P7C3 neuroprotective chemicals block axonal degeneration and preserve function after traumatic brain injury

Abstract

The P7C3 class of neuroprotective aminopropyl carbazoles has been shown to block neuronal cell death in models of neurodegeneration. We now show that P7C3 molecules additionally preserve axonal integrity after injury, before neuronal cell death occurs, in a rodent model of blast-mediated traumatic brain injury (TBI). This protective quality may be linked to the ability of P7C3 molecules to activate nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in nicotinamide adenine dinucleotide salvage. Initiation of daily treatment with our recently reported lead agent, P7C3-S243, 1 day after blast-mediated TBI blocks axonal degeneration and preserves normal synaptic activity, learning and memory, and motor coordination in mice. We additionally report persistent neurologic deficits and acquisition of an anxiety-like phenotype in untreated animals 8 months after blast exposure. Optimized variants of P7C3 thus offer hope for identifying neuroprotective agents for conditions involving axonal damage, neuronal cell death, or both, such as occurs in TBI.

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1. P7C3-S243 Preserves Memory after Blast-Mediated TBI

(A) Daily IP administration of P7C3-S243 for 11 days in divided daily doses for the total amount indicated dose-dependently preserved memory in the Barnes maze probe test in blast-injured mice, as measured by the most stringent measure of percent time in escape area (5 cm radius around the escape hole). Treatment with an intermediate dose (3 mg/kg/d) of the active (−)-P7C3-S243 enantiomer preserved normal performance to the level displayed by sham-injured mice. By contrast, mice treated with the same dose of the less active (+)-P7C3-S243 enantiomer showed the same deficit as injured mice that were treated with vehicle. (B) Daily administration of P7C3-S243 was initiated at later time periods after injury to define the window of therapeutic efficacy. Whereas both 3 and 30 mg/kg/d doses preserved normal function when treatment was initiated 24 hours after injury, only the 30 mg/kg/d dose was efficacious when treatment was initiated at 36 hours. When treatment was initiated 48 hours after injury, no protective efficacy was noted at any dose. (C) Oral administration of the highly active (−)-P7C3-S243 enantiomer preserved normal hippocampal dependent memory at 3, 10 and 30 mg/kg/day doses. Every group shown consisted of 25 male C57/Bl6 mice, aged 12–14 weeks, and data was collected and scored in an automated manner blind to treatment group. Data are represented as mean ± SEM. Significance was determined by two way ANOVA with Bonferroni post-hoc analysis. p-value labeled as *<0.05, **<0.01, ***<0.001, and ****<0.0001 compared to blast-injured animals treated with vehicle. See alsoFigure S1 and S2.

Figure 2. P7C3-S243 Preserves Hippocampal Synaptic Transmission after Blast-Mediated TBI

Treatment with P7C3-S243 rescued blast-injury-induced deficits in long-term potentiation (LTP) and paired-pulse facilitation (PPF) in the hippocampal CA1 Schaffer-collateral pathway. (A) LTP induced by 12 theta burst stimulation (TBS) is significantly decreased in animals that sustained blast-induced TBI 14 days prior to testing. (B) This deficit was not rescued by treatment with low dose P7C3-S243 (0.3 mg/kg/d) starting 24 hours after injury, but was rescued by treatment with higher doses of (C) 3 and (D) 30 mg/kg/d P7C3-S243. LTP 1 hour after 12 TBS is summarized by quantification of the initial slope in panel (E). (F) Blast-injury-induced PPF deficit of 50 ms inter-pulse interval was also rescued in animals treated with the two higher doses (3 and 30 mg/kg/d) of P7C3-S243. Data are represented as mean ± SEM. Statistics were determined by one-way ANOVA with Tukey’s post hoc test.

Figure 3. P7C3-S243 Blocks Axonal Degeneration after Blast-Mediated TBI

(A) Representative pictures from CA1 stratum radiatum show prominent silver staining of degenerating axons 12 days after blast-injury, in the absence of loss of NeuN of H&E staining. Images shown are representative of typical images from 5 animals in each group, and demonstrate that 3 and 30 mg/kg/day doses of P7C3-S243, initiated 24 hours after blast-injury, block axonal degeneration. Similar protective efficacy is seen in animals treated with 3 mg/kg/day of the highly active enantiomer (−)-P7C3-S243, but not in animals treated with the less active enantiomer (+)-P7C3-S243 (scale bar = 2.5µM). (B) Optical densitometry of light transmitted through silver-stained CA1 stratum radiatum from all animals in each group was used to quantify the protective effect. Signal was quantified for 18 sections for each of the 5 animals, spaced 480 µM apart. Agreater value indicates that more light passed unimpeded through the section by virtue of less silver staining, which reflects less axonal degeneration. Data are represented as mean ± SEM. P-value *<0.05, determined by-two way ANOVA with Bonferroni post-hoc analysis. See alsoFigure S3.

Figure 4. Toluidine Blue Staining and Transmission Electron Microscopy Visualization of Hippocampal Protection by orally administered (−)-P7C3-S243 after Blast-Injury

Daily oral administration of the highly active enantiomer (−)-P7C3-S243 for 14 days, starting 24 hours after injury, dose-dependently preserved CA1 morphology, as well as myelin and mitochondrial structures in the hippocampus after blast-injury. Two-weeks after either sham or blast-injury, animals were perfused and processed for ultrastructural pathology. Toluidine blue-stained semithin sections (left panel) of sham-injured mice treated with vehicle or (−)-P7C3-S243 showed normal CA1 histology, with densely packed neurons in the stratum pyramidale (1) and profuse dendritic profiles in the stratus radiatum (2; black arrows). Blast-injured animals treated with vehicle showed accumulation of chromatolytic and pyknotic neurons (white arrow) throughout the stratum pyramidale, as well as fewer dendrites in the stratum radiatum. There is no protection in CA1 morphology at the lowest concentration of blast-injured animals treated with 0.3 mg/kg/d of (−)-P7C3-S243. However, treatment with 3mg/kg/day (−)-P7C3-S243 lowered the abundance of chromatolytic and pyknotic neurons, and resulted in a more densely packed stratum pyramidale. At the highest concentration (30mg/kg/day) of (−)-P7C3-S243, there was complete preservation of CA1 morphology after blast-mediated TBI. Transmission electron micrographs (TEM; right panel) of immediately adjacent ultrathin sections showed normal myelin and axonal mitochondrial structures in the stratum radiatum of sham-injury mice treated with vehicle or (−)-P7C3-S243. Blastinjured mice treated with vehicle or 0.3mg/kg/day of (−)-P7C3-S243 showed degeneration of myelin sheath (red arrows), along with abnormal outer membrane and internal cristae structures within neuronal mitochondria (blue arrows). At 3 and 30mg/kg/day doses, however, both myelin and neuronal mitochondria were preserved. Pictures shown are representative of 4 animals per condition. Scale bars: Toluidine blue: 50uM; TEM: 500nm. See alsoFigure S4.

Figure 5. P7C3-S243 Blocks Cerebellar Axonal Degeneration and Preserves Balance and Coordination

(A) Representative pictures from the cerebellar molecular layer show prominent silver staining of degenerating axons 12 days after blast-injury, in the absence of loss of NeuN or H&E staining. Images shown are representative of typical images from 5 animals in each group, and demonstrate that 3 and 30 mg/kg/day doses of P7C3-S243, initiated 24 hours after blast-injury, block axonal degeneration. Similar protective efficacy is seen in animals treated with 3 mg/kg/day of the highly active enantiomer (−)-P7C3-S243, but not in animals treated with the less active enantiomer (+)-P7C3-S243. (scale bar = 2.5µM). (B) Optical densitometry of light transmitted through silver-stained cerebellar molecular layer from all animals in each group was used to quantify the protective effect. The specific tissue area was manually delineated, and signal was quantified for 18 sections for each of the 5 animals, spaced 480 µM apart. A greater value indicates that more light passed unimpeded through the section by virtue of less silver staining, which reflects less axonal degeneration. (C) Seven days after blast-injury, mice show a trend towards impaired balance and coordination with increased foot slips that did not achieve statistical significance. By 28 days, however, blast-injured mice showed a two-fold increase in the number of foot slips relative to sham-injured mice. When daily oral treatment with 6 mg/kg/day of the active enantiomer (−)-P7C3-S243 was initiated 24 hours after blast-injury, however, mice performed normally in this task. Every group shown consisted of 25 male C57/Bl6 mice, aged 12–14 weeks, and data was collected and scored in an automated manner blind to treatment group. Significance was determined by 2 way ANOVA with Bonferroni post-hoc analysis. Data are represented as mean ± SEM. p-value labeled as **<0.01 and ****<0.0001 compared to blast-injured animals treated with vehicle. See alsoFigure S5, S6.