bims-lysosi 2021-08-01 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021–08–01
34 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing

miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact proteotoxicity and muscle function during aging.
PICALM regulates cathepsin D processing and lysosomal function.
SNX19 restricts endolysosome motility through contacts with the endoplasmic reticulum.
Simultaneous determination of intraluminal lysosomal calcium and pH by dextran-conjugated fluorescent dyes.
A novel tool for detecting lysosomal membrane permeabilization by high-throughput fluorescence microscopy.
Leucyl-tRNA synthetase 1 is required for proliferation of TSC-null cells.
Competitive binding of extracellular accumulated heparan sulfate reduces lysosomal storage defects and triggers neuronal differentiation in a model of Mucopolysaccharidosis IIIB.
Lipid metabolism, inflammation, and foam cell formation in health and metabolic disorders: targeting mTORC1.
Hepatic mTORC1 signaling activates ATF4 as part of its metabolic response to feeding and insulin.
Ablation of microRNA-155 and neuroinflammation in a mouse model of CLN1-disease.
Gαq activation modulates autophagy by promoting mTORC1 signaling.
Wild-type GBA1 increases the α-synuclein tetramer-monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice.
Formation of Lipofuscin-Like Autofluorescent Granules in the Retinal Pigment Epithelium Requires Lysosome Dysfunction.
ubtor Mutation Causes Motor Hyperactivity by Activating mTOR Signaling in Zebrafish.
LRRK2 is required for CD38-mediated NAADP-Ca2+ signaling and the downstream activation of TFEB (transcription factor EB) in immune cells.
Lysosomal TPCN (two pore segment channel) inhibition ameliorates beta-amyloid pathology and mitigates memory impairment in Alzheimer disease.
Clinical Development of Cell Therapies to Halt Lysosomal Storage Diseases: Results and Lessons Learned.
Indices of Defective Autophagy in Whole Muscle and Lysosome Enriched Fractions From Aged D2-mdx Mice.
Platelet and myeloid cell phenotypes in a rat model of Fabry disease.
The role of mTOR in age-related diseases.
THAP1 modulates oligodendrocyte maturation by regulating ECM degradation in lysosomes.
Brain-specific inhibition of mTORC1 eliminates side effects resulting from mTORC1 blockade in the periphery and reduces alcohol intake in mice.
Endo-lysosomal Aβ concentration and pH trigger formation of Aβ oligomers that potently induce Tau missorting.
Mechanical Overloading Induced-Activation of mTOR Signaling in Tendon Stem/Progenitor Cells Contributes to Tendinopathy Development.
Host autophagy mediates organ wasting and nutrient mobilization for tumor growth.
DEPTOR inhibits lung tumorigenesis by inactivating the EGFR-mTOR signals.
Asymmetric organelle inheritance predicts human blood stem cell fate.
Targeting mTOR signaling overcomes acquired resistance to combined BRAF and MEK inhibition in BRAF-mutant melanoma.
Mitochondrial and Organellar Crosstalk in Parkinson's Disease.
Protein phosphatase 6 promotes neurite outgrowth by promoting mTORC2 activity in N2a cells.
The c.863A>G (p.Glu288Gly) variant of the CTSD gene is not associated with CLN10 disease.
Rewiring of the ubiquitinated proteome determines ageing in C. elegans.
Low-level mosaicism in tuberous sclerosis complex in four unrelated patients: Comparison of clinical characteristics and diagnostic pathways.
Knockdown of CNN3 Impairs Myoblast Proliferation, Differentiation, and Protein Synthesis via the mTOR Pathway.

Elife. 2021 Jul 27. pii: e66768. [Epub ahead of print]10

miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact proteotoxicity and muscle function during aging.

Isabelle Schiffer, Birgit Gerisch, Kazuto Kawamura, Raymond Laboy, Jennifer Hewitt, Martin Sebastian Denzel, Marcelo A Mori, Siva Vanapalli, Yidong Shen, Orsolya Symmons, Adam Antebi.

  Muscle function relies on the precise architecture of dynamic contractile elements, which must be fine-tuned to maintain motility throughout life. Muscle is also plastic, and remodeled in response to stress, growth, neural and metabolic inputs. The conserved muscle-enriched microRNA, miR-1, regulates distinct aspects of muscle development, but whether it plays a role during aging is unknown. Here we investigated Caenorhabditis elegans miR-1 in muscle function in response to proteostatic stress. mir-1 deletion improved mid-life muscle motility, pharyngeal pumping, and organismal longevity upon polyQ35 proteotoxic challenge. We identified multiple vacuolar ATPase subunits as subject to miR-1 control, and the regulatory subunit vha-13/ATP6V1A as a direct target downregulated via its 3'UTR to mediate miR-1 physiology. miR-1 further regulates nuclear localization of lysosomal biogenesis factor HLH-30/TFEB and lysosomal acidification. Our studies reveal that miR-1 coordinately regulates lysosomal v-ATPase and biogenesis to impact muscle function and health during aging.

Keywords:  C. elegans; genetics; genomics; lysosomal v-ATPase; miR-1; polyglutamine; proteostasis; vha-13

DOI:  https://doi.org/10.7554/eLife.66768
Biochem Biophys Res Commun. 2021 Jul 23. pii: S0006-291X(21)01061-5. [Epub ahead of print]570 103-109

PICALM regulates cathepsin D processing and lysosomal function.

Kathryn J Hattersley, Julian M Carosi, Leanne K Hein, Julien Bensalem, Timothy J Sargeant.

  Degradation and clearance of cellular waste in the autophagic and endo-lysosomal systems is important for normal physiology and prevention of common late-onset diseases such as Alzheimer's disease (AD). Phosphatidylinostol-binding clathrin assembly protein (PICALM) is a robust AD risk factor gene and encodes an endosomal protein clathrin-binding cytosolic protein, reduction of which is known to exacerbate tauopathy. Although PICALM is known to regulate initiation of autophagy, its role in maturation of lysosomal enzymes required for proteolysis has not been studied. We sought to determine the importance of PICALM for cellular degradative function by disrupting exon 1 of PICALM using CRISPR/Cas9 in HeLa cells. PICALM disruption increased numbers of early endosomes. Proteomic analysis of endosome-enriched samples showed that disrupting exon 1 of PICALM increased the abundance of lysosomal enzymes in these organelles, and western blotting revealed disruption to processing and maturation of the lysosomal protease, cathepsin D, and a deficit in autophagy. This study shows PICALM is important for the correct maturation of lysosomal enzymes and efficient proteolytic function in the lysosome.

Keywords:  Alzheimer's disease; Autophagy; Cathepsin D; Lysosomes; PICALM

DOI:  https://doi.org/10.1016/j.bbrc.2021.07.024
Nat Commun. 2021 07 27. 12(1): 4552

SNX19 restricts endolysosome motility through contacts with the endoplasmic reticulum.

Amra Saric, Spencer A Freeman, Chad D Williamson, Michal Jarnik, Carlos M Guardia, Michael S Fernandopulle, David C Gershlick, Juan S Bonifacino.

The ability of endolysosomal organelles to move within the cytoplasm is essential for the performance of their functions. Long-range movement involves coupling of the endolysosomes to motor proteins that carry them along microtubule tracks. This movement is influenced by interactions with other organelles, but the mechanisms involved are incompletely understood. Herein we show that the sorting nexin SNX19 tethers endolysosomes to the endoplasmic reticulum (ER), decreasing their motility and contributing to their concentration in the perinuclear area of the cell. Tethering depends on two N-terminal transmembrane domains that anchor SNX19 to the ER, and a PX domain that binds to phosphatidylinositol 3-phosphate on the endolysosomal membrane. Two other domains named PXA and PXC negatively regulate the interaction of SNX19 with endolysosomes. These studies thus identify a mechanism for controlling the motility and positioning of endolysosomes that involves tethering to the ER by a sorting nexin.

DOI: https://doi.org/10.1038/s41467-021-24709-1
Methods Cell Biol. 2021 ;pii: S0091-679X(21)00020-0. [Epub ahead of print]165 199-208

Simultaneous determination of intraluminal lysosomal calcium and pH by dextran-conjugated fluorescent dyes.

Philippe Pihán, Paula Nunes-Hasler, Nicolas Demaurex, Claudio Hetz.

  The lysosome is the main catabolic organelle in the cell, also serving as a signaling platform. Lysosomes maintain a low intraluminal pH where dozens of hydrolytic enzymes degrade a wide variety of macromolecules. Besides degradation of polymers, the lysosome is involved in various cellular processes, including energy metabolism, plasma membrane repair and antigen presentation. Recent work has shown that the lysosome is an important calcium store, modulating diverse cellular functions such as membrane fusion and fission, autophagy and lysosomal biogenesis. Precise measurement of free lysosomal calcium concentration has been hampered by its low luminal pH, since the affinity of most calcium probes decreases with higher proton concentration. Here we detailed an adapted protocol for the simultaneous measurement of lysosomal pH and calcium using dextran-conjugated ratiometric fluorescent dyes. As compared with indirect measurements of lysosomal calcium release using genetically-encoded calcium indicators (GECIs), the present method offers the possibility of obtaining pH-corrected, intraluminal calcium concentrations at single lysosome resolution. It also enables simultaneous temporal resolution of lysosomal calcium and pH.

Keywords:  Confocal microscopy; Lysosomal calcium; Lysosomal signaling; Ratiometric fluorescent probes

DOI:  https://doi.org/10.1016/bs.mcb.2021.02.007
Methods Cell Biol. 2021 ;pii: S0091-679X(20)30187-4. [Epub ahead of print]165 1-12

A novel tool for detecting lysosomal membrane permeabilization by high-throughput fluorescence microscopy.

Karla Alvarez-Valadez, Allan Sauvat, Hélène Fohrer-Ting, Christophe Klein, Oliver Kepp, Guido Kroemer, Mojgan Djavaheri-Mergny.

  Lysosomes are placed at the center of cellular trafficking and degradative pathways. They also function as a signaling platform for nutrient sensing and metabolic reprogramming. Lysosomes play crucial roles in cellular adaptation in response to stress and are tightly connected to a variety of cell death modalities. Several stimuli can initiate the permeabilization of the lysosome membrane, thus causing cell death when the cellular adaptive system fail to repair or replace damaged lysosomes. The induction of lysosomal membrane permeabilization (LMP) triggers the rapid translocation of Galectin 3/LGALS3 from the cytosol to the lysosomal lumen, making it a valuable marker of LMP. However, Galectin 3 can also be recruited to damaged endo/phagosomal membranes. To make sure that Galectin 3 labels damaged lysosomes, it is therefore important to verify its colocalization with lysosomal markers such as lysosome-associated membrane protein 1 (LAMP1). Here, we describe a simple, fast and robust protocol that allows the detection of LMP of individual lysosomes in U2OS cells expressing mCherry-tagged Galectin 3 and mGFP-tagged LAMP1. This method permits the high-throughput detection and quantification of damaged lysosomes by fluorescence microscopy. It also offers the advantage of studying, in the same experiment, the alterations in size, shape and subcellular localization of intact and damaged lysosomes.

Keywords:  Autophagy; Cell death; Galectin 3; High-throughput fluorescence microscopy; LAMP1; Lysosomal membrane permeabilization; Lysosomes; Stress responses

DOI:  https://doi.org/10.1016/bs.mcb.2020.10.004
Biochem Biophys Res Commun. 2021 Jul 26. pii: S0006-291X(21)01119-0. [Epub ahead of print]571 159-166

Leucyl-tRNA synthetase 1 is required for proliferation of TSC-null cells.

Ji-Hyun Bae, Jong Hyun Kim.

  Uncontrolled cell proliferation associated with cancer depends on the functional abrogation of at least one of tumor suppressor. In response to nutrient cue, tuberous sclerosis complex (TSC) works as a tumor suppressor which inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). However, the regulation mechanism of nutrient-dependent cell proliferation in TSC-null cells remains unclear. Here, we demonstrate that leucine is required for cell proliferation through the activation of leucyl-tRNA synthetase (LARS1)-mTORC1 pathway in TSC-null cells. Cell proliferation and survival were attenuated by LARS1 knock-down or inhibitors in TSC-null cells. In addition, either rapamycin or LARS1 inhibitors significantly decreased colony formation ability while their combined treatment drastically attenuated it. Taken together, we suggest that LARS1 inhibitors might considered as novel tools for the regression of tumor growth and proliferation in TSC-null tumor cells which regrow upon discontinuation of the mTORC1 inhibition.

Keywords:  Amino acids; Cell proliferation; LARS1; TSC; mTORC1

DOI:  https://doi.org/10.1016/j.bbrc.2021.07.080
Biochim Biophys Acta Mol Cell Res. 2021 Jul 27. pii: S0167-4889(21)00167-1. [Epub ahead of print] 119113

Competitive binding of extracellular accumulated heparan sulfate reduces lysosomal storage defects and triggers neuronal differentiation in a model of Mucopolysaccharidosis IIIB.

Valeria De Pasquale, Gianluca Scerra, Melania Scarcella, Massimo D'Agostino, Luigi Michele Pavone.

  Mucopolysaccharidoses (MPSs) are a group of inherited lysosomal storage disorders associated with the deficiency of lysosomal enzymes involved in glycosaminoglycan (GAG) degradation. The resulting cellular accumulation of GAGs is responsible for widespread tissue and organ dysfunctions. The MPS III, caused by mutations in the genes responsible for the degradation of heparan sulfate (HS), includes four subtypes (A, B, C, and D) that present significant neurological manifestations such as progressive cognitive decline and behavioral disorders. The established treatments for the MPS III do not cure the disease but only ameliorate non-neurological clinical symptoms. We previously demonstrated that the natural variant of the hepatocyte growth factor NK1 reduces the lysosomal pathology and reactivates impaired growth factor signaling in fibroblasts from MPS IIIB patients. Here, we show that the recombinant NK1 is effective in rescuing the morphological and functional dysfunctions of lysosomes in a neuronal cellular model of the MPS IIIB. More importantly, NK1 treatment is able to stimulate neuronal differentiation of neuroblastoma SK-NBE cells stable silenced for the NAGLU gene causative of the MPS IIIB. These results provide the basis for the development of a novel approach to possibly correct the neurological phenotypes of the MPS IIIB as well as of other MPSs characterized by the accumulation of HS and progressive neurodegeneration.

Keywords:  Heparan sulfate; Mucopolysaccharidosis; NAGLU; SK-NBE; neuronal differentiation

DOI:  https://doi.org/10.1016/j.bbamcr.2021.119113
J Mol Med (Berl). 2021 Jul 26.

Lipid metabolism, inflammation, and foam cell formation in health and metabolic disorders: targeting mTORC1.

So Yeong Cheon, KyoungJoo Cho.

  Metabolic homeostasis is important for maintaining a healthy lifespan. Lipid metabolism is particularly necessary for the maintenance of metabolic energy sources and their storage, and the structure and function of cell membranes, as well as for the regulation of nutrition through lipogenesis, lipolysis, and lipophagy. Dysfunctional lipid metabolism leads to the development of metabolic disorders, such as atherosclerosis, diabetes mellitus, and non-alcoholic fatty liver disease (NAFLD). Furthermore, dyslipidaemia causes inflammatory responses and foam cell formation. Mechanistic target of rapamycin (mTOR) signalling is a key regulator of diverse cellular processes, including cell metabolism and cell fate. mTOR complex 1 (mTORC1) is involved in lipid metabolism and immune responses in the body. Therefore, the mTORC1 signalling pathway has been suggested as a potential therapeutic target for the treatment of metabolic disorders. In this review, we focus on the roles of mTORC1 in lipid metabolism and inflammation, and present current evidence on its involvement in the development and progression of metabolic disorders.

Keywords:  Foam cells; Inflammation; Lipid metabolism; Mechanistic target of rapamycin (mTOR); Metabolic disorders

DOI:  https://doi.org/10.1007/s00109-021-02117-8
Mol Metab. 2021 Jul 22. pii: S2212-8778(21)00156-3. [Epub ahead of print] 101309

Hepatic mTORC1 signaling activates ATF4 as part of its metabolic response to feeding and insulin.

Vanessa Byles, Yann Cormerais, Krystle Kalafut, Victor Barrera, James E Hughes Hallett, Shannan Ho Sui, John M Asara, Christopher M Adams, Gerta Hoxhaj, Issam Ben-Sahra, Brendan D Manning.

   OBJECTIVE: The mechanistic target of rapamycin complex 1 (mTORC1) is dynamically regulated by fasting and feeding cycles in the liver to promote protein and lipid synthesis while suppressing autophagy. However, beyond these functions, the metabolic response of the liver to feeding and insulin signaling orchestrated by mTORC1 remains poorly defined. Here, we determine whether ATF4, a stress responsive transcription factor recently found to be independently regulated by mTORC1 signaling in proliferating cells, is responsive to hepatic mTORC1 signaling to alter hepatocyte metabolism.
METHODS: ATF4 protein levels and expression of canonical gene targets were analyzed in the liver following fasting and physiological feeding in the presence or absence of the mTORC1 inhibitor rapamycin. Primary hepatocytes from wild-type or liver-specific Atf4 knockout (LAtf4KO) mice were used to characterize the effects of insulin-stimulated mTORC1-ATF4 function on hepatocyte gene expression and metabolism. Both unbiased steady-state metabolomics and stable-isotope tracing methods were employed to define mTORC1 and ATF4-dependent metabolic changes. RNA-sequencing was used to determine global changes in feeding-induced transcripts in the livers of wild-type versus LAtf4KO mice.
RESULTS: We demonstrate that ATF4 and its metabolic gene targets are stimulated by mTORC1 signaling in the liver in response to feeding and in a hepatocyte-intrinsic manner by insulin. While we demonstrate that de novo purine and pyrimidine synthesis is stimulated by insulin through mTORC1 signaling in primary hepatocytes, this regulation was independent of ATF4. Metabolomics and metabolite tracing studies revealed that insulin-mTORC1-ATF4 signaling stimulates pathways of non-essential amino acid synthesis in primary hepatocytes, including those of alanine, aspartate, methionine, and cysteine, but not serine.
CONCLUSION: The results demonstrate that ATF4 is a novel metabolic effector of mTORC1 in liver, extending the molecular consequences of feeding and insulin-induced mTORC1 signaling in this key metabolic tissue to the control of amino acid metabolism.

Keywords:  ATF4; feeding; insulin; liver; mTORC1; methionine metabolism

DOI:  https://doi.org/10.1016/j.molmet.2021.101309
Biochem Biophys Res Commun. 2021 Jul 26. pii: S0006-291X(21)01094-9. [Epub ahead of print]571 137-144

Ablation of microRNA-155 and neuroinflammation in a mouse model of CLN1-disease.

Tamal Sadhukhan, Maria B Bagh, Sriparna Sadhukhan, Abhilash P Appu, Avisek Mondal, James R Iiben, Tianwei Li, Steven L Coon, Anil B Mukherjee.

  Infantile neuronal ceroid lipofuscinosis (INCL), also known as CLN1-disease, is a devastating neurodegenerative lysosomal storage disorder (LSD), caused by inactivating mutations in the CLN1 gene. The Cln1-/- mice, which mimic INCL, manifest progressive neuroinflammation contributing to neurodegeneration. However, the underlying mechanism of neuroinflammation in INCL and in Cln1-/- mice has remained elusive. Previously, it has been reported that microRNA-155 (miR-155) regulates inflammation and miR profiling in Cln1-/- mouse brain showed that the level of miR-155 was upregulated. Thus, we sought to determine whether ablation of miR-155 in Cln1-/- mice may suppress neuroinflammation in these mice. Towards this goal, we generated Cln1-/-/miR-155-/- double-knockout mice and evaluated the inflammatory signatures in the brain. We found that the brains of double-KO mice manifest progressive neuroinflammatory changes virtually identical to those found in Cln1-/- mice. We conclude that ablation of miR-155 in Cln1-/- mice does not alter the neuroinflammatory trajectory in INCL mouse model.

Keywords:  CLN1-Disease; Infantile neuronal ceroid lipofuscinosis; Lysosomal storage disease; Neuroinflammation; Palmitoyl-protein thioesterases-1

DOI:  https://doi.org/10.1016/j.bbrc.2021.07.057
Nat Commun. 2021 07 27. 12(1): 4540

Gαq activation modulates autophagy by promoting mTORC1 signaling.

Sofía Cabezudo, Maria Sanz-Flores, Alvaro Caballero, Inmaculada Tasset, Elena Rebollo, Antonio Diaz, Anna M Aragay, Ana María Cuervo, Federico Mayor, Catalina Ribas.

The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. Cells lacking Gαq/11 display higher basal autophagy, enhanced autophagy induction upon different types of nutrient stress along with a decreased mTORC1 activation status. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Conversely, stimulation of Gαq/11 promotes sustained mTORC1 pathway activation and reversion of autophagy promoted by serum or amino acids removal. Gαq is present in autophagic compartments and lysosomes and is part of the mTORC1 multi-molecular complex, contributing to its assembly and activation via its nutrient status-sensitive interaction with p62, which displays features of a Gαq effector. Gαq emerges as a central regulator of the autophagy machinery required to maintain cellular homeostasis upon nutrient fluctuations.

DOI: https://doi.org/10.1038/s41467-021-24811-4
Proc Natl Acad Sci U S A. 2021 Aug 03. pii: e2103425118. [Epub ahead of print]118(31):

Wild-type GBA1 increases the α-synuclein tetramer-monomer ratio, reduces lipid-rich aggregates, and attenuates motor and cognitive deficits in mice.

Kelly E Glajch, Tim E Moors, Yi Chen, Pascal A Bechade, Alice Y Nam, Molly M Rajsombath, Thomas D McCaffery, Ulf Dettmer, Andreas Weihofen, Warren D Hirst, Dennis J Selkoe, Silke Nuber.

  Loss-of-function mutations in acid beta-glucosidase 1 (GBA1) are among the strongest genetic risk factors for Lewy body disorders such as Parkinson's disease (PD) and Lewy body dementia (DLB). Altered lipid metabolism in PD patient-derived neurons, carrying either GBA1 or PD αS mutations, can shift the physiological α-synuclein (αS) tetramer-monomer (T:M) equilibrium toward aggregation-prone monomers. A resultant increase in pSer129+ αS monomers provides a likely building block for αS aggregates. 3K αS mice, representing a neuropathological amplification of the E46K PD-causing mutation, have decreased αS T:M ratios and vesicle-rich αS+ aggregates in neurons, accompanied by a striking PD-like motor syndrome. We asked whether enhancing glucocerebrosidase (GCase) expression could benefit αS dyshomeostasis by delivering an adeno-associated virus (AAV)-human wild-type (wt) GBA1 vector into the brains of 3K neonates. Intracerebroventricular AAV-wtGBA1 at postnatal day 1 resulted in prominent forebrain neuronal GCase expression, sustained through 6 mo. GBA1 attenuated behavioral deficits both in working memory and fine motor performance tasks. Furthermore, wtGBA1 increased αS solubility and the T:M ratio in both 3K-GBA mice and control littermates and reduced pS129+ and lipid-rich aggregates in 3K-GBA. We observed GCase distribution in more finely dispersed lysosomes, in which there was increased GCase activity, lysosomal cathepsin D and B maturation, decreased perilipin-stabilized lipid droplets, and a normalized TFEB translocation to the nucleus, all indicative of improved lysosomal function and lipid turnover. Therefore, a prolonged increase of the αS T:M ratio by elevating GCase activity reduced the lipid- and vesicle-rich aggregates and ameliorated PD-like phenotypes in mice, further supporting lipid modulating therapies in PD.

Keywords:  GBA; cathepsin; glucosylcerebrosidase; tetramer; α-synuclein

DOI:  https://doi.org/10.1073/pnas.2103425118
Invest Ophthalmol Vis Sci. 2021 Jul 01. 62(9): 39

Formation of Lipofuscin-Like Autofluorescent Granules in the Retinal Pigment Epithelium Requires Lysosome Dysfunction.

Cristina Escrevente, Ana S Falcão, Michael J Hall, Mafalda Lopes-da-Silva, Pedro Antas, Miguel M Mesquita, Inês S Ferreira, M Helena Cardoso, Daniela Oliveira, Ana C Fradinho, Thomas Ciossek, Paul Nicklin, Clare E Futter, Sandra Tenreiro, Miguel C Seabra.

Purpose: We aim to characterize the pathways required for autofluorescent granule (AFG) formation by RPE cells using cultured monolayers.
Methods: We fed RPE monolayers in culture with a single pulse of photoreceptor outer segments (POS). After 24 hours the cells started accumulating AFGs that were comparable to lipofuscin in vivo. Using this model, we used a variety of light and electron microscopical techniques, flow cytometry and Western blot to analyze the formation of AFGs. We also generated a mutant RPE line lacking cathepsin D by gene editing.
Results: AFGs seem to derive from incompletely digested POS-containing phagosomes and after 3 days are surrounded by a single membrane positive for lysosome markers. We show by various methods that lysosome-phagosome fusion is required for AFG formation, and that impairment of lysosomal pH or catalytic activity, particularly cathepsin D activity, enhances AF accumulation.
Conclusions: We conclude that lysosomal dysfunction results in incomplete POS degradation and enhanced AFG accumulation.

DOI: https://doi.org/10.1167/iovs.62.9.39
Neurosci Bull. 2021 Jul 26.

ubtor Mutation Causes Motor Hyperactivity by Activating mTOR Signaling in Zebrafish.

Tiantian Wang, Mingshan Zhou, Quan Zhang, Cuizhen Zhang, Gang Peng.

  Mechanistic target of rapamycin (mTOR) signaling governs important physiological and pathological processes key to cellular life. Loss of mTOR negative regulators and subsequent over-activation of mTOR signaling are major causes underlying epileptic encephalopathy. Our previous studies showed that UBTOR/KIAA1024/MINAR1 acts as a negative regulator of mTOR signaling, but whether UBTOR plays a role in neurological diseases remains largely unknown. We therefore examined a zebrafish model and found that ubtor disruption caused increased spontaneous embryonic movement and neuronal activity in spinal interneurons, as well as the expected hyperactivation of mTOR signaling in early zebrafish embryos. In addition, mutant ubtor larvae showed increased sensitivity to the convulsant pentylenetetrazol, and both the motor activity and the neuronal activity were up-regulated. These phenotypic abnormalities in zebrafish embryos and larvae were rescued by treatment with the mTORC1 inhibitor rapamycin. Taken together, our findings show that ubtor regulates motor hyperactivity and epilepsy-like behaviors by elevating neuronal activity and activating mTOR signaling.

Keywords:  Epilepsy; Hyperactivity; Ubtor; Zebrafish; mTOR

DOI:  https://doi.org/10.1007/s12264-021-00755-z
Autophagy. 2021 Jul 27. 1-19

LRRK2 is required for CD38-mediated NAADP-Ca2+ signaling and the downstream activation of TFEB (transcription factor EB) in immune cells.

Neel R Nabar, Christopher N Heijjer, Chong-Shan Shi, Il-Young Hwang, Sundar Ganesan, Mikael C I Karlsson, John H Kehrl.

  CD38 is a cell surface receptor capable of generating calcium-mobilizing second messengers. It has been implicated in host defense and cancer biology, but signaling mechanisms downstream of CD38 remain unclear. Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most common genetic cause of Parkinson disease; it is also a risk factor for Crohn disease, leprosy, and certain types of cancers. The pathogenesis of these diseases involves inflammation and macroautophagy/autophagy, processes both CD38 and LRRK2 are implicated in. Here, we mechanistically and functionally link CD38 and LRRK2 as upstream activators of TFEB (transcription factor EB), a host defense transcription factor and the master transcriptional regulator of the autophagy/lysosome machinery. In B-lymphocytes and macrophages, we show that CD38 and LRRK2 exist in a complex on the plasma membrane. Ligation of CD38 with the monoclonal antibody clone 90 results in internalization of the CD38-LRRK2 complex and its targeting to the endolysosomal system. This generates an NAADP-dependent calcium signal, which requires LRRK2 kinase activity, and results in the downstream activation of TFEB. lrrk2 KO macrophages accordingly have TFEB activation defects following CD38 or LPS stimulation and fail to switch to glycolytic metabolism after LPS treatment. In overexpression models, the pathogenic LRRK2G2019S mutant promotes hyperactivation of TFEB even in the absence of CD38, both by stabilizing TFEB and promoting its nuclear translocation via aberrant calcium signaling. In sum, we have identified a physiological CD38-LRRK2-TFEB signaling axis in immune cells. The common pathogenic mutant, LRRK2G2019S, appears to hijack this pathway.

Keywords:  Autophagy; B cell; LRRK2; TFEB; calcium; endocytosis; immunometabolism; innate immunity; lysosome; macrophage

DOI:  https://doi.org/10.1080/15548627.2021.1954779
Autophagy. 2021 Jul 27. 1-19

Lysosomal TPCN (two pore segment channel) inhibition ameliorates beta-amyloid pathology and mitigates memory impairment in Alzheimer disease.

Benjamin Chun-Kit Tong, Aston Jiaxi Wu, Alexis Shiying Huang, Rui Dong, Sandeep Malampati, Ashok Iyaswamy, Senthilkumar Krishnamoorthi, Sravan Gopalkrishnashetty Sreenivasmurthy, Zhou Zhu, Chengfu Su, Jia Liu, Juxian Song, Jia-Hong Lu, Jieqiong Tan, Weidong Pan, Min Li, King-Ho Cheung.

   ABBREVIATIONS: Aβ: β-amyloid; AD: Alzheimer disease; AIF1/IBA1: allograft inflammatory factor 1; ALP: autophagy-lysosomal pathway; APP: amyloid beta precursor protein; ATP6V1B1/V-ATPase V1b1: ATPase H+ transporting V1 subunit B1; AVs: autophagy vacuoles; BAF: bafilomycin A1; CFC: contextual/cued fear conditioning assay; CHX: Ca2+/H+ exchanger; CTF-β: carboxy-terminal fragment derived from β-secretase; CTSD: cathepsin D; fAD: familial Alzheimer disease; GFAP: glial fibrillary acidic protein; LAMP1: lysosomal associated membrane protein 1; LTP: long-term potentiation; MCOLN1/TRPML1: mucolipin 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPT: microtubule associated protein tau; MWM: Morris water maze; NFT: neurofibrillary tangles; PFC: prefrontal cortex; PSEN1: presenilin 1; SQSTM1/p62: sequestosome 1; TBS: theta burst stimulation; TEM: transmission electronic microscopy; TPCN2/TPC2: two pore segment channel 2; WT: wild-type; V-ATPase: vacuolar type H+-ATPase.

Keywords:  Alzheimer disease; autophagy-lysosomal pathway; calcium ion; presenilin-1; two pore segment channel 2

DOI:  https://doi.org/10.1080/15548627.2021.1945220
Curr Gene Ther. 2021 Jul 28.

Clinical Development of Cell Therapies to Halt Lysosomal Storage Diseases: Results and Lessons Learned.

Valeria Graceffa.

  Although cross-correction was discovered more than 50 years ago, and held the promise of drastically improving disease management, still no cure exists for lysosomal storage diseases (LSDs). Cell therapies hold the potential to halt disease progression: either a subset of autologous cells can be ex vivo/ in vivo transfected with the functional gene or allogenic wild type stem cells can be transplanted. However, majority of cell-based attempts have been ineffective, due to the difficulties in reversing neuronal symptomatology, in finding appropriate gene transfection approaches, in inducing immune tolerance, reducing the risk of graft versus host disease (GVHD) when allogenic cells are used and that of immune response when engineered viruses are administered, coupled with a limited secretion and uptake of some enzymes. In the last decade, due to advances in our understanding of lysosomal biology and mechanisms of cross-correction, coupled with progresses in gene therapy, ongoing pre-clinical and clinical investigations have remarkably increased. Even gene editing approaches are currently under clinical experimentation. This review proposes to critically discuss and compare trends and advances in cell-based and gene therapy for LSDs. Systemic gene delivery and transplantation of allogenic stem cells will be initially discussed, whereas proposed brain targeting methods will be then critically outlined.

Keywords:  GVHD.; cell therapies; cross-correction; gene therapy; lysosomal enzymes; lysosomal storage diseases

DOI:  https://doi.org/10.2174/1566523221666210728141924
Front Physiol. 2021 ;12 691245

Indices of Defective Autophagy in Whole Muscle and Lysosome Enriched Fractions From Aged D2-mdx Mice.

Swathy Krishna, Hannah R Spaulding, Tiffany S Quindry, Matthew B Hudson, John C Quindry, Joshua T Selsby.

  Duchenne muscular dystrophy (DMD) is a fatal, progressive muscle disease caused by the absence of functional dystrophin protein. Previous studies in mdx mice, a common DMD model, identified impaired autophagy with lysosomal insufficiency and impaired autophagosomal degradation as consequences of dystrophin deficiency. Thus, we hypothesized that lysosomal abundance would be decreased and degradation of autophagosomes would be impaired in muscles of D2-mdx mice. To test this hypothesis, diaphragm and gastrocnemius muscles from 11 month-old D2-mdx and DBA/2J (healthy) mice were collected. Whole muscle protein from diaphragm and gastrocnemius muscles, and protein from a cytosolic fraction (CF) and a lysosome-enriched fraction (LEF) from gastrocnemius muscles, were isolated and used for western blotting. Initiation of autophagy was not robustly activated in whole muscle protein from diaphragm and gastrocnemius, however, autophagosome formation markers were elevated in dystrophic muscles. Autophagosome degradation was impaired in D2-mdx diaphragms but appeared to be maintained in gastrocnemius muscles. To better understand this muscle-specific distinction, we investigated autophagic signaling in CFs and LEFs from gastrocnemius muscles. Within the LEF we discovered that the degradation of autophagosomes was similar between groups. Further, our data suggest an expanded, though impaired, lysosomal pool in dystrophic muscle. Notably, these data indicate a degree of muscle specificity as well as model specificity with regard to autophagic dysfunction in dystrophic muscles. Stimulation of autophagy in dystrophic muscles may hold promise for DMD patients as a potential therapeutic, however, it will be critical to choose the appropriate model and muscles that most closely recapitulate findings from human patients to further develop these therapeutics.

Keywords:  D2-mdx; Duchenne muscular dystrophy; diaphragm; dystrophin; gastrocnemius; mouse model

DOI:  https://doi.org/10.3389/fphys.2021.691245
FASEB J. 2021 Aug;35(8): e21818

Platelet and myeloid cell phenotypes in a rat model of Fabry disease.

Adam J Kanack, Kazuhiro Aoki, Michael Tiemeyer, Nancy M Dahms.

  Fabry disease results from a deficiency of the lysosomal enzyme ⍺-Galactosidase-A (⍺-Gal A) and is estimated to occur in approximately 1:4100 live births. Characteristic of the disease is the accumulation of α-Gal-A substrates, primarily the glycosphingolipids (GSLs) globotriaosylceramide and globotriaosylsphingosine. Thrombotic events are a significant concern for Fabry patients, with strokes contributing to a significant decrease in overall lifespan. Currently, the mechanisms underlying the increased risk of thrombotic events experienced by Fabry patients are incompletely defined. Using a rat model of Fabry disease, we provide an improved understanding of the mechanisms linking GSL accumulation to thrombotic risk. We found that ⍺-Gal A-deficient rats accumulate myeloid-derived leukocytes at sites of GSL accumulation, including in the bone marrow and circulation, and that myeloid-derived leukocyte and megakaryocyte populations were prominent among cell types that accumulated GSLs. In the circulation, ⍺-Gal A-deficient rats had increases in cytokine-producing cell types and a corresponding elevation of pro-inflammatory cytokines. Lastly, circulating platelets from ⍺-Gal A-deficient rats accumulated a similar set of ⍺-Galactosidase-A substrates as was observed in megakaryocytes in the bone marrow, and exhibited increased platelet binding to fibrinogen in microfluidic and flow cytometric assays.

Keywords:  animal model; glycobiology; glycosphingolipids; lysosomal storage disease

DOI:  https://doi.org/10.1096/fj.202001727RR
J Enzyme Inhib Med Chem. 2021 Dec;36(1): 1679-1693

The role of mTOR in age-related diseases.

Zofia Chrienova, Eugenie Nepovimova, Kamil Kuca.

  The ageing population is becoming a significant socio-economic issue. To address the expanding health gap, it is important to deepen our understanding of the mechanisms underlying ageing in various organisms at the single-cell level. The discovery of the antifungal, immunosuppressive, and anticancer drug rapamycin, which possesses the ability to extend the lifespan of several species, has prompted extensive research in the areas of cell metabolic regulation, development, and senescence. At the centre of this research is the mTOR pathway, with key roles in cell growth, proteosynthesis, ribosomal biogenesis, transcriptional regulation, glucose and lipid metabolism, and autophagy. Recently, it has become obvious that mTOR dysregulation is involved in several age-related diseases, such as cancer, neurodegenerative diseases, and type 2 diabetes mellitus. Additionally, mTOR hyperactivation affects the process of ageing per se. In this review, we provide an overview of recent insights into the mTOR signalling pathway, including its regulation and its influence on various hallmarks of ageing at the cellular level.

Keywords:  Ageing; age-related disease; mTORC1; mTORC2; rapamycin

DOI:  https://doi.org/10.1080/14756366.2021.1955873
Proc Natl Acad Sci U S A. 2021 Aug 03. pii: e2100862118. [Epub ahead of print]118(31):

THAP1 modulates oligodendrocyte maturation by regulating ECM degradation in lysosomes.

Dhananjay Yellajoshyula, Samuel S Pappas, Abigail E Rogers, Biswa Choudhury, Xylena Reed, Jinhui Ding, Mark R Cookson, Vikram G Shakkottai, Roman J Giger, William T Dauer.

  Mechanisms controlling myelination during central nervous system (CNS) maturation play a pivotal role in the development and refinement of CNS circuits. The transcription factor THAP1 is essential for timing the inception of myelination during CNS maturation through a cell-autonomous role in the oligodendrocyte lineage. Here, we demonstrate that THAP1 modulates the extracellular matrix (ECM) composition by regulating glycosaminoglycan (GAG) catabolism within oligodendrocyte progenitor cells (OPCs). Thap1 -/- OPCs accumulate and secrete excess GAGs, inhibiting their maturation through an autoinhibitory mechanism. THAP1 controls GAG metabolism by binding to and regulating the GusB gene encoding β-glucuronidase, a GAG-catabolic lysosomal enzyme. Applying GAG-degrading enzymes or overexpressing β-glucuronidase rescues Thap1 -/- OL maturation deficits in vitro and in vivo. Our studies establish lysosomal GAG catabolism within OPCs as a critical mechanism regulating oligodendrocyte development.

Keywords:  CNS myelination; extracellular matrix; neurodevelopmental disease

DOI:  https://doi.org/10.1073/pnas.2100862118
Nat Commun. 2021 07 27. 12(1): 4407

Brain-specific inhibition of mTORC1 eliminates side effects resulting from mTORC1 blockade in the periphery and reduces alcohol intake in mice.

Yann Ehinger, Ziyang Zhang, Khanhky Phamluong, Drishti Soneja, Kevan M Shokat, Dorit Ron.

Alcohol Use Disorder (AUD) affects a large portion of the population. Unfortunately, efficacious medications to treat the disease are limited. Studies in rodents suggest that mTORC1 plays a crucial role in mechanisms underlying phenotypes such as heavy alcohol intake, habit, and relapse. Thus, mTORC1 inhibitors, which are used in the clinic, are promising therapeutic agents to treat AUD. However, chronic inhibition of mTORC1 in the periphery produces undesirable side effects, which limit their potential use for the treatment of AUD. To overcome these limitations, we designed a binary drug strategy in which male mice were treated with the mTORC1 inhibitor RapaLink-1 together with a small molecule (RapaBlock) to protect mTORC1 activity in the periphery. We show that whereas RapaLink-1 administration blocked mTORC1 activation in the liver, RapaBlock abolished the inhibitory action of Rapalink-1. RapaBlock also prevented the adverse side effects produced by chronic inhibition of mTORC1. Importantly, co-administration of RapaLink-1 and RapaBlock inhibited alcohol-dependent mTORC1 activation in the nucleus accumbens and attenuated alcohol seeking and drinking.

DOI: https://doi.org/10.1038/s41467-021-24567-x
Nat Commun. 2021 Jul 30. 12(1): 4634

Endo-lysosomal Aβ concentration and pH trigger formation of Aβ oligomers that potently induce Tau missorting.

Marie P Schützmann, Filip Hasecke, Sarah Bachmann, Mara Zielinski, Sebastian Hänsch, Gunnar F Schröder, Hans Zempel, Wolfgang Hoyer.

Amyloid-β peptide (Aβ) forms metastable oligomers >50 kDa, termed AβOs, that are more effective than Aβ amyloid fibrils at triggering Alzheimer's disease-related processes such as synaptic dysfunction and Tau pathology, including Tau mislocalization. In neurons, Aβ accumulates in endo-lysosomal vesicles at low pH. Here, we show that the rate of AβO assembly is accelerated 8,000-fold upon pH reduction from extracellular to endo-lysosomal pH, at the expense of amyloid fibril formation. The pH-induced promotion of AβO formation and the high endo-lysosomal Aβ concentration together enable extensive AβO formation of Aβ42 under physiological conditions. Exploiting the enhanced AβO formation of the dimeric Aβ variant dimAβ we furthermore demonstrate targeting of AβOs to dendritic spines, potent induction of Tau missorting, a key factor in tauopathies, and impaired neuronal activity. The results suggest that the endosomal/lysosomal system is a major site for the assembly of pathomechanistically relevant AβOs.

DOI: https://doi.org/10.1038/s41467-021-24900-4
Front Cell Dev Biol. 2021 ;9 687856

Mechanical Overloading Induced-Activation of mTOR Signaling in Tendon Stem/Progenitor Cells Contributes to Tendinopathy Development.

Daibang Nie, Yiqin Zhou, Wang Wang, Jianying Zhang, James H-C Wang.

  Despite the importance of mechanical loading in tendon homeostasis and pathophysiology, the molecular responses involved in the mechanotransduction in tendon cells remain unclear. In this study, we found that in vitro mechanical loading activated the mammalian target of rapamycin (mTOR) in rat patellar tendon stem/progenitor cells (TSCs) in a stretching magnitude-dependent manner. Application of rapamycin, a specific inhibitor of mTOR, attenuated the phosphorylation of S6 and 4E-BP1 and as such, largely inhibited the mechanical activation of mTOR. Moreover, rapamycin significantly decreased the proliferation and non-tenocyte differentiation of PTSCs as indicated by the reduced expression levels of LPL, PPARγ, SOX-9, collagen II, Runx-2, and osteocalcin genes. In the animal studies, mice subjected to intensive treadmill running (ITR) developed tendon degeneration, as evidenced by the formation of round-shaped cells, accumulation of proteoglycans, and expression of SOX-9 and collagen II proteins. However, daily injections of rapamycin in ITR mice reduced all these tendon degenerative changes. Collectively, these findings suggest that mechanical loading activates the mTOR signaling in TSCs, and rapamycin may be used to prevent tendinopathy development by blocking non-tenocyte differentiation due to mechanical over-activation of mTOR in TSCs.

Keywords:  mTOR; mechanical loading; rapamycin; tendon stem cells; treadmill running

DOI:  https://doi.org/10.3389/fcell.2021.687856
EMBO J. 2021 Jul 26. e107336

Host autophagy mediates organ wasting and nutrient mobilization for tumor growth.

Rojyar Khezri, Petter Holland, Todd Andrew Schoborg, Ifat Abramovich, Szabolcs Takáts, Caroline Dillard, Ashish Jain, Fergal O'Farrell, Sebastian Wolfgang Schultz, William M Hagopian, Eduardo Martin Quintana, Rachel Ng, Nadja Sandra Katheder, Mohammed Mahidur Rahman, José Gerardo Teles Reis, Andreas Brech, Heinrich Jasper, Nasser M Rusan, Anne Hope Jahren, Eyal Gottlieb, Tor Erik Rusten.

  During tumor growth-when nutrient and anabolic demands are high-autophagy supports tumor metabolism and growth through lysosomal organelle turnover and nutrient recycling. Ras-driven tumors additionally invoke non-autonomous autophagy in the microenvironment to support tumor growth, in part through transfer of amino acids. Here we uncover a third critical role of autophagy in mediating systemic organ wasting and nutrient mobilization for tumor growth using a well-characterized malignant tumor model in Drosophila melanogaster. Micro-computed X-ray tomography and metabolic profiling reveal that RasV12 ; scrib-/- tumors grow 10-fold in volume, while systemic organ wasting unfolds with progressive muscle atrophy, loss of body mass, -motility, -feeding, and eventually death. Tissue wasting is found to be mediated by autophagy and results in host mobilization of amino acids and sugars into circulation. Natural abundance Carbon 13 tracing demonstrates that tumor biomass is increasingly derived from host tissues as a nutrient source as wasting progresses. We conclude that host autophagy mediates organ wasting and nutrient mobilization that is utilized for tumor growth.

Keywords:   Drosophila ; autophagy; cancer cachexia; muscle; tumor; wasting

DOI:  https://doi.org/10.15252/embj.2020107336
Cancer Lett. 2021 Jul 25. pii: S0304-3835(21)00356-6. [Epub ahead of print]

DEPTOR inhibits lung tumorigenesis by inactivating the EGFR-mTOR signals.

Longyuan Gong, Jianfeng Shu, Xiaoyu Chen, Hui Pan, Guoan Chen, Yanli Bi, Danrui Cui, Xufan Li, Dian Liu, Linchen Wang, Yue Wang, Pengyuan Liu, Xiufang Xiong, Yongchao Zhao.

  DEPTOR plays vital roles in the regulation of cell proliferation and survival by directly modulating the activity of mTORC1/2. However, the physiological role of DEPTOR in lung tumorigenesis, as well as its clinical significance, remains elusive. In this study, we revealed that decreased DEPTOR expression correlated with increased tumor size, poor differentiation, and worse survival in patients with lung cancer. DEPTOR depletion promoted cell proliferation, survival, migration, and invasion in human lung cancer cells. Mechanistically, DEPTOR bound to the kinase domain of EGFR via its PDZ domain to inactivate EGFR signal. Thus, DEPTOR depletion not only directly activated mTORC1/2, but also relieved the inhibition of EGFR to subsequently activate mTOR signals, leading to the induction of cell proliferation and survival. Additionally, activated EGFR-mTOR signals upregulated the expression of ZEB1 and SLUG to induce epithelial-mesenchymal transition, resulting in enhanced migration and invasion. Importantly, Deptor deletion accelerated KrasG12D;p53fl/fl-induced lung tumorigenesis and shortened mouse life span via the activation of EGFR-mTOR signals. Collectively, our study demonstrated that DEPTOR acts as a tumor suppressor in lung tumorigenesis, and its reduction may advance the progression of human lung cancer.

Keywords:  DEPTOR; EGFR; SLUG; ZEB1; mTOR

DOI:  https://doi.org/10.1016/j.canlet.2021.07.031
Blood. 2021 Jul 27. pii: blood.2020009778. [Epub ahead of print]

Asymmetric organelle inheritance predicts human blood stem cell fate.

Dirk Loeffler, Florin Schneiter, Weijia Wang, Arne Wehling, Tobias Kull, Claudia Lengerke, Markus G Manz, Timm Schroeder.

Understanding human hematopoietic stem cell fate control is important for their improved therapeutic manipulation. Asymmetric cell division, the asymmetric inheritance of factors during division instructing future daughter cell fates, was recently described in mouse blood stem cells. In human blood stem cells, the possible existence of asymmetric cell division remained unclear due to technical challenges in its direct observation. Here, we use long-term quantitative single-cell imaging to show that lysosomes and active mitochondria are asymmetrically inherited in human blood stem cells and that their inheritance is a coordinated, non-random process. Furthermore, multiple additional organelles, including autophagosomes, mitophagosomes, autolysosomes and recycling endosomes show preferential asymmetric co-segregation with lysosomes. Importantly, asymmetric lysosomal inheritance predicts future asymmetric daughter cell cycle length, differentiation and stem cell marker expression, while asymmetric inheritance of active mitochondria correlates with daughter metabolic activity. Hence, human hematopoietic stem cell fates are regulated by asymmetric cell division, with both mechanistic evolutionary conservation and differences to the mouse system.

DOI: https://doi.org/10.1182/blood.2020009778
Oncogene. 2021 Jul 24.

Targeting mTOR signaling overcomes acquired resistance to combined BRAF and MEK inhibition in BRAF-mutant melanoma.

Beike Wang, Wei Zhang, Gao Zhang, Lawrence Kwong, Hezhe Lu, Jiufeng Tan, Norah Sadek, Min Xiao, Jie Zhang, Marilyne Labrie, Sergio Randell, Aurelie Beroard, Eric Sugarman, Vito W Rebecca, Zhi Wei, Yiling Lu, Gordon B Mills, Jeffrey Field, Jessie Villanueva, Xiaowei Xu, Meenhard Herlyn, Wei Guo.

Targeting MAPK pathway using a combination of BRAF and MEK inhibitors is an efficient strategy to treat melanoma harboring BRAF-mutation. The development of acquired resistance is inevitable due to the signaling pathway rewiring. Combining western blotting, immunohistochemistry, and reverse phase protein array (RPPA), we aim to understanding the role of the mTORC1 signaling pathway, a center node of intracellular signaling network, in mediating drug resistance of BRAF-mutant melanoma to the combination of BRAF inhibitor (BRAFi) and MEK inhibitor (MEKi) therapy. The mTORC1 signaling pathway is initially suppressed by BRAFi and MEKi combination in melanoma but rebounds overtime after tumors acquire resistance to the combination therapy (CR) as assayed in cultured cells and PDX models. In vitro experiments showed that a subset of CR melanoma cells was sensitive to mTORC1 inhibition. The mTOR inhibitors, rapamycin and NVP-BEZ235, induced cell cycle arrest and apoptosis in CR cell lines. As a proof-of-principle, we demonstrated that rapamycin and NVP-BEZ235 treatment reduced tumor growth in CR xenograft models. Mechanistically, AKT or ERK contributes to the activation of mTORC1 in CR cells, depending on PTEN status of these cells. Our study reveals that mTOR activation is essential for drug resistance of melanoma to MAPK inhibitors, and provides insight into the rewiring of the signaling networks in CR melanoma.

DOI: https://doi.org/10.1038/s41388-021-01911-5
ASN Neuro. 2021 Jan-Dec;13:13 17590914211028364

Mitochondrial and Organellar Crosstalk in Parkinson's Disease.

Bipul Ray, Abid Bhat, Arehally Marappa Mahalakshmi, Sunanda Tuladhar, Muhammed Bishir, Surapaneni Krishna Mohan, Vishnu Priya Veeraraghavan, Ramesh Chandra, Musthafa Mohamed Essa, Saravana Babu Chidambaram, Meena Kishore Sakharkar.

  Mitochondrial dysfunction is a well-established pathological event in Parkinson's disease (PD). Proteins misfolding and its impaired cellular clearance due to altered autophagy/mitophagy/pexophagy contribute to PD progression. It has been shown that mitochondria have contact sites with endoplasmic reticulum (ER), peroxisomes and lysosomes that are involved in regulating various physiological processes. In pathological conditions, the crosstalk at the contact sites initiates alterations in intracellular vesicular transport, calcium homeostasis and causes activation of proteases, protein misfolding and impairment of autophagy. Apart from the well-reported molecular changes like mitochondrial dysfunction, impaired autophagy/mitophagy and oxidative stress in PD, here we have summarized the recent scientific reports to provide the mechanistic insights on the altered communications between ER, peroxisomes, and lysosomes at mitochondrial contact sites. Furthermore, the manuscript elaborates on the contributions of mitochondrial contact sites and organelles dysfunction to the pathogenesis of PD and suggests potential therapeutic targets.

Keywords:  Parkinson’s disease; endoplasmic reticulum; lysosome; mitochondria; mitochondrial contact sites; peroxisome

DOI:  https://doi.org/10.1177/17590914211028364
J Biochem. 2021 Jul 26. pii: mvab028. [Epub ahead of print]

Protein phosphatase 6 promotes neurite outgrowth by promoting mTORC2 activity in N2a cells.

Nao Kitamura, Nobuyuki Fujiwara, Koji Hayakawa, Takashi Ohama, Koichi Sato.

  Understanding the molecular mechanism of neuronal differentiation is important to overcome the incurable diseases caused by nervous system damage. Neurite outgrowth is prerequisite for neuronal differentiation and regeneration, and cAMP response element-binding protein (CREB) is one of the major transcriptional factors positively regulating this process. Neuronal differentiation stimuli activate mammalian target of rapamycin (mTOR) complex 2 (mTORC2)/Akt signaling to phosphorylate CREB, however, the precise molecular mechanism of this event has not been fully understood. In this manuscript, we show that neuronal differentiation stimuli increased a protein level of protein phosphatase 6 (PP6), a member of type 2A Ser/Thr protein phosphatases. PP6 knockdown suppressed mTORC2/Akt/CREB signaling and results in failure of neurite outgrowth. SIN1 is a unique component of mTORC2 that enhances mTORC2 activity toward Akt when it is in dephosphorylated form. We found PP6 knockdown increased SIN1 phosphorylation. These data suggest that PP6 may positively regulate neurite outgrowth by dephosphorylating SIN1 to activate mTORC2/Akt/CREB signaling.

Keywords:  N2a cell; Neurite outgrowth; PP6; SIN1; mTORC2

DOI:  https://doi.org/10.1093/jb/mvab028
Mol Genet Genomic Med. 2021 Jul 31. e1777

The c.863A>G (p.Glu288Gly) variant of the CTSD gene is not associated with CLN10 disease.

Juan Yang, Xiaoting Ding, Shasha Meng, Jinhua Cai, Weihui Zhou.

   BACKGROUND: Cathepsin D is a lysosomal aspartic protease encoded by the CTSD gene. It plays important roles in many biological processes. Biallelic loss-of-function mutation of CTSD is considered a cause of CLN10 disease. CLN10 is a rare autosomal recessive disorder that is one of 14 types of neuronal ceroid lipofuscinoses (NCLs). To date, only a few cases of CLN10 and 12 disease-causing mutations have been reported worldwide.
METHODS: Exome sequencing was performed on a 15-year-old girl with pervasive brain developmental disorder. The effects of the identified variants were investigated through multiple functional experiments.
RESULTS: There were no differences in mRNA and protein expression, intracellular localization, maturation, and proteolytic activity between the cells with the mutant CTSD gene and those with the wild-type CTSD gene.
CONCLUSION: These results suggest that the c.863A>G (p.Glu288Gly) homozygous variant is not a pathogenic variation, but a benign variant.

Keywords:   CTSD ; CLN10 disease; Cathepsin D; neuronal ceroid lipofuscinoses; variation

DOI:  https://doi.org/10.1002/mgg3.1777
Nature. 2021 Jul 28.

Rewiring of the ubiquitinated proteome determines ageing in C. elegans.

Seda Koyuncu, Rute Loureiro, Hyun Ju Lee, Prerana Wagle, Marcus Krueger, David Vilchez.

Ageing is driven by a loss of cellular integrity1. Given the major role of ubiquitin modifications in cell function2, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome3, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament4 and the EPS-8 modulator of RAC signalling5. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.

DOI: https://doi.org/10.1038/s41586-021-03781-z
Am J Med Genet A. 2021 Jul 30.

Low-level mosaicism in tuberous sclerosis complex in four unrelated patients: Comparison of clinical characteristics and diagnostic pathways.

Héctor Hugo Manzanilla-Romero, Denisa Weis, Simon Schnaiter, Sabine Rudnik-Schöneborn.

  Tuberous sclerosis complex (TSC) is an autosomal dominant neurocutaneous syndrome caused by either TSC1 or TSC2 gene mutations. About 15% of TSC patients remain without genetic diagnosis by conventional analysis despite clinical evidence. It is important to identify somatic mosaics, as therapeutic options are now available in patients with TSC1 or TSC2 mutations. Here, we describe the clinical and genetic characteristics of four male TSC patients with low-level mosaicism. Patients presented at ages between 9 months and 32 years. Clinical manifestations varied considerably and included brain lesions in all four patients, cardiac rhabdomyomas in two young patients, skin involvement in two patients, and retinal hamartomas and renal angiomyolipomas in three patients. One patient presented with epileptic seizures and psychomotor delay. Low levels of mosaicism for TSC1 or TSC2 mutation were found in different tissue samples employing next generation sequencing and multiple ligation-dependent probe amplification. The five disease-associated variants, including one second-hit mutation, include three truncating mutations and one deletion in TSC2, and one truncating mutation in TSC1. Sanger sequencing, allele-specific oligonucleotide PCR (ASO-PCR), and droplet digital PCR were used to confirm and quantify the disclosed mutations. Genetic identification of low-level mosaicism for TSC remains challenging but is important for optimal surveillance and management.

Keywords:  genetic diagnosis; next generation sequencing; somatic mosaicism; tuberous sclerosis complex

DOI:  https://doi.org/10.1002/ajmg.a.62433
Front Physiol. 2021 ;12 659272

Knockdown of CNN3 Impairs Myoblast Proliferation, Differentiation, and Protein Synthesis via the mTOR Pathway.

Yanling She, Cheng Li, Ting Jiang, Si Lei, Shanyao Zhou, Huacai Shi, Rui Chen.

   Background: Myogenesis is a complex process that requires optimal outside-in substrate-cell signaling. Calponin 3 (CNN3) plays an important role in regulating myogenic differentiation and muscle regeneration; however, the precise function of CNN3 in myogenesis regulation remains poorly understood. Here, we investigated the role of CNN3 in a knockdown model in the mouse muscle cell line C2C12.
Methods: Myoblast proliferation, migration, differentiation, fusion, and protein synthesis were examined in CNN3 knockdown C2C12 mouse muscle cells. Involvement of the mTOR pathway in CNN3 signaling was explored by treating cells with the mTOR activator MHY1485. The regulatory mechanisms of CNN3 in myogenesis were further examined by RNA sequencing and subsequent gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA).
Results: During proliferation, CNN3 knockdown caused a decrease in cell proliferation and migration. During differentiation, CNN3 knockdown inhibited myogenic differentiation, fusion, and protein synthesis in C2C12 cells via the AKT/mTOR and AMPK/mTOR pathways; this effect was reversed by MHY1485 treatment. Finally, KEGG and GSEA indicated that the NOD-like receptor signaling pathway is affected in CNN3 knockdown cell lines.
Conclusion: CNN3 may promote C2C12 cell growth by regulating AKT/mTOR and AMPK/mTOR signaling. The KEGG and GSEA indicated that inhibiting CNN3 may activate several pathways, including the NOD-like receptor pathway and pathways involved in necroptosis, apoptosis, and inflammation.

Keywords:  C2C12 myoblasts; CNN3; differentiation; mTOR pathway; proliferation; protein synthesis

DOI:  https://doi.org/10.3389/fphys.2021.659272