In a number of my posts describing the potential hazards posed by gene therapy techniques I explained that discoveries made by the ENCODE project research teams disclosed that much more than just specifically targeted genes were involved in the transcription process -- that the complete RNA template was comprised of a combination of gene segments and non-genetic DNA components constructed around the primary gene for the purpose of synthesizing proteins. I reported that at least one death and the development of a number of leukemia's had directly been attributed to the gene therapy process whereby a viral vector was used to deliver the "fixed" gene into the cell and where it was left to the cell machinery to incorporate the gene into its DNA. I concluded that we had much to learn before incorporating this otherwise elegant method of correcting "broken" DNA into an effective gene therapy for healing genetically inherited diseases and other disorders involving mutant alleles. Now experiments conducted on mice which had been treated to develop rapid onset of Alzheimer's demonstrate that ex vivo manipulation of the genetic information to generate amyloid plaque-ridding enzymes in the brain cells of the mice may prove that method to be a safe and workable therapy.
The procedure involves making genetic changes to cells taken from the patient and then putting back the cells. Introducing modified cells into the brains of the mice showed "..robust clearance of plaques in the brains of the mice" in the experiments carried out by Dennis Selkoe, Vincent and Stella Coates Professor of Neurologic Diseases at Harvard Medical School. In the Harvard study a gene for the enzyme neprilysin was introduced into skin cells taken from the mice which were subsequently planted in the brain.
The technique has not as yet been tried on humans. Apparently logistical problems involving the blood-brain barrier can be overcome but humans have a much larger brain than mice, making effective treatment using this method somewhat problematic since this requires more precise delivery of the modified cells to the affected areas where the amyloid plaques have gathered.
The body's own immune response to the buildup of amyloid plaques in the AD brain has been studied. Microglia are the phagocytes of the brain -- that is, these are the cells that engulf and absorb waste material, harmful microorganisms, or other foreign bodies including plaque material in the brain. Their efficacy is notable only in the early stages of AD and it is assumed that their inflammatory responses are ultimately detrimental because their action can't keep up with plaque production. They do have the unique property of being able to target the plaques directly in a process in which the amyloid beta release a chemotactic protein which serves as a homing beacon for migrating microglia.
It is tempting to think of a scenario whereby an AD patient's microglia might be harvested and then the gene for the enzyme neprilysin introduced into them thereby leaving the issue of targeting the plaques to the body's own defenses. This may or may not be possible as the microglia may not have those benign properties which make the skin cell such a suitable candidate. On the other hand, the homing properties of the microglia might be incorporated into the patient's modified skin cells. Either way, I admit that I am being purely speculative and can offer only ideas.
"Reality leaves a lot to the imagination"
- John Lennon
Tuesday, January 22, 2008
Ex vivo genetic intervention to treat Alzheimer's
Monday, January 21, 2008
Misfolded proteins, evolution, and coffee
In a previous post I discussed the nature of the disease we know as Alzheimer's. The culprit of this tragic affliction is the prematurely cleaved linear amino acid peptide, the beta amyloid precursor protein whose mutated form has become known as the prion (pronounced prē'ŏn). The process which gives these peptides their mutant characteristic was discovered by the late great Vernon Ingram. His studies on the action upon the precursor protein within the hydrophobic layer of the cell wall should not be dismissed. It has been estimated that 30-40% of encoded DNA codes for membrane proteins, thereby offering a large probability of divergence from normal translation to those proteins.
The prions that give rise to the development of Alzheimer's disease have been included in the class of misfolded proteins which contribute to some twenty known disorders related to the accumulation of beta amyloid fibrils or plaques in organs such as the brain, liver or spleen. These include Type II Diabetes and Huntington's disease. It is no mystery then that sufferers of diabetes have a 65 per cent higher risk of developing Alzheimer’s disease, according to a study published in the Archives of Neurology. Alzheimer's is estimated to be the most expensive medical problem in the Western world.
Recent advances in the understanding of the nature of protein misfolding has revealed that this trait is a generic property of polypeptide chains and that this indicates that"..a key feature of biological evolution has been the selection of protein sequences with inherent resistance to aggregation, coupled with mechanisms that normally ensure effective control and regulation of misfolding and aggregation in the cell."
The above extract was taken from a paper by Christopher M. Dobson of the University of Cambridge, Department of Chemistry. He describes the associated diseases as "becoming increasingly prevalent in the modern world". What are also becoming increasingly prevalent in the modern world are the advanced technologies that are used to make these discoveries. For example, data from highly advanced mass spectrometers are now being used to characterize particular protein components from the spectra of intact ribosomes (RNAs are transcribed from DNA and the RNAs are translated into proteins by ribosomes).
A protein is synthesized from the information represented by the base sequences of the particular RNA molecule which determine which amino acid is next in the assembly process. The final product is the protein, which normally folds into the three-dimensional shape as determined by their molecular arrangement. Competition in the intracellular environment determines the effectiveness of this process. The protein may misfold or degrade. It has been observed for example that the crowding effect of high concentrations of macromolecules in the cytoplasm of the cell can have a detrimental effect. Some of these macromolecules are actually represented by any of at least twenty different families of molecular "chaperones", which facilitate the at times complex folding process. Others are enzymes which catalyze some of the slower processes involved in folding. Evolution is an ongoing process. The research team at the Oxford Centre for Molecular Sciences has observed that one of the roles of molecular chaperones is to prevent the aggregation of partially folded proteins.
At least four different approaches to possible therapies which address the root causes of the prion diseases and the possible eradication of the misfolded proteins are being examined based on the knowledge derived from ongoing research. These were described at a workshop held at St Catherine's College, Oxford, from March 25–28, 1998, attracting participants from 32 nations. Some of the information contained above is culled from a report produced by Christopher M. Dobson and R. John Ellis, of the Oxford Centre for Molecular Sciences.
In a more recent development (updated: October 6, 2006), at the risk of stepping into controversial territory it should be noted that U.S. FDA approved medications currently available for treating Alzheimer's disease do not appear as effective in inhibiting formation of amyloid plaque as does Tetrahydrocannabinol or THC, the active component found in Marijuana. This was discovered by researchers at the Scripps Research Institute.
And research on mice conducted at the Byrd Alzheimer's Institute in Florida has revealed that coffee may have a beneficial effect on patients with Alzheimer's. Regular intake of caffeinated water by mice with prions in their brain cells showed reduced numbers of those prions compared to a control group, and memory studies on Alzheimer mice after ingestion of caffeinated water showed results equal to a control group.
Friday, January 18, 2008
Prion is pronounced prē'ŏn
Most people are under the impression that we do not know what causes the debilitating disease called Alzheimer's. We used to call it Senility. Doctors called it Dementia, and at one time considered it a normal part of aging. Some 50% of the population will display symptoms of this tragic disease after age eighty.
The disease was first described in 1906 by Dr. Alois Alzheimer, after whom the disease is named, after he autopsied the brain of a 51 year-old patient who showed severe symptoms of dementia and brain dysfunction and who died completely unresponsive to treatment. He noticed the characteristic 'plaques' and 'tangles' as he called them, evidence of the devastating destruction of what should have been a system of healthy neural networks in the brain of a woman too young to die.
Fast forward to 1976: Daniel Carlton Gajdusek wins the Nobel prize for his research on Kuru, a disease very similar to Alzheimer's, found primarily among the South Fore people of New Guinea, a native tribe which practised ritual cannibalism. It was found that females of the tribe were more likely to contract the disease, and he concluded that this was the result of eating the brains and other soft organs of their deceased. It was customary to allow the women and children first access to their dead relatives. The men would later indulge primarily in the fleshy portions. About eight times more women than men contracted the disease. Studies on the brains of the deceased victims of Kuru showed similar plaques and tangles. Obviously there was a connection between cannibalism and Kuru.
Fast forward again to 1997: Stanley B. Prusiner wins the Nobel prize in Physiology or Medicine for his discovery of the prion. Prions are the misfolded proteins that lie at the root cause of Alzheimer's disease, Creutzfeld-Jakob's disease, Kuru, and BSE, or Bovine Spongiform Encephalopathy as well as Scrapies, a similar disease affecting only sheep. He coined the term prion, which is a shortened form for 'proteinaceous infectious particle that lacks nucleic acid'.
Beef cattle farmers had for a long time supplemented the feed of their cattle with the reconstituted protein derived from the remains of sick and downed livestock, which were considered unfit for human consumption. Chicken farmers supplemented their chicken feed with the fecal remains of their own chickens, which was also high in protein. This practise made for a more robust, meatier animal, higher in monetary value than one whose diet was not supplemented in this way. By 1997 this practise was outlawed across the western world, and the link to BSE, or Mad Cow disease, and the resulting human form of the variant Creutzfeld-Jakob disease was officially recognized.
As stated, Alzheimer's disease is a prion disease. The prion is a mutant form of the peptides that are found throughout the body. In fact, there is no essential difference between the neural peptides found in the brain and the intestinal peptides. They are merely linear amino acid chains produced by the template action of genes found on chromosomes 11, 15, and 21. When a cell requires the raw materials for the normal synthesis of proteins, which are complex molecules constructed of amino acids, it pulls in a peptide through the hydrophobic cell wall, so they actually provide the vital service of supplying the raw materials for protein synthesis. They can themselves be considered simple proteins because all it takes to qualify for that definition is that they be a molecule constructed of amino acids.
The process by which these peptides become misfolded was described by the late great Vernon M. Ingram, professor of microbiology, in this article in which he traces the progression of Alzheimer's disease at the molecular level and which can be found in the July-August, 2003 issue of the journal American Scientist. I will briefly describe that process.
The prion which is known to cause Alzheimer's begins as a normal peptide. Enzyme activity within the hydrophobic layer of the neuron cell membrane cleaves the peptide at a location which results in the inability of the internal cellular machinery to properly identify the peptide as a source of amino acids and the peptide is discarded and evicted from the cell. This enzyme action is puzzling to microbiologists because enzymes can only do their work in the presence of water, and the term hydrophobic means 'fear of water'; ie. there is no water molecule present where cleavage was found to take place.
The evicted mutant peptide has 'sticky' properties and forms aggregates with others of its kind. These 'plaques' gather around the calcium ion channels of the cell wall and prop them open which results in a flood of calcium ions into the cell, which spells disaster. These calcium ions act on the tau protein in the neuron and their action compromises the integrity of the scaffolding which serves as the three dimensional skeletal structure of the cell. The cell collapses, and either performs apoptosis, which is cell suicide, or it just ruptures. Either way, a link in what might be an important chain of neurons in the network is now broken. The neurons have a backup mechanism in the form the neurotransmitter acetylcholine, which takes over the task of carrying information to the next cell in the chain, after which the acetylcholine molecule is broken down by the enzyme acetylcholine-esterase, a necessary measure to prevent the overabundance of neurotransmitters from upsetting the balance of messages sent and received by the neurons. This is why acetylcholine-esterase inhibitors form an important line of defense in the treatment of the progression of the disease.
As to how the original action on the peptide in the hydrophobic layer of the cell occurs in the absence of a water molecule might be explained by the following discussion, but keep in mind that this is completely my own theory and not officially acknowledged. For background information one would do well to view and study this slide show about how the recreational drug Ecstasy acts on the brain.
In short, when a receptor site on a host cell is overstimulated, it becomes tired and evicts the molecular blocker, which in the slide show example is serotonin, and then the receptor withdraws into the cell to regenerate. I believe that something similar happens to the receptor sites on the red blood cell. The red blood cell is not really a cell at all because it lacks a nucleus. It is a large molecule containing an iron ion at its center and at least four receptor sites on its surface to which bind oxygen or carbon dioxide molecules and which is responsible for completing the respiration process by transporting O2 and CO2 to and from the cells and lungs. These receptor sites are active 24 hours a day every day of one's life and I think that it is possible that these sites also sometimes get tired, and/or the iron ion loses its efficacy over time and an errant oxygen molecule will occasionally drop off. Oxygen is electrostatic and will form ionic bonds with certain places on certain molecules. I believe that the abnormal cleavage sites on the normal peptides are areas to which those oxygen molecules have bound themselves, and when the peptide passes through the hydrophobic layer the oxygen will combine with the hydrogen molecules there to form a water molecule, thereby facilitating the enzyme's action on the peptide.
So, in a sense the development of Alzheimer's disease in an individual of advanced age can indeed be considered part of the normal aging process. I believe that a radical treatment involving complete replacement of hemoglobin with rejuvenated blood product might be an effective treatment for prion related diseases but, admittedly, I speculate.
