Identification of potentially neuroprotective genes upregulated by neurotrophin treatment of CA3 neurons in the injured brain

Abstract

Specific neurotrophic factors mediate histological and/or functional improvement in animal models of traumatic brain injury (TBI). In previous work, several lines of evidence indicated that the mammalian neurotrophin NT-4/5 is neuroprotective for hippocampal CA3 pyramidal neurons after experimental TBI. We hypothesized that NT-4/5 neuroprotection is mediated by changes in the expression of specific sets of genes, and that NT-4/5-regulated genes are potential therapeutic targets for blocking delayed neuronal death after TBI. In this study, we performed transcription profiling analysis of CA3 neurons to identify genes regulated by lateral fluid percussion injury, or by treatment with the trkB ligands NT-4/5 or brain-derived neurotrophic factor (BDNF). The results indicate extensive overlap between genes upregulated by neurotrophins and genes upregulated by injury, suggesting that the mechanism behind neurotrophin neuroprotection may mimic the brain's endogenous protective response. A subset of genes selected for further study in vitro exhibited neuroprotection against glutamate excitotoxicity. The neuroprotective genes identified in this study were upregulated at 30 h post-injury, and are thus expected to act during a clinically useful time frame of hours to days after injury. Modulation of these factors and pathways by genetic manipulation or small molecules may confer hippocampal neuroprotection in vivo in preclinical models of TBI.

FIG. 1.

(A) Diagram of the area of lateral fluid percussion injury (arrow, LFP), and pump implantation (the cross denotes the cannula tip, and the circle represents the approximate area of neurotrophin diffusion; Royo et al., 2007). Diagram of the rat brain is modified from Paxinos and Watson, . (B) Photomicrographs of laser-capture microdissection of area CA3 neurons. CA3 neurons were captured onto a membrane with multiple laser pulses per membrane (panel B3), leaving behind the remaining tissue on the slide (panel B2). (C) Schematic representation of the experimental paradigm (LCM, laser capture microdissection; QC, quality control; DAVID, Database for Annotation, Visualization and Integrated Discovery [david.abcc.ncifcrf.gov]; NT-4/5, neurotrophin 4/5; BDNF, brain-derived neurotrophic factor).

FIG. 2.

Similar sets of genes are upregulated by TBI alone, NT-4/5 alone, or BDNF alone. Area-proportional Venn diagram of genes upregulated twofold or more relative to the SHAM-VEH group. Genes upregulated by TBI, by NT-4/5, and by BDNF are shown (TBI, traumatic brain injury; NT-4/5, neurotrophin 4/5; BDNF, brain-derived neurotrophic factor; SHAM-VEH, sham-injured vehicle-infused group).

FIG. 3.

Functional classifications of genes upregulated by TBI alone (black bars), NT-4/5 infusion alone (light grey bars), or BDNF infusion alone (dark grey bars), relative to SHAM-VEH (TBI, traumatic brain injury; NT-4/5, neurotrophin 4/5; BDNF, brain-derived neurotrophic factor; SHAM-NT-4/5, sham-injured NT-4/5-infused group; SHAM-BDNF, sham-injured BDNF-infused group; TBI-VEH, injured vehicle-infused group).

FIG. 4.

Possible mechanisms of action of neurotrophin neuroprotection in the injured brain. Functional classifications of genes differentially upregulated by NT-4/5 (light grey bars), or BDNF (dark grey bars) in injured versus sham-injured brains. Genes upregulated in the TBI-VEH group are shown for comparison (black bars; TBI, traumatic brain injury; NT-4/5, neurotrophin 4/5; BDNF, brain-derived neurotrophic factor; TBI-VEH, injured vehicle-infused group).

FIG. 5.

Neuroprotection against glutamate excitotoxicity in cultured neurons. (A) Transthyretin (Ttr) protection against 30 μM glutamate (white bars) or 100 μM glutamate (gray bars). (B) Thyrotrophin-releasing hormone (Trh) protection against 100 μM glutamate. (C) CCL-2 protection against 100 μM glutamate. (D) CCL-7 protection against 100 μM glutamate. Black bars in all panels are control cultures not treated with glutamate. Data are presented as mean ± standard error of 6–9 replicates from three independent experiments (^#p < 0.05 relative to control cultures that received neither glutamate nor protective agent [black bars], indicating neurotoxicity of glutamate; *p < 0.05 relative to the cultures that received glutamate but no neuroprotective agent, indicating neuroprotective efficacy against glutamate).