Deficiency of unc-51 like kinase 1 (Ulk1) protects against mice traumatic brain injury (TBI) by suppression of p38 and JNK pathway

https://doi.org/10.1016/j.bbrc.2018.04.154Get rights and content

Highlights

  • Ulk1-KO mice show reduced inflammation and apoptosis in hippocampus after TBI.

  • Ulk1-KO mice alleviate glial cells activation and autophagy in hippocampus of mice with TBI.

  • Ulk1 inhibition reduces inflammatory response, apoptosis and autophagy in LPS-stimulated astrocytes (AST) via p38/JNK pathway.

Abstract

Unc-51 like autophagy activating kinase 1 (Ulk1) is a serine/threonine kinase that plays a key role in regulating autophagy processes. We attempted to investigate the effects of Ulk1 on traumatic brain injury (TBI) progression by using wild type (WT) mice and Ulk1-knockout (KO) mice suffered with or not TBI. The results were verified using LPS-treated primary astrocyte (AST). Here, Ulk1 was over-expressed in hippocampus of WT mice after TBI, as well as in lipopolysaccharide (LPS)-stimulated AST. Ulk1-deletion improved cognitive ability and hippocampus histological changes in TBI mice. Nissl and neuronal nuclei (NeuN) staining indicated that Ulk1-deletion increased the number of surviving neurons in hippocampus of TBI mice. Ulk1-ablation alleviated neuroinflammation, as evidenced by the reduced expression of hippocampus pro-inflammatory cytokines in TBI mice. TBI-induced apoptosis was also ameliorated by Ulk1-ablation, as proved by the reduced number of TUNEL-staining cells, and cleaved Caspase-3 and poly (ADP-ribose) polymerase (PARP) expressions. Moreover, Ulk1-knockout suppressed TBI-stimulated activation of astrocytes and microglia cells. Additionally, hippocampus autophagy induced by TBI was attenuated by Ulk1-knockout. Further, TBI-activated p38/c-Jun N-terminal Kinase (JNK) pathway was repressed by Ulk1-deletion in hippocampus of mice. The findings above were confirmed in LPS-stimulated AST with or without Ulk1 siRNA transfection. Intriguingly, pre-treatment of p38 or JNK activator markedly abolished the anti-inflammation, anti-apoptosis and anti-autophagy effects of Ulk1-knockdown on LPS-incubated AST. In conclusion, our results demonstrated that Ulk1 might be a potential target for developing therapeutic strategy against TBI in future.

Introduction

Traumatic brain injury (TBI), a major cause of death and disability worldwide, is characterized by a direct injury to the head, resulting in tissue damage [1]. The function of brain is abnormal in patients with TBI, exhibiting neurological dysfunction, which is mainly caused by various pathological processes, such as inflammatory response, apoptosis and autophagy [2,3]. Despite the improved diagnosis and treatment methods, further study is still necessary to reveal the underlying mechanism by which TBI progresses and thus to develop effective therapeutic strategies.

Unc-51 like autophagy activating kinase 1 (Ulk1), as a serine/threonine kinase, plays a key role in autophagy induction, integrating signals from up-streaming sensors and transducing them to the down-streaming autophagy pathway [4]. In several cell lines, Ulk1 knockdown inhibits autophagy [5]. In addition, liver-specific deficiency of Ulk1 alleviates liver injury in animals through reducing autophagy under the regulation of JNK pathway [6]. Ulk1 is of great importance in the initial stages of autophagy and it is possible that genetic variation in this gene may lead to autophagy-mediated control of cellular processes, such as inflammation and apoptosis [7,8]. As reported previously, stress kinase p38 MAPK in microglia senses inflammatory cue LPS, directly activates Ulk1, reliving the autophagic inhibition on the inflammatory machinery, and therefore allowing for a full immune response [9]. Therefore, we hypothesized that Ulk1 might be also involved in TBI progression through regulating various cellular processes. And up until now, the effects of Ulk1 in TBI have not been reported.

In the present study, the wild type (WT) and Ulk1-knockout (KO) mice were employed to explore the role of Ulk1 in TBI development. We first found that Ulk1 was over-expressed in the hippocampus of WT mice. Ulk1-KO mice showed improved cognitive ability after TBI, accompanied with alleviated histological alterations of hippocampus. Ulk1-deletion reduced hippocampus inflammation and apoptosis in TBI mice. Further, the activation of astrocytes and microglial cells stimulated by TBI was also inhibited by Ulk1-KO. Autophagy, triggered by TBI, in hippocampus of mice was diminished in Ulk1-KO mice. The in vitro study using LPS-stimulated primary astrocytes confirmed that Ulk1 deficiency protected mice from TBI through p38/JNK pathway. These results supplied that Ulk1, at least partially, could be considered as an effective candidate for developing novel therapeutic strategies to suppress TBI progression.

Section snippets

Animals and treatment

Adult wild type (WT) male C57/BL6 and Ulk1-knockout (KO) male mice (6–8 weeks old), weighing approximately 20 ± 2 g, were purchased from Laboratory Animal Center of Shandong University (Shandong, China) and Cyagen Biosciences (Guangzhou, China), respectively. Animals were cultured in a pathogen free feeding center under controlled conditions (temperature 25 ± 2 °C and humidity 70 ± 5%) and a 12:12 h light: dark cycle. Mice were randomly divided into four groups (n = 8/group): WT/Sham, WT/TBI,

Ulk1 is highly expressed in hippocampus of mice after TBI and Ulk1-deficiency improves MWM performance in TBI mice

RT-qPCR and western blot analysis showed an evidently increased expression of Ulk1 in hippocampus of WT mice after TBI at D1, D3, D5 and D7 (Fig. 1A). Consistently, LPS treatment stimulated Ulk1 mRNA and protein expressions in a time-dependent manner (Fig. 1B). To further reveal the effects of Ulk1 in TBI, the WT mice and Ulk1-null mice (Ulk1-knockout/KO) mice were used for further research. MWM analysis was performed after TBI. The performance in probe trial of MWM on day 6 was determined

Discussion

Various molecular mechanisms or signaling pathways are involved in TBI, such as oxidative stress, inflammation, neuronal cell apoptosis and autophagy [[1], [2], [3]]. However, it's still not fully demonstrated. Ulk1 is reported to have a vital contribution in regulating autophagy [[4], [5], [6]]. And Ulk1 has been shown to be associated with brain injury; however, its role in TBI has never been elucidated. The present study evaluated the effects of Ulk1 on a mouse model of TBI. The major

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