EGR1 drives cell proliferation by directly stimulating TFEB transcription in response to starvation.

The stress-responsive transcription factor EB (TFEB) is a master controller of lysosomal biogenesis and autophagy and plays a major role in several cancer-associated diseases. TFEB is regulated at the posttranslational level by the nutrient-sensitive kinase complex mTORC1. However, little is known about the regulation of TFEB transcription. Here, through integrative genomic approaches, we identify the immediate-early gene EGR1 as a positive transcriptional regulator of TFEB expression in human cells and demonstrate that, in the absence of EGR1, TFEB-mediated transcriptional response to starvation is impaired. Remarkably, both genetic and pharmacological inhibition of EGR1, using the MEK1/2 inhibitor Trametinib, significantly reduced the proliferation of 2D and 3D cultures of cells displaying constitutive activation of TFEB, including those from a patient with Birt-Hogg-Dubé (BHD) syndrome, a TFEB-driven inherited cancer condition. Overall, we uncover an additional layer of TFEB regulation consisting in modulating its transcription via EGR1 and propose that interfering with the EGR1-TFEB axis may represent a therapeutic strategy to counteract constitutive TFEB activation in cancer-associated conditions.

Signals from the lysosome: a control centre for cellular clearance and energy metabolism.

For a long time, lysosomes were considered merely to be cellular 'incinerators' involved in the degradation and recycling of cellular waste. However, now there is compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signalling and energy metabolism. Furthermore, the essential role of lysosomes in autophagic pathways puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master regulator, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy has revealed how the lysosome adapts to environmental cues, such as starvation, and targeting TFEB may provide a novel therapeutic strategy for modulating lysosomal function in human disease.

Brain Disorders Due to Lysosomal Dysfunction.

Recent studies of autophagic and lysosomal pathways have significantly changed our understanding of lysosomes; once thought to be simple degradative and recycling centers, lysosomes are now known to be organelles capable of influencing signal transduction, via the mammalian target of rapamycin complex 1 (mTORC1), and regulating gene expression, via transcription factor EB (TFEB) and other transcription factors. These pathways are particularly relevant to maintaining brain homeostasis, as dysfunction of the endolysosomal and autophagic pathways has been associated with common neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's, and lysosomal storage disorders, a group of inherited disorders characterized by the intralysosomal buildup of partially degraded metabolites. This review focuses on the cellular biology of lysosomes and discusses the possible mechanisms by which disruption of their function contributes to neurodegeneration. We also review and discuss how targeting TFEB and lysosomes may offer innovative therapeutic approaches for treating a wide range of neurological conditions.

Investigation on lysosomal accumulation by a quantitative analysis of 2D phase-maps in digital holography microscopy.

Lysosomes are the terminal end of catabolic pathways in the cell, as well as signaling centers performing important functions such as the recycling of macromolecules, organelles, and nutrient adaptation. The importance of lysosomes in human health is supported by the fact that the deficiency of most lysosomal genes causes monogenic diseases called as a group Lysosomal Storage Diseases (LSDs). A common phenotypic hallmark of LSDs is the expansion of the lysosomal compartment that can be detected by using conventional imaging methods based on immunofluorescence protocols or overexpression of tagged lysosomal proteins. These methods require the alteration of the cellular architecture (i.e., due to fixation methods), can alter the behavior of cells (i.e., by the overexpression of proteins), and require sample preparation and the accurate selection of compatible fluorescent markers in relation to the type of analysis, therefore limiting the possibility of characterizing cellular status with simplicity. Therefore, a quantitative and label-free methodology, such as Quantitative Phase Imaging through Digital Holographic (QPI-DH), for the microscopic imaging of lysosomes in health and disease conditions may represent an important advance to study and effectively diagnose the presence of lysosomal storage in human disease. Here we proof the effectiveness of the QPI-DH method in accomplishing the detection of the lysosomal compartment using mouse embryonic fibroblasts (MEFs) derived from a Mucopolysaccharidosis type III-A (MSP-IIIA) mouse model, and comparing them with wild-type (WT) MEFs. We found that it is possible to identify label-free biomarkers able to supply a first pre-screening of the two populations, thus showing that QPI-DH can be a suitable candidate to surpass fluorescent drawbacks in the detection of lysosomes dysfunction. An appropriate numerical procedure was developed for detecting and evaluate such cellular substructures from in vitro cells cultures. Results reported in this study are encouraging about the further development of the proposed QPI-DH approach for such type of investigations about LSDs.

Cellular and Gene Expression Response to the Combination of Genistein and Kaempferol in the Treatment of Mucopolysaccharidosis Type I.

Flavonoids are investigated as therapeutics for mucopolysaccharidosis, a metabolic disorder with impaired glycosaminoglycan degradation. Here we determined the effects of genistein and kaempferol, used alone or in combination, on cellular response and gene expression in a mucopolysaccharidosis type I model. We assessed the cell cycle, viability, proliferation, subcellular localization of the translocation factor EB (TFEB), number and distribution of lysosomes, and glycosaminoglycan synthesis after exposure to flavonoids. Global gene expression was analysed using DNA microarray and quantitative PCR. The type and degree of flavonoid interaction were determined based on the combination and dose reduction indexes. The combination of both flavonoids synergistically inhibits glycosaminoglycan synthesis, modulates TFEB localization, lysosomal number, and distribution. Genistein and kaempferol in a 1:1 ratio regulate the expression of 52% of glycosaminoglycan metabolism genes. Flavonoids show synergy, additivity, or slight antagonism in all analysed parameters, and the type of interaction depends on the concentration and component ratios. With the simultaneous use of genistein and kaempferol in a ratio of 4:1, even a 10-fold reduction in the concentration of kaempferol is possible. Flavonoid mixtures, used as the treatment of mucopolysaccharidosis, are effective in reducing glycosaminoglycan production and storage and show a slight cytotoxic effect compared to single-flavonoid usage.

FGF signalling regulates bone growth through autophagy.

Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes.

Fingolimod phosphate inhibits astrocyte inflammatory activity in mucolipidosis IV.

Mucolipidosis IV (MLIV) is an orphan neurodevelopmental disease that causes severe neurologic dysfunction and loss of vision. Currently there is no therapy for MLIV. It is caused by loss of function of the lysosomal channel mucolipin-1, also known as TRPML1. Knockout of the Mcoln1 gene in a mouse model mirrors clinical and neuropathologic signs in humans. Using this model, we previously observed robust activation of microglia and astrocytes in early symptomatic stages of disease. Here we investigate the consequence of mucolipin-1 loss on astrocyte inflammatory activation in vivo and in vitro and apply a pharmacologic approach to restore Mcoln1-/- astrocyte homeostasis using a clinically approved immunomodulator, fingolimod. We found that Mcoln1-/- mice over-express numerous pro-inflammatory cytokines, some of which were also over-expressed in astrocyte cultures. Changes in the cytokine profile in Mcoln1-/- astrocytes are concomitant with changes in phospho-protein signaling, including activation of PI3K/Akt and MAPK pathways. Fingolimod promotes cytokine homeostasis, down-regulates signaling within the PI3K/Akt and MAPK pathways and restores the lysosomal compartment in Mcoln1-/- astrocytes. These data suggest that fingolimod is a promising candidate for preclinical evaluation in our MLIV mouse model, which, in case of success, can be rapidly translated into clinical trial.

SBDS-Deficient Cells Have an Altered Homeostatic Equilibrium due to Translational Inefficiency Which Explains their Reduced Fitness and Provides a Logical Framework for Intervention.

Ribosomopathies are a family of inherited disorders caused by mutations in genes necessary for ribosomal function. Shwachman-Diamond Bodian Syndrome (SDS) is an autosomal recessive disease caused, in most patients, by mutations of the SBDS gene. SBDS is a protein required for the maturation of 60S ribosomes. SDS patients present exocrine pancreatic insufficiency, neutropenia, chronic infections, and skeletal abnormalities. Later in life, patients are prone to myelodisplastic syndrome and acute myeloid leukemia (AML). It is unknown why patients develop AML and which cellular alterations are directly due to the loss of the SBDS protein. Here we derived mouse embryonic fibroblast lines from an SbdsR126T/R126T mouse model. After their immortalization, we reconstituted them by adding wild type Sbds. We then performed a comprehensive analysis of cellular functions including colony formation, translational and transcriptional RNA-seq, stress and drug sensitivity. We show that: 1. Mutant Sbds causes a reduction in cellular clonogenic capability and oncogene-induced transformation. 2. Mutant Sbds causes a marked increase in immature 60S subunits, limited impact on mRNA specific initiation of translation, but reduced global protein synthesis capability. 3. Chronic loss of SBDS activity leads to a rewiring of gene expression with reduced ribosomal capability, but increased lysosomal and catabolic activity. 4. Consistently with the gene signature, we found that SBDS loss causes a reduction in ATP and lactate levels, and increased susceptibility to DNA damage. Combining our data, we conclude that a cell-specific fragile phenotype occurs when SBDS protein drops below a threshold level, and propose a new interpretation of the disease.

gene2drug: a computational tool for pathway-based rational drug repositioning.

Motivation: Drug repositioning has been proposed as an effective shortcut to drug discovery. The availability of large collections of transcriptional responses to drugs enables computational approaches to drug repositioning directly based on measured molecular effects. Results: We introduce a novel computational methodology for rational drug repositioning, which exploits the transcriptional responses following treatment with small molecule. Specifically, given a therapeutic target gene, a prioritization of potential effective drugs is obtained by assessing their impact on the transcription of genes in the pathway(s) including the target. We performed in silico validation and comparison with a state-of-art technique based on similar principles. We next performed experimental validation in two different real-case drug repositioning scenarios: (i) upregulation of the glutamate-pyruvate transaminase (GPT), which has been shown to induce reduction of oxalate levels in a mouse model of primary hyperoxaluria, and (ii) activation of the transcription factor TFEB, a master regulator of lysosomal biogenesis and autophagy, whose modulation may be beneficial in neurodegenerative disorders. Availability and implementation: A web tool for Gene2drug is freely available at http://gene2drug.tigem.it. An R package is under development and can be obtained from https://github.com/franapoli/gep2pep. Contact: dibernardo@tigem.it. Supplementary information: Supplementary data are available at Bioinformatics online.

Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells.

Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease characterized by accumulation of cystine into lysosomes secondary to mutations in the cystine lysosomal transporter, cystinosin. The defect initially causes proximal tubular dysfunction (Fanconi syndrome) which in time progresses to end-stage renal disease. Cystinotic patients treated with the cystine-depleting agent, cysteamine, have improved life expectancy, delayed progression to chronic renal failure, but persistence of Fanconi syndrome. Here, we have investigated the role of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in conditionally immortalized proximal tubular epithelial cells derived from the urine of a healthy volunteer or a cystinotic patient. Lack of cystinosin reduced TFEB expression and induced TFEB nuclear translocation. Stimulation of endogenous TFEB activity by genistein, or overexpression of exogenous TFEB lowered cystine levels within 24 hours in cystinotic cells. Overexpression of TFEB also stimulated delayed endocytic cargo processing within 24 hours. Rescue of other abnormalities of the lysosomal compartment was observed but required prolonged expression of TFEB. These abnormalities could not be corrected with cysteamine. Thus, these data show that the consequences of cystinosin deficiency are not restricted to cystine accumulation and support the role of TFEB as a therapeutic target for the treatment of lysosomal storage diseases, in particular of cystinosis.