Latest revision as of 00:12, 22 October 2019

Excitatory Amino Acid Transporter 2 with C/D Box from Homo Sapiens

This part contains EAAT2 protein that may transport glutamate, which is an universal excitatory neurotransmitter, from cleft and extracellular matrix, and alleviate the excitotoxicity induced by the overhigh glutamate. The part also contains C/D box which may binds to L7Ae transfected and expressed on inner side of exosome membrane, and help hold the mRNA of EAAT2 on exosomes.

EAAT2-Functional Characterization

At the beginning of our project, we characterized the function of EAAT2 and its expression on pcDNA3.1(+), and simulated the inflammation-inducing environment for Neuro-2a cells to mimic the high-glutamate condition in neurodegenerative patients’ cerebrospinal fluid. In this part of test, we focused on two impacts our exosome medicine may bring to the patients: 1. Reducing the excitatory amino acids in cerebrospinal fluid. 2. Alleviate the cytotoxicity brought by over-high glutamate or the excitotoxicity induced.

Experiment data

1.Glutamate Assay

We tested two sets of cells: the EAAT2-transfected N2a cells and pcDNA3.1-transfected cells (negative control). For each set of N2a cells, the time interval for changing the DMEM medium to inflammation-inducing medium is three hours. There are four groups (approximately 0 hour, 3 hours, 6 hours and 9 hours) in total depending on the time to change the medium. The extracted cell medium was then applied with glutamate assay kit and the absorbance for each sample was read in plate reader. The distribution of each sample are displayed as below:

Figure 1: The glutamate concentration measured and calculated by comparing with calibration curve and plate blank, and its correlation to treated duration.

Figure 2: The comparison in glutamate concentration between EAAT2-transfected and pcDNA3.1(+)-transfected Neuro-2a cells, student’s t-test applied (*: p<0.05, **: p<0.01, ***: p<0.001).

The results of glutamate assay reveals that the expression of EAAT2 on cell membrane can maintain the concentration of glutamate in a relatively stable level, and significantly reduce the concentration of glutamate in the cell medium. Higher the glutamate concentration is, the more significant the EAAT2 may transport the glutamate in cells. In addition, the expression of EAAT2 will not over-transport the glutamate and make the glutamate concentration lower than the origin level.

2.WST-1 Cytotoxicity Test

To detect the cytotoxicity induced by the over-high glutamate concentration, we used WST-1 as an alternative of MTT in our test. The dehydrogenase in mitochondria can reduce the WST-1 to soluble and chromatic formazan, and a high absorbance measured indicates a low cytotoxicity induced by glutamate. The absorbance of these samples are measured at 450 nm and 690 nm as a reference wavelength. The distribution of each sample are displayed as below:

Figure 3: The absorbance (A450) measured after with reference absorbance (A690) and blank value subtracted, and its correlation to treated duration.

Figure 4: The comparison in A450- A690 between EAAT2-transfected and pcDNA3.1(+)-transfected Neuro-2a cells (after a two-hour incubation in 37 Celsius), student’s t-test applied (*: p<0.05, **: p<0.01, ***: p<0.001).

The results of WST-1 indicated that the expression of EAAT2 on cell membrane can significantly alleviate the excitotoxicity induced by glutamate. The cytotoxicity may have limited influence on cell proliferation even if the cells are cultured in inflammation-inducing (80 uM glutamate) for longer than 9 hours.

Further Application

Our primary goal is to save astrocytes from the high glutamate level at the early stage of neurodegenerative disease and strengthen the defence against the neurotoxicity. In details, we design this therapy to enhance the glutamate absorbing function by overexpression of EAAT2 on the astrocyte’s membrane. We use the mRNA-based vector for expression and protect the mRNA by exosome through the delivery process. We will further study the production, characterization, transportation, preservation and other related content of exosome therapy, and subsequently, carry out social activities related to exosome treatment to enhance our design.

Sequence and Features

Reference

Sahin, U., Karikó, K. and Türeci, Ö. (2014) ‘mRNA-based therapeutics — developing a new class of drugs’,Nature Publishing Group, 13(10), pp. 759–780. doi: 10.1038/nrd4278.