Protein synthetic machinery and mRNA in regenerating tips of spinal cord axons in lamprey - PubMed (original) (raw)
. 2016 Dec 1;524(17):3614-3640.
doi: 10.1002/cne.24020. Epub 2016 May 19.
Affiliations
- PMID: 27120118
- PMCID: PMC5050069
- DOI: 10.1002/cne.24020
Protein synthetic machinery and mRNA in regenerating tips of spinal cord axons in lamprey
Li-Qing Jin et al. J Comp Neurol. 2016.
Abstract
Polyribosomes, mRNA, and other elements of translational machinery have been reported in peripheral nerves and in elongating injured axons of sensory neurons in vitro, primarily in growth cones. Evidence for involvement of local protein synthesis in regenerating central nervous system (CNS) axons is less extensive. We monitored regeneration of back-labeled lamprey spinal axons after spinal cord transection and detected mRNA in axon tips by in situ hybridization and microaspiration of their axoplasm. Poly(A)+mRNA was present in the axon tips, and was more abundant in actively regenerating tips than in static or retracting ones. Target-specific polymerase chain reaction (PCR) and in situ hybridization revealed plentiful mRNA for the low molecular neurofilament subunit and β-tubulin, but very little for β-actin, consistent with the morphology of their tips, which lack filopodia and lamellipodia. Electron microscopy showed ribosomes/polyribosomes in the distal parts of axon tips and in association with vesicle-like membranes, primarily in the tip. In one instance, there were structures with the appearance of rough endoplasmic reticulum. Immunohistochemistry showed patches of ribosomal protein S6 positivity in a similar distribution. The results suggest that local protein synthesis might be involved in the mechanism of axon regeneration in the lamprey spinal cord. J. Comp. Neurol. 524:3614-3640, 2016. © 2016 Wiley Periodicals, Inc.
Keywords: axons; lamprey; mRNA; regeneration; ribosomes; spinal cord.
© 2016 Wiley Periodicals, Inc.
Conflict of interest statement
STATEMENT LQJ, CRP, MES are/were employees of Temple University. KSJ was an undergraduate student. WR is a graduate student. None of the authors have known or potential conflicts of interest, including any financial, personal, or other relationships with other people or organizations within 5 years of beginning the submitted work that could inappropriately influence, or be perceived to influence, this work.
Figures
Fig. 1. Selection of axon tips for EM analysis
A, live image of the tip (white star) of a large RS axon back-labeled with DTMR at 2 weeks post-TX. Body = body of the tip; fp = finger-like protrusion; CC = central canal. Blue Inset: a toluidine blue-stained 1 μm semithin section of the same tip, showing continuity of fp with body. B, EM of the area indicated by the white box in A. The green box is the area shown at higher magnification in Fig. 2. Parts of profiles a, c and d are included in Fig. 2. The large (approximately 1 μm), round osmophilic densities (white arrowheads) are intraglial lipid droplets (Lurie et al., 1994), and should not be confused with ribosomes, which are much smaller (20–29 nm). C, the same EM with outlines around areas that are probably contiguous. The yellow outline represents the boundary of space a, and includes the rER-like structure analyzed in Fig. 2. The blue outline encloses spaces b–d, which are probably part of the same axon tip (inset), although they appear to be separated from it in this plane of sectioning. They do not contain any of the structure analyzed in Fig. 2. Black/white inset: The original live image of the area covered by EM. In this and subsequent figures, rostral is left. A magenta-green copy is provided in supplementary Fig.S-1.
Fig. 2. EM evidence for rough endoplasmic reticulum (rER) in an axon tip
A, the boxed region illustrated in Fig. 1B and C shown at higher magnification. Note that it contains three of the related profiles (a, c, and d) of the axon tip. A membrane structure (blue arrowhead) divides compartment a into two spaces (sp1 and sp2). The enlargements in subsequent frames show that this structure is part of an assembly that resembles rER. The green, deep blue and light blue boxes are areas imaged at higher magnification in B, C, and D (framed with lines of corresponding colors), respectively. B, overall view of an assembly with the appearance of rER. The complete profile consists of membranous structures with Golgi apparatus-like appearance. Regions framed by yellow and red boxes are shown at higher magnification in E and F respectively (framed with lines of the same colors). In C&D and E&F, insets are magnifications of the boxed areas, to show the rER-like assembly associated with vacuoles or vesicles. Membranous structures studded with ribosome-like granules (yellow arrowheads) are shown in the respective insets. The dashed white line in B and the white arrow in F show a pathway connecting spaces sp1 and sp2. re = rER; nf = neurofilaments; mt = mitochondria; ga = Golgi apparatus-like structure; v = vesicles; co = cisternal organelle. A magenta-green copy is available as supplementary Fig.S-2.
Fig. 3. Polyribosomes near membranous inclusions in axon tips
DTMR-backfilled spinal cords were studied by EM at 2 weeks post-TX. A, EM of the junction between the body (bd) and a finger-like protrusion (fp) of an axon tip, outlined in the inset at the bottom left. The diffuse electron-dense material (black arrow) probably represents en face membrane or subaxolemmal cortex, and contains several ribosomes (small white box, enlarged in the inset). Both the bd and fp are filled with neurofilaments (nf; white arrows). B, a similar electron-dense plaque contains a crescent-shaped membranous inclusion. Polyribosomes (white arrowheads) are present in or adjacent to this plaque. Cisternal organelles (co) and vesicles (v) are found in the plaque. Another ribosome-containing plaque was located at the distal edge of the axon tip (black arrowhead in the bottom left inset; not shown by EM). C, for comparison, in a nearby unidentified cell, polyribosomes form rosettes (arrowheads), or are associated with membranous structures (arrows).
Fig. 4. Axon tips with ribosomes and polyribosomes
EMs of four transected axon tips, imaged at low magnification in the pale insets. The black-framed boxes in these insets indicate the region of the accompanying EM. A–C, the distal axon tips contain ribosomes/polyribosomes (arrowheads). When located in the body (bd) of an axon tip, far from membranous invaginations or vesicle-like structures (v), most ribosomes are present as single particles or duplets. nf = neurofilaments. D, clusters of ribosomes (arrowheads) near the axolemma at the tip of a small axon. Polyribosomes were often clustered near vesicle-like membranous structures (white boxed inset). The areas in the small white boxes are further enlarged in the white-framed insets.
Fig. 5. Immunohistochemical demonstration of ribosomal protein S6 in axon tips
Four tips are illustrated A, B, C and D. A1, B1, C1 and D1 show DTMR back-labeled axon tips at the time of fixation. B2, C2 and D2 show staining with an anti-S6 mAb; A2 is a negative control, with the primary antibody omitted. Note the absence of S6 label, even in the surrounding cells. A3, B3, C3 and D3 show panels A1/A2,B1/B2,C1/C2, and D1/D2 superimposed, respectively. The growth status of tip A was not determined. Tips B and C were actively growing at the time of fixation. Tip D was sessile. S6 label was distributed in areas just subjacent to the axolemma (green arrowheads), or in a linear formation penetrating the central part of the tip with the appearance of a microtubule bundle (yellow arrowhead in B2), and in surrounding cell bodies (blue arrowheads in B2, C2, and D2). Note the unstained region at the very caudalmost limit of the axon tip in B (white arrow in B1), and the absence of staining in the static tip (white stars in D2). E, the antibody recognized one major band of expected molecular weight on lamprey CNS homogenates. Scale: 50 μm. A magenta-green copy is available as supplementary Fig. S-3.
Fig. 6. Oligo(dT) staining is absent or very weak in uninjured axons
An oligo(dT) probe was used to label total mRNA by ISH in uninjured lamprey spinal cords. A, a transverse section of unlesioned spinal cord labeled with an oligo(dT) probe. Only cell bodies were stained. On the left side, the giant Müller RS axons of the ventral columns are indicated by *. cc, central canal; Mth, Mauthner axon. No staining was found in axons. Three pale green lines (dorsal to ventral) indicate the approximate levels of the plane of section shown in C, D, and E. B, a similar transverse section stained by IHC with LCM3, an anti-NF mAb that primarily labels axons (Jacobs et al., 1995; Merrick et al., 1995). C, D and E, three horizontal sections from a series through the ventral columns in an uninjured spinal cord, showing absence of labeling in the large reticulospinal axons (*), while neurons, glia and ependymal cells were labeled. In C, the central canal (cc) is bounded by columns of stained ependymal cells (e). The thicker columns of labeled cells above and below the central canal are the gray matter (gm). Scale: 100 μm. A magenta-green copy is available as supplementary Fig. S-4.
Fig. 7. Oligo(dT) staining is apparent only in injured axons and is stronger in growing than in static axon tips
In two DTMR-backfilled spinal cords at 2 weeks post-TX, a growing tip (1 in A–C) and a static tip (2 in D–F) were monitored and then stained with Oligo(dT). The top panels (A and D) are live fluorescent images taken at 0 hr. The middle panels (B and E) are live images taken 2 hours later. The bottom panels (C and F) are horizontal sections through the axon tips stained with Oligo(dT). Insets in A, B, D and E are enlargements of the areas in the white boxes containing the axon tips, used for defining the growth statuses of the tips (blue line), based on fiduciary landmarks (vertical green lines), using the “Find Edges” tool in Photoshop to define the leading edges. The arrow in C points to the distal-most edge of the growing axon tip 1, which contains label throughout most of its area (Percent Filled = 89.1), as compared to the absence of staining in adjacent axon shafts (*). A slender area of gray label to the right (distal) of the arrow makes it appear that the axon has regenerated even further, but careful microscopic focusing showed that it belongs to another axon. The arrow in F points to the distal-most edge of static tip 2 (Percent Filled = 3.3). G, correlation between growth status and the % of axon tip area occupied by ISH label. Percent Filled (mean ± sem) for growing, static, and retracting axon tips were 57.1 ± 8.1 (n = 14), 18.6 ± 3.7 (n = 15), and 6.8 ± 2.5 (n = 10), respectively. * indicates mean values significantly reduced compared to growing tips (p<0.05, Kruskal-Wallis one way-ANOVA). TX, side where transected; cc, central canal. A magenta-green copy is available as supplementary Fig. S-5.
Fig. 8. Aspiration of axoplasm/cytoplasm from axon tips/cell bodies
A–C, a spinal cord viewed under the dissecting fluorescence microscope before and after micro-aspiration. Two axon tips, 1 and 2 in A, were penetrated by separate micropipettes. Each tip disappeared after its axoplasm was aspirated (B and C, respectively). D–F, verification of transfer of cytoplasm to a micro-aspirating electrode. A large reticulospinal neuron, the left B1 (*), in the brainstem that had been back-labeled with DTMR was micro-aspirated and the transfer of cytoplasm into the electrode tip was observed by light (D) and fluorescence (E) microscopy. F is D and E superimposed, so that red fluorescence is seen within the glass pipet tip. Note: The electrode remained in the B1 cell after aspiration. G, global amplification of mRNAs. Microaspirated mRNAs in the cytoplasm of a Mauthner cell, and axoplasms of one individual and 6 pooled regenerating axon tips were amplified by RT-PCR with negative (10 pg total CNS RNA, no RT enzyme) and positive controls (10 and 100 pg total CNS RNA, with RT enzyme). H, After global amplification, target-specific PCR showed L-NFL mRNA in the cytoplasms and axoplasms. Cloned L-NFL was a positive control (L-NFL). Scale: 300 μm. A magenta-green copy is available as supplementary Fig. S-6.
Fig. 9. Differential expression of cytoskeletal protein mRNAs in growing and static axon tips
Target-specific primers for lamprey β-actin (A), β-tubulin (B), vimentin (C), L-NFL (D), NF95 (E), NF180 (F) and NF132 (not shown) were used for PCR. Distilled water was the negative control. G.tips = globally amplified cDNA from 6 pooled growing axon tips; S.tips = globally amplified cDNA from 6 pooled static tips; Total RNA = cDNA from total CNS RNA (100 pg); Library cDNA = lamprey CNS cDNA library. Both the growing and static tips contained mRNAs encoding parts of L-NFL (389 bp) and β-tubulin (505 bp), but they were sparser in static tips. β-actin (229 bp; loading volume was doubled) and NF95 (417 bp) mRNAs were sparse in growing tips and not detected in static tips. Vimentin (394 bp), NF180 (264 bp) and NF132 (287 bp, not shown) were not detected.
Fig. 10. Vimentin and L-NFL signals were undetectable in electrodes after sham aspiration
A/B and C/D, two axon tips (1 and 2) found in 2 spinal cords were micro-aspirated and collected in 2 tubes with RNase inhibitor. Spinal cords were imaged before and after the aspiration (A vs. B; C vs. D). E, a third spinal cord in a region with no axon tips was used as control for sham aspiration. F, penetration of spinal cord without entering axon tips (sham penetration). Green arrow: the tip of a sham aspiration electrode viewed by light microscopy. G and H, electrophoresis of target-specific PCR products in 1% agarosegel after amplification of vimentin (F) and L-NFL (G). Water = negative control. Vimentin = cloned lamprey vimentin gene (5 ng); L-NFL = cloned L-NFL gene (5 ng); 1/6 electrodes = reactions from single or 6 sham-aspiration tips; axoplasm(1)/(2) =axoplasm harvested from axon tip 1 (A and B) or axon tip 2 (C and D); * = no band detected in PCR product from samples of 1 or 6 pooled sham-aspiration electrodes. Scale: 300 μm. A magenta-green copy is available as supplementary Fig.S-7.
Fig. 11. High vimentin mRNA content in lamprey spinal cord
A, a back-labeled axon tip (*) visualized in the living spinal cord at 2 weeks post-TX. The right side of the spinal cord is partially obscured by muscle. B, paraffin section ISH with a vimentin cRNA antisense probe. The axon tip was not labeled by the probe (*). Staining was intense in unidentified adjacent cells (some are neurons – upper blue rectangle in B) and cell processes (some are presumed glial processes – both rectangles in B). C, widespread staining with a vimentin cRNA antisense probe in wholemounted lamprey brainstem. Note the unstained RS neurons, which look like bubbles against the background staining. Yellow arrowheads point to mesencephalic Müller cells M2 (upper) and M3 (lower); medial green arrowhead is the B1 Müller cell; lateral green arrowhead is the Mauthner cell. Scale bar: 300 μm. A magenta-green copy is available as supplementary Fig.S-8.
Fig. 12. ISH of axon tips with L-NFL probe
A–D, a tip that had grown 21.2 μm in 3.8 hours (A,B). The green line connects a stationary fiduciary landmark (small neuron) in the two frames. Insets in A/B and E/F (bottom left) are enlargements of the boxed areas as in Fig. 7. C, the growing tip from A/B is labeled intensely by ISH for L-NFL. Note the most intense label is found at the leading edge (red arrow) and in a narrow spine at the base of the tip (yellow arrowhead; presumably a microtubule bundle). More than 7 nearby axons (D) were not labeled by the probe (C). Binding of the L-NFL probe suppressed rhodamine fluorescence (* in D). E–H, a static axon tip monitored as in A–D. No tip displacement was discerned between the images in E and F (3.6 hrs apart). In G, this tip (red arrow) showed little or no labeling for L-NFL mRNA. * In F and H is a nearby axon tip that is not connected to the one that was monitored. Green arrowheads in C and G point to small neurons stained by the L-NFL probe. Scale: 200 μm. A magenta-green copy is available as supplementary Fig. S-9.
Fig. 13. β-tubulin ISH in axon tips
A/B, a growing (1; 31.83 μm/3.75 hr) and a static (2, white arrow) tip were identified in sequential live images at 2 weeks post-TX, and aligned against fiduciary landmarks (green line) to measure movement of the tip (blue and yellow lines). In A, * marks a 3rd axon tip, which was lost at the 2nd imaging. C, the spinal cord sectioned and stained with a biotin-labeled β-tubulin RNA probe, which labeled the distal end of the growing tip (red arrow) but not the static tip (white arrow). D, blockage of rhodamine fluorescence by presence of the β-tubulin RNA probe. E–H, a static axon tip. The β-tubulin probe bound to a broad area of the axon tip (G). Sometimes staining was seen even in axons (yellow arrowheads). Insets in A/B and E/F are enlargements of the boxed areas as in Fig. 7. H, DTMR-fluorescence of the section in G. Scale: 200 μm. A magenta-green copy is available as supplementary Fig. S-10.
Fig. 14. ISH of axon tips with a β-actin probe
A/B, two large RS axon tips had their growth status determined over a 4.5 hour interval. The green vertical line locates a fiduciary landmark. The blue and yellow vertical lines mark the distal edges of axon tips 1 and 2 in A. Tip 1 was growing (26.52 μm/4.5 hr). Tip 2 was static. Insets in A and B are magnifications of the areas in the white boxes as in Fig. 7. Yellow arrowhead in B shows the position of axon tip 2 relative to its position in A. C–F, in the same spinal cord, the β-actin probe stained the growing tip 1, (C and D), but not the static tip 2 (E and F). Green arrowheads point to lightly stained local neurons. D and F are fluorescent images of the sections in C and D, respectively. The red arrows in C and E point to the distal-most edges of the axon tips, based on the rhodamine backfilled images in D and F. Scale: 200 μm. A magenta-green copy is available as supplementary Fig. S-11.
Fig. 15. Localization of mRNAs for L-NFL and β-tubulin in some injured axons
Two fluorescently back-labeled spinal cords were stained with an L-NFL probe (A and C), and a probe for β-tubulin (E and G). DTMR fluorescent images of the same sections are shown immediately below them in B&D and F&H, respectively. In A/B and E/F, a small fraction of the injured axons were stained with the ISH probes (green stars). White stars identify unlabeled nearby axons. C/D and G/H show mRNA entering axon tips in the sub-axolemmal region (yellow arrowheads). The distal ends of the axon tips are indicated by green arrowheads (C,G). The yellow star in G identifies the positively stained axon just proximal to its stained, growing axon tip, which had been micro-aspirated and is partially collapsed. The green star in G marks a second positively stained axon, whose tip is not in the frame. Scale: 100 μm. A magenta-green copy is available as supplementary Fig. S-12.
References
- Baas PW. Microtubule transport in the axon. Int Rev Cytol. 2002;212:41–62. - PubMed
- Brady G, Barbara M, Iscove NN. Representative in vitro cDNA amplification from individual hemopoietic cells and colonies. Methods in Molecular and Cellular Biology. 1990;2:17–25.
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