CA2244412A1 - Nucleic acids and proteins related to alzheimer's disease, and uses therefor - Google Patents

Abstract

The present invention describes the identification, isolation, sequencing and characterization of several human genes which interact with the presenilins, mutations in which may lead to Familial Alzheimer's Disease. These presenilin-interacting protein genes may be involved in the pathways which, when affected by mutant presenilins, lead to the development of Alzheimer's Disease. In addition, mutations in the presenilin-interacting protein genes, even in the absence of defects in the presenilins, may be causative of Alzheimer's Disease. Nucleic acids and proteins comprising or derived from the presenilin-interacting proteins are useful in screening and diagnosing Alzheimer's Disease, in identifying and developing therapeutics for treatment of Alzheimer's Disease, and in producing cell lines and transgenic animals useful as models of Alzheimer's Disease.

Description

NUCLEIC ACIDS AND PROTEINS
R~LATED TO ~T 71:~F.Tl~IER'S DISEASE, AND USE~ THEREFOR
Field of the Invention The present invention relates generally to the field of neurological and physiological dysfunctions associated with Alzheimer's Disease. More particularly, the invention is concerned with the identification, isolation and cloning of genes which are associated with Alzheimer's Disease, as well as their corresponding transcripts and protein products. The present invention also relates to methods for detecting and diagnosing carriers of normal and mutant alleles of these genes, to methods for tietecting and diagnosing Alzheimer's Disease, to methods of identifying other genes and proteins related to, or interacting with, the genes and proteins of the invention, to methods of screening for potential therapeutics for Alzheimer's Disease, to methods of treatment for Alzheimer's Disease, and to cell lines and animal models useful in sclc~nil~g for and ev~ ting potentially useful therapies for Alzheimer's Disease.
Back~round of the Invention Alzheimer's Disease (AD) is a degenerative disorder of the human central nervous system characterized by progressive memory;~p~;....ent and cognitive andintellectual decline during mid to late adult life (K~t7m~n, 1986). The disease is accompanied by a constellation of neuro-pathologic Çe~Lul~s principal amongst which are the presence of extracellular amyloid or senile plaques, and neurofibrillary tangles in neurons. The etiology of this disease is complex, although in some families it appears to be inherited as an autosomal dominant trait. Linkage studies have identified three genes associated with the development of AD~ amyloid precursor protein (APP) (Chartier-Harlin et al., 1991; Goate et al., 1991; Murrell et al., 1991;
Karlinsky et al., 1992; Mullan et al., 1992), prt?s~nil;n-l (PS-1) (Sherrington, 1995), and presenilin-2 (PS-2) (Rogaev, 1995, and Levy-Lahad, 1995).
The presenilins are multi-sp~nning membrane proteins which were described in substantial detail in PCT Publication W096/34099, the entire disclosure of which is incorporated herein by reference. Although the functions of the presenilins are unknown, a number of autosomal dolllhl~ll presenilin mutations have been identified S~ UTE SHEET ~RULE 26) which are strongly associated with the development of early-onset, a~ essive, Familial Alzheimer's Disease (FAD).
The present disclosure describes the identification, isolation, sequencing and characterization of several human genes which interact with the presenilins, mutations 5 in which may lead to FAD. These prçsP!nilin-interacting protein genes may be involved in the pathways which, when affected by mutant presenilin~, lead to thedevelopment of ~17heim~r's Disease. In addition, mutations in the presP.nilin-interacting protein genes, even in the absence of defects in the pres~nilin~, may be causative of ~l~heimer's Disease.
Summarv of the Invention The present invention is based, in part, upon the identification, isolation, seql~çncing arld characterization of several hurnan genes, referred to herein ~
"presenilin~ ,LdcLillg protein genes" or "PS-interacting protein genes." The products of these genes are believed to interact in vivo with the human pres~.nilin-l proteins 15 and, therefore, are implicated in the bioeh~mic~l pathways which are affected in Al7h~im~r's Disease. Each of these genes, therefore, ples~llL~ a new therapeutic target for the treatrnent of ~ heimer's Disease. In addition, PS-interacting protein nucleic acids, PS-interacting proteins and peptides, antibodies to the PS-interacting proteins, cells L~ o~ ed with PS-interacting protein nucleic acids, and transgenic ~nim~l~20 altered with PS-interacting protein nucleic acids, all possess various utilities, as described herein, for the ~i~gnosi~ therapy and contimled investigation of ~l~hejmer's Disease and related disorders.

Thus, it is one object of the invention to provide isolated nucleic acids encoding at least a PS-interacting domain of a PS-interacting protein. These PS-25 interacting proteins include m~mm~ n S5a subunits of the 26S proteasome, theGT24 protein, the pO07 1 protein, the Rab l l protein, the retinoid X receptor-~, the cytoplasmic chaperonin, and several sequences identified herein as clones Y2H35,Y2H171, and Y2H41. Preferred nucleotide and amino acid sequences are provided herein. It is another object of the invention to provide probes and primers for these SlJ~ )TE SHEET (RULE 26) PS-interacting protein genes, and to provide nucleic acids which encode small antigenic det~-.-,i"~"l~ ofthese genes. Therefore, preferred embollim~nt~ include sequences of at least 10, 15 or 20 consecutive nucleotides selected from the disclosed se~uences.

Using the nucleic acid sequences and antibodies disclosed and enabled herein, methods for identifying allelic variants or heterospecific homologues of a human PS-interacting protein and gene are provided. The methods may be practicedusing nucleic acid hybridization or amplification techniques, immllnoch~mic:~l techniques, or any other technique known in the art. The allelic variants may include other nor~nal human alleles as well as mutant alleles of the PS-interacting protein genes which may be causative of Alzheimer's Disease. The heterospecific homologues may be from other m~mm~ n species, such as mice, rats, dogs, cats or non-hurnan primates, or may be from invertebrate species, such as Drosophila or C.
ele~ans. Thus, it is another object of the invention to provide nucleic acids which encode allelic or heterospecific variants of the disclosed sequences, as well as the allelic or heterospecific proteins encoded by them.

The it another object of the invention to provide vectors, and particularly cs:iion vectors, which include any of the above-described nucleic acids. ~t is afurther object of the invention to provide vectors in which PS-interacting protein nucleic acid sequences are operably joined to exogenous regulatory regions to produce altered patterns of ~ es~ion, or to exogenous coding regions to produce fusion proteins. Conversely, it is another object to provide nucleic acids in which PS-interacting protein regulatory regions are operably joined to exogenous coding regions, including standard marker genes, to produce constructs in which the regulation of PS-interacting protein genes may be studied and used in assays fortherapeutics.

It is another object of the invention to provide host cells and transgenic ~nim~l~ which have been transformed with any of the above-described nucleic acids S~:i 111 UTE SHEET (RULE 26) of the invention. The host cells may be prokaryotic or eukaryotic cells and, in particular, may be garnetes, zygotes, fetal cells, or stem cells useful in producing transgenic animal models. ~.

In particularly plbr~lled embodiments, the present invention provides a non-human animal model for Alzheimer's Disease, in which the genome of the animal, or an ancestor thereof, has been modified by at least one recombinant construct which has introduced one of the following modifications: ( l ) insertion of nucleotide sequences encoding at least a functional domain of a heterospecific normal PS-interacting protein, (2) insertion of nucleotide sequences encoding at least a 1~ functional domain of a heterospecific mut~nt PS-interacting protein, (3) insertion of nucleotide sequences encoding at least a functional domain of a conspecific homologue of a heterospecific mutant PS-interacting protein, and (4) inactivation of an endogenous PS-interacting protein gene. Preferred transgenic animal models are rats, mice, h~m~ters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-human prim~t~s, but in~ tes are also contemplated for certain utilities.

It is another object of the invention to provide methods for producing at least a fi~nctional domain of a PS-interacting protein using the nucleic acids of the invention. In addition, the present invention also provides subst~nti~lly pure preparations of such proteins, including short peptide sequences for used as 2û immunogens. Thus, the invention provides peptides comprising at least 10 or l 5 consecutive amino acid residues from the disclosed and otherwise enabled sequences.
The invention filrther provides subst~nti~lly pure plcp~lions of peptides which compnse at least a PS-interacting domain of a PS-interacting protein, as well assubstantially pure pr~al~ions of the entire proteins. ., Using the substantially pure peptides and proteins enabled herein, the .
invention also provides methods for producing antibodies which selectively bind to a PS-interacting protein, as well as cell lines which produce these antibodies.

SlJ~S 111 ~)TE SHEET (RULE 26) Another object of the present invention is to provide methods of identifying compounds which may have utility in the treatment of Al7heimer's Disease and related disorders. These methods include methods for identifying compounds which can modulate the c~ e:,sion of a PS-interacting protein gene, 5 methods for identifying compounds which can selectively bind to a PS-interacting protein, and methods of identifying compounds which can modulate activity of a PS-interacting protein. These methods may be con~ cte(l in vitro or in vivo~ and may employ the transformed cell lines and transgenic animal models of &e invention. The methods also may be part of a clinical trial in which compounds identified by the 10 methods of the invention are further tested in human subjects.

It is another object of the invention to provide methods of ~ no~ing or screening for inherited forrns of ~l~heimer's Disease by dcl~ if a subiect bears a mutant PS-interacting protein gene. Mutant PS-interacting genes may be detectec~
by assays including direct nucleotide seq~ cin~, probe specific hybri(li7~tion~
15 restriction enzyme digest and mapping, PCR mapping, ligase-m.o~i~tç~l PCR
detection, RNase protection, electrophoretic mobility shif~ detection, or chemic~l mi~m~trh cleavage. ~lt~rn~tively, mutant forms of a PS-interacting protein may be detected by assays including imml-no~ays, protease assays, or electrophoretic mobility assays.

It is also an obJect of the invention to provide pharmaceutical ~J~c~ Lions which may be used in the tre~tn Pnt of Alzheimer's Disease and related disorderswhich result from aberration in biochemic~ lW~S involving the PS-interacting proteins disclosed and enabled herein. Thus, the present invention also providespharmaceutical preparations co~ ing a ~ubsl~llially pure PS-i~ ;ldcli"g protein, an expression vector operably encoding a PS -interacting protein, an expression vector operably encoding a PS-interacting protein ~nticrn.~e sequence, an antibody which selectively binds to a mutant PS-interacting protein, or an antigenic f let~ n~nt of a mutant PS-interacting protein. These ph~ re~ltical preparations may be used to S~J~S 111 ~JTE SHEET (RULE 26) treat a patient bearing a mutant PS-interacting protein gene which is causative of ..
Alzheimer's Disease or related disorders.

These an other objects of the present invention are described more fully in the following specification and appended claims.

Detailed DescriPtion of the Invention I. Definitions In order to facilitate review of the various embo~1imentc of the invention, and an underst~n(1ing of the various elements and con.ctitl~pMt~ used in making and using the invention, the following definitions are provided for particular terms used in 10 the description and appended claims:
Presenilin. As used without filrther modification herein, the terrns "preserlilin" or 'presenilins" mean the pr~?s~onil;n-l (PS 1) and/or the presenilin-2 (PS2) genes/proteins. In particular, the unmodified terms "presenilin" or "presenilin.c" refer to the m~mm~ n PS 1 and/or PS2 genes/proteins and, ~ref~.dbly, the human PS l 15 and/or PS2 genes/proteins as described and disclosed in PCT Publication W096/34099.
Norrnal. As used herein with respect to genes, the terrn "normal" refers to a gene which encodes a no~nal protein. As used herein with respect to proteins, the term "normal" means a protein which perforrns its usual or normal physiological role 20 and which is not associated with, or causative of, a pathogenic condition or state.
Therefore, as used herein, the terrn "normal" is e~ t~ y synonymous with the usual meaning of the phrase "wild type." For any given gene, or corresponding protein, a multiplicity of normal allelic variants may exist, none of which is associated with the development of a pathogenic condition or state. Such norrnal allelic variants include, 25 but are not limited to, variants in which one or more nucleotide substitutions do not result in a change in the encoded amino acid sequence.
Mutant. As used herein with respect to genes, the term "mutant" refers to a gene which encodes a mutant protein. As used herein with respect to proteins, the term "Illu~ " means a protein which does not perform its usual or norrnal SU~S 111 UTE SHEET (RULE 26) W O g7t2~a96 PCT/CA97/OOOSl physiological role and which is associated with, or causative of, a pathogerlic condition or state. Therefore, as used herein, the term "mutant" is essentially synonymous with the terms "dysfunctional," "pathogenic," "disease-ca11~ing," and"deleterious." With respect to the presenilin and pres~niiin-interacting protein genes 5 and proteins of the present invention, the terrn "mutant" refers to genes/proteins bearing one or more nucleotide/amino acid substitutions, insertions and/or deletions which typically lead to the development ofthe syrnptoms of ~17heimer's Disease and/or other relevant inheritable phenotypes (e.g. cerebral hemorrhage, mental retardation, schizophrenia, psychosis, and depression) when expressed in h11m~n~10 This definition is understood to include the various mutations that naturally exist, including but not limited to those disclosed herein, as well as synthetic or recombinant mutations produced by human illlt;- v~ tion. The term "rnutant," as applied to these genes, is not int~n~ie-l to embrace sequence variants which, due to the degeneracy of the genetic code, encode proteins icl~ntic:~l to the normal sequences disclosed or 15 otherwise enabled herein; nor is it int~n(led to embrace sequence variants which, although they encode different proteins, encode proteins which are functionally equivalent to normal proteins.
SubstantiallY Pure. As used herein with respect to proteins (including antibodies) or other pl~dlions, the term "~j..l.s~ lly pure" means that the 20 preparation is ess~nti~lly free of other substances to an extent practical and ~r~iate for its int~nded use. In particular, a protein ~ d~ion is ~u1 ~ 1y pure if it is sufficiently free from other biological constituents so as to be useful in, for example, generating antibodies, sequencing, or producing pha~naceutical pL~aldlions. By techniques well known in the art, ~ul~ lly pure proteins or 25 peptides may be produced in light of the nucleic acid and amino acid sequences disclosed herein. In particular, in light of the nucleic acid and arnino acid sequences disclosed herein, one of ol-;lh~a. y skill in the art may, by application or serial application of well-known methods including HPLC or imm11nQ-affinity chromatography or electrophoretic separation, obtain proteins or peptides of any30 generally feasible purity. Preferably, but not necessarily, "subst~nti~lly pure"

SUt~ 111 IJTE SHEET ~RULE 26) dLions include at least 60% by weight (dry weight) the compound of interest.
More preferably the preparation is at least 75% or 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any app~ iatemethod, e.g., column chromatography, gel electrophoresis, or HPLC analysis. With5 respect to proteins, including antibodies, if a L)le~dlalion includes two or more different compounds of interest (e.g., two or more different antibodies, imrnunogens, functional domains, or other polypeptides of the invention)~ a "~ub~ ly pure"
~ ;p~Lion is preferably one in which the total weight (dry weight) of all the compounds of interest is at least 60% of the total dry weight. Similarly, for such 10 ~ preparations co.-lzt;.,i~-g two or more compounds of interest, it is preferred that the total weight of the compounds of interest be at least 75%, more plerel~ly at least 90%, and most preferably at least 99%, of the total dry weight of the preparation.
Finally, in the event that the protein of interest is mixed with one or more other proteins (e.g., serum albumin) or compounds ~e.g., flill1~nt~ excipients, salts,15 polys~c~-h~ri(1es~ sugars, lipids) for purposes of ~lmini~tration~ stability, storage, and the like, such other proteins or compounds may be ignored in calculation of the purity of the ~,lep~lion.
Isolated nucleic acid. As used herein, an "isolated nucleic acid" is a ribonucleic acid, deoxyribonucleic acid, or nucleic acid analog comprising a 20 polynucleotide sequence that is isolated or separate from sequences that are imme~ tely contiguous (one on the 5' end and one on the 3' end) in the naturallyoccurring genome of the organism from which it is derived. The te~n thel~,role includes, for example, a recombinant nucleic acid which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a 25 prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease trç~tment) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences and/or including exogenous re~gulatory elements.

SUtl~ 1 1 1 UTE SHEET ~RULE 26) W O 9~S2~296 PCT/CA971000Sl SubstantiallY identical sequence. As used herein, a "subst~nti~ly identical" amino acid se~uence is an amino acid sequence which differs only by constl ~aLive amino acid substitutions, for example, substitution of one amino acid for another of the sa~ne class ~e.g., valine for glycine, a~ginine ~or lysine, etc.) or by one 5 or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein (assayed, e.g., as described herein). Preferably, such a sequence is at least 85%, more preferably 90%, and most preferably 95% identical at the amino acid level to thesequence of the protein or peptide to which it is being compared. For nucleic acids, 10 the length of comparison sequences will generally be at least 50 nucleotides,preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably l 10 nucleotides. A "S11~St~nt~ Y identical" nucleic acid sequence codes for a ~ub~ lly identical amino acid sequence as defined above.
Transformed cell. As used herein, a "l.~lsrO,ll-ed cell" is a cell into which 15 (or into an ~noçstor of which) has been introduced, by means of recombinant DNA
techniques, a nucleic acid molecule of interest. The nucleic acid of interest will typically encode a peptide or protein. The transformed cell may express the sequence of interest or may be used only to propagate the sequence. The term "transformed"
may be used herein to embrace any method of introducing exogenous nucleic acids 20 including, but not limited to, LLdl1~rOI ~ tion~ transfection, electroporation, microinjection, viral-mediated transfection, and the like.
OperablY ioined. As used herein, a coding sequence and a regulatory region are said to be "operably joined" when they are covalently linked in such a way as to place the ~ s:jion or transcription of the coding sequence under the inflli~n~e or control 25 of the regulatory region. If it is desired that the coding sequences be translated into a fimctional protein, two DNA sequences are said to be operably joined if induction of promoter function results in the transcription of the coding se~uence and if the nature of the linkage between the t~vo DNA sequences does not (l) result in the introduction of a frame-shift mutation, (2) h~ r~- e with the ability of the regulatory region to 30 direct the ~ scliption of the coding sequences, or (3) interfere with the ability of the SU~ l UTE StlEET (RULE 26~

corresponding RNA transcript to be tr~n~l~te~l into a protein. Thus, a regulatory region would be operably joined to a coding sequence if the regulatory region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be tr~n~l~ted into the desired protein or polypeptide.
Strin~ent hYbridization conditions. Str-ngPnt hybridization conditions is a termof art understood by those of ordinary skill in the art. For any given nucleic acid sequence, slringpnt hybridization conditions are those conditions of temperature, chaotrophic acids, buffer, and ionic strength which will permit hybridization of that nucleic acid sequence to its complementary sequence and not to subst~nti~lly different 10 sequences. The exact conditions which constitute "stringent" conditions, depend upon the nature of the nucleic acid sequence, the length of the sequence, and the frequency of occurrence of subsets of that sequence within other non-identical sequences. By varying hybridization conditions from a level of stringency at which non-specific hybridization occurs to a level at which only specif~c hybridization is observed, one of 15 ordinary skill in the art can, without undue ~AI,C. j..-~nt~tion, cletermine conditions which will allow a given sequence to hybridize only with complementary sequences.
Suitable ranges of such stringency conditions are described in Krause and Aaronson (1991). Hybridization conditions, depending upon the length and commonality of asequence, may include tempe~ s o~20~C-65~C and ionic strengths from Sx to O.lx 20 SSC. Highly stringent hybridization conditions may include temperatures as low as 40-42~C (when d~l~Lu~ such as r~ ,alllide are included) or up to 60-65~C in ionic strengths as low as O.lx SSC. These ranges, however, are only illustrative arld,depending upon the nature of the target sequence, and possible future technological developments, may be more stringent than nt?ce~s~ry. Less than stringent conditions 25 are employed to isolate nucleic acid sequences which are substarltially similar, allelic or homologous to any given sequence.
Selectivelv binds. As used herein with respect to antibodies, an antibody is said to "selectively bind" to a target if the antibody recognizes and binds the target of interest but does not substantially recognize and bind other molecules in a sample, 30 e.g., a biological sample, which includes the target of interest. That is, the antibody SU~s ~ ITE SHEET (RUEE 263 W 097n7z96 PCT/CA97/00051 must bind to its target with sufficient specificity so as to distingt1ish the target from r essentially all of molecules which would reasonably be present in a biological sample including the target.
II. The Presenilins and Pres~nilin-Interactin~ Proteins The present invention is based, in part, upon the discovery of a family of m~mm~ n genes which, when ml7t~te-1, are associated with the development of Alzheimer's Disease. The discovery of these genes, designated presenilin- 1 (PS 1 ) and presP.nilin-2 (PS2), as well as the chara~ tion of these genes, their protein products, Illul~ v~lle~ldLehoInologues~ and possible functional roles, are described in PCT Publication W096/34099. The present invention is further based, in part, upon the discovery of a group of proteins which interact with the pres~nilin~
under physiological conditions and which, therefore, are believed to be involved in the biochemical pathways which are altered in .Al7heimçr's Disease. These proteins are referred to herein as presenilin-interacting (PS-interacting) proteins. Because mutations in the presenilin~ are known to be causative of Alzheimer's Disease, each of the PS-interacting genes and proteins disclosed and described herein presents a novel tOEget for therapeutic intervention in ~l7heim~r's Disease. That is, modulation of the hllela-;Lions of these proteins with the pre~s~nilin~, or modulation of the interactions of at least the PS-interacting domains of these PS-int~r~.ting proteins with at least the inter~ctin~ domains of the presenilins, provides a means of mod~ ting the activity and/or availability of the prçs~nilin~, or of mofl~ ting the activity and/or availability of the PS-interacting proteins. Furthennore, as aberrations in the interactions of mutant pres~nilin~ with one or more of these PS-interacting proteins is causative of Alzheimer's Disease, mutations in one or more of these PS-interacting proteins are also likely to be causative of ~ hejrner's Disease. Therefore, each of the PS-interacting genes and proteins disclosed and described herein presents a novel target for diagnosis of forms of .f~mili~tl and/or sporadic Alzheimer's Disease with anetiology independent of mutations in the pres.o.nilin~. Finally, as described more fully below, the PS-interacting genes and proteins described and disclosed herein provide for new assays for compounds which affect the interactions of the presenilins and PS-SU~:~ l 11 UTE SHEET (RULE 26) interacting proteins, assays for other members of the biochemical pathways involved in the etiology of Alzheimer's Disease, and new cell lines and transgenic animalmodels for use in such assays.

5 1. Presenilin Processin~
Employing the antibodies and protein-binding assays described and/or enabled in PCT Publication W096/34099, the processing and protein-protein interactions of both normal and mutant presenilins were investigated. It was found that mutations in the presenilins appear to lead to changes in both their intracellular 10 processing (e.g, endoproteolytic cleavage, ubiquitination, and clearance) and their intracellular interactions with other proteins expressed in human brain. As described below, knowledge of presenilin proce~in~ and interactions, and particularly changes in mutant pres~nilin processing and interactions, provides for new diagnostic and therapeutic targets for ~l7h~imer's Disease and related disorders.
Western blot analysis s~ Pst~ that the normal prPs~nilin~ undergo proteolytic cleavage to yield characteristic N- and C-tennin~l fr~ment~ As notedabove, the normal presenilin proteins have an expected molecular mass of 47-51 kDa depending, in part, upon rnRNA splice variations, electrophoretic conditions, etc.
Analysis of Western blots SllE~geSt~, however, that the normal presPnilin proteins undergo proteolytic cleavage to yield an approximately 35 kDa N-~Pnnin~l fragment and an approximately 18 kDa C-tPnnin~l fr~nPnt In particular, Western blots bearing lysates ~om wild-type native human fibroblasts, human neocortical brain tissue from control subjects, and neocortical brain tissue from non-transgenic and PSl transgenic mice using antibodies ("14.2") recognizing PS1-specific residues 1-25 at ~5 the N-terminu~ reveal the presence of a strong immlm-reactive band of approximately 35 kDa and, after longer exposures, a weaker band of approximately 45 kDa which presumably represents the full-length PS1 protein. Antibodies ("520"~ directed at residues 304-318 at the apex of the TM6~7 loop of PS 1, and antibodies ~"4627") directed at residues 457-467 in the ~-t~ormin-~c of PS1, both recognize the same strong band of approximately l 8 kDa. Antibodies 520 also recognize a weak band of 45 kDa Sll,~5 ~ l l IJTE SHEET (RULE 26) W097127296 PCT/CAg7/00051 coincident with the PSI band detected by 14.2. SeqllPncing ofthe major C-terminal fragrnent from PS 1-kansfected human embryonic kidney cells (EIEK 293) showed that the principal endoproteolytic cleavage occurs near M298 in the proximal portion of the TM6~7 loop, possibly by enzymes other than the proteasome. These 5 observations suggest that an endoproteolytic cleavage event occurs near the junction of exons 9 and 10 of PS 1. Full length PS 1 in these cells is quickly turned over (tl, 60 min.) by the proteasome.
To ~let~lmine whether mutations in the presenilin proteins result in alterations of their proteolytic cleavage, Western blots co~ lysates of fibroblast 10 and neocortical brain homogenates from normal subjects and subjects carrying PS1 mutations were investigated with the PS1 specific antibody Ab 14.2. In fibroblasts, there were no obvious di~ ces in the relative int~n.cities of the protein bands when Iysates from heterozygous carriers of the PS 1 mutations were cu~ art;d with normal homozygotes. In contrast, there appeared to be a difiference between PS 1 mutation 15 carriers and nnnn~lc in homo~enates of temporal neocortex from AD affected heterozygous carriers of either the PSl A246E or C410Y mutations (which are located in TM6 and TM7 respectively). In heterozygotes, a strongly i..,."l-"~reactive band of approximately 45 kDa was detecte :1 which initially appeared to correspond to the full-length PS 1 protein. Further analysis, however, revealed that this band ~cpres~ s an 20 ~lt~.rn~tively processed presenilin product. A similar band corresponding to this mutant processed PS 1 was observed in neocortical homogenates from some sporadiclate-onset AD patients. These data suggest that (1 ) some pathogenic PS 1 mutations associated with early-onset AD alter the way in which the presenilins are processed through endoproteolytic and proteasome pathways and (2) the presenilin proteins, and 25 charlges in the processing of the presenilins in the brain, are also implicated in late-onset and sporadic AD.

2. Presenilin-Interacting Proteins In order to identif~ proteins which may bind to or otherwise interact with 30 the presenilins in vivo. a yeast two-hybrid system was used as described below SlJl~ 111 UTE SHEET (RULE 26~

(Example 1). In particular, because mutations in the TM6~7 loop domains are known to be causative of AD, a yeast two-hybrid system was used to identify cellular proteins which may interact with normal and mutant pres~nilin TM6~7 loop domains. Yeast two-hybrid studies were also done with cDNAs corresponding to the5 C-te~rnin~l 18 kDa endoproteolytic cleavage fragment, and with cDNAs corresponding to the TM1~2 intr~ min~l loop domain, which is also the site oftheFAD associated Y1 15H missense mutation. In brief, cDNA sequences encoding the TM6~7 loop (i.e., residues 266 to 409 of PS1) were ligated in-frame to the GAL4 DNA-binding domain in the pAS2-1 yeast ~ c;ssion plasmid vector (Clontech).
10 This plasmid was then co-l~ ro~ ed into S. cerevisiae strain Y190 together with a library of human brain cDNAs ligated into the pACT2 yeast e~ s ,ion vector bearing the GAL4 activation domain (Clontech). After a~rupliate selection and re-screening, a number of clones were recovered and sequenced bearing human brain cDNAs encoding peptides which interacted with the normal pres~nilin TM6~7 dom~in To 15 ~et~rmine whether these pres~nilin interactions would be modified by AD related mutations within the TM6~7 loop, the yeast two-hybrid system was again used withTM6~7 loop peptides collt;~ the L286V, the L392V, and the exon 10 splicing When these mutant constructs were used as "bait" to re-screen the brain cDNA:GAL4 activation domain library, some but not all of the brain cDNA
20 sequences which interacted with the normal prçs~-nilin were recovered. In addition, several new clones were identified which interacted with the mutant but not the normal pr~s~nilin~ The clones collc;~ln~llding to the PS-interacting proteins with the highest presenilin affinity are described in Example 1 and below.
PS-interacting proteins, particularly those which interact selectively with 2~ either the normal or mutant presen;lin~, provide new targets for the identification of useful pharmaceuticals, new targets for diagnostic tools in the identification of individuals at risk, new se~uences for the production of transformed cell lines and transgenic animaI models, and new bases for therapeutic intervention in Alzheimer's Disease. In particular, the onset of AD may be associated with aberrant interactions 30 between mutant presenilin proteins and no~nal forms of PS-interacting proteins such SU~a l l l UTE SHEET ~RULE 26) WO 97/27296 PCT~CA97/00051 as those identified using the methods described herein. These changes may increase or decrease interactions present with normal PS l or may cause interaction with a novel mutation-specific PS-interacting protein. In addition, however, aberrant interactions may result from normal pres~onilin~ binding to mutant forms of the PS-ir~Ateracting proteins and, therefore, mutations in the PS-interacting proteins may also be causative of AD.
A. The S5a Subunit of the 26S Proteasome Two overlapping clones have been identified as repres~nting a portion of the human protein ~lt~rn~tively known as Antisecretory ~actor ("ASF") or the 10 Multiubiquitin chain-binding S5a subunit of the 26S proteasome ("S5a"). Theseclones, which together include residues 70-377 of S5a, were shown to interact with the normal presenitin TM~7 loop domain but only weakly with two TM6~7 loop domain mllt~n~ tested (L286V, L392V). The PS 1 :S5a interaction was confirmed byco-hllllluAloAv,eei~ tion studies, arld immlmocytochemical studies showed S5a and 15 PSl are c2rAAulcssed in contiguous intracell~ r compartments in brain cells typically affected by AD.
The interaction between PS1 and the proteasome could be relevant to the pathogenesis of AL7Jheimer's Disease (AD) through several possible mec.h~ni.cm~
First, most m~mm~ n cells seem to m~in~in very low levels of the PS l holoprotein.
20 A notable exception to this are cells c~AAvlc~ g the PS 1 Q290-3 l9 splicing mutation, which results in a mutant PS 1 holoplotcill which is not endoproteolytically cleaved and which is, therefore, readily cletect~hle. In the case of the ~290-319 splicing mutation at least, the presence of the mutant PS 1 holoprotein, or the absence or reduction in the 35 kDa N-termin~l and l 8 kDa C-t~nnin~l fr~ment.c, appears 25 sufficient to cause AD. It is possible, therefore, that even very subtle changes in the turnover of the mutant PS 1 holoprotein might have significant pathophysiological effects. I'hus, mutations in either the pres~nilin~ or S5a which perturb the PS 1 :S5a interaction in the m~mm~ n CNS may cause the presenilin holoprotein to be aberrantly processed and cause AD. Therefore, modulation of presenilin proteolytic 3û pathways might be applied therapeutically to enhance removal of mutant holoprotein.

SUv;i ~ JTE SHEET (RULE 26) To assess a potential in vivo relationship between PS 1 and the S5a subunit of the 26S proteasome, the effects of proteasome inhibitors on PS 1 metabolism were investigated. Short term organotypic cultures of neonatal rat hippocampus and carcinoma of colon (CaCo2) cells (which express high levels of both PSI and PS2)5 were ~lmini~tered either the specific, reversible proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinyl-H (LLnL) ~Rock et al., 19943, or the specific il~;velsible proteasome inhibitor lactacystin (Fenteany et al., 1995). Both agents caused an increase in the steady state levels of PS 1 holoprotein. Both agents also prolonged the half-life of the PS 1 holoprotein in pulse chase experiments in hippoc~mr~l slices from 10 ~15 minlltes to ~35 minutes As noted above, the PSl holoprotein appears to berapidly turned over in normal cells. However, even after four hours of metaboliclabelling, neither of the proteasome inhibitors affected the level of the 35 kDa N-tl-nninz~l PS 1 fr~ment or resulted in the apl,ea,~,ce of novel species. These studies imply that the majority of the PS 1 holoprotein is catabolized directly via a rapid, 15 proteasome dependent pathway in a manner similar to several other integral membrane proteins (e.g. Sec61 and CFTR). On the other hand, because the ~35 kDa and ~ 18 kDa t.-rm;n~l fr~rnent~ are still produced in the presence of proteasome inhibitors, this endoproteolytic cleavage of PS 1 is probably not mediated by the proteasome ~dLhw~y. Tll~,ro~, it appears that at least two proteolytic pathways act 20 upon the PS1 holoprotein.
An ~ltPrn~te possibility is that mutant PS 1 :S5a interactions may modify the function or the cellular regulation of S5a. To address this possibility, S5a levels were examined by Western blotting of lysates from postmortem temporal neocortex from non-AD neurologic controls (n - 8), sporadic AD (n = 8) and PS 1 -linked FAD
25 (n = 4). In the majority of non-AD brains, polyclonal anti-S5a antibodies specifically detectecl an S5a species with Mr of~ 50 kDa, which could be abolished by preabsorption of the antibody with recombinant His6-SSa or with extracts of myc-S5a transfected cells. In a subset of these control cases an additional S5a reactive band was observed at ~34 kDa. In contrast, in tissue from all subjects with sporadic late 30 onset AD, the predo~ l SSa reactive species was observed at ~ 40 kDa which was SIJ~;~ TE SHEET ~RULE 26) .

W o 97127296 PCT/CA97/00051 not seen in control tissue. The origin, and the functional significance of this altered electrophoretic mobility is unclear but indicates that SSa processing is altered in AD
brains, irrespective of whether the AD is presenilin-linked or sporadic.
Thus, the presenilin-proteasome interaction appears significant in several 5 respects. First, the facts that the normal presenilin TM6~7 loop domain interacts with the S5a protein, that the mutant prçs~nilin TM6~7 loop domains fail to interact (or interact very weakly) with the SSa protein, that pres~.nilin~ bearing mutations in the TM6~7 loop domain appear to be ~lirrelGlltly cleaved and multiubiquitinated, that proteasomes are known to be involved in the cleavage and clearance of a variety of 10 . proteins (particularly multiubiqnitin~te(i proteins), that inhibition of proteasome activity inhibits cleavage of the presenilin holoproteins, and that S5a processing is altered in AD brains, all suggest (l) that the S5a subunit and the 26S proteasome are involved in the no~mal processing of the presP-nilin~c and that mutations which disrupt this normal interaction may be ,~ ,nsible for the abnormal processing observed in 15 TM6~7 loop domain mllt~nt~; or (2) that the pr~ct~Milin-proteasome interaction may modulate the activity of PS 1, SSa, or both, with or without involving proteasome-mediated pr~s~nilin processing; or (3) that modulation of the norrnal quality control function of proteasome-mediated degradation of misfolded or mutant membrane proteins tr~ffickin~ through the ER and Golgi (such as APP, Notch, or Prion proteins), 20 and of misfolded, mutant, or ubiqllitin~tecl cytoplasmic proteins (including structural proteins such as tau, and short lived, proteasome processed ~i n~lin~ molecules such as NFkB). Thus, defective proteasome function might selectively cause these proteins (especially ~APP, tau, Prion) to be aberrantly metabolized. The latter would lead to the accumulation of neurotoxic, amyloidogenic protease-resistant derivatives such as 25 A,B and PrPsc, the accumulation of neurofibrilla~y tangles, and defective intracellular sign~ling functions. In support of these hypotheses, it should be noted that failure to clear hyperubiqlTitin~ted phosphorylated tau and other microtubule associated proteins is a prominent feature of Alzheimer's Disease (Kosik and Greenberg, l 994), suggesting a possible link between TM6~7 loop domain ~ x pres~nilin-30 proteasome interactions, tau-proteasome interactions, and the neurofibrillary tangles SIJ~;~ JTE SHEET (RULE 26) of tau protein in AD brains. Finally, proteasomes are known to be capable of degrading APP and of binding the A,B peptides which are associated with Alzheimer's Disease, suggesting a possible link between TM6~7 loop domain mllt~ntc, prçsenilin-proteasome interactions, APP-proteasome interactions, and the ~nyloid5 plaques characteristic of AD brains. Furthermore, ~lmini~tr~tion of proteasomeinhibitors such as LLnL and Lactacystin cause severe disturbances in ~APP
metabolism with increases in intracellular innm~tl]re N-glycosylated ,BAPP, and the secretion of much larger amounts of A,~42 isoforms into the media (Klafki, et al., 1996).
Therefore, pr~ nilin processing and the prçs~-ni~in-proteasome interaction are clear targets for the diagnosis as well as therapeutic intervention in AD. Thus~ as described below, assays may now be provided for drugs which affect the proteasome-mediated cleavage ofthe pre~P~ilin.~, which affect the ~lt~ tive endoproteolyticcleavage and ubiquitination of the mutant pr~senilinc, or which otherwise affect the 15 proce~.~inp and trafficking of the presenilin~ or the S5a subunit of the proteasome. In addition, as mutations in the 26S proteasome which disrupt the normal processing of the presenilin~ are likely to be causative of Alzheimer's Disease, additional diagnostic assays are provided for 11etectin~ mutations in the SSa or other subunits of theproteasome. Finally, additional transformed cell lines and transgenic models may20 now be provided which have been altered by the introduction of a normal or mutant sequence encoding at least a functional domain of the proteasome. The appearance of abnormal electrophoretic forms of S5a (and/or other proteasome subunits) in biologic tissues and fluids can be used as a clinical test for diagnosis and monitoring of disease activity in subjects with sporadic forms of AD.
2~ B. GT24: A Protein with "~ rlillo" RePeats Another PS-interacting protein, designated GT24, was identified from several over-lapping clones obtained using a PS l26ti409 domain as bait in the yeast two-hybrid system arld a human adult brain cDNA library. Six longer GT24 clones of ~3.8 kb in size were subsequently obtained by screening of conventional cDNA
30 libraries. The o~en reading frame within the longest GT24 clone obtained to date S~ TE StlEET ~RULE 26) (Accession number U81004~ suggests that GT24 is a protein of at least 1040 aminoacids with a uni~ue N-t~rminu~, and considerable homology to several ~
(~ repeat proteins at its C-t~nim~ Thus, for example, residues 440-862 of GT24 (numbering from Accession number U81004) have 32-56% identity (p=1.2e-l33) to residues 440-854 of murine p 120 protein (Accession number Z17804), and residues367-815 of GT24 have 26-42% identity (p=0.0017) to residues 245-465 of the D.
melano aster ~rm~lillo segment polarity protein (Accession number P18824). The (~T24 gene maps to chromosome ~pl5 near the anonymous microsatellite marker D5S748 and the Cri-du-Chat syndrome locus.
~Iybridization of unique 5' sequences of GT24 to Northern blots reveals that the GT24 gene is expressed as a range of L.~lS~iliplS varying in size between ~3.9 and 5.0 kb in several regions of human brain, and in several non-neurologic tissues such as heart. In addition, in situ hybridization studies using a 289 bp single copy fragment from the 5' end of GT24 in four month old murine brain reveal GT24 15 ll~lsc~il,tion closely parallels that of PS 1, with robust t;~ ssion in dentate and hippocampal neurons, in scattered neocortical neurons, and in cerebellar Purkinje cells. In day E13 murine embryos, GT24 is widely ~ essed at low levels, but is expressed at somewhat higher levels in somites and in the neural tube. A
physiological in vivo interaction between GT24 and PSl is ~u~po.led by co-20 immunop.~ci~ ion studies in HEK293 cells transiently transfected with a wild typehuman PS 1 cDNA, a c-mvc-tagged cDNA encoding residues 484- 1040 of GT24 (inciu-ling the C-t~rmin~l arm repeats~, or both cDNAs. Cell lysates were immunopl~ci~itated with anti-PS 1 antibodies and then investip;~te~1 for the presence of the mvc-GT24 protein by immuno-blotting. In PSl/mvc-GT~4 double transfected 25 cells, the imm~lnnprecipitates contained a robust anti-mvc reactive band of Mr ~60 kDa, which co-migrated with a mvc-GT24 control. In cells transfected with mYc-GT24 only, a very weak band was detectecl after long exposures, presumably reflecting interaction of the mvc-GT24 with low levels of endogenous PS 1. No mvc-reactive bands were detected in cells transfected with PS 1 alone, or in any of the 30 transfected cells imrnunoprecipitated with pre-immune serum. Taken together, these S~ l l ulTE SHEET (RULE 26) W 0 97/27296 PCT/CA97/OOOSl observations strongly suggest that the observed PS 1 :GT24 interaction is physiologically relevant.
To explore whether mutations in the TM6-TM7 loop of PS 1 might influence the PS 1 :GT24 interaction, we employed q~l~ntit~tive liquid 13-galactosidase 5 assays to directly compare the yeast-two-hybrid interaction of the C-t~-rmin~l residues 499-1040 of GT24 with wildtype and mutant PS12664og~ These studies revealed thatthe interaction of GT24499,o40 with a L286V mutant PS 1 domain was not significantly different from the interaction with the corresponding wild type PS 1 domain. In contrast, there was a sigIuficant reduction in the GT24499 lo40 interaction with the L392V mutant PS 1 construct. The absence of an effect of the L286V mutation, andthe presence of an effect with the L3g2V mutation, may suggest that some mutations may effect PS 1 :GT24 binding, while others may modulate the PS 1 response to GT24 binding.
The PS 1 :GT24 interaction could support several functions. The arm repeat 15 motif of GT24 has been detected in several proteins with diverse functions including ,B-catenin and its invertebrate homologue :~nn~(1illo plakoglobin, pl20, the adenomatous polyposis coli (APC) gene, ~u~ ssor of RNA polymerase 1 in yeast (SRP1), and smGDS. For example"B-c~t~nin, pl20 and plakoglobin play an essentialrole in intercellular adhesion. ~-catenin/~nn~1;llo is involved in transduction of 20 win,~less/Wnt signals during cell fate specification, and ,B-catenin and pl20 may play a role in other receptor mediated signal tr~nC~ f tion events including responses to trophic factors such as PDGF, EGF, CSF-l and NGF.
If the PS 1 :GT24 interaction is part of intercellular ~ign~lin~ pathways for trophic factors, or is involved in cell-cell adherence, disruption of the interaction may 25 be involved in the neurodegenerative processes in PS-linked ~AD brains, and in the increased sensitivity of PS 1 or PS2 transfected cells to apoptosis ~Wolozin et al., 1996). It is of note that at least one arm protein, smGDS, stim~ tes GDP/GTP
exchange on intracellular G-proteins (Kikuchi et al. 1992; Borguski et al., 1993), and that mutant forms of both ~APP and PS2 are thought to activate programmed cell SIJ~S 111 ~JTE SHEET (RULE 26) death pathways through me~h~ni~m~ involving heterotrimeric GTP/GDP proteins (Wolozin et al, 1996; Okamoto, et al., 1995; Yamatsuji, et al, 1996).
The interaction between PS 1 and GT24 may also be involved in some of the development~l phenotypes associated with homozygous PS1 knockouts in mice such as failed somitogenesis of the caudal embryo, short tail, and fatal cerebral hemorrhage at around day E13.5 (Wong et al., 1996). The resemblance ofthese skeletal phenotypes to those associated with null mutations in PAX1 and Notch, and the a~paLcllt suppressor effect of mutations in sell2 on Notch/linl2 mediated sign~ling in C. ele~ans suggest that the PS proteins fimction in the Notch sign~linf~
10 pathway. In addition, mice homozygous for a knockout of the Wnt-3a gene (Takada et al., 1994), and murine homozygotes for a spontaneous mutation, "vestigial tail" or y~, in the Wnt-3a gene ~Greco et al., 1996), have skeletal phenotypes of defective caudal somite and tail bud formation. The Wnt-3a knockouts are embryonic lethal by day 12.5. These phenotypes are sirnilar to those of homozygous knockouts of the murine PSl gene (Wong et al., 1996). The observation that GT24 binds to PS1, is expressed in embryonic somites, and contains the ~ lo repeat motif of other proteins used in the d~wll~Llc~ll signaling in the Win~less/Wnt pathway suggests that PS 1 is a dov~ can~ element in the GT24-Win~less/Wnt pathway. This can be exploited to create a bioassay for drugs affecting the GT24-PS 1 interaction directly, or 20 affecting u~sllc~ll or downstream components of that interaction pathway, and can therefore be used to monitor the effects of pres~nilin mutations. For example, cells transfected with normal or mutant presenilins may be exposed to soluble Wnt-3a protein (or other Wnt proteins such as Wnt-1) and assayed for changes which are specific to the Win~less/Wnt sign~ling pathway, or for any of the other changes 25 described herein for cell assays (e.g., intracellular ion levels, A,B processing, -apoptosis, etc.).
Thus, the GT24 protein also presents new targets for diagnosis as well as therapeutic intervention in AD. For exarnple, as mutations in the GT24 protein may also be causative of Alzheimer's Disease, additional diagnostic assays are provided for 30 detecting mutations in these sequences. Similarly, additional transformed cell lines Sl~ a 1 l l UTE SHEEl' (RULE 26) and transgenic models may now ~e provided which have been altered by introduction of a normal or mutant nucleic acid encoding at least a functional domain of the GT24 protein, and particularly the functional domains (e.g., residues 70-377) which interact with the pre~t~.nil;n.~ Such transformed cells and transgenics will have utility in assays 5 for compounds which modulate the presenilin-GT24 interactions.
C. pO071: A Protein with "~ lillo" Repeats Another independent clone isolated in the initial screening with the wild type PSI26,j409 "bait" also encodes a peptide with C-te~min~l a~rn repeats (clone Y2H25, Accession number U81005). A longer cDNA sequence corresponding to the Y2H25 clone has been deposited with GenBank as human protein pO071 (Accession number X81889) and is reproduced herein as SEQ ID NO: 5. Clone Y2H25 co,le~onds ~cc~nti~lly to nucleotide positions 1682- 1994 of SEQ ID NO: 5.
Comparison of the predicted sequence of the Y2H25/pO071 ORF with that of GT24 confirms that they are related proteins with 47% overall amino acid sequence identity, and with 70% identity between residues 346-862 of GT24 and residues 509-1022 of pO071. This suggests that PS 1 interacts with à novel class of arm repeat cc l It~
proteins. The broad ~4.5 kb hybridization signal obtained on Northern blots with the unique S' end of GT24 could reflect either ~ltP.rn~tive splicing/polyadenylation of GT24 or, less likely, the exi.~ten~e of additional members of this family with higher 2û degrees of N-t~ l homology to GT24 than pO071. Cells l~ r,l.l.ed with thesesequences, or transgenic ~nim~l~ including these sequences, will have additionalutility as animal models of AD and for use in screening for compounds which modulate the action of normal and mutant pres~onilin~
D. Rab 11 One clone (Y2H9), disclosed herein as SEQ ID NO: 5, was identified as interacting with the normal PS I TM6~7 loop domain and appears to correspond to a known gene, Rabl 1, available through Accession numbers X56740 and X53143.
Rabl 1 is believed to be involved in protein/vesicle trafficking in the ER/Golgi. Note the possible relationship to processing of membrane ploteills such as 13APP and Notch SUBSTITUTE SHEET (RULE 26~

with resultant overproduction of toxic A~ peptides (especially neurotoxic Al3l42(43 isoforms) (Scheuner, et al, 1995).
E. Retinoid X Receptor-,B
One clone (Y2H23b), disclosed herein as SEQ ID NO: 6, was identified as interacting with the no~nal PS 1 TM6~i loop domain and appears to correspond to a known gene, known variously as the retinoid X receptor-,B, nuclear receptor co-regulator, or MHC Class I regulatory element, and is available through Accessionnumbers M84820, X63522 and M81766. This gene is believed to be involved in intercellular si~n~lin~, suggesting a possible relationship to the intercellular signaling fimction mediated by C. ele~ans sell2 and Notch/lin-12 (transcription activator).
F. Cvtoplasmic Chaperonin One clone (Y2H27), disclosed herein as SEQ ID NO: 8, was identified as interacting with the normal PS 1 TM6~7 loop domain and appears to correspond to a known gene, a cytoplasmic chaperonin colllnilli~-g TCP-l, available through Accession numbers U17104 and X74801.
G. Clone Y2H35 One clone (Y2H35), disclosed herein as SEQ ID NO: 7, was identified as interacting with the normal PS 1 TM6~7 loop domain and appears to co~Tespond to a sequence that codes for a protein of unknown function, available through Accession number R12984, but which displays evolutionary conservation in yeast sequences.
H. Clone Y2H171 One clone (Y2H171), disclosed herein as SEQ ID NO: 9, was identified as interacting with the normal PS l TM6~7 loop domain and appears to cc,~ olld to aknown expressed repeat sequence available through Accession number D55326.
I. Clone Y2H41 One clone (Y2H41) was identified which reacts strongly with the TM6~7 loop domains of both PS 1 and PS2 as well as the mutant loop domains of PS 1. The sequence, disclosed as SEQ ID NO: 10, shows strong homology to an EST of unknown function (~ccession number T64843).

sut~ JTE SHEE~ (RIILE 26t III. Preferred Embodiments Based, in part, upon the discoveries disclosed and described herein, the following ~lert;.led embodiments of the present invention are provided.

l. Isolated Nucleic Acids In one series of embo~iment.c, the present invention provides isolated nucleic acids corresponding to, or relating to, the nucleic acid sequences disclosed herein, which encode at least the PS-interacting domain of a PS-interacting protein.
10 As described more fillly below, the disclosed and enabled sequences include normal sequences from humans and other m~mm~ n species, mutant sequences from hl~m~n~ and other m~mm~ n species, homologous sequences from non-m~mm~ n species such as Drosophila and C. ele~ans. subsets of these sequences useful as probes and PCR primers, subsets of these sequences encoding fr~ nt~ of the PS-interacting 15 proteins or corresponding to particular structural domains or polymorphic regions, complementary or :~nti~.o.n~e sequences corresponding to fragments of the PS-interacting protein genes, sequences in which the PS-interacting protein coding regions have been operably joined to exogenous regulatory regions, and sequencesencoding fusion proteins in which portions of the PS-interacting proteins are fused to 20 other proteins useful as m~rker.~ of ~ ion, as "tags" for pl-rific~tion, or in screens and assays for other proteins which interact with the PS-interacting proteins.
Thus, in a first series of embof~iment.c, isolated nucleic acid sequences are provided which encode at least a PS-interacting domain of a normal or mutant version of a PS-interacting protein. Examples of such nucleic acid sequences are disclosed 25 herein as SEQ ID NOs: l, 3, and 5-lO. In addition, given the sequences ofthe PS-interacting domains of the PS-interacting proteins disclosed herein, one of ordinary skill in the art is clearly enabled to obtain the entire genomic or cDNA sequence encoding the entire PS-interacting proteins. Thus, for example, based upon the initial clone ofthe GT24 protein obtained using the yeast two-hybrid system (Example l),30 the larger GT24 clone disclosed as SEQ ID NO: 3 was obtained by standard methods Sl,~S 111 ~JTE SHEET (RULE 26~
-known in the art. Complete cDNA or genomic clones of each of the genes encoding the disclosed sequences may be similarly obtained by one of ordinary skill in the art.
Therefore, the present invention provides complete genomic sequences as well as cDNA sequences corresponding to the PS-interacting protein genes of the invention.
5 Alternatively, the nucleic acids of the invention may comprise recombinant genes or 1'minigenes" in which all or some introns of the PS-interacting protein genes have been removed, or in which various combinations of introns and exons and local CiS-acting regulatory elements have been engineered in propagation or ~ ression constructs or vectors. ~or purposes of reducing the size of a recombinant PS-10 interacting protein gene, a cDNA gene may be employed, or various combinations ofintrons and untr~n~l~te~l exons may be removed from a DNA construct. These and many variations on these embo-liment~ are now enabled by the identification and description of the PS-interacting proteins provided herein.
In addition to the disclosed PS-interacting protein and gene sequences, one 15 of oldill~ y skill in the art is now enabled to identify and isolate nucleic acids representing PS-interacting genes or cDNAs which are allelic to the disclosed sequences or which are heterospecific homologues. Thus, the present invention provides isolated nucleic acids corresponding to these alleles and homologues, as well as the various above-described recombinant constructs derived from these sequences, 20 by means which are well known in the art. Briefly, one of ordinary skill in the art may now screen p-c~,~dlions of genomic or cDNA, including samples prepared firomindividual org;~ni~m~ (e.g., human AD patients or their family members) as well as bacterial, viral, yeast or other libraries of genomic or cDNA, using probes or PCR
primers to identify allelic or homologous sequences. Because it is desirable to 25 identify mutations in the PS-interacting proteins which may contribute to thedevelopment of AD or other disorders, because it is desirable to identify polymorphisms in the PS-interacting proteins which are not pathogenic, and because it is also desirable to create a variety of animal models which may be used to study AD and screen for potential therapeutics, it is particularly co~ l~lated that additional 30 PS-interacting protein sequences will be isolated from other plc~dlions or libraries S~l~;i 111 UTE SHEET (RULE 26) W O 97/27296 PCTtCA97/00051 of human nuc}eic acids and from ~ lions or libraries from ~nim~lc including rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-human primates. Furtherrnore, PS-interacting protein homologues from yeast or invertebrate species, including C. ele,~ans and other nematodes, as well as Drosophila and other 5 insects, may have particular utility for drug screening.
Standard hybridization screening or PCR techniques may be employed (as used, for example, in the identification of the mPS l gene disclosed in PCT
Publication W096/34099) to identify and/or isolate such allelic and homologous sequences using relatively short PS-interacting protein gene sequences. The 10 sequences may include 8 or fewer nucleotides depending upon the nature of the target sequences, the method employed, and the specificity required. Future technological developments may allow the advantageous use of even shorter sequences. With current technology, sequences of 9-50 nucleotides, and preferably about 18-24 are ~lc;f~ d. These sequences may be chosen from those disclosed herein, or may be 15 derived from other allelic or heterospecific homologues enabled herein. When probing mRNA or screening cDNA libraries, probes and primers from coding sequences (rather than introns) are l)refcldbly employed, and sequences which are omitted in ~lt~ tive splice variants typically are avoided unless it is speci~lcally desired to identify those variants. Allelic variants of the PS-interacting protein genes 20 may be expected to hybridize to the disclosed sequences under stringent hybridization conditions, as defined herein, whereas lower stringency may be employed to identify heterospecific homologues.
In another series of embo(liments, the present invention provides for isolated nucleic acids which include subsets of the PS-interacting protein sequences or 25 their complements. As noted above, such sequences will have utility as probes and PCR primers in the identification and isolation of allelic and homologous variants of the PS-interacting protein genes. Subsequences corresponding to polyrnorphic regions of the PS-interacting proteins, will also have particular utility in screening and/or genotyping individuals ~or diagnostic purposes, as described below. In 30 addition, and also as described below, such subsets will have utility for encoding (l) SUts;~ IJTE SHEET (RULE 26) fr~gment~ of the PS-interacting proteins for inclusion in fusion proteins, (2) fragm~nt~
which comprise functional domains of the PS-interacting proteins for use in binding studies, (3) fragments of the PS-interacting proteins which may be used as imml~nogens to raise antibodies against the PS-interacting proteins, and (4) fragments 5 of the PS-interacting proteins which may act as competitive inhibitors or as mimetics of the PS-interacting proteins to inhibit or mimic their physiological functions.
Final~y, such subsets may encode or lel)res~lll complement~T~ or ~nti~n~e sequences which can hybridize to the PS-interacting protein genes or PS-interacting protein mRNA transcripts under physiological conditions to inhibit the transcription or 10 translation of those sequences. Therefore, depending upon the int~nllecl use, the present invention provides nucleic acid subsequences of the PS-interacting protein genes which may have lengths varying from 8-lO nucleotides (e.g., for use as PCRprimers) to nearly the full size of the PS-interacting protein genomic or cDNAs.Thus, the present invention provides isolated nucleic acids comprising sequencesco~ ding to at least 8-10, preferably l5, and more preferably at least 20 consecutive nucleotides of the PS-interacting protein genes, as disclosed or otherwise enabled herein, or to their complements. As noted above, however, shorter sequences may be useful with different technologies.
In another series of embodiments, the present invention provides nucleic 20 acids in which the coding sequences for the PS-interacting proteins, with or without introns or recombinantly engineered as described above, are operably joined to endogenous or exogenous 5' and/or 3' regulatory regions. Using the present disclosure and standard genetic techniques (e.g., PCR extensions, targeting gene waLIcing), one of ordinary skill in the art is now enabled to clone the 5' and/or 3' endogenous regulatory 25 regions of any of the disclosed PS-interacting protein genes. Similarly, allelic variants of these endogenous regulatory regions, as well as endogenous regulatory regions from other m~mm~ n homologues, are similarly enabled without undue eXperim~nt~tion. Alternatively, exogenous regulatory regions (i.e., regulatory regions from a different conspecific gene or a heterospecific regulatory region) may be 30 operably joined to the PS-interacting protein coding sequences in order to drive S~ 1 1 UTE SHEET (RULE 26) e~ ion. Appropriate 5' regulatory regions will include promoter elements and may also include additional elements such as operator or enhancer sequences, ribosome binding sequences, RN~ capping sequences, and the like. The regulatory region may be selected from sequences that control the e~ ,s~ion of genes of 5 prokaryotic or eukaryotic cells, their viruses, and combinations thereof. Suchregulatory regions include, but are not limited to, the lac system, the trp system, the tac system, and the trc system; major operator and promoter regions of phage ~; the control region of the fd coat protein; early and late promoters of SV40; promoters derived from polyoma, adenovirus, retrovirus, baculovirus, and simian virus; 3-10 phosphoglycerate kinase promoter; yeast acid phosphatase promoters; yeast alpha-mating factors; promoter elements of other eukaryotic genes expressed in neurons or other cell types; and colllbillations thereof. In particular, regulatory elements may be chosen which are inducible or l~ressible (e.g., the ~-g~ to~ se promoter) to allow for controlled and/or manipulable ~pLcs~ion of the PS-interacting protein genes in 15 cells transformed with these nucleic acids. ~ltPrn~tively, the PS-interacting protein coding regions may be operably joined with regulatory elements which provide fortissue specific t;~lession in multicellular or~ni~m~. Such coll~LIucLs are particularly useful for the production of transgenic or~ni.cm~ to cause ~ es~ion of the PS-interacting protein genes only in al~plopl;ate tissues. The choice of ~lu~iate 20 regulatory regions is within the ability and discretion of one ûf or~ ~y skill in the art and the recombinant use of many such regulatory regions is now established in the art.
In another series of embo-1inn~nt~, the present invention provides for isolated nucleic acids encoding all or a portion of the PS-interacting proteins in the form of a fusion protein. In these embodiments, a nucleic acid regulatory region25 (endogenous or exogenous) is operably joined to a first coding region which is covalently joined in-frame to a second coding region. The second coding region optionally may be covalently joined to one or more additional coding regions and the last coding region is joined to a t~rmin~tion codon and, optionally, appropriate 3' regulatory regions (e.g., polyadenylation signals). The PS-interacting protein 30 sequences of the fusion protein may represent the first, second, or any additional SIJ~ 111 ~JTE S~IEET (RULE 26) CA 02244412 1998-0i-27 WO 97/27296 PC~/CA97/00051 coding regions. The PS-inter~cting protein sequences may be conserved or non-conserved domains and can be placed in any coding region of the fusion. The non-PS-interacting protein sequences of the fusion may be chosen according to the needs and discretion of the practitioner and are not limited by the present invention. Useful 5 non-PS-interacting protein sequences include, for example, short sequence "tags" such as antigenic ~let~- ., - it ~ or poly-His tags which may be used to aid in the i(lentifi~tion or purification of the resultant filsion protein. ~lt~rn~tively, the non-PS-interacting protein coding region may encode a large protein or protein fr~ment, such as an enzyme or binding protein which also may assist in the identifi~ tion and 10 purification of the protein, or which may be useful in an assay such as those described below. Particularly contemplated fusion proteins include poly-His and GST
~gl~lt~thione S-transferase) fusions which are useful in isolating and purifying the presenilins-interacting proteins, and the yeast two hybrid hlsions, described below, which are useful in assays to identify other proteins which bind to or interact with the 15 PS-interacting proteins.
In another series of embotlimentc the present invention provides isolated nucleic acids in the forrn of recombinant DNA constructs in which a marker or reporter gene (e.g., ,B-g;ll~rtocidase~ luciferase) is operably joined to the 5' regulatory region of a PS-interacting protein gene such that t;~lt;ssion of the marker gene is 20 under the control of those regulato~y sequences. Using the PS-interacting protein regulatory regions enabled herein, including regulatory regions from hurnan and other m~mm~ n species, one of ordinary skill in the art is now enabled to produce suchconstructs. As discussed more fillly below, such isolated nucleic acids may be used to produce cells, cell lines or transgenic ~nim~l~ which are useful in the identification of 25 compounds which can, directly or indirectly, differentially affect the expression of the PS-interacting proteins.
Finally, the isolated nucleic acids of the present invention include any of ~he above described se~uences when inc~ c1 in vectors. Appropriate vectors include cloning vectors and expression vectors of all types, including plasmids, phagemids, 30 cosmids, episomes, and the like, as well as integration vectors. The vectors may also SUBSTITUTE SHEET (RULE 26) CA 022444l2 l998-07-27 include various marker genes (e.g., antibiotic resi~t~nce or susceptibility genes) which are useful in identifying cells successfully transformed therewith. In addition, the vectors may include regulatory sequences to which the nucleic acids of the invention are operably joined, and/or may also include coding regions such that the nucleic 5 acids of the invention, when a~lu~.iateiy ligated into the vector, are expressed as fusion proteins. Such vectors may also include vectors for use in yeast "two hybrid,"
baculovirus, and phage-display systems. The vectors may be chosen to be useful for prokaryotic, eukaryotic or viral t;~L,ression, as needed or desired for the particular application. For example, vaccinia virus vectors or simian virus vectors with the 10 SV40 promoter (e.g., pSV2), or Herpes simplex virus or adeno-associated virus may be useful for l,~lsre-;Lion of m~mm~ n cells int~ in~ neurons in culture or in vivo, and the baculovirus vectors may be used in ~ re.;Lhlg insect cells (e.g., buLL~Iny cells). A great variety of di~.t;llL vectors are now commercially available and otherwise known in the art, and the choice of an ~lu~liate vector is within the 15 ability and discretion of one of o,dina~y skill in the art.

2. Substantiallv Pure Proteins The present invention provides for ~ub~l ;". I i~lly pure preparations of the PS-interacting proteins, fragrnt?nt.c of the PS-inleld~Ling proteins, and fusion proteins 20 including the PS-interacting proteins or fragments thereof. The proteins, fr~gmentc and fusions have utility, as described herein, in the generation of antibodies to normal and mutant PS-interacting proteins, in the identification of proteins (aside from the presçnilin.c) which bind to the PS-interacting proteins, and in diagnostic and therapeutic methods. Therefore, depending upon the intended use, the present 25 invention provides substantially pure proteins or peptides comprising amino acid sequences which are subsequences of the complete PS-interacting proteins and which may have lengths varying from 4-lO amino acids (e.g., for use as immunogens), or 10-lO0 amino acids (e.g., for use in binding assays~, to the complete PS-interacting proteins. Thus, the present invention provides subst~nti~lly pure proteins or peptides 30 comprising sequences corresponding to at least 4-5, preferably 6-lO, and more Sl-~ 111 l)TE SHEET (RULE 2Ei) w 097/27z96 PCT/CA97/00051 preferably at least 50 or 100 consecutive amino acids of the PS-interacting proteins, as disclosed or otherwise enabled herein.
The proteins or peptides of the invention may be isolated and purified by any of a variety of methods selected on the basis of the properties revealed by their 5 protein sequences. For example, the PS-interacting proteins may be isolated fiom cells in which the PS-interacting protein is normally highly expressed. ~ltPm~tively the PS-interacting protein, fusion protein, or fragment thereof, may be purified from cells transformed or transfected with w~ ssion vectors (e.g., baculovirus systems such as the pPbac and pMbac vectors (Str~t~gPne La Jolla, CA); yeast ~ es~,ion 10 systems such as the pYESHIS Xpress vectors (Invitrogen, San Diego, CA); eukaryotic ~Y.pl~sion systems such as pcDNA3 (Invitrogen, San Diego, CA) which has constantconstitutive ~ ylc;s~ion, or LacSwitch (str~t~gen~ La Jolla, CA) which is inducible;
or prokaryotic ~rcs~,ion vectors such as pKK233-3 (Clontech, Palo Alto, CA). ~n the event that the protein or ~agment integrates into the endoplasmic reticulum or 1~ plasma membrane of the recombinant cells (e.g., eukaryotic cells), the protein may be purified from the membrane fraction. ~Alt~ tively, if the protein aggregates in inclusion bodies within the recombinant cells (e.g., prokaryotic cells), the protein may be purified from whole Iysed cells or from solubilized inclusion bodies.
Purification can be achieved using standard protein purification procedures 20 including, but not limited to, gel-filtration chromatography, ion-e~ch~nge chromatography, high-performance liquid chromatography (RP-HPLC, ion-~ch~nge HPLC, size-exclusion HPLC, high-p~;.ro~ allce chromatofocusing chromatography, hydrophobic interactionchromatography, immllnf.~ ;ipilalion, orimmnnoafifinity pllrific~tion. Gel electrophoresis (e.g., PAGE, SDS-PAGE) can also be used to isolate 25 a protein or peptide based on its molecular weight, charge properties and hydrophobicity.
A PS-interacting protein, or a fragment thereof, may also be conveniently purified by creating a fusion protein including the desired PS-interacting protein sequence fused to another peptide such as an antigenic detc7~ t or poly-His tag 3Q ~e.g., QIAexpress vectors, QIAGEN Corp., Chal~wo~ , CA), or a larger protein (e.g., SU~ 111 ~JTE SHEET (RULE 263 GST using the pGEX-27 vector (Amrad, USA~ or green fluorescent protein using theGreen Lantern vector (GIBCO/BRL. Gaithersburg, MD). The fusion protein may be expressed and recovered from prokaryotic or eukaryotic cells and purified by anystandard method based upon the fusion vector se~uence. For example, the fusion 5 protein may be purified by immlm~affinity or irnmunoprecipitation with an antibody to the non-PS-interacting protein portion of the fusion or, in the case of a poly-His tag, by affinity binding to a niclcel column. The desired PS-interacting protein or ICragment may then be further purified from the fusion protein by enzymatic cleavage of the fusion protein. Methods for preparing and using such fusion constructs for the 10 purification of proteins are well known in the art and several kits are commercially available for this purpose. In light of the present disclosure, one is now enabled to employ such fusion constructs with the PS-interacting proteins.

3. Antibodies to the PS-interacting Proteins The present invention also provides antibodies, and methods of m~k;ng antibodies, which selectively bind to the PS-interacting proteins or fragm~-nt.c thereof.
Of particular importance, by identifying the PS-interacting domains of the PS-interacting proteins, and methods of identifying mutant forms of the PS-interacting proteins associated with ~l~h~imer's Disease, the present invention provides 2~ antibodies, and methods of m~king antibodies, which will selectively bind to and, thereby, identify and/or distinguish norrnal and mutant (i.e., pathogenic) forrns of the PS-interacting proteins. The antibodies of the invention have utility as laboratory reagents for, inter alia. immllnnaffinity purification of the PS-interacting proteins, Western blotting to identify cells or tissues ~ ,S::iillg the PS-interacting proteins, and 25 immunocytoch~ y or imrnunofluorescence techniques to establish the subcellular location of the proteins. In addition, as described below, the antibodies of theinvention may be used as diagnostics tools to identify carriers of AD-related PS-interacting protein alleles, or as thc,~ulic tools to selectively bind and inhibit pathogenic forrns of the PS-interacting proteins in vivo.

SUB5TITUTE SHEET (RU~ E 26~

WO 97/27296 P~TICA97/00051 The antibodies of the invention may be generated using the entire PS-interacting proteins of the invention, or using any PS-interacting protein epitope which is characteristic of that protein and which suhst~nti~lly distin~ hes it from other host proteins. Any method of choosing ~ntig~nic ~1etermin~nt.c known in the art 5 may, of course, be employed. Such epitopes may be identified by comr~ring sequences of, for example, 4-10 amino acid residues from a PS-interacting protein sequence to co,n~uLe. (l~t~b~es of protein sequences from the relevant host. In addition, larger fi~pment~ (e.g., 8-20 or, ~ re~bly, 9-15 residues) including one or more potential epitopes may also be employed. Antibodies to the PS-interacting 10 domains (identified by the yeast two-hybrid assays described below) are expected to have the greatest utility both diagnostically and therapeutically. On the other hand, antibodies against highly cons~ ed domains are expected to have the greatest utility for purification or iclenti fic~t;on of PS-interacting proteins.
PS-interacting protein immllnogen L)lcl)al~ions may be produced from 15 crude extracts (e.g., Iysates or membrane fractions of cells highly t;~LIJlt;SSillg the proteins), from proteins or peptides ~ub~lalllially purified from cells which naturally or recombinantly express them or, for short immlmogens, by chemical peptide synthesis. The immllnogens may also be in the form of a fusion protein in which the non-PS-interacting protein region is chosen ~or its adjuvant properties. As used20 herein, a PS-interacting protein immunogen shall be defined as a ~ Lion including a peptide comprising at least 4-8, and preferably at least 9-15 consecutive arnino acid residues of a PS-interacting proteins, as disclosed or otherwise enabled herein. Sequences of fewer residues may, of course, also have utility depending upon the intended use and future technological development~ Therefore, any PS-25 interacting protein derived sequences which are employed to generate antibodies tothe PS-interacting proteins should be regarded as PS-interacting protein imml]nogens.
The antibodies of the invention may be polyclonal or monoclonal, or may be antibody fr~grnPntc, including Fab fr~grnPntc F(ab')2, and single chain antibody fragments. In addition, after identifying useful antibodies by the method of the30 invention, recornbinant antibodies may be generated, including any of the antibody SU~:~ 111 UTE SHEET (RULE 26) -WO 97/27296 . PCT/CA97/00051 fragments listed above, as well as hllm~ni7ecl antibodies based upon non-human antibodies to the PS-interacting proteins. In light of the present disclosure, as well as the characterization of other PS-interacting proteins enabled herein, one of ordinary skill in the art may produce the above-described antibodies by any of a variety of standard means well known in the art. For an overview of antibody techniques, see Antibodv En~ineerin~: A Practical Guide. Borrebaek, ed., W.H. Freeman &
Company, NY (l 992~, or Antibody En~ineerin~. 2nd Ed., Borrebaek, ed., Oxford Ulliv~ y Press, Oxford (1995).
As a general matter, polyclonal antibodies may be generated by first 10 jmmllni7in~ a mouse, rabbit, goat or other suitable animal with the PS-interactin~
protein immunogen in a suitable carrier. To increase the immllnllgenicity of thepreparation, the immunogen may be coupled to a carrier protein or mixed with an adjuvant (e.g., Freund's adjuvant). Booster injections, although not necessary are recommen(1~ After an ~p.o~liate period to allow for the development of a humoral15 response, preferably several weeks, the ~nim~lc may be bled and the sera may be purified to isolate the immunoglobulin c(~ )ol,e~lt.
.~imil~rly, as a general matter, monoclonal anti-PS-interacting protein antibodies may be produced by first injecting a mouse, rabbit, goat or other suitable anirnal with a PS-i,lLel~clillg protein irnmunogen in a suitable carrier. As above, 20 carrier proteins or adjuv~l~s may be utilized and booster injections (e.g., bi- or tri-weekly over 8-l0 weeks) are lc;co"""~n~ 1 After allowing for development of a humoral response, the ~nim~l.c are sacrificed and their spleens are removed and resuspended in, for example, phosphate buffered saline {PBS). The spleen cells serve as a source of Iyrnphocytes, some of which are producing antibody of the a~propliate 25 specificity. These cells are then fused with an immortalized cell line (e.g., myeloma~, and the products of the fusion are plated into a number of tissue culture wells in the presence of a selective agent such as HAT. The wells are serially screened and replated, each time selecting cells making useful antibody. Typically, several screening and replating procedures are carried out until over 90% of the wells contain 30 single clones which are positive for antibody production. Monoclonal antibodies S~J~S l 1 1 UTE SHEET (RULE 26) - = =

w o 97/27296 PCT/CA97/U0051 produced by such clones may be purified by standard methods such as affinity chromatography using Protein A Sepharose, by ion-exchange chromatography, or by variations and combinations of these techniques.
The antibodies of the invention may be labelled or conjugated with other compounds or materials for diagnostic and/or therapeutic uses. For example, theymay be coupled to radionuclides, fluorescent compounds, or enzymes for im~ging or therapy, or to liposomes for the targeting of compounds contained in the liposomes to a specific tissue location.

10 4. Tlallsrollned Cell Lines The present invention also provides for cells or cell lines, both prokaryotic and eukaryotic, which have been tr~n.~formed or transfected with the nucleic acids of the present invention so as to cause clonal propagation of those nucleic acids and/or e~r~s~ion of the ~oteil,s or peptides encoded thereby. Such cells or cell lines will 15 have utility both in the propagation and production of the nucleic acids and proteins of the present invention but also, as further described herein, as model systems for diagnostic and thc;l~eu~ic assays. In particular, it is expected that cells co-transformed with PS-interacting protein sequences as well as pres~nilin sequences will have improved utility as models of the biochemical pathways which may be affected 20 in AD. For exarnple, cells co-l~ srolll~ed with the interacting domains of PS-interacting sequences and pres~Dnilini in yeast two-hybrid fusion constructs, will have utility in s~ .illg for compounds which either enh~n~e or inhibit interactions between these domains. Similarly, for cells transformed with a heterospecific pres~n;lin, co-~ldl~r,llllation with a similarly heterospecific PS-interacting protein, or 25 co-transformation and homologous recombination to introduce a similarly heterospecific PS-interacting domain of a PS-interacting protein (e.g., "hlTm~ni7ing" a non-human endogenous PS-interacting protein), will result in a better model system for studying the interactions of the preseni~in~ and the PS-interacting proteins. Cells transformed with only PS-interacting sequences will, of course, have utility of their SU~ ITE SHEET (RULE 26) own for studying the role of these proteins in the etiology of AD, and also as precursors for presenilin co-transformed cells.
As used herein, the te~ ro~ ed cell" is intende~l to embrace any cell, or the descendant of any cell, into which has been introduced any of the nucleic 5 acids of the invention, whether by l.~l~ro,l..ation, transfection, infection, or other means. Methods of producing ~ iate vectors, transforming cells with those vectors, and identifying transformants are well known in the art and are only briefly reviewed here ~see, for example, Sambrook et al. (1989) Molecular Clonin~: A
Laboratory ManuaL 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring 10 Harbor, New York~.
Prokaryotic cells useful for producing the transformed cells of the invention include members of the b~cteri~l genera Escherichia (e.g., E. coli), Psell-lom-~nas (e.g., P. aerll~inos~), and Bacillus (e.g., B. subtillus. B.
stearothermol~hilus), as well as many others well known and frequently used in the 15 art. Prokaryotic cells are particularly useful for the production of large quantities of the proteins or peptides of the invention (e.g., norrnal or mutant PS-interacting proteins, fr~ment~ of the PS-interacting proteins, fusion proteins of the PS-interacting proteins). Bacterial cells ~e.g., E. coli) may be used with a variety of ssion vector systems including, for example, plasmids with the T7 RNA
20 polymerase/promoter system, bacteriophage ~ regulatory sequences, or M13 Phage mGPI-2. B~ctt~n~l hosts may also be l~ r~l-ned with fusion protein vectors whichcreate, for example, lacZ, t~pE, maltose-binding protein, poly-His tags, or ~hlt~t1lione-S-transferase filsion proteins. All of these, as well as many other prokaryotic lci,sion systems, are well known in the art and widely available commercially 25 (e.g., pGEX-27 (Amrad, USA) for GST fusions).
Eukaryotic cells and cell lines useful for producing the l~ r~ ed cells of the invention include m~mm~ n cells and cell lines (e.g., PC12, COS, CHO, fibroblasts, myelomas, neurobl~tQm~s~ hybridomas, human embryonic kidney 293, oocytes, embryonic stem cells), insect cells lines (e.g., using baculovirus vectors such 30 as pPbac or pMbac (Stratagene, La Jolla, CA~), yeast (e.g., using yeast expression SUt~ I 11 UTE SHEET (RULE 26) W 0971~7296 PCT/C~97/00051 vectors such as pYESHIS (Invitrogen, CA)), and fungi. Eukaryotic cells are palticularly useful for embortimentc in which it is necessary that the PS-interacting proteins, or functional fragments thereof, pclro~ the functions and/or undergo the intracellular interactions associated with either the normal or mutant proteins. Thus, 5 for example, transfo~ned eukaryotic cells are preferred for use as models of PS-interacting protein function or interaction, and assays for screening candidate thefa~ lics preferably employ transformed eukaryotic cells.
To accomplish e~3les~ion in eukaryotic cells, a wide variety of vectors have been developed and are commercially available which allow inducible (e.g., i0 LacSwitch ~ ssion vectors, Stratagene, La Jolla, CA) or cognate ~e.g., pcDNA3vectors, Invitrogen, Chatsworlh, CA) e~les~ion of PS-interacting protein nucleotide sequences under the regulation of an artificial promoter element. Such promoter elements are often derived from CMV or SV40 viral genes, although other strong promoter etern~o-nt~ which are active in eukaryotic cells can also be employed to induce 15 transcription of PS-interacting protein nucleotide sequences. Typically, these vectors also contain an artificial polyadenylation sequence and 3' UTR which can also bederived from exogenous viral gene sequences or from other eukaryotic genes.
Furthermore, in some constructs, artificial, non-coding, spliceable introns and exons are included in the vector to enhance ~ les~ion of the nucleotide sequence of interest.
20 These e,~res~ion systems are commonly available from commercial sources and are typified by vectors such as pcDNA3 and pZeoSV (Invitrogen, San Diego, CA).
Innumerable commercially-available as well as custom-designed t;~)re~sion vectors are available from commercial sources to allow ~lGs~ion of any desired PS-interacting protein l~ s~;l;pt in more or less any desired cell type, either constitutively 2~ or after exposure to a certain exogenous stim~ (e.g., withdrawal of tetracycline or exposure to IPTG).
Vectors may be introduced into the recipient or "host" cells by various methods well known in the art including, but not limited to, calcium phosphate transfection, :jLlUlllilllll phosphate L~ r~ ion, DE~E dextran kansfection, 30 electroporation, lipofection (e.g., Dosper Liposomal transfection rea~ent, Boehringer Sll~:i 111 ~lTE SHEET (RULE 26) Mannheim, Germany), microinjection, ballistic insertion on micro-beads, protoplast fusion or, for viral or phage vectors, by infection with the recombinant virus or phage.

5. Trans~enic Animal Models The present invention also provides for the production of transgenic non-human animal models in which mutant or wild type PS-interacting protein se~uences are expressed, or in which the PS-interacting protein genes have been inactivated (e.g., "knock-out" deletions), for the study of Alzheimer's Disease, for the screening of candidate ph~rm~e~ltical compounds, for the creation of explanted m~mm~ n CNS
10 cell cultures (e.g., neuronal, glial, organotypic or mixed cell cultures), and for the evaluation of potential therapeutic h~ ions. Prior to the present invention, a partial animal model for Alzheimer's Disease existed via the insertion and over-es~ion of a mutant form of the human amyloid precursor protein gene as a minigene under the regulation of the platelet-derived growth factor ~ receptor 15 promoter element (Games et al., l995). This mutant (~APP7~7 Val~Ile) causes the appearance of synaptic pathology and amyloid ,B peptide deposition in the brain of transgenic ~nim~l~ bearing this transgene in high copy number. These changes in the brain of the transgenic animal are very similar to that seen in human AD (Games et al., l 995). It is, however, as yet unclear whether these ~nim~l~ become ~lementef1 but there is general consensus that it is now possible to recreate at least some aspects of AD in mice. In addition, transgenic animal models in which the presPnilin genes are gt~nçtic~lly ~ngineered are disclosed in PCT Publication W096/34099. These transgenic animal models have been shown to have altered A~ production and altered hippocampus-dependent memory function.
Animal species suitable for use in the animal models of the present invention include, but are not limited to, rats, mice, h~m~t~r.~, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-human primates (e.g., Rhesus monkeys, cl~illlpa,~es). For initial studies, transgenic rodents ~e.g., mice) may be ~lc;rt~ ,d due to their relative ease of ,~ rll~nce and shorter life spans. E~owever, transgenic yeast or invertebrates ~e.g., nematodes, insects) may be preferred for some studies because S~J~S 111 ~JTE SHEET (RUI E 26) W097127296 PCT/CA97/OOO~l they will allow for even more rapid and inexpensive screening. For example, in~ lebl~tes bearing mutant PS-interacting protein homologues (or m~mm~ n PS-interacting protein transgenes) which cause a rapidly occurring and easily scored phenotype (e.g., abnormal vulva or eye development after several days) can be used as 5 screens fior drugs which block the effect of the mutant gene. Such invertebrates may prove far more rapid and efficient for mass screenings than larger vertebrate ~nim~
Once lead compounds are found through such screens, they may be tested in higher~nim~ such a rodents. Ultim~t~ly, transgenic non-hurnan primates may be preferred for longer term studies due to their greater similarity to hl~m~n~ and their higher 10 cognitive abilities.
Using the nucleic acids disclosed and otherwise enabled herein, there are now several available approaches for the creation of a transgenic animal model for Alzheimer's Disease. Thus, the enabled animal models include: (I) Animals in which sequences encoding at least a functional domain of a noImal hurnan PS-interacting 15 protein gene have been recolllbi~ llly introduced into the ~enome of the animal as an additional gene, under the regulation of either an exogenous or an endogenous promoter element, and as either a m;nigene or a large genomic fr~gment; in whichsequences encoding at least a functional domain of a normal human PS-interactingprotein gene have been recombinantly substituted for one or both copies of the 20 animal's homologous PS-inl~ lg protein gene by homologous recombination or gene targeting; and/or in which one or both copies of one of the animalls homologous PS-interacting protein genes have been recombinantly 'Ihllm~ni7~ by the partial substitution of sequences encoding the human homologue by homologous recombination or gene l~gelillg. These animals are useful for evaluating the effects 25 of the transgenic procedures, and the effects of the introduction or substitution of a human or hllm~ni7~1 PS-interacting protein gene. (2) Animals in which sequences encoding at least a functional domain of a mutant (i.e., pathogenic~ human PS-interacting protein gene have been recombinantly introduced into the genome of the animal as an additional gene, under the regulation of either an exogenous or an 30 endogenous promoter element, and as either a minigene or a large genomic fragment;

SUBSTITUTE SHEET (RULE 26) in which sequences encoding at least a fimctional domain of a mutant human PS-interacting protein gene have been recombinantly substituted for one or both copies of the animal's homologous PS-interacting protein gene by homologous recombination or gene targeting, and/or in which one or both copies of one of the animal's 5 homologous PS-interacting protein genes have been recombinantly "hllnn~ni7ed" by the partial ~ul)s~i~u~ion of sequences encoding a mutant human homologue by homologous recombination or gene targeting. These ~nim~l.s are useful as models which will display some or all o~the characteristics, whether at the biochemical, physiological and/or behavioral level, of hllm~n~ carrying one or more alleles which 10 are pathogenic of ~17.heimer's Disease or other ~ e~es associated with mutations in the PS-interacting protein genes. (3) Animals in which sequences encoding at least a functional domain of a mutant version of one of that animal's PS-interacting protein genes (bearing, for exarnple, a specific mutation corresponding to, or similar to, one of the pathogenic mutations of the human PS-interacting proteins) have been 15 recombinantly introduced into the genome of the animal as an additional gene, under the regulation of either an exogenous or an endogenous promoter element, and as either a minigene or a large genomic fragrnent and/or in which sequences encoding at least a functional domain of a mutant version of one of that animal's PS-interacting protein genes (bearing, for example, a specific mutation corresponding to, or similar 20 to, one of the pathogenic mutations of the human PS-inter~t~tin~ proteins) have been recombinantly substituted for one or both copies of the animal's homologous PS-interacting protein gene by homologous recombination or gene targeting. These ~nim~ are also useful as models which will display some or all of the characteristics, whether at the biochemical, physiological and/or behavioral level, of hl-m~n~ carrying 2~ one or more alleles which are pathogenic of Alzheimer's Disease. (4) "Knock-out"
~nim~ in which one or both copies of one of the animal's PS-interacting protein genes have been partially or completely deleted by homologous recombination or gene targeting, or have been ina.;liv~ted by the insertion or substitution by homologous recombination or gene targeting of exogenous sequences (e.g., stop 30 codons, lox p sites). Such ~n;m~l~ are useful models to study the effects which loss of S~ I UTE SHEET (RULE 26J

PS-interacting protein gene ~yles~ion may have, to evaluate whether loss of function is preferable to continued expression of mutant forms, and to ex~min~ whether other genes can be recruited to replace a mutant PS-interacting protein or to intervene with the effects of other genes (e.g., PS1, PS2, APP or ApoE) causing AD as a tre~t~nent 5 for AD or other disorders. For example, a normal PS-interacting protein gene may be necçc.c~ry for the action of mutant pres~on;lin or APP genes to actually be expressed as AD and, therefore, transgenic PS-interacting protein animal models may be of use in elucidating such multigenic interactions.
In addition to transgenic animal models in which the ~ yl~s~ion of one or 10 more of the PS-interacting proteins is altered, the present invention also provides for the production of transgenic animal models in which the ~ ession of one or more of the presenilinc, APP, or ApoE is altered. The nucleic acids encoding the presçnilinc7 APP, and ApoE are known in the art, a methods for producing transgenic ~nim~lc with these sequences are also known (see, e.g., PCT Publication W096/34099, Games et 15 al., 1995). Indeed, because non-human ~nim~lc may differ from hllm~nc not only in their PS-interacting protein sequences, but also in the sequences of their pres~nilin, APP and/or ApoE homologues, it is particularly collL~ plated that transgenics may be produced which bear recombinant normal or mutant human sequences for at least one pres~nilin, APP and/or ApoE gene in addition to recombinant sequences for one or20 more PS-interacting proteins. Such co-transformed animal models would possessmore elements of the human molecular biology and, therefore, are expected to be better models of human disorders. Thus, in accordance with the present invention, transgenic animal models may be produced bearing norrnal or mutant sequences forone or more PS-interacting proteins, or interacting domains of these proteins. These 25 .~nim~lc will have utility in that they can be crossed with animals bearing a variety of mormal or mutant presenilin, APP or ApoE sequences to produce co-transformed animal models. Furthermore, as detailed below, it is expected that mutations in the PS-interacting genes, like mutations in the presenilinc themselves, may be causative of Alzheimer's Disease and/or other disorders as well (e.g., other cognitive, 30 intellectual, neurological or psychological disorders such as cere~ral hemorrhage, SlJ~S 1 l l ~JTE SHEET (RULE 26) schizophrenia, depression, mental retardation and epilepsy). Therefore, transgenic animal models bearing normal or mutant sequences corresponding to the PS-interacting proteins, absent transformation with any presenilin, APP or ApoE
sequences, will have utility of their own in the study of such disorders.
As detailed below, p,efel,~d choices for transgenic animal models transformed with PS-interacting proteins, or domains of PS-interacting proteins,include those transformed with nor~nal or mutant sequences corresponding to the clones identified and described in Example 1 and disclosed in SEQ ID NOs: 1-12.
These clones, which interact with normal or mutant PS 1 TM6~7 loop domains, were10 identified according to the methods described in Example 1, below, and PCT
Publication W096/34099. These clones, longer nucleic acid sequences comprising these clones, and other clones i~l~ntifie~l according to this and other methods of the invention (e.g., allelic and splice variants or heterospecific homologues of these clones) may all be employed in accordance with the present invention to produce 15 animal models which, with or without co-l-;-~r ll " ,~tion with presenilin, APP arld/or ApoE sequences, will have utility in the study of Alzheimer's Disease and/or other cognitive, intellectual, neurological or psychological disorders.
Thus, using the nucleic acids disclosed and otherwise enabled herein, one of oldilla, y skill in the art may now produce any of the following types of transgenic 20 animal models with altered PS-interacting protein ~.cs,ion~ Animals in which sequences encoding at least a functional domain of a normal human PS-interactingprotein gene have been recombinantly introduced into the genome of the animal as an additional gene, under the regulation of either an exogenous or an endogenous promoter element, and as either a minigene or a large genomic fr~gment in which 25 sequences encoding at least a functional domain of a normal human PS-interacting protein gene have been recombinantly substituted for one or both copies of the animal's homologous PS-interacting protein gene by homologous recombination or gene l~ ling; and/or in which one or both copies of one of the animal's homologous PS-interacting protein genes have been recombinantly "hl-m~ni7ed" by the partial30 substitution of sequences encoding the human homologue by homologous SlJ~;~ ITE SI~EET (RULE 26~

, CA 02244412 1998-0i-27 recombination or gene ~g~ g. These animals are particularly useful for providingtransgenic models which express human PS-interacting proteins as well as human presenilin proteins. They are also useful in evaluating the effects of the transgenic procedures, and the effects of the introduction or substitution of a human or 5 hl-m~ni7~ PS-interacting protein gene. (2) Animals in which sequences encoding at least a functional domain of a mutant (i.e., pathogenic) human PS-interacting protein gene have been recombinantly introduced into the genome of the animal as an additional gene, under the regulation of either an exogenous or an endogenous promoter element, and as either a minigene or a large genomic fr~grn~nt in which10 sequences encoding at least a functional domain of a mutant human PS-interacting protein gene have been recombinantly ~ub~LiLuLed for one or both copies of the animal's homologous PS-interacting protein gene by homologous recombination or gene targeting; and/or in which one or both copies of one of the animal's homologous PS-interacting protein genes have been lecol~ ina.~Lly "hllm~ni7~-1" by the partial 15 substitution of sequences encoding a mutant human homologue by homologous recombination or gene tal~,eti,lg. These animals are useful as models which willdisplay some or all of the characteristics, whether at the biochemic~l, physiological and/or behavioral level, of hnm~n~ carrying one or more alleles which are pathogenic of Alzheimer's Disease or other ~ e~c associated with mutations in these PS-20 interacting genes. (3) Animals in which sequences encoding at least a functionaldomain of a mutant version of one of that animal's PS-interacting protein genes (bearing, for example, a specific mutation corresponding to, or similar to, one of the pathogenic mutations of the human PS-interacting proteins) have been recombinantly introduced into the genome of the animal as an additional gene, under the regulation 25 of either an exogenous or an endogenous promoter element, and as either a minigene or a large genomic fr~nent; and/or in which sequences encoding at least a functional domain of a mutant version of one of that animal's PS-interacting protein genes (bearing, for example, a specific mutation corresponding to, or similar to, one of the pathogenic mutations of the hllm~n~ PS-interacting proteins) have been recombinantly 3Q substituted ror one or both copies of the animal's homologous PS-interacting protein S~ TE SHEET (RULE 2~

gene by homologous recombination or gene targeting. These ~mim~l.c are also useful as models which will display some or all of the characteristics, whether at the biochemical, physiological and/or behavioral level, of hlln~n~ car~ying one or more alleles which are pathogenic of Alzheimer's Disease. (4) "Knock-out" ~nim~l.c in5 which one or both copies of one of the animal's PS-interacting protein genes have been partially or completely deleted by homologous recombination or gene targeting, or have been inactivated by the insertion or substitution by homologous recombination or gene targeting of exogenous sequences (e.g., stop codons, lox psites). Such ~nim~l~ are useful models to study the effects which loss of PS-10 interacting protein gene c;~le~ion may have, to evaluate whether loss of function ispreferable to continue~ res~ion, and to ex~mine whether other genes can be recruited to replace a mutant PS-interacting protein or to i~ l V~llC with the effects of other genes (e.g., APP or ApoE) causing AD as a tre~trn~nt for AD or other disorders.
For example, a normal PS-interacting protein may be necessary for the action of 1~ mutant PS1, PS2 or APP genes to actually be expressed as AD and, therefore, transgenic PS-interacting protein animal models may be of use in elucidating such multigenic hlteld-;~ions.
In some ~re~led embodiments, transgenic animal models are produced in which just the PS-interacting domains of the PS-interacting proteins are introduced 20 into the genome of the animal by homologous recombination. Thus, for example,preferred embodiments include transgenic animals in which the PS~ te.~ g domains of PS-interacting proteins are "hl-m~ni7tod" by homologous recombinationwith sequences f~om human PS-interacting proteins. These ~nim~l~ may then be bred with transgenics in which normal or mutant prec~nilin sequences have been 25 introduced. The progeny of these ~nim~ls, having both human pr~sçnilin and human PS-interacting protein sequences, will provide improved animal models for Alzheimer's Disease.
To create an animal model (e.g., a tr~n~g~nic mouse), a normal or mutant PS-interacting gene ~e.g., normal or mutant S5a, GT24, pO071, Rabl 1, etc.), or a 30 normal or mutant version of a recombinant nucleic acid encoding at least a functional SIJCS~ 111 UTE SHEET ~RULE 26) , :

domain of a PS-interacting gene (e.g., the PS-interacting domains obtained in the yeast two-hybrid system), can be inserted into a germ line or stem cell using standard techniques of oocyte microin~ection, or transfection or microinjection into embryonic stem cells. Animals produced by these or similar processes are referred to as transgenic. Similarly, if it is desired to inactivate or replace an endogenous pres~n;l;n or PS-interacting protein gene, homologous recombination using embryonic stem cells may be employed. Animals produced by these or similar processes are referred to as "knock-out" (inactivation) or "knock-in" (replacement) models.
For oocyte injection, one or more copies of the recombinant DNA
10 constructs of the present invention may be inserted into the pronucleus of a just-fertilized oocyte. This oocyte is then reimplanted into a pseudo-~le~ foster mother. The liveborn ~n;m~l~ are screened for integrants using analysis of DNA (e.g., from the tail veins of offspring mice) for the presence of the inserted recombinant transgene sequences. The transgene may be either a complete genomic sequence 15 injected as a YAC, BAC, PAC or other chromosome DNA fr~mçnt a cDNA with either the natural promoter or a heterologous promoter, or a minigene Co.~ g all of the coding region and other elements found to be necess~ y for ol)limu~ c;ssion~Retroviral infection of early embryos can also be done to insert the recombinant DNA constructs of the invention. In this method, the transgene (e.g., a 20 normal or mutant S5a, GT24, pO071, Rab l l, etc., sequence~ is inserted into a lvvil~l vector which is used to infect embryos (e.g., mouse or non-human primateembryos) directly during the early stages of development to generate chimeras, some of which will lead to germline tr~ncmic~ion~
Homologous recombination using stem cells allows for the screening of 25 gene transfer cells to identify the rare homologous recombination events. Once identified, these can be used to generate chimeras by injection of blastocysts, and a l~r~3~(,lLion of the res~llting ~nim~lc will show germline tr~n.cmiccion from the recombinant line. This methodology is especially useful if inactivation of a gene is desired. For example, inactivation of the S5a gene in mice may be accomplished by 30 rlesigning a DNA fragment which contains sequences from an S5a coding region SIJ~ JTE SHEET (RULE 26~

fl~nking a selectable marker. Homologous recombination leads to the insertion of the marker sequences in the middle of the coding region, c~lleing inactivation of the SSa gene and/or deletion of intP!rn~l sequences. DNA analysis of individual clones can then be used to recognize the homologous recombination events.
The techniques of generating transgenic ~nim~c, as well ac the techniques for homologous recombination or gene targeting, are now widely accepted and practiced. A laboratory manual on the manipulation of the mouse embryo, for example, is available ~l.ot~iling standard laboratory techniques for the production of transgenic mice (Hogan et al., 1986). To create a ~ sg~l.e, the target sequence of 10 interest (e.g., norrnal or mutant presenilin sequences, normal or mutant PS-interacting protein sequences) are typically ligated into a cloning site located dowllsl~c~ll of some promoter element which will regulate the ~ ession of RNA from the sequence. Duwll~ ll of the coding sequence, there is typically an artificial polyadenylation sequence. ~n the trar~cgenic models that have been used to 15 successfully create ~nim~le which mimic aspects of inherited human neurodegenerative tliceslees~ the most sllccescfil1 promoter elements have been the platelet-derived growth factor rec~Lor~B gene subunit promoter and the h~mct~r prion protein gene promoter, although other promoter elements which direct e,~lession in central nervous system cells would also be usefill. An ~It~rn~te approach to creating a 20 tr~ncgto,ne is to use an endogenous pres~nilin or PS-interacting protein gene promoter and regulatory sequences to drive t;~les~ion of the tr~n~gene Finally, it is possible to create tr~n~g~n~ss using large genomic DNA fra~rnent.c such as YACs which contain the entire desired gene as well as its ap~ropliate regulatory sequences. Such consLIu~;ls have been s~lcces~fi~lly used to drive human APP ~ res~ion in kansgenic 25 mice (Lamb et al., 1993).
Animal models can also be created by targeting the endogenous presenilin or PS-interacting protein gene in order to alter the endogenous sequence by homologous recombination. These targeting events can have the effect of removingendogenous sequence ~knock-out) or altering the endogenous sequence to create an30 amino acid change associated with human disease or an otherwise abnormal sequence Sl,.,S 111 ~JTE St~EET (RULE 26) CA 022444l2 l998-07-27 (e.g., a sequence which is more like the human sequence than the original anirnal sequence) (knock-in animal models). A large nurnber of vectors are available to accomplish this and a~.~ro~iate sources of genomic DNA for mouse and other animal genomes to be targeted are comrnercially available from companies such as 5 GenomeSystems Inc. ~St. Louis, Missouri, USA). The typical feature of these ~geLillg vector constructs is that 2 to 4 kb of genomic DNA is ligated 5' to a selectable marker (e.g., a bacterial neomycin re~i~t~n~e gene under its own promoter element termed a "neomycin cassette"). A second DNA fragrnent from the gene of interest is then ligated do~llsllc~n of the neomycin c~sette but ~ sll~ll of a second 10 selectable marker ~e.g., thymidine kinase). The DNA fr~grnent~ are chosen such that mutant sequences can be introduced into the germ line of the targeted animal by homologous replacement of the endogenous sequences by either one of the sequences included in the vector. ~ltern~tively, the sequences can be chosen to cause deletion of sequences that would normally reside between the left and right arms of the vector 15 surrounding the neomycin c~ette The former is known as a knock-in, the latter is known as a knock-out. Again, innumerable model systems have been created, particularly for targeted knock-outs of genes including those relevant to neurodegenerative ~li.ce~.~es (e.g., targeted deletions of the murine APP gene by Zheng et al., 1995; targeted deletion of the murine prion gene associated with adult onset 20 human CNS degeneration by Bueler et al., 1996).
Finally, equivalents of transgenic ~nim~l~, including ~nim~l~ with m~lt~ted or inactivated pres~nilin genes, or mllt~ted or inactivated PS-interacting protein genes, may be produced using chemic~l or X-ray mllt~ nesis of g~mt~t~s, followed by fertilization. Using the isolated nucleic acids disclosed or otherwise enabled herein, 25 one of ordinary skill may more rapidly screen the r~s--lting offspring by, for exarnple, direct sequencing RFLP, PCR, or hybridization analysis to detect mllt~nt~, or Southern blotting to demonstrate loss of one allele by dosage.

6. Assa~s for Dru~s Which Affect PS-Interactin~ Protein Expression SlJ~ 11~ ~JTE SHEET (RULE 2~;) In another series of embodiments, the present invention provides assays for identifying small molecules or other compounds which are capable of inducing or inhibiting the eAIlre;~ion of the PS-interacting genes and proteins (e.g., S5a or GT24).
The assays may be pcLr~ ed in vitro using non-transformed cells, immortalized cell 5 lines, or recombinant cell lines, or in vivo using the transgenic animal models enabled herein.
In particular, the assays may detect the presence of increased or decreased e;AI~eS~iOn of S5a, GT24, pO071, Rab 11, or other PS-interacting genes or proteins on the basis of increased or decreased mRNA ~ es:~ion (using, e.g., the nucleic acid 10 probes disclosed and enabled herein), increased or decreased levels of PS-interacting proteins (using, e.g., the anti-PS-hllt; dclillg protein antibodies disclosed and enabled herein), or increased or decreased levels of ~A~le3~ion of a marker gene (e.g., ~-galactosidase or luciferase) operably joined to a PS-hl~ g protein 5' regulatory region in a recombinant construct.
Thus, for example, one may culture cells known to express a particular PS-interacting protein and add to the culture medium one or more test compounds. After allowing a sufficient period of time (e.g., 0-72 hours) for the compound to induce or inhibit the ~A~ ion of the PS-interacting protein, any change in levels of t;A~les~ion from an established baseline may be detected using any of the techniques described 20 above and well known in the art. In particularly plcr~ d embo~iment~, the cells are from an immortalized cell line such as a human neuroblastoma, glioblastoma or a hybridoma cell line. Using the nucleic acid probes and /or antibodies disclosed and enabled herein, detection of changes in the ~AI ,c;s~ion of a PS-interacting protein, and thus identifie~tion of the compound as an inducer or repressor of PS-interacting25 protein ~;A~lc;ssion, requires only routine experiment~tion.
In particularly preferred embodiments, a recombinant assay is employed in which a reporter gene such a ,B-galactosidase, green fluorescent protein, ~lk~ine phosphatase, or luciferase is operably joined to the S' regulatory regions of a PS-interacting protein gene. Preferred vectors include the Green T .~nteItl 1 vector 30 (GIBCO/BRL, Gaithersburg, MD) and the Great EScAPe pSEAP vector (Clontech, SUt~ JTE SHEFT ~RULE 26~

,~

wo 97/27Z96 PCT/CA97/00051 Palo Alto). The PS-interacting protein regulatory regions may be easily isolated and cloned by one of on~ ,y skill in the art in light of the present disclosure of coding regions from these genes. The reporter gene and regulatory regions are joined in-frame ~or in each of the three possible reading frames~ so that transcription and 5 translation of the reporter gene may proceed under the control of the PS-interacting protein regulatory elements. The recombinant construct may then be introduced into any a~plu~.,iate cell type, although m~rnm~ n cells are l~lere-l~d, and human cells are most pref~ ed. The transformed cells may be grown in culture and, after establishing the baseline level of ~I)res~ion of the reporter gene, test compounds may 10 be added to the medium. The ease of detection of the e~les~ion of the reporter gene provides for a rapid, high through-put assay for the i-l~ntification of inducers and r~esso~ o~the PS-interacting protein gene.
Compounds identified by ~is method will have potential utility in modifying the ~ s~ion of the PS-interacting protein genes in vivo. These 15 compounds may be filrther tested in the animal models disclosed and enabled herein to identify those compounds having the most potent in vivo effects. In addition, as described herein with respect to small molecules having binding activity for PS-interacting proteins, these molecules may serve as "lead compounds" for the further development of ph~ f ell~icals by, for example, subjecting the compounds to 20 sequential modifications, molecular modeling, and other routine procedures employed in rational drug design.

7. I~l~r~t;fic~tion of Compounds with PS-Interactin~ Protein Bindin~ CapacitY
In light of the present disclosure, one of ordinary skill in the art is enabled 25 to practice new screening methodologies which will be useiill in the identification of proteins and other compounds which bind to, or otherwise directly interact with, the PS-interacting pl()leills. The proteins and compounds will include endogenous cellular components, aside from the pres~nilin~, which interact with the PS-interacting proteins in vivo and which, therefore, provide new targets for phalmaceutical and 30 therapeutic interventions, as well as recombinant, synthetic and otherwise exogenous SUBS'rlTUTE SHEET (RULE 26) compounds which may have PS-interacting protein binding capacity and, therefore,may be candidates for ph~ ce ~tical agents. Thus, in one series of embo-limerlt~, cell Iysates or tissue homogenates (e.g., human brain homogenates, Iymphocyte Iysates) may be screened for proteins or other compounds which bind to one of the normal or mutant PS-interacting proteins. ~lt~.rn~tively, any of a variety of exogenous compounds, both naturally occurring and/or synthetic (e.g., libraries of small molecules or peptides), may be screened for PS-interacting protein bindingcapacity. Small molecules are particular plc;f~,~f~;d in this context because they are more readily absorbed after oral ~1mini~tration, have fewer potential antigenic ~let~rmin~ntc, and/or are more likely to cross the blood brain barrier than larger molecules such as nucleic acids or proteins. The methods of the present invention are particularly useful in that they may be used to identify molecules which selectively or preferentially bind to a mutant form of a PS-interacting protein (rather than a normal form) and, therefore, may have particular utility in treating cases of AD which arise 1~ from mutations in the PS-interacting ~ L~ins.
Once ide~tified by the methods described above, the c~n~ te compounds may then be produced in quantities sufficient for ph~rm~c elltical ~lmini~tration or testing (e.g."ug or mg or greater quantities), and form~ ted in a ph~,rm~ceutically acceptable carrier (see, e.g., Re~in~son's Pha~naceutical Sciences. Gennaro, A., ed., Mack Pub., 1990). These c~n~ 5e compounds may then be ~lmini~ctered to the olllled cells of the invention, to the transgenic animal models of the invention, to cell lines derived from the animal models or from human patients, or to Alzheimer's patients. The animal models described and enabled herein are of particular utility in further testing candidate compounds which bind to normal or mutant PS-interacting proteins for their thel~peuLic efficacy.
In addition, once identified by the methods described above, the candidate compounds may also serve as ~'lead compounds" in the design and development of new pharrnaceuticals. For example, as in well known in the art, se~uential modification of small molecules (e.g., amino acid residue replacement with peptides;
functional group replacement with peptide or non-peptide compounds) is a standard S~ JTE SHEET (RULE 26) -w o 97/27296 PCT/CA97/00051 approach in the pharmaceutical industry for the development of new ph~rm~eeuticals.
Such development generally proceeds from a "lead compound" which is shown to have at least some of the activity (e.g., PS-interacting protein binding or blocking ability) of the desired ph~ ce~l~ical. In particular, when one or more compoundshaving at least some activity of interest (e.g., modulation of PS-interacting protein activity) are identified, structural coml)~uison of the molecules can greatly inform the skilled practitioner by suggesting portions of the lead compounds which should be conserved and portions which may be varied in the design of new candidate compounds. Thus, the present invention also provides a means of identifying lead10 compounds which may be sequentially modified to produce new candidate compounds for use in the tre~ nt of Alzheimer's Disease. These new compounds then may be tested both for binding to PS-interacting proteins and/or blocking PS-interacting protein activity, and for therapeutic efficacy (e.g., in the animal models described herein). This procedure may be iterated until compounds having the desired 15 therapeutic activity and/or efficacy are identified.
In each of the present series of embo~lim~nt~, an assay is con~-cte-i to detect binding between a "PS-interacting protein component" and some other moiety.
Of particular utility will be sequential assays in which compounds are tested for the ability to bind to only normal or only mutant forms of the PS-interacting clom~in~ of 20 PS-illL~ld.;~ g proteins in the binding assays. Such compounds are expected to have the greatest therapeutic utilities, as described more fully below. The "PS-interacting protein component" in these assays may be a complete normal or mutant form of a PS-interacting protein (e.g., SSa, GT24, pO071, Rab l l, etc.) but need not be. Rather, particular functional domains of the PS-interacting proteins, particularly the PS-25 interacting domains as described above, may be employed either as separate molecules or as part of a fusion protein. For example, to isolate proteins or compounds that interact with these functional domains, s~l~ellillg may be carried out using fusion constructs and/or synthetic peptides corresponding to these regions.
Thus, for S5a, GST-fusion peptides may be made including sequences corresponding30 ~p~ ,ately to amino acids 70-377 of SEQ ID NO: 2 (included in clones Y2H29 SU~5 111 ~ITE SHEET (RULE 26~

and Y2H3 1, see Exarnple I ), approximately to amino acids 206-377 of SEQ ID NO: 2 (which includes protein-protein interaction motifs, see Ferrell et al., 1996), or to any other SSa domain of interest. Similarly, for GT24, GST- or other fusion peptides may be produced including sequences corresponding approximately to amino acids 440-815 of SEQ ID NO: 4 (including part of the ~rrn~-lillo repeat segrnent). Obviously, various combinations of fusion proteins and PS-interacting protein functional ~lom~in~
are possible and these are merely examples. In addition, the functional domains may be altered so as to aid in the assay by, for example, introducing into the functional domain a reactive group or amino acid residue (e.g., cysteine) which will facilitate I0 immobilization of the domain on a substrate (e.g., using sulfhydryl reactions). Thus, for example, the PS-interacting domain of S5a may be synthe~i7e~1 CO~ l;llg an additional C-t~rrnin~l cysteine residue to facilitate immobilization of the ~10m~in Such peptides may be used to create an affinity substrate for affinity chromatography (Sulfo-link; Pierce~ to isolate binding proteins for microsequencing. Sirnilarly, other 15 functional domain or antigenic fragments may be created with modified residues (see, e.g., Example 4).
The proteins or other compounds identified by these methods may be purified and characterized by any of the standard methods known in the art. Proteins may, for example, be purified and separated using electrophoretic (e.g., SDS-PAGE, 20 2D PAGE) or chromatographic (e.g., HPLC) techniques and may then be microsequenced. For proteins with a blocked N-t~ .,.i~.l.~, cleavage (e.g., by CNBr and/or trypsin) of the particular binding protein is used to release peptide fragments.
Further purification/characterization by HPLC and microseq~lenring and/or mass spectrometry by convellLional methods provides intPrn~l sequence data on such 25 blocked proteins. For non-protein compounds, standard organic chemical analysis ,.
techniques (e.g., IR, NMR and mass spectrometry; functional group analysis; X-ray crystallography) may be employed to ~let~nine their structure and identity.
Methods for screening cellular lysates, tissue homogenates, or small molecule libraries for candidate PS-interaction protein-binding molecules are well 30 known in the art and, in light of the present disclosure, may now be employed to S~ UTE SHEET(RULE 26) W 097/27296 PCT/CA97/OO~Sl identify compounds which bind to normal or mutant PS-interacting protein components or which modulate PS-interacting protein activity as defined by non-specific measures (e.g., changes in intracellular Ca2+, GTP/GDP ratio) or by specific measures (e.g., changes in A~ peptide production or ch~n~es in the ~ es~ion of 5 other do~ genes which can be monitored by differential display, 2D gel electrophoresis, differential hybridization, or SAGE methods). The pl~r~ d methods involve variations on the following techni~ues: (1) direct extraction by affinity chromatography; (2) co-isolation of PS-interacting protein components and bound proteins or other compounds by immllnoprecipitation; (3) the Biomolecular 10 Interaction Assay (BIAcore), and (4) the yeast two-hybrid systems. These and others are discussed separately below.
A. Affinitv Chromato~raphY
In light of the present disclosure, a variety of affinity binding techniques well known in the art may be employed to isolate proteins or other compounds which 15 bind to the PS-h~ d ;ling protein ~ ck~se-1 or otherwise enabled herein. In general, a PS-interacting protein component may be immobilized on a substrate (e.g., a column or filter) and a solution including the test compound(s) is contacted with the PS-interacting protein, fusion or fragment under conditions which are permissive for binding. The sub~llale is then washed with a solution to remove unbound or weakly 20 bound molecules. A second wash may then elute those compounds which strongly bound to the immobilized normal or mutant PS-interacting protein component.
~Itt~rn~ively, the test compounds may be imrnobilized and a solution cc,~ ,;.,g one or more PS-interacting protein components may be contacted with the column, filter or other substrate. The ability of the PS-interacting protein colllpollent to bind to the 25 test compouIlds may be det~rmined as above or a labeled form of the PS-interacting protein component (e.g., a radio-labeled or chemiluminescent functional domain) may be used to more rapidly assess binding to the substrate-immobilized compound(s). B. Co-Tmmnn~-pleci~,il;lLion Another well characterized technique for the isolation of PS-interacting 30 protein components and their associated proteins or other compounds is direct SUP'STITUTE SHEET ~RULE 26) CA 022444l2 l998-07-27 imm~ precipitation with antibodies. This procedure has been surcecsfill~y used, for example, to isolate many of the synaptic vesicle associated proteins (Phizicky and Fields, 1994). Thus, either normal or mutant, free or membrane-bound PS-interacting protein components may be mixed in a solution with the candidate compound(s) 5 under conditions which are permissive for binding, and the PS-interacting protein component may be immuno~leci~ ed. Proteins or other compounds which co-immun~ e with the PS-interacting protein component may then be identified by standard techniques as described above. General techniques for immnn~-precipitation may be found in, for exarnple, Harlow and Lane, (1988) 10 Antibodies: A LaboratorY ManuaL Cold Spring Harbor Press, Cold Spring Harbor, NY.
The antibodies employed in this assay, as described and enabled herein, may be polyclonal or monoclonal, and include the various antibody fragments (e.g., Fab, F(ab')2,) as well as single chain antibodies, and the like.
C. The Biomolecular Interaction Assay Another useful method for the detection and isolation of binding proteins is the Biomolecular Interaction Assay or "BLAcore" system developed by PharmaciaBiosensor and described in the m~nllf~ctllrer's protocol (LKB Ph~rm~ri~, Sweden). In light of the present disclosure, one of c,l-lh~ y skill in the art is now enabled to 20 employ this system, or a ~ub~ lial equivalent, to identify proteins or other compounds having PS-interacting protein binding capacity. The BIAcore system uses an affinity purified anti-GST antibody to irnmobilize GST-fusion proteins onto asensor chip. Obviously, other fusion proteins and corresponding antibodies may be substit~lte-l The sensor utilizes surface plasmon resonance which is an optical 25 phenomenon that detects changes in refractive indices. A homogenate of a tissue of interest is passed over the immobilized fusion protein and protein-protein interactions are registered as changes in the refractive index. This system can be used to det~nnine the kinetics of binding and to assess whether any observed binding is of physiological relevance.

.. .
SU~ 111 ~3TE SHEET (RULE 26) =

wo 97/27296 PCT/CA~7/00051 D. The Yeast Two-Hvbrid SYstem The yeast "two-hybrid" system takes advantage of transcriptional factors that are composed of two physically separable, functional domains (Phizicky and Fields, l 994). The most commonly used is the yeast GAL4 transcriptional activator 5 consi~ting of a DNA binding domain and a transcriptional activation domain. Two different cloning vectors are used to generate separate fusions of the GAL4 domains to genes encoding potential binding proteins. The fusion proteins are co-expressed, targeted to the nucleus and, if h~L~laclions occur, activation of a reporter gene (e.g., lacZ) produces a detect~le phenotype. For example, the Clontech ~tchm~k~r 10 System-2 may be used with the Clontech brain cDNA GAL4 activation domain fusion library with PS-interacting protein-GAL4 binding domain fusion clones (Clontech,Palo Alto, CA). In light of the disclosures herein, one of o~ ~ y skill in the art is now enabled to produce a variety of PS-interacting protein fusions, including fusions including either normal or mutant functional domains of the PS-interacting proteins, 15 and to screen such fuslon libraries in order to identify PS-interacting protein binding proteins.
E. Other Methods The nucleotide sequences and protein products, including both mutant and norrnal forms of these nucleic acids and their corresponding proteins, can be used with 2û the above techniques to isolate other interacting proteins, and to identify other genes whose ~ ssion is altered by the over-~Lpl~c;ssion of norrnal PS-interacting protein sequences, by the under-e~ sion of nonnal PS-interacting protein sequences, or by the ~ ion of mutant PS-interacting protein sequences. Identification of these other interacting proteins, as well as the if l~ntific~tion of other genes whose 25 e~ lcs~ion levels are altered in AD will identify other gene targets which have direct relevance to the pathogenesis of this disease in its clinical or pathological forms.
Specifically, other genes will be identified which may themselves be the site of other mutations causing Alzheimer's Disease, or which can themselves be targeted therapeutically (e.g., to reduce their ~r~s~ion levels to normal, or to 30 pharmacologically block the effects of their over-~ s~ion) as a potential treatment S~JC~ 111 ~JTE SHEET (RULE 26) for this disease. Specifically, these techniques rely on PCR-based and/or hybridization-based methods to identify genes which are differentially expressedbetween two conditions (a cell line e,~plessillg normal PS-interacting proteins colllp~hed to the same cell type ~Lc~illg a mutant PS-interacting protein). These 5 techniques include differential display, serial analysis of gene ~ c s~ion (SAGE), and mass-spectrometry of protein 2D-gels and subtractive hybridization (reviewed in Nowak, 1995 and Kahn, 1995).
As will be obvious to one of ol~dilldl y skill in the art, there are numerous other methods of screening individual proteins or other compounds, as well as large 10 libraries of proteins or other compounds (e.g., phage display libraries and'cloning systems from Stratagene, La Jolla, CA) to identify molecules which bind to normal or mutant PS-interacting protein components. All of these methods comprise the step of mixing a normal or mutant PS-interacting protein, filsion, or fragment with testcompounds, allowing for binding (if any), and assaying for bound complexes. All 15 such methods are now enabled by the present disclosure of substantially pure PS-hll~ .cting proteins, substantially pure Ps-int~r~rtin~ functional domain fragments, PS-interacting protein fusion proteins, PS~ f-- ~n. 1;1 Ig protein antibodies, and methods of m~king and using the same.

20 8. Disru~tin~ PS-Interactin~ Protein Interactions The ability to disrupt specific interactions of the PS-interacting proteins with the prec~nii;n.~, or with other ylvl~hls~ is potentially of great therapeutic value, and will be important in underst~n-ling the etiology of AD and in identif~ing additional targets for therapy. The methods used to identify compounds which disrupt 25 PS-interacting protein interactions may be applied equally well to interactions involving either normal or mutant PS-interacting proteins.
Assays for compounds which can disrupt PS-interacting protein interactions may be performed by any of a variety of methods well known in the art.
In essence, such assays will parallel those assays for identifying proteins and 30 compounds with binding activity toward the PS-interacting proteins. Thus, once a SlJ~ l 11 ~JTE StlEET (RULE 26~

compound with binding activity for a PS-interacting protein is identified by anymethod, that method or an equivalent method may be performed in the presence of c~n~ te compounds to identify compounds which disrupt the interaction. Thus, forexample, the assay may employ methods including (1) affinity chromatography; (2)imrnunoprecipitation; (3) theBiomolecularInteractionAssay (BI~core); or (4) me yeast two-hybrid systems. Such assays can be developed using either normal or mutant purified PS-interacting proteins, and/or either normal or mutant purifiedbinding proteins (e.g., normal or mutant pr~çnilin~).
For affinity methods, either the PS-interacting protein or its binding 10 partner may be affixed to a matrix, for example in a column, and the counterpart protein (e.g., the PS-interacting protein if pres~nilin or another binding partner is affixed to the matrix; or a pr~enilin or other binding partner if the PS-interacting protein is affixed to the matrix) is then exposed to the affixed protein/compound either before or after adding the candidate compound(s~. In the absence of a disruptive15 effect by the c~n~ te compoumd(s), the interaction between the PS-interactingprotein and its binding par~er will cause the counterpart protein to bind to the affixed protein. Any compound which disrupts the in~r~-~tion will cause release of the counterpart protein from the matrix. Release of the counterpart protein from thematrix can be measured using methods lmown in the art.
For PS-interacting protein interactions which are detectable by yeast two-hybrid systems, these assays may also be employed to identify compounds which disrupt the interaction. Briefly, a PS-interacting protein and its binding partner (or al~pl~liate structural domains of each) are employed in the fusion proteins of the system, and the cells are exposed to candidate compounds to det~otmin~ their effect 25 uponthe~ es~ionofthel~o.Lel gene. By~p~ liatechoiceofareportergene, such a system can be readily adapted for high through-put screening of large libraries of compounds by, for example, using a reporter gene which confers rçs;~t~nce to an antibiotic which is present in the medium, or which rescues an auxotrophic strain grown m mmlm~l medlum.

SU~S 111 ~lTE SHEET (RUEE 26) These assays may be used to screen many different types of compounds for their disruptive effect on the interactions of the PS-interacting proteins. For example, the compounds may belong to a library of synthetic molecules, or be specificallydesigned to disrupt the interaction. The compounds may also be peptides 5 corresponding to the interacting domain of either protein. This type of assay can be used to identify compounds that disrupt a specific interaction between a given PS-interacting protein variant and a given binding partner. In addition, compounds that disrupt all interactions with PS-interacting proteins may be identified. For exarnple, a compound that specifically disrupts the folding of PS-interacting proteins would be 10 expected to disrupt all interactions between PS-interacting proteins and other proteins.
,~lt~ tively, this type of disruption assay can be used to identify compounds which disrupt only a range of dir~clclll PS-interacting protein interactions, or only a single PS-interacting protein interaction.

15 9. Methods of IdentifYin~ Compounds Mo~ tin~ PS-Interactin~e Protein ActivitvIn another series of embo-lim~ntc, the present invention provides for methods of identifying compounds with the ability to modulate the activity of normal and mutant PS-interacting proteins. As used with respect to this series of embodiments, the term "activity" broadly includes gene and protein c~ c~,~,iOn, PS-20 interacting protein post-translation procçC~cin~, trafficking and loe~li7~tion~ and any fimctional activity (e.g., enzyrnatic, receptor-effector, binding, channel), as well as dowll ,llealn affects of any of these. It is known that Alzheimer's Disease is associated with increased production of the long form of A~ peptides, the appearance of amyloid plaques and neurofibrillary tangles, decreases in cognitive function, and apoptotic cell 25 death. Therefore, using the Ll~l~olllled cells and transgenic animal models ofthe present invention, cells obtained from subjects bearing norrnal or mutant PS-interacting protein genes, or ~nim~l~ or human subjects bearing naturally occurring normal or mutaIlt PS-interacting proteins, it is now possible to screen candidate ~h~ cellticals and treatments for their therapeutic effects by detecting changes in Sl,~;~ JTE SHEET ~RULE 26) -wo 97J27296 PCT/CA97/00051 one or more of these functional characteristics or phenotypic manifestations of normal or mutant PS-interacting protein e~le~iOn.
Thus, the present invention provides methods for screening or assaying for proteins, small molecules or other compounds which modulate PS-interacting protein 5 activity by contacting a cell in vivo or in vitro with a c:ln(li~l~te compound and assaying for a change in a marker associated with normal or mutant PS-interacting protein activity. The marker associated with PS-interacting protein activity may be any measurable biochemical, physiological, histological and/or behavioral characteristic associated with PS-interacting protein e~r~ion. In particular, useful 10 markers will include any measurable biochemical, physiological, histological and/or behavioral characteristic which distinguishes cells, tissues, ~nim~ or individuals bearing at least one mutant preePnilin or PS-interacting protein gene from their normal counterparts. In addition, the marker may be any specific or non-specific measure of pres.-nilin or PS-interacting protein activity. PS-interacting protein specific measures 15 include measures of PS-interacting protein ~ e~SiOn (e.g., PS-interacting protein rnRNA or protein levels) which may employ the nucleic acid probes or antibodies of the present invention. Non-specific measures include changes in cell physiology such as pH, intracellular calcium, cyclic AMP levels, GTP/GDP ratios, phosphatidylinositol activity, protein phosphorylation, etc., which can be monitored 20 on devices such as the cytosensor microphysiometer (Molecular Devices Inc., United States). The activation or inhibition of PS-interacting protein activity in its mutant or normal form can also be monitored by e~mining ch~n~es in the e~l~s~ion of other genes (e.g., the prese~ilin~) which are specific to the PS-interacting protein pathway leading to ~l7heimer's Disease. These can be assayed by such techniques as 25 dirr~ .Lial display, differential hybridization, and SAGE ~sequential analysis of gene expression), as well as by two (1imtqncional gel electrophoresis of cellular lysates. In each case, the dirrclelltially-~ essed genes can be ascertained by inspection ofi~enti~zll studies before and after application of the candidate compound.
Furthermore, as noted elsewhere, the particular genes whose ~ sion is modulated 30 by the a-lmini~tration of the candidate compound can be asc~ illed by cloning, 5~J~;i 111 ~ITE SHEET (RULE 26) wo 97/27296 PCT/CA97/00051 nucleotide sequencing, amino acid sequencing, or mass spectrometry (reviewed in Nowak, 1995).
In general, a cell may be contacted with a candidate compound and, a~er an ~l-lupliate period (e.g., 0-72 hours for most biochemical measures of cultured 5 cells), the marker of pr~s~nilin or PS-interacting protein activity may be assayed and compared to a baseline measurement. The baseline measurement may be made prior to contacting the cell with the candidate compound or may be an external baseline established by other ~c~ lents or known in the art. The cell may be a ll~l~rollned cell of the present invention or an explant from an animal or individual. ln particular, 10 the cell may be an explant from a carrier of a presenil;n or PS-interacting protein mutation (e.g., a hurnan subject with ~17heimer's Disease~ or an animal model of the invention (e.g., a ll~lSg~lliC nematode or mouse bearing a mutant pres~nilin or PS-interacting protein gene). To augment the effect of pre~nilin or PS-interacting protein mutations on the A13 ~dlllw~, transgenic cells or :~nim~1~ may be employed 15 which have increased A,3 pro(1~lction Preferred cells include those from neurological tissues such as neuronal, glial or mixed cell cultures; and cultured fibroblasts, liver, kidney, spleen, or bone marrow. The cells may be contacted with the candidate compounds in a culture in vitro or may be ?~rimini.ct~,red in vivo to a live animal or human subject. For live ~nim~l.c or human subjects, the test compound may be 20 ~1mini~t~ored orally or by any parenteral route suitable to the compound. For clinical trials of human subjects, measul~ ents may be conducted periodically (e.g., daily, weekly or monthly) for several months or years.
Because most individuals bearing a mutation in a particular gene are heterozygous at that locus (i.e., bearing one normal and one mutant allele), 25 compounds may be tested for their ability to modulate normal as well as mutant pres~nilin or PS-interacting protein activity. Thus, for example, compounds which enhance the function of normal prçcç~ilin~ or PS-interacting proteins may have utility in treating Alzheimer's Disease or related disorders. ~lt.om~tively, because ~u~ es~ion of the activity of both normal and mutant copies of a gene in a 30 heterozygous individual may have less severe clinical consequences than progression SU~;~ 111 UTE SHEET (RULE 26) of the associated fli~e~ce it may be desired to identify compound which inactivate or ~u~rt;SS all fo~ns of the presenilins, the PS-interacting proteins, or their interactions.
Preferably, however, compounds are identified which selectively or specifically inactivate or ~u~leS~ the activity of mutant presenilin or PS-interacting proteins 5 without disrupting the function of their normal counterparts.
In light of the identifir~tion~ characterization, and disclosure herein of a novel group of PS-interacting genes and proteins, the PS-interacting protein nucleic acid probes and antibodies, and the PS-interacting protein transformed cells andtr~ncg( nic ~nim~lc of the invention, one of ordinary skill in the art is now enabled by 10 perform a great variety of assays which will detect the modulation of presenilin and/or PS-interacting protein activity by c~n~ te compounds. Particularly ~lcr~l~d and contemplated embo-limentc are ~1iccllc~ed in some detail below.
A. PS-Interactin~ Protein Ex~ression In one series of embo-lim~nt.c, specific measures of PS-interacting protein 15 ~ c~ion are employed to screen candidate compounds for their ability to affect pres~nilin activity. Thus, using the PS-inter~çting protein nucleic acids and antibodies disclosed and otherwise enabled herein, one may use mRNA levels or protein levels as a marker for the ability of a candidate compound to modulate PS-interacting protein activity. The use of such probes and antibodies to measure gene and protein 20 t;2~ression is well known in the art and ~iccl-cce(l elsewhere herein. C~f particular interest may be the icl~ntification of compounds which can alter the relative levels of dirf~lelll variants (e.g., mutant and norrnal) of the PS-interacting proteins.
B. Intracellular Localization In another series of embo~limerltc~ compounds may be screened for their 25 ability to modulate the activity of the PS-interacting proteins based upon their effects on the kafficking and inkacellular localization of the PS-interacting proteins. The presenilins and some of the PS-interacting proteins (e.g., S5a) have been seen immnn~ cyto~ hemic~lly to be localized in membrane structures associated with the endoplasmic reticulum and Golgi a~pal~lus. Differences in localization of mutant and 30 normal pr~,c~nilin.c or PS-interacting proteins may, therefore, contribute to the etiology Sl,,.~ 111 ~Jl E SHEET (RULE 26) -of Al7heTmer's Disease and related disorders. C~ompounds which can affect the localization of these proteins may, therefore, be identified as potential therapeutics.
Standard techniques known in the art may be employed to detect the localization of the pres~nilin~ and PS-interacting proteins. Generally, these techniques will employ 5 the antibodies of the present invention, and in particular antibodies which selectively bind to one or more mutant PS-interacting proteins but not to normal proteins. As is well known in the art, such antibodies may be labeled by any'of a variety of techniques (e.g., fluorescent or radioactive tags, labeled secondary antibodies, avidin-biotin, etc.) to aid in vi~ li7ing the intr~ell~ r location of these proteins. The PS-10 interacting proteins may be co-localized to particular structures, as in known in the art, using antibodies to markers of those structures (e.g., TGN38 for the Golgi,kansferrin receptor for post-Golgi transport vesicles, LAMP2 for lysosomes).
Western blots of purified fractions from cell lysates enriched for different intracellular membrane bound organelles (e.g., lysosomes, synaptosomes, Golgi) may also be 15 employed.
B. Ion ~egulation/Metabolism In another series of embodiments, compounds may be screened for their ability to modulate the activity of the prPsenilin~ or PS-interacting proteins based upon measures in intracellular Ca2+, Na~ or K~ levels or metabolism. As noted above, 20 the presenilin~ are membrane associated proteins which may serve as, or interact with, ion lec~l~ or ion channels. Thus, compounds may be screened for their ability tomodulate pres~nilin and PS-interacting protein-related metabolism of calcium or other ions either in vivo or in vitro by, for example, measurements of ion channel fluxes and/or tr~n~m~mhrane voltage and/or current fluxes, using patch clamps, voltage 25 clamps or fluorescent dyes sensitive to intracellular ion levels or tr~n~memhrane voltage. Ion channel or receptor function can also be assayed by measurements ofactivation of second messengers such as cyclic AMP, cGMP tyrosine kin~ces, phosphates, increases in intracellular Ca2~ levels, etc. Recombinantly made proteins may also be reconstructed in artificial membrane systems to study ion channel 30 con~uctz~nce and, therefore, the "cell" employed in such assays may comprise an Sl,~ JTE SHEET (RULE 26) -W O 97ft7296 PCT/CA97/00051 artificial membrane or cell. Assays for changes in ion regulation or metabolism can be performed on cultured cells ~ es~ g endogenous normal or mutant presenilins and PS-interacting proteins. Such studies also can be performed on cells transfected with vectors capable of ~lCS~illg one of the presenilinc or PS~ L~l~cLi~lg proteins, or functional domains of one of the pres~nilin~ or PS-interacting proteins, in normal or mutant form. In addition, to enhance the signal measured in such assays, cells may be co-transfected with genes encoding ion channel proteins. For example, Xenopus oocytes or rat kidney (HEK293) cells may be co-transfected with sequences encoding rat brain Na+ ~1 subunits, rabbit skeletal muscle Ca2+ ~ ul)U~ , or rat heart K+ ,B 1 10 subunits. Changes in prç~enilin or PS-interacting protein-mediated ion channel activity can be measured by, for example, two-microelectrode voltage-clamp recordings in oocytes, by whole-cell patch-clamp recordings in HEK293 cells. or by equivalent means.
C. Apoptosis or Cell Death In another series of embo-lim~?nt~, compounds may be screened for their ability to modulate the activity of the pres~nilin~ or PS-interacting proteins based upon their effects on presen;lin or PS-hlLel~ ;lillg protein-related apoptosis or cell death. Thus, for example, baseline rates of apoptosis or cell death may be established for cells in culture, or the baseline degree of neuronal loss at a particular age may be established post-mortem for animal models or human subjects, and the ability of a candidate compound to ~U~ S~ or inhibit apoptosis or cell death may be measured.Cell death may be measured by standard microscopic techniques (e.g., light microscopy) or apoptosis may be measured more specifically by characteristic nuclear morphologies or DNA fr~nent~tion patterns which create nucleosomal ladders (see,e.g., Gavrieli et al., 1992; Jacobson et al., 1993; Vito et al., 1996). TUNEL may also be employed to evaluate cell death in brain (see, e.g., T ~m~nn et al., 1995). In preferred embo~iment~, compounds are screened for their ability to ~LIppleSS or inhibit neuronal loss in the transgenic animal models of the invention. Transgenic mice bearing, for example, a mutant human, mutant mouse, or hllm~ni7e-1 mutant presenilin or PS-interactin~ protein gene may be employed to identify or evaluate compounds S~ JTE SHEET (RULE 263 W 097/27296 PCT/CA971~0051 ~..

which may delay or arrest the neurodegeneration associated with Alzheimer's Disease. A similar transgenic mouse model, bearing a mutant APP gene, has recently been reported by Games et al. (1995).
D. A,B Peptide Production In another series of embodiments, compounds may be screened for their ability to modulate pres~nilin or PS-interacting protein-related ch~n~es in APP
processing. The A,13 peptide is produced in several isoforms rçsulting from differences in APP processing. The A,B peptide is a 39 to 43 amino acid derivative of ,BAPP
which is progressively deposited in diffuse and senile plaques and in blood vessels of subjects with AD. In human brain, A~ peptides are heterogeneous at both the N- and C-termini. Several observations, however, suggest that both the full length and N-t~ min~l trlm~ ~te~l forms of the long-tailed A~ peptides ending at residue 42 or 43 (i.e., A~ 1-42/43 and A~x-42/43) have a more important role in AD than do peptides ending at residue 40. Thus, A~ 42/43 and A,Bx-42/43 are an early and prominent feature of both senile plaques and diffuse plaques, while peptides ending at residue 40 (i.e., A,B 1 -40 and A,Bx-40) are predoll~illalllly associated with a subset of mature plaques and with amyloidotic blood vessels (see, e.g., Iwatsubo et al., 1995; Gravina et al., 1995; Tamaoka et al., 1995; Podlisny et al. 1995). Furthermore, the long-tailed isoforms have a greater ~lupellsil~ to fibril for nation, and are thought to be more neurotoxic than A,B1-40 peptides (Pike et al., 1993; Hilbich et al., 1991). Finally, mics.-nce mutations at codon 717 of the ,BAPP gene are associated with early onset FAD, and result in overproduction of long-tailed A,B in the brain of affected mutation carriers, in peripheral cells and plasma of both affected and presymptomatic carners, and in cell lines tr~ncfecterl with l3APP717 mutant cDNAs (T~ k~ et al., 1994;
26 Suzuki et al., 1994).
Thus, in one series of embo-limentc, the present invention provides methods for screening candidate compounds for their abil;ty to block or inhibit the increased production of long isoforms of the A,B peptides in cells or transgenic~nim~lc ~.fessi.lg a normal or mutant presenilin gene and/or a normal or mutant PS-interacting protein gene. In particular, the present invention provides such methods in S~ 111 UTE SHErT (RULE 26~

W 097/27296 PCT/CA97/UOQSl which cultured m~mm~ n cells, such as brain cells or fibroblasts, have been transformed according to the methods disclosed herein, or in which transgenic ~nlm~l~, such as rodents or non-human primates, have been produced by the methods disclosed herein, to express relatively high levels of a normal or mutant pres~nilin or 5 PS-interacting protein. Optionally, such cells or tr~n~g~nic ~nim~l~ may also be rc,lll,ed so as to express a normal or mutant form of the ,BAPP protein at relatively high levels.
In this series of embo(liment~, the candidate compound is ~1mini~t~red to the cell line or transgenic ~nim~l~ (e.g., by addition to the media of cells in culture; or 10 by oral or p~llLt;lal ~1mini~tration to an animal) and, after an ap~l~,pl;ate period (e.g., 0-72 hours for cells in culture, days or months for animal models), a biological sample is collected (e.g., cell culture supe~t~nt or cell lysate from cells in culture;
tissue homogenate or plasma from an animal) and tested for the level of the longisoforms of the A,B peptides. The levels of the peptides may be ~let~rmine-l in an 15 absolute sense (e.g., nMol/ml) or in a relative sense (e.g., ratio of long to short A,B
isoforms). The A,~ isoforms may be detecte~l by any means known in the art (e.g., electrophoretic separation and se~uencing) but, preferably, antibodies which arespecific to the long isoform are employed to ~1et~nnine the absolute or relative levels of the A~ l -42/43 or A,Bx-42/43 peptides. Candidate ph~rrn~cellticals or therapies 20 which reduce the absolute or relative levels of these long A~ isofolms, particularly in the transgenic animal models of the invention, are likely to have ~t;l~eutic utility in the treatment of Alzheimer's Disease, or other disorders caused by mutations in the presenilins or PS-interacting proteins, or by other aberrations in APP metabolism.
E. PhosPhorvlation of Microtubule Associated Proteins In another series of embodiments, c~n(lil1~tç compounds may be screened for their ability to modulate prest-nilin or PS-interacting protein activity by ~çs~ing ~ the effect of the compound on levels of phosphorylation of microtubule associated proteins (MAPs) such as tau. The abnormal phosphorylation of tau and other MAPs in the brains of victims of Alzheimer's Disease is well known in the art. Thus, 3~ compounds which prevent or inhibit the abnormal phosphorylation of MAPs may SUBSTITUTE SHEET (RULE 26) have utility in treating presenilin or PS-interacting protein-associated diseases such as AD. As above, cells from normal or mutant ~nim~l~ or subjects, or the transformed cell lines and animal models of the invention may be employed. Preferred assays will employ cell lines or animal models transformed with a mutant human or hllm~ni7f--1 5 mutant presenilin or PS-interacting protein gene. The baseline phosphorylation state of MAPs in these cells may be established and then ç~n~ te compounds may be tested for their ability to prevent, inhibit or counteract the hyperphosphorylation associated with 111~ The phosphorylation state ofthe MAPs may be determined by any standard method known in the art but, preferably, antibodies which bind 10 selectively to phosphorylated or unphosphorylated epitopes are employed. Suchantibodies to phosphorylation epitopes of the tau protein are known in the art (e.g., ALZ50).

10. Screening and Dia~nostics for Alzheimer's Disease A. General Dia~nostic Methods The PS-i-lt~dclil-g genes and gene products, as well as the PS-interacting protein derived probes, primers and antibodies, disclosed or otherwise enabled herein, are useful in the screening for carriers of alleles associated with Alzheimer's Disease, for (1 j~osi-c of victims of ~l7h-oimer's Disease, and for the screening and ~ no~ic of 20 related presenile and senile cl~m~nti~, psychiatric ~ e~cçs such as schizophrenia and depression, and neurologic ~ ez~es such as stroke and cerebral hemorrhage, all of which are seen to a greater or lesser extent in symptomatic human subjects bearing mutations in the PS 1 or PS2 genes or in the APP gene. Individuals at risk for Alzheimer's Disease, such as those with AD present in the family pedigree, or 25 individuals not previously known to be at risk, may be routinely screened using probes to detect the presence of a mutant PS-interacting protein gene or protein by a variety of techniques. Diagnosis of inherited cases of these diseases can be accomplished by methods based upon the nucleic acids (including genomic and mRNA/cDNA sequences), proteins, and/or antibodies disclosed and enabled herein, 30 including functional assays designed to detect failure or ~ nent~tion of the normal SU~ 111 UTE SHEET (RULE 263 w O 97/~7296 PCT/CA97/00051 presenilin or PS-interacting protein activity and/or the presence of specific new activities conferred by mutant PS-interacting proteins. Preferably, the methods and products are based upon the human nucleic acids, proteins or antibodies, as disclosed or otherwise enabled herein. As will be obvious to one of ordinary skill in the art, 5 however, the significant evolutionary co~ v~lion of large portions of nucleotide and amino acid sequences, even in species as diverse as hnm~n~, mice, C. ele~ans, and Drosophila, allow the skilled artisan to make use of non-human homologues of thePS-interacting proteins to produce useful nucleic acids, proteins and antibodies, even for applications directed toward human or other animal subjects. Thus, for brevitv of 10 exposition, but without limiting the scope of the invention, the following description will focus upon uses of the human homologues of PS-interacting proteins and genes.
It will be un(1erstQod~ however, that homologous sequences from other species will be equivalent for many purposes.
As will be appreciated by one of ordinary skill in the art, the choice of 15 diagnostic methods of the present invention will be jnfll~enfe-l by the nature of the available biological samples to be tested and the nature of the inforrnation required.
~l~heimer's Disease is, of course, primarily a disease of the brain, but brain biopsies are illV~lSiV~ and expensive procedures, particularly for routine screening. Other tissues which express the presPnilin~ or PS-interacting proteins at ~i nific~nt levels 20 may, therefore, be plere.,~d as sources for samples.
B. Protein Based Screens and Dia~nostics When a diagnostic assay is to be based upon PS-interacting proteins, a variety of approaches are possible. For example, diagnosis can be achieved by monitoringdifferences in the electrophoretic mobility of normal and mutant proteins. Such an 25 approach will be particularly useful in identifying mllt~nt~ in which charge substitutions are present, or in which insertions, deletions or substitutions have resulted in a significant change in the electrophoretic migration of the res--lt~nt protein. AltPrn~tively~ diagnosis may be based upon differences in the proteolytic cleavage pattems of normal and mutant proteins, differences in molar ratios of the -SU~ JTE SHEET (RULE 26) various amino acid residues, or by functional assays demonstrating altered function of the gene products.
In ~lef~ d embodiments, protein-based diagnostics will employ differences in the ability of antibodies to bind to normal and mutant PS-interacting proteins. Such ~ nostic tests may employ antibodies which bind to the normal proteins but not to mutant proteins, or vice versa. ~n particular, an assay in which a plurality of monoclonal antibodies, each capable of binding to a mutant epitope, may be employed. The levels of anti-mutant antibody binding in a sample obtained from atest subject (vi~ ed by, ~or example, radiolabelling, ELISA or chetn~ min~ccence) 10 may be compared to the levels of binding to a control sample. ~lt~tn~t;vely, antibodies which bind to normal but not mutant proteins may be employed, and decreases in the level of antibody binding may be used to distin~li~h homozygousnormal individuals from mutant heterozygotes or homozygotes. Such antibody diagnostics may be used for in situ immllnohi~toch~rni~try using biopsy samples of 15 CNS tissues obtained antemortem or l)o~ llem, including neuropathological structures associated with these diseases such as neurofibrillary tangles and amyloid plaques, or may be used with fluid samples such a cwe6l~ al fluid or with peripheral tissues such as white blood cells.
C. Nucleic Acid Based Screens and Di~nostics When the diagnostic assay is to be based upon nucleic acids from a sample, the assay may be based upon ml?NA, cDNA or genomic DNA. When mRNA is used from a sample, there are considerations with respect to source tissues and the possibility of alternative splicing. I'hat is, there may be little or no ~les~ion of transcripts unless ~lv~iate tissue sources are chosen or available, and alternative ~5 splicing may result in the loss of some inforrn~tion or difficulty in i~ yl~Lation~
Whether mRNA, cDNA or genomic DNA is assayed, standard methods well known in the art may be used to detect the presence of a particular sequence either in situ or in vitro (see, e.g., Sambrook et al., (1989) Molecular Clonin~: A Laboratorv Manual, 2nd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY). As a general matter,30 however, any tissue with nucleated cells may be ex~mined.

Sl,~a 1 l l UTE SHEFT (RULF 26) w ~97n729~ PCT/CA97/00051 Genomic DNA used for the diagnosis may be obtained from body cells, such as those present in the blood, tissue biopsy, surgical specimen, or autopsy material.
The DNA may be isolated and used directly for detection of a specific sequence or may be amplified by the polymerase chain reaction (PCR) pnor to analysis.
5 Similarly, RNA or cDNA may also be used, with or without PCR amplification. Todetect a specific nucleic acid sequence, direct nucleotide sequencing, hybridization using specific oligonucleotides, restriction enzyme digest and mapping, PCR
mapping, RNase protection, chemical mi~m~t~h cleavage, ligase-me~ ted detection,and various other methods may be employed. Oligonucleotides specific to particular lû sequences can be chemically syn1hesi7~1 and labeled ~adioactively or non-radioactively (e.g., biotin tags, ethidium bromide), and hybridized to individual samples immobilized on membranes or other solid-supports (e.g., by dot-blot or transfer from gels after electrophoresis), or in solution. The presence or absence of the target sequences may then be vicn~li7~1 using methods such ~ autoradiography, 15 fluorometry, or colorimetry. These procedures can be automated using rc(ll-ntl~nt, short oligonucleotides of known sequence fixed in high density to silicon chips.~I) Appropriate Probes and Primers Whether for hybridization, RNase protection, ligase-me~ te-1 detection, PCR
amplification or any other standards methods described herein and well known in the 20 art, a variety of subsequences of the PS-interacting protein sequences disclosed or otherwise enabled herein will be useful as probes and/or primers. These sequences or subsequences will include both normal sequences and deleterious mutant sequences.
In general, useful sequences will include at least 8-9, more preferably l 0-50, and most pleL~lably 18-24 consecutive nucleotides from introns, exons or intron/exon 25 boundaries. Depending upon the target sequence, the specificity required, and future technological developments, shorter sequences may also have utility. Therefore, any PS-interacting protein derived sequence which is employed to isolate, clone, amplify, identify or otherwise manipulate a PS-interacting protein sequence may be regarded as an ~ Liate probe or primer. Particularly contemplated as useful will be sequences SU~:I 111 ~JTE SHEET (RULE 26) W O 97/27296 PCTIC~97/00051 including nucleotide positions from the PS-interacting protein genes in which disease-causing mutations are lcnown to be present, or sequences which flank these positions.
(2) Hvbridization Screenin~
For in situ detection of a normal or mutant PS-interacting protein-related nucleic acid sequence, a sample of tissue may be prepared by standard techniques and then contacted with one or more of the above-described probes, preferably one which is labeled to facilitate detection, and an assay for nucleic acid hybridization is conducted under stringent conditions which perrnit hybridization only between the probe and highly or perfectly complementary sequences. Because many mutations 10 consist of a single nucleotide substitution, high stringency hybridization conditions may be required to ~ tingui~h normal sequences from most mutant sequences. When the PS-interacting protein genotypes of the subject's parents are known, probes may be chosen accordingly. Alternatively, probes to a variety of ,~ may be employed sequentially or in combination. Because most individuals carrying 15 mutations in the PS-interacting proteins will be heterozygous, probes to normal sequences also may be employed and homozygous normal individuals may be ~ictin~li~hed from mutant heterozygotes by the amount of binding (e.g., by intensity of radioactive signal). In another variation, competitive binding assays may be employed in which both normal and mutant probes are used but only one is labeled.
~3) RestrictionMappin~
Sequence alterations may also create or destroy fortuitous restriction enzyme recognition sites which are revealed by the use of a~l~l;ate enzyme digestion followed by gel-blot hybridization. DNA fr~ nt~ carrying the site (normal or mutant) are detected by their increase or reduction in size, or by the increase or 25 decrease of corresponding restriction fragment nurnbers. Such restriction fragment length polyrnorphism analysis (RF~P), or restriction mapping, may be employed with genomic DNA, mRNA or cDNA. The PS-interacting protein sequences may be amplified by PCR using the above-described primers prior to restriction, in which case the lengths of the PCR products may indicate the presence or absence of 30 particular restriction sites~ and/or may be subjected to restriction after amplification.

SUBSTITUTE SHEET (RULE 26) -w 0971Z7296 PCT/CA97/00051 The restriction fragments may be vi.~ ed by any convenient means (e.g., under W
light in the presence of ethidium bromide).

(4) PCR Mappin~
In another series of embodiment.s, a single base sTIhstitlltion mutation may be detected based on differential PCR product length or production in PCR. Thus, primers which span mutant sites or which, preferably, have 3' termini at mutation sites, may be employed to amplify a sample of genomic DNA, mRNA or cDNA ~om a subject. A mi~m~tch at a mutational site may be expected to alter the ability of the no~nal or mutant primers to promote the polymerase reaction and, thereby, result in product profiles which differ between normal subjects and heterozygous ~d/or homozygous ~ ; The PCR products of the normal and mutant gene may be differentially separated and detected by standard techniques, such as polyacrylamide or agarose gel electrophoresis and vi.~u~ tion with labeled probes, ethidium bromide or the like. Because of possible non-specific priming or readthrough of mutation sites, as well as the fact that most carriers of mutant alleles will beheterozygous, the power of this technique may be low.

(5) Electrophoretic Mobilitv Genetic testing based on DNA sequence dirr~ ces also may be achieved by detection of alterations in electrophoretic mobility of DNA, mRNA or cDNA
fragments in gels. Small sequence deletions and insertions, for example, can be visualized by high resolution gel ele~ ul~hore~is of single or double stranded DNA, ûr as changes in the migration pattern of DNA heteroduplexes in non-denaturing gel electrophoresis. Mutations or polyrnorphisms in the PS-interacting protein genes may also be rletectefT by methods which exploit mobility shifts due to single-skanded conformational polymorphisms (SSCP) associated with mRNA or single-stranded DNA secondary structures.
~6) ChemicalCleava eofMi~m~tches Mutations in the PS-interacting protein genes may also be detected by employing the chemical cleavage of mi~m~tch (CCM) method (see, e.g., Saleeba andCûtton~ 1993, and references therein). In this technique, probes (up to ~ 1 kb) may be SU~S 111 ~ITE SHEET (RULE 21i~

mixed with a sample of genomic DNA, cDNA or mRNA obtained from a subject.
The sample and probes are mixed and subjected to conditions which allow for heteroduplex formation (if any). Preferably, both the probe and sample nucleic acids are double-stranded, or the probe and sample may be PCR amplified together, to ensure creation of all possi~le micm~t~h heteroduplexes. Micm~tched T residues are reactive to osmium tetroxide and micm~tr.hed C residues are reactive to hydroxylamine. Because each micm~tched A will be accompanied by a micm~t-~hed T, and each mi.cm~tcheri G will be accompanied by a micm~tched C, any nucleotidedifferences bet~,veen the probe and sample (including small insertions or deletions~
I0 will lead to the formation of at least one reactive heteroduplex. After treatment with osmium tetroxide and/or hydroxylamine to modify any micm~tc h sites, the ~ ule is subjected to chemical cleavage at any modified micm~tch sites by, for example, reaction with piperidine. The l~ Lw e may then be analyzed by standard techniques such as gel electrophoresis to detect cleavage products which would in~lic~t~
mi.cm~tches between the probe and sample.

(7) Other Methods Various other methods of detecting PS-interacting protein mutations, based upon the sequences disclosed and othe~wise enabled herein, will be a~n~ to thoseof ordinary skill in the art. Any of these may be employed in accordance with the present invention. These in- IIT(ie, but are not limited to, nuclease protection assays (S l or ligase-me~ tçd), ligated PCR, cl~ . gradient gel electrophoresis (DGGE;
see, e.g., Fischer and T f nn~n, 1983), restriction ~n~lonllclease fin~ g combined with SSCP (REF-SSCP; see, e.g., Liu and Sommer, 1995~, and the like.
D. Other Screens and Dia~nostics In inherited cases, as the primary event, and in non-inherited cases as a secondary event due to the disease state, abnormal processing of the presenilinc, PS-interacting proteins, APP, or proteins reacting with the presenilins, PS-interacting proteins, or APP may occur. This can be detected as abnorrnal phosphorylation, glycosylation, glycation amidation or proteolytic c}eavage products in body tissues or fluids (e.g., CSF or blood).

S~J~a 111 ~ITE SHEET ~RULE 26) Diagnosis also can be made by observation of alterations in transcription, translation, and post-translational modification and processing, as well as alterations in the intracellular and extracellular trafficking of gene products in the brain and peripheral cells. Such changes will include alterations in the amount of messenger S RNA and/or protein, alteration in phosphorylation state, abnormal intracellular location/distribution, abnormal ext~acellular distribution, etc. Such assays will include: Northern Blots (e.g., with PS-interacting protein-specific and non-specific nucleotide probes), Westem blots and enzyme-linked immunosorbent assays (ELISA) (e.g., with antibodies raised specifically to a PS-interacting protein or PS-interacting 10 functional domain, including various post-translational modification states including glycosylated and phosphorylated isoforms). These assays can be performed on peripheral tissues (e.g., blood cells, plasma, cultured or other fibroblast tissues, etc.) as well as on biopsies of CNS tissues obtained ~nt~mf~rtem or postmortem, and upon cerebrospinal fluid. Such assays might also include in situ hybricli7~tion and 15 immlmohistochemistry (to localize mP~Pnger RNA and protein to specific subcellular cun~ ~ ents and/or within neuropathological structures associated with these diseases such as neurofibrillary tangles and amyloid plaques).
. Screenin~ and Diagnostic Kits In accordance with the present invention, (ii~gnos~ic kits are also provided 20 which will include the reagents necessary for the above-described diagnostic screens.
For example, kits may be provided which include antibodies or sets of antibodieswhich are specific to one or more mutant epitopes. These antibodies may, in particular, be labeled by any of the standard means which facilitate vi~u~li7~tion of binding. ~ltern~tively~ kits may be provided in which oligonucleotide probes or PCR
25 lprimers, as described above, are present for the detection and/or amplification of normal or mutant pr~enilin and/or PS-interacting protein nucleotide sequences.
Again, such probes may be labeled for easier detection of specific hybridization. As a~lo~liate to the various diagnostic embodiments described above, the oligonucleotide probes or antibodies in such kits may be immobilized to substrates 30 and ~p.opliate controls may be provided.

S~ 11 UTE SHEET (RULE 26 1 l . Methods of Treatment The present invention now provides a basis for therapeutic intervention in e~cec which are caused, or which may be caused, by mutations in the PS-5 interacting proteins. As noted above, mutations in the hPS 1 and hPS2 genes havebeen associated with the development of early onset forms of Al7heimer's Disease and, therefore, the present invention is particularly directed to the trP~tment of subjects ~i~gnosed with, or at risk of developing, ~l7heimer's Disease.
Without being bound to any particular theory of the invention, the effect of 10 the ~ heimer's Disease related mutations in the precPnilinc appears to be a gain of a novel function, or an acceleration of a normal function, which directly or indirectly causes aberrant processing of the Amyloid Precursor Protein (APP) into A,B peptide, abnormal phosphorylation homeost~cic, and/or abnormal apoptosis in the brain. Such a gain of function or acceleration of function model would be conci.ct~nt with the adult 15 onset of the sy,~ lol~s and the dominant inh~rit~nce of Alzheimer's Disease.
Nonetheless, the lllecl~ m by which mutations in the prPsenilin~ may cause theseeffects remains unknown.
The present invention, by identifying a set of PS-interacting proteins, provides new therapeutic targets for ill~ ing in the etiology of precenilin-related 20 AD. In addition, as mutations in the pres~-nilinc may cause AD, it is likely that mutations in the PS-interacting proteins may also cause AD. The fact that the PS-interacting protein SSa is alternately processed in the brains of victims of sporadic AD, as well as in the brains of victims of presenilin-linked AD, suggests that, at the very least, this PS-interacting protein is involved in the etiology of AD independent of 25 mutations in the presPnilinc It is likely that the other PS-interacting proteins also may be involved in non-presenilin-linked AD.
Therapies to treat PS-interacting protein-associated ~ cP~5 such as AD
may be based upon (1) ~dminictration of normal PS-interacting proteins, (2~ genetherapy with normal PS-interacting protein genes to compensate for or replace the 30 mutant genes, (3~ gene therapy based upon antisense sequences to mutant PS-SU~;i 1111 ITE SHEET (RULE 26~

interacting protein genes or which "knock out" the mutant genes, (4) gene therapy based upon sequences which encode a protein which blocks or corrects the deleterious effects of PS-interacting protein mutants, (5) irnrnunotherapy based upon antibodies to normal and/or mutant PS-interacting proteins, or (6) small molecules ~drugs) which 5 alter P~-interacting protein ~ ,s~ion, alter interactions between PS-interacting proteins and other proteins or ligands, or which otherwise block the aberrant function of mutant pres~nilin or PS-interacting proteins by ~lt~ring the structure ofthe mutant proteins, by enhancing their metabolic clearance, or by inhibiting their function.
A. Protein Thera~y Treatment of .AI7h~imer's Disease, or other disorders res-llting from PS-interacting protein mutations, may be ~rulllled by replacing the mutant protein with normal protein, by modlll~tin~ the function of the mutant protein, or by providing an excess of normal protein to reduce the effect of any aberrant function of the mutant proteins.
To accomplish this, it is n~c~ ry to obtain, as described and enabled herein, large amounts of substantially pure PS-interacting protein from cultured cell systems which can express the protein. Delivery of the protein to the affected brain areas or other tissues can then be accomplished using a~ru~l;ate p~ck~ing or ~lmini.ctration systems including, for example, liposome mediated protein delivery to 2û the target cells.
B. Gene Therapv Tn one series of embodiments, gene therapy may be employed in which normal copies of a PS-interacting protein gene are introduced into patients to code sl~cces~llly for normal protein in one or more different affected cell types. The gene 25 must be delivered to those cells in a form in which it can be taken up and code for sufficient protein to provide effective function. Thus, it is plcr~ l~c;d that the recombinant gene be operably joined to a strong promoter so as to provide a highlevel of e~l!L~ssion which will compensate for, or out-compete, the mutant proteins.
As noted above, the recombinant construct may contain endogenous or exogenous SUBSTITUTE SHEET ~RULE 26) WO 97/2729~i PCT/CA97/00051 regulatory elements, inducible or repressible regulatory elements, or tissue-specific regulatory elements.
In another series of embodiments, gene therapy may be employed to replace the mutant gene by homologous recombination with a recombinant construct.
5 The recombinant construct may contain a normal copy of the targeted PS-interacting protein gene, in which case the defect is coll~.;led in situ, or may contain a "knock-out" construct which introduces a stop codon, mi~sPnce mutation, or deletion which abolished function of the mutant gene. It should be noted in this respect that such a construct may knock-out both the normal and mutant copies of the targeted gene in a 10 heterozygous individual, but the total loss of gene function may be less deleterious to the individual than contimle-l progression of the disease state.
In another series of embo~lim~ntc, :mti~cpnce gene therapy may be employed. The ~nticence therapy is based on the fact that sequence-specific ~uyp~es~ion of gene ~ ,res~ion can be achieved by intracell~ r hybridization between 15 mRNA or DNA and a complement~ ntic~n~ce species. The formation of a hybrid duplex may then interfere with the Ll~ls~ ,Lion of the gene and/or the pr~c~ccin~, transport, translation and/or stability of the target rnRNA. Anticen.ce strategies may use a variety of approaches including the ~flminictration of ~nti.e-once oligonucleotides or antisense oligonucleotide analogs (e.g., analogs with phosphorothioate backbones) 20 or l~ reclion with ~ntictonce RNA ~yles~ion vectors. Again, such vectors may include exogenous or endogenous regulatory regions, inducible or ~ ible regulatory elements, or tissue-specific regulatory elements.
II1 another series of embodiments, gene therapy may be used to introduce a recombinant construct encoding a protein or peptide which blocks or otherwise 25 corrects the aberrant fimction caused by a mutant presenilin or PS-interacting protein gene. In one embodiment, the recombinant gene may encode a peptide which corresponds to a domain of a PS-interacting which has been found to abnormally interact with another cell protein or other cell ligand (e.g., a mutant prçsenilin). Thus, for example, if a mutant PS l TM6~7 domain is found to interact with a PS-30 interacting protein but the corresponding normal TM6~7 domain does not undergo S~ ~111 lJTE SHEET (RULE 26~

CA 02244412 l998-07-27 this interaction, gene therapy may be employed to provide an excess of the mutant TM6~7 domain which may compete with the mutant prçsloni1in protein and inhibit or b}ock the aberrant interaction. ~1t~ tively, the PS-interacting domain of a PS-interacting protein which interacts with a mutant, but not a normal, presenilin may be 5 encoded and expressed by a recombinant construct in order to compete with, and thereby inhibit or block, the aberrant interaction.
Retroviral vectors can be used for somatic cell gene therapy especially because of their high efficiency of infection and stable integration and ~;~pl~ssion. A
full length PS-interacting protein gene, subsequences encoding fimctional domains of 10 ~ these proteins, or any of the other therapeutic peptides described above, can be cloned into a retroviral vector and ~ es~ion may be driven fiom its endogenous promoter, from the retroviral long tt-rmin~1 repeat, or from a promoter specific for the target cell type of interest (e.g., neurons). Other viral vectors which can be used include adeno-associated virus, vaccinia virus, bovine papilloma virus, or a herpes virus such as 15 Epstein-Barr virus.
C. Tmmlmothera~y Irnmunotherapy is also possible for ~17h~imer's Disease. Antibodies may be raised to a norrnal or mutant PS-interacting protein (or a portion thereof) arrd are 2~-1min;etered to the patient to bind or block an aberrant interaction ~e.g., with a mutant 20 pr~eenilin) and y~ rellL its deleterious effects. Simn1t~n~ously, ~ ssion of the normal protein product could be encouraged. ~1t~rn~tively, antibodies may be raised to specific complexes between mutant or wild-type PS-interacting proteins and their interaction partners.
A further approach is to stim~ te endogenous antibody production to the 25 desired arltigen. A-lminietration could be in the forrn of a one time imm1mngenic r~l~lion or vaccine immunization. The PS-interacting protein or other antigen may be mixed with ph~rm~ceutically acceptable caIriers or excipients compatible with the protein. The immtmogenic composition and vaccine may fi~rther contain auxiliary substances such as emulsifying agents or adiuvants to enhance effectiveness.

S~Jt~5 ~ ITE SHEET (RULE 26) -7~-Tmmllnngenic compositions and vaccines may be ~lmini.~tered palel,LeLdlly by injection subcutaneously or i~ uscularly.
D. Small Molecule Thera~eutics As described and enabled herein, the present invention provides for a 5 number of methods of identifying small molecules or other compounds which may be useful in the treatment of Alzheimer's Disease or other disorders caused by mutations in the presPnilin~ or PS-interacting proteins. Thus, for example, the present invention provides for methods of identifying proteins which bind to normal or mutant PS-interacting proteins (aside from the presenilins). The invention also provides for 10 . methods of identifying small molecules which can be used to disrupt aberrant interactions between mutant presenilin~ and/or Ps-hll~ lg proteins and such other binding proteins or other cell components.

Exam~les Example 1. Isolation of PS-interacting ~roteins bv t~,vo-hybrid Yeast system.
To identify proteins interacting with the pres~nilin proteins, a commercially available yeast two-hybrid kit ("M~t~hm~kPr System 2" from Clontech, Palo Alto, CA) was employed to screen a brain cDNA library for clones which interact with fùnctional domains of the pres~nilinc. In view of the likelihood that the TM6~7 loop domains of the pres~nilin~ are important functional domains, partial cDNA sequences encoding either residues 266-409 of the normal PS l protein or residues 272-390 of the normal PS2 protein were ligated in-frame into the EcoRI and BamHI sites of the pAS2- I fusion-protein expression vector (Clontech). The reSlllt~nt fusion proteins contain the GAL4 DNA binding domain coupled in-frame either to the TM6~7 loop of the PS 1 protein or to the TM6~7 loop of the PS2 protein. These ion plasmids were co-transformed into S. cerevisiae skain Yl90 together with a library of human brain cDNAs ligated into the pACT2 yeast fusion-protein ~ression vector (Clontech) bearing the GAL4 activation domain using modified lithium acetate protocols of the "M~t-~hm~ker System 2" yeast two-hybrid kit (Clontech, Palo Alto, CA). Yeast clones bearing human brain cDNAs which interact SUBSTlTUTE SHEET (RULE 26) with the TM6~7 loop domain were selected for His- rc~ict~nce by plating on SD
minim~l medium lacking hi~ti(lin~ a~d for ,Bgal+ activation by color selection. The His+ ,Bgal~ clones were then purged of the pAS2-1 "bait" construct by culture inlO~lg/ml cyclohexamide and the unknown "trapped" inserts of the human brain 5 cDNAs encoding PS-interacting proteins were isolated by PCR and sequenced. Of 6 million initial transformants, 200 positive clones were obtained af[er His- selection, and 42 after ,Bgal+ color selection, carried out in accordance with the m~nnf~rturer's protocol for selection of positive colonies. Of these 42 clones there were several independent clones represen~ing the same genes.
To address the likelihood that mutations in the pr~c~nilin~ cause ~D
through the acquisition of a novel but toxic function (i.e., dc~ gain of function mutation) which is meAi~teA by a novel interaction between the mutant proteins and one or more other cellular proteins, the human brain cDNA libra~y cloned into the pACT2 ~ s~ion vector (Clontech) was re-screened using mutant TM6~7 loop domain sequences as described above and according to m~nllf~-~tllrer's protocols. In particular, mutant pre~e~ilin sequences corresponding to residues 260-409 of PS l TM6~7 loop domains bearing mutations L286V, L392V and ~290-3 l 9 were ligated in-frame into the GAL4 DNA-binding domain of the pAS2- l vector (Clontech3 and used to screen the human brain cDNA:GAL4 activation domain library of pACT
vectors (Clontech). Yeast were co-transformed, positive colonies were selected, and "trapped" sequences were recovered and sequenced as described above. In addition to some of the same sequences recovered with the normal TM6~7 loop domains, several new sequences were obtained which reflect aberrant interactions of the mutant pr~sPnitin~ with normal cellular proteins.
The recovered and sequenced clones corresponding to these PS-interacting proteins were colllp~ed to the public sequence databases using the BL:ASTN
algoli~ll.,l via the NCBI e-mail server. Descriptions of several of these clones follow:
Antisecretorv Factor/ Proteasome SSa Subunit. Two overlapping clones (Y2H29 and Y2H3 1) were identified which correspond to a C-t--~min~l fragment of a protein ~lt~rn~tively identified as Antisecretory Factor ("ASF") or the Multiubiquitin SUBSTITUTE SHEET (RULE 26~

-~0-chain binding S5a subunit of the 26S proteasome ("SSa") (Johansson et al. 1995;
Ferrell et al., 1996). The complete nucleotide and amino acid sequences of the S5a subunit are avaiIable through the public databases under Accession number U51007and are reproduced here as SEQ ID NO: 1 and SEQ ID NO: 2. The nucleotide 5 sequences of the Y2H29 and Y2H31 clones include nucleotides 351-1330 of SEQ IDNO: 1 and amino acid residues 70-377 of SEQ ID NO: 2. Thus, residues 70-377 of the full SSa subunit include the PS-interacting domain ofthis protein. l~e~ es 206-377 of S5a contain certain motifs that are important for protein-protein interactions (Ferrell et al., 1996).
The PS l-S5a subunit h~ a iLion was directly re-tested for both wild type and mutant PS1 TM6~7 loop (residues 260-409) by tlallsrollllillg Y187 yeast cells with the ay~lopliate wild type or mutant (L286V, L392V or ~290-319~ cDNA ligatedin-frarne to the GAL4-DNA binding domain of pACT2. The ~290-319 mutant fusion construct displayed autonomous ,Bgal activation in the absence of any S5a ''target sequence" and, therefore, could not be filrther analyzed. In contrast, both the L286V
and L392V mutant constructs interacted specifically with the S5a construct.
Q~ ntit~tive assays, however, showed that these interactions were weaker than those involving the wild type PS 1 260409 sequence and that the degree of interaction was crudely correlated with the age of onset of FAD. The difference in ,Bgal activation was not attributable to instability of the mutant PS 126a4O9 construct mRNAs or fusion proteins because Western blots of Iysates of transformed yeast showed equivalentquantities of mutant or wild-type fusion proteins.
Because one of the putative functions of S5a is to bind multi-ubiq l;tin~t~-l proteins, the PS 1 :S5a interaction observed in S. cerevisiae could arise either through yeast-dependent ubiquitination ofthe PS1260409 construct, or by direct interaction. The former would reflect a degradative pathway, a functional and perhaps reciprocal interaction between PS1 and S5a, or both. A direct interaction is favored by the fact that the PS 1 :SSa interaction is decreased rather than increased by the presence of the L286V and L392V mutations, and by the fact that neither of these mutations affect ubiquitin conjugation sites in the PS 1260409 loop (i.e., K265, K311, K314 or K395).

S~J~ 111 UTE SHEET (RULE 26) W 097~7296 PCT/CA97100051 To further examine this possibility, we investigated the direct interaction of recombinant His-tagged fusion proteins corresponding to full length SSa and the PSl260"09 loop. Partially purified recombinant His-tagged PSl260409100p and His-tagged SSa proteins and ~~ iate controls were mixed in phosphate buffered saline.
5 The ~ Ul~ was then subjected to size exclusion cl~ul~ ography, and eluates were e~-nine~1 by SDS-PAGE and Western blotting using anti-His-tag monoclonal antibodies ~Quiagen). In the crude PSl260409100p ~ Lalion alone, the PSl26040~ loop eluted from the size exclusion colurnn as a broad peak at 35 l~i....(es In the crude SSa l~l~al~lion alone, S5a eluted at 25 mlnllt.os However, when the crude PS126o~,og lOop 10 and S5a pl~udlions were mixed~ there was a significant shift in the elution of PS 126o 409 toward a higher molecular weight complex. Co-elution of S5a and PS126o~og in the same fraction was confinned by SDS-PAGE and Western blotting of fractions using the anti-~Iis-tag antibody. These results are con.~i~tent with a ubiquitin-independent and, therefore, possibly functional il~ eLion.
GT24 and related ~enes with homology to pl20/Plako~lobin familY. Five over-lapping clones (Y2H6, Y2HlOb, Y2H17h2, Y2H24, and Y2H25) were obtained which interact with the normal PS l TM6~7 loop domain and which appear to represent at least one novel gene. The Y2H24 clone was also found to interact with the mutant PS l TM6~7 loop domains. Note that it appears that more than one member of the gene family was isolated, suggesting a family of genes interactingdifferentially with different presenilins. The most complete availsble cDNA
corresponding to these clones was d~ei~l~t~-l GT24 and is disclosed herein as SEQ ID
NO: 3 and has been deposited with GenBank as Accession number U8 l O04. The open reading frame suggests that GT24 is a protein of at least 1040 amino acids with a unique N-Le. .-,;",~ and considerable homology to several ~ lillo (~) repeat proteins at its C-tl-,nnimls The predicted amino acid sequence of GT24 is disclosed herein as SEQ ID NO: 4. Thus, for example, residues 440-862 of GT24 have 32-56%
identity (p=1.2e-l33) to residues 440-854 of murine pl20 protein (Accession number Z17804), and residues 367-815 of GT24 have 26-42% identity (p=0.00 l 7~ to residues 245-465 of the D. melano~aster ~ tiillo segment pol~rity protein (Accession SUBSTITUTE SHEET (RULE 26) , number P18824). The C~T24 gene maps to chromosome SplS near the anonymous microsatellite marker DSS748 and the Cri-du-Chat syndrome locus. This sequence is also nearly identical to portions of two human ESTs of unknown function (i.e., nucleotides 2701 -3018 of Accession number F08730 and nucleotides 2974-3348 of 5 Accession number T18858). These clones also show lower degrees of homology with other partial cDNA and gDNA sequences (e.g., H17245, T06654, T77214, H24294, M62015, T87427 and G04019).
pO071 ~ene. An additional His-, ~gal~ clone isolated in the initial screening with wild type PS 1266409 "bait" had a similar nucleotide sequence to GT24 (target clone Y2H25; Accession nurnber U81005), and would also be predicted to encode a peptide with C~-t~rrnin~l arm repeats. A longer cDNA sequence closely corresponding to the Y2H25 clone has been deposited in GenBank as human protein pO071 (Accession number X81889). The nucleotide and collc;spollding amino acid sequences of pO071 are reproduced herein as SEQ ID NOs: 5 and 6. Comparison of the predicted se(luence of the pO071 ORF with that of GT24 confirms that they are related proteins with 47% overall amino acid sequence identity, and with 70% identity between residues 346-862 of GT24, and residues 509-1022 of pO071 (which includesresidues encoded by the Y2H25 cDNA). The latter result strongly suggests that PS 1 interacts with a novel class of arm repeat co~ proteins. The broad ~ 4 kb hybridization signal obtained on Northern blots with the unique 5' end of GT24 could reflect either alternate splicing/polyadenylation of GT24, or the ~ ten~e of additional members of this family with higher degrees of N-t~nnin~l homology to GT24 than pO071.
Rabl 1 ~ene. This clone (Y2H9), disclosed herein as SEQ ID NO: 7, was identified as interacting with the normal PS 1 TM6~7 loop domain and appears to correspond to a known gene, Rab l l, available through Accession numbers X56740 and X53143. Rabl 1 is believed to be involved in protein/vesicle traffickin~ in the ER/Golgi. Note the possible relationship to processing of membrane proteins such as BAPP and Notch with r~slllt~nt overproduction of toxic A13 peptides (especially neurotoxic A~ 2(43) isoforms) (Scheuner, et al, 1995).

Sl~ts~ ITE SHEET (RULE 26) Retinoid X receptor-B ~ene. This clone (Y2H23b~, disclosed herein as SEQ ID NO: 8, was identified as interacting with the normal PSl TM6~7 loop domain and appears to correspond to a known gene, known variously as the retinoid X
receptor-,B, nuclear receptor co-regulator or MHC Class I regulatory element, and available through Accession numbers M84820, X63522 and M81766. This gene is believed to be involved in intercellular ~ 1ing, suggesting a possible relationship to the intercellular sign~ling function metii~tec~ by C. ele~ans sell2 and Notchllin-12 (transcription activator).
CYtoplasmic chal~ero.lill ~ene. This clone (Y2H27), disclosed herein as 10 SEQ ID NO: 9, was identified as interacting with the nor nal PSl T~6~7 loop domain and appears to correspond to a known gene, a cytoplasmic chaperonin col-t~...io~ TCP-1, available through Accession numbers U17104 and X74801.
Unknown gene (~2H35). This clone (Y2H35), disclosed herein as SEQ
ID NO: 10, was identified as interacting with the normal PS 1 TM6~7 loop domain and appears to correspond to a known gene of unknown function, available throughAccession number R12984, which shows conservation down through yeast.
Unknown gene ~Y2H171). This clone (Y2H171), disclosed herein as SEQ
ID NO: 11, was identified as interacting with the normal PSl TM6~7 loop domain and appears to correspond to a known expressed repeat sequence available throughAccession ~ b-,l D55326.
Unknown gene (Y2H41). This clone (Y2H41) was identified which reacts strongly with the TM6~7 loop domains of both PSl and PS2 as well as the mutant loop domains of PSl. The sequence, disclosed as SEQ ID NO: 12, shows strong homology to an EST of unknown function (Accession number T64843).
Exarnple 2. Isolation of pres~nilin bindin~ proteins by affinitY chromato~raphy.To identify the proteins which may be involved in the biochemical - function of the pres~nilin~, PS-interacting proteins were isolated using affinity chromatography. A GST-fusion protein co..~ .g the PSl TM6~7 loop, prepared as described in Example 3, was used to probe human brain extracts, prepared by 30 homogenizing brain tissue by Polytron in physiological salt solution. Non-specific SUt~a ~ ITE SHEET (RULE 26) binding was elimin~tç(l by pre-clearing the brain homogenates of endogenous GST-binding components by incubation with glutathione-Sepharose beads. These GST-free homogenates were then incubated with the GST-PS fusion proteins to produce the desired complexes with functional binding proteins. These complexes were then 5 recovered using the affinity glutathione-Sepharose beads. Af'~er extensive washing with phosphate buffered saline, the isolated collection of proteins was separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE; Tris-tricine gradient gel 4-20%). Two major bands were observed at ~14 and 20 kD in addition to several weaker bands ranging from 50 to 60 kD.
The same approach may now be used to identi~y proteins which have binding activity for the PS-interacting proteins and, thereby, to further elucidate the etiology of AD and to identify additional thc.dl)~;ulics targets for intervention in AD
and related disorders.
Example 3. Eukarvotic and prokarvotic ~ ession vector svstems.
Constructs suitable for use in eukaryotic and prokaryotic t;~ ;s~ion systems have been generated using different classes of PS 1 nucleotide cDNA
sequence inserts. In the first class, termed full-length constructs, the entire PS 1 cDNA
sequence is inserted into the ~ es~ion plasmid in the correct orientation, and includes both the natural 5' UTR and 3' UTR sequences as well as the entire openreading frame. The open reading frames bear a nucleotide se~uence c~sette which allows either the wild type open reading frame to be in~ ded in the ~ t;s~ion system or altc;lllalively, single or a combination of double mutations can be inserted into the open reading frame. This was accomplished by removing a restriction fragment from the wild type open reading frame using the enzymes NarI and PflmI and replacing it with a similar fragment generated by reverse L~ scli~l~se PCR and bearing the nucleotide sequence encoding either the M146L mutation or the H163R mutation. A
second restriction fragment was removed from the wild type normal nucleotide sequence for the open reading frame by cleavage with the enzyrnes PflmI and NcoIand replaced with a restriction fragment beanng the nucleotide sequence encoding the A246E mutation, the A260V mutation, the A285V mutation, the L2~6V mutation, the SUBSTlTUTE SHEET (RULE 26) = _ L392V mutation or the C410Y mutation. A third variant, bearing a combination of either the M146L or H163R mutation in tandem with one ofthe re~ i"i"g mutations,was made by linlcing a NarI-PflmI fragment bearing one of the former mutations and a PflmI-NcoI fragment bearing one of the latter mutations.
The second class of cDN~ inserts, termed truncated constructs, was constructed by removing the 5' UTR and part of the 3' I~R sequences from full length wild type or mutant cDNA sequences. The 5' UTR sequence was replaced witha synthetic oligonucleotide cu"t~,"i--g a KpnI restriction site (GGTAC/C) and a small sequence (GCCACC) to create a Kozak initiation site around the ATG at the lQ beginning of the PS 1 ORF. The 3' UTl? was replaced with an oligonucleotide with an artificial EcoRI site at the 5' end. Mutant vanants of this construct were then made by inserting the mutant sequences described above at the NarI-PflmI and PsImI-NcoI
sites as described above.
For eukaryotic ~ ion, these various cDNA constructs bearing wild 15 type and mutant sequences, as described above, were cloned into the ~ ,ssion vector pZeoSV in which the SV60 promoter cassette had been removed by restriction digestion and replaced with the CMV promoter element of pcDNA3 (Invitrogen). Forprokaryotic ~ s~,ion, constructs have been made using the glutathione S-l~ 7r~ldse (GST) fusion vector pGEX-kg. The inserts which have been ~tt~chefl to the GST
20 fusion nucleotide sequence are the same nucleotide sequences described above bearing either the normal open reading frame nucleotide sequence, or bearing a combination of single and double mutations as described above. These GST fusion constructs allow ~ ssion of the partial or full-length protein in prokaryotic cell systems as mutant or wild type GST fusion proteins, thus allowing purification of the 25 ffill-length protein followed by removal of the GST fusion product by thrombin digestion. A filrther cDNA construct was made with the GST fusion vector, to allow the production of the amino acid sequence corresponding to the hydrophilic acidic loop domain between TM6 and TM7 of the full-length protein, either as a wild type nucleotide sequence or as a mutant sequence bearing either the A285V mutation, the 30 L286V mutation or the L392V mutation. This was accomplished by recovering wild SUc~ JTE SHEET (RULE 26) type or mutant sequence from a~plopliate sources of RNA using a ~' oligonucleotide primer with a 5' BamHI restriction site (G/GATCC), and a 3' primer with a 5' EcoRI
restriction site (G/AATTC). This allowed cloning of the ~plopl;ate mutant or wild type nucleotide sequence corresponding to the hydrophilic acidic loop domain at the BamHI and the EcoRI sites within the pGEX-KG vector.
The PS-interacting protein genes may be similarly manipulated by recombinant means for ~ ssion in prokaryotic or eukaryotic hosts. In particular,GST or other fusion proteins may be produced which will be useful in assays (e.g., yeast two-hybrid studies) for therapeutics.
10 Exam~le 4. AntibodY Production.
Peptide antigens corresponding to portions of the PS l protein were synth~i7e-1 by solid-phase techniques and purified by reverse phase high P1~;7~;L11e liquid chro~ ography. Peptides were covalently linked to keyhole limpet hemocyanin (KLH) via di~lllfide linkages that were made possible by the addition of a 15 cysteine residue at the peptide C-t~rmimls of the pr~s~nilin fr~nent This additional residue does not appear nonn~lly in the protein sequence and was included only to f~f~ilit~te linkage to the KLH molecule.
A total ofthree New ~e~nd white rabbits were i.l~ ed with peptide-KLH complexes for each peptide antigen in combination with Freund's adjuvant and20 were subsequently given booster injections at seven day intervals. Antisera were collected for each peptide and pooled and IgG ~ ci~ ted with ammonium sulfate.
Antibodies were then affinity purified with Sulfo-link agarose (Pierce) coupled with the a~plo~liate peptide. This final pllrifi~fion is required to remove non-specific interactions of other antibodies present in either the pre- or post-immllne serum.
The specificity of each antibody was confirrned by three tests. First, each detected single predominant bands of the approxirnate size predicted for pres~nilin-l on Western blots of brain homogenate. Second, each cross-reacted with recombinant fusion proteins bearing the ayplol~liate sequence. Third each could be specifically blocked by pre-absorption with recombinant PS l or the i ~ i7ing peptide.

SIJ~ 111 UTE SHEET (RULE 26) -Antibodies to peptides derived from the PS-interacting proteins may be produced by similar means.
Example 5. Trans~enic mice.
A series of wild type and mutant PS 1 and PS2 genes were constlucted for use in the ~ lion of l,~lsgenic mice. Mutant versions of PS 1 and PS2 were generated by site-directed mutagenesis of the cloned cDNAs using standard techniques.
The cDNAs and their mutant versions were used to prepare two classes of mutant and wild type PS 1 and PS2 cDNAs, as described in Example 3. The first 10 class, referred to as "full-length" cDNAs, were prepared by removing approximately 200 bp of the 3' untr~ncl~ted region imm~ t~ly before the polyA site by digestion with EcoRI (PS1) or PvuII (PS2). The second class, referred to as "I~ n~efl"
cDNAs, were prepared by replacing the 5' untr~n~l~te(l region with a ribosome binding site (Kozak COll5ell~US sequence) placed immediately 5' of the ATG start15 codon.
Various full length and t~mr~te~i wild type and mutant PSl and PS2 cDNAs, p.c;par~d as described above, were introduced into one or more of the following vectors and the rçslllting constructs were used as a source of gene for the production of transgenic mice.
The cos.TET ~ s~ion vector: This vector was derived from a cosmid clone co~ ;..;..g the Syrian h~m~tPr PrP gene. It has been described in detail by Scott et aL (1992) and Hsiao et al. (1995). PSl and PS2 cDNAs (full length or trlm-,~te~) were inserted into this vector at its SalI site. The final constructs contain 20 kb of 5' sequence fl~nk;ng the inserted cDNA. This 5' fl~nkin~ sequence includes the PrP
25 gene promoter, 50 bp of a PrP gene 5' untr~n~ te~l region exon, a splice donor site, a I
kb intron, and a splice acceptor site located immediately adjacent to the SalI site into - which the PS 1 or PS2 cDNA was inserted. The 3' sequence fl~nking the inserted cDNA includes an approximately 8 kb segment of PrP 3' untr~n~l~te-l region including a polyadenylation signal. Digestion of this construct with NotI (PS 1 ) or 30 FseI (PS2) released a fir~rnent C~ i 1 ~g a mutant or wild type PS gene under the S~ JTE SHEET (RULE 26) CA 022444l2 l998-07-27 control ofthe PrP promoter. The released fragment was gel purified and injected into the pronuclei of fertilized mouse eggs using the method of Hsiao et al. (1995).
Platelet-derived ~rowth factor receptor ~-subunit constructs: PS cDNAs were also introduced between the SalI (full length PS 1 cDNAs) or HindIII (trllnf~tefl 5 PS1 cDNAs, full length PS2 cDNAs, and tr ~neate~l PS2 cDNAs) at the 3' end of the human platelet derived growth factor receptor ~-subunit promoter and the EcoRI site at the'5' end of the SV40 polyA sequence and the entire r.~csette was cloned into the pZeoSV vector (Invitrogen, San Diego, CA.). Fr~m~nt~ rele~ed by ScaI/BamHI
digestion were gel purified and injected into the pronuclei of fertilized mouse eggs 10 using the method of Hsiao et al. (1995).
Human R-actin constructs: PS 1 and PS2 cDNAs were inserted into the SalI
site of pBAcGH. The construct produced by this insertion includes 3.4 kb of the human ~ actin 5' fl~nking sequence (the human ,B actin promoter, a spliced 78 bphuman ~ actin 5' untr~n~l~te~l exon and intron3 and the PS 1 or PS2 insert folIowed by 15 2.2 kb of human growth horrnone ~enl)mic sequence co~ .g several introns and exons as well as a polyadenylation signal. SffI was used to release a PS-cul.l;1;.-i..g fragment which was gel purified and injected into the pronuclei of fertilized mouse eggs using the method of Hsiao et al. (1995).
Phospho~lycerate lcinase constructs: PS1 and PS2 cDNAs were introduced 20 into the pL~90 vector. The cDNAs were inserted between the KpnI site downstream of the human phosphoglycerate kinase promoter and the XbaI site u~sl1ealll of the 3' untr~n~l~terl region of the hurnan phosphoglycerate kinase gene. PvuII/~in~lTTT (PS 1 cDNAs) or PvuII (PS2 cDNAs) digestion was used to release a PS-co~
fragment which was then gel purified and injected into the pronuclei of fertilized 25 mouse eggs as described above.
Analvsis of A~ in trans~enic murine hippocampus: To analyze the effect of a mutant human PS 1 transgene in mice, a PS 1 mutation observed in conjunction with a particularly severe fonn of early-onset PSl-linked Alzheimer's disease was used, namely the M146L mis~n~e mutation (!~helTington et al., 1995). The ~nim~
30 which were het ~erozygous ~or the PS 1 mutant transgene on a mixed F~B-C57BL/6 SU~ UTE SHEET (RUEE 26) -W ~97127296 PCT/CA97/00051 strain background, were cross-bred with similar mice bearing the human wild-type,~APP69s cDNA under the same Syrian h~m~t-~.r PrP promoter similar to those ~nim~l~
recently described by Hsiao et al., l995. These cross breedings were done because it is thought that human A13 is more susceptible to the forrnation of aggregates than are 5 murine A,B peptides.
The progeny ofthese PS1MI46L x ,BAPPWT cross-breedings were then genotyped to identify ~nim~le that contained both the human wild-type ,BAPP69s transgene and also the mutant human PS1MI46L transgene. These mice were aged until two to three months of age and then sacrificed, with the hippocatnpus and neocortex 10 being ~ secte~1 rapidly from the brain and frozen. Litter mates of these mice, which contained only the wild-type human ~APP695 transgene were also sacrificed, and their hippocampi and neocortices were ~ secte~l and rapidly frozen as well.
The concentration of both total A~ peptides (A,BX 4" and A~X 42 (43)) as well as the subset of A,B peptides ending on residues 42 or 43 (long-tailed A~42 peptides) 15 were then measured using a two-sandwich ELISA as described previously (Tamaoka et al., l 994; Suzuki et al., l 994). These results convincingly showed a small increase in total A,B peptides in the double ll~lsgel~ic ~nim~l~ bearing wild-type human ~APP69s and mutant human PSlM,46L transgenes compared to the wild-type human ~APP695 controls. More hllpl~s~ ely, these mea~ul~lllents also showed that there was 20 an increase in the amount of long-tailed A~ peptides ending on residues 42 or 43 (A~42). In contrast, litter mates bearing only the wild-type human ~APP69s transgene had A,B42 long-tailed peptide values which were below the limit of qll~ntit~tion("BLQ").
These observations therefore confirrn that the construction of transgenic 25 ~nim~1~ can lecapiLulate some of the bioehemie~1 features of human ~17heimer's disease (namely the overproduction of A,B peptide and, in particular, overproduction - of long-tailed isoforms of A~ peptide). These observations thus prove that the transgenic models are in fact useful in exploring therapeutic targets relevant to the tre~tment and pr~ ion of Alzheimer's disease.

SU~S l 11 ~JTE SHEET ~RULE 21i) W 097/27296 PCT/C~97/0~051 Analvsis of hiPPocampus dependent memorv fimctions in PS1 trans~enic mice: Fourteen transgenic C57BL/6 x FVB mice bearing the human PS1MI46V mutant transgene under the PrP promoter (as described) above and 12 wild type litter mates aged 2.5-3 months of age (both groups were balanced for age, weight, and sex) were 5 investigated for behavioral differences attributable to the mutant transgene. Also the qualitative observation of murine behavior in their home cages did not indicate bimodal distribution of behaviors in the sample of ~nim~l~
Exneriment 1. To test for subtle differences in exploratory behavior (e.g. locomotion, sç~nnin~ of the ellvilvlllllent through rearing, and patterns 10 of investigation of l-nf~n~ r environment), both PS 1M~46V and wild type litter mates were tested in the open-field (Janus, et al. 1995). The results of the test revealed no .cignific~nt differences bt;lwet;ll ll ,.. .~g5~., ic ~ and controls in exploration of a new environment measured by mice locomotor behaviors (walking, p~ ing, wall le~ning,rearing, grooming), (F(1,24~ = .98, NS). Thus, dirrel~llces any in behavior on the 15 Morris water maze test (see below) cannot be attributed to dirr~ ces in locomotor abilities, etc.
Experiment 2. One week after the open-field test, the PS1MI46V
mutant transgenic mice and their litter mates were trained in the Morris water maze.
In this test, a mouse has to swim in a pool in order to find a submerged escape 20 platform. The animal solves that test through learning the location of the platform using the available extra-maze spatial cues (Morris, 1990). This test was chosenbecause there is strong evidence that the hippocampal -formation is involved in this form of le~rning The hippocampus is also a major site of AD neuropathology in hl-m~n.c and defects in spatial learning (geographic disorientation, losing objects, 25 wandering, etc.) are prominent early features of human AD. As a result the test is likely to detect early changes equivalent to those seen in human AD. The Morris test is conducted in three phases. In the first phase (the learning acquisition phase), the mouse has to learn the spatial position of the platform. In the second phase (the probe trial), the platform is removed from the pool and the mouse's search for the platform 30 is recorded. In the final phase (the learning transfer phase), the platform is replaced in SU~;~ ITE SHEET (RULE 26) a new position in the pool, and the mouse has to learn that new spatial position of the platform.
Transgenic and wild type mice did not differ in their latencies to find the platform during learning acquisition (F(1,24) = 0.81, NS), and both groups showed 5 rapid learning across trials (F(10,15) - 11.57, p < 0.001). During the probe trial phase, mice from both groups searched the quadrant of the pool which originally contained the platform si~nific~ntly longer than other areas of the pool which had not contained the platfolm (F(3,22) = 28.9, p C 0.001). However, the wild type controls showed a trend which was not ~uite st~ti~tiC~llly significant (t(24) = 1.21, p = 0.24) for an increased nurnber of crossings of the exact previous position of the platform. In the learning transfer test, both groups showed the same latency of finding the new position of the platform in the initial block of trials (t(24) = 1.11, NS). Such long latency to find the new spatial position is expected because the mice spent most of their time searching for the platform in the old spatial position. However, in later trials in the learning I~ l phase, the wild type mice showed shorter swim latencies to the new position of the platform cO~ d to the PS 1M~46V mutant transgenics (F~1,24) 2.36, p = 0.14). The results indicate that PS1MI46V mutant ll~llSg~lliC mice were less flexible in transferring learned information to a new situation and tended to persevere in their search for the platform in the old location.
Thus, although no differences were found in the spontaneous exploration of a new enviromnent and in the acquisition of new spatial information between the wild type and the PS1MI46V mutant transgenic mice, the PS1MI46V mutant transgenic mice were hllpail ed in switching and/or adapting this knowledge in later situations.
Electro~hysiolo~ical Recordin~es in the hippocam~us of mutant trans~enic mice: Five to six months old litter mate control and human PS1MI46V mutant transgenic mice on the same C57BL/6 x FVB strain backgrounds as above were used ~ to study long term potentiation (LTP) as an electrophysiologic correlate of learning and memory in the hippocampus. Recordings were carried out on 400 ,um thick hippocampal slices according to conventional techni~ues. Briefly, brains were removed and transverse sections cont~ining hippocampi were obtained within 1 min.

SUBSTITUTE SHEET (RULE 26~

after mice were decapitated under halothane anesthesia. Slices were kept at roomtemperature in oxygenated artificial cerebrospinal fluid for one hour prior to recording. One slice at a time was transferred to the recording chamber, where they were m~int~ined at 32 ~C in an interface between oxyg~n~ted artificial cerebrospinal 5 fluid and humidified air. Slices were then allowed to equilibrate in the recording chamber for another hour.
Extracellular field recordings were carried out in the CAl subfield of the hippocampus at the Schaeffer collateral-pyramidal cell synapse. Synaptic responses were induced by the stim~ tir n of Schaeffer collaterals at a frequency of 0.03 Hz and 10 an intensity of 30-50 % of m~im~l response. Tetani to evoke long-term potentiation consisted of ~ trains of l 00 Hz stimulation lasting for 200 ms at an illLc~ llail~ interval of 10 seconds. Field potentials were recorded using an Axopatch 200B amplifier (Axon Instrument). Glass pipettes were fa~ricated ~om borosilicate glass with anouter diameter of l.S mm, and pulled with a two step Narishige puller. Data were15 acquired on a 486-IBM compatible co~ ,ul~. using PCLAMP6 software (Axon Instrument).
To test for any abnormality in presynaptic function, we investigated the dirr~ lces in paired-pulse f~rilit~tion, which is an example of use-dependent increase in synaptic efficacy and is considered to be presynaptic in origin. In hippoc~ll~us, 20 when two stimuli are delivered to the Schaeffer collaterals in rapid sllccçssion, paired-pulse facilitation manifests itself as an enh~nce~ dendritic response to the second stiTnlllu~ as the interstimulus interval gets shorter. In three pairs of wild-type/transgenic mice, we did not observe any difference in the paired-pulse facilitation over an illLt;l~lilllulus interval range of 20 ms to l sec. These data suggest that in 25 PSIMI46V mutant tr~n~enic mice, the excitability of Sch~ef~Pr collateral fibers and neuro~ er release are likely to be normal.
Tetanic stim~ tion induced a long-lasting increase in the synaptic strength in both control (n = 3~ and PS1MI46V mutant transgenic mice (n = 2). In slices obtained from the PS1MI46V mutant transgenic mice, long-lasting increase in the synaptic 30 strength was 30 % more than that obtained from control mice.

SU~S 1 l l ulTE SHEET (RULE 26) Although preferred embot1iment~ of the invention have been described herein in detail, it will be understood by those sl~illed in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the 5 appended claims.

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(1) GENERAL INFORMATION:
~i~ APPLICANT: ST. GEORGE-HYSLOP, PETER H.
ROMMENS, JOHANNA M.
FRASER, PAUL E.
(ii) TITLE OF INVENTION: NUCLEIC ACIDS AND PROTEINS RELATED TO
ALZHEIMER'S DISEASE, AND USES THEREFOR
(iii~ NUMBER OF SEQUENCES: 12 (iv) CO~E;PnNDENCE ADDRESS:
.', ..D)hESSEE: Sim & McBurney T~FET: 330 University Avenue, 6TH Floor ' I ~.IT': Toronto ~ ~T..TE: Ontario ., ro NTRY: Canada ,'l :I': M5G lR7 (v3 COMPUT';l -EADABLE FORM:
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(l SO-T ~RE: .~atentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: 27-JAN-1997 (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A~ APPLICATION NUMBER: US 08/592,591 (B) FILING DATE: Z6-JAN-1996 (vii) PRIOR APPLICATION DATA:
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(A) APPLICATION NUMBER: US 60/029,895 (B) FILING DATE: 08-NOV-1996 (vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: DOCKET# CAN-006PR(d) (B) FILING DATE: 02-JAN-1997 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: RAE, Patricia A.
(iX) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (416) 595-1155 (B) TELEFAX: (416) 595-1163 - -(2) INFORMATION FOR SEQ ID NO:1:
(i) SE~UE~C. CHARACTERISTICS:
(..) _E ~GTH: 1330 base pairs ( ) TY E: nucleic acid (.) 'T~ANDEDNESS: single ~ O_'OLOGY: linear (ix)-FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 145..1275 (D) OTHER INFORMATION: /product= "S5a"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Met Val Leu Glu Ser Thr Met Val Cys Val Asp Asn Ser Glu Tly5 Met Arg Asn Gly A2po Phe Leu Pro Thr Ar2 Leu Gln Ala Gln Gln Asp Ala Val Asn Ile Val Cys His Ser Lys Thr SU~SIII~TE SHEET(RULF 26) g er Asn Pro Glu Asn Asn Val GGlGyC LTT AT C AcA CTG GCT AAT GAC 315 Cys Glu Val Leu Thr Thr Leu Thr Pro Asp Thr Gly Ar~ Ile Leu Ser Lys Le5u His Thr Val Gln Pro Lys Gly Lys Ile T~r Phe Cys Thr Gly I e Arg Val Ala His Leu Ala Leu Lys His ArOg Gln Gly Lys Asn Hloi5 Lys Met Arg Ile le Ala Phe Val Gly Sle5r Pro Val Glu Asp Alz0n Glu Lys Asp Leu V1215 Lys Leu Ala Lys Arg Leu Lys Lys Glu Lys Val Asn Val Asp Ile Ile Asn Phe Gly Glu Glu Glu Val Asn Thr Glu Lys Leu Thr Ala Phe Val Asn Thr Leu Asn Gly Lys Asp G y Thr Gly Ser His L17e0u Val Thr Val Pro lP7r5o Gly Pro Ser Leu A a Asp Ala Leu Ile Ser Ser Yro Ile ~eu A a Gly Glu Gly Gly Ala Met Leu Gly Leu Gly Ala Ser Asp Phe G u Phe Gly Val Asp Pro Ser Ala Asp Pro Glu Leu Ala Leu Ala Leu Arg Val Ser Met Glu Glu Gln Arg Gln 2A3r0g Gln Glu Glu Glu 2A13a5 Arg Arg Ala Ala Ala Ala Ser Ala Ala Glu Ala Gly Ile Ala 25h0r Thr Gly Thr Glu 2As55p Ser Asp Asp Ala 2Le60u Leu Lys Met Thr 26e5 Ser Gln Gln Glu Phe Gly Arg Thr Gly Leu Pro Asp Leu Ser Ser Met Thr Glu Glu Glu Gln Ile Ala Tyr Ala Met Gln Met Ser Leu Gln Gly Ala Glu 3Poho Gly Gln Ala Glu Ser Ala Asp Ile Asp A}a Ser Ser Ala Met 3Aslp Thr Ser Glu Pro 3A2a0 Lys Glu Glu Asp 3A25p Tyr Asp Val Met 3G310n Asp Pro Glu Phe 3Leu Gln Ser Val Leu Glu Asn Leu Pro Gly Va Asp Pro Asn Asn G u Ala Ile Arg Asn A a Met Gly Ser Leu A a Ser Gln Ala Thr 3L6y5s Asp Gly Lys Lys 3A70p Lys Lys Glu Glu 3A75p Lys Lys ~2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 377 amino a~ids IB) TYPE: amino acid (D~ TOPOLOGY: linear (ii) MOLECU~E TYPE: prote1n Sl~ 111 UTE SHEET (RULE 26) (xi) SEQUENCE DESCP~IPTION: SEQ ID NO:2:
Met Val Leu Glu Ser Thr Met Val Cys Val Asp Asn Ser Glu Tyr Met Arg Asn Gly Asp Phe Leu Pro Thr Arg Leu Gln Ala Gln Gln Asp Ala Val Asn Ile Val Cys His Ser Lys Thr Arg Ser Asn Pro Glu Asn Asn Val Gly Leu Ile Thr Leu Ala Asn Asp Cys Glu Val Leu Thr Thr Leu Thr Pro Asp Thr Gly Arg Ile Leu Ser Lys Leu Bis Thr Val Gln Pro Lys Gly Lys Ile Thr Phe Cys Thr Gly Ile Arg Val Ala His Leu Ala Leu Lys His lAr00g Gln Gly Lys Asn His Lys Met Arg Ile le Ala Phe Lys lA3r0g Leu Lys Lys Glu Lys Val Asn Val Asp Ile Ile Asn Phe Gly G u Glu Glu Val Asn Thr Glu Lys Leu Thr Ala Phe Val Asn Thr Leu Asn Gly Lys Asp Gly Thr Gly Ser His Leu Val Thr Val Pro lP7r5o Gly Pro Ser Leu Ala Asp Ala Leu Ile Ser Ser Pro Ile Leu lAla0 Gly Glu Gly Gly A a Met Leu Gly Leu Gly Ala Ser Asp Phe Glu Phe Gly Val Asp Pro Ser Ala Asp Pro Glu Leu Ala Leu Ala 2Le20u Arg Val Ser Met Glu Glu Gln Arg Gln Arg Gln Glu Glu Glu A15a Arg Arg Ala Ala 2A10a Ala Ser Ala Ala G u Ala Gly Ile Ala Thr Thr Gly Thr Glu A5s5p Ser Asp Asp Ala Leu Leu Lys Met Thr Ile Ser Gln Gln Glu Phe Gly Arg Thr Gly 2L7eu5 Pro Asp Leu Ser Ser Met Thr Glu Glu Glu Gln Ile Ala Tyr Ala Met Gln Met Ser Leu Gln Gly Ala Glu Phe Gly Gln Ala Glu Ser Ala Asp Ile Asp Ala Ser Ser Ala Met Asp Thr Ser Glu Pro Ala Lys Glu Glu Asp 3A25p Tyr Asp Val Met Gln Asp Pro Glu Phe Leu Gln Arg Asn A355a Met Gly Ser Leu 3A160a Ser Gln Ala Thr Ly65 Asp Gly Lys Lys 3A7s0p Lys Lys Glu Glu 3A75p Lys Lys ~2) INFORMATION FOR SEQ ID N3:3:
~i) SEQUE~:-. CHARACT-.RISTICS
GTH: 384_ base pairs PE: nucle.c acid ) ~TRANDEDNES : single ~ O'OLOGY: l_near (ix) FEATURE-~A) NAMEtREY: CDS
~B) LOCATION: 2..3121 ~ix) FEATURE-~A) NAME/KEY: misc ~eature ~B) LOCATION: 1..3~41 ~D) OTHER INFORMATION: /note= ~GT24"

SU~;~ JTE SHEET (RULE 26~

Wo 97/Z7296 PCT/CA97/00051 _99 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Ser Gln Leu Pro Ala Arg Gly Thr Gln A a Arg Xaa Thr Gly G n Ser Phe Ser Gln G y Thr Thr Ser Arg Ala Gly His Leu Ala G31y Pro Glu Pro Ala Pro Pro Pro Pro Pro Xaa Pro Arg Glu Pro Phe Ala Pro Ser Leu Gly Ser Ala Phe His Leu Pro Asp Ala Pro Pro Ala Ala Ala GCC GCC GCG CTC TAC TAC TCC A~C TCC ACG CTG CCC GCG CCG CCG CGC 238 Ala Ala Ala Leu Tyr Tyr Ser Xaa Ser Thr Leu Pro Ala Pro Pro Arg G80y Gly Ser Pro Leu A a Ala Pro Gln Gly G y Ser Pro Thr Lys Leu Gln Arg Gly Gly Ser Ala Pro Glu Gly A a Thr Tyr Ala Ala Pro Arg Gly Ser Ser Pro Lys Gln Ser Pro Ser Arg Leu Ala Lys Ser Tyr Ser Thr Ser Ser Pro Ile Asn Ile Val Val Ser Ser Ala Gly Leu Ser Pro Ile Arg Val Thr Ser Pro Pro Thr Val Gln Ser Thr Ile Ser Ser Ser Pro Ile His Gln Leu Ser Ser Thr Ile Gly Thr Tyr Ala Thr Leu Ser Pro Thr Lys Arg Leu Val Hls Ala Ser G85u Gln Tyr Ser Lys 190 ser Gln Glu Leu lTgyr5 Ala Thr Ala Thr Leu Gln Arg Pro Gly Ser Leu Ala Glu Leu Arg Ala Leu Gln Ser Pro Glu His His 235e Asp Pro Ile Tyr Glu Asp Arg Val Tyr Gln Lys Pro Pro Met Arg Ser Leu Ser Gln Ser Gln Gly Asp Pro 2Le60u Pro Pro Ala His Th65r Gly Thr Tyr Arg 2Th0r Ser Gly Ser G n His Gly Pro Gln Asn Ala Ala Ala Ala Thr Phe Gln Arg Ala Ser Tyr Ala Ala Gly Pro Ala Ser Asn Tyr 3A15a Asp Pro Tyr Arg Gln Leu Gln Tyr Cys Pro Ser Val Glu Ser Pro Tyr Ser Lys Ser Gly Pro Ala Leu Pro Pro Glu Gly Thr Leu Ala Arg Ser Pro Ser le Asp Ser Ile Gln 3L5y5s Asp Pro Arg Glu Phe Gly Trp Arg Asp Pro Glu Leu Sl~ 111 UTE SHEFT (RULE 26) Pro Glu Val Ile Gln Met Leu GLn His Gln Phe Pro Ser Val Gln Ser AAC GCG GCA GCC TAC TTG GAnA CHiASC Leu Cys Phe Gly Asp Asn Ly 1198 4LoyOs Ala Glu Ile Ary Arg Gln Gly Gly Ile Gln Leu Leu Val Asp Leu Leu Asp Hls Arg Met Thr Glu Val His A2rg Ser Ala Cys Gly 4A30a Leu Arg Asn Leu Val Tyr Gly Lys Ala Asn Asp Asp Asn Lys I e Ala Leu Lys Asn 4c5yO Gly Gly Ile Pro A a Leu Val Arg Leu Leu Arg Lys Thr Thr Asp Leu Glu Ile Arg Glu Leu Val Thr Gly Va Leu Trp Asn Leu 4S8eOr Ser Cys Asp Ala L8e5u Lys Met Pro Ile Ile Gln Asp Ala Leu Ala Val Leu Thr Asn Ala Val Ile Ile Pro H s Ser Gly Trp Glu Asn Ser Pro Leu Gln 5Als5p Asp Arg Lys Ile Gln Leu His Ser Ser Gln Val Leu Arg Asn 5Ala Thr Gly Cys Leu Arg Asn Val Ser Ser A a Gly Glu Glu Ala 5A4r5y Arg Arg Met Arg 5G150u Cys Asp Gly Leu 5T5h5r Asp Ala Leu Leu Tyr Val Ile Gln Ser Ala Leu Gly Ser Ser G710u Ile Asp Ser Lys 5T7h5r Val Glu Asn Cys Val Cys Ile Leu Arg Asn Leu Ser Tyr Arg 5LgeOu Ala Ala Glu Thr Ser Gln Gly Gln His Met Gly Thr Asp Glu L6eOu5 Asp Gly Leu Leu C~s Gly Glu Ala Asn G y Lys Asp Ala Glu S6er Ser Gly Cys Trp G625y Lys Lys Lys Lys L3yo Lys Lys Ser Gln As~ Gln Trp Asp Gly Va Gly Pro Leu Pro Asp Cys Ala Glu Pro Pro Lys Gly Ile Gln ~et Leu Trp His Pro Ser Ile Val Lys Pro Tyr Leu Thr Leu Leu Ser Glu Cys Ser Asn P67r5o Asp Thr Leu Glu Gly Ala Ala Gly Ala Leu Gln Asn Leu Ala Ala Gly Ser Trp Lys rp Ser Val Tyr Ile Arg Ala Ala Val Arg L70ys5 Glu Lys Gly Leu P7rl0o Ile Leu Val Glu Leu Leu Arg Ile Asp 7A20n Asp Arg Val Val Cys Ala Val Ala Thr A a Leu Arg Asn Met Ala Leu Asp Val Arg Asn Lys Glu Leu Ile Gly Lys Tyr Ala Met Arg Asp SUBSTITUTE SHEET (RULE 26) W 097~7296 PCT/CA97/00~51 CTA GTC CAC AGG CTT CCA GGA GGG AAC ~AC AGC AAC AAC ACT GCA AGC 2302 Leu Val His Arq Leu Pro Gly Gly Asn Asn Ser Asn Asn Thr Ala Ser Lys Ala Met Ser Asp ASp Thr Val Thr Ala Val Cys Cys Thr Leu His ~ GAA GTG ATT ACC AAG AAC ATG GAG AAC GCC AAG GCC TTA CGG GAT GCC 2398 Glu Va Ile Thr Lys Asn et Glu Asn Ala Lys Ala Leu Arg Asp Ala Glooy Gly Ile Glu Lys Le5u Val Gly Ile Ser Lys Ser Lys Gly Asp Lys His Ser Pro Lys Val Val Lys Ala Ala Ser Gln Val Leu Asn Ser Met Trp Gln Tyr A83rg5 Asp Leu Arg Ser 8Le4uO Tyr Lys Lys Asp 3G45y Trp Ser Gln Tyr His Phe Val Ala Ser Ser Ser Thr Ile Glu Arc Asp Arg Gln Arg Pro Tyr Ser Ser Ser Arg Thr Pro Ser Ile Ser Pro Val Arg Val Ser Pro Asn Asn Arg Ser Ala Ser Ala Pro Ala Ser Pro Arg Glu Met Ile Ser Leu Lys Glu Arg Lys Thr Asp Tyr Glu Cys Thr Gly Ser Asn Ala Thr Tyr His Gly Gly Lys Gly Glu His Thr Ser Arg Lys Asp Ala Met Thr Ala Gln Asn Thr Gly I e Ser Thr Leu Tyr Arg Asn Ser Tyr Gly Ala Pro Ala Glu Asp Ile Lys His Asn Gln Val Ser Ala Gln Pro Val Pro Gln Glu Pro Ser Arg Lys Asp Tyr Glu Thr Tyr Gln Pro Phe Gln Asn Ser Thr Arg Asn Tyr Asp Glu Ser Phe Phe Glu Asp Gln Val ~ 980 985 99o His His Arg Pro Pro Ala Ser Glu Tyr Thr Met His Leu Gly Leu Lys Ser Thr lG01yOAsn Tyr Val Asp lPhe Tyr Ser Ala Ala Arg Pro Tyr Ser Glu Leu Asn Tyr Glu Thr Ser His Tyr Pro Ala Ser Pro Asp Ser Trp gTG TGAGGAGCAG GGCACAGGCG CTCCGGGAAA CAGTGCATGT GCATGCATAC 3171 ~ AAATGAATCG CAAGTTAACT TGGAAATCAG TAGAAAGCTA AAGTGATCCT AAATATGACA 3351 SUBSTITUTESHEET(RULE26) AAAACTCGAG

(2~ INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 1040 amino acids (B) TYPE: amino acld (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: protein (xi) SEQVENCE DESCRIPTION: SEQ ID NO:4:
Ser Gln Leu Pro Ala Arg Gly Thr Gln Ala Arg Xaa Thr Gly Gln Ser Phe Ser Gln G y Thr Thr Ser Arg Ala Gly His Leu Ala Gly Pro Glu Pro Ala Pro Pro Pro Pro Pro Xaa Pro Arg Glu Pro Phe Ala Pro Ser Leu Gly Ser Ala Phe His Leu Pro Asp Ala Pro Pro Ala Ala Ala Ala Ala Ala Leu Tyr Tyr Ser Xaa Ser Thr Leu Pro Ala Pro Pro Arg G y Gly Ser Pro Leu Ala Ala Pro Gln Gly Gly Ser Pro Thr Lys Leu Gln Arg Gly Gly Ser Ala Pro Glu Gly Ala Thr Tyr Ala Ala lPlr0o Arg Gly Ser Ser lPlr5o Lys Gln Ser Pro Ser Arg Leu Ala Lys Ser Tyr Ser Thr Ser Ser Pro Ile Asn Ile Val Val Ser Ser Ala Gl~ Leu Ser Pro Ile lA4rg5 Val Thr Ser Pro PrOo Thr Val Gln Ser Thr Ile Ser Ser Ser Pro Ile His Gln Leu Ser Ser Thr Ile Gly Thr Tyr Ala Thr Leu Ser Pro Thr Lys Arg Leu Val His Ala Ser Glu Gln Tyr Ser Lys His Ser Gln Glu Leu Tyr Ala Thr Ala Thr Leu Gln Arg Pro Gly 2S0e5r Leu Ala Ala Gly Ser Arg Ala Ser Tyr Ser Ser Gln His Gly His Leu Gly Pro Glu Leu Arg Ala Leu Gln Ser Pro Glu His His 215e Asp Pro Ile Tyr 2Glou Asp Arg Val Tyr Gln Lys Pro Pro Met Arg Ser Leu Ser Gln Ser Gln Gly Asp Pro Leu Pro Pro Ala His Thr Gly Thr Tyr Arg Thr Ser Thr Ala Pro Ser Ser Pro Gly Val Asp Ser Val Pro Leu Gln Arg Thr Gly Ser Gln His Gly Pro Gl~ Asn Ala Ala Ala Ala Thr Phe Gln Arg Ala S3e0r5 Tyr Ala Ala Gly 3Plro Ala Ser Asn Tyr Ala Asp Pro Tyr Arq Gln Leu Gln Tyr Cys 3P2ro5 Ser Val Glu Ser Pro Tyr Ser Lys Ser Gly Pro Ala Leu Pro Pro Glu Gly Thr Leu A31a5 Arg Ser Pro Ser 350e Asp Ser Ile Gln 3L5y5 Asp Pro Arg Glu Ph-e Gly Trp Arg Asp Pro Glu Leu Pro Glu Va Ile Gln Met Leu Gln His Gln Phe Pro Ser Val Gln Ser Asn Ala Ala Ala Tyr Leu Gln His Leu Cys Phe G 5y Asp Asn Lys Ile L~s Ala Glu Ile Arg 4A0r5g Gln Gly Gly Ile G n Leu Leu Val Asp Leu Leu S~ JTE !iHEET (RULE 26~

-W O 97~27~96 PCT/CA97100051 Asp His Arg Met Thr Glu Val His Arg Ser Ala Cys Gly Ala Leu Arg Asn Leu Va Tyr Gly Ly3 Ala Asn Asp Asp Asn Lys Ile Ala Leu Lys Asn Cys Gly Gly Ile Pro A a Leu Val Arg Leu Leu Arg Lys Thr Thr Asp Leu Glu Ile Arg Glu Leu Val Thr Gly Val Leu Trp Asn Leu Ser Ser Cys Asp Ala Leu Lys Met Pro Ile e Gln Asp Ala Leu A a Val Leu Thr Asn Ala Val Ile Ile Pro H s Ser Gly Trp Glu Asn Ser Pro Leu Gln 5A~5p Asp Arg Lys Ile G n Leu His Ser Ser Gln Val Leu Arg Asn 5A30a Thr Gly Cys Leu 5A3g Asn Val Ser Ser A a Gly Glu Glu Ala 5A4r5g Arg Arg Met Arg 5Glou Cys Asp Gly Leu 5T5h5r Asp Ala Leu Leu 5Ty60r Val Ile Gln Ser A a Leu Gly Ser Ser Glu Ile Asp Ser Lys Thr Val Glu Asn Cys 58aO Cys Ile Leu Arg Asn Leu Ser Tyr Arg Leu Ala Ala Glu Thr Ser Gln Gly Gln His M8t Gly Thr Asp Glu 6Lg5u Asp Gly Leu Leu C61yO Gly Glu Ala Asn G615y Lys Asp Ala Glu 6S2eOr Ser Gly Cys Trp 6 3 0 Y S e r G l n 6A 3 5p G l n T r p A s p G l y V a Gly Pro Leu Pro Asp Cys Ala Glu Pro Pro Lys Gly Ile Gln Met Leu Trp E~is Pro S6e60 Ile Val Lys Pro T66Y5r Leu Thr Leu Leu S67eOr Glu Cys Ser Asn 6P7r5o Asp Thr Leu Glu G61y Ala Ala Gly Ala Leu Gln Asn Leu Ala A690a Gly Ser Trp Lys T69r5p Ser Val Tyr Ile Ar~ Ala Ala Val Arg Lys Glu Lys Gly Leu Pro Ile Leu Val Glu Leu Leu Arg Ile Asp Asn Asp Arg Val Val 7C2ys5 Ala Val Ala Thr Ala Leu Arg Asn Met Ala Leu Asp Val Arg Asn Lys Glu Leu Ile G y Lys Tyr Ala Met Arg ASp Leu Val His 7A5r5g Leu Pro Gly Gly 7A6n Asn Ser Asn Asn Thr Ala Ser Lys Ala M77eOt Ser Asp Asp Thr Val Thr Ala Val Cys Cys Thr Leu His Glu Va Ile Thr Lys Asn Met Glu Asn Ala Lys A a Leu Arg Asp Ala G y Gly Ile Glu Lys Lgu Val Gly Ile Ser Lys Ser Lys Gly Asp Lys His Ser Pro Lys Va Val Lys Ala Ala Ser Gln Val Leu Asn Ser Met Trp Tyr Hi5s Phe Val Ala Ser Ser Ser Thr Ile Glu Arg Asp Arg Gln Arg Pro Tyr Ser Ser Ser Arg Thr Pro Ser Ile Ser Pro Val Arg Val Ser Pro Asn Asn Arg Ser Ala Ser Ala Pro A a Ser Pro Arg Glu Met Ile Ser Leu Lys Glu Arg Lys Thr Asp Tyr Glu Cys Thr Gly Ser Asn Ala Thr Tyr s Gly Gly Lys Gly G u His Thr Ser Arg Lys Asp Ala Met SUcs~ JTE SHEET (RULE 26) Thr A a Gln Asn Thr Gly I e Ser Thr Leu Tyr-Arg Asn Ser Tyr Gly Ala Pro Ala Glu Asp Ile Lys His Asn Gln Val Ser Ala Gln Pro Val Pro Gln Glu Pro 9e65r Arg Lys Asp Tyr Glu Thr Tyr Gln Pro Phe Gln Asn Ser Thr A93r0g Asn Tyr Asp Glu Ser Phe Phe Glu Asp Gln Val ~is His Arg gPgr5o Pro Ala Ser Glu lT0y0r0Thr Met His Leu lG100y5Leu Lys Ser Thr Gly Asn Tyr Val Asp Phe Tyr Ser Ala Ala Arg Pro Tyr Ser Glu Leu Asn Tyr Glu Thr Ser His Tyr Pro Ala Ser Pro Asp Ser Trp Val ~2) INFORMATION FOR SEQ ID NO:5:
(i) SE~'UE;iC- CHARACT:RISTICS:
~) LE GTH: 390 base pairs ) TY E: nucle c acid () 'TXANDEDNES : single .n) ~-O~OLOGY: l_near (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 142..3777 (D) OTHER INFORMATION: /note= "pO071"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Pro Ala Pro Glu Gln Ala Ser Leu Val Glu Glu Gly Gln Pro Gln Thr Arg Gln Glu Ala Ala Ser Thr Gly Pro Gly Met Glu Pro Glu Thr Thr Ala Thr Thr Ile Leu Ala Ser Val Lys Glu Gln Glu Leu Gln Phe Gln Arg Leu Thr Arg Glu Leu Glu Val Glu Arg G n Ile Val Ala Ser G n Leu Glu Arg Cys Arg Leu Gly Ala Glu Ser Pro Ser Ile Ala Ser Thr Ser Ser Thr Glu Lys Ser Phe Pro Trp Arg Ser Thr Asp Val Pro Asn Thr Gly Val Ser Lys Pro Arg V10al5 Ser Asp Ala Val lGln0 Pro Asn Asn Tyr Leu Ile Arg Thr Glu Pro Glu Gln Gly Thr lL2e5u Tyr Ser Pro Glu G30n Thr Ser Leu His G u Ser Glu Gly Ser Leu Gly Asn Ser Arg Ser Ser Thr Gln Met Asn Ser Tyr Ser Asp Ser Gly Tyr Gln Glu A a Gly Ser Phe His Asn Ser Gln Asn Val Ser Lys Ala Asp Asn A7r5g Gln Gln His Ser Phe Ile Gly Ser Thr Asn Asn Hls Val Val Arg A-sn Ser Arg Ala Glu Gly Gln Thr Leu Val Gln Pro S~ 111 UTE SHEET (RULE 26~

Ser Val Ala Asn Arg Ala Met Arg Arg Val Ser Ser Val Pro Ser Arg Ala Gln Ser Pro Ser Tyr Val Ile Ser Thr Gly 2V3aO Ser Pro Ser Arg 2G3$y Ser Leu Arg Thr 2S4eOr Leu Gly Ser Gly 2P4h5e Gly Ser Pro Ser 25aO

Thr Asp Pro Arg Pro Leu Asn Pro Ser A a Tyr Ser Ser Thr Thr Leu Pro Ala Ala 2A7rOg Ala Ala Ser Pro Tyr Ser Gln Arg Pro Ala Ser Pro Thr Ala I e Arg Arg Ile Gly Ser Val Thr Ser Arg Gln Thr Ser Asn Pro 3AOOn Gly Pro Thr Pro 3GlOn Tyr Gln Thr Thr Ala Arg Val Gly Ser Pro Leu Thr Leu Thr Asp Ala Gln Thr Arg Val Ala Ser Pro Ser G n Gly Gln Val Gly 3S3e5r Ser Ser Pro Lys 3ArOg Ser Gly Met Thr A15a Val Pro Gln His 350u Gly Pro Ser Leu Gln Arg Thr Val His ASp Met Glu Gln Phe 3G6y Gln Gln Gln Tyr Asp Ile Tyr Glu Arg Met Val Pro Pro Arg Pro Asp Ser Leu Thr Gly Leu Arg Ser ser 3T9yOr Ala Ser Gln His Ser Gln Leu Gly Gln Asp Leu Arg Ser Ala Val Ser Pro Asp Leu 4H1sO

Ile Thr Pro Ile 4Tly5r Glu Gly Arg Thr 4Tyr Tyr Ser Pro Val Tyr Arg Ser Pro Asn 43s0 Gly Thr Val Glu Leu Gln Gly Ser Gln Thr Ala Leu Tyr Arg 4T4hSr Gly Val Ser Gly 5e Gly Asn Leu Gln Arg Thr Ser Ser Gln Arg Ser Thr Leu Thr T6y5r Gln Arg Asn Asn T47yOr Ala Leu Asn Thr 4T7h5r Ala Thr Tyr Ala 4G81uo Pro Tyr Arg Pro I18e5 Gln Tyr Arg Val 4GlgOn Glu Cys Asn Tyr A95n Arg Leu Gln HiS AlOa Val Pro Ala Asp 5ASop5 Gly ACC ACA AGA TCC CCA TCA IAlTeA AsAp SAer Ile Gln Lys Asp 5p2rOo A g 1707 CC TGG CGT GAT CCT GlAG LTTG CprcOT Glu gal Ile 53i5 Met Le 1755 510n Phe Pro Ser Val G n AlAa AAA5Tn AlCA GCG GCC TAC CTG CAG CAC 1803 SU~ JTE SHEET (RULE 26) W O 97/27296 PCT/CA97/000~1 Gly Ile Lys His Leu Val Asp Leu Leu Asp His Arg Val Leu Glu Val Gln Lys Asn Ala Cys Gly Ala Leu Arg Asn Leu Val Phe Gly Lys Ser Thr Asp Glu Asn Lys Ile Ala Met Lys Asn Val Gly Gly Ile Pro Ala Leu L62eOu Arg Leu Leu Arg Lys Ser Ile Asp Ala Glu Val Arg Glu Leu Val Thr Gly Val Leu Trp Asn Leu Ser Ser Cys Asp Ala Val Lys Met Thr Ile Ile Arg Asp Ala Leu Ser Thr Leu Thr Asn Thr Val Ile Val Pro His Ser Gly Trp Asn Asn Ser Ser Phe Asp Asp Asp His Lys Ile Lys Phe Gln Thr Ser Leu Val Leu Arg Asn Thr Thr Gly Cys Leu Arg Asn Leu Thr Ser Ala Gly G u Glu Ala Arg Lys Gln Met Arg Ser Cys 7Glu5 Gly Leu Val Asp 52r Leu Leu Tyr Val Ile ~is Thr Cys Val Asn Thr Ser Asp Tyr Asp Ser Lys Thr Val Glu Asn Cys Val Cys Thr Leu Arg Asn Leu Ser Tyr Arg Leu Glu Leu Glu Val Pro Gln Ala Arg Leu Leu Gly Leu Asn Glu Leu Asp Asp Leu Leu Gly Lys 7G75u Ser Pro Ser Lys 7As3pO Ser Glu Pro ser 7C8ys Trp Gly Lys Lys 7LgyO Lys Lys Lys Lys Ar3 Thr Pro Gln Glu Asp Gln Trp Asp Gly Val Gly Pro Ile Pro Gly 79 80v 805 810 CTG TCG AAG TCC CCC AAA GGG GTT GAG ATG CTG TGG CAC CCA TCG GTG 26}9 Leu Ser Lys Ser Pro Lys Gly Val Glu Met Leu Trp His Pro Ser Val Val Lys Pro Tyr Leu Thr Leu Leu Ala Glu Ser Ser Asn Pro Ala Thr Leu Glu Gly Ser Ala Gly Ser Leu Gln Asn Leu Ser Ala Ser Asn Trp Lys Phe Ala Ala Tyr Ile Arg Gly Gly Arg Pro 8L7yO Arg Lys Gly Leu Pro Ile Leu Val Glu Leu Leu Arg Met Asp Asn Asp Arg Val Val Ser Ser Gly Ala Thr Ala Leu Arg Asn Met Ala Leu Asp Val Arg Asn Lys Glu Leu Ile G y Lys Tyr Ala Met Arg Asp Leu Val Asn Arg Leu Pro Gly Gly Asn Gly Pro Ser Val Leu Ser Asp Glu Thr Met Ala Ala Ile Cys Cys Ala Leu His Glu Val Thr Ser Lys Asn Met Glu Asn Ala Lys SUBSTITUTE SHEET (RULE 26) W ~ 97127296 PCT/CA97/00051 A a Leu Ala Asp Ser G y Gly Ile Glu Lys Leu Val Asn Ile Thr Lys Gly ~rg Gly Asp Ar~ Ser Ser Leu Lys Val Val Lys Ala Ala Ala Gln 97~ 980 985 ~ GTC TTG AAT ACA TTA TGG CAA TAT CGG GAC CTC CGG AGC ATT TAT AAA 3147Val Leu Asn Thr Leu Trp Gln Tyr Ar~ Asp Leu Arg Ser Ile Tyr Lys Lys Asp Gloy Trp Asn Gln Asn His Phe Ile Thr,Pro Va Ser Thr Leu Glu Arg Asp Arg Phe Lys Ser His Pro Ser Leu Ser Thr Thr Asn Gln Gln Met Ser Pro Ile Ile Gln Ser Val Gly Ser Thr Ser Ser Ser Pro Ala Leu Leu Gly Ile Arg Asp Pro Arg Ser Glu Tyr Asp Arg Thr Gln Pro Pro Met lG17n0Tyr Tyr Asn Ser Gln Gly Asp Ala Thr HLS LYS Gly Le,u Tyr Pro Gly Ser Ser Lys lP0ro0Ser Pro Ile Tyr 10e Ser Ser Tyr Ser Ser Pro Ala Arg Glu Gln Asn Arg Arg Leu Gln His Gln Gln Leu Tllylr5Tyr Ser Gln Asp lA1~0Ser Asn Arg Lys lAls2n5Phe Asp Ala Tyr Allr3g0 Leu Tyr Leu Gln Ser Pro His Ser Tyr Glu Asp Pro Tyr Phe Asp Asp Arg Val Hia lPlh5e0Pro Ala Ser Thr lA15p5Tyr Ser Thr Gln lTlyr60Gly Leu Tyr lAlr~0Ala Glu Gln Tyr lPlr8o5Gly Ser Pro Asp lSlegr0Trp Val Tyr Asp Gln Asp Ala Gln Gln Arg Asn Ser Phe Phe Leu Thr Leu Phe Arg Leu (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 1212 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: protein ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Pro Ala Pro Glu Gln Ala Ser Leu Val Glu Glu Gly Gln Pro Gln Thr Arg Gln Glu Ala Ala Ser Thr Gly Pro Gly Met Glu Pro Glu Thr Thr Ala Thr Thr Ile Leu Ala Ser Val Lys Glu Gln Glu Leu Gln Phe Gln Arg Leu Thr Arg Glu Leu Glu Val Glu Arg Gln Ile Val Ala Ser SU~ 111 ~JTE SHEET (RULE 26~

Gln Leu Glu Arg Cys Arg Leu Gly Ala Glu Ser Pro Ser Ile Ala Ser Thr Ser Ser Thr Glu Lys Ser Phe Pro Trp Arg Ser Thr Asp Val Pro Asn Thr Gly Va Ser Lys Pro Arg Va Ser Asp Ala Val Glr, Pro Asn Asn Tyr Lle5u Ile Arg Thr Glu PrOo Glu Gln Gly Thr Leu Tyr Ser Pro Glu Gln Thr Ser Leu His Glu Ser Glu Gly Ser LeU Gly Asn Ser Arg Ser Ser Thr Gln Met Asn Ser Tyr Ser Asp Ser Gly Tyr Gln Glu Ala Gly Ser Phe His lAs65n Ser Gln Asn Val lS7erO Lys Ala Asp Asn A17r5g Gln Gln His Ser Phe Ile Gly Ser Thr Asn Asn His Val Val Arg Asn Ser Arg Ala G u Gly Gln Thr Leu Va Gln Pro Ser Val A a Asn Arg Ala ~et Arg Arg Val Ser Ser Val Pro Ser Arg Ala Gln Ser Pro Ser Tyr Val Ile Ser Thr Gly Val Ser Pro Ser Arg 2G3y Ser Leu Arg Thr 2S4eOr Leu Gly Ser Gly Phe Gly Ser Pro Ser Val Thr Asp Pro Arg Pro Leu Asn Pro Ser Ala Tyr Ser Ser Thr Th5r Leu Pro Ala Ala 2A7rOg Ala Ala Ser Pro Tyr Ser Gln Arg Pro Ala Ser Pro Thr Ala le Arg Arg Ile Gly Ser Val Thr Ser Arg Gln Thr Ser Asn Pro Asn Gly Pro Thr Pro 3Gln5 Tyr Gln Thr Thr AlOa Arg Val Gly Ser Pro Leu Thr Leu Thr Asp Ala Gln Thr Arg Va Ala Ser Pro Ser G n Gly Gln Val Gly Ser Ser Ser Pro Lys 3A4rOg Ser Gly Met Thr Ala Val Yro Gln His Leu Gly Pro Ser Leu G355n Arg Thr Val His Asp Met Glu Gln Phe Gly Gln Gln Gln Tyr 3A750p Ile Tyr Glu Arg 3Me75 Val Pro Pro Arg P3rOo Asp Ser Leu Thr Gly Leu Arg Ser Ser Tyr Ala Ser Gln His Ser Gln Leu Gly Gln Asp Leu Arg Ser Ala 4VOal5 Ser Pro Asp Leu 4Hilo Ile Thr Pro Ile 4Tlyr Glu Gly Arg Thr 4T2yO Tyr Ser Pro Val Tyr Arg Ser Pro Asn His Gly Thr Val Glu 4Le3u5 Gln Gly Ser Gln Thr Ala Leu Tyr Arg Thr Gly Val Ser Gly I50 Gly Asn Leu Gln A4r5g5 Thr Ser Ser Gln A4r60g Ser Thr Leu Thr T46y5r Gln Arg Asn Asn 4T7yOr Ala Leu Asn Thr 4T7h5r Ala Thr Tyr Ala GloU
Pro Tyr Arg Pro le Gln Tyr Arg Val Gln Glu Cys Asn Tyr Asn Arg Leu Gln His AOa Val Pro Ala Asp 5AOsp5 Gly Thr Thr Arg 51erO Pro Ser Glu Leu Pro Glu Val Ile His ~et Leu Glu His Gln Phe Pro Ser Val Gln Ala Asn Ala Ala A a Tyr Leu Gln His 5L55u Cys Phe Gly Asp 5As50n Lys Val Lys Met G~u Val Cys Arg Leu Gly Gly Ile Lys His Leu Val SUBSTITUTE SHEET (RULE 26) W~97127296 PCT/CA97/00051 Asp Leu Leu Asp His Arg Val Leu G u Val Gln Lys Asn 5AgOa Cys Gly Ala Leu Arg Asn Leu Val Phe Gly Lys Ser Thr Asp Glu Asn Lys Ile ~ Ala Met Lys Asn Val Gly G y Ile Pro Ala Leu L20u Arg Leu Leu Arg 6L2ys Ser Ile Asp Ala Glu Val Arg Glu Leu Va Thr Gly Val Leu Trp Asn Leu Ser Ser Cys Asp Ala Val Lys ~et Thr Ile Ile Arg Asp Ala Leu Ser Thr L u Thr Asn Thr Val e Val Pro His Ser Gly Trp Asn Asn Ser Ser Phe Asp Asp Asp His Lys Ile Lys Phe G68Sn Thr Ser Leu Val Leu Arg Asn Thr Thr Gly Cys Leu Arg Asn Leu Thr Ser Ala Gly Glu Glu Ala Arg Lys G n Met Arg Ser Cys G u Gly Leu Val Asp 72erO
Leu Leu Tyr Val Ile His Thr Cys Val Asn Thr Ser Asp Tyr Asp Ser Lys Thr Val Glu Asn Cys Val Cys Thr Leu Arg Asn Leu S5eOr Tyr Arg Leu Glu Leu Glu Val Pro Gln A a Arg Leu Leu Gly Leu Asn Glu Leu Asp Asp Leu Leu Gly Lys Glu Ser Pro Ser Lys A78sOp Ser Glu Pro Ser C78y5s Trp Gly Lys Lys 7Lys Lys Lys Lys Lys Arg Thr Pro Gln Glu Asp Gln Trp Asp Gly Val Gly Pro Ile Pro Gly Leu Ser Lys Ser 81rSo Lys Gly Val Glu 82eO Leu Trp His Pro Ser Val Val Lys Pro Tyr Leu Thr Ser Leu Gln Asn Leu Ser Ala Ser Asn Trp Lys Phe Ala Ala Tyr Ile Ar65g Gly Gly Arg Pro 8L7yO Arg Lys Gly Leu 8P7ro5 Ile Leu Val Glu 8L8eOu Leu Arg Met Asp Asn Asp Arg Val Val Ser Ser Gly Ala Thr A a Leu Arg Asn Met A a Leu Asp Val Arg Asn Lys Glu Leu Ile Gly Lys Tyr Ala Met Arg Asp Leu Val Asn Arg Leu Pro Gly Gly Asn Gly Pro Ser Val Leu Ser Asp Glu Thr Met Ala Ala Ile Cys Cys Ala Leu His Glu Val Thr Ser Lys Asn Met Glu Asn Ala Lys Ala Leu Ala Asp Ser Gly Gly Ile Glu Lys Leu Val Asn Ile Thr Lys Gly Arg Gly Asp Arg Ser Ser Leu Lys Val Val Lys Ala Ala Ala Gln Val Leu Asn Thr Leu Trp Gln Tyr Aggr5g Asp Leu Arg Ser le Tyr Lys Lys Asp Gly Trp Asn Gln Asn His Phe Ile Thr Pro Val Ser Thr Leu Glu lAOr2gOAsp Arg Phe Lys Ser His Pro Ser Leu Ser Thr Thr Asn Gln Gln Met Ser 2ro Ile e Gln Ser Val Gly Ser Thr Ser Ser Ser Pro Ala Leu Leu Gly Ile Arg Asp Pro Arg lSeO6rOGlu Tyr Asp Arg TlOhr65Gln Pro Pro Me~ GlOn70TYr Tyr SU~ 11 UTE SHEET (RULE 26) Asn Ser lG07n5Gly Asp Ala Thr His8 Lys Gly Leu Tyr Pro Gly Ser Ser Lys Pro Ser Pro Ile Tyr I e Ser Ser Tyr Ser Ser Pro Ala Arg Glu lGlOn5Asn Arg Arg Leu G n His Gln Gln Leu Tyr Tyr Ser Gln Asp Asp Ser Asn Arg Lys Asn Phe Asp Ala Tyr Arg Leu Tyr Leu Gln Ser Pro His Ser Tyr lG14uoAsp Pro Tyr Phe lAslp45Asp Arg Val His lPhe5 Pro Ala Ser Thr lA1~5Tyr Ser Thr Gln Tyr Gly Leu Lys Ser Thr Thr Asn Tyr Val Asp Phe Tyr Ser Thr Lys Arg Pro Ser Tyr Arg Ala Glu Gln Tyr Pro Gly Ser Pro Asp Ser Trp Val Tyr Asp Gln Asp Ala Gln Gln Ar Asn Ser Phe Phe Leu Thr Leu Phe Arg Leu Arg (2) INFORMATION FOR SEQ ID NO:7:
(i) S~QUENC3 CHARACTERISTICS:
..l _E~GTH: 370 base pairs 'T'~ANDnEDNCESS single :\ l'O'OLOGY: linear (ix) FEATURE:
(A) NAME/KEY: misc feature (B) LOCATION: 1..970 (D) OTHER INFORMATION: /note~ "Y2H9"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUE~C CHARACTERISTICS:
..) _E GTH: 264 base pairs ) ~Y E: nucleic acid ) T~ANDEDNESS: single D) ~O?OLOGY: linear (ix) FEAT~RE:
(A) NAME/KEY: misc feature (B~ LOCATION: 1..2~4 ~D) OTHER INFORMATION: /note- "Y2H23b"

S~ JTE SHEET (RULE 26~

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

(2) INFOR~ATION FOR SEQ ID NO:9:
(i) SEQUENC CHARACTERISTICS:
(..) LE GTH: 340 base pairs (.) TY E: nucleic acid (C) STRANDEDNESS: single (~) TO,'OLOGY: linear (ix) FEATURE:
(A) NAME/KEY: misc ~eature (B) LOCATION: 1..3~0 (D) OTHER INFORMATION: /note= "Y2H27"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

(2) INFORMATION FOR SEQ ID NO:10:
(i) S_QUENC]- CHARACTERISTICS:
E GTH: 404 base pairs . TY'E: nucleic acid ~T~ANDEDNESS: single ~ 'O?OLOGY: linear (ix) FEATURE:
(A) NAME/KEY: misc ~eature (B) LOCATION: 1..4~4 (D) OTHER INFORMATION: /note= "Y2H35"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

(2) INFORMATION FOR SEQ ID NO:11:
(i) SE~-~UENCE CHARACTERISTICS:
E GTH: 350 base pairs ~) TY E: nucleic acid TRANDEDNESS: single 'O?OLOGY: linear (ix) FEATURE:
(A) NAME/KEY: misc ~eature (B) LOCATION: 1..3~0 (D) OTHER INFORMATION: /note- "Y2H171"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

SUBSTITUTE SHEET (RULI~ 26) AATTGACCTG CCCG~GAAGA GGCGGGCATG ACACAGCAAG ACGAGAAGAC CCTATGGAGC 300 (2~ INFORMATION FOR SEQ ID NO:12:
(i) S:~UE~C- CHARACTERISTICS:
E GTH: 3S0 base pairs ,) TY'E: nucleic acid r~ TRANDEDNESS: single D) _O'OLOGY: linear (ix) FEATURE:
(A) NA~E/KEY: misc feature (B) LOCATION: 1..3~0 (D) OTHER INFOR~ATION: /note= "Y2H41"
~xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

S~ ~S 111 IJTE SHEET (RULE 26)

Claims (83)

What is claimed is:

1. An isolated nucleic acid comprising a nucleotide sequence encoding at least a presenilin-interacting domain of a presenilin-interacting protein selected from the group consisting of a mammalian S5a (approximately residues 70-377 of SEQ IDNO: 2), GT24 (approximately residues 346-862 of SEQ ID NO: 4), p0071 (approximately residues 509-1022 of SEQ ID NO: 6), Rab11 (SEQ ID NO: 7), retinoid X receptor-.beta. (SEQ ID NO:8), cytoplasmic chaperonin (SEQ ID NO: 9),Y2H35 (SEQ ID NO: 10), Y2H171 (SEQ ID NO: 11), and a Y2H41 (SEQ ID NO:
12) presenilin-interacting domain.

2. An isolated nucleic acid comprising a nucleotide sequence of at least 10consecutive nucleotides selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO:
10, SEQ ID NO: 11, SEQ ID NO: 12, GenBank Accession Numbers F08730, T18858, X81889, X56740, X53143, M84820, X63522, M81766, U17104, X74801, R12984, D55326, and T64843, and a sequence complementary to any of these sequences.

3. An isolated nucleic acid as in claim 2 comprising a nucleotide sequence of at least 15 consecutive nucleotides selected from said group.

4. An isolated nucleic acid as in claim 2 comprising a nucleotide sequence of at least 20 consecutive nucleotides selected from said group.

5. An isolated nucleic acid comprising a nucleotide sequence encoding an antigenic determinant of a presenilin-interacting protein selected from the group consisting of a mammalian S5a, GT24, p0071, Rab11, retinoid X receptor-.beta., cytoplasmic chaperonin, Y2H35, Y2H171, and Y2H41 protein.

6. A method for identifying allelic variants or heterospecific homologues of a human presenilin-interacting protein gene comprising choosing a nucleic acid probe or primer capable of hybridizing to a human presenilin-interacting protein gene sequence under stringent hybridization conditions;
mixing said probe or primer with a sample of nucleic acids which may contain a nucleic acid corresponding to said variant or homologue;
detecting hybridization of said probe or primer to said nucleic acid corresponding to said variant or homologue.

7. A method as in claim 6 wherein said sample comprises a sample of nucleicacids selected from the group consisting of human genomic DNA, human mRNA, and human cDNA.

8. A method as in claim 6 wherein said sample comprises a sample of nucleicacids selected from the group consisting of mammalian genomic DNA, mammalian mRNA, and mammalian cDNA.

9. A method as in claim 6 wherein said sample comprises a sample of nucleicacids selected from the group consisting of invertebrate genomic DNA, invertebrate mRNA, and invertebrate cDNA.

10. A method as in claim 6 further comprising the step of isolating said nucleic acid corresponding to said variant or homologue.

11. A method as in claim 6 wherein said nucleic acid is identified by hybridization.

12. A method as in claim 6 wherein said nucleic acid is identified by PCR
amplification.

13. A method for identifying allelic variants or heterospecific homologues of a human presenilin-interacting protein gene comprising choosing an antibody capable of selectively binding to a human presenilin-interacting protein;
mixing said antibody with a sample of proteins which may contain a protein corresponding to said variant or homologue;
detecting binding of said antibody to said protein corresponding to said variant or homologue.

14. A method as in claim 13 wherein said sample comprises a sample of proteins selected from the group consisting of human proteins, human fusion proteins, and proteolytic fragments thereof.

15. A method as in claim 13 wherein said sample comprises a sample of proteins selected from the group consisting of mammalian proteins, mammalian fusion proteins, and proteolytic fragments thereof.

16. A method as in claim 13 wherein said sample comprises a sample of proteins selected from the group consisting of invertebrate proteins, invertebrate fusion proteins, and proteolytic fragments thereof.

17. A method as in claim 13 further comprising the step of substantially purifying said protein corresponding to said variant or homologue.

18. An isolated nucleic acid comprising an allelic variant or a heterospecific homologue of a human presenilin-interacting protein gene.

19. An isolated nucleic acid encoding an allelic variant or heterospecific homologue of a human presenilin-interacting protein.

20. An isolated nucleic acid comprising a recombinant vector including a nucleotide sequence of any one of claims 1-19.

21. An isolated nucleic acid as in claim 20 wherein said vector is an expression vector and said presenilin-interacting protein nucleotide sequence isoperably joined to a regulatory region.

22. An isolated nucleic acid as in claim 21 wherein said expression vector may express said presenilin-interacting protein sequence in mammalian cells.

23. An isolated nucleic acid as in claim 22 wherein said cells are selected from the group consisting of fibroblast, liver, kidney, spleen, bone marrow and neurological cells.

24. An isolated nucleic acid as in claim 21 wherein said vector is selected from the group consisting of vaccinia virus, adenovirus, retrovirus, neurotropic viruses and Herpes simplex.

25. An isolated nucleic acid as in claim 21 wherein said expression vector encodes at least a presenilin-interacting domain of a presenilin-interacting protein selected from the group consisting of a mammalian S5a, GT24, p0071, Rab11, retinoid X receptor-.beta., cytoplasmic chaperonin, Y2H35, Y2H171, and Y2H41 protein.

26. An isolated nucleic acid as in claim 21 wherein said vector further comprises sequences encoding an exogenous protein operably joined to said presenilin-interacting protein sequence and whereby said vector encodes a presenilin-interacting protein fusion protein.

27. An isolated nucleic acid as in claim 26 wherein said exogenous protein is selected from the group consisting of lacZ, trpE, maltose-binding protein, a poly-His tag, glutathione-S-transferase, a GAL4-DNA binding domain, and a GAL4 activationdomain.

28. An isolated nucleic acid comprising a recombinant expression vector including nucleotide sequences corresponding to an endogenous regulatory region of a presenilin-interacting protein gene.

29. An isolated nucleic acid as in claim 28 wherein said endogenous regulatory region is operably joined to a marker gene.

30. A host cell transformed with an expression vector of any one of claims 20-29, or a descendant thereof.

31. A host cell as in claim 30 wherein said host cell is selected from the group consisting of bacterial cells and yeast cells.

32. A host cell as in claim 30 wherein said host cell is selected from the group consisting of fetal cells, embryonic stem cells, zygotes, gametes, and germ line cells.

33. A host cell as in claim 30 wherein said cell is selected from the group consisting of fibroblast, liver, kidney, spleen, bone marrow and neurological cells.

34. A host cell as in claim 30 wherein said cell is an invertebrate cell.

35. A non-human animal model for Alzheimer's Disease, wherein a genome of said animal, or an ancestor thereof, has been modified by at least one recombinant construct, and wherein said recombinant construct has introduced a modification selected from the group consisting of (1) insertion of nucleotide sequences encoding at least a functional domain of a heterospecific normal presenilin-interacting protein, (2) insertion of nucleotide sequences encoding at least a functional domain of aheterospecific mutant presenilin-interacting protein, (3) insertion of nucleotide sequences encoding at least a functional domain of a conspecific homologue of a heterospecific mutant presenilin-interacting protein, and (4) inactivation of anendogenous presenilin-interacting protein gene.

36. An animal as in claim 35 wherein said modification is insertion of a nucleotide sequence encoding at least a functional domain of a normal human presenilin-interacting protein selected from the group consisting of a mammalian S5a, GT24, p0071, Rab11, retinoid X receptor-.beta., cytoplasmic chaperonin, Y2H35, Y2H171, and Y2H41 protein.

37. An animal as in claim 35 wherein said modification is insertion of a nucleotide sequence encoding at least a functional domain of a mutant human presenilin-interacting protein selected from the group consisting of a mammalian S5a, GT24, p0071, Rab11, retinoid X receptor-.beta., cytoplasmic chaperonin, Y2H35, Y2H171, and Y2H41 protein.

38. An aninal as in claim 35 wherein said animal is selected from the group consisting of rats, mice, hamsters, guinea pigs, rabbits, dogs, cats, goats, sheep, pigs, and non-human primates.

39. An animal as in claim 35 wherein said animal is an invertebrate.

40. A method for producing at least a functional domain of a presenilin-interacting protein comprising culturing a host cell of any of claims 30-34 under suitable conditions to produce said presenilin by expressing said nucleic acid.

41. A substantially pure preparation of a protein selected from the group consisting of a mammalian S5a, GT24, p0071, Rab11, retinoid X receptor-.beta., cytoplasmic chaperonin, Y2H35, Y2H171, and Y2H41 protein.

42. A substantially pure preparation of a polypeptide comprising an amino acid sequence of at least 10 consecutive amino acid residues selected from the group consisting SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and GenBank Accession Numbers F08730, T18858, X81889, X56740, X53143, M84820, X63522, M81766, U17104, X74801, R12984, D55326, and T64843.

43. A substantially pure preparation of a polypeptide as in claim 42 comprising an amino acid sequence of at least 15 consecutive amino acid residues selected from said group.

44. A substantially pure preparation of a polypeptide comprising at least a presenilin-interacting domain of a presenilin-interacting protein selected from the group consisting of a mammalian S5a, GT24, p0071, Rab11, retinoid X receptor-.beta., cytoplasmic chaperonin, Y2H35, Y2H171, and Y2H41 protein.

45. A substantially pure preparation of a polypeptide comprising an antigenic determinant of a presenilin-interacting protein selected from the group consisting of a mammalian S5a, GT24, p0071, Rab11, retinoid X receptor-.beta., cytoplasmic chaperonin, Y2H35, Y2H171, and Y2H41 protein.

46. A method of producing antibodies which selectively bind to a presenilin-interacting protein comprising the steps of administering an immunogenically effective amount of a presenilin-interacting protein immunogen to an animal, allowing said animal to produce antibodies to said immunogen; and obtaining said antibodies from said animal or from a cell culture derived therefrom.

47. A substantially pure preparation of an antibody which selectively binds to an antigenic determinant of a presenilin-interacting protein selected from the group consisting of a mammalian S5a, GT24, p0071, Rab11, retinoid X receptor-.beta., cytoplasmic chaperonin, Y2H35, Y2H171, and Y2H41 protein.

48. A substantially pure preparation of an antibody as in claim 47 wherein said antibody selectively binds to an antigenic determinant of a mutant presenilin-interacting protein and fails to bind to a normal presenilin-interacting protein.

49. A cell line producing an antibody of any one of claims 47-48.

50. A method for identifying compounds which can modulate the expression of a presenilin-interacting protein gene comprising contacting a cell with a test candidate wherein said cell includes a regulatory region of a presenilin-interacting protein gene operably joined to a coding region; and detecting a change in expression of said coding region.

51. A method as in claim 50 wherein said change comprises a change in a level of an mRNA transcript encoded by said coding region.

52. A method as in claim 50 wherein said change comprises a change in a level of a protein encoded by said coding region.

53. A method as in claim 50 wherein said change is a result of an activity of a protein encoded by said coding region.

54. A method as in claim 50 wherein said coding region encodes a marker protein selected from the group consisting of .beta.-galactosidase, alkaline phosphatase, green fluorescent protein, and luciferase.

55. A method for identifying compounds which can selectively bind to a presenilin-interacting protein comprising the steps of providing a preparation including at least one presenilin-interacting protein component;
contacting said preparation with a sample including at least one candidate compound; and detecting binding of said presenilin-interacting protein component to said candidate compound.

56. The method in 55 wherein said binding to said presenilin-interacting component is detected by an assay selected from the group consisting of: affinity chromatography, co-immunoprecipitation, a Biomolecular Interaction Assay, and a yeast two-hybrid system.

57. A method of identifying compounds which can modulate activity of a presenilin-interacting protein comprising the steps of providing a cell expressing a normal or mutant presenilin-interacting protein gene;
contacting said cell with at least one candidate compound; and detecting a change in a marker of said activity.

58. A method as in claim 57 wherein measurement of said marker indicates a difference between cells bearing an expressed mutant presenilin-interacting protein gene and otherwise identical cells free of an expressed mutant presenilin-interacting protein gene.

59. A method as in claim 57 wherein said change comprises a change in a non-specific marker of cell physiology selected from the group consisting of pH;intracellular Ca++, Na+, or K+; cyclic AMP levels; GTP/GDP ratios;
phosphatidylinositol activity; and protein phosphorylation.

60. A method as in claim 57 wherein said change comprises a change in expression of said presenilin-interacting protein.

61. A method as in claim 57 wherein said change comprises a change in intracellular concentration or flux of an ion selected from the group consisting of Ca2+, Na+ and K+.

62. A method as in claim 57 wherein said change comprises a change in occurrence or rate of apoptosis or cell death.

63. A method as in claim 57 wherein said change comprises a change in production of A.beta. peptides.

64. A method as in claim 57 wherein said change comprises a change in phosphorylation of at least one microtubule associated protein.

65. A method as in claim 57 wherein said cell is a cell cultured in vitro.

66. A method as in claim 65 wherein said cell is a transformed host cell of any one of claims 30-34.

67. A method as in claim 65 wherein said cell is explanted from a host bearing at least one mutant presenilin-interacting protein gene.

68. A method as in claim 65 wherein said cell is explanted from a transgenicanimal of any one of claims 35-39.

69. A method as in claim 57 wherein said cell is a cell in a live animal.

70. A method as in claim 69 wherein said cell is a cell of a transgenic animal of any one of claims 35-39.

71. A method as in claim 57 wherein said cell is in a human subject in a clinical trial.

72. A diagnostic method for determining if a subject bears a mutant presenilin-interacting protein gene comprising the steps of providing a biological sample of said subject;
detecting in said sample a mutant presenilin-interacting protein nucleic acid, a mutant presenilin-interacting protein, or a mutant presenilin-interacting protein activity.

73. A method as in claim 72, wherein a mutant presenilin-interacting proteinnucleic acid is detected by an assay selected from the group consisting of direct nucleotide sequencing, probe specific hybridization, restriction enzyme digest and mapping, PCR mapping, ligase-mediated PCR detection, RNase protection, electrophoretic mobility shift detection, and chemical mismatch cleavage.

74. A method as in claim 72, wherein a mutant presenilin-interacting protein is detected by an assay selected from the group consisting of an immunoassay, a protease assay, and an electrophoretic mobility assay.

75. A pharmaceutical preparation comprising a substantially pure presenilin-interacting protein and a pharmaceutically acceptable carrier.

76. A pharmaceutical preparation comprising an expression vector operably encoding a presenilin-interacting protein, wherein said expression vector may express said presenilin-interacting protein in a human subject, and a pharmaceutically acceptable carrier.

77. A pharmaceutical preparation comprising an expression vector operably encoding a presenilin-interacting protein antisense sequence, wherein said expression vector may express said presenilin-interacting protein antisense sequence in a human subject, and a pharmaceutically acceptable carrier.

78. A pharmaceutical preparation comprising a substantially pure antibody, wherein said antibody selectively binds to a mutant presenilin-interacting protein, and a pharmaceutically acceptable carrier.

79. A pharmaceutical preparation as in claim 78 wherein said preparation is essentially free of an antibody which selectively binds a normal presenilin-interacting protein.

80. A pharmaceutical preparation comprising a substantially pure preparationof an antigenic determinant of a mutant presenilin-interacting protein.

81. A pharmaceutical preparation as in claim 80 wherein said preparation is essentially free of an antigenic determinant of a normal presenilin-interacting protein.

82. A method of treatment for a patient bearing a mutant presenilin-interacting protein gene comprising the step of administering to said patient a therapeutically effective amount of the pharmaceutical preparation of any one of claims 75-81.

83. A method as in claim 82, wherein said pharmaceutical preparation is targeted to a cell type is selected from the group consisting of heart, brain, lung, liver, skeletal muscle, kidney, pancreas and neurological cells.