This Is MS Multiple Sclerosis Community: Knowledge & Support

AHA! Sounds like some things are coming together!

Polyamines, heat shock, MHC. AND the probability of you being able to do some actual experiments! This is gaining momentum now. Way to go!!

Ok..........post when you can, Wesley! I'm sure many of us are anxious to hear more and keep track of your progress in this! I know I am.

Deb

Wesley had asked for me to post a couple photos for him... I'm not certain you wanted them in this thread Wesley, so I'll put the URL below them and you can use them as you wish.


http://www.thisisms.com/images/upload/biodocfl2.JPG


http://www.thisisms.com/images/upload/biodocfl1.JPG

Thanks Arron. I will probably have two more in a few days.

The first picture is just a mockup of what I used to see when studying the Xi. I didn't want to use real pictures from the project since that is really property of that lab. I did have a lot better examples but this gives an idea of what it looks like. I would first incubate the cells (human XX fibroblasts) on slides with the primary antibody (here against macroH2A) and then incubate with a secondary antibody (goat anti-human IgG, IgM, IgA conjugated with FITC, a fluorescent tag). Then I would look at them under a fluroescent light microscope or somtimes a confocal microscope.

The second picture, the histones are in blue, 8 per nucleosome all bound together. The DNA in the nucleosome is B-DNA (right handed) but it is in a left handed supercoil over the surface of the histones giving storage of a negative supercoil.

Wesley

Okay, the ovarian cancer grant application stuff I was doing is done for now so I can get back to explaining my hypothesis again because there is still a lot to discuss. In my last big post, last Sunday, I got into nucleosomes and DNA supercoiling stress. Pretty complicated stuff but I felt it was necessary to go into that in order to explain my ideas about the chromosome fragmenting that I think may be involved in autoimmune diseases. Arron put some pictures up for me. I'll try to get some more up soon. What I was trying to point out is the packaging of DNA to keep it organized, compact, and protected. However, the DNA is not very well protected, even when packaged in nucleosomes.
I should probably reiterate the hypothesis again before I start up. Remember, it is just hypothesis and so you should keep in mind that it may be wrong in some areas once we get more research done to prove or disprove points. It is kind of a best guess on my part based on what we know at present.
DNA damage occurs and does not get repaired in a timely manner. This can lead to chromosome breaks so that there can be fragmentation of chromosomes. There can then be reactivation of previously sequestered genes and possibly uneven distribution of chromatin to daughter cells when the parent cell with the damage divides. As a consequence, there can be loss of epigenetic control leading to expression and overexpression of previously sequestered genes. The example I used most was the inactive X chromosome because it has some interesting genes on it, but it could be the active X or other chromosomes (autosomes) that suffer the fragmentation. The particular genes on the inactive X that I cited were polyamine genes: spermine synthase and spermidine/spermine N1-acetyltransferase. Overexpression of these genes is not immediately a problem since their substrate, primarily spermidine, is usually bound to DNA, RNA, or lipids. It is when there is a stress on the cell that there is newly synthesized, unbound polyamines and decarboxylated S-adenosylmethionine (needed for polyamine synthesis). Increased synthesis of polyamines can begin to have many detrimental effects on the cell and there can be an imbalance in the spermidine/spermine ratio. Spermine can interfere with ion channels such as calcium channels and it can alter the blood-brain barrier. An increase in spermine at the expense of spermidine could reduce the spermidine needed in myelin formation and translation efficiency. There can be induction of apoptosis in some cells and disruption of chromatin in other cells leading to expression of previously sequestered genes, such as endogenous reverse transcriptases. There can also be creation of autoantigens, in some cases perhaps due to polyamines in abundance binding to otherwise normal endogenous material, altering the epitopes on them. The work I do is in drug discovery to find inhibitors of polyamine enzymes, but I am in cancer research because I feel the study of polyamines is further along with regards to cancer and the research environment is more open to new ideas. There should be carryover to autoimmune diseases if we find good drugs and the hypothesis is right on polyamine involvement in autoimmune diseases.
I did that all in one breathe. I need to explain about the DNA damage, fragmentation, and the reverse transcriptases and some of the possible autoantigens that could occur.
So the DNA is in nucleosomes primarily; one nucleosome every 200 base pairs on average in humans. The nucleosomes compact the DNA lengthwise about 7x. The nucleosomes can then stack on top of each other so that the beads on a string appearance of nucleosomes can be stacked into somewhat irregular helical stacks giving further compaction. These stacks of nucleosomes may have roughly 300 nucleosomes in them which are attached to a nuclear matrix. There is a lot of supercoiling stress stored in there so they are anchored about every 300 nucleosomes to lock in the stress. If one of these stacks suffers a break in the strands, that stack will puff out as the strands are no longer constrained and the stretch goes into the beads on a string appearance giving it accessibility to DNA repair enzymes. That is what should happen in theory but it would depend on how tight the stacking is, the added hold by ions in the local environment, and the extent of the DNA damage. Since DNA repair occurs more readily in open chromatin stretches, the efficiency of repair will correlate to the accessibility. Supposedly there are checkpoints to halt cell cycle progression but these are mainly around S phase when the DNA is replicated. Once the cell has committed to mitosis, there may not be sufficient means and time to repair DNA damage deep in heterochromatin, especially the inactive X with its peripheral location.
Although histones and DNA are in the chromatin in equal amounts mass to mass (gram to gram), the histones do not provide adequate protection to damaging agents, even deep in the chromatin. Heavy metal ions and mutagens can work their way into the chromatin and cause DNA damage. The histones themselves provide no protection to the DNA from UV light, particularly UVB light. The bases in DNA are referred to as aromatic structures because their big flat structure of double bonded carbons and nitrogens not only have electrons shared between two atoms in each bond, but there are additional electrons circulating the length of the structure. The length of the base structure is such that it can resonate, or absorb UVB light (photons) and get excited electrons. These can then react with neighboring bases causing crosslinking or perhaps causing the base itself to break away from the ribose ring, giving what is called an abasic site. There are approximately 10,000 abasic sites formed per cell per day but they are usually repaired. Repair requires access to the strand and strand separation. The correct base can be ligated in to correspond to the base in the other strand. The base stacking in that area is lost until it is repaired so there is some local instability. Another type of damage is strand breaks in the phosphate backbone. This can then release the local supercoiling stress and lead to disruption of neighboring nucleosomes. These sites also need to be repaired promptly, but should be manageable in a healthy cell.
The reason I say that histones/nucleosomes don't provide protection to DNA from UVB light is because the histones are notoriously void of aromatic amino acids in their structure. This would be phenylalanine, tyrosine, and tryptophan. These are bulky amino acids so 'evolution' probably eliminated them as too bulky for the tight packaging needed in the histone to make a tightly compacted nucleosome core. So the histones do not absorb UVB to protect the DNA and, depending how tightly packaged the heterochromatin is, may even hinder prompt repair.
Here is the interesting thing. As if the cell knows it has damage and needs to do repair work, polyamine synthesis is invoked by UVB light in a stress response, particularly ornithine decarboxylase, the first of the polyamine synthesis genes. This then provides the precursor putrescine for the rest of the polyamine synthesis. The polyamines induced by the UVB stimulation can then stabilize the chromatin by either helping to hold nucleosomes together in internucleosomal links that might be possible in closely packed heterochromatin, or the polyamines may help open the nucleosomes in a stack by stealing any available supercoiling stress and twisting the nucleosomes apart momentarily enough to start unstacking the stack to make it more accessible. Who knows what goes on at that tiny of a level. It probably depends locally on the DNA sequence, extent of packaging and modifications, ionic milieu, supercoiling stress, and the extent of damage.
Now think, if this is a normal process where some sunlight exposure on a daily basis temporarily induces polyamine synthesis in skin fibroblasts, those polyamines then raise the person's entire levels of polyamines and help each cell seek out any hidden, unrepaired DNA damage it might have. Without a regular up tick in polyamine levels as a youth, perhaps there could be an accumulation of unrepaired damage hidden away.
One thing about DNA damage, particularly with regards to fragile sites, DNA damage is often found to be clustered 80% of the time. This means that if there are a number of DNA sites being damaged, 80% of them will be within a few helical turns of the DNA strands from each other, even within a few base pairs from each other. If they are in opposite strands, the only thing holding them together might be a few hydrogen bonds, which isn't much strength. If the strands come apart giving a double strand break, it is very difficult for the cell to recover at that site and religate the strands. This then could be the chromosome fragmentation that leads to the problems I've described. Even if the cell reacts quickly to a damage site, repairing one site may put strain into neighboring sites hindering their repair. We should also consider that some sites may not be easily accessed even if the stack of nucleosomes tries to unravel. There may be crosslinking of proteins, another type of chromatin damage, that keeps some of the chromatin clumped up and interferes with repair. In a time consuming process the chromatin is ribosylated, putting large trees of ribose groups around the site of damage. This can help lock in the chromatin so the disorder does not spread and the ribose trees can help compete histones off the DNA to give more accessibility. I don't know all the details about DNA repair. It's been quite a while since I have studied it, but it is several different cumbersome processes, some of which are stop-loss type processes. One autoantigen mentioned a lot in lupus is the Ku antigen. I believe one function attributed to this is to bind to double strand ends of DNA, as if it plays a part in stabilizing DNA damage.
So what I am saying is that DNA damage can be difficult to repair, particularly when it is accumulating without sufficient regular flexing/breathing/scanning of the chromatin that might be provided by daily stimulation systemically of polyamine synthesis and circulation. If there is an accumulation of unrepaired DNA damage, it may pass the point where it can be repaired properly and then a surge in polyamines may simply expedite the consequences of double strand breaks. What I am having trouble imagining is the time frame in which all this might occur, if it is at the root of MS development. Is this something that only occurs over many years accumulating unrepaired DNA damage and then shows up as an adult, or is it something that can occur in a matter of days in an adult? How long has DNA damage been hidden and has it spread to daughter cells and through how many generations of daughter cells?
Lupus bouts can be instigated by UVB light so that is known. Lupus, of course, is known for its light-sensitivity. But, since I am not an MSer and am somewhat new to studying it, I would like to know what you folks know about the light sensitivity in MS. I have heard through the forums that some people are greatly fatigued by sunlight while others say it makes them feel better. Can anybody explain that? That is why I am wondering about how quickly DNA damage could accumulate in an MSer and then show up as a bout, if in fact my idea of UVB light exposure and MS is valid. Is it insufficient exposure overtime (years) as a child or is it individual exposures (days) as an adult? MS really is a puzzler.
I'll try to get into the reverse transcriptases later this weekend.
Wesley

Hi, Wesley.

Are you sure that when some MSers are saying they get fatigued by sunlight, they aren't really referring to the "heat" instead?

Bottom line is that a lack of sunlight (and I'm not talking about heat, though - just light) is part of the problem in MS. It goes along, also, with the Vitamin D theory. Vitamin D has been shown to be helpful (not curative, but helpful in MS) Your body also gets Vitamin D from sunlight.

If some people are talking about photosensitivity, then that's different yet again. That is more a "sensory" symptomatic reaction due to the body's damage from the MS disease process, but is not indicative of any modification, change or progression of the disease itself.

I think you are finding the problem with describing the disease in the first place. There are two totally separate things going on. Symptoms and the disease process itself. That's why modification therapies of the disease itself rarely shows any affect on the symptoms of the disease. But the average layperson with MS will describe it as one in the same to you. (Of course.) They won't be able to "feel" the difference. But as you know, there is one.

Damage from MS can be going on without ANY symptoms showing up at all in a patient, and having a lot of symptoms doesn't necessarily mean that the disease process itself is progressing at all. See what I mean?

So, try not to get the two mixed up when doing your research on MS. The problem with MS is how it is affecting the brain (and the brain's interpretations of inside physiological processes and of outside stimuli.) Depending on where the damage is, the brain can get mixed up and totally misrepresent on the outside what is going on on the inside, and vice versa. A patient wouldn't necessarily be able to realize that at all. The brain is the complex part of this whole picture because of how skewed a picture, shall we say, that it can give you (i.e. a patient). You can swear you are in actual pain, but it could be entirely "sensory" pain (i.e. you only THINK you are in pain - which to a patient doesn't matter, but to a clinician, it's a huge difference), which is why most OTC drugs won't touch it.

I hope this helps. That's why with any of the disease modifying drugs that are currently on the market for MS, they have to "watched" not based on the patient's interpretation on whether they "think or feel" that the drug is working, but by actually trying to see what is going on inside the body (via MRI, blood tests, etc.). All a patient can do is let a doctor know whether "symptomatic treatment" by a certain drug helps them to feel better or not.

Remember, there is a difference in MS between "symptomatic treatment" and "disease modifying therapy". A huge difference in MS. In most other diseases, the symptoms themselves usually correspond more closely to the disease process itself. That is NOT the case in MS. There is no way you can really tell how MS is progressing or not progressing based on a patient's description of symptoms. They just aren't related.

Deb

I've actually tried to avoid getting too deep into symptoms in thinking about MS and lupus because I was afraid that might bias my thinking about how to explain the hypothesis with regards to one symptom and dwelling on the symptoms of one patient might bias my thinking towards a set of symptoms that are infrequent and possibly have a different unrelated cause. MS really is complicated but I think I understand about the pain as perhaps being a 'phantom' pain, or possibly being real.
Wesley

Yea, you've got the concept, Wesley.

Philosophically speaking for only ONE second here. What is "perception" anyway? Ya know? Exactly what IS reality, and how can you really tell the difference? (These are rhetorical questions only.)

Anyway, getting back to pure science. Yes, you're understanding MS.

Deb

This is the last big post I need to do to finish the hypothesis, at least in how it relates to MS. There is a lot more relative to lupus though. This gets into how endogenous reverse transcriptases could be expressed and the consequences.
Nucleosomes occur every 200 base pairs on average in humans. They even occur in active genes but somehow the RNA polymerase is able to transcribe the gene through them. Nucleosomes do not have specific sequences where they bind but they do have some preferences. How tightly they hold those preferences depends on several things including what modifications are on the histones. We speak of positioned nucleosomes, especially around the start of a gene. Usually an active gene has an open area just upstream of its start site. This is referred to as the promoter region of the gene. This can be a length of several hundred to several thousand base pairs where there are few nucleosomes. The promoter region is open so that transcription factors, hormone receptors, and chromatin modifying enzymes can attach to the DNA and build a transcription initiation complex. These complexes will be assembled and then an RNA polymerase binds and begins transcribing at the start site and moving into exon 1 and so on. This is the classic description of a gene, its promoter, and transcription by an RNA polymerase. It best describes RNA polymerase II which does transcription of the greatest variety of genes. It is a description that also fits RNA polymerase I and III for the most part. RNA polymerase III, however, can have a much simpler method. In some RNA pol III transcribed genes and pseudogenes, there is a very simple initiation complex formed, only about three components, and it does not form in a promoter region outside of the actual gene (ie. before exon 1). RNA pol III transcription initiation can occur in exon 1 (intrageneic initiation). Therefore, there is only a little length of DNA sequence needed to build the RNA pol III initiation complex and then recruit an RNA pol III. It is as little as 70 base pairs, less than half of a nucleosome's DNA (145 base pairs). Therefore, if a positioned nucleosome that has been positioned where it suppresses transcription from a gene is removed or simply slides 70 base pairs, it could give greater access to the gene. This could facilitate transcription from a previously sequestered RNA pol III gene, especially if the cell is having difficulties in keeping its DNA methylated and packaged.
So what genes could be involved? SINEs and LINEs are short and long interspersed nontranslated elements. These are repetitive sequences found throughout the genome. One of the main SINEs is the Alu sequence. This is a pseudogene, formerly a useful gene sequence that has degenerated through mutations and evolution so that it no longer is functional for its original purpose. Alu sequences are 80 to 410 bases long, but transcription can some times go beyond this giving a transcript of greater length and unknown consequences. The Alu sequences are GC rich, 9x the average of the overall genome. They are usually heavily methylated. About 99% of Alu sequences are inactive. They are found frequently in introns of transcribed RNA pol II genes. Introns are portions of an RNA transcript that are spliced out of the initial RNA message giving the final messenger RNA, mRNA. There are an estimated 500,000 copies of Alu sequences throughout the genome! So there would be many on the X chromosome. So we have polyamines stimulating histone acetylation, which loosens the nucleosome's hold on DNA. Polyamine synthesis is also competing for S-adenosylmethionine, which is needed for DNA methylation. The scenario I have been describing where there is over expression of polyamines could lead to loosening or loss of nucleosomes and methylation in RNA pol III transcribed genes and pseudogenes. I am proposing that this can lead to inappropriate transcription from previously sequestered RNA pol III transcribed genes. As evidence that this can occur, in cancer there is an observed increase in RNA pol III activity (White RJ RNA polymerase III transcription and cancer. Oncogene 2004; 23:3208-16). And of course it is known that in many cancers there is an increase in polyamine levels and synthesis. Another thing about Alu sequences, it has been proposed by others that Alu sequences can serve as alternate DNA replication initiation sites. How this happens is not known. But this would be generating DNA that needs to be methylated.
So now there is a vast increase in Alu RNA transcripts being created. Is that a problem? There have been autoantibodies found in lupus specifically against Alu RNA-protein complexes. I don't have that reference in front of me but I listed it and explain this whole RNA pol III activity in two previous publications (Brooks WH. Autoimmune diseases may result from inappropriate RNA polymerase III transcription. Arthritis Rheum 2002; 46:1412-3. and Brooks WH. Systemic lupus erythematosus and related autoimmune diseases are antigen-driven, epigenetic diseases. Med Hypotheses 2002; 59:736-41). So you can find the references to all this in those articles.
Charles Steinman did research on the free DNA in serum of lupus patients and he found that it was GC rich and was 55% Alu sequences. Alu sequences are normally 13% of the genomic DNA. So he said there was either selective fragmentation and degradation of DNA that left Alu sequences, or perhaps there was some sort of retroposon activity going on. I think his work was just before we started finding out there were endogenous human reverse transcriptases although we knew that there were reverse transcriptase in other species. So if a cell is transcribing an abundance of Alu RNA, how does it get to be an abundance of Alu DNA and why is it autoantigenic?
The LINE L1 sequence is the remnant of a reverse transcriptase. There are an estimated 20,000-50,000 LINE L1 sequences throughout the human genome and an estimated 30-60 still have functional reverse transcriptase activity. LINE L1 sequences can be transcribed by RNA pol III. And LINE L1 sequences are on the X chromosome 2x as frequently as in other chromosomes (and 3x in the Y chromosome). Mary Lyon who first came up with the idea of X inactivation has recently proposed that LINE sequences somehow serve as way stations or anchor points along the X for the XIST RNA that inactivates the X. A good proposal but I think it is still needing some research to prove it.
So if one or more of the functional LINE L1 reverse transcriptases is transcribed and translated due to the increase in polyamines disrupting the chromatin, then one type of RNA that it would find in abundance is the Alu RNA. Therefore, it could be creating hundreds or thousands of short Alu DNA fragments, some of which might extend on into other sequences. These fragments need to be methylated since that is how the immune system normally sees them but there is not sufficient S-adenosylmethionine due to the demands of polyamine synthesis. And, because the Alu DNA is not in its normal epigenetic context, the cell has to use the less efficient de novo methyltransferase to methylate them. Since there is no pattern to copy and not enough S-adenosylmethionine, a lot of the Alu DNA will remain hypomethylated. When these fragments get out of the cell, the immune system will not recognize them as self due to the hypomethylation and it will think that it is bacterial DNA. Therefore, there is an autoimmune reaction. This may be like the 'danger signal' that Polly Metzinger at NIH proposed comes from stressed cells. Of course there could be other endogenous reverse transcriptases involved if they can be transcribed by RNA pol III or if they happen to be next to an Alu sequence that gets transcribed. Plus, with all this additional disruption going on, some RNA pol II gene promoters that are normally sequestered could be opened up.
All of this probably isn't going on in MS. I am thinking more of lupus, but again I think there are parallels in the diseases. That amount of it that is occurring in MS may remain inaccessible to the immune system due to the blood-brain and blood-nerve barriers so that it is not noticed as much. There are some reports of reverse transcriptases and related proteins being found in MS.
Now, guilt by association or epitope spreading. Again, this is primarily with regards to lupus. The newly synthesized Alu DNA that is out of its normally epigenetic context will probably associate with histones. Those histones will not receive the appropriate modifications that would normally be found in the Alu sequences (probably methylation of some of the histones) so the histones would be out of their normal epigenetic context and bound to autoantigenic DNA. Eventually the immune system would consider the histones with DNA, and then histones alone to be autoantigens. Also, the Ku antigen associates with double strand DNA ends and it would probably be found attached to the Alu DNA fragments. Also, Ro and La, two other lupus autoantigens are thought to function normally in maturation and directing of RNA pol III transcripts. So they would be considered as autoantigens sometimes in lupus due to their associations with the Alu RNA complexes and reverse transcription complexes. As far as the splicing components that are targeted sometimes in lupus, they may be associated with Alu sequences that don't have the complete start and end signals for splicing so the splicing components are stuck there, not able to complete the splicing and perhaps complexed with polyamines, again giving autoepitopes.
This is mainly with regards to lupus but, in lupus, there is more access by the immune system to the abnormal complexes. If the blood-brain barrier were completely removed, perhaps this might occur in MS also. We know that some MSers also have lupus so perhaps the autoantigens in MS are only a small subset of what could occur and relate mainly to myelin formation and problems occurring in that formation, myelin being one of the larger components encountered in the nervous system when the blood-brain barrier is compromised.
Well, I think I've covered most of the hypothesis and then some. Probably too much on lupus but that's what got me interested in the autoimmune problems originally. We should consider MS and lupus as having similarities and yet, the cell types, accessibility to the immune system, accessibility to triggers, and our ability to view the consequences in a timely and thorough manner give quite different patterns to the diseases and different patterns in individuals.
I have taken a lot of criticism for these ideas (the ideas have even been referred to as 'wacky') but I have not been or will not be dissuaded from continuing to work on them. I am continually trying to get that criticism, as a matter of fact, as long as it is constructive so that I can continue to refine the hypothesis. I welcome any questions. I have not seen any theories that go into as much detail and that is something that I think is needed.
Thanks for reading all this.
Wesley