Evidence has shown a distinct correlation between aging and Alzheimer’s Disease (AD); however, age is not the only trigger of AD. Scientists have narrowed down and discovered a connection between Alzheimer’s Disease and an adrenergic receptor named Alpha2a-AR (Sun L, et al, 2010). Our results mainly consist of two parts.

The first part is to construct GST-α2A-AR-CT plasmid and Str-KDEL_SBP-α2A-AR-Rluc8 plasmid, and express GST-α2A-AR-CT protein in E.coli BL21. The second part is to transfect the Str-KDEL_SBP-α2A-AR-Rluc8 plasmid into HEK293 cell line to express Alpha2a-AR and construct RUSH-BRET platform to monitor movement α2A-AR between plasma membrane and endoplasmic reticulum.

 

Target1: Plasmids construction

· GST-α2A-AR-CT plasmid construction

In this part of the experiment, we used the following primers to amplify α2A-AR fragment (TIFNHDFRRAFKKILCRGDRKRIV) from α2A-AR-GFP and inserted into the vectors pGEX-4T-1 to obtain GST-α2A-AR-CT plasmid.

GST-α2A-AR-CT-Forward: 5’-GATCGGATCCACCATCTTCAACCACG-3’

GST-α2A-AR-CT-Reverse: 5’-GATCCTCGAGTCACACGATCCGCTTCC-3’

 

· Str-KDEL_SBP-α2A-AR-Rluc8 plasmid construction

First, using the human α2A-AR-Rluc8 plasmid as a template, point mutation at Sda1 to eliminate Sda1 enzyme restriction site in order to get mutant α2A-AR-Rluc8 plasmid. The primer is as follows:

α2A-Sda1 mutant-F: 5’-CACCCCTTACTCCCTACAAGTGACGCTGACGCTG-3’

α2A-Sda1 mutant-R: 5’-CAGCGTCAGCGTCACTTGTAGGGAGTAAGGGGTG-3’

 

Second, mutant α2A-AR-Rluc8 plasmid as a template to amplify target sequence by using the following primers. Gel DNA Extraction purified 2106 bp product.

KDEL-SBP-2A-Rluc8-F:5'-GATCCCTGCAGGTATGGGCTCCCTGCAGCCG-3'

KDEL-SBP-2A-Rluc8-R:5'-GATCTAGATTACTGCTCGTTCTCAGCACGCG-3'

 

Third, The plasmid Str-KDEL_SBP-EGFP-Ecadherin (purchased from Addgene) was double digested with Sda1 and Xba1 and purified the product at 5234 bp to obtain the Str-KDEL_SBP vector. we inserted the α2A-AR-Rluc8 fragment to Str-KDEL_SBP vector to obtain Str-KDEL_SBP-α2A-AR-Rluc8 plasmid.

 

To confirm whether our experiment is successful. We ran the gel electrophoresis. We use this method to obtain relatively pure bands of target genes. According to the gel below, we can see that the strip of α2A-AR-Rluc8 is located at 2322 bp around and the strip of Str-KDEL_SBP-EGFP-Ecadherin is located at 5210 bp around. In that way, we could basically confirm that we have obtained the expected plamids then we can recycle the strips for following experiments.

Figure1. Gel electrophoresis results of colony PCR. 1,2: α2A-AR-Rluc8; 3,4: Str-KDEL_SBP-α2A-AR-Rluc8; M: Marker-15K

 

· Colony PCR

In the past 12 days, we have inoculated 17 media regarding colony PCR. By using agarose gel electrophoresis and contrasting with marker, yes, the first and last one with a letter M is our marker. So, after we test the existence of the DH5alpha which is a kind of E. coli transfected by special plasmid, we found that only three results NO.7, 10, and 11 are in line with our expectations (700 bp around). In this way, we would say these three ones are our target strips.

Figure2. Gel electrophoresis results of colony PCR: Marker-15K; 1,19,20-Negative control; 2-18-Clony PCR samples and 7,10,11 are confirmed positive

 

· Detecting base pairs of target plasmid

Finding the base pairs of our target DNA is shown below:

DNA ligation, transfer DH5a, colony PCR, choose numbered 71011. Inoculation of LB. plasmid extraction. Picked 1 of the three for Sanger sequencing. The three products were sent to a company for Sanger sequencing and we got the following alignment and base pairs which confirmed that we have obtained the expected plasmid.

 

Figure3. Global sequence alignment of sequencing

 

Target2: Expression and purification of GST-α2A-AR-CT protein

· Expression

The next part of our experiments is to transform the GST-α2A-AR-CT plasmid into E.coli BL21 which is commonly used in plasmids transformation, and then to verify the protein expression in it.

· Purification

We ran a PAGE gel of the precipitate, cell lysate, flow through, wash and elution to test proteins. Our target proteins GST-α2A-AR-CT molecular weight is in the range of 25 kDa. The Figure 4 showed that GST-α2A-AR-CT protein is found on column E, marker 25, indicating GST-α2A-AR-CT is purified from E.coli BL21. At the end of the experiment, we completely captured the pure GST-α2A-AR-CT protein by purification, and we compared it with the sample from each process during purification by using protein gel electrophoresis. After the electrophoresis process our target protein now is pure and we finally identified that we got the right protein.

 

Figure4. SDS-PAGE analysis of GST-α-AR-CT

 

Target3: IP-MS to screen for candidate proteins

Through the GO data, we can know that our target gene is enriched at the biological process level, with the highest number of genes enriched in translation, and our differential genes are most significantly enriched in this regulation of translation. Through the KEGG data, we can know that the number of genes enriched on Ribosome is the highest, and our differential genes are most significantly enriched in Thermogenesis.

Based on the IP-MS results in figure 5 and 6, we could further find our candidate proteins that have the potential ability to affect the α-AR-CT transportation to the membrane and the detailed result could be found in this EXCEL.

IP-MS was completed by Professor Shen's research group at Nanjing University of Traditional Chinese Medicine, providing IP-MS testing service and data analysis service.

 

Figure5. KEGG Pathways for Significantly Expressed Proteins

 

Figure6. GOBP for Significantly Expressed Proteins

 

Target 4: RUSH-BRET platform establishment in host cell lines

We established the RUSH-BRET platform in host cell lines. Since the BRET instrument (Spark® Cyto PLATE READER WITH LIVE CELL IMAGING AND REAL-TIME CYTOMETRY) is only allowed for trained users, our BRET was completed by SubCat Academy in ATLATL lab and the measurement wavelength is 535nm/485nm.

The raw data can be found in Table 1. We select the V/R data from Table 1 and  calculate the average and difference values to create a fluorescence intensity of RUSH-BRET in different time in Table 2.

Table1. Fluorescence intensity of RUSH-BRET assays

 

 

Table2. Fluorescence intensity of RUSH-BRET in different time

 

The entire curve is smooth and the error bar is small, which is consistent with our ideal expectations. These increasing trends mean that the Str-KDEL_SBP-α2A-AR-Rluc8 structure has been disturbed by the biotin and α2A-AR-Rluc8 move from the endoplasmic reticulum to cell membrane. Rluc8 of SBP-α2AR -Rluc8 replace the RAS of the Venus-K-RAS on the cell membrane and produced BRET signal that represents the amount of the protein α2A-AR on the membrane.

Figure7. The surface expression of α2A-AR was measured by RUSH-based BRET assays

 

There are a wide variety of medications available for the treatment of Alzheimer's disease. Most of these drugs only have a blocking effect on a certain part of AD and do not block the course of AD, which is why these drugs can improve symptoms in early and middle stages of AD, but are less effective in late stages of AD. Therefore, it is more important to find multi-target drugs that can block the progression of AD.

 

Since we have made some progress in our RUSH-BRET system establishment and basic function test.  Next move we will design more inquiry experiments to evaluate the system and evaluate the candidate proteins we targeted from IP-MS results.  Based on our discussion with our instructors, we will test those candidate proteins by building gene knock-out cells to check if they possess the ability to affect the transportation of α2A-AR-CT to the membrane.  In the future, we will try to develop more effective drugs targeted for Alzheimer's disease by targeting the alpha2A-adrenergic receptor in collaboration with pharmaceutical companies.

 

 [1] Sun Lei, Yang Ying, Dou Caiyan. Research progress in the treatment of Alzheimer's disease [J]. Medical Information (Junten-Journal),2010,5(07):1969-1970.