Use of compounds to induce axonal growth

The present invention relates to a use of one or more microtubule stabilizing compounds for the preparation of a pharmaceutical composition for the treatment of lesions of CNS axons wherein the pharmaceutical composition is administered locally directly into the lesion or immediately adjacent thereto, whereby the one or more compound(s) is/are selected from the group consisting of taxanes, epothilones, laulimalides, sesquiterpene lactones, sarcodictyins, diterpenoids, peloruside A, discodermolide, dicoumarol, ferulenol, NSC12983, taccalonolide A, taccalonolide E, Rhazinilam, nordihydroguaiaretic acid (NDGA), GS-164, borneol esters, Synstab A, Tubercidin and FR182877 (WS9885B). Furthermore, the invention relates to a method for the treatment of lesions of CNS axons the method comprising the step of administering to a patient in the need thereof a

„ pharmaceutical composition comprising (i) one or more microtubule stabilizing compounds selected from the group consisting of taxanes, epothilones, laulimalides, sesquiterpene lactones, sarcodictyins, diterpenoids, discodermolide, dicoumarol, ferulenol, NSC12983, taccalonolide A, taccalonolide E, Rhazinilam, nordihydroguaiaretic acid (NDGA), GS-164, borneol esters, Synstab A, Tubercidin and FR182877 (WS9885B) and, optionally, (ii) further comprising suitable formulations of carrier, stabilizers and/or excipients, wherein the pharmaceutical composition is administered locally directly into the lesion or immediately adjacent thereto.

A variety of documents is cited throughout this specification. The disclosure content of said documents is herewith incorporated by reference.

The loss of sensorimotor functions observed after spinal cord injury follows directly from the limited capacities of the central nervous system (CNS) to regrow its cut

axons. The CNS axons fail to regenerate for two reasons. First, the scarring process and the myelin debris create an environment surrounding the damaged area that inhibits axonal growth (4, 22) and, second, the neurons show a poor intrinsic axonal growth in response to injury (1 , 16, 23).

Several extra-cellular outgrowth inhibitors have been identified, including myelin proteins such as Nogo, myelin-associated glycoprotein (MAG) and various chondroitin sulfate proteoglycans (CSPGs) (2). Their inhibitory effects occur through neuron specific morphological changes which are characterized by a growth cone collapse and halt of neurite outgrowth (2-4). At the intracellular level, most of these growing inhibitory signals are integrated by changing the activity state of the Rho family of GTPases (2). The members of the Rho GTPases include Cdc42, Rac and Rho. These proteins are involved in remodelling the actin cytoskeleton (5). Axonal growth cone are primarily composed of filamentous actin and microtubules. The microtubules are extending along the entire shaft of the axon and end in the central part of the growth cone. The role of the microtubules in axonal growth will be addressed below and is the major part of our invention. Actin filaments set up the peripheral part of the growth cone as finger-like filopodia and centrally as a thick meshwork called lamellipodia (6). Cdc42 and Rac act on the filopodial and lamellipodial extension respectively, while Rho mediates actin stress fiber formation. In neurons, the activation of Rho leads to growth cone collapse (5). Inversely, inactivation of the whole Rho GTPases with the bacterial toxin B causes a dramatic disruption of the actin network and promotes neurite elongation (7).

The correlation between extra-cellular inhibitors effects and change of Rho GTPases activity state has clearly been recognized. Myelin activates Rho, leading to growth inhibition (8). Conversely, the inactivation of the Rho signalling pathway either by the C3-exoenzyme (9) or by a dominant negative mutation of Rho, promotes neurites growth of neurons cultured on myelin or CSPGs inhibitory substrates (10, 11). Moreover, in vivo, C3-exoenzyme treatment enhances axon regeneration and allows a functional recovery in different models of spinal cord

injury (10, 12, 13). Yet, the Rho-GTPases have various other functions ranging from apoptosis to radical formation (14, 15). It is the present understanding in the field, that lesioned axons in the CNS do not regrow as the corresponding axons in the PNS because the extrinsic inhibitory factors described above activate signalling pathways within the cells that cause the cell to stop axonal growth. The intracellular mechanism underlying axonal growth and axonal halt are not clearly understood.

The initial neuronal polarization of dissociated embryonic hippocampal neurons in cell culture has been analyzed. It was known in the art that shortly after plating four to five neurites per cell form that all of have the potential to become the axon ⁽ J, 24, 25). However, only one of the neurites elongates rapidly whereas the other neurites only will grow at a later stage (24). The other neurites are actively restrained from growing: depolymerization of the actin cytoskeleton causes instead of one axon multiple axons to grow (7, 17). Thus it appears that the actin filaments restrain the axons from growing, whereas if the actin filaments are very dynamic and less stable, microtubule can protrude and, thus, allow axonal elongation. Similarly, if the Rho-GTPases are inhibited using the bacterial toxin B hippocampal neurons also form multiple axons per cell (7). Interestingly, as discussed above very similar processes appear to function in lesioned CNS axons (10, 12, 13). Inhibition of Rho using the bacterial toxin C3-exoenzyme causes cut axons to regrow their axon. Indeed using the bacterial toxin C3 exoenzyme is a promising method that probably will move into clinical trials phase I during the years 2004/5. However, a problem with this approach could be that C3 induced depolymerization of the actin filaments may destabilize the tight junctions of the blood brain barrier thereby causing problems.

Even without the understanding of the mechanism underlying axonal growth and axonal halt it has been reported that the treatment of incomplete spinal cord injuries with taxol and methylprednisolone given shortly after a compression injury improve functional outcome in an animal model (41). However, in the treatment required an administration of taxol in a daily dose which was close to the half of the

LD10 for daily intraperitoneal doses according to a study by the National Cancer Institute of the USA (42). The problem of the toxicity of taxol in the central nervous system was e.g. confirmed in a recent report in which neuronal death induced by taxol by an induction of apoptotic cell death in an in vivo model was discussed (43).

Adlard et al. (2000) describe the effects of taxol on the CNS response to physical injury. Adlard et al. (2000) report that in the short term, taxol may be stabilising neuronal microtubules and reducing reactive alterations in axons. At the same time the authors find that after longer periods, taxol causes an abnormally prolonged neuronal reaction to trauma.

Mercado-Gomez et al. (2004) saw a need to address taxol toxicity in the CNS. Their results show that taxol induces dose-dependent neuronal death.

In view of this prior art, the skilled person was in severe doubt as to whether microtubule stabilizing compounds such as taxol would at all provide a therapeutic window where therapeutic effects prevail and toxic side effects are acceptable.

WO 2005/014783 describes methods and compositions for reducing degeneration of an axon predetermined to be subject to degenerative neuropathy in a term patient. The described compositions comprise as active agent(s) an inhibitor of the ubiquitin-proteasome systems, and optionally a microtubule stabilizer like taxol.

WO 98/24427 describes compositions and methods for treating or preventing inflammatory diseases. Paclitaxel, which is comprised in one embodiment, is described as a compound disrupting microtubule formation.

However, treatments aiming at reducing degeneration of an axon or at the disruption of microtubule formation may be insufficient for treating lesions of CNS axons.

Thus, the technical problem underlying the present invention was to provide means and methods for the treatment of CNS lesions.

The solution to said technical problem is achieved by providing the embodiments characterized in the claims.

Accordingly, the present invention relates to a use of one or more microtubule stabilizing compounds for the preparation of a pharmaceutical composition for the treatment of lesions of CNS axons wherein the pharmaceutical composition is administered locally directly into the lesion or immediately adjacent thereto, whereby the one or more compound(s) is/are selected from the group consisting of: taxanes, epothilones, laulimalides, sesquiterpene lactones, sarcodictyins, diterpenoids, peloruside A, discodermolide, dicoumarol, ferulenol, NSC12983, taccalonolide A, taccalonolide E, Rhazinilam, nordihydroguaiaretic acid (NDGA), GS-164, borneol esters, Synstab A, Tubercidin and FR182877 (WS9885B)

The following list provides preferred representatives of microtubule stabilizing compounds envisaged for the use according to the present invention.

• Taxanes: o Taxol® (Paclitaxel) (Tax-11-en-9-one, 5beta,20-epoxy- 1 ,2alpha,4,7beta, 10beta,13alpha- hexahydroxy-, 4,10-diacetate 2- benzoate, 13-ester with (2R,3S)-N-benzoyl-3-phenylisoserine (8Cl)) (Schiff et ah, 1979; Schiff and Horwitz, 1980; Wani et al., 1971) o IDN5190 (Ortataxel; Hexanoic acid, 3-(((1 ,1-dimethylethoxy) carbonyl)amino)-2-hydroxy-5-methyl-,

(SaS^RJR.δaS.θS.IOaR.^aS.^bR, 13S,13aS)-7,12a- bis(acetyloxy)-13-(benzoyloxy)-3a,4,7,8,8a,9, 10, 10a, 12, 12a, 12b, 13- dodecahydro-9-hydroxy-5,8a, 14,14-tetramethyl-2,8-dioxo-6, 13a- methano-13aH-oxeto(2",3":5',6 ^I )benzo(1',2 ^I :4,5)cyclodeca(1 ,2-d)-1 _J 3-

dioxoI-4-yl ester, (2R.3S)-) (Nicoletti et al., 2000; Ojima et al., 1996) o IDN 5390 (13-(N-BOC-βisobutylisoserinoyl)-10-dehydro-10-deacetyl- C-secobaccatin) (Taraboietti et al., 2002) o BMS-188797 (semi-synthetic derivate of Taxol) (Rose et al. , 2001 a) o BMS-184476 (7-((Methylthio)methyl)paclitaxei) (Altstadt et al., 2001 ;

Rose et al., 2001a) o BMS-185660 (semi-synthetic derivate of Taxol) (Rose et al., 1997; Rose et al., 2000) o RPR109881A (Taxane derivate) (Kurata et al., 2000) o TXD258(RPR11625A) (Taxane derivate) (Bissery, 2001 ; Cisternino et al., 2003) o BMS-275183 (orally active taxane) (Rose et al., 2001 b) o DJ-927 (orally active taxane) (Shionoya et al., 2003; Syed et al., 2004) o Butitaxel analogues (AIi et al., 1997) o Macrocyclic Taxane analogues (Tarrant et al., 2004) o 7-deoxy-9beta-dihydro-9,10-O-acetal taxanes (Takeda et al., 2003) p 10-deoxy-IO-C-morpholinoethyl docetaxel analogues (limura et al., 2001) o RPR 116258A (Fumoleau et al., 2001 ; Goetz et al., 2001; Lorthoraly ef a/., 2000) o CT-2103 (Xyotax, PG-TXL) (conjugated poly(L-glutamic acid)- paclitaxel) (Langer, 2004; Li et al., 2000; Li et al., 1998; Multani et al., 1997; Oldham et al., 2000; Todd et al., 2001) o polymeric micellar paclitaxel (Leung et al., 2000; Ramaswamy et al.,

1997; Zhang ef al., 1997a; Zhang et al., 1997b) o Genexol-PM, Cremophor-Free, Polymeric Micelle-Formulated

Paclitaxel (Kim et ai, 2004) o Docosahexaenoic acid-conjugated Taxol (Taxoprexin; Paclitaxel 2'- (all-cis-4,7,10,13,16,19-docosahexaenoate)) (Bradley et ai, 2001a; Bradley et ai, 2001b; Wolff et ai, 2003) o PTX-DLPC (Taxol encapsulated into dilauroylphosphatidylcholine ^' liposomal formulations) (Koshkina et ai, 2001) o PNU-TXL (PNU 166945) (a water-soluble polymeric drug conjugate of Taxol) (Meerum Terwogt et ai, 2001) o MAC-321 (5beta, 20-epoxy-1, 2alpha-, A-, 7beta-, lObeta-, 13alpha- hexahydroxytax-11-en-9-one 4 acetate 2 benzoate 7-propionate 13- ester with (2R,3S)-N-tertbutoxycarbonyl-3-(2-furyl)isoserine) (Sampath et ai, 2003) o Protaxols (water soluble compounds releasing Taxol at basic pH / in plasma) several compounds mentioned in (Nicolaou et ai, 1993) o photoaffinity analogues of Taxol

^■ 3'-(p-azidobenzamido)taxol (Rao et ai, 1994)

^■ 2-(m-azidobenzoyl)taxol (Rao et ai, 1995)

^■ 7-(benzoyIdihydrocinnamoyl)Taxol (Rao et ai, 1999)

^■ 5-azido-2-nitrobenzoic acid C-7 photoaffinity analogue of Taxol as described in (Carboni et ai, 1993) o photoactivatable Taxol (2'-(4,5-dimethoxy-2-nitrobenzyl) carbonate of Taxol) (Buck and Zheng, 2002) o Docetaxel / Taxotere® (Benzenepropanoic acid, beta-(((1 ,1- dimethylethoxy) carbonyl)amino)-alpha-hydroxy-, (2aR,4S _> 4aS,6R _J 9S,11S,12S,12aR,12bS)12b(acetyloxy)-12-

(benzoyloxy)-2a,3,4,4a,5 _f 6,9,10 _I 11 _I 12,12a _l 12b-dodθcahydro-4,6 _I 11- trihydroxy-4a,8,1313-tetramethyl-5-oxo-7,11-methano-1H- cyclodeca(3,4)benz(1 ,2-b)oxet-9-yl ester, trihydrate, (alphaR.betaS)-

(Ringel and Horwitz, 1991))

• Epothilones (Goodin et al., 2004): o epothilone A (Bollag et al. , 1995; Kowalski et al. , 1997b) o epothilone B (Patupilone, EPO906) (Bollag et al., 1995; Kowalski et al., 1997b) o dEpoB (Chou et al., 2001; Chou et al., 1998a; Chou et.al., 1998b) o BMS-247550 (aza-EpoB) (Chou et al., 2001 ; Stachel et al., 2000) o Fs-deH-dEpoB (Chou et al., 2003) o epothilone D (KOS-862 / NSC-703147) (Dietzmann et al., 2003;

Kolman, 2004) o dEpoF (Chou ef a/., 2001) o BMS-310705 (21-Aminoepothilone B) (Uyar et al., 2003)

• Sesquiterpene lactones : o Parthenolide (Germacra-1(10),11(13)-dien-12-oic acid, 4,5-alpha- epoxy-6-beta-hydroxy-, gamma-lactone) (Miglietta et al., 2004) o Costunolide (Germacra-1(10),4,11(13)~trien-12-oic acid, 6-alpha- hydroxy-, gamma-lactone, (E ₁ E)-) (Bocca et al., 2004)

• Sarcodictyins: o Sarcodictyin A (Hamel et al., 1999) o Sarcodictyin B (Hamel et al., 1999)

• Eleutherobins (Lindel et al., 1997), caribaeoside and caribaeolin (Cinel et al., 2000)

• Peloruside A (secondary metabolite isolated from a New Zealand marine sponge, Mycale hentscheli) (Hood et al., 2002)

• Laulimalide and isolaulimalide (Mooberry et al. , 1999)

Discodermolide (Hung et al., 1996; Kowalski et al., 1997a; Lindel et al., 1997; Martello et al., 2000; ter Haar et al., 1996) (a marine-derived polyhydroxylated alkatetraene lactone, used as a immunosuppressive compound).

Also envisaged is the use of discodermolide analogues as described in the following publications:

(a) Hung, D. T.; Nerenberg, J. B.; Schreiber, S. L. Chem. Biol. 1994, /, 67-71.

(b) Hung, D. T.; Nerenberg, J. B.; Schreiber, S. L. J. Am. Chem. Soc. 1996, 118, 11054-11080.

(c) Paterson, I.; Florence, G. J. Tetrahedron Lett. 2000, 41, 6935-6939.

(d) Gunasekera, S. P.; Longley, R. E.; Isbrucker, R. A. J. Nat. Prod. 2001 , 64, 171-174.

(e) Isbrucker, R. A.; Gunasekera, S. P.; Longley, R. E. Cancer Chemother. Pharmacol. 2001, 49, 29-36. (f) Martello, L. A.; LaMarche, M. J.; He, L; Beauchamp, T. J.; Smith, A. B., Ill; Horwitz,

S. B. Chem. Biol. 2001, 8, 843-855. As they were not included in the original paper, full experimental details for the compounds reported therein are provided in the Supporting Information of this work.

(g) Curran, D. P.; Furukawa, T. Org. Lett. 2002, 4, 2233-2235. (h) Gunasekera, S. P.; Longley, R. E.; Isbrucker, R. A. J. Nat. Prod. 2002, 65, 1830-

1837.

(i) Gunasekera, S. P.; Paul, G. K.; Longley, R. E.; Isbrucker, R. A.; Pomponi, S. A. J. Nat. Prod. 2002, 6, 1643-1648.

G) Minguez, J. M.; Giuliano, K. A.; Balachandran, R.; Madiraju, C; Curran, D. P.; Day _s B. W. MoI. Cancer Ther. 2002, /, 1305-1313.

(k) Shin, Y.; Choy, N.; Balachandran, R.; Madiraju, C; Day, B. W.; Curran, D. P. Org. Lett. 2002, 4, 4443-4446.

(I) Choy, N.; Shin, Y.; Nguyen, P. Q.; Curran, D. P.; Balachandran, R.; Madiraju, C; Day, B. W. J. Med. Chem. 2003, 46, 2846-2864.

(m) Minguez, J. M.; Kim, S.-Y.; Giuliano, K. A.; Balachandran, R.; Madirajϋ, C; Day, B. W.; Curran, D. P. Bioorg. Med. Chem. 2003, //, 3335-3357.

(n) Paterson, !.; Delgado, O. Tetrahedron Lett. 2003, 44, 8877-8882.

(o) Burlingame, M. A.; Shaw, S. J.; Sundermann, K. F.; Zhang, D.; Petryka, J.; Mendoza, E.; Liu, F.; Myles, D. C; LaMarche, M. J.; Hirose, T.; Freeze, B. S.; Smith,

A. B., III. Bioorg. Med. Chem. Lett. 2004, 14, 2335-2338.

(p) Gunasekera, S. P.; Mickel, S. J.; Daeffler, R.; Niederer, D.; Wright, A. E.; Linley, P.; Pitts, T. J. Nat. Prod. 2004, 67, 749-756.

(q) Smith, A.B., 3rd, et al., Design, synthesis, and evaluation of analogues of (+)-14- normethyldiscodermolide. Org Lett, 2005. 7(2): p. 315-8.

(r) Smith, A.B., 3rd, et al., Design, synthesis, and evaluation of carbamate-substituted analogues of (+)-discodermoiide. Org Lett, 2005. 7(2): p. 311-4.

• Dicoumarol (3,3'-Methylen-bis(4-hydroxy-coumarin)) (Madari et al., 2003)

• Ferulenol (prenylated 4-hydroxycoumarin ; 2H-1-Benzopyran-2-one, 4- hydroxy-3-(3,7,11-trimethyl-2,6,10-dodecatrienyl)-) (Bocca et al., 2002))

• NSC12983 (Wu ef a/., 2001)

• Taccalonolides: o taccalonolide A (Tinley et al., 2003) o taccalonolide E (Tinley et al., 2003)

• Rhazinilam (lndolizino(8,1-ef)(1)benzazonin-6(5H)-one, 8a-ethyl- y.δ.δa.θ.iO.H-hexahydro-, (8aR-(8aR*,14aR*))-) (a plant-derived alkaloid) (David et al., 1994)

• NDGA and derivates: o Nordihydroguaiaretic acid (NDGA) (4,4'-(2,3-Dimethyl-1 ,4- butanediyl)bis-1 ,2-benzenediol) (Nakamura et al., 2003; Smart et al.,

1969) o Tetra-O-methyl nordihydroguaiaretic acid (Heller eif al., 2001)

• GS-164 (Shintani et al., 1997) (a small synthetic compound)

• Synstab A (Haggarty et al., 2000) (a small synthetic compound)

• borneol esters (from (Klar et al., 1998), e.g. compound 19a described therein)

• Tubercidin (7-beta-CD-ribofuranosyl-7H-pyrrolo(2,3-d)pyrimidin-4-amine) (a nucleoside analog) (Mooberry et al., 1995)

• FR182877 (WS9885B) (Sato et al., 2000a; Sato et al., 2000b) (derived from a strain of Streptomyces sp. No.9885)

The term ..microtubule stabilizing .compound" defines in context with the present invention compounds which promote microtubule assembly or inhibit microtubule depolymerization.

The microtubule stabilizing effect of microtubule stabilizing compounds of the invention will generally be concentration dependent. More specifically, at lower concentrations and/or dosages a stabilizing effect occurs which at the same time permits the (further) growth of microtubules by the addition of further tubulin molecules. At higher concentrations and/or dosages the stabilizing effect is more pronounced to such an extent that the growth of microtubules can no longer occur and only existing structures are preserved. The range of higher concentrations may be accompanied by unwanted and/or toxic side effects which result from the microtubules being prevented from growing. The treatment of lesion of CNS axons, however, may require, depending on the type of lesion, not only a stabilization of microtubules, but additionally conditions where microtubules can grow. Successful

treatment of such lesions requires concentrations/dosages in the above defined lower range. The determination of said concentrations/dosages is within the skills of the skilled artisan. For example, suitable concentrations/dosages may be determined in a first step in a suitable animal model, preferably rodent model (see also the Examples enclosed herewith). Data obtained in a rodent model can be used for an extrapolation to the corresponding concentrations/dosages to be applied in humans (see, e.g., Harkness and Wagner (1989)). Preferred concentrations and dosages are disclosed herein below.

The term "lesions of CNS axons" defines in the context of the present invention injuries of CNS axons which comprise spinal cord injuries, neurotraumatic injuries, cut, shot and stab wounds in the nervous system and injuries subsequent of neurodegenerative diseases, multiple sclerosis or stroke.

Multiple sclerosis (MS) causes gradual destruction of myelin (demyelination) and transection of neuron axons in patches throughout the brain and spinal cord. While demyelination is a hallmark of the early stages of MS, are later stages characterized by the transection and fragmentation of CNS axons. As a consequence, the later stages of MS are not or only to a limited extent to a treatment with microtubule stabilizing compounds in concentration/dosage ranges which prevent the growth of microtubules. Rather, it is preferred that uses and methods of the invention, when applied in the treatment of late stages of MS, involve the use of concentrations/dosages which, while stabilizing microtubules, concomitantly permit the growth of microtubules. These considerations apply mutatis mutandis to all conditions requiring both stabilization and growth of microtubules. Suitable concentrations/dosages falling into the such defined therapeutic window are referred to as "lower" herein above and are disclosed in concrete terms below.

The local administration directly into the lesion or immediately adjacent thereto has the advantage that severe side effects known to occur concurrently with the

systemic administration of microtubule stabilizing compounds do not occur or are observed to a significantly lesser extent.

In accordance with the present invention, the term "pharmaceutical composition" relates to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition is an composition to be administered locally directly into the lesion or immediately adjacent thereto. It is in particular preferred that said pharmaceutical composition is administered to a patient via infusion or injection. Administration of the suitable compositions may also be effected by alternative ways, e.g., by a gelfoam, use of ethylene-vinylacetate copolymers such as Elvax, or by formulation in liposomes, microspheres, nanoparticles or biodegradable polymers.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, organic solvents including DMSO etc. Compositions comprising such carriers can be formulated by well known conventional methods. These . pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. The therapeutically effective amount for a given situation will readily be determined by routine experimentation and is within the skills and judgement of the ordinary clinician or physician. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 5 g units per day. However, a more preferred dosage for continuous infusion might be in the range of 0.01 μg to 2 mg, preferably 0.01 μg to 1 mg, more preferably 0.01 μg to 100 μg, even more preferably 0.01 μg to 50 μg and most preferably 0.01 μg to 10 μg units

per point of administration (lesion) per hour.

Other preferred dosage ranges that are particularly suitable when minimization or complete avoidance of toxic side effects of the microtubule stabilizing compound(s) according to the invention is to be achieved, include a range from 25 pg to 250 ng per day. More preferred dosages are in a range from 50 pg/d and 50 ng/d, yet more preferred in a range from 50 pg/d to 5 ng/d. These lower and upper limits may be combined with the lower and upper limits disclosed herein above. The dosages may be applied continuously, for example, by continuous infusion. These values refer to dosages to be administered locally, i.e. per point of administration (lesion). A preferred class of microtubule stabilizing compounds according to the invention to be administered within these dosage ranges are taxanes. Particularly preferred is Taxol (Paclitaxel).

Specific dosages and dosage ranges suitable for the treatment of a specific patient, while preferably falling into the intervals disclosed above, can be determined by the skilled person, when provided with the teaching of the present invention, without further ado. In particular, the dosage will depend on the length and the thickness of the neuronal tissue which has undergone a lesion. For example, in case of a lesion of the spinal chord, the region to be treated may be considered as being of cylindric shape. The dosage to be applied (locally, and preferably continuously) will depend on the volume of the region to be treated. The dependency is preferably linear, i.e. the dosage is proportional to i^x π x L, wherein r is the radius of the spinal chord and L is the length of the region to be treated. Assuming that the therapeutic dosage to be applied per volume unit exhibits only a weak dependency on the species to be treated, the volume of the region that has undergone a lesion in a rodent model on the one side and the volume of the region to be treated in a human subject can be used to calculate the therapeutic human dose according to D (human) = D (rat) x V (human) / V (rat), wherein D indicates the therapeutic dosages in human and rat, respectively, and V indicate the volumes of the neural tissue (e.g. spinal chord) to be treated. This approach

requires the determination of the therapeutic dosage in the model, e.g. D (rat) in a first step.

Preferably the dosage of the microtubule stabilizing compounds envisaged for the use according to the invention is such that the concentrations achieved upon local administration in other body parts, i.e. body parts other than the region to which the local administration is to be effected, do not or not significantly exceed the concentrations in said other body parts achieved upon systemic administration of the exemplary dosages provided in the list below. Thereby it is ensured that toxic side effects known in the art of microtubule stabilizing compounds do not occur or occur in significantly reduced form as compared to the application of doses required for systemic administration in the treatment of CNS lesions (see 41, 43). The skilled person is aware of or capable of determining without further ado the dosages for local administration provided with the information given below. A frequently used indicator is the plasma concentration of a compound. Furthermore, animal experiments are suitable and established in the art to determine said dosage for local administration, wherein said dosage is such that toxic concentrations are not reached in other body parts. Together with an exemplary dosage for systemic administration the list below also provides corresponding publications.

• Taxanes

Taxol Prescription dose 175 mg/m ² i.v. for Paclitaxel ®

IDN519Q

60-90 mg/kg i.v. or p.o. mice

Polizzi, D., Pratesi, G., Tortoreto, M., Supino, R., Riva, A., Bombardelli, E., and Zunino, F. (1999). A novel taxane with improved tolerability and therapeutic activity in a panel of human tumor xenografts. Cancer Res 59,

1036-1040.

IDN5390

90 mg/kg i.v., s.c. or p.o. mice

Petrangolini G, Cassinelli G, Pratesi G, Tortoreto M, Favini E, Supino R, Lanzi C, Belluco S, Zunino F. Antitumour and antiangiogenic effects of IDN 5390, a novel C-seco taxane, in a paclitaxel-resistant human ovarian tumour xenograft. Br J Cancer. 2004 Apr 5;90(7): 1464-8.

BMS 188797

50 mg/m ² i.v. human

Advani R, Fisher GA, Jambalos C, Yeun A, Cho C, Lum BL, et al. Phase I study of BMS 188797, a new taxane analog administered weekly in patients with advanced malignancies. Proceedings of ASCO 2001 ; 20 : 422 (abstr.).

BMS 184476

60 mg/m ² i.v. human

Hidalgo M, Aleysworth C, Hammond LA, Britten CD, Weiss G, Stephenson J Jr, et al. Phase I and pharmacokinetic study of BMS 184476, a taxane with greater potency and solubility than paclitaxel. J Clin Oncol 2001 ; 19 : 2493-

503.

Camps C, FeNp E, Sanchez JM, Massuti B, Artal A, Paz-Ares L, Carrato A,

Alberola V, Blasco A, Baselga J, Astier L, Voi M, Rosell R. Phase II trial of the novel taxane BMS-184476 as second-line in non-small-cell lung cancer.

Ann Oncol. 2005 Jan 31

BMS 185660

48 mg/kg i.v. mice Rose WC, Clark JL, Lee FY, Casazza AM. Preclinical antitumor activity of water-soluble paclitaxel derivatives. Cancer Chemother Pharmacol.

1997;39(6):486-92.

RPR 109881 A

60-75 mg/m ² i.v. human Kurata T, Shimada Y, Tamura T, Yamamoto N, Hyoda I, Saeki T, et al.

Phase I and pharmacokinetic study of a new taxoid, RPR 109881 A, given as a 1-hour infusion in patients with advanced solid tumors. J Clin Oncol 2000 ; 18 : 3164-71.

Sessa C, Cuvier C, Caldiera S, Vernillet L, Perard D, Riva A, et al. A clinical and pharmacokinetic (PK) phase I study of RPR 109881 A, a new taxoid administerd as a three hour intravenous infusion to patients with advanced solid tumors. Proceedings of ASCO 1998; 17 : 728 (abstr.).

TXD258(RPR11625A) 40 mg/kg i.v. mice

Gueritte-Voegelein F, Guenard D, Lavelle F, Le Goff MT, Mangatal L, Potier - P. Relationships between the structure of taxol analogues and their antimitotic activity. J Med Chem. 1991 Mar;34(3):992-8.

BMS 275183

6.5-60 mg/kg i.v., p.o. mice

Rose WC, Long BH, Fairchild CR, Lee FY, Kadow JF.Preclinical pharmacology of BMS-275183, an orally active taxane. Clin Cancer Res.

2001 Jul;7(7):2016-21.

DJ-927

27 mg/m ² p.o. in human

Syed, S. K., Beeram, M., Takimoto, C. H., Jakubowitz, J., Kimura, M.,

Ducharme, M., Gadgeel, S., De Jager, R., Rowinsky, E., and Lorusso, P. (2004). Phase I and Pharmacokinetics (PK) of DJ-927, an oral taxane, in patients (Pts) with advanced cancers. J Clin Oncol (Meeting Abstracts) 22,

2028.

9.8 mg/kg/day (8 days) i.v. or p.o. in mice

Shionoya, M., Jimbo, T., Kitagawa, M., Soga, T., and Tohgo, A. (2003). DJ- 927, a novel oral taxane, overcomes P-glycoprotein-mediated multidrug resistance in vitro and in vivo. Cancer Sci 94, 459-466.

Butltaxel analogues

AIi SM, Hoemann MZ, Aube J, Georg Gl, Mitscher LA, Jayasinghe LR. Butitaxel analogues: synthesis and structure-activity relationships. J Med

Chem. 1997 Jan 17;40(2):236-41.

Macrocvclic Taxane analogues

100-200 mg/kg i.v. mice Tarrant, J. G., Cook, D., Fairchild, C, Kadow, J. F., Long, B. H., Rose, W.

C, and Vyas, D. (2004). Synthesis and biological activity of macrocyclic taxane analogues. Bioorg Med Chem Lett 14, 2555-2558.

9β-dihydrobaccatin-9,10-acetals derivative taxanes

5.3-40 mg/kg p.o., i.v. mice

Takeda Y, Yoshino T, Uoto K, Chiba J, lshiyama T, Iwahana M, Jimbo T, Tanaka N, Terasawa H, Soga T. New highly active taxoids from 9beta- dihydrobaccatin-9,10-acetals. Part 3. Bioorg Med Chem Lett. 2003 Jan 20; 13(2): 185-90.

10-deoxy-10-C-morphoIinoethyl docetaxel analogues

112.5-600 mg/kg i.v., p.o. mice limura S, Uoto K, Ohsuki S, Chiba J, Yoshino T, Iwahana M, Jimbo T, Terasawa H, Soga T. Orally active docetaxel analogue: synthesis of 10- deoxy-10-C-morpholinoethyl docetaxel analogues. Bioorg Med Chem Lett.

2001 Feb 12;11(3):407-10.

RPR 116258A

8-25 mg/m ² i.v. human Lorthoraly A, Pierga JY, Delva R, Girre V, Gamelin E, Terpereau A, et al.

Phase I and pharmacokinetic (PK) study of RPR 116258A given as a 1-hour infusion in patients (pts) with advanced solid tumors. Proceedings of the 11th NCI-EORTC-AACR Symposium 2000 (resume 569) : 156-7.

Goetz AD, Denis LJ, Rowinsky EK, Ochoa L, Mompus K, Deblonde B, et al. Phase I and pharmacokinetic study of RPR 116258A, a novel taxane derivative, administered intravenously over 1 hour every 3 weeks. Proceedings of ASCO 2001 ; 20 : 419 (abstr.), 106a.

Fumoleau P, Trigo J, Campone M, Baselga J, Sistac F, Gimenez L, et al. Phase I and pharmacokinetic (PK) study of RPR 116258A, given as a weekly 1-hour infusion at day 1 , day 8, day 15, day 22 every 5 weeks in patients (pts) with advanced solid tumors. Proceedings of the 2001 AACR- NCI-EORTC International Conference. Resume 282 : 58.

PG-TXL =CT-2103 = XYOTAX 175 mg/m ² i.v. human

Langer CJ. CT-2103: a novel macromolecular taxane with potential advantages compared with conventional taxanes. Clin Lung Cancer. 2004 Dec;6 Suppl 2:S85-8.

Polymeric micellar paclitaxel 25 mg/kg i.v. mice

Zhang X, Burt HM, Mangold G, Dexter D ₁ Von Hoff D, Mayer L, Hunter WL. Anti-tumor efficacy and biodistribution of intravenous polymeric micellar paclitaxel. Anticancer Drugs. 1997 Aug;8(7):696-701.

Genexol-PM, Cremophor-Free. Polymeric Micelle-Formulated Paclitaxel

135-390 mg/m ² i.v. human

Kim TY, Kim DW, Chung JY ₁ Shin SG, Kim SC, Heo DS, Kim NK, Bang YJ. Phase I and pharmacokinetic study of Genexol-PM, a cremophor-free, polymeric micelle-formulated paclitaxel, in patients with advanced malignancies. Clin Cancer Res. 2004 Jun 1 ;10(11):3708-16.

Docosahexaenoic acid-conjugated Taxol (Taxoprexin)

60-120 mg/kg i.v. mice Bradley MO, Webb NL, Anthony FH, Devanesan P, Witman PA, Hemamalini

S, Chander MC, Baker SD, He L, Horwitz SB, Swindell CS. Tumor targeting by covalent conjugation of a natural fatty acid to paclitaxel. Clin Cancer Res. 2001 Oct;7(10):3229-38.

PTX-DLPC (aerosol)

5mg/kg inhalation mice

Koshkina NV, Waldrep JC, Roberts LE, Golunski E, Melton S, Knight V. Paclitaxel liposome aerosol treatment induces inhibition of pulmonary metastases in murine renal carcinoma model. Clin Cancer Res. 2001 Oct;7(10):3258-62.

PNU-TXL

80-196 mg/m ² i.v. human

Meerum Terwogt JM, ten Bokkel Huinink WW, Schellens JH, Schot M, Mandjes IA, Zurlo MG, Rocchetti M, Rosing H, Koopman FJ, Beijnen JH. Phase I clinical and pharmacokinetic study of PNU 166945, a novel water-soluble polymer-conjugated prodrug of paclitaxel. Anticancer Drugs. 2001 Apr; 12(4):315-23.

MAC-321 10-70mg/kg i.v. in mice

Lethal dose 140mg/kg

Sampath, D., Discafani, C. M., Loganzo, F., Beyer, C, Liu, H., Tan, X., Musto, S., Annable, T., Gallagher, P., Rios, C, and Greenberger, L. M. (2003). MAC-321, a novel taxane with greater efficacy than paclitaxel and docetaxel in vitro and in vivo. MoI Cancer Ther 2, 873-884.

Docetaxel/Taxotere

Prescription dose 100 mg/m ² i.v. for Taxotere®

75 mg/m ² i.v. human according to Kulke MH, Kim H, Stuart K, Clark JW, Ryan DP, Vincitore M, Mayer RJ, Fuchs CS. A phase Il study of docetaxel in patients with metastatic carcinoid tumors. Cancer Invest. 2004;22(3):353-9.

Epothilon

Epothilone B (Patupilone, EPO906)

2-4 mg/kg mice in combination with the protein kinase inhibitor imatinib (STI 571 , GIivec)

O'Reilly, T., Wartmann, M., Maira, S. M., Hattenberger, M., Vaxelaire, J., Muller, M., Ferretti, S., Buchdunger, E., Altmann, K. H., and McSheehy, P. M. (2005). Patupilone (epothilone B, EPO906) and imatinib (STI571 , Glivec) in combination display enhanced antitumour activity in vivo against experimental rat C6 glioma. Cancer Chemother Pharmacol 55, 307-317.

Desoxyepothilone B 15-60 mg/kg i.v. mice

Chou TC, Zhang XG, Harris CR, Kuduk SD, Balog A, Savin KA, Bertino JR, Danishefsky SJ. Desoxyepothilone B is curative against human tumor xenografts that are refractory to paclitaxel. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26): 15798-802.

BMS-310705 (Aza-EpoB)

40 mg/m ² i.v. human

Eng C, Kindler HL, Nattam S, Ansari RH, Kasza K, Wade-Oliver K, Vokes EE.A phase Il trial of the epothilone B analog, BMS-247550, in patients with previously treated advanced colorectal cancer. Ann Oncol. 2004 Jun;15(6):928-32.

F3-deH-dEpoB

20-30 mg/kg i.v. mice

Chou TC, Dong H, Rivkin A, Yoshimura F, Gabarda AE, Cho YS, Tong WP, Danishefsky SJ. Design and total synthesis of a superior family of epothilone analogues, which eliminate xenograft tumors to a nonrelapsable state.

Angew Chem lnt Ed Engl. 2003 Oct 13;42(39):4762-7.

Epothilone D

Kolman, A. (2004). Epothilone D (Kosan/Roche). Curr Opin Investig Drugs 5, 657-667.

dEpoF

15-30 mg/kg

Chou TC, O'Connor OA, Tong WP, Guan Y, Zhang ZG, Stachel SJ, Lee C, Danishefsky SJ. The synthesis, discovery, and development of a highly promising class of microtubule stabilization agents: curative effects of desoxyepothilones B and F against human tumor xenografts in nude mice. Proc Natl Acad Sci U S A. 2001 JuI 3;98(14):8113-8.

Sesquiterpene lactones

Parthenolide

1-5 mg/day (tablets) p.o. human

Curry EA 3rd, Murry DJ, Yoder C, Fife K, Armstrong V, Nakshatri H, O'Connell M, Sweeney CJ. Phase I dose escalation trial of feverfew with standardized doses of parthenolide in patients with cancer. Invest New

Drugs. 2004 Aug;22(3):299-305.

Costunolide

50mg/kg p.o. rats Matsuda H, Shimoda H, Ninomiya K, Yoshikawa M. Inhibitory mechanism of costunolide, a sesquiterpene lactone isolated from Laurus nobilis, on blood- ethanol elevation in rats: involvement of inhibition of gastric emptying and increase in gastric juice secretion. Alcohol Alcohol. 2002 Mar-Apr;37(2):121- 7.

• Dicoumarol

34 mg/kg i.p. mice

Begleiter A, Leith MK, Thliveris JA, Digby T. Dietary induction of NQO1 increases the antitumour activity of mitomycin C in human colon tumours in vivo. Br J Cancer. 2004 Oct 18;91(8):1624-31.

• Ferulenol

2-319 mg/kg i.p. or p.o. mice toxicity test...

Fraigui O, Lamnaouer D, Faouzi MY. Acute toxicity of ferulenol, a 4- hydroxycoumarin isolated from Ferula communis L. Vet Hum Toxicol. 2002

Feb;44(1):5-7.

• NDGA derivative Tetra-O-methyl nordihydroguaiaretic acid 20 mg intra-tumoral mice Heller JD, Kuo J, Wu TC, Kast WM, Huang RC. Tetra-O-methyl nordihydroguaiaretic acid induces G2 arrest in mammalian cells and exhibits tumoricidal activity in vivo. Cancer Res. 2001 Ju1 15;61(14):5499-504.

• Tubercidin 10-50 mg/kg p.o., i.v. mice

Olsen DB, Eldrup AB, Bartholomew L ₁ Bhat B, Bosserman MR, Ceccacci A,

Colwell LF, Fay JF, Fiores OA, Getty KL, Grobler JA, LaFernina RL, Markel EJ, Migliaccio G, Prhavc M, Stahlhut MW, Tomassini JE, MacCoss M, Hazuda DJ, Carroll SS. A 7-deaza-adenosine analog is a potent and selective inhibitor of hepatitis C virus replication with excellent pharmacokinetic properties. Antimicrob Agents Chemother. 2004

Oct;48(10):3944-53. .

Jaffe JJ, Doremus HM, Meymarian E. Activity of tubercidin against immature Fasciola hepatica in mice. J Parasitol. 1976 Dec;62(6):910-3.

• FR182877

Sato B, Muramatsu H, Miyauchi M ₁ Hori Y, Takase S, Hino M, Hashimoto S, Terano H. A new antimitotic substance, FR182877. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities. J Antibiot (Tokyo). 2000 Feb;53(2): 123-30. Sato, B., Nakajima, H., Hori, Y., Hino, M., Hashimoto, S., and Terano, H.

(2000b). A new antimitotic substance, FR182877. II. The mechanism of action. J Antibiot (Tokyo) 53, 204-206.

Particularly preferred dosages are recited herein above. Progress can be monitored by periodic assessment.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable organic esters such as ethyl oleate, and further organic solvents including DMSO. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, fixed oils or organic solvents including DMSO. Intravenous vehicles include fluid and nutrient replenishes, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating

agents, and inert gases and the like. In addition, the pharmaceutical composition might comprise proteinaceous carriers, like, e.g., serum albumin or immunoglobulin, preferably of human origin. It is envisaged that the pharmaceutical composition of the might comprise, in addition -to the microtubule stabilizing compound, further biologically active agents, depending on the intended use of the pharmaceutical composition. Such agents might be drugs acting as cytostatica, drugs preventing hyperurikemia (decreased renal excretion of uric acid), drugs inhibiting immunereactions (e.g. corticosteroids), and/or drugs acting on the circulatory system. The administration of the pharmaceutical compound described herein is defined as locally directly into the lesion or immediately adjacent thereto. This definition is understood in the context of the present invention as an opposite to a more widespread or systemic administration. Such local administration may be effected e.g. by an injection or continuous infusion of the pharmaceutical composition directly into the lesion or immediately adjacent thereto. Alternatively, the composition may be administered e.g. by means of gelfoam or Elvax which are known in the art, inter alia from (44).

It has been surprisingly found that the microtubules of the axon in polarized neurons during initial axon formation show a higher ratio of acetylated/ tyrosinated tubulin and that the microtubules in the future axons are more resistant to nocodazol depolymerization. Furthermore, stabilization of microtubules using taxol, a drug used in cancer treatment, causes multiple axons to grow.

Beside of this effect of microtubule stabilizing which is the unique feature of the above recited compounds, taxol treated cultures show a drastic reduction in astrocyte numbers in these cultures. This induction of cell death was understood to be the effect of taxol to kill dividing cells. Without being bound by theory, the combination of the microtubule stabilizing effect of taxol with the effect of drastic reduction in astrocyte numbers may result in an additional improvement of the treatment of CNS lesions. This additional benefit

may be explained by an induction of intrinsic growth capacity (stabilizing the microtubules of the axons and thereby inducing axonal regrowth) and, second, a reduction of the inhibitory environment of the glial scar by reducing the astrocytes at the lesion site.

The present invention furthermore relates to a method for the treatment of lesions of CNS axons the method comprising the step of administering to a patient in the need thereof a pharmaceutical composition comprising

(i) one or more microtubule stabilizing compounds selected from the group consisting of taxanes, epothilones, laulimalides, sesquiterpene lactones, sarcodictyins, diterpenoids, peloruside A, discodermolide, dicoumarol, ferulenol, NSC12983, taccalonolide A, taccalonolide E, Rhazinilam, nordihydroguaiaretic acid (NDGA), GS-164, borneol esters, Synstab A, Tubercidin and FR182877 (WS9885B) and, optionally, (ii) further comprising suitable formulations of carrier, stabilizers and/or excipients, wherein the pharmaceutical composition is administered locally directly into the lesion or immediately adjacent thereto.

According to the use and the method of the invention it is preferred that

(a) the taxane(s) is/are selected from the group consisting of IDN5190, IDN 5390, BMS-188797, BMS-184476, BMS-185660, RPR109881A, TXD258(RPR11625A), BMS-275183, PG-TXL, CT-2103, polymeric micellar paclitaxel, Taxoprexin, PTX-DLPC, PNU-TXL (PNU166945), MAC-321 , Taxol, Protaxols, photoaffinity analogues of Taxol, photoactivatable Taxol and

Docetaxel/Taxotere;

(b) the epothilone(s) is/are selected from the group consisting of epothilone A, epothilone B, dEpoB, BMS-247550 (aza-EpoB), epothilone D, dEpoF and BMS-310705; (c) the sesquiterpene lactone(s) is/are Parthenolide or Costunolide;

(d) the sarcodictyin(s) is/are selected from sarcodictyin A and sarcodictyin B;

(e) the diterpenoid(s) is/are selected from the group consisting of Eleutherobins, caribaeoside and caribaeolin; or

(f) the discodermolide is/are a marine-derived polyhydroxylated alkatetraene lactone, used as a immunosuppressive compound. Protaxols are known as water soluble compounds releasing taxol at basic pH/in plasma (Nicolaou et al., 1993). Photoaffinity analogues of taxol are described in Rao et al., 1999; Rao et al., 1994. Photoactivatable taxol is known from Buck and Zheng, 2002. Docetaxel/Taxotere is known from Ringel and Horwitz, 1991. Parthenolide is known from Miglietta et al., 2004. Eleutherobin is known from Long et al., 1998. epothilones A and B are known from Ojima et al., 1999 and epothilone D from Dietzmann et al., 2003. Dicoumarol is known from Madari et al., 2003. Ferulenol (a prenylated 4-hydroxycoumarin) is known from Bocca et al.,

2002. Nordihydroguaiaretic acid (NDGA) is known from Nakamura et al., 2003. BMS-310705 is known from Uyar et al., 2003. NSC12983 (synthetic steroid derivative) is known from Wu et al., 2001. IDN 5390 is known from Taraboletti et al., 2002. Sarcodictyin A and B is known from Hamel et al., 1999. GS-164 (a small synthetic compound) is known from Shintani et al., 1997. Borneol esters suitable as inhibitors of microtubule depolymerization are known from Klar et al., 1998. Particularly envisaged for the use according to the present invention is compound 19a described in Klar et al., 1998. Compounds isolated from the octocoral Erythropodium caribaeorum such as eleutherobin, the new antimitotic diterpenoids desmethyleleutherobin, desacetyleleutherobin, isoeleutherobin A, Z-eleutherobin, caribaeoside, and caribaeolin are known from Cinel et al., 2000. Tubercidin (7- deazaadenosine) is known from Mooberry et al., 1995. Taccalonolides (plant- derived steroids with microtubule-stabilizing activity) are known from Tinley et al.,

2003. Rhazinilam, a plant-derived alkaloid, is known from David et al., 1994. Costunolide, a sesquiterpene lactone found in medicinal herbs, is known from Bocca et al., 2004.

As described herein above, the pharmaceutical composition may be locally administered by different means and methods. According to a preferred

embodiment of the invention the pharmaceutical composition is administered using an osmotic pump. Osmotic pumps are infusion pumps for continuos dosing. Osmotic pumps and their uses are generally known to the person skilled in the art. Osmotic pumps may be obtained from a variety of manufacturers including Alzet (www.alzet.com). Alternatively, administration is to be effected by local syringe injection. The envisaged routes of administration imply appropriate formulations. The skilled person is aware of formulations suitable for the above mentioned routes of administration.

In a further preferred embodiment, said pharmaceutical composition comprises gelfoam, Elvax, liposomes, microspheres, nanoparticles and/or biodegradable polymers.

In a more preferred embodiment of the invention the one or more taxanes comprised in the pharmaceutical composition is/are in a final concentration in a range of 0,1 pM and 1mM. More preferably, said final concentration is in a range of 0.1 pM to 100μM, 0.1pM to 1μM, 0.1pM to 10OnM, 0.1pM to 1OnM, 0.1pM to 1nM, or 1OpM to 1nM, and most preferred between 10OpM and 1nM.

In a preferred embodiment, one or more microtubule stabilizing compounds are the only active agent(s) comprised in said pharmaceutical composition.

It is also envisaged by the present invention that the pharmaceutical composition further comprises a compound selected from the group of c3-exoenzyme, chondroitinase, an iron chelator, cAMP and its derivatives such as dibutyryl cAMP (as described in Neumann et al., 2002), and substances that increase the intracellular cAMP level, either by stimulating the adenyl cyclase or by inhibiting phosphodiesterase, including rolipram. Said chelator is to be administered at the lesion site. cAMP and its derivatives such as dibutyryl cAMP and phosphdoesterase inhibitors such as rolipram may be administered locally at the lesion site or at the location of the brain nuclei or ganglia (location of the cell body) of the neurnal class to be treated, or systemically by infusion. Optionally, the

pharmaceutical composition may further comprise suitable formulations of carrier, stabilizers and/or excipients.

The beneficial effects of c3-exoenzyme and chondroitinase on axonal growth are known in the art (see references 10 and 12 as regards c3-exoenzyme and Bradbury et al., 2002 and Moon et al., 2001 as regards chondroitinase) and the compounds are the subject of clinical studies. Iron chelator therapy and cAMP therapy are also known to the person skilled the art e.g. from Hermanns et al., 2001 (iron chelator therapy) and from Nikulina et al., 2004; Pearse et al., 2004; and Neumann et al., 2002 (therapy with cAMP derivatives and/or rolipram).

The Figures show:

Figure 1 : Different distribution of acetylated and tyrosinated microtubules in hippocampal neurons (A-H) Stage 3 (A-D) and stage 2 (E-H) rat hippocampal neurons were fixed and stained with antibodies recognizing acetylated (B, F) and tyrosinated (C, G) oc-Tubulin. In stage 3 neurons, an enrichment of acetylated α-Tubulin is found in microtubules in the axon (arrow) in comparison to microtubules of minor neurites (arrowheads) (D and I). In ~50% of stage 2 neurons, the ratio acetylated/tyrosinated α-Tubulin is significantly increased in one of the minor neurites (white arrowhead with asterisk) (H and J).

(I, J) Ratio quantitation of fluorescence intensities of acetylated and tyrosinated α-Tubulin in microtubules of stage 2 (J) and stage 3 (I) neurons. In the medial part of each process, the average fluorescence intensity of both acetylated and tyrosinated oc-Tubulin was measured in a square of 5x5 pixel of pictures taken with 63x magnification and the ratio of the values was calculated.

Figure 2: Stability difference of microtubules in axons and minor neurites

Stage 3 (B-E) and stage 2 (G-J) rat hippocampal neurons were treated with 0.075% DMSO (B ₁ C; G ₁ H) or Nocodazole (3.3 or 5μM) (D ₁ E; I ₁ J) for 5 min, fixed and immunostained with an anti-α-Tubulin antibody and Rhodamine-coupled Phalloidin. In DMSO-treated control cells, microtubules reach close to the distal end (defined by the actin cytoskeleton) of axon (arrow) and minor neurites (arrowheads) (C, H). In Nocodazole-treated cells, microtubules retract towards the cell body (E, J). Retraction of axonal microtubules is significantly lower than retraction of microtubules in minor neurites (E, M). In a subpopulation of stage 2 neurons one minor neurite (arrowhead with asterisk) shows enhanced resistance to disruption by Nocodazole (J).

(K, L) Microtubules containing acetylated α-Tubulin show increased resistance to Nocodazole-induced depolymerization.

(M, N) Quantitation of the microtubule retraction of stage 3 (M) and stage 2 (N)

neurons.

Figure 3: Microtubule destabilization selectively impairs outgrowth of minor neurites (A) After 1 DIV, low concentrations of Nocodazole or DMSO were added to the culture medium of rat hippocampal neurons. After 2 further DIV, neurons were fixed and stained for the axonal marker Tau-1.

(B, C) After 3 DIV, neurons have formed one axon (black arrow) and several minor neurites (black arrowheads) under control conditions (B). The axon is positive for the axonal marker Tau-1 (white arrow) and shows the typical Tau-1 gradient towards the distal part of the axon, while minor neurites are Tau-1 -negative (white arrowheads) (C).

(D, E) While the number of minor neurites is reduced under growth conditions which slightly destabilize microtubules (D), neurons are still able to form an axon (E, white arrow).

(F) Nocodazole reduces the number of minor neurites formed in a concentration dependent manner.

(G) Neurite extension per se is not blocked by low concentrations of Nocodazole. Neurons are still growth-competent, as total neurite length increases under all growth conditions from day 1 to day 3 in vitro.

Figure 4: Taxol-induced microtubule stabilization triggers formation of multiple axons

(A) After 1 DIV, rat hippocampal neurons were treated with low concentrations of Taxol or DMSO. After 2 further DIV, neurons were fixed and stained for the axonal marker Tau-1.

(B, C) Taxol induces the formation of multiple elongated processes (B, black arrows) which are positive for the axonal marker Tau-1 (C, white arrows).

(D, E) Neurons grown under control conditions have formed one Tau-1 positive axon (arrow) and several minor neurites (arrowheads).

(F) Effects of Taxol on neurite extension: The number of neurites longer than 60μm

is increased after 2 days of Taxol-treatment, even exceeding the effect of the positive control Cytochalasin D, a drug also known to induce multiple axons (Bradke and Dotti, 1999).

(G) The number of cells with more than one Tau-1 -positive process increases in a concentration-dependent manner in Taxol-treated neurons.

(H) In later stages of neuronal development cells form a dense network which makes it very hard to assign processes to the cell they originate from. To circumvent this problem and follow individual neurons, a mouse hippocampal neuron culture containing 5% of neurons which express green fluorescent protein (GFP) under the control of the Actin-promoter was used for long term experiments. The mixed culture was treated with 1OnM Taxol or DMSO after 1 DIV, incubated till day 7 in vitro, fixed and immunostained for the dendritic marker MAP2. (I, J) Taxol induces the formation of multiple long processes (I) which have no or a weak MAP2-signal (J) resembling the axons of control cells (compare L). (K, L) The axon of cells grown under control conditions is MAP2-negative, the dendrites have a strong MAP2-signal.

Figure 5: Taxol induces the formation of axon-like processes with increased microtubule stability (A) Stage 2 rat hippocampal neurons were treated with 1OnM Taxol after 1 DIV. (B-. H) On day 2 in vitro, cells had formed multiple axon-like processes (B, G, black arrows) and were treated with 0.02% DMSO (H) or 5μM Nocodazole (C-E) for 5 min, PHEM-fixed and immunostained with an anti-α-Tubulin antibody (C) and Rhodamine-coupled Phalloidin (D) or antibodies recognizing acetylated and tyrosinated α-Tubulin (H).

(C) In neurons pretreated with 1OnM Taxol for 1 day, only little microtubule retraction occurred (C, white arrows).

(F) Retraction of microtubules in Taxol-treated neurons is significantly lower than retraction of microtubules in minor neurites (p<0.001) but does not differ significantly from retraction of axonal microtubules (p=0.11).

(G, H) Taxol-induced processes (G, black arrows) show an increased ratio of

acetylated to tyrosinated alpha-Tubulin like axons (H, white arrows; compare Figure 1D).

Figure 6: Retraction bulbs but not growth cones increase in size over time. GFP-M mice were anesthetized to injure either the sciatic nerve or the dorsal columns, the mice were then sacrificed at various post injury times, and the tissue was fixed and analyzed exploiting the GFP-signal of the mouse strain using confocal microscopy. a-d, Growth cones at 1 day (a) and 1 week (b) after sciatic nerve crush; Higher magnifications in (c) and (d), respectively. e-j, Retraction bulbs at 1day (e), 1 week (f) and 5 weeks (g) after dorsal column lesion; Higher magnifications in (h), (i) and (j), respectively. Note that some of the axons possess some minor swelling on the shaft in addition to the big terminal bulbs (i and j). k, Size comparison of growth cones and retraction bulbs by tip / axon shaft ratio shows the size increase of the retraction bulbs while growth cone size

(morphology) remains constant.

I, Absolute size increase of retraction bulbs over time analyzed by surface area quantification. All values are (mean ± s.d). Asterisks indicate p < 0.001 in (k) and (I), pi: post injury.

Scale bars, 75 μm (a, b, e-g), 5 μm (c, d, h-j).

Figure 7: Retraction bulbs contain mitochondria and frans-Golgi-Network (TGN) derived vesicles. a-d, Electron microscopy (EM) was used to analyze the morphology and quantity of vesicles and mitochondria. EM images of a growth cone (a) at 2 days after sciatic nerve crush and a retraction bulb (c) at 4 days after dorsal column lesion showing the mitochondria and vesicle accumulation in the end structures, b, d, Higher magnification of marked areas in (a) and (c), respectively. Black arrows show some of the vesicles and black arrowheads some of the mitochondria in the

end structures. e-j, Localization of vesicles shown by the immunofluorescent labeling of vesicles with anti- golgin-160 antibody specific to TGN derived vesicles. GFP positive end structures (green) (e, h); labeled vesicles (red) (f, i); overlay of GFP and golgin-160 signal (g, j). White arrows indicate the vesicles that accumulate in the end structures, and white arrowheads indicate vesicles in the axon shaft. k,l, Concentration of the mitochondria (k) and vesicles (I) in the growth cones at 2 days and in retraction bulbs at various time points post injury as calculated from EM images. GCs: Growth Cones and RBs: Retraction Bulbs.

Single asterisk indicate p < 0.05 between retraction bulbs at that time point and growth cones at 2 days pi (n - 7-9 EM images for each data set). Scale bars, 1 μm (a, c), 5 μm (g, j).

Figure 8: Retraction bulbs have dispersed and disorganized microtubules, a-f, lmmunostaining of end structures with anti-Glu-tubulin antibody recognizing the detyrosinated alpha-tubulin subunits already assembled into microtubule filaments to visualize the organization of microtubules, a-c, Growth cones possess tightly bundled microtubules parallel to the axonal axis. GFP positive growth cone (green) (a), anti-Glu-tubulin staining (red) (b) and overlay (c). d-f, Retraction bulbs have highly dispersed and disorganized microtubules. GFP positive retraction bulb (green) (d), anti-Glu-tubulin staining (red) (e) and overlay

(f)-

Yellow arrows in (f) indicate dispersed microtubules that are highly deviated from the axonal axis and positioned almost vertical. White arrow indicates where microtubules are densely accumulated and white arrowhead indicates areas with fewer microtubules. g-o, The electron microscopy images of retraction bulbs and growth cones were analyzed to quantify the microtubule angle deviations. Unlesioned central axons were also quantified as a control. A representative unlesioned CNS axon (g), growth cone (j) and retraction bulb (m). h, k, n, the microtubules in the images

were traced in black to indicate overall microtubule organization in the end structures, i, I, o, higher magnifications of the marked areas in (h, k and n) respectively. The angles between the pseudo-colored microtubule filaments and the axonal axis were quantified in all images as it is shown in the representative images. The left site of the image in m-o is distal to the cell body. p, results of angle quantification. In the graph, each dot represents a single microtubule filament (at least 7 EM image analyzed and pooled for each condition). The microtubules are significantly more vertical in the retraction bulbs compared to both growth cones and unlesioned central axons (p < 0.0001). Scale bars, 5 μm (a, d), 1 μm (g, j, m).

Figure 9: Nocodazole application converts a growth cone into a retraction bulb like structure.

One day after sciatic nerve injury, GFP-M mice were reanesthetized and their sciatic nerves exposed. The exposed sciatic nerves were treated with 10μl of either

330μM nocodazole (a-c; h-m) or 5% DMSO as control (d-f). The effects on the morphology and the cytoskeletal structure were assessed 24 hours after treatment. a, After treatment with nocodazole (dissolved in 5 % DMSO) 45% ± 13% of the axons form bulbs at their tips instead of growth cones. b, c, Higher magnifications of the peripheral bulbs marked with arrowheads in (a). d, The morphology of the growth cones did not change under control conditions. Growth cones treated with 5% DMSO resemble untreated growth cones. e, f, Higher magnifications of the growth cones marked with arrowheads in (d). g, Quantification of peripheral bulb sizes. The axon / tip ratio of the peripheral bulbs is significantly higher than of both DMSO treated growth cones and untreated growth cones (Asterisks indicate p < 0.001 for each condition). h-m, The nocodazole treated peripheral bulbs (h and k, green) were immunostained with anti-Glu-tubulin antibody (i and I, red) to assess the underlying microtubule organization. The white arrowheads in merged images (j and m) point to some of the microtubules which are dispersed in a similar way as observed in retraction bulbs (compare with Fig. 3d-f).

Scale bars: 50 μm in (a, d); 5 μm in (b, c, e, f); 2μm (h and k).

Figure 10: Stabilization of microtubules increases the length and number of sprouts. a-h, The effect of the taxol on dorsal column axons which already formed retraction bulbs has been observed by live imaging. 10 μl of saline (control) (a-d) or 10 μl of 5 μM taxol (e-h) applied repetitively (once per hour, 4 or 5 times in total) starting from 24 hours post injury. The lesion sites were visualized by live imaging technique. Overview images: before lesion: control (a), taxol (e); immediately after injury: control (b), taxol (f); 24 hours after injury, just before treatments: control (c), taxol (g); 48 hours after injury, 24 hours after treatments: control (d), taxol (h). (c' ₅ d', g', h') are the higher magnifications of the marked areas in (c, d, g, h) respectively. The white arrow heads in (h') show some of the regenerating sprouts that were formed after taxol treatment. i-j, High resolution confocal images of the regions in (d') and (h') respectively. After capturing the live images at 48 hours post injury, the animals were perfused for the confocal microscopy. The white arrow heads in (i) and (j) indicate the sprouts. Note that, there are many more sprouts in taxol applied animals compared to controls. In addition, the lengths of the sprouts in taxol conditions are longer than the controls. Although the slim growth cone and retraction bulb like morphologies are detectable at the terminal of sprouts from both control and taxol applied axons, the spiky terminals full of many filopodia like extensions are only detectable after taxol treatment. k, Quantification of the sprout lengths. All the sprout lengths coming from one axon have been quantified and summed up to indicate the total length of sprouts from one axon. Microtubule stabilization by taxol significantly induced longer sprouts compared to controls (p < 0.01). Each dot represents the quantifications from one animal. I, Quantification of the numbers of sprouts coming from one axon. Microtubule stabilization by taxol significantly increased the number of sprouts compared to controls (p < 0.05). Each dot represents the quantifications from one animal.

Scale bars, 500 μm (a-h), 100 μm (c\ d', g' _? h'), 50 μm (i, j).

Figure 11: End-structures of PNS and CNS axons following lesion. a, Cartoon of the spinal cord depicting the DRG neurons and their axons in the spinal cord and sciatic nerve. A representative DRG neuron is highlighted; the cell body residing in the dorsal root is shown in blue, the central branch of the axon in green and the peripheral branch in red. The site of dorsal column transection to lesion the central axonal branches and the site of sciatic nerve crush to lesion the peripheral axonal branches are indicated by arrows. b, c, Saggital section (b) and cross section (c) through the thoracic spinal cord, showing the GFP positive axons in the dorsal columns from a GFP-M transgenic mouse. The dashed rectangles encircle the central axonal branches of primary sensory neurons which localize superficially within the dorsal column of the spinal cord. These axons were targeted for lesioning in this study. d-f, Horizontal section of the sciatic nerve showing PNS axons: unlesioned (d) and 2 days after sciatic nerve lesion (e). f, higher magnification of the marked area in (e). White arrowheads in (e) show the growth cones formed at the proximal tip of the cut peripheral axonal braches. g-i, Horizontal section of the dorsal column showing CNS axons: unlesioned (g) and 2 days after dorsal column lesion (h). i, higher magnification of the marked area in (h). Arrowheads in (h) show the retraction bulbs formed at the proximal tips of the lesioned central axonal branches. Arrow in (h) marks a retraction bulb observed on the axon shaft of a lesioned neuron. Scale bars: 75 μm (b, d, e, g and h); 300 μm (c); 5 μm (f and i).

Figure 12: Typical appearance of in vitro growth cones of DRG neurons, a, DRG neuronal culture has beed prepared as described ³³ before and stained by tuj-1 antibody (red) 30 hours after culturing on laminin substrate. Since DRG neurons do not form dendrites in vivo as well as in vitro, all the axonal tips observed in the image can be considered growth cones. b-g, Higher magnifications of the pointed axonal tips in (a). Most of the growth

cones have several filopodia (b, c, e, f, and g). Some of the growth cones are outspreaded on the substrate (c, e and g) like typical hand-like morphology of the cultured hippocampal neurons. Those in vitro growth cones of DRG neurons are different in many aspects than in vivo growth cones: 1- in vitro they have several filopodia but in vivo not, 2- in vitro they grow in almost all directions by making some turns or forming branches but in vivo they grow in a parallel way without any bending and branching, 3- in vitro most of the times they are broad but in vivo they have a streamlined shape.

Figure 13: Taxol application interferes with the formation of retraction bulbs. a, b, In vivo live imaging has been used to identify the effect of the taxol on dorsal column after a small lesion. 10 μl of 1 μM taxol (a) or saline (b) applied repetitively (once per hour) starting from immediately after injury. The lesion sites before and after injury (up to 6 hours post injury) in taxol (a) and saline (b) applications were visualized. One hour after injury the first big retraction bulbs form in control animals but not in the taxol applied animals. Although most of the axons form retraction bulbs in the saline application over 6 hours (71.3% ± 9.1%; mean ± s.d.; n=18 animals) only a minority of taxol treated animals show axons forming retraction bulbs (22.8% ± 13.1%; mean ± s.d.; n=11 animals), (compare also 6 hour time point of (a) and (b)). c, d, Confocal image of the lesion sites at 6 hours after injury from the same animals shown in a and b, respectively. The dash lines indicate the injury site. e-j, Higher magnifications of the pointed retraction bulbs (the biggest ones from the samples); e-f from the taxol applied and h-j from the control. Note that the retraction bulbs of the taxol applied axons are smaller than the controls. k, The percentage of the axons with retraction bulbs from the taxol applied axons (n= 11 animals) compared to saline applied control axons (n=18 animals). From 1 hour post injury time point on, there are significantly less bulbs in taxol applied animals (*p < 0.05, *** p < 0.001). Scale bars: 100 μm in (a, b, c, d); 20 μm in (e, f, g, h, i, j).

Figure 14: Model for retraction bulb formation versus growth cone mediated regeneration, a, d, Before injury: PNS and CNS axons are connected to their targets and are functional. An injury to a CNS axon (a) or PNS axon (d) results in disconnection of the axon from the target tissue. The red flashes in (a) and (g) represent the injury. b, e, A few days after injury: The axonal part distal from the lesion site dies back (not shown). The CNS axon proximal to axotomy responds to injury by forming a retraction bulb (b) which is characterized by dispersed microtubules and accumulated internal structures including post-Golgi derived vesicles and mitochondria as a result of lack of regrowth. Stabilization of the microtubules starting immediately after injury (from a) can inhibit formation of these retraction bulbs.

The PNS axon forms a growth cone following a lesion (e) which efficiently uses membrane trafficking, energy and an intact cytoskeleton for elongation. Microtubules align straight to serve as a road for the transported materials and also to support the rapidly elongating growth cone membrane, and to form the backbone of the growing axon. Destabilization of microtubules at this level can convert the growth cones into retraction bulb like structures containing dispersed microtubule organization. Conversely, stabilization of microtubules at this level can increase the regeneration capacity of the lesioned CNS axons and produce more and longer sprouts. c, f, A few weeks after injury: The Retraction bulb still enlarges with the accumulation of continuous flow of membrane traffic (c). In contrast, the PNS axon continues its rapid elongation until finding and connecting to the target (f).

The invention will now be described by reference to the following biological examples which are merely illustrative and are not to be construed as a limitation of scope of the present invention.

Example 1 : Procedures and Material

Ce// Culture

Primary hippocampal neurons derived from rat embryos were cultured following the protocol of Goslin and Banker (1991) and de Hoop et al. (1997). In brief, the hippocampi of E18 rats were dissected, trypsinized, and physically dissociated. The cells were then washed in HBSS, and 1.0-1.3x10 ⁵ cells were plated onto poly- lysine-treated glass coverslips in 6 cm petri dishes containing minimal essential medium (MEM) and 10% heat-inactivated horse serum. The cells were kept in 5% CO2 at 36.5°C. After 12-18 hr, the coverslips were transferred to a 6 cm dish containing astrocytes in MEM and N2 supplements.

Handling Living Cells on the Microscope Stage

Neurons were kept alive and observed at the microscope as described in detail in Bradke and Dotti (1997). For short observation times, we used glass bottom dishes (MatTek Corporation, Ashland, MA) filled with 3ml HEPES-buffered HBSS, which permitted the fitting of a 12mm Cellocate coverslip (Eppendorf, Hamburg, Germany) and allowed observation with 32x and 4Ox objectives. Cells were kept at 36°C on the microscope stage by heating the room to an adequate temperature. Cells were illuminated with a 100 W, 12 V halogen light, which was set to minimal intensity to avoid phototoxicity.

Videomicroscopy

Living cells were analyzed using a Zeiss Axiovert 135. The microscope was equipped with LD A-Plan 32x (NA 0.4), Plan-Apochromat 4Ox (NA 1.0) and Plan- Apochromat 63x (NA 1.4) objectives. Images were captured using a camera from the 4912 series (Cohu, San Diego, CA). The camera was connected to a Hamamatsu CCD camera C 2741 control panel. Pictures were recorded on the

hard disc of a personal computer equipped with an image grabber (LG3 image grabber, Scion, Frederick, MD).

Cells grown on Cellocate coverslips (Eppendorf, Hamburg, Germany) were transferred into a petri dish filled with prewarmed HEPES-buffered HBSS and fixed onto a microscope stage. Cells were localized on the coverslip grid using a 32x long-distance working lens or a 4Ox oil immersion objective.

Drug treatment

For long term incubation, 0.1 to 3OnM Taxol (Sigma), 0.1 to 1 μM cytochalasin D (Sigma) or 3nM to 10μM Nocodazole (Sigma) were added to culture medium after 1 day in culture, and cells were further incubated at 36.5°C. Drugs were kept as stock solutions in DMSO at -20 ⁰ C (Taxol: 5mM; Cytochalasin D: 1OmM; Nocodazole: 6,67mM).

Analyzing short term microtubule depolymerization, the fate of individual neurons was followed. Cells localized on the grid of a Cellocate coverslip were photographed. The coverslip was then transferred into HEPES-buffered HBSS containing Nocodazole and incubated for 5 min at 36.5°C in the incubator. [As

Nocodazole is insoluble in aqueous solutions, special care was taken to ensure equal distribution of Nocodazole in HBSS.] After short term incubation, neurons were PHEM-fixed to eliminate depolymerized tubulin subunits and allow clear visualization of microtubules.

lmmunocytochemistry

To detect proteins by immunohistochemistry, cells were fixed in 4% para- formaldehyde for 15 min at 37°C (20 min for Tau-1 stainings), aldehyde groups were quenched in 50 mM ammonium chloride for 10 min, and the cells were then extracted with 0.1 % Triton X-100 for 3 min.

To visualize microtubules clearly, an alternative fixation method (PHEM-fixation) was used if required. To get rid of unpolymerized tubulin subunits, cells were simultaneously fixed and permeabilized for 15 min at 37°C in PHEM buffer (60 mM

PIPES and 25 mM HEPES) containing 3.7% paraformaldehyde, 0.25%

glutaraldehydθ, 5 mM EGTA, 1mM MgCI, 3.7% sucrose, and 0.1% Triton X-100 (adapted from Smith, 1994) and quenched as above.

The neurons were then blocked at room temperature for 1 hr in a solution containing 2% fetal bovine serum (Gibco, Grand Island, NY, USA), 2% bovine serum albumin (BSA, Sigma), and 0.2% fish gelatine (Sigma) dissolved in phosphate-buffered saline. The cells were then incubated with primary antibodies diluted in 10% blocking solution. Primary antibodies used were: Tau-1 (Chemicon, Temecuia/CA, USA, 1 :5.000), anti-MAP2 (Chemicon, 1 :6.000), anti-αTubulin (clone B-5-1-2; Sigma, Mϋnchen, Germany; 1 :20.000), anti-acetylated Tubulin (clone 6-11B-1; Sigma, 1 :50.000), or anti-tyrosinated Tubulin YL1/2 (Abeam, Cambridge, UK, 1 :40.000).

For visualization of F ^' Actin, Rhodamine-coupled Phalloidin (stored as a methanol stock solution at -20 ⁰ C) was used (Molecular Probes, Leiden, Netherlands; 4 U/ml). As secondary antibodies, Alexa Fluor 488, 555 or 568conjugated goat anti-mouse, anti-rabbit or anti-rat IgG antibodies (Molecular probes, 1:500) were used.

Image Analysis and Quantitation

Length and intensity measurements were done using Scion Image Beta 4.0.2 for MS Windows (based on NIH-image). For length measurements, cVisTec Professional 1.0 (cVisTec, Munich, Germany) was additionally used. To determine the ratio of acetylated to tyrosinated α-Tubulin, the fluorescence signals of different channels of pictures taken with 63x magnification were compared. In the medial part of each process, the average fluorescence intensity of both acetylated and tyrosinated α-Tubulin was measured in a square of 5x5 pixel and the ratio of the values was determined. For minor neurites of the same cell, the average of the ratios was calculated in polarized neurons and used for comparison to the ratio of the corresponding axon. For unpolarized neurons, the highest ratio was compared to the average of the ratios of the remaining neurites. To measure microtubule retraction after Nocodazole treatment, fixed and stained neurons were relocated on the Cellocate coverslips. Pictures were taken of both the actin cytoskeleton (visualized by staining with Rhodamine-coupled Phalloidin)

and the microtubules (stained with an antibody recognizing α-Tubulin). The actin cytoskeleton outlining the neuron was used as a reference point to measure retraction of microtubules from the distal part of the processes towards the cell body. Pictures were processed using Adobe Photoshop 7.0, Deneba Canvas 8.0, and Scion Image Beta 4.0.2.

Mice

We used GFP-M and YFP-H transgenic mice (8-12 weeks, 15-30 grams) expressing GFP/YFP under the control of the neuron specific Thy-1 promoter. All animal experiments were performed in accordance with the animal handling laws of the government (Regierung von Oberbayern, No: 209.1/211-2531-115/02).

Dorsal Column Lesions and Postoperative Care The mice were anesthetized with a mixture of midazolam (Dormicum, 2 mg/kg), medetomidine (Domitor, 0.15 mg/kg), and fentanyl (0.05 mg/kg) injected intraperitoneal^. The lamina at the level of T8/9 was removed by laminectomy to expose the dorsal columns at the thoracic level. Fine irridectomy scissors (FST) was used to transect the dorsal columns bilaterally. The lamina was closed and the skin stapled. Two to three hours after surgery animals were woken up by subcutaneous injection of a mixture of flumazenil (Antisedan, 0.2 mg/kg), atipamezole (Anexate, 0.75 mg/kg), and naloxone (Narcanti, 0.12 mg/kg). The animals were kept on a heating pad for the following 24 hours. Post surgical animal care was done as follows: the bladders were expressed everyday twice; antibiotic (10μl of 7.5% Borgal solution [Hoechst Russel Vet]) was given subcutaneously everyday once and buprenorphine (0.15mg/kg) once every 12 hours after surgery in total 3 times.

Sciatic Nerve Lesions The mice were anesthetized as described. The sciatic nerve was exposed at around 2.5 cm from the DRG cell bodies by a small incision and crushed with

forceps for ten seconds. The incision was sutured and the skin stapled. The anesthesia was reversed and the animals were transferred to a heating pad for the following 24 hours.

Sacrifice and Sectioning

Animals were sacrificed and perfused at the defined times. In brief, animals were anesthetized with 5% chloral hydrate solution (prepared in saline) and perfused intracardially with 0.1 M phosphate buffer solution (PBS) for 5 minutes at a speed of 3ml/min and followed immediately by 4% paraformaldehyde (PFA) in PBS for 20 minutes for immunostainings, 45 minutes for morphology analysis. The spinal cord or sciatic nerve lesion sites were carefully dissected and post fixed in 4% PFA (30 minutes for immunostainings at room temperature, overnight at 4 ⁰ C for morphology analysis). The tissues were transferred to a 15% sucrose solution for 4 hours at room temperature and subsequently to 30% sucrose solution for overnight incubation. The tissues were frozen in optimum cutting temperature (OCT) and sectioned longitudinally by a cryostat (Leica CM 3050) at 10 μm. For size quantifications, we either made thick cryostat sections (40 μm) or whole mount spinal cord tissue for confocal observation.

lmmunohistochemistry

Tissue sections were first washed with PBS twice for 10 minutes, then incubated with blocking solution containing 10% goat serum and 0.3-0.5% Triton X-100 in PBS for 1 hour at room temperature, washed with PBS, incubated with primary antibody (in blocking solution with 5% goat serum) overnight at 4 ⁰ C. The primary antibodies used were anti-Glu-tubulin (rabbit) 1:500 dilution (Chemicon) and anti- golgin-160 (rabbit) 1:200 (a gift from Dr. Francis Barr). The sections were washed with PBS and incubated with an anti-rabbit secondary antibody Alexa 568 (Molecular Probes). The sections were mounted with water based mounting medium (Polysciences Inc, Warrington, PA).

Confocal Imaging

We acquired confocal images with a Leica SP2 confocal microscope system in sequential scanning mode.

Electron Microscopy

Animals were perfused as described above by using Lewis Shute fixative instead of 4% PFA. The tissue was dissected without drying and post fixed in Lewis Shute fixative and then in Osmium tetroxide. Afterwards, the tissue was dehydrated and embedded in araldite, cut by an ultramicrotome (LKB) at 50 nm and post stained with leadcitrate and uranylacetate by an ultrastainer (LKB). EM images of the sections were acquired by using a Zeiss EM 10 electron microscope.

Nocodazole Treatment

Sciatic nerve lesion was performed as described. After 24 hours animals were re- anesthetized and 10 μl of 330 μM of nocodazole (Sigma-Aldrich, diluted in 5% DMSO in PBS) or 5% DMSO alone was applied onto lesion site with a pipette. The injury site was closed and animals were perfused 24 hours after nocodazole or DMSO application for staining and imaging.

In vivo Imaging

We used an Olympus SZX-12 fluorescent stereomicroscope equipped with 1x Plan Apochromat objective and 1.6x Plan Fluorite objective (Olympus). The ColorView Il camera integrated to the microscope was used to capture images through Analysis FIVE software (Soft Imaging System). Animals were anesthetized as described. After removing the laminae at T11/12 level, a small unilateral lesion was applied with the vannas spring scissors (FST) by transecting only the superficial axons of the dorsal columns. We imaged the injury site before and after lesion by acquiring serial images. The animals were kept on a heating pad during the imaging session.

Taxol Treatment

A small unilateral spinal cord lesion at T12 level was performed as described. 10 μl

of 1 μM or 5 μM taxol (Sigma-AIdrich, diluted in Saline) or saline as control was applied onto the lesion site each hour starting immediately after or 24 hours after lesioning. The lesion sites were visualized by in vivo imaging as described. The animals' anesthesia was boosted after 3 hours. At the end of the observation period animals were perfused and prepared for the confocal imaging as described.

Size, Distance and Sprouting Quantifications

The size of retraction bulbs and growth cones was quantified as follows: the maximum diameter of the retraction bulb and growth cone at the tip of the axon was measured and divided by the axon diameter of the same end structure where the axon looked normal.

The distance of taxol and saline treated axons to the lesion site was quantified as follows: The lesion site was outlined from the in vivo binocular images. We then measured the distance of the axonal tips to the proximal edge of the lesion. The length of the sprouts quantified on confocal images. The length of all sprouts including sprouts emerging from sprouts measured and summed up for final value. As number of sprouts, only primary sprouts emerging from directly axonal shaft were quantified.

Image Processing and Data Evaluation

Angle deviations, numbers of vesicle and mitochondria, size of end structures and distance to the lesion site were quantified with Analysis FIVE software. The images were assembled with Photoshop (Adobe) and Canvas (ACD Systems). The cartoons in Figures were drawn with Illustrator (Adobe). The statistical analysis was performed with Excel (Microsoft) and statistical significance (p < 0.05) was calculated using two-tailed, unpaired t-test.

Example 2: Different distribution of acetylated and tyrosinated microtubules in hippocampal neurons Microtubules, along with the actin cytoskeleton and intermediate filaments one of the major components of the cellular cytoskeleton, are formed by polymerization of

heterodimers of α- and β-TubuIin. Numerous studies have revealed interactions between these different components of cytoskeletal architecture (for review see 28- 31). While intermediate filaments are predominantly involved in the formation of stable structures, the actin and microtubule cytoskeletons have additional roles in highly dynamic processes including cell migration, nuclear movement, cell division and cell polarity (for review see 31, 32). With the importance of the actin cytoskeleton in neuronal polarization becoming more and more evident (for review see 33), we hypothesized a role for microtubules in this complex process as well. As a first means to study a putative role of microtubules in initial neuronal polarization, we examined the distribution of different posttranslational modifications of α-Tubulin in axons and minor neurites, two types of neuronal processes in early developmental stages.

Newly synthesized α-Tubulin carries a C-terminal tyrosine residue, however, when integrated into microtubules removal of this residue from α-Tubulin occurs (34, 35). Tyrosinated α-Tubulin found in microtubules is therefore usually associated with recent assembly and serves as a marker for dynamic microtubules (36). In contrast, stable microtubular structures carry a different posttranslational modification. α-Tubulin present in such microtubules tends to be acetylated. Acetylation hence serves as a marker for stable microtubules (37). To assess the distribution of these different posttranslational modifications in hippocampal neurons, we fixed stage 3 neurons [in 4% PFA/Sucrose] and stained for acetylated and tyrosinated α-Tubulin. Further analysis was performed using fluorescence microscopy and CCD imaging. By means of a double band pass filter we observed an enrichment of acetylated α-Tubulin in axonal microtubules in comparison to microtubules in minor neurites in 83.5% (±1.0%; n=709) of stage 3 neurons (2 days in vitro DIV) (Figure 1A-D). In contrast, tyrosinated α-Tubulin was predominant in growth cones of all processes, reflecting their dynamic state required for steering (38) and extension. For a more quantitative analysis, we compared the ratio of the fluorescence intensities of acetylated and tyrosinated α-Tubulin in axons and minor neurites. In the axonal shaft, the ratio was increased 3.24-fold (±0.8; n=106) compared to

minor neurites (Figure 1H).

To examine whether such differences already occur in unpolarized neurons, we fixed hippocampal neurons after 1 DIV and performed a similar assessment. In 54.2% (±7.8%; n=107) of stage 2 neurons, the ratio of acetylated to tyrosinated α-Tubulin of the minor neurite with the highest ratio was significantly different from the remaining minor neurites (p-value<0.01), on average the ratio was increased 1.87-fold (±0.59) in the minor neurite with the highest ratio (p-valύe<0.001) (Figure 11).

Thus the axon of stage 3 neurons and one minor neurite of approximately 50% of stage 2 neurons show the typical markers of lower microtubule turnover.

Example 3: Stability difference of microtubules in axons and minor neurites

Our next step was to address whether the different distribution of posttranslational modifications reflects an actual stability difference of microtubules in axons and minor neurites.

To test the resistance of microtubules to depolymerization, we applied the drug Nocodazole to stage 3 neurons, followed by fixation [in 3.7% PFA/Sucrose, 0.25% Glutaraldehyde, 1x PHEM-buffer and 0.1 % Triton X-100] after a brief incubation (Figure 2 A). Nocodazole is known to disrupt microtubules by binding to β-Tubulin and preventing formation of one of the two interchain disulfide linkages, thus inhibiting microtubule dynamics. After fixation, neurons were doublestained for F-Actin and α-Tubulin or acetylated and tyrosinated α-Tubulin. Fluorescence microscopy and CCD imaging was used to quantify retraction of microtubules from the distal end of processes or distribution of posttranslational modifications of α-Tubulin after partial depolymerization of the microtubule cytoskeleton.

In control cells (Figure 2 B, C) treated with DMSO microtubules reached close to the distal end of processes (Figure 2 C). However, in cells briefly treated with Nocodazole (Figure 2 D, E), we observed a retraction of microtubules towards the cell body (Figure 2 E) which was significantly higher in minor neurites compared to axons (p-value O.001) in 60% of the cells (59.3±6.9% and 60%±5.1%, n=59 and 60, for 3.3 and 5μM Nocodazole, respectively) (Figure 2M). Thus, axonal

microtubules obviously show a higher resistance to depolymerization than microtubules in minor neurites.

To scrutinize whether such a microtubule stabilization possibly precedes axon formation, we fixed hippocampal neurons after 1 DIV and performed a similar analysis. In control cells (Figure 2 G, H) treated with DMSO, microtubules reached close to the distal end of all minor neurites (Figure 2 H). In contrast, in neurons briefly treated with 3.3μM Nocodazole, the difference between microtubule retraction in the minor neurite with the lowest retraction and the average retraction of the remaining minor neurites was highly significant (4.50±1.27μm and 13.71±2.84μm, respectively; n=34; p<0.001) (Figure 2 N). Obviously the microtubules of one minor neurite show increased stability in otherwise morphologically unpolarized neurons.

In line with the distribution of stability markers in untreated stage 3 neurons (Figure 1D) these results point to an increased stability of axonal microtubules and a possible role for microtubule stability in initial neuronal polarization.

Example 4: Microtubule destabilization selectively impairs outgrowth of minor neurites So far we had shown that axonal microtubules have a higher ability to resist depolymerization under harsh microtubule-destabilizing conditions. If there was a putative stability difference in axons and minor neurites, we hypothesized that particular growth conditions should allow formation of axons but not minor neurites. To assess the effect of modest microtubule destabilization on process formation, we treated hippocampal neurons after 1 DIV with low concentrations of Nocodazole. After 2 further DIV under such growth conditions cells were fixed [with 4% PFA/Sucrose] and stained for the axonal marker Tau-1. Using fluorescence microscopy, we evaluated effects on the formation of axons and minor neurites. In control cells, the number of processes almost doubled from day 1 to day 3 in vitro (2.22±0.24, 3.96+0.11 , and 3.99±0.14 processes per cell after 1 DIV untreated, 3 DIV untreated or 3 DIV DMSO-treated; n=820, 807, and 760 respectively) (Figure 3F), reflecting the formation of minor neurites and an axon

(Figure 3B and C). In contrast, in cells grown in the presence of low concentration of the microtubule destabilizing drug Nocodazole the number of formed processes decreased in a concentration-dependent manner (Figure 3F). Cells were still able to form an axon, however, the number of minor neurites was reduced (3.58±0.08, 2.64±0.08, and 2.20±0.18; n=800, 831 and 784 for 15, 45 and 75nM Nocodazole, respectively) (Figure 3D and E). This reduction in the number of processes was not due to a general inhibition of neurite outgrowth by Nocodazole. Total neurite length increased 4-fold from day 1 to day 3 in vitro under control conditions (62.7μm to 238.91μm, n=129 and 177, respectively). When treated with 45 or 75nM Nocodazole, total neurite length still increased more than 3- or 2-fold, respectively (197.31μm and 134.89μm; n=191 and 228 for 45 or 75nM Nocodazole, respectively) (Figure 3G). The clear increase of total neurite length under all conditions shows that neurons were still in a growth competent state, even when subject to slight destabilization of microtubules. Thus, microtubule destabilization selectively impairs formation of minor neurites in early neuronal development in vitro.

Example 5: Taxol-induced microtubule stabilization triggers formation of multiple axons

So far, our results pointed to an increased stability of microtubules during development and maintenance of the axon in early developmental stages. Therefore we next wanted to asses whether microtubule stabilization itself is sufficient to induce axon formation. To test the influence of microtubule stabilization on axon formation we treated hippocampal neurons after 1 DIV with low concentrations of the microtubule- stabilizing drug Taxol (Figure 4A and H). Taxol binds to the N-terminal region of β-Tubulin and promotes the formation of highly stable microtubules (39, 40). Taxol (also known as Paclitaxel) is successfully used as an anti-cancer drug so far as it arrests dividing cells in the G2-M-phase of cell division. Neurons grown in the presence of low concentrations of Taxol were fixed [with 4% PFA/sucrose] after 3

to 7 DIV and stained for the axonal marker Tau-1 or the dendritic marker MAP2. Under control conditions, neurons had formed one axon and several minor neurites (Figure 4D and E). However, Taxol-treated neurons formed several elongated processes which were positive for the axonal marker Tau-1 (Figure 4B and C) and showed an increased ratio of acetylated to tyrosinated α-Tubulin like axons (Figure 5G and H, compare Figure 1 D). The number of processes which exceeded 60μm had increased more than 2-fold by Taxol treatment (DMSO 1.53±0.05, 3nM Taxol 2.93±0.46, 1OnM Taxol 3.58±0.54) (Figure 4F). Similarly, the number, of cells with more than one Tau-1 -positive process went up about 4-fold in a concentration- dependent manner in Taxol-treated neurons (from 20.19±3.19% in DMSO-treated neurons to 80.81 ±1.71 % neurons treated with 1OnM Taxol) (Figure 4G). In addition to this, Taxol-induced processes had a weak MAP2-signal resembling characteristics of axons in control neurons (Figure 4 I/J and KJL).

Example 6: Taxol induces the formation of axon-Iike processes with increased microtubule stability

To test whether the low Taxol concentrations used were actually able to stabilize microtubules, we treated hippocampal neurons (1 DIV) with 1OnM Taxol and performed the microtubule stability assay as described before (see 2.) after 24h incubation (Figure 5A). After brief incubation with 5μM Nocodazole, axonal microtubules had retracted on average 8.89μm (±3.23μm; n=60) which was significantly different from the retraction of minor neurites (12.47±2.22μm; p-value<0.001). In contrast, in Taxol-induced axon-Iike processes little microtubule retraction occurred (Figure 5B to E) (7.48±1.76μm; n=67), which did not differ significantly from axons (p-value=0.11) (Figure 5F). Thus, stability of microtubules present in Taxol-induced processes was increased in comparison to microtubules of minor neurites and resembled the stability of axonal microtubules.

As Taxol-induced processes show typical axonal characteristics -increased length and microtubule stability, Tau-1 -positive, MAP2-negative- we concluded that Taxol triggers the formation of multiple axons in hippocampal neurons.

Example 7: Role of microtubule stabilization in mediating axonal growth in an inhibitory environment

The in vitro setup consists on primary culture of cerebellar granule neurons (CGN) obtained from the granular layer of the cerebellum of P7-day-old rats. In culture, these cells exhibit, over a period of 5 stages, a specific pattern of polarity development. They display a first unipolar stage with just one extended axon followed by a bipolar morphology. Then one of the emerged neurites extends two distal ramifications leading to the "T-shaped" axon morphology observed in situ. Cells finally become multipolar with the development of short and thick dendrites around the cell body ²⁰ .

This example refers to the unipolar stage and addresses the effect of microtubule stabilization on the axonal growth capacity on permissive and non-permissive substrates. CNS-myelin contains inhibitory factors which contribute to the lack of regeneration ²² . This inhibitory environment is reproduced by plating the cultured cells on CNS-myelin according to the protocol routinely used in laboratory ¹⁶ . On the basis of morphological and molecular criteria it is determined whether the growth processes of cerebellar granule neurons (CGN) is influenced by microtubule stabilization. To this end, P7 rats CGN are dissected, dissociated and plated on myelin in the presence or absence of the microtubule stabilizing drugs, including taxol. The cells are exposed to taxol (concentration range 0.1 pM - 100μM) for various time periods ⁷ . Concomitantly, a set of cells plated and treated with C3- exoenzyme serves as growth enhancing reference ²¹ . The C3-exoenzyme is commercially available. After 36 hours of culture, the cells are fixed and stained with a neuron-specific beta tubulin antibody. Pictures are made from the neurons and axonal length is measured using the Scion Image software (NIH, Bethesda, USA).

Example 8: Effects of microtubulule stabilization with taxol on anatomical and functional outcomes

This example describes the assessment of whether a microtubule stablizing compound such as taxol promotes axonal growth in vivo and improves the clinical outcome of spinal cord injury. The injury is induced by a dorsal hemi-section of the spinal cord at thoracic level (T8) ²³ . This bilateral hemi-section interrupts the multiple motor control projections and so leads to a diminution of the locomotor performances. Of particular interest is the corticospinal tract (CST) and the raphaespinal tract. The interruption of these tracts depresses the locomotor function ¹² (and Saruhashi et al., 1996; Antri et al., 2002; Kim et al., 2004 and Lee et al., 2004).

First, the lesion site is characterized in the used spinal cord injury paradigm. The consequences of the hemi-section on the spinal tracts and on the locomotor's functions are also evaluated. Then it is examined whether taxol induces change in the lesion site, axonal outgrowth and eventually improves the functional recovery.

Taxol or its vehicle is continuously and locally delivered at the site of injury via a catheter connected to an osmotic minipump (2004, Alzet) and inserted into the intrathecal space of the spinal cord. To determine the optimal dose for the administration of treatment, first taxol is perfused at concentrations ranging from 0.01 pM to 100 μM/hour. The treatment is started one week following injury. In case the treatment interferes with scarring and causes additional damage at the lesion, then it is delayed until two weeks following injury. Delayed post-injury deliveries were chosen because it is the more significant clinical paradigm: the glial scar is already installed at these times ⁴ and therefore it allows to focus on the regenerative process.

- Lesion site. First, a qualitative characterization of the different lesion paradigms is

done. Sham-operated (laminectomy only) and spinal-injured are fixed at 2- 4 and 6 weeks post-lesion. Coronal and horizontal serial sections of the cords are carried out where histological staining is performed and the following immunohistochemical markers are analyzed. First sections are stained with Hematoxilin Eosin to assess the extent of the necrotic cavities and of the scar tissue around the lesion site induced by the injury. The horizontal sections are stained with Luxol Fast Blue to identify the myelinated white matter and the amount of the spared fibers at the lesion site. Then address the cellular response to injury is addressed by immuno-labelling the coronal sections on the adjacent slides series. Based on the change of the morphology of the cells, the astroglial and microglial reactivity are examined which will reflect the occurring inflammatory process using glial fibrillary acidic protein (GFAP) and 0X42 antibody respectively ⁴ . Additionally, it is evaluated if the model leads to the proliferation of oligodendrocyte progenitors by testing O4 marker immunoreactivity (Ishii et al., 2001).

To test whether taxol causes changes at the lesion site, the effect of taxol on the lesion paradigms defined above is studied.

- Axonal growth. The effect of taxol on axonal growth is evaluated by labelling the CST and the Raphae spinal tract. This technique is first applied in spinal cord- injured and non-injured rats to control the efficiency of the transection induced by the hemi-section. Then the effect on taxol on the transected neurons is evaluated. To study the growth velocity of axonal growth in detail, the animals are analysed after different time points ranging from 3 days to 6 weeks post-treatment. Tthe transected neurons of the CST are labelled with the anterograde tracer biotinylated dextran amine (BDA). This anterograde tracer permits to completely reconstruct the axonal arbors of the CST (Bareyre et al., 2004). The CST is running from the sensorimotor areas of the cortex through the brainstem to the spinal cord and is involved in the voluntary motor functions (Smith and Bennett, 1987; Tracey (1995)). It can be anterogradely labelled and in murine animals, the majority of its fibers are localized in the ventral part the dorsal column, dorsal to the central canal.

Then this tract can be a useful tool for regeneration examination. BDA is applied bilaterally by stereotaxic injections at different sites of the sensorimotor cortex (Paxinos and Watson, 1982). After 2 weeks, the time required for the tracer to be transported along the CST pathway ¹³ (and Lee et al., 2004; Reiner et al., 2000), the rats are perfused with fixative. The spinal cords are dissected and cut into 2 cm lengths. BDA stain is then visualized using the avidin-biotinylated HRP procedure on serial horizontal sections (Reiner et al., 2000).

The anatomic part of the study is completed by examining the effect of taxol on the Raphe nuclei projections into the spinal cord with serotonin immuno-labelling. The raphaespinal tract as well as the CST strongly participated to the locomotor functions (Schmidt and Jordan, 2000). Its fibers can be also easily visualized through the spinal cord by the serotonin immuno-labelling. At the thoracic level a dense network of serotonin immuno-reactivity is present in the spinal cord dorsolateral funiculus which is partially affected by the hemi-section (Paxinos and Watson, 1982; Schmidt and Jordan, 2000).

- Functional recovery. Effect of taxol on functional recovery are examined by measuring the hindlimb performances weekly for the length of the study using the Basso - Beattie - Bresnahan (BBB) locomotor rating scale. The locomotor deficit is a first estimate on control injured compared to sham animals. To test whether taxol treatment contributes to a functional improvement, compare the locomotor behaviour of the vehicle- and taxol-treated animals is compared. The spinal hemi-section performed at the thoracic level dramatically impaired the hindlimb functions leading to a complete hindlimb paralysis during the first days post-injury. Afterwards, the hindlimb performances gradually improve. Therefore, locomotion constitutes a clinical relevant behaviour to predict neurological recovery. The BBB scale which covers a wide range of motor deficits is accurately designed to assess qualitative hindlimbs recovery after spinal cord injury. The animals of each group are carefully handled and placed on the open-field and observed during 5 minutes. From the observation of the articulation movement of

the 3 joints, (hip, knee and ankle) an individual score for each leg is attributed from 0 (paralysis) to 21 (normal walking). The components of the hindlimb movement include weight support, plantar and dorsal stepping, forelimb-hindlimb coordination, paw rotation, toe clearance, trunk stability and tail placement. The BBB scale defines 3 level of recovery. The first level (between 0 and 8) describes the early phase of recovery by scoring the small or large movements of the 3 joints of the hindlimb. Scores from 9 to 13 defines the intermediate phase of recovery focused on weight support capacity. The last level (from 14 to 21) indicates the progressive improvements in the coordinated walking ability (Basso et al., 1995).

Example 9: Manipulation of microtubules using Taxol increases the regeneration capacity of the CNS axons after a lesion in vivo

The peripheral axonal branches of the dorsal root ganglion (DRG) neurons run in the peripheral nervous system (PNS) where they generate growth cones and regrow after nerve injury. In contrast, the central axonal branches coursing in the dorsal columns of spinal cord do not regrow upon lesioning. The lesiόned proximal axonal terminals form retraction bulbs. To better understand the underlying differences between retraction bulbs and growth cones in vivo, we induced growth cone formation by lesioning the sciatic nerve which contains the peripheral axonal branches from the lumbar 4 and 5 DRG neurons. Conversely, retraction bulb formation was induced by lesioning the dorsal columns at the T8/T9 level. These experiments were performed with transgenic mice expressing GFP in a subset of neurons and are, therefore, a good system to study individual axons' responses upon injury ¹¹ ' ¹² (described in Fig. 11).

Peripherally axotomized DRG neurons in vivo generated a slim growth cone that morphologically remained constant over time (Fig. 6a-d). The maximal width of the growth cone was comparable to the diameter of the axonal shaft: The growth cone (tip) / shaft ratio was 1.31 ± 0.14 at one day (n=113 growth cones), 1.51 ± 0.15 four days (n=48 growth cones) and 1.20 ± 0.20 one week after sciatic lesion (n=21

growth cones), {mean ± s.d.; n > 7 mice for each time point [Fig. 6k]). Later time points were difficult to visualize because of the high velocity of axonal regeneration in the PNS. While growth cones of DRG neurons in cell culture have filopodia (Fig. 12), their growth cones rarely show such structures in vivo.

After axotomizing the central axonal branches, retraction bulbs formed at the proximal tips. These bulbs were typically round or oval with a much larger diameter than their axons and lacking any kind of extensions (Fig. 6e-j). Retraction bulbs increased about three times in size from 1 day to 5 weeks post injury (Fig. 6e-j). The retraction bulb (tip) / axon shaft ratio was 4.09 ± 0.73 at one day (Fig. 6e, h; n=109 retraction bulbs), 8.49 ± 0.51 one week (Fig. 6f, i; n=111 retraction bulbs) and 11.79 ± 0.49 five weeks post injury (Fig. 6g, j; n=92 retraction bulbs), (mean ± s.d.; n > 8 mice for each time point [Fig. 6k]). In contrast to the slim growth cones that were continuous with the peripheral axonal shaft, the bulbous structures allowed to quantify the surface of the retraction bulbs: also the absolute size of retraction bulbs increased over time (Fig. 6I). Some of the axons formed smaller retraction bulbs on their shafts in addition to the bigger terminal bulbs (arrow in Fig. 11h, Fig. 11i, j).

What may cause the generation of retraction bulb morphology and stalling of axons? Various activities including synthesis of cytoskeleton monomers and polymerization of monomers into filaments at the growth cone require a high energy supply from mitochondria to sustain axonal growth ² ' ¹³ ' ¹⁴ . Using electron microscopy we analyzed the mitochondria morphology and density in growth cones and retraction bulbs at various time points. The mitochondria of the retraction bulbs (Fig. 7c, d) were indistinguishable from the mitochondria of growth cones (Fig. 7a, b). They also did not show characteristics of degenerating mitochondria that typically contain vacular swollen membranes and loss of cristae ¹⁵ ' ¹⁶ . Mitochondria density in retraction bulbs at 4 days, 2 weeks and 5 weeks post injury (3.63 ± 0.61 , 3.9 ± 0.62, 4.75 ± 0.66 [number / μm ² ] respectively, mean ± s.e.m.; n > 7 EM retraction bulbs per time point) were significantly ( p < 0.05, Fig. 7k) higher than in

growth cones (1.76 ± 0.47, mean ± s.e.m.; n=8 EM growth cones). Thus, as far as it is deducible from the morphological analysis, sufficient energy appears to be provided in the retraction bulbs to potentially support axonal growth.

Vesicular trafficking from the cell body to the growth cone is necessary to integrate additional membrane at the distal axonal tip into the plasma membrane to permit axon elongation ³ ' ⁴ ' ¹⁷ ' ¹⁸ . We therefore investigated whether a lack of sufficient membrane traffic underlies both retraction bulb formation and axonal halt after CNS injury. We assessed post-Golgi-vesicular trafficking by co-immunostaining GFP labeled growth cones and retraction bulbs using the trans-Go\g\ derived vesicle marker golgin-160 ¹⁹ . Post-Golgi vesicles accumulated both in the growth cones (Fig. 7e-g) and retraction bulbs (Fig. 7h-j). Similar results were obtained by electron microscopy (compare Fig. 7a and Fig. 7c; arrows in higher magnifications: Fig. 7b and Fig. 7d, respectively): the density of vesicles was significantly (p < 0.05, Fig. 7I) higher in the retraction bulbs at 4 days, 2 weeks and 5 weeks post injury (Fig. 7I; 24.6 ± 3.86, 35.7 ± 6.54, 51.17 ± 6.52 [number / μm ² ], respectively; mean ± s.e.m. n > 7 EM retraction bulbs per time point) compared to growth cones (12.15 ± 2.5, mean ± s.e.m, n=8 EM growth cones). Additionally, vesicle density in the retraction bulbs significantly increased over time (from 1 day to 5 weeks post injury, Fig. 7I, p < 0.01). Thus, it appears that vesicles are transported from cell body through the injured central axon to the retraction bulb.

Microtubules are bundled in the core domain of growth cones ⁷ ' ²⁰ from which they proceed into the outward bound parts of the growth cone to initiate steering events and axon elongation ⁷ ' ²¹ ' ²² . To assess the distribution of microtubules, we co- immunostained GFP-labeled axonal shafts, growth cones and retraction bulbs with an antibody recognizing tubulin. Microtubules within the growth cones and their axons were tightly bundled and parallel aligned (Fig. 8a-c). In contrast, in retraction bulbs the microtubules were highly disorganized (Fig. 8d-f).

We further quantified the deviation of microtubules from the axonal axis by analyzing EM images. In unlesioned controls the microtubule filaments were aligned parallel to the axonal axis: 99.1 % of the microtubules deviated less than 30° from the axonal axis (a representative image is shown in Fig. 8g, the microtubules of the same image were pseudo-colored in black in Fig. 8h, higher magnification of the marked area shown with quantification of angle deviations in Fig. 8i, [n=7 EM axonal shafts]). In growth cones, 97.0% of the microtubules deviated less than 30° showing that they were mostly parallel to the axonal axis as in the unlesioned controls (Fig. 8j-l, [n=8 EM growth cones]). In retraction bulbs, only 51.3% of the microtubules deviated less than 30°, 35.3% between 30° and 60° and 13.3% more than 60° from the axonal axis (Fig. 8m-o [n=9 EM retraction bulbs]). These results showed that microtubules in retraction bulbs were highly disorganized and dispersed compared to unlesioned axons and growth cones (p < 0.0001 , quantifications of the data shown in Fig. 8p). This observation led us to the question whether microtubule destabilization of a growth cone would be sufficient to transform its morphology to a retraction bulb.

We crushed the sciatic nerve of GFP-M mice to generate growth cones and applied 10μl of the microtubule disrupting drug nocodazole (330 μM) to the site of the Iesioned sciatic nerve 24 hours later. Upon evaluation of the sciatic nerve another 24 hours after nocodazole treatment, we found that 42.3% ± 13.2% (mean ± s.d.; n=165 axons from 11 mice) of the axons transformed their growth cones into a bulb (Fig. 9a-c). Application of DMSO which served as a negative control did not cause bulb formation (Fig. 9d-f). The tip / axon shaft ratio of nocodazole induced retraction bulbs (4.25 ± 0.56, mean ± s.d.; n=70 from 12 mice) was significantly higher than both Iesioned, untreated (1.45 ± 0.13, mean ± s.d.; n=53 from 8 mice) and Iesioned, DMSO treated (1.64 ± 0.46, mean ± s.d.; n=62 from 12 mice) controls (Fig. 9g, p < 0.0001 for both). Those peripheral bulbs showed a similar size increase as observed 1 day after spinal cord injury (4.09 ± 0.73; Fig. 6e, h, k). When we analyzed the nocodazole induced peripheral bulbs, we found a dispersed microtubule organization similar to that in retraction bulbs of the CNS (Fig. 9 h-m;

compare to Fig. 8e, f). Thus, destabilization of microtubules is sufficient to generate retraction bulb like structures from a growth cone characterized by morphological and cytoskeletal criteria.

If dispersing microtubules was responsible for the retraction bulb formation, microtubule stabilization should prevent axons from forming retraction bulbs. A small lesion in the dorsal column at T12 level of GFP-M mice was made by cutting the superficially lying central axons from the DRG neurons. The injury site was then bathed in either the microtubule stabilizing drug taxol or saline (control). We then continuously observed the axons' behaviour during the subsequent 6 hours by using a binocular equipped with fluorescent light and a digital camera ¹² . In control conditions, retraction bulbs started to form 1-2 hours after the lesion (Fig. 10b, k). At 6 hours post injury already 71.3% ± 9.1 % (mean ± s.d.; n=18 mice) of the axons formed retraction bulbs at their tips (Fig. 13b, d, k). However, when 10 μl of 1 μM taxol, was repetitively applied (once per hour) starting immediately after the injury, only 22.8% ± 13.1 % (mean ± s.d.; n=11 mice) of the axons developed retraction bulbs (defined by a tip / axon ratio > 2) in the first 6 hours (Fig. 13a, c, k). In addition, the retraction bulbs formed during taxol treatment were smaller (Fig. 13e- g; tip / axon ratio: 2.53 ± 0.54 mean ± s.d.; for the 5 biggest retraction bulbs per animal) than the control ones (Fig. 13h-j; 4.14 ± 0.44 mean ± s.d.; n=11 mice for each condition; p < 0.001). These results suggest that stabilization of microtubules prevents formation of retraction bulbs. Interestingly, the axons of taxol treated animals also showed little retraction from the lesion site. In 6 out of 11 taxol treated animals the proximal axonal stumps were as close as 25 μm to the injury site 6 hours after injury whereas only 1 out of 8 controls showed such little retraction (compare Fig. 13c and d).

We then wanted to examine the effect of microtubule stabilization on the lesioned

CNS axons which already formed retraction bulbs. If the microtubule stabilization would transform the non-regenerating retraction bulbs into growth cones, this should increase the limited regeneration of capacity the CNS axons. To test this,

we performed a laminectomy at T12 (Fig. 10a, e), applied a small lesion (Fig. 10b, f) as described above, and waited 24 hours to let the mice to form the retraction bulbs (Fig. 10c, g). At 24 hours of post-injury, we applied either 10 μl of 5 μM taxol or saline (control) once each hour in total 4-5 times. During the experiments, we imaged the living animals: before lesion to be sure that there is not any nonspecific injury; after lesion to confirm that the target axons are cut; before treatments to confirm that target axons already have not formed long sprouts; finally 24 after treatment to identify the sprouts forming after treatments. 24 hours after treatments (48 hours post injury), the animals wee perfused and confocal images captured to quantify the regenerating sprouts. We analyzed the sprouts emerging from one axon (the one having the most sprouting) in both conditions (Fig. 10d, h). In control conditions, the total length of the sprouts from one axon was 292.17 μm ± 56.87 μm (mean ± s.e.m.; n=12 mice; Fig. 10i, k) and the total number of the sprouts was 3.50 ± 0.70 (mean ± s.e.m.; n=12 mice; Fig. 10i, I). However, in taxol applied animals, our preliminary analysis shows that the total length of sprouts was 693.03 μm ± 123.63 (mean ± s.e.m.; n=12 mice; Fig. 10j, k) and the total number of the sprouts was 7.83 ± 1.79 (mean ± s.e.m.; n=12 mice; Fig. 10j, I) which both were significantly higher than control conditions (p < 0.01 and p < 0.05 respectively). When we analyzed the terminals of the sprouts, 3 different types of morphology were noticeable after taxol treatment: 1- a slim growth cone similar to those formed at the periphery in vivo 57% ± 20% (mean ± s.d.; n=90 from 12 mice), (yellow arrow in Fig. 10j) 2- a small retraction bulb 41.8% ± 16.6% (mean ± s.d.; n=66 from 12 mice), (blue arrow in Fig. 10j) 3- an extended retraction bulb which possesses several filopodia like extensions which has been observed in 4 out of 12 taxol treated animals, 8.9% ± 5.0% (mean ± s.d.; n=5 from 4 mice), (red arrow in Fig. 10j). Conversely, in control conditions, the sprout terminals tended to form only 1 ^st and 2 ^nd type of morphologies: a slim growth cone in 50.1 % ± 15.6% of sprouts (mean ± s.d.; n=52 from 11 mice), (yellow arrow in Fig. 10i) or a retraction bulb in 49.9% ± 15.6% of sprouts (mean ± s.d.; n=56 from 11 mice), (blue arrow in Fig. 10i). None of the control treated axons showed the 3 ^rd type of morphology.

One of the key questions in the regeneration field is why axons of the PNS grow after injury, whereas axons of the CNS stall. We show that microtubules in the retraction bulbs are disorganized. Conversely, microtubule destabilization is sufficient to produce retraction bulb like structures derived from growth cones formed after peripheral injury.

These findings provide a simple explanation why axons containing retraction bulbs cannot regrow. It is a classical finding that destabilization of microtubules using microtubule depolymerizing drugs inhibits axon outgrowth ^23'26 . While the core domain of the growth cones and the axonal shaft contain bundled microtubules that give rigidity to the distal tip to support axonal elongation, the disorganized microtubules found in retraction bulbs apparently lack such characteristics Hence, stabilization of microtubules in lesioned CNS axons both hinders retraction bulb formation and appear to increase the regeneration capacity of the axons (summarized in Fig. 14).

Microtubules are active players in axon elongation and guidance regulated by external signals ⁷ ' ²² ' ²⁷ . The known inhibitory cues present in the CNS myelin and the glial scar including MAG, Nogo, OMgP and Versican converge downstream of their receptors onto the Rho signaling pathway ^28'31 which, beside of regulating the actin cytoskeleton, also affect microtubule stability and dynamics ³² . Interestingly, we recently showed that stabilization of microtubules causes non-growing minor neurites to change to growing axons using hippocampal neurons in cell culture as a model system (Witte and Bradke, submitted). We expect that, as this study suggests, stabilization of microtubules could be a potential therapeutic approach to enhance regeneration of injured CNS axons.

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Abbreviations:

DIV days in vitro

PFA Paraformaldehyde

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