bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2021‒06‒20
thirty-five papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing


  1. Nat Methods. 2021 Jun 14.
      Lysosomes are critical for cellular metabolism and are heterogeneously involved in various cellular processes. The ability to measure lysosomal metabolic heterogeneity is essential for understanding their physiological roles. We therefore built a single-lysosome mass spectrometry (SLMS) platform integrating lysosomal patch-clamp recording and induced nano-electrospray ionization (nanoESI)/mass spectrometry (MS) that enables concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes. The accuracy and reliability of this technique were validated by supporting previous findings, such as the transportability of lysosomal cationic amino acids transporters such as PQLC2 and the lysosomal trapping of lysosomotropic, hydrophobic weak base drugs such as lidocaine. We derived metabolites from single lysosomes in various cell types and classified lysosomes into five major subpopulations based on their chemical and biological divergence. Senescence and carcinoma altered metabolic profiles of lysosomes in a type-specific manner. Thus, SLMS can open more avenues for investigating heterogeneous lysosomal metabolic changes during physiological and pathological processes.
    DOI:  https://doi.org/10.1038/s41592-021-01182-8
  2. Autophagy. 2021 Jun 16.
      The autophagosome has two lipid bilayer membranes. The outer membrane fuses with the lysosome, while the inner membrane is degraded to release autophagic contents for degradation. It remains unclear how the inner vesicle of the autophagosome (called the autophagic vesicle) is disintegrated after autophagosome-lysosome fusion. Here, we identified C. elegans LPLA-2/M05B5.4 as a key enzyme that degrades membranous material in lysosomes. LPLA-2 is homologous to human PLA2G15, a lysosomal phospholipase A2 family protein that catalyzes cleavage of membrane phospholipids. We found that loss of LPLA-2 causes accumulation of large membrane whorls in enlarged lysosomes and both phenotypes are suppressed by blocking macroautophagy/autophagy. Moreover, autophagic vesicles persisted in enlarged lysosomes in PLA2G15 knockdown cells and lpla-2(lf) mutants, which suggests that the breakdown of the inner autophagosomal membrane in lysosomes is impaired. lpla-2(lf) mutants exhibit severe defects in both embryonic and larval development. Our data suggest that disintegration of the inner autophagosomal membrane by LPLA-2 promotes the release and subsequent degradation of autophagic contents in lysosomes, which is essential for C. elegans development.
    Keywords:  Autophagic vesicle, C. elegans; development; inner autophagosomal membrane; lysosomal phospholipase A2; lysosome
    DOI:  https://doi.org/10.1080/15548627.2021.1943178
  3. Sci Immunol. 2021 Jun 18. pii: eabg0791. [Epub ahead of print]6(60):
      The nutrient-sensing mammalian target of rapamycin (mTOR) is integral to cell fate decisions after T cell activation. Sustained mTORC1 activity favors the generation of terminally differentiated effector T cells instead of follicular helper and memory T cells. This is particularly pertinent for T cell responses of older adults who have sustained mTORC1 activation despite dysfunctional lysosomes. Here, we show that lysosome-deficient T cells rely on late endosomes rather than lysosomes as an mTORC1 activation platform, where mTORC1 is activated by sensing cytosolic amino acids. T cells from older adults have an increased expression of the plasma membrane leucine transporter SLC7A5 to provide a cytosolic amino acid source. Hence, SLC7A5 and VPS39 deficiency (a member of the HOPS complex promoting early to late endosome conversion) substantially reduced mTORC1 activities in T cells from older but not young individuals. Late endosomal mTORC1 is independent of the negative-feedback loop involving mTORC1-induced inactivation of the transcription factor TFEB that controls expression of lysosomal genes. The resulting sustained mTORC1 activation impaired lysosome function and prevented lysosomal degradation of PD-1 in CD4+ T cells from older adults, thereby inhibiting their proliferative responses. VPS39 silencing of human T cells improved their expansion to pertussis and to SARS-CoV-2 peptides in vitro. Furthermore, adoptive transfer of CD4+ Vps39-deficient LCMV-specific SMARTA cells improved germinal center responses, CD8+ memory T cell generation, and recall responses to infection. Thus, curtailing late endosomal mTORC1 activity is a promising strategy to enhance T cell immunity.
    DOI:  https://doi.org/10.1126/sciimmunol.abg0791
  4. J Clin Invest. 2021 Jun 15. pii: 146870. [Epub ahead of print]
      Disordered lysosomal/autophagy pathways initiate and drive pancreatitis, but the underlying mechanisms and links to disease pathology are poorly understood. Here, we show that mannose-6-phosphate (M6P) pathway of hydrolase delivery to lysosomes critically regulates pancreatic acinar cell cholesterol metabolism. Ablation of the Gnptab gene coding for a key enzyme in M6P pathway disrupted acinar cell cholesterol turnover, causing accumulation of non-esterified cholesterol in lysosomes/autolysosomes, its' depletion in the plasma membrane, and upregulation of cholesterol synthesis and uptake. We found similar dysregulation of acinar cell cholesterol, and a decrease in GNPTAB levels, in both WT experimental pancreatitis and human disease. The mechanisms mediating pancreatic cholesterol dyshomeostasis in Gnptab-/- and experimental models involve disordered endolysosomal system, resulting in impaired cholesterol transport through lysosomes and blockage of autophagic flux. By contrast, in Gnptab-/- liver the endolysosomal system and cholesterol homeostasis were largely unaffected. Gnptab-/- mice developed spontaneous pancreatitis. Normalization of cholesterol metabolism by pharmacologic means alleviated responses of experimental pancreatitis, particularly trypsinogen activation, the disease hallmark. The results reveal the essential role of M6P pathway in maintaining exocrine pancreas homeostasis and function, and implicate cholesterol disordering in the pathogenesis of pancreatitis.
    Keywords:  Autophagy; Cell Biology; Gastroenterology; Lysosomes; Mitochondria
    DOI:  https://doi.org/10.1172/JCI146870
  5. FASEB J. 2021 Jul;35(7): e21732
      Acid ceramidase (murine gene code: Asah1) (50 kDa) belongs to N-terminal nucleophile hydrolase family. This enzyme is located in the lysosome, which mediates conversion of ceramide (CER) into sphingosine and free fatty acids at acidic pH. CER plays an important role in intracellular sphingolipid metabolism and its increase causes inflammation. The mammalian target of rapamycin complex 1 (mTORC1) signaling on late endosomes (LEs)/lysosomes may control cargo selection, membrane biogenesis, and exosome secretion, which may be fine controlled by lysosomal sphingolipids such as CER. This lysosomal-CER-mTOR signaling may be a crucial molecular mechanism responsible for development of arterial medial calcification (AMC). Torin-1 (5 mg/kg/day), an mTOR inhibitor, significantly decreased aortic medial calcification accompanied with decreased expression of osteogenic markers like osteopontin (OSP) and runt-related transcription factor 2 (RUNX2) and upregulation of smooth muscle 22α (SM22-α) in mice receiving high dose of Vitamin D (500 000 IU/kg/day). Asah1fl/fl /SMCre mice had markedly increased co-localization of mTORC1 with lysosome-associated membrane protein-1 (Lamp-1) (lysosome marker) and decreased co-localization of vacuolar protein sorting-associated protein 16 (VPS16) (a multivesicular bodies [MVBs] marker) with Lamp-1, suggesting mTOR activation caused reduced MVBs interaction with lysosomes. Torin-1 significantly reduced the co-localization of mTOR vs Lamp-1, increased lysosome-MVB interaction which was associated with reduced accumulation of CD63 and annexin 2 (exosome markers) in the coronary arterial wall of mice. Using coronary artery smooth muscle cells (CASMCs), Pi -stimulation significantly increased p-mTOR expression in Asah1fl/fl /SMCre CASMCs as compared to WT/WT cells associated with increased calcium deposition and mineralization. Torin-1 blocked Pi -induced calcium deposition and mineralization. siRNA mTOR and Torin-1 significantly reduce co-localization of mTORC1 with Lamp-1, increased VPS16 vs Lamp-1 co-localization in Pi -stimulated CASMCs, associated with decreased exosome release. Functionally, Torin-1 significantly reduces arterial stiffening as shown by restoration from increased pulse wave velocity and decreased elastin breaks. These results suggest that lysosomal CER-mTOR signaling may play a critical role for the control of lysosome-MVB interaction, exosome secretion and arterial stiffening during AMC.
    Keywords:  arterial stiffness; exosome; mTOR; sphingolipids; vascular calcification
    DOI:  https://doi.org/10.1096/fj.202100385R
  6. Nat Commun. 2021 06 16. 12(1): 3660
      The mechanistic target of rapamycin complex 1 (mTORC1) integrates cellular nutrient signaling and hormonal cues to control metabolism. We have previously shown that constitutive nutrient signaling to mTORC1 by means of genetic activation of RagA (expression of GTP-locked RagA, or RagAGTP) in mice resulted in a fatal energetic crisis at birth. Herein, we rescue neonatal lethality in RagAGTP mice and find morphometric and metabolic alterations that span glucose, lipid, ketone, bile acid and amino acid homeostasis in adults, and a median lifespan of nine months. Proteomic and metabolomic analyses of livers from RagAGTP mice reveal a failed metabolic adaptation to fasting due to a global impairment in PPARα transcriptional program. These metabolic defects are partially recapitulated by restricting activation of RagA to hepatocytes, and revert by pharmacological inhibition of mTORC1. Constitutive hepatic nutrient signaling does not cause hepatocellular damage and carcinomas, unlike genetic activation of growth factor signaling upstream of mTORC1. In summary, RagA signaling dictates dynamic responses to feeding-fasting cycles to tune metabolism so as to match the nutritional state.
    DOI:  https://doi.org/10.1038/s41467-021-23857-8
  7. Am J Med Genet A. 2021 Jun 17.
      Niemann-Pick disease type C (NPC) is a rare and fatal lysosomal storage disorder characterized by neurodegeneration and hepatic involvement. Mutations in either NPC1 or NPC2, two genes encoding lysosomal proteins, lead to an intracellular accumulation of unesterified cholesterol and sphingolipids in late endosomes/lysosomes. Early cholestatic disease is considered a hallmark of patients with early disease onset. This can potentially result in liver failure shortly after birth or subclinical hepatic inflammation. Previous reports suggest an association between NPC and hepatocellular carcinoma, a cancer that is rare during childhood. We present a 12-year-old male with a known diagnosis of NPC1 disease who was found to have a stage III hepatocellular carcinoma, underwent surgical resection with adjuvant chemotherapy, and subsequently died from metastatic disease. This report provides evidence of an increased risk of hepatocellular carcinoma in NPC patients, suggesting a need for screening in this patient population.
    Keywords:  NPC1; Niemann-Pick disease type C; hepatocellular carcinoma
    DOI:  https://doi.org/10.1002/ajmg.a.62382
  8. Mol Genet Metab Rep. 2021 Sep;28 100773
      Drug-induced lysosomal storage disease (DILSD) caused by cationic amphiphilic drugs (CADs), which exhibits toxic manifestations and pathological findings mimicking Fabry disease (α-galactosidase A deficiency), has attracted the interests of clinicians and pathologists. Although the affected region is lysosomes in both the diseases, DILSD is characterized by intralysosomal accumulation of phospholipids and Fabry disease that of globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3). However, it is unknown whether administration of CADs affects the catabolism of Gb3 and Lyso-Gb3 in Fabry disease. In this study, we independently administered hydroxychloroquine/amiodarone to wild-type and Fabry mice and examined the effects of the drugs on the enzyme activity and substrates accumulated in organs and tissues. The results revealed that the administration of the drugs induced accumulation of phosphatidylcholine in both the wild-type and Fabry mice. However, reduction of α-galactosidase A activity in the organs and tissues of the wild-type mice was not found, and the storage of Gb3 and Lyso-Gb3 was not accelerated by these drugs in the Fabry mice. This suggests that hydroxychloroquine/amiodarone do not have any significant impact on the catabolism of Gb3 and Lyso-Gb3 in organs and tissues of both wild-type and Fabry mice.
    Keywords:  Amiodarone; CAD, cationic amphiphilic drug; DILSD, drug-induced lysosomal storage disease; Drug-induced lysosomal storage disease; Fabry disease; Gb3, globotriaosylceramide; Globotriaosylceramide; Globotriaosylsphingosine; Hydroxychloroquine; ILV, intralysosomal luminal vesicle; LC, liquid chromatography; Lyso-Gb3, globotriaosylsphingosine; MRM, multiple reaction monitoring; MS/MS, tandem mass spectrometry; PhC, phosphatidylcholine; Phospholipid; α-Gal, α-galactosidase A; α-Galactosidase A
    DOI:  https://doi.org/10.1016/j.ymgmr.2021.100773
  9. Nutr Metab (Lond). 2021 Jun 12. 18(1): 61
      BACKGROUND: Previous work in HEK-293 cells demonstrated the importance of amino acid-induced mTORC1 translocation to the lysosomal surface for stimulating mTORC1 kinase activity and protein synthesis. This study tested the conservation of this amino acid sensing mechanism in human skeletal muscle by treating subjects with chloroquine-a lysosomotropic agent that induces in vitro and in vivo lysosome dysfunction.METHODS: mTORC1 signaling and muscle protein synthesis (MPS) were determined in vivo in a randomized controlled trial of 14 subjects (10 M, 4 F; 26 ± 4 year) that ingested 10 g of essential amino acids (EAA) after receiving 750 mg of chloroquine (CHQ, n = 7) or serving as controls (CON, n = 7; no chloroquine). Additionally, differentiated C2C12 cells were used to assess mTORC1 signaling and myotube protein synthesis (MyPS) in the presence and absence of leucine and the lysosomotropic agent chloroquine.
    RESULTS: mTORC1, S6K1, 4E-BP1 and rpS6 phosphorylation increased in both CON and CHQ 1 h post EAA ingestion (P < 0.05). MPS increased similarly in both groups (CON, P = 0.06; CHQ, P < 0.05). In contrast, in C2C12 cells, 1 mM leucine increased mTORC1 and S6K1 phosphorylation (P < 0.05), which was inhibited by 2 mg/ml chloroquine. Chloroquine (2 mg/ml) was sufficient to disrupt mTORC1 signaling, and MyPS.
    CONCLUSIONS: Chloroquine did not inhibit amino acid-induced activation of mTORC1 signaling and skeletal MPS in humans as it does in C2C12 muscle cells. Therefore, different in vivo experimental approaches are required for confirming the precise role of the lysosome and amino acid sensing in human skeletal muscle. Trial registration NCT00891696. Registered 29 April 2009.
    Keywords:  Amino acid sensing; Chloroquine; Muscle protein turnover; mTOR signaling
    DOI:  https://doi.org/10.1186/s12986-021-00585-w
  10. ACS Sens. 2021 Jun 14.
      Here, we have designed and synthesized acidic pH-activatable visible to NIR switchable ratiometric pH-sensitive fluorescent dye. The design consists of a cell-permeable organic probe containing a lysosome targeting morpholine functionality and an acidic pH-activatable oxazolidine moiety. The visible closed oxazolidine form (λabs 418 nm) can be switched to the highly conjugated NIR Cy-7 form (λabs 780 nm) through ring opening of the oxazolidine moiety at acidic pH. This switching of the ratiometric fluorescent probe is highly reversible and can be controlled by pH. NMR, UV/vis, and fluorescence spectroscopies allowed monitoring of pH switching behavior of the probe. This bioresponsive in situ acidic organelle activatable fluorophore showed reversible pH-switchable ratiometric optical properties, high photostability, huge bathochromic emission shift of 320 nm from basic to acidic pH, off-to-on narrow NIR absorption and emission bands with enhanced molar extinction coefficient at lysosomal pH, good quantum yield, low cytotoxicity, and targeted imaging ability of live cell lysosomes with ideal pKa. The report demonstrated ratiometric imaging with improved specificity of the acidic lysosome while minimizing signals at the NIR region from nontargeted neutral or basic organelles in human carcinoma HeLa and A549 as well as rat healthy H9c2(2-1) live cells, which is monitored by confocal laser scanning microscopy.
    Keywords:  cyanine dye; live-cell imaging; lysosome targeting; pH switching; ratiometric probe
    DOI:  https://doi.org/10.1021/acssensors.1c00961
  11. Mol Biol Rep. 2021 Jun 16.
      Glioblastoma multiforme (GBM) is one of the aggressive brain cancers with patients having less survival period upto 12-15 months. Mammalian target of rapamycin (mTOR) is a serine/threonine kinase, belongs to the phosphatidylinositol 3-kinases (PI3K) pathway and is involved in various cellular processes of cancer cells. Cancer metabolism is regulated by mTOR and its components. mTOR forms two complexes as mTORC1 and mTORC2. Studies have identified the key component of the mTORC2 complex, Rapamycin-insensitive companion of mammalian target of rapamycin (Rictor) plays a prominent role in the regulation of cancer cell proliferation and metabolism. Apart, growth factor receptor signaling such as epidermal growth factor signaling mediated by epidermal growth factor receptor (EGFR) regulates cancer-related processes. In EGFR signaling various other signaling cascades such as phosphatidyl-inositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR pathway) and Ras/Raf/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) -dependent signaling cross-talk each other. From various studies about GBM, it is very well established that Rictor and EGFR mediated signaling pathways majorly playing a pivotal role in chemoresistance and tumor aggressiveness. Recent studies have shown that non-coding RNAs such as microRNAs (miRs) and long non-coding RNAs (lncRNAs) regulate the EGFR and Rictor and sensitize the cells towards chemotherapeutic agents. Thus, understanding of microRNA mediated regulation of EGFR and Rictor will help in cancer prevention and management as well as a future therapy.
    Keywords:  EGFR; Glioblastoma multiforme (GBM); HDAC; Rictor; Temozolomide (TMZ); mTOR
    DOI:  https://doi.org/10.1007/s11033-021-06462-2
  12. Nat Commun. 2021 06 16. 12(1): 3653
      The Mechanistic Target Of Rapamycin Complex 1 (mTORC1) pathway controls several aspects of neuronal development. Mutations in regulators of mTORC1, such as Tsc1 and Tsc2, lead to neurodevelopmental disorders associated with autism, intellectual disabilities and epilepsy. The correct development of inhibitory interneurons is crucial for functional circuits. In particular, the axonal arborisation and synapse density of parvalbumin (PV)-positive GABAergic interneurons change in the postnatal brain. How and whether mTORC1 signaling affects PV cell development is unknown. Here, we show that Tsc1 haploinsufficiency causes a premature increase in terminal axonal branching and bouton density formed by mutant PV cells, followed by a loss of perisomatic innervation in adult mice. PV cell-restricted Tsc1 haploinsufficient and knockout mice show deficits in social behavior. Finally, we identify a sensitive period during the third postnatal week during which treatment with the mTOR inhibitor Rapamycin rescues deficits in both PV cell innervation and social behavior in adult conditional haploinsufficient mice. Our findings reveal a role of mTORC1 signaling in the regulation of the developmental time course and maintenance of cortical PV cell connectivity and support a mechanistic basis for the targeted rescue of autism-related behaviors in disorders associated with deregulated mTORC1 signaling.
    DOI:  https://doi.org/10.1038/s41467-021-23939-7
  13. J Immunol. 2021 Jun 16. pii: ji2001364. [Epub ahead of print]
      Insufficient autophagic degradation has been implicated in accelerated cellular senescence during chronic obstructive pulmonary disease (COPD) pathogenesis. Aging-linked and cigarette smoke (CS)-induced functional deterioration of lysosomes may be associated with impaired autophagy. Lysosomal membrane permeabilization (LMP) is indicative of damaged lysosomes. Galectin-3 and tripartite motif protein (TRIM) 16 play a cooperative role in recognizing LMP and inducing lysophagy, a lysosome-selective autophagy, to maintain lysosome function. In this study, we sought to examine the role of TRIM16-mediated lysophagy in regulating CS-induced LMP and cellular senescence during COPD pathogenesis by using human bronchial epithelial cells and lung tissues. CS extract (CSE) induced lysosomal damage via LMP, as detected by galectin-3 accumulation. Autophagy was responsible for modulating LMP and lysosome function during CSE exposure. TRIM16 was involved in CSE-induced lysophagy, with impaired lysophagy associated with lysosomal dysfunction and accelerated cellular senescence. Airway epithelial cells in COPD lungs showed an increase in lipofuscin, aggresome and galectin-3 puncta, reflecting accumulation of lysosomal damage with concomitantly reduced TRIM16 expression levels. Human bronchial epithelial cells isolated from COPD patients showed reduced TRIM16 but increased galectin-3, and a negative correlation between TRIM16 and galectin-3 protein levels was demonstrated. Damaged lysosomes with LMP are accumulated in epithelial cells in COPD lungs, which can be at least partly attributed to impaired TRIM16-mediated lysophagy. Increased LMP in lung epithelial cells may be responsible for COPD pathogenesis through the enhancement of cellular senescence.
    DOI:  https://doi.org/10.4049/jimmunol.2001364
  14. Front Cell Dev Biol. 2021 ;9 656604
      Skeletal muscle protein synthesis is a highly complex process, influenced by nutritional status, mechanical stimuli, repair programs, hormones, and growth factors. The molecular aspects of protein synthesis are centered around the mTORC1 complex. However, the intricacies of mTORC1 regulation, both up and downstream, have expanded overtime. Moreover, the plastic nature of skeletal muscle makes it a unique tissue, having to coordinate between temporal changes in myofiber metabolism and hypertrophy/atrophy stimuli within a tissue with considerable protein content. Skeletal muscle manages the push and pull between anabolic and catabolic pathways through key regulatory proteins to promote energy production in times of nutrient deprivation or activate anabolic pathways in times of nutrient availability and anabolic stimuli. Branched-chain amino acids (BCAAs) can be used for both energy production and signaling to induce protein synthesis. The metabolism of BCAAs occur in tandem with energetic and anabolic processes, converging at several points along their respective pathways. The fate of intramuscular BCAAs adds another layer of regulation, which has consequences to promote or inhibit muscle fiber protein anabolism. This review will outline the general mechanisms of muscle protein synthesis and describe how metabolic pathways can regulate this process. Lastly, we will discuss how BCAA availability and demand coordinate with synthesis mechanisms and identify key factors involved in intramuscular BCAA trafficking.
    Keywords:  AMPK (5′-AMP activated kinase); BCKD; branch chain amino acids; branched-chain α-ketoacid dehydrogenase; mammalian target of rapamycin; protein synthesis; skeletal muscle
    DOI:  https://doi.org/10.3389/fcell.2021.656604
  15. Mol Genet Metab. 2021 May 07. pii: S1096-7192(21)00702-2. [Epub ahead of print]
      Gangliosidoses are inherited lysosomal storage disorders caused by reduced or absent activity of either a lysosomal enzyme involved in ganglioside catabolism, or an activator protein required for the proper activity of a ganglioside hydrolase, which results in the intra-lysosomal accumulation of undegraded metabolites. We hereby describe morphological, ultrastructural, biochemical and genetic features of GM2 gangliosidosis in three captive bred wild boar littermates. The piglets were kept in a partially-free range farm and presented progressive neurological signs, starting at 6 months of age. Animals were euthanized at approximately one year of age due to their poor conditions. Neuropathogens were excluded as a possible cause of the signs. Gross examination showed a reduction of cerebral and cerebellar consistency. Central (CNS) and peripheral (PNS) nervous system neurons were enlarged and foamy, with severe and diffuse cytoplasmic vacuolization. Transmission electron microscopy (TEM) of CNS neurons demonstrated numerous lysosomes, filled by parallel or concentric layers of membranous electron-dense material, defined as membranous cytoplasmic bodies (MCB). Biochemical composition of gangliosides analysis from CNS revealed accumulation of GM2 ganglioside; furthermore, Hex A enzyme activity was less than 1% compared to control animals. These data confirmed the diagnosis of GM2 gangliosidosis. Genetic analysis identified, at a homozygous level, the presence of a missense nucleotide variant c.1495C > T (p Arg499Cys) in the hexosaminidase subunit alpha gene (HEXA), located within the GH20 hexosaminidase superfamily domain of the encoded protein. This specific HEXA variant is known to be pathogenic and associated with Tay-Sachs disease in humans, but has never been identified in other animal species. This is the first report of a HEXA gene associated Tay-Sachs disease in wild boars and provides a comprehensive description of a novel spontaneous animal model for this lysosomal storage disease.
    Keywords:  GM2; HEXA; Lysosomal storage disease; Swine; Tay-Sachs disease; Whole-genome sequencing; Wild boar
    DOI:  https://doi.org/10.1016/j.ymgme.2021.05.001
  16. Nat Commun. 2021 Jun 18. 12(1): 3786
      Mutation of C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD), which is attributed to both a gain and loss of function. C9orf72 forms a complex with SMCR8 and WDR41, which was reported to have GTPase activating protein activity toward ARF proteins, RAB8A, and RAB11A. We determined the cryo-EM structure of ARF1-GDP-BeF3- bound to C9orf72:SMCR8:WDR41. The SMCR8longin and C9orf72longin domains form the binding pocket for ARF1. One face of the C9orf72longin domain holds ARF1 in place, while the SMCR8longin positions the catalytic finger Arg147 in the ARF1 active site. Mutations in interfacial residues of ARF1 and C9orf72 reduced or eliminated GAP activity. RAB8A GAP required ~10-fold higher concentrations of the C9orf72 complex than for ARF1. These data support a specific function for the C9orf72 complex as an ARF GAP. The structure also provides a model for the active forms of the longin domain GAPs of FLCN and NPRL2 that regulate the Rag GTPases of the mTORC1 pathway.
    DOI:  https://doi.org/10.1038/s41467-021-24081-0
  17. Bioessays. 2021 Jun 14. e2100093
      Ferroptosis, a form of regulated cell death triggered by lipid hydroperoxide accumulation, has an important role in a variety of diseases and pathological conditions, such as cancer. Targeting ferroptosis is emerging as a promising means of therapeutic intervention in cancer treatment. Polyunsaturated fatty acids, reactive oxygen species, and labile iron constitute the major underlying triggers for ferroptosis. Other regulators of ferroptosis have also been discovered recently, among them the mechanistic target of rapamycin complex 1 (mTORC1), a central controller of cell growth and metabolism. Inhibitors of mTORC1 have been used in treating diverse diseases, including cancer. In this review, we discuss recent findings linking mTORC1 to ferroptosis, dissect mechanisms underlying the establishment of mTORC1 as a key ferroptosis modulator, and highlight the potential of co-targeting mTORC1 and ferroptosis in cancer treatment. This review will provide valuable insights for future investigations of ferroptosis and mTORC1 in fundamental biology and cancer therapy.
    Keywords:  GPX4; SLC7A11; autophagy; cancer therapy; ferroptosis; lipid peroxidation; mTOR; mTORC1; oncogene
    DOI:  https://doi.org/10.1002/bies.202100093
  18. Front Genet. 2021 ;12 667283
      Discovered in 2009, the DEP-domain containing mTOR-interacting protein, DEPTOR, is a known regulator of the mechanistic target of rapamycin (mTOR), an evolutionarily conserved kinase that regulates diverse cellular processes in response to environmental stimuli. DEPTOR was originally identified as a negative regulator of mTOR complexes 1 (mTORC1) and 2 (mTORC2). However, recent discoveries have started to unravel the roles of DEPTOR in mTOR-independent responses. In the past few years, mTOR emerged as an important regulator of skeletal development, growth, and homeostasis; the dysregulation of its activity contributes to the development of several skeletal diseases, both chronic and genetic. Even more recently, several groups have reported on the relevance of DEPTOR in skeletal biology through its action on mTOR-dependent and mTOR-independent pathways. In this review, we summarize the current understanding of DEPTOR in skeletal development and disease.
    Keywords:  DEPTOR; bone; cartilage; mTOR; skeleton
    DOI:  https://doi.org/10.3389/fgene.2021.667283
  19. Bio Protoc. 2021 Apr 20. 11(8): e3989
      Mammalian target of rapamycin (mTOR) controls many crucial cellular functions, including protein synthesis, cell size, energy metabolism, lysosome and mitochondria biogenesis, and autophagy. Consequently, deregulation of mTOR signaling plays a role in numerous pathological conditions such as cancer, metabolic disorders and neurological diseases. Developing new tools to monitor mTOR spatiotemporal activation is crucial to better understand its roles in physiological and pathological conditions. However, the most widely used method to report mTOR activity relies on the quantification of specific mTOR-phosphorylated substrates by western blot. This approach requires cellular lysate preparation, which restricts the quantification to a single time point. Here, we present a simple protocol to study mTOR activity in living cells in real time using AIMTOR, an intramolecular BRET-based (bioluminescence resonance energy transfer) biosensor that we recently designed ( Bouquier et al., 2020 ). We describe transfection of AIMTOR in the C2C12 cell line and procedures to monitor BRET in a cell population using a plate reader and in single cells by microscopy. Importantly, this protocol is transposable to any cell line and primary cells. In addition, several subcellular compartment-specific versions of AIMTOR have been developed, enabling compartmentalized assessment of mTOR activity. This protocol describes how to use the sensitive AIMTOR biosensor to investigate mTOR signaling dynamics in living cells. Graphic abstract: AIMTOR protocol overview from seeding cells to live BRET recording.
    Keywords:  BRET; Kinase activity; Living cells; Real time imaging; mTOR signaling; mTORC1 biosensor
    DOI:  https://doi.org/10.21769/BioProtoc.3989
  20. EMBO J. 2021 06 14. e105985
      Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK-dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK-dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation-independent accumulation of ULK substrates and kinase activity-regulated recruitment of autophagy proteins to ubiquitin-positive structures.
    Keywords:  PIK3R4; PRKAG2; ULK1; VPS15; p62
    DOI:  https://doi.org/10.15252/embj.2020105985
  21. Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Jun 08. pii: S1388-1981(21)00116-5. [Epub ahead of print]1866(9): 158988
      Neutral lipases-mediated lipolysis and acid lipases-moderated lipophagy are two main processes for degradation of lipid droplets (LDs). However, the individual and interactive roles of these metabolic pathways are not well known across vertebrates. This study explored the roles of lipolysis and lipophagy from the aspect of neutral and acid lipases in zebrafish. We established zebrafish strains deficient in either adipose triglyceride lipase (atgl-/-; AKO fish) or lysosomal acid lipase (lal-/-; LKO fish) respectively, and then inhibited lipolysis in the LKO fish and lipophagy in the AKO fish by feeding diets supplemented with the corresponding inhibitors Atglistatin and 3-Methyladenine, respectively. Both the AKO and LKO fish showed reduced growth, swimming activity, and oxygen consumption. The AKO fish did not show phenotypes in adipose tissue, but mainly accumulated triacylglycerol (TAG) in liver, also, they had large LDs in the hepatocytes, and did not stimulate lipophagy as a compensation response but maintained basal lipophagy. The LKO fish reduced total lipid accumulation in the body but had high cholesterol content in liver; also, they accumulated small LDs in the hepatocytes, and showed increased lipolysis, especially Atgl expression, as a compensatory mechanism. Simultaneous inhibition of lipolysis and lipophagy in zebrafish resulted in severe liver damage, with the potential to trigger mitophagy. Overall, our study illustrates that lipolysis and lipophagy perform individual and interactive roles in maintaining homeostasis of TAG and cholesterol metabolism. Furthermore, the interactive roles of lipolysis and lipophagy may be essential in regulating the functions and form of mitochondria.
    Keywords:  Interaction; Lipid catabolism; Lipolysis; Lipophagy; Mitochondria; Zebrafish
    DOI:  https://doi.org/10.1016/j.bbalip.2021.158988
  22. Contact (Thousand Oaks). 2021 Jan-Dec;4:4 25152564211016608
      Cellular adaptation to stress and metabolic cues requires a coordinated response of different intracellular compartments, separated by semipermeable membranes. One way to facilitate interorganellar communication is via membrane contact sites, physical bridges between opposing organellar membranes formed by an array of tethering machineries. These contact sites are highly dynamic and establish an interconnected organellar network able to quickly respond to external and internal stress by changing size, abundance and molecular architecture. Here, we discuss recent work on nucleus-vacuole junctions, connecting yeast vacuoles with the nucleus. Appearing as small, single foci in mitotic cells, these contacts expand into one enlarged patch upon nutrient exhaustion and entry into quiescence or can be shaped into multiple large foci essential to sustain viability upon proteostatic stress at the nuclear envelope. We highlight the remarkable plasticity and rapid remodelling of these contact sites upon metabolic or proteostatic stress and their emerging importance for cellular fitness.
    Keywords:  NVJ; Snd3; glucose; metabolism; quiescence; stress response
    DOI:  https://doi.org/10.1177/25152564211016608
  23. Proc Natl Acad Sci U S A. 2021 Jun 22. pii: e2022115118. [Epub ahead of print]118(25):
      In 2006, GRN mutations were first linked to frontotemporal dementia (FTD), the leading cause of non-Alzheimer dementias. While much research has been dedicated to understanding the genetic causes of the disease, our understanding of the mechanistic impacts of GRN deficiency has only recently begun to take shape. With no known cure or treatment available for GRN-related FTD, there is a growing need to rapidly advance genetic and/or small-molecule therapeutics for this disease. This issue is complicated by the fact that, while lysosomal dysfunction seems to be a key driver of pathology, the mechanisms linking a loss of GRN to a pathogenic state remain unclear. In our attempt to address these key issues, we have turned to the nematode, Caenorhabditis elegans, to model, study, and find potential therapies for GRN-deficient FTD. First, we show that the loss of the nematode GRN ortholog, pgrn-1, results in several behavioral and molecular defects, including lysosomal dysfunction and defects in autophagic flux. Our investigations implicate the sphingolipid metabolic pathway in the regulation of many of the in vivo defects associated with pgrn-1 loss. Finally, we utilized these nematodes as an in vivo tool for high-throughput drug screening and identified two small molecules with potential therapeutic applications against GRN/pgrn-1 deficiency. These compounds reverse the biochemical, cellular, and functional phenotypes of GRN deficiency. Together, our results open avenues for mechanistic and therapeutic research into the outcomes of GRN-related neurodegeneration, both genetic and molecular.
    Keywords:  Caenorhabditis elegans; frontotemporal dementia; progranulin
    DOI:  https://doi.org/10.1073/pnas.2022115118
  24. Semin Cancer Biol. 2021 Jun 14. pii: S1044-579X(21)00183-8. [Epub ahead of print]
      Iron, the most abundant metal in human brain, is an essential microelement that regulates numerous cellular mechanisms. Some key physiological roles of iron include oxidative phosphorylation and ATP production, embryonic neuronal development, formation of iron-sulfur clusters, and the regulation of enzymes involved in DNA synthesis and repair. Because of its physiological and pathological importance, iron homeostasis must be tightly regulated by balancing its uptake, transport, and storage. Endosomes and lysosomes (endolysosomes) are acidic organelles known to contain readily releasable stores of various cations including iron and other metals. Increased levels of ferrous (Fe2+) iron can generate reactive oxygen species (ROS) via Fenton chemistry reactions and these increases can damage mitochondria and genomic DNA as well as promote carcinogenesis. Accumulation of iron in the brain has been linked with aging, diet, disease, and cerebral hemorrhage. Further, deregulation of brain iron metabolism has been implicated in carcinogenesis and may be a contributing factor to the increased incidence of brain tumors around the world. Here, we provide insight into mechanisms by which iron accumulation in endolysosomes is altered by pH and lysosome membrane permeabilization. Such events generate excess ROS resulting in mitochondrial DNA damage, fission, and dysfunction, as well as DNA oxidative damage in the nucleus; all of which promote carcinogenesis. A better understanding of the roles that endolysosome iron plays in carcinogenesis may help better inform the development of strategic therapeutic options for cancer treatment and prevention.
    Keywords:  Brain; Carcinogens; Endolysosomes; Iron; ROS
    DOI:  https://doi.org/10.1016/j.semcancer.2021.06.013
  25. PLoS Genet. 2021 Jun 14. 17(6): e1009583
      Ribosome biogenesis lies at the nexus of various signaling pathways coordinating protein synthesis with cell growth and proliferation. This process is regulated by well-described transcriptional mechanisms, but a growing body of evidence indicates that other levels of regulation exist. Here we show that the Ras/mitogen-activated protein kinase (MAPK) pathway stimulates post-transcriptional stages of human ribosome synthesis. We identify RIOK2, a pre-40S particle assembly factor, as a new target of the MAPK-activated kinase RSK. RIOK2 phosphorylation by RSK stimulates cytoplasmic maturation of late pre-40S particles, which is required for optimal protein synthesis and cell proliferation. RIOK2 phosphorylation facilitates its release from pre-40S particles and its nuclear re-import, prior to completion of small ribosomal subunits. Our results bring a detailed mechanistic link between the Ras/MAPK pathway and the maturation of human pre-40S particles, which open a hitherto poorly explored area of ribosome biogenesis.
    DOI:  https://doi.org/10.1371/journal.pgen.1009583
  26. JCI Insight. 2021 Jun 17. pii: 137708. [Epub ahead of print]
      The acute respiratory distress syndrome (ARDS) is a highly lethal condition that impairs lung function and causes respiratory failure. Mechanical ventilation maintains gas exchange in patients with ARDS, but exposes lung cells to physical forces that exacerbate lung injury. Our data demonstrate that mTOR complex 1 (mTORC1) is a mechanosensor in lung epithelial cells and that activation of this pathway during mechanical ventilation impairs lung function. We found that mTORC1 is activated in lung epithelial cells following volutrauma and atelectrauma in mice and humanized in vitro models of the lung microenvironment. mTORC1 is also activated in lung tissue of mechanically ventilated patients with ARDS. Deletion of Tsc2, a negative regulator of mTORC1, in epithelial cells impairs lung compliance during mechanical ventilation. Conversely, treatment with rapamycin at the time mechanical ventilation is initiated improves lung compliance without altering lung inflammation or barrier permeability. mTORC1 inhibition mitigates physiologic lung injury by preventing surfactant dysfunction during mechanical ventilation. Our data demonstrate that in contrast to canonical mTORC1 activation under favorable growth conditions, activation of mTORC1 during mechanical ventilation exacerbates lung injury and inhibition of this pathway may be a novel therapeutic target to mitigate ventilator-induced lung injury during ARDS.
    Keywords:  Pulmonary surfactants; Pulmonology; Signal transduction
    DOI:  https://doi.org/10.1172/jci.insight.137708
  27. Autophagy. 2021 Jun 16.
      Primary dysfunction of autophagy due to Mendelian defects affecting core components of the autophagy machinery or closely related proteins have recently emerged as an important cause of genetic disease. This novel group of human disorders may present throughout life and comprises severe early-onset neurodevelopmental and more common adult-onset neurodegenerative disorders. Early-onset (or congenital) disorders of autophagy often share a recognizable "clinical signature," including variable combinations of neurological, neuromuscular and multisystem manifestations. Structural CNS abnormalities, cerebellar involvement, spasticity and peripheral nerve pathology are prominent neurological features, indicating a specific vulnerability of certain neuronal populations to autophagic disturbance. A typically biphasic disease course of late-onset neurodegeneration occurring on the background of a neurodevelopmental disorder further supports a role of autophagy in both neuronal development and maintenance. In addition, an associated myopathy has been characterized in several conditions. The differential diagnosis comprises a wide range of other multisystem disorders, including mitochondrial, glycogen and lysosomal storage disorders, as well as ciliopathies, glycosylation and vesicular trafficking defects. The clinical overlap between the congenital disorders of autophagy and these conditions reflects the multiple roles of the proteins and/or emerging molecular connections between the pathways implicated and suggests an exciting area for future research. Therapy development for congenital disorders of autophagy is still in its infancy but may result in the identification of molecules that target autophagy more specifically than currently available compounds. The close connection with adult-onset neurodegenerative disorders highlights the relevance of research into rare early-onset neurodevelopmental conditions for much more common, age-related human diseases.
    DOI:  https://doi.org/10.1080/15548627.2021.1943177
  28. Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Jun 07. pii: S1388-1981(21)00113-X. [Epub ahead of print]1866(9): 158985
      Phosphatidylinositol is the parent lipid for the synthesis of seven phosphorylated inositol lipids and each of them play specific roles in numerous processes including receptor-mediated signalling, actin cytoskeleton dynamics and membrane trafficking. PI synthesis is localised to the endoplasmic reticulum (ER) whilst its phosphorylated derivatives are found in other organelles where the lipid kinases also reside. Phosphorylation of PI to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane and to phosphatidylinositol 4-phosphate (PI4P) at the Golgi are key events in lipid signalling and Golgi function respectively. Here we review a family of proteins, phosphatidylinositol transfer proteins (PITPs), that can mobilise PI from the ER to provide the substrate to the resident kinases for phosphorylation. Recent studies identify specific and overlapping functions for the three soluble PITPs (PITPα, PITPβ and PITPNC1) in phospholipase C signalling, neuronal function, membrane trafficking, viral replication and in cancer metastases.
    Keywords:  Golgi; Lipid transfer; Membrane traffic; PITPNC1; Phosphatidic acid; Phosphatidylinositol; Phospholipase C
    DOI:  https://doi.org/10.1016/j.bbalip.2021.158985
  29. EMBO J. 2021 Jun 14. e106871
      Low-density lipoprotein (LDL)-cholesterol delivery from late endosomes to the plasma membrane regulates focal adhesion dynamics and cell migration, but the mechanisms controlling it are poorly characterized. Here, we employed auxin-inducible rapid degradation of oxysterol-binding protein-related protein 2 (ORP2/OSBPL2) to show that endogenous ORP2 mediates the transfer of LDL-derived cholesterol from late endosomes to focal adhesion kinase (FAK)-/integrin-positive recycling endosomes in human cells. In vitro, cholesterol enhances membrane association of FAK to PI(4,5)P2 -containing lipid bilayers. In cells, ORP2 stimulates FAK activation and PI(4,5)P2 generation in endomembranes, enhancing cell adhesion. Moreover, ORP2 increases PI(4,5)P2 in NPC1-containing late endosomes in a FAK-dependent manner, controlling their tubulovesicular trafficking. Together, these results provide evidence that ORP2 controls FAK activation and LDL-cholesterol plasma membrane delivery by promoting bidirectional cholesterol/PI(4,5)P2 exchange between late and recycling endosomes.
    Keywords:  cholesterol trafficking; focal adhesion kinase; oxysterol-binding protein-related protein; phosphoinositides; recycling
    DOI:  https://doi.org/10.15252/embj.2020106871
  30. Sci Rep. 2021 Jun 16. 11(1): 12689
      Centrosomal P4.1-associated protein (CPAP) plays a critical role in restricting the centriole length in human cells. Here, we report a novel, positive regulatory influence for CPAP on endocytic vesicular transport (EVT) and lysosome targeting of internalized-cell surface receptor EGFR. We observed that higher CPAP levels cause an increase in the abundance of multi-vesicular body (MVB) and EGFR is detectable in CPAP-overexpression induced puncta. The surface and cellular levels of EGFR are higher under CPAP deficiency and lower under CPAP overexpression. While ligand-engagement induced internalization or routing of EGFR into early endosomes is not influenced by cellular levels of CPAP, we found that targeting of ligand-activated, internalized EGFR to lysosome is impacted by CPAP levels. Transport of ligand-bound EGFR from early endosome to late endosome/MVB and lysosome is diminished in CPAP-depleted cells. Moreover, CPAP depleted cells appear to show a diminished ability to form MVB structures upon EGFR activation. These observations suggest a positive regulatory effect of CPAP on EVT of ligand-bound EGFR-like cell surface receptors to MVB and lysosome. Overall, identification of a non-centriolar function of CPAP in endocytic trafficking provides new insights in understanding the non-canonical cellular functions of CPAP.
    DOI:  https://doi.org/10.1038/s41598-021-91818-8
  31. Front Cell Dev Biol. 2021 ;9 648384
      Tumor progression involves invasion, migration, metabolism, autophagy, exosome secretion, and drug resistance. Cargos transported by membrane vesicle trafficking underlie all of these processes. Rab GTPases, which, through coordinated and dynamic intracellular membrane trafficking alongside cytoskeletal pathways, determine the maintenance of homeostasis and a series of cellular functions. The mechanism of vesicle movement regulated by Rab GTPases plays essential roles in cancers. Therefore, targeting Rab GTPases to adjust membrane trafficking has the potential to become a novel way to adjust cancer treatment. In this review, we describe the characteristics of Rab GTPases; in particular, we discuss the role of their activation in the regulation of membrane transport and provide examples of Rab GTPases regulating membrane transport in tumor progression. Finally, we discuss the clinical implications and the potential as a cancer therapeutic target of Rab GTPases.
    Keywords:  GDP; GEF; Rab GTPase; cancer biology; membrane trafficking
    DOI:  https://doi.org/10.3389/fcell.2021.648384
  32. Front Oncol. 2021 ;11 667495
      Spliceosomes are large RNA-protein molecular complexes which mediate splicing of pre-mRNA in eukaryotic cells. Their function is frequently altered in cancer, providing opportunities for novel therapeutic approaches. The ubiquitin specific protease 39 (USP39) is a highly conserved deubiquitylation family member that plays an essential role in pre-mRNA splicing where it serves to assemble the mature spliceosome complex. Previous studies have reported that USP39 acts in an oncogenic manner where it contributes to cancer progression and predicts poor prognosis in various human tumor types. Here we report that USP39 is differentially upregulated in human esophageal squamous cell carcinoma (ESCC) and its expression is significantly associated with clinicopathological characteristics including differentiation status and TNM stage. We found the USP39 upregulation was maintained in ESCC cell lines where it functioned to promote cancer cell growth in vitro and in xenografts. RNA-seq analyses identified that mTOR pathway activation was affected by shRNA-mediated silencing of USP39. Subsequent biochemical analyses demonstrated that USP39 regulates the activity of mTORC2 by selectively enhancing the splicing and maturation of Rictor mRNA, although not other key mTORC components. Together, our report proposes USP39 as a biomarker and oncogenic factor in ESCC, with a potential for targeting the USP39/mTOR2/Rictor axis as a therapeutic strategy. Furthermore, our study adds ESCC to the list of cancers where USP39 contributes to tumorigenesis and progression.
    Keywords:  RNA splicing; esophageal squamous cell carcinoma (ESCC); mammalian target of rapamycin (mTOR); rictor; ubiquitin specific protease 39 (USP39)
    DOI:  https://doi.org/10.3389/fonc.2021.667495
  33. Neoplasia. 2021 Jun 11. pii: S1476-5586(21)00032-4. [Epub ahead of print]23(7): 643-652
      Ribonucleotide reductase (RNR) is the key enzyme that catalyzes the production of deoxyribonucleotides (dNTPs) for DNA replication and it is also essential for cancer cell proliferation. As the RNR inhibitor, Gemcitabine is widely used in cancer therapies, however, resistance limits its therapeutic efficacy and curative potential. Here, we identified that mTORC2 is a main driver of gemcitabine resistance in non-small cell lung cancers (NSCLC). Pharmacological or genetic inhibition of mTORC2 greatly enhanced gemcitabine induced cytotoxicity and DNA damage. Mechanistically, mTORC2 directly interacted and phosphorylated RNR large subunit RRM1 at Ser 631. Ser631 phosphorylation of RRM1 enhanced its interaction with small subunit RRM2 to maintain sufficient RNR enzymatic activity for efficient DNA replication. Targeting mTORC2 retarded DNA replication fork progression and improved therapeutic efficacy of gemcitabine in NSCLC xenograft model in vivo. Thus, these results identified a mechanism through mTORC2 regulating RNR activity and DNA replication, conferring gemcitabine resistance to cancer cells.
    Keywords:  DNA replication stress; Gemcitabine; Ribonucleotide reductase; mTORC2
    DOI:  https://doi.org/10.1016/j.neo.2021.05.007
  34. Cell Rep. 2021 Jun 15. pii: S2211-1247(21)00623-9. [Epub ahead of print]35(11): 109259
      Dysfunction of the endolysosomal-autophagy network is emerging as an important pathogenic process in Alzheimer's disease. Mutations in the sorting receptor-encoding gene SORL1 cause autosomal-dominant Alzheimer's disease, and SORL1 variants increase risk for late-onset AD. To understand the contribution of SORL1 mutations to AD pathogenesis, we analyze the effects of a SORL1 truncating mutation on SORL1 protein levels and endolysosome function in human neurons. We find that truncating mutation results in SORL1 haploinsufficiency and enlarged endosomes in human neurons. Analysis of isogenic SORL1 wild-type, heterozygous, and homozygous null neurons demonstrates that, whereas SORL1 haploinsufficiency results in endosome dysfunction, complete loss of SORL1 leads to additional defects in lysosome function and autophagy. Neuronal endolysosomal dysfunction caused by loss of SORL1 is relieved by extracellular antisense oligonucleotide-mediated reduction of APP protein, demonstrating that PSEN1, APP, and SORL1 act in a common pathway regulating the endolysosome system, which becomes dysfunctional in AD.
    Keywords:  Alzheimer's disease; SORL1; amyloid precursor protein; autophagy; endosome; iPSC; lysosome
    DOI:  https://doi.org/10.1016/j.celrep.2021.109259
  35. Nat Commun. 2021 06 14. 12(1): 3606
      Membrane fusion is fundamental to biological processes as diverse as membrane trafficking or viral infection. Proteins catalyzing membrane fusion need to overcome energy barriers to induce intermediate steps in which the integrity of bilayers is lost. Here, we investigate the structural features of tightly docked intermediates preceding hemifusion. Using lipid vesicles in which progression to hemifusion is arrested, we show that the metastable intermediate does not require but is enhanced by divalent cations and is characterized by the absence of proteins and local membrane thickening. Molecular dynamics simulations reveal that thickening is due to profound lipid rearrangements induced by dehydration of the membrane surface.
    DOI:  https://doi.org/10.1038/s41467-021-23722-8