
Aging is one of the biggest problems in human society at the moments with the rapidly growing elderly population and the research on aging as well as geriatric diseases has uppermost importance. Therefore, delineating the mechanisms of aging-related changes in cellular functions would be the first step to understand aging and to devise the methods to regulate aging and aging-related diseases. It is obvious that each cell types in the brain including neurons and astrocytes would undergo cell type-specific aging process. Therefore, it is both necessary and important to study the aging process and the mechanism underlying it at the level of each cell types. However, most of the aging studies involving central nervous system (CNS) have been focused on neurons or microglia. This may pose a problem in the interpretation of aging process in the brain considering the magnitude of the cell population and proliferation potency of the other important cell types, i.e. astrocytes. For example, a decrease in brain volume is observed as one of the characteristics of brain aging, but Freeman
With the multitude of the function served by the astrocytes, functional dysregulation of astrocytes with aging may also affect various other brain cells resulting in the dysregulation of the maintenance of the optimal brain environment (Dossi
In this study, we developed an
The materials used in this study are as follows: Dulbecco’s modified Eagle medium (DMEM)/F12, Penicillin-Streptomycin (P/S), 0.25% trypsin-EDTA, and 10% Fetal Bovine Serum (FBS) were from Gibco BRL (Grand Island, NY, USA); Tween® 20 and ECLTM Western blotting detection reagent were from Amersham Life Science (Arlington Heights, IL, USA); anti-β Actin was obtained from Sigma (St. Louis, MO, USA); anti-iNOS and senescence detection kit were the product of Abcam (Cambridge, UK); minoxidil was obtained from Sigma; Agilent Seahorse XF Cell Mito Test Kit was from Agilent Technologies (CA, USA); Alexa Fluor® 594 conjugated
Animal maintenance and experimental processes were performed following the rules and conditions approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University (KU19017). Sprague-Dawley (SD) rats were purchased from Samtako, Inc. (Osan, Korea). Astrocytes were cultured from brain cortex of postnatal day 2 (P2) SD rats as described previously (Bang
Pregnant Sprague-Dawley (SD) rats were purchased from ORIENT (Seongnam, Korea). The primary neurons were dissected from the cortex of embryonic day 18 (E18) SD rats. The isolated cortical neurons were mechanically triturated and seeded on the poly-D-lysine-coated plate (50 μg/mL). The seeded cells were maintained in NBM with B27 supplement and L-glutamine in a 95% CO2 incubator at 37°C. The media were half-changed every 3 days.
SA-β-gal positive cells were detected using Senescence Detection Kit (Abcam)
Cells were incubated on a poly-D-lysine-coated coverslip and were fixed by 4% paraformaldehyde (PFA) for 10 min at 37°C. After then, the cells were permeabilized by 0.1% Triton X-100 for 20 min at room temperature. The fixed cells were stained by DAPI (4′,6-diamidino-2-phenylindole) for 10 min at room temperature. The samples were mounted with GEL/MOUNT (Biomeda Corp., CA, USA) and visualized by a digital microscope (CELENA, Logos Biosystems, Anyang, Korea).
The RNA isolation was conducted with TRIzol reagent (Invitrogen). The concentration of extracted RNA was measured using a spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). The RNA was used for the synthesis of cDNA with RT reaction mixture containing RevertAid Reverse transcriptase reaction buffer (Thermo Fisher Scientific) and dNTP (Promega, WI, USA). cDNA was used as template for PCR amplification with following protocol: [94°C, 30 s; 60°C, 1 min; 72°C, 30 s]×30 cycles, then 72°C for 10 min; or [94°C, 30 s; 60°C, 1 min; 72°C, 30 s]×23 cycles, then 72°C for 10 min. The sequences of primers used in this study are as follows:
Western blot experiment was conducted following a previously described procedure (Bang
The plasminogen activator inhibitor-1 (PAI-1) activity was measured through inverse casein zymography following previously described methods (Ko
Confluent cells were scratched 700 nm-wide using a certified Essen Bioscience automated 96-wound-makerTM (Essen Bioscience, Hertfordshire, UK). Wound width and density were measured using the IncuCyte ZOOM system (Essen Bioscience, MI, USA) and were analyzed using the IncuCyte ZOOM microscope software 2015A (Essen Bioscience).
Alexa FluorTM 594 conjugated
The cells were incubated with 100 nM TMRM staining solution in light protection conditions for 30 min at 37°C. After then, the cells were rinsed with phosphate-buffered saline (PBS) and imaged by the IncuCyte ZOOM system (Essen Bioscience). The images were analyzed by the IncuCyte ZOOM microscope software 2015A (Essen Bioscience).
The measurement of mitochondrial OCR was proceeded following the Agilent Seahorse XF Cell Mito Test Kit user guide. Briefly, the sensor cartridges were incubated overnight with a calibrant buffer in a non-CO2 incubator at 37°C. Mitochondrial modulators such as 1.0 μM oligomycin, 1.0 μM FCCP, or 0.5 μM rotenone/0.5 μM antimycin A were put in each port of cartridge as instructed. The cells were incubated with Seahorse XF Base Medium containing 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose for about 1 h. Mitochondrial OCR was measured by Agilent Seahorse XFe96 Analyzer. The results were analyzed by Wave Desktop 2.6 software (Santa Clara, CA, USA).
Astrocyte cultured media (ACM) were harvested from astrocyte cultured for 24 h in serum free condition. The rat cortical neurons were incubated for 2 days and added with ACM obtrained from young and aged culture. The length of the neurite was measured using the IncuCyte® Live-Cell Analysis system (Essen BioScience, Inc., Ann Arbor, MI, USA).
Astrocytes were incubated for 2 or 12 weeks and sub-cultured. The sub-cultured astrocytes were treated with vehicle (DPBS; Dulbecco’s Phosphate-Buffered Saline) or 10 ng/mL lipopolysaccharide (LPS) and harvested with trizol. The samples were used for RNA-sequencing by a commercial sequencing company (Macrogen, Seoul, Korea).
The cells were incubated with 5 μM MitoSOXTM (Invitrogen) for 10 min at 37°C, protected from light. The samples were washed gently 3 times. The level of mitochondrial superoxide was measured using the IncuCyte® Live-Cell Analysis system.
All the experimental data were presented as the mean ± SEM and the statistical analyses were performed using GraphPad Prism version 5 software (GraphPad Software Inc., CA, USA). The group comparisons were conducted using two-way ANOVA followed by Bonferroni’s post-test. Two-sample comparison was analyzed with unpaired t-test. A
Astrocytes were incubated for 2 (short-term cultured astrocytes) or 12 (long-term cultured astrocytes) weeks. Cells were stained with SA-β-gal to confirm whether long-term cultured astrocytes show cellular senescence phenotype. Usually, astrocytes are cultured for 2 weeks and are used for experiments. To establish senescent cells, astrocytes were cultured for 4, 8, 10, 12, 16 and 20 weeks (data not shown). While astrocyte cultures exceeding 16-week period separated from the culture plate, cells cultured for less than 10 weeks did not show a senescent phenotype. As a result, we found that 12-week cultured astrocytes could be maintained for the longest time without being separated from the culture plate. Long-term cultured astrocytes displayed SA-β-gal positive cells 11 times more than short-term cultured astrocytes (Fig. 1A). Senescent cells exhibit nucleus enlargement phenotype (Yoon
To determine whether long-term cultured astrocytes show the molecular phenotypes of senescent cells as well, we first performed RT-PCR to detect mRNA expression, which has been implicated in cellular senescence. Those senescence-related factors include immune- and cell cycle-related factors as well as protease-related factors. In this study, the expression level of
To identify functional significance of the astrocyte senescent phenotypes, astrocyte migration, phagocytosis, and mitochondrial function were examined. Migration of cells was investigated through wound-scratch assay. After making scratch wound with a width of about 700-800 μm on the confluent astrocyte cultures, the width and density of wound were automatically determined using computer-aided analytical tools (Incucyte, Essen Bioscience). Wound closure of long-term cultured astrocytes was significantly slower than cells cultured for short-term (Fig. 3A). The rate of decrease in wound width was slower in long-term cultured cells and the relative cell density in wound, which represents the cell density in the wound area compared to the cell density outside the wound area that changes over time, was higher in the short-term cultured cells compared to the long-term cultured cells. Next, phagocytosis function of astrocytes was investigated using
Astrocytes and neurons interact closely, and consequently, we reasoned that dysfunction of astrocytes in long-term culture could adversely affect neuronal function such as neurite outgrowth and mitochondrial OCR (Lee
RNA-seq was performed to examine the gene expression profile of long-term cultured astrocytes and comparative transcriptome analysis was performed according to the workflow in Fig. 5A. The gene ontology of the selected target gene was identified through GENE MENIA and GOrilla. Genes which showed a significant change (38 up-regulated genes, and 64 down-regulated genes (|log2
In addition, CMap was performed to find perturbagens showing opposite phenotype to the changes in gene expression in long-term cultured astrocytes. Among the perturbagens identified through CMap, candidates having the potential to regulate changes in long-term cultured astrocytes were selected and further investigated (Fig. 5B).
We confirmed whether the functional dysregulation of long-term cultured astrocytes can be modulated by selected perturbagens identified with CMap analysis (Fig. 5B). We preliminarily screened several candidates and minoxidil showed most significant functional changes in long-term cultured astrocytes. During the long-term culture period, minoxidil was administered for the last 2 weeks, and it was examined whether the functional changes of astrocytes by long-term culture can be reversed. The number of SA-β-gal positive cells increased during long-term culture, which was significantly reduced by minoxidil treatment (Fig. 6A). In addition, the size of the nucleus was enlarged by long-term culture that was significantly reduced in the minoxidil-treated group (Fig. 6B). The size of nucleus in minoxidil treated group was exhibited similar size with short-term cultured astrocytes (Fig. 6B). In the zymography experiments, the increased PAI-1 activity was normalized by minoxidil treatment (Fig. 7). The mitochondrial function was examined using MitoSOX™. The increased production of mitochondrial superoxide by long-term culture was mitigated in the minoxidil-treated group (Fig. 8). Taken together, the dysregulation of astrocyte functions by long-term culture was normalized by treatment with minoxidil.
In this study, cellular senescence of astrocytes was induced through long-term culture. We have previously reported the induction of cellular senescence of astrocytes using several different methods such as drug treatment or high-passage subculture
Senescent cells release senescence-associated secretory phenotype (SASP) such as cytokines, chemokines and growth factors (Elkhattouti
p16 is a tumor suppressor that slows cell division by inhibiting CDK and is considered as a biomarker of cellular senescence (Hall
Functional examination of aged astrocytes in this study showed prominent defects in cell migration. In this regard, it is noteworthy that tPA and PAI-1 are well known targets and regulators of proliferation and migration pathways (Czekay
Mitochondrial dysfunction including mtDNA mutations, decreased mitochondrial potential. Actually, the imbalance in mitochondrial dynamics is regarded as one of the indicators of aging. (Sugrue and Tatton, 2001; Seo
Transcriptomic profiling using GENE MANIA and GOrilla, revealed major changes in cytokines/chemokines and related functions as well as regulations of extracellular regions. This is consistent with the previous research suggesting the up-regulation of immune-related factors such as SASP with aging (Coppe
Through the CMap analysis and subsequent validation studies, minoxidil has been identified as a potential candidate to reverse or prevent astrocyte aging. Minoxidil is a pyrimidine derivative drug used to treat high blood pressure and hair loss. Interestingly, minoxidil has been implicated in neuroprotection, induction of cell growth factor, and stimulation of cell proliferation (Messenger and Rundegren, 2004; Chen
In conclusion, we demonstrated that long-term culture can be used as an appropriate cell model for astrocyte aging research and for screening of anti-aging drugs. Genetic profiling analysis using aged astrocytes may provide us chances to excavate a potential therapeutic candidate against aging and various neurodegenerative diseases that is, in this case, minoxidil.
This work was supported by the Brain Research Program (2019M3C7A1031455) and the Basic Science Research Program (2022R1A2C1005917) through the National Research Foundation (NRF) of Korea funded by the Ministry of Science, ICT & Future Planning, and the NRF grant funded by the Korea government (MSIT) (2016R1A5A2012284, 2017M3A9G2077568 and 2020M3E5D9080165).
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