WO2014059091A2 - Methods for modulating mitochondrial function via inf2 - Google Patents

Description

Methods for Modulating Mitochondrial Function via INF2 Introduction

[0001] This invention was made with government support under contract numbers R01 GM069818 and R01 DK88826 awarded by the National Institutes of Health. The government has certain rights in the invention.

Background of the Invention

[0002] Mitochondrial function extends far beyond that of ATP generation, since mitochondria act as sensors of metabolic homeostasis and are key players in cell death pathways (Nunnari & Suomalainen (2012) Cell 148:1145; Martinou & Youle (2011) Developmental Cell 21:92) . Important to mitochondrial function is their dynamic ability to undergo fusion, fission and move in cells, with defects implicated in many neurodegenerative diseases (Chen & Chan (2009) Hum. Mol . Genet. 18:R169; Correia, et al .

(2012) Adv. Exp. Med. Biol. 724:205) . During fission, a key step is oligomerization of Drpl (Dnml in yeast) into a helical ring around the outer mitochondrial membrane, followed by ring constriction. The mechanism for Drpl recruitment to fission sites, however, is unclear in mammals. One issue is that the diameter of the Drpl ring is significantly narrower (100-130 nm for Dnml) than an unconstricted mitochondrion (Mears, et al . (2011) Nature Struct. Mol. Bol . 18:20) , suggesting that prior constriction is necessary. Recent findings show that mitochondrial fission occurs preferentially at endoplasmic reticulum (ER) contact sites, with ER circumscribing mitochondria (Friedman, et al . (2011) Science 334:358) . Mitochondria are constricted at these ER contact sites even in the absence of Drpl activity (Friedman, et al . (2011) supra) , supporting previous evidence for Drpl/Dnml- independent constriction in C. elegans (Labrousse, et al . (1999) Mol. Cell 4:815) and budding yeast (Legesse-Miller, et al. (2003) Mol. Biol. Cell 14:1953) .

[0003] INF2 (inverted formin-2, FH2 and H2 domain containing protein) is a metazoan formin protein with intriguing biochemical, cellular, and physiological characteristics. Biochemically, INF2 accelerates both actin polymerization and depolymerization (Chhabra & Higgs (2006) J. Biol. Che . 281:26754) . In cells, INF2 exists as two isoforms differing in C-terminal sequence: the CAAX isoform, which is tightly bound to ER (Chhabra, et al . (2009) J^". Cell Sci. 122:1430) has a C-terminal sequence of KRPSRNQEEFVPDSDDIKAKRLCIVQ (SEQ ID N0:1); and the non-CAAX isoform, which is cytoplasmic (Ramabhadran, et al . (2011) Molecular biology of the cell 22, 4822 (Dec, 2011) and has a C-terminal sequence of KRPSRNQEGLRSRPKAK (SEQ ID NO : 2 ) . Suppression of INF2-nonCAAX in culture cells causes Golgi dispersal. In contrast, the cellular function of INF2-CAAX is not described in the art, since its suppression has no apparent effect on ER structure or dynamics (Chhabra, et al . (2009) J. Cell Sci. 122:1430) . Physiologically, mutations in INF2 are linked to two human diseases: focal and segmental glomerulosclerosis, a degenerative kidney disease (Brown, et al . (2010) Nat. Genet. 42:72; and Charcot-Maire-Tooth disease (CMTD) , a peripheral neuropathy (Boyer, et al . (2011) New England J. Med. 365:2377) .

Summary of the Invention

[0004] It has now been found that INF2-CAAX mediates mitochondrial fission. Therefore, this invention is a method for identifying an agent that modulates INF2-CAAX activity or INF2 -CAAX-mediated mitochondrial function. The method involves contacting a eukaryotic cell with a test agent, wherein the eukaryotic cell includes an INF2-CAAX polypeptide; and determining the effect of the test agent on INF2-CAAX activity or INF2 -CAAX-mediated mitochondrial function, wherein a decrease or increase in INF2-CAAX activity or INF2 -CAAX-mediated mitochondrial function, compared to INF2-CAAX activity or INF2 -CAAX-mediated mitochondrial function in the absence of the test agent, indicates that the test agent modulates INF2-CAAX) activity or INF2 -CAAX-mediated mitochondrial function. In one embodiment, the determining step includes real-time imaging of the cell to detect a change in mitochondrial constriction, fission or motility. In another embodiment, the eukaryotic cell has been genetically modified with an expression vector that includes a nucleotide sequence encoding an INF2-CAAX polypeptide. An agent identified by the screening method and a method of using such agents in the amelioration of a disease associated with mitochondrial dysfunction {e.g., neurodegenerative disease, cancer, diabetes or cardiac disease) are also provided.

Detailed Description of the Invention

[0005] Mitochondrial fission is fundamentally important to cellular physiology. It has now been shown that act in polymerization through the ER-localized formin INF2 is required for efficient mitochondrial fission in mammalian cells. Specifically, it was observed that inhibition of INF2-CAAX using interfering RNAs (RNAi) causes mitochondria to elongate, while a constitutively active INF2-CAAX mutant causes mitochondria to shorten. Actin filaments accumulate between mitochondria and INF2 -enriched ER membranes at constriction sites. Based upon these results it is believed that INF2 -induced actin filaments drive initial mitochondrial constriction, allowing Drpl-driven secondary constriction. Based on these findings, the present invention includes the use of IFN2 effector molecules to modulate the activity of IFN2-CAAX and to ameliorate a variety of neurodegenerative diseases such as Charcot - Marie-Tooth disease (CMTD) , Alzheimer's, Huntington's, Parkinson's, and amyotrophic lateral sclerosis (ALS) , as well as cardiac disease and cancer, by targeted therapies that either activate or inhibit INF2-CAAX. In addition, the invention also includes the use of INF2-CAAX in screening assay to identify effector molecules that alter mitochondrial fission and ameliorate neurodegenerative diseases .

[0006] When used in screening assays, INF2-CAAX can be exogenously or endogenously expressed by a host cell . Therefore, this invention provides isolated cells harboring a nucleic acid encoding INF2-CAAX. The cells typically include a nucleic acid having a nucleotide sequence encoding INF2-CAAX; and a regulatory element (s) operably linked to the nucleotide sequence encoding the INF2-CAAX. The cells produce INF2-CAAX polypeptides, and are useful in screening methods for identifying agents that modulate INF2-CAAX activity and/or INF2 -CAAX-mediated mitochondrial constriction .

[0007] Nucleotide and amino acid sequences of ER-localized INF2 are known in the art. Human INF2-CAAX has the amino acid sequence set forth under GENBANK Accession No. NP_071934 and is readily distinguished from the cytoplasmic form by the C-terminal sequence KRPSRNQEEFVPDSDDIKAKRLCIVQ

(SEQ ID NO:l) . Human INF2-CAAX has the nucleotide sequence set forth under GENBANK Accession No. NM_022489. The sequence of the mouse INF2-CAAX is found under GENBANK accession number NP_940802. In some embodiments, the INF2- CAAX polypeptide is a full-length polypeptide.

[0008] For exogenous expression, any suitable expression vector can be used including, but are not limited to, baculovirus vectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral vectors (e.g., viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, and the like), Pl-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest. Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available. For eukaryotic host cells, the following vectors are provided by way of example; pXTl, pcDNA3.1, pSG5 (Stratagene) , pSVK3 , pBPV, pMSG, and pSVLSV40 (Pharmacia) . However, any other plasmid or other vector may be used so long as it is compatible with the host cell.

[0009] Depending on the host/vector system used, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see, e.g., Bitter, et al . (1987) Meth. Enzymol . 153:516-544) . In some embodiments, regulatory elements include regulatory elements that result in neuronal cell-specific expression of the operably linked INF2-CAAX polypeptide -encoding nucleic acid. Neuronal cell -specific regulatory elements

(including promoters, enhancers, and the like) are known to those skilled in the art. Examples of neuronal cell- specific regulatory elements include those from a neuron- specific enolase (NSE) gene (Hannas-Dj ebarra , et al . (1997) Brain Res. Mol . Brain Res. 46:91-99; a PDGF gene; a Thl gene {e.g., mouse Thyl .2 (Caroni, et al . (1997) J^". Neurosci. Methods 71:3-9); a neurofilament gene (e.g., NF- L, NF-M, and NF-L) ; a glial filament acidic protein (GFAP) gene; a myelin basic protein gene; a microtubule-associated protein gene; a synaptophysin gene; a tyrosine hydroxylase gene; and the like. Thus, a suitable neuronal cell-specific regulator region includes, e.g., an NSE promoter; a PDGF promoter; an aromatic amino acid decarboxylase (7AADC) promoter; a neurofilament promoter (see, e.g., GENB/A K Accession No. L0 147) ; a synapsin promoter (see, e.g., GENBANK Accession No. M55301) ; a thy-1 promoter (see, e.g., Chen, et al . (1987) Cell 51:7-19); a serotonin receptor promoter (see, e.g., GENBANK Accession No. S62283); a tyrosine hydroxylase promoter (TH) ; a GnRH promoter (see, e.g., Radovick, et al . (1991) Proc . Natl. Acad. Sci . USA 88:3402-3406); an L7 promoter (see, e.g., Oberdick, et al . (1990) Science 248:223-226); · a DNMT promoter (see, e.g., Bartge, et al . (1988) Proc. Natl. Acad. Sci. USA 85:3648- 3652); an enkephalin promoter (see, e.g., Comb, et al . (1988) EMBO J. 17:3793-3805); a myelin basic protein (MBP) promoter; a GFAP promoter; and a CMV enhancer/platelet - derived growth factor-β promoter (see, e.g., Liu, et al . (2004) Gene Therapy 11:52-60) .

[ 00010 ] In some embodiments, the INF2-CAAX polypeptide- encoding nucleotide sequence is operably linked to an inducible promoter. Suitable inducible promoters include, but are not limited to, the pL of bacteriophage λ; Plac; Ptrp; Ptac (Ptrp-lac hybrid promoter) ; an isopropyl -beta-D- thiogalactopyranoside (IPTG) -inducible promoter, e.g., a lacZ promoter; a tetracycline- inducible promoter; an arabinose inducible promoter, e.g., PBAD (see, Guzman, et al . (1995) J. Bacteriol. 177:4121-4130); a xylose-inducible promoter, e.g., Pxyl (see, Kim, et al . (1996) Gene 181:71- 76) ; a GAL1 promoter; a tryptophan promoter; a lac promoter; an alcohol -inducible promoter, e.g., a methanol- inducible promoter, an ethanol - inducible promoter; a raffinose- inducible promoter; a heat-inducible promoter, e.g., heat inducible lambda PL promoter, a promoter controlled by a heat-sensitive repressor (e.g., CI857- repressed lambda-based expression vectors; see Hoffinann, et al. (1999) FEMS Microbiol. Lett. Ill (2 ) : 327 -3 ) ; and the like.

[ 00011 ] To generate a genetically modified host cell, a construct including a nucleotide sequence encoding an INF2- CAAX polypeptide is introduced stably or transiently into a host cell, using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, DEAE-dextran-mediated transfection, liposome-mediated transfection, heat shock in the presence of lithium acetate, and the like. For stable transformation, a nucleic acid will generally further include a selectable marker, e.g., any of several well- known selectable markers such as neomycin resistance, ampicillin resistance, tetracycline resistance, chloramphenicol resistance, kanamycin resistance, and the like.

[ 00012 ] Suitable host cells include mammalian cells, including primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. Suitable mammalian cell lines include, but are not limited to, HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096) , U20S cells, 293 cells

(e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658) , Huh-7 cells, BHK cells (e.g., ATCC No. CCL10) , PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651) , RATI cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573) , HLHepG2 cells, and the like.

[00013] In some embodiments, the cells used in the screening assay herein are mammalian cells that normally produce INF2-CAAX. Examples of such cells include neuronal cells, microglial cells, and astrocytes. Immortalized neuronal cells, microglial cells, and astrocytes are also of interest. Suitable immortalized cells include, but are not limited to, neuro-2A cells; B103; PC12 ; NT2 ; and the like. PC12 cells are available from ATCC under deposit number CRL-1721. Neuro-2a cells are available from ATCC under deposit number CCL-131.

[00014] In some embodiments, the cell is a neuronal cell or a neuronal -like cell. The cells can be of human, non-human primate, mouse, or rat origin, or derived from a mammal other than a human, non-human primate, rat, or mouse. Suitable cell lines include, but are not limited to, a human glioma cell line, e.g., SVGpl2 (ATCC CRL-8621) , CCF- STTG1 (ATCC CRL-1718) , SW 1088 (ATCC HTB-12) , S 1783 (ATCC HTB-13) , LLN-18 (ATCC CRL-2610) , LNZTA3WT4 (ATCC CRL- 11543), LNZTA3WT11 (ATCC CRL-11544), U-138 MG (ATCC HTB- 16) , U-87 MG (ATCC HTB-14) , H4 (ATCC HTB-148) , and LN-229

(ATCC CRL-2611); a human medulloblastoma-derived cell line, e.g., D342 Med (ATCC HTB-187), Daoy (ATCC HTB-186), D283 Med (ATCC HTB-185) ; a human tumor-derived neuronal - like cell, e.g., PFSK-1 (ATCC CRL-2060) , SK-N-DZ (ATCC CRL- 2149), SK-N-AS (ATCC CRL-2137), SK-N-FI (ATCC CRL-2142), IMR-32 (ATCC CCL-127) , etc.; a mouse neuronal cell line, e.g., BC3H1 (ATCC CRL-1443), EOCi (ATCC CRL-2467) , C8-D30

(ATCC CRL-2534) , C8-S (ATCC CRL-2535) , Neuro-2a (ATCC CCL- 131) , NB41A3 (ATCC CCL-147) , SW10 (ATCC CRL-2766) , NG108-15 (ATCC HB-12317) ; a rat neuronal cell line, e.g., PC-12 (ATCC CRL-1721) , CTX TNA2 (ATCC CRL-2006) , C6 (ATCC CCL- 107), F98 (ATCC CRL-2397) , RG2 (ATCC CRL-2433), B35 (ATCC CRL-2754) , R3 (ATCC CRL-2764) , SCP (ATCC CRL-1700) , and OA1 (ATCC CRL-6538) .

[ 00015 ] Using the cells of this invention in screening assays, agents that modulate INF2-CAAX activity and/or INF2 -CAAX-mediated mitochondrial function (e.g., constriction, fission or motility) can be identified. The agents so identified are candidate agents for treating or ameliorating neurodegenerative diseases or other diseases associated with dysfunctional mitochondria. In some embodiments, the assays are in vitro cell -based screening methods for identifying compounds that modulate INF2-CAAX activity and/or mitochondrial function. In some embodiments, a subject screening assay involves contacting a eukaryotic cell that produces an INF2-CAAX polypeptide with a test agent; and determining the effect, if any, of the test agent on INF2-CAAX activity and/or mitochondrial function. A change in INF2-CAAX activity and/or mitochondrial function, compared to INF2-CAAX activity and/or mitochondrial function in the absence of the test agent, indicates that the test agent modulates INF2-CAAX activity and/or mitochondrial function. A test agent of interest is an agent that reduces or increases INF2-CAAX activity and/or mitochondrial function (e.g., constriction, fission or motility) by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, or more, compared to the level of INF2-CAAX activity and/or mitochondrial function in the absence of the test agent .

[ 00016 ] The terms "candidate agent," "test agent," "agent," "substance," "effector" and "compound" are used interchangeably herein. Candidate agents encompass numerous chemical classes, typically synthetic, semi-synthetic, or naturally-occurring inorganic or organic molecules. Candidate agents include those found in large libraries of synthetic or natural compounds. For example, synthetic compound libraries are commercially available from Maybridge Chemical Co. (Trevillet, Cornwall, UK), ComGenex (South San Francisco, CA) , and MicroSource (New Milford, CT) . A rare chemical library is available from Aldrich (Milwaukee, WI) . Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from Pan Labs (Bothell, WA) or are readily producible.

[ 00017 ] Candidate agents may be small organic or inorganic compounds having a molecular weight of more than 50 and less than about 10,000 daltons, e.g., from about 50 daltons to about 100 daltons, from about 100 daltons to about 500 daltons, from about 500 daltons to about 1000 daltons, from about 1000 daltons to about 5000 daltons, or from about 5000 daltons to about 10,000 daltons. Candidate agents may have functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and may include at least an amine, carbonyl , hydroxyl or carboxyl group, and may contain at least two of the functional chemical groups. The candidate agents may include cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, antibodies, nucleic acids, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[ 00018 ] Assays of the invention include controls, where suitable controls include a sample (e.g., a sample including the test cell) in the absence of the test agent. Generally a plurality of assay mixtures is run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection .

[ 00019 ] Screening may be directed to known pharmacologically active compounds and chemical analogs thereof, or to new agents with unknown properties such as those created through rational drug design. Efficacious candidates can be identified by phenotype, i.e., an arrest or reversal of particular cognitive behaviors in a suitable animal model for a neurodegenerative disease.

[ 00020 ] Agents that have an effect in an assay method of the invention may be further tested for cytotoxicity, bioavailability, and the like, using well-known assays. Agents that have an effect in an assay method of the invention may be subjected to directed or random and/or directed chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Such structural analogs include those that increase bioavailability, and/or reduced cytotoxicity. Those skilled in the art can readily envision and generate a wide variety of structural analogs, and test them for desired properties such as increased bioavailability and/or reduced cytotoxicity and/or ability to cross the blood- brain barrier. [00021] A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc. that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti -microbial agents, etc. may be used. The mixture of components is added in any order that provides for the requisite binding.

[00022] Incubations are performed at any suitable temperature, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Typically between 0.1 and 1 hour will be sufficient.

[00023] A candidate agent is assessed for any cytotoxic activity it may exhibit toward the cell used in the assay, using well-known assays, such as trypan blue dye exclusion, an MTT ( [3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyl-2 H- tetrazolium bromide]) assay, and the like. Agents that do not exhibit significant cytotoxic activity are considered candidate agents.

[00024] In accordance with some embodiments of the screening assay, the determining step includes contacting cells expressing an INF2-CAAX polypeptide with an indicator agent that is an indicator of mitochondrial function. Indicator agents will in many embodiments include a fluorescent dye. Suitable indicator agents include, but are not limited to, dihydrorhodamine 123; MITOTRACKER mitochondrial function indicator Orange CM-H₂ TMRos ; MITOTRACKER mitochondrial function indicator CMTMRos; MITOTRACKER mitochondrial function indicator Red CM-H₂XRos; MITOTRACKER mitochondrial function indicator Red CMXRos; rhodamine 123; 5,5' ,6,6'- tetrachloro- 1 , 1 ' , 3 , 3 ' -tetraethylbenzimidazolylcarbocyanine iodide; tetramethylrhodamine , ethyl ester, perchlorate; and tetramethylrhodamine , methyl ester, perchlorate. The effect, if any, of the test agent on mitochondrial function is in some embodiments determined by detecting a change in the indicator agent .

[ 00025 ] In other embodiments, a screening assay involves contacting a cell that expresses an INF2-CAAX polypeptide and also includes in a mitochondrial indicator protein that provides a detectable signal with a test agent. In these embodiments, the effect, if any, of the test agent on mitochondrial function is determined by analyzing the cells by real-time imaging. Real-time imaging can detect, e.g., a change in mitochondrial motility. Proteins that provide for detectable signals include, e.g. GFP . In many embodiments, the mitochondrial indicator protein includes a mitochondrial localization signal.

[ 00026 ] In still other embodiments, a screening assay involves contacting a cell that expresses an INF2-CAAX polypeptide with a test agent and determining whether the activity of INF2-CAAX is modulated. The effect, if any, of the test agent on INF2-CAAX activity is determined by, e.g., analyzing actin polymerization.

[ 00027 ] A test agent that INF2-CAAX activity and/or mitochondrial function is a candidate agent for treating a disease associated with dysfunctional mitochondria, including, e.g., a neurodegenerative disease or cancer. A candidate agent identified can be further evaluated, in a secondary screen, for efficacy in vivo, using an appropriate animal model of the disease or condition. For example, when the disease is a neurodegenerative disease, such secondary screens can employ any phenomena associated learning impairment, dementia or cognitive disorders that can be readily assessed in an animal model . The screening can include assessment of phenomena including, but not limited to: 1) assessment behavioral symptoms associated with memory and learning; and 2) detection of neurodegeneration characterized by progressive and irreversible differentiation of the limbic system, association neocortex, and basal forebrain

(neurodegeneration can be measured by, for example, detection of synaptophysin expression in brain tissue)

(see, e.g., Games, et al . (1995) Nature 373:523-7) . These phenomena may be assessed in the screening assays either singly or in any combination.

[00028] Generally, the screen will include control values (e.g., the extent of neuronal and/or behavioral deficits in the test animal in the absence of test compound (s) ) . Test substances which are considered positive, i.e., likely to be beneficial in the treatment, will be those which have a substantial effect upon neuronal and behavioral deficits, and associated disorders.

[00029] Methods for assessing these phenomena, and the effects expected of a candidate agent for treatment are known in the art. For example, methods for using transgenic animals in various screening assays for, for example, testing compounds for an effect on Alzheimer's disease

(AD), are found in WO 9640896; WO 9640895; and WO 9511994. Examples of assessment of these phenomena are provided below, but are not meant to be limiting.

[00030] Behavioral tests designed to assess learning and memory deficits can be employed. An example of such as test is the Morris Water maze. In this procedure, the animal is placed in a circular pool filled with water, with an escape platform submerged just below the surface of the water. A visible marker is placed on the platform so that the animal can find it by navigating toward a proximal visual cue. Alternatively, a more complex form of the test in which there are no formal cues to mark the platform's location will be given to the animals. In this form, the animal must learn the platform's location relative to distal visual cues. Alternatively, or in addition, memory and learning deficits can be studied using a 3 runway panel for working memory impairment (attempts to pass through two incorrect panels of the three panel-gates at four choice points) (Ohno, et al . (1997) Pharmacol. Biochem. Behav. 57:257- 261) .

[ 00031] This invention also provides therapeutic agents that modulate (i.e., increase or decrease) INF2-CAAX activity and/or INF2 -CAAX-mediated mitochondrial function; as well as compositions, including pharmaceutical compositions, containing the agents. The therapeutic agents may be identified by the screening method described herein, by design, or by agents described herein that have the intended effect of increasing or decreasing INF-CAAX expression or activity.

[ 00032 ] Suitable agents include small organic or inorganic compounds as described above, as well as biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. In some embodiments, a suitable agent is a peptide or polypeptide that inhibits the interaction of a INF2-CAAX polypeptide with ER or mitochondria. In accordance with this embodiment, the polypeptide is a splice variant -specific antibody or antibody fragment against human INF2-CAAX (acetyl -EEVPPDSDDNKTKKLC-amide; SEQ ID NO:9), which blocks the interaction of INF2-CAAX polypeptide with the ER thereby inhibiting INF2-CAAX activity and increasing mitochondrial length. In another embodiment, the agent is a siRNA molecule that inhibits INF2-CAAX expression. In accordance with this embodiment, oligonucleotides of the siRNA molecule include 5 ' -ACA AAG AAA CTG TGT GTG A-3' (SEQ ID NO:5) or 5'-CCC TGA TTC TGA TGA TAA T-3' (SEQ ID NO:6), which inhibit the expression of INF2-CAAX thereby inhibiting INF2-CAAX activity and increasing mitochondrial length. In yet other embodiments, the agent is a constitutively activate INF2-CAAX protein, e.g., harboring a A149D mutation, which decreases mitochondrial length and increased ER/mitochondrial association.

[ 00033 ] In some embodiments, the invention provides compositions containing an agent that modulates INF2-CAAX activity and/or INF2 -CAAX-mediated mitochondrial function; and at least one other therapeutic agent. Therapeutic agents that can be formulated together with an agent that modulates INF2-CAAX activity and/or mitochondrial function include, but are not limited to, agents that are used to treat individuals with AD, including, but not limited to, acetylcholinesterase inhibitors, including, but not limited to, ARICEPT (donepezil) , EXELON (rivastigmine) , metrifonate, and TACRINE (Cognex) ; non-steroidal antiinflammatory agents, including, but not limited to, ibuprofen and indomethacin; cyclooxygenase-2 (Cox2) inhibitors such as CELEBREX; and monoamine oxidase inhibitors, such as SELEGILENE (Eldepryl or Deprenyl) . Any known inhibitor of chymotrypsin-like serine proteases can be formulated together with another therapeutic agent used to treat AD. Dosages for each of the above agents are known in the art, and can be used in a pharmaceutical preparation with an agent that modulates INF2-CAAX activity and/or mitochondrial function. For example, ARICEPT is generally administered at 50 mg orally per day for 6 weeks, and, if well tolerated by the individual, at 10 mg per day thereafter .

[ 00034 ] The invention also provides formulations, including pharmaceutical formulations, containing an agent that modulates INF2-CAAX activity and/or mitochondrial function. In general, a formulation includes an effective amount of an agent that modulates INF2-CAAX activity and/or mitochondrial function. An "effective amount" means a dosage sufficient to produce a desired result, e.g., a reduction or increase in mitochondrial motility, reduction in impairment of mitochondrial function, reduction or increase in mitochondrial fission, an improvement in learning, memory, etc.

[ 00035 ] In the methods of this invention, the active agent (s) may be administered to the host using any convenient means capable of resulting in the desired modulation of INF2-CAAX activity and/or mitochondrial function, reduction in a neurological disorder, etc. Thus, the agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents of this invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols.

[ 00036 ] In pharmaceutical dosage forms, the agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

[ 00037 ] For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose ; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.

[ 00038 ] The agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers , isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.

[ 0003 9 ] The agents can be utilized in aerosol formulation to be administered via inhalation. The compounds of this invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane , propane, nitrogen and the like.

[ 00040 ] Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature .

[00041] Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful , tablespoonful , tablet or suppository, contains a predetermined amount of the composition containing one or more agents. Similarly, unit dosage forms for injection or intravenous administration may include the agents in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.

[00042] The term "unit dosage form, " as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of this invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or yehicle. The specifications for the novel unit dosage forms of the invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host .

[00043] Other modes of administration will also find use in this invention. For instance, an agent of the invention can be formulated in suppositories and, in some cases, aerosol and intranasal compositions. For suppositories, the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10%

(w/w) , preferably about 1% to about 2%.

[00044] Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.

[ 00045 ] An agent of the invention can be administered as injectables. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.

[ 00046 ] Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington (2000) The Science and Practice of Pharmacy, A. R. Gennaro, Lippincott, Williams & Wilkins. The composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.

[ 00047 ] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public . [00048] An agent of this invention can be administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration. Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, intratumoral , subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral and other parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. The composition can be administered in a single dose or in multiple doses.

[00049] The blood-brain barrier limits the uptake of many therapeutic agents into the brain and spinal cord from the general circulation. Accordingly, in some embodiments, an agent that modulates INF2-CAAX activity and/or mitochondrial function may delivered in such a manner as to avoid the blood-brain barrier. An agent that modulates INF2-CAAX activity and/or mitochondrial function may be formulated and/or modified to enable the agent to cross the blood-brain barrier. Delivery of therapeutic agents to the CNS can be achieved by several methods.

[00050] One method relies on neurosurgical techniques. In the case of gravely ill patients such as accident victims or those suffering from various forms of dementia, surgical intervention is warranted despite its attendant risks. For instance, therapeutic agents can be delivered by direct physical introduction into the CNS, such as intraventricular or intrathecal injection of drugs. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Methods of introduction may also be provided by rechargeable or biodegradable devices. Another approach is the disruption of the blood-brain barrier by substances which increase the permeability of the blood-brain barrier. Examples include intra-arterial infusion of poorly diffusible agents such as mannitol, pharmaceuticals which increase cerebrovascular permeability such as etoposide, or vasoactive agents such as leukotrienes . See, e.g., Neuwelt & Rappoport (1984) Fed. Proc. 43:214-219; Baba, et al . (1991) J. Cereb. Blood Flow Metab. 11:638-643; and Gennuso et al . (1993) Cancer Invest. 11 : 638-643.

[ 00051] Therapeutic compounds can also be delivered by using pharmacological techniques including chemical modification or screening for an analog which will cross the blood-brain barrier. The compound may be modified to increase the hydrophobicity of the molecule, decrease net charge or molecular weight of the molecule, or modify the molecule, so that it will resemble one normally transported across the blood-brain barrier. See, e.g., Levin (1980) J. Med. Chem. 23:682-684; and Kostis, et al . (1994) J. Clin. Pharmacol. 34:989-996.

[ 00052 ] Encapsulation of the drug in a hydrophobic environment such as liposomes is also effective in delivering drugs to the CNS . For example WO 91/04014 describes a liposomal delivery system in which the drug is encapsulated within liposomes to which molecules have been added that are normally transported across the blood-brain barrier .

[ 00053 ] Another method of formulating the drug to pass through the blood-brain barrier is to encapsulate the drug in a cyclodextrin . Any suitable cyclodextrin which passes through the blood-brain barrier may be employed, including, but not limited to, J-cyclodextrin, K-cyclodextrin and derivatives thereof. See, e.g., U.S. Patent Nos . 5,017,566, 5,002,935 and 4,983,586. Such compositions may also include a glycerol derivative as described by U.S. Patent No. 5, 153, 179.

[ 00054 ] Delivery may also be obtained by conjugation of a therapeutic agent to a transportable agent to yield a new chimeric transportable therapeutic agent. For example, vasoactive intestinal peptide analog (VlPa) exerted its vasoactive effects only after conjugation to a monoclonal antibody (Mab) to the specific carrier molecule transferrin receptor, which facilitated the uptake of the VIPa-Mab conjugate through the blood-brain barrier. See, e.g., Bickel, et al . (1993) Proc . Natl. Acad Sci . USA 90:2618- 2622. Several other specific transport systems have been identified, these include, but are not limited to, those for transferring insulin, or insulin-like growth factors I and II. Other suitable, non-specific carriers include, but are not limited to, pyridinium, fatty acids, inositol, cholesterol, and glucose derivatives. Certain prodrugs have been described whereby, upon entering the central nervous system, the drug is cleaved from the carrier to release the active drug. See, U.S. Patent No. 5,017,566.

[ 00055 ] This invention also provides a method of treating or ameliorating a disease associated with dysfunctional mitochondria in an individual. The method generally involves administering to an individual having a disease associated with dysfunctional mitochondria an effective amount of an agent that modulates INF2-C7AAX activity and/or INF2 -CAAX-mediated mitochondrial function. An "effective amount" of an agent that modulates INF2-CAAX activity and/or INF2 -CAAX-mediated mitochondrial function is an amount that modulates INF2-CAAX activity and/or mitochondrial function by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more, compared to the level of modulates INF2- CAAX activity and/or mitochondrial function in the absence of the agent .

[ 00056 ] By treatment is meant at least an amelioration of the symptoms associated with the pathological condition afflicting the host, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the pathological condition being treated, such as an INF2 -CAAX-associated neurological disorder and pain associated therewith. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are completely inhibited, e.g. prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the pathological condition, or at least the symptoms that characterize the pathological condition .

[ 00057 ] A variety of hosts (wherein the term "host" is used interchangeably herein with the terms "subject" and

"patient") are treatable according to the subject methods. Generally such hosts are "mammals" or "mammalian," where these terms are used broadly to describe organisms that are within the class Mammalia, including the orders Carnivore

(e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys) . In many embodiments, the hosts will be humans.

[ 00058 ] A variety of subjects are suitable for treatment with an agent of this invention. Suitable subjects include any individual, particularly a human, who has a disease associated with dysfunctional mitochondria, who is at risk for developing a disease associated with dysfunctional mitochondria, who has had a disease associated with dysfunctional mitochondria and is at risk for recurrence of the disease, or who is recovering from a disease associated with dysfunctional mitochondria.

[00059] Such subjects include, but are not limited to, individuals who have been diagnosed as having Alzheimer's disease; individuals who have been diagnosed as having CMTD, Huntington's, Parkinson's, or ALS; individuals who have suffered one or more strokes; individuals who have suffered traumatic head injury; individuals who have high serum cholesterol levels; individuals who have Αβ deposits in brain tissue; individuals who have had one or more cardiac events; subjects undergoing cardiac surgery; subjects with multiple sclerosis; and individuals who have been diagnosed with other conditions associated with mitochondrial dysfunction. See, e.g., Nunnair & Suomalainen (2012) Cell 148:1145; Martinou & Youle (2011) Dev. Cell 21:92; Chen & Chan (2009) Human. Mol . Genet. 18:R169; and Correia, et al . (2012) Adv. Exp. Med. Biol. 724:205.

[00060] In particular embodiments, the methods and compositions of the invention are used for the treatment of neurodegenerative disorders. For example, Alzheimer's disease is linked to mitochondrial activity as it has been shown that tau overexpression indirectly elongates mitochondria, which then malfunction and cause cell death (DuBoff, et al. (2012) Neuron 75:618-632) . The mitochondrial protein encoded by PINK 1 has also provided a direct link between mitochondria and Parkinson's disease (Valente, et al . (2004) Science 304:1158) . Huntington's Disease has been associated with defects in energy metabolism that appear to be widespread, affecting both the brain and peripheral tissues, and arising from mitochondrial dysfunction (Leegwater-Kim, et al . (2004) NeuroRx 1 : 128) .

[00061] The methods and compositions of the invention can be also be used for the treatment of diabetes and metabolic disease. The central role of mitochondria in metabolism of carbohydrates and fatty acids gives this organelle an important function in diabetes (Maechler, et al . (2001) Nature 414:807) . A mouse knockout of an abundant mitochondrial transcription factor has provided a model for 13-cell ablation in juvenile diabetes (Silva, et al . (2000) Nat. Genet. 26:335) Insulin release depends on mitochondrial function as influenced by the expression of the membrane transporter UCP2 (Petersen, et al . (2003) Science 300:1140; Zhang, et al . (2001) Cell 105:745) . Furthermore, cancer is connected with mitochondrial malfunction (the Warburg effect; Kim, et al . (2009) IBC 1:1-7), such that an agent of this invention can be used in the treatment of cancer. In addition, mitochondrial defects, which accumulate naturally during the course of a lifetime, have been found in diseased human heart tissue

(Murray, et al . (2007) Curr. Opin. Clin. Nutr. Metab. Care 10:704-11) . Therefore, an agent of this invention can also be used in the treatment of cancer.

[00062] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1: Materials and Methods

[00063] Plasmids and siRNA Oligonucleotides . The human full length GFP- INF2 -CAAX construct has been described

(Ramabhadran, et al . (2011) Mol . Biol. Cell 22:4822) . All point mutations were made using the QUIKCHA GE mutagenesis kit (Stratagene, Santa Clara, CA) . The GFP-ER construct, containing the ER-targeting sequence (amino acids 233-250) of budding yeast UBC6 is described in the art (Wozniak, et al. (2009) J^". Cell Sci . 122:1979). CFP-Sec61p, Mito-dsRed and CFP-Drpl K38E are also known in the art (Stefanovic & Hedge (2007) Cell 128:1147-59; Rizzuto, et al . (1995) Curr. Biol. 5:635-642; Breckenridge , et al . (2003) J^". Cell Biol. 160 : 1115-1127) .

[ 00064 ] Oligonucleotides for human total INF2 siRNA were synthesized by IDT Oligo against target sequence 5'-GGA UCA ACC UGG AGA UCA UCC GC- 3 ' (siRNA#l; SEQ ID NO : 3 ) , and 5 ' - GCA GUA CCG CUU CAG CAU UGU CA- 3 ' (siRNA#2 ; SEQ ID NO: 4). Oligonucleotides for INF2-CAAX isoform were 5 ' -ACA AAG AAA CTG TGT GTG A-3' (siRNA#l; SEQ ID NO:5), and 5'-CCC TGA TTC TGA TGA TAA T- 3 ' (siRNA#2; SEQ ID NO:6). Oligonucleotides for human Drpl siRNA were synthesized by IDT Oligo against target sequence 5'-GCC AGC UAG AUA UUA ACA ACA AGA A-3' (siRNA#l; SEQ ID NO: 7) and 5'-GGA ACG CAG AGC AGC GGA AAG AGC T-3' (siRNA#2; SEQ ID NO:8). As a control, Silencer Negative Control #1 siRNA (Ambion) was used.

[ 00065 ] Cell Culture, Transfection and Drug Treatment. NIH 3T3 cells and U20S cell lines were grown in DMEM

(Invitrogen) supplemented with 10% calf serum (Atlanta Biologicals) . Cells were seeded at 2xl0⁵ cells per well of a 6-well dish -16 hours prior to transfection . Plasmid transfections were performed in OPTI-MEM media (Invitrogen) with 2 pL LIPOFECTAMINE 2000 (Invitrogen) per well. For all experiments, the following amounts of DNA were transfected per well: 30 ng mito-dsRed; 60 ng CFP-Sec61 ; 100 ng for all INF2 constructs; 100 ng Drpl K38E. In the case of simultaneous INF2 and Drpl transfection, 100 ng for each plasmid was used. For RNAi transfections , cells were plated on 6-well plates with 30-40% density, and 2 μΐ RNAmax

(Invitrogen) and 63 pg of siRNA were used per one well. Cells were analyzed 24 hours and 72-80 hours post- transfeetion for DNA and RNAi , respectively. When needed, cells were treated with MITOTRACKER Red CMXRos (Invitrogen) at 100 nM in DMEM for 20 minutes prior to fixation. Preparation of U20S cells stably expressing GFP-INF2 -CAAX was according to known methods (Ramabhadran, et al . (2011) Mol. Biol. Cell 22:4822) . For Latrunculin B (LatB) treatment, cells were plated on poly-L-lysine-coated coverslips (0.1 mg/ml of Sigma-Aldrich >300,000 MW) , and transfected with INF2-A149D or INF2-A149D/I643A plasmid the day before treatment. Cells were incubated with medium containing 0.5 μΜ LatB (from a 2 mM stock in DMSO, medium was pre-equilibrated for temperature and C0₂ content before use) for 30 or 60 minutes, with DMSO used as the negative control .

[ 00066 ] Antibodies . Polyclonal antibodies against human INF2 N-terminus (amino acids 1-424) or FH1-FH2-C (amino acids 469-1249, CAAX) were raised in rabbits (Covance; Denver, PA) , and affinity purified using DID construct (amino acids 1-269) or FH1-FH2 (amino acids 469-940) coupled to Sulfolink (Thermo/Pierce) . Production of splice variant- specific antibodies against human INF2-CAAX (acetyl - EEVPPDSDDNKTKKLC-amide; SEQ ID NO: 9) or non-CAAX (acetyl - CQEGLRPRPKAK; SEQ ID NO: 10) peptides was as described

(Ramabhadran, et al . (2011) supra). Anti-tubulin (DM1 -a, Sigma) was used at 1:10,000 dilution. Drpl was detected using a rabbit monoclonal antibody (Cell Signaling) at 1:50 dilution. GM130 (Abeam) was stained using a rabbit polyclonal antibody at 1:250 dilution. Secondary antibodies used were Cy5- or Fluorescein-conj ugated anti-rabbit IgG,

(Jackson Immunoresearch and Vector Laboratories, respectively) at 1:300 dilution. [00067] Immunofluorescence Microscopy. Cells were fixed with 4% formaldehyde (Electron Microscopy Sciences, PA) in phosphate-buffered saline (PBS) for 30 minutes at room temperature. After washing with PBS, cells were permeabilized with 0.1% .TRITON X-100 in PBS for 15 minutes. Cells were then washed with PBS, blocked with 0.5% bovine serum albumin (BSA) in PBS for 1 hour, and incubated with primary antibodies in PBS for 1 hour at room temperature. After washing with PBS, secondary antibodies were applied for 1 hour at room temperature. When needed, 500 nM ALEXAFLUOR660 -phalloidin (Invitrogen) or 100 nM TRITC- phalloidin (Sigma/Aldrich) , and 10 μΜ , 6-diamidino-2 - phenylindole (DAPI) were added to secondary antibody solution. Samples were mounted on polyvinyl alcohol -DABCO .

[00068] Live Imaging and Confocal Microscopy. Imaging of live and fixed cells was performed using spinning disk or laser scanning confocal systems. For live imaging, cells grown on 18 mm coverslips were mounted into Rose chambers, then onto a Wave FX spinning disk confocal microscope (Quorum Technologies, Inc., Guelph, Canada, on a NIKON ECLIPSE Ti microscope) with Bionomic Controller (20/20 Technology, Inc.) temperature-controlled stage set to 37°C. After equilibrating to temperature for 10 minutes, cells were imaged with the 6 Ox 1.4 NA Plan Apo objective (NIKON) using the 403 nm and 450/50 filter for DAPI, 491 nm laser and 525/20 filter for GFP, and the 561 nm laser and 593/40 filter for mRFP . To visualize dynamic structures in volume and time, 5-11 z-stacks of 0.2 μπι were collected for each color and each time point, at 5 sec intervals for 4-7 minutes. Maximum intensity projections from best focus Z slices were assembled using Metamorph software and processed using NIKON ELEMENTS and PHOTOSHOP CS (ADOBE, San Jose, CA) . [00069] For fixed samples, the laser scanning NIKON AlRSi Confocal Workstation with PMT DU4 and Galvano scanner, and lasers 405, 488, 561 and 639.5 nm was used, attached to NIKON ECLIPSE Ti inverted microscope. Images were taken as 1024x1024 pixels with PlanApoVC 60x oil objective (NA 1.4), and a pinhole of 1 airy unit. 3D reconstructions were generated in NIKON NIS-Elements AR (3.22.11) from 9 consecutive z-steps of 0.12 ym using the alpha blending option .

[00070] Immunoprecipitation and Western Blotting. For immunoprecipitations (IP), U20S cells (100 mm plate at 90% confluence) were lysed at +4°C in 2.5 mL of cold PBS containing Roche Complete Protease inhibitors, 10 mM DTT and 4% SDS . Lysates were immediately boiled 5 minutes, cooled for 1 minute in 23 °C water, then l/l0th volume of 300 mM N--ethylmaleimide (NEM, Thermo/Pierce. Freshly made in water) was added. "Thesit" (nonaethylene glycol monododecyl ether, Sigma) was added to 9% from a 20% stock in water, then the sample was centrifuged at 100,000 rpm for 20 minutes in a TLA120 rotor (Beckman) . The resulting homogenates were pre-cleared using Protein A SEPHAROSE beads (GE Biosciences) for 2 hours at 4°C. The IPs were carried out overnight at 4°C using 2 g of the appropriate antibody and 25 pL of Protein A SEPHAROSE beads per mL homogenate. The beads were washed multiple times with PBS (lacking the SDS and DTT but containing 1% thesit) before being processed for SDS-PAGE and western blot analysis.

[00071] To prepare samples for SDS-PAGE, cells were grown on 6-well plate, trypsinized, washed with PBS and resuspended 50 pL PBS. Fifty pL were mixed with 34 L of 10% SDS and 1 L of 1 M DTT, boiled 5 minutes, cooled to 23°C, then 17 μΐ of 300 mM of freshly made NEM in water was added. Just before SDS-PAGE, the protein sample was mixed 1:1 with 2xDB (250 mM Tris-HCl pH 6.8 , 2 mM EDTA, 20% glycerol, 0.8% SDS, 0.02% bromophenol blue, 1000 mM NaCl , 4 M urea) . Proteins were separated by 7.5% SDS-PAGE and transferred to a PVDF membrane (polyvinylidine difluoride membrane, Millipore) . The membrane was blocked with TBS-T (20 mM Tris-HCl, pH 7.6, 136 mM NaCl, and 0.1% TWEEN-20) containing 3% BSA

(Research Organics) for 1 hour, then incubated with the primary antibody solution at 4°C overnight. After washing with TBS-T, the membrane was incubated with horseradish peroxidase (HRP) -conjugated secondary antibody (Bio-Rad) for 1 hour at room temperature. Signals were detected by Chemiluminescence (Pierce) .

[ 00072 ] Mitochondria Length and Drpl Puncta Analysis . To measure mitochondrial length, maximum intensity projections of z-series with 0.2 μπι increments for red channel (MITOTRACKER or mito-dsRed) were created. The flat regions of cells with clearly resolved mitochondria were selected, and 25-30 mitochondria per cell were measured using the line tool in NIKON ELEMENTS software. Drpl puncta were counted on fixed cells labeled with anti-Drpl antibody samples. Only mitochondria-associated puncta were quantified, and their association with mitochondria was verified on consecutive z-planes. Statistic analysis was performed in EXCEL (MICROSOFT) , data presented as mean ± standard error or standard deviation from at least two experiments. Unpaired Student's t-test was used to compare values with p<0.01 considered significant.

[ 00073 ] Microtubule Pelleting Assays. Microtubule pelleting assays were conducted following methods known in the art

(Gaillard, et al . (2011) Mol . Biol. Cell 22:4575) . INF2- FH1FH2C constructs (amino acids 469-1249 of human CAAX variant) for WT and I643A mutant were expressed in bacteria and purified. Pelleting assays using taxol -stabilized porcine brain microtubules (0.5 μΜ) were conducted at 23 C in 10 mM imidazole pH 7 , 50 mM KC1 , 1 mM EGTA, 1 m MgCl₂, 1 mM DTT, 0.1 mM GTP, 20 μΜ paclitaxel (Calbiochem, La Jolla CA) and 0.5 mM thesit . Centrifugat ion at 23 °C and 60,000 rpm for 15 minutes in a TLA- 100 rotor (Beckman-Coulter , Brea CA) .

Example 2 : Role of INF2 in Mitochondrial Fission

[00074] It has been shown that mitochondrial fission takes place at ER contact sites and other proteins mutated in CMTD affect mitochondrial dynamics (Cartoni & Martinou (2009) Exp. Neurol. 218:268; Niemann, et al . (2005) J. Cell Biol. 170:1067) . Therefore, a possible role of INF2 in mitochondrial fission was analyzed. Using siRNA-mediated INF2 suppression in either U20S or NIH 3T3 cells, it was observed that INF2 suppression resulted in significant increases in mitochondrial length. It was therefore determined whether specific suppression of INF2-CAAX in U20S cells would result in similar mitochondrial elongation. When U20S cells were treated with two distinct siRNAs that specifically suppressed INF2-CAAX, mitochondrial length increased 2.5-fold. However, INF2-CAAX did not cause Golgi expansion, an effect attributable to INF2-nonCAAX (Ramabhadran, et al . (2011) supra) . U20S cells express considerably less INF2-CAAX than 3T3 cells (Ramabhadran, et al . (2011) supra) , but do express detectable INF2-CAAX protein levels. Thus, suppression of INF2-CAAX, which localizes to ER, caused an increase in mitochondrial length.

[00075] It was subsequently determined whether INF2-CAAX over- expression would cause the opposite mitochondrial effect to INF2-CAAX suppression. A GFP-fusion construct of INF2-CAAX (called INF2-WT) localized to ER in U20S cells (Ramabhadran, et al . (2011) supra) . However, this construct did not cause a significant change in mitochondrial length. It was reasoned that INF2-WT might be autoinhibited, since INF2 possesses autoinhibitory sequences similar to other formins (Chhabra, et al . (2009) J. Cell Sci . 122:1430) . To test this, A149 was mutated to aspartic acid, since a similar mutation in the formin mDial causes constitutive activation (Otomo, et al . (2005) Mol . Cell 18:273) . INF2- A149D caused a 2.2 -fold decrease in mitochondrial length. Also apparent was the extensive association between ER and mitochondria in INF2-A149D cells, with ER often circumscribing mitochondria. Thus, constitutively active INF2-CAAX caused a decrease in mitochondrial length and increased ER/mitochondrial association.

[ 00076 ] INF2-A149D effects were examined in more detail by live-cell microscopy using ER-green (Wozniak, et al . (2009) J. Cell Sci. 122:19799) as a negative control. In ER-green cells, infrequent fission events occurred. In contrast, fission events were 2 -fold more frequent in INF2-A149D cells, often occurring at multiple places on a single mitochondrion. These fission events invariably corresponded to regions where INF2 circumscribed the mitochondrion. In addition, mitochondria were significantly less mobile in INF2-A149D cells than in ER-green or INF2-WT cells. This feature was most clearly evident in temporal overlays of mitochondria. In control cells, mitochondria were highly dynamic, changing position on a timescale of seconds. However, mitochondrial motility dramatically decreased in INF2-A149D cells. A 5-fold decrease in mitochondrial fusion was also observed in INF2-A149D cells, possibly due to mitochondrial immobility. Thus, constitutively active INF2- CAAX decreased mitochondrial motility, resulting in an increase in fission and a decrease in fusion. [ 00077 ] Since Drpl acts in mitochondrial fission, a potential relationship between INF2 and Drpl was examined. Drpl localized to cytoplasm and to mitochondrially- associated puncta in U20S cells. Suppression of Drpl dramatically reduced mitochondrially-associated puncta, in addition to causing mitochondrial elongation. Drpl puncta also decreased upon INF2 suppression. One explanation is that INF2 suppression reduces overall Drpl protein levels. However, western blot analysis of Drpl in cell lysates indicated this was not the case. In contrast, INF2-A149D expression caused dense Drpl puncta associated with mitochondria. It was concluded that INF2 facilitates interaction of Drpl with mitochondria. If Drpl acts downstream of INF2 , Drpl inhibition should inhibit INF2- A149D-induced mitochondrial fission. Both Drpl suppression by siRNA and the dominant -negative (GTPase-deficient ) K38E Drpl construct reversed the effect of INF2-A149D on mitochondria size, with the K38E mutant producing a stronger effect. Thus, INF2 -mediated mitochondrial fission occurred through Drpl .

[ 00078 ] INF2 has biochemical effects on both actin and microtubules (Gaillard, et al . (2011) Mol . Biol. Cell 22:4575), and its effects on mitochondria could be mediated through either cytoskeletal element. The actin monomer sequestering drug latrunculin B (LatB) was used to test a role for actin in mitochondrial fission. LatB significantly increased mitochondrial length in U20S cells, similar to other cell types (De Vos, et al . (2005) Curr. Biol. 15:678) . Furthermore, LatB antagonized INF2 -A149D- induced mitochondrial shortening. To further test the relevance of actin filaments to INF2Ds mitochondrial effects, a key actin polymerization residue, 1643, was mutated. Interestingly, the I643A mutation antagonized the effect of INF2-A149D, as INF2 -A149D/I643A caused no significant change in mitochondrial length and did not decrease mitochondrial mobility. This mutation did not alter INF2- microtubule interactions, indicating that its effects are specific to actin regulation. These results showed that INF2 affected mitochondrial length and ER/mitochondrial interaction in an actin-dependent manner.

[00079] Direct evidence for actin filament accumulation around mitochondria during fission was sought by examining cells transfected by INF2-A149D using laser scanning confocal microscopy. Both polymerized actin and INF2 accumulated at mitochondrial constriction sites. This accumulation was particularly interesting when viewed in 3D reconstruction, as the peak of the polymerized actin staining occurred between the mitochondrial and INF2 staining. These results showed that actin polymerized at the ER/mitochondrial interface during constriction/fission.

[00080] These results provide a mechanism for the finding that ER association stimulates mitochondrial fission (Friedman, et al . (2011) Science 334:358) . Upon mitochondria-ER interaction, INF2 is activated to polymerize actin. Actin polymerization between ER and mitochondrion generates force to drive initial mitochondrial constriction, which enhances Drpl ring assembly at the constriction site. Drpl activity further constricts the mitochondrion, resulting in fission. INF2Ds severing/depolymerization activity might rapidly remove actin filaments after fission. Additional molecules might be required to convert actin polymerization force into mitochondrial membrane deformation, including: actin bundling molecules, actin filament pointed (minus) end binding molecules on the mitochondrion, and molecules mediating ER/mitochondria interaction. This model supports findings suggesting that Drpl oligomeric rings are narrower than unconstricted mitochondria (Mears, et al . (2011) Nature Struct. Mol. Biol. 18:20), and that mitochondria can constrict in a Drpl - independent manner (Friedman, et al . (2011) supra; Labrousse, et al . (1999) Mol. Cell 4:815; Legesse-Miller, et al . (2003) Mol. Biol. Cell 14:1953). The process superficially resembles yeast endocytosis, which requires both actin polymerization and dynamin (Liu, et al . (2010) Curr. Opin. Cell Biol. 22:36), but has two different features. First, filament pointed ends contact the deforming mitochondrial membrane, due to INF2Ds tight association with the ER membrane and its association with filament barbed ends. In contrast, filament barbed ends contact the deforming endocytic membrane. Second, actin polymerization occurs before Drpl activity, whereas polymerization occurs simultaneously with or after dynamin activity during endocytosis.

[ 00081 ] The findings herein tie mitochondrial dynamics to neurodegenerative disease and mutations of three mitochondrial dynamics proteins (INF2, Mfn2 , and GDAP1) , which lead to CMTD (Boyer, et al . (2011) New Engl. J. Med. 365:2377_/ Cartoni & Martinou (2009) Exp. Neurol. 218:268; Niemann, et al . (2005) J^". Cell. Biol. 170:1067) . More generally, mitochondrial dynamics defects are linked to Alzheimer's, Huntington's and Parkinson's (Chen & Chan

(2009) Hum. Mol. Genet. 18:R169; Correia, et al . (2012) Adv. Exp. Med. Biol. 724:205) .

Claims

What is claimed is:

1. A method for identifying an agent that modulates inverted formin-2, FH2 and WH2 domain containing protein, isoform 2 (INF2-CAAX) activity or INF2 -CAA -mediated mitochondrial function comprising

contacting a eukaryotic cell with a test agent, wherein the eukaryotic cell comprises an INF2-CAAX polypeptide; and

determining the effect of the test agent on INF2-CAAX activity or INF2 -CAAX-mediated mitochondrial function,

wherein a decrease or increase in INF2-CAAX activity or INF2 -CAAX-mediated mitochondrial function, compared to INF2-CAAX activity or INF2 -CAAX-mediated mitochondrial function in the absence of the test agent, indicates that the test agent modulates INF2-CAAX) activity or INF2 -CAAX- mediated mitochondrial function.

2. The method of claim 1, wherein said determining step comprises real-time imaging of the cell to detect a change in mitochondrial constriction, fission or motility.

3. The method of claim 1, wherein the eukaryotic cell has been genetically modified with an expression vector that comprises a nucleotide sequence encoding an INF2-CAAX polypeptide .

4. An agent identified by the method of claim 1.

5. A method for ameliorating a disease associated with mitochondrial dysfunction comprising administering to a subject in need of treatment and agent that modulates INF2- CAAX activity or INF2 -CAAX-mediated mitochondrial function so that the subject's disease is ameliorated.

6. The method of claim 5, wherein the disease comprises a neurodegenerative disease, cancer, diabetes or cardiac disease.