WO1999021995A1

WO1999021995A1 - Treatment and prevention of neurodegenerative diseases using modulators of the interaction of app and a polypeptide defining a pi domain

Info

Publication number: WO1999021995A1
Application number: PCT/US1998/022523
Authority: WO
Inventors: Shasta Sabo; Joseph Buxbaum; Paul Greengard
Original assignee: The Rockefeller University
Priority date: 1997-10-24
Filing date: 1998-10-23
Publication date: 1999-05-06
Also published as: AU1197999A

Abstract

Modulators that affect the interaction of a polypeptide defining a PI domain with the cytoplasmic domain of amyloid precursor protein decrease overall production of beta amyloid (Aβ) by a cell, and thus provide therapeutics for the treatment and/or prevention of neurodegenerative diseases, such as Alzheimer's disease and dementia.

Description

TREATMENT AND PREVENTION OF NEURODEGENERATIVE DISEASES USES MODULATORS OF THE INTERACTION OF APP AND A POLYPEPTIDE DEFINING A PI DOMAIN

GOVERNMENT RIGHTS CLAUSE

This invention was made with government support under Grant No. NIH AG09464 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION Amyloid plaques are a major pathological hallmark of Alzheimer's disease. The principle component of amyloid plaques, Aβ,¹ is derived by proteolytic processing of the Alzheimer amyloid protein precursor (APP)^2,3. APP is a Type I integral membrane protein expressed as three major alternatively spliced isoforms of 695, 751 and 770 amino acids in length. The 695 amino acid isoform is most abundant in neurons. APP is processed in two pathways by at least three unidentified proteases known as the α- ,β- and γ-secretases.^4'6 Aβ is generated from cleavage by both β- and γ-secretase. α- secretase cleaves APP within the Aβ domain, precluding Aβ formation. The majority of processed APP is cleaved by -secretase, releasing the large extracellular domain referred to as APPs. Both APPs and Aβ are secreted by normal cells.^4"8 However, the cells in Alzheimer patients abnormally allow the aggregation and accumulation of Aβ, and thus facilitate the formation of the amyloid plaques.

What is presently needed is a facile, reliable screening assay to screen agents which have activity in the inhibition and/or prevention of Alzheimer's Disease.

SUMMARY OF THE INVENTION The present invention relates to applicants' discovery that a polypeptide defining a Phosphotyrosine Interaction (PI) domain, conserved variants thereof, or fragments thereof, interacts with the cytoplasmic domain of APP, and that this binding regulates secretion of APP fragments. By intervening in this interaction, APP processing and trafficking, and hence the progression and/or onset of Alzheimer's disease, can be inhibited or prevented.

Hence, the present invention extends to a cell line for screening agents capable of modulating the interaction of a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof with APP, wherein said cell line over-expresses both APP and a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof. A cell line of the present invention can be derived from Madin-Darby canine kidney (MDCK) cells. However, cell lines of the present invention can also be derived from other mammalian cell lines presently known, or hereinafter discovered. In one embodiment of the invention, the cell line over expresses both APP and rat Fe65 (SEQ ID NO: 7), conserved variants thereof, or fragments thereof, as an example of a polypeptide defining a Pl-domain.

The present invention further extends to a method for screening for agents capable of modulating the interaction of a polypeptide defining a Pl-domain, conserved variants thereof, or fragments thereof, and the cytoplasmic domain of amyloid precursor protein (APP), wherein the method comprises:

(a) providing a mammalian cell line which over-expresses both APP and a polypeptide defining a Pl-domain, conserved variants thereof, or fragments thereof, in culture;

(b) optionally, detectably labeling APP and the polypeptide defining a PI- domain, conserved variants thereof, or fragments thereof, produced by the mammalian cells during anabolism; then (c) allowing the mammalian cells to continue metabolizing in a suitable label-free environment;

(d) contacting the mammalian cells at the start, or during step (c) with an agent suspected of being capable of modulating the interaction between the optionally detectably labeled polypeptide defining a PI domain, conserved variants thereof or fragments thereof, and the cytoplasmic domain of APP;

(e) lysing the mammalian cells;

(f) measuring optionally labeled APPs and Aβ produced; and

(g) comparing the measurements of step (f) with control cells not treated with the suspected agent.

Any polypeptide defining a PI domain, conserved variants thereof, or fragments thereof, has applications in the present invention. The PI was initially described by Bork, P. And Margolis, B. , Cell 80:693-694 (March, 1995), which is incorporated by reference herein in its entirety. The domain is characterized by: having at least 150 amino acid residues; having a putative secondary structure generally comprising seven β-sheets and, in most PI domains, at least two α-helices in the order cd ' (when present), βl, αl, β2- 7, α2; and having a putative globular tertiary structure composed of a β-sandwich with two nearly orthogonal antiparallel β-sheets and at least two α-helices.

Examples of polypeptides defimng a Pl-domain, conserved variants thereof, or fragments thereof, which have applications in the present invention include, but are not limited to: human She comprising an amino acid sequence of Figure 3 (SEQ ID NO:l), conserved variants thereof or fragments thereof; murine She comprising an amino acid sequence of Figure 4 (SEQ ID NO: 2) conserved variants thereof or fragments thereof; human Sck comprising an amino acid sequence of Figure 5 (SEQ ID NO: 3) conserved variants thereof or fragments thereof; murine xl 1 comprising an amino acid sequence of Figure 6 (SEQ ID NO:4) conserved variants thereof or fragments thereof; human xl 1 comprising an amino acid sequence of Figure 7 (SEQ ID NO:5) conserved variants thereof or fragments thereof;

Numb comprising an amino acid sequence of Figure 8 (SEQ ID NO:6), conserved variants thereof, or fragments thereof; rat Fe65 comprising an amino acid sequence of Figure 9 (SEQ ID NO:7), conserved variants thereof, or fragments thereof;

Disabled comprising an amino acid sequence of Figure 10 (SEQ ID NO:8), conserved variants thereof or fragments thereof human DOC-2 comprising an amino acid sequence of Figure 11 (SEQ ID NO:9), conserved variants thereof, or fragments thereof; murine p96 comprising an amino acid sequence of Figure 12 (SEQ ID NO:10), conserved variants thereof or a fragment thereof; human Fe65 comprising an amino acid sequence of Figure 13 (SEQ ID NO:l 1), conserved variants thereof or fragments thereof; human FE65-like protein comprising an amino acid of Figure 14 (SEQ ID NO: 12), conserved variants thereof, or a fragment thereof; rat Fe65L2 protein comprising an amino acid sequence of figure 15 (SEQ ID NO: 13), conserved variants thereof, or fragments thereof; or human IRS-1 protein comprising an amino acid sequence of Figure 16 (SEQ ID NO: 14), conserved variants thereof, or fragments thereof.

In a preferred embodiment of the present invention, a polypeptide defining a PI domain, conserved variants thereof or fragments thereof is human Fe65, comprising an amino acid sequence of Figure 14 (SEQ ID NO:12), conserved variants thereof or fragments thereof. Moreover, the present invention extends to a mammalian cell line which over expresses a polypeptide defining a PI domain, conserved variants thereof or fragments thereof, and APP. In one embodiment of the present invention, the mammalian cell line comprises MDCK cells, which over-express APP and rat Fe65 (SEQ ID NO:7), conserved variants thereof, or fragments thereof.

Methods which can be used to measure optionally labeled APP and polypeptide defining a PI domain conserved variants thereof or fragments thereof include, but are not limited to, immunoprecipitation, or immunoassays, such as sandwich ELISA.

A further aspect of the present invention involves a therapeutic composition comprising an agent which modulates the interaction of a polypeptide defining a PI domain conserved variants thereof or fragments thereof, and the cytoplasmic domain of an amyloid precursor protein (APP), and a pharmaceutically acceptable carrier thereof.

The present invention further extends to a therapeutic composition as described above, wherein the agent is a non-peptidyl agent.

Naturally, the present invention extends to a method for treating and/or preventing neurodegenerative disease in a mammal in need of such therapy, which comprises administering a therapeutically effective amount of a therapeutic composition comprising an agent capable of modulating the interaction of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof, and the cytoplasmic domain of amyloid precursor protein (APP), and a pharmaceutically acceptable carrier thereof. Moreover, the agent can be a non-peptidyl agent.

Examples of a neurodegenerative disease which can be treated or prevented with the present invention include, but are not limited to, Alzheimer's disease or dementia. Furthermore, a method for treating and/or preventing neurodegenerative disease, as disclosed herein, extends to any agent that modulates the binding of any polypeptide defining a PI domain, conserved variants thereof or fragments thereof, to the cytoplasmic domain of an amyloid precursor protein. The PI domain is characterized by: having at least 150 amino acid residues; having a putative secondary structure generally comprising seven β-sheets and, in most PI domains, at least two α-helices in the order αl ' (when present), βl, αl, β2- 7, α2; and having a putative globular tertiary structure composed of a β-sandwich with two nearly orthogonal antiparallel β-sheets and at least two α-helices.

Examples of polypeptides defining a PI domain, conserved variants thereof, or fragments thereof, comprises: human She comprising an amino acid sequence of Figure 3 (SEQ ID NO:l), conserved variants thereof or fragments thereof; murine She comprising an amino acid sequence of Figure 4 (SEQ ID NO: 2) conserved variants thereof or fragments thereof; human Sck comprising an amino acid sequence of Figure 5 (SEQ ID NO: 3) conserved variants thereof or fragments thereof; murine xl 1 comprising an amino acid sequence of Figure 6 (SEQ ID NO:4) conserved variants thereof or fragments thereof; human xl 1 comprising an amino acid sequence of Figure 7 (SEQ ID NO:5) conserved variants thereof or fragments thereof;

Numb comprising an amino acid sequence of Figure 8 (SEQ ID NO:6), conserved variants thereof, or fragments thereof; rat Fe65 comprising an amino acid sequence of Figure 9 (SEQ ID NO:7), conserved variants thereof, or fragments thereof; Disabled comprising an amino acid sequence of Figure 10 (SEQ ID NO:8), conserved variants thereof or fragments thereof human DOC-2 comprising an amino acid sequence of Figure 11 (SEQ ID NO:9), conserved variants thereof, or fragments thereof; murine p96 comprising an amino acid sequence of Figure 12 (SEQ ID NO: 10), conserved variants thereof or a fragment thereof; human Fe65 comprising an amino acid sequence of Figure 13 (SEQ ID NO:l 1), conserved variants thereof or fragments thereof; human FE65-like protein comprising an amino acid of Figure 14 (SEQ ID NO : 12), conserved variants thereof, or a fragment thereof; rat Fe65L2 protein comprising an amino acid sequence of figure 15 (SEQ ID NO: 13), conserved variants thereof, or fragments thereof; or human IRS-1 protein comprising an amino acid sequence of Figure 16 (SEQ ID NO: 14), conserved variants thereof, or fragments thereof.

Accordingly, one object of the present invention is to exploit Applicants' discovery that intervening in the binding of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof with the cytoplasmic domain of APP decreases APP processing, and hence decreases the amount of Aβ produced so that the progression and/or onset of Alzheimer's disease can be inhibited or prevented.

Another object of the present invention is to provide a reliable method for screening agents which can interfere with the protein-protein interaction of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof and APP. Such agents have applications in the prevention and/or inhibition of a neurodegenerative disease, such as Alzheimer's Disease or dementia.

Yet another object of the present invention is to provide a mammalian cell line which over-expresses APP and a polypeptide defining a PI domain, conserved variants thereof or fragments thereof so as to provide a facile and convenient method for identifying agents which modulate the interaction between APP and a polypeptide defining a PI domain, conserved variants thereof or fragments thereof.

Yet still another object is to provide a therapeutic composition for treating and/or preventing Alzheimer's disease, comprising an agent which can modulate the protein- protein interaction of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof, with APP, along with a pharmaceutically acceptable carrier thereof.

Still yet another object of the present invention is to provide a method for treating and/or preventing a neurodegenerative disease, such as Alzheimer's Disease, comprising administering a therapeutically effective amount of a therapeutic composition of the present invention.

Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing description which proceeds with references to the following illustrative drawings.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is a graph showing that over-expression of Fe65 causes an increase in secretion of APPs from MDCK cells. In these experiments, cells were pulsed with [³⁵S] -methionine for two hours, followed by a two-hour chase. APPs was immunoprecipitated from the chase medium with the monoclonal antibody, 6E10. Immunoprecipitated APPs was quantified by Phosphorlmager. Values were normalized to total labeled cellular holoAPP immunoprecipitated, with the polyclonal antibody 369, from lysates collected before the chase. The data shown represents the mean +/-SE pooled from three experiments (n= ll). Figure 2A is a graph showing that over-expression of Fe65 causes an increase in Aβ secretion from MDCK cells. ELISA of conditioned medium from MDCK cells reveals an increase in Aβ secretion upon Fe65 expression. For these experiments, cells were incubated in serum-free medium for four hours at 37C. Then, the medium was collected and subjected to a sandwich ELISA for Aβ using 6E10 as the capture antibody and 4G8 as the detection antibody. The values obtained are normalized to total cellular holoAPP determined by immunoblotting with 369. The results plotted represent the mean +/-S.E. (n= 12) for two experiments.

Figure 2B is a gel showing the effect of Fe65 on Aβ secretion in the MDCK cells was confirmed by immunoprecipitation. These experiments were performed essentially as described above for APPs except Aβ was immunoprecipitated with both 6E10 and 4G8. The data shown are from a representative experiment.

Figure 3 is the amino acid sequence of human She.

Figure 4 is the amino acid sequence of murine She.

Figure 5 is the amino acid sequence of human Sck.

Figure 6 is the amino acid sequence of murine xll.

Figure 7 is the amino acid sequence of human xl l.

Figure 8 is the amino acid sequence of Numb.

Figure 9 is the amino acid sequence of rat Fe65.

Figure 10 is the amino acid sequence of Disabled.

Figure 11 is the amino acid sequence of human DOC-2.

Figure 12 is the amino acid sequence of murine p96.

Figure 13 is the amino acid sequence of human Fe65.

Figure 14 is the amino acid sequence of human Fe65-like protein.

Figure 15 is the amino acid sequence of rat Fe65L2 protein.

Figure 16 is the amino acid sequence of human IRS-1 protein. DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employed conventional prognostic, diagnostic, pharmaceutical and biological techniques within the skill of the art. Such techniques are explained fully in the literature.

The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to prevent, and preferably reduced by at least about 30 percent, more preferably by at least 50 percent, most preferably by at least 90 percent, the occurrence of amyloid formation.

The term "domain" is used herein to describe any amino acid sequence which represents a fragment of a larger polypeptide, which fragment has a characteristic sequence, structure, and/or activity.

The amino acid residues described herein are preferred to be in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin-binding is retained by the polypeptide. NH₂ refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, /. Biol. Chem. , 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence: TABLE OF CORRESPONDENCE

SYMBOL AMINO ACID

1 -Letter 3-Letter

Y Tyr tyrosine

G Gly glycine

F Phe phenylalanine

M Met methionine

A Ala alanine

S Ser serine

I He isoleucine

L Leu leucine

T Thr threonine

V Val valine

P Pro proline

K Lys lysine

H His histidine

Q Gin glutamine

E Glu glutamic acid

W Trp tryptophan

R Arg arginine

D Asp aspartic acid

N Asn asparagine

C Cys cysteine

It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino- terminus to carboxy -terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein.

Mutations can be made in a DNA sequence encoding a polypeptide defining a PI domain such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein.

The following is one example of various groupings of amino acids:

Amino acids with nonpolar R groups

Alanine

Valine

Leucine

Isoleucine

Proline Phenylalanine Tryptophan Methionine

Amino acids with uncharged polar R groups

Glycine Serine Threonine Cysteine Tyrosine Asparagine Glutamine

Amino acids with charged polar R groups (negatively charged at pH 6.0)

Aspartic acid Glutamic acid

Basic amino acids (positively charged at pH 6.0)

Lysine Arginine Histidine (at pH 6.0)

Another grouping may be those amino acids with phenyl groups:

Phenylalanine Tryptophan Tyrosine Another grouping may be according to molecular weight (i.e., size of R groups):

Glycine 75

Alanine 89

Serine 105 Proline 115

Valine 117

Threonine 119

Cysteine 121

Leucine 131 Isoleucine 131

Asparagine 132

Aspartic acid 133

Glutamine 146

Lysine 146 Glutamic acid 147

Methionine 149

Histidine (at pH 6.0) 155

Phenylalanine 165

Arginine 174 Tyrosine 181

Tryptophan 204

Particularly preferred substitutions are:

- Lys for Arg and vice versa such that a positive charge may be maintained;

- Glu for Asp and vice versa such that a negative charge may be maintained; - Ser for Thr such that a free -OH can be maintained; and

- Gin for Asn such that a free NH₂ can be maintained. Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly "catalytic" site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces β-turns in the protein's structure.

Two amino acid sequences are "substantially homologous" when at least about 70% of the amino acid residues (preferably at least about 80% , and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions.

The screening assays of the present invention are based on cell lines which over-express both APP and a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof derived from a mammalian cell line. Any mammalian cell line presently known or subsequently discovered can be used as the basis for a cell line of the present invention. In one embodiment, a cell line of the present invention is derived from Madin-Darby canine kidney (MDCK) cells.

More particularly, cell lines of the present invention can be used to examine the effects of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof on processing of APP. In an embodiment of the present invention, the mammalian cell line comprises Madin-Darby canine kidney (MDCK) cells stably expressing the 695 amino acid isoform of APP¹² (MDCK-APP) which are transfected with cDNA encoding a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof and selected for puromycin resistance. Furthermore, a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof, whose effect on processing of APP was studied in this embodiment of the present invention, and is used in a method to screen for agents that modulate the interaction of a polypeptide defining a PI domain and the APP protein of the present invention is Fe65, a brain enriched protein of unknown function which binds to the NPTY sequence in the cytoplasmic tail of APP. Fe65 contains two types of protein-protein interaction domains, a WW domain, and a PI domain. Binding of Fe65 to APP occurs through the second PI domain of Fe65. The amino acid sequence of Fe65 is set forth in Figure 10 (SEQ ID NO:8). However, as explained above, any polypeptide defining a PI domain, conserved variants thereof, or fragments thereof, has applications in the present invention.

In this embodiment of the present invention, doubly stable clonal cell lines (MDCK- APP/Fe65) were isolated using cloning rings, and cells expressing high levels of Fe65 were identified by immunoblotting with antibodies raised against the WW domain of human Fe65.

Immunoprecipitation of APPs from conditioned medium of [³⁵S] -methionine pulse- labeled MDCK-APP and MDCK-APP/Fe65 cells shows that over-expression of Fe65 increases secretion of APPs (Figure 1). Secretion of APPs derived from newly synthesized APP is elevated by 67% (+/-14%) in an MDCK-APP/Fe65 cell line. Similar results were seen with another MDCK cell line over-expressing Fe65 when compared to transfection with empty vector alone. Interestingly, stable over-expression of Fe65 also caused enhanced APPs secretion from an MDCK cell line transiently transfected with APP-751 (data not shown). Thus, Fe65 causes an increase in APPs secretion from both neuronal and non-neuronal isoforms of APP.

Since the release of APP increased, it was expected that Aβ secretion would decrease. Surprisingly, Aβ secretion increased approximately four-fold from MDCK-APP/Fe65 cells in both sandwich ELISA (Figure 2A) and immunoprecipitation (Figure 2B) experiments when compared to cells over-expressing APP alone. Similar results were seen in another MDCK cell line over-expressing Fe65 (data not shown). Disclosed herein is the first demonstration of an effect of a direct protein-protein interaction with APP on the proteolytic processing of APP. The results presented are contrary to conventional wisdom: the effects on Aβ and APPs are similar, whereas they were expected to be in opposite directions. Since the plasma membrane is a subcellular compartment at or near which both APPs and Aβ are thought to be produced, it is possible that a polypeptide defining a PI domain, conserved variants thereof or fragments thereof such as Fe65, may either increase the amount of APP transported from the Golgi to the plasma membrane or the amount of APP recycled back to the cell surface after endocytosis.

Moreover, since the over-expression of a polypeptide defining a PI domain causes a profound increase in Aβ secretion, agents or drugs that inhibit the interaction of a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof, can be used to inhibit Aβ secretion in the brain, preventing and/or retarding the formation of plaques. In addition, agents suspected of having the desired activity can be screened for both effects on the interaction of APP with polypeptide defining a PI domain, such as Fe65, conserved variants thereof or fragments thereof, and effects on the Fe65- dependent increase in secretion of Aβ. These agents or drugs are typically formulated in compositions for administration to mammalian patients in need of such therapy.

The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e. , carrier, or vehicle.

The therapeutic compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, and the severity of the condition under treatment. Precise amount of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosages may range from about 0.1 to 20, preferably about 0.5 to about 10, and more preferably 1 to 5 milligrams of active ingredient per kilogram body weight of individual per day, and depend on the route of administration. Typically, the unit dosage form contains from about 0.5 mg to about 750 mg depending on the activity of the particular Fe65 modulator being utilized as the active ingredient.

EXAMPLES The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

Methods

Constructs of polypeptides defining a PI domain. In this Example, rat Fe65 (SEQ ID NO: 7) was selected as a non-limiting example of a polypeptide defining a PI domain. As explained above, any polypeptide defining a PI domain, conserved variants thereof, or fragments thereof, have applications in the present invention. Rat Fe65 was subcloned into the mammalian expression vector, pcDNA3 (Invitrogen) by PCR using rat Fe65 in pGEX as a template. The 5' PCR primer encoded an amino terminal FLAG epitope tag.

Stable Cell Lines. MDCK-695 cells¹² were transfected in 10 cm diameter plates using the calcium phosphate transfection system (Gibco) essentially following the manufacturers instructions. Each plate was transfected with 15 g of rat Fe65 in pcDNA3 and 5 μg of pPUR (Clontech), a selection vector containing a puromycin resistance gene. After selection with 2.5 μg/ml puromycin (Clontech), individual clones were isolated using cloning rings. Cell lines expressing high levels of rat Fe65 were identified by immunoblotting with polyclonal antibodies raised against a GST- fusion protein of amino acids 201-240 of rat Fe65. These cells are maintained in DMEM containing 200 μg/ml of G418 (Gibco) and 1 μg/ml of puromycin.

Immunoprecipitation. Immunoprecipitation was performed essentially as described previously.¹³ Briefly, cells were plated at a density of 5 X 10* cells/cm². After washing, they were incubated with [³⁵S]-methionine (NEN) in methionine-free

Dulbeco's Modified Eagle Medium (DMEM, Gibco) for two hours at 37° C, followed by a two-hour chase at 37° C in complete DMEM. APPs generated by α-cleavage and Aβ were immunoprecipitated from the chase medium with monoclonal antibodies 6E10 and 4G8 and agarose-linked goat anti-mouse IgG (American Qualex). Immunoprecipitates were separated by SDS-PAGE and quantified by Phosphorlmager (Molecular Dynamics). Sister cultures were lysed with 1 % NP40 in phosphate- buffered saline (PBS) immediately after labeling. Values obtained for APPs and Aβ were normalized to total labeled cellular holo/APP immunoprecipitated from these lysates with the polyclonal antibody, 369, raised against the cytoplasmic domain of APP.¹⁴

ELISA. For ELISAs, the cell medium was changed to fresh serum-free DMEM and incubated for four hours at 37 °C. Then the conditioned medium was collected and subjected to sandwich ELISA for Aβ using 6E10 as the capture antibody and 4G8 as the detection antibody. A more detailed description of the ELISA can be found elsewhere.¹⁵ Samples were incubated with biotinylated antibody and anti-biotin alkaline phosphatase conjugate. After 1 hour, alkaline phosphatase substrate was added and Aβ was quantified using the Dynatech Plate Reader. The values obtained were normalized to total cellular holoAPP determined by immunoblotting with 369 and [¹²⁵I] -protein A (Amersham) followed by Phosphorlmager quantification.

While the invention has been described and illustrated herein by reference to various specific material, procedures and examples, it is understood that the invention is not restricted to the particular material combinations of material, and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art.

The following is a list of documents related to the above disclosure and particularly to the experimental procedures and discussions. The documents and all other documents cited throught this Application should be considered as incorporated by reference in their entirety.

REFERENCES

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2. Goldgaber, D.; Lerman, M.I.; McBride, O.W.; and Saffiotti, U.X., D.C. , Science, 235, 877-880 (1987).

3. Kang, J.; Lamaire, H.-G. ; Unterbeck, A. et al., Nature, 325, 733-736 (1987).

4. Weidmann, A.; Konig, G.; Bunke, D. ; Fischer, P.; Salbaum, J.M.X. ; Masters, C.L. ; and Beyreuther, K., Cell 57, 115-126 (1989).

5. Esch, F.S. ; Keim, P.S.; Beattie, E.C. et al., Science 248, 1122-1124 (1990).

6. Seubert, P.; Vigo-Pelfrey, C; Esch, F. et al, Nature 359, 325-327 (1992).

7. Haass, C; Koo, E.H.; Mellon, A.; Hung, A.Y.; and Selkoe, D.J., Nature 357, 500-502 (1992).

8. Shoji, M. ; Golde, T.E. ; Ghisco, J. et al., Science 258, 126-129 (1992).

9. Duilio, A. ; Zambrano, N.; Mogavero, A.R. ; Ammendola, R.; Cimino, F. ; and Russo, T., Nucleic Acids Research 19, 5269-5274 (1991).

10. Fiore, F.; Zambrano, N. ; Minopoli, G. ; Donini, V.; Duilio, A.; and Russo, T., Journal of Biological Chemistry 270, 30853-30856 (1995).

11. Zambrano, N. ; Buxbaum, J.D.; Minopoli, G.X.; Fiore, F. et al., Journal of Biological Chemistry 272, 6399-6405 (1997). 12. Haass, C; Koo, E.H. ; Teplow, D.B.; and Selkoe, D.J. , Proceedings of the National Academy of Sciences of the United States of America 91, 1564-1568 (1994).

13. Buxbaum, J.D.; Koo, E.H.; and Greengard, P. , Proceedings of the National Academy of Sciences of the United States of America 90, 9195-9198 (1993).

14. Caporaso, G.L. ; Gandy, S.L.; Buxbaum, J.D.; and Greengard, P. , PNAS 89, 2252-2256 (1992).

15. Barelli, H.; Lebeau, A. ; Vizza Vona, J. et al., Molecular Medicine (in press).

Many other variations and modifications of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The above-described embodiments and examples are, therefore, intended to merely exemplary, and all such variations and modifications are intended to be included within the scope of the invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A cell line for screening agents capable of modulating the interaction of a polypeptide defining a PI domain, conserved variants thereof, or fragments thereof with APP, wherein said cell line over-expresses both APP and polypeptide defining a PI domain, conserved variants thereof, or fragments thereof.

2. The cell line of Claim 1, wherein said cell line is derived from Madin-Darby canine kidney (MDCK) cells.

3. The cell line of Claim 1, wherein said PI domain is characterized by: a) having at least 150 amino acid residues; b) having a putative secondary structure generally comprising seven β- sheets; c) having at least two α-helices in the order αl ' (when present), βl, αl, β2-7, α2; and d) having a putative globular tertiary structure composed of a β- sandwich with two nearly orthogonal antiparallel β-sheets and at least two α-helices.

4. The cell line of Claim 1, wherein said polypeptide defining a PI domain, conserved variants thereof, or fragments thereof is selected from the group consisting of: human She comprising an amino acid sequence of Figure 3 (SEQ ID NO: l), conserved variants thereof or fragments thereof; murine She comprising an amino acid sequence of Figure 4 (SEQ ID NO: 2) conserved variants thereof or fragments thereof; human Sck comprising an amino acid sequence of Figure 5 (SEQ ID NO: 3) conserved variants thereof or fragments thereof; murine xl 1 comprising an amino acid sequence of Figure 6 (SEQ ID NO:4) conserved variants thereof or fragments thereof; human xl 1 comprising an amino acid sequence of Figure 7 (SEQ ID NO: 5) conserved variants thereof or fragments thereof;

Disabled comprising an amino acid sequence of Figure 10 (SEQ ID NO:8), conserved variants thereof or fragments thereof human DOC-2 comprising an amino acid sequence of Figure 11 (SEQ ID NO:9), conserved variants thereof, or fragments thereof; murine p96 comprising an amino acid sequence of Figure 12 (SEQ ID NO: 10), conserved variants thereof or a fragment thereof; human Fe65 comprising an amino acid sequence of Figure 13 (SEQ ID NO:l 1), conserved variants thereof or fragments thereof; human FE65-like protein comprising an amino acid of Figure 14 (SEQ ID

NO: 12), conserved variants thereof, or a fragment thereof; rat Fe65L2 protein comprising an amino acid sequence of figure 15 (SEQ ID NO: 13), conserved variants thereof, or fragments thereof; and human IRS-1 protein comprising an amino acid sequence of Figure 16 (SEQ ID NO: 14), conserved variants thereof, or fragments thereof.

5. A method for screening for agents capable of modulating the interaction of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof, and the cytoplasmic domain of amyloid precursor protein (APP), wherein the method comprises: (a) providing a mammalian cell line which over-expresses both APP and said polypeptide defining a PI domain, conserved variants thereof or fragments thereof in culture;

(b) optionally, radioactively labeling said APP and said polypeptide defining a PI domain, conserved variants thereof or fragments thereof produced by said cells during anabolism; then (c) allowing the mammalian cells to continue metabolizing in a suitable label-free environment;

(d) contacting the mammalian cells at the start, or during step (c) with an agent suspected of being capable of modulating the interaction between said polypeptide defining a PI domain, conserved variants thereof or fragments thereof and the cytoplasmic domain of amyloid precursor protein;

(e) lysing the mammalian cells;

(f) measuring optionally labeled APPs and Aβ produced; and

6. The method for screening agents of Claim 5, wherein said PI domain of said polypeptide defining a PI domain, or fragment thereof defining a PI domain, is characterized by: a) having at least 150 amino acid residues; b) having a putative secondary structure generally comprising seven β- sheets; c) having at least two α-helices in the order αl ' (when present), βl, αl, β2-7, α2; and d) having a putative globular tertiary structure composed of a β- sandwich with two nearly orthogonal antiparallel β-sheets and at least two α-helices.

7. The method for screening agents of Claim 5, wherein said polypeptide defining a PI domain, conserved variants thereof, or fragments thereof, is selected from the group consisting of: human She comprising an amino acid sequence of Figure 3 (SEQ ID NO: l), conserved variants thereof or fragments thereof; murine She comprising an amino acid sequence of Figure 4 (SEQ ID NO: 2) conserved variants thereof or fragments thereof; human Sck comprising an amino acid sequence of Figure 5 (SEQ ID NO: 3) conserved variants thereof or fragments thereof; murine xl 1 comprising an amino acid sequence of Figure 6 (SEQ ID NO:4) conserved variants thereof or fragments thereof; human xl 1 comprising an amino acid sequence of Figure 7 (SEQ ID NO:5) conserved variants thereof or fragments thereof; Numb comprising an amino acid sequence of Figure 8 (SEQ ID NO: 6), conserved variants thereof, or fragments thereof; rat Fe65 comprising an amino acid sequence of Figure 9 (SEQ ID NO:7), conserved variants thereof, or fragments thereof;

Disabled comprising an amino acid sequence of Figure 10 (SEQ ID NO:8), conserved variants thereof or fragments thereof human DOC-2 comprising an amino acid sequence of Figure 11 (SEQ ID NO:9), conserved variants thereof, or fragments thereof; murine p96 comprising an amino acid sequence of Figure 12 (SEQ ID NO: 10), conserved variants thereof or a fragment thereof; human Fe65 comprising an amino acid sequence of Figure 13 (SEQ ID

NO:l 1), conserved variants thereof or fragments thereof; human FE65-like protein comprising an amino acid of Figure 14 (SEQ ID NO: 12), conserved variants thereof, or a fragment thereof; rat Fe65L2 protein comprising an amino acid sequence of figure 15 (SEQ ID NO: 13), conserved variants thereof, or fragments thereof; and human IRS-1 protein comprising an amino acid sequence of Figure 16 (SEQ ID NO: 14), conserved variants thereof, or fragments thereof.

8. The method for screening agents of Claim 5, wherein said mammalian cell line is derived from Madin-Darby canine kidney (MDCK) cells.

9. The method for screening agents of Claim 8, wherein said polypeptide defining a PI domain, conserved variants thereof, or fragments thereof, is rat Fe65 comprising an amino acid sequence of SEQ ID NO:7.

10. The method according to Claim 5 wherein the measurement of step (f) is by sandwich ELISA.

11. The method according to Claim 5 wherein the measurement of step (f) is by immunoprecipitation .

12. A therapeutic composition comprising: an agent capable of modulating the interaction of a polypeptide defining a PI domain, conserved variants thereof or fragments thereof, and the cytoplasmic domain of an amyloid precursor protein (APP); and a pharmaceutically acceptable carrier thereof.

13. The therapeutic composition of Claim 12, wherein said agent is non- peptidyl.

14. The therapeutic composition of Claim 12, wherein said PI domain is characterized by:

a) having at least 150 amino acid residues: b) having a putative secondary structure generally comprising seven β- sheets; c) having at least two α-helices in the order αl ' (when present), βl, αl, β2-7, α2; and d) having a putative globular tertiary structure composed of a β- sandwich with two nearly orthogonal antiparallel β-sheets and at least two α-helices.

15. The therapeutic composition of Claim 12, wherein said polypeptide defining a PI domain is selected from the group consisting of: human She comprising an amino acid sequence of Figure 3 (SEQ ID NO:l), conserved variants thereof or fragments thereof; murine She comprising an amino acid sequence of Figure 4 (SEQ ID NO: 2) conserved variants thereof or fragments thereof; human Sck comprising an amino acid sequence of Figure 5 (SEQ ID NO: 3) conserved variants thereof or fragments thereof; murine xl 1 comprising an amino acid sequence of Figure 6 (SEQ ID NO:4) conserved variants thereof or fragments thereof; human xl 1 comprising an amino acid sequence of Figure 7 (SEQ ID NO:5) conserved variants thereof or fragments thereof;

Disabled comprising an amino acid sequence of Figure 10 (SEQ ID NO:8), conserved variants thereof or fragments thereof human DOC-2 comprising an amino acid sequence of Figure 11 (SEQ ID

NO:9), conserved variants thereof, or fragments thereof; murine p96 comprising an amino acid sequence of Figure 12 (SEQ ID NO: 10), conserved variants thereof or a fragment thereof; human Fe65 comprising an amino acid sequence of Figure 13 (SEQ ID NO: 11), conserved variants thereof or fragments thereof; human FE65-like protein comprising an amino acid of Figure 14 (SEQ ID NO: 12), conserved variants thereof, or a fragment thereof; rat Fe65L2 protein comprising an amino acid sequence of figure 15 (SEQ ID NO: 13), conserved variants thereof, or fragments thereof; and human IRS-1 protein comprising an amino acid sequence of Figure 16 (SEQ

ID NO: 14), conserved variants thereof, or fragments thereof.

16. The therapeutic composition of Claim 12, wherein said polypeptide defining a PI domain, conserved variants thereof, or fragments thereof is rat Fe65 (SEQ ID NO: 7), conserved variants thereof or fragments thereof.

17. A method for treating and/or preventing neurodegenerative disease in a mammal in need of such therapy which comprises administering a therapeutically effective amount of a therapeutic composition of Claim 12.