Revision as of 12:23, 11 October 2018

OriginALS

Results

Technical Experiments

Calcium Phosphate Transfection

Title: Transfection to microglia and astrocytes via “calcium phosphate” method.

Conducted by: Sagi Angel

Date: 3-7.6.18

Aim: In this experiment we have tried to use the calcium phosphate protocol in order to transfect microglia astrocytes and HEK as control in GFP gene. The use of HEK cells as control, is due to its ability to be transfected relatively easily by the various techniques, including calcium phosphate method.

Importance: This experiment was carried out in parallel with experiments using different methods of transfection (different reagents and electroporation) in order to find an efficient way of inserting our plasmids into astrocytes and microglia for the continuation of the project.

Design:
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Expectations:

The expected results were the illumination of GFP under a fluorescent microscope. It could be assumed that if the experiment was conducted well, the HEK cells would be able to insert the GFP plasmid easily and the illumination would be high, and one could hope that some of the experimental cells, astrocytes and microglia, would also show fluorescence. If there was a problem during the experiment or in the quantities of the materials, it would not be possible to see fluorescence in the HEK cells or we would see a high cell mortality rate.

Results:

All of the cell types survived the transfection process –no significant death rate was noticed
In the HEK cells used as control indeed there was an expression of the GFP gene under the fluorescence microscope the expression wasn’t high
No GFP was detected in the microglia and astrocytes cells in both option of transfection

[ADD results Photo]

Discussion:

The purpose of the experiment was to introduce a fluorescent gene into microglia and astrocytes using the HEK cells as control. Because fluorescence illumination is obtained in the control cells, it is assumed that the transduction process worked properly, but the specific cells of the microglia and astrocytes do not respond well to the process in a manner similar to the control cells. It should be noted that the percentage of infection in the control cells was not particularly high, which may indicate a process that is not done optimally or on problems in concentrations of DNA.

Conclusion:

The process of transfusion using calcium chloride is simple and inexpensive, but does not allow high penetration rates in our target cells - astrocytes and microglia.

While there was fluorescence illumination in the control cells, it was lower than expected, which means that conditions may be improved to increase efficiency, and this may also could have effect on the target cells.

Since there are many materials that make up the buffer, it is possible that a change in quantity or lack of accurate preparation may affect the quality of the results.

It should be concluded that this method, especially in a short period of time that does not allow many adjustments and repetitions, is not suitable for the continuation of work in the project and has indeed been abandoned in favor of the use of ready-made reagents that showed higher efficiency in control cells and astrocytes and microglia.

Electroporation Transfection

Title: Knockout of IKK-β gene in microglia cell-line.

Conducted by: Einan Farhi and Mor Pasy

Date: 15.7.18

Experiment goal and significance: The objective is to create a stable cell-line of the BV2 cells with a knockout mutation of the Inhibitor of Nuclear Factor kappa-B Kinase subunit beta (IKBKB) gene. With which, we aim to demonstrate how targeting the Nuclear Factor kappa B (NFκB) pathway will diminish the amount of inflammation promoting cytokines produced by the immune representative cells of the brain.

Design:

Each transfection mixture was pipetted evenly into 6 wells of a 24 well-plate. DNA quantities that were used are as following: 2.5, 5, 9 μg of DNA. As a positive control, BV-2 cells were transfected with 5 μg pAc-GFP and as a negative control cells were electroporated with no plasmid and also seeded without electroporation. Here is a schematic diagram of the wells mentioned:

BV2 electroporation with px601-f4/80-g2; 2.5x10⁶ cells per cuvette; A-030 program
2.5 μg DNA	2.5 μg DNA	2.5 μg DNA	2.5 μg DNA	2.5 μg DNA	2.5 μg DNA
5 μg DNA	5 μg DNA	5 μg DNA	5 μg DNA	5 μg DNA	5 μg DNA
9 μg DNA	9 μg DNA	9 μg DNA	9 μg DNA	9 μg DNA	9 μg DNA
No DNA	No DNA	No DNA	No DNA	No DNA	No DNA
No transfection	No transfection	No transfection	No transfection	No transfection	No transfection

Expectations:

We expected to observe a GFP signal in all the wells that were successfully transfected with the px601-f4/80-g2 plasmid, while we did not expect to observe any GFP signal from the wells in the “No DNA” or “No transfection” groups.

Results:

In the following 3 days, the culture was monitored using a fluorescent microscope.

Unfortunately, no GFP positive cells were observed under the microscope lens. As the positive control showed a successful transfection in the technical sense, we concluded that there is a problem in transfecting this particular cell-line with such a large construct. This is plausible because the transfection efficiency was low even with a small construct such as pAc-GFP with only ~5% transfection efficiency (Figure1).

[add results photo]

Figure 1 - BV-2 cells, transfected with 5 μg pAc-GFP,
captured under the microscope with a green fluorescent filter.

Discussion:

As we didn’t manage to observe the GFP in the cells that were transfected with our vector, there was no reason to continue to the next step of the experiment. However, in our discussion, we arrived to an alternative explanation of No GFP result. We suggested that maybe the cells were successfully transfected and had the plasmids in them but maybe the F4/80 promoter was too weakly expressed so that a GFP was below our detection limit.

Conclusion:

The experiment with our plasmid px601-f4/80-g2 (and the rest of our plasmids with the same goal but with different guide sequences) failed. We concluded that continuing with the attempted method and plasmid would be time consuming and we would have to check for many different reasons as of why did the transfection failed. Therefore we made a change in our course of action and decided to try to accomplish the inhibition of the NFκB pathway with a different approach using Lentiviral vectors.

Transfection by transfection-reagent

Title: Transfection of the C8-D30 astrocytes and BV-2 microglia cell lines with PUC-GFP vector.

Conducted by:Liat Tsoran and Ori Tulchinsky

Date:

Aim: In this experiment we have tried to use jetPEI-Macrophage and jetPRIME transfection reagent in order to transfect microglia cells (BV2) and astrocyte cells (C8-D30), respectively, while using HEK-cell as a positive control. The use of HEK cell-line as a positive control, is due to its transfectability by the various techniques.

Importance: This experiment was carried out in parallel with experiments using different methods of transfection (different reagents, calcium phosphate and electrophoresis) in order to find an efficient way of inserting our plasmids into microglia for the continuation of the project.

Design:

Experiment 1 – Microglia (BV2) transfection:

[add photo]

Experiment 2 – Astrocyte (C8-D30) transfection:

[add photo]

Expectations:

The expected results were to detect GFP fluorescence under a fluorescent microscope. It could be assumed that if the experiment was conducted well, the HEK cells would be able to harbor the GFP containing plasmid and fluorescence would be high. One could have hoped that some of the cell-lines of our experimetns would also show fluorescence. If there was a problem during the experiment or in the quantities of the materials, it would not be possible to see fluorescence in the HEK cells or we would see a high cell mortality rate.

Results:

All the cell types survived the transfection process –no significant death rate was noticed.
In the HEK cells used as control indeed there was an expression of the GFP.
No GFP was detected in the microglia nor the astrocyte cells in all groups of transfection.

Discussion:

The purpose of the experiment was to introduce a fluorescent gene into microglia and astrocytes using the HEK cells as control. Because fluorescence illumination is obtained in the control cells, it is assumed that the transfection process worked properly, although that microglia and astrocytes did not respond well to the process in a manner similar to the control cells.

Conclusion:

The process of transfection using jetPEI-Macrophage transfection reagent for microglia cells and jetPRIME for astrocyte cells was unsuccessful. For this reason, we switched to other methods - prior to electrophoresis and when this method was not successful we transferred all our vectors to Lantivirus vectors and we used the Lentivirus infection method.

Microglia Experiments

Cytokines Inhibition Assay

Title: Validation of IKKB knockdown through measurement of cytokine TNFa and IL1a expression in BV2 cells.
Conducted by: Avital Bailen and Daniel Deitch.
Date: 20.9.18-11.10.18
Aim: Quantify the expression of IL1a and TNFa through qPCR using cell lines infected with the shIKK vector using a Lenti-virus packaging.
Importance: We predict that reducing the cytokines, IL-1α and TNF-α, will prevent the creation of new Reactive Astrocytes in the brain (Liddelow et al.). We predict that upon microglial IKKb knockdown reduction in the secretion of the mentioned cytokines will occur thus new reactive astrocytes formation will be prevented, this way, preventing further damage to motor neurons.

Experiments	Protocols	Notebook
BV2 infection
Cytokine inhibition Assay (qPCR)
Cytokine inhibition Assay (ELISA)

Theoretical background:
The synthesis of IL1α and TNFα cytokines is mediated by the NF-kB transcription factor (Tak et al.). NF-kB activation in microglia causes motor neurons death in vitro as well as in vivo. Heterozygous inhibition of NF-kB in microglia substantially delayed disease progression in ALS mice models (Ashley et al.). In the experiment we used a commercial shikkb viral vector. This vector expresses a short hairpin RNA targeting the IKKβ mRNA. An RNA interference with the expression of IKK-β should inhibit the activation of the NFκB pathway which would produce a weaker expression of its target genes¹, among them: Interleukin 1 subunit α (Il1α)² and Tumor Necrosis Factor subunit α (TNFα)³.

Procedure:

Infect BV2 microglia cells with shIKKb plasmid.
Perform selection of transfected cells with Puromycin.
Grow BV2 cells (wild type and infected) in 6-well plates to 80% confluence.
Activate the cells with LPS for 2 hours to induce microglia activation and cytokine secretion.
Extract RNA and create cDNA.
Run qPCR.

Experimental Design:

[add table of design]

+shIKK - BV2 microglia cells infected with shIKK

-shIKK -BV2 microglia cells which underwent the infection process without a plasmid

No treatment – WT BV2 microglia

Strengths and weaknesses:

Strengths:

Sensitivity – detects synthesized product at very low concentrations.
Provides quantitative results.
Easily reproducible procedure.

Weaknesses:

As the qPCR machine is very sensitive, the experiments require a certain amount of technical ability, therefore it may take several experiments before achieving reliable results.

References:

Wang, X. , Li, H. , Xu, K. , Zhu, H. , Peng, Y. , Liang, A. , Li, C. , Huang, D. and Ye, W. (2016), SIRT1 expression is refractory to hypoxia and inflammatory cytokines in nucleus pulposus cells: Novel regulation by HIF‐1α and NF‐κB signaling. Cell Biol Int, 40: 716-726.
Mori N and Prager D. (1996). Blood, 87.
Shakhov AN, Collart MA, Vassalli P, Nedospasov SA and Jongeneel CV. (1990). J. Exp. Med., 171.
Das, Amitabh, et al. “Transcriptome Sequencing Reveals That LPS-Triggered Transcriptional Responses in Established Microglia BV2 Cell Lines Are Poorly Representative of Primary Microglia.” Journal of Neuroinflammation, vol. 13, no. 1, 2016.

Astrocyte Experiments

Astrocyte Activation

Title: Activation of astrocyte cell line C8-D30 using lipopolysaccharide (LPS) and pro-inflammatory cytokines.
Conducted by: Mor Sela

Date: 29.7.18-2.8.18

Aim: Activation of astrocytes was performed to confirm that our C8D30 astrocyte cell line can accurately model resting and reactive astrocytes for our experimental design.

Importance:Our project is based on the assumption that reactive astrocytes are a main factor in ALS and therefore our product is designed to specifically disarm them. Without a “reactive astrocyte” experimental group, we can not test the efficiency and specificity of our product in reactive astrocytes when compared to other cells in the system.

Experiments	Protocols	Notebook
ELISA
Western Blot
Staining using Dino antigens

Theoretical background:
Reactive astrocytes are found in several forms, including forms called A1 and A2. Gene expression analysis in reactive astrocytes has shown that reactive astrocytes type A1 express many genes that are detrimental to synapses (such as complement cascade genes)¹. Studies show that A1 reactive astrocytes are produced as a result of an NFkB protein signal. A process termed nuclear factor kappa light- chain enhancer of activated B cells². Reactive astrocytes type A2 over-express neurotropic factors, which promote synapse repair. Meaning A1 reactive astrocytes are harmful while A2 reactive astrocytes are helpful to the central nervous system (CNS) and brain¹.

Recently, researchers have developed a model of exclusively A1 reactive astrocytes in cell culture which allows for targeted research. In this model, researchers quickly extract astrocytes from brain tissue which has not yet been damaged. Then these astrocytes are grown in tissue culture with the relevant medium. Finally, a cocktail of cytokines, taken from the medium of activated microglia, is added to the astrocyte culture. At this point, the astrocytes exhibit an A1 reactive astrocytes phenotype¹.

Microglia can be activated in vivo by inducing chronic CNS damage or by injection of lipopolysaccharides (LPS). Activated microglia secrete three cytokines which induce A1 reactive astrocytes: interleukin 1 alpha (IL1a), tumor necrosis factor alpha (TNFa), and complement component 1q (C1q). Adding LPS or these three cytokines in vitro produces A1 reactive astrocytes with a genetic profile very similar to A1 reactive astrocytes in vivo.¹

Experiments on this model have shown that A1 reactive astrocytes lose almost all functions displayed by resting astrocytes. A1 reactive astrocytes have very low ability to create synapses, can not perform phagocytosis, and do not induce neuronal rehabilitation or growth.

Single cell data has shown that the complement component C3 is a preferred reactive astrocytes marker to glial fibrillary acidic protein (GFAP). C3 is specific to A1 reactive astrocytes, over A2 reactive astrocytes and resting astrocytes³. Meaning A1 reactive astrocytes secrete many classical complement cascade components which accelerate synapse degeneration and other toxic substances which cause damage to neurons and oligodentrocytes^,4,5,6.

Procedure:

Activation of Microglia (BV2 cell line) by adding LPS to their medium inducing the secretion of cytokines^7,8
After 24 hours – transfer Microglia medium (cytokines +) to the “resting” Astrocyte (C8D30) wells (Activation step)¹
Adding commercial cytokines (IL-1a, TNF, C1q) to different “resting” Astrocyte wells (Activation step).¹
Validation of astrocyte reactivity - using ELISA and Western Blot to measure the expression of C3 protein in the samples⁹

[procedure img]

Design:

Experiment 1 – Measurement of C3 in activated astrocytes using ELISA

C8D30 who grow with ACM + LPS (from microglia plate)	C8D30 who grow with MCM + LPS (from microglia plate) + ACM	C8D30 who grow with MCM (from microglia plate) without LPS + ACM Negative control	MCM without Microglia cells + LPS (from microglia plate) + ACM + C8D30 Negative control
V	V	V	V	Biological repetition 1
V	V	X	X	Biological repetition 2
X	V	X	X	Biological repetition 3
X	V	X	X	Biological repetition 4

Experiment 2 – Measurement of C3 in activated astrocytes using Western Blot

ACM + 3 cytokines + C8D30 after 48hr.

ACM + 3 cytokines + C8D30 after 24 hr.

ACM + C8D30

Negative control

C8D30 who grow with ACM + LPS (from microglia plate) after 48 hr.

C8D30 who grow with ACM + LPS (from microglia plate) after 24 hr.

C8D30 who grow with MCM + LPS (from microglia plate) after 48 hr.

C8D30 who grow with MCM + LPS (from microglia plate) after 24 hr.

MCM without LPS + C8D30

Negative control

V

Biological repetition 1

V

X

V

X

Biological repetition 2

Strengths and weaknesses:

Strengths:

Adding LPS to microglia medium mimics the in vivo conditions and process better than adding commercial cytokines directly to astrocyte medium
This activation process is fast and simple.
Western Blot Analysis:

Sensitivity – detect protein at very low concentrations (0.1 ng protein per sample).
Specificity- Gel electrophoresis sorts each protein sample according to size, shape, and charge. The observed bands give an indication as to the size of the protein or polypeptide. Additionally, as the detection is based on antibody binding, the process can locate a specific protein in a sample of over 300,000 different proteins.

ELISA (Enzyme-Linked Immunosorbent Assay)¹⁰

Relatively cheap reagents with a long shelf life.
Sensitivity and specificity higher than western blot analysis.
No radiation, as opposed to exposure during binding the antibody to the protein or disposing of chemicals from western blot analysis.
Faster and easier procedure than western blot analysis.
The results are quantitative rather than qualitative (as in western blot analysis).
Applicable for a wide range of proteins.

Weaknesses:

In the article Liddelow, 2017¹ the activation is induced by injecting mouse models with LPS rather than adding LPS to cell culture. Therefore, the strength of the activation may be lower in our experiment. Additionally, we do not measure the concentration of the cytokines produced after the microglia are incubated with LPS overnight. Meaning that the astrocyte activation is induced with an unknown concentration of cytokines, as well as other unknown factors found in the medium.
Activation of astrocytes in this way does not fully mimic the process in vivo. Although there is evidence that these three cytokines are enough to induce reactive astrocytes, it is possible that other factors are involved in vivo which may affect the phenotype.
In Liddelow, 2017¹ primary astrocytes are activated, while we are working with C8D30 cell lines. Therefore, the protocol may not correspond exactly.

References:

Liddelow, S.A., Guttenplan, K.A., Clarke, L.E., Bennett, F.C., Bohlen, C.J., Schirmer, L., Bennett, M.L., M€unch, A.E., Chung, W.S., Peterson, T.C., et al.(2017). Neurotoxic reactive astrocytes are induced activated microglia. Nature541, 481–487.
Lian, H., Yang, L., Cole, A., Sun, L., Chiang, A.C.-A., Fowler, S.W., Shim, D.J., Rodriguez-Rivera, J., Taglialatela, G., Jankowsky, J.L., et al. (2015). NFkB activated astroglial release of complement C3 compromises neuronal morphology and function associated with Alzheimer’s disease. Neuron 85, 101–115.
"Reactive Astrocytes: Production, Function, and Therapeutic Potential" Shane A. Liddelow1,* and Ben A. Barres1,* 1Department of Neurobiology, Stanford University School of Medicine, Stanford, CA 94305, USA *Correspondence: liddelow@stanford.edu (S.A.L.), barres@stanford.edu (B.A.B.)
Stevens, B., Allen, N.J., Vazquez, L.E., Howell, G.R., Christopherson, K.S., Nouri, N., Micheva, K.D., Mehalow, A.K., Huberman, A.D., Stafford, B., et al. (2007). The classical complement cascade mediates CNS synapse elimination. Cell 131, 1164–1178.
Hong, S., Beja-Glasser, V.F., Nfonoyim, B.M., Frouin, A., Li, S., Ramakrishnan, S., Merry, K.M., Shi, Q., Rosenthal, A., Barres, B.A., et al. (2016). Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352, 712–716.
Sekar, A., Bialas, A.R., de Rivera, H., Davis, A., Hammond, T.R., Kamitaki, N., Tooley, K., Presumey, J., Baum, M., Van Doren, V., et al. (2016). Schizophrenia risk from complex variation of complement component 4. Nature 530,177–183.
"Activation of BV2 microglia by lipopolysaccharide triggers an inflammatory reaction in PC12 cell apoptosis through a toll-like receptor 4-dependent pathway" Xiao-jing Dai, Na Li, Le Yu, Zi-yang Chen, Rong Hua, Xia Qin, and Yong-Mei Zhang
"Development of an Insert Co-culture System of Two Cellular Types in the Absence of Cell-Cell Contact." Renaud J1, Martinoli MG2.
Lindblom, Rickard PF, et al. "Unbiased expression mapping identifies a link between the complement and cholinergic systems in the rat central nervous system." The Journal of Immunology 192.3 (2014): 1138 1153.‏
"Advantages, Disadvantages and Modifications of Conventional ELISA" Samira Hosseini, Patricia Vázquez Villegas,Marco Rito-Palomares,Sergio O. Martinez-Chapa 31 December 2017

Timp1 and Steap4 Promoter Assay

Title: Promoter assay for Timp1 and Steap4 promoters in reactive astrocytes.

Conducted by: Nitzan Keidar and Mor Sela

Date: 24.9.18-28.9.18

Aim: Our goal in this experiment is to assess the strength and the specificity of the promoters Timp1 and Steap4 by quantifying the amount of luminescence produced by the Luciferase enzyme cloned downstream of these promoters, under our experimental conditions.

Importance: Our project is based on the assumption that reactive astrocytes can be targeted based on specific genetic markers (e.g Timp1 and Steap4). Non-specific expression can lead to off target activity such as healthy resting astrocytes, microglia or other neighboring brain cells.

Experiments	Protocols	Notebook
Promoter assay in reactive astrocytes

Theoretical background:

“Reactive astrocytes" change their gene expression profile relative to quiescent astrocytes. Two such distinguishing genetic markers are Steap4 and Timp1 genes, expressed exclusively in reactive astrocytes^1-4. Genetic reporter systems are widely used to study eukaryotic gene expression and cellular physiology.

Our promoter assay kit is a "Dual-Luciferase® Reporter Assay System" of Promega. The term “dual reporter” refers to the simultaneous expression and measurement of two individual reporter enzymes within a single system.

Typically, the “experimental” reporter is correlated with the effect of specific experimental conditions, while the activity of the co-transfected “control” reporter provides an internal control that serves as the baseline response. Normalizing the activity of the experimental reporter to the activity of the internal control minimizes experimental variability caused by differences in cell viability or transfection efficiency.

Thus, dual-reporter assays often allow more reliable interpretation of the experimental data by reducing external influences.

We used pGL3 series of firefly and Renilla luciferase vectors for the DLR™ Assay Systems. Our vectors are:
[Add picture of experiment plasmids]

Procedure:

Co-transfect cells with plasmids pGL3+Timp1/ PGL3+Steap4 and Renilla+T7
Allow translation of Luciferese enzyme (48 hours).
Cell lysis to release Luc enzymes

In luminometer:

Provide enzymes with substrate and co-factors to produce light.
Measure light emission against controls. Renilla Luc correspond to efficiency of transfection, Firefly Luc correspond to strength of promoter.

[picture of experimental procedure]