Elsevier

Neuroscience

Volume 460, 15 April 2021, Pages 107-119
Neuroscience

Research Article
Reduction of Autophagosome Overload Attenuates Neuronal Cell Death After Traumatic Brain Injury

https://doi.org/10.1016/j.neuroscience.2021.02.007Get rights and content

Highlights

  • Autophagosomes accumulate in ipsilateral cerebral cortical neurons after TBI.

  • SAR405 is a highly specific VSP34 inhibitor that reduces autophagosome overload.

  • SAR405 attenuates apoptosis, ameliorates brain edema, and neurological defects induced by TBI.

  • CQ inhibits autophagosome degradation, reversing the protective effects of SAR405.

  • Autophagy inhibition by VSP34 gene knockout reduces neuronal cell death after TBI.

Abstract

Previous studies have shown that alterations in autophagy-related proteins exist extensively after traumatic brain injury (TBI). However, whether autophagy is enhanced or suppressed by TBI remains controversial. In our study, a controlled cortical impact was used to establish a model of moderate TBI in rats. We found that a significant increase in protein levels of LC3-II and SQSTM1 in the injured cortex group. However, there were no significant differences in protein levels of VPS34, Beclin-1, and phosphor-ULK1, which are the promoters of autophagy. Lysosome dysfunction after TBI might lead to autophagosome accumulation. In addition, the highly specific autophagy inhibitor SAR405 administration reduced TBI-induced apoptosis-related protein cleaved caspase-3 and cleaved caspase-9 levels in the ipsilateral cortex, as well as brain edema and neurological defects accessed by mNSS. Furthermore, chloroquine treatment reversed the beneficial effects of SAR405 by increasing the accumulation of autophagosomes. Finally, our data showed that autophagy inhibition by VPS34 gene knockout method attenuated cell death after TBI. Our findings indicate that impaired autophagosome degradation is involved in the pathological reaction after TBI, and the inhibition of autophagy contributes to attenuate neuronal cell death and functional defects.

Graphical abstract

Targets of SAR405 and chloroquine (CQ) on autophagy pathways. VPS34 is involved in autophagy initiation complex (VPS15-VPS34-ATG14L-Beclin-1) and inhibited by SAR405. CQ inhibits the pathway as it disrupts vesicle fusion between the lysosome and autophagosomes. *The figure was created and exported with BioRender.com under a paid subscription.

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Introduction

Traumatic brain injury (TBI) is a major medical problem worldwide. It leads to disability and death in adults, causing a public burden on modern society (Zeng et al., 2020a, Zeng et al., 2020b). Reducing secondary brain damage is the key to saving patient lives and improving neurological function. Many pathological processes, including apoptosis, inflammation, and oxidative stress, lead to aggravated secondary brain damage (Zhang and Wang, 2018). Although secondary brain damage may be reduced by various treatments, the curative effect and prognosis of TBI patients are poor (Anthony Jalin et al., 2019). Therefore, new treatment strategies are urgently needed to reduce secondary brain injury.

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conservative process in which cells mediate the retention and turnover of organelles and cytoplasm through lysosome-dependent pathways. In the process of autophagy, autophagosomes separate components for autophagy by forming a closed bilayer membrane structure and transporting it to lysosomes for degradation (Grishchuk et al., 2011, Cui et al., 2017a, Cui et al., 2017b). Some studies have shown that Autophagy is associated with apoptosis, neuroinflammatory response, and neurological deficiency in various neurological conditions, including TBI (Grishchuk et al., 2011, Xing et al., 2012, Zhang et al., 2017a, Zhang et al., 2017b). Liang et al. found that the autophagy marker protein, Beclin-1, interacts with Bcl-2 to regulate apoptosis (Liang et al., 1998). Luo et al. further found that reducing the Beclin-1/Bcl-2 ratio might reduce TBI-induced neuronal cell apoptosis (Luo et al., 2011). A growing body of research has proved that some drugs may reduce nerve cell damage by regulating the autophagy pathway (Smith et al., 2011, Zhang et al., 2017a, Zhang et al., 2017b). However, there remains a lack of direct evidence on the process and role of autophagy after TBI.

Previous researches have shown that autophagy is activated after TBI (Sadasivan et al., 2008, Luo et al., 2011). TBI was observed to induce an increase in Beclin-1 and LC3-II (the phosphatidyl ethanolamine-conjugated form) levels. Conversely, Sarkar et al. found that autophagy might be inhibited after TBI (Sarkar et al., 2014). They found that LC3-II levels were increased during the early stage after TBI due to autophagosome accumulation, but Beclin-1 levels were not altered. More evidence is needed to detect the state of autophagy after TBI before exploring its effect on secondary brain injury.

The effect of autophagy on the apoptotic signalling pathway in TBI remains unclear probably because existing studies employ non-selective drugs to regulate autophagy. For example, the widely used autophagy inhibitor, 3-methyladenine (3-MA), is thought to reduce neuronal apoptosis in TBI mice by inhibiting autophagy activation (Luo et al., 2011). On the contrary, Jin et al. showed that 3-MA might weaken the protective effect of mild hypothermia in brain injury treatment (Jin et al., 2016). Additionally, 3-MA inhibits both VPS34/PIK3C3 and PIK3C1. Wu et al. reported that continuous treatment with 3-MA might significantly increase autophagy by inhibiting PIK3C1 (Wu et al., 2010). Therefore, the use of selective inhibitors that target VPS34 might provide more convincing evidence on the role of autophagy in TBI.

SAR405 is a highly selective molecular mass kinase inhibitor that has been demonstrated to inhibit VPS34 without off-target activity on PIK3C1 kinases (Pasquier, 2015). Besides, the application of autophagy-related gene knockdown methods in TBI animal models has not been explored.

In the present study, we investigated the role and state of autophagy flux in rats after TBI and the precise regulation of autophagy using highly specific inhibitors and autophagy gene knockdown methods that target VPS34.

Section snippets

Animals preparation

All the rats were purchased from the Chengdu Dashuo Experimental Animal Co. Ltd. All surgical procedures and animal experiments were approved by the Institutional Animals Ethics Committees (IAEC) at Southwest Medical University, Luzhou, China (permit number: SYXK (Chuan) 2018-065), and complied with the Guidelines of the National Institutes of Health on the Care and Use of Laboratory Animals. A total of 228 adult (11–16 weeks) male Sprague-Dawley (SD) rats weighing 320 ± 10 g were used. They

Autophagosomes accumulated in the ipsilateral cerebral cortical neurons after TBI

To examine the state of autophagy after injury, we determined the time course of autophagy marker protein levels in the ipsilateral cortex. VPS34/PIK3C3 (phosphatidylinositol 3-kinase, catalytic subunit type 3)-Beclin-1 complex and ULK1 (unc-51 like autophagy activating kinase 1) complex are regarded as the markers of autophagy initiation, ATG12 (autophagy-related 12)–ATG5 (autophagy-related 5) conjugation initiates autophagy independently of Beclin-1. Western blot analysis showed that there

Discussion

In the present study, we established rat models of moderate brain injury and found that: (1) levels of upstream autophagy regulators, such as VPS34-Beclin-1 complex, ULK1 complex and ATG12–ATG5 conjugate remained unchanged in the cortex at all examined time points after TBI; (2) a significant increase in LC3-II and SQSTM1 protein levels, which peaked 1 d after TBI, was observed, and GFP-LC3 signal was mainly located in RBFOX3-positive neuronal cells; (3) the increase of apoptotic protein was

Author contributions

Xingyun Quan, Li Song, and Xiaomei Zheng designed this research; Huaqiang Ding, Sijing Li, Guanghui Xu. and Xin Li. performed experiment; Xingyun Quan and Liang Liu. analyzed the data; Xingyun Quan and Li Song wrote the paper.

Acknowledgements

This work was supported by grants from the Project of Sichuan Provincial Health Department (110371); the Project of Sichuan Medical Association (S17074); the Key projects of Education Department of Sichuan (12ZA075); the Luzhou Science and Technology Bureau Project (2017-S-40).

Conflict of interest

The authors declare no conflict of interest.

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    These authors contributed equally to this work.

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