A pyrazole curcumin derivative restores membrane homeostasis disrupted after brain trauma

Abstract

We have assessed potential mechanisms associated with the deleterious effects of TBI on the integrity of plasma membranes in the hippocampus, together with consequences for behavioral function. In addition, we have investigated the efficacy of a dietary intervention based on a pyrazole curcumin derivative with demonstrated bioactivity and brain absorption, to re-establish membrane integrity. We report that moderate fluid percussion injury (FPI) increases levels of 4-Hydroxynonenal (HNE), an intermediary for the harmful effects of lipid peroxidation on neurons. A more direct action of FPI on membrane homeostasis was evidenced by a reduction in calcium-independent phospholipase A2 (iPLA₂) important for metabolism of membrane phospholipids such as DHA, and an increase in the fatty acid transport protein (FATP) involved in translocation of long-chain fatty acids across the membrane. A potential association between membrane disruption and neuronal function was suggested by reduced levels of the NR2B subunit of the transmembrane NMDA receptor, in association with changes in iPLA2 and syntaxin-3 (STX-3, involved in the action of membrane DHA on synaptic membrane expansion). In addition, changes in iPLA2, 4-HNE, and STX-3 were proportional to reduced performance in a spatial learning task. In turn, the dietary supplementation with the curcumin derivative counteracted all the effects of FPI, effectively restoring parameters of membrane homeostasis. Results show the potential of the curcumin derivative to promote membrane homeostasis following TBI, which may foster a new line of non-invasive therapeutic treatments for TBI patients by endogenous up-regulation of molecules important for neural repair and plasticity.

Figure 1

(A) Effects of FPI on lipid peroxidation as evidenced by changes in 4-HNE in the hippocampus. Western blot analysis revealed that the levels of 4-HNE were significantly increased after FPI, whereas dietary curcumin derivative supplementation significantly reduced the levels of 4-HNE in FPI rats. Representative Western blot for 4-HNE from different groups in hippocampus are depicted, multiple bands were recognized. For quantification purpose, multiple bands of HNE-bound proteins were grouped and quantified using NIH Image software as described elsewhere (Wu, et al., 2004a, Wu, et al., 2004b). The values were converted to percent of RD sham group (mean ± SEM). ANOVA followed by post-hoc tests with Bonferroni’s comparisons, *p < 0.05; ***p < 0.001. (B) Immunostaining showed 4-HNE labeling throughout the hippocampal formation but with preferential distribution in pyramidal layer as shown in the high magnifications photographs (arrows, C). FPI increased 4-HNE immunostaining but dietary curcumin derivative counteracted the effect of FPI. Scale bars (B, C) = 400 µm and 50 µm respectively. RD: regular diet; FPI: fluid percussion injury.

Figure 2

(A–B) Effects of FPI and curcumin derivative treatment on molecular systems important for maintenance of plasma membrane homeostasis, and cognitive abilities. FPI decreased iPLA2 levels (A) while increased FATP levels (B) in the hippocampus. Representative Western blots for iPLA2 and FATP from all experimental groups in hippocampus are shown here. Dietary curcumin derivative supplementation significantly enhanced the levels of iPLA2 after FPI. (C) Slope of escape latency (the learning speed) to find the platform in training trial during Morris water maze test for each group of animals. FPI significantly decreased the learning speed in regular diet animals but the dietary curcumin derivative supplementation significantly improved the learning speed after FPI. (D) Correlation of slope of latency with the hippocampal levels of iPLA2. Slope of latency changed in inverse proportion to iPLA2 levels. The values were converted to percent of RD sham group (mean ± SEM). *p < 0.05, **p< 0.01, ***p< 0.001, ANOVA followed by post-hoc tests with Bonferroni’s comparisons. RD, regular diet; FPI, fluid percussion injury

Figure 3

Effects of FPI and curcumin derivative treatments on molecular systems important for plasma membrane expansion such as syntaxin-3 and GAP-43. (A) FPI decreased the levels of STX-3 whereas dietary curcumin derivative supplementation significantly counteracted these effects. Representative Western blots for STX-3 from all experimental groups in hippocampus are shown here. (B) Levels of the lipid peroxidation marker 4-HNE changed in inverse proportion to STX-3 levels. (C) The levels of STX-3 changed in inverse proportion to the slope of latency. (D) FPI decreased the levels of GAP-43 in regular diet group (RD/FPI), dietary curcumin derivative supplementation significantly counteracted the FPI effects (CD/FPI). The values were converted to percent of RD sham group (mean ± SEM). *p < 0.05, ***p< 0.001, ANOVA followed by post-hoc tests with Bonferroni’s comparisons. RD, regular diet; FPI, fluid percussion injury

Figure 4

Effects of FPI and curcumin derivative treatment on levels of NR2B, and their associations with cognitive abilities and membrane homeostasis. (A) The levels of NR2B were significantly decreased after FPI, and dietary curcumin derivative supplementation counteracted these effects. Representative Western blots for NR2B from different groups in hippocampus. (B) The levels of NR2B changed in inverse proportion to the slope of latency. (C–D) The levels of NR2B changed in direct proportion to iPLA2 levels and STX-3 levels. The values were converted to percent of RD sham group (mean ± SEM). *p< 0.05, **p< 0.01. ANOVA followed by post-hoc tests with Bonferroni’s comparisons. RD, regular diet; FPI, fluid percussion injury

Figure 5

Possible events by which TBI can disrupt plasma membrane homeostasis. TBI increases free radical formation in the mitochondria, thereby promoting oxidation of membrane lipids, as detected by the lipid peroxidation marker 4-HNE. These events may lead to membrane dysfunction as evidenced by reduced levels of iPLA2. These events may lower membrane function and expansion capacity as evidenced by reduced levels of STX-3 and GAP-43. The loss of membrane function and flexibility may compromise performance of membrane embedded receptors such as the subunit NR2B of the NMDA receptor. These alterations can result in abnormal neuronal signaling, which can reduce learning capacity and other functions that rely on synaptic plasticity and neuronal excitability. The curcumin derivative curcumin derivative administration had a comprehensive effect on most of these alterations. The observed effects of curcumin derivative after TBI may be primarily due to its ability to counteract lipid peroxidation, thereby, preserving membrane homeostasis, and reducing TBI induced cognitive deficits.