紫杉醇減少脊髓損傷后疤痕形成并促進軸突再生

1、Title：Microtubule Stabilization Reduces Scarring and Causes AxonRegeneration After Spinal Cord Injury Farida Hellal et al. Science 331, 928 (2011); DOI: 10.1126/science.1201148,Reporter :***Supervisor : ***Secondary

2、 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

3、proliferation, migration, and differentiation as well as intracellular trafficking and secretion of extracellular matrix (ECM) molecules (1, 2) . Moreover, moderate microtubule stabilization prevents axonal retraction a

4、nd swelling of the axon tip after central nervous system (CNS) injury (3) and stimulates axon growth of cultured neurons (4) , enabling them to overcome the growth inhibitory effect of CNS myelin (3) .,Assumptions,They h

5、ypothesized 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 growth .,M

6、ethods,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 t

7、he injury site using an intrathecal catheter connected to an osmotic minipump.,What happened 7 days later ？,,Farida Hellal et al. Science 2011;331:928-931,Published by AAAS,Fig. 1 Taxol decreases scarring induced by spin

8、al 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) Taxol (256 ng/day) 7 days after injury. Scale bars, 300 μm. (D)

9、 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

10、 size (F) (n = 7 to 10 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 IVac

11、cumulation induced by spinal cord injury (A)Representation of lesioned spinal cord (box). (B-D) Mid-saggital sections of lesion site from rat treated with Vehicle or Taxol at 7 days post-injury. Taxol decreases ECM de

12、posit 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 µm.,,(A)Representation of lesioned s

13、pinal 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 µm. (D) Taxol does not aff

14、ect cell proliferation induced by spinal cord injury. Results expressed 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 do

15、es 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 treated with Vehicle or Taxol stained with TUNEL at 3 (B) and 7 (

16、C) daysafter dorsal hemisection injury. Scale bar, 150 µm. (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

17、test (n= 7 animals per group),Conclusion 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

18、hinders TGF- β signaling and attenuates fibrogenesis .,A key event in fibrotic scarring after CNS injury is the activation of transforming growth factor–β (TGF-β) signaling. Following SCI, TGF-β expression dramatically i

19、ncreases, which favors fibrosis (10–12). Integrity of the microtubule network is crucial for the transduction of this signal (13) . Smad2, the downstream effector of the TGF- β pathway, binds to microtubules through conv

20、entional kinesin-1 (14) .,Fig. 2. Taxol dampens TGF-β signaling. (A) Taxol treatment increases total tubulin and decreases tyrosinated tubulin in the lesion site，enabling kinesin-1 to bind tightly to microtubules (15)

21、.(B) Kinesin-1 enrichment in microtubule fraction of Taxol-treated lesion site. (C) His-Smad2 binds to kinesin-1 and endogenous Smad2 coimmunoprecipitates with kinesin-1 (D) of brain and spinal cord extracts.,Fig. 2. T

22、axol dampens TGF-β signaling. (E and F) Taxol alters microtubule-based cargo transport. (E) Overlay of colorcoded time series of red fluorescent protein (RFP)–labeled peroxisomes bound to the kinesin-1 (KIF5) motor do

23、main 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 int

24、ensity for KIF5- 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 traffick

25、ing.,Indeed, in TGF-β1–stimulated astrocytes, Taxol caused Smad2/3 to localize persistently to microtubules (fig. S4) and inhibited 70% of its translocation to the nucleus (Fig. 2, G and H),Figure S4: Taxol sequesters Sm

26、ad2 onto microtubules Astrocytes treated with DMSO or Taxol (100 nM) 30 minutes prior to TGF-ß1 (2ng/ml) stimulation. Cells were permeabilised during fixation to extract solublecomponents of the cytoplasm and sta

27、ined with Smad2/3 antibody. Taxol induces Smad2/3 co-localization with microtubules. Scale bar, 10 µm.,Indeed, in TGF-β1–stimulated astrocytes, Taxol caused Smad2/3 to localize persistently to microtubules (fig. S4)

28、 and inhibited 70% of its translocation to the nucleus (Fig. 2, G and H),Fig. 2. Taxol dampens TGF-β signaling. (G and H) In cultured astrocytes, Taxolcounteracts the TGF-β1–induced nuclear translocation of Smad2/3 (a

29、rrowheads) causing cytoplasmic Smad2/3 accumulation (arrow). Results in (H) are mean T SD [three independent experiments; *P = 0.041;one-way analysis of variance (ANOVA)].,In time-lapse microscopy, overexpressed Smad2 f

30、used 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

31、 signaling. (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,

32、20 mm. BSA, bovine serum albumin; DAPI, 4´,6´-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide.,Indeed, in cultured meningeal cells, Taxol reduced the TGF-β1–stimulated production of fibronectin (Fig. 3A) and

33、 impaired TGF-β1–stimulated migration (Fig.3, B and C).,Fig. 3. Taxol decreases meningeal cell migration and glycosaminoglycan release in vitro and in vivo.(A)Taxol decreases fibronectin deposits induced by TGF-b1 in me

34、ningeal 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

35、T SD from three independent experiments;*P = 0.003; one-way ANOVA.,Conclusion 2:,Low doses of Taxol prevent fibrotic scarring after SCI by interfering with Smad dependent TGF-β signaling and reducing extracellular matrix

36、 secretion and cell migration.,3. The 3rd question is whether Taxol decreases CSPGs after SCI.,TGF-β signaling also regulates the production of the axon growth inhibitory chondroitin sulfate proteoglycans (CSPGs) (10) .

37、,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 from rat

38、 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 outli

39、ne the lesion site. Scale bar, 150 µm.,At 7 days after injury, Taxol decreased the amount of NG2, one of the most abundant CSPGs (17) (fig. S1).,Fig. 3. Taxol decreases meningeal cell migration and glycosaminoglycan

40、 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, E and F)The conditioned medium of cultured meningeal cells

41、 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 scaf

42、folding the cells as observed 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 be

43、comes permissive for regenerating axons in vivo by evaluating the regenerative response of dorsal root ganglion (DRG) neurons.,The procedure: These neurons are set into a growth-competent state by injuring their peripher

44、al 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 permissive for conditioned axons to gro

45、w. 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 regene

46、ration and functional recovery. (A)Spinal 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). S

47、cale bars, 200 mm.(B) Longest regenerating axon per animal ± SEM (**P = 0.002; two-tailed t test). The longest axons per animal grew 1199 ± 250 mm in the Taxol-treated group versus 176 ± 225 mm in the ve

48、hicle-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

49、also enhances 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

50、.,Figure S5: Taxol promotes axonal outgrowth of primary neurons plated on inhibitory substrates .(A) Cerebellar granule neurons (CGNs) plated on laminin or CSPGs at 2 DIV. (B)Taxol increases neurite growth of CGNs pla

51、ted 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) Taxol increases neurite outgrowth of prima

52、ry 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, respectiv

53、ely; one way ANOVA).,Fig. 4. Taxol promotesaxonal 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 t

54、he cord is enriched in serotonergic fibers (C,arrow heads). Scale bars, 75 mm. (D) Quantification of 5-HT–positive fibers caudal to the lesion ±SEM after dorsal hemisection [n = 16 rats per group;***P = 0.0001; tw

55、otailed 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 trea

56、ted animals 4 weeks after injury. Scale bar, 10 µm.,Taxol induces growth of 5-HT axons after dorsal hemisection.,Conclusion 5:,6 . They last examined whether Taxol treatment leads to functional recovery after moder

57、ate spinal cord contusion (24) .,Fig. 4. Taxol promotes axonal regeneration and functional recovery. (E) Quantification of 5-HT–positive fibers caudal to the lesion ± SEM after contusion injury [n = 10 rats per gro

58、up; **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 , Taxol- and vehicle-treated a

59、nimals 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% mis

60、step frequency after 6 and 8 weeks, resulting in improvement by a factor of 3.4 (Fig. 4F and movies S3 and S4).,Taxol-induced functional recovery correlates with its axon growth–inducing effect.,Conclusion 6:,That is to

61、say , moderate stabilization of microtubules counteracted various cellular processes that prevent axon regeneration. Thus, Taxol has the potential to offer a multitargeted therapy for SCI .,1 . At low doses, Taxol reduce

62、d 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-β signaling and reducing extracellular m

63、atrix 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 axo

64、ns .,5 . Taxol induces growth of 5-HT axons after dorsal hemi-section.,6 . Taxol-induced functional recovery correlates with its axon growth–inducing effect.,Conclusions:,References and Notes,Thanks for your attention !,