The Elusive Cause of Alzheimer's Disease, Found At Last?

Alzheimer’s disease is an incurable, degenerative form of dementia which eventually leads to death in those affected. Symptoms include loss of memory pertaining to recent events, confusion, irritability, aggression, mood swings, linguistic difficulties, and, in later stages of the disease, long-term memory loss. These symptoms result from accumulation of amyloid plaques and neurofibrillary tangles in the brain. Amyloid plaques are caused by deposits of amyloid-β protein in the brain. This leads to damage to membranes of axons and dendrites of neurons, which combine with amyloid-β protein to form amyloid plaques which gather in between healthy neurons and affect brain function. On the other hand neurofibrillary tangles are formed when a faulty version of tau protein—a protein that supports the structure of neuron—is produced in the brain, resulting in the collapse of neuronal structures.

As can be seen from the picture, the language and memory regions of the brain have atrophied, which leads to the linguistic difficulty and memory loss symptoms of Alzheimer's.
https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgzUqsv_SzlCE-YHnqXiMC4NtF8ZxXTayNeSQJkBLvizHLpmk1m0X-ajy-Kqwb7Uyy63NMVurClbN1O8DhioZCEeGpbQNeIi5dsSxoumvZPuD8ffHaZciowuydKf0Mb4iSfJw0kjjGqyXvR/s1600/alzheimersbrain.jpg

Neurofibrillary tangles form in the neurons, while amyloid plaques form in between neurons, both contributing to decreased brain functionality.
http://www.ahaf.org/assets/images/plaques_and_tangles_border.jpg

Almost all of us have heard of Alzheimer’s disease before. We know its general symptoms, and some of us might even have relatives who suffer from it. However, what has eluded us in the past is what causes Alzheimer’s.  Although a family history of Alzheimer's has been identified as a risk factor, other factors appear to be in play as well, as not all members of families with histories of Alzheimer's are affected by it. So what differentiates one person from another in terms of whether or not they will develop the disease, if not the nucleotide sequences in their genes? The study “Epigenetic Differences in Cortical Neurons from a Pair of Monozygotic Twins Discordant for Alzheimer’s Disease,” conducted by Diego Mastroeni, Ann McKee, Andrew Grover, Joseph Rogers, and Paul D. Coleman, purported to answer just that.

"EPIGENETIC DIFFERENCES IN CORTICAL NEURONS FROM A PAIR OF MONOZYGOTIC TWINS DISCORDANT FOR ALZHEIMER'S DISEASE"

In the past, studies have been done to identify certain genes related to Alzheimer’s. But, although certain genes have been found to be associated with the disease, these genes only indicated the probability of Alzheimer’s, and so could not be the sole cause of the disease. Therefore, Mastroeni and his colleagues toyed with the idea that epigenetic modifications, which result in phenotypic differences in monozygotic twins, could also determine the onset of Alzheimer’s disease. They hypothesized that these epigenetic mechanisms may be responsible for mediating the effects of the environment—external factors—on Alzheimer’s risk. In order to investigate this possible cause, the study examined DNA methylation, in which a methyl group is added to the 5^th carbon of the cytosine pyrimidine ring or 6^th carbon of the adenine purine ring to turn the gene in which methylation occurs "off", altering gene expression without changing the nucleotide sequence itself.

FIGURE 1: A methyl group (CH₃) is added to the C5 on cytosine to block the transcription of the gene, locking the gene in “off” mode so that it is not expressed.

http://www.hgu.mrc.ac.uk/img/researchers_img/meehan/DNA_Methylation_in_Vertebrates_a.jpg

                As in all controlled experiments, there was both a control and experimental condition. Since this approach to finding the cause of Alzheimer’s deals with DNA, the scientists had to find subjects who had identical genetic material in order to ensure that the only differences in gene expression would be due to epigenetic modifications. Therefore, their subjects were a pair of monozygotic twins—twins that were born from the same pregnancy and developed from one oocyte (egg cell)—who were discordant for Alzheimer’s disease.

                Both twins were evaluated before and after their deaths by neurologists as well as neuropathologists in order to diagnose any neurological diseases. Ante-mortem evaluations showed that one twin was positive for AD, while the other was neurologically normal. Both twins were white male chemical engineers, however the twin who developed Alzheimer’s had extensive exposure to pesticides in his work. The affected twin experienced his first symptoms of Alzheimer’s at age 60, his memory loss increasing in severity over the course of the next 16 years, until he passed away at age 76. On the other hand, his identical twin, who underwent the same education and had the same job, but had a different working environment, developed prostate cancer and passed away at age 79, with his cognitive facilities still intact.

                The tissue processing protocols and facility in which the twins were autopsied were the same, with both twins frozen on aluminum plates at -80°C on dry ice immediately after their bodies were recovered, and then transferred to a freezer of the same temperature for storage. The post-mortem examinations of brain tissue for the non-Alzheimer’s twin showed a scant amount of Thioflavin S plaque (top left) and neurofibrillary tangle (bottom left). On the other hand, the Alzheimer’s twin showed high amounts of both (right column), which led to impaired cognitive function.

FIGURE 2: The brain tissues of both twins were analyzed for Thioflavin S plaque and neurofibrillary tangle, with high levels of both indicating Alzheimer’s, whereas low levels indicate normal brain tissue.

"Epigenetic Differences in Cortical Neurons from a Pair of Monozygotic Twins Discordant for Alzheimer's Disease "

Immunohistochemistry, antigen detection through the use of various antibodies which bind to their specific corresponding antigens, was also used to analyze the twins’ brain tissues, specifically, their temporal neo-cortex, which was cut into 1-cm thick layers. These slabs were then washed, treated with several chemicals, and then further sliced into 40 micrometer pieces, which were then washed again and incubated in various antibody solutions. Afterwards, the brain tissue was again treated with chemicals, dried, rinsed with alcohol, de-fatted, and mounted. Special microscopes that utilized laser scanning were then used to examine the immunostained tissue sections. Under the microscope, the specific immunoreacted substances which were being tested for (in this case, 5-methylcytosine) would exhibit a fluorescence due to the chemicals with which the tissue was treated (ex. fluorophore-conjugated secondary antibody solution). In statistically analyzing the samples, the fluorescence intensities of 5-methylcytosine, the methylated version of the cytosine nitrogen base in DNA, were evaluated (the intenser the fluorescence from the sample, the more methylation of DNA in that region). The anterior temporal neocortex, which is affected by Alzheimer’s, as well as the cerebellum, which is not affected, were processed using antibody concentrations which would detect 5-methylcytosine. As was expected, there was little difference between the cell layers of the cerebellum in both twins (bottom row of fig. 3) and a visible difference between their anterior temporal neocortex cell layers (top row of fig. 3).

FIGURE 3: Immunoreactivity (reactions between an antigen and its particular antibody) for DNA methylation in the anterior temporal neocortex (top row) and cerebellum (bottom row) are shown. The left column shows the results for the non-demented twin, and the right column shows those for the Alzheimer’s twin.
"Epigenetic Differences in Cortical Neurons from a Pair of Monozygotic Twins Discordant for Alzheimer's Disease "

The results of the DNA methylation immunoreactivity show that there is decreased methylation in the anterior temporal neocortex of the Alzheimer’s twin relative to the unaffected twin, due to the disease’s impact on the region. Using a T-test, Mastroeni found that all markers had significant decreases (P<0.0001) in imunoreactivity in the twin affected by Alzheimer’s than in the non-Alzheimer’s twin, meaning that there was less DNA methylation in the Alzheimer's twin. The implications of this finding can be applied to previous studies on Alzheimer’s disease, which reported either increases or decreases in the expression of certain genes in Alzheimer’s patients. As many genes have methylation sites, the explanation of Alzheimer’s resulting from hypomethylation (decrease in methylation) could perhaps account for the complexity of the disorder and serve as an all-encompassing explanation for the various biological characteristics of the disease. These findings also show that epigenetic mechanisms may very well also be the means by which life events (ex. exposure to pesticides, or work environment) are translated into effects on our physiology (ex. increased risk for developing Alzheimer’s).

OTHER RESEARCH:

Prior to the aforementioned study, scientists had already established quite an extensive database of Alzheimer’s related research. One risk factor in developing Alzheimer’s is genetics. Two kinds of genes were found to be associated with Alzheimer’s which increased the risk of the disease being passed down in the family—ApoE4, which increases risk of Alzhiemer’s, and deterministic genes, which are very rare but, if inherited, inevitably result in early onset Alzheimer’s. These genes can contain mutations which result in heightened risk for Alzheimer’s, but even this does not explain the whole picture, which is why the investigation into possible epigenetic causes for Alzheimer’s was needed to further illuminate the issue.

 A research study published two years after Mastroeni’s study further explored epigenetics as a marker of Alzheimer’s disease. As ribosomal insufficiencies have also been observed in Alzheimer’s patients, Pietrzak hypothesized that genes which code for rRNA (which is required in the synthesis of ribosomes, which make protein) are being methylated and thus turned “off”, effectively decreasing ribosome production. The findings of the study supported his hypothesis that rDNA (DNA which codes for rRNA) hypermethylation could be another epigenetic marker of Alzheimer’s disease. This again puts an emphasis on the complexity of Alzheimer's disease, as it is characterized by the hypomethylation of certain genes and the hypermethylation of others.

A 2012 research study on mice with Alzheimer’s disease showed that the memory loss typical to Alzheimer’s cases result from methylation of genes involved in neuronal communication. By blocking the enzyme which catalyzes the methylation of the genes, HDAC2, scientists were able to restore the function of the neurons which had been turned “off.” If drugs could be made to inhibit the enzyme in humans, it could possibly be used as a treatment for the disease in the future. In this instance, as well as the studies mentioned above, an epigenetic approach was taken to understanding Alzheimer’s.

These recent studies go to show the importance of gene regulation in Alzheimer’s disease, and, although we are still not 100% sure as to the cause of Alzheimer’s disease, much progress has been made and the beginnings of a potential treatment have appeared. The next steps the scientific community must take include bringing this drug which could hinder the effects of Alzheimer’s into existence, and continuing its research into the causes of Alzheimer’s. Although we definitely know more about the disease than we did 10 years ago, we must know even more in order to identify a cure for the currently incurable disease, and eradicate today’s most prevalent form of dementia.


For MORE information about DNA methylation, please view 24:20-28:19 of this youtube video: 

BIBLIOGRAPHY:

1.       "Alzheimer's & Dementia Causes." Alzheimer's Association. Alzheimer's Association, 2011. Web. 28 Apr 2012. <http://www.alz.org/research/science/alzheimers_disease_causes.asp>.

2.       "Alzheimer's Disease." Science Daily. Science Daily, n.d. Web. 19 Apr 2012. <http://www.sciencedaily.com/articles/a/alzheimer's_disease.htm>.

3.       Mastroeni, Diego, Ann McKee, Andrew Grover, Joseph Rogers, and Paul Coleman. "Epigenetic Differences in Cortical Neurons from a Pair of Monozygotic Twins Discordant for Alzheimer's Disease."Epigenetic Differences in Cortical Neurons from a Pair of Monozygotic Twins Discordant for Alzheimer's Disease 4.8 (2009): 1-6. PLoS One. Web. 16 Apr 2012.

4.       Pietrzak, Maciej, Grzegorz Rempala, Peter Nelson, Jing-Juan Zheng, and Michal Hetman. "Epigenetic Silencing of Nucleolar rRNA Genes in Alzheimer's Disease." Epigenetic Silencing of Nucleolar rRNA Genes in Alzheimer's Disease 6.7 (2011): 1-6.PLoS One. Web. 18 Apr 2012.

5.       Tsai, Li-Huei. "HHMI News: REAWAKENING NEURONS - Researchers Find an Epigenetic Culprit of Memory Decline." HHMI. Howard Hughes Medical Institute, 29 Feb 2012. Web. 27 Apr 2012. <http://www.hhmi.org/news/tsai20120229.html>.