Literature Research
EAAT2
Glutamate, the primary excitatory amino acid neurotransmitter, in the central nervous system (CNS) is also a powerful neurotoxin that may result in the nerve cells’ death. It must be taken up by the synaptic cleft when participating in the signaling process from glutamatergic nerve endings to various types of glutamatergic receptors (Kanai and Hediger, 2003). Otherwise, a large number of detrimental consequences such as calcium homeostasis dysfunction, increased nitric oxide (NO) production, activation of proteases, an increase in cytotoxic transcription factors and increased free radicals will be caused after glutamate receptors receive prolonged stimulation (Wang and Qin, 2010). Therefore, many neurological disorders such as epilepsy, stroke and neurodegenerative diseases, and dysfunctional glutamate transporters have a strong association with increasing glutamate levels, which are likely to initiate the brain injury (Kim et al., 2011).
The glutamate concentration in the synaptic cleft and its post-synaptic activity of glutamate receptors are highly related to the dynamic equilibrium between the release and clearance of glutamate (Kanai and Hediger, 2003). In order to terminate synaptic transmission, glutamate transporters control glutamate within a low level by immediately removing it from the extracellular space after releasing caused by an action potential (Beart and O’Shea, 2007).
The high-affinity sodium-dependent transporters are regarded as the predominant mechanism in the brain for maintaining the stable concentration glutamate by absorbing this excitotoxic amino acid. The EAATs located on the membrane of astroglia or in glia which closely resemble ion channels are known to be the notable transporters to treat the prolonged exciting problem (Kim et al., 2011).
The EAATs are symporters which means that the transporter protein transport a substrate by the co- and counter-transport of ions (Robinson,1998). The initiation of transport cycle is triggered by binding of a substrate molecule, three sodium ions and a proton to form an outward-facing EAAT conformation. Afterwards, the substrates, sodium ions and protons and are released into the cytoplasm of the cell, thereby resulting in an inward-facing conformation formed. Subsequently, after the counter-transport of a potassium, the new substrate molecule become accessible again and the transporter will also return to its outward-facing conformation (Levy et al., 1998).
Since the activities of EAAt2 are regulated both transcriptionally and post-transcriptionally, up-regulation of EAAT2 are likely to be observed at both transcriptional and translational level.
For transcriptional example: NF-kB is primarily a transcriptional activator, which can also act as a transcriptional repressor in some cases. It mediate the expression of many essential molecules such as epithelial cell adhesion molecule (EpCAM) and cytochrome P-4501A1. According to the study of Sitcheran et al. (2005), NF-kB directly binds to the EAAT2 promoter, and regulates its transcription. The studies with pharmacological and genetic inhibitors suggest that the EAAT2 expression depend on the signaling pathways through PI3K and NFkB (Kim et al., 2011).
For post-transcriptional example： A study have previously found that a SUMO1- conjugated C-terminal fragment of EAAT2 (termed CTESUMO1) accumulates in ALS (Foran et al., 2014). Small ubiquitin modifier 1 (SUMO1) is a posttranslational modifier protein, which attaches to lysine residues via an isopeptide bond. (Foran et al., 2014). EAAT2s modified with SUMO1 have been changed in localization, trafficking, and half-life due to this ubiquitination modification. Since EAAT2 with this modification can no longer be maintained at the cell membrane, it would lost the transport fuction immediately and be degenerated in a short period (Foran et al., 2014).
According to previous research, dysfunction or decreased expression of EAAT2 associated with impaired glutamate uptake has been implicated in many pathogenesis of diseases. In extracellular fluid, down-regulation of EAAT2 followed by accumulation of glutamate leading to neuronal death have been recorded in diverse aetiology, including Alzheimer’s disease, several types of dementia, etc (Liu et al., 2017)
Cx43
Connexin 43 (Cx) proteins can be assembled into a six-protein oligomer called a connexon. Connexons are inserted into the plasma membrane (Falk et al., 2016). During cell-cell contact, the hemichannels can dock head-on with the hemichannels from another cell, which is exosomes in our project. These connexons can then cluster to form a gap junction (GJ) plaque. The plaque can be used for rapid material exchange between exosomes and target cells (Falk et al., 2016). Once the plaque is no longer needed for cell-cell communication, the central portion of the plaque or the entire plaque would be internalized to form an annular gap junction. The annular gap junction may then be degraded via lysosomal degradation (Falk et al., 2016). The free connexin proteins in cytoplasmic membranes that are released during vesicle degradation may then be recycled back to the cell surface to participate in the formation of new, or the addition to existing gap junction plaques (Falk et al., 2016).
The consequences of this rapid turnover showing that these proteins generate subcellular structures that are in a constant state of flux. In a normal situation, the size and longevity of individual gap junctions can be balanced through the accretion of “new” protein and internalization of the oldest protein (Solan and Lampe, 2018).
The phosphorylation of Ser 368 is required for conformational alteration of the Connexon, which is also likely to play an essential role in internalization. When gap junction channels aged (permanently closed) and move inward towards plaque centres, the phosphorylation of S368 allows ZO-1-bound to be transited into ZO-1-unbound gap junction channels. The gap junction channels would be closed and be internalized (Falk et al., 2016). However, in our project, we used the S368A mutant of Cx43. Without the serine in 368 positions, the ZO-1 would be kept binding to gap junction channels, and the gap junction plaques can not be internalized. The gap junction plaques would be kept at the efficient transition state (Falk et al., 2016).
IVT delivery system
Injection of in vitro transcribed (IVT) mRNA could be used to express encoded proteins in the injected tissue in living organisms for delivering genetic information. Structural assembling of natural mRNA could be engineered to synthetic mRNA for transient expression of proteins in the transfected cells (Sahin et al., 2014). Contrast to traditional DNA therapies that requires access of exogenous DNA into the nucleus for RNA transcription, IVT mRNA could deliver its function by only reaching the cytoplasm and translating the mRNA without entering nucleus. Therefore it is free of risks of insertion mutagenesis into cell genome and induce potential ethical problems. Traditional therapeutic DNA treatments also depends it functionality on nuclear envelope breakdown during mitosis, which could be a limit of functional efficiency (Sahin et al., 2014).
IVT mRNA also have advantages over most pharmaceutical therapies due to its transient activity and fast degradation in vivo. Synthetic mRNA is also cost less to produce, and could functionality could be easily modified with knowledge of related genetic materials. In different clinical areas, IVT mRNA is either at preclinical stages or has reached Phase III clinical testing (in cancer immunotherapy) (Heiser et al., 2002, Morse et al., 2002). When used for ex vivo cell therapeutic applications, IVT mRNA therapy would be challenged by industrialization hurdles for cell therapies, but in the case of in vivo usae, IVT mRNA has the advantage of low cost, easily manufactural and free of many mutagenesis risks (Sahin et al., 2014).
Experiment design
EAAT2-Functional Characterization
1. Transfection of EAAT2-C/D Box-pcDNA3.1(+)
To characterize our part designed for coding expressing EAAT2, we transfected the part BBa_k3030002 on pcDNA3.1(+) using engreen the transfecting reagent Engreen to P4 of N2a cells, which have multiple branches that mimic appear in normal neurons and glial cells. All of the Neuro-2a cells were incubated in 5% FBS-added DMEM medium with anti-anti in a 24-well plate until the cells were grown to approximately 60% confluence. In this part of experiment, the empty plasmid pcDNA3.1(+) was used as the negative control plasmid, and transfected into another 12 wells, the distribution of the samples in each well is displayed as below:
Our operation in this experiment follows the protocol for 24-well engreen transfection: Protocol link.
2. Cell Nurturing: Inflammation-inducing Condition
12 hours after the transfection of BBa_k3030002 and BBa_k3030004, the medium of N2a cells were was changed to the normal exosome-free DMEM medium.
To induce inflammation in the N2a cells, we changed the medium to phenol-free RPMI1640 medium with 5% exosome-free FBS, and additional glutamate were was added to ensure that the concentration of glutamate is was 80 uM.
The time interval of changing DMEM medium to high glutamate -containinged medium is was controlled to three hours, and for both of the EAAT2-transfected and pcDNA-transfected N2a cells, there are were four groups of cells to be manipulated, and each group has was present in three wells. The distribution of sample in each well is displayed as below:
3. WST-1
To test the cytotoxicity induced by glutamate to in N2a cells, we used Beyotime WST-1 Cell Proliferation and Cytotoxicity Assay Kit. In each well, we added 20 ul WST-1 in 200 ul medium in each well of the 24-well plate. After incubating the WST-1-added cells in 37 ℃ for 2 hours, we measured the absorbance at 450 nm, and 620 nm was used as reference wavelength in a 96-well plate.
Our operation in this experiment follows the protocol for WST-1 Test: Protocol link.
4. Glutamate Assay
To detect the exact glutamate concentration in the cell medium that mimics the cerebrospinal fluid, we used the Enzychrom Glutamate Assay Kit. The cell medium from each well was extracted, while 20 ul in each sample was used in the glutamate assay. The measurement of absorbance at 565 nm was conducted twice respectively: at the moment immediately after adding the working reagent solution and after a 30-minute incubation at room temperature.
Our operation in this experiment follows the protocol for Glutamate Assay: Protocol link.
EAAT2-Exosome Delivery
1. Cx43 Transfection
To ensure the exosome to specifically combine and deliver cargo to neurons and glial cells containing Cx43 on membrane surface, we transfected Cx43 coding sequence on pcDNA3.1 (+) into the P3 HEK293T cells (exosome donor) and P4 N2a cells (exosome receptor) using engreen transfection kit. Both of the HEK293T and N2a cells were respectively incubated in DMEM medium with 5% exosome-free FBS and 1% anti-anti in two T25 flasks. In this part of experiment, the empty pcDNA3.1(+) was used as the negative control plasmid, and transfected in the other two T25 flasks which share the same incubating condition with the Cx43-trasfected cells.
Our operation in this experiment follows the protocol for T25 plate engreen transfection: Protocol link.
2. Ultracentrifugation: Exosome Isolation
12 hours after we finished the transfection of Cx43 and pcDNA3.1(+) in two HEK293T flasks, the medium was changed to exosome-free DMEM medium for cell culture. After 24 hours, the cell medium was extracted for ultracentrifugation. The extracted exosome pellets were resuspended in 50 ul cold sterile PBS and well-stored in a -80 ℃ freezer.
Our operation in this experiment follows the protocol for ultracentrifugation: Protocol link.
3. Endo-free Miniprep of EAAT2-C/D Box-pcDNA3.1(+)
Before transfecting the mRNA into the exosomes (and the N2a cells), we need to get the endotoxin-free plasmids which can be transcribed to mRNA. Thus, we used Axygen Endo-free Plasmid Kit for DNA isolation of DH5a that transformed with part BBa_k3030002 and with BBa_k3030004. The isolated plasmid was then stored in a -20 ℃ freezer.
Our operation in this experiment follows the protocol for Endo-free Miniprep: Protocol link.
4. In Vitro Transcription
We used MAXIscript Kit from Ambion for in vitro transcription of our isolated EAAT2-C/D Box-pcDNA3.1(+) and empty pcDNA3.1(+). To prevent the premature termination, we extend the reaction time to 2 hours, decline the reaction temperature to 15 ℃, and rise the concentration of each NTP to 500 M. After accomplishing the IVT, we added 1 ul TURBO DNase each sample to remove the template DNA in each sample, and incubated at 37 ℃for 15 minutes.
Our operation in this experiment follows the protocol for In Vitro Transcription: Protocol link.
5. Polyadenylation
In order to protect the naked mRNA transcribed, we used Poly[A] Enzyme from Thermo Fisher Advanced miRNA Assay Kit to add poly-A tails on the 3’ ends.
Our operation in this experiment follows the protocol for Polyadenylation: Protocol link.
6. RT-PCR
To calculate the approximate length of mRNA in vitro transcribed, we used universal RT primers and RT-PCR master mix in Taqman Advanced miRNA cDNA Synthesis Kit to produce cDNA of the transcribed mRNA. Before conducting the reverse transcription, the sample were incubated at 85 ℃ for 3 minutes to inactivate the TURBO DNase added in step 4. For each reaction, the reagent mix is added as followed instruction (15 ul sample were added in each reaction):
7. DNA Agarose Gel Electrophoresis
When we apply MAXIscript Kit for the IVT of EAAT2-C/D Box-pcDNA3.1(+) and empty pcDNA3.1(+), we adjust the protocol to prevent the premature termination of transcription. To investigate if the prematuration is well-prevented, we run a DNA agarose gel electrophoresis and used 5 ul 1 kb DNA ladder as a length reference. We added 2 ul 6x DNA loading dye, 2 ul sample cDNA (1498.5 ng/ul), and add RNase-free water to 12 ul in each reaction. We made a 1.0% agarose gel using 1 g agarose powder and 100 ml 1x TAE and 10 ul 10000x Gel-red. After the gel is cold, we loaded 10 ul sample in each well for electrophoresis.
8. Exo-fect Exosome Transfection
To transfect our mRNA into exosomes, we used SBI Exo-fect Transfection Kit, due to the limitation of reaction times, we used 30 ul Exo-fect solution to transfect 13.5 ug mRNA into approximately 9.37 x 107 exosomes. In order to thoroughly mix the reagents while the concentration of added exosomes are 10 times of the recommended, we extend the shaking time to 15 minutes and rise the shaking speed to 120 rpm. The transfected exosomes were then centrifuged at 21100 xg for 5 minutes, and 900 ul cold sterile PBS was added to resuspend the exosome pellet. The exosome solution was added to two N2a-culturing flasks with 2 ml DMEM exosome-free medium.
Our operation in this experiment follows the protocol for Exo-fect Transfection: Protocol link.
9. qRT-PCR
24 hours after adding the exosome solution, we discarded the medium of two N2a cell-containing flasks, and washed them twice by using warm sterile PBS. The isolation of mRNA from those cells were conducted with application of Beyotime RNAeasy RNA Isolation Kit. The mRNA were then used as template in the following RT-PCR and qPCR procedures by applying the Cells-to-CT™ 1-Step Power SYBR™ Green Kit from Thermo Fisher. The results were then analyzed in Quantstudio 5 and relevant apps.
Our operation in this experiment follows the protocol for mRNA isolation: Protocol link.
Our operation in this experiment follows the protocol for qRT-PCR: Protocol link.
EAAT2-RNA Demonstration
This set of experiments is based on the completion of the exosome-delivery-relevant experiments.
1. Cx43 Transfection
In this part of experiment, the mass expression of Cx43 in both HEK293T and N2a cells was still essential for conducting the following procedures. We transfected Cx43-pcDNA3.1(+) both to HEK293T 3rd generations of cells and N2a 4th generations of cells in the same way (Engreen transfection) as that in the exosome-delivery experiments, which ensures the exosome to specifically deliver their cargo to neurons and glial cells expressing Cx43 on the membrane surface. Both of HEK293T and N2a cells were incubated in DMEM medium with 5% exosome-free FBS and 1% Penicillin-Streptomycin in two T25 flasks.
Our operation in this experiment follows the protocol for T25 plate engreen transfection: Protocol link.
2. Ultracentrifugation: Exosome Isolation
This procedure of isolating exosomes is the same as the ultracentrifugation method in the exosome-delivery experiments. 12 hours after the transfection of Cx43 in two HEK293T flasks, the medium was changed to exosome-free DMEM medium for cell culture. After 24 hours, the cell medium was removed for ultracentrifugation. The extracted exosome pellets were resuspended in 50 ul cold sterile PBS and well-stored in a -80 ℃ freezer.
Our operation in this experiment follows the protocol for ultracentrifugation: Protocol link.
3. In Vitro Transcription
We used MAXIscript Kit from Ambion for in vitro transcription of our isolated EAAT2-C/D Box-pcDNA3.1(+) and pcDNA3.1(+) (Endotoxin-free). The Ribo m7G Cap Analog (Promega) was also added in the reagent solution, which protects the naked mRNA and facilitate the translation of mRNA in N2a cells. After accomplishing the IVT, we added 1 ul TURBO DNase each sample to remove the template DNA in each sample, and incubated at 37 ℃ for 15 minutes.
Our operation in this experiment follows the protocol for in vitro transcription:Protocol link.
4. Polyadenylation
In order to protect the naked mRNA transcribed and facilitate the translation of mRNA in eukaryotes, we used Poly[A] Enzyme from Thermo Fisher Advanced miRNA Assay Kit to add poly-A tails on the 3’ ends.
Our operation in this experiment follows the protocol for Polyadenylation:Protocol link.
5. Transfection of EAAT2-C/D Box-pcDNA3.1(+) (mRNA)
To ensure that the RNA transcribed from EAAT2-C/D Box-pcDNA3.1(+) can be well-expressed in N2a, we transfected the mRNA of part BBa_k3030002 on pcDNA3.1(+) using engreen to P4 N2a cells. All of the Neuro-2a cells were incubated in 5% FBS-added DMEM medium with anti-anti in a 24-well plate until the cells were approximately 60% confluence. To avoid the wrong results in WST-1 test caused by cytotoxicity induced by engreen, the empty pcDNA3.1(+) was used as the negative control plasmid, and transfected into another 12 wells, the distribution of sample in each well is displayed as below:
Our operation in this experiment follows the protocol for 24-well engreen transfection: Protocol link.
6. Cell Nurturing: Inflammation-inducing Condition
This procedure of isolating exosome is the same as the inflammation-inducing step in exosome-delivery experiments. 12 hours after the transfection of both mRNA, the medium of N2a cells were changed to normal exosome-free DMEM medium. To induce inflammation in the N2a cells, we changed the medium to phenol-free RPMI1640 medium with 5% exosome-free FBS, and additional glutamate were added to ensure that the concentration of glutamate is 80 uM. The time interval of changing DMEM medium to glutamate-contained medium is controlled to three hours, and for both of the EAAT2-transfected and pcDNA-transfected N2a cells, there are four groups of cells to be manipulated, and each group has three wells. The distribution of sample in each well is displayed as below:
7. WST-1
The excitotoxicity induced by glutamate to N2a cells were detected by applying Beyotime WST-1 Cell Proliferation and Cytotoxicity Assay Kit. We added 20 ul WST-1 in each 200 ul-medium-containing well of the 24-well plate. After incubating the WST-1-added cells in 37 ℃ for 2 hours, we measured the absorbance at 450 nm, and 620 nm as reference wavelength in a 96-well plate.
Our operation in this experiment follows the protocol for WST-1 Test: Protocol link.
8. Glutamate Assay
The measurement of glutamate’s concentration in cell medium is also the same as the glutamate assay procedure in exosome-delivery experiments. To detect the exact glutamate concentration in the cell medium that mimics the cerebrospinal fluid, we used Enzychrom Glutamate Assay Kit. The cell medium from each well was extracted, while 20 ul in each sample was used in the glutamate assay. The measurement of absorbance at 565 nm was conducted twice respectively: at the moment adding working reagent solution and after a 30-minute incubation at room temperature.
Our operation in this experiment follows the protocol for Glutamate Assay: Protocol link.
Measurement-Part Improvement
1. Transformation of 14O and 16A to DH5a
After the plasmid containing BBa_k598009, BBa_k598010 from PKU 2011 part list, and our newly designed BBa_k3030016, BBa_k3030017 were well received, we conducted the transformation of these plasmids to DH5a competent cells respectively. 100 ul transformed cells were used to coat on LB plates and incubated at 37 ℃ overnight (12 hours approximately).
Our operation in this experiment follows the protocol for Transformation: Protocol link.
2. Miniprep
To amplify the plasmid we received for the following experiments, we selected three colonies on LB-0.1% ampicillin selective plates respectively for the four plasmids. And incubated the 12 colonies in 10 ml LB-Amp medium for overnight. Before the miniprep, we tested the OD600 for each colony using spectrometer, and the OD value were all above 2. Then we used Qiagen Miniprep DNA Isolation Kit to conduct the miniprep. The isolated DNA were then eluted using ddH2O, and stored in 200 ul centrifuge tubes in -20 ℃ freezers. For each plasmids, we used one tube (50 ul in approximately 150 ng/ul) of isolated DNA in the following transformation step.
Our operation in this experiment follows the protocol for Miniprep: Protocol link.
3. Transformation of 14O and 16A to DE3
The transformation of these four plasmids to DE3 was conducted after the miniprep of DE3 respectively. 100 ul transformed cells were extract and then incubated overnight (12 hours approximately) on LB-Amp plates.
Our operation in this experiment follows the protocol for Transformation: Protocol link.
4. Detection of GFP in induction system
After the overnight incubation, the transformed DE3 colonies were selected and incubated in Sangon ready-for-use M9 minimal antibiotic-containing medium with MgSO4, CaCl2 and 20% glucose added. The incubation lasted for approximately 7 hours, and the OD600 reached 0.4. The remaining steps were conducted as the protocol says for measurement and part improvement, and the distribution of the samples are displayed as below:
Our operation in this experiment follows the protocol for Measurement/Part Improvement: Protocol link.
Notebook
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Protocols
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References
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