EPPS treatment attenuates traumatic brain injury in mice by reducing Aβ burden and ameliorating neuronal autophagic flux

Abstract

Beta-amyloid (Aβ) burden and impaired neuronal autophagy contribute to secondary brain injury after traumatic brain injury (TBI). 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS) treatment has been reported to reduce Aβ aggregation and rescue behavioral deficits in Alzheimer's disease-like mice. Here, we investigated neuroprotective effects of EPPS in a mouse model of TBI. Mice subjected to controlled cortical impact (CCI) were treated with EPPS (120 mg/kg, orally) immediately after CCI and thereafter once daily for 3 or 7 days. We found that EPPS treatment profoundly reduced the accumulation of beta-amyloid precursor protein (β-APP) and Aβ over a widespread area detected in the pericontusional cortex, external capsule (EC), and hippocampal CA1 and CA3 at 3 days after TBI, accompanied by significant reduction in the TBI-induced diffuse axonal injury identified by increased immunoreactivity of SMI-32 (an indicator for axonal damage). We also found that EPPS treatment ameliorated the TBI-induced synaptic damage (as reflected by enhanced postsynaptic density 95, PSD-95), and impairment of autophagy flux in the neurons as reflected by reduced autophagy markers (LC3-II/LC3-I ratio and p62/SQSTM1) and increased lysosomal enzyme cathepsin D (CTSD) in neurons detected in the cortex and hippocampal CA1. As a result, EPPS treatment significantly reduced the TBI-induced early neuronal apoptosis (assessed by active caspase-3), and eventually prevented cortical tissue loss and hippocampal neuronal loss at 28 days after TBI. Additionally, we found that inhibition of autophagic flux with chloroquine by decreasing autophagosome-lysosome fusion significantly reversed the decreased expressions of neuronal p62/SQSTM1 and apoptosis by EPPS treatment. These data suggest that the neuroprotection by EPPS is, at least in part, related to improved autophagy flux. Finally, we found that EPPS treatment significantly improved the cortex-dependent motor and hippocampal-dependent cognitive deficits associated with TBI. Taken together, these findings support the further investigation of EPPS as a treatment for TBI.

Keywords: Autophagy; Axonal injury; Beta-amyloid; Neuroprotection; Traumatic brain injury.

Figure 1.

(A) Schematic diagram describing the experimental design. IHC, immunohistochemistry; WB, western blot. (B) Experimental design showing the administration of EPPS (120 mg/kg) and/or chloroquine (CQ, 10 mg/kg) or vehicle. (C) Schematic diagram showing the regions of interest (ROI) selected for immunohistochemical image acquisition and quantitative analysis: from the cortex (a), corpus callosum (b), external capsule (c), hippocampal CA1 (d) and CA3 (e).

Figure 2.. EPPS treatment reduces β-APP and Aβ accumulation and SMI-32 expression at 3 days after TBI.

Representative images of immunofluorescence staining showing (A) SMI-32 (red) and β-APP (green), and (B) SMI-32 (red) and Aβ (green). Images were taken from the pericontusional cortex, external capsule (EC), and hippocampal CA1 of the indicated groups. Semi-quantification of relative fluorescence intensity for β-APP, Aβ and SMI-32 in the pericontusional (C) cortex, (D) EC, and (E) hippocampal CA1 of the indicated groups. β-APP, beta-amyloid precursor protein; Aβ, beta-amyloid; SMI-32, nonphosphorylated neurofilament-H used as a marker of axonal damage. Scale bar = 50 μm. All data was expressed as mean ± S.E.M. and analyzed using the Kruskal-Wallis followed by Mann-Whitney U test. n=5 mice per group. *p<0.05 and **p<0.01 vs. vehicle.

Figure 3.. EPPS treatment reduces neuronal autophagosome accumulation and synaptic damage at 3 days after TBI.

(A) Representative images of Western blots showing the protein levels of autophagosomal markers, microtubule-associated protein 1 light chain 3 (LC3) and p62/SQSTM1 (sequestosome 1), as well as postsynaptic density 95 (PSD-95), in the injured cerebral cortex of the indicated groups. (B, C, D) Semi-quantitation of immunoblots was analyzed by densitometry, and data are expressed as the ratio of LC3-II to LC3-I (B), p62 level normalized to GAPDH (C), and PSD-95 level normalized to β-actin (D) from three independent experiments. (E, G) Representative images of immunofluorescence staining showing the expression and co-localization of the neuronal marker NeuN (red) and p62/SQSTM1 (green). Images were taken from the pericontusional (E) cortex and (G) hippocampal CA1 of the indicated groups. The number of overall p62-positive cells and p62-positive neurons in the pericontusional (F) cortex and (H) hippocampal CA1 were counted as described in the Methods section. Scale bar = 50 μm. All data was expressed as mean ± S.E.M. and analyzed using the Kruskal-Wallis followed by Mann-Whitney U test. n=5 mice per group. ^#p<0.05 and ^##p<0.01 vs. sham and *p<0.05 and **p<0.01 vs. vehicle.

Figure 4.. EPPS treatment ameliorates the impairment of the autophagy-lysosome pathway in neurons at 3 days after TBI.

(A, C) Representative images of immunofluorescence staining showing the expression and co-localization of the neuronal marker NeuN (red) and lysosomal enzyme cathepsin D (CTSD, green). Images were taken from the pericontusional (A) cortex and (C) hippocampal CA1 of the indicated groups. The number of overall CTSD-positive cells and CTSD-positive neurons in the pericontusional (C) cortex and (D) hippocampal CA1 were counted as described in the Methods section. Scale bar = 50 μm. (E) Representative images of Western blots showing the CTSD precursor form (53 kDa) and mature active form (30 kDa) in the injured cerebral cortex in the indicated groups. (F) Semi-quantitation of immunoblots was analyzed by densitometry. Data are expressed as fold changes of the two CTSD isoforms normalized to GAPDH (F) from three independent experiments. All data was expressed as mean ± S.E.M. and analyzed using the Kruskal-Wallis followed by Mann-Whitney U test. n=5 mice per group. ^#p<0.05 and ^##p<0.01 vs. sham and *p<0.05 and **p<0.01 vs. vehicle.

Figure 5.. EPPS treatment attenuates autophagic neuronal cell death at 3 days after TBI.

(A, B) Representative images of immunofluorescence staining showing the expression and co-localization of the neuronal marker NeuN (red) and active caspase-3 (A. casp-3). Images were taken from the pericontusional (A) cortex and (B) hippocampal CA1 of the indicated groups. (C) The number of A. casp-3-positive neurons in the pericontusional cortex and hippocampal CA1 were counted as described in the Methods section. (D) Representative images of immunofluorescence staining showing the expression and co-localization of the autophagosomal marker p62/SQSTM1 (red) and active caspase-3 (A. casp-3, green) in the indicated groups. (E) The number of overall A. casp-3-positive cells and the p62 and A. casp-3 double-positive cells in the pericontusional cortex were counted as described in the Methods section. Scale bar = 50 μm. All data was expressed as mean ± S.E.M. and analyzed using the Kruskal-Wallis followed by Mann-Whitney U test. n=5 mice per group. ^#p<0.05 and ^##p<0.01 vs. sham and *p<0.05 and **p<0.01 vs. vehicle.

Figure 6.. Inhibition of autophagic flux with chloroquine (CQ) reverses the EPPS-induced decrease in autophagosome accumulation at 3 days after TBI.

(A) Representative images of immunofluorescence staining showing the expression and co-localization of the neuronal marker NeuN (red) and autophagosomal marker p62/SQSTM1 (green) in the pericontusional cortex and hippocampal CA1 of the indicated groups. The number of p62-positive neurons in the pericontusional (B) cortex and (C) hippocampal CA1 were counted as described in the Methods section. Scale bar = 50 μm. All data was expressed as mean ± S.E.M. and analyzed using the Kruskal-Wallis followed by Mann-Whitney U test. n=4 mice per group. ^#p<0.05 and ^##p<0.01 vs. sham, *p<0.05 and **p<0.01 vs. vehicle, and ^§p<0.05 vs. EPPS+CQ.

Figure 7.. EPPS treatment reduces damages to the hippocampus and white matter and contusion volumes at 28 days after TBI.

(A) Representative H&E images showing the morphology of viable neurons in the hippocampal CA1 and CA3 in the indicated groups. Scale bar = 100 μm. (B) Quantification of the number of viable neurons in the indicated groups. (C) Representative Luxol fast blue (LFB) images of sham, vehicle- and EPPS-treated mice taken from ipsilateral hemisphere and external capsule (EC) for white matter integrity assessment. Scale bar = 100 μm. (D) Semi-quantification of relative LFB intensity in the EC of sham, vehicle- and EPPS-treated mice. ^#p<0.05 and ^##p<0.01 vs. sham and *p<0.05 vs. vehicle. (E) Representative H&E images showing whole brain coronal sections (−1.0, −1.5, −2.0 and −2.5 mm relative to bregma) in the vehicle- and EPPS-treated mice. Scale bar = 1 mm. (F) The contusion volumes in vehicle- and EPPS-treated groups. All data was expressed as mean ± S.E.M. and analyzed using the Kruskal-Wallis followed by Mann-Whitney U test. n=5 mice per group. ^#p<0.05 and ^##p<0.01 vs. sham and *p<0.05 vs. vehicle.

Figure 8.. EPPS treatment improves the short-term motor function and long-term cognitive function after TBI.

(A) Beam walking (left) and beam balance (right) tests were conducted at 0 to 5 days after TBI to evaluate the motor function in vehicle- (close circle) and EPPS-treated (open circle) mice. (B) Barnes maze test was conducted at 24 to 28 days after TBI, and (C) novel object recognition was conducted at 28 days after TBI in sham, vehicle- and EPPS-treated mice. All data was expressed as mean ± S.E.M. Data for beam-balance, beam-walking and Barnes maze latencies were analyzed using two-way repeated measures analysis of variance (ANOVA). Data for number of errors head pokes and novel object recognition index were analyzed using one-way ANOVA followed by the Bonferroni post hoc tests. n=9 mice per group. ^#p<0.05 vs. sham and *p<0.05 vs. vehicle.