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First published online September 20, 2006
doi: 10.1242/10.1242/jcs.03168

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

¹ Neuropathology Unit, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy
² Department of Neurology and INSPE, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy
³ Neurophysiology Unit, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy
⁴ Università Vita-Salute San Raffaele, 20132 Milan, Italy
⁵ Waisman Center and Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI 53706, USA

View larger version (115K): [in a new window]   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 µm for A,B; 8 µm for C,D; 20 µm for E-J; 5 µm for K.

View larger version (80K): [in a new window]   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; ß-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 µm for A-C; 20 µm for D-F and H-K.

View larger version (70K): [in a new window]   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 points. Electron microscopy images of t10 (ten days after crushing) nerve samples are shown in (A1) for control and (BI,BII) 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,AI), whereas in nerves of mutant mice Schwann cells prevailed that were still sorting axons (B,BI) and bands of Bungner (BII). 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 diameter. Error bars represent the +s.e.m. Bar in F, 10 µm for A-F; 6 µm for AI; 3 µm for BI,BII. *P<0.05.

View larger version (40K): [in a new window]   Fig. 4. Delayed nerve regeneration in nerves of GFAP-null mice measured by neurophysiology and motor neuron retrograde labeling with GFP-conjugated cholera toxin subunit B. (A) The total number of labeled motor neurons in the lumbar enlargement 48 hours after injection of fluorescent cholera toxin subunit B in the gastrocnemius is significantly reduced in GFAP-null mice compared with control mice. (B) Distal cMAP recordered in nerves of GFAP-null mice always showed amplitudes of half-values compared with controls, whereas NCVs did not show significant differences; *P<0.05. Bar, 50 µm.

View larger version (79K): [in a new window]   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 (blue). The dedifferentiated Schwann cells showed expression of the above molecules as in wild type. Bar, 30 µm. (I,J) Protein extracts from the distal stump of crushed nerves from wild-type (I) and GFAP-null mice (J) at different time points were immunoblotted with an anti-vimentin antibody. Sample loading was normalized against ß-tubulin. Ratio of vimentin to ß-tubulin was measured by densitometry and expressed in the bottom line as times of increase at each time point T (in days) relative to T zero. Compared with control mice, vimentin is similarly upregulated in nerves of GFAP-null mice.

View larger version (73K): [in a new window]   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 NI, 40 µm for A-D; 80 µm for F-I and K-N.

View larger version (30K): [in a new window]   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, ß8, 5 and ß1. GFAP co-precipitated with the integrin subunits v and ß8 but not ß1, whereas vimentin co-precipitated with the integrin subunits 5 and ß1 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.