Astrocytes are known to be the most abundant cells in the brain that play an important role in maintaining the brain environment. Astrocytes maintain the physical structure of the brain, the ion homeostasis, and the secretion of extracellular matrix proteins to support the maintenance of neuronal cells and the brain environment (Simard and Nedergaard, 2004; Burda
Astrocytes in aged subjects show diverse dysfunction and altered phenotypes. In the brains of the elderly with Alzheimer’s disease, there are increased expressions of aged astrocytes and changes in astrocyte morphology (Bhat
The concept of serial passage cultivation was first described in a study back in 1961 using human diploid cells (Hayflick and Moorhead, 1961). Primary cells as well as stem cells can undergo successive passages. Interestingly, aging-related phenotypes have been observed during late passages of primary cells. For instance, late passage-cultivated mesenchymal stem cells (MSCs) displayed up-regulated SA-β-gal activity and reduced cell migration ability at (Hong
The aging of astrocytes or brain cells can be caused by various factors such as oxidative stress, shortened or dysfunctional telomeres, DNA damage, mitochondrial dysfunction, cellular senescence, and oncogenic mutations which could lead to diverse results (Munoz-Najar and Sedivy, 2011; Zhu
The materials used in this study are the following: Dulbecco’s modified Eagle medium (DMEM)/F12, Penicillin-Streptomycin (P/S), 0.25% trypsin-EDTA, and 10% Fetal Bovine Serum (FBS) from Gibco BRL (Grand Island, NY, USA); Tween® 20 and ECLTM Western blotting detection reagent from Amersham Life Science (Arlington Heights, IL, USA); anti-β Actin from Sigma (St. Louis, MO, USA); anti-iNOS and senescence detection kit from Abcam (Cambridge, UK); Agilent Seahorse XF Cell Mito Test Kit from Agilent Technologies (CA, USA); Alexa Fluor® 594 conjugated Escherichia coli (K-12 strain) BioParticles® from Thermo Fisher Scientific (MA, USA); Tetramethylrhodamine Methyl Ester (TMRM) from Thermo Fisher Scientific.
Animal care and experimental procedures were executed following the protocols and approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University (Seoul, Korea) (KU18050). Sprague-Dawley (SD) rats were obtained from Samtako, Inc (Gyeonggi, Korea). Astrocytes were cultured in the brain cortex of postnatal day 2 (P2) SD rats as described previously (Bang
Sprague-Dawley (SD) rats were obtained from ORIENT (Gyeonggi, Korea). The neurons were isolated from the cerebral cortex of embryonic day 18 (E18) SD rats. The isolated primary cortical neurons were seeded on the poly-D-lysine-coated plate (50 μg/mL) and maintained in NBM with B27 and L-glutamine in a 95% CO2 incubator at 37°C for 10 days and the media were half-changed with fresh ones every 3 days.
SA-β-gal staining was performed using Senescence Detection Kit (Abcam) to detect SA-β-gal positive cells
The grown cells on a poly-D-lysine-coated coverslip were fixed by 4% paraformaldehyde (PFA) for 10 min at 37°C. After then, the cells were permeabilized with 0.1% Triton X-100 for 20 min and washed 3 times with PBS at room temperature. The fixed samples were stained for 10 min at room temperature with DAPI (4′,6-diamidino-2-phenylindole). After then, the samples are mounted and visualized by a digital microscope (CELENA, Logos Biosystems, Gyeonggi, Korea). To measure the size of the nucleus, the indicating blue color pixels in the captured images by the digital microscope were measured using the Image-J software (NIH, MD, USA).
The expressions of mRNAs in astrocytes such as IL-1β, IL-6, TNFα, iNOS, COX2, and GAPDH were measured using RT-PCR. The RNA was isolated with TRIzol reagent (Invitrogen) and the RNA concentration was measured using a spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). cDNA was synthesized using RNA and RT reaction mixture with RevertAid Reverse transcriptase reaction buffer (Thermo Fisher Scientific) and dNTP (Promega, WI, USA). A total of 0.5 µg of cDNA was used for PCR amplification under the following cycle parameters: [94°C, 30 s; 60°C, 1 min; 72°C, 30 s]×30 cycles, then 72°C for 10 min to detect IL-1β, IL-6, TNFα, iNOS and COX2; and [94°C, 30 s; 60°C, 1 min; 72°C, 30 s]×23 cycles, then 72°C for 10 min to detect GAPDH. The primers were designed as the following: IL-1β (sense: 5’-AAA ATG CCT CGT GCT GTC TG-3’/ antisense: 5’-CTA TGT CCC GAC CAT TGC TG-3’), IL-6 (sense: 5’-TTG TGC AAT GGC AAT TCT GA-3’/ antisense: 5’-TGG AAG TTG GGG TAG GAA GG-3’), TNFα (sense: 5’-TAG CCC ACG TCG TAG CAA AC-3’/ antisense: 5’- GGA GGC TGA CTT TCT CCT GG-3’), iNOS (sense: 5’-CTG GCT GCC TTG TTC AGC TA-3’/ antisense: 5’-AGT GTA GCG TTT CGG GAT CT-3’), COX2 (sense: 5’-TGC ATG TGG CTG TGGATG TCA TCA A-3’/ antisense: 5’-CAC TAA GAC AGA CCC GTC ATC TCC A-3’), and GAPDH (sense: 5’-GTG AAG GTC GGT GTG AAC GGA TTT-3’/ antisense: 5’-CAC AGT CTT CTG AGT GGC AGT GAT-3’). The PCR products were electrophoresed with 1.2% agarose gel and imaged with ethidium bromide (Sigma). The bands were measured using the Image-J software (NIH). Each band intensity was normalized by GAPDH mRNA.
Western blot was analyzed following a previously published protocol (Bang
The cells were seeded on poly-D-lysine-coated 96-well plates at a density of 2.5×105 cells/mL and maintained for 4 days. After then, a 700 nm-wide scratch was made in each well using a certified Essen Bioscience automated 96-wound-makerTM (Essen Bioscience, MI, USA). Wound width was detected using the IncuCyte ZOOM system (Essen Bioscience) by imaging each well every 3 h for 72 h and were analyzed using the IncuCyte ZOOM microscope software 2015A (Essen Bioscience).
NO was detected as described previously by measuring the nitrite, a stable oxidation product of NO (Green
The mitochondrial OCR of cells was measured following the Agilent Seahorse XF Cell Mito Test Kit user guide (Agilent Technologies). Briefly, the cartridges were hydrated overnight with a calibrant buffer in a non-CO2 incubator at 37°C. Either of the 1.0 μM oligomycin, 1.0 μM FCCP, or 0.5 μM rotenone/0.5 μM antimycin A were put in the cartridge of each port A to C, respectively. The cell medium was changed to Seahorse XF Base Medium containing 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose and then maintained for 45 min to 1 h. The assay was performed using Agilent Seahorse XFe96 Analyzer (Agilent Technologies). The results were obtained using Wave Desktop 2.6 software (Agilent Technologies) and calculated using the Agilent Seahorse XF Cell Mito Test Kit user guide (Agilent Technologies).
Alexa FluorTM 594 conjugated
TMRM is a dye that penetrates the cells and accumulates in the mitochondria which have active membrane potentials. The cells were maintained in light protection conditions with 100 nM TMRM for 30 min in a 95% CO2 incubator at 37°C. After then, the cells were washed with phosphate-buffered saline (PBS) and visualized by a digital microscope (CELENA, Logos Biosystems). The red particles of the captured images were analyzed using the IncuCyte ZOOM microscope software 2015A (Essen Bioscience).
For the neurite outgrowth measurement, neurons were seeded on a PDL-coated well plate at 7×105 cells/mL in the culture media. The plated neurons were maintained for 2 days and were added with astrocyte cultured medium with 1/3 medium. Astrocyte cultured medium was obtained from astrocytes incubated for 24 h in serum-free conditions. We captured and measured the neurite outgrowth image using the IncuCyte® Live-Cell Analysis system by the NeuroTrack Scan Type (Essen Bioscience). We obtained the images every 12 h for 72 h.
All the experimental data were expressed as the mean ± SEM and the statistical analyses were conducted using GraphPad Prism version 5 software (GraphPad Software Inc., CA, USA). The group comparisons were performed using two-way ANOVA followed by Bonferroni’s post-test. Two-sample analyses were conducted with unpaired t-test. A
To investigate whether serial passage cultivation induces astrocyte senescence, passage 7 (P7) considered as late passage astrocytes were compared with passage 1 (P1) as early passage astrocytes. We performed SA-β-gal staining in P1 and P7 astrocytes to confirm the differences in phenotypes and the effects of serial passage. Late passage astrocytes showed increased SA-β-gal positive cell number about 1.3 times more than early passage astrocytes (Fig. 1A). The black arrows represent SA-β-gal-positive cells. The late passage astrocytes (P7) showed a significantly increased nuclear size of about 12% more than P1 (Fig. 1B). These results indicate that the P7 serial passage cultivated astrocytes exhibits the phenotypes of aged cells.
To evaluate whether the expressions of immune-related genes are changed in the late passage astrocytes, the mRNA expressions of IL-1β, IL-6, iNOS, TNFα, and COX2 using RT-PCR were measured with or without LPS. Astrocytes were treated with 0 (vehicle), 1, and 10 ng/mL of LPS for 24 h. The higher dose of LPS increased the mRNA expression of immune-related genes at least three times more in P1 astrocytes than the vehicle group including IL-1β, IL-6, iNOS and TNFα, but not COX2. Late passage astrocytes (P7 astrocytes) were detected with down-regulated mRNA expression of immune-related genes including IL-1β, IL-6, iNOS, TNFα, and COX2. Immune stimulation through LPS treatment did not have any change in the mRNA expression of the immune-related mediators in late passage astrocytes (Fig. 2A). The protein level of iNOS was also not affected by LPS treatment in late passage astrocytes (Fig. 2B). Additionally, the determination of nitrite in late passage astrocytes shows no difference in the non-stimulus group and LPS-stimulated group (Fig. 2C). These data demonstrate that late passage astrocytes may have reduced immune responses, even with LPS stimulation.
To investigate the change of astrocytic function in late passage astrocytes, we evaluated the wound healing ability and phagocytic capacity. The wound healing ability was measured using a wound-scratch assay in late passage astrocytes (P7) compared with early passage astrocytes (P1). Late passage astrocytes show a wider wound width and lower relative wound density than early passage astrocytes (P1) and 36 h after making the wound scratch, the wound of P1 was closed while the wound width of P7 was still about 260 μm (Fig. 3A, 3B). The wound healing ability was also significantly decreased in the late passage astrocytes. To confirm the phagocytotic capacity in late passage astrocytes,
To investigate the mitochondrial function in late passage astrocytes, we measured the mitochondrial membrane potential through TMRM and the mitochondrial oxygen consumption rate (OCR) in the late passage astrocytes (P7) and early passage astrocytes (P1). Late passage astrocytes (P7) showed decreased TMRM objective area and total TMRM intensity about 80% less than early passage astrocytes (P1) (Fig. 4A). Doxorubicin was used as a positive control. Mitochondrial OCR was experimented using Agilent Seahorse XFe96 Analyzer (Agilent Technologies) showing that late passage astrocytes have reduced ATP production (Fig. 4B). These results show that late passage astrocytes have decreased mitochondrial function, which indicates reduced energy metabolism.
The results above demonstrated that late passage astrocytes have decreased astrocytic functions (Fig. 3, 4). To determine the effects of late passage astrocytes on neurons, astrocyte conditioned media (ACM) was added on neurons with 1/3 medium. The ACM was harvested in serum-free conditions. The primary cortical neurons in DIV2 were treated with ACM, and the neurite outgrowth was measured every 12 h for 72 h. P1 ACM-treated neurons have increased neurite outgrowth compared with vehicle-treated neurons (no ACM). The late passage (P7) astrocyte conditioned media (P7 ACM)-treated neurons presented a down-regulated neurite outgrowth of about 30% shorter neurite in comparison with early passage (P1) ACM-treated neurons (Fig. 5). These results demonstrate that late passage astrocytes have lesser supportive functions on neurons than P1.
Astrocytes play important physical and molecular roles in the brain. Any disruption of their normal physiological function can lead to the pathology of CNS disorders. Therefore, the aging of astrocytes has the potential to affect the brain environment and function. As an important mechanism of aging, cellular senescence has been considered as an inducing factor of age-related neurodegenerative disorders. However, little is known about the importance or effect of astrocyte aging in the brain. Therefore, we attempted to make an
Astrocytes that underwent serial passage cultivation for several times showed aged astrocyte phenotypes. Various studies have reported nuclear enlargement of aged primary astrocytes (Yoon
We also examined the molecular and functional changes in late passage astrocytes. Astrocytes along with microglia are known to have immune-related functions in the brain. All neuroinflammatory and regulatory processes in the CNS are generally initiated to prevent the disruption of cellular homeostasis. The acute inflammatory response in the CNS induces the repair of damaged brain regions and is rapidly triggered by activated glial cells. However, sustained secretion of inflammatory mediators by dysregulated astrocytes can induce chronic inflammation which can lead to brain degeneration (Sochocka
Immunosenescence, a term first introduced by Walford in 1969, is characterized by a quantitative reduction in the adequate immune responses, a process that decreases responsiveness and increases vulnerability to extrinsic factors such as bacterial, viral, and fungal pathogens (Rosenstiel
In addition to the morphological and molecular features of aged astrocytes, we investigated the role of late passages on brain function including wound healing, phagocytosis, mitochondrial energy metabolism, and neurite outgrowth. Our results showed that late passage cultivated astrocytes exhibited functional changes manifested by decreased wound healing capacity. These results are consistent with our previous reports showing the decreased wound healing capacity in aging-induced astrocytes through tenovin-1 or etoposide treatment (Bang
Astrocytes show a reduced phagocytic capacity during reactive astrogliosis, while the microglia have increased phagocytosis following various stimuli and aging (Jung and Chung, 2018). Our results demonstrated that aged astrocytes through late passage cultivation have decreased phagocytotic capacity. There are some regulatory factors related to phagocytosis in astrocytes. Drosophila glial cells in aged brains have decreased phagocytosis through a decreased translation regulator,
Glial cells including astrocytes and microglia play a central role in Aβ regulation and clearance (Ries and Sastre, 2016). Microglia and astrocytes can generate Aβ degrading proteases such as neprilysin (NEP), endothelin-converting enzyme (ECE), cathepsin B (CAT-B), and matrix metalloproteases (MMPs) (Eckman
Mitochondrial dysfunction of aged astrocytes was confirmed through mitochondrial membrane potential and mitochondrial OCR. Aged astrocytes by replicative senescence showed glutamate uptake and decreased mitochondrial activity (Pertusa
Although there is no direct study on the effects of aged astrocytes on neurons, one study isolated astrocytes and neurons from old rats (24 months) and co-cultured both for 5 days. Astrocytes from old rats supported the neurite outgrowth less than astrocytes from young rats (3 months) (Rozovsky
This paper was supported by Konkuk University in 2020.
There are no conflict of interest.