TRPMLs (or mucolipins) constitute a family of endosomal cation channels with homology to the transient receptor potential superfamily. In mammals, the TRPML family includes three members: TRPML1-3. Although TRPML1 and TRPML3 have been well characterized, the cellular function of TRPML2 has remained elusive. To address TRPML2 function in a physiologically relevant cell type, we first analyzed TRPML2 expression in different mouse tissues and organs and found that it was predominantly expressed in lymphoid organs and kidney. Quantitative RT-PCR revealed tight regulation of TRPML2 at the transcriptional level. Although TRPML2 expression was negligible in resting macrophages, TRPML2 mRNA and protein levels dramatically increased in response to TLR activation both in vitro and in vivo. Conversely, TRPML1 and TRPML3 levels did not change upon TLR activation. Immunofluorescence analysis demonstrated that endogenous TRPML2 primarily localized to recycling endosomes both in culture and primary cells, in contrast with TRPML1 and TRPML3, which distribute to the late and early endosomal pathway, respectively. To better understand the in vivo function of TRPML2, we generated a TRPML2-knockout mouse. We found that the production of several chemokines, in particular CCL2, was severely reduced in TRPML2-knockout mice. Furthermore, TRPML2-knockout mice displayed impaired recruitment of peripheral macrophages in response to i.p. injections of LPS or live bacteria, suggesting a potential defect in the immune response. Overall, our study reveals interesting differences in the regulation and distribution of the members of the TRPML family and identifies a novel role for TRPML2 in the innate immune response.

The dry (nonneovascular) form of age-related macular degeneration (AMD), a leading cause of blindness in the elderly, has few, if any, treatment options at present. It is characterized by early accumulation of cellular waste products in the retinal pigmented epithelium (RPE); rejuvenating impaired lysosome function in RPE is a well-justified target for treatment. It is now clear that amino acids and vacuolar-type H+ -ATPase (V-ATPase) regulate the mechanistic target of rapamycin, complex 1 (mTORC1) signaling in lysosomes. Here, we provide evidence for the first time that the amino acid transporter SLC36A4/proton-dependent amino acid transporter (PAT4) regulates the amino acid pool in the lysosomes of RPE. In Cryba1 (gene encoding βA3/A1-crystallin) KO (knockout) mice, where PAT4 and amino acid levels are increased in the RPE, the transcription factors EB (TFEB) and E3 (TFE3) are retained in the cytoplasm, even after 24 h of fasting. Consequently, genes in the coordinated lysosomal expression and regulation (CLEAR) network are not activated, and lysosomal function remains low. As these mice age, expression of RPE65 and lecithin retinol acyltransferase (LRAT), two vital visual cycle proteins, decreases in the RPE. A defective visual cycle would possibly slow down the regeneration of new photoreceptor outer segments (POS). Further, photoreceptor degeneration also becomes obvious during aging, reminiscent of human dry AMD disease. Electron microscopy shows basal laminar deposits in Bruch's membrane, a hallmark of development of AMD. For dry AMD patients, targeting PAT4/V-ATPase in the lysosomes of RPE cells may be an effective means of preventing or delaying disease progression.

Endolysosomes are dynamic, intracellular compartments, regulating their surface-to-volume ratios to counteract membrane swelling or shrinkage caused by osmotic challenges upon tubulation and vesiculation events. While osmosensitivity has been extensively described on the plasma membrane, the mechanisms underlying endolysosomal surface-to-volume ratio changes and identities of involved ion channels remain elusive. Endolysosomes mediate endocytosis, exocytosis, cargo transport, and sorting of material for recycling or degradation. We demonstrate the endolysosomal cation channel TRPML2 to be hypotonicity/mechanosensitive, a feature crucial to its involvement in fast-recycling processes of immune cells. We demonstrate that the phosphoinositide binding pocket is required for TRPML2 hypotonicity-sensitivity, as substitution of L314 completely abrogates hypotonicity-sensitivity. Last, the hypotonicity-insensitive TRPML2 mutant L314R slows down the fast recycling pathway, corroborating the functional importance of hypotonicity-sensitive TRPML2. Our results highlight TRPML2 as an accelerator of endolysosomal trafficking by virtue of its hypotonicity-sensitivity, with implications in immune cell surveillance and viral trafficking.

Beta-coronaviruses have emerged as a severe threat to global health. Undercovering the interplay between host and beta-coronaviruses is essential for understanding disease pathogenesis and developing efficient treatments. Here we report that the transcription factors TFEB and TFE3 translocate from the cytosol to the nucleus in response to beta-coronavirus infection by a mechanism that requires activation of calcineurin phosphatase. In the nucleus, TFEB and TFE3 bind to the promoter of multiple lysosomal and immune genes. Accordingly, MHV-induced upregulation of immune regulators is significantly decreased in TFEB/TFE3-depleted cells. Conversely, over-expression of either TFEB or TFE3 is sufficient to increase expression of several cytokines and chemokines. The reduced immune response observed in the absence of TFEB and TFE3 results in increased cellular survival of infected cells but also in reduced lysosomal exocytosis and decreased viral infectivity. These results suggest a central role of TFEB and TFE3 in cellular response to beta-coronavirus infection.

Dysregulation of alternative splicing in prostate cancer is linked to transcriptional programs activated by AR, ERG, FOXA1, and MYC. Here, we show that FOXA1 functions as the primary orchestrator of alternative splicing dysregulation across 500 primary and metastatic prostate cancer transcriptomes. We demonstrate that FOXA1 binds to the regulatory regions of splicing-related genes, including HNRNPK and SRSF1. By controlling trans-acting factor expression, FOXA1 exploits an "exon definition" mechanism calibrating alternative splicing toward dominant isoform production. This regulation especially impacts splicing factors themselves and leads to a reduction of nonsense-mediated decay (NMD)-targeted isoforms. Inclusion of the NMD-determinant FLNA exon 30 by FOXA1-controlled oncogene SRSF1 promotes cell growth in vitro and predicts disease recurrence. Overall, we report a role for FOXA1 in rewiring the alternative splicing landscape in prostate cancer through a cascade of events from chromatin access, to splicing factor regulation, and, finally, to alternative splicing of exons influencing patient survival.

There is growing evidence supporting the role of fibroblasts in all stages of atherosclerosis, from the initial phase to fibrous cap and plaque formation. In the arterial wall, as with macrophages and vascular smooth muscle cells, fibroblasts are exposed to a myriad of LDL lipids, including the lipid species formed during the oxidation of their polyunsaturated fatty acids of cholesteryl esters (PUFA-CEs). Recently, our group identified the final oxidation products of the PUFA-CEs, cholesteryl hemiesters (ChE), in tissues from cardiovascular disease patients. Cholesteryl hemiazelate (ChA), the most prevalent lipid of this family, is sufficient to impact lysosome function in macrophages and vascular smooth muscle cells, with consequences for their homeostasis. Here, we show that the lysosomal compartment of ChA-treated fibroblasts also becomes dysfunctional. Indeed, fibroblasts exposed to ChA exhibited a perinuclear accumulation of enlarged lysosomes full of neutral lipids. However, this outcome did not trigger de novo lysosome biogenesis, and only the lysosomal transcription factor E3 (TFE3) was slightly transcriptionally upregulated. As a consequence, autophagy was inhibited, probably via mTORC1 activation, culminating in fibroblasts' apoptosis. Our findings suggest that the impairment of lysosome function and autophagy and the induction of apoptosis in fibroblasts may represent an additional mechanism by which ChA can contribute to the progression of atherosclerosis.

TFEB and TFE3 are transcriptional regulators of the innate immune response, but the mechanisms regulating their activation upon pathogen infection are poorly elucidated. Using C. elegans and mammalian models, we report that the master metabolic modulator 5'-AMP-activated protein kinase (AMPK) and its negative regulator Folliculin (FLCN) act upstream of TFEB/TFE3 in the innate immune response, independently of the mTORC1 signaling pathway. In nematodes, loss of FLCN or overexpression of AMPK confers pathogen resistance via activation of TFEB/TFE3-dependent antimicrobial genes, whereas ablation of total AMPK activity abolishes this phenotype. Similarly, in mammalian cells, loss of FLCN or pharmacological activation of AMPK induces TFEB/TFE3-dependent pro-inflammatory cytokine expression. Importantly, a rapid reduction in cellular ATP levels in murine macrophages is observed upon lipopolysaccharide (LPS) treatment accompanied by an acute AMPK activation and TFEB nuclear localization. These results uncover an ancient, highly conserved, and pharmacologically actionable mechanism coupling energy status with innate immunity.

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.

To reestablish homeostasis and mitigate stress, cells must activate a series of adaptive intracellular signaling pathways. The participation of the transcription factors TFEB and TFE3 in cellular adaptation to starvation is well established. Here, we show that TFEB and TFE3 also play an important role in the cellular response to ER stress. Treatment with ER stressors causes translocation of TFEB and TFE3 to the nucleus in a process that is dependent on PERK and calcineurin but not on mTORC1. Activated TFEB and TFE3 enhance cellular response to stress by inducing direct transcriptional upregulation of ATF4 and other UPR genes. Under conditions of prolonged ER stress, TFEB and TFE3 contribute to cell death, thus revealing an unexpected role for these proteins in controlling cell fate. This work evidences a broader role of TFEB and TFE3 in the cellular response to stress than previously anticipated and reveals an integrated cooperation between different cellular stress pathways.

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.

Regenerative medicine holds great promise for both degenerative diseases and traumatic tissue injury which represent significant challenges to the health care system. Hearing loss, which affects hundreds of millions of people worldwide, is caused primarily by a permanent loss of the mechanosensory receptors of the inner ear known as hair cells. This failure to regenerate hair cells after loss is limited to mammals, while all other non-mammalian vertebrates tested were able to completely regenerate these mechanosensory receptors after injury. To understand the mechanism of hair cell regeneration and its association with regeneration of other tissues, we performed a guided mutagenesis screen using zebrafish lateral line hair cells as a screening platform to identify genes that are essential for hair cell regeneration, and further investigated how genes essential for hair cell regeneration were involved in the regeneration of other tissues. We created genetic mutations either by retroviral insertion or CRISPR/Cas9 approaches, and developed a high-throughput screening pipeline for analyzing hair cell development and regeneration. We screened 254 gene mutations and identified 7 genes specifically affecting hair cell regeneration. These hair cell regeneration genes fell into distinct and somewhat surprising functional categories. By examining the regeneration of caudal fin and liver, we found these hair cell regeneration genes often also affected other types of tissue regeneration. Therefore, our results demonstrate guided screening is an effective approach to discover regeneration candidates, and hair cell regeneration is associated with other tissue regeneration.

Recently, it has been shown that targeted mutagenesis using zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to generate knockout zebrafish lines for analysis of their function and/or developing disease models. A number of different methods have been developed for the design and assembly of gene-specific ZFNs and TALENs, making them easily available to most zebrafish researchers. Regardless of the choice of targeting nuclease, the process of generating mutant fish is similar. It is a time-consuming and multi-step process that can benefit significantly from development of efficient high throughput methods. In this study, we used ZFNs assembled through either the CompoZr (Sigma-Aldrich) or the CoDA (context-dependent assembly) platforms to generate mutant zebrafish for nine genes. We report our improved high throughput methods for 1) evaluation of ZFNs activity by somatic lesion analysis using colony PCR, eliminating the need for plasmid DNA extractions from a large number of clones, and 2) a sensitive founder screening strategy using fluorescent PCR with PIG-tailed primers that eliminates the stutter bands and accurately identifies even single nucleotide insertions and deletions. Using these protocols, we have generated multiple mutant alleles for seven genes, five of which were targeted with CompoZr ZFNs and two with CoDA ZFNs. Our data also revealed that at least five-fold higher mRNA dose was required to achieve mutagenesis with CoDA ZFNs than with CompoZr ZFNs, and their somatic lesion frequency was lower (<5%) when compared to CopmoZr ZFNs (9-98%). This work provides high throughput protocols for efficient generation of zebrafish mutants using ZFNs and TALENs.

Noncanonical mechanistic target of rapamycin (mTOR) pathways remain poorly understood. Mutations in the tumor suppressor folliculin (FLCN) cause Birt-Hogg-Dubé syndrome, a hamartomatous disease marked by mitochondria-rich kidney tumors. FLCN functionally interacts with mTOR and is expressed in most tissues, but its role in fat has not been explored. We show here that FLCN regulates adipose tissue browning via mTOR and the transcription factor TFE3. Adipose-specific deletion of FLCN relieves mTOR-dependent cytoplasmic retention of TFE3, leading to direct induction of the PGC-1 transcriptional coactivators, drivers of mitochondrial biogenesis and the browning program. Cytoplasmic retention of TFE3 by mTOR is sensitive to ambient amino acids, is independent of growth factor and tuberous sclerosis complex (TSC) signaling, is driven by RagC/D, and is separable from canonical mTOR signaling to S6K. Codeletion of TFE3 in adipose-specific FLCN knockout animals rescues adipose tissue browning, as does codeletion of PGC-1β. Conversely, inducible expression of PGC-1β in white adipose tissue is sufficient to induce beige fat gene expression in vivo. These data thus unveil a novel FLCN-mTOR-TFE3-PGC-1β pathway-separate from the canonical TSC-mTOR-S6K pathway-that regulates browning of adipose tissue.

Mammalian TFEB and TFE3, as well as their ortholog in Caenorhabditis elegans HLH-30, play an important role in mediating cellular response to a variety of stress conditions, including nutrient deprivation, oxidative stress, and pathogen infection. In this study, we identify a novel mechanism of TFEB/HLH-30 regulation through a cysteine-mediated redox switch. Under stress conditions, TFEB-C212 undergoes oxidation, allowing the formation of intermolecular disulfide bonds that result in TFEB oligomerization. TFEB oligomers display increased resistance to mTORC1-mediated inactivation and are more stable under prolonged stress conditions. Mutation of the only cysteine residue present in HLH-30 (C284) significantly reduced its activity, resulting in developmental defects and increased pathogen susceptibility in worms. Therefore, cysteine oxidation represents a new type of TFEB post-translational modification that functions as a molecular switch to link changes in redox balance with expression of TFEB/HLH-30 target genes.

Gene therapy is under advanced clinical development for several lysosomal storage disorders. Pompe disease, a debilitating neuromuscular illness affecting infants, children, and adults with different severity, is caused by a deficiency of lysosomal glycogen-degrading enzyme acid α-glucosidase (GAA). Here, we demonstrated that adeno-associated virus-mediated (AAV-mediated) systemic gene transfer reversed glycogen storage in all key therapeutic targets - skeletal and cardiac muscles, the diaphragm, and the central nervous system - in both young and severely affected old Gaa-knockout mice. Furthermore, the therapy reversed secondary cellular abnormalities in skeletal muscle, such as those in autophagy and mTORC1/AMPK signaling. We used an AAV9 vector encoding a chimeric human GAA protein with enhanced uptake and secretion to facilitate efficient spread of the expressed protein among multiple target tissues. These results lay the groundwork for a future clinical development strategy in Pompe disease.

The transcription factors TFE3 and TFEB cooperate to regulate autophagy induction and lysosome biogenesis in response to starvation. Here we demonstrate that DNA damage activates TFE3 and TFEB in a p53 and mTORC1 dependent manner. RNA-Seq analysis of TFEB/TFE3 double-knockout cells exposed to etoposide reveals a profound dysregulation of the DNA damage response, including upstream regulators and downstream p53 targets. TFE3 and TFEB contribute to sustain p53-dependent response by stabilizing p53 protein levels. In TFEB/TFE3 DKOs, p53 half-life is significantly decreased due to elevated Mdm2 levels. Transcriptional profiles of genes involved in lysosome membrane permeabilization and cell death pathways are dysregulated in TFEB/TFE3-depleted cells. Consequently, prolonged DNA damage results in impaired LMP and apoptosis induction. Finally, expression of multiple genes implicated in cell cycle control is altered in TFEB/TFE3 DKOs, revealing a previously unrecognized role of TFEB and TFE3 in the regulation of cell cycle checkpoints in response to stress.

Signal-transducing adaptor molecules (STAMs) are involved in growth factor and cytokine signaling as well as receptor degradation, and they form complexes with a number of endocytic proteins, including Hrs and Eps15. In this study, we demonstrate that STAM proteins also localize prominently to early exocytic compartments and profoundly regulate Golgi morphology. Upon STAM overexpression in cells, the Golgi apparatus becomes extensively fragmented and dispersed, but when STAMs are depleted, the Golgi becomes highly condensed. Under both scenarios, vesicular stomatitis virus G protein-green fluorescent protein trafficking to the plasma membrane is markedly inhibited, and recovery of Golgi morphology after Brefeldin A treatment is substantially impaired in STAM-depleted cells. Furthermore, STAM proteins interact with coat protein II (COPII) proteins, probably at endoplasmic reticulum (ER) exit sites, and Sar1 activity is required to maintain the localization of STAMs at discrete sites. Thus, in addition to their roles in signaling and endocytosis, STAMs function prominently in ER-to-Golgi trafficking, most likely through direct interactions with the COPII complex.

The MAL protein is an essential component of the specialized machinery for apical targeting in epithelial cells. The src family kinase Lck plays a pivotal role in T cell signaling. We show that MAL is required in T cells for efficient expression of Lck at the plasma membrane and activation of IL-2 transcription. To investigate the mechanism by which MAL regulates Lck targeting, we analyzed the dynamics of Lck and found that it travels to the plasma membrane in specific transport carriers containing MAL. Coimmunoprecipitation experiments indicated an association of MAL with Lck. Both carrier formation and partitioning of Lck into detergent-insoluble membranes were ablated in the absence of MAL. Polarization of T cell receptor for antigen (TCR) and microtubule-organizing center to immunological synapse (IS) were also defective. Although partial correction of the latter defects was possible by forced expression of Lck at the plasma membrane, their complete correction, formation of transport vesicles, partitioning of Lck, and restoration of signaling pathways, which are required for IL-2 transcription up-regulation, were achieved by exogenous expression of MAL. We concluded that MAL is required for recruitment of Lck to specialized membranes and formation of specific transport carriers for Lck targeting. This novel transport pathway is crucial for TCR-mediated signaling and IS assembly.

Mutations in the gene AK2 are responsible for reticular dysgenesis (RD), a rare and severe form of primary immunodeficiency in children. RD patients have a severely shortened life expectancy and without treatment die, generally from sepsis soon after birth. The only available therapeutic option for RD is hematopoietic stem cell transplantation (HSCT). To gain insight into the pathophysiology of RD, we previously created zebrafish models for Ak2 deficiencies. One of the clinical features of RD is hearing loss, but its pathophysiology and causes have not been determined. In adult mammals, sensory hair cells of the inner ear do not regenerate; however, their regeneration has been observed in several non-mammalian vertebrates, including zebrafish. Therefore, we used our RD zebrafish models to determine whether Ak2 deficiency affects sensory organ development and/or hair cell regeneration. Our studies indicated that Ak2 is required for the correct development, survival and regeneration of sensory hair cells. Interestingly, Ak2 deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in the hair cells. Finally, we show that glutathione treatment can partially rescue hair cell development in the sensory organs in our RD models, pointing to the potential use of antioxidants as a therapeutic treatment supplementing HSCT to prevent or ameliorate sensorineural hearing deficits in RD patients.

Human genetic diversity can reveal critical factors in host-pathogen interactions. This is especially useful for human-restricted pathogens like Salmonella enterica serovar Typhi (S. Typhi), the cause of typhoid fever. One key defense during bacterial infection is nutritional immunity: host cells attempt to restrict bacterial replication by denying bacteria access to key nutrients or supplying toxic metabolites. Here, a cellular genome-wide association study of intracellular replication by S. Typhi in nearly a thousand cell lines from around the world-and extensive follow-up using intracellular S. Typhi transcriptomics and manipulation of magnesium availability-demonstrates that the divalent cation channel mucolipin-2 (MCOLN2 or TRPML2) restricts S. Typhi intracellular replication through magnesium deprivation. Mg2+ currents, conducted through MCOLN2 and out of endolysosomes, were measured directly using patch-clamping of the endolysosomal membrane. Our results reveal Mg2+ limitation as a key component of nutritional immunity against S. Typhi and as a source of variable host resistance.