CA2424577A1 - Use of huntingtin and related compounds for modulating cell survival - Google Patents

Abstract

The invention provides methods and reagents for modulating cell death and cell proliferation, More specifically, the invention provides huntingtin proteins having a selected phosphorylation state and a biologically-active fragments and variants thereof for modulating cell survival.

Description

USE OF NUNTINC:TIN AND RELATED (.'OMPOUNDS
FOR MODULATING C'EI.1. SURVIVAL
FIELE) OF THE INVENTION
The invention is, in general, in the field ofcell growth regulation. More specifically, the invention provides methods and reagents for modulating cell death and cell proliferation.
BA('KGRt>ll'xIDOi~'!1'HE INVENTION
('ell growth is intimately asst>ciated with cell death, anal cells can vary between excess proliferation, normal proliferation, steady state, normal cell senescence and abnormal cell death (FIG. 1 ). Diseases and di~;orders often result from a perturbation in the balance between cell growth and death Thus, cancers may be caused by excessive cell growth, out of proportion to cell deatlo, resulting ~n rapid prolifi;ration. Bv contrast, excessive cell death, out of proportion to cell regeneration., can result in the destruction of crucial areas of tissue as observed iu degenerative diseases inch as Yarkinscm's Disease. Amyotrophic Lateral Sclerosis. fllzheirner's disease, or I-luntin~~ton~s Disease (III)).
IID is a devastating neurodeg~ner~rtive disease that usually presents in mid-adult life, affects approximately 1 in 10,(~OU inc;ividuals, and results in psychiatric disturbance, involuntary movement disorder, and ~;,ognitive decline associated with inexorable progression to death, typically t 7 ye;asrs t<>llowin~ onset. The lesion in HD
has been traced to the expansion of a C'AG trinucieotidc repeat in the first exon ol~the HD gene, ITIS, located on chromosome 4 (4p l6. 3) (Hr.rntin~=ton Disease C'ollaborativc Research Group, 19')3).
Alleles containing expansions ~~fgreoter than 35 C"AO repeats are associated with the clinical pl~eno~ype of I-1D, with an earlier age of onset occurring with higher (.~'AG
repeat sizes (i~ndrcw et al. ( 1993) Nat. CJenet. 4, ;'-)8-403).
The mutation in the HD gene ~-osults in a protein, huntingtin (htt), with an expanded polyglutamine tract. Htt is a large pr~~tein of uncertain function that is ubiquitously expressed w many tissues ofthe bod,~, hut which has the highest levels in brain and testis (Sharp and Ross, ( 1996) Neurclbiol. I )is. >, 3-15). Proteolytic cleavage of htt, possibly by caspases, produces N-terminal htt fragments containing the expanded polyglutamine tract (Goldherg ct al. ( 1 ~)9C~) Nat. Genet. ~ ?'~, 442-449 Wellington et al. ( 1998) .I. Biol. Chem.
27 3, <) l 58-'.rl (~7; Wellington et al. (2n00) .l. Biol. C'hem. 275, t 0831-( 9838). N-terminal fragments of mutant expanded htt haze altered cellular interactions ( Li et al. ( 1995) Nat.
Genet. 25, 385-389; Burke et al. t 19~)O) Hart. Med. '?, 347-:349; Bao et al.
( 199(>) Pro~~. Nat.
Acid. Sci. (JSA 93, 5037-5042: blanker et al. ( 1996) Hum. Mul. Genet. 6, 487-495;
Kalchm~rr~ et al. ( 19961 .1. Biol. C"hem. 2 i"1, 19385-19394; Kalchman et al.
(1997) Nat.
Genet. I U, =14-53), nuclear localisation (Davies et nl. ( 1997 ) Cell 90, S
37-548; DiFi~lia et al.
I 9970 Science 277, 1990-199.;; Beclrc:r et al. ( i 998) Neurobiol. Dis. 4, 387-397; Hackam et trl. ( 1998) ('ell. Biol. 141, 109;'-1 1054 Schillin g et al. ( 1999) Hum.
Mos. Genet. 8, 397-407;
Hodgson et al. ( 1999) Neuron ?3, 18 . -192; Gutekunst et al. ( 1 f)99) ,1.
Neurosci. 19, 2522-x534; Wheeler et al. (2000) Hum. Mos. Genet. 9, 503-513; l.i et al. (2000) Nat. Genet. 25, 335-389), and are directly toxic to nc~rronal cells in a variety of in vitro model systems ( Martindale et al. ( 1998) Nat. (tenet. 18, I 5()-154; Sandou et al. ( 1998) Cell 95, 55-(iEi;
Hackam et al. (1998) ('ell. Biol. 14'1, 1097-1 1(151. These htt fragments are also prone to intracellular aggregation and inclusion for7nation (Hackam et al. ( 1998) Cell. Biol; 141, I tO)?- ; 1 ()5; Martindalc: et al. ( I 998 ) "tat. (::ienet. 18, 150-154;
Wang et al. ( 1999) 'Veuroreport. 10, 2435-2438, (:"ooper et al. (1998) I-sum. Mol Genet. 7, 783-790), altLrough tl7e relevance uf'htt ag,~re~;ation to th~.~ pathogenesis of f-iD remains unclear (reviewed in Sisoda ( 1998) (.dell 95,1-4). TI-re expansion of polyglutamine residues in htt has been proposed to result in a novel toxic; )a;n ol' function of the rmutant protein (MacDonald and (:ruselia ( 1996) C~'urr. Opin. Nee-rrubiol. (i, 638-643). Htt h<rs also been implicated in hacmatolooiesis (Metzlcr et al. (2000y Hum Mul l~enet 9, 387-~)4), and a lower incidence of cancer ~rmon~ patients with H1:), which appears to be related to intrinsic biologic factors, has been ol7scrved (Sorensen et al. ( 199~r) Carrrcer 8(i, 1342-~l 34(i) Wild type htt leas anti-apoptutic properties and has bc-en im~~licated as a cancer promoting protein (PCT WO
()1;79''8 3).
lice homozygous for targeted disruption of Hc:lh (-/-), the marine homologue of the EvD gene, clie at embryonic day 7.5 (Nasir et al. (1995) Cell 81, 811-823;
Duyao et al. (1995) Science 269, 4t>7-41(>; ?eitlin et al. ( 1995) Nat. (uenet. 1 1, 155-162).
Mice with decreased levels ofhtl following targeted insert non of a neo construct into the Hdh gene have ~uberrant brain development ar2d perinatal leth..rlity ( White et ai. ( I 997) 'Vat.
Genes. 17, 404-410).
Mice Ireterozvgorrs for targeted disruption of the I~dln ~;en~ ( +i-) express half the normal levels ofendogcnous htt and develop neuronal degeneration in the basal ganglia in adulthood ((J'K~rsky et al. ( 1999) Brain. Ices. 818, 4(i8-479).

Mice containing yeast 4trti6ci:d chromosome (YA(') transgenic for the entire ~enotT~ic region of the human 11D gene, including all its regulatory sequences (Hod~;son et al. ( 1999 ) Neuron 23, I 81 ~-192 ), have peen generated to study the phenotypic effect; of varying endogenous htt levels ~m tnicc expressing transgenic htt. 'The appropriate expression of human lttt Burin" development itt 1'A(' trrtnsgen~c mice was demonstrated by the ability of tic human transgenc in rescue the embryonic lethality of Hdh nullizygous mice (-i-) (llodgson et al. ( l99(i) Hum. Mc3(. Crenet. 5, 1875--185). In addition, YAC transgenic mice that express human l7tt mth 18 j~oly~;lutamines (YA('18) corresponding to a GAG
repeat length observed in t.tnaflected persons, 46 polyglutamines (YAC'46) corresponding to a (. AC! repeat length observed in adult-onset HD patients, and 72 polyglutamines (~'AC72) corresponding to a repeat length caus.ingjuvet~ile-onset HD (Hodgson et al.
(1999) Neuron 23, 1 ~ 1-192) express similar levels of~transgenic human htt differing only in polygltttatnine expansion length. Y'.A(: 18 mice have do observable phenotype up to 24 months of age, indicating that human htt with a holyglutamine tract of not~rna) length is not pathogenic in mice. However, mice traosgenic for mutant htt with an expanded polyglutamine develop a progressive phenotype characterised by behavior atl, cellular and neuropathologic abnormalities similar to those observn~d in HD (Elodgson ct al. ( 1999) Neuron 23, lEl-192).
(.given the devastating effects of conditions assc7ciated with aberrant cell growth regulation, in diseases and discjrders ;~xernplificd by cancer or 11D, it would be useful to have reagents and methods for prop noting or inhibiting cell survival SI1MMARY OF'fHE INVENTI(>N
In various alternative aspects. t:he invention provides methods and reagents for modulating cell survival. In one aspect, the invention provides the use of an exogenous huntingtin protein having a selected lhosphorylation state or a biologically-active fragment or variant thereof for modulating cell survival, where the huntingtin protein may be phospl~orylated huntingtin protein, unphosphoryl~tted huntington protein, or constitutively phosphorylated huntingtin protein. to an alternative aspect, the invention provides the use of an exogcnr>us huntingtin protein havs~~g a selected phosphorylation state or a biologically-active fragment or variant thereof for lrcatment or pro phylaxis of a cell degenerative disease, where the l~untingtin protein is phospllorylated huntingtin protein or a constitutively phosphorylated huntingtin protein. In an alternative aspect, the invention provides the use of an exogenous huntingtin protein having a selected phosphoryl~ttion state or a biologically-active fragment or variant thereof for treatment or prophylaxis of a cell proliferation disease, where the huntingtin protein is unph«sphorylated huntingtin protein. In some embodiments, the invention provides for the modul;rtion of call death or apoptosis, or of cell proliferation.
In alternative embodinnents, toe invention provides for the use of a nucleic a~~id nnoleculc encoding a huntingtin protein or a biologically-active li-agment thereof, or for the use of a nucleic acid nrolcculc complementary to a nucleic aci<l encoding a huntingtin protein or a biologically-active fragment thereof. In alternative embodiments, the invention provides for use of an antibody that specifically hinds :z huntingtin protein or a biologically-active fragment thereon.
In alternative embodiments, tine cell degenerative disease may be an apoptotic disease. a neurodegenerative disease. Huntington's disuse, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis;, multiple sclerosis, polyr;lutamine diseases, spinocercbellar ataxias, autosornal dcpminant cerebellar ataxia with retinal degeneration, spinobulbar muscular atrophy (SBM ~), dentatorubralpallidoluysian atrophy (DRPL.A), Machado-.loseph disease, stroke, epi'iepsy, spinal cord injury, physical trauma, or retinal degeneration.
In alternative embodirrrents, the cell prolilerative disease may be cancer, testicular cancers, embryonic cancers, leukemiars, lraematopoietic diseases, psoriasis, atherosclerosis, inflammatory diseases, or derrnatolo,~ical diseases.
In alternative embodiments oi~various asloects of the invention, the variant rrray be an agonist of a huntingtin protein or an :u~tagonist ol~a huntingtin protein. 1n alternative embodiments, the huntingtin protein may he full-lenf;th huntingtin protein;
wild-type huntingtin hroteio; or mutant huntinytin protein; ~u~d/or be phosphorylated on serine=421, tlrreonine 1024, or threonine 2t)68 0l the: human huntingtin protein (SEQ ID
NO: 1 ).
Irr alternative aspects, the invention provides a method of assaying a test cc»-npound, by providing a svstenr including a hlrntingtin protein or biologically-active fragment, contacting the system with the test c~ympound, anti determining whether the test compound modulates the phosphorylatiors of thu huntingtin protein or fra;~ment. In alternative emboclinreats, the assaying is clone in virr-n and further includes providing a kinase, for examlolc AK~I , capable ol'phosphorylating the huntingtin protein or biologically-active ti~agn,ent.
In alternative aspects, the invention provides a method of assaying a test compound, by providing a system including an exogenous huntin~,tin protein having a selected phosphorylation state or biologically-<~ctive fragn7ent, where the huntingtin protein may be phosphorylated huntingtin protein, us7phosphorylated huntingtin protein, or constitutively phosphorylated huntingtin protein, c~.>ntacting the system with the test compound, and determining whether the test compound modulates a function ofthe huntingtin protein or fragment, where the function rnay be apoptosis inhibition. aggregation inhibition, o~~ cell proliferation.
In altci-native aspects, the invention provides the use of an exogenous huntin.gtin protein having a selected phosphorylation state or a biologically-active fragment or variant for ameliorating the cytotoxie effects of a therapeutic con7pound, for example, a pro-apoptotic compound such as tamoxi I'en, by co-administering it with the therapeutic compound.
"C:ell survival" refers to a reduction or decrease in cell death, or a pron7otion or increase in cell proliferation. ''Cell death" or "apoptosis.'~ defines a specific execution of programmed cell death that call be try;gered by several factors (Kramn7er et al. (1991 ) "Apoptosis in the AP()-1 Systcn7", ~lpoptosis: ~ he Mcilecular Basis of ('ell Death, pp. 87-99 (Fold Spring I larbor Laboratory 1'res:;). C)i7e of the I~rctors triggering apoptosis is loss of cell anchorage (Meredith, .l. I?., Jr.. B. IarLeli, of al. ( 1')93). ''1'l7e extracellular matrix as a cell survival factor." Molecular Biology {~f the ("ell. ~1(9): 953-61 ). a phenomenon known as "anoikis-' ~ lirisch, ~. M. and H. l'ranG;is I 1994'1. "Disruption of epithelial cell-matrix interactions induces apoptosis." .lournal of Cell I3iolc>gy. 124(~l ): 619-?6). "Cell proliferation- refers to excessive or tAberrant cell browth. W'hcn the normal function of cell survival go awry, the cause or the result can be cell degenerative or cell proliferativ~~
diseases, including cancer, viral infe~aions, autoimmune diseaselallergies, cardiovascular cllS(:~ISeS, Ilerlr'Odege17e1'at1017, etC.
"Modulating," or wmodulates~ means changing, by either increase or decrease.
~S "huntingtin'" or "htt' protejt7 or polypeptide is a protein, the mutant form of which has been shown to be associated with l-Juntington's disease (Hl)). While the invention encontpassas l7tt from various species. such as n7ouse, rat. 1)rc~.sr~~lrilcr, (". ele~~czrrs, etc., an exemplary htt protein from humans lias ait amino a.tcid seduenc:e (SEQ ID NO:
1), listed on (JcnBanl: as Accession Number NP ()02102, and is encoded by a nucleic acid sequence (SIQ II) NO: ?), listed on C:renl3ank as ;~ceession Number NM 0021 1 I . The "C.' terminus"
of htt rnclicatcs, generally, the half o~ 1:1e wild-type or mutant ht1 protein that includes the C-terminus, and possesses biological a~aivity as described herein. The term is used b synonymously with C-terminal fragment or C-terminal domain. C-terminal fragments of htt also include amino acids 58S-x144 oi~Sl:(;> ID NO: l, and any biologically-active fragments thereof. flue "N Lel'I11111uS'~ or °W-terwin~tl fragment" indicates.
generally, the half of"the wild-type htt protein that includes th~; N-tcrrninus arid any biologically-active fragrraents thereof. A "full-length"" lttt protein leas tltc entire amino acid sequence of a htt protein that is naturally found irt an organism of thu species that normally expresses that htt protein. For example, a full length human Irtt pro~.ein nnay have tl7e amino acid sequence of SEQ ID NO:
1. !1 "wild type"' htt protein possesses, ~:enerally. a polyglutamine tract of less than 35 glutantinesw and is not associated with the development of l~f~ A "mutant" htt protein possesses, generally, a polyglutamin~.: tract of rttorc than 35 glutamines, and is associated with the development of HD of varying severity, depending on the length of the polyglutamine tract. A htt protein ac-cording to the invention rnay have a sequence substantially identical to drat of SEQ ID 'NO: I .
.=1 "phosphorylated"' htt. protein is post-translationally modified on any amino acid residue catpable of being phosl:nhoryluted in viva. A htt protein hats a "selected phosphorylation state"' if the pi~osph«ry~lation state oi'that protein is determinable. In alternative embodiments, a lltt protein may have a selected phosphorylation state in that it is phosphorylated at higher, same, or lc~w~n levels than a wild type htt protein.
In alternative embodiments. a htt protein may also have a selected phosphorylation state in that it is unphosphorylatecl or constitutively phospltorylated. 1'ltosphorylated htt proteins may be phosphorylated, for example, ern serine 4'? 1, threonine 1()?4, or threonine 2068. An "unphosphctrv~latcd"" hit protein may he incapable of being phc>sphorylated on an amino acid residue capable of being phosl,horyl<~ted i~t vivo, for example, by mutation of that residue to an amino acid that is not capable of being phosphorylated. A mutation of a serine to an alanine in a polyloeptide sequence, tear exa.l.mple, results in a pr~>tcin that is not capable of being pltosphorylatcd at that p,.rrticulx~r position in the polypeptide sequence. A htt ~~rotein that possesses an alanine at position -t21 c>f SEQ lD NO: 1 instead of~a serine is such an "unphosphorylated" htt proteilt. An ~.mpltusphorvlatcd htt promin may also be a protein that is capable of being phosphorylated irr ~~ir=a, but is not phosphorvlated due to, for ex~.mple, the presencr of an inhibitor, for example, a kiltasc inhibitor; due to an antibody that interferes with the lohosphorylation site; or due to the activity of~at phospltatase. A
constitutive°ly phosphorylated"' htt protein is a protein that possesses a mutation at an amino acid residue that is capable of being plnospltorylated in viva, where the mutation mimics phosphorylation at that residue, and the resultant polypeptide possesses the biological activity of a phosphorylated polypeptide. ~' ener.rlly. mutation of a phosphorylatable residue to a glutamic acid or aspartic acid residuL results in constitutive pltosphorylation. A
constitutivcly phosphorylated htt prc3tcin may also be phosphorylated by a kinase.
1 "biologically-active fragment'' of a htt protein includes an amino acid sec;uence found in a naturally-occurring htt pr~.~tein that is capable of modulating apoptosis or cell deati~, cell proliferation, or proucin a~;gre~;ation, iur c;xample, aggregation caused bw the expansion of a polyglutamine tract, os described herein or known to those of ordinary skill in the art. A ''variant'' of a htt protein includes a modification, for example.
by deletion, addition, or substitution, of an amin~a acid sequence found in a naturally-occurring '~~tt protein that is capable o1'modulating apoptosis c»-cell death, cell proliferation, or protein aggregation, as described herein or known to those of ordinary skill in the art.
,~ ..protein,"' "peptide'' or "p«lypeptidc'' is any chain of two or more amino acids, including naturally occurring or non-naturally occurring amino acids or amino acid analogues, regardless of post-cranslalional moditication (e.g., glycosylation or phoshhorvlation). ,An "amino acid si~qucnce'", ''polypeptidc", .'peptide" or "protein'' of the invention may include peptides or proteins that have abnormal linkages, cross links and end caps, non-peptidvl bonds or alternatme modifying groups. Such modified peptides are also within Ilte scope of the invention. Tale term "modifying group" is intended to include structures that are directly attached to the peptidic structure (e.~;., by covalent coupling), as well as those that are indirectly attached to the peptidic struct~.tre (e.g., by a stable non-c;ovalent association or by covalent ~:oupling to additional amino acid residues, or rnimetics, analogues or derivatives hereof, which stay flank tl~e core peptidic structure). For example, the modifying group can be coupled to the amino-terminus or carboxy-terminus of a peptidic structure, <tr to a peptidic or poptidon~imetic region flanking the core domain. Alternatively, the modifying group can he coupled to a side chain of at least one amino acid residue of a peptidic structure, or to a heptidic or peptide- rmimetic region Ranking the core domain (e.g., through the epsilon amino group of a lysyl residue(s), through the carboxyl group of an asparuc acid residtte(s) or a glutami<v acid residue(s), through a hydroxy group of a tyrosyl residue(s), a serine residues) or a threonine residues) or other suitable reactive group 011 all amino acid side chain). Modilying ;groups covalcntly coupled to the peptidic structi_tre can be attached by means and using methods well known is the art for linking chemical structures, including, for example, amide, alkyiamino, c,:u-btunate or urea herds. A
protein may be "exogenous" if it has been subjected to some corm oi'human manipulation, for example, by the alteration or determination oi~a phosphorylation state.
A "substantially identical'" se'.luertce is an amigo acid e'r nucleotide sequencf: that differs from a reference: sequence only by one or snore conservative substitutions, as discussed herein, or by one or more anon-conserwativc substitutions, deletion, or insertions located ;:rt positions of the sequence: that do not destroy htt biological function as described herein. Such a sequence c;an he at le<~st (~()'%~ or 7j'%n or more ~;cnerally at least 80°~u, 85°/,, ~)0'%, gr t)5%, or as mach as 9'~'%~ identical at the 4urtino acid or nucleotide level to the sequence used for comparison Sequence identity can be readily measured using publicly available sequence analysis software (e.~;., Sequence Analysis Software Package of the Genetics Computer (,coup, Universi~y of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, gr B!_.AS~I~ softwa.tre ~rvailalole from sloe National Li~~rary of Medicine). Exatmples of useful scatty are include the programs, Pile-rtp and PrettyBox. Such softwarre matches similar sequences icy assigning degrees oi~homology to various substitutions, deletions, stubsti-ution~,. and otherrnodrfications.
,1n "agonist"' is a compound, including a loeptidc c7r peptide analogue, that possesses the biological activities of a n~rturall~.~-occurring htt protein, and triggers biochemical responses that would be triggered by a naturally-occurring htt protein. Thus an agonist, according to the invention, may moclulat.e cell survival by, for example, inhibiting cell death car apoptosis, c>r promoting cell proli ~~eratiim. An atgonist comp gund will have biological activit.v if it competes with a htt protein, peptide or peptide analogue, as described luerein, in its ability to inhibit an apoptotic or aggrc;~;ation response, or promote a proliferative response when compared to a htt protei:~, peptide or peptide analogue. Generally, an agonist con upgund wilt exhibit at least 2()'ia ~oodulation. or at least :30",=., to SO'%' modulation, or even over 80",~ or over 100°.. modulation when compared to a htt protein, peptide or peptide analo;;ue.
:1n "antal;onist'" is a compound, including a peptide or peptide analogue, that nullifies the biological activities of'a naturally-occurring htt protein, and is incapable of triggering the biocheanical response:: that would be elicited by a naturally-occurring htt protein. Thus an antagoroist, according to the invention, may modulate cell survival by, for ~xamhlc. promoting cell deatl-' or apoptosis, or inhibiting cell proliferation. An ant~rgonist compound will have biological actin it.y if it compc;te;s with a htt protc;in, peptide or peptide analogue, as described herein. in its ability to modulate a apoptotic, aggregation, or proliferative response when cc°nnpar~d to a htt protein, peptide c5r peptide analogue.
C~ca~erally, an antagonist compound will exhibit at least 2()°,%~, modulation, or at least 30"io to ~0'io modulation, or even over 8()% ~)r over 100''~~~ n ~rodulation when compared to a htt lorotein, pelotide or peptide analogue- Antisense oligc>nucleotidc molecules, or antibodies that inierferc with htt function, may be antagonists. An antagonist n oay also be a biologically inactive form, c>r fragment, of Intt pr<}ti;in that interferes with tl~~~
action of the wild-type protein, such as a dominant negative mutant of a htt protein.
:~\ "nucleic acid molecule" is and chain of two or more nucleotides including;
naturally occurrin g or non-naturally occurring nucleotides or nucleotide analogues. A
nucleic acid rnc>lecule is "compl~ment~rrv" to another nucleic rucid molecule if it hybridizes, r.~nder conditions of high strin~;eocy, witl°r the second nucleic acid molecule.
,vu antibody ''specifically bilTds'" an antigen when it recognises and binds the antigen, but does not substantially recognise .rncl hind other molecules in a sample, having fir example an affinity for the antigen which is 1 (), 10U, I00() or I ()000 times greater than the affinity of the antibody for another rLference molecule in a sa~trple.
~ "cell degen erative disease" is a disorder or condition characterised by the larogrcssiv~: or acute loss of cells or tissue. A progressive cell degenerative disease could result from, for example, a hereditar~,~ deficiency or environmental factors leading to a gradual Ions of cells or tissue, often over a period of years. An acute cell degenerative disease could result from trautna, su~~h as physical injury or stroke.
A "cell proliferative disease' is a disorder or condition characterised by excessive or aberrant cell growth.
-\ "test compound" is .my chemical comp<our~d, be it naturally-occurring or artificially-~derivcd. 'fe;st compounds may include, without Iin nitation, peptides, pol~,~peptides, synthesisec.i c.>rganic noolecules, rnaturally occurring; organic mi.>lecules, alzd nucleic .acid rnotccrctca. ~\ test compound can "camp ete" wlth a kn()wn eol7lpOUIld such as a htt protein or fi-agn~ent thereof by, for example, interfering with modulatiol~ of apoptosis or cell death, cell proliferation. or protein aggregation by pat or the known compound, or by interfering; with any biological respi»~se induced by ota or the known compound. Generally, a test c~~mpound will exhibit at least 20':% modulation, or at least 30%f to St>°r~, modulation, or even over 80'%
or ovi;r l0t)'%; modul~t.ion when con uparcd to a l7tt protein, peptide or peptide analo;~ue.
13y "contacting" is n~e~rnt to submit an animal, cell, lysate, extract, or molecule derived ti-om a cell to a test Corl1pollnd.

By ~'determinin~" is meant analyzing the effect of a test compound on the test system. 'fhe means for analyzing may include. without limitation, antibody labeling;, cell proliferation assays, imnn anoprecipitation, in x~iv« and irz nit~-o phosphorylation assays, cell death assays, anoikis assays, ultrastructural analysis, histological analysis, or any other methods known to those skilled in tl~e art.
()then features and advantages of the invention will be apparent from the following description of the drawings grad the invention, and from the claims.
BRIEF DES('RII'7fION OF ~(HE DRAWINGS
I~I(i. 1 is an autoradiot;raph :~,howing phosphorylation of serine 421 of htt bar AKT' in intro.
IvIO. 2 is a schematic diagram of the strategy used to construct the 5421 mutants.
FI(J. 3 shows photographs d~hicting stable rctroviral transfection of NIH3T3 cells.
I1 I( l. :~ is a bar graph showing soft-agar assay results w kith retrovirally transiected N1H i-f3 cells.
Fl(;. :~ is a bar graph showing htt expression in gastric cancer.
E~I(i. t is a bar graph showin;y htt expression in breast cancer.
hI(i. " is a bar graph showing floc effect ofhtt on NIHi3TS cells in an anoikis assay.
FIGS. ~A-B are bar graphs snowing the effect of phosphorylation of htt on J_~IBI~-100 cells in an anoil:is assay.
l~'IGS. ~)A-B slow the effect 3.~f phosphorylation o(~htt on HBL-100 cells in an anoikis assay I' I(J. 10 is an amino acid seduence of human huntingtin (GenBank Accessicm No.
NP 0021()2) (SEQ ID NO: 1 ~.
FL(iS. I 1 A-C is a nucleic acid seduence of human huntingtin (GenBank Accession No. NM ()021 I 1 ) (SEQ ID NO: 2) DETAILED DI-SC"RIPT1ON Oh 'THE INVENTION
In general, the invention pre>vidcs methods and uses for htt in modulating cell survival. It l7as been found th;zt phosphorylation of htt plays a role in its cell survival function, and that dephosphorylated or unphosphorylatable htt is capable of promos=ing or increasing cell death or apoptosis, acid inhibiting or decreasing cell proliferation.
(: onverselv, lohosphorylated htt inhioits apoptosis and promotes cell proliferation.

fhosphorylation also decreases the c.-e.llular toxicity oi~htt. Various alternative embodiments of the invention are described below . ~Chese embodiments include, without limitation, use of htt to modulate cell survival, to diagnose cell proliferation, or to assay test compounds.
Use of Htt to Modulate ('ell Survival I'he methods of~tle in~entiou may be used to promote cell survival, for example, by inhibiting cell death or al~optosis, or by promoting cell proliferation. The methods of the invention may also be used to inhib~l cell survival by promoting cell death or apoptosis, or by iniohiting cell proliferation. Since htt is ubiquitously expressed, the methods and reagents according to the invention m~ty be u~~ed in a wide range of cells types to modulate cell survival in a variety of cell proliferative or cell degenerative disorders.
The subject or patient to he treated rnay be a patient that is not suffering from HD, or a patient having an l7'l:~ allele that is not associated with a 11D disease state (such as an ITlS
allele hacking 35 or fewer CA(i repeats in the first exon of the HI) gene). Htt can be used to treat an "2.poptotic disease" state. defined for example as a condition characterized by the occurrence of undesirably high levels of ape>ptosis. for cxarznple certain neurodegenerative diseasca such as Alzheimer's disease, Parkinson's disuse, amyotrophic. lateral sclerosis, multiple sclerosis, restenosis, stroke, and ischemic brain injury and other diseases in which neuronal cells undergo undesired apoptotic cell death.
Wild-type htt proteins, phosl:>horylated htt proteins, or li-agments or agonists thereof, or antagonists of dephosphorvlated <:>r unphosphorylatable htt proteins, or fragments thereof, may he used to promote cell ~:urvival and. for example, treat diseases or disorders that result in inappropriate cell death, includin., chronic or progressme cell death, or acute cell death.
Such diseases or disorders may incl;rde, without limitation, neurodegenerative diseases, such as HI), Alaheimer's disease, l'arkin~~.on's disease. amyotrophic lateral sclerosis, multiple sclerosis; polyglutamine diseases, such as HL>, spinocerebellar ataxias, autosomal dominant cerebellar ataxia with retinal cie;~enc°ration, spinobulbar muscular atrophy (SBMA), or dcntatorubralpallidoluysian atrophy (DllfL.A), or Machado-,loseph disease;
stroke.. epilepsy, spinal cord injury; phvsieal trauma; or retinal degeneration.
Dephosphorylated or r.rnpho:>phorylatable htt 1_>roteins, or fragments or agonists thereof, or antagonists of~ wild-type htt proteins, phosphorylated htt proteins, or fra,~ments thereon, may he used to inhibit cell ;survival and, for example, zreat diseases or disorders that result in inappropriate cell proliferation, Such diseases or disorders may include, without limitation, cancer, such as testicular can cers, embryonic cancers, leukemias;
haemat:opoietic diseases; psoriasis, atherosclerosis; dermatological diseases, such as petmphigus vulgaris and pemphigus foleaceus; or inflammatory disorders.
~'on117ounds to one aspect, con ~pouods uc~:ordiug to the IIIVC;IItIOII 111Clude nOI1-pathOgerllC htt polypeptides, for example, the wild-type human Irtt protein (SEQ ID N(~:1), as well as homoio~.a, such as the mouse, rat, car zebrulish homologs, and ti-a~ments thereof. A skilled perscm will recognise that non-patloo~;enic htt polypeptides include those proteins that contain less than about 35 conse~;uti~-e glutamin ts, and thus may differ from the protein of SEQ ID N(): l in tile length oC the pcEiyglutamine traca. ('ompotrnds within the. scope of the invv nrion rnclude htt polypept~dcs that are phosphorylated or llnphosphorylated, rnc:ludrn g polypcptid~s that are constitutivcly Ishoshhorylatcd, or that arc unphosphorylatable.
C"ompounds within the scope c>f the invention also include Iraglnents of htt polypeptides, for example, ("-tc;nninal polypeptides (e.g., a fragment that corresponds to amino acids ~85-144 of St::v:Q ID N(): 1, or that corrc-sponds to about the ('-terminal half of SEQ ID NU: I ).
N-terminal fragments, for example, fragments corresponding to about the N-terminal half of Sf:Q 1D N(): 1 are also within the scope of"the invention.
In alternative embodiments, ' compound according to the invention can be ;:r non-peptide molecule as well as a aeptid:: or- peptide analogue. A rron-peptide molecule carp be any rrrol~culc: that exhibits biological activity as described herein.
E3iological activity can, for example, be measured Ill t~nllS o1 ability to elicit a apoptosis, aggregation, or cell-prolifcr<rtion response. Compounds cap~ible of increasing wild-type htt expression levels are also within the scope of the invention.
("ompounds can be prepared by, 9~or example, replacing, deleting, or inserting an amlncl acid residue of a htt protein, l~rptide or peptide analogue, as described herein, with other conservative anwino acid residues, i.~., residues having similar physical, biological, or chemical properties, and screening 1~r biological function.
ft tS w'ell knowtl irl the art th<rt Sl)Ille nlodItICat1011'i aIICI changes can be made in the structure of a polypeptide; without sllbstalttially altering the biological function of that peptide, to obtain a biologically equivalent polypcptide. In one aspect of the invention, polypeptides of the present invention also extend to biologically equivalent peptides that differ from a portion of the sequenc.> of the polypeptides of tl-re present invention by conservartive amino acid substitutiotls. .As used herein, the tcnn "conserved amino ~~cid substitutions" refers to the substituti~m ofone amino acid for ~ulother at a given location in thi; pepttdc, where the substitution c~u1 be made without substantial loss of the relevant Iiu lotion. In making such changes, substieutions ofliki; amino acid residues can be made on the basis of rciative similarity of side-chain substituents, for example, their sire, charge, hydrophobicity, hydrclphilicitv, anti 311e like, argil such substitutions may be assayed for their effect orl the function of the peptide by routine testing.
~s used herein, the term "amino acids" means those L-amino acids commonly found in tlatur<tllv occurring proteins, D-amino acids and such amiml acids when they have been rnoditieef. .Accclrclingly, aminc7 acids of the invention May include, for example: 2-Aminoadipic acid; 3-:Aminoa<lipic acid; beta-Alanine; beta-Aminopropionic acid; ?-Anlinohrttyr-ic acid; 4-Arninohutyric acid; piperidinic acid; O-.Anlinocaproic acid; 2-Aminoheptanoic acid; 2-Amilroisobc.ltyric acid; a-Alninoisobrrtyric acid; 2-Aminopimelic acid, ~,~ Diamu~obutyric acid; Dcstltosinc; 2,2'-Diaminopimelic acid; 2,3-Diaminopropionic acid, N-Ethyiglycitle; N-Cthyiasp~rrayine; Hydroxylysine; allo-1-lydroxylysine; 3-Hydroxyproline; 4-H;ydroxyproline; Isodusmosinc; allo-Isolcncine; N-Methylglycine;
sarcosinc; N-Methylisoleucine; (i-N-methyllysinc; N-Methylvaline; Norvalinc;
Norfeucin e;
~rl1d ~)1-IllthInC.
In some; e;mbodiments~ conserved armino acid substitutions may be made where an anuno acid residue is substituted fot another having a similar hydrophilicity value Ie.g., within a v,rlue oi~plus or minus ?.()). where the following play he an amino acid having a hyclropathic index of about -1.6 sucdl as l'yr (-1. Z) or 1'ro (-1.r'~)s arc assigned to amino acid residues (as detailed in United States Patent No. ~,534,1()I, incorporated herein by reference) Arg (+3.0); Lys (-~ 3.0); ;\sp ( ~ ~.()); ( ilu (a 3.C)); Sc:r (~-0.3); Asn (+0.2); Gln (~-0.2); (.:ily (0); Pro (-t).5); Tllr (-0.4); Ala (-0.~); His (-().5); ('ys (-1.()); Met (-1.3); ~'al (_ 1.S); Lcu (-1.81; Ile (-l.8 ); Tyr (-2.3 ): I'he. (-2.5); and I'rp (-3.~).
In altcrntttive emhodirnents, conserved amino acid substitutions may be made where an ammo acid residue is substituted for mother having a similar hydropathic index (e.g., wlthln a value of plus or minus ?.0). In 511611 en"1110(1111reI1tS, each amino acid residue may be assigned a hvdropathie index on the basis of '.its llydrophc>bicity and charge characteristics, as follows: Ile ( ~=L5); Val t+4.2 t; Leu (+_s.8); Plle (+2.8); C'ys (-+?.5); Met (+1.~)); Ala (+l .8);
Gly (-0.4); Thr (-0.7); Scr (-0.8); Trp (-().~)); 'I~yr. (-I3); Pro (-I .6);
His (-3.2); Glu I--3.5); Gln (-3.5 ~; Asp (.-3.5); Asn (- 3.5); Lys ( 3.~)); and Arg (-4_5).

In alternative embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic :rnd r~cutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, t~lu; basic: I.ys, Arg, His: neutral:
Gly, Ser, Thr, Cys, Asn, ( iln, 1'yr.
('onservative amino acid changes can include the substitution of an L-amino acid by the ec>rresponding D-amino acid., by a conservative L)-amino acid, or by a naturally-occurring, non-genetically encoded form of amina acid, as well as a conservative substitution of~arn L,-amino acid. Naturally-occurring; non-genetically encoded amino acids include beta-alanine, .;-amino-prapievnic acid, 2,_~-diamino propionic acid, alpha-aminc7isobutyric acid, 4-aminc~~-butyric acid, N-methylglycine (sarcosinc), hydroxyproline, ornithine, citrulline, t-butylalanine, t-butylglycinc, N-methylisoleucine, phenylglycine, cyclc~l~cxylalttnine, norleucine. norvaline, ?-napthyfalanine, pyridylalanine, 3-benzcthienyl alaninc, 4-chloropl7enylalanine, '?-tluorophenylalanine, 3-Iluorophenylalanine, 4-(luoropl7enylaclanine, loenicilla~~~inc, ~,2,,,4-tetrahydro-isoquir~oline-3-carboxylix acid, beta-2-thienylalantne, methionine sr.tltoxi~_le, Itomoarginine, N-acetyl lysit7e;, 2-amino bulyr-ic acid, 2.-amino butyric acid, 2,4,-diamino butyric; acid, p-aminophenylalanine, N-n~ethylvalin~, homocysteinc, homoscrine, cysteic acid, cpsilon~amino hc;xanoic acid, delta-amino valcric acid, or ?,3-diaminobuiyric acid.
In alternative embodiments, ~:onst.rvativc ar~~ino acid changes include changes based on cansidcrations of hydrophilicity c~r hydrophohicity, size or volume, or charge. ~.mino acids can be generally characterized as lryclrophohic or hydrolohilic, depending primarily on the properties of the amino acFd side churn. A hydrophobic amino acid exhibits a hydroplaobieity of greater than zero, and ~r hydrophilic amino acid exhibits a hydroyhilieity of less than zero, based on the narm:rlized COIISeIISUS hydrophobicity scale of Eisenberg et al.
(J. ~Inl. Bin. 179:1 ~5~-147, l8xl.). Genetically encoded hydrophobic amino acids include Gly, Ala, 1'he, Val, 1_eu, lle, Pro, Mlet anc'l'p, and genetically encoded hydrophilic amino acids include fhr, His, Glu. (Jln, App, Ark., Scr, and Lys. Non-genetically encoded hydrophobic amino acids include t-but-ylalanine, ~.~~hilv non-gencticaliy encoded hydrophilic amigo acids include citrulline and hor~~ocysteir~e ljydrophobic or hydroploilic amino acids can be ft.rrthcr subdivided based on the;
characteristics oi~their side chains. For example, am aromatic amino acid is a hydrophobic ~unincr acid with a side chain coniair~ing at least one aromatic or heteroaromatic rin;~, which may contain one or more substituent.5 such as -Ol-l, -SH, -C'N. -F, -C1, -Br, -1, -NOZ, -NO, -NH~, -NHR, -NRR, -('(O)R, -l'(O)C>Fl, -('(O)OR, -C'(O)NH2, -C~'(O)NHR, -C(O)NF;R, etc., where R is independently (Ci-C,>) ~tlk.yl, srrbstitutcd (("i-C~,) alkyl, (C;'i-C~,) alkenyl, substituted (( ',-C',,) alkenyl, (C'~-C~~) alkynyl, substituted (C'i-C'r>) alkynyl, (C;-C?o) aryl, substituted (C";-(',r,) aryl, (Cr,-('-~,,) all:aryl, substituted (C',,-C'~~,1 alkaryl, ~-20 membc:red hetercaryl, substituted 5-'?0 m~mb~r~.xl hetcroaryl, 6-2to mcmbered alkheteroaryl or substituted 6-?O rnembered alkhetar-c~ary-1. Genetically encoded aromatic amino acids include t'he, i yr, and -fryp, wi~ile non-genetically encoded aromatic amino acids include phenylglycine, 2-napthylalanitre. betu~-2-t.hienylalanine, 1,2,~3,.I-tCtrahydro-isoquinoline-3-car-boxylic acid, ~!-chlorophenylalaninc, ?-fluorophenylalunine~3-tluorophenylalanine, and 4-tluorophenylalanine.
;1n apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH and which has hands in which a pair of electrons shared in common by two atoms is generally held equally lay each of the two atoms ( i.e., the side chain is not polar). Genetically encoded apolar ar~~inc> acids include C~ly, L.cu, Val, Ile, Ala, and Met, while: ne>n-genetically encoded apolar amino acids include cyclohexylalanine.
Apolar amino acids .an be lur~ther subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic° hyctrc?carbon side chain. <enetically encoded aliph~.tic amino adds include Ala, Leu, Val, and Ile, while: non-genetically encoded aliphatic amino acids include norleucine.
.-1 polar amino acid is a hydrmphiiic amine acid with a side chain that is uncharged at physiological pH, but which has one bond in which the pair ol~electrons shared in common by two atoms is held more closely by one of the atoms. (~enctically encoded polar amino acids include Ser, Thr, Asn, acrd GII~., while; non-~;enctically errroded polar amino acids include citrullinc, N-acetyl lysine, and metllionine srrlloxide.
.-1n acidic amigo acid is a hylrophilic amino acid with a side chain pKa value of less than ~. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of~a hydrogen ion. Gc;netic;ally encoded acidic arming acids include .asp and (11u. .A basic amino acid is a hydrophilic amino acid with a side chain pKa value of~greater than ~ . Basic amino acids typically loav~ laositivi;ly charged side chains at physiological pH
due to assc:ociation with hydrorrium ion. (i~netically encoded basic amino acids include Arg, 1_-vs, and Ilis_ while non-genetically cncocled basic amino acids include the non-cyclic a1171r10 awls or7~ithinc. 2,3,-diaminopropiorwtc acid, ?,4-cliaminobutyric acid, and homoarginine.

It will be appreciated by one skilled in the art that the above classifications are not absolute and that an amino acid may be classified in more than one category.
In addition, amino acids can be classified based cvn known behaviour and or characteristic chemical, physical, or biological properties based nn specified assays or as compared with previously identified amino acids. Amine acids c:an also include bifunctiunal moieties having amino acid-l3kc side chains.
('oascrvativ a charges can also include the substitution of a chemically derivatised moiety for a non-derivatised residue, by for example, reaction of a functional side group of an amino acid. Thus, these suostitut;ems can include compounds whose free amino groups have been derivatised to amine hydrt>chlorides, p--toluc;nc sultonyl groups, carbobenzoxy groups, t-butvloxycarhonyl groups, c hloroacetyl groups or formyl groups.
Similarly, free carboxyl groups can be derivatized t:? form salts, methyl and ethyl esters or other types of esters or hydrazides, and side chains can be derivatize<i to forr~~ O-acyl or O-alkyl derivatives for free I~ydroxyl groups or N-im-be~7zylhistidine for the imidazole nitrogen of histidine.
Peptide <.rnidogucs also include; amino acids that l~avc loeen chemically altered, for example, by rnethylation, by amidation of the ~'-terminal amino acid by an alkyl amine such a_s ethylamine, ethanolamine, or utbylerke; diamine, or arylation or methylation of an amino acid side chain t-such as acylation c~f the epsilon amino group of lysine). Peptide analogues can also include replacement of the amine linkage in the peptide with a substituted amide (for example, groups of the f«rmula--('((>)-NR, where K is (C,-Ct,) alkyl, (C,-C~,) alkenyl, (C'i-('~,) all<ynyl, substituted (Ci-C,,) alkyl, substituted (C',-C,,) alkcnyl, or substituted (Ci-C~,) al l:ynyl) or isostere of an amide linkage ( for example, -Cfi~Nl l-, -CH~S, -CH~CH.~-, -('I-l=('H- leis and traps), -C:'(O)CH~-. -(: fI(OH)C'li~-. or-C'H~SO-).
hhe compound can be covalc°ntlv linked. for example, by polymerisation or conjugation, to form homopolymers or heteropolymers. 'ipacers and linkers, typically composed of small neutral molecule, such as amino acids that are uncharged under pl~ysiulogical conditions, can be usel. linkages can be achieved in a number of ways. For example, cysteine residues eao be acidecl at the peptide tennina, and multiple peptides can be covalcntly bonded by controlled oxi~.lation. Alternatively, heterobifunctional agent, such as disultidc/amide forming agents or thiocther/amide forming agents can be used.
The compound can also be linked ro a another compound that can modulate an apoptotic, aggro<~ativ~, or proliferative rospons;.. The comportnd can also be constrained, for .°xample, by having cyclic portions.

Peptides or peptide analogues can be synthesised by standard chemical techniques, for example, by automated synthesi4, using solution or solid phGtsc synthesis methodology.
Automated peptide synthesisers are commercially available and use techniques well known in the art. Peptides and peptide analogues can also be prepared using recombinant DNA
technology using standard methods ~~uch as those described in, for example, Sambrook, et crl.
(Molecular Cloning: .A Laboratory l~~lanual. ~"'t ed., Cold Spring Harbor Laboratory, Cold Spring f-larbcar Laboratory Press, Cold Spring Harbor, N.Y., 1 ~)$~)) or Ausubel et al. (Current Protocols tn !~lofecul<~r Biolo';y, Jol=n Wiley Xc Sons, 1~)O4).
('ompounds, such as peptides (or ,analogues thereoi~ can be identified by routine cxperimentalion by, for example, mothfying residues within htt proteins or polypeptides;
introducing single or multiple aminca acid substitutions, deletions, or insertions, and identifying those compounds that retain biologicrtl activity, e.,~., those compounds that have the ability to modulate an apoptotic, ag~~regative, or proliferative response.
Compounds according, to the invention include nucleic acid molecules, for example, ~:I)N;~, RNA, genomte DNA, or ant; sense molecules. Nucleic acid molecules can be used, for example, for gene therapy using standard techniques.
In general, candidate compounds for modulating cell survival, or for the prevention or treatment of cell degenerat;vc or :;c:ll proliferative disorders are identified from large libraries of both natural product or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known m the urt. l~hose, skilled in tl~e field of drug discovery and development will understand that tlov precise source of test extracts or compounds is not critical to the method(s) of the, inveiaion. Accordingly, virluallv any number of chemical extracts or compounds can be screeuc;d using the exemplary methods described herein.
E~;amples of such extracts or compounds include, but arc not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fe~r-mcntation broths, arid synthetic compounds, as well as mc~di (ication of existing compounds. Numerous methods arc also available for generating rand«tn or directed synthesis t e.g., ~.emi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-hawed compounds. Synthetic ccrrnpor-tnd libraries are commercially available.
Alternatively, libraries of natural connpounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, l-.'K), Xen ova (Slough, UK I, Harbor Branch Oceanow~raphic Institute (Ft. fierce, Fla.), and PharmaMar, l .S.A (t''an:briclge, Mass.). In addition, natural and synthel.ically produced libraries of, for example, pancreatic beta cell precursor polypeptides containing if°ader sequences, are produced, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthcrn~are, it desired, any library <>r compound is readily modified using standard chemical, physical, or biochemical mcahods.
When a crude extract ins found to ntodulatc cell survival, further fractionation of the positive lead extr<~et is necessary to i:alate chf°mical constituents responsible For the observed effect. Thus, the goat of the extraction, ti-actionatian, and purification process is the careful characterization and identiiic;~tion of a chemical entity within the crude extract having cell proliferation, -preventati~~e, or -palliative activities. The same assays described herein for the detection of activities in mixtures afcompounds can be used to purify the active component and to lest derivatives thereat Melhods of fractionation and purification of such heterogeneous extracts are 1mown in the art. if desired, cc7mpounds shown to be useful agents for tr°emment arc chemically t.uodiiied according to methods known in the art.
Compounds identified as being of therapeutic value may he subsequently analyzed using a n~amnraliao HD model, or any other ~rnin~al model for a degenerative or proliferative disorder.
t'andidate test compounds can be first assayed for their ability to modulate the apoptc~tic, aggregative, proliferative or other response. The cells or cell preparations used in tl~e assays c:an be obtained fi-orn cell 'fines or can be isolated from patients or animal models fir degenerative or cell proliferative diseases or disorders, using standard techniques. The assays can be performed using stand~.rrd assays as described herc;in, or known to those of ordinary skill in the art. 'best compounds that modulate apoptotic, aggregative, proliferative responses can then be used for further analysis. 1'est compounds identified as being nwduiators of apoptotic, aggregative, or proliferativc responses can be further tested in animal models afcell degeneration c~r pruliferatic>n, using standard techniques.
Antibodies f( he compounds ofthe invention can be used to prepare antibodies to htt polypeptides or analogues thereof, t.tsing standard techniques of preparation as, for example, described in Harlalv and Lane (Antibodies; A Lal-~oratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.l'., 1988), or known to those skilled in the art. Antibodies can be tailored to rnir~irmisc~ adverse host immctne re~wponsa by, far example, using chimeric antibodies contain an antigen binding domain from one species and the Fc portion from another species, or by using antibodies made. from Izybridomas of the appropriate species.
Pharn~~rceutical C'ompositions._Dosa~es,,And Administration t'ornpounds ol~the invention ~.an be provided alone or ~n combination with other compounds ( for example, nucleic acu molecules. small molecules, peptides, or peptide analogues), in the presence of a liposomc, an adjuvant, or any pharmaceutically acceptable carrier, in a form suitable for administration to humans. Ifdesrred, treatment with a compound according to the in~~ention m~rv be combined with more traditional and existing tlnerapics for degenerative or proliferative diseases.
('onvcntional phar-rnaceutical practice may be employed to provide suitable fc>rmulatiorrs or compositions to administer the compounds to patients suffering from or presyz-nptomatic for type I diabetes. Arzv appropriate route of administration may be:
employed, for example, parenteral, iotravrnous, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventric~.rlar, intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal, aeroscal, or orarl administration. Therapeutic formulations may be in the form o(~ liquid solutions or suspensions; Ior oral administration, formulations may be in tine form o(~ tablets or capsules; an~.f for intr~rnasal fomulatior~s, in the form of po~,vders, nasal drops, or aerosols.
l~-1ethads well known in the art for making formulations are found in, for example, ~'Remm~ton's Pharmaceutical Sciences" ( 19'x' edition), ed. A. e~c:nnaro, 1995, Mack Publishing Company, Laston, Pa. Fc~rmulatio:ns for parenteral administration may, for exarnlnle, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetabC~; origin, or hydrogenated napthalenes.
Biocompatible, biodo~;r~rdable lactide polymer, l;zcticle/glycolide copolymer, or polyoxyethylene-polyoxypropylenc copolymers may t}c used to control the release of the compounds. Other potentially useful parenteral delivery systems for modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, inrplantablcv infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for Lxample, polyoxyethylene-9-lauryl ether, glycocholate and dcoxycholate, or may he oily solutions for administration in the form of nasal drops, or as4rg~,l.
'I 9 For therapeutic or prophylactic; compositions, the compounds are administered to an individual in an amount sufficient to stop or slow cell degeneration or cell proliferation, depending on the disorder. An "effective amount" of a compound according to the invention includes <r therapeutically effective amount or a prophvlactically effective amount. A
"therapeutically effective amount" r~°t'crs to an amount efiectivc, at dosages and for periods of time necessary, to achieve tire desired therapeutic result, such as reduction of eel, death or apoptosis, or promotion of'cell proliferation, for a cell degenerative disease, or the promotion of cell death car apoptosis, or rc~ductic~n of cell proliferation, for a cell proliferative disease. A
therapeutically effective amount of a compound may vary according to factors such as the disease state, age, sex.. and weight of the individual, and the ability of the compound to elicit a desircct response in the indiv~drral. I_~osage regimens may be adjusted to provide the optimum tl-rerapeutic response A thcr~rpeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A -'prophylactically et~fectiv~ amount" refers to an ru~ount effective, at dosages and for per iods of time necessary, to achieve the desired prophylactic result, such as reduction of cell death or apoptosis, or promotion ofccll proliferation, for ~r cell degenerative disease, or the promotion of cell death or apoptc~sis, or reduction of cell proliferation, for a cell prolifcrative disease. 'typically, a prophylactic: dose is used in subjects prior to or at an earlier stage oi~discase, sr~ that a prophylacaically ef~feetive amount may be less than a therapeutically chfective amount. A l;referred range for therapeutically or prophylactically effective arnourrts of a compound rnay be 0.1 rrM-0.1 M, ().l nh1-O.OSM, 0.05 nM-l:~hM or 0.()1 iaM-1()lrM.
It is to be noted that dosage values may vary with the severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individu;rl recd and the professional.judgement oftUe person administering or supervisins the administratic»~ of she compositions. Dosage ranges set forth herein are exemplary only and do not limit the ~.iosa~~e ranges that may be selected by medical practitioners. 'The amount of «ctive uompounc.l in the composition may vary according to factors such as the disease stag, age, sex, and weight of the individual.
Dosage regimens may be adjusted to provide the optinrrnm therapeutic response. For example, a single bolus may be ~rdmirvistered, several divided doses may be administered over time or the d~~se may be proportionally reduced or increased as indicatc;d by the exigencies of the therapeutic situation. It rnay be advantageous to formulate parenteral compositions in dosage unit form for ease oi~administration anct unifornity of dosage.
In the case of vaccine formulations, an immunogcnic;dly effect amount of a compound of the invention can be provided, alone or in combination with other compounds, with an acljuvant, for example, hreuud's incomplete adjuvant or aluminum hydroxide. 'the comlouncl ntay also Ioe; linked with ar carrier rnc>lecule, such as bovine serum albumin or lccyhole limpet hemocyanin to enhance immunogenicity.
In general, compounds of th~:. invention should be used without causing substantial toxicity Toxicity of the eom~~ound~" of the ir~verttion can be cletcnnined using standard techniques, for example, by testing ~n cell cultures or experimental animals and del.ermining I,lto therapeutic index., i.e., the ratio fretween the LD50 (the dose lethal to 50% of the pc>puiation) ~tnd the LD100 (toe dose lethal to 1t10'% of~thc population). (n some circumstaltces how ever, such as in severe disease COIldIt1o11S, it may be necessary to aaclminister st.ibstantial excesses o1~tlac compositions.
hl~~ Co117pUrlndS ()t thf' IrlvCIlIloll Inlay bl'. provided in a phartnaceutieal pack or kit comprising; one or more c;ontas.incrs filled with orrc or more; of the ingredients of the pharmaceutical compositions of the invention. Associated with such containers) can be a notice in the form prescribed oy a g'>vernmental agency regulating the manufacture, use, or sale of pharmaceuticals err biological products, which notice ret7cets approval by the agency of manufacture, use, or sale fc~r human administration.
Assavs Various assays may bc: performed to determine biological activity of a test compound. I~or exannple, modulatic:'n ol~~rpoptosis, aggregation, or cell proliferation may be tested as described herein or as known by one of ordinary skill in the art.
~iurvi vcrl .4sswv.~.s Screening for the pro-surviv,il effect of'a candidate compound, for example, a htt protein ti-agrrlent can be performed resins cell litres transfcetect with the gene encoding the candidate ti-agment. 'fhe transtecteci cells may he treated with a pro-apoptotic drug and one of several cellular markers oh viability measured. These markers may include, without lil~~itation: (1 ) cell death, measured lay cell morpholcagy; (2) mitochondrial viability, measured by enzyme activity; and (_~) aS~gregate formation, measured by z~

immunofluorescence staining. In vi~-o eti~ects of~htt earn be assessed by: (1) observation of cellular morphology by microscopy; and (2) DNA fragmentation using 'CUNEL
staining.
Additionally, cell lines can be created that stably express a bir~li>gically active fragment of htt.
that can be used as a reagent f.>r screening the effectiveness ol~the fragment in protf:cting against multiple; pro-cell cleatl~ stimr~ii.
('ell 1)ecrth o'- Apc~ptosis Assrws.
('ells and samples may be obtained from a variety of sources including from experimental animals or human patients, from cell tin es rmade using recombinant techniques, or from depositories such as ArfC'C. Alternatively, htt may be introduced into cells using standard transient transfection techniques.
('ells, for example, human nurrronai precursor cell line NT2 cells, may be transtected with loll or control cDNAs using standard techniques. Cell death can be quantified in NT2 cells by co-transfection of the expression constructs with a plasmid containing a marker gene, for example, the LacZ gene at an appropriate ratio, and the cells may be stain;:d for (3-galac;tosidase activity at 24 hr loost-t~ansEcetion using standard procedures.
The pre-survival effect of the candidate protein may lane assessed by incubating transfected cells with a pro-apoptotic clru~;, tin- example, t;urroxi yen at various concentrations. An apoptotic morphology may be scored as blur-stainini; ells that ~u~e rounded up, blebbed and condensed, which may be clearly distinguished ti-orn viable cells that are flat and have neurite-like extensions.
,~irochortdricrl Viubilitv (,ells, for example, IILK 29~?'T cells m.ay be lransfected with htt or a control DNA, for example, Lac'Z. DNA using standard techniques. Transfccted cells may be treated at 4~
hours post--transfer-lion with a various concentrations of tamoxifcn or other apoptosis induces ('ell viability may n reasured by MTT assay at 24 hr post-lransfection by incubating the cells for ? hr in a 1:111 dilution of WST-1 reagent (Boehringer Mannheim) and release of lormaran tr'om mitochondria rnay br quantified at 45() nm using an ELISA plate reader.
Mock transfected, vector only, and l,acZ transfected cells serm as controls for- tran~~fection-related toxicity. One way ANOVA irnd Newman-Keels test may be used for statistical analysis. 'statistical analyses of the tell death data in NT2 and HEK cells may be performed using one-way ANOVA and Newm.w-Kculs post-comparison tests.

.~lg~,Trogate t~~orrncatiorT.
('ells, for example, human embryonic kidney cells (HEK 29sT) may be transfected using standard technidues. At 48 h ,cost-transfection, the cells may be treated with tamoxifen to induce ug~regate formation, then processed for inm~unotluorescence. The cells pan be fixed, permeabilised, then incubated with anti-htt antibody. Secondary antibodies conjugated to a marker, such us F1T~' with the use of DAPI (~',C'-diamindino-2-phenviindole) as a nuclear cormter-stain. Appropriate control experiments rnay be performed to determine the sl>eciGcrty oi'the antibodies, rncludin g secondary antib~~dy only and nu:>ck transfected cells. 'I he eel is may be viewed with microscope, digitally captured ~,vith a (.'CD camera and the irna~es may be colourised and overlapped. The proportion of cells with rrggre~ates is presented a~: a percent of~ihe total number ofcells expressing htt.
C'c~ll l'roli~ercatior~ .Assays 1'arious cell prolifer-atron as~;ays, such as those described herein or known to one of ordinary skill in the art may be use. Such assays include M'T'f assays;
contact inlri'~oition assays, conducted on soft agae~ or in an animal model, for example, to detern~ine tumour growth; as well as multiple commercially-available cell proliferation assays.
fninrcrl ~9oclels Various ~rnimal models of call degenerative or- cell proliferative diseases exist. In addition to the YA(~' transgemc htt model described herein, the familial ALS-SODI
crans~;euc arouse model may he used as an amyotropic lateral sclerosis model;
the ids mouse may he used as a model for retinal c9e~eneration; the I:AE? mouse may be used as an animal model of human multiple sclerosis; sac. These animal models may be used as source cells or tissue for the assays of the invention;. Test compounds may also be assayed in these models.
The following examples are intended to illustrate various embodiments and aspects eh~the irwcntion, and do not limit th:~ invention in any way.
EX:1MI'L,ES
Pho~orylation.of Htt and its Dole in t._,'cllular Proliferation The protein kinasc AKT is involved in signaling pathways known to be involved in causing cancer. AK'1~ is a Scrine (~)i~fhreonine (T) kinase which preferentially lohosphorylates S/T residues lying ir= RXRXXS/T' sequences, where X is any amino acid, and SiT means either S or T. ~Chia, or a ~:loscly related, motif may be found in human htt at rlilllllo aClll poSltlOtls:
5421 (serine at amino acid position -I21 ) Sequence = GGRSRSGSIVE1JIAG, f20O'3 (threonine at amino acid position ?06~) Sequence = LDRFRLSTMQDSI_SP, f1024 (threonine at amino acid position 1 U24) Sequence -=TSTTRAL'TFGCCEAL, where residues predicted to oe phosphorylated are indicated in bold font.
5421 mutants of full-length iltt (unphosphorylatablc and constitutively phospllorylated at serine 421) were ~.:onstructed (FIG. 2). PC'IZ mutagenesis was perfol~lrled to replace serine 421 with alanine (5421 A, which is unphosphorylatable) and aspartate or glutamate (5421 D or 5421 E, vvllich mimic constitutive phospllorylation), followed by cloning into .;~)4~) construct c<)ntainillg truncated huntingiin protein. The resulting constructs were then clclned into full length htt and into the commercially available MSCV mnlrine retroviral vector. Sequence analysis conducted across the mutated region and acro;~s the polyglutamine region of the constructs, as well as transient transfeetions of the constnlcts into O3'T cells, which resulted ill proteins of the expected malls, coll~nned the integrity of the constructs.
Serinc 421 of huntirlgtin can be llhosphorylated by purified recombinant active AKT
irr ltr-o ( F1G. 1 ). lmmunoprecipitatcd rcc;ombinant protein containing the N-terminal one-third of htt was used as a sub~trute for AKT. Pllosphc)rylation was detected by autoradio'raphy since radiolabelled ATP was a component o1'the i~t vitro kinase reaction.
Vfhen scrine 421 was nlutatec( to alanine (S421A), a non-phosphorylatable amino acid, 64°/, of the phosphorylation signal on hurltingtin was ablated. These results demonstratf~ that AKT can phosphorylate htt in letro and that this reaction is specific for serine 421. Other sites for AK'f phosphorylation are tllreonines lt>24 <uld 2Uc78.
Retroviral introduction of the 5421 mutants into N1H ~~f3 cells was used to examine the oncogenic (proliferative) effects of phosphorylation on serine 421. Stable retroviral transfection of NIH3~1~3 cells with wild-type huntingtin leads to malignant, morphologic transtonnation (FIG. 3). To test the oncogenic potential of htt and to examine the affect of phosphorylation of htt at S42 I on cc.~ll proliferation, a soft agar assay was conducted in V1H ~~f3 cells t Fl(i 41. Comparison of tile colony lelrmation capabilities of NIH3T3 cells transfcctecl with wild-type htt, control vector, mutant htt, or oncogene (c.~l., RAS) indicated that wild-type htt expression has a :4 fold increase in number oi~colonies (more colonies =

more like cancer cells) over control c.~e;lls (about 1 ~'ia, compared to about 5%), but IeSS than a dramatic once>gene like RAS (which loads to about 4C1",% colonies). Mutant htt (HD138) tr-anstection leads to no (t)°~o) soft agar colonies.
Screening human tumors for increased hit expression levels, either through screening human lymphoma samples, screening Nt'1 (t() cell lysates, or screening cancer tissue arrays, indicated that increased lc;vels ohhtt arc fi,und in some forms u(~human tumors such as gastric arid breast tumars (FIGS. 5 a;rd ti), Accordingly, measurement of htt levels may be used as a diagnostic for cancer ar cell proliferation, where an increased level of lltt, compare:cl to normal tissue, is an ind~catiar~ of ~ihcr-ran1. cell pr~rliferation or cancer.
E~tt Mediates Anoikis Resistance lJsing the marine transforn~ecl Iibroblast cell line, NIHi'3T3, the anoikis-resistance properties ot' wild type htt ( l 5 glutainin c repeat) and mutant htt ( 138 glutamine repeat) were investigated. NIHi37 3 cells require a matrix fur- attachment in order to experience optimum growong concfitians. When deiac:hed fi~onr the suppar-ling matrix, they experience a high rate of ap optasis. NIH!~rI'3 cells were trur~slected with a mammalian expression vector <;ontaining wild type lttt I 15 glutamirte repeat) or mutant i~tt ( 1 p8 ~lutan nine repeat).
Untransfecteal and transfected NIH/.:i'I"3 calls were growr7 on cell culture plates, whose plastic surl'aee served as a substrate Cc.~r matrix clcposition and subsequent cellular attachment, or on poly-HEMA c;oatecl cell culture plates. The nonionic nature of p~aly-1-IEMA inhibits both matrix deposition and subsequent cell attachment. A poly-HEMA
concentration of 1 t7 mg/ml ( 1 mgicmo'~) w ~as used and has been shown to be adequate in preventing cull adhesion to tissue culture plastic. ~fhe cells were cultured in DMEM with 1 U'%, serum for 8 hours under attached colldltlorls an plastic plates or under detacl~rc:d cond~tiotts on poly-HEMA-cc>atcd Mastic plates. Cells were harvested, counted and viability was assi;ssed via annexin-V-FITC and propidium iodide (PI) staining.
Protection from cell c~eatl~ induced by anoikis is shown to 3't'3/l Swt and 3'1"3~ 138wt (*p ~().()5, **p<0.05 with respect to parental cells) (FICF. ?).
Ail cells grew well ors normal cell culture plates, as evident by a low rate of apoptosis, but both wild-type and nnutant htt-c;xpressing NIH;'3T3 cells grown in poly-HElV1.A coatec.l plates also grew well, indicating a significant measure of anoikis-resistance.
I:ntransfectecl NIH/3T"3 cells experienced high rates of apoptasis (~-45°,jo) when grown in poly-HEMA coated plates, as detachment resulted in anaikis directed apoptosis.

Phoshhorylation of Htt Mediates Atyoikis Resistance hhe human br~easl cell litre f IB1_.-100, transformed by the SV40 vinrs large 'T antigen with epithelial like morphology. was used to investigate the importance of the huntingtin serinc residue at position 421 with r;~spect to phosphorylation towards anoikis resistant, anchorage independent cell growth. :HBI: 10!) calls were transfected with a 3949-1 Swt huntingtin vector construct, o~~ a similar vector in which the noi-n~al serine at position 421 was mutated to an alanine residue. This mutation prevents plwsphorylation from occun-ing at that site, which is I»-esumed to be mediated by the kinase Afs:T/PKB. Cells were cultured in DMFM with ~% serum for 24 hours either on untreated tissue culture plastic or on poly-HEMA-coated plastic: plates. Cells were harvested, counted and viability was assessed via ,.rr~ncxin-V-1~IIC' arid loropidium iodide (1'I) staining.
tJnce again. nornoal HBh-1(e0 cells grew well in normal cell culture plates ~,v~here attachment was facilitated, but cxpei-ienced a high degree of arnoikis mediated apoytosis when grown on poly-HEMA coated plates where attachment was inhibited (FIGS. 8A-B).
HBL-lt)0 cells transfected with the noi-rnal hrmtingtin construct displayed pro-survival, anti-apoptotic properties as they grew w~;ll whether attached to a inatrix or in a detached state.
f he pro-survival effect of huntingtiro expression was negat~;d when the serine at position 421 was mutated to ate ~rlanin~, residue (f~lG. ~B). HBh-10() cells expressing the 3949-15(S-A) construca hel~aved sirnilarly to untransfected cells, indicating drat the anoikis-resistant, anti-apoptotic signal due to huntingtin w as mediated by phosphorylation of 5421 of the lmntingtin protein, which may, in scn-ue embodiments, be mediated by AKT/PKB.
Kinase Inhibition Studies F'or Anoikis Mediated Si>;nal ~I ransduction.
HBL-l00 ells were transiently translccted with a FLAG-tagged 3949-ISwt htt, or a FLAt=,,-ta'~,ged (S-A) unphosphorylatable mutant htt. Wild-type cells were also treated with LY294()0~', a specific; PI-3 kiirase inhibitor, at a concentration cof 10 LrM, to determine wheti~er inhibition of kinase liniction could abrogate the induction of anoikis. Western blot analysis continnfng the expression ~>f~both full length and 39x9 huntingtin in the FLAG-tagged constructs (Flti.'aB). The pxv;sencc of endogenous huntingtin in the parental cells is also sh~}wo. The pro-survival effect of Iruntingtin is observed in the wild-type (*p~~ 0.0001 withi respect to parental, I_Y-treated. and mutant cells) (FIt~. 9A).

lJntransfected HBL-1 I)0 cells and the 5421 A mutant ~rcw well when allowed to a.tttaclt to cell culture plates, h~~wever both grew poorly in a dc;tached state. By comparison, the. normal huntingtin construct grew well irrespective ofattac:lu ent. These findings i:vidence the utility of phosphorylation, for example at position 421, of~the huntingCin protein in order for that protein to mediate the anti-apoptotic, anoikis resistant signal transduction. In scomc embodiments, the specific phc>phoinositol-a (PI-3) kinase inhibitor, LY294002 (LY), may be used to block phosphorylation of the i~uniingtin serinc residue at position 421.
Blocking of f'1-3 kirtase by Ll' may prevent activation of AK1~/PKB, tl7LIS
preventing the phosphorylation of se;rine; 421 o(~humin~;tin. fndcud L~' prevented anoil:is resistant cell growth oi~detached HBI.-lOt> cells m a manner nearly identical to the huntingtin S421 A
construct. Accordingly, in some enooodin~ents AK'f!PKB mediated phosphorylation of huntingtin at the serine residue positioned at 421 may be used to allow cells to grow independent of cell attachment, and therefore subvert the anoikis signal transduction pathway that promotes apoptosis.
Whip the invention has been described in connection with specific embodiments thereof: it will be understood that it is caloable of fur-tlrer modifications and this application is intended to cover any variations, us~,a, or adaptations of the invention following, in general, the principles of the invcntiotr and including such departures ii-om the present discosurc that come within known or customary pa-actice within the art to which the invention pertains, and may be applied to the essential teatores set forth herein and in the scope; of the appended claims.
All patents, patent aplolicati<~ns, ~rnd publications referred to herein are hereby incorporated by reference in their entirety to the same extent as if each individual patent, patent aplolication, cor publication w;:rs specifically an c1 individually indicated to be incorporatecj by reference in its entirety.

Claims (25)

1. Use of an exogenous huntingtin protein having a selected phosphorylation state or a biologically-active fragment or variant thereof for modulating cell survival in a host, wherein said huntingtin protein is selected from the group consisting of phosphorylated huntingtin protein, unphosphorylated huntingtin protein, and constitutively phosphorylated huntingtin protein.

2. Use of an exogenous huntingtin protein having a selected phosphorylation state or a biologically-active fragment or variant thereof for treatment or prophylaxis of a cell degenerative disease, wherein said huntingtin protein is phosphorylated huntingtin protein or constitutively phosphorylated huntingtin protein.

3. Use of an exogenous huntingtin protein having a selected phosphorylation state or a biologically-active fragment or variant thereof for treatment or prophylaxis of a cell proliferation disease, wherein said huntingtin protein is unphosphorylated huntingtin protein.

4. The use of any one of claims 1 through 3, wherein said variant is selected from the group consisting of an agonist of a huntingtin protein, and an antagonist of a huntingtin protein.

5. The use of claim 1, wherein said modulating comprises modulating cell death or apoptosis.

6. The use of claim 1, wherein said modulating comprises modulating cell proliferation.

7. The use of any one of claims 1 through 3, wherein said huntingtin protein is selected from the group consisting of full-length huntingtin protein, wild-type huntingtin protein, and mutant huntingtin protein.

8. The use of any one of claims 1, 2 or 4 through 7, wherein said phosphorylated huntingtin protein is phosphorylated on serine 421 of the human huntingtin protein (SEQ ID
NO: 1).

9. The use of any one of claims 1, 2, or 4 through 7, wherein said phosphorylated huntingtin protein is phosphorylated on threonine 1024 or threonine 2068 of the human huntingtin protein (SEQ ID NO: 1).

10. The use of a nucleic acid molecule encoding a huntingtin protein according to any one of claims 1 through 9, or a biologically-active fragment thereof.

11. The use of an antibody that specifically binds said huntingtin protein according to any one of claims 1 through 9, or a biologically-active fragment thereof.

12. The use of a nucleic acid molecule complementary to a nucleic acid encoding said huntingtin protein according to any one of claims 1 through 9, or a biologically-active fragment thereof.

13. The use of claim 2, wherein said cell degenerative disease is selected from the group consisting of apoptotic diseases, neurodegenerative diseases, Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, polyglutamine diseases, spinocerebellar ataxias, autosomal dominant cerebellar ataxia with retinal degeneration, spinobulbar muscular atrophy (SBMA), dentatorubralpallidoluysian atrophy (DRPLA), Machado-Joseph disease, stroke, epilepsy, spinal cord injury, physical trauma, and retinal degeneration.

14, The use of claim 3, wherein said cell proliferative disease is selected from the group consisting of cancer, testicular cancers, embryonic cancers, leukemias, haematopoietic diseases, psoriasis, atherosclerosis, inflammatory diseases, and dermatological diseases.

15. A method of assaying a test compound, said method comprising:
(a) providing a system comprising a huntingtin protein or biologically-active fragment thereof, (b) contacting said system with said test compound, and (c) determining whether said test compound modulates the phosphorylation of said huntingtin protein or fragment thereof.

16. The method of claim 15, wherein said assaying is done in vitro and further comprises providing a kinase capable of phosphorylating said huntingtin protein or biologically-active fragment thereof.

17. The method of claim 15, wherein said kinase is AKT.

18. A method of assaying a test compound, said method comprising:
(a) providing a system comprising a huntingtin protein having a selected phosphorylation state or a biologically-active thereof, wherein said huntingtin protein is selected from the group consisting of phosphorylated huntingtin protein, unphosphorylated huntingtin protein, and constitutively phosphorylated huntingtin protein;
(b) contacting said system with said test compound; and (c) determining whether said test compound modulates a function of said huntingtin protein or fragment thereof, wherein said function is selected from the group consisting of apoptosis inhibition, aggregation inhibition, and cell proliferation.

19. The method of claim 15 or 18, wherein said test compound is selected from the group consisting of an agonist of a huntingtin protein, and an antagonist of a huntingtin protein.

20. The method of claim 15 or 18, wherein said huntingtin protein is selected from the group consisting of full length huntingtin protein, wild-type huntingtin protein, and mutant huntingtin protein.

21. The method of claim 18, wherein said phosphorylated huntingtin protein is phosphorylated on serine 421 of the human huntingtin protein (SEQ ID NO: 1).

22. The method of claim 18, wherein said phosphorylated huntingtin protein is phosphorylated on threonine 1024 or threonine 2068 of the human huntingtin protein (SEQ
ID NO: 1).

23. Use of an exogenous huntingtin protein having a selected phosphorylation state or a biologically-active fragment or variant thereof for ameliorating the cytotoxic effects of a therapeutic compound, wherein said huntingtin protein or a biologically-active fragment or variant thereof is administered together with said therapeutic compound.

24. The use of claim 23, wherein said therapeutic compound is a pro-apoptotic compound.

25. The use of claim 24, wherein said pro-apoptotic compound is tamoxifen.