Intra-axonal translation of Khsrp mRNA slows axon regeneration by destabilizing localized mRNAs

Abstract

Axonally synthesized proteins support nerve regeneration through retrograde signaling and local growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional regulation of neuronal mRNAs, but only a limited number of axonal RBPs are known. We used targeted proteomics to profile RBPs in peripheral nerve axons. We detected 76 proteins with reported RNA binding activity in axoplasm, and levels of several change with axon injury and regeneration. RBPs with altered levels include KHSRP that decreases neurite outgrowth in developing CNS neurons. Axonal KHSRP levels rapidly increase after injury remaining elevated up to 28 days post axotomy. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP is through local translation of Khsrp mRNA in axons. KHSRP can bind to mRNAs with 3'UTR AU-rich elements and targets those transcripts to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of KHSRP target mRNAs, Gap43, Snap25, and Fubp1, following sciatic nerve injury and these mice show accelerated nerve regeneration in vivo. Together, our data indicate that axonal translation of the RNA binding protein Khsrp mRNA following nerve injury serves to promote decay of other axonal mRNAs and slow axon regeneration.

Graphical Abstract

Intra-axonal translation of Khsrp mRNA slows axon regeneration.

Figure 1.

Peripheral nerve injury changes the axonal RNA binding protein populations.(A, B) Sciatic nerve axoplasm harvested proximal to the injury site from 3–28 days post-crush lesion was trypsin-digested and processed for liquid chromatography and mass spectrometry using parallel reaction monitoring (PRM) to detect proteins with known RNA binding activity. Levels of proteins from spectral counts relative to uninjured (naïve) axoplasm shown are Log₂ fold-change as indicated in (A) (N = 3 for each time point). (B) shows volcano plot for PRM results for 7 days crush versus naïve samples graphed as log₂ fold-change versus negative log₁₀P value for differences. Also see Supplemental Figure S1 for graphical representation of full time course. (C) Representative immunoblot for naïve and 7 days injured sciatic nerve axoplasm confirms the increase in FXR1, hnRNP A3, hnRNP AB, hnRNP H1 and KHSRP. ERK 1/2 and GAPDH show relatively equivalent loading of the lysates.(D, E)Representative immunoblots for kinetics of KHSRP elevation in sciatic nerve axoplasm over 0–28 days post-crush lesion are shown in (D). Quantification of KHSRP immunoreactivity across multiple animals is shown in (E) as mean fold-change relative to naïve ± standard error of the mean (SEM; N = 3 mice for each time point; *** P ≤ 0.001 for versus 0 day, †P ≤ 0.05, †††P ≤ 0.005, †††P ≤ 0.001 and ††††P ≤ 0.0005 versus 3 days, &&&P ≤ 0.001 and &&&&P ≤ 0.0005 versus 7 days, and $$ P ≤ 0.01 and $$$$P ≤ 0.0005 versus 14 days by one-way ANOVA with Tukey's post-hoc analysis). (F) Representative confocal images for KHSRP protein in naïve and 7 days post-crush sciatic nerve. Upper panels of each pair show XY images of merged neurofilament (NF) + KHSRP and DAPI + KHSRP; lower panels show KHSRP signals overlapping with NF or DAPI in individual Z planes projected as an XYZ image [scale bar = 5 μm].

Figure 2.

KHSRP depletion enhances axonal growth from naïve but not injury-conditioned DRG neurons. (A) Representative images of 24 h DRG neuron cultures from naïve and 7 days injury-conditioned Khsrp^+/+ and Khsrp^–/– mice immunostained for NF are shown [scale bar = 100 μm]. (B) Quantification of total axon length per neuron from cultures as in (A) shows significantly greater axon growth in Khsrp^–/– compared to Khsrp^+/+ DRGs. However, there is no significant difference in axon length between the genotypes when cultures were prepared 7 days after nerve crush injury (i.e. ‘injury-conditioned’ neurons). Data are expressed as mean ± SEM; refer to Supplemental Figure S2 for axon branching analyses (N ≥ 75 neurons analyzed per condition in three independent culture preparations; * P ≤ 0.05 and ** P ≤ 0.01 by one-way ANOVA with Tukey's post-hoc analysis). (C) Representative immunoblot shows signal for KHSRP in cell body and axonal preparations from DRG cultures from indicated mice. There is no KHSRP band in the Khsrp^–/– cell body or axonal preparations, despite GAPDH showing more protein in those samples than in the Khsrp^+/+ samples.

Figure 3.

KHSRP deletion increases in vivo axon regeneration after a conditioning sciatic nerve injury. (A) Regeneration indices calculated as fraction of SCG10 axonal profiles at injury site (0 μm) are shown as mean ± SEM as indicated. There was no significant differences between Khsrp^–/– and Khsrp^+/+ mice at 7 and 10 days post-injury, but significant differences are seen between the genotypes at 14 days post-injury (n = 6 mice per genotype and timepoint, * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001 by two-way ANOVA with Bonferroni post-hoc analysis). (B) Representative SCG10 immunostained images of sciatic nerves after single (3 days; upper panels) or injury-conditioned (7 + 3 days; lower panels) sciatic nerve crush injuries for Khsrp^+/+ and Khsrp^–/– are shown. Proximal is on the left and distal on the right; the dashed line indicates the injury site, with the second injury for the double crush injured animals placed at ∼0.5 cm proximal to the initial injury site [scale bar = 500 μm]. (C) Regeneration indices calculated as fraction of SCG10 axonal profiles relative to the injury site (0 μm) are shown as mean ± SEM as indicated. There was no significant difference in the regeneration after the single injury, but the injury-conditioned Khsrp^–/– mice show significantly higher regeneration indices. Refer to Supplemental Figure S3B for regeneration index comparisons of naïve versus injury-conditioned nerves within genotypes (N = 5 mice per genotype and condition; * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001 by two-way ANOVA with Bonferroni post-hoc analysis). (D) Confocal XYZ images of gastrocnemius muscles of the injury-conditioned Khsrp^+/+ and Khsrp^–/– mice at 14 days after second nerve crush are shown. NMJs are detected by post-synaptic (α-bungarotoxin; green) and pre-synaptic markers (cocktail of anti-NF, -synapsin I and -synaptophysin; red) signals showing higher matching of pre- and post-synaptic markers in Khsrp^–/– than Khsrp^+/+ mice. Inset panels on lower right of both rows show higher magnification of the NMJs outlined by dashed boxes [scale bars = 20 μm for main panels and 5 μm for insets]. (E) Quantification of NMJ occupancy (% presynaptic area/postsynaptic area) shows significantly greater occupancy in the injury-conditioned Khsrp^–/– than in Khsrp^+/+ mice but no difference between genotypes was seen with the single crush lesion (injury-conditioned = 7 + 14 days; single nerve crush = 14 days). Data are expressed as mean ± SEM (N ≥ 15 NMJs quantified in three animals per condition per genotype; ** P ≤ 0.01 by Student's t test).

Figure 4.

Khsrp mRNA is transported into PNS axons. (A) Representative confocal images for smFISH/IF for Khsrp mRNA (grey) and NF (magenta) in dissociated DRG cultures are shown as indicated. There is a clear signal for Khsrp mRNA in the cell body (left and middle columns) and distal axons (right column) of DRGs from Khsrp^+/+ mice. Axons of Khsrp^–/– DRGs show Khsrp mRNA signals comparable to the scrambled probe; however, there was faint Khsrp signal in the soma of the Khsrp^–/– cultures that was consistently above the scrambled probe signal [scale bar = 10 μm].(B, C)Quantification smFISH signals for Khsrp mRNA in soma (B) and axons (C) is shown as mean ± SEM for scramble (Khsrp^+/+) and Khsrp mRNA (Khsrp^+/+ and Khsrp^–/–) probes; in each case, scramble probe was hybridized to Khsrp^+/+ DRG cultures as in panel A (N ≥ 16 neurons over three separate cultures; *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 by one way ANOVA with Tukey's post-hoc analysis). (D) Representative confocal images for smFISH/IF for Khsrp mRNA (grey) and NF (magenta) in uninjured sciatic nerve are shown as indicated. Left column shows XYZ projections from eight optical planes taken at 0.2 μm Z step intervals; right column shows ‘axon only’ Khsrp mRNA signals generated by extracting FISH signals overlapping with NF in individual Z sections and projecting those as an ‘Axonal Khsrp mRNA’ XYZ image [scale bar = 5 μm]. (E) Quantification of axonal Khsrp mRNA signals from (D) are shown as mean ± SEM (N = 6 animals per genotype; ** P ≤ 0.01 by Student's t-test). (F) Representative matched exposure smFISH/IF images for Khsrp mRNA, NF, GFAP and DAPI in naïve versus 7 days post-crush injured sciatic nerves as indicated. Upper row shows merged signals as single XY planes; lower two rows show XYZ projections for Khsrp mRNA colocalizing with NF (middle row) and GFAP (lower row). Representative matched exposure images for scramble probe are shown in Supplemental Figure S3E [scale bar = 5 μm]. (G) Quantification of axonal smFISH signals for Khsrp mRNA in naive and 7 days regenerating sciatic nerve axons (N = 6 animals; no significant differences detected by Student's t-test).

Figure 5.

Khsrp mRNA is rapidly translated in axons after injury. (A) Schematic of nerve ligation model used for panels B and C. Proximal nerve is on the left and distal on the right as indicated. The nerve was ligated and then immediately crushed ∼1 cm distal to the ligation.(B, C) Confocal images for KHSRP protein in naïve (B) and post-crush injury (3 and 16 h; (C). Upper rows of image pairs show XYZ projections of merged signals for KHSRP (grey) and NF (magenta), while lower rows show ‘axonal KHSRP’ signals as from individual Z planes that were projected as an XYZ image. As in Figure 1E, the strong signals that are outside of the axons are in Schwann cell nuclei based on DAPI co-labeling (data not shown). Representative images for ligation efficiency and KHSRP signals proximal and distal to ligation are shown in Supplemental Figure S4 [scale bar = 5 μm]. (D) Quantification of the axonal KHSRP immunoreactivity from ligation proximal and distal and crush sites are shown as mean ± SEM (N = 3 animals per time point; * P ≤ 0.05 and ** P ≤ 0.01 for indicated time points versus naïve nerve, ††P ≤ 0.01 for indicated time points versus ligation proximal, and && P ≤ 0.01 for indicated time points versus ligation distal by Student's t-test; ligation proximal versus distal have no significance). (E) Representative immunoblots for ex vivo puromycinylated naïve versus crushed sciatic nerve segments are shown as indicated. Protein synthesis inhibition with anisomycin completely blocks the puromycinylation of KHSRP in axoplasm samples extruded from the nerve segments, and GAPDH shows relatively equivalent protein loading across the lanes. Note that total KHSRP levels also increases with crush injury and this was attenuated by anisomycin. (F) Quantification of puromycinylated KHSRP signals from (D) is shown as mean ± SEM. Crush injury significantly increases axonal KHSRP synthesis and this blocked by anisomycin (N = 3; *** P ≤ 0.001 by one way ANOVA with Tukey's post-hoc analysis). (G) FRAP analyses for distal axons of neurons transfected with GFP^MYR5’/3’khsrp (schematic above graph) is shown as normalized average % recovery ± SEM. Pre-treatment with anisomycin or cycloheximide significantly attenuates the GFP recovery, indicating protein synthesis dependent recovery for GFP^MYR fluorescence in the axons (N ≥ 10 neurons over three culture preparations; * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.005 by two-way ANOVA with Bonferroni post-hoc analyses for indicated time points versus control). Representative images sequences for FRAP are shown in Supplemental Figure S5. (I) Schematic for proposed signaling pathway for axotomy induced increase in axoplasmic Ca²⁺ leading to eIF2α phosphorylation is shown. GSK260614 was used as a specific PERK inhibitor and Sephin1 as an inhibitor of eIF2α dephosphorylation. (H) FRAP analyses for distal axons of neurons transfected with GFP^MYR5’/3’khsrp reporter is as normalized average % recovery ± SEM. Treatment with thapsigargin increased and BAPTA-AM decreased recovery over control conditions. The thapsigargin-induced increase was blocked by GSK260614, while the BAPTA-AM-induced decrease was partially blocked by Sephin1 (N ≥ 17 neurons over three culture preparations; # P ≤ 0.05, ## P ≤ 0.01, ### P ≤ 0.005 and #### P ≤ 0.001 for thapsigargin or BAPTA-AM versus corresponding control time points and +P ≤ 0.05, ++P ≤ 0.01, +++P ≤ 0.005 and ++++P ≤ 0.001 for thapsigargin or BAPTA-AM versus corresponding thapsigargin + GSK260614 or BAPTA-AM + Sephin1 time points by two-way ANOVA with Bonferroni post-hoc analyses for indicated time points versus control; for data points appearing to error bars, the SEM is too small to show).

Figure 6.

Increased levels of KHSRP target mRNAs in Khsrp^–/– neurons. (A) Sciatic nerve levels of Gap43, Snap25 and Fubp1 mRNAs are increased 7 days after sciatic nerve crush in Khsrp^–/– compared to Khsrp^+/+ mice. In contrast, sciatic nerve Hmgb1 mRNA levels show no change comparing the Khsrp^–/– versus Khsrp^+/+ mice. Values shown as mean ± SEM (N = 5 mice per genotype; * P ≤ 0.05, ** P ≤ 0.01 and NS for indicated pairs across genotype within condition and & P ≤ 0.05 and && P ≤ 0.01 for crush versus naïve within genotype by Student's t-test). (B) Schematic of expression constructs used for rescue experiments shown in panels C–F. (C) Analyses of soma and axon Gap43 and Fubp1 mRNA levels in Khsrp^–/– DRG neurons transfected with GFP, GFP-KHSRP and GFP-KHSRPΔKH4 is shown as mean mRNA copies/ng of total RNA ± SEM after normalization to mitochondrial 12S RNA. Supplemental Figure S6A shows expression levels for GFP, GFP-KHSRP and GFP-KHSRPΔKH4 and Supplemental Figure S6B shows RNA levels for transfected Khsrp^+/+ DRG cultures (N = 3 per condition; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 and NS = not significant for indicated pairs by Student's t test).(D, E)Representative images of DRG neurons transfected with GFP, GFP-KHSRP or GFP-KHSRPΔKH4 are shown for Khsrp^+/+ and Khsrp^–/– (schematics for constructs above image columns) are shown in (D) as indicated. Quantification of total axon length/neuron for transfected DRG neurons is shown in (E) as mean ± SEM. Expression of GFP-KHSRP decreases axon outgrowth in both Khsrp^+/+ and Khsrp^–/– DRGs, but GFP-KHSRPΔKH4 had no significant effect on Khsrp^+/+ and only a modest decrease in axon length in Khsrp^–/– neurons (N ≥ 30 neurons over three different culture preparations; * P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001 by one-way ANOVA with Tukey's post-hoc analysis for indicated comparisons) [scale bar = 100 μm].

Figure 7.

Axon growth promotion from loss of neuronal KHSRP. (A–C) DRGs cultured from Khsrp^fl/fl mice in (A) show reduced Khsrp mRNA by RTddPCR after transduction with AAV2-CMV-GFP-Cre compared to AAV2-CMV-GFP. AAV2-CMV-GFP-Cre transduction of wild type DRGs had no effect on Khsrp mRNA levels. By immunofluorescence where only neuronal KHSRP levels were assessed in (B), the AAV2-CMV-GFP-Cre transduced Khsrp^fl/fl DRGs showed greater than 80% reduction in KHSRP signals. Representative immunofluorescent images in (C) show relative absence of KHSRP signals in neuronal cell body and axons of DRGs, but prominent signals in adjacent Schwann cells of the AAV2-CMV-GFP-Cre transduced Khsrp^fl/fl dissociated DRG culture (N = 6 culture preparations for each condition for (A) and N = 29 neurons in three separate transfections for (B); *** P ≤ 0.005 and **** P ≤ 0.001 for indicated pairs by Students t-test) [scale bar = 10 μm].(D, E)Representative immunofluorescent images for NF in AAV2-CMV-GFP versus AAV2-CMV-GFP-Cre transduced Khsrp^fl/fl mice is shown in (D). Quantification of axon length and axon branching (E) as mean ± SEM indicate show significantly increased axon growth with Cre-driven deletion of KHSRP in the Khsrp^fl/fl mouse DRGs (N ≥ 75 neurons over three separate culture preparations/transductions for each condition; *** P ≤ 0.005 and NS = not significant by Student's t-test) [scale bar = 100 μm]. (F) Schematic with time line for viral transduction of Khsrp^fl/fl mice followed by double sciatic nerve crush lesion as used in Figure 3. Not that the AAV2-CMV-GFP-Cre injection site is separated from both nerve crush sites by ∼0.75 and ∼1.25 cm. The animals were transduced on day 0, underwent distal nerve crush on day 10 (crush # 1), and underwent proximal nerve crush on day 17 (crush # 2). Regeneration was evaluated 3 days later. (G, H) Representative confocal images for KHSRP and NF with DAPI staining of sciatic nerve from AAV2-CMV-GFP-Cre transduced wild type and Khsrp^fl/fl mice after nerve crush injury # 2 are shown in (G) as indicated. (H) shows regeneration indices for AAV2-CMV-GFP-Cre transduced wild type and Khsrp^fl/fl mice 3 days after nerve crush injury # 2. Supplemental Figure S7 shows SCG10 and GFP immunofluorescence to compare regeneration between the AAV2-CMV-GFP-Cre transduced wild type and Khsrp^fl/fl mice (N = 6 mice per genotype; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.005 and **** P ≤ 0.001 by two-way ANOVA with Bonferroni post-hoc analysis) [scale bar = 10 μm].