WO2004108079A2 - Parkin-interacting proteins - Google Patents

Abstract

The present invention relates to a novel interaction between FLRF/Nrdp1 and parkin and the implications of this interaction for diagnosis and treatment of Parkinson's Disease. Methods of screening for compounds and tools for use in the diagnosis and treatment of Parkinson's Disease are provided. Compounds and methods for use in the diagnosis and treatment of Parkinson's Disease are also provided.

Description

PARKIN-INTERACTING PROTEINS

CLAIM OF PRIORITY

This application claims priority under 35 USC § 119(e) to U.S. Patent Application Serial No. 60/474,242, filed on May 30, 2003, the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to research reagents, methods of screening drug candidates, and screening and development of therapies for Parkinson's Disease (PD).

BACKGROUND OF THE INVENTION

Parkinson's Disease (PD) is the second most common neurodegenerative disorder after Alzheimer's Disease (AD). More than 1% of the population over 50 years old suffer from PD, with approximately 500,000 PD patients in the U.S. alone. PD patients experience slowness of movement, rigidity, tremor, and difficulty with balance, with variable manifestations of dementia occurring in about 40% of PD patients, some of whom develop an AD-like dementia in the latter stages of the disease. At present, PD cannot be prevented; however, palliative care with dopamine replacement therapy is available. Such therapy is not without side effects and is not always effective in treating the symptoms of PD.

The main pathological features of PD are the loss of dopaminergic neurons in the substantia nigra and the presence of abnormal protein aggregates that form filamentous inclusions in the neuronal cytoplasm, termed Lewy Bodies (LBs) or Lewy neurites (nerve fibres) in PD brains. (Galvin et al., Arch. Neurol. 58(2): 186-90 (2001); Lang and Lozano, N Engl J. Med. 339(15):1044-53 (1998); Lang and Lozano, N Engl. J. Med. 339(16): 1130-43 (1998)). Degeneration of dopaminergic nigral neurons leads to the loss of dopaminergic projections to the striatum, which represents the primary neurochemical deficit in PD. Immunohistochemical examination shows that LBs contain many different proteins. For example, neurofilament and ubiquitin have consistently been described as present in the LBs (Gai et al., Brain 118(6): 1447-59 (1995); Takeda, Am. J Pathol. 152(2):367-72 (1998)). More recently, α-synuclein and parkin were identified as predominant proteins in LBs (Baba et ύ., Am. J Pathol. 152(4):879-84 (1998)). The majority of PD is sporadic. Rare familial forms of PD are either autosomal recessive or autosomal dominant, suggesting that the etiology of the disorder could be complicated (Baba et al., Am. J. Pathol. 152(4):879-84 (1998); Lang and Lozano, N Engl. J. Med. 339(15):1044-53 (1998)). The discovery of genetic linkages for PD to several loci has enabled the identification of genetic factors and mutations in several genes including ubiquitin carboxy-terminal hydrolase (UCH)-Ll (Leroy, et al., Nature 395(6701):451-2 (1998)) and α-synuclein were identified to cause autosomal dominant PD. Autosomal recessive juvenile Parkinsonism (AR-JP) was mapped to the long arm of chromosome 6 (6q25.2-q27) and is linked strongly to the markers D6S305 and D6S253 (Matsumine et al, Am. J. Hum. Genet. 60(3):588-96 (1997)). D6S305 is deleted in one Japanese patient (Matsumine et al., Genomics 49(l):143-6 (1998)). Using the positional cloning strategy combined with the exon-trapping technology and cDΝA library screening, Kitada et al. identified a gene named parkin, in which exons 3-7 were deleted in the same Japanese patient (Kitada et al., Nature 392(6676):605-8 (1998)). Kitada et al. also described four other AR-JP patients from three unrelated families with a deletion of exon 4 in the parkin gene, confirming that mutations in the parkin gene appear to be responsible for the pathogenesis of AR-JP. Although most mutations in the parkin gene are thought to inactivate the gene via exon deletions (Matsumine et al., Genomics 49(l):143-6 (1998); Kitada et al., Nature 392(6676):605-8 (1998)); Leroy et al., Hum. Genet. 103(4):424-27 (1998); Lucking et al., Lancet 352(9137):1355-6 (1998)), missense point mutations were also identified in some of the AR-JP patients (Abbas, et al., Hum. Mol. Genet. 8(4):567-74 (1999); Hattori et al., Biochem. Biophys. Res. Comm., 249(3):754-8 (1998)), further confirming the involvement of parkin in the pathogenesis of at least some occurrences of PD.

The parkin gene is one of the largest genes in the human genome. It spans 1.5 megabases and has 12 exons and unusually large introns. A 4.5 kilobase transcript is expressed in many human tissues, but is abundant in the brain. The parkin gene encodes a protein of 465 amino acids with a molecular weight of about 52 kD (Kitada et al. (1998) supra). The functions of parkin are not completely understood. The deduced amino acid sequence of parkin shows similarity to ubiquitin at the Ν-terminus. Further studies have shown that parkin is a ubiquitin ligase (i.e., E3 ubiquitin ligase) (hnai et al., J. Biol. Chem. 275(46):35661-4 (2000); Shimura et al., Nat. Genet. 25(3):302-5 (2000); and Zhang et al, Proc. Natl. Acad. Set USA 97(24): 13354-9 (2000)) and that α-synuclein, the Pael receptor and CRCel interact with parkin and are substrates for ubiquitination by parkin (rmai et al., Cell 105(7):891-902 (2001); Shimura et al., Science 293(5528):263-269 (2001)). The results are consistent with the findings that mutations in (UCH)-L1 contribute to a dominant form of PD and that ubiquitin is present in Lewy bodies, suggesting that the ubiquitin pathway is indeed involved in the pathogenesis of PD. In fact, in PC12 neuronally differentiated by nerve growth factor, Parkin overproduction protected against cell death mediated by ceramide (Darios et al., Hum Mol Genet, 2003, 12(5):517-26).

Ubiquitination of proteins is a regulated process that is responsible for the selective degradation of specific proteins. Subtle perturbations in the ubiquitination pathway can lead to inappropriate stabilization or degradation of important signaling molecules, which in turn can result in pathology. Ubiquitin protein conjugation is an ATP-dependent process that is mediated by three distinct classes of enzymes, ubiquitin-activating enzyme (El), ubiquitin- conjugating enzymes (E2s), and ubiquitin protein ligases (E3s). El activates ubiquitin and forms a thioester bond with ubiquitin. Ubiquitin is then transferred to E2. Finally E3 ligases recognize specific target proteins and facilitate the transfer of ubiquitin from E2 to the substrate. Poly-ubiquitinated substrates are targeted for degradation in the proteasome. h PD patients, parkin's ability to ubiquitinate its substrate proteins, including α-synuclein, is diminished, typically by mutations or deletions in the gene (loss of function mutations). This leads to accumulation and aggregation of these proteins, resulting in the death of nigral neurons.

Like ubiquitin, parkin immunoreactivity is also present in Lewy bodies of sporadic PD. AR-JP patients with mutations in parkin show a severe loss of DA neurons and the absence of Lewy bodies, possibly due to the loss of parkin function. The C-terminus of parkin contains two RING (Really Interesting New Gene) finger motifs and an IBR (in between ring finger) domain. The RING finger and IBR domains could function as protein interaction and/or transcriptional activator domains. The Pael receptor and α-synuclein were recently described as parkin interactors as well as substrates (hnai et al. (2001), supra; Shimura et al. (2001), supra) for ubiquitination. However, the -synuclein snάparkin genes are responsible for only a small percentage of PD patients.

Another protein, FLRF/Nrdpl (Fetal Liver Ring Finger/Neuregulin receptor degradation protein-1) has been implicated in blood cell development and differentiation (Abdullah et al, Blood Cells Mol. Dis. 27(l):320-33 (2001)). FLRF/Nrdpl binds to Erb3 (neuregulin receptor) and causes its degradation (Qiu and Goldberg, Proc. Natl. Acad. Sci. USA, 99(23): 14843-14848 (2002); Diamonti et al., Proc. Natl. Acad. Sci. USA 99(5):2866- 2871, (2002)). FLRF/Nrdpl contains a RING finger domain motif typically found in E3 ligases (Figure 1); FLRF/Nrdpl possesses E3 ligase activity (Qiu and Goldberg (2002), supra). E3 ligases participate in the ubiquitination process by binding to E2s (ubiquitin- conjugating enzymes) via this RING finger domain prior to ubiquitin transfer. FLRF/Nrdpl is predominantly expressed in heart, skeletal muscle and certain regions of the brain, including those that are most affected in Parkinson's disease.

SUMMARY

The present invention is based, at least in part, on the discovery that FLRF/Nrdpl (fetal liver RING fmger/neuregulin receptor degradation protein-1) interacts with parkin, and this interaction results in the ubiquitination and subsequent degradation of parkin. Reduced levels of parkin have been associated with Parkinson's Disease. Thus, the present invention provides for reagents, compounds, compositions, animals, and methods relating to the newly discovered interactions of FLRF/Nrdpl and parkin.

One aspect of the invention provides methods to identify potential parkin-interacting proteins, hi some embodiments, the parkin-interacting protein is FLRF/Nrdpl or a homolog or variant thereof.

The invention provides methods for identifying a compound that modulates an interaction of FLRF/Nrdpl with parkin. The methods include providing a sample comprising FLRF/Nrdpl or a parkin binding fragment of FLRF/Nrdpl, and parkin or a FLRF/Nrdpl binding fragment of parkin; contacting the sample with one or more test compounds; and evaluating one or more of (i) binding of FLRF/Nrdpl to parkin or (ii) FLRF/Nrdpl - dependent degradation of parkin, in the presence and the absence of the test compound. A test compound that inhibits or enhances one or more of (i) binding of FLRF/Nrdpl to parkin or (ii) FLRF/Nrdpl -dependent degradation of parkin is a compound that modulates an interaction of FLRF/Nrdpl with parkin. hi another aspect, the invention relates to methods for identifying candidate compounds for the treatment of Parkinson's Disease. The methods include providing a sample including FLRF/Nrdpl or a parkin-binding fragment thereof, and parkin or a FLRF/Nrdpl -binding fragment thereof; contacting the sample with one or more test compounds; and evaluating an interaction of FLRF/Nrdpl with parkin in the presence and the absence of the test compound. A test compound that inhibits an interaction of FLRF/Nrdpl with parkin is a candidate compound for the treatment of Parkinson's Disease.

In some embodiments, the interaction of FLRF/Nrdpl with parkin is (i) binding of FLRF/Nrdpl to parkin or (ii) FLRF/Nrdpl -dependent degradation of parkin, hi some embodiments, the interaction is inhibited, e.g., statistically significantly inhibited, or enhanced, e.g., statistically significantly enhanced, i some embodiments, the parkin and FLRF/Nrdpl (or fragments thereof) are Drosophila proteins or polypeptides. In an alternative embodiment, the parkin and FLRF/Nrdpl (or fragments thereof) are human proteins or polypeptides.

In some embodiments, the methods further include providing a non-human animal model exhibiting a symptom associated with Parkinson's Disease; administering to the animal model a candidate compound that modulates an interaction of FLRF/Nrdpl with parkin; and monitoring the animal for an improvement in a symptom associated with Parkinson's Disease, e.g., an improvement in motor function, or a reduction in loss of dopaminergic neurons (e.g., reduction in the rate of loss). A desirable improvement in a symptom indicates that the candidate compound is a candidate therapeutic agent for the treatment of Parkinson's Disease, h some embodiments, the non-human animal model is Drosophila melanogaster. In other embodiments, the non-human animal model is a rodent, e.g., a rat or mouse. In some embodiments, the non-human animal model is a transgenic animal whose somatic and/or germ cells comprise a FLRF/Nrdpl inhibitory transgene, e.g., a non-functional allele of FLRF/Nrdpl.

The invention also provides methods of preparing pharmaceutical compositions to treat Parkinson's Disease. The methods include obtaining a candidate compound that inhibits an interaction of FLRF/Nrdpl with parkin; determining whether the candidate compound improves a symptom of PD in an animal model, wherein an improvement indicates that candidate compound is a candidate therapeutic agent; and formulating the candidate therapeutic agent with a pharmaceutically acceptable carrier to prepare the pharmaceutical composition. In some embodiments, the methods include optimizing the candidate compound, hi some embodiments, the methods include testing the candidate compound in a clinical trial. The test compounds utilized in the assays and methods described herein can be, ter alia, nucleic acids, small molecules, organic or inorganic compounds, antibodies or antigen- binding fragments thereof, proteins, or polypeptides. For example, FLRF/Nrdpl polypeptides or polynucleotides (e.g., FLRF/Nrdpl variants including truncation mutants, deletion mutants, and point mutants as described herein; sense, antisense, and small inhibitory RNAs (siRNAs) including short hairpin RNAs (shRNAs); and ribozymes) can be used as test compounds in the methods described herein. Alternatively, compounds or compositions that mimic the parkin-binding portion of FLRF/Nrdpl can be used. FLRF/Nrdpl proteins or fragments thereof with mutations, e.g., in the RING finger domain, coiled coil, and/or B Box (see Fig. 1), are also included in the present invention. A test compound that has been screened by an in vitro method described herein and determined to have a desired activity, e.g., inhibition of FLRF/Nrdpl activity or expression, can be considered a candidate compound. A candidate compound that has been screened, e.g., in an in vivo model, and determined to have a desirable effect on one or more symptoms of a disorder, can be considered a candidate therapeutic agent. Candidate therapeutic agents, once screened in a clinical setting, are therapeutic agents, which can then be optionally optimized ( e.g., by derivatization), and formulated with physiologically acceptable excipients to form pharmaceutical compositions.

In some embodiments, the compounds are optimized to improve their therapeutic index, i.e., increase therapeutic efficacy and/or decrease unwanted side effects. Thus, in some embodiments, the methods described herein include optimizing the test or candidate compound, hi some embodiments, the methods include formulating a therapeutic composition including a test or candidate compound (e.g., an optimized compound) and a pharmaceutically acceptable carrier therefor, hi some embodiments, the compounds are optimized by derivatization.

The invention also provides non-human transgenic animals, whose somatic and/or germ cells comprise a FLRF/Nrdpl inhibitory transgene, wherein the animals exhibit one or more symptoms associated with Parkinson's Disease (PD), e.g., loss of motor function and loss of dopaminergic neurons. An "inhibitory transgene" is a gene whose expression causes a reduction in levels of expression or activity of FLRF/Nrdpl. Thus, the invention provides for transgenic animals for the study of PD, e.g., transgenic non-human mammals such as rodents, e.g., mice or rats, or transgenic flies, e.g., Drosophila melanogaster. hi one embodiment, the FLRF/Nrdpl gene is knocked out in the animal. In some embodiments, the inhibitory transgene comprises a non-functional allele of FLRF/Nrdpl, a conditional allele of FLRF/Nrdpl, a spatially- and/or temporally-limited FLRF/Nrdpl transgene (e.g., a transgene whose effect is limited to neuronal tissues in the adult animal), or a systemic, constitutively expressed FLRF/Nrdpl transgene. In some embodiments, the FLRF/Nrdpl gene is knocked out in a tissue-specific fashion, h some embodiments, the specific tissue is neural tissue. In some embodiments, the neural tissue comprises substantia nigra neurons. In another embodiment, the FLRF/Nrdpl gene is overexpressed, e.g., in a tissue-specific fashion. Such animals are useful for elucidating the role of the FLRF/parkin interaction in PD and for identifying compounds or compositions that affect the FLRF/parkin interaction. hi some embodiments, the animal also includes one or more additional transgenes, e.g., non-FLRF/Nrdpl transgenes, e.g., a parkin transgene.

In some embodiments of the above methods, the FLRF/Nrdpl or a parkin-binding fragment thereof, is attached to a solid substrate. The methods described herein may be practiced using standard binding assay techniques. These include, z^'nter alia, yeast two- hybrid, GST-fusion protein interaction assays, co-immunoprecipitation, and radiolabel binding assays, such as those demonstrated in the Examples.

The invention also provides methods of modulating a FLRF/Nrdpl -parkin interaction, e.g., in vitro or in vivo, e.g., in a subject, by administering to the subject a composition that modulates a FLRF/Nrdpl -parkin interaction. In one embodiment, the composition includes a nucleic acid molecule that recognizes and binds to a FLRF/Nrdpl polynucleotide. In another embodiment, the composition is a FLRF/Nrdpl -binding fragment of parkin, or a parkin- binding fragment of FLRF/Nrdpl. In another embodiment, the composition inhibits FLRF/Nrdpl binding to parkin, hi an alternative embodiment, the composition enhances binding of FLRF/Nrdpl to parkin.

Also within the invention is the use of an inhibitor of FLRF/Ndrpl expression or activity in the treatment of Parkinson's disease, and the use of an inhibitor of FLRF/Nrdpl in the manufacture of a medicament for the treatment of Parkinson's disease.

In a further aspect, the invention provides methods of treating a subject having PD by administering to the subject a therapeutically effective amount of a compound that modulates an interaction of FLRF/Nrdpl with parkin, e.g., a compound identified by a method described herein, hi some embodiments, the compositions include one or more compounds selected from the group consisting of FLRF/Nrdpl interacting proteins, a parkin-binding fragment of FLRF/Nrdpl, or a FLRF/Nrdpl -binding fragment of parkin, and polynucleotide molecules that recognize and bind to a nucleic acid encoding FLRF/Nrdpl. In one embodiment, the composition inhibits FLRF/Nrdpl binding to parkin, hi some embodiments, the composition includes one or more small molecules, peptides, nucleic acids, or polypeptides that modulate an interaction of FLRF/Nrdpl with parkin. Thus in one aspect, the invention relates to methods for treating subjects having PD by administering to the subject a therapeutically effective amount of a compound that inhibits FLRF/Nrdpl expression or activity. In some embodiments, the FLRF/Nrdpl inhibitor is selected from the group consisting of an antisense nucleic acid, siRNA, or ribozyme that selectively binds to a nucleic acid encoding FLRF/Nrdpl.

As used here, the term "therapeutically effective amount" or "amount effective to treat PD" refers to an amount of a compound or composition effective to reduce one or more pathophysiological and/or physical manifestations of PD, such as preventing or slowing the loss of dopaminergic neurons in the substantia nigra, preventing or slowing the formation of filamentous intraneuronal inclusions (Lewy bodies), and reducing or reversing extrapyramidal movement disorders including tremor, rigidity, etc.

The present invention also provides methods for diagnosing a subject with PD. The methods include measuring activity or expression levels of FLRF/Nrdpl in the subject and comparing the measured levels to a reference, e.g., a control subject or control population without PD. If levels of FLRF/Nrdpl activity or expression are increased as compared to the control, the subject can be diagnosed with PD or a likelihood of developing PD. Levels of FLRF/Nrdpl expression or activity can be assessed using methods known in the art, including, but not limited to, protein or mRNA measurements. In some embodiments, the methods include isolating a gene encoding FLRF/Nrdpl from a subject with increased or decreased levels of FLRF/Nrdpl expression or activity and determining the presence of any mutations in the sequence of FLRF/Nrdpl in the subject. The presence of such a mutation can be correlated with a disease state, e.g., the presence of PD, in the patient.

In some embodiments, the subject is a vertebrate, but can be an invertebrate organism such as an arthropod (e.g., a fly). In some embodiments, the subjects are mammals. Mammalian subjects include canine, feline, ovine, primate, equine, porcine, caprine, camelid, avian, bovine, and murine organisms. In some embodiments, the subjects are humans, h some embodiments, the subjects are non-human mammals.

The present invention also provides antibodies and antigen-binding fragments thereof that bind to FLRF/Nrdpl, e.g., that bind to a parkin-interacting fragment or the RING Finger domain, coiled coil, and/or B Box of FLRF/Nrdpl. hi one embodiment, the antibody or antigen-binding fragment thereof binds to human FLRF/Nrd l. In another embodiment, the immuno globulin or immunoglobulin fragment binds to Drosophila FLRF/Nrdpl. hi some embodiments, the antibody or antigen-binding fragment thereof prevents binding of FLRF/Nrdpl to parkin.

The invention also provides test compounds, candidate compounds, candidate therapeutic compounds and therapeutic agents identified by the disclosed methods, h some embodiments, the invention provides therapeutic compositions including one or more therapeutic agents, and a pharmaceutically acceptable excipient and/or carrier therefor. Compositions described herein can include one or more active agents, e.g., polypeptide sequences of FLRF/Nrdpl, including sequences of FLRF/Nrdpl including the parkin-binding fragment of FLRF/Nrdpl, and a carrier therefor. The compounds and compositions described herein that decrease levels of FLRF/Nrdpl expression or activity are useful as therapeutics or diagnostics for PD and/or as important research tools for elucidating the mechanisms underlying PD, e.g., as targets for the development of therapeutic interventions. Additionally, FLRF/Nrdpl polypeptides/protein or polynucleotides (e.g., sense, antisense, small inhibitory RNAs (siRNAs) including short hairpin RNAs (shRNAs), and ribozymes) are useful therapeutic agents for PD. For example, mutations in the FLRF/Nrdpl protein can be corrected via gene therapy. Alternatively, compounds or compositions that mimic the parkin-binding portion of FLRF/Nrdpl can be used to overcome a loss-of function mutation or block the interaction between FLRF/Nrdpl and parkin. FLRF/Nrdpl proteins with mutations, e.g., in the RING finger domain, coiled coil, or B Box,, are also included in the present invention, hi some embodiments, the compounds are derivatized to optimize their therapeutic index, i.e., increase therapeutic efficacy and/or decrease unwanted side effects.

In addition, the invention provides compositions for use in treating PD. The compositions include an active compound that modulates (e.g., inhibits) an interaction between FLRF/Nrdpl and a pharmaceutically acceptable excipient and or carrier therefor, hi one embodiment, the compound is FLRF/Nrdpl or a parkin-binding fragment or variant thereof. In another embodiment, the compound is an antibody or antigen-binding fragment thereof that binds to FLRF/Nrdpl, or binds to a parkin-binding fragment of FLRF/Nrdpl. Other compounds include nucleic acid molecules that encode FLRF/Nrdpl or a parkin- binding fragment thereof, or nucleic acid molecules that recognize and bind to endogenous polynucleotides encoding FLRF/Nrdpl, including, but not limited to, antisense, siRNA, and ribozymes.

A further object of the invention is to provide methods of modulating the expression of a nucleic acid encoding a FLRF/Nrdpl protein in an organism, e.g., to reduce levels of expression or activity of FLRF/Nrdpl, to treat PD. The methods include administering to the organism an effective amount of a composition that modulates the expression of a nucleic acid encoding FLRF/Nrdpl. In one embodiment, the composition increases expression of FLRF/Nrdpl. In another embodiment, the composition decreases expression of FLRF/Nrdpl. Such compositions can comprise antisense, siRNA, or ribozyme molecules that recognize and bind to a nucleic acid encoding the FLRF/Nrdpl protein, hi some embodiments, the expression of FLRF/Nrdpl is modulated in a neural cell, hi some embodiments, the neural cell is a substantia nigra neuron.

The identification of FLRF/Nrdpl as a binding partner for parkin and as possessing E3 ligase activity (protein degradation problems are associated with PD) make it a potential locus of PD-causing mutations. Thus, provided herein are methods of screening for genetic mutations for diagnosis or to identify at-risk factors associated with PD by analyzing mutations in the FLRF/Nrdpl gene in PD patients. Also provided herein are methods for diagnosing PD and/or determining the cause of PD. The methods include assessing whether mutations or deletions are present in the FLRF/Nrdpl gene of the subject; and if mutations or deletions are present, diagnosing the subject with PD and/or determining the cause of PD in the subject as due to mutations or deletions in the FLRF/Nrdpl gene of the subject.

In one embodiment, the RING Finger domain, coiled coil, and/or B Box of FLRF/Nrdpl contains mutations, e.g., deletions, h another embodiment, the parkin-binding fragment of FLRF/Nrdpl contains mutations, e.g., deletions, h another embodiment, the mutation or deletion affects the ubiquitin E3 ligase activity of FLRF/Nrdpl. In one embodiment, the E3 ligase activity is increased.

Also described herein are methods for screening for neural-specific substrates of FLRF/Nrdpl. The methods include identifying a neural-specific binding partner of FLRF/Nrdpl from a neural cell; and assessing the ability of FLRF/Nrdpl to ubiquitinate the neural-specific binding partner via E3 ligase activity, h some embodiments, the neural- specific binding partner is identified from a substantia nigra neuron. The identification of substrates of FLRF/Nrdpl in neural cells will provide useful information related to the etiology of PD.

Also described herein are inhibitors and enhancers of the E3 ligase activity of FLRF/Nrdpl, which also provide effective research tools and therapeutic agents for PD.

In general, terms in the present application are used consistent with the manner in which those terms are understood in the art. To aid in the understanding of the specification and claims, the following definitions are provided.

"Gene" refers to a DNA sequence that encodes through its template or messenger RNA a sequence of amino acids characteristic of a specific polypeptide. The term "gene" includes intervening, non-coding regions, as well as regulatory regions, and can include 5' and 3' ends.

As used herein, the term "nucleic acid" includes single-stranded and double-stranded nucleic acids including, but not limited to, DNAs, RNAs (e.g., mRNA, tRNAs, rRNAs, siRNAs, and shRNAs), cDNAs, recombinant DNA (rDNA), antisense nucleic acids, oligonucleotides, oligomers, and polynucleotides. The term encompasses both sense and antisense strands. The term may also include hybrids such as triple-stranded regions of RNA and/or DNA or double-strand RNA:DNA hybrids. The term is also contemplated to include modified nucleic acids, e.g., biotinylated nucleic acids, tritylated nucleic acids, fluorophore labeled nucleic acids, inosine, and the like.

The nucleic acids described herein can be derived from a variety of sources, including DNA, cDNA, synthetic DNA, synthetic RNA, or combinations thereof. Such sequences can comprise genomic DNA that may or may not include naturally occurring introns. Moreover, such genomic DNA may be obtained associated with promoter regions and/or poly(A) sequences. The sequences, genomic DNA or cDNA may be obtained in any of several ways. Genomic DNA can be extracted and purified from suitable cells by means well-known in the art. Alternatively, mRNA can be isolated from a cell and used to produce cDNA by reverse transcription or other means. In some embodiments, an "isolated gene" or "isolated nucleic acid" excludes genomic sequences found upstream or downstream of a gene.

"Expression" refers to the transcription of a gene sequence and subsequent processing steps, such as translation of a resultant mRNA to produce the final end product of a gene. The end product may be a protein (such as an enzyme or receptor) or a nucleic acid (such as tRNA, antisense RNA, or other regulatory factor).

A "promoter region" includes a promoter as well as other sequences necessary for the initiation of transcription of a gene. The presence of a promoter region is sufficient to cause the expression of a gene sequence operably linked to the promoter region. A "promoter" is a DNA sequence located 5' to a gene that can be recognized by an RNA polymerase and indicates the site for transcription initiation. The presence of such a sequence permits the RNA polymerase to bind and initiate transcription of operably linked gene sequences. Many different promoters are known in the art that direct expression of a gene in certain cell types. Tissue-specific promoters can comprise nucleic acid sequences that cause a greater (or decreased) level of expression in cells of a certain tissue type. Tissue-specific promoters also encompass "leaky" promoters, which regulate expression of a selected nucleic acid primarily in one tissue, but can cause at least low level expression in other tissues as well. A promoter is "operably linked" to a sequence of DNA if upon introduction into a host cell the promoter controls the transcription of the DNA sequence(s) into one or more species of RNA. .

A "vector" is a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors capable of directing the expression of nucleic acids to which they are linked are referred to herein as "expression vectors." In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids, which are generally circular double-stranded DNA not bound to a chromosome. The present methods and compositions encompass other forms of expression vectors that serve equivalent functions. Numerous expression vectors are known in the art and can be used to express the polynucleotides and/or polypeptides described herein.

The term "animal" is used herein to include all vertebrate and invertebrate animals, particularly those with a central nervous system. It includes an individual animal in all stages of development, including embryonic and fetal stages. Animals include higher eukaryotes such as avians; mammals including primates (e.g., humans), lagomorphs (e.g., rabbits and hares), and rodents (e.g., mice, rats, chinchillas, guinea pigs, hamsters, and the like). "Animal" also includes invertebrates, e.g., insects such as Drosophila.

A "transgenic animal" is an animal containing one or more somatic and/or germ cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation or by inheritance from a manipulated progenitor at a subcellular levels, such as by microinjection or infection with a recombinant viral vector (e.g., adenovirus, retro virus, herpes virus, adeno-associated virus, lentivirus). This introduced DNA molecule can be integrated within a chromosome, or it can be extra-chromosomally replicating DNA. In one embodiment, the cells of the transgenic animal express a recombinant form of FLRF/Nrd l protein, e.g., either agonistic or antagonistic form, h some embodiments, the cells of the transgenic animal do not express a functional FLRF/Nrdpl protein and/or a function parkin protein (e.g., the cells express a FLRF/Nrdpl inhibitory transgene). Transgenic animals in which the FLRF/Nrdpl gene is conditionally silenced, e.g., by FLP or CRE recombinase dependent constructs, are also encompassed by the present invention.

"Embryonic stem cells" or "ES cells" are cells or cell lines usually derived from embryos that are pluripotent, meaning that they are undifferentiated cells. These cells are also capable of incorporating exogenous DNA by homologous recombination and subsequently developing into any tissue in the body when incorporated into a host embryo. It is possible to isolate pluripotent cells from sources other than embryonic tissue by methods that are well understood in the art.

The term "somatic cell" indicates any animal or human cell that is not a sperm or egg cell or is capable of becoming a sperm or egg cell. The term "germ cell" or "germ-line cell" refers to any cell that is either a sperm or an egg cell or is capable of developing into a sperm or an egg cell and can therefore pass its genetic information to offspring. The term "germ cell-line transgenic animal" refers to a transgenic animal in which the genetic information was incorporated in a germ line cell, thereby conferring the ability to transfer the information to the offspring. If such offspring in fact possess some or all of that information, then they, too, are transgenic animals.

The genetic alteration of genetic information may be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient. In the last case, the altered or introduced gene may be expressed differently than the native gene.

"Antibody" refers to polyclonal, monoclonal and/or monospecific antibodies and fragments thereof, and antigen-binding fragments thereof, that can bind to the FLRF protein and fragments thereof. The term antibody is used to refer both to a homogeneous molecular entity, or a mixture such as a serum product made up of a plurality of different molecular entities. Methods for making antibodies are known in the art. The term "antibody" is meant to include, but is not limited to, polyclonal, monoclonal, chimeric, human, humanized, bispecific, multispecific, and Primatized® antibodies, includes synthetically or recombinantly produced molecules that bind to a target protein. Antigen-binding fragments include single chain antibodies (ScFv), Fab and F(ab)₂.

By "modulate" is meant to increase or decrease the wild-type activity or binding of an enzyme or other protein. Modulation can be effected by affecting the concentration or subcellular localization of a biologically active protein, i.e., by regulating expression or degradation, or by direct agonistic or antagonistic effect, e.g., through inhibition, activation, binding, or release of binding partners, modification (either chemically or structurally) or by direct or indirect interaction that can involve additional factors. Modulated activities of FLRF/Nrdpl can include binding to other proteins, especially parkin; E3 ligase activity; and binding to antibodies.

Substantially complementary oligonucleotide sequences are greater than about 80 percent complementary to the full length of the corresponding target sequence to which the oligonucleotide binds, hi some embodiments, the substantially complementary oligonucleotide sequences will be greater than about 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to the corresponding target sequence, or will be 100% complementary to the corresponding target sequence.

Substantially identical oligonucleotide or amino acid sequences are greater than about 90 percent identical to the reference sequence, hi some embodiments, the identical oligonucleotide sequences will be greater than about 95%, 96%, 97%, 98%, 99% identical, or will be 100% identical, to the reference sequence.

As used herein, "parkin" refers to the human and Drosophila parkin proteins, as well as any parkin homologs existing in these and other species. As the parkin gene has been found in such genetically diverse organisms as humans and Drosophila, one skilled in the art will appreciate that many species of animals are expected to have a homologous gene. Parkin homologs have been identified in human, mouse, rat, and Drosophila. The expression of parkin protein in other species such as bird and Xenopus has been investigated, however a cDNA for a parkin homolog has not yet been identified in these species. As used herein, "dparkin" refers to Drosophila parkin and "hparkin" refers to human parkin. The sequence of the parkin gene and parkin protein from various species can be found by searching GenBank or other comparable database.

As used herein, a "FLRF/Nrdpl -binding fragment" of parkin means a fragment of the parkin protein that retains the ability to bind to FLRF/Nrdpl. hi some embodiments, the FLRF/Nrdpl -binding fragment of parkin includes the N terminus of parkin, e.g., a fragment corresponding to about the N-terminal 50, 75, 80, 85, 90, 98, or 100 amino acids of hparkin. In some embodiments, the FLRF/Nrdpl -binding fragment of parkin includes the U-Like Domain (see Fig. 2C).

By "parkin-interacting protein" is meant a protein that physically binds to the parkin protein, hi some embodiments, the parkin interacting protein is FLRF/Nrdpl, also referred to herein as FLRF or Nrdpl .

As used herein, a "parkin binding fragment" of FLRF/Nrdpl means a fragment of the FLRF/Nrdpl protein that retains the ability to bind parkin, hi some embodiments, a parkin binding fragment of FLRF/Ndrpl includes the N-terminus of FLRF/Nrdpl, e.g., a fragment corresponding to about the N-terminal 50, 75, 80, 85, 90, 100, 125,130, 133, or 150 amino acids of FLRF/Ndrpl, e.g., including one or more of the RING finger and/or two zinc finger domains, hi some embodiments, the parkin-binding fragment of FLRF/Nrdpl includes one or more of the E3 ligase domain, the RING domain, the B box, and/or the coiled-coil domain (see Fig. 1).

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

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

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a representation of the amino acid sequence and molecular characterization of the structure of human FLRF/Nrdpl (SEQ ID NO:l) and the Drosophila homologue (SEQ ID NO:2), including the location of a RING finger domain (SEQ ID NOs:3 and 4, for the human and drosophila sequences, respectively), a B-box domain (a zinc finger motif of around 40 amino acids; SEQ ID NOs:5 and 6, human and drosophila, respectively), and a coiled coil domain (SEQ ID NOs:7 and 8, human and drosophila, respectively).

FIG. 2 A is a reproduction of a gel showing the results of HA-Nrdpl or Myc-hparkin immunoprecipitated using anti-HA antibody 12CA5 or anti-Myc antibody 9E10. ³⁵S- methionine labeled HA-Nrdpl, Myc-hparkin and luciferase proteins obtained by in vitro TNT translation, and incubated in binding buffer. Precipitates were analyzed on SDS-PAGE and then on a phosphorimager.

FIG. 2B is a reproduction of a gel showing the results of in vitro binding assays of GST or GST-Nrdpl fusion proteins with full length hparkin, hparkin N-terminus (hp-NT), and hparkin C-terminus (hp-CT) (leftmost six lanes), and hparkin and Nrdpl to hparkin N- terminus (hp-NT). Bound proteins were analyzed on a phosphorimager. Ten percent of input is shown.

FIG. 2C is a schematic representation of the structure of the full length hparkin protein (top), N-terminal fragment of hparkin (hp-NT, middle), and C-terminal fragment of hparkin (hp-CT, bottom) that were used in the experiments whose results are shown in Figs 2A and 2B. Numbers represent amino acids. ULD: Ubiquitin like domain; IBR: In-between RING sequence.

FIGs. 3A-3B are reproductions of gels showing the results of in vivo co- immunoprecipitation of parkin and FLRF/Nrdpl from C33A cells co-transfected with plasmids of HA-Nrdpl and Myc-hparkin. FIG. 3A, Anti-Myc antibody 9E10 was used to pull down Myc-hparkin. HA-Nrdpl was detected using anti-HA antibody 12CA5. Two percent of the input is shown. FIG. 3B, Anti-HA antibody 12AC5 was used to pull down HA-Nrdpl. Myc-parkin was detected using anti-Myc antibody 9E10. Two percent of the input is shown. He: heavy chain; Lc: light chain.

FIG. 4 A is a Western blot probed with anti-Myc 9E10 of C33A cells co-transfected with Myc-parkin and Nrdpl -FLAG or a control vector. 24 hours post-transfection, cells were treated with or without 5 mM of MG132 for 8 hours.

FIG. 4B is a bar graph illustrating the results of real time RT-PCR carried out to determine the relative mRNA and protein levels of Myc-parkin, quantitated from three independent experiments (lane 3 in panel B is 100%). mRNA levels were normalized by using b-actin.

FIG. 4C is a reproduction of a gel showing the results of an immunoprecipitation experiment carried out with Myc 9E10 antibody. Transfected C33A cells were labeled with

S methionine for 3 hours and chased up to 16 hours (h) as indicated. Precipitated Myc- parkin was separated on SDS-PAGE.

FIG. 4D is a line graph illustrating the quantitation by phosphorimager of the immunoprecipitation experiment shown in FIG. 5C. Three separate experiments were carried out. Nrdpl-CT: Nrdpl C-terminus.

FIG.4E is a reproduction of a Western blot showing expression of parkin in C33A cells transfected with Nrdpl or vector control. Parkin expression in vector transfected cells is 100%.

FIG.4F is a bar graph illustrating the quantification of the results of experiment shown in FIG. 4A. Three independent experiments were plotted.

FIG. 5A is a line graph illustrating the quantitation by phosphorimager of the immunoprecipitation experiment shown in FIG. 5B.

FIG. 5B is a reproduction of a gel showing the results of an immunoprecipitation experiment in C33 A cells co-transfected with Nrdpl -FLAG, Myc-hparkin and FLAG- r

CDCrel-1 constructs or controls. Cells were labeled with S methionine for 3 hours and then chased up to 16 hours (h), as indicated. Immunoprecipitation was carried out with M2 antibody. Precipitated FLAG-CDCrel-1 was resolved on SDS-PAGE.

FIG. 5C is a line graph illustrating the quantitation by phosphorimager of the Western Blot shown in FIG. 5D.

FIG. 5D is a reproduction of a Western blot showing CDCrel-1 expression in transfected cells (as described in FIG. 6A) treated with 12 μM of cycloheximide (CHX) for 0 to 16 hours (h). CDCrel-1 was detected using anti-FLAG antibody M2.

FIG. 6A is a schematic illustration of the dNrdpl intron-exon structure.

FIGs. 6B-6D are schematic illustrations of an inverted construct for dNrdpl RNAi with dNrdpl genomic DNA as the first half and a reversed dNrdpl cDNA fragment as the second half into the pUAST vector (FIG. 6B) and two controls, an anti-sense construct (FIG. 6C) and a sense construct (no A in ATG) (FIG. 6D).

DETAILED DESCRIPTION

As described herein, parkin interacts with RING finger protein FLRF/Nrdpl. This interaction provides evidence of a link between FLRF/Nrdpl activity and Parkinson's Disease (PD). FLRF/Nrdpl is a ubiquitin E3 ligase, a member of a subfamily of RING finger domain-containing proteins called the tripartite motif family or RBCC for RING, B-box, coiled-coil. This subfamily includes a number of proteins thought to be involved in developmental and cellular processes, and mutation or rearrangement of some RBCC genes is associated with human disease. RING fingers are zinc-binding domains believed to mediate a variety of protein-protein interactions and are found in a subclass of E3 ubiquitin ligases. B-boxes, also known as TRAF-type zinc fingers, are zinc-binding domains of unknown function. Coiled-coil regions also mediate protein-protein interactions, and are often involved in homodimerization of proteins. See Diamonti et al., Proc. Nat. Acad. Sci. USA 99(5):2866-2871 (2002).

Thus, the present invention relates to methods of modulating an interaction between the FLRF/Nrdpl and parkin proteins, methods to identify test compounds and compositions that modulate the interaction between the FLRF/Nrdpl and parkin proteins, and compounds and compositions identified by the methods described herein. The present methods are useful to identify compounds that can be used to treat and/or prevent PD, and to identify novel targets for compounds that can be used to treat and or prevent PD.

The present disclosure shows that targeting of the FLRF/Nrdpl -parkin interaction is a therapeutic intervention point for PD. A therapeutic compound as described herein can be a small molecule, peptide or nucleic acid, immunoglobulin, or other peptide/protein, etc., that modulates an FLRF/Nrdpl interaction. Methods of reducing FLRF/Nrdpl expression can also be therapeutic, e.g., using methodologies such as: RNA interference, antisense oligonucleotides, morpholino oligonucleotides, and immunoglobulins or fragments thereof that bind to FLRF/Nrdpl .

Alternatively, it may be therapeutically beneficial in conditions associated with abnormally decreased FLRF/Nrdpl activity or expression to increase the expression of FLRF/Nrdpl or to facilitate expression of a non-mutated FLRF/Nrdpl, e.g., in the case of a loss-of-function mutation.

Alternative embodiments provide methods for screening candidate drugs and therapies directed to treating and/or preventing PD, and to identifying novel FLRF/NRrdpl- or parkin-interacting proteins that are targets for drugs and therapies for treating and/or preventing PD. One skilled in the art will understand that the present invention provides important research tools to develop an effective model of PD, useful in developing methods of diagnosing, prognosticating, predicting, and treating PD.

Nucleic Acid Molecules

The methods and compositions describe herein include the use of nucleic acid molecules that modulate the interaction between FLRF/Nrdpl and parkin. The nucleic acid molecules include those identified in the methods and assays described herein. Other embodiments include polynucleotides encoding FLRF/Nrdpl and parkin polypeptides and fragments thereof, particularly fragments including regions involved in the binding between FLRF/Nrdpl and parkin and in the E3 ligase activity of FLRF/Nrdpl. Another embodiment encompasses antisense, ribozyme, and siRNA molecules that target the FLRF/Nrdpl gene or mRNA. hi some embodiments, polynucleotides described herein encode an FLRF/Nrdpl polypeptide with at least about 80% identity with human or Drosophila FLRF/Nrdpl. In some embodiments, the polypeptides have about 85%, 90%, or 95% identity, hi some embodiments, the FLRF/Nrdpl polynucleotides encode polypeptides or fragments thereof possessing parkin-binding activity, h some embodiments, the polynucleotides encode FLRF/Nrd l polypeptides or fragments thereof possessing E3 ligase activity. In some embodiments, the polynucleotides encode FLRF/Nrdpl polypeptides or fragments thereof possessing parkin-binding activity, but not possessing E3 ligase activity. In some embodiments, the polynucleotides encode FLRF/Nrdpl polypeptides or fragments thereof possessing E3 ligase activity, but not possessing parkin-binding activity.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 85%, 90%, 95% or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J Mol. Biol. 48:444-453 ) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using the default parameters, e.g., a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Specifically contemplated are isolated portions of genomic DNA, cDNA, mRNA, antisense molecules, siRNAs, and ribozymes, as well as polynucleotides based on an alternative backbone or including alternative bases, whether derived from natural sources or synthesized. Such hybridizing or complementary polynucleotides may hybridize under appropriate stringency conditions to a polynucleotide encoding a polypeptide according to the present invention.

The ability of two nucleotide sequences to hybridize to each other is based upon the degree of complementarity of the two nucleotide sequences, which in turn is based on the fraction of matched complementary nucleotide pairs. The more nucleotides in a given sequence that are complementary to another sequence, the greater the degree of hybridization of one to the other. The degree of hybridization also depends on the conditions of stringency which include temperature, solvent ratios, salt concentrations and the like. In particular, "selective hybridization" pertains to conditions in which the degree of hybridization of a polynucleotide described herein to the target would require complete or nearly complete complementarity. The complementarity must be sufficiently high so as to assure that the polynucleotide described herein will bind specifically to the target nucleotide sequence relative to the binding of other nucleic acids present in the hybridization medium. With selective hybridization, complementarity will be at least 95-100%. In some embodiments, the complementarity is about 97-100%, e.g., about 100%.

As used herein, the term "hybridizes under stringent conditions" describes conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous methods are described in that reference and either can be used. As used herein, high stringency conditions are 0.5M sodium phosphate, 7% SDS at 65°C, followed by one or more washes at 0.2X SSC, 1% SDS at 65°C.

The polypeptide encoding or antisense polynucleotide molecules described herein may further be modified to contain a detectable label for diagnostic and probe purposes. A variety of such labels are known in the art and can readily be employed with the polynucleotide molecules described herein. Suitable labels include, but are not limited to, biotin, radiolabeled nucleotides, and the like. A skilled artisan can employ any of the labels known in the art to obtain a labeled polynucleotide.

Antisense and ribozyme molecules corresponding to the polypeptide coding or complementary sequence can be prepared. Methods of making antisense molecules that bind to mRNA, form triple helices, or are enzymatically active and cleave RNA and single- stranded DNA (ssDNA) are known in the art. See, e.g., Antisense and Ribozyme Methodology:Laboratory Companion (Ian Gibson, ed., Chapman & Hall, 1997) and Ribozyme Protocols: Methods in Molecular Biology (Phillip C. Turner, ed., Humana Press, Clifton, NJ, 1997).

Also contemplated is the use of compounds that mediate posttranscriptional gene silencing (PTGS), quelling, and RNA interference (RNAi). RNAi compounds typically are about 21 to about 25 nucleotides in length and are also known as short interfering RNAs or short inhibitory RNAs (siRNAs). In some embodiments, the siRNAs are produced from an initiating double stranded RNA (dsRNA). In some embodiments, 5' phosphorylated siRNAs are used; in other embodiments, hydroxylated forms can be utilized. See, e.g., Lipardi et al., 2001 Cell 107(3): 297-307; Boutla et al., 2001 Curr. Biol. 11(22): 1776-80; Djikeng et al., 2001 RNA 7(11): 1522-30; Elbashir et al., 2001 EMBO J. 20(23): 6877-88; Harborth et al., 2001 J Cell. Sci. 114(Pt. 24): 4557-65; Hutvagner et al., 2001 Science 293(5531): 811-3; and Kalidas, S., and Smith, D. P. 2002 Neuron 33, 177-184. h one embodiment, the siRNA is directed to a FLRF/Nrdpl -encoding mRNA. In another embodiment, the siRNA is directed towards a protein that binds to and modulates the activity of or is modulated by FLRF/Nrdpl, e.g., parkin. Thus, the invention includes animal models of PD in which expression of endogenous FLRF/Nrdpl is suppressed by administration of RNAi. In some embodiments, the animal model is a Drosophila, wherein the endogenous Drosophila Nrdpl is suppressed by RNAi. In some embodiments, the animal model is a transgenic animal in which the expression of parkin, synuclein or Pael-R has been suppressed or enhanced, and the endogenous Drosophila Nrdpl is suppressed by RNAi. The invention also includes cells and tissues from wild type and transgenic animals ( e.g., transgenic animals lacking or overexpressing parkin, synuclein or Pael-R), in which endogenous Drosophila Nrdpl is suppressed by RNAi. As an alternative, existing Drosophila strains with p-element insertions adjacent to dNrdpl gene can be isolated and characterized (Kretzschmar et al., Embo J. 11(7):2531-9 (1992)).

Polypeptides

Polypeptides contemplated for use in the methods described herein include those that modulate FLRF/Nrdpl and parkin interactions, e.g., polypeptides identified in the methods and assays described herein.

Also contemplated are FLRF/Nrdpl polypeptides and fragments thereof, particularly fragments including all or part of the RING Finger domain, coiled coil, B Box, and those regions involved in E3 ligase activity and parkin interaction. For example, the fragments can comprise a RING finger domain (SEQ ID NOs:3 and 4, for the human and drosophila sequences, respectively), a B-box domain (a zinc finger motif of around 40 amino acids; SEQ ID NOS:5 and 6, human and drosophila, respectively), and a coiled coil domain (SEQ ID NOs:7 and 8, human and drosophila, respectively). Variants of the FLRF/Nrdpl protein can be generated by mutagenesis, such as by the introduction of discrete point mutations, or by truncation, hi one embodiment, FLRF/Nrdpl can be mutated to destroy the binding to parkin and/or E3 ligase activity, hi other embodiments, FLRF/Nrdpl can be mutated to increase the ability to bind to parkin or E3 ligase activity. Such mutants can be used as therapeutics as well as tools, e.g., in methods to for study the etiology or mechanisms of PD. Polypeptides for use in the methods described herein include FLRF/Nrdpl polypeptides at least about 80% identity with human or Drosophila FLRF/Nrdpl . In some embodiments, the FLRF/Nrdpl polypeptides have about 90%, 95%, or 100% identity with human or Drosophila FLRF/Nrdpl. In some embodiments, the FLRF/Nrdpl polypeptide or fragment thereof possesses parkin-binding activity, hi some embodiments, the FLRF Nrdpl polypeptide or fragment thereof possesses E3 ligase activity, hi some embodiments, the FLRF/Nrdpl polypeptide or fragment thereof possesses parkin-binding activity, but does not posses E3 ligase activity. In some embodiments, the FLRF/Nrdpl polypeptide or fragment thereof possesses E3 ligase activity, but does not posses parkin-binding activity. In one embodiment, the FLRF/Nrdpl is the human protein FLRF/Nrdpl (also known as hypothetical SBBI03 protein, and sometimes referred to herein as or hNrdpl) found in the GenBank database under Accession Nos. AAC27647 and AF077599, as well as FLRF/Nrdpl homologs from other species. The mouse homologue sequence (Accession No. AF305730) named FLRF/Nrdpl was identified by Abdullah and colleagues, (Abdullah et al., Blood Cells, Molecules, and Diseases 27(l):320-333 (2001)), which permitted identification of the corresponding human homolog in the GenBank sequence database.

In another embodiment, the parkin-interacting protein is Drosophila FLRF/Nrdpl, sometimes referred to herein as dNrdpl (CG17033 protein) (GenBank database Accession Nos. AE003529, AAF49566, NP_648816; cDNA sequence Accession No. NM_140559). FLRF/Nrdpl homologs have also been identified in Zebrafish, Xenopus laevis, and mouse. FLRF/Nrdpl homologs in additional species may be obtained by standard cloning techniques or by using, e.g., a BLAST search of a sequence database based on one or more of the above mentioned species FLRF gene or protein sequences. One skilled in the art will appreciate that FLRF/Nrdpl proteins from different species can be interchangeably used in the assays, methods, and compositions described herein; sequences of FLRF/Nrdpl genes and FLRF/Nrdpl proteins from various species can be found by searching GenBank or other comparable database, or by homology cloning using known methodology.

Antibodies

Polyclonal and monoclonal antibodies and fragments of these antibodies that bind to FLRF/Nrdpl or parkin can be prepared using methods known in the art. For example, suitable host animals can be immunized using appropriate immunization protocols and the peptides, polypeptides, or proteins described herein. Peptides for use in immunization are typically about 8-40 residues long. If necessary or desired, the polypeptide immunogens can be conjugated to suitable carriers. Methods for preparing immunogenic conjugates with carriers such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or other carrier proteins are well known in the art (See, Harlow et al., 1988). In some circumstances, direct conjugation using, for example, carbodiimide reagents, may be effective; in other instances linking reagents such as those supplied by Pierce Chemical Co., Rockford, IL, may be desirable to provide accessibility to the polypeptide or hapten. The hapten peptides can be extended at either the amino or carboxy terminus with a cysteine residue or interspersed with cysteine residues, for example, to facilitate linking to a carrier. Administration of the immunogens is conducted generally by injection over a suitable time period and with use of suitable adjuvants, as is generally understood in the art. During the immunization schedule, titers of antibodies are taken to determine adequacy of antibody formation.

Antigenic proteins or polypeptides for use as immunogens can be prepared synthetically in a protein synthesizer and optionally coupled to a carrier molecule and injected over several months into rabbits. Rabbit sera is tested for immunoreactivity. Monoclonal antibodies can be made by injecting mice with FLRF/Nrdpl proteins, or antigenic fragments thereof. Monoclonal antibodies can be screened by ELISA and tested for specific immunoreactivity. Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988) and Using Antibodies: A Laboratory Manual Harlow and Lane, Eds., Cold Spring Harbor Press (1999). These antibodies are useful in assays and as pharmaceuticals and diagnostics.

Thus, anti-peptide antibodies can be generated using synthetic peptides, for example, the peptides derived from the sequence of FLRF/Nrdpl from human, Drosophila, or other species. Synthetic antigenic peptides can be as small as 2-3 amino acids in length, but are typically at least about 3, 5, 10, or 15 or more amino acid residues long (or any range in between). Such peptides can be determined using programs such as DNAStar. The peptides can be coupled to KLH using standard methods and can be immunized into animals such as rabbits. Polyclonal peptide antibodies can then be purified, for example using Actigel beads containing the covalently bound peptide.

While the polyclonal antisera produced in this way may be satisfactory for some applications, for pharmaceutical compositions, monoclonal preparations are typically used. Immortalized cell lines that secrete the desired monoclonal antibodies may be prepared using the standard method of Kohler and Milstein or modifications that effect immortalization of lymphocytes or spleen cells, as is generally known (See, e.g., Harlow et al., 1988 and 1998). The immortalized cell lines secreting the desired antibodies can be screened by immunoassay in which the antigen is the peptide hapten, polypeptide or protein. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured either in vitro or by production in ascites fluid.

The desired monoclonal antibodies are then recovered from the culture supernatant or from the ascites supernatant. Fragments of the monoclonal antibodies that contain the immunologically significant portion can be used as agonists or antagonists of FLRF/Nrdpl - parkin or FLRF/Nrdpl E3 ligase activity. In some embodiments, the use of immunologically reactive (i.e., antigen binding) fragments, such as the Fab, scFv, Fab', of F(ab')₂ fragments is preferable, especially in a therapeutic context, as these fragments are generally less immunogenic than the whole immunoglobulin.

The antibodies or fragments may also be produced by recombinant means. Regions that bind specifically to the desired regions of FLRF/Nrdpl or parkin can also be produced in the context of chimeras with multiple species origin. Immunoglobulin reagents so created are contemplated for use diagnostically or as stimulants or inhibitors of FLRF/Nrdpl -parkin binding or FLRF/Nrdpl E3 ligase activity.

In one embodiment, antibodies or antigen-binding fragments bind FLRF/Nrdpl with high affinity, i.e., ranging from 10^"5 to 10^"9 M. hi some embodiments, the antibody or antigen-binding fragment will comprise or be derived from (e.g., a portion of) a chimeric, primate, Primatized®, human or humanized antibody. Also, the invention embraces the use of antigen-binding fragments, e.g., Fabs, Fvs, Fabs, F(ab')₂, and aggregates thereof.

Another embodiment contemplates chimeric antibodies that recognize FLRF/Nrdpl, parkin, or other FLRF Nrdpl interactor. A chimeric antibody can be, e.g., an antibody with non-human variable regions and human constant regions, most typically rodent variable regions and human constant regions.

A Primatized® antibody refers to an antibody with primate variable regions, e.g., CDR's, and human constant regions. In some embodiments, such primate variable regions are derived from an Old World monkey.

A humanized antibody refers to an antibody with substantially human framework and constant regions, and non-human complementarity-determining regions (CDRs). "Substantially" refers to the fact that humanized antibodies typically retain at least several donor framework residues (i.e., of non-human parent antibody from which CDRs are derived).

Methods for producing chimeric, primate, Primatized®, humanized and human antibodies are well known in the art. See, e.g., U.S. Patent 5,530,101, issued to Queen et al.; U.S. Patent 5,225,539, issued to Winter et al.; U.S. Patents 4,816,397 issued to Boss et al., and 4,816,567, issued to Cabilly et al., all of which are incorporated by reference in their entirety for all purposes. The selection of human constant regions may be significant to the therapeutic efficacy of the subject anti-FLRF/Nrdpl, parkin, or other FLRF/Nrdpl interactor. In some embodiments, the subject anti-FLRF/Nrdpl, parkin, or other FLRF/Nrdpl interactor immunoglobulin will comprise human γl, or γ3 constant regions. In some embodiments, the subject anti-FLRF/Nrdpl, parkin, or other FLRF/Nrdpl interactor immunoglobulin will comprise human γl constant regions.

Methods for making human immunoglobulins are also known and include, by way of example, production in SCID mice, and in vitro immunization.

For example, antibodies to F:RF/Nrdpl proteins or peptides can be prepared in the following fashion. cDNAs can be expressed as six-histidine fusion proteins in E. coli, and purified using nickel chelate chromatography, or are expressed as GST-fusion proteins purified using glutathione beads. Purified proteins can be injected into rabbits and antisera generated. Anti-peptide antisera can also be generated using techniques known in the art. Antibodies can be affinity purified if necessary. Antibodies to the candidate parkin- interacting proteins can be used to determine cellular localization and for co- immunoprecipitation experiments. Expression and cellular localization of endogenous proteins in mammalian cells and S2 cells as well as in Drosophila can be characterized by immunofluorescence co-localization.

Human brain libraries can be used to screen for parkin-interacting proteins, as PD is a brain specific disease. Specific brain interactions can provide information on why only neurons are affected in PD. A second approach involves identifying parkin-interacting proteins from Drosophila. Drosophila is easily manipulated genetically and PD models have already been generated in Drosophila using mutant synuclein. Human homologues of dparkin-interacting proteins identified from Drosophila represent new leads for PD genetics. Suppressors and enhancers of parkin and parkin-interacting proteins can be identified genetically in Drosophila.

Immunocytochemistry can be used to determine if interacting proteins co-localize with parkin and synuclein in PD brains. FLRF/Nrdpl interactors can be tested to determine if they are substrates of parkin or FLRF/Nrdpl E3 ligases.

The expression of Pael-R in dopaminergic neurons in Drosophila induces increased cell death, an effect mitigated by parkin co-expression. (Yang et al., Neuron 37:911-924 (2003); Feany and Pallanck, Neuron 38:13-16 (2003)). Mitochondrial pathology and muscular degeneration are observed in parkin null mutants in Drosophila. (Greene et al., Proc. Natl. Acad. Sci. USA 100(7):4078-4083 (2003)). Transgenic lines eliciting overexpression of FLRF/Nrdpl and FLRF/Nrdpl knockout lines of Drosophila are crossed with Drosophila of varying parkin activity. Such crossings may result in the exacerbation or amelioration of the phenotypes observed in the Yang et al. and Greene et al. publications.

Methods to Identify Parkin Binding Partners and Modulators of the Flrf Nrdpl -Parkin Binding Interaction

Described herein are methods of identifying binding partners of the parkin protein, such as FLRF/Nrdpl. These methods can also be used to identify additional binding partners of FLRF/Nrdpl. In some embodiments, the methods include binding assays carried out using neural cells, hi some embodiments, the neural cells are substantia nigra neurons. As FLRF/Nrdpl is an E3 ligase, binding assays can be used to identify potential substrates of the E3 ligase activity. Ubiquitination assays can also be performed to determine if binding partners of FLRF/Nrdpl are E3 ligase substrates. Any of these methods can be used to identify modulators of the interaction between FLRF/Nrdpl and parkin, e.g., modulators that affect the binding of FLRF/Nrdpl to parkin and the E3 ligase activity. h some embodiments, an in vitro binding assay for FLRF Nrdpl -parkin interaction modulators can include contacting a sample including FLRF/Nrdpl, or a parkin-binding domain thereof, and/or parkin, or a FLRF/Nrdpl -binding domain thereof, and screening one or more test compounds, including other candidate targets or substrates. In some embodiments, the method includes screening a library of candidate targets or substrates, particularly neural targets, for compounds that modulate (e.g., statistically significantly increase or decrease) a FLRF/Nrdpl -parkin interaction.

The methods to determine binding partners can be performed using a variety of art- recognized techniques. Many of these techniques are exemplified below, including, but not limited to, yeast two-hybrid (Y2H), radiolabel binding assays, co-immunoprecipitation, and GST-fusion protein assays.

In the present invention, suitable cells are used for preparing assays, for the expression of FLRF/Nrdpl, parkin, or other FLRF/Nrdpl -interacting proteins. The cells may be made or derived from mammals, yeast, fungi, or viruses. A suitable cell for the purposes of this invention is one that includes, but is not limited to a cell that can exhibit a detectable FLRF/Nrdpl -parkin interaction. Suitable cells include, but are not limited to, human cervical cancer C33a cells, P19 rat neuronal cells, SKN-MC neuroblastoma cells, HEK293 cells, SY5Y neuroblastoma cells, PC 12 pheochromocytoma cells, NT2 cells, fly S2 cells, and primary cells (i.e., dissected cells). In some embodiments, neural cells are used. In some embodiments, substantia nigra cells are used.

To identify and isolate a binding partner, full length FLRF/Nrd l and/or parkin protein can be used. The GenBank accession numbers for human and Drosophila nucleotide and amino acid sequences are described herein. The sequence of FLRF Nrdpl from other species may be used and a number of such sequences are available in publicly available sequences databases, including mouse, Zebrafish, and Xenopus. Alternatively, a polypeptide fragment of the FLRF/Nrdpl or parkin protein can be used. Suitable fragments of the protein include at least about 10 contiguous amino acid residues of any FLRF/Nrdpl or parkin sequence, h some embodiments, the fragment comprises at least about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 residues, or any length in between. Sequences of FLRF/Nrdpl include portions or all of the region involved in E3 ligase activity or parkin interaction or the RING Finger domain, coiled coil and/or B Box,. Likewise, the FLRF/Nrdpl -binding portion of parkin may be used.

In some embodiments, the methods include a binding assay to identify FLRF/Nrdpl E3 ligase substrates, by providing a FLRF/Nrdpl polypeptide, or a fragment thereof (e.g., the E3 ligase substrate binding domain portion, e.g., the RING finger domain, coiled coil, and/or the B Box (see Fig. 1)), and screening a library of candidate targets or substrates, particularly neural targets, for binding to the polypeptide or fragment. Other binding partners of FLRF Nrd l can also be identified by using the entire protein or other portions of the molecule (e.g., other than the parkin-binding domain or E3 ligase portion). After incubating the mixture under appropriate conditions, one would determine whether FLRF/Nrdpl or a fragment thereof bound with the candidate modulator or substrate present. For cell-free binding assays, one or more components usually comprises or is coupled to a label. The label may provide for direct detection, such as radioactivity, luminescence, optical or electron density, or indirect detection such as an epitope tag or an enzyme. A variety of methods may be employed to detect the label depending on the nature of the label and other assay components. For example, the label may be detected bound to the solid substrate or a portion of the bound complex containing the label may be separated from the solid substrate, and the label thereafter detected. Fluorescence resonance energy transfer may be utilized to monitor the interaction of two labeled molecules. Alternatively, an instrument, such as a surface plasmon resonance detector manufactured by BIAcore (Uppsala, Sweden), may be used to observe interactions with a fixed target.

Alternatively, the nucleic acid molecules described herein can be used in a yeast two- hybrid (Y2H) system. The two-hybrid system is extremely useful for studying protei protein interactions; the system has been used to identify other protein partner pairs and can readily be adapted to employ the nucleic acid molecules described herein. Methods of performing and using Y2H systems are known. See, e.g., Finley et al, "Two-Hybrid Analysis of Genetic Regulatory Networks," in The Yeast Two-Hybrid System (Paul L. Bartel et al., eds., Oxford, 1997); M. Yang, "Use of a Combinatorial Peptide Library in the Two- Hybrid Assay," in The Yeast Two-Hybrid System (Paul L. Bartel et al., eds., Oxford, 1997); Yeast Hybrid Technologies (Li Zhu and Gregory J. Hannen, eds., Eaton Publ. (2000); Gietz et al., Mol. & Cell. Biochem. 172: 67-9 (1997); Young, Biol. Reprod. 58: 302-311 (1998); R. Brent et a\., Annu. Rev. Genet. 31:663-704 (1997) and U.S. Patents Nos. 5,989,808; 6,251 ,602; and 6,284,519. The FLRF/Nrdp 1 protein was identified in the present invention using the Y2H interacting system with a portion of parkin as bait.

Variations of the system are available for screening yeast phagemid (see, e.g., Harper, Cellular Interactions and Development: A Practical Approach, 153-179 (1993); Elledge et al., Proc. Natl Acad. Sci. USA 88:1731-5 (1991)) orplasmid (Bartel, Cell 14:920-4 (1993)); Finley et al, Proc. Natl Acad. Sci. USA 91:12980-4 (1994)) cDNA libraries to clone interacting proteins, as well as for studying known protein pairs.

The success of the two-hybrid system relies upon the fact that the DNA binding and polymerase activation domains of many transcription factors, such as GAL4, can be separated and then rejoined to restore functionality (Morin et al., Nuc. Acids Res. 21:2157-63 (1993)). While these examples describe two-hybrid screens in the yeast system, it is understood that a two-hybrid analogous screen may be conducted in other systems, such as mammalian cell lines. The invention is therefore not limited to the use of a yeast two-hybrid system, but encompasses such alternative systems.

Yeast strains with integrated copies of various reporter gene cassettes, such as for example GAL.fwdarw.LacZ, GAL.fwdarw.HIS3 or GAL.fwdarw.URA3 (Bartel, in Cellular Interactions and Development: A Practical Approach, 153-179 (1993); Harper et al., Cell 75:805-16 (1993); Fields et al, Trends Genetics 10:286-92 (1994)) are co-transformed with two plasmids, each expressing a different fusion protein. One plasmid encodes a fusion between protein "X" and the DNA binding domain of, for example, the GAL4 yeast transcription activator (Brent et al, Cell 43:729-36 (1985); Ma et al, Cell 48:847-53 (1987); Keegan et al, Science 231:699-704 (1986)), while the other plasmid encodes a fusion between protein "Y" and the RNA polymerase activation domain of GAL4 (Keegan et al, 1986). The plasmids are transformed into a strain of the yeast that contains a reporter gene, such as lacZ, whose regulatory region contains GAL4 binding sites. If proteins X and Y interact, they reconstitute a functional GAL4 transcription activator protein by bringing the two GAL4 components into sufficient proximity to activate transcription. It is well understood that the role of bait and prey proteins may be alternatively switched and thus the embodiments of this invention contemplate and encompass both alternative arrangements.

Either hybrid protein alone must be unable to activate transcription of the reporter gene, the DNA-binding domain hybrid, because it does not provide an activation function, and the activation domain hybrid, because it cannot localize to the GAL4 binding sites. Interaction of the two test proteins reconstitutes the function of GAL4 and results in expression of the reporter gene. The reporter gene cassettes consist of minimal promoters that contain the GAL4 DNA recognition site (Johnson et al, Mol. Cell. Biol. 4:1440-8 (1984); Lorch et al, J. Mol. Biol. 186:821-824 (1984)) cloned 5' to their TATA box. Transcription activation is scored by measuring either the expression of β-galactosidase or the growth of the transformants on minimal medium lacking the specific nutrient that permits auxotrophic selection for the transcription product, e.g., URA3 (uracil selection) or HIS3 (histidine selection). See, e.g., Bartel, 1993; Durfee et al, Genes & Devel 7:555-569 (1993); Fields et al, Trends Genet. 10:286-292 (1994); and U.S. Pat. No. 5,283,173.

Generally, these methods include two proteins to be tested for interaction that are expressed as hybrids in the nucleus of a yeast cell. One of the proteins is fused to the DNA- binding domain (DBD) of a transcription factor and the other is fused to a transcription activation domain (AD). If the proteins interact, they reconstitute a functional transcription factor that activates one or more reporter genes that contain binding sites for the DBD. An exemplary two-hybrid assay that has been used for FLRF/Nrdpl and parkin are presented in the Examples below. However, the yeast two-hybrid assay can also be used to screen for compounds or compositions that disrupt the binding of two known interactors. For instance, compounds or compositions that disrupt the interaction of FLRF/Nrdpl and parkin. Thus, the yeast two-hybrid screen presents an excellent system for screening for compounds or compositions that may serve as therapeutics for PD.

Additional methods of preparing two hybrid assay systems for FLRF Nrdpl or parkin interactors can be prepared based on the information presented herein. See, e.g., Finley et al, "Two-Hybrid Analysis of Genetic Regulatory Networks," in The Yeast Two-Hybrid System (Paul L. Bartel et al, eds., Oxford, 1997); Meijia Yang, "Use of a Combinatorial Peptide Library in the Two-Hybrid Assay," in The Yeast Two-Hybrid System (Paul L. Bartel et al, eds., Oxford, 1997); Gietz et al, "Identification of proteins that interact with a protein of interest: Applications of the yeast two-hybrid system," Mol. & Cell. Biochem. 172:67-9 (1997); K. H. Young, "Yeast Two-Hybrid: So Many h teractions,(in) so Little Time," Biol. Reprod. 58:302-311 (1998); R. Brent et al, "Understanding Gene and Allele Function with Two-Hybrid Methods," Annu. Rev. Genet. 31:663-704 (1997). It will be appreciated that protein networks can be elucidated by performing sequential screens of activation domain- fusion libraries.

Chemical Libraries

Compounds and compositions that are assayed by these methods can be randomly selected or rationally selected or designed. As used herein, a compound is said to be randomly selected when the compound is chosen randomly without considering the specific sequences involved in the association of FLRF/Nrdpl alone, FLRF/Nrdpl interacting proteins alone, or with their associated substrates, binding partners, etc. An example of randomly selected compounds is the use of a chemical library or a peptide combinatorial library, or a growth broth of an organism.

The compounds can be, for example, peptides, small molecules, vitamin derivatives, or carbohydrates. A skilled artisan can readily recognize that there is no limit to the structural nature of the compounds.

Peptide compounds can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art. In addition, the DNA encoding these peptides can be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. The production of polypeptides using solid phase peptide synthesis is necessitated if non- nucleic acid-encoded amino acids are to be included.

Uses for Compounds and Compositions That Modulate FLRF/Nrdpl -Parkin Interactions or FLRF/Nrdpl E3 Ligase Activity

FLRF/Nrdpl binds to parkin, a protein known to play a role in the occurrence of Parkinson's Disease (PD). Compounds that modulate (e.g., decrease) the expression of FLRF/Nrdpl, bind to FLRF/Nrdpl, block the interaction of FLRF Nrdpl and parkin, or inhibit FLRF Nrdpl E3 ligase activity can be used to modulate the biological and pathological processes associated with PD, e.g., to treat, prevent, or delay the development or progression of PD in a subject. The methods include administering a therapeutically effective amount of the compound to the subject. In addition, as FLRF/Nrdpl has been identified as having E3 ligase activity and protein degradation errors are believed to play a role in PD, FLRF/Nrdpl has significance in helping to determine the mechanisms underlying PD apart from its parkin interaction. As mentioned above, compounds that affect a FLRF/Nrdpl function, e.g., binding to parkin, and compounds that bind to FLRF/Nrdpl, can be used as therapeutic and/or diagnostic compounds for PD (e.g., assessing mutations in FLRF/Nrdpl), or as research tools. In some embodiments, the methods include treating, prevent, or delaying the development or progression of a subject who has PD by administering a therapeutically effective amount of an inhibitor of FLRF/Nrdpl expression or activity, e.g., a FLRF/Nrdpl antisense, antibody, RNAi, or other inhibitor identified by a method described herein.

The compounds described herein can be administered alone, or in combination with other compounds that modulate a particular pathological process, or other modes of treatment (e.g., physical therapy or other standard or experimental treatments for PD). As used herein, two compounds are said to be administered in combination when the two compounds are administered simultaneously or are administered independently in a fashion such that the compounds will act at substantially the same time, or over the same period of time. Alternatively, the compounds described herein can be administered before or after another compound or mode of treatment. hi general, the compounds described herein can be administered in a therapeutically effective amount by any of the accepted modes of administration for compounds that serve similar utilities. The compounds can be administered by a variety of routes including, but not limited to, parenteral (e.g., subcutaneous (sc), intravenous (iv), intramuscular (im), or intraperitoneal (ip)), transdermal, or mucosal (e.g., oral, sublingual, pulmonary (e.g., inhalation), rectal or vaginal) routes. In some embodiments, the compounds are administered by direct application to neural tissues, e.g., injection into a CNS fluid or infusion into the substantia nigra, e.g., by local injection or implantation of a device for long-term release of the compound. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, severity of the disease, and the nature of the effect desired.

The present invention further provides compositions containing one or more active compounds that modulate expression or at least one activity of FLRF/Nrdpl as described herein. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. Typical dosages of the active compound winch modulate FLRF/Nrdpl -parkin interaction or FLRF/Nrdpl E3 ligase activity comprise from about 0.0001 to about 50 mg/kg body weight. In some embodiments, the dosages comprise from about 0.001 to about 50 mg/kg body weight. In some embodiments, the dosages comprise from about 0.1 to about 1 mg/kg body weight. In an average human of 70 kg, the range would be from about 7 μg to about 3.5 g, e.g., about 0.5 mg to about 5 mg. hi addition to the active compound, the compositions described herein can be formulated to contain suitable pharmaceutically acceptable carriers, e.g., carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically for delivery to the site of action. Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts, h addition, suspensions of the active compounds as appropriate injection suspensions may be administered. In some embodiments, the injection suspension is an oily suspension; suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, (e.g., ethyl oleate or triglycerides). Aqueous injection suspensions can contain substances that increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol and or dextran. Optionally, the suspension can also contain stabilizers. Liposomes and other viral and non- viral vectors can also be used to encapsulate or otherwise prepare the compound for delivery into the cell. The pharmaceutical composition for systemic administration according to the invention may be formulated for enteral, parenteral, or topical administration, hi some embodiments, two or more types of formulations can be used simultaneously to achieve systemic administration of the active ingredient.

Suitable formulations for oral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups or inhalations and controlled release forms thereof.

Once a compound (or agent) of interest has been identified, standard principles of medicinal chemistry can be used to produce derivatives of the compound. Derivatives can be screened for improved pharmacological properties, for example, efficacy, pharmaco-kinetics, stability, solubility, and clearance. The moieties responsible for a compound's activity in the assays described above can be delineated by examination of structure-activity relationships (SAR) as is commonly practiced in the art. A person of ordinary skill in pharmaceutical chemistry could modify moieties on a candidate compound or agent and measure the effects of the modification on the efficacy of the compound or agent to thereby produce derivatives with increased potency. For an example, see Nagarajan et al, J. Antibiot. 41:1430-8 (1988). Furthermore, if the biochemical target of the compound (or agent) is known or determined, the structure of the target and the compound can inform the design and optimization of derivatives. Molecular modeling software is commercially available (e.g., Molecular Simulations, h e.) for this purpose. The derivatives can be evaluated for inhibitory activity, therapeutic activity, and therapeutic efficacy in vivo and/or in vitro, e.g., using a method described herein. Methods described herein, and methods known in the art, can be used to select a compound with a favorable therapeutic profile, i.e., a compound that has the greatest desirable effect with the least unwanted side effects. For example, therapeutic efficacy and toxicity of such compounds and compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LDso/ED₅o. Compounds and compositions that exhibit large therapeutic indices are preferred.

Potentially, any compound that modulates an FLRF/Nrdpl -parkin interaction (e.g., binding) or FLRF/Nrdpl E3 ligase activity can be a therapeutic compound. Transgenic Animals

Transgenic animal models can be created that overexpress, conditionally express, or do not express, active FLRF/Nrdpl, e.g., express greater or lesser amounts of FLRF/Nrdpl as compared to a wildtype animal. These animals can be used to study the physiological effects of compounds that modulate the FLRF/Nrdpl -parkin interaction and/or the interaction of FLRF/Nrdpl and other FLRF/Nrdpl -interacting proteins, e.g., those identified by the methods described herein. Alternatively, transgenic animals can be created that express a transgenic form of FLRF/Nrdpl alone or in addition to a transgenic form of parkin or express FLRF/Nrdpl interacting proteins alone or in addition to a transgenic form of FLRF/Nrdpl. Transgenic animals expressing parkin can be crossed with transgenic animals expressing FLRF/Nrdpl or FLRF/Nrdpl -interacting proteins to obtain heterozygote as well as homozygote animals that express both desired transgenes.

Animal models can be used as a model to determine the efficacy of candidate compounds that modulate the FLRF/Nrdpl -FLRF/Nrdpl -interacting protein interaction or FLRF/Nrdpl -parkin interaction activity in vitro. The transgenic animals described herein can also be used as models of disease, e.g., of PD. Thus, the animals can be used to evaluate a therapeutic effect of administration of a candidate compound, e.g., a candidate compound identified by a method described herein, that modulates a FLRF/Nrdpl -parkin interaction. See, e.g., Pendleton et al, JPEr300(l):91-96 (2002), e.g., an effect on motor activity including climbing response and geotactic response, or an effect on the loss, rate of loss, or severity of loss, of dopaminergic neurons in the dorsomedial group.

Transgenic animals, such as transgenic mice or flies, can be created using known methods to express, for example, human FLRF/Nrdpl or a FLRF/Nrdpl -interacting protein, or fragments or variants thereof, or do not express functional FLRF/Nrdpl, i.e., FLRF/Nrdpl knockouts (KO) of all or part of the gene encoding mouse FLRF/Nrdpl.

For example, embryonic stem cells in mice and other animals can be used to select transgenic cells and perform gene targeting. This allows more genetic engineering than is possible with other transgenic techniques. For example, mouse ΕS cells are relatively easy to grow as colonies in vitro. The cells can be transfected by standard procedures and transgenic cells clonally selected by antibiotic resistance. See, e.g., Doetschman et al, Gene transfer in embryonic stem cells. In Pinkert (Εd.) Transgenic Animal Technology: A Laboratory Handbook, Academic Press, New York, pp. 115-146 (1994). Furthermore, the efficiency of this process is such that sufficient transgenic colonies (hundreds to thousands) can be produced to allow a second selection for homologous recombinants. For example, mouse ES cells can be combined with a nonnal host embryo and, because they retain their potency, can develop into all the tissues in the resulting chimeric animal, including the germ cells. The transgenic modification can then be transmitted to subsequent generations.

Methods for deriving embryonic stem (ES) cell lines in vitro from, for example, early preimplantation mouse embryos are well known. See, e.g., Evans et al, Nature 29:154-6 (1981) and Martin, Proc. Natl. Acad. Sci. USA 78:7634-8 (1981). ES cells can be passaged in an undifferentiated state, provided that a feeder layer of fibroblast cells or a differentiation inhibiting source is present.

In some embodiments, the transgenic animals are Drosophila strains that over-express human or Drosophila Nrdpl, or have suppressed endogenous Drosophila Nrdpl expression. The transgenic animals can be conditional or constitutive. Knock-in animals include animals wherein genes have been introduced, and animals wherein a gene that was previously knocked-out is reintroduced into the animal, e.g., under control of a regulatable element. Other transgenic animals can be created with inducible forms of FLRF/Nrdpl or a FLRF/Nrdpl -interacting protein to study the effects of the gene on PD. These animals can also be used to study long-term effects of FLRF/Nrdpl or FLRF/Nrdpl -interacting protein modulation. The transgenic vectors may direct expression in a tissue-specific manner by the use of tissue-specific promoters. In some embodiments, the FLRF/Nrdpl expression is regulated in a neural-specific fashion, particularly regulated in substantia nigra cells. Such systems can provide a tissue-specific knock-out of FLRF/Nrdpl or FLRF/Nrdpl -interacting protein activity.

General methods for creating transgenic animals are known in the art, and are described in, for example, Strategies in Transgenic Animal Science (Glenn M. Monastersky and James M. Robl eds., ASM Press; Washington, DC, 1995); Transgenic Animal Technology: A Laboratory Handbook (Carl A. Pinkert ed., Academic Press 1994); Trans enic Animals (Louis Marie Houdebine, ed., Harwood Academic Press, 1997); Overexpression and Knockout of Cytokines in Transgenic Mice (Chaim O. Jacob, ed., Academic Press 1994); Microinjection and Transgenesis: Strategies and Protocols (Springer Lab Manual) (Angel Cid-Arregui and Alejandro Garcia-Carranca, eds., Springer Verlag 1998); and Manipulating the Mouse Embryo: A Laboratory Manual (Brigid Hogan et al, eds., Cold Spring Harbor Laboratory Press 1994). Methods for making transgenic flies include the P-lacW and the P-element mobilization method described by Bier et al, Genes Dev. 3, 1273-1287 (1989); Pirrotta, V. (1986) in Drosophila: A Practical Approach, ed. Roberts, D. B. (IRL, Oxford), pp. 83-110. pUAST is a well-characterized system for the generation of transgenic flies that allows transgenes to be induced to a relatively high level of expression at different stages and in different tissues. However, if expression of transgenes driven by Gal 4 is not sufficient, as an alternative, transgenic flies can be generated using another promoter, e.g., using a heat- shock promoter that has been shown to effectively induce expression of transgenes. Furthermore, it has been recently reported that expression of parkin is low at early stages and increases with age (Greene et al. Proc Natl Acad Sci USA 100, 4078-4083 (2003). The heat shock promoter may provide additional leverage to investigate parkin functions at late stages. Further, to efficiently generate Drosophila models, RNAi inverted constructs can be tested in S2 cells before injection for transgenic models. The constructs can be modified by, for example, replacing introns with artificial introns if necessary. The constructs with the most robust inhibition can be selected for further experiments in Drosophila models.

A. Nrdpl Transgenic Crosses

In some embodiments, the methods include crossing a FLRF/Nrdpl knockout animal with another transgenic animal to breed animals that lack FLRF/Nrdpl as well as one or more other genes. Thus, once generated, the Nrdpl transgenic animals described herein can be crossed with other transgenic animals, e.g., animals transgenic for parkin (Goldberg et al, J Biol Chem. 278(44):43628-35 (2003)), α-synuclein (see, e.g., Feany and Bender, Nature 404(6776):394-8 (2000); Auluck et al, Science 295, 865-868 (2002), or Pael-R (Yang et al, Neuron 37, 911-924 (2003).

To further examine the effects of Nrdpl in specific tissues or during specific stages of development, transgenic fly strains for Nrdpl generated as described herein can be crossed with Gal4 expressing flies. Many fly strains with Gal4 expression have been developed. They include tissue-specific strains such as the neuron specific strain ELAV-Gal4 (Lin and Goodman, Neuron 13, 507-523 (1994), developmental stage specific strains or heat-shock strains in which gene expression can be manipulated at any stage (Zecca et al, Cell 87(5): 833-844 (1996); Nishimura et al, Cell 116(5): 671-82 (2004)). In particular, the neuron specific strains can be used to study interactions between dparkin and dNrdpl, as the central nervous system (CNS) is the primary target for PD.

B. Characterization of Nrdpl Transgenic Phenotypes

To evaluate the role of Nrdpl in PD, the phenotype of animals transgenic for Nrdpl can be evaluated. Phenotypes obtained from systemic suppression or overexpression of Nrdpl, and spatially and/or temporally targeted suppression or overexpression of Nrdpl, in neural and non-neural tissues, can provide clues on functions of targeted proteins. The phenotypes of animals transgenic for Nrdpl and one or more other genes (e.g., parkin, Pael- R, α-synuclein, and/or Gal4) can also be evaluated. In particular, phenotypes associated with Parkinson's disease in humans can be evaluated, including, but not limited to, dopaminergic neurodegeneration, intracytoplasmic neuronal inclusion bodies containing α-synuclein (resembling the Lewy bodies seen in humans suffering from PD), and progressive locomotor dysfunction. See, e.g., Feany and Bender, Nature 404:394-398 (2000); Haass and Kahle, Nature 404(6776): 341, 343 (2000). For example, the evaluation can include monitoring an effect on motor activity including climbing response and geotactic response, or an effect on the loss, (e.g., the rate or severity of loss), of dopaminergic neurons in the dorsomedial group.

If the phenotypes caused by dNrdpl overexpression or suppression include lethality, rescue and other studies can be performed, e.g., by over-expressing wild type or mutant hNrdpl in these flies, or mutant forms of dNrdpl. Next, Drosophila models for Nrdpl over- expression or dNrdpl suppression can be crossed with dparkin and/or hparkin overexpressing or knockout/knockdown strains, as well as Drosophila strains expressing α-synuclein (Auluck et al, Science 295:865-868 (2002)) or Pael-R. Visible phenotypes, such as viability, wing formation and patterns, bristles, eye colors, and segmentation changes are observed. In particular, the number and morphology of dopaminergic neurons, which have been shown to be affected by parkin and its substrates α-synuclein and Pael-R, are examined (Yang et al, Neuron 37:911-924 (2003)). This can be done by immunostaining of tyrosine hydroxylase (TH) in transgenic fly brains as described (Yang et al, Id.). Drosophila lacking dparkin manifest abnormal mitochondria (Greene et al, Proc Natl Acad Sci USA 100:4078-4083 (2003)). Effects on mitochondria caused by Nrdpl can also be examined in Drosophila models that overexpress Nrdpl. This information will provide clues on which pathways parkin Nrdpl may regulate. Furthermore, pathological studies can be carried out to determine the functions of interactions between parkin and Nrdpl in brain and other tissues. The growth, differentiation, and viability during development of the CNS in dNrdpl transgenic flies, with and without knockout and/or expression of parkin, α-synuclein, and/or Pael-R, can be investigated, e.g., as previously described (Yang et al, Neuron 37:911-924 (2003)). Finally, locomotor dysfunction in these flies can be tested, e.g., as described (Feany and Bender, Nature 404:394-398 (2000)).

In addition to the specific materials and methods referred to herein, one of skill in the art is directed to the references cited in this application as well as the several Current Protocol guides, which are continuously updated, widely available and published by John Wiley and Sons (New York). In the life sciences, Current Protocols publishes comprehensive manuals in Molecular Biology, Immunology, Human Genetics, Protein Science, Cytometry, Neuroscience, Pharmacology, Cell Biology, Toxicology, and Nucleic Acid Chemistry. Additional sources are known to one of skill in the art.

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

EXAMPLES

Example 1. Yeast Two-Hybrid Screen

To identify parkin-interacting proteins, the N-terminal of Drosophila parkin cDNA was cloned into pGKBT7 vector (Gal4 DNA binding domain) (Matchmaker III from Clontech) and used as a bait to screen a Drosophila PACT2-1 cDNA library (Clontech) using a yeast two hybrid assay. Plasmid DNA from the libraries was transformed into yeast cells expressing a dparkin bait.

The N-terminus of the Drosophila parkin cDNA (dparkin) (Fruitfly database CGI 0523: 1-576 bps) was amplified by PCR with primers that added EcoRI and BamHI sites to the 5' and 3' ends and ligated into pGKBT7 to encode a hybrid protein containing the DNA-binding domain of Gal4 fused to dparkin protein. Expression of the chimeras in yeast was determined by Western blot analysis. Yeast cells of the AH 109 reporter strain were sequentially transformed with the dparkin/pGKBT7 construct and then with a Drosophila expression library containing cDNAs fused to the sequence of the GAL4-transactivation domain in pACT2 vector (Clontech). Briefly, the Gal4-dparkin bait was transformed into yeast host AH109. Positive clones were selected and picked from Trp-SD selection medium and tested for chimera protein expression. Plasmid DNA from the Drosophila library was then transformed into cells expressing the dparkin bait and plated on SD-Ade/-His/-Leu/-Trp selection medium. After a few days, positive clones were selected and tested for β- galactosidase and α-galactosidase activities.

One of the three prey tested was retested by using the yeast two hybrid assay and confirmed as a possible parkin-interacting protein. Sequencing analysis of the clone revealed a cDNA with an open reading frame of 951 base pairs that encodes a 317 amino acid protein, which shares 45% homology with human FLRF/Nrdpl as shown in Figure 1. The new protein was termed dFLRF. The most distinct feature of FLRF/Nrdpl is a RING finger domain, a characteristic of ubiquitin E3 ligases. FLRF/Nrdpl is an E3 ligase that ubiquitinates and degrades at least two substrates, ErbB3 and ErbB4. (Diamonti et al. (2002), supra; and Qiu and Goldberg (2002), supra).

Example 2. In Vitro Interactions

To validate the interaction between FLRF/Nrdpl and parkin discovered in the yeast two-hybrid assay, in vitro binding assays were performed. Yeast two-hybid and co- immunoprecipitation assays do not prove direct binding. However, in vitro assays using bacterially synthesized proteins can demonstrate a direct interaction.

FLRF/Nrdpl and parkin were inserted into the pcDNA 3.1 vector and used for coupled in vitro transcription/translation in rabbit reticulocyte lysates using the TNT kit (Promega, Madison, WI). Myc-hparkin, ΕLA-Nrdpl, and luciferase peptides labeled with ³⁵S methionine were generated using the Promega TNT kit, and mixed and rocked in binding buffer (10 mM Tris-HCl, pH8.0; 200 mM NaCl; 5 mM EDTA, 0.5% NP-40, 1 mM DTT, 3 mg/ml of BSA and the proteinase inhibitors). Anti-Myc or anti-HA antibody was employed to precipitate either Myc-hparkin or HA-Nrdpl and the co-immunoprecipitated FLRF/Nrdpl or hparkin was separated by SDS-PAGE and then analyzed by a phosphorimager. In a separate experiment, bacterially expressed GST- FLRF/Nrdpl (GST= glutathione S- transferase) was produced and purified. Glutathione-Sepharose beads bound to GST-fusion proteins were washed with binding buffer, and rocked with aliquots of in vitro translated S methionine-labeled hparkin proteins for 1 hour at 4°C in binding buffer. The beads were washed with binding buffer, and boiled in SDS-PAGE sample buffer. The proteins binding to GST-FLRF/Nrdpl beads were resolved on SDS-PAGE and detected by a phosphorimager. Domains of hparkin that bind to Nrdpl were mapped with hparkin C-terminal or N-terminal TNT translated peptides (Fig. 2C; numbers refer to the amino acid sequence of human parkin, GenBank Accession No. BAA25751). In vitro immunoprecipitation analysis was performed from binding mixtures of hparkin and FLRF/Nrdpl or controls. Co-precipitated proteins were then separated by SDS-PAGE and analyzed using a phosphoimager.

GST-parkin and GST-FLRF/Nrdpl fusion proteins were expressed and purified. Translation of plasmids in the presence of ³⁵S-methionine generated radiolabeled peptides for these proteins as well as the luciferase control (Figure 2A-2B, TNT). The FLRF/Nrdpl band was clearly observed from hparkin precipitates while hparkin was pulled down by HA-Nrdpl (Figure 2A). Similar results were obtained when FLAG was tagged onto a FLRF/Nrdpl c- terminal construct. An in vitro interaction between hparkin and FLRF/Nrdpl was also demonstrated by mixing equivalent aliquots of the radiolabeled hparkin with purified GST or GST- FLRF/Nrdpl fusion protein (Figure 2B). GST or GST- FLRF/Nrdpl fusion proteins were adsorbed to glutathione-agarose beads. In vitro translated hparkin was specifically retained on the beads by the GST- FLRF/Nrdpl fusion protein, but not parental GST peptide, and the luciferase control binds to neither GST nor GST-FLRF/Ndrpl, indicating specific binding between hparkin and FLRF/Nrdpl (Figure 2B). Further experiments using in vitro binding assays mapped the binding between hparkin and FLRF/Nrdpl to the hparkin N- terminus (hp-NT) (Figure 2B), consistent with the results from the yeast two hybrid assays.

These results demonstrate that FLRF/Nrdpl and parkin do interact in vitro.

Example 3. In Vivo Co-Immunoprecipitation

To investigate interactions between parkin and FLRF/Nrdpl in vivo, human cervical cancer C33A cells were utilized for co-immunoprecipitation. Myc-tagged parkin cDNA and HA-tagged FLRF/Nrdpl cDNA were cloned into a myc vector or pcDNA3.1, respectively. Myc-parkin and HA-FLRF/Nrdpl plasmids were co-transfected into C33A cells for co- immunoprecipitation experiments.

First, anti-Myc 9E10 antibody was used to precipitate parkin protein from C33A cell lysates. The negative control cells transfected with Myc- vector and HA- FLRF/Nrdpl showed no detectable HA- FLRF/Nrdpl protein pulled down, while a clear band of HA- FLRF/Nrdpl was detected from the anti-Myc precipitates in cells co-transfected with HA- FLRF/Nrdpl and Myc-hparkin (Figure 3A).

In a separate experiment, anti-HA (12CA5) antibody was used to precipitate Nrdpl protein from C33A cell lysates. Co-immunoprecipitated hparkin was detected from cells co- transfected with Myc-hparkin and HA-Nrdpl, but not from control cells that were co- transfected with Myc-hparkin and a HA-vector (Fig. 3B). demonstrating interactions between parkin and FLRF/Nrdpl in vivo.

The results with C33A cells demonstrate that the interaction between FLRF/Nrdpl and parkin can occur in non-neuronal cell lines. Thus, co-immunoprecipitation experiments can be performed in both non-neuronal and neuronal cell lines. Along this line, PC 12 cells, SKN-MC cells, neuroblastoma cell lines, SY5Y cells, NT2 cells and fly S2 cells, among other cell lines as would be evident to the skilled artisan, can be used to examine the interaction between parkin and FLRF/Nrdpl.

Example 4. Immunofluorescence Co-Localization

To further characterize interactions between parkin and FLRF/Nrdpl, C33A and PC 12 cells were cultured on glass cover slips and co-transfected with Myc-hparkin and FLRF/Nrdpl-FLAG.

Forty-eight hours after transfection, cells were treated with retinoic acid for an additional 48 hours to induce the cells to differentiate. Cells treated with or without retinoic acid were fixed in 4% paraformaldehyde for 20 minutes, washed 3 times with PBS, permeabilized in 0.1% Triton x-100 in PBS for 20 minutes, and blocked with 3% milk and 3% goat serum in PBS for 1 hour. The cells were incubated with primary antibodies of HA and Flag that have been conjugated with fluorescence overnight at 4°C. The localization of proteins was visualized by fluorescence and confocal microscopy.

Immunofluorescence analysis was carried out with fluorescence conjugated anti-Myc and anti-FLAG antibodies or anti-parkin antibody and then a fluorescence labeled secondary antibody in cells treated or untreated with nerve growth factor (NGF).

Co-localization of hparkin and Nrdpl was visualized in cell bodies as well as neuronal processes in an aggregate-like structure. As one theory, interactions between hparkin and FLRF/Nrdpl may occur in synaptic vesicles (SV) where parkin has been localized (Huynh et al, Hum. Mol Genet. 12(20) :2587-97 (2003)). Example 5. Effects of Parkin on FLRF/Nrdpl Half-Life

The effects of parkin on the stability of FLRF/Nrdpl were evaluated in a transfected cell model.

A. Transfection ofC33A cells

C33A cells were transfected with or without MG132, a proteasome inhibitor (to inhibit protein degradation). No apparent difference was observed for parkin protein among cells transfected with parkin, parkin + FLRF/Nrdpl, or FLRF/Nrdpl. However, in cells transfected with parkin and FLRF/Nrdpl without MG132 treatment, FLRF/Nrdpl was barely detectable, while in cells transfected with FLRF/Nrdpl alone and without treatment with MG132, FLRF/Nrdpl was readily detected. In both cases of treatment with MG132, high levels of expression of FLRF/Nrdpl mRNA were detected by Northern blot analysis. These results suggest that FLRF/Nrdpl protein is stabilized by MG132 and that parkin facilitates the degradation of FLRF/Nrdpl.

B. Pulse Chase Analysis

Transfected cells were incubated and radio-labeled with ³⁵S-Met for three hours and then washed. Cell lysates were collected at 0, 0.5, 2, 5, and 16 hours after washing. Parkin or FLRF/Nrdpl was immunoprecipitated with antibodies against Flag or Myc that tagged parkin or FLRF/Nrdpl . Precipitated parkin or FLRF/Nrdpl was analyzed by SDS-PAGE separation and exposed to X-ray film. The half-lives of ³⁵S-labeled proteins were determined. Without parkin co-transfection, the half-life of FLRF/Nrdpl is >10 hours. With parkin co-transfection, the half-life of FLRF/Nrdpl is <5 hours. Thus, parkin likely participates in the degradation of FLRF/Nrdpl in the cell. Ubiquitination studies may be performed to confirm the mechanism of FLRF/Nrdpl degradation by parkin. Methods of determining ubiquitination are well known in the art, including for use with parkin. See, e.g., hnai et al. J. Biol. Chem. 275(46):35661-4 (2000); Shimura et al Nat. Genet. 25(3):302-5 (2000); Zhang et al. Proc. Natl Acad. Sci. USA 97(24): 13354-9 (2000); Imai et al. Cell 105(7):891-902 (2001); Shimura et al. Science 293(5528):263-9 (2001); Diamonti et al. Proc. Natl. Acad. Sci. USA 99(5):2866-71 (2002); Qiu and Goldberg, Proc. Natl. Acad. Sci. USA 99(23): 14843-8 (2002).

Example 6: Effect of FLRF/Νrdpl on Parkin Expression Levels Since both parkin and FLRF/Nrdpl are E3 ligases (Shimura et al. Nat. Genet. 25(3):302-5 (2000); Zhang et al. Proc. Natl. Acad. Sci. USA 97(24): 13354-9 (2000); hnai et al, Cell 105(7):891-902 (2001); Shimura et al, Science 293(5528): 263-9 (2001); Diamonti et al, Proc. Natl. Acad. Sci. USA 99(5):2866-71 (2002); Qiu and Goldberg, Proc. Natl. Acad. Sci. USA 99(23): 4843-8 (2002)), the role of Νrdpl or parkin as a substrate or an E3 ligase in the FLRF/Νrdpl/parkin complex was evaluated.

C33A cells that were co-transfected with Myc-hparkin and FLRF/Νrdpl -FLAG or a control were treated with or without the proteasome inhibitor, MG132. Cell lysates were subjected to Western blot analysis with anti-Myc or anti-FLAG antibodies. A relatively much lower level of hparkin expression (<25%) was detected in the presence of FLRF/Νrdpl than in the absence of FLRF/Νrdpl (Figs. 4A-B, compare lanes 1 and 3).

To demonstrate that the discrepancy of hparkin protein levels was not caused by transfection efficiency, real-time PCR was performed.

Briefly, total R A from transfected C33A cells was isolated using Trizol Reagent (Invitrogen) and used for reverse transcription (Superscript III, Life Technologies). PCR reaction was carried out in a 25 ml final volume containing regular PCR components, realtime PCR Enhancer (5X: lmg/ml Bovine Serum, 750 mM Trehalose, 1% Tween-20), SYBER Green™, forward and reverse primers designed from pcDΝA3.1 vector and parkin gene respectively (160 bps PCR product) and 1.25 units of hot master Taq DNA polymerase with either cDNA or standard dilutions as templates. PCR reaction was performed on DNA Engine Opticon™ 2 System (MJ Research Incorporated, Waltham, MA). mRNA levels were calculated and expressed in relative copy numbers normalized against b-actin mRNA as described (Overbergh et al, Cytokine, 11:305-312 (1999)).

The real-time PCR assays demonstrated that the discrepancy of hparkin protein levels was not caused by transfection efficiency, because levels of mRNAs transcribed from pcDNA3.1 -Myc-hparkin plasmid in all transfected cells were statistically equal (Fig. 4B, lanes 1 and 3), indicating that FLRF/Nrdpl affects hparkin stability (Fig. 4B). Further experiments demonstrated that the level of hparkin expression was dramatically elevated (>4 fold) when cells were co-transfected with FLRF/Nrdpl and treated with MG132 (Figs. 4A-B, compare lanes 1 and 2). On the other hand, consistent with previous reports (Choi et al, Neuroreport 12(13):2839-43 (2000); Shimura et al, Nat. Genet. 25(3): 302-5 (2000)), in the absence of FLRF/Nrdpl, MG132 treatment did not significantly change the level of hparkin accumulation (Figs. 4A-B, compare lanes 3 and 4).

Levels of endogenous parkin protein in cells overexpressing FLRF/Nrdp-1 were also evaluated. In this experiment, C33A cells were transfected with FLRF/Nrdpl, or control vector, and levels of endogenous parkin were analyzed by Western blotting. As shown in Figs. 4E-F, over-expression of FLRF/Nrdpl in C33A cells reduced the level of endogenous parkin protein; over-expression of FLRF/Nrdpl in C33A reduced parkin protein to 40% (Fig. 4F). These results indicate that FLRF/Nrdpl affects parkin stability and degradation, via a proteasome dependent pathways.

To further characterize the effects of FLRF/Nrdpl on parkin metabolism, a pulse- chase experiment was performed. C33A cells co-transfected with plasmids (myc-hparkin and FLRF/Nr i-FLAG, FLRF/Nrφi-CT-FLAG or a control plasmid) were metabolically labeled with [³⁵S] -methionine for 3 hours. [³⁵S]-labeled proteins were then chased for 0, 1.5, 3, 5 hours up to 16 hours. Myc-hparkin was immunoprecipitated using 9E10 antibody and analyzed on SDS-PAGE gels and then a phosphorimager (Fig. 4C-D). Expression of Myc- parkin was quantitated (Fig. 4D).

The results show that half-life of hparkin is about 5 hours in the absence of FLRF/Νrdpl in C33A cells, consistent with the previous reports (Zhang et al, Proc. Nαtl Acαd. Sci. USA 97(24): 13354-9 (2000); whereas overexpression of FLRF/Νrdpl reduces parkin half-life to 2 hours (Fig.4D), indicating that FLRF/Νrdpl accelerates parkin degradation. However, in contrast to the previous report (Qiu et al, Proc. Nαtl. Acαd. Sci. U SA 99(23):14843-8 (2002), the C-terminus of FLRF/Νrdpl does not have dominant effects on degradation of parkin.

Example 7: FLRF/Νrdpl Affects hparkin Activity on Down-Stream Substrates

The effect of FLRF/Νrdp-1 over-expression on degradation of hparkin downstream substrate CDCrel-1 was evaluated using a pulse-chase experiment.

Briefly, plasmids of FLAG-CDCreZ-1, Myc-hpαrkin, FLRF/Νrdpl -FLAG were transfected in C33A cells individually or in combination. Cells were labeled with [³⁵S]- methionine and then chased up to 16 hours. FLAG-CDCrel-1 was immunoprecipitated using anti-FLAG antibody M2, resolved on SDS-PAGE gels and then quantitated in a phosphorimager (Fig. 5A-B). In the absence of parkin expression, the half-life of CDCrel-1 is approximately 8 hours, while over-expression of parkin accelerated CDCrel-1 turnover with a half-life of 5 hours. However, CDCrel-1 was re-stabilized when both hparkin and FLRF/Nrdpl were co-transfected (Fig. 5A-B), indicating that FLRF/Nrdpl affected CDCrel- 1 turnover not directly but rather via modulating parkin activity. Similar results were obtained in C33A cells when protein inhibitor cycloheximide was used in co-transfected C33A cells (Fig. 5C-D).

Thus, accelerated degradation of hparkin downstream substrate CDCrel-1 was abrogated with Nrdpl over-expression (Figs. 5A-D). These results demonstrate that Nrdpl not only affects hparkin stability but also hparkin activity on its down-stream substrates.

Example 8. Sequencing of FLRF/Nrdpl cDNAs from PD Patients

FLRF Nrdpl cDNAs are amplified by RT-PCR from PD patients' tissues or cells, e.g., blood, cheeks, or neural cells. These cDNAs are sequenced to determine if mutations in the gene are associated with PD. Single strand conformational polymorphism (SSCP) has been widely used to detect base changes.

In this technique, PCR products of the gene (cDNA or genomic DNA) with or without mutations are denatured at 94 °C, cooled on ice to prevent hybrid formation, then electrophoresed on standard non-denaturing polyacrylamide gels. The two strands of the PCR product will usually run with very different mobilities, and base changes can further alter the mobility of each strand. Thus, what appears to be a single PCR product by standard analysis, can split into four different bands on an SSCP gel if the original DNA sample was heterozygous for base changes that altered the mobility of both strands.

Various studies have analyzed pairs of PCR products known to differ by single base substitutions to obtain an estimate of the fraction that can be distinguished by the SSCP protocol. In one such study, 80% of 228 variant PCR products were distinguishable (Sheffield et al, Genomics 16:325-332 (1993)); in another, the rate of detection was 80-90% (Michaud et al, Genomics 13:389-394 (1992)). However, when three different electrophoretic conditions were utilized for analysis, a 100% detection rate was achieved.

Example 9: dFLRF/dNrdpl Transgenic Flies

If drosophila FLRF/Ndrpl (dFLRF/dNrdpl) functions in flies as human FLRF/hNrd l functions in human cell lines, then phenotypes in dNrd l transgenic flies or FLRF/dNrdpl mutant flies are possible. It is likely that phenotypes induced in Drosophila models with overexpression of parkin, α-synuclein or Pael-R will be modulated by FLRF/Nrdpl expression since FLRF/Nrdpl affects parkin stability.

To test this hypothesis, fly strains that over-express FLRF/Nrdpl are generated using the pUAST system (Brand and Perrimon Development 118:401-415 (1993). pUAST consists of a Gal4UAS promoter and a "white" gene in a P-element. Genes of interest are cloned into the multiple-cloning-lihker downstream of a Gal4UAS so that the expression of the gene will be GAL4 inducible. By microinjection of a pUAST construct and helper plasmid DNA that expresses a transposase into fly embryos, part of the pUAST plasmid DNA between 3'p and 5'p (from Gal4UAS to the end of white gene) is incorporated into the fly chromosome (s). Transgenic flies are then selected by eye color changes (white gene marker). This technique has been used to study presenilin 1 and SMN proteins (Miguel-Aliaga et al, FEBSLett. 486(2):99-102 (2000); Noll et al, De.vBiol. 227(2):450-64 (2000); Chan, et al, Hum. Mol. Genet. 12(12):1367-76 (2003)). Since the C-terminal of FLRF/Nrdpl exhibits dominant effects on the full length of FLRF/Nrdpl for ErbB3/ErbB4, a total of 6 different transgenic strains that include FLRF/dNrdpl, d-CT- FLRF/Nrφi (C-terminus), d-ΝT-Νrdp (Ν-terminus), hFLRF/hΝrdpl, h-CT- FLRF/Nr i and h- Νt- FLRF/Nrφi are generated. Expression of trans-genes is tested by RT-PCR and Western blot analyses. Phenotypes are examined as described below. Transgenic flies can be generated by injection of constructs into Drosophila embryos, and transgenic flies are established as described herein.

The expression of transgenes for dFLRF/dΝrdpl is examined during generation and establishment of the transgenic animals using standard methods including real-time RT-PCR. If positive results are obtained, Western blot, histocytochemistry and in situ hybridization are used to evaluate the localization and expression of the transgenes in different cell types will be carried out. The effect of dsRΝA inhibition of dFLRF/dΝrdpl expression in RΝAi Drosophila is also be evaluated by these methods.

Example 10: R A Interference of Νrdpl in Drosophila

Overexpression of parkin reduces the severity of the phenotypic effects such as reduction of dopaminergic neurons, or overexpression of synuclein or Pael-R (Yang et al, Neuron. 37(6):911-24 (2003)). Preliminary data suggested that interactions between parkin and FLRF/Νrdpl regulate parkin stability. Therefore, if endogenous dFLRF/dΝrdpl is suppressed, levels of both endogenous dparkin and transgenic parkin proteins will be elevated as a result. Consequently, phenotypes induced by overexpression of α-synuclein or Pael-R may be modulated.

To evaluate the role of dFLRF/dNrdpl, fly strains with no or low expression of dFLRF/dNrdpl are generated using RNA interference (RNAi). The dFLRF/dNrdpl intron-exon structure is shown in Fig. 6A. An inverted construct for dFLRF/dNrdpl RNAi is cloned using a recently described approach (Kalidas and SmithNeuron 33:177-184 (2002) within dFLRF/dΝrdpl genomic DΝA as the first half and a reversed complementary dFLRF/dΝrdpl cDΝA fragment as the second half in the pUAST vector (FIG. 6B). Two controls, the anti-sense construct (FIG. 6C) and the sense construct (No A in ATG) (FIG. 6D) are also generated. The transgenic flies are generated by injection of constructs into Drosophila embryos by the Duke University Model System Genomics Unit, and transgenic flies are established as described herein.

OTHER EMBODIMENTS

While the invention has been described and illustrated above by reference to various specific materials, procedures and examples, it is understood that the invention is not restricted to the particular materials, combinations of materials, and procedures selected for that purpose. In addition, it is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

We claim:

1. A method of identifying a compound that modulates an interaction of FLRF/Nrdp 1 with parkin, the method comprising: providing a sample comprising FLRF/Nrdpl or a parkin binding fragment of FLRF/Nrdpl, and parkin or a FLRF/Nrdpl binding fragment of parkin; contacting the sample with one or more test compounds; and evaluating one or more of (i) binding of FLRF/Nrdpl to parkin or (ii) FLRF/Nrdpl -dependent degradation of parkin, in the presence and the absence of the test compound, wherein a test compound that inhibits or enhances one or more of (i) binding of FLRF/Nrdpl to parkin or (ii) FLRF/Nrdpl -dependent degradation of parkin is a compound that modulates an interaction of FLRF/Nrdpl with parkin.

2. A method of identifying a candidate compound for the treatment of Parkinson's Disease, the method comprising: providing a sample comprising FLRF/Nrdpl or a parkin-binding fragment thereof, and parkin or a FLRF/Nrdpl -binding fragment thereof; contacting the sample with one or more test compounds; and evaluating an interaction of FLRF/Nrdpl with parkin in the presence and the absence of the test compound, wherein a test compound that inhibits an interaction of FLRF/Nrdpl with parkin is a candidate compound for the treatment of Parkinson's Disease.

3. The method of claim 1 or 2, wherein the interaction of FLRF/Nrdpl with parkin is (i) binding of FLRF/Nrdpl to parkin or (ii) FLRF/Nrdpl -dependent degradation of parkin.

4. The method of claim 1, wherein the interaction is inhibited.

5. The method of claim 1, wherein the interaction is enhanced.

6. The method of claim 2, further comprising: providing a non-human animal model exhibiting a symptom associated with Parkinson's Disease; administering to the animal model a candidate compound that modulates an interaction of FLRF/Nrdpl with parkin; and monitoring the animal for an improvement in a symptom associated with Parkinson's Disease, wherein an improvement in a symptom indicates that the candidate compound is a candidate therapeutic agent for the treatment of Parkinson's Disease.

7. The method of claim 6, wherein the non-human animal model is Drosophila melanogaster.

8. The method of claim 6, wherein the non-human animal model is a transgenic animal whose somatic and or germ cells comprise a FLRF/Nrdpl inhibitory transgene.

9. The method of claim 8, wherein the FLRF/Nrdpl transgene comprises a nonfunctional allele of FLRF/Nrdpl.

10. The method of claim 6, wherein the symptom associated with Parkinson's Disease is selected from the group consisting of loss of motor function and loss of dopaminergic neurons.

11. The method of claim 10, wherein the effect on the symptom is an improvement in motor function.

12. The method of claim 10, wherein the effect on the symptom is a reduction in loss of dopaminergic neurons.

13. The method of claim 2, further comprising optimizing the candidate compound.

14. The method of claim 13, further comprising formulating a therapeutic composition comprising the optimized candidate compound and a pharmaceutically acceptable carrier therefor.

15. The method of claim 2, further comprising formulating a therapeutic composition comprising the candidate compound and a pharmaceutically acceptable carrier therefor.

16. The method of any one of claims 1-15, wherein the parkin binding fragment of FLRF/Nrdpl comprises one or more of the RING finger domain, coiled coil, or B Box.

17. The method of any one of claims 1-15, wherein the FLRF/Nrdp 1 binding fragment of parkin comprises the first 50 amino acids of the N-terminus of parkin.

18. A method of preparing a pharmaceutical composition to treat Parkinson's Disease, the method comprising: obtaining a candidate compound that inhibits an interaction of FLRF/Nrdpl with parkin; determining whether the candidate compound improves a symptom of PD in an animal model, wherein an improvement indicates that candidate compound is a candidate therapeutic agent; and formulating the candidate therapeutic agent with a pharmaceutically acceptable carrier to prepare the pharmaceutical composition.

19. The method of claim 18, further comprising optimizing the candidate compound.

20. The method of claim 18 or 19, further comprising testing the candidate compound in a clinical trial.

21. A non-human transgenic animal, whose somatic and/or germ cells comprise a FLRF/Nrdpl inhibitory transgene, wherein the animal exhibits one or more symptoms associated with Parkinson's Disease.

22. The non-human transgenic animal of claim 21, wherein the animal is Drosophila melanogaster.

23. The non-human transgenic animal of claim 21 , wherein the animal is a mouse.

24. The non-human transgenic animal of claim 21 , wherein the inhibitory transgene comprises a non-functional allele of FLRF/Nrdpl.

25. The non-human transgenic animal of claim 21, wherein the one or more symptoms associated with Parkinson's Disease are selected from the group consisting of loss of motor function and loss of dopaminergic neurons.

26. A method of treating a subject having Parkinson's Disease, the method comprising administering to the subject a therapeutically effective amount of a compound that inhibits FLRF/Nrdpl expression or activity.

27. The method of claim 26, wherein the FLRF/Nrdpl inhibitor is selected from the group consisting of an antisense nucleic acid, siRNA, or ribozyme that selectively binds to a nucleic acid encoding FLRF/Nrdpl.

28. The use of an inhibitor of FLRF/Ndrpl expression or activity in the treatment of Parkinson's disease.

29. The use of an inhibitor of FLRF/Nrdpl in the manufacture of a medicament for the treatment of Parkinson's disease.