bims-lysosi 2021-10-10 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021‒10‒10
forty-one papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Tubular lysosomes harbor active ion gradients and poise macrophages for phagocytosis.
Context-specific regulation of lysosomal lipolysis through network-level diverting of transcription factor interactions.
A novel neuron-specific regulator of the V-ATPase in Drosophila.
Monomethyl branched-chain fatty acid mediates amino acid sensing upstream of mTORC1.
Mapping of mTOR drug targets: Featured platforms for anti-cancer drug discovery.
Glucocerebrosidase 1 and leucine-rich repeat kinase 2 in Parkinson disease and interplay between the two genes.
mTOR-Activating Mutations in RRAGD are Causative for Kidney Tubulopathy and Cardiomyopathy.
Microglia regulate brain Progranulin levels through the endocytosis-lysosomal pathway.
Clinical Trials for Gene Therapy in Lysosomal Diseases With CNS Involvement.
Chemoenzymatic glycan-selective remodeling of a therapeutic lysosomal enzyme with high-affinity M6P-glycan ligands. Enzyme substrate specificity is the name of the game.
Molecular markers of brain cholesterol homeostasis are unchanged despite a smaller brain mass in a mouse model of cholesteryl ester storage disease.
Iduronate-2-sulfatase transport vehicle rescues behavioral and skeletal phenotypes in a mouse model of Hunter syndrome.
LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons.
Direct targeting of wild-type glucocerebrosidase by antipsychotic quetiapine improves pathogenic phenotypes in Parkinson's disease models.
Mefloquine as a dual inhibitor of glioblastoma angiogenesis and glioblastoma via disrupting lysosomal function.
Intracellular ion changes induced by the exposure to beta-amyloid can be explained by the formation of channels in the lysosomal membranes.
Primary cells derived from Tuberous Sclerosis Complex patients show autophagy alteration in the haploinsufficiency state.
Rheb-regulated mitochondrial pyruvate metabolism of Schwann cells linked to axon stability.
A plastid two-pore channel essential for inter-organelle communication and growth of Toxoplasma gondii.
A late endosome signaling hub that couples PI3Kα and WNT/β-catenin signaling in breast cancer.
The concerted action of oncogenic driver mutations directs global translation in intestinal epithelial cells.
A Hybrid Nanogel to Preserve Lysosome Integrity for Fluorescence Imaging.
Hepatic Lysosomal Acid Lipase Overexpression Worsens Hepatic Inflammation in Mice Fed a Western Diet.
Evaluation of Strategies for Measuring Lysosomal Glucocerebrosidase Activity.
The key role of the central cavity in sodium transport through ligand-gated two-pore channels.
Muscle-specific Cand2 is translationally upregulated by mTORC1 and promotes adverse cardiac remodeling.
Dihydroartemisinin ameliorates retinal vascular dysfunction in diabetes mellitus via FASN/Kmal-mTOR/SREBP1 feedback loop.
Coordinated glucose-induced Ca2+ and pH responses in yeast Saccharomyces cerevisiae.
Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex.
Functions of the (pro)renin receptor (Atp6ap2) at molecular and system levels: pathological implications in hypertension, renal and brain development, inflammation, and fibrosis.
Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease.
NHE6-depletion corrects ApoE4-mediated synaptic impairments and reduces amyloid plaque load.
Chronic Exposure to Palmitic Acid Down-regulates AKT in Beta-Cells through Activation of mTOR.
The Effect of A2E on the Uptake and Release of Calcium in the Lysosomes and Mitochondria of Human RPE Cells Exposed to Blue Light.
Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer.
Blue Light Activated Rapamycin for Optical Control of Protein Dimerization in Cells and Zebrafish Embryos.
Whole-Exome Sequencing Identified a Novel Homozygous Frameshift Mutation of HPS3 in a Consanguineous Family with Hermansky-Pudlak Syndrome.
ATP-citrate lyase promotes axonal transport across species.
Measurement of LRRK2 Kinase Activity by Proximity Ligation Assay.
Author Correction: Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth.
Role of autophagy in muscle disease.

Proc Natl Acad Sci U S A. 2021 Oct 12. pii: e2113174118. [Epub ahead of print]118(41):

Tubular lysosomes harbor active ion gradients and poise macrophages for phagocytosis.

Bhavyashree Suresh, Anand Saminathan, Kasturi Chakraborty, Matthew Zajac, Chang Cui, Lev Becker, Yamuna Krishnan.

  Lysosomes adopt dynamic, tubular states that regulate antigen presentation, phagosome resolution, and autophagy. Tubular lysosomes are studied either by inducing autophagy or by activating immune cells, both of which lead to cell states where lysosomal gene expression differs from the resting state. Therefore, it has been challenging to pinpoint the biochemical properties lysosomes acquire upon tubulation that could drive their functionality. Here we describe a DNA-based assembly that tubulates lysosomes in macrophages without activating them. Proteolytic activity maps at single-lysosome resolution revealed that tubular lysosomes were less degradative and showed proximal to distal luminal pH and Ca2+ gradients. Such gradients had been predicted but never previously observed. We identify a role for tubular lysosomes in promoting phagocytosis and activating MMP9. The ability to tubulate lysosomes without starving or activating immune cells may help reveal new roles for tubular lysosomes.

Keywords:  DNA nanotechnology; MMP9; lysosomes; macrophages; phagocytosis

DOI:  https://doi.org/10.1073/pnas.2113174118
Proc Natl Acad Sci U S A. 2021 Oct 12. pii: e2104832118. [Epub ahead of print]118(41):

Context-specific regulation of lysosomal lipolysis through network-level diverting of transcription factor interactions.

Vinod K Mony, Anna Drangowska-Way, Reka Albert, Emma Harrison, Abbas Ghaddar, Mary Kate Horak, Wenfan Ke, Eyleen J O'Rourke.

  Plasticity in multicellular organisms involves signaling pathways converting contexts-either natural environmental challenges or laboratory perturbations-into context-specific changes in gene expression. Congruently, the interactions between the signaling molecules and transcription factors (TF) regulating these responses are also context specific. However, when a target gene responds across contexts, the upstream TF identified in one context is often inferred to regulate it across contexts. Reconciling these stable TF-target gene pair inferences with the context-specific nature of homeostatic responses is therefore needed. The induction of the Caenorhabditis elegans genes lipl-3 and lipl-4 is observed in many genetic contexts and is essential to survival during fasting. We find DAF-16/FOXO mediating lipl-4 induction in all contexts tested; hence, lipl-4 regulation seems context independent and compatible with across-context inferences. In contrast, DAF-16-mediated regulation of lipl-3 is context specific. DAF-16 reduces the induction of lipl-3 during fasting, yet it promotes it during oxidative stress. Through discrete dynamic modeling and genetic epistasis, we define that DAF-16 represses HLH-30/TFEB-the main TF activating lipl-3 during fasting. Contrastingly, DAF-16 activates the stress-responsive TF HSF-1 during oxidative stress, which promotes C. elegans survival through induction of lipl-3 Furthermore, the TF MXL-3 contributes to the dominance of HSF-1 at the expense of HLH-30 during oxidative stress but not during fasting. This study shows how context-specific diverting of functional interactions within a molecular network allows cells to specifically respond to a large number of contexts with a limited number of molecular players, a mode of transcriptional regulation we name "contextualized transcription."

Keywords:  FOXO/DAF-16; TFEB/HLH-30; fasting; fat; oxidative stress

DOI:  https://doi.org/10.1073/pnas.2104832118
eNeuro. 2021 Oct 06. pii: ENEURO.0193-21.2021. [Epub ahead of print]

A novel neuron-specific regulator of the V-ATPase in Drosophila.

Amina Dulac, Abdul-Raouf Issa, Jun Sun, Giorgio Matassi, Célia Jonas, Baya Chérif-Zahar, Daniel Cattaert, Serge Birman.

  The V-ATPase is a highly conserved enzymatic complex that ensures appropriate levels of organelle acidification in virtually all eukaryotic cells. While the general mechanisms of this proton pump have been well studied, little is known about the specific regulations of neuronal V-ATPase. Here, we studied CG31030, a previously uncharacterized Drosophila protein predicted from its sequence homology to be part of the V-ATPase family. In contrast to its ortholog ATP6AP1/VhaAC45 which is ubiquitous, we observed that CG31030 expression is apparently restricted to all neurons, and using CRISPR/Cas9-mediated gene tagging, that it is mainly addressed to synaptic terminals. In addition, we observed that CG31030 is essential for fly survival and that this protein co-immunoprecipitates with identified V-ATPase subunits, and in particular ATP6AP2. Using a genetically-encoded pH probe (VMAT-pHluorin) and electrophysiological recordings at the larval neuromuscular junction, we show that CG31030 knockdown induces a major defect in synaptic vesicle acidification and a decrease in quantal size, which is the amplitude of the postsynaptic response to the release of a single synaptic vesicle. These defects were associated with severe locomotor impairments. Overall, our data indicate that CG31030, which we renamed VhaAC45-related protein (VhaAC45RP), is a specific regulator of neuronal V-ATPase in Drosophila that is required for proper synaptic vesicle acidification and neurotransmitter release.Significance StatementIn this study, we provide evidence that a previously uncharacterized Drosophila protein, CG31030, is necessary for fly survival and expressed specifically in neurons, where it interacts with constitutive and accessory subunits of the V-ATPase. Physiologically, we show that this protein is required for synaptic vesicle acidification and to ensure proper synaptic transmission at the neuromuscular junction. This implies that CG31030, alias VhaAC45RP, is a novel synaptic protein essential to nervous system functioning and neurotransmitter release. This work therefore provides a new step towards a more exhaustive understanding of the regulations of neuronal V-ATPase and their potential repercussions on synaptic transmission and neurological diseases.

Keywords:  ATP6AP1L/ATP6AP1; CG31030/VhaAC45RP; Drosophila melanogaster; neuronal V-ATPase; quantal size; synaptic vesicle acidification

DOI:  https://doi.org/10.1523/ENEURO.0193-21.2021
Dev Cell. 2021 Oct 01. pii: S1534-5807(21)00728-0. [Epub ahead of print]

Monomethyl branched-chain fatty acid mediates amino acid sensing upstream of mTORC1.

Mengnan Zhu, Fukang Teng, Na Li, Li Zhang, Shuxian Zhang, Fan Xu, Jing Shao, Haipeng Sun, Huanhu Zhu.

  Animals have developed various nutrient-sensing mechanisms for survival under fluctuating environmental conditions. Although extensive cell-culture-based analyses have identified diverse mediators of amino acid sensing upstream of mTOR, studies using animal models to examine intestine-initiated amino acid sensing mechanisms under specific physiological conditions are lacking. Here, we developed a Caenorhabditis elegans model to examine the impact of amino acid deficiency on development. We discovered a leucine-derived monomethyl branched-chain fatty acid and its downstream metabolite, glycosphingolipid, which critically mediates the overall amino acid sensing by intestinal and neuronal mTORC1, which in turn regulates postembryonic development at least partly by controlling protein translation and ribosomal biogenesis. Additional data suggest that a similar mechanism may operate in mammals. This study uncovers an amino-acid-sensing mechanism mediated by a lipid biosynthesis pathway.

Keywords:  C. elegans; amino acid sensing; developmental arrest; developmental control; glucosylceramide; mTOR; mTORC1; mmBCFA; nutrient sensing; sphingolipid

DOI:  https://doi.org/10.1016/j.devcel.2021.09.010
Pharmacol Ther. 2021 Oct 05. pii: S0163-7258(21)00214-X. [Epub ahead of print] 108012

Mapping of mTOR drug targets: Featured platforms for anti-cancer drug discovery.

Raef Shams, Yoshihiro Ito, Hideyuki Miyatake.

  The mammalian/mechanistic target of rapamycin (mTOR) is a regulatory protein kinase involved in cell growth and proliferation. mTOR is usually assembled in two different complexes with different regulatory mechanisms, mTOR complex 1 (mTORC1) and mTORC2, which are involved in different functions such as cell proliferation and cytoskeleton assembly, respectively. In cancer cells, mTOR is hyperactivated in response to metabolic alterations and/or oncogenic signals to overcome the stressful microenvironments. Therefore, recent research progress for mTOR inhibition involves a variety of compounds that have been developed to disturb the metabolic processes of cancer cells through mTOR inhibition. In addition to competitive or allosteric inhibition, a new inhibition strategy that emerged mTOR complexes destabilization has recently been a concern. Here, we review the history of mTOR and its inhibition, along with the timeline of the mTOR inhibitors. We also introduce prospective drug targets to inhibit mTOR by disrupting the complexation of the components with peptides and small molecules.

Keywords:  Cancer therapy; Drug target; mTOR; mTOR inhibitors generations; mTORC1/2 destabilizers

DOI:  https://doi.org/10.1016/j.pharmthera.2021.108012
J Neurochem. 2021 Oct 07.

Glucocerebrosidase 1 and leucine-rich repeat kinase 2 in Parkinson disease and interplay between the two genes.

Chiao-Yin Lee, Elisa Menozzi, Kai-Yin Chau, Anthony H V Schapira.

  The glucocerebrosidase 1 gene (GBA1), bi-allelic variants of which cause Gaucher Disease (GD), encodes the lysosomal enzyme glucocerebrosidase (GCase) and is a risk factor for Parkinson Disease (PD). GBA1 variants are linked to a reduction in GCase activity in the brain. Variants in Leucine-Rich Repeat Kinase 2 (LRRK2), such as the gain-of-kinase-function variant G2019S, cause the most common familial form of PD. In patients without GBA1 and LRRK2 mutations, GCase and LRRK2 activity are also altered, suggesting that these two genes are implicated in all forms of PD and that they may play a broader role in PD pathogenesis. In this review, we review the proposed roles of GBA1 and LRRK2 in PD, focussing on the endolysosomal pathway. In particular, we highlight the discovery of Ras-related in brain (Rab) guanosine triphosphatases (GTPases) as LRRK2 kinase substrates and explore the links between increased LRRK2 activity and Rab protein function, lysosomal dysfunction, alpha-synuclein accumulation and GCase activity. We also discuss the discovery of RAB10 as a potential mediator of LRRK2 and GBA1 interaction in PD. Finally, we discuss the therapeutic implications of these findings, including current approaches and future perspectives related to novel drugs targeting LRRK2 and GBA1.

Keywords:  GBA1; Gaucher disease; LRRK2; Parkinson disease; glucocerebrosidase; neurodegeneration

DOI:  https://doi.org/10.1111/jnc.15524
J Am Soc Nephrol. 2021 Oct 04. pii: ASN.2021030333. [Epub ahead of print]

mTOR-Activating Mutations in RRAGD are Causative for Kidney Tubulopathy and Cardiomyopathy.

Karl-Peter Schlingmann, François Jouret, Kuang Shen, Anukrati Nigam, Francisco Arjona, Claudia Dafinger, Pascal Houillier, Deborah Jones, Felix Kleinerüschkamp, Jun Oh, Nathalie Godefroid, Mehmet Eltan, Tülay Güran, Stéphane Burtey, Marie-Christine Parotte, Jens König, Alina Braun, Caro Bos, Maria Ibars Serra, Holger Rehmann, Fried Zwartkruis, Kirsten Renkema, Karin Klingel, Eric Schulze-Bahr, Bernhard Schermer, Carsten Bergmann, Janine Altmüller, Holger Thiele, Bodo Beck, Karin Dahan, David Sabatini, Max Liebau, Rosa Vargas-Poussou, Nine Knoers, Martin Konrad, Jeroen de Baaij.

Background Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all tubulopathy patients lack a genetic diagnosis. Methods We performed whole-exome and genome sequencings of a patient cohort with a novel inherited salt-losing tubulopathy, hypomagnesemia, and dilated cardiomyopathy. We also conducted subsequent functional analyses in vitro of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). Results In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD encoded by RRAGD plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro Conclusions Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.

DOI: https://doi.org/10.1681/ASN.2021030333
JCI Insight. 2021 Oct 07. pii: e136147. [Epub ahead of print]

Microglia regulate brain Progranulin levels through the endocytosis-lysosomal pathway.

Tingting Dong, Leon Tejwani, Youngseob Jung, Hiroshi Kokubu, Kimberly Luttik, Terri M Driessen, Janghoo Lim.

  Genetic variants in Granulin (GRN), which encodes the secreted glycoprotein Progranulin (PGRN), are associated with several neurodegenerative diseases including frontotemporal lobar degeneration, neuronal ceroid lipofuscinosis, and Alzheimer's disease. These genetic alterations manifest in pathological changes due to a reduction of PGRN expression; therefore, identifying factors that can modulate PGRN levels in vivo would enhance our understanding of PGRN in neurodegeneration, and could reveal novel potential therapeutic targets. Here, we report that modulation of the endocytosis-lysosomal pathway via reduction of Nemo-like kinase (Nlk) in microglia, and not neurons, can alter total brain Pgrn levels in mice. We demonstrate that Nlk reduction promotes Pgrn degradation by enhancing its trafficking through endocytosis-lysosomal pathway, specifically in microglia. Furthermore, genetic interaction studies in mice showed that Nlk heterozygosity in Grn haploinsufficient mice further reduces Pgrn levels and induces neuropathological phenotypes associated with PGRN deficiency. Our results reveal a new mechanism for Pgrn level regulation in the brain through the active catabolism by microglia and provide insights into the pathophysiology of PGRN-associated diseases.

Keywords:  Dementia; Mouse models; Neurodegeneration; Neuroscience

DOI:  https://doi.org/10.1172/jci.insight.136147
Front Mol Biosci. 2021 ;8 624988

Clinical Trials for Gene Therapy in Lysosomal Diseases With CNS Involvement.

Caroline Sevin, Kumaran Deiva.

  There are over 70 known lysosomal storage disorders (LSDs), most caused by mutations in genes encoding lysosomal hydrolases. Central nervous system involvement is a hallmark of the majority of LSDs and, if present, generally determines the prognosis of the disease. Nonetheless, brain disease is currently poorly targeted by available therapies, including systemic enzyme replacement therapy, mostly (but not only) due to the presence of the blood-brain barrier that restricts the access of orally or parenterally administered large molecules into the brain. Thus, one of the greatest and most exciting challenges over coming years will be to succeed in developing effective therapies for the treatment of central nervous system manifestations in LSDs. Over recent years, gene therapy (GT) has emerged as a promising therapeutic strategy for a variety of inherited neurodegenerative diseases. In LSDs, the ability of genetically corrected cells to cross-correct adjacent lysosomal enzyme-deficient cells in the brain after gene transfer might enhance the diffusion of the recombinant enzyme, making this group of diseases a strong candidate for such an approach. Both in vivo (using the administration of recombinant adeno-associated viral vectors) and ex vivo (auto-transplantation of lentiviral vector-modified hematopoietic stem cells-HSCs) strategies are feasible. Promising results have been obtained in an ever-increasing number of preclinical studies in rodents and large animal models of LSDs, and these give great hope of GT successfully correcting neurological defects, once translated to clinical practice. We are now at the stage of treating patients, and various clinical trials are underway, to assess the safety and efficacy of in vivo and ex vivo GT in several neuropathic LSDs. In this review, we summarize different approaches being developed and review the current clinical trials related to neuropathic LSDs, their results (if any), and their limitations. We will also discuss the pitfalls and the remaining challenges.

Keywords:  Adeno-associated virus; CNS-central nervous system; gene therapy; lentival vector; lysosomal diseases

DOI:  https://doi.org/10.3389/fmolb.2021.624988
Chem Sci. 2021 Sep 29. 12(37): 12451-12462

Chemoenzymatic glycan-selective remodeling of a therapeutic lysosomal enzyme with high-affinity M6P-glycan ligands. Enzyme substrate specificity is the name of the game.

Xiao Zhang, Huiying Liu, Naresh Meena, Chao Li, Guanghui Zong, Nina Raben, Rosa Puertollano, Lai-Xi Wang.

Functionalization of therapeutic lysosomal enzymes with mannose-6-phosphate (M6P) glycan ligands represents a major strategy for enhancing the cation-independent M6P receptor (CI-MPR)-mediated cellular uptake, thus improving the overall therapeutic efficacy of the enzymes. However, the minimal high-affinity M6P-containing N-glycan ligands remain to be identified and their efficient and site-selective conjugation to therapeutic lysosomal enzymes is a challenging task. We report here the chemical synthesis of truncated M6P-glycan oxazolines and their use for enzymatic glycan remodeling of recombinant human acid α-glucosidase (rhGAA), an enzyme used for treatment of Pompe disease which is a disorder caused by a deficiency of the glycogen-degrading lysosomal enzyme. Structure-activity relationship studies identified M6P tetrasaccharide oxazoline as the minimal substrate for enzymatic transglycosylation yielding high-affinity M6P glycan ligands for the CI-MPR. Taking advantage of the substrate specificity of endoglycosidases Endo-A and Endo-F3, we found that Endo-A and Endo-F3 could efficiently deglycosylate the respective high-mannose and complex type N-glycans in rhGAA and site-selectively transfer the synthetic M6P N-glycan to the deglycosylated rhGAA without product hydrolysis. This discovery enabled a highly efficient one-pot deglycosylation/transglycosylation strategy for site-selective M6P-glycan remodeling of rhGAA to obtain a more homogeneous product. The Endo-A and Endo-F3 remodeled rhGAAs maintained full enzyme activity and demonstrated 6- and 20-fold enhanced binding affinities for CI-MPR receptor, respectively. Using an in vitro cell model system for Pompe disease, we demonstrated that the M6P-glycan remodeled rhGAA greatly outperformed the commercial rhGAA (Lumizyme) and resulted in the reversal of cellular pathology. This study provides a general and efficient method for site-selective M6P-glycan remodeling of recombinant lysosomal enzymes to achieve enhanced M6P receptor binding and cellular uptake, which could lead to improved overall therapeutic efficacy of enzyme replacement therapy.

DOI: https://doi.org/10.1039/d1sc03188k
Lipids. 2021 Oct 07.

Molecular markers of brain cholesterol homeostasis are unchanged despite a smaller brain mass in a mouse model of cholesteryl ester storage disease.

Amal A Aqul, Charina M Ramirez, Adam M Lopez, Dennis K Burns, Joyce J Repa, Stephen D Turley.

  Lysosomal acid lipase (LAL), encoded by the gene LIPA, facilitates the intracellular processing of lipids by hydrolyzing cholesteryl esters and triacylglycerols present in newly internalized lipoproteins. Loss-of-function mutations in LIPA result in cholesteryl ester storage disease (CESD) or Wolman disease when mutations cause complete loss of LAL activity. Although the phenotype of a mouse CESD model has been extensively characterized, there has not been a focus on the brain at different stages of disease progression. In the current studies, whole-brain mass and the concentrations of cholesterol in both the esterified (EC) and unesterified (UC) fractions were measured in Lal-/- and matching Lal+/+ mice (FVB-N strain) at ages ranging from 14 up to 280 days after birth. Compared to Lal+/+ controls at 50, 68-76, 140-142, and 230-280 days of age, Lal-/- mice had brain weights that averaged approximately 6%, 7%, 18%, and 20% less, respectively. Brain EC levels were higher in the Lal-/- mice at every age, being elevated 27-fold at 230-280 days. Brain UC concentrations did not show a genotypic difference at any age. The elevated brain EC levels in the Lal-/- mice did not reflect EC in residual blood. An mRNA expression analysis for an array of genes involved in the synthesis, catabolism, storage, and transport of cholesterol in the brains of 141-day old mice did not detect any genotypic differences although the relative mRNA levels for several markers of inflammation were moderately elevated in the Lal-/- mice. The possible sites of EC accretion in the central nervous system are discussed.

Keywords:  brain mass; cholesterol-esterifying enzymes; esterified cholesterol sequestration; lysosomal acid lipase; residual blood volume; unesterified cholesterol

DOI:  https://doi.org/10.1002/lipd.12325
JCI Insight. 2021 Oct 08. pii: e145445. [Epub ahead of print]6(19):

Iduronate-2-sulfatase transport vehicle rescues behavioral and skeletal phenotypes in a mouse model of Hunter syndrome.

Annie Arguello, René Meisner, Elliot R Thomsen, Hoang N Nguyen, Ritesh Ravi, Jeffrey Simms, Iris Lo, Jessica Speckart, Julia Holtzman, Thomas M Gill, Darren Chan, Yuhsiang Cheng, Chi-Lu Chiu, Jason C Dugas, Meng Fang, Isabel A Lopez, Hilda Solanoy, Buyankhishig Tsogtbaatar, Yuda Zhu, Akhil Bhalla, Kirk R Henne, Anastasia G Henry, Anthony Delucchi, Simona Costanzo, Jeffrey M Harris, Dolores Diaz, Kimberly Scearce-Levie, Pascal E Sanchez.

  Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficiency of the iduronate-2-sulfatase (IDS) enzyme, resulting in cellular accumulation of glycosaminoglycans (GAGs) throughout the body. Treatment of MPS II remains a considerable challenge as current enzyme replacement therapies do not adequately control many aspects of the disease, including skeletal and neurological manifestations. We developed an IDS transport vehicle (ETV:IDS) that is engineered to bind to the transferrin receptor; this design facilitates receptor-mediated transcytosis of IDS across the blood-brain barrier and improves its distribution into the brain while maintaining distribution to peripheral tissues. Here we show that chronic systemic administration of ETV:IDS in a mouse model of MPS II reduced levels of peripheral and central nervous system GAGs, microgliosis, and neurofilament light chain, a biomarker of neuronal injury. Additionally, ETV:IDS rescued auricular and skeletal abnormalities when introduced in adult MPS II mice. These effects were accompanied by improvements in several neurobehavioral domains, including motor skills, sensorimotor gating, and learning and memory. Together, these results highlight the therapeutic potential of ETV:IDS for treating peripheral and central abnormalities in MPS II. DNL310, an investigational ETV:IDS molecule, is currently in clinical trials as a potential treatment for patients with MPS II.

Keywords:  Behavior; Neurological disorders; Neuroscience; Therapeutics; Translation

DOI:  https://doi.org/10.1172/jci.insight.145445
Autophagy. 2021 Oct 06. 1-29

LGALS3 (galectin 3) mediates an unconventional secretion of SNCA/α-synuclein in response to lysosomal membrane damage by the autophagic-lysosomal pathway in human midbrain dopamine neurons.

Kevin Burbidge, David J Rademacher, Jessica Mattick, Stephanie Zack, Andrea Grillini, Luc Bousset, Ochan Kwon, Konrad Kubicki, Alexander Simon, Ronald Melki, Edward M Campbell.

  Numerous lines of evidence support the premise that the misfolding and subsequent accumulation of SNCA/α-synuclein (synuclein alpha) is responsible for the underlying neuronal pathology observed in Parkinson disease (PD) and other synucleinopathies. Moreover, the cell-to-cell transfer of these misfolded SNCA species is thought to be responsible for disease progression and the spread of cellular pathology throughout the brain. Previous work has shown that when exogenous, misfolded SNCA fibrils enter cells through endocytosis, they can damage and rupture the membranes of their endocytotic vesicles in which they are trafficked. Rupture of these vesicular membranes exposes intralumenal glycans leading to galectin protein binding, subsequent autophagic protein recruitment, and, ultimately, their introduction into the autophagic-lysosomal pathway. Increasing evidence indicates that both pathological and non-pathological SNCA species undergo autophagy-dependent unconventional secretion. While other proteins have also been shown to be secreted from cells by autophagy, what triggers this release process and how these specific proteins are recruited to a secretory autophagic pathway is largely unknown. Here, we use a human midbrain dopamine (mDA) neuronal culture model to provide evidence in support of a cellular mechanism that explains the cell-to-cell transfer of pathological forms of SNCA that are observed in PD. We demonstrate that LGALS3 (galectin 3) mediates the release of SNCA following vesicular damage. SNCA release is also dependent on TRIM16 (tripartite motif containing 16) and ATG16L1 (autophagy related 16 like 1), providing evidence that secretion of SNCA is mediated by an autophagic secretory pathway.

Keywords:  Autophagy; Parkinson disease; alpha-synuclein (synuclein alpha); extracellular vesicles; galectins; induced pluripotent stem cells; lysosomes; tripartite motif proteins; unconventional secretion

DOI:  https://doi.org/10.1080/15548627.2021.1967615
JCI Insight. 2021 Oct 08. pii: e148649. [Epub ahead of print]6(19):

Direct targeting of wild-type glucocerebrosidase by antipsychotic quetiapine improves pathogenic phenotypes in Parkinson's disease models.

Lena F Burbulla, Jianbin Zheng, Pingping Song, Weilan Jiang, Michaela E Johnson, Patrik Brundin, Dimitri Krainc.

  Current treatments for Parkinson's disease (PD) provide only symptomatic relief, with no disease-modifying therapies identified to date. Repurposing FDA-approved drugs to treat PD could significantly shorten the time needed for and reduce the costs of drug development compared with conventional approaches. We developed an efficient strategy to screen for modulators of β-glucocerebrosidase (GCase), a lysosomal enzyme that exhibits decreased activity in patients with PD, leading to accumulation of the substrate glucosylceramide and oxidized dopamine and α-synuclein, which contribute to PD pathogenesis. Using a GCase fluorescent probe and affinity-based fluorescence polarization assay, we screened 1280 structurally diverse, bioactive, and cell-permeable FDA-approved drugs and found that the antipsychotic quetiapine bound GCase with high affinity. Moreover, quetiapine treatment of induced pluripotent stem cell-derived (iPSC-derived) dopaminergic neurons from patients carrying mutations in GBA1 or LRRK2 led to increased wild-type GCase protein levels and activity and partially lowered accumulation of oxidized dopamine, glucosylceramide, and α-synuclein. Similarly, quetiapine led to activation of wild-type GCase and reduction of α-synuclein in a GBA mutant mouse model (Gba1D409V/+ mice). Together, these results suggest that repurposing quetiapine as a modulator of GCase may be beneficial for patients with PD exhibiting decreased GCase activity.

Keywords:  Drug screens; Neurodegeneration; Neuroscience; Parkinson disease

DOI:  https://doi.org/10.1172/jci.insight.148649
Biochem Biophys Res Commun. 2021 Sep 27. pii: S0006-291X(21)01371-1. [Epub ahead of print]580 7-13

Mefloquine as a dual inhibitor of glioblastoma angiogenesis and glioblastoma via disrupting lysosomal function.

Bing Wan, Zhenghong Wu, Xiulan Zhang, Birun Huang.

  Angiogenesis, the formation of new blood vessels from the pre-existing ones, is a hallmark characteristic of glioblastoma, making it an appealing target for treatment development. Given potent anti-cancer efficacy of mefloquine, FDA-approved anti-malarial drug, there is increasing interest in repurposing mefloquine for treatment of cancers, including glioblastoma. In line with these efforts, our work is the first to demonstrate that mefloquine is also an inhibitor of glioblastoma angiogenesis. Using glioblastoma microvascular endothelial cell (GMEC) isolated from glioblastoma patients, we show that mefloquine at clinically achievable concentration inhibits GMEC differentiation, capillary network formation, adhesion to Matrix, growth and survival. Mefloquine also inhibits growth and induces apoptosis in glioblastoma cells regardless of cellular origin and genetic background. We further show that mefloquine significantly inhibits glioblastoma growth but not formation, and this is associated with decreased glioblastoma angiogenesis in mice. Mechanistically, mefloquine disrupted lysosomal integrity and function in GMECs, leading to oxidative stress and lysosomal lipid damage. Rescue studies confirm that mefloquine acts on GMECs in a lysosomal disruption-dependent manner. Our findings demonstrate the anti-angiogenic activity of mefloquine via disrupting lysosomal function. The dual inhibitory role of mefloquine in glioblastoma angiogenesis and glioblastoma displays its advantage over other anti-cancer drugs for glioblastoma treatment. Our work also highlights the essential role of lysosome in both glioblastoma and its angiogenesis.

Keywords:  Angiogenesis; GMECs; Lysosome; Mefloquine

DOI:  https://doi.org/10.1016/j.bbrc.2021.09.069
Biochim Biophys Acta Mol Cell Res. 2021 Oct 01. pii: S0167-4889(21)00199-3. [Epub ahead of print] 119145

Intracellular ion changes induced by the exposure to beta-amyloid can be explained by the formation of channels in the lysosomal membranes.

Dmitry V Zaretsky, Maria V Zaretskaia.

  In this manuscript, we reassess the data on beta-amyloid-induced changes of intracellular ions concentrations published previously by Abramov et al. (2003, 2004). Their observations made using high-resolution confocal microscopy with fast temporal resolution of images formed by fluorescent ion-sensitive fluorescent probes in living cells represent an unequivocal support for the amyloid channel theory. However, closer look reveals multiple facts which cannot be explained by channel formation in plasma membrane. Recently proposed amyloid degradation toxicity hypothesis provides the interpretation to these facts by considering that channels are formed in the lysosomal membranes.

Keywords:  beta-amyloid toxicity; calcium homeostasis; intracellular ion disturbances; intracellular pH; lysosome; membrane channel; proteolytic digestion

DOI:  https://doi.org/10.1016/j.bbamcr.2021.119145
Genet Mol Biol. 2021 ;pii: S1415-47572021000600701. [Epub ahead of print]44(4): e20200475

Primary cells derived from Tuberous Sclerosis Complex patients show autophagy alteration in the haploinsufficiency state.

Clévia Rosset, Mariane da Cunha Jaeger, Eduardo Filippi-Chiela, Larissa Brussa Reis, Ivaine Taís Sauthier Sartor, Cristina Brinckmann Oliveira Netto, Caroline Brunetto de Farias, Rafael Roesler, Patricia Ashton-Prolla.

Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field.

DOI: https://doi.org/10.1590/1678-4685-GMB-2020-0475
Dev Cell. 2021 Sep 29. pii: S1534-5807(21)00731-0. [Epub ahead of print]

Rheb-regulated mitochondrial pyruvate metabolism of Schwann cells linked to axon stability.

Lanlan Jia, Maoxing Liao, Aidi Mou, Quanzhen Zheng, Wanchun Yang, Zongyan Yu, Yiyuan Cui, Xiaoqiang Xia, Yue Qin, Mina Chen, Bo Xiao.

  The metabolic coupling of Schwann cells (SCs) and peripheral axons is poorly understood. Few molecules in SCs are known to regulate axon stability. Using SC-specific Rheb knockout mice, we demonstrate that Rheb-regulated mitochondrial pyruvate metabolism is critical for SC-mediated non-cell-autonomous regulation of peripheral axon stability. Rheb knockout suppresses pyruvate dehydrogenase (PDH) activity (independently of mTORC1) and shifts pyruvate metabolism toward lactate production in SCs. The increased lactate causes age-dependent peripheral axon degeneration, affecting peripheral nerve function. Lactate, as an energy substrate and a potential signaling molecule, enhanced neuronal mitochondrial metabolism and energy production of peripheral nerves. Albeit beneficial to injured peripheral axons in the short term, we show that persistently increased lactate metabolism of neurons enhances ROS production, eventually damaging mitochondria, neuroenergetics, and axon stability. This study highlights the complex roles of lactate metabolism to peripheral axons and the importance of lactate homeostasis in preserving peripheral nerves.

Keywords:  ROS; Rheb; Schwann cells; axon degeneration; lactate shuttle; mTORC1; metabolic coupling; oxidative stress; peripheral axons; pyruvate metabolism

DOI:  https://doi.org/10.1016/j.devcel.2021.09.013
Nat Commun. 2021 Oct 04. 12(1): 5802

A plastid two-pore channel essential for inter-organelle communication and growth of Toxoplasma gondii.

Zhu-Hong Li, Thayer P King, Lawrence Ayong, Beejan Asady, Xinjiang Cai, Taufiq Rahman, Stephen A Vella, Isabelle Coppens, Sandip Patel, Silvia N J Moreno.

Two-pore channels (TPCs) are a ubiquitous family of cation channels that localize to acidic organelles in animals and plants to regulate numerous Ca2+-dependent events. Little is known about TPCs in unicellular organisms despite their ancient origins. Here, we characterize a TPC from Toxoplasma gondii, the causative agent of toxoplasmosis. TgTPC is a member of a novel clad of TPCs in Apicomplexa, distinct from previously identified TPCs and only present in coccidians. We show that TgTPC localizes not to acidic organelles but to the apicoplast, a non-photosynthetic plastid found in most apicomplexan parasites. Conditional silencing of TgTPC resulted in progressive loss of apicoplast integrity, severely affecting growth and the lytic cycle. Isolation of TPC null mutants revealed a selective role for TPCs in replication independent of apicoplast loss that required conserved residues within the pore-lining region. Using a genetically-encoded Ca2+ indicator targeted to the apicoplast, we show that Ca2+ signals deriving from the ER but not from the extracellular space are selectively transmitted to the lumen. Deletion of the TgTPC gene caused reduced apicoplast Ca2+ uptake and membrane contact site formation between the apicoplast and the ER. Fundamental roles for TPCs in maintaining organelle integrity, inter-organelle communication and growth emerge.

DOI: https://doi.org/10.1038/s41467-021-25987-5
Mol Cell Oncol. 2021 ;8(4): 1954470

A late endosome signaling hub that couples PI3Kα and WNT/β-catenin signaling in breast cancer.

Samuel J Rodgers, Sabryn A Hamila, Christina A Mitchell, Lisa M Ooms.

  AKT is the central phosphoinositide 3-kinase (PI3K) signaling effector, however, PIK3CA (p110α subunit of PI3Kα)-mutant estrogen receptor-positive (ER+) breast cancers exhibit minimal AKT activation and the downstream signaling is poorly characterized. We discovered that a subset of PIK3CA-mutant ER+ breast cancers exhibit increased inositol polyphosphate 4-phosphatase type II (INPP4B) expression, which promotes late endosome formation and glycogen synthase kinase 3 beta (GSK3β) trafficking, leading to enhanced Wingless-related integration site (WNT)/catenin beta 1 (β-catenin) activation.

Keywords:  INPP4B; PIK3CA; WNT/β-catenin; cell proliferation; late endosome

DOI:  https://doi.org/10.1080/23723556.2021.1954470
Mol Cell Oncol. 2021 ;8(4): 1879614

The concerted action of oncogenic driver mutations directs global translation in intestinal epithelial cells.

Wouter Smit, Jarom Heijmans.

  Oncogenic transformation of colorectal cancer cells is driven by a set of mutations that cause aberrant signaling of growth factor-receptor pathways. Using organoids, we demonstrate that the most frequent driver mutations in APC, KRAS, SMAD4, and TP53 are enhancers of the global mRNA translational capacity, which is linked to intestinal cell growth in an mTOR-dependent manner.

Keywords:  Colorectal cancer; driver mutations; global translation; mTOR signaling; protein synthesis

DOI:  https://doi.org/10.1080/23723556.2021.1879614
ACS Nano. 2021 Oct 06.

A Hybrid Nanogel to Preserve Lysosome Integrity for Fluorescence Imaging.

Weiqi Zhang, Baoji Du, Menghan Gao, Ching-Hsuan Tung.

  Fluorescence imaging of lysosomes provides a powerful tool to probe the lysosome physiology in living cells, yet the continuous light exposure inevitably causes lysosome damage and phototoxicity, which remains a formidable challenge. Here the long-term lysosome tracking with minimized photodamage was realized using a multifunctional nanoprobe, a platinum nanoparticle, and a quinacrine co-loaded nanogel. To construct the hybrid nanogel, cisplatin first functioned as cross-linker to withhold all components and then was reduced to a platinum nanoparticle in situ by ethanol. The platinum nanoparticle enabled a long-term quinacrine fluorescence imaging of lysosome by scavenging the light induced reactive oxygen species which could damage lysosomal membranes.

Keywords:  fluorescence imaging; hyaluronan; lysosome; nanogel; platinum nanoparticles

DOI:  https://doi.org/10.1021/acsnano.1c05864
J Lipid Res. 2021 Oct 05. pii: S0022-2275(21)00115-2. [Epub ahead of print] 100133

Hepatic Lysosomal Acid Lipase Overexpression Worsens Hepatic Inflammation in Mice Fed a Western Diet.

Michael W Lopresti, Wenqi Cui, Breann Abernathy, Gavin Fredrickson, Fanta Barrow, Arnav S Desai, Xavier S Revelo, Douglas Mashek.

  Non-alcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipid droplets (LD) in hepatocytes. NAFLD development and progression is associated with an increase in hepatic cholesterol levels and decreased autophagy and lipophagy flux. Previous studies have shown that the expression of lysosomal acid lipase (LAL, encoded by the gene LIPA), which can hydrolyze both triglyceride and cholesteryl esters, is inversely correlated with the severity of NAFLD. In addition, ablation of LAL activity results in profound NAFLD. Based on this, we predicted that overexpressing LIPA in the livers of mice fed a Western diet would prevent the development of NAFLD. As expected, mice fed the Western diet exhibited numerous markers of NAFLD, including hepatomegaly, lipid accumulation, and inflammation. Unexpectedly, LAL overexpression did not attenuate steatosis and had only minor effects on neutral lipid composition. However, LAL overexpression exacerbated inflammatory gene expression and infiltration of immune cells in mice fed the Western diet. LAL overexpression also resulted in abnormal phagosome accumulation and lysosomal lipid accumulation depending upon the dietary treatment. Overall, we found that hepatic overexpression of LAL drove immune cell infiltration and inflammation and did not attenuate the development of NAFLD, suggesting that targeting LAL expression may not be a viable route to treat NAFLD in humans.

Keywords:  Cholesterol/Cell and tissue; Dietary fat; Inflammation; Lipase/Hepatic; Liver; Lysosomal acid lipase; NAFLD; autophagy; immune infiltration

DOI:  https://doi.org/10.1016/j.jlr.2021.100133
Mov Disord. 2021 Oct 06.

Evaluation of Strategies for Measuring Lysosomal Glucocerebrosidase Activity.

Daniel Ysselstein, Tiffany J Young, Maria Nguyen, Shalini Padmanabhan, Warren D Hirst, Nicolas Dzamko, Dimitri Krainc.

  Mutations in GBA1, which encode for the protein glucocerebrosidase (GCase), are the most common genetic risk factor for Parkinson's disease and dementia with Lewy bodies. In addition, growing evidence now suggests that the loss of GCase activity is also involved in onset of all forms of Parkinson's disease, dementia with Lewy bodies, and other dementias, such as progranulin-linked frontal temporal dementia. As a result, there is significant interest in developing GCase-targeted therapies that have the potential to stop or slow progression of these diseases. Despite this interest in GCase as a therapeutic target, there is significant inconsistency in the methodology for measuring GCase enzymatic activity in disease-modeling systems and patient populations, which could hinder progress in developing GCase therapies. In this review, we discuss the different strategies that have been developed to assess GCase activity and highlight the specific strengths and weaknesses of these approaches as well as the gaps that remain. We also discuss the current and potential role of these different methodologies in preclinical and clinical development of GCase-targeted therapies. © 2021 International Parkinson and Movement Disorder Society.

Keywords:  GCase; GCase enzyme activity; Parkinson's disease; glucocerebrosidase

DOI:  https://doi.org/10.1002/mds.28815
Phys Chem Chem Phys. 2021 Sep 14. 23(34): 18461-18474

The key role of the central cavity in sodium transport through ligand-gated two-pore channels.

Stefan Milenkovic, Igor V Bodrenko, Armando Carpaneto, Matteo Ceccarelli.

Subcellular and organellar mechanisms have manifested a prominent importance for a broad variety of processes that maintain cellular life at its most basic level. Mammalian two-pore channels (TPCs) appear to be cornerstones of these processes in endo-lysosomes by controlling delicate ion-concentrations in their interiors. With evolutionary remarkable architecture and one-of-a-kind selectivity filter, TPCs are an extremely attractive topic per se. In the light of the current COVID-19 pandemic, hTPC2 emerges to be more than attractive. As a key regulator of the endocytosis pathway, it is potentially essential for diverse viral infections in humans, as demonstrated. Here, by means of multiscale molecular simulations, we propose a model of sodium transport from the lumen to the cytosol where the central cavity works as a reservoir. Since the inhibition of hTPC2 is proven to stop SARS-CoV2 in vitro, shedding light on the hTPC2 function and mechanism is the first step towards the selection of potential inhibiting candidates.

DOI: https://doi.org/10.1039/d1cp02947a
EMBO Rep. 2021 Oct 04. e52170

Muscle-specific Cand2 is translationally upregulated by mTORC1 and promotes adverse cardiac remodeling.

Agnieszka A Górska, Clara Sandmann, Eva Riechert, Christoph Hofmann, Ellen Malovrh, Eshita Varma, Vivien Kmietczyk, Julie Ölschläger, Lonny Jürgensen, Verena Kamuf-Schenk, Claudia Stroh, Jennifer Furkel, Mathias H Konstandin, Carsten Sticht, Etienne Boileau, Christoph Dieterich, Norbert Frey, Hugo A Katus, Shirin Doroudgar, Mirko Völkers.

  The mechanistic target of rapamycin (mTOR) promotes pathological remodeling in the heart by activating ribosomal biogenesis and mRNA translation. Inhibition of mTOR in cardiomyocytes is protective; however, a detailed role of mTOR in translational regulation of specific mRNA networks in the diseased heart is unknown. We performed cardiomyocyte genome-wide sequencing to define mTOR-dependent gene expression control at the level of mRNA translation. We identify the muscle-specific protein Cullin-associated NEDD8-dissociated protein 2 (Cand2) as a translationally upregulated gene, dependent on the activity of mTOR. Deletion of Cand2 protects the myocardium against pathological remodeling. Mechanistically, we show that Cand2 links mTOR signaling to pathological cell growth by increasing Grk5 protein expression. Our data suggest that cell-type-specific targeting of mTOR might have therapeutic value against pathological cardiac remodeling.

Keywords:  Cand2; cardiac; hypertrophy; mTOR

DOI:  https://doi.org/10.15252/embr.202052170
Pharmacol Res. 2021 Oct 04. pii: S1043-6618(21)00455-2. [Epub ahead of print] 105871

Dihydroartemisinin ameliorates retinal vascular dysfunction in diabetes mellitus via FASN/Kmal-mTOR/SREBP1 feedback loop.

Chufeng Gu, Xinping She, Chuandi Zhou, Tong Su, Shuai He, Chunren Meng, Qing Gu, Dawei Luo, Zhi Zheng, Qinghua Qiu.

  Microvascular dysfunctions are the primary etiology of visual impairment caused by diabetic retinopathy (DR). Dihydroartemisinin (DHA), the active metabolite of the antimalarials artemisinins, exhibits antiangiogenic properties in numerous diseases. Here, we investigated the function and mechanisms of DHA as a vascular protective agent in DR. DHA exerted its protective effect on vascular injuries in diabetic mice and inhibited cell proliferation and tube formation in human retinal microvascular endothelial cells by decreasing the level of fatty acid synthase (FASN), enhancing the malonylation of mTOR at lysine 1218 (K1218) and attenuating the activation of mTOR complex 1 (mTORC1). Impressively, chemosynthetic small interfering RNA against FASN and mutagenesis of K1218 of mTOR shown therapeutic potential in suppressing cell proliferation and tube formation induced by high glucose. Notably, suppression of mTORC1 kinase activity further inhibited FASN by reducing p70S6K phosphorylation and resulting in reduced expression of sterol regulatory element binding protein 1, which interacted directly with the FASN promoter at nucleotide positions -64 and -55. In conclusion, our study elucidated the promising effect of FASN and malonylation in vascular injuries of DR and indicated the great potential of DHA as a therapeutic approach.

Keywords:  Diabetic retinopathy; Dihydroartemisinin; Dihydroartemisinin (PubChem CID: 3000518); Fatty acid synthase; Mechanistic target of rapamycin; Posttranslational modifications; Vascular dysfunction

DOI:  https://doi.org/10.1016/j.phrs.2021.105871
Cell Calcium. 2021 Sep 26. pii: S0143-4160(21)00133-0. [Epub ahead of print]100 102479

Coordinated glucose-induced Ca2+ and pH responses in yeast Saccharomyces cerevisiae.

Tien-Yang Ma, Marie-Anne Deprez, Geert Callewaert, Joris Winderickx.

  Ca2+ and pH homeostasis are closely intertwined and this interrelationship is crucial in the cells' ability to adapt to varying environmental conditions. To further understand this Ca2+-pH link, cytosolic Ca2+ was monitored using the aequorin-based bioluminescent assay in parallel with fluorescence reporter-based assays to monitor plasma membrane potentials and intracellular (cytosolic and vacuolar) pH in yeast Saccharomyces cerevisiae. At external pH 5, starved yeast cells displayed depolarized membrane potentials and responded to glucose re-addition with small Ca2+ transients accompanied by cytosolic alkalinization and profound vacuolar acidification. In contrast, starved cells at external pH 7 were hyperpolarized and glucose re-addition induced large Ca2+ transients and vacuolar alkalinization. In external Ca2+-free medium, glucose-induced pH responses were not affected but Ca2+ transients were abolished, indicating that the intracellular [Ca2+] increase was not prerequisite for activation of the two primary proton pumps, being Pma1 at the plasma membrane and the vacuolar and Golgi localized V-ATPases. A reduction in Pma1 expression resulted in membrane depolarization and reduced Ca2+ transients, indicating that the membrane hyperpolarization generated by Pma1 activation governed the Ca2+ influx that is associated with glucose-induced Ca2+ transients. Loss of V-ATPase activity through concanamycin A inhibition did not alter glucose-induced cytosolic pH responses but affected vacuolar pH changes and Ca2+ transients, indicating that the V-ATPase established vacuolar proton gradient is substantial for organelle H+/Ca2+ exchange. Finally, a systematic analysis of yeast deletion strains allowed us to reveal an essential role for both the vacuolar H+/Ca2+ exchanger Vcx1 and the Golgi exchanger Gdt1 in the dissipation of intracellular Ca2+.

Keywords:  Ca(2+) homeostasis; Glucose signaling; Saccharomyces cerevisiae; Yeast; pH homeostasis

DOI:  https://doi.org/10.1016/j.ceca.2021.102479
EMBO J. 2021 Oct 06. e107958

Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex.

Dimitrios Papagiannidis, Peter W Bircham, Christian Lüchtenborg, Oliver Pajonk, Giulia Ruffini, Britta Brügger, Sebastian Schuck.

  Cells dynamically adapt organelle size to current physiological demand. Organelle growth requires membrane biogenesis and therefore needs to be coordinated with lipid metabolism. The endoplasmic reticulum (ER) can undergo massive expansion, but the underlying regulatory mechanisms are largely unclear. Here, we describe a genetic screen for factors involved in ER membrane expansion in budding yeast and identify the ER transmembrane protein Ice2 as a strong hit. We show that Ice2 promotes ER membrane biogenesis by opposing the phosphatidic acid phosphatase Pah1, called lipin in metazoa. Specifically, Ice2 inhibits the conserved Nem1-Spo7 complex and thus suppresses the dephosphorylation and activation of Pah1. Furthermore, Ice2 cooperates with the transcriptional regulation of lipid synthesis genes and helps to maintain cell homeostasis during ER stress. These findings establish the control of the lipin phosphatase complex as an important mechanism for regulating ER membrane biogenesis.

Keywords:  Opi1; endoplasmic reticulum; lipid droplets; lipin; organelle biogenesis

DOI:  https://doi.org/10.15252/embj.2021107958
Pharmacol Res. 2021 Oct 01. pii: S1043-6618(21)00506-5. [Epub ahead of print] 105922

Functions of the (pro)renin receptor (Atp6ap2) at molecular and system levels: pathological implications in hypertension, renal and brain development, inflammation, and fibrosis.

Nadin Hoffmann, Jörg Peters.

  The (pro)renin receptor [(P)RR, Atp6ap2] was initially discovered as a membrane-bound binding partner of prorenin and renin. A soluble (P)RR has additional paracrine effects and is involved in metabolic syndrome and kidney damage. Meanwhile it is clear that most of the effects of the (P)RR are independent of prorenin. In the kidney, (P)RR plays an important role in renal dysfunction by activating proinflammatory and profibrotic molecules. In the brain, (P)RR is expressed in cardiovascular regulatory nuclei and is linked to hypertension. (P)RR is known to be an essential component of the v-ATPase as a key accessory protein and plays an important role in kidney, brain and heart via regulating the pH of the extracellular space and intracellular compartments. V-ATPase and (P)RR together act on WNT and mTOR signalling pathways, which are responsible for cellular homeostasis and autophagy. (P)RR through its role in v-ATPase assembly and function is also important for fast recycling endocytosis by megalin. In the kidney, megalin together with v-ATPase and (P)RR is crucial for endocytic uptake of components in the RAS and their intracellular processing. In the brain, (P)RR, v-ATPases and megalin are important regulators both during development and in the adult. All three proteins are associated with diseases such as XLMR, XMRE, X-linked parkinsonism and epilepsy, cognitive disorders with Parkinsonism, spasticity, intellectual disability, and Alzheimer's Disease which are characterized by impaired neuronal function and/or neuronal loss. The present review focusses on the relevant effects of Atp6ap2 without assigning them necessarily to the RAS. Mechanistically, many effects can be well explained by the role of Atp6ap2 for v-ATPase assembly and function. Furthermore, application of a soluble (P)RR analogue as new therapeutic option is discussed.

Keywords:  (Pro)renin receptor; autophagy; endocytosis; hypertension; inflammation; kidney and brain; megalin; renin-angiotensin-system; v-ATPase

DOI:  https://doi.org/10.1016/j.phrs.2021.105922
EMBO Mol Med. 2021 Oct 04. e14434

Correction of oxidative stress enhances enzyme replacement therapy in Pompe disease.

Antonietta Tarallo, Carla Damiano, Sandra Strollo, Nadia Minopoli, Alessia Indrieri, Elena Polishchuk, Francesca Zappa, Edoardo Nusco, Simona Fecarotta, Caterina Porto, Marcella Coletta, Roberta Iacono, Marco Moracci, Roman Polishchuk, Diego Luis Medina, Paola Imbimbo, Daria Maria Monti, Maria Antonietta De Matteis, Giancarlo Parenti.

  Pompe disease is a metabolic myopathy due to acid alpha-glucosidase deficiency. In addition to glycogen storage, secondary dysregulation of cellular functions, such as autophagy and oxidative stress, contributes to the disease pathophysiology. We have tested whether oxidative stress impacts on enzyme replacement therapy with recombinant human alpha-glucosidase (rhGAA), currently the standard of care for Pompe disease patients, and whether correction of oxidative stress may be beneficial for rhGAA therapy. We found elevated oxidative stress levels in tissues from the Pompe disease murine model and in patients' cells. In cells, stress levels inversely correlated with the ability of rhGAA to correct the enzymatic deficiency. Antioxidants (N-acetylcysteine, idebenone, resveratrol, edaravone) improved alpha-glucosidase activity in rhGAA-treated cells, enhanced enzyme processing, and improved mannose-6-phosphate receptor localization. When co-administered with rhGAA, antioxidants improved alpha-glucosidase activity in tissues from the Pompe disease mouse model. These results indicate that oxidative stress impacts on the efficacy of enzyme replacement therapy in Pompe disease and that manipulation of secondary abnormalities may represent a strategy to improve the efficacy of therapies for this disorder.

Keywords:  N-acetylcysteine; Pompe disease; alpha-glucosidase; enzyme replacement therapy; oxidative stress

DOI:  https://doi.org/10.15252/emmm.202114434
Elife. 2021 Oct 07. pii: e72034. [Epub ahead of print]10

NHE6-depletion corrects ApoE4-mediated synaptic impairments and reduces amyloid plaque load.

Theresa Pohlkamp, Xunde Xian, Connie H Wong, Murat S Durakoglugil, Gordon Chandler Werthmann, Takaomi C Saido, Bret M Evers, Charles L White, Jade Connor, Robert E Hammer, Joachim Herz.

  Apolipoprotein E4 (ApoE4) is the most important and prevalent risk factor for late-onset Alzheimer's disease (AD). The isoelectric point of ApoE4 matches the pH of the early endosome (EE), causing its delayed dissociation from ApoE receptors and hence impaired endolysosomal trafficking, disruption of synaptic homeostasis and reduced amyloid clearance. We have shown that enhancing endosomal acidification by inhibiting the EE-specific sodium-hydrogen exchanger 6 (NHE6) restores vesicular trafficking and normalizes synaptic homeostasis. Remarkably and unexpectedly, loss of NHE6 (encoded by the gene Slc9a6) in mice effectively suppressed amyloid deposition even in the absence of ApoE4, suggesting that accelerated acidification of early endosomes caused by the absence of NHE6 occludes the effect of ApoE on amyloid plaque formation. NHE6 suppression or inhibition may thus be a universal, ApoE-independent approach to prevent amyloid buildup in the brain. These findings suggest a novel therapeutic approach for the prevention of AD by which partial NHE6 inhibition reverses the ApoE4 induced endolysosomal trafficking defect and reduces plaque load.

Keywords:  mouse; neuroscience

DOI:  https://doi.org/10.7554/eLife.72034
Am J Pathol. 2021 Oct 04. pii: S0002-9440(21)00433-8. [Epub ahead of print]

Chronic Exposure to Palmitic Acid Down-regulates AKT in Beta-Cells through Activation of mTOR.

Richa Aggarwal, Zhechu Peng, Ni Zeng, Joshua Silva, Lina He, Jingyu Chen, Anketse Debebe, Taojian Tu, Mario Alba, Chien-Yu Chen, Eileen X Stiles, Handan Hong, Bangyan L Stiles.

  High circulating lipids occurring in obese individuals and insulin resistant patients are considered a contributing factor to Type 2 Diabetes (T2D). Exposure to high lipids is proposed to both protect and damage beta-cells under different circumstances. Here, by feeding mice high fat diet (HFD) for 2 weeks up to 14 months, we showed that HFD initially causes the beta-cells to expand in population whereas long-term exposure to HFD is associated with failure of beta-cells and the inability of animals to respond to glucose challenge. To prevent the failure of beta-cells and the development of T2D, we explored the molecular mechanisms that underlie this biphasic response of beta-cells to lipid exposure. Using palmitic acid (PA) in cultured beta-cells and islets, we demonstrated that chronic exposure to lipids leads to reduced viability and inhibition of cell cycle progression concurrent with downregulation of a pro-growth/survival kinase AKT, independent of glucose. This AKT downregulation by PA is correlated with the induction of mTOR/S6K activity. Inhibiting mTOR activity with rapamycin induces raptor and restores AKT activity, allowing beta-cells to gain proliferation capacity that are lost after HFD exposure. In summary, we elucidated a novel mechanism for which lipid exposure may cause the dipole effects on beta-cell growth, where mTOR acts as a lipid sensor. These mechanisms can be novel targets for future therapeutic developments.

Keywords:  Beta-cells; Free Fatty Acid (FFA); High Fat Diet (HFD); Islet; Palmitic Acid; Rapamycin; S6K; mTOR; p-AKT

DOI:  https://doi.org/10.1016/j.ajpath.2021.09.008
J Ophthalmol. 2021 ;2021 5586659

The Effect of A2E on the Uptake and Release of Calcium in the Lysosomes and Mitochondria of Human RPE Cells Exposed to Blue Light.

Mao-Mei Luo, Lin Chen, Shu Wang, Chun Zeng, De-Zhi Li, YeGe Bi, Long-Qian Liu, Shan-Jun Cai.

We aimed to explore the effect of N-retinylidene-N-retinylethanolamine (A2E) on the uptake and release of calcium in lysosomes and mitochondria by establishing a model of human retinal pigment epithelial (RPE) cell injury induced by exposure to blue light. Primary human RPE cells were cultured from passages 4 to 6 and exposed to blue light at an intensity of 2000 ± 500 lux for 6 hours. After blue light exposure, the culture was maintained for 24 hours. A2E at a final concentration of 25 μM was added to the culture 2 hours before light exposure, and nifedipine at a final concentration of 10-4 M was added 1 hour before light exposure. The levels of Ca2+ in the cytosol (CaTM/2AM), mitochondria (Rhod/2AM), and lysosomes (LysoTracker Red and Fluo-3/AM) were determined. In order to measure the calcium levels in the different organelles, RPE were imaged using a laser scanning confocal microscope. Moreover, changes in the mitochondrial membrane potential were detected by flow cytometry analysis of JC-1-stained cells. The obtained results revealed that blue light illumination increased the calcium fluorescence intensity in the cytoplasm, mitochondria, and lysosomes of human RPE cells when compared with the control cells (P < 0.05). After A2E treatment, the fluorescence intensity of the calcium in the cytoplasm was further increased (P < 0.05), while that in the mitochondria and lysosomes decreased (P < 0.05). In addition, we observed that nifedipine reduced the fluorescence intensity of calcium in the RPE cells. Our results also showed that the mitochondrial membrane potential in the RPE treated with blue light and A2E was lower than that in the control, blue light, and A2E-treated cells (P < 0.05). Blue light increased calcium levels in the cytoplasm, lysosomes, and mitochondria of RPE cells. A2E damages the lysosomal and mitochondrial membranes, resulting in calcium release into the cytoplasm. Finally, our results demonstrated that both blue light and A2E treatments reduced mitochondrial membrane potential, increasing cytosolic Ca2+ levels, which can contribute to the activation of RPE death.

DOI: https://doi.org/10.1155/2021/5586659
Cell Death Dis. 2021 Oct 07. 12(10): 918

Identification of phosphoenolpyruvate carboxykinase 1 as a potential therapeutic target for pancreatic cancer.

Xiao-Ren Zhu, Shi-Qing Peng, Le Wang, Xiao-Yu Chen, Chun-Xia Feng, Yuan-Yuan Liu, Min-Bin Chen.

Pancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.

DOI: https://doi.org/10.1038/s41419-021-04201-w
ACS Chem Biol. 2021 Oct 05.

Blue Light Activated Rapamycin for Optical Control of Protein Dimerization in Cells and Zebrafish Embryos.

Taylor M Courtney, Kristie E Darrah, Trevor J Horst, Michael Tsang, Alexander Deiters.

Rapamycin-induced dimerization of FKBP and FRB is the most commonly utilized chemically induced protein dimerization system. It has been extensively used to conditionally control protein localization, split-enzyme activity, and protein-protein interactions in general by simply fusing FKBP and FRB to proteins of interest. We have developed a new aminonitrobiphenylethyl caging group and applied it to the generation of a caged rapamycin analog that can be photoactivated using blue light. Importantly, the caged rapamycin analog shows minimal background activity with regard to protein dimerization and can be directly interfaced with a wide range of established (and often commercially available) FKBP/FRB systems. We have successfully demonstrated its applicability to the optical control of enzymatic function, protein stability, and protein subcellular localization. Further, we also showcased its applicability toward optical regulation of cell signaling, specifically mTOR signaling, in cells and aquatic embryos.

DOI: https://doi.org/10.1021/acschembio.1c00547
Biomed Res Int. 2021 ;2021 4535349

Whole-Exome Sequencing Identified a Novel Homozygous Frameshift Mutation of HPS3 in a Consanguineous Family with Hermansky-Pudlak Syndrome.

Zhao-Xia Wang, Yi-Hui Liu, Yi Dong, Ya-Li Li, Tie-Yu Tang, Liang-Liang Fan.

Hermansky-Pudlak syndrome (HPS) is a rare genetic disorder with an autosomal recessive inherited pattern. It is mainly characterized by deficiencies in lysosome-related organelles, such as melanosomes and platelet-dense granules, and leads to albinism, visual impairment, nystagmus, and bleeding diathesis. A small number of patients will present with granulomatous colitis or fatal pulmonary fibrosis. At present, mutations in ten known genetic loci (HPS1-11) have been identified to be the genetic cause of HPS. In this study, we enrolled a consanguineous family who presented with typical HPS phenotypes, such as albinism, visual impairment, nystagmus, and bleeding diathesis. Whole-exome sequencing and Sanger sequencing were applied to explore the genetic lesions of the patient. A novel homozygous frameshift mutation (NM_032383.5, c.1231dupG/p.Aps411GlyfsTer32) of HPS3 was identified and cosegregated in the family members. Furthermore, real-time PCR confirmed that the mutation decreased the expression of HPS3, which has been identified as the disease-causing gene of HPS type 3. According to ACMG guidelines, the novel mutation, resulting in a premature stop codon at amino acid 442, is a pathogenic variant. In summary, we identified a novel mutation (NM_032383.5, c.1231dupG/p.Aps411GlyfsTer32) of HPS3 in a family with HPS. Our study expanded the variant spectrum of the HPS3 gene and contributed to genetic counseling and prenatal genetic diagnosis of the family.

DOI: https://doi.org/10.1155/2021/4535349
Nat Commun. 2021 Oct 07. 12(1): 5878

ATP-citrate lyase promotes axonal transport across species.

Aviel Even, Giovanni Morelli, Silvia Turchetto, Michal Shilian, Romain Le Bail, Sophie Laguesse, Nathalie Krusy, Ariel Brisker, Alexander Brandis, Shani Inbar, Alain Chariot, Frédéric Saudou, Paula Dietrich, Ioannis Dragatsis, Bert Brone, Loïc Broix, Jean-Michel Rigo, Miguel Weil, Laurent Nguyen.

Microtubule (MT)-based transport is an evolutionary conserved process finely tuned by posttranslational modifications. Among them, α-tubulin acetylation, primarily catalyzed by a vesicular pool of α-tubulin N-acetyltransferase 1 (Atat1), promotes the recruitment and processivity of molecular motors along MT tracks. However, the mechanism that controls Atat1 activity remains poorly understood. Here, we show that ATP-citrate lyase (Acly) is enriched in vesicles and provide Acetyl-Coenzyme-A (Acetyl-CoA) to Atat1. In addition, we showed that Acly expression is reduced upon loss of Elongator activity, further connecting Elongator to Atat1 in a pathway regulating α-tubulin acetylation and MT-dependent transport in projection neurons, across species. Remarkably, comparable defects occur in fibroblasts from Familial Dysautonomia (FD) patients bearing an autosomal recessive mutation in the gene coding for the Elongator subunit ELP1. Our data may thus shine light on the pathophysiological mechanisms underlying FD.

DOI: https://doi.org/10.1038/s41467-021-25786-y
Bio Protoc. 2021 Sep 05. 11(17): e4140

Measurement of LRRK2 Kinase Activity by Proximity Ligation Assay.

Matthew T Keeney, Eric K Hoffman, Timothy J Greenamyre, Roberto Di Maio.

  Missense mutations in leucine rich-repeat kinase 2 (LRRK2) cause forms of familial Parkinson's disease and have been linked to 'idiopathic' Parkinson's disease. Assessment of LRRK2 kinase activity has been very challenging due to its size, complex structure, and relatively low abundance. A standard in the field to assess LRRK2 kinase activity is to measure the level of substrate phosphorylation (pThr73-Rab10) or autophosphorylation of serine 1292 (i.e., phosphoserine 1292; pS1292). The levels of pS1292 have typically been assessed by western blotting, which limits cellular and anatomical resolution. Here, we describe the method for a novel proximity ligation assay (PLA) that can detect endogenous LRRK2 kinase activity (PLA LRRK2) in situ at cellular and subcellular resolutions. PLA is a fluorescence- or chromogen-based assay that can be used to either (1) detect protein-protein interactions or (2) detect and amplify post-translational modifications on proteins. We used PLA for in situ detection and amplification of LRRK2 autophosphorylation levels at serine 1292. Our findings demonstrate that PLA LRRK2 is a highly sensitive and specific assay that can be used for assessing kinase activity in cultured cells and postmortem tissues.

Keywords:  Fluorescence; LRRK2; Parkinson’s disease; Proximity ligation assay

DOI:  https://doi.org/10.21769/BioProtoc.4140
Nat Chem Biol. 2021 Oct 06.

Author Correction: Selective inhibitors of mTORC1 activate 4EBP1 and suppress tumor growth.

Bianca J Lee, Jacob A Boyer, G Leslie Burnett, Arun P Thottumkara, Nidhi Tibrewal, Stacy L Wilson, Tientien Hsieh, Abby Marquez, Edward G Lorenzana, James W Evans, Laura Hulea, Gert Kiss, Hui Liu, Dong Lee, Ola Larsson, Shannon McLaughlan, Ivan Topisirovic, Zhengping Wang, Zhican Wang, Yongyuan Zhao, David Wildes, James B Aggen, Mallika Singh, Adrian L Gill, Jacqueline A M Smith, Neal Rosen.

DOI: https://doi.org/10.1038/s41589-021-00905-4
Mol Aspects Med. 2021 Oct 05. pii: S0098-2997(21)00101-1. [Epub ahead of print] 101041

Role of autophagy in muscle disease.

Anais Franco-Romero, Marco Sandri.

  Beside inherited muscle diseases many catabolic conditions such as insulin resistance, malnutrition, cancer growth, aging, infections, chronic inflammatory status, inactivity, obesity are characterized by loss of muscle mass, strength and function. The decrease of muscle quality and quantity increases morbidity, mortality and has a major impact on the quality of life. One of the pathogenetic mechanisms of muscle wasting is the dysregulation of the main protein and organelles quality control system of the cell: the autophagy-lysosome. This review will focus on the role of the autophagy-lysosome system in the different conditions of muscle loss. We will also dissect the signalling pathways that are involved in excessive or defective autophagy regulation. Finally, the state of the art of autophagy modulators that have been used in preclinical or clinical studies to ameliorate muscle mass will be also described.

Keywords:  Atrophy; Autophagy; Disuse; Muscle wasting; Proteostasis; Skeletal muscle

DOI:  https://doi.org/10.1016/j.mam.2021.101041