Increase in Mitochondrial Biogenesis in Neuronal Cells by RNS60, a Physically-Modified Saline, via Phosphatidylinositol 3-Kinase-Mediated Upregulation of PGC1α
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This study highlights a novel approach to upregulate mitochondrial biogenesis in neuronal cells. RNS60 is a 0.9% saline solution containing oxygenated nanobubbles that is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. RNS60, but not NS (normal saline), PNS60 (saline containing a comparable level of oxygen without the TCP modification), or RNS10.3 (TCP-modified normal saline without excess oxygen), increased the expression of Nrf1, Tfam, Mcu, and Tom20 (genes associated with mitochondrial biogenesis) and upregulated mitochondrial biogenesis in MN9D dopaminergic neuronal cells. Similarly RNS60 also increased mitochondrial biogenesis in primary dopaminergic neurons and in the nigra of MPTP-intoxicated mice. However, RNS60 had no effect on lysosomal biogenesis. Interestingly, we found that RNS60 upregulated PGC1α and siRNA knockdown of PGC1α abrogated the ability of RNS60 to increase mitochondrial biogenesis. Furthermore, we delineated that RNS60 increased the transcription of Pgc1a via type IA phosphatidylinositol (PI) 3-kinase-mediated activation of cAMP-response element-binding protein (CREB). Accordingly, knockdown of the PI3K – CREB pathway suppressed RNS60-mediated mitochondrial biogenesis. These results describe a novel property of RNS60 of enhancing mitochondrial biogenesis via PI 3-kinase-CREB-mediated up-regulation of PGC1α, which may be of therapeutic benefit in different neurodegenerative disorders.
KeywordsMitochondrial biogenesis Neurons PGC1α Modified saline Nanobubbles PI3 kinase
Mitochondrion is the most important organelle in terms of energy metabolism and neurons in the central nervous system (CNS) have an intense demand for mitochondrial function. Recent studies indicate alteration in mitochondrial homeostasis in the CNS of patients with different neurodegenerative diseases. While complex I activity is reduced in I in Parkinson’s disease (PD), activities of cytochrome oxidase and multiple electron transport chain (ETC) enzymes are decreased in Alzheimer’s disease (AD) and Huntington’s disease, respectively (Mizuno et al. 1995; Hirai et al. 2001; Baloyannis 2006; Surmeier and Sulzer 2013; Guedes-Dias et al. 2015). Mitochondrial dysfunction has also been reported in amyotrophic lateral sclerosis and progressive supranuclear palsy (Albers and Beal 2002; Tafuri et al. 2015). Leber’s hereditary optic neuropathy, a focal degeneration of the optic nerves, is known to arise due to mutations in mtDNA-encoded complex I genes and is associated with complex I dysfunction (Carelli et al. 2004). Mitochondrial dysfunction also participates in the pathogenesis of epilepsy (Zsurka and Kunz 2015). Since different neurodegenerative diseases are associated with mitochondrial malfunction, it is important to focus on developing methods to repair and restore mitochondria. Enhancing mitochondrial biogenesis may have therapeutic importance for different neurodegenerative diseases.
The diverse functionality of mitochondria requires a very complex and coordinated regulation of its activity with transcription factors NRFs/PPARs/ERRs that activate target genes encoding enzymes of fatty acid metabolism, oxidative phosphorylation and anti-oxidant defense (Scarpulla 2002; Fan and Evans 2015). By co-activating and regulating the expression of this network, PGC1α, the master regulator of mitochondrial biogenesis, directly links external physiological stimuli to the regulation of mitochondrial biogenesis and function (Wu and Boss 2007; Wareski et al. 2009). However, mechanisms by which PGC1α is activated and mitochondrial biogenesis is upregulated are poorly understood. RNS60 is physically modified saline that contains no active pharmaceutical ingredients. It is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure (Khasnavis et al. 2012; Mondal et al. 2012; Roy et al. 2014). Recently, we have demonstrated that RNS60 exerts anti-inflammatory effects in glial cells via suppression of nuclear factor kappa B (NF-κB) activation (Khasnavis et al. 2012). RNS60 also protects dopaminergic neurons in MPTP mouse model of PD (Khasnavis et al. 2014) and hippocampal neurons in a mouse model of AD (Modi et al. 2015). Here, we delineate that RNS60 stimulated mitochondrial biogenesis and increased the expression of Nrf1, Tfam, Mcu, and Tom20 (genes associated with mitochondrial biogenesis) in dopaminergic neuronal cells. However, RNS60 did not have any such stimulatory effect on lysosomal biogenesis. Furthermore, we demonstrate that RNS60 induced the activation of type IA PI3K and that RNS60 increased mitochondrial biogenesis via type IA PI3K-CREB-mediated upregulation of PGC1α. Finally, MPTP intoxication reduced the expression of PGC1α and decreased mitochondrial biogenesis, which were increased by RNS60 treatment. Our studies suggest that this physically-modified saline may be of therapeutic value in the treatment of PD and other neurodegenerative disorders in which mitochondrial abnormality plays an important role.
MN9D cells were obtained from Dr. A. Heller (University of Chicago, Chicago, IL, USA). Cells were maintained in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 3.7 g/L NaHCO3, 50 U/mL penicillin, and 50 μg/mL streptomycin in an incubator with an atmosphere of 7% CO2 at 37°C. These cells express abundant tyrosine hydroxylase, synthesize dopamine (DA) and also quantitatively release DA (Choi et al. 1991; Perez et al. 2002; Dong et al. 2008).
Hank’s balanced salt solution (HBSS) and 0.05% trypsin were purchased from Mediatech (Washington, DC). Fetal bovine serum (FBS) was obtained from Atlas Biologicals (Fort Collins, CO). Antibiotic-antimycotic mixture was obtained from Sigma-Aldrich (St. Louis, MO).
Isolation of Mouse Primary Dopaminergic Neurons
Nigra was dissected as a thin slice of ventral mesencephalon tissue from E12.5 to 14 days old fetus and homogenized with 1 ml of trypsin for 5 min at 37°C followed by neutralization of trypsin as described earlier (Roy and Pahan 2013). The single cell suspension of nigral tissue was plated in the poly-d-lysine pre-coated 75 mm flask and was allowed to differentiate fully for 9–10 days before treatment (Roy and Pahan 2013).
Animals and MPTP Intoxication
Six- to eight-weeks old C57BL/6 mice were purchased from Harlan, Indianapolis, IN. Animal maintenance and experiments were in accordance with National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use committee of the Rush University Medical Center, Chicago, IL. For acute MPTP intoxication, mice received four intraperitoneal (i.p.) injections of MPTP-HCl (18 mg/kg of free base; Sigma Chemical Co., St. Louis, MO) in saline at 2-h intervals (Ghosh et al. 2007; Ghosh et al. 2009; Roy et al. 2012). Control animals received only saline.
Preparation of RNS60
NS, normal saline from the same manufacturing batch. This saline contacted the same device surfaces as RNS60 and was bottled in the same way.
RNS10.3, a saline that was processed with TCP flow in the absence of any excess oxygen.
PNS60, saline with the same oxygen content as RNS60 (55 ± 5 ppm) that was prepared inside of the same device but was not processed with TCP flow.
Careful analysis demonstrated that all four fluids were chemically identical (Khasnavis et al. 2012). Liquid chromatography quadrupole time-of-flight mass spectrometric analysis also showed no difference between RNS60 and other control solutions (Khasnavis et al. 2012). Atomic force microscopy showed that RNS60 displayed different surface nanobubble nucleation characteristics relative to that of control saline solutions (Khasnavis et al. 2012) even when they contained similar levels of dissolved oxygen.
RNS60/NS Treatment of Mice
Mice were treated with RNS60 or NS (300 μl/mouse/d) via intraperitoneal (i.p.) injection from 3 h after the last injection of MPTP for 7 days as described (Khasnavis et al. 2014).
Rabbit polyclonal anti-PGC1α, mouse monoclonal anti-Nrf1 and rabbit polyclonal anti-TFAM antibodies were obtained from Abcam (Cambridge, MA). Anti-tyrosine hydroxylase (TH), anti-p300 and anti-NeuN antibodies were obtained Millipore (Billerica, MA). Rabbit monoclonal antibodies against phospho-CREB, total CREB and CREB-binding protein (CBP) were purchased from Cell Signaling (Beverly, MA). Cy2- and Cy5-conjugated antibodies were obtained from Jackson Immuno Research Laboratories (West Grove, PA).
Assay of PI3K Activation
Upon activation, PI3K subunits translocate to the plasma membrane. Therefore, after different time of treatment with RNS60 and NS, subunits of PI3K were monitored in plasma membrane. Briefly, cells were dissociated in 100 mM sodium bicarbonate buffer (pH 11.5) and spun in an ultracentrifuge at 40,000 rpm for 1 h at 4°C. The resultant supernatant was aspirated and the pellet was immersed in double-distilled water and SDS and stored at −80°C overnight. The following day, the pellet was resuspended by repeated grinding and boiling, and processed for Western blot using antibodies against p110α, p110β and p110γ (Khasnavis et al. 2014).
Western Blot Analysis
Western blotting was conducted as described earlier (Corbett et al.; Saha et al. 2006) with modifications. Briefly, cells were scraped in 1X RIPA buffer, protein was measured using Bradford reagent and sodium dodecyl sulfate (SDS) buffer was added and electrophoresed on NuPAGE® Novex® 4–12% Bis-Tris gels (Life Technologies, Grand Island, NY) and proteins transferred onto a nitrocellulose membrane (Bio-Rad) using the Thermo-Pierce Fast Semi-Dry Blotter. The membrane was then washed for 15 min in TBS plus Tween 20 (TBST) and blocked for 1 h in TBST containing BSA. Next, membranes were incubated overnight at 4°C under shaking conditions with primary antibodies. The next day, membranes were washed in TBST for 1 h, incubated in secondary antibodies against for 1 h at room temperature, washed for one more hour and visualized under the Odyssey® Infrared Imaging System (Li-COR, Lincoln, NE).
Semi-Quantitative RT-PCR Analysis
NRF1: Sense: 5′-CTTCATGGAGGAGCACGGAG-3′
TFAM: Sense: 5′-CAGTAGCCTTGTGGGCTTTC-3′
TOMM20: Sense: 5′-GTGCCATCTTGACGGGAGAT-3′
MCU: Sense: 5′-GCCACCAAAGAGAGACCTCC-3′
PGC1α: Sense: 5′-TCTCAGTAAGGGGCTGGTTG-3′
TFEB: Sense: 5′-AAC AAA GGC ACC ATC CTC AA -3′
LAMP2: Sense: 5′-GGT GCT GGT CTT TCA GGC TTG ATT-3′
GAPDH: Sense: 5′-GGTGAAGGTCGGTGTGAACG-3′
Real-Time PCR Analysis
The mRNA quantification was performed using the ABI-Prism7700 sequence detection system (Applied Biosystems, Foster City, CA) using SYBR Select master mix (Applied Biosystems). The mRNA expression of the targeted genes was normalized to the level of Gapdh mRNA and data was processed by the ABI Sequence Detection System 1.6 software as described earlier (Jana et al. 2007; Brahmachari et al. 2009; Ghosh et al. 2009; Khasnavis and Pahan 2012).
Quantification of Mitochondrial DNA (mtDNA)
Total DNA was isolated from MN9D cells using QIAamp DNA mini kit (Qiagen). The mtDNA content relative to nuclear DNA was assessed by real-time PCR using the ABI7500 (Applied Biosystems).
Primers of the single copy nuclear gene Ndufv1:
Relative mtDNA was determined using ΔCT method.
Monitoring Mitochondrial Membrane Potential
By Microplate Reader
Briefly, cells were plated in 96-well microplates and treated with RNS60 and NS for 18 h under serum-free condition. Cells were incubated with 500 nM TMRE for 30 min at 37°C. Media was replaced with 100 uL PBS/0.2% BSA. Plate was read on a PerkinElmer fluorescence plate reader with Ex/Em 549/575 nm range.
By FACS Analysis
Briefly, 105 cells were plated and treated with RNS60 and NS for 18 h under serum-free condition. Cells were incubated with 100 nM TMRE for 30 min at 37°C. Media was replaced with PBS/0.2% BSA for flow cytometry analysis. TMRE signal was detected in PE-FL2 channel in a BD FACSCanto II Flow Cytometer.
Chromatin Immunoprecipitation Assay
The PCRs were repeated by using varying cycle numbers and different amounts of templates to ensure that results were in the linear range of PCR. Quantitative real-time PCR was also performed using the same primers and SYBR select MasterMix. Data were normalized to input and nonspecific IgG, and fold increase versus control was calculated.
Immunostaining of Cells
Immunocytochemistry was performed as described earlier (Khasnavis and Pahan). Briefly, after treatment, cells were fixed with chilled Methanol (Fisher Scientific, Waltham, MA) overnight, followed by two brief rinses with filtered PBS. Samples were blocked with 2% BSA (Fisher Scientific) in PBS containing Tween 20 (Sigma) and Triton X-100 (Sigma) for 30 min and incubated at room temperature under shaking conditions for 2 h in PBS containing the following anti-mouse primary antibodies: TFAM (1:500), PGC1α (1:300), Nrf1 (1:300), and NeuN (1:500). After four 15 min washes in filtered PBS, the slides were further incubated with Cy2 or Cy5-labeled secondary antibodies (all 1:200; Jackson ImmunoResearch, West Grove, PA) for 1 h under similar shaking conditions. Following four 15 min washes with filtered PBS, cells were incubated for 4–5 min with 4′, 6-diamidino-2-phenylindole (DAPI, 1:10,000; Sigma). The samples were run in an EtOH and Xylene (Fisher) gradient, mounted, and observed under Olympus BX41 fluorescence microscope.
Immunostaining of Tissue Sections
After treatment, mice were sacrificed and their brains fixed, embedded, and processed. Sections were made from ventral midbrain regions and for immunofluorescence staining on fresh frozen sections, anti-mouse TFAM (1:300), anti-mouse Nrf1 (1:200), anti-mouse PGC1α (1:200), and anti-mouse NeuN (1:500) were used (Ghosh et al. 2015). The samples were mounted and observed under Olympus BX41 fluorescence microscope (Dasgupta et al. 2004).
Electron Microscopy and Counting of Mitochondria and Lysosomes
After treatment, cells were fixed with paraformaldehyde (2%) and glutaraldehyde (2.5%) mixture. After primary fixation, samples were prepared in the Electron Microscopy core facility of the University of Illinois at Chicago Research Resources Center. To stabilize cell components, samples were treated with 1% osmium tetroxide in phosphate buffer. Next, the samples were dehydrated through an increasing concentration of ethanol, passed through propylene oxide and then infiltrated and embedded in a liquid resin. Resin block is then sectioned by ultramicrotomy with 50–70 nm thickness and collected on metal mesh ‘grids’ followed by staining these grids with electron dense stains before observation in the TEM (JEOL JEM-1220).
Counting analysis was performed using Olympus Microsuite V software with the help of a touch counting module. Before counting cells, the entire image area was calibrated with the help of a rectangular box available in the touch counting panel. Once the area of the image was measured, the touch counting program was applied to count the number of mitochondria or lysosomes using a simple mouse click method. Next, the total number of signals in a given area was normalized by the total area of the image and presented as number of cells per square millimeter unit. Counting was done for 20 fields in each treatment group.
Cells cultured to 70–80% confluence were subjected to different stimuli under serum-free condition followed by incubation with 75 nM MitoTracker Red (Thermo Fisher Scientific, Waltham, MA) for 45mins. Cells were then washed thoroughly with filtered PBS and mounted on glass slides and viewed under BX41 fluorescence microscope. Cells were visualized with a 100× objective and MitoTracker signals were counted as described above using Olympus Microsuite V software.
All values are expressed as means ± SEM. One-way ANOVA was performed while analyzing dose-dependent effect of RNS60 on mRNA expression of Nrf1, Tfam, Mcu, and Tom20 in neuronal cells. In other cases, Student’s t-test was used to compare outcome between two groups (e.g. control vs MPTP, MPTP vs RNS60 etc.).
RNS60 Increases the Biogenesis of Mitochondria, but Not Lysosomes, in MN9D Dopaminergic Neuronal Cells
RNS60 Increases the Expression of Genes Associated with Mitochondrial Biogenesis in MN9D Dopaminergic Neuronal Cells
RNS60 Increases Mitochondrial Biogenesis in Primary Mouse Dopaminergic Neurons
RNS60 Treatment Protects and/or Stimulates Mitochondrial Biogenesis in the Nigra of MPTP-Intoxicated Mice
Upregulation of PGC1α in Dopaminergic Neurons by RNS60
RNS60 Increases Mitochondrial Biogenesis in MN9D Dopaminergic Neuronal Cells via PGC1α
RNS60 Stimulates the Transcription of Pgc1a in MN9D Dopaminergic Neuronal Cells via Class IA PI3K-Mediated Activation of CREB
Next, to delineate whether PI3K is involved in RNS60-mediated upregulation of PGC1α, cells were pretreated with different concentrations of LY294002, a morpholine-containing chemical compound that strongly inhibits PI3K, for 30 min followed by stimulation with 10% RNS60 for 2 h and analysis of Pgc1a mRNA. As expected, RNS60 treatment increased the mRNA expression of Pgc1a (Fig. 8c–d). However, pretreatment with LY294002 abrogated RNS60-mediated increase in Pgc1a expression in neuronal cells (Fig. 8c–d). To further confirm, we performed TH:PGC1α double labeling. Although RNS60 did not modulate TH, marked increase in PGC1α protein was seen after RNS60 treatment (Fig. 8e). However, LY294002 strongly suppressed RNS60-mediated upregulation of PGC1α (Fig. 8e). These results suggest that RNS60 upregulates PGC1α in MN9D neuronal cells via PI3K.
Since RNS60 increased the production of ATP (Fig. 2d), we examined if RNS60 required the PI3K-PGC1α pathway for stimulating ATP production in neurons. Suppression of RNS60-induced ATP production by LY294002 suggests the involvement of PI3K in RNS60-mediated ATP generation. Similarly, PGC1α siRNA, but not control siRNA, also reduced the production of ATP in RNS60-stimulated neuronal cells, indicating the involvement of PGC1α in this process. These results were not due to any change in viability as evident from MTT and LDH release assays (Fig. 8g–h and j–k). Although RNS60 protected neurons from Aβ1–42-mediated toxicity (Modi et al. 2014), RNS60 was unable to stimulate the survival of normal neurons (Fig. 8g–h and j–k).
RNS60 Stimulates Mitochondrial Biogenesis in Dopaminergic Neurons
The mitochondria is the most important organelle in cells that not only participates in energy homeostasis and metabolism but also play significant roles in other biological processes like signal transduction, aging, oxidative stress, and apoptosis (Mizuno et al. 1995; Baloyannis 2006; Zsurka and Kunz 2015). Accordingly, the abundance, morphology, and functional properties of mitochondria are finely tuned to meet cell-specific energetic, metabolic, and signaling demands. As a result, decreased mitochondrial biogenesis and function are seen in many pathological conditions including neurodegenerative disorders (Dong et al. 2015; Wang et al. 2016). Therefore, the ability to manipulate or influence mitochondrial biogenesis in neurons may be of therapeutic importance for neurodegenerative diseases. Although there may be other approaches to stimulate mitochondrial biogenesis, here we introduce a simple saline-based agent to achieve this. RNS60 is an electrokinetically modified saline that contains charge-stabilized nanobubbles, but no active pharmaceutical ingredients. Due to Taylor-Couette-Poiseuille (TCP) turbulence, RNS60 is proposed to contain charge-stabilized nanostructures consisting of an oxygen nanobubble core surrounded by an electrical double-layer at the liquid/gas interface (Khasnavis et al. 2012). Several lines of evidence presented in this manuscript clearly demonstrate that RNS60 is capable of upregulating mitochondrial biogenesis in dopaminergic neurons. Our conclusion is based on the following findings. First, we observed that RNS60, but not NS, RNA10.3 or PNS60, stimulated mitochondrial biogenesis in MN9D dopaminergic neuronal cells. Interestingly, RNS60 did not increase lysosomal biogenesis in MN9D cells. Second, RNS60, but not NS, RNS10.3 or PNS60, upregulated the expression of Nrf1, Tfam, Mcu, and Tom20, genes associated with mitochondrial biogenesis, in MN9D cells. However, RNS60 did not modulate the expression of Tfeb and Lamp2, genes associated with lysosomal biogenesis. Third, electron microscopy also revealed increase in mitochondria, but not lysosomes, by RNS60 in MN9D cells. Fourth, RNS60 also increased mitochondrial biogenesis in primary mouse dopaminergic neurons. Fourth, while the level of TFAM decreased in the nigra of MPTP mouse model of PD, RNS60 treatment upregulated and/or protected nigral TFAM from MPTP toxicity. Recently we have delineated protection of dopaminergic neurons and restoration of locomotor activities in MPTP-intoxicated mice by RNS60 (Khasnavis et al. 2014). Since mitochondrial dysfunction is known to contribute to nigrostriatal pathology in PD patients and in animal models of PD (Mizuno et al. 1995; Surmeier and Sulzer 2013), our current results suggest that this mitochondria-boosting effect of RNS60 may play a role in RNS60-mediated protection of the nigrostriatum in MPTP mouse model.
RNS60 Requires PGC1α to Stimulate Mitochondrial Biogenesis
Mitochondrial biogenesis is a complex process that requires a coordinated regulation of synthesis, import, and incorporation of proteins and lipids to the existing mitochondrial reticulum, as well as replication of the mitochondrial DNA. A major breakthrough in our understanding of how mitochondrial biogenesis is coordinately regulated was the identification of NRF-1, NRF-2 and PPARγ, transcription factors that are responsible for mitochondrial biogenesis. Accordingly, it has been found that PPARγ coactivator 1α (PGC1α), functioning as a coactivator of NRF-1, NRF-2 and PPARγ, is capable of integrating various physiological signals and enhancing mitochondrial biogenesis (Wu and Boss 2007; Wareski et al. 2009; Villena 2015). Accordingly, we observed upregulation of PGC1α by RNS60 in MN9D neuronal cells. Peripheral administration of RNS60 also increased PGC1α in THir neurons as well as other brain cells in vivo in the nigra of MPTP intoxicated mice. Suppression of Nrf1, Tfam, Mcu, and Tom20 gene expression and attenuation of mitochondrial biogenesis in RNS60-stimulated neuronal cells by siRNA knockdown of PGC1α suggests that RNS60 stimulates mitochondrial biogenesis via PGC1α.
RNS60 Upregulates PGC1α in Neuronal Cells via Class IA PI3K-Mediated Activation of CREB
Mechanisms by which PGC1α is upregulated are poorly understood. Recently we have delineated that RNS60 increases IκBα in microglia via the activation of class IA PI3K p110α/β, but not class IB PI3K p110γ (Khasnavis et al. 2012). PI3K is a key signaling molecule implicated in the regulation of a broad array of biological responses including metabolism, cell growth, cell survival, and apoptosis (Koyasu 2003). In case of class IA PI3K, the p85 regulatory subunit interacts with the IRS-1 through its SH2 domain and thus recruits the p110 catalytic subunit (p110α/β) to the cell membrane, resulting in the activation of downstream signaling molecules like Akt/protein kinase B and p70 ribosomal S6 kinase (Koyasu 2003). While, for class IB PI3K, p110γ is activated by the engagement of G-protein coupled receptors. The p110γ then catalyzes the release of phosphatidylinositol (3,4,5)-triphosphate as the second messenger from phosphatidylinositol (4,5)-bisphosphate and activates downstream signaling molecules (Franke et al. 1997). Here we demonstrate that RNS60 induces the activation of both the subunits of class IA PI3K (p110α and p110β) without modulating class IB PI3K p110γ in MN9D neurons, suggesting the specific activation of class IA p110α/β PI3K in dopaminergic neuronal cells. Earlier by using Atomic Force Microscopy, we have demonstrated that RNS60 contains nanobubbles and that nanobubble composition of RNS60 is different from other control saline solutions (Khasnavis et al. 2012). Nanobubbles may interact with cells based upon their charge and size as these charged entities possess the necessary energetics to affect the membrane. Consistent to the presence of activated PI3K at the membrane, RNS60 containing charge-stabilized nanobubbles induced the recruitment of PI3K subunits to the membrane of neuronal cells. Abrogation of RNS60-mediated increase in PGC1α and upregulation of mitochondrial biogenesis in neuronal cells by inhibitors of PI3K suggest the involvement of PI3K in RNS60-mediated increase in mitochondrial biogenesis. Furthermore, we have seen that a consensus CRE is present in the Pgc1a promoter near the transcription start site and that RNS60 induced the recruitment of CREB and CBP to this CRE in Pgc1a promoter. Suppression of RNS60-induced CREB activation by inhibition of PI3K suggests that RNS60 induces the activation of CREB in neuronal cells via PI3K. Recently, we have delineated that Akt, the downstream mediator of PI3K, is responsible for the activation of CREB in RNS60-treated microglial cells (Khasnavis et al. 2012). Therefore, it is possible that a similar pathway exists in dopaminergic neurons as well. Furthermore, attenuation of RNS60-mediated increase in PGC1α and mitochondrial biogenesis by siRNA knockdown of CREB suggests that RNS60 increases the transcription of Pgc1a in neuronal cells via class IA PI3K-mediated activation of CREB, resulting in increased mitochondrial biogenesis.
In summary, we have demonstrated that RNS60 upregulates mitochondrial biogenesis in neuronal cells via class IA PI3K-CREB-mediated upregulation of PGC1α. These results highlight a novel mitochondria boosting property of RNS60 and suggest that this simple modified saline may be explored to rejuvenate degenerating neurons in PD and other neurodegenerative disorders where impaired mitochondrial function serves as a causative factor.
This study was supported by Revalesio Corporation.
Compliance with Ethical Standards
Conflict of Interest
- Carelli V, Rugolo M, Sgarbi G, Ghelli A, Zanna C, Baracca A, Lenaz G, Napoli E, Martinuzzi A, Solaini G (2004) Bioenergetics shapes cellular death pathways in Leber's hereditary optic neuropathy: a model of mitochondrial neurodegeneration. Biochim Biophys Acta 1658:172–179CrossRefPubMedGoogle Scholar
- Ghosh A, Roy A, Liu X, Kordower JH, Mufson EJ, Hartley DM, Ghosh S, Mosley RL, Gendelman HE, Pahan K (2007) Selective inhibition of NF-kappaB activation prevents dopaminergic neuronal loss in a mouse model of Parkinson's disease. Proc Natl Acad Sci U S A 104:18754–18759CrossRefPubMedPubMedCentralGoogle Scholar
- Ghosh A, Jana M, Modi K, Gonzalez FJ, Sims KB, Berry-Kravis E, Pahan K (2015) Activation of peroxisome proliferator-activated receptor alpha induces lysosomal biogenesis in brain cells: implications for lysosomal storage disorders. J Biol Chem 290:10309–10324CrossRefPubMedPubMedCentralGoogle Scholar
- Guedes-Dias P, Pinho BR, Soares TR, de Proenca J, Duchen MR, Oliveira JM (2016) Mitochondrial dynamics and quality control in Huntington's disease. Neurobiol Dis 90:51–57Google Scholar
- Hirai K, Aliev G, Nunomura A, Fujioka H, Russell RL, Atwood CS, Johnson AB, Kress Y, Vinters HV, Tabaton M, Shimohama S, Cash AD, Siedlak SL, Harris PL, Jones PK, Petersen RB, Perry G, Smith MA (2001) Mitochondrial abnormalities in Alzheimer's disease. J Neurosci 21:3017–3023CrossRefPubMedGoogle Scholar
- Wang W, Wang X, Fujioka H, Hoppel C, Whone AL, Caldwell MA, Cullen PJ, Liu J, Zhu X (2016) Parkinson's disease-associated mutant VPS35 causes mitochondrial dysfunction by recycling DLP1 complexes. Nat Med 90:51–57Google Scholar
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