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Loss of glial fibrillary acidic protein (GFAP) impairs Schwann cell proliferation and delays nerve regeneration after damage

Loss of glial fibrillary acidic protein (GFAP) impairs Schwann cell proliferation and delays... Research Article 3981 Loss of glial fibrillary acidic protein (GFAP) impairs Schwann cell proliferation and delays nerve regeneration after damage 1 1 1 1,2 3 Daniela Triolo , Giorgia Dina , Isabella Lorenzetti , MariaChiara Malaguti , Paolo Morana , 2,3 2,4 5 1,2 1,2, Ubaldo Del Carro , Giancarlo Comi , Albee Messing , Angelo Quattrini and Stefano C. Previtali * 1 2 3 Neuropathology Unit, Department of Neurology and INSPE, and Neurophysiology Unit, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy Università Vita-Salute San Raffaele, 20132 Milan, Italy Waisman Center and Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA *Author for correspondence (e-mail: [email protected]) Accepted 14 July 2006 Journal of Cell Science 119, 3981-3993 Published by The Company of Biologists 2006 doi:10.1242/jcs.03168 Summary Axonal loss causes disabling and permanent deficits in that GFAP and the other Schwann-cell-intermediate many peripheral neuropathies, and may result from filament vimentin physically interact in two distinct inefficient nerve regeneration due to a defective signaling pathways involved in proliferation and nerve relationship between Schwann cells, axons and the regeneration. GFAP binds integrin v8, which initiates extracellular matrix. These interactions are mediated by mitotic signals soon after damage by interacting with surface receptors and transduced by cytoskeletal fibrin. Consistently, ERK phosphorylation was reduced in molecules. We investigated whether peripheral nerve crushed GFAP-null nerves. Vimentin instead binds integrin regeneration is perturbed in mice that lack glial fibrillary 51, which regulates proliferation and differentiation acidic protein (GFAP), a Schwann-cell-specific cytoskeleton later in regeneration, and may compensate for the absence constituent upregulated after damage. Peripheral nerves of GFAP in mutant mice. GFAP might contribute to form develop and function normally in GFAP-null mice. macro-complexes to initiate mitogenic and differentiating However, axonal regeneration after damage was delayed. signaling for efficient nerve regeneration. Mutant Schwann cells maintained the ability to dedifferentiate but showed defective proliferation, a key Key words: Cytoskeleton, Transgenic mice, Extracellular matrix, event for successful nerve regeneration. We also showed Nerve regeneration, Adhesion Introduction extensively documented and consist of a series of stereotyped Axonal loss and defective axonal regeneration is responsible steps (Griffin and Hoffman, 1993; Scherer and Salzer, 2001). for severe and permanent deficits in peripheral neuropathy. The segments of axons distal to the site of injury degenerate, Although axonal loss might not affect the life span of a patient, and macrophages penetrate and remove the disrupted myelin it results in severe disability with progressive muscle atrophy sheets, while Schwann cells dedifferentiate, re-enter the cell- and weakness, sensory deficits, and foot and leg abnormalities. proliferation cycle and provide a substrate for axonal regrowth. Any improvement in regeneration would benefit these patients. The onset of mitogenesis is synchronous with a peak 3-5 days Efficient axonal regeneration relies on the pathogenetic after injury and requires a prelude phase of intense mechanism that caused the axonal degeneration. If the disease rearrangement of the Schwann-cell cytoplasm. During this primarily affects the neuronal cell body (sensory or motor process Schwann cells acquire again the expression of surface neurons), the damage mostly prevents recovery. Conversely, molecules characteristic of embryonic development, such as when the neuronal cell body is preserved, the regeneration neural cell adhesion molecule (NCAM), L1-adhesion molecule NTR capacity should be maintained but still depends on the and p75 , and upregulate cytoskeletal constituents, such as permissive environment in the nerve. In the latter case, the glial fibrillary acidic protein (GFAP) and vimentin (Jessen et nerve regeneration results from balanced interaction of al., 1990; Martini, 1994; Neuberger and Cornbrooks, 1989). Schwann cells, the environment – the extracellular matrix After proliferation, Schwann cells interact with molecules in (ECM) – and regrowing axons (Scherer and Salzer, 2001; Stoll the extracellular environment to reorganize the basement and Muller, 1999). membrane and to rearrange themselves into bands of Bungner. All the events involved in axonal regeneration can be These bands are rail-track-like structures upon which axons recapitulated in the model of Wallerian degeneration- can efficiently regenerate. Further steps in nerve regeneration regeneration, which is induced in rodents by traumatic crush include Schwann cells that surround bundles of regenerating injury of the sciatic nerve. The spatial-temporal events axons, segregate larger axons into 1:1 relationship, enwrap associated with degeneration and regeneration have been them and form myelin sheaths. Journal of Cell Science 3982 Journal of Cell Science 119 (19) Most of these latter events reproduce what occurs in First we confirmed that homozygous mutant mice did not development (Webster, 1993), and rely on the interaction of synthesize GFAP in Schwann cells. We performed Schwann cell surface receptors with molecules that normally immunohistochemistry and western blot analysis of the sciatic form the ECM endoneurium or infiltrate the nerve because of nerve. Both experiments showed absence of the GFAP protein the blood-nerve barrier disruption (Akassoglou et al., 2002; (Fig. 1A,B, and data not shown). Feltri et al., 2002; Lefcort et al., 1992; Patton et al., 1997; Then, we investigated whether peripheral nerve Previtali et al., 2003b). These complex interactions result in a development was impaired. We compared semi-thin and ultra- continuous reorganization of the Schwann cell cytoskeleton thin sections from the sciatic nerve of postnatal day (P) P1, P7, that operates either downstream the outside-in or upstream the P14, P28 and P60 GFAP-null mice to age-matched controls. inside-out signaling pathway (Previtali et al., 2001). No significant differences in axonal sorting, Schwann-cell GFAP is a glial-specific member of the intermediate filament axon relationship and myelination were observed (Fig. 1C-J). family, which includes cell-type-specific filamentous proteins Myelin-forming and non-myelin-forming Schwann cells did with similar structure and function as scaffold for cytoskeleton not show cyto-architectural abnormalities; in particular, we did assembly and maintenance (Coulombe and Wong, 2004). not observe filament aggregates or any other abnormalities in During development, Schwann cells express two other the basement membrane (Fig. 1K). intermediate filaments: nestin and vimentin (Dong et al., 1999; We then determined the absolute number of myelinated Jessen and Mirsky, 1991). GFAP appears at a relatively late fibers and performed morphometric analysis comparing the stage in Schwann cell development, essentially when immature sciatic nerve of GFAP-null mice those of wild type. The total Schwann cells are formed, and is downregulated in those number of myelinated fibers, fiber diameter and myelination Schwann cells that form myelin (Jessen et al., 1990). After (g-ratio) was not significantly different in GFAP-null mice and birth, only non-myelin-forming Schwann cells and Schwann wild-type littermates (Fig. 1L,M). cells that dedifferentiate after nerve injury express GFAP, To confirm morphological data we performed functional whereas myelin-forming Schwann cells express vimentin tests. Mutant mice appeared normal from birth to 15 months (Jessen et al., 1990; Mirsky and Jessen, 2005). of age. They walk, run, climb and reproduce similarly to age- GFAP is also expressed in astrocytes, a Schwann cell matched controls. At 3 and 6 months, the GFAP null mice and counterpart in the central nervous system (CNS). The wild-type littermates showed no significant difference in upregulation of GFAP, together with vimentin, was thought to rotarod testing (Fig. 1M). Consistent with behavioral analysis, be a crucial step for astrocyte activation in response to brain GFAP-null mice showed normal nerve conduction velocity damage. However, deletion of GFAP in mutant mice did not (NCV) (35±0.5 m/second) and compound motor action result in any gross CNS abnormality (Gomi et al., 1995; potentials (cMAP) (37±0.7 mV) in the neurophysiological tests Liedtke et al., 1996; McCall et al., 1996; Pekny et al., 1995) (Fig. 1N). or in defective response to CNS injury (Pekny et al., 1999). Impaired astrocyte reaction to injury was only observed in The absence of GFAP is probably compensated by double GFAP-vimentin-deficient mice (Pekny et al., 1999). other intermediate filaments and modifies the By contrast, there are no extensive reports on the effects of endoneurial ECM composition GFAP deletion on peripheral nerve development and function, We investigated whether other intermediate filaments can or on the possible consequences for nerve regeneration. Here, compensate for the absence of GFAP in mutant mice, thus we examined the peripheral nervous system of GFAP-null explaining the normal phenotype. In fact, during development, mice. We observed normal nerve development and adult nerve two other intermediate filaments are described in Schwann function. No morphological abnormalities were detected cells: nestin and vimentin (Dong et al., 1999; Jessen and despite different composition of the Schwann cell cytoskeleton Mirsky, 1991). and endoneurial ECM. However, the lack of GFAP delayed First, we performed qualitative analysis by nerve regeneration after damage, probably due to defective immunohistochemistry. Results showed similar expression of Schwann cell proliferation. We also found that GFAP and vimentin in non-myelin-forming Schwann cells, as depicted by vimentin associate with two different adhesion pathways. colocalization with L1, in GFAP-null mice and controls GFAP complexes with integrin v8, which binds fibrin (compare Fig. 2A-C with 2G-I). Accordingly, vimentin was and modulates Schwann cell proliferation after damage. similarly expressed in myelin-forming Schwann cells, Consistently, we found reduced phosphorylation of ERK1/2 in identified by myelin-associated glycoprotein (MAG), in both injured sciatic nerves of GFAP-null mice. Vimentin, instead, mutants and controls (compare Fig. 2D-F with 2J-L). Nestin associates with integrin 51 and fibronectin, and regulates the was similarly expressed in myelin-forming and non-myelin subsequent steps of Schwann cell proliferation and nerve forming Schwann cells in both GFAP-null and wild-type regeneration. controls (data not shown). To test whether the absence of GFAP alters expression of the other intermediate filaments Results quantitatively, we performed western blot analysis. Western Loss of GFAP does not impair peripheral nerve blots of total nerve lysate did not show significant differences development and function in the amount of vimentin and nestin in nerves of GFAP-null The effects of GFAP deletion have been previously analyzed mice compared with controls (Fig. 2M). in the CNS (reviewed in Messing and Brenner, 2003; Privat, Manipulation of the cytoskeleton might alter outside-in and 2003), whereas consequences on the peripheral nervous system inside-out signaling as shown in the CNS of GFAP-vimentin have not been investigated in detail. To this aim, we analyzed double mutants (Menet et al., 2001). We therefore investigated peripheral nerve development and function in GFAP null mice. the expression of surface receptors and ECM molecules Journal of Cell Science GFAP and nerve cell regeneration 3983 Fig. 1. Morphological and functional analysis of the sciatic nerve in GFAP-null mice. (A,B) Staining for GFAP in nerves of wild-type and GFAP- null sciatic mice; GFAP is absent in nerves of the mutant. Ultra-thin (C,D) and semi-thin (E-J) section analysis of sciatic nerves from control and GFAP- null mice at 1, 7, 14 and 60 days after birth. No significant differences were observed. (K) Ultra-thin sections of the sciatic nerve from 2-months old GFAP-null mice. Both myelin- forming and non-myelin forming Schwann cells showed normal features. (L) Myelin- fiber density in adult sciatic nerve from control and GFAP- null mice. No significant differences were observed. (M) Morphometry of myelinated axons in adult sciatic nerve from control and mutant mice. No significant differences were observed per number and size distribution. (N) Rotarod test analysis and (O) electrophysiological analysis performed in mutant and control mice of 2 months of age. No significant differences were observed. Bar in J, 30 m for A,B; 8 m for C,D; 20 m for E-J; 5 m for K. previously described in the peripheral nerve (Previtali et al., contribution and is associated with upregulation of 2001; Previtali et al., 2003b). By immunohistochemistry, we intermediate filaments in Schwann cells, we examined nerve did not observe differences in the expression of integrins (1, regeneration in GFAP-null mice. Sciatic nerves from GFAP- 2, 3, 6, 7, 1, 4), -dystroglycan, L1 and NCAM in null mice and age-matched controls were crushed and Schwann cells of GFAP-null mice, or of collagen IV and examined 3 mm and 10 mm distal to the site of injury 3, 7, 10, laminin chains 2, 4, 1 and 1 in the endoneurium. 15, 21 and 45 days after injury. Results showed a clear delay Similarly, we did not observe differences in the expression of in nerve regeneration and remyelination in GFAP-null mice. collagen IV, fibronectin, vitronectin and laminin chains 1, 5, Data for both distances were concordant. 2, 1 and 3 in the perineurium and blood vessels. Only Three days after injury both wild-type and GFAP-null mice fibronectin showed higher expression in the endoneurium of showed diffuse signs of degeneration, including myelinolysis, GFAP-null mice compared with controls (compare Fig. 2N,O myelin debris and axonal fragmentation (data not shown). with 2P,Q, and data not shown). Western blot confirmed the Seven and ten days after injury, in wild-type nerves, we increased amount of fibronectin, whereas other ECM observed some features of axonal degeneration, some components, such as collagen IV or laminins were expressed macrophages and some bands of Bungner. However, the at similar levels (Fig. 2R). prominent aspect was the presence of many regrowing axons, some organized in clusters of regeneration, some arranged in Regeneration of sciatic nerve after injury is impaired in a 1:1 ratio with Schwann cells, and few ensheathed with thin GFAP-null mice myelin, indicating remyelination (Fig. 3A,A ). By contrast, in Since efficient nerve regeneration requires Schwann cells nerves of GFAP-null mice we observed diffuse signs of Journal of Cell Science 3984 Journal of Cell Science 119 (19) Fig. 2. Expression of intermediate filaments and ECM constituents in the sciatic nerve of GFAP-null mice. (A-C) Double staining for L1 and vimentin (merge in C) shows vimentin expression in non-myelin-forming Schwann cells. (D-F) Double-staining for MAG and vimentin (merge in F) shows vimentin expression in myelin forming Schwann cells. (G) Western blot analysis shows comparable levels of nestin and vimentin in mutants compared with wild type; -tubulin was used to normalize the samples. (H,I) Fibronectin expression in sciatic nerves of wild-type and GFAP-null mice; an increased amount of fibronectin was observed in the endoneurium in mutants. (J,K) Collagen IV expression in sciatic nerves of wild-type and GFAP-null mice. Mutants and controls showed comparable levels of collagen IV. (L) Western blot analysis confirmed an increased amount of fibronectin in the sciatic nerve of GFAP-null mice compared with controls, whereas collagen IV and laminins were present at equal amounts. SDS-PAGE gels were 7.5% except when analyzing laminins (5%). Bar in L, 60 m for A-C; 20 m for D-F and H- K. ongoing axonal degeneration, invading macrophages and 30% less fibers than controls (P=0.05 at 3 mm and P<0.01 at several Schwann cells arranged into Bungner bands (Fig. 10 mm; Fig. 3G,H). Fiber diameter distribution confirmed a I II 3B,B ,B ). Few clusters of regeneration and fibers in a 1:1 ratio reduced number of fibers at each diameter in nerves of GFAP- and, rarely, thinly myelinated fibers were observed (Fig. null mice, especially for those >3 m (Fig. 3K,L). I II 3B ,B ). To quantify differences, we performed morphometric Forty-five days after injury, a morphologically normal analysis counting all the fibers with a diameter >1 m (fibers situation was essentially achieved in control nerves, whereas that were expected to undergo myelination). The average GFAP-null mice still showed signs of ongoing regeneration number of axons in nerves of GFAP-null mice was significantly (Fig. 3E,F). Morphometric analysis confirmed that nerves of reduced compared with those of wild type, by 33% at 3 mm mutant mice contained roughly 20% less fibers than controls and 53% at 10 mm to the site of injury (P<0.05 and P<0.01 (P<0.05; Fig. 3G,H and 3M,N). We also detected thickening respectively; Fig. 3G,H). Interestingly, we found a higher of the myelin sheath – represented as the g-ratio – which was percentage of myelinated vs non-myelinated fibers (59% vs significantly higher in nerves of GFAP-null mice compared 41%) in controls, whereas this relationship was the opposite in with those of control animals (0.73±0.08 vs 0.69±0.09). nerves of GFAP-null mice (25% myelinated vs 75% non- To confirm delayed nerve regeneration in nerves of GFAP- myelinated fibers). Moreover, fiber-diameter distribution null mice, we performed two functional tests: motor-neuron confirmed a reduced number of fibers in nerves of GFAP-null retrograde labeling using fluorochrome-conjugated cholera mice at almost any diameter (Fig. 3I,J). toxin subunit B and neurophysiological analysis. A preliminary Fifteen and 21 days after injury, nerves of both wild-type trial was performed in control mice to identify the first time and GFAP-null mice showed increased signs of regeneration point after sciatic crush injury at which fluorescent cholera (Fig. 3C,D, and data not shown). However, signs of toxin injected in gastrocnemius was detected in motor neurons. degeneration and invading macrophages were preeminent in In control mice, motor neurons were labeled by fluorescent mutant nerves. The overall number of fibers was significantly cholera toxin when injected 12 days after injury and sacrificed reduced in nerves of GFAP-null mice compared with those of 48 hours later, at day 14. We therefore analyzed at this time wild type. Twenty days after injury, mutant nerves showed 11- point three GFAP-null mice and 3 age-matched controls. We Journal of Cell Science GFAP and nerve cell regeneration 3985 Fig. 3. Delayed nerve regeneration in the sciatic nerves of GFAP-null mice revealed by morphologic and morphometric analysis. (A-F) Light- microscopy images of injured nerves of GFAP-null mice, 3 mm distal to the lesion, compared with age-matched controls at different time 1 I II points. Electron microscopy images of t10 (ten days after crushing) nerve samples are shown in (A ) for control and (B ,B ) for mutant mice. Ten days after crushing, nerves of control mice showed several fibers in 1:1 ratio as well as thinly myelinated fibers (A,A ), whereas in nerves I II of mutant mice Schwann cells prevailed that were still sorting axons (B,B ) and bands of Bungner (B ). Twenty-one days after crushing, maturation of nerves in control mice was evident (C) whereas nerves of mutant mice still showed several degenerating fibers and clusters of regeneration (D). Forty-five days after crush, nerves of controls were nearly normal (E) whereas nerves of GFAP-null mice contained degenerating and thinly myelinated fibers (F). (G-N) Morphometric analysis of regenerating nerves at different time points, comparing data obtained 3 mm and 10 mm distally to the site of injury. (G,H) Diagram of the total number of fibers at 10, 21 and 45 days after injury; nerves of GFAP-null mice always showed significantly reduced number of fibers. (J-N) Diagram of the regenerating nerves at different time points subdivided per fiber diameter; results show reduced number of regenerating fibers in nerves of mutant mice, primarily those with larger I I II diameter. Error bars represent the +s.e.m. Bar in F, 10 m for A-F; 6 m for A ; 3 m for B ,B . *P<0.05. observed in GFAP-null mice that only 6% of motor neurons (5.6±2 vs 10.4±2 mV; P=not significant) and NCVs 20% were labeled by the fluorescent dye, whereas in control mice slower (16±1 vs 20±1 m/second; P=not significant) although it was 20% (P<0.001; Fig. 4A). differences were not significant. Consistent with the above results, GFAP-null mice showed neurophysiological signs of delayed regeneration (Fig. 4B). Loss of GFAP does not affect Schwann cell Ten days after crushing, we did not detect any signal in either dedifferentiation and cytoskeleton rearrangement GFAP-null or control mice, in agreement with motor neuron To obtain efficient nerve regeneration after damage, Schwann dye labeling, which showed target-muscle innervation only cells have to dedifferentiate, proliferate, and provide a after day 12. Fifteen days after injury, amplitudes of distal favorable environment for axonal regrowth. A defect in one of cMAP in GFAP-null mice were 40% of those in control mice these Schwann cell functions that implies the continuous (0.8±0.4 vs 1.4±0.1 mV; P=0.02), whereas NCVs were similar rearrangement of the cytoskeleton may explain the delay of (9.5±1 vs 10±2 m/second). At 21 days after crushing, regeneration in GFAP mutants. amplitudes in GFAP-null mice were still 50% of those in First, we evaluated whether GFAP-null Schwann cells controls (0.9±0.1 vs 1.9±0.3 mV; P=0.01) with similar NCVs normally dedifferentiate and modulate the cytoskeleton in (11.4±1 vs 12.5±2 m/second). Finally, 45 days after injury, response to crush injury. After damage Schwann cell GFAP-null mice showed amplitudes 40% of those in controls dedifferentiation results in the downregulation of myelin genes Journal of Cell Science 3986 Journal of Cell Science 119 (19) and the re-expression of genes such as those encoding NCAM, NTR L1-molecule and p75 , along with the upregulation of GFAP and vimentin (Jessen et al., 1990; Martini, 1994; Neuberger and Cornbrooks, 1989). In GFAP-null mice, by 3 days after damage, we observed that mutant Schwann cells regularly NTR dedifferentiated and expressed NCAM, L1 p75 and vimentin (compare Fig. 5A,C,E,G with 5B,D,F,H respectively). Then we evaluated whether vimentin was adequately upregulated in the sciatic nerve of GFAP null mice. We performed western blot analysis of the distal stump of the sciatic nerve and measured the amount of vimentin at 3, 6, 15, 21 and 45 days after injury in GFAP-null and control mice. Results showed that GFAP-null mice upregulate vimentin in injured nerves similarly to controls (Fig. 5I,J). Hence, defective regeneration was not the consequence of impaired Schwann cell dedifferentiation or insufficient vimentin upregulation. Delayed nerve regeneration is probably due to reduced Schwann cell proliferation in GFAP-null mice Since Schwann cell proliferation is a crucial step to initiate and obtain efficient nerve regeneration (Chen et al., 2005), and GFAP has been associated with cell mitosis (Yasui et al., 1998), we then focused our attention on Schwann cell proliferation. Previous reports showed that Schwann cells mainly proliferate in the first week after nerve damage, with a peak at day 3 (Clemence et al., 1989; Oaklander and Spencer, 1988; Pellegrino et al., 1986). Therefore, we performed crush injury of the sciatic nerve of six GFAP-null and six wild-type littermates. Three and 6 days after damage the animals were pulsed with BrdU to label cells in the DNA-synthesis phase of the cell cycle and then killed to perform BrdU staining. DAPI- Fig. 5. Vimentin upregulation is maintained in nerves of GFAP-null mice after sciatic nerve injury. (A-H) Longitudinal sections of 3- day-old injured sciatic nerves from wild-type (A,C,E,G) and GFAP- null mice (B,D,F,H) double-stained for vimentin and neurofilaments (A,B), NCAM and neurofilaments (C,D), L1 and neurofilaments (E,F), p75NTR and neurofilaments (G,H). DAPI staining of nuclei Fig. 4. Delayed nerve regeneration in nerves of GFAP-null mice (blue). The dedifferentiated Schwann cells showed expression of the measured by neurophysiology and motor neuron retrograde labeling above molecules as in wild type. Bar, 30 m. (I,J) Protein extracts with GFP-conjugated cholera toxin subunit B. (A) The total number from the distal stump of crushed nerves from wild-type (I) and of labeled motor neurons in the lumbar enlargement 48 hours after GFAP-null mice (J) at different time points were immunoblotted injection of fluorescent cholera toxin subunit B in the gastrocnemius with an anti-vimentin antibody. Sample loading was normalized is significantly reduced in GFAP-null mice compared with control against -tubulin. Ratio of vimentin to -tubulin was measured by mice. (B) Distal cMAP recordered in nerves of GFAP-null mice densitometry and expressed in the bottom line as times of increase always showed amplitudes of half-values compared with controls, at each time point T (in days) relative to T zero. Compared with whereas NCVs did not show significant differences; *P<0.05. Bar, control mice, vimentin is similarly upregulated in nerves of GFAP- 50 m. null mice. Journal of Cell Science GFAP and nerve cell regeneration 3987 positive and S100-positive Schwann cell nuclei (DAPI and Schwann cell proliferation in the nerves of mutant mice both S100 ) labeled with BrdU were counted in the distal stump of 3 days (5.5±1.2% vs 2.6±0.7%, P<0.001) and 6 days the sciatic nerve. Results showed a significant reduction in (3.5±0.8% vs 2.1±1.0%, P=0.04) after injury (Fig. 6F-J). Schwann cell proliferation in GFAP-null mice at day 3 However, we did not observe significant differences in (8.3±0.5% vs 12.2±1.4%, P=0.04) and an almost significant Schwann cell apoptosis at days 3 and 6 (Fig. 6K-O). reduction at day 6 (6.9±1.0% vs 9.7±1.0%, P=0.06) (Fig. 6A- E). To confirm these data, we performed similar double GFAP and vimentin constitute different target molecules staining for phosphorylated histone H3 with an antibody that for integrin receptors involved in Schwann cell recognizes only proliferating cells in the mitotic phase of proliferation and nerve regeneration the cell cycle. Results confirmed a significant reduction of Schwann cell proliferation and migration after nerve damage Fig. 6. Schwann cells in the distal stump of nerves of GFAP-null mice after injury show reduced proliferation but normal apoptosis. (A-D) Nuclei staining with DAPI (blue) and BrdU (green) on longitudinal sections of the distal stump of the sciatic nerve at 3 and 6 days (t3 and t6, respectively) after injury. The number of BrdU-positive nuclei is reduced at both t3 and t6 in nerves of mutant mice. (E) Quantitative analysis shows that the percentage of BrdU-positive nuclei is significantly decreased at t3 (*P=0.04) and consistently but not significantly reduced at t6 (P=0.06). (F-I) Staining of nuclei with DAPI (blue) and of phosphorylated histone H3 (green) on longitudinal sections of the distal stump of the sciatic nerve at days 3 and 6 after injury. The number of nuclei positive for phosphorylated histone H3 is reduced at both t3 and t6 in nerves GFAP-null mice. (J) Quantitative analysis shows that the percentage of phosphorylated histone H3 nuclei is significantly reduced at both t3 (**P<0.001) and t6 (*P=0.04). (K-N). S100 (green) and TUNEL (red) staining on longitudinal sections of the distal stump of the sciatic nerve at t3 and t6 after crushing. TUNEL staining shows a similar number of positive nuclei in mutant and control nerves at both time points. (O) Quantitative analysis shows no significant difference in the percentage of positive nuclei in mutants and controls. Error bars represent the ±s.e.m. Bar in N , 40 m for A-D; 80 m for F-I and K-N. Journal of Cell Science 3988 Journal of Cell Science 119 (19) is induced and modulated by complex interactions of ECM or vimentin. The immunoprecipitates were analyzed by receptors and ECM molecules. They include the ECM western blotting using antibodies against integrin v, 5, 1 molecules normally expressed in the endoneurium and those and 8. Two different anti-GFAP antibodies (mouse and rat) molecules that infiltrate the nerve as the result of blood-nerve but not anti-vimentin co-precipitated integrin subunits v and barrier disruption, such as fibrin. Disruption of this signaling 8 (Fig. 7A). By contrast, two different anti-vimentin pathway interferes with Schwann cell proliferation. antibodies (rabbit and mouse) but not anti-GFAP co- We investigated whether known adhesion complexes precipitated integrin subunits 5 and 1 (Fig. 7A). To confirm involved in Schwann cell proliferation can interact with GFAP that vimentin cannot associate with v integrin in the absence or vimentin. Although Schwann cells express several ECM of GFAP, we performed further experiments with nerves of receptors during development and adult life (Milner et al., mutant and control mice. In the homogenate of nerves from 1997; Previtali et al., 2003b), two adhesion pathways have been GFAP-null mice, the anti-vimentin antibody did again not proposed to function after nerve injury. First, fibrin interacts precipitate v integrin, whereas v integrin was with integrin v8, which signals the occurred damage and immunoprecipitated by GFAP in nerve homogenates of control stimulate Schwann cell proliferation (Akassoglou et al., 2003; mice (Fig. 7B). Akassoglou et al., 2002; Chernousov and Carey, 2003). Then, It has been reported that the fibrin–v8-integrin pathway the reorganization of the ECM allows fibronectin-integrin modulates Schwann cell proliferation by phosphorylating 51 interaction that carries on signaling for Schwann cell ERK1/2 [p44/42 MAP kinase (MAPK)] (Akassoglou et al., proliferation and differentiation to complete regeneration 2002). We first verified that ERK1/2 phosphorylation is the (Chernousov and Carey, 2003; Haack and Hynes, 2001; cause and not the consequence for reduced Schwann cell Lefcort et al., 1992). We therefore investigated whether GFAP proliferation. The sciatic nerves of ten 3-months old mice were and/or vimentin participate in the fibrin-v8 and/or crushed. Five mice were treated with the MAP kinase kinase fibronectin-51 complex. The homogenate of rat sciatic (MEK) inhibitor PD098059, which blocks ERK1/2 nerve was immunoprecipitated by using antisera against GFAP phosphorylation and compared to the other five mice treated Fig. 7. Characterization of integrin binding by GFAP and vimentin in the peripheral nerve, and of ERK1/2 phosphorylation. (A) Rat sciatic nerve lysate was immunoprecipitated with anti-GFAP or anti- vimentin antibody. The immunoprecipitated proteins were separated in SDS-PAGE (7.5%) under reducing conditions and blotted with antibodies against integrin subunits v, 8, 5 and 1. GFAP co- precipitated with the integrin subunits v and 8 but not 1, whereas vimentin co-precipitated with the integrin subunits 5 and 1 but not v. (B) Sciatic nerve lysate of GFAP-null mice was immunoprecipitated as described above with anti-vimentin antibody and blotted with anti-integrin v antibody; similarly the wild-type sciatic nerve lysate was immunoprecipitated with anti-GFAP antibody and blotted with anti-integrin v antibody. Vimentin still did not co-precipitate with integrin v in GFAP-null mice lysate, whereas integrin v again co-precipitated with GFAP in lysate of control-mice nerves. (C) Protein extracts form the distal stamp of wild-type mice treated with DMSO and wild-type mice treated with the MEK inhibitor PD098059 at 3 days (T3) after injury were immunoblotted with antibody against total ERK1/2 or phosphorylated ERK1/2. By densitometry the ratio of totalERK1/2 to pERK1/2 was measured and is stated below the blot as a number, indicates the phosphorylation state. Mice treated with PD098059 showed reduced ERK phosphorylation (D) Protein extracts from the distal stump of crushed nerves from wild-type and GFAP-null mice at T3 and T6 were immunoblotted with antibody against total ERK1/2 or phosphorylated ERK1/2. By densitometry, the ratio of totalERK1/2 to pERK1/2 was measured as described above. Nerves of GFAP-null mice showed less phosphorylated Erk1/2 compared with controls at both time points after injury. Journal of Cell Science GFAP and nerve cell regeneration 3989 with the vehicle (DMSO 10%). At 3 days post-injury, BrdU mature nerves, only non-myelin-forming Schwann cells and labeling of S100-positive cells showed Schwann cell Schwann cells that dedifferentiate after nerve injury express proliferation reduced by 50% in mice treated with the GFAP (Jessen et al., 1990). Hence, most of the time developing PD098059 (9.5%±1.4 vs 4.5%±1.5, P=0.0003; data not Schwann cells do not express GFAP, whereas its expression is shown). In the same animals, the homogenate of the restricted to a short, temporary window. The role of GFAP, crushed controlateral nerve confirmed reduced ERK1/2 being mostly unknown and related to the cytoskeleton phosphorylation by 25% (Fig. 7C) organization, may be therefore skipped and seems insignificant Since we were able to show that GFAP is part of the in the developing Schwann cells. fibrin–v8-integrin pathway, we investigated whether As to the second point, redundancy of and/or compensation defective Schwann cell proliferation in GFAP-null mice was by other intermediate filaments may mask GFAP deficiency in associated with reduced ERK1/2 phosphorylation. The Schwann cell development. Schwann cells express two other homogenates of the distal stump of T3 and T6 crushed nerve intermediate filaments, nestin and vimentin, during their from GFAP-null and wild-type controls were compared by embryonic development, and vimentin is maintained at high using an antibody specific for the phosphorylated form of levels also in mature Schwann cells (Dong et al., 1999; ERK1/2. At both time points, the sciatic nerves of GFAP-null Jessen and Mirsky, 1991). We confirmed by qualitative and mice showed a reduction of ERK1/2 phosphorylation, by 27% quantitative analyses that vimentin and nestin are expressed at at T3 and 48% at T6 compared with wild-type controls (Fig. similar amounts in mature GFAP-null Schwann cells compared 7D). The levels of total ERK1/2 were similar in GFAP-null with controls. Hence, redundancy might explain the absence of mice and controls. Hence, the difference in ERK1/2 phenotype in the peripheral nerve of GFAP-null mice. Finally, phosphorylation was not the consequence of different amounts mutant mice showed an increased amount of fibronectin in the of total ERK1/2. endoneurium. Fibronectin is a potent promoter of peripheral neurite outgrowth both during development and regeneration Discussion (Lefcort et al., 1992). The increased expression of fibronectin This study shows that GFAP modulates the Schwann cell might further favor nerve development in mutant mice response for tissue recovery after peripheral nerve injury. We independently by the presence of GFAP. provide evidence for the first time that, (1) GFAP is involved in Schwann cell proliferation, (2) GFAP is the cytoskeleton Loss of GFAP affects early Schwann cell proliferation component of the previously identified pathway originated by thus causing delayed nerve regeneration fibrin that drives Schwann cell proliferation after damage, (3) Although PNS function and development appeared normal in GFAP and vimentin constitute two different pathways that link the absence of GFAP, nerve regeneration was delayed in GFAP the Schwann cell cytoskeleton to the ECM, both of which mutants. In fact, the demand for intermediate filaments is involved in proliferation and differentiation. Finally, our study highly increased in Schwann cell after damage. Both GFAP shows that GFAP is not necessary for the development of and vimentin are upregulated, perhaps to provide an efficient the peripheral nerve, probably compensated for by other cytoskeleton rearrangement necessary for proliferation and intermediate filaments. Disruption or defective function of the differentiation (Gillen et al., 1995; Neuberger and Cornbrooks, GFAP pathway may therefore interfere with the regenerative 1989; Thomson et al., 1993). Thus, compensatory mechanisms capacity of the peripheral nerve that, in chronic conditions, might no longer be sufficient for the Schwann cell to support might determine severe degenerative defects and axonal loss. the loss of GFAP in an acute crisis, such as after injury. Accordingly, nerve regeneration in vivo and neurite outgrowth Vimentin and nestin probably compensate for the in vitro was described as being delayed in vimentin-null mice absence of GFAP during PNS development (Perlson et al., 2005). Whether delayed regeneration in It was shown previously by gene targeting inactivation that vimentin-null mice depends exclusively on impaired retrograde GFAP is not overtly required for normal mouse CNS transport of the perk–vimentin–dynein–importin- complex in development (Gomi et al., 1995; Liedtke et al., 1996; McCall the damaged axons, or is the consequence of impaired function et al., 1996; Pekny et al., 1995). Although these reports were of vimentin-null Schwann cells, needs further investigation. focused on the CNS, we confirmed here that also the PNS The observation that GFAP-null mice had a delay in nerve develops normally. We investigated nerve development from regeneration might be due to different scenarios: (1) defective birth to adulthood, and observed in GFAP-null mice normal Schwann cell dedifferentiation, (2) impaired proliferation, (3) timing of Schwann-cell–axon interactions, normal cyto- defective organization into bands of Bungner or, (4) impaired architecture of both myelin-forming and non-myelin-forming interaction between Schwann cells and regrowing axons. The Schwann cells, and regular fiber-type distribution. Peripheral regular upregulation of vimentin and the coherent expression nerves also showed normal function in neurophysiological and of markers of dedifferentiation, such as vimentin, L1, NCAM NTR functional tests. The lack of an evident phenotype might be the and p75 , in GFAP-null Schwann cells after damage consequence of at least two events: (1) GFAP has no main role suggested a normal dedifferentiation process. Moreover, in Schwann cell development and nerve function and/or, (2) GFAP-null Schwann cells could organize Bungner bands, other molecules are redundant or can compensate for loss of which appeared morphologically normal – although only with GFAP. a delay. Finally, we observed – although not in detail – normal Regarding the first point, GFAP appears at a relative late Schwann cell-axon interaction by morphological analysis and stage in Schwann cell development, basically when immature expression of L1/NCAM/NF markers. Schwann cells have formed already, and it is downregulated in Our results provided evidence that defective regeneration those Schwann cells that form myelin (Jessen et al., 1990). In is probably the consequence of reduced Schwann cell Journal of Cell Science 3990 Journal of Cell Science 119 (19) proliferation after damage. Soon after nerve injury, with cytoskeleton constituents including intermediate filaments dedifferentiated Schwann cells enter the cell cycle to provide (Herrmann and Aebi, 2000; Kreis et al., 2005; Rutka et al., a sufficient substrate for nerve regrowth. Previous studies 1997). Intermediate filaments participating in these cross- showed that Schwann cells highly proliferate in the first week bridges probably constitute a link between cell surface and post injury, with a peak at day 3 (Clemence et al., 1989; nucleus (Maniotis et al., 1997). This interaction provides a Oaklander and Spencer, 1988; Pellegrino et al., 1986). Our structural framework to facilitate intracellular responses, 2+ results of BrdU (labeling cells in G1-S-M phase) and histone including protein phosphorylation, intracellular pH and Ca H3 (labeling cells in M phase) analyses in nerves of control modification, and the activation of MAP kinase cascades mice confirmed data previously reported on Schwann cell (Turner, 2000). These signaling events culminate in the proliferation after injury, and showed a significant reduction in reorganization of the cytoskeleton necessary for motility, the number of mitotic Schwann cells in GFAP mutants. proliferation and gene expression. Reduced proliferation was statistically significant at days 3 and Our results suggested that the interaction of intermediate 6 for histone H3 and at day 3 for BrdU, and very close to filaments and integrins was specific and segregated. GFAP significance at day 6 for BrdU (P=0.06). Apoptosis was not associated with integrin v8 but not integrin 51, whereas modified by the absence of GFAP, suggesting a defect in vimentin associated with integrin 51 but not integrin v8, proliferation and not in cell survival. Accordingly we found a thus also excluding non-specific binding. However, we do not reduction in ERK1/2 phosphorylation in nerves of mutant know whether this is a direct interaction or whether it is mice. Activation of the MAP-kinase pathway regulates mediated by docking molecules. In the absence of GFAP, transcription of genes associated with proliferation and vimentin still associates with integrin 51 and not with differentiation in several cell types (Cowley et al., 1994; Kotch, integrin v8, therefore explaining why vimentin can not 2000), including Schwann cells (Akassoglou et al., 2002). As compensate for the absence of GFAP in transducing the confirmed by our results with the ERK inhibitor, the MAP- fibrin–integrin-v8 signaling that is initiated early after kinase pathway is at least one of the pathways that regulate damage. The Fibrin–integrin-v8–GFAP complex probably Schwann cell proliferation after injury. In fact, we found that drives Schwann cell proliferation only in close relation to nerve Schwann cell proliferation and ERK1/2 phosphorylation was damage, whereas other pathways modulate Schwann cell reduced but not abolished in nerves of mutant mice. Several mitosis in other situations. For example, in embryogenesis receptors and molecular pathways sustain proliferation. We integrin 51 and fibronectin control the proliferation of probably interfered with only one of these pathways, which Schwann cell progenitors, which express vimentin and not requires fibrin deposition and integrin v8 activation, and is GFAP (Haack and Hynes, 2001; Lefcort et al., 1992; Peters and specifically active in the first steps of nerve regeneration Hynes, 1996). Similarly, in the advanced phase of nerve (Akassoglou et al., 2003; Akassoglou et al., 2002). regeneration Schwann cell proliferation is controlled by A potential role for GFAP in cell proliferation has also been fibronectin and integrin 51 (Akassoglou et al., 2003; suggested previously (Yasui et al., 1998; Kawajiri et al., 2003). Akassoglou et al., 2002). Our results would fit with these Cytoskeleton reorganization and cytoplasm segregation is a previous observations. After nerve damage, the immediate crucial step in cell proliferation and division. In particular, repair reaction is sustained by the temporary ECM matrix several intermediate filaments are phosphorylated during formed by blood-derived fibrin, which immediately activates mitosis and continuously shift from an assembled Schwann cell proliferation via the integrin-v8–GFAP (filamentous) to a disassembled (soluble) state. Rho-kinases pathway. The absence of GFAP in our mutants would therefore phosphorylate GFAP, thereby causing their disassembly to block this early signal for Schwann cell proliferation. Later on, accomplish cytokinesis (Yasui et al., 1998). Similarly, Aurora- a mature ECM-scar is formed by fibronectin, which substitutes B kinase, required for chromosome segregation and mitosis, fibrin to sustain Schwann cell proliferation and initiates tasks was recently reported to phosphorylate a number of for differentiation. The presence of different pathways that intermediate filaments including GFAP (Kawajiri et al., 2003). regulate Schwann cell proliferation between regeneration and Our findings suggest that the role of GFAP in proliferation is development is not surprising. For example, cyclin D1 cell-specific or, more likely, specific to the cell environment regulates Schwann cell proliferation in nerve regeneration but and stimuli. Increased astrocyte proliferation and GFAP not in development (Atanasoski et al., 2001; Kim et al., 2000). overexpression is also observed in reactive astrocytosis in Finally, although the MAP-kinase pathway that drives cell CNS. However, when GFAP is deleted reactive astrocytosis is proliferation might be directly initiated by integrins (Aplin et not impaired and BrdU-pulse investigation failed to al., 2001), this pathway is more likely the consequence of demonstrate proliferative abnormalities in GFAP mutant collaborative signaling, in which integrin-mediated events are astrocytes (Pekny et al., 1999). initiated by other types of receptors, primarily tyrosine-kinase growth-factor receptors (Assoian and Schwartz, 2001; Howe et GFAP and vimentin link ECM to the Schwann cell al., 1998). For example, ErbB receptors and TGF- have been cytoskeleton via two distinct pathways already associated with Schwann cell proliferation in vivo and We found that GFAP and vimentin bind to integrin v8 and in vitro, and in different steps of nerve development (reviewed integrin 51, respectively, two ECM-receptors involved in in Mirsky and Jessen, 2005). It is tempting to speculate that Schwann cell proliferation and nerve regeneration (Akassoglou an extracellular fibrin (or fibronectin) network helps to et al., 2003; Akassoglou et al., 2002; Chernousov and Carey, accumulate integrin receptors in a restricted area of the 2003; Haack and Hynes, 2001; Lefcort et al., 1992). This Schwann cell surface, whereas intracellularly the GFAP (or was not a surprise. As transmembrane receptors, integrins vimentin) cytoskeleton helps to cluster the tyrosine-kinase physically interact, directly or through cytolinker molecules, receptors to initiate the MAP-kinase signaling pathway. Journal of Cell Science GFAP and nerve cell regeneration 3991 Can defective GFAP explain impaired regeneration in rod was measured in subsequent trials (four trials in the first 2 days and one trial on each of 3 consecutive days). peripheral neuropathy? Overall, our data showed that GFAP-null mice have delayed Neurophysiological analysis nerve regrowth and functional recovery after injury. Five 3-month-old GFAP-null mice and five 3-month-old control littermates were Impairment in nerve regrowth was more evident in the first 2 analyzed, as described (Bolino et al., 2004), before crush injury and 10, 15, 21 and 45 days after crush injury. Mice were anesthetized with avertin and placed under a weeks after damage, probably due to a reduced capacity of heating lamp to avoid hypothermia. The sciatic nerve conduction velocity (NCV) Schwann cells to proliferate and therefore to organize the was obtained by stimulating the nerve with steel monopolar needle electrodes. A regenerative scar. However, morphological and functional pair of stimulating electrodes was inserted subcutaneously near the nerve at the ankle. A second pair of electrodes was placed at the sciatic notch, to obtain two differences were still present 45 days after injury, at a time distinct sites of stimulation, proximal and distal along the nerve. The muscular when repair is almost complete in control mice. The mutant response to the electrical nerve stimulation, compound motor action potentials mice showed reduced numbers of regrowing axons at all (cMAP), was recorded with a pair of needle electrodes; the active electrode was inserted in muscles in the middle of the paw, while the reference was placed in the times and at 45 days still 20% of fibers were missing. skin between the first and second digit. Moreover, the difference in regrowth was more significant for larger axons. Antibodies and Immunohistochemistry Our data sustain that nerve repair is partially impaired but Antibodies used for immunohistochemistry and/or western blotting are listed in Table 1. Immunofluorescence on cryosections was performed as described (Previtali not abolished in GFAP mutants, whereas nerve development et al., 2003b), and examined with confocal (Biorad MRC 1024) or fluorescent and function is not affected. This raises the possibility that microscope (Olympus BX). GFAP mutations may affect the peripheral nerve. From this point of view, we may envisage three scenarios. (1) Gfap Inhibitor of ERK1/2 phosphorylation The MAP kinase kinase (MEK) inhibitor PD098059 (Sigma) was dissolved in mutations give rise to peripheral neuropathy. These are dimethyl sulfoxide (DMSO) and stored in aliquots at –80° C. The compound was probably not loss-of-function but gain-of-function mutations, diluted in saline (NaCl) immediately before use to a final concentration of 1 mg/kg. because GFAP-null mice did not show peripheral neuropathy. Gain-of-function mutations in human are responsible for a Table 1. List of the antibodies severe leukodystrophy, Alexander disease (Messing and Goldman, 2004). The severity of Alexander disease might Antigen Species Clone Source mask a more modest peripheral neuropathy, in a way similar BrdU Mouse BMC9318 Roche to what occurred with the neuropathy associated with Collagen IV Rabbit Chemicon -Dystroglycan Rabbit AP83 K. Campbell congenital muscular dystrophy in mutations in the laminin 2 ERK1/2 Rabbit Cell Signaling gene (LAMA2) (Shorer et al., 1995). (2) GFAP dysfunction is pERK1/2 Mouse E10 Cell Signaling not the consequence of genomic mutation but due to post- Fibronectin Rabbit Chemicon translational defects, i.e. phosphorylation or glycosylation. GFAP Mouse GA5 Chemicon GFAP Rat 2.2B10 Zymed Something similar has been described for defective pHistone H3 Rabbit Ser10 Upstate dystroglycan glycosylation. Mutations in genes that encode Integrin 1 Rabbit Chemicon proteins that glycosylate the -dystroglycan cause congenital Integrin 2 Rabbit Chemicon muscular dystrophies in human and/or neuropathy in mice Integrin 3 Rabbit Chemicon (Levedakou et al., 2005; Muntoni et al., 2004). (3) GFAP Integrin 5 Rabbit Chemicon Integrin 6 Rat GoH3 A. Sonnenberg mutations do not cause a peripheral neuropathy, but reduce the Integrin v Rabbit Chemicon capacity of nerve repair in course of genetic or acquired Integrin 1 Rat Mb1.2 Chemicon neuropathies. In this case, the modest delay in regeneration we Integrin 1 Rabbit Chemicon saw in GFAP-mutant mice after a single pathogenetic event, Integrin 1 Mouse 2B1 Chemicon Integrin 4 Rabbit Chemicon may become more relevant by adding the delay in regeneration Integrin 8 Goat G17 Santa Cruz of several fibers in the presence of a prolonged and/or L1 Rat 324 Chemicon continuous damage. Laminin EHS Rabbit Sigma Laminin 1 Rabbit H300 Santa Cruz Laminin 1 Rat AL1 Chemicon Materials and Methods Laminin 2 Rat 4H8-2 Alexis Generation of GFAP-null mice Laminin 4 Rabbit H-194 Santa Cruz Generation of GFAP-null mice and characterization of their CNS has been described Laminin 5 Mouse 4C7 Chemicon before (McCall et al., 1996); mice have been subsequently made congenic on an Laminin 1 Rat LT3 Chemicon inbred C57BL/6 background. Animals were generated from our colony and genotyped by PCR analysis of genomic DNA from tail clips. All experiments were Laminin 2 Rabbit 1117+ R. Timpl performed following the institutional guidelines. Laminin 1 Rat A5 Chemicon Laminin 3 Rabbit H140 Santa Cruz Sciatic nerve crush-lesion MAG Mouse Chemicon Adult mice were anesthetized with avertin (trichloroethanol, 0.02 ml/g of body NCAM Rabbit Chemicon weight) and crush injury was performed as described (Quattrini et al., 1996). After Nestin Mouse ab6142 abcam skin incision, the sciatic nerve was exposed and crushed distal to the sciatic notch Neurofilament-HRabbit Chemicon for 20 seconds with fine forceps previously cooled in dry ice. To identify the site Neurofilament-M Mouse NN18 Chemicon of injury, forceps were previously dropped into vital carbon. The nerve was replaced p75NTR Rabbit Chemicon under the muscle and the incision sutured. S100 Mouse SH-b1 Sigma S100 Rabbit Chemicon Rotarod analysis -Tubulin Mouse Tub2.1 Sigma Five 3-month-old GFAP-null mice and five 3-month-old control littermates were Vimentin Rabbit Chemicon placed on a round metal bar, first rotating at four rotations per minute and then Vimentin Mouse LN-6 Sigma accelerating at 7.2 rpm (Ugo Basile, Como, Italy). The animals were allowed to stay Vitronectin Rabbit H270 Santa Cruz on the rod for a maximum of 700 seconds and the time they stayed on the rotating Journal of Cell Science 3992 Journal of Cell Science 119 (19) PD098059 (0.4 ml volume), or an equivalent volume of the vehicle (DMSO 10%), number GGP030193) and Ricerca finalizzata (AQ, grant number was injected i.p. 2 hours after nerve injury and every 12 hours until mice were killed RF2003/171). after 3 days for BrdU and western blotting experiments. Immunoprecipitation and immunoblotting References Akassoglou, K., Yu, W. M., Akpinar, P. and Strickland, S. (2002). Fibrin inhibits Proteins were isolated from snap-frozen sciatic nerves of adult mice as described (Previtali et al., 2003a; Previtali et al., 2000). For western blot nerves suspended in peripheral nerve remyelination by regulating Schwann cell differentiation. Neuron 33, 861-875. Tris-buffered SDS lysis buffer (95 mM NaCl, 25 mM Tris-HCl pH 7.4, 10 mM Akassoglou, K., Akpinar, P., Murray, S. and Strickland, S. (2003). Fibrin is a regulator EDTA, 2% SDS, protease or phosphatase inhibitors), sonicated and boiled. For of Schwann cell migration after sciatic nerve injury in mice. Neurosci. Lett. 338, 185- immunoprecipitation, nerves were suspended in Igepal (Sigma, Milano, Italy) lysis buffer plus protease inhibitors and sonicated. Immunoprecipitations were performed Aplin, A. E., Stewart, S. A., Assoian, R. K. and Juliano, R. L. (2001). Integrin-mediated with anti-GFAP (mouse or rat) and anti-vimentin (rabbit or mouse) for 3 hours at adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1. J. Cell 4°C. The immune complexes were collected by 90 minutes incubation with protein- Biol. 153, 273-282. A or -G agarose beads (Sigma). After washing, antigens were separated by heating Assoian, R. K. and Schwartz, M. A. (2001). Coordinate signaling by integrins and in reducing SDS sample buffer, and analyzed by SDS-polyacrilamide (PAGE) gel receptor tyrosine kinale in the regulation of G1 phase cell-cycle progression. Curr. (8.5%). For western blotting, equal amounts of homogenates (5 g) were diluted in Opin. Genet. Dev. 11, 48-53. 8M urea / 0.05M DTT, separated in sample buffer on 5 or 7.5% SDS-PAGE gel and Atanasoski, S., Shumas, S., Dickson, C., Scherer, S. S. and Suter, U. (2001). transferred to PVDF (Millipore, Roma, Italy) or nitrocellulose membrane (Biorad, Differential cyclin D1 requirements of proliferating schwann cells during development Segrate, Italy). Blots were blocked in PBS (0.05% Tween-5% dry milk) and and after injury. Mol. Cell. Neurosci. 18, 581-592. incubated with the appropriate primary + peroxidase-conjugated secondary antibody Bolino, A., Bolis, A., Previtali, S. C., Dina, G., Bussini, S., Dati, G., Amadio, S., Del (Sigma) and visualized by ECL (Amersham, Cologno M., Italy). The intensity of Carro, U., Mruk, D. D., Feltri, M. L. et al. (2004). Disruption of Mtmr2 produces the bands was quantified by densitometry and the ratio between each antibody and CMT4B1-like neuropathy with myelin outfolding and impaired spermatogenesis. J. -tubulin, or phosphorylated ERK1/2 on total ERK1/2, was determined. Cell Biol. 167, 711-721. Chen, Y. Y., McDonald, D., Cheng, C., Magnowski, B., Durand, J. and Zochodne, D. W. (2005). Axon and Schwann cell partnership during nerve regrowth. J. Neuropathol. Retrograde labeling of motor neurons Exp. Neurol. 64, 613-622. Five 3-months old GFAP-null mice and five age-matched controls were Chernousov, M. A. and Carey, D. J. (2003). alphaVbeta8 integrin is a Schwann cell anesthetized, the gastrocnemius muscle exposed, and 5 l of 1% cholera toxin receptor for fibrin. Exp. Cell Res. 291, 514-524. subunit B (Alexa Fluor-488 conjugate, Invitrogen, Burlingame, CA) in distilled Clemence, A., Mirsky, R. and Jessen, K. R. (1989). 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BrdU, phosphoryated histone H3 and TUNEL analysis (1999). Schwann cell development in embryonic mouse nerves. J. Neurosci. Res. 56, BrdU (Roche, Monza, Italy) incorporation was performed as described (Feltri et al., 334-348. 2002). We injected i.p. with 100 g BrdU per g body weight 4 and 2 hours before Feltri, M., Graus-Porta, D., Previtali, S., Nodari, A., Migliavacca, B., Cassetti, A., killing six mice (three GFAP-null mice and three wt mice) 3 days after injury and Littlewood-Evans, A., Reichardt, L., Messing, A., Quattrini, A. et al. (2002). a further six mice (three GFAP-null mice and three wt mice) 6 days after injury. Conditional disruption of beta1 integrin in Schwann cells impedes interactions with The sciatic nerve was then processed for immunohistochemistry. Longitudinal nerve axons. J. Cell Biol. 156, 199-209. cryosections were first incubated with anti-BrdU antibody and anti-S100 antibody, Gillen, C., Gleichmann, M., Spreyer, P. and Muller, H. W. (1995). then with secondary antibody, and nuclei were labeled with DAPI (Vector Differentially expressed genes after peripheral nerve injury. J. Neurosci. Res. 42, 159- Laboratories, San Giuliano Milanese, Italy). Only rod-shaped nuclei associated with nerves were counted and the fraction of BrdU-positive nuclei was determined. At Gomi, H., Yokoyama, T., Fujimoto, K., Ikeda, T., Katoh, A., Itoh, T. and Itohara, S. least 900 nuclei were examined. For histone H3 staining a further 6+6 animals were (1995). Mice devoid of the glial fibrillary acidic protein develop normally and are analyzed. Staining was performed similarly as described for BrdU. For terminal susceptible to scrapie prions. Neuron 14, 29-41. transferase dUTP nick-end-labeling (TUNEL) assay, the sciatic nerves were Griffin, J. W. and Hoffman, P. N. (1993). Degeneration and regeneration in the dissected, fixed 1 hour in 4% paraformaldehyde, and cryopreserved for Peripheral Neuropathy (ed. P. Dyck, P. Thomas, J. peripheral nervous system. In immunohistochemistry. Sections were treated with acetone, stained with anti-S100 Griffin, P. Low and J. Poduslo), pp. 361-376. Philadelphia: Saunders Co. antibody and then processed for TUNEL as described (Grinspan et al., 1996). Nuclei Grinspan, J. B., Marchionni, M. A., Reeves, M., Coulaloglou, M. and Scherer, S. S. (1996). Axonal interactions regulate Schwann cell apoptosis in developing were identified by DAPI staining. For quantification, DAPI-positive nuclei peripheral nerve: neuregulin receptors and the role of neuregulins. J. Neurosci. 16, associated with nerves were counted and the fraction of TUNEL-positive nuclei was 6107-6118. determined. Haack, H. and Hynes, R. (2001). Integrin receptors are required for cell survival and proliferation during development of the peripheral glial lineage. Dev. Biol. 233, 38-55. Light- and electron-microscopy Herrmann, H. and Aebi, U. (2000). Intermediate filaments and their associates multi- Morphological studies of semi-thin and ultra-thin nerve sections were performed as talented structural elements specifying cytoarchitecture and cytodynamics. Curr. Opin. described (Previtali et al., 2000), and examined using a light- (Olympus BX51) or Cell Biol. 12, 79-90. electron microscope (Zeiss CEM 902). Howe, A., Aplin, A. E., Alahari, S. K. and Juliano, R. L. (1998). Integrin signaling and cell growth control. Curr. Opin. Cell Biol. 10, 220-231. Morphometry Jessen, K. R. and Mirsky, R. (1991). Schwann cell precursors and their development. Digitalized images of fiber cross sections from corresponding levels of the sciatic Glia 4, 185-194. nerve were obtained with a digital camera (Leica DFC300F) using a 100 objective. Jessen, K. R., Morgan, L., Stewart, H. J. and Mirsky, R. (1990). 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Loss of glial fibrillary acidic protein (GFAP) impairs Schwann cell proliferation and delays nerve regeneration after damage

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Research Article 3981 Loss of glial fibrillary acidic protein (GFAP) impairs Schwann cell proliferation and delays nerve regeneration after damage 1 1 1 1,2 3 Daniela Triolo , Giorgia Dina , Isabella Lorenzetti , MariaChiara Malaguti , Paolo Morana , 2,3 2,4 5 1,2 1,2, Ubaldo Del Carro , Giancarlo Comi , Albee Messing , Angelo Quattrini and Stefano C. Previtali * 1 2 3 Neuropathology Unit, Department of Neurology and INSPE, and Neurophysiology Unit, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy Università Vita-Salute San Raffaele, 20132 Milan, Italy Waisman Center and Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA *Author for correspondence (e-mail: [email protected]) Accepted 14 July 2006 Journal of Cell Science 119, 3981-3993 Published by The Company of Biologists 2006 doi:10.1242/jcs.03168 Summary Axonal loss causes disabling and permanent deficits in that GFAP and the other Schwann-cell-intermediate many peripheral neuropathies, and may result from filament vimentin physically interact in two distinct inefficient nerve regeneration due to a defective signaling pathways involved in proliferation and nerve relationship between Schwann cells, axons and the regeneration. GFAP binds integrin v8, which initiates extracellular matrix. These interactions are mediated by mitotic signals soon after damage by interacting with surface receptors and transduced by cytoskeletal fibrin. Consistently, ERK phosphorylation was reduced in molecules. We investigated whether peripheral nerve crushed GFAP-null nerves. Vimentin instead binds integrin regeneration is perturbed in mice that lack glial fibrillary 51, which regulates proliferation and differentiation acidic protein (GFAP), a Schwann-cell-specific cytoskeleton later in regeneration, and may compensate for the absence constituent upregulated after damage. Peripheral nerves of GFAP in mutant mice. GFAP might contribute to form develop and function normally in GFAP-null mice. macro-complexes to initiate mitogenic and differentiating However, axonal regeneration after damage was delayed. signaling for efficient nerve regeneration. Mutant Schwann cells maintained the ability to dedifferentiate but showed defective proliferation, a key Key words: Cytoskeleton, Transgenic mice, Extracellular matrix, event for successful nerve regeneration. We also showed Nerve regeneration, Adhesion Introduction extensively documented and consist of a series of stereotyped Axonal loss and defective axonal regeneration is responsible steps (Griffin and Hoffman, 1993; Scherer and Salzer, 2001). for severe and permanent deficits in peripheral neuropathy. The segments of axons distal to the site of injury degenerate, Although axonal loss might not affect the life span of a patient, and macrophages penetrate and remove the disrupted myelin it results in severe disability with progressive muscle atrophy sheets, while Schwann cells dedifferentiate, re-enter the cell- and weakness, sensory deficits, and foot and leg abnormalities. proliferation cycle and provide a substrate for axonal regrowth. Any improvement in regeneration would benefit these patients. The onset of mitogenesis is synchronous with a peak 3-5 days Efficient axonal regeneration relies on the pathogenetic after injury and requires a prelude phase of intense mechanism that caused the axonal degeneration. If the disease rearrangement of the Schwann-cell cytoplasm. During this primarily affects the neuronal cell body (sensory or motor process Schwann cells acquire again the expression of surface neurons), the damage mostly prevents recovery. Conversely, molecules characteristic of embryonic development, such as when the neuronal cell body is preserved, the regeneration neural cell adhesion molecule (NCAM), L1-adhesion molecule NTR capacity should be maintained but still depends on the and p75 , and upregulate cytoskeletal constituents, such as permissive environment in the nerve. In the latter case, the glial fibrillary acidic protein (GFAP) and vimentin (Jessen et nerve regeneration results from balanced interaction of al., 1990; Martini, 1994; Neuberger and Cornbrooks, 1989). Schwann cells, the environment – the extracellular matrix After proliferation, Schwann cells interact with molecules in (ECM) – and regrowing axons (Scherer and Salzer, 2001; Stoll the extracellular environment to reorganize the basement and Muller, 1999). membrane and to rearrange themselves into bands of Bungner. All the events involved in axonal regeneration can be These bands are rail-track-like structures upon which axons recapitulated in the model of Wallerian degeneration- can efficiently regenerate. Further steps in nerve regeneration regeneration, which is induced in rodents by traumatic crush include Schwann cells that surround bundles of regenerating injury of the sciatic nerve. The spatial-temporal events axons, segregate larger axons into 1:1 relationship, enwrap associated with degeneration and regeneration have been them and form myelin sheaths. Journal of Cell Science 3982 Journal of Cell Science 119 (19) Most of these latter events reproduce what occurs in First we confirmed that homozygous mutant mice did not development (Webster, 1993), and rely on the interaction of synthesize GFAP in Schwann cells. We performed Schwann cell surface receptors with molecules that normally immunohistochemistry and western blot analysis of the sciatic form the ECM endoneurium or infiltrate the nerve because of nerve. Both experiments showed absence of the GFAP protein the blood-nerve barrier disruption (Akassoglou et al., 2002; (Fig. 1A,B, and data not shown). Feltri et al., 2002; Lefcort et al., 1992; Patton et al., 1997; Then, we investigated whether peripheral nerve Previtali et al., 2003b). These complex interactions result in a development was impaired. We compared semi-thin and ultra- continuous reorganization of the Schwann cell cytoskeleton thin sections from the sciatic nerve of postnatal day (P) P1, P7, that operates either downstream the outside-in or upstream the P14, P28 and P60 GFAP-null mice to age-matched controls. inside-out signaling pathway (Previtali et al., 2001). No significant differences in axonal sorting, Schwann-cell GFAP is a glial-specific member of the intermediate filament axon relationship and myelination were observed (Fig. 1C-J). family, which includes cell-type-specific filamentous proteins Myelin-forming and non-myelin-forming Schwann cells did with similar structure and function as scaffold for cytoskeleton not show cyto-architectural abnormalities; in particular, we did assembly and maintenance (Coulombe and Wong, 2004). not observe filament aggregates or any other abnormalities in During development, Schwann cells express two other the basement membrane (Fig. 1K). intermediate filaments: nestin and vimentin (Dong et al., 1999; We then determined the absolute number of myelinated Jessen and Mirsky, 1991). GFAP appears at a relatively late fibers and performed morphometric analysis comparing the stage in Schwann cell development, essentially when immature sciatic nerve of GFAP-null mice those of wild type. The total Schwann cells are formed, and is downregulated in those number of myelinated fibers, fiber diameter and myelination Schwann cells that form myelin (Jessen et al., 1990). After (g-ratio) was not significantly different in GFAP-null mice and birth, only non-myelin-forming Schwann cells and Schwann wild-type littermates (Fig. 1L,M). cells that dedifferentiate after nerve injury express GFAP, To confirm morphological data we performed functional whereas myelin-forming Schwann cells express vimentin tests. Mutant mice appeared normal from birth to 15 months (Jessen et al., 1990; Mirsky and Jessen, 2005). of age. They walk, run, climb and reproduce similarly to age- GFAP is also expressed in astrocytes, a Schwann cell matched controls. At 3 and 6 months, the GFAP null mice and counterpart in the central nervous system (CNS). The wild-type littermates showed no significant difference in upregulation of GFAP, together with vimentin, was thought to rotarod testing (Fig. 1M). Consistent with behavioral analysis, be a crucial step for astrocyte activation in response to brain GFAP-null mice showed normal nerve conduction velocity damage. However, deletion of GFAP in mutant mice did not (NCV) (35±0.5 m/second) and compound motor action result in any gross CNS abnormality (Gomi et al., 1995; potentials (cMAP) (37±0.7 mV) in the neurophysiological tests Liedtke et al., 1996; McCall et al., 1996; Pekny et al., 1995) (Fig. 1N). or in defective response to CNS injury (Pekny et al., 1999). Impaired astrocyte reaction to injury was only observed in The absence of GFAP is probably compensated by double GFAP-vimentin-deficient mice (Pekny et al., 1999). other intermediate filaments and modifies the By contrast, there are no extensive reports on the effects of endoneurial ECM composition GFAP deletion on peripheral nerve development and function, We investigated whether other intermediate filaments can or on the possible consequences for nerve regeneration. Here, compensate for the absence of GFAP in mutant mice, thus we examined the peripheral nervous system of GFAP-null explaining the normal phenotype. In fact, during development, mice. We observed normal nerve development and adult nerve two other intermediate filaments are described in Schwann function. No morphological abnormalities were detected cells: nestin and vimentin (Dong et al., 1999; Jessen and despite different composition of the Schwann cell cytoskeleton Mirsky, 1991). and endoneurial ECM. However, the lack of GFAP delayed First, we performed qualitative analysis by nerve regeneration after damage, probably due to defective immunohistochemistry. Results showed similar expression of Schwann cell proliferation. We also found that GFAP and vimentin in non-myelin-forming Schwann cells, as depicted by vimentin associate with two different adhesion pathways. colocalization with L1, in GFAP-null mice and controls GFAP complexes with integrin v8, which binds fibrin (compare Fig. 2A-C with 2G-I). Accordingly, vimentin was and modulates Schwann cell proliferation after damage. similarly expressed in myelin-forming Schwann cells, Consistently, we found reduced phosphorylation of ERK1/2 in identified by myelin-associated glycoprotein (MAG), in both injured sciatic nerves of GFAP-null mice. Vimentin, instead, mutants and controls (compare Fig. 2D-F with 2J-L). Nestin associates with integrin 51 and fibronectin, and regulates the was similarly expressed in myelin-forming and non-myelin subsequent steps of Schwann cell proliferation and nerve forming Schwann cells in both GFAP-null and wild-type regeneration. controls (data not shown). To test whether the absence of GFAP alters expression of the other intermediate filaments Results quantitatively, we performed western blot analysis. Western Loss of GFAP does not impair peripheral nerve blots of total nerve lysate did not show significant differences development and function in the amount of vimentin and nestin in nerves of GFAP-null The effects of GFAP deletion have been previously analyzed mice compared with controls (Fig. 2M). in the CNS (reviewed in Messing and Brenner, 2003; Privat, Manipulation of the cytoskeleton might alter outside-in and 2003), whereas consequences on the peripheral nervous system inside-out signaling as shown in the CNS of GFAP-vimentin have not been investigated in detail. To this aim, we analyzed double mutants (Menet et al., 2001). We therefore investigated peripheral nerve development and function in GFAP null mice. the expression of surface receptors and ECM molecules Journal of Cell Science GFAP and nerve cell regeneration 3983 Fig. 1. Morphological and functional analysis of the sciatic nerve in GFAP-null mice. (A,B) Staining for GFAP in nerves of wild-type and GFAP- null sciatic mice; GFAP is absent in nerves of the mutant. Ultra-thin (C,D) and semi-thin (E-J) section analysis of sciatic nerves from control and GFAP- null mice at 1, 7, 14 and 60 days after birth. No significant differences were observed. (K) Ultra-thin sections of the sciatic nerve from 2-months old GFAP-null mice. Both myelin- forming and non-myelin forming Schwann cells showed normal features. (L) Myelin- fiber density in adult sciatic nerve from control and GFAP- null mice. No significant differences were observed. (M) Morphometry of myelinated axons in adult sciatic nerve from control and mutant mice. No significant differences were observed per number and size distribution. (N) Rotarod test analysis and (O) electrophysiological analysis performed in mutant and control mice of 2 months of age. No significant differences were observed. Bar in J, 30 m for A,B; 8 m for C,D; 20 m for E-J; 5 m for K. previously described in the peripheral nerve (Previtali et al., contribution and is associated with upregulation of 2001; Previtali et al., 2003b). By immunohistochemistry, we intermediate filaments in Schwann cells, we examined nerve did not observe differences in the expression of integrins (1, regeneration in GFAP-null mice. Sciatic nerves from GFAP- 2, 3, 6, 7, 1, 4), -dystroglycan, L1 and NCAM in null mice and age-matched controls were crushed and Schwann cells of GFAP-null mice, or of collagen IV and examined 3 mm and 10 mm distal to the site of injury 3, 7, 10, laminin chains 2, 4, 1 and 1 in the endoneurium. 15, 21 and 45 days after injury. Results showed a clear delay Similarly, we did not observe differences in the expression of in nerve regeneration and remyelination in GFAP-null mice. collagen IV, fibronectin, vitronectin and laminin chains 1, 5, Data for both distances were concordant. 2, 1 and 3 in the perineurium and blood vessels. Only Three days after injury both wild-type and GFAP-null mice fibronectin showed higher expression in the endoneurium of showed diffuse signs of degeneration, including myelinolysis, GFAP-null mice compared with controls (compare Fig. 2N,O myelin debris and axonal fragmentation (data not shown). with 2P,Q, and data not shown). Western blot confirmed the Seven and ten days after injury, in wild-type nerves, we increased amount of fibronectin, whereas other ECM observed some features of axonal degeneration, some components, such as collagen IV or laminins were expressed macrophages and some bands of Bungner. However, the at similar levels (Fig. 2R). prominent aspect was the presence of many regrowing axons, some organized in clusters of regeneration, some arranged in Regeneration of sciatic nerve after injury is impaired in a 1:1 ratio with Schwann cells, and few ensheathed with thin GFAP-null mice myelin, indicating remyelination (Fig. 3A,A ). By contrast, in Since efficient nerve regeneration requires Schwann cells nerves of GFAP-null mice we observed diffuse signs of Journal of Cell Science 3984 Journal of Cell Science 119 (19) Fig. 2. Expression of intermediate filaments and ECM constituents in the sciatic nerve of GFAP-null mice. (A-C) Double staining for L1 and vimentin (merge in C) shows vimentin expression in non-myelin-forming Schwann cells. (D-F) Double-staining for MAG and vimentin (merge in F) shows vimentin expression in myelin forming Schwann cells. (G) Western blot analysis shows comparable levels of nestin and vimentin in mutants compared with wild type; -tubulin was used to normalize the samples. (H,I) Fibronectin expression in sciatic nerves of wild-type and GFAP-null mice; an increased amount of fibronectin was observed in the endoneurium in mutants. (J,K) Collagen IV expression in sciatic nerves of wild-type and GFAP-null mice. Mutants and controls showed comparable levels of collagen IV. (L) Western blot analysis confirmed an increased amount of fibronectin in the sciatic nerve of GFAP-null mice compared with controls, whereas collagen IV and laminins were present at equal amounts. SDS-PAGE gels were 7.5% except when analyzing laminins (5%). Bar in L, 60 m for A-C; 20 m for D-F and H- K. ongoing axonal degeneration, invading macrophages and 30% less fibers than controls (P=0.05 at 3 mm and P<0.01 at several Schwann cells arranged into Bungner bands (Fig. 10 mm; Fig. 3G,H). Fiber diameter distribution confirmed a I II 3B,B ,B ). Few clusters of regeneration and fibers in a 1:1 ratio reduced number of fibers at each diameter in nerves of GFAP- and, rarely, thinly myelinated fibers were observed (Fig. null mice, especially for those >3 m (Fig. 3K,L). I II 3B ,B ). To quantify differences, we performed morphometric Forty-five days after injury, a morphologically normal analysis counting all the fibers with a diameter >1 m (fibers situation was essentially achieved in control nerves, whereas that were expected to undergo myelination). The average GFAP-null mice still showed signs of ongoing regeneration number of axons in nerves of GFAP-null mice was significantly (Fig. 3E,F). Morphometric analysis confirmed that nerves of reduced compared with those of wild type, by 33% at 3 mm mutant mice contained roughly 20% less fibers than controls and 53% at 10 mm to the site of injury (P<0.05 and P<0.01 (P<0.05; Fig. 3G,H and 3M,N). We also detected thickening respectively; Fig. 3G,H). Interestingly, we found a higher of the myelin sheath – represented as the g-ratio – which was percentage of myelinated vs non-myelinated fibers (59% vs significantly higher in nerves of GFAP-null mice compared 41%) in controls, whereas this relationship was the opposite in with those of control animals (0.73±0.08 vs 0.69±0.09). nerves of GFAP-null mice (25% myelinated vs 75% non- To confirm delayed nerve regeneration in nerves of GFAP- myelinated fibers). Moreover, fiber-diameter distribution null mice, we performed two functional tests: motor-neuron confirmed a reduced number of fibers in nerves of GFAP-null retrograde labeling using fluorochrome-conjugated cholera mice at almost any diameter (Fig. 3I,J). toxin subunit B and neurophysiological analysis. A preliminary Fifteen and 21 days after injury, nerves of both wild-type trial was performed in control mice to identify the first time and GFAP-null mice showed increased signs of regeneration point after sciatic crush injury at which fluorescent cholera (Fig. 3C,D, and data not shown). However, signs of toxin injected in gastrocnemius was detected in motor neurons. degeneration and invading macrophages were preeminent in In control mice, motor neurons were labeled by fluorescent mutant nerves. The overall number of fibers was significantly cholera toxin when injected 12 days after injury and sacrificed reduced in nerves of GFAP-null mice compared with those of 48 hours later, at day 14. We therefore analyzed at this time wild type. Twenty days after injury, mutant nerves showed 11- point three GFAP-null mice and 3 age-matched controls. We Journal of Cell Science GFAP and nerve cell regeneration 3985 Fig. 3. Delayed nerve regeneration in the sciatic nerves of GFAP-null mice revealed by morphologic and morphometric analysis. (A-F) Light- microscopy images of injured nerves of GFAP-null mice, 3 mm distal to the lesion, compared with age-matched controls at different time 1 I II points. Electron microscopy images of t10 (ten days after crushing) nerve samples are shown in (A ) for control and (B ,B ) for mutant mice. Ten days after crushing, nerves of control mice showed several fibers in 1:1 ratio as well as thinly myelinated fibers (A,A ), whereas in nerves I II of mutant mice Schwann cells prevailed that were still sorting axons (B,B ) and bands of Bungner (B ). Twenty-one days after crushing, maturation of nerves in control mice was evident (C) whereas nerves of mutant mice still showed several degenerating fibers and clusters of regeneration (D). Forty-five days after crush, nerves of controls were nearly normal (E) whereas nerves of GFAP-null mice contained degenerating and thinly myelinated fibers (F). (G-N) Morphometric analysis of regenerating nerves at different time points, comparing data obtained 3 mm and 10 mm distally to the site of injury. (G,H) Diagram of the total number of fibers at 10, 21 and 45 days after injury; nerves of GFAP-null mice always showed significantly reduced number of fibers. (J-N) Diagram of the regenerating nerves at different time points subdivided per fiber diameter; results show reduced number of regenerating fibers in nerves of mutant mice, primarily those with larger I I II diameter. Error bars represent the +s.e.m. Bar in F, 10 m for A-F; 6 m for A ; 3 m for B ,B . *P<0.05. observed in GFAP-null mice that only 6% of motor neurons (5.6±2 vs 10.4±2 mV; P=not significant) and NCVs 20% were labeled by the fluorescent dye, whereas in control mice slower (16±1 vs 20±1 m/second; P=not significant) although it was 20% (P<0.001; Fig. 4A). differences were not significant. Consistent with the above results, GFAP-null mice showed neurophysiological signs of delayed regeneration (Fig. 4B). Loss of GFAP does not affect Schwann cell Ten days after crushing, we did not detect any signal in either dedifferentiation and cytoskeleton rearrangement GFAP-null or control mice, in agreement with motor neuron To obtain efficient nerve regeneration after damage, Schwann dye labeling, which showed target-muscle innervation only cells have to dedifferentiate, proliferate, and provide a after day 12. Fifteen days after injury, amplitudes of distal favorable environment for axonal regrowth. A defect in one of cMAP in GFAP-null mice were 40% of those in control mice these Schwann cell functions that implies the continuous (0.8±0.4 vs 1.4±0.1 mV; P=0.02), whereas NCVs were similar rearrangement of the cytoskeleton may explain the delay of (9.5±1 vs 10±2 m/second). At 21 days after crushing, regeneration in GFAP mutants. amplitudes in GFAP-null mice were still 50% of those in First, we evaluated whether GFAP-null Schwann cells controls (0.9±0.1 vs 1.9±0.3 mV; P=0.01) with similar NCVs normally dedifferentiate and modulate the cytoskeleton in (11.4±1 vs 12.5±2 m/second). Finally, 45 days after injury, response to crush injury. After damage Schwann cell GFAP-null mice showed amplitudes 40% of those in controls dedifferentiation results in the downregulation of myelin genes Journal of Cell Science 3986 Journal of Cell Science 119 (19) and the re-expression of genes such as those encoding NCAM, NTR L1-molecule and p75 , along with the upregulation of GFAP and vimentin (Jessen et al., 1990; Martini, 1994; Neuberger and Cornbrooks, 1989). In GFAP-null mice, by 3 days after damage, we observed that mutant Schwann cells regularly NTR dedifferentiated and expressed NCAM, L1 p75 and vimentin (compare Fig. 5A,C,E,G with 5B,D,F,H respectively). Then we evaluated whether vimentin was adequately upregulated in the sciatic nerve of GFAP null mice. We performed western blot analysis of the distal stump of the sciatic nerve and measured the amount of vimentin at 3, 6, 15, 21 and 45 days after injury in GFAP-null and control mice. Results showed that GFAP-null mice upregulate vimentin in injured nerves similarly to controls (Fig. 5I,J). Hence, defective regeneration was not the consequence of impaired Schwann cell dedifferentiation or insufficient vimentin upregulation. Delayed nerve regeneration is probably due to reduced Schwann cell proliferation in GFAP-null mice Since Schwann cell proliferation is a crucial step to initiate and obtain efficient nerve regeneration (Chen et al., 2005), and GFAP has been associated with cell mitosis (Yasui et al., 1998), we then focused our attention on Schwann cell proliferation. Previous reports showed that Schwann cells mainly proliferate in the first week after nerve damage, with a peak at day 3 (Clemence et al., 1989; Oaklander and Spencer, 1988; Pellegrino et al., 1986). Therefore, we performed crush injury of the sciatic nerve of six GFAP-null and six wild-type littermates. Three and 6 days after damage the animals were pulsed with BrdU to label cells in the DNA-synthesis phase of the cell cycle and then killed to perform BrdU staining. DAPI- Fig. 5. Vimentin upregulation is maintained in nerves of GFAP-null mice after sciatic nerve injury. (A-H) Longitudinal sections of 3- day-old injured sciatic nerves from wild-type (A,C,E,G) and GFAP- null mice (B,D,F,H) double-stained for vimentin and neurofilaments (A,B), NCAM and neurofilaments (C,D), L1 and neurofilaments (E,F), p75NTR and neurofilaments (G,H). DAPI staining of nuclei Fig. 4. Delayed nerve regeneration in nerves of GFAP-null mice (blue). The dedifferentiated Schwann cells showed expression of the measured by neurophysiology and motor neuron retrograde labeling above molecules as in wild type. Bar, 30 m. (I,J) Protein extracts with GFP-conjugated cholera toxin subunit B. (A) The total number from the distal stump of crushed nerves from wild-type (I) and of labeled motor neurons in the lumbar enlargement 48 hours after GFAP-null mice (J) at different time points were immunoblotted injection of fluorescent cholera toxin subunit B in the gastrocnemius with an anti-vimentin antibody. Sample loading was normalized is significantly reduced in GFAP-null mice compared with control against -tubulin. Ratio of vimentin to -tubulin was measured by mice. (B) Distal cMAP recordered in nerves of GFAP-null mice densitometry and expressed in the bottom line as times of increase always showed amplitudes of half-values compared with controls, at each time point T (in days) relative to T zero. Compared with whereas NCVs did not show significant differences; *P<0.05. Bar, control mice, vimentin is similarly upregulated in nerves of GFAP- 50 m. null mice. Journal of Cell Science GFAP and nerve cell regeneration 3987 positive and S100-positive Schwann cell nuclei (DAPI and Schwann cell proliferation in the nerves of mutant mice both S100 ) labeled with BrdU were counted in the distal stump of 3 days (5.5±1.2% vs 2.6±0.7%, P<0.001) and 6 days the sciatic nerve. Results showed a significant reduction in (3.5±0.8% vs 2.1±1.0%, P=0.04) after injury (Fig. 6F-J). Schwann cell proliferation in GFAP-null mice at day 3 However, we did not observe significant differences in (8.3±0.5% vs 12.2±1.4%, P=0.04) and an almost significant Schwann cell apoptosis at days 3 and 6 (Fig. 6K-O). reduction at day 6 (6.9±1.0% vs 9.7±1.0%, P=0.06) (Fig. 6A- E). To confirm these data, we performed similar double GFAP and vimentin constitute different target molecules staining for phosphorylated histone H3 with an antibody that for integrin receptors involved in Schwann cell recognizes only proliferating cells in the mitotic phase of proliferation and nerve regeneration the cell cycle. Results confirmed a significant reduction of Schwann cell proliferation and migration after nerve damage Fig. 6. Schwann cells in the distal stump of nerves of GFAP-null mice after injury show reduced proliferation but normal apoptosis. (A-D) Nuclei staining with DAPI (blue) and BrdU (green) on longitudinal sections of the distal stump of the sciatic nerve at 3 and 6 days (t3 and t6, respectively) after injury. The number of BrdU-positive nuclei is reduced at both t3 and t6 in nerves of mutant mice. (E) Quantitative analysis shows that the percentage of BrdU-positive nuclei is significantly decreased at t3 (*P=0.04) and consistently but not significantly reduced at t6 (P=0.06). (F-I) Staining of nuclei with DAPI (blue) and of phosphorylated histone H3 (green) on longitudinal sections of the distal stump of the sciatic nerve at days 3 and 6 after injury. The number of nuclei positive for phosphorylated histone H3 is reduced at both t3 and t6 in nerves GFAP-null mice. (J) Quantitative analysis shows that the percentage of phosphorylated histone H3 nuclei is significantly reduced at both t3 (**P<0.001) and t6 (*P=0.04). (K-N). S100 (green) and TUNEL (red) staining on longitudinal sections of the distal stump of the sciatic nerve at t3 and t6 after crushing. TUNEL staining shows a similar number of positive nuclei in mutant and control nerves at both time points. (O) Quantitative analysis shows no significant difference in the percentage of positive nuclei in mutants and controls. Error bars represent the ±s.e.m. Bar in N , 40 m for A-D; 80 m for F-I and K-N. Journal of Cell Science 3988 Journal of Cell Science 119 (19) is induced and modulated by complex interactions of ECM or vimentin. The immunoprecipitates were analyzed by receptors and ECM molecules. They include the ECM western blotting using antibodies against integrin v, 5, 1 molecules normally expressed in the endoneurium and those and 8. Two different anti-GFAP antibodies (mouse and rat) molecules that infiltrate the nerve as the result of blood-nerve but not anti-vimentin co-precipitated integrin subunits v and barrier disruption, such as fibrin. Disruption of this signaling 8 (Fig. 7A). By contrast, two different anti-vimentin pathway interferes with Schwann cell proliferation. antibodies (rabbit and mouse) but not anti-GFAP co- We investigated whether known adhesion complexes precipitated integrin subunits 5 and 1 (Fig. 7A). To confirm involved in Schwann cell proliferation can interact with GFAP that vimentin cannot associate with v integrin in the absence or vimentin. Although Schwann cells express several ECM of GFAP, we performed further experiments with nerves of receptors during development and adult life (Milner et al., mutant and control mice. In the homogenate of nerves from 1997; Previtali et al., 2003b), two adhesion pathways have been GFAP-null mice, the anti-vimentin antibody did again not proposed to function after nerve injury. First, fibrin interacts precipitate v integrin, whereas v integrin was with integrin v8, which signals the occurred damage and immunoprecipitated by GFAP in nerve homogenates of control stimulate Schwann cell proliferation (Akassoglou et al., 2003; mice (Fig. 7B). Akassoglou et al., 2002; Chernousov and Carey, 2003). Then, It has been reported that the fibrin–v8-integrin pathway the reorganization of the ECM allows fibronectin-integrin modulates Schwann cell proliferation by phosphorylating 51 interaction that carries on signaling for Schwann cell ERK1/2 [p44/42 MAP kinase (MAPK)] (Akassoglou et al., proliferation and differentiation to complete regeneration 2002). We first verified that ERK1/2 phosphorylation is the (Chernousov and Carey, 2003; Haack and Hynes, 2001; cause and not the consequence for reduced Schwann cell Lefcort et al., 1992). We therefore investigated whether GFAP proliferation. The sciatic nerves of ten 3-months old mice were and/or vimentin participate in the fibrin-v8 and/or crushed. Five mice were treated with the MAP kinase kinase fibronectin-51 complex. The homogenate of rat sciatic (MEK) inhibitor PD098059, which blocks ERK1/2 nerve was immunoprecipitated by using antisera against GFAP phosphorylation and compared to the other five mice treated Fig. 7. Characterization of integrin binding by GFAP and vimentin in the peripheral nerve, and of ERK1/2 phosphorylation. (A) Rat sciatic nerve lysate was immunoprecipitated with anti-GFAP or anti- vimentin antibody. The immunoprecipitated proteins were separated in SDS-PAGE (7.5%) under reducing conditions and blotted with antibodies against integrin subunits v, 8, 5 and 1. GFAP co- precipitated with the integrin subunits v and 8 but not 1, whereas vimentin co-precipitated with the integrin subunits 5 and 1 but not v. (B) Sciatic nerve lysate of GFAP-null mice was immunoprecipitated as described above with anti-vimentin antibody and blotted with anti-integrin v antibody; similarly the wild-type sciatic nerve lysate was immunoprecipitated with anti-GFAP antibody and blotted with anti-integrin v antibody. Vimentin still did not co-precipitate with integrin v in GFAP-null mice lysate, whereas integrin v again co-precipitated with GFAP in lysate of control-mice nerves. (C) Protein extracts form the distal stamp of wild-type mice treated with DMSO and wild-type mice treated with the MEK inhibitor PD098059 at 3 days (T3) after injury were immunoblotted with antibody against total ERK1/2 or phosphorylated ERK1/2. By densitometry the ratio of totalERK1/2 to pERK1/2 was measured and is stated below the blot as a number, indicates the phosphorylation state. Mice treated with PD098059 showed reduced ERK phosphorylation (D) Protein extracts from the distal stump of crushed nerves from wild-type and GFAP-null mice at T3 and T6 were immunoblotted with antibody against total ERK1/2 or phosphorylated ERK1/2. By densitometry, the ratio of totalERK1/2 to pERK1/2 was measured as described above. Nerves of GFAP-null mice showed less phosphorylated Erk1/2 compared with controls at both time points after injury. Journal of Cell Science GFAP and nerve cell regeneration 3989 with the vehicle (DMSO 10%). At 3 days post-injury, BrdU mature nerves, only non-myelin-forming Schwann cells and labeling of S100-positive cells showed Schwann cell Schwann cells that dedifferentiate after nerve injury express proliferation reduced by 50% in mice treated with the GFAP (Jessen et al., 1990). Hence, most of the time developing PD098059 (9.5%±1.4 vs 4.5%±1.5, P=0.0003; data not Schwann cells do not express GFAP, whereas its expression is shown). In the same animals, the homogenate of the restricted to a short, temporary window. The role of GFAP, crushed controlateral nerve confirmed reduced ERK1/2 being mostly unknown and related to the cytoskeleton phosphorylation by 25% (Fig. 7C) organization, may be therefore skipped and seems insignificant Since we were able to show that GFAP is part of the in the developing Schwann cells. fibrin–v8-integrin pathway, we investigated whether As to the second point, redundancy of and/or compensation defective Schwann cell proliferation in GFAP-null mice was by other intermediate filaments may mask GFAP deficiency in associated with reduced ERK1/2 phosphorylation. The Schwann cell development. Schwann cells express two other homogenates of the distal stump of T3 and T6 crushed nerve intermediate filaments, nestin and vimentin, during their from GFAP-null and wild-type controls were compared by embryonic development, and vimentin is maintained at high using an antibody specific for the phosphorylated form of levels also in mature Schwann cells (Dong et al., 1999; ERK1/2. At both time points, the sciatic nerves of GFAP-null Jessen and Mirsky, 1991). We confirmed by qualitative and mice showed a reduction of ERK1/2 phosphorylation, by 27% quantitative analyses that vimentin and nestin are expressed at at T3 and 48% at T6 compared with wild-type controls (Fig. similar amounts in mature GFAP-null Schwann cells compared 7D). The levels of total ERK1/2 were similar in GFAP-null with controls. Hence, redundancy might explain the absence of mice and controls. Hence, the difference in ERK1/2 phenotype in the peripheral nerve of GFAP-null mice. Finally, phosphorylation was not the consequence of different amounts mutant mice showed an increased amount of fibronectin in the of total ERK1/2. endoneurium. Fibronectin is a potent promoter of peripheral neurite outgrowth both during development and regeneration Discussion (Lefcort et al., 1992). The increased expression of fibronectin This study shows that GFAP modulates the Schwann cell might further favor nerve development in mutant mice response for tissue recovery after peripheral nerve injury. We independently by the presence of GFAP. provide evidence for the first time that, (1) GFAP is involved in Schwann cell proliferation, (2) GFAP is the cytoskeleton Loss of GFAP affects early Schwann cell proliferation component of the previously identified pathway originated by thus causing delayed nerve regeneration fibrin that drives Schwann cell proliferation after damage, (3) Although PNS function and development appeared normal in GFAP and vimentin constitute two different pathways that link the absence of GFAP, nerve regeneration was delayed in GFAP the Schwann cell cytoskeleton to the ECM, both of which mutants. In fact, the demand for intermediate filaments is involved in proliferation and differentiation. Finally, our study highly increased in Schwann cell after damage. Both GFAP shows that GFAP is not necessary for the development of and vimentin are upregulated, perhaps to provide an efficient the peripheral nerve, probably compensated for by other cytoskeleton rearrangement necessary for proliferation and intermediate filaments. Disruption or defective function of the differentiation (Gillen et al., 1995; Neuberger and Cornbrooks, GFAP pathway may therefore interfere with the regenerative 1989; Thomson et al., 1993). Thus, compensatory mechanisms capacity of the peripheral nerve that, in chronic conditions, might no longer be sufficient for the Schwann cell to support might determine severe degenerative defects and axonal loss. the loss of GFAP in an acute crisis, such as after injury. Accordingly, nerve regeneration in vivo and neurite outgrowth Vimentin and nestin probably compensate for the in vitro was described as being delayed in vimentin-null mice absence of GFAP during PNS development (Perlson et al., 2005). Whether delayed regeneration in It was shown previously by gene targeting inactivation that vimentin-null mice depends exclusively on impaired retrograde GFAP is not overtly required for normal mouse CNS transport of the perk–vimentin–dynein–importin- complex in development (Gomi et al., 1995; Liedtke et al., 1996; McCall the damaged axons, or is the consequence of impaired function et al., 1996; Pekny et al., 1995). Although these reports were of vimentin-null Schwann cells, needs further investigation. focused on the CNS, we confirmed here that also the PNS The observation that GFAP-null mice had a delay in nerve develops normally. We investigated nerve development from regeneration might be due to different scenarios: (1) defective birth to adulthood, and observed in GFAP-null mice normal Schwann cell dedifferentiation, (2) impaired proliferation, (3) timing of Schwann-cell–axon interactions, normal cyto- defective organization into bands of Bungner or, (4) impaired architecture of both myelin-forming and non-myelin-forming interaction between Schwann cells and regrowing axons. The Schwann cells, and regular fiber-type distribution. Peripheral regular upregulation of vimentin and the coherent expression nerves also showed normal function in neurophysiological and of markers of dedifferentiation, such as vimentin, L1, NCAM NTR functional tests. The lack of an evident phenotype might be the and p75 , in GFAP-null Schwann cells after damage consequence of at least two events: (1) GFAP has no main role suggested a normal dedifferentiation process. Moreover, in Schwann cell development and nerve function and/or, (2) GFAP-null Schwann cells could organize Bungner bands, other molecules are redundant or can compensate for loss of which appeared morphologically normal – although only with GFAP. a delay. Finally, we observed – although not in detail – normal Regarding the first point, GFAP appears at a relative late Schwann cell-axon interaction by morphological analysis and stage in Schwann cell development, basically when immature expression of L1/NCAM/NF markers. Schwann cells have formed already, and it is downregulated in Our results provided evidence that defective regeneration those Schwann cells that form myelin (Jessen et al., 1990). In is probably the consequence of reduced Schwann cell Journal of Cell Science 3990 Journal of Cell Science 119 (19) proliferation after damage. Soon after nerve injury, with cytoskeleton constituents including intermediate filaments dedifferentiated Schwann cells enter the cell cycle to provide (Herrmann and Aebi, 2000; Kreis et al., 2005; Rutka et al., a sufficient substrate for nerve regrowth. Previous studies 1997). Intermediate filaments participating in these cross- showed that Schwann cells highly proliferate in the first week bridges probably constitute a link between cell surface and post injury, with a peak at day 3 (Clemence et al., 1989; nucleus (Maniotis et al., 1997). This interaction provides a Oaklander and Spencer, 1988; Pellegrino et al., 1986). Our structural framework to facilitate intracellular responses, 2+ results of BrdU (labeling cells in G1-S-M phase) and histone including protein phosphorylation, intracellular pH and Ca H3 (labeling cells in M phase) analyses in nerves of control modification, and the activation of MAP kinase cascades mice confirmed data previously reported on Schwann cell (Turner, 2000). These signaling events culminate in the proliferation after injury, and showed a significant reduction in reorganization of the cytoskeleton necessary for motility, the number of mitotic Schwann cells in GFAP mutants. proliferation and gene expression. Reduced proliferation was statistically significant at days 3 and Our results suggested that the interaction of intermediate 6 for histone H3 and at day 3 for BrdU, and very close to filaments and integrins was specific and segregated. GFAP significance at day 6 for BrdU (P=0.06). Apoptosis was not associated with integrin v8 but not integrin 51, whereas modified by the absence of GFAP, suggesting a defect in vimentin associated with integrin 51 but not integrin v8, proliferation and not in cell survival. Accordingly we found a thus also excluding non-specific binding. However, we do not reduction in ERK1/2 phosphorylation in nerves of mutant know whether this is a direct interaction or whether it is mice. Activation of the MAP-kinase pathway regulates mediated by docking molecules. In the absence of GFAP, transcription of genes associated with proliferation and vimentin still associates with integrin 51 and not with differentiation in several cell types (Cowley et al., 1994; Kotch, integrin v8, therefore explaining why vimentin can not 2000), including Schwann cells (Akassoglou et al., 2002). As compensate for the absence of GFAP in transducing the confirmed by our results with the ERK inhibitor, the MAP- fibrin–integrin-v8 signaling that is initiated early after kinase pathway is at least one of the pathways that regulate damage. The Fibrin–integrin-v8–GFAP complex probably Schwann cell proliferation after injury. In fact, we found that drives Schwann cell proliferation only in close relation to nerve Schwann cell proliferation and ERK1/2 phosphorylation was damage, whereas other pathways modulate Schwann cell reduced but not abolished in nerves of mutant mice. Several mitosis in other situations. For example, in embryogenesis receptors and molecular pathways sustain proliferation. We integrin 51 and fibronectin control the proliferation of probably interfered with only one of these pathways, which Schwann cell progenitors, which express vimentin and not requires fibrin deposition and integrin v8 activation, and is GFAP (Haack and Hynes, 2001; Lefcort et al., 1992; Peters and specifically active in the first steps of nerve regeneration Hynes, 1996). Similarly, in the advanced phase of nerve (Akassoglou et al., 2003; Akassoglou et al., 2002). regeneration Schwann cell proliferation is controlled by A potential role for GFAP in cell proliferation has also been fibronectin and integrin 51 (Akassoglou et al., 2003; suggested previously (Yasui et al., 1998; Kawajiri et al., 2003). Akassoglou et al., 2002). Our results would fit with these Cytoskeleton reorganization and cytoplasm segregation is a previous observations. After nerve damage, the immediate crucial step in cell proliferation and division. In particular, repair reaction is sustained by the temporary ECM matrix several intermediate filaments are phosphorylated during formed by blood-derived fibrin, which immediately activates mitosis and continuously shift from an assembled Schwann cell proliferation via the integrin-v8–GFAP (filamentous) to a disassembled (soluble) state. Rho-kinases pathway. The absence of GFAP in our mutants would therefore phosphorylate GFAP, thereby causing their disassembly to block this early signal for Schwann cell proliferation. Later on, accomplish cytokinesis (Yasui et al., 1998). Similarly, Aurora- a mature ECM-scar is formed by fibronectin, which substitutes B kinase, required for chromosome segregation and mitosis, fibrin to sustain Schwann cell proliferation and initiates tasks was recently reported to phosphorylate a number of for differentiation. The presence of different pathways that intermediate filaments including GFAP (Kawajiri et al., 2003). regulate Schwann cell proliferation between regeneration and Our findings suggest that the role of GFAP in proliferation is development is not surprising. For example, cyclin D1 cell-specific or, more likely, specific to the cell environment regulates Schwann cell proliferation in nerve regeneration but and stimuli. Increased astrocyte proliferation and GFAP not in development (Atanasoski et al., 2001; Kim et al., 2000). overexpression is also observed in reactive astrocytosis in Finally, although the MAP-kinase pathway that drives cell CNS. However, when GFAP is deleted reactive astrocytosis is proliferation might be directly initiated by integrins (Aplin et not impaired and BrdU-pulse investigation failed to al., 2001), this pathway is more likely the consequence of demonstrate proliferative abnormalities in GFAP mutant collaborative signaling, in which integrin-mediated events are astrocytes (Pekny et al., 1999). initiated by other types of receptors, primarily tyrosine-kinase growth-factor receptors (Assoian and Schwartz, 2001; Howe et GFAP and vimentin link ECM to the Schwann cell al., 1998). For example, ErbB receptors and TGF- have been cytoskeleton via two distinct pathways already associated with Schwann cell proliferation in vivo and We found that GFAP and vimentin bind to integrin v8 and in vitro, and in different steps of nerve development (reviewed integrin 51, respectively, two ECM-receptors involved in in Mirsky and Jessen, 2005). It is tempting to speculate that Schwann cell proliferation and nerve regeneration (Akassoglou an extracellular fibrin (or fibronectin) network helps to et al., 2003; Akassoglou et al., 2002; Chernousov and Carey, accumulate integrin receptors in a restricted area of the 2003; Haack and Hynes, 2001; Lefcort et al., 1992). This Schwann cell surface, whereas intracellularly the GFAP (or was not a surprise. As transmembrane receptors, integrins vimentin) cytoskeleton helps to cluster the tyrosine-kinase physically interact, directly or through cytolinker molecules, receptors to initiate the MAP-kinase signaling pathway. Journal of Cell Science GFAP and nerve cell regeneration 3991 Can defective GFAP explain impaired regeneration in rod was measured in subsequent trials (four trials in the first 2 days and one trial on each of 3 consecutive days). peripheral neuropathy? Overall, our data showed that GFAP-null mice have delayed Neurophysiological analysis nerve regrowth and functional recovery after injury. Five 3-month-old GFAP-null mice and five 3-month-old control littermates were Impairment in nerve regrowth was more evident in the first 2 analyzed, as described (Bolino et al., 2004), before crush injury and 10, 15, 21 and 45 days after crush injury. Mice were anesthetized with avertin and placed under a weeks after damage, probably due to a reduced capacity of heating lamp to avoid hypothermia. The sciatic nerve conduction velocity (NCV) Schwann cells to proliferate and therefore to organize the was obtained by stimulating the nerve with steel monopolar needle electrodes. A regenerative scar. However, morphological and functional pair of stimulating electrodes was inserted subcutaneously near the nerve at the ankle. A second pair of electrodes was placed at the sciatic notch, to obtain two differences were still present 45 days after injury, at a time distinct sites of stimulation, proximal and distal along the nerve. The muscular when repair is almost complete in control mice. The mutant response to the electrical nerve stimulation, compound motor action potentials mice showed reduced numbers of regrowing axons at all (cMAP), was recorded with a pair of needle electrodes; the active electrode was inserted in muscles in the middle of the paw, while the reference was placed in the times and at 45 days still 20% of fibers were missing. skin between the first and second digit. Moreover, the difference in regrowth was more significant for larger axons. Antibodies and Immunohistochemistry Our data sustain that nerve repair is partially impaired but Antibodies used for immunohistochemistry and/or western blotting are listed in Table 1. Immunofluorescence on cryosections was performed as described (Previtali not abolished in GFAP mutants, whereas nerve development et al., 2003b), and examined with confocal (Biorad MRC 1024) or fluorescent and function is not affected. This raises the possibility that microscope (Olympus BX). GFAP mutations may affect the peripheral nerve. From this point of view, we may envisage three scenarios. (1) Gfap Inhibitor of ERK1/2 phosphorylation The MAP kinase kinase (MEK) inhibitor PD098059 (Sigma) was dissolved in mutations give rise to peripheral neuropathy. These are dimethyl sulfoxide (DMSO) and stored in aliquots at –80° C. The compound was probably not loss-of-function but gain-of-function mutations, diluted in saline (NaCl) immediately before use to a final concentration of 1 mg/kg. because GFAP-null mice did not show peripheral neuropathy. Gain-of-function mutations in human are responsible for a Table 1. List of the antibodies severe leukodystrophy, Alexander disease (Messing and Goldman, 2004). The severity of Alexander disease might Antigen Species Clone Source mask a more modest peripheral neuropathy, in a way similar BrdU Mouse BMC9318 Roche to what occurred with the neuropathy associated with Collagen IV Rabbit Chemicon -Dystroglycan Rabbit AP83 K. Campbell congenital muscular dystrophy in mutations in the laminin 2 ERK1/2 Rabbit Cell Signaling gene (LAMA2) (Shorer et al., 1995). (2) GFAP dysfunction is pERK1/2 Mouse E10 Cell Signaling not the consequence of genomic mutation but due to post- Fibronectin Rabbit Chemicon translational defects, i.e. phosphorylation or glycosylation. GFAP Mouse GA5 Chemicon GFAP Rat 2.2B10 Zymed Something similar has been described for defective pHistone H3 Rabbit Ser10 Upstate dystroglycan glycosylation. Mutations in genes that encode Integrin 1 Rabbit Chemicon proteins that glycosylate the -dystroglycan cause congenital Integrin 2 Rabbit Chemicon muscular dystrophies in human and/or neuropathy in mice Integrin 3 Rabbit Chemicon (Levedakou et al., 2005; Muntoni et al., 2004). (3) GFAP Integrin 5 Rabbit Chemicon Integrin 6 Rat GoH3 A. Sonnenberg mutations do not cause a peripheral neuropathy, but reduce the Integrin v Rabbit Chemicon capacity of nerve repair in course of genetic or acquired Integrin 1 Rat Mb1.2 Chemicon neuropathies. In this case, the modest delay in regeneration we Integrin 1 Rabbit Chemicon saw in GFAP-mutant mice after a single pathogenetic event, Integrin 1 Mouse 2B1 Chemicon Integrin 4 Rabbit Chemicon may become more relevant by adding the delay in regeneration Integrin 8 Goat G17 Santa Cruz of several fibers in the presence of a prolonged and/or L1 Rat 324 Chemicon continuous damage. Laminin EHS Rabbit Sigma Laminin 1 Rabbit H300 Santa Cruz Laminin 1 Rat AL1 Chemicon Materials and Methods Laminin 2 Rat 4H8-2 Alexis Generation of GFAP-null mice Laminin 4 Rabbit H-194 Santa Cruz Generation of GFAP-null mice and characterization of their CNS has been described Laminin 5 Mouse 4C7 Chemicon before (McCall et al., 1996); mice have been subsequently made congenic on an Laminin 1 Rat LT3 Chemicon inbred C57BL/6 background. Animals were generated from our colony and genotyped by PCR analysis of genomic DNA from tail clips. All experiments were Laminin 2 Rabbit 1117+ R. Timpl performed following the institutional guidelines. Laminin 1 Rat A5 Chemicon Laminin 3 Rabbit H140 Santa Cruz Sciatic nerve crush-lesion MAG Mouse Chemicon Adult mice were anesthetized with avertin (trichloroethanol, 0.02 ml/g of body NCAM Rabbit Chemicon weight) and crush injury was performed as described (Quattrini et al., 1996). After Nestin Mouse ab6142 abcam skin incision, the sciatic nerve was exposed and crushed distal to the sciatic notch Neurofilament-HRabbit Chemicon for 20 seconds with fine forceps previously cooled in dry ice. To identify the site Neurofilament-M Mouse NN18 Chemicon of injury, forceps were previously dropped into vital carbon. The nerve was replaced p75NTR Rabbit Chemicon under the muscle and the incision sutured. S100 Mouse SH-b1 Sigma S100 Rabbit Chemicon Rotarod analysis -Tubulin Mouse Tub2.1 Sigma Five 3-month-old GFAP-null mice and five 3-month-old control littermates were Vimentin Rabbit Chemicon placed on a round metal bar, first rotating at four rotations per minute and then Vimentin Mouse LN-6 Sigma accelerating at 7.2 rpm (Ugo Basile, Como, Italy). The animals were allowed to stay Vitronectin Rabbit H270 Santa Cruz on the rod for a maximum of 700 seconds and the time they stayed on the rotating Journal of Cell Science 3992 Journal of Cell Science 119 (19) PD098059 (0.4 ml volume), or an equivalent volume of the vehicle (DMSO 10%), number GGP030193) and Ricerca finalizzata (AQ, grant number was injected i.p. 2 hours after nerve injury and every 12 hours until mice were killed RF2003/171). after 3 days for BrdU and western blotting experiments. Immunoprecipitation and immunoblotting References Akassoglou, K., Yu, W. M., Akpinar, P. and Strickland, S. (2002). Fibrin inhibits Proteins were isolated from snap-frozen sciatic nerves of adult mice as described (Previtali et al., 2003a; Previtali et al., 2000). For western blot nerves suspended in peripheral nerve remyelination by regulating Schwann cell differentiation. Neuron 33, 861-875. Tris-buffered SDS lysis buffer (95 mM NaCl, 25 mM Tris-HCl pH 7.4, 10 mM Akassoglou, K., Akpinar, P., Murray, S. and Strickland, S. (2003). Fibrin is a regulator EDTA, 2% SDS, protease or phosphatase inhibitors), sonicated and boiled. For of Schwann cell migration after sciatic nerve injury in mice. Neurosci. Lett. 338, 185- immunoprecipitation, nerves were suspended in Igepal (Sigma, Milano, Italy) lysis buffer plus protease inhibitors and sonicated. Immunoprecipitations were performed Aplin, A. E., Stewart, S. A., Assoian, R. K. and Juliano, R. L. (2001). Integrin-mediated with anti-GFAP (mouse or rat) and anti-vimentin (rabbit or mouse) for 3 hours at adhesion regulates ERK nuclear translocation and phosphorylation of Elk-1. J. Cell 4°C. The immune complexes were collected by 90 minutes incubation with protein- Biol. 153, 273-282. A or -G agarose beads (Sigma). After washing, antigens were separated by heating Assoian, R. K. and Schwartz, M. A. (2001). Coordinate signaling by integrins and in reducing SDS sample buffer, and analyzed by SDS-polyacrilamide (PAGE) gel receptor tyrosine kinale in the regulation of G1 phase cell-cycle progression. Curr. (8.5%). For western blotting, equal amounts of homogenates (5 g) were diluted in Opin. Genet. Dev. 11, 48-53. 8M urea / 0.05M DTT, separated in sample buffer on 5 or 7.5% SDS-PAGE gel and Atanasoski, S., Shumas, S., Dickson, C., Scherer, S. S. and Suter, U. (2001). transferred to PVDF (Millipore, Roma, Italy) or nitrocellulose membrane (Biorad, Differential cyclin D1 requirements of proliferating schwann cells during development Segrate, Italy). Blots were blocked in PBS (0.05% Tween-5% dry milk) and and after injury. Mol. Cell. Neurosci. 18, 581-592. incubated with the appropriate primary + peroxidase-conjugated secondary antibody Bolino, A., Bolis, A., Previtali, S. C., Dina, G., Bussini, S., Dati, G., Amadio, S., Del (Sigma) and visualized by ECL (Amersham, Cologno M., Italy). The intensity of Carro, U., Mruk, D. D., Feltri, M. L. et al. (2004). Disruption of Mtmr2 produces the bands was quantified by densitometry and the ratio between each antibody and CMT4B1-like neuropathy with myelin outfolding and impaired spermatogenesis. J. -tubulin, or phosphorylated ERK1/2 on total ERK1/2, was determined. Cell Biol. 167, 711-721. Chen, Y. Y., McDonald, D., Cheng, C., Magnowski, B., Durand, J. and Zochodne, D. W. (2005). Axon and Schwann cell partnership during nerve regrowth. J. Neuropathol. Retrograde labeling of motor neurons Exp. Neurol. 64, 613-622. Five 3-months old GFAP-null mice and five age-matched controls were Chernousov, M. A. and Carey, D. J. (2003). alphaVbeta8 integrin is a Schwann cell anesthetized, the gastrocnemius muscle exposed, and 5 l of 1% cholera toxin receptor for fibrin. Exp. Cell Res. 291, 514-524. subunit B (Alexa Fluor-488 conjugate, Invitrogen, Burlingame, CA) in distilled Clemence, A., Mirsky, R. and Jessen, K. R. (1989). 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Thomas, J. peripheral nervous system. In immunohistochemistry. Sections were treated with acetone, stained with anti-S100 Griffin, P. Low and J. Poduslo), pp. 361-376. Philadelphia: Saunders Co. antibody and then processed for TUNEL as described (Grinspan et al., 1996). Nuclei Grinspan, J. B., Marchionni, M. A., Reeves, M., Coulaloglou, M. and Scherer, S. S. (1996). Axonal interactions regulate Schwann cell apoptosis in developing were identified by DAPI staining. For quantification, DAPI-positive nuclei peripheral nerve: neuregulin receptors and the role of neuregulins. J. Neurosci. 16, associated with nerves were counted and the fraction of TUNEL-positive nuclei was 6107-6118. determined. Haack, H. and Hynes, R. (2001). Integrin receptors are required for cell survival and proliferation during development of the peripheral glial lineage. Dev. Biol. 233, 38-55. Light- and electron-microscopy Herrmann, H. and Aebi, U. (2000). 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