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RISE Program

NPY-Induced Kinase Activation in C. elegans Effects on Signaling Nodes

Ricardo Chiesa1, Francisco Fuster1, Jessica Díaz Rivera1

1Department of Biology, University of Puerto Rico, Cayey Campus

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_Abstract

Neuropeptide Y (NPY) acts as neurotransmitter by activating multiple receptors thus regulating a number of physiological functions. Studies demonstrated that receptor 1 has anxiolytic effects in human body. To further investigate the effect of NPY, we studied the reactions that the proteins had with the presence of NPY and the changes that NPY causes on GABA receptor. No statistically significant effect was observed in the proteins with the presence of NPY, just Akt1. These results suggests that NPY has an up regulation in Akt1 protein.

Introduction

Neuropeptides are small proteins that act on the cell's surface receptors. They are secreted by neurons for communication with other cells. They adhere to the receptors and trigger actions on the cell. Sometimes these actions lead to a whole network of action and reaction induced pathways. We are working with the effects of neuropeptide Y (NPY), to monitor the changes of a proposed pathway that is supposedly related to anxiolytic effects, specifically receptor Y1. NPY is a neurotransmitter found in the brain and the rest of the nervous system. It occurs naturally in the human body and it is proposed that it regulates many anxiety related conditions, like excessive food intake, memory, and even in learning.The proteins will be extracted from the nematode Caenorhabditis elegans. The nematode is used because it produces many neurotransmitters that are comparable to the ones in the human nervous system. Even though it has a primitive nervous system it is found to be very useful because their hermaphroditic reproduction allows for a

fast renewal supply of these animals, also their maturity span is between 3 to 4 days. The method used to extract these proteins in mostly with the integration of the technique of heat shock. The animal is exposed to a dramatic change of temperature, causing permeability of its membrane, basically expelling the contents of its cells which include proteins. The proposed pathway includes a chain of events that occur after NPY is introduced. Once NPY adheres to the receptor on the cell surface, it stimulates the activation of phospholipase C (PLC). The lipase breaks a phosphate groups from the cell's membrane, separating the phosphorus molecule from the hydrophobic region of the phospholipid. This leads to the release of Ca+ from the endoplasmic reticulum, which activates protein kinase C (PKC). PKC then carries the phosphate group to Hras, which becomes phosphorylated, or activated. This then leads to yet another chain reaction between proteins that pass down this phosphate group, becoming phosphorylated, until the

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process reaches RSK 90. This molecule now has the phosphate group and passes it to CREB, which phosphorylates the gene GABRA. This process turned the gene, on for the synthesis of GABA. GABA is then synthesized and attached to the cell membrane, waiting for a substrate to adhere to activate yet another pathway, linked to anxiolysis. Our primary objectives are: to monitor changes in signaling nodes with the presence of NPY, monitor the effect that NPY causes on GABA synthesis and validate the proposed pathway. We hypothesize that in the presence of NPY, there will be an up regulation of the GABRA gene and increase in the levels of proteins of the proposed pathway. To monitor these changes we used the ELISA assay. This technique helps us detect the presence of a specific protein. It uses a primary layer of antibodies that adhere to the protein, then a secondary layer that adheres to the primary antibody which then creates a union between the structures. Each protein that is detected with ELISA has a specific function: Akt1 is a critical regulator role in diverse cellular processes including growth, survival and the cell cycle, p38 MAP kinase participates in a signaling cascade controlling the cellular response to pro-inflammatory cytokinases and a variety of cellular stresses and Stat3 is an important signaling molecule for many cytokines and growth receptors.

Methodology:

Cultivation of Caenorhabditis elegans:This cultivation involves two major changes of medium. First these nematodes grew in a solid medium of agar, with a similarity to its normal environment. They had plenty of bacteria to feed on, this way there are in no stress. The bacteria they normally feed on is E. coli which was added to the medium.The second medium in which they finished the process of cultivation is on a liquid medium. Here they have a liquid environment with many elements found on their normal aquatic environment in the roots of plants, including the bacteria to feed upon.

Protein extraction:This process involved the use of centrifuging to push all contents down. After this is done, the supernatant in the liquid is removed to concentrate all the other contents left on the bottom. Then next step involves the repetition of the last step to concentrate even more the amount of proteins in a small pellet at the bottom of the tube. To preserve the proteins place them on water at room temperature for 15 min and later store at -80 degrees Celsius.

Protein Quantification BCA:The BCA protein assay was based on a biuret reaction, which is the reduction of Cu2+ to Cu+ by proteins in an alkaline solution with concentration-dependent detection of the monovalent copper ions. Bicinchoninic acid is a chromogenic reagent that chelates the reduced copper, producing a purple complex with strong absorbance at 562 nm.

ELISA(Enzyme-linked Immunosorbent) AssayWe had to do a Reagent Preparation by preparing 1X Wash Buffer by diluting 20X

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Wash Buffer in equivalently purified water. Then we prepared the cell lysates by adding fresh media containing regulator for desired time. Removed media and rinse cells once with ice cold-PBS, to harvest cells under non-denaturing conditions. We removed the PBS and added 0.5 ml ice cold 1X Cell Lysis Buffer plus 1mM phenylmethylsulfony fluoride (PMSF) to each plate and then we put the plate to incubate for a period of 5 minutes. We scraped the cells to transfer to an appropriate tube. Microcentrifuging was using for 10 minutes at 4 degrees Celsius and transferred the supernatant (cell lysate) to a new tube to store at -80 degrees Celsius. Next step was the Test Procedure. We added 100 ul of Sample Diluent to a microcentrifuge tube. Transferred 100 ul of cell lysate into the tube and vortex for a few seconds. Then we added 100 ul of each diluted cell lysate to the appropriate well. The plate was sealed with tape and incubated it for 2 hours at 37 degrees Celsius. The tape was removed and all the wells were washed. We discarded plate contents into a receptacle and washed it 4 times with 1X Wash Buffer, 200 ul each time for each well. For each wash, strike plates on fresh towels hard enough to remove the residual solution in each well. We added 100 ul of Detection Antibody to each well, sealed with tape and incubated it for 1 hour at 37 degrees Celsius. The well wash procedure was repeated. The following step was to add 100 ul HRP-linked secondary antibody to each well, and again repeated wash wells procedure. Then we added 100 ul of TMB Substrate to each well, sealed with tape and incubated the plate for a period of 30 minutes at 37 degrees Celsius. The last step was to add 100 ul of STOP Solution to each well and shake gently for a few seconds. The initial color reaction was blue, which changed to yellow with the addition of the STOP Solution.

SpectrophotometryPlace the 96 well plate in the spectrophotometer. Choose the two blanks and the experimental wells using “Microplate Manager 5.2.1 Program”.

Results:

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Discussion:

The levels of total Akt-1 were higher (statistical significance) in the +NPY protein extracts. However, phospho-Akt-1 levels were the same both in the control and experimental group. We have to corroborate if a CRE element is present in the Akt-1 gene. If future experiments show an increase in the levels of phospho-CREB, this could account for the increase in the levels of total Akt-1. Phospho-Stat-3 and phospho-p38 levels are the same both in control group (no NPY) and the experimental group (with NPY).

References:

[1] Domschke K, Dannlowski U, Hohoff C, et all. (2010). Neuropeptide Y (NPY) gene: Impact on emotional processing and treatment response in anxious depression. Eur Neuropsychopharmacol, 5, 301-9.[2] Bacchi F, Mathe AA, Jimenez P, et. all. (2006). Anxiolytic-like effect of the selective Neuropeptide Y Y2 receptor antagonist BIIE0246 in the elevated plus-maze. Peptides,12, 3202-7.[3] Geary, T. G., Kubiak, T. M. (2005). Neuropeptide G-protein couple receptors, their cognate ligands and behavior in Caernorhabditis elegans. TRENDS in Pharmacological Sciencies, 26, 56-58.