Albano, Camilla

Bajetto, Greta

Biolatti, Matteo

DE ANDREA, Marco

Dell’Oste, Valentina

Gugliesi, Francesca

Landolfo, Santo

Pasquero, Selina

Porporato, Paolo

Trifirò, Linda

English

Institutional Research Information System University of Turin

EXPLOITING LIPID METABOLISM BY HSV-1: A CHALLENGE TO RETHINK NEW THERAPIES FOR ALZHEIMER'S DISEASEaDepartment of Public Health and Pediatric Sciences, University of Turin, Turin, Italy; bDepartment of Translational Medicine, University of Piemonte Orientale, Novara, Italy; cCAAD Center for Translational Research on Autoimmune and Allergic Disease, Novara, Italy; dDepartment of Analytical Chemistry, Gdańsk University of Technology, Gdańsk, Poland; eDepartment of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy; fDepartment of Immunobiology, University of Arizona, Tucson, Arizona, USA.Herpes simplex virus-1 (HSV-1) establishes a life-long latent infection and can enter the brain via retrograde axonal transport. Recurrent reactivation ofHSV-1 may lead to neurodegenerative disorders, including Alzheimer’s disease (AD), although the underlying mechanisms have not been fullyelucidated yet. Lipids constitute the bulk of the brain dry mass and alteration of lipid metabolism is a key component in AD. Considering that themechanisms for remodeling of metabolism by HSV-1 are still poorly understood, we aim at dissecting the host metabolic pathways modulated byinfection in a neuronal-like cell line to identify pathways that might be targeted to prevent AD. Specifically, we found an increase in both de novosynthesis and lipid storage following HSV-1 infection. In addition, anti-AD compounds targeting lipid metabolism (e.g. CMS121, C75) impaired HSV-1infectivity. Overall, our data unveil new aspects of HSV-1-AD interplay and uncover new potential targets to rethink new possible therapies.Camilla Albanoa, Selina Pasqueroa, Linda Trifiròa, Francesca Gugliesia, Gloria Griffanteb, Greta Bajettoa,c, Weronika Hewelt-Belkad, Erika Minae, Paolo Porporatoe, Dana M. Cairnsf, David L. Kaplanf, Adam Mullisf, Santo Landolfoa, Marco De Andreaa,d, Valentina Dell’Ostea, Matteo BiolattiaSH-SY5Y cells were infected with HSV-1 (MOI 1). (A) At 8, 24, and 32 hpi, totalRNA was isolated and subjected to RT-qPCR to measure mRNA expression levelsof the lipogenic enzymes ATP citrate lyase (ACL), Acetyl-CoA carboxylase (ACC1),and Fatty acid synthase (FAS). Values were normalized to the housekeepingglyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene mRNA and plotted asfold induction relative to mock-infected cells (set at 1). The results wereexpressed as the mean ± standard error of the mean (SEM) of three independentexperiments (*** P <0.001; ** P <0.01; *, P <0.05; unpaired t test) for thecomparison between mock- and infected cells. (B) Western blot analysis ofprotein lysates from mock- or infected cells using the indicated antibodies. Arepresentative blot is shown. (C) SH-SY5Y cells were infected with HSV-1 or HSV-1 UV (MOI 1). Western blot analysis of protein lysates from mock- or infectedcells using the indicated antibodies. A representative blot is shown.SH-SY5Y cells were treated with the anti-AD compounds (5 μM) CMS121 (upper panel) or C75 (lower panel) for two hours and then infectedwith HSV-1 (MOI 1). (A, E) At 48 hpi, the number of HSV-1 genomes released into the supernatants of the infected cells was measured byqPCR (bars show means and SEM from three independent experiments (* P <0.05; t-test unpaired). (B, F) The supernatants used for panel Aor E were used to determine the number of infectious units/mL by the viral yield test. The bars represent the means and the SEM of threeindependent experiments (* P <0.05; t-test unpaired). (C, G) The genome-to-infectious unit ratio has been determined and the values areabove the bars (* P <0.05, t-test unpaired). (D, H) SH-SY5Y cells were infected with HSV-1 (MOI 1) and treated with CMS121 or C75. At thetimes indicated, cells were harvested and subjected to Western blot analysis using the indicated antibodies. A representative blot is shown.ABSTRACTRESULTSBACKGROUNDSH-SY5Y cells were infected with HSV-1 (MOI 1). At 8, 24, and 32 hpi, totalRNA was isolated and subjected to RT-qPCR to measure mRNA expressionlevels of the AD-related genes: TAU, presenllin-1 (PS1), presenllin-2 (PS2), β-site APP cleaving enzyme 1 (BACE1), Hexokinase 1 (HK1), Hexokinase 2 (HK2),glycogen synthase kinase 3α (GSK-3α), glycogen synthase kinase 3β (GSK-3β).Values were normalized to the housekeeping glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene mRNA and plotted as fold induction relative tomock-infected cells (set at 1). The results were expressed as the mean ±standard error of the mean (SEM) of three independent experiments (*** P<0.001; ** P <0.01; *, P <0.05; unpaired t test) for the comparison betweenmock- and infected cells.Valentina Dell’OsteUniversity of TurinDept. of Public Health and Pediatric SciencesVia Santena, 9 – 10126 Turin (ITALY)valentina.delloste@unito.it[Laval K & Enquist LW Front Neurol 2021]AIM OF THE PROJECT(4) Priming of the brain to a similar inflammatory state following transduction ofinflammatory stimuli from the PNS. HSV-1 reactivation or new primary infection of thePNS will thus enhance inflammatory responses in the brain. These events are likely totrigger the production of APP and tau proteins in the brain and repeated inflammationwill lead to the accumulation of these proteins and the development of Aβ plaques thatcause AD.HSV-1 as a trigger for ADviplab.unito.itfacebook.com/viplabtorinotwitter.com/VIPLab6instagram.com/vip_lab_toModulation of AD-related genes by HSV-1AB CACL ACC1 FASCMS121 and C75 reduce the infectivity of HSV-1 viral particlesA B C DCMS121E F G HC75TAU PS1 PS2 BACE1GSK-3βHK1 HK2 GSK-3αSH-SY5Y cells were infected with HSV-1 (MOI 1). (A) At 8, 24, and 32 hpi, total RNA wasisolated and subjected to RT-qPCR to measure mRNA expression levels of enzymesinvolved in lipid accumulation: diacylglycerol O-acyltransferase 1 (DGAT1), diacylglycerolO-acyltransferase 1 (DGAT2), sterol O-acyl transferase 1 (SOAT1). Values were normalizedto the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene mRNAand plotted as fold induction relative to mock-infected cells (set at 1). The results wereexpressed as the mean ± standard error of the mean (SEM) of three independentexperiments (*** P <0.001; ** P <0.01; *, P <0.05; unpaired t test) for the comparisonbetween mock- and infected cells. (B) SH-SY5Y cells were infected with HSV-1 (MOI 1) for48 h. All cells were stained BioTracker 488 Green Lipid Droplets Dye to visualise LDs (green)and DAPI to visualise the cell nuclei (blue). LD numbers were analysed using ImageJanalysis software. The results were expressed as the mean ± standard error of the mean(SEM) of three independent experiments (*** P <0.001; unpaired t test) for thecomparison between mock- and infected cells.C75Lipidomic comparison of SH-SY5Y cells infected with HSV-1. (A) SH-SY5Ycells were infected with HSV-1 or left uninfected (MOCK) or treated withthe indicated compounds or vehicle (DMSO), and then infected with HSV-1(MOI of 1) and maintained in serum-free medium. At 24 or 48 hpi, totallipids from cells were extracted and analyzed by LC-MS/MS. The foldchanges of lipid species peak area are visualized as a heat map showing thelevels of upregulated (red, fold change > 1) and downregulated (blue, foldchange < 1) glycerophospholipids. PC, glycerophosphocholine. n = 2independent determinations. (B) Histogram reporting the total MS signalfor PC in the different conditions.AB HSV-1 infection increases de novo fatty acid synthesisthrough transactivation of lipogenic enzymes. HSV-1 infection increased lipid storage. The anti-AD compounds CMS121 and C75 act inhibitHSV-1 replication. Some genes involved in AD are deregulated followingHSV-1 infection.(1) HSV-1 replicates in the oralmucosa epithelial cells; (2) HSV-1initiates a productive infection in TGneurons; (3) Synaptic transmissionof the inflammatory responseaccompanied by the release ofneuropeptides, neurotransmittersand the propagation of specificaction potentials from the TG to themain sensory nucleus located in thebrainstem;CONTACTSCONCLUSIONS[Vastag L et al Plos Path 2011]Effects of HCMV and HSV-1 on central carbon metabolismSummary of major metabolite concentration and fluxchanges in response to HSV-1 infection of growth arrestedfibroblasts. Arrow colors denote flux changes and fontcolors denote metabolite level changes relative to themock-treated control (red-increased, green-decreased,grey-not detected).ABDGAT1 DGAT2 SOAT1HSV-1 triggers lipid accumulationHSV-1 induced lipogenesis24 hpi48 hpiDMEM Cytochalasin BBPTES11 0 411 0 511 0 611 0 711 0 811 0 9PFU/mLSH-SY5Y cells were infectedwith HSV-1 (MOI 1) andtreated with a non-toxicconcentration of CytochalasinB or BPTES (5 μM). The extentof HSV-1 replication wasassessed by titrating theinfectivity of supernatantsand cell-associated viruses bystandard plaque assay. Thebars show means and SEMsfrom three independentexperiments (unpaired t-test).Glucose is the main source of HSV-1 induced lipogenesis[Amin Sa et J Mole Struct 2017][Tilekar K et al Biochim Biophys Acta 2020]HSV-1 induces lipogenic enzymesLIPID NAME FC FCEtherPC_30:0 1,08 1,18EtherPC_30:1 iso 1 1,24 1,01EtherPC_30:1 iso 2 1,04 1,00EtherPC_32:0 1,12 1,41EtherPC_34:0 1,23 1,52EtherPC_34:1 1,06 1,06EtherPC_34:2 iso 1 0,92 1,10EtherPC_34:2 iso 2 1,18 1,09EtherPC_36:1 1,28 1,62EtherPC_36:2 iso 1 1,08 1,13EtherPC_36:2 iso 2 1,12 1,17EtherPC_36:3 iso 1 1,06 1,19EtherPC_36:3 iso 2 1,13 1,08EtherPC_38:2 1,12 1,32EtherPC_38:3 1,02 1,23EtherPC_38:4 iso 1 1,15 1,12EtherPC_38:4 iso 2 1,02 1,14EtherPC_38:5 1,06 1,20EtherPC_40:3 1,20 1,57EtherPC_40:4 iso 1 0,95 1,00EtherPC_40:4 iso 2 1,03 1,19EtherPC_40:4 iso 3 0,90 1,03LPC_18:1 0,54 1,07PC_28:0 0,91 0,71PC_28:1 iso 2 0,84 0,36PC_30:0_14:0_16:0 1,00 1,00PC_30:1 iso 3 1,03 0,89PC_31:0_15:0_16:0 1,07 1,07PC_32:0_16:0_16:0 0,92 1,15PC_32:1 iso 1 0,94 0,71PC_32:1 iso 2 1,14 0,94PC_32:1 iso 3 1,16 1,11PC_32:2 iso 3 0,91 0,76PC_33:0_16:0_17:0 1,03 1,28PC_33:1 iso 1 1,11 0,99PC_33:1 iso 2 1,10 0,96PC_34:1_16:0_18:1 0,92 0,94PC_34:3 iso 1 0,80 0,62PC_34:3 iso 2 0,73 0,52PC_35:2_iso 1 1,01 0,93PC_35:2_iso 2 1,06 0,92PC_36:1_18:0_18:1 1,23 1,26PC_36:2 1,01 0,89PC_36:3 iso 1 0,91 0,69PC_36:3 iso 2 1,02 0,77PC_36:4 1,16 1,06PC_38:1 1,16 1,31PC_38:2 1,13 1,07PC_38:3 iso 1 0,98 0,84PC_38:3 iso 2 1,05 0,84PC_38:3 iso 3 1,21 1,16PC_38:4 iso 1 0,81 0,59PC_38:4 iso 2 1,15 1,14PC_38:5 0,98 0,87PC_38:6 1,09 0,91PC_40:3 1,16 1,28PC_40:4 iso 1 0,84 0,70PC_40:4 iso 2 1,09 1,07PC_40:5 iso 1 0,85 0,74PC_40:5 iso 2 1,09 1,02PC_40:6_18:1_22:5 0,94 0,84PC_40:7_18:1_22:6 1,01 0,89PC_42:4 0,86 0,7624h 48h 24h 48h 24h 48hHSV-1/MOCK CMS121/DMSOFC FC1,37 1,351,19 1,251,01 1,150,99 0,991,18 1,191,12 1,230,87 1,020,90 1,031,61 1,650,84 0,980,87 1,000,96 0,840,85 0,921,12 1,110,95 0,910,82 0,900,98 0,970,97 0,951,12 1,110,86 0,911,03 1,020,59 0,692,02 1,251,03 1,060,70 0,870,90 0,950,80 0,920,80 0,960,78 0,861,02 1,100,75 0,840,80 0,920,78 0,840,94 1,050,97 1,100,79 0,911,11 1,100,62 0,660,76 0,801,01 1,060,85 0,761,09 1,321,00 1,100,81 0,870,70 0,780,78 0,551,04 1,200,91 1,080,88 1,000,64 0,720,75 0,870,64 0,740,69 0,790,69 0,730,62 0,620,69 0,780,60 0,700,66 0,780,63 0,700,66 0,710,62 0,720,58 0,620,60 0,75FC FC1,27 1,081,89 1,450,81 0,771,02 1,050,91 1,211,07 0,951,26 0,890,64 0,590,85 0,771,09 0,940,66 0,591,47 1,310,69 0,590,97 0,931,49 1,421,31 1,391,05 0,971,38 1,241,21 1,271,61 1,441,22 1,170,92 0,891,26 0,831,17 1,321,90 1,970,99 1,111,32 1,310,98 1,081,02 1,170,99 1,100,93 0,881,00 1,001,52 1,651,24 1,151,02 1,121,06 1,201,07 1,031,34 1,311,59 1,881,04 1,111,03 1,060,78 0,790,91 0,871,18 1,120,91 0,940,95 0,860,69 0,690,68 0,710,87 0,870,91 0,971,05 1,151,12 1,030,82 0,870,86 0,800,81 0,900,93 0,980,96 1,010,80 0,870,99 1,030,86 0,930,85 0,920,86 0,810,81 0,85C75/DMSOMOCK DMSO CMS121Total lipidsHSV-1

Exploiting lipid metabolism by HSV-1: a challenge to rethink new therapies for Alzheimer's disease.

https://iris.unito.it/bitstream/2318/1897332/1/Poster%20Dell%27Oste%20Spagna%202023.pdf

Exploiting lipid metabolism by HSV-1: a challenge to rethink new therapies for Alzheimer's disease.

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