bims-lysosi 2022-03-13 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2022‒03‒13
35 papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Small molecule C381 targets the lysosome to reduce inflammation and ameliorate disease in models of neurodegeneration.
Coordination of mitochondrial and lysosomal homeostasis mitigates inflammation and muscle atrophy during aging.
Celastrol, a TFEB (transcription factor EB) agonist, is a promising drug candidate for Alzheimer disease.
Lysosomal Changes in Mitosis.
Mitochondria-lysosome contact site dynamics and misregulation in neurodegenerative diseases.
Lysosomal ATP Transporter SLC17A9 Controls Cell Viability via Regulating Cathepsin D.
Lamp1 mediates lipid transport, but is dispensable for autophagy in Drosophila.
Annexins A1 and A2 are recruited to larger lysosomal injuries independently of ESCRTs to promote repair.
KAT7-mediated CANX (calnexin) crotonylation regulates leucine-stimulated MTORC1 activity.
Altered voltage-dependence of slowly activating chloride-proton antiport by late endosomal ClC-6 explains distinct neurological disorders.
Autophagy in the Neuronal Ceroid Lipofuscinoses (Batten Disease).
Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis.
Natural history of retinal degeneration in ovine models of CLN5 and CLN6 neuronal ceroid lipofuscinoses.
CFTR Modulators Restore Acidification of Autophago-Lysosomes and Bacterial Clearance in Cystic Fibrosis Macrophages.
mTOR-Mediated Regulation of Immune Responses in Cancer and Tumor Microenvironment.
Tsc2 knockout counteracts ubiquitin-proteasome system insufficiency and delays photoreceptor loss in retinitis pigmentosa.
AAV9-coGLB1 Improves Lysosomal Storage and Rescues Central Nervous System Inflammation in a Mutant Mouse Model of GM1 Gangliosidosis.
HPS6 Regulates the Biogenesis of Weibel-Palade Body in Endothelial Cells Through Trafficking v-ATPase to Its Limiting Membrane.
Mediobasal hypothalamic FKBP51 acts as a molecular switch linking autophagy to whole-body metabolism.
Role of Lysosomal Gene Variants in Modulating GBA-Associated Parkinson's Disease Risk.
The V-ATPase complex regulates non-canonical Atg8-family protein lipidation through ATG16L1 recruitment.
mTOR activity is essential for retinal pigment epithelium regeneration in zebrafish.
Alterations of Neuronal Lysosomes in Alzheimer's Disease and in APPxPS1-KI Mice.
α-synuclein and phosphoinositide-binding proteins: α-synuclein inhibits the association of PX- but not FYVE-containing proteins with vesicles in vivo.
Lysosomal cysteine proteases are mediators of cell death in macrophages following exposure to amorphous silica nanoparticles.
mTORC1 induces plasma membrane depolarization and promotes preosteoblast senescence by regulating the sodium channel Scn1a.
Lysosomal TRPML1 regulates mitochondrial function in hepatocellular carcinoma cells.
Contribution of Autophagy-Lysosomal Pathway in the Exosomal Secretion of Alpha-Synuclein and Its Impact in the Progression of Parkinson's Disease.
The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity.
A non-canonical tricarboxylic acid cycle underlies cellular identity.
Inhibition of PIKfyve ameliorates the proliferation, migration of vascular smooth muscle cells and vascular intima hyperplasia via reducing mTORC1 activity.
L-Acetylcarnitine causes analgesia in mice modeling Fabry disease by up-regulating type-2 metabotropic glutamate receptors.
mTOR inhibition downregulates glucose-6-phosphate dehydrogenase and induces ROS-dependent death in T-cell acute lymphoblastic leukemia cells.
Regulation of liver subcellular architecture controls metabolic homeostasis.
miRNA-Dependent Regulation of AKT1 Phosphorylation.

Proc Natl Acad Sci U S A. 2022 03 15. 119(11): e2121609119

Small molecule C381 targets the lysosome to reduce inflammation and ameliorate disease in models of neurodegeneration.

Ryan T Vest, Ching-Chieh Chou, Hui Zhang, Michael S Haney, Lulin Li, Nouf N Laqtom, Betty Chang, Steven Shuken, Andy Nguyen, Lakshmi Yerra, Andrew C Yang, Carol Green, Mary Tanga, Monther Abu-Remaileh, Michael C Bassik, Judith Frydman, Jian Luo, Tony Wyss-Coray.

  SignificanceNeurodegenerative diseases are poorly understood and difficult to treat. One common hallmark is lysosomal dysfunction leading to the accumulation of aggregates and other undegradable materials, which cause damage to brain resident cells. Lysosomes are acidic organelles responsible for breaking down biomolecules and recycling their constitutive parts. In this work, we find that the antiinflammatory and neuroprotective compound, discovered via a phenotypic screen, imparts its beneficial effects by targeting the lysosome and restoring its function. This is established using a genome-wide CRISPRi target identification screen and then confirmed using a variety of lysosome-targeted studies. The resulting small molecule from this study represents a potential treatment for neurodegenerative diseases as well as a research tool for the study of lysosomes in disease.

Keywords:  drug development; drug discovery; inflammation; lysosomes; neurodegenerative disease

DOI:  https://doi.org/10.1073/pnas.2121609119
Aging Cell. 2022 Mar 09. e13583

Coordination of mitochondrial and lysosomal homeostasis mitigates inflammation and muscle atrophy during aging.

Andrea Irazoki, Marta Martinez-Vicente, Pilar Aparicio, Cecilia Aris, Esmaeil Alibakhshi, Maria Rubio-Valera, Juan Castellanos, Luis Lores, Manuel Palacín, Anna Gumà, Antonio Zorzano, David Sebastián.

  Sarcopenia is one of the main factors contributing to the disability of aged people. Among the possible molecular determinants of sarcopenia, increasing evidences suggest that chronic inflammation contributes to its development. However, a key unresolved question is the nature of the factors that drive inflammation during aging and that participate in the development of sarcopenia. In this regard, mitochondrial dysfunction and alterations in mitophagy induce inflammatory responses in a wide range of cells and tissues. However, whether accumulation of damaged mitochondria (MIT) in muscle could trigger inflammation in the context of aging is still unknown. Here, we demonstrate that BCL2 interacting protein 3 (BNIP3) plays a key role in the control of mitochondrial and lysosomal homeostasis, and mitigates muscle inflammation and atrophy during aging. We show that muscle BNIP3 expression increases during aging in mice and in some humans. BNIP3 deficiency alters mitochondrial function, decreases mitophagic flux and, surprisingly, induces lysosomal dysfunction, leading to an upregulation of Toll-like receptor 9 (TLR9)-dependent inflammation and activation of the NLRP3 (nucleotide-binding oligomerization domain (NOD)-, leucine-rich repeat (LRR)-, and pyrin domain-containing protein 3) inflammasome in muscle cells and mouse muscle. Importantly, downregulation of muscle BNIP3 in aged mice exacerbates inflammation and muscle atrophy, and high BNIP3 expression in aged human subjects associates with a low inflammatory profile, suggesting a protective role for BNIP3 against age-induced muscle inflammation in mice and humans. Taken together, our data allow us to propose a new adaptive mechanism involving the mitophagy protein BNIP3, which links mitochondrial and lysosomal homeostasis with inflammation and is key to maintaining muscle health during aging.

Keywords:  aging; inflammation; lysosome; mitochondria; mitophagy; muscle

DOI:  https://doi.org/10.1111/acel.13583
Autophagy. 2022 Mar 06. 1-3

Celastrol, a TFEB (transcription factor EB) agonist, is a promising drug candidate for Alzheimer disease.

Wei Zhang, Jigang Wang, Chuanbin Yang.

  Alzheimer disease (AD) is the most common neurodegenerative disease. Unfortunately, current effective therapeutics for AD are limited and thus the discovery of novel anti-AD agents is urgently needed. A key pathological hallmark of AD is the accumulation of phosphorylated MAPT/tau (microtubule associated protein tau) aggregates to form neurofibrillary tangles. Autophagy is a conserved catabolic process that degrades protein aggregates or organelles via lysosomes. TFEB (transcription factor EB), a master regulator of autophagy, transcriptionally regulates multiple autophagy, and lysosomal-related genes. A compromised autophagy-lysosomal pathway (ALP) has been implicated in AD progression, and enhancing TFEB-mediated ALP to degrade MAPT/tau aggregates is a promising anti-AD strategy. In a recent study, we showed that celastrol, a natural small molecule with an anti-obesity effect, is a novel TFEB activator, which enhances autophagy and lysosomal biogenesis both in vitro and in animal brains. Consequently, celastrol promotes the degradation of phosphorylated MAPT/tau aggregates both in cells and in the brain of P301S MAPT/tau and 3XTg mice, two commonly used AD animal models. Interestingly, celastrol also alleviates memory deficits in these mice. Altogether, celastrol enhances TFEB-mediated autophagy and lysosomal biogenesis to ameliorate MAPT/tau pathology, suggesting that celastrol represents a novel anti-AD and other tauopathies drug candidate.Abbreviations: AD: Alzheimer disease; ALP: autophagy-lysosomal pathway; MAPT/tau: microtubule-associated protein tau; MTORC1: mechanistic target of rapamycin kinase complex 1; TFEB: transcription factor EB.

Keywords:  Alzheimer disease; MTOR; TFEB; autophagy; celastrol; lysosome; tau

DOI:  https://doi.org/10.1080/15548627.2022.2046437
Cells. 2022 Mar 03. pii: 875. [Epub ahead of print]11(5):

Lysosomal Changes in Mitosis.

Jonathan Stahl-Meyer, Lya Katrine Kauffeldt Holland, Bin Liu, Kenji Maeda, Marja Jäättelä.

  The recent discovery demonstrating that the leakage of cathepsin B from mitotic lysosomes assists mitotic chromosome segregation indicates that lysosomal membrane integrity can be spatiotemporally regulated. Unlike many other organelles, structural and functional alterations of lysosomes during mitosis remain, however, largely uncharted. Here, we demonstrate substantial differences in lysosomal proteome, lipidome, size, and pH between lysosomes that were isolated from human U2OS osteosarcoma cells either in mitosis or in interphase. The combination of pharmacological synchronization and mitotic shake-off yielded ~68% of cells in mitosis allowing us to investigate mitosis-specific lysosomal changes by comparing cell populations that were highly enriched in mitotic cells to those mainly in the G1 or G2 phases of the cell cycle. Mitotic cells had significantly reduced levels of lysosomal-associated membrane protein (LAMP) 1 and the active forms of lysosomal cathepsin B protease. Similar trends were observed in levels of acid sphingomyelinase and most other lysosomal proteins that were studied. The altered protein content was accompanied by increases in the size and pH of LAMP2-positive vesicles. Moreover, mass spectrometry-based shotgun lipidomics of purified lysosomes revealed elevated levels of sphingolipids, especially sphingomyelin and hexocylceramide, and lysoglyserophospholipids in mitotic lysosomes. Interestingly, LAMPs and acid sphingomyelinase have been reported to stabilize lysosomal membranes, whereas sphingomyelin and lysoglyserophospholipids have an opposite effect. Thus, the observed lysosomal changes during the cell cycle may partially explain the reduced lysosomal membrane integrity in mitotic cells.

Keywords:  cell cycle; lipidome; lysosomal leakage; lysosome; mitosis

DOI:  https://doi.org/10.3390/cells11050875
Trends Neurosci. 2022 Mar 03. pii: S0166-2236(22)00017-0. [Epub ahead of print]

Mitochondria-lysosome contact site dynamics and misregulation in neurodegenerative diseases.

Jasmine Cisneros, Tayler B Belton, George C Shum, Catherine G Molakal, Yvette C Wong.

  Neurons rely heavily on properly regulated mitochondrial and lysosomal homeostasis, with multiple neurodegenerative diseases linked to dysfunction in these two organelles. Interestingly, mitochondria-lysosome membrane contact sites have been identified as a key pathway mediating their crosstalk in neurons. Recent studies have further elucidated the regulation of mitochondria-lysosome contact dynamics via distinct tethering/untethering protein machinery. Moreover, this pathway has been shown to have additional functions in regulating organelle network dynamics and metabolite transfer between lysosomes and mitochondria. In this review, we highlight recent advances in the field of mitochondria-lysosome contact sites and their misregulation across multiple neurodegenerative disorders, which further underscore a potential role for this pathway in neuronal homeostasis and disease.

Keywords:  Charcot-Marie-Tooth disease; Parkinson’s disease; inter-organelle contact sites; lysosomal storage disorders; lysosomes; mitochondria

DOI:  https://doi.org/10.1016/j.tins.2022.01.005
Cells. 2022 Mar 04. pii: 887. [Epub ahead of print]11(5):

Lysosomal ATP Transporter SLC17A9 Controls Cell Viability via Regulating Cathepsin D.

Peng Huang, Qi Cao, Mengnan Xu, Xian-Ping Dong.

  SLC17A9 (solute carrier family 17 member 9) functions as an ATP transporter in lysosomes as well as other secretory vesicles. SLC17A9 inhibition or silence leads to cell death. However, the molecular mechanisms causing cell death are unclear. In this study, we report that cell death induced by SLC17A9 deficiency is rescued by the transcription factor EB (TFEB), a master gene for lysosomal protein expression, suggesting that SLC17A9 deficiency may be the main cause of lysosome dysfunction, subsequently leading to cell death. Interestingly, Cathepsin D, a lysosomal aspartic protease, is inhibited by SLC17A9 deficiency. Heterologous expression of Cathepsin D successfully rescues lysosomal dysfunction and cell death induced by SLC17A9 deficiency. On the other hand, the activity of Cathepsin B, a lysosomal cysteine protease, is not altered by SLC17A9 deficiency, and Cathepsin B overexpression does not rescue lysosomal dysfunction and cell death induced by SLC17A9 deficiency. Our data suggest that lysosomal ATP and SLC17A9 play critical roles in lysosomal function and cell viability by regulating Cathepsin D activity.

Keywords:  ATP transporter; lysosome; solute carrier family 17 member 9 (SLC17A9); vesicular nucleotide transporter (VNUT)

DOI:  https://doi.org/10.3390/cells11050887
Autophagy. 2022 Mar 10. 1-16

Lamp1 mediates lipid transport, but is dispensable for autophagy in Drosophila.

Norin Chaudhry, Margaux Sica, Satya Surabhi, David Sanchez Hernandez, Ana Mesquita, Adem Selimovic, Ayesha Riaz, Laury Lescat, Hua Bai, Gustavo C Macintosh, Andreas Jenny.

  The endolysosomal system not only is an integral part of the cellular catabolic machinery that processes and recycles nutrients for synthesis of biomaterials, but also acts as signaling hub to sense and coordinate the energy state of cells with growth and differentiation. Lysosomal dysfunction adversely influences vesicular transport-dependent macromolecular degradation and thus causes serious problems for human health. In mammalian cells, loss of the lysosome associated membrane proteins LAMP1 and LAMP2 strongly affects autophagy and cholesterol trafficking. Here we show that the previously uncharacterized Drosophila Lamp1 is a bona fide ortholog of vertebrate LAMP1 and LAMP2. Surprisingly and in contrast to lamp1 lamp2 double-mutant mice, Drosophila Lamp1 is not required for viability or autophagy, suggesting that fly and vertebrate LAMP proteins acquired distinct functions, or that autophagy defects in lamp1 lamp2 mutants may have indirect causes. However, Lamp1 deficiency results in an increase in the number of acidic organelles in flies. Furthermore, we find that Lamp1 mutant larvae have defects in lipid metabolism as they show elevated levels of sterols and diacylglycerols (DAGs). Because DAGs are the main lipid species used for transport through the hemolymph (blood) in insects, our results indicate broader functions of Lamp1 in lipid transport. Our findings make Drosophila an ideal model to study the role of LAMP proteins in lipid assimilation without the confounding effects of their storage and without interfering with autophagic processes.Abbreviations: aa: amino acid; AL: autolysosome; AP: autophagosome; APGL: autophagolysosome; AV: autophagic vacuole (i.e. AP and APGL/AL); AVi: early/initial autophagic vacuoles; AVd: late/degradative autophagic vacuoles; Atg: autophagy-related; CMA: chaperone-mediated autophagy; Cnx99A: Calnexin 99A; DAG: diacylglycerol; eMI: endosomal microautophagy; ESCRT: endosomal sorting complexes required for transport; FB: fat body; HDL: high-density lipoprotein; Hrs: Hepatocyte growth factor regulated tyrosine kinase substrate; LAMP: lysosomal associated membrane protein; LD: lipid droplet; LDL: low-density lipoprotein; Lpp: lipophorin; LTP: Lipid transfer particle; LTR: LysoTracker Red; MA: macroautophagy; MCC: Manders colocalization coefficient; MEF: mouse embryonic fibroblast MTORC: mechanistic target of rapamycin kinase complex; PV: parasitophorous vacuole; SNARE: soluble N-ethylmaleimide sensitive factor attachment protein receptor; Snap: Synaptosomal-associated protein; st: starved; TAG: triacylglycerol; TEM: transmission electron microscopy; TFEB/Mitf: transcription factor EB; TM: transmembrane domain; tub: tubulin; UTR: untranslated region.

Keywords:  Autophagy; Drosophila; LAMP proteins; lipid transport; lysosome

DOI:  https://doi.org/10.1080/15548627.2022.2038999
FEBS Lett. 2022 Mar 11.

Annexins A1 and A2 are recruited to larger lysosomal injuries independently of ESCRTs to promote repair.

Willa Wen-You Yim, Hayashi Yamamoto, Noboru Mizushima.

  Damaged lysosomes can be repaired by calcium release-dependent recruitment of the ESCRT machinery. However, the involvement of annexins, another group of calcium-responding membrane repair proteins, has not been fully addressed. Here, we show that although all ubiquitously expressed annexins (ANXA1, A2, A4, A5, A6, A7, and A11) localize to damaged lysosomes, only ANXA1 and ANXA2 are important for repair. Their recruitment is calcium-dependent, ESCRT-independent, and selective towards lysosomes with large injuries. Lysosomal leakage was more severe when ANXA1 or ANXA2 was depleted compared to that of ESCRT components. These findings suggest that ANXA1 and ANXA2 constitute an additional repair mechanism that serves to minimize leakage from damaged lysosomes.

Keywords:  ESCRT; lysosome; membrane permeabilization; membrane repair

DOI:  https://doi.org/10.1002/1873-3468.14329
Autophagy. 2022 Mar 10. 1-18

KAT7-mediated CANX (calnexin) crotonylation regulates leucine-stimulated MTORC1 activity.

Guokai Yan, Xiuzhi Li, Zilong Zheng, Weihua Gao, Changqing Chen, Xinkai Wang, Zhongyi Cheng, Jie Yu, Geng Zou, Muhammad Zahid Farooq, Xiaoyan Zhu, Weiyun Zhu, Qing Zhong, Xianghua Yan.

  Amino acids play crucial roles in the MTOR (mechanistic target of rapamycin kinase) complex 1 (MTORC1) pathway. However, the underlying mechanisms are not fully understood. Here, we establish a cell-free system to mimic the activation of MTORC1, by which we identify CANX (calnexin) as an essential regulator for leucine-stimulated MTORC1 pathway. CANX translocates to lysosomes after leucine deprivation, and its loss of function renders either the MTORC1 activity or the lysosomal translocation of MTOR insensitive to leucine deprivation. We further find that CANX binds to LAMP2 (lysosomal associated membrane protein 2), and LAMP2 is required for leucine deprivation-induced CANX interaction with the Ragulator to inhibit Ragulator activity toward RRAG GTPases. Moreover, leucine deprivation promotes the lysine (K) 525 crotonylation of CANX, which is another essential condition for the lysosomal translocation of CANX. Finally, we find that KAT7 (lysine acetyltransferase 7) mediates the K525 crotonylation of CANX. Loss of KAT7 renders the MTORC1 insensitivity to leucine deprivation. Our findings provide new insights for the regulatory mechanism of the leucine-stimulated MTORC1 pathway.

Keywords:  CANX; KAT7; LAMP2; MTORC1; leucine; lysine crotonylation; ragulator

DOI:  https://doi.org/10.1080/15548627.2022.2047481
J Physiol. 2022 Mar 09.

Altered voltage-dependence of slowly activating chloride-proton antiport by late endosomal ClC-6 explains distinct neurological disorders.

Giovanni Zifarelli, Michael Pusch, Peying Fong.

  KEY POINTS: Ionic composition and pH within intracellular compartments, such as endo-lysosomes, rely on the activity of chloride/proton transporters including ClC-6. Distinct CLCN6 mutations previously were found in individuals with neurodegenerative disease, and also putatively associated with neuronal ceroidal lipofuscinosis. Limited knowledge of wild-type ClC-6 transport function impedes understanding of mechanisms underlying these conditions. We resolved transient and transport currents that permit measurement of voltage- and pH- dependences, as well as kinetics, for wild-type and disease-associated mutant ClC-6s. These findings define wild-type ClC-6 function robustly, and reveal how alterations of the slow activation gating of the transporter cause different kinds of neurological diseases.ABSTRACT: ClC-6 is an intracellularly localized member of the CLC family of chloride transport proteins. It presumably functions in the endo-lysosomal compartment as a chloride-proton antiporter, despite a paucity of biophysical studies in direct support. Observations of lysosomal storage disease, as well as neurodegenerative disorders, emerge with its disruption by knockout or mutation, respectively. An incomplete understanding of wild type ClC-6 function obscures clear mechanistic insight into disease etiology. Here, high-resolution recording protocols that incorporate extreme voltage pulses permit detailed biophysical measurement and analysis of transient capacitive, as well as ionic transport currents. This approach reveals that wild type ClC-6 activation and transport require depolarization to voltages beyond 140 mV. Mutant Y553C associated with early-onset neurodegeneration exerts gain-of-function by shifting the half-maximal voltage for activation to less depolarized voltages. Moreover, we show that the E267A proton glutamate mutant conserves transport currents, albeit reduced. Lastly, the positive shift in activation voltage shown by V580M, a mutant identified in a patient with late- onset lysosomal storage disease, can explain loss-of-function leading to disease. Abstract figure legend CLC transport proteins comprise both channels and transporters. Vesicular CLC transporters function to regulate compartmental ionic homeostasis and acidification. ClC-6 is a vesicular CLC that localizes to the endo-lysosomal compartment. Functional plasma membrane overexpression of GFP-tagged ClC-6 in HEK293 cells surmounted spatial inaccessibility, and rapid whole cell patch recording protocols enabling resolution of fast capacitive transients, as well as ionic transport currents, provided details of wild-type ClC-6 biophysical properties including voltage-dependence, pH-dependence, and kinetics. Clearly defined wild-type ClC-6 function permitted subsequent comparative analysis of mutants, including but not limited to those pertinent to disease. These range from one causing severe, early-onset neurodegeneration, to two variants previously identified in Kufs disease, a late-onset lysosomal storage disease characterized by neuronal ceroid lipofuscinosis. These findings further inform models whereby disruption of ClC-6 biophysical properties set the stage for dysregulated compartmental homeostasis and hence, disease. This article is protected by copyright. All rights reserved.

Keywords:  Kuf disease; anion transport; endosome; organellar homeostasis; vesicular CLC

DOI:  https://doi.org/10.1113/JP282737
Front Cell Dev Biol. 2022 ;10 812728

Autophagy in the Neuronal Ceroid Lipofuscinoses (Batten Disease).

William D Kim, Morgan L D M Wilson-Smillie, Aruban Thanabalasingam, Stephane Lefrancois, Susan L Cotman, Robert J Huber.

  The neuronal ceroid lipofuscinoses (NCLs), also referred to as Batten disease, are a family of neurodegenerative diseases that affect all age groups and ethnicities around the globe. At least a dozen NCL subtypes have been identified that are each linked to a mutation in a distinct ceroid lipofuscinosis neuronal (CLN) gene. Mutations in CLN genes cause the accumulation of autofluorescent lipoprotein aggregates, called ceroid lipofuscin, in neurons and other cell types outside the central nervous system. The mechanisms regulating the accumulation of this material are not entirely known. The CLN genes encode cytosolic, lysosomal, and integral membrane proteins that are associated with a variety of cellular processes, and accumulated evidence suggests they participate in shared or convergent biological pathways. Research across a variety of non-mammalian and mammalian model systems clearly supports an effect of CLN gene mutations on autophagy, suggesting that autophagy plays an essential role in the development and progression of the NCLs. In this review, we summarize research linking the autophagy pathway to the NCLs to guide future work that further elucidates the contribution of altered autophagy to NCL pathology.

Keywords:  Batten disease; autophagosome; autophagy; lysosome; mTOR; model system; neurodegeneration; neuronal ceroid lipofucinosis

DOI:  https://doi.org/10.3389/fcell.2022.812728
PLoS Pathog. 2022 Mar;18(3): e1010322

Reovirus infection is regulated by NPC1 and endosomal cholesterol homeostasis.

Paula Ortega-Gonzalez, Gwen Taylor, Rohit K Jangra, Raquel Tenorio, Isabel Fernandez de Castro, Bernardo A Mainou, Robert C Orchard, Craig B Wilen, Pamela H Brigleb, Jorna Sojati, Kartik Chandran, Martin Sachse, Cristina Risco, Terence S Dermody.

Cholesterol homeostasis is required for the replication of many viruses, including Ebola virus, hepatitis C virus, and human immunodeficiency virus-1. Niemann-Pick C1 (NPC1) is an endosomal-lysosomal membrane protein involved in cholesterol trafficking from late endosomes and lysosomes to the endoplasmic reticulum. We identified NPC1 in CRISPR and RNA interference screens as a putative host factor for infection by mammalian orthoreovirus (reovirus). Following internalization via clathrin-mediated endocytosis, the reovirus outer capsid is proteolytically removed, the endosomal membrane is disrupted, and the viral core is released into the cytoplasm where viral transcription, genome replication, and assembly take place. We found that reovirus infection is significantly impaired in cells lacking NPC1, but infection is restored by treatment of cells with hydroxypropyl-β-cyclodextrin, which binds and solubilizes cholesterol. Absence of NPC1 did not dampen infection by infectious subvirion particles, which are reovirus disassembly intermediates that bypass the endocytic pathway for infection of target cells. NPC1 is not required for reovirus attachment to the plasma membrane, internalization into cells, or uncoating within endosomes. Instead, NPC1 is required for delivery of transcriptionally active reovirus core particles from endosomes into the cytoplasm. These findings suggest that cholesterol homeostasis, ensured by NPC1 transport activity, is required for reovirus penetration into the cytoplasm, pointing to a new function for NPC1 and cholesterol homeostasis in viral infection.

DOI: https://doi.org/10.1371/journal.ppat.1010322
Sci Rep. 2022 Mar 07. 12(1): 3670

Natural history of retinal degeneration in ovine models of CLN5 and CLN6 neuronal ceroid lipofuscinoses.

S J Murray, N L Mitchell.

Neuronal ceroid lipofuscinoses (NCL; Batten disease) are a group of inherited neurodegenerative diseases with a common set of symptoms including cognitive and motor decline and vision loss. Naturally occurring sheep models of CLN5 and CLN6 disease display the key clinical features of NCL, including a progressive loss of vision. We assessed retinal histology, astrogliosis, and lysosomal storage accumulation in CLN5 affected (CLN5-/-) and CLN6 affected (CLN6-/-) sheep eyes and age-matched controls at 3, 6, 12, and 18 months of age to determine the onset and progression of retinal pathology in NCL sheep. The retina of CLN5-/- sheep shows progressive atrophy of the outer retinal layers, widespread gliosis, and accumulation of lysosomal storage in retinal ganglion cells late in disease. In contrast, CLN6-/- retina shows significant atrophy of all retinal layers, progressive gliosis, and earlier accumulation of lysosomal storage. This study has highlighted the differential vulnerability of retinal layers and the time course of retinal atrophy in two distinct models of NCL disease. This data will be valuable in determining potential targets for ocular therapies and the optimal timing of these therapies for protection from retinal dysfunction and degeneration in NCL.

DOI: https://doi.org/10.1038/s41598-022-07612-7
Front Cell Infect Microbiol. 2022 ;12 819554

CFTR Modulators Restore Acidification of Autophago-Lysosomes and Bacterial Clearance in Cystic Fibrosis Macrophages.

Asmaa Badr, Mostafa Eltobgy, Kathrin Krause, Kaitlin Hamilton, Shady Estfanous, Kylene P Daily, Arwa Abu Khweek, Ahmad Hegazi, Midhun N K Anne, Cierra Carafice, Frank Robledo-Avila, Youssra Saqr, Xiaoli Zhang, Tracey L Bonfield, Mikhail A Gavrilin, Santiago Partida-Sanchez, Stephanie Seveau, Estelle Cormet-Boyaka, Amal O Amer.

  Cystic fibrosis (CF) human and mouse macrophages are defective in their ability to clear bacteria such as Burkholderia cenocepacia. The autophagy process in CF (F508del) macrophages is halted, and the underlying mechanism remains unclear. Furthermore, the role of CFTR in maintaining the acidification of endosomal and lysosomal compartments in CF cells has been a subject of debate. Using 3D reconstruction of z-stack confocal images, we show that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Using several complementary approaches, we report that CF macrophages display defective lysosomal acidification and degradative function for cargos destined to autophagosomes, whereas non-autophagosomal cargos are effectively degraded within acidic compartments. Notably, treatment of CF macrophages with CFTR modulators (tezacaftor/ivacaftor) improved the autophagy flux, lysosomal acidification and function, and bacterial clearance. In addition, CFTR modulators improved CFTR function as demonstrated by patch-clamp. In conclusion, CFTR regulates the acidification of a specific subset of lysosomes that specifically fuse with autophagosomes. Therefore, our study describes a new biological location and function for CFTR in autophago-lysosomes and clarifies the long-standing discrepancies in the field.

Keywords:  Burkholderia cenocepacia clearance; CFTR modulators; autophago-lysosomes; autophagosomes; autophagy; cystic fibrosis; lysosomal acidification; macrophages

DOI:  https://doi.org/10.3389/fcimb.2022.819554
Front Immunol. 2021 ;12 774103

mTOR-Mediated Regulation of Immune Responses in Cancer and Tumor Microenvironment.

Sahar Mafi, Behzad Mansoori, Shahram Taeb, Hossein Sadeghi, Reza Abbasi, William C Cho, Davoud Rostamzadeh.

  The mechanistic/mammalian target of rapamycin (mTOR) is a downstream mediator in the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways, which plays a pivotal role in regulating numerous cellular functions including cell growth, proliferation, survival, and metabolism by integrating a variety of extracellular and intracellular signals in the tumor microenvironment (TME). Dysregulation of the mTOR pathway is frequently reported in many types of human tumors, and targeting the PI3K/Akt/mTOR signaling pathway has been considered an attractive potential therapeutic target in cancer. The PI3K/Akt/mTOR signaling transduction pathway is important not only in the development and progression of cancers but also for its critical regulatory role in the tumor microenvironment. Immunologically, mTOR is emerging as a key regulator of immune responses. The mTOR signaling pathway plays an essential regulatory role in the differentiation and function of both innate and adaptive immune cells. Considering the central role of mTOR in metabolic and translational reprogramming, it can affect tumor-associated immune cells to undergo phenotypic and functional reprogramming in TME. The mTOR-mediated inflammatory response can also promote the recruitment of immune cells to TME, resulting in exerting the anti-tumor functions or promoting cancer cell growth, progression, and metastasis. Thus, deregulated mTOR signaling in cancer can modulate the TME, thereby affecting the tumor immune microenvironment. Here, we review the current knowledge regarding the crucial role of the PI3K/Akt/mTOR pathway in controlling and shaping the immune responses in TME.

Keywords:  PI3K/Akt/mTOR signaling pathway; T cell; Tumor microenvironment; cancer; immune response; mTOR

DOI:  https://doi.org/10.3389/fimmu.2021.774103
Proc Natl Acad Sci U S A. 2022 Mar 15. 119(11): e2118479119

Tsc2 knockout counteracts ubiquitin-proteasome system insufficiency and delays photoreceptor loss in retinitis pigmentosa.

Yixiao Wang, Claudio Punzo, John D Ash, Ekaterina S Lobanova.

  SignificanceStudies in multiple experimental systems have demonstrated that an increase in proteolytic capacity of post-mitotic cells improves cellular resistance to a variety of stressors, delays cellular aging and senescence. Therefore, approaches to increase the ability of cells to degrade misfolded proteins could potentially be applied to the treatment of a broad spectrum of human disorders. An example would be retinal degenerations, which cause irreversible loss of vision and are linked to impaired protein degradation. This study suggests that chronic activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway in degenerating photoreceptor neurons could stimulate the degradation of ubiquitinated proteins and enhance proteasomal activity through phosphorylation.

Keywords:  mTORC1; photoreceptor; proteasome; protein phosphorylation; retinal degeneration

DOI:  https://doi.org/10.1073/pnas.2118479119
Curr Gene Ther. 2022 Mar 04.

AAV9-coGLB1 Improves Lysosomal Storage and Rescues Central Nervous System Inflammation in a Mutant Mouse Model of GM1 Gangliosidosis.

Sichi Liu, Wenhao Ma, Yuyu Feng, Yan Zhang, Xuefang Jia, Chengfang Tang, Fang Tang, Xiaobing Wu, Yonglan Huang.

  BACKGROUND: GM1 gangliosidosis (GM1) is an autosomal recessive disorder characterized by deficiency of beta-galactosidase (β-gal), a ubiquitous lysosomal enzyme that catalyzes the hydrolysis of GM1 ganglioside.OBJECTIVE: To explore the application of the AAV9-coGLB1 for effective treatment in a GM1 gangliosidosis mutant mouse model.
METHODS: We designed a novel adeno-associated virus 9 (AAV9) vector expressing β-gal (AAV9-coGLB1) to treat GM1 gangliosidosis. The vector, injected via the caudal vein at 4 weeks of age, drove the widespread and sustained expression of β-gal for up to 32 weeks in the Glb1G455R/G455R mutant mice (GM1 mice).
RESULTS: The increased levels of β-gal reduced the pathological damage occurring in GM1 mice. Histological analyses showed that myelin deficits and neuron-specific pathology were reduced in cerebral cortex region of AAV9-coGLB1-treated mice. Immunohistochemical staining showed that the accumulation of GM1 ganglioside was also reduced after gene therapy. The reduction of the storage in these regions was accompanied by a decrease in activated microglia. In addition, AAV9 treatment reversed the blockade of autophagic flux in GM1 mice.
CONCLUSION: These results show that AAV9-coGLB1 reduces the pathological signs of GM1 gangliosidosis in a mouse model.

Keywords:  AAV9; GM1 gangliosidosis; autophagic flux; central nervous system inflammation; gene therapy; mouse model

DOI:  https://doi.org/10.2174/1566523222666220304092732
Front Cell Dev Biol. 2021 ;9 743124

HPS6 Regulates the Biogenesis of Weibel-Palade Body in Endothelial Cells Through Trafficking v-ATPase to Its Limiting Membrane.

Jiran Lu, Jing Ma, Zhenhua Hao, Wei Li.

  The Weibel-Palade body (WPB) is one of the lysosome-related organelles (LROs) in endothelial cells, whose main content is von Willebrand factor (vWF). The biogenesis of LROs is regulated by the Hermansky-Pudlak syndrome (HPS) protein-associated complexes through transporting cargo proteins to WPBs. Our previous studies have shown that HPS6, a subunit of BLOC-2 complex, is likely involved in the maturation of WPBs. However, the underlying mechanism remains unknown. In this study, we found that the knockdown of HPS6 in human umbilical vein endothelial cells (HUVECs) resulted in misshaped WPBs, decreased WPB number, and impaired vWF tubulation, which are similar to the characteristics of HPS6-deficient mouse endothelial cells. We observed similar morphological changes of WPBs in HUVECs after the knockdown of ATP6V0D1 (a subunit of v-ATPase). Furthermore, we found that HPS6 interacted with ATP6V0D1, suggesting that HPS6 transports ATP6V0D1 to the WPB limiting membrane for the assembly of the v-ATPase complex to maintain its acidic luminal pH, which is critical for the formation of vWF tubules during WPB maturation. In conclusion, HPS6 likely regulates the biogenesis of WPBs by participating in the trafficking of v-ATPase to the WPB membrane.

Keywords:  HPS6; Hermansky–Pudlak syndrome; Weibel–Palade body; lysosome-related organelle; v-ATPase; von Willebrand factor

DOI:  https://doi.org/10.3389/fcell.2021.743124
Sci Adv. 2022 Mar 11. 8(10): eabi4797

Mediobasal hypothalamic FKBP51 acts as a molecular switch linking autophagy to whole-body metabolism.

Alexander S Häusl, Thomas Bajaj, Lea M Brix, Max L Pöhlmann, Kathrin Hafner, Meri De Angelis, Joachim Nagler, Frederik Dethloff, Georgia Balsevich, Karl-Werner Schramm, Patrick Giavalisco, Alon Chen, Mathias V Schmidt, Nils C Gassen.

The mediobasal hypothalamus (MBH) is the central region in the physiological response to metabolic stress. The FK506-binding protein 51 (FKBP51) is a major modulator of the stress response and has recently emerged as a scaffolder regulating metabolic and autophagy pathways. However, the detailed protein-protein interactions linking FKBP51 to autophagy upon metabolic challenges remain elusive. We performed mass spectrometry-based metabolomics of FKBP51 knockout (KO) cells revealing an increased amino acid and polyamine metabolism. We identified FKBP51 as a central nexus for the recruitment of the LKB1/AMPK complex to WIPI4 and TSC2 to WIPI3, thereby regulating the balance between autophagy and mTOR signaling in response to metabolic challenges. Furthermore, we demonstrated that MBH FKBP51 deletion strongly induces obesity, while its overexpression protects against high-fat diet (HFD)-induced obesity. Our study provides an important novel regulatory function of MBH FKBP51 within the stress-adapted autophagy response to metabolic challenges.

DOI: https://doi.org/10.1126/sciadv.abi4797
Mov Disord. 2022 Mar 09.

Role of Lysosomal Gene Variants in Modulating GBA-Associated Parkinson's Disease Risk.

Letizia Straniero, Valeria Rimoldi, Edoardo Monfrini, Salvatore Bonvegna, Giada Melistaccio, Julie Lake, Giulia Soldà, Massimo Aureli, Shankaracharya, Pamela Keagle, Tatiana Foroud, John E Landers, Cornelis Blauwendraat, Anna Zecchinelli, Roberto Cilia, Alessio Di Fonzo, Gianni Pezzoli, Stefano Duga, Rosanna Asselta.

  BACKGROUND: To date, variants in the GBA gene represent the most frequent large-effect genetic factor associated with Parkinson's disease (PD). However, the reason why individuals with the same GBA variant may or may not develop neurodegeneration and PD is still unclear.OBJECTIVES: Therefore, we evaluated the contribution of rare variants in genes responsible for lysosomal storage disorders (LSDs) to GBA-PD risk, comparing the burden of deleterious variants in LSD genes in PD patients versus asymptomatic subjects, all carriers of deleterious variants in GBA.
METHODS: We used a custom next-generation sequencing panel, including 50 LSD genes, to screen 305 patients and 207 controls (discovery cohort). Replication and meta-analysis were performed in two replication cohorts of GBA-variant carriers, of 250 patients and 287 controls, for whom exome or genome data were available.
RESULTS: Statistical analysis in the discovery cohort revealed a significantly increased burden of deleterious variants in LSD genes in patients (P = 0.0029). Moreover, our analyses evidenced that the two strongest modifiers of GBA penetrance are a second variation in GBA (5.6% vs. 1.4%, P = 0.023) and variants in genes causing mucopolysaccharidoses (6.9% vs. 1%, P = 0.0020). These results were confirmed in the meta-analysis, where we observed pooled odds ratios of 1.42 (95% confidence interval [CI] = 1.10-1.83, P = 0.0063), 4.36 (95% CI = 2.02-9.45, P = 0.00019), and 1.83 (95% CI = 1.04-3.22, P = 0.038) for variants in LSD genes, GBA, and mucopolysaccharidosis genes, respectively.
CONCLUSION: The identification of genetic lesions in lysosomal genes increasing PD risk may have important implications in terms of patient stratification for future therapeutic trials. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson Movement Disorder Society.

Keywords:  Parkinson's disease; GBA; lysosomal genes; mutation burden

DOI:  https://doi.org/10.1002/mds.28987
Autophagy. 2022 Mar 08. 1-2

The V-ATPase complex regulates non-canonical Atg8-family protein lipidation through ATG16L1 recruitment.

Lewis Timimi, Carmen Figueras-Novoa, Elena Marcassa, Oliver Florey, J Kenneth Baillie, Rupert Beale, Rachel Ulferts.

  Conjugation of the Atg8 (autophagy related 8) family of ubiquitin-like proteins to phospholipids of the phagophore is a hallmark of macroautophagy/autophagy. Consequently, Atg8 family members, especially LC3B, are commonly used as a marker of autophagosomes. However, the Atg8 family of proteins are not found solely attached to double-membrane autophagosomes. In non-canonical Atg8-family protein lipidation they become conjugated to single membranes. We have shown that this process is triggered by recruitment of ATG16L1 by the vacuolar-type H+-translocating ATPase (V-ATPase) proton pump, suggesting a role for pH sensing in recruitment of Atg8-family proteins to single membranes.

Keywords:  ATG16L1; Atg4; Atg8; CASM; SopF; V-ATPase; influenza; lipidation; non-canonical autophagy

DOI:  https://doi.org/10.1080/15548627.2022.2029233
PLoS Genet. 2022 Mar 10. 18(3): e1009628

mTOR activity is essential for retinal pigment epithelium regeneration in zebrafish.

Fangfang Lu, Lyndsay L Leach, Jeffrey M Gross.

The retinal pigment epithelium (RPE) plays numerous critical roles in maintaining vision and this is underscored by the prevalence of degenerative blinding diseases like age-related macular degeneration (AMD), in which visual impairment is caused by progressive loss of RPE cells. In contrast to mammals, zebrafish possess the ability to intrinsically regenerate a functional RPE layer after severe injury. The molecular underpinnings of this regenerative process remain largely unknown yet hold tremendous potential for developing treatment strategies to stimulate endogenous regeneration in the human eye. In this study, we demonstrate that the mTOR pathway is activated in RPE cells post-genetic ablation. Pharmacological and genetic inhibition of mTOR activity impaired RPE regeneration, while mTOR activation enhanced RPE recovery post-injury, demonstrating that mTOR activity is essential for RPE regeneration in zebrafish. RNA-seq of RPE isolated from mTOR-inhibited larvae identified a number of genes and pathways dependent on mTOR activity at early and late stages of regeneration; amongst these were components of the immune system, which is emerging as a key regulator of regenerative responses across various tissue and model systems. Our results identify crosstalk between macrophages/microglia and the RPE, wherein mTOR activity is required for recruitment of macrophages/microglia to the RPE injury site. Macrophages/microglia then reinforce mTOR activity in regenerating RPE cells. Interestingly, the function of macrophages/microglia in maintaining mTOR activity in the RPE appeared to be inflammation-independent. Taken together, these data identify mTOR activity as a key regulator of RPE regeneration and link the mTOR pathway to immune responses in facilitating RPE regeneration.

DOI: https://doi.org/10.1371/journal.pgen.1009628
J Alzheimers Dis. 2022 Mar 07.

Alterations of Neuronal Lysosomes in Alzheimer's Disease and in APPxPS1-KI Mice.

Brainbank NeuroCEB Neuropathology Network

  BACKGROUND: The cellular and molecular alterations associated with synapse and neuron loss in Alzheimer's disease (AD) remain unclear. In transgenic mouse models that express mutations responsible for familial AD, neuronal and synaptic losses occur in populations that accumulate fibrillar amyloid-β 42 (Aβ 42) intracellularly.OBJECTIVE: We aimed to study the subcellular localization of these fibrillar accumulations and whether such intraneuronal assemblies could be observed in the human pathology.
METHODS: We used immunolabeling and various electron microscopy techniques on APP x presenilin1 - knock-in mice and on human cortical biopsies and postmortem samples.
RESULTS: We found an accumulation of Aβ fibrils in lipofuscin granule-like organelles in APP x presenilin1 - knock-in mice. Electron microscopy of human cortical biopsies also showed an accumulation of undigested material in enlarged lipofuscin granules in neurons from AD compared to age-matched non-AD patients. However, in those biopsies or in postmortem samples we could not detect intraneuronal accumulations of Aβ fibrils, neither in the lipofuscin granules nor in other intraneuronal compartments.
CONCLUSION: The intralysosomal accumulation of Aβ fibrils in specific neuronal populations in APPxPS1-KI mice likely results from a high concentration of Aβ 42 in the endosome-lysosome system due to the high expression of the transgene in these neurons.

Keywords:  Alzheimer’s disease; amyloid-β; electron microscopy; lysosome

DOI:  https://doi.org/10.3233/JAD-215692
Biochem Biophys Res Commun. 2022 Mar 04. pii: S0006-291X(22)00133-4. [Epub ahead of print]603 7-12

α-synuclein and phosphoinositide-binding proteins: α-synuclein inhibits the association of PX- but not FYVE-containing proteins with vesicles in vivo.

Santhanasabapathy Rajasekaran, Dhaval Patel, Stephan N Witt.

  By an unknown mechanism, alpha-synuclein (α-syn) inhibits autophagy in yeast and human cells. Herein, using the yeast Saccharomyces cerevisiae, we tested the hypothesis that α-syn disrupts autophagy by inhibiting the required association of sorting nexin 4 (Snx4) with phagophores. Snx4 contains a phox (PX) homology domain that selectively binds membranes enriched in phosphatidylinositol 3-phosphate (PI3P). Using fluorescence microscopy, we show that upon nitrogen starvation, 70% of the cells exhibited green puncta (phagophores); whereas identically treated cells expressing α-syn exhibited a significantly lower percentage of cells (30%) with such puncta. Our interpretation is that α-syn outcompetes Snx4 for binding to membranes enriched in PI3P, resulting in fewer phagophores and consequently inefficient induction of autophagy. As a control, we tested whether α-syn disrupts the binding of Vps27-GFP to late endosomes/multivesicular bodies (MVBs). Vps27 contains a PI3P-binding domain called FYVE. α-Syn did not disrupt the binding of Vps27-GFP to late endosomes. α-Syn likely inhibits the binding of PX- but not FYVE-containing proteins to PI3P because FYVE domains bind more than two-orders of magnitude tighter than PX domains. We propose that in all cells, whether yeast or human, α-syn has the potential to inhibit protein trafficking pathways that are dependent on PX-domain proteins such as sorting nexins.

Keywords:  Autophagy; Multivesicular body; Parkinson's disease; Retromer; Sorting nexin; α-synuclein

DOI:  https://doi.org/10.1016/j.bbrc.2022.01.101
Chem Biol Interact. 2022 Mar 06. pii: S0009-2797(22)00087-4. [Epub ahead of print]356 109882

Lysosomal cysteine proteases are mediators of cell death in macrophages following exposure to amorphous silica nanoparticles.

Fatima Ishaq, Naphannop Sereesongsaeng, Michael C Johnston, Christopher J Scott, Roberta E Burden.

  Increasing use of nanomaterials in everyday products such as cosmetics, medicines and food packaging is of grave concern given the lack of understanding with regards the impact such materials have on biological systems. The aim of this study is to examine cell death induced by cationic amorphous silica nanoparticles and determine the involvement of lysosomal cysteine proteases in this process. We report that multiple forms of cell death including apoptosis and pyroptosis are elicited following exposure to amorphous silica nanoparticles and that lysosomal cysteine proteases are involved in both cell death pathways in macrophages. Interestingly, lysosomal cysteine protease mRNA expression and release into the extracellular environment is induced following exposure to amorphous silica nanoparticles. Previously, the determination of nanoparticle-induced toxicity has focused on cytokine readouts, but the work presented here demonstrates that changes to normal protease biology should also be considered when evaluating the molecular mechanisms by which nanoparticulate matter causes cellular inflammation and death.

Keywords:  Cell death; Cysteine protease; Lysosome; Nanoparticle; Nanotoxicity; Silica

DOI:  https://doi.org/10.1016/j.cbi.2022.109882
Bone Res. 2022 Mar 08. 10(1): 25

mTORC1 induces plasma membrane depolarization and promotes preosteoblast senescence by regulating the sodium channel Scn1a.

Ajuan Chen, Jian Jin, Shasha Cheng, Zezheng Liu, Cheng Yang, Qingjing Chen, Wenquan Liang, Kai Li, Dawei Kang, Zhicong Ouyang, Chenfeng Yao, Xiaochun Bai, Qingchu Li, Dadi Jin, Bin Huang.

Senescence impairs preosteoblast expansion and differentiation into functional osteoblasts, blunts their responses to bone formation-stimulating factors and stimulates their secretion of osteoclast-activating factors. Due to these adverse effects, preosteoblast senescence is a crucial target for the treatment of age-related bone loss; however, the underlying mechanism remains unclear. We found that mTORC1 accelerated preosteoblast senescence in vitro and in a mouse model. Mechanistically, mTORC1 induced a change in the membrane potential from polarization to depolarization, thus promoting cell senescence by increasing Ca2+ influx and activating downstream NFAT/ATF3/p53 signaling. We further identified the sodium channel Scn1a as a mediator of membrane depolarization in senescent preosteoblasts. Scn1a expression was found to be positively regulated by mTORC1 upstream of C/EBPα, whereas its permeability to Na+ was found to be gated by protein kinase A (PKA)-induced phosphorylation. Prosenescent stresses increased the permeability of Scn1a to Na+ by suppressing PKA activity and induced depolarization in preosteoblasts. Together, our findings identify a novel pathway involving mTORC1, Scn1a expression and gating, plasma membrane depolarization, increased Ca2+ influx and NFAT/ATF3/p53 signaling in the regulation of preosteoblast senescence. Pharmaceutical studies of the related pathways and agents might lead to novel potential treatments for age-related bone loss.

DOI: https://doi.org/10.1038/s41413-022-00204-1
J Cell Sci. 2022 Mar 11. pii: jcs.259455. [Epub ahead of print]

Lysosomal TRPML1 regulates mitochondrial function in hepatocellular carcinoma cells.

Wei Xiong Siow, Yaschar Kabiri, Rachel Tang, Yu-Kai Chao, Eva Plesch, Carola Eberhagen, Florian Flenkenthaler, Thomas Fröhlich, Franz Bracher, Christian Grimm, Martin Biel, Hans Zischka, Angelika M Vollmar, Karin Bartel.

  Liver cancers, including hepatocellular carcinoma (HCC), are the second most lethal cancers worldwide and novel therapeutic strategies are still highly needed. Recently, the endolysosomal cation channel TRPML1 has gained focus in cancer research representing an interesting novel target. We utilized the recently developed isoform-selective TRPML1 activator ML1-SA1 and the CRISPR/Cas9 system to generate tools for over-activation and loss-of-function studies on TRPML1 in HCC. After verification of our tools, we investigated the role of TRPML1 in HCC by studying proliferation, apoptosis, and proteomic alterations. Further, we analyzed mitochondrial function in detail, facilitating confocal and transmission electron microscopy, combined with SeahorseTM and Oroboros® functional analysis. We report that TRPML1 over-activation by a novel, isoform-selective, low-molecular activator induces apoptosis by impairing mitochondrial function calcium dependently. Additionally, TRPML1 loss-of-function deregulates mitochondrial renewal, which leads to proliferation impairment. Thus, our study reveals a novel role for TRPML1 as regulator of mitochondrial function and its modulators as promising molecules for novel therapeutic options in HCC therapy.

Keywords:  Calclium; Cancer; Hepatocellular Carcinoma; Lysosome; Mitochondria; TRPML1

DOI:  https://doi.org/10.1242/jcs.259455
Front Mol Neurosci. 2022 ;15 805087

Contribution of Autophagy-Lysosomal Pathway in the Exosomal Secretion of Alpha-Synuclein and Its Impact in the Progression of Parkinson's Disease.

Denisse Sepúlveda, Marisol Cisternas-Olmedo, Javiera Arcos, Melissa Nassif, René L Vidal.

  Parkinson's disease (PD) is caused by the degeneration of dopaminergic neurons due to an accumulation of intraneuronal abnormal alpha-synuclein (α-syn) protein aggregates. It has been reported that the levels of exosomal α-syn of neuronal origin in plasma correlate significantly with motor dysfunction, highlighting the exosomes containing α-syn as a potential biomarker of PD. In addition, it has been found that the selective autophagy-lysosomal pathway (ALP) contributes to the secretion of misfolded proteins involved in neurodegenerative diseases. In this review, we describe the evidence that supports the relationship between the ALP and α-syn exosomal secretion on the PD progression and its implications in the diagnosis and progression of this pathology.

Keywords:  Parkinson’s disease progression; autophagy-lysosomal pathway; biomarker; degradation; α-syn exosomal secretion

DOI:  https://doi.org/10.3389/fnmol.2022.805087
J Cell Biol. 2022 Apr 04. pii: e202010065. [Epub ahead of print]221(4):

The E3 ligase TRIM1 ubiquitinates LRRK2 and controls its localization, degradation, and toxicity.

Adrienne E D Stormo, Farbod Shavarebi, Molly FitzGibbon, Elizabeth M Earley, Hannah Ahrendt, Lotus S Lum, Erik Verschueren, Danielle L Swaney, Gaia Skibinski, Abinaya Ravisankar, Jeffrey van Haren, Emily J Davis, Jeffrey R Johnson, John Von Dollen, Carson Balen, Jacob Porath, Claudia Crosio, Christian Mirescu, Ciro Iaccarino, William T Dauer, R Jeremy Nichols, Torsten Wittmann, Timothy C Cox, Steve Finkbeiner, Nevan J Krogan, Scott A Oakes, Annie Hiniker.

Missense mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD); however, pathways regulating LRRK2 subcellular localization, function, and turnover are not fully defined. We performed quantitative mass spectrometry-based interactome studies to identify 48 novel LRRK2 interactors, including the microtubule-associated E3 ubiquitin ligase TRIM1 (tripartite motif family 1). TRIM1 recruits LRRK2 to the microtubule cytoskeleton for ubiquitination and proteasomal degradation by binding LRRK2911-919, a nine amino acid segment within a flexible interdomain region (LRRK2853-981), which we designate the "regulatory loop" (RL). Phosphorylation of LRRK2 Ser910/Ser935 within LRRK2 RL influences LRRK2's association with cytoplasmic 14-3-3 versus microtubule-bound TRIM1. Association with TRIM1 modulates LRRK2's interaction with Rab29 and prevents upregulation of LRRK2 kinase activity by Rab29 in an E3-ligase-dependent manner. Finally, TRIM1 rescues neurite outgrowth deficits caused by PD-driving mutant LRRK2 G2019S. Our data suggest that TRIM1 is a critical regulator of LRRK2, controlling its degradation, localization, binding partners, kinase activity, and cytotoxicity.

DOI: https://doi.org/10.1083/jcb.202010065
Nature. 2022 Mar 09.

A non-canonical tricarboxylic acid cycle underlies cellular identity.

Paige K Arnold, Benjamin T Jackson, Katrina I Paras, Julia S Brunner, Madeleine L Hart, Oliver J Newsom, Sydney P Alibeckoff, Jennifer Endress, Esther Drill, Lucas B Sullivan, Lydia W S Finley.

The tricarboxylic acid (TCA) cycle is a central hub of cellular metabolism, oxidizing nutrients to generate reducing equivalents for energy production and critical metabolites for biosynthetic reactions. Despite the importance of the products of the TCA cycle for cell viability and proliferation, mammalian cells display diversity in TCA-cycle activity1,2. How this diversity is achieved, and whether it is critical for establishing cell fate, remains poorly understood. Here we identify a non-canonical TCA cycle that is required for changes in cell state. Genetic co-essentiality mapping revealed a cluster of genes that is sufficient to compose a biochemical alternative to the canonical TCA cycle, wherein mitochondrially derived citrate exported to the cytoplasm is metabolized by ATP citrate lyase, ultimately regenerating mitochondrial oxaloacetate to complete this non-canonical TCA cycle. Manipulating the expression of ATP citrate lyase or the canonical TCA-cycle enzyme aconitase 2 in mouse myoblasts and embryonic stem cells revealed that changes in the configuration of the TCA cycle accompany cell fate transitions. During exit from pluripotency, embryonic stem cells switch from canonical to non-canonical TCA-cycle metabolism. Accordingly, blocking the non-canonical TCA cycle prevents cells from exiting pluripotency. These results establish a context-dependent alternative to the traditional TCA cycle and reveal that appropriate TCA-cycle engagement is required for changes in cell state.

DOI: https://doi.org/10.1038/s41586-022-04475-w
J Cardiovasc Pharmacol. 2022 Mar 11.

Inhibition of PIKfyve ameliorates the proliferation, migration of vascular smooth muscle cells and vascular intima hyperplasia via reducing mTORC1 activity.

Min Gu, Zhen Wang, Feifei Feng, Yongjian Yang, Xiongshan Sun, Dachun Yang.

ABSTRACT: The current study was designed to investigate the role and mechanism of Phosphatidylinositol 3-phosphate 5-kinase type III (PIKfyve) in the proliferation, migration of vascular smooth muscle cells (VSMCs) and vascular intima hyperplasia. We firstly observed increased protein levels of PIKfyve, phospho (p)-S6 Ribosomal Protein (S6)Ser235/236, p-4EBP1Thr37/46 in VSMCs after 24 h of platelet derived growth factor (PDGF)-BB treatment. By using cell counting kit-8 assay, Ki-67 immunofluorescence staining and wound healing assay, we found that PIKfyve inhibition ameliorated the enhanced activity of VSMC proliferation and migration induced by PDGF-BB. Silencing PIKfyve also suppressed the phosphorylation of S6 and 4EBP1 (two major effectors of mTORC1), glucose consumption, activity of HK and LDH in PDGF-BB-challenged VSMCs. After rescuing the phosphorylation of S6 and 4EBP1 by silencing Tsc1, the suppressive effects of PIKfyve inhibition on glucose utilization, proliferation and migration in VSMCs were abolished. The animal model of vascular restenosis was established in C57BL/6J mice by wire injury. We found the expression of PIKfyve was increased in carotid artery at day 28 after injury. Reducing the activity of PIKfyve alleviated vascular neointima hyperplasia after injury. In conclusion, targeting PIKfyve might be a novel effective method to reduce the proliferation, migration of VSMCs and vascular restenosis by affecting mTORC1-mediated glucose utilization.

DOI: https://doi.org/10.1097/FJC.0000000000001243
Mol Pain. 2022 Mar 07. 17448069221087033

L-Acetylcarnitine causes analgesia in mice modeling Fabry disease by up-regulating type-2 metabotropic glutamate receptors.

Francesco Formaggio, Roberto Rimondini, Cecilia Delprete, Leonardo Scalia, Emilio Merlo Pich, Rocco Liguori, Ferdinando Nicoletti, Marco Caprini.

  Fabry disease (FD) is a X-linked lysosomal storage disorder caused by deficient function of the alpha-galactosidase A (α-GalA) enzyme. α-GalA deficiency leads to multisystemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide (Gb3). A hallmark symptom of FD patients is neuropathic pain that appears in the early stage of the disease as a result of peripheral small fiber damage. Previous studies have shown that Acetyl-L-carnitine (ALC) has neuroprotective, neurotrophic, and analgesic activity in animal models of neuropathic pain. To study the action of ALC on neuropathic pain associated with FD, we treated α-GalA gene null mice (α-GalA(-/0)) with ALC for 30 days. In α-Gal KO mice ALC treatment induced acute and long-lasting analgesia, which persisted 1 month after drug withdrawal. This effect was antagonized by single administration of LY341495, an orthosteric antagonist of mGlu2/3 metabotropic glutamate receptors. We also found an up-regulation of mGlu2 receptors in cultured DRG neurons isolated from 30-day ALC treated α-GalA KO mice. However, the up-regulation of mGlu2 receptors was no longer present in DRG neurons isolated 30 days after the end of treatment. Taken together, these findings suggest that ALC induces analgesia in an animal model of FD by up-regulating mGlu2 receptors, and that analgesia is maintained by additional mechanisms after ALC withdrawal. ALC might represent a valuable pharmacological strategy to reduce pain in FD patients.

Keywords:  Fabri disease; Ion channels; Mouse model; Pain; mGlu Receptors

DOI:  https://doi.org/10.1177/17448069221087033
Redox Biol. 2022 Feb 24. pii: S2213-2317(22)00040-4. [Epub ahead of print]51 102268

mTOR inhibition downregulates glucose-6-phosphate dehydrogenase and induces ROS-dependent death in T-cell acute lymphoblastic leukemia cells.

Micol Silic-Benussi, Evgenyia Sharova, Francesco Ciccarese, Ilaria Cavallari, Vittoria Raimondi, Loredana Urso, Alberto Corradin, Harel Kotler, Gloria Scattolin, Barbara Buldini, Samuela Francescato, Giuseppe Basso, Sonia A Minuzzo, Stefano Indraccolo, Donna M D'Agostino, Vincenzo Ciminale.

  mTOR activation is a hallmark of T-cell acute lymphoblastic leukemia (T-ALL) and is associated with resistance to glucocorticoid (GC)-based chemotherapy. We previously showed that altering redox homeostasis primes T-ALL cells to GC-induced apoptosis. Here we investigated the connection between the mTOR pathway and redox homeostasis using pharmacological inhibitors and gene silencing. In vitro studies performed on T-ALL cell lines and CG-resistant patient-derived T-ALL xenograft (PDX) cells showed that the mTOR inhibitor everolimus increased reactive oxygen species (ROS) levels, augmented lipid peroxidation, and activated the ROS-controlled transcription factor NRF2. These effects were accompanied by a decrease in the levels of NADPH and of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway (PPP), which is a major source of cytosolic NADPH needed for maintaining the cellular ROS-scavenging capacity. The mTOR inhibitor everolimus induced mitochondrial inner membrane depolarization and dose-dependent apoptosis of T-ALL cells, but did not kill normal T-cells. Importantly, the combination of everolimus and the GC dexamethasone had a synergistic effect on killing T-ALL cells. The effects of mTOR inhibition were blunted by ROS scavengers and phenocopied by siRNA-mediated G6PD silencing. In vivo studies of NOD/SCID mice inoculated with refractory T-ALL PDX demonstrated that everolimus overcame dexamethasone resistance in conditions of high tumor burden that mimicked the clinical setting of acute leukemia. These findings provide insight into the crosstalk between mTOR and ROS homeostasis in T-ALL cells and furnish mechanistic evidence to support the combination of glucocorticoids with mTOR inhibitors as a therapeutic avenue for treating refractory T-ALL.

Keywords:  G6PD; ROS; T-ALL; mTOR

DOI:  https://doi.org/10.1016/j.redox.2022.102268
Nature. 2022 Mar 09.

Regulation of liver subcellular architecture controls metabolic homeostasis.

Güneş Parlakgül, Ana Paula Arruda, Song Pang, Erika Cagampan, Nina Min, Ekin Güney, Grace Yankun Lee, Karen Inouye, Harald F Hess, C Shan Xu, Gökhan S Hotamışlıgil.

Cells display complex intracellular organization by compartmentalization of metabolic processes into organelles, yet the resolution of these structures in the native tissue context and their functional consequences are not well understood. Here we resolved the three-dimensional structural organization of organelles in large (more than 2.8 × 105 µm3) volumes of intact liver tissue (15 partial or full hepatocytes per condition) at high resolution (8 nm isotropic pixel size) using enhanced focused ion beam scanning electron microscopy1,2 imaging followed by deep-learning-based automated image segmentation and 3D reconstruction. We also performed a comparative analysis of subcellular structures in liver tissue of lean and obese mice and found substantial alterations, particularly in hepatic endoplasmic reticulum (ER), which undergoes massive structural reorganization characterized by marked disorganization of stacks of ER sheets3 and predominance of ER tubules. Finally, we demonstrated the functional importance of these structural changes by monitoring the effects of experimental recovery of the subcellular organization on cellular and systemic metabolism. We conclude that the hepatic subcellular organization of the ER architecture are highly dynamic, integrated with the metabolic state and critical for adaptive homeostasis and tissue health.

DOI: https://doi.org/10.1038/s41586-022-04488-5
Cells. 2022 Feb 26. pii: 821. [Epub ahead of print]11(5):

miRNA-Dependent Regulation of AKT1 Phosphorylation.

Mallory I Frederick, Tarana Siddika, Pengcheng Zhang, Nileeka Balasuriya, Matthew A Turk, Patrick O'Donoghue, Ilka U Heinemann.

  The phosphoinositide-3-kinase (PI3K)/AKT pathway regulates cell survival and is over-activated in most human cancers, including ovarian cancer. Following growth factor stimulation, AKT1 is activated by phosphorylation at T308 and S473. Disruption of the AKT1 signaling pathway is sufficient to inhibit the epithelial-mesenchymal transition in epithelial ovarian cancer (EOC) cells. In metastatic disease, adherent EOC cells transition to a dormant spheroid state, characterized previously by low S473 phosphorylation in AKT1. We confirmed this finding and observed that T308 phosphorylation was yet further reduced in EOC spheroids and that the transition from adherent to spheroid growth is accompanied by significantly increased levels of let-7 miRNAs. We then used mechanistic studies to investigate the impact of let-7 miRNAs on AKT1 phosphorylation status and activity in cells. In growth factor-stimulated HEK 293T cells supplemented with let-7a, we found increased phosphorylation of AKT1 at T308, decreased phosphorylation at S473, and enhanced downstream AKT1 substrate GSK-3β phosphorylation. Let-7b and let-7g also deregulated AKT signaling by rendering AKT1 insensitive to growth factor simulation. We uncovered let-7a-dependent deregulation of PI3K pathway components, including PI3KC2A, PDK1, and RICTOR, that govern AKT1 phosphorylation and activity. Together, our data show a new role for miRNAs in regulating AKT signaling.

Keywords:  miRNA; oncogenic kinase; posttranslational modification; protein phosphorylation; signaling

DOI:  https://doi.org/10.3390/cells11050821