Gervasi C et al. (2003), Increased expression of multiple neurofilament ...

XB-ART-5364

J Comp Neurol 2003 Jun 23;4612:262-75. doi: 10.1002/cne.10695.

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Increased expression of multiple neurofilament mRNAs during regeneration of vertebrate central nervous system axons.

Gervasi C , Thyagarajan A , Szaro BG .

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Characteristic changes in the expression of neuronal intermediate filaments (nIFs), an abundant cytoskeletal component of vertebrate axons, accompany successful axon regeneration. In mammalian regenerating PNS, expression of nIFs that are characteristic of mature neurons becomes suppressed throughout regeneration, whereas that of peripherin, which is abundant in developing axons, increases. Comparable changes are absent from mammalian injured CNS; but in goldfish and lamprey CNS, expression of several nIFs increases during axon regrowth. To obtain a broader view of the nIF response of successfully regenerating vertebrate CNS, in situ hybridization and video densitometry were used to track multiple nIF mRNAs during optic axon regeneration in Xenopus laevis. As in other successfully regenerating systems, peripherin expression increased rapidly after injury and expression of those nIFs characteristic of mature retinal ganglion cells decreased. Unlike the decrease in nIF mRNAs of regenerating PNS, that of Xenopus retinal ganglion cells was transient, with most nIF mRNAs increasing above normal during axon regrowth. At the peak of regeneration, increases in each nIF mRNA resulted in a doubling of the total amount of nIF mRNA, as well as a shift in the relative proportions contributed by each nIF. The relative proportions of peripherin and NF-M increased above normal, whereas proportions of xefiltin and NF-L decreased and that of XNIF remained the same. The increases in peripherin and NF-M mRNAs were accompanied by increases in protein. These results are consistent with the hypothesis that successful axon regeneration involves changes in nIF subunit composition conducive to growth and argue that a successful injury response differs between CNS and PNS.

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Species referenced: Xenopus laevis
Genes referenced: ctdspl ina neff1 nefl nefm nfs1 prph
???displayArticle.antibodies??? Nefm Ab1 Nefm Ab4 Prph Ab1 Prph Ab2 Prph Ab3 phospho-Nefm Ab3

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	Fig. 1. Increases in NF-M and peripherin mRNAs at 9 days postcrush demonstrated by RNase protection. Ten g of total RNA pooled from 12 eyes were hybridized simultaneously with 32P-labeled RNA probes against Xenopus NF-M and peripherin in one tube, and 3 g of RNA were hybridized with a probe to Elongation Factor 1- (EF1- ) in a separate tube (B,C,D). A: Probe hybridized against yeast RNA, demonstrating that the RNase digestion went to completion. B: RNA from naive, unoperated eyes. C: RNA from the contralateral control eye, 9 days after optic nerve crush. D: RNA from the operated eye, 9 days postcrush. After correcting for a slight increase (10%) in the expression of EF1- mRNA, the ratios of RNA expression between the operated and contralateral control eyes (D,C) were 1.3 for NF-M and 2.3 for peripherin.
	Fig. 2. Expression of nIF mRNA in the contralateral, unoperated control eye at late stages of regeneration (A–E) and in the operated eye 6 (F–J) and 12 (K–O) days after optic nerve crush. Neighboring transverse sections were hybridized with digoxigenin-labeled cRNA probes to peripherin (A,F,K), xefiltin (B,G,L), XNIF (C,H,M), NF-M (D,I,N), and NF-L (E,J,O). A representative example of a section from an operated eye hybridized with a sense probe to peripherin showed no label above background (P). i, inner nuclear layer; p, retinal pigmented epithelium (naturally pigmented and not labeled); r, retinal ganglion cell (RGC) layer. Scale bar 100 m in P and applies to all panels.
	Fig. 3. Frequency of RGC labeling with probes to the five nIF mRNAs in control and operated eyes. Labeled RGC layer cells were scored according to intensity of labeling as weak, moderate, or strong. Percent labeled cells in each category are represented cumulatively as the mean SE. A: Values pooled from 10 contralateral control eyes at late stages of regeneration represent normal, unoperated eyes. Values in contralateral control (B), and operated eyes (C), 6 days after crush. Values in contralateral control (D) and operated eyes (E), 9 days after crush. The total percentages of labeled RGCs in the operated eye at this time were not significantly different between peripherin, xefiltin, XNIF, and NF-M (single factor ANOVA, df 6, P 0.48). Figure legend in A applies to all panels.
	Fig. 4. Differences in nIF mRNA levels between the operated (OE) and unoperated, contralateral control (UE) RGCs, as measured by video densitometry from in situ hybridizations performed under standardized conditions. For each nIF and time period after optic nerve crush, the difference in the mean intensity/pixel was determined between the UE and OE RGCs and then averaged among animals from a given time period (mean SE; n 4 animals for 3–6 days and 9–12 days; 5 for 18–21 days; 2 for 35 days). Levels of nIF mRNA remain relatively stable from 3–6 days postcrush, when regenerating axons cross the lesion and enter the optic nerve, from 9–12 days, when they have reached the optic chiasm, and from 18–21 days, when they reach and cover the tectum. Data within each of these time windows were therefore pooled.
	Fig. 5. Characterization of peripherin antisera on Western blots of total spinal cord homogenates. 1, preimmune serum for lane 2, diluted 1:500. 2, Xenopus peripherin N-terminal head domain peptide, rabbit antiserum #1, diluted 1:500. 3, preimmune serum for lane 4, diluted 1:500. 4, Xenopus peripherin N-terminal head domain peptide rabbit antiserum #2, diluted 1:2,000. 5, Previously characterized Xenopus peripherin C-terminal peptide rabbit antiserum, diluted 1:2,000.
	Fig. 6. Immunocytochemical localization of peripherin in regenerating axons. Transverse sections through the brains of frogs sacrificed 28 days (A–D) and 21 days (E,F) after optic nerve crush were immunostained with antibodies to a C-terminal peptide derived from Xenopus peripherin (diluted 1:1000: A,C,E) and to nonphosphorylated NF-M (S8 after phosphatase treatment, 1:1000: B,D; RMO270, 1:100: F). Dorsal side is up and the left side is at the left. A: At the optic chiasm, peripherin-containing fibers run longitudinally in the left, crushed optic nerve (arrow). In contrast, in the right, uncrushed optic nerve, peripherin is found only in clusters of glia cells (g). B: On a neighboring section, regenerating fibers growing outside the degenerating core (asterisk) stain strongly for NF-M. The degenerating core was not visible in A because of the level of sectioning. C: A more posterior section of the same animal as in A and B shows peripherin containing fibers in the contralateral optic tract of the diencephalon (arrow). D: A neighboring section to that in C shows the distribution of NF-M for comparison. E: In the operated optic nerve close to the chiasm, peripherin is present in the youngest regenerating axons (arrows), which are located close to the glia limitans, but is absent in the oldest regenerating axons, which are found close to the degenerating core (asterisk). F: On a section adjacent to that shown in E, NF-M is present in older regenerating axons, but is absent from young regenerating axons close to the glial limitans (dots). g, glia; oc, optic chiasm; ot, optic tract; p, pial surface; v, ventricle. Scale bars 300 m in D (applies also to A–C) and 100 m in F (applies also to E).