bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022–10–09
38 papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing



  1. Biol Pharm Bull. 2022 ;45(10): 1426-1431
      Vacuolar-type ATPase (V-ATPase) shares its structure and rotational catalysis with F-type ATPase (F-ATPase, ATP synthase). However, unlike subunits of F-ATPase, those of V-ATPase have tissue- and/or organelle-specific isoforms. Structural diversity of V-ATPase generated by different combinations of subunit isoforms enables it to play diverse physiological roles in mammalian cells. Among these various roles, this review focuses on the functions of lysosome-specific V-ATPase in bone resorption by osteoclasts. Lysosomes remain in the cytoplasm in most cell types, but in osteoclasts, secretory lysosomes move toward and fuse with the plasma membrane to secrete lysosomal enzymes, which is essential for bone resorption. Through this process, lysosomal V-ATPase harboring the a3 isoform of the a subunit is relocated to the plasma membrane, where it transports protons from the cytosol to the cell exterior to generate the acidic extracellular conditions required for secreted lysosomal enzymes. In addition to this role as a proton pump, we recently found that the lysosomal a3 subunit of V-ATPase is essential for anterograde trafficking of secretory lysosomes. Specifically, a3 interacts with Rab7, a member of the Rab guanosine 5'-triphosphatase (GTPase) family that regulates organelle trafficking, and recruits it to the lysosomal membrane. These findings revealed the multifunctionality of lysosomal V-ATPase in osteoclasts; V-ATPase is responsible not only for the formation of the acidic environment by transporting protons, but also for intracellular trafficking of secretory lysosomes by recruiting organelle trafficking factors. Herein, we summarize the molecular mechanism underlying secretory lysosome trafficking in osteoclasts, and discuss the possible regulatory role of V-ATPase in organelle trafficking.
    Keywords:  organelle trafficking; osteoclast; proton pump; secretory lysosome; vacuolar-type ATPase
    DOI:  https://doi.org/10.1248/bpb.b22-00371
  2. Front Mol Neurosci. 2022 ;15 944883
      Neuronopathic Gaucher disease (nGD) is an inherited neurodegenerative disease caused by mutations in GBA1 gene and is associated with premature death. Neuroinflammation plays a critical role in disease pathogenesis which is characterized by microgliosis, reactive astrocytosis, and neuron loss, although molecular mechanisms leading to neuroinflammation are not well-understood. In this report, we developed a convenient tool to quantify microglia proliferation and activation independently and uncovered abnormal proliferation of microglia (∼2-fold) in an adult genetic nGD model. The nGD-associated pattern of inflammatory mediators pertinent to microglia phenotypes was determined, showing a unique signature favoring pro-inflammatory chemokines and cytokines. Moreover, highly polarized (up or down) dysregulations of mTORC1 signaling with varying lysosome dysfunctions (numbers and volume) were observed among three major cell types of nGD brain. Specifically, hyperactive mTORC1 signaling was detected in all disease-associated microglia (Iba1high) with concurrent increase in lysosome function. Conversely, the reduction of neurons presenting high mTORC1 activity was implicated (including Purkinje-like cells) which was accompanied by inconsistent changes of lysosome function in nGD mice. Undetectable levels of mTORC1 activity and low Lamp1 puncta were noticed in astrocytes of both diseased and normal mice, suggesting a minor involvement of mTORC1 pathway and lysosome function in disease-associated astrocytes. These findings highlight the differences and complexity of molecular mechanisms that are involved within various cell types of the brain. The quantifiable parameters established and nGD-associated pattern of neuroinflammatory mediators identified would facilitate the efficacy evaluation on microgliosis and further discovery of novel therapeutic target(s) in treating neuronopathic Gaucher disease.
    Keywords:  Gaucher disease; Lamp1; Tmem119; disease-associated microglia; mTOR; neurodegenerative disorders; neuroinflammation; reactive astrocytes
    DOI:  https://doi.org/10.3389/fnmol.2022.944883
  3. NPJ Parkinsons Dis. 2022 Oct 06. 8(1): 126
      Mutations in the GBA gene that encodes the lysosomal enzyme β-glucocerebrosidase (GCase) are a major genetic risk factor for Parkinson's disease (PD). In this study, we generated a set of differentiated and stable human dopaminergic cell lines that express the two most prevalent GBA mutations as well as GBA knockout cell lines as a in vitro disease modeling system to study the relationship between mutant GBA and the abnormal accumulation of α-synuclein. We performed a deep analysis of the consequences triggered by the presence of mutant GBA protein and the loss of GCase activity in different cellular compartments, focusing primarily on the lysosomal compartment, and analyzed in detail the lysosomal activity, composition, and integrity. The loss of GCase activity generates extensive lysosomal dysfunction, promoting the loss of activity of other lysosomal enzymes, affecting lysosomal membrane stability, promoting intralysosomal pH changes, and favoring the intralysosomal accumulation of sphingolipids and cholesterol. These local events, occurring only at a subcellular level, lead to an impairment of autophagy pathways, particularly chaperone-mediated autophagy, the main α-synuclein degradative pathway. The findings of this study highlighted the role of lysosomal function and lipid metabolism in PD and allowed us to describe a molecular mechanism to understand how mutations in GBA can contribute to an abnormal accumulation of different α-synuclein neurotoxic species in PD pathology.
    DOI:  https://doi.org/10.1038/s41531-022-00397-6
  4. Nat Commun. 2022 Oct 07. 13(1): 5924
      Haploinsufficiency of GRN causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), which is cleaved to lysosomal granulin polypeptides. The function of lysosomal granulins and why their absence causes neurodegeneration are unclear. Here we discover that PGRN-deficient human cells and murine brains, as well as human frontal lobes from GRN-mutation FTD patients have increased levels of gangliosides, glycosphingolipids that contain sialic acid. In these cells and tissues, levels of lysosomal enzymes that catabolize gangliosides were normal, but levels of bis(monoacylglycero)phosphates (BMP), lipids required for ganglioside catabolism, were reduced with PGRN deficiency. Our findings indicate that granulins are required to maintain BMP levels to support ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD due to PGRN deficiency and other neurodegenerative diseases.
    DOI:  https://doi.org/10.1038/s41467-022-33500-9
  5. Mol Neurobiol. 2022 Oct 04.
      The mechanistic target of the rapamycin (mTOR) pathway is involved in cortical development. However, the efficacy of mTOR inhibitors in malformations of cortical dysplasia (MCD) outside of the tuberous sclerosis complex is unknown. We selected the MCD rat model with prenatal MAM exposure to test the efficacy of mTOR inhibitors in MCDs. We explored the early cortical changes of mTOR pathway protein expression in rats aged P15. We also monitored the early treatment effect of the mTOR inhibitor, rapamycin, on N-methyl-D-aspartate (NMDA)-induced spasms at P15 and their behavior in the juvenile stage. In vivo MR spectroscopy was performed after rapamycin treatment and compared with vehicle controls. There was no difference in mTORC1 pathway protein expression between MAM-exposed MCD rats and controls at P15, and prolonged treatment of rapamycin had no impact on NMDA-induced spasms despite poor weight gain. Prenatal MAM-exposed juvenile rats treated with rapamycin showed increased social approaching and freezing behavior during habituation. MR spectroscopy showed altered neurometabolites, including Gln, Glu+Gln, Tau, and Cr. Despite behavioral changes and in vivo neurometabolic alteration with early prolonged rapamycin treatment, rapamycin had no effect on spasms susceptibility in prenatal MAM-exposed infantile rats with MCD without mTORC1 activation. For MAM-exposed MCD rats without mTORC1 activation, treatment options outside of mTOR pathway inhibitors should be explored.
    Keywords:  Malformations of cortical development (MCD); Methylazoxymethanol (MAM); N-methyl-D-aspartate (NMDA); Rapamycin; mTOR
    DOI:  https://doi.org/10.1007/s12035-022-03033-9
  6. Mol Nutr Food Res. 2022 Oct 03. e2200186
       SCOPE: Mechanistic target of rapamycin (mTOR) serves as a central signaling node in the coordination of cell growth and metabolism, and it functions via two distinct complexes, namely, mTORC1 and mTORC2. mTORC1 plays a crucial role in sensing amino acids, whereas mTORC2 involves in sensing growth factors. However, it remains largely unclear whether mTORC2 can sense amino acids and the mechanism by which amino acids regulate mTORC2 has not been studied.
    METHODS AND RESULTS: After treating cells with indicated concentration of amino acids for different time, it is found that the mTORC2 activation was significantly increased in response to amino acids stimulation, especially cystine. Particularly, knockdown SLC7A11 by siRNA showed that SLC7A11-mediated cystine uptake is responsible for activating mTORC2. Mechanistically, we found that p38 is activated in response to cystine stimulation, and co-immunoprecipitation experiments suggested that p38 regulated the assembly of components within mTORC2 by mediating the phosphorylation of the mTORC2 subunit Sin1 in a cystine-dependent manner. Finally, combined with inducers and inhibitors of ferroptosis and cell viability assay, we observed that cystine-mediated regulation of the p38-Sin1-mTOR-AKT pathway induced resistance to ferroptosis.
    CONCLUSION: These results indicate that cystine-induced activation of the p38-Sin1-mTORC2-AKT pathway suppresses ferroptosis. This article is protected by copyright. All rights reserved.
    Keywords:  Cystine; Ferroptosis; Sin1; mTORC2; p38
    DOI:  https://doi.org/10.1002/mnfr.202200186
  7. Structure. 2022 Oct 06. pii: S0969-2126(22)00355-0. [Epub ahead of print]30(10): 1369-1371
      In this issue of Structure, Armstrong and colleagues probe the structure of human fucosidase FucA1. Their work resolves an ongoing debate around the enzyme's catalytic mechanism and provides a valid structural template to guide the design of drugs alleviating the rare, yet severe, lysosomal storage disease fucosidosis.
    DOI:  https://doi.org/10.1016/j.str.2022.09.002
  8. Biol Pharm Bull. 2022 ;45(10): 1419-1425
      Vacuolar-type H+-ATPase (V-ATPase) was first identified as an electrogenic proton pump that acidifies the lumen of intracellular organelles. Subsequently, it was observed that the proton pump also participates in the acidification of extracellular compartments. V-ATPase plays important roles in a wide range of cell biological processes and physiological functions by generating an acidic pH; therefore, it has attracted much attention not only in basic research but also in pathological and clinical aspects. Emerging evidence indicates that the luminal acidic endocytic organelles and their trafficking may function as important hubs that connect and coordinate various signaling pathways. Various pharmacological analyses have suggested that acidic endocytic organelles are important for the maintenance of cell polarity. Recently, several studies using genetic approaches have revealed the involvement of V-ATPase in the establishment and maintenance of apico-basal polarity. This review provides a brief overview of the relationship between the polarity of epithelial cells and V-ATPase as well as V-ATPase driven luminal acidification.
    Keywords:  cell polarity; epithelial cell; organelle acidification; vacuolar-type H+-ATPase; vesicle trafficking
    DOI:  https://doi.org/10.1248/bpb.b22-00205
  9. Mol Ther. 2022 Oct 03. pii: S1525-0016(22)00606-2. [Epub ahead of print]
      Krabbe disease (KD) is a lysosomal storage disease (LSD) caused by mutations in the galc gene. There are over 50 monogenetic LSDs, which largely impede the normal development of children, and often lead to premature death. At present, there are no cures for LSDs and the available treatments are generally insufficient, short acting, and not without co-morbidities nor long-term side effects. The last 30 years have seen significant advances in our understanding of LSD pathology as well as treatment options. Two gene therapy-based clinical trials, NCT04693598 and NCT04771416, for KD were recently started based on those advances. This review will discuss how our knowledge of KD got to where it is today, focusing on preclinical investigations, and how what was discovered may prove beneficial for treatment of other LSDs.
    Keywords:  Krabbe disease; Monogenetic diseases; adeno-associated viral vectors; demyelination; lysosomal storage disease; psychosine; substrate reduction therapies
    DOI:  https://doi.org/10.1016/j.ymthe.2022.09.017
  10. Oncogene. 2022 Oct 04.
      Cancer progression is associated with metabolic reprogramming and causes significant intracellular stress; however, the mechanisms that link cellular stress and growth signalling are not fully understood. Here, we identified a mechanism that couples the mitochondrial stress response (MSR) with tumour progression. We demonstrated that the MSR is activated in a significant proportion of human thyroid cancers via the upregulation of heat shock protein D family members and the mitokine, growth differentiation factor 15. Our study also revealed that MSR triggered AKT/S6K signalling by activating mTORC2 via activating transcription factor 4/sestrin 2 activation whilst promoting leucine transporter and nutrient-induced mTORC1 activation. Importantly, we found that an increase in mtDNA played an essential role in MSR-induced mTOR activation and that crosstalk between MYC and MSR potentiated mTOR activation. Together, these findings suggest that the MSR could be a predictive marker for aggressive human thyroid cancer as well as a useful therapeutic target.
    DOI:  https://doi.org/10.1038/s41388-022-02484-7
  11. CNS Neurosci Ther. 2022 Oct 02.
      The autophagy-lysosomal pathway (ALP) is involved in the degradation of protein aggregates and damaged organelles. Transcription factor EB (TFEB), a major regulator of ALP, has emerged as a leading factor in addressing neurodegenerative disease pathology, including Alzheimer's disease (AD), Parkinson's disease (PD), PolyQ diseases, and Amyotrophic lateral sclerosis (ALS). In this review, we delineate the regulation of TFEB expression and its functions in ALP. Dysfunctions of TFEB and its role in the pathogenesis of several neurodegenerative diseases are reviewed. We summarize the protective effects and molecular mechanisms of some TFEB-targeted agonists in neurodegenerative diseases. We also offer our perspective on analyzing the pros and cons of these agonists in the treatment of neurodegenerative diseases from the perspective of drug development. More studies on the regulatory mechanisms of TFEB in other biological processes will aid our understanding of the application of TFEB-targeted therapy in neurodegeneration.
    Keywords:  TFEB agonist; autophagy; autophagy-lysosomal pathway; neurodegenerative disease; transcription factor EB
    DOI:  https://doi.org/10.1111/cns.13985
  12. Mol Genet Metab. 2022 Sep 23. pii: S1096-7192(22)00402-4. [Epub ahead of print]137(3): 239-248
      Niemann-Pick disease Type C (NPC) is a lysosomal storage disorder caused by mutation of the NPC1/NPC2 genes, which ultimately results in the accumulation of unesterified cholesterol (UEC) in lysosomes, thereby inducing symptoms such as progressive neurodegeneration and hepatosplenomegaly. This study determines the effects of 6-O-α-maltosyl-β cyclodextrin (Mal-βCD) on lipid levels and synthesis in Npc1-deficient (Npc1-KO cells) and vehicle CHO cells. Compared to vehicle cells, Npc1-KO cells exhibited high level of UEC, and low levels of esterified cholesterols (ECs) and long-chain fatty acids (LCFAs). The difference in lipid levels between Npc1-KO and CHO cells was largely ameliorated by Mal-βCD administration. Moreover, the effects of Mal-βCD were reproduced in the lysosomes prepared from Npc1-KO cells. Stable isotope tracer analysis with extracellular addition of D4-deuterated palmitic acid (D4-PA) to Npc1-KO cells increased the synthesis of D4-deuterated LCFAs (D4-LCFAs) and D4-deuterated ECs (D4-ECs) in a Mal-βCD-dependent manner. Simultaneous addition of D6-deuterated UEC (D6-UEC) and D4-PA promoted the Mal-βCD-dependent synthesis of D6-/D4-ECs, consisting of D6-UEC and D4-PA, D4-deuterated stearic acid, or D4-deuterated myristic acid, in Npc1-KO cells. These results suggest that Mal-βCD helps to maintain normal lipid metabolism by restoring balance among UEC, ECs, and LCFAs through acting on behalf of NPC1 in Npc1-KO cells and may therefore be useful in designing effective therapies for NPC.
    Keywords:  6-O-α-Maltosyl-β cyclodextrin; Esterified cholesterol; Long-chain fatty acid; Niemann-Pick disease type C; Stable isotope tracer analysis; Unesterified cholesterol
    DOI:  https://doi.org/10.1016/j.ymgme.2022.09.007
  13. Cell Death Discov. 2022 Oct 06. 8(1): 409
      Although malignant gliomas frequently show aberrant activation of the mammalian target of rapamycin (mTOR), mTOR inhibitors have performed poorly in clinical trials. Besides regulating cell growth and translation, mTOR controls the initiation of autophagy. By recycling cellular components, autophagy can mobilize energy resources, and has thus been attributed cancer-promoting effects. Here, we asked whether the activation of autophagy represents an escape mechanism to pharmacological mTOR inhibition in glioma cells, and explored co-treatment with mTOR and autophagy inhibitors as a therapeutic strategy. Mimicking conditions of the glioma microenvironment, glioma cells were exposed to nutrient starvation and hypoxia. We analyzed autophagic activity, cell growth, viability and oxygen consumption following (co-)treatment with the mTOR inhibitors torin2 or rapamycin, and autophagy inhibitors bafilomycin A1 or MRT68921. Changes in global proteome were quantified by mass spectrometry. In the context of hypoxia and starvation, autophagy was strongly induced in glioma cells and further increased by mTOR inhibition. While torin2 enhanced glioma cell survival, co-treatment with torin2 and bafilomycin A1 failed to promote cell death. Importantly, treatment with bafilomycin A1 alone also protected glioma cells from cell death. Mechanistically, both compounds significantly reduced cell growth and oxygen consumption. Quantitative proteomics analysis showed that bafilomycin A1 induced broad changes in the cellular proteome. More specifically, proteins downregulated by bafilomycin A1 were associated with the mitochondrial respiratory chain and ATP synthesis. Taken together, our results show that activation of autophagy does not account for the cytoprotective effects of mTOR inhibition in our in vitro model of the glioma microenvironment. Our proteomic findings suggest that the pharmacological inhibition of autophagy induces extensive changes in the cellular proteome that can support glioma cell survival under nutrient-deplete and hypoxic conditions. These findings provide a novel perspective on the complex role of autophagy in gliomas.
    DOI:  https://doi.org/10.1038/s41420-022-01195-y
  14. Nat Commun. 2022 Oct 05. 13(1): 5655
      Aging is considered to be accelerated by insulin signaling in lower organisms, but it remained unclear whether this could hold true for mammals. Here we show that mice with skeletal muscle-specific double knockout of Akt1/2, key downstream molecules of insulin signaling, serve as a model of premature sarcopenia with insulin resistance. The knockout mice exhibit a progressive reduction in skeletal muscle mass, impairment of motor function and systemic insulin sensitivity. They also show osteopenia, and reduced lifespan largely due to death from debilitation on normal chow and death from tumor on high-fat diet. These phenotypes are almost reversed by additional knocking out of Foxo1/4, but only partially by additional knocking out of Tsc2 to activate the mTOR pathway. Overall, our data suggest that, unlike in lower organisms, suppression of Akt activity in skeletal muscle of mammals associated with insulin resistance and aging could accelerate osteosarcopenia and consequently reduce lifespan.
    DOI:  https://doi.org/10.1038/s41467-022-33008-2
  15. Curr Mol Pharmacol. 2022 Oct 05.
      Mechanistic/Mammalian target of rapamycin (mTOR) orchestrates cellular homeostasis by controlling cell growth, proliferation, metabolism, and survival by integrating various growth factors, nutrients, amino acids. Eccentric synchronization of mTOR has been incriminated in various diseases/disorders like cancer, neurodegenerative disorders, and diabetes mellitus and its complications. Recent reports also highlight the role of mTOR in diabetes and its associated complications. This review tries to fathom the role of mTOR signaling in diabetes mellitus and its complications- diabetic cardiomyopathy, diabetic nephropathy, and diabetic retinopathy and highlights mTOR as a putative target for the development of novel anti-diabetic drug candidates.
    Keywords:  Apoptosis; Diabetes mellitus; Diabetic cardiomyopathy; Diabetic nephropathy; Diabetic retinopathy; Insulin resistance; mTOR
    DOI:  https://doi.org/10.2174/1874467215666221005123919
  16. Exp Dermatol. 2022 Oct 04.
      Transient Receptor Potential Vanilloid subtype 3 (TRPV3) is a non-selective cation channel that is known to be activated by physiological temperature and endogenous ligands. Involvement of TRPV3 in different skin functions has been reported. In this work we demonstrate that activation of TRPV3 by FPP, an endogenous ligand enhances skin wound healing and bacterial clearance there. We report for the first time that TRPV3 is endogenously expressed in macrophages and activation of TRPV3 results in efficient bacterial clearance. At the subcellular level, TRPV3 is present in the lysosome and also in the nucleolus. We demonstrate that pharmacological modulation of TRPV3 protects lysosomal functions at hyperthermic shock conditions. The localization of TRPV3 at the nucleolus is specific, more in case of LPS-treatment and dynamic with respect to the cell signaling. We demonstrate that at certain conditions, the nucleolar localization of TRPV3 is correlated with the presence of TRPV3 at the lysosome and with the cellular stress in general. We propose that TRPV3 act as a lysosomal regulator and sensor for cellular stress. These findings may have broad implications in understanding the cellular stress and TRPV3-induced channelopathies and may have clinical relevance to skin infection treatment.
    Keywords:  Lysosome function; Skin infection; Wound healing; hyperthermic shock; nucleolar localization
    DOI:  https://doi.org/10.1111/exd.14683
  17. Mol Genet Metab Rep. 2022 Dec;33 100919
      Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency of α-galactosidase A and subsequent accumulation of glycosphingolipids with terminal α-D-galactosyl residues. The molecular process through which this abnormal metabolism of glycosphingolipids causes multisystem dysfunction in Fabry disease is not fully understood. We sought to determine whether dysregulated DNA methylation plays a role in the development of this disease. In the present study, using isogenic cellular models derived from Fabry patient endothelial cells, we tested whether manipulation of α-galactosidase A activity and glycosphingolipid metabolism affects DNA methylation. Bisulfite pyrosequencing revealed that changes in α-galactosidase A activity were associated with significantly altered DNA methylation in the androgen receptor promoter, and this effect was highly CpG loci-specific. Methylation array studies showed that α-galactosidase A activity and glycosphingolipid levels were associated with differential methylation of numerous CpG sites throughout the genome. We identified 15 signaling pathways that may be susceptible to methylation alterations in Fabry disease. By incorporating RNA sequencing data, we identified 21 genes that have both differential mRNA expression and methylation. Upregulated expression of collagen type IV alpha 1 and alpha 2 genes correlated with decreased methylation of these two genes. Methionine levels were elevated in Fabry patient cells and Fabry mouse tissues, suggesting that a perturbed methionine cycle contributes to the observed dysregulated methylation patterns. In conclusion, this study provides evidence that α-galactosidase A deficiency and glycosphingolipid storage may affect DNA methylation homeostasis and highlights the importance of epigenetics in the pathogenesis of Fabry disease and, possibly, of other lysosomal storage disorders.
    Keywords:  DNA methylation; Deoxygalactonojirimycin; Fabry disease; Globotriaosylceramide; Substrate reduction therapy; α-Galactosidase A
    DOI:  https://doi.org/10.1016/j.ymgmr.2022.100919
  18. J Cell Biol. 2022 Dec 05. pii: e202201114. [Epub ahead of print]221(12):
      Rab5 and Rab7 are known to regulate endosome maturation, and a Rab5-to-Rab7 conversion mediated by a Rab7 activator, Mon1-Ccz1, is essential for progression of the maturation process. However, the importance and mechanism of Rab5 inactivation during endosome maturation are poorly understood. Here, we report a novel Rab5-GAP, TBC1D18, which is associated with Mon1 and mediates endosome maturation. We found that increased active Rab5 (Rab5 hyperactivation) in addition to reduced active Rab7 (Rab7 inactivation) occurs in the absence of Mon1. We present evidence showing that the severe defects in endosome maturation in Mon1-KO cells are attributable to Rab5 hyperactivation rather than to Rab7 inactivation. We then identified TBC1D18 as a Rab5-GAP by comprehensive screening of TBC-domain-containing Rab-GAPs. Expression of TBC1D18 in Mon1-KO cells rescued the defects in endosome maturation, whereas its depletion attenuated endosome formation and degradation of endocytosed cargos. Moreover, TBC1D18 was found to be associated with Mon1, and it localized in close proximity to lysosomes in a Mon1-dependent manner.
    DOI:  https://doi.org/10.1083/jcb.202201114
  19. Front Genet. 2022 ;13 1013266
      Lysosomal acid lipase (LAL), encoded by the gene LIPA, is the sole neutral lipid hydrolase in lysosomes, responsible for cleavage of cholesteryl esters and triglycerides into their component parts. Inherited forms of complete (Wolman Disease, WD) or partial LAL deficiency (cholesteryl ester storage disease, CESD) are fortunately rare. Recently, LAL has been identified as a cardiovascular risk gene in genome-wide association studies, though the directionality of risk conferred remains controversial. It has also been proposed that the low expression and activity of LAL in arterial smooth muscle cells (SMCs) that occurs inherently in nature is a likely determinant of the propensity of SMCs to form the majority of foam cells in atherosclerotic plaque. LAL also likely plays a potential role in fatty liver disease. This review highlights the nature of LAL gene mutations in WD and CESD, the association of LAL with prediction of cardiovascular risk from genome-wide association studies, the importance of relative LAL deficiency in SMC foam cells, and the need to further interrogate the pathophysiological impact and cell type-specific role of enhancing LAL activity as a novel treatment strategy to reduce the development and induce the regression of ischemic cardiovascular disease and fatty liver.
    Keywords:  Cholesteryl Ester Storage Disease; GWAS; LIPA; Wolman Disease; atherosclerosis; lysosomal acid lipase; nonalcoholic fatty liver disease; smooth muscle cells
    DOI:  https://doi.org/10.3389/fgene.2022.1013266
  20. JCI Insight. 2022 Oct 04. pii: e159247. [Epub ahead of print]
      Neuropathic pain is a refractory condition that involves de novo protein synthesis in the nociceptive pathway. The mechanistic target of rapamycin (mTOR) is a master regulator of protein translation; however, mechanisms underlying its role in neuropathic pain remain elusive. Using the spared nerve injury-induced neuropathic pain model, we found that mTOR was preferentially activated in large-diameter dorsal root ganglion (DRG) neurons and spinal microglia. However, selective ablation of mTOR in DRG neurons, rather than microglia, alleviated acute neuropathic pain in mice. We showed that injury-induced mTOR activation promoted the transcriptional induction of Npy likely via signal transducer and activator of transcription 3 (STAT3) phosphorylation. NPY further acted primarily on Y2 receptors (Y2R) to enhance neuronal excitability. Peripheral replenishment of NPY reversed pain alleviation upon mTOR removal, whereas Y2R antagonists prevented pain restoration. Our findings reveal an unexpected link between mTOR and NPY/Y2R in promoting nociceptor sensitization and neuropathic pain.
    Keywords:  NPY; Neuroscience; Pain
    DOI:  https://doi.org/10.1172/jci.insight.159247
  21. Nat Commun. 2022 Oct 04. 13(1): 5851
      TAPL is a lysosomal ATP-binding cassette transporter that translocates a broad spectrum of polypeptides from the cytoplasm into the lysosomal lumen. Here we report that, in addition to its well-known role as a peptide translocator, TAPL exhibits an ATP-dependent phosphatidylserine floppase activity that is the possible cause of its high basal ATPase activity and of the lack of coupling between ATP hydrolysis and peptide efflux. We also present the cryo-EM structures of mouse TAPL complexed with (i) phospholipid, (ii) cholesteryl hemisuccinate (CHS) and 9-mer peptide, and (iii) ADP·BeF3. The inward-facing structure reveals that F449 protrudes into the cylindrical transport pathway and divides it into a large hydrophilic central cavity and a sizable hydrophobic upper cavity. In the structure, the peptide binds to TAPL in horizontally-stretched fashion within the central cavity, while lipid molecules plug vertically into the upper cavity. Together, our results suggest that TAPL uses different mechanisms to function as a peptide translocase and a phosphatidylserine floppase.
    DOI:  https://doi.org/10.1038/s41467-022-33593-2
  22. Aging Dis. 2022 Oct 01. 13(5): 1562-1575
      Tuberous sclerosis complex (TSC) is an autosomal dominant disease caused by inactivating mutations in TSC1 or TSC2.Patients with TSC often require organ transplantation after organ failure. TSC1 serves as an important control node in immune cell development and responses; however, its effect on T cells in transplant immunity has not yet been explored. Here, we characterized the effect of TSC1 deficiency in T cells on acute allograft rejection using a mouse cardiac transplantation model. We observed compromised allograft survival in mice with TSC1-deficient T cells. Notably, the allografts in mice transferred with TSC1-deficient CD8+T cells showed accelerated acute allograft rejection. TSC1 deficiency triggered the increased accumulation of CD8+ T cells in allografts due to augmented infiltration caused by increased CXCR3 expression levels and elevated in-situ proliferation of TSC1-deficient CD8+ T cells. Compared to CD8+ T cells from wild-type (WT) mice, TSC1-deficient CD8+ T cells exhibited enhanced cell proliferation and increased expression levels of interferon-γ and granzyme B after alloantigen stimulation. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), is used to treat patients with TSC and prevent rejection after solid-organ transplantation. Although rapamycin induced most cardiac allografts to survive beyond 100 d in WT mice, rapamycin-treated cardiac allografts in TSC1-deficient mice were rejected within 60 d. These results suggest that TSC1-deficient recipients may be more resistant to rapamycin-mediated immunosuppression during organ transplantation. Collectively, TSC1 significantly accelerates acute allograft rejection by enhancing the alloreactivity of CD8+ T cells, making them more resistant to mTOR inhibitor-mediated immunosuppression.
    Keywords:  acute rejection; cardiac transplantation; tuberous sclerosis complex 1
    DOI:  https://doi.org/10.14336/AD.2022.0224
  23. Cell. 2022 Sep 30. pii: S0092-8674(22)01123-0. [Epub ahead of print]
      The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca2+ transients on the outer surface of the ER membrane, whose amplitude, frequency, and duration are controlled by the metazoan-specific ER transmembrane autophagy protein EPG-4/EI24. Persistent Ca2+ transients/oscillations on the cytosolic ER surface in EI24-depleted cells cause accumulation of FIP200 autophagosome initiation complexes on the ER. This defect is suppressed by attenuating ER Ca2+ transients. Multi-modal SIM analysis revealed that Ca2+ transients on the ER trigger the formation of dynamic and fusion-prone liquid-like FIP200 puncta. Starvation-induced Ca2+ transients on lysosomes also induce FIP200 puncta that further move to the ER. Multiple FIP200 puncta on the ER, whose association depends on the ER proteins VAPA/B and ATL2/3, assemble into autophagosome formation sites. Thus, Ca2+ transients are crucial for triggering phase separation of FIP200 to specify autophagosome initiation sites in metazoans.
    Keywords:  ATG9; Ca(2+) transient; EI24; ER; FIP200; autophagosome formation; liquid-liquid phase separation; lysosome
    DOI:  https://doi.org/10.1016/j.cell.2022.09.001
  24. J Therm Biol. 2022 Oct;pii: S0306-4565(22)00140-1. [Epub ahead of print]109 103326
      Injury to the intestinal epithelial cells and loss of the intestinal barrier are critical to heatstroke. To reveal the mechanism through which heatstroke leads to intestinal epithelial injury, the relationship between reactive oxygen species (ROS), c-Jun NH2-terminal kinase (JNK), and lysosomes were studied in intestinal epithelial cells subjected to heat stress. Cells of heat stress groups were incubated at 43 °C for 1 h, then incubated at 37 °C as indicated. Control group cells were incubated at 37 °C. Cell-counting kit-8 assay was used to assess cell viability. Cells were labeled with 2'-7'dichlorofluorescin diacetate and acridine orange (AO) staining, respectively, the total ROS and AO were detected by confocal laser scanning microscopy and flow cytometry. Apoptosis was analyzed by flow cytometry using annexin V-fluorescein isothiocyanate/prodium iodide staining, the expressions of mitogen-activated protein kinases were detected by western blotting. Heat stress induced apoptosis and inhibited cell viability, the production of ROS, and lysosomal injury in IEC-6 cells. After pretreatment with the lysosomal cathepsin inhibitor E64, the JNK inhibitor SP600125, or the ROS scavenger NAC, the effect of heat stress on apoptosis or lysosomal injury was significantly attenuated. In conclusion, heat stress induced apoptosis, lysosomal injury, and the accumulation of ROS in IEC-6 cells; mechanistically, this occurred through the ROS-induced activation of JNK signaling, which mediated the lysosomal injury and ultimately apoptosis.
    Keywords:  Apoptosis; Heat stress; JNK; Lysosome; ROS
    DOI:  https://doi.org/10.1016/j.jtherbio.2022.103326
  25. Biochem Biophys Res Commun. 2022 Sep 24. pii: S0006-291X(22)01334-1. [Epub ahead of print]631 130-137
      The mammalian target of rapamycin (mTOR)-composed of multiple complexes, including mTOR complex 1/2 (mTORC1/2)-is a serine-threonine kinase that regulates embryonic development. The transcription factor, hypoxia-inducible factor-1α (HIF-1α), is also involved in embryonic development. As the relationship between mTOR and HIF-1α during embryonic development remains unclear, we investigated the relationship between the two using ex vivo submandibular salivary gland organ cultures. When the expression of HIF-1α increased under hypoxic conditions (1% O2), the expression of mTOR signaling pathway-related proteins decreased. Conversely, when the expression of HIF-1α decreased, the expression of mTOR signaling pathway-related proteins increased. These results indicate a strong relationship between HIF-1α and the mTOR signaling pathway. For the first time, we clarified that HIF-1α negatively regulates the mTOR signaling pathway and suppresses salivary gland development under 1% O2 using small molecules. Our research provides new insights into the relationship between HIF-1α and the mTOR signaling pathway in embryonic organ development.
    Keywords:  Development; HIF-1α; Hypoxia; Salivary glands; mTOR signaling
    DOI:  https://doi.org/10.1016/j.bbrc.2022.09.078
  26. Rejuvenation Res. 2022 Oct 07.
      Oncogene-induced senescence (OIS), characterized by irreversible cell cycle arrest by oncogene activation, plays an important role in the pathogenesis of aging and age-related diseases. Recent research indicates that OIS is driven by activation of mitogen-activated protein kinase (MAPK). However, it is not apparent whether MAPK inhibition helps to recover senescence. In our previous study, we uncovered p38 MAPK inhibitor, SB203580, as an effective agent to reduce ROS and increase proliferation in premature senescent cells. In this study, we evaluated whether SB203580 could ameliorate senescence in normal senescent cells. The senescence-improving effect was observed in the results that SB203580 treatment restored lysosomal function, as evidenced by a decrease in lysosomal mass and an increase in autophagic vacuoles. Then, SB203580-mediated lysosomal function restoration triggered the clearance of damaged mitochondria, leading to metabolic reprogramming necessary for amelioration of senescence. Indeed, p38 MAPK inhibition by SB203580 improved key senescent phenotypes. Our findings suggest a novel mechanism by which modulation of p38 MAPK activity leads to senescence improvement through functional restoration of lysosome and mitochondria.
    DOI:  https://doi.org/10.1089/rej.2022.0043
  27. Life Sci Alliance. 2022 Nov;pii: e202201530. [Epub ahead of print]5(11):
      Selective neuronal vulnerability is common in neurodegenerative diseases but poorly understood. In genetic prion diseases, including fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD), different mutations in the Prnp gene manifest as clinically and neuropathologically distinct diseases. Here we report with electroencephalography studies that theta waves are mildly increased in 21 mo old knock-in mice modeling FFI and CJD and that sleep is mildy affected in FFI mice. To define affected cell types, we analyzed cell type-specific translatomes from six neuron types of 9 mo old FFI and CJD mice. Somatostatin (SST) neurons responded the strongest in both diseases, with unexpectedly high overlap in genes and pathways. Functional analyses revealed up-regulation of neurodegenerative disease pathways and ribosome and mitochondria biogenesis, and down-regulation of synaptic function and small GTPase-mediated signaling in FFI, implicating down-regulation of mTOR signaling as the root of these changes. In contrast, responses in glutamatergic cerebellar neurons were disease-specific. The high similarity in SST neurons of FFI and CJD mice suggests that a common therapy may be beneficial for multiple genetic prion diseases.
    DOI:  https://doi.org/10.26508/lsa.202201530
  28. J Cell Biol. 2022 Dec 05. pii: e202203013. [Epub ahead of print]221(12):
      Retromer-dependent endosomal recycling of membrane receptors requires Rab7, sorting nexin (SNX)-retromer, and factors that regulate endosomal actin organization. It is not fully understood how these factors cooperate to form endosomal subdomains for cargo retrieval and recycling. Here, we report that WDR91, a Rab7 effector, is the key factor that specifies the endosomal retrieval subdomain. Loss of WDR91 causes defective recycling of both intracellular and cell surface receptors. WDR91 interacts with SNXs through their PX domain, and with VPS35, thus promoting their interaction with Rab7. WDR91 also interacts with the WASH subunit FAM21. In WDR91-deficient cells, Rab7, SNX-retromer, and FAM21 fail to localize to endosomal subdomains, and endosomal actin organization is impaired. Re-expression of WDR91 enables Rab7, SNX-retromer, and FAM21 to concentrate at WDR91-specific endosomal subdomains, where retromer-mediated membrane tubulation and release occur. Thus, WDR91 coordinates Rab7 with SNX-retromer and WASH to establish the endosomal retrieval subdomains required for retromer-mediated endosomal recycling.
    DOI:  https://doi.org/10.1083/jcb.202203013
  29. Proc Natl Acad Sci U S A. 2022 Oct 11. 119(41): e2205874119
      ATB[Formula: see text] (SLC6A14) is a member of the amino acid transporter branch of the SLC6 family along with GlyT1 (SLC6A9) and GlyT2 (SLC6A5), two glycine-specific transporters coupled to 2:1 and 3:1 Na[Formula: see text]:Cl[Formula: see text], respectively. In contrast, ATB[Formula: see text] exhibits broad substrate specificity for all neutral and cationic amino acids, and its ionic coupling remains unsettled. Using the reversal potential slope method, we demonstrate a 3:1:1 Na[Formula: see text]:Cl[Formula: see text]:Gly stoichiometry for ATB[Formula: see text] that is consistent with its 2.1 e/Gly charge coupling. Like GlyT2, ATB[Formula: see text] behaves as a unidirectional transporter with virtually no glycine efflux at negative potentials after uptake, except by heteroexchange as remarkably shown by leucine activation of NMDARs in Xenopus oocytes coexpressing both membrane proteins. Analysis and computational modeling of the charge movement of ATB[Formula: see text] reveal a higher affinity for sodium in the absence of substrate than GlyT2 and a gating mechanism that locks Na[Formula: see text] into the apo-transporter at depolarized potentials. A 3:1 Na[Formula: see text]:Cl[Formula: see text] stoichiometry justifies the concentrative transport properties of ATB[Formula: see text] and explains its trophic role in tumor growth, while rationalizing its phylogenetic proximity to GlyT2 despite their extreme divergence in specificity.
    Keywords:  NMDAR; electrophysiology; glycine transporters; ion-coupled transporter; thermodynamic
    DOI:  https://doi.org/10.1073/pnas.2205874119
  30. Elife. 2022 Oct 03. pii: e79128. [Epub ahead of print]11
      The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis, and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage. However, a direct role of the mTORC1-S6K1 signaling pathway in DNA repair and the mechanism by which this signaling pathway regulates DNA repair is unknown. In this study, we discovered a novel role for S6K1 in regulating DNA repair through the coordinated regulation of the cell cycle, homologous recombination (HR) DNA repair (HRR) and mismatch DNA repair (MMR) mechanisms. Here, we show that S6K1 orchestrates DNA repair by phosphorylation of Cdk1 at serine 39, causing G2/M cell cycle arrest enabling homologous recombination and by phosphorylation of MSH6 at serine 309, enhancing MMR. Moreover, breast cancer cells harboring RPS6KB1 gene amplification show increased resistance to several DNA damaging agents and S6K1 expression is associated with poor survival of breast cancer patients treated with chemotherapy. Our findings reveal an unexpected function of S6K1 in the DNA repair pathway, serving as a tumorigenic barrier by safeguarding genomic stability.
    Keywords:  CDK1; DNA repair; MSH2; MSH6; S6K1; biochemistry; cancer biology; cdk1(cdc2); chemical biology; dna repair; mTORC1; msh2; msh6; s6k1
    DOI:  https://doi.org/10.7554/eLife.79128
  31. NPJ Parkinsons Dis. 2022 Oct 06. 8(1): 127
      Uncontrolled microglial activation is pivotal to the pathogenesis of Parkinson's disease (PD), which can secrete Cathepsin L (CTSL) to affect the survival of neurons in the PD patients; however, the precise mechanism has yet to be determined. We demonstrated for the first time that CTSL was mostly released by exosomes derived from α-Syn-activated microglia, resulting in neuronal damage and death. The elevation of CTSL activity was blocked by GW4869, suggesting a critical role for exosomes in mediating CTSL release. Furthermore, the P2X7R/PI3K/AKT signalling pathway was identified as the underlying molecular mechanism since specific antagonists of this signalling pathway, P2X7R knockdown and exosome release inhibitors significantly reduced the injury to cultured mouse cortical neurons. Our study suggests that increased extracellular release of CTSL from α-Syn-activated microglia through exosomes amplifies and aggravates of the neurotoxic effect of microglia, implying that CTSL may be involved in a fresh mechanism of PD pathogenesis, and serve as a potential biomarker and a target for PD drug development.
    DOI:  https://doi.org/10.1038/s41531-022-00394-9
  32. Histopathology. 2022 Oct 08.
      Low-grade oncocytic tumor (LOT) of the kidney has recently emerged as a potential novel tumor type. Despite similarity to oncocytoma or eosinophilic chromophobe renal cell carcinoma, it shows diffuse keratin 7 immunohistochemistry (IHC) and negative KIT (CD117), which differs from both. We aimed to identify the molecular characteristics of these tumors. Seventeen tumors (1 man, 16 women, 9 previously published) fitting the original description of this entity (solid eosinophilic cell morphology, often with areas of tumor cells loosely stretched in edematous stroma, and the above IHC features), were analyzed with a next-generation sequencing panel of 324 cancer-associated genes from formalin-fixed, paraffin-embedded tissue. All tumors harbored at least one alteration in either TSC1 (n= 7, 41%), TSC2 (n=2, 12%), MTOR (n=5, 29%), or PIK3CA (n=4, 24%). Four tumors harbored a second alteration, including two NF2, one each in conjunction with MTOR and TSC2 alterations, one PTEN with TSC1 alteration, and one tumor with both MTOR and TSC1 alterations. No other renal cancer-related or recurring gene alterations were identified. In addition to the previously described IHC findings, 16/16 were positive for GATA3. Eleven patients with follow-up had no metastases or recurrent tumors. Recurrent tuberous sclerosis / MTOR pathway gene alterations in LOT supports its consideration as a distinct morphologic, immunohistochemical, and genetic entity. PIK3CA is another pathway member that may be altered in these tumors. Further study will be necessary to determine whether tumor behavior or syndromic associations differ from those of oncocytoma and chromophobe carcinoma, warranting different clinical consideration.
    Keywords:  MTOR; PIK3CA; TSC1; TSC2; low-grade oncocytic tumor; oncocytoma
    DOI:  https://doi.org/10.1111/his.14816
  33. Front Cardiovasc Med. 2022 ;9 899283
      Danon disease is a rare disease caused by glycogen storage lysosomal disorder. It is related to the pathogenic mutation of the LAMP2 gene. In this case report, we present a patient with a novel pathogenic mutation (c.764_765insGA) with cardiac-only symptoms. Her family members do not carry the same mutation she does, suggesting this is a de novo mutation. Further tests revealed vacuoles and glycogen disposition in the patient's heart tissue and a significant decrease in LAMP2 protein expression. Protein structure remodeling of LAMP2 predicted that the mutant protein has conformational change lacking an important transmembrane domain, subsequently causing protein destabilization.
    Keywords:  Danon disease; LAMP2; Sanger sequencing; genetic diagnosis; hypertrophy
    DOI:  https://doi.org/10.3389/fcvm.2022.899283