Affecting 4 million people nationwide, Alzheimer’s disease has become the fourth highest killer in the United States. It robs a person of their identity while affecting entire families. With compassion and a sense of duty, researchers set out nearly a century ago to cure this disease. Though much advancement has been made, scientists have yet to discover the one miracle cure. One possible treatment that scientists have suggested is using embryonic stem cells, differentiated into neurons to replace dead ones in the brain. These “blank” cells are capable of being transformed into 300 different somatic cells. Scientists now have hope of creating human tissue in the laboratory; stem cells could potentially help thousands of patients waiting for donor tissue or organs through therapeutic cloning.

Alzheimer patients are hopeful about this possibility: it means progress and the ability to gain former identity. However, critics worry about the ethics of this method of medical aid. They state that the therapeutic cloning of today will become the reproductive cloning of tomorrow. Dissenters fret over demoralizing human identity with clones and of committing murder by not allowing a potential human life to develop. Embryonic stem cells and the abundance of possibilities they bring forth give rise to new questions about the value of human life and the ethics it entails: Is it justified to prolong a life that already is, while ending one that could have been?

On November 4, 1906, Bavarian neuropsychiatrist Alois Alzheimer described a form of dementia he had closely observed and followed to other psychiatrists in Tubingen, Germany. With a light microscope and a recently invented silver stain, he had performed a postmortem examination on his patient’s brain, which revealed thousands of clusters of a substance sporadically placed over the cerebral cortex. Along with this, he observed neurons that were chocked by the large mass of fibrils they possessed. It was the first time any biological proof had been established for a form of insanity. August D. was fairly young when this disorder first took over her, which fueled the belief for many years that this was a rare disease that attacked before old-age (Ingram 2003). In the next few decades that followed, especially with the aid of a new powerful electron microscope in the early 1960’s (EM), researchers dove into trying to solve the mystery of what the clusters of substance and the fibrils were: if they, in fact, caused the dementia or if they formed after neurons started dying due to the disease.

Researchers concluded that the substances littered across the brain were clusters of amyloid, an insoluble, wax-like material that formed around neurons and blocked out any means of nourishment to get to the cell, essentially suffocating it. Amyloid consisted of amyloid fibrils closely intertwined with glial cells, supporting brain cells, attached to their outer fringes. The neurofibrillary tangles inside nerve cells still remained a mystery. Researchers also noticed that the brain chemical acetylcholine, which was produced in the forebrain and supported memory region in other parts of the brain, dropped 50 to 90 percent in Alzheimer’s patients.

In 1968, British researchers Bernard Tomlinson, Gary Blessed, and Martin Roth, dismissed the myth that this disease only strikes younger people by examining tissues from the deceased elderly and finding 62 percent had the very same lesions. It proved for the first time that Alzheime’s was not a rarity, that it is a disease that strikes both older and younger people and is not just insanity or simple a due process of aging to show Alzheimer-like symptoms (Tanzi and Parson, 2000).

Since the beginning of Alzheimer study, debate often arose in the scientific community of the true perpetrator of the disease: The amyloid forming “A-beta” or the neurofibrillary tangles. The Tauists believe that the mutation of the tau protein, a subunit of the tangles inside neurons, was the primary cause of the disease. In a normal state, tau is necessary to a cell’s infrastructure. It essentially forms microtubules that are laid down in the cell’s cytoplasm to act like railway tracks to transport other proteins around the cell. However, in an individual with Alzheimer’s disease, this usually helpful protein twists and transforms into an insoluble substance. This decreases the amount of microtubules produced, which slows down the flow of protein cargo around the cell, forming tangles inside the cell and lost synapses between the neurons (St George-Hyslop, 2000).

In 1981, scientist Dennis Selkoe discovered that this transformed tau was even more insoluble than amyloid, making the process of isolating and understanding its genetic structure nearly impossible. However, as the tangles had never actually been sequenced, the very existence of the tau protein in the tangles was still speculation. It wasn’t until 1991, however, that scientists verified the significant role of the tau protein by purifying and sequencing a short strip of tangle filament. With more researchers leaning towards plaques, and a majority of the government funding being funneled into amyloid-related discoveries and prevention, the Tauists are making slow progress.

Tauists also face problems with the Alzheimer’s disease induced mice. The animals grow plaques similar to that of a human Alzheimer patient, but very occasionally do related neurofibrillary tangles appear inside mice nerve cells. In 1998, tangle research was reignited with the discovery of mutations on the tau gene. By now, more than twelve different mutation sites have been found on the gene, each responsible for the choking tangle formations. Tauists turned their attention towards researching the how and when of the mutations, in hopes that, eventually, a prevention could be found (Gotz, 2001).

In June 1983, pioneering researchers George Glenner and Cai’ne Wong accomplished what, until then, seemed impossible: isolation of brain amyloid. Unlike others who had attempted to draw the amyloid from between degenerating neurons and failed, Glenner and Wong instead realized that the amyloid clinging to the walls of the brain’s blood vessels was more accessible. Over the next few months, the two scientists took off the meninges, the outer protective layers of the brain replete with blood vessels, ground them up, and extracted the connective tissue between the vessels so that the end product they had left was amyloid. By adding a Congo red stain to the grayish substance, they made it glow a bright green, making it easier to observe its components (Glenner, 1972).

The task was now to try and read the amino acid sequence of the protein. This, however, proved difficult. The beta-pleated structure of amyloid resisted almost all attempts of dissolving it. Glenner then decided to immerse amyloid in water containing a chaotrope, which is a specialized salt. He hoped to change the bonding of H20 along with chemical aspects of amyloid in hopes of increasing its solubility. The test was successful, yielding, surprisingly, two proteins suspended in the solution. Both of these were very short sequences, but genes usually don’t make such short proteins. Hence, they decided it had to be a peptide, a part of a longer protein. They decided to pursue the second peptide, which they named beta, while putting the alpha peptide on hold. Finally, they were able to read amyloid’s short, twenty-four amino acid sequence. It was a landmark discovery; researchers now knew at least a part of the supposedly insoluble substance (Tanzi and Parson, 2000).

The peptide Glenner and Wong first found was later named “A-beta” for amyloid beta. A-beta is a fragment that escapes from the amyloid-beta precursor protein (APP). Individual A-beta fragments free-float in the brain until they come in contact with other A-beta peptides and form free-floating fibrils, long strands of the fragments. As these fibrils interact with each other, they tend to settle and accumulate into amyloid plaques between neurons in the brain. On the outer fringes of the amyloid plaques lie glial cells, supporting brain cells, while extending from them were swollen neurites, the “arms” of neurons that relay messages between brain cells (Tanzi and Parson, 2000).

Neuritic plaque density in normal humans is two neuritic plates per square millimeter, while Alzheimer’s disease patients have more than eight neuritic plaques per square millimeter (Religa, 2003). This causes severe problems since plaques sever normal communication between cells rendering them unable to perform normal functions. Signals from one cell to another become lost in the insoluble folded protein. In humans, A-beta is constantly made and secreted by healthy brain and body cells as a by-product of APP. Normally, as plaques accrue, glial cells rush to the affected area to digest the A-beta and clean the plaque debris. However, in an AD patient, the glial cells activate the release of more A-beta, which results in an increase in plaque formation, attracting even more glial cells attempting to digest A-beta, thus turning into a vicious circle.

Three main proteases, enzymes that act like scissors to cut other proteins, are responsible for A-beta being released from APP: alpha-secretase, beta-secretase, and gamma-secretase (Tanzi and Parson, 2000). In 1987, researchers found a protease inhibitor on the APP gene. The purpose of this inhibitor is to hinder the formation of beneficial proteases that destroy A-beta before it aggregates into plaques. A-beta is cut in sizes ranging from 39 amino acid sequence to a 42 amino acid sequence. Of these, A-beta 40 is the most prominent, but A-beta 42, being denser, has more chances to amass (Religa, 2003). Though not certain yet, researchers believe it acts as the origin point for other sizes of A-beta fragments to build around.

By 1989, at least one mutation had been found on the APP gene on chromosome 21. A short distance from the A-beta region of APP, a point mutation occurs where Thymine is replaced by the base C, which meant an isoleucine amino acid is replaced by valine. In following years, similar point mutation DNA base change mutations would be discovered on chromosome 21. However, by 1990, scientists found Alzheimer related mutations on both chromosome 1 and chromosome 14. On the latter lay the presenilin gene (PS1), which, researchers thought, escorted the APP protein inside a cell into the path of the three proteases. Further research is slowly leaning towards another hypothesis: mutations on presenilin 1, which accounts for most of the early on-set Alzheimer’s cases, might cause the PS1 protein it to function as a protease itself, directly releasing A-beta from the APP protein (Zekanowski, 2003).

To obtain in vivo information about how PS1 mutations cause Alzheimer’s disease at such early ages, scientists characterized the neuropathological phenotype of four PS1-FAD (hereditary Alzheimer’s disease caused by mutation on chromosome 14) patients from a large Colombian family bearing the codon 280 Glu to Ala substitution (Glu280Ala) PS1 mutation (Lemere 1996). Using antibodies specific to A-beta, researchers detected massive deposition of A-beta 42. Their results in brain tissue are consistent with recent biochemical evidence of increased A-beta 42 levels in PS1-FAD patients and strongly suggest that mutant PS1 proteins alter APP to favor production of A-beta 42. Further DNA sequencing revealed another mutation of GCT to GGT in code 136 of PS-1, leading to the substitution of amino acid Ala with Gly (Xu E, 2002).

Shortly after the discoveries made with PS-1, an Alzheimer’s related mutation was detected on chromosome 1. It was an A-to-T base change, leading to an isoleucine (I) rather than an asparagine (N) amino acid in the 141st codon (N141I). This gene shared the same function as its sister gene, PS2. Both presenilins are still being researched (Taisuke 1997). Scientists have realized that these two genes are highly conserved in any organism higher than yeast and bacteria, meaning that they are playing an intricate part of human survival or else would have been weeded out by evolution by now. However, as opposed to PS1, PS2 affects only a small handful of the early-onset cases that account for 40% for all Alzheimer cases worldwide. As with APP, both presenilins have minimal bearing on old-age cases (Chandak, 2001).

With three genes related specifically to early cases, scientists still had to find the culprit responsible for late onset Alzheimer’s disease. They found it in the autumn of 1992 with the discovery of apolipoprotein E, a gene residing on chromosome 19. The problem with APOE was that lacked the definite quality of the previous three. It had three variants that differed from each other by few bases, E-2, E-3 and E-4. If APOE-4 was inherited, it appeared to contain a polymorphism (similar to a mutation because it has the ability to alter bases but unlike one because it is common in populations and doesn’t necessarily mean disease) that increased risk of late onset Alzheimer’s disease (Tanzi and Parson, 2000).

It is thought that APOE related Alzheimer’s disease accounts for 50 percent of late onset cases. Tauists believe that APOE-4 promotes tangles in an indirect way and causes neuronal death. This hypothesis, though still pursued by Tauists, was rejected by many Baptists (those who believe Beta-Amyloid Protein is responsible for Alzheimer’s disease) since APOE is secreted out of cells, while tangles form inside of them. Research is still continuing on the significance of APOE-4 and the role it plays in Alzheimer’s disease (Lambert and Mann, 2001).

With all these uncertainties about Alzheimer’s prevention, some researchers feel that the most beneficial method of cure lies with embryonic stem cells. Experiments are currently being conducted using a broad topic of neurodegenerative diseases, specifically targeted at mice with reduced motor abilities, as seen in many Alzheimer’s disease patients also. In a study conducted by Miguel Ramlho-Santos in 2002, three different types of mouse stem cells were compared to chart any similarities between them. These were: hematopoietic, embryonic, and neural stem cells. The embryonic and neural stem cells showed the most similarities at transcription. These transcriptions were taken from the lateral ventricles of the brain.

Through detailed observation of the neural stem cells and the embryonic stem cells, the scientists found between these two groups was a great overlap of genes enriched in both. This great similarity between them shows that neural development, or creating neurons to cure different brain diseases, might not be quite as difficult as previously thought to be. Embryonic cells do not need numerous signals or altercations to become part of the nervous system because of a plethora of like traits (Ramlho-Santos 2002).

Japanese scientist Shunmei Chiba recently conducted embryonic stem cell differentiation into neurons and implanted them in neurological disease induced mice. He observed considerable improvement. Chuba used one hundred and thirty-six mice as transplant recipients. The mice were anesthetized and then a burr hole mark was made in the cranium, 3.0 mm lateral to the sagittal suture. Then, a metal probe that was chilled with liquid nitrogen was applied and incision made by a force of 100 grams for thirty seconds four times. When mice received this cryogenic injury three times or less, motor deficiencies varied in the group. Twenty percent showed spontaneous recovery, while 80% developed hemiplegia, total or partial paralysis of one side of the body that results from disease or injury to the motor centers of the brain. Two and five days later, he tested motor functions of the mice and, excluding any that made functional recovery and showed insufficient signs of cyroinjury, he randomly separated them into recipients of neuron-like cells and the control group (Chiba, 2004).

In order to create these “neuron-like” cells, Chiba used two Embryonic Stem (ES) lines, E14.1 and R-CMTI-1. Embryonic stem cells were maintained on gelatin-coated dishes with mitomycin C (a complex of antibiotic substances) treated mouse fetal fibroblasts (connective tissue that secretes proteins and especially molecular collagen, a protein that occurs in vertebrates as the chief constituent of connective tissue fibrils). Actual neural differentiation began in the presence of all-trans retinoic acid (RA) four days later, which has been shown in the past to promote differentiation of neurons. After being cultured for another four days, this time with the RA, the cells formed floating cell aggregates, or clumps, called Embryoid bodies.

These were transferred onto fresh Petri dishes, and 1 micro-Molar RA was added at days four and six (Chiba, 2004). On day 8, the cells showed several neural differentiation markers, including the mRNA of glia-specific glial fibrillary acidic protein (GFAP), and it was decided that this cell population, with RA-treated EB at day 8, would be the graft for neural tissue transplantation. Additionally, he knew these near-neurons would transform fully inside the brain because with some RA-treated EB, he recovered them and then immersed them into further culture until day 15 when they began to grow neurites, proving a full transformation from stem cell to neuron (Chiba, 2004).

They found significant responses in the mice to the new nerve cells. ES cells taken from the E14.1 cell line promoted behavioral recovery. The animal had been trained to walk along a narrow wooden beam that was 6 mm wide and 120 mm long, suspended 300 mm above a 100mm soft pad prior to cryogenic injury. Less than five mistakes every fifty steps are considered normal. In mice with right hemiplegia, many scored fifty mistakes in fifty steps. Mice transplanted with the neuron-like cells gradually improved motor performance; after day 11, the number of mistakes they made was similar to the numbers in healthy mice. What this experiment essentially means is that stem cells can be used in order to aid neurodegenerative diseases (Chiba, 2004). However, research has only extended to mice thus far, but hopes are high for humans also. Additionally, scientists are excited about the in-vitro produced neurons’ ability to travel to the diseased region in a brain even when inserted at great distances (Blakeslee, 2000). This, along with the craze for rising discoveries, bolsters the hope that embryonic stem cells are the safest, most effective answer to prevent and cure fatal neurological diseases.

Humans have an irrepressible hunger for individual value; every person wants to know that they are unique and admired because of that individuality. Human cloning provides a threat to that uniqueness, because though a clone can never be exactly like he twin in character, just having someone with identical physical traits can chip away at a human’s sense of self (Brock, 2002). Another fear is mass producing humans only for their genome, and are not valued or seen as contributing members to society. Humans produced in-vitro may be looked down upon by those born through sexual reproduction, and then questions would arise as to what society standards and morals applied to them, though they may be human just like everyone else. Also, a child clone of a dead child given to parents set on repeating their former child’s life would restrict the clone, because their future is already planned for them. They would live their entire lives being compared and judged according to the clone (Brock, 2002).

The United Nations also debated whether to ban embryonic stem cell research or not, in one unified decision as opposed to the present situation where scientists travel from one country that doesn’t accept stem cell research to another that does and simply start where they left off in the new surroundings. Many pushed for an all-out ban, stating it was murder to kill a potential human life. The United States National Academy of Sciences endorses a ban on any reproductive cloning, but encourages countries to allow research on beneficial therapeutic cloning. However, officials from countries such as Ethiopia state that a partial ban would leave doors open to abuses ultimately leading back to reproductive cloning, with the emergence of a black market in embryos provided by impoverished women. The clones of these embryos could, in theory, be implanted into the womb of a carrier and develop there into a human baby (Semple, 2003).

Affecting millions worldwide, Alzheimer’s Disease poses a dire threat to today’s society. Many hope that use of embryonic stem cells through therapeutic cloning can be the next panacea to cure neurodegenerative disease such as AD. Though the question still exists about the ethics behind having to kill a potential human being in order to save a living one, ultimately, whatever can be done to aid the Alzheimer’s disease patient must be, for it is only through the preservation of past memories that we can confidently step forward and change our future.

References

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Human Embryos Created Through Cloning. Gina Kolata. HYPERLINK “http://www.nytimes.com/2004/02/12/science/12CELL.htm“. Updated 12 Feb. 2004; cited 12 Feb. 2004.

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Ramlho-Santos, Miguel. Yoon, Soonsang. Matsuzaki, Yumi. Mulligan, Richard C., Melton, Douglas A. (2002). ‘Stemness’: Transcriptional Profiling of Embryonic and Adult Stem Cells. Science, 298, 597.

Religa, Dorota. “Amyloid (beta) Pathology in Alzheimer’s Disease.” The American Journal of Psychiatry 160 (2003): 867.

Taisuke, Tomita. 1997 March 4. The presenilin 2 mutation (N141I) linked to familial Alzheimer disease (Volga German families) increases the secretion of amyloid beta protein ending at the 42nd (or 43rd) residue. Neurobiology. 94 (5): 2025–2030.

Tanzi, Rudolph E. and Ann. B Parson. 2000. Decoding Darkness. Perseus Publishing. Cambridge, Massachusetts. 281 p.

U.N. to Consider Whether to Ban Some, or All, Forms of Cloning Human Embryos, Semple, Kirk, HYPERLINK 3 Nov. 2003; cited 2 Nov. 2003.

Zekanowski, Cezary, and Maria Styczy. Dec. 2003. Mutations in presenilin 1, presenilin 2 and amyloid precursor protein genes in patients with early-onset Alzheimer’s Disease. Experimental Neurology [online version]. 184: 991-996.

Zhonghua Yi Xue Za Zhi. 2002 Nov 25. Mutation site of presenilin-1 gene in familial Alzheimer’s disease. National Library of Medicine.82(22):1518-20.

By Nida Faheem, Brain Blogger Intern Writer