Exp Neurobiol 2021; 30(5): 341-355
Published online October 31, 2021
© The Korean Society for Brain and Neural Sciences
Shinrye Lee, Yu-Mi Jeon, Myungjin Jo and Hyung-Jun Kim*
Dementia Research Group, Korea Brain Research Institute (KBRI), Daegu 41062, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-53-980-8380, FAX: 82-53-980-8389
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Sirtuin 3 (SIRT3), a well-known mitochondrial deacetylase, is involved in mitochondrial function and metabolism under various stress conditions. In this study, we found that the expression of SIRT3 was markedly increased by oxidative stress in dopaminergic neuronal cells. In addition, SIRT3 overexpression enhanced mitochondrial activity in differentiated SH-SY5Y cells. We also showed that SIRT3 overexpression attenuated rotenoneor H2O2-induced toxicity in differentiated SH-SY5Y cells (human dopaminergic cell line). We further found that knockdown of
Dopaminergic neurons are the major source of dopamine (DA) in the CNS. Dopaminergic neurons play an important role in the control of brain functions including movement, cognition, learning, and emotion. Previous studies have suggested that oxidative stress, neuroinflammation, and ageing significantly exacerbate dopaminergic neuronal death [1-3]. Many clinical and experimental studies have suggested that the death of dopaminergic neurons is linked to PD [4, 5]. Interestingly, several proteins related with Parkinson's Disease (PD), such as
Mitochondria are major intracellular organelles that modulate various cellular processes, such as cell death, autophagy, differentiation, cell cycle, and proliferation . Many studies have reported that mitochondrial dysfunction is caused by the dysregulation of mitochondrial proteins under various stress conditions . Moreover, mitochondrial dysfunction induces excessive ROS generation and ATP deficits, leading to dopaminergic neuronal death . Recent studies have suggested that mitochondrial dysfunction is associated with many metabolic diseases, including neurodegenerative diseases, immune diseases, cancer, diabetes, and obesity [10-14]. These results demonstrate that mitochondrial dysfunction may play a crucial role in dopaminergic neuronal death.
Sirtuin 3 (SIRT3) is a member of the class III histone deacetylase family, and it exhibits deacetylase activity in mitochondria. Several studies have shown that SIRT3 plays an important role in regulating ATP production, antioxidant mechanisms, inflammation, autophagy, cell death, and metabolism [15-18]. SIRT3 is predominantly localized in the mitochondria and nucleus [19, 20] and is primarily expressed in metabolically active tissues, including the brain, muscle, liver, kidney, and heart . Additionally, the level of SIRT3 is modulated by mitochondrial stress, oxidative stress, proteotoxic stress, and starvation conditions [15, 22, 23]. SIRT3 prevents dopaminergic neuronal loss cause by oxidative stress in
In the present study, we demonstrated that SIRT3 expression in dopaminergic neurons was upregulated by oxidative stress and that overexpression of SIRT3 attenuated oxidative stress-induced neuronal toxicity. Moreover, we found that SIRT3 regulated the cell death caused by LPS/IFN-γ treatment in astrocytes. By studying the dopaminergic neuronal system in a
The following reagents were purchased from the indicated providers: dimethyl sulfoxide (DMSO; Sigma, D8418), all-
The SH-SY5Y human neuroblastoma cell line was maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, 11995-065) supplemented with 10% heat-inactivated foetal bovine serum (FBS, Gibco, 16000-044) and 50 µg/ml penicillin-streptomycin (Gibco, 15140-122). Cells were grown at 37℃ in a humidified atmosphere containing 5% CO2. For differentiation, SH-SY5Y cells were grown to confluence, and the growth medium (DMEM+10% FBS) was exchanged for differentiation medium (1:1 DMEM/Ham’s F12 supplemented with 1% FBS, 1% MEM-NEAA, 50 μg/ml P/S, and 10 μM all-
SH-SY5Y cells in 6-well plates (40×104 cells/ml) were transfected with 4 µg of human
Differentiated SH-SY5Y cells in 6-well plates (40×104 cells/well) were transfected with control siRNA (Santa Cruz; sc-37007), human
Primary cell cultures Primary cultures of dissociated cerebral cortical neurons were prepared from 16-day-old embryonic C57/BL6 mice as described previously [29, 30]. Briefly, mouse embryos were decapitated, and the brains were rapidly removed and placed in a culture dish containing HBSS (Gibco). Cortices were isolated, transferred to a conical tube and washed twice in HBSS (Gibco). Cortical tissues were enzymatically digested with prewarmed papain (20 units/ml) (Worthington Biochemical Corporation) and DNase I (0.005%) for 30 min at 37℃. The tissues were mechanically dissociated (triturated) with 1,000 μl and 200 μl pipette tips to achieve complete tissue homogenization. The cortical cells were centrifuged at 130×g for 10 min at room temperature, and the dissociated cells that were obtained were seeded onto plates coated with poly-D-lysine (Sigma-Aldrich) in neurobasal media containing 2 mM glutamine (Gibco), N2 supplement (Gibco), B27 supplement (Gibco), and 50 μg/ml penicillin-streptomycin (P/S, Gibco). The culture media were changed initially after 5 days and every 3 days thereafter, and the cells were used after being cultured for 14~21 days.
Primary astrocyte cultures were prepared from 1- to 2-day-old C57/BL6 mice as described previously . Briefly, whole brains were homogenized and passed through a 70-μm strainer. The cells were seeded in T75 culture flasks. The cells were grown at 37℃ in a humidified atmosphere containing 5% CO2. The culture medium was changed initially after 5 days and every 2 days thereafter, and the cells were used after being cultured for 14~21 days. Secondary pure astrocyte cultures were obtained by shaking mixed glial cultures at 250 rpm for 4 h; then, the culture medium was discarded. Astrocytes were dissociated using trypsin-EDTA (Life Technologies) and then centrifuged at 800×g for 30 min. The astrocytes obtained were seeded onto plates in DMEM (Life Technologies) supplemented with 10% heat-inactivated FBS and 50 μg/ml P/S. The purity of the cells in culture was determined by immunocytochemistry, which indicated that the cultures contained over 93% GFAP-positive cells. The animals used in the current research were acquired and cared for in accordance with the guidelines published in the National Institutes of Health
Differentiated SH-SY5Y cells (8×104 cells/ml) were grown in 96-well plates and treated with rotenone/H2O2 as indicated for 24 h. DMSO was used as a negative control. To measure cytotoxicity, Cell Counting Kit-8 (CCK-8, Enzo Life Science, ALX-850-039-KI02) was used according to the manufacturer’s instructions. Briefly, 10 µl of CCK-8 reagent was added to each well, and the plate was incubated at 37℃ for 2 h. The absorbance at 450 nm was measured by using a microplate reader (Tecan). Cell viability was expressed as a percentage of the control. All experiments were performed in triplicate.
CMFDA fluorescence-based assay was used to investigate additional neuronal toxicity. Differentiated SH-SY5Y cells and primary cortical neurons were labeled with a 5 μM concentration of CellTracker Green CMFDA Dye (Invitrogen) for 30 min using the manufacturer’s protocol. Then, the dye solution was aspirated, and the cells were incubated with dye-free medium for 45 min. The samples were mounted and observed with a microscope. Photomicrographs from three randomly chosen fields were obtained and the number of CMFDA-positive cells was counted. The quantification of CMFDA-positive cells was expressed as a percentage of control.
Cells were homogenized in Cell Lysis Buffer (Cell Signaling, 9803) containing protease and phosphatase inhibitor cocktails. The protein concentrations of the cell lysates were determined by BCA protein assay (Thermo Fisher Scientific, 23225). Next, the protein extracts were mixed with 4× Bolt LDS Sample Buffer (Invitrogen) and 10× Bolt Sample Reducing Agent (Invitrogen), and then they were boiled at 95℃ for 5 min. An equal amount of protein from each sample was separated on Bolt 4~12% Bis-Tris gels (Invitrogen, NW04120BOX) or NuPAGE 3~8% Tris-Acetate gels (Invitrogen, EA0378BOX) and then transferred to polyvinylidene difluoride (PVDF, Invitrogen, LC2005) membranes. After blocking the membranes with 5% skim milk in TBS with 0.025% Tween 20, the blots were probed with antibodies as indicated and detected with an ECL Prime Kit (GE Healthcare, RPN2232). Samples from three independent experiments were used, and the relative expression levels were determined using a Fusion-FX Imaging System (Viber Lourmat).
RNA was extracted from cells and fly heads by using a TRIzol Plus RNA Purification Kit (Invitrogen, 12183-555) according to the manufacturer’s instructions. cDNA synthesis was performed at 37℃ for 120 min from 100 ng of RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). Quantitative RT-PCR was performed using a one-step SYBR® PrimeScript RT-PCR Kit (Takara Bio Inc., RR420A) according to the manufacturer’s instructions, which was followed by detection using an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems).
For assessment of neuronal mitochondrial dysfunction, differentiated SH-SY5Y cells (7×104 cells/ml) were seeded into XF 24-well culture plates (Seahorse Bioscience). The cells were washed twice with XF Base Medium supplemented with 2 mM L-glutamine, 10 mM D-glucose and 1 mM sodium pyruvate (pH 7.4) and incubated at 37℃ in a non-CO2 incubator for 1 h. Mitochondrial dysfunction was evaluated using the XF Cell Mito Stress Test Kit (Seahorse Bioscience) according to the manufacturer’s instructions, followed by measurement using an XF24 Extracellular Flux Analyser (Seahorse Bioscience). The 24-well utility plate was hydrated, treated with 2 µM oligomycin, 2 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), 0.5 µM antimycin A+rotenone, and thenused to calibrate the analyser. . The basal oxygen consumption rate (OCR), ATP production, maximum reserve and respiratory capacity were calculated as previously described , with averages calculated from 4 wells per condition in each individual experiment. The OCR was normalized to the total protein concentration (OD). After Seahorse analysis, the plate was centrifuged at 280×g for 5 min. The media was aspirated, and the plate was washed twice with PBS. The cells were lysed in RIPA buffer. Protein concentrations in the cell lysates were determined using a BCA Assay Kit.
Primary astrocytes were transfected with human SIRT3 expression construct or mouse
Adult males (0 to 1 day old) were separated and transferred into experimental vials containing fly media mixed with rotenone (500 µM) at a density of 20 flies per vial (
Adult flies were dissected in PBS and fixed in 4% PFA in PBS for 1 day at 4℃. The brains were then washed six times with PBS and pre-incubated in PBS-T for 1 day at 4℃. After blocking with 5% normal goat serum in PBS-T (0.3% Triton X-100) for 1 hr. The DAPI (4’, 6-diamidino-2-phenylindole dihydrochloride) were incubated with 5% normal goat serum in PBS-T for 2 days at 4℃. The samples were mounted and observed with fluorescence confocal microscope (Leica). Photomicrographs of dopaminergic neurons labeled with mCD8-GFP were acquired through confocal Z-stacks. Numbers of dopaminergic neurons were counted per brain for PPL1clusters.
Data were analyzed by unpaired Student’s
To determine the effects of SIRT3 on oxidative stress-induced neurotoxicity, we first examined the levels of SIRT3 protein in dopaminergic neuronal cells under various stress conditions such as MG132 (ubiquitin proteasome system inhibitor), rapamycin (autophagy inducer), H2O2 (reactive oxygen species) and rotenone (mitochondrial electron transport chain inhibitor). To do this, we induced the differentiation process of SH-SY5Y cells. Previous studies indicated that differentiated SH-SY5Y cells show dopaminergic neuron-like properties, such as neurite extension and the expression of dopaminergic neuronal markers [34-36]. Several studies showed that the protein level of SIRT3 is upregulated by oxidative stress. Moreover, it is already known that MG132 and rapamycin can induce oxidative stress and cell death [37, 38]. In subsequent experiments, we used rotenone and H2O2 as stimuli to induce oxidative stress. To examine the effect of the SIRT3 on the oxidative stress-induced cell death and mitochondrial dysfunction, we generated stable SH-SY5Y cell lines expressing Flag-tagged human SIRT3. We observed whether SIRT3 modulates mitochondrial activity in differentiated SH-SY5Y cells. We monitored the cellular OCR in real time as a measure of mitochondrial respiration and glycolysis using a Seahorse XF24 Extracellular Flux Analyser and a Mitochondrial Stress Test Kit (Seahorse Bioscience). Sequential injections of oligomycin, FCCP, and antimycin A+rotenone enable the measurement of basal respiration, ATP production, maximal respiration, and spare respiratory capacity. Interestingly, we found that the basal respiration, ATP production, and maximal respiration parameters were markedly decreased in Flag-expressing cells compared to SIRT3-overexpressing cells, but the spare respiratory capacity was not altered (Fig. 1C, 1D). Moreover, OCR values were normalized to the total cellular protein concentration for each group. These findings reveal that SIRT3 may play a critical role in the modulation of mitochondrial activity in dopaminergic neuronal cells.
We next investigated whether overexpression of SIRT3 regulates rotenone- or H2O2-induced neuronal toxicity in differentiated SH-SY5Y cells. We showed that rotenone or H2O2 treatment of differentiated SH-SY5Y cells induced cytotoxicity in a dose-dependent manner, and SIRT3 overexpression attenuated rotenone- or H2O2-induced neuronal toxicity (Fig. 2A, 2B). We also confirmed the CCK-8 assay data using CMFDA staining (fluorescent cell tracker). Oxidative stress-induced reduction of CMFDA-labeled cells was notably lower in SIRT3-overexpressiong cells compared to Flag-expressing cells (Fig. 2C). Moreover, we performed subcellular fractionation to measure the SIRT3 protein levels in both the mitochondria and the cytosol. The levels of both mitochondrial and cytosolic SIRT3 protein were significantly increased in SIRT3-overexpressing cells compared to Flag-expressing cells (Fig. 2D). Taken together, these findings suggest that SIRT3 overexpression suppresses rotenone- or H2O2-induced toxicity in dopaminergic neuronal cells.
To further confirm that knockdown of
In mammals, calorie restriction induces upregulation of SIRT3 . To check whether Sirt2 function in
In various neurodegenerative diseases, proinflammatory reactive astrocytes contribute to neuronal death by secreting neurotoxic factors such as proinflammatory cytokines and chemokines. Previous studies demonstrated that reactive astrocyte induced neuronal defect could be a major characteristic of PD [40-42]. Moreover, glial activation is implicated in the dopaminergic neuronal loss in α-synuclein and MPTP-based PD animal models [43, 44]. In addition, reactive astrocytes were accumulated in the substantia nigra pars compacta of PD patients [45, 46]. SIRT3 is expressed both neurons and astrocytes. Thus, SIRT3 may act not only as a cell-intrinsic factor modulating neuronal death but also as a factor regulating non-cell-autonomous neuronal death through glia. To investigate the relevance of SIRT3 to astrocytes mediated neurotoxicity, we used the astrocyte/neuron coculture model. Previous study showed that LPS/IFN-γ-stimulated astrocytes led to a significant neuronal toxicity in a coculture model . We first transfected primary astrocytes with
We demonstrated that SIRT3 is a major regulator of oxidative stress-induced neuronal toxicity in dopaminergic neurons. Oxidative stress is currently well known as an inducing factor of neuronal toxicity. In this study, we demonstrated that oxidative stress increased SIRT3 protein levels in differentiated SH-SY5Y cells. We also found that SIRT3 overexpression suppressed oxidative stress-induced neuronal toxicity in differentiated SH-SY5Y cells. Furthermore, downregulation of
Understanding the mitochondrial function of SIRT3 in neurons can lead to new therapeutic agents to prevent and treat mitochondria-related neurodegenerative diseases. Previous studies have suggested that SIRT3 deficiency significantly induces mitochondrial dysfunction and neuronal damage [49, 50]. SIRT3 is associated with proteotoxic- and mitochondrial stress-mediated cell death in cancer cells . In this study, SIRT3 were significantly increased by oxidative stress in mammalian neuronal cell and
Several studies have shown that starvation can induce the oxidative stress . Starvation-induced oxidative stress is closely related to starvation-induced cell toxicity [58-61]. Furthermore, Sirt3 deficiency leads to mitochondrial dysfunction and increase the vulnerability of cells to oxidative stress [62-65]. Thus, we investigated the effects of starvation on the oxidative stresses-related genes in
Many studies have shown that LPS/IFN-γ-induced proinflammatory cytokines in astrocytes lead to neuronal death in astrocyte/neuron coculture models. Interestingly, these proinflammatory cytokines are significantly elevated in the plasma of PD patients [66, 67]. Moreover, a recent meta-analysis revealed that IL-6, TNF-α, and IL-1β levels were significantly increased in the peripheral blood of PD patients . In this study, we found that the SIRT3 overexpression mitigated neuronal toxicity caused by LPS/IFN-γ stimulated astrocytes (Fig. 5). The number of CMFDA-positive neurons was notably decreased in co-cultures with LPS/IFN-γ-treated astrocytes, and SIRT3 overexpression significantly attenuated the neurotoxicity of LPS/IFN-γ-stimulated astrocytes (Fig 5). Furthermore, we found that the neurotoxicity of LPS/IFN-γ-stimulated astrocytes was enhanced by knock-down of
In the present study, we demonstrated that SIRT3 expression in dopaminergic neurons was upregulated by oxidative stress. We identified that SIRT3 played a critical role in the neurotoxicity induced by oxidative stress by regulating mitochondrial function in mammalian cells as well as a
This work was supported by the KBRI Research Program of the Ministry of Science, ICT & Future Planning (21-BR-02-15); the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT & Future Planning (NRF-2020R1A2C4002366 and NRF-2021R1C1C1008688); and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, South Korea (grant number: HI14C1135).