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Mitochondrial Dysfunction in Gnrh Neurons Impaired Gnrh Production

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Mitochondrial Dysfunction in Gnrh Neurons Impaired Gnrh Production Biochemical and Biophysical Research Communications 530 (2020) 329e335 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Mitochondrial dysfunction in GnRH neurons impaired GnRH production * Yoshiteru Kagawa a, , Banlanjo Abdulaziz Umaru a, Subrata Kumar Shil a, Ken Hayasaka a, Ryo Zama a, Yuta Kobayashi a, b, Hirofumi Miyazaki a, Shuhei Kobayashi a, Chitose Suzuki c, Yukio Katori b, Takaaki Abe c, Yuji Owada a a Department of Organ Anatomy, Tohoku University Graduate School of Medicine, Sendai, 980-8575, Japan b Department of Otolaryngology Head and Neck Surgery, Tohoku University Graduate School of Medicine, Sendai, 980-8574, Japan c Department of Nephrology, Endocrinology, and Vascular Medicine, Tohoku University Graduate School of Medicine, Sendai, 980-8574, Japan article info abstract Article history: The onset establishment and maintenance of gonadotropin-releasing hormone (GnRH) secretion is an Received 13 July 2020 important phenomenon regulating pubertal development and reproduction. GnRH neurons as well as Accepted 18 July 2020 other neurons in the hypothalamus have high-energy demands and require a constant energy supply Available online 7 August 2020 from their mitochondria machinery to maintain active functioning. However, the involvement of mito- chondrial function in GnRH neurons is still unclear. In this study, we examined the role of NADH De- Keywords: hydrogenase (Ubiquinone) FeeS protein 4 (Ndufs4), a member of the mitochondrial complex 1, on GnRH Mitochondria neurons using Ndufs4-KO mice and Ndufs4-KO GT1-7 cells. Ndufs4 was highly expressed in GnRH Ndufs4 GnRH neuron neurons in the medial preoptic area (MPOA) and NPY/AgRP and POMC neurons in the arcuate (ARC) fi GnRH nucleus in WT mice. Conversely, there was a signi cant decrease in GnRH expression in MPOA and Transcription median eminence of Ndufs4-KO mice, followed by impaired peripheral endocrine system. In Ndufs4-KO GT1-7 cells, Gnrh1 expression was significantly decreased with or without stimulation with either kisspeptin or NGF, whereas, stimulation significantly increased Gnrh1 expression in control cells. In contrast, there was no difference in cell signaling activity including ERK and CREB as well as the expression of GPR54, TrkA and p75NTR, suggesting that Ndufs4 is involved in the transcriptional regu- lation system for GnRH production. These findings may be useful in understanding the mitochondrial function in GnRH neuron. © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction neuroplasticity. This may explain the pathological phenotypes, but detailed underlying mechanisms are still unknown. Mitochondria are vital cytoplasmic organelles that play crucial NADH Dehydrogenase (Ubiquinone) FeeS Protein 4 (Ndufs4) is roles in generation of cellular energy in the form of ATP by the one of the compartments of mitochondrial complex 1, the primary process of oxidative phosphorylation [1]. Given this essential entry point for electrons into the electron transport chain (ETC). function, mitochondria defects in tissues with high energy re- Ndufs4 is one of the key molecules involved in Leigh syndrome quirements can lead to a wide variety of diseases. Notably, several because its mutation in patients shows several phenotypes such as neurodegenerative diseases including Parkinson’s disease, Alz- retarded growth, developmental delay, visual defects, muscular heimer’s disease, and Leigh syndrome are triggered by mitochon- hypotonia, encephalomyopathy and failure to thrive, leading to dria dysfunction in neurons [2e4]. Neurons demand high levels of early death [6]. Consistently, mice with a global deletion of Ndufs4 ATP for their functional activity in signal transduction and release show developmental delay, motor alterations, respiratory deficits of neurotransmitters [5] and a disruption in energy may alter and epilepsy [7]. Experimentally, it has been reported that Ndufs4 inactivation in Vglut2-expressing glutamatergic neurons leads to decreased neuronal firing, motor and respiratory deficits, and early * Corresponding author. 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, death [8]. In addition, Ndufs4 deletion in GABAergic neurons causes Japan. basal ganglia inflammation, hypothermia and severe epileptic E-mail address: [email protected] (Y. Kagawa). https://doi.org/10.1016/j.bbrc.2020.07.090 0006-291X/© 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 330 Y. Kagawa et al. / Biochemical and Biophysical Research Communications 530 (2020) 329e335 seizure preceding death [8]. These results suggest that Ndufs4 2.3. Image analysis performs different functions in the maintenance of CNS homeo- stasis depending on the subtype of neurons. Consistently, although Quantitative analysis for intensity of GnRH-positive fluores- in patients, all neurons carry the mutation responsible for the Leigh cence was performed using Imaris software (Carl Zeiss). Briefly, the syndrome, not every neuron is harmed by the mutation. limited area in ME was cropped as shown in Supplemental Fig. 4. Among the hypothalamic neurons, numerous studies already The sum of fluorescence intensity was evaluated and area was used demonstrate the role of mitochondria in proopiomelanocortin for normalization. Analysis for all samples was blindly performed in (POMC) and neuropeptide Y (NPY)/agouti-related protein (AgRP) same condition (gamma setting: 0.70, threshold setting: 60.0). neurons, which are closely associated with obesity. They show that the loss of mitofusin protein leads to altered function of POMC and 2.4. Cell culture and treatment NPY/AgRP neurons [9,10]. Additionally, the neuronal activity of both POMC and NPY/AgRP neurons is modified by reactive oxygen GT1-7 cell (mouse hypothalamic GnRH neuronal cell line) was species, which is generated by mitochondria during the process of obtained from Merck Millipore (MA, USA), and maintained by oxidative phosphorylation [11,12], suggesting that the mitochon- passage in Dulbecco’s modified Eagle’s medium (DMEM, Thermo drial function in hypothalamic neurons is critically essential for Fisher Scientific Inc.) containing 10% FBS (Thermo Fisher Scientific homeostasis. Inc.). Cells were treated with 300 nM kisspeptin (Tocris Bioscience, At the apex of the hypothalamicepituitaryegonadal (HPG) axis, Bristol, England) and 100 ng/ml NGF (R&D systems, MN, USA) for the gonadotropin-releasing hormone (GnRH) neuron in the hypo- 4 h as reported previously [16e18]. thalamus is the master regulator of pubertal development and reproduction [13]. GnRH neurons located in the medial preoptic area (MPOA) with their fibers projecting to the arcuate (ARC) nu- 2.5. Crispr/Cas9 editing cleus and the median eminence (ME). Stimulation of GnRH secre- tion results in the release of gonadotropins, luteinizing hormone To construct the sgRNA expression plasmid, we selected target (LH) and follicle-stimulating hormone (FSH) from the anterior pi- sites within exon 1 of mouse Ndufs4 gene using CHOPCHOP soft- tuitary gland [14]. This triggers the onset of puberty as well as ware (https://chopchop.cbu.uib.no/). The following oligonucleo- maintenance of the reproductive system. Although previous tides were used: gRNA_1; 50-TGGAACTCTACAGACGGAAA-30 and 50- studies suggest GnRH secretion to be regulated by many factors TTTCCGTCTGTAGAGTTCCA-30, gRNA_2; 50-CGCTGAGA- such as kisspeptin [15] and nerve growth factor (NGF) [16], the CAGGCGATGTTG-30 and 50-CAACATCGCCTGTCTCAGCG-30. The involvement of mitochondrial function in generation/secretion of double-stranded oligonucleotides were synthesized and inserted GnRH in GnRH neurons is still unclear. into pGuide-it-ZsGreen1 vector (Takara, Tokyo, Japan). The con- In this study, we explored the function of Ndufs4 in GnRH structed vector was transfected into cells using Lipofectamine® neurons using Ndufs4-KO mice and GnRH-secreting neuronal cell 2000 Reagent (Thermo Fisher Scientific Inc.). Successfully trans- line (GT1-7). Current findings provide a preliminary reference for fected cells, which were GFP-positive, were sorted using FACS Aria the exploration of mitochondrial function in GnRH neuron and are II (BD bioscience, NJ, USA). Sequencing analysis was performed to beneficial for therapeutic guidance for reproduction and for mito- screen genomic mutation using the above mentioned forward chondrial diseases. primer. 2.6. Isolation of mitochondria 2. Materials and methods GT1-7 cells were scrapped using mitochondrial isolation buffer 2.1. Animals (0.25 M sucrose, 20 mM Tris-HCl (pH 8.0), 0.1 mM EDTA) and ho- mogenized using dounce homogenizer. Homogenate was centri- Ndufs4-KO mice (B6.129S4-Ndufs4tm1.1Rpa/J) were obtained fuged at 600ÂG for 10 min and its supernatant was centrifuged from the Jackson Laboratory (ME, USA). All experimental protocols again at 12,000ÂG for 15 min. The pellet was dissolved and protein were reviewed by the Ethics Committee for Animal Experimenta- concentration was measured by BCA Protein Assay Kit (Thermo tion of Tohoku University Graduate School of Medicine and carried Fisher Scientific Inc.). out under the law and notification requirements of the Japanese government. 2.7. Quantitative real-time PCR 2.2. Histological studies Total RNA was extracted using an RNeasy plus Mini kit (Qiagen, Netherlands).
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