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Abnormal mitochondrial dynamics and impaired mitochondrial biogenesis in trigeminal ganglion neurons in a rat model of migraine
Neuroscience Letters 636 (2017) 127–133
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Abnormal mitochondrial dynamics and impaired mitochondrial biogenesis in trigeminal ganglion neurons in a rat model of migraine Xin Dong a,1 , Xinying Guan a,1 , Keyan Chen a , Shanquan Jin a , Chengyun Wang b , Lanyun Yan a , Zhaochun Shi a , Xue Zhang a , Ling Chen c , Qi Wan a,∗ a
Department of Neurology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu Province, 210029, China Department of Neurology, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, Jiangsu Province, 223300, China c Department of Physiology, Nanjing Medical University, 818 Tianyuan East Road, Nanjing, Jiangsu Province, 210029, China b
h i g h l i g h t s • Defective mitochondrial morphology was detected in TG neurons in a rat model of migraine. • Mitochondrial dynamics was shifted towards fission in TG neurons in a rat model of migraine. • Mitochondrial biogenesis was suppressed in TG neurons in a rat model of migraine.
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Article history: Received 15 September 2016 Received in revised form 26 October 2016 Accepted 27 October 2016 Available online 29 October 2016 Keywords: Migraine Mitochondrial dynamics Mitochondrial biogenesis Trigeminal ganglion
a b s t r a c t Accumulating evidence has demonstrated a possible role of mitochondrial dysfunction in migraine pathophysiology. Migraine sufferers exhibit impaired metabolic capacity, with an increased formation of reactive oxygen species (ROS). Mitochondrial dynamics and mitochondrial biogenesis are key processes regulating mitochondrial homeostasis. The aim of this study was to explore the alterations of mitochondrial regulatory networks in a rat model of migraine induced by repeated dural stimulation with inflammatory soup (IS). Ultrastructural, protein, gene and mitochondrial DNA analysis were applied to assess mitochondrial dynamics and biogenesis in trigeminal ganglion (TG) neurons. Mitochondria in TG neurons exhibited small and fragmented morphology after repeated dural stimulation. Further investigations showed that mitochondrial fission protein dynamin-related protein 1 (Drp1) was increased while mitochondrial fusion protein Mitofusin1 (Mfn1) was reduced in TG neurons. In addition, our results also presented that mitochondrial DNA copy number in TG neurons was reduced significantly, accompanied by alterations in mRNA and protein levels of regulatory factors related to mitochondrial biogenesis including peroxisome proliferator-activated receptor-gamma coactivator-1a (PGC-1␣) and its downstream regulators in TG neurons in the IS-induced migraine model. These findings suggest that the mitochondrial dynamic regulatory networks are maladjusted in TG neurons in a rat model of migraine. Regulation of mitochondrial dynamics and biogenesis signaling may indicate a new mitochondria-targeted therapeutic strategy for migraine. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Abbreviations: ROS, reactive oxygen species; TG, trigeminal ganglion; Drp1, Dynamin related protein 1; Mfn1, Mitofusin1; mtDNA, mitochondrial DNA; PGC-1a, peroxisome proliferator-activated receptor-gamma coactivator-1a; NRF1, nuclear respiratory factor 1; NRF2, nuclear respiratory factor 2; TFAM, mitochondrial transcription factor A; IS, inflammatory soup. ∗ Corresponding author at: Department of Neurology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu Province, 210029, China. E-mail address: qi [email protected] (Q. Wan). 1 These authors contribute equally to this work. http://dx.doi.org/10.1016/j.neulet.2016.10.054 0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.
Migraine is a complex neurological disorder affecting about 12% of the population worldwide and is found to be the sixth most prevalent cause of disability [1]. Prophylactic pharmacotherapy for migraine headaches is less than satisfactory, due to the poor efficacy and debilitating adverse effects. Previous investigations have suggested a possible role of mitochondrial dysfunction in migraine pathophysiology [2]. Further elucidation of the metabolism signaling in the primary afferent neurons in the headache circuit will
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facilitate our understanding of the pathophysiology of migraine and promote the development of new therapeutics. Mitochondria are the primary energy-generating system in eukaryotic cells. Due to the high dependence on energy for neural activity, aberrant mitochondrial morphology and function may cause a series of neurological diseases [3]. Most recently, substantial amounts of evidence have demonstrated a contribution of mitochondria to pain processing in neuropathic and inflammatory pain states [4]. Mechanical stimulation of trigeminal sensory neurons can increase the production of reactive oxygen spices (ROS) in the spinal trigeminal nucleus [5]. Biochemical and genetic studies have identified a decreased activity of mitochondrial respiratory chain enzymes with an increased formation of ROS in migraine sufferers and detected specific mitochondrial DNA (mtDNA) variants responsible for migraine susceptibility [2,6]. Mitochondrial quality control is achieved by the dynamic interplay of fusion, fission and mitochondrial biogenesis. Fission is a process dividing the damaged mitochondria into a healthy and an abnormal portion while fusion results in a mixing of membranes and matrix content among mitochondria [7]. Mitochondrial fission and fusion processes are mediated by Dynamin-related protein 1 (Drp1) and Mitofusin1 (Mfn1), respectively. Mitochondrial dynamics including fission and fusion plays a significant role in maintaining mitochondrial morphology and function when cells experience stress [8]. Mitochondrial biogenesis responds to metabolic signals by increasing the actual mass of mitochondria with mtDNA replication [9]. It is tightly regulated by peroxisome proliferator-activated receptor-gamma coactivator-1a (PGC-1a) and its downstream regulators including nuclear respiratory factor 1 (NRF1), nuclear respiratory factor 2 (NRF2) and mitochondrial transcription factor A (TFAM) [10]. Low expression of PGC-1␣ can lead to a decrease in ATP production and excessive reactive oxygen species (ROS) formation during stress response [11]. We hypothesize that mitochondrial regulatory networks are disrupted in migraine, which may produce abnormal mitochondrial function. Thus, in the present study, we established a rat model of migraine induced by repeated inflammatory stimulation of the dura and investigated the alterations of mitochondrial regulatory networks including mitochondrial fission, fusion and mitochondrial biogenesis in the primary afferent neurons of migraine model rats.
affix it to the skull. After surgery, rats were housed separately and recovered for at least 3 days before the subsequent experiments. 2.3. Infusion of inflammatory soup or saline Rats were placed in a glass chamber in which they can move freely during the infusion. 10 l of inflammatory soup (IS) containing 2 mM histamine, serotonin, bradykinin, and 0.2 mM prostaglandin E2 in phosphate-buffered saline (PBS) at pH 7.4 (adapted from Strassman et al.) was delivered onto the dura through a microinfusion pump which was attached to the opening of PE10 tube [14]. Control rats were subjected to the same surgical procedure, but the IS was replaced with saline. The infusion was conducted once daily at the same time of each day for up to 7 days. 2.4. Assessment of mechanical sensitivity The baseline periorbital mechanical thresholds were determined by applying the von Frey monofilaments (Stoelting Co., Wood Dale, IL, USA) to the left side of the face over the rostral portion of the eye, as described by Oshinsky and Gomonchareonsiri [13]. Rats were tested for baseline mechanical thresholds once daily before they received IS or saline infusion for up to 7 days. The von Frey stimuli was applied to the skin 5 times for 5 s each in a sequential ascending or descending order to determine the thresholds of response. The mechanical thresholds were defined as the stimuli evoking a positive response in 3 out of 5 trials of the von Frey monofilament. Rats that did not respond to the 26 g stimulus were assigned 26 g as their threshold for analysis. 2.5. Transmission electron microscopy After 7 days of dural IS infusion, rats were anesthetized with 10% chloral hydrate (4 ml/kg intraperitoneally) and perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 2.5% glutaraldehyde for fixation. The left trigeminal ganglion was then dissected, subsequently postfixed for 1 h in 1% osmium tetroxide, dehydrated in graded ethanol, and embedded in plastic resin. Ultrathin sections (50–60 nm) were cut on a LKB ultramicrotome, stained with uranyl acetate and lead citrate, and then observed under a JEM-1011 transmission electron microscope.
2. Materials and methods
2.6. Western blot analysis
2.1. Animals
Rats were sacrificed with an overdose of chloral hydrate after various periods (3days, 5days, 7days) of dural IS stimulation. The left trigeminal ganglion (TG) was isolated immediately for further protein and gene analysis. The protein concentration was determined with BCA Protein Assay Kit (Pierce Biotechnology Inc., USA). Equivalent amounts of protein samples were separated by 10% SDS polyacrylamide gel and electrotransferred onto polyvinylidene fluoride membranes. The membrane bands were then incubated overnight at 4 ◦ C with antibodies directly against Drp1 (1:1000, Cell Signaling Technology), Mfn1 (1:1000, Abcam), PGC-1a (1:500, Cell Signaling Technology), NRF1 (1:1000, Abcam), NRF2 (1:1000, Abcam), TFAM (1:200, Santa Cruz Biotechnology) and -actin (1:2000, Millipore). Antibody binding was visualized via enhanced chemiluminescence (Millipore, Billerica, MA, USA). The blots were scanned and analyzed with the image analysis software package (NIH Image, Bethesda, MD, USA).
A total number of fifty-six adult male Sprague-Dawley rats weighing 180–200 g were used for the experiments. The animals were maintained in a temperature-controlled (21–22 ◦ C) environment under a 12-h light/dark cycle with free access to food and water. All experimental procedures on the animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
2.2. Surgical procedure The surgical procedures were performed as previously reported [12,13]. Briefly, under anesthesia with 10% chloral hydrate (4 ml/kg intraperitoneally), the rats were placed in a stereotactic frame (KOPF instruments, Tujunga, CA, USA). After an incision to expose the skull completely, a 3 mm diameter craniotomy was performed above the superior sagittal sinuses in the left frontal bone using a burr drill (+3 mm posterior to Bregma and +1.5 mm lateral to the midline), taking care not to damage the dura. Afterwards, we proceeded to place a polyethylene tube (PE10) above the dura and
2.7. Quantitative real-time PCR Total RNA was extracted from TG tissues with Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions and cDNA was synthesized via PrimeScriptTM RT
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3.2. Repeated dural stimulation causes defective mitochondrial morphology We used transmission electron microscopy to assess the morphology of mitochondria in TG neurons. The electron micrographs of mitochondrial from saline-treated rats displayed normal appearing cristae and electron-density matrix (Fig. 2a and c). In contrast, dural IS stimulation for 7 days produced many small, fragmented and asymmetrical mitochondria (Fig. 2b). Closer analysis revealed altered ultrastructure characterized by few remaining cristae and appearance of vacuoles (Fig. 2d). These fragmented mitochondria were rarely observed in the control group. 3.3. Repeated dural stimulation causes imbalanced mitochondrial dynamics Fig. 1. The baseline von Frey periorbital mechanical thresholds decrease after repeated dural IS infusion. The baseline periorbital mechanical thresholds were assessed before the rats received IS or saline infusion on each day. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the control group. n = 12 per group.
reagent Kit (Takara, Tokyo, Japan). Quantitative real-time PCR was performed on StepOnePlusTM Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using the SYBR Green Master Mix (Vazyme Biotech, China).The primers are listed in supplementary Table 1. Relative expression of genes was normalized to the endogenous reference GAPDH using 2−CT method. 2.8. Determination of mitochondrial DNA content Total DNA was extracted from TG with QIAamp DNA mini kit (QIAGEN, Duesseldorf, Germany). Quantitative real-time PCR was performed on StepOnePlusTM Real-Time PCR System (Applied Biosystems, Carlsbad, CA). Relative levels of mtDNA copy number were determined by comparing the mitochondrial specific ND1 gene to the nuclear -actin gene by 2−CT method. The primers are listed in supplementary Table 1. 2.9. Statistics analysis Data were retrieved and processed with the software Microcal Origin 8.0 (Vazyme Biotech, China). The results were presented as the means ± standard error of the mean (SEM). Significance of differences in mechanical thresholds was analyzed with two-way analysis of variance (ANOVA) and significance of other variables was analyzed with one-way ANOVA followed by Tukey’s multiple comparison tests using SPSS software, version 21.0 (SPASS Inc, Chicago, IL, USA). Differences were considered statistically significant at p< 0.05. 3. Results 3.1. Decreased baseline mechanical thresholds induced by repeated dural stimulation
The mitochondrial mass is determined by mitochondrial dynamics including fission and fusion. Therefore, we further examined the regulating signals in mitochondrial fission. The levels of mitochondrial fission proteins Drp1 and fusion protein Mfn1 in TG neurons were detected using western blot analysis. The protein levels of Drp1 were significantly increased after 5 days of dural infusion (Fig. 3a, p < 0.05) and then continued to rise after 7 days of stimulation (Fig. 3a, p < 0.01). A remarkable reduction in protein levels of Mfn1 was detected in rats after the 7 days’ infusion of IS compared with saline-treated rats (Fig. 3b, p < 0.01). These results suggested that repeated dural stimulation induces imbalanced mitochondrial dynamics in favor of fission in TG neurons. 3.4. Repeated dural stimulation causes impaired mitochondrial biogenesis To directly assess whether mitochondrial biogenesis is affected, we measured mtDNA copy number in TG neurons harvested from rats received saline and IS infusion respectively. The level of mtDNA copy number decreased slightly after 3 or 5 days of dural stimulation. Significantly decreased mtDNA copy number was observed in TG neurons in rats treated with IS for 7 days in comparison to the control group (Fig. 4a, p < 0.01). We further investigated the expression levels of key regulators responsible for mitochondrial biogenesis in TG neurons. Quantitative PCR analysis found that repeated IS infusion over the dura for 5 days significantly downregulated the mRNA levels of PGC-1␣ (Fig. 4b, p < 0.05), NRF1 (Fig. 4b, p < 0.05), NRF2 (Fig. 4b, p < 0.05) and TFAM (Fig. 4b, p < 0.05). The mRNA levels of these regulators continued to decline in rats infused with IS for 7 days in comparison to saline-treated rats. Consistently, a significant decrease in the protein levels of PGC-1␣ (Fig. 5a, p < 0.05) and its downstream regulators including NRF1 (Fig. 5b, p < 0.05), NRF2 (Fig. 5c, p < 0.05) and TFAM (Fig. 5d, p < 0.01) was detected in rats after 5 days of dural inflammatory stimulation compared with saline-treated rats by western blot analysis. Dural infusion of IS for 7 consecutive days produced a continuous decline in the protein levels of these regulators. These results suggested that mitochondrial biogenesis was definitely impaired in TG neurons in rats stimulated by repeated dural infusion of IS. 4. Discussion
To investigate whether repeated IS infusion over the dura induces significant mechanical allodynia, we measured the baseline periorbital pressure thresholds with von Frey monofilament before the infusion of IS or saline onto the dura on each day. As indicated in Fig. 1, there was no difference in periorbital mechanical thresholds between rats in two groups before infusion. After the fifth infusion of IS, the baseline periorbital mechanical threshold was significantly decreased below 2 g Von Frey force (Fig. 1), suggesting the establishment of mechanical allodynia.
The present study is the first to investigate alterations of mitochondrial regulatory networks including mitochondrial dynamics and biogenesis in a rat model of migraine induced by dural inflammatory stimulation. Repeated dural stimulation with inflammatory soup caused an excess of mitochondrial fission protein Drp1, creating many small and fragmented mitochondria in TG neurons. In addition, mitochondrial biogenesis is impaired in response to repeated dural stimulation as evidenced by decreased mtDNA copy
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Fig. 2. Transmission electron microscopy analysis shows defective mitochondrial morphology in TG neurons of rats after repeated dural stimulation. Representative micrographs of mitochondria in TG neurons from saline-treated rats (a, c) and rats after 7 d of dural IS infusion (b, d). Arrows indicate mitochondria. Scale bars: a–b, 500 nm; c–d, 200 nm. n = 4 per group.
Fig. 3. Mitochondrial dynamics is significantly altered in TG neurons of rats after repeated dural stimulation. Western blot analysis of mitochondrial fission protein Drp1 (a) and mitochondrial fusion protein Mfn1 (b) in TG neurons from saline-treated rats and rats after 3 d, 5 d, 7 d of dural IS infusion. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the control group. n = 4 per group.
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Fig. 4. MtDNA copy number and transcription levels of genes related to mitochondrial biogenesis are significantly downregulated in TG neurons of rats after repeated dural stimulation. (a) MtDNA copy number measured by real-time quantitative PCR in TG neurons from control group and rats after 3 d, 5 d, 7 d of dural IS infusion. (b) Real-time quantitative PCR analysis of PGC-1␣, NRF1, NRF2 and TFAM mRNA levels in TG neurons from saline-treated rats and rats after 3 d, 5 d, 7 d of dural IS infusion. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the control group. n = 4 per group.
number and downregulated mRNA and protein expression levels of regulators involved in mitochondrial biogenesis signaling. It is commonly believed that chemical stimulation of the dura activates the trigeminovascular system, which may play a crucial role in the pathophysiology of migraine [14,15]. Repeated infusion of inflammatory soup over the dura produces a chronic state of trigeminal hypersensitivity and it has been developed as a commonly used animal model for studying migraine [12,13]. Accordingly, our present study used repeated IS infusion over the dura to model the repeated activation of dural afferents and trigeminovascular systems in patients with recurrent migraine. Rats that received IS infusion for up to 5 days developed low baseline periorbital mechanical thresholds below 2 g Von Frey force, suggesting the establishment of mechanical allodynia. The state of mechanical allodynia is a manifestation of trigeminovascular sensitization, which indicates the underlying pathogenesis of migraine [15]. Previous evidence of deficit in mitochondrial morphology came from the findings of subsarcolemmal clusters of abnormal mitochondria in migraine sufferers in 1988 [2]. In the present study, ultrastructural studies using transmission electron microscopy revealed small and fragmented mitochondria in TG neurons in the IS-induced migraine model. The morphological changes
represent, in fact, the manner that mitochondria utilize to respond to a metabolic perturbation in migraine. Mitochondrial morphology was mainly determined by mitochondrial fission and fusion [16]. Therefore, we further investigate the regulatory signaling in mitochondrial fission and fusion. The results showed that the mitochondria exhibited increased fission and decreased fusion in trigeminal ganglion neurons of migraine model rats, suggesting that mitochondrial dynamics are shifted towards mitochondrial fission. Consistently, previous studies have shown that a highly selective Drp1 inhibitor can attenuate mechanical allodynia in preclinical models of inflammatory and neuropathic pain, providing support for a substantial role of mitochondrial fission in pain processing [17,18]. Mitochondrial biogenesis increases mitochondrial number to accommodate the energy demands in response to different physiologic and pathologic conditions [9]. It is a complex process demanding coordinated execution of mtDNA replication and translation, synthesis and import of nuclear DNA encoded proteins to the existing mitochondrial reticulum, and recruitment of membrane for a certain threshold of mitochondrial membrane potential [9]. Mitochondrial biogenesis is tightly regulated by PGC-1␣ and its downstream regulators including NRF-1, NRF-2 and TFAM [10]. TFAM is responsible for the regulation of mtDNA replication and
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Fig. 5. Protein levels of mitochondrial biogenesis signaling pathway are reduced in TG neurons of rats after repeated dural stimulation determined by western blot analysis. Representative blots and quantification of protein levels of PGC-1␣ (a), NRF1 (b), NRF2 (c) and TFAM (d) in TG neurons from saline-treated rats and rats after 3 d, 5 d, 7 d of dural IS infusion. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the control group. n = 4 per group.
transcription. The level of PGC-1␣ was significantly decreased in dorsal root ganglion from rats of diabetic neuropathy [19]. During HIV painful neuropathy, there is an increase in mtDNA damage and a loss of mitochondria in distal sensory axons [20]. Consistently, our studies also showed that repeated dural IS infusion induced an impairment of mitochondrial biogenesis, as indicated by decreased mtDNA content and downregulated signaling pathway related to mitochondrial biogenesis. It is previously shown that changes in mtDNA integrity and number in neurons alter the levels of key mitochondrial electron transport chain proteins and cause energy production defects [21], which may represent an underlying mechanism for migraine pathogenesis [2]. The overload production of reactive oxygen species (ROS) due to mitochondrial dysfunction was reported in migraine sufferers [6]. Oxidative stress induces mitochondrial fission and causes disturbances in mitochondrial biogenesis [22,23]. Disturbances in mitochondrial dynamics and biogenesis in turn lead to the increased production of ROS [11,24]. ROS participates in the initiation of cortical spreading depression, a wave of neuronal depolarization that occurs in migraine attack [25].
Additionally, ROS can activate nociceptive signaling via activation of transient receptor potential cation channel TRPA1 [26]. The uncoupling protein UCP5 which decreases the mitochondrial membrane potential and in turn reduces ROS production may exert a neuroprotective effect following CSD induction [27]. Therefore we postulated that impaired dynamic mechanisms regulating mitochondrial quality control may indicate a decreased ability to cope with neuronal metabolic load, which possibly represent a novel mechanism for mitochondrial dysfunction in migraine headache. Nevertheless, our current results are limited to elucidate an impairment of mitochondrial dynamics and biogenesis signaling in the rat model of migraine. Questions regarding whether and how disrupted mitochondrial regulatory networks influence the pathogenesis of migraine still require further investigation. In addition, a comparison with effects of IS administration to other trigeminally-innervated organ can better reveal the correlation between maladjusted mitochondrial regulatory networks and migraine pathophysiology. In conclusion, our results suggest that the dynamic mechanisms regulating mitochondrial quality control is maladjusted in
X. Dong et al. / Neuroscience Letters 636 (2017) 127–133
TG neurons in a rat model of migraine. Repeated dural stimulation shifts the balance of mitochondrial dynamics in favor of fission and suppresses the mitochondrial biogenesis signaling in TG neurons. Manipulation of mitochondrial dynamics and biogenesis signaling may indicate a new mitochondria-targeted strategy to attenuate the metabolic defects in migraine. Nevertheless, further studies are urgently required to more precisely define the role of mitochondrial dynamics and biogenesis in the pathophysiology of migraine. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors sincerely thank Professor Ling Chen for the technical assistance. This work was supported by National Natural Science Foundation of China (81070896). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2016.10. 054. References [1] C. Global Burden of Disease Study, Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013, Lancet 386 (2015) 743–800. [2] W.R. Hardison, H.H. Yorns Jr., Mitochondrial dysfunction in migraine, Semin. Pediatr. Neurol. 20 (2013) 188–193. [3] M.T. Lin, M.F. Beal, Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases, Nature 443 (2006) 787–795. [4] S.J. Flatters, The contribution of mitochondria to sensory processing and pain, Prog. Mol. Biol. Transl. Sci. 131 (2015) 119–146. [5] A. Viggiano, U. Nicodemo, E. Viggiano, G. Messina, A. Viggiano, M. Monda, B. De Luca, Mastication overload causes an increase in O2- production into the subnucleus oralis of the spinal trigeminal nucleus, Neuroscience 166 (2010) 416–421. [6] M. Neri, A. Frustaci, M. Milic, V. Valdiglesias, M. Fini, S. Bonassi, P. Barbanti, A meta-analysis of biomarkers related to oxidative stress and nitric oxide pathway in migraine, Cephalalgia 35 (2015) 931–937. [7] F. Sanchis-Gomar, F. Derbre, Mitochondrial fission and fusion in human diseases, N. Engl. J. Med. 370 (2014) 1073–1074. [8] R.J. Youle, A.M. van der Bliek, Mitochondrial fission, fusion, and stress, Science 337 (2012) 1062–1065. [9] C. Ploumi, I. Daskalaki, N. Tavernarakis, Mitochondrial biogenesis and clearance: a balancing act, FEBS J. (2016).
133
[10] R.C. Scarpulla, R.B. Vega, D.P. Kelly, Transcriptional integration of mitochondrial biogenesis, ABBV Trends Endocrinol. Metab. 23 (2012) 459–466. [11] J. St-Pierre, S. Drori, M. Uldry, J.M. Silvaggi, J. Rhee, S. Jager, C. Handschin, K. Zheng, J. Lin, W. Yang, D.K. Simon, R. Bachoo, B.M. Spiegelman, Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators, Cell 127 (2006) 397–408. [12] A. Melo-Carrillo, A. Lopez-Avila, A chronic animal model of migraine induced by repeated meningeal nociception, characterized by a behavioral and pharmacological approach, Cephalalgia 33 (2013) 1096–1105. [13] M.L. Oshinsky, S. Gomonchareonsiri, Episodic dural stimulation in awake rats: a model for recurrent headache, Headache 47 (2007) 1026–1036. [14] A.M. Strassman, S.A. Raymond, R. Burstein, Sensitization of meningeal sensory neurons and the origin of headaches, Nature 384 (1996) 560–564. [15] C. Bernstein, R. Burstein, Sensitization of the trigeminovascular pathway: perspective and implications to migraine pathophysiology, J. Clin. Neurol. 8 (2012) 89–99. [16] C.A. Galloway, H. Lee, Y. Yoon, Mitochondrial morphology-emerging role in bioenergetics, Free Radic. Biol. Med. 53 (2012) 2218–2228. [17] L.F. Ferrari, A. Chum, O. Bogen, D.B. Reichling, J.D. Levine, Role of Drp1, a key mitochondrial fission protein, in neuropathic pain, J. Neurosci. 31 (2011) 11404–11410. [18] H. Kanda, S. Liu, T. Iida, H. Yi, W. Huang, R.C. Levitt, D.A. Lubarsky, K.A. Candiotti, S. Hao, Inhibition of mitochondrial fission protein reduced mechanical allodynia and suppressed spinal mitochondrial superoxide induced by perineural human immunodeficiency virus gp120 in rats, Anesth. Analg. 122 (2016) 264–272. [19] S.K. Roy Chowdhury, D.R. Smith, A. Saleh, J. Schapansky, A. Marquez, S. Gomes, E. Akude, D. Morrow, N.A. Calcutt, P. Fernyhough, Impaired adenosine monophosphate-activated protein kinase signalling in dorsal root ganglia neurons is linked to mitochondrial dysfunction and peripheral neuropathy in diabetes, Brain 135 (2012) 1751–1766. [20] H.C. Lehmann, W. Chen, J. Borzan, J.L. Mankowski, A. Hoke, Mitochondrial dysfunction in distal axons contributes to human immunodeficiency virus sensory neuropathy, Ann. Neurol. 69 (2011) 100–110. [21] V. Carelli, D.C. Chan, Mitochondrial DNA: impacting central and peripheral nervous systems, Neuron 84 (2014) 1126–1142. [22] H.C. Lee, Y.H. Wei, Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress, Int. J. Biochem. Cell Biol. 37 (2005) 822–834. [23] S. Wu, F. Zhou, Z. Zhang, D. Xing, Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins, FEBS J. 278 (2011) 941–954. [24] A.H. Bhat, K.B. Dar, S. Anees, M.A. Zargar, A. Masood, M.A. Sofi, S.A. Ganie, Oxidative stress, mitochondrial dysfunction and neurodegenerative diseases; a mechanistic insight, Biomed. Pharmacother. 74 (2015) 101–110. [25] A. Malkov, A.I. Ivanov, I. Popova, M. Mukhtarov, O. Gubkina, T. Waseem, P. Bregestovski, Y. Zilberter, Reactive oxygen species initiate a metabolic collapse in hippocampal slices: potential trigger of cortical spreading depression, J. Cereb. Blood Flow Metab. 34 (2014) 1540–1549. [26] A. Shatillo, K. Koroleva, R. Giniatullina, N. Naumenko, A.A. Slastnikova, R.R. Aliev, G. Bart, M. Atalay, C. Gu, R. Khazipov, B. Davletov, O. Grohn, R. Giniatullin, Cortical spreading depression induces oxidative stress in the trigeminal nociceptive system, Neuroscience 253 (2013) 341–349. [27] E. Viggiano, V. Monda, A. Messina, F. Moscatelli, A. Valenzano, D. Tafuri, G. Cibelli, B. De Luca, G. Messina, M. Monda, Cortical spreading depression produces a neuroprotective effect activating mitochondrial uncoupling protein-5, Neuropsychiatr. Dis. Treat. 12 (2016) 1705–1710.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Research article
Abnormal mitochondrial dynamics and impaired mitochondrial biogenesis in trigeminal ganglion neurons in a rat model of migraine Xin Dong a,1 , Xinying Guan a,1 , Keyan Chen a , Shanquan Jin a , Chengyun Wang b , Lanyun Yan a , Zhaochun Shi a , Xue Zhang a , Ling Chen c , Qi Wan a,∗ a
Department of Neurology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu Province, 210029, China Department of Neurology, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, Jiangsu Province, 223300, China c Department of Physiology, Nanjing Medical University, 818 Tianyuan East Road, Nanjing, Jiangsu Province, 210029, China b
h i g h l i g h t s • Defective mitochondrial morphology was detected in TG neurons in a rat model of migraine. • Mitochondrial dynamics was shifted towards fission in TG neurons in a rat model of migraine. • Mitochondrial biogenesis was suppressed in TG neurons in a rat model of migraine.
a r t i c l e
i n f o
Article history: Received 15 September 2016 Received in revised form 26 October 2016 Accepted 27 October 2016 Available online 29 October 2016 Keywords: Migraine Mitochondrial dynamics Mitochondrial biogenesis Trigeminal ganglion
a b s t r a c t Accumulating evidence has demonstrated a possible role of mitochondrial dysfunction in migraine pathophysiology. Migraine sufferers exhibit impaired metabolic capacity, with an increased formation of reactive oxygen species (ROS). Mitochondrial dynamics and mitochondrial biogenesis are key processes regulating mitochondrial homeostasis. The aim of this study was to explore the alterations of mitochondrial regulatory networks in a rat model of migraine induced by repeated dural stimulation with inflammatory soup (IS). Ultrastructural, protein, gene and mitochondrial DNA analysis were applied to assess mitochondrial dynamics and biogenesis in trigeminal ganglion (TG) neurons. Mitochondria in TG neurons exhibited small and fragmented morphology after repeated dural stimulation. Further investigations showed that mitochondrial fission protein dynamin-related protein 1 (Drp1) was increased while mitochondrial fusion protein Mitofusin1 (Mfn1) was reduced in TG neurons. In addition, our results also presented that mitochondrial DNA copy number in TG neurons was reduced significantly, accompanied by alterations in mRNA and protein levels of regulatory factors related to mitochondrial biogenesis including peroxisome proliferator-activated receptor-gamma coactivator-1a (PGC-1␣) and its downstream regulators in TG neurons in the IS-induced migraine model. These findings suggest that the mitochondrial dynamic regulatory networks are maladjusted in TG neurons in a rat model of migraine. Regulation of mitochondrial dynamics and biogenesis signaling may indicate a new mitochondria-targeted therapeutic strategy for migraine. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Abbreviations: ROS, reactive oxygen species; TG, trigeminal ganglion; Drp1, Dynamin related protein 1; Mfn1, Mitofusin1; mtDNA, mitochondrial DNA; PGC-1a, peroxisome proliferator-activated receptor-gamma coactivator-1a; NRF1, nuclear respiratory factor 1; NRF2, nuclear respiratory factor 2; TFAM, mitochondrial transcription factor A; IS, inflammatory soup. ∗ Corresponding author at: Department of Neurology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu Province, 210029, China. E-mail address: qi [email protected] (Q. Wan). 1 These authors contribute equally to this work. http://dx.doi.org/10.1016/j.neulet.2016.10.054 0304-3940/© 2016 Elsevier Ireland Ltd. All rights reserved.
Migraine is a complex neurological disorder affecting about 12% of the population worldwide and is found to be the sixth most prevalent cause of disability [1]. Prophylactic pharmacotherapy for migraine headaches is less than satisfactory, due to the poor efficacy and debilitating adverse effects. Previous investigations have suggested a possible role of mitochondrial dysfunction in migraine pathophysiology [2]. Further elucidation of the metabolism signaling in the primary afferent neurons in the headache circuit will
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facilitate our understanding of the pathophysiology of migraine and promote the development of new therapeutics. Mitochondria are the primary energy-generating system in eukaryotic cells. Due to the high dependence on energy for neural activity, aberrant mitochondrial morphology and function may cause a series of neurological diseases [3]. Most recently, substantial amounts of evidence have demonstrated a contribution of mitochondria to pain processing in neuropathic and inflammatory pain states [4]. Mechanical stimulation of trigeminal sensory neurons can increase the production of reactive oxygen spices (ROS) in the spinal trigeminal nucleus [5]. Biochemical and genetic studies have identified a decreased activity of mitochondrial respiratory chain enzymes with an increased formation of ROS in migraine sufferers and detected specific mitochondrial DNA (mtDNA) variants responsible for migraine susceptibility [2,6]. Mitochondrial quality control is achieved by the dynamic interplay of fusion, fission and mitochondrial biogenesis. Fission is a process dividing the damaged mitochondria into a healthy and an abnormal portion while fusion results in a mixing of membranes and matrix content among mitochondria [7]. Mitochondrial fission and fusion processes are mediated by Dynamin-related protein 1 (Drp1) and Mitofusin1 (Mfn1), respectively. Mitochondrial dynamics including fission and fusion plays a significant role in maintaining mitochondrial morphology and function when cells experience stress [8]. Mitochondrial biogenesis responds to metabolic signals by increasing the actual mass of mitochondria with mtDNA replication [9]. It is tightly regulated by peroxisome proliferator-activated receptor-gamma coactivator-1a (PGC-1a) and its downstream regulators including nuclear respiratory factor 1 (NRF1), nuclear respiratory factor 2 (NRF2) and mitochondrial transcription factor A (TFAM) [10]. Low expression of PGC-1␣ can lead to a decrease in ATP production and excessive reactive oxygen species (ROS) formation during stress response [11]. We hypothesize that mitochondrial regulatory networks are disrupted in migraine, which may produce abnormal mitochondrial function. Thus, in the present study, we established a rat model of migraine induced by repeated inflammatory stimulation of the dura and investigated the alterations of mitochondrial regulatory networks including mitochondrial fission, fusion and mitochondrial biogenesis in the primary afferent neurons of migraine model rats.
affix it to the skull. After surgery, rats were housed separately and recovered for at least 3 days before the subsequent experiments. 2.3. Infusion of inflammatory soup or saline Rats were placed in a glass chamber in which they can move freely during the infusion. 10 l of inflammatory soup (IS) containing 2 mM histamine, serotonin, bradykinin, and 0.2 mM prostaglandin E2 in phosphate-buffered saline (PBS) at pH 7.4 (adapted from Strassman et al.) was delivered onto the dura through a microinfusion pump which was attached to the opening of PE10 tube [14]. Control rats were subjected to the same surgical procedure, but the IS was replaced with saline. The infusion was conducted once daily at the same time of each day for up to 7 days. 2.4. Assessment of mechanical sensitivity The baseline periorbital mechanical thresholds were determined by applying the von Frey monofilaments (Stoelting Co., Wood Dale, IL, USA) to the left side of the face over the rostral portion of the eye, as described by Oshinsky and Gomonchareonsiri [13]. Rats were tested for baseline mechanical thresholds once daily before they received IS or saline infusion for up to 7 days. The von Frey stimuli was applied to the skin 5 times for 5 s each in a sequential ascending or descending order to determine the thresholds of response. The mechanical thresholds were defined as the stimuli evoking a positive response in 3 out of 5 trials of the von Frey monofilament. Rats that did not respond to the 26 g stimulus were assigned 26 g as their threshold for analysis. 2.5. Transmission electron microscopy After 7 days of dural IS infusion, rats were anesthetized with 10% chloral hydrate (4 ml/kg intraperitoneally) and perfused with 0.1 M phosphate-buffered saline (PBS, pH 7.4) followed by 2.5% glutaraldehyde for fixation. The left trigeminal ganglion was then dissected, subsequently postfixed for 1 h in 1% osmium tetroxide, dehydrated in graded ethanol, and embedded in plastic resin. Ultrathin sections (50–60 nm) were cut on a LKB ultramicrotome, stained with uranyl acetate and lead citrate, and then observed under a JEM-1011 transmission electron microscope.
2. Materials and methods
2.6. Western blot analysis
2.1. Animals
Rats were sacrificed with an overdose of chloral hydrate after various periods (3days, 5days, 7days) of dural IS stimulation. The left trigeminal ganglion (TG) was isolated immediately for further protein and gene analysis. The protein concentration was determined with BCA Protein Assay Kit (Pierce Biotechnology Inc., USA). Equivalent amounts of protein samples were separated by 10% SDS polyacrylamide gel and electrotransferred onto polyvinylidene fluoride membranes. The membrane bands were then incubated overnight at 4 ◦ C with antibodies directly against Drp1 (1:1000, Cell Signaling Technology), Mfn1 (1:1000, Abcam), PGC-1a (1:500, Cell Signaling Technology), NRF1 (1:1000, Abcam), NRF2 (1:1000, Abcam), TFAM (1:200, Santa Cruz Biotechnology) and -actin (1:2000, Millipore). Antibody binding was visualized via enhanced chemiluminescence (Millipore, Billerica, MA, USA). The blots were scanned and analyzed with the image analysis software package (NIH Image, Bethesda, MD, USA).
A total number of fifty-six adult male Sprague-Dawley rats weighing 180–200 g were used for the experiments. The animals were maintained in a temperature-controlled (21–22 ◦ C) environment under a 12-h light/dark cycle with free access to food and water. All experimental procedures on the animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
2.2. Surgical procedure The surgical procedures were performed as previously reported [12,13]. Briefly, under anesthesia with 10% chloral hydrate (4 ml/kg intraperitoneally), the rats were placed in a stereotactic frame (KOPF instruments, Tujunga, CA, USA). After an incision to expose the skull completely, a 3 mm diameter craniotomy was performed above the superior sagittal sinuses in the left frontal bone using a burr drill (+3 mm posterior to Bregma and +1.5 mm lateral to the midline), taking care not to damage the dura. Afterwards, we proceeded to place a polyethylene tube (PE10) above the dura and
2.7. Quantitative real-time PCR Total RNA was extracted from TG tissues with Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions and cDNA was synthesized via PrimeScriptTM RT
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3.2. Repeated dural stimulation causes defective mitochondrial morphology We used transmission electron microscopy to assess the morphology of mitochondria in TG neurons. The electron micrographs of mitochondrial from saline-treated rats displayed normal appearing cristae and electron-density matrix (Fig. 2a and c). In contrast, dural IS stimulation for 7 days produced many small, fragmented and asymmetrical mitochondria (Fig. 2b). Closer analysis revealed altered ultrastructure characterized by few remaining cristae and appearance of vacuoles (Fig. 2d). These fragmented mitochondria were rarely observed in the control group. 3.3. Repeated dural stimulation causes imbalanced mitochondrial dynamics Fig. 1. The baseline von Frey periorbital mechanical thresholds decrease after repeated dural IS infusion. The baseline periorbital mechanical thresholds were assessed before the rats received IS or saline infusion on each day. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the control group. n = 12 per group.
reagent Kit (Takara, Tokyo, Japan). Quantitative real-time PCR was performed on StepOnePlusTM Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) using the SYBR Green Master Mix (Vazyme Biotech, China).The primers are listed in supplementary Table 1. Relative expression of genes was normalized to the endogenous reference GAPDH using 2−CT method. 2.8. Determination of mitochondrial DNA content Total DNA was extracted from TG with QIAamp DNA mini kit (QIAGEN, Duesseldorf, Germany). Quantitative real-time PCR was performed on StepOnePlusTM Real-Time PCR System (Applied Biosystems, Carlsbad, CA). Relative levels of mtDNA copy number were determined by comparing the mitochondrial specific ND1 gene to the nuclear -actin gene by 2−CT method. The primers are listed in supplementary Table 1. 2.9. Statistics analysis Data were retrieved and processed with the software Microcal Origin 8.0 (Vazyme Biotech, China). The results were presented as the means ± standard error of the mean (SEM). Significance of differences in mechanical thresholds was analyzed with two-way analysis of variance (ANOVA) and significance of other variables was analyzed with one-way ANOVA followed by Tukey’s multiple comparison tests using SPSS software, version 21.0 (SPASS Inc, Chicago, IL, USA). Differences were considered statistically significant at p< 0.05. 3. Results 3.1. Decreased baseline mechanical thresholds induced by repeated dural stimulation
The mitochondrial mass is determined by mitochondrial dynamics including fission and fusion. Therefore, we further examined the regulating signals in mitochondrial fission. The levels of mitochondrial fission proteins Drp1 and fusion protein Mfn1 in TG neurons were detected using western blot analysis. The protein levels of Drp1 were significantly increased after 5 days of dural infusion (Fig. 3a, p < 0.05) and then continued to rise after 7 days of stimulation (Fig. 3a, p < 0.01). A remarkable reduction in protein levels of Mfn1 was detected in rats after the 7 days’ infusion of IS compared with saline-treated rats (Fig. 3b, p < 0.01). These results suggested that repeated dural stimulation induces imbalanced mitochondrial dynamics in favor of fission in TG neurons. 3.4. Repeated dural stimulation causes impaired mitochondrial biogenesis To directly assess whether mitochondrial biogenesis is affected, we measured mtDNA copy number in TG neurons harvested from rats received saline and IS infusion respectively. The level of mtDNA copy number decreased slightly after 3 or 5 days of dural stimulation. Significantly decreased mtDNA copy number was observed in TG neurons in rats treated with IS for 7 days in comparison to the control group (Fig. 4a, p < 0.01). We further investigated the expression levels of key regulators responsible for mitochondrial biogenesis in TG neurons. Quantitative PCR analysis found that repeated IS infusion over the dura for 5 days significantly downregulated the mRNA levels of PGC-1␣ (Fig. 4b, p < 0.05), NRF1 (Fig. 4b, p < 0.05), NRF2 (Fig. 4b, p < 0.05) and TFAM (Fig. 4b, p < 0.05). The mRNA levels of these regulators continued to decline in rats infused with IS for 7 days in comparison to saline-treated rats. Consistently, a significant decrease in the protein levels of PGC-1␣ (Fig. 5a, p < 0.05) and its downstream regulators including NRF1 (Fig. 5b, p < 0.05), NRF2 (Fig. 5c, p < 0.05) and TFAM (Fig. 5d, p < 0.01) was detected in rats after 5 days of dural inflammatory stimulation compared with saline-treated rats by western blot analysis. Dural infusion of IS for 7 consecutive days produced a continuous decline in the protein levels of these regulators. These results suggested that mitochondrial biogenesis was definitely impaired in TG neurons in rats stimulated by repeated dural infusion of IS. 4. Discussion
To investigate whether repeated IS infusion over the dura induces significant mechanical allodynia, we measured the baseline periorbital pressure thresholds with von Frey monofilament before the infusion of IS or saline onto the dura on each day. As indicated in Fig. 1, there was no difference in periorbital mechanical thresholds between rats in two groups before infusion. After the fifth infusion of IS, the baseline periorbital mechanical threshold was significantly decreased below 2 g Von Frey force (Fig. 1), suggesting the establishment of mechanical allodynia.
The present study is the first to investigate alterations of mitochondrial regulatory networks including mitochondrial dynamics and biogenesis in a rat model of migraine induced by dural inflammatory stimulation. Repeated dural stimulation with inflammatory soup caused an excess of mitochondrial fission protein Drp1, creating many small and fragmented mitochondria in TG neurons. In addition, mitochondrial biogenesis is impaired in response to repeated dural stimulation as evidenced by decreased mtDNA copy
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Fig. 2. Transmission electron microscopy analysis shows defective mitochondrial morphology in TG neurons of rats after repeated dural stimulation. Representative micrographs of mitochondria in TG neurons from saline-treated rats (a, c) and rats after 7 d of dural IS infusion (b, d). Arrows indicate mitochondria. Scale bars: a–b, 500 nm; c–d, 200 nm. n = 4 per group.
Fig. 3. Mitochondrial dynamics is significantly altered in TG neurons of rats after repeated dural stimulation. Western blot analysis of mitochondrial fission protein Drp1 (a) and mitochondrial fusion protein Mfn1 (b) in TG neurons from saline-treated rats and rats after 3 d, 5 d, 7 d of dural IS infusion. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the control group. n = 4 per group.
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Fig. 4. MtDNA copy number and transcription levels of genes related to mitochondrial biogenesis are significantly downregulated in TG neurons of rats after repeated dural stimulation. (a) MtDNA copy number measured by real-time quantitative PCR in TG neurons from control group and rats after 3 d, 5 d, 7 d of dural IS infusion. (b) Real-time quantitative PCR analysis of PGC-1␣, NRF1, NRF2 and TFAM mRNA levels in TG neurons from saline-treated rats and rats after 3 d, 5 d, 7 d of dural IS infusion. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the control group. n = 4 per group.
number and downregulated mRNA and protein expression levels of regulators involved in mitochondrial biogenesis signaling. It is commonly believed that chemical stimulation of the dura activates the trigeminovascular system, which may play a crucial role in the pathophysiology of migraine [14,15]. Repeated infusion of inflammatory soup over the dura produces a chronic state of trigeminal hypersensitivity and it has been developed as a commonly used animal model for studying migraine [12,13]. Accordingly, our present study used repeated IS infusion over the dura to model the repeated activation of dural afferents and trigeminovascular systems in patients with recurrent migraine. Rats that received IS infusion for up to 5 days developed low baseline periorbital mechanical thresholds below 2 g Von Frey force, suggesting the establishment of mechanical allodynia. The state of mechanical allodynia is a manifestation of trigeminovascular sensitization, which indicates the underlying pathogenesis of migraine [15]. Previous evidence of deficit in mitochondrial morphology came from the findings of subsarcolemmal clusters of abnormal mitochondria in migraine sufferers in 1988 [2]. In the present study, ultrastructural studies using transmission electron microscopy revealed small and fragmented mitochondria in TG neurons in the IS-induced migraine model. The morphological changes
represent, in fact, the manner that mitochondria utilize to respond to a metabolic perturbation in migraine. Mitochondrial morphology was mainly determined by mitochondrial fission and fusion [16]. Therefore, we further investigate the regulatory signaling in mitochondrial fission and fusion. The results showed that the mitochondria exhibited increased fission and decreased fusion in trigeminal ganglion neurons of migraine model rats, suggesting that mitochondrial dynamics are shifted towards mitochondrial fission. Consistently, previous studies have shown that a highly selective Drp1 inhibitor can attenuate mechanical allodynia in preclinical models of inflammatory and neuropathic pain, providing support for a substantial role of mitochondrial fission in pain processing [17,18]. Mitochondrial biogenesis increases mitochondrial number to accommodate the energy demands in response to different physiologic and pathologic conditions [9]. It is a complex process demanding coordinated execution of mtDNA replication and translation, synthesis and import of nuclear DNA encoded proteins to the existing mitochondrial reticulum, and recruitment of membrane for a certain threshold of mitochondrial membrane potential [9]. Mitochondrial biogenesis is tightly regulated by PGC-1␣ and its downstream regulators including NRF-1, NRF-2 and TFAM [10]. TFAM is responsible for the regulation of mtDNA replication and
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Fig. 5. Protein levels of mitochondrial biogenesis signaling pathway are reduced in TG neurons of rats after repeated dural stimulation determined by western blot analysis. Representative blots and quantification of protein levels of PGC-1␣ (a), NRF1 (b), NRF2 (c) and TFAM (d) in TG neurons from saline-treated rats and rats after 3 d, 5 d, 7 d of dural IS infusion. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 compared with the control group. n = 4 per group.
transcription. The level of PGC-1␣ was significantly decreased in dorsal root ganglion from rats of diabetic neuropathy [19]. During HIV painful neuropathy, there is an increase in mtDNA damage and a loss of mitochondria in distal sensory axons [20]. Consistently, our studies also showed that repeated dural IS infusion induced an impairment of mitochondrial biogenesis, as indicated by decreased mtDNA content and downregulated signaling pathway related to mitochondrial biogenesis. It is previously shown that changes in mtDNA integrity and number in neurons alter the levels of key mitochondrial electron transport chain proteins and cause energy production defects [21], which may represent an underlying mechanism for migraine pathogenesis [2]. The overload production of reactive oxygen species (ROS) due to mitochondrial dysfunction was reported in migraine sufferers [6]. Oxidative stress induces mitochondrial fission and causes disturbances in mitochondrial biogenesis [22,23]. Disturbances in mitochondrial dynamics and biogenesis in turn lead to the increased production of ROS [11,24]. ROS participates in the initiation of cortical spreading depression, a wave of neuronal depolarization that occurs in migraine attack [25].
Additionally, ROS can activate nociceptive signaling via activation of transient receptor potential cation channel TRPA1 [26]. The uncoupling protein UCP5 which decreases the mitochondrial membrane potential and in turn reduces ROS production may exert a neuroprotective effect following CSD induction [27]. Therefore we postulated that impaired dynamic mechanisms regulating mitochondrial quality control may indicate a decreased ability to cope with neuronal metabolic load, which possibly represent a novel mechanism for mitochondrial dysfunction in migraine headache. Nevertheless, our current results are limited to elucidate an impairment of mitochondrial dynamics and biogenesis signaling in the rat model of migraine. Questions regarding whether and how disrupted mitochondrial regulatory networks influence the pathogenesis of migraine still require further investigation. In addition, a comparison with effects of IS administration to other trigeminally-innervated organ can better reveal the correlation between maladjusted mitochondrial regulatory networks and migraine pathophysiology. In conclusion, our results suggest that the dynamic mechanisms regulating mitochondrial quality control is maladjusted in
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TG neurons in a rat model of migraine. Repeated dural stimulation shifts the balance of mitochondrial dynamics in favor of fission and suppresses the mitochondrial biogenesis signaling in TG neurons. Manipulation of mitochondrial dynamics and biogenesis signaling may indicate a new mitochondria-targeted strategy to attenuate the metabolic defects in migraine. Nevertheless, further studies are urgently required to more precisely define the role of mitochondrial dynamics and biogenesis in the pathophysiology of migraine. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments The authors sincerely thank Professor Ling Chen for the technical assistance. This work was supported by National Natural Science Foundation of China (81070896). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet.2016.10. 054. References [1] C. Global Burden of Disease Study, Global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries, 1990–2013: a systematic analysis for the Global Burden of Disease Study 2013, Lancet 386 (2015) 743–800. [2] W.R. Hardison, H.H. Yorns Jr., Mitochondrial dysfunction in migraine, Semin. Pediatr. Neurol. 20 (2013) 188–193. [3] M.T. Lin, M.F. Beal, Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases, Nature 443 (2006) 787–795. [4] S.J. Flatters, The contribution of mitochondria to sensory processing and pain, Prog. Mol. Biol. Transl. Sci. 131 (2015) 119–146. [5] A. Viggiano, U. Nicodemo, E. Viggiano, G. Messina, A. Viggiano, M. Monda, B. De Luca, Mastication overload causes an increase in O2- production into the subnucleus oralis of the spinal trigeminal nucleus, Neuroscience 166 (2010) 416–421. [6] M. Neri, A. Frustaci, M. Milic, V. Valdiglesias, M. Fini, S. Bonassi, P. Barbanti, A meta-analysis of biomarkers related to oxidative stress and nitric oxide pathway in migraine, Cephalalgia 35 (2015) 931–937. [7] F. Sanchis-Gomar, F. Derbre, Mitochondrial fission and fusion in human diseases, N. Engl. J. Med. 370 (2014) 1073–1074. [8] R.J. Youle, A.M. van der Bliek, Mitochondrial fission, fusion, and stress, Science 337 (2012) 1062–1065. [9] C. Ploumi, I. Daskalaki, N. Tavernarakis, Mitochondrial biogenesis and clearance: a balancing act, FEBS J. (2016).
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[10] R.C. Scarpulla, R.B. Vega, D.P. Kelly, Transcriptional integration of mitochondrial biogenesis, ABBV Trends Endocrinol. Metab. 23 (2012) 459–466. [11] J. St-Pierre, S. Drori, M. Uldry, J.M. Silvaggi, J. Rhee, S. Jager, C. Handschin, K. Zheng, J. Lin, W. Yang, D.K. Simon, R. Bachoo, B.M. Spiegelman, Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators, Cell 127 (2006) 397–408. [12] A. Melo-Carrillo, A. Lopez-Avila, A chronic animal model of migraine induced by repeated meningeal nociception, characterized by a behavioral and pharmacological approach, Cephalalgia 33 (2013) 1096–1105. [13] M.L. Oshinsky, S. Gomonchareonsiri, Episodic dural stimulation in awake rats: a model for recurrent headache, Headache 47 (2007) 1026–1036. [14] A.M. Strassman, S.A. Raymond, R. Burstein, Sensitization of meningeal sensory neurons and the origin of headaches, Nature 384 (1996) 560–564. [15] C. Bernstein, R. Burstein, Sensitization of the trigeminovascular pathway: perspective and implications to migraine pathophysiology, J. Clin. Neurol. 8 (2012) 89–99. [16] C.A. Galloway, H. Lee, Y. Yoon, Mitochondrial morphology-emerging role in bioenergetics, Free Radic. Biol. Med. 53 (2012) 2218–2228. [17] L.F. Ferrari, A. Chum, O. Bogen, D.B. Reichling, J.D. Levine, Role of Drp1, a key mitochondrial fission protein, in neuropathic pain, J. Neurosci. 31 (2011) 11404–11410. [18] H. Kanda, S. Liu, T. Iida, H. Yi, W. Huang, R.C. Levitt, D.A. Lubarsky, K.A. Candiotti, S. Hao, Inhibition of mitochondrial fission protein reduced mechanical allodynia and suppressed spinal mitochondrial superoxide induced by perineural human immunodeficiency virus gp120 in rats, Anesth. Analg. 122 (2016) 264–272. [19] S.K. Roy Chowdhury, D.R. Smith, A. Saleh, J. Schapansky, A. Marquez, S. Gomes, E. Akude, D. Morrow, N.A. Calcutt, P. Fernyhough, Impaired adenosine monophosphate-activated protein kinase signalling in dorsal root ganglia neurons is linked to mitochondrial dysfunction and peripheral neuropathy in diabetes, Brain 135 (2012) 1751–1766. [20] H.C. Lehmann, W. Chen, J. Borzan, J.L. Mankowski, A. Hoke, Mitochondrial dysfunction in distal axons contributes to human immunodeficiency virus sensory neuropathy, Ann. Neurol. 69 (2011) 100–110. [21] V. Carelli, D.C. Chan, Mitochondrial DNA: impacting central and peripheral nervous systems, Neuron 84 (2014) 1126–1142. [22] H.C. Lee, Y.H. Wei, Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress, Int. J. Biochem. Cell Biol. 37 (2005) 822–834. [23] S. Wu, F. Zhou, Z. Zhang, D. Xing, Mitochondrial oxidative stress causes mitochondrial fragmentation via differential modulation of mitochondrial fission-fusion proteins, FEBS J. 278 (2011) 941–954. [24] A.H. Bhat, K.B. Dar, S. Anees, M.A. Zargar, A. Masood, M.A. Sofi, S.A. Ganie, Oxidative stress, mitochondrial dysfunction and neurodegenerative diseases; a mechanistic insight, Biomed. Pharmacother. 74 (2015) 101–110. [25] A. Malkov, A.I. Ivanov, I. Popova, M. Mukhtarov, O. Gubkina, T. Waseem, P. Bregestovski, Y. Zilberter, Reactive oxygen species initiate a metabolic collapse in hippocampal slices: potential trigger of cortical spreading depression, J. Cereb. Blood Flow Metab. 34 (2014) 1540–1549. [26] A. Shatillo, K. Koroleva, R. Giniatullina, N. Naumenko, A.A. Slastnikova, R.R. Aliev, G. Bart, M. Atalay, C. Gu, R. Khazipov, B. Davletov, O. Grohn, R. Giniatullin, Cortical spreading depression induces oxidative stress in the trigeminal nociceptive system, Neuroscience 253 (2013) 341–349. [27] E. Viggiano, V. Monda, A. Messina, F. Moscatelli, A. Valenzano, D. Tafuri, G. Cibelli, B. De Luca, G. Messina, M. Monda, Cortical spreading depression produces a neuroprotective effect activating mitochondrial uncoupling protein-5, Neuropsychiatr. Dis. Treat. 12 (2016) 1705–1710.