JP2010506569A - Cells expressing α-synuclein and uses thereof - Google Patents

Abstract

Disclosed are screening methods for identifying mammalian neuronal cells that express alpha synuclein and compounds that reduce toxicity induced by alpha synuclein expression. Using compounds identified by such screening, Parkinson's disease, familial Parkinson's disease, Lewy body disease, Lewy body variants of Alzheimer's disease, Lewy body dementia, multiple system atrophy, or Guam Synucleinopathies such as Parkinson dementia complex can be treated or prevented.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 60 / 829,320, filed Oct. 13, 2006. The entire contents of this prior application are incorporated herein by reference.

TECHNICAL FIELD The present invention relates to α-synuclein expressing cells and screening methods for identifying compounds that reduce toxicity induced by α-synuclein expression.

Background Parkinson's disease is a neurodegenerative disorder that is pathologically characterized by the presence of Lewy bodies in the cytoplasm (Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer, Berlin, pp. 920-933, 1912). (Non-patent document 1); Pollanen et al., J. Neuropath. Exp. Neurol. 52: 183-191, 1993 (non-patent document 2)), the main component of cytoplasmic Lewy bodies is an α-amino acid of 140 amino acids. A fiber composed of synuclein protein (Spillantini et al., Proc. Natl. Acad. Sci. USA 95: 6469-6473, 1998 (non-patent document 3); Arai et al., Neurosci. Lett. 259: 83-86 , 1999 (Non-Patent Document 4); Ueda et al., Proc. Natl. Acad. Sci. USA 90: 11282-11286, 1993 (Non-Patent Document 5)). Three dominant mutations in α-synuclein that cause familial early-onset Parkinson's disease have been described, suggesting that Lewy bodies contribute mechanistically to neuronal degeneration in Parkinson's disease and related disorders (Polymeropoulos et al. al., Science 276: 2045-2047, 1997 (non-patent document 6); Kruger et al., Nature Genet. 18: 106-108, 1998 (non-patent document 7); Zarranz et al., Ann. Neurol. 55 : 164-173, 2004 (non-patent document 8)). Triple and double mutations in the α-synuclein gene have been linked to early onset of Parkinson's disease (Singleton et al., Science 302: 841, 2003; Chartier-Harlin at al. Lancet 364: 1167-1169, 2004 (Non-Patent Document 10); Ibanez et al., Lancet 364: 1169-1171, 2004 (Non-Patent Document 11)).

  Because α-synuclein is commonly involved in a range of diseases, including Parkinson's disease, Lewy body dementia, multiple system atrophy, and Lewy body variants of Alzheimer's disease, these diseases It is classified under the generic term “synucleinopathy”. Protecting neurons from the toxic effects of α-synuclein is a promising strategy for treating these diseases. Therefore, there is a need for a system that allows the identification of compounds and compositions that block or suppress alpha-synuclein-induced toxicity in neurons. Such compounds and compositions are useful in treating or preventing synucleinopathies.

Lewy in Handbuch der Neurologie, M. Lewandowski, ed., Springer, Berlin, pp. 920-933, 1912 Pollanen et al., J. Neuropath. Exp. Neurol. 52: 183-191, 1993 Spillantini et al., Proc. Natl. Acad. Sci. USA 95: 6469-6473, 1998 Arai et al., Neurosci. Lett. 259: 83-86, 1999 Ueda et al., Proc. Natl. Acad. Sci. USA 90: 11282-11286, 1993 Polymeropoulos et al., Science 276: 2045-2047, 1997 Kruger et al., Nature Genet. 18: 106-108, 1998 Zarranz et al., Ann. Neurol. 55: 164-173, 2004 Singleton et al., Science 302: 841, 2003 Chartier-Harlin at al. Lancet 364: 1167-1169, 2004 Ibanez et al., Lancet 364: 1169-1171, 2004

Overview The present invention is toxic to cells by the expression of α-synuclein alone, without the need to treat the cells simultaneously with additional compositions (e.g., toxicity-inducing agents or neural differentiation factors). Thus, α-synuclein is based in part on the discovery that an inducible expression system can be stably expressed in cells of mammalian neural origin.

  The present invention focuses on mammalian neural cells comprising a stably integrated expression construct comprising an inducible promoter operably linked to a nucleic acid encoding a protein comprising human alpha synuclein, in which toxicity induction Inducing expression of the nucleic acid without simultaneously treating the cells with agents or neural differentiation factors reduces cell viability. The α-synuclein can be, for example, wild-type α-synuclein, or mutant α-synuclein (eg, A53T mutant human α-synuclein, A30P mutant human α-synuclein, or E46K mutant human α-synuclein). . Alpha synuclein can be full length alpha synuclein or a functional fragment of alpha synuclein. The mammalian nerve cell can be a human nerve cell. Mammalian neurons are neuronal cell lines (e.g., H4 cell line, PC12 cell line, SK-N-SH cell line, SH-SY5Y cell line, Neuro-2a cell line, U87 MG cell line, or Any other neuronal cell line described) or derived from a neuronal cell line. The protein can be a fusion protein comprising a detectable protein such as, for example, a fluorescent protein, an enzyme, or an epitope. In some embodiments, the fluorescent protein can be a red fluorescent protein, a green fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein, and a cyan fluorescent protein. In some embodiments, the enzyme can be β-galactosidase, luciferase, alkaline phosphatase, or horseradish peroxidase. In some embodiments, the epitope can be a FLAG, HA, His6, AU1, Tap, protein A, or MYC epitope. Mammalian neurons can be isolated cells or non-human mammals (e.g., mouse, rat, rabbit, guinea pig, goat, dog, cat, horse, cow, whale, or monkey) or non- It can be present on a human mammal.

  In some embodiments, the mammalian neuronal cell can comprise a stably integrated repressor construct that constitutively expresses the repressor protein, wherein the (i) inducer is exogenous. The repressor protein binds to the inducible promoter to suppress nucleic acid expression, and (ii) when the inducer is added exogenously, the repressor protein binds to the inducer, Nucleic acid is expressed. The repressor can be a Tet repressor, such as a reverse tetracycline-regulated transactivator (rtTA). The inducer can be tetracycline or a tetracycline derivative or analog such as doxycycline.

  The present specification also focuses on non-human mammals containing any of the mammalian mammalian cells described above. The non-human mammal can be any of the non-human mammals described herein.

  The present invention also provides a method of inducing toxicity in mammalian neurons, wherein the method is toxic to cells of a nucleic acid (eg, a nucleic acid comprising an α-synuclein coding sequence) in a mammalian neuron. Inducing a certain level of expression. The mammalian neural cell can be any of the mammalian neural cells described above. Thus, the α-synuclein can be any of the α-synucleins described herein.

  Further provided are methods of inducing toxicity in mammalian neurons. This method involves the induction of any of the above neurons with an exogenously added amount effective to induce expression and toxicity of a nucleic acid (eg, a nucleic acid comprising an α-synuclein coding sequence) in the cell. Contacting with a substance. The α-synuclein can be any α-synuclein described herein.

  The present specification also focuses on methods for identifying compounds that block or inhibit alpha-synuclein-induced toxicity, which includes (i) cells in the presence of and in the absence of a candidate agent. Culturing any of the mammalian neurons described above under conditions that allow expression of the nucleic acid at a level sufficient to induce toxicity in said cell; (ii) cell viability in the presence of said candidate agent; And (iii) comparing cell viability measured in the presence of said candidate agent with cell viability in the absence of said candidate agent, wherein cell viability is said candidate agent A candidate drug is identified as a compound that inhibits or suppresses alpha-synuclein-induced toxicity when it is higher in the presence of the candidate drug compared to in the absence of.

  Also provided are methods for identifying compounds that block or suppress α-synuclein-induced Golgi fragmentation, the method comprising (i) Golgi fragment in a cell in the presence of and in the absence of the candidate agent. Culturing any of the above mammalian neural cells in the presence of an exogenously added inducer in an amount effective to induce expression of the nucleic acid at a level that induces oxidization; (ii) Measuring Golgi fragmentation in cells in the presence of the candidate agent; and (iii) Golgi fragmentation in cells measured in the presence of the candidate agent, and Golgi fragmentation in the absence of the candidate agent. A candidate agent that inhibits α-synuclein-induced Golgi fragmentation when Golgi fragmentation is less in the presence of the candidate agent compared to the absence of the candidate agent Or suppress It is identified as an object.

  Also noted are methods for identifying compounds that increase vesicular transport from the endoplasmic reticulum to the Golgi. The method comprises (i) an amount effective to induce nucleic acid expression at a level that reduces vesicular transport from the endoplasmic reticulum to the Golgi in the presence of the candidate agent and in the absence of the candidate agent. Culturing the cell according to any one of claims 15 to 17 in the presence of an exogenously added inducer; (ii) small endoplasmic reticulum to Golgi in the cell in the presence of the candidate agent; Measuring vesicular transport; and (iii) vesicle transport from the endoplasmic reticulum to the Golgi in the cells measured in the presence of the candidate drug, and from the endoplasmic reticulum measured in the absence of the candidate drug to the Golgi. Vesicle transport in a cell in the presence of the candidate agent as compared to vesicle transport from the endoplasmic reticulum to the Golgi in the absence of the candidate agent. Increase in vesicle transport from the Golgi to the Golgi Identified as a compound that increases vesicular transport from the endoplasmic reticulum to the Golgi.

  We also looked at ways to identify compounds that increase vesicle docking and fusion, which includes (i) reducing vesicle docking and fusion in cells in the presence of and in the absence of a candidate agent. Culturing any of the above mammalian neuronal cells in the presence of an exogenously added inducer in an amount effective to induce expression of the nucleic acid at a level to allow; (ii) the candidate agent Measuring vesicle docking and fusion in cells in the presence of; and (iii) vesicle docking and fusion in cells in the presence of the candidate agent The method comprises the steps of: docking and fusion of a vesicle in the presence of the candidate agent as compared to docking and fusion of the vesicle in the absence of the candidate agent. Increase By, the candidate agent is identified as a compound that increases the docking and fusion of the vesicles.

  The invention also provides a method for identifying a compound that increases secretion of a protein from a cell. The method was (i) added exogenously in an amount effective to induce nucleic acid expression at a level that reduces protein secretion from the cell in the presence and in the absence of the candidate agent. Culturing any of the above mammalian neuronal cells in the presence of an inducer; (ii) measuring protein secretion from the cells in the presence of the candidate agent; and (iii) of the candidate agent Comparing protein secretion from the cell in the presence to protein secretion in the absence of the candidate agent, the method comprising comparing the protein secretion in the absence of the candidate agent An increase in protein secretion from the cell in the presence of the candidate agent identifies the candidate agent as a compound that increases protein secretion from the cell.

  Also noted is a method of treating synucleinopathies, wherein the method is identified in an individual diagnosed as having synucleinopathies (e.g., a human patient) by any of the methods described herein. Administering a pharmaceutical composition comprising a therapeutically effective amount of the compound. Optionally, the method may also include selecting and / or diagnosing the individual as having an synucleinopathy or at risk of developing a synucleinopathy. The synucleinopathy is, for example, Parkinson's disease, familial Parkinson's disease, Lewy body disease, Lewy body variant of Alzheimer's disease, Lewy body dementia, multiple system atrophy, or Guam Parkinson dementia complex obtain. A synucleinopathy can be a synucleinopathy caused by a mutation in α-synuclein, such as any of the mutations described herein.

  “Toxicity inducer” means toxicity in cells expressing α-synuclein that exceeds the level of toxicity (if any) caused by expressing only α-synuclein in the cell or contacting the cell only with a toxicity-inducing agent. Is a composition that induces levels of A “toxicity-inducing agent” can be a compound (eg, a proteasome inhibitor) that is administered at a level that is not itself toxic to cells, but induces toxicity when combined with the expression of α-synuclein. In some embodiments, a “toxicity inducing agent” is one or more that induces a mammalian cell for cytotoxicity or otherwise sensitizes a mammalian cell when combined with α-synuclein expression. It can be a genetic element (eg, a mutation such as a gene inactivating mutation). For example, the mutation may be a ts41 mutation in APPBP1, resulting in a defect in the ubiquitin-proteasome pathway.

  As used herein, a “neural differentiation factor” is a protein that induces differentiation of nerve cells. Examples of neural differentiation factors include NGF, BDNF, neurotrophin-3, and neurotrophin-4.

  The advantage of the neuronal cells described herein is that they allow stable and regulated expression of α-synuclein (including wild-type α-synuclein), as α-synuclein expression alone is toxic to cells. is there. Thus, compounds identified to rescue the toxicity that occurs in these α-synuclein expressing cells, for example, rather than act on toxicity-inducing agents or some other cofactor used to induce cytotoxicity. -It is thought to mediate its beneficial effects through synuclein-related pathways.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

  Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

FIG. 4 is a schematic diagram showing tetracycline-regulated conditional expression of wild-type α-synuclein in H4 cells. Tetracycline-regulated expression of α-synuclein was due to the use of regulatory elements derived from the tetracycline (Tet) resistant operon encoded by E. coli Tn10. Two expression cassettes expressing Tet repressor (TR) or α-synuclein (Syn) were integrated into the host cell genome by a lentiviral system. The Tet repressor is constitutively transcribed under the CMV promoter. Two tetracycline response elements (TRE) were inserted into the promoter region. When the Tet repressor binds to TRE, α-synuclein transcription is stopped. In the presence of tetracycline (ie, inducing conditions), binding of the tetracycline to the Tet repressor releases this protein from the TRE, resulting in the expression of α-synuclein. FIG. 3 is a photograph of an immunoblot showing conditional overexpression of wild type α-synuclein in TS217 cells. TS217 cell lysates were collected before induction with tetracycline (-) or after induction for 1 day (1d) or 3 days (3d). Parental H4 (C) cell lysates were also collected to show endogenous levels of α-synuclein protein. Lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and proteins were detected using antibodies specific for α-synuclein or actin shown on the left. FIG. 6 is a line graph showing progressive cytotoxicity in H4 cells overexpressing wild-type α-synuclein. Α-synuclein was induced by adding tetracycline for 3-6 days. Cytotoxicity was analyzed by measuring cellular ATP levels. As an index of cytotoxicity induced by α-synuclein, relative cell viability was calculated as the ratio of induced cells to control cells. The Y axis represents relative viability and the X axis represents days. Error bars represent the standard deviation of 8 iterations. It is a photograph of live TS217 cells. TS217 H4 cells were plated on glass tissue culture chamber slides coated with poly-L-lysine. Cells were cultured for an additional 5 days with or without induction of α-synuclein tetracycline (control). Live cell calcein AM staining was imaged with a fluorescence microscope (Olympus) equipped with a digital camera controlled by the Metamorph program (Universal Imaging, West Chester, Pa.). 4B is a bar graph showing the quantification of cytotoxicity determined by the calcein imaging of FIG. 4A and quantified using Metamorph software. ^Unmatched t test, ^*** P <0.0001. Figure 2 is a line sigmoidal curve-fit showing the concentration response of tetracycline in a cell viability assay. Stable H4 clones were incubated with various concentrations of tetracycline for 6 days in 96 well plates. Cells were lysed and cellular ATP was measured. The medium alone was used as a control. Error bars represent the standard deviation of 8 iterations. The X-axis represents the logarithm of tetracycline concentration (μg / mL), and the Y-axis represents ATP concentration expressed as relative light units (RLU). It is a photograph of a cell and shows Golgi fragmentation in wild type α-synuclein overexpressing cells. TS217 H4 cells were plated on glass tissue culture chamber slides coated with poly-L-lysine. Cells were cultured for an additional 6 days with or without α-synuclein tetracycline induction (control) or with (induction). GM130 immunoreactive Golgi complex was imaged. FIG. 6B is a bar graph showing quantitative analysis of cells with normal Golgi complex (according to FIG. 6A). The intact Golgi structure was captured by eliminating the small focal point representing the fragmented Golgi. ^Unmatched t test, ^*** P <0.0001. It is a photograph of the cultured cell which shows double immunofluorescence staining. H4 cells were plated on glass tissue culture chamber slides coated with poly-L-lysine. Cells were cultured for an additional 6 days with or without (control) or tetracycline induction of α-synuclein. Cells were stained using antibodies specific for GM130 or mannosidase II, respectively. A high magnification field of view of the Golgi apparatus clearly showed changes in Golgi morphology in α-synuclein overexpressing cells. It is a photograph of cultured cells stained with mannosidase II or calnexin showing the integrity of the endoplasmic reticulum (ER) in tetracycline treated cells. TS217 H4 cells were plated on glass tissue culture chamber slides coated with poly-L-lysine. Cells were cultured for an additional 5 days with or without induction of α-synuclein tetracycline (control). Calnexin immunoreactive ER and mannosidase II immunoreactive Golgi complex were analyzed. 1 is a bar graph showing rescue of toxicity induced by α-synuclein by compounds. TS217 cells were treated with tetracycline to induce α-synuclein expression for 5 days. During 5 days induction, cells were treated with various concentrations of 1-t-butyl-3- (4-chloro-phenyl) -1H-pyrazolo [3,4-d] pyrimidin-4-ylamine (Cmp J) (0 , 0.08 μM, 0.15 μM, and 0.3 μM). Relative viability was determined by measuring ATP concentration in each cell set, and relative viability was the ratio of induction to control as described above. The Y axis represents relative viability and the X axis represents the concentration of the compound. The asterisk indicates the result of the Kruskal-Wallis test: ^* , ^** , ^*** : P <0.05, 0.01, and 0.001, respectively. 2 is a bar graph showing the influence of plating density on cytotoxicity induced by α-synuclein. Serially diluted TS217 cells were plated into 96-well plates at dilutions of 250, 500, 1000, 2000, and 4000 cells / well. Cells were incubated for 5 days in the presence of tetracycline. Following incubation, cells were harvested and lysed to determine the intracellular ATP concentration. Relative cell viability was calculated as the ratio of induced cells to control cells. Error bars represent the standard deviation of 6 iterations. FIG. 4 is a scatter plot showing inhibition of α-synuclein-induced cytotoxicity in TS217 cells by forskolin (FSK). The X axis represents the dose of FSK in micromolar (μM). The Y axis represents the relative concentration of ATP (expressed as relative light units (RLU)) in TS217 cells as a function of cell viability.

Detailed description
α-Synuclein Nucleic Acids and Proteins The compositions and methods disclosed herein use proteins that include α-synuclein polypeptides. The term “alpha synuclein” encompasses native alpha synuclein sequences (eg, natural human wild type and mutant alpha synuclein) and functional variants thereof.

によってコードされる。 Human alpha synuclein has the following nucleotide sequence:

Coded by.

を有する。「変種」という用語は、本明細書において、機能的な断片、変異体、および誘導体を含むよう用いられる。例えば、「変種」は、特定の部位において、天然および非天然アミノ酸を含む天然アミノ酸の置換(例えば、保存的アミノ酸置換)を含み得るがそれに限定されるわけではない。いくつかの態様において、αシヌクレインタンパク質は、SEQ ID NO:2のアミノ酸配列と少なくとも80%、85%、90%、95%、または98%同一であり、α-シヌクレイン機能を保持する。 Human α-synuclein has the following amino acid sequence:

Have The term “variant” is used herein to include functional fragments, variants, and derivatives. For example, a “variant” can include, but is not limited to, substitution of natural amino acids, including natural and unnatural amino acids (eg, conservative amino acid substitutions) at a particular site. In some embodiments, the alpha synuclein protein is at least 80%, 85%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 2 and retains alpha-synuclein function.

  As used herein, α-synuclein `` activity '' or `` function '' refers to cytotoxicity (e.g., loss of Golgi integrity and / or induction of cell death (e.g., Including, but not limited to, the ability to cause (measured by a decrease in cellular ATP concentration or apoptosis). Without being limited by any particular mechanism of action, cytotoxicity can be caused by the formation of inclusion bodies or aggregates in the cell, or impaired proteasome activity.

  In some embodiments, full-length α-synuclein protein can be used. The term “full length” refers to an α-synuclein protein comprising all amino acids encoded by α-synuclein cDNA. In other embodiments, different lengths of α-synuclein protein may be used. For example, only functional active domains of proteins can be used. Thus, almost any length protein fragment can be used if it is functional.

  In certain embodiments, variants of α-synuclein protein can be used. Such variants can include biologically active fragments of α-synuclein protein. These include proteins with amino acid substitutions with α-synuclein activity. Α-synuclein mutants that can be expressed in mammalian cells include A53T mutants (including substitution of alanine for threonine at position 53) and A30P mutants (substitution of alanine for proline at position 30). Included).

  In certain embodiments, a fusion protein comprising at least a portion of an α-synuclein protein may be used. For example, a portion of α-synuclein protein can be fused to the second domain. The second domain of the fusion protein can be selected from the group consisting of an immunoglobulin element (eg, an Fc fragment of an immunoglobulin molecule), a dimerization domain, a targeting domain, a stabilization domain, and a purification domain. Alternatively, a portion of the α-synuclein protein can be fused with a heterologous molecule such as a detection protein. Exemplary detection proteins include: (1) fluorescent proteins such as green fluorescent protein (GFP), cyan fluorescent protein (CFP), or yellow fluorescent protein (YFP); (2) β-galactosidase or alkaline phosphatase (AP), etc. And (3) an epitope such as glutathione-S-transferase (GST) or hemagglutinin (HA). For example, α-synuclein protein can be fused to GFP at the N-terminus or C-terminus or other portion of the α-synuclein protein. These fusion proteins provide a method for the rapid and easy detection and identification of α-synuclein proteins in recombinant host cells exemplified herein by mammalian cells (e.g., mammalian neural cells). To do.

  The present specification also describes a method of preparing a nucleic acid encoding an α-synuclein protein and introducing it into a mammalian cell so that the cell can express the α-synuclein protein. The term “alpha synuclein nucleic acid” encompasses a nucleic acid comprising the sequence shown in SEQ ID NO: 1 and a nucleic acid encoding any of the alpha-synuclein variants described herein. Exemplary α-synuclein nucleic acids include those that encode wild type human α-synuclein or an A53T or A30P variant protein.

  The term “nucleic acid” generally refers to at least one molecule or strand of DNA, RNA, or a derivative or mimetic thereof, comprising at least one nucleobase such as, for example, a natural purine base or pyrimidine base found in DNA or RNA. Point to. In general, the term “nucleic acid” refers to at least one single-stranded molecule, but in certain embodiments is partially, substantially, or completely complementary to the at least one single-stranded molecule. Also includes at least one additional strand. Thus, a nucleic acid also includes at least one double-stranded molecule or at least one triple-stranded molecule that includes one or more complementary strands of a particular sequence, i.e., "complement", that includes one strand of the molecule. May be.

Nucleic acid vectors for inducing and expressing α-synuclein in mammalian cells Non-limiting examples of using nucleic acid vectors including plasmids, linear nucleic acid molecules, artificial chromosomes, and viral vectors to transfer α-synuclein nucleic acid into mammalian cells. Can be The vector may include an α-synuclein nucleic acid, any of the α-synuclein variants or variants described herein as a transgene.

  Once the vector or nucleic acid molecule containing the construct has been prepared for expression, a variety of suitable means such as transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, etc. In any case, the DNA construct can be introduced into an appropriate host cell. If the vector is a viral vector, the vector can be delivered to the cell by direct infection. Preferably, the vector is stably integrated into the host genome. Methods for generating cell lines containing stably integrated nucleic acids (e.g., vectors) are well known in the art (e.g., Sambrook et al. In Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY , Vol. 1, 2, 3 (1989)). Briefly, a number of antibiotics including, for example, G418, neomycin, or hygromycin B can be used to select cells transfected with nucleic acids. In general, the nucleic acid to be transfected contains the appropriate antibiotic resistance gene or is co-transfected with a vector containing the appropriate antibiotic resistance gene. Thus, when cultured in antibiotics, only cells and their progeny that contain a stably integrated nucleic acid encoding an antibiotic resistance gene will survive.

  α-synuclein can be expressed under the control of an inducible promoter. In general, inducible expression vectors are composed of one or more copies of an inducer response element, which includes (i) a mammalian transcription promoter (e.g., a CMV containing a DNA docking site for the TATA box promoter element-transcription machinery, for example). A promoter) and (ii) an operably linked nucleic acid encoding the transgene of interest (eg, α-synuclein). Within the cell containing the expression vector, there is also a transcriptional repressor protein that can bind to the inducer response element in the absence of the inducer. The repressor can be expressed endogenously or exogenously in trans from nucleic acid introduced into the cell. Transcriptional repressors can bind to inducers in addition to DNA binding, and when the inducer binds to the repressor, the repressor dissociates from the DNA (see FIG. 1). Thus, in the absence of an inducer (eg, a compound such as doxycycline, tetracycline, or tamoxifen), the repressor protein binds to one or more inducer response elements and prevents transgene transcription. However, when the repressor protein binds to the inducer, it dissociates from the inducer response element, allowing transgene transcription and subsequent expression. In general, transgene expression after treatment with an inducer depends on the dosage of the inducer (eg, the higher the concentration of inducer applied, the higher the expression of the transgene). Examples of such inducible expression vectors include, but are not limited to, the Tet-On vector system as described below, and the ecdysone inducible expression vector as manufactured by Stratagene Inc. (La Jolla, Calif.). The system is included.

  Alternatively, the cell containing the expression vector may contain a transcriptional activator that can bind to the inducer response element only in the presence of the inducer. Thus, when an inducer is applied to a cell, the expression of the transgene is still “switched on” by inducing the binding of a positive transcription factor to the inducer response element. Examples of such vectors and regulatory systems include the estrogen / tamoxifen-regulated estrogen receptor vector system.

  In some embodiments, α-synuclein polypeptides can be expressed under the control of a “Tet-On” system as illustrated in the examples below (see also FIG. 1). The Tet-on vector contains a tetracycline response element (TRE) to which a tetracycline-regulated transrepressor, rtTA, binds. RtTA, a variant of the wild-type tet repressor (TetR), is a fusion polypeptide composed of a TetR repressor and the herpes simplex virus type 1 VP16 transactivation domain. Four amino acid changes in the wild-type TetR DNA binding domain alter rtTA DNA binding properties so that the TetO sequence in the target transgene tetracycline response element (TRE) can be recognized only in the presence of tetracycline or doxycycline effectors is doing. Thus, in the Tet-On system, transcription of TRE-regulated target genes is promoted by rtTA only in the presence of tetracycline or doxycycline.

  Methods for assessing the induction of α-synuclein after administration of inducers (e.g., tetracycline or doxycycline) include Western blotting using antibodies specific for α-synuclein, or mRNA expression of α-synuclein. RT-PCR or Northern blotting techniques for detection can be mentioned. Such methods are well known to those skilled in the art and are described in detail in Sambrook et al. (In Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY, Vol. 1, 2, 3 (1989)). . Expression of α-synuclein is about 1 hour after induction (e.g., about 30 minutes, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours, about 12 hours, about 12 hours) , About 16 hours, about 20 hours, about 24 hours, about 36 hours, about 48 hours or more) (see Example 1). Maximum induction levels are often observed approximately 12-24 hours after induction. In some embodiments, changes in expression over time can be determined (evaluated) after induction. For example, the expression level of α-synuclein prior to induction (ie, before adding an inducer (eg, doxycycline or tamoxifen)) can be determined at various time points (eg, 1, 4, 8, and 12 hours after induction; 4 , 8, 12, and 16 hours; 8, 12, 16, and 20 hours; 12, 24, 36, and 48 hours).

  A suitable starting concentration of inducer is about 0.001 μM (eg, about 0.01 μM, about 0.1 μM, about 1.0 μM, about 10.0 μM, about 100 μM). The concentration of inducer can be optimized for a particular experiment and can depend, for example, on the cell line, transgene, or culture conditions in which the cells are cultured (eg, low serum conditions).

  In some embodiments, a mammalian cell (e.g., a mammalian neural cell) comprising a nucleic acid vector encoding α-synuclein under the control of an inducible promoter is a non-human mammal (e.g., a mouse, rat, or guinea pig). ) Vectors can be introduced into mammalian cells (e.g., neuronal cells) ex vivo, i.e., cells are transfected in vitro and then implanted or otherwise delivered (e.g., surgically). Can be transplanted). Alternatively, non-human transgenic animals can be established using any of a variety of techniques known in the art (eg, Manson et al. (2001) Exp. Rev. Mol. Med. 11; and See Hofker et al. Trangenic Mouse: Methods and Protocols (Methods in Molecular Biology) Humana Press, Clifton, NJ, Vol. 29 (2002)).

  Induction of α-synuclein expression in an animal can be achieved by administering an appropriate amount of an inducer to the animal. The inducer can be delivered to the mammal as part of the food or water (ie, included in the food or water), or can be administered intravenously or parenterally (eg, subcutaneous injection).

  Appropriate dosages of inducers in animal models and methods for detecting induction of transgenes are described, for example, in Teng et al. (2002) Physiol. Genomics 11: 99-107; Kim et al. (2003) Am J. Pathol. 162 (5): 1693-1707; and Zabala et al. (2004) Cancer Res. 64: 2799-2804.

  It will be appreciated that any of the above in vitro or in vivo aspects of mammalian cells can be used in the following screening methods.

Screening Methods The present invention focuses on methods for screening and identifying “candidate agents” (eg, compounds or drugs) that prevent or inhibit cytotoxicity caused by overexpression of α-synuclein in mammalian neurons. Thus, a `` candidate agent '' as referred to herein has the potential to reduce, prevent or suppress (i.e. prevent or suppress) cytotoxicity caused by overexpression of α-synuclein in mammalian neurons. Any material provided. Cytotoxicity caused by over-expression of α-synuclein in mammalian neurons can be, for example, apoptosis or necrosis, including but not limited to impaired proteasome activity in the cell, or inclusion / aggregate formation in the cytoplasm. possible. However, regardless of the exact mechanism of action, the agents identified by the screening methods herein include, but are not limited to, Parkinson's disease, familial Parkinson's disease, Lewy body disease, and Lewy body mutations in Alzheimer's disease. It may be useful in treating synucleinopathies in subjects (eg, human patients) such as type, Lewy body dementia, multiple system atrophy, or Guam Parkinson dementia complex.

  Includes, but is not limited to, nucleic acids, polypeptides, small molecule compounds, macromolecular compounds, peptidomimetics, or any other compound described herein (e.g., see `` compounds '' below). Various types of candidate agents that are not necessarily screened can be screened by the methods described herein. In some cases, the candidate agent is a genetic factor that reduces, prevents or suppresses the cytotoxicity caused by overexpression of α-synuclein in mammalian neurons. For example, cDNA libraries containing coding sequences for various genes can be screened to identify promising therapeutic genes for the diseases described herein. Alternatively, screening can be performed to identify genetic elements that contribute to the cytotoxicity caused by α-synuclein expression in mammalian cells. For example, a library of siRNA or antisense oligonucleotides can be screened so that the level or amount of cytotoxicity in the absence of one or more genes can be determined. In another example, mammalian nerve cells can be mutated to inactivate one or more genes prior to performing a screening assay. In these examples, in the absence of a gene (by mutational inactivation or silencing), the level of cytotoxicity induced by α-synuclein in the cell is reduced, thereby inducing the gene into α-synuclein. It is shown to contribute to cytotoxicity. Thus, siRNA or antisense oligonucleotides that target genes can be useful, for example, in treating synucleinopathies.

  Screening methods to identify drugs that can block or suppress the cytotoxicity caused by overexpression of α-synuclein include (i) inducing toxicity in cells in the presence and in the absence of the candidate drug. (Ii) measuring cell viability in the presence of the candidate agent; and (iii) culturing the cell under conditions allowing expression of the nucleic acid encoding α-synuclein at a level sufficient for ) Comparing the cell viability measured in the presence of the candidate agent with the cell viability in the absence of the candidate agent, wherein the cell viability is compared with the absence of the candidate agent. When higher in the presence of the candidate agent, the candidate agent is identified as a compound that blocks or inhibits alpha-synuclein-induced toxicity.

  The screening assay can include mammalian cells containing a stably integrated nucleic acid encoding α-synuclein under the control of an inducible promoter. The cell can be any mammalian cell, but preferably the cell is a neuronal cell (e.g., a primary neuron or PC12, H4, SK-N-SH, SH-SY5Y, Neuro-2a, SVG p12, CCF- STTG1, SW 1088, SW 1783, LN-18, A172, U-138 MG, T98G, U-87 MG, U-118 MG, Hs 683, M059K, MO59J, H4, LN-229, Daoy, or PFSK-1 (Further cell lines are available from the American Type Culture Collection (ATCC), Manassas, VA)). The α-synuclein can be under the control of a tetracycline inducible promoter (as described in the examples) or any other suitable inducible promoter.

  Cells containing α-synuclein stably incorporated under the control of an inducible promoter (eg, a tetracycline inducible promoter) are cultured in a tissue culture plate, ideally a multiwell assay plate such as a 96 well culture plate. It is possible. The cells can be cultured in the absence (non-induction) or presence (induction) of an inducing agent (eg, tetracycline or doxycycline) to induce expression of α-synuclein. Non-induced cells and induced cells can also be treated with a candidate compound (eg, a dose of a candidate agent such as a compound). Induced cells or non-induced cells cultured in the absence of the compound can optionally be treated with a similar amount or concentration of vehicle (eg, DMSO) that delivered the candidate agent. The assay may include a cell or set of cells as follows: (i) induced cells treated with the candidate compound; (ii) induced cells untreated with the candidate compound; (iii) uninduced cells treated with the candidate compound; And (iv) non-induced cells untreated with the candidate compound. In order to determine whether a candidate compound prevents or suppresses α-synuclein-induced cytotoxicity in mammalian cells, the amount of toxicity present in cell set (i) is determined according to the cytotoxicity present in cell set (ii). Can be compared. If the cytotoxicity present in cell set (ii) is greater than the cytotoxicity present in cell set (i), this will prevent or inhibit the cytotoxicity induced by α-synuclein in the cell. Indicates. Cell sets (iii) and (iv) can be used as controls to normalize the amount of cytotoxicity in cells due to culture conditions, for example, regardless of the inducer or compound, respectively. For example, the amount of toxicity present in cell set (iii) may indicate that the candidate agent is cytotoxic by itself.

  For example, if one wishes to determine concentration dependence or EC50 (see below), the cells can be treated with two or more concentrations of the compound. Suitable concentrations of candidate compounds for the assay include, for example, about 0.01 μM to 1 mM agent (e.g., about 0.01 μM to 0.1 μM, about 0.1 μM to 1 μM, about 1 μM to 10 μM, about 10 to 1 mM, or about 100 μM to 1 mM).

  It will be appreciated that some optimization may be required to determine the appropriate amount of inducer or compound for this method. Such optimization can be based, for example, on the type of cell used, the details of the compound, the amount of alpha-synuclein induction, or the time required before induction of alpha-synuclein.

  Methods for assessing the effectiveness of an agent to prevent or suppress alpha-synuclein-induced cytotoxicity can be quantitative, semi-quantitative, or qualitative. Thus, for example, the activity of a drug can be determined as an individual value. An example of a quantitative determination of a drug is the 50% effective concentration, or EC50 value, which is the molar concentration of drug (eg, compound) that gives half of the drug's maximum response. Alternatively, drug efficacy can be assessed using various semi-quantitative / qualitative systems known in the art. Thus, the effectiveness of an agent to block or suppress alpha-synuclein-induced cytotoxicity in mammalian cells is, for example, (a) “excellent”, “good”, “good”, “impossible”, and / or One or more of “poor”; (b) one or more of “very high”, “high”, “average”, “low”, and / or “very low”; or ( c) one of "+++++", "++++", "+++", "++", "+", "+/-", "and / or"-" Or they can be expressed as a plurality.

  A method for determining the efficacy of an agent (eg, a compound such as any of the compounds described herein) in preventing or suppressing α-synuclein-induced cytotoxicity in mammalian cells. The cells are generally plated on a solid support matrix (eg, a plastic tissue culture plate or a multiwell (96 or 386 well) tissue culture plate) and cultured in a suitable medium. The cells are then contacted with a serial dilution of a candidate agent, generally at a concentration of, for example, 10 μM to 0.1 μM. In many cases, control compounds (eg, known concentrations of known inhibitors) are also added to one cell set as an internal standard. In many cases, a set of cells is cultured in the presence of a carrier, buffer, or vehicle that delivers the compound. Cells are cultured for various times such as 1-3 days (1 day, 2 days, 3 days, 4 days, 1 week, 2 weeks) in the presence or absence of the test compound and then remain on the plate Test for the number of cells to survive or the viability of the cells remaining on the plate. There are numerous methods for detecting (eg, determining or measuring) the degree of cytotoxicity induced by α-synuclein in the presence or absence of an agent and are well known to those skilled in the art. These methods can include, for example, measuring the intracellular ATP concentration. The amount of ATP present in a cell or cell population is proportional to the number of living cells in that population. In one example, the ATP concentration can be determined enzymatically, for example, by using luciferase / luciferin. These enzymes produce a light signal in reactions that require ATP hydrolysis. Therefore, the more ATP present in the sample, the more light is generated. In this method, cells are first collected and lysed. The cell lysate is then incubated with luciferase / luciferin and the amount of ATP-dependent light generated from the sample is measured using a luminometer (e.g. Turner BioSystems TD-20 / 20 Luminometer, Turner Biosystems, Sunnyvale, CA). Can be used to detect and / or quantify. In this case, to determine the effectiveness of a given agent in blocking or suppressing α-synuclein-induced cytotoxicity, the amount of light signal generated from the induced cell in the presence of the compound is: It can be compared to the light signal generated from the induced cells in the absence of the drug. If more light signal is generated from a lysate of cells cultured in the presence of the drug compared to cells cultured in the absence of the drug, this indicates that the compound prevents or inhibits cytotoxicity Indicates to do. Further examples of this method are described in the examples.

  Other methods suitable for determining the effectiveness of an agent in preventing or suppressing alpha-synuclein-induced cytotoxicity are, for example, cells that remain after an induction period in the absence or presence of the agent Counting the number. In this method, cells are trypsinized from the plate, washed, stained with a dye (e.g., trypan blue), and microscopic or mechanical cell counter (Beckman-Coulter Z1 (TM) Series COULTER COUNTER (R) Cell and Particle Counter). Since dyes such as trypan blue are taken up only by dead or dying cells, this method allows discrimination between live and non-viable cells in the population (ie, blue or white cells). Another method for determining the inhibition or suppression of α-synuclein induced cytotoxicity by a post-treatment agent (e.g., any one of the compositions described herein) is a metabolic assay, For example, the MTT metabolic assay (Invitrogen, USA). MTT diphenyltetrazolium bromide is a tetrazolium salt (yellowish) that is decomposed into formazan crystals by a succinate dehydrogenase system that belongs to the mitochondrial respiratory chain and is active only in living cells. Mitochondrial succinate dehydrogenase reduces MTT crystals to purple formazan in the presence of an electron coupling reagent. When cells are exposed to MTT reagent after treatment of the cells with compounds, the more live cells in the well, the more formazan dye is produced. The amount of formazan dye can be measured using, for example, a spectrophotometer.

Commonly used to test the inhibition or suppression of cytotoxicity in cells (e.g., cytotoxicity caused by overexpression of α-synuclein in mammalian cells) by agents (e.g., compounds or compositions described herein) Other methods that are involved include monitoring of DNA synthesis in the cell. For example, induced cells cultured in the presence or absence of a drug are also treated with nucleotide analogs that can be incorporated into the DNA of the cell upon cell division. Examples of such nucleotide analogues include, for example, BrdU or ³ H-thymidine. In each case, the amount of label incorporated into the induced cells (cultured in the presence and absence of a given agent) is quantified, but the amount of label incorporation is directly proportional to the amount of cell proliferation in the cell population. . The amount of label incorporated into the induced cells in the presence and absence of the drug can be normalized to the amount of label incorporated into the non-induced cells. More signals (ie, more DNA synthesis) are seen in the set of induced cells treated with the drug compared to induced cells not treated with the drug, indicating that the drug is induced by α-synuclein Indicates to block or suppress toxicity.

  Other methods suitable for assessing the prevention or suppression of α-synuclein-induced cytotoxicity by drugs include the detection of apoptosis in cells. Such methods of detecting or measuring apoptosis include, for example, monitoring DNA fragmentation, caspase activation, or annexin V expression.

  The present invention also focuses on screening methods for identifying compounds that block or suppress α-synuclein-induced Golgi fragmentation. For example, induced cells cultured in the presence and absence of drug are (i) fixed (eg, with formaldehyde or paraformaldehyde); (ii) detectably labeled with a Golgi-specific protein marker Treated with a primary antibody; (iii) the signal produced by the detectable label can be detected using any of several methods, including fluorescence-assisted cell sorting (FACS) and confocal microscopy . In one example, the Golgi border can be specifically detected using a first antibody against the membrane-bound Golgi localization protein, GM130. In some cases, an intermittent pattern indicating an intact Golgi is detected. More diffuse Golgi staining generally indicates a disruption of the organelle integrity. Exemplary methods for determining Golgi integrity are described in the Examples, such as those described in Gosavi et al. (2002) J. Biol. Chem. 277 (50): 48984-48992. .

The detectable label can be bound to a first antibody (a primary antibody that specifically recognizes a Golgi-specific protein marker) or a secondary antibody that can bind to the first antibody. Alternatively, the first antibody can be bound to a first member of a binding pair (ie, streptavidin or biotin) and the second member of the binding pair can be linked to a detectable moiety. The detectable moiety includes a radioactive label (e.g., ¹²⁵ I, ³⁵ S, ³³ P, or ³² P), a fluorescent label (e.g., Texas Red, fluorescein), a luminescent moiety (e.g., a lanthanide), or one of the FRET pairs. One or more members may be included. Methods for detecting these detectable markers are well known in the art and are described in the examples below.

  Blockade of ER to Golgi vesicle trafficking has been observed following induction of α-synuclein expression (see PCT Publication WO 2006/073734, which is incorporated by reference in its entirety). Assays suitable for monitoring vesicular traffic between the ER and the Golgi generally involve monitoring the transport of specific proteins between these two organelles. For example, the protein can be an endogenous protein that is to be secreted and can be detected, for example, by any of the immobilization and antibody-based staining methods described herein. Alternatively, the one or more proteins can be a detectable protein (eg, a green fluorescent protein, or a protein bound to a fluorescent protein), where the protein can be visualized directly in the cell. These assays monitor the subcellular localization of proteins transported through the ER to Golgi pathway. Because α-synuclein expression blocks protein transport from the ER to the Golgi, candidate agents that promote or increase protein transport from the ER to the Golgi thus increase vesicle transport from the ER to the Golgi Identified. These compounds are believed to be candidate therapeutics for reducing α-synuclein-mediated toxicity and treating synucleinopathies (eg, any of the synucleinopathies described herein). . Exemplary methods for detecting ER to Golgi transport for use in the methods described herein are described, for example, by Kawano et al. (2006) J. Biol. Chem. 281 (40): 30279- 30288; Kumagai et al. (2005) J. Biol. Chem. 280 (8): 6488-6495; and Giussani et al. (2006) Mol. Cell. Biol. 26 (13): 5055-5069. it can.

  Perform screening methods to identify compounds that modulate donor vesicle (e.g., ER or synaptic vesicle) fusion to receptor membranes (e.g., Golgi or presynaptic membranes) in α-synuclein expressing cells You can also Compounds that increase donor vesicle fusion to the receptor membrane in α-synuclein-expressing cells are considered candidate therapeutics to reduce α-synuclein-mediated toxicity and to treat synucleinopathies. Exemplary methods for detecting vesicle docking and fusion for use in the methods described herein are described, for example, in Hibbert et al. (2006) Int. J. Biochem. Cell Biol. 38 (3) : 461-471; Tsuboi et al. (2005) J. Biol. Chem. 280 (47): 39253-39259; and Huang et al. (2005) Mol. Biol. Cell 16 (6): 2614-2623 be able to.

  The present invention also discloses methods for identifying compounds that increase protein secretion from cells. It will be appreciated that such methods can also be used to assess α-synuclein-induced cytotoxicity in mammalian cells (eg, neuronal cells). For example, the method comprises (i) culturing induced cells in the presence and absence of a candidate agent; (ii) determining the amount of one or more proteins secreted from the cell in the presence of the candidate agent. And (iii) determining the amount of one or more proteins secreted from the cell in the presence of the candidate agent by the amount of one or more proteins secreted in the absence of the candidate agent Including a step of comparing. Increased (increased) secretion of one or more proteins in the presence of the candidate agent compared to the absence of the candidate agent, thereby causing the candidate agent to secrete one or more proteins. It is shown to be a compound that can be increased. Suitable methods for detecting protein expression from cells are well known in the art. For example, media from cultured cells can be collected and analyzed for the presence or amount of protein, eg, by Western or dot blotting, or enzyme linked immunosorbent assay (ELISA). If the protein is secreted at low levels, the medium can optionally be concentrated. Alternatively, when the secreted protein to be detected is, for example, an enzyme, the enzyme activity in the cell culture medium can be detected or quantified by using, for example, a chromogenic substrate. In some cases, secreted proteins remain bound to the cell surface and can be detected using, for example, FACS or confocal microscopy techniques. In some cases, secreted proteins can be detected in situ (i.e., intracellularly) during migration to the cell surface, e.g., by immobilization and antibody-based staining as described above, or when the protein can be detected directly. (Eg, fluorescent proteins) can detect or quantify proteins by FACS or microscopy techniques.

  It is understood that the screening methods described herein can also be used as secondary screens or cell-based screens to identify compounds useful in treating synucleinopathies. For example, the present screening methods can be used after a primary screen in which compounds are initially selected based on their ability to inhibit alpha-synuclein-induced toxicity, for example in another system (eg, yeast).

  For example, in any of the screening methods described herein, exemplary compounds useful as positive controls include 1-t-butyl-3- (4-chloro-phenyl) -1H-pyrazolo [3, 4-d] pyrimidin-4-ylamine and forskolin (see Example 5) and those found, for example, in PCT Publication WO 2006/034003, which is incorporated by reference in its entirety.

  Screening assays can be performed in any format that allows rapid preparation, processing, and analysis of multiple reactions. This can be done, for example, in a multiwell assay plate (eg 96 well or 386 well). Stock solutions of various drugs can be made manually or mechanically, and subsequent pipetting, dilution, mixing, dispensing, washing, incubation, sample reading, data collection, and analysis are all commercially available analysis software, Robotics and robotic control using detection instruments that can detect signals generated from the assay can be performed. Examples of such detectors include, but are not limited to, spectrophotometers, luminometers, fluorometers, and devices that measure radioisotope decay.

Compounds to be screened or identified using any of the methods described herein can include a variety of chemical types, but typically include small organic molecules having a molecular weight of 50-2,500 daltons. Molecules can be included. These compounds can contain functional groups necessary for structural interaction (e.g., hydrogen bonding) with proteins, typically at least one amine group, carbonyl group, hydroxyl group, or carboxyl group. And preferably contains at least two of the functional chemical groups. These compounds often contain cyclic carbon or heterocyclic structures and / or aromatic or polycyclic aromatic structures (eg, purine cores) substituted with one or more of the above functional groups.

  In another embodiment, the compounds include peptides, polypeptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives or structural analogs thereof, polynucleotides, Biomolecules can also be included, including but not limited to nucleic acid aptamers and polynucleotide analogs.

  Compounds can be identified from a number of potential sources including chemical libraries, natural product libraries, and combinatorial libraries of random peptides, oligonucleotides, or organic molecules. Chemical libraries consist of diverse chemical structures, some of which are analogs of known compounds, or analogs or compounds identified as “hits” or “leads” in other drug discovery screens, and others One comes from natural products, and the other comes from non-directed synthetic organic chemistry. Natural product libraries are used to create screening mixtures by (1) fermentation and extraction of cultures from soil, plant or marine microorganisms, or (2) extraction of plants or marine organisms. Collection of microorganisms, animals, plants, or marine organisms. Natural product libraries include polypeptides, non-ribosomal peptides, and variants (non-natural) thereof. For a review, see Science 282: 63-68 (1998). Combinatorial libraries are composed of multiple peptides, oligonucleotides, or organic compounds as a mixture. These libraries are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning, or proprietary synthesis methods. Of particular interest are non-peptide combinatorial libraries. Still other libraries of interest include peptides, proteins, peptidomimetics, multiparallel synthetic collections, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created from it, see Myers, Curr. Opin. Biotechnol. 8: 701-707 (1997). Once test compounds have been identified through the use of the various libraries herein, the test compounds “hits” or “leads” can subsequently be blocked or inhibited from α-synuclein-induced cytotoxicity in mammalian cells. The “hits” or “leads” can be modified to optimize performance.

  The compounds identified above can be synthesized by any chemical or biological method. The compounds identified above may be pure or in a heterogeneous composition (e.g., a pharmaceutical composition), and may be in a physiologically or pharmaceutically acceptable diluent or It can be prepared in a carrier (see pharmaceutical composition and method of treatment below).

Pharmaceutical compositions Agents that have been found to prevent or suppress alpha-synuclein-induced cytotoxicity in mammalian neurons are eg Parkinson's disease, familial Parkinson's disease, Lewy body disease, Alzheimer's disease Formulated as a pharmaceutical composition for administration to a subject to treat synucleinopathies such as Lewy body variants, Lewy body dementia, multiple system atrophy, or Guam Parkinson dementia complex It is possible. Typically, the pharmaceutical composition includes a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. . The composition may comprise a pharmaceutically acceptable salt, such as an acid addition or base addition salt (see, for example, Berge et al., J. Pharm. Sci. 66: 1-19, 1977). .

  The drug can be formulated according to standard methods. Pharmaceutical formulation is a well-established technique, for example, Gennaro (ed.), Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams & Wilkins (2000) (ISBN: 0683306472); et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott Williams & Wilkins Publishers (1999) (ISBN: 0683305727); and Kibbe (ed.), Handbook of Pharmaceutical Excipients American Pharmaceutical Association, 3rd ed. (2000 ) (ISBN: 091733096X).

  In one embodiment, the agent that blocks or inhibits α-synuclein-induced cytotoxicity in mammalian cells is shaped like sodium chloride, disodium phosphate heptahydrate, monosodium phosphate, and stabilizers It can be formulated with drug substance. The drug can be provided, for example, in a buffer solution at a suitable concentration and can be stored at 2-8 ° C.

  The pharmaceutical composition may be in various forms. These include, for example, liquid dosage forms such as solutions (eg, injections and infusions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories, semisolid dosage forms, and solid dosage forms Shape is included. The preferred form may depend on the intended mode of administration and therapeutic application. Typically, the pharmaceutical compositions described herein are in the form of injections or infusions.

  Such compositions can be administered by a parenteral mode (eg, intravenous, subcutaneous, intraperitoneal, or intramuscular injection). As used herein, the phrases “parenteral administration” and “parenteral administration” refer to methods of administration, generally by injection, other than enteral administration and topical administration, including but not limited to intravenous. Internal, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, epidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, Intracerebral, intracranial, intracarotid, and intrasternal injections and infusions are included. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to stable storage at high concentration. Sterile injectable solutions can be prepared by incorporating the required amount of a drug described herein in a suitable solvent, optionally with one or a combination of the above components, followed by filter sterilization. . Generally, dispersions are prepared by incorporating the agents described herein in a sterile medium that contains the underlying dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for preparing sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization, whereby the drug described herein and any further desired An ingredient powder is obtained. The proper fluidity of the solution is maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. Is possible. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

  In certain embodiments, the agent can be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for preparing such formulations are patented or generally known. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

  An agent identified as an agent that blocks or inhibits α-synuclein-induced cytotoxicity in mammalian cells, eg, its stability and / or retention in circulation such as blood, serum, or other tissues, eg Modifications can be made with at least a 1.5, 2, 5, 10, or 50-fold improvement. The modified drug can be evaluated to determine if it can reach the treatment site of interest (e.g., the location of the Lewy body) as may occur with synucleinopathies such as Parkinson's disease. There is (for example, by using a labeled drug).

  For example, the agent can be associated with a polymer, eg, a substantially non-antigenic polymer such as polyalkylene oxide or polyethylene oxide. Suitable polymers will vary substantially by weight. Polymers having a number average molecular weight of about 200 to 35,000 daltons (or about 1,000 to about 15,000, and 2,000 to about 12,500) can be used. For example, the drug can be conjugated to a water soluble polymer such as a hydrophilic polyvinyl polymer such as polyvinyl alcohol or polyvinyl pyrrolidone. A non-limiting list of such polymers is that the water solubility of polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycol, polyoxyethylenated polyols, copolymers thereof, and block copolymers is maintained. These block copolymers are included under certain conditions. Further useful polymers include polyoxyalkylenes such as polyoxyethylene, polyoxypropylene, and block copolymers of polyoxyethylene and polyoxypropylene (Pluronics); polymethacrylic acid; carbomers; and branched polysaccharides or unbranched Examples include polysaccharides. When an agent (e.g., a compound) is used in combination with a second agent (e.g., any additional therapy for synucleinopathies, such as an acetylcholinesterase inhibitor), the two agents are separated separately or Both can be formulated. For example, each pharmaceutical composition can be mixed and administered together, eg, just prior to administration, or can be administered separately, eg, simultaneously or at different times.

Agents that can block or inhibit α-synuclein-induced cytotoxicity in administered mammalian cells can be administered to a subject, eg, a human subject, by a variety of methods. For many applications, the route of administration is one of the following: intravenous injection or infusion (IV), subcutaneous injection (SC), intraperitoneal injection (IP), or intramuscular injection. In some cases, it may be administered directly to the CNS, for example, intrathecally, intraventricularly (ICV), intracerebral, or intracranial. The drug can be administered as a fixed dose or in a mg / kg dose. In other examples, administration can be oral (eg, inhalation), transdermal (topical), transmucosal, or rectal.

  Oral compositions generally include an inert diluent or edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, eg, gelatin capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch or lactose Disintegrants such as hydrating agents, alginic acid, Primogel, or corn starch; lubricants such as magnesium stearate or Sterote; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin Or flavoring agents such as peppermint, methyl salicylate, or orange flavor.

  Powders and tablets contain 1% to 95% (w / w) of active compound. In certain embodiments, the active compound ranges from 5% to 70% (w / w). Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like. The term “preparation” is intended to include a formulation of an active compound and an encapsulant as a carrier to provide a capsule, in which the active compound may be a carrier with or without other carriers. And is thus associated with the carrier. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.

  Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use disperse the finely divided active ingredient in water with viscous materials such as natural or synthetic rubbers, resins, methylcellulose, sodium carboxymethylcellulose, and other well known suspending agents. It is possible to make it.

  When administered by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

  Systemic administration by transmucosal or transdermal means is also possible. For transmucosal or transdermal administration, penetrants appropriate to permeate the barrier are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, surfactants, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished using nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

  The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

  The dose can also be selected to reduce or avoid production of antibodies to the drug, if the drug is a polypeptide or otherwise antigenic. The route of administration and / or method of administration of the drug can also be tailored to individual cases.

  Dosage regimens are adjusted to provide the desired response, eg, a therapeutic response or a combined therapeutic effect. The dosing regimen, for example, prevents or inhibits α-synuclein-induced cytotoxicity in one or more affected cells of a mammal having synucleinopathies. In general, a dose can be provided to a subject using a fixed dose of drug (eg, compound), optionally formulated separately or together with an appropriate dose of a second therapeutic agent. Suitable dosages and / or dosage ranges of the drug include an amount sufficient to prevent or inhibit alpha-synuclein induced cytotoxicity in the subject.

  The dose of agent required to prevent or suppress alpha-synuclein-induced cytotoxicity can depend on a variety of factors including, for example, the age, sex, and weight of the subject to be treated. Other factors that affect the dose administered to a subject include, for example, the type or severity of synucleinopathies. For example, patients with advanced Alzheimer's disease may require different doses of drugs that prevent or inhibit alpha-synuclein-induced cytotoxicity than patients with milder forms of Alzheimer's disease. Other factors include, for example, other disorders that the patient suffers simultaneously or previously suffered, the patient's general health, the patient's genetic predisposition, dietary habits, administration time, excretion rate, drug Combinations and any other additional therapeutic agents administered to the patient can be included. It should be understood that the specific dosage and treatment regimen for any particular patient will depend on the judgment of the treating physician. The amount of active ingredient will also depend on the particular compound as well as the presence and nature of additional therapeutic agents in the composition.

  As used herein, dosage unit form or “fixed dose” refers to a physically discrete unit suitable as a unit dosage for the subject to be treated; each unit is a necessary pharmaceutical carrier. A predetermined amount of active compound calculated to provide the desired therapeutic effect (e.g., prevention or suppression of α-synuclein-induced cytotoxicity) in the context of and, optionally in the context of other drugs including. Appropriate dosing frequencies are described elsewhere herein.

  The pharmaceutical composition may comprise a therapeutically effective amount of an agent that has been found to prevent or inhibit alpha-synuclein-induced cytotoxicity as described herein. Such an effective amount can be determined based on the effect of the drug to be administered or, if multiple drugs are used, the combined effect of the drug and the second drug. A therapeutically effective amount of an agent is determined by the individual's disease state, age, sex, and weight, and the desired response, e.g., improvement of at least one disorder parameter, e.g., at least one symptom of synucleinopathy (e.g., memory impairment It can also vary depending on factors such as the ability of the compound to induce improvement in forgetting) in an individual. A therapeutically effective amount is also the amount by which a therapeutically beneficial effect exceeds any toxic or adverse effects.

  The following are examples of the present invention. These examples should in no way be construed as limiting the scope of the invention.

Example
Example 1. Conditional overexpression of α-synuclein A TS217 cell line was generated from H4 human glioma cells stably expressing α-synuclein polypeptide under the control of a tetracycline-on promoter (Fig. 1). To test for induction of wild-type α-synuclein expression, TS217 cells were treated with 0.1 μg / mL tetracycline (“-”) or with 0.1 μg / mL tetracycline for 1 day (1d) and 3 days (3d). (See Figure 2). Whole cell lysates were prepared at various time points from treated or untreated TS217 cells, and cellular proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). To show endogenous levels of α-synuclein protein, whole cell lysates from parental H4 (C) were also prepared and subjected to SDS-PAGE. The expression of α-synuclein was confirmed by Western blotting using an antibody specific for α-synuclein protein (FIG. 2). α-synuclein expression increased at least 2-fold by day 1 after induction, and increased 3-fold or more than 4-fold by day 3 after induction. From these results, it was shown that the expression of α-synuclein is regulated by tetracycline.

Example 2. Cytotoxicity in cells overexpressing α-synuclein In some experimental systems, overexpression of α-synuclein is a toxicity inducer or low dose proteasome inhibition such as serum deprivation, dopamine, and lactacystin It has been shown to sensitize cells to cell death induced by drug-like conditions (Smith et al. (2005) Hum. Mol. Genet. 14 (24): 3801-3811; Tabrizi et al (2000) Hum. Mol. Genet. 9 (18): 2683-2689; and Ostreova et al. (1999) J. Neurosci. 19 (14): 5782-5791). To determine the effect of wild-type α-synuclein overexpression on cell viability using a stable induction system, TS217 cells were treated with 0.1 μg / mL tetracycline for 3-6 days to obtain α-synuclein Expression was induced. By measuring cellular ATP levels in cell lysates using the ViaLight® Plus Bioassay kit (Cambrex, Rockland, Maine), the relative viability of the cells was determined at 1-day intervals (day 3, 4 Day, day 5 and day 6). As an indicator of α-synuclein-induced cytotoxicity, relative cell viability was calculated as the ratio of induced cells to control cells (cells untreated with tetracycline) (see FIG. 3). In the absence of any other toxicity-inducing agent, relative cell viability decreases by more than 50% from day 3 to day 6, and expression of α-synuclein alone in these cells can cause cell death It was shown that.

  Cytotoxicity of α-synuclein overexpression in TS217 cells was also assessed using a membrane permeable dye, calcein AM. Expression of α-synuclein by plating TS217 cells onto glass tissue culture chamber slides coated with poly-L-lysine and culturing in the absence (control) or presence (induction) of 0.1 μg / mL tetracycline for 5 days Induced. Cells were treated with 2 μM calcein AM, and fluorescence of enzyme-activated calcein AM (intracellular) was measured using a fluorescence microscope (Olympus, Pennsylvania) with a digital camera controlled by the Metamorph program (Universal Imaging, Westchester, PA). (State, Center Valley). Calcein positive cells were quantified using Metamorph software (Universal Imaging, West Chester, PA) (FIG. 4A). After 5 days of treatment with tetracycline (and induction of α-synuclein), viable cell counts decreased by approximately 60% (Figure 4B), further showing that overexpression of α-synuclein in these cells leads to cell death. It was done.

  The effect of plating density on the toxicity induced by α-synuclein was also investigated. TS217 cells were plated into 96-well plates at dilutions of 250, 500, 1000, 2000, and 4000 cells / well. Cells were incubated for 5 days in the presence of tetracycline, then harvested and lysed (as described above) and the intracellular ATP concentration was measured. More cell death is observed at lower cell densities (e.g., 250 cells / well) than at higher cell densities (e.g., 4000 cells / well), and plating density affects alpha-synuclein-induced toxicity (FIG. 10).

Example 3. Tetracycline dose dependence on cell viability To determine if the effect of tetracycline on cell viability is concentration dependent, TS217 cells cultured in 96 well plates were mixed with various concentrations of tetracycline. Incubated for days. After treatment, each experimental cell group was lysed as described above, and cellular ATP was measured. The medium alone (without tetracycline) was used as a control (FIG. 5). Relative cell viability as a function of intracellular ATP concentration decreased in a dose-dependent manner as tetracycline concentration increased (see FIG. 5).

Example 4. Golgi fragmentation in α-synuclein overexpressing cells. TS217 cells were plated on glass tissue culture chamber slides coated with poly-L-lysine in the absence of 0.1 μg / mL tetracycline (control) or Cultured in the presence (induction) for 6 days to induce α-synuclein expression. After the treatment, the cells were fixed with 4% formaldehyde supplemented with 4% sucrose in phosphate buffered saline, permeabilized with PBS containing 0.2% Triton, and stained with an antibody specific for cis-Golgi tethered protein GM130 ( FIG. 6A). The number of cells containing intact Golgi decreased by almost 80% after 6 days induction of α-synuclein (FIG. 6B).

  To further evaluate the effect of forced overexpression of α-synuclein on Golgi integrity, TS217 cells treated in the absence (control) and presence (induction) of tetracycline for 6 days (see above) Double staining with antibodies specific for GM130 and membrane-bound Golgi enzyme mannosidase II, respectively. Control cells showed an intermittent colocalization pattern for both GM130 and mannosidase II, whereas the induced cells had a diffuse delocalized Golgi pattern and α-synuclein overexpression was Golgi in these cells. It was further shown that it caused fragmentation (Figure 7).

  To determine whether α-synuclein has a similar disruptive effect on the endoplasmic reticulum (ER), TS217 cells were plated on glass tissue culture slides as before and 0.1 μg / mL tetracycline was absent. Incubation under (control) and presence (induction) for 5 days induced α-synuclein expression. Cells were fixed and stained with antibodies against the ER-localized Ca2 + binding protein calnexin, or mannosidase II as a control (FIG. 8). While the Golgi apparatus showed significant fragmentation (see also FIG. 7), macroscopic changes in ER morphology were not observed in cells overexpressing α-synuclein (FIG. 8).

Example 5. Dose-dependent rescue of cell viability To determine whether selected compounds can inhibit α-synuclein-induced cytotoxicity, TS217 cells plated in 96-well tissue culture plates were 1-t-butyl-3- (4-chloro-phenyl) -1H-pyrazolo [3,4-d] pyrimidin-4-ylamine (Compound J (Cmp J)) (0.08 μM, 0.15 μM, and 0.3 μM) Or in the presence of DMSO as a control (Figure 9), or forskolin (0.3 μM, 1 μM, 3 μM, and 10 μM) or in the presence of DMSO as a control (Figure 11) for 5 days with 0.1 μg / mL tetracycline Cultured. After 5 days of treatment, cells were lysed and assayed for intracellular ATP concentration as a function of cell viability (as above).

Other Embodiments While the invention has been described in conjunction with the detailed description thereof, it is to be understood that the foregoing description is for purposes of illustration and is not intended to limit the scope of the invention. It is. Other aspects, advantages, and modifications of the invention are within the scope of the claims set forth below.

Claims (29)

  A mammalian neuronal cell comprising a stably integrated expression construct comprising an inducible promoter operably linked to a nucleic acid encoding a protein comprising human alpha synuclein, a toxicity-inducing agent Alternatively, mammalian neuronal cells whose cell viability decreases when the expression of the nucleic acid is induced without simultaneously treating the cells with a neuronal differentiation factor.   2. The cell according to claim 1, wherein the α-synuclein is wild-type α-synuclein.   2. The cell according to claim 1, wherein the α-synuclein is a mutant α-synuclein.   4. The cell according to claim 3, wherein the mutant α-synuclein is mutant human α-synuclein A53T.   4. The cell according to claim 3, wherein the mutant α-synuclein is mutant human α-synuclein A30P.   4. The cell according to claim 3, wherein the mutant α-synuclein is mutant human α-synuclein E46K.   The cell according to any one of claims 1 to 6, wherein the α-synuclein is full-length α-synuclein.   The cell according to any one of claims 1 to 7, which is a human nerve cell.   The cell according to any one of claims 1 to 8, which is derived from a neuronal cell line.   The cell according to any one of claims 1 to 7, which is derived from an H4 cell line.   The cell according to any one of claims 1 to 7, which is derived from a PC12 cell line.   12. The cell according to any one of claims 1 to 11, wherein the protein is a fusion protein comprising a detectable protein.   13. A cell according to claim 12, wherein the detectable protein is a fluorescent protein, an enzyme, or an epitope.   14. The cell according to claim 13, wherein the detectable protein is a fluorescent protein selected from the group consisting of a red fluorescent protein, a green fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein, and a cyan fluorescent protein.   15. The cell according to any one of claims 1 to 14, which is an isolated cell. Further comprising a stably integrated repressor construct that constitutively expresses the repressor protein;
(i) When the inducer is not added exogenously, the repressor protein binds to the inducible promoter and suppresses nucleic acid expression, and (ii) When the inducer is added exogenously, the repressor protein The cell according to any one of claims 1 to 15, wherein the nucleic acid is expressed by binding to an inducer.   17. The cell of claim 16, wherein the repressor protein is a Tet repressor.   The cell according to claim 16 or 17, wherein the inducer is tetracycline or doxycycline.   A non-human mammal comprising the nerve cell according to any one of claims 1 to 7.   16. A method of inducing toxicity in a mammalian neuron comprising the step of inducing a level of expression of a nucleic acid that is toxic to the cell in the cell of any one of claims 1-15.   A method of inducing toxicity in a mammalian neuronal cell, wherein the cell according to any one of claims 16-18 is applied in an amount of exogenous effective to induce nucleic acid expression and toxicity in the cell. The method comprising the step of contacting with an inducer added to the method. 16. The cell of any one of claims 1-15, under conditions that permit expression of the nucleic acid in the presence of the candidate agent and in the absence of the candidate agent at a level sufficient to induce toxicity in the cell. Culturing
Measuring cell viability in the presence of the candidate agent; and comparing cell viability measured in the presence of the candidate agent with cell viability in the absence of the candidate agent; A method for identifying a compound that inhibits or suppresses toxicity induced by α-synuclein, comprising:
A candidate drug is identified as a compound that blocks or inhibits alpha-synuclein-induced toxicity when cell viability is higher in the presence of the candidate drug compared to in the absence of the candidate drug ,Method. Claims in the presence of a candidate agent and in the presence of an exogenously added inducer in an amount effective to induce nucleic acid expression at a level that induces toxicity in cells in the absence of the candidate agent. Culturing the cell according to any one of 16 to 18;
Measuring cell viability in the presence of the candidate agent; and comparing cell viability measured in the presence of the candidate agent with cell viability in the absence of the candidate agent; A method for identifying a compound that inhibits or suppresses toxicity induced by α-synuclein, comprising:
A candidate drug is identified as a compound that blocks or inhibits alpha-synuclein-induced toxicity when cell viability is higher in the presence of the candidate drug compared to in the absence of the candidate drug ,Method. An amount of exogenously added inducer effective in inducing nucleic acid expression at a level that induces Golgi fragmentation in cells in the presence and in the absence of the candidate agent. Culturing the cell of any one of claims 16 to 18 in the presence;
Measuring Golgi fragmentation in cells in the presence of the candidate agent; and comparing Golgi fragmentation in cells measured in the presence of the candidate agent to Golgi fragmentation in the absence of the candidate agent A method for identifying a compound that inhibits or suppresses Golgi fragmentation induced by α-synuclein, comprising:
Identification of a candidate drug as a compound that blocks or inhibits α-synuclein-induced Golgi fragmentation when Golgi fragmentation is less in the presence of the candidate drug compared to the absence of the candidate drug The way it is. Exogenously added induction in an amount effective to induce nucleic acid expression at a level that reduces vesicle transport from the endoplasmic reticulum to the Golgi in the presence of the candidate agent and in the absence of the candidate agent. Culturing the cell according to any one of claims 16 to 18 in the presence of a substance;
Measuring vesicle transport from the endoplasmic reticulum to the Golgi in a cell in the presence of the candidate agent; and vesicle transport from the endoplasmic reticulum to the Golgi in the cell measured in the presence of the candidate agent. A method of identifying a compound that increases vesicle transport from the endoplasmic reticulum to the Golgi, comprising comparing to vesicle transport from the endoplasmic reticulum to the Golgi measured in the absence of
By increasing vesicle transport from the endoplasmic reticulum to the Golgi in cells in the presence of the candidate agent as compared to vesicle transport from the endoplasmic reticulum to the Golgi in the absence of the candidate agent, the candidate A method wherein the agent is identified as a compound that increases vesicular transport from the endoplasmic reticulum to the Golgi. Existence of exogenously added inducer in an amount effective to induce nucleic acid expression at a level that reduces vesicle docking and fusion in the cell in the presence of the candidate agent and in the absence of the candidate agent A step of culturing the cells according to any one of claims 16 to 18 below;
Measuring docking and fusion of vesicles in cells in the presence of the candidate agent; and docking and fusion of vesicles in cells in the presence of the candidate agent to vesicles in the absence of the candidate agent A method of identifying a compound that increases vesicle docking and fusion comprising the steps of:
Increased vesicle docking and fusion in the presence of the candidate agent compared to vesicle docking and fusion in the absence of the candidate agent allows the candidate agent to dock and fuse vesicles A method identified as a compound that increases. In the presence of the candidate agent and in the presence of an exogenously added inducer in an amount effective to induce expression of the nucleic acid at a level that reduces protein secretion from the cell in the absence of the candidate agent, A step of culturing the cell according to any one of claims 16 to 18;
Measuring protein secretion from a cell in the presence of the candidate agent; and comparing protein secretion from the cell in the presence of the candidate agent to protein secretion in the absence of the candidate agent. A method for identifying a compound that increases the secretion of a protein from a cell comprising:
Identification of the candidate agent as a compound that increases protein secretion from the cell by increasing protein secretion from the cell in the presence of the candidate agent as compared to protein secretion in the absence of the candidate agent The way it is.   A method according to any one of claims 22 to 27 for treating synucleinopathy, wherein the individual has synucleinopathy or is at risk of developing synucleinopathy. Administering a pharmaceutical composition comprising a therapeutically effective amount of the compound identified by.   The synucleinopathy is Parkinson's disease, familial Parkinson's disease, Lewy body disease, Lewy body variant of Alzheimer's disease, Lewy body dementia, multiple system atrophy, or Guam Parkinson dementia complex Item 28. The method according to Item 28.

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