Biochemical and Biophysical Research Communications 530 (2020) 329e335

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Biochemical and Biophysical Research Communications

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Mitochondrial dysfunction in GnRH 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 (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 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 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 [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 (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, 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 600G for 10 min and its supernatant was centrifuged from the Jackson Laboratory (ME, USA). All experimental protocols again at 12,000G 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). Quantitative real-time PCR (qPCR) was performed Mice were transcardially perfused with 4% paraformaldehyde in using following TaqMan probes: Mm00656176_m1 for Ndufs4, phosphate buffer, and paraffin sections (4 mm) were prepared from Mm00600697_m1 for Gfap, Mm01248771_m1 for Rbfox3 (NeuN), brain and pituitary gland. Hematoxylin and eosin (H&E) staining Mm01266402_m1 for Mbp, Mm00434455_m1 for Itgam (Cd11b), was performed using standard methods. For DAB (3,30-Dia- Mm02619580_g1 for Actb, Mm01315604_m1 for Gnrh1. Quantifi- minobenzidine) staining and immunofluorescence staining, sec- cation was normalized with cycle threshold values to Actb. qPCR for tions were incubated with primary antibodies shown in TrkA and Gpr54 was performed using SYBR® Premix Ex Taq™ II Supplemental Table 1 and the appropriate secondary antibodies (Takara). Target gene primers are as follows: TrkA, Fw50-ATATC- shown in Supplemental Table 1. Visualisation was performed using TAGCCAGCCTGCACTTTGT-30 and Rv 50-TGCTCATGCCAAAGTCTCCA- Vectastain ABC reagent (Vector Laboratories, ON, Canada). For 30,p57NTR, Fw50-GCACATACTCAGATGAAGCCAACCA-30, Rv50- immunofluorescence staining, diamidino-2-phenylindole (DAPI, GGGGCGTAGACCTTGTGATCC-30, Actb, Fw50-CATCCGTAAA- Thermo Fisher Scientific Inc., MA, USA) is used for counterstaining. GACCTCTATGCCAAC-30 and Rv50-ATGGAGCCACCGATCCACA-30, Observation was performed using confocal laser microscopy (Zeiss Gpr54, Fw50-GCAGCCCATGGTTGAAGCTAA-30 and Rv50-AGC- LSM800, Carl Zeiss, Oberkochen, Germany). TATGCCCGGATGACAGAAG-30. Y. Kagawa et al. / Biochemical and Biophysical Research Communications 530 (2020) 329e335 331

2.8. Western blotting 2.11. Hormone concentrations of serum

The cell lysates were resolved by SDS-PAGE and transferred to a Blood samples were collected via retinal venous plexus and polyvinylidene difluoride membrane (Merck Millipore, MA, USA). serum was prepared. The measurement of LH and FSH were per- The membrane was incubated with primary antibody (as shown in formed using ELISA kit (LifeSpan BioScience, Inc. WA USA) ac- Supplemental Table 1) overnight at 4 C followed by incubation cording to manufacturer’s protocol. Testosterone measurement was with secondary antibody (as shown in Supplemental Table 1). performed with liquid chromatographyetandem mass spectrom- Detection was performed with the ECL Detection Kit (Thermo etry (LC-MS/MS) by ORIENTAL YEAST Co., Ltd. (Tokyo, Japan). Fisher Scientific Inc.). 2.12. Statistical analysis

All the data were expressed as means and error bars were 2.9. Mitochondrial respiration assay attached by standard error (SE). Data were compared by a two- tailed unpaired Student’s t-test or one-way analysis of variance Oxygen consumption rate (OCR) of GT1-7 cells was measured for multiple comparisons. Differences were considered significant using the XF24 analyzer (Agilent Technologies, CA, USA). GT1- at P values < 0.05 in all of the cases. 7 cells were seeded at a concentration of 80,000 cells/well and incubated for 24 h. Following incubation, medium was changed with assay medium containing 25 mM glucose, 2 mM L-glutamine, 3. Results 1 mM sodium pyruvate. The basal OCR measurements were recorded before oligomycin addition. Simultaneously, the extra- 3.1. Ndufs4 is expressed in the neurons of MPOA and ARC nucleus cellular acidification rate (ECAR) was measured. Ndufs4-KO mice were smaller than wild-type mice (WT) by postnatal day 21 (P21) when they grow up with their mother as shown in the previous report [7]. After weaning at P21, those dif- 2.10. Mitochondrial membrane potential measurement ferences became completely apparent as the mice got older by P49 (Fig. 1A). Furthermore, a significant difference in cortex size was Mitochondrial membrane potential was measured by JC1 observed between WT and Ndufs4-KO mice at P49, but at P21, membrane potential dye (Dojindo, Kumamoto, Japan) according to cortex size was same (Fig. 1B and Supplemental Fig. 1A). Consis- manufacturer’s protocol. Live cell images were captured in incu- tently, the size of testes in Ndufs4-KO mice were smaller than WT at bation chamber of Zeiss LSM 800 microscope (Carl Zeiss). P21 (Supplemental Figs. 1B and 1C), suggesting that the

Fig. 1. Ndufs4 is expressed in the neurons of MPOA and ARC nucleus. (A) The graph shows the body weight of WT and Ndufs4-KO mice at P21 and P49 (n ¼ 6). (B) The graph shows cortex size of WT and Ndufs4-KO mice at P21 and P49 (n ¼ 6) (C) H&E staining in MPOA and ARC region of WT and Ndufs4-KO mice (P21). Scale bar: 200 mm (D) DAB staining of Ndufs4 in MPOA and ARC region of WT and Ndufs4-KO mice (P21). Scale bar: 100 mm (E) Co-immunofluorescence staining of Ndufs4 (green) with MAP2 (red) in MPOA and ARC region of WT and Ndufs4-KO mice (P21). Nuclei are stained with DAPI (blue). Scale bar: 50 mm. 332 Y. Kagawa et al. / Biochemical and Biophysical Research Communications 530 (2020) 329e335 development of Ndufs4-KO mice were impaired. localized in the ARC nucleus. We investigated the expression of Given these phenotypes as well as patients of Ndufs4-mutation Ndufs4 in each cell body and found that GnRH neurons as well as [6], we hypothesized that Ndufs4 deficiency may impair the hy- NPY/AgRP and POMC expressed Ndufs4 (Fig. 2A, E and F). Notably, pothalamic system, which is the master regulator of biological immunofluorescence staining revealed decreased expression of homeostasis through both endocrine and autonomic nervous sys- GnRH in MPOA and ME of Ndufs4-KO (Fig. 2B and C). Consistently, tem. First, we compared morphological changes between WT and mRNA expression of GnRH from punched-out hypothalamus tissue Ndufs4-KO focusing on MPOA, the surroundings of organum vas- was also decreased in Ndufs4-KO mice (Fig. 2D), suggesting that the culosum laminae terminalis (OVLT), and ARC nucleus in the hypo- population of GnRH neurons or expression of GnRH may be thalamus at P21 in H&E stained sections, but no apparent impaired in Ndufs4-KO mice. On the other hand, there was no difference in morphology was observed (Fig. 1C). Interestingly, a difference in the expression of both NPY and POMC between WT small and diffuse population of Ndufs4 expressed cells was clearly and Ndufs4-KO mice (Fig. 2E and F). observed in MPOA and as well as ARC nucleus (Fig. 1D). Thus, to determine the type of cells in MPOA and ARC nucleus showing high 3.3. Ndufs4-KO impairs the functions of hormone-related expression of Ndufs4 at P21, we performed co-immunostaining of peripheral organs in mice Ndufs4 with several cell markers and found that Ndufs4 co- localized with MAP2-immunolabeled neurons in MPOA and ARC In the apex of HPG, the system of GnRH neuron-LH and FSH- nucleus (Fig. 1E). Distribution of GFAP was not observed in both testosterone is well studied [13]. In this regard, we examined the MPOA and ARC at P21 (Supplemental Fig. 2A). MBP was observed in role of Ndufs4 in several phenotypes of HPG. Although H&E staining MPOA co-localizing with Ndufs4, but could not be observed in ARC demonstrated no apparent difference in the morphology of anterior nucleus (Supplemental Fig. 2B). Next, we evaluated the differences pituitary gland between WT and Ndufs4-KO mice (Supplemental in expression of NeuN, GFAP, MBP, and CD11b (marker for micro- Fig. 5), DAB staining notably revealed that the expression of both glia) by qPCR using punctured hypothalamic tissue, however, there LH and FSH in anterior pituitary gland was significantly increased in was no difference between WT and Ndufs4-KO in all marker Ndufs4-KO mice compared to WT mice (Fig. 3A). In addition, the (Supplemental Figs. 3A and 3B), suggesting that there might not be levels of serum LH and FSH were significantly decreased in Ndufs4- change in the population of each cell type. KO mice (Fig. 3B and C) as well as the level of testosterone (Fig. 3D). This suggests that the decrease of GnRH in Ndufs4-KO mice may 3.2. GnRH expression in hypothalamus is decreased in Ndufs4-KO trigger the altered expression/secretion system of LH and FSH from mice anterior pituitary gland, followed by the decreased secretion of testosterone from the peripheral tissue. Granted, we cannot defini- Among hypothalamic neurons, the cell body of GnRH neurons is tively conclude on this because Ndufs4 is somatically deleted in the localized in the MPOA, and the cell body of NPY/AgRP and POMC is mice used in this study.

Fig. 2. GnRH expression in hypothalamus is decreased in Ndufs4-KO mice. (A) Co-immunofluorescence staining of Ndufs4 (green) with GnRH (red) in MPOA of WT mice (P21). Nuclei were stained with DAPI (blue). The area surrounded by a white square is highlighted as high magnification. Scale bar of low magnification: 100 mm, of high magnification: 20 mm. (B) Immunostaining of GnRH (green) in MPOA and ARC/ME region of WT (P21) and Ndufs4-KO mice (P21). Nuclei were stained with DAPI (blue). Scale bar: 200 mm. (C) The intensity of green fluorescence in limited area in ME was analyzed with Imaris software as shown in Supplemental Fig. 4. Stained sections are obtained from WT and Ndufs4-KO mice (P21) (n ¼ 6). (E, F) Co-immunofluorescence staining of Ndufs4 (green) with NPY (E) and POMC (F) in ARC region of WT and Ndufs4-KO mice (P21). Nuclei were stained with DAPI (blue). Data shown are the means ± s.e.m. *p < 0.05. Y. Kagawa et al. / Biochemical and Biophysical Research Communications 530 (2020) 329e335 333

Fig. 3. The functions of hormone-related peripheral organs were impaired in Ndufs4-KO mice. (A) DAB staining of FSH and LH in anterior pituitary gland of WT and Ndufs4-KO mice (P21). Scale bar: 50 mm (B, C) Quantitative analysis of serum LH (B) and FSH (C) by ELISA. Serum was obtained from WT (P21) (n ¼ 8) and Ndufs4-KO (P21) (n ¼ 6). (D) Quantitative analysis of testosterone by LC-MS/MS in peripheral serum of WT (P21) (n ¼ 8) and Ndufs4-KO mice (P21) (n ¼ 6). Data shown are the means ± s.e.m. *p < 0.05.

3.4. Gnrh1 production was significantly suppressed in Ndufs4-KO ECAR was at the same levels (Supplemental Fig. 6), meaning that GT1-7 cells glycolytic metabolism under normal state may not be impaired. Consistently, staining with JC1 revealed an increased level of green To determine the role of Ndufs4 in GnRH neuron, we established fluorescence in Ndufs4-KO GT1-7 cells, indicating a decreased level two clones of Ndufs4-KO GT1-7 cells created using CRISPR/Cas9 of mitochondrial membrane potential (Fig. 4E). These data sug- methods. After confirmation of Ndufs4 loss in CRISPR/Cas9 edited gested that loss of Ndufs4 mainly induced altered mitochondria cells (Fig. 4A), we measured the mitochondrial bioenergetics respiration, followed by altered level of ATP production. function and found that basal OCR and ATP production were Since kisspeptin and NGF are well known to induce Gnrh1 significantly decreased in Ndufs4-KO cells (Fig. 4B, C and D). expression in GT1-7 cells, we then evaluated the effects of loss of Rotenone and antimycin A; mitochondrial ETC complex I and III Ndufs4 on Gnrh1 expression with or without stimulation. While the inhibitor, respectively, blocked OCR to basal levels in both control treatment with both kisspeptin and NGF induced Gnrh1 expression and Ndufs4-KO GT1-7 cells, suggesting that OCR in GT1-7 cells is in control cells, Gnrh1 expression was significantly decreased in almost dependent on mitochondrial ETC and Ndufs4 directly reg- Ndufs4-KO cells and stimulation did not affect Gnrh1 expression in ulates OCR through mitochondrial ETC. On the other hand, basal these KO cells (Fig. 4F). Conversely, there was no difference in cell

Fig. 4. Gnrh1 production was significantly suppressed in Ndufs4-KO GT1-7 cells. (A) Western blot to confirm the expression of Ndufs4 in Ndufs4-KO GT1-7 cells created using CRISPR/Cas9 methods. Voltage-dependent anion channel (VDAC) is used as endogenous control. T: total cell lysate, M: isolated mitochondrial lysate. (B, C, D) The assay for mitochondrial bioenergetics function using the XF24 analyzer. (E) The assay for mitochondrial membrane potential using JC1 dye. Scale bar: 50 mm (F) qPCR analysis for Gnrh1 expression with kisspeptin and NGF for 4 h, respectively. b- was used as endogenous control. (G) Western blot for phosphorylated ERK and CREB, and total levels of ERK and CREB. Data shown are the means ± s.e.m. and representative of 3 independent experiments. *p < 0.05, **P < 0.01. 334 Y. Kagawa et al. / Biochemical and Biophysical Research Communications 530 (2020) 329e335 signaling activity including extracellular signal-regulated kinase fact, AgRP-specific leptin receptor knock-out female mice exhibited (ERK) and cAMP response element binding protein (CREB) between a significant delay in the pubertal onset of estrous cycles compared control and Ndufs4-KO cells (Fig. 4G). Furthermore, there was no with control animals, but no significant differences in male puberty difference in the expression NGF receptors, tropomyosin receptor onset or adult fecundity in either sex were observed [22]. Further kinase A (TrkA) and low-affinity NGF factor receptor (p75NTR), and detailed mechanisms should be assessed using Ndufs4 conditional kisspeptin receptors, G-protein coupled receptor (Gpr54), between knock-out mice using POMC-cre and NPY/AgRP-Cre which can control and Ndufs4-KO GT1-7 cells with or without treatments provide us clearer evidence. Nonetheless, our in vitro experiments (Supplemental Fig. 7A, 7B and 7C) as well as between WT and suggest that loss of Ndufs4 in GnRH neuron itself may be partially Ndufs4-KO hypothalamus (Supplemental Figs. 7D, 7E and 7F). involved in the impaired system of HPG axis. Overall, these data suggests that Ndufs4 is involved in the tran- scriptional system for Gnrh1 expression. Declaration of competing interest

4. Discussion The authors declare no competing interests.

Ndufs4 is one of the key molecular determinants for Leigh Acknowledgments syndrome, which is one of the mitochondrial diseases. Patients with Leigh syndrome show many phenotypes including severe This work was supported by Japan Society for the Promotion of muscle and movement problems, inability of growth, and failure to Science (JSPS) KAKENHI Grant (No. 19H04026 and No. 18K19723 to thrive. Recently, Bolea et al. demonstrated that loss of Ndufs4 in Y. O.). Vglut2-expressing glutamatergic neurons leads to decreased neuronal firing, motor and respiratory deficits, and early death [8]. Appendix A. Supplementary data They also showed that loss of Ndufs4 in GABAergic neurons causes basal ganglia inflammation, hypothermia and severe epileptic Supplementary data to this article can be found online at seizure preceding death [8], suggesting that neuronal Ndufs4 https://doi.org/10.1016/j.bbrc.2020.07.090. deletion/mutation found in patients with Leigh syndrome may be the main source for these phenotypes and Ndufs4 functions differently in each type of neurons. In this study, for the first time References we demonstrated the expression of Ndufs4 in the cell body of [1] I. Martinez-Reyes, N.S. Chandel, Mitochondrial TCA cycle metabolites control several hypothalamic neurons including GnRH neurons in MPOA, physiology and disease, Nat. Commun. 11 (2020) 102, https://doi.org/10.1038/ and NPY/AgRP and POMC neurons in ARC nucleus. Notably, GnRH s41467-019-13668-3. production in GnRH neurons of Ndufs4-KO mice was impaired, [2] S. Ciccone, E. Maiani, G. Bellusci, M. Diederich, S. Gonfloni, Parkinson’s disease: a complex interplay of mitochondrial DNA alterations and oxidative stress, Int. followed by altered endocrine system in the peripheral tissue. J. Mol. Sci. 14 (2013) 2388e2409, https://doi.org/10.3390/ijms14022388. Altered functions of GnRH neurons lead to an impaired repro- [3] N.J. Lake, M.J. Bird, P. Isohanni, A. Paetau, Leigh syndrome: neuropathology duction system. It has been reported that GnRH neuron-specific and pathogenesis, J. Neuropathol. Exp. Neurol. 74 (2015) 482e492, https:// doi.org/10.1097/NEN.0000000000000195. deletion of Gpr54 in both male and female mice showed infer- [4] X. Li, L. Wang, M. Cykowski, T. He, T. Liu, J. Chakranarayan, A. Rivera, H. Zhao, tility, failure to go through puberty and exhibiting markedly S. Powell, W. Xia, S.T.C. Wong, OCIAD1 contributes to neurodegeneration in reduced gonadal size and FSH levels [19]. Consistent with these Alzheimer’s disease by inducing mitochondria dysfunction, neuronal vulner- ability and synaptic damages, EBioMedicine 51 (2020) 102569, https:// data, Ndufs4-KO mice exhibited impaired secretion of gonado- doi.org/10.1016/j.ebiom.2019.11.030. trophs and testosterone, and the delayed development of testis. [5] I.H. Jain, L. Zazzeron, O. Goldberger, E. Marutani, G.R. Wojtkiewicz, T. Ast, These data thus suggests that mitochondria in GnRH neurons are H. Wang, G. Schleifer, A. Stepanova, K. Brepoels, L. Schoonjans, P. Carmeliet, partially involved in the reproductive systems and these findings A. Galkin, F. Ichinose, W.M. Zapol, V.K. Mootha, Leigh syndrome mouse model can be rescued by interventions that normalize brain hyperoxia, but not HIF may reasonably explain the failure to thrive in Leigh syndrome. activation, Cell Metabol. 30 (2019), https://doi.org/10.1016/ GnRH-neuron specific Ndufs4-KO mice will provide further j.cmet.2019.07.006, 824-832 e823. detailed mechanisms. [6] V. Petruzzella, R. Vergari, I. Puzziferri, D. Boffoli, E. Lamantea, M. Zeviani, S. Papa, A nonsense mutation in the NDUFS4 gene encoding the 18 kDa GnRH production is regulated by many factors such as kiss- (AQDQ) subunit of complex I abolishes assembly and activity of the complex peptin from kisspeptin neurons in ARC nucleus [20], and several in a patient with Leigh-like syndrome, Hum. Mol. Genet. 10 (2001) 529e535, neuropeptides released from POMC and NPY/AgRP neurons [21]as https://doi.org/10.1093/hmg/10.5.529. [7] S.E. Kruse, W.C. Watt, D.J. Marcinek, R.P. Kapur, K.A. Schenkman, R.D. Palmiter, well as NGF [16]. 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Horvath, Mitochondrial dynamics controlled by 2þ activation of ERK and Ca signaling, we could not find any differ- mitofusins regulate Agrp neuronal activity and diet-induced obesity, Cell 155 ence in activation between control and Ndufs4-KO cells (Fig. 4G). (2013) 188e199, https://doi.org/10.1016/j.cell.2013.09.004. For an indepth analysis, we evaluated the expression of Gpr54, TrkA [10] M. Schneeberger, M.O. Dietrich, D. Sebastian, M. Imbernon, C. Castano, NTR A. Garcia, Y. Esteban, A. Gonzalez-Franquesa, I.C. Rodriguez, A. Bortolozzi, and p75 , but there was no difference between control and P.M. Garcia-Roves, R. Gomis, R. Nogueiras, T.L. Horvath, A. Zorzano, M. Claret, Ndufs4-KO cells (Supplemental Fig. 7), suggesting that Ndufs4 may Mitofusin 2 in POMC neurons connects ER stress with leptin resistance and e be involved in the process of Gnrh1 transcription. 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