TRPML1: Role In Autophagy And Potential Target To Treat Lysosomal Storage Disorders

The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+ signaling mechanism controls the activities of the phosphatase calcineurin and of its substrate TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+ release via mucolipin 1 (TRPML1) activates calcineurin, which binds and de-phosphorylates TFEB, thus promoting its nuclear translocation. Induction of autophagy and lysosomal biogenesis via TFEB required TRPML1-mediated calcineurin activation, linking lysosomal calcium signaling to both calcineurin regulation and autophagy induction. In addition to the role of TRPML1 on sustaining transcriptional autophagy program, through the activation of TFEB, we also found that TRPML1-activation induces the recruitment of PtdIns(3)P-binding proteins to the nascent autophagosome, whereas genetic or pharmacological inhibition of TRPML1 channel inhibits autophagy initiation. Importantly, alteration of this function has pathological consequences, and thus we found that autophagosome formation is impaired in human fibroblasts from patients affected of mucolipidosis IV (MLIV; a severe lysosomal storage disorder caused by mutations in TRPML1). By using specific compound inhibitors during starvation, we found that TRPML1-mediated induction of autophagosome biogenesis requires calmodulin, CaMKKβ, and the PtdIns(3)-generating enzyme VPS34. Therefore, we hypothezise that during starvation, TRPML1 activation releases lysosomal calcium that activates a calcium-depedent pathway involving CaMKKβ and the induction of two essential protein complexes involved in autophagy initiation such as ULK1 and PIK3C3 complexes. In parallel studies, we used high content (HC) screening approaches to identify small molecules able to ameliorate the MLIV phenotype. In one of them, we tested whether previously identified drugs (150 FDA compounds) inducing TFEB translocation and cellular clearance might be active in MLIV patient cells. This screening resulted in the identification of 3 drugs able to induce TFEB nuclear translocation confirming the importance of the TRPML1-mediated signalling to promote TFEB activity. In addition, only one of these hits were able to reduce the pathological accumulation of autophagic substrates such as p62 and NBR1 in MLIV human fibroblasts. In a second independent HC-screening, we developed a cell-based assay to identify FDA-drugs able to reduce cholesterol accumulation in MLIV cells. We identify 8 small molecules able to reduce cholesterol accumulation in MLIV human fibroblasts that will need further characterization to define their ability to ameliorate the phenotype of this devastating disease. In summary, we found two novel signaling pathways triggered by TRPML1-dependent lysosomal calcium release that regulate cellular homeostasis by both promoting autophagy initiation and sustaining transcriptional programs inducing autophagic and lysosomal genes during starvation. Finally, we use part of this knowledge to develop cell-based high content screening assays that identified 9 FDA-approved compounds able to ameliorate the autophagic impairment and reduce lipid storage in MLIV disease cells

Dalla siderurgia al turismo. Un'analisi empirica sull'area di Piombino.

Scopo della ricerca: lo scopo di questa dissertazione è quello di indagare sulla possibilità di far coesistere turismo e industria nella città di Piombino, nota principalmente come polo siderurgico e come punto di partenza per l’Isola d’Elba. Si può trasformare un territorio come Piombino in città turistica? Ha un futuro l’industria siderurgica? Qual è l’opinione dei cittadini al riguardo? Come si può combattere la mentalità operaia di questo paese? Quali sono le iniziative proposte dagli opinion leaders? Che tipo di turismo si può sviluppare? Queste sono solo alcune delle domande cui daremo risposta nel corso della trattazione. Metodologia: la ricerca è stata effettuata tramite analisi sia di tipo quantitativo, con l’utilizzo di questionari auto – somministrati ai cittadini, sia attraverso analisi qualitative, con interviste face to face a esponenti del Comune, ristoratori, albergatori e proprietari di strutture extra – alberghiere. In questo modo si è potuto analizzare quello che è il pensiero degli abitanti, direttamente coinvolti in questa diatriba, ma anche il pensiero di coloro che impiegano tempo e risorse nell’attività turistica e di chi ha fatto del turismo il proprio lavoro. Risultati: analizzando i dati è emerso come la possibilità di far diventare Piombino città turistica abbia un effetto positivo sui giovani in cerca di un lavoro dinamico e non scandito dai turni dell’industria. Tuttavia, nonostante le elevate potenzialità della città, si pongono degli ostacoli dovuti alla mentalità dei più anziani, abituati ad avere l’utilizzo esclusivo del territorio (il turismo a Piombino è nato solo agli inizi degli anni ’90), nonché delle poche risorse messe a disposizione dei Comuni per poter investire in un settore in crescita come il turismo. Limitazioni della ricerca: non avendo a disposizione gli strumenti di marketing di cui dispone una società di ricerche di mercato, ho cercato di svolgere il mio lavoro nel modo più accurato possibile, cercando di tener conto di tutti i soggetti coinvolti in questa delicata tematica. Il tema dell’industria a Piombino è sempre più spesso al centro dei dibattiti, per cui ho cercato di sviluppare l’argomento in tutta la sua essenza, senza farmi coinvolgere in opinioni politiche che avrebbero potuto deviare lo scopo per cui la ricerca è stata condotta. Implicazioni pratiche: i risultati ci suggeriscono che la cosa migliore per il territorio di Piombino sia quella di far coesistere turismo e industria, far diventare Piombino anche città turistica, non solo città – fabbrica. Questo è però un processo che si andrà delineando nel corso degli anni, e di certo non di facile attuazione. Il consiglio finale dato alla fine di questa trattazione è quello di puntare sul turismo sostenibile e sulla tutela dell’ambiente. Siamo tutti fiduciosi nelle potenzialità di questa città, che potrebbe offrire molto di più ai suoi visitatori e ai cittadini stessi

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). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease

Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009; Guo et al., 2017; Jha et al., 2014; Ruas et al., 2015; Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+-mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand

Agonist-mediated switching of ion selectivity in TPC2 differentially promotes lysosomal function

Ion selectivity is a defining feature of a given ion channel and is considered immutable. Here we show that ion selectivity of the lysosomal ion channel TPC2, which is hotly debated (Calcraft et al., 2009;Guo et al., 2017;Jha et al., 2014;Ruas et al., 2015;Wang et al., 2012), depends on the activating ligand. A high-throughput screen identified two structurally distinct TPC2 agonists. One of these evoked robust Ca2+-signals and non-selective cation currents, the other weaker Ca2+-signals and Na+-selective currents. These properties were mirrored by the Ca2+ mobilizing messenger, NAADP and the phosphoinositide, PI(3,5)P-2, respectively. Agonist action was differentially inhibited by mutation of a single TPC2 residue and coupled to opposing changes in lysosomal pH and exocytosis. Our findings resolve conflicting reports on the permeability and gating properties of TPC2 and they establish a new paradigm whereby a single ion channel mediates distinct, functionally-relevant ionic signatures on demand

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). Here, tamoxifen exerts its action through a mechanism that involves activation of the transcription factor EB (TFEB), a master gene of lysosomal function and autophagy. In vivo administration of tamoxifen to the CLN7Δex2 mouse model reduced the accumulation of Gb3 and SCMAS, decreased neuroinflammation, and improved motor coordination. These data strongly suggest that tamoxifen may be a suitable drug to treat some types of Batten disease.This work was funded by the European Union’s Horizon 2020 research and innovation programme (BATCure, grant No. 666918 to DLM, JPB, SEM, TB and SS). JPB is funded by the Agencia Estatal de Investigación (PID2019-105699RB-I00/ AEI / 10.13039/501100011033 and RED2018-102576-T), Plan Nacional sobre Drogas (2020I028), Junta de Castilla y León (Escalera de Excelencia CLU-2017-03), Ayudas Equipos Investigación Biomedicina 2017 Fundación BBVA and Fundación Ramón Areces. SS was funded by a grant from the Mila’s Miracle Foundation. TB was supported by German Research Council (DFG) grant FOR2625. SM benefits from MRC funding to the MRC Laboratory for Molecular Cell Biology University Unit at UCL (award code MC_U12266B) towards laboratory and office space. We acknowledge Marcella Cesana for providing the TFEB virus. Graphical abstract was created using BioRender.com

An exploratory study of the association between online gaming addiction and enjoyment motivations for playing massively multiplayer online role-playing games

Massively multiplayer online role-playing games (MMORPGs) are a popular form of entertainment used by millions of gamers worldwide. Potential problems relating to MMORPG play have emerged, particularly in relation to being addicted to playing in such virtual environments. In the present study, factors relating to online gaming addiction and motivations for playing in MMORPGs were examined to establish whether they were associated with addiction. A sample comprised 1167 gamers who were surveyed about their gaming motivations. Latent Class Analysis revealed seven classes of motivations for playing MMORPGs, which comprised: (1) novelty; (2) highly social and discovery-orientated; (3) aggressive, anti-social and non-curious; (4) highly social, competitive; (5) low intensity enjoyment; (6) discovery-orientated; and (7) social classes. Five classes of gaming addiction-related experiences were extracted including: (1) high risk of addiction, (2) time-affected, (3) intermediate risk of addiction, (4) emotional control, and (5) low risk of addiction classes. Gender was a significant predictor of intermediate risk of addiction and emotional control class membership. Membership of the high risk of addiction class was significantly predicted by belonging to a highly social and competitive class, a novelty class, or an aggressive, anti-social, and non-curious class. Implications of these findings for assessment and treatment of MMORPG addiction are discussed

Lung emphysema and impaired macrophage elastase clearance in mucolipin 3 deficient mice

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease

TRPML1: The Ca(2+)retaker of the lysosome

Efficient functioning of lysosome is necessary to ensure the correct performance of a variety of intracellular processes such as degradation of cargoes coming from the endocytic and autophagic pathways, recycling of organelles, and signaling mechanisms involved in cellular adaptation to nutrient availability. Mutations in lysosomal genes lead to more than 50 lysosomal storage disorders (LSDs). Among them, mutations in the gene encoding TRPML1 (MCOLN1) cause Mucolipidosis type IV (MLIV), a recessive LSD characterized by neurodegeneration, psychomotor retardation, ophthalmologic defects and achlorhydria. At the cellular level, MLIV patient fibroblasts show enlargement and engulfment of the late endo-lysosomal compartment, autophagy impairment, and accumulation of lipids and glycosaminoglycans. TRPML1 is the most extensively studied member of a small family of genes that also includes TRPML2 and TRPML3, and it has been found to participate in vesicular trafficking, lipid and ion homeostasis, and autophagy. In this review we will provide an update on the latest and more novel findings related to the functions of TRPMLs, with particular focus on the emerging role of TRPML1 and lysosomal calcium signaling in autophagy. Moreover, we will also discuss new potential therapeutic approaches for MLIV and LSDs based on the modulation of TRPML1-mediated signaling