WO1995006062A1 - Novel proteins isolated from nerve cells, dna sequences encoding same and usages thereof - Google Patents

Abstract

The present invention is based on the identification of four novel protein tyrosine kinases (PTKs) isolated from the postsynaptic density (PSD). These PTKs, whose molecular weights were approximately 166, 90, 66, and 50 kDa, as shown in the figure, were found to be resistant to Genistein, did not crossreact with antibodies which bind to other PTKs known in the art. The present invention further discloses previously unidentified proteins, isolated from the PSD, which have molecular weights of approximately 110, 120 and 270 kDa. These proteins were found to crossreact with an antibody which is selective for dystrophin.

Description

NOVEL PROTEINS ISOLATED FROM NERVE CELLS, DNA SEQUENCES ENCODING SAME AND USAGES

THEREOF

This application is a continuation of U.S. Serial No. 08/188,422, filed January 24, 1994, which is a continuation-in-part of U.S. Serial No.

08/110,501, filed August 23, 1993, the contents of both of which are incorporated herein by reference.

Field of the Invention

The present invention relates to the field of neurobiology . Specifically, the present invention discloses novel proteins isolated from nerve cells, DNA sequences encoding novel neuronal proteins, and usages of the novel proteins and DNA sequences for the design of useful pharmaceutical compounds and identifying critical regions in the brain.

Background of the Invention PROTEIN KINASES

During the past decade, protein tyrosine kinases (PTKs) have been intensively studied because of their possible involvement in many important cellular processes (for review see Cantley et al., (1991)). It is generally accepted that protein phosphorylation at tyrosine group is associated with cellular growth and differentiation in the nervous system. Nonetheless, recent findings showing the presence of high level of PTKs activity in adult rat brain and their involvement in LTP suggest additional roles of these kinases in brain function, such as signal transduction. Previous studies indicate that PTKs could be classified in two classes: (i) receptor tyrosine kinases that transduce signals from growth factor and neurotrophins, and (ii) nonreceptor tyrosine kinases that are associated with and could be activated by various transmembrane signaling molecules. A number of PTKs, including c-yes, c- fyn, c-abl, trkB, c-src and its neuron specific homologue c-sre⁺, has been identified in nervous system (Sudol et al, Oncogene Res. 2:345-355 (1988), Neillette et al, Oncogenes, Benz et al. eds. pp. 121-142, Kluwer Academic Publishers, Νorwell, Mass. (1989); Letwin et /., Oncogene 5:621-627 (1988) and Klein et al, EMBO J. 8:3701-3709 (1989)). In addition to PTKs, ΝMDARl has also become one of the loci of research interests recently because of its possible involvement in induction of LTP.

Despite their potential roles in CΝS, little is known about the identity or the exact function of specific PTKs in the postsynaptic density (PSD) even though growing evidence suggest that the PSD, a disc-shaped proteinaceous structure attached to the inner surface of postsynaptic membrane of chemical synapses, is crucial to synaptic function (Siekevitz, Proc. Natl. Acad. Sci. USA 84:8687-8691 (1987)).

DYSTROPHIN

Duchenne muscular dystrophy (DMD), which is characterized by a progressive muscle degeneration, is a fatal X-linked inherited disease affecting one in 3500 boys (for review see Hoffman and Kunkel, Neuron 2:1019-1029 (1989) and Ahn and Kunkel, Nature Genetics 3:283-291 (1993)). A significant number of DMD patients also suffers from moderate, non- progressive cognitive impairment of unknown pathology (Rosman and Kakulas, Brain 89:769-788 (1966); Moser, H. , Hum. Genet. 66: 17-40 (1984);

Emery, A.E.H., Duchenne Muscular Dystrophy, Oxford University Press, Oxford, pp. 99-103 (1988)). Dystrophin is defective in DMD patients and the protein is encoded by a 14-kilobase (kb) mRΝA transcribed from the giant DMD gene, which spans more than 2.5 milling base pairs (Koenig et al. , Cell 50:509-517 (1987), Hoffman et al, Cell 57:919-928 (1987); Bonilla et al,

Cell 54:447-452 (1988)). Dystrophin is expressed as 427 kilodalton (kDa) polypeptide predominantly in skeletal and a cardiac muscle, where it has been localized in sarcolemma membrane (Arahata et al, Nature (Lond.) 333:861- 863 (1988); Bonilla et al, Cell 54:447-452 (1988), Zubrycka Gaarn et al, Nature (Lond). 333:466-469 (1988); Byers et al, J. Cell. Biol. 775:411-421 (1991)). The 427-kDa dystrophin is a membrane cytoskeletal protein which consists of four distinct domains based on deduced amino acid sequence; a N- terminal actin binding domain, a spectrin-like rod domain with repetitive triple-helix structures, a cysteine-rich domain showing homology with the calcium-binding domain of α-actinin from Dictyostelium, and a unique, most conserved C-terminal domain (Hammonds, Jr. , Cell 57: 1 (1987); Davision and Critchley, Cell 52:159-160 (1988), Koenig et al , Cell 53:219-228 (1988); Byers et al, J. Cell Biol. 709:1633-1641 (1989)). A brain specific 427-kDa dystrophin is transcribed from an alternate promoter, which is located upstream from the muscle-type dystrophin promoter, generating a different N- terminal end (Chelly et al , Nature (Lond) 344:64-65 (1990); Boyce et al , Proc. Natl. Acad. Sci. USA 88:1276-1280 (1991)). Brain is the only non- muscle tissue which express significant level of 427 kDa dystrophin. In the brain, dystrophin is expressed in the postsynaptic region of selective neuronal populations (Lidov et al, Nature (London) 348:725-728 (1990)).

Recent studies indicated that the major mRNA isoforms from the DMD gene in brain and other non-muscle tissues thus far identified are generated from the distal promoter and are alternatively spliced (Feener et al. , Nature (Lond) 338:509-511 (1989); Geng et al 1991; Hugnot et al, Proc. Natl. Acad. Sci. USA 89:7506-7510 (1992), Lederfien et al. , Proc. Natl. Acad. Sci.

USA 89:5346-5350 (1992); Bies et al, Nucleic Acids Res. 20:1725-1731 (1992); Byers etal, Nature Genetics 4:77-81 (1993)). One of these isoforms, so called Dpi 16, has been well characterized and exhibit peripheral nerve- specific expression (Byers et al, Nature Genetics 4:77-81 (1993)). Multiple mRNAs generated from the C-terminal region of dystrophin gene were known to be the most abundant dystrophin transcript but protein products of these transcripts have never been identified in brain (Lederfien et al. , Proc. Natl. Acad. Sci. USA 89:5346-5350 (1992)). Predicted protein products generated from these spliced variants indicated that these dystrophin isoforms have very similar molecular weights (Lederfein et al , Proc. Natl Acad. Sci. USA

89:5346-5350 (1992); Bies et al, Nucleic Acid Res. 20:1725-1731 (1992)). To begin examining potential role of these multiple dystrophin isoforms in brain, two different antibodies against C-termim of dystrophin were used to determine subcellular localization and expression in 427 kDa dystrophin- deficient mdx and DMD brains. We focused on the PSD, a proteinaceous, disc-shaped subcellular structure located in the inner surface of the postsynaptic neuron in mammalian synapses. The PSD appears to be central to synaptic function (Siekevitz, Proc. Natl Acad. Sci. USA 82:3494-3498 (1985); Wu and Black, Proc. Natl. Acad. Sci. USA 84:8687-8691 (1987); Wu and Black, Proc. Natl Acad. Sci. USA 85:6207-6210 (1988); Wu and Black, J. Cognitive Neurosci. 7:194-200 (1989); Lisman and Goldring, Proc. Natl.

Acad. Sci. USA 85:5320-25324 (1988)).

We have previously shown that the 427-kDa brain dystrophin is a component of the isolated postsynaptic density (PSD) fraction and this molecule is absent in the mdx mouse and human DMD patient (Kim et al , Proc. Natl Acad. Sci. USA 89: 11642-11644 (1992); Kim et al., Deficiency of

Synaptic Dystrophin in Human Duchenne Muscular Dystrophy Brain, submitted for publication). We now report that the multiple dystrophin isoforms are colocalized with the 427 kDa brain dystrophin in the PSD of rat and human cerebral cortex. These proteins were differently regulated during cortical synaptic development and were expressed normally in the mdx mouse and human DMD cerebral cortex. Our studies suggest that these brain dystrophin isoforms also participate in synaptic functions as the 427 kDa dystrophin, raising the possibility of their roles in functional compensation in dystrophin deficiency of DMD brain. SUMMARY OF THE INVENTION

The present invention is based on the identification, purification, and characterization, of novel proteins selectively produced by nerve cells, novel DNA sequences expressed in nerve cells, and methods of using these novel protein and DNA compositions.

In one aspect of the present invention, the identification, purification, and characterization of four novel PTKs isolated from the PSD is disclosed. These PTKs are expressed within the rat brain and have been shown to have human homologues (Example 1). In detail, using enriched samples of PSDs, novel PTK of approximate molecular weights of 166, 90, 66, and 50 kDa have been identified, isolated, and characterized. Unlike most other PTKs, the PTKs of the present invention are not sensitive to Genistein (Akiyama et al, Methods in Enzymology 207:362-370 (1991). In addition, the PTKs of the present invention were found not to cross-react, at least at any significantly detectable level, with the anti-

PTK antibodies; anti-yes, anti-blk, anti-lyn, anti-hck, anti-fyn2, anti-lck, anti- fgr and anti-src Mabs which are known in the art. Our studies revealed that the only antibody which has shown any degree of cross reactivity is the anti- fyn antibody which cross reacts (weakly) with the 166 kDa PTK of the present invention.

Therefore, in one embodiment, the present invention provides a substantially pure PTK, isolated from nerve cells, or enriched PSDs, which has a molecular weight selected from the group consisting of approximately 166, 90, 66, and 50 kDa. These PTKs are insensitive to Genistein and do not cross-react with any significant degree with many known anti-PTK antibodies.

The isolation of the first members of this class of PTKs, which are preferentially expressed in the PSD, now enables a screening assay which can be used in the identification of agonists (PTK simulators) and antagonists (PTK blockers) of the PTKs of the present invention, and human homologues thereof. The present invention further provides methods for assaying for the expression of one or more of the PTKs of the present invention using polyacrylamide gel electrophoresis (see Example 1). In general, such methods comprise isolating a protein fraction from an isolated population of cells and then resolving the isolated proteins, for example via PAGE, to determine if there is a PTK present in the sample that has a molecular weight selected from the group consisting of 166, 90, 66, 50 kDa.

Since antibodies can now readily be generated which selectively bind one or more of the PTKs of the present invention, or a human homologue thereof, the present invention further provides methods for assaying for the expression of one or more of the PTKs of the present invention, or a human homologue thereof, using antibody based detection systems. In general, these methods comprise incubating tissue, a cell, or cellular extract with one or more antibodies which bind one or more of the PTKs of the present invention, or a human homologue thereof, and then determining whether the antibody binds the tissue, cell, or cellular extract.

In another aspect of the present invention, novel 110, 120 and 270 kDa dystrophin cross-reactive proteins are described (hereinafter the 110, 120 and 270-DMD proteins, respectively, see Examples 2 and 3). These proteins were found to be present in brain tissue isolated from normal adults but was almost completely absent in brain tissue isolated from patients with Duchenne Muscular Dystrophy (DMD). Two of these DMD proteins, the 110 and 120 DMD proteins, have been found in normal, and at lower levels in DMD brain tissue and are described elsewhere (Kim et al., Proc. Natl. Acad. Sci. USA 89:11642-11644 (Dec 1992) and in Example 2).

In detail, using enriched samples of PSDs, novel 110, 120 and 270 kDa proteins which cross-reacts with anti-427 dystrophin antibodies have been identified, isolated, and characterized. Therefore, in one embodiment, the present invention provides substantially pure 110, 120 and 270-DMD proteins, isolated from nerve cells, or enriched PSDs, which have a molecular weight of approximately 110, 120 and 270 kDa, respectively. The DMD proteins of the present invention can be used in a variety of fashions. For example, the DMD proteins can be used to generate protein specific antibodies. These antibodies can be used; (1) to assay for the temporal and tissue specific expression of the DMD proteins, (2) as an affinity matrix to isolate additional DMD protein, and (3) to screen for DNA sequences encoding the 110, 120 and DMD protein via expression cloning.

The present invention further provides methods for assaying for the expression of the DMD proteins of the present invention using polyacrylamide gel electrophoresis (see Examples 2 and 3). In general, these methods comprise isolating a protein fraction from an isolated population of cells and then resolving the isolated proteins, for example via PAGE, to determine if there are DMD proteins present in the sample with a molecular weight of approximately 110, 120 and 270 kDa.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Detection of autophosphorylated PTKs in the PSD.

Figure 2. Localization, regional expression, and enrichment of dystrophin and dystrophin-related proteins in the PSD. (A) Western blot analysis of dystrophin in PSDs isolated from cerebral cortex (CTX), olfactory bulb (Olf-B), and cerebellum (CBL). Anti-dystrophin antibody (anti 6-10) specifically detected three distinct proteins of about 400 kDa (arrow 1)

(dystrophin), 120 kDa (arrow 2) and 110 kDa (arrow 3), dystrophin-related proteins. The three proteins exhibited differential regional expression. Molecular size markers are at left. (B) Western blot analysis of dystrophin in total tissue homogenate (H), SM, and PSD. In the above three brain regions examined, dystrophin and dystrophin-related proteins were expressed to a much greater extent in the PSD than in either H or SM.

Figure 3 (Panels a and b). Differential expression of dystrophin and dystrophin related proteins in the PSD during development of the cerebral cortex. (A) Western blot analysis of dystrophin in PSD fractions isolated from cerebral cortex on postnatal days 3,5,7,10,14,28, and 60. Dystrophin and dystrophin-related proteins are designated by arrows 1-3 as in Fig. 2. (B) Densitometric scan of the immunoblotted 400-kDa dystrophin bands from A. Results are expressed in arbitrary units. Figure 4. Expression of brain dystrophin in normal rat (lane 1), normal mouse (lane 2), and mdx mouse (lane 3). Dystrophin and dystrophin- related proteins are designated by arrows 1-3 as in Figs. 2 and 3. While the 400-kDa brain dystrophin was absent in the mdx mouse (lane 3), the 110- and 120-kDa proteins were still present but were reduced. Normal rat (lane 1) and mouse (lane 2) brains contained the three proteins specifically detected by anti 6-10.

Figure 5. Expression of the 427 kDa dystrophin and the related 270 kDa dystrophin related antigen in normal and DMD brain.

Figure 6 (Panels a and b). Western blot of postmortem degradation of the rat 427 kDa dystrophin.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is based on the identification, purification, and characterization, of novel proteins produced by nerve cells, novel DNA sequences expressed in subpopulations of nerve cells, and methods of using these novel protein and DNA compositions.

In one aspect of the present invention, the identification, purification, and characterization of four novel PTKs, isolated from the PSD is disclosed. These PTKs have human homologues (Example 1).

In detail, using enriched samples of PSDs, novel protein tyrosine kinases of approximate molecular weights of 166, 90, 66, and 50 kDa have been identified, isolated, and characterized. Unlike most other PTKs, the PTKs of the present invention are not sensitive to Genistein (Akiyama et al, Methods in Enzymology 207:362-370 (1991)). In addition, the PTKs of the present invention were found not to cross-react, at least at any significantly detectable level, with the anti-PTK antibodies; anti-yes, anti-blk, anti-lyn, anti- hck, anti-fyn2, anti-lck and anti-src antibodies (except for anti-src antibodies, all other are polyclonal antibodies raised in rabbits) which are known in the art. The only antibody which has shown any degree of cross reactivity is the anti-fyn antibody which cross reacts (weakly) with the 166 kDa PTK.

Therefore, in one embodiment, the present invention provides a substantially pure PTK, isolated from PSD of cerebral cortex, which has a molecular weight selected from the group consisting of approximately 166, 90, 66, and 50 kDa. These PTKs are insensitive to Genistein and do not cross- react with any significant degree with many known anti-PTK antibodies.

As used herein, a PTK is said to be substantially pure if the PTK is the only protein in the sample with PTK activity. Therefore, a substantially pure PTK may not necessarily be purified to homogeneity so long as only one protein within the sample has PTK activity. In one application of this embodiment, the PTK is purified to homogeneity to be used in protein sequencing, cDNA isolation and antibody generation. In another application, the PTK is only purified to the level of substantially purity so that it can be used to assay for antagonist and agonist of PTK activity. One skilled in the art will readily purify the PTKs of the present to the level of purity that is needed for the purpose for which it is to be put.

The PTKs of the present invention can be purified by a variety of techniques known in the art. Such techniques include, but are not limited to, preparative polyacrylamide gel electrophoresis (PAGE), ion-exchange chromatography, size exclusion chromatography, HPLC, affinity chromatography, and immunoprecipitation. One skilled in the art will recognize that any one of a number of different techniques can be employed either individually or in combination in order to purify the PTKs of the present invention to the desired level of purity. Due to the insoluble nature of the PSD, the preferred method for purification is PAGE. During the purification, the PTK activity can be monitored using one of a number of techniques known in the art. As described in Example 1 , a modification of the procedure of Ferrel et al (Methods of Enzymology 200:430-435 (1991)) was used to assay for PTK activity. Alternatively, an antibody based detection system employing readily generatable anti-166 kDa PTK, anti-90 kDa PTK, anti-66 kDa PTK, or anti-55 kDa PTK antibodies can be employed to monitor the purification procedure.

A variety of source material can be used to obtain the PTKs of the present invention (and as described below, the human homologues of the PTKs herein described). The preferred source is neurological tissue, the most preferred source being isolated PSDs. Once isolated, the PTKs of the present invention can be used in a variety of fashions. For example, the isolated PTKs can be used to generate protein specific antibodies. These antibodies can be used; (1) to assay for the temporal and tissue specific expression of the PTKs of the present invention, (2) as an affinity matrix to isolate additional PTK, (3) to screen for DNA sequences encoding the PTK via expression cloning, and (4) used to design inhibitors and activators of the PTK activity.

Techniques for generating antibodies are known in the art (for example see Harlow et al, Antibodies: a laboratory manual, Cold Spring Harbor Press (1989)). Therefore, a skilled artisan can readily obtain antibodies specific for the PTKs of the present invention without undue experimentation.

The PTKs of the present invention can further serve as targets for the development of neuropharmaceutical agents. Once isolated, an important protein such as the PTKs of the present invention provides a new target that a skilled artisan can use in designing and developing pharmaceutical agents. Since the PTKs of the present invention are resistant to Genistein, this class of PTKs are valuable in drug development.

Further, the PTKs of the present invention serve as a valuable source of important amino acid sequence information. One skilled in the art can readily employ known techniques such as Edman degradation in a commercially available amino acid sequencer in order to determine the amino acid sequence of the PTK's herein identified and described. The amino acid sequence, being an inherent property of the PTK, can be determined without undue experimentation.

The amino acid sequence of the PTKs of the present invention can be used to generate small peptide fragments for binding or activity studies as well as for use as an immunogen. Further, the amino acid sequence of the PTKs, taken with the genetic code, provides skilled artisan with the various DNA sequences which can encode the PTKs disclosed herein.

In addition to providing all the potential DNA sequences encoding the PTKs of the present invention, fragments of the amino acid sequence of the PTKs of the present invention can serve as a source for generating oligonucleotide probes which can be employed in known recombinant DNA methodology to obtain the actual sequences used by the rat or humans to encode the PTKs of the present invention.

As well as using rat brain as the source of PTKs, the present invention is based on the identification of human homologues of the PTKs described herein. As used herein, a human homologue of the PTKs of the present invention is a PTK isolated from humans that has approximately the same molecular weight of the PTKs described herein. In general, a human homologue will possess at least an 80% homology in amino acid sequence with the rat PTKs and possess similar biological activity.

Therefore, the present invention provides substantially pure human homologues of the PTKs of the present invention.

The present invention further provides methods of obtaining isolated DNA sequences encoding human homologues of the PTKs of the present invention. Specifically, a DNA molecule encoding a human homologues of the

PTKs of the present invention can be isolated from humans by using the obtained PTK encoding sequences, or fragments thereof, as hybridization probes to screen a DNA library, or as an amplification primer in a reaction such as PCR. In detail, using the sequences encoding the rat PTKs of the present invention, or fragments thereof, a DNA library can be screened for DNA sequences encoding a human homologue of the PTKs of the present invention by: a) screening a cDNA or genomic library prepared from humans using fragments of the rat PTK encoding sequence as a probe; b) identifying members within the library which contain sequences which hybridize to the probe; and c) isolating the DNA molecules from the identified members of step (b).

Alternatively, using fragments of the sequences encoding the rat PTKs of the present invention as amplification primers, DNA sequences encoding a human homologue of the PTKs disclosed herein can be produced by the steps of: a) isolating mRNA, DNA, or cDNA from human; b) amplifying nucleic acid sequences encoding a human homologue of the PTKs disclosed herein using amplification primers derived from sequences encoding the rat PTKs to prime the amplification; c) isolating the amplified sequences of step (b).

When using DNA probes derived from the rat sequences which encode the PTKs of the present invention for screening a DNA library via colony/plaque hybridization, one skilled in the art will recognize the need to employ moderately high stringency condition, for example, hybridization from about 50-65°C, 5X SSC, 50% formamide, wash at 50-65°C, 0.5X SSC, in order to obtain sequences having regions which are greater than 80% homologous to the rat PTKs of the present invention. Any tissue can be used as the source for the genomic DNA or RNA encoding human homologues of the PTKs of the present invention. However, with respect to RNA, the most preferred source are tissues which express elevated levels of the desired PTK. In the preferred embodiment, the RNA used for generating a cDNA library will be isolated from nerve cell. The human sequences from the above-described gene library which are found to hybridize the rat PTK probes described above are then analyzed to determine the extent and nature of sequences encoding the PTK human homologue.

The isolation of the first members of this class of PTKs, which are preferentially expressed in the PSD, now makes possible a screening assay which enables the identification of agonists (PTK simulators) and antagonists

(PTK blockers) of the PTKs of the present invention, and human homologues thereof.

In initially evaluating potential antagonist and agonist of the PTKs of the present invention, or human homologues thereof, one or more members of the PTKs disclosed herein are tested for their ability to bind the agent which is being screened. An assay for PTK activity on a transferred blot as described in Example 1, or in transfected cells (for example, oocytes or COS cells) is then, or alternatively, used to determine the functionality of the PTK in the presence of the agent and thus makes possible the identification of agents that interfere with its regulation and activity.

The agents screened in the assays disclosed herein can be, but are not limited to, peptides, carbohydrates, vitamin derivatives, or other pharmaceutical agents. These agents can be selected and screened at random or rationally selected or designed using protein modeling techniques. For random screening, agents such as peptides, carbohydrates, pharmaceutical agents and the like are selected at random and are assayed for their ability to bind to or stimulate/block the activity of a member of the PTKs as outlined in Example 1 or in any other method of assaying PTK activity known in the art. Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be "rationally selected or designed" when the agent is chosen based on the configuration of the PTKs of the present invention. For example, one skilled in the art can readily adapt currently available procedures to generate antibodies, peptides, pharmaceutical agents and the like which are capable of binding to a specific peptide sequence of the PTK protein, for example see Hurby et al. (Application of Synthetic Peptides: Antisense Peptides", In Synthetic Peptides, A User's Guide, W.H. Freeman, NY, pp. 289-307 (1992)), Kaspczak et al (Biochemistry 28:9230-8 (1989)) and Harlow (Antibodies, Cold Spring Harbor Press, NY (1990)).

In one application of the herein disclosed screening assay, antagonist and agonist which stimulate or block the activity of PTKs of the present invention, or human homologues thereof, can be identified by: a) isolating one or more of the PTKs of the present invention, or a human homologue thereof; b) incubating the PTK of step (a) with an agent to be tested; and c) assaying the incubated PTK of step (b) for the activity of the

PTK by measuring the agents effect on PTK activity.

In one application of this assay, the PTK(s) is immobilized on a solid support while in other embodiments the assay is conducted on isolated PSD or

PTK. In another application of the herein disclosed screening assay, antagonist and agonist which stimulate or block the activity of PTKs of the present invention, or human homologues thereof, can be identified by: a) altering an expression host such that it expresses one or more of the PTKs of the present invention, or a human homologue thereof; b) incubating the expression host of step (a) with an agent to be tested; and c) assaying the expression host (b) for the activity of the PTK by measuring the agents effect on PTK activity.

Any expression host can be used in the above assay so long as it expresses a functional form of the PTK protein and the PTK activity can be measured as described below in the examples. Such hosts can be modified to contain DNA sequences encoding the PTKs, or human homologues thereof, using routine procedures known in the art. Alternatively, one skilled in the art can introduce mRNA encoding the PTK directly into a host such as Xenopus oocyte. The present invention further provides methods for assaying for the expression of one or more of the PTKs of the present invention using polyacrylamide gel electrophoresis (see Example 1). In general, these methods comprise isolating a protein fraction from an isolated population of cells and then resolving the isolated proteins, for example via PAGE, to determine if there is a PTK present in the sample that has a molecular weight selected from the group consisting of 166, 90, 66, 50 kDa.

Since antibodies can now readily be generated which selectively bind one or more of the PTKs of the present invention, or a human homologue thereof, the present invention further provides methods for assaying for the expression of one or more of the PTKs of the present invention, or a human homologue thereof using antibody based detection systems. In general, these methods comprise incubating tissue, a cell, or cellular extract with one or more antibodies which bind one or more of the PTKs of the present invention, or a human homologue thereof, and then determining whether the antibody binds the tissue, cell, or cellular extract.

In detail, the expression of one or more of the PTKs of the present invention, or a human homologue thereof, can be detected by:

(a) incubating tissue, a cell, or an extract thereof, with a composition containing an antibody, or fragment thereof, which binds to one or more of the PTKs of the present invention, or a human homologue thereof; and

(b) detecting whether the antibody becomes bound to the tissue, cell, or cellular extract. In addition to protein based detection system, the presence of cells expressing one or more of the PTKs of the present invention may be detected through the use of hybridization probes, such as mRNA, cDNA, genomic DNA, or synthetic oligonucleotide probes which bind to a PTK gene sequence, or to a PTK encoding mRNA sequence. Specifically, a probe derived from all or part of the sequence encoding one of the PTKs of the present invention, or derived from the sequences encoding the human homologue of the PTK, can be used as a probe or an amplification primer to detect cells which express a message homologous to the probe or amplification primer. One skilled in the art can readily adapt currently available nucleic acid amplification or detection techniques so that it employs probes or primers which are based on a sequence encoding a member of the PTKs of the present invention, for example, see

Sambrook et al., In: Molecular Cloning, a Laboratory Manual, Coldspring Harbor, NY (1982), and by Haymes, et al, In: Nucleic Acid Hybridization, a Practical Approach, IRL Press, Washington, DC (1985).

In general, the expression of one or more of the PTKs of the present invention can be identified by:

(a) incubating tissue, a cell, or an extract of a cell, with a nucleic acid molecule which hybridizes to mRNA encoding one of the PTKs of the present invention, or a human homologue thereof; and

(b) determining whether the nucleic acid molecule has become hybridized to a complementary nucleic acid molecule present in the tissue, cell, or cellular extract.

Alternatively, the expression of one or more of the PTKs of the present invention can be identified by:

(a) incubating tissue, a cell, or an extract of a cell, with amplification primers derived from the sequences encoding the PTKs of the present invention, or a human homologue thereof;

(b) amplifying nucleic acids within the tissue, cell, or cellular extract which are complementary to the sequence encoding the PTK; and

(c) determining whether there is an amplification of a sequence within the tissue, cell, or cellular extract.

The tissues, or cells used in the assays described above include, but are not limited to, cells, protein or membrane extracts of cells, or biological fluids such as cerebral fluid. The sample used in the above-described assays will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is capable with the system utilized.

Conditions for incubating an antibody or a probe/primer with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody or probe/primer used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats (such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or rocket immunofluorescent assays) can readily be adapted to employ the antibodies of the present invention. Examples of such assays can be found in Chard, T. "An Introduction to Radioimmunoassay and Related Techniques" Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, et al, "Techniques in Immunocytochemistry, " Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., "Practice and Theory of

Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular

Biology, " Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

The detection of foci of PTK expression, through the use of protein separation techniques, labeled antibodies, or nucleic acid probes, can be used to diagnose certain classes of cells of different neurological origin. In one diagnostic embodiment, samples of tissue are removed from a subject and are incubated in the presence of antibodies which are detectably labeled. In another preferred embodiment, this technique is done in a non-invasive manner through the use of magnetic imaging, fluorography, etc. In another aspect of the present invention, novel 110, 120 and 270 kDa dystrophin antibody cross-reactive protein are described (hereinafter the DMD proteins, and the 110-DMD, 120-DMD and 270-DMD proteins, respectively, see Examples 2 and 3). These proteins were present in brain tissue isolated from normal adults but were almost completely absent in brain tissue isolated from patients with Duchenne Muscular Dystrophy. Two of these DMD proteins, the 110 and 120 DMD proteins, are described elsewhere (Kim et al, Proc. Natl Acad. Sci. USA 89:11642-11644 (Dec, 1992)).

In detail, using an enriched PSD preparation, novel 110, 120 and 270 kDa proteins which cross-react with anti-427 kDa dystrophin antibodies have been identified, isolated, and characterized. Therefore, in one embodiment, the present invention provides substantially pure 110-DMD, 120-DMD and 270- DMD protein, isolated from nerve cells, or enriched PSDs, which have a molecular weight of approximately 110, 120 and 270 kDa, respectively.

As used herein, a DMD protein is said to be substantially pure if the DMD protein is the only protein in the sample which cross reacts with an anti-

427 kDa dystrophin selective antibody. Therefore, substantially pure DMD protein may not necessarily be purified to homogeneity so long as only one protein within the sample cross-reacts with an anti-dystrophin antibody. In one application of this embodiment, the DMD protein is purified to homogeneity so as to be used in protein sequencing, cDNA isolation and antibody generation. In another application, the DMD protein is purified only to the level of substantially purity so that it can be used to assay for the function of the DMD protein. One skilled in the art will readily purify the isolated DMD proteins of the present invention to the level of purity that is needed for the purpose to which it is to be put.

The DMD proteins of the present invention can be purified by a variety of techniques known in the art. Such techniques include, but are not limited to, preparative polyacrylamide gel electrophoresis (PAGE), ion-exchange chromatography, size exclusion chromatography, HPLC, affinity chromatography, and immunoprecipitation. One skilled in the art will recognize that any one of a number of different techniques can be employed either individually or in combination in order to obtain the DMD proteins of the present invention. Due to the insoluble nature of the PSD, the preferred method for purification is preparative PAGE. During the purification, the DMD proteins activity can be monitored using one of a number of techniques known in the art. As described in Examples 2 and 3, antibody based detection system can be employed.

A variety of source material can be used to obtain the DMD proteins of the present invention. The preferred source is neurological tissue, the most preferred source being isolated PSDs.

Once isolated, the DMD proteins of the present invention can be used in a variety of fashions. For example, the DMD proteins can be used to generate protein specific antibodies. These antibodies can be used; (1) to assay for the temporal and tissue specific expression of the DMD proteins, (2) as an affinity matrix to isolate additional DMD protein, and (3) to screen for DNA sequences encoding the DMD proteins via expression cloning.

Techniques for generating antibodies are known in the art (for example see Harlow et al., Antibodies: a laboratory manual Cold Spring Harbor Press (1989)). Therefore, a skilled artisan can readily obtain antibodies specific for the DMD proteins of the present invention without undue experimentation.

The DMD proteins of the present invention can further serve as a target for the development of neuropharmaceutical agents. Once isolated, an important protein such as the DMD proteins of the present invention provide new targets that a skilled artisan can use in designing and developing pharmaceutical agents.

Since the DMD proteins of the present invention are differentially expressed in normal and DMD brain, the DMD proteins of the present invention can be used as markers of various disease states. Examples 2 and 3 demonstrate the correlation of the loss of the DMD proteins with the appearance of Duchenne Muscular Dystrophy.

The DMD proteins of the present invention further serves as a source of important amino acid sequence information. One skilled in the art can readily employ known techniques such as Edman degradation in a commercially available amino acid sequencer in order to determine the amino acid sequence of the DMD proteins herein identified and described. The amino acid sequence is an inherent property of the DMD proteins and can be determined without undue experimentation.

The amino acid sequence of the DMD proteins can be used to generate small peptide fragments for binding or activity studies as well as for use as an immunogen. Further, the amino acid sequence of the DMD proteins, taken with the genetic code, provides a skilled artisan with the various DNA sequences which can encode the DMD proteins disclosed herein.

In addition to providing all the potential DNA sequences encoding the DMD proteins of the present invention, fragments of the amino acid sequences of the DMD proteins can serve as a source for generating oligonucleotide probes which can be employed in known recombinant DNA methodology to obtain the actual sequences encoding the DMD proteins of the present invention.

The present invention additionally provides methods for assaying for the expression of the DMD proteins of the present invention using polyacrylamide gel electrophoresis (see Example 3). In general, these methods comprise isolating a protein fraction from a population of cells and then resolving the isolated proteins, for example via PAGE, to determine if there is a DMD protein present in the sample that has a molecular weight of approximately 110, 120 or 270 kDa.

Since antibodies can now readily be generated which selectively bind to the DMD proteins of the present invention, the present invention further provides methods for assaying for the expression of the 110-DMD, 120-DMD, and 270-DMD proteins using antibody based detection systems. In general, these methods comprise incubating tissue, a cell, or cellular extract with one or more antibodies which bind to one or more of the DMD proteins of the present invention and then determining whether the antibody binds the tissue, cell, or cellular extract.

For example, the expression of a DMD protein of the present invention can be detected by: (a) incubating tissue, a cell, or an extract thereof, with a composition containing an antibody, or fragment thereof, which binds to one or more of the 110-DMD, 120-DMD and 270-DMD proteins of the present invention; and (b) detecting whether the antibody becomes bound to the tissue, cell, or cellular extract.

In addition to protein based detection system, the presence of cells expressing one or more of the DMD proteins of the present invention may be detected through the use of hybridization probes, such as mRNA, cDNA, genomic DNA, or synthetic oligonucleotide probes which bind to a DMD protein gene sequence, or to a DMD protein encoding mRNA sequence. Specifically, a probe derived from all or part of the sequence encoding the 110-DMD, 120-DMD or 270-DMD protein can be used as a probe or an amplification primer to detect cells which express a message homologous to the probe or amplification primer. One skilled in the art can readily adapt currently available nucleic acid amplification or detection techniques so that it employs probes or primers which are based on a sequence encoding the DMD proteins, for example, see Sambrook, et al, In: Molecular Cloning, a Laboratory Manual, Coldspring Harbor, NY (1982), and by Haymes et al, In: Nucleic Acid Hybridization, a Practical Approach, IRL Press,

Washington, DC (1985).

For example, the expression of one or more of the DMD proteins of the present invention can be identified by:

(a) incubating tissue, a cell, or an extract of a cell, with a nucleic acid molecule which hybridizes to mRNA encoding the 110-DMD, 120-DMD or

270-DMD proteins of the present invention; and

(b) determining whether the nucleic acid molecule has become hybridized to a complementary nucleic acid molecule present in the tissue, cell, or cellular extract. Alternatively, the expression of a DMD protein can be identified by: (a) incubating tissue, a cell, or an extract of a cell, with amplification primers derived from the nucleotide sequences encoding the 110-DMD, 120- DMD or 270-DMD proteins of the present invention, or a human homologue thereof; (b) amplifying nucleic acids within the tissue, cell, or cellular extract which are complementary to the sequence encoding the DMD protein; and

(c) determining whether there is an amplification of a sequence within the tissue, cell, or cellular extract.

The tissues, or cells used in the assays described above include, but are not limited to, cells, protein or membrane extracts of cells, or biological fluids such as cerebral fluid. The sample used in the above-described assays will vary based on the assay format, nature of the detection method and the tissues, cells or extracts used as the sample to be assayed. Methods for preparing protein extracts or membrane extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is capable with the system utilized.

Conditions for incubating an antibody or a probe/primer with a test sample vary. Incubation conditions depend on the format employed in the assay, the detection methods employed, and the type and nature of the antibody or probe/primer used in the assay. One skilled in the art will recognize that any one of the commonly available hybridization, amplification or immunological assay formats (such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or rocket immunofluorescent assays) can readily be adapted to employ the antibodies of the present invention. Examples of such assays can be found in Chard, T.

"An Introduction to Radioimmunoassay and Related Techniques" Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock et al , "Techniques in Immunocytochemistry, " Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., "Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular

Biology, " Elsevier Science Publishers, Amsterdam, The Netherlands (1985). The detection of foci of DMD protein expression, through the use of protein separation techniques, labeled antibodies, or nucleic acid probes, can be used to diagnose the presence of DMD, or certain classes of cells of different neurological origin. In one diagnostic embodiment, samples of tissue are removed from a subject and are incubated in the presence of antibodies which are detectably labeled. In another preferred embodiment, this technique is done in a non-invasive manner through the use of magnetic imaging, fluoro- graphy, etc.

Having now generally described the invention, the agents and methods of obtaining same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention, unless specified.

EXAMPLES

EXAMPLE 1. Protein Tyrosine Kinases (PTKs) in the Postsynaptic Density (PSD) And Phosphorylation of the NMDARI

Materials

[γ-³²P] ATP was obtained from Amersham. Anti-phosphotyrosyl antiserum was purchased from Zymed. PY20, a monoclonal antibody against phosphotyrosine, was a product of ICN. Anti-NMDARl antibodies are gifts from Dr. Robert J. Wenthold of National Institute of Health. The other chemicals are of the highest grade from commercial sources.

Methods

Experimental Animals and Dissection. Sprague-Dawley rats were used in the present study. The animals were maintained in a temperature- controlled room as described (Wu et al , Proc. Natl. Acad. Sci. USA 84:8687- 8691 (1987)). Animals were killed by exposure to CO₂ vapor. Tissues were removed and stored at -80°C until use.

Subcellular Fractions. Synaptic membranes (SM) and highly purified PSD fractions were obtained as described (Wu et al, J. Neurochem. 46:831- 841 (1986); Wu et al, Mol Brain Res. 7:167-184 (1986)).

Protein Determination. Protein content was determined by the procedures of Lowry et al. (J. Biol. Chem. 793:265-275 (1951)), with bovine serum albumin as standard.

SDS/PAGE and Western Blot Analysis. SDS-PAGE, using a 7.5-12 % acrylamide gradient slab gel, was performed according to Wu et al. (Mol.

Brain Res. 7:167-184 (1986)). Proteins in the gel were electrophoretically transferred to Immobilon-P (Millipore) as described (Towbin et al , Proc. Natl Acad. Sci. USA 76:4350-4354 (1979)). Western blot analysis was carried out using a commercial available detection system (Amersham). Detection of Tyrosine Phosphorylation. Protein (50 μg each) was incubated with a mixture of ATP and [γ-³²P] ATP (0.013 mM, 3 μCi/ml) in the presence or absence of 10 mM MnCl₂ and 50 μM sodium ortho-Nanadate at 25 °C for 15 minutes. Then, the reaction mixture was subjected to SDS- PAGE. The destained gel was incubated with 1M KOH at 50°C for 2 hours, dried and analyzed by autoradiography.

Detection of PTKs Blotted in the PVDF Membrane. This method is based on the procedures as described (Ferrel et al , Method in Enzymology 200:430-435 (1991) with some modification. After transferring the proteins from gel to PVDF membrane (Millipore), the blotted membrane was rinsed with water briefly. Then, the membrane was incubated with renaturation buffer (140 mM ΝaCl, 10 mM Tris-HCl, pH 7.4, 2 mM DTT, 2 mM EDTA, 1 % BSA, 0.1 % ΝP-40) at 4°C for 3 days. Subsequently, autophosphorylation was carried out by incubating the membrane with a buffer containing 3% glycerol, 0.1M NaCl, 0.02 M HEPES, pH 17.0, 0.5 mM DTT, 0.7 mg BSA/ml, 10 mM MnCl₂ and |_/y-³²P] ATP (mixed with cold ATP to a final concentration of 0.013 mM, 20μCi/ml) at 25°C for overnight. After the reaction, the protein blot was washed as described (Ferrel et al. , Method in

Enzymology 200:430-435 (1991) and was analyzed by autoradiography.

Phosphorylation and Immunoprecipitation of NMDAR1 in the PSD. The PSD (50 μg each) was incubated with 13 μM ATP in a PTK buffer

(20 mM HEPES/KOH, pH 7.0, containing 3% glycerol, 0.1 M NaCl and 0.5 mM DTT) in the presence and absence of 10 mM Mn²⁺ and 50 μM o- Vanadate at 25 °C for 15 minutes. The reaction mixture was treated with 1 % SDS and was incubated at 80°C for 10 minutes. Then the mixture was diluted to 0.1 % SDS with a dilution buffer (10 mM PMSF and 0.5 % NP-40).

Immunoprecipitation of phosphotyrosyl proteins was carried out by incubating with a monoclonal anti-phosphotyrosine antibody linked to agarose (p-Tyr Agarose conjugate, Santa Cruz Biotechnology, CA) at room temperature for 2 hours, according to Sambrook et al. (1989). The immunoprecipitated proteins were subjected to SDS-PAGE and Western blotting analysis, using specific anti-NMDARl antibodies.

Occurrence and Enrichment of Protein Tyrosine Kinases And Their Substrates in the PSD

Same amount of homogenate (H), synaptic membrane (SM) and postsynaptic density (PSD) was incubated with [γ-³²P] ATP in the presence

(+) or absence (-) of tyrosine kinase activator, Mn²⁺/o-Van, as described previously. The reaction mixture was resolved by SDS-PAGE, treated with

1 M KOH and analyzed autoradiographically. Molecular weight marker proteins are shown in the left panel. Immunochemical Demonstration of the Occurrence of Phosphotyrosyl

Proteins In the PSD

The PSD fractions were isolated from cerebral cortex (CTX), olfactory bulb (OB) and cerebellum (CBL) of adult rat brains. The proteins were subjected to SDS-PAGE for Western blotting analysis using specific antibodies raised against the phosphotyrosyl proteins. Molecular weight marker proteins are shown in the right panel. Detection Of PTKs In The Cerebral Cortical PSD By Autophosphorylation

The PSD (50μg each) from cerebral cortex of adult rat brain was assayed for PTKs by autophosphorylation in the presence of Mn²⁺ as described previously. The autophosphorylation kinases were analyzed by autoradiography without (-) and with (+) treatment with IM KOH. Molecular weight markers are shown in the right panel.

Results

Occurrence and Enrichment of PTKs and Their Substrates in the PSD

To demonstrate the existence of PTKs in the complex synaptic structure, same amounts of PSD, synaptic membrane and homogenate were incubated with [τ-³²P] ATP in the presence or absence of PTK activator,

Mn²⁺/o-Van. Then, the reaction mixtures were subjected to SDS-PAGE, treated with or without IM KOH, and analyzed by autoradiography. The results showed that several proteins (M.W. = 300, 110, 74, 66, 50 kDa) in the PSD, synaptic membrane and homogenate were dramatically enhanced by the activator. In addition, these proteins were greatly enriched in the PSD as compared with those in the SM or H from which the density was derived. Immunoblotting Analysis of Phosphotyrosyl Proteins In The PSD

From Various Brain Regions

To examine the regional expression of phosphotyrosyl proteins, same amount of PSD isolated from cerebral cortex, olfactory bulb and cerebellum were subjected to Western blot analysis, using polyclonal antibody which specifically recognizes phosphotyrosyl proteins. The results thus obtained revealed that seven phosphotyrosyl proteins (M.W. = 215, 180, 150, 80, 74,

32, 18 kDa) were detectable in the PSD from all the three brain regions examined. Further, these phosphotyrosyl proteins were expressed apparently in a region-specific manner. Direct Detection of PTKs in the PSD

The above results indicate that the Mn²⁺/o-Van activates the phosphorylation of several proteins in the PSD. These proteins are presumably PTKs and their substrates. It is well known that a prominent property of PTK is its capability of autophosphorylation. Consequently, we attempted to identify PTKs by detecting the autophosphorylation of PTKs in the PSD. While four autophosphorylated proteins (M.W. of 166, 90, 66 and 50 kDa) were detected in the presence of Mn²⁺/o-Van, control experiment without Mn²⁺/o-Van showed that no autophosphorylation was detected. Tyrosine Phosphorylation of NMDA RI in the PSD

Recently, it has been reported that NMDAR1, a subclass of NMDA receptors, which are known to be crucial for induction of LTP, are highly enriched in the PSD (Suen et al, 1993). The involvement of tyrosine phosphorylation in induction of LTP and enrichment of PTKs in the PSD raise the possibility that the NMDA receptor may be regulated by tyrosine phosphorylation. To examine this speculation, immunoprecipitation experiment was carried. Same amount of PSDs were incubated with ATP in the presence or absence of Mn²⁺/o-Van. After phosphorylation, the PSD were solubilized and incubated with a monoclonal antibody specific to phosphotyrosine residue to precipitate the phosphorylated proteins. The immunoabsorbed proteins were resolved by SDS-PAGE and analyzed by immunoblotting to detect the NMDAR1 using anti-NMDARl antibody.

The result of the Western blotting analysis of NMDAR1 showed that the 120 kDa NMDAR1 was precipitated by the anti-phosphotyrosine antibody, indicating that the receptor was phosphorylated by the PTK(s) in the PSD.

Recently, PTKs are implicated to be involved in induction of LTP, as PTK inhibitor blocks the initiation of LTP in hippocampal cells. The results of our studies raise the possibility that the PTKs in the PSD may directly regulate the NMDAR1 function by tyrosine phosphorylation. Discussion

In this study we demonstrated that PTKs and their substrate were highly concentrated in PSD as compared with those in synaptic membrane or homogenate. Furthermore, our results of Mn ⁺/°Nan-dependent autophosphorylation of blotted proteins revealed the presence in cortical PSD of at least four PTKs. These PTKs are phosphorylatable at the tyrosine(s) and were further supported by the results of phosphoamino acid analysis and Western blotting analysis using specific antibodies against phosphotyrosine (data not shown). Although a number of PTKs have been found in nervous tissue, only the nonreceptor tyrosine kinases encoded by proto-oncogene src (Cudmore et al, J. Neurochem. 57:1240-1248 (1991)), fyn and yes (Grant et al, Science 258: 1903-1910 (1992)) were reported to be localized in the PSD. Of these four PTKs detected in the cortical PSD, only one (with molecular weight of 66 kDa) shares the similar molecular weight with that of src family PTKs.

The relationship of the 66 kDa PTK and those of the src family is currently under investigation. The other three PTKs (M.W. = 0, 90 and 166) were, based on molecular sizes, probably unique PSD proteins and deserve further investigation. PTKs in the postsynaptic cell appear to contribute to the induction rather than the maintenance stage of LTP (O'Dell et al , Nature 353:558-560 (1991)). This idea is consistent with the evidence that PTKs and their substrates are enriched in the PSD. Evidence that LTP induced by pairing the synaptic input with strong postsynaptic depolarization is fully blocked by the tyrosine kinase inhibitor genistein, whereas LTP in fyn mutant mice still can be obtained by paring strong postsynaptic depolarization, indicates that PTKs other than fyn participate in LTP (Grant et al, Science 258: 1903-1910 (1992)). This finding may give a clue to the function of those PTKs detected in the PSD. Further, our finding of tyrosine phosphorylation of the ΝMDAR1 suggests that PTKs in the PSD may play roles in synaptic plasticity. The physiological roles of PTKs in the PSD remain to be determined. To elucidate exact function of these PTKs, we are currently trying to purify and will then perform biochemical and functional characterization of the proteins. Since to serve a regulatory function tyrosine phosphorylation in the PSD must be regulated, it is equally important to define how the enzymes are controlled.

In summary, several PTKs and substrates were selectively localized to the PSD, potentially allowing these synaptic molecules to serve postsynaptic mechanisms. Regardless of the specific roles of the PTKs in the synapses, one such function may be the regulation of NMDAR1 through tyrosine phosphorylation of the receptor.

EXAMPLE 2.

Materials And Methods

Experimental Animals and Dissection. Sprague-Dawley rats were used in the present study. Control (C57BL/10) and madx (C57BL/10 mdx/mdx) mice were obtained from The Jackson Laboratory. The animals were maintained in a temperature-controlled room as described (Wu et al, Proc. Natl. Acad. Sci. USA 84:8687-8691 (1987)). Animals were killed by exposure to CO₂ vapor. Tissues were removed and stored at -80°C until use. Subcellular Fractions. Synaptic membrane (SM) and highly purified

PSD fractions were obtained as described (Wu et al, J. Neurochem. 46:831- 841 (1986); Wu et al, Mol. Brain Res. 7:167-184 (1986)).

Protein Determination. Protein content was determined by the procedure of Lowry et al. (/. Biol Chem. 793:265-275 (1951)), with bovine serum albumin as standard.

Antibody. The high-affinity anti-dystrophin antibody, anti 6-10, was a generous gift from Timothy 1. Byers and Louis M. Kunkel (Children's Hospital and Harvard Medical School, Boston). The production of anti 6-10 was described in refs. 19 and 28. SDS/PAGE and Western Blotting Analysis. SDS/PAGE analysis using a 3.5-12% acrylamide gradient slab gel was performed according to Wu et al. (Mol. Brain Res. 7:167-184 (1986)). Proteins were electrophoretically transferred to Immobilon-P (Millipore) as described (Towbin et al., Proc. Natl. Acad. Sci. USA 76:4350-4354 (1979)). Western blotting analysis was carried out with a commercial Western blot detection system (Amersham).

Results And Discussion

Occurrence and Regional Expression of Dystrophin and Dystrophin- Related Proteins in the PSD. To localize brain dystrophin within the synaptic apparatus and to define regional distribution, we performed Western blot analysis using the specific high-affinity anti-dystrophin antibody anti 6-10 (Lidov, H.G.W. et al, Nature (London) 348:725-728 (1990)). PSDs isolated from adult rat cerebral cortex, cerebellum, and olfactory bulb were examined. The cortex, cerebellum, and olfactory bulb have been associated with cognitive function, motor coordination, and olfactory information processing, respectively. Anti 6-10 specifically recognized three distinct proteins, including a 400-kDa brain dystrophin and two smaller, previously unidentified dystrophin-related proteins with apparent molecular masses of 110 kDa and 120 kDa (Fig. 2 Panel A). Densitometric measurements revealed that relative concentrations of these proteins in each brain region differed markedly (data not shown). In the cerebrocortical PSD, the 400-kDa protein was twice as abundant as either the 120-kDa or the 110-kDa protein. In the PSDs from cerebellum and olfactory bulb, however, the 120-kDa protein was predominant, followed by the 400- kDa and then the 110-kDa protein. Differential regional concentration of these proteins suggests that they are differentially expressed dystrophin isoforms. It is unlikely that the 110-kDa and 120-kDa proteins are proteolytic products of dystrophin, since proteolysis would have yielded comparable relative concentrations in the different brain areas. Enrichment of Dystrophin and Dystrophin-Related Proteins in the PSD.

To more definitively localize brain dystrophin, distribution was examined in total homogenate, SM, and PSDs isolated from the different brain regions. In all regions examined, the concentration of the 400-kDa dystrophin was 10-fold higher in the PSD than in whole tissue homogenate or the SM fraction (Fig. 2 Panel B). The dystrophin-related proteins also exhibited significant enrichment in the PSDs. Our results suggest that dystrophin and related proteins are PSD components, raising the possibility that the proteins play roles in synaptic function through postsynaptic mechanisms.

Developmental Expression of Dystrophin and Dystrophin Related Proteins in the Cerebral Cortex of the Rat.

Since DMD, including associated cognitive deficits (Hoffman et al.,

Neuron 2:1019-1029 (1989); Rosman et al, Brain 89 :769-788 (1966); Moser, H., Hum. Genet. 66:17-40 (1984); Emery, A.E.H. , Duchenne Muscular

Dystrophy, Oxford Univ. Press Oxford, pp. 99-103 (1988)), exhibits a characteristic developmental pattern, we defined the developmental expression of dystrophin in the cortical PSD. The 400-kDa and 110-kDa proteins exhibited dramatic 9-fold developmental increases from postnatal day 7 to day 10, while the 120-kDa protein did not change significantly (Fig. 3). Three separate experiments yielded similar results. These observations further suggest that the three molecules are independently expressed and that the 400- kDa and 110-kDa proteins are involved in synaptogenesis or synaptic maturation. Expression of Brain Dystrophins in the mdx Mouse.

To begin defining the pathogenetic significance of brain dystrophin, we examined the mdx mouse (Bulfield et al, Proc. natl Acad. Sci. USA 87: 1189- 1192 (1984); Sieinski et al, Science 244:1578-1580 (1989)), an X-linked mutant model of human DMD that exhibits muscle dystrophin deficiency. In fact, the 400-kDa dystrophin was undetectable in PSDs isolated from mdx mouse brain (Fig. 4). In contrast, the 120-kDa and 110-kDa proteins were decreased but detectable in brains of mdx mice. Another marker associated with the PSDs,the major PSD protein (Wu et al, Proc. Natl. Acad. Sci. USA 84:8687-8691 (1987); Wu et al, Proc. Natl Acad. Sci. USA 85:6207-6210 (1988); Wu et al, J. Cognitive Neurosci. 7:194-200 (1989); Wu et al, Proc. Natl Acad. Sci. USA 89:3015-3019 (1992)) derived from mdx brain was unaffected as judged from calmodulin binding (data not shown). The decreased expression of the 120- and 110-kDa molecular species in mdx brains was apparently specific, since equal amounts of protein were loaded on each lane, and since other species, such as the band at 70 kDa, for example, were identical in mdx and normals (Fig. 4). Interestingly, the 120- and 110-kDa species were expressed less in mouse than in rat brain (Fig. 4). Disproportionate deficiency of the 400-kDa dystrophin protein in the mdx brain is consistent with a pathogenetic role in associated brain dysfunction. Moreover, selective deficiency further supports the contention that expression of dystrophin and the expression of related proteins are differentially regulated.

EXAMPLE 3. Case Report of Dystrophin Expression in DMD

The DMD case patient was an 8 year old boy, the product of a full term uncomplicated pregnancy and normal early development. At 14 months, he could not rise from the floor except by using a Gower's maneuver. At 2 years of age, a CPK was elevated at 10,000 IQ. A muscle biopsy showed changes consistent with Duchenne' s muscular dystrophy (data not shown). Maternal CPK was normal and there was no family history of DMD. Subsequent evaluation at Children's Hospital showed no dystrophin on Western blotting and no detectable genomic deletion (thus presumably a point mutation or mutation in a regulatory or processing sequence). The patient had multiple heel cord lengthening procedures and was ambulatory with assistance during childhood.

He did not exhibit any symptom of mental impairment. He had no problems related to aspiration or swallowing and had no evidence of congestive heart failure. At age seven, an echocardiogram showed normal left ventricular function and a resting heart rate of 100. Several months later, he underwent an orthopedic procedure following which he never resumed ambulation. A Holter monitor EKG showed frequent unifocal premature ventricular contractions.

Ten days prior to death, the patient developed vomiting, diarrhea and symptoms consistent with a viral illness. He had an acute and fulminant courses involving chest pain, pulmonary infiltrates, respiratory failure, hypotension and death. At autopsy, he had diffuse alveolar damage, evidence of multiorgan acute hypotensive injury, and skeletal and cardiac muscle necrosis and fibrosis consistent with the diagnosis of Duchenne muscular dystrophy. The brain showed evidence of edema but was otherwise unremarkable. Tissue specimens were obtained after a postmortem intervals of 6 hours.

Methods

Patient and Preparation of Tissues

Age-matching control brain tissue was obtained from an 8 and half year old boy who developed a myelodysplastic syndrome which evolved into acute myelogenous leukemia. He was in good health prior to his initial diagnosis and received a bone marrow transplant at age 8, and had a stormy post transplant course with multiple medical complications. Five months later, he developed gastrointestinal symptoms, candidemia, renal failure, and finally respiratory failure. At autopsy, he had widespread hemorrhagic lesions and evidence of chronic graft-versus-host disease. He had several small cerebral hemorrhages, but otherwise the brain was histologically normal. Tissue specimens were obtained at 12 hours postmortem. The tissues of both DMD and control cases were obtained in the autopsy room and frozen immediately in liquid nitrogen. Subcellular Fractionation

Synaptic membrane (SM) and highly purified PSD fractions were prepared as previously described (Wu et al., Mol. Brain Res. 7:167-184 (1986); Wu et al, J. Neurochem. 46:831-841 (1986)). Protein concentration was determined by the procedure of Lowry et al , with bovine serum albumin as standard (J. Biol Chem. 793:265-275 (1951)). Antibody

The anti 6-10 is a highly specific rabbit polyclonal antibody against the rod domain of dystrophin. The preparation and specificity of the anti 6-10 has been well described (Kim et al, Proc. Natl Acad. Sci. USA 89:11642-11644

(1992); Lidov et al, Nature 348:725-728 (1990); Byers et al, J. Cell Biol 775:411-421 (1991)). Immunoblot Analysis

Protein samples were fractionated by SDS-polyacrylamide gels and electrophoretically transferred to Immobilon-P membrane (Millipore) as described (Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press, pp. 479-489 (1988)). The blots were blocked with 5% non-fat dry milk in TBS-T buffer (25 mM Tris, 137 mM NaCl, 0.1 % Tween-20, pH 7.6) for overnight in cold room and were incubated at room temperature with the anti 6-10 (1:2500 dilution) for

2 hours and then with goat anti-rabbit secondary antibody linked to horseradish peroxidase for 1 hour. Between each step, the blots were extensively washed with the TBS-T buffer. Finally, development of immunoblotted bands were carried out with ECL immunoblot detection system (Amersham).

Results

Deficiency of Synaptic Dystrophin in the DMD Brain

To examine the expression of the brain dystrophin within the synaptic structures of human cerebral cortex, we performed immunoblot analysis using the highly specific anti-dystrophin antibody, anti 6-10, in total homogenate (H), synaptic membrane (SM) and postsynaptic density (PSD) fractions from the cerebral cortex of DMD and the age-matched control patients. Similar to those found in rat and mouse brains (Kim et al, Proc. Natl Acad. Sci USA 89:11642-11644 (1992)), the human 427 kDa dystrophin exhibited significant enrichment in the PSD, indicating that the human brain dystrophin is also the component of the PSD. The 427 kDa dystrophin was absent in the PSD from the DMD brain, but was normally expressed in the PSD from an age-matched control brain (Fig. 5), suggesting that this synaptic protein is affected by the DMD mutation. The anti 6-10 also detected two immunologically-related proteins with apparent molecular weights of 120 and 110 kDa in the PSDs from both control and DMD cerebral cortex, reproducing findings in the rat brain (See Example 2). Postmortem Effect on Dystrophin Degradation

In addition to the 427 kDa dystrophin, a previously unidentified, 270 kDa protein was specifically detected by the anti 6-10 in the PSD of control brain but not in the DMD brain. To determine whether the 270 kDa molecular species was a degradation product of the larger 427 kDa dystrophin, we examined the effect of postmortem time on detectable dystrophin species using the PSD from adult rat cerebral cortex. The 270 kDa protein was undetectable in 10 hour postmortem, suggesting that it was not a degradation product (Fig. 6 Panel A). However, the 427 kDa dystrophin started to degrade rapidly with generation of protein bands ranging 85 to 100 kDa after 5 postmortem hours (Fig. 6 Panel B).

Discussion We demonstrated here that the 427 kDa dystrophin was normally expressed in the PSD isolated from cerebral cortex of adult human brain. The protein, however, was absent in the PSD of DMD brain. Unexpectedly, a previously unidentified protein of 270 kDa was found in the human PSD. The 270 kDa protein was detectable in control human PSD but was undetectable in rat PSD obtained 10 hours postmortem, suggesting that this synaptic protein may be a dystrophin isoform linked to the DMD gene rather than a degradation product of the higher molecular weight dystrophin. Recent studies indicated that the brain dystrophin transcripts spliced differently from muscle, raising the possibility that this 270 kDa molecular species may be the protein product of the alternatively spliced form of the 427 kDa dystrophin mRNA

(Feener et al, Nature 338:509-511 (1989)).

The exact function of dystrophin in brain remains to be determined. However, series of evidence suggests that the protein may play roles in normal cognitive process and DMD pathology. First, dystrophin may play a role in synaptic formation, based on the previous findings that the rat brain dystrophin exhibited selective developmental increase during postnatal day 7 to 14, a critical period of neuronal synaptogenesis (Example 2). Dystrophin, known to be a cytoskeletal protein in muscle (Hoffman et al., Neuron 2: 1019-1029 (1989)), may serve the similar structural role in the PSD, especially during synaptogenesis or synaptic maturation. Defect of synaptic dystrophin may cause abnormal synaptic function due to the altered synaptic structure. Second, dystrophin may be involved in the regulation of the postsynaptic calcium. Recent studies showed an increased calcium influx in dystrophic muscle and increased Ca ⁺ level in cultured cerebral cell from mdx mice (Hopf et al, Brain Res. 578:49-54 (1992)). The increase in Ca²⁺ was thought to be achieved through muscle-type nitrendipine-sensitive Ca²⁺ channel (or L-type Ca²⁺ channel), which has been detected in the brain PSD using nitrendipine binding assay (Wu et al, Mol. Brain Res. 7:167-184 (1986); Turner et al, J. Cell Biol 115: 1701-1712 (1991)). These observations raise the possibility that dystrophin may play a role in synaptic calcium homeostasis and defect of synaptic dystrophin may lead to Ca²⁺-related excitotoxic damage or increase vulnerability of neuron to at least one form of physiological stress, hypoxia- induced damage (Mehler et al, Proc. Natl. Acad. Sci. USA 89:2461-2465 (1992)). Consequently, dystrophin deficiency in the brain may cause abnormal synaptic function resulting from functional alteration of the other synaptic components physiologically associated with dystrophin. Taken together, results from our previous and present studies suggest that dystrophin may play a critical role in synaptic function through postsynaptic mechanism, further raising the possibility that deficiency of this synaptic protein may underlie etiology of Duchenne muscular dystrophy. Defect of brain dystrophin may lead to general reduction in intelligence and mental retardation in all or some DMD patients.

Claims

WHAT IS CLAIMED IS:

1. An isolated, substantially pure protein tyrosine kinase, wherein said protein tyrosine kinase is selectively expressed in the postsynaptic density, and said protein tyrosine kinase has an approximate molecular weight selected from the group consisting of 166, 90, 66, and 50 kDa.

2. The protein tyrosine kinase of claim 1 wherein said protein tyrosine kinase is resistant to Genistien.

3. An isolated, substantially pure protein with an approximate molecular weight selected from the group consisting of 110, 120 and 270 kDa, wherein said protein is selectively expressed in the postsynaptic density and said protein cross reacts with an anti-427 kDa dystrophin antibody.