Targeting p75 neurotrophin receptors ameliorates spinal cord injury-induced detrusor sphincter dyssynergia in mice

Abstract

Aims: To determine the role of p75 neurotrophin receptor (p75^NTR ) and the therapeutic effect of the selective small molecule p75^NTR modulator, LM11A-31, in spinal cord injury (SCI) induced lower urinary tract dysfunction (LTUD) using a mouse model.

Methods: Adult female T₈ -T₉ transected mice were gavaged daily with LM11A-31 (100 mg/kg) for up to 6 weeks, starting 1 day before, or 7 days following injury. Mice were evaluated in vivo using urine spot analysis, cystometrograms (CMGs), and external urethral sphincter (EUS) electromyograms (EMGs); and in vitro using histology, immunohistochemistry, and Western blot.

Results: Our studies confirm highest expression of p75^NTRs in the detrusor layer of the mouse bladder and lamina II region of the dorsal horn of the lumbar-sacral (L₆ -S₁ ) spinal cord which significantly decreased following SCI. LM11A-31 prevented or ameliorated the detrusor sphincter dyssynergia (DSD) and detrusor overactivity (DO) in SCI mice, significantly improving bladder compliance. Furthermore, LM11A-31 treatment blocked the SCI-related urothelial damage and bladder wall remodeling.

Conclusion: Drugs targeting p75^NTRs can moderate DSD and DO in SCI mice, may identify pathophysiological mechanisms, and have therapeutic potential in SCI patients.

Keywords: LM11A-31; lower urinary tract dysfunction/symptoms (LUTD/LUTS); neurodegeneration; neurogenic bladder dysfunction; proneurotrophins.

FIGURE 1

Schematics of p75^NTR signaling pathways. A, The p75^NTR signals through dimerization with sortilin or Trk receptors. p75^NTR -sortilin complexes will preferentially bind proBDNF/proNGF that activate apoptotic signaling cascades. Conversely, p75^NTR-Trk receptors bind to mature neurotrophins to activate cell survival pathways. LM11A-31 is reported to be a dual-action drug that can downregulate apoptotic JNK signaling though the p75^NTR-sortlin dimer by blocking proneurotrophin binding, while promoting activation of AKT mediated survival pathways through disinhibition and/or activation of the TrkA/BNTR-p75 dimer. B, Western blot analysis demonstrates that LM11A-31 inhibits phosphorylation of JNK in UROtsa cells challenged with protease resistant proNGF

FIGURE 2

Urine spot analysis and the beneficial effect of LM11A-31 in SCI mice. Tests were conducted on control or SCI mice, receiving vehicle (water) or LM compound (n ≥ 4). A, Normal voiding pattern from a mouse with an intact spinal cord receiving water. B, A control mouse receiving LM11A-24 (100 mg/kg/day). C, A control mouse receiving LM11A-31 (100 mg/kg/day). D, Voiding pattern of a SCI mouse receiving water. E, SCI mouse receiving LM11A-24. F, SCI mouse receiving LM11A-31

FIGURE 3

LM11A-31 ameliorates SCI-induced DSD and NDO. A, Control mouse. The inset (A1, right) shows the boxed area of the expanded time-base. B, SCI mouse two weeks post-injury, receiving vehicle (water). C, SCI mouse receiving LM11A-31 starting 1 week post-SCI (n ≥ 4). D, SCI mouse receiving LM11A-24 starting 1 week post-SCI. E, Control mouse, CMG recording. F, SCI mouse 6 weeks postinjury, receiving vehicle (water). G, SCI mouse 6 weeks post-injury, receiving LM11A-31 starting from the day prior to injury

FIGURE 4

Pretreatment with LM11A-31 or -24 prevents SCI-induced adverse morphological changes in the bladder wall. A, H&E stained section of a control mouse bladder. B, Bladder section of a SCI mouse (1 day post-injury) receiving water. C, Bladder section of a SCI mouse (1 day post-injury) administered LM11A-31. D, Bladder section of a SCI mouse (10 days post-injury) receiving water. E, Bladder section of a SCI mouse bladder (10 days post-injury) receiving LM11A-31. (A–E., n ≥ 3). F, Value of TER in bladder wall sheets from control and SCI mice (1 day post-injury) with or without LM11A-31 or LM11A-24 treatment. All treatments commenced one day prior to injury. Mean data ± SD; P ≤ 0.05 versus control (n ≥ 4, unpaired Student’s t-test). G, p75^NTR ECD levels measured by ELISA (Biosensis) from mouse urine. These values dramatically increased 1 day following SCI (*P < 0.05, n = 4) in comparison to control animals (n = 6). At this time point, pretreatment with LM11A-31 significantly decreased the amount of cleaved p75^NTR ECD (**P < 0.05, n = 4). The p75^NTR ECD levels measured three days post-transection, were comparatively lower, and the levels were sustained until 7 days post-injury. The LM11A-31 treatment decreased the sustained p75^NTR ECD levels in the urine at 3 and 7 days following SCI. H, Western blot analysis of p75^NTRs, proBDNF, proNGF, and β-actin in the mucosa and detrusor of control, SCI, and LM11A-31 treated mice 7 days post-injury. The full-length p75^NTR protein (75 kDa band) was highest in control samples and significantly decreased in both mucosa and detrusor layers following SCI. Daily treatment with LM11A-31 prevented the decrease in full-length p75^NTR. The antibody used for p75^NTR detection also detected multiple smaller weight bands resulting from proteolytic cleavage following receptor activation. I) Relative expression of p75NTR, proBDNF, and proNGF normalized to β-actin. *P < 0.05, versus control samples

FIGURE 5

Immunofluorescent labelling of p75^NTRs, their colocalization with Calcitonin Gene-Related Peptide(CGRP) and Tyrosine Hydroxylase (TH)-positive nerves-effect of LM11A-31. A, p75NTRs (green) were expressed on TH-positive structures (red) in spinal cord (SC) intact mouse bladders. B, Seven days post-SCI. C, p75NTRs and their colocalization with TH-positive nerves was present in bladders of SCI mice (7 days post-injury) pretreated with LM11A-31. The colocalization between SC intact, SCI, and SCI with LM11A-31 treatment were not significantly different; P > 0.05, One-way ANOVA with Tukey’s multiple comparison test. D, p75^NTRs also colocalized with bladder CGRP-positive nerves (red) in SC intact animals. Colocalization is indicated by merged yellow fluorescence and highlighted with yellow arrows. E, Seven days post-SCI: p75^NTR-immunoreactivity (green) decreased compared to SC intact mice, the % area colocalized with CGRP (red) did not significantly change. F, Expression level of p75^NTR and colocalization of p75^NTRs with CGRP-positive nerves was not found to differ from vehicle treated SCI mice (DT = detrusor; LP = lamina propria; and UT = urothelium). G, p75^NTR expression localized to the dorsal horn (white outlined region) of L6-S1 spinal cord segments in SC intact mice. H, p75^NTR labeling in 7 day post-SCI mouse with decreased expression in lamina-II region. I, SCI mice with LM11A-31 for 7 days. J, Colocalization analysis of p75^NTR CGRP and K) TH immunofluorescence. The % area that overlapped between p75^NTR and CGRP or p75^NTR and TH immunofluorescence were quantified using MATLAB software within the total bladder wall area as defined by DAPI staining. L, p75^NTR and CGRP expressing fiber in SC intact mouse urothelium (white bordered region). M) p75^NTR expression in urothelial basal layer of SCI mouse bladders which was not found in SC intact mice. Similar urothelial staining profile was also seen with SCI + LM11A-31 and SCI + LM111A-24 treated mice. Graphical representation of % area expression of p75^NTR in the bladder wall and L6-S1 dorsal horn are shown in Supplemental Figure S12