MicroRNA-5572 Is Associated with Endoplasmic Reticulum Stress Responses in Low Zinc Treated and SOD1 G85R-Transfected HEK293 Cells

Abstract

Amyotrophic lateral sclerosis (ALS) is a fetal neurodegenerative disease. The mechanism of sporadic ALS onset remains unclarified in detail. Disruption of zinc homeostasis could be related to sporadic ALS. Previously, we first reported miR-5572 as a microRNA (miRNA) among those identified in the spinal cords of patients with sporadic ALS. However, since its function in ALS remained unknown, this study further examined the role of miR-5572 in low-zinc status and ALS model cells which transfected with causative gene, Cu/Zn superoxide dismutase 1 (SOD1) G85R mutant vector. The miR-5572 level was increased by low-zinc condition accompanied by increase of endoplasmic reticulum (ER) stress. In addition, increase of miR-5572 enhanced the cellular toxicity induced by low-zinc treatment. The expression of miR-5572 was also increased, which was accompanied by an increase of ER stress markers associated with SOD1 aggregation formation. Cell death and ER stress makers levels induced by tunicamycin treatment were further increased in miR-5572 mimic-transfected cells. This study showed that miR-5572 exacerbated ER stress toxicity associated with low-zinc status and mutant SOD1 aggregates in ALS.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown cause, in which the upper and lower motor neurons selectively degenerate. It is accompanied by severe muscle atrophy and weakness. Furthermore, it progresses rapidly, after which the affected patient dies of respiratory muscle paralysis within approximately 3 to 5 years after onset. Although roughly 95% of patients with ALS have sporadic cases, the cause of this disease remains unknown. The remaining 5% of affected patients had familial cases in Japan. Several causative genes, such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 kDa (TDP43), fused in sarcoma (FUS), and chromosome 9 open reading frame 72 (C9orf72), have also been identified among these familial cases.¹⁾ Nevertheless, although various possible causes of sporadic ALS have been suggested, intracellular stress factors, including oxidative stress, endoplasmic reticulum (ER) stress, dyshomeostasis of metals, and so on, have been proposed to be involved in the onset of ALS.^2,3)

Among disruption of the metal metabolism, there have been reports on zinc in relation to ALS. Increased zinc levels in cerebrospinal fluid⁴⁾ and decreased expression of metallothionein and zinc transporters in the spinal cord have been reported in patients of sporadic ALS.^5,6) Although it has been suggested that ER stress caused by low zinc may alter zinc transporters and contribute to the development of ALS,^7,8) the mechanism remains to be elucidated.

The relationship between the onset of diseases and epigenetics has been of great focus. Epigenetics research the mechanisms that regulate gene transcription without causing changes in the genomic sequence. Among the typical epigenetic mechanisms, in addition to DNA methylation and histone modifications, microRNA (miRNA) has also been determined. MiRNAs are evolutionarily conserved and short non-coding RNAs of about 20 bases that bind to the 3′-untranslated region of target genes to suppress transcription and translation.^9,10) Thus, as miRNA analysis has also been progressing to understand the pathogenesis of neurodegenerative diseases, results are expected to help elucidate the disease’s pathogenic mechanism and as a biomarker.^11,12) Therefore, epigenetics is essential in evaluating the pathogenic mechanisms accounting for sporadic ALS.¹³⁾

Previously, we first identified miR-5572 in ALS patient samples.¹⁴⁾ In addition, recently some studies have been showed the association of miR-5572 with diseases, for instance, Sjögren’s Syndrome,¹⁵⁾ psoriasis,¹⁶⁾ prostate cancer¹⁷⁾ and gastric cancer.¹⁸⁾ However, the biological function of miR-5572 and its role in zinc homeostasis and the pathogenesis of ALS remains unknown. Therefore, in this study, we investigated the role of miR-5572 in zinc homeostasis and ALS.

MATERIALS AND METHODS

Cell Culture

The human embryonic kidney 293 (HEK293) cell was purchased from American Type Culture Collection (Manassas, VA, U.S.A.). HEK293 was cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO, U.S.A.) supplemented with 10% fetal bovine serum (FBS) under 5% CO₂ at 37 °C.

Preparation of Zinc Free DMEM and Low Zinc Condition

Zinc free DMEM was prepared as the previous studies.^19–21) Zinc in FBS was derived using Chelex^®100 resin (BioRad, Hercules, CA, U.S.A.). Removal of zinc was determined by atomic absorption spectrophotometry (SHIMADZU, Kyoto, Japan). N,N,N′,N′-Tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) (Dojindo, Kumamoto, Japan) was added to zinc free DMEM to prepare low zinc condition. Four micromolar of ZnSO₄ (Nacalai Tesquse, Kyoto, Japan) was used as control and cells were treated with TPEN 0, 0.1, 0.5, and 1 µM.

Cell Viability under Low-Zinc Condition

HEK293 cells were seeded at a density of 1.0 × 10⁴ cells/well in a 96-well multiplate and cultured for 24 h. Cells were treated with ZnSO₄ 4 µM or TPEN 0, 0.1, 0.5 and 1 µM. At 24 and 48 h after treatment, cell viability was measured using a cell counting kit-8 colorimetric assay (Dojindo) based on the WST-8 assay.

Determination of Intracellular Zinc Level

HEK293 cells were seeded at a density of 3.0 × 10⁵ cells/well in a 12-well multiplate and cultured for 24 h. Cells were treated with ZnSO₄ 4 µM or TPEN 0, 0.1 and 0.5 µM for 24 h. Cells were stained using fluorescent probe, ZINPYR-1 (Cayman, Ann Arbor, MI, U.S.A.), to detect intracellular free zinc. Pictures of images were obtained using confocal imaging system (Zeiss LSM 700, Carl Zeiss, Oberkochen, Germany). Fluorescent level of probe was normalized by numbers of cells in each picture to determine intracellular zinc level using ImageJ software (NIH, Bethesda, MD, U.S.A.). Analysis was performed in 5 pictures at each experimental group.

RNA Extraction

Total RNA, including miRNA, was extracted from HEK293 cells using miRNeasy Mini Kit (Qiagen, Venlo, the Netherland). During extraction, we followed the manufacturer’s protocol.

Quantitative RT-PCR for miRNA

Total RNA (5 ng), including miRNA, was subjected to a reverse transcript reaction, using a TaqMan™ MicroRNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, U.S.A.) and RT primers specific to miR-5572 miRNA or U6 snRNA sequences supplied in TaqMan™ MicroRNA assays; hsa-miR-5572 (Assay ID: 471859_mat) or U6 snRNA (Assay ID: 001973) (Applied Biosystems), to make cDNA. We followed the manufacturer’s protocol during this procedure. A cDNA aliquot specific for miR-5572 or U6 was then subjected to real-time RT-PCR, where amplification was conducted using a TaqMan^® Fast Advanced Master Mix (Applied Biosystems), and miR-5572 TaqMan probes or U6 supplied in TaqMan™ MicroRNA assays. A StepOne Real-Time PCR System (Applied Biosystems) was used for RT-PCR under the following conditions: 50 °C/2 min × one cycle; 95 °C/10 min × one cycle; 95 °C/15 s, 60 °C/60 s, ×40 cycles. Amplification of U6 cDNA in the sample was used as an internal control for PCR amplification reactions.

MicroRNA Mimic Experiments

miRNA overexpression using a miRNA mimic experiment was conducted using AccuTarget™ “human” miRNA Mimic “hsa-miR-5572” [Accession: MIMAT0022260] (BIONEER, Daejeon, South Korea), and an AccuTarget™ miRNA mimic Negative control #1 (BIONEER). First, HEK293 cells were seeded at a density of 7.0 × 10⁵ cells/well in a 6-well multiplate for real-time RT-PCR and Western blotting (WB), or a density of 1.0 × 10⁴ cells/well in a 96-well multiplate for WST-8 assay, after which culturing was conducted for 24 h. Next, the miR-5572 mimic or negative control miRNA mimic (NC), and the SOD1 WT mCherry or SOD1 G85R mCherry vectors were co-transfected into HEK293 cells using Lipofectamine2000 (Invitrogen, Waltham, MA, U.S.A.) in Opti-MEM (Invitrogen). At 24h after transfection, transfected cells were treated with tunicamycin (0.5, 1 µg/mL), or a dimethyl sulfoxide (DMSO) vehicle control set never to exceed 0.1%, and ZnSO₄ 4 µM or TPEN 0, 0.1 and 0.5 µM. Then, following the manufacturer’s instructions (Dojindo), cell viability was measured by WST-8 assay. Real-time RT-PCR was also conducted using HEK293 cells transfected with NC or miR-5572 mimics and treated with 1 µg/mL tunicamycin for 24 h.

Quantitative RT-PCR for mRNA

First, total RNA was isolated using Tripure Isolation Reagents (Sigma-Aldrich), following the manufacturer’s protocols. Likewise, cDNA was prepared with ReverTra Ace^® qPCR RT Master Mix (TOYOBO, Osaka, Japan) from 1 µg total RNA, following the manufacturer’s protocols. Next, real-time RT-PCR was conducted with a THUNDERBIRD^® SYBR qPCR Mix (TOYOBO) and amplified using a StepOne Real-Time PCR System under the following conditions: 95 °C/60 s × one cycle; 95 °C/15 s, 60 °C/45 s, ×40 cycles. Primers used for real-time RT-PCR are as follows: BIP (NM_005347) (forward: 5′-TGTTACAATCAAGGTCTATGAAGGTG-3′; reverse: 5′-CAAAGGTGACTTCAATCTGTGG-3′), CHOP (NM_004083) (forward: 5′-AATCAGAGCTGGAACCTGAGGA-3′; reverse: 5′-TGCTTTCAGGTGTGGTGATGTATG-3′), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (NM_002046) (forward: 5′-TGGTGAAGACGCCAGTGGA-3′; reverse: 5′-GCACCGTCAAGGCTGAGAAC-3′). All primer sets were designed using Primer3. Also, the amplification of GAPDH cDNA in the sample was used as an internal control for all PCR amplification reactions.

SOD1 Aggregation

HEK293 cells were seeded at a density of 5.0 × 10⁴ cells/well in a 12-well multiplate and cultured for 24 h. Then, SOD1 WT mCherry vector or SOD1 G85R mCherry vector, which were constructed previously,²²⁾ were transfected to cells using Lipofectamine2000 (Invitrogen) in Opti-MEM (Invitrogen). Afterward, SOD1 aggregations were determined at 24–72 h post-transfection, using a confocal imaging system (Zeiss LSM 700) or an IN Cell Investigator (GE Healthcare Life Sciences, Chicago, IL, U.S.A.).

Sodium Dodecyl Sulfate (SDS)-Polyacrylamide Gel Electrophoresis (PAGE) and WB

SDS-PAGE and WB were performed according to the a slightly modified methods of previous papers.^23,24) Cells were lysed in RIPA buffer and protein was extracted. The information of primary antibodies were the following: mouse anti-β-actin antibody (Sigma-Aldrich) (1 : 1000 dilution) (RRID:AB_476692), mouse anti-CHOP antibody (Cell Signaling Technology, Danvers, MA, U.S.A.) (1 : 1000 dilution) (RRID:AB_2089254), and rabbit anti-BiP antibody (Cell Signaling Technology) (1 : 1000 dilution) (RRID:AB_2119845). Second antibodies used for WB in this study were following; goat anti-mouse immunoglobulin G (IgG) antibody, peroxidase conjugated, H + L (1 : 5000 dilution) (Merck KGaA, Darmstadt, Germany) (RRID:AB_90456), goat anti-rabbit IgG antibody, peroxidase conjugated, H + L (1 : 5000 dilution) (Merck KGaA) (RRID:AB_90264). The band intensity was determined by ImageJ software (NIH).

Statistical Analysis

All results were expressed as mean ± standard errors. Furthermore, statistical analysis was conducted using IBM SPSS Statistics ver. 19.0 (IBM, Westchester, NY, U.S.A.) or StatView (Abacus, Baltimore, MD, U.S.A.).

RESULTS

Determination of Cell Viability under Low-Zinc Conditions

Cell viability under low-zinc conditions was determined. There was not significant change in cell viability at 24 h after TPEN treatment (Fig. 1A). The cell viability was significantly decreased to about 70% by 0.1 and 0.5 µM TPEN, and 14% by 1.0 µM TPEN treatment compared to control (ZnSO₄ 4 µM) at 48 h after TPEN treatment (Fig. 1A). The level of intracellular zinc was significantly decreased by 0, 0.1, and 0.5 µM TPEN treatment (Fig. 1B).

Fig. 1. Cell Viability under Low-Zinc Condition and Intracellular Zinc Level Were Determined

(A) Cell viability was determined at 24 and 48 h after TPEN treatment (biological replicates n = 4/group). (B) Fluorescence level of intracellular zinc was determined using ZNPYR-1 probe at 24 h after TPEN treatment. Scale bar is 20 µm. Data of intracellular zinc level was calculated as fluorescence level in total cells divided by number of nuclear, and this data was calculated per picture (n = 5 pictures/group). Data are presented as mean ± standard error. Statistical significance was determined using one-way ANOVA, followed by post hoc Bonferroni’s test (* p < 0.05).

Increase of ER Stress Markers by Low-Zinc Conditions

The level of BIP mRNA was significantly increased at 24 and 48 h after 0.5 µM TPEN treatment (Fig. 2A). The level of BIP protein was significantly increased at 48 h after 0.5 µM TPEN treatment (Fig. 2B, Supplementary Figs. 1, 2). Meanwhile, the level of CHOP mRNA was significantly increased at 24 h after 0.1 and 0.5 µM TPEN treatment, and at 48 h after 0, 0.1, and 0.5 µM TPEN treatment (Fig. 2C). The level of CHOP protein was significantly increased at 24 h after 0.5 µM TPEN treatment, and at 48 h after 0.1 and 0.5 µM TPEN treatment (Fig. 2D, Supplementary Figs. 3, 4).

Fig. 2. The Levels of BIP and CHOP mRNA and Protein Were Determined in TPEN-Treated HEK293 Cells

(A) The level of BIP mRNA was determined at 24 and 48 h after TPEN treatment (biological replicates n = 4/group). (B) The level of BIP protein was determined at 24 and 48 h after TPEN treatment (biological replicates n = 3/group). (C) The level of CHOP mRNA was determined at 24 and 48 h after TPEN treatment (biological replicates n = 4/group). (D) The level of CHOP protein was determined at 24 and 48 h after TPEN treatment (biological replicates n = 3/group). Data are presented as mean ± standard error. Statistical significance was determined using one-way ANOVA, followed by post hoc Bonferroni’s test (* p < 0.05).

MiR-5572 Level Was Increased by Low-Zinc Condition and Accelerated Toxicity of Low-Zinc Condition

The level of miR-5572 was significantly increased at 24 h after 0.5 µM TPEN treatment, and showed increasing trend but no significant change at 48 h after TPEN treatment (Fig. 3A). The miR-5572 mimic transfection significantly decreased cell viability under 0, 0.1 and 0.5 µM TPEN treatment, compared to the TPEN-treated NC group (Fig. 3B).

Fig. 3. The Levels of miR-5572 under Low-Zinc Conditions and Cell Viability by Low-Zinc Conditions in miR-5572 Mimic-Transfected HEK293 Cells Were Determined

(A) The level of miR-5572 was determined at 24 and 48 h after TPEN treatment (biological replicates n = 4/group). Data are presented as mean ± standard error. Statistical significance was determined using one-way ANOVA, followed by post hoc Bonferroni’s test (* p < 0.05). (B) Cell viability was determined in HEK293 cells transfected with the negative control (NC) or miR-5572 mimic and treated with TPEN for 48 h (biological replicates n = 4/group). Data are presented as mean ± standard error. Statistical significance was determined using two-way ANOVA, followed by post hoc Bonferroni’s test (* p < 0.05, # p < 0.05 vs. NC-Zn 4 µM, † p < 0.05 vs. miR-5572 mimic-Zn 4 µM).

The Human ALS Model Using SOD1 Mutant Was Confirmed

Since miR-5572 was reported only in the human genome, we tried to establish a human ALS model using HEK293 cells transfected with SOD1 WT or SOD1 G85R vectors. As observed, levels of cellular SOD1 aggregation were significantly increased in the SOD1 G85R group than the SOD1 WT group at 48 and 72 h after transfection (Figs. 4A, 4B). These cells were therefore used as the ALS model in subsequent experiments.

Fig. 4. SOD1 Aggregations, miR-5572 Level, BIP and CHOP Level Were Determined in HEK293 Cells Transfected with SOD1 WT or SOD1 G85R mCherry Vectors

(A) Fluorescence images of SOD1 in HEK293 cells transfected with SOD1 WT or SOD1 G85R mCherry vectors obtained at 24, 48, and 72 h after vector transfection. Arrowheads show SOD1 aggregations. (B) The level of SOD1 aggregation was quantified using an IN Cell Investigator in SOD1 WT or SOD1 G85R-transfected HEK293 cells (counted at least 750 cells from each biological replicates n = 3/group). (C) The levels of miR-5572 (biological replicates n = 5/group), (D) BIP mRNA (biological replicates n = 4/group) and (E) CHOP mRNA (biological replicates n = 4/group) were determined in SOD1 WT or SOD1 G85R-transfected HEK293 cells at 24, 48, and 72 h after vector transfection. (F) The levels of BIP and CHOP protein were determined in SOD1 WT or SOD1 G85R-transfected HEK293 cells at 72 h after vector transfection (biological replicates n = 3/group). Data are presented as mean ± standard error. Statistical significance at each time points between SOD1 WT and SOD1 G85R groups was determined using Student’s t-test (* p < 0.05).

The Level of miR-5572 Was Increased in ALS Model Cells

Results showed that the level of miR-5572 was significantly increased in the SOD1 G85R group than the SOD1 WT group at 72 h after SOD1 transfection (Fig. 4C). Additionally, the level of mRNA of ER stress markers (BIP and CHOP) were increased in the SOD1 G85R group compared with the SOD1 WT group at 72 h after transfection (Figs. 4D, 4E). And the level of BIP and CHOP protein were increased in the SOD1 G85R group compared with the SOD1 WT group at 72 h after transfection (Fig. 4F, Supplementary Fig. 5).

MiR-5572 Enhanced the Toxicity of Endoplasmic Reticulum Stress

The miR-5572 mimic transfection significantly decreased cell viability at 24 h after 0.5 and 1 µg/mL tunicamycin treatment compared to the tunicamycin-treated NC group (Fig. 5A). The level of miR-5572 was significantly increased by tunicamycin treatment (Fig. 5B). BIP, and CHOP mRNA and protein levels were significantly increased by tunicamycin treatment in both the NC and miR-5572 mimic groups (Figs. 5C, 5D). Additionally, the BIP and CHOP mRNA and protein level in the miR-5572 mimic group treated with tunicamycin was significantly increased compared with mRNA in the NC group treated with tunicamycin (Figs. 5C, 5D, Supplementary Fig. 6).

Fig. 5. Determination of miR-5572’s Function during Endoplasmic Reticulum Stress Response

(A) Cell viability was determined in HEK293 cells transfected with negative control (NC) or miR-5572 mimics and treated with tunicamycin for 24 h (biological replicates n = 6/group). Data are presented as mean ± standard error. Statistical significance was determined using two-way ANOVA, followed by post hoc Bonferroni’s test (* p < 0.05). (B) The level of miR-5572 was determined at 4 h after tunicamycin treatment (biological replicates n = 4/group). (C) The levels of endoplasmic reticulum stress markers, BIP and CHOP mRNA were determined in HEK293 cells transfected with NC or miR-5572 mimics and treated with 1 µg/mL tunicamycin for 24 h (biological replicates n = 3/group). (D) The levels of BIP and CHOP protein were determined in HEK293 cells transfected with NC or miR-5572 mimics and treated with 1 µg/mL tunicamycin for 24 h (biological replicates n = 3/group). Data are presented as mean ± standard error. Statistical significance was determined using two-way ANOVA, followed by post hoc Bonferroni’s test (* p < 0.05, # p < 0.05 vs. NC-tunicamycin 0 µg/mL, † p < 0.05 vs. miR-5572 mimic-tunicamycin 0 µg/mL).

DISCUSSION

The pathogenic mechanism accounting for the onset of sporadic ALS remains to be elucidated. In a previous study, we focused on microRNA among epigenetics and found an increase of miR-5572 in sporadic ALS spinal cords.¹⁴⁾ Previously, we had also reported that a target gene of miR-5572 was SLC30A3 which had been found to be decreased in patients with ALS.¹⁴⁾ Furthermore, we speculated an intracellular dyshomeostasis of zinc could induce existed in these patients that induced ER stress. It has also been reported that one miRNA can regulate up to 400 genes.²⁵⁾ Therefore, to study the pathophysiological function of miR-5572 related to zinc homeostasis and ALS, we used cells under low-zinc condition or ALS model cells into which mutant SOD1 G85R was introduced.

First, we determined the relationship between low-zinc and miR-5572 function. We used HEK293 cells because efficiency of transfection of foreign genes in HEK293 cells is relatively higher than that in neuronal cell lines. It should be important to use human neuronal cell line from the view of ALS. However, in that case we have to overcome the problems of low efficiency of gene transfection in human neuronal cell lines to determine the mechanism of molecules such as miR-5572 or to establish in vitro ALS model using foreign genes (plasmid or miRNA mimics). The miR-5572 level was increased by low-zinc condition accompanied by increase of ER stress (Figs. 2, 3A). In addition, increase of miR-5572 enhanced the cellular toxicity induced by low-zinc treatment (Fig. 3B). These results suggested that elevation of miR-5572 level via ER stress is related to low-zinc induced toxicity. The previous study showed that low-zinc status modified the structure of naïve SOD1 to that of SOD1 mutant via increase of ER stress in HEK293 cells.⁴⁾ This phenomenon could be occurred in this study, and there is the possibility that increase of miR-5572 level via ER stress induced by low-zinc could interact with the mechanism of conversion of naïve SOD1 to mutant-like SOD1 structure. Further study to determine the effects of miR-5572 on SOD1 structure conversion induced by low-zinc using miRNA inhibitor or mimic should be required.

Second, HEK293 cells with SOD1 mutant were applied to examine functions of miR-5572 in ALS model. Although the level of intracellular SOD1 aggregation was increased at 48 h after vector transfection (Figs. 4A, 4B), levels of ER stress markers were increased at 72 h post-transfection (Figs. 4D–F). As a result, the expression of miR-5572 was also increased, which was accompanied by an increase of ER stress markers associated with SOD1 aggregation formation (Fig. 4C). These results, therefore, propose that ER stress and miR-5572 expression increased due to the increase in aggregates caused by mutant SOD1.

As shown above, increase of miR-5572 was related to ER stress in commonly both low-zinc treated cells and ALS model cells. Intracellular stress factors, such as oxidative stress and ER stress [11], are crucial in the pathogenesis of ALS. Although reports on changes in miRNA due to intracellular stress, such as oxidative stress and ER stress,²⁶⁾ no accounts on changes in miR-5572 expression influencing these stresses are available and we focused on ER stress in this study. Hence, when the quantity of miR-5572 in cells was increased using a miRNA mimic, enhancement of cell death due to ER stress, including enhancement of ER stress markers, were observed (Figs. 5A, 5C, 5D). In addition, the level of miR-5572 was significantly increased by tunicamycin treatment (Fig. 5C). These results, therefore, suggested miR-5572 as a toxicity modifier during ER stress. Thus, intracellular turbulence of zinc is proposed to cause ER stress and neuronal motor death in patients with ALS.

Although we were unable to present the results of cell death by SOD1WT and G85R in this study, based on a report of SOD1G85R transfection in a cell line other than HEK293,²⁷⁾ it is expected that cell survival is reduced by the SOD1 G85R mutation SOD1 G85R mutation may reduce cell viability. The G85R mutation is also thought to abolish the Cu/Zn holding capability of SOD1,²⁸⁾ and the protein structure of SOD1 could be changed to apo form. It is also possible that apo-SOD1 aggregates more than holo-SOD1 when zinc is low. Although not investigated in this study, it has been reported that a low zinc state converts SOD1 WT to a mutant-like structure,⁸⁾ suggesting that zinc is important for SOD1 structural maintenance and is closely related to ALS pathogenesis.

There were limitations of this study. As a true ALS model cell, it is necessary to use a motor neuron or nerve cell line, but we were unable to prepare such an experimental system due to difficulties in gene transfection. Furthermore, miR-5572 is a miRNA that is expressed only in human species, so it was necessary to use human-derived cells. This led us to select HEK293 cells, a cell line that is both easy to transfect gene and of human origin. In the future, although the hurdle is high, it will be necessary to establish motor neurons differentiated from human iPS cells derived from ALS patients or human nerve-derived cell lines with ALS-causing genes such as SOD1 to confirm the function of miR-5572. In this study, although we did not perform the knockdown experiments to determine the contribution of endogenous miR-5572, miR-5572 may be a toxic modifier for low zinc conditions and vesicular stress. We believe that the experiments with miR-5572 mimic suggested that increased miR-5572 could be a toxicity modifier for low zinc conditions and endoplasmic reticulum stress. We think that the contribution of endogenous miR-5572 should also be investigated in the future by establishing a knockdown and knockout experimental system for miR-5572.

ER stress is thought to be involved in the final common pathway leading to ALS onset.

This study observed that ER stress via low-zinc status or SOD1 G85R mutant increased miR-5572 level. Previous studies showed that SOD1 G85R or SOD1 G93A mutants increased mature miRNA level by modulating miRNA biogenesis process,²⁹⁾ zinc deficiency modulated several miRNA in esophageal carcinoma,³⁰⁾ and ER stress inducer, thapsigargin, increased miR-708 level.³¹⁾ Although some previous studies including this study showed miRNA levels were modulated by low-zinc or ALS-related gene mutation which were related ER stress, a few reports exist that propose ER stress affecting the maturation of miRNAs. A previous report showed that ER-bound kinase/endoribonuclease, inositol-requiring enzyme-1 (IRE1), regulated miR-2137 maturation.³²⁾ There is possible that ER stress could affect the miRNA including miR-5572 maturation process. In a previous report, it was reported that the G85R mutation triggers an ER stress response via the PERK, IRE1 and ATF6 pathways.³³⁾ Although it is possible that these pathways were also activated in this study, we could not be examined in the present study. Further investigation of the involvement of specific endoplasmic reticulum stress pathways will be necessary to determine interaction between miR-5572 and ER stress pathway in the future.

Regarding endoplasmic reticulum stress and zinc dynamics, previous reports have reported that intracellular zinc is upregulated³⁴⁾ or decreased³⁵⁾ in SOD1 mutant mice have been reported to have increased zinc accumulation.³⁶⁾ Thus, it is highly likely that endoplasmic reticulum stress and SOD1 mutations alter intracellular zinc dynamics, but this study was inconclusive. To elucidate the relationship between cell death caused by endoplasmic reticulum stress and other factors and zinc dynamics, it would be important to measure zinc levels in individual cell organelles rather than zinc levels in the whole cell, which could not be achieved in the present study. This is an important issue that should be considered in the future. In addition, we determined that SLC30A3 mRNA, the target of miR-5572, was reduced at the same time that ER stress markers were raised after introduction of the SOD1 G85R mutation (Supplementary Fig. 7). Although we could not determine how this change affects zinc dynamics, further investigation of changes in other zinc transporters and changes in zinc dynamics at the cell organelle level will be necessary. It will also be necessary to search for other target genes of miR-5572 besides SLC30A3.

In the case of miRNA gene encodes an intron, expression of the intronic sequence can be induced with transcription of the host gene, which is called an intronic miRNA.³⁷⁾ Regarding miR-5572, although the gene is encoded in the intron of the human ARNT2 gene, it is unknown whether the induction mechanism is due to intronic miRNA. Therefore, further investigation is necessary.

Conclusively, based on the results obtained, miR-5572 is proposed to be associated with ER stress induced by low-zinc or causative ALS-related mutant as the pathogenic mechanism accounting for ALS onset, which acts as a vicious cycle, thereby worsening the pathological condition. However, although miR-5572 is a vital molecule accounting for the onset or progression of ALS, it can become a biomarker for therapeutic targets and diseases in the future.

Acknowledgments

The Grant-in-Aid of The Nakabayashi Trust for ALS Research, Tokyo, Japan and Mishima Kaiun Memorial Foundation, and Suzuken Memorial Foundation to H.K., a Grant-in-Aid of AMED-SICORP (Grant Number: JP23jm0210097) to A.K., and the Collaborative Research Project (2019–23) of the Brain Research Institute, Niigata University and a Grant-in-Aid for Scientific Research on Innovative Areas JSPS KAKENHI (JP19H05767A02) to I.H., was partly used to support this study.

Author Contributions

H.K., N.H., S.Y., A. Okuda, T.M., and K.O. designed experiments, performed experiments and analyzed data. H.K., N.H., S.Y., K.O., A. Ogata, H.Y., A.K., I.H., and M.I. interpreted and discussed results. H.K. wrote the initial draft of manuscript. H.K. supervised and conceived project. K.O., A. Ogata, H.Y., A.K., I.H., and M.I. edited and revised manuscript. All authors approved the final version.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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