A novel inhibitor of p75-neurotrophin receptor improves functional outcomes in two models of traumatic brain injury

Abstract

The p75 neurotrophin receptor is important in multiple physiological actions including neuronal survival and neurite outgrowth during development, and after central nervous system injury. We have discovered a novel piperazine-derived compound, EVT901, which interferes with p75 neurotrophin receptor oligomerization through direct interaction with the first cysteine-rich domain of the extracellular region. Using ligand binding assays with cysteine-rich domains-fused p75 neurotrophin receptor, we confirmed that EVT901 interferes with oligomerization of full-length p75 neurotrophin receptor in a dose-dependent manner. Here we report that EVT901 reduces binding of pro-nerve growth factor to p75 neurotrophin receptor, blocks pro-nerve growth factor induced apoptosis in cells expressing p75 neurotrophin receptor, and enhances neurite outgrowth in vitro Furthermore, we demonstrate that EVT901 abrogates p75 neurotrophin receptor signalling by other ligands, such as prion peptide and amyloid-β. To test the efficacy of EVT901 in vivo, we evaluated the outcome in two models of traumatic brain injury. We generated controlled cortical impacts in adult rats. Using unbiased stereological analysis, we found that EVT901 delivered intravenously daily for 1 week after injury, reduced lesion size, protected cortical neurons and oligodendrocytes, and had a positive effect on neurological function. After lateral fluid percussion injury in adult rats, oral treatment with EVT901 reduced neuronal death in the hippocampus and thalamus, reduced long-term cognitive deficits, and reduced the occurrence of post-traumatic seizure activity. Together, these studies provide a new reagent for altering p75 neurotrophin receptor actions after injury and suggest that EVT901 may be useful in treatment of central nervous system trauma and other neurological disorders where p75 neurotrophin receptor signalling is affected.

Keywords: EVT901; TBI; neuron; oligodendrocyte; p75NTR.

The p75 neurotrophin receptor (p75NTR) is a member of the TNF-receptor superfamily. Delbary-Gossart, Lee et al. describe the neuroprotective effects of a novel piperizine derivative that blocks p75NTR. In two rodent models of traumatic brain injury, EVT901 protects neurons and glia, reduces inflammation and seizure susceptibility, and improves neurological outcomes.

Figure 1

Mechanism of action through the CRD1 domain of p75NTR: cellular response and induction of neuritogenesis. (A) EVT901 inhibits the binding of AP-p75NTR to the receptor p75NTR. Binding experiments on SKN-BE-p75NTR with soluble AP-p75NTR (10 nM) in the presence of increasing concentrations of EVT901. Data are reported as mean % inhibition of binding, representative of three independent experiments. (B) Competition of ¹²⁵I-NGF binding with EVT901. Binding of ¹²⁵I-NGF (0.3 nM) to SKNBE-p75NTR in the presence of increasing concentrations of EVT901. Results are mean % inhibition of specific binding, representative of three independent experiments. (C) HEK293T cells transiently co-transfected with the same amount of DNA originating from the p75NTR full-length tagged either in C-terminal with HA (HA- p75NTR) or in N-terminal with Flag (Flag-p75NTR) and then treated or not with the compound EVT901 at 100 nM for 24 h. Oligomerization evaluated using an ELISA assay with flag-coated plates and anti-HA-HRP antibody [two-way ANOVA, EVT901 dose effect, F(4,94) = 46.85, P < 0.0001; Tag, F(3,94) = 394.40, P < 0.0001; EVT901 dose × Tag, F(12,94) = 46.82, P < 0.0001]. ^***P < 0.0001 compared to 0. Data are indicated as mean ± SEM from six independent experiments. (D and E) HEK293T cells transiently transfected for 2 days with the same amount of DNA originating from the indicated constructs: empty vector (control), full-length of p75NTR (p75NTR), short-form of p75NTR (CRD1-p75NTR), full-length of CD40 (CD40), or a chimera of CD40 and the CRD1 domain of p75NTR (CD40-CRD1-p75NTR) (E), and then treated or not with the compound EVT901 at 100 nM for 24 h. Apoptosis quantified using an ELISA kit and data are expressed as mean number of oligonucleosomes/10⁶ cells ± SEM, three independent experiments. Two-way ANOVA [EVT901 dose effect, F(1,68) = 53.2, P < 0.0001; constructs, F(4,68) = 20.77, P < 0.0001; EVT901 effect × constructs, F(4,68) = 12.32, P < 0.0001]. ^**P < 0.005, ^***P < 0.001 (EVT901 versus untreated), ^###P < 0.001 (untreated control versus untreated constructs). (F) HEK293T cells transiently transfected for 24 h with the indicated constructs: empty vector (control), full-length of p75NTR (p75NTR), FAS, TNFRI, TNFRII, HVEM, LTβR, 4.1BB, or BCMA receptors, and then treated or not with the compound EVT901 at 100 nM for 24 h. Apoptosis was measured and expressed as mean number of oligonucleosomes/10⁶ cells ± SEM. Data are representative of two independent experiments. Two-way ANOVA [EVT901 dose effect, F(3,134) = 8.75, P < 0.0001; constructs, F(8,134) = 28.34, P < 0.0001; EVT901 dose effects × constructs, F(6,134) = 6.75, P < 0.0001]. ^***P < 0.001 (EVT901 versus untreated; ^###P < 0.001 (untreated control versus untreated constructs). (G) Inhibition of specific p75NTR ligand-induced apoptosis. Subconfluent monolayers of SH-SY5Y cells stimulated for 48 h with pro-NGF (10 ng/ml; solid blue line), PrP_106–126 (10 μM; solid black line), or amyloid-β_1–40 (10 μM; dashed black line), in addition to increasing concentrations of EVT901 (from 0.01 to 100 nM). Apoptosis was measured and reported as % inhibition of apoptosis, representative of three independent experiments. (H) Induction of neuritogenesis through TrkA phosphorylation. SHSY-5Y cells serum-deprived, and treated with rhNGF (10 ng/ml) or EVT901 (0.01 to 10 nM), and then maintained in the same media for 3 days. The neurite length was visualized after staining with May-Grunwald-Giemsa and digitally photographed from randomly selected image fields (magnification ×10). (I) Confluent SHSY-5Y cells serum-deprived, and treated with NGF (10 ng/ml) or EVT901 (100 nM) for different times (1–3 days). Quantification of levels of total TrkA or phosphorylated TrkA (Tyr490) expressed as OD₄₅₀ and representative of three independent experiments. Four-way ANOVA [time effect, F(2,18) = 21.0, P < 0.001; NGF effect, F(1,18) = 152.75, P < 0.0001; EVT901 effect, F(1,18) = 4.32, P = 0.052; pTrkA, not significant]. Note that there are significant interactions: time × NGF, F(2,18) = 33.81, P < 0.0001; time × EVT901, F(2,8) = 40.19, P < 0.0001; time × pTrkA, F(2,18) = 5.99, P = 0.01; NGF × pTrkA, F(1,18) = 167.68, P = 0.0001; EVT901 × pTrkA, F(1,18) = 43.79, P = 0.00010; time × NGF × pTrkA, F(2,18) = 50.54, P = 0.0001; time × EVT901 × pTrkA, F(2,18) = 25.23, P = 0.0001. ^***P < 0.001 compared to untreated control. (J) Chemical structure of EVT901; 1-(phenyl-3, 6-dihydro-2H-pyridinyl)-2-(aryl-bridged piperazines)-ethanone.

Figure 2

EVT901 blocks proNGF-mediated caspase activity and p75NTR expression in OPCs and oligodendrocytes in vitro and reduces proNGF-induced cell death. (A) Immunostaining showed that treatment with pro-NGF (50 ng/ml) induced caspase-3 activity in OPCs in vitro. (B and C) Caspase activity was measured by counting positive cells using the FLICA assay. Pro-NGF (50 ng/ml) increased caspase activity in cultured OPCs and oligodendrocytes. However, EVT901 added to the medium reduced the percentage of caspase-positive cells in a dose-dependent manner for OPCs and oligodendrocytes [one-way ANOVA, F(7,67) = 10.02, P < 0.0001 for OPCs and F(7,65) = 10.55, P < 0.0001 for oligodendrocytes]. (D–F) Both OPCs and oligodendrocytes expressed p75NTR in a small percentage of cells in vitro, this was increased significantly by the addition of pro-NGF, and co-incubation with EVT901 reduced or inhibited p75NTR expression. Note that mature NGF (mNGF) had no effect on apoptosis or p75NTR expression. One-way ANOVA, F(7,105) = 20.30, P < 0.0001 and F(7,98) = 5.68, P < 0.0001 for OPCs and oligodendrocytes, respectively. ^***P < 0.0001 compared to cells alone; ^#P < 0.05, ^##P < 0.005, ^###P < 0.0001 compared to 0 nM EVT901. Scale bar = 30 μm.

Figure 3

Induction of p75NTR and proNGF in the white matter (corpus callosum) after CCI-TBI. (A–D) Immunoblot analysis showed that both p75NTR and proNGF protein levels are increased after TBI in the ipsilateral white matter as compared to uninjured (UN) and sham injured tissue. Two-way ANOVA, (A) for p75NTR: side, F(1,12) = 92.42, P < 0.0001; time, F(5,12) = 32.32, P < 0.0001; (C) for proNGF: side, F(1,12) = 126.54, P < 0.0001; time, F(5,12) = 18.18, P < 0.001. In ipsilateral white matter, p75NTR and proNGF are significantly increased [F(5,12) = 32.55, P < 0.0001 and F(5,12) = 11.72, P < 0.0001, respectively]. (B) P75NTR immunoreactivity is co-localized with CC1 expression in cells with oligodendrocyte morphologies (arrowheads). Scale bar = 20 μm. (D) ProNGF immunoreactivity is co-localized in CD11b-positive and GFAP-positive cells (arrowheads), suggesting that microglia and astrocytes are responsible for proNGF expression in the corpus callosum at 1 day after TBI. Scale bars = 50 μm. (E) Immunoblot analysis showed that TBI increased the cleaved caspase-3 expression on the ipsilateral side compared to the contralateral side at 8 days after injury [side, F(1,12) = 6.78, P < 0.05; time, F(5,12) = 6.02, P < 0.01]. In the ipsilateral white matter, cleaved caspase-3 protein level is significantly increased [F(5,12) = 6.02, P < 0.01]. *P < 0.05, ^**P < 0.005, ^***P < 0.0005, ^##P < 0.01 compared to sham; n = 3/group; Error bars indicate ±SEM. (F) Cleaved caspase-3 immunoreactivity is co-localized in CC1 positive oligodendrocytes (arrowheads). Scale bar = 50 μm. *P < 0.05, ^**P < 0.005, ^***P < 0.0005, ^##P < 0.01 compared to sham.

Figure 4

EVT901 treatment reduced atrophy in grey and white matter at 8 days post injury. (A) The region of interest (ROI) included tissue from 3 mm anterior to 3 mm posterior to bregma. (B and C) Illustration of representative brain figures of the hemisphere (B) and cerebral cortex and white matter (C), where stereological methods were used to estimate tissue volume by counting Cavalieri markers using Stereo Investigator. (C) The region of interest included the cerebral cortex from the midline to the rhinal fissure (small arrowhead); the ipsilateral cerebral cortex is indicated in red versus the contralateral cerebral cortex in blue, and ipsilateral white matter is indicated in pink whereas light blue indicates contralateral white matter. (D) Representative brain tissues stained with Cresyl Violet. Scale bar = 1 mm. (E) Total estimated volume was measured in each hemisphere. Total volume did not change between the ipsilateral and contralateral hemispheres at 8 days post-injury. (F) Total cortical volume estimates revealed significant atrophy of cortex on the ipsilateral but not contralateral side after TBI [two-way ANOVA, side, F(1,45) = 101.09, P < 0.001]; *P < 0.05 compared to sham. However, EVT901 treatment was protective on the ipsilateral side [dose × side, F(4,45) = 2.69, P < 0.05]; n = 8–12 per group. (G) Total white matter volume was also significantly reduced ipsilateral to the injury [side effect, F(1,45) = 17.51, P < 0.001; dose effect, F(4,45) = 2.22, P > 0.05]. EVT901 treatment preserved white matter volume on the ipsilateral side [side × dose, F(4,45) = 2.98, P < 0.05]. *P < 0.05 compared to sham, ^#P < 0.05 compared to 0 mg/kg. Error bar indicates ± SEM.

Figure 5

EVT901 treatment inhibits cell death in the cerebral cortex and underlying white matter at 8 days after CCI-TBI. (A) Stereological sampling of total number of oligodendrocytes (CC1-positive cells, arrowheads in the inset) in white matter was evaluated on DAB immunostained sections. (B) Cell numbers were estimated using unbiased stereology by optical fractionation (see ‘Materials and methods’ section; Stereo Investigator). TBI significantly reduced the number of CC1-positive cells in the white matter ipsilateral to the TBI. Two-way ANOVA, side, F(1,28) = 17.51, P < 0.0001. EVT901 treatment preserved the number of oligodendrocytes ipsilateral to the lesion [dose × side, F(4,28) = 2.69, P = 0.05]. n = 5–7 per group. Error bar indicates ± SEM. A subsequent t-test showed a significant difference between 0 and 1 mg/kg on the ipsilateral side (P < 0.05). (C) Neuronal loss was evaluated in sections immunostained for NeuN using DAB; cell staining in the ipsilateral perilesional cortex is shown for representative vehicle and EVT901-treated cases. Arrows indicate individual neurons. (D) Unbiased stereological analysis by optical fractionation was used to count NeuN-positive cells (see ‘Materials and methods’ section; Stereo Investigator). TBI caused a marked reduction in the number of cortical neurons counted ispilaterally versus contralaterally. Two-way ANOVA, side, F(1,6) = 28.65, P < 0.01. However, EVT901 treatment (1 mg/kg) protects NeuN-positive cells on the ipsilateral side [F(1,6) = 5.89, P = 0.05]. *P < 0.05 compared to vehicle, n = 5. Scale bar = 10 μm.

Figure 6

EVT901 treatment spares myelin and reduces microglial activation at 8 days after CCI-TBI. (A and B) Eriochrome cyanine staining showed a dramatic loss of myelin staining after CCI-TBI that was significantly reversed by 7 days of EVT901 treatment (1 mg/kg). Intensity measurements of staining in the white matter (outlined area) ipsilateral to the injury as a proportion of the same area on the contralateral side, revealed a significant effect of EVT901 treatment. n = 3–5/group. Two tailed t-test, *P < 0.05. Scale bar in A = 1 mm. (C) Brain tissue sections were immuno-stained with OX-42 to reveal the presence of microglia. Representative sections showing staining in the corpus callosum are shown for vehicle and EVT901-treated (1 mg/kg) subjects. Both DAB (middle) and fluorescent (OX-42 in red, DAPI in blue) immunostained sections are shown. Scale bars = 50 μm. (D) Microglial activation, measured by proportional area of OX-42 staining, was increased ipsilateral to the lesion; EVT901 treatment reduced OX-42 staining [two way ANOVA, side, F(1,8) = 44.96, P < 0.002; dose, F(1,18) = 10.11, P < 0.05]. There was also a significant dose × side interaction in microglia activation with EVT901 treatment [F(1,8) = 40.85, P < 0.001]. ^##P < 0.01 compared to vehicle; n = 3–5/group. Bars represent mean ± SEM.

Figure 7

Acute expression of p75NTR after FPI-TBI and reduction of neurodegeneration with EVT901 in vivo. (A) Immunohistochemical expression of p75NTR at 1–2 days in the dentate gyrus of TBI rats using specific anti-p75NTR antibody. (B) Representative photomicrographs (original magnification ×10) of Fluoro-Jade® staining of damaged neurons in the granular layer of the dentate gyrus in FPI-TBI rats 1 week post-injury. (C) Representative photomicrographs of Fluoro-Jade®-stained sections from the thalamus 14-days post-trauma (ipsilateral versus contralateral to the injury). (D) Quantification of Fluoro-Jade® B-positive degenerating cells in the thalamus of FPI-TBI rats after EVT901 treatment. Following Fluoro-Jade staining, the digital images were collected and the damaged cells were quantified using Explora Nova Software. Three mg/kg/day of EVT901 protected neurons in the thalamus after TBI [one-way ANOVA, EVT901 effect, F(3,18) = 8.46, P = 0.001]. ^#P < 0.05 and ^##P < 0.005 compared to sham. ^**P < 0.01 compared to vehicle. n = 5–7/group. Bars represent mean ± SEM.

Figure 8

EVT901 improves functional recovery at 23 days after FPI-TBI and reduces post-TBI seizure activity. (A–C) Improvement in cognitive function after FPI-TBI evaluated by the object recognition test. Rats with a FPI-TBI or sham surgery were treated orally with vehicle alone or different doses of EVT901 (0.1, 0.3, 1 and 3 mg/kg) at 24 h post-surgery and once daily for 23 days. Recognition memory was evaluated using the Novel Object Recognition Test. (A) Time spent exploring novel (N) objects was recorded and expressed in seconds. There is a significant EVT901 effect on the novel object recognition test [two-way ANOVA with repeated measurement, F(4,52) = 2.87, P < 0.05]. *P < 0.05 compared to 0 mg/kg. (B) There is no effects on familiar object recognition test [F(4,52) = 1.10, P = 0.37]. (C) The novelty index (N – F/N + F) was significantly reduced by TBI [F(1,52) = 12.73, P < 0.001]. EVT901 treatment improved recognition memory function as measured by the novelty index [F(4,52) = 6.59, P < 0.0001] ^#P < 0.05 compared to sham; *P < 0.05 and ^**P < 0.005 compared to 0 mg/kg; n = 9–10/group. (D) Improvement in recovery after TBI evaluated by the conditioned freezing response. TBI significantly reduced freezing responses [F(1,55) = 13.84; P < 0.001], while treatment with EVT901 preserved the freezing response [F(3,55) = 5.68: P < 0.01]; ^##P < 0.005 compared to sham; ^**P < 0.005 compared to 0 mg/kg; n = 12/group. (E) Inhibition of kainate-induced seizure hypersensitivity after TBI by EVT901 (1 mg/kg). Evaluation of the increased susceptibility of rats to seizure induction by kainate was determined after a single injection of a sub-convulsant dose of kainate (8 mg/kg, intraperitoneally) in the different groups at 6 weeks after FPI-TBI. Seizure intensity was measured by Racine’s score from 45 min to 3 h 45 min post-kainate injection. n = 10/group. There were significant time, EVT901, and time × EVT901 effects [two-way ANOVA with repeated measurement, time effect; F(11,297) = 24.90 P < 0.0001, EVT901 effect; F(2,27) = 11.78, P < 0.0001, time × EVT901 effect; F(22,297) = 6.16, P < 0.0001]. ^###P < 0.0001 compared to sham, *P < 0.05 compared to TBI. (F) For each group, we defined a status epilepticus (SE) corresponding to the per cent of rats that reached continuous seizure activity in the different groups. Note that kainate treatment induced high numbers of seizure incidence (90%) in TBI-vehicle group, while EVT901 treatment reduced it to 40%. Results are given as mean ± SEM.