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Published as doi: 10.1096/fj.05-5339fje.
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(The FASEB Journal. 2006;20:1939-1941.)
© 2006 FASEB

TACE-induced cleavage of NgR and p75NTR in dorsal root ganglion cultures disinhibits outgrowth and promotes branching of neurites in the presence of inhibitory CNS myelin

Zubair Ahmed, Gordon Mazibrada, Ruth J. Seabright, Russell G. Dent, Martin Berry and Ann Logan1

Molecular Neuroscience Group, Division of Medical Sciences, University of Birmingham, Birmingham, UK

1Correspondence: Molecular Neuroscience Group, Division of Medical Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK. E-mail: a.logan{at}bham.ac.uk

ABSTRACT

After binding, central nervous system (CNS) myelin-derived axon growth inhibitory ligands, the Nogo-66 receptor (NgR), complexes with LINGO-1 and either the low-affinity neurotrophin receptor (p75NTR) or TROY to initiate growth cone collapse via a Rho-A inhibitory signaling pathway and/or Ca2+-dependent activation of epidermal growth factor receptor (EGFR) through an unknown signaling pathway. We have shown that axon growth through CNS myelin is disinhibited after neurotrophic factor administration by 1) initiating intramembranous proteolysis (RIP) of p75NTR, leading to cleavage of the extracellular (p75ECD) and intracellular domains (p75ICD) by {alpha}- and {gamma}-secretase, respectively, thereby paralyzing inhibitory signaling; 2) shedding of soluble NgRECD, which acts as a competitive antagonist to NgR for binding of inhibitory ligands; and 3) antagonizing NgR/p75NTR clustering by competitive p75ECD/NgR interaction. Here, we report that TNF-alpha converting enzyme (TACE) (a disintegrin and metalloproteinase 17, ADAM17) induces disinhibition of FGF2-stimulated neurite outgrowth of dorsal root ganglion neurons (DRGN) cultured in the presence of a predetermined concentration of inhibitory CNS myelin-derived ligands. After addition of TACE (which has {alpha}-secretase activity) to mitotically arrested adult rat mixed DRG cultures, we demonstrate 1) NgRECD shedding; 2) release of p75ECD and p75ICD by RIP of p75NTR; 3) blockade of Rho-A activation; 4) reduced EGFR phosphorylation; and 5) increased FGF2-stimulated DRGN neurite outgrowth and branching in the presence of CNS myelin-derived inhibitory ligands. Thus, TACE-induced cleavage of NgR and RIP of p75NTR abrogates axon growth inhibitory signaling, thereby disinhibiting CNS axon/neurite growth.—Ahmed, Z., Mazibrada, G., Seabright, R. J., Dent, R. G., Berry, M., Logan, A. TACE-induced cleavage of NgR and p75NTR in dorsal root ganglion cultures disinhibits neurite outgrowth and promotes branching of neurites in the presence of inhibitory CNS myelin.


Key Words: ectodomain shedding • disinhibited DRGN neurite outgrowth • RIP of p75NTR

SHEDDING OF THE extracellular domain (ECD) of membrane-anchored proteins is a post-translational mechanism for regulating receptor function. For example, ECD shedding is essential for proper signaling of both epidermal growth factor receptor (EGFR) ligands (1 , 2) and notch-mediated lateral inhibition (3 , 4) , and for both limiting inflammatory reactions (5) and regulating axonal guidance mediated by tumor necrosis factor receptor (TNFR) (6) . "Sheddases" proteolytically cleave ECD from membrane-bound proteins (7 , 8) and include tumor necrosis factor (TNF)-{alpha} converting enzyme (TACE), a member of the metalloprotease-disintegrin family (ADAM 17), the activity of which is specifically inhibited by the tissue inhibitor of metalloproteinase-3 (TIMP-3) (9 10 11) . TACE releases TNF-{alpha} from cells by liberating pro-TNF ECD (12 , 13) and also cleaves the ECD from other proteins, including transforming growth factor alpha (TGF{alpha}) (14) , NgR (15 , 16) , and the low-affinity neurotrophin receptor p75NTR (11 12 13) . The latter binds all known neurotrophins and mediates multiple functions, including cell survival, axon growth cone collapse, and apoptosis (17 18 19) . In releasing soluble 55 kDa p75ECD extracellularly, TACE initiates regulated intramembranous proteolysis (RIP) of the remaining 32 kDa cytoplasmic transmembrane fragment (p75CTF) of p75NTR by triggering {gamma}-secretase-mediated cytosolic shedding of the 25 kDa intracellular domain (ICD), p75ICD, leaving the intramembranous domain (p75IMD) in situ (20 21 22) .

p75NTR/TROY (23 24 25 26) independently complex with the Nogo receptor (NgR) and LINGO-1 (27) , signaling inhibition of axon growth through growth cone collapse after NgR binding of the CNS myelin-derived inhibitory ligands, myelin-associated glycoprotein (MAG), oligodendrocyte-derived myelin glycoprotein (OMgp), and Nogo-A (26 , 28 29 30 31 32) . Activated p75NTR/TROY triggers conversion of Rho-GDP to Rho-GTP, leading to sequential ROCK/LIM kinase/cofilin-mediated actin filament depolymerization and growth cone collapse (25 26 27) . EGFR is also required for CNS axon growth inhibition by CNS myelin through NgR-dependent activation of Ca2+-dependent phosphorylation of EGFR by an as yet undefined downstream signaling pathway (33) . In the adult rat myelinated optic nerve, intravitreal sciatic nerve grafting (34 , 35) and neurotrophic factor (NTF) administration (36) both promote robust retinal ganglion cell axon (RGC) axon regeneration, correlated with NTF-induced RIP of p75NTR, leading to shedding of NgRECD, fragmentation of TROY, and suppressed activation of both EGFR and Rho-A. Moreover, siRNA targeted knockdown NgR, p75NTR, and Rho-A disinhibits both DRGN and RGC neurite outgrowth in the presence of CNS myelin (37 , 38) . Accordingly, we hypothesized that RIP-induced abrogation of inhibitory signaling leading to growth cone collapse explained the paradoxical regeneration of retinal ganglion cell (RGC) axons through the axon growth inhibitory environment of the CNS myelin-rich optic nerve. In the present in vitro study, we used active TACE to disinhibit FGF2-stimulated DRGN neurite outgrowth in the presence of inhibitory CNS myelin ligands by cleavage of NgRECD, initiation of RIP of p75NTR, and suppression of Rho-A and EGFR activation. In light of our in vivo optic nerve and in vitro siRNA findings (34 35 36 37 38) , we predicted that the neurites of TACE-treated cultured DRGN would be insensitive to growth inhibitory CNS myelin ligands when stimulated with FGF2 and grow normally in their presence.

MATERIALS AND METHODS

Adult DRG cultures
Dissociated adult rat (6- to 8-wk-old) DRG cells were cultured in 4-well plates at a density of 1500 DRGN/well on glass coverslips precoated with 100 µg/ml poly-D-lysine, followed by 20 µg/ml Laminin-I (both from Sigma, Dorset, UK) in Neurobasal-A containing B27 supplement (Invitrogen, Paisley, UK), as described elsewhere (37) . To limit non-neuronal cell proliferation, the mixed DRG cultures were treated with 30 µM 5-fluoro-2-deoxyuridine (Sigma, St. Louis, MO, USA) daily throughout the culture period (39) . DRG cells were cultured throughout in either the presence or absence of adult rat CNS myelin extracts at 37°C in a humidified atmosphere containing 5% CO2. Other test reagents were added on the day of culture preparation, with samples and measurements taken after 3 or 6 days in culture.

Preparation of CNS myelin
CNS myelin was prepared according to our earlier published method (37) . Briefly, adult Sprague-Dawley rat brains were homogenized in 0.32 M sucrose, 1 mM EDTA, pH7.0 at 4°C, and centrifuged. The supernatant was resuspended in 0.9 M sucrose, overlain with 1–2 ml of 0.32M sucrose, and centrifuged at 20,000 g for 60 min. The CNS myelin at the interface of the two sucrose layers was collected, dispersed in 20 volumes of 0.32 M sucrose, and centrifuged at 13,000 g for 25 min. CNS myelin extract was then diluted in 25 volumes of water and centrifuged at 20,000 g for 25 min. The final white pellet was resuspended in a small volume of water, freeze dried overnight, and protein content was determined using the Pierce bicinchoninic acid (BCA) assay (Bio-Rad, Hercules, CA, USA). Western blot of the CNS myelin extracts confirmed the presence of Nogo-A, MAG, OMgp, and chondroitin sulfate proteoglycan (CSPG) (37) . The CNS myelin extract was added to DRG cultures at a protein concentration of 200 µg/ml, previously determined to be optimally inhibitory to FGF2-stimulated DRGN neurite outgrowth (37) .

Treatment of DRGN with FGF2 and TACE
FGF2 (Peprotech, London, UK) was added at 10 ng/ml (predetermined to cause optimal stimulation of DRGN neurite outgrowth) and active TACE (R & D Systems, Abingdon, UK) was added at 10 ng/ml (predetermined to cause optimal p75NTR peptide cleavage) to DRG culture plates in triplicates. DRG cultures were also treated with either 25 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma) to activate endogenous TACE production (20 , 22) , or TIMP3 (50 nM, Chemicon, Hampshire, UK) a specific inhibitor of TACE activity (9 10 11) . Cells were treated for 72 h before harvesting for Western blot and subsequent quantitative assessment of proteins by densitometry as described below.

Antibodies
Monoclonal ß-III tubulin antibody (Ab) (Sigma) was used at 1:100 to label DRGN neurites by immunocytochemistry (ICC). Polyclonal anti-p75NTR Ab was used to identify and localize p75NTR, p75CTF, p75ICD and p75ECD (Promega, Madison, WI, USA; 1:500 dilution for both Western blots and ICC). Microtubule associated protein-1B (MAP1B) (Abcam Ltd, Cambridge, UK) was used at 1:500 dilution for Western blots and ICC. Antimouse NgR (Autogen Bioclear, Wiltshire, UK) was used at 1:500 dilution for Western blots and ICC. A second NgR Ab, goat anti-human NgR (Santa Cruz Biotechnology, San Diego, USA), was used at 1:500 dilution to confirm the results obtained with antimouse NgR. A goat polyclonal anti-human TROY Ab was used to detect TROY in Western blots and ICC (1:500), whereas a goat polyclonal anti-human Ab was used at 1:400 dilution to detect phosphorylated EGFR in Western blots (both from Santa Cruz Biotechnology). Monoclonal anti-EGFR was used to detect total EGFR in Western blots (1:400; Novocastra, Newcastle on Tyne, UK). A rabbit polyclonal antibody (pAb) raised against the 1–23 amino-terminal of rat FGF2 was used to detect FGF2 in Western blots at a 1:500 dilution (gift from Andrew Baird, University of Birmingham).

Immunocytochemistry
DRG cultures were fixed in 4% paraformaldehyde for 10 min (TAAB Laboratories, Berkshire, UK) before washing x 3 in PBS, then blocked in PBS containing 0.5% BSA (Sigma) and 1% Triton X-100 (Sigma), and incubated with the relevant primary Ab diluted 1:100 in PBS containing 0.5% BSA and 0.5% Tween-20 (Sigma) for 1 h at room temperature in a humidified chamber. Cells were then washed X3 in PBS and incubated with either AlexaFluor 488 (Green), or Texas Red (Red) (both from Invitrogen), diluted 1:100 in PBS-T-BSA for 1 h at room temperature. After further washes in PBS, coverslips were mounted in FluorSave (Calbiochem, San Diego, CA, USA) and viewed under a fluorescent microscope (Carl Zeiss, Welwyn-Garden City, UK).

Measurement of DRGN neurite outgrowth
Photomicrographs of ßIII-tubulin+ immunostained DRGN neurites were captured using Axiovision Software (Carl Zeiss, Hertfordshire, UK) from 30 randomly selected DRGN/coverslip and neurite lengths measured using Axiovision (Carl Zeiss) and represented graphically as means ± SD.

DRGN branching analysis
The numbers of primary ßIII-tubulin+ DRGN neurites emerging directly from DRGN somata were counted and from each neuritic tree, the total number of branches exceeding 20 µm in length (40) recorded using Axiovision software (Carl Zeiss) and represented graphically as means ± SD.

Counting of DRGN and non-neuronal cells
To quantify the numbers of DRGN vs. non-neuronal cells present in the mixed DRG cultures, cultures were double stained with ßIII-tubulin (to identify all neurons) and 4',6'-diam idino-2-phenylidole (DAPI) (to identify all cell nuclei). Each coverslip (n=3, 3 independent experiments) was partitioned into nine equal quadrants and, in each, random photomicrographs of merged images from ßIII-tubulin/DAPI-stained DRG cultures were captured using Axiovision Software (Carl Zeiss, Hertfordshire, UK), as described above, and the relative proportions of ßIII-tubulin+ DRGN to DAPI-stained cells counted. Numbers of non-neuronal cells were calculated by subtracting the number of DRGN from the total number of DAPI-stained cells and the counts were represented graphically as means ± SD. The same method was used to count the number of TROY+, p75NTR+, and NgR+ DRGN at day 1 in culture.

Protein extraction and Western blot
To determine fragmentation of NgR and RIP of p75NTR into p75ECD, p75CTF, and p75ICD after addition of TACE, DRG cultures were washed twice with PBS and incubated for 15 min at 37°C with 0.25% trypsin/EDTA (Invitrogen), followed by trituration and centrifugation at 1300 rpm for 5 min. The cell pellets were resuspended in ice-cold lysis buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.5 mM EGTA, 150 mM NaCl, 1% Nonidet P-40 (Sigma), and protease inhibitor cocktail (Sigma) and incubated on ice for 30 min, centrifuged at 13,000 rpm at 4°C, and cell lysates were normalized for protein concentration using a colorimetric protein assay (Bio-Rad). Proteosome inhibitors were not used prior to cell lysis. To determine the levels of shed NgR and p75ECD, cell culture media were collected and concentrated using microconcentrators (Millipore, Bedford, MA, USA). Each sample (40 µg total protein) was incubated with 2x Laemmli loading buffer at 90°C for 4 min and separated on a 12% SDS-polyacrylamide gel to probe for NgR and p75NTR (Invitrogen) and 6% SDS gels for MAP1B. Proteins were transferred to PVDF membranes (Millipore UK, Gloucestershire, UK), and blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 and 5% nonfat milk. Membranes were blotted overnight with the relevant Ab. To detect protein bands, an enhanced chemiluminescence (ECL) system (Amersham, Buckinghamshire, UK) and HRP-conjugated secondary Ab (1:1000; Amersham) were used. Each blot was stripped and reprobed with additional relevant antibodies thereafter. Each experiment was performed six times.

Rho activation assay
GTP-bound Rho was assayed from DRG cell culture lysates using a commercially available Rho activation assay kit, following the manufacturer’s instructions (Upstate Biotechnology, Milton Keynes, UK).

Densitometry
Western blots were scanned into Adobe Photoshop, keeping all scanning parameters the same for each blot, to provide a densitometric value for bands of interest. TIFF files were then analyzed in ScionImage (version 4.0.2, Scion Corp., Frederick, MD, USA) using built-in gel plotting macros. The integrated density of each band in each lane was calculated for six separate blots from six independent experiments, and a mean value was plotted ± SD.

Statistical analysis
Sample means were calculated and differences analyzed for significance using GraphPad Prism (GraphPad Software Inc., Version 4.0, San Diego, CA, USA) by 1 way ANOVA, followed by post hoc testing with Dunnett’s method to identify statistically significant groups.

RESULTS

Characterization of DRG cultures
The DRG cultures used in these experiments were mixed primary cultures prepared from dissociated adult rat DRG, and comprised DRGN along with numerous non-neuronal cells including fibroblast, endothelial, satellite, and Schwann cells (Fig. 1 A–D). Since glia also produce TACE (41) , we first determined the relative proportions of DRGN and non-neuronal cells in FGF2-stimulated cultures. The number of ßIII-tubulin+ DRGN/culture well was determined as 1500 at 0 day, and their number remained constant over a 6 day culture period (Fig. 1B, D, E ). The initial number of non-neuronal cells (~7500/culture well) increased significantly to 20,000 over a 6 day culture period (Fig. 1A, E ). Hence, throughout this study DRG cells were routinely cultured with the mitotic inhibitor 5-fluoro-2'-deoxyuridine (5-FDU), which arrested proliferation of non-neuronal cells to a level equivalent to that at 0 days (Fig 1C, E ), thus keeping the contribution of non-neuronal cells static. When cells from TACE + FGF2-treated cultures were lysed at 3 days, titers of full-length p75NTR changed by 7% in the presence/absence of mitotic inhibitor (not shown), suggesting that most of the p75NTR fragmentation observed related to changes in DRGN. Immunocytochemistry of DRG cultures revealed that almost 100%, 70%, and 30% of DRGN were immunopositive for TROY, p75NTR, and NgR, respectively (Fig. 2 A, B).


Figure 1
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  		Figure 1. A–D) Photomicrographs of mixed adult rat DRG cultures at 3 days to demonstrate DAPI-stained nuclei (A, C) and ßIII-tubulin+ (B, D) -stained DRG neurons in the presence of FGF2, with and without treatment with the mitotic inhibitor 5-fluro-2'-deoxyuridine (5-FDU). E) Quantification of the mean number of DRGN and non-neuronal cells in mixed adult rat DRG cultures at 0 days, 3 days, and 6 days with and without FDU treatment.


Figure 2
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  		Figure 2. Immunocytochemistry to show TROY/p75NTR/NgR/ß-III-tubulin immunostaining in mixed DRG cultures (A) and quantification of the relative proportions of immunopositive DRGN (B). Note a DRGN that was negative for NgR immunostaining (arrow).

TACE-induced RIP of p75NTR in DRG cultures
Addition of FGF2 to mitotically arrested DRG cultures in the presence or absence of inhibitory CNS myelin extracts induced limited fragmentation of p75NTR compared to untreated controls (Fig. 3 A, B), while addition of either active TACE, alone or active TACE + FGF2 cleaved most of the p75NTR present, eliminating full-length p75NTR protein (Fig. 3A, B ) both with and without CNS myelin. PMA, which activates endogenous TACE (20 , 22) , also cleaved p75NTR in FGF2-stimulated cultures, but fragmentation was less than that observed with the addition of exogenous active TACE. In each case, p75ECD accumulated in the culture media after p75NTR fragmentation, and p75CTF and p75ICD appeared in the cell lysates in a reciprocally coordinated manner (Fig. 3A, B ). TACE-induced RIP of p75NTR coincided with a significant reduction in the levels of Rho-GTP and phosphorylated EGFR (pEGFR) within DRG cultures only in the presence of CNS myelin, whereas total Rho and EGFR levels did not change (Fig. 3A, B ). The further addition of TIMP3 blocked TACE-mediated fragmentation of p75NTR in the presence of FGF2 and restored the levels of Rho-GTP and pEGFR to those seen in FGF-2-treated cultures with CNS myelin extracts (Fig. 3A, B ). Similar levels of p75NTR fragmentation were observed in the absence of CNS myelin; however, Rho-GTP and pEGFR were not detected confirming that, without the presence of CNS myelin inhibitory ligands, there was no activation of either Rho-A or EGFR (Fig. 3A, B ).


Figure 3
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  		Figure 3. TACE-induced RIP of p75NTR and suppression of Rho and EGFR activation in the presence/absence of CNS myelin ligands. A) Representative Western blot/Rho immunoprecipitation in cell lysates and media of p75NTR, p75CTF, p75ICD, p75ECD, Rho-GTP, total Rho, phosphorylated EGFR (pEGFR), total EGFR, and ß-actin in mitotically arrested mixed adult rat DRG cultures after FGF2, TACE, PMA, and TIMP3 treatments singly, and in combination. B) Mean (±SD) integrated optical density of the protein bands from lysates and media seen in panel A (mean integrated density) and % reduction in Rho-GTP and pEGFR compared to control for treatments of DRG cultures with TACE, TACE + FGF2, PMA + FGF2, and TACE + FGF2 + TIMP3. ß-actin was used as a loading control. ***P < 0.0001.

TACE induced fragmentation of NgR but not of TROY
Addition of TACE to mitotically arrested DRG cultures either in the presence or absence of CNS myelin also caused fragmentation of NgR, as has been reported (15 , 16) , yielding a shed 48 kDa band in the cell culture medium (Fig. 4 ). However, when the same blots were stripped and reprobed, no fragmentation of TROY was detected (Fig. 4) , suggesting receptor-specific TACE cleavage.


Figure 4
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  		Figure 4. TACE-induced fragmentation of NgR. Representative Western blot to show that addition of TACE to mitotically arrested mixed adult rat DRG cultures induced fragmentation of NgR to release a 50 kDa fragment of NgRECD that is detectable in the media, while TROY was not fragmented by TACE.

TACE-induced RIP of p75NTR promoted DRGN neurite outgrowth in the presence of inhibitory CNS myelin ligands
In the absence of CNS myelin, FGF2 promoted significant DRGN neurite outgrowth whereas addition of either FGF2 + PMA, or FGF2 + TACE + TIMP3 did not affect neurite outgrowth. The addition of either TACE alone, or PMA alone did not promote DRGN neurite outgrowth (Fig. 5 A, B).


Figure 5
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  		Figure 5. TACE-induced RIP of p75NTR enhanced DRGN neurite outgrowth in the presence/absence of CNS myelin ligands. A) Representative ßIII-tubulin immunocytochemistry in DRG cultures after exposure to FGF2, FGF2 + CNS myelin, TACE + CNS myelin, and FGF2 + TACE + CNS myelin. B) Quantification of mean longest DRGN neurite lengths (±SD) demonstrated significantly enhanced FGF2-stimulated DRGN neurite outgrowth after addition of TACE + FGF + CNS myelin. Addition of TIMP3 blocked TACE-mediated DRGN neurite outgrowth. ***P < 0.0001. *P < 0.05. C) Representative Western blot to show the presence of FGF2 in CNS myelin extracts.

After the addition of a predetermined concentration of CNS myelin protein extract, DRGN neurite outgrowth was completely blocked in the presence of FGF2 (Fig. 5A, B ), while TACE and PMA significantly disinhibited FGF2-stimulated DRGN neurite outgrowth compared to that seen without CNS myelin and with TACE or PMA alone (P<0.0001, Fig. 5A, B ). Addition of TIMP3 blocked TACE activity in FGF-2-stimulated DRG cultures and restored the inhibition of DRGN neurite outgrowth mediated by CNS myelin to control levels (Fig. 5B ). A neuritogenic effect of CNS myelin extract on DRGN neurite outgrowth and branching was revealed in the presence of TACE and FGF2 (Fig. 5B , Fig. 6 A, B). This was probably the result of the activity of contaminating FGF2 present in the CNS myelin extracts (Fig. 5C ).


Figure 6
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  		Figure 6. TACE-induced RIP of p75NTR enhanced neurite number and branching in DRGN in the presence of CNS myelin ligands. The mean (±SD) number of A) primary DRGN neurites and B) branches after 3 days in culture after exposure to FGF2, FGF2 + myelin, TACE + CNS myelin, and FGF2 + TACE + CNS myelin increased significantly compared to controls. Treatment with TIMP3 restored the inhibitory potential of CNS myelin ligands and blocked DRGN neurite outgrowth. Representative ßIII-tubulin immunocytochemistry of DRGN in 3-day-old cultures (C–E) demonstrates branching after exposure of cultures to FGF2 with no CNS myelin, TACE + FGF2 + CNS myelin, and PMA + FGF2 + CNS myelin. **P < 0.001.

Addition of TACE enhanced DRGN branching
When TACE was added to mitotically arrested, FGF2-stimulated DRG cells cultured in the presence of CNS myelin, the number of DRGN primary neurites and their branching frequency was increased (Fig. 6A, B ). After treatment with FGF2 without CNS myelin, the majority of DRGN had 3–4 primary neurites, each with 1–4 branches (Fig. 6A-C ). Addition of CNS myelin to the mitotically arrested cultures completely suppressed neurite outgrowth. However, the addition of TACE and FGF2 in the presence of CNS myelin allowed the majority of DRGN to grow >5 primary neurites, each with >10 branches (Fig. 6A, B, D ). Treatment of CNS myelin-exposed DRGN with PMA + FGF2 similarly increased the numbers of primary neurites and their branches compared to DRGN treated with PMA alone (Fig. 6A, B, E ). TIMP3 restored the inhibitory potential of CNS myelin extracts in the presence of TACE, leading to suppressed neurite outgrowth and branching of FGF2-stimulated DRGN (Fig. 6A, B ).

TACE-mediated branching of DRGN was correlated with up-regulation of MAP1B
When lysates of mitotically arrested DRG cultures were probed for MAP1B, there was significant up-regulation of MAP1B protein in cultures treated with TACE + FGF2 in both the presence and absence of CNS myelin (Fig. 7 ). MAP1B was also up-regulated in DRG cultures treated with PMA + FGF2 in the presence of CNS myelin compared to PMA alone, but levels were significantly less than those observed with TACE + FGF2 (P<0.001 TACE+FGF2 vs. PMA+FGF2, Fig. 7 ). In TIMP3-treated, mitotically arrested DRG cultures, levels of MAP1B were similar to those in control cultures (Fig. 7) .


Figure 7
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  		Figure 7. TACE-induced RIP of p75NTR correlated with enhanced MAP1B levels (a measure of neurite branching) in the presence of CNS myelin. Representative Western blots and subsequent densitometry of MAP1B levels (mean±SD) in lysates of cells from control cultures and cultures exposed to FGF2 and TACE alone, and to combinations of TACE + FGF2, PMA + FGF2, and TACE + FGF2 + TIMP3. Exposure to TACE + FGF2 in the presence of CNS myelin enhanced MAP1B levels significantly compared to other treatments, while addition of TIMP3 restored MAP1B levels to those observed in controls. ß-actin was used as a loading control. ***P < 0.0001, **P < 0.001.

DISCUSSION

There is good evidence for a role of CNS myelin-derived axon growth inhibitors and their receptor signaling complex (NgR/p75NTR/Lingo-1) in the failure of DRGN axon regeneration, although the relative contribution of non-myelin inhibitors is less well defined (38 , 43) .

RIP is initiated by TACE ({alpha}-secretase)
In this study, we show that addition of TACE to mitotically arrested mixed DRG cultures induces fragmentation of both NgR and p75NTR, leading to blocked Rho-A activation, reduced EGFR phosphorylation, and disinhibition of FGF2-stimulated DRGN neurite outgrowth and branching in the presence of inhibitory CNS myelin extracts. TACE-induced NgR and p75NTR fragmentation was blocked by the addition of TIMP3, a specific inhibitor of TACE activation, reinstating myelin-related inhibition of DRGN neurites. Conversely, NgR and p75NTR fragmentation was promoted by the TACE activator, PMA, in mitotically arrested DRG cultures, suggesting endogenous TACE was activated (22) . TACE activation by PMA is PKC-mediated (1) , and the reported effects of PKC on axonal growth are controversial, with both neuritogenic and neurite growth inhibitory effects described. For example, PKC is reported to act as a common downstream mediator for several neuritogenic factors, including NCAM and NGF (44) . By contrast, others have reported that PKC mediated the inhibitory activity of CNS myelin and CSPG on axonal outgrowth (44 , 45) . Nevertheless, we have shown that PMA simultaneously induced TACE activation, RIP of p75NTR, and enhanced neurite outgrowth in both RGC (38) and DRGN (this paper), which suggests that the neuritogenic and disinhibitory effects of PMA on TACE activated NgR and p75NTR cleavage predominate.

A growing number of transmembrane growth factor receptors are emerging as RIP targets after ligand binding and/or ectodomain shedding (21) . p75NTR serves as a coreceptor for NTF by forming a high-affinity heteromeric complex with Trk receptors (46) , but disassembly into p75ECD, p75CTF, and p75ICD fragments after RIP (20 , 22 , 47) abolished Trk/p75NTR receptor interactions (21) . In this study, the non-Trk binding ligand FGF2 stimulated only limited TACE-induced RIP of p75NTR in mitotically arrested DRG cultures, suggesting that Trk/p75NTR ligand binding may normally block accessibility of TACE to p75NTR substrates through either steric hindrance, or conformational change. It remains to be seen whether other p75NTR-independent NTF cause TACE-induced RIP of p75NTR in DRGN. The addition of exogenous TACE to mitotically arrested DRG cultures cleaved the majority of p75NTR, whereas simultaneous addition of the TACE activation blocker TIMP3 (9 , 10) prevented the generation of p75CTF and p75ICD, confirming that p75ECD removal is a prerequisite for {gamma}-secretase-mediated generation of p75ICD (20 , 21 , 47) .

The robust nature of the NTF-induced p75NTR RIP phenomenon we are reporting is reflected in these and other experiments, where NTF induced RIP of p75NTR (36 , 38 , 48) . p75ICD was detectable in protein extracts without the use of proteosome inhibitors prior to cell lysis. Although p75ICD trafficks to the nucleus, where it induces apoptosis by activation of the transcription factor NF{kappa}B (22) , titers are tightly controlled and undergo rapid removal by proteosomal degradation (21 , 22) . This probably explains why we did not observe enhanced apoptosis of DRGN in any of our cultures.

Our results conflict with those of a recent in vitro study that proposes that the induction of TACE activity and RIP of p75NTR is a prerequisite for inhibitory Rho-A signaling in immature rat cerebellar granule cells cocultured with MAG-Fc expressing CHO cells (45) . By contrast, here we show, in mitotically arrested primary adult rat DRG cultures, that TACE-induced p75NTR fragmentation disinhibits DRGN neurite outgrowth in the presence of inhibitory CNS myelin. In a related paper, we also describe NTF-induced RIP of p75NTR in regenerating RGC both in vivo and in vitro (38) . In additional in vivo experiments, Logan et al. (36) showed that intravitreally implanted engineered fibroblasts expressing single and combined NTF genes synergistically induced RIP of p75NTR, correlated with suppressed Rho activation and enhanced RGC axon regeneration. Moreover, in vitro combinations of NTF synergistically promoted RGC neurite outgrowth in the presence of inhibitory CNS myelin, which also correlated with TACE activation, RIP of p75NTR, and reduced Rho activation (36) . Blocking EGFR phosphorylation disinhibited axon growth mediated by CNS myelin and CSPG (33) . In all of our in vivo and in vitro experiments, EGFR phosphorylation was also blocked after NTF-induced RIP of p75NTR, disinhibiting DRGN and RGC neurite outgrowth/axon regeneration in the presence of CNS myelin. In our studies that show RIP of p75NTR associated with enhanced NTF-induced axon/neurite outgrowth, we have used physiological in vivo and in vitro models relevant to the phenomena of adult CNS injury, which have a number of key differences to the Domeniconi et al. (45) in vitro paradigms, including 1) robust in vivo activation of p75NTR RIP in NTF-stimulated adult neurons; 2) addition of NTF to cultured adult RGC (36 , 38) and DRGN (37 , 48) , and this paper), which potentiates RIP of p75NTR; 3) reciprocal change between full-length p75NTR and its fragments in vivo and in vitro; 4) detection of high titers of p75ICD in the absence of proteosome inhibitors both in vivo and in vitro; 5) induction of neurite growth disinhibition in cultured primary adult neurons by adding CNS myelin extracts (containing Nogo-A, MAG, OMgp, and CSPG (48) ) directly to media rather than by the exclusive in vitro use of MAG-Fc expressing CHO cells and immature CNS neurons.

Fate of TROY and NgR
TROY can initiate the inhibitory signaling complex in the absence of p75NTR (23 , 24) . The pattern of TACE-induced TROY and NgR cleavage in DRG cultures reported here was different from that seen in retinal cultures, where fragmentation of TROY but not NgR occurred (38) . In mitotically arrested DRG cultures, TROY was not fragmented by TACE whereas NgR was, confirming earlier reports that TACE fragments NgR (15 , 16) . NgRECD shedding is expected to disinhibit neurite outgrowth since shed NgRECD is able to bind and sequester all the CNS myelin-related inhibitory ligands, which would then be unavailable for association with p75NTR/TROY/LINGO-1, thereby blocking inhibitory Rho-A signaling. Fragmentation of NgR by TACE would contribute to the disinhibition of FGF2-stimulated DRGN neurite growth in the presence of CNS myelin and enhance DRGN neurite branching. Similarly, others have shown that inhibition of NgR signaling either using a dominant negative mutant (49) , or transgenically with a soluble function-blocking NgR fragment (50) , enhances NTF-stimulated axon regeneration.

Suppression of Rho-A and EGFR activation
Disinhibition of CNS axon regeneration may be induced by several experimental approaches. For example, blocking activation of Rho-A and ROCK with the antagonists C3 transferase or Y-27632 enhanced NTF-stimulated axonal outgrowth on CNS myelin substrates in vitro and in vivo (51 52 53) . However, the modes of delivery of these antagonists may determine their potency, since C3 was not effective in all in vivo studies (53) . Single gene ligand knockout (e.g., of Nogo-A) does not promote CNS axon regeneration (54 , 55) , probably because multiple alternative ligands remain active (29 , 32) .

In vivo, the failure of CNS axons to regenerate after injury is attributable in part to the presence of axon outgrowth inhibitors including MAG, OMgp, CSPG, and Nogo-A (29 , 31 , 32 , 56) . We have previously shown that siRNA-mediated knockdown of p75NTR and its fragments in adult rat DRG cell cultures blocked CNS myelin-induced Rho-A activation and disinhibited NTF-stimulated neurite outgrowth (37) . The effect is similar to that of TACE-mediated RIP of p75NTR, which disinhibited FGF2-stimulated DRGN neurite outgrowth in the presence of inhibitory CNS myelin ligands by suppressing inhibitory signaling through pEGFR and Rho-GTP. The accompanying suppression of EGFR activation is probably a result of attenuated Ca2+ influx after RIP of p75NTR and NgRECD shedding. Although the exact mechanism for the activation of EGFR phosphorylation by CNS myelin-derived ligands is unknown, activation of p75NTR does lead to increased uptake of Ca2+ (57) .

DRGN neurite growth requires NTF stimulation
TACE treatment almost completely fragmented p75NTR in the mitotically arrested mixed DRG cultures both with and without CNS myelin, but there was no accompanying DRGN neurite outgrowth in the presence of CNS myelin unless FGF2 was added. Therefore, DRGN require stimulation into an "activated growth state" through either NTF, or cAMP administration in order to drive neurite/axon growth (49 , 58 , 59) . The significant enhancement of FGF2-stimulated neurite outgrowth by disinhibited DRGN in the presence of CNS myelin suggests that the NTF-stimulated growth is normally held in check by inhibitory ligands. Since TROY and NgR were not completely fragmented in our experiments, there is still likely to be residual inhibitory signaling in DRGN even in the absence of full-length p75NTR. The limited fragmentation of p75NTR and NgR induced by FGF2 in DRG cultures probably reflects limited endogenous TACE activation by this growth factor when acting in isolation. Logan et al. (36) report that a synergistic NTF activation of endogenous TACE is required for robust cleavage of p75NTR and NgR.

An apparent neuritogenic effect of the CNS myelin extract was observed in mitotically arrested DRG cultures treated with TACE and FGF2 (Figs. 5 and 6) . In our experiments, preparations of rat brain myelin were used as a source of inhibitory ligands (48) . While the myelin extracts were demonstrably inhibitory to neurite growth, the preparations also contained significant levels of contaminating FGF2 (Fig. 5C ). FGF2 is a myelin binding NTF whose neuritogenic effects were masked by the activity of the inhibitory ligands in the CNS myelin extracts. It seems that when the actions of the CNS myelin-related inhibitory ligands were blocked by RIP of p75NTR, the neurotrophic actions of the contaminating FGF2 were revealed (Fig 5B, C ).

Disinhibition of DRGN neurite growth in presence of CNS myelin-derived ligands
TACE treatment of mitotically arrested DRG cultures enhanced DRGN neurite number and branching measured by quantitative neurite analysis and by raised MAP1B levels. MAP1B is normally down-regulated in adult neurons, but is re-expressed in regenerating axons and their growth cones (42 , 60) . It is unusual that DRGN constitutively express high levels of MAP1B, possibly associated with central sprouting and regeneration of their peripheral axon projections (42 , 60) . Cultured neurons from MAP1B hypomorphic mutant mice exhibit defects in axonal outgrowth (61) , whereas DRGN from MAP1B–/– mice exhibit a "curled" neuritic outgrowth and increased frequency of branching (40) .

CONCLUSIONS

Our results suggest that TACE-induced NgR fragmentation and RIP of p75NTR suppresses Rho-A activation and EGFR phosphorylation, paralyzes the signaling cascade-mediating growth cone collapse and disinhibits NTF-stimulated DRGN neurite outgrowth in the presence of inhibitory CNS myelin ligands. In addition, the shed ECD fragments of NgR and p75NTR have the potential to act as inhibitory signaling antagonists, so that p75ECD competitively blocks NgR/p75NTR clustering and NgRECD binds all CNS myelin-derived ligands, further disinhibiting DRGN neurite outgrowth. TACE-induced receptor shedding and RIP may therefore beexploited therapeutically to disinhibit axon growth, thereby enhancing the efficacy of NTF treatments.

ACKNOWLEDGMENTS

This work was supported by the BBSRC, Grant 918986, the Wellcome Trust, Grant No. 065920 and the International Spinal Research Trust (NRB 070).

Received for publication January 17, 2006. Accepted for publication April 17, 2006.

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