WO2007135570A2

WO2007135570A2 - Pink1 deficient animals, screening methods, and related therapeutics

Info

Publication number: WO2007135570A2
Application number: PCT/IB2007/002527
Authority: WO
Inventors: Jongkyeong Chung
Original assignee: Genexel-Sein, Inc.
Priority date: 2006-04-19
Filing date: 2007-04-18
Publication date: 2007-11-29
Also published as: WO2007135570A3; WO2007135570A8

Abstract

The present invention provides PINK1 deficient animals, screening methods, and related therapeutics. In particular, the present invention provides transgenic animals with a disruption in their endogenous PINK1 gene causing the cells in the animal to exhibit a mitochondrial related dysfunction phenotype and/or causing the animal to exhibit symptoms of neurodegenerative disease. The present invention also provides methods of using PINK1 deficient cells or animals to screen candidate compounds, as well as therapeutics agents useful for treating PINK1 deficient subjects.

Description

PBVKl DEFICIENT ANEVIALS, SCREENING METHODS, AND RELATED

THERAPEUTICS

The present Application claims priority to U.S. Provisional Application Serial Number 60/793,082, filed April 19, 2006, which is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to PlNKl deficient animals, screening methods, and related therapeutics. In particular, the present invention relates to transgenic animals with a disruption in their endogenous PINKl gene causing the cells in the animal to exhibit a mitochondrial related dysfunction phenotype and/or causing the animal to exhibit symptoms of neurodegenerative disease. The present invention also relates to the use of PINKl deficient cells or animals to screen candidate compounds, as well as therapeutics agents useful for treating PINKl deficient subjects.

BACKGROUND OF THE INVENTION

Parkinson's Disease is a progressive neurological disease affecting as many as 1 ,500,000 Americans. Parkinson's Disease occurs when certain nerve cells (neurons) in the part of the brain called the substantia nigra die or become impaired. Normally, these cells produce a vital chemical known as dopamine. Dopamine allows smooth, coordinated function of the body's muscles and movement. Generally, when approximately 80% of the dopamine-producing cells are damaged, the symptoms of Parkinson's Disease appear.

Parkinson's Disease affects both men and women in almost equal numbers. It shows no social, ethnic, economic or geographic boundaries. In the United States, it is estimated that 60,000 new cases are diagnosed each year. While the condition usually develops after the age of 65, 15% of those diagnosed are under 50. Idiopathic Parkinson's Disease is by far the most common, and includes the rare genetic forms caused by mutations in the genes for alpha-synuclein and parkin. Known environmental causes include the very rare cases of poisoning by MPTP (l-methyl-4-phenyl-4-propionoxypiperidine), carbon monoxide, and manganese, as well as recurrent head trauma. Neuroleptic exposure, on the other hand, is a relatively common cause of drug-induced Parkinsonism (and is reversible).

The incidence of Parkinson's Disease increases with age. The median age of onset for all forms of Parkinson syndrome is 61.6 years, with median idiopathic Parkinson's Disease onset at 62.4 years. Onset before age 30 is rare, but up to 10% of cases of idiopathic Parkinson's Disease begin by age 40. In a recent study in the United States, the incidence of Parkinson's was 10.9 cases per 100,000 person years in the general population, and 49.7 per 100,000 person-years for those over age 50. The incidence is growing as the population ages. Prevalence is estimated to be approximately 300 per 100,000 in the United States and Canada, but with the important caveat that perhaps 40% of cases may be undiagnosed at any given time.

Symptoms such as bradykinesia are slowness in voluntary movements. It produces difficulty initiating movement as well as difficulty completing movement once it is in progress. The delayed transmission from the brain to the skeletal muscles, due to diminished dopamine, produces bradykinesia. Tremors in the hands, fingers, forearm, or foot tend to occur when the limb is at rest, but not when performing tasks. Tremor may occur in the mouth and chin as well. Rigidity, or stiff muscles, may produce muscle pain and an expressionless, mask-like face. Rigidity tends to increase during movement. Poor balance is due to the impairment or loss of the reflexes that adjust posture in order to maintain balance. Falls are common in people with Parkinson's. The Parkinsonian gait is the distinctive unsteady walk associated with Parkinson's Disease. There is a tendency to lean unnaturally backward or forward, and to develop a stooped, head-down, shoulders- drooped stance. Arm swing is diminished or absent and people with Parkinson's tend to take small shuffling steps (called Destination). Someone with Parkinson's may have trouble starting to walk, appear to be falling forward as they walk, freeze in mid-stride, and have difficulty making a turn.

As such, what is needed are methods and compositions for identifying agents to treat Parkinson's disease, and agents to treat neurodegenertive diseases like Parkinson's.

SUMMARY OF THE INVENTION

The present invention provides PINKl deficient animals, screening methods, and related therapeutics. In particular, the present invention provides transgenic animals with a disruption in their endogenous PINKl gene causing the cells in the animal to exhibit a mitochondrial related dysfunction phenotype and/or causing the animal to exhibit symptoms of neurodegenerative disease. The present invention also provides methods of using PINKl deficient cells or animals to screen candidate compounds, as well as therapeutics agents useful for treating PINKl deficient subjects. In some embodiments, the present invention provides methods of screening comprising: a) contacting a PINKl deficient cell with a candidate compound, and b) detecting: i) the presence or absence of parkin mRNA or protein up-regulation in the cell, or ii) the presence or absence of reduced mitochondrial related dysfunction in the cell. The mitochondrial related dysfunction may be any measurable parameter related to dysfunction of mitochondria in cell. In certain embodiments, the mitochondrial related dysfunction is evidenced by a phenotype selected from the group consisting of: reduced mitochondrial DNA expression; reduced mitochondrial protein expression; reduced ATP levels in the cell; irregular arrangement of myofibrils; enlarged mitochondria; reduced number of mitochondria organelles in the cell; a decreased anti-tyrosine hydroxylase staining intensity; and a reduced level of dopamine levels. In further embodiments, the parkin mRNA or protein regulation is increased expression compared to a control PINKl deficient cell not exposed to the candidate compound.

In particular embodiments, the detecting comprises comparison to a control PINKl deficient cell not contacted with the candidate compound. In additional embodiments, the presence of parkin protein up-regulation, or the presence of reduced mitochondrial related dysfunction in the cell identifies the candidate compound as a therapeutic compound for treating neurodegenerative disease. In some embodiments, the neurodegenerative disease is Parkinson's disease. In certain embodiments, the neurodegenerative disease is autosomal recessive juvenile Parkinson's. In other embodiments, the cell is a DA neuron cell.

In some embodiments, the detecting comprises quantitating the amount of parkin mRNA present in the cell and comparing to a control PINKl deficient cell not contacted with the candidate compound. In other embodiments, the detecting comprises quantitating the amount of parking protein present in the cell.

In some embodiments, the present invention provides methods of screening comprising: a) contacting a PINKl deficient animal with a candidate compound (or causing expression of an exogenous gene such a Parkin or BcI-I), and b) detecting: i) the presence or absence of parkin protein or mRNA up-regulation in cells of the animal, or ii) the presence or absence of reduced mitochondrial related dysfunction in cells of the animal. In further embodiments, the mitochondrial related dysfunction is evidenced by a phenotype selected from the group consisting of: reduced mitochondrial DNA expression; reduced mitochondrial protein expression; reduced ATP levels in the cells; irregular arrangement of myofibrils; enlarged mitochondria; reduced number of mitochondria organelles in the cells; a decreased anti -tyrosine hydroxylase staining intensity; a reduced level of dopamine expression; muscle cell apoptosis; defects in DA neurons in either the DLl 10 or DM cluster. In particular embodiments, the animal is a mouse, rat, fly, hamster, dog, monkey, or other experimental animal. In other embodiments, the animal is Drosophila melanogaster, and the mitochondrial related dysfunction is evidenced by a phenotype selected from the group consisting of: abnormally positioned wings; a crushed thorax, impaired flight ability, reduced climbing rate; and complete male sterility due to defective Nebenkem.

In certain embodiments, the animal exhibits at least one neurodegenerative symptom similar to human Parkinson's disease. In particular embodiments, the animal is Drosophila melanogaster and the symptom is selected from the group consisting of: indirect flight muscle (IFM) degeneration; reduced climbing ability; complete male sterility due to impaired sperms with swelled Nebenkem; downturned wing phenotype with rigidity; reduced life-span; a defect in flight ability; a crushed looking thorax in the mid-anterior and/or antero-lateral regions; disorganized muscle fibers, enlarged mitochondria; irregular arrangement of myofibrils in the indirect flight muscle; swollen mitochondria with loss of the outer membrane in the indirect flight muscle; and apoptosis in muscles cells.

In other embodiments, the detecting comprises comparison to a control PINKl deficient animal not contacted with the candidate compound. In particular embodiments, the presence of parkin protein up-regulation, or the presence of reduced mitochondrial related dysfunction in the cells of the animal identifies the candidate compound as a therapeutic compound for treating neurodegenerative disease.

In particular embodiments, the neurodegenerative disease is parkinson's disease. In other embodiments, the neurodegenerative disease is autosomal recessive juvenile Parkinson's.

In some embodiments, the present invention provides methods of treatment comprising: contacting a cell that is PDMKl deficient with an agent that upregulates parkin expression or that reduces mitochondrial related dysfunction in the cell. In other embodiments, the agent comprises a vector (e.g., expression vector), wherein the vector is configured to cause the expression of PINKl in the cell. In particular embodiments, the agent comprises a vector (e.g., expression vector), wherein the vector is configured to cause the expression of Bcl-2 in the cell. In further embodiments, the agent comprises a vector (e.g., expression vector), wherein the vector is configured to cause the expression parkin in the cell. In some embodiments, the mitochondrial related dysfunction has a phenotype selected from the group consisting of: reduced mitochondrial DNA expression; reduced mitochondrial protein expression; reduced ATP levels in the cell; irregular arrangement of myofibrils; enlarged mitochondria; reduced number of mitochondria organelles in the cell; a decreased anti-tyrosine hydroxylase staining intensity, and a reduced level of dopamine expression. In certain embodiments, the cell is a DA neuron cell.

In additional embodiments, the present invention provides methods of treatment comprising; administering to an animal that is PINKl deficient an agent that upregulates parkin expression or that reduces mitochondrial related dysfunction in cells of the animal. In certain embodiments, the agent comprises a vector, wherein the vector is configured to cause the expression of PINKl in cells of the animal. In other embodiments, the agent comprises a vector, wherein the vector is configured to cause the expression of Bcl-2 in cells of the animal. In further embodiments, the agent comprises a vector, wherein the vector is configured to cause the expression parkin in cells of the animal.

In some embodiments, the present invention provides a transgenic animal whose genome comprises a gene disruption in its endogenous PINKl gene, and wherein at least some of the cells of the animal exhibit a mitochondrial related dysfunction phenotype. In further embodiments, the mitochondrial related dysfunction phenotype is selected from the group consisting of: reduced mitochondrial DNA expression; reduced mitochondrial protein expression; reduced ATP levels in the cells; irregular arrangement of myofibrils; enlarged mitochondria; reduced number of mitochondria organelles in the cells; a decreased anti- tyrosine hydroxylase staining intensity; a reduced level of dopamine expression; muscle cell apoptosis; defects in DA neurons in either the DLl 10 or DM cluster. In other embodiments, the PINKl disruption reduces or eliminates the expression of a functional Parkin protein. In certain embodiments, the animal is Drosophila melanogaster.

In some embodiments, the present invention provides a transgenic animal whose genome comprises a gene disruption in its endogenous PINKl gene, and wherein the animal exhibits at least one neurodegenerative symptom similar to human Parkinson's disease. In particular embodiments, the symptom is selected from the group consisting of: indirect flight muscle (IFM) degeneration; reduced climbing ability; complete male sterility due to impaired sperms with swelled Nebenkem; downtumed wing phenotype with rigidity; reduced life-span; a defect in flight ability; a crushed looking thorax in the mid-anterior and/or antero-lateral regions; disorganized muscle fibers, enlarged mitochondria; irregular arrangement of myofibrils in the indirect flight muscle, swollen mitochondria in the indirect flight muscle; and apoptosis in the muscle cells.

In additional embodiments, the present invention provides compositions comprising an agent (e.g., compound, medicament, or other agent) that mimics the activity of PINKl in vivo and causes increased parkin expression in PINKl deficient subjects. The present invention also contemplates the design, and screening, of compounds that treat PINKl deficient subjects that readily traverse the blood brain barrier. Brain uptake of drugs can be improved via prodrug formation. Prodrugs are pharmacologically inactive compounds that result from transient chemical modifications of biologically active species. The chemical change is usually designed to improve some deficient physicochemical property, such as membrane permeability or water solubility. After administration, the prodrug, by virtue of its improved characteristics, is brought closer to the receptor site and is maintained there for longer periods of time. Here it gets converted to the active form, usually via a single activating step. For example, esterifϊ cation or amidation of hydroxy-, amino-, or carboxylic acid- containing drugs, may greatly enhance lipid solubility and, hence, entry into the brain. Drugs may be adapted for CNS delivery through the use of lipophilic analogs, liposomes, PEGylated derivitives, immunoliposomes, redox delivery systems, carrier mediated delivery systems, receptor or vector mediated delivery, osmotic blood brain barrier disruption, biochemical blood brain barrier disruption, or olfactory delivery. Alternatively, delivery could also be achieved via invasive procedures such as intraventricular or intrathecal delivery, injections, catheters, pumps, biodegradable polymer wafers, microspheres, nanoparticles, or delivery from biological tissues. (Mishra, A. et al., 2003, J Pharm Pharmaceut Sci, 6(2):252-273). Additionally, compounds or intervention may be applied with the compounds of the present invention to increase uptake to desired tissues (e.g., brain tissue). Such methods include, but are not limited to, those described in U.S. Pat. Appln. Ser. Nos. 20030162695, 20030129186, 20020038086, and 20020025313, herein incorporated by reference in their entireties.

DESCRIPTION OF THE FIGURES

Figure 1 shows characterization of various PINKl mutants. Figure Ia shows exons of PINKl (CG4523) indicated by boxes, and coding regions that are colored black. The deleted regions for D3 (PINKl⁰³, 379bp) and B9 (PINKl⁸⁹, 570bp) are also presented. The gray bar indicates the region used as a probe for Northern blotting. Figures lb-c show the determination of the expression level of PINKl by Northern (b) and Western (c) blotting. Asterisk indicates a nonspecific band. Figure Id shows immunostaining of the nucleus of sperms in 3-day-old male testis (dark gray in upper panel, Hoechst33258; light gray, α- tubulin; upper panels). TEM analysis of the spermatozoa of testis of males hatched within 12 hr (middle and bottom panels). Ax, axoneme; N, Nebenkern. White bar, 5 μm; black bar, 5 μm; gray bar, 500 nm. Figure Ie shows downturned wing phenotypes in PINKl mutants. Figure If shows a comparison of flight ability. Figure Ig shows a comparison of the climbing rate (*, P=4.59x\0^Λ; **, P=ISIxW⁴; ***, P=UOxIO^'6; ****, P=3A6χ\0^'5). Figure Ih shows collapsed-thorax phenotypes (Black arrows) in PINKl mutants right after eclosion. Figure Ii show percent of collapsed-thorax phenotypes. Bars indicate mean ± standard deviation (S.D.).

Figure 2 shows mitochondrial defects in PINKl mutants. Figure 2a shows longitudinal sections of thoraces. Black bar, 200 μm; gray bar, 20 μm. Figure 2b shows TEM analysis of IFM of 2-day-old males. White bar, 5 μm; gray bar, 2 μm. Figure 2c shows merged images of apoptotic cells (TUNEL, light gray) and nuclei (Hoechst33258, dark gray) of the IFM. Gray bar, 20 μm. Figure 2d shows quantification of the mtDN A of thoraces (only the P values for Co III are shown: *,

***, P=AAIxIO^'2). n=3. Figure 2e shows a comparison of the ATP content of thoraces (*, P=I.20x 10^{^}*; **, P=I.1 Ox 10³; ***, P=3.08x 10^'2). Figure 2f shows the percent of defective thorax and wing phenotypes. Bars indicate mean ± S.D.

Figure 3 shows DA neuronal degeneration in PlNKl mutants. Figure 3 a shows whole-mount adult male brains (30-day-old) showing DA neuron clusters marked by anti- TH antibody (Green). White bar, 100 μm. Figure 3b shows a graph showing the number of DA neurons in each cluster at 30 days (*, P=3.68xlO^"8; **, P=2.73xlO^'9). n=20. Figure 3c shows the amount of dopamine in adult head (*, /*=6.85xlO^~3; **, P=1.19xlO^"3). n=4. Figure 3d shows the examination of the mitochondria in DA neurons within DLl cluster of 3-day-old adult brain. Gray bar, 10 μm. Figure 3e shows a graph showing the percentage of the number of DA cells with mitochondria larger than 2 μm in diameter over the total number of DA cells in each cluster (*,

****,

n=l 0. Figure 3f shows a TEM analysis of the DA neurons in DLl cluster of 20-day-old adult brain. Dark gray arrows indicate the mitochondria in DA neurons, n, nucleus. White bar, 2 μm; gray bar, 1 μm. Bars indicate mean ± S.D. Figure 4 shows in vivo interaction between PINKl and Parkin. Figure 4a shows the percentage of defective thorax and wing phenotypes. Figure 4b shows longitudinal sections of thoraces (upper and middle panels), and merged images (bottom panels) of TUNEL (light gray) and Hoechst33258 (dark gray) staining of the thoraces. Black bar, 200 μm; gray bar, 20 μm. Figure 4c shows a western blot of mtProtein cytochrome c oxidase subunit III (Co III) of thoraces. n=3. Figure 4d shows an examination of the mitochondria in DA neurons within DLl cluster of 3-day-old adult brain (left). Gray bar, 10 μm. Graph showing the percentage of the number of DA cells with mitochondria larger than 2 μm in diameter over the total number of DA cells in each cluster (right; *,

***, .P=4.79^χ 10 ^s). n=l 0. Figure 4e shows a graph showing the number of DA neurons in each cluster at 30 days (*,

Figure 4f shows a comparison of the climbing rate (*, P=4.59x 10^"*; **, P=2.40x 10^"2). Figure 4g shows a comparison of the flight ability. Figure 4h shows the percentage of defective thorax and wing phenotypes. Figure 4i shows the longitudinal sections of thoraces (upper panels), and merged images (bottom panels) of TUNEL (light gray) and Hoechst33258 (dark gray) staining of the thoraces. Gray bar, 20 μm. Bars indicate mean ± S.D.

GENERAL DESCRIPTION OF THE INVENTION

The present invention provides PINKl deficient animals, screening methods, and related therapeutics. In particular, the present invention provides transgenic animals with a disruption in their endogenous PINKl gene causing the cells in the animal to exhibit a mitochondrial related dysfunction phenotype and/or causing the animal to exhibit symptoms of neurodegenerative disease. The present invention also provides methods of using PINKl deficient cells or animals to screen candidate compounds, as well as therapeutics agents useful for treating PINKl deficient subjects.

Autosomal recessive juvenile parkinsonism (AR-JP) is an early-onset form of Parkinson's disease characterized by motor disturbances and dopaminergic (DA) neurodegeneration¹"². To address its underlying molecular pathogenesis, loss-of-function mutants of Drosophila PTEN-induced putative kinase 1 (PINKl)³, a novel AR-JP -linked gene⁴, were generated and characterized. It was shown that PINKl mutants exhibit indirect flight muscle (IFM) and DA neuronal degeneration accompanied by locomotive defects. Furthermore, transmission electron microscopy (TEM) analysis and a rescue experiment with Drosophila Bcl-2 demonstrated that mitochondrial dysfunction accounts for the degenerative changes in all phenotypes of PINKl mutants. Surprisingly, it was also found that PESfKl mutants share striking phenotypic similarities with parkin mutants. Transgenic expression of Parkin dramatically ameliorated all PINKl loss-of-function phenotypes, but not vice versa, suggesting that Parkin functions downstream of PINKl. Taken together, this genetic evidence clearly establishes that Parkin and PINKl act in a common pathway in maintaining mitochondrial integrity and function in both muscles and DA neurons. The invention herein includes compositions and methods useful for the discovery, testing, design and use of medicaments, identification of new drug targets, and diagnosis relating to neurodegenerative conditions such as Parkinson's disease (e.g. in humans).

DEFINITIONS

To facilitate understanding of the invention, a number of terms are defined below.

As used herein, a "PINKl deficient cell" refers to a cell that has reduced levels of PINKl mRNA, PINKl protein, or both, compared to wild-type levels. This definition includes any reason that a cell may be PINKl deficient, including, for example, a disruption in the one or both of the PINKl alleles in the cell (e.g., mutation, insertion, truncation, point mutation, etc.), or mutation in another gene (e.g., transcription factor) that causes lower levels of expression (or no expression) of PENKl mRNA or protein.

As used herein, the phrase "mitochondrial related dysfunction" refers to any loss of normal mitochondrial related activity in a cell, compared to wild-type levels, that is caused by the cell being PINKl deficient. Examples of phenotypes associated with mitochondrial related dysfunction include, but are not limited to: reduced mitochondrial DNA expression; reduced mitochondrial protein expression; reduced ATP levels in the cell; irregular arrangement of myofibrils; enlarged mitochondria; reduced number of mitochondria organelles in the cell; a decreased anti-tyrosine hydroxylase staining intensity; and a reduced level of dopamine levels.

As used herein, the term "antisense" is used in reference to RNA sequences that are complementary to a specific RNA sequence (e.g., mRNA). Included within this definition are antisense RNA ("asRNA") molecules involved in gene regulation by bacteria. Antisense RNA may be produced by any method, including synthesis by splicing the gene(s) of interest in a reverse orientation to a viral promoter that permits the synthesis of a coding strand. Once introduced into an embryo, this transcribed strand combines with natural mRNA produced by the embryo to form duplexes. These duplexes then block either the further transcription of the mRNA or its translation. In this manner, mutant phenotypes (e.g., PINKl deficient mutants) may be generated.

As used herein, the term "vector" is used in reference to nucleic acid molecules that transfer DNA segment(s) from one cell to another. The term "vehicle" is sometimes used interchangeably with "vector."

The term "expression vector" as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host organism. Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.

The term "test compound" or "candidate compound" refers to any chemical entity, pharmaceutical, drug, and the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample. Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present invention. A "known therapeutic compound" refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.

DETAILED DESCRIPTION OF THE INVENTION

Drosophila PINKl gene (CG4523) encodes a polypeptide of 721 amino acids with a molecular weight of about 80 kDa. Similar to human PINKl⁴ (which has a nucleotide accession number of NM 032409; and an amino acid accession number of AAQ89316; both sequences of which are herein incorporated by reference), structural analysis of Drosophila PINKl protein also revealed two characteristic motifs, a mitochondrial targeting motif (MTM) and a serine/threonine kinase domain. The kinase domain exhibited a 60% similarity (42% identity) with that of human PINKl . Consistent with the localization of human PINKl 4, Drosophila PINKl was also found localized in mitochondria.

As part of the development of the present invention, the expression pattern of PINKl was observed and it was found that its transcripts are ubiquitously expressed throughout all developmental stages. Its spatial expression in adults was broadly distributed over all segments, but particularly higher in the thoraces. PINKl loss-of-function mutant flies were generated to reveal in vivo roles of PINKl in Drosophila, including PINKl loss- of-function mutant flies, PINKl D3 and PINKl B9, as well as the revertants (PINKlRV) (Fig. 1 a), and those alleles were confirmed by conducting Southern, Northern, and Western blot analysis and genetic analysis with PINKl-RNAi lines (Fig. Ib, c). PINKl mutants were viable and developed to adulthood. However, they displayed shorter longevity and complete male sterility due to impaired sperms with swelled Nebenkern, a specialized mitochondrial derivative (Fig. Id). Moreover, at the age of 3 days, about 65% of PINKl mutants exhibited a downtumed wing phenotype with rigidity, and this percentage slightly increased thereafter (Fig. Ie). To confirm whether this wing phenotype is caused by loss of PINKl function, UAS-HA-PINKl transgenic lines were generated. When PINKl expression was induced using the hs-GAL4 driver in the PINKl mutant background, this wing phenotype was remarkably restored (Fig. Ie). The discovery of the downturned rigid wing phenotype in PINKl mutants led to a search for defects in locomotion. As expected, the flies showed complete defects in flight ability and slower climbing speed, and those phenotypes were all rescued by PINKl expression (Fig. If, g, respectively).

Another apparent phenotype was found in the thoraces of PINKl mutants right after eclosion (—85%), which were crushed particularly in the mid-anterior and antero-lateral regions (Fig. Ih, i), and the percentage of flies with this crushed thorax phenotype progressively increased over time (-95% at the age of 30 days, Fig. Ii). Cross thoracic sections of PINKl mutants showed abnormal structure and reduced content of IFM (Fig. 2a, first row). In the magnified sections of the mutants, disorganized muscle fibers and enlarged mitochondria were found with highly decreased staining intensity (Fig. 2a, second row). Consistently, examination of the IFM ultrastructure of PINKl mutants revealed irregular arrangement of myofibrils and immensely swollen mitochondria with loss of the outer membrane (Fig. 2b).

Given the reduced muscle content and mitochondrial impairment in the mutants, it was then investigated whether apoptosis is apparent in the muscles of PINKl mutants by performing TUNEL assay. TUNEL signals were indeed ubiquitiously detected in the IFM of PINKl mutants, but neither in the controls (Fig. 2c). These phenotypes shown in PINKl mutants were all rescued by PINKl expression (Fig. Ii, 2a, c). Collectively, these results demonstrate that loss of PINKl induces IFM degeneration and mitochondrial impairment. To further confirm mitochondrial impairment in the muscles (Fig.2b), the abundance of mitochondria in the mutants was quantified. A marked reduction in the level of mitochondrial DNA (mtDNA) and protein (mtProtein) was observed in PINKl mutants when compared to the controls (Fig. 2d), and those levels were almost completely restored when PINKl was expressed back in the mutants (Fig. 2d). ATP quantification assays were also conducted with the thoraces of the mutants to determine whether altered mitochondrial morphology and amount are correlated with its functional loss. Compared to the controls, PINKl mutants showed a more than 2-fold reduction in the ATP level, and this was also markedly restored by PINKl expression (Fig. 2e).

Because substantial evidence indicated that anti-apoptotic Bcl-2 families are involved in the protection of mitochondrial integrity and function⁵'⁶ it was believed that BcI- 2 expression may suppress the mitochondrial dysfunction and other apparent phenotypes of PINKl mutants. As a result, a remarkable recovery of the level of mtDNA (Fig. 2d), mtProtein and ATP (Fig. 2e) was observed by Buffy, a sole Drosophila Bcl-2 homolog⁷, expression under the hs-GAL4 driver, while its expression in the wildtype background did not affect the mitochondria. Consistently, all of the PINKl mutant phenotypes except for the flight defects were markedly restored by Buffy expression (Fig. 2a, f). Overall, these results strongly suggest that mitochondrial dysfunction is the main cause for those aberrant phenotypes of PINKl mutants.

Furthermore, because DA neurodegeneration is one of the major characteristics shown in AR-JP patients², it was examined whether DA neurons in the adult brains of the mutants are also impaired. Using the method of immunostaining with tyrosine hydroxylase (TH) antibody, the number of DA neurons was counted in a blind fashion. As a result, the number of DA neurons within each of the major clusters including dorsomedial (DM), dorsolateral (DL) 1 and 2, and posteriomedial (PM) clusters of PINKl mutants was not changed at 3 days (data not shown). However, at 30 days, the mutants exhibited a small but significant decrease (-10%) in the number of DA neurons in the DM and DLl regions of the mutants compared to the wildtype and revertants (Fig. 3a, b). This was further confirmed by using another method of expressing nuclear-lacZ in DA neurons. In addition to this neuronal loss at 30 days, the mutants exhibited a markedly decreased anti-TH staining intensity in their DA neurons (Fig. 3a), which was consistent with their reduced level of dopamine (Fig. 3 c). Then, to examine whether the cause of this phenotype is correlated with mitochondrial impairment, TH>mitoGFP lines were generated to express mitochondria- targeted GFP in DA neurons. In PINKl mutants, a number of mitochondria were found profoundly enlarged in DA neurons in all of the clusters, most severe in DLl (Fig. 3d, e), and the mitochondrial size progressively increased with age (data not shown). Consistent with this result, TEM analysis also revealed grossly enlarged mitochondria in DA neurons of the mutants (Fig. 3f). In addition, enlarged mitochondria were observed in other neurons such as surrounding cell bodies of DA neurons in posterior protocerebrum and neuropils and serotonergic neurons but not in photoreceptor cells, insulin-secreting cells and circadian pacemaker cells. These results indicate that PINKl is a critical factor required in DA neurons for maintaining mitochondrial integrity as well as neuronal function.

Surprisingly, it was noticed that all of the phenotypes of PINKl mutants were highly reminiscent of parkin mutants generated in the art^8"11 (e.g., abnormally positioned wings crushed thoraces (Fig. Ih, i), disorganized muscle fibers with enlarged mitochondria (Fig. 4i), muscle cell apoptosis (Fig. 4i), impaired flight ability and highly reduced climbing rate complete male sterility due to defective Nebenkern (Fig. Id) and defects in DA neurons in two particular clusters, DLl 10 and DM⁸'⁹). Therefore, any possible interactions between these two genes was tested by first expressing Parkin in PINKl mutants. As a result, the crushed-thorax and drooped- wing phenotypes of PINKl mutants were dramatically restored by Parkin expression (Fig. 4a). Muscle sections showed intact structure of muscles and mitochondria in Parkin-expressed PINKl mutants (Fig. 4b). Moreover, the mtDNA and mtProtein contents as well as the ATP level in the IFM were markedly rescued by Parkin expression. Consistent with these findings, TUNEL signals in the IFM were almost completely disappeared (Fig. 4b). In addition, the loss of DA neurons in the DM and DLl regions of the PINKl mutants was significantly restored by Parkin expression (Fig. 4e), and the enlarged mitochondria in those neurons within the DM, DLl, and DL2 clusters were also remarkably rescued (Fig. 4d). Furthermore, the rescued flies were able to climb up faster and even fly (Fig. 4f, g, respectively). These results indicate that Parkin can compensate for the mitochondrial dysfunction produced by loss of PINKl in both muscles and DA neurons and the consequent defective phenotypes.

Furthermore, it was found that Parkin could not block the apoptosis induced by well- known cell death molecules suggesting that the suppression of PINKl loss-of-function phenotypes by Parkin expression is not resulted from its general protective role against apoptotic insults but rather resulted from its specific protective role against mitochondrial dysfunction induced by loss of PINKl.

However, genetic analysis vice versa showed that the apparent phenotypes as well as the mitochondrial morphology and apoptosis in parkin mutants could not be recovered by increased PINKl expression (Fig. 4h, I). These results indicate that Parkin acts downstream of PINKl to maintain mitochondrial integrity and function. Moreover, in PINKl and parkin double mutants it was found that they do not show further severity in the phenotypes (Fig. 4i) compared to the either single mutants, indicating that PINKl and Parkin indeed converge in a common genetic pathway. Furthermore, mitochondrial localization of both of these molecules supports our idea of their functional interaction in that organelle.

It has been previously demonstrated that inactivation of the JNK pathway prevents impaired morphology and decreased staining intensity of DA neurons exhibited in parkin mutants⁸. Together with the knowledge of the in vivo interaction between PINKl and Parkin, it was hypothesized that the JNK pathway may be activated in the IFM of PINKl mutants. Indeed, using puc (a JNK target gene¹²)-lacZ line, strong ectopic puc expressions was found in the muscles of the PINKl mutants, but not in the wildtype. Furthermore, hep (a Drosophila MKK7 homologl3) and PINKl double mutants showed significant restoration of the apparent phenotypes and markedly reduced apoptosis, while the mitochondria remain swelled. Collectively, these data suggest that the apoptosis induced by mitochondrial impairment resulted from PINKl ablation is mediated by ectopic activation of the JNK pathway.

The present invention provides genetic evidence that PINKl plays a key role in mitochondria, and its dysfunction contributes to the degeneration of high-energy demanding cells including DA neurons, muscles, as well as sperms. However, there appeared to be some differences in vulnerability to mitochondria impairment among the tissues and DA neuron clusters, which could be explained by the different threshold levels for mitochondrial dysfunction-induced cell death among the types of tissues and cells.

Intriguingly, all of the phenotypes shown in PINKl mutants were strikingly similar to those of parkin mutants. The results from the rescue experiment with parkin transgene provide definitive evidence for the functional interaction between PINKl and Parkin, and this indicates that Parkin also has a crucial role in maintaining mitochondrial integrity and function. Moreover, through epistatic analysis, Parkin was found to act downstream of PINKl. This novel and important finding of the convergence of Parkin and PINKl in a pathway involved in the protection of mitochondrial integrity provides invaluable clues for understanding the central pathogenic mechanism of AR-JP and related diseases. Taken together, the methods, systems, and compositions of the present invention are useful to investigate how PINKl and Parkin protect mitochondrial integrity and will allow identification of the upstream and downstream molecules involved in this pathway. The present invention will allow the development of better and effective treatment strategies for AR-JP and other forms of PD, which should, for example, be aimed specifically against mitochondrial dysfunction.

The present invention provides transgenic animals, and cells, having somatic and/or germ cells in which at least one allele of an endogenous PINKl gene is functionally disrupted (e.g., the animals have PINKl deficient cells). The present invention also provides drug-screening assays employing PINKl deficient cells and animals.

Transgenic Animals lacking functional PlNKl Genes and Homologs, Mutants, and Variants Thereof

Preferred embodiments of the present invention are illustrated below using the example of a Drosophilα model for mutant PINKl. It is understood that other non-human animals may be generated using methods known in the art. PINKl target sequences for disruption from various non-human animals (e.g., mice, rats) are readily identified from public sequence databases. For example, the nucleic acid sequence for Mus musculus is accession number NM_026880, which is herein incorporated by reference.

The animal may be heterozygous or, more preferably, homozygous for the PINKl gene disruption. As used herein, the term "gene disruption" refers to any genetic alteration that prevents normal production of PINKl protein (e.g., prevents expression of a PlNKl gene product, expression of normal PINKl gene product, or prevents expression of normal amounts of the PINKl gene product). In some embodiments, the gene disruption comprises a deletion of all or a portion of the PINKl gene. In other embodiments, the gene disruption comprises an insertion or other mutation of the PINKl gene. In still other embodiments, the gene disruption is a genetic alteration that prevents expression, processing, or translation of the PINKl gene. In certain embodiments, both PINKl gene alleles are functionally disrupted such that expression of the PINKl gene product is substantially reduced or absent in cells of the animal. The term "substantially reduced or absent" is intended to mean that essentially undetectable amounts of normal PINKl gene product are produced in cells of the animal. This type of mutation is also referred to as a "null mutation" and an animal carrying such a null mutation is also referred to as a "knockout animal." In preferred embodiments, the transgenic animals display a Parkinson's disease phenotype similar to that observed in humans.

A preferred embodiment of the present invention is based upon a non-mammalian animal model for PD in the fruit fly Drosophila melanogaster (hereafter referred to as Drosophila). One of the most profound and surprising biological discoveries in the last two decades is that most animals across the animal kingdom, including humans, possess many of the same genes that function in similar ways in cells, tissues and organs. In fact, only 94 of an estimated 1,278 human gene families are vertebrate-specific. Furthermore, at least 77% of known human disease genes have at least one counterpart within the genome of Drosophila, a model organism and workhorse in the study of genetics (Reiter et al., Gen. Res. 111:1114 (2001)). Many genes implicated in human diseases, including signaling pathways and effectors of tissue- and cell-specification, were originally identified and characterized in the fruit fly. Thus, genes within most human disease-associated networks are present in the fruit fly genome and have comparable roles in fly biology.

Utilizing transgenic animals for genetic screens

In some embodiments, the PINKl animals of the present invention are crossed with other transgenic models or other stains of animals to generate Fl hybrids for additional disease models. In another embodiment, a disease condition is induced by breeding an animal of the invention with another animal genetically prone to a particular disease. For example, in some embodiments, the PINKl animal is crossed with knockout animals models of other genes associated with PD or related conditions.

In some embodiments, the PINKl animals are used to generate animals with an active PINKl gene from another species (a "heterologous" PINKl gene). In preferred embodiments, the gene from another species is a human gene. In some embodiments, the human gene is transiently expressed. In other embodiments, the human gene is stably expressed (e.g., the PINKl null animals are used to generate animals that are transgenic for human PINKl). Such animals find use to identify agents that restore the expression of parkin.

Identification of Binding Partners and Genetic Assays In some embodiments, binding partners of PINKl amino acids are identified. In some embodiments, the PINKl nucleic acid or fragments thereof are used in fly two-hybrid screening assays and yeast two-hybrid screening assays. For example, in some embodiments, the nucleic acid sequences are subcloned into pGPT9 (Clontech, La Jolla, CA) to be used as a bait in a yeast-2-hybrid screen for protein-protein interaction of a human fetal kidney cDNA library (Fields and Song Nature 340:245, (1989); herein incorporated by reference). In other embodiments, phage display is used to identify binding partners (Parmley and Smith Gene 73 : 305, (1988); herein incorporated by reference). Binding partners identified by in vitro methods may be expressed (e.g., overexpressed) or regulated in the animals models of the present invention, in vivo, to identify biological effects in the context of the PINKl animal model.

Drug Screening in transgenic animals

The present invention provides methods and compositions for using transgenic animals as a target for screening drugs that can alter, for example, interaction between PINKl and binding partners (e.g., those identified using the above methods). Drugs or other agents (e.g., from compound libraries) are exposed to the transgenic animal model and changes in phenotypes or biological markers are observed or identified. For example, drugs are tested for the ability to improve neurological function or phenotypes associated with loss of neurological function.

The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone, which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckennann et al., J. Med. Chem. 37: 2678-85 (1994))); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are preferred for use with peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, Proc. Natl. Acad. Sci. U.S.A. 90:6909 (1993); Erb et al, Proc. Nad. Acad. Sci. USA 91 :11422 (1994); Zuckermann et al, J. Med. Chem. 37:2678 (1994); Cho et al, Science 261:1303 (1993); Carrell et al, Angew. Chem. Int. Ed. Engl. 33.2059 (1994); Carell et al, Angew. Chem. Int. Ed. Engl. 33:2061 (1994); and Gallop et al, J. Med. Chem. 37:1233 (1994).

Therapeutic Agents

The present invention further provides agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein (e.g., PINKl transgenic knockout animal, hybrid of a PINKl transgenic knockout animal, progeny of PINKl transgenic knockout animal, neuronal modulating agent or PINKl mimetic, etc.) in an appropriate animal model (such as those described herein) to determine the efficacy, toxicity, side effects, or mechanism of action, of treatment with such an agent. Furthermore, agents identified by the above- described screening assays can be used for treatments of neurologically related disease (e.g., including, but not limited to, Parkinson's disease).

EXPERIMENTAL

The following example is provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

EXAMPLE

This example describes the materials and methods used for the results reported above in the Detailed Description and Figures 1-4.

Fly stocks

From the GenExel (GenExel-Sein, Inc., South Korea) library, a P-element insertion line (PINK1^GE271) was isolated in the second exon of the gene (Fig. Ia). PINKl⁰³ and PINKl alleles were generated through imprecise excision of the P-element of PINKl and found to be loss-of- function mutants for PINKl (Fig. Ib, c). Also generated was a revertant allele (PINK1^RV) by precise excision (but has 40bp of P-element), which showed almost the same amount of transcripts and proteins as to that of the wildtype (Fig. Ib, c). The generation of park! mutants and IJAS-parkin has been previously described⁸. The UAS-Buffy fly line was a generous gift from Dr. H. Richardson (Peter MacCallum Cancer Centre, Australia)⁷ and the UAS-mitoGFP line from Dr. H. J. Bellen (Baylor College of Medicine)¹⁴ (generated by Drs. A. Pilling and W. Saxton (Indiana University)). The TH- GAL4 fly line was a gift from Dr. S. Birman (CNRS-INSERM-Universite de Ia Mediterranee, France)¹⁵. The hs-GPΛΛ line was obtained from the Bloomington Stock Center (Bloomington, IN).

Northern blotting, mtDNA-PCR and immunoblot analysis

Northern blotting was conducted as previously described¹⁶ with purified mRNA using 504bp fragment of PINKl ORF (Fig. Ia) as a probe. For mtDNA-PCR, total DNA of thoraces of 2-day-old flies was extracted and subjected to PCR. The genomic DNA level of rp49 of each sample was also examined by PCR and used as a loading control. Results are expressed as fold change relative to the control. Immunoblot analysis was conducted as previously described¹⁷ with the thoraces of 2-day-old flies. Mouse monoclonal anti- cytochrome c oxidase subunit III (Co III, yeast) antibody (Molecular Probes), anti-β-tubulin (E7) mouse antibody (DSHB) and rabbit anti-PINKl antibody were used at 1 : 1,000 dilutions. The polyclonal antibody to Drosophila PINKl was generated in rabbit by injecting glutathione-S-transferase (GST) fused PINKl (amino acids 480-960) and further purified.

Immunostaining and TUNEL assay

Adult brain and testis were fixed with 4% paraformaldehyde and blocked in TBST with 2% BSA. The primary antibodies used in this study: anti-TH rabbit antibody (1 :50, Pel-Freez) and anti-cc -tubulin mouse antibody (1 :100, DSHB, University of Iowa). Hoechst33258 (Sigma) was used to visualize the nucleus of sperms. For adult brains, Z- series images (images for x200 were sectioned at 1 μm intervals and x 1,000 or χ3,000 at 0.3 μm) were obtained by LSM510 confocal microscope. For TUNEL assay, apoptosis in the thoraces of 2-day-old flies was detected using the in situ cell death detection kit (Roche).

Muscle section and TEM

Muscle sections were carried out as previously described¹ ', but with some modifications. The samples embedded in Spurr's resin were trimmed and sectioned from the lateral side of the thorax (at a thickness of 5 μm between 150 μm and 300 μm in depth), and the serial sections were then stained with toluidine blue dye. About ten thoraces of 2- day-old flies were observed for each genotype. The sections were observed at two magnifications (xlOO and x 1,000) of light microscope (Leica). For TEM analysis, samples were prepared as previously described⁹.

ATP assay

Five thoraces of 2-day-old flies were dissected and homogenized in 100 μl of 6 M guanidine-HCl in Extraction buffer (100 raM Tris and 4 mM EDTA, pH 7.8) to inhibit ATPases¹⁸, followed by fast-freezing in liquid nitrogen and boiling for 3 min. The samples were then centrifuged to collect the supernatant, which was then 1/750 diluted with Extraction buffer and mixed with luminescent solution (Enliten kit, Promega). The luminescence was measured by a luminometer (Berthold Technologies, USA), and the results were compared to the standards. The relative ATP level was then calculated by dividing the luminescence by the total protein concentration, which was determined by Bradford method. For Bradford assay, samples were 1/30 diluted with Extraction buffer. Average ± SD is from n=3 experiments.

Dopamine enzyme immunoassay (EIA)

Dopamine EIA (LDN, Germany) was conducted according to the manufacturer's instruction, but fly samples were prepared as follows: Fifty fly heads per genotype were dissected and homogenized in PBS with Assay buffer (1 M HCl). Then, after adding extract buffer, they were incubated for 20 min and then were centrifuged at 13,000 rpm for 30 min. The supernatant was collected and assayed.

Behavioral assays

For climbing speed assay, groups often 3-day-old males were transferred into 18 cm-long vials and incubated for 1 hr at room temperature for environmental acclimation. After tapping the flies completely down to the bottom, their climbing time was marked at the 15 cm finish line when more than five flies have arrived. Five trials were performed for each group and repeated with four more different groups. The average climbing time ± SD was calculated for each genotype. Flight assay was performed as previously described¹¹ with 3-day-old males (n>100). Quantification of wing and thorax phenotypes

For quantification, % of defective thorax and wing phenotypes of 3-day-old males were measured (n>300).

Generation and characterization of PINKl transgenic flies

Drosophila PINKl EST (clone #GH06623) was obtained from DGRC. The entire open reading frame (ORP) was subcloned into N-terminally HA-tagged pUAST vector. This generated construct was subjected to DNA sequencing for validation and then microinjected into wlll8 embryos for generation of transgenic flies. Then, their expressions were checked by crossing with hs-GAlΛ and thereafter conducting immunoblot analysis with anti-HA antibody (data not shown) and also with our generated anti-PINKl rabbit antibody (Fig. Ic).

Characterization of PINKl mutants

The PINKl^Di allele has lost 379bp, a deletion containing most of the second and third exons, including ATG, and the PINKl⁸⁹ 570bp, from the second exon to the first fifty- five nucleotides of the fourth exon.

Male sterility in PINKl mutants

PINKl mutants did not contain matured sperms determined by linear morphology of nucleus stained by Hoechst33258 in the pouch called vas deferens in 3-day-old male testis. Due to sterility and its gene locus in X chromosome, males were the only mutant progenies obtainable. However, PINKl mutant female (generated by mating heterogenous PINKl⁸⁹ female with transgene rescued PINKl mutant male) was fertile.

Transgenic rescue experiments

All transgenic rescue experiments with the Λ_?-GAL4 driver were conducted at 25°C to minimize the transgene expression, which was high enough to rescue the phenotypes of PINKl mutants.

Muscle sections Thorax sections of the controls stained with toluidine blue show striated muscle fibers intactly attached together and their magnified sections show a clear vision of lines of dark blue mitochondrial spots between elongated muscle fiber bands in light blue color. The PINKl³⁹ mutants, however, show reduced muscle content and near-invisibility of mitochondria.

IFM TEM analysis

The revertants show electron-dense mitochondria intactly surrounded by regularly arranged myofibrils whereas PINK1^B9 mutants show grossly swollen mitochondria.

DA neuronal clusters

DM, dorsomedial; DL, dorsolateral; PM, posteromedial clusters.

Quantification analysis

All statistical analyses were performed using one-way ANOVA. For quantification of DA neurons, 20 brains of each genotype were observed by all experimenters in a blind fashion to eliminate bias. To quantify DA cells with enlarged mitochondria, we calculated the percentage of the number of DA cells with mitochondria larger than 2 μm in diameter over the total number of DA cells in each cluster from 10 brains of each genotype.

Mitochondrial genes used for the quantification ofmtDNA level

Co I and Co III: cytochrome c oxidase subunit I and III, respectively, Cyt B: cytochrome b.

Genotypes

Genotypes: WT (will 8); RV (PINK1^RVZY); D3 (PINKl⁰³ZY); B9 (PINKl⁸⁹ZY); hs>PINKl (UAS-PINKIZY; hs-GAlA/+); B9, hs>PINKl (PINKl⁸⁹, OAS-PINKIZY; hs- G AIAt+); park' (+IY;; park¹1 park¹); B9, hs>Buffy (PINKl⁸⁹ZY; hs-G AlAZU AS-Buβy); WT, TH>mitoGFP (+ZY;; TH-GAlA, UAS-mitoGFPZ+); B9, TH>mitoGFP (PINKl⁸⁹ZY;; TH- GAL4, UAS-mitoGFPZ+); B9, hs>parJdn (PINKl⁸⁹, UAS-parkinZY; hs-GAlAI+); B9, TH>parkin, TH>mitoGFP (PINKl⁸⁹, UAS-parkinZY;; TH-GAlA, UAS-mitoGFP/+);park^I, hs>PINKl (+ZY; hs-G AlAfU AS-PINKl; park¹ Ipark¹); B9, park¹ (PINKl⁸⁹ZY;; park¹ /park¹). Additional Fly stock Information

For validation of the PINKl mutants, PINKl RNAi lines were generated. The plasmid construct for this carries the C-terminal region of PINKl (l,801bp~end in ORP) in pSymp vector. \JAS-domi (a Drosophila homolog of HtrA2/omi) transgenic flies were generated by subcloning their cDNA into the pUAST vector, followed by microinjection and their expressions were confirmed by immunoblot analysis. The TPH- (serotonergic neuron-specific) GAL4 was generated by Dr. J. Kim (KAIST, Korea, unpublished). puc^E69, the puckered-lacZ reporter fly strain, was kindly provided by Dr. T. Adachi-Yamada (Kobe University, Japan)¹². The mef2-GALA fly line was kindly provided by Dr. E. N. Olson (University of Texas Southwestern Medical Center at Dallas)¹⁹. The hep' fly line was a gift from Dr. S. Noselli (CNRS-University of Nice Sophia-Antipolis, France)¹³. The dilp2- (insulin-secreting cell-specific) GAL4 fly line was a generous gift from Dr. E. J. Rulifson (Stanford University School of Medicine)²⁰. The OAS-p53 fly line was kindly provided by Dr. G. M. Rubin (University of California at Berkeley)²¹. The fly lines for UAS-/αcZ and 24B-, gmr-, pdf- (circadian pacemaker cell-specific) and da-GALA were obtained from the Bloomington Stock Center (Bloomington, IN).

Southern blotting and Quantitative PCR

For Southern blot analysis, 10 μg of total genomic DNA for each genotype was prepared by the conventional method, and was cut with EcoRI (KOSCHEM, Korea). 410bp (Supplementary Fig. SIf) fragment of PINKl ORF was used as a probe. For Real-Time PCR, total RNA was extracted by the Easy-Blue System (Intron, Korea), and reverse- transcribed using Oligo-dT Reverse Transcription Kit (Promega, WI). Then, PCR was performed using SYBR Premix Ex Taq (Takara) on iCycler iQ Multicolor Real-Time PCR Detection System (Bio-Rad). rp49 levels were measured for internal control. Results are expressed as fold change compared to the first indicated sample.

S2 and HEK 293T cell culture, transfection, and immunocytochemistry

The plasmid constructs for S2 cell transfection were generated by subcloning N- terminally HA-tagged PINKl cDNA into pHym vector. S2 cells were grown in M3 media (Sigma S8398) supplemented with 10% IMS (Sigma 17267) at 25°C and were transiently transfected by DDAB. The cells were preincubated with 5 μg/mL MitoTracker Red CMXRos (Molecular Probes) for 30 min at 25°C and then subjected to the standard immunocytochemistry. For HEK 293T transfection, PINKl cDNA ligated into pcDNA3- FLAG vector was tranfected with Lipofectamine Plus method (Invitrogen). Immunocytochemistry was performed with anti-rabbit DYKDDDDK (SEQ ID NO:1) Tag (FLAG) antibody (Cell Signalling) and anti-mouse monoclonal MTC02 (mitochondria- specific) antibody (Abeam).

Immunostaining andpuc-lacZ reporter assay

Adult brain, thorax muscle, and eye disc were fixed with 4% paraformaldehyde and blocked in TBST with 2% BSA. The primary antibodies used in this study: anti-HA monoclonal mouse antibody (1 :100, 6E2, Cell Signaling), anti-Myc monoclonal mouse antibody (1 : 100, 9El 0), anti-TH rabbit antibody (1 :50, Pel-Freez), anti-_/ff-gal mouse antibody (1:100, DSHB, University of Iowa), and anti-tryptophan hydroxylase sheep antibody (1 :100, Pel-Freez). Hoechst33258 (Sigma) was used to visualize the nucleus of muscles, and TRITC-labeled phalloidin (Sigma) to mark actin. Alexa 488-conjugated streptavidin (1 :100, Molecular Probes) was used to mark mitochondria in IFM. puc-lacZ reporter assay was conducted as previously described¹⁶.

Longevity and fertility assays

Longevity and fertility assays were performed as previously described²². n=300 for longevity assay and n=50 for fertility assay.

REFERENCES

1. Shen, J. & Cookson, M. R. Mitochondria and dopamine: new insights into recessive parkinsonism. Neuron 43, 301-304 (2004); herein incorporated by reference in its entirety.

2. Moore, D. J., West, A. B., Dawson, V. L. & Dawson, T. M. Molecular pathophysiology of Parkinson's disease. Annu. Rev. Neurosci. 28, 57-87 (2005); herein incorporated by reference in its entirety.

3. Unoki, M. & Nakamura Y. Growth-suppressive effects of BPOZ and EGR2, two genes involved in the PTEN signaling pathway. Oncogene 20, 4457-4465 (2001); herein incorporated by reference in its entirety. 4. Valente, E. M. et al. Hereditary early-onset Parkinson's disease caused by mutations in PINKl . Science 304, 1 158-1160 (2004); herein incorporated by reference in its entirety.

5. Vander Heiden, M. G., Chandel, N. S., Williamson, E. K., Schumacker, P. T. & Thompson, C. B. Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria. Cell 91, 627-637 (1997); herein incorporated by reference in its entirety.

6. Vander Heiden, M. G. & Thompson, C. B. Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis? Nat. Cell. Biol. 1, E209-E216 (1999); herein incorporated by reference in its entirety.

7. Quinn, L. et al. Buffy, a Drosophila Bcl-2 protein, has anti-apoptotic and cell cycle inhibitory functions. EMBO J. 22, 3568-3579 (2003); herein incorporated by reference in its entirety.

8. Cha, G.. H. et al. Parkin negatively regulates JNK pathway in the dopaminergic neurons of Drosophila. Proc. Natl. Acad. Sci. USA 102,10345-10350 (2005); herein incorporated by reference in its entirety.

9. Greene, J. C. et al. Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. Proc. Natl. Acad. Sci. USA 100, 4078-4083 (2003); herein incorporated by reference in its entirety.

10. Whitworth, A. J. et al. Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease. Proc. Natl. Acad. Sci. USA 102, 8024-8029 (2005); herein incorporated by reference in its entirety.

11. Pesah, Y. et al. Drosophila parkin mutants have decreased mass and cell size and increased sensitivity to oxygen radical stress. Development 131, 2183-2194 (2004); herein incorporated by reference in its entirety.

12. Adachi-Yamada, T., Fujimura-Kamada, K., Nishida, Y. & Matsumoto, K. Distortion of proximodistal information causes JNK-dependent apoptosis in Drosophila wing. Nature 400, 166-169 (1999); herein incorporated by reference in its entirety.

13. Glise, B., Bourbon, H. & Noselli, S. hemipterous encodes a novel Drosophila MAP kinase kinase, required for epithelial cell sheet movement. Cell 83, 451-461 (1995); herein incorporated by reference in its entirety.

14. Verstreken, P. et al. Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions. Neuron 47, 365-378. (2005); herein incorporated by reference in its entirety. 15. Friggi-Grelin, F. et al. Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase. J. Neurobiol. 54, 618-627 (2003); herein incorporated by reference in its entirety.

16. Lee, S. B., Park, J., Jung, J. U. & Chung, J. Nef induces apoptosis by activating JNK signaling pathway and inhibits NF-kappaB-dependent immune responses in Drosophila. J. Cell. Sci. 118, 1851-1859 (2005); herein incorporated by reference in its entirety.

17. Kim, S., Jee, K., Kim, D., Koh, H. & Chung, J. Cyclic AMP inhibits Akt activity by blocking the membrane localization of PDKl . J. Biol. Chem. 276, 12864-12870 (2001); herein incorporated by reference in its entirety.

18. Schwarze, S. R., Weindruch, R. & Aiken, J. M. Oxidative stress and aging reduce COX I RNA and cytochrome oxidase activity in Drosophila. Free Radic. Biol. Med. 25, 740-747 (1998); herein incorporated by reference in its entirety.

19. Ranganayakulu, G., Schulz, R. A. & Olson, E. N. Wingless signaling induces nautilus expression in the ventral mesoderm of the Drosophila embryo. Dev Biol 176, 143— 148 (1996); herein incorporated by reference in its entirety.

20. Rulifson, E. J., Kim, S. K. & Nusse, R. Ablation of insulin-producing neurons in flies: growth and diabetic phenotypes. Science 296, 1 118-1120 (2002); herein incorporated by reference in its entirety.

21. Brodsky, M. H. et al. Drosophila p53 binds a damage response element at the reaper locus. Cell 101, 103-113 (2000); herein incorporated by reference in its entirety.

22. Lee, J. H. et al. In vivo p53 function is indispensable for DNA damage- induced apoptotic signaling in Drosophila. FEBS Lett. 28, 5-10 (2003); herein incorporated by reference in its entirety.

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods, compositions, and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry and molecular biology or related fields are intended to be within the scope of the claims.

Claims

CLAIMSI claim:

1. A screening method compri sing: a) contacting a PINKl deficient cell with a candidate compound, and b) detecting: i) the presence or absence of parkin mRNA or protein up- regulation in said cell, or ii) the presence or absence of reduced mitochondrial related dysfunction in said cell.

2. The method of Claim 1, wherein said mitochondrial related dysfunction is evidenced by a phenotype selected from the group consisting of: reduced mitochondrial DNA expression; reduced mitochondrial protein expression; reduced ATP levels in said cell; irregular arrangement of myofibrils; enlarged mitochondria; reduced number of mitochondria organelles in said cell; a decreased anti- tyrosine hydroxylase staining intensity; and a reduced level of dopamine levels.

3. The method of Claim 1, wherein said detecting comprises comparison to a control PINKl deficient cell not contacted with said candidate compound.

4. The method of Claim 1 , wherein the presence of parkin protein up-regulation, or the presence of reduced mitochondrial related dysfunction in said cell identifies said candidate compound as a therapeutic compound for treating neurodegenerative disease.

5. The method of Claim 4, wherein said neurodegenerative disease is Parkinson's disease.

6. The method of Claim 4, wherein said neurodegenerative disease is autosomal recessive juvenile Parkinson's.

7. The method of Claim 1, wherein said cell is a DA neuron cell.

8. The method of Claim 1, wherein said detecting comprises quantitating the amount of parkin mRNA present in said cell.

9. The method of Claim 1, wherein said detecting comprises quantitating the amount of parking protein present in said cell.

10. A screening method comprising: a) contacting a PINKl deficient animal with a candidate compound, and b) detecting: i) the presence or absence of parkin protein or mRNA up- regulation in cells of said animal, or ii) the presence or absence of reduced mitochondrial related dysfunction in cells of said animal.

11. The method of Claim 10, wherein said mitochondrial related dysfunction is evidenced by a phenotype selected from the group consisting of: reduced mitochondrial DNA expression; reduced mitochondrial protein expression; reduced ATP levels in said cells; irregular arrangement of myofibrils; enlarged mitochondria; reduced number of mitochondria organelles in said cells; a decreased anti- tyrosine hydroxylase staining intensity; a reduced level of dopamine expression; muscle cell apoptosis; defects in DA neurons in either the DLl 10 or DM cluster.

12. The method of Claim 10, wherein said animal is Drosophila melanogaster, and said mitochondrial related dysfunction is evidenced by a phenotype selected from the group consisting of: abnormally positioned wings; a crushed thorax, impaired flight ability, reduced climbing rate; and complete male sterility due to defective Nebenkern.

13. The method of Claim 10, wherein said animal exhibits at least one neurodegenerative symptom similar to human Parkinson's disease.

14. The method of Claim 13, wherein said animal is Drosophila melanogaster.

15. The method of Claim 14, wherein said symptom is selected from the group consisting of: indirect flight muscle (IFM) degeneration; reduced climbing ability; complete male sterility due to impaired sperms with swelled Nebenkern; downturned wing phenotype with rigidity; reduced life-span; a defect in flight ability; a crushed looking thorax in the mid-anterior and/or antero-lateral regions; disorganized muscle fibers, enlarged mitochondria; irregular arrangement of myofibrils in the indirect flight muscle; swollen mitochondria with loss of the outer membrane in said indirect flight muscle; and apoptosis in muscles cells.

16. The method of Claim 10, wherein detecting comprises comparison to a control PINKl deficient animal not contacted with said candidate compound.

17. The method of Claim 10, wherein the presence of parkin protein up- regulation, or the presence of reduced mitochondrial related dysfunction in the cells of said animal identifies said candidate compound as a therapeutic compound for treating neurodegenerative disease.

18. The method of Claim 17, wherein said neurodegenerative disease is parkinson's disease.

19. The method of Claim 17, wherein said neurodegenerative disease is autosomal recessive juvenile Parkinson's.

20. A method of treatment comprising; contacting a cell that is PINKl deficient with an agent that upregulates parkin expression or that reduces mitochondrial related dysfunction in said cell.

21. The method of Claim 20, wherein said agent comprises a vector, wherein said vector is configured to cause the expression of PINKl in said cell.

22. The method of Claim 20, wherein said agent comprises a vector, wherein said vector is configured to cause the expression if Bcl-2 in said cell.

23. The method of Claim 20, wherein said agent comprises a vector, wherein said vector is configured to cause the expression of parkin in said cell.

24. The method of Claim 20, wherein mitochondrial related dysfunction has a phenotype selected from the group consisting of: reduced mitochondrial DNA expression; reduced mitochondrial protein expression; reduced ATP levels in said cell; irregular arrangement of myofibrils; enlarged mitochondria; reduced number of mitochondria organelles in said cell; a decreased anti- tyrosine hydroxylase staining intensity, and a reduced level of dopamine expression.

25. The method of Claim 20, wherein said cell is a DA neuron cell.

26. A method of treatment comprising; administering to an animal that is PINKl deficient an agent that upregulates parkin expression or that reduces mitochondrial related dysfunction in cells of said animal.

27. The method of Claim 26, wherein said agent comprises a vector, wherein said vector is configured to cause the expression of PINKl in cells of said animal.

28. The method of Claim 26, wherein said agent comprises a vector, wherein said vector is configured to cause the expression of Bcl-2 in cells of said animal.

29. The method of Claim 26, wherein said agent comprises a vector, wherein said vector is configured to cause the expression of parkin in cells of said animal.

30. A transgenic animal whose genome comprises a gene disruption in its endogenous PINKl gene, and wherein at least some of the cells of said animal exhibit a mitochondrial related dysfunction phenotype.

31. The animal of Claim 30, wherein said mitochondrial related dysfunction phenotype is selected from the group consisting of: reduced mitochondrial DNA expression; reduced mitochondrial protein expression; reduced ATP levels in said cells; irregular arrangement of myofibrils; enlarged mitochondria; reduced number of mitochondria organelles in said cells; a decreased anti- tyrosine hydroxylase staining intensity; a reduced level of dopamine expression; muscle cell apoptosis; defects in DA neurons in either the DLl 10 or DM cluster.

32. The animal of Claim 30, wherein said PINKl disruption reduces or eliminates the expression of a functional Parkin protein.

33. The animal of Claim 30, wherein said animal is Drosophila melanogaster.

34. A transgenic animal whose genome comprises a gene disruption in its endogenous PINKl gene, and wherein said animal exhibits at least one neurodegenerative symptom similar to human Parkinson's disease.

35. The animal of Claim 34, wherein said symptom is selected from the group consisting of: indirect flight muscle (IFM) degeneration; reduced climbing ability; complete male sterility due to impaired sperms with swelled Nebenkern; downturned wing phenotype with rigidity; reduced life-span; a defect in flight ability, a crushed looking thorax in the mid-anterior and/or antero-lateral regions; disorganized muscle fibers, enlarged mitochondria, or highly decreased staining intensity; irregular arrangement of myofibrils in the indirect flight muscle, swollen mitochondria in said indirect flight muscle; and apoptosis in the muscle cells.

36. A composition comprising a medicament that mimics the activity of PINKl in vivo and causes increased parkin expression in PINKl deficient subjects.