Inhibiting phosphatase and actin regulator 1 expression is neuroprotective in the context of traumatic brain injury

Abstract

Studies have found that the phosphatase actin regulatory factor 1 expression can be related to stroke, but it remains unclear whether changes in phosphatase actin regulatory factor 1 expression also play a role in traumatic brain injury. In this study we found that, in a mouse model of traumatic brain injury induced by controlled cortical impact, phosphatase actin regulatory factor 1 expression is increased in endothelial cells, neurons, astrocytes, and microglia. When we overexpressed phosphatase actin regulatory factor 1 by injection an adeno-associated virus vector into the contused area in the traumatic brain injury mice, the water content of the brain tissue increased. However, when phosphatase actin regulatory factor 1 was knocked down, the water content decreased. We also found that inhibiting phosphatase actin regulatory factor 1 expression regulated the nuclear factor kappa B signaling pathway, decreased blood-brain barrier permeability, reduced aquaporin 4 and intercellular adhesion molecule 1 expression, inhibited neuroinflammation, and neuronal apoptosis, thereby improving neurological function. The findings from this study indicate that phosphatase actin regulatory factor 1 may be a potential therapeutic target for traumatic brain injury.

Keywords: apoptosis; aquaporin 4; blood brain barrier; intercellular adhesion molecule 1; neuroinflammation; nuclear factor kappa B; occludin; phosphatase and actin regulator-1; traumatic brain injury; zonula occludens 1.

Figure 1

PHACTR-1 expression in the brain after TBI. (A, B) PHACTR-1 protein expression was significantly increased in the TBI group compared with the sham group, and the increased expression was maintained for at least 7 days. Values were normalized to the sham group and are expressed as mean ± SD (n = 3/group). *P < 0.05, **P < 0.01, vs. sham group (one-way analysis of variance followed by Tukey’s honestly significant difference post hoc test). (C) Immunofluorescence staining for PHACTR-1 (Alexa Fluor 488, green) in endothelial cells (CD31-positive cells, Alexa Fluor 555, red), astrocytes (GFAP-positive cells, Alexa Fluor 594, red), microglial cells (Iba1-positive cells, Alexa Fluor 647, red), and neurons (NeuN-positive cells, Alexa Fluor 594, red). PHACTR-1 expression was significantly increased in these cells 3 days after TBI. White arrows indicate co-staining. Scale bar: 25 μm. GFAP: Glial fibrillary acidic protein; Iba: ionized calcium-binding adapter molecule; PHACTR: phosphatase and actin regulator; TBI: traumatic brain injury.

Figure 2

Effect of AAV targeting PHACTR-1 on the contusion point after TBI. (A) Contusion point (blue area) and AAV injection sites (red arrows) in the peri-contusion area. The fluorescence image shows transfected AAV (green) in the peri-contusion area. Scale bar: 50 μm. (B, C) Western blot analysis of PHACTR-1 expression in the different groups 3 days after TBI. Values were normalized to the sham group and are expressed as mean ± SD (n = 3/group). **P < 0.01, vs. TBI group (one-way analysis of variance followed by Tukey’s honestly significant difference post hoc test). AAV: Adeno-associated virus; PHACTR: phosphatase and actin regulator; TBI: traumatic brain injury.

Figure 3

Effect of PHACTR-1 on brain edema and BBB integrity 3 days after TBI. (A) Brain water content in the different groups (*P < 0.05, **P < 0.01, vs. TBI) (one-way analysis of variance followed by Tukey’s Honestly Significant Difference post hoc test). (B, C) The EB concentration was significantly decreased in the TBI + PHACTR-1-RNAi group compared with the TBI + NC-RNAi group. (D–F) Expression of the TJ proteins ZO-1 and occludin (both Alexa Fluor 488, green) in endothelial cells (CD31) (Alexa Fluor 555, red) was significantly increased in the TBI + PHACTR-1-RNAi group compared with the TBI + NC-RNAi group, and measurement of quantification by western blotting (normalized to the sham group). Scale bar: 25 μm. Data are expressed as mean ± SD (n = 5/group). *P < 0.05, **P < 0.01, vs. TBI + NC-RNAi group (Student’s t-test). BBB: Blood-brain barrier; EB: Evans Blue; PHACTR: phosphatase and actin regulator; TBI: traumatic brain injury; TJ: tight junction; ZO: zonula occludens.

Figure 4

Effect of PHACTR-1 on AQP-4 and ICAM-1 expression in injured brain tissues 3 days after TBI. (A–C) AQP-4 (Alexa Fluor 647, red) expression levels were significantly decreased after PHACTR-1 knockdown. (D–F) ICAM-1 (Alexa Fluor 647, red) expression levels were significantly decreased after PHACTR-1 knockdown. Scale bar: 35 μm in A, 60 μm in D. Data are expressed as mean ± SD (n = 5/group). **P < 0.01, vs. TBI + NC-RNAi group (Student’s t-test). AQP: Aquaporin; ICAM: intercellular cell adhesion molecule; PHACTR: phosphatase and actin regulator; TBI: traumatic brain injury.

Figure 5

Effect of PHACTR-1 on neuroinflammation in injured brain tissues 3 days after TBI. (A) Immunofluorescence staining showed that astrocyte (GFAP-positive cells, Alexa Fluor 594, red) and M1 microglial cell (CD16/32 (Alexa Fluor 488, green)/Iba1 (Alexa Fluor 647, red)-positive cells) activation was increased after TBI but reduced by PHACTR-1 knockdown. The opposite effect was seen for M2 microglial cells (CD206 (Alexa Fluor 488, green)/Iba1 (Alexa Fluor 647, red)-positive cells). Scale bars: 60 μm. (B, C) After TBI, expression of the inflammatory cytokines TNF-α, IL-1β, and IL-6 was decreased by PHACTR-1 knockdown, while expression of the anti-inflammatory cytokines IL-4 and IL-10 was increased by PHACTR-1 knockdown, as detected by real-time polymerase chain reaction. Data are expressed as mean ± SD (n = 5/group). *P < 0.05, **P < 0.01, vs. TBI + NC-RNAi group (Student’s t-test). GFAP: Glial fibrillary acidic protein; Iba: ionized calcium-binding adapter molecule; IL: interleukin; PHACTR: phosphatase and actin regulator; TBI: traumatic brain injury, TNF: tumor necrosis factor alpha.

Figure 6

Effect of PHACTR-1 on neuronal apoptosis and brain volume loss 7 days after TBI. (A–C) The number of apoptotic nerve cells and neurons (NeuN positive cell, Alexa Fluor 488, green) observed after TBI was significantly reduced by PHACTR-1 knockdown. Scale bars: 25 μm. (D, E) Nissl staining showed that the brain volume loss induced by TBI was attenuated by PHACTR-1 knockdown. Data are expressed as mean ± SD (n = 5/group). *P < 0.05, **P < 0.01, vs. TBI + NC-RNAi group (Student’s t-test). DAPI: 4′,6-Diamidino-2-phenylindole; PHACTR: phosphatase and actin regulator; TBI: traumatic brain injury; TUNEL: TdT-mediated dUTP nick-end labeling.

Figure 7

Effect of PHACTR-1 on NF-κB/p65 expression in injured brain tissue 3 days after TBI. (A–C) Expression of NF-κB/p65 (Alexa Fluor 594, red) in both the cytoplasm and nucleus, as well as translocation of p65 to the nucleus, induced by TBI were significantly decreased by PHACTR-1 knockdown. Scale bar: 25 μm. Data are expressed as mean ± SD (n = 5/group). *P < 0.05, **P < 0.01, vs. TBI + NC-RNAi group (Student’s t-test). DAPI: 4′,6-Diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; NF-κB: nuclear factor kappa B; PHACTR: phosphatase and actin regulator; TBI: traumatic brain injury.

Figure 8

Effect of PHACTR-1 on neurological function after TBI. (A, B) After PHACTR-1 knockdown, the mNSS decreased significantly from day 3 after TBI, and rotarod latency increased from day 7 after TBI. Data are expressed as mean ± SD (n = 12/group). *P < 0.05, **P < 0.01, vs. TBI + NC-RNAi group (Student’s t-test). mNSS: Modified neurological severity score; PHACTR: phosphatase and actin regulator; TBI: traumatic brain injury.