human chromosome 21 genes allowed us to evaluate the contribution of single genes to the trisomic mouse ES cell transcriptome. In addition, for the clones overexpressing the Runx1 gene, we compared the transcriptome changes with the corresponding protein changes by mass spectroscopy analysis.

Conclusions

We determined that only a subset of genes produces a strong transcriptional response when overexpressed in mouse ES cells and that this effect can be predicted taking into account the basal gene expression level and the protein secondary structure. We showed that the human chromosome 21-mouse ES cell bank is an important resource, which may be instrumental towards a better understanding of Down syndrome and other human aneuploidy disorders.

Sik1 (salt inducible kinase 1) is a serine/threonine kinase that belongs to the stress- and energy-sensing AMP-activated protein kinase family. During murine embryogenesis, sik1 marks the monolayer of future myocardial cells that will populate first the primitive ventricle, and later the primitive atrium suggesting its involvement in cardiac cell differentiation and/or heart development. Despite that observation, the involvement of sik1 in cardiac differentiation is still unknown. We examined the sik1 function during cardiomyocyte differentiation using the ES-derived embryoid bodies. We produced a null embryonic stem cell using a gene-trap cell line carrying an insertion in the sik1 locus. In absence of the sik1 protein, the temporal appearance of cardiomyocytes is delayed. Expression profile analysis revealed sik1 as part of a genetic network that controls the cell cycle, where the cyclin-dependent kinase inhibitor p57Kip2 is directly involved. Collectively, we provided evidence that sik1-mediated effects are specific for cardiomyogenesis regulating cardiomyoblast cell cycle exit toward terminal differentiation.

The European Mouse Mutagenesis Consortium is the European initiative contributing to the international effort on functional annotation of the mouse genome. Its objectives are to establish and integrate mutagenesis platforms, gene expression resources, phenotyping units, storage and distribution centers and bioinformatics resources. The combined efforts will accelerate our understanding of gene function and of human health and disease.

Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

Disease gene identification has made enormous strides in the past twenty years through functional, positional and candidate gene approaches, and more recently by the exploitation of genome-wide strategies. However, although pathogenic mutations in over 2000 genes have been identified as causative of human diseases, much less is known about the relationship between the molecular defects and mechanisms that lead to disease pathology and symptoms. Recent advances in diverse fields such as genomics, proteomics, cell biology, as well as studies on transgenic animals have greatly accelerated our understanding of the biochemical and cellular basis of many diseases but much still remains to be discovered. The current challenge is to understand the molecular and metabolic pathways by which a particular pathogenic variation leads to a specific phenotype. The study of abnormal conditions is of crucial importance for the understanding of normal physiology and often provides us with the rationale for the development of novel therapeutic strategies.

The protein product of the ocular albinism type 1 gene, named OA1, is a pigment cell-specific G protein-coupled receptor exclusively localized to intracellular organelles, namely lysosomes and melanosomes. Loss of OA1 function leads to the formation of macromelanosomes, suggesting that this receptor is implicated in organelle biogenesis, however the mechanism involved in the pathogenesis of the disease remains obscure. We report here the identification of an unexpected abnormality in melanosome distribution both in retinal pigment epithelium (RPE) and skin melanocytes of Oa1-knock-out (KO) mice, consisting in a displacement of the organelles from the central cytoplasm towards the cell periphery. Despite their depletion from the microtubule (MT)-enriched perinuclear region, Oa1-KO melanosomes were able to aggregate at the centrosome upon disruption of the actin cytoskeleton or expression of a dominant-negative construct of myosin Va. Consistently, quantification of organelle transport in living cells revealed that Oa1-KO melanosomes displayed a severe reduction in MT-based motility; however, this defect was rescued to normal following inhibition of actin-dependent capture at the cell periphery. Together, these data point to a defective regulation of organelle transport in the absence of OA1 and imply that the cytoskeleton might represent a downstream effector of this receptor. Furthermore, our results enlighten a novel function for OA1 in pigment cells and suggest that ocular albinism type 1 might result from a different pathogenetic mechanism than previously thought, based on an organelle-autonomous signalling pathway implicated in the regulation of both membrane traffic and transport.

Background

The TRIM family is composed of multi-domain proteins that display the Tripartite Motif (RING, B-box and Coiled-coil) that can be associated with a C-terminal domain. TRIM genes are involved in ubiquitylation and are implicated in a variety of human pathologies, from Mendelian inherited disorders to cancer, and are also involved in cellular response to viral infection.

Results

Here we defined the entire human TRIM family and also identified the TRIM sets of other vertebrate (mouse, rat, dog, cow, chicken, tetraodon, and zebrafish) and invertebrate species (fruitfly, worm, and ciona). By means of comparative analyses we found that, after assembly of the tripartite motif in an early metazoan ancestor, few types of C-terminal domains have been associated with this module during evolution and that an important increase in TRIM number occurred in vertebrate species concomitantly with the addition of the SPRY domain. We showed that the human TRIM family is split into two groups that differ in domain structure, genomic organization and evolutionary properties. Group 1 members present a variety of C-terminal domains, are highly conserved among vertebrate species, and are represented in invertebrates. Conversely, group 2 is absent in invertebrates, is characterized by the presence of a C-terminal SPRY domain and presents unique sets of genes in each mammal examined. The generation of independent sets of group 2 genes is also evident in the other vertebrate species. Comparing the murine and human TRIM sets, we found that group 1 and 2 genes evolve at different speeds and are subject to different selective pressures.

Conclusion

We found that the TRIM family is composed of two groups of genes with distinct evolutionary properties. Group 2 is younger, highly dynamic, and might act as a reservoir to develop novel TRIM functions. Since some group 2 genes are implicated in innate immune response, their evolutionary features may account for species-specific battles against viral infection.

Shroom is a recently-described regulator of cell shape changes in the developing nervous system. This protein is a member of a small family of related proteins that are defined by sequence similarity and in most cases by some link to the actin cytoskeleton. At present these proteins are named Shroom, APX, APXL, and KIAA1202. In light of the growing interest in this family of proteins, we propose here a new standard nomenclature.

In an effort to make transgenesis more flexible and reproducible, we developed a system based on novel 5′ and 3′ ‘gene trap’ vectors containing heterospecific Flp recognition target sites and the corresponding ‘exchange’ vectors allowing the insertion of any DNA sequence of interest into the trapped locus. Flp-recombinase-mediated cassette exchange was demonstrated to be highly efficient in our system, even in the absence of locus-specific selection. The feasibility of constructing a library of ES cell clones using our gene trap vectors was tested and a thousand insertion sites were characterized, following electroporation in ES cells, by RACE–PCR and sequencing. We validated the system in vivo for two trapped loci in transgenic mice and demonstrated that the reporter transgenes inserted into the trapped loci have an expression pattern identical to the endogenous genes. We believe that this system will facilitate in vivo studies of gene function and large-scale generation of mouse models of human diseases, caused by not only loss but also gain of function alleles.

The identification and study of evolutionarily conserved genomic sequences that surround disease-related genes is a valuable tool to gain insight into the functional role of these genes and to better elucidate the pathogenetic mechanisms of disease. We created the DG-CST (Disease Gene Conserved Sequence Tags) database for the identification and detailed annotation of human–mouse conserved genomic sequences that are localized within or in the vicinity of human disease-related genes. CSTs are defined as sequences that show at least 70% identity between human and mouse over a length of at least 100 bp. The database contains CST data relative to over 1088 genes responsible for monogenetic human genetic diseases or involved in the susceptibility to multifactorial/polygenic diseases. DG-CST is accessible via the internet at http://dgcst.ceinge.unina.it/ and may be searched using both simple and complex queries. A graphic browser allows direct visualization of the CSTs and related annotations within the context of the relative gene and its transcripts.

Melanogenesis is the process that regulates skin and eye pigmentation. Albinism, a genetic disease causing pigmentation defects and visual disorders, is caused by mutations in genes controlling either melanin synthesis or melanosome biogenesis. Here we show that a common transcriptional control regulates both of these processes. We performed an analysis of the regulatory region of Oa1, the murine homolog of the gene that is mutated in the X-linked form of ocular albinism, as Oa1's function affects melanosome biogenesis. We demonstrated that Oa1 is a target of Mitf and that this regulatory mechanism is conserved in the human gene. Tissue-specific control of Oa1 transcription lies within a region of 617 bp that contains the E-box bound by Mitf. Finally, we took advantage of a virus-based system to assess tissue specificity in vivo. To this end, a small fragment of the Oa1 promoter was cloned in front of a reporter gene in an adeno-associated virus. After we injected this virus into the subretinal space, we observed reporter gene expression specifically in the retinal pigment epithelium, confirming the cell-specific expression of the Oa1 promoter in the eye. The results obtained with this viral system are a preamble to the development of new gene delivery approaches for the treatment of retinal pigment epithelium defects.

Mmutations in paraplegin, a putative mitochondrial metallopeptidase of the AAA family, cause an autosomal recessive form of hereditary spastic paraplegia (HSP). Here, we analyze the function of paraplegin at the cellular level and characterize the phenotypic defects of HSP patients' cells lacking this protein. We demonstrate that paraplegin coassembles with a homologous protein, AFG3L2, in the mitochondrial inner membrane. These two proteins form a high molecular mass complex, which we show to be aberrant in HSP fibroblasts. The loss of this complex causes a reduced complex I activity in mitochondria and an increased sensitivity to oxidant stress, which can both be rescued by exogenous expression of wild-type paraplegin. Furthermore, complementation studies in yeast demonstrate functional conservation of the human paraplegin–AFG3L2 complex with the yeast m-AAA protease and assign proteolytic activity to this structure. These results shed new light on the molecular pathogenesis of HSP and functionally link AFG3L2 to this neurodegenerative disease.

In several neurodegenerative diseases, axonal degeneration occurs before neuronal death and contributes significantly to patients’ disability. Hereditary spastic paraplegia (HSP) is a genetically heterogeneous condition characterized by selective degeneration of axons of the corticospinal tracts and fasciculus gracilis. HSP may therefore be considered an exemplary disease to study the local programs mediating axonal degeneration. We have developed a mouse model for autosomal recessive HSP due to mutations in the SPG7 gene encoding the mitochondrial ATPase paraplegin. Paraplegin-deficient mice are affected by a distal axonopathy of spinal and peripheral axons, characterized by axonal swelling and degeneration. We found that mitochondrial morphological abnormalities occurred in synaptic terminals and in distal regions of axons long before the first signs of swelling and degeneration and correlated with onset of motor impairment during a rotarod test. Axonal swellings occur through massive accumulation of organelles and neurofilaments, suggesting impairment of anterograde axonal transport. Retrograde axonal transport is delayed in symptomatic mice. We speculate that local failure of mitochondrial function may affect axonal transport and cause axonal degeneration. Our data suggest that a timely therapeutic intervention may prevent the loss of axons.