1  

HIV-1 Nef Gene’s Effect on the Production of Beta Amyloid

Parth Patel

Life Sciences

Woodstock High School

2010 Towne Lake Hills South Dr.

Woodstock, GA 30189

      2  

Synopsis

HIV-1 Nef Gene’s Effect on the Production of Amyloid

HIV/AIDS is a pandemic that kills millions of people. HIV-1 may induce Alzheimer-like

dementia. Alzheimer’s destroys the lives of millions people through the slow decay of brain cells

causing memory loss. In Alzheimer’s the protein beta amyloid is responsible for producing

plaques, which lead to inflammation and cell death. Studies have shown beta amyloid peptides

secreted from cells in association with exosomes. Exosomal proteins were also found to

accumulate in plaques, suggesting a role for exosomes in the pathogenesis of Alzheimer’s. HIV

Nef protein has shown to be secreted in exosomes and enhances the production of exosomes. The

hypothesis is “If beta amyloid is expressed in association with Nef exosomes, and I transfect

NS20Y cells with the nef gene, collect that supernatant, and place it on un-transfected NS20Y

cells, then the production of beta amyloid by the treated cells should be enhanced”. Nef-GFP was

transfected into NS20Y cells to produce Nef exosomes. Medium, containing exosomes, was

collected and placed onto untreated NS20Y cells. ELISA and Western blot measured beta

amyloid production from treated cells. Results show that Nef exosomes (applied exogenously)

enhance the secretion of beta amyloid peptides.

      3  

Table of Contents

Title Page Page 1

Synopsis Page 2

Table of Contents Page 3

Introduction Page 4

Purpose, Procedure, and Materials Page 6

Results Page 8

Conclusion Page 9

Literature Cited Page 12

Appendices Page 13

Acknowledgments Page 17

      4  

Introduction

HIV/AIDS infection is a worldwide epidemic and according to  According to UNAIDS

there were 1.7 million AIDS deaths in 2011, which is actually down from 2.3 million in 2005.

This shows that treatments for HIV/AIDS are improving and more lives are being saved.

However, one complication of HIV-1 infection known as HIV Associated Dementia (HAD),

which is a form of dementia similar to that found in Alzheimer’s patients, has been increasing in

incidence even with modern drug treatments according to an HIV associated neurological

incidence study.

The interesting thing about HAD which was sought to exploit through the research was

its similarities to Alzheimer’s disease. Alzheimer’s is another worldwide epidemic that is

destroying the lives of millions, and in Alzheimer’s disease, there is a protein called beta

amyloid which is responsible for producing plaques (See Figure 1.0) on brain cells, which leads

to inflammation and ultimately cell death resulting in effects like memory loss.

Figure 1.0 – Beta Amyloid Plaque Formation

("Beta Amyloid Formation")

Previous studies have shown that beta amyloid peptides can be secreted from cells in

association with exosomes according to study done specifically on the association between beta

amyloid and exosome excretion; exosomes are small vesicles that have the ability to carry viral

and cellular components (See Figures 2.0).

      5  

Figure 2.0 – Exosome Basics

(Théry)

Subsequently, exosomal proteins were also found to accumulate in the plaques of

Alzheimer’s patient brains, which could be suggesting a role for exosomes in the pathogenesis of

Alzheimer’s disease. Interestingly, HIV Nef, a product of the nef gene (one of six accessory

genes), has shown to be secreted in exosomes and enhances the production of exosomes, so if

more exosomes are associated with the enhanced produciton of beta amyloid, then in turn the nef

gene could be possibly enhancing the secretion of beta amyloid.

      6  

Purpose, Procedure, and Materials

The purpose of this experiment was to study the pathogenesis of HAD, and the possible

implications of the pathogenesis Alzheimer’s disease being related to that of HAD by testing to

see if Nef exosomes are taken up brain cells, then it would enhance the secretion of beta

amyloid. Simultaneously, the purpose was to observe whether or not beta amyloid is expressed

along with exosomes from the nef gene. This experiment is the first of many because its main

purpose was to determine whether or not the nef gene enhances the production of beta amyloid.

Furthermore, the experiment was conducted using a mouse brain cell line called NS20Y, and the

brain cells were transfected with the nef gene. This means the results of this experiment may not

be true for humans, beside the fact that mouse brain cells were used, because HIV has only been

known to infect blood cells. However, if this hypothesis proves to be true, then further studies

may be conducted using human brain and blood cells, eventually leading to method of blocking

the pathogenesis of HAD.

First, the NS20Y cells were split; the media consisted of 10% FBS, DMEM, 1% Glutamax,

1% Antibiotic, and Antymycotic. Then, Nef-GFP was transfected into NS20Y cells, using the

Lipofectamine 2000 Kit, to produce Nef exosomes. A mock and Nef-GFP flasks were created.

The mock was given 20 uL of Lipofectamine, and the Nef-GFP flask was given 10 ug (20.78 uL)

of Nef-GFP (Nef is tagged with GFP in order to observe transfects rate) solution Next, the cells

were split into a 6-well plate and triplicated with .5mL, 1mL, and 1.5mL of Nef-GFP and Mock

supernatants (See Figure 3.0).

      7  

Figure 3.0- Samples

(Created by Researcher)

The resulting conditioned medium was then micro-centrifuged at 16.1 rpm for 10

minutes. Then, the conditioned medium, containing the Nef exosomes, was collected and placed

onto untreated NS20Y cells, and a fluorescent microscope was used to observe whether or not

the brain cells took up the exosomes in the supernatant. Lastly, beta amyloid production from the

treated cells was measured by ELISA using the Mouse/Rat Beta Amyloid (1-40) High Specific

Assay, and beta amyloid precursor protein was measured by Western blot techniques with gels

that were developed for two, eight, and sixty minutes. Next summer, the plans are to continue

research and repeat the same experiment on a different mouse brain cell line, ATT20wt, and on

human brain cells.

      8  

Results

The .5mL NS20Y control (mock) treated cells showed no green fluorescent glow, but the

.5mL Nef-GFP treated cells did express a green fluorescent glow as a result of the nef gene being

tagged with GFP, which indicated that an uptake of vesicles (exosomes) occurred. Additionally,

the 1mL NS20Y control treated cells showed no green fluorescent glow while the 1ml Nef-GFP

treated cells showed a greater uptake of exosomes occurred. However, the 1.5mL NS20Y Nef-

GFP treated cells showed a decrease in the uptake of exosomes while the NS20Y control treated

cells showed no green fluorescent glow, which could be suggesting that there is a limit to how

many exosomes can be taken up.

The ELISA results for the Mouse/Rat Beta Amyloid (1-40) High Specific Assay showed

the amount of beta amyloid found in the supernatant of the mock cells and the nef-transfected

cells. The original or stock solution of the mock-transfected cells secreted 211pg/mL, and the

nef-transfected cells secreted 641pg/mL. The .5mL solution of mock-transfected cells secreted

179pg/mL while the nef-transfected cells secreted 49pg/mL, which was the least. The second

most secreted was 177pg/mL by the 1.5mL mock-transfected cells and 235 by the 1.5ml nef-

transfected cells. The greatest amount of beta amyloid secreted was 263pg/mL by the 1mL

mock-transfected cells and 300pg/Ml by the 1mL nef-transfected cells (See Figure 4.0).

Figure 4.0 – Mouse/Rat Beta Amyloid (1-40) High Specific Assay Results

(Created by Researcher)

      9  

The sixty minute developed western blot showed the clearest results and was used to

observe the amount of beta amyloid precursor protein found within the lysate mock and nef-

transfected cells. The western blot results showed that the 1mL nef-transfect cells had the

greatest trace or amount of beta amyloid precursor protein; however, other specific results are

not presentable because the data is too difficult to examine (See Figure 5.0).

Figure 5.0 – Western Blot Results

(Created by Researcher)

Conclusion

The experiment has supported the hypothesis that the uptake of Nef exosomes by brain

cells would enhance the secretion of beta amyloid.  Nef-GFP vesicles produced from transfected

NS20Y cells appear to be able to change the amount of beta amyloid secreted when applied to

un-transfected cells, and therefore, it can be concluded that Nef may play a role in HIV

Associated Dementia by modulating the secretion of beta amyloid. However, in the ELISA and

western blot results there may have been a mix up with the .5mL supernatant solution for the

      10  

mock-transfacted and nef-transfected cells. However, this does not the affect the conclusion that

the uptake of exosomes by brain cells enhances the production of beta amyloid. As a result,

further conclusions cannot be drawn until more trials are conducted and experimentation is

conducted in consideration of the human body’s immune system response.

In the continuation of this project, there are various paths that may be taken. This

summer, the plan is to conduct this same experiment on human brain cells and then with human

blood cells because HIV cannot directly infect brain cells because of the blood barrier, but it is

possible that HIV hijacks a pathway to the brain and sends exosomes to infect the brain cells.

This seems logical and plausible because the vesicles would be able to bypass the brain’s blood

barrier. As a result, the Nef exosomes would be mimicking how HIV attacks T-cells (See Figure

6.0).

Figure 6.0 – HIV T-cell Infection

(Created by Dr. Andrea Raymond)

      11  

However, it is extremely difficult to obtain live human brain cells, but once this obstacle

is overcome, blocking the Nef enhancement of beta amyloid production could represent a new

treatment for AIDS dementia.

      12  

Literature Cited

United Nations. UNAIDS. 2012 UNAIDS Report on the Global AIDS Epidemic. N.p.: Joint United Nations Programme on HIV/AIDS (UNAIDS), 2012. Print. Sacktor, N. HIV-associated neurologic disease incidence changes: Multicenter AIDS Cohort Study, 1990–1998. Comp. R. H. Lyles et al. N.p.: n.p., 2001. Print. Planck, Max. Alzheimer's disease beta-amyloid peptides are released in association with exosomes. Comp. L. Rajendran et al. N.p.: n.p., 2006. Print. "Beta Amyloid Formation." Elements4Health. Elements4Health, 2014. Web. 17 June 2013. <http://www.elements4health.com/images/stories/ amyloid-plaque-formation.jpg>. Théry, Clotilde. "Exosome Basics." TheScientist. The Scientist, 1 July 2011. Web. 12 June 2013. <http://www.the-scientist.com/?articles.view/ articleNo/30791/title/Exosome-Basics/>.

      13  

Appendix

Figure 7.0- .5mL Control Treated Cells

Figure 7.1- .5mL Nef-GFP Treated Cells

      14  

Figure 8.0 – 1mL Control Treated Cells

Figure 8.1 – 1mL Nef-GFP Treated Cells

      15  

Figure 9.0 – 1.5mL Control Treated Cells

Figure 9.1 – 1.5mL Nef-GFP Treated Cells

      16  

Figures 7.1, 8.1, and 9.1 are not the pictures of the NS20Y cells from this experiment

because the pictures from this experiment were lost. However, these pictures are accurate

measurements in the ratio of the amount NS20Y cells that took up the exosomes and displayed

green fluorescent glow.

      17  

Acknowledgments

This experiment and research was conducted at Morehouse School of Medicine in

Biosafety Hazard Level 2 laboratory for six weeks throughout June and July of 2013.

Dr. Michael D. Powell as the primary investigator expertly taught the biological part of

the experiment and presented various types of literature consisting of background information

regarding the research. This tremendously helped in grasping the concepts firmly and quickly.

Dr. Powell also provided assistance in formulating the hypothesis and drawing conclusions,

making sure none of the conclusions were invalid or extrapolation. Dr. Powell was a great

mentor, teacher, and friend throughout the duration of the research. He is also responsible for

retrieving financial sponsorship and providing the brain cells, Lipofectamine Kit, media, and

Mouse/Rat Beta Amyloid (1-40) High Specific Assay Kit.

Mrs. Mahfuz Khan, the research assistant, assisted in the experimental design,

demonstrated how special equipment, such as the NanoDrop and fluorescent microscope, were to

be used, and taught various lab techniques that were vital to gathering the most accurate data.

She also demonstrated how to conduct ELISA and western blot tests.

Lastly, the Morehouse School of Medicine was generous enough to allow access to its

equipment and providing this research experience through the Vivien Thomas Summer Research

Program.