Sarah Stofel's CIAP Aptamer Project (2019)

RNA Aptamer Against Calf Intestinal Alkaline Phosphatase Used in an ELONA to Detect Amyloid-Beta Protein for Early Alzheimer’s Diagnosis

Throughout 2019, an estimated $290 billion dollars will be spent on Alzheimer’s detection and care. This is an alarming but unsurprising number considering that there are currently 5.8 million reported cases of Alzheimer’s Disease in the United States, a rate that is projected to more than double by 2050 (Alzheimer’s Association, 2019). Unless a cure forAlzheimer’s is created, or a cheaper diagnostic tool is invented, the amount of money spent on Alzheimer’s and the amount of people affected will continue to rise. Early Alzheimer’s detection would not only help advance current research efforts searching for an ongoing cure, but improve current treatment options as well.

Alzheimer’s is categorized as a neurodegenerative disease pathologically identified by the increased presence of amyloid – beta (A) containing plaques and neurofibrillary tangles, figure 2 displays an example of this (Han, 2017). It’s hypothesized that the neurodegenerative effects of Alzheimer’s are due to the aggregation of A plaques that occur from a mutation in amyloid precursor protein as well as in apolipoprotein E4 (O’Brien, 2011). The current process of diagnosing a patient with Alzheimer’s is inefficient, time consuming, and incapable of early detection. Diagnostic efforts rely on documentation of cognitive decline, a symptom that only appears once neurodegeneration has begun, in order to perform the tests necessary to determine with reasonably high accuracy whether or not Alzheimer’s dementia is the cause of those symptoms. However, Alzheimer’s is only diagnosed with complete certainty after death (Mayo Clinic). If the amyloid hypothesis is correct and Alzheimer’s disease is mediated by, at least in part, amyloid – beta protein, Alzheimer’s could be diagnosed using this protein as a biological marker saving thousands of dollars and possibly thousands of lives (Pike, 1993).

The normal function of amyloid – beta is still unknown. It is believed that amyloid – beta peptides are a subunit of amyloid precursor protein which is supposedly responsible for the regulation and formation of synaptic transmission between neurons. Current research for an inhibitory aptamer against beta-secretase 1 (BACE1) shows that beta – secretase as well as gamma secretase help cleave amyloid precursor protein into amyloid – beta peptides (Liang, 2015). Through this research it has also been determined that the mutant form of APP which causes an overabundance of amyloid – beta peptides can be differentiated from the wildtype APP by its resistance to beta-secretase (Xu, 2016). Knowing how to differentiate between APP proteins can allow us to detect these elevated levels in simple assays with the help of a reporter molecule.

Calf Intestinal Alkaline Phosphatase (CIAP) is an enzyme capable of dephosphorylating both the 3’ and 5’ ends of RNA and DNA to prevent self-ligation (Thermo Fisher Scientific). CIAP is also used as a reporter molecule in various applications, such as in an ELONA. ELONA’s are a plate based assay that uses either an antibodies, aptamers, or both an antibody and aptamer to help in detecting certain molecules. An antibody could lay the foundation for the ELONA, and CIAP could be used as a secondary reporter in the assay in order to detect the elevated levels of amyloid – beta.

An aptamer is a unique oligonucleotide sequence that will bind with high specificity to a target protein. Aptamers can be used as a replacement for antibodies in some applications, such as the ELONA. They are more stable, cost effective, versatile, and easier to obtain-making aptamers more beneficial than antibodies. An aptamer selected against CIAP could be used as a detection method for the beta-amyloid protein that is a precursor to Alzheimer’s allowing for early diagnosis. This could be done with the help of an ELONA. Elevated levels of amyloid – beta protein can be tested for by using either blood, cerebrospinal fluid, or urine samples (Anoop, 2010). Current research speculates that A42 would be the best biomarker for Alzheimer’s due to its pathogenesis in the disease (Perrin, 2009).

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References

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Anoop, A., Singh, P., Jacob, R., & Maji, S. (2010). CSF Biomarkers for Alzheimer's Disease Diagnosis. NCBI. Retrieved May 3, 2019, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2915796/.

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CIAP (Calf Intestinal Alkaline Phosphatase), 20 U/L - Thermo Fisher Scientific. (n.d.). Retrieved April 10, 2019, from https://www.thermofisher.com/order/catalog/product/18009019

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Liang, H., Shi, Y., Kou, Z., Peng, Y., Chen, W., Li, X., . . . Zhang, X. (2015). Inhibition of BACE1 Activity by a DNA Aptamer in an Alzheimer’s Disease Cell Model. Plos One,10(10). doi:10.1371/journal.pone.0140733

O'Brien, R. J., & Wong, P. C. (2015). Amyloid Precursor Protein Processing and Alzheimer’s Disease. PubMed. Retrieved April 5, 2019, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174086/.

Perrin, R. J., Fagan, A. M., & Holtzman, D. M. (2009). Multimodal techniques for diagnosis and prognosis of Alzheimers disease. Nature,461(7266), 916-922. doi:10.1038/nature08538

Pike, C., Burdick, D., Walencewicz, A., Glabe, C., & Cotman, C. (1993). Neurodegeneration induced by beta-amyloid peptides in vitro: The role of peptide assembly state. The Journal of Neuroscience,13(4), 1676-1687. doi:10.1523/jneurosci.13-04-01676.1993

Punsky, K. (2016, March 31). Mayo Clinic Researchers Find Way to Prevent Accumulation of Amyloid Plaque, a Hallmark of Alzheimer's Disease. Retrieved November 3, 2019, from https://newsnetwork.mayoclinic.org/discussion/mayo-clinic-researchers-find-way-to- prevent-accumulation-of-amyloid-plaque-a-hallmark-of-alzheimers-disease/.

Balansethupathy, B. (2017, September 5). ELONA: the evolution of ELISA. Retrieved

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“Reporter Enzymes - Horseradish Peroxidase and Alkaline Phosphatase.”

Research Inc. https://www.jacksonimmuno.com/technical/products/conjugate-selection/enzymes.

Vassar, R. J. (2016). Bace1, The Beta-Secretase Enzyme: A Leading Therapeutic Target For Alzheimers Disease. Alzheimers & Dementia,12(7). doi:10.1016/j.jalz.2016.06.289

Xu, T., Yan, Y., Kang, Y., Jiang, Y., Melcher, K., & Xu, H. (2016). Alzheimer’s disease- associated mutations increase amyloid precursor protein resistance to γ-secretase cleavage and the Aβ42/Aβ40 ratio. Nature. Retrieved May 5, 2019, from https://www.nature.com/articles/celldisc201626.