WO2014092227A1 - Composition for treatment of diseases related to removal of extracellular alpha-synuclein and method for screening therapeutic agent for said diseases - Google Patents

Abstract

The present invention relates to a composition for treatment of diseases related to the removal of extracellular alpha-synuclein and to a method for screening a therapeutic agent for said diseases.

Description

Compositions for the treatment of diseases associated with extracellular α-synuclein removal and methods for screening the same

The present invention relates to compositions for treating diseases associated with extracellular α-synuclein removal and methods for screening the same.

Parkinson's disease (PD) is a chronic progressive motor nervous system disorder. About 50,000 Americans are diagnosed with PD every year. The main symptoms of these neurodegenerative disorders are tremor, stiffness, motor slowness and balance disorder. Many PD patients also suffer from a variety of other symptoms, including emotional changes, memory loss, speech disorders, or difficulty sleeping.

PD is caused by the specificity and progressive neuronal loss of midbrain dopamine (DA) neurons. Primarily, these neurons produce dopamine, a chemical transporter responsible for signal transduction between melanocytes and striatum, resulting in smooth and intentional muscle activity. However, the loss of dopamine excites the nerve cells of the striatum so that they can be impaired, impairing their ability to direct and control their movements.

Currently used therapies for PD rely heavily on supplementing dopamine by oral administration of L-DOPA (L-dihydroxyphenylalanine), a dopamine precursor, to a patient. Such therapy requires an increase in dosage as treatment continues, resulting in serious side effects. There is a need for other therapies for PD.

As a related prior patent, Korean Patent Publication No. 1020080106928 is administered by administering 7-chloro-4-aminoquinoline compounds such as amodiaquine or glafenine. Methods and kits for treating Parkinson's disease or inhibiting Parkinson's disease are characterized.

Meanwhile, diseases with accumulation of α-synuclein, such as Parkinson's disease (PD) and Lewy body dementia (DLB), are a common cause of dementia in motor disorders and the elderly population. Under physiological conditions, α-synuclein is a protein present in the protoplasts located at presynaptic sites (Iwai A, Masliah E, Yoshimoto M, Ge N, Flanagan L, de Silva HA, Kittel A, Saitoh T (1995). Neuron 14: 467-475).

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a novel Parkinson's disease (PD) and Lewy body dementia (DLB) therapeutic composition.

Another object of the present invention is to screen candidate candidates for Parkinson's Disease (PD) and Lewy Body Dementia (DLB).

In order to achieve the above object, the present invention provides an antibody that specifically binds to the α-synuclein peptide set forth in SEQ ID NO: 1.

In another aspect, the present invention provides a composition for preventing or treating Parkinson's disease, comprising the antibody of the present invention as an active ingredient.

In another aspect, the present invention provides a composition for preventing or treating Lewy Body Dementia (DLB) comprising the antibody of the present invention as an active ingredient.

In addition, the present invention corresponds to a case where the candidate substance and the extracellular α-synuclein protein are contacted with the microglial cell under an environment suitable for the two components to interact, and the uptake and degradation rate of the α-synuclein protein are increased. Provided is a method for identifying a candidate for Parkinson's disease or Lewy body dementia (DLB) treatment, wherein the candidate is determined to be a candidate for Parkinson's disease or Lewy Body Dementia (DLB).

In one embodiment of the present invention, the candidate is preferably an antibody antisense oligonucleotide or a compound, but is not limited thereto.

As used herein, the term "epitope" refers to a protein determinant capable of specifically binding to an antibody. Epitopes usually consist of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics. Three-dimensional epitopes and non-stereo epitopes are distinguished in that the binding to the former is lost but not to the latter in the presence of a denatured solvent.

In a preferred embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more antibodies of the invention in combination with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant do. Preferably, the acceptable formulation material is nontoxic at the dosages and concentrations used for the recipient. In a preferred embodiment, there is provided a pharmaceutical composition comprising a therapeutically effective amount of an antibody of the invention.

In certain embodiments, preferably acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed.

In certain embodiments, the pharmaceutical composition modifies, maintains or preserves, for example, the pH, osmolarity, viscosity, purity, color, isotonicity, flavor, sterilization, stability, dissolution or release rate, adsorption or permeation of the composition. Formulation materials for In such embodiments, suitable formulation materials include, but are not limited to: amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); Antimicrobial agents; Antioxidants (eg, ascorbic acid, sodium sulfate or sodium hydrogen sulfide); Buffer solutions (eg, borate, bicarbonate, tris-HCl, citrate, phosphate or other organic acids); Volume control agents (eg mannitol or glycine); Chelating agents (eg ethylenediamine tetraacetic acid (EDTA); complexing agents (eg caffeine, polyvinylpyrrolidone, β-cyclodextrin or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; Disaccharides; and other carbohydrates (e.g. glucose, mannose, or dextrins); proteins (e.g. serum albumin, gelatin or immunoglobulins); pigments, anti-microbial and diluents; emulsifiers; hydrophilic polymers (e.g. poly Vinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g. sodium); preservatives (e.g. benzalkonium chloride, benzoic acid, salicylic acid, chimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine Sorbic acid or hydrogen peroxide); solvents (e.g. glycerin, propylene glycol or polyethylene glycol); sugar alcohols (e.g. mannitol or sorbitol); suspending agents; surfactants or Wetting agents (e.g., polysorbates such as pluronic, PEG, sorbitan esters, polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol and tiloxapal); stability enhancers ( For example sucrose or sorbitol); enteric enhancers (e.g. alkali metal halide compounds, preferably sodium chloride, potassium chloride, mannitol sorbitol); transport excipients; diluents; prostheses; and / or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Gennaro, ed.), 1990, obtained from Publishing Company.

In certain embodiments, the optimal pharmaceutical composition will be determined by one of ordinary skill in the art, for example, according to the intended route of administration, mode of transport and dosage required. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES above. In certain embodiments, the composition affects the rate of in vivo clearance, rate of in vivo release, stability, and physical state of the antibodies of the invention.

In certain embodiments, the primary excipient or carrier in the pharmaceutical composition is aqueous or non-aqueous in nature. For example, a suitable excipient or carrier may be an injectable, physiological saline or artificial cerebrospinal fluid that is generally capable of supplementing other substances in the composition for parenteral administration. Neutral buffered saline or physiological saline mixed with serum albumin is further typical excipients. In a preferred embodiment, the pharmaceutical composition of the present invention comprises tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which further comprises sorbitol, sucrose, Tween-20 and / or Suitable substituents thereof. In certain embodiments of the present invention, the antibody compositions of the present invention may be formulated with selected compositions having the desired purity and optimal formulation formulations.

Were prepared for storage in the form of lyophilized cake or aqueous solution by mixing (see REMINGTON'S PHARMACEUTICAL SCIENCES cited above).

The pharmaceutical compositions of the invention can be selected for parenteral administration. Optionally, the composition may be selected for inhalation or transport through the digestive tract, such as oral. Preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation component is preferably present at an acceptable concentration at the site of administration. In certain embodiments, buffers are used to maintain the composition at physiological or slightly lower pH, generally in the range of about pH 5 to about pH 8.

The formulations can also be administered orally. Antibodies of the invention administered in this manner may be formulated with or without a carrier generally used in the preparation of solid pharmaceutical formulations such as tablets and capsules. In certain embodiments, the capsule may be designed to release the effective portion of the formulation at a gastrointestinal tract location where the bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate uptake of the antibodies of the invention. Diluents, flavors, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used.

Pharmaceutical compositions of the invention are preferably provided comprising an effective amount of one or multiple antibodies of the invention in a mixture with non-toxic excipients suitable for the manufacture of tablets. By dissolving the tablets in sterile water or other suitable excipients, solutions may be prepared in unit pharmaceutical formulations. Suitable excipients include, but are not limited to: inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; Or binders such as starch, gelatin or acacia; Or lubricants such as magnesium stearate, stearic acid or talc.

Pharmaceutical compositions used for administration in vivo are generally provided as sterile preparations. It can be sterilized by filtration through sterile filtration membranes. When the composition is lyophilized, it can be sterilized using the method before or after lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or as a solvent. Parenteral compositions are generally stored in containers with sterile access ports, such as vials or intravenous solution bags with stoppers that can be inserted into subcutaneous needles.

Once the pharmaceutical composition has been formulated, it is stored in sterile vials in the form of solvents, suspensions, gels, emulsions, solids or dehydrogenated or lyophilized powders. Such formulations are stored in an easy-to-use form or in a form that requires reconstitution (eg, lyophilized) prior to administration.

An effective amount of a pharmaceutical composition containing an antibody of the invention to be used therapeutically depends, for example, on the therapeutic situation and the subject. Those skilled in the art will appreciate that dosage levels suitable for treatment may be partially determined by the transport molecule, signs of the use of the antibody of the present invention, route and size of administration (weight, body surface area or organ size) and / or symptoms of the patient (age and overall health). Will vary depending on the condition. In certain embodiments, the clinician may titrate the dosage and alter the route of administration to obtain the optimal therapeutic effect. Typical dosages range from about 0.1 μg / kg up to about 30 mg / kg or more, depending on the factors mentioned above. In a preferred embodiment, the dosage range is from 0.1 μg / kg to about 30 mg / kg; More preferably from 1 μg / kg to about 30 mg / kg; Even more preferably from 5 μg / kg to about 30 mg / kg.

The number of administrations will depend on the pharmacokinetic parameters of the antibodies of the invention of the formulation used. In general, the clinician has administered the composition until the dosage achieves the desired effect. Therefore, the compositions were administered by a single administration or by two or more administrations (with or without the same amount of the desired molecule) or by continuous infusion via an insertion device or catheter over time. Moreover, refining at a suitable dosage is an area of common work typically made by a person skilled in the art and done by a person skilled in the art. Appropriate doses were identified using appropriate dose-response data. In certain embodiments, antibodies of the invention can be administered to a patient for an extended period of time. Routine administration of the antibodies of the present invention minimized immune adverse reactions or allergic reactions generally associated with antibodies to human antigens, such as non-complete human antibodies produced in non-human species, in non-human animals.

The route of administration of the pharmaceutical composition is according to known methods, including, for example: oral; Injection by intravenous, intraperitoneal, intracranial (brain parenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional routes; Sustained release system; Or injection using an insertion device. In certain embodiments, the composition may be administered by injection or infusion or infusion device continuously.

The composition can be administered topically through the insertion of a membrane, sponge or other suitable material in which the required molecules are absorbed or encapsulated. In certain embodiments, when using an insertion device, the device is inserted into an appropriate tissue or organ and the required molecules are transported by diffusion, sustained-release concentrates or continuous administration.

Hereinafter, the present invention will be described.

We show that antibodies against α-synuclein specifically target extracellular α-synuclein proteins and aid in their removal by microglia, thus inhibiting their action on adjacent cells. Antibody-assisted clearance occurs mainly in microglia via Fcγ receptors, but not in neurons or astrocytes. Stereotaxic administration of antibodies to the brain of α-synuclein tg mice inhibits the transfer of α-synuclein to astrocytes in neurons resulting in an increase of α-synuclein and its antibodies in microglial cells . Passive immunization with α-synuclein antibodies also reduces neuronal and glial accumulation of α-synuclein and mitigates behavioral disorders and neurodegeneration associated with α-synuclein overexpression. These results provide a mechanism for immunotherapy against PD / DLB and suggest an extracellular form of α-synuclein as a therapeutic target.

Hereinafter, the present invention will be described in detail.

Antibody-Mediated Removal of Extracellular α-synuclein Aggregates by Microglia

To investigate the therapeutic effect of antibodies against α-synuclein, we prepared antibodies against monoclonal human α-synuclein and tested their effects on the removal of extracellular α-synuclein aggregates from microglia. .

We produce oligomers and fibrils from recombinant human α-synuclein; Electron microscopy (EM), size exclusion chromatography (SEC), and gel electrophoresis confirmed their physical properties. [Alpha] -Synuclein oligomers were spherical, curvy, and short elongated structures on EM. It appears as a heterogeneous mixture (Fig. 1C-F). Based on SEC data, about half of the proteins in the oligomers were high molecular weight oligomers and the other half were monomers (FIG. 1G, H). By silver staining and western blotting we found that fibrils and oligomers contain both SDS-resistant aggregates (multiple high molecular weights) and SDS soluble aggregates, showing 16 kDa molecular weight as monomers (FIG. 1H, I). ).

We first evaluated the effect of monoclonal monoclonal antibodies on the uptake of α-synuclein fibrils in BV-2 microglia cell line. Of these, two clones 169 and 274 promoted uptake, while the other two clones 62 and 171 were ineffective (FIGS. 2A, B). The effect of these antibodies is dose dependent and reaches the point of saturation (FIG. 2C). Based on this preliminary characterization, 274 antibodies were selected and used in the present invention. The 274 antibody is specific for α-synuclein of the human type, with an epitope at the C terminus of the protein. We compared the uptake and degradation rates of α-synuclein aggregates in BV-2 microglia in the presence of 274 antibody or control IgG. For the uptake experiments, 0.2 μM α-synuclein aggregates previously incubated with 5 μg / ml of 274 antibody or control IgG were added to the cells. Cells were collected at the indicated time after incubation at 37 ° C. For digestion experiments, the aggregates were preincubated for 10 and 5 minutes for fibrils and oligomers respectively, and the cells were washed and incubated at 37 ° C. in fresh culture medium for the stated time. In the presence of 274 antibody, the time required to reach the maximum level of internalized fibril α-synuclein was reduced from 30 to 10 minutes (FIG. 3A) and the half-life of imported fibrils α-synuclein Decreased from 77 to 11 minutes (FIG. 3B). For removal of oligomer α-synuclein aggregates, the time required for absorbed α-synuclein oligomers to reach maximum levels was 11 to 4 minutes in the presence of 274 antibodies and the half-life of the absorbed oligomer was 18 to 6 minutes. Decreased (Figure 3C, D). The addition of 62 antibodies (FIG. 2) on the other hand had no effect on the uptake and degradation rates of α-synuclein fibrils or oligomers in BV-2 cells (data not shown), suggesting that this effect is antibody specific. . In contrast to the fibrils and oligomers, the uptake rate for the monomers was unchanged by the addition of 274 antibody in the microglia (not shown). These results indicate that in the presence of 274 antibody, uptake and degradation of extracellular α-synuclein aggregates, but not monomers, are promoted in microglia.

In order to further regenerate the brain cell environment, the inventors cultured rat primary microglia to determine the rate at which these microglia uptake the α-synuclein protein secreted from neuronal cells. For this experiment, we used conditioned media obtained from differentiated SH-SY5Y neuroblastoma cells that overexpress human α-synuclein. The conditioned medium contains both monomeric and SDS stable aggregates (FIG. 1I). In comparison to cells treated with control IgG, uptake of a-synuclein secreted from neuronal cells reached faster and higher levels in 274 antibody treated primary microglia (FIG. 3E, F), which antibody Suggests that it helps microglia in the removal of neuronal secreted extracellular a-synuclein.

We next describe the rate of uptake and degradation of exogenous α-synuclein fibrils in differentiated SH-SY5Y human neuroblastoma cells as well as rat primary astrocytes and cortical neurons. The effect of the antibody was investigated. In contrast to microglia, no change in the uptake or degradation rate of extracellular α-synuclein fibrils was observed in these cells (FIGS. 4A-F). Similarly, uptake of monomer was not affected by the antibody in astrocytes or neurons (not shown). Thus, antibody mediated removal of extracellular α-synuclein is a selective process for microglia and only applies to aggregates, not monomers.

Antibody-assisted clearance of α-synuclein aggregates is mediated by Fcγ receptors resulting in rapid delivery to lysosomes

To assess the role of the Fcγ receptor in antibody mediated α-synuclein removal, monoclonal antibodies against CD16 / CD32 that block Fcγ receptors II and III (Wijngaarden S, van Roon JA, van de Winkel JG, Bijlsma) JW, Lafeber FP (2005) Rheumatology 44: 729-734) and inhibited. Blockage of the Fcγ receptor eliminated the effect of 274 antibodies on uptake of the α-synuclein oligomer (FIG. 5A). Fcγ receptors were present with oligomer α-synuclein on the surface of BV-2 cells only in the presence of 274 antibodies, not in the presence of control IgG or Fab fragments of 274 antibodies (FIG. 5B), which was responsible for the uptake process of Fcγ receptors. Prove your involvement. Fcγ receptors were markedly expressed in BV-2 microglia and rat primary microglia but were not detected in astrocytic or differentiated neuroblastoma cells (FIG. 5C). Type II and Type III (CD32 / CD16) Fcγ receptors are low affinity receptors for IgG and require a multivalent antigen-antibody complex for high avidity (Ravetch JV, Bolland S (2001) Annu Rev Immunol 19: 275-290). It has a high affinity and cannot bind to these Fcγ receptors. To confirm the uptake of the α-synuclein-antibody immune complexes, control IgG or monoclonal antibody 274 was precultured with various amounts of α-synuclein fibrils and added to BV-2 cells. The level of absorbed antibody was directly proportional to the amount of foreign α-synuclein aggregates (FIG. 5D). α-synuclein and pre-cultured control IgG were not taken up by the cells, as did the foreign β-synuclein and 274 antibodies pre-cultured (FIG. 5D). In addition, immunofluorescence analysis showed a large increase in antibody uptake in the presence of foreign α-synuclein aggregates, and co-existence of α-synuclein and antibody was demonstrated in the plasma after absorption (FIGS. 5E, F). Overall, these results suggest specific uptake of α-synuclein-antibody complexes in microglia.

The inventors next examined the intracellular trafficking pathway of the α-synuclein-antibody immune complex as compared to the pathway performed by α-synuclein alone. To this end, we have described low density lipoproteins (LDL) fluorescently labeled with markers of clathrin-mediated endocytosis, and cholera toxin B subunit (CTB) (Sharma DK,) as markers for lipid raft mediated endocytosis. Brown JC, Choudhury A, Peterson TE, Holicky E, Marks DL, Simari R, Parton RG, Pagano RE (2004) Mol Biol Cell 15: 3114-3122). In the presence of the control antibody, the absorbed α-synuclein aggregates alone did not exist extensively in common with either marker (FIG. 6A), which suggests a new transport mechanism. However, when the α-synuclein aggregate was applied with the 274 antibody, a large portion of the absorbed α-synuclein aggregate coexisted with the CTB (FIG. 6A).

To confirm these results, we performed an immunolocalization experiment. Absorbed α-synuclein aggregates did not co-exist with the Golgi apparatus marker GM130, which suggests a deficiency overlapping with the biosynthetic pathway (not shown). On the other hand, when incubated with 274 antibodies, about 38.6% of the absorbed α-synuclein aggregates were found in the positive zone for caveolin-1, whereas in the presence of control IgG they were rarely present with caveolin-1. (Figures 6B, C).

This is consistent with the result that 274 antibody alters the transport pathway of absorbed α-synuclein performed by CTB (FIG. 6A). Changes in the transport pathway lead to increased coexistence in lysosomes; In the presence of 274 antibody, the co-existence of the lysosomal marker cathepsin D with α-synuclein was increased from 28 to 93% (Figure 6D, E). Overall, these results indicate that, when complexed with antibodies, α-synuclein aggregates are absorbed through Fcγ receptors, perform different intracellular hydrogen pathways, and are delivered to lysosomes more effectively than free α-synuclein aggregates. Suggests that it can decompose faster.

274 Antibodies Reduce Neurodegenerative Diseases, Behavioral Disorders and Cell Delivery in Cells of α-Synuclein in a tg Mouse Model

To determine whether antibody-assisted removal of extracellular α-synuclein aggregates inhibits intracellular aggregate delivery, we injected 274 antibodies into the hippocampus of tg mice expressing human α-synuclein and The amount of transfer from neurons to astrocytes was analyzed.

Injection of control IgG showed no effect on α-synuclein delivery from neurons to astrocytes; Both the ipsilateral and contralateral sides of the hippocampus showed the same degree of astroglial α-synuclein accumulation (FIGS. 7A, E, K). In contrast, injection of the 274 antibody resulted in a significant decrease in astrocyte glial accumulation of α-synuclein (FIGS. 7B, F, L). Injection of the antibody had no effect on α-synuclein expression in neurons (FIG. 7C, D) and did not change the number of astrocytic cells (FIG. 7G, H), thus antibody treatment resulted in cell transfer in cells of α-synuclein. Suggests that Injection of control IgG showed no effect on microglia (FIG. 7I); However, both ipsilateral and contralateral sides of the hippocampus injected with 274 antibodies showed a slight increase in microglial activation (FIG. 7J).

To confirm the involvement of microglia in the blockade of cell delivery in the cells, we used the dual immunofluorescence staining to investigate the position of human α-synuclein and 274 antibodies in microglia.

The number of microglia containing human α-synuclein showed a significant increase ipsilaterally in mice injected with 274 antibodies (FIGS. 8A, B). The injected antibody was also detected in microglia in ipsilaterally in tg mice injected with 274 antibodies (FIG. 8C, D), but not in mice injected with control IgG (FIG. 8D). The co-location of α-synuclein and injected antibodies was consistently observed in tg mice injected with 274 antibodies (FIG. 8E), but not in mice injected with control IgG. These results suggest that α-synuclein antibodies promote the removal of neuron-derived α-synuclein by microglia to inhibit cell delivery in cells of α-synuclein aggregates.

To assess kinetics for microglia supported removal of α-synuclein following 274 antibody injections, double labeling with antibodies to α-synuclein and Iba-1 was performed at various time points (FIG. 8F). After 1 day of injection, about 20% of the microglia showed α-synuclein accumulation, while on day 7 and 14 about 10% of the microglia had α-synuclein and at about 5% on day 28 Decreased (FIG. 8G).

To confirm whether the 274 antibody inhibited the transfer of α-synuclein from neurons and astroglia, a dual labeling study was performed with NeuN and GFAP antibodies.

Confocal supported images showed that α-synuclein present in the same position as the pyramidal NeuN-positive neurons represented about 20% of the cells (FIG. 8H) and noticeable differences between the IgG and 274 antibody groups were observed. (FIG. 8I). In contrast, the α-synuclein-positive cells with fusiforms do not exist simultaneously with NeuN, but rather with GFAP (FIG. 8J). About 15% of GFAP cells contained α-synuclein in the control group. However, treatment with 274 antibody resulted in a decrease in GFAP / α-synuclein-positive cells at the injection site (FIG. 8K).

Image analysis of GFAP and Iba-1-positive cells was performed in the hippocampus underneath the site of the needle tread to avoid the nonspecific effect of the scar along the tread. Along with the injection traces, astrogliosis lasted 4 weeks after injection, while the microglial response along the traces was transient and began to decrease after 2 weeks. Similarly, the number of activated microglia containing α-synuclein in the hippocampus (under injection marks) decreased with time (FIG. 8F, G).

To further assess the effect of antibody-assisted clearance of extracellular α-synuclein aggregates on functional and neuropathological parameters, passive immunoassays were performed by intraperitoneal injection of control IgG or 274 antibodies with non-tg and tg mice. It was. Analysis of functional motor coordination in the pole test compared to IgG and 274-treated non-tg, IgG-treated tg mice took longer to complete the paradigm (FIG. 9A). . In contrast, tg mice treated with 274 antibodies performed similarly to the non-tg group (FIG. 9A). In the open field, IgG-treated tg mice showed some hypersensitivity compared to non-tg (FIG. 9B). In α-synuclein tg mice this effect was reversed by treatment of 274 antibody (FIG. 9B). Other activities in the open field, including total distance and breeding, did not differ among the four groups (Figures 9C, D).

With cross-species polyclonal antibodies, α-synuclein immunostaining was restricted to neuropil in non-tg mice with no differences observed between the control IgG and 274 antibody groups (FIG. 9E-G). ). In IgG-treated α-synuclein tg mice, of astrocytes (FIG. 9F) and neuronal cells (FIG. 9G) in α-synuclein (FIG. 9E) in neocortex (FIG. 9H) and hippocampus (FIG. 9I). There was extensive accumulation. In contrast, α-synuclein tg mice treated with 274 antibodies (FIG. 9E) were found to have astrocytes (FIG. 9F) and neurons α-synuclein (FIG. 9G) in neocortex (FIG. 9H) and hippocampus (FIG. 9I). There was a significant decrease in the accumulation of.

We also analyzed the striatum of passively immunized mice and found that the levels of α-synuclein were reduced to a lower extent compared to the cortex and hippocampus.

274 in the cortex and hippocampus reduced the level of α-synuclein to about 70-80% (FIG. 9H, I); In the striatum, the level was reduced to 30-35% (FIG. 9J), suggesting differential site-specific transport of antibodies.

We performed ELISA for α-synuclein in CSF and brain of control and antibody treated α-synuclein mice. The level of α-synuclein in brain homogenates of vehicle treated α-synuclein tg mice was 13.57 ± 1.610 ng / ml (n = 8), whereas α-synuclein tg treated with 274 antibody was 5.031. ± 1.011 ng / ml (n = 8). However, the level of α-synuclein in CSF is very low under the detection limits of the ELISA system of the present invention.

α-synuclein-containing microglia were observed mainly in neural networks and rarely around the blood vessels.

To further assess the effect of 274 antibodies on the elimination of α-synuclein in passive immunization, a double labeling study was conducted with antibodies to NeuN, GFAP, and Iba-1. Confocal image analysis indicated that the proportion of pyramidal NeuN-positive cells showing α-synuclein immunoreactivity was decreased in the group injected with 274 antibodies (FIG. 9K, L). In addition, compared to α-synuclein tg mice treated with IgG, treatment with 274 antibody resulted in the reduction of fusiform GFAP positive astroglia including α-synuclein (FIG. 9M, N).

In contrast, treatment with 274 antibodies resulted in an increase in Iba-1-positive microglia with α-synuclein, as compared to α-synuclein tg mice immunized with IgG alone (FIG. 9 O, P). We also performed double labeling assays for α-synuclein and cathepsin D and found that they were present in the same position between the two markers in microglia of animals immunized with 274 (FIG. 9Q, R).

Immunohistochemistry and image analysis were performed to study the effects of passive immunity on neurodegeneration and neuroinflammatory pathology. As antibodies to NeuN, IgG-treated α-synuclein tg mice showed a decrease in stereoscopic neuronal evaluation in the hippocampus compared to non-tg controls (FIGS. 10A, B). The loss of neurons was evident at the CA3 site of the hippocampus (FIG. 10A, arrow). In contrast, α-synuclein tg mice treated with 274 antibodies showed hippocampal NeuN cell counts corresponding to the non-tg control (FIGS. 10A, B).

 Synaptic changes were also analyzed. We found a 28% reduction in synaptophysin in IgG-treated tg, and this synaptic damage was significantly alleviated by treatment with 274 antibody (FIG. 10C, D). Immunohistochemistry with antibodies to GFAP compared to non-tg controls, α-synuclein tg mice increased astrocytes in the deep layers of neocortex (Figure 10E, inset) and hippocampus (Figures 10E, F). It was shown that it showed astrogliosis. Passive immunization with 274 antibody reduced levels of GFAP immunoreactivity in the neocortex (FIG. 10E, inset) and hippocampus (FIG. 10E, F) in α-synuclein tg mice. Finally, microglial counts were performed with an antibody against the microglia marker, Iba-1, which resulted in similar levels of microglia treated with IgG and 274 antibodies as well as a-synuclein tg mice treated with IgG controls. Was detected between non-tg mice (FIG. 10G). However, α-synuclein tg mice treated with 274 antibodies showed a moderate increase in the level of Iba-1 immunostaining (FIG. 10G, H). Consistent with this, α-synuclein tg mice treated with IgG alone showed increased levels of pro-immune cytokines, TNFα (FIG. 10 I, J), and IL-6 (FIG. 10K, L), whereas 274 antibodies Low immunization reduced that level to baseline. This result is consistent with the result that 274 antibodies promote the removal of extracellular α-synuclein.

Taken together, these results show that immunization with 274 antibodies reduces functional damage in α-synuclein tg mice, which is accompanied by reduction in accumulation of α-synuclein in glial and neuronal cells and alleviation of neurodegeneration.

As can be seen through the present invention, the antibody against α-synuclein specifically promotes the removal of extracellular α-synuclein aggregates by microglia and forms α-synuclein when complexed with specific antibodies. Synuclein aggregates are absorbed through Fcγ receptors on the surface of microglial cells and after absorption their immune complexes are transported along the transport pathway and cause more efficient delivery to lysosomes. Removal of α-synuclein mediated by antibodies also inhibited the transfer of α-synuclein from cell to cell in the tg mouse model. These results suggest that α-synuclein immunotherapy can perform therapeutic and prophylactic effects through the promotion of microglia mediated removal of extracellular α-synuclein.

1 shows the properties of α-synuclein fibrils and oligomers. AF , EM image of fibrils ( A ), sonicated fibrils ( B ), and oligomers ( CF ). Scale bar: AC , 0.5 μm; DF , 30 nm. G , size exclusion chromatography of oligomers. Asterisks (7 ml fractions) indicate oligomeric fractions. Monomers are present in 13-15 ml fractions. Silver stained image of H , SEC fractions. I , Western blot analysis of several forms of α-synuclein. M, monomers; F, fibrils; O, oligomers; CM, conditioned medium of differentiated SH-SY5Y cells expressing α-synuclein.

Figure 2 shows the effect of monoclonal antibodies on the uptake of extracellular α-synuclein aggregates. A , uptake of α-synuclein fibrils in the presence of described monoclonal antibodies against α-synuclein or control IgG (-) in BV-2 microglia. 5 μg / ml α-synuclein fibrils (0.2 μM) Antibodies against control IgG or α-synuclein were preincubated for 5 minutes at room temperature and treated with BV-2 cells at 37 ° C. for 5 minutes. The amount of α-synuclein absorbed was analyzed by Western blot. B , the amount of α-synuclein absorbed was quantified and normalized to the level of beta-actin. The number of y-axes indicates the amount of α-synuclein relative to the control. n = 4, * p <0.05. C , antibody dose dependent uptake of α-synuclein fibrils (n = 3, p <0.05) *, 274; #, 169.

3 shows the effect of 274 antibodies on uptake and degradation of extracellular α-synuclein in microglia. Rate of uptake ( A , C ) and degradation ( B , D ) of α-synuclein fibrils ( A , B ) and oligomers ( C , D ) in AD , BV-2 cells. Adsorbed α-synuclein aggregates were analyzed by western blotting at the time stated. Graphs in A and C show the amount of α-synuclein compared to the maximum absorbed level with control IgG. Graphs in B and D show the amount of α-synuclein remaining compared to that at time O. All values were normalized to beta-actin. The curves of each graph are significantly different (n = 4, p <0.05). E , F , show the effect of 274 antibodies on uptake of neuron secreted α-synuclein in primary microglia. E , primary rat microglia were treated with conditioned medium containing α-synuclein secreted from differentiated SH-SY5Y cells in the presence of control IgG or 274 antibodies. Quantitative analysis of data at F and E. The curves are significantly different (p <0.05, n = 3). Control blot: beta-tubulin.

4 shows the lack of the effect of 274 antibodies on uptake and degradation of α-synuclein fibrils in primary astrocytes and neurons. A , B , primary astrocytic cells (n = 4). C , D , primary cortical neurons (n = 3). E , F , differentiated SH-SY5Y cells (n = 4). A , C , E , uptake rate. The graph shows the amount of α-synuclein relative to the level of maximum absorption with control IgG. B , D , F , decomposition rate. The graph shows the amount of α-synuclein remaining compared to that at time O. Pre-incubation time with α-synuclein fibrils was 12 hours ( B , D , F ). All α-synuclein data was normalized to beta-actin data. The curve of the graph is not significantly different.

5 shows the role of Fcγ receptors in the uptake of α-synuclein-antibody immune complexes. A , Effect of Fcγ receptor blocking antibodies on uptake of α-synuclein oligomers in BV-2 cells. The absorbed α-synuclein oligomers were analyzed by Western blot and normalized to beta-actin. The y-axis of the graph shows the amount of α-synuclein relative to the level of maximum absorption with control IgG. The red curve is significantly different from the others (n = 7, p <0.05). Immunofluorescence images of B , Fcγ receptors and α-synuclein. Arrows indicate the co-existence of α-synuclein and Fcγ receptors on the surface of the cells. Scale bar: 20 μm. C , Expression of Fcγ receptors in several cell types. For BV-2, rat microglia, and rat astrocytes, anti-CD16 / CD32 antibodies specific to rodents were used, whereas for SH-SY5Y, anti-CD32 antibodies specific to human proteins were used. Expression of CD16 has not been reported in brain neurons in previous studies. Red, Fcγ receptors; Blue, nuclear. Scale bar: 20 μm. D , uptake of antibody into BV-2 cells in the presence of several different amounts of α-synuclein fibrils or α-synuclein (0 -330 nM). Absorbed antibody was analyzed with anti-mouse IgG antibody. The bottom panel is beta-actin. E , Immunotype and microscopic results of α-synuclein fibrils and antibodies taken up in BV-2 cells. Scale bar: 20 μm. Quantification of fluorescence from antibodies absorbed at F , E. 150 cells were analyzed in three independent experiments (50 cells in each experiment). * p <0.0001.

6 shows altered intracellular transport and increased transport of lysosomes of uptake of α-synuclein aggregates. A , Altered intracellular transport of α-synuclein fibrils uptake in BV-2 cells. Fluorescently labeled tracers were treated with BV-2 microglia cells for 5 min at 37 ° C. with control IgG-α-synuclein mixture or α-synuclein-274 immune complex. Arrows indicate coexistence between α-synuclein and CTB in the presence of 274 antibody. Scale bar: 5 μm. BE , localization of α-synuclein aggregates absorbed in the endocytic compartment. Immune complexes were treated with BV-2 cells for 5 minutes. The number of C , E and α-synuclein points (puncta) is co-located with caveolin-1 ( C ) and quantified cathepsin D ( E ). 3000 points were analyzed from three independent experiments (1000 points for each experiment). Scale bar: 5 μιη. * p <0.005.

7 shows astrocytic delivery in reduced neurons of α-synuclein in vivo following alone injection of 274 antibody. A , B , low-pan (20X) view of the cortex and hippocampus of PDGF-α-synuclein tg mice (line M) comparing the patterns of α-synuclein immunoreactivity. Scale bar: 200 μm. CJ , hippocampal CA1 site ( C , D ) showing α-synuclein accumulation in neurons and dentate gyrus site ( EJ ) ( E , F ) showing α-synuclein accumulation in glial cells, GFAP of astrocytes High Power View (200X) of presence ( G , H ), and presence of Iba1 microglia ( I , J ). Scale bar: 25 μm. Image analysis of the number of K , L , α-synuclein-positive neurons and glial cells. N = 8, 9 months per group. * Both Student's t test p <0.01, unpaired.

FIG. 8 shows increased localization of α-synuclein and IgG in microglia cells in vivo after one injection of antibody. PDGF-α-synuclein tg mice (line M) were given for injection in the hippocampus as shown in FIG. 7. Images in the AE are obtained from the injection, ipsilateral region and dentate gyrus. Scale bar, 10 μm. A , double labeling with antibodies to α-synuclein (red) and microglia marker (Iba1, green). B , Image analysis for double label for measurement of proportion of Iba-1-positive cells co-located with α-synuclein. N = 8 per group, 9 months of age. * Both Student's t test p <0.01, unpaired. Double labeling with antibodies against C , mouse IgG (red) and Iba-1 (green). D , Image analysis for double label for measurement of the proportion of Iba-1-positive cells co-located with mouse IgG. N = 8 per group, 9 months of age. * Both Student's t test p <0.01, unpaired. E , double label with antibody to mouse IgG (red) and α-synuclein (green). Arrows indicate the coexistence of two markers in each panel. All images in FK are obtained from the hippocampus at the injection and ipsilateral site. Example of microglia double labeled with antibodies against F , α-synuclein (red) and Iba-1 (green). G , percent of Iba-1-positive cells with α-synuclein after 1, 7, 14, and 28 days after injection. Double label with antibody to H , α-synuclein (red) and neuronal marker (NeuN, green). Arrows indicate coexistence at the top, while arrows at the bottom indicate spindle-like α-synuclein positive cells that do not coexist with NeuN. I , Image analysis for double labeling for proportional measurement of positive cells co-existing with α-synuclein. N = 8 per group, 9 months of age. J , double label with antibody against α-synuclein (red) and GFAP (green). Arrowheads indicate α-synuclein-positive neurons that do not coexist with GFAP; arrows indicate α-synuclein-positive cells coexist with GFAP. K , Image analysis for double labeling for the ratio of GFAP-positive cells co-existing with antibodies to α-synuclein (n = 8, 9 months of age per group; * One-way ANOVA, post hoc Tukey-Kramer <0.05). Scale bar: H , J , 25 μm.

9 shows improved behavior and reduced α-synuclein accumulation in α-synuclein tg mice after passive immunization with 274 antibody. A , the total time traveled down the pole test. B , open field total spontaneous activity (number of crosses of photocell). C , Open field Total distance traveled 5 minutes after voluntary active testing. D , Open field Total number of roundings after 5 minutes of spontaneous active testing. P <0.05 by unilateral ANOVA with post hoc Dunnet's compared to AD , * non-tg and α-synuclein tg; # p <0.05 by unilateral ANOVA with post hoc compared to IgG and 274 antibodies in α-synuclein tg. E , low magnification view (x20) of α-synuclein immunoreactivity in cortex and hippocampus. Squares show magnified sites in F and G. Scale bar, 250 μm. F , G , high magnification (x630) views ( F , astrocytes; G , neurons). Scale bar, 20 μm. H- sh, respectively neocortex, hippocampus, image analysis of glial and neuronal cells can be estimated (dissection method) representing the α- synuclein in the striatum. * Student's t by test p <0.001. K , double labeled with antibody against α-synuclein (red) and neuronal marker (NeuN, green) in hippocampus. Arrow heads indicate coexistence. L , Image analysis for double labeling to assess the proportion of NeuN-positive cells co-existing with α-synuclein. M , double labeled with antibodies against α-synuclein (red) and GFAP (green) in the hippocampus. Arrowheads indicate α-synuclein-positive cells coexisting with astrocytes. N , Image analysis for double labeling for evaluation of the proportion of GFAP positive cells co-existing with antibodies to α-synuclein. O , double labeled with antibodies against α-synuclein (red) and Iba-1 (green) in hippocampus. Arrowheads indicate α-synuclein-positive cells coexist with microglia. P , Image analysis for double labeling for evaluation of the proportion of Iba-1-positive cells co-existing with antibodies to α-synuclein. Q , double labeled with antibodies against α-synuclein (red) and cathepsin D (green) in the hippocampus. Arrow heads indicate coexistence. R , Image analysis for double labeling for evaluation of the proportion of cathepsin-D-positive cells co-existing with antibodies to α-synuclein. Scale bar, 10 μιη. * 10-month-old mice at p <0.01 unpaired, .n = 8- by both Student's t tests.

10 shows reduced neurodegenerative and pre-inflammatory cytokines after passive immunization with 274 antibodies. Low magnification view (x20) of sections immunostained with antibodies against NeuN in α-synuclein tg and non-tg mice treated with A , IgG or 274 antibodies. Α-synuclein tg mice treated with IgG show neuronal loss in the hippocampus (arrow). Scale bar, 250 μm. B , Stereoscopic Analysis of NeuN in Hippocampus (Anatomy Method). C , high magnification views (x630), scale bars, 20 μm of sections immunostained with antibodies to synaptophysin in α-synuclein tg and non-tg mice treated with IgG or 274 antibodies, respectively. D , stereoscopic analysis of synaptophysin in the hippocampus (anatomist method). Low magnification views (x20) of sections immunostained with antibodies against GFAP in α-synuclein tg and non-tg mice treated with E , IgG or 274 antibodies. Α-synuclein tg mice treated with IgG show increased astrocytes in the neocortex and hippocampus. Scale bar, 250 μm. F , Image analysis of GFAP immune response expressed at absorbance. The measurement is performed on the entire hippocampus. G , high magnification view (x630), scale bar, 20 μm of hippocampal sections immunostained with antibodies against Iba-1 in α-synuclein tg and non-tg mice treated with IgG or 274 antibodies, respectively. H , Image analysis of the estimated number of microglia in the hippocampus. * p <0.05 by unilateral ANOVA with post hoc Dunnet s compared to non-tg and α-synuclein tg; p <0.05 by unilateral ANOVA with post hoc Fisher compared to IgG and 274 antibodies at # α-synuclein tg. IL , sections were immunolabeled with antibodies against TNFα and IL-6 and microdensitometric analysis was performed in the hippocampus. In α-synuclein tg mice treated with IgG compared to non-tg controls, the levels of TNFα ( I , J ) and IL-6 ( K , L ) were increased, but treatment with 274 antibody was not affected by α-synuclein. reduced levels of these psychocaine in tg mice. * P <0.05 by unilateral ANOVA, Fisher and post hoc Dunnet's when comparing treatment groups and comparing non-tg and α-synuclein tg.

The present invention is described in more detail with reference to the following non-limiting examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as being limited by the following examples.

Antibodies used in the present invention include: α-synuclein monoclonal antibody (BD Biosciences, # 610787), α-synuclein polyclonal antibody (Cell Signaling Technology, # 2642), myc polyclonal antibody (Abcam, # ab9106), CD32 Polyclonal antibodies (United States Biological, # c2384-0B), and CD16 / CD32 monoclonal antibodies (Abcam, # ab25235), GM130 monoclonal antibodies (BD Biosciences, # G65120), cathepsin D monoclonal antibodies (Abcam, # ab6313), and caveolin-1 monoclonal antibody (BD Biosciences, # C13620). FITC-labeled cholera toxin B subunit (CTB) was purchased from Sigma. Bodipy-labeled GM1, Bodipy-FL LDL, and Alexa Fluor 568-attached Dextran were purchased from Invitrogen. UltraLink immobilized protein A / G, Gentle Ag / Ab binding buffer pH 8.0, and IgG prep elution buffer from Piercef.

Example 1 Antibody Production

The production and properties of monoclonal antibodies against α-synuclein are described in Lee et al., 2011b. All antibodies against α-synuclein are monoclonal antibodies of the IgG2a isotype. Epitopes of 62 and 274 antibodies are present at the C-terminus of α-synuclein (120-140), but epitopes of 169 and 171 antibodies require both C-terminal regions (120-140) and intermediate regions (61-95) Shall. Species reactivity tests indicate that 169, 274, and 171 antibodies respond only to human α-synuclein, while 62 antibodies are reactive to both human and mouse α-synuclein. The antibodies used in the present invention do not distinguish between different forms. In particular 274 antibodies show immunoreactivity to both the extracellular and intraplasma forms of α-synuclein and to the monomer and aggregate forms. Mixtures of control IgG, mouse IgG isotypes were prepared from normal mouse serum collected using protein A / G columns.

Example 2: Purification of α-synuclein and production of fibrils and oligomers

Wild-type human α-synuclein was purified as described by Lee HJ, Bae EJ, Jang A, Ho DH, Cho ED, Suk JE, Yun YM, Lee SJ (2011b) J Neurosci Methods 199: 249-257. For fibrillation, α-synuclein (3 mg / ml in PBS) was incubated at 37 ° C. for 2 weeks with continued stirring at 250 rpm. After short sonication, α-synuclein was incubated for another week. After incubation, the protein was centrifuged at 100,000 × g for 1 hour and the pellet was resuspended in PBS. In some experiments, small fibril sections prepared by sonication were used. α-synuclein oligomers were prepared by the methods described in Danzer KM, Haasen D, Karow AR, Moussaud S, Habeck M, Giese A, Kretzschmar H, Hengerer B, Kostka M (2007) J Neurosci 27: 9220-9232. Lyophilized α-synuclein was dissolved in 50 mM sodium phosphate, pH 7.0 with 20% ethanol to a final concentration of 0.1 mg / ml and stirred at 250 rpm for 4 hours at room temperature. α-synuclein was lyophilized again and resuspended in 50 mM sodium phosphate, pH 7.0 with 10% ethanol of 1/2 starting volume. The protein was incubated at room temperature for 24 hours with the lid open for evaporation of residual ethanol.

Example 3: Cell Culture

SH-SY5Y cells were maintained and differentiated as described in Lee HJ, Khoshaghideh F, Patel S, Lee SJ (2004) J Neurosci 24: 1888-1896. BV-2 microglia cell lines were maintained in DMEM with 5% fetal bovine serum (FBS) and penicillin and streptomycin. Hybridoma cells were cultured in DMEM with 10% FBS and 100 μM hypoxanthine supplement (Invitrogen). Rat primary glial cultures were prepared as described in Lee HJ, Suk JE, Bae EJ, Lee SJ (2008) Biochem Biophys Res Commun 372: 423-428.

Primary cortical neurons were obtained from embryos on day 15-16 (E15-16) of Sprague Dawley rats according to the method described in Lee HJ, Patel S, Lee SJ (2005) J Neurosci 25: 6016-6024.

Example 4: Preparation of Conditioned Media

Differentiated SH-SY5Y cells were injected with adeno / α-syn. Two days after injection, cells were washed three times with DMEM and incubated in serum-free DMEM. After 18 hours of incubation at 37 ° C, the conditioned medium was collected and 4 ° C, 250xgin After centrifugation for 10 minutes, the supernatant was removed at 10,000 x 4 ° C to remove cellular debris.gin Centrifuge for 10 minutes. The supernatant was concentrated using an Amicon 10K MWCO filter (Millipore).

Example 5: Uptake and Degradation of α-Synuclein Aggregates and Monomers in Cells

BV-2, microglia and astrocytes were divided into petri dishes the day before the experiment. After washing the cells twice with cold PBS, 0.2 μM α-synuclein fibrils or oligomers previously incubated with 5 μg / ml normal mouse IgG or α-synuclein antibodies at room temperature were added to the culture medium and cells. Monomer was added at 1 μM. Conditioned medium (5 × concentrated) was preincubated with antibody for 5 minutes. Cells were then incubated at 37 ° C. and collected at the time indicated. To determine the rate of degradation, aggregates were taken up as above and cells were washed twice with cold PBS. Fresh culture medium was added and culture of cells was performed at 37 ° C. for the time stated.

Example 6 Uptake of α-Synuclein Antibodies

BV-2 cells treated with 5 μg / ml of 274 antibody or control IgG were preincubated with various amounts of α-synuclein fibrils (0, 2.66, 13.3, 66.5, 133, and 330 nM). To rule out the possibility of nonspecific adsorption of the 274 antibody, 5 μg / ml of 274 antibody preincubated with various amounts of α-synuclein monomer (0, 66.5, and 330 nM) was added to the cells. Levels of absorbed antibodies were detected with anti-mouse IgG antibodies.

Example 7: Preparation of Cell Extracts

Cells were washed with PBS and disrupted in extraction buffer [PBS, 1% Triton X-100, 1% (v / v) Protease Inhibitor Mixture (Sigma)]. After incubation on ice for 10 minutes, the cell lysate was centrifuged at 16,000 x g for 10 minutes. Triton X-100 insoluble pellets were resuspended in Laemmli sample buffer and sonicated briefly.

Example 8: Western Blotting

Western blotting was performed as described in Lee HJ, Lee SJ (2002) J Biol Chem 277: 48976-48983. Images were obtained and quantified using Fujifilm Luminescent Image Analyzer LAS-3000 and Multi Gauge (v3.0) software (Fujifilm).

For quantification, all lanes were analyzed from the start of the running gel to the α-synuclein monomer band.

Example 9: Immunofluorescence Staining of Cultured Cells

The procedure for cell staining was performed as described in Lee HJ, Lee SJ (2002) J Biol Chem 277: 48976-48983.

In summary, cells cultured on poly-L-lysine-coated coverslips were immobilized in 4% paraformaldehyde in PBS and taken up in 0.1% Triton X-100. Cells were incubated in blocking solution (5% bovine serum albumin / 3% goat serum in PBS) prior to addition of the primary antibody diluted in blocking solution. After washing, the cells were incubated with fluorescent dye attached secondary antibody. Nuclei were stained with TOPRO-3 iodide (Invitrogen) and coverslips mounted on slide glass using antifade reagent (Invitrogen). Olympus FV1000 confocal laser scanning microscope was used for the observation of the cells.

Example 10 Intracerebral Injection of Tg Mouse Main and Antibodies

For the present invention, mouse (line M) overexpressing α-synuclein derived from the platelet-derived growth factor-beta (PDGF-beta) promoter was used (Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M Takeda A, Sagara Y, Sisk A, Mucke L (2000) Science 287: 1265-1269). This model was chosen because mice derived from these lines generate α-synuclein aggregates in both neuronal and glial cells distributed throughout the renal cortex, hippocampus, and striatum, similar to those described in Lewy body disease. In these mice, α-synuclein accumulates in neural networks and neuronal cell bodies in the deep layers of the neocortex and hippocampus. Α-synuclein begins to accumulate in neurons at 3 months of age and has maximum accumulation at 12 months of age. Thus, in this tg mouse model, α-synuclein pathology proceeds in a very constant and spatiotemporal order. All animal testing procedures were approved by the Experimental Animal Ethics Committee. All experiments were performed in female mice. Eight mice per group were used for each experiment. A total of 32 mice were used for this experiment; 16 mice were α-synuclein tg mice and the other 16 non-tg animal controls (9 months of age). Mice from each group were treated with 3 μl of non-immune IgG controls ( n = 8 non-tg and n = 8 α-synuclein tg) or antibodies against α-synuclein (clone 274; 1 mg / ml) ( n = 8 non -tg and n = 8 α-synuclein tg) were injected into hippocampus (0.25 μl / min using a 5 μl Hamilton syringe). In summary, mice placed on Koft stereotactic devices and coordinates (hippocampus: AP: 2.0 mm, outer 1.5 mm, depth 1.3 mm) under anesthesia were placed in Franklin and Paxinos (Franklin KBJ, Paxinos G, (2008) The mouse brain in stereotaxic coordinates , Ed 3. Burlington, MA: Elsevier). Hamilton syringes connected to a hydraulic system were used for antibody delivery for injection of the solution. For diffusion of the solution into brain tissue, the needles were held for 5 minutes after completion of injection.

Mice were unilaterally injected (right) for comparison with the contralateral side. Mice were survived 4 weeks after antibody injection. An additional group of twelve α-synuclein tg mice (3 months old) were unilaterally injected into the hippocampus with antibodies against α-synuclein (clone 274; 1 mg / ml) and at 1, 7, 14, and 28 days after injection Sacrificed for analysis. Mice were anesthetized with chloral hydrate and perfused to the heart with 0.9% saline according to the National Institutes of Health guidelines for human treatment of animals. The brain was removed and fixed for 48 hours at 4 ℃ in phosphate-buffer 4% paraformaldehyde, pH 7.4 for neuropathology analysis.

Example 11: Passive Immunization and Behavioral Studies

A total of 32 mice were used in this experiment, of which 16 were α-synuclein tg mice (line M, 10 months old) and the remaining 16 were non-tg animal controls (10 months old). Mouse monoclonal antibodies ( n = 8 non) against non-immune IgG controls ( n = 8 non-tg and n = 8 α-synuclein tg) or α-synuclein (clone 274; 1 mg / ml) in each group -tg and n = 8 α-synuclein tg) were given intraperitoneally at a concentration of 1 mg / ml (100 μl / week, for 4). Mice were survived for 1 month after the first injection and as described in Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L (2000) Science 287: 1265-1269. In the pole test a test for motor function and a spontaneous activity in the open field were performed. Behavioral tests were performed to perform mouse neuropathology analysis.

Example 12 Immunohistochemical Analysis of Neuropathology and α-Synuclein Accumulation

Analysis of α-synuclein accumulation was performed on a series of sections, pre-floating, derived from non-tg and α-synuclein tg mice treated with antibodies to IgG or α-synuclein (clone 274). Sections were overnight incubated with anti-α-synuclein antibody (1: 500, affinity purified rabbit polyclonal; Millipore Bioscience Research Reagents) at 4 ° C., followed by biotinylated goat anti-earth IgG (1: 100; Vector Laboratories) After treatment with Avidin D-horseradish peroxidase (1: 200, ABC Elite; Vector Laboratories), it was detected with diaminobenzidine (DAB). Sections were imaged with an Olympus brightfield digital microscope. For each case, three sections were analyzed by dissection analysis using the Stereo-Investigator System (MBF Bioscience) and the results were averaged and expressed in number / mm ³ . To analyze the effect of antibodies on neurodegenerative and neuroinflammatory responses, sections were analyzed for NeuN (Millipore), glial fibrillary acidic protein (GFAP) (Millipore), Iba1 (Wako), IL6 (Novus Biologicals), and tumor necrosis factor Mouse monoclonal antibody against Abcam) and detected with DAB. Sections immunostained with antibody GFAP and Iba-1 were imaged with an Olympus brightfield digital microscope and absorbance and cell counts were analyzed with an Image Pro-Plus system. Sections immunostained with antibodies to NeuN were analyzed by dissection analysis using Stereo-Investigator System (MBF Bioscience) and the results were averaged and expressed in number / mm ³ .

Example 13: Dual Immunohistochemistry and Confocal Analysis

To determine co-location, 40-μm-thick vibratomic fragments were prepared using α-synuclein (Millipore Bioscience Research Reagents, affinity purified polyclonal, 1: 500), mouse IgG (to detect 274 antibody distribution), and Double labeling with a combination of antibodies to Iba-1 (microglia marker). α-synuclein immunoreactive structures were detected by the Tyramide Signal Amplification-Direct (Red) system (1: 100; NEN Life Sciences) while mouse IgG or Iba-1 were fluorescein isothiocyanate (FITC) -attached horse anti-mouse antibodies (1:75; Vector Laboratories).

All sections were run simultaneously under the same conditions and two experiments were performed to evaluate the reproducibility of the results. Sections were imaged with a Zeiss 63 x (NA 1.4) objective on an Axiovert 35 microscope (Zeiss) attached with an MRC1024 LSCM system (Bio-Rad). For each section, a series of optical sections were obtained in a 10 μm plane and used for the analysis of the percentage of Iba-1-positive cells representing α-synuclein-immunoreactive material. From each case, an average of 120 Iba-1-positive cells was analyzed (30 per segment). Further studies to determine the co-existence of α-synuclein in neurons or astroglia after immunization include NeuN (neuron marker; Millipore, mouse monoclonal, 1: 1000) or GFAP (astroglia marker; Millipore, mouse monoclonal, 1). : 2000) and fragments double-labeled with antibodies to α-synuclein (rabbit polyclonal; Millipore, 1: 5000). α-synuclein was detected by the Tyramide Signal Amplification-Direct (Red) system (1: 100; NEN Life Sciences), while NeuN or GFAP was detected by FITC.

Example 14 Statistical Analysis

Values shown in the figures are expressed as mean ± SEM. P value for determining the statistical significance of the deviation Paired or unpaired, both Student's using GraphPad InStat version 3.05 softwarettest, one-way ANOVA with apost hocCalculated via Dunnet or one-way ANOVA with Tukey post test. For statistical analysis of the absorption kinetics data, the best-fit relative values of α-synuclein obtained through independent experiments were analyzed by an extra addition sum sum F test using Graph-Pad InStat version 3.05 software.

Claims (6)

1. An antibody that specifically binds to the α-synuclein peptide set forth in SEQ ID NO: 1.
2. Parkinson's disease prevention or treatment composition comprising the antibody of claim 1 as an active ingredient.
3. A composition for preventing or treating Lewy Body Dementia (DLB) comprising the antibody of claim 1 as an active ingredient.
4. When a candidate substance and extracellular α-synuclein protein are contacted with microglia in an environment suitable for the two components to interact, and the rate of uptake and degradation of the α-synuclein protein in the microglia is increased. A method for identifying candidates for Parkinson's disease or Lewy body dementia (DLB) treatment, characterized in that the candidate substance is determined as a candidate for Parkinson's disease or Lewy body dementia (DLB) treatment.
5. 5. The method of claim 4, wherein the candidate is an antibody. The method of identifying candidates for Parkinson's disease or Lewy Body Dementia (DLB).
6. 5. The method of claim 4, wherein the microglia express Fcγ receptors. 6.

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