bims-lysosi 2020-10-25 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2020‒10‒25
twenty-six papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Mammalian Atg8-family proteins are upstream regulators of the lysosomalsystem by controlling MTOR and TFEB.
Visualisation of cholesterol and ganglioside GM1 in zebrafish models of Niemann-Pick type C disease and Smith-Lemli-Opitz syndrome using light sheet microscopy.
Pharmacological intervention to restore connectivity deficits of neuronal networks derived from ASD patient iPSC with a TSC2 mutation.
In Vivo Remodeling of Altered Autophagy-Lysosomal Pathway by a Phosphopeptide in Lupus.
Crosstalk of Hedgehog and mTORC1 Pathways.
Deregulation of signalling in genetic conditions affecting the lysosomal metabolism of cholesterol and galactosyl-sphingolipids.
Role of AMPK/mTOR-independent autophagy in clear cell renal cell carcinoma.
Newborn Screening for Pompe Disease.
An autophagy-dependent tubular lysosomal network synchronizes degradative activity required for muscle remodeling.
Fine-tuning of AMPK-ULK1-mTORC1 regulatory triangle is crucial for autophagy oscillation.
An inventory of lysosomal ABC transporters.
FUS contributes to mTOR-dependent inhibition of translation.
Diclofenac impairs autophagic flux via oxidative stress and lysosomal dysfunction: Implications for hepatotoxicity.
Loss of ULK1 Attenuates Cholesterogenic Gene Expression in Mammalian Hepatic Cells.
Rab GTPases in Osteoclastic Bone Resorption and Autophagy.
Roles of Src family kinase, Ras, and mTOR signaling in intestinal epithelial homeostasis and tumorigenesis.
MRI-visible perivascular spaces as an imaging biomarker in Fabry disease.
An iPSC-Derived Neuron Model of CLN3 Disease Facilitates Small Molecule Phenotypic Screening.
Oxidative Stress and Alterations in the Antioxidative Defense System in Neuronal Cells Derived from NPC1 Patient-Specific Induced Pluripotent Stem Cells.
Lysosomal-associated transmembrane protein 4B regulates ceramide-induced exosome release.
Possible Novel Therapeutic Targets in Lymphangioleiomyomatosis Treatment.
Lysosomal cathepsin creates chimeric epitopes for diabetogenic CD4 T cells via transpeptidation.
Mir214-3p and Hnf4a/Hnf4α reciprocally regulate Ulk1 expression and autophagy in nonalcoholic hepatic steatosis.
A Human TSC1 Variant Screening Platform in Gabaergic Cortical Interneurons for Genotype to Phenotype Assessments.
Translational control during cellular senescence.
Mutations in circulating tumor DNA predict primary resistance to systemic therapies in advanced hepatocellular carcinoma.

Autophagy. 2020 Oct 19.

Mammalian Atg8-family proteins are upstream regulators of the lysosomalsystem by controlling MTOR and TFEB.

Suresh Kumar, Ashish Jain, Seong Won Choi, Gustavo Peixoto Duarte da Silva, Lee Allers, Michal H Mudd, Ryan Scott Peters, Jan Haug Anonsen, Tor-Erik Rusten, Michael Lazarou, Vojo Deretic.

  Macroautophagy/autophagy delivers cytoplasmic cargo to lysosomes for degradation. In yeast, the single Atg8 protein plays a role in the formation of autophagosomes whereas in mammalian cells there are five to seven paralogs, referred to as mammalian Atg8s (mAtg8s: GABARAP, GABARAPL1, GABARAPL2, LC3A, LC3B, LC3B2 and LC3C) with incompletely defined functions. Here we show that a subset of mAtg8s directly control lysosomal biogenesis. This occurs at the level of TFEB, the principal regulator of the lysosomal transcriptional program. mAtg8s promote TFEB's nuclear translocation in response to stimuli such as starvation. GABARAP interacts directly with TFEB, whereas RNA-Seq analyses reveal that knockout of six genes encoding mAtg8s, or a triple knockout of the genes encoding all GABARAPs, diminishes the TFEB transcriptional program. We furthermore show that GABARAPs in cooperation with other proteins, IRGM, a factor implicated in tuberculosis and Crohn disease, and STX17, are required during starvation for optimal inhibition of MTOR, an upstream kinase of TFEB, and activation of the PPP3/calcineurin phosphatase that dephosphorylates TFEB, thus promoting its nuclear translocation. In conclusion, mAtg8s, IRGM and STX17 control lysosomal biogenesis by their combined or individual effects on MTOR, TFEB, and PPP3/calcineurin, independently of their roles in the formation of autophagosomal membranes.

Keywords:  Crohn’s disease; GABARAP; HIV; LC3; MTOR; Mycobacterium tuberculosis; TFEB; autophagy; lysosome; metabolism

DOI:  https://doi.org/10.1080/15548627.2020.1837423
Histochem Cell Biol. 2020 Oct 20.

Visualisation of cholesterol and ganglioside GM1 in zebrafish models of Niemann-Pick type C disease and Smith-Lemli-Opitz syndrome using light sheet microscopy.

Sophie R Cook, Cerys Bladen, Johanna Smith, Emily Maguire, Jordan Copner, Gareth D Fenn, Kim Wager, Helen Waller-Evans, Emyr Lloyd-Evans.

  Lysosomal storage diseases are the most common cause of neurodegeneration in children. They are characterised at the cellular level by the accumulation of storage material within lysosomes. There are very limited therapeutic options, and the search for novel therapies has been hampered as few good small animal models are available. Here, we describe the use of light sheet microscopy to assess lipid storage in drug and morpholino induced zebrafish models of two diseases of cholesterol homeostasis with lysosomal dysfunction: First, Niemann-Pick type C disease (NPC), caused by mutations in the lysosomal transmembrane protein NPC1, characterised by intralysosomal accumulation of cholesterol and several other lipids. Second, Smith-Lemli-Opitz syndrome (SLOS), caused by mutations in 7-dehydrocholesterol reductase, which catalyses the last step of cholesterol biosynthesis and is characterised by intralysosomal accumulation of dietary cholesterol. This is the first description of a zebrafish SLOS model. We find that zebrafish accurately model lysosomal storage and disease-specific phenotypes in both diseases. Increased cholesterol and ganglioside GM1 were observed in sections taken from NPC model fish, and decreased cholesterol in SLOS model fish, but these are of limited value as resolution is poor, and accurate anatomical comparisons difficult. Using light sheet microscopy, we were able to observe lipid changes in much greater detail and identified an unexpected accumulation of ganglioside GM1 in SLOS model fish. Our data demonstrate, for the first time in zebrafish, the immense potential that light sheet microscopy has in aiding the resolution of studies involving lysosomal and lipid disorders.

Keywords:  Cholesterol; Ganglioside GM1; Light sheet microscopy; Niemann–Pick type C; Smith–Lemli–Opitz syndrome

DOI:  https://doi.org/10.1007/s00418-020-01925-2
Mol Autism. 2020 Oct 19. 11(1): 80

Pharmacological intervention to restore connectivity deficits of neuronal networks derived from ASD patient iPSC with a TSC2 mutation.

Mouhamed Alsaqati, Vivi M Heine, Adrian J Harwood.

BACKGROUND: Tuberous sclerosis complex (TSC) is a rare genetic multisystemic disorder resulting from autosomal dominant mutations in the TSC1 or TSC2 genes. It is characterised by hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) pathway and has severe neurodevelopmental and neurological components including autism, intellectual disability and epilepsy. In human and rodent models, loss of the TSC proteins causes neuronal hyperexcitability and synaptic dysfunction, although the consequences of these changes for the developing central nervous system are currently unclear.METHODS: Here we apply multi-electrode array-based assays to study the effects of TSC2 loss on neuronal network activity using autism spectrum disorder (ASD) patient-derived iPSCs. We examine both temporal synchronisation of neuronal bursting and spatial connectivity between electrodes across the network.
RESULTS: We find that ASD patient-derived neurons with a functional loss of TSC2, in addition to possessing neuronal hyperactivity, develop a dysfunctional neuronal network with reduced synchronisation of neuronal bursting and lower spatial connectivity. These deficits of network function are associated with elevated expression of genes for inhibitory GABA signalling and glutamate signalling, indicating a potential abnormality of synaptic inhibitory-excitatory signalling. mTORC1 activity functions within a homeostatic triad of protein kinases, mTOR, AMP-dependent protein Kinase 1 (AMPK) and Unc-51 like Autophagy Activating Kinase 1 (ULK1) that orchestrate the interplay of anabolic cell growth and catabolic autophagy while balancing energy and nutrient homeostasis. The mTOR inhibitor rapamycin suppresses neuronal hyperactivity, but does not increase synchronised network activity, whereas activation of AMPK restores some aspects of network activity. In contrast, the ULK1 activator, LYN-1604, increases the network behaviour, shortens the network burst lengths and reduces the number of uncorrelated spikes.
LIMITATIONS: Although a robust and consistent phenotype is observed across multiple independent iPSC cultures, the results are based on one patient. There may be more subtle differences between patients with different TSC2 mutations or differences of polygenic background within their genomes. This may affect the severity of the network deficit or the pharmacological response between TSC2 patients.
CONCLUSIONS: Our observations suggest that there is a reduction in the network connectivity of the in vitro neuronal network associated with ASD patients with TSC2 mutation, which may arise via an excitatory/inhibitory imbalance due to increased GABA-signalling at inhibitory synapses. This abnormality can be effectively suppressed via activation of ULK1.

DOI: https://doi.org/10.1186/s13229-020-00391-w
Cells. 2020 Oct 20. pii: E2328. [Epub ahead of print]9(10):

In Vivo Remodeling of Altered Autophagy-Lysosomal Pathway by a Phosphopeptide in Lupus.

Fengjuan Wang, Inmaculada Tasset, Ana Maria Cuervo, Sylviane Muller.

  The phosphopeptide P140/Lupuzor, which improves the course of lupus disease in mice and patients, targets chaperone-mediated autophagy (CMA), a selective form of autophagy that is abnormally upregulated in lupus-prone MRL/lpr mice. Administered intravenously to diseased mice, P140 reduces the expression level of two major protein players of CMA, LAMP2A and HSPA8, and inhibits CMA in vitro in a cell line that stably expresses a CMA reporter. Here, we aimed to demonstrate that P140 also affects CMA in vivo and to unravel the precise cellular mechanism of how P140 interacts with the CMA process. MRL/lpr mice and CBA/J mice used as control received P140 or control peptides intravenously. Lysosome-enriched fractions of spleen or liver were prepared to examine lysosomal function. Highly purified lysosomes were further isolated and left to incubate with the CMA substrate to study at which cellular step P140 interacts with the CMA process. The data show that P140 effectively regulates CMA in vivo in MRL/lpr mice at the step of substrate lysosomal uptake and restores some alterations of defective lysosomes. For the first time, it is demonstrated that by occluding the intralysosome uptake of CMA substrates, a therapeutic molecule can attenuate excessive CMA activity in a pathological pro-inflammatory context and protect against hyperinflammation. This recovery effect of P140 on hyperactivated CMA is not only important for lupus therapy but potentially also for treating other (auto)inflammatory diseases, including neurologic and metabolic disorders, where CMA modulation would be highly beneficial.

Keywords:  MRL/lpr mice; P140 peptide; chaperone-mediated autophagy; lupus; lysosomes

DOI:  https://doi.org/10.3390/cells9102328
Cells. 2020 Oct 18. pii: E2316. [Epub ahead of print]9(10):

Crosstalk of Hedgehog and mTORC1 Pathways.

Lasse Jonsgaard Larsen, Lisbeth Birk Møller.

  Hedgehog (Hh) signaling and mTOR signaling, essential for embryonic development and cellular metabolism, are both coordinated by the primary cilium. Observations from cancer cells strongly indicate crosstalk between Hh and mTOR signaling. This hypothesis is supported by several studies: Evidence points to a TGFβ-mediated crosstalk; Increased PI3K/AKT/mTOR activity leads to increased Hh signaling through regulation of the GLI transcription factors; increased Hh signaling regulates mTORC1 activity positively by upregulating NKX2.2, leading to downregulation of negative mTOR regulators; GSK3 and AMPK are, as members of both signaling pathways, potentially important links between Hh and mTORC1 signaling; The kinase DYRK2 regulates Hh positively and mTORC1 signaling negatively. In contrast, both positive and negative regulation of Hh has been observed for DYRK1A and DYRK1B, which both regulate mTORC1 signaling positively. Based on crosstalk observed between cilia, Hh, and mTORC1, we suggest that the interaction between Hh and mTORC1 is more widespread than it appears from our current knowledge. Although many studies focusing on crosstalk have been carried out, contradictory observations appear and the interplay involving multiple partners is far from solved.

Keywords:  4E-BP1; GLI1; GLI2; GLI3; Mammalian target of rapamycin; S6K; TSC; autophagy; eIF4E; primary cilia

DOI:  https://doi.org/10.3390/cells9102316
Neurobiol Dis. 2020 Oct 17. pii: S0969-9961(20)30417-4. [Epub ahead of print]146 105142

Deregulation of signalling in genetic conditions affecting the lysosomal metabolism of cholesterol and galactosyl-sphingolipids.

S Gowrishankar, S M Cologna, M I Givogri, E R Bongarzone.

  The role of lipids in neuroglial function is gaining momentum in part due to a better understanding of how many lipid species contribute to key cellular signalling pathways at the membrane level. The description of lipid rafts as membrane domains composed by defined classes of lipids such as cholesterol and sphingolipids has greatly helped in our understanding of how cellular signalling can be regulated and compartmentalized in neurons and glial cells. Genetic conditions affecting the metabolism of these lipids greatly impact on how some of these signalling pathways work, providing a context to understand the biological function of the lipid. Expectedly, abnormal metabolism of several lipids such as cholesterol and galactosyl-sphingolipids observed in several metabolic conditions involving lysosomal dysfunction are often accompanied by neuronal and myelin dysfunction. This review will discuss the role of lysosomal biology in the context of deficiencies in the metabolism of cholesterol and galactosyl-sphingolipids and their impact on neural function in three genetic disorders: Niemann-Pick type C, Metachromatic leukodystrophy and Krabbe's disease.

Keywords:  Akt; Cholesterol; Endosome; Exosome; Krabbe's disease; Lipid raft; Lipids; Lysosomes; Metabolism; Metachromatic leukodystrophy; Myelin; Niemann-Pick type C disease; Psychosine; Signalling; Sulfatides

DOI:  https://doi.org/10.1016/j.nbd.2020.105142
J Investig Med. 2020 Oct 21. pii: jim-2020-001524. [Epub ahead of print]

Role of AMPK/mTOR-independent autophagy in clear cell renal cell carcinoma.

Milan Radovanovic, Sasenka Vidicevic, Jelena Tasic, Nina Tomonjic, Zeljka Stanojevic, Predrag Nikic, Aleksandar Vuksanovic, Zoran Dzamic, Uros Bumbasirevic, Aleksandra Isakovic, Vladimir Trajkovic.

  We examined the status and role of autophagy, a process of lysosomal recycling of cellular material, in clear cell renal cell carcinoma (ccRCC). Paired samples of tumor and adjacent non-malignant tissue were collected from 20 patients with ccRCC after radical nephrectomy. The mRNA levels of apoptosis (BAD, BAX, BCL2, BCLXL, BIM) and autophagy (ATG4, BECN1, GABARAP, p62, UVRAG) regulators were measured by RT-qPCR. The protein levels of autophagosome-associated LC3-II, autophagy receptor p62, apoptotic marker PARP, as well as phosphorylation of autophagy initiator Unc 51-like kinase 1 (ULK1), its activator AMP-activated protein kinase (AMPK) and 4EBP1, the substrate of ULK1 inhibitor mechanistic target of rapamycin (mTOR), were analyzed by immunoblotting. The mRNA levels of pro-apoptotic BAX, anti-apoptotic BCLXL and pro-autophagic ATG4, p62 and UVRAG were higher in ccRCC tumors. Autophagy induction was confirmed by an increase in phospho-ULK1 and degradation of the autophagic target p62, while apoptotic PARP cleavage was unaltered. AMPK phosphorylation was reduced and 4EBP1 phosphorylation was increased in ccRCC tissue. The expression of apoptosis regulators did not correlate with clinicopathological features of ccRCC. Conversely, high mRNA levels of ATG4, GABARAP and p62 were associated with lower tumor stage, as well as with smaller tumor size and better disease-specific 5-year survival (ATG4 and p62). Accordingly, low p62 protein levels, corresponding to increased autophagic flux, were associated with lower tumor stage, reduced metastasis and improved 5-year survival. These data demonstrate that transcriptional induction of autophagy in ccRCC is accompanied by AMPK/mTOR-independent increase in ULK1 activation and autophagic flux, which might slow tumor progression and metastasis independently of apoptosis.

Keywords:  apoptosis; carcinoma; cell death; kidney; prognosis

DOI:  https://doi.org/10.1136/jim-2020-001524
Int J Neonatal Screen. 2020 Jun;6(2): 31

Newborn Screening for Pompe Disease.

Takaaki Sawada, Jun Kido, Kimitoshi Nakamura.

  Glycogen storage disease type II (also known as Pompe disease (PD)) is an autosomal recessive disorder caused by defects in α-glucosidase (AαGlu), resulting in lysosomal glycogen accumulation in skeletal and heart muscles. Accumulation and tissue damage rates depend on residual enzyme activity. Enzyme replacement therapy (ERT) should be started before symptoms are apparent in order to achieve optimal outcomes. Early initiation of ERT in infantile-onset PD improves survival, reduces the need for ventilation, results in earlier independent walking, and enhances patient quality of life. Newborn screening (NBS) is the optimal approach for early diagnosis and treatment of PD. In NBS for PD, measurement of AαGlu enzyme activity in dried blood spots (DBSs) is conducted using fluorometry, tandem mass spectrometry, or digital microfluidic fluorometry. The presence of pseudodeficiency alleles, which are frequent in Asian populations, interferes with NBS for PD, and current NBS systems cannot discriminate between pseudodeficiency and cases with PD or potential PD. The combination of GAA gene analysis with NBS is essential for definitive diagnoses of PD. In this review, we introduce our experiences and discuss NBS programs for PD implemented in various countries.

Keywords:  Pompe disease; genotype-phenotype correlation; newborn screening; pseudodeficiency; treatment and follow-up

DOI:  https://doi.org/10.3390/ijns6020031
J Cell Sci. 2020 Oct 19. pii: jcs.248336. [Epub ahead of print]

An autophagy-dependent tubular lysosomal network synchronizes degradative activity required for muscle remodeling.

Tadayoshi Murakawa, Amy A Kiger, Yuriko Sakamaki, Mitsunori Fukuda, Naonobu Fujita.

  Lysosomes are compartments for the degradation of both endocytic and autophagic cargoes. The shape of lysosomes changes with cellular degradative demands, however, there is limited knowledge about the mechanisms or significance that underlies distinct lysosomal morphologies. Here, we found an extensive tubular autolysosomal network in Drosophila abdominal muscle remodeling during metamorphosis. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein, Syntaxin 17, and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling.

Keywords:  Atrophy; Autolysosome; Drosophila; Metamorphosis; Muscle; Syntaxin17

DOI:  https://doi.org/10.1242/jcs.248336
Sci Rep. 2020 Oct 20. 10(1): 17803

Fine-tuning of AMPK-ULK1-mTORC1 regulatory triangle is crucial for autophagy oscillation.

Marianna Holczer, Bence Hajdú, Tamás Lőrincz, András Szarka, Gábor Bánhegyi, Orsolya Kapuy.

Autophagy is an intracellular digestive process, which has a crucial role in maintaining cellular homeostasis by self-eating the unnecessary and/or damaged components of the cell at various stress events. ULK1, one of the key elements of autophagy activator complex, together with the two sensors of nutrient and energy conditions, called mTORC1 and AMPK kinases, guarantee the precise function of cell response mechanism. We claim that the feedback loops of AMPK-mTORC1-ULK1 regulatory triangle determine an accurate dynamical characteristic of autophagic process upon cellular stress. By using both molecular and theoretical biological techniques, here we reveal that a delayed negative feedback loop between active AMPK and ULK1 is essential to manage a proper cellular answer after prolonged starvation or rapamycin addition. AMPK kinase quickly gets induced followed by AMPK-P-dependent ULK1 activation, whereas active ULK1 has a rapid negative effect on AMPK-P resulting in a delayed inhibition of ULK1. The AMPK-P → ULK1 ˧ AMPK-P negative feedback loop results in a periodic repeat of their activation and inactivation and an oscillatory activation of autophagy, as well. We demonstrate that the periodic induction of self-cannibalism is necessary for the proper dynamical behaviour of the control network when mTORC1 is inhibited with respect to various stress events. By computational simulations we also suggest various scenario to introduce "delay" on AMPK-P-dependent ULK1 activation (i.e. extra regulatory element in the wiring diagram or multi-phosphorylation of ULK1).

DOI: https://doi.org/10.1038/s41598-020-75030-8
FEBS Lett. 2020 Oct 24.

An inventory of lysosomal ABC transporters.

Gergely Szakacs, Rupert Abele.

  ABC transporters fulfill diverse physiological functions in different cellular localizations ranging from the plasma membrane to intracellular membranous compartments. Several ABC transporters have been spotted in the endolysosomal system, which consists of endosomes, autophagosomes, lysosomes and lysosome-related organelles. In this review we present an overview of lysosomal ABC transporters including ABCA2, ABCA3, ABCA5, ABCB6, ABCB9 and ABCD4, discussing their trafficking routes, putative substrates, potential physiological functions and associated diseases. In addition, we offer a critical evaluation of the literature linking ABC transporters to lysosomal drug sequestration, examining pitfalls associated with in vitro models of drug resistance.

Keywords:  ABC transporters; cholesterol; drug resistance; endolysosomal system; lipid; lysosomes; peptide transport; surfactant; trafficking; vitamin B12

DOI:  https://doi.org/10.1002/1873-3468.13967
J Biol Chem. 2020 Oct 20. pii: jbc.RA120.013801. [Epub ahead of print]

FUS contributes to mTOR-dependent inhibition of translation.

Myriam Sévigny, Isabelle Bourdeau Julien, Janani Priya Venkatasubramani, Jeremy B Hui, Paul A Dutchak, Chantelle F Sephton.

  The amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD)-linked RNA-binding protein called fused in sarcoma (FUS) has been implicated in several aspects of RNA regulation, including mRNA translation. The mechanism by which FUS affects the translation of polyribosomes has not been established. Here we show that FUS can associate with stalled polyribosomes and that this association is sensitive to mTOR (mammalian Target of Rapamycin) kinase activity. Specifically, we show that FUS association with polyribosomes is increased by Torin1 treatment or when cells are cultured in nutrient-deficient media, but not when cells are treated with rapamycin, the allosteric inhibitor of mTORC1. Moreover, we report that FUS is necessary for efficient stalling of translation as FUS-deficient cells are refractory to the inhibition of mTOR-dependent signaling by Torin1. We also show that ALS-linked FUS mutants, R521G and P525L, associate abundantly with polyribosomes and decrease global protein synthesis. Importantly, the inhibitory effect on translation by FUS is impaired by mutations that reduce its RNA binding affinity. These findings demonstrate that FUS is an important RNA-binding protein that mediates translational repression through mTOR-dependent signaling, and that ALS-linked FUS-mutants can cause a toxic gain-of-function in the cytoplasm by repressing the translation of mRNA at polyribosomes.

Keywords:  RNA binding protein; Torin1; amyotrophic lateral sclerosis (ALS) (Lou Gehrig disease); fragile X mental retardation (FMRP) 1 (FMRP); frontotemporal degeneration (FTD); fused in sarcoma (FUS); mammalian target of rapamycin (mTOR); polyribosome; protein synthesis; ribosome; translation regulation

DOI:  https://doi.org/10.1074/jbc.RA120.013801
Redox Biol. 2020 Oct 12. pii: S2213-2317(20)30956-3. [Epub ahead of print]37 101751

Diclofenac impairs autophagic flux via oxidative stress and lysosomal dysfunction: Implications for hepatotoxicity.

Seung-Hwan Jung, Wonseok Lee, Seung-Hyun Park, Kang-Yo Lee, You-Jin Choi, Soohee Choi, Dongmin Kang, Sinri Kim, Tong-Shin Chang, Soon-Sun Hong, Byung-Hoon Lee.

  Treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with various side effects, including cardiovascular and hepatic disorders. Studies suggest that mitochondrial damage and oxidative stress are important mediators of toxicity, yet the underlying mechanisms are poorly understood. In this study, we identified that some NSAIDs, including diclofenac, inhibit autophagic flux in hepatocytes. Further detailed studies demonstrated that diclofenac induced a reactive oxygen species (ROS)-dependent increase in lysosomal pH, attenuated cathepsin activity and blocked autophagosome-lysosome fusion. The reactivation of lysosomal function by treatment with clioquinol or transfection with the transcription factor EB restored lysosomal pH and thus autophagic flux. The production of mitochondrial ROS is critical for this process since scavenging ROS reversed lysosomal dysfunction and activated autophagic flux. The compromised lysosomal activity induced by diclofenac also inhibited the fusion with and degradation of mitochondria by mitophagy. Diclofenac-induced cell death and hepatotoxicity were effectively protected by rapamycin. Thus, we demonstrated that diclofenac induces the intracellular ROS production and lysosomal dysfunction that lead to the suppression of autophagy. Impaired autophagy fails to maintain mitochondrial integrity and aggravates the cellular ROS burden, which leads to diclofenac-induced hepatotoxicity.

Keywords:  Diclofenac; Hepatotoxicity; Lysosomal dysfunction; Mitophagy; Nonsteroidal anti-inflammatory drugs; Reactive oxygen species

DOI:  https://doi.org/10.1016/j.redox.2020.101751
Front Cell Dev Biol. 2020 Sep 30. 8 523550

Loss of ULK1 Attenuates Cholesterogenic Gene Expression in Mammalian Hepatic Cells.

Sangam Rajak, Liliana F Iannucci, Jin Zhou, B Anjum, Nelson George, Brijesh K Singh, Sujoy Ghosh, Paul M Yen, Rohit A Sinha.

  The hepatic mevalonate (MVA) pathway, responsible for cholesterol biosynthesis, is a therapeutically important metabolic pathway in clinical medicine. Using an unbiased transcriptomics approach, we uncover a novel role of Unc-51 like autophagy activating kinase 1 (ULK1) in regulating the expression of the hepatic de novo cholesterol biosynthesis/MVA pathway genes. Genetic silencing of ULK1 in non-starved mouse (AML-12) and human (HepG2) hepatic cells as well as in mouse liver followed by transcriptome and pathway analysis revealed that the loss of ULK1 expression led to significant down-regulation of genes involved in the MVA/cholesterol biosynthesis pathway. At a mechanistic level, loss of ULK1 led to decreased expression of SREBF2/SREBP2 (sterol regulatory element binding factor 2) via its effects on AKT-FOXO3a signaling and repression of SREBF2 target genes in the MVA pathway. Our findings, therefore, discover ULK1 as a novel regulator of cholesterol biosynthesis and a possible druggable target for controlling cholesterol-associated pathologies.

Keywords:  FOXO3A transcription factor; SREBF2; ULK1; cholesterogenesis; mevalonate pathway

DOI:  https://doi.org/10.3389/fcell.2020.523550
Int J Mol Sci. 2020 Oct 16. pii: E7655. [Epub ahead of print]21(20):

Rab GTPases in Osteoclastic Bone Resorption and Autophagy.

Michèle Roy, Sophie Roux.

  Small guanosine triphosphate hydrolases (GTPases) of the Rab family are involved in plasma membrane delivery, fusion events, and lysosomal and autophagic degradation pathways, thereby regulating signaling pathways and cell differentiation and function. Osteoclasts are bone-resorbing cells that maintain bone homeostasis. Polarized vesicular trafficking pathways result in the formation of the ruffled border, the osteoclast's resorptive organelle, which also assists in transcytosis. Here, we reviewed the different roles of Rab GTPases in the endomembrane machinery of osteoclasts and in bone diseases caused by the dysfunction of these proteins, with a particular focus on autophagy and bone resorption. Understanding the molecular mechanisms underlying osteoclast-related bone disease development is critical for developing and improving therapies.

Keywords:  Rab GAP; Rab GTPases; autophagy; bone resorption; endomembrane machinery; osteoclasts

DOI:  https://doi.org/10.3390/ijms21207655
Cancer Sci. 2020 Oct 18.

Roles of Src family kinase, Ras, and mTOR signaling in intestinal epithelial homeostasis and tumorigenesis.

Takashi Matozaki, Takenori Kotani, Yoji Murata, Yasuyuki Saito.

  The turnover of intestinal epithelial cells (IECs) is relatively rapid (3 to 5 days in mouse and human), and this short existence and other aspects of the homeostasis of IECs are tightly regulated by various signaling pathways including Wnt-β-catenin signaling. Dysregulation of IEC homeostasis likely contributes to the development of intestinal inflammation and intestinal cancer. The roles of receptor protein tyrosine kinases and their downstream signaling molecules such as Src family kinases, Ras, and mammalian target of rapamycin (mTOR) in homeostatic regulation of IEC turnover have recently been evaluated. These signaling pathways have been found to promote not only the proliferation of IECs but also the differentiation of progenitor cells into secretory cell types such as goblet cells. Of note, signaling by SFKs, Ras, and mTOR has been shown to oppose the Wnt-β-catenin signaling pathway and thereby to limit the number of Lgr5+ intestinal stem cells or of Paneth cells. Such cross talk of signaling pathways is important not only for proper regulation of IEC homeostasis but for the development of intestinal tumors and potentially for anticancer therapy.

Keywords:  Intestinal epithelial cell (IEC); Ras; Src; Wnt; mTORC1

DOI:  https://doi.org/10.1111/cas.14702
J Neurol. 2020 Oct 19.

MRI-visible perivascular spaces as an imaging biomarker in Fabry disease.

D Lyndon, I Davagnanam, D Wilson, F Jichi, A Merwick, F Bolsover, H R Jager, L Cipolotti, C Wheeler-Kingshott, D Hughes, E Murphy, R Lachmann, D J Werring.

  INTRODUCTION: Fabry disease (FD) is an X-linked lysosomal storage disorder resulting in vascular glycosphingolipid accumulation and increased stroke risk. MRI findings associated with FD include white matter hyperintensities (WMH) and cerebral microbleeds (CMBs), suggesting the presence of cerebral small vessel disease. MRI-visible perivascular spaces (PVS) are another promising marker of small vessel disease associated with impaired interstitial fluid drainage. We investigated the association of PVS severity and anatomical distribution with FD.PATIENTS AND METHODS: We compared patients with genetically proven FD to healthy controls. PVS, WMH, lacunes and CMBs were rated on standardised sequences using validated criteria and scales, blinded to diagnosis. A trained observer (using a validated rating scale), quantified the total severity of PVS. We used logistic regression to investigate the association of severe PVS with FD.
RESULTS: We included 33 FD patients (median age 44, 44.1% male) and 20 healthy controls (median age 33.5, 50% male). Adjusting for age and sex, FD was associated with more severe basal ganglia PVS (odds ratio (OR) 5.80, 95% CI 1.03-32.7) and higher total PVS score (OR 4.03, 95% CI 1.36-11.89). Compared with controls, participants with FD had: higher WMH volume (median 495.03 mm3 vs 0, p = 0.0008), more CMBs (21.21% vs none, p = 0.04), and a higher prevalence of lacunes (21.21% vs. 5%, p = 0.23).
CONCLUSIONS: PVS scores are more severe in FD than control subjects. Our findings have potential relevance for FD diagnosis and suggest that impaired interstitial fluid drainage might be a mechanism of white matter injury in FD.

Keywords:  Fabry disease; Magnetic resonance imaging; Neurovascular disease; Perivascular spaces

DOI:  https://doi.org/10.1007/s00415-020-10209-7
ACS Pharmacol Transl Sci. 2020 Oct 09. 3(5): 931-947

An iPSC-Derived Neuron Model of CLN3 Disease Facilitates Small Molecule Phenotypic Screening.

Nihar Kinarivala, Ahmed Morsy, Ronak Patel, Angelica V Carmona, Md Sanaullah Sajib, Snehal Raut, Constantinos M Mikelis, Abraham Al-Ahmad, Paul C Trippier.

The neuronal ceroid lipofuscinoses (NCLs) are a family of rare lysosomal storage disorders. The most common form of NCL occurs in children harboring a mutation in the CLN3 gene. This form is lethal with no existing cure or treatment beyond symptomatic relief. The pathophysiology of CLN3 disease is complex and poorly understood, with current in vivo and in vitro models failing to identify pharmacological targets for therapeutic intervention. This study reports the characterization of the first CLN3 patient-specific induced pluripotent stem cell (iPSC)-derived model of the blood-brain barrier and establishes the suitability of an iPSC-derived neuron model of the disease to facilitate compound screening. Upon differentiation, hallmarks of CLN3 disease are apparent, including lipofuscin and subunit c of mitochondrial ATP synthase accumulation, mitochondrial dysfunction, and attenuated Bcl-2 expression. The model led to the identification of small molecules that cleared subunit c accumulation by mTOR-independent modulation of autophagy, conferred protective effects through induction of Bcl-2 and rescued mitochondrial dysfunction.

DOI: https://doi.org/10.1021/acsptsci.0c00077
Int J Mol Sci. 2020 Oct 16. pii: E7667. [Epub ahead of print]21(20):

Oxidative Stress and Alterations in the Antioxidative Defense System in Neuronal Cells Derived from NPC1 Patient-Specific Induced Pluripotent Stem Cells.

Alexandra V Jürs, Christin Völkner, Maik Liedtke, Katharina Huth, Jan Lukas, Andreas Hermann, Moritz J Frech.

  Oxidative stress (OS) represents a state of an imbalanced amount of reactive oxygen species (ROS) and/or a hampered efficacy of the antioxidative defense system. Cells of the central nervous system are particularly sensitive to OS, as they have a massive need of oxygen to maintain proper function. Consequently, OS represents a common pathophysiological hallmark of neurodegenerative diseases and is discussed to contribute to the neurodegeneration observed amongst others in Alzheimer's disease and Parkinson's disease. In this context, accumulating evidence suggests that OS is involved in the pathophysiology of Niemann-Pick type C1 disease (NPC1). NPC1, a rare hereditary neurodegenerative disease, belongs to the family of lysosomal storage disorders. A major hallmark of the disease is the accumulation of cholesterol and other glycosphingolipids in lysosomes. Several studies describe OS both in murine in vivo and in vitro NPC1 models. However, studies based on human cells are limited to NPC1 patient-derived fibroblasts. Thus, we analyzed OS in a human neuronal model based on NPC1 patient-specific induced pluripotent stem cells (iPSCs). Higher ROS levels, as determined by DCF (dichlorodihydrofluorescein) fluorescence, indicated oxidative stress in all NPC1-deficient cell lines. This finding was further supported by reduced superoxide dismutase (SOD) activity. The analysis of mRNA and protein levels of SOD1 and SOD2 did not reveal any difference between control cells and NPC1-deficient cells. Interestingly, we observed a striking decrease in catalase mRNA and protein levels in all NPC1-deficient cell lines. As catalase is a key enzyme of the cellular antioxidative defense system, we concluded that the lack of catalase contributes to the elevated ROS levels observed in NPC1-deficient cells. Thus, a restitution of a physiological catalase level may pose an intervention strategy to rescue NPC1-deficient cells from the repercussions of oxidative stress contributing to the neurodegeneration observed in NPC1.

Keywords:  NPC1; ROS; SOD1; SOD2; catalase; iPSC

DOI:  https://doi.org/10.3390/ijms21207667
FASEB J. 2020 Oct 08.

Lysosomal-associated transmembrane protein 4B regulates ceramide-induced exosome release.

Kohei Yuyama, Hui Sun, Daisuke Mikami, Tetsuo Mioka, Katsuyuki Mukai, Yasuyuki Igarashi.

  Exosomes are extracellular vesicles that mediate the transport of intracellular molecules, including neurodegenerative agents. Exogenously administrated ceramides have been implicated in the acceleration of exosome production by neurons; however, the molecular machinery involved in this process is unknown. Here, we found that ceramides, especially those consisting of long fatty acids, were internalized into the endocytic pathway in neuroblastoma SH-SY5Y cells to induce exosome secretion through lysosome-associated protein transmembrane 4B (LAPTM4B). Knockdown of LAPTM4B inhibited the ceramide-mediated increase in exosome release completely. Fluorescence microscopy observations indicated that exogenous ceramides promote the transport of multivesicular bodies to the plasma membranes in a LAPTM4B-dependent manner. Similarly, inhibition of acid ceramidase, which tends to induce intracellular ceramide accumulation, increased exosome production by SH-SY5Y cells in a LAPTM4B-dependent manner. Furthermore, the level of amyloid-ß protein (Aß) was decreased in neuronal cells following treatment with exogenous ceramide or inhibition of acid ceramidase, and this effect was attributed to the LAPTM4B-dependent efflux of Aß-containing exosomes. Overall, these findings reveal the novel machinery involved in exosome secretion regulated by ceramides and LAPTM4B, and may contribute to efforts to ameliorate the cellular accumulation of neurodegenerative agents such as Aß.

Keywords:  LAPTM4B; amyloid‐ß; ceramide; exosome

DOI:  https://doi.org/10.1096/fj.202001599R
Front Med (Lausanne). 2020 ;7 554134

Possible Novel Therapeutic Targets in Lymphangioleiomyomatosis Treatment.

Xixi Song, Hui Cai, Chengyu Yang, Xiaomin Xue, Jian Wang, Yuqing Mo, Mengchan Zhu, Guiping Zhu, Ling Ye, Meiling Jin.

  Lymphangioleiomyomatosis (LAM) is a rare systemic neoplastic disease that exclusively happens in women. Studies focusing on LAM and tuberous sclerosis complex (TSC) have made great progress in understanding the pathogenesis and searching for treatment. The inactive mutation of TSC1 or TSC2 is found in patients with LAM to activate the crucial mammalian target of rapamycin (mTOR) signaling pathway and result in enhanced cell proliferation and migration. However, it does not explain every step of tumorigenesis in LAM. Because cessation of rapamycin would break the stabilization of lung function or improved quality of life and lead to disease recurrent, continued studies on the pathogenesis of LAM are necessary to identify novel targets and new treatment. Researchers have found several aberrant regulations that affect the mTOR pathway such as its upstream or downstream molecules and compensatory pathways in LAM. Some therapeutic targets have been under study in clinical trials. New methods like genome-wide association studies have located a novel gene related to LAM. Herein, we review the current knowledge regarding pathogenesis and treatment of LAM and summarize novel targets of therapeutic potential recently.

Keywords:  lymphangioleiomyomatosis; mTOR; pathogenesis; target; treatment

DOI:  https://doi.org/10.3389/fmed.2020.554134
J Exp Med. 2021 Feb 01. pii: e20192135. [Epub ahead of print]218(2):

Lysosomal cathepsin creates chimeric epitopes for diabetogenic CD4 T cells via transpeptidation.

Brendan Reed, Frances Crawford, Ryan C Hill, Niyun Jin, Janice White, S Harsha Krovi, Philippa Marrack, Kirk Hansen, John W Kappler.

The identification of the peptide epitopes presented by major histocompatibility complex class II (MHCII) molecules that drive the CD4 T cell component of autoimmune diseases has presented a formidable challenge over several decades. In type 1 diabetes (T1D), recent insight into this problem has come from the realization that several of the important epitopes are not directly processed from a protein source, but rather pieced together by fusion of different peptide fragments of secretory granule proteins to create new chimeric epitopes. We have proposed that this fusion is performed by a reverse proteolysis reaction called transpeptidation, occurring during the catabolic turnover of pancreatic proteins when secretory granules fuse with lysosomes (crinophagy). Here, we demonstrate several highly antigenic chimeric epitopes for diabetogenic CD4 T cells that are produced by digestion of the appropriate inactive fragments of the granule proteins with the lysosomal protease cathepsin L (Cat-L). This pathway has implications for how self-tolerance can be broken peripherally in T1D and other autoimmune diseases.

DOI: https://doi.org/10.1084/jem.20192135
Autophagy. 2020 Oct 20. 1-17

Mir214-3p and Hnf4a/Hnf4α reciprocally regulate Ulk1 expression and autophagy in nonalcoholic hepatic steatosis.

Da-Hye Lee, So-Hyun Park, Jiyun Ahn, Seung Pyo Hong, Eunyoung Lee, Young-Jin Jang, Tae-Youl Ha, Yang Hoon Huh, Seung-Yeon Ha, Tae-Il Jeon, Chang Hwa Jung.

  Macroautophagy/autophagy, a self-degradative process, regulates metabolic homeostasis in response to various stress conditions and is a therapeutic target for nonalcoholic fatty liver disease. We found that autophagic activity was inhibited as a result of a significant reduction in the expression of autophagy-related genes such as Ulk1 in a mouse model and patients with fatty liver. This downregulation was caused by increased Mir214-3p levels and decreased Hnf4a/Hnf4α mRNA levels in hepatocytes. Mir214-3p suppressed Ulk1 expression through direct binding at a 3' untranslated region sequence. Hnf4a directly activated transcription of Ulk1. We investigated lipid accumulation and the expression of autophagy-related genes in the livers of mice treated with anti-Mir214-3p. Hepatic steatosis was alleviated, and Ulk1 mRNA levels were significantly increased by locked nucleic acid-mediated Mir214-3p silencing. Additionally, autophagosome formation and MAP1LC3/LC3-II protein levels were increased, indicating an increase in autophagic activity. Interestingly, suppression of Mir214-3p did not ameliorate fatty liver under Ulk1 suppression, suggesting that reduced Mir214-3p levels mitigate hepatic steatosis through upregulation of Ulk1. These results demonstrate that inhibition of Mir214-3p expression ameliorated fatty liver disease through increased autophagic activity by increasing the expression of Ulk1. Thus, Mir214-3p is a potential therapeutic target for nonalcoholic fatty disease. Abbreviations: AMPK: adenosine monophosphate-activated protein kinase; ATG: autophagy-related; ChIP: chromatin immunoprecipitation; CTSB: cathepsin B; CTSL: cathepsin L; CQ: chloroquine; HFD: high-fat diet; HNF4A: hepatocyte nuclear factor 4, alpha; IF: immunofluorescence; IHC: immunohistochemistry; LDs: lipid droplets; Leup: leupeptin; LFD: low-fat diet; LNA: locked nucleic acid; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; miRNA: microRNA; MTOR: mechanistic target of rapamycin kinase; NAFLD: non-alcoholic fatty liver disease; NASH: non-alcoholic steatohepatitis; PCR: polymerase chain reaction; TEM: transmission electron microscopy; TF: transcription factor; TLDA: TaqMan low-density array; ULK1: unc-51 like kinase 1; UTR: untranslated region.

Keywords:  autophagosome; high-fat diet; lysosome; microRNA; nonalcoholic fatty liver disease; transcription factor

DOI:  https://doi.org/10.1080/15548627.2020.1827779
Front Mol Neurosci. 2020 ;13 573409

A Human TSC1 Variant Screening Platform in Gabaergic Cortical Interneurons for Genotype to Phenotype Assessments.

Dean Wundrach, Luis E Martinetti, April M Stafford, Stephanie M Bilinovich, Kartik Angara, Jeremy W Prokop, Shane R Crandall, Daniel Vogt.

  The TSC1 and TSC2 genes are connected to multiple syndromes from Tuberous Sclerosis Complex (TSC) to autism spectrum disorder (ASD), with uncertainty if genetic variants cause all or subsets of phenotypes based on the location and type of change. For TSC1, few have addressed if non-TSC associated genetic variants have direct contributions to changes in neurological genotype-to-phenotype impacts, including elevated rates of ASD and seizures. Dominant variants cause TSC, yet TSC1 has many heritable variants not dominant for TSC that are poorly understood in neurological function, with some associated with ASD. Herein, we examined how missense variants in TSC1, R336W, T360N, T393I, S403L, and H732Y, impacted the development of cortical inhibitory interneurons, cell-types whose molecular, cellular, and physiological properties are altered after the loss of mouse TSC1. We found these variants complemented a known phenotype caused by loss of TSC1, increased cell size. However, distinct variants, particularly S403L showed deficits in complementing an increase in parvalbumin levels and exhibited smaller amplitude after hyperpolarizations. Overall, these data show that subtle phenotypes can be induced by some TSC1 missense variants and provide an in vivo system to assess TSC1 variants' neurological impact better.

Keywords:  GABA; TSC1; autism (ASD); cortical interneuron; variant

DOI:  https://doi.org/10.3389/fnmol.2020.573409
Mol Cell Biol. 2020 Oct 19. pii: MCB.00512-20. [Epub ahead of print]

Translational control during cellular senescence.

Matthew J Payea, Carlos Anerillas, Ravi Tharakan, Myriam Gorospe.

Senescence is a state of long-term cell-cycle arrest that arises in cells that have incurred sub-lethal damage. While senescent cells no longer replicate, they remain metabolically active and further develop unique and stable phenotypes that are not present in proliferating cells. On one hand, senescent cells increase in size, maintain an active mTORC1 complex, and produce and secrete a substantial amount of inflammatory proteins as part of the senescence associated secretory phenotype (SASP). On the other hand, these pro-growth phenotypes contrast with the p53-mediated growth arrest typical of senescent cells that is associated with nucleolar stress and an inhibition of rRNA processing and ribosome biogenesis. In sum, translation in senescent cells paradoxically comprises both a global repression of translation triggered by DNA damage and a select increase in the translation of specific proteins, including SASP factors.

DOI: https://doi.org/10.1128/MCB.00512-20
Oncogene. 2020 Oct 23.

Mutations in circulating tumor DNA predict primary resistance to systemic therapies in advanced hepatocellular carcinoma.

Johann von Felden, Amanda J Craig, Teresa Garcia-Lezana, Ismail Labgaa, Philipp K Haber, Delia D'Avola, Amon Asgharpour, Douglas Dieterich, Antoinette Bonaccorso, Miguel Torres-Martin, Daniela Sia, Max W Sung, Parissa Tabrizian, Myron Schwartz, Josep M Llovet, Augusto Villanueva.

Little is known about the mutational landscape of advanced hepatocellular carcinoma (HCC), and predictive biomarkers of response to systemic therapies are lacking. We aimed to describe the mutational landscape of advanced HCC and to identify predictors of primary resistance to systemic therapies using circulating tumor DNA (ctDNA). We prospectively enrolled 121 patients between October 2015 and January 2019. We performed targeted ultra-deep sequencing of 25 genes and Digital Droplet PCR of TERT promoter, including sequential samples throughout treatment. Primary endpoint was progression-free survival (PFS) stratified by mutation profiles in ctDNA. Secondary endpoints were overall survival and objective response rate. The most frequent mutations in ctDNA of advanced HCC were TERT promoter (51%), TP53 (32%), CTNNB1 (17%), PTEN (8%), AXIN1, ARID2, KMT2D, and TSC2 (each 6%). TP53 and CTNNB1 mutations were mutually exclusive. Patients with mutations in the PI3K/MTOR pathway had significantly shorter PFS than those without these mutations after tyrosine kinase inhibitors (2.1 vs 3.7 months, p < 0.001), but not after immune checkpoint inhibition (CPI). WNT pathway mutations were not associated with PFS, overall survival, or objective response after CPI. Serial profiling of ctDNA in a subset correlated with treatment response. Mutation profiling of ctDNA in advanced HCC shows similar mutation frequencies for known HCC drivers compared to early stages and identifies predictive biomarkers of response to systemic therapies.

DOI: https://doi.org/10.1038/s41388-020-01519-1