bims-lysosi 2022-05-08 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2022–05–08
38 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Acat1/Soat1 knockout extends the mutant Npc1 mouse lifespan and ameliorates functional deficiencies in multiple organelles of mutant cells.
V-ATPase is a universal regulator of LC3-associated phagocytosis and non-canonical autophagy.
PAT2 regulates vATPase assembly and lysosomal acidification in brown adipocytes.
VAMP8 phosphorylation regulates lysosome dynamics during autophagy.
Abnormal triaging of misfolded proteins by adult neuronal ceroid lipofuscinosis-associated DNAJC5/CSPα mutants causes lipofuscin accumulation.
Pathogenic variants of Valosin Containing Protein induce lysosomal damage and transcriptional activation of autophagy regulators in neuronal cells.
Proteotoxic stress disrupts epithelial integrity by inducing MTOR sequestration and autophagy overactivation.
Coping with starvation: Cysteine keeps mTORC1 suppressed to ensure survival.
Experimental Therapeutic Approaches for the Treatment of Retinal Pathology in Neuronal Ceroid Lipofuscinoses.
Plasma membrane H+-ATPases promote TORC1 activation in plant suspension cells.
The V-ATPases in cancer and cell death.
Isolation and characterization of novel plekhm1 and def8 mutant alleles in Drosophila.
Rare Diseases in Glycosphingolipid Metabolism.
Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model.
mTORC1 under the control of CB1R.
SHIP164 is a chorein motif lipid transfer protein that controls endosome-Golgi membrane traffic.
Off-target effects of the lysosomal acid lipase inhibitors Lalistat-1 and Lalistat-2 on neutral lipid hydrolases.
Mechanisms of VPS35-Mediated Neurodegeneration in Parkinson's Disease.
The Dictyostelium Model for Mucolipidosis Type IV.
Pathways of integrins in the endo-lysosomal system.
Nucleoporin POM121 signals TFEB-mediated autophagy via activation of SIGMAR1/sigma-1 receptor chaperone by pridopidine.
Lactose-Appended Hydroxypropyl-β-Cyclodextrin Lowers Cholesterol Accumulation and Alleviates Motor Dysfunction in Niemann-Pick Type C Disease Model Mice.
Cathepsin B Relocalization in Late Membrane Disrupted Neurons Following Diffuse Brain Injury in Rats.
Targeting MCOLN1/TRPML1 channels to protect against ischemia-reperfusion injury by restoring the inhibited autophagic flux in cardiomyocytes.
STING controls energy stress-induced autophagy and energy metabolism via STX17.
Distinctive molecular features of regenerative stem cells in the damaged male germline.
Tollip-deficient zebrafish display no abnormalities in development, organ morphology or gene expression in response to lipopolysaccharide.
Suppression of lysosomal-associated protein transmembrane 5 ameliorates cardiac function and inflammatory response by inhibiting the NF-κB pathway after myocardial infarction in mice.
Drug Development in the Field of Sphinogolipid Metabolism.
A racemosin B derivative, C25, suppresses breast cancer growth via lysosomal membrane permeabilization and inhibition of autophagic flux.
Correction: mTORC1 in the orbitofrontal cortex promotes habitual alcohol seeking.
A novel superfamily of bridge-like lipid transfer proteins.
Trans-omics analysis of insulin action reveals a cell growth subnetwork which co-regulates anabolic processes.
Cholesterol determines the cytosolic entry and seeded aggregation of tau.
Differential expression of gene co-expression networks related to the mTOR signaling pathway in bipolar disorder.
Structural insights into Ras regulation by SIN1.
Remofuscin induces xenobiotic detoxification via a lysosome-to-nucleus signaling pathway to extend the Caenorhabditis elegans lifespan.
Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Proc Natl Acad Sci U S A. 2022 May 03. 119(18): e2201646119

Acat1/Soat1 knockout extends the mutant Npc1 mouse lifespan and ameliorates functional deficiencies in multiple organelles of mutant cells.

Maximillian A Rogers, Catherine C Y Chang, Robert A Maue, Elaina M Melton, Andrew A Peden, William S Garver, Junghoon Lee, Peter Schroen, Mitchell Huang, Ta-Yuan Chang.

  SignificanceNiemann-Pick type C disease (NPCD) is an incurable genetic neurological disorder. Cells with NPC mutations fail to export cholesterol from endosomal organelle to multiple other organelles. ACAT1 is an enzyme that converts cholesterol to cholesteryl esters for storage. In mutant NPC cells, cholesterol storage still occurs, although at reduced rate. Here we show that in mutant NPC cells, ACAT1 blockade (A1B) decreases cholesterol storage such that it can be utilized to fulfill cholesterol needs in multiple organelles. In mutant NPC1 mice, Acat1 gene knockout reduces pathological onset and prolongs the lifespan by 34%. This work identifies ACAT1 as a target to treat NPCD and may help to explain why A1B has been reported to ameliorate preclinical models for Alzheimer's disease.

Keywords:  Niemann-Pick disease type C; acyl-coenzyme A:cholesterol acyltransferase; cholesterol esterification

DOI:  https://doi.org/10.1073/pnas.2201646119
J Cell Biol. 2022 Jun 06. pii: e202105112. [Epub ahead of print]221(6):

V-ATPase is a universal regulator of LC3-associated phagocytosis and non-canonical autophagy.

Kirsty M Hooper, Elise Jacquin, Taoyingnan Li, Jonathan M Goodwin, John H Brumell, Joanne Durgan, Oliver Florey.

Non-canonical autophagy is a key cellular pathway in immunity, cancer, and neurodegeneration, characterized by conjugation of ATG8 to endolysosomal single membranes (CASM). CASM is activated by engulfment (endocytosis, phagocytosis), agonists (STING, TRPML1), and infection (influenza), dependent on K490 in the ATG16L1 WD40-domain. However, factors associated with non-canonical ATG16L1 recruitment and CASM induction remain unknown. Here, using pharmacological inhibitors, we investigate a role for V-ATPase during non-canonical autophagy. We report that increased V0-V1 engagement is associated with, and sufficient for, CASM activation. Upon V0-V1 binding, V-ATPase recruits ATG16L1, via K490, during LC3-associated phagocytosis (LAP), STING- and drug-induced CASM, indicating a common mechanism. Furthermore, during LAP, key molecular players, including NADPH oxidase/ROS, converge on V-ATPase. Finally, we show that LAP is sensitive to Salmonella SopF, which disrupts the V-ATPase-ATG16L1 axis and provide evidence that CASM contributes to the Salmonella host response. Together, these data identify V-ATPase as a universal regulator of CASM and indicate that SopF evolved in part to evade non-canonical autophagy.

DOI: https://doi.org/10.1083/jcb.202105112
Mol Metab. 2022 May 02. pii: S2212-8778(22)00077-1. [Epub ahead of print] 101508

PAT2 regulates vATPase assembly and lysosomal acidification in brown adipocytes.

Jiefu Wang, Yasuhiro Onogi, Martin Krueger, Josef Oeckl, Ruth Karlina, Inderjeet Singh, Stefanie M Hauck, Regina Feederle, Yongguo Li, Siegfried Ussar.

   OBJECTIVE: Brown adipocytes play a key role in maintaining body temperature as well as glucose and lipid homeostasis. However, brown adipocytes need to adapt their thermogenic activity and substrate utilization to changes in nutrient availability. Amongst the multiple factors influencing brown adipocyte activity, autophagy is an important regulatory element of thermogenic capacity and activity. Nevertheless, a specific sensing mechanism of extracellular amino acid availability linking autophagy to nutrient availability in brown adipocytes is unknown.
METHODS: To characterize the role of the amino acid transporter PAT2/SLC36A2 in brown adipocytes, loss or gain of function of PAT2 were studied with respect to differentiation, subcellular localization, lysosomal activity and autophagy. Activity of vATPase was evaluated by quenching of EGFP fused to LC3 or FITC-dextran loaded lysosomes in brown adipocytes upon amino acid starvation, whereas the effect of PAT2 on assembly of the vATPase was investigated by Native-PAGE.
RESULTS: We show that PAT2 translocates from the plasma membrane to the lysosome in response to amino acid withdrawal. Loss or overexpression of PAT2 impair lysosomal acidification and starvation induced S6K re-phosphorylation, as PAT2 facilitates the assembly of the lysosomal vATPase, by recruitment of the cytoplasmic V1 subunit to the lysosome.
CONCLUSION: PAT2 is an important sensor of extracellular amino acids and regulator of lysosomal acidification in brown adipocytes.

Keywords:  Brown adipocytes; Lysosomal acidification; Proton-coupled amino acid transporter; Transporter translocation across membranes; V-ATPase assembly

DOI:  https://doi.org/10.1016/j.molmet.2022.101508
Autophagy Rep. 2022 ;1(1): 79-82

VAMP8 phosphorylation regulates lysosome dynamics during autophagy.

Lei Wang, Jiajie Diao.

  In the final critical step for autophagic degradation, lysosomes fuse with autophagosomes to form autolysosomes. Although recent research has suggested that soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are important for lysosome-autophagosome fusion, neither the architecture of the prefusion state nor the regulatory mechanisms have been identified. In our study, using structured illumination microscopy, we observed that lysosomes formed clusters around individual autophagosomes, thereby setting the stage for membrane fusion. Moreover, VAMP8 (vesicle-associated membrane protein 8) assists in forming the prefusion state of these clusters. We also found that VAMP8 phosphorylation reduces spontaneous lysosome-autophagosome fusion, whereas its dephosphorylation promotes fusion events between lysosomes and autophagosomes in both normal and autophagy-induced conditions. Our data thus suggest a key role of VAMP8 phosphorylation in the regulation of lysosome-autophagosome fusion.

Keywords:  Autophagy; VAMP8; fusion; lysosomes; phosphorylation

DOI:  https://doi.org/10.1080/27694127.2022.2031378
Autophagy. 2022 May 04. 1-20

Abnormal triaging of misfolded proteins by adult neuronal ceroid lipofuscinosis-associated DNAJC5/CSPα mutants causes lipofuscin accumulation.

Juhyung Lee, Yue Xu, Layla Saidi, Miao Xu, Konrad Zinsmaier, Yihong Ye.

  Mutations in DNAJC5/CSPα are associated with adult neuronal ceroid lipofuscinosis (ANCL), a dominant-inherited neurodegenerative disease featuring lysosome-derived autofluorescent storage materials (AFSMs) termed lipofuscin. Functionally, DNAJC5 has been implicated in chaperoning synaptic proteins and in misfolding-associated protein secretion (MAPS), but how DNAJC5 dysfunction causes lipofuscinosis and neurodegeneration is unclear. Here we report two functionally distinct but coupled chaperoning activities of DNAJC5, which jointly regulate lysosomal homeostasis: While endolysosome-associated DNAJC5 promotes ESCRT-dependent microautophagy, a fraction of perinuclear and non-lysosomal DNAJC5 mediates MAPS. Functional proteomics identifies a previously unknown DNAJC5 interactor SLC3A2/CD98hc that is essential for the perinuclear DNAJC5 localization and MAPS but dispensable for microautophagy. Importantly, uncoupling these two processes, as seen in cells lacking SLC3A2 or expressing ANCL-associated DNAJC5 mutants, generates DNAJC5-containing AFSMs resembling NCL patient-derived lipofuscin and induces neurodegeneration in a Drosophila ANCL model. These findings suggest that MAPS safeguards microautophagy to avoid DNAJC5-associated lipofuscinosis and neurodegeneration.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AFSM: autofluorescent storage materials; ANCL: adult neuronal ceroid lipofuscinosis; Baf. A1: bafilomycin A1; CLN: ceroid lipofuscinosis neuronal; CLU: clusterin; CS: cysteine string domain of DNAJC5/CSPα; CUPS: compartment for unconventional protein secretion; DN: dominant negative; DNAJC5/CSPα: DnaJ heat shock protein family (Hsp40) member C5; eMI: endosomal microautophagy; ESCRT: endosomal sorting complex required for transport; GFP: green fluorescent protein; HSPA8/HSC70: heat shock protein family A (Hsp70) member 8; INCL: infant neuronal ceroid lipofuscinosis; JNCL: juvenile neuronal ceroid lipofuscinosis; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAPTM4B: lysosomal protein transmembrane 4 beta; LN: linker domain of DNAJC5/CSPα; MAPS: misfolding-associated protein secretion; mCh/Ch: mCherry; mCi/Ci: mCitrine; MTOR: mechanistic target of rapamycin kinase; NCL: neuronal ceroid lipofuscinosis; PPT1: palmitoyl-protein thioesterase 1; PQC: protein quality control; SBP: streptavidin binding protein; SGT: small glutamine-rich tetratricopeptide repeat; shRNA: short hairpin RNA; SLC3A2/CD98hc: solute carrier family 3 member 2; SNCA/α-synuclein: synuclein alpha; TMED10: transmembrane p24 trafficking protein 10; UV: ultraviolet; VPS4: vacuolar protein sorting 4 homolog; WT: wild type.

Keywords:  CLN4; DNAJC5/CSPα; ESCRT; cysteine string protein α; lysobody; lysosome/endolysosome; microautophagy/eMI; misfolding-associated protein secretion/MAPS; neuronal ceroid lipofuscinosis/NCL; protein quality control/PQC; unconventional protein secretion/UPS

DOI:  https://doi.org/10.1080/15548627.2022.2065618
Neuropathol Appl Neurobiol. 2022 May 02. e12818

Pathogenic variants of Valosin Containing Protein induce lysosomal damage and transcriptional activation of autophagy regulators in neuronal cells.

Veronica Ferrari, Riccardo Cristofani, Maria E Cicardi, Barbara Tedesco, Valeria Crippa, Marta Chierichetti, Elena Casarotto, Marta Cozzi, Francesco Mina, Mariarita Galbiati, Margherita Piccolella, Serena Carra, Thomas Vaccari, Angele Nalbandian, Virginia Kimonis, Tyler R Fortuna, Udai B Pandey, Maria C Gagliani, Katia Cortese, Paola Rusmini, Angelo Poletti.

   AIM: Mutations in the Valosin-containing protein (VCP) gene cause various lethal proteinopathies that mainly include inclusion body myopathy with Paget's disease of bone frontotemporal dementia (IBMPFD) and amyotrophic lateral sclerosis (ALS). Different pathological mechanisms have been proposed. Here, we define the impact of VCP mutants on lysosomes and how cellular homeostasis is restored by inducing autophagy in the presence of lysosomal damage.
METHODS: By electron microscopy, we studied lysosomal morphology in VCP animal and motoneuronal models. Using western blotting, RT-qPCR, immunofluorescence, and filter trap assay, we evaluated the effect of selected VCP mutants in neuronal cells on lysosome size and activity, lysosomal membrane permeabilization, and their impact on autophagy.
RESULTS: We found that VCP mutants induced aberrant multilamellar organelles in VCP animal and cell models similar to those found in patients with VCP mutations or with lysosomal storage disorders. In neuronal cells, we found altered lysosomal activity characterized by membrane permeabilization with galectin-3 redistribution and activation of PPP3CB. This selectively activated the autophagy/lysosomal transcriptional regulator TFE3, but not TFEB, and enhanced both SQSTM1/p62 and lipidated MAP 1LC3B levels inducing autophagy. Moreover, we found that WT VCP, but not the mutants, counteracted lysosomal damage induced either by trehalose or by a mutant form of SOD1 (G93A), also blocking the formation of its insoluble intracellular aggregates. Thus, chronic activation of autophagy might fuel the formation of multilamellar bodies.
CONCLUSION: Together, our findings provide insights into the pathogenesis of VCP-related diseases, by proposing a novel mechanism of multilamellar body formation induced by VCP mutants that involves lysosomal damage and induction of lysophagy.

Keywords:  ALS; PQC; TFE3; lysosome; neurodegeneration; p97

DOI:  https://doi.org/10.1111/nan.12818
Autophagy. 2022 May 06. 1-15

Proteotoxic stress disrupts epithelial integrity by inducing MTOR sequestration and autophagy overactivation.

Xiaoxiang Cheng, Pei Zhang, Hongyu Zhao, Hui Zheng, Kai Zheng, Hong Zhang, Hongjie Zhang.

  Macroautophagy/autophagy, an evolutionarily conserved degradation system, serves to clear intracellular components through the lysosomal pathway. Mounting evidence has revealed cytoprotective roles of autophagy; however, the intracellular causes of overactivated autophagy, which has cytotoxic effects, remain elusive. Here we show that sustained proteotoxic stress induced by loss of the RING and Kelch repeat-containing protein C53A5.6/RIKE-1 induces sequestration of LET-363/MTOR complex and overactivation of autophagy, and consequently impairs epithelial integrity in C. elegans. In C53A5.6/RIKE-1-deficient animals, blocking autophagosome formation effectively prevents excessive endosomal degradation, mitigates mislocalization of intestinal membrane components and restores intestinal lumen morphology. However, autophagy inhibition does not affect LET-363/MTOR aggregation in animals with compromised C53A5.6/RIKE-1 function. Improving proteostasis capacity by reducing DAF-2 insulin/IGF1 signaling markedly relieves the aggregation of LET-363/MTOR and alleviates autophagy overactivation, which in turn reverses derailed endosomal trafficking and rescues epithelial morphogenesis defects in C53A5.6/RIKE-1-deficient animals. Hence, our studies reveal that C53A5.6/RIKE-1-mediated proteostasis is critical for maintaining the basal level of autophagy and epithelial integrity.Abbreviations: ACT-5: actin 5; ACTB: actin beta; ALs: autolysosomes; APs: autophagosomes; AJM-1: apical junction molecule; ATG: autophagy related; C. elegans: Caenorhabditis elegans; CPL-1: cathepsin L family; DAF: abnormal dauer formation; DLG-1: Drosophila discs large homolog; ERM-1: ezrin/radixin/moesin; EPG: ectopic P granule; GFP: freen fluorescent protein; HLH-30: helix loop helix; HSP: heat shock protein; LAAT-1: lysosome associated amino acid transporter; LET: lethal; LGG-1: LC3, GABARAP and GATE-16 family; LMP-1: LAMP (lysosome-associated membrane protein) homolog; MTOR: mechanistic target of rapamycin kinase; NUC-1: abnormal nuclease; PEPT-1/OPT-2: Peptide transporter family; PGP-1: P-glycoprotein related; RAB: RAB family; RIKE-1: RING and Kelch repeat-containing protein; SLCF-1: solute carrier family; SQST-1: sequestosome related; SPTL-1: serine palmitoyl transferase family.

Keywords:  Autophagy; C. elegans; LET-363/MTOR; endosomal degradation; epithelial morphogenesis; proteostasis

DOI:  https://doi.org/10.1080/15548627.2022.2071381
Mol Cell. 2022 May 05. pii: S1097-2765(22)00375-6. [Epub ahead of print]82(9): 1613-1615

Coping with starvation: Cysteine keeps mTORC1 suppressed to ensure survival.

Dohun Kim, Gerta Hoxhaj.

Jouandin et al. (2022) show that lysosomal-derived cysteine serves as a signal to promote the tricarboxylic acid (TCA) cycle and suppress TORC1 signaling for Drosophila to endure starvation periods.

DOI: https://doi.org/10.1016/j.molcel.2022.04.018
Front Neurol. 2022 ;13 866983

Experimental Therapeutic Approaches for the Treatment of Retinal Pathology in Neuronal Ceroid Lipofuscinoses.

Udo Bartsch, Stephan Storch.

  The neuronal ceroid lipofuscinoses (NCLs) are a group of childhood-onset neurodegenerative lysosomal storage disorders mainly affecting the brain and the retina. In the NCLs, disease-causing mutations in 13 different ceroid lipofuscinoses genes (CLN) have been identified. The clinical symptoms include seizures, progressive neurological decline, deterioration of motor and language skills, and dementia resulting in premature death. In addition, the deterioration and loss of vision caused by progressive retinal degeneration is another major hallmark of NCLs. To date, there is no curative therapy for the treatment of retinal degeneration and vision loss in patients with NCL. In this review, the key findings of different experimental approaches in NCL animal models aimed at attenuating progressive retinal degeneration and the decline in retinal function are discussed. Different approaches, including experimental enzyme replacement therapy, gene therapy, cell-based therapy, and immunomodulation therapy were evaluated and showed encouraging therapeutic benefits. Recent experimental ocular gene therapies in NCL animal models with soluble lysosomal enzyme deficiencies and transmembrane protein deficiencies have shown the strong potential of gene-based approaches to treat retinal dystrophies in NCLs. In CLN3 and CLN6 mouse models, an adeno-associated virus (AAV) vector-mediated delivery of CLN3 and CLN6 to bipolar cells has been shown to attenuate the retinal dysfunction. Therapeutic benefits of ocular enzyme replacement therapies were evaluated in CLN2 and CLN10 animal models. Since brain-targeted gene or enzyme replacement therapies will most likely not attenuate retinal neurodegeneration, there is an unmet need for treatment options additionally targeting the retina in patients with NCL. The long-term benefits of these therapeutic interventions aimed at attenuating retinal degeneration and vision loss in patients with NCL remain to be investigated in future clinical studies.

Keywords:  Batten disease; NCL; enzyme replacement therapy; gene therapy; lysosomal storage disorder; neuronal ceroid lipofuscinoses; retinal degeneration

DOI:  https://doi.org/10.3389/fneur.2022.866983
iScience. 2022 May 20. 25(5): 104238

Plasma membrane H+-ATPases promote TORC1 activation in plant suspension cells.

Cecilia Primo, Catherine Navarre, François Chaumont, Bruno André.

  The TORC1 (Target of Rapamycin Complex 1) kinase complex plays a pivotal role in controlling cell growth in probably all eukaryotic species. The signals and mechanisms regulating TORC1 have been intensely studied in mammals but those of fungi and plants are much less known. We have previously reported that the yeast plasma membrane H+-ATPase Pma1 promotes TORC1 activation when stimulated by cytosolic acidification or nutrient-uptake-coupled H+ influx. Furthermore, a homologous plant H+-ATPase can substitute for yeast Pma1 to promote this H+-elicited TORC1 activation. We here report that TORC1 activity in Nicotiana tabacum BY-2 cells is also strongly influenced by the activity of plasma membrane H+-ATPases. In particular, stimulation of H+-ATPases by fusicoccin activates TORC1, and this response is also observed in cells transferred to a nutrient-free and auxin-free medium. Our results suggest that plant H+-ATPases, known to be regulated by practically all factors controlling cell growth, contribute to TOR signaling.

Keywords:  Biological sciences; Molecular plant pathology; Plant biology

DOI:  https://doi.org/10.1016/j.isci.2022.104238
Cancer Gene Ther. 2022 May 03.

The V-ATPases in cancer and cell death.

Fangquan Chen, Rui Kang, Jiao Liu, Daolin Tang.

Transmembrane ATPases are membrane-bound enzyme complexes and ion transporters that can be divided into F-, V-, and A-ATPases according to their structure. The V-ATPases, also known as H+-ATPases, are large multi-subunit protein complexes composed of a peripheral domain (V1) responsible for the hydrolysis of ATP and a membrane-integrated domain (V0) that transports protons across plasma membrane or organelle membrane. V-ATPases play a fundamental role in maintaining pH homeostasis through lysosomal acidification and are involved in modulating various physiological and pathological processes, such as macropinocytosis, autophagy, cell invasion, and cell death (e.g., apoptosis, anoikis, alkaliptosis, ferroptosis, and lysosome-dependent cell death). In addition to participating in embryonic development, V-ATPase pathways, when dysfunctional, are implicated in human diseases, such as neurodegenerative diseases, osteopetrosis, distal renal tubular acidosis, and cancer. In this review, we summarize the structure and regulation of isoforms of V-ATPase subunits and discuss their context-dependent roles in cancer biology and cell death. Updated knowledge about V-ATPases may enable us to design new anticancer drugs or strategies.

DOI: https://doi.org/10.1038/s41417-022-00477-y
Biol Futur. 2022 May 04.

Isolation and characterization of novel plekhm1 and def8 mutant alleles in Drosophila.

Tamás Maruzs, Enikő Lakatos, Dalma Feil-Börcsök, Péter Lőrincz, Gábor Juhász.

  Lysosomal degradation of cytoplasmic components by autophagy ensures the continuous turnover of proteins and organelles and aids cellular survival during nutrient deprivation and other stress conditions. Lysosomal targeting of cytoplasmic proteins and organelles requires the concerted action of several proteins and multisubunit complexes. The core components of this machinery are conserved from yeast to humans and many of them are well-characterized; however, novel molecular players have been recently discovered and are waiting for detailed analysis. The osteopetrosis-linked PLEKHM1 protein is a lysosomal adaptor involved in autophagosome and endosome to lysosome fusion events and its role in lysosomal positioning in osteoclasts was reported together with its proposed binding partner, the relatively uncharacterized DEF8 protein. Here, we report the generation and subsequent analysis of novel mutant alleles of Drosophila plekhm1 and def8. Interestingly, the CRISPR-generated null mutations of these genes do not have any obvious effects on autophagy in Drosophila tissues, even though RNAi knockdown of these genes seems to perturb autophagy. Although these results are quite surprising and raise the possibility of compensatory changes in the case of null mutants, the new alleles will be valuable tools in future studies to understand the cellular functions of Drosophila Plekhm1 and Def8 proteins.

Keywords:  Autophagy; Def8; Drosophila; Lysosome; Plekhm1

DOI:  https://doi.org/10.1007/s42977-022-00118-3
Adv Exp Med Biol. 2022 ;1372 189-213

Rare Diseases in Glycosphingolipid Metabolism.

Hongwen Zhou, Zhoulu Wu, Yiwen Wang, Qinyi Wu, Moran Hu, Shuai Ma, Min Zhou, Yan Sun, Baowen Yu, Jingya Ye, Wanzi Jiang, Zhenzhen Fu, Yingyun Gong.

  Sphingolipidoses is a cluster of genetic rare disorders regarding glycosphingolipid metabolism, classified as lysosomal storage disorders (LSD). Here, we focus on eight inheritable diseases, including GM1 gangliosidosis, GM2 gangliosidosis, Fabry disease, Gaucher's disease, metachromatic leukodystrophy, Krabbe disease, Niemann-Pick disease A and B, and Farber disease. Mostly, pathogenic mutations in the key enzyme are loss-function, resulting in accumulation of substrates and deficiency of products. Thus, cellular overload of substrates causes lipotoxicity, which is deleterious to cellular and organ function. In the terms of clinical manifestations in sphingolipidoses, multiple systems and organs, especially central nervous system (CNS) are usually affected. As for diagnosis strategy, enzymatic activity assay and genetic sequencing are helpful. Up till now, limited treatment approaches have approved for treating sphingolipidoses, with some potential strategies for further evaluation. In general, enzyme replacement therapy (ERT), substrate reduction therapy (SRT), and molecular chaperones are feasible choices for enzyme deficiency disorders, but these therapies are limited to relieve CNS lesions and symptoms due to prevention from blood-brain barrier. Other possible treatments such as gene therapy, bone marrow transplantation (BMT), and hematopoietic stem cell transplantation (HSCT) need further evaluation.

Keywords:  Fabry disease; Farber disease; GM1 gangliosidosis; GM2 gangliosidosis; Gaucher’s disease; Glycosphingolipid; Krabbe disease; Lysosomal storage disorder; Metachromatic leukodystrophy; Niemann–Pick disease A and B; Sphingolipidoses

DOI:  https://doi.org/10.1007/978-981-19-0394-6_13
ACS Chem Neurosci. 2022 May 06.

Phenotypic Screening Using High-Content Imaging to Identify Lysosomal pH Modulators in a Neuronal Cell Model.

Marcus Y Chin, Kean-Hooi Ang, Julia Davies, Carolina Alquezar, Virginia G Garda, Brendan Rooney, Kun Leng, Martin Kampmann, Michelle R Arkin, Aimee W Kao.

  Lysosomes are intracellular organelles responsible for the degradation of diverse macromolecules in a cell. A highly acidic pH is required for the optimal functioning of lysosomal enzymes. Loss of lysosomal intralumenal acidity can disrupt cellular protein homeostasis and is linked to age-related diseases such as neurodegeneration. Using a new robust lysosomal pH biosensor (FIRE-pHLy), we developed a cell-based fluorescence assay for high-throughput screening (HTS) and applied it to differentiated SH-SY5Y neuroblastoma cells. The goal of this study was twofold: (1) to screen for small molecules that acidify lysosomal pH and (2) to identify molecular targets and pathways that regulate lysosomal pH. We conducted a screen of 1835 bioactive compounds with annotated target information to identify lysosomal pH modulators (both acidifiers and alkalinizers). Forty-five compounds passed the initial hit selection criteria, using a combined analysis approach of population-based and object-based data. Twenty-three compounds were retested in dose-response assays and two compounds, OSI-027 and PP242, were identified as top acidifying hits. Overall, data from this phenotypic HTS screen may be used to explore novel regulatory pathways of lysosomal pH regulation. Additionally, OSI-027 and PP242 may serve as useful tool compounds to enable mechanistic studies of autophagy activation and lysosomal acidification as potential therapeutic pathways for neurodegenerative diseases.

Keywords:  high-content analysis; lysosomal pH; lysosomes; neurons; pH biosensor; phenotypic screening

DOI:  https://doi.org/10.1021/acschemneuro.1c00804
Nat Rev Nephrol. 2022 May 04.

mTORC1 under the control of CB1R.

Monica Wang.

DOI: https://doi.org/10.1038/s41581-022-00582-z
J Cell Biol. 2022 Jun 06. pii: e202111018. [Epub ahead of print]221(6):

SHIP164 is a chorein motif lipid transfer protein that controls endosome-Golgi membrane traffic.

Michael G Hanna, Patreece H Suen, Yumei Wu, Karin M Reinisch, Pietro De Camilli.

Cellular membranes differ in protein and lipid composition as well as in the protein-lipid ratio. Thus, progression of membranous organelles along traffic routes requires mechanisms to control bilayer lipid chemistry and their abundance relative to proteins. The recent structural and functional characterization of VPS13-family proteins has suggested a mechanism through which lipids can be transferred in bulk from one membrane to another at membrane contact sites, and thus independently of vesicular traffic. Here, we show that SHIP164 (UHRF1BP1L) shares structural and lipid transfer properties with these proteins and is localized on a subpopulation of vesicle clusters in the early endocytic pathway whose membrane cargo includes the cation-independent mannose-6-phosphate receptor (MPR). Loss of SHIP164 disrupts retrograde traffic of these organelles to the Golgi complex. Our findings raise the possibility that bulk transfer of lipids to endocytic membranes may play a role in their traffic.

DOI: https://doi.org/10.1083/jcb.202111018
Mol Metab. 2022 Apr 30. pii: S2212-8778(22)00079-5. [Epub ahead of print] 101510

Off-target effects of the lysosomal acid lipase inhibitors Lalistat-1 and Lalistat-2 on neutral lipid hydrolases.

Ivan Bradić, Katharina B Kuentzel, Sophie Honeder, Gernot F Grabner, Nemanja Vujić, Robert Zimmermann, Ruth Birner-Gruenberger, Dagmar Kratky.

   OBJECTIVES: Lysosomal acid lipase (LAL) is the key enzyme, which degrades neutral lipids at an acidic pH in lysosomes. The role of LAL in various cellular processes has mostly been studied in LAL-knockout mice, which share phenotypical characteristics with humans suffering from LAL deficiency. In vitro, the cell-specific functions of LAL have been commonly investigated by using the LAL inhibitors Lalistat-1 and Lalistat-2.
METHODS: We performed lipid hydrolase activity assays and serine hydrolase-specific activity-based labeling combined with quantitative proteomics to investigate potential off-target effects of Lalistat-1 and -2.
RESULTS: Pharmacological LAL inhibition but not genetic loss of LAL impairs isoproterenol-stimulated lipolysis as well as neutral triglyceride and cholesteryl ester hydrolase activities. Apart from LAL, Lalistat-1 and -2 also inhibit major cytosolic lipid hydrolases responsible for lipid degradation in primary cells at neutral pH through off-target effects. Their binding to the active center of the enzymes leads to a decrease in neutral lipid hydrolase activities in cells overexpressing the respective enzymes.
CONCLUSIONS: Our findings are critically important since they demonstrate that commonly used concentrations of these inhibitors are not suitable to investigate the role of LAL-specific lipolysis in lysosomal function, signaling pathways, and autophagy. The interpretation of their effects on lipid metabolism should be taken with caution and the applied inhibitor concentrations in cell culture studies should not exceed 1 μM.

Keywords:  ATGL; HSL; LAL; Lipid hydrolase activity; Lipolysis; MGL

DOI:  https://doi.org/10.1016/j.molmet.2022.101510
Int Rev Mov Disord. 2021 ;2 221-244

Mechanisms of VPS35-Mediated Neurodegeneration in Parkinson's Disease.

Dorian Sargent, Darren J Moore.

  Parkinson's disease is a sporadic and common neurodegenerative movement disorder resulting from the complex interplay between genetic risk, aging and environmental exposure. Familial forms of PD account for ~10% of cases and are known to result from the inheritance of mutations in at least 15 genes. Mutations in the vacuolar protein sorting 35 ortholog (VPS35) gene cause late-onset, autosomal dominant familial PD. VPS35 is a key suunit of the pentameric retromer complex that plays a role in the retrograde sorting and recycling of transmembrane cargo proteins from endosomes to the plasma membrane and trans-Golgi network. A single heterozygous Asp620Asn (D620N) mutation in VPS35 has been identified in multiple families that segregates with PD, and a number of experimental cellular and animal models have been developed to understand its pathogenic effects. At the molecular level, the D620N mutation has been shown to impair the interaction of VPS35 with the WASH complex, that plays an accessory function in retromer-dependent sorting. In addition, the D620N mutation has been linked to the abnormal sorting of retromer cargo, including CI-M6PR, AMPA receptor subunits, MUL1, LAMP2a and ATG9A, as well as to LRRK2 hyperactivation. At the cellular level, data support an impact of D620N VPS35 on mitochondrial function, the autophagy-lysosomal pathway, Wnt signaling and neurotransmission via altered endosomal sorting. The relevance of abnormal retromer sorting and cellular pathways to PD-related neurodegenerative phenotypes induced by D620N VPS35 in rodent models is not yet clear. There is also uncertainty regarding the mechanism-of-action of the D620N mutation and whether it manifests pathogenic effects in animal models and PD through a gain-of-function and/or a partial dominant-negative mechanism. Here, we discuss the emerging molecular and cellular mechanisms underlying PD induced by familial VPS35 mutations, going from structure to cellular function to neuropathology. We further discuss studies linking reduced retromer function to other neurodegenerative diseases and potential therapeutic strategies to normalize retromer function to mitigate disease.

Keywords:  Golgi; LRRK2; Lysosome; Mitochondria; Parkinson’s disease (PD); VPS35; endosome; retromer; vesicular sorting

DOI:  https://doi.org/10.1016/bs.irmvd.2021.08.005
Front Cell Dev Biol. 2022 ;10 741967

The Dictyostelium Model for Mucolipidosis Type IV.

Claire Y Allan, Paul R Fisher.

  Mucolipidosis type IV, a devastating neurological lysosomal disease linked to mutations in the transient receptor potential channel mucolipin 1, TRPML1, a calcium permeable channel in the membranes of vesicles in endolysosomal system. TRPML1 function is still being elucidated and a better understanding of the molecular pathogenesis of Mucolipidosis type IV, may facilitate development of potential treatments. We have created a model to study mucolipin function in the eukaryotic slime mould Dictyostelium discoideum by altering expression of its single mucolipin homologue, mcln. We show that in Dictyostelium mucolipin overexpression contributes significantly to global chemotactic calcium responses in vegetative and differentiated cells. Knockdown of mucolipin also enhances calcium responses in vegetative cells but does not affect responses in 6-7 h developed cells, suggesting that in developed cells mucolipin may help regulate local calcium signals rather than global calcium waves. We found that both knocking down and overexpressing mucolipin often, but not always, presented the same phenotypes. Altering mucolipin expression levels caused an accumulation or increased acidification of Lysosensor Blue stained vesicles in vegetative cells. Nutrient uptake by phagocytosis and macropinocytosis were increased but growth rates were not, suggesting defects in catabolism. Both increasing and decreasing mucolipin expression caused the formation of smaller slugs and larger numbers of fruiting bodies during multicellular development, suggesting that mucolipin is involved in initiation of aggregation centers. The fruiting bodies that formed from these smaller aggregates had proportionately larger basal discs and thickened stalks, consistent with a regulatory role for mucolipin-dependent Ca2+ signalling in the autophagic cell death pathways involved in stalk and basal disk differentiation in Dictyostelium. Thus, we have provided evidence that mucolipin contributes to chemotactic calcium signalling and that Dictyostelium is a useful model to study the molecular mechanisms involved in the cytopathogenesis of Mucolipidosis type IV.

Keywords:  acidic vesicles; aggregation; calcium signalling; endocytosis; growth; lysosomes; mucolipidosis IV; plasma membrane

DOI:  https://doi.org/10.3389/fcell.2022.741967
Biol Futur. 2022 May 02.

Pathways of integrins in the endo-lysosomal system.

Márton Molnár, Ármin Sőth, Zsófia Simon-Vecsei.

  In this review, we present recent scientific advances about integrin trafficking in the endo-lysosomal system. In the last few years, plenty of new information has emerged about the endo-lysosomal system, integrins, and the mechanism, how exactly the intracellular trafficking of integrins is regulated. We review the internalization and recycling pathways of integrins, and we provide information about the possible ways of lysosomal degradation through the endosomal and autophagic system. The regulation of integrin internalization and recycling proved to be a complex process worth studying. Trafficking of integrins, together with the regulation of their gene expression, defines cellular adhesion and cellular migration through bidirectional signalization and ligand binding. Thus, any malfunction in this system can potentially (but not necessarily) lead to tumorigenesis or metastasis. Hence, extensive examinations of integrins in the endo-lysosomal system raise the possibility to identify potential new medical targets. Furthermore, this knowledge can also serve as a basis for further determination of integrin signaling- and adhesion-related processes.

Keywords:  Endo-lysosomal system; Integrin; Internalization and recycling pathway; Lysosomal degradation

DOI:  https://doi.org/10.1007/s42977-022-00120-9
Autophagy. 2022 May 04. 1-26

Nucleoporin POM121 signals TFEB-mediated autophagy via activation of SIGMAR1/sigma-1 receptor chaperone by pridopidine.

Shao-Ming Wang, Hsiang-En Wu, Yuko Yasui, Michal Geva, Michael Hayden, Tangui Maurice, Mauro Cozzolino, Tsung-Ping Su.

  Macroautophagy/autophagy is an essential process for cellular survival and is implicated in many diseases. A critical step in autophagy is the transport of the transcription factor TFEB from the cytosol into the nucleus, through the nuclear pore (NP) by KPNB1/importinβ1. In the C9orf72 subtype of amyotrophic lateral sclerosis-frontotemporal lobar degeneration (ALS-FTD), the hexanucleotide (G4C2)RNA expansion (HRE) disrupts the nucleocytoplasmic transport of TFEB, compromising autophagy. Here we show that a molecular chaperone, the SIGMAR1/Sigma-1 receptor (sigma non-opioid intracellular receptor 1), facilitates TFEB transport into the nucleus by chaperoning the NP protein (i.e., nucleoporin) POM121 which recruits KPNB1. In NSC34 cells, HRE reduces TFEB transport by interfering with the association between SIGMAR1 and POM121, resulting in reduced nuclear levels of TFEB, KPNB1, and the autophagy marker LC3-II. Overexpression of SIGMAR1 or POM121, or treatment with the highly selective and potent SIGMAR1 agonist pridopidine, currently in phase 2/3 clinical trials for ALS and Huntington disease, rescues all of these deficits. Our results implicate nucleoporin POM121 not merely as a structural nucleoporin, but also as a chaperone-operated signaling molecule enabling TFEB-mediated autophagy. Our data suggest the use of SIGMAR1 agonists, such as pridopidine, for therapeutic development of diseases in which autophagy is impaired.Abbreviations: ALS-FTD, amyotrophic lateral sclerosis-frontotemporal dementiaC9ALS-FTD, C9orf72 subtype of amyotrophic lateral sclerosis-frontotemporal dementiaCS, citrate synthaseER, endoplasmic reticulumGSS, glutathione synthetaseHRE, hexanucleotide repeat expansionHSPA5/BiP, heat shock protein 5LAMP1, lysosomal-associated membrane protein 1MAM, mitochondria-associated endoplasmic reticulum membraneMAP1LC3/LC3, microtubule-associated protein 1 light chain 3NP, nuclear poreNSC34, mouse motor neuron-like hybrid cell lineNUPs, nucleoporinsPOM121, nuclear pore membrane protein 121SIGMAR1/Sigma-1R, sigma non-opioid intracellular receptor 1TFEB, transcription factor EBTMEM97/Sigma-2R, transmembrane protein 97.

Keywords:  ALS/FTD; KPNB1/importinβ1; SIGMAR1; TFEB; c9orf72; chaperone; nucleocytoplasmic transport; nucleoporin POM121; pridopidine; sigma-1 receptor

DOI:  https://doi.org/10.1080/15548627.2022.2063003
ACS Appl Bio Mater. 2022 May 04.

Lactose-Appended Hydroxypropyl-β-Cyclodextrin Lowers Cholesterol Accumulation and Alleviates Motor Dysfunction in Niemann-Pick Type C Disease Model Mice.

Takumi Nishida, Ryoma Yokoyama, Yuto Kubohira, Yuki Maeda, Toru Takeo, Naomi Nakagata, Hiroki Takagi, Kandai Ishikura, Kazunori Yanagihara, Shogo Misumi, Naoki Kishimoto, Yoichi Ishitsuka, Yuki Kondo, Tetsumi Irie, Minami Soga, Takumi Era, Risako Onodera, Taishi Higashi, Keiichi Motoyama.

  Niemann-Pick disease type C (NPC) is characterized by the accumulation of glycolipids such as free cholesterol, sphingomyelin, and gangliosides in late endosomes/lysosomes (endolysosomes) due to abnormalities in the membrane proteins NPC1 or NPC2. The main symptoms of NPC caused by free cholesterol accumulation in various tissues vary depending on the time of onset, but hepatosplenomegaly and neurological symptoms accompanied by decreased motor, cognitive, and mental functions are observed in all age groups. However, the efficacy of NPC treatment remains limited. Herein, we have fabricated lactose-appended hydroxypropyl-β-cyclodextrin (Lac-HPβCD) and evaluated its lowering effects on cholesterol accumulation in NPC model mice. We reveal that Lac-HPβCD lowers cholesterol accumulation in the liver and spleen by reducing the amount of free cholesterol. Moreover, Lac-HPβCD reduces the amount of free cholesterol in the cerebrum and slightly alleviates motor dysfunction. These results suggest that Lac-HPβCD has potential for the treatment of NPC.

Keywords:  Niemann−Pick disease type C; cholesterol; cyclodextrin; liver targeting; spleen

DOI:  https://doi.org/10.1021/acsabm.2c00233
ASN Neuro. 2022 Jan-Dec;14:14 17590914221099112

Cathepsin B Relocalization in Late Membrane Disrupted Neurons Following Diffuse Brain Injury in Rats.

Martina L Hernandez, Michael Marone, Karen M Gorse, Audrey D Lafrenaye.

  Traumatic brain injury (TBI) has consequences that last for years following injury. While TBI can precipitate a variety of diffuse pathologies, the mechanisms involved in injury-induced neuronal membrane disruption remain elusive. The lysosomal cysteine protease, Cathepsin B (Cath B), and specifically its redistribution into the cytosol has been implicated in cell death. Little is known about Cath B or neuronal membrane disruption chronically following diffuse TBI. Therefore, the current study evaluated Cath B and diffuse neuronal membrane disruption over a more chronic post-injury window (6 h-4 w). We evaluated Cath B in adult male Sprague-Dawley rats following central fluid percussion injury (CFPI). Expression of Cath B, as well as Cath B-associated pro (Bak and AIF) and anti-apoptotic (Bcl-xl) proteins, were assessed using western blot analysis. Cath B activity was also assessed. Localization of Cath B was evaluated in the membrane disrupted and non-disrupted population following CFPI using immunohistochemistry paired with quantitative image analysis and ultrastructural verification. There was no difference in expression or activity of Cath B or any of the associated proteins between sham and CFPI at any time post-injury. Immunohistological studies, however, showed a sub-cellular re-localization of Cath B at 2 w and 4 w post-injury in the membrane disrupted neuronal population as compared to the time-point matched non-disrupted neurons. Both membrane disruption and Cath B relocalization appear linked to neuronal atrophy. These observations are indicative of a late secondary pathology that represents an opportunity for therapeutic treatment of these neurons following diffuse TBI. Summary Statement Lysosomal cathepsin B relocalizes to the cytosol in neurons with disrupted plasmalemmal membranes weeks following diffuse brain injury. Both the membrane disrupted and cathepsin B relocalized neuronal subpopulations displayed smaller soma and nucleus size compared to non-pathological neurons, indicating atrophy.

Keywords:  AIF; Bak/Bcl-XL; cathepsin B; membrane disruption; neuronal atrophy; traumatic brain injury

DOI:  https://doi.org/10.1177/17590914221099112
Autophagy. 2022 May 06. 1-3

Targeting MCOLN1/TRPML1 channels to protect against ischemia-reperfusion injury by restoring the inhibited autophagic flux in cardiomyocytes.

Zhongheng Sui, Meng-Meng Wang, Yanhong Xing, Jiansong Qi, Wuyang Wang.

  Accumulating evidence suggests that macroautophagy/autophagy dysfunction plays a critical role in myocardial ischemia-reperfusion (I/R) injury. However, the underlying mechanisms responsible for malfunctional autophagy in cardiomyocytes subjected to I/R are poorly understood. As a result, there are no effective therapeutic options that target autophagy to prevent myocardial I/R injury. We recently revealed that MCOLN1/TRPML1, a lysosomal cationic channel, directly contributes to the inhibition of autophagic flux in cardiomyocytes post I/R. We found that MCOLN1 is activated secondary to reactive oxygen species (ROS) elevation following I/R, which in turn induces the release of lysosomal zinc into the cytosol. This ultimately blocks autophagic flux in cardiomyocytes by disrupting the fusion between autophagosomes containing engulfed mitochondria and lysosomes. Furthermore, we discovered that the MCOLN1-mediated inhibition of autophagy induced by I/R impairs mitochondrial function, which results in further detrimental ROS release that directly contributes to cardiomyocyte death. More importantly, restoration of blocked autophagic flux in cardiomyocytes subjected to I/R achieved by blocking MCOLN1 channels significantly rescues cardiomyocyte death in vitro and greatly improves cardiac function of mice subjected to I/R in vivo. Therefore, targeting MCOLN1 represents a novel therapeutic strategy to protect against myocardial I/R injury.Abbreviations: I/R: ischemia-reperfusion; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MCOLN1/TRPML1: mucolipin TRP cation channel 1; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1.

Keywords:  Autophagy inhibition; MCOLN1; cardiomyocyte death; ischemia-reperfusion injury; mitochondria turnover

DOI:  https://doi.org/10.1080/15548627.2022.2072657
J Cell Biol. 2022 Jul 04. pii: e202202060. [Epub ahead of print]221(7):

STING controls energy stress-induced autophagy and energy metabolism via STX17.

Yueguang Rong, Shen Zhang, Nilay Nandi, Zhe Wu, Linsen Li, Yang Liu, Yuehan Wei, Yuan Zhao, Weigang Yuan, Chuchu Zhou, Guanghua Xiao, Beth Levine, Nan Yan, Shan Mou, Liufu Deng, Zaiming Tang, Xiaoxia Liu, Helmut Kramer, Qing Zhong.

The stimulator of interferon genes (STING) plays a critical role in innate immunity. Emerging evidence suggests that STING is important for DNA or cGAMP-induced non-canonical autophagy, which is independent of a large part of canonical autophagy machineries. Here, we report that, in the absence of STING, energy stress-induced autophagy is upregulated rather than downregulated. Depletion of STING in Drosophila fat cells enhances basal- and starvation-induced autophagic flux. During acute exercise, STING knockout mice show increased autophagy flux, exercise endurance, and altered glucose metabolism. Mechanistically, these observations could be explained by the STING-STX17 interaction. STING physically interacts with STX17, a SNARE that is essential for autophagosome biogenesis and autophagosome-lysosome fusion. Energy crisis and TBK1-mediated phosphorylation both disrupt the STING-STX17 interaction, allow different pools of STX17 to translocate to phagophores and mature autophagosomes, and promote autophagic flux. Taken together, we demonstrate a heretofore unexpected function of STING in energy stress-induced autophagy through spatial regulation of autophagic SNARE STX17.

DOI: https://doi.org/10.1083/jcb.202202060
Nat Commun. 2022 May 06. 13(1): 2500

Distinctive molecular features of regenerative stem cells in the damaged male germline.

Hue M La, Jinyue Liao, Julien M D Legrand, Fernando J Rossello, Ai-Leen Chan, Vijesh Vaghjiani, Jason E Cain, Antonella Papa, Tin Lap Lee, Robin M Hobbs.

Maintenance of male fertility requires spermatogonial stem cells (SSCs) that self-renew and generate differentiating germ cells for production of spermatozoa. Germline cells are sensitive to genotoxic drugs and patients receiving chemotherapy can become infertile. SSCs surviving treatment mediate germline recovery but pathways driving SSC regenerative responses remain poorly understood. Using models of chemotherapy-induced germline damage and recovery, here we identify unique molecular features of regenerative SSCs and characterise changes in composition of the undifferentiated spermatogonial pool during germline recovery by single-cell analysis. Increased mitotic activity of SSCs mediating regeneration is accompanied by alterations in growth factor signalling including PI3K/AKT and mTORC1 pathways. While sustained mTORC1 signalling is detrimental for SSC maintenance, transient mTORC1 activation is critical for the regenerative response. Concerted inhibition of growth factor signalling disrupts core features of the regenerative state and limits germline recovery. We also demonstrate that the FOXM1 transcription factor is a target of growth factor signalling in undifferentiated spermatogonia and provide evidence for a role in regeneration. Our data confirm dynamic changes in SSC functional properties following damage and support an essential role for microenvironmental growth factors in promoting a regenerative state.

DOI: https://doi.org/10.1038/s41467-022-30130-z
FEBS Open Bio. 2022 May 03.

Tollip-deficient zebrafish display no abnormalities in development, organ morphology or gene expression in response to lipopolysaccharide.

Lidia Wolińska-Nizioł, Karolina Romaniuk, Karolina Wojciechowska, Krzysztof Surga, Maciej Kamaszewski, Hubert Szudrowicz, Marta Miączyńska.

  Tollip is a multifunctional adaptor protein implicated in innate immunity, lysosomal trafficking/autophagy of protein aggregates and various signaling processes in mammalian models. To verify evolutionary conservation of these functions, we used CRISPR/Cas9 editing to construct a zebrafish line bearing a stable tollip knockout. In contrast to previously reported tollip morphants, Tollip-deficient fish display normal development until adulthood, are fertile and have no apparent physiological defects. When challenged with lipopolysaccharide (LPS), inflammatory gene expression is unaffected. Moreover, Tollip deficiency does not aggravate swimming deficiency resulting from lysosomal dysfunction and proteotoxicity in a fish model of Gaucher disease. Thus, individual functions of Tollip may be organism-specific or manifest only upon certain conditions/challenges or disease backgrounds.

Keywords:  CRISPR/Cas9; Gaucher disease; LPS; Tollip; innate immunity; zebrafish

DOI:  https://doi.org/10.1002/2211-5463.13423
Exp Anim. 2022 Apr 28.

Suppression of lysosomal-associated protein transmembrane 5 ameliorates cardiac function and inflammatory response by inhibiting the NF-κB pathway after myocardial infarction in mice.

Zhanchun Song, Xiaozeng Wang, Lianqi He, Liang Chen, Zhichao Ren, Siyu Song.

  Myocardial infarction (MI) as the remarkable presentation of coronary artery disease is still a reason for morbidity and mortality in worldwide. Lysosomal-associated protein transmembrane 5 (LAPTM5) is a lysosomal-related protein found in hematopoietic tissues and has been confirmed as a positive regulator of pro-inflammatory pathways in macrophages. However, the role of LAPTM5 in MI remains unknown. In this study, we found that both mRNA and protein expression levels of LAPTM5 were significantly elevated in MI mice. Suppression of LAPTM5 in myocardial tissues decreased cardiac fibrosis and improved cardiac function after MI. At the molecular level, downregulated LAPTM5 dramatically suppressed the macrophage activation and inflammatory response via inhibiting the activation of the NF-κB pathway. Collectively, suppression of LAPTM5 in myocardial tissues inhibits the pro-inflammatory response and the cardiac dysfunction caused by MI. This study indicated that LAPTM5 as a pro-inflammatory factor plays a crucial role in MI disease.

Keywords:  NF-κB pathway; inflammatory response; lysosomal-associated protein transmembrane 5 (LAPTM5); myocardial infarction

DOI:  https://doi.org/10.1538/expanim.22-0008
Adv Exp Med Biol. 2022 ;1372 169-188

Drug Development in the Field of Sphinogolipid Metabolism.

Zhibei Qu, Lu Zhou.

  Sphingolipids are the major lipid components on cellular membranes especially on lipid raft regions, intermediating various important biological functions for eukaryotic cells. Sphingolipid metabolism pathways can utilize sugar, protein, nucleic acid, and other metabolites participating lipid transport in the circulation, play an essential role in maintaining cell homeostasis and are related to a variety of different diseases including lysosomal storage disorders (LSDs), Gaucher disease, etc. The dynamic balance of sphingolipid levels in organisms is regulated by a series of sphingolipid synthases, hydrolases, and metabolic enzymes, such as sphingomyelinase (SMase), sphingomyelin synthase (SMS), serine palmitoyltransferase (SPT), ceramide synthase (CerS), glucosylceramide synthase (GCS), etc. Thus, sphingolipids and its related enzymes are potential targets for drug discoveries and receive great research interests by medicinal chemist. In this chapter, we will discuss the relationship between sphingolipids and the regulating enzymes involved in sphingolipid metabolisms, and systematically summarize the advances in the development of new drugs in the field.

Keywords:  Ceramide synthase; Drug development; Serine palmitoyl transferase; Sphingolipid metabolism; Sphingomyelin synthase; Sphingomyelinase

DOI:  https://doi.org/10.1007/978-981-19-0394-6_12
Biochem Pharmacol. 2022 May 02. pii: S0006-2952(22)00154-X. [Epub ahead of print] 115060

A racemosin B derivative, C25, suppresses breast cancer growth via lysosomal membrane permeabilization and inhibition of autophagic flux.

Xiao Xiao, Philip E D Chung, Mei Xu, Anling Hu, Yangju Ju, Xinmei Yang, Jialei Song, Jinrui Song, Chunlin Wang, Eldad Zacksenhaus, Sheng Liu, Zhixu He, Yaacov Ben-David.

  Breast cancer is the most common malignancy among women worldwide. As conventional therapies are only partially successful in eradicating breast cancer, the development of novel strategies is a top priority. We previously showed that C25, a new racemosin B derivative, exerts its anti-cancer activity through inhibition of autophagy, but the underlying mechanism remained unknown. Here we show that C25 inhibits the growth of diverse breast cancer cell subtypes and effectively suppresses tumor progression in a xenotransplantation model of triple negative breast cancer. C25 acts as a lysosomotropic agent to induce lysosomal membrane permeabilization and inhibit autophagic flux, resulting in cathepsin release and cell death. In accordance, RNA sequencing and gene set enrichment analysis revealed that C25 induces pathways consistent with autophagy inhibition, cell cycle arrest and senescence. Interestingly, knockdown of TFEB or SQSTM1 reduced cell death induced by C25 treatment. Finally, we show that C25 synergizes with the chemo-therapeutics etoposide and paclitaxel to further limit breast cancer cell growth. Thus, C25 alone or in combination with other anti-neoplastic agents offers a novel therapeutic strategy for aggressive forms of breast cancer and possibly other malignancies.

Keywords:  2-a]carbazoles; C25; autophagy; indolo[3; lysosomal membrane; permeabilization, cell cycle, breast cancer, therapy

DOI:  https://doi.org/10.1016/j.bcp.2022.115060
Elife. 2022 May 04. pii: e79782. [Epub ahead of print]11

Correction: mTORC1 in the orbitofrontal cortex promotes habitual alcohol seeking.

Nadege Morisot, Khanhky Phamluong, Yann Ehinger, Anthony L Berger, Jeffrey J Moffat, Dorit Ron.



Keywords:  neuroscience; rat

DOI:  https://doi.org/10.7554/eLife.79782
Trends Cell Biol. 2022 Apr 28. pii: S0962-8924(22)00083-6. [Epub ahead of print]

A novel superfamily of bridge-like lipid transfer proteins.

Sarah D Neuman, Tim P Levine, Arash Bashirullah.

  Lipid transfer proteins mediate nonvesicular transport of lipids at membrane contact sites to regulate the lipid composition of organelle membranes. Recently, a new type of bridge-like lipid transfer protein has emerged; these proteins contain a long hydrophobic groove and can mediate bulk transport of lipids between organelles. Here, we review recent insights into the structure of these proteins and identify a repeating modular unit that we propose to name the repeating β-groove (RBG) domain. This new structural understanding conceptually unifies all the RBG domain-containing lipid transfer proteins as members of an RBG protein superfamily. We also examine the biological functions of these lipid transporters in normal physiology and disease and speculate on the evolutionary origins of RBG proteins in bacteria.

Keywords:  AlphaFold; Vps13; autophagy; lipid transfer proteins; lipids; membrane contact sites

DOI:  https://doi.org/10.1016/j.tcb.2022.03.011
iScience. 2022 May 20. 25(5): 104231

Trans-omics analysis of insulin action reveals a cell growth subnetwork which co-regulates anabolic processes.

Akira Terakawa, Yanhui Hu, Toshiya Kokaji, Katsuyuki Yugi, Keigo Morita, Satoshi Ohno, Yifei Pan, Yunfan Bai, Andrey A Parkhitko, Xiaochun Ni, John M Asara, Martha L Bulyk, Norbert Perrimon, Shinya Kuroda.

  Insulin signaling promotes anabolic metabolism to regulate cell growth through multi-omic interactions. To obtain a comprehensive view of the cellular responses to insulin, we constructed a trans-omic network of insulin action in Drosophila cells that involves the integration of multi-omic data sets. In this network, 14 transcription factors, including Myc, coordinately upregulate the gene expression of anabolic processes such as nucleotide synthesis, transcription, and translation, consistent with decreases in metabolites such as nucleotide triphosphates and proteinogenic amino acids required for transcription and translation. Next, as cell growth is required for cell proliferation and insulin can stimulate proliferation in a context-dependent manner, we integrated the trans-omic network with results from a CRISPR functional screen for cell proliferation. This analysis validates the role of a Myc-mediated subnetwork that coordinates the activation of genes involved in anabolic processes required for cell growth.

Keywords:  In silico biology; Omics; Systems biology

DOI:  https://doi.org/10.1016/j.isci.2022.104231
Cell Rep. 2022 05 03. pii: S2211-1247(22)00540-X. [Epub ahead of print]39(5): 110776

Cholesterol determines the cytosolic entry and seeded aggregation of tau.

Benjamin J Tuck, Lauren V C Miller, Taxiarchis Katsinelos, Annabel E Smith, Emma L Wilson, Sophie Keeling, Shi Cheng, Marina J Vaysburd, Claire Knox, Lucy Tredgett, Emmanouil Metzakopian, Leo C James, William A McEwan.

  Assemblies of tau can transit between neurons, seeding aggregation in a prion-like manner. To accomplish this, tau must cross cell-limiting membranes, a process that is poorly understood. Here, we establish assays for the study of tau entry into the cytosol as a phenomenon distinct from uptake, in real time, and at physiological concentrations. The entry pathway of tau is cell type specific and, in neurons, highly sensitive to cholesterol. Depletion of the cholesterol transporter Niemann-Pick type C1 or extraction of membrane cholesterol renders neurons highly permissive to tau entry and potentiates seeding even at low levels of exogenous tau assemblies. Conversely, cholesterol supplementation reduces entry and almost completely blocks seeded aggregation. Our findings establish entry as a rate-limiting step to seeded aggregation and demonstrate that dysregulated cholesterol, a feature of several neurodegenerative diseases, potentiates tau aggregation by promoting entry of tau assemblies into the cell interior.

Keywords:  Alzheimer’s disease; CP: Metabolism; CP: Neuroscience; Niemann-Pick disease; cholesterol; endocytosis; neurodegeneration; seeded aggregation; tau

DOI:  https://doi.org/10.1016/j.celrep.2022.110776
Transl Psychiatry. 2022 May 04. 12(1): 184

Differential expression of gene co-expression networks related to the mTOR signaling pathway in bipolar disorder.

Sung Woo Park, Mi Kyoung Seo, Maree J Webster, Jung Goo Lee, Sanghyeon Kim.

Bipolar disorder (BPD) is a severe mental illness characterized by episodes of depression and mania. To investigate the molecular mechanisms underlying the pathophysiology of bipolar disorder, we performed transcriptome studies using RNA-seq data from the prefrontal cortex (PFC) of individuals with BPD and matched controls, as well as data from cell culture and animal model studies. We found 879 differentially expressed genes that were also replicated in an independent cohort of post-mortem samples. Genes involving the mechanistic target of rapamycine (mTOR) pathway were down-regulated, while genes interrelated with the mTOR pathway such as Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway were up-regulated. Gene co-expression network analyses identified a module related to the mTOR pathway that was up-regulated in BPD and also enriched for markers of endothelial cells. We also found a down-regulated co-expression module enriched for genes involved in mTOR signalling and in mTOR related pathways and enriched with neuronal markers. The mTOR related modules were also replicated in the independent cohort of samples. To investigate whether the expression of the modules related to mTOR signalling pathway could be differentially regulated in different cell types we performed comparative network analyses in experimental models. We found both up-regulated modules in the PFC significantly overlapped with an up-regulated module in the brain endothelial cells from mice treated with lipopolysaccharides (LPS) and mTOR related pathways such as JAK-STAT, PI3K-Akt and ribosome were enriched in the common genes. In addition, the down-regulated module in the PFC significantly overlapped with a down-regulated module from neurons treated with the mTOR inhibitor, Torin1 and mTOR signalling, autophagy, and synaptic vesicle cycles were significantly enriched in the common genes. These results suggest that co-expression networks related to mTOR signalling pathways may be up- or down-regulated in different cell types in the PFC of BPD. These results provide novel insights into the molecular mechanisms underlying the pathophysiology of BPD.

DOI: https://doi.org/10.1038/s41398-022-01944-8
Proc Natl Acad Sci U S A. 2022 May 10. 119(19): e2119990119

Structural insights into Ras regulation by SIN1.

Yuyuan Zheng, Lei Ding, Xianhui Meng, Meg Potter, Alison L Kearney, Jie Zhang, Jie Sun, David E James, Guang Yang, Chun Zhou.

  SignificanceBoth the mTORC2 and Ras-ERK pathways respond to growth factor stimulation and play critical roles in cell growth and proliferation, disarray of these pathways leads to many diseases, especially cancer. These two signaling pathways crosstalk at many levels; recently it's become clear that the SIN1 component of mTORC2 could interact with Ras family small GTPases, but how these two proteins interact at the molecular level and the functional outcomes of this interaction remain to be addressed. In this work we determined the high-resolution structure of Ras-SIN1 complexes and revealed the detailed interaction mechanism. We also showed that Ras-SIN1 association inhibits insulin-induced ERK activation. Insights from this work could improve our understanding of the disease-causing mechanism of errant mTORC2 or Ras proteins.

Keywords:  PI3K; Ras; SIN1; insulin; mTORC2

DOI:  https://doi.org/10.1073/pnas.2119990119
Sci Rep. 2022 May 03. 12(1): 7161

Remofuscin induces xenobiotic detoxification via a lysosome-to-nucleus signaling pathway to extend the Caenorhabditis elegans lifespan.

Miae Oh, Jiah Yeom, Ulrich Schraermeyer, Sylvie Julien-Schraermeyer, Young-Hee Lim.

Lipofuscin is a representative biomarker of aging that is generated naturally over time. Remofuscin (soraprazan) improves age-related eye diseases by removing lipofuscin from retinal pigment epithelium (RPE) cells. In this study, the effect of remofuscin on longevity in Caenorhabditis elegans and the underlying mechanism were investigated. The results showed that remofuscin significantly (p < 0.05) extended the lifespan of C. elegans (N2) compared with the negative control. Aging biomarkers were improved in remofuscin-treated worms. The expression levels of genes related to lysosomes (lipl-1 and lbp-8), a nuclear hormone receptor (nhr-234), fatty acid beta-oxidation (ech-9), and xenobiotic detoxification (cyp-34A1, cyp-35A1, cyp-35A2, cyp-35A3, cyp-35A4, cyp-35A5, cyp-35C1, gst-28, and gst-5) were increased in remofuscin-treated worms. Moreover, remofuscin failed to extend the lives of C. elegans with loss-of-function mutations (lipl-1, lbp-8, nhr-234, nhr-49, nhr-8, cyp-35A1, cyp-35A2, cyp-35A3, cyp-35A5, and gst-5), suggesting that these genes are associated with lifespan extension in remofuscin-treated C. elegans. In conclusion, remofuscin activates the lysosome-to-nucleus pathway in C. elegans, thereby increasing the expression levels of xenobiotic detoxification genes resulted in extending their lifespan.

DOI: https://doi.org/10.1038/s41598-022-11325-2
PLoS Biol. 2022 May;20(5): e3001621

Correction: Mutations in LRRK2 linked to Parkinson disease sequester Rab8a to damaged lysosomes and regulate transferrin-mediated iron uptake in microglia.

Adamantios Mamais, Jillian H Kluss, Luis Bonet-Ponce, Natalie Landeck, Rebekah G Langston, Nathan Smith, Alexandra Beilina, Alice Kaganovich, Manik C Ghosh, Laura Pellegrini, Ravindran Kumaran, Ioannis Papazoglou, George R Heaton, Kirsten Harvey, Rina Bandopadhyay, Nunziata Maio, Changyoun Kim, Matthew J LaVoie, David C Gershlick, Mark R Cookson.

[This corrects the article DOI: 10.1371/journal.pbio.3001480.].

DOI: https://doi.org/10.1371/journal.pbio.3001621