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Experiments for the Invention of Drugs for Alzheimer's Disease

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Proposed Study

Aim 1: HSV-1 upregulation effects on mRNA-146a

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In this initial aim of the study, we propose to show the effects of HSV-1 in the brain of Alzheimer’s patients and its production of infection towards inflammation in the brain. We hypothesize that HSV-1 should induce upregulation of mRNA-146a in the brain of Alzheimer’s patients, which is the main RNA associated with pro-inflammation in the brain. Understanding this mechanism could help relate to our hypothesis on how we can reduce or inhibit inflammation in the brain of AD patients to avoid further complication with the disease. We will need this knowledge to understand how our mRNA works and its pathway. It also will help with future remedies and further research purposes.

Proposed Experiments

We will use a set of neural cells from AD infected human brain and test our hypothesis on a petri dish. Human neural cells will grow in a medium for two weeks. We propose that they will grow into neurons, glias or some form of cell related to the central nervous system. After two weeks, the cells will show a fair distribution between neurons and glia. We will then take samples of the cells and place them in a separate petri dish to extract the RNA and protein. Then infect the neural cells with HSV-1 every 24 hours and record the progression of the infection each day. After two weeks of culture, we will then homogenize the tissues for 3 minutes. We also will try other strains of viruses and bacteria to see if they have the same effects as HSV-1 does for mRNA-146a in Alzheimer’s disease (AD). We will use chlamydia pneumoniae as a control, which has been previously shown to have a role in the pathogenesis of AD (Kamer, 2008). We propose that this bacteria strain would not have an effect on mRNA-146a or induce it to cause an inflammation in the brain. The same procedure will be done and ran for two weeks. Western blot immunoblot on down regulated proteins of mRNA – 146a was also done. ie CFH and COX-2 with the use of ẞ-actin as our control.

Predicted Results

We propose that there will be a change of structure in human neural cells and the morphology of the cells will be infected. After each day, there should be progression of infected cells, since our study watched the virus for two weeks. We also should see an increase in number of the cells infected, with an increase of at least 45% from the original state that it was in. In our proposed experiments, we predicted that the virus would take over the state of the cells and results from the days of infected cells will prove this prediction correctly. On the otherhand, bacteria cells might show a slight change in the cells but we propose it should not be as drastic as the virus. If the virus, does in fact change the neural cells, it will indicate to our proposal that we are on the right track and also give us information that HSV-1 might induce miRNA-146a. The replications of cells will happen but not in a health way. If that occurs, we can hypothesize that CFH if being downregulated and brain cells will have inflammation.

Aim 2: Effects of PDTC on inflammation in mice hippocampus

For our second aim, we propose if pyrrolidine dithiocarbamate (PDTC) is able to reduce inflammation in AD. We hypothesize that PDTC is able to attenuate inflammation by using this inhibitor as a blocker to transverse the cell membrane. We will be testing the effects of PDTC in mice infected with Lipopolysaccharide (LPS) and mice that are later treated with PDTC from infections of LPS. LPS is known as an activator that stimulates proinflammatory cytokines in mice and human brains (Zhang, 2018). We will be using PDTC to test for reduction in inflammation in mice brain by injecting PDTC in the hippocampus area.

Proposed Experiments

The mixture of LPS and PDTC will be administered together then we will dilute it with saline. This mixture will be injected into the mouse intraperitoneally (IP) and our control will be administered with only saline

We will be injecting the first group with saline only, the second group with LPS, the third group with PDTC, then the fourth group will be injected with PDTC after 1hr of injecting LPS into the mouse. The mouse will be monitored for 24hrs in the cage and we will record its behavior. We will take blood samples from the tail veins of the mouse to measure protein levels and collect samples of hippocampus cells in each group of mice that we test. Then run it through western blot analysis.

We will want to make sure the first results are validated when done, so a second method will be done to validate.

We will split eighteen mice into three groups. The first group we will injected IP with the same control- saline, the second group with LPS and the third with LPS+PDTC. Blood samples will be gathered again, as well as hippocampus cells and ran through an ELISA analysis.

We will be using ELISA (Enzyme-linked Immunosorbent Assay) to measure levels of IL-6, TNF-a (these are cytokines in the brain) serum and hippocampus cells.

Then we will buffer our hippocampus cells of the mouse, centrifuge and load for electrophoresis. We will then place the membrane that includes the transfer proteins into an incubator to incubate overnight.

While our proteins are prepping, we will take hare anti-NF-kB antibody and anit-B-actin antibody at 4° C and will refrigerate them. We will use horseradish conjugated secondary antibodies as well to detect immunoreactivity of the protein.

Predicted Results

We propose that PDTC could potentially work on those proteins and mRNA-146a pathway that upregulate inflammation in the hippocampus through the Nf-kB pathway. We propose that our results will show PDTC inhibition in the hippocampus area.

As we compare our study proposed to the controls, we should see an increase in the cytokine protein levels in the serum and hippocampus homogenate with mouse infected with LPS. This will show that due to no inhibition of PDTC, mRNA-146a is able to be induce and bring about inflammation in AD brains.

In our second experiment, we should also see the same effects of PDTC in mice who are infected with LPS.

We propose that mice with no treatment would have an increase in LPS. We potentially should see a decrease in mice with PDTC+ LPS. This could be due to LPS functioning in advance before PDTC was injected, with this understanding we propose that since LPS was in the mice system before hand, inflammation would still occur but PDTC could only limit the reaction. If this proposal turns out correctly, it will further shows that PDTC is working as an inhibitor. Lastly, mice with just PDTC should show greater inhibition and reduction of inflammation.

If after all we propose, the experiment does not work out, it shows that PDTC is not inhibiting mRNA-146a in the hippocampus and further analogies we have hypothesized would not work.

Aim 3: Pyrrolidine dithiocarbamate effect on proinflammatory genes leads to NF-kB inhibition

In this third aim, we will work on a deeper level from our second aim but focusing more on the genes regulated in proinflammation and how it corresponds in the pathway of NF-kb. We will be using the same inhibitor – pyrrolidine dithiocarbamate to test the effect on the NF-kB pathway specifically.

This should help us understand what we are inhibiting, and to ensure we are targeting the right pathway, RNA’s and genes altogether.

From the understanding of previous aims, we see that HSV-1 might affect neural cells, so we further hypothesize that maybe we could check those neural cells in a specific area of the brain- the hippocampus. If our previous proposed experiments do correlate and work efficiently. This last stage would help tie every hypothesis together and completely block a pathway with no effects on the RNA’s or genes while inhibiting inflammation in Alzheimers Disease.

Proposed Experiments

We will use cells from male mice. We will categorize our models into three groups. In each one

of the following groups we will be injecting concentrations and a control. The Injection of Lipopolysaccharide (LPS) or Salmonella will be used, since we used LPS in our second aim we will go ahead and make use of salmonella in this study. There is not much difference in the two agents. LPS is imbedded in bacteria’s, so using bacteria would work just as well. We will focus on using the Northern blot method to carry out our experiment.

The category of mice will be (i) injection of PDTC + LPS, having different concentrations of PDTC, (ii) IP with only PDTC and GADPH will be our control. We will slowly kill the mice by exsanguination.

The mice injected with PDTC will be killed between two to five hours. This will help us get enough products retained with PDTC in the mice. Then we will proceed in taking blood samples from the mice after 5 hours to obtain plasma genes ie TNF-a, CINC and ICAM-1.

We will look into taking mice probes for the inflammatory genes; COX-2, CINC and ICAM-1 and amplify them in a RT-PCR using RNA from our second aim in rats treated with LPS. Then proceed to design primers corresponding to the mice genes that trigger proinflammation – ie COX-2, CINC and ICAM-1, and then test our hypothesis on a Northern blot. When we run our primers through the northern we propose to see bands that correspond to the proinflammatory genes discussed previously. If the bands happen not to show then

Predicted Results

In conclusion with our experiments above, we propose that inhibition of pro-inflammatory genes that are regulated through the NF-kB pathway will be inhibited by the presence of PDTC.

As we proposed, we focus on the inflammatory genes. The proposed experiment should result in less inhibition with LPS + PDTC and the different kinds of concentration and the genes should not be completely inhibited. This could be because of the LPS that is already involved, just as we proposed in 2 aim. We propose that the second mice with only PDTC will have increased inhibition with all the genes.

Overall, if our proposed experiments do work out and we see an inhibition of the proinflammatory genes i.e TNF-a, COX-2, CINC, ICAM-1 then we would have shown PDTC’s effect on proinflammatory genes and how this can benefit the pathway (NF-kB) towards inflammation in AD.

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