Neuroprotective pentapeptide CN-105 is associated with reduced sterile inflammation and improved functional outcomes in a traumatic brain injury murine model

Abstract

At present, there are no proven pharmacological treatments demonstrated to improve long term functional outcomes following traumatic brain injury(TBI). In the setting of non-penetrating TBI, sterile brain inflammatory responses are associated with the development of cerebral edema, intracranial hypertension, and secondary neuronal injury. There is increasing evidence that endogenous apolipoprotein E(apoE) modifies the neuroinflammatory response through its role in downregulating glial activation, however, the intact apoE holoprotein does not cross the blood-brain barrier due to its size. To address this limitation, we developed a small 5 amino acid apoE mimetic peptide(CN-105) that mimics the polar face of the apoE helical domain involved in receptor interactions. The goal of this study was to investigate the therapeutic potential of CN-105 in a murine model of closed head injury. Treatment with CN-105 was associated with a durable improvement in functional outcomes as assessed by Rotarod and Morris Water Maze and a reduction in positive Fluoro-Jade B stained injured neurons and microglial activation. Administration of CN-105 was also associated with reduction in mRNA expression of a subset of inflammatory and immune-related genes.

Conflict of interest statement

The patent for CN-105 is held by Duke University, and D.T. La, B.J.K., and H.N.D. are co-inventors. D.T. La is an officer of Aegis-CN, which supplied drug for these studies, but had no role in experimental design or writing of this manuscript.

Figures

Figure 1. CN-105 improves vestibulomor and cognitive function.

Following TBI, intravenous administration of 0.05 mg/kg of CN-105 was the lowest effective dose resulting in improved vestibulomotor functional performance, as assessed by Rotarod(A) and cognitive function as assessed by the Morris Water Maze(B) and the MWM probe trial(C). There was no difference in swim speed on the MWM(D). CN-105(0.05 mg/kg) administered 4 hours post-injury was also associated with a significant and durable improvement in Rotarod latency(E) and by reduced deficit on MWM(F) and the MWM probe trial(G) No significant difference in speed was detected between the two treatment groups(H). For comparison purposes results from sham mice are shown(dotted lines) in panels A and B but are not included in statistical calculations. Asterisks denote significant differences in performance as measured by ANOVA; *p < 0.05 and ** p < 0.01.

Figure 2. CN-105 reduces microgliosis and neuronal injury.

Comparison images of activated F4/80 immunostained microglial in the hippocampus 10 days post-TBI, brain slices from vehicle(A,C,E,G) and CN-105(B,D,F,H) treated mice, unbiased stereology confirms that treatment with CN-105 is associated with a reduction in microgliosis(p = 0.0002, ***)(I). Higher powered images of microglia in the CA3 region(C,D) in the polymorphic region(E,F) and in corpus callosum and fimbria of the periventricular region(G,H). Microglia are indicated in with arrows and phagocytic microglia with arrowheads in E and F. Images C-H are at 20x magnification and the black bar in G represents 40 μm. Images A and B are at 4x magnification and the white bar in A represents 250 μm. Comparison images of FJB-stained brain slices 24 hours post-TBI show degenerating neurons in the hilus of the dorsal dentate gyrus of TBI vehicle treated mice(J) which are significantly reduced by treatment with CN-105(K), quantification p < 0.0001(L).

Figure 3. CN-105 ameliorates changes in inflammatory gene expression.

One day post-injury, differential inflammatory gene expression analysis demonstrated changes in gene expression relative to sham vehicle treated controls for TBI CN-105 treated and untreated mice. Results are organized by upregulated(A) and downregulated(B) expression of genes from vehicle treated TBI mice relative to vehicle treated sham mice. Results from CN-105 treated TBI and sham mice relative to vehicle treated sham mice are also included. Only genes from TBI CN-105 treated mice that showed more than a 3 fold change when compared to TBI vehicle treated mice are presented.

Figure 4. Pharmacokinetic studies of CN-105 demonstrate CNS bioavailability.

Concentration of [14C]- radioactivity in the plasma and central nervous system of male CD-1 mice following an intravenous dose of [14C]-radiolabeled CN-105 peptide(A). For clarity, the concentration of [14C]- radioactivity during the first 6 hours only is depicted in(B).

Figure 5. TLR signaling is downregulated by CN-105.

(A) A schematic of the signaling pathway proposed for sterile inflammation post injury. Green arrows indicate upregulation and red arrows downregulation of genes 24 hours after TBI.(B) Graphic representation of genes 24 hours post TBI demonstrating fold change compared to sham control.(C) CN-105 modifies the post-injury inflammatory response. Arrows indicate reduced upregulation(green) and reduced downregulation(red) of gene expression when TBI injured mice are treated with CN-105 as compared to vehicle.(D) Graphic representation of differential gene expression 24 hours post TBI demonstrating fold change in gene expression of CN-105 treated mice normalized to vehicle treated TBI mice.