A Look into Alzheimer’s Disease: RNA Sequencing and Immunocytochemistry

By Julia Kim | Commack High School, New York, United States

I. Introduction

Alzheimer’s Disease (AD) is a neurodegenerative disease diagnosed in humans that results in damaged memory function. Most commonly, AD is found in elderly individuals ages 60 and older, and the illness may also be hereditary (“Alzheimer’s Disease-” 2020). (1) The number of people with AD has been increasing, with an estimated 14 million cases of AD in the year 2060 (“Alzheimer’s Disease-” 2020). (1) Patients who suffer from AD have tau protein neurofibrillary tangles in their brains (Ewald, 2010)3 along with the buildup of beta-amyloid plaques; these plaques and neurofibrillary tangles form in certain areas of the brain including the hippocampus, which correlates to the decline in memory function as a result of AD. While beta-amyloid and tau are both essential proteins in the brain, as beta-amyloid aids in neural growth and tau stabilize microtubules, too much of either is considered to be a sign of AD development (“What happens-” 2017).9 While there is no current treatment for AD, many studies have been carried out regarding AD and related components. For example, metals such as zinc, copper, and iron have all been proven to show some correlation with plaque development in the brain, despite being necessary for the human body (“Metals, aluminum-” 2017).6 Studies such as those that have proven a correlation between AD and metal intake have contributed to the knowledge available on AD, although a cure has not yet been found.

Keywords: Alzheimer's Disease; RNA Sequencing; Immunocytochemistry; MicroRNA Biomarkers; Histone Deacetylase 2 (HDAC2)  

II. Methods and Techniques

Many researchers have looked into factors that may influence the severity of AD development. A study conducted by Peña-Bautista et al. looked into plasma microRNA (miRNA) biomarkers that may aid in identifying AD. From analyzing 11 miRNA samples, this study concluded that 3 specific miRNAs, miRNA-92a-3p, miRNA-486-5p, and miRNA-29a-3p, could be biomarkers of AD (Peña-Bautista, 2022). (7) While further research with greater sample sizes is needed to solidify this conclusion, this result gives a better understanding of the beginning stages of AD and how to detect the disease with 3 miRNA biomarkers. This information, along with more research on its applications, can possibly stall AD growth in several patients in the future. RNA sequencing, which is the process of finding the sequence of a piece of RNA, was an essential technique used in this study. In RNA sequencing, genotypes in different parts of the human body are identified along with healthy or unhealthy cells (Edwards, 2022). (2) This technique starts with RNA extraction from the cells, then moves onto transcription for complementary DNA (cDNA). cDNA is made with mRNA and reverse transcriptase, an enzyme that aids in reverse transcription (Shchelochkov, 2023). (8) Next-generation sequencing (NGS), which allows researchers to sequence DNA on a significantly large scale, is also part of this process, and it ultimately reveals the original RNA sequence (Edwards, 2022). (2) This technique is used throughout this study as a method to analyze miRNA samples and later quantify them using quantitative PCR. Out of the 11 miRNA samples analyzed and isolated through RNA sequencing, 8 were quantified while the rest were not found (Peña-Bautista, 2022). (7) Researchers were then able to perform further analyses and narrow the 8 possible miRNA biomarkers to 3. This study utilized RNA sequencing to find more information on AD biomarkers. AD has also been studied by Gräff et al. for an epigenetic blockade caused by the gene histone deacetylase 2 (HDAC2). In the brain, learning ability and memory are supported by histone acetylation, a function that helps gene expression and the cell cycle (Gujral, 2020). (5) This study found that an increase of HDAC2 in certain areas of the brain, specifically the hippocampus where memory function is determined, results in a reduction of histone acetylation; therefore, since histone acetylation is essential to the brain’s memory and learning capabilities, HDAC2 is detrimental to memory and leads to AD. Additionally, it was found that the brain’s capacity may not be permanently damaged from neurodegenerative diseases such as AD, however, it could be simply damaged. This provides a wide range of possibilities and brings researchers closer to the treatment for AD as a whole. Knowing that the disease can be reversed or lessened, many potential findings open up for curing AD. Throughout conducting this study, several experimental techniques were used to analyze and collect data on a molecular level, including the process of immunocytochemistry. Immunocytochemistry works by incorporating antibodies into cell samples with the hope of detecting an antigen. This method of research first associates the antibody with a visual indicator, such as a colored dye or enzyme, that appears when a binding has taken place between the antibody and antigen. If the antigen is absent, no color change will be shown. This technique allows researchers to identify antigens, and, in real-life applications, diagnose certain illnesses and diseases, cancer being one of them. Immunocytochemistry was used in the study by Gräff et al. for the purpose of observing the causes for HDAC2 to grow or decline in the brain. The effects of hydrogen peroxide and beta-amyloid proteins, both related to AD growth, were studied on HDAC2 in neurons using immunocytochemistry. It was found that both hydrogen peroxide and beta-amyloid proteins increased HDAC2 levels, therefore allowing researchers to come to the conclusion that HDAC2 is involved in AD aggregation since it has a direct relationship with the increase of hydrogen peroxide and beta-amyloid levels (Gräff, 2012). (4) This research technique allowed these results to be confirmed further by its researchers.

III. Conclusion

The conclusions reached by both studies were accurate based on the results reached through various experimental techniques. RNA sequencing used in the study by Peña-Bautista et al. showed that out of 11 miRNA samples 3 were undetected in the AD brain; 3 other miRNAs were proven to show significant outcomes: miRNA-92a-3p, miRNA-486-5p, and miRNA-29a-3p. In the study carried out by Gräff et al., immunocytochemistry played a large role in clarifying how HDAC2 levels shifted based on other neurodegenerative factors, specifically hydrogen peroxide and beta-amyloid proteins. HDAC2, which is detrimental to cognitive function, increases as other neurodegenerative factors increase. These results are helpful in understanding AD and may contribute greatly to future research. With miRNAs, the conclusions reached showed to be important, however, the study should be repeated with a larger sample size to confirm the accuracy of the results. Other miRNAs can be searched for as well, as only three specific samples were found to correlate with early AD development. For the discovery of HDAC2, future research may consist of methods to permanently stop the increase of HDAC2 in the brain, as it was proven to contribute to neurodegeneration as well. Additionally, potential attempts to reverse cognitive decline may also be studied.

IV. References

1. Alzheimer’s disease and related dementias. (2020). Centers for Disease Control and Prevention. Retrieved July 4, 2023, from https://www.cdc.gov/aging/aginginfo/alzheimers.htm

2. Edwards, O. (2022). What is RNA sequencing? Yourgenome. Retrieved on July 6, 2023, from https://www.yourgenome.org/facts/what-is-rna-sequencing/.

3. Ewald, C. Y. and Li, C. (2010). Understanding the molecular basis of Alzheimer’s disease using a Caenorhabditis elegans model system. Brain Structure and Function, 214, 263-283.

4. Gräff, J., Rei, D., Guan, J. S., Wang, W. Y., Seo, J., Hennig, K. M., ... Tsai, L. H. (2012). An epigenetic blockade of cognitive functions in the neurodegenerating brain. Nature, 483(7388), 222-226.

5. Gujral, P., Mahajan, V., Lissaman, A. C., and Ponnampalam, A. P. (2020). Histone acetylation and the role of histone deacetylases in normal cyclic endometrium. Reproductive Biology and Endocrinology, 18, 1-11.

6. Metals, aluminum, and dementia. (2019). Alzheimer’s Society. Retrieved on July 4, 2023, from https://www.alzheimers.org.uk/about-dementia/risk-factors-and-prevention/metals-and-dementia

7. Peña-Bautista, C., Tarazona-Sánchez, A., Braza-Boils, A., Balaguer, A., Ferré-González, L., Cañada-Martínez, A. J., ... Cháfer-Pericás, C. (2022). Plasma microRNAs as potential biomarkers in early Alzheimer disease expression. Scientific Reports, 12(1), 1-10.

8. Shchelochkov, O. A. (2023). cDNA (copy DNA). National Human Genome Research Institute. Retrieved on July 7, 2023, from https://www.genome.gov/genetics-glossary/Copy-DNA

9. What happens to the brain in Alzheimer’s Disease?. (2017). National Institutes of Health. Retrieved on July 4, 2023, https://www.nia.nih.gov/health/what-happens-brain-alzheimers-disease

The Scientific Review Article published, “A Look into Alzheimer’s Disease: RNA Sequencing and Immunocytochemistry,” was received on July 11, 2023, and was reviewed and accepted on July 15, 2023. To contact editors and reviewers please click here.