Bex1 is involved in the regeneration of axons after injury

Abstract

Successful axonal regeneration is a complex process determined by both axonal environment and endogenous neural capability of the regenerating axons in the central and the peripheral nervous systems. Numerous external inhibitory factors inhibit axonal regeneration after injury. In response, neurons express various regeneration-associated genes to overcome this inhibition and increase the intrinsic growth capacity. In the present study, we show that the brain-expressed X-linked (Bex1) protein was over-expressed as a result of peripheral axonal damage. Bex1 antagonized the axon outgrowth inhibitory effect of myelin-associated glycoprotein. The involvement of Bex1 in axon regeneration was further confirmed in vivo. We have demonstrated that Bex1 knock-out mice showed lower capability for regeneration after peripheral nerve injury than wild-type animals. Wild-type mice could recover from sciatic nerve injury much faster than Bex1 knock-out mice. Our findings suggest that Bex1 could be considered as regeneration-associated gene.

Fig. 1. Bex1 expression in axons

MNs from E13-14 mouse embryos were fixed at 3 DIV and stained with antibodies specific for Bex1 (green), MAP2 (red) and the Tau-1 antibody (blue). The scale bar is 40μm.

Fig. 2. Bex1 upregulation in MNs after axonal injury

a) MNs from the spinal cord of MPZ-KO and MAG-KO mice and mice subjected to SN-crush injury or demyelination were dissected by Laser capture microdissection and their total RNA was used for quantitative real-time PCR. n =3, error bars represent SEM, * p < 0.001, ** p < 0.005, ANOVA compared to MNs derived from un-injured WT mice b) To monitor the expression of Bex1 protein, the tissue sections from lumbar-spinal cord of MPZ-KO and MAG-KO mice and mice subjected to sciatic-nerve crush-injury or demyelination were stained with anti Bex1 (green) and anti-nonphosphorylated neurofilament (SMI32; a marker of neurons) (red) antibodies. DAPI (blue) was used for nuclear staining. Scale bar is 50 μm. Note elevation of Bex immunostaining following crush injury.

Fig. 2. Bex1 upregulation in MNs after axonal injury

a) MNs from the spinal cord of MPZ-KO and MAG-KO mice and mice subjected to SN-crush injury or demyelination were dissected by Laser capture microdissection and their total RNA was used for quantitative real-time PCR. n =3, error bars represent SEM, * p < 0.001, ** p < 0.005, ANOVA compared to MNs derived from un-injured WT mice b) To monitor the expression of Bex1 protein, the tissue sections from lumbar-spinal cord of MPZ-KO and MAG-KO mice and mice subjected to sciatic-nerve crush-injury or demyelination were stained with anti Bex1 (green) and anti-nonphosphorylated neurofilament (SMI32; a marker of neurons) (red) antibodies. DAPI (blue) was used for nuclear staining. Scale bar is 50 μm. Note elevation of Bex immunostaining following crush injury.

Fig. 3. The function of Bex1 under MAG signalling on axonal growth

a) MNs prepared from E13-14 WT or Bex-KO mice embryos were cultured on PLL substrate or PLL + MAG (4μg/ml). WT- MNs were transfected 2 h after plating with expression vectors for GFP, or GFP-Bex1, and Bex-KO- MNs were transfected 2 h after plating with expression vectors for GFP. Transfected cells (GFP-positive) were analyzed at 3 DIV. The scale bar is 40 μm. b) The average length of the axons are measured from the soma along the process to its tip by using ImageJ software (released by NIH) (n=3, means ± SEM; * p < 0.01, ANOVA compared to neurons cultured on just PLL).

Fig. 4. Retrograde labelling of spinal cord MNs in vivo

Retrograde labelling was performed for the quantification of spinal-MN perikarya within the ventral horn that projects to the sciatic nerve. 7 and 14 days after sciatic-nerve injury, the sciatic nerve was exposed again and a small crystal of DiI was placed 7 mm distal to the crush lesion site. The dye was transported retrogradely to the MN perikarya. In the sham-operated controls, the sciatic nerve was exposed but not injured. After 2 days, spinal cords were removed and sectioned. The serial sections were analyzed by microscope (a) and the number of labelled MNs was counted on each section (b), data are presented as average ± SEM. Repeated-measures ANOVA with Bonferroni-Holm post hoc analysis was completed to assess statistical differences.

Fig. 5. Behavioral analysis illustrates functional recovery following SN injury

a) Rotarod test was used to assess the motor coordination and balance of the animals on days 7, 14, 21 and 28 after operation. Rotation was started at a speed of 4 rpm and accelerated to 40 rpm in 270 sec. The latency period until the mice fell off the apparatus was monitored for 300 sec. b) Gait analysis was used to measure Sciatic Functional Index (SFI). The hind feet of mice were painted and the mice allowed to walk freely on a blank strip of paper. The SFI was calculated after measuring the distance to Opposite Foot (TOF), Print Length (PL), Total Toes Spreading (TS) and distance between Intermediary Toes (IT). c) Toe-pinch reflex was used to assess the recovery of sensory function by pinching the most distal portion of the last three toes of the injured hindlimb with a flattened forceps. Foot withdrawal was recorded as positive responses indicative of recovery. n = 3, data are presented as average ± SEM. Repeated-measures ANOVA with Bonferroni-Holm post hoc analysis was completed to assess statistical differences.