Publications

( 0 )
Antonucci, A;Reggente, M;Roullier, C;Gillen, AJ;Schuergers, N;Zubkovs, V;Lambert, BP;Mouhib, M;Carata, E;Dini, L;Boghossian, AA.
Nature nanotechnology,
(2022)
The distinctive properties of single-walled carbon nanotubes (SWCNTs) have inspired the development of many novel applications in the field of cell nanobiotechnology. However, studies thus far have not explored the effect of SWCNT functionalization on transport across the cell walls of prokaryotes. We explore the uptake of SWCNTs in Gram-negative cyanobacteria and demonstrate a passive length-dependent and selective internalization of SWCNTs decorated with positively charged biomolecules. We show that lysozyme-coated SWCNTs spontaneously penetrate the cell walls of a unicellular strain and a multicellular strain. A custom-built spinning-disc confocal microscope was used to image the distinct near-infrared SWCNT fluorescence within the autofluorescent cells, revealing a highly inhomogeneous distribution of SWCNTs. Real-time near-infrared monitoring of cell growth and division reveal that the SWCNTs are inherited by daughter cells. Moreover, these nanobionic living cells retained photosynthetic activity and showed an improved photo-exoelectrogenicity when incorporated into bioelectrochemical devices.
Armbruster, M;Naskar, S;Garcia, JP;Sommer, M;Kim, E;Adam, Y;Haydon, PG;Boyden, ES;Cohen, AE;Dulla, CG.
Nature neuroscience,
25,
(5),
607-616,
(2022)
Astrocytes are glial cells that interact with neuronal synapses via their distal processes, where they remove glutamate and potassium (K+) from the extracellular space following neuronal activity. Astrocyte clearance of both glutamate and K+ is voltage dependent, but astrocyte membrane potential (Vm) is thought to be largely invariant. As a result, these voltage dependencies have not been considered relevant to astrocyte function. Using genetically encoded voltage indicators to enable the measurement of Vm at peripheral astrocyte processes (PAPs) in mice, we report large, rapid, focal and pathway-specific depolarizations in PAPs during neuronal activity. These activity-dependent astrocyte depolarizations are driven by action potential-mediated presynaptic K+ efflux and electrogenic glutamate transporters. We find that PAP depolarization inhibits astrocyte glutamate clearance during neuronal activity, enhancing neuronal activation by glutamate. This represents a novel class of subcellular astrocyte membrane dynamics and a new form of astrocyte-neuron interaction.
Torromino, G;Loffredo, V;Cavezza, D;Sonsini, G;Esposito, F;Crevenna, AH;Gioffrè, M;De Risi, M;Treves, A;Griguoli, M;De Leonibus, E.
Nature communications,
13,
(1),
4194,
(2022)
Incidental memory can be challenged by increasing either the retention delay or the memory load. The dorsal hippocampus (dHP) appears to help with both consolidation from short-term (STM) to long-term memory (LTM), and higher memory loads, but the mechanism is not fully understood. Here we find that female mice, despite having the same STM capacity of 6 objects and higher resistance to distraction in our different object recognition task (DOT), when tested over 1 h or 24 h delays appear to transfer to LTM only 4 objects, whereas male mice have an STM capacity of 6 objects in this task. In male mice the dHP shows greater activation (as measured by c-Fos expression), whereas female mice show greater activation of the ventral midline thalamus (VMT). Optogenetic inhibition of the VMT-dHP pathway during off-line memory consolidation enables 6-object LTM retention in females, while chemogenetic VMT-activation impairs it in males. Thus, removing or enhancing sub-cortical inhibitory control over the hippocampus leads to differences in incidental memory.
Antonica, F;Santomaso, L;Pernici, D;Petrucci, L;Aiello, G;Cutarelli, A;Conti, L;Romanel, A;Miele, E;Tebaldi, T;Tiberi, L.
Nature communications,
13,
(1),
4767,
(2022)
Pediatric and adult high-grade gliomas are the most common primary malignant brain tumors, with poor prognosis due to recurrence and tumor infiltration after therapy. Quiescent cells have been implicated in tumor recurrence and treatment resistance, but their direct visualization and targeting remain challenging, precluding their mechanistic study. Here, we identify a population of malignant cells expressing Prominin-1 in a non-proliferating state in pediatric high-grade glioma patients. Using a genetic tool to visualize and ablate quiescent cells in mouse brain cancer and human cancer organoids, we reveal their localization at both the core and the edge of the tumors, and we demonstrate that quiescent cells are involved in infiltration of brain cancer cells. Finally, we find that Harmine, a DYRK1A/B inhibitor, partially decreases the number of quiescent and infiltrating cancer cells. Our data point to a subpopulation of quiescent cells as partially responsible of tumor invasiveness, one of the major causes of brain cancer morbidity.
Hegazi, S;Cheng, AH;Krupp, JJ;Tasaki, T;Liu, J;Szulc, DA;Ling, HH;Rios Garcia, J;Seecharran, S;Basiri, T;Amiri, M;Anwar, Z;Ahmad, S;Nayal, K;Sonenberg, N;Liu, BH;Cheng, HM;Levine, JD;Cheng, HM.
Nature communications,
13,
(1),
1594,
(2022)
Ubiquitin ligases control the degradation of core clock proteins to govern the speed and resetting properties of the circadian pacemaker. However, few studies have addressed their potential to regulate other cellular events within clock neurons beyond clock protein turnover. Here, we report that the ubiquitin ligase, UBR4/POE, strengthens the central pacemaker by facilitating neuropeptide trafficking in clock neurons and promoting network synchrony. Ubr4-deficient mice are resistant to jetlag, whereas poe knockdown flies are prone to arrhythmicity, behaviors reflective of the reduced axonal trafficking of circadian neuropeptides. At the cellular level, Ubr4 ablation impairs the export of secreted proteins from the Golgi apparatus by reducing the expression of Coronin 7, which is required for budding of Golgi-derived transport vesicles. In summary, UBR4/POE fulfills a conserved and unexpected role in the vesicular trafficking of neuropeptides, a function that has important implications for circadian clock synchrony and circuit-level signal processing.
Maqdasy, S;Lecoutre, S;Renzi, G;Frendo-Cumbo, S;Rizo-Roca, D;Moritz, T;Juvany, M;Hodek, O;Gao, H;Couchet, M;Witting, M;Kerr, A;Bergo, MO;Choudhury, RP;Aouadi, M;Zierath, JR;Krook, A;Mejhert, N;Rydén, M.
Nature metabolism,
4,
(2),
190-202,
(2022)
The mechanisms promoting disturbed white adipocyte function in obesity remain largely unclear. Herein, we integrate white adipose tissue (WAT) metabolomic and transcriptomic data from clinical cohorts and find that the WAT phosphocreatine/creatine ratio is increased and creatine kinase-B expression and activity is decreased in the obese state. In human in vitro and murine in vivo models, we demonstrate that decreased phosphocreatine metabolism in white adipocytes alters adenosine monophosphate-activated protein kinase activity via effects on adenosine triphosphate/adenosine diphosphate levels, independently of WAT beigeing. This disturbance promotes a pro-inflammatory profile characterized, in part, by increased chemokine (C-C motif) ligand 2 (CCL2) production. These data suggest that the phosphocreatine/creatine system links cellular energy shuttling with pro-inflammatory responses in human and murine white adipocytes. Our findings provide unexpected perspectives on the mechanisms driving WAT inflammation in obesity and may present avenues to target adipocyte dysfunction.
Carvelli, A;Setti, A;Desideri, F;Galfrè, SG;Biscarini, S;Santini, T;Colantoni, A;Peruzzi, G;Marzi, MJ;Capauto, D;Di Angelantonio, S;Ballarino, M;Nicassio, F;Laneve, P;Bozzoni, I.
The EMBO journal,
e108918,
(2022)
The transition from dividing progenitors to postmitotic motor neurons (MNs) is orchestrated by a series of events, which are mainly studied at the transcriptional level by analyzing the activity of specific programming transcription factors. Here, we identify a post-transcriptional role of a MN-specific transcriptional unit (MN2) harboring a lncRNA (lncMN2-203) and two miRNAs (miR-325-3p and miR-384-5p) in this transition. Through the use of in vitro mESC differentiation and single-cell sequencing of CRISPR/Cas9 mutants, we demonstrate that lncMN2-203 affects MN differentiation by sponging miR-466i-5p and upregulating its targets, including several factors involved in neuronal differentiation and function. In parallel, miR-325-3p and miR-384-5p, co-transcribed with lncMN2-203, act by repressing proliferation-related factors. These findings indicate the functional relevance of the MN2 locus and exemplify additional layers of specificity regulation in MN differentiation.
Musardo, S;Therin, S;Pelucchi, S;D'Andrea, L;Stringhi, R;Ribeiro, A;Manca, A;Balducci, C;Pagano, J;Sala, C;Verpelli, C;Grieco, V;Edefonti, V;Forloni, G;Gardoni, F;Meli, G;Di Marino, D;Di Luca, M;Marcello, E.
Molecular therapy : the journal of the American Society of Gene Therapy,
(2022)
The development of new therapeutic avenues that target the early stages of Alzheimer’s disease (AD) is urgently necessary. A disintegrin and metalloproteinase domain 10 (ADAM10) is a sheddase that is involved in dendritic spine shaping and limits the generation of amyloid-β. ADAM10 endocytosis increases in the hippocampus of AD patients, resulting in the decreased postsynaptic localization of the enzyme. To restore this altered pathway, we developed a cell-permeable peptide (PEP3) with a strong safety profile that is able to interfere with ADAM10 endocytosis, upregulating the postsynaptic localization and activity of ADAM10. After extensive validation, experiments in a relevant animal model clarified the optimal timing of the treatment window. PEP3 administration was effective for the rescue of cognitive defects in APP/PS1 mice only if administered at an early disease stage. Increased ADAM10 activity promoted synaptic plasticity, as revealed by changes in the molecular compositions of synapses and the spine morphology. Even though further studies are required to evaluate efficacy and safety issues of long-term administration of PEP3, these results provide preclinical evidence to support the therapeutic potential of PEP3 in AD.
Sun, Q;Wang, Y;Ji, H;Sun, X;Xie, S;Chen, L;Li, S;Zeng, W;Chen, R;Tang, Q;Zuo, J;Hou, L;Hosaka, K;Lu, Y;Liu, Y;Ye, Y;Yang, Y.
Cell death & disease,
13,
(8),
724,
(2022)
Nasopharyngeal carcinoma (NPC) clinical trials show that antiangiogenic drugs (AADs) fail to achieve the expected efficacy, and combining AAD with chemoradiotherapy does not show superiority over chemoradiotherapy alone. Accumulating evidence suggests the intrinsic AAD resistance in NPC patients with poorly understood molecular mechanisms. Here, we describe NPC-specific FGF-2 expression-triggered, VEGF-independent angiogenesis as a mechanism of AAD resistance. Angiogenic factors screening between AAD-sensitive cancer type and AAD-resistant NPC showed high FGF-2 expression in NPC in both xenograft models and clinical samples. Mechanistically, the FGF-2-FGFR1-MYC axis drove endothelial cell survival and proliferation as an alternative to VEGF-VEGFR2-MYC signaling. Genetic knockdown of FGF-2 in NPC tumor cells reduced tumor angiogenesis, enhanced AAD sensitivity, and reduced pulmonary metastasis. Moreover, lenvatinib, an FDA recently approved multi-kinase inhibitor targeting both VEGFR2 and FGFR1, effectively inhibits the tumor vasculature, and exhibited robust anti-tumor effects in NPC-bearing nude mice and humanized mice compared with an agent equivalent to bevacizumab. These findings provide mechanistic insights on FGF-2 signaling in the modulation of VEGF pathway activation in the NPC microenvironment and propose an effective NPC-targeted therapy by using a clinically available drug.
Meka, DP;Kobler, O;Hong, S;Friedrich, CM;Wuesthoff, S;Henis, M;Schwanke, B;Krisp, C;Schmuelling, N;Rueter, R;Ruecker, T;Betleja, E;Cheng, T;Mahjoub, MR;Soba, P;Schlüter, H;Fornasiero, EF;Calderon de Anda, F.
Cell reports,
39,
(3),
110686,
(2022)
Microtubule (MT) modifications are critical during axon development, with stable MTs populating the axon. How these modifications are spatially coordinated is unclear. Here, via high-resolution microscopy, we show that early developing neurons have fewer somatic acetylated MTs restricted near the centrosome. At later stages, however, acetylated MTs spread out in soma and concentrate in growing axon. Live imaging in early plated neurons of the MT plus-end protein, EB3, show increased displacement and growth rate near the MTOC, suggesting local differences that might support axon selection. Moreover, F-actin disruption in early developing neurons, which show fewer somatic acetylated MTs, does not induce multiple axons, unlike later stages. Overexpression of centrosomal protein 120 (Cep120), which promotes MT acetylation/stabilization, induces multiple axons, while its knockdown downregulates proteins modulating MT dynamics and stability, hampering axon formation. Collectively, we show how centrosome-dependent MT modifications contribute to axon formation.
Duan, S;Sawyer, TW;Sontz, RA;Wieland, BA;Diaz, AF;Merchant, JL.
Cellular and molecular gastroenterology and hepatology,
(2022)
Efforts to characterize the signaling mechanisms that underlie gastroenteropancreatic neoplasms (GEP-NENs) are precluded by a lack of comprehensive models that recapitulate pathogenesis. Investigation into a potential cell-of-origin for gastrin-secreting NENs revealed a non-cell autonomous role for loss of menin in neuroendocrine cell specification, resulting in an induction of gastrin in enteric glia. Here, we investigated the hypothesis that cell autonomous Men1 inactivation in glial fibrillary acidic protein (GFAP)-expressing cells induced neuroendocrine differentiation and tumorigenesis.Transgenic GFAPΔMen1 mice were generated by conditional GFAP-directed Men1 deletion in GFAP-expressing cells. Cre specificity was confirmed using a tdTomato reporter. GFAPΔMen1 mice were evaluated for GEP-NEN development and neuroendocrine cell hyperplasia. Small interfering RNA-mediated Men1 silencing in a rat enteric glial cell line was performed in parallel.GFAPΔMen1 mice developed pancreatic NENs, in addition to pituitary prolactinomas that phenocopied the human MEN1 syndrome. GFAPΔMen1 mice exhibited gastric neuroendocrine hyperplasia that coincided with a significant loss of GFAP expression. Men1 deletion induced loss of glial-restricted progenitor lineage markers and an increase in neuroendocrine genes, suggesting a reprogramming of GFAP+ cells. Deleting Kif3a, a mediator of Hedgehog signaling, in GFAP-expressing cells attenuated neuroendocrine hyperplasia by restricting the neuroendocrine cell fate. Similar results in the pancreas were observed when Sox10 was used to delete Men1.GFAP-directed Men1 inactivation exploits glial cell plasticity in favor of neuroendocrine differentiation.
Sun, Q;Ye, Y;Gui, A;Sun, X;Xie, S;Zhan, Y;Chen, R;Yan, Y;Gu, J;Qiu, S;Liu, W;Zuo, J;Zhang, Q;Yang, L.
Cancer letters,
537,
215678,
(2022)
Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin’s lymphoma, with the combination of rituximab and chemotherapy being the standard treatment for it. Although rituximab monotherapy has a remarkable response rate, drug resistance with unclear mechanisms and lack of effective second-line therapy limit the survival benefits of patients with lymphoma. Here, we report that MORTALIN is highly expressed and correlates with resistance to rituximab-based therapy and poor survival in patients with DLBCL. Mechanistically, gain- and loss-of-function experiments revealed that the voltage-dependent anion channel 1-binding protein, MORTALIN, regulated Ca2+ release from the endoplasmic reticulum through mitochondria-associated membrane, facilitating AP1-mediated cell proliferation and YY-1-mediated downregulation of FAS in DLBCL cells. These dual mechanisms contribute to rituximab resistance. In mouse models, genetic depletion of MORTALIN markedly increased the antitumor activity of rituximab. We shed mechanistic light on MORTALIN-Ca2+-CaMKII-AP1-mediated proliferation and MORTALIN-Ca2+-CaMKII-inhibited death receptor in DLBCL, leading to rituximab resistance, and propose MORTALIN as a novel target for the treatment of DLBCL.
Hori, T;Eguchi, K;Wang, HY;Miyasaka, T;Guillaud, L;Taoufiq, Z;Mahapatra, S;Yamada, H;Takei, K;Takahashi, T.
eLife,
11,
(2022)
Elevation of soluble wild-type (WT) tau occurs in synaptic compartments in Alzheimer’s disease. We addressed whether tau elevation affects synaptic transmission at the calyx of Held in slices from mice brainstem. Whole-cell loading of WT human tau (h-tau) in presynaptic terminals at 10-20 µM caused microtubule (MT) assembly and activity-dependent rundown of excitatory neurotransmission. Capacitance measurements revealed that the primary target of WT h-tau is vesicle endocytosis. Blocking MT assembly using nocodazole prevented tau-induced impairments of endocytosis and neurotransmission. Immunofluorescence imaging analyses revealed that MT assembly by WT h-tau loading was associated with an increased MT-bound fraction of the endocytic protein dynamin. A synthetic dodecapeptide corresponding to dynamin 1-pleckstrin-homology domain inhibited MT-dynamin interaction and rescued tau-induced impairments of endocytosis and neurotransmission. We conclude that elevation of presynaptic WT tau induces de novo assembly of MTs, thereby sequestering free dynamins. As a result, endocytosis and subsequent vesicle replenishment are impaired, causing activity-dependent rundown of neurotransmission.
Lattmann, E;Deng, T;Walser, M;Widmer, P;Rexha-Lambert, C;Prasad, V;Eichhoff, O;Daube, M;Dummer, R;Levesque, MP;Hajnal, A.
PLoS biology,
20,
(2),
e3001317,
(2022)
Cell invasion is an initiating event during tumor cell metastasis and an essential process during development. A screen of C. elegans orthologs of genes overexpressed in invasive human melanoma cells has identified several components of the conserved DNA pre-replication complex (pre-RC) as positive regulators of anchor cell (AC) invasion. The pre-RC genes function cell-autonomously in the G1-arrested AC to promote invasion, independently of their role in licensing DNA replication origins in proliferating cells. While the helicase activity of the pre-RC is necessary for AC invasion, the downstream acting DNA replication initiation factors are not required. The pre-RC promotes the invasive fate by regulating the expression of extracellular matrix genes and components of the PI3K signaling pathway. Increasing PI3K pathway activity partially suppressed the AC invasion defects caused by pre-RC depletion, suggesting that the PI3K pathway is one critical pre-RC target. We propose that the pre-RC, or a part of it, acts in the postmitotic AC as a transcriptional regulator that facilitates the switch to an invasive phenotype.
Liu, J;Knopp, KA;Rackaityte, E;Wang, CY;Laurie, MT;Sunshine, S;Puschnik, AS;DeRisi, JL.
mBio,
13,
(3),
e0020522,
(2022)
Lymphocytic choriomeningitis virus (LCMV) is a well-studied mammarenavirus that can be fatal in congenital infections. However, our understanding of LCMV and its interactions with human host factors remains incomplete. Here, host determinants affecting LCMV infection were investigated through a genome-wide CRISPR knockout screen in A549 cells, a human lung adenocarcinoma line. We identified and validated a variety of novel host factors that play a functional role in LCMV infection. Among these, knockout of the sialomucin CD164, a heavily glycosylated transmembrane protein, was found to ablate infection with multiple LCMV strains but not other hemorrhagic mammarenaviruses in several cell types. Further characterization revealed a dependency of LCMV entry on the cysteine-rich domain of CD164, including an N-linked glycosylation site at residue 104 in that region. Given the documented role of LCMV with respect to transplacental human infections, CD164 expression was investigated in human placental tissue and placental cell lines. CD164 was found to be highly expressed in the cytotrophoblast cells, an initial contact site for pathogens within the placenta, and LCMV infection in placental cells was effectively blocked using a monoclonal antibody specific to the cysteine-rich domain of CD164. Together, this study identifies novel factors associated with LCMV infection of human tissues and highlights the importance of CD164, a sialomucin that previously had not been associated with viral infection. IMPORTANCE Lymphocytic choriomeningitis virus (LCMV) is a human-pathogenic mammarenavirus that can be fatal in congenital infections. Although frequently used in the study of persistent infections in the field of immunology, aspects of this virus’s life cycle remain incomplete. For example, while viral entry has been shown to depend on a cell adhesion molecule, DAG1, genetic knockout of this gene allows for residual viral infection, implying that additional receptors can mediate cell entry. The significance of our study is the identification of host factors important for successful infection, including the sialomucin CD164, which had not been previously associated with viral infection. We demonstrated that CD164 is essential for LCMV entry into human cells and can serve as a possible therapeutic target for treatment of congenital infection.
Heckman, CA;Ademuyiwa, OM;Cayer, ML.
Cell communication and signaling : CCS,
20,
(1),
130,
(2022)
For many cell types, directional locomotion depends on their maintaining filopodia at the leading edge. Filopodia lack any Ca2+-binding structural protein but respond to store-operated Ca2+ entry (SOCE).SOCE was induced by first replacing the medium with Ca2+-free salt solution with cyclopiazonic acid (CPA). This lowers Ca2+ in the ER and causes stromal interacting molecule (STIM) to be translocated to the cell surface. After this priming step, CPA was washed out, and Ca2+ influx restored by addition of extracellular Ca2+. Intracellular Ca2+ levels were measured by calcium orange fluorescence. Regulatory mechanisms were identified by pharmacological treatments. Proteins mediating SOCE were localized by immunofluorescence and analyzed after image processing.Depletion of the ER Ca2+ increased filopodia prevalence briefly, followed by a spontaneous decline that was blocked by inhibitors of endocytosis. Intracellular Ca2+ increased continuously for ~ 50 min. STIM and a transient receptor potential canonical (TRPC) protein were found in separate compartments, but an aquaporin unrelated to SOCE was present in both. STIM1- and TRPC1-bearing vesicles were trafficked on microtubules. During depletion, STIM1 migrated to the surface where it coincided with Orai in punctae, as expected. TRPC1 was partially colocalized with Vamp2, a rapidly releasable pool marker, and with phospholipases (PLCs). TRPC1 retreated to internal compartments during ER depletion. Replenishment of extracellular Ca2+ altered the STIM1 distribution, which came to resemble that of untreated cells. Vamp2 and TRPC1 underwent exocytosis and became homogeneously distributed on the cell surface. This was accompanied by an increased prevalence of filopodia, which was blocked by inhibitors of TRPC1/4/5 and endocytosis.Because the media were devoid of ligands that activate receptors during depletion and Ca2+ replenishment, we could attribute filopodia extension to SOCE. We propose that the Orai current stimulates exocytosis of TRPC-bearing vesicles, and that Ca2+ influx through TRPC inhibits PLC activity. This allows regeneration of the substrate, phosphatidylinositol 4,5 bisphosphate (PIP2), a platform for assembling proteins, e. g. Enabled and IRSp53. TRPC contact with PLC is required but is broken by TRPC dissemination. This explains how STIM1 regulates the cell’s ability to orient itself in response to attractive or repulsive cues. Video Abstract.
Bai, Q;Yang, C;Yang, M;Pei, Z;Zhou, X;Liu, J;Ji, H;Li, G;Wu, M;Qin, Y;Wang, Q;Wu, L.
Analytical chemistry,
94,
(6),
2901-2911,
(2022)
Elucidating the intrinsic relationship between mitochondrial pH (pHm) fluctuation and lipid droplets (LDs) formation is vital in cell physiology. The development of small-molecular fluorescent probes for discrimination and simultaneous visualization of pHm fluctuation toward LDs has not yet been reported. In this work, utilizing pH-driven polarity-reversible hemicyanine and rhodamine derivatives, a multifunctional fluorescent probe is developed for selectively identifying mitochondria and LDs under specific pH values via dual-emission channels. This rapid-response probe, Hcy-Rh, has two distinct chemical structures under acidic and alkaline circumstances. In acidic conditions, Hcy-Rh exhibits good hydrophilicity that can target mitochondria and display an intense red fluorescence. Conversely, the probe becomes lipophilic under weakly alkaline conditions and targets LDs, showing a strong blue emission. In this manner, Hcy-Rh can selectively label mitochondria and LDs, exhibiting red and blue fluorescence, respectively. Moreover, this ratiometric probe is applied to map pHm changes in living cells under the stimulus with FCCP, NAC, and H2O2. The interplay of LD-mitochondria under oleic acid treatment and starvation-induced autophagy has been studied using this probe at different pH values. In a word, Hcy-Rh is a potential candidate for further exploring mitochondria-LD interaction mechanisms under pHm fluctuation. Moreover, the polarity-dependent strategy is valuable for designing other functional biological probes in imaging multiple organelles.
Tamura, T;Torii, S;Kajiwara, K;Anzai, I;Fujioka, Y;Noda, K;Taguwa, S;Morioka, Y;Suzuki, R;Fauzyah, Y;Ono, C;Ohba, Y;Okada, M;Fukuhara, T;Matsuura, Y.
PLoS pathogens,
18,
(6),
e1010593,
(2022)
Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of Erns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.
Allen, SL;Seabright, AP;Quinlan, JI;Dhaliwal, A;Williams, FR;Fine, NHF;Hodson, DJ;Armstrong, MJ;Elsharkaway, AM;Greig, CA;Lai, YC;Lord, JM;Lavery, GG;Breen, L.
Cells,
11,
(7),
(2022)
Sarcopenia is a common complication affecting liver disease patients, yet the underlying mechanisms remain unclear. We aimed to elucidate the cellular mechanisms that drive sarcopenia progression using an in vitro model of liver disease. C2C12 myotubes were serum and amino acid starved for 1-h and subsequently conditioned with fasted ex vivo serum from four non-cirrhotic non-alcoholic fatty liver disease patients (NAFLD), four decompensated end-stage liver disease patients (ESLD) and four age-matched healthy controls (CON) for 4- or 24-h. After 4-h C2C12 myotubes were treated with an anabolic stimulus (5 mM leucine) for 30-min. Myotube diameter was reduced following treatment with serum from ESLD compared with CON (-45%) and NAFLD (-35%; p < 0.001 for both). A reduction in maximal mitochondrial respiration (24% and 29%, respectively), coupling efficiency (~12%) and mitophagy (~13%) was identified in myotubes conditioned with NAFLD and ESLD serum compared with CON (p < 0.05 for both). Myostatin (43%, p = 0.04) and MuRF-1 (41%, p = 0.03) protein content was elevated in myotubes treated with ESLD serum compared with CON. Here we highlight a novel, experimental platform to further probe changes in circulating markers associated with liver disease that may drive sarcopenia and develop targeted therapeutic interventions.
Vignoli, B;Canossa, M.
Cells,
11,
(9),
(2022)
Neurons release and respond to brain-derived neurotrophic factor (BDNF) with bursts of brain activity. BDNF action is known to extend to peri-synaptic astrocytes, contributing to synaptic strengthening. This implies that astrocytes have a set of dynamic responses, some of which might be secondary to activation of the tropomyosin tyrosine kinase B (TrkB) receptor. Here, we assessed the contribution of BDNF to long-term synaptic potentiation (LTP), by specifically deleting TrkB in cortical astrocytes. TrkB deletion had no effect on LTP induction, stabilization and maintenance, indicating that TrkB signaling in astrocytes is extraneous to transducing BDNF activity for synaptic strengthening.