Ndufa4 Regulates the Proliferation and Apoptosis of Neurons via miR-145a-5p/Homer1/Ccnd2

The Dandy–Walker malformation (DWM) is characterized by neuron dysregulation in embryonic development; however, the regulatory mechanisms associated with it are unclear. This study aimed to investigate the role of NADH dehydrogenase 1 alpha subcomplex 4 (NDUFA4) in regulating downstream signaling cascades and neuronal proliferation and apoptosis. Ndufa4 overexpression promoted the proliferation of neurons and inhibited their apoptosis in vitro, which underwent reverse regulation by the Ndufa4 short hairpin RNAs. Ndufa4-knockout (KO) mice showed abnormal histological alterations in the brain tissue, in addition to impaired spatial learning capacity and exploratory activity. Ndufa4 depletion altered the microRNA expressional profiles of the cerebellum: Ndufa4 inhibited miR-145a-5p expression both in the cerebellum and neurons. miR-145a-5p inhibited the proliferation of neurons and promoted their apoptosis. Ndufa4 promoted and miR-145a-5p inhibited the expression of human homer protein homolog 1 and cyclin D2 in neurons. Thus, Ndufa4 promotes the proliferation of neurons and inhibits their apoptosis by inhibiting miR-145a-5p, which directly targets and inhibits the untranslated regions of Homer1 and Ccnd2 expression. Supplementary Information The online version contains supplementary material available at 10.1007/s12035-023-03239-5.


Introduction
The Dandy-Walker malformation (DWM), or Dandy-Walker syndrome, is a severe congenital posterior fossa anomaly characterized by vermis agenesis and hypoplasia, cystic enlargement of the fourth ventricle, meningeal anomalies, occipital skull defects, and hydrocephalus [1][2][3]. The incidence rate of DWM is between 1/200 and 1/35000 in different regions, with a mortality rate of > 12%, accounting for ~ 4% of hydrocephalus cases in the USA [1,2,4]. DWM originates during embryonic development of the cerebellum and fourth ventricle, and patients commonly present with non-neurologic comorbidities such as mental health and learning disability, endocrine and metabolic diseases, eye and ear disorders, circulatory system disorders, and even neoplasms [5]. Clinical treatment available at present mainly involves surgery, including ventriculoperitoneal shunting, cystoperitoneal shunting, and endoscopic third ventriculostomy, to alleviate hydrocephalus, posterior fossa symptoms, and other associated comorbidities [1,6]. DWM is a sporadic disorder and can be caused by atresia of the Luschka and Magendie foramina and developmental abnormalities of the rhombencephalon [1,7]. These result in vermian development arrest and fourth ventricle foramina fenestration failure, which is also associated with chromosomal abnormalities, congenital infections, and environmental exposure [1,7]. However, the molecular mechanisms underlying DWM development are still unclear.
NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 (NDUFA4) is a subunit of complex I in the mitochondrial respiratory chain, which is involved in the assembly and functioning of cytochrome c oxidase (COX, complex IV) during mitochondrial electron transport chain and aerobic metabolism [8][9][10]. NDUFA4 plays an essential role in mitochondrial function and energy metabolism, and NDUFA4 expression and mutations are involved in the development of various cellular processes and human disorders such as gastric cancer, clear-cell renal cell carcinoma, colorectal cancer, and diabetes mellitus [11][12][13][14]. For instance, the overexpression of NDUFA4 promotes the proliferation of gastric cancer cells and inhibits their apoptosis, which mediates long noncoding RNA macrophage migration inhibitory factor antisense RNA1-regulated pathogenesis of gastric cancer [12]. In addition, NDUFA4 expression promotes the proliferation, migration, invasion, and apoptosis of colorectal cancer cells [11]. NDUFA4 is also characterized as one interacting protein of DJ1 (Parkinson disease protein 7) linked with epigenetic regulation and apoptosis pathways in multiple sclerosis development [15]. Therefore, NDUFA4 is a critical regulator of cell proliferation and apoptosis associated with the pathogenesis of many human disorders.
NDUFA4 also has a crucial role in neuronal functions and the development of neurological diseases. Genetic association analysis of > 1500 clinical samples showed that NDUFA4 mutation is closely associated with the development of Alzheimer's disease (AD) [16]. Quantitative proteomic analysis of mitochondrial proteomes showed that NDUFA4 levels in the brain tissue of patients with AD were substantially altered compared with those in healthy individuals [17]. In addition, homozygous splice donor site mutations are characterized in patients with Leigh syndrome by neurological phenotypes such as dystonia, ataxia, bulbar dysfunction, and intermittent encephalopathy [18]. In our previous studies, array-based comparative genomic hybridization (array-CGH) based on three DWM fetuses showed that the chromosome 7p21.3 region containing NDUFA4 was critically associated with DWM [19]. Additionally, DWM fetuses frequently have NDUFA4 haploinsufficiency and copy number variations (CNVs) [20]. NDUFA4 was found to effectively enhance the growth and inhibit the apoptosis of neurons by promoting B-cell lymphoma-2 (BCL-2) expression and inhibiting caspase-3 cleavage and cytochrome c (Cyt C) expression and release [21]. Moreover, NDUFA4 expression was found to considerably modulate the regulatory effects of cyclosporin A on neuron growth and apoptosis [21]. All the above studies confirmed that NDUFA4 expression may participate in DWM pathogenesis. However, how NDUFA4 affects neuron growth and apoptosis in embryonic development remains unclear.
Epigenetic regulations mediated by microRNAs have an essential role in various cellular biological processes and pathogenic conditions [22,23]. However, little is known about the role of Ndufa4-regulated microRNAs. This study investigated the potential functions of Ndufa4-regulated microRNAs in neuronal proliferation and apoptosis, in addition to downstream target genes and signaling cascades, using a cellular neuronal differentiation model, which was established by treating pluripotent mouse embryonal carcinoma cells (P19 cell line) with all-trans-retinoid acid (RA). Our data would reveal novel clues regarding neuron growth and apoptosis in embryonic development.

Cell Culture and Modeling
We purchased P19 cells from the American Type Culture Collection. Cells were cultivated with Minimal Essential Medium-alpha modification (αMEM #12,571,048; Thermo Fishier Scientific, Waltham, MA, USA) containing 2 mM L-glutamine, 10% fetal bovine serum (Gibco), 100 units/mL of penicillin, and 100 μg/μL of streptomycin in a humidified 5% CO 2 atmosphere at 37 °C. Cell authentication was done using a short tandem repeat DNA profiling assay. Neural differentiation of cultured P19 cells was induced, as previously described [24]. Briefly, we cultured P19 cells under normal conditions in αMEM supplemented with 1 μM RA (#R4643; Sigma-Aldrich, St. Louis, MO, USA) for 4 d at 37 °C.

Cell Apoptosis
The P19-derived neurons were processed using the Annexin V-APC/7-aminoactinomycin D (7-AAD) Apoptosis Kit (#KA3808; Abnova, Taipei, Taiwan) according to the manufacturer's instructions. Briefly, ~ 1 × 10 5 P19-derived neurons were harvested by centrifugation, washed with PBS, resuspended in 100 µL of binding buffer, and incubated with gentle vortexing in 5 μL of Annexin V-APC and 5 μL of 7-AAD solution for 30 min at room temperature in the dark. Finally, the P19-derived neurons were washed again with PBS and the percentage of apoptotic P19derived neurons was computed by flow cytometry.

Ndufa4-Knockout (KO) Mice
The whole body knockout of Ndufa4 had its limitations, but conditional knockout mice were difficult to obtain due to its long construction cycle, so our experiment was still conducted with the whole body knockout mice.
The Ndufa4-KO mouse model established by the Cre/ LoxP system, as previously described, was purchased from Cyagen Biosciences (Guangzhou, China). The methods of generating Ndufa4-KO mice are shown as follows or in Supplemental Figure S1.
The mouse Ndufa4 gene (NM_010886.3) contains four exons. Exon 3 and 4 were selected as the constitutive KO region. Homologous arms containing upstream and downstream sequences of exon 3 and 4 were amplified by polymerase chain reaction (PCR) using the template DNA extracted from a BAC clone (4E12) to engineer the targeting vector. Then, the homologous arms were sequentially assembled to the 5' and 3' of a loxP-flanking PGK-neo cassette for positive selection. A diphtheria toxin A cassette for negative selection was located upstream of the 5' homologous arm. The targeting vector linearized with NotI was electroporated into C57BL/6 ES cells, followed by G418 antibiotic selection, PCR, and Southern blot validation. After correctly confirming targeted ES clones via Southern blotting, two clones were selected for blastocyst microinjection to produce the F0 generation. The F0 was bred with EIIa-cre mice from the Jackson Laboratory (strain #: 003,724) to delete the PGK-neo cassette. Homozygous F2 was acquired by mating the F1 heterozygotes. The mice were validated using PCR with the primers listed below. All mice were kept in a specific pathogen-free-grade atmosphere in a 12/12 h day/night cycle at 20 °C-26 °C. They were fed a standard diet after sterilization, with free access to drinking water. Finally, the mice were euthanized using intraperitoneal 4% chloral hydrate, and mouse brain tissue was collected surgically.
All experimental procedures using mice were approved by the Experimental Animal Care and Ethics Committee of the Forevergen Medical Laboratory Animal Center, Guangzhou, China (Approval no: IACUC-G16051).

Mouse Behavior Evaluation
The Morris water maze and open-field tests were used to assess the effects of Ndufa4 KO on mouse behaviors, as previously described [25,26]. The Morris water maze test analyzed the spatial learning capacity of Ndufa4-KO mice. Briefly, the mice were placed at one of four starting spots in a pool and their latency time (s), path length (mm), times on the platform, and time in target quadrants (s) were recorded using the EthoVision system version 2.3 (Noldus, the Netherlands). Next, after dark-adapting the mice for 25 min for the open-field test, they were placed in a 50 × 50 cm open-field arena. To evaluate their exploratory activities, the total distance traveled (mm), number of crossings, center distance (mm), and center time (s) was recorded using the EthoVision system.

Hematoxylin and Eosin (H&E) Staining
The histological alterations in the brain tissue were analyzed using H&E staining with a commercialized H&E staining kit (#ab245880; Abcam, Cambridge, UK) according to the manufacturer's instructions. Briefly, the brain sections were deparaffinized, hydrated in distilled water (DW), and incubated in Mayer's hematoxylin for 6 min at room temperature. Next, they were rinsed twice with DW, incubated in bluing reagent for 15 s at room temperature, and incubated again in Eosin Y solution (Modified Alcoholic; Abcam) for 3 min. Finally, these sections were rinsed and dehydrated with absolute alcohol and then cleared and mounted with synthetic resin.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labeling Assay (TUNEL) Staining
The apoptosis in brain tissue was analyzed using TUNEL staining with a commercialized TUNEL staining kit (#C1086; Beyotime Biotechnology, Shanghai, China) following the manufacturer's instructions. Briefly, the sections of brain tissue were deparaffinized and hydrated, then incubated with protease K (20 µg/ml) for 20 min. Next, these sections were rinsed thrice with PBS and incubated in the TUNEL solution for 60 min, avoiding light at 37 °C. Finally, the sections were rinsed thrice with PBS, followed by mounting with an antifade mounting medium (#P0128S; Beyotime Biotechnology). The signals were captured using a fluorescence microscope.

Transmission Electron Microscopy (TEM)
The subcellular structures of brain tissue were observed using TEM. Briefly, the fresh brain tissue was fixed in TEM fixative solution for 2 h at 4 °C, washed thrice with 0.1 M PBS for 15 min, and dehydrated for 15 min using a graded series of ethanol solution (50-100%), followed by 100% acetone for 15 min. Subsequently, the brain tissue was embedded in Spurr's EPON 812 Resin (#02,660-AB; Emicron, Egypt) by heating it at 60 °C for 48 h and then sliced into 60-nm-thick sections. Finally, these sections were stained with 2% alcoholsaturated uranium acetate solution for 15 min, incubated in lead citrate for 15 min, dried, and observed using TEM.

Transcriptome Profiling and Bioinformatics
Differentially expressed microRNAs and messenger RNA (mRNA) profiles were detected in mouse brain tissues caused by Ndufa4 KO using next-generation deep sequencing. Briefly, the total RNA samples from the brain tissue were isolated using the MagMAXmirVana Total RNA Isolation Kit (#A27828; Thermo Fishier Scientific) according to the manufacturer's instructions. Next, the samples were analyzed using a NanoDrop 2000 spectrophotometer (Thermo Fishier Scientific) to evaluate the RNA quality and concentration, separated using polyacrylamide gel electrophoresis (PAGE), and the isolated RNA bands were arranged in 18-30 nt using the Small RNA PAGE extraction kit (KA4434; Abnova) according to the manufacturer's instructions. Subsequently, ligation was performed with 3′-and 5′-adaptors and reverse transcription (RT)-PCR to construct a sequencing complementary DNA (cDNA) library. The quality of the cDNA library was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), which was then denatured to single-stranded DNA (ssDNA) and sequenced using the Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA) for 52 cycles. Next, the reads that were obtained were filtered using SolexaSolexa CHASTITY to select clean reads, which were used for subsequent adaptor trimming and alignment with the miRbase database. Tag counts were applied to evaluate the expressional levels of microRNA or mRNA. Significantly different microRNA and mRNA expression was defined as a fold-change (FC) of > 1.5 and P < 0.05. The microRNA target genes and their interaction networks were predicted using Targetscan software release 3.1 (www. targe tscan. org/ mamm_ 31/) [27]. Lastly, hierarchical clustering of differentially expressed microRNAs or mRNAs was completed using the R software.

Western Blotting
Total protein samples were prepared from cultured P19-derived neurons or brain tissue using a radio immunoprecipitation assay buffer (#R0010; Solarbio Life Science, Beijing, China) according to the manufacturer's instructions. Bicinchoninic acid assay was used to measure the protein concentration. Approximately 30 μg of total protein was boiled in loading buffer for 5 min at 100 °C, separated using 10-12% sodium dodecyl sulfate (SDS)-PAGE, and transferred onto polyvinylidene dif luoride (PVDF) membranes (Merck Millipore, Burlington, MA, USA). Subsequently, the PVDF membranes were incubated in 5% bovine serum albumin for 2-3 h at room temperature, incubated overnight in diluted primary antibodies at 4 °C, washed with Trisbuffered saline with 0.1% Tween® 20 detergent, and incubated again in horseradish peroxidase-conjugated secondary antibodies for 1-2 h at room temperature. Next, protein bands were developed using enhanced electrochemiluminescence substrates (#32,106; Thermo Fishier Scientific), and protein band intensities were scanned to compare protein abundance.

Ndufa4 Depletion Causes Abnormal Histologic and Cellular Alterations in the Mouse Brain Tissue
To study the role of Ndufa4 in neurons in vivo, Ndufa4-KO mice were established using the Cre/LoxP system. Real time-qPCR showed that the Ndufa4 mRNA levels in the cortex, hippocampus, cerebellum, and whole brain were significantly lower in the Ndufa4-KO mice compared with wild-type (WT) mice ( Fig. 1A and Supplemental Figure S2 A). Western blotting results showed that Ndufa4 KO significantly decreased NDUFA4 protein levels in the cortex, hippocampus, cerebellum, and whole brain (Fig. 1B). H&E staining showed normal histological structures of the cerebellum in WT mice, including a regular cell lining of the cerebellum molecular layer, granular cell layer, and Purkinje cells (Fig. 1C). However, compared with WT mice, Ndufa4-KO mice showed enhanced basophilia in the Purkinje cells and substantial white calcification nodules (Fig. 1C). In addition, TEM analysis showed massive neuronal edema and swelling in the cortex, in addition to considerable plasma membrane destruction, decreased organelle density, severe mitochondria damage, and rough endoplasmic reticulum extension (Fig. 1D). Similar neuronal edema, mitochondria damage, and subcellular organelle destruction was observed in the hippocampus and cerebellum of Ndufa4-KO mice compared with WT mice (Fig. 1D). These results indicated that Ndufa4 expression is critical for maintaining normal histological structures and mitochondrial functions in the mouse cortex, hippocampus, and cerebellum.

Ndufa4 Depletion Impairs the Spatial Learning Capacity and Exploratory Activity in Mice
The body, whole brain, and cerebellum weights of Ndufa4-KO mice were slightly lower than those of WT mice ( Fig. 2A). To gain a better understanding of the physiological functions of Ndufa4, the behavioral alterations of Ndufa4-KO mice were compared with those of WT mice. The Morris water maze test showed that the latency time, path length, times on the platform, and time in quadrants were lower for Ndufa4-KO mice than for WT mice, indicating that Ndufa4 depletion impairs the spatial learning capacity (Fig. 2B), although it could not completely rule out the influence of loss of respiratory chain components caused by the whole body knockout. Similarly, the open-field test showed decreased total distance traveled, number of crossings, center distance, and center time in Ndufa4-KO mice, indicating poor exploratory activity (Fig. 2C). Furthermore, TUNEL staining showed increased apoptosis in the brain tissues of Ndufa4-KO mice compared with WT mice (Fig. 2D). In addition, western blotting showed that NDUFA4, Bcl-2, and Bcl-XL levels in the cerebellum considerably decreased in Ndufa4-KO mice compared with WT mice (Fig. 2E). In contrast, Ndufa4 KO significantly increased Bax, cleaved caspase-3, and cleaved caspase-9 levels were observed in in the cerebellum of Ndufa4-KO mice (Fig. 2E). These results showed that Ndufa4 KO causes apoptosis of the neurons in the cerebellum and substantially impairs mouse behaviors.

Ndufa4 Overexpression Promotes Neuronal Proliferation and Inhibits Neuron Apoptosis, Whereas Ndufa4 KO Has the Opposite Effect in P19-Derived Neurons
To evaluate the effects of Ndufa4 expression on neuron functions, P19 cells were differentiated into neurons by treating them with RA as introduced in the "Materials and Methods" section. Subsequently, the Ndufa4 expression in induced neurons was altered by transfection with shNdufa4 or LV003-Ndufa4 (Ndufa4). Real-time qPCR showed that shNdufa4 was considerably inhibited, but LV0003-Ndufa4 vectors increased Ndufa4 mRNA expression in P19-derived neurons ( Fig. 3A and Supplemental Figure S2 B). The MTS assay showed that shNdufa4 was considerably decreased, but Ndufa4 overexpression considerably promoted the proliferation of P19derived neurons compared with the control group (Fig. 3B). Conversely, shNdufa4 was considerably promoted, but Ndufa4 overexpression considerably inhibited neuron apoptosis in P19derived neurons (Fig. 3C). Consistent with this result, western blotting showed that shNdufa4 was subbstantially decreased, but Ndufa4 overexpression substantially increased NDUFA4, Bcl-2, and Bcl-XL levels in P19-derived neurons (Fig. 3D). In addition, shNdufa4 was substantially upregulated, but Ndufa4 overexpression substantially downregulated the Bax, cleaved caspase-3, and cleaved caspase-9 levels in P19-derived neurons (Fig. 3D). These results indicated that Ndufa4 overexpression promoted the proliferation and inhibited the apoptosis of P19derived neurons. After knockout or overexpression of Ndufa4 in mouse neural stem cell line NE-4C, cell proliferation is also inhibited or promoted. (Supplemental Figure S3).

Ndufa4 Modulates microRNA Expression and Inhibits miR-212-5p and miR-145a-5p in the Cerebellum and Neurons
To determine the molecular mechanism underlying Ndufa4 functions, a large-scale identification of differentially expressed microRNAs in the cerebellum was conducted between WT mice and Ndufa4-KO mice using next-generation deep sequencing, and the potential mechanism of Ndufa4 in the cortex and hippocampus will be shown in a future study. In all, 50 microRNAs were differentially expressed between the WT and Ndufa4-KO mice cerebellum. Of these, 40 were upregulated and 10 downregulated in Ndufa4-KO mice (FC > = 1.5; Fig. 4A). Real-time qPCR and droplet digital PCR showed that mmu-miR-212-5p, mmu-miR-139a-5p, and mmu-miR-145a-5p expression considerably increased; however, mmu-miR-205a-5p and mmu-miR-196-5p expression considerably decreased in the cerebellum of Ndufa4-KO mice ( Fig. 4B and Supplemental Figure S4 A). In addition, shNdufa4 considerably increased but Ndufa4 overexpression inhibited mmu-miR-212-5p, mmu-miR-145a-5p, and mmu-miR-196-5p expression in P19-derived neurons ( Fig. 4C and Supplemental Figure S4 B). These results indicated that Ndufa4 regulated neuronal functions and mouse brain development and behaviors by inhibiting downstream microRNAs such as mmu-miR-212-5p and mmu-miR-145a-5p.

miR-145a-5p Inhibits the Proliferation of Neurons and Promotes Their Apoptosis
For cellular function analysis of miR-145a-5p, the miR-145a-5p expression in P19-derived neurons was altered by transfection with miR-145a-5p inhibitors or mimics. Real time-qPCR and droplet digital PCR confirmed that miR-145a-5p inhibitors considerably downregulated but miR-145a-5p mimics upregulated the miR-145a-5p expression in neurons (Fig. 5A and Supplemental Figure S4 C). Alterations in miR-145a-5p expression did not induce any change in the Ndufa4 mRNA content in P19-derived neurons (Fig. 5A and Supplemental Figure S2 C). The MTS assay showed that miR-145a-5p inhibitors substantially promoted but miR-145a-5p mimics substantially inhibited the proliferation of P19-derived neurons compared with negative controls (Fig. 5B). Flow cytometry showed that miR-145a-5p mimics substantially increased but miR-145a-5p inhibitors did not affect the percentage of apoptotic P19-derived neurons compared with the control group (Fig. 5C).

Discussion
Neuronal function dysregulation and the resultant vermian developmental arrest and fourth ventricle foramina fenestration failure are major clinical manifestations in patients with DWM [1,7]. However, the molecular mechanisms underlying the regulation of neuronal functional alterations, such as proliferation and apoptosis, during the pathogenesis of DWM are unclear. NDUFA4 is an essential subunit of the mitochondrial respiratory chain associated with neuronal functions and various neurological disorders [16,19]. Our previous studies showed that NDUFA4 was closely implicated in the development of DWM [19][20][21]. This study used the P19-derived cellular neuron model to show that Ndufa4 effectively promotes the proliferation of neurons and inhibits their apoptosis. Ndufa4-KO substantially impaired the histological structures and cellular functions of the brain, inducing considerable impairment in the spatial learning capacity and exploratory activity of mice. In addition, Ndufa4 inhibited miR-145a-5p expression, which in turn inhibited the proliferation of neurons and promoted their apoptosis. The roles of Ndufa4 and miR-145a-5p in regulating neuronal functions are mediated by their targeting of Homer1 and Ccnd2 through the direct binding of miR-145a-5p to Homer1 and Ccnd2 3′ UTRs. Overall, our results provided a novel insight into the neuronal function regulation mediated by the Ndufa4/miR-145a5p/ target gene axis.
The pathogenesis of DWM is closely associated with mutation and expressional alterations in various functional genes, such as forkhead transcription factor (FOXC1), fibroblast growth factor 17 (FGF17), and Ndufa4 [19,20,29]. However, their pathogenic roles in DWM development are still unclear owing to a lack of extensive functional investigations. The present study elucidated the neuronregulating roles of NDUFA4 by silencing or overexpressing Ndufa4 in neurons induced from P19 cells using RA treatment, which showed substantial proliferation-promoting and apoptosis-inhibiting functions of NDUFA4 in neurons. Using Ndufa4-KO mice, the contribution of NDUFA4 to maintaining structural homeostasis in the brain, spatial learning capacity, and exploratory activity was elucidated. Direct evidence of alterations in NDUFA4 expression alterations that cause considerable behavioral abnormalities in the mouse model was found. These cellular and animal results convincingly established Ndufa4 as a critical regulator of neuronal development, and behavior.
Ndufa4 mutation and expression was also implicated in other neurological disorders, such as AD, and neurological symptoms in Leigh syndrome [16,18]. The mediating roles of NDUFA4-regulated neuronal proliferation and apoptosis in the pathogenesis of these neurological disorders should be investigated further.
The important biological functions of microRNAs are mediated by their inhibition of functional gene expression, mainly via binding with 3′ UTRs of target genes. For instance, the pathogenic role of miR-145a-5p in nasopharyngeal carcinoma is mediated by its inhibition of NUAK Family SNF1-like Kinase 1 (NUAK1) expression [36]. In this study, next-generation deep sequencing of differentially expressed microRNAs in the cerebellum of Ndufa4-KO mice was performed to characterize the miR-145a-5p target genes in neurons, combined with bioinformatic prediction. Homer1, a postsynaptic density protein, is a key regulator of neuronal synaptic activity and neurological disease pathogenesis [37,38]. In contrast, Ccnd2 is a member of the highly conserved cyclin family that regulates cell cycle progression, cell proliferation, and apoptosis in distinct contexts whose expression is considerably regulated by microRNAs [39,40]. In this study, miR-145a-5p could effectively inhibit Homer1 and Ccnd2 expression in neurons, which was significantly promoted by Ndufa4 in neurons. In addition, Ndufa4 3′ UTR overexpression was found to considerably promote Homer1 and Ccnd2 expression in neurons. In addition, miR-145a-5p was found to directly bind to Homer1 and Ccnd2 3′ UTRs. These results indicated that Homer1 and Ccnd2 expression was considerably regulated by Ndufa4/miR-145a-5p associated with neuronal functioning.
However, our research has limitations. First, the whole body knockout of Ndufa4 cannot completely avoid its influence on the behavioral phenotype of mice as a component of the respiratory chain. Regional specific deletion may be more appropriate, but at present, we are unable to obtain these mice. Second, the detection of O 2 consumption rate and ATP generation in neurons may provide more powerful evidence for miR-145 mediated effects, which will be further explored in our future research.
In summary, the mitochondrial respiratory chain component protein NDUFA4 was found to promote the proliferation of neurons and inhibit their apoptosis by inhibiting miR-145a-5p to enhance Homer1 and Ccnd2 expression. The inhibitory effects of Ndufa4 on miR-145a-5p functioning could be mediated by the direct association of the Ndufa4 3′ UTR with miR-145a-5p. These findings reveal novel clues regarding the neuron growth and apoptosis in embryonic development and other neurological disorders.
Code Availability Not applicable.

Declarations
Ethics Approval The animal study was reviewed and approved by the Experimental Animal Ethics Committee of Forevergen Medical Laboratory Animal Center (Approval No: IACUC-G16051), Guangzhou, China.

Consent to Participate Not applicable.
Consent for Publication Not applicable.

Competing Interests
The authors declare no competing interests.
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