Form 3
Report for Joint/Usage Research Program for Endocrine/Metabolism(Fiscal Year 2022)

Date：(Year)/(Month)/(Day)To Director of Institute for Molecular and Cellular Regulation, Gunma University
Principal Applicant
Institution

Ajou University

Position

Associate professor

Name

Bum-HO, Bin

We report on the results of joint research in fiscal 2022 as below.
（Program No.

）

１．Research Title

Golgi stress as a source of aging
Anti-aging research has been conducted from various perspectives. This study
proposes a new aging mechanism that has not been tried so far, suggests ananti-aging strategy based on this, and seeks to discover related materials. In
detail, attention is paid to the aging of the Golgi apparatus, which acts as a major
pathway of senescence-associated secretory phenotype (SASP), which is seen
when cells are aged. We reveal whether the aging of the Golgi apparatus progresses during aging, and confirms the adverse effects on cells caused by agingof the Golgi apparatus. Furthermore, factors involved in Golgi aging are identified, and related genes/proteins are discovered to reveal the mechanism of Golgi
aging. Based on the Golgi aging research method found in this way, we present
a method for screening anti-aging materials, discover active ingredients, andobserve toxicity and healthy lifespan extension in animal models. The ultimate
goal of these studies is to contribute to extending the healthy lifespan of mankind.

２．Purpose andSignificance of theresearch project● Although it is well known that senescent cells secrete numerous substances,
there is no known Golgi aging model that is directly linked to extracellularsecretion.
● The reliability and universal applicability of Golgi senescence are increased
by linking experiments using cells/animals and related cells or transcriptomes ofpatients.
● The stacking change seen in aging of the Golgi apparatus can be related to
the nutrient deficiency according to aging. So, it can be easily applied to thedevelopment of anti-aging foods by linking the actual life-long phenomenon withfood.
● It is possible to utilize ZIP13 deficient mice produced and reported previously
by paying attention to ZIP13, the Golgi zinc transporter, which we studied as agene that regulates aging of the Golgi apparatus.

３．Period of The Program

April 1, 2022 ～ March 31, 2023

４．Project Members
Name

Age

Sex

Affiliation

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Position

Role

Institute for Molecular andCellular Regulation IMCR
Gunma University

（Principal Applicant）

Bum-HO, Bin

41

M

University of Ajou, Department of biological sciences

Associate professor

Project director

30

M

University of Ajou, Department of biological sciences

Ph. D. candidate

Cell analysis（Research Collaborators）

Byungsun,Cha※If additional space is required, please attach a separate sheet.
５．Collaborating Researcher Name ofDevelopmental Biolof IMCR
Laboratory
ogy and Metabolism

Name

Ayako Fukunaka

６． Research Plans● Presenting a mouse / human Golgi aging model-Golgi apparatus-related protein changes in aging mouse / human skin and liver: WB (Western blotting) andtissue staining analysis using GM130 antibody and TGN46 antibody- Analysis of Golgi apparatus-related genes in aging mouse / human skin and liver: Q-PCR (Quantitative
PCR) analysis using Arf4, Uap1/1, Hsp47, Mt1 primers-Golgi apparatus-related genetic changes in passage-aged human fibroblasts: analysis of organelle-related
genes in Golgi cells through transcriptome analysis-Identification of aging-related genes using transcriptome data generated from multiple aging models.
Specifically, published in Nature in 2020, produced and published single-cell transcriptome data from 23
tissues and organs in mouse models of different stages of aging. From this data through systems biology
techniques, the expression dynamics of genes constituting the Golgi apparatus network and how Golgi
stress changes in each tissue according to the aging process are traced (A single-cell transcriptomic atlascharacterizes aging tissues in the mouse, Nature, 2021).
- Development of a Golgi apparatus prediction machine learning model using expression information of
Golgi apparatus-related genes. To this end, key predictive variables for model development are selectedfrom the transcriptome data of the model with reduced Golgi function from the open database, and theaforementioned single-cell transcriptome data are used as training and cross-validation data. The developed model was finally verified for its performance by utilizing the transcript data of other aging diseases
that were published.
-Golgi apparatus protein changes in passage-aged human fibroblasts: WB analysis using GM130 antibody
and TGN46 antibody-Golgi body lipid changes in passage-aged human fibroblasts: FACS (Fluorescence-activated cell sorting)analysis after staining with BODIPY-TR ceramide-Golgi body morphological changes in passage-aged human fibroblasts: Confocal microscopy/airy scan
analysis using GM130 antibody and TGN46 antibody-Changes in Golgi apparatus protease function of passage-aged human fibroblasts: WB analysis using
CREB3 antibody for S1P-dependent processing of CREB3 after BFA treatment-Changes in Golgi stress responsiveness of passage-aged human fibroblasts:
Analysis of the activationof the Golgi stress-regulating transcription factor CREB3 after induction of Golgi stress with GCA, BFA,
Momensine, etc. (WB: CREB3, Q-PCR: Arf4, Uap1/1, Hsp47, Mt1)
● Identification of the mechanism of abnormal polymerization of microtubules due to aging of the Golgi
apparatus-Analysis of microtubule polymerization of senescent cells: After treatment with Nocodazole, the degree ofrecombination, after fluorescence staining with GM130 antibody, TGN46 antibody, and alpha-tubulin antibody, image analysis using confocal microscopy-Analysis of microtubule polymerization due to Golgi stress: After Golgi stress induction with GCA, BFA,
Momensine, etc., Nocodazole treatment, recombination level, GM130 antibody, TGN46 antibody, alpha-tubulin antibody after fluorescence staining, image using confocal microscopy analyze-Aging Golgi apparatus microtubule polymerization change: analysis of Golgi apparatus-microtubule complex using Tubulin polymerization assay kit (Cytoskeleton, Inc. BK006P)-Changes in microtubules in the aged crude perinuclear fraction: WB analysis of proteins using GM130

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Institute for Molecular andCellular Regulation IMCR
Gunma University

antibody and TGN46 antibody in the non-solubilized fraction of surfactant NP40
-Changes in nuclear migration of microtubule-dependent epigenetic enzymes in senescent cells: WB
analysis using antibodies for p300 and HDAC1/2 proteins that migrated in the nucleus after separating thecytoplasm and nucleus using a sucrose cushion-Microtubule-dependent changes in nuclear migration of p53 in senescent cells: WB analysis using antibody-based p53 protein after separating the cytoplasm and nucleus using a sucrose cushion-Changes in nuclear migration of the transcription factor SMAD2/3 involved in collagen production in aging
fibroblasts: After TGF-beta treatment, fluorescence staining using SMAD2/3 antibody, and confocal microscopy/airy scan analysis
７．Research results:
Please describe the details of the contribution of the joint research with IMCR in obtaining the results.

<Result 1. Golgi stress increases during aging>
To investigate the effect of aging on the function of the Golgi apparatus, the expression levels
of Golgi proteins and proteins that change during aging were initially analyzed. The expression
levels of collagen, MT1 (a cellular zinc indicator), and histone 3 acetylation at lysine on position
18 (H3K18ac) and 27 (H3K27ac) were markedly reduced during senescence. However, the expression of the Golgi proteins GM130 and TGN46 increased (Figures 1A). In accordance, senescent cells showed expanded and loosely stacked trans-Golgi/TGN and cis-Golgi/CGN structures(Figure 1B) with increased volume (Figures 1C), indicating increased Golgi stress. Moreover, toevaluate Golgi enzyme activity, the CREB3-dependent stress response, which requires cleavageof the CREB3 protein by zinc metalloprotease S2P in the Golgi, was monitored after pharmacological disruption of the Golgi was induced by BFA. In senescent cells, CREB3 cleavage did not
progress properly, indicating Golgi dysfunction (Figure 1D). Considering the loosely packed
structure of the Golgi and reduced zinc levels in senescent cells, zinc-dependent Golgi stacking
proteins GRASP55 and Golgin45 were monitored by confocal microscopy, which showed a decrease in the co-stained area in the dispersed Golgi cisterna of senescent cells (Figure 1E). These
results indicate that Golgi stress increases during aging, and that aging, zinc deficiency, and Golgi
stress are associated with a disturbed Golgi morphology.

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Institute for Molecular andCellular Regulation IMCR
Gunma University

Figure 1. Aging-induced Golgi stress disturbs Golgi function and stacking.
(A) Western blotting of young (P6), semi-aged (P14), and aged (P32) human primary fibroblasts
with each antibody. (B) Immunostaining of young and aged human primary fibroblasts with anti-TGN46 and anti-GM130 antibodies. (C) Golgi volume analysis of young and aged human primary fibroblasts stained with BODIPY™ TR Ceramide by FACS. (D) Western blotting of young
and aged human primary fibroblasts with anti-CREB3 antibody after BFA treatment (3 µM) for4 / 12

Institute for Molecular andCellular Regulation IMCR
Gunma University

the indicated intervals. FL, Full-length; CL, Cleaved. Right panel indicates relative CREB3
cleavage level comparing to control. (E) Immunostaining of young and aged human primary fibroblasts with anti-GRASP55 and anti-Golgin45. Nuclei were stained with DAPI. C, Compact; F,
Fragmented.

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Institute for Molecular andCellular Regulation IMCR
Gunma University

<Result 2. Golgi stress disrupts perinuclear microtubule assembly and impaired protein nuclear translocation>
The Golgi apparatus is a major non-centrosomal center for microtubule nucleation and anchorage. Golgi-derived microtubules are involved in stack organization after cell division, vesicle
trafficking, cell migration, and regulation of durotaxis. Accordingly, we next isolated Golgi-associated microtubule complexes. Though the protein levels of tubulin were comparable inwhole cell lysates, Golgi-associated microtubules were markedly decreased in aged cells, as wellas in young cells treated with the Golgi stressors GCA and monensin (Figure 2A). In addition,
analysis of the nuclear and perinuclear fraction revealed decreased protein levels of tubulin inaged cells and Golgi stressor-treated young cells (Figure 2B), indicating that Golgi stress leads to
disruption of the structures of Golgi-associated perinuclear microtubules. Confocal microscopic
analysis revealed a loosely packed and fragmented Golgi in both GCA-treated young and senescent cells, but not in young cells (Figure 2C). Furthermore, in cells treated with NOC to induce
microtubule depolymerization, both GCA-treated and senescent cells showed impaired microtubule repolymerization after wash-off of NOC (NOC relief; Figure 2D), which is inconsistent withthe results observed in young cells.
Since microtubules facilitate the nuclear translocation of proteins, we speculated whether Golgi stress-mediated microtubule misassembly would affect this event. Therefore, we analyzed thenuclear content of proteins that have been reported to be involved in nuclear translocation. The
results showed that the levels of the tumor suppressor protein p53 and the epigenetic enzymes
p300 and HDAC1/2 were evidently reduced in the nuclear fraction isolated from GCA-treatedyoung cells (Figure 2E) and senescent cells (Figure 2F). To clarify the association between Golgi
microtubules and nuclear translocation, CAMSAP2, a protein that stabilizes the minus ends ofGolgi-associated microtubules in pigmented cells, was depleted by siRNA treatment in a melanocyte cell line to disrupt Golgi-associated microtubule structure.
Taken together, Golgi stress impairs the nuclear translocation of proteins by disrupting the

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Institute for Molecular andCellular Regulation IMCR
Gunma University

Golgi-associated perinuclear microtubule structures.

Figure 2. Golgi stress disrupts Golgi-associated microtubule assembly and inhibits nuclear
translocation.
(A) Western blotting of Golgi-associated microtubule complex fractions isolated from young and7 / 12

Institute for Molecular andCellular Regulation IMCR
Gunma University

aged human primary fibroblasts, and young human primary fibroblasts treated with GCA (5µM/12 h) and monensin (100 nM/12 h) for analysis of the indicated antibodies. (B) Western blotting of the nuclear and perinuclear fraction of young, young GCA- and BFA-treated, and aged
human primary fibroblasts at the indicated concentrations for 12 h for analysis of anti-αTubulin.
(C) Immunostaining of young, young GCA-treated (5 µM/12 h), and aged human primary fibroblasts with anti-αTubulin. N, Nucleus. (D) Immunostaining of young, young GCA-treated (5µM/12 h), and aged human primary fibroblasts with anti-αTubulin. Cells were treated with nocodazole (NOC; 1 µM/1 h) and then washed with PBS (NOC relief; 15 min). N, Nucleus. (E and F)
Western blotting of isolated cytosolic and nuclear fractions of young, young GCA-treated (5µM/12 h), and aged human primary fibroblasts with the indicated antibodies.

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Institute for Molecular andCellular Regulation IMCR
Gunma University

<Contribution>
Animal experiments (IMCR + Ajou univ.)
Young (4 weeks old) and old (40 weeks old) C57BL/6JN mice were obtained from JA BIO(Suwon, Republic of Korea). Zip13-/- transgenic C57BL/6JN mice backcrossed for more thanseven generations were provided by Gunma University, Japan. Mice were housed and handled at
the Department of Meridian and Acupoint of the College of Korean Medicine, Semyung University (smecae 201001), according to the guidelines approved by the Institutional Animal Care and
Use Committee (IACUC). All mice were housed in a specific pathogen-free barrier facility,
maintained under a 12-h light-dark cycle, provided water ad libitum, and fed a standard AIN-76A
rodent diet (Doo Yeol Biotech, Seoul, Republic of Korea). Because Zip13-/- mice showed hypodontia, they were fed a powdered diet (Doo Yeol Biotech) to provide appropriate nutrition. Micein the zinc-deficient diet experiment were fed an AIN-76A Zn-deficient diet for 4 weeks (DooYeol Biotech).

Cell culture and materials (IMCR + Ajou univ.)
Human primary fibroblasts were purchased from the American Type Culture Collection(ATCC; Manassas, VA, USA), and cultured until young (P6), semi-old (P14), or aged (P32).
Mouse wild-type (Zip13+/+) and Zip13-deficient (Zip13-/-) dermal fibroblasts were obtained aspreviously described [55]. All cells were maintained in Dulbecco Modified Eagle Medium(DMEM)-high glucose culture medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; GenDEPOT, Katy, TX, USA) and 1% penicillin and streptomycin (P/S;
Welgene, Gyeongsan, Korea). The cells were then incubated in an atmosphere of 5% CO2 at
37 °C. N,N,N′,N′-Tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), golgicide A (GCA),
monensin (Mon), brefeldin A (BFA), nocodazole (NOC), etoposide (Eto), nutlin-3, doxorubicin(Dox) (Sigma-Aldrich, St. Louis, Missouri, USA), and H2O2 (Junsei Chemical Co., Tokyo, Japan)

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Institute for Molecular andCellular Regulation IMCR
Gunma University

were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich) and maintained at −36 °C until
further use. Antisense LNA GapmeR in vivo Large Scale (LNA) (QIAGEN, Germantown, MD,USA) was used according to the manufacturer’s instructions. Recombinant mouse TGF-β (R&D
Systems, Minneapolis, MN, USA) was reconstituted in HCl containing bovine serum albumin(BSA), according to the manufacturer’s instructions, and maintained at −80 °C. Cell irradiation
experiments were performed using Stratalinker UV 1800 crosslinker (Stratagene, San Diego, CA,
USA).

Western blotting (IMCR + Ajou univ.)
The cells were lysed with RIPA buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.25% sodium deoxycholate, 1% Triton X-100, and 1 mM EGTA) for 5 min, and the lysate was centrifuged at 13,000 rpm for 15 min. The supernatant was collected, and the sample protein concentration was determined using Bicinchoninic Acid (BCA) Protein Assay Kit (P8100BCA; GenDEPOT). Samples were prepared in sample buffer and subjected to polyacrylamide gel electrophoresis, and a wet transfer was performed. Blots were blocked with 1% BSA in Tris-bufferedsaline Tween 20 (TBST) for 1 h and rinsed thrice with TBST for 5 min each time. The membranes were incubated with the primary antibodies (Table S1) overnight at 4 °C. After rinsing
thrice with TBST for 5 min each time, the membranes were incubated with HRP-conjugated polyclonal secondary antibodies (GenDEPOT) diluted at 1/10000 for 1 h at room temperature. Blot
images were acquired using Fusion Solo S chemiluminescence imaging system (Vilber, Collégien, France).

Immunofluorescence staining of cells (Ajou univ.)
Cells were cultured on Lab-Tek chamber slides (Thermo Fisher Scientific), fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS), and permeabilized with 0.1% Triton X-100 in
PBS containing 1% BSA for 5 min. Subsequently, the cells were stained with antibodies (Abcam,

10 / 12Institute for Molecular andCellular Regulation IMCR
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Cambridge, UK) and 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). Secondary staining
was performed using Alexa Fluor 488-conjugated F(ab’)2 Fragment fluorescent dye (Invitrogen,
Carlsbad, CA, USA). Fluorescence was detected using LSM 800 confocal microscope (Carl
Zeiss, Jena, Germany).

Immunoprecipitation (Ajou univ.)
The assay was performed using MagListo™ Protein G Kit (Bioneer, Daejeon, Republic ofKorea). Briefly, AccuNanoBead™ Protein G Magnetic Nanobeads were washed twice with
Binding & Washing buffer using MagListo™-2 Magnetic Separation Rack (Bioneer). Subsequently, anti-Golgin45 antibody (ab155510; Abcam) was added to 500 μL of Binding & Washingbuffer and incubated in a Roto-Minim rotator (Benchmark Scientific, Sayreville, NJ, USA) for 10
min at room temperature, and the supernatant was discarded. The cells previously cultivated in a
60-mm dish at 80% confluency were lysed with NP40 lysis buffer (150 mM NaCl, 1% NP40, and
50 mM Tris-HCl (pH 7.5)), centrifuged at 15,000 rpm for 5 min, and the supernatant was transferred to a 1.5-mL tube containing the bead complex. The samples were incubated in a rotator
overnight at 4 °C. The following day, the tube was placed on the magnetic rack for 1 min andwashed twice with 1 mL NP40 lysis buffer. Next, NP40 buffer and Elution buffer were added at aratio of 1:1 to the bead complex, gently mixed, and incubated at 70 °C for 10 min. After removalof the magnetic beads, the eluted fraction was transferred to a new tube, and sample buffer wasadded.

Isolation of Golgi-associated microtubule complex fractions (Ajou univ.)
The assay was performed using the Microtubules/Tubulin In Vivo Assay Kit (Cat. #BK038;
Cytoskeleton Inc., Denver, CO, USA), according to the manufacturer’s instructions. Cells werelysed using LMS2 buffer at 37 °C, homogenized using Dounce homogenizer, and centrifuged at
3,000 rpm for 5 min at 37 °C. The pellets containing large complexes of microtubules attached to

11 / 12Institute for Molecular andCellular Regulation IMCR
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the nucleus and Golgi apparatus were diluted in sample buffer.

Isolation of soluble and nuclear and perinuclear area fractions (Ajou univ.)
The cells were lysed using NP40 lysis buffer and centrifuged at 13,000 rpm for 15 min at 4 °C.
The supernatant was collected as a soluble fraction. The pellet was collected as an insoluble fraction containing a crude nuclear and perinuclear fraction and sonicated for 5 min. Protein concentration was determined by BCA assay and samples were dissolved in sample buffer.

８．Present status of academic conference presentations and research papers associated with the resultsof the joint research, and exchange of information on the joint research with the collaborating researcher at
IMCR.
(As much as possible, please state papers that include the names of the collaborating researcher at IMCR
or papers stating that the research was supported by the Joint Research Program with IMCR.
Regarding papers, please send a PDF file together with the report to the email address of the general affairs section of the Institute.) Office of General Affairs: kk-msomu4@jimu.gunma-u.ac.jp① Please list the publications that include the name of the collaborating researcher from IMCR and send a

reprint of each publication to IMCR.
-In preparation② Please list the publications that include a description that the research was supported by the Joint
Research Program with IMCR and send a reprint of each publication to IMCR.
-In preparation

List up to 3 conferences (name of conference, date of conference, and title of the presentation).
-None

③

Exchange of information exchange with collaborating researcher from IMCR (please list main points ofcommunication).
-Golgi stress increases during aging-Golgi stress disrupts perinuclear microtubule assembly and impaired protein nuclear translocation④

12 / 12Institute for Molecular andCellular Regulation IMCR
Gunma University