The overexpression of RBM3 alleviates TBI-induced behaviour impairment and AD-like tauopathy in mice

Abstract

The therapeutic hypothermia is an effective tool for TBI-associated brain impairment, but its side effects limit in clinical routine use. Hypothermia up-regulates RNA-binding motif protein 3 (RBM3), which is verified to protect synaptic plasticity. Here, we found that cognitive and LTP deficits, loss of spines, AD-like tau pathologies are displayed one month after TBI in mice. In contrast, the deficits of LTP and cognitive, loss of spines and tau abnormal phosphorylation at several sites are obviously reversed in TBI mice combined with hypothermia pre-treatment (HT). But, the neuroprotective role of HT disappears in TBI mouse models under condition of blocking RBM3 expression with RBM3 shRNA. In other hand, overexpressing RBM3 by AAV-RBM3 plasmid can mimic HT-like neuroprotection against TBI-induced chronic brain injuries, such as improving LTP and cognitive, loss of spines and tau hyperphosphorylation in TBI mouse models. Taken together, hypothermia pre-treatment reverses TBI-induced chronic AD-like pathology and behaviour deficits in RBM3 expression dependent manner, RBM3 may be a potential target for neurodegeneration diseases including Alzheimer disease.

Keywords: RNA-binding motif protein 3; cognitive deficits; hypothermia pre-treatment; tau phosphorylation; traumatic brain injury.

FIGURE 1

Hypothermia pre‐treatment reduces TBI‐induced AD‐like tau phosphorylation. The mice were performed hypothermia or sham treatment and then underwent TBI or sham. One mother after TBI or sham treatment, the hippocampal or cortex was homogenized in RIPA buffer and targets protein were measured by Western blotting. The expression of the pT231, pS396 and Tau5 in the extracts of cerebral cortex (A) or hippocampi (C) and quantitative analysis (B and D). The band used for quantification was specified at 55 Kda. The size of molecular weight in WB is marked in the revised manuscript. The five consecutive sections from each brain were stained with avidin horseradish peroxidase‐labelled antibodies and visualized with the diaminobenzidine tetrachloride system. The representative immunostaining image (E) and quantitative analysis (F). One‐way ANOVA. All data were expressed as the mean ± SEM (n = 10, male). *P < .05 vs. Sham; ^# P < .05 vs. TBI

FIGURE 2

Hypothermia pre‐treatment induces RBM3 expression in mice. The expression of the RBM3 in the extracts of hippocampi of mice was detected by Western blotting (A) and quantitative analysis (B). The representative immunostaining image (C) 1 m after TBI or sham treatment and quantitative analysis (D). One‐way ANOVA. All data were expressed as the mean ± SEM (n = 10, male). **P < .01 vs. Sham; ^# P < .05 vs. TBI

FIGURE 3

Blocking RBM3 expression reduces protection of hypothermia pre‐treatment against impairment of spatial learning and memory. The AAV‐shRNA‐RBM3 or empty vector (AAV‐control) of 1.5 μL was injected into the hippocampal CA3 area with microinjector, respectively, and then, the mice underwent a time sequence treatment including sham, hypothermia or TBI. The representative image of green fluorescence protein showed the plasmid expression of adeno‐associated virus carrying RBM3 or shRNA plasmid or empty plasmid (A). The expression of the RBM3 in the extracts of hippocampi (B) of mice was detected by Western blotting and quantitative analysis (C) 1 m after TBI or sham treatment. Mice were trained to find the platform for 6 consecutive days (three trials per day) and probe trial on day 7. The escape latency time (sec) (D), the latency to find the platform (E), the count of platform crossings (F), the time spent in the target quadrant (G), the average swimming speed during the probe test (H). Student's t test; two tailed. All data were expressed as the mean ± SEM (n = 10, male). *P < .05, **P < .01, ***P < .001 vs. TBI + HT+AAV‐control

FIGURE 4

HT improves TBI‐induced impairment of LTP and spine caused by TBI via RBM3. The coronal slices (400 μm) were cut with the vibratome and incubated for 1 h in a continuously flowing artificial cerebrospinal fluid (ACSF). Slices were transferred to a recording chamber submerged in ACSF and stimulated in CA3 using glass microelectrodes, and fEPSPs were recorded in the stratum radiatum of the CA1. The slope of fEPSP after HFS is normalized by the baseline. The onset of HFS at 0 min; the traces are average fEPSPs from five sweeps before (−30 min‐0 min) and after (0 min‐60 min) LTP induction (A). Quantitative analyses for fEPSPs measured 40‐60 min after HFS relative to baseline (B). The brains stained by silver nitrate were sliced by the vibratome at a thickness of 40 μm. Golgi staining neuronal spine densities were randomly determined in segments of dendrites at a distance of 90 μm from the soma and counted in z‐stacks by manual scrolling of the images. The images were obtained under bright‐field microscopy (Axioplan 2; Zeiss, Brighton, MI, USA). The dendritic spines from at least 60 neurons per group were counted with Neurolucida software (MicroBrightField, Williston, VA, USA). All tracings and analyses were performed in a blind manner. The representative image of spine density in the hippocampus was measured via Golgi stain (C). Quantitative analysis of the spine numbers (at least 15 neurons, three dendritic branches per neuron, from three mice, were used for the analysis) (D). The expression of the pT231, pS396 and Tau5 in the extracts of hippocampi of mice was detected by Western blotting (E) and quantitative analysis (F) 1 m after TBI or sham treatment. Student's t test; two tailed. All data were expressed as the mean ± SEM, (n = 10, male). *P < .05, **P < .01 vs. TBI + HT+AAV‐control

FIGURE 5

Overexpression of RBM3 restores capacity of learning and memory of TBI mice. The AAV‐RBM3 or empty vector (AAV‐control) of 1.5 μl was injected into the hippocampal CA3 area with microinjector, respectively, and then, the mice underwent a time sequence treatment including sham or TBI. The expression of the RBM3 in the extracts of hippocampi of mice was detected by Western blotting (A) and quantitative analysis (B) 1 m after treatment. Mice were trained to find the platform for 6 consecutive days (three trials per day) and probe trial on day 7. The escape latency time (sec) (C), the latency to find the platform (D), the count of platform crossings (E), the time spent in the target quadrant (F), the average swimming speed (G) during the probe test. Student's t test; two tailed. All data were expressed as the mean ± SEM (n = 10, male). ^# P < .05, ^### P < .001 vs. TBI + AAV‐control

FIGURE 6

Overexpression of RBM3 improves TBI‐induced impairment of LTP and spine, and tau hyperphosphorylation. The traces are average fEPSPs from five sweeps before (−30 min‐0 min) and after (0 min‐60 min) LTP induction (A). Quantitative analyses for fEPSPs measured 40‐60 min after HFS relative to baseline (B). Golgi staining neuronal spine densities were randomly determined in segments of dendrites at a distance of 90 μm from the soma and counted in z‐stacks by manual scrolling of the images. The representative image of spine density in the hippocampus was measured via Golgi stain (C). Quantitative analysis of the spine numbers (D). The expression of the pT231, pS396 and Tau5 in the extracts of hippocampi of mice was detected by Western blotting (E) and quantitative analysis (F) 1 m after TBI or sham treatment. Student's t test; two tailed. All data were expressed as the mean ± SEM, (n = 10, male). ^# P < .05, ^## P < .01 vs. TBI + HT + AAV‐control