Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury - PubMed (original) (raw)

Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury

Maritza Oñate et al. Sci Rep. 2016.

Erratum in

Abstract

Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury.

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Figures

Figure 1

Figure 1. Unfolded protein response is activated after sciatic nerve injury.

Wild-type mice were injured and at different days post-injury (dpi) a 5 mm segment of the sciatic nerve was removed in the injured segment (site of injury or middle, M) and proximal (P) and distal (D) for posterior analysis. BiP protein levels were evaluated at (A) 24 h post injury (hpi) and at (B,C) 8 and 21 days post injury (dpi) in P, M and D segments and compared to contralateral uninjured nerves (label as C). Protein levels were quantified by densitometry and normalized with Hsp90 expression (bottom panel). (D) Xbp1s mRNA expression was quantified by real-time PCR in D segment at 2, 8 and 14 dpi. (E) Xbp1 spliced (Xbp1s) and unspliced (XBP1u) forms mRNA levels were analyzed in D segment at 14 dpi by RT-PCR followed by PstI digestion. Actin levels were used as loading control. (F) Wfs1, Atf3, (G) Chop and Gadd34, expression were analyzed from sciatic nerves by real-time PCR in uninjured conditions and at 14 dpi in D segments. Data is expressed as mean ± S.E.M.; *p < 0.05, ***p < 0.001, n.s.: non significant. Statistical differences were analyzed using student’s t-test (n = 3 animals per group).

Figure 2

Figure 2. ER stress responses after sciatic nerve crush.

Wild-type mice were crush injured in the right sciatic nerve and sham operated in the left sciatic nerve. After 8 days, nerves were removed for histological analysis. (A) KDEL staining (red) was performed using indirect immunofluorescence and co-stained with S100 (Schwann cells), NF-M (axons), MBP (myelin), and a Cd11b (macrophages) in green. Cell nuclei were counter stained using DAPI (blue). Co-localization is denoted using white arrows and S100 negative KDEL positive cells with asterisk. Scale bar: 20 μm. (B) DRGs and were collected from animals described in A and KDEL staining (red) was performed together with the neuronal marker NF-M (green). Scale bars: 100 μm. (C) Thoracic spinal cord from animals described in A was analyzed for KDEL staining (red). Scale bars: 100 μm (low magnification) and 40 μm (high magnification).

Figure 3

Figure 3. XBP1 deficiency decreases myelin removal, axonal regeneration and locomotor recovery after sciatic nerve injury.

(A) XBP1WT and XBP1Nes−/− mice were crush injured in the right sciatic nerve and sham operated in the left sciatic nerve. Locomotor recovery was evaluated using the SFI analysis before (0 day) and at indicated time points. (B) Electron microscopy of uninjured and distal segments of 14 dpi from XBP1WT and XBP1Nes−/− mice. White arrowheads indicate intact myelinated axons, black arrowheads, intact myelins, and asterisks unmyelinated axons. Black arrows point to degenerated myelins and white arrows, to remyelinated axons. Scale bar: 2 μm. (C) Transversal semi-thin sections of sciatic nerves stained with toluidine blue from uninjured and at 14 dpi XBP1WT and XBP1Nes−/− mice. Sections were obtained 3 mm distal to crush segment. Black and white arrows indicate demyelinated and regenerated fibers, respectively (left panel). Scale bar: 10 μm. Quantification of degenerated myelins and remyelinated axons density is presented (right panel). Data are shown as mean ± S.E.M.; *p < 0.05; **p < 0.01. SFI data were analyzed by repeated measures ANOVA followed by Bonferroni’s post hoc test (n = 7 animals per group). Morphological data was analyzed at each time point by student’s t-test (n = 3 animals per group).

Figure 4

Figure 4. ATF4 deficiency does not affect axonal regeneration or locomotor recovery after sciatic nerve injury.

(A) Wild-type (WT) and ATF4−/− mice were crush injured in the right sciatic nerve and sham operated in the left sciatic nerve. Locomotor recovery was evaluated using the SFI analysis before (0 day) and at indicated time points. (B) Electron microscopy of uninjured and distal segments of 14 dpi from WT and ATF4−/− mice. White arrowheads indicate intact myelinated axons and black arrowheads, intact myelins. Black arrows point to degenerated myelins and white arrows, to remyelinated axons. Scale bar: 2 μm. (C) Transversal semi-thin sections of sciatic nerves stained with toluidine blue from uninjured and at 14 dpi WT and ATF4−/− mice. Sections were obtained 3 mm distal to crush segment. Black and white arrows indicate demyelinated and regenerated fibers, respectively (left panel). Scale bar: 10 μm. Quantification of degenerated myelins and remyelinated axons density is presented (right panel). Data are shown as mean ± S.E.M.; SFI data were analyzed by repeated measures ANOVA followed by Bonferroni’s post hoc test (n = 7 animals per group). Morphological data was analyzed at each time point by student’s t-test (n = 3 animals per group).

Figure 5

Figure 5. Overexpression of XBP1s increases myelin removal, axonal regeneration and locomotor recovery after peripheral nerve injury.

(A) TgXBP1s and Non-Tg mice were damaged in the right sciatic nerve and sham operated in the left side. Locomotor performance was evaluated using the SFI before at the indicated time points. (B) Uninjured and distal segments of 14 dpi from TgXBP1s and Non-Tg mice were analyzed by EM. In control conditions black arrowheads indicate compact myelin, white arrowheads, myelinated axons and asterisk, unmyelinated axons. At 14 dpi, white and black arrows point remyelinated fibers and degenerated myelins, respectively. Scale bar: 2 μm. (C). TgXBP1s and Non-Tg sciatic nerves from uninjured and at 14 dpi transverse semi-thin sections were obtained 3 mm distal to the crush segment and stained with toluidine blue. Black and white arrows indicate demyelinated and regenerated fibers, respectively (left panel). Scale bar: 10 μm. Quantification of the density of degenerated myelins and remyelinated axons was performed from transverse semi-thin sections of each mouse strain (right panel). Data are shown as mean ± S.E.M.; *p < 0.05; **p < 0.01. SFI data were analyzed by repeated measures ANOVA followed by Bonferroni’s post hoc test (n = 7 animals per group). Morphological data was analyzed at each time point by student’s t-test (n = 3 animals per group).

Figure 6

Figure 6. XBP1 expression in the nervous system enhances macrophage infiltration in injured sciatic nerves.

(A) Sciatic nerves from XBP1Nes−/− and XBP1WT littermates were processed for immunofluorescence from uninjured conditions and at 14 dpi distal sciatic nerves were analyzed for Cd11b (green) to evaluate macrophages and MBP (red) to stain myelin sheaths. Nuclei were counterstained using DAPI (blue, left panel). The staining density for Cd11b was quantified at 14 dpi in XBP1Nes−/− and XBP1WT mice (right panel). (B) TgXBP1s and non-Tg sciatic nerves were analyzed as described in A. Mcp-1 expression was analyzed in sciatic nerves of XBP1Nes−/− and XBP1WT mice (C) or in TgXBP1s and non-Tg sciatic nerves (D) by real-time PCR at 2 dpi. Data are shown as mean ± S.E.M.; *p < 0.05; **p < 0.01. Data were analyzed by student’s t-test at each time point (n = 3 animals per group). Scale bar: 20 μm.

Figure 7

Figure 7. XBP1s overexpression in neurons enhances axonal regeneration in vivo.

(A) Schematic representation of the experimental design. Wild-type mice were injected with 1μl of AAV EGFP, AAV XBP1s/EGFP or AAV shXBP1/EGFP in L3 and L4 DRGs. EGFP expression was used to identify transduced neurons. At 7 days post-injection the sciatic nerve was crushed at the sciatic notch level (yellow light bolt) and at 14 dpi the nerves were dissected and analyzed in transverse sections 3 mm distal to the injury (black arrow) and normalized to EGFP-positive axons 6 mm proximal to the crush (C-labeled box). (B) EGFP expression (green) of infected neurons from DRGs, 7 days after injection in uninjured nerves. NF-M immunostaining (red) was used to identify neuronal somas and axons. Scale bar: 100 μm. (C) Cross section of an uninjured nerve 7 days after AAV injection. EGFP fluorescence in green, immunostained for NF-M (red) and counterstained using DAPI (blue). Transduced somas and axons are indicated with white arrowheads in the insets of B and C. Scale bar: 100 μm. (D) Axonal regeneration in the distal sciatic nerve was evaluated in AAV-EGFP (upper panel), AAV-XBP1/EGFP (middle panel) and AAV-shXBP1/EGFP (lower panel) injected DRGs at 14 dpi. Scale bar: 20 μm. (E) Quantification of mean EGFP+/NF-M+ axons in distal segment normalized to proximal EGFP+/NF-M+ axons of the same sciatic nerve from AAV-EGFP, AAV-XBP1s/EGFP and AAV-shXBP1/EGFP injected mice. Data are expressed as mean ± S.E.M. *p < 0.05. Student’s t-test was performed for statistical analysis against control EGFP condition (n = 3 per condition).

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