bims-lysosi 2022-04-17 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2022–04–17
37 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

Inhibition of nonalcoholic fatty liver disease in mice by selective inhibition of mTORC1.
The HOPS tethering complex is required to maintain signaling endosome identity and TORC1 activity.
c-Abl Activation Linked to Autophagy-Lysosomal Dysfunction Contributes to Neurological Impairment in Niemann-Pick Type A Disease.
Glucocerebrosidase Mutations Cause Mitochondrial and Lysosomal Dysfunction in Parkinson's Disease: Pathogenesis and Therapeutic Implications.
Endomembrane damage sensing by V-ATPase recruits ATG16L1 for LC3 lipidation in situ.
Complex regulation of fatty liver disease.
Extralysosomal cathepsin B in central nervous system: Mechanisms and therapeutic implications.
Establishing spatial control over TORC1 signaling.
The Role of mTOR and eIF Signaling in Benign Endometrial Diseases.
Targeting calcium-mediated inter-organellar crosstalk in cardiac diseases.
Comparison between Nanoparticle Encapsulation and Surface Loading for Lysosomal Enzyme Replacement Therapy.
Ursolic Acid Derivative UA232 Promotes Tumor Cell Apoptosis by Inducing Endoplasmic Reticulum Stress and Lysosomal Dysfunction.
Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique.
Repurposing Dipyridamole in Niemann Pick Type C Disease: A Proof of Concept Study.
Yeast cell death pathway requiring AP-3 vesicle trafficking leads to vacuole/lysosome membrane permeabilization.
Dynamics and distribution of endosomes and lysosomes in dendrites.
The GBA1 D409V mutation exacerbates synuclein pathology to differing extents in two alpha-synuclein models.
Lysosomes in Stem Cell Quiescence: A Potential Therapeutic Target in Acute Myeloid Leukemia.
What's new and what's next for gene therapy in Pompe disease?
Multifaceted targeted protein degradation systems for different cellular compartments.
Crosstalk between dihydroceramides produced by Porphyromonas gingivalis and host lysosomal cathepsin B in the promotion of osteoclastogenesis.
Author Correction: The AUTOTAC chemical biology platform for targeted protein degradation via the autophagy-lysosome system.
Defects of Nutrient Signaling and Autophagy in Neurodegeneration.
Aggregation chimeras provide evidence of in vivo intercellular correction in ovine CLN6 neuronal ceroid lipofuscinosis (Batten disease).
Urine-Derived Kidney Progenitor Cells in Cystinosis.
Regulation of Aging and Longevity by Ion Channels and Transporters.
Differential Oxygen Exposure Modulates Mesenchymal Stem Cell Metabolism and Proliferation through mTOR Signaling.
Entamoeba histolytica EHD1 Is Involved in Mitosome-Endosome Contact.
mTOR Signaling Pathway Regulates the Release of Proinflammatory Molecule CCL5 Implicated in the Pathogenesis of Autism Spectrum Disorder.
PEN2: Metformin's new partner at lysosome.
Condensation of Ede1 promotes the initiation of endocytosis.
Arabidopsis HOPS subunit VPS41 carries out plant-specific roles in vacuolar transport and vegetative growth.
VCP suppresses proteopathic seeding in neurons.
SNX27-Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling.
Characterization of neurological disease progression in a canine model of CLN5 neuronal ceroid lipofuscinosis.
Sphingolipids control dermal fibroblast heterogeneity.
On the Connections between TRPM Channels and SOCE.

Science. 2022 Apr 15. 376(6590): eabf8271

Inhibition of nonalcoholic fatty liver disease in mice by selective inhibition of mTORC1.

Bridget S Gosis, Shogo Wada, Chelsea Thorsheim, Kristina Li, Sunhee Jung, Joshua H Rhoades, Yifan Yang, Jeffrey Brandimarto, Li Li, Kahealani Uehara, Cholsoon Jang, Matthew Lanza, Nathan B Sanford, Marc R Bornstein, Sunhye Jeong, Paul M Titchenell, Sudha B Biddinger, Zoltan Arany.

Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) remain without effective therapies. The mechanistic target of rapamycin complex 1 (mTORC1) pathway is a potential therapeutic target, but conflicting interpretations have been proposed for how mTORC1 controls lipid homeostasis. We show that selective inhibition of mTORC1 signaling in mice, through deletion of the RagC/D guanosine triphosphatase-activating protein folliculin (FLCN), promotes activation of transcription factor E3 (TFE3) in the liver without affecting other mTORC1 targets and protects against NAFLD and NASH. Disease protection is mediated by TFE3, which both induces lipid consumption and suppresses anabolic lipogenesis. TFE3 inhibits lipogenesis by suppressing proteolytic processing and activation of sterol regulatory element-binding protein-1c (SREBP-1c) and by interacting with SREBP-1c on chromatin. Our data reconcile previously conflicting studies and identify selective inhibition of mTORC1 as a potential approach to treat NASH and NAFLD.

DOI: https://doi.org/10.1126/science.abf8271
J Cell Biol. 2022 May 02. pii: e202109084. [Epub ahead of print]221(5):

The HOPS tethering complex is required to maintain signaling endosome identity and TORC1 activity.

Jieqiong Gao, Raffaele Nicastro, Marie-Pierre Péli-Gulli, Sophie Grziwa, Zilei Chen, Rainer Kurre, Jacob Piehler, Claudio De Virgilio, Florian Fröhlich, Christian Ungermann.

The endomembrane system of eukaryotic cells is essential for cellular homeostasis during growth and proliferation. Previous work showed that a central regulator of growth, namely the target of rapamycin complex 1 (TORC1), binds both membranes of vacuoles and signaling endosomes (SEs) that are distinct from multivesicular bodies (MVBs). Interestingly, the endosomal TORC1, which binds membranes in part via the EGO complex, critically defines vacuole integrity. Here, we demonstrate that SEs form at a branch point of the biosynthetic and endocytic pathways toward the vacuole and depend on MVB biogenesis. Importantly, function of the HOPS tethering complex is essential to maintain the identity of SEs and proper endosomal and vacuolar TORC1 activities. In HOPS mutants, the EGO complex redistributed to the Golgi, which resulted in a partial mislocalization of TORC1. Our study uncovers that SE function requires a functional HOPS complex and MVBs, suggesting a tight link between trafficking and signaling along the endolysosomal pathway.

DOI: https://doi.org/10.1083/jcb.202109084
Front Cell Dev Biol. 2022 ;10 844297

c-Abl Activation Linked to Autophagy-Lysosomal Dysfunction Contributes to Neurological Impairment in Niemann-Pick Type A Disease.

Tamara Marín, Andrés E Dulcey, Fabián Campos, Catalina de la Fuente, Mariana Acuña, Juan Castro, Claudio Pinto, María José Yañez, Cristian Cortez, David W McGrath, Pablo J Sáez, Kirill Gorshkov, Wei Zheng, Noel Southall, Maria Carmo-Fonseca, Juan Marugán, Alejandra R Alvarez, Silvana Zanlungo.

  Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the deficiency in acid sphingomyelinase (ASM) activity. NPA patients present severe and progressive neurodegeneration starting at an early age. Currently, there is no effective treatment for this disease and NPA patients die between 2 and 3 years of age. NPA is characterized by an accumulation of sphingomyelin in lysosomes and dysfunction in the autophagy-lysosomal pathway. Recent studies show that c-Abl tyrosine kinase activity downregulates autophagy and the lysosomal pathway. Interestingly, this kinase is also activated in other lysosomal neurodegenerative disorders. Here, we describe that c-Abl activation contributes to the mechanisms of neuronal damage and death in NPA disease. Our data demonstrate that: 1) c-Abl is activated in-vitro as well as in-vivo NPA models; 2) imatinib, a clinical c-Abl inhibitor, reduces autophagy-lysosomal pathway alterations, restores autophagy flux, and lowers sphingomyelin accumulation in NPA patient fibroblasts and NPA neuronal models and 3) chronic treatment with nilotinib and neurotinib, two c-Abl inhibitors with differences in blood-brain barrier penetrance and target binding mode, show further benefits. While nilotinib treatment reduces neuronal death in the cerebellum and improves locomotor functions, neurotinib decreases glial activation, neuronal disorganization, and loss in hippocampus and cortex, as well as the cognitive decline of NPA mice. Our results support the participation of c-Abl signaling in NPA neurodegeneration and autophagy-lysosomal alterations, supporting the potential use of c-Abl inhibitors for the clinical treatment of NPA patients.

Keywords:  Niemann-Pick disease; autophay-lysosomal pathway; c-Abl kinase; lysosomal storage disorder (LSD); neurodegeneration

DOI:  https://doi.org/10.3389/fcell.2022.844297
Front Aging Neurosci. 2022 ;14 851135

Glucocerebrosidase Mutations Cause Mitochondrial and Lysosomal Dysfunction in Parkinson's Disease: Pathogenesis and Therapeutic Implications.

Wei Zheng, Dongsheng Fan.

  Parkinson's disease (PD) is the second most common neurodegenerative disease and is characterized by multiple motor and non-motor symptoms. Mutations in the glucocerebrosidase (GBA) gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), which hydrolyzes glucosylceramide (GlcCer) to glucose and ceramide, are the most important and common genetic PD risk factors discovered to date. Homozygous GBA mutations result in the most common lysosomal storage disorder, Gaucher's disease (GD), which is classified according to the presence (neuronopathic types, type 2 and 3 GD) or absence (non-neuronopathic type, type 1 GD) of neurological symptoms. The clinical manifestations of PD in patients with GBA mutations are indistinguishable from those of sporadic PD at the individual level. However, accumulating data have indicated that GBA-associated PD patients exhibit a younger age of onset and a greater risk for cognitive impairment and psychiatric symptoms. The mechanisms underlying the increased risk of developing PD in GBA mutant carriers are currently unclear. Contributors to GBA-PD pathogenesis may include mitochondrial dysfunction, autophagy-lysosomal dysfunction, altered lipid homeostasis and enhanced α-synuclein aggregation. Therapeutic strategies for PD and GD targeting mutant GCase mainly include enzyme replacement, substrate reduction, gene and pharmacological small-molecule chaperones. Emerging clinical, genetic and pathogenic studies on GBA mutations and PD are making significant contributions to our understanding of PD-associated pathogenetic pathways, and further elucidating the interactions between GCase activity and neurodegeneration may improve therapeutic approaches for slowing PD progression.

Keywords:  Gaucher’s disease (GD); Parkinson’s disease (PD); glucocerebrosidase (GBA); lysosomal; mitochondrial

DOI:  https://doi.org/10.3389/fnagi.2022.851135
Autophagy. 2022 Apr 12. 1-3

Endomembrane damage sensing by V-ATPase recruits ATG16L1 for LC3 lipidation in situ.

Yue Xu, Jingjin Ding.

  LC3 lipidation-mediated selective macroautophagy/autophagy helps eukaryotes to defend against endogenous dangers and foreign invaders. However, LC3 activation mechanisms of selective autophagy are still elusive. We previously determined that the V-ATPase-ATG16L1 axis is critical for LC3 recruitment to bacteria-residing vacuoles, whereas the Salmonella effector SopF directly targets V-ATPase to disrupt ATG16L1 interaction. Here we show that host ARF GTPase binding causes SopF-dependent ADP-ribosylation of the Gln124 site of the ATP6V0C/V0C subunit of V-ATPase. Furthermore, LC3 activation by pH perturbation of endolysosomes and the Golgi apparatus is also abolished by SopF or a ATP6V0CQ124A mutation, illustrating that disruption of the proton gradient in acidic compartments is a universal signal that triggers V-ATPase-ATG16L1-induced LC3 lipidation.

Keywords:  ARF; ATG16L1; SopF; V-ATPase; VAIL; endomembrane damage; pH disturbance; selective autophagy

DOI:  https://doi.org/10.1080/15548627.2022.2062889
Science. 2022 Apr 15. 376(6590): 247-248

Complex regulation of fatty liver disease.

Henry N Ginsberg, Arya Mani.

Hepatic lipogenesis is fine-tuned by mechanistic target of rapamycin (mTOR) signaling.

DOI: https://doi.org/10.1126/science.abp8276
Brain Pathol. 2022 Apr 12. e13071

Extralysosomal cathepsin B in central nervous system: Mechanisms and therapeutic implications.

Junjun Ni, Fei Lan, Yan Xu, Hiroshi Nakanishi, Xue Li.

  Cathepsin B (CatB) is a typical cysteine lysosomal protease involved in a variety of physiologic and pathological processes. It is expressed in most cell types and is primarily localized within subcellular endosomal and lysosomal compartments. Emerging scientific evidence indicates that lysosomal leaked CatB is involved in mitochondrial stress, inflammasome activation, and nuclear senescence, but without the acidic environment. CatB is also secreted as a myokine, which is involved in muscle-brain cross talk and neuronal dendritic remodeling. Lysosomal-leaked and cellular-secreted CatB functions are dependent on its enzymatic activity at a neutral pH. In the present review, we summarize the available experimental evidence that mechanistically links extralysosomal CatB to physiological and pathological functions in central nervous system, and their potential for use in therapeutic approaches.

Keywords:  cathepsin B; central nervous system; leakage; lysosome; neurodegeneration; neuron development

DOI:  https://doi.org/10.1111/bpa.13071
J Cell Biol. 2022 May 02. pii: e202203136. [Epub ahead of print]221(5):

Establishing spatial control over TORC1 signaling.

Oliver Schmidt, Mariana E G de Araujo.

Target-of-rapamycin complex 1 resides on lysosomes/vacuoles and additionally on signaling endosomes. Gao et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202109084) set out to define the molecular identity of signaling endosomes, along with players required for the formation and maintenance of this endosomal subpopulation.

DOI: https://doi.org/10.1083/jcb.202203136
Int J Mol Sci. 2022 Mar 22. pii: 3416. [Epub ahead of print]23(7):

The Role of mTOR and eIF Signaling in Benign Endometrial Diseases.

Tatiana S Driva, Christoph Schatz, Monika Sobočan, Johannes Haybaeck.

  Adenomyosis, endometriosis, endometritis, and typical endometrial hyperplasia are common non-cancerous diseases of the endometrium that afflict many women with life-impacting consequences. The mammalian target of the rapamycin (mTOR) pathway interacts with estrogen signaling and is known to be dysregulated in endometrial cancer. Based on this knowledge, we attempt to investigate the role of mTOR signaling in benign endometrial diseases while focusing on how the interplay between mTOR and eukaryotic translation initiation factors (eIFs) affects their development. In fact, mTOR overactivity is apparent in adenomyosis, endometriosis, and typical endometrial hyperplasia, where it promotes endometrial cell proliferation and invasiveness. Recent data show aberrant expression of various components of the mTOR pathway in both eutopic and ectopic endometrium of patients with adenomyosis or endometriosis and in hyperplastic endometrium as well. Moreover, studies on endometritis show that derangement of mTOR signaling is linked to the establishment of endometrial dysfunction caused by chronic inflammation. This review shows that inhibition of the mTOR pathway has a promising therapeutic effect in benign endometrial conditions, concluding that mTOR signaling dysregulation plays a critical part in their pathogenesis.

Keywords:  adenomyosis; eIFs; endometriosis; endometritis; mTOR signaling; typical endometrial hyperplasia

DOI:  https://doi.org/10.3390/ijms23073416
Expert Opin Ther Targets. 2022 Apr 15.

Targeting calcium-mediated inter-organellar crosstalk in cardiac diseases.

Mohit M Hulsurkar, Satadru K Lahiri, Jason Karch, Meng C Wang, Xander H T Wehrens.

IntroductionAbnormal calcium signaling between organelles such as the sarcoplasmic reticulum (SR), mitochondria and lysosomes is a key feature of various heart diseases. Calcium serves as a secondary messenger mediating inter-organellar crosstalk, which is essential for maintaining the overall cardiomyocyte function.Areas coveredThis article examines the available literature related to various calcium channels and transporters involved in inter-organellar calcium signaling. The SR calcium-release channels known as ryanodine receptor type-2 (RyR2) and inositol 1,4,5-trisphosphate receptor (IP3R), as well as calcium-transporter SR/ER-ATPase 2a (SERCA2a) are illuminated. The roles of mitochondrial voltage-dependent anion channels (VDAC), the mitochondria Ca2+ uniporter complex (MCUC), and the lysosomal H+/Ca2+ exchanger, two pore channels (TPC), and transient receptor potential mucolipin (TRPML) are discussed. Furthermore, recent studies showing calcium-mediated crosstalk between the SR, mitochondria, and lysosomes as well as how this crosstalk is dysregulated in cardiac diseases are placed under the spotlight.Expert opinionEnhanced SR calcium release via RyR2 and reduced SR reuptake via SERCA2a, increased VDAC and MCUC-mediated calcium-uptake into mitochondria, enhanced MPTP opening, and enhanced lysosomal calcium-release via lysosomal TPC and TRPML may all contribute to aberrant calcium homeostasis causing heart disease. While mechanisms of this crosstalk need to be studied further, interventions targeting these calcium channels or combinations thereof might represent a promising therapeutic strategy.

DOI: https://doi.org/10.1080/14728222.2022.2067479
Int J Mol Sci. 2022 Apr 06. pii: 4034. [Epub ahead of print]23(7):

Comparison between Nanoparticle Encapsulation and Surface Loading for Lysosomal Enzyme Replacement Therapy.

Eameema Muntimadugu, Marcelle Silva-Abreu, Guillem Vives, Maximilian Loeck, Vy Pham, Maria Del Moral, Melani Solomon, Silvia Muro.

  Poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) enhance the delivery of therapeutic enzymes for replacement therapy of lysosomal storage disorders. Previous studies examined NPs encapsulating or coated with enzymes, but these formulations have never been compared. We examined this using hyaluronidase (HAse), deficient in mucopolysaccharidosis IX, and acid sphingomyelinase (ASM), deficient in types A-B Niemann-Pick disease. Initial screening of size, PDI, ζ potential, and loading resulted in the selection of the Lactel II co-polymer vs. Lactel I or Resomer, and Pluronic F68 surfactant vs. PVA or DMAB. Enzyme input and addition of carrier protein were evaluated, rendering NPs having, e.g., 181 nm diameter, 0.15 PDI, -36 mV ζ potential, and 538 HAse molecules encapsulated per NP. Similar NPs were coated with enzyme, which reduced loading (e.g., 292 HAse molecules/NP). NPs were coated with targeting antibodies (> 122 molecules/NP), lyophilized for storage without alterations, and acceptably stable at physiological conditions. NPs were internalized, trafficked to lysosomes, released active enzyme at lysosomal conditions, and targeted both peripheral organs and the brain after i.v. administration in mice. While both formulations enhanced enzyme delivery compared to free enzyme, encapsulating NPs surpassed coated counterparts (18.4- vs. 4.3-fold enhancement in cells and 6.2- vs. 3-fold enhancement in brains), providing guidance for future applications.

Keywords:  ICAM-1 targeting; encapsulation; enzyme therapeutics; in vivo biodistribution; lysosomal delivery; poly(lactic-co-glycolic acid) nanoparticles; surface loading

DOI:  https://doi.org/10.3390/ijms23074034
Int J Biol Sci. 2022 ;18(6): 2639-2651

Ursolic Acid Derivative UA232 Promotes Tumor Cell Apoptosis by Inducing Endoplasmic Reticulum Stress and Lysosomal Dysfunction.

Wenfeng Gou, Na Luo, Bing Yu, Hongying Wu, Shaohua Wu, Chen Tian, Jianghong Guo, Hongxin Ning, Changfen Bi, Huiqiang Wei, Wenbin Hou, Yiliang Li.

  Due to increased drug and radiation tolerance, there is an urgent need to develop novel anticancer agents. In our previous study, we performed a series of structural modifications of ursolic acid (UA), a natural product of pentacyclic triterpenes, and found UA232, a derivative with stronger anti-tumor activity. In vitro experiments showed that UA232 inhibited proliferation, induced G0/G1 arrest, and promoted apoptosis in human breast cancer and cervical cancer cells. Mechanistic studies revealed that UA232 promoted apoptosis and induced protective autophagy via the protein kinase R-like endoplasmic reticulum kinase/activating transcription factor 4/C/EBP homologous protein-mediated endoplasmic reticulum stress. In addition, we also found that UA232 induced lysosomal biogenesis, increased lysosomal membrane permeability, promoted lysosomal protease release, and led to lysosome-dependent cell death. Furthermore, UA232 suppressed tumor growth in a mouse xenograft model. In conclusion, our study revealed that UA232 exerts multiple pharmacological effects against breast and cervical cancers by simultaneously triggering endoplasmic reticulum stress and lysosomal dysfunction. Thus, UA232 may be a promising drug candidate for cancer treatment.

Keywords:  ER stress; apoptosis; autophagy; lysosomal membrane permeability

DOI:  https://doi.org/10.7150/ijbs.67166
STAR Protoc. 2022 Jun 17. 3(2): 101263

Identification of the interacting partners of a lysosomal membrane protein in living cells by BioID technique.

Dat Nguyen-Tien, Takehiro Suzuki, Toshihiko Kobayashi, Noriko Toyama-Sorimachi, Naoshi Dohmae.

  The purpose of this protocol is to screen and identify the physiologically relevant interactors of a lysosomal protein in living cells. Here, we describe how to identify solute carrier family 15 member 4 (SLC15A4)-interacting proteins by BioID and mass spectrometry analysis. This protocol utilizes fusion of SLC15A4 with a mutant form of biotin ligase, BirA. The fusion protein can promiscuously biotinylate the proteins proximal to SLC15A4. The biotinylated endogenous proteins are pulled down by magnetic streptavidin beads and detected by mass spectrometry analysis. For complete details on the use and execution of this protocol, please refer to Kobayashi et al. (2021).

Keywords:  Cell-based Assays; Mass Spectrometry; Molecular Biology; Protein Biochemistry; Proteomics

DOI:  https://doi.org/10.1016/j.xpro.2022.101263
Int J Mol Sci. 2022 Mar 22. pii: 3456. [Epub ahead of print]23(7):

Repurposing Dipyridamole in Niemann Pick Type C Disease: A Proof of Concept Study.

Rita Pepponi, Roberta De Simone, Chiara De Nuccio, Sergio Visentin, Andrea Matteucci, Antonietta Bernardo, Patrizia Popoli, Antonella Ferrante.

  Niemann Pick type C disease (NPC) is a rare disorder characterized by lysosomal lipid accumulation that damages peripheral organs and the central nervous system. Currently, only miglustat is authorized for NPC treatment in Europe, and thus the identification of new therapies is necessary. The hypothesis addressed in this study is that increasing adenosine levels may represent a new therapeutic approach for NPC. In fact, a reduced level of adenosine has been shown in the brain of animal models of NPC; moreover, the compound T1-11, which is able to weakly stimulate A2A receptor and to increase adenosine levels by blocking the equilibrative nucleoside transporter ENT1, significantly ameliorated the pathological phenotype and extended the survival in a mouse model of the disease. To test our hypothesis, fibroblasts from NPC1 patients were treated with dipyridamole, a clinically-approved drug with inhibitory activity towards ENT1. Dipyridamole significantly reduced cholesterol accumulation in fibroblasts and rescued mitochondrial deficits; the mechanism elicited by dipyridamole relies on activation of the adenosine A2AR subtype subsequent to the increased levels of extracellular adenosine due to the inhibition of ENT1. In conclusion, our results provide the proof of concept that targeting adenosine tone could be beneficial in NPC.

Keywords:  A2A receptor; Niemann Pick type C disease; adenosine; dipyridamole

DOI:  https://doi.org/10.3390/ijms23073456
Cell Rep. 2022 Apr 12. pii: S2211-1247(22)00399-0. [Epub ahead of print]39(2): 110647

Yeast cell death pathway requiring AP-3 vesicle trafficking leads to vacuole/lysosome membrane permeabilization.

Zachary D Stolp, Madhura Kulkarni, Yining Liu, Chengzhang Zhu, Alizay Jalisi, Si Lin, Arturo Casadevall, Kyle W Cunningham, Fernando J Pineda, Xinchen Teng, J Marie Hardwick.

  Unicellular eukaryotes have been suggested as undergoing self-inflicted destruction. However, molecular details are sparse compared with the mechanisms of programmed/regulated cell death known for human cells and animal models. Here, we report a molecular cell death pathway in Saccharomyces cerevisiae leading to vacuole/lysosome membrane permeabilization. Following a transient cell death stimulus, yeast cells die slowly over several hours, consistent with an ongoing molecular dying process. A genome-wide screen for death-promoting factors identified all subunits of the AP-3 complex, a vesicle trafficking adapter known to transport and install newly synthesized proteins on the vacuole/lysosome membrane. To promote cell death, AP-3 requires its Arf1-GTPase-dependent vesicle trafficking function and the kinase Yck3, which is selectively transported to the vacuole membrane by AP-3. Video microscopy revealed a sequence of events where vacuole permeability precedes the loss of plasma membrane integrity. AP-3-dependent death appears to be conserved in the human pathogenic yeast Cryptococcus neoformans.

Keywords:  AP-3; CP: Cell biology; Yck3; cryptococcus; lysosome; programmed cell death; regulated cell death; vacuolar membrane permeabilization; vacuole; vesicle trafficking; yeast

DOI:  https://doi.org/10.1016/j.celrep.2022.110647
Curr Opin Neurobiol. 2022 Apr 07. pii: S0959-4388(22)00031-9. [Epub ahead of print]74 102537

Dynamics and distribution of endosomes and lysosomes in dendrites.

Chan Choo Yap, Ashley J Mason, Bettina Winckler.

All cells are filled with membrane-bound organelles which are responsible for the synthesis and transport as well as degradation of membrane proteins. The localization of these organelles inside cells is highly regulated. The regulation of organelle positioning has been widely studied in many cell types. In neurons, organelle positioning and its regulation is of particular interest because of the enormous size of neurons and the high spatial heterogeneity of different functional domains, such as axons, proximal and distal portions of dendrites, and synapses. We will discuss new discoveries with regard to the dynamic positioning of endosomes and lysosomes between soma and along dendrites. Just as the "how" of dynamic endosome/lysosome positioning is still being investigated, the "why" is also being explored. An exciting possibility is that synaptic activity influences organelle behaviors. We will discuss what is currently known about the how and the why of endosome/lysosome dynamics in dendrites.

DOI: https://doi.org/10.1016/j.conb.2022.102537
Dis Model Mech. 2022 Apr 14. pii: dmm.049192. [Epub ahead of print]

The GBA1 D409V mutation exacerbates synuclein pathology to differing extents in two alpha-synuclein models.

Nicole K Polinski, Terina N Martinez, Sylvie Ramboz, Michael Sasner, Mark Herberth, Robert Switzer, Syed O Ahmad, Lee J Pelligrino, Sean W Clark, Jacob N Marcus, Sean M Smith, Kuldip D Dave, Mark A Frasier.

  Heterozygous mutations in the GBA1 gene-encoding lysosomal glucocerebrosidase (GCase)--are the most common genetic risk factor for Parkinson's disease (PD). Experimental evidence suggests a correlation between decreased GCase activity and accumulation of alpha-synuclein (aSyn). To enable a better understanding of the relationship between aSyn and GCase activity, we developed and characterized two mouse models that investigate aSyn pathology in the context of reduced GCase activity. The first model uses constitutive overexpression of wild type human aSyn in the context of the homozygous GCase activity-reducing D409V mutant form of GBA1. While increased aSyn pathology and grip strength reductions are observed in this model, the nigrostriatal system remains largely intact. The second model involves injection of aSyn preformed fibrils (PFFs) into the striatum of the homozygous GBA1 D409V KI model. The GBA1 D409V mutation did not exacerbate pathology induced by aSyn PFF injection. This study sheds light on the relationship between aSyn and GCase in mouse models, highlighting the impact of model design on the ability to model a relationship between these proteins in PD-related pathology.

Keywords:  Alpha-Synuclein; GCase; Parkinson's disease

DOI:  https://doi.org/10.1242/dmm.049192
Cancers (Basel). 2022 Mar 23. pii: 1618. [Epub ahead of print]14(7):

Lysosomes in Stem Cell Quiescence: A Potential Therapeutic Target in Acute Myeloid Leukemia.

Vaibhav Jain, Swaroop Bose, Awadhesh K Arya, Tasleem Arif.

  Lysosomes are cellular organelles that regulate essential biological processes such as cellular homeostasis, development, and aging. They are primarily connected to the degradation/recycling of cellular macromolecules and participate in cellular trafficking, nutritional signaling, energy metabolism, and immune regulation. Therefore, lysosomes connect cellular metabolism and signaling pathways. Lysosome's involvement in the critical biological processes has rekindled clinical interest towards this organelle for treating various diseases, including cancer. Recent research advancements have demonstrated that lysosomes also regulate the maintenance and hemostasis of hematopoietic stem cells (HSCs), which play a critical role in the progression of acute myeloid leukemia (AML) and other types of cancer. Lysosomes regulate both HSCs' metabolic networks and identity transition. AML is a lethal type of blood cancer with a poor prognosis that is particularly associated with aging. Although the genetic landscape of AML has been extensively described, only a few targeted therapies have been produced, warranting the need for further research. This review summarizes the functions and importance of targeting lysosomes in AML, while highlighting the significance of lysosomes in HSCs maintenance.

Keywords:  AML; HSCs; NSCs; apoptosis; lysosomes; mitochondria

DOI:  https://doi.org/10.3390/cancers14071618
Expert Opin Biol Ther. 2022 Apr 15.

What's new and what's next for gene therapy in Pompe disease?

Angela L Roger, Ronit Sethi, Meredith L Huston, Evelyn Scarrow, Joy Bao-Dai, Elias Lai, Debolina D Biswas, Léa El Haddad, Laura M Strickland, Priya S Kishnani, Mai K ElMallah.

   INTRODUCTION: Pompe disease is an autosomal recessive disorder caused by a deficiency of acid-⍺-glucosidase (GAA), an enzyme responsible for hydrolyzing lysosomal glycogen. A lack of GAA leads to accumulation of glycogen in the lysosomes of cardiac, skeletal, and smooth muscle cells, as well as in the central and peripheral nervous system. Enzyme replacement therapy has been the standard of care for 15 years and slows disease progression, particularly in the heart, and improves survival. However, there are limitations of ERT success, which gene therapy can overcome.
AREAS COVERED: Gene therapy offers several advantages including prolonged and consistent GAA expression and correction of skeletal muscle as well as the critical CNS pathology. We provide a systematic review of the preclinical and clinical outcomes of adeno-associated viral mediated gene therapy and alternative gene therapy strategies, highlighting what has been successful.
EXPERT OPINION: Although the preclinical and clinical studies so far have been promising, barriers exist that need to be addressed in gene therapy for Pompe disease. New strategies including novel capsids for better targeting, optimized DNA vectors, and adjuctive therapies will allow for a lower dose, and ameliorate the immune response.

Keywords:  AAV; GAA; Pompe disease; acid alpha-glucosidase; gene therapy; neuromuscular disorder

DOI:  https://doi.org/10.1080/14712598.2022.2067476
Bioessays. 2022 Apr 13. e2200008

Multifaceted targeted protein degradation systems for different cellular compartments.

Cornelia E Zorca, Armaan Fallahi, Sophie Luo, Mohamed A Eldeeb.

  Selective protein degradation maintains cellular homeostasis, but this process is disrupted in many diseases. Targeted protein degradation (TPD) approaches, built upon existing cellular mechanisms, are promising methods for therapeutically regulating protein levels. Here, we review the diverse palette of tools that are now available for doing so throughout the gene expression pathway and in specific cellular compartments. These include methods for directly removing targeted proteins via the ubiquitin proteasome system with proteolysis targeting chimeras (PROTACs) or dephosphorylation targeting chimeras (DEPTACs). Similar effects can also be achieved through the lysosomal system with autophagy-targeting chimeras (AUTACs), autophagosome tethering compounds (ATTECs), and lysosome targeting chimeras (LYTACs). Other methods act upstream to degrade RNAs (ribonuclease targeting chimeras; RIBOTACs) or transcription factors (transcription factor targeting chimeras; TRAFTACs), offering control throughout the gene expression process. We highlight the evolution and function of these methods and discuss their clinical implications in diverse disease contexts.

Keywords:  lysosomal degradation; proteasome; targeted protein degradation; ubiquitin

DOI:  https://doi.org/10.1002/bies.202200008
J Cell Mol Med. 2022 Apr 16.

Crosstalk between dihydroceramides produced by Porphyromonas gingivalis and host lysosomal cathepsin B in the promotion of osteoclastogenesis.

Carolina Duarte, Chiaki Yamada, Christopher Garcia, Juliet Akkaoui, Anny Ho, Frank Nichols, Alexandru Movila.

  Emerging studies indicate that intracellular eukaryotic ceramide species directly activate cathepsin B (CatB), a lysosomal-cysteine-protease, in the cytoplasm of osteoclast precursors (OCPs) leading to elevated RANKL-mediated osteoclastogenesis and inflammatory osteolysis. However, the possible impact of CatB on osteoclastogenesis elevated by non-eukaryotic ceramides is largely unknown. It was reported that a novel class of phosphoglycerol dihydroceramide (PGDHC), produced by the key periodontal pathogen Porphyromonas gingivalis upregulated RANKL-mediated osteoclastogenesis in vitro and in vivo. Therefore, the aim of this study was to evaluate a crosstalk between host CatB and non-eukaryotic PGDHC on the promotion of osteoclastogenesis. According to a pulldown assay, high affinity between PGDHC and CatB was observed in RANKL-stimulated RAW264.7 cells in vitro. It was also demonstrated that PGDHC promotes enzymatic activity of recombinant CatB protein ex vivo and in RANKL-stimulated osteoclast precursors in vitro. Furthermore, no or little effect of PGDHC on the RANKL-primed osteoclastogenesis was observed in male and female CatB-knock out mice compared with their wild type counterparts. Altogether, these findings demonstrate that bacterial dihydroceramides produced by P. gingivalis elevate RANKL-primed osteoclastogenesis via direct activation of intracellular CatB in OCPs.

Keywords:   Porphyromonas gingivalis ; cathepsin B; lysosomes; osteoclast; phosphoglycerol dihydroceramide

DOI:  https://doi.org/10.1111/jcmm.17299
Nat Commun. 2022 Apr 12. 13(1): 2108

Author Correction: The AUTOTAC chemical biology platform for targeted protein degradation via the autophagy-lysosome system.

Chang Hoon Ji, Hee Yeon Kim, Min Ju Lee, Ah Jung Heo, Daniel Youngjae Park, Sungsu Lim, Seulgi Shin, Srinivasrao Ganipisetti, Woo Seung Yang, Chang An Jung, Kun Young Kim, Eun Hye Jeong, Sun Ho Park, Su Bin Kim, Su Jin Lee, Jeong Eun Na, Ji In Kang, Hyung Min Chi, Hyun Tae Kim, Yun Kyung Kim, Bo Yeon Kim, Yong Tae Kwon.

DOI: https://doi.org/10.1038/s41467-022-29845-w
Front Cell Dev Biol. 2022 ;10 836196

Defects of Nutrient Signaling and Autophagy in Neurodegeneration.

Jon Ondaro, Haizea Hernandez-Eguiazu, Maddi Garciandia-Arcelus, Raúl Loera-Valencia, Laura Rodriguez-Gómez, Andrés Jiménez-Zúñiga, Julen Goikolea, Patricia Rodriguez-Rodriguez, Javier Ruiz-Martinez, Fermín Moreno, Adolfo Lopez de Munain, Ian James Holt, Francisco Javier Gil-Bea, Gorka Gereñu.

  Neurons are post-mitotic cells that allocate huge amounts of energy to the synthesis of new organelles and molecules, neurotransmission and to the maintenance of redox homeostasis. In neurons, autophagy is not only crucial to ensure organelle renewal but it is also essential to balance nutritional needs through the mobilization of internal energy stores. A delicate crosstalk between the pathways that sense nutritional status of the cell and the autophagic processes to recycle organelles and macronutrients is fundamental to guarantee the proper functioning of the neuron in times of energy scarcity. This review provides a detailed overview of the pathways and processes involved in the balance of cellular energy mediated by autophagy, which when defective, precipitate the neurodegenerative cascade of Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis or Alzheimer's disease.

Keywords:  AMPK; autophagy; glucose metabolism; lipid metabolism; mTORC1 (mechanistic target of rapamycin complex 1); mitochondrial metabolism; neurodegenerative diseases; nutrient sensing pathways

DOI:  https://doi.org/10.3389/fcell.2022.836196
PLoS One. 2022 ;17(4): e0261544

Aggregation chimeras provide evidence of in vivo intercellular correction in ovine CLN6 neuronal ceroid lipofuscinosis (Batten disease).

Lucy Anne Barry, Graham William Kay, Nadia Lesley Mitchell, Samantha Jane Murray, Nigel P Jay, David Norris Palmer.

The neuronal ceroid lipofuscinoses (NCLs; Batten disease) are fatal, mainly childhood, inherited neurodegenerative lysosomal storage diseases. Sheep affected with a CLN6 form display progressive regionally defined glial activation and subsequent neurodegeneration, indicating that neuroinflammation may be causative of pathogenesis. In this study, aggregation chimeras were generated from homozygous unaffected normal and CLN6 affected sheep embryos, resulting in seven chimeric animals with varied proportions of normal to affected cells. These sheep were classified as affected-like, recovering-like or normal-like, based on their cell-genotype ratios and their clinical and neuropathological profiles. Neuropathological examination of the affected-like animals revealed intense glial activation, prominent storage body accumulation and severe neurodegeneration within all cortical brain regions, along with vision loss and decreasing intracranial volumes and cortical thicknesses consistent with ovine CLN6 disease. In contrast, intercellular communication affecting pathology was evident at both the gross and histological level in the normal-like and recovering-like chimeras, resulting in a lack of glial activation and rare storage body accumulation in only a few cells. Initial intracranial volumes of the recovering-like chimeras were below normal but progressively recovered to about normal by two years of age. All had normal cortical thicknesses, and none went blind. Extended neurogenesis was evident in the brains of all the chimeras. This study indicates that although CLN6 is a membrane bound protein, the consequent defect is not cell intrinsic. The lack of glial activation and inflammatory responses in the normal-like and recovering-like chimeras indicate that newly generated cells are borne into a microenvironment conducive to maturation and survival.

DOI: https://doi.org/10.1371/journal.pone.0261544
Cells. 2022 Apr 06. pii: 1245. [Epub ahead of print]11(7):

Urine-Derived Kidney Progenitor Cells in Cystinosis.

Koenraad Veys, Sante Princiero Berlingerio, Dries David, Tjessa Bondue, Katharina Held, Ahmed Reda, Martijn van den Broek, Koen Theunis, Mirian Janssen, Elisabeth Cornelissen, Joris Vriens, Francesca Diomedi-Camassei, Rik Gijsbers, Lambertus van den Heuvel, Fanny O Arcolino, Elena Levtchenko.

  Nephropathic cystinosis is an inherited lysosomal storage disorder caused by pathogenic variants in the cystinosin (CTNS) gene and is characterized by the excessive shedding of proximal tubular epithelial cells (PTECs) and podocytes into urine, development of the renal Fanconi syndrome and end-stage kidney disease (ESKD). We hypothesized that in compensation for epithelial cell losses, cystinosis kidneys undertake a regenerative effort, and searched for the presence of kidney progenitor cells (KPCs) in the urine of cystinosis patients. Urine was cultured in a specific progenitor medium to isolate undifferentiated cells. Of these, clones were characterized by qPCR, subjected to a differentiation protocol to PTECs and podocytes and assessed by qPCR, Western blot, immunostainings and functional assays. Cystinosis patients voided high numbers of undifferentiated cells in urine, of which various clonal cell lines showed a high capacity for self-renewal and expressed kidney progenitor markers, which therefore were assigned as cystinosis urine-derived KPCs (Cys-uKPCs). Cys-uKPC clones showed the capacity to differentiate between functional PTECs and/or podocytes. Gene addition with wild-type CTNS using lentiviral vector technology resulted in significant reductions in cystine levels. We conclude that KPCs present in the urine of cystinosis patients can be isolated, differentiated and complemented with CTNS in vitro, serving as a novel tool for disease modeling.

Keywords:  cell model; cystinosis; gene therapy; kidney progenitors

DOI:  https://doi.org/10.3390/cells11071245
Cells. 2022 Mar 31. pii: 1180. [Epub ahead of print]11(7):

Regulation of Aging and Longevity by Ion Channels and Transporters.

Kartik Venkatachalam.

  Despite significant advances in our understanding of the mechanisms that underlie age-related physiological decline, our ability to translate these insights into actionable strategies to extend human healthspan has been limited. One of the major reasons for the existence of this barrier is that with a few important exceptions, many of the proteins that mediate aging have proven to be undruggable. The argument put forth here is that the amenability of ion channels and transporters to pharmacological manipulation could be leveraged to develop novel therapeutic strategies to combat aging. This review delves into the established roles for ion channels and transporters in the regulation of aging and longevity via their influence on membrane excitability, Ca2+ homeostasis, mitochondrial and endolysosomal function, and the transduction of sensory stimuli. The goal is to provide the reader with an understanding of emergent themes, and prompt further investigation into how the activities of ion channels and transporters sculpt the trajectories of cellular and organismal aging.

Keywords:  ER; aging; calcium; ion channels; lifespan; longevity; lysosomes

DOI:  https://doi.org/10.3390/cells11071180
Int J Mol Sci. 2022 Mar 29. pii: 3749. [Epub ahead of print]23(7):

Differential Oxygen Exposure Modulates Mesenchymal Stem Cell Metabolism and Proliferation through mTOR Signaling.

Inês Moniz, João Ramalho-Santos, Ana F Branco.

  Mesenchymal stem cells reside under precise hypoxic conditions that are paramount in determining cell fate and behavior (metabolism, proliferation, differentiation, etc.). In this work, we show that different oxygen tensions promote a distinct proliferative response and affect the biosynthetic demand and global metabolic profile of umbilical cord-mesenchymal stem cells (UC-MSCs). Using both gas-based strategies and CoCl2 as a substitute for the costly hypoxic chambers, we found that specific oxygen tensions influence the fate of UC-MSCs differently. While 5% O2 potentiates proliferation, stimulates biosynthetic pathways, and promotes a global hypermetabolic profile, exposure to <1% O2 contributes to a quiescent-like cell state that relies heavily on anaerobic glycolysis. We show that using CoCl2 as a hypoxia substitute of moderate hypoxia has distinct metabolic effects, when compared with gas-based strategies. The present study also highlights that, while severe hypoxia regulates global translation via mTORC1 modulation, its effects on survival-related mechanisms are mainly modulated through mTORC2. Therefore, the experimental conditions used in this study establish a robust and reliable hypoxia model for UC-MSCs, providing relevant insights into how stem cells are influenced by their physiological environment, and how different strategies of modulating hypoxia may influence experimental outcomes.

Keywords:  cobalt chloride; hypoxia; mTOR; mesenchymal stem cells; metabolism

DOI:  https://doi.org/10.3390/ijms23073749
mBio. 2022 Apr 11. e0384921

Entamoeba histolytica EHD1 Is Involved in Mitosome-Endosome Contact.

Herbert J Santos, Yuki Hanadate, Kenichiro Imai, Haruo Watanabe, Tomoyoshi Nozaki.

  Interorganellar cross talk is often mediated by membrane contact sites (MCSs), which are zones where participating membranes come within 30 nm of one another. MCSs have been found in organelles, including the endoplasmic reticulum, Golgi bodies, endosomes, and mitochondria. Despite its seeming ubiquity, reports of MCS involving mitochondrion-related organelles (MROs) present in a few anaerobic parasitic protozoa remain lacking. Entamoeba histolytica, the etiological agent of amoebiasis, possesses an MRO called the mitosome. We previously discovered several Entamoeba-specific transmembrane mitosomal proteins (ETMPs) from in silico and cell-biological analyses. One of them, ETMP1 (EHI_175060), was predicted to have one transmembrane domain and two coiled-coil regions and was demonstrated to be mitosome membrane integrated based on carbonate fractionation and immunoelectron microscopy (IEM) data. Immunoprecipitation analysis detected a candidate interacting partner, EH domain-containing protein (EHD1; EHI_105270). We expressed hemagglutinin (HA)-tagged EHD1 in E. histolytica, and subsequent immunofluorescence and IEM data indicated an unprecedented MCS between the mitosome and the endosome. Live imaging of a green fluorescent protein (GFP)-EHD1-expressing strain demonstrated that EHD1 is involved in early endosome formation and is observed in MCS between endosomes of various sizes. In vitro assays using recombinant His-EHD1 demonstrated ATPase activity. MCSs are involved in lipid transfer, ion homeostasis, and organelle dynamics. The serendipitous discovery of the ETMP1-interacting partner EHD1 led to the observation of the mitosome-endosome contact site in E. histolytica. It opened a new view of how the relic mitochondria of Entamoeba may likewise be involved in organelle cross talk, a conserved feature of mitochondria and other organelles in general. IMPORTANCE Membrane contact sites (MCSs) are key regulators of interorganellar communication and have been widely demonstrated between various organelles. However, studies on MCSs involving mitochondrion-related organelles (MROs), present in some anaerobic parasitic protozoans, remain scarce. Entamoeba histolytica, the etiological agent of amoebiasis, possesses an MRO called the mitosome. This organelle is crucial for cellular differentiation and disease transmission, thereby significantly contributing to the amoeba's parasitic lifestyle. Our recent discovery of the interaction between the Entamoeba-specific transmembrane mitosomal protein (ETMP1) and EH domain-containing protein (EHD1) showcases a newly found mitosome-endosome contact site in E. histolytica. This finding reflects the idea that despite their substantially divergent and reduced nature, MROs like mitosomes conserve mechanisms for interorganellar cross talk. We posit lipid and ion transport, mitosome fission, and quality control as potential processes that are mediated by the ETMP1-EHD1-tethered mitosome-endosome contact site in E. histolytica.

Keywords:  EH domain; Entamoeba histolytica; endosome; membrane contact site; mitochondrion-related organelles; mitosome

DOI:  https://doi.org/10.1128/mbio.03849-21
Front Immunol. 2022 ;13 818518

mTOR Signaling Pathway Regulates the Release of Proinflammatory Molecule CCL5 Implicated in the Pathogenesis of Autism Spectrum Disorder.

Baojiang Wang, Yueyuan Qin, Qunyan Wu, Xi Li, Dongying Xie, Zhongying Zhao, Shan Duan.

  Autism spectrum disorder (ASD) is a complex pervasive neurodevelopmental disorder and neuroinflammation may contribute to the pathogenesis of ASD. However, the exact mechanisms of abnormal release of proinflammatory mediators in ASD remain poorly understood. This study reports elevated plasma levels of the proinflammatory chemokine (C-C motif) ligand 5 (CCL5) in children with ASD, suggesting an aberrant inflammatory response appearing in the development of ASD. Mining of the expression data of brain or blood tissue from individuals with ASD reveals that mTOR signaling is aberrantly activated in ASD patients. Our in vitro study shows that suppression of mTOR reduces the gene expression and release of CCL5 from human microglia, supporting that CCL5 expression is regulated by mTOR activity. Furthermore, bacterial lipopolysaccharide (LPS)-induced CCL5 expression can be counteracted by siRNA against NF-κB, suggests a determining role of NF-κB in upregulating CCL5 expression. However, a direct regulatory relationship between the NF-κB element and the mTOR signaling pathway was not observed in rapamycin-treated cells. Our results show that the phosphorylated CREB can be induced to suppress CCL5 expression by outcompeting NF-κB in binding to CREB-binding protein (CREBBP) once the mTOR signaling pathway is inhibited. We propose that the activation of mTOR signaling in ASD may induce the suppression of phosphorylation of CREB, which in turn results in the increased binding of CREBBP to NF-κB, a competitor of phosphorylated CREB to drive expression of CCL5. Our study sheds new light on the inflammatory mechanisms of ASD and paves the way for the development of therapeutic strategy for ASD.

Keywords:  CCL5; NF-κB; autism spectrum disorder; mTOR; neurodevelopmental disorder; proinflammatory cytokines

DOI:  https://doi.org/10.3389/fimmu.2022.818518
Cell Res. 2022 Apr 13.

PEN2: Metformin's new partner at lysosome.

Kei Sakamoto, Niels Jessen.

DOI: https://doi.org/10.1038/s41422-022-00661-7
Elife. 2022 Apr 12. pii: e72865. [Epub ahead of print]11

Condensation of Ede1 promotes the initiation of endocytosis.

Mateusz Kozak, Marko Kaksonen.

  Clathrin-mediated endocytosis is initiated by a network of weakly interacting proteins through a poorly understood mechanism. Ede1, the yeast homologue of mammalian Eps15, is an early-arriving endocytic protein and a key initiation factor. In the absence of Ede1, most other early endocytic proteins lose their punctate localization and endocytic uptake is decreased. We show that in yeast cells, cytosolic concentration of Ede1 is buffered at a critical level. Excess amounts of Ede1 form large condensates which recruit other endocytic proteins and exhibit properties of phase-separated liquid droplets. We demonstrate that the central region of Ede1, containing a coiled-coil and a prion-like region, is essential for both the condensate formation and the function of Ede1 in endocytosis. The functionality of Ede1 mutants lacking the central region can be partially rescued by an insertion of heterologous prion-like domains. Conversely, fusion of a heterologous lipid-binding domain with the central region of Ede1 can promote clustering into stable plasma membrane domains. We propose that the ability of Ede1 to form condensed networks supports the clustering of early endocytic proteins and promotes the initiation of endocytosis.

Keywords:  S. cerevisiae; cell biology

DOI:  https://doi.org/10.7554/eLife.72865
Plant Physiol. 2022 Apr 13. pii: kiac167. [Epub ahead of print]

Arabidopsis HOPS subunit VPS41 carries out plant-specific roles in vacuolar transport and vegetative growth.

Dong Jiang, Yilin He, Xiangui Zhou, Zhiran Cao, Lei Pang, Sheng Zhong, Liwen Jiang, Ruixi Li.

  The homotypic fusion and protein sorting (HOPS) complex is a conserved, multi-subunit tethering complex in eukaryotic cells. In yeast and mammalian cells, the HOPS subunit vacuolar protein sorting-associated protein 41 (VPS41) is recruited to late endosomes after Ras-related protein 7 (Rab7) activation and is essential for vacuole fusion. However, whether VPS41 plays conserved roles in plants is not clear. Here, we demonstrate that in the model plant Arabidopsis (Arabidopsis thaliana), VPS41 localizes to distinct condensates in root cells in addition to its reported localization at the tonoplast. The formation of condensates does not rely on the known upstream regulators but depends on VPS41 self-interaction and is essential for vegetative growth regulation. Genetic evidence indicates that VPS41 is required for both homotypic vacuole fusion and cargo sorting from the AP-3, Rab5, and Golgi-independent pathways but is dispensable for the Rab7 cargo inositol transporter 1 (INT1). We also show that VPS41 has HOPS-independent functions in vacuolar transport. Taken together, our findings indicate that Arabidopsis VPS41 is a unique subunit of the HOPS complex that carries out plant-specific roles in both vacuolar transport and developmental regulation.

Keywords:  HOPS complex; VPS41; condensates; vacuolar transport

DOI:  https://doi.org/10.1093/plphys/kiac167
Mol Neurodegener. 2022 Apr 12. 17(1): 30

VCP suppresses proteopathic seeding in neurons.

Jiang Zhu, Sara Pittman, Dhruva Dhavale, Rachel French, Jessica N Patterson, Mohamed Salman Kaleelurrrahuman, Yuanzi Sun, Jaime Vaquer-Alicea, Gianna Maggiore, Christoph S Clemen, William J Buscher, Jan Bieschke, Paul Kotzbauer, Yuna Ayala, Marc I Diamond, Albert A Davis, Conrad Weihl.

   BACKGROUND: Neuronal uptake and subsequent spread of proteopathic seeds, such as αS (alpha-synuclein), Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery participating in this process is poorly understood. One proteinopathy called multisystem proteinopathy (MSP) is associated with dominant mutations in Valosin Containing Protein (VCP). MSP patients have muscle and neuronal degeneration characterized by aggregate pathology that can include αS, Tau and TDP-43.
METHODS: We performed a fluorescent cell sorting based genome-wide CRISPR-Cas9 screen in αS biosensors. αS and TDP-43 seeding activity under varied conditions was assessed using FRET/Flow biosensor cells or immunofluorescence for phosphorylated αS or TDP-43 in primary cultured neurons. We analyzed in vivo seeding activity by immunostaining for phosphorylated αS following intrastriatal injection of αS seeds in control or VCP disease mutation carrying mice.
RESULTS: One hundred fifty-four genes were identified as suppressors of αS seeding. One suppressor, VCP when chemically or genetically inhibited increased αS seeding in cells and neurons. This was not due to an increase in αS uptake or αS protein levels. MSP-VCP mutation expression increased αS seeding in cells and neurons. Intrastriatal injection of αS preformed fibrils (PFF) into VCP-MSP mutation carrying mice increased phospho αS expression as compared to control mice. Cells stably expressing fluorescently tagged TDP-43 C-terminal fragment FRET pairs (TDP-43 biosensors) generate FRET when seeded with TDP-43 PFF but not monomeric TDP-43. VCP inhibition or MSP-VCP mutant expression increases TDP-43 seeding in TDP-43 biosensors. Similarly, treatment of neurons with TDP-43 PFFs generates high molecular weight insoluble phosphorylated TDP-43 after 5 days. This TDP-43 seed dependent increase in phosphorlyated TDP-43 is further augmented in MSP-VCP mutant expressing neurons.
CONCLUSION: Using an unbiased screen, we identified the multifunctional AAA ATPase VCP as a suppressor of αS and TDP-43 aggregate seeding in cells and neurons. VCP facilitates the clearance of damaged lysosomes via lysophagy. We propose that VCP's surveillance of permeabilized endosomes may protect against the proteopathic spread of pathogenic protein aggregates. The spread of distinct aggregate species may dictate the pleiotropic phenotypes and pathologies in VCP associated MSP.

Keywords:  Alpha-synuclein; CRISPR screen; Frontotemporal dementia; Seeding; TDP-43

DOI:  https://doi.org/10.1186/s13024-022-00532-0
PLoS Biol. 2022 Apr 13. 20(4): e3001601

SNX27-Retromer directly binds ESCPE-1 to transfer cargo proteins during endosomal recycling.

Boris Simonetti, Qian Guo, Manuel Giménez-Andrés, Kai-En Chen, Edmund R R Moody, Ashley J Evans, Mintu Chandra, Chris M Danson, Tom A Williams, Brett M Collins, Peter J Cullen.

Coat complexes coordinate cargo recognition through cargo adaptors with biogenesis of transport carriers during integral membrane protein trafficking. Here, we combine biochemical, structural, and cellular analyses to establish the mechanistic basis through which SNX27-Retromer, a major endosomal cargo adaptor, couples to the membrane remodeling endosomal SNX-BAR sorting complex for promoting exit 1 (ESCPE-1). In showing that the SNX27 FERM (4.1/ezrin/radixin/moesin) domain directly binds acidic-Asp-Leu-Phe (aDLF) motifs in the SNX1/SNX2 subunits of ESCPE-1, we propose a handover model where SNX27-Retromer captured cargo proteins are transferred into ESCPE-1 transport carriers to promote endosome-to-plasma membrane recycling. By revealing that assembly of the SNX27:Retromer:ESCPE-1 coat evolved in a stepwise manner during early metazoan evolution, likely reflecting the increasing complexity of endosome-to-plasma membrane recycling from the ancestral opisthokont to modern animals, we provide further evidence of the functional diversification of yeast pentameric Retromer in the recycling of hundreds of integral membrane proteins in metazoans.

DOI: https://doi.org/10.1371/journal.pbio.3001601
Dev Neurobiol. 2022 Apr 15.

Characterization of neurological disease progression in a canine model of CLN5 neuronal ceroid lipofuscinosis.

Elizabeth J Meiman, Grace Robinson Kick, Cheryl A Jensen, Joan R Coates, Martin L Katz.

  Golden Retriever dogs with a frameshift variant in CLN5 (c.934_935delAG) suffer from a progressive neurodegenerative disorder analogous to the CLN5 form of neuronal ceroid lipofuscinosis (NCL). Five littermate puppies homozygous for the deletion allele were identified prior to the onset of disease signs. Studies were performed to characterize the onset and progression of the disease in these dogs. Neurological signs that included restlessness, unwillingness to cooperate with the handlers, and proprioceptive deficits first became apparent at approximately 12 months of age. The neurological signs progressed over time and by 21 to 23 months of age included general proprioceptive ataxia, menace response deficits, aggressive behaviors, cerebellar ataxia, intention tremors, decreased visual tracking, seizures, cognitive decline and impaired prehension. Due to the severity of these signs, the dogs were euthanized between 21 and 23 months of age. Magnetic resonance imaging revealed pronounced progressive global brain atrophy with a more than 7-fold increase in the volume of the ventricular system between 9.5 and 22.5 months of age. Accompanying this atrophy were pronounced accumulations of autofluorescent inclusions throughout the brain and spinal cord. Ultrastructurally, the contents of these inclusions were found to consist primarily of membrane-like aggregates. Inclusions with similar fluorescence properties were present in cardiac muscle, but the contents of these inclusions were homogenously finely granular. Similar to other forms of NCL, the affected dogs had low plasma carnitine concentrations, suggesting impaired carnitine biosynthesis. These data on disease progression will be useful in future studies using the canine model for therapeutic intervention studies. Significance statement: CLN5 neuronal ceroid lipofuscinosis (NCL) is a rare hereditary disorder characterized by progressive neurodegeneration, usually with childhood onset. The disorder results from deleterious sequence variants in the gene encoding a protein involved in normal lysosomal function. There is currently no effective treatment for this disease. Naturally occurring canine disease models have played a key role in developing therapies for similar disorders. To establish a canine model for CLN5 disease, research was performed to characterize the disease progression in a naturally occurring form of this disorder in Golden Retriever dogs. This article is protected by copyright. All rights reserved.

Keywords:  Batten disease; carnitine; hereditary disorder; lysosomal storage disease; neurodegeneration

DOI:  https://doi.org/10.1002/dneu.22878
Science. 2022 Apr 15. 376(6590): eabh1623

Sphingolipids control dermal fibroblast heterogeneity.

Laura Capolupo, Irina Khven, Alex R Lederer, Luigi Mazzeo, Galina Glousker, Sylvia Ho, Francesco Russo, Jonathan Paz Montoya, Dhaka R Bhandari, Andrew P Bowman, Shane R Ellis, Romain Guiet, Olivier Burri, Johanna Detzner, Johannes Muthing, Krisztian Homicsko, François Kuonen, Michel Gilliet, Bernhard Spengler, Ron M A Heeren, G Paolo Dotto, Gioele La Manno, Giovanni D'Angelo.

Human cells produce thousands of lipids that change during cell differentiation and can vary across individual cells of the same type. However, we are only starting to characterize the function of these cell-to-cell differences in lipid composition. Here, we measured the lipidomes and transcriptomes of individual human dermal fibroblasts by coupling high-resolution mass spectrometry imaging with single-cell transcriptomics. We found that the cell-to-cell variations of specific lipid metabolic pathways contribute to the establishment of cell states involved in the organization of skin architecture. Sphingolipid composition is shown to define fibroblast subpopulations, with sphingolipid metabolic rewiring driving cell-state transitions. Therefore, cell-to-cell lipid heterogeneity affects the determination of cell states, adding a new regulatory component to the self-organization of multicellular systems.

DOI: https://doi.org/10.1126/science.abh1623
Cells. 2022 Apr 01. pii: 1190. [Epub ahead of print]11(7):

On the Connections between TRPM Channels and SOCE.

Guilherme H Souza Bomfim, Barbara A Niemeyer, Rodrigo S Lacruz, Annette Lis.

  Plasma membrane protein channels provide a passageway for ions to access the intracellular milieu. Rapid entry of calcium ions into cells is controlled mostly by ion channels, while Ca2+-ATPases and Ca2+ exchangers ensure that cytosolic Ca2+ levels ([Ca2+]cyt) are maintained at low (~100 nM) concentrations. Some channels, such as the Ca2+-release-activated Ca2+ (CRAC) channels and voltage-dependent Ca2+ channels (CACNAs), are highly Ca2+-selective, while others, including the Transient Receptor Potential Melastatin (TRPM) family, have broader selectivity and are mostly permeable to monovalent and divalent cations. Activation of CRAC channels involves the coupling between ORAI1-3 channels with the endoplasmic reticulum (ER) located Ca2+ store sensor, Stromal Interaction Molecules 1-2 (STIM1/2), a pathway also termed store-operated Ca2+ entry (SOCE). The TRPM family is formed by 8 members (TRPM1-8) permeable to Mg2+, Ca2+, Zn2+ and Na+ cations, and is activated by multiple stimuli. Recent studies indicated that SOCE and TRPM structure-function are interlinked in some instances, although the molecular details of this interaction are only emerging. Here we review the role of TRPM and SOCE in Ca2+ handling and highlight the available evidence for this interaction.

Keywords:  Ca2+ signaling; ORAI channels; SOCE; TRPM channels

DOI:  https://doi.org/10.3390/cells11071190