WO2009114700A2 - Cell model of neurodegenerative disease and methods of use thereof for identifying inhibitors of mitochondrial fusion - Google Patents

Cell model of neurodegenerative disease and methods of use thereof for identifying inhibitors of mitochondrial fusion Download PDF

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WO2009114700A2
WO2009114700A2 PCT/US2009/036973 US2009036973W WO2009114700A2 WO 2009114700 A2 WO2009114700 A2 WO 2009114700A2 US 2009036973 W US2009036973 W US 2009036973W WO 2009114700 A2 WO2009114700 A2 WO 2009114700A2
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cell
mitochondrial
protein
compound
gene
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WO2009114700A3 (en
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Asa Abeliovich
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The Trustees Of Columbia University In The City Of New York
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    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
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    • G01N2800/00—Detection or diagnosis of diseases
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    • G01N2800/2814—Dementia; Cognitive disorders
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    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • Models of neurodegenerative diseases are essential for the development and validation of effective therapies to threat such diseases.
  • Cell-based disease models are desirable because they are readily manipulated with genetic and pharmacological interventions, and can be miniaturized for high-throughput screening of drugs.
  • a method for determining whether a compound enhances mitochondrial fission in a mammalian cell comprising: (a) inducing mitochondrial fusion in a mammalian cell, wherein the inducing comprises (i) reducing activity or expression of a mitochondrial fission gene or protein; or (ii) increasing activity or expression of a mitochondrial fusion gene or protein; (b) contacting the cell with a candidate compound; (c) determining whether mitochondrial fusion is reduced in the cell of (b) compared to a cell of (a) in the absence of the candidate compound, wherein a determination of a reduction in mitochondrial fusion indicates that the compound enhances mitochondrial fission.
  • the inducing comprises both (i) reducing activity or expression of a mitochondrial fission gene or protein; and (ii) increasing activity or expression of a mitochondrial fusion gene or protein.
  • a method for determining whether a compound will be useful for treating a neurodegenerative disease comprising: (a) inducing mitochondrial fusion in a mammalian cell, wherein the inducing comprises (i) reducing activity or expression of a mitochondrial fission gene or protein; or (ii) increasing activity or expression of a mitochondrial fusion gene or protein; (b) contacting the cell with a candidate compound; (c) determining whether mitochondrial fusion is reduced in the cell of (b) compared to a cell of (a) in the absence of the candidate compound, wherein a determination of a reduction in mitochondrial fusion indicates that the compound will be useful for treating a neurodegenerative disease.
  • the inducing comprises both (i) reducing activity or expression of a mitochondrial fission gene or protein; and (ii) increasing activity or expression of a mitochondrial fusion gene or protein.
  • the neurodegenerative disease is Parkinson's disease, Alzheimer's disease or amyotrophic lateral sclerosis.
  • the mitochondrial fission gene or protein is Dnml or Fisl .
  • the mitochondrial fusion gene or protein is Opal, mitofusinl or mitofusin2.
  • the reducing comprises using a dominant negative mutant of the mitochondrial fission gene or protein.
  • the reducing comprises using a nucleic acid that specifically binds to a nucleic acid encoding the mitochondrial fission gene, and inhibits protein expression from the nucleic acid.
  • the nucleic acid is a siRNA, shRNA or antisense RNA.
  • the increasing comprises expressing the mitochondrial fusion gene or protein from a nucleic acid vector.
  • the compound comprises mfisi-1 or a structural analog thereof.
  • the compound is a GTPase inhibitor.
  • the inhibitor is dynasore or a structural analog thereof.
  • the compound comprises a PINKl protein or a peptide fragment thereof.
  • the cell is a mammalian cell. In another embodiment, the cell is a neuronal cell. [0011] In one embodiment, the determining comprises assessing cell death. In one embodiment, the determining comprises microscopy. In one embodiment, the determining comprises detecting mitochondria. In one embodiment, the cell of further comprises a nucleic acid vector encoding a detectable mitochondria-specific protein. In one embodiment, the determining comprises using an antibody that specifically binds to a mitochondria- specific protein. In one embodiment, the antibody comprises a detectable label.
  • the method is carried out in a high throughput manner.
  • FIG. 1 Mitochondrial fusion was induced in COS7 cells by expression of DrpS637D, a dominant negative mutant of Dnmll. Overexpression of PINKl promotes mitochondrial fission. Also see Example 1.
  • the disclosure provides a cell-based method for determining whether a candidate compound may be a useful therapeutic compound for treating Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), or Alzheimer's disease (AD).
  • PD Parkinson's disease
  • ALS amyotrophic lateral sclerosis
  • AD Alzheimer's disease
  • PD Parkinson's disease
  • ALS amyotrophic lateral sclerosis
  • AD Alzheimer's disease
  • the cell-based method provided herein is a novel mammalian cell model of PD and other neurodegenerative disorders. The cell model is based on the assessment of mitochondrial fusion and fission.
  • the cell model is used to identify inhibitors of the mitochondrial fusion phenotype induced in cells by manipulation of the cell machinery (for example, genes, proteins, and signaling pathways) that regulates mitochondrial fusion and fission.
  • Compounds identified as inhibitors of the mitochondrial fusion phenotype will be highly valued candidates for treatment of PD and other neurodegenerative disorders.
  • Mitochondria have been implicated in PD by the presence of pathological changes in patient brains, by the finding that mitochondrial toxins can recapitulate some aspects of the disease, and by the recent identification of Pinkl (used interchangeably with PINKl herein) mutations in PD (Beal, 2003). Pinkl encodes a mitochondrial kinase and deletion/loss of function is associated with familial forms of PD (Abeliovich and Flint Beal, 2006). Drosophila with loss of Pinkl show some evidence of excessive mitochondrial fusion (Poole et al, 2008), but it is unclear if this is a primary event. In mice, loss of Pinkl does not lead to obvious mitochondrial changes. Because these data are indirect and not carried out in a mammalian system, the methods lack usefulness for identification and development of therapeutic compounds to treat neurodegenerative diseases in humans.
  • a cell-based assay for the identification of compounds, factors or agents that function in a similar manner to Pinkl and suppress the excessive fusion phenotype of DNMIl inhibition or other forms of mitochondrial fusion.
  • Such mitochondrial fusion could be induced either by decreasing the activity of fission genes, such as Dnmll and Fisl; or by increasing the activity of fusion genes, such as Opal, mitofusinl and mitofusin2.
  • Such manipulations will lead to excessive fusion and subsequently cell death, both of which can be assayed by techniques known in the art, for example, microscopy (see Example 1) or by any cell death assay.
  • Dmnll Based on the known nucleotide and amino acid sequences of Dmnll (see, for example, NCBI Accession Nos. BC024590 and AB209070), Fisl (see, for example, NCBI Accession Nos. BC003540 and AAH03540), Opal (see, for example, NCBI Accession Nos. AK022522 and AAH75805), mitofusinl (see, for example, NCBI Accession Nos. U95822 and AB043588), and mitofusin2 (see, for example, NCBI Accession Nos. CAI19087 and AY028429), one having ordinary skill in the art would know how to generate an expression vector and express the protein of interest in a cell.
  • Vectors that may be used in the disclosed methods include non-viral nucleic acid vectors, such as plasmids, as well as viral vectors.
  • Non-limiting examples of viral vectors include lentiviral, adeno-associated virus-2 (AAV-2), adenoviral, retroviral, polio viral, murine Maloney-based, alpha viral vector, pox viral, herpes viral, vaccinia viral, baculoviral, parvoviral, or any combination thereof.
  • Cells that may be used in the disclosed methods include any mammalian cell, for example a neuronal cell.
  • the neuronal cell may be a primary neuronal cell, for example, a post-mitotic neuron.
  • Non- limiting examples of post-mitotic neurons include cortical neurons, dopamine neurons and sympathetic neurons.
  • Mitochondria are known to be important in PD. Studies have focused on the role of oxidative stress and mitochondrial energetics in PD. Morphology of mitochondria, including fusion and fission, plays a role in oxidative stress and energetics. Thus, the methods provided herein can also be used to identify compounds that are generally protective to mitochondria, for example, compounds that regulate oxidative stress and energetics through morphological regulation of mitochondria. In addition to identifying compounds that decrease mitochondrial fusion, the disclosed methods may also be used to identify compounds or conditions that increase mitochondrial fusion. Normal cells maintain a balance of mitochondrial fusion and fission, thereby maintaining proper generation of cellular energy. Mitochondria also play a role in other cellular processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth, all of which may also be affected by proper or improper regulation of mitochondrial morphology.
  • a fusion protein can be expressed in a cell, where the fusion protein is a mitochondrial-specific protein fused to a detectable marker.
  • the detectable marker can be a fluorescent protein or peptide which is directly detected by fluorescence microscopy.
  • pDsRed2-Mito is a mammalian expression vector that encodes a fusion of red fluorescent protein and the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase.
  • the expression of a fluorescent protein such as green fluorescent protein or red fluorescent protein, enables visualization mitochondrial morphology. Fluorescence can be detected directly (e.g., detection of a fluorescent protein) or indirectly (e.g., detection of an antibody with a fluorescent label or tag, wherein the antibody specifically binds a protein of interest, such as a mitochondrial marker protein).
  • Non-limiting examples of mitochondrial marker proteins that can be detected include cytochrome c oxidase, manganese superoxide dismutase and mitochondrial porin. Additional examples of mitochondrial-specific antibodies are available from MitoSciences.
  • any compound, factor or agent that functions similarly to overexpression of wildtype Pinkl and suppresses the excessive fusion phenotype would be a potentially therapeutic molecule for PD therapy or other neurodegenerative disorders such as ALS and AD.
  • Candidate compounds that can be used in the disclosed methods include compounds that inhibit or suppress proteins and genes involved in mitochondrial fusion, such as Opal, mitofusinl and mitofusin2.
  • Other non-limiting examples of candidate compounds include, but are not limited to, GTPase inhibitors, such inhibitors can be readily screened for by methods known in the art.
  • An exemplary compound which can be assessed in the disclosed methods is the GTPase competitive inhibitor called dynasore, a competitive inhibitor of the GTPase dynamin (Macia E. et al., 2006), or structural analogs thereof.
  • Further examples of compounds that can be assessed using the disclosed methods are mfisi-1 and structural analogs thereof as described in U.S. Patent Application Publication No.
  • the methods disclosed herein can be carried out in a high throughput manner, such as for 100 compounds, 1000 compounds, or more than 1000 compounds.
  • COS7 cells were co-transfected with MitoDSRed 0.25 ⁇ g (ClonTech), pGW-DrpS637D (0.35 ⁇ g) (dominant negative mutant of Dnmll) (Chang and Blackstone, 2007) and pLenti6-DEST-PINKl-V5 wildtype (human PINKl expression from a lentiviral vector) at 0.1, 0.3, 1.0 ⁇ g (Addgene, Cookson), and a green fluorescent protein (GFP) marker plasmid (0.1 ⁇ g) as control.
  • the dominant negative mutant of Dnmll (Drps637D) induced an excessive mitochondrial fusion phenotype in the COS7 cells.
  • the mitochondria were analyzed for changes in morphology by immunofluorescence microscopy at 36 hours post-transfection.
  • the data in Figure 1 are reported as a ratio of the number of cells with fused, elongated mitochondria to the total cells expressing DSRed in mitochondria.
  • the data were analyzed by one-way analysis of variance. Three random areas per well were imaged and each section had approximately 150 cells. The trans fection efficiency was approximately 40%.

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Abstract

The disclosure provides a method for identifying compounds that may be used in the treatment of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. The disclosed methods provide a novel mammalian cell-based model of neurodegenerative diseases based on mitochondrial fusion and fission.

Description

CELL MODEL OF NEURODEGENERATIVE DISEASE AND METHODS OF USE THEREOF FOR IDENTIFYING INHIBITORS OF MITOCHONDRIAL FUSION
[0001] This application claims priority to U.S. Provisional Application No. 61/035,839 filed on March 12, 2008, which is hereby incorporated by reference in its entirety.
[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
[0003] A portion of the disclosure of this patent document contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
BACKGROUND
[0004] Models of neurodegenerative diseases are essential for the development and validation of effective therapies to threat such diseases. Cell-based disease models are desirable because they are readily manipulated with genetic and pharmacological interventions, and can be miniaturized for high-throughput screening of drugs.
[0005] Presently, lack of a reliable cell model for neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis, is a major limitation in research and drug development. Mammalian cell models are needed to develop new therapeutic compounds and treatment strategies for treating neurodegenerative disorders.
SUMMARY
[0006] A method is provided for determining whether a compound enhances mitochondrial fission in a mammalian cell, the method comprising: (a) inducing mitochondrial fusion in a mammalian cell, wherein the inducing comprises (i) reducing activity or expression of a mitochondrial fission gene or protein; or (ii) increasing activity or expression of a mitochondrial fusion gene or protein; (b) contacting the cell with a candidate compound; (c) determining whether mitochondrial fusion is reduced in the cell of (b) compared to a cell of (a) in the absence of the candidate compound, wherein a determination of a reduction in mitochondrial fusion indicates that the compound enhances mitochondrial fission. In one embodiment, the inducing the comprises both (i) reducing activity or expression of a mitochondrial fission gene or protein; and (ii) increasing activity or expression of a mitochondrial fusion gene or protein.
[0007] A method is provided for determining whether a compound will be useful for treating a neurodegenerative disease, the method comprising: (a) inducing mitochondrial fusion in a mammalian cell, wherein the inducing comprises (i) reducing activity or expression of a mitochondrial fission gene or protein; or (ii) increasing activity or expression of a mitochondrial fusion gene or protein; (b) contacting the cell with a candidate compound; (c) determining whether mitochondrial fusion is reduced in the cell of (b) compared to a cell of (a) in the absence of the candidate compound, wherein a determination of a reduction in mitochondrial fusion indicates that the compound will be useful for treating a neurodegenerative disease. In one embodiment, the inducing the comprises both (i) reducing activity or expression of a mitochondrial fission gene or protein; and (ii) increasing activity or expression of a mitochondrial fusion gene or protein.
[0008] In one embodiment, the neurodegenerative disease is Parkinson's disease, Alzheimer's disease or amyotrophic lateral sclerosis. In one embodiments, the mitochondrial fission gene or protein is Dnml or Fisl . In one embodiment, the mitochondrial fusion gene or protein is Opal, mitofusinl or mitofusin2. In one embodiment, the reducing comprises using a dominant negative mutant of the mitochondrial fission gene or protein. In one embodiment, the reducing comprises using a nucleic acid that specifically binds to a nucleic acid encoding the mitochondrial fission gene, and inhibits protein expression from the nucleic acid. In one embodiment, the nucleic acid is a siRNA, shRNA or antisense RNA. In one embodiment, the increasing comprises expressing the mitochondrial fusion gene or protein from a nucleic acid vector.
[0009] In one embodiment, the compound comprises mfisi-1 or a structural analog thereof. In one embodiment, the compound is a GTPase inhibitor. In one embodiment, the inhibitor is dynasore or a structural analog thereof. In one embodiment, the compound comprises a PINKl protein or a peptide fragment thereof.
[0010] In one embodiment, the cell is a mammalian cell. In another embodiment, the cell is a neuronal cell. [0011] In one embodiment, the determining comprises assessing cell death. In one embodiment, the determining comprises microscopy. In one embodiment, the determining comprises detecting mitochondria. In one embodiment, the cell of further comprises a nucleic acid vector encoding a detectable mitochondria-specific protein. In one embodiment, the determining comprises using an antibody that specifically binds to a mitochondria- specific protein. In one embodiment, the antibody comprises a detectable label.
[0012] In one embodiment, the method is carried out in a high throughput manner.
BRIEF DESCRIPTION OF THE FIGURES
[0013] Figure 1: Mitochondrial fusion was induced in COS7 cells by expression of DrpS637D, a dominant negative mutant of Dnmll. Overexpression of PINKl promotes mitochondrial fission. Also see Example 1.
DETAILED DESCRIPTION
[0014] The disclosure provides a cell-based method for determining whether a candidate compound may be a useful therapeutic compound for treating Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), or Alzheimer's disease (AD). Currently, there are no validated cell models for neurodegenerative diseases, such as PD, that are of high value for the identification of or discovery of therapeutic compounds that may be useful in the treatment of neurodegenerative diseases. The cell-based method provided herein is a novel mammalian cell model of PD and other neurodegenerative disorders. The cell model is based on the assessment of mitochondrial fusion and fission. The cell model is used to identify inhibitors of the mitochondrial fusion phenotype induced in cells by manipulation of the cell machinery (for example, genes, proteins, and signaling pathways) that regulates mitochondrial fusion and fission. Compounds identified as inhibitors of the mitochondrial fusion phenotype will be highly valued candidates for treatment of PD and other neurodegenerative disorders.
[0015] Mitochondria have been implicated in PD by the presence of pathological changes in patient brains, by the finding that mitochondrial toxins can recapitulate some aspects of the disease, and by the recent identification of Pinkl (used interchangeably with PINKl herein) mutations in PD (Beal, 2003). Pinkl encodes a mitochondrial kinase and deletion/loss of function is associated with familial forms of PD (Abeliovich and Flint Beal, 2006). Drosophila with loss of Pinkl show some evidence of excessive mitochondrial fusion (Poole et al, 2008), but it is unclear if this is a primary event. In mice, loss of Pinkl does not lead to obvious mitochondrial changes. Because these data are indirect and not carried out in a mammalian system, the methods lack usefulness for identification and development of therapeutic compounds to treat neurodegenerative diseases in humans.
[0016] The subtle nature of PD, in that the disease does not arise for the initial decades, also underscores the difficulty in generating a disease model. A novel cell-based disease model is described herein.
[0017] Provided herein is a cell-based assay for the identification of compounds, factors or agents that function in a similar manner to Pinkl and suppress the excessive fusion phenotype of DNMIl inhibition or other forms of mitochondrial fusion. Such mitochondrial fusion could be induced either by decreasing the activity of fission genes, such as Dnmll and Fisl; or by increasing the activity of fusion genes, such as Opal, mitofusinl and mitofusin2. Such manipulations will lead to excessive fusion and subsequently cell death, both of which can be assayed by techniques known in the art, for example, microscopy (see Example 1) or by any cell death assay. Based on the known nucleotide and amino acid sequences of Dmnll (see, for example, NCBI Accession Nos. BC024590 and AB209070), Fisl (see, for example, NCBI Accession Nos. BC003540 and AAH03540), Opal (see, for example, NCBI Accession Nos. AK022522 and AAH75805), mitofusinl (see, for example, NCBI Accession Nos. U95822 and AB043588), and mitofusin2 (see, for example, NCBI Accession Nos. CAI19087 and AY028429), one having ordinary skill in the art would know how to generate an expression vector and express the protein of interest in a cell.
[0018] One of skill in the art know how to generate a vector to express a protein of interest in a cell using well-known molecular biology techniques. Vectors that may be used in the disclosed methods include non-viral nucleic acid vectors, such as plasmids, as well as viral vectors. Non- limiting examples of viral vectors include lentiviral, adeno-associated virus-2 (AAV-2), adenoviral, retroviral, polio viral, murine Maloney-based, alpha viral vector, pox viral, herpes viral, vaccinia viral, baculoviral, parvoviral, or any combination thereof. Cells that may be used in the disclosed methods include any mammalian cell, for example a neuronal cell. The neuronal cell may be a primary neuronal cell, for example, a post-mitotic neuron. Non- limiting examples of post-mitotic neurons include cortical neurons, dopamine neurons and sympathetic neurons.
[0019] Mitochondria are known to be important in PD. Studies have focused on the role of oxidative stress and mitochondrial energetics in PD. Morphology of mitochondria, including fusion and fission, plays a role in oxidative stress and energetics. Thus, the methods provided herein can also be used to identify compounds that are generally protective to mitochondria, for example, compounds that regulate oxidative stress and energetics through morphological regulation of mitochondria. In addition to identifying compounds that decrease mitochondrial fusion, the disclosed methods may also be used to identify compounds or conditions that increase mitochondrial fusion. Normal cells maintain a balance of mitochondrial fusion and fission, thereby maintaining proper generation of cellular energy. Mitochondria also play a role in other cellular processes, such as signaling, cellular differentiation, cell death, as well as the control of the cell cycle and cell growth, all of which may also be affected by proper or improper regulation of mitochondrial morphology.
[0020] Regulation of mitochondrial fission and fusion can be detected by assessing mitochondrial morphology or by assessing cell death. Too much mitochondrial fission or too much mitochondrial fusion can be toxic to cells and lead to cell death. Mitochondrial morphology can be visualized using microscopic methods. For example, a fusion protein can be expressed in a cell, where the fusion protein is a mitochondrial-specific protein fused to a detectable marker. For example, the detectable marker can be a fluorescent protein or peptide which is directly detected by fluorescence microscopy. For example, pDsRed2-Mito (Clontech) is a mammalian expression vector that encodes a fusion of red fluorescent protein and the mitochondrial targeting sequence from subunit VIII of human cytochrome c oxidase. The expression of a fluorescent protein, such as green fluorescent protein or red fluorescent protein, enables visualization mitochondrial morphology. Fluorescence can be detected directly (e.g., detection of a fluorescent protein) or indirectly (e.g., detection of an antibody with a fluorescent label or tag, wherein the antibody specifically binds a protein of interest, such as a mitochondrial marker protein). Non-limiting examples of mitochondrial marker proteins that can be detected include cytochrome c oxidase, manganese superoxide dismutase and mitochondrial porin. Additional examples of mitochondrial-specific antibodies are available from MitoSciences.
[0021] In a cell-based assay such as the one provided herein, any compound, factor or agent that functions similarly to overexpression of wildtype Pinkl and suppresses the excessive fusion phenotype would be a potentially therapeutic molecule for PD therapy or other neurodegenerative disorders such as ALS and AD.
[0022] Candidate compounds that can be used in the disclosed methods include compounds that inhibit or suppress proteins and genes involved in mitochondrial fusion, such as Opal, mitofusinl and mitofusin2. Other non-limiting examples of candidate compounds include, but are not limited to, GTPase inhibitors, such inhibitors can be readily screened for by methods known in the art. An exemplary compound which can be assessed in the disclosed methods is the GTPase competitive inhibitor called dynasore, a competitive inhibitor of the GTPase dynamin (Macia E. et al., 2006), or structural analogs thereof. Further examples of compounds that can be assessed using the disclosed methods are mfisi-1 and structural analogs thereof as described in U.S. Patent Application Publication No. US 2005/0038051. As shown in Example 1, overexpression of Pinkl suppresses the induced excessive mitochondrial fusion phenotype. Fragments of Pinkl may also be used in the disclosed methods. One of skill in the art would know how to make such fragments of Pinkl, based on the known nucleotide and amino acid sequences of Pinkl . For example, see NCBI Accession Nos. NP_115785, AAH28215, AB053323.
[0023] The methods disclosed herein can be carried out in a high throughput manner, such as for 100 compounds, 1000 compounds, or more than 1000 compounds.
EXAMPLES
EXAMPLE 1
PINKl OVEREXPRESSION SUPPRESSES EXCESSIVE MITOCHONDRIAL FUSION IN
CELLS EXPRESSING A DNMlL DOMINANT NEGATIVE MUTANT [0024] Mitochondrial fusion and fission, dynamics of mitochondrial morphology, are highly regulated and play a key role in mitochondrial energetics and in apoptosis (Griffin et al., 2006). Reducing the activity of a gene essential for mitochondrial fission, DNMIl (dynamin 1 -like) (also known as DRPl) using a dominant negative mutant allele (Chang and Blackstone, 2007; Cribbs and Strack, 2007), leads to elongated mitochondria and toxicity (Figure 1).
[0025] In this experiment, COS7 cells were co-transfected with MitoDSRed 0.25 μg (ClonTech), pGW-DrpS637D (0.35 μg) (dominant negative mutant of Dnmll) (Chang and Blackstone, 2007) and pLenti6-DEST-PINKl-V5 wildtype (human PINKl expression from a lentiviral vector) at 0.1, 0.3, 1.0 μg (Addgene, Cookson), and a green fluorescent protein (GFP) marker plasmid (0.1 μg) as control. The dominant negative mutant of Dnmll (Drps637D) induced an excessive mitochondrial fusion phenotype in the COS7 cells. The mitochondria were analyzed for changes in morphology by immunofluorescence microscopy at 36 hours post-transfection. The data in Figure 1 are reported as a ratio of the number of cells with fused, elongated mitochondria to the total cells expressing DSRed in mitochondria. The data were analyzed by one-way analysis of variance. Three random areas per well were imaged and each section had approximately 150 cells. The trans fection efficiency was approximately 40%.
[0026] As shown here, the phenotype of the Dnmll dominant negative (excessive fusion) is suppressed by overexpressing wildtype Pinkl, this result is not observed with PD-associated mutant alleles of Pinkl. Thus, Pinkl activity promotes mitochondrial fission and suppresses the phenotype of reduced DNMIl activity, and thus prevents excessive mitochondrial fusion.
[0027] Although the invention has been described and illustrated in the foregoing illustrative embodiments, it is understood that the present disclosure has been made only by way of example, and that numerous changes in the details of implementation of the invention can be made without departing from the spirit and scope of the invention, which is limited only by the claims that follow. Features of the disclosed embodiments can be combined and rearranged in various ways within the scope and spirit of the invention.
REFERENCES
Abeliovich. A. and Flint BeaJ, M. (2006). Parkinsonism genes: culprits and clues. Journal of
Neurøchemistry 99. 1062-1072. Beal, MF. (2003 ) Mitochondria, oxidative damage, and inflammation in Parkinson's disease.
Ann N Y Acad Sci 991 , 120- 131.
Chang, CR and Blackstone C. (2007) Cyclic AMP-dcpcndcnt protein kinase phosphorylation of DRpI regulates its GTPase activity and mitochondrial morphology. Journal of Biological Chemistry 282, 21583-21587.
Cribbs Jl and Strack S. (2007) Reversible phosphorylation of Dtp 1 by cyclic AMP- dependent protein kinase and calcineurin regulates mitochondrial fission and cell death. EMBO Rep 8, 939-944.
Griffin EE, ϋetmcr SA and Chan DC (200b). Molecular mechanism of mitochondrial membrane fusion. Biocbemica Biophysica Acta 1763, 482-489. Macia E et al. (2006) Dynasore, a cell-permeable inhibitor or dynaniin. Dev Cell 10, 839- 850. Poolc AC et al. (2008) The PINIvI /parkin pathway regulates mitochondrial morphology, Proe
Natl Λcad Sci USA 105, 1638-1&43.

Claims

What is claimed is:
1. A method for determining whether a compound enhances mitochondrial fission in a mammalian cell, the method comprising:
(a) inducing mitochondrial fusion in a mammalian cell, wherein the inducing comprises (i) reducing activity or expression of a mitochondrial fission gene or protein; or (ii) increasing activity or expression of a mitochondrial fusion gene or protein;
(b) contacting the cell with a candidate compound;
(c) determining whether mitochondrial fusion is reduced in the cell of (b) compared to a cell of (a) in the absence of the candidate compound,
wherein a determination of a reduction in mitochondrial fusion indicates that the compound enhances mitochondrial fission.
2. A method for determining whether a compound will be useful for treating a neurodegenerative disease, the method comprising:
(a) inducing mitochondrial fusion in a mammalian cell, wherein the inducing comprises (i) reducing activity or expression of a mitochondrial fission gene or protein; or (ii) increasing activity or expression of a mitochondrial fusion gene or protein;
(b) contacting the cell with a candidate compound;
(c) determining whether mitochondrial fusion is reduced in the cell of (b) compared to a cell of (a) in the absence of the candidate compound,
wherein a determination of a reduction in mitochondrial fusion indicates that the compound will be useful for treating a neurodegenerative disease.
3. The method of claim 2, wherein the neurodegenerative disease is Parkinson's disease, Alzheimer's disease or amyotrophic lateral sclerosis.
4. The method of claim 1 or 2, wherein the mitochondrial fission gene or protein is Dnml or Fisl.
5. The method of claim 1 or 2, wherein the mitochondrial fusion gene or protein is Opal, mitofusinl or mitofusin2.
6. The method of claim 1 or 2, wherein the reducing comprises using a dominant negative mutant of the mitochondrial fission gene or protein.
7. The method of claim 1 or 2, wherein the reducing comprises using a nucleic acid that specifically binds to a nucleic acid encoding the mitochondrial fission gene, and inhibits protein expression from the nucleic acid.
8. The method of claim 7, wherein the nucleic acid is a siRNA, shRNA or antisense RNA.
9. The method of claim 1 or 2, wherein the increasing comprises expressing the mitochondrial fusion gene or protein from a nucleic acid vector.
10. The method of claim 1 or 2, wherein the compound comprises mfisi-1 or a structural analog thereof.
11. The method of claim 1 or 2, wherein the compound is a GTPase inhibitor.
12. The method of claim 11, wherein the inhibitor is dynasore or a structural analog thereof.
13. The method of claim 1 or 2, wherein the compound comprises a PINKl protein or a peptide fragment thereof.
14. The method of claim 1 or 2, wherein the cell is a mammalian cell.
15. The method of claim 1 or 2, wherein the cell is a neuronal cell.
16. The method of claim 1 or 2, wherein the determining comprises assessing cell death.
17. The method of claim 1 or 2, wherein the determining comprises microscopy.
18. The method of claim 1 or 2, wherein the determining comprises detecting mitochondria.
19. The method of claim 1 or 2, wherein the cell of (a) further comprises a nucleic acid vector encoding a detectable mitochondria-specific protein.
20. The method of claim 1 or 2, wherein the determining comprises using an antibody that specifically binds to a mitochondria-specific protein.
21. The method of claim 20, wherein the antibody comprises a detectable label.
22. The method of claim 1 or 2, wherein the method is carried out in a high throughput manner.
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