bims-lysosi 2021-06-13 papers

bims-lysosi

Biomed News

on Lysosomes and signaling

Issue of 2021‒06‒13
thirty-four papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

Rab11 is required for lysosome exocytosis through the interaction with Rab3a, Sec15 and GRAB.
Altered heparan sulfate metabolism during development triggers dopamine-dependent autistic-behaviours in models of lysosomal storage disorders.
An interdomain hydrogen bond in the Rag GTPases maintains stable mTORC1 signaling in sensing amino acids.
UDP-GlcNAc-1-phosphotransferase is a clinically important regulator of human and mouse hair pigmentation.
The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II.
Genetically Encoded, pH-Sensitive mTFP1 Biosensor for Probing Lysosomal pH.
mTOR pathway and DNA damage response: A therapeutic strategy in cancer therapy.
Maturing Autophagosomes are Transported Towards the Cell Periphery.
Complete Correction of Brain and Spinal Cord Pathology in Metachromatic Leukodystrophy Mice.
Triamterene induces autophagic degradation of lysosome by exacerbating lysosomal integrity.
PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis.
Lipid Droplet Contacts With Autophagosomes, Lysosomes, and Other Degradative Vesicles.
Glucose starvation induces autophagy via ULK1-mediated activation of PIKfyve in an AMPK-dependent manner.
Amyloid-β oligomers block lysosomal targeting of miRNPs to prevent miRNP recycling and target repression in glial cells.
RIP3 impedes transcription factor EB to suppress autophagic degradation in septic acute kidney injury.
αB-crystallin/HSPB2 is critical for hyperactive mTOR-induced cardiomyopathy.
The BPAN and intellectual disability disease proteins WDR45 and WDR45B modulate autophagosome-lysosome fusion.
Anderson-Fabry Disease: From Endothelial Dysfunction to Emerging Therapies.
A multifaceted role of progranulin in regulating amyloid-beta dynamics and responses.
Click and count: specific detection of acid ceramidase activity in live cells.
Rheb-mTOR activation rescues Aβ-induced cognitive impairment and memory function by restoring miR-146 activity in glial cells.
Hyperactive LRRK2 kinase impairs the trafficking of axonal autophagosomes.
Acid ceramidase promotes senescent cell survival.
Regulation of the one carbon folate cycle as a shared metabolic signature of longevity.
Macrophage LAMTOR1 Deficiency Prevents Dietary Obesity and Insulin Resistance Through Inflammation-Induced Energy Expenditure.
Arginine Metabolism and Its Potential in Treatment of Colorectal Cancer.
Plasma methionine metabolic profile is associated with longevity in mammals.
Decreased glucocerebrosidase activity and substrate accumulation of glycosphingolipids in a novel GBA1 D409V knock-in mouse model.
Structural analysis of the full-length human LRRK2.
Amino acid transporters as emerging therapeutic targets in cancer.
The mechanism of full activation of tumor suppressor PTEN at the phosphoinositide-enriched membrane.
AIE-based nanoaggregate tracker: high-fidelity visualization of lysosomal movement and drug-escaping processes.
Effect of rapamycin on mitochondria and lysosomes in fibroblasts from patients with mtDNA mutations.
The mammalian cholesterol synthesis enzyme squalene monooxygenase is proteasomally truncated to a constitutively active form.

J Cell Sci. 2021 Jun 01. pii: jcs246694. [Epub ahead of print]134(11):

Rab11 is required for lysosome exocytosis through the interaction with Rab3a, Sec15 and GRAB.

Cristina Escrevente, Liliana Bento-Lopes, José S Ramalho, Duarte C Barral.

  Lysosomes are dynamic organelles, capable of undergoing exocytosis. This process is crucial for several cellular functions, namely plasma membrane repair. Nevertheless, the molecular machinery involved in this process is poorly understood. Here, we identify Rab11a and Rab11b as regulators of Ca2+-induced lysosome exocytosis. Interestingly, Rab11-positive vesicles transiently interact with lysosomes at the cell periphery, indicating that this interaction is required for the last steps of lysosome exocytosis. Additionally, we found that the silencing of the exocyst subunit Sec15, a Rab11 effector, impairs lysosome exocytosis, suggesting that Sec15 acts together with Rab11 in the regulation of lysosome exocytosis. Furthermore, we show that Rab11 binds the guanine nucleotide exchange factor for Rab3a (GRAB) as well as Rab3a, which we have previously described to be a regulator of the positioning and exocytosis of lysosomes. Thus, our study identifies new players required for lysosome exocytosis and suggest the existence of a Rab11-Rab3a cascade involved in this process.

Keywords:  Exocytosis; GRAB, RAB3IL1; Lysosome; Rab11; Rab3a; Sec15

DOI:  https://doi.org/10.1242/jcs.246694
Nat Commun. 2021 06 09. 12(1): 3495

Altered heparan sulfate metabolism during development triggers dopamine-dependent autistic-behaviours in models of lysosomal storage disorders.

Maria De Risi, Michele Tufano, Filomena Grazia Alvino, Maria Grazia Ferraro, Giulia Torromino, Ylenia Gigante, Jlenia Monfregola, Elena Marrocco, Salvatore Pulcrano, Lea Tunisi, Claudia Lubrano, Dulce Papy-Garcia, Yaakov Tuchman, Alberto Salleo, Francesca Santoro, Gian Carlo Bellenchi, Luigia Cristino, Andrea Ballabio, Alessandro Fraldi, Elvira De Leonibus.

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.

DOI: https://doi.org/10.1038/s41467-021-23903-5
J Biol Chem. 2021 Jun 08. pii: S0021-9258(21)00661-X. [Epub ahead of print] 100861

An interdomain hydrogen bond in the Rag GTPases maintains stable mTORC1 signaling in sensing amino acids.

Shawn B Egri, Kuang Shen.

  Cellular growth and proliferation are primarily dictated by the mechanistic target of rapamycin complex 1 (mTORC1), which balances nutrient availability against the cell's anabolic needs. Central to the activity of mTORC1 is the RagA-RagC GTPase heterodimer, which under favorable conditions recruits the complex to the lysosomal surface to promote its activity. The RagA-RagC heterodimer has a unique architecture in that both subunits are active GTPases. To promote mTORC1 activity, the RagA subunit is loaded with GTP and the RagC subunit is loaded with GDP, while the opposite nucleotide loading configuration inhibits this signaling pathway. Despite its unique molecular architecture, how the Rag GTPase heterodimer maintains the oppositely loaded nucleotide state remains elusive. Here, we applied structure-function analysis approach to the crystal structures of the Rag GTPase heterodimer, and identified a key hydrogen bond that stabilizes the GDP-loaded state of the Rag GTPases. This hydrogen bond is mediated by the backbone carbonyl of Asn30 in the nucleotide binding domain (NBD) of RagA or Lys84 of RagC, and the hydroxyl group on the side chain of Thr210 in the C-terminal roadblock domain (CRD) of RagA or Ser266 of RagC, respectively. Eliminating this interdomain hydrogen bond abolishes the ability of the Rag GTPase to maintain its functional state, resulting in a distorted response to amino acid signals. Our results reveal that this long-distance interdomain interaction within the Rag GTPase is required for the maintenance and regulation of the mTORC1 nutrient-sensing pathway.

Keywords:  Rag GTPase; amino acid; enzyme mechanism; hydrogen bond; mTOR complex 1 (mTORC1); nutrient sensing

DOI:  https://doi.org/10.1016/j.jbc.2021.100861
J Invest Dermatol. 2021 Jun 08. pii: S0022-202X(21)01319-1. [Epub ahead of print]

UDP-GlcNAc-1-phosphotransferase is a clinically important regulator of human and mouse hair pigmentation.

Stephan Tiede, Jennifer E Hundt, Ralf Paus.

UDP-GlcNAc-1-phosphotransferase, a product of two separate genes (GNPTAB, GNPTG), is essential for sorting and transport of lysosomal enzymes to lysosomes. GNPTAB gene defects cause extracellular missorting of lysosomal enzymes resulting in lysosomal storage diseases, namely mucolipidosis (ML) type II, which is associated with hair discoloration. Yet, the physiological functions of GNPTAB in the control of hair follicle (HF) pigmentation remain unknown. To elucidate these, we have silenced GNPTAB- in organ-cultured human HFs as a human ex vivo-model for MLII. GNPTAB-silencing profoundly inhibited intrafollicular melanin production, the correct sorting of melanosomes, tyrosinase activity and HMB45 expression in the HF pigmentary unit, and altered HF melanocyte morphology in situ. In isolated primary human HF melanocytes, GNPTAB knockdown significantly reduced melanogenesis, tyrosinase activity, and correct tyrosinase protein sorting as well as proopiomelanocortin (POMC) expression, and caused the expected lysosomal enzyme missorting in vitro. Moreover, transgenic mice overexpressing an inserted missense-mutation corresponding to human MLII showed significantly reduced HF pigmentation, thus corroborating the in vivo relevance of our ex vivo and in vitro findings in the human system. This identifies GNPTAB as a clinically important enzymatic control of human HF pigmentation, likely by directly controlling tyrosinase sorting and POMC-transcription in HF melanocytes.

DOI: https://doi.org/10.1016/j.jid.2021.04.028
Nat Commun. 2021 06 07. 12(1): 3333

The lysosomal Ragulator complex plays an essential role in leukocyte trafficking by activating myosin II.

Takeshi Nakatani, Kohei Tsujimoto, JeongHoon Park, Tatsunori Jo, Tetsuya Kimura, Yoshitomo Hayama, Hachiro Konaka, Takayoshi Morita, Yasuhiro Kato, Masayuki Nishide, Shyohei Koyama, Shigeyuki Nada, Masato Okada, Hyota Takamatsu, Atsushi Kumanogoh.

Lysosomes are involved in nutrient sensing via the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 is tethered to lysosomes by the Ragulator complex, a heteropentamer in which Lamtor1 wraps around Lamtor2-5. Although the Ragulator complex is required for cell migration, the mechanisms by which it participates in cell motility remain unknown. Here, we show that lysosomes move to the uropod in motile cells, providing the platform where Lamtor1 interacts with the myosin phosphatase Rho-interacting protein (MPRIP) independently of mTORC1 and interferes with the interaction between MPRIP and MYPT1, a subunit of myosin light chain phosphatase (MLCP), thereby increasing myosin II-mediated actomyosin contraction. Additionally, formation of the complete Ragulator complex is required for leukocyte migration and pathophysiological immune responses. Together, our findings demonstrate that the lysosomal Ragulator complex plays an essential role in leukocyte migration by activating myosin II through interacting with MPRIP.

DOI: https://doi.org/10.1038/s41467-021-23654-3
ACS Sens. 2021 Jun 08.

Genetically Encoded, pH-Sensitive mTFP1 Biosensor for Probing Lysosomal pH.

Marcus Y Chin, Anand R Patwardhan, Kean-Hooi Ang, Austin L Wang, Carolina Alquezar, Mackenzie Welch, Phi T Nguyen, Michael Grabe, Anna V Molofsky, Michelle R Arkin, Aimee W Kao.

  Lysosomes are important sites for macromolecular degradation, defined by an acidic lumenal pH of ∼4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein (FP)-based pH biosensor called Fluorescence Indicator REporting pH in Lysosomes (FIRE-pHLy). This biosensor was targeted to lysosomes with lysosomal-associated membrane protein 1 (LAMP1) and reported lumenal pH between 3.5 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan pH-sensitive FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content quantitative imaging. We expressed FIRE-pHLy in several cellular models and quantified the alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust, and versatile lysosomal pH biosensor, that has broad applications for investigating pH dynamics in aging- and lysosome-related diseases, as well as in lysosome-based drug discovery.

Keywords:  high-content analysis; lysosomes; neurons; pH biosensor; ratiometric imaging

DOI:  https://doi.org/10.1021/acssensors.0c02318
DNA Repair (Amst). 2021 Jun 03. pii: S1568-7864(21)00098-7. [Epub ahead of print]104 103142

mTOR pathway and DNA damage response: A therapeutic strategy in cancer therapy.

Romina Danesh Pazhooh, Parnia Rahnamay Farnood, Zatollah Asemi, Liaosadat Mirsafaei, Bahman Yousefi, Hamed Mirzaei.

  The mammalian target of rapamycin (mTOR) is a conserved serine/threonine-protein kinase, comprising two subunit protein complexes: mTORC1 and mTORC2. In response to insult and cancer, the mTOR pathway plays a crucial role in regulating growth, metabolism, cell survival, and protein synthesis. Key subunits of mTORC1/2 catalyze the phosphorylation of various molecules, including eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1), ribosomal protein S6 kinase β-1 (S6K1). The DNA damage response (DDR) maintains genomic stability and provides an opportunity for treating tumors with defects caused by DNA damaging agents. Many mTOR inhibitors are utilized for the treatment of cancers. However, several clinical trials are still assessing the efficacy of mTOR inhibitors. This paper discusses the role of the mTOR signaling pathway and its regulators in developing cancer. In the following, we will review the interaction between DDR and mTOR signaling and the innovative therapies applied in preclinical and clinical trials for treating cancers.

Keywords:  Cancer; DNA damage; DNA repair; Therapy; mTOR

DOI:  https://doi.org/10.1016/j.dnarep.2021.103142
Cell Mol Neurobiol. 2021 Jun 09.

Maturing Autophagosomes are Transported Towards the Cell Periphery.

Anna Hilverling, Eva M Szegö, Elisabeth Dinter, Diana Cozma, Theodora Saridaki, Björn H Falkenburger.

  Autophagosome maturation comprises fusion with lysosomes and acidification. It is a critical step in the degradation of cytosolic protein aggregates that characterize many neurodegenerative diseases. In order to better understand this process, we studied intracellular trafficking of autophagosomes and aggregates of α-synuclein, which characterize Parkinson's disease and other synucleinopathies. The autophagosomal marker LC3 and the aggregation prone A53T mutant of α-synuclein were tagged by fluorescent proteins and expressed in HEK293T cells and primary astrocytes. The subcellular distribution and movement of these vesicle populations were analyzed by (time-lapse) microscopy. Fusion with lysosomes was assayed using the lysosomal marker LAMP1; vesicles with neutral and acidic luminal pH were discriminated using the RFP-GFP "tandem-fluorescence" tag. With respect to vesicle pH, we observed that neutral autophagosomes, marked by LC3 or synuclein, were located more frequently in the cell center, and acidic autophagosomes were observed more frequently in the cell periphery. Acidic autophagosomes were transported towards the cell periphery more often, indicating that acidification occurs in the cell center before transport to the periphery. With respect to autolysosomal fusion, we found that lysosomes preferentially moved towards the cell center, whereas autolysosomes moved towards the cell periphery, suggesting a cycle where lysosomes are generated in the periphery and fuse to autophagosomes in the cell center. Unexpectedly, many acidic autophagosomes were negative for LAMP1, indicating that acidification does not require fusion to lysosomes. Moreover, we found both neutral and acidic vesicles positive for LAMP1, consistent with delayed acidification of the autolysosome lumen. Individual steps of aggregate clearance thus occur in dedicated cellular regions. During aggregate clearance, autophagosomes and autolysosomes form in the center and are transported towards the periphery during maturation. In this process, luminal pH could regulate the direction of vesicle transport. (1) Transport and location of autophagosomes depend on luminal pH: Acidic autophagosomes are preferentially transported to the cell periphery, causing more acidic autophagosomes in the cell periphery and more neutral autophagosomes at the microtubule organizing center (MTOC). (2) Autolysosomes are transported to the cell periphery and lysosomes to the MTOC, suggesting spatial segregation of lysosome reformation and autolysosome fusion. (3) Synuclein aggregates are preferentially located at the MTOC and synuclein-containing vesicles in the cell periphery, consistent with transport of aggregates to the MTOC for autophagy.

Keywords:  Amphisomes; Autolysosomes; Autophagy; Lysosomes; MTOC; Time-lapse microscopy; α-Synuclein

DOI:  https://doi.org/10.1007/s10571-021-01116-0
Front Mol Neurosci. 2021 ;14 677895

Complete Correction of Brain and Spinal Cord Pathology in Metachromatic Leukodystrophy Mice.

Emilie Audouard, Valentin Oger, Béatrix Meha, Nathalie Cartier, Caroline Sevin, Françoise Piguet.

  Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder characterized by accumulation of sulfatides in both glial cells and neurons. MLD results from an inherited deficiency of arylsulfatase A (ARSA) and myelin degeneration in the central and peripheral nervous systems. Currently, no effective treatment is available for the most frequent late infantile (LI) form of MLD after symptom onset. The LI form results in rapid neurological degradation and early death. ARSA enzyme must be rapidly and efficiently delivered to brain and spinal cord oligodendrocytes of patients with LI MLD in order to potentially stop the progression of the disease. We previously showed that brain gene therapy with adeno-associated virus serotype rh10 (AAVrh10) driving the expression of human ARSA cDNA alleviated most long-term disease manifestations in MLD mice but was not sufficient in MLD patient to improve disease progression. Herein, we evaluated the short-term effects of intravenous AAVPHP.eB delivery driving the expression of human ARSA cDNA under the control of the cytomegalovirus/b-actin hybrid (CAG) promoter in 6-month-old MLD mice that already show marked sulfatide accumulation and brain pathology. Within 3 months, a single intravenous injection of AAVPHP.eB-hARSA-HA resulted in correction of brain and spinal cord sulfatide storage, and improvement of astrogliosis and microgliosis in brain and spinal cord of treated animals. These results strongly support to consider the use of AAVPHP.eB-hARSA vector for intravenous gene therapy in symptomatic rapidly progressing forms of MLD.

Keywords:  aav; gene therapy; intravenous injection (i.v.); lysosomal storage disease; metachromatic leukodystrophy; sulfatide accumulation

DOI:  https://doi.org/10.3389/fnmol.2021.677895
Arch Pharm Res. 2021 Jun 07.

Triamterene induces autophagic degradation of lysosome by exacerbating lysosomal integrity.

Na Yeon Park, Doo Sin Jo, Yong Hwan Kim, Ji-Eun Bae, Joon Bum Kim, Hyun Jun Park, Ji Yeon Choi, Ha Jung Lee, Jeong Ho Chang, Heeyoun Bunch, Hong Bae Jeon, Yong-Keun Jung, Dong-Hyung Cho.

  The maintenance of lysosomal integrity is essential for lysosome function and cell fate. Damaged lysosomes are degraded by lysosomal autophagy, lysophagy. The mechanism underlying lysophagy remains largely unknown; this study aimed to contribute to the understanding of this topic. A cell-based screening system was used to identify novel lysophagy modulators. Triamterene (6-phenylpteridine-2,4,7-triamine) was identified as one of the most potent lysophagy inducers from the screening process. We found that triamterene causes lysosomal rupture without affecting other cellular organelles and increases autophagy flux in HepG2 cells. Damaged lysosomes in triamterene-treated cells were removed by autophagy-mediated pathway, which was inhibited by depletion of the autophagy regulator, ATG5 or SQSTM1. In addition, treatment of triamterene decreased the integrity of lysosome and cell viability, which were rescued by removing the triamterene treatment in HepG2 cells. Hence, our data suggest that triamterene is a novel lysophagy inducer through the disruption of lysosomal integrity.

Keywords:  Autophagy; HepG2 cells; LLOMe; Lysophagy; Lysosomal integrity; Triamterene

DOI:  https://doi.org/10.1007/s12272-021-01335-5
Nat Commun. 2021 06 08. 12(1): 3444

PRMT5-mediated arginine methylation activates AKT kinase to govern tumorigenesis.

Shasha Yin, Liu Liu, Charles Brobbey, Viswanathan Palanisamy, Lauren E Ball, Shaun K Olsen, Michael C Ostrowski, Wenjian Gan.

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function.

DOI: https://doi.org/10.1038/s41467-021-23833-2
Contact (Thousand Oaks). 2020 Jan 01. 3 1-13

Lipid Droplet Contacts With Autophagosomes, Lysosomes, and Other Degradative Vesicles.

Kristina Drizyte-Miller, Micah B Schott, Mark A McNiven.

  Lipid droplets (LDs) are dynamic fat-storage organelles that interact readily with numerous cellular structures and organelles. A prominent LD contact site is with degradative vesicles such as autophagosomes, lysosomes, autolysosomes, and late endosomes. These contacts support lipid catabolism through the selective autophagy of LDs (i.e., lipophagy) or the recruitment of cytosolic lipases to the LD surface (i.e., lipolysis). However, LD-autophagosome contacts serve additional functions beyond lipid catabolism, including the supply of lipids for autophagosome biogenesis. In this review, we discuss the molecular mediators of LD contacts with autophagosomes and other degradative organelles as well as the diverse cellular functions of these contact sites in health and disease.

Keywords:  autophagosome; autophagy; cell biology; endosome; lipid droplet; lysosome

DOI:  https://doi.org/10.1177/2515256420910892
Dev Cell. 2021 Jun 01. pii: S1534-5807(21)00439-1. [Epub ahead of print]

Glucose starvation induces autophagy via ULK1-mediated activation of PIKfyve in an AMPK-dependent manner.

Cansu Karabiyik, Mariella Vicinanza, Sung Min Son, David C Rubinsztein.

  Autophagy is an essential catabolic process induced to provide cellular energy sources in response to nutrient limitation through the activation of kinases, like AMP-activated protein kinase (AMPK) and ULK1. Although glucose starvation induces autophagy, the exact mechanism underlying this signaling has yet to be elucidated. Here, we reveal a role for ULK1 in non-canonical autophagy signaling using diverse cell lines. ULK1 activated by AMPK during glucose starvation phosphorylates the lipid kinase PIKfyve on S1548, thereby increasing its activity and the synthesis of the phospholipid PI(5)P without changing the levels of PI(3,5)P2. ULK1-mediated activation of PIKfyve enhances the formation of PI(5)P-containing autophagosomes upon glucose starvation, resulting in an increase in autophagy flux. Phospho-mimic PIKfyve S1548D drives autophagy upregulation and lowers autophagy substrate levels. Our study has identified how ULK1 upregulates autophagy upon glucose starvation and induces the formation of PI(5)P-containing autophagosomes by activating PIKfyve.

Keywords:  AMPK; PI(5)P; PIKfyve; ULK1; autophagy; glucose starvation; mTOR; phagophore

DOI:  https://doi.org/10.1016/j.devcel.2021.05.010
J Cell Sci. 2021 Jun 01. pii: jcs258360. [Epub ahead of print]134(11):

Amyloid-β oligomers block lysosomal targeting of miRNPs to prevent miRNP recycling and target repression in glial cells.

Dipayan De, Suvendra N Bhattacharyya.

  Upon exposure to amyloid-β oligomers (Aβ1-42), glial cells start expressing proinflammatory cytokines, despite an increase in levels of repressive microRNAs (miRNAs). Exploring the mechanism of this potential immunity of target cytokine mRNAs against repressive miRNAs in amyloid-β-exposed glial cells, we have identified differential compartmentalization of repressive miRNAs in glial cells that explains this aberrant miRNA function. In Aβ1-42-treated cells, whereas target mRNAs were found to be associated with polysomes attached to endoplasmic reticulum (ER), the miRNA ribonucleoprotein complexes (miRNPs) were found to be present predominantly with endosomes that failed to recycle to ER-attached polysomes, preventing repression of mRNA targets. Aβ1-42 oligomers, by masking Rab7a proteins on endosomal surfaces, affected Rab7a interaction with Rab-interacting lysosomal protein (RILP), restricting the lysosomal targeting and recycling of miRNPs. RNA-processing body (P-body) localization of the miRNPs was found to be enhanced in amyloid-β-treated cells as a consequence of enhanced endosomal retention of miRNPs. Interestingly, depletion of P-body components partly rescued the miRNA function in glial cells exposed to amyloid-β and restricted the excess cytokine expression. This article has an associated First Person interview with the first author of the paper.

Keywords:  Endosome–lysosome interaction; RNA processing bodies; mRNA compartmentalization; miRNA-mediated translation repression; miRNP recycling

DOI:  https://doi.org/10.1242/jcs.258360
Cell Death Dis. 2021 Jun 08. 12(6): 593

RIP3 impedes transcription factor EB to suppress autophagic degradation in septic acute kidney injury.

Ruizhao Li, Xingchen Zhao, Shu Zhang, Wei Dong, Li Zhang, Yuanhan Chen, Zhilian Li, Huan Yang, Ying Huang, Zhiyong Xie, Weidong Wang, Chunling Li, Zhiming Ye, Zheng Dong, Xinling Liang.

Autophagy is an important renal-protective mechanism in septic acute kidney injury (AKI). Receptor interacting protein kinase 3 (RIP3) has been implicated in the renal tubular injury and renal dysfunction during septic AKI. Here we investigated the role and mechanism of RIP3 on autophagy in septic AKI. We showed an activation of RIP3, accompanied by an accumulation of the autophagosome marker LC3II and the autophagic substrate p62, in the kidneys of lipopolysaccharide (LPS)-induced septic AKI mice and LPS-treated cultured renal proximal tubular epithelial cells (PTECs). The lysosome inhibitor did not further increase the levels of LCII or p62 in LPS-treated PTECs. Moreover, inhibition of RIP3 attenuated the aberrant accumulation of LC3II and p62 under LPS treatment in vivo and in vitro. By utilizing mCherry-GFP-LC3 autophagy reporter mice in vivo and PTECs overexpression mRFP-GFP-LC3 in vitro, we observed that inhibition of RIP3 restored the formation of autolysosomes and eliminated the accumulated autophagosomes under LPS treatment. These results indicated that RIP3 impaired autophagic degradation, contributing to the accumulation of autophagosomes. Mechanistically, the nuclear translocation of transcription factor EB (TFEB), a master regulator of the lysosome and autophagy pathway, was inhibited in LPS-induced mice and LPS-treated PTECs. Inhibition of RIP3 restored the nuclear translocation of TFEB in vivo and in vitro. Co-immunoprecipitation further showed an interaction of RIP3 and TFEB in LPS-treated PTECs. Also, the expression of LAMP1 and cathepsin B, two potential target genes of TFEB involved in lysosome function, were decreased under LPS treatment in vivo and in vitro, and this decrease was rescued by inhibiting RIP3. Finally, overexpression of TFEB restored the autophagic degradation in LPS-treated PTECs. Together, the present study has identified a pivotal role of RIP3 in suppressing autophagic degradation through impeding the TFEB-lysosome pathway in septic AKI, providing potential therapeutic targets for the prevention and treatment of septic AKI.

DOI: https://doi.org/10.1038/s41419-021-03865-8
J Cell Physiol. 2021 Jun 08.

αB-crystallin/HSPB2 is critical for hyperactive mTOR-induced cardiomyopathy.

Lianmei Wang, Fang Wang, Kemei Liu, Caifeng Long, Yi Chen, Chunjia Li, Li Li, Fangming Liu, Xinyu Zhang, Yanling Jing, Yanan Wang, Aihua Liang, Hongbing Yan, Hongbing Zhang.

  Even though aberrant mechanistic target of rapamycin (mTOR) signaling is known to cause cardiomyopathy, its underlying mechanism remains poorly understood. Because augmentation of αB-crystallin and hspB2 was presented in the cortical tubers and lymphangioleiomyomatosis of tuberous sclerosis complex patients, we deciphered the role of αB-crystallin and its adjacent duplicate gene, hspB2, in hyperactive mTOR-induced cardiomyopathy. Cardiac Tsc1 deletion (T1-hKO) caused mouse mTOR activation and cardiomyopathy. Overexpression of αB-crystallin and hspB2 was presented in the hearts of these mice. Knockout of αB-crystallin/hspB2 reversed deficient Tsc1-mediated fetal gene expression, mTOR activation, mitochondrial damage, cardiomyocyte vacuolar degeneration, cardiomyocyte size, and fibrosis of T1-hKO mice. These cardiac-Tsc1; αB-crystallin; hspB2 triple knockout (tKO) mice had improved cardiac function, smaller heart weight to body weight ratio, and reduced lethality compared with T1-hKO mice. Even though activated mTOR suppressed autophagy in T1-hKO mice, ablation of αB-crystallin and hspB2 failed to restore autophagy in tKO mice. mTOR inhibitors suppressed αB-crystallin expression in T1-hKO mice and rat cardiomyocyte line H9C2. Starvation of H9C2 cells activated autophagy and suppressed αB-crystallin expression. Since inhibition of autophagy restored αB-crystallin expression in starved H9C2 cells, autophagy is a negative regulator of αB-crystallin expression. mTOR thus stimulates αB-crystallin expression through suppression of autophagy. In conclusion, αB-crystallin and hspB2 play a pivotal role in Tsc1 knockout-related cardiomyopathy and are therapeutic targets of hyperactive mTOR-associated cardiomyopathy.

Keywords:  TSC1; cardiomyopathy; hspB2; mTOR; αB-crystallin

DOI:  https://doi.org/10.1002/jcp.30465
Autophagy. 2021 Jun 09. 1-2

The BPAN and intellectual disability disease proteins WDR45 and WDR45B modulate autophagosome-lysosome fusion.

Cuicui Ji, Yan G Zhao.

  WDR45 and WDR45B are β-propeller proteins belonging to the WIPI (WD repeat domain, phosphoinositide interacting) family. Mutations in WDR45 and WDR45B are genetically linked with beta-propeller protein-associated neurodegeneration (BPAN) and intellectual disability (ID), respectively. WDR45 and WDR45B are homologs of yeast Atg18. Atg18 forms a complex with Atg2 for autophagosome biogenesis, probably by transferring lipids from the ER to phagophores. We revealed that WDR45 and WDR45B are critical for autophagosome-lysosome fusion in neural cells. WDR45 and WDR45B, but not their disease-related mutants, bind to the tether protein EPG5 and facilitate its targeting to late endosomes/lysosomes. In Wdr45 Wdr45b-deficient cells, the formation of tether-SNARE fusion machinery is compromised. The macroautophagy/autophagy deficiency in wdr45 wdr45b DKO cells is ameliorated by suppression of O-GlcNAcylation, which promotes autophagosome maturation. Thus, our results provide insights into the pathogenesis of WDR45- and WDR45B-related neurological diseases.

Keywords:  Autophagy; BPAN; ID; WDR45; WDR45B; autophagosome maturation

DOI:  https://doi.org/10.1080/15548627.2021.1924039
Adv Pharmacol Pharm Sci. 2021 ;2021 5548445

Anderson-Fabry Disease: From Endothelial Dysfunction to Emerging Therapies.

Cosimo A Stamerra, Rita Del Pinto, Paolo di Giosia, Claudio Ferri, Amirhossein Sahebkar.

The Anderson-Fabry disease is a rare, X-linked, multisystemic, progressive lysosomal storage disease caused by α-galactosidase A total or partial deficiency. The resulting syndrome is mainly characterized by early-onset autonomic neuropathy and life-threatening multiorgan involvement, including renal insufficiency, heart disease, and early stroke. The enzyme deficiency leads to tissue accumulation of the glycosphingolipid globotriaosylceramide and its analogues, but the mechanisms linking such accumulation to organ damage are only partially understood. In contrast, enzyme replacement and chaperone therapies are already fully available to patients and allow substantial amelioration of quality and quantity of life. Substrate reduction, messenger ribonucleic acid (mRNA)-based, and gene therapies are also on the horizon. In this review, the clinical scenario and molecular aspects of Anderson-Fabry disease are described, along with updates on disease mechanisms and emerging therapies.

DOI: https://doi.org/10.1155/2021/5548445
Life Sci Alliance. 2021 Jul;pii: e202000874. [Epub ahead of print]4(7):

A multifaceted role of progranulin in regulating amyloid-beta dynamics and responses.

Huan Du, Man Ying Wong, Tingting Zhang, Mariela Nunez Santos, Charlene Hsu, Junke Zhang, Haiyuan Yu, Wenjie Luo, Fenghua Hu.

Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). PGRN polymorphisms are associated with Alzheimer's disease. PGRN is highly expressed in the microglia near Aβ plaques and influences plaque dynamics and microglial activation. However, the detailed mechanisms remain elusive. Here we report that PGRN deficiency reduces human APP and Aβ levels in the young male but not female mice. PGRN-deficient microglia exhibit increased expression of markers associated with microglial activation, including CD68, galectin-3, TREM2, and GPNMB, specifically near Aβ plaques. In addition, PGRN loss leads to up-regulation of lysosome proteins and an increase in the nuclear localization of TFE3, a transcription factor involved in lysosome biogenesis. Cultured PGRN-deficient microglia show enhanced nuclear translocation of TFE3 and inflammation in response to Aβ fibril treatment. Taken together, our data revealed a sex- and age-dependent effect of PGRN on APP metabolism and a role of PGRN in regulating lysosomal activities and inflammation in plaque-associated microglia.

DOI: https://doi.org/10.26508/lsa.202000874
Chem Sci. 2020 Oct 22. 11(48): 13044-13051

Click and count: specific detection of acid ceramidase activity in live cells.

Mireia Casasampere, Eduardo Izquierdo, Josefina Casas, José Luís Abad, Xiao Liu, Ruijuan Xu, Cungui Mao, Young-Tae Chang, Antonio Delgado, Gemma Fabrias.

The use of intact cells in medical research offers a number of advantages over employing cell-free systems. In diagnostics, cells isolated from liquid biopsies can be directly used, speeding up the time of analysis and diminishing the risk of protein degradation by sample manipulation. In drug discovery, studies in live cells take into account aspects neglected in cell-free systems, such as uptake, metabolization, and subcellular concentration by compartmentalization of potential drug candidates. Therefore, probes for studies in cellulo are of paramount importance. Acid ceramidase (AC) is a lysosomal enzyme that hydrolyses ceramides into sphingoid bases and fatty acids. The essential role of this enzyme in the outburst and progress of several diseases, some of them still incurable, is well sustained. Despite the great clinical relevance of AC as a biomarker and therapeutic target, the specific monitoring of AC activity in live cells has remained elusive due to the concomitant existence of neutral and alkaline ceramidases. In this work, we report that 1-deoxydihydroceramides are exclusively hydrolysed by AC. Using N-octanoyl-18-azidodeoxysphinganine as a probe and a BODIPY-substituted bicyclononyne, we show the click-reliant predominant staining of lysosomes, with extra-lysosomal labeling also occurring in some cells. Importantly, using pharmacological and genetic tools together with high resolution mass spectrometry, we demonstrate that both lysosomal and extra-lysosomal staining are AC-dependent. These findings are translated into the specific flow cytometry monitoring of AC activity in intact cells, which fills an important gap in the field of diseases linked to altered AC activity.

DOI: https://doi.org/10.1039/d0sc03166f
Mol Ther Nucleic Acids. 2021 Jun 04. 24 868-887

Rheb-mTOR activation rescues Aβ-induced cognitive impairment and memory function by restoring miR-146 activity in glial cells.

Dipayan De, Ishita Mukherjee, Subhalakshmi Guha, Ramesh Kumar Paidi, Saikat Chakrabarti, Subhas C Biswas, Suvendra N Bhattacharyya.

  Deposition of amyloid beta plaques in adult rat or human brain is associated with increased production of proinflammatory cytokines by associated glial cells that are responsible for degeneration of the diseased tissue. The expression of these cytokines is usually under check and is controlled at the post-transcriptional level via several microRNAs. Computational analysis of gene expression profiles of cortical regions of Alzheimer's disease patients' brain suggests ineffective target cytokine mRNA suppression by existing micro-ribonucleoproteins (miRNPs) in diseased brain. Exploring the mechanism of amyloid beta-induced cytokine expression, we have identified how the inactivation of the repressive miR-146 miRNPs causes increased production of cytokines in amyloid beta-exposed glial cells. In exploration of the cause of miRNP inactivation, we have noted amyloid beta oligomer-induced sequestration of the mTORC1 complex to early endosomes that results in decreased Ago2 phosphorylation, limited Ago2-miRNA uncoupling, and retarded Ago2-cytokine mRNA interaction in rat astrocytes. Interestingly, constitutive activation of mTORC1 by Rheb activator restricts proinflammatory cytokine production by reactivating miR-146 miRNPs in amyloid beta-exposed glial cells to rescue the disease phenotype in the in vivo rat model of Alzheimer's disease.

Keywords:  Alzheimer’s disease; Astrocyte; Endosome; Rheb; amyloid beta; cognition; cytokines; mTORC1 activation; miRNAs; neuroinflammation

DOI:  https://doi.org/10.1016/j.omtn.2021.04.008
Autophagy. 2021 Jun 10. 1-3

Hyperactive LRRK2 kinase impairs the trafficking of axonal autophagosomes.

C Alexander Boecker, Erika L F Holzbaur.

  Parkinson disease (PD)-causing mutations in the LRRK2 (leucine rich repeat kinase 2) gene hyperactivate LRRK2 kinase activity. Here, we discuss our recent work linking LRRK2 hyperactivation to defective axonal autophagosome transport in neurons. In three different models, we observed that expression of the most common causative mutation for PD, LRRK2G2019S, disrupts processive autophagosome transport in a kinase-dependent manner. Mechanistically, we found that hyperactive LRRK2 recruits SPAG9/JIP4, a motor adaptor known to bind to LRRK2-phosphorylated RAB proteins, to the autophagosomal membrane. Increased SPAG9/JIP4 levels induce abnormal recruitment and activation of kinesin-1, which we propose results in an unproductive tug-of-war between anterograde and retrograde motors bound to autophagosomes. Disruption of autophagosome transport correlates with defective autophagosome maturation, suggesting that hyperactive LRRK2 may impair efficient degradation of autophagosomal cargo. Our work demonstrates that LRRK2 hyperactivation is sufficient to induce defects in autophagosome transport and maturation, further establishing a role of defective autophagy in the pathogenesis of PD.

Keywords:  Autophagy; JIP4; LRRK2; Parkinson’s disease; axonal transport

DOI:  https://doi.org/10.1080/15548627.2021.1936933
Aging (Albany NY). 2021 Jun 08. 13(undefined):

Acid ceramidase promotes senescent cell survival.

Rachel Munk, Carlos Anerillas, Martina Rossi, Dimitrios Tsitsipatis, Jennifer L Martindale, Allison B Herman, Jen-Hao Yang, Jackson A Roberts, Vijay R Varma, Poonam R Pandey, Madhav Thambisetty, Myriam Gorospe, Kotb Abdelmohsen.

  Cellular senescence is linked to chronic age-related diseases including atherosclerosis, diabetes, and neurodegeneration. Compared to proliferating cells, senescent cells express distinct subsets of proteins. In this study, we used cultured human diploid fibroblasts rendered senescent through replicative exhaustion or ionizing radiation to identify proteins differentially expressed during senescence. We identified acid ceramidase (ASAH1), a lysosomal enzyme that cleaves ceramide into sphingosine and fatty acid, as being highly elevated in senescent cells. This increase in ASAH1 levels in senescent cells was associated with a rise in the levels of ASAH1 mRNA and a robust increase in ASAH1 protein stability. Furthermore, silencing ASAH1 in pre-senescent fibroblasts decreased the levels of senescence proteins p16, p21, and p53, and reduced the activity of the senescence-associated β-galactosidase. Interestingly, depletion of ASAH1 in pre-senescent cells sensitized these cells to the senolytics Dasatinib and Quercetin (D+Q). Together, our study indicates that ASAH1 promotes senescence, protects senescent cells, and confers resistance against senolytic drugs. Given that inhibiting ASAH1 sensitizes cells towards senolysis, this enzyme represents an attractive therapeutic target in interventions aimed at eliminating senescent cells.

Keywords:  SASP; post-transcriptional; senescent cell metabolism; senotherapy; translational control

DOI:  https://doi.org/10.18632/aging.203170
Nat Commun. 2021 06 09. 12(1): 3486

Regulation of the one carbon folate cycle as a shared metabolic signature of longevity.

Andrea Annibal, Rebecca George Tharyan, Maribel Fides Schonewolff, Hannah Tam, Christian Latza, Markus Max Karl Auler, Adam Antebi.

The metabolome represents a complex network of biological events that reflects the physiologic state of the organism in health and disease. Additionally, specific metabolites and metabolic signaling pathways have been shown to modulate animal ageing, but whether there are convergent mechanisms uniting these processes remains elusive. Here, we used high resolution mass spectrometry to obtain the metabolomic profiles of canonical longevity pathways in C. elegans to identify metabolites regulating life span. By leveraging the metabolomic profiles across pathways, we found that one carbon metabolism and the folate cycle are pervasively regulated in common. We observed similar changes in long-lived mouse models of reduced insulin/IGF signaling. Genetic manipulation of pathway enzymes and supplementation with one carbon metabolites in C. elegans reveal that regulation of the folate cycle represents a shared causal mechanism of longevity and proteoprotection. Such interventions impact the methionine cycle, and reveal methionine restriction as an underlying mechanism. This comparative approach reveals key metabolic nodes to enhance healthy ageing.

DOI: https://doi.org/10.1038/s41467-021-23856-9
Front Cell Dev Biol. 2021 ;9 672032

Macrophage LAMTOR1 Deficiency Prevents Dietary Obesity and Insulin Resistance Through Inflammation-Induced Energy Expenditure.

Lingwen Ying, Mingliang Zhang, Xiaojing Ma, Yiming Si, Xiaoya Li, Jiaorong Su, Jun Yin, Yuqian Bao.

  Here, we studied the metabolic function of LAMTOR1 from macrophages using LAMTOR1 macrophage-specific knockout (MKO) mice. LAMTOR1 MKO mice showed resistance to high-fat diet (HFD)-induced obesity, lipid steatosis, and glucose metabolic disorders, with elevated levels of pro-inflammatory cytokines. The energy expenditure, oxygen consumption, and CO2 production increased significantly in HFD-fed MKO vs. wild-type (WT) mice. HE and immunohistochemistry staining showed a remarkable CD68+ Kupffer cell accumulation in the liver. Additionally, flow cytometry revealed that the proportion of macrophages and monocytes increased significantly in the liver of MKO mice. Of note, these macrophages were probably derived from the bone marrow since the proportion of CD11b+ cells as well as the proliferative activity was also increased in the context of femoral bone marrow cells. In addition, the Kupffer cells of both WT and KO mice were double-positive for the M1 (CD86) and M2 (CD206) markers. However, the expression of both M1 and M2 macrophage-related genes was increased in the liver of HFD-fed KO mice. Murine primary hepatocytes and Kupffer cells were further isolated and incubated with oleic acid for 24 h. The glucose output of primary hepatocytes from MKO mice was not affected. However, decreased lipid tolerance was observed in LAMTOR1-deficient Kupffer cells. Overall, our results suggest that LAMTOR1 deficiency in macrophages prevents obesity and metabolic disorders via the accumulation of Kupffer cells in the liver and the consequent hyper-inflammation and increased energy expenditure. Therefore, our results provide a new perspective for macrophage-derived LAMTOR1 in the context of systemic metabolism.

Keywords:  Kupffer cell; inflammation; late endosomal/lysosomal membrane adaptor p18; lipotoxicity; liver steatosis

DOI:  https://doi.org/10.3389/fcell.2021.672032
Front Cell Dev Biol. 2021 ;9 658861

Arginine Metabolism and Its Potential in Treatment of Colorectal Cancer.

Tao Du, Junyi Han.

  Colorectal cancer is the leading cause of death from cancer globally. The current treatment protocol still heavily relies on early detection and surgery. The molecular mechanisms underlying development of colorectal cancer are clinically important and determine the prognosis and treatment response. The arginine metabolism pathway is hyperactive in colorectal cancer and several molecules involved in the pathway are potential targets for chemoprevention and targeted colorectal cancer therapy. Endothelial nitric oxide synthase (eNOS), argininosuccinate synthetase and ornithine decarboxylase (ODC) are the main enzymes for arginine metabolism. Limiting arginine-rich meat consumption and inhibiting ODC activity largely reduces polyamine synthesis and the incidence of colorectal cancer. Arginine transporter CAT-1 and Human member 14 of the solute carrier family 6 (SLC6A14) are overexpressed in colorectal cancer cells and contributes to intracellular arginine levels. Human member 9 of the solute carrier family 38 (SLC38A9) serves as a component of the lysosomal arginine-sensing machinery. Pharmaceutical inhibition of single enzyme or arginine transporter is hard to meet requirement of restoring of abnormal arginine metabolic network. Apart from application in early screening for colorectal cancer, microRNA-based therapeutic strategy that simultaneously manipulating multiple targets involved in arginine metabolism brings promising future in the treatment of colorectal cancer.

Keywords:  arginine metabolism; colorectal cancer; signal pathway; stem cells; transporters protein

DOI:  https://doi.org/10.3389/fcell.2021.658861
Commun Biol. 2021 Jun 11. 4(1): 725

Plasma methionine metabolic profile is associated with longevity in mammals.

N Mota-Martorell, M Jové, R Berdún, R Pamplona.

Methionine metabolism arises as a key target to elucidate the molecular adaptations underlying animal longevity due to the negative association between longevity and methionine content. The present study follows a comparative approach to analyse plasma methionine metabolic profile using a LC-MS/MS platform from 11 mammalian species with a longevity ranging from 3.5 to 120 years. Our findings demonstrate the existence of a species-specific plasma profile for methionine metabolism associated with longevity characterised by: i) reduced methionine, cystathionine and choline; ii) increased non-polar amino acids; iii) reduced succinate and malate; and iv) increased carnitine. Our results support the existence of plasma longevity features that might respond to an optimised energetic metabolism and intracellular structures found in long-lived species.

DOI: https://doi.org/10.1038/s42003-021-02254-3
PLoS One. 2021 ;16(6): e0252325

Decreased glucocerebrosidase activity and substrate accumulation of glycosphingolipids in a novel GBA1 D409V knock-in mouse model.

Nicole K Polinski, Terina N Martinez, Alexander Gorodinsky, Ralph Gareus, Michael Sasner, Mark Herberth, Robert Switzer, Syed O Ahmad, Mali Cosden, Monika Kandebo, Robert E Drolet, Peter D Buckett, Weisong Shan, Yi Chen, Lee J Pellegrino, Gregory D Ellsworth, Leo B Dungan, Warren D Hirst, Sean W Clark, Kuldip D Dave.

Multiple mutations have been described in the human GBA1 gene, which encodes the lysosomal enzyme beta-glucocerebrosidase (GCase) that degrades glucosylceramide and is pivotal in glycosphingolipid substrate metabolism. Depletion of GCase, typically by homozygous mutations in GBA1, is linked to the lysosomal storage disorder Gaucher's disease (GD) and distinct or heterozygous mutations in GBA1 are associated with increased Parkinson's disease (PD) risk. While numerous genes have been linked to heritable PD, GBA1 mutations in aggregate are the single greatest risk factor for development of idiopathic PD. The importance of GCase in PD necessitates preclinical models in which to study GCase-related mechanisms and novel therapeutic approaches, as well as to elucidate the molecular mechanisms leading to enhanced PD risk in GBA1 mutation carriers. The aim of this study was to develop and characterize a novel GBA1 mouse model and to facilitate wide accessibility of the model with phenotypic data. Herein we describe the results of molecular, biochemical, histological, and behavioral phenotyping analyses in a GBA1 D409V knock-in (KI) mouse. This mouse model exhibited significantly decreased GCase activity in liver and brain, with substantial increases in glycosphingolipid substrates in the liver. While no changes in the number of dopamine neurons in the substantia nigra were noted, subtle changes in striatal neurotransmitters were observed in GBA1 D409V KI mice. Alpha-synuclein pathology and inflammation were not observed in the nigrostriatal system of this model. In summary, the GBA1 D409V KI mouse model provides an ideal model for studies aimed at pharmacodynamic assessments of potential therapies aiming to restore GCase.

DOI: https://doi.org/10.1371/journal.pone.0252325
Cell. 2021 Jun 03. pii: S0092-8674(21)00601-2. [Epub ahead of print]

Structural analysis of the full-length human LRRK2.

Alexander Myasnikov, Hanwen Zhu, Patricia Hixson, Boer Xie, Kaiwen Yu, Aaron Pitre, Junmin Peng, Ji Sun.

  Mutations in leucine-rich repeat kinase 2 (LRRK2) are commonly implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 regulates critical cellular processes at membranous organelles and forms microtubule-based pathogenic filaments, yet the molecular basis underlying these biological roles of LRRK2 remains largely enigmatic. Here, we determined high-resolution structures of full-length human LRRK2, revealing its architecture and key interdomain scaffolding elements for rationalizing disease-causing mutations. The kinase domain of LRRK2 is captured in an inactive state, a conformation also adopted by the most common PD-associated mutation, LRRK2G2019S. This conformation serves as a framework for structure-guided design of conformational specific inhibitors. We further determined the structure of COR-mediated LRRK2 dimers and found that single-point mutations at the dimer interface abolished pathogenic filamentation in cells. Overall, our study provides mechanistic insights into physiological and pathological roles of LRRK2 and establishes a structural template for future therapeutic intervention in PD.

Keywords:  LRRK2; LRRK2 dimer; LRRK2 mutations; Parkinson's disease; kinase

DOI:  https://doi.org/10.1016/j.cell.2021.05.004
Cancer Sci. 2021 Jun 06.

Amino acid transporters as emerging therapeutic targets in cancer.

Yasuhiro Saito, Tomoyoshi Soga.

  Amino acids are indispensable nutrients for both normal and cancer cells. Cancer cells are unable to synthesize essential amino acids as well as some non-essential amino acids adequately to support rapid proliferation, and must uptake amino acids from the surroundings. In order to meet the increased demand for amino acids needed for proliferation, high levels of amino acid transporters are expressed on the surface of cancer cells. Cancer cells utilize amino acids to synthesize proteins and nucleotides, as well as to obtain energy. In addition, amino acids are known to play pathological roles in cancer cells. Interestingly, breast cancer cells limit the use of amino acids for cell proliferation according to amino acid availability, which depends on estrogen receptor status. Here, we present a summarized literature review of novel amino acid functions in cancer cells. This review organizes the knowledge available on two amino acid transporters, SLC7A5 and SLC7A11, which are considered essential for breast cancer cell growth in a cell-dependent manner. In particular, we propose the glutamine recycling model to clarify the mechanism underlying aberrant SLC7A5 activation. Finally, we overview the pathological significances of SLC7A5 and SLC7A11 in cancer tissues.

Keywords:  Amino acid transporter; Breast cancer; Cell proliferation; Cystine uptake; Leucine uptake

DOI:  https://doi.org/10.1111/cas.15006
iScience. 2021 May 21. 24(5): 102438

The mechanism of full activation of tumor suppressor PTEN at the phosphoinositide-enriched membrane.

Hyunbum Jang, Iris Nira Smith, Charis Eng, Ruth Nussinov.

  Tumor suppressor PTEN, the second most highly mutated protein in cancer, dephosphorylates signaling lipid PIP3 produced by PI3Ks. Excess PIP3 promotes cell proliferation. The mechanism at the membrane of this pivotal phosphatase is unknown hindering drug discovery. Exploiting explicit solvent simulations, we tracked full-length PTEN trafficking from the cytosol to the membrane. We observed its interaction with membranes composed of zwitterionic phosphatidylcholine, anionic phosphatidylserine, and phosphoinositides, including signaling lipids PIP2 and PIP3. We tracked its moving away from the zwitterionic and getting absorbed onto anionic membrane that harbors PIP3. We followed it localizing on microdomains enriched in signaling lipids, as PI3K does, and observed PIP3 allosterically unfolding the N-terminal PIP2 binding domain, positioning it favorably for the polybasic motif interaction with PIP2. Finally, we determined PTEN catalytic action at the membrane, all in line with experimental observations, deciphering the mechanisms of how PTEN anchors to the membrane and restrains cancer.

Keywords:  Cancer; In Silico Biology; Structural Biology

DOI:  https://doi.org/10.1016/j.isci.2021.102438
Chem Sci. 2020 Aug 28. 11(47): 12755-12763

AIE-based nanoaggregate tracker: high-fidelity visualization of lysosomal movement and drug-escaping processes.

Zhenxing Liu, Qi Wang, Zhirong Zhu, Ming Liu, Xiaolei Zhao, Wei-Hong Zhu.

High-fidelity imaging and long-term visualization of lysosomes are crucial for their functional evaluation, related disease detection and active drug screening. However, commercial aggregation-caused quenching probes are not conducive to precise lysosomal imaging because of their inherent drawbacks, like easy diffusion, short emission and small Stokes shift, let alone their long-term tracing due to rapid photobleaching. Herein we report a novel aggregation-induced emission (AIE)-based TCM-PI nanoaggregate tracker for direct visualization of lysosomes based on the building block of tricyano-methylene-pyridine (TCM), wherein introduced piperazine (PI) groups behave as targeting units to lysosomes upon protonation, and the self-assembled nanostructure contributes to fast endocytosis for enhanced targeting ability as well as extended retention time for long-term imaging. The piperazine-stabilized TCM-PI nanoaggregate shifts the emission maximum to 677 nm in an aqueous environment, and falls within the desirable NIR region with a large Stokes shift of 162 nm, thereby greatly reducing biological fluorescent background interference. In contrast with the commercially available LysoTracker Red, the essential AIE characteristic of high photostability can guarantee three-dimensional high-fidelity tracing with low photobleaching, and little diffusion from lysosomes, and especially overcome the AIE bottleneck to target specificity. Consequently, the AIE-based nanoaggregate tracker successfully achieves the high-fidelity and long-term tracing of lysosomal movement and even monitors the drug-escaping process from lysosomes to cell nuclei, which provides a potential tool to benefit drug screening.

DOI: https://doi.org/10.1039/d0sc04156d
Am J Physiol Cell Physiol. 2021 06 09.

Effect of rapamycin on mitochondria and lysosomes in fibroblasts from patients with mtDNA mutations.

Nashwa J Cheema, Jessie M Cameron, David A Hood.

  Maintaining mitochondrial function and dynamics is crucial for cellular health. In muscle, defects in mitochondria result in severe myopathies where accumulation of damaged mitochondria causes deterioration and dysfunction. Importantly, understanding the role of mitochondria in disease is a necessity to determine future therapeutics. One of the most common myopathies is mitochondrial encephalopathy lactic acidosis stroke-like episodes (MELAS), which has no current treatment. Recently, MELAS patients treated with rapamycin exhibited improved clinical outcomes. However, the cellular mechanisms of rapamycin effects in MELAS patients are currently unknown. In this study, we used cultured skin fibroblasts as a window into the mitochondrial dysfunction evident in MELAS cells, as well as to study the mechanisms of rapamycin action, compared to control, healthy individuals. We observed that mitochondria from patients were fragmented, had a 3-fold decline in the average speed of motility, a 2-fold reduced mitochondrial membrane potential and a 1.5-2-fold decline in basal respiration. Despite the reduction in mitochondrial function, mitochondrial import protein Tim23 was elevated in patient cell lines. MELAS fibroblasts exhibited increased MnSOD levels and lysosomal function when compared to healthy controls. Treatment of MELAS fibroblasts with rapamycin for 24 hrs resulted in increased mitochondrial respiration compared to control cells, a higher lysosome content, and a greater localization of mitochondria to lysosomes. Our studies suggest that rapamycin has the potential to improve cellular health even in the presence of mtDNA defects, primarily via an increase in lysosomal content.

Keywords:  human fibroblasts; lysosomes; mitochondrial health; mitochondrial myopathies

DOI:  https://doi.org/10.1152/ajpcell.00471.2020
J Biol Chem. 2021 Apr 29. pii: S0021-9258(21)00520-2. [Epub ahead of print] 100731

The mammalian cholesterol synthesis enzyme squalene monooxygenase is proteasomally truncated to a constitutively active form.

Hudson W Coates, Isabelle M Capell-Hattam, Andrew J Brown.

  Squalene monooxygenase (SM, also known as squalene epoxidase) is a rate-limiting enzyme of cholesterol synthesis that converts squalene to monooxidosqualene and is oncogenic in numerous cancer types. SM is subject to feedback regulation via cholesterol-induced proteasomal degradation, which depends on its lipid-sensing N-terminal regulatory domain. We previously identified an endogenous truncated form of SM with a similar abundance to full-length SM, but whether this truncated form is functional or subject to the same regulatory mechanisms as full-length SM is not known. Here, we show that this truncated SM differs from full-length SM in two major ways: it is cholesterol-resistant and adopts a peripheral rather than integral association with the endoplasmic reticulum membrane. However, truncated SM retains full squalene monooxygenase activity and is therefore constitutively active. Truncation of SM occurs during its endoplasmic reticulum-associated degradation and requires the proteasome, which partially degrades the SM N-terminus and disrupts cholesterol-sensing elements within the regulatory domain. Furthermore, truncation relies on a ubiquitin signal that is distinct from that required for cholesterol-induced degradation. Using mutagenesis, we demonstrate that partial proteasomal degradation of SM depends on both an intrinsically disordered region near the truncation site and the stability of the adjacent catalytic domain, which escapes degradation. These findings uncover an additional layer of complexity in the post-translational regulation of cholesterol synthesis and establish SM as the first eukaryotic enzyme found to undergo proteasomal truncation.

Keywords:  cholesterol; endoplasmic reticulum-associated protein degradation (ERAD); proteasome; protein degradation; squalene monooxygenase; ubiquitylation (ubiquitination)

DOI:  https://doi.org/10.1016/j.jbc.2021.100731