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.

RagD auto-activating mutations impair MiT/TFE activity in kidney tubulopathy and cardiomyopathy syndrome.

Heterozygous mutations in the gene encoding RagD GTPase were shown to cause a novel autosomal dominant condition characterized by kidney tubulopathy and cardiomyopathy. We previously demonstrated that RagD, and its paralogue RagC, mediate a non-canonical mTORC1 signaling pathway that inhibits the activity of TFEB and TFE3, transcription factors of the MiT/TFE family and master regulators of lysosomal biogenesis and autophagy. Here we show that RagD mutations causing kidney tubulopathy and cardiomyopathy are "auto- activating", even in the absence of Folliculin, the GAP responsible for RagC/D activation, and cause constitutive phosphorylation of TFEB and TFE3 by mTORC1, without affecting the phosphorylation of "canonical" mTORC1 substrates, such as S6K.

Drug screening identifies tazarotene and bexarotene as therapeutic agents in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD, MIM #272200) results from pathogenic variants in the SUMF1 gene that impair proper function of the formylglycine-generating enzyme (FGE). FGE is essential for the posttranslational activation of cellular sulfatases. MSD patients display reduced or absent sulfatase activities and, as a result, clinical signs of single sulfatase disorders in a unique combination.

Structure of the lysosomal mTORC1-TFEB-Rag-Ragulator megacomplex.

The transcription factor TFEB is a master regulator of lysosomal biogenesis and autophagy. The phosphorylation of TFEB by the mechanistic target of rapamycin complex 1 (mTORC1) is unique in its mTORC1 substrate recruitment mechanism, which is strictly dependent on the amino acid-mediated activation of the RagC GTPase activating protein FLCN. TFEB lacks the TOR signalling motif responsible for the recruitment of other mTORC1 substrates.

Differential dysregulation of granule subsets in WASH-deficient neutrophil leukocytes resulting in inflammation.

Dysregulated secretion in neutrophil leukocytes associates with human inflammatory disease. The exocytosis response to triggering stimuli is sequential; gelatinase granules modulate the initiation of the innate immune response, followed by the release of pro-inflammatory azurophilic granules, requiring stronger stimulation. Exocytosis requires actin depolymerization which is actively counteracted under non-stimulatory conditions.

Repurposing of tamoxifen ameliorates CLN3 and CLN7 disease phenotype.

Batten diseases (BDs) are a group of lysosomal storage disorders characterized by seizure, visual loss, and cognitive and motor deterioration. We discovered increased levels of globotriaosylceramide (Gb3) in cellular and murine models of CLN3 and CLN7 diseases and used fluorescent-conjugated bacterial toxins to label Gb3 to develop a cell-based high content imaging (HCI) screening assay for the repurposing of FDA-approved compounds able to reduce this accumulation within BD cells. We found that tamoxifen reduced the lysosomal accumulation of Gb3 in CLN3 and CLN7 cell models, including neuronal progenitor cells (NPCs) from CLN7 patient-derived induced pluripotent stem cells (iPSC).

Altered heparan sulfate metabolism during development triggers dopamine-dependent autistic-behaviours in models of lysosomal storage disorders.

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function.

A substrate-specific mTORC1 pathway underlies Birt-Hogg-Dubé syndrome.

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases.

mTOR-dependent phosphorylation controls TFEB nuclear export.

During starvation the transcriptional activation of catabolic processes is induced by the nuclear translocation and consequent activation of transcription factor EB (TFEB), a master modulator of autophagy and lysosomal biogenesis. However, how TFEB is inactivated upon nutrient refeeding is currently unknown. Here we show that TFEB subcellular localization is dynamically controlled by its continuous shuttling between the cytosol and the nucleus, with the nuclear export representing a limiting step.

mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy.

The mammalian target of rapamycin complex 1 (mTORC1) kinase promotes cell growth by activating biosynthetic pathways and suppressing catabolic pathways, particularly that of macroautophagy. A prerequisite for mTORC1 activation is its translocation to the lysosomal surface. Deregulation of mTORC1 has been associated with the pathogenesis of several diseases, but its role in skeletal disorders is largely unknown.

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.

Munc13-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses.

The molecular mechanisms that regulate late endosomal maturation and function are not completely elucidated, and direct evidence of a calcium sensor is lacking. Here we identify a novel mechanism of late endosomal maturation that involves a new molecular interaction between the tethering factor Munc13-4, syntaxin 7, and VAMP8. Munc13-4 binding to syntaxin 7 was significantly increased by calcium.

Impairment of chaperone-mediated autophagy leads to selective lysosomal degradation defects in the lysosomal storage disease cystinosis.

Metabolite accumulation in lysosomal storage disorders (LSDs) results in impaired cell function and multi-systemic disease. Although substrate reduction and lysosomal overload-decreasing therapies can ameliorate disease progression, the significance of lysosomal overload-independent mechanisms in the development of cellular dysfunction is unknown for most LSDs. Here, we identify a mechanism of impaired chaperone-mediated autophagy (CMA) in cystinosis, a LSD caused by defects in the cystine transporter cystinosin (CTNS) and characterized by cystine lysosomal accumulation.

Upregulation of the Rab27a-dependent trafficking and secretory mechanisms improves lysosomal transport, alleviates endoplasmic reticulum stress, and reduces lysosome overload in cystinosis.

Cystinosis is a lysosomal storage disorder caused by the accumulation of the amino acid cystine due to genetic defects in the CTNS gene, which encodes cystinosin, the lysosomal cystine transporter. Although many cellular dysfunctions have been described in cystinosis, the mechanisms leading to these defects are not well understood. Here, we show that increased lysosomal overload induced by accumulated cystine leads to cellular abnormalities, including vesicular transport defects and increased endoplasmic reticulum (ER) stress, and that correction of lysosomal transport improves cellular function in cystinosis.

MUNC13-4 protein regulates the oxidative response and is essential for phagosomal maturation and bacterial killing in neutrophils.

Neutrophils use diverse mechanisms to kill pathogens including phagocytosis, exocytosis, generation of reactive oxygen species (ROS), and neutrophil extracellular traps. These mechanisms rely on their ability to mobilize intracellular organelles and to deliver granular cargoes to specific cellular compartments or into the extracellular milieu, but the molecular mechanisms regulating vesicular trafficking in neutrophils are not well understood. MUNC13-4 is a RAB27A effector that coordinates exocytosis in hematopoietic cells, and its deficiency is associated with the human immunodeficiency familial hemophagocytic lymphohistiocytosis type 3.

Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFC1/Slp1 and the RhoA-GTPase-activating protein Gem-interacting protein.

Cytoskeleton remodeling is important for the regulation of vesicular transport associated with exocytosis, but a direct association between granular secretory proteins and actin-remodeling molecules has not been shown, and this mechanism remains obscure. Using a proteomic approach, we identified the RhoA-GTPase-activating protein Gem-interacting protein (GMIP) as a factor that associates with the Rab27a effector JFC1 and modulates vesicular transport and exocytosis. GMIP down-regulation induced RhoA activation and actin polymerization.

Increased survival and reduced neutrophil infiltration of the liver in Rab27a- but not Munc13-4-deficient mice in lipopolysaccharide-induced systemic inflammation.

Genetic defects in the Rab27a or Munc13-4 gene lead to immunodeficiencies in humans, characterized by frequent viral and bacterial infections. However, the role of Rab27a and Munc13-4 in the regulation of systemic inflammation initiated by Gram-negative bacterium-derived pathogenic molecules is currently unknown. Using a model of lipopolysaccharide-induced systemic inflammation, we show that Rab27a-deficient (Rab27a(ash/ash)) mice are resistant to lipopolysaccharide (LPS)-induced death, while Munc13-4-deficient (Munc13-4(jinx/jinx)) mice show only moderate protection.

Munc13-4 restricts motility of Rab27a-expressing vesicles to facilitate lipopolysaccharide-induced priming of exocytosis in neutrophils.

LPS is an efficient sensitizer of the neutrophil exocytic response to a second stimulus. Although neutrophil exocytosis in response to pathogen-derived molecules plays an important role in the innate immune response to infections, the molecular mechanism underlying LPS-dependent regulation of neutrophil exocytosis is currently unknown. The small GTPase Rab27a and its effector Munc13-4 regulate exocytosis in hematopoietic cells.

Functional characterization of Wiskott-Aldrich syndrome protein and scar homolog (WASH), a bi-modular nucleation-promoting factor able to interact with biogenesis of lysosome-related organelle subunit 2 (BLOS2) and gamma-tubulin.

The Arp2/3 complex is essential for actin filament nucleation in a variety of cellular processes. The activation of the Arp2/3 complex is mediated by nucleation-promoting factors, such as the Wiskott-Aldrich syndrome family proteins, which share a WCA (WH2 domain, central region, acidic region) catalytic module at the C-terminal region, required for Arp2/3 activation, but diverge at the N-terminal region, required for binding to specific activators. Here, we report the characterization of WASH, a new member of the WAS family that has nucleation-promoting factor activity and recently has been demonstrated to play a role in endosomal sorting.

NESCA: a new NEMO/IKKgamma and TRAF6 interacting protein.

NEMO/IKKgamma is the essential regulatory subunit of the IkB Kinase (IKK) complex, required for the activation of Nuclear Factor kB (NF-kB) in many physiological processes such as inflammation, immunity, apoptosis, or development. NEMO works at a converging point of the NF-kB pathway as it interacts with upstream signaling molecules to orchestrate its activation. Here we report on the identification of a novel NEMO-interacting protein, NESCA, an adapter molecule previously shown to be involved in the NGF-pathway via the TrkA receptor.

MRX87 family with Aristaless X dup24bp mutation and implication for polyAlanine expansions.

Background: Cognitive impairments are heterogeneous conditions, and it is estimated that 10% may be caused by a defect of mental function genes on the X chromosome. One of those genes is Aristaless related homeobox (ARX) encoding a polyA-rich homeobox transcription factor essential for cerebral patterning and its mutations cause different neurologic disorders. We reported on the clinical and genetic analysis of an Italian family with X-linked mental retardation (XLMR) and intra-familial heterogeneity, and provided insight into its molecular defect.