HERV INHIBITORS FOR USE IN TREATING TAUOPATHIES
20220332799 · 2022-10-20
Inventors
- Ina Maja Vorberg (Bonn, DE)
- Shu Liu (Bonn, DE)
- Philip Denner (Bonn, DE)
- Stefan Lichtenthaler (Graefelfing, DE)
- Stephan Mueller (Munchen, DE)
Cpc classification
G01N2333/15
PHYSICS
G01N2800/2835
PHYSICS
C07K2317/76
CHEMISTRY; METALLURGY
A61P25/28
HUMAN NECESSITIES
C12N2740/10022
CHEMISTRY; METALLURGY
C12Q1/6883
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
C12Q1/6883
CHEMISTRY; METALLURGY
Abstract
The present invention relates to inhibitors of HERV proteins comprising HERV Env and/or Gag, or fragments thereof, for use in treating a tauopathy, Parkinson's disease, or ALS (Amyothrophic Lateral Sclerosis). The present invention further relates to inhibitors of receptors which bind HERV Env proteins for use in treating a tauopathy, Parkinson's disease, or ALS (Amyothrophic Lateral Sclerosis). The present invention further relates to molecules binding to HERV Env and/or Gag, or fragments thereof, or to a nucleic acid molecule encoding said HERV Env and/or Gag, or fragments thereof, for use in diagnosing a tauopathy, Parkinson's disease, or ALS.
Claims
1. Inhibitor of HERV proteins comprising HERV Env and/or Gag, or fragments thereof, for use in treating a tauopathy, or Parkinson's disease.
2. Inhibitor of claim 1, wherein said inhibitor inhibits maturation or expression of said HERV Env and/or Gag proteins, and/or binding of said HERV Env protein to a receptor.
3. Inhibitor of claim 1 or 2, wherein said inhibitor inhibits maturation of said HERV Env and/or Gag proteins, and wherein said inhibitor is a HERV protease inhibitor.
4. Inhibitor of claim 1 or 2, wherein said inhibitor inhibits expression of said HERV Env and/or Gag proteins, and wherein said inhibitor is a nucleic acid molecule hybridizing to at least a portion of the nucleic acid sequence encoding said HERV Env and/or Gag proteins, respectively.
5. Inhibitor of claim 1 or 2, wherein said inhibitor inhibits binding of said HERV Env protein to a receptor.
6. Inhibitor of claim 5, which is an anti-HERV Env protein-antibody.
7. Inhibitor of a receptor binding a HERV Env protein for use in treating tauopathy, or Parkinson's disease.
8. Inhibitor of claim 7, wherein said inhibitor inhibits maturation or expression of said receptor, and/or binding of HERV Env protein to said receptor.
9. Inhibitor of claim 7 or 8, which is a nucleic acid molecule complementary to at least a portion of the nucleic acid sequence encoding said receptor.
10. Inhibitor of claim 7 or 8, which is an antibody binding to said receptor or a fragment thereof.
11. Inhibitor of any one of claims 7 to 10, wherein said receptor is selected from the group consisting of SLC1A4 and SLC1A5.
12. Molecule binding to HERV Env protein or a fragment thereof, or to a nucleic acid molecule encoding said HERV Env protein or a fragment thereof, for use in diagnosing a tauopathy, or Parkinson's disease.
13. Molecule of claim 12, which is an anti-HERV Env protein-antibody.
14. Molecule of claim 12, which is a nucleic acid molecule binding to the nucleic acid molecule encoding HERV Env protein or a fragment thereof.
15. Inhibitor of any one of claims 1 to 11 or molecule of any one of claims 12 to 14, wherein said tauopathy is selected from the group consisting of Alzheimer's Disease (AD), Argyrophilic Grain Disease (AGD), Cortical Basal Degeneration (CBD), Progressive Supranuclear Palsy (PSP), Pick's Disease (PiD), and Frontotemporal Dementia with Parkinsonism related to chromosome 17 (FTDP-17).
Description
FIGURES
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[0218] The present invention is further illustrated by the following examples. Yet, the examples and specific embodiments described therein must not be construed as limiting the invention to such specific embodiments.
EXAMPLES
Example 1
[0219] Upregulation of Endogenous Retrovirus Increases Intercellular Protein Aggregate Induction
[0220] The prion domain NM of the Saccharomyces cerevisiae prion protein Sup35 stably expressed in the cytosol of mouse neuroblastoma N2a cells was induced to aggregate by exposure to amyloid fibrils of recombinant NM protein (Krammer et al., PNAS (2009), 106: 462-467). To study cellular mechanisms of protein aggregate spreading, subclone N2a s2E was used, selected by two rounds of limiting dilution cloning. This clone was selected due to its ability to potently induce NM aggregation in cells expressing soluble NM upon coculture and EV addition (Liu et al., mBio (2016), 7:00915-00916). Donor clone s2E was cocultured with recipient cell line N2a expressing soluble NM-GFP (NM-GFP.sup.sol) and the percentage of recipient cells with induced NM-GFP aggregates was subsequently determined by automated microscopy (
[0221] It was determined whether the highly efficient aggregate induction by coculture or by EVs from donor cells of later passage was associated with active endogenous retroviral particles present in the exosomal fraction. To this end, the reverse transcriptase (RT) activity of released particles from donor cell clone s2E (P16) was compared with N2a NM-HA.sup.agg cell clones 1C and 3B that exhibit low aggregate induction rates in recipient cells (Hofmann et al., PNAS (2013), 10: 5951-5956; Liu et al., MBio (2016), 7). Only N2a NM-HA.sup.agg clone s2E released particles with increasing RT activity upon prolonged culture (
[0222] It was determined if endogenous retroviral particles or vesicles released by the donor cells contained infectious NM seeds. To separate EVs from viral particles, an Optiprep velocity gradient previously used to separate HIV-1 virions from non-viral extracellular vesicles was employed (Dettenhofer et al., J Virol (1999), 73: 1460-1467). Western blot analyses revealed the presence of NM-HA predominately in fractions that contained exosomal marker Alix (
Example 2
[0223] ERV Gene Products are Required for Intercellular Aggregate Induction Via EVs
[0224] Experiment 1 showed that upregulated ERV Env and Gag proteins in donor cells are associated with EVs and facilitate efficient EV-mediated aggregate transmission to recipient cells. To further investigate if EV-mediated NM aggregate induction depends on the fusogenic activity of Env, anti-HIV-1 drugs were screened for their effects on NM aggregate induction in coculture, as well on EV- and fibril-mediated NM aggregate induction in recipient cells (
[0225] Neutralization experiments with antibodies targeting Env protein revealed a dose-dependent reduction of NM-GFP aggregate positive recipient cells cocultured with donor cells (
[0226] It was evaluated whether treatment with DNA methyl transferase inhibitors 5-Azacytidine (Aza) and Decitabine (Dec), capable of erasing epigenetic marks and thereby inducing ERV expression (Chiappinelli et al., Cell (2015), 162:974-986; Ramos et al., Epigenetics Chromatin (2015), 8:11) would result in increased intercellular aggregate induction efficiency. Clone s2E with low MuERVs expression (P1) was chosen for the experiment. Indeed, treatment of the cell clone s2E for three days with the epigenetic drugs and subsequent culture in the absence of the drugs for 5 days resulted in increased expression of total env and gag mRNA (
[0227] Alignment analysis showed substantial similarity of P10404 with MCF247, a polytropic MuLV (
[0228] XPR1 is a multiple-membrane spanning receptor with eight putative transmembrane domains and four extracellular loops (ECL) (Battini et al., PNAS (1999), 96: 1385-1390). Polymorphisms in ECL 3 and 4 affect the entry of certain X/P-MuLV subtypes. Analysis of XPR1 of N2a cells demonstrated that its Env recognition domain differed at 9 residues within ECL 3 and 4 from XPR1 expressed by HEK cells (
[0229] To examine this effect on Tau aggregate spreading, an N2a s2E cell clone stably propagating aggregated Tau-GFP.sup.CBD was produced (
Example 3
[0230] Fusogenic Viral Glycoproteins Drastically Increase Intercellular Transmission of Proteinaceous Seeds
[0231] The foregoing experiments demonstrated that upregulation of endogenous retroviruses drastically increased intercellular aggregate transmission via receptor-ligand interactions. It was tested whether the expression of unrelated viral glycoproteins that target specific membrane proteins on recipient cells might also be able to increase intercellular aggregate transmission and induction. The vesicular stomatitis virus glycoprotein VSV-G is routinely used to pseudotype viral particles for efficient uptake by a broad spectrum of target cells expressing the LDL receptor. Recently, VSV-G has been successfully used to pseudotype EVs for enhanced protein delivery to recipient cells (Meyer et al., Int J Nanonmed (2017), 12: 3153-3170). It was tested if ectopic VSV-G expression also increased intercellular spreading of proteinaceous seeds. The N2a NM-HA.sup.agg clone 2E (precursor clone of s2E) and HEK NM-HA.sup.agg clone C3, two cell lines that are characterized by poor NM aggregate induction rates when cocultured with recipient cells, were transfected with plasmids coding for VSV-G. The presence of VSV-G on EVs isolated from both donor cell clones (
[0232] It was then tested if viral glycoproteins could also promote spreading of pathogenic protein aggregates between cells. Thus, the effect of VSV-G expression on the intercellular spreading of transmissible spongiform encephalopathy (TSE) agents was evaluated. TSE agents, the so far only bona fide mammalian prions, are composed of misfolded cellular prion protein PrP. The conversion of cellular (PrP.sup.C), a protein tethered to the cell membrane by a glycosylphosphatidyl-anchor, into its infectious aggregated isoform (PrP.sup.Sc), occurs on the cell surface or along the endocytic pathway. It has previously been shown that N2a cells release prion infectivity associated with EVs. N2a cells persistently infected with TSE strain 22L (N2a.sup.22L) were transiently transfected with control plasmid or a plasmid coding for VSV-G. EVs were isolated from medium of transfected cells containing VSV-G (
[0233] We further tested if VSV-G expression also increased the intercellular transmission of Tau aggregates and subsequent induction of Tau aggregation in a reporter cell line. To this end, we established a Tau cell model that had been described previously by Diamond and coworkers (Sanders et al., Neuron (2014), 82:1271-1288). HEK cells were engineered to stably express the aggregation competent Tau core spanning amino acid residues 244-372 with two point mutations P301L/V337M fused to GFP (hereafter termed Tau-GFP). Cells were exposed to brain homogenates from patients who had suffered from Alzheimer's disease (AD), cortical basal degeneration (CBD), progressive supranuclear palsy (PSP) or frontotemporal lobar degeneration (FTLD). Upon limiting dilution cloning, cell clones HEK Tau-GFP.sup.AD, Tau-GFP.sup.FTLD Tau-GFP.sup.PSP and Tau-GFP.sup.CBD stably producing Tau aggregates were established (
Example 4
[0234] Human Endogenous Retrovirus Proteins Contribute to Intercellular Protein Aggregate Transmission
[0235] To examine the effect of endogenous HERVs on protein aggregate transmission, T47D human breast tumor cells which exhibit highly increased HERV-K expression upon stimulation with female steroid hormones were used (Ono et al., J Virol (1987), 61: 2059-2062). It was first tested if HERV-K proteins contribute to the intercellular transmission of the model prion NM described above. To this end, a T47D cell clone stably expressing soluble NM-GFP (T47D NM-GFP.sup.sol) was exposed to in vitro formed NM fibrils for one day. The resulting T47D NM-GFP.sup.agg bulk cell population was cocultured with recipient HEK NM-mCherry.sup.sol cells in the presence or absence of Amprenavir, shown to also be effective against HERV-K (Tyagi et al., Retrovirology (2017), 14:21) (
[0236] To examine the effect of HERV-K proteins on Tau aggregate spreading, a T47D donor cell line stably expressing Tau-GFP.sup.sol was generated. As exposure of T47D cells to brain homogenates resulted in poor Tau-GFP aggregation (less than 0.5% of recipient cells), VSV-G pseudotyped EVs derived from HEK Tau-GFP.sup.AD and Tau-GFP.sup.FTLD cells (see
[0237] Elevated Transcripts of Distinct HERV Families in Postmortem Brains from Different Tauopathy Patients
[0238] The foregoing experiments indicated that murine and human ERV proteins expressed by donor cells facilitate efficient cell-to-cell and EV-mediated spreading of proteopathic seeds from donor to recipient cells. To test if HERV env expression is upregulated in tauopathies, quantitative real-time PCR was performed using predesigned primer sets against env sequences of nine HERV family members (de Parseval et al., J Virol (2003), 77:10414-10422; Strissel et al., Oncotarget (2012), 3:1204-1219). These primer sets locate in the coding elements that detect the expression of the coding copies of the env genes. It was found that transcripts of distinct HERVs were elevated in postmortem brain samples from individuals suffering from different tauopathies (
[0239] Methods
[0240] Human Brain Samples
[0241] Frozen brain tissue samples from neuropathologically confirmed cases of AD, CBD, PSP and controls were provided by Brain Bank Tubingen.
[0242] Ethics Statement
[0243] For all the patient sample experiments, the ethical approval has been obtained from ‘Medizinische Fakultät Ethik-Kommission, Rheinische Friedrich-Wilhelms-Universität, Project no. 236/18(2018)’.
[0244] Molecular Cloning
[0245] For the expression of lentiviral constructs Tau-GFP and Tau-FusionRed, the four repeat domain 4RN1 of human Tau (amino acid residues 244 to 372) containing the mutations P301L and V337M was fused aminoterminally to GFP or FusionRed (Evrogen) with an 18-amino acid flexible linker (EFCSRRYRGPGIHRSPTA), as described previously (Woerman et al., PNAS (2016), 113:E8187-E8196). Coding regions were cloned into the lentiviral vector pRRL.sin.PPT.hCMV.Wpre via BamHI and SalI (Hofmann et al., PNAS (2013), 10: 5951-5956). Murine and human receptor XPR1 were amplified from cDNA of N2a or HEK cells, respectively. The coding region of murine XPR1 tagged aminoterminally with a hemagglutinin epitope (HA) was cloned into a PiggyBac expression vector PB510B-1 (System Biosciences) using XbaI and NotI restriction sites.
[0246] Cell Lines
[0247] N2a, Hela, L929, CAD5 and HEK293T cells are from ATCC and were cultured in Opti-MEM (Gibco) supplemented with glutamine, 10% (v/v) fetal bovine serum (FCS) (PAN-Biotech GmbH) and antibiotics. Melan-a cells are from Wellcome Trust Functional Genomics Cell Bank and were cultured in RPMI 1640 (Gibco) with 2 mM glutamine, 10% FCS, antibiotics and 200 nM 12-0-tetradecanoyl phorbol acetate PMA and incubated at 37° C. and 10% CO.sub.2. T47D cells were cultured in DMEM (Gibco) supplemented with 2 mM Glutamine and 10 (v/v) FCS. Cells were incubated at 37° C. and 5% CO.sub.2. The total numbers of viable cells and the viability of cells were determined using the Vi-VELL™XR Cell Viability Analyzer (Beckman Coulter).
[0248] Isolation of Cortical Neurons
[0249] Preparation of cortical neurons was performed using postnatal day 13 SWISS pups as described previously (Hofmann et al., PNAS (2013), 10: 5951-5956). Neurons were transduced with lentivirus 2 days post preparation on 96 well plates or Sarstedt 8 slice chambers. After 2 days, EVs were added and neurons were incubated for 2 days. Subsequently, neurons were fixed for microscopy and imaging analysis.
[0250] Production and Transduction with Lentiviral Particles
[0251] HEK293T cells were cotransfected with plasmids pRSV-Rev, pMD2.VSV-G, pMDI.g/pRRE (all plasmids were published in Dull T, Zufferey R, Kelly M, Mandel R J, Nguyen M, Trono D, Naldini L A third-generation lentivirus vector with a conditional packaging system. J Virol. 1998 November 72(11):8463-71), and pRRI.sin.PPT.hCMV.Wpre (plasmid published in Follenzi, A. and L. Naldini (2002) HIV-based vectors. Preparation and use. Methods in molecular medicine 69: 259-274) containing Tau-GFP/FusionRed. Supernatants were harvested 30 and 54 h later and concentrated using PEG according to published protocols (Follenzi et al., Methods Mol Med (2002), 69:259-274). Cell lines and primary neurons were transduced with lentivirus, and stable cell clones expressing Tau-GFP/-FusionRed were produced by limiting dilution cloning (Krammer et al., PNAS (2009), 106:462-467).
[0252] EV Isolation
[0253] To prepare EV-depleted medium, FCS was ultracentrifuged at 100,000×g for 20 h at 4° C. Medium supplemented with the EV-depleted FCS and antibiotics was subsequently filtered through 0.22 μM and a 0.1 μM filter-sterilization devices (Millipore). For EV isolation, 2-4×10.sup.6 cells were seeded in T175 flasks in 35 ml EV-depleted medium to reach confluence after 3 days. Cells and cell debris were pelleted by differential centrifugation (300×g, 10 min; 2,000×g, 20 min; 16,000×g, 30 min, 4° C.). The remaining supernatant (conditioned medium) was subjected to ultracentrifugation at 100,000×g for 1 h at 4° C. using rotors Ti45 or SW32Ti (Beckman Coulter). The pellet was rinsed in PBS and spun again using rotor SW55Ti at 100,000×g for 1 h at 4° C.
[0254] Aggregate Induction Assay
[0255] Recipient cells were cultured on CellCarrier-96 plates or 384 black microplates (PerkinElmer) at appropriate cell numbers for 1 h, and then treated with 5-10 μl of prepared samples (isolated EVs or recombinant NM fibrils). For aggregate induction by coculture, recipient and donor cells were mixed at different ratios based on the population doubling time of donor and recipient cells, and a total of 10.sup.4 cells/per well was plated. After additional incubation for 16 h or 72 h (NM or Tau, respectively), cells were fixed in 4% paraformaldehyde and nuclei were counterstained with 4 μM Hoechst for 15 min. Cells were imaged with the automated confocal microscope CellVoyager CV6000 (Yokogawa Inc.) using a 20× or 40× objective. Maximum intensity projections were generated from Z-stacks. Images from 16 fields per well were taken. On average, a total of 3-4×10.sup.4 cells per well and at least 3 wells per treatment were analyzed.
[0256] Sample Preparation for Mass Spectrometry
[0257] Cell pellets from five s2E cell culture replicates, and six replicates of EV pellets harvested from conditioned medium of s2E cells at passages 7 and 16 were collected for a quantitative proteomics analysis. Cell pellets were lysed in 150 μL SDT buffer (4% SDS (w/v), 100 mM Tris/HCl pH 7.6, 0.1 M DTT) by homogenization with a dounce tissue grinder and heated for 3 min at 95° C. Samples were sonicated 5 times for 30 s with intermediate cooling using a vialtweeter sonifier (amplitude 100%, duty cycle 50%; Hielscher, Germany). EV pellets were lysed in 100 μL STET lysis buffer (150 mM NaCl, 50 mM TrisHCl pH 7.5, 2 mM EDTA, 1% Triton X-100) on ice for 30 min with intermediate vortexing. Cell debris was removed by centrifugation at 16,000×g for 5 min. The protein concentration was determined using the colorimetric 660 nm assay (Thermo Fisher Scientific). For cell lysates, the assay solution was supplemented with the ionic detergent compatibility reagent (Thermo Fisher Scientific). A protein amount of 30 μg per sample for cell lysates and 10 μg for EV lysates was subjected to proteolytic digestion using the filter aided sample preparation (FASP) protocol (Wisniewski et al., Nat Methods (2009), 6:359-362) with 30 kDa Vivacon spin filters (Sartorius, Germany). Proteolytic peptides were desalted by stop and go extraction (STAGE) with C18 tips (Rappsilber et al., Anal Chem (2003), 75:663-670). The purified peptides were dried by vacuum centrifugation. Peptides from cell lysates and EV samples were dissolved in 40 or 20 μL of 0.1% formic acid, respectively.
[0258] LC-MS/MS Analyses
[0259] Samples were analyzed by LC-MS/MS for relative label free protein quantification. A peptide amount of approximately 1 μg per sample was separated on a nanoLC system (EASY-nLC 1000, Proxeon—part of Thermo Fisher Scientific) using in-house packed C18 columns (50 cm or 30 cm×75 μm ID, ReproSil-Pur 120 C18-AQ, 1.9 μm, Dr. Maisch GmbH, Germany) with a binary gradient of water (A) and acetonitrile (B) containing 0.1% formic acid at 50° C. column temperature and a flow rate of 250 nl/min. Peptides from cell lysates were separated on a 50 cm column using a gradient of 250 min length, whereas a 183 min gradient on a 30 cm column was used for peptides from EV samples (250 min. gradient: 0 min., 2% B; 5 min., 5% B; 185 min., 25% B; 230 min., 35% B; 250 min., 60% B; 183 min. gradient: 0 min., 2 B; 3:30 min., 5% B; 137:30 min., 25% B; 168:30 min., 35% B; 182:30 min., 60% B). The nanoLC was coupled online via a nanospray flex ion source (Proxeon—part of Thermo Fisher Scientific) equipped with a PRSO-V2 column oven (Sonation, Germany) to a Q-Exactive mass spectrometer (Thermo Fisher Scientific). Full MS spectra were acquired at a resolution of 70,000. The top 10 peptide ions were chosen for Higher-energy C-trap Dissociation (HCD) with a normalized collision energy of 25%. Fragment ion spectra were acquired at a resolution of 17,500. A dynamic exclusion of 120 s was used for peptide fragmentation.
[0260] Data Analysis and Label Free Quantification
[0261] The raw data was analyzed by the software Maxquant (maxquant.org, Max-Planck Institute Munich) version and 1.5.5.1 (Cox et al., Mol Cell Proteomics (2014), 13:2513-2526). The MS data was searched against a fasta database of Mus musculus from UniProt including also non-reviewed entries supplemented with databases of lentiviruses and murine leukemia viruses (download: Dec. 9, 2017, 52041+712+43 entries). Trypsin was defined as protease. Two missed cleavages were allowed for the database search. The option first search was used to recalibrate the peptide masses within a window of 20 ppm. For the main search, peptide and peptide fragment mass tolerances were set to 4.5 and 20 ppm, respectively. Carbamidomethylation of cysteine was defined as static modification. Acetylation of the protein N-term as well as oxidation of methionine were set as variable modifications. The false discovery rate for both peptides and proteins was adjusted to less than 1%. Label free quantification (LFQ) of proteins required at least two ratio counts of razor peptides. Only unique and razor peptides were used for quantification.
[0262] The LFQ values were log.sub.2 transformed and a two sided Student's t-test was used to evaluate statistically significant changed abundance of proteins between cell lysates from passages 16 and 7 as well as EV lysates from passages 15 and 6. A p-value less than 5% was set as significance threshold. Additionally, a permutation based false discovery rate estimation was used to account for multiple hypotheses (Tusher et al., PNAS (2001), 98:5116-5121).
[0263] OptiPrep Density Gradient
[0264] For separating EVs and virus, the discontinuous iodixanol gradient in 1.2% increments ranging from 6 to 18% were prepared as previously described (Dettenhofer et al., J Virol (1999), 73:1460-1467). The 100,000×g pellet from 1050 ml culture supernatant (30 T175 flasks) was resuspended in 1 ml PBS and overlaid onto the gradient. The gradient was subjected to high-speed centrifugation at 100,000×g for 2 h at 4° C. using a SW41Ti rotor (Beckman Coulter). 12 fractions of 1 ml each were collected from the top of the gradient, diluted with PBS in 5 ml, and centrifuged at 100,000×g for 1 h at 4° C. The pelleted fractions were resuspended in 100 μl PBS, and then used for further experiments. The reverse transcriptase activity of the viruses was measured by using a colorimetric reverse transcriptase assay (Roche).
[0265] Determination of Extracellular Vesicles Size and Number
[0266] ZetaView PMX 110-SZ-488 Nano Particle Tracking Analyzer (Particle Metrix GmbH) was used to determine the size and number of isolated extracellular vesicles. The instrument captures the movement of extracellular particles by utilizing a laser scattering microscope combined with a video camera. For each measurement, the video data is calculated by the instrument, resulting in a velocity and size distribution of the particles. For nanoparticle tracking analysis, the Brownian motion of the vesicles from each sample was followed at 22° C. with properly adjusted equal shutter and gain. At least six individual measurements of 11 subvolumes (positions) within the measurement cell and around 2200 traced particles in each measurement were detected for each sample.
[0267] Electron Microscopy (EM)
[0268] EM imaging of extracellular vesicle preparations was performed as previously described (Thery et al., Curr Protoc Cell Biol (2006), Chapter3:Unit3 22). Briefly, the 100,000×g pellets from conditioned medium were fixed in 2% paraformaldehyde, loaded on glow discharged Formvar/carbon-coated EM grids (Plano GmbH), contrasted in uranyl-oxalat (pH 7) for 5 min and embedded in uranyl-methylcellulose for 5 min. Samples were examined using a JEOL JEM-2200FS transmission electron microscope at 200 kV (JEOL).
[0269] Infectivity Assay
[0270] The infectivity assay was performed as previously described (Pothlichet et al., Int J Cancer (2006), 119:815-822). Briefly, melan-a cells were exposed to conditioned medium from different cell clones at either low or high passsages in the presence of 4 μg polybrene/ml for 24 h. The medium was then replaced with normal culture medium. After five days, cells were lysed for western blot analysis of retroviral Env and Gag proteins.
[0271] Drug Treatments
[0272] The treatment of cells with Amprenavir (10 μM; Santa Cruz) and DMSO was performed for 72 h in EV-depleted medium in T175 flasks. Afterwards, the total numbers of viable cells and the viability upon drug treatment were determined using the Vi-VELL™ XR Cell Viability Analyzer (Beckman Coulter). EVs were isolated from the conditioned medium via ultracentrifugation and processed for the aggregate induction assay as described above. NM aggregate induction by coculture of donor and recipient cells or by exposure of recipient cells to donor-derived EVs was performed in the absence of the drugs. For coculture and EV treatment of recipient Tau-FusonRed cells, donor s2E P21 or T47D cells with Tau-GFP aggregates were pretreated as above. Isolated EVs or pretreated donor cells were then incubated with recipient cells in the presence of compounds for 72 h.
[0273] To inhibit methyltransferases, s2E P1 donor cells were treated for three days with methyltransferase inhibitors 5-Azacytidine (Aza) 200 nM, Decitabine (Dec) 100 nM or DMSO as solvent control. Subsequently, the cells were cultured in the absence of the drugs for 5 days. Pre-treated donor cells were subsequently cocultured with recipient cells as described above to monitor aggregate induction efficiency in recipient N2a NM-GFP.sup.sol cells. Cell lysates of donor cells were also analyzed for MuERV Env and Gag expression levels by western blot. To increase DNA methylation, the s2E donor clone (P21) was treated with methyl group donors L-methionine (L-M) 80 mM, Betaine (B) 80 mM, Choline chloride (CC) 20 mM or medium control for 6 days. MuERV Env and Gag protein levels were analyzed by western blot. Subsequently, cells were cocultured with recipient cells for 16 h. The percentage of aggregate containing recipient cells was compared to the percentage of aggregate bearing recipients cocultured with solvent-treated donors.
[0274] Neutralization Assay
[0275] To block MuLVs Env on the surface of the donor cell clones s2E and s2E Tau-GFP.sup.CBD and on secreted EVs, mAb83A25, reactive against a broad range MuLVs (Evans et al., J Virol (1990), 64:6176-6183) was incubated with either EVs or donor cells in serial dilutions for 1 h at 37° C. with rotation at 20 rpm. Donor cells were subsequently mixed with recipient cells for 16 h. Alternatively, antibody-treated and untreated EVs were added to recipient cells for 16 h (NM) or 3 days (Tau) incubation time.
[0276] Transfection of siRNAs and Plasmids
[0277] To transiently knock-down the upregulated specific MuLV Env and Gag genes in s2E clones, custom-designed Silencer select siRNAs (Thermo Fisher Scientific) against AA037244.2 (env) and AID54952 (gag) were used. Pre-designed siRNAs against murine XPR1 and mCat-1 genes were used to knock-down genes coding for putative receptors. For transfection, 2-4×10.sup.5 cells/well were seeded on 6 well plates. The next day, 30 nM siRNA or plasmid DNA was transfected using Lipofectamine RNAiMAX or Lipofectamine2000 transfection reagent, respectively, according to the manufacturer's instructions (Thermo Fisher Scientific). After 2 days, transfected cells were harvested for aggregate induction assays and qRT-PCR, western blot analysis.
[0278] PK Treatment for Detection of PrP.sup.Sc
[0279] Cells from one well of 6 well plate were lysed in 1 ml lysis buffer. 900 μl of lysates were digested with 20 μg/ml proteinase K (PK) at 37° C. for 30 min for PrP.sup.Sc detection. Proteolysis was terminated by adding 0.5 mM Pefabloc. To make the pellet visible, 10 μl blue dextran was added to each sample and the samples were centrifuged at 20,817×g for 1 h. Proteins in 100 μl untreated lysates were precipitated with 4× methanol overnight at −20° C. and pelleted at 2,120×g for 25 min at 4° C. Untreated samples were analysed with the PK-treated pellets for total PrP and PrPSc by western blot using monoclonal anti-PrP antibody 4H11.
[0280] Sedimentation Tau Polymers
[0281] The sedimentation assay was performed as described previously (Sanders et al., Neuron (2014), 82:1271-1288). Briefly, cell pellets were lysed in lysis buffer (150 mM NaCl (w/v), 50 mM (v/v) Tris-HCl, pH7.5, 1% (v/v) NP-40, protease inhibitor) on ice for 30 min. Cleared cell lysates were separated from cell debris by centrifugation at 2650×g for 2 min at 4° C. Cleared cell lysates adjusted to 100 μg total protein were subjected to centrifugation at 100,000×g for 1 h, 4° C. Pellets were washed with 1.5 ml PBS and insoluble material was pelleted again at 100,000×g for 30 min. Proteins in the supernatant fractions were precipitated with 4× methanol overnight at −20° C. and pelleted at 2,120×g for 25 min at 4° C. (soluble fraction). The pellet (insoluble fraction) and ⅓ of the soluble fraction dissolved in RIPA buffer with 4% SDS were loaded for western blot analysis.
[0282] Pronase digestion of Tau
[0283] The resistance of Tau aggregates to pronase treatment was probed as described previously (Sanders et al., Neuron (2014), 82:1271-1288). Briefly, 18 μl cleared cell lysates or brain homogenates (containing a total protein concentration of 20-100 μg dependening on Tau aggregates content) were incubated with 2 μl 1 mg/ml pronase (Roche) at 37° C. for 1 h. Afterwards, samples were boiled in 4× sample buffer with 1% SDS final. Pronase-resistant Tau bands were detected by western blot as described below with rabbit anti-Tau ab64193 (Abcam).
[0284] Preparation of Brain Homogenates
[0285] Frozen human brain samples were homogenized in complete OptiMEM culture medium (for cell culture), QIAzol lysis reagent (for RNA isolation) or lysis buffer (PBS with 1% Triton-X and protease, phosphotase inhibitors for protein analysis) using the Precellys® 24 (Bertin Instruments) with 1.4 mm ceramic beads at 4° C. for 4 cycles 5500 rpm 20 sec. For 10 brain homogenates for aggregate induction in cell cultures, crude homogenates were cleared of cell debris at 872×g for 5 min at 4° C. Supernatants were sonicated at 50% power for 6 min and stored at −80° C. RNA was isolated using the Qiagen RNeasy Lipid Tissue Mini Kit combined with genomic DNA digestion as described in the manufacturer's instruction. For protein analysis, brain homogenates were cleared of cell debris at 15000×g for 15 min, 4° C.
[0286] Tau Aggregate Induction Using Patient-Derived Brain Homogenate
[0287] To test the Tau aggregate induction by brain homogenates from different tauopathy patients, HEK Tau-GFP.sup.sol cells were plated on a CellCarrier-96 black microplate (PerkinElmer) at 2000 cells/well in 50 μl complete medium. The next day, 6 μl 10% brain homogenate and 0.2 μl lipofectamine2000 were diluted into OptiMEM without antibiotics (final 60 μl) for 20 min at RT. Brain homogenate-liposome mixtures were added to recipient cells for 5 h and 50 μl complete medium were added to cells afterwards. The induced cells were fixed 3 days later in 4% paraformaldehyde. Nuclei were counterstained with Hoechst. Cells were imaged using the automated confocal microscope CellVoyager CV6000 (Yokogawa Inc.) and a 40× objective.
[0288] qRT-PCR
[0289] Total RNA from cell pellets or brain samples was isolated using the RNeasy Mini Kit or RNeasy Lipid Tissue Mini Kit (Qiagen). RNA concentration and quality were determined using the Agilent 2100 Bioanalyzer System. For a 20 μl reaction, 1 μg RNAs were reversely transcribed to cDNA using the iScript™ cDNA Synthesis Kit (Bio-Rad). For a 20 μl qRT-PCR reaction, 2 μl of synthesized cDNA was used as template. For qRT-PCR of murine env AA037244.2 and gag AID54952, custom designed TaqMan probes were used (Thermo Fisher Scientific). Pre-designed TaqMan probes by the company for murine pan-env, xpr1, mcat-1 and gapdh as housekeeping control and TaqMan™ Gene Expression Master Mix (Thermo Fisher Scientific) were used. qRT-PCR using TaqMan probes was performed as described in the manufacturer's instruction. For qRT-PCR analyses of HERV family members, primers were designed using the corresponding cDNA sequences (cf. SEQ ID NOs. 73-92). PowerUP SYBR™ Green Master Mix (Thermo Fisher Scientific) was mixed with different cDNAs and corresponding primers as indicated in the instruction. The fast cycling mode was used for all primers.
[0290] Western Blotting
[0291] For Western blot analysis, protein concentrations were measured using the Quick Start™ Bradford Protein assay (Bio-Rad). Proteins were separated on NuPAGE®Novex® 4-12 Bis-Tris Protein Gels (Life Technologies) followed by transfer onto a PVDF membrane (GE Healthcare). Western blot analysis was performed using rat hybridoma anti-MuERV Env mAb83A25; anti-xenotropic MuLV virus antibody ABIN457298 for detecting both Env and Gag (antibodies-online); mouse anti-MuERV Gag ab100970 (Abcam); mouse anti-Alix (1:1000; BD Bioscience); rat anti-HA 3F10 (1:1000; Roche); mouse anti-GAPDH 6C5 (1:5000; Abcam); mouse anti-Hsc/Hsp70 N27F3-4 (1:1000; ENZO); mouse anti-VSV-G A5977 (Sigma); rabbit anti-Tau ab64193 (Abcam); mouse anti-HERV K Env HERM-1811-5 (Amsbio); mouse anti-HERV K Gag HERM-1841-5 (Amsbio). The membrane was incubated with Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) according to the manufacturer's recommendations.
[0292] Image Analysis
[0293] The image analysis was performed using the CellVoyager Analysis support software. An image analysis routine was developed for single cell segmentation and aggregate identification (Yokogawa Inc.). The total number of cells was determined based on the Hoechst signal, and recipient cells were detected by their GFP/FusionRed signal. Green aggregates were identified via morphology and intensity characteristics. The percentage of recipient cells with aggregated NM-GFP or Tau-FusionRed/Tau-GFP was calculated as the number of aggregate-positive cells to total recipient cells set to 100%. False positive recipient cells were detected due to the heterogeneity of Tau-GFP/-FusionRed expression of individual cells. The mean percentage of false positives determined in control recipient cells was subtracted from all samples. Of note, negative values were sometimes obtained when no induction was observed. For data presentation, the minimum of the Y Axis was set to 0.
[0294] Immunofluorescence Staining and Confocal Microscopy Analysis of Prion-Infected Cells
[0295] Cells were fixed in 4% paraformaldehyde for 20 min at 37° C. and permeabilized in 0.1 Triton X-100 for 10 min at RT. For PrP.sup.Sc staining, proteins were denatured in 6 M guanidine hydrochloride for 10 min at RT to reduce PrP.sup.C staining and increase detection of PrP.sup.Sc (Taraboulos et al., J Cell Biol (1990), 110:2117-2132). Cells were rinsed with PBS, blocked in 0.2% gelatine for 1 h and incubated for 2 h with anti-PrP 4H11 antibody hybridoma solution diluted 1:10 in blocking solution (Ertmer et al., JBC (2004), 279:41918-41927). After three washing steps in PBS, cells were incubated for 1 h with Alexa Fluor 488-conjugated anti-Mouse IgG secondary antibody diluted 1:800 in blocking solution (Thermo Fisher Scientific) and nuclei were counterstained for 15 min with 4 μg/ml Hoechst 33342 (Molecular Probes). 96 well plate was scanned with CellVoyager CV6000 (Yokogawa Inc.). Confocal laser scanning microscopy was performed on a Zeiss LSM 800 laser-scanning microscope with Airyscan (Carl Zeiss).
[0296] Statistical Analysis
[0297] All analyses were performed using the Prism 6.0 (GraphPad Software v.7.0c). Statistical inter-group comparisons were performed using the one-way ANOVA with a Bonferroni post-test or Student's t test. p values smaller than 0.05 were considered significant. All experiments were performed in triplicates or sextuplicates and repeated at least three times. Error bars represent the standard deviation (SD).
Example 5
[0298] Downregulation of HERV-W Env Syncytin-1 Reduces Intercellular Aggregate Spreading
[0299] Human endogenous retroviruses (HERV) are usually silenced but become de-repressed during aging and in several human malignancies, including cancer, inflammatory diseases and neurodegeneration. To assess if HERV expression could affect intercellular spreading of protein aggregation, the inventors first made use of two cancer cell lines known to overexpress HERV. Human breast cell line MCF-7 was engineered to stably express Tau-GFP and exposed to AD brain homogenate to isolate clones propagating Tau-GFPAD. Cells were incubated with or without 5-Aza-2-deoxycytidine (Aza) for HERV de-repression and subsequently cocultured with recipient HEK Tau-FRsol cells (
[0300] HIV Protease Inhibitor Known to Inhibit HERV-K Maturation Reduces Intercellular Aggregate Spreading
[0301] The inventors further genetically engineered human A375 melanoma cells to express Tau-GFP and exposed them to AD brain homogenate to isolate a clone propagating Tau-GFPAD. Cells were treated with 10 μM Lopinavir, an HIV protease inhibitor shown to inhibit HERV-K protease required for HERV protein maturation. Upon coculture, the inventors observed a significant reduction of recipient cells with aggregates (
[0302] HERV Env/Receptor Interactions Contribute to the Spreading of Proteopathic Seeds
[0303] To assess if HERV Env can mediate contact between donor and recipient membranes and thereby contribute to proteopathic seed spreading, the inventors overexpressed HERV-W Syncytin-1 in two HEK cell models propagating either aggregated NM-HA (HEK NM-HAagg) or aggregated Tau-GFP (HEK Tau-GFPAD) (
[0304] Materials and Methods
[0305] Molecular Cloning
[0306] To generate the expression vector coding for SLC1A4 or SLC1A5, the corresponding cDNA for SLC1A4 (cataloge nr. #EX-A3396-Lv213; GeneCopoeia) or SLC1A5 (cataloge nr. #EX-Z2810-Lv213; GeneCopoeia) was cloned into cataloge nr. #PB510B-1 vector (SBI) under the CMV promoter. To generate the phCMV-Syncytin-1-100UTR plasmid, Syncytin-1 cDNA tagged with a Myc epitope sequence (cataloge nr. #EX-T0264-Lv213; GeneCopoeia) was cloned into phCMV-EcoENV (Addgene #15802) using EcoRI and XhoI to replace EcoENV. The 100 bp sequence from 3′-UTR of Syncytin-1 shown to enhance gene expression was amplified using primers (SEQ ID NO: 95 forward: 5′-CCGCTCGAGAGCGGTCGTCGGCCAAC-3′/ SEQ ID NO: 96 reverse: 5′-GAAGATCTCCTTCCCAGCTAGGCTTAGGG-3′) and genomic DNA from MCF-7 cells as template. The sequence was cloned into phCMV-Syncytin-1 using XhoI and BglII restriction sites. The three point mutations R314A, N315A and K316A, shown to destroy fusogenic activity, were introduced using the Q5 site-directed mutagenesis Kit (NEB).
[0307] Cell Lines
[0308] MCF-7 (ATCC HTB-22) cells were cultured in MEM (Gibco) with 10% FCS, P/S, 10 nM estrogen and 0.01 mg/ml human recombinant insulin. A375 (ATCC CRL-1619) cells were cultured in DMEM (Gibco) with 10% FCS, P/S.
[0309] Brain Homogenate Preparation and Clarification
[0310] Frozen human brain samples were homogenized in lysis buffer (for protein analysis) via Precellys® 24 (Bertin Instruments) with 1.4 mm ceramic beads at 4° C. for 4 cycles 5500 rpm 20 s. To prepare 10% (w/v) clear brain homogenate for aggregate induction, crude homogenates were centrifuged at 872×g for 5 min at 4° C., and then the supernatants were sonicated with 50% power for 6 min. These homogenates were frozen at −80° C. until use. For protein analysis, cleared supernatants were prepared by centrifugation of the crude homogenates at 15,000×g for 15 min.
[0311] Tau Aggregate Induction by Brain Homogenate and Liposomes
[0312] To induce Tau aggregation in MCF7/A375 Tau-GFPsol cells with brain homogenates from AD patients, cells were plated on 6-well plates at 1×10.sup.6 cells/well in 2 ml complete medium one day before. Next day, 200 μl 10% brain homogenates and 4 μl lipofectamine2000 were incubated for 20 min and added to recipient cells to have final 1% brain homogenates on cells. After 3 days, cells were split and further expanded for limited dilution clone selection as previously described.
[0313] Production and Transduction with Lentiviral Particles
[0314] HEK293T cells were cotransfected with plasmids pRSV-Rev, pMD2.VSV-G, pMDI.g/pRRE (all plasmids were published in Dull T, Zufferey R, Kelly M, Mandel R J, Nguyen M, Trono D, Naldini L A third-generation lentivirus vector with a conditional packaging system. J Virol. 1998 November 72(11):8463-71), and pRRI.sin.PPT.hCMV.Wpre (plasmid published in Follenzi, A. and L. Naldini (2002) HIV-based vectors. Preparation and use. Methods in molecular medicine 69: 259-274) containing Tau-GFP for fluorescence tagged Tau expression or pSIH-shRNA-Syn GGCCCTCCCTTATCATATT (SEQ ID NO: 97) with the CTTCCTGTCAGA (SEQ ID NO: 98) loop sequence to silence Syncytin-1 expression, pSIH-puro-control (Addgene #26597) was used to produce control shRNA lentivirus. Supernatants were harvested and concentrated with PEG according to published protocols. MCF-7 and A375 cell lines were transduced with Tau-GFP lentivirus to produce MCF-7 and A375 Tau-GFPsol cells. MCF-7 Tau-GFPAD clones were transduced with shRNA-Syn or control lentiviruses, and selected with 2 μg/ml puromycin for 2 weeks.
[0315] Drug Treatment
[0316] To inhibit methyltransferase, MCF-7 Tau-GFPAD cells were treated with 2 μM Aza or DMSO for 4 d. Thereafter, the pretreated donor cells were cocultured with recipient HEK Tau-FRsol in the absence of the drugs for 3 d. The treatment of A375 melanoma cells with Lopinavir (10 μM; Selleckchem) and DMSO was performed for 72 h in EV-depleted medium in T175 flasks. Afterwards, the total numbers of viable cells and the viability upon drug treatments were determined using the Vi-VELLTMXR Cell Viability Analyzer (Beckman Coulter). EV were isolated from the conditioned medium via ultracentrifugation and processed for the assays as described above.
[0317] Transfection with siRNAs or Plasmids
[0318] To transiently knock-down specific genes, custom-designed Silencer select siRNAs from Thermo Fisher were used. Pre-designed siRNAs were used to knock-down genes. For transfection, cells were pre-seeded on 6 well plate one d before at 2×10.sup.5 cells/well. The next day, either a final 60 nM (1:1 SLC1A4 (#s12914)/SLC1A5 (#s12918)) siRNAs (Lifetechnologies) was mixed with 1:20 diluted Lipofectamine RNAiMAX for siRNAs or 2 μl plasmid was mixed with 4 μl TransIT-2020 (Mirusbio) diluted in Opti-MEM for 30 min before addition to cells. After 1-3 d, transfected cells were harvested for aggregate induction assays, qRT-PCR or Western blot analysis.
[0319] qRT-PCR
[0320] Total RNAs from cell pellets were isolated using the RNeasy Mini Kit or RNeasy Lipid Tissue Mini Kit (Qiagen). RNA concentration and quality were determined with Agilent 2100 Bioanalyzer System. RNAs were reversely transcribed to cDNA using the iScript™ cDNA Synthesis Kit (Bio-Rad). For mRNA analysis, pre-designed TaqMan assays for human SLC1A4 (Hs00983079_m1), SLC1A5 (Hs01056542_m1), GAPDH (Hs02786624_g1) or ACTB (Hs01060665_g1) as housekeeping control were utilized with TaqMan™ Gene Expression Master Mix (Thermo Fisher).
[0321] Western Blotting
[0322] For Western blot analysis, protein concentrations were measured by Quick Start™ Bradford Protein assay (Bio-Rad) and proteins were separated on NuPAGE®Novex® 4-12% Bis-Tris Protein Gels (Life Technologies) followed by transfer onto a PVDF membrane (GE Healthcare) in a wet blotting chamber. Western blot analysis was performed using rabbit anti-Flotillin-1 ab133497 (Abcam); rat anti-HA 3F10 (1:1000; Roche); mouse anti-GAPDH 6C5 (1:5000; Abcam); mouse anti-Hsp70/72 N27F3-4 (1:1000; ENZO); rabbit anti-Tau ab64193 (Abcam); rat anti-c-myc-HRP 130-092-113 (Miltenyi Biotec). The membrane was incubated with Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) according to the manufacturer's recommendations.
[0323] Image Analysis
[0324] The image analysis was performed using the CellVoyager Analysis support software. An image analysis routine was developed for single cell segmentation and aggregate identification (Yokogawa Inc.) The total number of cells was determined based on the Hoechst signal, and recipient cells were detected by their GFP/-FR signal. Green aggregates were identified via morphology and intensity characteristics. The percentage of recipient cells with aggregated NM-GFP or Tau-FR/Tau-GFP was calculated as the number of aggregate-positive cells per total recipient cells set to 100%. False positive induced recipient cells were detected due to the heterogeneity in GFP/FR expression of individual cells. The mean percentage of false positives determined in control recipient cells was subtracted from all samples. Of note, negative values were sometimes obtained when no induction was observed. For data presentation, the minimum range of Y Axis was set to 0.
[0325] Statistical Analysis
[0326] All analyses were performed using the Prism 6.0 (GraphPad Software v.7.0c). Statistical inter-group comparisons were performed using the one-way ANOVA with a Bonferroni post-test or unpaired Student's t test. p values smaller than 0.03 (*), 0.002 (**) and 0.0002 (***) were considered significant. All experiments were performed in triplicates or sextuplicates and repeated at least two times. Error bars represent the standard deviation (SD).
REFERENCES
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