bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2022–02–06
forty-five papers selected by
Stephanie Fernandes, Max Planck Institute for Biology of Ageing
  1. Leucine retention in lysosomes is regulated by starvation.
  2. LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function.
  3. MEK/ERK-mediated oncogenic signals promote secretion of extracellular vesicles by controlling lysosome function.
  4. Vps501, a novel vacuolar SNX-BAR protein cooperates with the SEA complex to regulate TORC1 signaling.
  5. Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation.
  6. Quantitative and Time-Resolved Monitoring of Organelle and Protein Delivery to the Lysosome with A Tandem Fluorescent Halo-GFP reporter.
  7. Defects in the autophagy lysosomal pathway in a cell model of disease-associated tau.
  8. Fluoxetine ameliorates Mucopolysaccharidosis type IIIA.
  9. Deficiency of the lysosomal exonuclease PLD3 impacts the degradative route.
  10. Synaptic stimulation induces tau clearance by enhancing autophagosomal/lysosomal degradation.
  11. Molecular Features of CA-074 pH-Dependent Inhibition of Cathepsin B.
  12. DNAJC5 affects the endo-lysosomal pathway, APP processing, and AD pathology in vitro and in vivo.
  13. Defining the role of PLD3 in Alzheimer disease pathology.
  14. Differential gene expression analysis for the identification of potential biomarkers for Niemann-Pick type C disease.
  15. Differential regulation of progranulin derived granulin peptides.
  16. Environmental control of Pub1 (NEDD4 family E3 ligase) in Schizosaccharomyces pombe is regulated by TORC2 and Gsk3.
  17. Lysosomal subcellular distribution in neurons: Are there synaptic lysosomes?
  18. Assessing stimulation-dependent changes in LRRK2 and GCase expression/activity and convergence at the lysosome in cryopreserved monocytes.
  19. E3 ligase RNF167 and deubiquitinase STAMBPL1 modulate mTOR and cancer progression.
  20. Defective proteostasis in patient-derived iPSC astrocytes and neurons carrying a MAPT IVS10+16 mutation.
  21. Developing human cellular models to understand biological mechanisms linked to AD genetic risk.
  22. LRRK2, GBA and their interaction in the regulation of autophagy: implications on therapeutics in Parkinson's disease.
  23. Lysosomal permeabilization as a mechanism of amyloid degradation toxicity in Alzheimer's disease.
  24. The Effects of Combined Therapy With Metformin and Hydroxypropyl-β-Cyclodextrin in a Mouse Model of Niemann-Pick Disease Type C1.
  25. Interaction between TRPML1 and p62 in Regulating Autophagosome-Lysosome Fusion and Impeding Neuroaxonal Dystrophy in Alzheimer's Disease.
  26. Contribution of Alzheimer's disease risk genes in the maintenance of endolysosomal transport regulation.
  27. Lysosphingolipid urine screening test using mass spectrometry for the early detection of lysosomal storage disorders.
  28. Quantitation of a plasma biomarker profile for the early detection of Gaucher disease type 1 patients.
  29. Minimum Effective Dose to Achieve Biochemical Correction With AAV Vector-Mediated Gene Therapy in Mice With Pompe Disease.
  30. A farnesyltransferase inhibitor restores cognitive deficits in Tsc2+/- mice through inhibition of Rheb1.
  31. Lysosomal exocytosis of HSP70 stimulates monocytic BMP6 expression in Sjögren's syndrome.
  32. Neuroprogenitor Cells From Patients With TBCK Encephalopathy Suggest Deregulation of Early Secretory Vesicle Transport.
  33. ER-phagy requires the assembly of actin at sites of contact between the cortical ER and endocytic pits.
  34. Beyond Protein Synthesis; The Multifaceted Roles of Tuberin in Cell Cycle Regulation.
  35. Dual Inhibition of mTORC1/2 Reduces Migration of Cholangiocarcinoma Cells by Regulation of Matrixmetalloproteinases.
  36. SNARE proteins: zip codes in vesicle targeting?
  37. PARK2 regulates eIF4B-driven lymphomagenesis.
  38. Living in α-syn: Tackling aggregates in Parkinson's disease.
  39. HSP90 inhibitors induce GPNMB cell-surface expression by modulating lysosomal positioning and sensitize breast cancer cells to glembatumumab vedotin.
  40. Profiling and functional characterization of maternal mRNA translation during mouse maternal-to-zygotic transition.
  41. ALFY localizes to early endosomes and cellular protrusions to facilitate directional cell migration.
  42. Retromer stabilizing compounds rescue enlarged endosome phenotypes in hiPSC-derived models of AD.
  43. Cathepsin D as a potential therapeutic target to enhance anticancer drug-induced apoptosis via RNF183-mediated destabilization of Bcl-xL in cancer cells.
  44. Loss of TSC1/TSC2 sensitizes immune checkpoint blockade in non-small cell lung cancer.
  45. Sortilin restricts secretion of apolipoprotein B-100 by hepatocytes under stressed but not basal conditions.
  1. Proc Natl Acad Sci U S A. 2022 Feb 08. pii: e2114912119. [Epub ahead of print]119(6):
    Leucine retention in lysosomes is regulated by starvation.
    Urmi Bandyopadhyay, Pavlina Todorova, Natalya N Pavlova, Yuma Tada, Craig B Thompson, Lydia W S Finley, Michael Overholtzer.
      Cells acquire essential nutrients from the environment and utilize adaptive mechanisms to survive when nutrients are scarce. How nutrients are trafficked and compartmentalized within cells and whether they are stored in response to stress remain poorly understood. Here, we investigate amino acid trafficking and uncover evidence for the lysosomal transit of numerous essential amino acids. We find that starvation induces the lysosomal retention of leucine in a manner requiring RAG-GTPases and the lysosomal protein complex Ragulator, but that this process occurs independently of mechanistic target of rapamycin complex 1 activity. We further find that stored leucine is utilized in protein synthesis and that inhibition of protein synthesis releases lysosomal stores. These findings identify a regulated starvation response that involves the lysosomal storage of leucine.
    Keywords:  leucine; lysosome; mTOR
    DOI:  https://doi.org/10.1073/pnas.2114912119
  2. EMBO J. 2022 Jan 31. e108119
    LAMTOR1 inhibition of TRPML1-dependent lysosomal calcium release regulates dendritic lysosome trafficking and hippocampal neuronal function.
    Jiandong Sun, Yan Liu, Xiaoning Hao, Weiju Lin, Wenyue Su, Emerald Chiang, Michel Baudry, Xiaoning Bi.
      Lysosomes function not only as degradatory compartments but also as dynamic intracellular calcium ion stores. The transient receptor potential mucolipin 1 (TRPML1) channel mediates lysosomal Ca2+ release, thereby participating in multiple cellular functions. The pentameric Ragulator complex, which plays a critical role in the activation of mTORC1, is also involved in lysosomal trafficking and is anchored to lysosomes through its LAMTOR1 subunit. Here, we report that the Ragulator restricts lysosomal trafficking in dendrites of hippocampal neurons via LAMTOR1-mediated tonic inhibition of TRPML1 activity, independently of mTORC1. LAMTOR1 directly interacts with TRPML1 through its N-terminal domain. Eliminating this inhibition in hippocampal neurons by LAMTOR1 deletion or by disrupting LAMTOR1-TRPML1 binding increases TRPML1-mediated Ca2+ release and facilitates dendritic lysosomal trafficking powered by dynein. LAMTOR1 deletion in the hippocampal CA1 region of adult mice results in alterations in synaptic plasticity, and in impaired object-recognition memory and contextual fear conditioning, due to TRPML1 activation. Mechanistically, changes in synaptic plasticity are associated with increased GluA1 dephosphorylation by calcineurin and lysosomal degradation. Thus, LAMTOR1-mediated inhibition of TRPML1 is critical for regulating dendritic lysosomal motility, synaptic plasticity, and learning.
    Keywords:  LTD; LTP; calcineurin; calcium; dynein
    DOI:  https://doi.org/10.15252/embj.2021108119
  3. Cancer Sci. 2022 Feb 02.
    MEK/ERK-mediated oncogenic signals promote secretion of extracellular vesicles by controlling lysosome function.
    Tomoya Hikita, Ryo Uehara, Reina E Itoh, Fumie Mitani, Mamiko Miyata, Takuya Yoshida, Rui Yamaguchi, Chitose Oneyama.
      Cancer cells secrete large amounts of extracellular vesicles (EVs) originating from multivesicular bodies (MVBs). Mature MVBs fuse either with the plasma membrane for release as EVs often referred as to exosomes or with lysosomes for degradation. However, the mechanisms regulating MVB fate remain unknown. Here, we investigated the regulators of MVB fate by analyzing the effects of signaling inhibitors on EV secretion from cancer cells engineered to secrete luciferase-labeled EVs. Inhibition of the oncogenic MEK/ERK pathway suppressed EV release and activated lysosome formation. MEK/ERK-mediated lysosomal inactivation impaired MVB degradation, resulting in increased EV secretion from cancer cells. Moreover, MEK/ERK inhibition prevented c-MYC expression and induced the nuclear translocation of MiT/TFE transcription factors, thereby promoting the activation of lysosome-related genes, including the gene encoding a subunit of vacuolar-type H+ -ATPase, which is responsible for lysosomal acidification and function. Furthermore, c-MYC upregulation was associated with lysosomal genes downregulation in MEK/ERK-activated renal cancer cells/tissues. These findings suggest that the MEK/ERK/c-MYC pathway controls MVB fate and promotes EV production in human cancers by inactivating lysosomal function.
    Keywords:  MEK/ERK; c-MYC; extracellular vesicles; lysosome; renal cancer
    DOI:  https://doi.org/10.1111/cas.15288
  4. Traffic. 2022 Jan 30.
    Vps501, a novel vacuolar SNX-BAR protein cooperates with the SEA complex to regulate TORC1 signaling.
    Shreya Goyal, Verónica A Segarra, Nitika, Aaron M Stetcher, Andrew W Truman, Adam M Reitzel, Richard J Chi.
      The sorting nexins (SNX), constitute a diverse family of molecules that play varied roles in membrane trafficking, cell signaling, membrane remodeling, organelle motility and autophagy. In particular, the SNX-BAR proteins, a SNX subfamily characterized by a C-terminal dimeric Bin/Amphiphysin/Rvs (BAR) lipid curvature domain and a conserved Phox-homology domain, are of great interest. In budding yeast, many SNX-BARs proteins have well-characterized endo-vacuolar trafficking roles. Phylogenetic analyses allowed us to identify an additional SNX-BAR protein, Vps501, with a novel endo-vacuolar role. We report that Vps501 uniquely localizes to the vacuolar membrane and has physical and genetic interactions with the SEA complex to regulate TORC1 inactivation. We found cells displayed a severe deficiency in starvation-induced/nonselective autophagy only when SEA complex subunits are ablated in combination with Vps501, indicating a cooperative role with the SEA complex during TORC1 signaling during autophagy induction. Additionally, we found the SEACIT complex becomes destabilized in vps501Δsea1Δ cells, which resulted in aberrant endosomal TORC1 activity and subsequent Atg13 hyperphosphorylation. We have also discovered that the vacuolar localization of Vps501 is dependent upon a direct interaction with Sea1 and a unique lipid binding specificity that is also required for its function. This article is protected by copyright. All rights reserved.
    Keywords:  Atg27; SEA complex; SNX-BAR; TORC1; autophagy; retromer
    DOI:  https://doi.org/10.1111/tra.12833
  5. Proc Natl Acad Sci U S A. 2022 Feb 08. pii: e2121494119. [Epub ahead of print]119(6):
    Structure of the Mon1-Ccz1 complex reveals molecular basis of membrane binding for Rab7 activation.
    Björn U Klink, Eric Herrmann, Claudia Antoni, Lars Langemeyer, Stephan Kiontke, Christos Gatsogiannis, Christian Ungermann, Stefan Raunser, Daniel Kümmel.
      Activation of the GTPase Rab7/Ypt7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 marks organelles such as endosomes and autophagosomes for fusion with lysosomes/vacuoles and degradation of their content. Here, we present a high-resolution cryogenic electron microscopy structure of the Mon1-Ccz1 complex that reveals its architecture in atomic detail. Mon1 and Ccz1 are arranged side by side in a pseudo-twofold symmetrical heterodimer. The three Longin domains of each Mon1 and Ccz1 are triangularly arranged, providing a strong scaffold for the catalytic center of the GEF. At the opposite side of the Ypt7-binding site, a positively charged and relatively flat patch stretches the Longin domains 2/3 of Mon1 and functions as a phosphatidylinositol phosphate-binding site, explaining how the GEF is targeted to membranes. Our work provides molecular insight into the mechanisms of endosomal Rab activation and serves as a blueprint for understanding the function of members of the Tri Longin domain Rab-GEF family.
    Keywords:  GEF; GTPase; cryo-EM; endosome; trafficking
    DOI:  https://doi.org/10.1073/pnas.2121494119
  6. Mol Biol Cell. 2022 Feb 02. mbcE21100526
    Quantitative and Time-Resolved Monitoring of Organelle and Protein Delivery to the Lysosome with A Tandem Fluorescent Halo-GFP reporter.
    M Rudinskiy, T J Bergmann, M Molinari.
      Lysosomal degradative compartments hydrolyze macromolecules to generate basic building blocks that fuel metabolic pathways in our cells. They also remove misfolded proteins and control size, function and number of cytoplasmic organelles via constitutive and regulated autophagy. These catabolic processes attract interest because their defective functioning is linked to human disease and their molecular components are promising pharmacologic targets. The capacity to quantitatively assess them is highly sought for. Here, we present a tandem-fluorescent reporter consisting of a HaloTag-GFP chimera appended at the C- or at the N-terminus of select polypeptides to monitor protein and organelle delivery to the lysosomal compartment. The Halo-GFP changes color upon fluorescent pulse with cell-permeable HaloTag ligands and, again, upon delivery to acidic, degradative lysosomal compartments, where the fluorescent ligand-associated HaloTag is relatively stable, whereas the GFP portion is not, as testified by loss of the green fluorescence and generation of a protease-resistant, fluorescent HaloTag fragment. The Halo-GFP tandem fluorescent reporter presented in our study allows quantitative and, crucially, time-resolved analyses of protein and organelle transport to the lysosomal compartment by high resolution confocal laser scanning microscopy, antibody-free electrophoretic techniques and flow cytometry.
    DOI:  https://doi.org/10.1091/mbc.E21-10-0526
  7. Alzheimers Dement. 2021 Dec;17 Suppl 3 e051303
    Defects in the autophagy lysosomal pathway in a cell model of disease-associated tau.
    Saskia J Pollack, Dina Dakkak, Tong Guo, Patricia Gómez-Suaga, Wendy Noble, Diane P Hanger.
       BACKGROUND: Intracellular accumulation of insoluble tau is an important hallmark of Alzheimer's disease (AD) and related tauopathies. We have previously identified in human tauopathy brain a truncated tau species (Tau35), comprising the C-terminal half with four microtubule-binding repeats. Minimal Tau35 expression in transgenic mice results in a progressive tauopathy phenotype including tau phosphorylation and aggregation, cognitive and behavioural abnormalities and impaired protein clearance. The autophagy-lysosomal pathway (ALP) plays a crucial role in the clearance of protein aggregates and defects in ALP are associated with the pathogenesis of AD. We sought to explore the effect of Tau35 expression on the ALP and whether autophagy is disrupted due to lysosomal dysfunction.
    METHODS: Chinese hamster ovary (CHO) cells stably expressing Tau35 (CHO-Tau35) or full-length human 2N4R tau (CHO-FL) were generated. Primary cortical neurons from Tau35 transgenic and wild-type mice were cultured for 14 days in vitro(DIV) and brain homogenates were prepared from mice aged 4 and 12 months. The effect of Tau35 on the ALP was examined using immunofluorescence and western blots.
    RESULT: Nuclear translocation of transcription factor EB (TFEB), a key mediator of lysosomal biogenesis, was significantly reduced in both CHO-FL and CHO-Tau35 cells. However, only CHO-Tau35 cells exhibited disrupted mammalian target of rapamycin complex 1 (mTORC1) activity and autophagic flux. Expression of ALP markers, including LC3-I/II, LAMP1, LAMP2 and cathepsin D, were also reduced in CHO-Tau35 cells and in post-symptomatic Tau35 transgenic mice.
    CONCLUSIONS: Our findings suggest that N-terminally cleaved tau damages both lysosomal clearance of cellular proteins and lysosomal biogenesis. The Tau35-expressing cultured neurons will provide a useful tool to explore molecular mechanisms underlying tau-induced lysosomal dysfunction, which may lead to the identification of novel therapeutic targets for dementia.
    DOI:  https://doi.org/10.1002/alz.051303
  8. Mol Ther. 2022 Feb 01. pii: S1525-0016(22)00076-4. [Epub ahead of print]
    Fluoxetine ameliorates Mucopolysaccharidosis type IIIA.
    Capuozzo Antonella, Montefusco Sandro, Cacace Vincenzo, Sofia Martina, Esposito Alessandra, Napolitano Gennaro, Nusco Eduardo, Polishchuk Elena, Pizzo Maria Teresa, De Risi Maria, De Leonibus Elvira, Sorrentino Nicolina Cristina, Medina Diego Luis.
      Mucopolysaccharidosis type IIIA is an autosomal recessive disorder caused by mutations in SGSH involved in the degradation of heparan sulfate. MPS-IIIA presents severe neurological symptoms like progressive developmental delay and mental retardation, for which there is currently no treatment. Brain targeting represents the main challenge for the therapeutics to treat MPS-IIIA, and the development of small molecule-based treatments able to reach the CNS could be a relevant advance for therapy. Using cell-based high content imaging to survey clinical-approved drugs in MPS-IIIA cells, we identified Fluoxetine, a selective serotonin reuptake inhibitor. Fluoxetine increases lysosomal and autophagic functions via TFEB activation through a RagC-dependent mechanism. Mechanistically, Fluoxetine increases lysosomal exocytosis in mouse embryonic fibroblasts from MPS-IIIA mice, suggesting that this process may be responsible for heparan sulfate clearance. In vivo, Fluoxetine ameliorates somatic and brain pathology in a mouse model of MPS-IIIA by decreasing the accumulation of glycosaminoglycans and aggregated autophagic substrates, reducing inflammation, and slowing down cognitive deterioration. Altogether, we repurposed Fluoxetine for potential therapeutics to treat human MPS-IIIA disease.
    DOI:  https://doi.org/10.1016/j.ymthe.2022.01.037
  9. Alzheimers Dement. 2021 Dec;17 Suppl 3 e050868
    Deficiency of the lysosomal exonuclease PLD3 impacts the degradative route.
    Zoë P Van Acker, Marine Bretou, Ragna Sannerud, Markus Damme, Wim Annaert.
       BACKGROUND: Phospholipase D3 (PLD3) is a 5'-3' exonuclease that resides in lysosomes where it is implicated in the regulation of inflammatory responses by degrading ssDNA. Polymorphisms in the PLD3 gene have been linked to late-onset Alzheimer's disease (LOAD; Cruchaga et al., PMID: 24336208), but contradictory data exist on the impact of PLD3 deficiency on APP processing. Given PLD3 is primarily expressed in neurons within the brain, we were interested in exploring its role in neuronal endolysosomal homeostasis.
    METHOD: We generated PLD3-/- neuroblastoma SH-SY5Y clonal cell lines, using Crispr/cas9 gene editing, that were subsequently stably rescued with lentiviral vector technology. For this, the wild-type PLD3 sequence was compared to variants harbouring LOAD-associated risk single-nucleotide polymorphisms (SNPs), including M6R, K228R, V232M, N236S, N284S and T426A. PLD3 exonuclease kinetics towards different substrates were measured using EFQO assays (Cappel et al., PMID: 33288674). Autophagy and mitophagy dysregulations, as possible important catalysts for DNA delivery to lysosomes, were further scrutinized with biochemical assays and confocal imaging. In parallel, we investigated the endolysosomal morphology by electron microscopy.
    RESULT: Firstly, most SNP variants, except the M6R and T426A variants, display a significantly reduced exonuclease activity; indicating a loss-of-function for these variants. Overall, knock-out of PLD3 leads to elevations in lysosomal and autophagosomal components, including a defect in mitophagy, lysosomal cholesterol accumulation and an accretion of the uncleaved/inactive CatD. These aberrant features can be rescued by re-introducing wild-type PLD3, but not PLD3 SNP-variants. This underscores a critical role for PLD3 in lysosomal homeostasis, likely mediated through its exonuclease activity. Given different variants elicit different exonuclease kinetics towards different ssDNA substrates, this suggests that PLD3 contributes to upholding lysosomal DNA levels within a narrow range, which is required for a normal lysosomal proteostasis.
    CONCLUSION: PLD3 depletion and exonuclease dysfunction-causing SNPs promote lysosomal impairment and, as such, a congestion of the degradative route. We hypothesize that this could relate to a dysfunctional exonuclease activity of PLD3 in lysosomes. Given the observation of engulfed mitochondria within autophagosomes, the substrates for PLD3 could potentially come from mtDNA.
    DOI:  https://doi.org/10.1002/alz.050868
  10. Alzheimers Dement. 2021 Dec;17 Suppl 3 e051678
    Synaptic stimulation induces tau clearance by enhancing autophagosomal/lysosomal degradation.
    Yvette Akwa, Chiara Di Malta, Elise Gondard, Anne Boiret, Andres M Lozano, Thomas Vaccari, Carmine Settembre, Etienne E Baulieu, Davide Tampellini.
       BACKGROUND: Alterations of synaptic excitability and reduced brain metabolism are some of the earliest changes associated Alzheimer's disease (AD) pathogenesis (Reiman et al., 2004; Sperling et al., 2009). Among different approaches for therapeutics, the stimulation of synaptic activity exerts protective effects in models of AD, and deep brain stimulation (DBS) has been shown to ameliorate AD symptoms in certain patients (Sankar et al., 2015; Swaab and Bao, 2010; Tampellini, 2015). Such positive effects might be the result of changes occurring at cellular level upon activity induction, which promote cellular mechanisms correcting neuronal and synaptic dysfunctions. We have demonstrated that synaptic stimulation, via DBS or other methods, is protective in mouse models of AD and frontotemporal dementia (FTD) by enhancing autophagy and lysosomal degradation of pathologic tau, and by protecting synapses (Akwa et al., 2018; Mann et al., 2018). Our more recent investigations are revealing the involvement of Transcription factor EB (TFEB), a pivotal player in lysosomal biogenesis and autophagy (Sardiello et al., 2009), and its downstream genes in the enhancement of lysosomal activity upon stimulation.
    METHOD: 3xTg mice received deep brain stimulation by electrode implantation in the entorhinal cortex (Mann et al., 2018). Cultured neurons were prepared from E15 PS19 mouse embryos (Akwa et al., 2018) and stimulated at 14 days in vitro (Ehlers, 2003). Confocal immunofluorescence, Western blot and statistical analyses were performed as described (Akwa et al., 2018). RT-qPCR was performed as described (Napolitano et al., 2018).
    RESULT: DBS was able to reduce levels of hyperphosphorylated and oligomeric (but not total) tau restoring levels of synaptic proteins back to wild-type in 3xTg mice. Pathological tau clearance occurred via autophagic pathway and lysosomal activity, the latest being enhanced by synaptic stimulation. TFEB was required for tau clearance as its inhibition prevented activity-induced reduction of pathological tau. In addition, RT-qPCR data analyses revealed increase expressions of TFEB downstream genes, including ATP6-V1H, ATP6-V0D1, and mucolipin-1 in neurons during synaptic stimulation.
    CONCLUSION: Protective effects of DBS/synaptic stimulation against pathological tau include the involvement TFEB and the enhancement of autophagy and lysosomal degradation.
    DOI:  https://doi.org/10.1002/alz.051678
  11. Biochemistry. 2022 Feb 04.
    Molecular Features of CA-074 pH-Dependent Inhibition of Cathepsin B.
    Michael C Yoon, Mitchell P Christy, Von V Phan, William H Gerwick, Gregory Hook, Anthony J O'Donoghue, Vivian Hook.
      CA-074 is a selective inhibitor of cathepsin B, a lysosomal cysteine protease. CA-074 has been utilized in numerous studies to demonstrate the role of this protease in cellular and physiological functions. Cathepsin B in numerous human disease mechanisms involves its translocation from acidic lysosomes of pH 4.6 to neutral pH 7.2 of cellular locations, including the cytosol and extracellular environment. To gain in-depth knowledge of CA-074 inhibition under these different pH conditions, this study evaluated the molecular features, potency, and selectivity of CA-074 for cathepsin B inhibition under acidic and neutral pH conditions. This study demonstrated that CA-074 is most effective at inhibiting cathepsin B at an acidic pH of 4.6 with nM potency, which was more than 100-fold more potent than its inhibition at a neutral pH of 7.2. The pH-dependent inhibition of CA-074 was abolished by methylation of its C-terminal proline, indicating the requirement for the free C-terminal carboxyl group for pH-dependent inhibition. Under these acidic and neutral pH conditions, CA-074 maintained its specificity for cathepsin B over other cysteine cathepsins, displayed irreversible inhibition, and inhibited diverse cleavages of peptide substrates of cathepsin B assessed by profiling mass spectrometry. Molecular docking suggested that pH-dependent ionic interactions of the C-terminal carboxylate of CA-074 occur with His110 and His111 residues in the S2' subsite of the enzyme at pH 4.6, but these interactions differ at pH 7.2. While high levels of CA-074 or CA-074Me (converted by cellular esterases to CA-074) are used in biological studies to inhibit cathepsin B at both acidic and neutral pH locations, it is possible that adjusted levels of CA-074 or CA-074Me may be explored to differentially affect cathepsin B activity at these different pH values. Overall, the results of this study demonstrate the molecular, kinetic, and protease specificity features of CA-074 pH-dependent inhibition of cathepsin B.
    DOI:  https://doi.org/10.1021/acs.biochem.1c00684
  12. Alzheimers Dement. 2021 Dec;17 Suppl 3 e054177
    DNAJC5 affects the endo-lysosomal pathway, APP processing, and AD pathology in vitro and in vivo.
    Niko Nykanen, Zhaohua Wang, Todd A Davis, Marie Nunez, Kevin O'Dell, John R Cirrito, Mark Sands, Bruno A Benitez.
       BACKGROUND: The DnaJ heat shock protein family member C5 (DNAJC5) gene encodes Cysteine String Protein-alpha (CSPα). CSPα is a key endo-lysosomal element of the misfolding-associated protein secretion (MAPS) machinery. MAPS eliminates misfolded cytosolic proteins, including alpha-synuclein, tau, TDP-43, huntingtin. Mutations in the DNAJC5 gene cause rare early-onset dementia called adult-onset Neuronal ceroid lipofuscinosis (ANCL). Data from CSPα-deficient mice and flies suggest that CSPα is critical for preventing age-dependent neurodegeneration. The endo-lysosome plays an essential role in normal and abnormal Amyloid-beta precursor protein (APP) processing and subsequent β-amyloidogenesis in Alzheimer's disease (AD). However, the role of CSPα in APP processing, trafficking, and amyloidogenesis is not well understood.
    METHODS: We used histological staining and whole transcriptome data from ANCL, AD patients, and age-matched pathology-free controls. Differential expression (DE) analysis was performed using DESeq2 software. We performed histological analysis of 5XFAD mouse models crossed with DNAJC5 mice. We used mouse neuroblastoma (N2A) cells stably expressing wild-type human APP695 (N2A695) and human wild-type (WT) and mutant DNAJC5. We used ELISA to quantify Aβ40 and Aβ42 in cell culture media and human brain lysates.
    RESULTS: CSPα co-localizes with endo-lysosomal and synaptic markers in N2A695 cells. CSPα overexpression affects lysosomal function and SNAP29-mediated exocytosis. Overexpression and knockdown of hCSPα-WT in N2A695 cells significantly affect extracellular Aβ40, Aβ42, full-length APP, and APP C-terminal fragments (CTF). N2A-APP cells expressing a gain-of-function DNAJC5 mutant displayed a significant increase in lysosomal and autophagy (LC3-II and p62) proteins, lysosomal exocytosis, and secreted levels of Aβ40 and Aβ42. ANCL brains showed considerable neuronal Aβ accumulation. ANCL brains exhibit a significant reduction of soluble and insoluble Aβ4 and Aβ42. Transcriptome analysis from ANCL brains shows changes in the mTOR pathway. DNAJC5 transcript levels are significantly reduced in AD cases compared to controls. A mouse AD model exhibits an inverse correlation between DNAJC5 transcript levels and Aβ plaques. 5XFAD mice haploinsufficient for DNAJC5 gene significantly increased the Aβ plaque burden and decreased Aβ plaque latency.
    CONCLUSIONS: Our results provide evidence of the novel and unexpected role of CSPα in endo-lysosomal function, lysosomal exocytosis, β-amyloidogenesis both in vitro and in vivo.
    DOI:  https://doi.org/10.1002/alz.054177
  13. Alzheimers Dement. 2021 Dec;17 Suppl 3 e054611
    Defining the role of PLD3 in Alzheimer disease pathology.
    Matthew J Rosene, Simon Hsu, Rita Martinez, Joanne Norton, Ping Yan, John R Cirrito, Jin-Moo Lee, Ana Maria Cuervo, Alison M Goate, Carlos Cruchaga, Celeste M Karch.
       BACKGROUND: Alzheimer's disease (AD) is characterized by the accumulation of amyloid-β (Aβ) in the brain. We recently identified coding variants in the phospholipase D3 (PLD3) gene that double the risk for late onset AD.
    METHOD: We examined the impact of PLD3 risk variants on PLD3 and Aβ metabolism using CRISPR/Cas9 in induced pluripotent stem cells (iPSC). We then modeled the PLD3 expression patterns observed in AD brains in immortalized cell and AD mouse models. Lysosomal function was assessed in human brain tissue.
    RESULT: PLD3 A442A disrupts a splicing enhancer binding site and reduces PLD3 splicing in human brains. Differentiation of PLD3 A442A and isogenic control iPSCs into cortical neurons produced cells that were morphologically similar. At the molecular level, PLD3 A442A neurons displayed a similar defect in PLD3 splicing as was observed in human brains and a significant increase in Aβ42/Aβ40 compared with isogenic control lines. Thus, PLD3 A442A is sufficient to alter PLD3 splicing and Aβ metabolism. PLD3 expression was significantly lower in AD brains compared with controls, and PLD3 expression was highly correlated with expression of lysosomal genes. Thus, we sought to determine whether PLD3 contributes to Aβ accumulation in AD via disrupted Aβ metabolism. We found that overexpression of PLD3 in immortalized cells decreased Aβ levels while shRNA silencing of PLD3 increased Aβ levels. In an AD mouse model, overexpression of PLD3 in hippocampal neurons produced decreased interstitial fluid (ISF) Aβ levels and accelerated Aβ turnover. Conversely, knocking out PLD3 increased ISF Aβ, reduced Aβ turnover, and increased APP protein levels. Thus, reduced turnover of ISF Aβ along with increase APP substrate may lead to Aβ accumulation. To begin to determine whether PLD3 influences Aβ turnover via the lysosome, we isolated lysosomal fractions from human AD and control brains. PLD3 was enriched in lysosomal subfractions and PLD3 distribution in these subfractions was altered in AD. Furthermore, PLD3 stability in the lysosomal fractions was disrupted in AD brains.
    CONCLUSION: Together, our findings demonstrate that PLD3 promotes Aβ clearance through pathways involving lysosomal degradation.
    DOI:  https://doi.org/10.1002/alz.054611
  14. Alzheimers Dement. 2021 Dec;17 Suppl 3 e056185
    Differential gene expression analysis for the identification of potential biomarkers for Niemann-Pick type C disease.
    Tamara Patricia Pradel-Bernal, Jared Rivera-Osorio, Maria Del Carmen Cardenas-Aguayo.
       BACKGROUND: Niemann-Pick disease (NP) is a rare neurovisceral lysosomal lipid storage disorder. NPC (NP type C) is characterized by the accumulation of unesterified cholesterol and other lipids within the cell, the function of lysosomes usually is impaired as well as the autophagy flux. To better understand the molecular pathways affected in this disease, we perform a bioinformatic study.
    METHOD: We used in silico methods to identify genes affected in this disease. The first approach was an analysis of microarray data from the public archive GEO2, accession number GSE124283. We studied gene expression of skin fibroblast from 55 individuals, 22 healthy controls (13 males and 9 females) and other 23 with NPC (12 males and 11 females), 16 patients suffering from infantile NPC, 5 juvenile NPC and 2 adults. The differential expression analysis was performed using limma package, genes with significative adjusted p value and fold change > 0.5 and < -0.5 were selected, we used topGO package to obtain the Gene Ontology.
    RESULT: The analysis revealed that cellular components enriched were vesicle membrane, vacuolar lumen lysosome, cytoplasm vesicle, cytoskeletal fiber, nucleus and nucleoplasm. For molecular function were affected catalytic activity, cadherin binding and enzyme binding, and the biological process enriched were the phagosome maturation, lysosome organization, vesicular transport, apoptotic process, gene expression, proteosome-mediated ubiquitin, cell cycle, cellular macromolecule biosynthetic process and regulation of cellular response to stress.
    CONCLUSION: Our results were consistent with changes in cell homeostasis reported from NPC patients. Abnormal protein degradation and metabolism might contribute to the higher level of mortality in this disease. We propose that some of the DEGs that are more related to NPC disease according to our study, could have value as potential biomarkers of this disease helping to unravel the molecular mechanisms involve in this disease.
    DOI:  https://doi.org/10.1002/alz.056185
  15. Mol Neurodegener. 2022 Feb 04. 17(1): 15
    Differential regulation of progranulin derived granulin peptides.
    Tingting Zhang, Huan Du, Mariela Nunez Santos, Xiaochun Wu, Mitchell D Pagan, Lianne Jillian Trigiani, Nozomi Nishimura, Thomas Reinheckel, Fenghua Hu.
       BACKGROUND: Haploinsufficiency of progranulin (PGRN) is a leading cause of frontotemporal lobar degeneration (FTLD). PGRN is comprised of 7.5 granulin repeats and is processed into individual granulin peptides in the lysosome. However, very little is known about the levels and regulations of individual granulin peptides due to the lack of specific antibodies.
    RESULTS: Here we report the generation and characterization of antibodies specific to each granulin peptide. We found that the levels of granulins C, E and F are regulated differently  compared to granulins A and B in various tissues. The levels of PGRN and granulin peptides vary in different brain regions and the ratio between granulins and PGRN is highest in the cortical region in the adult male mouse brain. Granulin-A is localized in the lysosome in both neurons and microglia and its levels in microglia increase under pathological conditions. Interestingly,  the levels of granulin A in microglia change correspondingly with PGRN in response to stroke but not demyelination. Furthermore, deficiency of lysosomal proteases and the PGRN binding partner prosaposin leads to alterations in the ratios between individual granulin peptides. Granulins B, C and E are heavily glycosylated and the glycosylation patterns can be regulated under.
    CONCLUSION: Our results support that the levels of individual granulin peptides are differentially regulated under physiological and pathological conditions and provide novel insights into how granulin peptides function in the lysosome.
    Keywords:  Cathepsin; Frontotemporal lobar degeneration (FTLD); Glycosylation; Granulin; Lysosome; Progranulin (PGRN)
    DOI:  https://doi.org/10.1186/s13024-021-00513-9
  16. Life Sci Alliance. 2022 May;pii: e202101082. [Epub ahead of print]5(5):
    Environmental control of Pub1 (NEDD4 family E3 ligase) in Schizosaccharomyces pombe is regulated by TORC2 and Gsk3.
    Tingting Wang, Philip Woodman, Sean J Humphrey, Janni Petersen.
      Cells respond to changing nutrient environments by adjusting the abundance of surface nutrient transporters and receptors. This can be achieved by modulating ubiquitin-dependent endocytosis, which in part is regulated by the NEDD4 family of E3 ligases. Here we report novel regulation of Pub1, a fission yeast Schizosaccharomyces pombe member of the NEDD4-family of E3 ligases. We show that nitrogen stress inhibits Pub1 function, thereby increasing the abundance of the amino acid transporter Aat1 at the plasma membrane and enhancing sensitivity to the toxic arginine analogue canavanine. We show that TOR complex 2 (TORC2) signalling negatively regulates Pub1, thus TORC2 mutants under nutrient stress have decreased Aat1 at the plasma membrane and are resistant to canavanine. Inhibition of TORC2 signalling increases Pub1 phosphorylation, and this is dependent on Gsk3 activity. Addition of the Tor inhibitor Torin1 increases phosphorylation of Pub1 at serine 199 (S199) by 2.5-fold, and Pub1 protein levels in S199A phospho-ablated mutants are reduced. S199 is conserved in NEDD4 and is located immediately upstream of a WW domain required for protein interaction. Together, we describe how the major TORC2 nutrient-sensing signalling network regulates environmental control of Pub1 to modulate the abundance of nutrient transporters.
    DOI:  https://doi.org/10.26508/lsa.202101082
  17. Alzheimers Dement. 2021 Dec;17 Suppl 3 e053844
    Lysosomal subcellular distribution in neurons: Are there synaptic lysosomes?
    César Augusto Vogado Cunha, Tatiana Burrinha, Claudia G Almeida.
       BACKGROUND: Despite Alzheimer's disease's first description 114 years ago1 , the mechanisms driving the most common late-onset forms' molecular etiology and neuropathophysiology remain elusive. While considering its elaborate display of pathological hallmarks, recent advances have described synapse loss as the most accurate correlate of disease progression2 . A wide array of data, including insights from human genetics, preclinical AD models, clinical research, and trials, has continuously linked a dysfunctional lysosomal network to pathological aging and disease onset. However, evidence correlating lysosomal dysfunction with early synapse loss is still lacking. Here, we aim to characterize the subcellular distribution of neuronal lysosomes, hypothesizing that these complex organelles localize to both presynaptic and postsynaptic terminals.
    METHOD: We have analyzed the subcellular distribution of synaptic and non-synaptic associated LAMP-1+ organelles in our established mature primary hippocampal and cortical mouse neuronal in vitro model and adult mouse brain synaptosomes. We applied quantitative single-cell analysis using ICY and ImageJ software for analytical purposes.
    RESULT: Our results show that the subcellular distribution of (endo)lysosomes is highly compartmentalized and dynamic. Based on the % of (endo)lysosomes in the neuronal soma, we documented that roughly 42% of (endo)lysosomes distribute along dendrites and 37% along the axon initial segment (AIS). Remarkably, we report that close to 8% of the total (endo)lysosomal pool was associated with synapsin, a presynaptic compartment marker. We also reveal that about 26% of (endo)lysosomes colocalized with PSD-95, a postsynaptic compartment marker, constituting 60% of the dendritic (endo)lysosomes. Moreover, we found LAMP-1 enriched in mouse brain synaptosomes against nuclear, non-nuclear, non-synaptosomal and membrane fractions. We are currently pushing to characterize these synaptic (endo)lysosomes' function.
    CONCLUSION: These results suggest that (endo)lysosomes are present at synapses. In the future we would like to establish whether its dysfunctional activity and trafficking act as a catalyst in the very early stages of synaptic dysfunction in the aging brain, potentially contributing to LOAD etiology and development. References: (1) Alzheimer, A., 1907. Uber einen eigenartigc Erkrankung der Hirnrinde. Allgemeine Zeitschrift fur Psyciatrie und Psychisch-Gerichtliche Medizin 64, 3. (2) Tampellini, D., Gouras, G., 2010. Synapses, synaptic activity and intraneuronal Aβ in Alzheimer's disease. Front Aging Neurosci. 2, 13. DOI: 10.3389/fnagi.2010.00013.
    DOI:  https://doi.org/10.1002/alz.053844
  18. Alzheimers Dement. 2021 Dec;17 Suppl 3 e054214
    Assessing stimulation-dependent changes in LRRK2 and GCase expression/activity and convergence at the lysosome in cryopreserved monocytes.
    Rebecca Wallings, Laura Hughes, Nicholas Dzamko, Malu Gamez Tansey.
       BACKGROUND: Both leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GCase) are promising targets for the treatment of Parkinson's disease (PD). Evidence suggests that both proteins are involved in similar biological pathways, including lysosomal function. However, studies to date have largely investigated the enzymes in isolation and any relationship between LRRK2 and GCase remains unclear. Recent data suggest that it is the response to immunological challenges that is altered in PD and that circulating baseline cytokine levels are not useful diagnostic or predictive biomarkers in PD. Taken together, this project aims to optimize a blood collection and cryopreservation protocol to facilitate the collection and study of monocytes from PD patients and healthy controls, with a particular focus on measuring stimulation-dependent changes in LRRK2 and GCase protein activity levels and lysosomal function.
    METHOD: Human PBMCs were isolated from whole blood and plated fresh or post-cryopreservation. PBMCs were treated with MLi2 or CBE +/- IFN-γ. Treatment effects on cathepsin activity, LRRK2 and GCase enzymatic activity, and antigen presentation were assessed in monocytes via flow cytometry. Media was collected RESULT: We first assessed the effects of cryopreservation on our read-outs. Despite cryopreservation having a small effect at baseline, both fresh and cryopreserved monocytes gated from total PBMCs exhibited comparable changes in response to IFN-γ. We observed that LRRK2 kinase inhibition decreases cathepsin activity in monocytes in a stimulation-dependent manner. This was coupled with increased LRRK2 expression and alterations in immune cell activation with LRRK2 kinase inhibition. Similarly, an increase in GCase activity levels were observed in monocytes upon IFN-γ treatment.
    CONCLUSION: These results show that cryopreservation of PBMCs doesn't affect our ability to measure stimulation-dependent changes in both LRRK2 and GCase expression/activity levels. These assays are therefore to be repeated in idiopathic PD PBMCs. Furthermore, these results suggest a fundamental role of LRRK2, specifically LRRK2 kinase activity, in lysosomal function in immune cells. Given the parallel increases in GCase activity, future research aims to investigate how these two enzymes interact and converge on biological pathways regulating inflammation and the lysosome in patient monocytes.
    DOI:  https://doi.org/10.1002/alz.054214
  19. Mol Cell. 2022 Jan 25. pii: S1097-2765(22)00002-8. [Epub ahead of print]
    E3 ligase RNF167 and deubiquitinase STAMBPL1 modulate mTOR and cancer progression.
    Dong Wang, Chenchen Xu, Wenyu Yang, Jie Chen, Yuhui Ou, Yuanyuan Guan, Jialiang Guan, Ying Liu.
      The mTOR complex 1 (mTORC1) is an essential metabolic hub that coordinates cellular metabolism with the availability of nutrients, including amino acids. Sestrin2 has been identified as a cytosolic leucine sensor that transmits leucine status signals to mTORC1. In this study, we identify an E3 ubiquitin ligase RING finger protein 167 (RNF167) and a deubiquitinase STAMBPL1 that function in concert to control the polyubiquitination level of Sestrin2 in response to leucine availability. Ubiquitination of Sestrin2 promotes its interaction with GATOR2 and inhibits mTORC1 signaling. Bioinformatic analysis reveals decreased RNF167 expression and increased STAMBPL1 expression in gastric and colorectal tumors. Knockout of STAMBPL1 or correction of the heterozygous STAMBPL1 mutation in a human colon cancer cell line suppresses xenograft tumor growth. Lastly, a cell-permeable peptide that blocks the STAMBPL1-Sestrin2 interaction inhibits mTORC1 and provides a potential option for cancer therapy.
    Keywords:  Sestrin2; amino acid sensing; colorectal cancer; mTOR; tumorigenesis; ubiquitination
    DOI:  https://doi.org/10.1016/j.molcel.2022.01.002
  20. Alzheimers Dement. 2021 Dec;17 Suppl 3 e054448
    Defective proteostasis in patient-derived iPSC astrocytes and neurons carrying a MAPT IVS10+16 mutation.
    Sidhartha Mahali, Rita Martinez, Miwei Hu, Jacob Marsh, Sally Temple, Celeste M Karch.
       BACKGROUND: Impaired proteostasis is associated with normal aging and is accelerated in neurodegeneration. This impairment may lead to the toxic accumulation of protein. In a subset of frontotemporal dementia (FTD) cases, mutations in the microtubule-associated protein tau (MAPT) that alter the relative levels of tau isoforms are sufficient to cause tau inclusions in neurons and astroglia and neurodegeneration without the presence of mutated protein (e.g. MAPTIVS10+16). However, the pathogenic events triggered by the expression of the alternatively spliced tau remain poorly understood.
    METHOD: To determine whether altered tau splicing induced from MAPT IVS10+16 mutations is sufficient to alter proteostasis in neurons and glia, we used human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes from patients carrying the MAPT IVS10+16 mutation and CRISPR/Cas9, isogenic corrected controls.
    RESULT: We found that neurons from MAPT IVS10+16 carriers exhibited significantly higher levels of tau containing 4 microtubule binding repeats (4R tau), deficits in lysosomal trafficking, and acidity relative to isogenic-control neurons. Conversely, astrocytes from MAPT IVS10+16 carriers exhibited morphologically an increase in acidic lysosomes compared to isogenic-control astrocytes. Astrocytes from MAPT IVS10+16 carriers were also larger in size, consistent with cellular hypertrophy observed in brains from FTD-tau patients.
    CONCLUSION: Our findings suggest that altered tau splicing induced by the MAPT IVS10+16 mutation is sufficient to cause impaired lysosomal function and altered proteostasis in a cell-type specific manner.
    DOI:  https://doi.org/10.1002/alz.054448
  21. Alzheimers Dement. 2021 Dec;17 Suppl 3 e049943
    Developing human cellular models to understand biological mechanisms linked to AD genetic risk.
    Jessica E Young.
       BACKGROUND: Mechanisms underlying late-onset sporadic Alzheimer's disease are genetically and biologically diverse. There is an outstanding need to identify modifiable cellular pathways associated with loci that increase AD risk. Furthermore, these pathways likely function differently in the diverse cell types of the CNS. Human induced pluripotent stem cells (hiPSCs) are an attractive model to address these challenges. hiPSCs can be differentiated to multiple cell types in two and three-dimensional formats. Using living human cells, novel cellular phenotypes can be uncovered and employed in therapeutic screens.
    METHOD: We have applied this methodology to understand the role of the AD risk gene, SORL1 in endo-lysosomal trafficking in human neurons and microglia. SORL1 is a well-established AD risk gene and functions as an endosomal sorting receptor, known for regulating APP processing. Our data suggests multiple roles for SORL1 in endolysosomal trafficking beyond regulating amyloid beta peptide levels in neurons. We generated hiPSC lines with SORL1 AD-associated variants as well as loss of SORL1 function.
    RESULTS: In neurons and microglia derived from hiPSCs, we document changes in endosome morphology, sequestration of neurotrophin and synaptic receptors in early and recycling endosomes, impairments in phagocytosis and mis-trafficking of substrates to lysosomes. We also demonstrate that treatment with small molecules that enhance endosomal trafficking are able to ameliorate some of these phenotypes.
    CONCLUSION: Collectively are data shows that distinct cellular phenotypes can be modeled by manipulation of an AD risk genes and that strategies enhancing endosomal trafficking should be considered as therapeutic targets for AD.
    DOI:  https://doi.org/10.1002/alz.049943
  22. Transl Neurodegener. 2022 01 31. 11(1): 5
    LRRK2, GBA and their interaction in the regulation of autophagy: implications on therapeutics in Parkinson's disease.
    Shirley Yin-Yu Pang, Rachel Cheuk Nam Lo, Philip Wing-Lok Ho, Hui-Fang Liu, Eunice Eun Seo Chang, Chi-Ting Leung, Yasine Malki, Zoe Yuen-Kiu Choi, Wing Yan Wong, Michelle Hiu-Wai Kung, David Boyer Ramsden, Shu-Leong Ho.
      Mutations in leucine-rich repeat kinase 2 (LRRK2) and glucocerebrosidase (GBA) represent two most common genetic causes of Parkinson's disease (PD). Both genes are important in the autophagic-lysosomal pathway (ALP), defects of which are associated with α-synuclein (α-syn) accumulation. LRRK2 regulates macroautophagy via activation of the mitogen activated protein kinase/extracellular signal regulated protein kinase (MAPK/ERK) kinase (MEK) and the calcium-dependent adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathways. Phosphorylation of Rab GTPases by LRRK2 regulates lysosomal homeostasis and endosomal trafficking. Mutant LRRK2 impairs chaperone-mediated autophagy, resulting in α-syn binding and oligomerization on lysosomal membranes. Mutations in GBA reduce glucocerebrosidase (GCase) activity, leading to glucosylceramide accumulation, α-syn aggregation and broad autophagic abnormalities. LRRK2 and GBA influence each other: GCase activity is reduced in LRRK2 mutant cells, and LRRK2 kinase inhibition can alter GCase activity in GBA mutant cells. Clinically, LRRK2 G2019S mutation seems to modify the effects of GBA mutation, resulting in milder symptoms than those resulting from GBA mutation alone. However, dual mutation carriers have an increased risk of PD and earlier age of onset compared with single mutation carriers, suggesting an additive deleterious effect on the initiation of PD pathogenic processes. Crosstalk between LRRK2 and GBA in PD exists, but its exact mechanism is unclear. Drugs that inhibit LRRK2 kinase or activate GCase are showing efficacy in pre-clinical models. Since LRRK2 kinase and GCase activities are also altered in idiopathic PD (iPD), it remains to be seen if these drugs will be useful in disease modification of iPD.
    Keywords:  Autophagy; GBA; GCase; Interaction; LRRK2; Mutation; Parkinson’s disease; α-Synuclein
    DOI:  https://doi.org/10.1186/s40035-022-00281-6
  23. Alzheimers Dement. 2021 Dec;17 Suppl 3 e057555
    Lysosomal permeabilization as a mechanism of amyloid degradation toxicity in Alzheimer's disease.
    Dmitry Zaretsky, Maria Zaretskaia, Yaroslav Molkov.
       BACKGROUND: The amyloid channel theory readily explains primary molecular damage induced by beta-amyloid (Aβ) but cannot interpret multiple major phenomena associated with Alzheimer's Disease such as autophagy failure and decreased metabolism. To explain them, the amyloid degradation toxicity hypothesis suggests that the cytotoxicity is initiated by the channel formation in lysosomal membranes by amyloid fragments (such as Aβ25-35 ) produced by the digestion of endocytosed Aβ. One amyloid channel is sufficient to neutralize lysosomal content and inactivate proteases; therefore, undigested amyloid accumulates, and autophagy stalls. Inadequate mitophagy results in an increased generation of reactive oxygen species and decreased energy production. Huge electrical conductance of amyloid channels prompted us to estimate the maximal size of the macromolecules which can leak from permeabilized lysosomes through amyloid channels.
    METHODS: To estimate the channels' ability to leak molecules of various molecular weights, we modeled the channels as saline-filled cylinders in the non-conductive membranes passing spheres with a density of average globular proteins. The histograms of single channel conductance at various pH were constructed from the previously published experimental dataset. An approximation with a power function was used to extrapolate the probabilities of channels with high molecular weight cut-offs (MWCO).
    RESULTS: A variability of channel sizes suggests that channel-forming barrel-shaped aggregates can have various number of monomers. Extrapolation of the channel size distribution predicts that a significant number of channels has MWCO exceeding 10 kDa, while acidic environment disproportionally promotes their formation. This range of MWCO is sufficient for the leakage of cathepsins (20-30 kDa) implicated in the induction of necrosis and/or apoptosis. Channels with MWCO above 1000 Da can release into the cytoplasm the amyloid fragments which can form membrane channels in the plasma membrane by entering from the internal leaflet. The time needed for endocytosis and proteolytic digestion explains why cellular responses to Aβ exposure are taking significant time (at least several minutes).
    CONCLUSION: While dissipation of the proton gradient by small amyloid channel readily explains lysosomal failure, relatively rare events of lysosomal permeabilization to large macromolecules by amyloid channels can be an alternative mechanism of cellular death induced by the exposure to Aβ.
    DOI:  https://doi.org/10.1002/alz.057555
  24. Front Pharmacol. 2021 ;12 825425
    The Effects of Combined Therapy With Metformin and Hydroxypropyl-β-Cyclodextrin in a Mouse Model of Niemann-Pick Disease Type C1.
    Jiang Du, Xinlei Liu, Yan Zhang, Xiaojing Han, Chunya Ma, Yanli Liu, Lihong Guan, Liang Qiao, Juntang Lin.
      Niemann-Pick disease type C1 (NPC1) is a neurodegenerative disorder characterized by lysosomal storage of free cholesterol. 2-Hydroxypropyl-β-cyclodextrin (HPβCD) is a cyclic oligosaccharide derivative that is being developed to treat NPC1. Recently, metformin was reported to be beneficial in various neurodegenerative diseases, such as Alzheimer's and Huntington's diseases. In this study, we examined the effects of combined treatment with HPβCD and metformin on Npc1 -/- mice. Unfortunately, body weight and survival rates showed that cotreatment with metformin did not extend survival time and increase the body weight of HPβCD-treated Npc1 -/- mice. However, cotreatment with metformin reduced inflammatory response and inhibited the proinflammatory cytokine release in the brain, liver and spleen of HPβCD-treated Npc1 -/- mice. Furthermore, metformin did not reduce the free cholesterol levels in Npc1 -/- brain tissue or fibroblasts. In conclusion, our results demonstrate that metformin does not show beneficial effects on body weight or survival time but reduced the inflammatory response in a mouse model of NPC1 when combined with HPβCD.
    Keywords:  HPβCD; NPC1 disease; cholesterol accumulation; combined therapy; metformin
    DOI:  https://doi.org/10.3389/fphar.2021.825425
  25. Oxid Med Cell Longev. 2022 ;2022 8096009
    Interaction between TRPML1 and p62 in Regulating Autophagosome-Lysosome Fusion and Impeding Neuroaxonal Dystrophy in Alzheimer's Disease.
    Lu Zhang, Yu Fang, Xuan Cheng, Yajun Lian, Hongliang Xu.
      The loss of transient receptor potential mucolipin 1 (TRPML1), an endosomal and lysosomal Ca2+-releasing channel, has been implicated in neurodegenerative disorders. Mounting evidence have shown that TRPML1 could clear intraneuronal amyloid-β (Aβ), which triggers a hypothesis that TRPML1 activation may be beneficial for axonal transport in Alzheimer's disease (AD). In this work, the functional roles of TRPML1 were studied in the APP/PS1 transgenic mice and Aβ1-42-stimulated hippocampal neurons HT22. We found that lentivirus-mediated overexpression of TRPML1 was shown to promote an accumulation of autolysosomes and increase brain-derived neurotrophic factor (BDNF) transportation to the nucleus, suggesting an axon-protective function. More importantly, we found that TRPML1 also increased p62 that interacted with dynein. Lentivirus-mediated knockdown of p62 or inhibition of dynein by ciliobrevin D stimulation was found to reduce autolysosome formation and nuclear accumulation of BDNF in HT22 cells with Aβ1-42 stimulation. Inhibition of p62 by XRK3F2 stimulation was observed to promote the death of hippocampal neurons of the APP/PS1 transgenic mice. TRPML1 recruited dynein by interacting with p62 to promote the autophagosome-lysosome fusion to mediate BDNF nuclear translocation to impede axon dystrophy in mice with Alzheimer-like phenotypes. In summary, these results demonstrate the presence of a TRPML1/p62/dynein regulatory network in AD, and activation of TRPML1 is required for axon protection to prevent neuroaxonal dystrophy.
    DOI:  https://doi.org/10.1155/2022/8096009
  26. Alzheimers Dement. 2021 Dec;17 Suppl 3 e053723
    Contribution of Alzheimer's disease risk genes in the maintenance of endolysosomal transport regulation.
    Marine Bretou, Ragna Sannerud, Zoë P Van Acker, Wendy Vermeire, David Demedts, Wim Annaert.
       BACKGROUND: Alzheimer's disease (AD) is the most common form of dementia of which the incidence increases with age. AD has a strong genetic component, for which further study is essential to identify pathways that can be targeted therapeutically, as no cure is available yet. While most studies have focused over the years on the early onset risk factors (i.e. APP, PSEN1 and PSEN2), those represent only a minor fraction of all cases. GWAS studies have identified ≈30 late-onset AD (LOAD) susceptibility loci that, when functionally clustered, are directly or indirectly linked to cellular trafficking routes and functioning. These include genes linked to cholesterol metabolism, endocytic transport regulation and lysosomal processes. The genetic findings concur with the appearance of endolysosomal abnormalities at preclinical stages, before the appearance of senile plaques and tangles.
    METHOD: Using a CRISPR-Cas9 based approach, knock-out (KO) of various risk factors from early (PSEN1) or late (PICALM, PLD3) onset risk factors were realized in fibroblasts and neuronal SH-SY5Y cells. Analysis of the endolysosomal pathway and lysosomal functions were carried out using biochemical assays, lipidomic analysis of isolated organelles, confocal imaging as well as electron microscopy.
    RESULT: Both the deficiency of presenilins and late risk factors led to endolysosomal dysfunctions, as characterized by an enlargement of late endosomes/lysosomes that accumulate diverse types of lipids such as cholesterol or glycerophospholipids. Further focusing on PSENs, their depletion led to a delay in the endosomal maturation, resulting in an accumulation of endosomal cargoes and adapters such as the retromer subunit VPS35. Re-expression of either catalytic or inactive PSEN1 was sufficient to correct these aberrant accumulations. Whereas the endolysosomal abnormalities could be connected to the accumulation of APP-CTFs, the nature of γ-secretase-independent contribution remains enigmatic. Activation of recycling significantly alleviated endolysosomal defects observed in PSENdKO cells.
    CONCLUSION: Altogether, our results underscore that different upstream mechanisms could lead to similar endolysosomal dysfunctions. Our observations are also shedding light on the potential of modulating/restoring endosomal recycling regulation as a strategy to alleviate observed early endolysosomal dysfunctions at preclinical stages of AD pathogenesis.
    DOI:  https://doi.org/10.1002/alz.053723
  27. Bioanalysis. 2022 Feb 04.
    Lysosphingolipid urine screening test using mass spectrometry for the early detection of lysosomal storage disorders.
    Marcel A Kelkel, Michel Boutin, Filipa Curado, Peter Bauer, Éliane Beauregard-Lacroix, François E Mercier, Bruno Maranda, Iskren Menkovic, Tristan Martineau, Christiane Auray-Blais.
      Background: Sphingolipidoses are caused by a defective sphingolipid catabolism, leading to an accumulation of several glycolipid species in tissues and resulting in neurotoxicity and severe systemic manifestations. Methods & results: Urine samples from controls and patients were purified by solid-phase extraction prior to the analysis by ultra-high-performance liquid chromatography (UPLC) combined with MS/MS. A UPLC-MS/MS method for the analysis of 21 urinary creatinine-normalized biomarkers for eight diseases was developed and validated. Conclusion: Considering the growing demand to identify patients with different sphingolipidoses early and reliably, this methodology will be applied for high-risk screening to target efficiently patients with various sphingolipidoses.
    Keywords:  analog; biomarker; creatinine; lysosomal storage diseases; lysosphingolipid; screening; sphingolipidosis; tandem mass spectrometry; ultra-high-performance liquid chromatography; urine
    DOI:  https://doi.org/10.4155/bio-2021-0259
  28. Bioanalysis. 2022 Feb 04.
    Quantitation of a plasma biomarker profile for the early detection of Gaucher disease type 1 patients.
    Iskren Menkovic, Michel Boutin, Abdulfatah Alayoubi, Filipa Curado, Peter Bauer, François E Mercier, Georges-Étienne Rivard, Christiane Auray-Blais.
      Aim: Gaucher disease (GD) is caused by a deficiency of the lysosomal enzyme acid β-glucocerebrosidase. Recent metabolomic studies highlighted several new metabolites increased in the plasma of GD patients. We aimed to develop and validate a UPLC-MS/MS method allowing a relative quantitation of lyso-Gb1 and lyso-Gb1 analogs -28, -12, -2, +14, +16 and +18 Da in addition to sphingosylphosphorylcholine, N-palmitoyl-O-phosphocholine to study potential correlations with clinical manifestations. Methodology & results: Following solid-phase extraction, plasma samples were evaporated and resuspended in 100 μl of resuspension solution. Three microliter is injected into the UPLC-MS/MS for analysis. Conclusion: All biomarkers studied were increased in GD patients. Significant correlations were observed between specific analogs and hematological, and visceral complications, as well as overall disease severity.
    Keywords:  Gaucher disease; N-palmitoyl-O-phosphocholineserine; biomarker; glucosylsphingosine; lyso-Gb1; lyso-Gb1 analogs; plasma; sphingosylphosphorylcholine; tandem mass spectrometry
    DOI:  https://doi.org/10.4155/bio-2021-0242
  29. Hum Gene Ther. 2022 Feb 01.
    Minimum Effective Dose to Achieve Biochemical Correction With AAV Vector-Mediated Gene Therapy in Mice With Pompe Disease.
    Sangoh Han, Dorothy Brooke Gheorghiu, Songtao Li, Hye Ri Kang, Dwight Koeberl.
      Pompe disease is an autosomal recessive lysosomal storage disorder caused by deficiency of acid α-glucosidase (GAA), resulting in skeletal muscle weakness and cardiomyopathy. Muscle weakness progresses despite currently available therapy, which has prompted the development of gene therapy with adeno-associated virus (AAV) type 2 vectors cross-packaged as AAV8 (2/8). Preclinical studies of gene therapy demonstrated that the minimum effective dose for biochemical correction with AAV2/8-LSPhGAA was approximately 2 x 1011 vector genomes (vg)/kg body weight. The current study examined the transduction of AAV2/8-LSPeGFP vector in adult GAA-KO mice with Pompe disease, and correlated that degree of transduction with the biochemical correction achieved by the same dose of AAV2/8-LSPhGAA. The minimum effective dose was found to be approximately 2 x 1011 vg/kg, with all hepatocytes variably transducing at this dose. At this dose, liver GAA significantly increased, while liver glycogen significantly decreased. The 2 x 1011 vg/kg dose was sufficient to significantly decrease diaphragm glycogen. However, the heart, diaphragm, and quadriceps all required a four-fold higher dose to achieve correction of GAA deficiency in association with significant clearance of stored glycogen, which correlated with increased serum GAA activity. These data indicate that AAV2/8-LSPeGFP transduced all hepatocytes when the 2 x 1011 vg/kg dose was administered, which correlated with partial biochemical correction from the equivalent dose of AAV2/8-LSPhGAA. Together these data support the conclusion that substantial transduction of the liver is required to achieve biochemical correction from AAV2/8-LSPhGAA.
    DOI:  https://doi.org/10.1089/hum.2021.252
  30. J Neurosci. 2022 Feb 04. pii: JN-RM-0449-21. [Epub ahead of print]
    A farnesyltransferase inhibitor restores cognitive deficits in Tsc2+/- mice through inhibition of Rheb1.
    Hiroko Sugiura, Tadayuki Shimada, Keiko Moriya-Ito, Jun-Ichi Goto, Hiroki Fujiwara, Rie Ishii, Hiroshi Shitara, Choji Taya, Satoshi Fujii, Toshiyuki Kobayashi, Okio Hino, Paul F Worley, Kanato Yamagata.
      Tuberous sclerosis complex (TSC) is caused by mutations in Tsc1 or Tsc2, whose gene products inhibit the small G-protein Rheb1. Rheb1 activates mTORC1, which may cause refractory epilepsy, intellectual disability and autism. The mTORC1 inhibitors have been used for TSC patients with intractable epilepsy. However, its effectiveness for cognitive symptoms remains unclear. We found a new signaling pathway for synapse formation through Rheb1 activation, but not mTORC1. Here, we show that treatment with the farnesyltransferase inhibitor lonafarnib increased unfarnesylated (inactive) Rheb1 levels and restored synaptic abnormalities in cultured Tsc2+/- neurons, whereas rapamycin did not enhance spine synapse formation. Lonafarnib treatment also restored the plasticity-related Arc expression in cultured Tsc2+/- neurons. Lonafarnib action was partly dependent on the Rheb1 reduction with syntenin. Oral administration of lonafarnib increased unfarnesylated protein levels without affecting mTORC1and MAP kinase signalings, and restored dendritic spine morphology in the hippocampi of male Tsc2+/- mice. In addition, lonafarnib treatment ameliorated contextual memory impairments and restored memory-related Arc expression in male Tsc2+/- mice in vivo Heterozygous Rheb1 knockout in male Tsc2+/- mice reproduced the results observed with pharmacological treatment. These results suggest that the Rheb1 activation may be responsible for synaptic abnormalities and memory impairments in Tsc2+/- mice, and its inhibition by lonafarnib could provide insight into potential treatment options for TSC-associated neuropsychiatric disorders (TANDs).SIGNIFICANCE STATEMENTTuberous sclerosis complex (TSC) is an autosomal dominant disease that causes neuropsychiatric symptoms, including intractable epilepsy, intellectual disability (ID) and autism. No pharmacological treatment for ID is reported so far. To develop a pharmacological treatment for ID, we investigated the mechanism of TSC and found Rheb1 activation is responsible for synaptic abnormalities in TSC neurons. To inhibit Rheb1 function, we used farnesyltransferase inhibitor lonafarnib, because farnesylation of Rheb1 is required for its activation. Lonafarnib treatment increased inactive Rheb1, and recovered proper synapse formation and plasticity-related Arc expression in TSC neurons. Furthermore, in vivo lonafarnib treatment restored contextual memory and Arc induction in TSC mice. Taken together, Rheb1 inhibition by lonafarnib could provide insight into potential treatments for TSC-associated ID.
    DOI:  https://doi.org/10.1523/JNEUROSCI.0449-21.2022
  31. J Clin Invest. 2022 Feb 03. pii: e152780. [Epub ahead of print]
    Lysosomal exocytosis of HSP70 stimulates monocytic BMP6 expression in Sjögren's syndrome.
    Ying-Qian Mo, Hiroyuki Nakamura, Tsutomu Tanaka, Toshio Odani, Paola Perez, Youngmi Ji, Benjamin N French, Thomas Jf Pranzatelli, Drew G Michael, Hongen Yin, Susan S Chow, Maryam Khalaj, Sandra A Afione, Changyu Zheng, Fabiola Reis Oliveira, Ana Carolina F Motta, Alfredo Ribeiro-Silva, Eduardo M Rocha, Cuong Q Nguyen, Masayuki Noguchi, Tatsuya Atsumi, Blake M Warner, John A Chiorini.
      BMP6 is a central cytokine in the induction of Sjögren's syndrome (SS)-associated secretory hypofunction. However, the upstream initiation leading to the production of this cytokine in SS is unknown. In this study, RNA in situ hybridization on salivary gland sections taken from SS patients indicated monocytic lineage cells as a cellular source of BMP6. RNA sequencing data from human salivary glands suggested TLR4 signaling was an upstream regulator of BMP6, which was confirmed by in vitro cell assays and single-cell transcriptomics of human PBMCs. Further investigation showed HSP70 was an endogenous natural TLR4 ligand that stimulated BMP6 expression in SS. Release of HSP70 from epithelial cells could be triggered by overexpression of lysosome-associated membrane protein 3 (LAMP3), a protein also associated with SS in several transcriptome studies. In vitro studies supported HSP70 was released as a result of lysosomal exocytosis initiated by LAMP3 expression, and reverse transcription PCR on RNA from minor salivary glands of SS patients confirmed a positive correlation between BMP6 and LAMP3 expression. BMP6 expression could be experimentally induced in mice by overexpression of LAMP3, which developed an SS-like phenotype. The newly identified LAMP3/HSP70/BMP6 axis provided an etiological model for SS gland dysfunction and autoimmunity.
    Keywords:  Autoimmune diseases; Autoimmunity; Innate immunity; Rheumatology
    DOI:  https://doi.org/10.1172/JCI152780
  32. Front Cell Neurosci. 2021 ;15 803302
    Neuroprogenitor Cells From Patients With TBCK Encephalopathy Suggest Deregulation of Early Secretory Vesicle Transport.
    Danielle de Paula Moreira, Angela May Suzuki, André Luiz Teles E Silva, Elisa Varella-Branco, Maria Cecília Zorél Meneghetti, Gerson Shigeru Kobayashi, Mariana Fogo, Merari de Fátima Ramires Ferrari, Rafaela Regina Cardoso, Naila Cristina Vilaça Lourenço, Karina Griesi-Oliveira, Elaine Cristina Zachi, Débora Romeo Bertola, Karina de Souza Weinmann, Marcelo Andrade de Lima, Helena Bonciani Nader, Andrea Laurato Sertié, Maria Rita Passos-Bueno.
      Biallelic pathogenic variants in TBCK cause encephaloneuropathy, infantile hypotonia with psychomotor retardation, and characteristic facies 3 (IHPRF3). The molecular mechanisms underlying its neuronal phenotype are largely unexplored. In this study, we reported two sisters, who harbored biallelic variants in TBCK and met diagnostic criteria for IHPRF3. We provided evidence that TBCK may play an important role in the early secretory pathway in neuroprogenitor cells (iNPC) differentiated from induced pluripotent stem cells (iPSC). Lack of functional TBCK protein in iNPC is associated with impaired endoplasmic reticulum-to-Golgi vesicle transport and autophagosome biogenesis, as well as altered cell cycle progression and severe impairment in the capacity of migration. Alteration in these processes, which are crucial for neurogenesis, neuronal migration, and cytoarchitecture organization, may represent an important causative mechanism of both neurodevelopmental and neurodegenerative phenotypes observed in IHPRF3. Whether reduced mechanistic target of rapamycin (mTOR) signaling is secondary to impaired TBCK function over other secretory transport regulators still needs further investigation.
    Keywords:  GM130; STAM; autophagy; clathrin; early secretory pathway; iPSC-neurodevelopmental disease modeling; mTOR; vesicle trafficking
    DOI:  https://doi.org/10.3389/fncel.2021.803302
  33. Proc Natl Acad Sci U S A. 2022 Feb 08. pii: e2117554119. [Epub ahead of print]119(6):
    ER-phagy requires the assembly of actin at sites of contact between the cortical ER and endocytic pits.
    Dongmei Liu, Muriel Mari, Xia Li, Fulvio Reggiori, Susan Ferro-Novick, Peter Novick.
      Fragments of the endoplasmic reticulum (ER) are selectively delivered to the lysosome (mammals) or vacuole (yeast) in response to starvation or the accumulation of misfolded proteins through an autophagic process known as ER-phagy. A screen of the Saccharomyces cerevisiae deletion library identified end3Δ as a candidate knockout strain that is defective in ER-phagy during starvation conditions, but not bulk autophagy. We find that loss of End3 and its stable binding partner Pan1, or inhibition of the Arp2/3 complex that is coupled by the End3-Pan1 complex to endocytic pits, blocks the association of the cortical ER autophagy receptor, Atg40, with the autophagosomal assembly scaffold protein Atg11. The membrane contact site module linking the rim of cortical ER sheets and endocytic pits, consisting of Scs2 or Scs22, Osh2 or Osh3, and Myo3 or Myo5, is also needed for ER-phagy. Both Atg40 and Scs2 are concentrated at the edges of ER sheets and can be cross-linked to each other. Our results are consistent with a model in which actin assembly at sites of contact between the cortical ER and endocytic pits contributes to ER sequestration into autophagosomes.
    Keywords:  actin; autophagy; endoplasmic reticulum
    DOI:  https://doi.org/10.1073/pnas.2117554119
  34. Front Cell Dev Biol. 2021 ;9 806521
    Beyond Protein Synthesis; The Multifaceted Roles of Tuberin in Cell Cycle Regulation.
    E Fidalgo da Silva, J Fong, A Roye-Azar, A Nadi, C Drouillard, A Pillon, L A Porter.
      The ability of cells to sense diverse environmental signals, including nutrient availability and conditions of stress, is critical for both prokaryotes and eukaryotes to mount an appropriate physiological response. While there is a great deal known about the different biochemical pathways that can detect and relay information from the environment, how these signals are integrated to control progression through the cell cycle is still an expanding area of research. Over the past three decades the proteins Tuberin, Hamartin and TBC1D7 have emerged as a large protein complex called the Tuberous Sclerosis Complex. This complex can integrate a wide variety of environmental signals to control a host of cell biology events including protein synthesis, cell cycle, protein transport, cell adhesion, autophagy, and cell growth. Worldwide efforts have revealed many molecular pathways which alter Tuberin post-translationally to convey messages to these important pathways, with most of the focus being on the regulation over protein synthesis. Herein we review the literature supporting that the Tuberous Sclerosis Complex plays a critical role in integrating environmental signals with the core cell cycle machinery.
    Keywords:  TSC; cell cycle; cell growth; mTOR; tuberin; tuberous sclerosis complex
    DOI:  https://doi.org/10.3389/fcell.2021.806521
  35. Front Cell Dev Biol. 2021 ;9 785979
    Dual Inhibition of mTORC1/2 Reduces Migration of Cholangiocarcinoma Cells by Regulation of Matrixmetalloproteinases.
    Katharina Joechle, Huda Jumaa, Kerstin Thriene, Claus Hellerbrand, Birte Kulemann, Stefan Fichtner-Feigl, Sven A Lang, Jessica Guenzle.
      Cholangiocarcinoma (CCA) is a rare but highly aggressive tumor entity for which systemic therapies only showed limited efficacy so far. As OSI-027-a dual kinase inhibitor targeting both mTOR complexes, mTORC1 and mTORC2 - showed improved anti-cancer effects, we sought to evaluate its impact on the migratory and metastatic capacity of CCA cells in vitro. We found that treatment with OSI-027 leads to reduced cell mobility and migration as well as a reduced surviving fraction in colony-forming ability. While neither cell viability nor proliferation rate was affected, OSI-027 decreased the expression of MMP2 and MMP9. Moreover, survival as well as anti-apoptotic signaling was impaired upon the use of OSI-027 as determined by AKT and MAPK blotting. Dual targeting of mTORC1/2 might therefore be a viable option for anti-neoplastic therapy in CCA.
    Keywords:  MTORC1/2; cholangiocarcinoma; invasion; matrix metalloproteinases; migration
    DOI:  https://doi.org/10.3389/fcell.2021.785979
  36. Biochem J. 2022 Feb 11. 479(3): 273-288
    SNARE proteins: zip codes in vesicle targeting?
    Seiichi Koike, Reinhard Jahn.
      Membrane traffic in eukaryotic cells is mediated by transport vesicles that bud from a precursor compartment and are transported to their destination compartment where they dock and fuse. To reach their intracellular destination, transport vesicles contain targeting signals such as Rab GTPases and polyphosphoinositides that are recognized by tethering factors in the cytoplasm and that connect the vesicles with their respective destination compartment. The final step, membrane fusion, is mediated by SNARE proteins. SNAREs are connected to targeting signals and tethering factors by multiple interactions. However, it is still debated whether SNAREs only function downstream of targeting and tethering or whether they also participate in regulating targeting specificity. Here, we review the evidence and discuss recent data supporting a role of SNARE proteins as targeting signals in vesicle traffic.
    Keywords:  membrane fusion; snare proteins; trafficking
    DOI:  https://doi.org/10.1042/BCJ20210719
  37. Mol Cancer Res. 2022 Feb 01. pii: molcanres.MCR-21-0729-A.2021. [Epub ahead of print]
    PARK2 regulates eIF4B-driven lymphomagenesis.
    Bandish B Kapadia, Anirban Roychowdhury, Forum Kayastha, Nahid Nanaji, Ronald B Gartenhaus.
      Patients with high-risk diffuse large B-cell lymphoma (DLBCL) have poor outcomes following first-line cyclophosphamide, doxorubicin, vincristine, prednisone, and rituximab (R-CHOP); thus, treatment of this fatal disease remains an area of unmet medical need and requires identification of novel therapeutic approaches. Dysregulation of protein translation initiation has emerged as a common downstream node in several malignancies, including lymphoma. Ubiquitination, a prominent post-translational modification associated with substrate degradation, has recently been shown to be a key modulator of nascent peptide synthesis by limiting several translational initiation factors. While a few deubiquitinases have been identified, the E3-ligase responsible for the critical ubiquitination of these translational initiation factors is still unknown. In this study, using complementary cellular models along with clinical readouts, we establish that PARK2 ubiquitinates eIF4B and consequently regulates overall protein translational activity. The formation of this interaction depends on upstream signaling, which is negatively regulated at the protein level of PARK2. Through biochemical, mutational, and genetic studies, we identified PARK2 as a mTORC1 substrate. mTORC1 phosphorylates PARK2 at Ser127, which blocks its cellular ubiquitination activity, thereby hindering its tumor suppressor effect on eIF4B's stability. This resultant increase of eIF4B protein level helps drive enhanced overall protein translation. These data support a novel paradigm in which PARK2-generated eIF4B ubiquitination serves as an anti-oncogenic intracellular inhibitor of protein translation, attenuated by mTORC1 signaling. Implications: Our data implicates the FASN/mTOR-PARK2-eIF4B axis as a critical driver of enhanced oncogene expression contributing to lymphomagenesis.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-21-0729
  38. Neuron. 2022 Feb 02. pii: S0896-6273(22)00055-1. [Epub ahead of print]110(3): 351-352
    Living in α-syn: Tackling aggregates in Parkinson's disease.
    Christopher J Griffey, Ai Yamamoto.
      How do protein aggregates contribute to neurodegenerative disorders, and can they be therapeutically targeted? In this issue of Neuron, Stojkovska et al. (2022) show that aggregated α-synuclein disrupts ER and lysosomal function in Parkinson's disease patient-derived neurons and that combined enhancement of multiple arms of the proteostasis network improves these defects.
    DOI:  https://doi.org/10.1016/j.neuron.2022.01.016
  39. Oncogene. 2022 Feb 02.
    HSP90 inhibitors induce GPNMB cell-surface expression by modulating lysosomal positioning and sensitize breast cancer cells to glembatumumab vedotin.
    Marco Biondini, Alex Kiepas, Leeanna El-Houjeiri, Matthew G Annis, Brian E Hsu, Anne-Marie Fortier, Geneviève Morin, José A Martina, Isabelle Sirois, Adriana Aguilar-Mahecha, Tina Gruosso, Shawn McGuirk, April A N Rose, Unal M Tokat, Radia M Johnson, Ozgur Sahin, Eric Bareke, Julie St-Pierre, Morag Park, Mark Basik, Jacek Majewski, Rosa Puertollano, Arnim Pause, Sidong Huang, Tibor Keler, Peter M Siegel.
      Transmembrane glycoprotein NMB (GPNMB) is a prognostic marker of poor outcome in patients with triple-negative breast cancer (TNBC). Glembatumumab Vedotin, an antibody drug conjugate targeting GPNMB, exhibits variable efficacy against GPNMB-positive metastatic TNBC as a single agent. We show that GPNMB levels increase in response to standard-of-care and experimental therapies for multiple breast cancer subtypes. While these therapeutic stressors induce GPNMB expression through differential engagement of the MiTF family of transcription factors, not all are capable of increasing GPNMB cell-surface localization required for Glembatumumab Vedotin inhibition. Using a FACS-based genetic screen, we discovered that suppression of heat shock protein 90 (HSP90) concomitantly increases GPNMB expression and cell-surface localization. Mechanistically, HSP90 inhibition resulted in lysosomal dispersion towards the cell periphery and fusion with the plasma membrane, which delivers GPNMB to the cell surface. Finally, treatment with HSP90 inhibitors sensitizes breast cancers to Glembatumumab Vedotin in vivo, suggesting that combination of HSP90 inhibitors and Glembatumumab Vedotin may be a viable treatment strategy for patients with metastatic TNBC.
    DOI:  https://doi.org/10.1038/s41388-022-02206-z
  40. Sci Adv. 2022 Feb 04. 8(5): eabj3967
    Profiling and functional characterization of maternal mRNA translation during mouse maternal-to-zygotic transition.
    Chunxia Zhang, Meng Wang, Yisi Li, Yi Zhang.
      Translational regulation plays an important role in gene expression and function. Although the transcriptional dynamics of mouse preimplantation embryos have been well characterized, the global mRNA translation landscape and the master regulators of zygotic genome activation (ZGA) remain unknown. Here, by developing and applying a low-input ribosome profiling (LiRibo-seq) technique, we profiled the mRNA translation landscape in mouse preimplantation embryos and revealed the translational dynamics during mouse preimplantation development. We identified a marked translational transition from MII oocytes to zygotes and demonstrated that active translation of maternal mRNAs is essential for maternal-to-zygotic transition (MZT). We further showed that two maternal factors, Smarcd2 and Cyclin T2, whose translation is activated in zygotes, are required for chromatin reprogramming and ZGA, respectively. Our study thus not only filled in a knowledge gap on translational regulation during mammalian preimplantation development but also revealed insights into the critical function of maternal mRNA translation in MZT.
    DOI:  https://doi.org/10.1126/sciadv.abj3967
  41. J Cell Sci. 2022 Jan 31. pii: jcs.259138. [Epub ahead of print]
    ALFY localizes to early endosomes and cellular protrusions to facilitate directional cell migration.
    Kristiane Søreng, Serhiy Pankiv, Camilla Bergsmark, Ellen M Haugsten, Anette K Dahl, Laura R de la Ballina, Ai Yamamoto, Alf H Lystad, Anne Simonsen.
      Cell migration is a complex process underlying physiological and pathological processes such as brain development and cancer metastasis. The autophagy-linked FYVE protein (ALFY), an autophagy adaptor protein known to promote clearance of protein aggregates, has been implicated in brain development and neural migration during cerebral cortical neurogenesis in mice. However, a specific role of ALFY in cell motility and extracellular matrix adhesion during migration has not been investigated. Here we reveal a novel role for ALFY in the endocytic pathway and in cell migration. We show that ALFY localizes to RAB5 and EEA1 positive early endosomes in a PtdIns(3)P dependent manner and is highly enriched in cellular protrusions at the leading and lagging edge of migrating cells. We find that cells lacking ALFY have reduced attachment and altered protein levels and glycosylation of integrins, resulting in the inability to form a proper leading edge and loss of directional cell motility.
    Keywords:  ALFY; Endosome; Focal adhesion; Integrin; Migration; WDFY3
    DOI:  https://doi.org/10.1242/jcs.259138
  42. Alzheimers Dement. 2021 Dec;17 Suppl 3 e055236
    Retromer stabilizing compounds rescue enlarged endosome phenotypes in hiPSC-derived models of AD.
    Allison Knupp, Refugio A Martinez, Suman Jayadev, Jessica E Young.
       BACKGROUND: The SORL1 gene encodes for the protein SorLA, a sorting receptor involved in retromer-related endosomal traffic. Many SORL1 genetic variants increase Alzheimer's disease (AD) risk, and rare loss-of-function truncation mutations have been found to be causal of AD. Previous work has shown that SORL1 depletion results in enlarged early endosomes in hiPSC-derived neurons.
    METHOD: We used CRISPR/Cas9 technology to insert SORL1 AD-risk variants in human induced pluripotent stem cells (hiPSCs) to test the hypothesis that risk variants in the VPS10 domain of SORL1 (SORL1 VPS10 variants) contribute to AD pathogenesis by leading to dysfunction in endosomal trafficking.
    RESULTS: We report that SORL1 VPS10 variants in hiPSC-derived neurons lead to early endosome enlargement, a cellular phenotype that is indicative of 'traffic jams' and is now considered a hallmark cytopathology AD. We determine that the retromer stabilizing compound TPT-260 reduces early endosome enlargement in SORL1 VPS10 variants.
    CONCLUSION: Our data, together with recent findings, underscores how sporadic AD pathways that regulate endosomal trafficking, and autosomal-dominant AD pathways that regulate APP cleavage, independently converge on AD's defining cytopathology. Moreover, demonstrating a partial rescue of cellular phenotypes in SORL1 VPS10 variants will contribute to the development of new and precision treatments for AD.
    DOI:  https://doi.org/10.1002/alz.055236
  43. Cell Death Dis. 2022 Feb 04. 13(2): 115
    Cathepsin D as a potential therapeutic target to enhance anticancer drug-induced apoptosis via RNF183-mediated destabilization of Bcl-xL in cancer cells.
    Seung Un Seo, Seon Min Woo, Seung-Soon Im, Younghoon Jang, Eugene Han, Sang Hyun Kim, Hongchan Lee, Hyun-Shik Lee, Ju-Ock Nam, Edward Gabrielson, Kyoung-Jin Min, Taeg Kyu Kwon.
      Cathepsin D (Cat D) is well known for its roles in metastasis, angiogenesis, proliferation, and carcinogenesis in cancer. Despite Cat D being a promising target in cancer cells, effects and underlying mechanism of its inhibition remain unclear. Here, we investigated the plausibility of using Cat D inhibition as an adjuvant or sensitizer for enhancing anticancer drug-induced apoptosis. Inhibition of Cat D markedly enhanced anticancer drug-induced apoptosis in human carcinoma cell lines and xenograft models. The inhibition destabilized Bcl-xL through upregulation of the expression of RNF183, an E3 ligase of Bcl-xL, via NF-κB activation. Furthermore, Cat D inhibition increased the proteasome activity, which is another important factor in the degradation of proteins. Cat D inhibition resulted in p62-dependent activation of Nrf2, which increased the expression of proteasome subunits (PSMA5 and PSMB5), and thereby, the proteasome activity. Overall, Cat D inhibition sensitized cancer cells to anticancer drugs through the destabilization of Bcl-xL. Furthermore, human renal clear carcinoma (RCC) tissues revealed a positive correlation between Cat D and Bcl-xL expression, whereas RNF183 and Bcl-xL expression indicated inverse correlation. Our results suggest that inhibition of Cat D is promising as an adjuvant or sensitizer for enhancing anticancer drug-induced apoptosis in cancer cells.
    DOI:  https://doi.org/10.1038/s41419-022-04581-7
  44. Sci Adv. 2022 Feb 04. 8(5): eabi9533
    Loss of TSC1/TSC2 sensitizes immune checkpoint blockade in non-small cell lung cancer.
    Qingyuan Huang, Fei Li, Hai Hu, Zhaoyuan Fang, Zhendong Gao, Guozhan Xia, Wai-Lung Ng, Alireza Khodadadi-Jamayran, Ting Chen, Jiehui Deng, Hua Zhang, Christina Almonte, Kristen Labbe, Han Han, Ke Geng, Sittinon Tang, Gordon J Freeman, Yuan Li, Haiquan Chen, Kwok-Kin Wong.
      Tuberous sclerosis complex subunit 1 (TSC1) and 2 (TSC2) are frequently mutated in non-small cell lung cancer (NSCLC), however, their effects on antitumor immunity remained unexplored. A CRISPR screening in murine KrasG12D/Trp53-/- (KP) model identified Tsc1 and Tsc2 as potent regulators of programmed cell death ligand 1 (Pd-l1) expression in vitro and sensitivity to anti-programmed cell death receptor 1 (PD-1) treatment in vivo. TSC1 or TSC2 knockout (KO) promoted the transcriptional and membrane expression of PD-L1 in cell lines. TSC2-deficient tumors manifested an inflamed microenvironment in patient samples and The Cancer Genome Atlas dataset. In syngeneic murine models, KP-Tsc2-KO tumors showed notable response to anti-PD-1 antibody treatment, but Tsc2-wild-type tumors did not. Patients with TSC1/TSC2-mutant NSCLC receiving immune checkpoint blockade (ICB) had increased durable clinical benefit and survival. Collectively, TSC1/TSC2 loss defines a distinct subtype of NSCLC characterized as inflamed tumor microenvironment and superior sensitivity to ICB.
    DOI:  https://doi.org/10.1126/sciadv.abi9533
  45. J Clin Invest. 2022 Feb 03. pii: e144334. [Epub ahead of print]
    Sortilin restricts secretion of apolipoprotein B-100 by hepatocytes under stressed but not basal conditions.
    Donna M Conlon, Carolin V Schneider, Yi-An Ko, Amrith Rodrigues, Kathy Guo, Nicholas J Hand, Daniel J Rader.
      Genetic variants at the SORT1 locus in humans causing increased SORT1 expression in liver are significantly associated with reduced plasma levels of LDL cholesterol and apolipoprotein B (apoB). However, the role of hepatic sortilin remains controversial, as genetic deletion of sortilin in mice has yielded variable and conflicting effects on apoB secretion. Sort1 knockout mice on a chow diet and several Sort1-deficient hepatocyte lines displayed no difference in apoB secretion. When these models were challenged with high fat or ER stress, the loss of Sort1 expression resulted in a significant increase in apoB-100 secretion. Sort1 overexpression studies yielded reciprocal results. Importantly, diabetic carriers of SORT1 variant have larger decreases in plasma apoB, TG, and VLDL and LDL particle number as compared to non-diabetics with the same variants. We conclude that under basal non-stressed conditions, loss of sortilin has little effect on hepatocyte apoB secretion, but that in the setting of lipid-loading or ER stress, sortilin deficiency leads to increased apoB secretion. These results are consistent with the directionality of effect in human genetics studies and suggest that under stress conditions, hepatic sortilin directs apoB toward lysosomal degradation rather than secretion, potentially serving as a quality control step in the apoB secretion pathway in hepatocytes.
    Keywords:  Genetics; Lipoproteins; Vascular Biology
    DOI:  https://doi.org/10.1172/JCI144334
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