CA2304981A1 - Rho family antagonists and their use to block inhibition of neurite outgrowth - Google Patents

Abstract

This invention provides for the use of antagonists of Rho, or proteins related to Rho as therapeutic targets for agents designed to block growth inhibition by myelin or myelin proteins. One embodiment pertains to the use of Rho antagonists that foster axon regeneration in the central nervous system. The therapeutic agent or antagonist can be small molecules, proteins or peptides, or any agent that binds to Rho or its family members to inactivate this patheway. Embodiments include: the use of the Rho regulatory pathway as a target for Rho antagonists; the use of GDP dissociation inhibitors (GDIs) inhibit the dissociation of GDP from Rho, and thereby prevent the binding of GTP necessary for the activation of Rho; the use of Rho specific GTPase activating protein (GAPs) as targets for the regulation of Rho activity; the use of agents that promote Rho binding to GDI, and block Rho binding to the plasma membrane are also considered within the scope of this invention; the use of C3 transferase and related toxins such as A and B, with related Rho-inhibitory activity to inactivate Rho and stimulate axon growth; the use of dominant negative forms of Rho, used to inactivate Rho, to foster axon growth.

Description

RHO FAMILY ANTAGONISTS AND THEIR USE TO
BLOCK INHIBITION OF NEURTTE OUTGROWTH
FIELD OF INVENTION
This invention relates to the regulation of growth of neurons in the Central Nervous System.
BACKGROUND
Followingtraulnaintheadutcentralnervous system(CNS) ofmarnmals, injuredneurons donotregenerate their transected axons. An important barrier to regeneration is the axon growth inhibitory activity that is 1-'. present in CNS myelin and that is also associatedwith the plasmamembrane of oligodendrocytes, the cells that synthesize myelin in the CNS (see Schwab M.E., et al., ( 1993) Ann. Rev.
Neurosci.,16. 565-595, forreview) . The growth inhibitory properties of CNS myelin have been demonstrated in anumber of differeatlaboratories by awide variety of techniques, including plating neurons on myelin substrates or cryostatsecctions ofwhite mater, and observations of axon contactwithmature oligodendrocytes (Schwab, 2t) M.E., et al., ( 1993) Annu. Rev. N~urosci. 6 565-595). Therefore, it is well documented that adult neurons cannot extend n.eurites over CNS myelin in vitro.
Ithas also beenwell docurnentedthatremoving myelin in vivo improves the success of regenerative growth over the native terrain of the CNS. h,egeneration occurs after irradiation of newbornra~s, aprocedurethat 2.5 kills oligodendrocytes and prevents the appearance of myelin proteins (Savio and Schwab, (1990) Neurobiology 87, 4130-4133). After such aprocedure in rats and combinedwith a corticospinal tract lesion, some crnticospinal axons regrow long distances beyondthe lesions.
Also, in a chickmodel of spinal cordrepair, the onset of myelination correlates with aloss of its regenerative ability of cut axons (Keizstead, et al., (1992) Proc. Nat. Acad. Sci. (USA) 89 11664-11668). The removal of myelin with 3 0 anti-galactocerebroside and complement inthe embryonic chick spinal cord extends the permissive period SUBSTITUTE SHEET (RULE 26) for axonalregeneration. These experiments demonstrate agood correlationbetween myelination and the failure of axons to regenerate in the CNS.
Myelininhibits axongrovrthbecauseitcontains atleastseveraldifferentgrowthinhibitoryproteins.Ithas been well documentedby us and by others thatmyelin-associated glycoprotein (MAG) has potent growth inhibitory activity, both in vitro and in vivo (McKerracher, L., et al., (1994) Neuron 13 805-81 l;
Mukhopadhyay, G., et al., (1994;) Neuron 13 805-811; Li, M., et al., (1996) J.
Neurosci. Res. 46, 404-414;Schafer,M.,etal.,(1996)Neuron 6 1107-1113.).Ahighmolecularweightinhibitoryactivity has been characterizedby Schwab and collaborators, and neutralization of this activity with the IN-I
antibody allows some a~cons to regenerate in white matter (Schwab, M.E., et al., ( 1993 Ann. Rev.
Neurosci.,16 565-595; Hregman, B., et al., ( 1995) Nature378, 498-501.). We also have evidencethat there is an additional growth inhibitory protein in myelin (Xiao, Z., et al., ( 1997) Soc. Neurosci. Absts.
23 1994) . Clearly, there are multiple inhibitory proteins that stop axon regeneration in mammalian CNS
myelin.
In additionto themyelin-deriveditxhibitors there are also othergrowthinhibitorymolecules expressedin theadultmammalianCNS.
Tenacinisagrowthinhibitoryproteinthatisexpressedinsomeunmyelinated regions of the CNS (Bartsch,U., et al., ( 1994) J. Neurosci. 4 4756 - 4768) and after lesion tenascin is expressedby astrocytes thatborderthe lesion site (Aj emain and David ( 1994) J. Comp.Neurol. 340.
233-242). Also growth iinhibitory proteins that are proteoglycans are expressedby reactive astrocytes, andthese proteins form abarrier to regeneration at the glial scar (McKeon and Silver ( 1995) Exp. Neurol.
136, 32 - 43).
While axons damagedin the CNS in vivo do not typically regrow, there have been some reports of long distanceaxonextensioniinadultwhitematter.Suchgrowthhasbeenobservedfollowingtran splantationof graftedneural tissue (W'ictorin, K:., et al., (1990) Nature 347, 556-558;
Davies, S.J.A., et al., ( 1994) J.
Neurosci. 4 1596-1612.; Isacson, 0. and Deacon, T.W. (1996) Neuroscience 75 827-837), SUBSTITUTE SHEET (RULE 26) suggesting that embryonic; neurons primed forrapid extension of axons may be less susceptible to growth inhibition. Some embryonic neurans are not susceptible to MAG (Mukhopadhyay, G., et al., ( 1994}
Neuron 13, 805-811 ), but most embryonic neurons are inhibitedby the othermyelin inhibitors (Schwab, M.E., et al., ( 1993) Ann. ,Rev. Neurosci., 16, 565-595). Therefore, in the cases when axons are able to .'i extend onmyelin, signaling through. intracellularpathways may play an important role in stimulating, or blocking the inhibition oivaxon growth. For example, it is known that laminin is able to stimulate rapid neurite growth (Kahn, T. B., et al., ( 1995) Neuron 14, 275-285), and we have documented that when laminin is present in sufficient concentration, neurites can extend directly onmyelin substrates. These fmdingssuggestthepossibilitythatthestimulationoftheintegrins,thereceptorsforlam inin, issufficientto lc) allowaxongrowthonmyelin.Similarly,ithasbeendocumentedthatwhentheadhesionmol~cul eLlis expressed ectopically on astrocytes, it can partially overcome theirnon permissive substrate properties (Mohajeri, M.H., et al., (1996) Eur. J Neurosci. 8 1085-1097). Therefore, neurons can, under appropriate conditions, grow axons on inhibitory substrates, suggesting that the balance of positive to negative growth cues is a critical determinant for the success or failure of axon regrowth after injury.
Growth inhibitory proteins typically cause growth cone collapse, a process thax causes dramatic rearrangements to the growth cone cytoskeleton {Bandtlow, C.E., et al., ( 1993) Science 259; 80-83; Fan, J., etal., (1993) J. Cell RYoI.121, 867-878; Li, M., etal., (1996) J.
Neurosci. Res. 46 404-414). One family of proteins that has been implicatedin receptor mediated signaling to the cytoskeleton is the small GTPases of the Rho family (Hall, A. (1996) Ann. Rev. Cell Biol. 10, 31-54). In non-neuronal cells it has been clearly docummtedthat mutations in Rho family members that include Rho, Rac and cdc42, affectadhesion,actinpolyzr>erization, andthefomlationof lamellipodiaandfilopodia,whichareallprocess importanttomotility(Nobes,C.D.andHall,A.R.(1995) Ce1181 53-62.).Thereisnowgoodevidence thatmembersoftheRhofamilyregulateaxonoutgrocvthindevelopment. MutationsinRho-relatedfamily members blockthe extension of axons in Drosophila (Lao, L., et al., (1994) Genes Dev. 8,1787-1802) anddisruptaxonalpathfindinginC.elegans(Zipkin,LL.,etal.,(1997) Ce1190 883-894.).Morerecently it has been shown that the guidance molecule collapsin acts through a Rac-dependentmechanism {Jin, Z.
suBSTrruTE sHeFr tRU~ Zs~

_ WO 99/23113 PCT/CA98/01013 andStrittmatter, S.M. (1997) J. Neurosci.17 6256-6263).
Intransgenicmicethatexpress constitutively active Rac in Purkinj a cells, there are alterations in the development of axon terminals and dendritic arborizations (Luo, L., etal., (1996) Nature379, 837-840.). Consistentwiththe observations in vivo, itwas foundthatdominant:negativekac expressed inPCl2 cells disrupts neurite outgrowthinresponse to NGF {Hutchens, J.A., et al., ( 1997) Molec. Biol. Cell 8, 481-500.). Also, treatment of PC 12 cells withlysophosphatidicacid, amitogenicphospholipid, causesneuriteretrac~tionthatismediatedbyRho (Tigyi, G., et al., ( 1996) J. Neurochem. 66 537-548.). Therefore, different members of the Rho family can exert distinct effects on neurite growth, and in PC 12 cells the activation of Rho is correlatedwith growth cone collapse. In nan-neuronal cells, Rhoparticipates in integrin-dependent signalling (Laudanna, C., et al., ( 1996) Science 271, 98 i-983.; Udagawa, T. andMclntyre, B.W. ( 1996) J. Biol. them. 271, 12542-12548). ThepossibilitythatRhomightplayarolewithinthemyelin-derivedgrowthinhibitory system has been studied (:lin, Z. and Strittmatter, S.M. ( 1997) J. Neurosci.
17 6256-6263). It was concluded, however, that the inhibitory effects of myelin are not mediated by Rho family members.
A needremains for ameans of inactivating the multiple inhibitory proteins present in myelin that prevent axonal regrowth after injiuy in the CNS.
This backgroundinformati~onis providedforthepurpose ofmakingknown informationbelievedbythe applicanttobeofpossiblerelevancetothepresentinvention.
Noadmissionisnecessarilyintended,nor shouldbeconst<ued,thaxanyoftheprecedinginfotmationconstitutespriorartagainstthe presentinvention.
SUMMARY OF THE INVENTION
Thepresentinventionrelatesto antagonists andinhibitors tomembers oftheRho family of proteins and diagnostic,therapeutic, andresearchuses for eachofthese aspects. Inparticular, members of the Rho su~ssHE~r tRU~ zs~

family of proteins serve as atherapeutic targetto fosterregrowth of injured or degenerating axons in the CNS.
In accordancewith thepresent invention, apreferred embodimentrelates to antagonists andinhibitors of members of the Rho family of proteins andtheiruse as ameans of blocking a common signalingpaxhway us edby the diverse growth inhibitory molecules. The antagonists and inhibitors may be mutated fom~s of Rho andbiologically activf; (Rho farr~ly-inhibitory) fragments, peptides, C3 andbiologically active (Rho family-inhibitory) fragments, or small molecules such as Y-27632.
I 0 In yet a further aspect of tb.e present invention, Rho family memberproteins can be usedto deign small molecules that antagonizf; and inhibitRho family proteins, to blockinhibition of neurite outgrowth. In another aspect of the pres f;nt invention Rho family members can be used to design antagonist agents that suppress the myelin growth inhibitory system. These antagonist agents can be used to promote axon regrowth and recovery from trauma or neurodegenerative disease.
1 '~
This invention provides f ~r the use of Rho, orproteins related to Rho as therapeutic targets for agents designedto block growth vahibition by myelin ormyelin proteins. One embodiment pertains to the use of Rho antagonists that foster axon regeneration in the central nervous system.
The therapeutic agent or antagonist can be small molecules, proteins or peptides, or any agent that binds to Rho or its family 2(l members to inactivate this pathway.
Another embodiment pertains to the us a of the Rho regulatory pathway as atarget for Rho antagonists .
This pathway involves the GDPIGTP exchange proteins(GEPs). Rho has two interconvertible forms, GDP-boundinactive, and GTP-bound active forms. The GEPs promote the exchange of nucleotides and 25 thereby constitute targets for regulating the activity of Rho.
In another embodiment G DP dissociation inhibitors (GDIs) inhibitthe dissociation of GDP fromRho, and SU9STiTUTE SHEET (RULE 26) thereby preventthe binding of GTP necessary forthe activation of Rho.
Therefore, GDIs are targets for agents thatregulate Rho a~rtivity. The GTP bound active Rho can be convertedto the GDP-found inactive form by a GTPase reaction that is facilitated by its specific GTPas a activating protein {GAP). Thus, another embodiment pertains to the use of GAPs as targets for the regulation of Rho activity. Such inhibitors could block exchange of the GTP/GDP cycle of Rho activationlinactivation.
Another embodiment pertains to the factthat Rho is found in the cytoplasm complexedwith a GTPase inhbitingprotein (GDI). 'Tobecame active, Rhobinds GTP andis translocatedtothemembrane. Thus, agents that promote Rho binding to GDI, and block Rho binding to the plasma membrane are also considered within the scope of this invention.
Yet another embodimentperrains to the observation that abacterial mon-ADP
ribosyltransferase, C3 transferase, ribosylates ltho to inactivate the protein. Thus this embodiment pertains to the use of C3 lxansferasetoinactivateRhoandstimulateaxongrowth.
Likewise,otherbacterialtoxins,suchastoxins A and B, with related Rho-inhibitory activity are considered to be within the scope of this invention.
Moreover, various mutations of the Rho protein can create dominantnegative Rho, which can interferewith thebiological activity of Endogenous R.ho inneurons. Thus, yet a further embodiment of this invention pertains to the use of dominant negative forms of R.ho, used to inactivate Rho, to foster axon growth.
~!0 In accordancewith another aspect of the present invention, there is provided an assay methoduseful to identify R.ho family member antagonist agents that suppress inhibition of neuron growth, comprising the steps of a) culturingneuronsonagrowthpem~issivesubstratethatincorporatesagrowth-inhibitingamount ?S of a Rho family member; and b) exposing the cultuzedneurons of step a) to acandidate Rho family member antagonist agent in an amount and for a period sufficient prospectively to permit growth of the neurons;
SUBSTITUTE SHEET (RULE 28) thereby identifying as Rho family antagonists the candidates of step b) which elicitneurite outgrowth from the cultured neurons of step a).
In accordance with another aspect of the present invention, there is provided amethod to suppress the inhibition of neuron, comprising the steps of delivering, to the nerve growth environment, a Rho family antagonist in an amount effective to reverse said inhibition.
In another embodiment, ~anases activated by Rho, such as Rho-associated kinase, are antagonist candidates. Thus, compounds such as Y-27632 (U.S. PatentNo. 04997834), that blockRho-associated kinase activity, thereby inactivating the Rho signaling pathway, are also embodiments of thi~'invention.
Thus, the use other compounds withinthis family of compounds as desaibedinU.S.
PatentNo. 04997834 that inhibit Rho kinase are also considered within the scope of this invention.
In yet another embodiment, akit is provided comprising components necessary to conduct the assay method useful to screen F;ho family antagonist agents.
Various other obj ects and advantages of the presentinvention will become apparent from the detailed description of the invention.
BRIEF DESCRIPTION OF FIGURES
Figure 1 shows results of tn°atmentwith C3 to stimulate neurite outgrowth on inhibitory MAG substrates.
A)PCl2cellsplatedon~L~Gremainedroundedanddidnotextendneurites.
B)CellsplatedonMAG
in the presence of C3 grew neurites,. C) PC 12 cells plated onpolylysine (PLL) substrates as apositive control.
SUBSTITUTE SHEET (RULE 26) Figure 2 shows therole of inb~grins in overriding growth inhibition by myelin.
The anti- aI integrin function blocking antibody, 3A3,wasusedto determineifintegrinfunctionis necessary forlamininto override groovthinln'bitionbymyelinorMAG. Forexpelimentsonmyelinsubstrates(A-D),cellswerefluorescently labelledwithDil, andplated onmyelin {A), polylysine (B), ormyelin+1 gg laminin (C andD). Cantml IgG
was added to samples A-C, the 3A3 antibody to D. Neurites do not extend on myelin but grow on laminin ormixedlaminin/myelinsubstrates. When3A3 is added, lamininnolongeroverrides growthinhibitionby myelin. Panels (E-1~ show by phase contrast cells plated on recombinant MAG
(E), laminin (F), or reco~nbinantMAGplus lanunin (G andI~, with control antibody (E-G) orwith 3A3 ()~. Integrin function is needed to override growth inhibition by MAG.
IO
Figure 3 presents the results of studies in which PC 12 cells traps fected with dominant negative Rho extend short neurites on MAG substrates. Mock-transfected PCI2 cells {a,c,e) or cells transfected with dominant negative Rho (b,d,~ were ptated on larninin (a,b) or MAG (c-~. MAG
inhibits neurite outgrowth {c), but dominant negative Rho cells spread on MAG andsome cells extend shortneurites (d). Treatment with C3 further stimulates neurite outgrowth on MAG from both lines of cells (e,f).
Figure 4 shows activation. of Rho an MAG substrates. Activated Rho is associated with the plasma membrane. To determine i:f activated Rho was detectedunder conditions where PC
12 cells do notgrow neurites,cellsweregrowniinsuspensionorplatedonMAGorcollagensubstrates.
Twohourslaterthe _ plasmamembranes were purified, the proteins separatedby SDS PAGE, andthe proteins transferedto nitrocelluloseandstainedwithPonceauS.
RhoAwasdetectedontheblotsbyimmunoreactivitywithanti-RhoA antibody. hn~moreactivity was strongestwhen cells were grown in suspension orwhen cells were plated on MAG. Therefore, Rho A is more activewhen cells are keptin suspension orplated on MAG
than when plated on growth-permissive collagen.
25;
V
Figure 5 shows treatment of retinal neurons with C3 stimulates neurite growth on polylysine and MAG
substrates. On nMAG substrates neurite growth is inhibited (a), but after C3 treatment retinal neurons SUBSTITUTE SHEET (RULE 28) platedonnMAGsubstrates exteudneurites (b). Growth ofneurites fromretinalneuronsplatedonPLL(c).
Bar, 50 p,m.
Figure 6 demonstrates ALiP-ribosylation of Rho by C3 detectedin cultured cells. PC12 cells orretinal neurons were cultured in the presence (+) or absence of C3 (-) for two days.
The cells were lysed, and ~,g of protein from each sample was separated on a 11 % acrylamide gel. The proteins were transfered tonitrocellulose,probedwiithmouse anti-RhoA antibody and anti-mouse-HRP
antibody, andrevealedby a chemiluminescentreaction (top panel). The membranes werethenreprobedwithrabbbit anti-Cdc42 and anti-rabbitallcalinephosphatase andrevealedwithNTBBCIP colorreaction.
Treatment of cells withC3 10 resulats in anADP-ribosylation-induced decrease inthe mobility of RhoA. The mobility of ~dc42 does not change with C3 treatment.
Figure 7 illustrates methods usedto study the effect of C3 on injured opticnerve. Figure 7ashows the optic nerve was removed from the sheath prior to crushing with 10.0 sutures (top) and C3 was applied 1 _'~ in Gelfoam and Elvax tuL~es (redbars) immediately following optic nerve crush (huddle). The retinal ganglion cell axons were d~ctected by anteroglade labeling with cholera toxin and timmunodetection of the choleratoxin in longitudinal sections of the optic nerve (bottom). Figures 7c, 7d, 7e, and7f showtreatment of crushed optic nerve with C3 stimulates regenerative growth of retinal ganglion cell axons. (c) Longitudinal 15 ~,msection of abufiver-treated control optic nerve showing the failure of the RGC axons 2(l to cross the injuzedregion; (d,e ) Longitudinal 15 ~m sections of two different optic nerves treatedwith C3 showing anterogradely-lalxled axon extending past the crush (arrows) . The site of crush is indicatedwith arrowheads; (f) Higherma~cationview of {e) showingthe twisted growth of regenerating axons. Bar, 100 lun (c,d,e) and 50 lun in f. Figure 7b shows quantitation of axon regeneration across the site of lesion.
Representationofregeneration observedindifferentanimals. Foreach animal,thema~~imumnumberof 2:5 axonsobservedinasinglel4pmsectionwascountedatdifferentdistancesfromthesiteofthe crush.Each point represents one animal, but animals with growth past 500 pm are also represented atthe shorter distances. Large numbers of regenerating fibers (> l0/s ection) were observedto cross the lesion after C3 SUBSTITUTE SHEET (RULE 28) treatment compared to treatment with PBS.
DETAILED :DESCRIPTION OF THE INVENTION
This invention arises fromthe discovery that Rho family members are key molecules in regulating inhibition by myeiinproteins, andby rvIAG. Thw, this inveutionprovides the advantage of identifying an intracellular target, Rho family member., for all of the multiple inhibitory proteins that mustbe inactivatedto allow for growth on myelin. This uivention provides antagonists of Rho family members, that permit axon 10 regeneration. The method of this invention provides for inactivation of Rho family members, thereby stimulatingneurite growth on growth inhibitory substrates. Therefore, antagonists thatinactivate Rho family members in vivo allow axon regeneration in the injured or diseased CNS.
This invention provides for the use of Rho, or proteins related to Rho as therapeutic targets for agents designedtoblockgrowthinhibitionbymyelinormyelinproteins.Oneembodimentpertainsto theuseof Rho antagonists that foster axon regeneration in the central nervous system.
The therapeutic agent or antagonist can be small molecules, proteins or peptides, or any agent that binds to Rho or its family members to inactivate this pathway. Another embodiment pertains to the use of the Rho regulatory pathway as atargetforRho antagonists. This pathway involves the GDP/GTP
exchangeproteins(GEPs). .
Rho has two interconvertible fornis, GDP bound inactive, and GTP-bound active forms. The GEPs promote the exchange of nucleotides and thereby constitute targets for regulating the activity of Rho. In another embodiment GDP dissociation inhibitors (GDIs) inhibit the dissociation of GDP fromRho, and thereby preventthe binding of GTP necessary forthe activation of Rho.
Therefore, GDIs are targets for agents thaxregulate Rho a~rivity. The GTP-boundactive Rho canbe convertedto the GDP-boundinactive fomn by a GTPase reactio:a that is facilitated by its specific GTPas a activating protein (GAP). Thus, another embodiment pertains to the us a of GAPs as targets for the regulation of Rho activity. Another embodunent pertains to the fact that Rho is found inthe cytoplasm complexedwith a GTPase inhibiting SUBSTITUTE SHEET (RULE 2E) protein (GDI). To become active, Rho binds GTP and is translocated to the membrane. Thus, agents that promoteRhobindingtoGDI, andblockRho bindingto theplasmamembrane arealso consideredwithin the scope of this invention. Yet another embodiment pertains to the observation that abacterial ADP
ribosyltransferase, C3 transferase, ribosylates Rho to inactivatethe protein.
Thus this embodimentpertains :i to the use of C3 transferase to inactivate Rho and stimulate axon growth.
Likewise, other bacterial toxins, such as toxins A and B, with relatedRho-inhibitory activity are consideredto bewithinthe scope of this invention. Moreover, various mutations of the Rho protein can create dominantnegative Rho, which can interfere with the biological activity of endogenous Rho in neurons. Thus, yet a further embodiment of thfis invention pertains to the we of dorinantnegative forms of Rho, usedto inactivate Rho, to foster axon growth.
"Antagonist" refers to aphartaceutical agentwhich in accordance with the present inveationwhich inh~its at least one biological activity normally associatedwith Rho family members, that is blocking or suppressing theinhibitionofneurongrowth.
Antagonistswhichmaybeusedinaccordancewiththepresentinvention includewithoutlimitation, one ormoreRhofamilymembers fragment, adeiivative ofRho family member or of a Rho family members fragment, an analog of Rho family members or of a Rho family members fragment or of said derivative, and apharmaceutical agent, and is further characterizedby the properly of suppressing Rho family members mediated inhibition of neurite outgrowth.
Preferred antagonists include:
mutated forn~s of Rho, such as Rho wherein the effector domain, A-37, has been mutaxedto prevent GTP _ exchange; theADP-ribos;rl transferase C3 and biologically effective fragments that antagonise Rho family members in one of the assays of this invention; and compounds such as Y-27632 that antagonise Rho-associatedkinase(Somiyo,1997,Nature,389:908-910;Uehata,eta1.,1997,Nature,389:990-994;U.S.
Patent No. 4,997,834). As described above, other antagonists include GDP
dissociation inhibitors (GDIs), such as Rho GDl'-dissociationinhibitar 1 (RhoGDI fromHomo sapiens) inhibitthe dissociation of GDP fromRho, and thereby prevent the binding of GTP necessary for the activation ofRho (see, for example, Takahashi, K.., J. Biol. Chem., (I997), 272:23371-5; Gosser, Y.Q., et al., Nature (1997) 387:814; Adra, et al., (1997) Proc. Natl. Acad. Sci., 94:4279-4284.
SI~BS'TiTUTE SHEET (RULE 26) The antagonist of Rho family members in accordance with thepresern invention is not limitedto Rho family members orits derivatives, butalso includes the therapeutic application of all agents, referredherein as pharmaceutical agents, which alter the biological activity of the Rho family members protein such that inhibition of neurons or their axon is suppressed.
The term "effective amount' or "growth promoting amount" refers to the amount of pharmaceutical agent requiredto produce a desired antagonist effect of the Rho family members biological activity. The precise effective amountwill vary withthenature of phamlaceutical agentus ed andmay be deterrninedby one or ordinary skill in the art with only :routine experimentation.
As used herein, the Rho family of proteins comprises, but is not limited to rho, rac, cdc42 and their isotypes, such as RhoA, RlaoB, RhoC, as well as Rho-associatedkinase that are expressedin neural tissue.
Othermembers of the Rho family that are detemlined andwhose inhibition of activity allows forneurite outgrowth are comtemplated to be pact of this invention. (See, for example, Katoh, H., et al., J. Biol.
1-'i Chem, 273:2489-2492,1998; van Leeuwen, F., et al, J. Cell Biol.,139:797-807,1997; Matsui et al., EMBO J.15:2208-2216,1996; Amano et al., Science, 275:1308; Ishizaki, T. et al., (1997) FEBS Lett., 404:118-I24).
As used herein, the terms "Rho farruly member biological activity" refers to cellular events triggered by, _ being of either biochemica or biophysical natur e. The following list is provided, without limitation, which discloses some of the known activities associated with contact-mediated gr owth inhibition of neurite outgrowth, adhesion to neuronal cells, andpromotion of neurite out growth fromnew born dorsal root ganglion neurons.
2 S As usedherein, the term"biologically active", orreference to the biological activity of Rho family members or, orpolypeptide fragmentthereof, refers to apolypeptide that is able to produce one of the functional characteristics exhibited~by Rho family members or its receptors describedherein. In one embodim~t, SUBSTITUTE SHEET (RULE 26) biologically activeproteins are those that demonstrate inhibitory growth activities central nervous system neurons. Such activity may be assayed by any method known to those of skill in the art.
ThetermC3referstoC3ADP-ribosyltransferase,aspecificRhoinactivator.
Apreferredrepresentative example is C3 ADP-ribosyl.transferase, a 23 KDa exoenzyme secreted from certain strains of types C and D from Clostddiumbotulin~um, which specifically ADP-ribosylates the rho family of these GTP binding proteins. ThisADP-ribosyl;ationoccursataspecificasparagineresidueintheirputativeeffectordomain, andpresumablyinterferesw~iththeirinteractionwithaputative effectormoleculedownstreaminsignal transduction. Nuinelvus references describing these compounds can be foundin Methods in Enzymology, Vo1256, Part B, Eds.: W.E. Balch, C.H. Der, andA. Hall; AcademicPress,1995, foreg. Pgs:~ 196-206, 207 et seq, 184-189, and 174 et seq..
B aced on the present evidence that Rho family members can affect growth inhibitory protein signals in myelin, the means exist to idlentify agents and then spies that suppress myelin-mediatedinhibition of nerve growth. Further, one can e~:ploit the yowth inhibiting properties of Rho family members, or Rho family members agonists, to suppress undesired nerve growth. Without the critical finding that Rho family members has growth inhibitory properties, these strategies would not be developed.
Rho Family Member Antagonists and Assay Methods to Identify Rho family members _ Antagonists In one embodiment, Rho family memberantagonistswill be inhibitors of GTPase activity. The GTPI GDP
cycle of Rho family member activation/inactivation is regulated by a number of exchange factors.
Compounds thatblock exchange, thereby inactivatingRho familymembers are preferred embodiments of this invention.
in another embodiment suitable Rho family member antagonist candidates are developed comprising SUBSTITUTE SHEET (RULE 26) fragments, analogs anddarivatives of Rho family member. Sequences farRho family members arekaown, such as those described: Chardin, l'., etal., (1988) Nucleic Acids Research,16:271?; Yeramian, etal., ( 1987) NucleicAcids Research,1:5:1869). Such candidates may interferewith Rho fam;ly members-mediated growth inhibiticm as competitive butnon functional mimics of endogenous Rho family member.
Fromthe amino acid sequence of Rho family members and fromthe clonedDNA coding forit, itwill be appreciated that Rho family member fragments can be produced either by peptide synthesis or by recombinantDNA expression of either atruncated domain of Rho family member, or of intact Rho family members couldbepreparc;dusingstandardrecombinantprocedures,thatcanthenbedigestedenzymic ally in either arandom or a site-selective manner. Analogs of ltho family members or Rho family members fragments can be generated also by recombinant DNA techniques or by peptide synthesis, and will incorporateoneormore,~e.g.1-S,L,-orD-aminoacidsubstitutions.
Derivativesof)thofamilymembers, Rho family members fragments and Rho family member analogs can be generated by chemical reaction of the parent substance to incorporate the desired derivatizing group, such as N-terminal, C-terminal and infra-residue modifying groups that have the effect of masking or stabilizing the substance or target amino acids within it.
In specific embodiments of the invention, candidate Rho family member antagonists include those that are derived from a determination of the functionally active regi on(s) of aRho family member. Antibodies ca be prepared using techniques known in the against epitopes in Rho family member, which, when found 2 0 to be function-blocking in in vitro assays, can be used to map the active regions of the polypeptide as has been reported for other proteins (for example, see Fahrig, et al., ( 1993) Europ. .I. Neurosci., 5 1118-1126; Tropak, et al., (1994) J. Neurochem., 62, 854-862). Thus, it canbe determinedwhichregions ofRhofamilyrnemberGTPasesrecognizedbysubstratemoleculesthatareinvolvedininhibit ionofneutite outgrowth. When those ar a known, synthetic peptides can be prepared to be assayed as candidate :!5 antagonistsoftheRhofa~~nilymemberseffect.
Derivativesofthesecanbeprepared,inciudingthosewith selected amino acid substitutions to provide desirable propeues to enhance their effectiveness as antagonists of the Rho family members candidate functional regions of Rho familymember can also be SUBSTITUTE SHEET (RULE 28) detenninedby thepreparation of altered forms of the Rho family members domains using recombinant DNAtechnologies toproducedeletion orinsertionmutantsthat canbe expressedinvarious cell types as chimericproteins. All of tl~e above forms of Rho familymembers, and forms thatmay be generatedby technologies not limited to the above, can be tested for the presence of functional regions that inhibit or suppress neurite outgrowth, and can be used to design and prepare peptides to serve as antagonists.
In accordance with an aspect of the invention, the Rho family member antagonist is formulated as a pharmaceutical composition which contains the Rho family member antagonist in an amount effective to suppress R.ho family member-mediated inhibition of nerve growth, in combination with a suitable 10 pharmaceutical carrier. Such compositions are useful, in accordance with another aspect of the invention, to suppress Rho family member-inhibitedneme growthinpatients diagnosedwith avariety of neurological disorder, conditions and ailments of the PNS andthe CNS where ti eatmentto increase neurite extension, growth, orregeneration is desired, e. g., in patients with nervous system damage. Patients suffering from traumatic disorders (including but not limited to spinal cord inj vies, spinal cord lesions, surgical nerve 15 lesions or other CNS pathway lesions) damage secondary to infarction, infection, exposure to toxic agents, malignancy, paraneoplastic synd~~omes, or patients with various types of degenerative disorders of the central nervous system can be treated with such Rho family members antagonists. Examples of such disordersincludebutarenotlimitedtoStrokes,Alzheimer'sdisease,Down'ssyndrome,Cre utzfeldt Jacob disease, kuru, Gerstman-S~xausslersyndrome, scrapie; transmissiblemink encephalopathy, Huntington's .
disease, Riley-Day familial dysautonomia, multiple systematrophy, amylotrophic lateral sclerosis orLou Gehrig's disease, progressive supranuclear palsy, Pwkinson's disease and the like. The Rho family members antagonists maybe usedto plrnnote the regeneration of CNS pathways, fiber systems andtracts.
Inaparticular embodiment: of the invention, the Rho family members antagonist is usedto promote the regeneration of nerve fibers over long distances following spinal cord damage.
2_'~
In another embodiment, the invention provides an assay method adapted to identify R.ho family member antagonists, thatis agents thatblock or suppress the growth-inhibiting action of Rho family members. In SUBSTITUTE SHEET (RULE 26) 16 .
its most convenient form, the assay is a tissue culture assay that measures neurite out-growth as a convenient end-point, and. accordingly uses nerve cells that extend neurites when grown on apermissive substrate.
Nervecellssuit<~bleinthisregardincludeneuroblastomacellsoftheNG1081ineage,sucha s NGl 08-15, as well as otht;rneuronal cell lines such as PC 12 cells (American Type Culture Collection, 12301 Parklawn Drive, i~ockville, MD 20852 USA, ATCC Accession No. CRL 1721), human neuroblastoma cells, and larimary cultures of CNS or PNS neurons taken from embryonic, postnatal or adult animals. The nerve cells, for instance about 103 cells-microwell or equivalent, are cultured on a growth permissive substrate, such as polylysine or laminin, that is over-layed with a growth-inhibiting amount of Rho family members. The Rho family members incoiporatedin the culture is suitably myelin-1 (:~ egtractedRho familymembers, although forms of Rho family members otherthan endogenou3 forms can be usedprovidedthey exhibitthe Rho family members properly of inhibiting neuron growth when added to a substrate that is otherwise gr owth permissive.
Inthis assay, candidate Rho family member antagonists, i. e., compounds thatblock the growth-inhibiting 1-'i effectofRhofamilymembers,areaddedtotheRhofamilymember-containingtissueculturepreferablyin amount cuff cientto neutralize the Rho family member growth-inhibiting activity, that is between 1.5 and 15 beg of Rho family memberv, antagonist per well containing a density of 1000 NGl 08-15 cellslwell cultured for 24 hr. in Dulbecco's nunimal essential medium. After culturing for a pen od sufficient for neurite outgrowth, e.g. 3-7 days, the culture is evaluated for neurite outgrowth, and antagonists are thereby .
20 revealedasthosecandidata~whichelicitneuriteoutgrowth.
Desirably,candidatesselectedasRhofamily memberantagonists arethosewhich elicitneurite outgrowthto astatisticallysignificant extent, e.g., in at least 50%, more desirably at least 60%, e.g. 70%, per 1,000 culturedneurons.
t~therassayteststhatcouldbeusedincludewithoutlimitationthefollowing:1) Thegrowthconecollapse 2.5 assay thatis used to asses s growth inhibitory activity of collapsin (Raper, J.A., andKapfhammer, J.P., (1990) Neuron, 2 21-29; Luo, L., et al., (1993) Ce1175 217-227) and of various other inhibitory molecules (Igarashi, M..,. et al., ( 1993) Science 259, 77-79) when eby the test substance is addedto the St~BSTfrUTE SHEET (RULE 26) culture medium and a loss of elaborate growth cone morphology is scored. 2) The use of patterned substrates to assess substrate preference (Walter, J. et al., (1987) Development 101, 909-913; Stahl, et al., (1990) Neuron 5 7'.35-743) or avoidance of test substrates (Ethell, D.W,. et al., (1993) Dev.
Brain Res. ?2,1-8). 3) Thf; expression of recombinantproteins on aheterologous cell surface, andthe transfectedcellsareusedi:nco-cultt~reexperiments.
Theabilityoftheneuronstoextendneuritesonthe transfeetedcells is assessed (Mukhopadhyay et al., ( 1994) Neuron 13, 757-767). 4) Theuse of sections oftissue, suchas sections of CNS whitematter, to assess molecules thatmay modulategrowthinhibition (Carbonetto, S., et al., (1987) J. Neuroscience 7 610-620; Savlo, T. and Schwab, M.E., (1989) J.
Neurosci., 9:1126-1133). 5) Nem-ite retraction assays whereby test substrates are applied to differentiatedneural cells f ntheirabiiity to induce or inhibitthe retraction of previously extended neurites (Jalnink, et al., (1994) ,i'. Cell Bio. 126, 801-810; Sudan, H.S.; et al., (1992) Neuron 8 363-375;
Smalheiser, N., ( 1993) J. Neuroclzem. 61; 340-342). 6) The repulsion of cell-cell interactions by cell aggregation assays (Kelm, S., et al., (1994) Current Biology 4, 965-972; Brady-Kainay, S., et al., { 1993) J. Cell Biol. 4, 96'1-972). 7) The use of nitrocellulose to prepare substrates for growth assays 1 _'. to assess the ability of neural cells to extendneurites on the test substrate (Laganeur, C. andLemmon, V., (1987) PNAS 84 7753-7757; Dou, C-L and Levine, J.M., (1994) J. Neuroscience 14. 7616-7628).
Diagnostic, Therapeutic' and Research Uses for Rho Family MemberAntagonists Rho familymember antagonists haveuses in diagnostics. Such molecules canbe used in assays to detect, prognose, diagnose, or rn~onitor various conditions, diseases, and disorders affecting neurite growth extension,invasiveness, mdregeneration. Alternatively,theRhofamilymembe~-antagonistsmaybeused tomonitortherapies forth;>eases andconditionswhichultimatelyresultinnervedamage; such diseases and conditions include but are not limitedto CNS trauma, (e.g. spinal cordinjuries), infarction, infection, 2.5 malignancy, exposure to toxic agents, nutritional deficiency, paraneoplastic synd~bmes, and degenerative nervediseases (includingbutnotlimitedtoAlzheimer's disease, Parkinson's disease, Huntington's Chorea, amyotrophic lateral sclerosis, progressive supra-nuclear palsy, and other dementias). In a specific SUBSTITUTE SHEET (RULE 26) embodiment, suchmoleculm may be vsedto detect anincxuase in neurite outgrowth as an indicator of CNS
fiberregeneration. For example, in specific embodiments, altered levels of Rho family members activity in apatient sample containing CNS myelin canbe adiagnosticmarkerforthe presence of amalignancy, includingbutnotlimitedtoglioblastoma,neuroblastoma, andmelanoma, oraconditioninvolvingnerve growth, invasiveness, or regeneration in a patient.
Useful fornerve growth suppression are pharmaceutical compositions that contain, roan amount effective to suppress nerve growth, Rho family member antagonist in combination with an acceptable carrier.
Candidate Rho family members antagonists include fragments of Rho family members that incorporate the ectodomain, including the c;ctodomain perse and otherN- andlor C-tem~inally truncated fr~ag~nts of Rho family members or the ectodomain, as well as analogs thereof in which amino acids, e. g. from 1 to 10 residues, are substituted, particularly conservatively, and derivatives of Rho family members or Rho family members fragments inwhichtheN- ~andlorC-terminalresidues are derivatizedby chemicalstabilizing groups.
IS
In apreferred embodimer.~t, mutated forms of Rho family members are used as antagonists. One key example is Rho with amutated effector domain, A-37, which prevents GTP
exchange. Various other mutations of the Rho protein that create dominate negative Rho which can interfere with the biological activity of endogenous Rtro inneurons are considered as antagonists withinthe scope of this inventionto .
inactivate Rho, thereby fostering growth of neurons.
In another preferred embodiment GDP dissociationinhibitrn~s (GDIs) which inhibitthe dissociation of GDP
from Rho, and thereby prevent the binding of GTP necessary for the activation of Rho are used as antagonists.
2:i Inyetanotherpreferred embodiment, GTPase activating protein (GAP) which facilitates the conversion of the GTP-bound active Rho to the GDP-bound inactive form forms the target for regulation of Rho SUBSTITUTE SHEET (RULE 26) activity. Thus, cornpo~mds thaactivate GAP, thereby facilitating the conversion of active Rho into inactive Rho would be candidates for promoting neuronal growth.
In still another preferred embodiment, compounds that affect Rho bindingto the plasmamembrane, thereby decreasing the activity ofRho are also c;onsideredRho antagonists of this invention. Inthis case, thetarget designis basedontheknowledgethatl~hois foundinthe cytoplasmcomplexedwithaGTPaseinhibiting protein (GDIJ. Tobecome active, Rho binds GTP andis translocaxedto the membrane. Thus, agents that promote GDI activity andblakRho binding to the plasmamembranewould decreas a Rho activity, they eby serving as Rho antagonists. that would permit neuron growth.
In specific embodiments of the invention, candidate Rho family members antagonists include specific regions ofthe Rho family members molecule, ~rnd analogs or derivatives of these. These canbe identifiedbyusing the same technologies described above for identification of Rho family members regions that serve as inhibitors of neurite outgrowth.
The Rho family members relaxed derivatives, analogs, and fi~agrnents of the invention can be producedby various methods known in t~.e art. The manipulations which result in their production can occur at the gene or protein level. For example, Rho family members-encoding DNA can be modifiedby any of numerous strategies known in the art (~~Ianiatis e~ al., Molecular Cloning,Al,aboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.'~.,1982), such as by cleavage at appropriate sites withrestric~tion endonuclease(s), subjected to enzymatic modifications if desired, isolated, and ligated in-vitro.
Additionally, the Rho family members-encoding gene can be mutated in-vitro or in-vivo forinstance in the manner applied frn~ production of the ectodomain, to create andlor destroy translation, initiation, andlor termination sequences, orto create variations in codingregions and/or formnew restriction endonuclease sites or destroy preexisting cures, to facilitate further in-vitro modification. Anytechnique formutagenesis knownintheartcabbeused,includingbutnotiimitedto, in-vitro sitedirectedmutagenesis(Hutchinson, SU~STtTUTE SHEET (RULE 26) WO 99/23113 PCf/CA98/01013 et al., (1978) J. Biol. Chem. 253, 6551), use of TABTM linkers (Pharmacia), etc.
For delivery of Rho family members antagonists, vatzous known delivery systems can be used, such as encapsulationinliposomes cxsemipenneablemembranes, expressioninsuitablytransformedortransfectia~a glialcells,oligodendroglialcells,fibroblasts,etc.accordingtotheprocedureknownto thoseskilledinthe are(Lindvall, etal.,(1994)Curr.OpinionNeurobiol.4,752-757).
Linkagetoligandssuchasantibodies can be us edto target delivery to myelin and to other therapeutically relevant sites in-vivo. Methods of introduction include, but ~~re not limited to, intraderrnal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, andintiranasalroutes, andtransfusionintoventricles orasite of operation (e.g. forspinal 1 G cordlesions) ortumorremoval. Likewise, cells secreting Rho family members antagonist'dcti'vity, for example, and not by way of limitation., hybridoma cells encapsulatedin asuitable biological membrane may be implanted in a patient so as to provide a continuous source of Rho family members inhibitor.
Therapeutic Uses of Rho family Antagonists l :i In a~n embodiment, antagonists, derivatives, analogs, inhibitors of Rho family members can be used in regimens where anincrease inneurite extension, growth, orregeneration is desired, e.g., in patients with nervous system damage. Patients suffering from traumatic disorders (including but not limitedto spinal cord injuries, spinal cord lesions, or other CNS pathway lesions), surgical nerve lesions, damage secondary to 2() infarction, infection, exposure to toxic agents, malignancy, paraneoplastic syndromes, orpatients with various types of degenerative disorders of the central nervous system can be treated with such inhibitory protein antagonists. Examples of such disorders include but are not limited to Alzheimer's Disease, Parkinsons'Disease,Huntington's (:horea, amyotrophiclateralsclerosis,progressivesupranuclearpalsy and other demential. Such antagonists may beusedto promote the regeneration of CNS pathways, fiber 2:5 systems and tracts . Administration of antibodies directedto an epitope of, (or the binding portion thereof, or cells secreting such as antibodies) can also beusedto inhibitRho familymembersprotein functionin patients. In aparticular embodiment of the invention, antibodies directed to Rho family members may be SUBSTITUTE SHEET (RULE 26) used to promote the regeneration of nerve fibers over long distances following spinal cord damage.
Various delivery systems we known ~~nd can be us ed for delivery of antagonists or inhf bitors of Rho family members andrelatedmole~:ules, e.g., enca~ulationinliposomes orsemipelmeablemembranes, expression by bacteria, etc. Linkage; to ligands such as antibodies can be used to target myelin associated protein-relatedmolecules to therapeutically desirable sites in vivo. Methods of introduction include but are not limitedto intradermal, :intramuscular, intraperitoneal, intravenous, subcutaneous, oral, andintranasal routes, and infusion into ventricles or a site of operation (e.g. for spinal cord lesions) or tumor removal.
In addition, any methodv~hich results in decreased synthesis of Rho family members malt be used to diminishtheirbiological fimcrion. For example, andnotby way of limitation, agents toxicto the cells which synthesize Rho family members and/or its receptors (e.g. oligodendrocytes) may be usedto decrease the concentration of inhibitory proteins to promote regeneration of neurons.
1.'i EXAMPLES

This example demonstratex in vitro evidence that Rho family members are responsible forregulating the 2s) neuronal response to MA.G. In particular, this demonstrates thatthe small GTPase Rho regulates the responsetoMAG.
PCl2cellswereplatedonpolylysine(PLL),laminin,orMAGsubstratesandexposed to NGF to stimulate neurite growth. P C 12 cells differentiated neurites on PLL and laminin substrates, but on MAG substt~ates the cells rem~uned rounded and did not grow neurites.
2.5 The addition of the ADP-ribosyl transferase C3 from Clostridium botulinum, that efficiently inactivates Rho familymembers witboutaffeceting Rac and cdc42 (Udagawa, T. and McIntyre, B. W. (1996) J. Biol.
Chem.2_71,12542-12548),allowedthecellstoextendneuritesonMAGsubstrates.
inadditionthis sues~rrru~ sHEEr ~RUC.s zs~

example demanst<atesneurite growthfromPCl2 cells transfectedwith adominantnegativeNl9RhoA
construct. On laminin and PLL substrates the N 19 RhoA P C 12 cells grew neurites thatwere longerthan the mock-transfected controls. Moreover, N19 RhoA PC 12 cells were able to extend neurites when plated on MAG substrates. Therefore, the inactivation of Rho stimulates neurite outgrowth and allows neurite extension on MAG;~ubstrates. These results implicate Rho in signaling growth inhibition by MAG
Cell Cultare We obtained PC 12 cells :from three different sources: from Dr. Phil Barker (Montreal Neurological Institute); from the ATCC (obtained from W. Mushinsky, McGill), and from Gabor Tigyi, (University of Tennessee) andwe foundihat all lines of cells were inhibitedby bothmyelin andMAG. PC12 cellswere grown in Dulbecco's modified eagle's medium (DMEM) with 10 % horse serum and 5 % fetal bovine serum.
PC12ce11sstablytransfectedwithconstitutivelyactiveanddominantnegativeRhoAconstr ucts were kindly providedby Dr. G. Tigyi (University of Tennessee, Memphis, USA).
Thethree cell lines used included smock transfectc;d cell line;, a constitutively active RhoA (V
l4GRhoA) cell line, and a dominant negativeRhoA(Nl9TRhoA)cellline.
Transfe~ctedPCl2celllinesweremaintainedinthegrowthmedium containing 400 mg/L G4~18. For cell differentiation experiments, cells were plated on appropriate substrates in DMEM with 1 % fetal bovine serum and 100 ng/ml nerve growth factor. For experiments on mixedsubstrata (laminin~MAGorlaminin/myelin), PCl2wereplatedinDMEMwith 1%
lipidfree-BSA.
in the presence or the absence of 50pg/ml of an irrelevant antibody or of a purified function blocking antibody (clone 3A3) against the rat a 1 ~i 1 integrin (a gift of S.Carbonetto). PC12 cell differentiation experiments were done i;n 96 well plates in duplicate, and each experimentwas repeatedaminimum of three times.
To culture cerebellar granule cells, 3 - 4 rats from P3 to P7 were decapitated. The cerebellum was removed and placed in ll~iEM-HEPES where underlying tissue and the meninges was removed The cerebellumwas cut into small pieces and treatedwith 0.125 % trypsin in MEM-HEPES for 20' at 37°C.
S~UBSTrTUTE SHEET (RULE 26) Thetissuewasthentrituratedwithafirepolishedpasteurpipettetobreakupanyclumpsofti ssue. The cells were spun down at 1500 rpm for 10', and the pellet was resuspended in MEM-HEPES with 2mM
EDTA. The cell suspensionwas placed on an iso-osmotic percoll gradientwith 60%
and3 5% percoll, centrifugedforl5'at2300rpm,andtheinterfacebetweenthe60%and35%percollwascollecte d. Cells werewashedonce, andresu;~pendedinDMEMwithl0%FBS,vitamins,andpeniciljinlstreptomycinin the pres ence or absence of 20 p,glml C3 transferase. Cells were placed in 4-chamber, chamber slides coated withpoly-1-lysine or laminin andtreatedwith spots of MAG ormyelin.
200,000 cells per chamber were plated.
Preparation of browth substrates Poly-1-lysine was obtained. from Sigma (St. Louis, Mo). Laminin was prepared from EHS tumors (Paulsson and Lindblom ( 1994}. Cell biology: A laboratory handbook, Academic Press, pp589-594) and collagen fromrattails (Greene, et al., ( 1987) Meth. Enzymolo~y 147, 207-216).
Myelinwasmadefrom bovine brain corpus callosum, and native MAG was pwified from myelin after extraction in 1%
octylglucoside and separation by ion exchange chromatography (McKerracher, L., et al., ( 1994) Neuron 13, 805-811}. This nativeMAGhas some additional proteins, including some tenascin (Xiao, Z., et al., (1997} Neurosci. Abstr. ;?3, 1994). Recombinant MAG was made in baculovirus as described (McKemacher, L., et al., (1994) Neuron 13 805-811).
Test substrate were preparf;d as uniform substrates in 96-well plates or 4-chambered slides, or as spots on 18 mm glass coverslips. First, poly-L-lysine was coated by incubation of 100 p,glml for 3 hours at 37°C,andthewellsorcove~~slipswerewashedwithwaterandch-ied.
Lamininsubstrateswereprepared byincubating25pg/mllamininonpoly-L-lysinecoateddishesfor3hoursat37°C.
SolidMAGormyelin substrateswerepreparedb;tdryingdownMAGovernight,orincubatingalOmglmlmyelinsolut ionfor 3hoursonpolylysinecoatedsubstrates.For96-wellpiates,1-4 ugofeitherrecombinantMAG(rMAG) ~ofnaativeMAGpelwellwaused FormixedlamininlmyelinorlamininIMA.Gsubstrata, 8 pgofinh~itory SUBSTITUTE SHEET (RULE 2B) proteins and 10 ~,g of laminin were dried down on 96-well plates precoated with polylysine. For 4-chamberedchamberslides, 40 p,gMAGperchamberwasused, andfor 100mmplates 0.6-1 mgofMAG
was drieddown. Spots of MAG on coverslips were generatedby plaxing of 2 mg/ml recombinant MAG
on polylysine for 3-4 hour in ahumid chamber at 37°C. Collagen substrates were made by incubating 10-15 ~,glml of rat tail collagen fo:r 3 hours at 37°C.
Immunocytochemistry PC 12 cells were visualized by phase contrast microscopy, or following labelling with the lipophilic fluorescent dye, DiI (McKt;rracher, L., et al., ( 1994) Neecron 13 805-811 ).
Granule cells werevisua>;zed byimmunocytochemistry, Following l2-24hoursinculture,cellswerefixedfor30'atroomtemperatiu~e in 4% parafomialdehyde, 0. 5% glutaraldehyde, 0.1 M phosphate buffer.
Following fixation, cells were washed 3 X 5' with PBS and then blocked for 1 hour at room temperature in 3 %BSA, 0.1 % Triton-X
100. Granule cell cultureswe<~e incubatedovelnightwith a polyclonal anti-rMAG
antibody (called 57A++) to label MAGspots. The IvIAG antibody was detected using anFITC conjugated secondary antibody.
Rhodamine conjugated ph;alloidin was diluted 1: 200 with the s econdaiy antibody to label granule cell actin filaments.
C3 transferase preparation and use TheplasmidpGEX2T-C3 codingforthe GST-C3 fusionproteinwas obtainedfromA. Hall (London).
Recombinant C3 was puri~~ed as described by Dillon and Feig (Met. Enzymology, ( 1994), 256, pp 174-184).Afterfusionproteincleavageby thrombin,thrombinwasremovedbyincubatingtheproteinsolution 1 hour onice wifih 100 ~.1 of°p-aminobenzamidine agarose-beads (Sigma).
The C3 solutionwas desalted 2:i on PD 10 column (Pharmacia) with PBS, and sterilizedthmugh a 0.22 E.un filter. The C3 concentrationwas evaluatedby Lowry assay (DC protein assay, Bio-Rad) andtoxinpuritywas controlledby SDS-PAGE
analysis.
SUBSTtTU?'E SHEET (RULE 26) TotesttheeffectofC3ont~ieoutgrowthonPCl2cells, C3transferasewasscrapeloadedintothecells beforeplating on appropriatesubstrates. Cellswere grownto confluenceinserumcontainingmediain 6 well plates. Cells werewaslred once with scraping buffer ( 114mM KCI, l 5mM
NaCI, 5.5 mM MgCl2, l OmM Tris-HCl). Cells wme then scrapedwith arubber policeman into 0.5 ml scraping buffer inthe presence or absence of 20~ p,gJml C'.3 transferase. The cells were pelleted, and resuspended in 2 ml DMEM, l % FBS, and 50 n.g/ml nerve growth factorbefore plating. 10 p,g/ml C3 was addedto scrape loaded cells. Cells were ~~ifferentiated for 48 hours then fixed in 4 %
paraformaldehyde, 0.5 glutaraldehyde, 0.1 M P04 buffer.
10 Membrane Translocation Assay for IthoA
PC 12 cells were collected amdresuspended in DMEM, 0.1 % B SA, SOnglml NGF, then plated on 100 mm dishes coatedwith collagen or MAG, or left in suspension. Two hours later, cells werewashedwith ice cold PBS + protease inhibitors (1 pg/ml aprotinin, l p,g/ml leupeptin, l p,g/ml antipain, l ~tglml 15 pepstatin).
Cellswerethenscrapedinto5mlPBS+proteaseinhibitors,andthecellswerepelletedand washedwith PBS + protease inhibitors. The cell pellets weremechanically homogenizedby 25 strokes in a glass-teffon homogenizer, the homogenate centrifuged for 20 min at 8,000 rpm, andthe cell debris inthe pelletwas discarded. The s~upernatantwas centrifuged for 1 hour at 100,000 x g to separate membrane and cytosolic fractions. ll~Iembrane pellets were washed 1 X with PBS +
protease inhibitors and _ 20 resuspendedinPBS with0..5 % SDS, and 50-100 pg ofmembraneproteinwas analyzedby SDS-PAGE
on 12 % gels. Gels were tr~rnsferredto Protran nitrocellulose membrane and stainedwith Ponceau S.
Blotswereblockedfor 1 horrrin 5 % skimmilkinTBS, andprobedovemightwithRhoA
antibody diluted 1:200 in 1.5 % skim milk in TBS. Rho A antibody was detected by using an alkaline phosphatase conjugated secondary antaibody and an alkaline phosphatase detection kit (Gibco-BRL).
Growth inhibition of PC,'12 cells and its modulation byNGF and laminin SUBSTmJTE SHEET (RULE 2B) PC 12 cells typically extend neurites in respons a to NGF, butwhen plated on myelin substrates the cells remainroundand donotextendneurites (Moskowitz, P.F., etal., (1997) J.
Neurosci. Res. 34,129-134.) (Fig. 2). MAG is apotentinhibitorofaxon growthpresentinmyelin. We observedthatPC 12 cells plated onsubstrates ofMAGalsoremained.rounded{Fig.l), afinding in contrastto areportthatPCl2 cells are :i not responsive to MAG (:Bartsch,1:J., et al., (1995) Neuron 15,1375-1381).
To further examine the response of PC12 cells to MAG, we plated three different lines of PC12 cells on both native and recombinantMAGsubstrates inthepresenceofNGF. All ofthelines ofPC 12 cells showedreducedcell spreading, andmost cellsn~mainedroundedwithoutneu~ites.
However,withincreasingtime, someneurites were able to extend on MAGsubstrates (see below). We also observedthat different preparations of MAGcandifferintheirpotencytoinhibitneuritegrowth,andthattheactivityofMAGisredi~
cedorlost upon freeze-thaw.
Laminin is known to override completely, growth inhibition of NG 108 cells by myelin (David, S., et al., (1995)J. Neurosci. Res.42,594-602).Similarly,wefoundthatPCl2cellsareabletoextendneurites on mixedrnyelin andlami:nin substrates or on mixed laminin/MAG substrates {Fig.2}. To determine if signalling throughintegrins is responsible for overriding growth inhibition by myelin, we used the integrin funetionblockingantibocfiy3A3raisedagainstthealsubunitextracellulardomain.
Previousstudieshave documented that a 1131 inte;grin is the dominant integnn expressedby PC 12 cells, andthat the 3A3 antibody blocks PC12 cell neuiite growth on laminin (Tomaselli, K.J., et al., (1990) Neuron 5, 651-662). We.
plated PC 12 cells onmixe;dmyelin and laminin substrates, in the pr esence of the 3A3 antibody, orwith a non-specific IgG antibody as a control. The 3A3 antibody blockedneurite extension on both andlaminin andthe mixed myelinllanninin substrates (Fig. 2). On MAG or onmyelin substrates the cells remained rounded. The observation that the 3A3 antibody restores growth inhibition on mixed substrates demonstrates that laminin does not override growth inhibition by masldngthe inhibitory domain of MAG, 2.5 but that signals elicited through integrins receptors are responsible.
Effect of C3 Transferase on PC12 cells SUBSTITUTE SHEET (RULE 26) To investigatepossi'ble intracellulartargets thatmay override growth inhibition by myelin andby MAG, we focused onthe small GTP~~s a Rho which is knownto play arole in convergent signalling pathways that affectmorphologyandmotility(Hall,A.,(1996)Ann. Rev. CellBiol. 10,31-54).WeinactivatedRho in PC12 cells by scrape loading them with the bacterial toxin C3 before plating the cells on the test substrates. C3 is known to inactivate Rho through ADP ribosylation (Udagawa, T. andMcIntyre, B. W.
(1996)J.Biol.Chem.271,12542-12548).Oncontrolsubstratesofpolylysineandlaminin,treatmentwith C3 potentiatedboththenurnberofceliswithneurites andthelengthofneurites fromcells (Fig.3). OnMAG
andmyelin substrates where neurite formation is inhibited, C3 has a dramatic effect on the ability to extend neurites (Fig 3). When treated with C3, abouthalf of the PC 12 cells plated on eitherrMAG orn~tive MAG
hadneurites of approximately 1 celhbody diameter. In contrast, the untreated cells remainedroundedand clumped. Similarly, PC12 cellsplatedonmyelinremainedrounded, butthe additionof C3 allowedneurites to extend directly on the rriyelin substrate. These results demonstt~ate that C3 treatment elicits neurite growth from PC12 cells plated on growth inhibitory myelin or MAG substrates.
Growth of dominant-nE:gative Rho-transfected cells on MAG substrates PC 12 cells transfectedwith constitutively active RhoA (V l4GRhoA), and PC 12 cells transfectedwith dominant negarive RboA (l~ 19TRhoA), and the mock-transfected cells, were examined fortheir ability toextendneuritesondifferenttestsubstrates.Cellswiththeconstitutivelyactivemutat ion,Vl4GRhoA
cells, differentiatedpoorly on all substrates, including poly-L-lysine andlaminin. Thetreatment of the V l4GRhoA cells with C3 allowedthe growth of some shoitneurites on all of the test substrates, including MAG.
2.'> Inthe same series of experiments the response of dominantnegative Rho-transfected cells, N l9TRhoA
cells; to MAG and myelin substrates was examined. When Nl9TRhoA cells were plated on MAG
substrates, they spread arid did not remain rounded as did the mock iransfected PC 12 cells. A small su~srrru~ sH~ (RUt.s Zs~

number of cells had short neurites, an effect that was observed on both the rMAG and native MAG
substrates (Fig.3}.
C3 treatment of mock transfi;cted andN l 9TRhoA cells had a dramatic effect of neurite outgrowth as most cells had extensive neurites (Fig.3). 'The effect of C3 onNl9TRhoA cells was muchmore markedthan theeffectonthemocktransfectedcells.
Therefore,thecombinationofC3treatmentandtransfectionof dominant negative Rho elicited excellent outgrowth of neurites from PC 12 cells plated on inhibitory MAG
(and myelin) substrates.
Effect of C3 on Primary Cells To test the involvement of Rho in the response of primary neurons to MAG and to myelin substrates, cerebellar granule neurons were plated on test substrates andtreatedwith C3.
Neurite outgrowth from these cells was known to be; inhibited by MAG {Li, VL, et al., ( 1996) J.
Neurosci. Res. 46 404-414) and the C3 stimulated growth of neurites from the granule cells on both permissive and inhibitory substrates.
The growth substrate influences the cellular location of Rho Rho is associated with the plLasma membrane when it is in an acrivatedstate, andit moves into the cytosolic fraction when it is in the GI~P-bound inactive state. To determine if the growth substrate influences the cellular localization of Rho, cells were either left in suspension or platedon MAG or collagen substrates, andpreparedmembranes fr~omthe cells two hours later. Itwas shown that Rho was principally localized inthe cytosolic fiactionwhen cells wereplated on collagen, agrovvthpermissive substrate. However, Rho was associatedwith the ph~sma membrane when cells where grown in suspension andwhen cells were plated on MAG (Fig. 4}.
E:KAMPLE II: IN'VIVO DEMONSTRATIONS
SUBSTITUTE SHEET (RULE 26) WO 99/23113 PCT/CA9$/01013 1. Effect of C3 on cultured retinal neurons To testthe involvement of Rho in the response of primary neurons to MAG andto myelin substrates, we purifiedretinalneuronsandltreatedthemwithC3.
Neuriteoutgrowthfromthesecellswasinhibitedby MAG (Fig. 5a). As with PC.12 cells, tr. eatment of retinal neurons cells with C3 allowed neurite extension on the growth inhibitory MAG substrates to an extent similar to that observed on control substrates (Fig.
Sb and S) To ensure that the effect of C3 treatment resulted from uptake of C3 into the cells, we ex~nined by Western blot the electrophoreti c mobility of Rho in PC 12 cells and retinal neurons treatedwith C3 (Fig.

6). It has previously been shown thatADP-ribosylation of Rho results in decreas edrnobility of Rho on SDS-acrylamide gels (MethodEnzymol. Vof 256, Chapter22 pg198). For our experiments, PC 12 cells were scrape-loadedwith (:3 orwith scrape-loading buffer as a control, and cell lysates were prepared after 48 hours in culture. VVestem blots of the lysates with anti-RhoA
antibody revealedan increase inthe apparentmolecularweight.in cells treatedwith C3. As a control forthe specificity of the effect, weprobed the same blots for another small GT'Pase of the Rho family, Cdc42. Cdc42 didnotshow any change in mobility upon treatment with C3.
2(i Tocultureretinalneurons,retinasw~,~reremovedfiomPl-PSratpups,andthecells weredissociatedwith 12.5 U papain/ml in Hanks balanced salts solution, 0.2 mgJml DL cysteine and 20 ~g/ml bovine serum albumin. The dissociated cells were plated on test substrates in the presence of 50 ~,glml BDNF in DMEM with 10% FBS, vitamins, aradpenicillin/streptomycin in the presence or abs ence of 50 p,g/ml C3 transferase. Neurons were visualized by fluorescent microscopy with anti-~iIII
tubulin antibody.
2:i Z. Effects of C3 on retunal ganglion cell axon growth in vivo SUBSTITUTE SHEET (RULE 2B) _ WO 99123113 PCT/CA98/01013 To explore the possibility that treatment of damaged axons with C3 might fosterregeneration in vivo, we examined regeneration of retinal ganglion cell (RGC) axons in the optic nerve 2 weeks after optic nerve crush. Recently, it has been shown that microlesions in the CNS reduce the extent of the glial scar and allow axons access to CNS white matter distal to the lesion (Davies, S.J.A., et al. (1997) Nature 390, 680-683). To make microlesions of optic nerve, 10.0 sutures were used to axotomize RGC axons by constriction (Fig. 7a). Retrograde labeling of RGCs from the superior colliculus (not shown), as well as anterograde labeling techniques (eg., Fig. 7a) verified that RGC axons were effectively axotomized To apply C3 to crushed nerves, Gelfoam soaked with 2 mglml C3 was wrapped aroundthe left optic nerve at the crush site, and two F?lvax tubes, each loaded with 20 ~.g of C3 were positioned for sustained slow 1 (I release (Fig. 7a). Twelve ~~nimals were treatedwith C3, and a further 8 animals weretreatedv~ith PBS as controls. Crushed and regenerating axons were visualized by anterograde labeling with choleratoxin injected into the eye 12 days after optic nerve crush (Fig. ?a). Fourteen days after optic nerve crush, longitudinal cryostat sections of the optic nerves were examined by fluorescent microscopy for immunoreactivity to cholera toxin to detect anterogradely labeled RGC axons.
l:i In control optic nerves that: received optic nerve crash alone, no RGC axons extendedpastthe crushsite (n= 3 animals). In control animals treated with PBS- Elvax pellets and gelfoam, the crush site was easily detectedwhere most anten~gradely labeled axons stopped abmptly (Fig. 7c).
However, in these animals, afewaxonsdidextendpastthecrush(Fig.7c,arrows),andthenumberofaxonsthatregenerate dvaried 20 firm animal to animal. The application of Gelfoam and Elvax tubes may have altered the responseto injury.
Nonetheless, the respon:;e to C3 treatment applied with this lesion paradigm was dramatic.
We observedthat C3 treatment allowed many RGC axons to grow past the region of the lesion. In 7 of 12 C3-treated animals, the; lesion site was not clearly deli ned because of the large numbers of axons that 2 S extended through the site. (Fig. 7d and e). Many of the axons that extendedpastthe lesion site showed a twisted path of growth, supporting their identification as regenerating axons (Fig. 7f). A quantitative comparison of C3 andPBS treated animals revealedthatmore fibers grew pastthe lesionsite after C3 suesmuTe sHEEr cRU~,.s zs~

treatmentthan after PBS treatment (Fig. 7b). For this analys is we made a cons ervative estimate of the lesion site based on morphology, and counted the number of fibers in the distal optic nerve in 14 ~m sections. Seven of I2 C3-treated animals showedatleastonesectionwith 10-20 axons extending250 E.~m past the crush, compared with 1 of 8 of the PBS-treated controls (Fig.

7). In some animals regenerating axons were observed up to 1 mm from the crush, an extent of regeneration similar to that observedinmouseopticnerveaftertreatmentwithlN-1 antibodytoblockmyelininhibitorswherefibers extended up to 750 E,u~n ((Bartsch., U., et al., (1995) Neuron 15 1375-1381).
C3 treatment of crushed optic nerve in adult rats Rats were anesthetizedwif h 0.6 ml/kg hypnom~, 2. 5 mg/kg diazepan and 3 5 mg/kg ketamin. TAe left optic nervewas expos edby asupraorbital. approach, the optic nerve sheath slit longitudinally, the optic nerve liftedoutandcrushed 1 mmfromthe giobeby constrictionwith a lO.Osutureheldfor60seconds (Fig.4a).
For C3 treatment andbufffx controls, Gelfoam soaked in PBS or 2 mg/ml C3 transferasewas placed on thenerve atthe lesion site.. Two 3 mmlong tubes of Elvax (Sefton, et al., ( 1984)) loadedwithbufferor 1_'~
20~,gC3wereinsertedintheGelfoamnearthenerveforcontinuedslowreleaseofC3(Fig.4b).
Twelve days after crush, 5 ~,1 of 1 °ro cholera toxin ~i subunit (ListBiological laboratories, Inc., Cambell, CA) was injectedintothevitreousto anterogr'adely label retinal ganglioncell axons (Fig. 4c). Twoweeks afteroptic nerve crushthe animals were fixedby perfusionwith 4% paraformaldehyde, andthe eyewith attached optic nerve was removed ~andpostfixed in 4% paraformaldeliyde. Longitudinal cryostat sections were processedforimmunorea.ctivitytocholeratoxinwithgoatanti-choleratoxinatl:I2,000(ListBiol.Labs Inc, CA), followed byrabb~it anti-goat biotinylated antibody ( 1:200, Vector Labs, Burlingame, CA), ~d DTAF-streptavidin (1: 500, Jackson Immunoresearch Laboratories).
Discussion 2:i Here we reportthat the small GTP binding protein Rho is likely to be a key intermediate in the neuronal response to neurite growth inhibitory signals. Treatment of cultured PC 12 cells, retinal neurons, and cerebellar granule cells with C3 enzyme to inactivate Rho allowedneurites to extend directly on inhibitory SUBSTITUTE SHEET (RULE 28) substi ates of MAG or myelin . Als o, P C I 2 cells trans fe cted with dominant negative RhoA extended neurites on MAG substrab~. Therefore, inactivation of Rho was sufficient to allow neurite growth on MAG
or myelin substrates when neurons were grown in the presence of neurotr~ophic factors.
Further, our observations of microlesioned optic nerves after treatmentwith C3 provide the first evidence that the inactivation of R.ho in axons and non-neuronal cells near the site of lesion can help foster regeneration after injury. ~VVhile the in vitro experiments showed that C3 can affect directly the growth of neurites fromretinal cells, itis likelythatthe effects we observed after application of C3 to the opticnerve in vivo we more complex. C3 may affect other non-neuronal cells, such as macrophages and astrocytes, andthesepossibilitiesneed.tobefiutYterexamined.Nonetheless,ourdataprovidecompel linge~hdencethat C3 can promote neurite growth on inhibitory substrates irt virro, and helps to overcome growth inhibition in vivo.
Regulation of neurite l;rowth by Itilo family mernbcrs I 5 Not all of the myelin-derived inhibitory molecules we known to date. and less is known aboutthe neuronal receptors for growth inhibitory molecules. Several different MAG receptou hav a been identified (Collins et al. 1997; Yang etal.1996), and additional neuronal receptors to myelin inhibitors are likely to exist.
Targeting intracellular siyaling mechanisms converging to Rho rather than indivi dual receptors may be the mostpractical way to overcome growth inhibition irr vi vo. The advantage of inactivating Rho to stimulate 0 regeneration is that axons can regenerate directly on the native ten ain of the CNS, and thus may be more likely to fmd their natmval target'.
Both MAG and the other myelin-derived yowth inhibitory p~nteins block axon extension by causing growth cone collapse (Li, M., Eat al., (1996) J. Neurosci. Res. 46, 404-414;
Bandtlow, C.E., et al., (1993) 2 5 Science 259, 80-83). These finding suggested to us that growth cone collapse by the myelin-derived inhibitors might be regulated by ltho. Moreover, in non-nem~onal cells, Rho participates in integrin-dependent signaling (Laudanna, C., et al., ( 1996) Science 27I, 98 I-983.;
Udagawa. T. and McIntyre, SUBSTITUTE SHEET (RULE 28) B.W. {1996) J. Biol. Chem. 271,12542-12548.). Togetherwith the observationthatlaminin can override myelin-derivedinhibition;, wehypothesizedthat small GTPases of the Rho family might play arole in integrating signaling from positive and negative gr owth cues. To investigate this possibility, u~ehavemade use of the ADP--ribosyl transferase C3 fi om Clostridium botul inum that efficiently inactivates Rho without affecting Rac and Cdc42, tu~o othermemheu of the Rho family ( Udagawa; T. and McIntyre, B. W. (1996) J. Biol. Chem. 271,12542-12548) andfoundthat C3 treatment fosters neurite growthinthepresence of growthinlu'bitors. Moreovw, immunocytochemicalobsemations indicatethatRhoproteinis concentrated at the f-tlopodial tips of groocrth cones ian adhes ion struct<u~es called point contacts (Renaudin et a1.1998).
Therefore, our in vitro results suggestthe Rho signaling pathway is a key target forregulating growth cone motility and stimulating regeneration.
Moreover, this datais relevantto the finding of Song et al (Song et al.
Science? 81:1515-1518 ( I998)) who report that growth cone repulsion by MAG can be converted into attraction by elevation of intracellular cAMP levels to activate protein kinase A (PKA). Experiments with non-neuronal cells has implicated cAMP inthe regulation of Rho because elevation of cAIVIP inhibits Rho activation (Laudanna, C., etal., (1996) Science 2T 1, 981-983). In PKA deficient PC 12 cells, elevation of cAMP fails toprotect from the activation of Rho by lysophosphatidic acid (Tigyi, G.; et al., (1996) J. Neccrochem. 66, 537-548), afindingthatsu~;gests thatPKA.-dependentregulation ofRho occurs inneural cells aswell.
Therefore, the cAMP-dependentregulationis likely to be upstr eam of Rho (Laudanna, C., et al., ( 1996) _ Science 271; 981-983).
The non-neuronal response to optic nerve injury Remarkably, we observed that RGC axons crossed the lesion site to enter the distal optic nerve after treatment of injured optic nerve with C3. Some axons gr ew up to I mmpastthe site of lesion. This distance is comparabletothemaxi:maldistancesobservedfollowingtreatmentofopticnervewithIN-1 antibody (Bartsch, U., et al., ( 1995) Neuron 1 5,1375-1381) Themoststtiking feature of ourresults was the large SUBSTITUTE SHEET (RULE 2B) number of axons thatwere; able to cross the lesion site compared to PBS-treated controls (see Fig. 7).
Therefore, it is appears that C3 was also able to promote axon growth on inhibitory proteins pr went at the filial scar, indicatingthattayeting the Rho signalingpathvay as widespread efficacy in stimulating axon regeneration after injury.
SUBSTITUTE SHEET (RULE 2fi)

Claims (22)

We Claim:

1. An antagonist of one or more of Rho family members characterized by the ability to elicit neurite outgrowth from cultured neurons in an assay method, comprising the steps of:
(a) culturing neurons on a growth permissive substrate that incorporates a growth-inhibiting amount of a Rho family member; and (b) exposing the cultured neurons of step a) to a candidate Rho family member antagonist agent in an amount and for a period sufficient prospectively to permit growth of the neurons;
thereby identifying as Rho family antagonists the candidates of step b) which elicit neurite outgrowth from the cultured neurons of step a).

2. The antagonist according to clam 1, wherein said Rho family members we selected from the group comprising RhoA, RhoB. RhoC, Rac, cdc42 and Rho-associated protein kinase.

3. The antagonist according to claim 1, wherein said interaction with the Rho regulatory pathway is via interaction with GTP/GDP cycle.

4. The antagonist according to claim 3, wherein the interaction with the GTP/GDP cycle involves GTP/GDP exchange proteins (GEP's); GDP dissociation inhibitors (GDI's); or GTPase activating protein (GAP) to regulate Rho activity.

5. The use of antagonists of one or more Rho family members to promote neural growth by inhibiting Rho family members in the central nervous system.

6. The use of ADP-ribosyl transferase C3, or other closely related toxins, to promote neural growth by inhibiting one or more Rho family members in the central nervous system.

7. The use of a GTPase activating protein that is specific to Rho to convert GTP-bound active Rho to GDP-bound inactive Rho.

8. Theuse of ADP-ribosyl transferase C3 according to claim 6,wherein said related toxins are toxins A or B.

9. The use of biologically active fragments of ADP-ribosyl transferase a C3, analogs and derivatives thereof, to promote neural growth by inhibiting one or more Rho family members in the central nervous system.

10. The use of Y27632,orrelated compounds,to promote neural growth by inhibiting Rho-associated kinase in the central nervous system.

11. The use of genetically mutated forms of Rho, to promote neural growth by inhibiting one or more Rho family members in the central nervous system.

12. The use of dominant negative Rho to inactivate Rho, to foster axon growth in the central nervous system.

13. The genetically mutated form of Rho according to claim 11, wherein the mutation is in the effector domain, A-37, thereby preventing GTP exchange.

14. Theuse of GDP dissociation inhibitors, orstimulation thereof, to inhibit the dissociation of GDP
from Rho and thereby prevent the binding of GTP necessary for the activation of Rho.

15. The use of compounds that promote Rho binding to GTPase inhibiting protein (GDI), thereby antagonizing the ability of Rho to be translocated to the plasma membrane.

16. A method for producing Rho antagonists from Rho family members, fragments, analogs of derivatives by peptide synthesis or by recombinant DNA expression of either a truncated domain of Rho family members, incorporating one ormore L- or D-amino acid substitutions, or of intact Rho family members using standard recombinant procedures and selecting antagonist characterized by the ability to elicit neurite outgrowth from culturedneurons in an assay method, comprising the steps of:
(a) culturing neurons on a growth permissive substrate that incorporates a growth-inhibiting amount of a Rho family member; and (b) exposing the cultured neurons of step a) to a candidate Rho family member antagonist agent in an amount and for a period sufficient prospectively to permit growth of the neurons;
thereby identifying as Rho family antagonists the candidates of step b) which elicitneurite outgrowth from the cultured neurons of step a).

17. The antagonist according to claim 1, wherein derivatives of Rho family members, Rho family members fragments and Rho family members analogs can be generated by chemical reaction of the parentsubstance to incorporate the desired derivitizing group, such as N-ternunal, C-terminal and infra-residue modifying groups that have the effect of masking or stabilizing the substance or target amino acids within it.

18. An antagonist of one or more of Rho family members, characterized by following properties:
(a) blocks growth inhibition of neurites by myelin or myelin proteins; and (b) interferes with Rho family members-mediated growth inhibition as competitive but non-functional mimics of endogenous Rho family members;

19. A composition comprising a therapeutically effective amount of the composition of claim 1 in a suitable pharmacologic carrier.

20. An assay methodauseful to identify Rho family member antagonist agents that suppress inhibition of neuron growth, comprising the steps of:

(a) culturing neurons on a growth permissive substrate that incorporates agrowth-inhibiting amount of a Rho family member, and (b) exposing the culturedneurons of step a) to a candidate Rho family member antagonist agent in an amount and for a period sufficient prospectively to permit growth of the neurons;
thereby identifying as Rho family antagonists the candidates of step b) which elicitneurite outgrowth from the cultured neurons of step a).

21. A kit to test for Rho family antagonists that can be used to promote neurite growth comprising the components necessary to work the method of claim 16, in a suitable container.

22. A method to suppress the inhibition of neuron, comprising the steps of delivering, to the nerve growth environment, a Rho family antagonist in an amount effective to reverse myelin inhibition.