Downregulated miR-18b-5p triggers apoptosis by inhibition of calcium signaling and neuronal cell differentiation in transgenic SOD1 (G93A) mice and SOD1 (G17S and G86S) ALS patients

Background MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level and are key modulators in neurodegenerative diseases. Overexpressed miRNAs play an important role in ALS; however, the pathogenic mechanisms of deregulated miRNAs are still unclear. Methods We aimed to assess the dysfunction of RNAs or miRNAs in fALS (SOD1 mutations). We compared the RNA-seq of subcellular fractions in NSC-34 WT (hSOD1) and MT (hSOD1 (G93A)) cells to find altered RNAs or miRNAs. We identified that Hif1α and Mef2c were upregulated, and Mctp1 and Rarb were downregulated in the cytoplasm of NSC-34 MT cells. Results SOD1 mutations decreased the level of miR-18b-5p. Induced Hif1α which is the target for miR-18b increased Mef2c expression as a transcription factor. Mef2c upregulated miR-206 as a transcription factor. Inhibition of Mctp1 and Rarb which are targets of miR-206 induces intracellular Ca2+ levels and reduces cell differentiation, respectively. We confirmed that miR-18b-5p pathway was also observed in G93A Tg, fALS (G86S) patient, and iPSC-derived motor neurons from fALS (G17S) patient. Conclusions Our data indicate that SOD1 mutation decreases miR-18b-5p, which sequentially regulates Hif1α, Mef2c, miR-206, Mctp1 and Rarb in fALS-linked SOD1 mutation. These results provide new insights into the downregulation of miR-18b-5p dependent pathogenic mechanisms of ALS.

Calcium signaling is a ubiquitous system that is involved in the regulation of cellular processes such as cell proliferation and apoptosis [29,30]. Intracellular Ca 2+ levels are tightly controlled by transporters, and binding proteins [30]. The multiple-C2 domain transmembrane protein 1 (Mctp1), which has Ca 2+ binding-affinity C2 domains, is essential for neuronal calcium signaling [30,31]. High cellular Ca 2+ concentration leads to apoptosis through mitochondrial dysfunction [29]. According to a recent report, intracellular Ca 2+ levels are not only increased, but Ca 2+ buffering is also perturbed following SOD1 mutation in ALS [32].
Cell growth and differentiation are regulated by retinoids (vitamin A derivatives) and play a prominent role in neuronal cells [33]. Retinoic acid (RA) is a biologically active form of vitamin A and it regulates cell proliferation and differentiation [33]. During this process, retinoic acid receptor beta (Rarb), a transcriptional co-regulator with retinoic X receptor (RXR) mediates RA response [34]. Overexpression of mutated human SOD1 in MNs, such as NSC-34 cells, proves impaired cell differentiation and induced apoptosis [35,36]. Dysregulation of calcium signaling and neuronal cell differentiation are related to apoptosis and are representative events in ALS pathogenesis [36][37][38][39][40].
In this study, we performed whole transcriptome analysis to explain the role of SOD1 mutation by studying the subcellular fractionation of NSC-34 hSOD1 (wtNSC-34) and hSOD1 (G93A) (mtNSC-34) cells. With respect to our RNA-seq results, we found several altered RNAs [hypoxia inducible factor 1 alpha (Hif1α), myocyte specific enhancer factor 2c (Mef2c), Mctp1, and Rarb] in mtNSC-34 cells. The RNA levels of Hif1α and Mef2c were upregulated in the nucleus and the cytoplasm of mtNSC-34 cells. Specifically, the cytoplasmic RNAs of Hif1α and Mef2c were higher in number than nuclear RNAs in mtNSC-34 cells. Furthermore, Mctp1 and Rarb transcripts were highly expressed in the nucleus, but were decreased in the cytoplasm of mtNSC-34 cells. For the reason that Hif1α, Mef2c, Mctp1, and Rarb were observed to be different in the cytoplasm of mtNSC-34 cells, we hypothesized that these genes were posttranscriptionally regulated in mtNSC-34 cells. To identify the post-transcriptional regulation of Hif1α, Mef2c, Mctp1, and Rarb, we found that miR-18b-5p was involved in the regulation of Hif1α, and miR-206 regulated both Mctp1 and Rarb. To determine whether or not miR-18b-5p is related to SOD1 mutation in ALS, we validated the expression of miR-18b-5p, miR-206, Hif1α, Mef2c, Mctp1, and Rarb in vitro and in vivo. Our results indicate that a new molecular pathway for miR-18b-5p, which sequentially regulates Hif1α, Mef2c, miR-206, Mctp1, and Rarb is involved in the pathogenic mechanisms of ALS-linked SOD1 mutations.

Animals
Animal studies were carried out in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines of Seoul National University for the care and use of laboratory animals. Transgenic mice expressing the human G93A-mutated SOD1 gene (B6SJL-Tg (SOD1-G93A) 1 Gur/J; Jackson Laboratory, Bar Harbor, Me, USA) were used in this study. WT and SOD1-G93A mice used for biochemical analyses were sacrificed 120 days after birth.

Annexin V and PI analysis by flowcytometry
NSCs were seeded in 6-well tissue culture plates. For using Annexin-V-FITC and PI Apoptosis Detection Kit (556,547, BD Bioscience, Eugene, NJ, USA), the adherent NSCs were detached with TripleExpress (12605-010, GIBCO, NY 14072 USA). The culture medium was then added to inactivate trypsin. The supernatant was removed after centrifuging for 5 min at 1500×g. and cells were stained with Annexin V-FITC and PI according to the manufacturer's instructions. The cells were analyzed immediately after staining using a FACSCalibur (BD Biosciences, San Jose, CA). For each measurement, at least 20,000 cells were counted. Fluorescence was evaluated using the green or red channel, and the data were analyzed using Flowwing Software (Version 2.5.1, Unversity of Turku, Filand).

Intracellular Ca 2+ assay
The day before the experiment, plate the cells overnight in growth medium using 4 × 10 4 to 8 × 10 4 cells per well at a plating volume of 100 μl per well for 96-well plates. After 48 h, FLUOFORTE Dye-Loading Solution added to each well and incubated the cell plates for 45 min at 37°C and 15 min at room temperature. Then, fluorescence was measured at 490/525 nm using a fluorescence plate reader. The changes in intracellular calcium levels of each group were measured by quantifying the fold change of the fluorescence level of Fluo-4 with respect to the basal level.

Lactate dehydrogenase (LDH) release assay
Cell culture medium was collected and briefly centrifuged. The supernatants were transferred into wells in 96-well plates. Equal amounts of lactate dehydrogenase assay substrate (MAK066, SIGMA, Burlington MA USA), enzyme and dye solution were mixed. A Half volume of the above mixture was added to one volume of medium supernatant. After incubating at room temperature for 30 min, the reaction was terminated by the addition of 1/10 volume of 1 N HCl to each well. Spectrophotometrical absorbance was measured at a wavelength of 490 nm and reference wavelength of 690 nm.

Subcellular fractionation
wt and mtNSC-34 cells were grown in a 10 cm dish and they were harvested in 450 ul of ice-cold buffer A (10 mM HEPES at pH 7.9, 10 mM KCl, 1 mM dithiothreitol [DTT], and 0.1 mM EDTA at pH 8.0). NSC-34 WT and MT cells dispersed by pipetting and incubated for 25 min on ice. Then 5 μl of 10% NP-40 was added, and cells were incubated for 2 min on ice. The nuclei were precipitated by centrifugation at 5000 rpm for 3 min at 4°C. The supernatant was taken as the cytoplsamic fraction.

RNA-seq
Three sets of wt and mtNSC-34 cells were grown and harvested with each set from a separate passage of single cell line. Following subcellular fractionation, transcriptomes of 12 samples were analyzed by RNA-seq (Macrogen Inc.), the Illumina standard kit was used according to the manufacturer's protocol. Briefly, 3 μg of each total RNA sample was used for polyA mRNA selection using streptavidin-coated magnetic beads, followed by thermal mRNA fragmentation. The fragmented mRNA was subjected to cDNA synthesis using reverse transcriptase (SuperScript II) and random primers. The cDNA was further converted into double stranded cDNA and, after an end repair process (Klenow fragment, T4 polynucleotide kinase and T4polymerase), was finally ligated to Illumina paired end (PE) adaptors. Size selection was performed using a 2% agarose gel, generating cDNA libraries ranging in size from 200 to 250 bp. Finally, the libraries were enriched using 10 cycles of PCR and purified by the QIAquick PCR purification kit (28,106, Qiagen, PL Venlo Netherlands). The enriched libraries were diluted with Elution Buffer to a final concentration of 10 nM. Each library was run at a concentration of 8 pM on one Genome Analyzer (GAIIx) lane using 53 bp sequencing. Reads were then processed and aligned to the mouse genome UCSC build mm9 using GSNAP. The unit of measurement is Reads Per Kilobase of exon per Million fragments mapped (RPKM).

Differential expression analysis
The expressed transcripts was quantified using Kallisto. DESeq2 and edgeR were used to identify transcripts that were differentially expressed between wt and mtNSC-34 cells. Different expression level of each transcript was defined as those that satisfied 2 criteria: |log 2 (foldchange)| > 1 and p < 0.01 after the Benjamini-Hochberg correction in DEseq2 and edgeR.

Reverse transcription quantitative PCR (RT-qPCR)
Total RNA was extracted from wt and mtNSC-34 cells by TRIzol reagent (5741, MRC, Cincinnati OH USA). RNA was measured in a spectrophotometer at 260-nm absorbance. RNA analysis was conducted as follows. Fifty nanograms of RNA were used as a template for quantitative RT-PCR amplification, using SYBR Green Real-time PCR Master Mix (QPK201, Toyobo, Osaka Japan). Primers were standardized in the linear range of cycle before the onset of the plateau. Primer sequences are given in supplementary table 3 and 4. Mouse and human GAPDH was used as an internal control. Twostep PCR thermal cycling for DNA amplification and real-time data acquisition were performed with an ABI StepOnePlus™ Real-Time PCR System using the following cycle conditions: 95°C for 1 min × 1 cycle, and 95°C for 15 s, followed by 62°C for 1 min × 50 cycles. Fluorescence data were analyzed by the ABI StepOnePlus software and expressed as C t the number of cycles needed to generate a fluorescent signal above a predefined threshold. The ABI StepOnePlus software set baseline and threshold values. Expression of each gene was normalized to Gapdh and expression of each miRNA was normalized to U6. The fold change in mRNA and miR-NAs expression versus controls calculated using the 2 (−ΔΔCT) method. For miRNAs RT-qPCR, 50 ng of total RNA was reverse transcribed, using GenoExplore micro-RNA RT-qPCR Kit (2001, Geno Sensor Corporation, Tempe, Arizona 85,282 USA), and subsequently quantified using specific primers (GenoExplorer microRNA RT-qPCR primer sets (2003, Geno Sensor Corporation, Tempe, Arizona 85,282 USA)) for U6, miR-18b-5p and miR-206 (mouse and human).

Human spinal cord samples
Post mortem spinal cord specimens from six normal controls and one with fALS (G86S) were used (supplementary Table S6). Control spinal cord samples were obtained from The Netherlands Brain Bank and the guidelines by The Netherlands Brain Bank were followed. Post mortem spinal cord specimens from fALS (G86S) spinal cord and blood samples from fALS (G17S) were analyzed with Institutional permission under Review Board in Seoul National University Hospital.

Generation of neural stem cell from iPSC
Colonies were detached by 2 mg/ml dispase and transferred in embryoid body (EB) medium that contains Essential 6 Medium supplemented with 15% knockout SR (10828-028, Gibco, Grand Island NY USA), 50 U/ml of penicillin, 50 μg/ml streptomycin (15140-122, GIBCO Grand Island NY USA) to 60-mm incoated bacterial plate at 37 for 5-7 days with change of medium every single day. And then, formed EBs transferred to Cell Start coated-35-mm culture dish. The culture method has been used previously [43]. When EBs attached to dish after 2-3 days, changed 0.5% N2 in DMEM/F12 supplemented with 1% nonessential amino acids, 50 U/ ml of penicillin, 50μg/ml streptomycin and 0.1 mM 2mercaptoethanol to the same based-medium plus 1% N2 supplement and 40 ng/ml b-fibroblast growth factor every other day until neural structures appeared. These neural structures were mechanically isolated and cultured floating in the medium and then became spheres. These spheres were fragmented mechanically and cultured onto Cell start-coated culture dished for 1 day and treated accutase (A11105-01, Gibco, Grand Island NY USA) at 37°C incubator or 1 h. Neural stem cells were cultured in DMEM/F12 supplemented with 1% nonessential amino acids, 50 U/ml of penicillin, 50 μg/ml streptomycin and 0.1 mM 2-mercaptoethanol plus 0.5% N2 supplement and 40 ng/ml b-fibroblast growth factor onto the Cell Start-coated plates.

Confocal microscopy
Immunofluorescence staining and confocal microscopy was used to determine mouse anti-Chat (AB144P, Chemicon, Tumecula CA USA). Images were analyzed using a spinning disk confocal microscope (Leica, Buffalo Grove IL USA). Deconvolution and 3-dimensional construction of the confocal image was performed by AQI-X-COMBO-CWF program (Media cybernetics Inc., Rockville, MD, USA). Control experiments were performed in the absence of primary antibody or in the presence of blocking peptide.

Statistical analysis
The data are presented as the mean ± standard error of the mean (SEM). Data analysis was performed by Student's t test or one-way ANOVAs followed by Mann-Whitney and Kruskal-Wallis tests. Differences were considered statistically significant when p < 0.05.

SOD1 mutation (G93A) induces apoptosis by aberrant gene expression
To study the mechanism of RNA biogenesis by SOD1 mutation, we performed transcriptome analysis to identify RNA processing variation via subcellular fractionized RNAs in NSC-34 hSOD1 (wtNSC-34) and hSOD1 (G93A) (mtNSC-34) stable cell lines (Fig. 1a). By performing comparative RNA-seq analysis between the nucleus and cytoplasm of WT and mtNSC-34 cells, we identified significant changes in Mctp1 and Rarb expression (Fig. 1a). The heat map shows that Mctp1 and Rarb were upregulated in the nucleus, but greatly downregulated in the cytoplasm of mtNSC-34 cells (Fig. 1a). To validate Mctp1and Rarb transcripts, we carried out reverse transcriptase PCR (RT-PCR) analysis and confirmed the presence of Mctp1 and Rarb mRNAs (Fig.  1b). To understand how Mctp1 and Rarb transcripts are regulated in mtNSC-34 cells, we performed revers transcription quantitative PCR (RT-qPCR) analysis in WT and mtNSC-34 cells (Fig. 1c). We found that Mctp1 and Rarb mRNAs were low in mtNSC-34 cells (Fig. 1c). These results led us to assume that Mctp1 and Rarb transcripts might be post-transcriptionally regulated or deficiently transported in the cytoplasm of mtNSC-34 cells. For the reason that miRNAs are one of the most representative the post-transcriptional regulators [45][46][47], we focused on the post-transcriptional regulation and identified that Mctp1 and Rarb mRNAs are targeted by miR-206 (Additional file 1: Fig. S1A). Furthermore, we discovered that miR-206 was remarkably upregulated in mtNSC-34 cells (Fig. 1d). Moreover, we investigated whether or not Mctp1 and Rarb were related to calcium signaling and neuronal differentiation, respectively, and measured intracellular Ca 2+ levels and total neurite length in WT and mtNSC-34 cells (Fig. 1e and f). As expected, the intracellular Ca 2+ levels increased, and the neurite outgrowth decreased significantly in mtNSC-34 cells with the aggregation of SOD1 (G93A) (Fig. 1e and  f). These results showed that the downregulated Mctp1 and Rarb could stimulate the alteration of calcium signaling and cell differentiation in mtNSC-34 cells, respectively. From the RNA-seq results, we also identified that Hif1α and Mef2c, which are regulated by Hif1α [48], increased in the nucleus and cytoplasm of mtNSC-34 cells (Fig. 1a). Interestingly, Mef2c has been reported to regulate miR-206 as a transcription factor [49]. We validated that Hif1α and Mef2c expression was significantly elevated in mtNSC-34 cells (Fig. 2a and b). From these results, we also hypothesized that an unknown target miRNA of Hif1α might be downregulated, and that the upregulated Hif1α could upregulate Mef2c expression serially. We also identified that the miR-18b-5p was target of Hif1α mRNAs (Additional file 1: Fig. S1B) [50]. RT-qPCR analysis confirmed that miR-18b-5p was significantly reduced in mtNSC-34 cells (Fig. 2e). We also confirmed that only mtSOD1 (G93A) were associated with miR-18b-5p, Hif1α, Mef2c, Mctp1 and Rarb in NSC-34 (control, wtSOD1, and mtSOD1) stable cell lines (Fig. 2a-e). Owing to the fact that SOD1 mutations cause apoptosis [51], we measured the Significantly different at *, p < 0.05; **, p < 0.005. The experiments were replicated 3 times levels of Bax and Bcl2 as pro-and anti-apoptotic markers. Bax expression was increased, while Bcl2 expression was decreased in mtNSC-34 cells (Fig. 2a, b, and g). We then measured the amount of lactate dehydrogenase (LDH) released to observe apoptosis in NSC-34 cell lines. As expected, LDH release increased in mtNSC-34 cells (Fig. 2f). These results suggested that downregulated miR-18b-5p might sequentially control Hif1α, Mef2c, miR-206, Mctp1, and Rarb expression in SOD1 mutation.

Hif1α directly regulates Mef2c expression and apoptosis
As we had mentioned previously in this article that decreased miR-18b-5p upregulated Hif1α, which subsequently increased Mef2c, we confirm that Hif1α regulated Mef2c. We applied RNAi to decrease Hif1α, and observed the regulation of Mef2c, miR-206, Mctp1, and Rarb in mtNSC-34 cells (Additional file 3: Fig. S3). The expression of Mef2c was reduced by siHif1α in mtNSC-34 cells (Additional file 3: Fig. S3A-C). Mctp1 and Rarb expressions were also increased (Additional file 3: Fig. S3A, D, and E). To prove that Hif1α restored apoptosis, we observed the downregulation of Bax and upregulation of Bcl2 by siHif1α in mtNSC-34 cells (Additional file 3: Fig. S3A, F, and G). We also indirectly identified that Mef2c, inhibited by siHif1α, decreased miR-206 (Additional file 3: Fig.   S3H). In order to observe the reduced apoptosis, we applied the LDH assay and, as we expected, LDH release was reduced by siHif1α in mtNSC-34 cells (Additional file 3: Fig. S3I). These results suggested that Hif1α directly regulated Mef2c and contributed to apoptosis.

miR-206 not only controls post-transcriptional regulation of both Mctp1 and Rarb, but also induces apoptosis
To further investigate the roles of miR-206, we carried out a luciferase reporter assay using the 3′ UTR of Mctp1 and Rarb. Mctp1 was significantly downregulated and intracellular Ca 2+ levels were increased by overexpressed miR-206 (Fig. 4a-d). Rarb levels were also decreased by miR-206 ( Fig. 4d and e). The posttranscriptionally downregulation of Rarb by miR-206 also caused the neuronal differentiation (Fig. 4e). We then observed that overexpressed miR-206 induced apoptosis, because Bax expression was upregulated, while Bcl2 expression was downregulated by miR-206 ( Fig. 4f and g). Overexpression of miR-206 was confirmed by RT-qPCR analysis and miR-206 considerably induced LDH release ( Fig. 4h and i). We opted to have it reconfirmed by performing flow cytometry analysis of mNSCs overexpressing miR-206 ectopically. As a result, apoptosis was markedly induced (Fig. 4j) Fig. S4B and C). In addition, the LDH assay using anti-206 (anti-miR-206) in mtNSC-34 cells showed that anti-206 (anti-miR-206) restored cell death (Additional file 4: Fig. S4F). These findings suggested that miR-206 directly regulates Mctp1 and Rarb, and then induces apoptosis.
To confirm that miR-206 posttranscriptionally regulates Mctp1 and Rarb, we deleted miR-206 binding sites from 3'UTR of Mctp1 and Rarb. We carried out a luciferase reporter assay using the mutation 3′ UTR of Mctp1 and Rarb. It did not show any significant change in miR-206 expression (Additional file 4: Fig. S4H and I).
Altogether, these gain-and loss-of-function studies concerning Mctp1 and Rarb imply that calcium signaling and neuronal cell differentiation are involved in important pathogenic mechanisms associated to SOD1 (G93A) mutations.

Downregulation of the miR-18b-5p signaling pathway is involved in diverse SOD1 mutations and in vivo studies
To identify the pivotal role of the miR-18b-5p pathway in other SOD1 mutations, we ectopically caused the overexpression of SOD1 (G85R and D90A) in contNSC-34 cells. Transfected G85R and D90A demonstrated that Hif1α and Mef2c expression was increased and Mctp1 and Rarb expression was decreased (Additional file 6: Fig. S6A-C). G85R and D90A also enhanced apoptosis by difference the expression of Bax and Bcl2 (Additional file 6: Fig. S6A and D). RT-qPCR analysis results showed that miR-18b-5p was downregulated and miR-206 was upregulated by G85R and D90A (Additional file 6: Fig.  S6E-G). Furthermore, we sought to verify the miR-18b-5p pathway in G93A Tg and fALS (G86S) patient. We first compared the miR-18b-5p pathway in the spinal cords of WT and G93A Tg. The expression of Hif1α and Mef2c was highly increased, yet that of Mctp1 and Rarb was significantly decreased in G93A Tg ( Fig. 6a and b). Increased Bax and decreased Bcl2 indicated the apoptosis occurring in G93A Tg ( Fig. 6a and b). RT-qPCR results also showed that miR-18b-5p was downregulated and miR-206 was upregulated in G93A Tg (Fig. 6c). Further, we confirmed the miR-18b-5p pathway in the spinal cord of the G86S patient. Hif1α and Mef2c expression significantly increased, while the expression of Mctp1 and Rarb decreased in the G86S patient ( Fig. 6d and e). The expression of Bax and Bcl2 was induced and reduced respectively in the G86S patient ( Fig. 6d and e). RT-qPCR analysis showed decreased miR-18-5p and increased miR-206 in the G86S patient (Fig. 6f). These results strongly demonstrated that the miR-18b-5p pathway was generally involved in SOD1 mutation. In the G86S patient study, we could not compare the normal lumber to G86S patient lumber tissues (Fig. 6d). However, we clearly confirmed miR-18b-5p pathway in cervical tissues. To support that miR-18b-5p pathway play a pivotal role in fALS patient motor neurons, we developed hiPSCs derived from WT and fALS SOD1 (G17S) patient blood. WT and G17S iPSCs were confirmed using pluripotency markers and RT-PCR ( Fig. 7a and Additional file 7: Fig. S7A). We also generated NSCs, and the immunocytochemical staining demonstrated that SOX2 and Nestin expression were induced in WT and G17S NSCs (Fig. 7b). To validate the variation in miR-18b-5p, Hif1α, Mef2c, miR-206, Mctp1, and Rarb transcripts, we induced MNs from NSCs which were characterized by HB9 and ChAT (Fig. 7c). We confirmed that Hif1α and Mef2c transcripts significantly increased in G17S MNs (Fig. 7d).

Discussion
Recently, various pathological pathways and mechanisms have gradually been discovered for ALS and FTLD [13]. SOD1, FUS, and TDP-43 are representatively associated with ALS pathogenesis [2][3][4][5][6][7][8][9]. Specifically, RNA processing studies are based on the pathogenic mechanisms of FUS and TDP-43 because they have several functional and structural similarities [52,53]. Although SOD1, which is the first discovered ALS-causing mutated gene and is linked only with the ALS phenotype, does not have much functional correlation with FUS and TDP-43, many researchers have attempted to study dysregulated RNA biogenesis with regard to SOD1 mutations [9,13] [30,31] and that Rarb is associated with cell differentiation [37]. Indeed, we found that the downregulation of Mctp1 and Rarb in mtNSC-34 cells and the intracellular Ca 2+ levels increased and the neurite outgrowth was reduced. Previous research introduced us the idea that Mef2c could regulate miR-206 expression [49] and that Hif1α could also induce Mef2c as a transcription factor [48]. In this research, we identified that both Hif1α and Mef2c were increased in mtNSC-34 cells. Earlier studies also shed some light on Hif1α regulation by miR-18b-5p [49]. Importantly, we first discovered that miR-18b-5p expression was significantly decreased in mtNSC-34 cells. Clues pertaining to altered miR-18b-5p, Hif1α, Mef2c, miR-206, Mctp1, and Rarb were perfectly correlated to each other and were related to apoptosis in mtNSC-34 cells.   We proved that the miR-18b-5p pathway was functional in vitro, but whether or not the downregulated miR-18b-5p pathway could be revealed in the G93A Tg, G86S patient and G17S human MNs was not clear. We also reconfirmed the downregulated miR-18b-5p pathway in the G93A Tg, G86S patient and G17S MNs. As observed in our in vitro studies, miR-18b-5p was incredibly downregulated and miR-206 expression was upregulated in the G93A Tg, G86S patient and G17S MNs. The mRNAs of Hif1α, Mef2c, Mctp1 and Rarb were the same as those observed in vitro studies of the G93A Tg, G86S patient and G17S MNs. Intracellular Ca 2+ levels were enhanced and MN differentiation was significantly inhibited in G17S MNs. Increased Bax and decreased Bcl2 RNAs also indicated that apoptosis by downregulated miR-18b-5p was elevated in the G93A Tg, G86S patient, and G17S MNs.
According to the recent reports, apoptotic cell death of motor neurons (including SOD1, TDP-43, and FUS) [2][3][4][5][6][7][8][9]54] and abnormal RNA metabolism (mRNA transcription and miRNAs) [9,14,55] in ALS are so controversial issues because non-apoptotic features have been found in ALS patients [54]. Besides, the crucial role of apoptotic cell death by abnormal RNA metabolism is still unclear. We, for the first time, have discovered that downregulated miR-18b-5p, which may be one of the important pathogenic mechanisms in ALS associated SOD1 mutants (D90A, G17S, G85R, G86S and G93A) is associated with the sequential regulation of Hif1α, Mef2c, miR-206, Mctp1, and Rarb. Indeed, downregulated Mctp1 directly increased Ca 2+ levels, and decreased Rarb significantly reduced cell differentiation in all investigated SOD1 mutations. The causes of down regulated miR-18b-5p by SOD1 mutants need to be further examined. It will provide novel insights into undescribed cellular processes and support to understand that miRNAs are related to important pathogenic mechanisms of sporadic and familial ALS.