Note: Descriptions are shown in the official language in which they were submitted.
CA 02214841 1997-10-31
RHO FAMILY ANTAGONISTS AND l H~l~ USE TO
BLOCK INHIBITION OF NEURITE OUTGROWTH
FIELD OF INVENTION
This invention relates to the regulation of growth of neurons in the Central Nervous System.
BACKGROUND
Following trauma in the adult central nervous system (CNS) of m~mm~l~, injured neurons do not
regenerate their transected axons. An important barrier to regeneration is the axon growth inhibitory
activity that is present in CNS myelin and that is also associated with the plasma membrane of
oligodendrocytes, the cells that synthesize myelin in the CNS (see Schwab, ef al., Ann. Rev.
Neurosci.,16,565 595,1993forreview). ThegrowthinhibitorypropertiesofCNSmyelinhavebeen
demonstrated in a number of di~el~ laboratories by a wide variety oftechniques, including plating
neurons on myelin substrates or cryostat sections of white matter, and observations of axon contact
with mature oligodendrocytes (Schwab et al., 1993). Therefore, it is well documented that adult
neurons cannot extend neurites over CNS myelin in vitro.
It has also been well documented that removing myelin in vivo improves the success of regenerative
growth over the native terrain of the CNS. Regeneration occurs after irradiation of newborn rats,
a procedure that kills oligodendrocytes and prevents the appearance of myelin proteins (Savio and
Schwab, Neurobiology, 87, 4130-4133, 1990). After such a procedure in rats and combined with a
corticospinal tract lesion, some corticospinal axons regrow long distances beyond the lesions. Also,
in a chick model of spinal cord repair, the onset of myelination correlates with a loss of its
regenerative ability of cut axons (Keirstead, et al., Proc. Nat. Acad. Sci. (USA), 89, 11664-11668,
1992). The removal of myelin with anti-galactocerebroside and complement in the embryonic chick
spinal cord extends the permissive period for axonal regeneration. These experiments demonstrate
a good correlation between myelination and the failure of axons to regenerate in the CNS.
CA 02214841 1997-10-31
Myelin inhibits axon growth because it contains at least several dirrelelll growth inhibitory proteins.
It has been well documented by us and by others that myelin-associated glycoprotein (MAG) has
potent growth inhibitory activity, both in vitro and in vivo (McKerracher et al. 1994; Mukhopadhyay
et al. 1994; Li et al. 1996; Schafer et al. 1996). A high molecular weight inhibitory activity has been
characterized by Schwab and collaborators, and neutralization ofthis activity with the IN-l antibody
allows some axons to regenerate in white matter (Schwab et al. 1993; Bregman et al. 1995). We also
have evidence that there is an additional growth inhibitory protein in myelin (Xiao et al. 1997).
Clearly, there are multiple inhibitory proteins that stop axon legellel~ion in m~mm~ n CNS myelin.
While axons damaged in the CNS in vivo do not typically regrow, there have been some reports of
long distance axon extension in adult white matter. Such growth has been observed following
transplantation of grafted neural tissue (Wictorin et al. 1990; Davies et al. 1994; Isacson and Deacon,
1996), suggesting that embryonic neurons primed for rapid extension of axons may be less susceptible
to growth inhibition. Some embryonic neurons are not susceptible to MAG (Mukhopadhyay et al.
1994), but most elllblyonic neurons are inhibited by the other myelin inhibitors (Schwab et al. 1993).
Therefore, in the cases when axons are able to extend on myelin, sign~lin~ through intracellular
p~hw~y~ may play an important role in stim~ ting or blocking the inhibition of axon growth. For
example, it is known that lam~nin is able to stim~ te rapid neurite growth (Kuhn et al. 1995), and we
have docllm~nted that when laminin is present in sufficient concentration, neurites can extend directly
on myelin substrates. These fin~lin~ suggest the possibility that the stimulation of the integrins, the
receptors for l~minin, is sufficient to allow axon growth on myelin. Similarly, it has been documented
that when the adhesion molecule Ll is expressed ectopically on astrocytes, it can partially overcome
their non-permissive substrate properties (Mohajeri et al. 1996). 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 that causes dramatic
rearrangements to the growth cone cytoskeleton (Bandtlow et al. 1993; Fan et al. 1993; Li et al.
CA 02214841 1997-10-31
1996). One family of proteins that has been implicated in receptor-mediated sign~ling to the
cytoskeleton is the small GTPases of the Rho family (Hall, 1996). In non-neuronal cells it has been
clearly documented that mutations in Rho family members that include Rho, Rac and cdc42, affect
adhesion, actin polymerization, and the formation of lamellipodia and filopodia, which are all process
important to motility (Nobes and Hall, 1995). There is now good evidence that members of the Rho
family regulate axon outgrowth in development. Mutations in Rho-related family members block the
extension of axons in Drosophila (Luo et al. 1994) and disrupt axonal p~tl~fin(ling in C. elegans
(Zipkin et al. 1997). More recently it has been shown that the guidance molecule collapsin acts
through a Rac-dependent mech~ni~m (Jin and Strittmatter, 1997). In transgenic mice that express
constitutively active Rac in Purkinje cells, there are alterations in the development of axon t~rmin~l~
and dendritic arborizations (Luo et al. 1996). Consistent with the observations in vivo, it was found
that dominant negative Rac expressed in PC12 cells disrupts neurite outgrowth in response to NGF
(Hutchens et al. 1997). Also, treatment of PC12 cells with Iysophosphatidic acid, a mitogenic
phospholipid, causes neurite retraction that is mediated by Rho (Tigyi et al. 1996). Therefore,
di~ere,ll members of the Rho family can exert distinct effects on neurite growth, and in PC12 cells
the activation of Rho is correlated with growth cone collapse. In non-neuronal cells, Rho participates
in integrin-dependent ~ign~lling (T ~ nn~ et al. 1996, Udagawa and McIntyre, 1996). The
possibility that Rho might play a role within the myelin-derived growth inhibitory system has been
studied (Jin and Strittm~tter (1997) J. Neurosci. 17:6256-6263). It was concluded, however, that
the inhibitory effects of myelin are not mediated by Rho family members.
A need remains for a means of inactivating the multiple inhibitory proteins present in myelin that
prevent axonal regrowth after injury in the CNS.
This background information is provided for the purpose of making known information believed by
the applicant to be of possible relevance to the present invention. No admission is necessarily
int~n~lerl, nor should be construed, that any of the preceding information con~itutes prior art against
the present invention.
CA 02214841 1997-10-31
SUMMARY OF THE INVENTION
The present invention relates to antagonists and inhibitors to members ofthe Rho family of proteins,
antibodies directed against the components of this system and diagnostic, therapeutic, and research
uses for each of these aspects. In particular, members of the Rho family of proteins serve as a
therapeutic target to foster regrowth of injured or degenerating axons in the CNS.
In accordance with the present invention, a pl erell~d embodiment relates to antagonists and inhibitors
of members of the Rho family of proteins and their use as a means of blocking a common sign~ling
pathway used by the diverse growth inhibitory molecules. The antagonists and inhibitors may be
either peptides or small molecules.
In yet a further aspect of the present invention, Rho family members protein can be used to design
small molecules that antagonize and inhibit Rho family proteins, to block inhibition of neurite
outgrowth. In another aspect of the present 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.
In a further aspect of the present invention, inhibitors of the Rho family of proteins can be used to
block inhibition of neurite outgrowth and to suppress the myelin growth inhibitory system. Such
inhibitors could block exchange of the GTP/GDP cycle of Rho activation/inactivation.
A further embodiment involves a method of suppressing the inhibition of neuron growth, comprising
the steps of delivering to the nerve growth environment, antibodies directed against Rho family
members in an amount effective to reverse said inhibition.
In accordance with another aspect of the present invention, there is provided an assay method useful
to identify Rho farnily member antagonist agents that suppress inhibition of neuron growth,
comprising the steps of:
CA 02214841 1997-10-31
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).
In accordance with another aspect of the present invention, there is provided a method to suppress
the inhibition of neuron, colllplising the steps of delivering, to the nerve growth environment, a Rho
family antagonist in an amount effective to reverse said inhibition.
In another embodiment, the nucleic acids encoding Rho family members can be used in antisense
techniques and therapies.
In yet another embodiment, a kit is provided comprising components necessary to conduct the assay
method useful to screen Rho family antagonist agents.
Various other objects and advantages ofthe present invention will become apparent from the detailed
description of the invention.
BRIEF DESCRIPTION OF FIGURES
Figure 1. Treatment with C3 stim~ tes neurite out~rowth on inhibitory MAG substrates. A) PC 12
cells plated on MAG remained rounded and did not extend neurites. B) Cells plated on MAG in the
presence of C3 grew neurites. C) PC12 cells plated on polylysine (PLL) substrates as a positive
control.
Figure 2. Role of integrins in overriding growth inhibition by myelin. The anti-al integrin function
CA 02214841 1997-10-31
blocking antibody, 3A3, was used to determine if integrin function is necessary for laminin to override
growth inhibition by myelin or MAG. For experiments on myelin substrates (A-D), cells were
fluorescently labelled with DiI, and plated on myelin (A), polylysine (B~, or myelin +1 ~lg laminin (C
and D). Control IgG was added to samples A-C, the 3A3 antibody to D. Neurites do not extend on
myelin but grow on laminin or mixed laminin/myelin substrates. When 3A3 is added, laminin no
longer overrides growth inhibition by myelin. Panels (E-H) show by phase contrast cells plated on
recolllbh~ MAG (E), laminin (F), or recombinant MAG plus laminin (G and H), with control
antibody (E-G) orwith 3A3 (H). Integrin function is needed to override growth inhibition by MAG.
Figure 3. PC12 cells transfected with dominant negative Rho extend short neurites on MAG
substrates. Mock-transfected PC12 cells (a,c,e) or cells transfected with dominant-negative Rho
(b,d,f) were plated on laminin (a,b) or MAG (c-f). MAG inhibits neurite outgrowth (c), but dominant
negative Rho cells spread on MAG and some cells extend short neurites (d). Treatment with C3
further stim~ tes neurite outgrowth on MAG from both lines of cells (e,f).
Figure 4. Activation of Rho on MAG substrates. Activated Rho is associated with the plasma
membrane. To determine if activated Rho was detected under conditions where PC 12 cells do not
grow neurites, cells were grown in suspension or plated on MAG or collagen substrates. Two hours
later the plasma lllellll)l~1es were purified, the proteins separated by SDS PAGE, and the proteins
transfered to nitrocellulose and stained with Ponceau S. Rho A was detected on the blots by
immunoreactivity with anti-RhoA antibody. Immunoreactivity was strongest when cells were grown
in suspension or when cells were plated on MAG. Therefore, Rho A is more active when cells are
kept in suspension or plated on MAG than when plated on growth-permissive collagen.
DETAILED DESCRIPTION OF T~E INVENTION
This invention arises from the discovery that Rho family members are key molecules in re~ ting
inhibition by myelin proteins, and by MAG. Thus, this invention provides the advantage of identifying
CA 02214841 1997-10-31
an intracellular target, Rho family members, for all of the multiple inhibitory proteins that must be
inactivated to allow for growth on myelin. The method of this invention provides for inactivation of
Rho family members, thereby stim~ tin~ neurite growth on growth inhibitory substrates. Therefore,
antagonists that inactivate Rho family members in vivo should allow axon regeneration in the injured
or diseased CNS.
"Antagonist" refers to a pharm~ce~ltical agent which in accordance with the present invention which
inhibits at least on biological activity normally associate with Rho family members, that is blocking
or suppres~illg the inhibition of neuron growth. Antagonists which may be used in accordance with
the present invention include without limitation a Rho family members antibody or a binding fragment
of said antibody, a Rho family members fragment, a derivative of Rho family members 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 a pharm~ce~ltical agent, and is further characterized by the property of
suppressing Rho family members-mediated inhibition of neurite outgrowth.
The antagonist of Rho family members in accordance with the present invention is not limited to Rho
family members or its derivatives, but also includes the therapeutic application of all agents, referred
herein as pharmaceutical agents, which alter the biological activity ofthe 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 pharrnaceutical
agent required to produce a desired antagonist effect of the Rho family members biological activity.
The precise effective amount will vary with the nature of pharmaceutical agent used and may be
dete~mined by 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, RhoB, and RhoC. Other members ofthe Rho family that are d~ l;lled and
whose inhibition of activity allows for neurite outgrowth are comtemplated to be part of this
invention.
CA 02214841 1997-10-31
As used herein, the terms "Rho family member biological activity" refers to cellular events triggered
by, being of either biochemical or biophysical nature. The following list is provided, without
limitation, which discloses some of the known activities associated with contact-mediated growth
inhibition of neurite outgrowth, adhesion to neuronal cells, and promotion of neurite out growth from
new born dorsal root ganglion neurons.
As used herein, the term "biologically active", or reference to the biological activity of Rho family
members or, or polypeptide fragment thereof, refers to a polypeptide that is able to produce one of
the functional characteristics exhibited by Rho family members or its receptors described herein. In
one embodiment, biologically active proteins 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.
Based on the present evidence that Rho family members can affect growth inhibitory protein signals
in myelin, the means exist to identify agents and therapies that suppress myelin-mediated inhibition
of nerve growth. Further, one can exploit the growth 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 member antagonists will be inhibitors of GTPase activity. The
GTP/GDP GyGle of ~o family members activation/inactivation is re~ll~ted by a number of exchange
factors. Compounds that block exchange, thereby inactivating Rho family members are pleîelled
embodiments of this invention.
In another embodiment suitable Rho family member antagonist candidates are developed comprising
fragments, analogs and derivatives of Rho family members. Such candidates may interfere with Rho
CA 02214841 1997-10-31
family members-mediated growth inhibition as competitive but non-functional mimics of endogenous
Rho family members. From the amino acid sequence of Rho family members and from the cloned
DNA coding for it, it will be appreciated that Rho family members fragments can be produced either
by peptide synthesis or by recoll.l)inalll DNA expression of either a truncated domain of Rho family
members, or of intact Rho family members could be prepared using standard recombinant
procedures, that can then be digested enzymically in either a random or a site-selective manner.
Analogs of Rho family members or Rho farnily members fragments can be generated also by
recoll.l)inalll DNA techniques or by peptide synthesis, and will incorporate one or more, e.g. 1-5, I,-
or D-amino acid substitutions. Derivatives of Rho family members, Rho family members fragments
and Rho family members analogs can be generated by chemical reaction of the pare~t substance to
incorporate the desired derivatizing group, such as N-terminal, C-terminal and intra-residue modifying
groups that have the effect of m~ckin~; or stabilizing the substance or target amino acids within it.
In specific embodiments ofthe invention, candidate Rho family member antagonists include those that
are derived from a dete~ .nalion of the functionally active region(s) of a Rho family member. The
antibodies mentioned above and any others to be prepared against epitopes in Rho family members,
when found 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.,
J. Neurosci., 5: 1118-1126; Tropak et al., (1994) J. Neurochem., 62: 854-862). Thus, it can be
determined which regions of Rho family members GTPases recognized by substr~te molecules that
are involved in inhibition of neurite outgrowth. When those are known, synthetic peptides can be
prepared to be assayed as candidate antagonists of the Rho family members effect. Derivatives of
these can be prepared, inr,lllding those with selected amino acid substitutions tD pr~le desirable
properties to enhance their effectiveness as antagonists of the Rho family members candidate
functional regions of Rho family members can also be determined by the prepa~ ~lion of altered forms
of the Rho family members domains using recolllbhlall~ DNA technologies to produce deletion or
insertion m~lt~nts that can be expressed in various cell types as chimeric proteins that contain the Fc
portion of immunoglobulin G (Kelm et al., (1994) Curr. Biol., 4: 965-972). Alternatively, candidate
mutant forms of Rho family members can be expressed on cell surfaces by tralisfection ~-various
CA 02214841 1997-10-31
cultured cell types. All of the above forms of Rho family members, and forms that may be generated
by 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
pharm~ce~1tical composition which contains the Rho family member antagonist in an amount effective
to suppress Rho family l.~llll)el -mediated inhibition of nerve growth, in colllbinalion with a suitable
pharmaceutical carrier. Such compositions are useful, in accordance with another aspect of the
invention, to suppress Rho family member-inhibited nerve growth in patients diagnosed with a variety
of neurological disorder, conditions and ~ilments of the PNS and the CNS where treatment to
increase neurite extension, growth, or regeneration is desired, e.g., in patients with nervous system
damage. Patients suffering from traumatic disorders (including but not limited to spinal cord injuries,
spinal cord lesions, surgical nerve lesions or other CNS pathway lesions) damage secondary to
infarction, infection, exposure to toxic agents, m~lign~ncy, paraneoplastic syndromes, or patients with
various types of degenerative disorders ofthe central nervous system (Cutler, (1987) In: Scientific
AmericanMedicines,vol. 2, ScientificAmericanInc.,N.Y.,pp. 11-1-11-13)canbetreatedwithsuch
Rho family members antagonists. Examples of such disorders include but are not limited to Strokes,
Alzheimer's disease, Down's syndrome, Creutzfeldt-Jacob disease, kuru, Gerstman-Straussler
syndrome, scrapie, tr~n~mi~sihle mink encephalopathy, Hllntington's disease, Riley-Day familial
dysautonomia, multiple system atrophy, amylotrophic lateral sclerosis or Lou Gehrig's disease,
progressive supranuclear palsy, Parkinson's disease and the like. The Rho family members antagonists
may be used to promote the regeneration of CNS paLllw~ys, fiber systems and tracts. A(lmini~tration
of antibodies directed to an epitope of a Rho family member, or the binding portion thereof, or cells
secreting such antibodies can also be used to inhibit Rho family member function in patients. In a
particular embodiment of the invention, the Rho family members antagonist is used to promote the
regeneration of nerve fibers over long distances following spinal cord damage.
In another embodiment, the invention provides an assay method adapted to identify Rho family
CA 02214841 1997-10-31
member antagonists, that is agents that block or suppress the growth-inhibiting action of Rho family
members. In 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 a permissive substrate. Nerve cells suitable in this regard include neuroblastoma cells of the
NG108 lineage, such as NG108- 15, as well as other neuronal cell lines such as PC 12 cells ~American
Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 USA, ATCC accession NO.
CRL 1721), human neuroblastoma cells, and p.hllaly cultures of CNS or PNS neurons taken from
embryonic, postnatal or adult ~nim~l~ The nerve cells, for instance about 103 cells-microwell or
equivalent, are cultured on a growth permissive substrate, such as polylysine or l~minin, that is over-
layed with a ~rowth-inhibiting amount of Rho family members. The Rho family members
incorporated in the culture is suitably myelin-extracted Rho family members, although forms of Rho
family members other than endogenous forrns can be used provided they exhibit the Rhofamily
members property of inhibiting neuron growth when added to a substrate that is otherwise growth
permissive.
In this assay, c~n~i~late Rho family member antagonists, i.e., compounds that block the growth-
inhibiting effect of Rho family members, are added to the Rho family member-cont~ining tissue
culture pl ~rt;l ~bly in amount sufficient to neutralize the Rho family member growth-inhibiting activity,
that is between 1.5 and 15 ,ug of Rho family members antagonist per well co~ ining a density of
1000 NG108-15 cells/well cultured for 24 hr. in Dulbecco's minim~l essential medium. After
culturing for a period suff1cient for neurite outgrowth, e.g. 3-7 days, the culture is evaluated for
neurite outgrowth, and antagonists are thereby revealed as those candidates which elicit neurite
outgrowth. Desirably, candidates selected as Rho family member antagonists are those which elicit
neurite outgrowth to a statistically significant extent, e.g., in at least 50%, more desirably at least
60%, e.g. 70%, per 1,000 cultured neurons.
Other assay tests that could be used include without limitation the following: 1) The growth cone
collapse assay that is used to assess growth inhibitory activity of collapsin (Raper, J.A., and
Kapfhammer, J.P., (1990)Neuron, 2:21-29; Luo et al., (1993) Cell, 75:217-227) and of various other
CA 02214841 1997-10-31
inhibitory molecules agarashi, M. et al., (1993) Science, 259:77-79) whereby the test substance is
added to the culture medium and a loss of elaborate growth cone morphology is scored. 2) The use
of patterned substrates to assess substrate pl ~rerence (Walter, J. et al., (1987) Developmenlt, 101: 909-
913; Stahl et al., (1990) Neuron, 5:735-743) or avoidance of test substrates (Ethell, D.W. et al.,
(1993) Dev. BrainRes., 72: 1-8). 3) The expression of recolllbinalll proteins on a heterologous cell
surface, and the transfected cells are used in co-culture expelhll~ . The ability ofthe neurons to
extend neurites on the transfected cells is assessed (Mukhopadhyay et al., (1994) Neuron, 13:757-
767). 4) The use of sections of tissue, such as sections of CNS white matter, to assess molecules
that may modulate growth inhibition (Carbonetto et al., (1987) J. Neuroscience, 7:610-620; Savlo,
T. and Schwab, M.E., (1989) J. Neurosci., 9: 1126-1133). 5) Neurite retraction assays whereby test
substrates are applied to di~erel-liated neural cells for their ability to induce or inhibit the retraction
of previously extended neurites (Jalnink et al., (1994) J. Cell Bio., 126: 801 -810; Sudan, H. S. et al.,
(1992)Neuron, 8:363-375; Smalheiser, N. (1993) J. Neurochem., 61:340-342). 6) The repulsion
of cell-cell interactions by cell aggregation assays (Kelm, S. et al., (1994) Currenl Biology, 4:965-
972; Brady-Kainay, S. et al., (1993) J. Cell Biol., 4:961-972). 7) The use of nitrocellulose to
prepare substrates for growth assays to assess the ability of neural cells to extend neurites on the test
substrate (Laganeur, C. and Lemrnon, V., (1987)PNAS, 84:7753-7757; Dou, C-L and Levine, J.M.,
(1994) J. Neuroscience, 14:7616-7628).
Useful Rho family member antagonists include antibodies to Rho family members and the binding
fragments ofthose antibodies. Antibodies which are either monoclonal or polyclonal can be produced
which recognize Rho family members and its various epitopes using now routine procedures. For the
raising of antibody, various host animals can be immlmi~ed by injection with Rho family members or
~agment thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used
to increase the immunological response, depending on the host species, and including but not limited
to Freund's (complete and incomplete), mineral gels such as ~lllmimlm hydroxide, surface active
substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, d-~ - ophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-
Guerin).
CA 02214841 1997-10-31
Produ~tion of Antibodies Aga~nst the Components of Rho Fumily Members
Antibodies can be produced which recognize members of the Rho family. Such antibodies can be
polyclonal or monoclonal.Various procedures known in the art may be used for the production of
polyclonal antibodies to epitopes of Rho family members. For the production of antibody, various
host animals can be imm~lni7e~ by injection with a neurite growth regulatory factor protein, or a
synthetic protein, or fragment thereof, including but not limited to rabbits, mice, rats, etc. Various
adjuvants may be used to increase the immunological response, depending on the host species, and
including but not limited to Freund's (complete and incomplete), mineral gels such as all1mimlm
hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such
as BCG (bacille Calmette-Guerin) and corynebacterium parvum. A monoclonal antibody to an
epitope of a Rho family member can be prepared by using any technique which provides for the
production of antibody molecules by continuous cell lines in culture. These include but are not limited
to the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497),
and the more recent human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today
4: 72) and EBV-hybridoma technique (Cole et al.,1985, Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc., pp. 77-96). In a particular embodiment, the procedure described . may be used to
obtain mouse monoclonal antibodies which recognize Rho family members.
The monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric
human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies may be made
by any of numerous techniques known in the art (® . q., Teng et al., 1983, Proc. Natl. Acad. Sci.
U.S.A. 80:7308-7312; Kozbor et al., 1983, Imrnunology Today 4:72-79; Olsson et al., 1982, Meth.
Enzymol. 92:3-16). Chimeric antibody molecules may be prepared cont~ining a mouse
antigen-binding domain with human constant regions (Morrison et al., 1984, Proc. Natl. Acad. Sci.
U.S.A.81:6851,Takedaetal.,1985,Nature314:452). Amolecularcloneofanantibodytoaneurite
growth regulatory factor epitope can be prepared by known techniques. Recombinant DNA
CA 02214841 1997-10-31
14
methodology (see e.g., Maniatis et al., 1982, Molecular Cloning, A Laboratory Manual, Cold Spring
Harbor Laboratory, Cold Spring Harbor, N.Y.) may be used to construct nucleic acid sequences
which encode a monoclonal antibody olecule, or antigen binding region thereof.
A monoclonal antibody to an epitope of a Rho family member can be prepared by using any technique
which provides for the production of antibody molecules by continuous cell lines in culture. These
include but are not limited to the hybridoma technique originally described by Koler and Milstein
((1975) Nature, 256:495-497), and the more recent human B cell hybridoma technique (Kozbor et
al., (1983) Immunology Today, 4:72) and EBV-hybridoma technique (Cole et al., (1985) In
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp 77-96). In a particular
embodiment,theproceduredescribedbyNobile-Orazioetal.((1984)Neurology,34:1336-1342)may
be used to obtain antibodies which recognize recolllbil~allL Rho family members (for example of
techniques, see Attia S. et al., (1993)J. Neurochem., 61: 718-726).
The monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric
human-mouse (or other species) monoclonal antibodies. Human monoclonal antibodies maybe made
by any of numerous techniques known in the art (e.g. Tan et al., (1983) Proc. Natl. Acad. Sci. U.S.A.,
80: 7308-7312; Kozbor et al., (1983) Immunology Today, 4: 72-79; Olsson et al., (1982) Meth.
Enzymol., 92: 3-16). Chimeric antibody molecules may be prepared cont~ining a mouse antigen-
binding domain with human contact regions (Morrision et al., (1984) Proc. Natl. Acad. Sci. U.S.A.,
81: 6851, Takeda et al., (1985) Nature, 314: 452).
A molecular clone of an antibody to a Rho family member epitope can be prepared by known
techniques. I~ecombinant DNA methodology may be used to construct nucleic acid sequences which
encode a monoclonal antibody molecule, or antigen binding region thereof (see e.g., Maniatis et al.,
(1982) InMolecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y.).
For use, Rho family member antibody molecules may be purified by known techniques, such as
CA 02214841 1997-10-31
immunoabsorption or immunoaffinity chromatography, chromotographic methods such as HPLC
(high pe~ ance liquid chl ollla~ography), or a combination thereof, etc.
Rho family member antibody fragments which contain the idiotype ofthe molecule can be generated
by known techniques. For example, such fragments include but are not limited to: the F (ab')~
fragment which can be produced by pepsin digestion of the antibody molecule; the Fab, fragments
which cen be generated by reducing the disulfide bridges of the F (ab')2 fr~gment, and the two Fab
or Fab fragments which can be generated by treating the antibody molecule with papain and a
reducing agent.
Monoclonal antibodies known to react with human Rho family members may be tested for their
usefulness to serve as Rho family member antagonists (Nobile-Orazio et al., (1984) Neurology, 34:
1336-1342; Doberson et al., (1985) Neurochem. Res., 10: 499-513).
Antibody molecules may be purified by known techniques, e.g., immunoabsorption or immunoaffinity
chromatography, chromatographic methods such as HPLC (high performance liquid
chromatography), or a colllbhlalion thereof, etc.Antibody fragments which contain the idiotype of
the molecule can be generated by known techniques. For example, such fragments include but are not
limited to: the F(ab')2 fragment which can be produced by pepsin digestion of the antibody
molecule; the Fab, fr~gments which can be generated by reducing the disulfide bridges of the
F(ab')2 fragment, and the 2 Fab or Fab fragments which can be generated by treating the
antibody molecule with papain and a reducing agent.
Diagnos*c, Th~,; ,7~ . ~ and Research Uses f or Rho Family MemberAntagonists and Anh~odies
Rho family member antagonists and antibodies have uses in diagnostics. Such molecules can be used
in assays such as immunoassays to detect, prognose, diagnose, or monitor various conditions,
diseases, and disorders affecting neurite growth extension, invasiveness, and regeneration.
Alternatively, the Rho family member antagonists and antibodies may be used to monitor therapies
CA 022l484l l997-lO-3l
16
for diseases and conditions which ultim~tely result in nerve damage; such diseases and conditions
include but are not limited to CNS trauma, (e.g. spinal cord injuries), infarction, infection,
m~lign~ncy, exposure to toxic agents, nutritional deficiency, paraneoplastic syndromes, and
degenerative nerve diseases (including but not limited to Alzheimer's disease, Parkinson's disease,
ntin tonls Chorea, amyotrophic lateral sclerosis, progressive supra-nuclear palsy, and other
dementias). In a specific embodiment, such molecules may be used to detect an increase in neurite
o~ gruwlh as an indicator of CNS fiber regeneration. For example, in specific embodiments, altered
levels of Rho family members in a patient sample co~ inillg CNS myelin can be a diagnostic marker
for the presence of a m~lign~ncy, including but not limited to glioblastoma, neuroblastoma, and
melanoma, or a condition involving nerve growth, invasiveness, or regeneration in a patient. In a
particular embodiment, altered levels of Rho family members can be detected by means of an
immunoassay in which the lack of any binding to anti-inhibitory protein antibodies is observed. The
immunoassays which can be used include but are not limited to competitive and non-competitive
assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent
assay), "sandwich" immunoassays, precipitation reactions, gel diffusion precipitation reactions,
immllnodiffusion assays, a~l~ltin~tion assays, complement-fixation assays, immunoradiometric
assays, fluorescent immunoassays, protein A immunoassays, immunoelectrophoresis assays, and
immunohistochemistry on tissue sections, to name but a few.
Useful for nerve growth suppression are pharm~ceutical compositions that contain, in an amount
effective to suppress nerve growth, either Rho family member antagonist or antibody in combination
with an acceptable carrier. Candidate Rho family members antagonists include fragments of Rho
familymem~ers that incorporate the ectodomain, includin~ the ectodomainper se and otherN- and/or
C-terminally truncated fragments 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 in which the N- and/or C-
terminal residues are derivatized by chemical stabilizing groups. Such Rho family members
antagonists can also include anti-idiotypes of Rho family members antibodies and their binding
CA 02214841 1997-10-31
fragments.
In specific embodiments ofthe invention, c~n.1i.1~te Rho family members antagonists include specific
regions of the Rho family members molecule, and analogs or derivatives of these. These can be
identified by using the same technologies described above for identification of Rho family members
regions that serve as inhibitors of neurite outgrowth.
The Rho family members related derivatives, analogs, and fragments ofthe invention can be produced
by various methods known in the 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 modified by
any of numerous strategies known in the art (Maniatis et a/., Molecular Cloning, A Laboratory
Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982), such as by cleavage at
appropriate sites with restriction endonuclease(s), subjected to enzymatic modifications if desired,
isolated, and ligated in-vitro.
Additionally, the Rho family members-encoding gene can be mllt~ted in-vitro or in-vivo for instance
in the manner applied for production of the ectodomain, to create and/or destroy translation,
initiation, and/or te~ ina~ion sequences, or to create variations in coding regions and/or form new
restriction endonuclease sites or destroy preexisting ones, to facilitate further in-vitro modification.
Any technique for mutagenesis known in the art cab be used, including but not limited to, in-vitro site
directed mutagenesis (H~ltçhin~on, etal., J. Biol. Chem., 253, 6551, 1978), use of TABTM linkers
(Pharmacia), etc.
For delivery of Rho family members antagonists, various known delivery systems can be used, such
as encapsulation in liposomes or semipermeable membranes, expression in suitably transformed or
transfection glial cells, oligodendroglial cells, fibroblasts, etc. according to the procedure known to
those skilled in the are (Lindvall et a/., Curr. Opinion Neurobiol., J 752-757, 1994). Linkage to
ligands such as antibodies can be used to target delivery to myelin and to other therapeutically
relevant sites in-vivo. Methods of introduction include, but are not limited to, intradermal,
CA 022l484l l997-lO-3l
18
intr~mllsclll~r, intraperitoneal, intravenous, subcutaneous, oral, and intranasal routes, and transfusion
into ventricles or a site of operation (e.g. for spinal cord lesions) or tumor removal. Likewise, cells
secreting Rho family members antagonist activity, for example, and not by way of limitation,
hybridoma cells encapsulated in a suitable biological membrane may be implanted in a patient so as
to provide a continuous source of Rho family members inhibitor.
'rherapeu~ic Uses of Rho family members
CNS myelin associated inhibitory proteins ofthe present invention can be therapeutically useful in the
treatment of patients with m~lign~nt tumors including, but not limited to melanoma or tumors of
nerve tissue (e.g. neuroblastoma). In one embodiment, patients with neuroblastoma can be treated
with Rho family members or analogs, derivatives, or subsequences thereof, and the human functional
equivalents thereof, which are inhibitors of neurite extension.
In an alternative embodiment, antagonists, derivatives, analogs, inhibitors, or antibodies to Rho family
members can be used in re imen~ where an increase in neurite extension, growth, or regeneration is
desired, e.g., in patients with nervous system damage. Patients suffering from traumatic disorders
(inclll~ling but not limited to spinal cord injuries, spinal cord lesions, or other CNS pa~llway lesions),
surgical nerve lesions, damage secondary to infarction, infection, exposure to toxic agents,
m~lign~ncy, paraneoplastic syndromes, or patients with various types of degenerative disorders of
the central nervous system (Cutler, 1987, In: Scientific American Medicines v. 2, Scientific American
Inc., N.Y., pp. 1 1-1-1 1-13) can be treated with such inhibitory protein antagonists. Examples of such
disorders include but are not limited to Alzheimer's Disease, Parkinsons' Disease, Hlmtington's
Ch~rea, arnyotropl~c lateral sclerosis, progressive supranuclear palsy and other dementias. Such
antagonists may be used to promote the regeneration of CNS pathways, fiber systems and tracts.
~-lmini~tration of antibodies directed to an epitope of, (or the binding portion thereof, or cells
secreting such as antibodies) can also be used to inhibit Rho family members protein function in
patients. In a particular embodiment ofthe invention, antibodies directed to Rho family members may
be used to pl u.l.ol~ the regeneration of nerve fibers over long distances following spinal cord damage.
CA 02214841 1997-10-31
19
Various delivery systems are known and can be used for delivery of antagonists or inhibitors of Rho
family members, related molecules, or antibodies thereto, e.g., encapsulation in liposomes or
semipermeable membranes, expression by bacteria, etc. Linkage to ligands such as antibodies can be
used to target myelin associated protein-related molecules to therapeutically desirable sites in vivo.
Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, oral, and intranasal routes, and infusion into ventricles or a site of
operation (e.g. for spinal cord lesions) or tumor removal. Likewise, cells secreting CNS myelin
inhibitory protein antagonist activity, for example, and not by way of limitation, hybridoma cells,
encapsulated in a suitable biological membrane may be implanted in a patient so as to provide a
continuous source of anti-CNS myelin inhibiting protein antibodies.
In addition, any method which results in decreased synthesis of Rho family members may be used to
~'imini~h their biological function. For example, and not by way of limitation, agents toxic to the cells
which synthesize Rho family members and/or its receptors (e.g. oligodendrocytes) may be used to
decrease the concentration of inhibitory proteins to promote regeneration of neurons.
EXAMPLES
EXAMPLE 1
This example demonstrates in vitro evidence that Rho family members are responsible for re~ ting
the neuronal response to MAG. In particular, this demonstrates that the small GTPase Rho regulates
the response to MAG. PC12 cells were plated on polylysine (PLL), l~minin7 or MAG substrates and
exposed to NGF to stiml71~7te neurite growth. PC12 cells di~ iated neurites on PLL and laminin
substrates, but on MAG substrates the cells remained rounded and did not grow neurites.
The addition of the ADP-ribosyl transferase C3 from Clostridium botulinum, that efficiently
inactivates Rho family members without affecting Rac and cdc42 (Udagawa and McIntyre, 1996),
allowed the cells to extend neurites on MAG substrates. In addition this example demonstrates
neurite growth from PC 12 cells ~ r~;~d with a dominant negative Nl 9RhoA construct. On laminin
CA 02214841 1997-10-31
and PLL substrates the N19 RhoA PC12 cells grew neurites that were longer than 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 stim~ tes neurite outgrowth and allows neurite
extension on MAG substrates. These results implicate Rho in sign~ling growth inhibition by MAG
Cell Culture
We obtained PC12 cells from three dinrelelll sources: from Dr. Phil Barker (Montreal Neurological
Institute), from the ATCC (obtained from W. Mllehin~ky, McGill), and from Gabor Tigyi,
(University of Tennessee) and we found that all lines of cells were inhibited by both myelin and MAG.
PC12 cells were grown in Dulbecco's modified eagle's medium (DMEM) with 10 % horse serum and
5 % fetal bovine serum. PC12 cells stably transfected with constitutively active and dominant
negative RhoA constructs were kindly provided by Dr. G. Tigyi (University of Tennessee, Memphis,
USA). The three cell lines used included a mock transfected cell line, a constitutively active RhoA
(V14GRhoA) cell line, and a dominant negative RhoA (N19TRhoA) cell line. Transfected PC12 cell
lines were m~int~ined in the growth medium cont~ining 400 mg/L G418. For cell diJre-~nliation
experiments, cells were plated on applop-iate substrates in DMEM with 1 % fetal bovine serum and
100 ng/ml nerve growth factor. For experiments on mixed substrata (laminin/MAG or
laminin/myelin), PC12 were plated in DMEM with 1% lipid free-BSA in the presence or the absence
of 50~g/ml of an irrelevant antibody or of a purified function blocking antibody (clone 3A3) against
the rat al ,~ 1 integrin (a gift of S .Carbonetto). PC 12 cell di~el en~iation experiments were done in 96-
well plates in duplicate, and each experiment was repeated a minim~lm of three times.
To culture cerebellar granule cells, 3 - 4 rats from P3 to P7 were decapitated. The cerebellum was
removed and placed in MEM-HEPES where underlying tissue and the meninges was removed. The
cerebellum was cut into small pieces and treated with 0.125% trypsin in MEM-HEPES for 20' at
37~C. The tissue was then triturated with a fire polished pasteur pipette to break up any clumps of
tissue. The cells were spun down at 1500 rpm for 10', and the pellet was resuspended in MEM-
HEPES with 2mM EDTA. The cell suspension was placed on an iso-osmotic percoll gradient with
CA 02214841 1997-10-31
60% and 35% percoll, centrifuged for 15' at 2300 rpm, and the interface between the 60% and 35%
percoll was collected. Cells were washed once, and resuspended in DMEM with 10% FBS,
vit~min~, and penicillin/streptomycin in the presence or absence of 20 ~g/ml C3 transferase. Cells
were placed in 4-chamber, chamber slides coated with poly-l-lysine or laminin and treated with spots
of MAG or myelin. 200,000 cells per chamber were plated.
Preparation of growth substrates
Poly-l-lysine was obtained from Sigma (St. Louis, Mo). T,~minin was prepared from EHS tumors
(Paulsson and Lindblom (1994). Cell biology: A laboratory handbook, Ac,~d~ mic Press, pp~89-594)
and collagen from rat tails (Greene et al (1987) Meth. Enzymology 147:207-216). Myelin was made
from bovine brain corpus callosum, and native MAG was purified from myelin after extraction in
1% octylglucoside and separation by ion exchange chromatography (McKerracher et al (1994)
Neuron 13:801). This native MAG has some additional proteins, including some tenascin (Xiao et
al !997) Neurosci. Abstr vol 23: 1994). Recombinant MAG was made in baculovirus as described
(McKerracher et al, IBID).
Test substrate were prepared 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 llg/ml for 3
hours at 37~C, and the wells or coverslips were washed with water and dried. T,~minin substrates
were prepared by incllb~ting 25 ~lg/ml laminin on poly-L-lysine coated dishes for 3 hours at 37~C.
Solid MAG or myelin substrates were prepared by drying down MAG overnight, or incubating a 10
mg/ml myelin solution for 3 hours on polylysine coated substrates. For 96-well plates, 1-4 ~lg of
either recombinant MAG (rMAG) or of native MAG per well was used. For mixed laminin/myelin
or laminin/MAGsubstrata, 8~1g of inhibitory proteins and 10 ~lg of laminin were dried down on 96-
well plates precoated with polylysine. For 4-chambered chamber slides, 40 ~lg MAG per chamber
was used, and for 100 mm plates 0.6-1 mg of MAG was dried down. Spots of MAG on coverslips
were generated by plating of 2 mg/ml recombinant MAG on polylysine for 3-4 hours in a humid
chamber at 37~C. Collagen substrates were made by incubating 10-15 ~lg/ml of rat tail collagen for
CA 02214841 1997-10-31
3 hours at 37~C.
Immunocytochemistry
PC12 cells were visualized by phase contrast microscopy, or following labelling with the lipophilic
fluorescent dye, DiI (McKerracher et al, 1994). Granule cells were visualized byimmllnocytochemistry. Following 12-24 hours in culture, cells were fixed for 30' at room
teln~e~ re in 4% palafo-.llaldehyde, 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 1 00. Granule cell cultures were incubated overnight with a polyclonal anti-
rMAG antibody (called 57A++) to label MAG spots. The MAG antibody was detected using an
FITC conjugated secondary antibody. Rhodamine conjugated phalloidin was diluted 1:200 with the
secondary antibody to label granule cell actin filaments.
C3 transferase preparation and use
The plasmid pGEX2T-C3 coding for the GST-C3 fusion protein was obtained from A. Hall
(London). Recol~lbh1all~ C3 was purified as described byDillon and Feig (Met. Enzymology, (1994),
256, pp 174-184). A~cer fusion protein cleavage by ~ in, thrombin was removed by incubating
the protein solution 1 hour on ice with 100~11 of p-aminobenzamidine agarose-beads (Sigma). The C3
solutionwasdesaltedonPDlOcolumn(Pharmacia)withPBS,andsterilizedthroughaO.2211mfilter.
The C3 concentration was evaluated by Lowry assay (DC protein assay, Bio-Rad) and toxin purity
was controlled by SDS-PAGE analysis.
To test the effect of C3 on the outgrowth on PC 12 cells, C3 transferase was scrape loaded into the
cells before plating on appropliate substrates. Cells were grown to confiuence in serum cont~ining
media in 6 well plates. Cells were washed once with scraping buffer ( I 1 4mM KCI, 1 5mM NaCl, 5 . 5
mM MgCI2, lOmM Tris-HCl). Cells were then scraped with a rubber policeman into 0.5 ml scraping
buffer in the presence or absence of 20 llg/ml C3 transferase. The cells were pelleted, and
CA 02214841 1997-10-31
resuspended in 2 ml DMEM, 1% FBS, and 50 ng/ml nerve growth factor before plating. 10 ~Lg/ml
C3 was added to scrape loaded cells. Cells were di~ele.-~iated for 48 hours then fixed in 4 %
pal~rol..laldehyde, 0.5 % glutaraldehyde, 0.1 M PO4 buffer.
Membrane Translocation Assay for RhoA
PC 12 cells were collected and resuspended in DMEM, 0.1 % BSA, 50ng/ml NGF, then plated on
100 mm dishes coated with collagen or MAG, or left in suspension. Two hours later, cells were
washed with ice cold PBS + protease inhibitors (1 ~lg/ml aplo~ in, 1 ~g/ml leupeptin, 1 llg/ml
~ntip~in,1 ~g/ml pepstatin). Cells were then scraped into 5ml PBS + protease inhibitors, and the cells
were pelleted and washed with PBS + protease inhibitors. The cell pellets were mechanically
homogenized by 25 strokes in a glass-teflon homogenizer, the homogenate centrifuged for 20 min
at 8,000 rpm, and the cell debris in the pellet was discarded. The supe...ala..~ was centrifuged for 1
hour at 100,000 x g to separate membrane and cytosolic fractions. Membrane pellets were washed
1 X with PBS + protease inhibitors and resuspended in PBS with 0.5 % SDS, and 50-100 llg of
membrane protein was analyzed by SDS-PAGE on 12 % gels. Gels were l-~nsrelled to Protran
nitrocellulose membrane and stained with Ponceau S. Blots were blocked for 1 hour in 5 % skim
milk in TBS, and probed overnight with Rho A antibody diluted 1:200 in 1.5 % skim milk in TBS.
Rho A antibody was detected by using an alkaline phosphatase conjugated secondary antibody and
an alkaline phosphatase detection kit (Gibco-BRL).
Growth inhibition of PC12 cells and its modulation by NGF and laminin
PC12 cells typically extend neurites in response to NGF, but when plated on myelin substrates the
cells remain round and do not extend neurites (Moskowitz et al. 1997) (Fig. 2). MAG is a potent
inhibitor of axon growth present in myelin. We observed that PC 12 cells plated on substrates of MAG
also remained rounded (Fig.1), a finding in contrast to a report that PC12 cells are not responsive to
MAG (Bartsch et al. 1995). To further examine the response of PC12 cells to MAG, we plated three
dilrelenl lines of PC12 cells on both native and recoll.binalll MAG substrates in the presence of NGF.
CA 02214841 1997-10-31
24
All of the lines of PC12 cells showed reduced cell spreading, and most cells remained rounded
without neurites. However, with increasing time, some neurites were able to extend on MAG
substrates (see below). We also observed that di~elell~ prepal~lions of MAG can differ in their
potency to inhibit neurite growth, and that the activity of MAG is reduced or lost upon freeze-thaw.
T ~minin is known to override completely, growth inhibition of NG108 cells by myelin (David et al.
1995). Similarly, we found that PC12 cells are able to extend neurites on mixed myelin and laminin
substrates or on mixed laminin/MAG substrates (Fig.2). To determine if ~ign~lling through integrins
is responsible for overriding growth inhibition by myelin, we used the integrin function blocking
antibody 3A3 raised against the al subunit extracellular domain. Previous studies have docllmentecl
that all31 integrin is the dominant integrin expressed by PC12 cells, and that the 3A3 antibody blocks
PC12 cell neurite growth on laminin (Tomaselli et al. 1990). We plated PC12 cells on mixed myelin
and laminin substrates, in the presence of the 3A3 antibody, or with a non-specific IgG antibody as
a control. The 3A3 antibody blocked neurite extension on both and laminin and the mixed
myelin/laminin substrates (Fig. 2). On MAG or on myelin 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 m~king the inhibitory domain of MAG, but that
signals elicited through integrins receptors are responsible.
Effect of C3 Transferase on PC12 cells
To investigate possible intracellular targets that may override growth inhibition by myelin and by
MAG, we focused on the small GTPase Rho which is known to play a role in convergent signalling
p~lhw~ys that affect morphology and motility (Hall, 1996). We inactivated Rho 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 and McIntyre, 1996). On control
substrates of polylysine and l~minin, treatment with C3 potentiated both the number of cells with
neurites and the length of neurites from cells (Fig. 3). On MAG and myelin substrates where neurite
formation is inhibited, C3 has a dramatic effect on the ability to extend neurites (Fig 3). When treated
CA 02214841 1997-10-31
with C3, about half of the PC12 cells plated on either rMAG or native MAG had neurites of
approximately 1 cell body diameter. In contrast, the untreated cells remained rounded and clumped.
Similarly, PC12 cells plated on myelin remained rounded, but the addition of C3 allowed neurites to
extend directly on the myelin substrate. These results demonstrate that C3 treatment elicits neurite
growth from PC12 cells plated on growth inhibitory myelin or MAG substrates.
Growth of dominant . ~gali.~e Rho-transfected cells on MAG substrates
PC 12 cells transfected with constitutively active RhoA (V 1 4GRhoA), and PC 12 cells transfected with
dominant negative RhoA (N19TRhoA), and the mock-transfected cells, were examined for their
ability to extend neurites on di~erenl test substrates. Cells with the constitutively active mutation,
V14GRhoA cells, di~ele~ ted poorly on all substrates, including poly-L-lysine and l~ninin The
tre~trnent of the V14GRhoA cells with C3 allowed the growth of some short neurites on all of the
test substrates, including MAG.
In the same series of expelillle~ the response of don~illalll negative Rho- transfected cells,
N19TRhoA cells, to MAG and myelin substrates was examined. WhenN19TRhoA cells were plated
on MAG substrates, they spread and did not remain rounded as did the mock transfected PC 12 cells.
A small 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 transfected and N19TRhoA cells had a dramatic effect of neurite outgrowth
as most cells had extensive neurites (Fig.3). The effect of C3 on N19TRhoA cells was much more
mar~ed than the effect on the mock transfected cells. Therefore, the conlbillaLion of C3 treatment
and transfection of domilla ll negative Rho elicited excellent outgrowth of neurites from PC12 cells
plated on inhibitory MAG (and myelin) substrates.
Effect of C3 on Primary Cells
CA 022l484l l997-lO-3l
26
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 and treated with C3. Neurite outgrowth
from these cells was known to be inhibited by MAG (Li et al. 1996) and the C3 stim~ te~ growth
of neurites from the granule cells on both permissive and inhibitory substrates.
The growth substrate infl~ c~ the cellular location of Rho
Rho is associated with the plasma membrane when it is in an activated state, and it moves into the
cytosolic fraction when it is in the GDP-bound inactive state. To determine if the growth substrate
influences the cellular localization of Rho, cells were either left in suspension or plated on MAG or
collagen substrates, and plepared membranes from the cells two hours later. It was shown that Rho
was principally localized in the cytosolic fraction when cells were plated on collagen, a growth
permissive substrate. However, Rho was associated with the plasma membrane when cells where
grown in suspension and when cells were plated on MAG (Fig. 4).
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CA 02214841 1997-10-31
From the foregoing description, one skilled in the art can easily ascertain the essential characteristics
ofthis invention, and without departing from the spirit and scope thereof, can make various changes
and modifications to the invention to adapt it to various usages and conditions. Such changes and
modifications are properly, equitably, and intenfled to be within the full range of equivalence of the
~llowing claims.
