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Doctor of Science
A study on the survival regulation of neural stem cells by miR-351-5p/Miro2 and discovering
Alzheimer’s disease treatment targets
miR-351-5p/Miro2 에 의한 신경줄기세포의 생존조절 연구와
알츠하이머병 치료 타겟 발굴
The Graduate School University of Ulsan
Department of Medical Science Sujeong Park
[UCI]I804:48009-200000506790 [UCI]I804:48009-200000506790
A study on the survival regulation of neural stem cells by miR-351-5p/Miro2 and discovering Alzheimer ’ s
disease treatment targets
Sujeong Park
Supervisor: Heuiran Lee
A dissertation Submitted to
the Graduate School of the University of Ulsan in partial fulfillment of
the requirements for the degree of
Doctor of Science by
Sujeong Park
Department of Medical Science Ulsan, Korea
August 2021
A study on the survival regulation of neural stem cells by miR-351-5p/Miro2 and discovering
Alzheimer ’ s disease treatment targets
This certifies that the dissertation of Sujeong Park is approved.
Department of Medical Science Ulsan, Korea
August 2021
i
Abstract
Background
The neurogenesis process in the adult hippocampal region serves to maintain the plasticity of brain structure and function. The importance of maintaining adult neural stem cells in the hippocampal region has been confirmed in observations of patients with Alzheimer’s disease (AD) where decreases in hippocampal neural stem (HCN) cells have been observed. Though the abnormal expression of specific microRNAs (miRNAs) has been reported in AD, the role of miRNAs in HCN cells is not yet known. In recent studies, I discovered miR-351-5p, which regulates the survival of HCN cells, and used the bioinformatics tools to excavate the mitochondrial GTPase Miro2 as a target gene.
Aim
This study attempts to elucidate the mechanism that regulates the cell death of HCN cells through miR-351-5p and its target gene, Miro2.
Results
First, I ascertained autophagy-dependent cell death (ADCD) in HCN cells by overexpressing miR-351-5p or suppressing the expression of Miro2 using Miro2-specific siRNA.
Additionally, I showed that cell death was inhibited by overexpressing Miro2 using adenovirus.
Pink1-Parkin-dependent mitophagy occurs excessively during this cell death process, and mitochondrial dysfunction was demonstrated by mitochondrial membrane depolarization, reduced ATP production capacity, and ROS overproduction. In particular, I showed that the dramatic increase of the fragmented mitochondria is a crucial event leading to ADCD using a mitochondrial fission blocker Mdivi-1. Moreover, using in situ hybridization, real-time PCR, and immunohistochemistry, the expression of miR-351-5p was observed to significantly increase and the expression of Miro2 substantially decrease in the AD mouse model.
ii Conclusion
The data suggest that the reduction of Miro2 due to the overexpression of miR-351-5p leads to excessive fission and mitophagy stress of the mitochondria, which in turn negatively affects HCN cell survival. As enhancing HCN cell viability is expected to compensate for deficient neurogenesis in patients with AD, the confirmation of miR-351-5p’s dysregulation is an important step in therapeutic developments.
Keywords: Hippocampal neural stem cells, miR-351-5p, Miro2, cell death, autophagy, mitophagy, fission, Alzheimer’s disease
iii
Contents
Abstract ... i Contents ... iii List of abbreviations ... v Introduction ... １ Material and Methods ... ３ 1. Adult hippocampal neural stem cell
2. MiRNAs, small interfering RNAs (siRNAs), and transfection 3. Cell viability assay
4. Western blotting and immunohistochemistry 5. Animals
6. Autophagic flux assay 7. Mitophagy assay
8. Mitochondrial function analysis 9. miRNA quantification
10. 3′UTR luciferase reporter assay 11. miRNA in situ hybridization 12. Statistical analysis
Results ... ８ 1. miR-351-5p explicitly induces cell death in HCN cells
iv 2. miR-351-5p induces ADCD in HCN cells
3. Mitochondrial GTPase Miro2 is the direct target of miR-351-5p 4. Miro2 regulates cell death in HCN cells
5. Effect of miR-351-5p and Miro2 expression on PINK1-Parkin-dependent mitophagy 6. Suppression of Miro2 by miR-351-5p induces mitochondrial dynamics and dysfunction 7. Inhibition of mitochondrial fission blocked ADCD induced by miR-351-5p/Miro2 by restoring mitochondrial function
8. Overexpression of Miro2 adequately recovered cell death and mitochondrial fission induced by miR-351-5p or insulin withdrawal on HCN cells
9. The hippocampus’s expression of miR-351-5p and its target candidate, Miro2, is associated with AN and AD pathophysiology
10. miR-351-5p and Miro2 expression showed an opposite correlation in AD model mice 11. The schematic mechanism of the miR-351-5p/Miro2 axis in AN
Discussion ... ５０ References ... ５３ 국문 초록 ... ６０
v
List of abbreviations
AD Alzheimer’s disease
HCN Hippocampal neural stem
miRNA MicroRNA
ADCD Autophagy-dependent cell death
AN Adult neurogenesis
SGZ Subgranular zone
DG Dentate gyrus
siRNA Small interfering RNAs
mRFP Monomeric red fluorescent protein
GFP Green fluorescent protein
tfLC3 Tandem fluorescent tagged LC3
MMP Mitochondrial membrane potential
LC3 Light chain 3 (LC3)
１
Introduction
Until the 1990s, a central dogma of neuroscience was the “no new neurons” doctrine1 23. All neurons were assumed to be generated only in prenatal development and early postnatal life;
in an adult brain, neurogenesis was absent. Today, however, this dogma has been challenged experimentally, and it has finally been overturned that the production of new neurons in the adult brain is limited but progressive4. The number and maturation of these new neurons progressively declined as neurodegeneration diseases advanced, especially AD5-7.
The common cause of AD is neuronal loss in the brain, particularly in the hippocampus, which can lead to disabilities in memory and learning8. Thus, the decline in neurogenesis in AD suggests that it is an early contributor to disease progression9-11. Moreover, enhancing the cell viability of HCN cells can contribute to overcoming neurodegenerative diseases caused by a reduced neural stem cell pool12,13. Adult neurogenesis (AN) occurs in specific areas of the adult brain; the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) and the subventricular zone (SVZ) of the lateral ventricle 14 15,16. In the adult SGZ, HCN cells can proliferate and migrate a short distance into the granule cell in the DG1718. Furthermore, each HCN cell can differentiate into neurons, astrocytes, and oligodendrocytes and functionally integrate into existing neuronal networks in vivo19. Recently, it was reported that the cells undergo autophagic cell death in the absence of insulin, which is known as a growth factor regulating proliferation20,21.
Recent data indicate that regulation of mitochondrial function and morphology through fusion and fission events is essential to control the development and sustain maturation in new neurons during adulthood22,23 24. Furthermore, mitochondrial fission also functions by segregating dysfunctional mitochondria for degradation through mitochondrial autophagy, termed mitophagy25,26. However, excessive fission will deplete the bioenergetics reserve in the
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remaining mitochondria and leads to subsequent cell death27-29. So, maintaining a healthy mitochondrial pool is vital for neuronal health30,3132.
MiRNAs are endogenous small non-coding RNAs that function as posttranscriptional regulator of gene expression with critical functions in cell death and mitochondrial function33-
3637. The miRNA binds to its complementary sequences in the target mRNA, thereby block its translation or degrade the target mRNA38,39.
In the previous study, I investigated miRNAs abundantly present in the state of HCN cell death induced in insulin-deprived conditions40. Interestingly, several miRNAs have been observed to be increased in these conditions41. Among these miRNAs, I found that rno-miR-351-5p specifically induces HCN cell death. Furthermore, the target of miR-351-5p was predicted using miRNA biological target prediction tools. As a result, I acknowledged that Miro2 is the target protein of miR-351-5p. Miro2 is an atypical GTPase within the Ras superfamily, localized on the outer mitochondrial membrane42,43. It is well known for playing a vital role in mitochondrial axonal transport, mitochondrial dynamics (fission and fusion), and Mito-Ca2+
homeostasis44-47. Also, Miro2 is activated by PINK1 phosphorylation, followed by the ubiquitinated-degradation by Parkin48-50. To know how the mitochondrial function of Miro2 can affect HCN cell death, I examined various mitochondrial functional assays when Miro2 expression is down or overexpression of miR-351-5p at the upstream mechanism.
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Material and Methods
1. Adult hippocampal neural stem cell
Cells were cultured in Dulbecco’s modified Eagle’s medium nutrient mixture F-12 (Invitrogen) supplemented with 1.27 g/L of sodium bicarbonate (Invitrogen), 100 mg/L of transferrin (Sigma), 16 mg/L of putrescine dihydrochloride (Sigma), 20-nM progesterone, 30-nM sodium selenite (Sigma), 100 μg/ml of streptomycin, and 100 U/ml penicillin (Invitrogen). Also, all plastic and glassware for HCN cell culture were coated with 10 mg/L of poly-L-ornithine and 1 mg/L of laminin.
2. MiRNAs, small interfering RNAs (siRNAs), and transfection
MiRNA mimics and anti-miRNAs were constructed by Genolution, whereas siRNA duplexes were purchased from Bioneer. HCN cells were transfected with miRNAs or siRNAs using a lipofectamine 2000 following the manufacturer’s instructions.
3. Cell viability assay
HCN cells were seeded in a 24-well plate. Cell death was assayed using Hoechst 33342 (Invitrogen) and propidium iodide (PI; Sigma) staining. After 5 min, the observed fields were randomly selected (>3 areas, >300 cells), and collected images were analyzed using ImageJ software.
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4. Western blotting and immunohistochemistry
HCN cells were harvested and lysed in M-per buffer (Thermo Fisher) with protease cocktail inhibitors (Merck) and phosphatase inhibitors (Merck). After lysis, cell lysates were measured using the Pierce BCA protein assay kit (Thermo Fisher). Furthermore, samples were loaded with 15–20 μg of proteins for immunoblotting analysis. The primary antibodies were used as follows: Miro2 from Proteintech; light chain 3 (LC3) and β-actin from Sigma, PINK1 from Novus Biologicals, Drp1 from Cell signaling, Mfn2 from Abcam, p62 from Abnova, and Nestin from Millipore. Also, peroxidase-conjugated secondary antibodies were bound to the primary antibodies, and their binding was detected using a chemiluminescence detection kit (Thermo Fisher).
4. Transmission electron microscopy (TEM)
Cells were fixed with 2% paraformaldehyde and 2.5% glutaraldehyde in a sodium cacodylate buffer at 4°C. The fixed samples were dehydrated using an ethanol gradient (50%, 60%, 70%, 80%, 90%, and 100%) for 30 min each. Ultra-thin sections were cut from an uncoated copper grid and stained with lead nitrate and 2% uranyl acetate. The samples were then examined under TEM.
5. Animals
AD model 5xFAD mice (APP KM670/671NL (Swedish), APP I716V (Florida), APP V717I (London), PSEN1 M146L (A > C), PSEN1 L286V), and 3xTg-AD mice (APP Swedish, tauP301L, PSEN1 M146V) were purchased from Jackson Laboratory. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee of Sungkyunkwan University (#2018-01-01-1).
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6. Autophagic flux assay
HCN cells were co-treated with a monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 (tfLC3) constructs and miR-351-5p.
After 24 h, images were obtained with a confocal microscope (LSM780).
7. Mitophagy assay
HCN cells treated with miR-351-5p or siRNA Miro2 were stained with MitoTracker (Invitrogen) and LysoTracker (Thermo Fisher) and observed under a confocal microscope. The co-localization of the MitoTracker and Lysotrackers was observed using a confocal fluorescence microscope. HCN cells transfected with miR-351-5p or siMiro2 were stained with mitophagy dye (Dojindo) to confirm the degree of mitophagy according to the manufacturer's instructions. Also, HCN cells were treated with a concentration of 10 μM CCCP to induce mitophagy. At 24 h, HCN cells were washed twice. The intensity of the mitophagy dye was measured using the ZEN software.
8. Mitochondrial function analysis
To determine the mitochondrial membrane potential (MMP), HCN cells were prepared in 6- well plates and transfected with miRNAs and siRNA for 24 h. Cells were then stained using JC-1 fluorescent dye (Abcam) for 1 h at 37°C, and the cells were harvested and analyzed using a flow cytometer using FACS Canto Plus Software (BD Biosciences). Fluorescent signals for J-aggregates and J-monomers were read at the excitation and emission wavelengths of 535 and 595 nm and 485 and 535 nm, respectively. Finally, the J-aggregates/J-monomers ratio was measured. And ATP levels of mitochondria were examined using the Mitochondrial ToxGlo Assay (Promega). To assay the mitochondrial activity, treated HCN cells were incubated in a white 96-well plate in a glucose- or galactose-containing medium for 90 min. Then, 100 μl of
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Mitochondrial Tox-Glo assay medium was added to each well. The HCN cells were then incubated at 37°C for 30 min, and their luminescence was measured to detect ATP production.
To quantify mitochondrial fission, cells were treated with MitoTracker and imaged with a confocal microscope. A minimum of 100 randomly selected cell regions in each treatment group was analyzed for mitochondrial length using ImageJ software.
9. miRNA quantification
RNA was isolated from each tissue sample using TRIzol reagent (Invitrogen). To quantify miR-351-5p expression, reverse transcription was performed using the Mir-X miRNA first- strand synthesis kit (Clontech), followed by PCR amplification using IQ SyBr Green I mix (BioRad) on the CFX96 Real-Time System (BioRad). GAPDH was used as the reference gene for relative quantification. The primer used for miR-351-5p was 5′- TCCCTGAGGAGCCCTTTGAGCCTG-3′. For quantification of the Miro2 mRNA level, reverse transcription was performed using oligo dT primers. GAPDH was also used as the reference gene. The primers used for Miro2 and GAPDH were as follows: Miro2 sense, 5′-
GAGAAGATCCGAACCAAGTGG-3′, and anti-sense, 5′-
ACACAGCCTCTATGGTACTCC-3′; GAPDH sense, 5′-TGCACCACCAACTGCTTAGC-3′, and anti-sense, 5′-GCATGGACTGTGGTCATGAG-3′.
10. 3′UTR luciferase reporter assay
Wild-type 3′-UTR of Miro2 (WT-Miro2) and mutant-type 3′-UTR of Miro2 (MT-Miro2) were constructed into psiCHECK-2 at the 3′-end of the coding sequence of the Renilla luciferase by the manufacturer. For the luciferase reporter analysis, two types of DNAs were transfected using Lipofectamine 2000 along with the miR-351-5p or miR-Con mimics into HCN cells.
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The activities of firefly and renilla luciferases were measured 24 h after transfection. The dual- luciferase assay was performed using the Dual-Luciferase Reporter Assay System (Promega).
11. miRNA in situ hybridization
miRNA in situ hybridization was performed as described51. Briefly, miRCURY LNA detection probes (5′ and 3′ DIG-labeled) against miR-351-5p and U6 snRNA were purchased from Exiqon. Brain sections were treated with 1 µg/ml proteinase K at 37°C for 30 min. Following sections, each LNA probe was attached and incubated overnight. Sections were incubated with anti-DIG-AP antibody (Roche) and NBT/BCIP (Sigma Aldrich) to detect small RNAs.
12. Statistical analysis
All experiments were conducted three times independently. Furthermore, all quantitative results are represented as means ± standard deviation (SD). Statistical analysis was performed with Prism v.5 software (GraphPad). The differences between means were considered statistically significant at values of *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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Results
miR-351-5p explicitly induces cell death in HCN cells
In a previous study, miRNA microarray revealed that miR-351-5p is one of the most increased miRNAs in cell death-inducing conditions by insulin withdrawal. To explore the physiological roles of the upregulated miR-351-5p, I transfected its mimic to HCN cells. As a result, HCN cells began to exhibit signs of severe cytotoxicity, featuring cellular process loss, cell shrinkage, and even cell death, whereas a negative control did not (Fig. 1A). Furthermore, the extent of cell death was measured using PI staining, which helps to observe an increased cell death pattern (Fig. 1B). Moreover, to verify the specificity of cell death-inducing activity of miR- 351-5p on HCN cells, cells were treated with miR-351-5p and anti-miR-351-5p which specifically target miR-351-5p. miR-351-5p sharply induced HCN cell death. Alternatively, anti-miR-351-5p co-treatment with miR-351-5p specifically inhibited HCN cell death caused by miR-351-5p (Fig. 1A).
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Fig. 1. Overexpression of miR-351-5p induced cell death in HCN cells.
(A and B) HCN cells were transfected with miR-Con, miR-351-5p, anti-miR-Con, anti-miR- Con, or anti-miR-351-5p. After staining with Hoechst 33342 and PI at 24 h after transfection, cell death was quantified by counting PI-positive cells. Anti-miR-351-5p specifically inhibited miR-351-5p-induced cell death dose-dependently.
１０ miR-351-5p induces ADCD in HCN cells
miR-351-5p treated HCN cells featured many autophagic vacuoles, as clearly identified and quantified using TEM (data not shown). When HCN cells were treated with miR-351-5p, I observed LC3 conversion and degradation of p62 as established autophagy indicators.
Additionally, combinational treatment with miR-351-5p and bafilomycin A1 (Baf-A), an inhibitor of lysosomal proteolysis, was restored entirely LC3 conversion. In this circumstance, decreased expression of p62 was also significantly rescued (Fig. 2A–C).
To accurately monitor autophagic activity, it is essential to determine autophagic flux, which is determined as the amount of autophagosome structure. Then I used an mRFP-mGFP-LC3 tfLC3 plasmid. When autophagic flux is going, the fluorescence of GFP is quenched because of low lysosomal pH, whereas that of mRFP is stable. Formation of autophagosome increases the number of mRFP-positive-LC3 puncta, whereas it became GFP-positive/mRFP-positive- LC3 (yellow) puncta when miR351 and Baf-A had treated together (Fig. 2D and E). These data presented that autophagic flux is fully processed during HCN cell death induced by miR- 351-5p.
Furthermore, I treated late-phase autophagy inhibitors, such as chloroquine (CQ) and Baf-A, after transfection with miR-351-5p. Because both CQ and Baf-A blocked the autolysosomal stage, they rescued HCN cell death induced by miR-351-5p (Fig. 2F and G). Therefore, I investigated whether genetic approaches could prevent cell death or not.
Autophagy-related 5 (ATG5) and autophagy-related 7 (ATG7) are considered to be essential molecules for the induction of autophagy. As a genetic approach, ATG5 and ATG7 are well known for the critical molecule necessary for phagophore membrane elongation.
For preventing autophagic death of HCN cells, ATG5/7 siRNA was co-transfected with miR- 351-5p. Cell death induced by miR-351-5p was effectively suppressed in the presence of ATG5/7 siRNA. Also, the cytoprotective effect of ATG5/7 siRNA was monitored and quantified (Fig. 2H and I). These indicate that miR-351-5p specifically induces ADCD in HCN cells
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Fig. 2. Overexpression of miR-351-5p induces ADCD in HCN cells
(A) After treatment with miR-351-5p or miR-Con, cells were treated with Baf-A (10 or 20 nM) for 1 h before harvesting. Cell lysates were prepared and analyzed through Western blotting using anti-LC3 and anti-p62. β-actin was used as a loading control. (B and C) Quantitation of the LC3 conversion and expression of p62. The ratio of LC3-II/LC3-I and p62 expression was analyzed using ImageJ software. (D) Autophagic flux was monitored using the mRFP-GFP tandem fluorescent‑tagged LC3 (tfLC3) plasmid. At 24 h after transfection with miR-351-5p or miR-Con, cells were observed under the confocal microscope. Baf-A (20 nM) treatment was applied 1 h before observation. (E) Quantitation of red LC3 puncta from (D). (n = 39 for miR-Con, n=32 for miR-351-5p, and n=84 for miR-351-5p + Baf-A for three independent experiments). (F–I) Cells were treated with autophagy inhibitors, CQ and Baf-A, and siRNA of ATG5 and ATG7. At 24 h after transfection, cell death was assessed using the method described here. Cell death induced by miR-351-5p was effectively suppressed in the presence of autophagy inhibitors.
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Mitochondrial GTPase Miro2 is the direct target of miR-351-5p
To investigate the predicted target gene of the miR-351-5p, I used TargetScan, DIANA, and miRanda. Among the 12 genes identified, Miro2 was selected to explore its effect on HCN cells (Fig. 3A). A dual-luciferase reporter assay was used to reveal the direct targeting of miR- 351-5p and Miro2 (Fig. 3B). The results showed that miR-351-5p significantly attenuated the luciferase activity of the reporter vector containing the targeting sequence of Miro2 3'UTR, whereas this effect was abrogated when the 3'UTR binding sequence was mutated (Fig. 3C).
Given that Miro2 was significantly downregulated due to miR-351-5p treatment in HCN cells, I observed that the miR-351-5p overexpression could induce endogenous Miro2 inactivation in HCN cells. As such, I examined the downregulated endogenous level of Miro2 protein and mRNA in HCN cells (Fig. 3D and E). These findings indicate that miR-351-5p directly suppressed Miro2 expression by binding to the 3'UTR of Miro2.
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１８ Fig. 3. Miro2 is a direct target of miR-351-5p
(A) The Venn diagram displays the putative target genes of miR-351-5p as TargetScan, miRanda, and DIANA predicted. There were 12 candidates by all three algorithms. (B) Sequence alignment of miR-351-5p with the 3’-UTR of Miro2. Luciferase assay constructs with either WT or MT 3'UTR of Miro2 were prepared. (C) 293T cells were transfected with miR-351-5p, miR-Con, or anti-miR-351-5p with luciferase assay constructs. miR-351-5p inhibited luciferase expression by directly targeting the 3'UTR of Miro2. (D) HCN cells were transfected with miR-351-5p or miR-Con. WB analysis indicated that miR-351-5p downregulated Miro2 protein. (E) Downregulated Miro2 was analyzed using WB and RT- qPCR. miR-351-5p specifically downregulated Miro2 at both the mRNA and protein levels.
The data represent the mean ± SD values (n > 3). ***, p < 0.001.
１９ Miro2 regulates cell death in HCN cells
To verify the specificity of HCN cell death-inducing activity of Miro2, I treated siRNA of Miro2. As a result, siMiro2-transfected cells displayed increased cell death than the controls (Fig. 4A and B).
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Fig. 4. Suppression of Miro2 induced cell death in HCN cells
(A and B) HCN cells were transfected with siCon or siMiro2. After 24 h, cell death was determined using the method described herein. Suppression of Miro2 significantly induced cell death in HCN cells. The graph data represent the mean ± SD values (n > 3). ** p < 0.01.
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Effect of miR-351-5p and Miro2 expression on PINK1-Parkin-dependent mitophagy To further investigate the role of Miro2 in mitochondrial dynamics such as mitochondrial fission, fusion, and mitophagy, I observed the ultrastructure of HCN cells using TEM. HCN cells treated with miR-351-5p or siMiro2 have many autophagosomes containing mitochondria (Fig. 5A and B). miR-351-5p- or siMiro2-treated HCN cells were stained with the MitoTracker and LysoTracker. The merged image shows the co-localization of both markers in the miR-351-5p- or siMiro2-treated cells, indicating positive mitophagy (Fig. 5C).
After confirming that mitophagy was completed the process of mitophagy by comparing the relative amount of overlap between the mitochondrial marker and lysosomal markers via confocal microscopy. Under normal pH conditions, the mitochondrial dye exhibits fluorescence. However, when an autophagosome and lysosome are fused in an acidic environment, the dye's fluorescence intensity will increase. The fluorescence intensity of the mitophagy dye in miR-351-5p- or siMiro2-treated cells was significantly higher than that of the controls. Additionally, cells with miR-351-5p mimic and anti-miR-351-5p demonstrated a decreased signal in the intensity of the mitophagy marker (Fig. 5D and E).
To further verify whether the PINK1/Parkin pathway is involved in Miro2 suppression by miR-351-5p induced mitophagy, the protein expression of PINK1 and Parkin was measured in the HCN cell death groups. Compared with a control group, the expression of PINK1 and Parkin was significantly increased in HCN cells treated with miR-351-5p or siMiro2 (Fig. 5F and G). Therefore, these data suggest that the PINK1/Parkin pathway is activated during mitophagy, induced by inhibiting Miro2 resulting from miR-351-5p overexpression. Moreover, cell death caused by miR-351-5p was effectively suppressed in the presence of siPINK1 and siParkin. (Fig. 5H and I).
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Fig. 5. Suppression of Miro2 induces mitophagy in HCN cells.
(A and B) Cells were transfected with miR-351-5p, miR-Con, or siMiro2 and observed using TEM. The number of autophagosomes containing mitochondria per cell has been quantified (n > 4). miR-351-5p and siMiro2 significantly increased the number of autophagic vesicles relative to the control group. (C) To assess mitophagy induction, HCN cells transfected with miR-Con, miR-351-5p, or siMiro2 were treated with MitoTracker (red) and LysoTracker (green). Under the confocal microscope, colocalized regions emerged as yellow puncta. (D and E) HCN cells transfected with miR-Con, miR-351-5p, or siMiro2 were treated with mitophagy dye whose fluorescence was altered through mitophagy progression. (F and G) Western blotting revealed that PINK1 and Parkin were accumulated in the condition of miR- 351-5p- and siMiro2-induced mitophagy. Anti-miR-351-5p suppressed the accumulation of PINK1 and Parkin induced by miR-351-5p. (H and I) Cells were co-treated with the siRNA of PINK1 or Parkin and miR-351-5p or miR-Con. At 24 h after transfection, cell death was assessed using the method as described. Cell death induced by siMiro2 and miR-351-5p was effectively suppressed in the presence of siRNA of PINK1 or Parkin. The table data represent the mean ± SD values (n > 3). ** p < 0.01, *** p < 0.001.
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Suppression of Miro2 by miR-351-5p induces mitochondrial dynamics and dysfunction Along with including mitophagy, both miR-351-5p and siMiro2 affect the disruption of mitochondrial function in HCN cells. One of the methods for predicting potential mitochondrial dysfunction is measuring the ATP level. ATP levels were measured after transfection with miR-351-5p or siMiro2 and the level decreased to half relative to controls (Fig. 6A). To evaluate the membrane potential of mitochondria, JC-1 staining was performed.
Cells with high MMP promoted red fluorescent JC-1 aggregates, whereas cells with low membrane potential showed a diffuse green fluorescence. The vast majority of the HCN cells from controls showed evident red fluorescence. In contrast, cells treated with either miR-351- 5p or siMiro2 were observed a diffuse green fluorescence, indicating altered MMP (Fig. 6B and C).
Notably, in response to miR-351-5p and siMiro2, the number of fragmented mitochondria was drastically increased (Fig. 6D and E). Mitochondrial dynamics-related proteins, such as Drp1 and Mfn2, were altered upon treatment with miR-351-5p and siMiro2 (Fig. 6F and G). Thus, the suppression of Miro2 expression by miR-351-5p triggers excessive mitochondrial fission, accompanied by mitochondrial dysfunction.
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Fig. 6. Suppression of Miro2 caused mitochondrial fission and dysfunction
(A) Mitochondrial ATP production was analyzed via the Tox-Glo assay. miR-351-5p- and siMiro2-transfected cells were incubated in galactose-containing media, and ATP production level was quantified. miR-351-5p and siMiro2 decreased mitochondrial ATP synthesis.
Relative quantification was performed based on miR-Con-treated cells. Mitochondrial uncoupler CCCP was used as an inducer of mitochondrial dysfunction. (B and C) Mitochondrial membrane potential was measured via JC-1. Decreased mitochondrial potential in miR-351-5p- and siMiro2-treated HCN cells revealed a high ratio of green/red fluorescence of JC-1. (D and E) HCN cells transfected with miR-351-5p and siMiro2 were stained with MitoTracker Red. High-resolution images of HCN cells revealed excessive mitochondrial fission in miR-351-5p and siMiro2-treated cells. The observed mitochondrial fission was quantitated. (F and G) Mitochondrial fission factor Drp1 and mitochondrial fusion factor Mfn2 were analyzed through Western blotting after treatment with miR-351-5p and siMiro2. β-actin was used as a loading control. Anti-miR-351-5p restored mitochondrial dysfunction. The graph data represent the mean ± SD values (n > 3). * p < 0.05, ** p < 0.01, *** p < 0.001
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Inhibition of mitochondrial fission blocked ADCD induced by miR-351-5p/Miro2 by restoring mitochondrial function
To determine whether excessive mitochondrial fission contributes to ADCD in HCN cells, I used mitochondrial fission inhibitor Mdivi-1, specifically targeting Drp1. Pretreatment of HCN cells with Mdivi-1 before treatment with miR-351-5p or siMiro2 reduced the degree of mitochondrial division and elongated their morphology (Fig. 7A and B). After treating with Mdivi-1, HCN cells stained with JC-1 had a high red fluorescence signal (Fig. 7C and D).
Besides, inhibition of mitochondrial fission completely blocked cell death induced by miR- 351-5p (Fig. 7E and F).
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Fig. 7. Inhibition of mitochondrial fission suppressed ADCD induced by miR-351-5p via Miro2 by re-establishing mitochondrial function.
(A and B) Inhibitory effects of Mdivi-1 on mitochondrial fission induced by miR-351-5p or siMiro2 were observed and quantitated. (C and D) After treatment of Mdivi-1, mitochondrial membrane potential was measured via JC-1. (E and F) Mdivi-1 inhibited ADCD induced by miR-351-5p and siMiro2 in a dose-dependent manner. Cell death was analyzed as described above. The graph data represent the mean ± SD values (n > 3). ** p < 0.01, *** p < 0.001.
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Overexpression of Miro2 recovered cell death and mitochondrial fission induced by miR- 351-5p or insulin withdrawal on HCN cells
The ability of Miro2 to affect cell viability in HCN cells was confirmed using recombinant adenovirus (rAD-Miro2) along with miR-351-5p. For this purpose, HCN cells were treated with rAD-GFP and rAD-Miro2. Then, Miro2 protein expression was dose-dependently upregulated, and almost all cells expressed green fluorescence (Fig. 8A and B). rAD-Miro2 effectively enhanced cell viability in the miR-351-5p- or siMiro2-treated cells, whereas rAD- Con did not affect cell viability (Fig. 8C and D). Furthermore, even in mitochondrial dynamics, rAD-Miro2 restored the extent of excessive mitochondrial fission. (Fig. 8E and F).
I observed these mitochondrial morphologies to examine whether HCN cell death induced by insulin withdrawal shows any treatment features with rAD-Miro2. The mitochondrial morphology recovered as a tubular network. Furthermore, overexpression of Miro2 completely inhibited cell death and mitochondrial fission triggered by insulin withdrawal (Fig.
8G and H). These data demonstrated that Miro2 regulates cell death through mitochondrial fission, subsequently inducing mitophagy.
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３９
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Fig. 8. rAD-Miro2 restored mitochondrial fission induced by miR-351-5p
(A and B) Adenovirus expressing Miro2 was prepared. The infection efficiency of rAD in HCN cells was confirmed using rAD-GFP. The expression levels of Miro2 were investigated through Western blotting. β-actin was used as a loading control. (C and D) Cells were transfected with miR-Con, miR-351-p, or siMiro2, with either rAD-Con or rAD-Miro2. After 24 h, cells were stained with PI and observed. Cell death was calculated by counting PI- positive cells. (E and F) Mitochondrial morphology was monitored and quantitated using MitoTracker after co-treatment with rAD-Miro2 and miR-351-5p. The graph data represent the mean ± SD values (n > 3). * p < 0.05, ** p < 0.01, *** p < 0.001. (G and H) HCN cells were cultured in insulin (-) medium in the presence of rAD-GFP or rAD-Miro2 for 24 h. Cell death was measured by counting PI-positive cells. Mitochondrial morphology was monitored using MitoTracker under the confocal microscope.
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The hippocampus’ expression of miR-351-5p and Miro2, is associated with AN and AD pathophysiology
Recently, many studies have found that the occurrence of adult hippocampal neurogenesis (AHN) progressively declined in patients with AD. By analyzing human brain samples obtained under tightly controlled conditions and processing methods, I used RNA-seq data from the Allen Brain Atlas, a database of Aging, Dementia, and TBI. According to the metadata regarding the hippocampus, I found that the group of patients with AD showed a slightly decreased expression in Miro2 compared with the non-dementia group (Fig. 9A). To confirm this result, I examined the Miro2 expression in hippocampal progenitor cells of the AD mice brain. In mice, Nestin expression occurs during most of the differentiation stages of AHN, and the population of Nestin+ cells, in consequence, exhibits varying degrees of maturation. Thus, hippocampal specimens from WT and the triple-transgenic model (3xTG) were examined for Miro2 expression in Nestin-positive cells. The immunohistochemical analysis presented that the expression of Miro2 in hippocampal cells was diminished compared with that of WT mice (Fig. 9B and C).
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Fig. 9. Expression of miR-351-5p and Miro2 in the hippocampus is associated with AN and AD pathophysiology.
(A) RNA sequencing data from the Aging, Dementia and TBI Study at the Allen Brain Institutes were analyzed for Miro2 expression. The AD group (n = 24) neuropathological features of AD, including amyloid β secretion and the presence of amyloid plaques; non- dementia groups (n = 38) were formed. (B and C) To analyze the expression of Miro2 in proliferating cells in the hippocampus of a mouse brain, 15-week-old WT, and 3xTG AD model mice were examined via immunohistochemistry using anti-Miro2 and anti-Nestin antibodies. Images were observed using a confocal microscope, and levels of Miro2 were quantified specifically in Nestin+ cells using ImageJ software. ML is the molecular layer, and GCL is the granule cell layer in the dentate gyrus. The graph data represent the mean ± SD values (n = 16 cells for WT, 18 cells for 3xTG from three independent experiments). * p <
0.05; ***, p < 0.001.
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miR-351-5p and Miro2 expression showed an opposite correlation in AD model mice To confirm the expression level of miR-351-5p and Miro2, I used real-time PCR analysis of miRNA and mRNA in the hippocampus of 3xTG and 5xFAD mice. In the AD model, Miro2 expression was decreased (Fig. 10A and B). Similarly, immunohistochemical analysis revealed a decreased Miro2 expression in the 5xFAD hippocampal DG (Fig. 10C and D).
Finally, I performed in situ investigation of miRNA expression in the brain sections of 5XFAD.
The in situ hybridization is a powerful tool for studying miRNA detection of low-abundance in brain tissue. In the hippocampus, the major of neurons expressed miR-351-5p-positive.
Furthermore, the miR-351-5p signal was higher in the hippocampus of the AD mice brain (Fig.
10E). Based on these observations, the potential involvement of dysregulated miR-351-5p and its target Miro2 expression in the survival of HCN cells in AD.
４５
４６
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Fig. 10. miR-351-5p and Miro2 expression showed an opposite correlation in AD model mice.
(A and B) To compare the expression levels of miR-351-5p and Miro2, the hippocampus obtained from 10-month-old WT and 3xTG AD model mice was examined by quantitative PCR. (C and D) In addition, cryo-sections of the hippocampus were stained with anti-Miro (green) and anti-amyloid β antibody (red). ML is the molecular layer, and GCL is the granule cell layer in the dentate gyrus. The hippocampal expression level of Miro was measured using ImageJ software compared with cortex expression. The graph data represent the mean ± SD values (n > 3). ** p < 0.01; *** p < 0.001. (E) hippocampus sections were examined by in situ hybridizations using probes for miR-351-5p and U6.
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The schematic mechanism of the miR-351-5p/Miro2 axis in AN
The study reveals that miR-351-5p/Miro2 axis might play a critical role during HCN cell death derived from the overall data of this study. Upon abnormal stimuli, an increased level of miR- 351-5p downregulated Miro2 directly. Reversely, the insufficient level of Miro2 triggers excessive mitochondrial fragmentation, finally causing mitophagy and even HCN cell death, possibly leading to AD (Fig. 11).
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Fig. 11. Schematic mechanism of miR-351-5p/Miro2 axis in AN
By an unknown mechanism, abnormal stimuli increase miR-351-5p, which sequentially downregulates Miro2 in HCN cells. Aberrant low Miro2 levels readily induced mitochondrial fission, resulting in excessive mitophagy and cell death in the HCN population, which can contribute to the pathological condition in AD.
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Discussion
After discovering that neurogenesis steadily occurs in the adult hippocampus, many studies have presented that diseases such as AD, which are attributed to neuronal damage, have fewer adult neural stem cells52-54. This finding is further supported by the fact that it is smaller than the stem cell pool of older patients with AD or that AHN in patients with AD declines sharply55. The Allen Brain Institute’s Aging, Dementia, and the TBI study showed that the expression of Miro2 was notably decreased in the AD and dementia groups. Given that AD is a gradual disease that progresses over a long time, minor differences in Miro2 expression significantly impact maintaining hippocampal neurogenesis. Also, the decreased expression of Miro2 in proliferating cells in the hippocampus of AD model mice suggests that Miro2 plays a vital role in AN. Moreover, according to the current results, miR-351-5p increases, and target Miro2 located downstream decreases because the miR-351-5p/Miro2 axis maintains a pool of HCN cells. It implies that it plays an important role. Some studies suggested that neurogenesis in the adult hippocampus improves cognition and mood 56 11 57. They argued that finding the main factor of neurogenesis in the adult hippocampus shows promise as a therapeutic target for neurological diseases 58 59. Therefore, the miR-351-5p/Miro2 axis could be a promising target for maintaining the neural stem cell pool in neurodegenerative brain diseases.
Dysregulation of mitochondrial dynamics has been implicated in the pathogenesis of various human diseases, including neurological disorders60 61 62,63. Mitochondria are vital sites for energy production and are essential organelles that regulate cell survival, death, and metabolic homeostasis64. Mitochondria precisely regulate their homeostasis through various dynamic phenomena, including fission, fusion, and mitophagy. The results showed that the suppression of Miro2 expression by miR-351-5p induces excessive mitochondrial fission. Under normal physiological conditions, mitochondrial fission is an essential phenomenon to maintain mitochondrial quality by removing damaged mitochondria through mitophagy. Thus, the result
５１
is that excessive mitochondrial fission cause unexpected mitophagic events, which leads to loss of normal mitochondria, and eventually, HCN cell death. In this process, when the fission was blocked using Mdivi-1, an essential regulator of mitochondrial fission, the effect of preventing apoptosis was also seen. Overexpression of Miro2 inhibited ADCD induced by insulin withdrawal. These results demonstrate that Miro2 plays an essential role in the ADCD of HCN cells through excessive mitochondrial fission.
Miro protein, known to have a subset of Miro1 and Miro2, contributes to maintaining mitochondrial dynamics while regulating mitochondrial fission, fusion, and mitophagy6566 67. Under normal conditions, Miro GTPase and VIMAR/RAP1GDS1 complex regulate mitochondrial fission through Drp1. In our study, miR-351-5p levels were increased due to migration to mitophagy in response to mitochondrial fission and loss of normal levels of Miro2 protein. Interestingly, Miro1 and Miro2 are believed to have functional redundancy in knockout mice, despite slight phenotypic differences. Recently, several studies have revealed functional differences between Miro1 and Miro2 68 69. Our results suggest that miR-351-5p preferentially downregulates Miro2 over Miro1. Miro1 excludes the miR-351-5p target sequence in the 3'UTR (data not shown). Furthermore, I also confirmed that cell death was observed only in siMiro2, not in siMiro1, in HCN cells (data not shown). Also, Miro1 expression levels did not differ from the RNA sequencing data of the Allen Brain Institute’s Aging, Dementia, and TBI studies (data not shown). Many questions about the functional differences between Miro1 and Miro2 have not been resolved. However, these results show that Miro2 plays a potentially different role compared with Miro1 in the mitochondrial fission and fusion maintenance of HCN cells.
HCN cell death was known to be an ADCD and met the criteria proposed by Shen and Codogno 40 70. Cell death induced by miR-351-5p occurred separately from apoptosis, increased autophagic flux, and suppressed by autophagy inhibitors. In particular, the miR-351- 5p/Miro2 axis induced PINK1-Parkin-mediated mitophagy. The correlation between the Miro
５２
protein and the PINK-Parkin mediated mitophagy has been previously reported 71. Here, Miro1 is phosphorylated by PINK1 at Ser65, then ubiquitinated by Parkin and degraded by the proteasome. In addition, Miro1 knockdown suppresses mitophagy through translocation in mitochondrial Parkin. HCN cell death-mediated by the miR-351-5p/Miro2 axis was characterized by ADCD here; however, little is known about the molecular basis for HCN cell death, so an additional investigation is needed.
Several miRNAs, such as miR-9, miR-19, miR-124, and miR-137, are associated with neurogenesis development7273 74 75 76. However, these miRNAs are known to be involved in embryonic neurogenesis or neuronal fate determination, maturation, and migration. Current results indicate that miR-351-5p is a new specific miRNA for directly determining cell survival and death among HCN cells. To date, most knowledge of HCN cell survival and death has been obtained from rodent models, providing only limited information about the physiological regulation of AN. Physiological or pathological stimuli that upregulate miR-351-5p in HCN cells are still unknown, and their regulatory mechanisms require further investigation.
Overall, our study shows the aberrant expression of miR-351-5p and mitochondrial GTPase Miro2 in the hippocampus of human patients with AD and AD model mice. Therefore, these results suggest that the miR-351-5p/Miro2 axis can play an essential role in the hippocampus and contribute to the onset or progression of AD. miR-351-5p induces uncontrolled mitochondrial fragmentation through Miro2 repression, which is a critical determinant of mitophagy-dependent cell death in adult HCN cells of rats. Collectively, I showed that these miR-351-5p and Miro2 are essential regulators of HCN cell death, suggesting an association with hippocampal neurogenesis dysregulation in AD.
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