Ndufs4 ablation decreases synaptophysin expression in hippocampus

Altered function of mitochondrial respiratory chain in brain cells is related to many neurodegenerative diseases. NADH Dehydrogenase (Ubiquinone) Fe-S protein 4 (Ndufs4) is one of the subunits of mitochondrial complex I and its mutation in human is associated with Leigh syndrome. However, the molecular biological role of Ndufs4 in neuronal function is poorly understood. In this study, upon Ndufs4 expression confirmation in NeuN-positive neurons, and GFAP-positive astrocytes in WT mouse hippocampus, we found significant decrease of mitochondrial respiration in Ndufs4-KO mouse hippocampus. Although there was no change in the number of NeuN positive neurons in Ndufs4-KO hippocampus, the expression of synaptophysin, a presynaptic protein, was significantly decreased. To investigate the detailed mechanism, we silenced Ndufs4 in Neuro-2a cells and we observed shorter neurite lengths with decreased expression of synaptophysin. Furthermore, western blot analysis for phosphorylated extracellular regulated kinase (pERK) revealed that Ndufs4 silencing decreases the activity of ERK signalling. These results suggest that Ndufs4-modulated mitochondrial activity may be involved in neuroplasticity via regulating synaptophysin expression.

www.nature.com/scientificreports/ retardation, seizures, respiratory insufficiency and failure to thrive leading to early death 18,19 . Similarly, global Ndufs4-KO mouse showed retarded growth, loss of motor ability, breathing abnormalities, and death 20 . Moreover, previous studies have demonstrated the importance of Ndufs4 in neuronal function. In recent study, glutamatergic neuron specific Ndufs4 loss in mice showed a significant impairment of neuronal firing, motor deficit, brain stem inflammation and region specific marked astrogliosis 21 . Furthermore, GABAergic neurons specific Ndufs4 loss exhibited basal ganglia inflammation and severe epileptic seizures preceding death 21 . Furthermore, dopaminergic neuron specific Ndufs4-KO mouse showed the impairment of dopamine release in striatum, amygdala without loss of neuron [22][23][24] . These evidence suggest that Ndufs4 loss in neurons might be a key factor for the development of encephalopathy. However, the molecular biological role of Ndufs4 in neural function is still unknown.
In this study, we explored how Ndufs4 ablation affects neuron function focusing on neurite growth and synaptic plasticity and found that synaptophysin, a presynaptic protein, expression decreases significantly with a decreased activity of ERK signalling. This novel finding may provide more insights in hippocampal pathophysiology of neurodegenerative disease and may be useful in designing an effective treatment strategy.

Results
Expression and localization of Ndufs4 in cerebral cortex and hippocampus. We first explored Ndufs4 expression and localization in the brain. Western blot analysis using isolated mitochondria from brain confirmed Ndufs4 expression (Fig. 1A). To determine the expression level in different brain regions, we punched out the tissue from the cerebral cortex, hippocampus, and cerebellar cortex. qPCR analysis demonstrated that Ndufs4 was expressed in all regions of the brain although these expression levels were lower compared to the   www.nature.com/scientificreports/ expression in the heart tissue (Supplemental Fig. 1A,B). Furthermore, enzyme-based immunohistochemistry also confirmed the expression of Ndufs4 in cerebral cortex and CA1, CA2 and CA3 region of hippocampus (Fig. 1B). To investigate which cell types express Ndufs4, we performed co-immunostaining of Ndufs4 with several cell markers such as neuronal nuclei (NeuN) for neuron, glial fibrillary acidic protein (GFAP) for astrocyte and myelin basic protein (MBP) for oligodendrocyte. We found that Ndufs4 co-localized with all cell markers in hippocampus and cerebral cortex with equal fluorescence ( Fig. 1C and Supplemental Fig. 1C-F). Then, to explore the mitochondrial functionality, we measured complex I respiration using the isolated mitochondria from punch out hippocampus tissue and found a significant decrease in basal respiration and maximal respiration in Ndufs4-KO compared to WT, but ATP production was not changed (Fig. 1D-F).
Elevated GFAP expression was observed in Ndufs4-KO hippocampus, but not in primary cultured Ndufs4-KO astrocytes. To evaluate whether Ndufs4 deficiency alters the expression of cell markers and cell population, immunostaining and Western blot were performed using hippocampus of WT and Ndufs4-KO mice. Although there was no difference in expression of NeuN-positive neuron and MBP-positive oligodendrocyte ( Fig. 2A,C,D), interestingly, an increased number of hypertrophic GFAP-positive astrocytes with increased number of processes were found in Ndufs4-KO hippocampus ( Fig. 2B; Supplemental Fig. 2A,B). Elevated GFAP expression in Ndufs4-KO hippocampus was also confirmed by Western blot (Fig. 2D; Supplemental Fig. 2C). This led us to hypothesize that reactive gliosis, characterized by hypertrophy of the astrocyte cellular processes and high levels of expression of GFAP, may be occurring in Ndufs4-KO brain. As such, to investigate whether Ndufs4 deficiency in astrocytes affects the function of astrocytes, we prepared primary cultured astrocytes from Ndufs4-KO brain (Supplemental Fig. 2D), but we could not find gliosis character such as increased GFAP expression and increased cell proliferation (Fig. 2E,F; Supplemental Fig. 2E) in Ndufs4-KO astrocytes, suggesting that the gliosis character observed in Ndufs4-KO mouse brain may be due to altered neuroplasticity in hippocampus.

Ndufs4 deficiency decreased synaptophysin expression in hippocampus.
To ascertain whether any neuronal dysfunction occurred in hippocampus due to Ndufs4 deficiency, we counted the number of NeuN positive cells in hippocampus, but we did not find a significant difference between WT and Ndufs4-KO mouse brain (Supplemental Fig. 3A). Because previous reports have demonstrated the presence of neuron apoptosis in the olfactory bulb of Ndufs4-KO mouse 20 , supportively, we evaluated neuron apoptosis, but we could not find cleaved caspase 3 positive cells in both WT and Ndufs4-KO hippocampus (Fig. 3A). Then, we investigated the synaptic protein expression in hippocampus because mitochondrial function is closely associated with the synthesis of synaptic protein 25 . Notably, synaptophysin expression was significantly decreased in Ndufs4-KO . We also confirmed that Ndufs4 silencing impaired mitochondrial function including basal respiration, maximum respiration, as well as ATP production (Supplemental Fig. 4D). Using these cells, we found that Ndufs4 silencing decreased the average neurite length compared to control ( To confirm the direct relation between ERK activity and neurite growth/synaptophysin expression, we performed a rescue experiment using a pERK agonist Carbamylcholine Chloride (carbachol). We found a significantly higher neurite growth and synaptophysin expression in carbachol treated Ndufs4 knockdown cells, and even in control cells (Supplemental Fig. 5A-D). Considering that carbachol is a nonspecific cholinergic agonist and acts on nicotinic and muscarinic receptors, and as well, activates many downstream signalling pathways [26][27][28] , it may activate other cell signalling outside of the Ndufs4 cascade, but there is a strong possibility that drug-induced-increased pERK activity in Ndufs4 knockdown cells partially rescued neurite growth and synaptophysin expression. Taken together, these data suggest that Ndufs4-modulated mitochondrial function may be involved in neuroplasticity through the ERK signalling followed by alteration of neurite growth and synaptophysin expression.

Discussion
Mitochondrial function in neuron is quite essential for electrical activity, axon extension, regeneration, branching, synaptic formation, and synaptic neurotransmission 5,29,30 , and altered mitochondrial function leads to neurodegenerative diseases [31][32][33] . Ndufs4 is the gene responsible for Leigh syndrome 17 . It has been reported that loss of Ndufs4 reduces maximum CI dependent oxidative phosphorylation in synaptosomal mitochondria of olfactory bulb, brainstem and cerebellum 33 . Furthermore, striatal medium spiny neurons specific Ndufs4 loss progressively impairs the motor function without loss of neuronal number 22 . However, the molecular function of Ndufs4 in neuron is still not well deciphered. In this study, we first demonstrated Ndufs4 expression in hippocampal neurons in WT and then showed mitochondrial dysfunction with decreased expression of synaptophysin without loss of neuronal cells in Ndufs4 deficient mouse hippocampus. Mitochondria are intimately involved in cell signalling pathways such as growth factor signalling, differentiation, cell death signalling, and autophagy through regulating the levels of intracellular signalling molecules such as Ca 2+ , ATP and ROS 34 . Notably, mitochondrial dysfunction promotes p38 mitogen-activated protein kinase activity, resulting in neuronal intracellular responses including inflammation and apoptosis [35][36][37] . Similarly, decreased activity of ERK signalling inhibits mitochondrial fusion and stimulates apoptotic mitochondrial www.nature.com/scientificreports/ membrane permeabilization through phosphorylation of mitochondrial fusion protein, mitofusin 1, in rat primary cortical neurons thereby regulating cell death 38 . Furthermore, pharmacological inhibition of c-Jun N-terminal kinases results in a decreased mitochondrial membrane potential in motor neurons, leading to cell degeneration 39 . These previous studies thus suggest the involvement of mitochondria in the cell signalling cascade. In this study, although there was no difference in the phosphorylation level of ERK between WT and Ndufs4 KO hippocampus (Supplemental Fig. 4F,G), silencing of Ndufs4 decreased the levels of phosphorylation of ERK. These results may reflect the close association between Ndufs4 in neuron and ERK signalling. It is well known that intracellular cell signalling is responsible for presynaptic formation. For example, altered ERK signalling has been shown to impair synaptic plasticity in the hippocampus [40][41][42] . It has also been reported that ERK is responsible for the presynaptic modulation of synaptic plasticity in the hippocampal CA3 region that requires synapsin 1, a presynaptic protein 43 . Similarly, another study also demonstrated that pharmacological blockade of ERK activity prevents stimulation-induced dendritic spine formation in hippocampal CA1 pyramidal neuron in ex vivo slice systems 44 . In this study, we demonstrated a decreased activity of ERK signalling and decrease of synaptophysin expression, suggesting that Ndufs4 is regulating the synaptophysin expression through modulating ERK signalling. Furthermore, a rescue experiment with carbachol demonstrated that a significantly higher neurite growth and synaptophysin expression in carbachol treated Ndufs4 knockdown cells, and even in control cells. Considering that carbachol is a nonspecific cholinergic agonist and acts on nicotinic and muscarinic receptors, and as well, activates many downstream signalling pathways [26][27][28] , it may also activate other cell signalling outside of the Ndufs4 cascade, but there is a strong possibility that drug-induced-increased pERK activity in Ndufs4 knockdown cells partially rescued the synaptophysin expression. Further experiments using a selective ERK agonist such a mutant of ERK will clarify this issue.
Reactive gliosis is commonly observed with neuron loss or dysfunction in CNS disorders. Previous research shows a marked gliosis with significant loss of synapse in cerebral cortex of Alzheimer's patients 45 . Similarly, another study shows astroglial reactivity in hippocampus in a mouse model of traumatic axonal injury 46 . Notably, it has been reported that glutamatergic neuron specific Ndufs4-KO mouse shows elevated GFAP expression in vestibular nucleus, cerebellar fastigial nucleus and inferior olive 21 . Similarly, GABAergic neuron specific Ndufs4-KO mouse shows elevated GFAP in basal ganglia, substantia nigra pars reticulata and olfactory bulb 21  www.nature.com/scientificreports/ Furthermore, Nestin-driven Ndufs4-KO mouse shows elevated GFAP in olfactory bulb, cerebellum, and vestibular nuclei 20 suggesting neuron dysfunction may trigger astrogliosis. In this study, gliosis character was observed in in vivo Ndufs4-KO hippocampus with decreased synaptophysin expression with no neuron loss. In addition, primary cultured astrocytes from Ndufs4-KO hippocampus did not show any gliosis character. These results indicate that altered neuroplasticity including impaired synaptic formation may be the inducing factor of gliosis. Further detailed study may reveal the mechanism underlying the astrocyte reactivity in Ndufs4-KO mouse.
In summary, we provide new insights on the effect of Ndufs4 loss in the function of hippocampal neurons. Our work highlighted the involvement of Ndufs4 in neuroplasticity by regulating synaptophysin expression through phosphorylation of ERK. This knowledge may be useful in understanding the pathophysiological aspect of neurodegenerative diseases but, further clear understanding of molecular mechanism of Ndufs4 is quite essential.

Materials and methods
Animals. Ndufs4-KO mice (B6.129S4-Ndufs4 tm1.1Rpa /J) were purchased from The Jackson Laboratory (Maine, USA). The animals were housed in standard cages in a temperature and humidity-controlled room with a 12 h light-dark cycle and ad libitum access to food and water. Animal experiments were carried out in compliance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). All animal care procedures were approved by the Animal Care and Use Committee of Tohoku University Graduate School of Medicine and under the law and notification of the Japanese government. Efforts were taken to minimize animal sufferings. The heterozygous mice were inter-crossed to get the homozygous KO mouse pups and then genotyped to identify the individual mouse.
Tissue collection and quantitative real-time PCR. The brain was removed quickly under anaesthesia and made the serial coronal sections, followed by punching out the hippocampal tissue. Total RNA was extracted by RNeasy Mini Kit (Qiagen, Hilden, Netherlands). Transcriptor High Fidelity cDNA Synthesis Kit (Roche, Basel, Switzerland) was used for reverse transcription. qPCR for Ndufs4 was performed using StepOnePlus Real-Time PCR system (Applied Biosystems, California, USA) using following mouse specific TaqMan Probes: Ndufs4:Mm0656176_ml, Actb: Mm02619580_gl. Actb was used as endogenous control. Relative gene expression was calculated using the 2 −∆∆Ct method.
Immunostaining. The male mice at p21 were transcardially perfused with glyoxal (ALTFiX, Falma Co., Ltd. Tokyo, Japan) under anaesthesia. Brains were removed, post-fixed in glyoxal for overnight at 4 °C and subsequent dehydration by gradient concentration of alcohol, cleaned by xylene, infiltrated, and embedded in paraffin. Brain samples were cut in a serial 4 μm of coronal sections using a sliding microtome (Leica Biosystems, Wetzlar, Germany).
For enzyme-based immunohistochemistry, the sections were deparaffinized, rehydrated in phosphate buffered saline (PBS) and permeabilized with 0.3% Triton X-100 in PBS, blocked in 5% fetal bovine serum (FBS) followed by overnight incubation of primary antibody shown in Table 1. After primary antibody incubation, sections were washed with PBS and incubated with biotin conjugated goat anti mouse IgG. Then, sections were incubated with ABC reagent (VECTASTAIN Elite ABC kit, Vector Laboratories Inc., Cat. No. PK-6100, California, USA). Haematoxylin stain was used as nuclear counterstaining.
For immunofluorescence, sections were deparaffinised, hydrated followed by permeabilization with 0.3% Tritox X-100. Sections were incubated in primary antibodies shown in Table 1. Respective secondary antibodies and DAPI (Thermo Fisher Scientific Inc., Massachusetts, USA) reaction were allowed. Images were acquired using a confocal laser scanning microscope (LSM780; Carl Zeiss, Oberkochen, Germany). Number of astrocytes and astrocyte processes were counted using cell counter plugin of ImageJ (NIH, USA). Actual fluorescence intensity was measured after subtracting the background intensity using ImageJ software.
For immunofluorescence using cryosection, brain was removed, post fixed with 4% paraformaldehyde (PFA) and cryoprotected in 15% sucrose followed 30% sucrose in PBS until the tissue sink. Then a cryostat (Tissue-Tek OCT Compound, Sakura Finetek, Cat. No. 4583, Osaka, Japan) was used to cut 14 µm section. Sections were blocked with 20% FBS in 0.3% Triton X-100 in PBS for 1 h followed by overnight incubation of primary antibody (Table1) in 4 °C. Respective secondary antibodies were used, and images were acquired using a confocal laser scanning microscope (LSM780).
Immunocytochemistry of synaptophysin in Neuro-2a cells neurites was performed according to described previously 47 . Cells were fixed with 4% PFA and permeabilized with 0.1% Triton X-100 and incubated by primary antibodies shown in Table 1. Respective secondary antibody with DAPI reaction was allowed. Images were captured by confocal laser scanning microscope LSM780. Synaptophysin stained red puncta was counted as presynapse and respective neurite length was measured using ImageJ software.
Purified astrocyte culture. Primary astrocytes were prepared from cerebral cortices and hippocampi of 0-to 1-day-old WT and littermate Ndufs4-KO mice as described previously 48 . Briefly, the cerebral cortices and hippocampi were isolated carefully with removing the olfactory bulbs and the meninges. Tissues were transferred in Dulbecco's Modified Eagle's Medium (DMEM) (Sigma Aldrich, Missouri, USA) supplemented with 10% FBS, 20 mM d-glucose, 1% v/v penicillin/streptomycin and treated with trypsin (Life Technologies, California, USA) at a final concentration of 0.25% for 20 min in 37 °C. The harvested cells were resuspended in DMEM containing 10% v/v heat-inactivated FBS, 1% v/v penicillin/streptomycin and filtered using a 100 μm cell strainer (Falcon, New York, USA). Finally, the cells from individual brain were seeded in respective T-75 flasks (BD Falcon, Schaffhausen, Switzerland). Medium was replaced every three days interval. After 11-12 days, the cells www.nature.com/scientificreports/ reached confluency and the flasks were shaken for 24 h at 200 rpm to remove microglia and oligodendrocyte progenitor cells. Following a recovery day, the remaining astrocytes on the adherent monolayer were detached using 0.1% w/v trypsin with 0.02% w/v of ethylenediaminetetraacetic acid (EDTA) and passaged in dishes or plates for further analysis.

Isolation of mitochondria from tissue and cultured cells.
Mitochondria were isolated from brain and heart as described before 49 with modifications. Briefly, isolated brain and heart were trypsinized for 20 min with shaking for every 5 min. www.nature.com/scientificreports/ antimycin A through the ports in assay cartridge to a final concentration of 1 μg/ml, 1 μM and 5 μM, respectively. This allowed determination of the basal level of oxygen consumption, level of ATP production and maximal respiration.  Images were taken and cells were fixed by 4% followed by co-immunofluorescence staining.
Statistical analysis. Data were presented as mean ± SEM (standard error of mean) of at least six independent experiments with at least three biological replicates. Comparisons were made by two tailed unpaired Student's t test. Differences were considered significant at P values < 0.05. Analysis was performed using the Microsoft Excel.