Inhibition of Drp1 after traumatic brain injury provides brain protection and improves behavioral performance in rats

https://doi.org/10.1016/j.cbi.2019.03.013Get rights and content

Highlights

  • Drp1 protein expression was increased in the ipsilateral hippocampus after TBI. But, TBI-induced phosphorylation of Drp1 were not observed within the first 24 h.

  • Mdivi-1 attenuated H2O2-induced mitochondrial membrane potential dissipation in PC-12 cells.

  • Mdivi-1 rescued neurogenesis impairment in DG and attenuated brain damage against TBI.

  • Mdivi-1 treatment post-injury was highly effective in animal model of TBI to improve cognitive deficits and anxiety-like behavior.

Abstract

The imbalance between mitochondrial fusion and fission has been implicated in cerebral ischemia and several neurodegenerative diseases. However, the role of mitochondrial fission in traumatic brain injury (TBI) remains poorly understood. Mitochondrial fission is mediated by dynamin-related protein 1 (Drp1), which is highly expressed in the nervous system. In the present study, we investigated the changes in Drp1 expression in the ipsilateral hippocampus of rats after TBI and the effects of Mdivi-1 (a selective inhibitor of Drp1) as a post-insult treatment for TBI. Our findings showed that the protein levels of Drp1 were increased at 6 h and peaked at 12 h post-TBI, but we did not observe Drp1 phosphorylation at Ser616, Ser637, Ser40 or Ser44 during this process. We examined the effect of Mdivi-1 on trauma-induced brain damage in both vitro and vivo. In cells, Mdivi-1 significantly attenuated H2O2-induced mitochondrial membrane potential (MMP) dissipation in PC-12 cells. Three days of Mdivi-1 treatment significantly reduced the cortical lesion volume, blood-brain barrier permeability, brain edema and oxidative stress. Mdivi-1 reduced activated caspase-3 release in the cortical border zone and hippocampal dentate gyrus three days after TBI. Furthermore, treatment with Mdivi-1 for 4 weeks rescued neurogenesis in DG and attenuated hippocampal atrophy. Regarding behavioral outcomes, Mdivi-1-treated TBI rats showed a significant improvement in water maze acquisition and retention compared with the saline-treated TBI rats. Moreover, Mdivi-1 treatment reduced anxiety-like behavior in an open-field test. Our results support the notion that Mdivi-1 provides brain protection and improves the behavioral performance in TBI rats.

Introduction

Traumatic brain injury (TBI) is a common cause of serious public health problems throughout the world. TBI patients impose a tremendous burden on their families and society and increase demands on the healthcare system. Survivors of TBI experience various problems, including sensory and motor dysfunction as well as cognitive, emotional, and behavioral problems, which can occur either individually or in combination [1,2]. TBI is a devastating injury that often results in lifelong cognitive deficits [3]. More than 70% of people who sustain TBI report memory deficits [4]. In a controlled cortical impact mouse model of TBI, cognitive impairments evidenced during the Morris water maze test increased with injury severity [5]. Other studies have shown selective behavioral deficits after neurological impairment mild traumatic brain injury model [6]. The hippocampus plays a crucial role in memory and cognition, but it is highly vulnerable to brain trauma [7]. TBI results in significant hippocampal atrophy in patients and a reduction in hippocampal volume, correlated with damaged verbal memory function [8]. Neurons in the rat hippocampus showed cell death features, such as condensed nuclei or fragmentation of nuclei, several hours after TBI [9]. Cognitive decline is displayed post-injury in mice and coincides with loss of neurons in the hippocampus [10,11]. Neurogenesis includes the proliferation of neural precursor cells and the survival of newborn neurons, which are strongly interrelated with hippocampus-dependent learning and memory [12]. Hippocampal neurogenesis plays a significant role in TBI-related pathologies; for example, it is implicated in higher cognitive function [10] and has been associated with post-traumatic seizure generation [13]. This suggests that damage to the hippocampus is widespread post-injury and may contribute to the cognitive deficits seen in TBI survivors. However, the molecular basis for the memory and behavioral deficits caused by TBI is unclear, and understanding these biochemical mechanisms could lead to new pharmacological therapies to improve cognition after TBI.

The brain is a highly aerobic, energy-demanding tissue and, as such, is dependent upon mitochondria to maintain cerebral function. Recent studies have proven that there is an imbalance between increased energy demand to repair cell damage and decreased energy production caused by mitochondrial dysfunction during the pathogenic process of TBI [14]. Mitochondria not only provide cellular energy through ATP synthesis, but also play an important role in intracellular calcium buffering, reactive oxygen species (ROS) production, and apoptosis. Post-traumatic perturbations in cellular mitochondrial function have been documented by several researchers, and may trigger or exacerbate damaging secondary intracellular cascades after the primary injury: altered glucose utilization, diminished energy production, functional mitochondrial changes [[15], [16], [17]]. Dysfunctions in the mitochondria post-TBI have been linked to the impairment of brain mitochondrial electron transfer chain and energy transduction attributed to the overloading of intracellular calcium ([Ca2+]i), oxidative damage, and disruption of synaptic homeostasis ultimately ensuing with cell death [18]. The disturbance of [Ca2+]i homeostasis and the extensive production of intracellular ROS can damage the mitochondrial membrane lipids, which is one of the inducers of mitochondrial membrane potential (MMP) reduction [19]. Mitochondria continuously undergo fission and fusion, which helps to maintain mitochondrial circuitry and homeostasis. These dynamic processes are involved in cell apoptosis [20,21] and are essential for synaptic functions [22,23]. Excessive fission can lead to reduced mitochondrial respiration and ATP production, increased ROS generation, and release of apoptogenic factors [24,25], changes similar to those seen in the second injury after TBI. Emerging evidence indicates that Drp1 is concomitantly involved in apoptosis with mitochondrial outer membrane permeabilization (MOMP) and that inhibition of Drp1 prevents partially intrinsic apoptosis [26]. It has been widely acknowledged that massive production of reactive oxygen species, such as superoxide anion (O2-) and hydrogen peroxide (H2O2), played a crucial role in the process of delayed neuronal death following TBI. PC-12 is a cell line derived from rat pheochromocytoma and has been widely used as an in vitro model for investigating neuronal apoptosis, oxygen sensor mechanism [27]. In this study, we tested the hypothesis that Mdivi-1 may have neuroprotective potential in H2O2-stimulated cell apoptosis model through ameliorating mitochondrial membrane potential dissipation.

Mitochondrial membrane fusion is known to be mediated by mitofusin-1 and mitofusin-2 (Mfn1 and Mfn2, respectively) and optic atrophy 1 (OPA1) [20]. Mitochondrial fission is mediated by mitochondrial fission 1 (Fis1) and dynamin-related protein 1 (Drp1) [28]. Drp1 is a large protein in the dynamin GTPase family that is critical for regulating mitochondrial division, size and shape. Drp1 assembles from the cytosol onto mitochondria at focal sites of mitochondrial fission. Disorders in the fusion/fission balance resulting from the upregulation of Drp1 in various diseases may limit mitochondrial motility, decrease energy production and promote oxidative stress, all of which induce cell dysfunction and death in a variety of cell types, including islet cells, hepatocytes, skeletal muscle cells, mononuclear blood cells, endothelial cells, and dorsal root ganglion neurons [22,29]. Compared to other cell types, neurons are highly susceptible to mitochondrial dysfunction because mitochondrial fission is essential for the axonal transport of organelles into areas with high metabolic demand, whereas mitochondrial fusion supports the substitution and regeneration of mitochondrial proteins [30]. Drp1 is highly expressed in the nervous system [[31], [32], [33], [34]]. Several recent studies reported abnormal Drp1 expression in the postmortem brains from APP cell lines, AD mouse models and AD patients [[35], [36], [37]]. Previous reports have suggested that excessive mitochondrial fission contributes to the pathogenesis of Alzheimer's disease (AD) via neuronal death and synaptic damage [38,39]. Moreover, recent studies have proven that inhibition of mitochondrial fission plays a protective role against neurodegeneration in the cerebellum [30] and against neurotoxicity [40]. However, the role of mitochondrial fission in the pathogenic process of TBI has not been illuminated; thus, our studies sought to evaluate the role of Drp1 in a TBI model.

Mitochondrial division inhibitor-1 (Mdivi-1) is a selective inhibitor of Drp1. It has been shown to selectively target Drp1 in mammalian cells by binding an allosteric site and suppressing the ability of Drp1 to catalyze GTP hydrolysis as well as its self-assembly into ring-like structures around the mitochondria [41,42]. As a selective inhibitor of mitochondrial division, most studies about Mdivi-1 so far have been focused on Drp1 inhibition, and there is little known regarding the relationship of Mdivi-1 with other signals. Mdivi-1 can induce the rapid and reversible formation of interconnected mitochondria without affecting other cellular structures such as the cytoskeleton and endoplasmic reticulum, suggesting its selectivity for mitochondrial fission. Inhibition of Drp1 by Mdivi-1 remarkably reduced the infarct volume and neurological deficits in MCAO mice [43]. Mdivi-1 also protected primary neurons against glutamate excitotoxicity and attenuated ischemic brain damage in a mouse model [44]. However, despite its well-known and multifaceted neuroprotective effects, Mdivi-1 has rarely been investigated as a post-insult treatment for TBI. In our study, the potential therapeutic effects of Mdivi-1 were tested in a model of moderate traumatic brain injury.

Drp1 is regulated by various posttranslational modifications, including phosphorylation, ubiquitylation, and sumoylation [31,45,46]. For example, phosphorylation of Drp1 at Ser616 by calmodulin-dependent protein kinase Iα could promote mitochondrial fission [33], and phosphorylation of Drp1 at Ser637 by cyclic adenosine monophosphate-dependent protein kinase could inhibit mitochondrial fission [32,45]. Recently, reports indicated that glycogen synthase kinase 3β (GSK-3β)-dependent Drp1 phosphorylation at Ser40 and Ser44 could increase Drp1 GTPase activity and mitochondrial fragmentation in neurons [47]. Furthermore, blocking GSK-3β-induced Drp1 phosphorylation in the hippocampal cornu ammonis 1 (CA1) region significantly reduced the Aβ burden and rescued memory deficits in AD transgenic mice [47]. Phosphorylation of Drp1 is an important posttranslational modification that affects the activity of the protein. However, whether phosphorylation of Drp1 contributed to TBI pathogenesis is still unclear. In our study, we investigated whether Mdivi-1 could rescue the cognitive deficits and anxiety-like behavior after TBI.

Section snippets

Animals and housing

Adult male Sprague Dawley rats (2-month-old males, 250─300 g) were individually housed in standard cages at 22 ± 2 °C under diurnal conditions (12 h light/dark cycle) and had access to water and food ad libitum. The rats were fasted for 8 h before they underwent surgery. The animals were divided into 4 groups (n = 10/group) for surgery and testing. After either sham surgery or TBI, all rats were transferred to single housing for the remainder of the study. All procedures were performed in

Drp1 but not Phosphorylation of Drp1 was enhanced in the hippocampus after TBI

In our studies, rats were subjected to TBI, and ipsilateral hippocampus tissue extracts prepared from animals at 3 h, 6 h, 12 h and 24 h post-injury were subjected to western blot analyses. The results indicated that the total Drp1 levels in the ipsilateral hippocampus in the first 24 h after TBI began to increase at 6 h (Fig. 1A, F(4,25) = 8.661, P = 0.029), peaked at 12 h (P < 0.01), and then slightly decreased afterward. Phosphorylation of Drp1 is an important posttranslational modification

Discussion

In both experimental and clinical traumatic brain injury, chronic neurological and psychological dysfunctions are common but have not been well addressed with current pharmacological treatments [57,58]. Our results provided some new insights into the protective effects of Mdivi-1 on TBI-induced neurological and psychological dysfunction. In this study, we investigated whether Drp1 was manipulated in the pathological processes of TBI and observed that TBI increased the expression of total Drp1,

Author contributions

LYC and YGL designed the study; YS, TL, ZGL, ZKX, ZLZ, and FL performed the experiments and analyzed the data. YS wrote the manuscript. All authors agreed to be accountable for the content of the work.

Conflict of interest

None.

Acknowledgments

The study was supported by the Promotive Research Fund for Young and Middle-Aged Scientists of Shandong Province (BS10YY026) and the Independent Innovation Foundation of Shandong University, IIFSDU (2012TS144, 2015TS017).

References (78)

  • N. Noshita et al.

    Neurobiol. Dis.

    (2002)
  • A. Pitkanen et al.

    Neurotherapeutics

    (2014)
  • J. Grohm et al.

    Brain Behav. Immun.

    (2010)
  • D.C. Chan

    Cell

    (2006)
  • P. Jezek et al.

    Int. J. Biochem. Cell Biol.

    (2009)
  • A. Jahani-Asl et al.

    J. Biol. Chem.

    (2011)
  • P.H. Reddy et al.

    Brain Res. Rev.

    (2011)
  • H. Chen et al.

    Cell

    (2007)
  • E. Bossy-Wetzel et al.

    Curr. Opin. Cell Biol.

    (2003)
  • A. Cassidy-Stone et al.

    Dev. Cell

    (2008)
  • A. Tanaka et al.

    Mol. Cell

    (2008)
  • Y.X. Zhao et al.

    CNS Neurosci. Ther.

    (2014)
  • C.R. Chang et al.

    J. Biol. Chem.

    (2007)
  • Z. Harder et al.

    Curr. Biol.

    (2004)
  • J. Yan et al.

    Neurobiol. Aging

    (2015)
  • L. Kong et al.

    Pharmacol. Res.

    (2015)
  • Y. Tang et al.

    Exp. Neurol.

    (2010)
  • X. Qiu et al.

    Neuroscience

    (2013)
  • Y. Cao et al.

    Brain Res.

    (2013)
  • N. Taguchi et al.

    J. Biol. Chem.

    (2007)
  • H. Yang et al.

    Acta Pharmacol. Sin.

    (2016)
  • M. Shapira et al.

    Mol. Cell Neurosci.

    (2007)
  • L.L. Lackner et al.

    Biochim. Biophys. Acta

    (2009)
  • M. Ariza et al.

    Neuropsychologia

    (2006)
  • M.A. Trivedi et al.

    Neurobiol. Learn. Mem.

    (2004)
  • W.H. Williams et al.

    J. Neurol. Neurosurg. Psychiatry

    (2010)
  • J.M. Silver et al.

    Am. J. Psychiatry

    (2009)
  • E. Zaloshnja et al.

    J. Head Trauma Rehabil.

    (2008)
  • H.L. Lew et al.

    J. Rehabil. Res. Dev.

    (2006)
  • P.M. Washington et al.

    J. Neurotrauma

    (2012)
  • A. Milman et al.

    J. Neurotrauma

    (2005)
  • F. Tomaiuolo et al.

    J. Neurol. Neurosurg. Psychiatry

    (2004)
  • E.D. Bigler et al.

    AJNR. Am. J. Neuroradiol.

    (1997)
  • R. Pullela et al.

    Dev. Neurosci.

    (2006)
  • S. Zhao et al.

    J. Neurotrauma

    (2016)
  • P. Hu et al.

    Hippocampus

    (2016)
  • J. Lifshitz et al.

    J. Cereb. Blood Flow Metab.

    (2003)
  • P.A. Casey et al.

    J. Neurotrauma

    (2008)
  • S. Scafidi et al.

    J. Neurochem.

    (2009)
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