bims-lysosi 2021-07-04 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021‒07‒04
thirty papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Vacuolar H+-ATPase dysfunction rescues intralumenal vesicle cargo sorting in yeast lacking PI(3,5)P2 or Doa4.
Amino Acid-Mediated Intracellular Ca2+ Rise Modulates mTORC1 by Regulating the TSC2-Rheb Axis through Ca2+/Calmodulin.
The Glucosylceramide Synthase Inhibitor PDMP Causes Lysosomal Lipid Accumulation and mTOR Inactivation.
Regulation of mTORC1 activity by the Golgi apparatus.
Lysosomal Storage Disorders: Molecular Basis and Therapeutic Approaches.
mTOR Driven Gene Transcription Is Required for Cholesterol Production in Neurons of the Developing Cerebral Cortex.
Analysis of cathepsin B and cathepsin L treatment to clear toxic lysosomal protein aggregates in neuronal ceroid lipofuscinosis.
Mammalian/mechanistic target of rapamycin (mTOR) complexes in neurodegeneration.
Acid ceramidase improves mitochondrial function and oxidative stress in Niemann-Pick type C disease by repressing STARD1 expression and mitochondrial cholesterol accumulation.
Accumulation of saposin in dystrophic neurites is linked to impaired lysosomal functions in Alzheimer's disease brains.
TM4SF5 knockout protects mice from diet-induced obesity partly by regulating autophagy in adipose tissue.
Targeting of Lysosomal Pathway Genes for Parkinson's Disease Modification: Insights From Cellular and Animal Models.
In Silico and In Cell Hybrid Selection of Nonrapalog Ligands to Allosterically Inhibit the Kinase Activity of mTORC1.
Plasma Proteomic Analysis in Morquio A Disease.
Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes.
Inhibition of iduronic acid biosynthesis by ebselen reduces GAG accumulation in MPS-I fibroblasts.
Brain Symptoms of Tuberous Sclerosis Complex: Pathogenesis and Treatment.
Chemical and Structural Strategies to Selectively Target mTOR Kinase.
Human α-Galactosidase A Mutants: Priceless Tools to Develop Novel Therapies for Fabry Disease.
Cathepsin D-Managing the Delicate Balance.
LRRK2 recruitment, activity, and function in organelles.
Glucosylceramide Associated with Gaucher Disease Forms Amyloid-like Twisted Ribbon Fibrils That Induce α-Synuclein Aggregation.
Genome editing in lysosomal disorders.
Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans.
Legionella pneumophila LegC7 effector protein drives aberrant ER:endosome contacts in yeast.
A Golgi-derived vesicle potentiates PtdIns4P to PtdIns3P conversion for endosome fission.
Functional and structural analyses of novel Smith-Kingsmore Syndrome-Associated MTOR variants reveal potential new mechanisms and predictors of pathogenicity.
The V-ATPase a3 Subunit: Structure, Function and Therapeutic Potential of an Essential Biomolecule in Osteoclastic Bone Resorption.
MPS I Gene Therapy.
Neu1 deficiency induces abnormal emotional behavior in zebrafish.

J Cell Sci. 2021 Jul 01. pii: jcs.258459. [Epub ahead of print]

Vacuolar H+-ATPase dysfunction rescues intralumenal vesicle cargo sorting in yeast lacking PI(3,5)P2 or Doa4.

Zachary N Wilson, Dalton Buysse, Matt West, Daniel Ahrens, Greg Odorizzi.

  Endosomes undergo a maturation process highlighted by a reduction in lumenal pH, a conversion of surface markers that prime endosome-lysosome fusion, and the sequestration of ubiquitinated transmembrane protein cargoes within intralumenal vesicles (ILVs). We investigated ILV cargo sorting in mutant strains of the budding yeast Saccharomyces cerevisiae that are deficient for either the lysosomal/vacuolar signaling lipid PI(3,5)P2 or the Doa4 ubiquitin hydrolase that deubiquitinates ILV cargoes. Disruption of PI(3,5)P2 synthesis or Doa4 function causes a defect in the sorting of a subset of ILV cargoes. We show that these cargo-sorting defects are suppressed by mutations that disrupt Vph1, which is a subunit of Vacuolar H+-ATPase (V-ATPase) complexes that acidify late endosomes and vacuoles. We further show that Vph1 dysfunction increases endosome abundance and disrupts vacuolar localization of Ypt7 and Vps41, two critical mediators of endosome-vacuole fusion. Because V-ATPase inhibition attenuates endosome-vacuole fusion and rescues the ILV cargo-sorting defects in yeast lacking PI(3,5)P2 and Doa4 activity, our results suggest that the V-ATPase performs a role in the coordination of ILV cargo sorting with the membrane fusion machinery.

Keywords:  Endosomes; Intralumenal vesicles; Membrane fusion; V-ATPase

DOI:  https://doi.org/10.1242/jcs.258459
Int J Mol Sci. 2021 Jun 27. pii: 6897. [Epub ahead of print]22(13):

Amino Acid-Mediated Intracellular Ca2+ Rise Modulates mTORC1 by Regulating the TSC2-Rheb Axis through Ca2+/Calmodulin.

Yuna Amemiya, Nao Nakamura, Nao Ikeda, Risa Sugiyama, Chiaki Ishii, Masatoshi Maki, Hideki Shibata, Terunao Takahara.

  Mechanistic target of rapamycin complex 1 (mTORC1) is a master growth regulator by controlling protein synthesis and autophagy in response to environmental cues. Amino acids, especially leucine and arginine, are known to be important activators of mTORC1 and to promote lysosomal translocation of mTORC1, where mTORC1 is thought to make contact with its activator Rheb GTPase. Although amino acids are believed to exclusively regulate lysosomal translocation of mTORC1 by Rag GTPases, how amino acids increase mTORC1 activity besides regulation of mTORC1 subcellular localization remains largely unclear. Here we report that amino acids also converge on regulation of the TSC2-Rheb GTPase axis via Ca2+/calmodulin (CaM). We showed that the amino acid-mediated increase of intracellular Ca2+ is important for mTORC1 activation and thereby contributes to the promotion of nascent protein synthesis. We found that Ca2+/CaM interacted with TSC2 at its GTPase activating protein (GAP) domain and that a CaM inhibitor reduced binding of CaM with TSC2. The inhibitory effect of a CaM inhibitor on mTORC1 activity was prevented by loss of TSC2 or by an active mutant of Rheb GTPase, suggesting that a CaM inhibitor acts through the TSC2-Rheb axis to inhibit mTORC1 activity. Taken together, in response to amino acids, Ca2+/CaM-mediated regulation of the TSC2-Rheb axis contributes to proper mTORC1 activation, in addition to the well-known lysosomal translocation of mTORC1 by Rag GTPases.

Keywords:  Rheb GTPase; TSC; amino acid; calcium; calmodulin; mTOR

DOI:  https://doi.org/10.3390/ijms22136897
Int J Mol Sci. 2021 Jun 30. pii: 7065. [Epub ahead of print]22(13):

The Glucosylceramide Synthase Inhibitor PDMP Causes Lysosomal Lipid Accumulation and mTOR Inactivation.

Pia Hartwig, Doris Höglinger.

  For many years, the biology of glycosphingolipids was elucidated with the help of glucosylceramide synthase (GCS) inhibitors such as 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP). Additionally, PDMP gained interest because of its chemosensitizing effects. Several studies have successfully combined PDMP and anti-cancer drugs in the context of cancer therapy. However, the mechanism of action of PDMP is not fully understood and seems to go beyond glycolipid inhibition. Here, we used a functionalized sphingosine analogue (pacSph) to investigate the acute effects of PDMP on cellular sphingolipid distribution and found that PDMP, but not other GCS inhibitors, such as ND-DNJ (also called Miglustat), induced sphingolipid accumulation in lysosomes. This effect could be connected to defective export from lysosome, as monitored by the prolonged lysosomal staining of sphingolipids as well as by a delay in the metabolic conversion of the pacSph precursor. Additionally, other lipids such as lysobisphosphatidic acid (LBPA) and cholesterol were enriched in lysosomes upon PDMP treatment in a time-dependent manner. We could further correlate early LBPA enrichment with dissociation of the mechanistic target of rapamycin (mTOR) from lysosomes followed by nuclear translocation of its downtream target, transcription factor EB (TFEB). Altogether, we report here a timeline of lysosomal lipid accumulation events and mTOR inactivation arising from PDMP treatment.

Keywords:  LBPA; cholesterol; clickable lipids; glycolipids; lysosomal biology; sphingolipids

DOI:  https://doi.org/10.3390/ijms22137065
Fac Rev. 2021 ;10 50

Regulation of mTORC1 activity by the Golgi apparatus.

Christian Makhoul, Paul A Gleeson.

  Mechanistic (or mammalian) target of rapamycin complex 1 (mTORC1) is a major signalling kinase in cells that regulates proliferation and metabolism and is controlled by extrinsic and intrinsic signals. The lysosome has received considerable attention as a major hub of mTORC1 activation. However, mTOR has also been located to a variety of other intracellular sites, indicating the possibility of spatial regulation of mTORC1 signalling within cells. In particular, there have been numerous recent reports of mTORC1 activation associated with the Golgi apparatus. Here, we review the evidence for the regulation of mTORC1 signalling at the Golgi in mammalian cells. mTORC1 signalling is closely linked to the morphology of the Golgi architecture; a number of Golgi membrane tethers/scaffolds that influence Golgi architecture in mammalian cells that directly or indirectly regulate mTORC1 activation have been identified. Perturbation of the Golgi mTORC1 pathway arising from fragmentation of the Golgi has been shown to promote oncogenesis. Here, we highlight the potential mechanisms for the activation mTORC1 at the Golgi, which is emerging as a major site for mTORC1 signalling.

Keywords:  Arf1; GAT4; GCC88; GOLPH3; Golgi architecture; Rab1A; actin; mTORC1; signalling; trans-Golgi network

DOI:  https://doi.org/10.12703/r/10-50
Biomolecules. 2021 Jun 30. pii: 964. [Epub ahead of print]11(7):

Lysosomal Storage Disorders: Molecular Basis and Therapeutic Approaches.

Enrico Moro.

  Lysosomal storage disorders (LSDs) are a group of 60 rare inherited diseases characterized by a heterogeneous spectrum of clinical symptoms, ranging from severe intellectual disabilities, cardiac abnormalities, visceromegaly, and bone deformities to slowly progressive muscle weakness, respiratory insufficiency, eye defects (corneal clouding and retinal degeneration), and skin alterations [...].

Keywords:  biomarkers; gene therapy; lysosomal storage disorders

DOI:  https://doi.org/10.3390/biom11070964
Int J Mol Sci. 2021 Jun 03. pii: 6034. [Epub ahead of print]22(11):

mTOR Driven Gene Transcription Is Required for Cholesterol Production in Neurons of the Developing Cerebral Cortex.

Martin Schüle, Tamer Butto, Sri Dewi, Laura Schlichtholz, Susanne Strand, Susanne Gerber, Kristina Endres, Susann Schweiger, Jennifer Winter.

  Dysregulated mammalian target of rapamycin (mTOR) activity is associated with various neurodevelopmental disorders ranging from idiopathic autism spectrum disorders (ASD) to syndromes caused by single gene defects. This suggests that maintaining mTOR activity levels in a physiological range is essential for brain development and functioning. Upon activation, mTOR regulates a variety of cellular processes such as cell growth, autophagy, and metabolism. On a molecular level, however, the consequences of mTOR activation in the brain are not well understood. Low levels of cholesterol are associated with a wide variety of neurodevelopmental disorders. We here describe numerous genes of the sterol/cholesterol biosynthesis pathway to be transcriptionally regulated by mTOR complex 1 (mTORC1) signaling in vitro in primary neurons and in vivo in the developing cerebral cortex of the mouse. We find that these genes are shared targets of the transcription factors SREBP, SP1, and NF-Y. Prenatal as well as postnatal mTORC1 inhibition downregulated expression of these genes which directly translated into reduced cholesterol levels, pointing towards a substantial metabolic function of the mTORC1 signaling cascade. Altogether, our results indicate that mTORC1 is an essential transcriptional regulator of the expression of sterol/cholesterol biosynthesis genes in the developing brain. Altered expression of these genes may be an important factor contributing to the pathogenesis of neurodevelopmental disorders associated with dysregulated mTOR signaling.

Keywords:  NF-Y; SP1; SREBP; cholesterol; mTOR; mTORC1; neurogenesis

DOI:  https://doi.org/10.3390/ijms22116034
Biochim Biophys Acta Mol Basis Dis. 2021 Jun 29. pii: S0925-4439(21)00138-1. [Epub ahead of print] 166205

Analysis of cathepsin B and cathepsin L treatment to clear toxic lysosomal protein aggregates in neuronal ceroid lipofuscinosis.

Alessandro Di Spiezio, André R A Marques, Lina Schmidt, Niklas Thießen, Lisa Gallwitz, Jens Fogh, Udo Bartsch, Paul Saftig.

  Proteolysis mediated by lysosomal cathepsin proteases maintains a physiological flow in autophagy, phagocytosis and endocytosis. Neuronal Ceroid Lipofuscinosis (NCL) is a childhood neurodegenerative disorder characterized by disturbed autophagic flow and pathological accumulation of proteins. We demonstrated a therapeutic clearance of protein aggregates after dosing NCL10 mice with recombinant human pro-cathepsin-D. Prompted by these results and speculating that cathepsins may act in a redundant and in an hierarchical manner we envisaged that a treatment with human recombinant cysteine proteases pro-cathepsin-L (proCTSL) and pro-cathepsin-B (proCTSB) could similarly be used to induce protein degradation. Both enzymes were taken up by mannose 6-phosphate receptor- and LRP-receptor-mediated endocytosis and processed to the lysosomal mature cathepsins. In murine NCL10 astrocytes an abnormal increase in LAMP1 and saposin expression was revealed. Although proCTSB application did not improve this phenotype, proCTSL treatment led to reduced saposin-C levels in this model as well as in an acute brain slice model. Intracerebral dosing in a NCL10 mouse model revealed cellular and lysosomal uptake of both enzymes. Only proCTSL mildly reduced saposin-C levels and attenuated reactive astrogliosis. Application of both proteases did not improve weight loss and mortality of mutant mice. Our data reveal that although recombinant lysosomal proteases can be efficiently delivered to neuronal lysosomes cysteine proteases are less efficient in protein aggregates clearance as compared to the cathepsin-D treatment. Our data including in vitro degradation assays support the idea that bulk proteolysis requires a hierarchical process in which both aspartyl and cysteine hydrolases play a role.

Keywords:  Cathepsin B; bulk proteolysis; cathepsin D; cathepsin L; enzyme replacement therapy; neuronal ceroid lipofuscinosis 10

DOI:  https://doi.org/10.1016/j.bbadis.2021.166205
Mol Neurodegener. 2021 Jul 02. 16(1): 44

Mammalian/mechanistic target of rapamycin (mTOR) complexes in neurodegeneration.

Henry Querfurth, Han-Kyu Lee.

  Novel targets to arrest neurodegeneration in several dementing conditions involving misfolded protein accumulations may be found in the diverse signaling pathways of the Mammalian/mechanistic target of rapamycin (mTOR). As a nutrient sensor, mTOR has important homeostatic functions to regulate energy metabolism and support neuronal growth and plasticity. However, in Alzheimer's disease (AD), mTOR alternately plays important pathogenic roles by inhibiting both insulin signaling and autophagic removal of β-amyloid (Aβ) and phospho-tau (ptau) aggregates. It also plays a role in the cerebrovascular dysfunction of AD. mTOR is a serine/threonine kinase residing at the core in either of two multiprotein complexes termed mTORC1 and mTORC2. Recent data suggest that their balanced actions also have implications for Parkinson's disease (PD) and Huntington's disease (HD), Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). Beyond rapamycin; an mTOR inhibitor, there are rapalogs having greater tolerability and micro delivery modes, that hold promise in arresting these age dependent conditions.

Keywords:  Akt; Alzheimer’s; Insulin signaling; Parkinson’s; Rapamycin; mTOR

DOI:  https://doi.org/10.1186/s13024-021-00428-5
Redox Biol. 2021 Jun 19. pii: S2213-2317(21)00211-1. [Epub ahead of print]45 102052

Acid ceramidase improves mitochondrial function and oxidative stress in Niemann-Pick type C disease by repressing STARD1 expression and mitochondrial cholesterol accumulation.

Sandra Torres, Estel Solsona-Vilarrasa, Susana Nuñez, Nuria Matías, Naroa Insausti-Urkia, Fernanda Castro, Mireia Casasempere, Gemma Fabriás, Josefina Casas, Carlos Enrich, José C Fernández-Checa, Carmen Garcia-Ruiz.

  Niemann-Pick type C (NPC) disease, a lysosomal storage disorder caused by defective NPC1/NPC2 function, results in the accumulation of cholesterol and glycosphingolipids in lysosomes of affected organs, such as liver and brain. Moreover, increase of mitochondrial cholesterol (mchol) content and impaired mitochondrial function and GSH depletion contribute to NPC disease. However, the underlying mechanism of mchol accumulation in NPC disease remains unknown. As STARD1 is crucial in intramitochondrial cholesterol trafficking and acid ceramidase (ACDase) has been shown to regulate STARD1, we explored the functional relationship between ACDase and STARD1 in NPC disease. Liver and brain of Npc1-/- mice presented a significant increase in mchol levels and STARD1 expression. U18666A, an amphiphilic sterol that inhibits lysosomal cholesterol efflux, increased mchol levels in hepatocytes from Stard1f/f mice but not Stard1ΔHep mice. We dissociate the induction of STARD1 expression from endoplasmic reticulum stress, and establish an inverse relationship between ACDase and STARD1 expression and LRH-1 levels. Hepatocytes from Npc1+/+ mice treated with U18666A exhibited increased mchol accumulation, STARD1 upregulation and decreased ACDase expression, effects that were reversed by cholesterol extraction with 2-hydroxypropyl-β-cyclodextrin. Moreover, transfection of fibroblasts from NPC patients with ACDase, decreased STARD1 expression and mchol accumulation, resulting in increased mitochondrial GSH levels, improved mitochondrial functional performance, decreased oxidative stress and protected NPC fibroblasts against oxidative stress-mediated cell death. Our results demonstrate a cholesterol-dependent inverse relationship between ACDase and STARD1 and provide a novel approach to target the accumulation of cholesterol in mitochondria in NPC disease.

Keywords:  Acid ceramiase; Cholesterol; Mitochondrial function; NPC disease; Oxidative stress

DOI:  https://doi.org/10.1016/j.redox.2021.102052
Mol Neurodegener. 2021 Jul 02. 16(1): 45

Accumulation of saposin in dystrophic neurites is linked to impaired lysosomal functions in Alzheimer's disease brains.

Md Golam Sharoar, Sarah Palko, Yingying Ge, Takaomi C Saido, Riqiang Yan.

  Neuritic plaques in Alzheimer's disease (AD) brains refer to β-amyloid (Aβ) plaques surrounded by dystrophic neurites (DNs), activated microglia and reactive astrocytes. Most recently, we showed that DNs form sequentially in three layers during plaque growth. Although lysosomal proteins such as LAMP1 are found in DNs, it is not clear how many and how early lysosomal proteins are involved in forming neuritic plaques. To answer this unmet question, we examined APP knock-in (APPNL-G-F), 5xFAD and APP/PS1ΔE9 mouse brains and found that the lysosomal activator proteins saposins (SAPs) and LAMP1 were accumulated to surround Aβ plaques at the earliest stage, namely the 1st layer of DNs. Noticeably, lysosomal hydrolases were not detectable in these early DNs, suggesting that DNs at this early stage likely enrich dysfunctional lysosomes. In old AD mouse brains and in the later stage of human AD brains, SAP-C+-DNs and LAMP1+-DNs were gradually reduced in concomitant with the growth of amyloid plaques. Remarkably, the observed LAMP1 immunoreactivity near plaques in aged AD mouse and human brains were actually associated with disease-associated microglia rather than neuronal sources, likely reflecting more severely impaired lysosomal functions in neurons. Western blot analyses showed increased levels of SAP-C in AD mouse brains, and Aβ oligomers induced elevated levels of SAP-C in cellular assays. The elevated protein levels of SAP-C in AD mouse brains during plaque growth potentially contributed lysosomal membrane leakage and loss of hydrolases. Together, our study indicates that lysosomal functions are impaired by being entrapped in DNs early during plaque growth, and this may viciously facilitate growth of amyloid plaques.

Keywords:  ATG9; Aging; Alzheimer’s disease; Dystrophic neurites; LAMP1 and galectin-3; Lysosomes; Prosaposin; Saposin-C

DOI:  https://doi.org/10.1186/s13024-021-00464-1
Diabetes. 2021 Jun 29. pii: db210145. [Epub ahead of print]

TM4SF5 knockout protects mice from diet-induced obesity partly by regulating autophagy in adipose tissue.

Cheoljun Choi, Yeonho Son, Jinyoung Kim, Yoon Keun Cho, Abhirup Saha, Minsu Kim, Hyeonyeong Im, Kyungmin Kim, Juhyeong Han, Jung Weon Lee, Je Kyung Seong, Yun-Hee Lee.

Transmembrane 4 L six family member 5 (TM4SF5) functions as a sensor for lysosomal arginine levels and activates the mammalian target of rapamycin complex 1 (mTORC1). While the mTORC1 signaling pathway plays a key role in adipose tissue metabolism, the regulatory function of TM4SF5 in adipocytes remains unclear. This study aimed to establish a TM4SF5 knockout (KO) mouse model and investigated the effects of TM4SF5 KO on mTORC1 signaling-mediated autophagy and mitochondrial metabolism in adipose tissue. TM4SF5 expression was higher in inguinal white adipose tissue (iWAT) than in brown adipose tissue and significantly upregulated by a high-fat diet (HFD). TM4SF5 KO reduced mTORC1 activation and enhanced autophagy and lipolysis in adipocytes. RNAseq analysis of TM4SF5 KO mouse iWAT showed that the expression of genes involved in peroxisome proliferator-activated receptor alpha signaling pathways and mitochondrial oxidative metabolism were upregulated. Consequently, TM4SF5 KO reduced adiposity and increased energy expenditure and mitochondrial oxidative metabolism. TM4SF5 KO prevented HFD-induced glucose intolerance and inflammation in adipose tissue. Collectively, our study demonstrated that TM4SF5 can regulate autophagy and lipid catabolism in adipose tissue and suggested that TM4SF5 could be therapeutically targeted for the treatment of obesity-related metabolic diseases.

DOI: https://doi.org/10.2337/db21-0145
Front Neurol. 2021 ;12 681369

Targeting of Lysosomal Pathway Genes for Parkinson's Disease Modification: Insights From Cellular and Animal Models.

Tetsuro Abe, Tomoki Kuwahara.

  Previous genetic studies on hereditary Parkinson's disease (PD) have identified a set of pathogenic gene mutations that have strong impacts on the pathogenicity of PD. In addition, genome-wide association studies (GWAS) targeted to sporadic PD have nominated an increasing number of genetic variants that influence PD susceptibility. Although the clinical and pathological characteristics in hereditary PD are not identical to those in sporadic PD, α-synuclein, and LRRK2 are definitely associated with both types of PD, with LRRK2 mutations being the most frequent cause of autosomal-dominant PD. On the other hand, a significant portion of risk genes identified from GWAS have been associated with lysosomal functions, pointing to a critical role of lysosomes in PD pathogenesis. Experimental studies have suggested that the maintenance or upregulation of lysosomal activity may protect against neuronal dysfunction or degeneration. Here we focus on the roles of representative PD gene products that are implicated in lysosomal pathway, namely LRRK2, VPS35, ATP13A2, and glucocerebrosidase, and provide an overview of their disease-associated functions as well as their cooperative actions in the pathogenesis of PD, based on the evidence from cellular and animal models. We also discuss future perspectives of targeting lysosomal activation as a possible strategy to treat neurodegeneration.

Keywords:  ATP13A2; GBA; LRRK2; VPS35; lysosome; α-synuclein

DOI:  https://doi.org/10.3389/fneur.2021.681369
J Med Chem. 2021 Jun 30.

In Silico and In Cell Hybrid Selection of Nonrapalog Ligands to Allosterically Inhibit the Kinase Activity of mTORC1.

Raef Shams, Akihiro Matsukawa, Yukari Ochi, Yoshihiro Ito, Hideyuki Miyatake.

Cancer-specific metabolic alterations hyperactivate the kinase activity of the mammalian/mechanistic target of rapamycin (mTOR) for overcoming stressful environments. Rapalogs, which allosterically inhibit mTOR complex 1 (mTORC1), have been approved as anticancer agents. However, the immunosuppressive side effect of these compounds results in the promotion of tumor metastasis, thereby limiting their therapeutic efficacy. We first report a nonrapalog inhibitor, WRX606, identified by a hybrid strategy of in silico and in cell selections. Our studies showed that WRX606 formed a ternary complex with FK506-binding protein-12 (FKBP12) and FKBP-rapamycin-binding (FRB) domain of mTOR, resulting in the allosteric inhibition of mTORC1. WRX606 inhibited the phosphorylation of not only the ribosomal protein S6 kinase 1 (S6K1) but also eIF4E-binding protein-1 (4E-BP1). Hence, WRX606 efficiently suppressed tumor growth in mice without promotion of metastasis. These results suggest that WRX606 is a potent lead compound for developing anticancer drugs discovered by in silico and in cell methods.

DOI: https://doi.org/10.1021/acs.jmedchem.1c00536
Int J Mol Sci. 2021 Jun 07. pii: 6165. [Epub ahead of print]22(11):

Plasma Proteomic Analysis in Morquio A Disease.

José V Álvarez, Susana B Bravo, María Pilar Chantada-Vázquez, Sofía Barbosa-Gouveia, Cristóbal Colón, Olalla López-Suarez, Shunji Tomatsu, Francisco J Otero-Espinar, María L Couce.

  Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal disease caused by mutations in the gene encoding the enzymeN-acetylgalactosamine-6-sulfate sulfatase (GALNS), and is characterized by systemic skeletal dysplasia due to excessive storage of keratan sulfate (KS) and chondroitin-6-sulfate in chondrocytes. Although improvements in the activity of daily living and endurance tests have been achieved with enzyme replacement therapy (ERT) with recombinant human GALNS, recovery of bone lesions and bone growth in MPS IVA has not been demonstrated to date. Moreover, no correlation has been described between therapeutic efficacy and urine levels of KS, which accumulates in MPS IVA patients. The objective of this study was to assess the validity of potential biomarkers proposed by other authors and to identify new biomarkers. To identify candidate biomarkers of this disease, we analyzed plasma samples from healthy controls (n=6) and from untreated (n=8) and ERT-treated (n=5, sampled before and after treatment) MPS IVA patients using both qualitative and quantitative proteomics analyses. The qualitative proteomics approach analyzed the proteomic profile of the different study groups. In the quantitative analysis, we identified/quantified 215 proteins after comparing healthy control untreated, ERT-treated MPSIVA patients. We selected a group of proteins that were dysregulated in MPS IVA patients. We identified four potential protein biomarkers, all of which may influence bone and cartilage metabolism: fetuin-A, vitronectin, alpha-1antitrypsin, and clusterin. Further studies of cartilage and bone samples from MPS IVA patients will be required to verify the validity of these proteins as potential biomarkers of MPS IVA.

Keywords:  biomarkers; enzyme replacement therapy; lysosomal disorders; proteomics

DOI:  https://doi.org/10.3390/ijms22116165
J Cell Biol. 2021 Sep 06. pii: e202005072. [Epub ahead of print]220(9):

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes.

Charlene E Lancaster, Aaron Fountain, Roaya M Dayam, Elliott Somerville, Javal Sheth, Vanessa Jacobelli, Alex Somerville, Mauricio R Terebiznik, Roberto J Botelho.

Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as "phagosome resolution." Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintaining the degradative power of macrophages to handle multiple waves of phagocytosis.

DOI: https://doi.org/10.1083/jcb.202005072
Glycobiology. 2021 Jun 29. pii: cwab066. [Epub ahead of print]

Inhibition of iduronic acid biosynthesis by ebselen reduces GAG accumulation in MPS-I fibroblasts.

Marco Maccarana, Emil Tykesson, Edgar M Pera, Nadège Gouignard, Jianping Fang, Anders Malmström, Giancarlo Ghiselli, Jin-Ping Li.

  Mucopolysaccharidosis type one (MPS-I) is a rare lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase which removes iduronic acid in both chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) and thereby contributes to the catabolism of glycosaminoglycans (GAGs). To ameliorate this genetic defect, the patients are currently treated by enzyme replacement and bone marrow transplantation, which have a number of drawbacks. This study was designed to develop an alternative treatment by inhibition of iduronic acid formation. By screening the Prestwick drug library, we identified ebselen as a potent inhibitor of enzymes that produce iduronic acid in CS/DS and HS. Ebselen efficiently inhibited iduronic acid formation during CS/DS synthesis in cultured fibroblasts. Treatment of MPS-I fibroblasts with ebselen not only reduced accumulation of CS/DS, but also promoted GAG degradation. In early Xenopus embryos, this drug phenocopied the effect of downregulation of DS-epimerase 1, the main enzyme responsible for iduronic production in CS/DS, suggesting that ebselen inhibits iduronic acid production in vivo. However, ebselen failed to ameliorate the CS/DS and GAG burden in MPS-I mice. Nevertheless, the results propose a potential of iduronic acid substrate reduction therapy for MPS-I patients.

Keywords:  ebselen; epimerases; mucopolysaccharidosis type I; schondroitin dermatan sulfate; substrate reduction therapy

DOI:  https://doi.org/10.1093/glycob/cwab066
Int J Mol Sci. 2021 Jun 22. pii: 6677. [Epub ahead of print]22(13):

Brain Symptoms of Tuberous Sclerosis Complex: Pathogenesis and Treatment.

Masashi Mizuguchi, Maki Ohsawa, Hirofumi Kashii, Atsushi Sato.

  The mammalian target of the rapamycin (mTOR) system plays multiple, important roles in the brain, regulating both morphology, such as cellular size, shape, and position, and function, such as learning, memory, and social interaction. Tuberous sclerosis complex (TSC) is a congenital disorder caused by a defective suppressor of the mTOR system, the TSC1/TSC2 complex. Almost all brain symptoms of TSC are manifestations of an excessive activity of the mTOR system. Many children with TSC are afflicted by intractable epilepsy, intellectual disability, and/or autism. In the brains of infants with TSC, a vicious cycle of epileptic encephalopathy is formed by mTOR hyperactivity, abnormal synaptic structure/function, and excessive epileptic discharges, further worsening epilepsy and intellectual/behavioral disorders. Molecular target therapy with mTOR inhibitors has recently been proved to be efficacious for epilepsy in human TSC patients, and for autism in TSC model mice, indicating the possibility for pharmacological treatment of developmental synaptic disorders.

Keywords:  TSC; autism; epilepsy; epileptic encephalopathy; intellectual disability; mTOR inhibitor; mTORopathy

DOI:  https://doi.org/10.3390/ijms22136677
ChemMedChem. 2021 Jun 11.

Chemical and Structural Strategies to Selectively Target mTOR Kinase.

Chiara Borsari, Martina De Pascale, Matthias P Wymann.

  Dysregulation of the mechanistic target of rapamycin (mTOR) pathway is implicated in cancer and neurological disorder, which identifies mTOR inhibition as promising strategy for the treatment of a variety of human disorders. First-generation mTOR inhibitors include rapamycin and its analogues (rapalogs) which act as allosteric inhibitors of TORC1. Structurally unrelated, ATP-competitive inhibitors that directly target the mTOR catalytic site inhibit both TORC1 and TORC2. Here, we review investigations of chemical scaffolds explored for the development of highly selective ATP-competitive mTOR kinase inhibitors (TORKi). Extensive medicinal chemistry campaigns allowed to overcome challenges related to structural similarity between mTOR and the phosphoinositide 3-kinase (PI3K) family. A broad region of chemical space is covered by TORKi. Here, the investigation of chemical substitutions and physicochemical properties has shed light on the compounds' ability to cross the blood brain barrier (BBB). This work provides insights supporting the optimization of TORKi for the treatment of cancer and central nervous system disorders.

Keywords:  BBB permeability; binding modes; chemical scaffolds; mTOR inhibitors; physicochemical properties

DOI:  https://doi.org/10.1002/cmdc.202100332
Int J Mol Sci. 2021 Jun 17. pii: 6518. [Epub ahead of print]22(12):

Human α-Galactosidase A Mutants: Priceless Tools to Develop Novel Therapies for Fabry Disease.

Andrea Modrego, Marilla Amaranto, Agustina Godino, Rosa Mendoza, José Luis Barra, José Luis Corchero.

  Fabry disease (FD) is a lysosomal storage disease caused by mutations in the gene for the α-galactosidase A (GLA) enzyme. The absence of the enzyme or its activity results in the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3), in different tissues, leading to a wide range of clinical manifestations. More than 1000 natural variants have been described in the GLA gene, most of them affecting proper protein folding and enzymatic activity. Currently, FD is treated by enzyme replacement therapy (ERT) or pharmacological chaperone therapy (PCT). However, as both approaches show specific drawbacks, new strategies (such as new forms of ERT, organ/cell transplant, substrate reduction therapy, or gene therapy) are under extensive study. In this review, we summarize GLA mutants described so far and discuss their putative application for the development of novel drugs for the treatment of FD. Unfavorable mutants with lower activities and stabilities than wild-type enzymes could serve as tools for the development of new pharmacological chaperones. On the other hand, GLA mutants showing improved enzymatic activity have been identified and produced in vitro. Such mutants could overcome several complications associated with current ERT, as lower-dose infusions of these mutants could achieve a therapeutic effect equivalent to that of the wild-type enzyme.

Keywords:  Fabry disease; alpha-galactosidase A; enzyme replacement therapy; pharmacological chaperones; rare diseases

DOI:  https://doi.org/10.3390/ijms22126518
Pharmaceutics. 2021 Jun 05. pii: 837. [Epub ahead of print]13(6):

Cathepsin D-Managing the Delicate Balance.

Olja Mijanovic, Anastasiia I Petushkova, Ana Brankovic, Boris Turk, Anna B Solovieva, Angelina I Nikitkina, Sergey Bolevich, Peter S Timashev, Alessandro Parodi, Andrey A Zamyatnin.

  Lysosomal proteases play a crucial role in maintaining cell homeostasis. Human cathepsin D manages protein turnover degrading misfolded and aggregated proteins and favors apoptosis in the case of proteostasis disruption. However, when cathepsin D regulation is affected, it can contribute to numerous disorders. The down-regulation of human cathepsin D is associated with neurodegenerative disorders, such as neuronal ceroid lipofuscinosis. On the other hand, its excessive levels outside lysosomes and the cell membrane lead to tumor growth, migration, invasion and angiogenesis. Therefore, targeting cathepsin D could provide significant diagnostic benefits and new avenues of therapy. Herein, we provide a brief overview of cathepsin D structure, regulation, function, and its role in the progression of many diseases and the therapeutic potentialities of natural and synthetic inhibitors and activators of this protease.

Keywords:  cathepsin D; diabetes; inhibitors; lysosome; malignant tumor; neurodegenerative disease; regulated cell death

DOI:  https://doi.org/10.3390/pharmaceutics13060837
FEBS J. 2021 Jul 01.

LRRK2 recruitment, activity, and function in organelles.

Luis Bonet-Ponce, Mark R Cookson.

  Protein coding mutations in Leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD) and non-coding variations around the gene increase the risk of developing sporadic PD. It is generally accepted that pathogenic LRRK2 mutations increase LRRK2 kinase activity, resulting in a toxic hyperactive protein that is inferred to lead to the PD phenotype. LRRK2 has long been linked to different membrane trafficking events, but the specific role of LRRK2 in these events has been difficult to resolve. Recently, several papers have reported the activation and translocation of LRRK2 to cellular organelles under specific conditions, which suggests that LRRK2 may influence intracellular membrane trafficking. Here, we review what is known about the role of LRRK2 at various organelle compartments.

Keywords:  Endomembranes; Leucine-rich repeat kinase 2; Lysosome; Membrane trafficking; Neurodegeneration; Parkinson’s disease

DOI:  https://doi.org/10.1111/febs.16099
ACS Nano. 2021 Jul 02.

Glucosylceramide Associated with Gaucher Disease Forms Amyloid-like Twisted Ribbon Fibrils That Induce α-Synuclein Aggregation.

Ashim Paul, Guy Jacoby, Dana Laor Bar-Yosef, Roy Beck, Ehud Gazit, Daniel Segal.

  A major risk factor for Gaucher's disease is loss of function mutations in the GBA1 gene that encodes lysosomal β-glucocerebrosidase, resulting in accumulation of glucosylceramide (GlcCer), a key lysosomal sphingolipid. GBA1 mutations also enhance the risk for Parkinson's disease, whose hallmark is the aggregation of α-synuclein (αSyn). However, the role of accumulated GlcCer in αSyn aggregation is not completely understood. Using various biophysical assays, we demonstrate that GlcCer self-assembles to form amyloid-like fibrillar aggregates in vitro. The GlcCer assemblies are stable in aqueous media of different pH and exhibit a twisted ribbon-like structure. Near lysosomal pH GlcCer aggregates induced αSyn aggregation and stabilized its nascent oligomers. We found that several bona fide inhibitors of proteinaceous amyloids effectively inhibited aggregation of GlcCer. This study contributes to the growing evidence of cross-talk between proteinaceous amyloids and amyloid-like aggregates of metabolites accumulated in diseases and suggests these aggregates as therapeutic targets.

Keywords:  Gaucher disease; Parkinson’s disease; amyloid-like aggregation; cross-seeding; glucosylceramide; α-synuclein

DOI:  https://doi.org/10.1021/acsnano.1c02957
Prog Mol Biol Transl Sci. 2021 ;pii: S1877-1173(21)00042-9. [Epub ahead of print]182 289-325

Genome editing in lysosomal disorders.

Luisa Natalia Pimentel-Vera, Edina Poletto, Esteban Alberto Gonzalez, Fabiano de Oliveira Poswar, Roberto Giugliani, Guilherme Baldo.

  Lysosomal disorders are a group of heterogenous diseases caused by mutations in genes that encode for lysosomal proteins. With exception of some cases, these disorders still lack both knowledge of disease pathogenesis and specific therapies. In this sense, genome editing arises as a technique that allows both the creation of specific cell lines, animal models and gene therapy protocols for these disorders. Here we explain the main applications of genome editing for lysosomal diseases, with examples based on the literature. The ability to rewrite the genome will be of extreme importance to study and potentially treat these rare disorders.

Keywords:  CRISPR-Cas9; Genome editing; Lysosomal disorders (LDs); TALENs; Zinc-finger nucleases

DOI:  https://doi.org/10.1016/bs.pmbts.2021.02.001
Sci Rep. 2021 Jun 30. 11(1): 13619

Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans.

Benjamin N Nelson, Savannah G Beakley, Sierra Posey, Brittney Conn, Emma Maritz, Janakiram Seshu, Karen L Wozniak.

Cryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

DOI: https://doi.org/10.1038/s41598-021-92991-6
Traffic. 2021 Jun 29.

Legionella pneumophila LegC7 effector protein drives aberrant ER:endosome contacts in yeast.

Nathan K Glueck, Kevin M O'Brien, Danielle C Seguin, Vincent J Starai.

  Legionella pneumophila is a facultative intracellular bacterial pathogen, causing the severe form of pneumonia known as Legionnaires' disease. Legionella actively alters host organelle trafficking through the activities of 'effector' proteins secreted via a TypeIVB secretion system, in order to construct the bacteria-laden Legionella-containing vacuole (LCV) and prevent lysosomal degradation. The LCV is created with membrane derived from host ER, secretory vesicles, and phagosomes, although the precise molecular mechanisms that drive its synthesis remain poorly understood. In an effort to characterize the in vivo activity of the LegC7/YlfA SNARE-like effector protein from Legionella in the context of eukaryotic membrane trafficking in yeast, we find that LegC7 interacts with the Emp46p/Emp47p ER-to-Golgi glycoprotein cargo adapter complex, alters ER morphology, and induces aberrant ER:endosome interactions, as measured by visualization of ER cargo degradation, reconstitution of split-GFP proteins, and enhanced oxidation of the ER lumen. LegC7-dependent toxicity, disruption of ER morphology, and ER:endosome fusion events were dependent upon endosomal VPS class C tethering complexes and the endosomal t-SNARE, Pep12p. This work establishes a model in which LegC7 functions to recruit host ER material to the bacterial phagosome during infection by driving ER:endosome contacts, potentially through interaction with host membrane tethering complexes and/or cargo adapters. This article is protected by copyright. All rights reserved.

Keywords:  LegC7; Legionella pneumophila; SNARE proteins; Saccharomyces cerevisiae; VPS class C tethering complexes; cargo adapters; membrane fusion

DOI:  https://doi.org/10.1111/tra.12807
Nat Cell Biol. 2021 Jun 28.

A Golgi-derived vesicle potentiates PtdIns4P to PtdIns3P conversion for endosome fission.

Bo Gong, Yuting Guo, Shihui Ding, Xiaohui Liu, Anming Meng, Dong Li, Shunji Jia.

Endosome fission is essential for cargo sorting and targeting in the endosomal system. However, whether organelles other than the endoplasmic reticulum (ER) participate in endosome fission through membrane contacts is unknown. Here, we characterize a Golgi-derived vesicle, the SEC14L2 compartment, that plays a unique role in facilitating endosome fission through ternary contacts with endosomes and the ER. Localized to the ER-mediated endosome fission site, the phosphatidylinositol transfer protein SEC14L2 promotes phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 3-phosphate (PtdIns3P) conversion before endosome fission. In the absence of SEC14L2, endosome fission is attenuated and more enlarged endosomes arise due to endosomal accumulation of PtdIns4P and reduction in PtdIns3P. Collectively, our data suggest roles of the Golgi network in ER-associated endosome fission and a mechanism involving ER-endosome contacts in the regulation of endosomal phosphoinositide conversion.

DOI: https://doi.org/10.1038/s41556-021-00704-y
PLoS Genet. 2021 Jul 01. 17(7): e1009651

Functional and structural analyses of novel Smith-Kingsmore Syndrome-Associated MTOR variants reveal potential new mechanisms and predictors of pathogenicity.

Aaron D Besterman, Thorsten Althoff, Peter Elfferich, Irma Gutierrez-Mejia, Joshua Sadik, Jonathan A Bernstein, Yvette van Ierland, Anja A Kattentidt-Mouravieva, Mark Nellist, Jeff Abramson, Julian A Martinez-Agosto.

Smith-Kingsmore syndrome (SKS) is a rare neurodevelopmental disorder characterized by macrocephaly/megalencephaly, developmental delay, intellectual disability, hypotonia, and seizures. It is caused by dominant missense mutations in MTOR. The pathogenicity of novel variants in MTOR in patients with neurodevelopmental disorders can be difficult to determine and the mechanism by which variants cause disease remains poorly understood. We report 7 patients with SKS with 4 novel MTOR variants and describe their phenotypes. We perform in vitro functional analyses to confirm MTOR activation and interrogate disease mechanisms. We complete structural analyses to understand the 3D properties of pathogenic variants. We examine the accuracy of relative accessible surface area, a quantitative measure of amino acid side-chain accessibility, as a predictor of MTOR variant pathogenicity. We describe novel clinical features of patients with SKS. We confirm MTOR Complex 1 activation and identify MTOR Complex 2 activation as a new potential mechanism of disease in SKS. We find that pathogenic MTOR variants disproportionately cluster in hotspots in the core of the protein, where they disrupt alpha helix packing due to the insertion of bulky amino acid side chains. We find that relative accessible surface area is significantly lower for SKS-associated variants compared to benign variants. We expand the phenotype of SKS and demonstrate that additional pathways of activation may contribute to disease. Incorporating 3D properties of MTOR variants may help in pathogenicity classification. We hope these findings may contribute to improving the precision of care and therapeutic development for individuals with SKS.

DOI: https://doi.org/10.1371/journal.pgen.1009651
Int J Mol Sci. 2021 Jun 28. pii: 6934. [Epub ahead of print]22(13):

The V-ATPase a3 Subunit: Structure, Function and Therapeutic Potential of an Essential Biomolecule in Osteoclastic Bone Resorption.

Anh Chu, Ralph A Zirngibl, Morris F Manolson.

  This review focuses on one of the 16 proteins composing the V-ATPase complex responsible for resorbing bone: the a3 subunit. The rationale for focusing on this biomolecule is that mutations in this one protein account for over 50% of osteopetrosis cases, highlighting its critical role in bone physiology. Despite its essential role in bone remodeling and its involvement in bone diseases, little is known about the way in which this subunit is targeted and regulated within osteoclasts. To this end, this review is broadened to include the three other mammalian paralogues (a1, a2 and a4) and the two yeast orthologs (Vph1p and Stv1p). By examining the literature on all of the paralogues/orthologs of the V-ATPase a subunit, we hope to provide insight into the molecular mechanisms and future research directions specific to a3. This review starts with an overview on bone, highlighting the role of V-ATPases in osteoclastic bone resorption. We then cover V-ATPases in other location/functions, highlighting the roles which the four mammalian a subunit paralogues might play in differential targeting and/or regulation. We review the ways in which the energy of ATP hydrolysis is converted into proton translocation, and go in depth into the diverse role of the a subunit, not only in proton translocation but also in lipid binding, cell signaling and human diseases. Finally, the therapeutic implication of targeting a3 specifically for bone diseases and cancer is discussed, with concluding remarks on future directions.

Keywords:  ATP6V0A3; ATP6V1C; OC-116 kDa; TCIRG1; V-ATPase; V-type proton ATPase 116 kDa subunit a3; anti-resorptive therapeutics; bone; osteoclasts; osteopetrosis; osteoporosis; signalosome

DOI:  https://doi.org/10.3390/ijms22136934
J Inherit Metab Dis. 2021 Jun 29.

MPS I Gene Therapy.

Sarah C Hurt, Patricia I Dickson, David T Curiel.

  Mucopolysaccharidoses type I (MPS I) is an inherited metabolic disease characterized by a malfunction of the α-l-iduronidase (IDUA) enzyme leading to the storage of glycosaminoglycans (GAGs) in the lysosomes. This disease has longtime been studied as a therapeutic target for those studying gene therapy and many studies have been done using various vectors to deliver the IDUA gene for corrective treatment. Many vectors have difficulties with efficacy and insertional mutagenesis concerns including adeno-associated viral vectors (AAV). Studies of AAVs treating MPS I have seemed promising, but recent deaths in gene therapy clinical trials for other inherited diseases using AAVs have left questions about their safety. Additionally, the recent modifications to adenoviral vectors leading them to target the vascular endothelium minimizing the risk of hepatotoxicity could lead to them being a viable option for MPS I gene therapy when coupled with gene editing technologies like CRISPR/Cas9. This article is protected by copyright. All rights reserved.

Keywords:  Adenovirus; CRISPR; Gene Editing; Gene Therapy; Mucopolysaccharidosis type I

DOI:  https://doi.org/10.1002/jimd.12414
Sci Rep. 2021 Jun 29. 11(1): 13477

Neu1 deficiency induces abnormal emotional behavior in zebrafish.

Asami Ikeda, Mayu Komamizu, Akito Hayashi, Chiharu Yamasaki, Keiji Okada, Momoko Kawabe, Masaharu Komatsu, Kazuhiro Shiozaki.

NEU1 sialidase hydrolyzes sialic acids from glycoconjugates in lysosomes. Deficiency of NEU1 causes sialidosis with symptoms including facial dysmorphism, bone dysplasia, and neurodegeneration. However, the effects of NEU1 deficiency on emotional activity have not been explored. Here, we conducted the behavioral analysis using Neu1-knockout zebrafish (Neu1-KO). Neu1-KO zebrafish showed normal swimming similar to wild-type zebrafish (WT), whereas shoaling was decreased and accompanied by greater inter-fish distance than WT zebrafish. The aggression test showed a reduced aggressive behavior in Neu1-KO zebrafish than in WT zebrafish. In the mirror and 3-chambers test, Neu1-KO zebrafish showed more interest toward the opponent in the mirror and multiple unfamiliar zebrafish, respectively, than WT zebrafish. Furthermore, Neu1-KO zebrafish also showed increased interaction with different fish species, whereas WT zebrafish avoided them. In the black-white preference test, Neu1-KO zebrafish showed an abnormal preference for the white region, whereas WT zebrafish preferred the black region. Neu1-KO zebrafish were characterized by a downregulation of the anxiety-related genes of the hypothalamic-pituitary-adrenal axis and upregulation of lamp1a, an activator of lysosomal exocytosis, with their brains accumulating several sphingoglycolipids. This study revealed that Neu1 deficiency caused abnormal emotional behavior in zebrafish, possibly due to neuronal dysfunction induced by lysosomal exocytosis.

DOI: https://doi.org/10.1038/s41598-021-92778-9