The Journal of Neuroscience, August 22, 2007, ():
Disorganized Microtubules Underlie the Formation of Retraction Bulbs and the Failure of Axonal Regeneration
J. Neurosci. Ertürk et al.
27: 9169
Supplemental Data
Files in this Data Supplement:
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Figure S1. End-structures of PNS and CNS axons following lesion.
(A, B) Saggital section (A) and cross section (B) through the thoracic spinal cord, showing the GFP positive axons in the dorsal columns from a GFP-M transgenic mouse. The dotted rectangles frame the central axonal branches of primary sensory neurons which localize superficially within the dorsal column of the spinal cord. These axons were targeted for lesioning in this study.
(C-E) Horizontal section of the sciatic nerve showing PNS axons: unlesioned (C) and 2 days after sciatic nerve lesion (D). White arrowheads in (D) show the growth cones formed at the proximal tip of the cut peripheral axonal branches. (E) higher magnification of the marked area in (D).
(F-H) Horizontal section of the dorsal column showing CNS axons: unlesioned (F) and 2 days after dorsal column lesion (G). Arrowheads in (G) show the retraction bulbs formed at the proximal tips of the lesioned central axonal branches. The arrow in (G) marks a swelling observed on the axon shaft of a lesioned neuron. (H) higher magnification of the marked area in (G).
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Figure S2. Typical appearance of growth cones of DRG Neurons in culture.
(A) Dissociated DRG neurons were prepared as described (Neumann et al., 2002), cultured on laminin and stained with the TuJ1 antibody (red). The axonal tips contain growth cones.
(B-G) Higher magnifications of the axonal tips indicated in (A). The growth cones have several filopodia. Some of the growth cones are spread out on the substrate (C, E and G) and have a fan-like morphology in contrast to their in vivo counterparts that have a more stream-lined shape.
Scale bar: 50 µm in (A).
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Figure S3. Retraction bulbs have dispersed and disorganized stable and dynamic microtubules
Growth cones and retraction bulbs were fixed and stained with an anti-Glu-tubulin antibody, which reveals relatively old microtubules, and an anti-Tyr-tubulin antibody, which show relatively newly polymerized microtubules.
(A-D) In retraction bulb tyrosinated microtubules (C) show a dispersed and disorganized distribution similar to de-tyrosinated microtubules (B). However, distribution of tyrosinated and de-tyrosinated microtubules differed within retraction bulbs. Whereas de-tyrosinated microtubules are present within most of the regions (B), tyrosinated microtubules show more restricted localizations (C) and do not enrich at the axonal tip. In (B) and (C), the yellow arrows point some of the highly deviated microtubules; the white arrowheads point the regions with few microtubule filaments and the white arrows show the regions of microtubule accumulations. (D) is the merge of de-tyrosinated (B) and tyrosinated tubulin channels (C).
(E-H) In growth cones tyrosinated microtubules (G) also align in parallel bundles within growth cones and show a similar distribution as de-tyrosinated microtubules (F). The yellow arrowheads in (F) and (G) show some of the microtubules which are organized in parallel bundles. (H) is the merge image of de-tyrosinated (F) and tyrosinated tubulin channels (G).
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Figure S4. Low concentrations of nocodazole neither caused retraction bulb formation nor inhibited the growth of injured peripheral axons
(A-I) In addition to 330 µM nocodazole, we also tested the effects of 33 µM and 3.3 µM concentrations of nocodazole on growth cones. 24 hours post sciatic nerve injury, the mice were re-anesthetized and their sciatic nerves exposed. We treated the injured sciatic nerve with 10 µl of 33 µM nocodazole (A), 3.3 µM nocodazole (D) or 0.5% DMSO (G). Unlike 330 µM nocodazole, 33 µM and 3.3 µM nocodazole do not cause formation of big bulbous axon terminals at the axons terminals. (B, C), (E, F) and (H, I) are higher magnifications of the marked axon terminals in (A), (D) and (G) respectively. The red brackets in (A, D and G) show the injury sites which were determined by delimitating the deformed tissue. In both relatively low doses of nocodazole treatments, many axons regenerate and grow beyond the crush site, as do the ones in the control (6 out of 6 animals for 33 ?M nocodazole and 5 out of 5 animals 3.3 ?M nocodazole).
(J) The axon / tip ratio quantification of axon terminals after treatments. Application of 33 µM nocodazole has a weak effect on the morphology of axon terminals (1.84 ± 0.12, average ± s.d.; n=43 from 6 mice); 3.3 µM Nocodazole does not show any significant change of the size of axon terminals (1.44 ± 0.04, average ± s.d.; n=29 from 5 mice) compared to control (1.21 ± 0.11, average ± s.d.; n=31 from 5 mice).
(K-P) In vivo live imaging shows that low doses of nocodazole treatments do not inhibit the growth of peripheral axons. The tips of the axons from 24 hours to 48 hours are followed by the green arrowheads in 33 µM nocodazole (K, L), 3.3 µM nocodazole (M, N) or control (O, P). The images are aligned with the purple arrows with respect to the landmarks, the vertical vessels, which remained at the same position during imaging. In all cases, the peripheral axons are able to grow a distance of about 0.5 to 1 mm within 24 hours of observation.
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Figure S5. Co-immuno-staining of in vitro growth cones and nocodazole induced bulbous ending with tyrosinated and de-tyrosinated tubulin antibodies
(A-D) A bulbous axon terminal (A) stained with both anti-Glu-tubulin antibody (B) and anti-tyrosinated-tubulin antibody (C). Tyrosinated microtubules are disorganized and dispersed in bulbous endings similar to de-tyrosinated microtubules.
(E-H) An expanded growth cone (E) stained with both with anti-Glu-tubulin antibody (F) and anti-Tyr-tubulin antibody (G). (H) is the merge image of stable (F) and dynamic microtubules (G) in this growth cone. Tyrosinated microtubules also align in parallel bundles within axonal shafts similar to de-tyrosinated microtubules. However, they extend into peripheral region of the growth cone.
(I-L) A slim growth cone (I) stained with both anti-Glu-tubulin antibody (J) and anti-Tyr-tubulin antibody (K) antibodies and their merge is shown in (L). Notably, dynamic and stable microtubules are tightly bundled and extended to the tip of the growth cone indicating that this is a fast growing growth cone similar to the ones seen in vivo at the injured peripheral axons of DGR neurons (see also Fig. 1B,C)
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Figure S6. Taxol treatment does not alter the growth capacity of the peripheral axons
In vivo imaging of injured sciatic nerve axons from 24 hours to 48 hours post-injury times after taxol or control treatments.
(A-D) 24 hours after the sciatic nerve injury, the mice were re-anesthetized and their sciatic nerves treated once with 10 µl of 1 µM taxol or DMSO control. Then we observed the growth of the axons from 24 to 48 hours post injury times for taxol (A, B) and control treatments (C, D). Taxol treatment does not affect the growth ability of the injured peripheral axons. The tips of the axons followed by the green arrowheads and the images are aligned with the purple-dashed lines with respect to the landmarks.
(E) The quantification of axon regeneration after the treatments. The axons grow 572 ± 277 µm (average ± s.d.; n= 8 mice) in taxol conditions and 510 ± 276 µm (average ± s.d.; n= 7 mice) in controls.
