TPC2 rescues lysosomal storage in mucolipidosis type IV , Niemann–Pick type C1, and Batten disease
2022; Springer Nature; Volume: 14; Issue: 9 Linguagem: Inglês
10.15252/emmm.202115377
ISSN1757-4684
AutoresAnna Scotto Rosato, Einar Krogsaeter, Dawid Jaślan, Carla Abrahamian, Sandro Montefusco, Chiara Soldati, Barbara Spix, Maria Teresa Pizzo, Giuseppina Emanuela Grieco, Julia Böck, Amanda Wyatt, Daniela Wünkhaus, Marcel Passon, Marc Stieglitz, Marco Keller, Guido Hermey, Sandra Markmann, Doris Gruber-Schoffnegger, Susan L. Cotman, Ludger Johannes, Dennis Crusius, Ulrich Boehm, Christian Wahl‐Schott, Martin Biel, Franz Bracher, Elvira De Leonibus, Elena Polishchuk, Diego L. Medina, Dominik Paquet, Christian Grimm,
Tópico(s)Adenosine and Purinergic Signaling
ResumoArticle5 August 2022Open Access Transparent process TPC2 rescues lysosomal storage in mucolipidosis type IV, Niemann–Pick type C1, and Batten disease Anna Scotto Rosato Anna Scotto Rosato Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis, Methodology Search for more papers by this author Einar K Krogsaeter Einar K Krogsaeter orcid.org/0000-0001-8232-5498 Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Dawid Jaślan Dawid Jaślan orcid.org/0000-0002-0685-7633 Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis, Methodology Search for more papers by this author Carla Abrahamian Carla Abrahamian orcid.org/0000-0002-0425-8004 Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Sandro Montefusco Sandro Montefusco Telethon Institute of Genetics and Medicine, Naples, Italy Contribution: Formal analysis, Investigation Search for more papers by this author Chiara Soldati Chiara Soldati Telethon Institute of Genetics and Medicine, Naples, Italy Contribution: Methodology Search for more papers by this author Barbara Spix Barbara Spix Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Maria Teresa Pizzo Maria Teresa Pizzo Telethon Institute of Genetics and Medicine, Naples, Italy Contribution: Data curation, Investigation Search for more papers by this author Giuseppina Grieco Giuseppina Grieco Telethon Institute of Genetics and Medicine, Naples, Italy Contribution: Formal analysis, Methodology Search for more papers by this author Julia Böck Julia Böck Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Amanda Wyatt Amanda Wyatt orcid.org/0000-0003-1107-3857 Experimental Pharmacology, Center for Molecular Signaling (PZMS), Saarland University School of Medicine, Homburg, Germany Contribution: Data curation, Methodology Search for more papers by this author Daniela Wünkhaus Daniela Wünkhaus Evotec AG, Hamburg, Germany Contribution: Methodology Search for more papers by this author Marcel Passon Marcel Passon orcid.org/0000-0002-9776-4992 Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation Search for more papers by this author Marc Stieglitz Marc Stieglitz Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation Search for more papers by this author Marco Keller Marco Keller orcid.org/0000-0003-4792-3980 Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Methodology Search for more papers by this author Guido Hermey Guido Hermey orcid.org/0000-0003-4762-5262 Center for Molecular Neurobiology Hamburg (ZMNH), Institute of Molecular and Cellular Cognition, UKE, Hamburg, Germany Contribution: Resources Search for more papers by this author Sandra Markmann Sandra Markmann Evotec AG, Hamburg, Germany Contribution: Methodology Search for more papers by this author Doris Gruber-Schoffnegger Doris Gruber-Schoffnegger Evotec AG, Hamburg, Germany Contribution: Methodology Search for more papers by this author Susan Cotman Susan Cotman orcid.org/0000-0003-3114-0543 Department of Neurology, Center for Genomic Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Contribution: Resources Search for more papers by this author Ludger Johannes Ludger Johannes orcid.org/0000-0002-2168-0004 Cellular and Chemical Biology Department, Institut Curie, U1143 INSERM, UMR3666 CNRS, PSL Research University, Paris, France Contribution: Methodology Search for more papers by this author Dennis Crusius Dennis Crusius Institute for Stroke and Dementia Research (ISD), Ludwig-Maximilians-University (LMU) Hospital, Munich, Germany Contribution: Methodology Search for more papers by this author Ulrich Boehm Ulrich Boehm orcid.org/0000-0003-2436-6907 Experimental Pharmacology, Center for Molecular Signaling (PZMS), Saarland University School of Medicine, Homburg, Germany Contribution: Funding acquisition, Methodology Search for more papers by this author Christian Wahl-Schott Christian Wahl-Schott Institute for Neurophysiology, Hannover Medical School, Hannover, Germany Contribution: Resources, Funding acquisition Search for more papers by this author Martin Biel Martin Biel Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Resources, Funding acquisition Search for more papers by this author Franz Bracher Franz Bracher Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition Search for more papers by this author Elvira De Leonibus Elvira De Leonibus Telethon Institute of Genetics and Medicine, Naples, Italy Institute of Biochemistry and Cell Biology (IBBC), CNR, Rome, Italy Contribution: Data curation, Formal analysis, Supervision, Investigation Search for more papers by this author Elena Polishchuk Elena Polishchuk Telethon Institute of Genetics and Medicine, Naples, Italy Contribution: Data curation, Formal analysis, Methodology Search for more papers by this author Diego L Medina Corresponding Author Diego L Medina [email protected] orcid.org/0000-0002-7347-2645 Telethon Institute of Genetics and Medicine, Naples, Italy Medical Genetics Unit, Department of Medical and Translational Science, Federico II University, Naples, Italy Contribution: Funding acquisition Search for more papers by this author Dominik Paquet Corresponding Author Dominik Paquet [email protected] orcid.org/0000-0003-2065-1639 Institute for Stroke and Dementia Research (ISD), Ludwig-Maximilians-University (LMU) Hospital, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University (LMU), Munich, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration, Writing - review & editing Search for more papers by this author Christian Grimm Corresponding Author Christian Grimm [email protected] orcid.org/0000-0002-0177-5559 Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing - original draft, Project administration Search for more papers by this author Anna Scotto Rosato Anna Scotto Rosato Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis, Methodology Search for more papers by this author Einar K Krogsaeter Einar K Krogsaeter orcid.org/0000-0001-8232-5498 Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Dawid Jaślan Dawid Jaślan orcid.org/0000-0002-0685-7633 Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis, Methodology Search for more papers by this author Carla Abrahamian Carla Abrahamian orcid.org/0000-0002-0425-8004 Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Sandro Montefusco Sandro Montefusco Telethon Institute of Genetics and Medicine, Naples, Italy Contribution: Formal analysis, Investigation Search for more papers by this author Chiara Soldati Chiara Soldati Telethon Institute of Genetics and Medicine, Naples, Italy Contribution: Methodology Search for more papers by this author Barbara Spix Barbara Spix Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Maria Teresa Pizzo Maria Teresa Pizzo Telethon Institute of Genetics and Medicine, Naples, Italy Contribution: Data curation, Investigation Search for more papers by this author Giuseppina Grieco Giuseppina Grieco Telethon Institute of Genetics and Medicine, Naples, Italy Contribution: Formal analysis, Methodology Search for more papers by this author Julia Böck Julia Böck Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation, Formal analysis Search for more papers by this author Amanda Wyatt Amanda Wyatt orcid.org/0000-0003-1107-3857 Experimental Pharmacology, Center for Molecular Signaling (PZMS), Saarland University School of Medicine, Homburg, Germany Contribution: Data curation, Methodology Search for more papers by this author Daniela Wünkhaus Daniela Wünkhaus Evotec AG, Hamburg, Germany Contribution: Methodology Search for more papers by this author Marcel Passon Marcel Passon orcid.org/0000-0002-9776-4992 Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation Search for more papers by this author Marc Stieglitz Marc Stieglitz Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Data curation Search for more papers by this author Marco Keller Marco Keller orcid.org/0000-0003-4792-3980 Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Methodology Search for more papers by this author Guido Hermey Guido Hermey orcid.org/0000-0003-4762-5262 Center for Molecular Neurobiology Hamburg (ZMNH), Institute of Molecular and Cellular Cognition, UKE, Hamburg, Germany Contribution: Resources Search for more papers by this author Sandra Markmann Sandra Markmann Evotec AG, Hamburg, Germany Contribution: Methodology Search for more papers by this author Doris Gruber-Schoffnegger Doris Gruber-Schoffnegger Evotec AG, Hamburg, Germany Contribution: Methodology Search for more papers by this author Susan Cotman Susan Cotman orcid.org/0000-0003-3114-0543 Department of Neurology, Center for Genomic Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Contribution: Resources Search for more papers by this author Ludger Johannes Ludger Johannes orcid.org/0000-0002-2168-0004 Cellular and Chemical Biology Department, Institut Curie, U1143 INSERM, UMR3666 CNRS, PSL Research University, Paris, France Contribution: Methodology Search for more papers by this author Dennis Crusius Dennis Crusius Institute for Stroke and Dementia Research (ISD), Ludwig-Maximilians-University (LMU) Hospital, Munich, Germany Contribution: Methodology Search for more papers by this author Ulrich Boehm Ulrich Boehm orcid.org/0000-0003-2436-6907 Experimental Pharmacology, Center for Molecular Signaling (PZMS), Saarland University School of Medicine, Homburg, Germany Contribution: Funding acquisition, Methodology Search for more papers by this author Christian Wahl-Schott Christian Wahl-Schott Institute for Neurophysiology, Hannover Medical School, Hannover, Germany Contribution: Resources, Funding acquisition Search for more papers by this author Martin Biel Martin Biel Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Resources, Funding acquisition Search for more papers by this author Franz Bracher Franz Bracher Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition Search for more papers by this author Elvira De Leonibus Elvira De Leonibus Telethon Institute of Genetics and Medicine, Naples, Italy Institute of Biochemistry and Cell Biology (IBBC), CNR, Rome, Italy Contribution: Data curation, Formal analysis, Supervision, Investigation Search for more papers by this author Elena Polishchuk Elena Polishchuk Telethon Institute of Genetics and Medicine, Naples, Italy Contribution: Data curation, Formal analysis, Methodology Search for more papers by this author Diego L Medina Corresponding Author Diego L Medina [email protected] orcid.org/0000-0002-7347-2645 Telethon Institute of Genetics and Medicine, Naples, Italy Medical Genetics Unit, Department of Medical and Translational Science, Federico II University, Naples, Italy Contribution: Funding acquisition Search for more papers by this author Dominik Paquet Corresponding Author Dominik Paquet [email protected] orcid.org/0000-0003-2065-1639 Institute for Stroke and Dementia Research (ISD), Ludwig-Maximilians-University (LMU) Hospital, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University (LMU), Munich, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Methodology, Project administration, Writing - review & editing Search for more papers by this author Christian Grimm Corresponding Author Christian Grimm [email protected] orcid.org/0000-0002-0177-5559 Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Validation, Visualization, Methodology, Writing - original draft, Project administration Search for more papers by this author Author Information Anna Scotto Rosato1,†, Einar K Krogsaeter1,†, Dawid Jaślan1, Carla Abrahamian1, Sandro Montefusco2, Chiara Soldati2, Barbara Spix1, Maria Teresa Pizzo2, Giuseppina Grieco2, Julia Böck1, Amanda Wyatt3, Daniela Wünkhaus4, Marcel Passon1, Marc Stieglitz5, Marco Keller5, Guido Hermey6, Sandra Markmann4, Doris Gruber-Schoffnegger4, Susan Cotman7, Ludger Johannes8, Dennis Crusius9, Ulrich Boehm3, Christian Wahl-Schott10, Martin Biel5, Franz Bracher5, Elvira De Leonibus2,11, Elena Polishchuk2, Diego L Medina *,2,12, Dominik Paquet *,9,13 and Christian Grimm *,1 1Faculty of Medicine, Walther Straub Institute of Pharmacology and Toxicology, Ludwig-Maximilians-Universität, Munich, Germany 2Telethon Institute of Genetics and Medicine, Naples, Italy 3Experimental Pharmacology, Center for Molecular Signaling (PZMS), Saarland University School of Medicine, Homburg, Germany 4Evotec AG, Hamburg, Germany 5Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany 6Center for Molecular Neurobiology Hamburg (ZMNH), Institute of Molecular and Cellular Cognition, UKE, Hamburg, Germany 7Department of Neurology, Center for Genomic Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 8Cellular and Chemical Biology Department, Institut Curie, U1143 INSERM, UMR3666 CNRS, PSL Research University, Paris, France 9Institute for Stroke and Dementia Research (ISD), Ludwig-Maximilians-University (LMU) Hospital, Munich, Germany 10Institute for Neurophysiology, Hannover Medical School, Hannover, Germany 11Institute of Biochemistry and Cell Biology (IBBC), CNR, Rome, Italy 12Medical Genetics Unit, Department of Medical and Translational Science, Federico II University, Naples, Italy 13Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University (LMU), Munich, Germany † These authors contributed equally to this work *Corresponding author. Tel: +39 08119230698; E-mail: [email protected] author. Tel: +49 89440046123; E-mail: [email protected] author. Tel: +49 89218073811; E-mail: [email protected] EMBO Mol Med (2022)14:e15377https://doi.org/10.15252/emmm.202115377 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Lysosomes are cell organelles that degrade macromolecules to recycle their components. If lysosomal degradative function is impaired, e.g., due to mutations in lysosomal enzymes or membrane proteins, lysosomal storage diseases (LSDs) can develop. LSDs manifest often with neurodegenerative symptoms, typically starting in early childhood, and going along with a strongly reduced life expectancy and quality of life. We show here that small molecule activation of the Ca2+-permeable endolysosomal two-pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with LSDs such as cholesterol or lipofuscin accumulation, or the formation of abnormal vacuoles seen by electron microscopy. Rescue effects by TPC2 activation, which promotes lysosomal exocytosis and autophagy, were assessed in mucolipidosis type IV (MLIV), Niemann–Pick type C1, and Batten disease patient fibroblasts, and in neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. For in vivo proof of concept, we tested TPC2 activation in the MLIV mouse model. In sum, our data suggest that TPC2 is a promising target for the treatment of different types of LSDs, both in vitro and in-vivo. Synopsis Mutations in proteins of the endolysosomal machinery such as lysosomal enzymes or lysosomal membrane proteins often result in severe neurodegenerative disease, including mucolipidosis type IV, Niemann Pick type C1, or Batten disease. Small molecule activation of the endolysosomal two-pore channel 2 (TPC2) results in an amelioration of cellular phenotypes associated with lysosomal storage diseases (LSDs), such as the accumulation of cholesterol, lactosylceramide, or lipofuscin, or the formation of abnormal vacuoles. Rescue effects of the TPC2 agonist are demonstrated in LSD patient fibroblasts and neurons derived from newly generated isogenic human iPSC models for MLIV and Batten disease. Mechanistically, the fully preserved capability of TPC2 to promote lysosomal exocytosis and autophagy in these diseases is demonstrated, suggesting rescue effects by clearing intracellular debris. Expression patterns of TPC2 in the brain are examined using a newly engineered TPC2-GFP reporter mouse model, complemented by qPCR analyses of human and mouse brain samples. In vivo efficacy of the PI(3,5)P2-mimetic TPC2 agonist TPC2-A1-P is shown, restoring pathological hallmarks in a mouse model of MLIV. The paper explained Problem Batten disease (JNCL), mucolipidosis type IV (MLIV), and Niemann–Pick type C1 (NPC1) are fatal neurodegenerative rare lysosomal storage diseases. There is currently no curative therapy available for either of these diseases. Results We show here that treatment with a PI(3,5)P2-mimetic small molecule agonist of the endolysosomal cation channel TPC2, TPC2-A1-P, ameliorates cellular disease phenotypes in patient fibroblasts and iPSC-derived neuronal models of MLIV, NPC1, and JNCL as well as disease phenotypes in the mouse model of MLIV in vivo. Impact Our data suggest that activation of TPC2 has the potential to serve as a novel approach to treat different lysosomal storage disorders, in particular those going along with a disturbed endolysosomal Ca2+ homeostasis. Introduction Lysosomal Ca2+ release is of significant physiological relevance. Lysosomal Ca2+ regulates several cellular processes, e.g., autophagy (Medina et al, 2015), membrane trafficking (Dong et al, 2010; Ruas et al, 2010; Cao et al, 2015), exocytosis (Samie et al, 2013; Davis et al, 2020), nutrient adaptation (Cang et al, 2013), membrane repair (Cheng et al, 2014), and cell migration (Bretou et al, 2017). Disruption of lysosomal Ca2+ content or Ca2+ release is associated with several diseases, particularly neurodegenerative lysosomal storage diseases (Kiselyov et al, 2010; Lloyd-Evans & Platt, 2011; Feng & Yang, 2016). Mucolipidosis type IV (MLIV) constitutes the most direct link between defective lysosomal Ca2+ release and neurodegeneration, caused by dysfunction of the lysosomal cation channel TRPML1 (also called MCOLN1) (Slaugenhaupt, 2002; Feng & Yang, 2016). TRPML1 signaling or TRPML1-mediated Ca2+ release is similarly impaired in other LSDs such as Niemann–Pick type C1 (NPC1) (Shen et al, 2012), Niemann–Pick type A (NPA; also called infantile neurovisceral form of acid sphingomyelinase (SMPD1) deficiency) (Zhong et al, 2016), and Fabry disease (Zhong et al, 2016). Pharmacological and genetic activation of TRPML1 ameliorates NPC1-associated lactosylceramide (LacCer) trafficking defects and cholesterol accumulation (Shen et al, 2012), while activation of the lysosomal big conductance Ca2+-activated potassium (BK) channel TRPML1 dependently rescues aberrant lysosomal storage in NPA and Fabry disease (Zhong et al, 2016). Furthermore, loss of FIG 4 (polyphosphoinositide phosphatase) and PYKfyve (FYVE finger-containing phosphoinositide kinase), which are both involved in the synthesis of the endogenous TRPML/two-pore channel (TPC) agonist PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate), is associated with neurological or neurodegenerative disease phenotypes (Chow et al, 2007; Zhang et al, 2007; Zou et al, 2015), and TRPML1 activation in FIG 4−/− cells rescues lysosomal storage phenotypes (Zou et al, 2015). While activation of TRPML1 in LSDs is gaining traction, effects of activating the related two-pore channel 2 (TPC2 or TPCN2) remain unexplored. TPC2 shares several features with TRPML1: both channels are permeable for Ca2+ and Na+ (Calcraft et al, 2009; Zong et al, 2009; Wang et al, 2012; Gerndt et al, 2020), reside in endolysosomal membranes (Pryor et al, 2006; Calcraft et al, 2009; Kim et al, 2009; Ruas et al, 2010), are activated by PI(3,5)P2 (Dong et al, 2010; Wang et al, 2012; Gerndt et al, 2020), are widely expressed in the CNS (Bae et al, 2014; Pereira et al, 2017; Foster et al, 2018; Minckley et al, 2019), cause trafficking defects when lost (Dong et al, 2010; Shen et al, 2012; Chen et al, 2014; Grimm et al, 2014; Nguyen et al, 2017), interact with mTOR/TFEB/autophagy pathways (Medina et al, 2011; Cang et al, 2013; Medina et al, 2015; Wang et al, 2015; Li et al, 2016; Ogunbayo et al, 2018; Scotto Rosato et al, 2019), and promote lysosomal exocytosis (Medina et al, 2011; Samie et al, 2013; Gerndt et al, 2020). We therefore hypothesized that TPC2 activation may modulate lysosomal Ca2+ signaling to rescue LSD phenotypes, particularly in LSDs where TRPML1 is impacted. In our study, we focused on MLIV and NPC1 on the one hand, LSDs that both have been shown before to be connected to disrupted lysosomal Ca2+ signaling and TRPML1 dysfunction (Shen et al, 2012). On the other hand, we focused on juvenile neuronal ceroid lipofuscinosis (JNCL) or Batten disease, caused by mutations in CLN3, an LSD which shows prominent retinal and neurodegenerative phenotypes with gradual vision loss and progressive cognitive decline as observed in MLIV, and with a similar age-dependent disease onset and evidence for disturbed lysosomal Ca2+ homeostasis (Chandrachud et al, 2015). By analyzing disease hallmarks in patient fibroblasts, novel CRISPR/Cas9-engineered iPSCs/iPSC-derived neurons, and the MLIV mouse upon treatment with a TPC2 small molecule agonist, TPC2-A1-P, we demonstrate that TPC2 activation ameliorates the phenotypes of these LSDs both in vitro and in vivo. Results TPC2 activation modulates LSD phenotypes in human patient fibroblasts Based on the concept that disrupted endolysosomal Ca2+ homeostasis constitutes a major pathomechanism underlying LSDs as evidenced by MLIV, we assessed the effect of our recently published PI(3,5)P2-mimetic TPC2 agonist, TPC2-A1-P (Gerndt et al, 2020), releasing both Ca2+ and Na+, on the phenotypes of the above-mentioned LSDs. For NPC1 and MLIV, lactosylceramide (LacCer) and cholesterol trafficking defects are reported (Shen et al, 2012; Chen et al, 2014). Hence, we started our study by assessing these defects in fibroblasts from NPC1 and MLIV patients compared to control (CTR) fibroblasts. The lipid LacCer is internalized clathrin independently and targeted to the Golgi apparatus in CTR cells, whereas in several LSD fibroblasts including NPC1 and MLIV it accumulates in late endosomes and lysosomes. Accordingly, we observed significant endolysosomal accumulation of LacCer in NPC1 and MLIV, and a range of other LSDs compared to CTR, but not for JNCL (CLN3Δ1.02kb/Δ1.02kb) and Gaucher, as reported previously (Vitner et al, 2010), demonstrating reproducibility of the assay (Fig 1A). We next assessed the effect of TPC2 activation in MLIV and NPC1 versus CTR fibroblasts. In MLIV fibroblasts, carrying the most common patient variation (MCOLN1IVS3-2A>G/Ex1-7del; GM02048) TPC2 activation by TPC2-A1-P significantly reduced lysosomal accumulation of LacCer (Mander's coefficient) and the number of LacCer puncta per area after incubation overnight (16 h) (Fig 1B and C), while in NPC1 cells (NPC1P237S/I1061T; GM03123), significant rescue was seen after 48 h incubation (Fig 1D and E). To assess maximal rescue effects, we tested overexpression of a gain-of-function variant of TPC2 (TPC2M484L/G734E; Chao et al, 2017) with and without TPC2-A1-P activation in MLIV fibroblasts (Fig 1F and G). Both TPC2 overexpression alone and overexpression in combination with TPC2-A1-P significantly reduced lysosomal accumulation of LacCer in MLIV cells, with a stronger effect seen in the combination. To exclude any potential toxic effects of TPC2-A1-P on fibroblasts, cell viability assays were performed (Fig EV1A). Commercially available drugs reported to activate TPC2 were examined alongside TPC2-A1-P (Zhang et al, 2019). In these tests, TPC2-A1-P showed no toxicity up to the maximal test concentration (100 μM; Fig EV1B). By using the Ca2+ chelator BAPTA-AM, we could further demonstrate that reduction in free intracellular Ca2+ induces a similar LacCer trafficking defect in CTR as in MLIV or NPC1 cells (Fig 1H and I), suggesting a relevant role of Ca2+ in the process. Furthermore, TPC2-A1-P rescued the lysosomal LacCer accumulation in mock, but not in siTPC2-treated NPC1 fibroblasts, corroborating the on-target effect of TPC2-A1-P (Fig 1J and K). LacCer trafficking is also affected by intracellular cholesterol levels (Pryor et al, 2006; Vitner et al, 2010; Shen et al, 2012; Chen et al, 2014). Cholesterol reduction reportedly restores proper LacCer trafficking to Golgi, whereas cholesterol overload redirects LacCer to endolysosomal compartments (Puri et al, 1999). We therefore next assessed endolysosomal cholesterol accumulation, which has been reported for both MLIV and NPC1 (Shen et al, 2012; Chen et al, 2014; Grimm et al, 2014). Altered cellular cholesterol homeostasis can conveniently be visualized using the polyene antibiotic filipin. While we could not detect cholesterol storage in JNCL cells, we could confirm that NPC1 and MLIV fibroblasts strongly accumulate cholesterol (Fig 2A and B). In both NPC1 and MLIV cells, accumulated cholesterol was efficiently reduced upon TPC2 activation with TPC2-A1-P (Fig 2C and D). While in MLIV cells, significant effects were seen already after 24 h treatment, again in NPC1 cells only after 48 h effects were significant (Fig EV2A and B). In a further set of experiments, we tested TPC2M484L/G734E overexpression with and without TPC2-A1-P activation, finding that only overexpression in combination with the agonist significantly reduced intracellular cholesterol (Fig 2E–G). Using BAPTA-AM, we could again demonstrate, in analogy to LacCer, that chelation of Ca2+ results in cholesterol accumulation (Fig EV2C and D), confirming free intracellular Ca2+ to play a role in the process. BAPTA-AM was also shown to blunt the effect of TPC2-A1-P (Fig EV2E and F). We further silenced TPC2 expression in healthy human fibroblasts, which resulted in cholesterol accumulation in siTPC2, but not in mock-treated cells (Fig 2H and I), in accordance with previous observations in murine TPC2 knockout fibroblasts (Grimm et al, 2014). Furthermore, TPC2-A1-P rescued the cholesterol accumulation in mock-treated, but not in siTPC2-treated NPC1 fibroblasts, corroborating the on-target effect of TPC2-A1-P (Fig 2J and K). Efficacy of the siRNAs was validated using qRT-PCR (Fig EV2G). We next used electron microscopy (EM) to assess ultrastructural changes following compound treatment. Gross alterations in endolysosomal morphology have previously been reported in MLIV and NPC1 fibroblasts (Garver et al, 2000; Vergarajauregui et al, 2008). We found an abundance of lysosomes with aberrant/lamellar structures in NPC1 and to a lesser extent in MLIV cells, but observed no changes in lysosomal morphology in JNCL fibroblasts (Fig 2L and M). NPC1 fibroblasts showed a stronger difference from CTR than MLIV fibroblasts and only for the former we found TPC2-A1-P trea
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