1、Reporter :*Supervisor : *Secondary Supervisor : *,Backgrounds,Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. Microtubule dynamics regulate key processes during scarring, including cell proliferation, migration, and differentiation as
2、well as intracellular trafficking and secretion of extracellular matrix (ECM) molecules (1, 2) . Moreover, moderate microtubule stabilization prevents axonal retraction and swelling of the axon tip after central nervous system (CNS) injury (3) and stimulates axon growth of cultured neurons (4) , ena
3、bling them to overcome the growth inhibitory effect of CNS myelin (3) .,Assumptions,They hypothesized that moderate microtubule stabilization with Taxol, an approved drug, would facilitate axonal regeneration after spinal cord injury (SCI) by decreasing scar formation and enhancing intrinsic axonal
4、growth .,Methods,They first examined whether Taxol treatment reduced scarring after SCI. Adult rats underwent a dorsal hemisection at the eighth thoracic spinal cord level; Taxol (256 ng/day ) was continuously delivered at the injury site using an intrathecal catheter connected to an osmotic minipum
5、p.,What happened 7 days later ？,Farida Hellal et al. Science 2011;331:928-931,Published by AAAS,Fig. 1 Taxol decreases scarring induced by spinal cord injury. (A)Representation of lesioned spinal cord (box). (B and C) Midsagittal sections of lesion site from rats treated with (B) vehicle or (C) Taxo
6、l (256 ng/day) 7 days after injury. Scale bars, 300 m. (D) Taxol significantly decreases fibrotic scarring (expressed as percentage of vehicle control; n = 12 rats per group; *P = 0.002; two-tailed t test) without affecting glial compaction (28 days after injury) (E) or injury size (F) (n = 7 to 10
7、rats per group; P = 0.951; two-tailed t test). Data expressed as mean SEM. GFAP, glial fibrillary acidic protein.,Figure S1: Taxol decreases fibronectin, NG2 proteoglycan and collagen IVaccumulation induced by spinal cord injury (A)Representation of lesioned spinal cord (box). (B-D) Mid-saggital sec
8、tions of lesion site from rat treated with Vehicle or Taxol at 7 days post-injury. Taxol decreases ECM deposit in the core of the lesion as assessed by fibronectin (B), collagen IV (C) and NG2 proteoglycan (D) immunostainings. The dashed lines outline the lesion site. Scale bar, 150 m.,(A)Representa
9、tion of lesioned spinal cord (box). (B-C) Mid-saggital sections of lesionsite from rats treated with Vehicle or Taxol (7 days post-injury) stained with DAPI (B)and phospho-histone H3 (C). Scale bar, 150 m. (D) Taxol does not affect cell proliferation induced by spinal cord injury. Results expressed
10、as mean SEM; p= 0.591; two-tailed t test (n= 7 animals per group).,Figure S2: Taxol does not interfere with proliferation,Figure S3: Taxol does not induce apoptosis in the injured spinal cord(A) Representation of lesioned spinal cord (box). (B-C) Mid-saggital sections of lesion site from rats treate
11、d with Vehicle or Taxol stained with TUNEL at 3 (B) and 7 (C) daysafter dorsal hemisection injury. Scale bar, 150 m. (D) Taxol does not affect the celldeath induced by spinal cord injury. Results expressed as mean SEM; 3dpi: p= 0.628;7dpi: p= 0.679; two-tailed t test (n= 7 animals per group),Conclus
12、ion 1:,At low doses, Taxol reduced fibrotic scarring by mechanisms independent of cell proliferation or apoptosis.,2. Then , they examined whether stabilizing the microtubule network hinders TGF- signaling and attenuates fibrogenesis .,A key event in fibrotic scarring after CNS injury is the activat
13、ion of transforming growth factor (TGF-) signaling. Following SCI, TGF- expression dramatically increases, which favors fibrosis (1012). Integrity of the microtubule network is crucial for the transduction of this signal (13) . Smad2, the downstream effector of the TGF- pathway, binds to microtubule
14、s through conventional kinesin-1 (14) .,Fig. 2. Taxol dampens TGF- signaling. (A) Taxol treatment increases total tubulin and decreases tyrosinated tubulin in the lesion site，enabling kinesin-1 to bind tightly to microtubules (15) .(B) Kinesin-1 enrichment in microtubule fraction of Taxol-treated le
15、sion site. (C) His-Smad2 binds to kinesin-1 and endogenous Smad2 coimmunoprecipitates with kinesin-1 (D) of brain and spinal cord extracts.,Fig. 2. Taxol dampens TGF- signaling. (E and F) Taxol alters microtubule-based cargo transport. (E) Overlay of colorcoded time series of red fluorescent protein
16、 (RFP)labeled peroxisomes bound to the kinesin-1 (KIF5) motor domain upon Rapalog addition. Blue marks the initial distribution; the red gradient shows the distribution over time (30 min). Scale bars, 10 mm. (F) Time traces of radius of circle enclosing 90% of total fluorescence intensity for KIF5-
17、or dynein adaptor (BICDN)linked peroxisomes. Mean T SEM of 5 to 8 COS-7 cells per condition (P = 0.004; two-tailed t test for both KIF5 and BICDN).,This suggesting that Taxol would hinder Smad2 trafficking.,Indeed, in TGF-1stimulated astrocytes, Taxol caused Smad2/3 to localize persistently to micro
18、tubules (fig. S4) and inhibited 70% of its translocation to the nucleus (Fig. 2, G and H),Figure S4: Taxol sequesters Smad2 onto microtubules Astrocytes treated with DMSO or Taxol (100 nM) 30 minutes prior to TGF-1 (2ng/ml) stimulation. Cells were permeabilised during fixation to extract solublecomp
19、onents of the cytoplasm and stained with Smad2/3 antibody. Taxol induces Smad2/3 co-localization with microtubules. Scale bar, 10 m.,Indeed, in TGF-1stimulated astrocytes, Taxol caused Smad2/3 to localize persistently to microtubules (fig. S4) and inhibited 70% of its translocation to the nucleus (F
20、ig. 2, G and H),Fig. 2. Taxol dampens TGF- signaling. (G and H) In cultured astrocytes, Taxolcounteracts the TGF-1induced nuclear translocation of Smad2/3 (arrowheads) causing cytoplasmic Smad2/3 accumulation (arrow). Results in (H) are mean T SD three independent experiments; *P = 0.041;one-way ana
21、lysis of variance (ANOVA).,In time-lapse microscopy, overexpressed Smad2 fused to PAGFP (photoactivatable green fluorescent protein ) moved into the nucleus within minutes after TGF-b1 stimulation, whereas Taxol treatment abolished this movement (movies S1 and S2) .,Fig. 2. Taxol dampens TGF-b signa
22、ling. (I)In vivo, 7 days after SCI, phosphorylated Smad2/3 translocated into the nucleus in 95% of vehicle treated animals, compared with only 30% of the Taxol-treated animals (n = 13 rats per group). Scale bar, 20 mm. BSA, bovine serum albumin; DAPI, 4,6-diamidino-2-phenylindole; DMSO, dimethyl sul
23、foxide.,Indeed, in cultured meningeal cells, Taxol reduced the TGF-1stimulated production of fibronectin (Fig. 3A) and impaired TGF-1stimulated migration (Fig.3, B and C).,Fig. 3. Taxol decreases meningeal cell migration and glycosaminoglycan release in vitro and in vivo.(A)Taxol decreases fibronect
24、in deposits induced by TGF-b1 in meningeal cells. Scale bar, 300 mm. (B and C) Three days in vitro,Taxol (1 and 10 nM) decreases meningeal cell migration induced by TGF-b1.Arrows indicate the initial gap size. Scale bar, 300 mm; results in (C) are means T SD from three independent experiments;*P = 0
25、.003; one-way ANOVA.,Conclusion 2:,Low doses of Taxol prevent fibrotic scarring after SCI by interfering with Smad dependent TGF- signaling and reducing extracellular matrix secretion and cell migration.,3. The 3rd question is whether Taxol decreases CSPGs after SCI.,TGF- signaling also regulates th
26、e production of the axon growth inhibitory chondroitin sulfate proteoglycans (CSPGs) (10) .,Figure S1: Taxol decreases fibronectin, NG2 proteoglycan and collagen IVaccumulation induced by spinal cord injury (A)Representation of lesioned spinal cord (box). (B-D) Mid-saggital sections of lesion site f
27、rom rat treated with Vehicle or Taxol at 7 days post-injury. Taxol decreases ECM deposit in the core of the lesion as assessed by fibronectin (B), collagen IV (C) and NG2 proteoglycan (D) immunostainings. The dashed lines outline the lesion site. Scale bar, 150 m.,At 7 days after injury, Taxol decre
28、ased the amount of NG2, one of the most abundant CSPGs (17) (fig. S1).,Fig. 3. Taxol decreases meningeal cell migration and glycosaminoglycan release in vitro and in vivo. (Fig. 3D)Lesion site extracts from Taxol-treated animals showed a significant reduction of GAGs compared with controls. (Fig. 3,
29、 E and F)The conditioned medium of cultured meningeal cells and astrocytes treated with 10 nM Taxol showed a 35% and 32% decrease of GAG levels, respectively.(Fig. 3G)Moreover, the CSPGs expressed in the Taxol treated animals localized to the intracellular space instead of scaffolding the cells as o
30、bserved in vehicle-treated animals.,Administration of low doses of Taxol decreases CSPGs at the lesion site after SCI.,Conclusion 3:,4 . Further , they asked whether the Taxol-treated lesion site becomes permissive for regenerating axons in vivo by evaluating the regenerative response of dorsal root
31、 ganglion (DRG) neurons.,The procedure: These neurons are set into a growth-competent state by injuring their peripheral axon (conditioning) that allows them to regenerate their CNS axon, but only in a scar-free environment (21) . They assessed whether the reduction of the scar induced by Taxol is p
32、ermissive for conditioned axons to grow. Taxol was delivered at the lesion for 4 weeks; 2 weeks after central injury, they conditioned the lumbar L4-6 DRG neurons by transecting the sciatic nerve.,What about the results ?,Fig. 4. Taxol promotes axonal regeneration and functional recovery. (A)Spinal
33、cord horizontal sections of L4-6 DRG axons labeled with cholera toxin B, 6 weeks after injury . Taxol treatment promotes regeneration of growth competent neurons (arrowheads). Scale bars, 200 mm.(B) Longest regenerating axon per animal SEM (*P = 0.002; two-tailed t test). The longest axons per anima
34、l grew 1199 250 mm in the Taxol-treated group versus 176 225 mm in the vehicle-treated animals (n = 13 animals per group; P = 0.002; two tailed t test),The Taxol-treated lesion site thus becomes favorable for regeneration of growth-competent axons .,Conclusion 4:,Question 5 : Because Taxol also enha
35、nces intrinsic axon growth (4) and the elongation of cultured neurons plated on CSPGs or CNS myelin components (3) (fig. S5), they assessed whether Taxol treatment alone could promote growth of injured CNS axons.,Figure S5: Taxol promotes axonal outgrowth of primary neurons plated on inhibitory subs
36、trates .(A) Cerebellar granule neurons (CGNs) plated on laminin or CSPGs at 2 DIV. (B)Taxol increases neurite growth of CGNs plated on laminin and CSPGs. Results are means SD from 3 independent experiments; n= 150-200 cells per condition; * p= 0.016; p= 0.038 respectively; two-tailed t test. (C) Tax
37、ol increases neurite outgrowth of primary cortical neurons plated on MAG, NogoA, Semaphorin 3A or CSPGs. Results are means SD from 3 independent experiments; n= 150-200 cells per condition; * p= 0.005, p=0.009, p=0.007, p=0.002 and p=0.001, respectively; one way ANOVA).,Fig. 4. Taxol promotesaxonal
38、regeneration andfunctional recovery. (C) Spinal cord sagittal sections stained with antibody to 5-HT 4 weeks after injury. After Taxol treatment, the caudal part of the cord is enriched in serotonergic fibers (C,arrow heads). Scale bars, 75 mm. (D) Quantification of 5-HTpositive fibers caudal to the
39、 lesion SEM after dorsal hemisection n = 16 rats per group;*P = 0.0001; twotailed t test (D).,Figure S6: Taxol promotes formation of growth cone-like structures at the tip ofthe 5HT-positive axons.Two representative examples of axonal tips of 5 HT-positive axons from Vehicle and Taxol treated animal
40、s 4 weeks after injury. Scale bar, 10 m.,Taxol induces growth of 5-HT axons after dorsal hemisection.,Conclusion 5:,6 . They last examined whether Taxol treatment leads to functional recovery after moderate spinal cord contusion (24) .,Fig. 4. Taxol promotes axonal regeneration and functional recove
41、ry. (E) Quantification of 5-HTpositive fibers caudal to the lesion SEM after contusion injury n = 10 rats per group; *P = 0.002; two-tailed t test (E). (F) Taxol treatment improves locomotor performance over time (*P = 0.004, *P = 0.0001; two-way ANOVA). n.s.,not significant. After 2 and 4 weeks , T
42、axol- and vehicle-treated animals performed equally on the grid walk ，while significantly different after 6 and 8 weeks .,However , while vehicle-treated animals did not show additional recovery, Taxol-treated animals improved further to a 5% misstep frequency after 6 and 8 weeks, resulting in impro
43、vement by a factor of 3.4 (Fig. 4F and movies S3 and S4).,Taxol-induced functional recovery correlates with its axon growthinducing effect.,Conclusion 6:,That is to say , moderate stabilization of microtubules counteracted various cellular processes that prevent axon regeneration. Thus, Taxol has th
44、e potential to offer a multitargeted therapy for SCI .,1 . At low doses, Taxol reduced fibrotic scarring by mechanisms independent of cell proliferation or apoptosis.,2 . Low doses of Taxol prevent fibrotic scarring after SCI by interfering with Smad dependent TGF- signaling and reducing extracellul
45、ar matrix secretion and cell migration.,3 . Administration of low doses of Taxol decreases CSPGs at the lesion site after SCI.,4 . The Taxol-treated lesion site thus becomes favorable for regeneration of growth-competent axons .,5 . Taxol induces growth of 5-HT axons after dorsal hemi-section.,6 . Taxol-induced functional recovery correlates with its axon growthinducing effect.,Conclusions:,References and Notes,Thanks for your attention !,
