LIGANDS BINDING TO PRION PROTEIN FOR USE IN THE TREATMENT OF SYNUCLEINOPATHIES
20200277390 ยท 2020-09-03
Inventors
Cpc classification
A61K9/0019
HUMAN NECESSITIES
A61K39/3955
HUMAN NECESSITIES
A61K9/0053
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
International classification
C07K16/28
CHEMISTRY; METALLURGY
A61K39/395
HUMAN NECESSITIES
A61P25/28
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
Abstract
The present invention provides ligands capable of binding to prion protein, such as anti-prion protein antibodies and antigen-binding fragment thereof, for the prevention and/or treatment of synucleinopathies, such as Parkinson's disease. The present invention also provides pharmaceutical compositions comprising such ligands and methods for treating synucleinopathies or for reducing the uptake of -synuclein fibrils.
Claims
1.-31. (canceled)
32. A method of preventing and/or treating a synucleinopathy in a subject, comprising: administering a ligand capable of binding to prion protein (PrP) to the subject.
33. The method according to claim 32, wherein the PrP is cellular prion protein (PrP.sup.C).
34. The method according to claim 32, wherein the ligand is capable of binding to the N-terminal part and/or to the C-terminal part of the prion protein.
35. The method according to claim 32, wherein the ligand does not bind to the charged cluster 2 region of the prion protein and does not bind to the helix 1 region of the prion protein.
36. The method according to claim 32, wherein the ligand is capable of binding to the octapeptide repeat region of the prion protein.
37. The method according to claim 32, wherein the ligand is capable of binding to two distinct epitopes of the prion protein.
38. The method according to claim 37, wherein the prion protein is cellular prion protein and wherein the ligand is capable of binding to an epitope in the N-terminal part of the cellular prion protein and to an epitope in the C-terminal part of the cellular prion protein.
39. The method according to claim 32, wherein the ligand is an anti-prion protein antibody, or an antigen-binding fragment thereof.
40. The method according to claim 39, wherein the ligand is a multispecific antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, or a monoclonal antibody or antigen binding fragment thereof.
41. The method according to claim 39, wherein the ligand is a human antibody or antigen-binding fragment thereof, or a humanized antibody or antigen-binding fragment thereof.
42. The method according to claim 32, wherein the synucleinopathy is selected from Parkinson's disease, dementia with Lewy bodies, and multiple systems atrophy.
43. The method according to claim 32, wherein the ligand is administered intravenously or intramuscularly.
44. The method of claim 32, further comprising: administration of an antiparkinson medication to the subject.
45. The method according to claim 44, wherein the ligand is administered intravenously or intramuscularly and the antiparkinson medication is administered orally.
46. The method according to claim 44, wherein the antiparkinson medication is selected from the group consisting of: dopaminergic precursors, COMT inhibitors, peripheral aromatic L-amino acid decarboxylase inhibitors, selective monoamine oxidase B inhibitors, dopamine receptor agonists, anticholinergics, positive allosteric modulators of mGluR4, and anti--synuclein antibodies.
47. The method according to claim 46, wherein the antiparkinson medication is a dopaminergic precursor, such as levodopa (L-DOPA).
48. A conjugate, comprising: the ligand as defined in claim 32; and an agent facilitating passage of the ligand across a blood-brain barrier of a subject having or at risk of having a synucleinopathy, the ligand conjugated to the agent.
49. A nucleic acid molecule, comprising: a polynucleotide encoding the ligand as defined in claim 32, wherein the ligand is a peptide or polypeptide, such as an anti-prion protein antibody or an antigen-binding fragment thereof.
50. A pharmaceutical composition, comprising: the ligand as defined in claim 32; and a pharmaceutically acceptable carrier, diluent and/or excipient.
51. A method for reducing uptake of -synuclein fibrils, comprising: administering to a subject the ligand as defined in claim 32.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0236] In the following a brief description of the appended figures will be given. The figures are intended to illustrate the present invention in more detail. However, they are not intended to limit the subject matter of the invention in any way.
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EXAMPLES
[0252] In the following, particular examples illustrating various embodiments and aspects of the invention are presented. However, the present invention shall not to be limited in scope by the specific embodiments described herein. The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. The present invention, however, is not limited in scope by the exemplified embodiments, which are intended as illustrations of single aspects of the invention only, and methods which are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become readily apparent to those skilled in the art from the foregoing description, accompanying figures and the examples below. All such modifications fall within the scope of the appended claims.
Example 1: Expression, Purification and Characterization of Recombinant -Syn Proteins
[0253] First, highly pure rec human and mouse -Syn protein were produced and subjected to the fibrillation process.
[0254] To this end, recombinant -Synuclein (-Syn) protein was purified from Escherichia coli BL21 (DE3) cells expressing mouse -Syn construct from the pET11a expression vector. E. coli cells were grown in minimal medium at 37 C. in the presence of ampicillin (100 g/mL) until OD600 of about 0.6, followed by induction with 0.6 mM IPTG for 5 hours. The protein was extracted from periplasm by osmotic shock as previously described (Huang C, Ren G, Zhou H, Wang C C. A new method for purification of recombinant human alpha-synuclein in Escherichia coli. Protein Expr Purif. 2005 July; 42(1):173-7), followed by boiling for 20 min and ammonium sulfate precipitation. The protein was next purified by anion exchange chromatography (HiTrap Q FF column, GE Healthcare) and fractions were analyzed by SDS-PAGE. Finally, the protein was dialyzed against water, lyophilized and stored at 80 C.
[0255] Prior to fibrillation, the protein was filtered with 0.22 m syringe filter and the concentration was determined by absorbance measured at 280 nm. Purified mouse -Syn (1.5 mg/mL) was incubated in the presence of 100 mM NaCl, 20 mM Tris-HCl pH 7.4 and 10 M ThioflavinT (ThT). Reactions were performed in black 96-well plates with a clear bottom (Perkin Elmer), in the presence of one 3-mm glass bead (Sigma) in a final reaction volume of 200 L. Plates were sealed and incubated in BMG FLUOstar Omega plate reader at 37 C. with cycles of 50 sec of shaking (400 rpm, double-orbital) and 10 sec of rest. ThT fluorescence measurements (excitation: 450 nm, emission 480 nm, bottom read) were taken every 15 min.
[0256] Obtained -Syn proteins and fibrils were analyzed and characterized as shown in
[0257] Finally, the amyloids were structurally characterized by atomic force microscopy (AFM). Atomic Force Microscopy (AFM) was used to acquire high resolution three-dimensional reconstructions of -Syn preparations. All AFM images were acquired using a commercially available microscope (Solver Pro AFM from NTMDTNT-MDT Co.MoscowRussia) endowed with a closed-loop scanner. Measurements were carried out in air at room temperature working in dynamic mode. Cantilevers, characterized by a resonant frequency of about 90 kHz and a force constant of about 1.74 nN/nm (NSG03 series from NT-MDTNT-MDT Co.MoscowRussia) were used working at low oscillation amplitudes with half free-amplitude set-point. High resolution images of 1010 m2 were 512512 pixels frames acquired at 1 lines/second scan speed. All AFM data were analysed using Gwyddion, free SPM data analysis software (D. Neas, P. Klapetek (2011), Gwyddion: an open-source software for SPM data analysis. Central European Journal of Physics 10, 181-188). Fibril length was evaluated as linear fibril's end-to-end distance and analyzed using commercial data analysis software (Igor Pro, Wavemetrics, US). Samples for AFM imaging were prepared by drop deposition of fibril solution on a ultra-flat mica surface. Briefly, 20 L of solution were spotted onto a freshly cleaved piece of Goodfellow mica (88 mm2 side size) and left to adhere for 20 minutes. Subsequently a 60 L drop of ethanol was placed on the sample to induce fibril precipitation for 5 minutes. Sample was thereafter blow-dried under a flow of nitrogen.
[0258] Results are shown in
Example 2: -Syn Uptake is Facilitated in Cells Expressing PrP.SUP.C
[0259] To assess whether PrP.sup.C expression may facilitate -Syn amyloid entrance in cells first an in vitro approach was used. To this end, N2a PrP.sup.+/+ and N2a PrP.sup./ neuroblastoma (N2a) cells were incubated with recombinant (rec) mouse -Syn amyloid fibrils for 24 h. Uptake of -Syn amyloid was compared in N2a PrP.sup.+/+ and N2a PrP.sup./ cells, i.e. in N2a cells that constitutively express PrP and in the same cell line ablated for PrP.sup.C (using CRISPR-Cas9-Based Knockout system) (M. Mehrabian et al. (2014), CRISPR-Cas9-based knockout of the prion protein and its effect on the proteome. PloS one 9, e114594).
[0260] To investigate whether mouse -Syn fibrils are internalized by N2a cells confocal microscopy was used and the percentage of N2a cells that were able to take-up the amyloids were quantitatively analyzed. Briefly, cells were cultured on coverslips and treated with -Syn fibrils (2 M), afterwards the cells were fixed with 4% formaldehyde in PBS for 30 min. Cells were then washed three times with PBS (1) followed by blocking in 5% Normal Goat Serum (NGS, ab7481, Abcam)/0.3% Triton X-100 for 1 h, at room temperature (R.T.). Cells were incubated with primary antibodies diluted in 1% of blocking buffer (anti-PrP Ab W226, 1:500, anti--Syn Ab C-20-R, Santa Cruz, 1:1,000), followed by three washings with PBS and secondary antibody incubation (goat anti-mouse Alexa488, and goat anti-rabbit Alexa594, Life Technologies). To ensure that -Syn preparations were within the cell cytoplasm a specific dye that labels the entire cell was used (HCS CellMask dye). Cells were mounted in Aqua Poly/Mount (Polysciences), and images were acquired using Leica confocal microscope (Leica TCS SP2, Wetzlar, Germany). The uptake quantification was performed in blind using Oil Immersion 63 objective on more than 200 cells per one single independent experiment (in total of N=3). Random fields per coverslips at 63 magnification were captured using Leica confocal microscope (Leica TCS SP2, Wetzlar, Germany). To observe internalized -Syn fibrils, the coverslips were double-labeled with anti--Syn antibody and whole cytoplasmic dye CellMask. Cells considered -Syn positive were those in which the aggregates were found in perinuclear zone. The images were acquired as 20-30 z-stacks of 0.22 m, 10241024, and analyzed using Orthogonal Views function in Image J (NIH). Data are represented as % of total cell counted in three independent experiments.
[0261] Results are shown in
[0262] N2a PrP.sup./ neuroblastoma (N2a) cells were transfected with full-length PrP (N2aPrPFL). As shown, in
[0263] Next, the effect of mouse -Syn preparations on endogenous PrP.sup.C and transfected PrPFL protein levels was determined in N2a cells. To this end, cell lysates of N2a PrP.sup.+/+ cells and of transfected N2aPrPFL cells were prepared and analyzed by SDS-PAGE and Western blot, using W226 anti-PrP antibody. Briefly, after 4 days of treatment with different -Syn preparations, medium was removed and the cells were washed twice with PBS 1 and lysed in lysis buffer. Total protein content of cell lysates was measured using bicinchoninic acid protein (BCA) quantification kit (Pierce) and stored at 20 C. until analysis. The total of 30 g/mL of cell lysates were resuspended in Laemmli loading loading buffer, and boiled for 10 min at 95 C. Subsequently the samples were loaded onto a 12% Tris-Glycine SDS-PAGE gel, and transferred onto nitrocellulose membrane (GE Healthcare), blocked using 5% non-fat milk (w/v) blocking solution for 1 h at room temperature with agitation followed by incubation with anti-PrP antibodies (W226, 1:1000; SAF43, 1:1000) or anti 3-actin (1:50000, A3854 Sigma-Aldrich) diluted in blocking solution. Membranes were washed with TBST (0.1% Tween 20 in TBS), and incubated in horseradish-peroxidaseconjugated (HRP) goat anti-mouse secondary Ab (diluted 1:2000) for 1 h. The membranes were washed in TBST and proteins were visualized following the manufacturer's instructions using Amersham ECL Western Blotting Detection Reagent (GE Healthcare) with UVITEC Cambridge. Quantitative densitometry analysis of proteins was performed using NIH Image software (Image 1.50a, USA).
[0264] Results are shown in
[0265] RT-PCR was performed to investigate whether or not the slight increase of protein levels were due to the mRNA increase. Total RNA extraction was performed using a ready-to-use TRIzol Reagent (Invitrogen) following the Manufacture's instruction. Briefly, the medium was removed from plates with control cells and those treated with different -syn preparations and the cells were washed twice with PBS 1. Subsequently, the cells were lysed using the TRIzol Reagent. Following RNA isolation, a DNase I digestion was performed using 1 unit of enzyme per g RNA for 10 min at room temperature, and RNA cleanup was implemented using RNeasy spin columns following the instructions. RNA concentration was determined using the NanoDrop system (Thermo Scientific). First-strand cDNA was synthesized using 4 g of total RNA in a 20 L reverse transcriptase reaction (RT+samples) mixture following the instructor manual. For each sample a negative control was carried along by omission of the reverse transcriptase (RT-control). The cDNA was diluted to 1 ng/L final concentration prior to Real-Time PCR reactions. Two ng RNA equivalent was added to the reaction mix including 2iQ SYBR Green Supermix (Bio-Rad Laboratories, Inc.), 400 nM of the corresponding forward and reverse primer (Sigma), and quantified in technical triplicates on an iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.). After initial denaturation for 3 min at 95 C., 45 cycles were performed at 95 C. for 10 sec and 60 C. for 1 min. Differential gene expression was normalized to GAPDH and ACTB expression. RT-controls were included in the plates for each primer pair and sample. The relative expression ratio was calculated using the CT method. Significance was calculated with the unpaired Student's t-test (p<0.05). The primers for Prnp-FW: 5-GAGACCGATGTGAAGATGATGGA-3 (SEQ ID NO: 5) and RV: 5-TAATAGGCCTGGGACTCCTTCTG-3 (SEQ ID NO: 6), ACTB-FW: 5-GTTGCGTTACACCCTTTCTTG-3 (SEQ ID NO: 7), GAPDH-FW: 5-CCTGCACCACCAACTGCTTA-3 (SEQ ID NO: 8).
[0266] Results are shown in
[0267] Next, the viability of the cell lines after exposure to exogenous -Syn amyloids for 24 hours was tested. Cell viability was determined by 3-(4,5-dimethyl-2-thizolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma Aldrich) assay following the manufacturer's instructions after 24 h treatment with -Syn amyloids. Thirty thousand cells/well were treated with 2 M (30 g/mL) of -syn fibrils in Costar 96-Well Plates (Thermo Fisher Scientific Inc.). Three independent experiments were performed in six technical replicas per condition (treated or un-treated). Water-insoluble colored formazan derivative was solubilized in DMSO:isopropanol (1:1). Absorbance of converted dye was measured at a wavelength of 570 nm. Viability was assessed in terms of % of control (untreated cells).
[0268] Results are shown in
[0269] Cellular localization of -Syn fibrils was investigated by confocal quadruple-labelled immunofluorescent imaging. Quadruple staining was carried out using 4% paraformaldehyde fixed cells. Nonspecific protein interactions were blocked with 10% normal goat serum (Sigma) and 0.3% Triton-X100 and incubated with the primary antibodies (D18 for PrPC, C-20-R for -Syn, from Santa Cruz, EEA1-endosomal, Calenexin-endoplasmatic reticulum, Lamp1-lysosomal and M6PR-Golgi markers, from Abcam), in a humidified chamber at 4 C. overnight. Following washes in PBS the cells were incubated with secondary antibodies conjugated to biotin (1:500, ThermoFisher) followed by incubation with Alexa Fluor 647 Streptavidin conjugate (1:500, ThermoFisher). Coverslips were mounted in Aqua Poly/Mount (Polysciences), and images were acquired using C1 Nikon confocal microscope. Hippocampal neurons grown for 6 days in vitro (DIV), were fixed with 4% paraformaldehyde/PBS and immuno-stained with monoclonal MAP-2 antibody (Abcam), anti -Syn antibody (C-20-R, Santa Cruz). Followed by the secondary antibody incubation (goat anti-mouse Alexa488, and goat anti-rabbit Alexa594, Life Technologies) and HCS CellMask dye (Thermo Fisher Scientific). Cells were mounted in Aqua Poly/Mount (Polysciences), and images were acquired using C1 Nikon confocal microscope.
[0270] As shown in
[0271] Next, -Syn amyloid internalization in primary cultures of hippocampal neurons was investigated. To this end, both, PrP wild-type and knock out mice (FVB Prnp+/+ and Pmp/) were used. Briefly, hippocampi were dissected from 0-1-day-old postnatal animals. The isolated tissue was quickly sliced and digested in a digestion solution containing Trypsin (Sigma-Aldrich) and DNAse (Sigma-Aldrich). The reaction was stopped with Trypsin inhibitor (Sigma-Aldrich) and cells were mechanically dissociated in a dissection medium containing DNAse. After centrifugation, the cell pellet was resuspended in the culture medium and distributed in a 12 well Multiwell (Falcon), on coverslips (12 mm diameter) previously coated with polyornithine (50 g/mL, Sigma-Aldrich) and Matrigel (2% (w/v), BD). Plating was carried out at a density of 100.000 cells per coverslip. Hippocampal neurons cultures were incubated at 37 C., in a humidified atmosphere with 5% CO2 in culture medium, consisting of MEM (Gibco), supplemented with 35 mM glucose (CarloErba Reagents), 1 mM Apo-Transferrin, 15 mM HEPES, 48 mM Insulin, 3 mM Biotin, 1 mM Vitamin B12 (Sigma-Aldrich) and 500 nM Gentamicin (Gibco) and 5-10% dialyzed FBS (Gibco). Cortical neurons cultures were incubated at 37 C., in a humidified atmosphere with 5% CO2 in culture medium, consisting of Neurobasal medium (Gibco) supplemented with B27 (Gibco). With the primary cultures -Syn uptake experiments were performed essentially as described above.
[0272] Results are shown in
Example 3: Recombinant PrP Binds -Syn Amyloids
[0273] Since confocal microscopy experiments revealed the co-localization between PrP.sup.C attached to the cell membrane and exogenously added -Syn amyloids, the nature of the molecular interaction between the two proteins was characterized in more detail. To this end, enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) experiments were performed.
[0274] For ELISA Nunc-Immuno 96 MicroWell solid plates (Falcon) were coated o/n at +4 C. with 50 uL (50 ng) of MoPrP(23-231) and MoPrP(89-231) proteins in PBS. The day after wells were washed seven times with PBS-T (1PBS+0.3% Tween-20) and blocked with 5% BSA/PBS for 1 hour at room temperature. After washing five times with PBS-T different forms of -Syn (monomeric, non-sonicated, sonicated and long sonicated -Syn fibrils) were added to MoPrP(23-231) and MoPrP(89-231) coated wells at different molar concentrations (1; 1, 1:3, 1:10) and incubated for 30 min at 37 C. Following -Syn incubation wells were rinsed with PBS and incubated 1 hr with C-20-R (Santa Cruz, 1:1000) and W226 Ab (1:1,000, for control wells). After washing seven times with PBS secondary goat anti rabbit HRP, and goat anti mouse HRP were incubated at RT for 45 min. HRP signal was visualized by determining the absorbance after sequential additions of 3,3,5,5-tetramethylbenzidine (TMB, Sigma-Aldrich, 100 L per well) and stopped with 100 l 1 N sulfuric acid. The resulting yellow end product was read on SpectraMaxM5 (Molecular Devices) at 450 nm wavelength.
[0275] The results reveal that PrPC binds fibrillary -Syn in vitro (
[0276] SPR experiments were performed to calculate binding constants. Biacore 2000 Surface Plasmon Resonance (SPR) instrument was used at a constant temperature of 25 C. First, sonicated -Syn fibrils (0.35 mg/mL diluted in 10 mM Na acetate, pH 4.0) were immobilized over the Biacore CM5 gold chip surface via amine coupling reaction (according to the Manufactures' instructions). Fibrils were injected in three distinct flow cells with different contact times in order to achieve different binding levels (3300 RU, 4200 RU and 5000 RU in fc2, fc3 and fc4 respectively). Underivatized fc1 was used as reference cell and PBS buffer was flowed (5 L/min) as running buffer over the surface. Binding affinity tests of -Syn monomer (5 M) and PrP (100 nM) were performed injecting analytes in running buffer at a flow rate of 50 l/min for 3 minutes (association phase) and afterwards flushing with running buffer (dissociation phase). Binding affinity parameters were determined using the BIAevaluation software and the scientific data analysis software Igor Pro.
[0277] To illustrate the binding first sonicated fibrils were immobilized on the surface of one flow cell of a CM5 biosensor chip and monomeric -Syn protein was added (
Example 4: Detection of Proteinase K-Resistant -Syn Deposits and Other Hallmarks of Synucleinopathy in Pmnp+/+ and Prn/ Mice
[0278] Prnp.sup./ mice neither propagate prions nor develop scrapie suggesting the central role of PrP.sup.C in the development of prion diseases (H. Bueler et al., Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356, 577-582 (1992); S. B. Prusiner et al., Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc Natl Acad Sci USA 90, 10608-10612 (1993)). The critical feature for the development of prion disease is a direct interaction of PrP.sup.C with PrP.sup.Sc which acts as a template for the conversion (L. Solforosi et al., Toward molecular dissection of PrPC-PrPSc interactions. J Biol Chem 282, 7465-7471 (2007)). However, there are no reports of role of PrP.sup.C in synucleinopathies.
[0279] Therefore it was assessed whether the in vitro results might be recapitulated in an in vivo mouse model. Thus, stereotaxic injections of -Syn amyloid fibrils were performed in Prnp.sup.+/+ and Prnp.sup./ FVB mice both in the Substantia Nigra pars compacta (SNpc) and in the striatum.
[0280] To this end, female inbred FVB/N (Friend virus B-type susceptibility-NIH) FVB Prnp.sup.+/+ and FVB Prnp.sup./ mice at 2 months-of-age were used. For stereotaxic surgery mice were subdivided into groups composed of 3 animals each and intraperitoneally anesthetized with a mixture of Xylazine (15 mg/kg) and Zoletil (15 mg/kg). Sonicated -Syn short fibrils (15 g) or sterile saline solution were stereotactically injected via a 10 L Hamilton syringe into the Substantia Nigra pars compacta (AP3.2, ML1.2, DV4.4 from Bregma) or in the striatum (AP+0.2, ML2, DV2.4 from Bregma) of the right hemisphere at a rate of 3 L for 1 min, 3 L for 2 min, 4 L for 5 min. The needle was withdrawn of one coordinate and left for further 2 min before being totally removed. After recovery from surgery, animals were regularly monitored and sacrificed at 5 months-post-inoculation (mpi) by an overdose of Xylazine/Zoletil and transcardially perfused with 4% paraformaldehyde (PFA, pH 7.4). Brains were post fixed ON in PFA and sunk in 30% sucrose prior to be embedded in the Killik medium (WO1030799, Bio-Optica) and stored at 80 C. until use. Brains were cut with the Microm 550 cryostat to generate series of 10 m slides thick coronal sections on Superfrost glass slides (Menzel-GIser Adhesion Slides SuperFrost Plus). Endogenous peroxidase inactivation was performed in 3% H.sub.2O.sub.2, 10% methanol in PBS for 10 minutes. For -Syn detection, 5 g/mL of Proteinase-K (PK) digestion was used to reveal aggregates. Blocking was performed in 0.05% Triton-X100, 5% normal goat serum (NGS, Sigma-Aldrich), 1% bovine albumin serum (BSA, Sigma-Aldrich) in PBS. Primary anti--Syn antibody (C20-R, Santa Cruz, 1:500) was incubated overnight. For phosphorylated -Syn (p--Syn) detection, slides were previously treated with 70% formic acid for 30 min. Anti-phosphorylated Ser129 -Syn antibody (P-Syn/81A, BioLegend, 1:700) was incubated overnight. Sections were then incubated with proper biotinylated-secondary antibodies (Sigma-Aldrich) followed by the VECTASTAIN ABC Kit. Antibody labeling was revealed using 3-diaminobenzidine (DAB; Sigma-Aldrich, SIGMAFAST) as a chromogen. Slides were dehydrated as follow: 1 min in EtOH 50%, 1 min in EtOH 70%, 1 min in EtOH 90%, 1 min in EtOH 100%, 1 min in EtOH/Xilene (1:1), 2 min in Xilene, and mounted with Eukitt mounting medium (Bio Optica). Quantification of PK-resistant -Syn aggregates was performed with Imagej software (Image) 1.50a).
[0281] Results are shown in
[0282] Notably, when Prnp.sup.+/+ mice were injected within the SNpc, -Syn aggregates were significantly higher (
[0283] Similarly, the stereotaxic injections in the striatum led to the formation of -Syn aggregates in the brain. However, Prnp.sup./ mice accumulated lower amount of aggregates compared to Prnp.sup.+/+ mice (
[0284] -Syn amyloid fibril injection in the SNpc induced strong front and hind limb clasping in Prnp.sup.+/+ mice, while in the case of injection within the striatum only one Prnp.sup.+/+ mouse was clasping. On the contrary, clasping was never observed in Prnp.sup./ or control mice.
[0285] Another hallmark of synucleinopathies is the presence of phosphorylated -Syn deposits at residue S129 (pS129) (29). As shown in
[0286] Next, astroglial activation was assessed. To this end, brain slices were blocked in 5% NGS, 1% BSA, 1% Triton-X100 in PBS and incubated ON with anti Glial Fibrillary Acidic Protein (GFAP, ab7260, 1:1000). Antibody staining was revealed after incubation with the appropriate secondary antibody Alexa 488 (Life Technologies, 1:500). 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, SIGMAFAST) was used for nuclear staining. Slides were coverslipped with VECTASHIELD Antifade Mounting Medium (H-1000, Vector Laboratories). Fluorescent images (10241024 pixels) were acquired with the C1 Nikon confocal. For the GFAP fluorescence a 20 objective was used and stacks of z-sections with an interval of 0.25 m were sequentially scanned, to obtain representative images of the hippocampus.
[0287] Results are shown in
[0288] To investigate levels of tyrosine hydroxylase (TH), brain slices were blocked in 5% NGS, 1% BSA, 1% Triton-X100 in PBS and incubated ON with the primary antibody anti Tyrosine Hydroxylase (TH, ab112, 1:1000). Antibody staining was revealed after incubation with the appropriate secondary antibody Alexa 488 (Life Technologies, 1:500). 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, SIGMAFAST) was used for nuclear staining. Slides were coverslipped with VECTASHIELD Antifade Mounting Medium (H-1000, Vector Laboratories). Fluorescent images (10241024 pixels) were acquired with the C1 Nikon confocal. TH labelled slides were sequentially scanned as 20 z-sections with an interval of 0.25 m for all the area of interest (distance from Bregma: 2.92). TH positive (TH+) cells were counted with an automatic protocol with the Volocity 5.4 3D imaging software (PerkinElmer, Coventry, United Kingdom).
[0289] Results shown in
Example 5: Inhibition of Uptake of -Syn Fibrils by Anti-PrP Antibodies
[0290] To investigate the influence of anti-PrP antibodies on binding of PrP to -Syn and on -Syn uptake, thirty thousand cells were cultured on coverslips and treated with non-specific mouse IgG or different amounts of W226 or SAF34 anti-PrP antibodies 30 min before addition of -Syn amyloids.
[0291] Cells were then treated with -Syn amyloid fibrils (2 M) for 24 h before quantification. For quantification cells were fixed with 4% formaldehyde in PBS for 30 min. Cells were then washed three times with PBS (1) followed by blocking in 5% Normal Goat Serum (NGS, ab7481, Abcam)/0.3% Triton X-100 for 1 h, at room temperature (R.T.) Cells were incubated with primary antibodies diluted in 1% of blocking buffer (anti--Syn Ab C-20-R, Santa Cruz, 1:1,000), followed by three washings with PBS and secondary antibody incubation (goat anti-mouse Alexa488, and goat anti-rabbit Alexa594, Life Technologies). To ensure that -Syn preparations were within the cell cytoplasm a specific dye that labels the entire cell was used (HCS CellMask dye). Cells were mounted in Aqua Poly/Mount (Polysciences), and images were acquired using Leica confocal microscope (Leica TCS SP2, Wetzlar, Germany).
[0292] The uptake quantification was performed in blind using Oil Immersion 63 objective on more than 200 cells per one single independent experiment (in total of N=3). Random fields per coverslips at 63 magnification were captured using Leica confocal microscope (Leica TCS SP2, Wetzlar, Germany). To observe internalized -Syn fibrils, the coverslips were double-labeled with anti--Syn antibody and whole cytoplasmic dye CellMask. Cells considered -Syn positive were those in which the aggregates were found in perinuclear zone. The images were acquired as 20-30 z-stacks of 0.22 m, 10241024, and analyzed using Orthogonal Views function in Image J (NIH). Data are represented as % of total cell counted in three independent experiments.
[0293] Results are shown in
Example 6: Inhibition of Uptake of -Syn Fibrils by an Anti-PrP Antibody Targeting the Octapeptide Repeat (OR) Region of PrP
[0294] This example shows the ability of anti-PrP antibody POM2, which binds to the octapeptide region (OR) of PrP (Polymenidou M. et al. (2008) The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes, PLoS one 3(12): e3872), to reduce -Syn uptake in a pre-incubation setting as well as in a co-incubation setting.
[0295] In the pre-incubation setting, neuronal cells (N2a) were cultured on coverslips and treated with anti-PrP antibody POM2 (at 15 g/ml). At 1 h after treatment start, POM2 was removed and -Synuclein fibrils (0.5 M, sonicated 5 min) were added (pre-incubation/pretreatment group; also referred to as 1 h).
[0296] In the co-incubation setting, neuronal cells (N2a) were cultured on coverslips and anti-PrP antibody POM2 (at 15 g/ml) and -Synuclein fibrils (0.5 M, sonicated 5 min) were added at about the same time (co-incubation group; also referred to as 24 h) and, in contrast to the pre-incubation setting, POM2 was not removed.
[0297] In both settings, cells were incubated with -Syn fibrils (0.5 M) for 24 h (with or without POM2). Thereafter, cells were quantified. To this end, cells were fixed with 4% formaldehyde in PBS for 30 min. Cells were then washed three times with PBS (1) followed by blocking in 5% Normal Goat Serum (NGS, ab7481, Abcam)/0.3% Triton X-100 for 1 h, at room temperature (R.T.) Cells were incubated with primary antibodies diluted in 1% of blocking buffer (anti--Syn Ab C-20-R, Santa Cruz, 1:1,000), followed by three washings with PBS and secondary antibody incubation (goat anti-mouse Alexa488, and goat anti-rabbit Alexa594, Life Technologies). To ensure that -Syn preparations were within the cell cytoplasm a specific dye that labels the entire cell was used (HCS CellMask dye). Cells were mounted in Aqua Poly/Mount (Polysciences), and images were acquired using Leica confocal microscope (Leica TCS SP2, Wetzlar, Germany).
[0298] The uptake quantification was performed in blind using Oil Immersion 63 objective on more than 200 cells per one single independent experiment (in total of N=3). Random fields per coverslips at 63 magnification were captured using Leica confocal microscope (Leica TCS SP2, Wetzlar, Germany). To observe internalized -Syn fibrils, the coverslips were double-labeled with anti--Syn antibody and whole cytoplasmic dye CellMask. Cells considered -Syn positive were those in which the aggregates were found in perinuclear zone. The images were acquired as 20-30 z-stacks of 0.22 m, 10241024, and analyzed using Orthogonal Views function in Image J (NIH).
[0299] Results are shown in
[0300] Accordingly, the data suggest that anti-PrP antibodies targeting the octapeptide repeat region can be useful in the treatment of synucleinopathies.
TABLE-US-00006 TABLEOFSEQUENCESANDSEQIDNUMBERS(SEQUENCELISTING): SEQIDNO Sequence Remarks SEQIDNO:1 MANLGCWMLVLFVATWSDLGLCKKRPKPGGW humanPrP NTGGSRYPGQGSPGGNRYPPQGGGGWGQP HGGGWGQPHGGGWGQPHGGGWGQPHG GGWGQGGGTHSQWNKPSKPKTNMKHMAGA AAAGAVVGGLGGYMLGSAMSRPIIHFGSDYED RYYRENMHRYPNQVYYRPMDEYSNQNNFVHD CVNITIKQHTVTTTTKGENFTETDVKMMERVVE QMCITQYERESQAYYQRGSSMVLFSSPPVILLISFL IFLIVG SEQIDNO:2 MANLGYWLLALFVTMWTDVGLCKKRPKPGGW mousePrP NTGGSRYPGQGSPGGNRYPPQGGTWGQPHG GGWGQPHGGSWGQPHGGSWGQPHGGGW GQGGGTHNQWNKPSKPKTNLKHVAGAAAAG AVVGGLGGYMLGSAMSRPMIHFGNDWEDRYY RENMYRYPNQVYYRPVDQYSNQNNFVHDCV NITIKQHTVTTTTKGENFTETDVKMMERVVEQM CVTQYQKESQAYYDGRRSSSTVLFSSPPVILLISFLI FLIVG SEQIDNO:3 KKRPKPGGWNTGGSRYPGQGSPGGNRYPPQG humanPrP,amino GGGWGQPHGGGWGQPHGGGWGQPHGG acids23-230 GWGQPHGGGWGQGGGTHSQWNKPSKPKT NMKHMAGAAAAGAVVGGLGGYMLGSAMSRP IIHFGSDYEDRYYRENMHRYPNQVYYRPMDEYS NQNNFVHDCVNITIKQHTVTTTTKGENFTETDV KMMERVVEQMCITQYERESQAYYQRGS SEQIDNO:4 TFFYGGSRGKRNNEKTEEY AN-2peptide SEQIDNO:5 GAGACCGATGTGAAGATGATGGA PrnpFWprimer SEQIDNO:6 TAATAGGCCTGGGACTCCTTCTG PrnpRVprimer SEQIDNO:7 GTTGCGTTACACCCTTTCTTG ACTBFWprimer SEQIDNO:8 CCTGCACCACCAACTGCTTA GAPDHFWprimer SEQIDNO:9 GQPHGGX.sub.1W PrPoctapeptide whereinX.sub.1isGorS SEQIDNO:10 GQPHGGGW PrPoctapeptide