Fall 2012 Hours

Inhibition of Beta-secretase Amyloid Cleaving Enzyme (BACE1) by Aptamers, having therapeutic possibilities for Alzheimer’s Disease.

Faraz Damghani

Abstract:

            Alzheimer’s Disease (AD) is a neurodegenerative disease (disrupts the neuron cell’s ability to correctly transmit electrical current) that normally occurs when the human brain becomes elderly. Over time, the disease destroys memory, thinking skills and the brain’s ability to communicate to the rest of the body. Currently, there is no ultimate cure for AD and no definitive explanation for the cause of AD. Nevertheless, strong scientific evidence suggests that the buildup of beta amyloid plaques in the brain serve as the cause of AD. Beta amyloid plaques consist of cleaved sections of Amyloid Precursor Protein (APP) which is normally located in the membrane of neuron cells [1]. 

Figure 1: BACE1 initiates the cleaving of APP. Gamma secretase cleaves the other section of APP leading to beta amyloid formation [3].

Enzymes BACE1 and gamma-secretase amyloid cleave APP and the sections that were cleaved, build up causing amyloid plaques. Since BACE1 initiates the cleavage/formation of beta amyloid plaques, it logically makes sense that an aptamer that prevents BACE1 from cleaving APP will prevent beta amyloid plaques from forming [2]. When beta amyloid plaques are not formed than the neuron cells are able to keep their roles, thus, possibly preventing AD and its symptoms to occur.

Initially BACE1 cleaves APP close to the membrane in the extracellular region and then gamma scretase cleaves APP in the transmembrane region (figure 1). The amyloid beta is left behind and plaques start to form [2].

Specific Aim #1: To identify an aptamer against BACE1 through the SELEX process. The process requires several rounds of selection which help to find an aptamer with high affinity. It makes sense to use filter selection because the protein is not biotinylated and has a high molecular weight.  Other researchers have been successful in binding RNA aptamers to BACE1.

Specific Aim #2: The development of an aptamer for BACE1 can inhibit BACE1’s cleaving function. When APP is not cleaved than beta amyloid plaques are also not formed. Since it’s assumed that beta amyloid plaques have a huge role in AD, an aptamer that prevents plaques from happening can have therapeutic possibilities.

BACE1 with no tag (catalogue # BA1-H5213 ) can be ordered from ACROBIOSYSTEMS USA  at $280 per 100 micrograms [5]. The molecular weight of the enzyme is 70 kDa.   ACROBIOSYSTEMS can be reached via telephone at 301-825-5518. 

References:

1.Rentmeister,A., Bill,A., Wahle,T. (2006) “RNA aptamers selectively modulated protein

       

     recruitment to the cytoplasmic domain of B-secretase BACE1 invitro.” RNA 9:

     1650-1660.

2. Arnold, Steve and Missailidis, Sotiris (2009). Development of aptamers that bind to

      BACE1. Alzheimer's & Dementia, 5(4 Sup.), P416-P416.

3. Lammich S, Kojro E, Postina R, Gilbert S, Pfeiffer R, Jasionowski M, Haass C,

     Fahrenholz F. (1999). Constitutive and regulated alpha-secretase cleavage of

     Alzheimer's amyloid precursor protein by a disintegrin metalloprotease.

4. Mattson, M. Nature. 422, 385-387 (2003)

5. http://www.acrobiosystems.com/P234-Human-BACE1-Protein-Native.html#.UFaXdY1lSSo

  

Link to full proposal:
https://www.dropbox.com/s/patwcrfnfgqosvh/Damghani_Faraz-Target%20proposal.docx
Link to progress report 1:
https://www.dropbox.com/s/xqwzswh01nqci04/Damghani_Faraz-Fall%202012%20progress%20report%201.docx
Link to progress report 2:
https://www.dropbox.com/s/f9zmi4gor53d01l/Damghani_Faraz-Fall%202012%20progress%20report%202.docx
Link to final manuscript:
https://www.dropbox.com/s/rt8boeu1cdsjqg2/Damghani_Faraz-Fall%202012%20final%20manuscript.docx

Nucleic Acid Aptamer Selection Against Angiotensin II

Nucleic Acid Aptamer Selection Against Angiotensin II

Owais Jamil
Nucleic Acid Pool: N50 RNA Pool
Target: Angiotensin II

Hypertension or more commonly referred to as high blood pressure, is a condition in which the blood pressure in the arteries increases, increasing a person’s risk of heart disease. This includes life threatening ailments such as stroke or heart failure (1). According to the Center for Disease Control, cardiovascular disease is the leading cause of death in the United States, affecting over two million each year. Deterrence of hypertension by means of would eliminate the risk of patients experiencing subsequent cardiovascular events and preventing long term damage to vital organs or death.

One of the causes of hypertension is an abnormality in the renin-angiotensin- aldosterone system (RAAS), a hormone system that regulates cardiac function by controlling blood pressure (2). Angiotensin II is a peptide hormone that is a part of this system that stimulates the release of aldosterone, a steroid hormone that causes blood pressure to increase by narrowing blood vessels. A patient suffering from hypertension would have an overly active RAAS and large quantities of aldosterone, and angiotensin II in their bloodstream. Inhibition of angiotensin II could prevent unsafe rises in blood pressure, thus preventing further complications in a patient’s condition.

A treatment for hypertension would be to use an RNA aptamer with a high specificity for angiotensin II. An aptamer is a short strand of oligonucleotides that has a high binding affinity for a specific macromolecular target (3). Aptamers can be used for a variety of functions, one of which includes inhibiting the function of its target. Thus, an aptamer could be an effective treatment by inhibiting the function of angiotensin II.

Specific Aim 1: Perform the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method to select an RNA aptamer against angiotensin II. 

Specific Aim 2: Modify aptamer for inhibition of angiotensin II. After selecting for an aptamer with a high binding affinity for angiotensin II, it can be modified for use as an inhibitor, as angiotensin II is very abundant in patients with high blood pressure. This would prevent the release of aldosterone, the hormone subsequently leading to a drop in blood pressure (4).

Figure 1 Specific Aim 2. By using an aptamer to inhibit the function of angiotensin II, aldosterone will not be released and blood pressure can be stabilized. 

Biotinylated Angiotensin II (MW = 1.3kDa) can be ordered from AnaSpec.
Catalog Number: 60276-1
Cost for 1mg: $66
Cost per round:  $0.01

References

1. Constantino, I., Gorelick P. B. 2003 “Hypertension, Angiotensin, and Stoke: Beyond Blood Pressure” Stroke (35). 348-350

2. Peach, M. 1977 “Renin-Angiotensin System: Biochemistry and Mechanisms of Action” Physiological Review (57). 313-370

3. Elligton, A.D., Szostak J.W. 1990 “In vitro selection of RNA molecules that bind to specific ligands” Nature (346). 818-822

4. Crowley, S.D., Gurley, S.B., Herrera, M. J., Ruiz, P., Griffiths, R., Kumar, A.P., Hyung-Suk, K., Smithies, O., Le, T. H., Coffman, T. M. 2006 “Angiotensin II causes hypertension and cardiac hypertrophy through its receptors in the kidney” PNAS (103). 17985-17990

Click here to view full target proposal.
(http://dl.dropbox.com/u/106599935/Owais%20Jamil%20Target%20Proposal.pdf)

Click here to view the first progress report.
(http://dl.dropbox.com/u/106599935/Owais%20Jamil%20Progress%20Report%201.pdf)

Click here to view the second progress report.
(http://dl.dropbox.com/u/106599935/Owais%20Jamil%20Progress%20Report%202.pdf)

Click here to view the final manuscript
(http://dl.dropbox.com/u/106599935/Owais%20Jamil%20Final%20Manuscript%20Fall%202012.pdf)

Nucleic Acid Aptamers in the Study of Fibroblast Growth Factor 8b

Robert (Robby) Bedenbaugh


Fibroblast Growth Factor 8 (FGF-8) is a member of the growth factor family that plays pivotal roles in a number of developmental processes. FGF-8’s many functions can be attributed to alternative splicing, which generates eight different isoforms in mice(1). One isoform that has become a focus of many studies is Fibroblast Growth Factor 8b (FGF-8b). This isoform is most active during embryogenesis when it helps organize and induce development. For example, FGF-8b is extremely important in early neural development and differentiation of cells (2). Moreover, it has been shown to function in the isthmus where its activation of the Ras-ERK signaling pathway induces midbrain-hindbrain differentiation and the development of the cerebellum (3).

Although a wealth of information is know about FGF-8b there is still a great deal to be discovered. Despite many attempts, an antibody that sufficiently binds FGF-8b has not been developed. Therefore, protein concentrations have been assumed to be proportional to mRNA concentrations determined during RT-PCR studies(4). Not only could this assumption be inaccurate but also since the protein itself cannot be tagged and studied it is possible that the protein is not being produced and secreted despite the presence of FGF-8b mRNA in the cell. Moreover, it is not known whether or not the protein is sequestered within the cell before being secreted or how far it diffuses after secretion.

Aptamers provide an innovative solution to the afore mentioned problems.  If a high specificity, high affinity RNA ligand were developed to bind to FGF-8b it would be a valuable research tool in the study of the protein. This aptamer could be used to tag the protein and indicate which cells it is present in, where it is most concentrated, and even how far it is able to diffuse after being secreted from the cells. This and other information could be used to elucidate how the protein functions in development.

Specific Aim 1: Produce an aptamer via systematic evolution of ligands by exponential enrichment (SELEX) that will bind FGF-8b with high specificity. The SELEX method is a cycle that consists of the basic steps of partitioning the nucleic acid species that bind to the target from those that don’t, eluting these species, and then replicating them. This method starts with a random oligonucleotide library of around 10¹⁴ unique sequences, and after many rounds produces an Aptamer

Specific Aim 2: Employ the Aptamer to help determine which cells FGF-8b is produced in, where it is concentrated, and what roles it plays in development.

The Recombinant Mouse FGF8b that is necessary for the completion of this project will be ordered from R&D systems where its catalog number is 423-F8/CF. The protein is priced at $2000 dollars for .5 milligrams, which makes the cost per round for the target 18 dollars.

1.      Gemel, J. et al. (1996) “Structure and Sequence of Human FGF8.” Genomics 35(1):253-257

2.      Alam, A., Suzuki, H., Tsukahara, T. (2009)”Expression Analysis of FGF8a and FGF8b in Early Stage of P19 Cells During Neural Differentiation.” Cell Biology International. 33(9):1032-7.

3.      Suzuki, A., Harada, H., Nakamura, H. (2012) “Nuclear Translocation of FGF8 and its Implication to Induce Sprouty2.” Growth Development and Differentiation. 54(4):463-73.

4.      Abu-Issa, R. et al. (2002) “FGF8 is Required for Pharyngeal Arch and Cardiovascular Development in the Mouse.” Development. 129(19): 4613-4625.

Proposal
Progress Report 1

Progress Report 2
Final Report