Introduction

Chris Weihl led this live discussion on 28 May 1999. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.
 

Article under discussion

van Leeuwen FW, de Kleijn DPV, van den Hurk HH, Neubauer A, Sonnemans MAF, Sluijs JA, Koycu S, Ramdjielal RDJ, Salehi A, Martens GJM, Grosveld FG, Peter J, Burbach H, Hol EM. Frameshift mutants of β amyloid precursor protein and ubiquitin-B in Alzheimer's and Down's syndrome patients. Science 1998 Jan 9;279 (5348): 242-247. Abstract

Transcript:
Live discussion held 28 May 1998.

Participants: Fred van Leeuwen, Elly Hol, Ahmad Salehi, Paul Coleman (moderator), Howard Federoff, Reinko Roelofs, Ruth Perez, Rachel Aronoff, Chris Weihl, Marc Paradis, Jill Weimer, J. Shioi, Keith Crutcher, Kieran Breen.

Note: Transcript has been edited for clarity and accuracy.

Asalehi: Welcome to Ichat about frameshift mutations of beta amyloid precursor protein and ubiquitin-B in AD and DS

Howard Federoff: Good morning (US EST) from Howard Federoff.

Fred van Leeuwen: Hello Howard, How are you?

Asalehi: Greetings from Amsterdam. Is 14.40 here in Amsterdam.

Howard Federoff: I am well thanks Fred, how are you?

Fred van Leeuwen: I am fine, thank you, the traffic is quite slow.

Elly Hol: Good afternoon Paul and Howard. Hi Reinko.

Asalehi: Greetings from Amsterdam Paul.

Howard Federoff: Good day to all.

Fred van Leeuwen: Hello Paul, welcome to the site and thanks for chairing the session.

Fred van Leeuwen: Howard, do you already have a burning question?

Howard Federoff: Fred: I am wondering whether the dinucleotide deleting may be a general phenomena?

Fred van Leeuwen: Yes, we think. As indicated in the Science paper ref.30 we have found similar events outside the brain in a vasopressin transgene. We hope to submit this manuscript very soon.

Paul Coleman: Fred, I am wondering if the +1 immunoreactivity in IHC is limited to cells that are Alz 50 or Bodian or MAC-1 positive.

Fred van Leeuwen: We found colocalization of +1 proteins as a sub population of Alz-50, Bodian and MC1 positive neurons.

Howard Federoff: Is there evidence in other AD-derived material for other mRNA modifications?

Elly Hol: Howard, we are currently checking other AD tissues if +1 proteins are present. Data are in progress.

Howard Federoff: My line of inquiry is more general: Is there evidence that a systematic alteration in mRNA biogenesis exists in AD?

Fred van Leeuwen: We presently do not know. At the moment we are checking whether it is present in more transcripts, indicating that we are dealing with a general mechanism.

Paul Coleman: How do you relate the limited IR of +1 proteins, particularly the ubiquitin, to the more widespread IR for lysosomal enzymes shown by Randy Nixon's group?

Fred van Leeuwen: Good morning Rachel.

Rachel: Hello!

Howard Federoff: Rachel- how are you?

Paul Coleman: Rachel - good to have this chance to chat.

Rachel: Hi, again. It's an early morning, and El Nino isn't over yet.

Rachel: I'm sorry to come in the middle.

Paul Coleman: Actually, you are on time. We just jumped the gun.

Fred van Leeuwen: Ubiquitin is as you know a different protein degradation pathway. How they are exactly related is not yet clear to our knowledge.But can you rephrase the question?

Paul Coleman: Hello Keith.

Howard Federoff: On the subject of a general mechanism: Do you have thoughts as to whether some pressure exerted at the cellular level may account for the propensity of vasopressinergic and AD neurons to undergo nucleotide deletion?

Fred van Leeuwen: Yes we know it is the transcriptional activity as is clear from the Brattleboro rat but also the Down syndrome patients.

Paul Coleman: Hi June.

June Kinoshita: Sorry I'm late.. Hi all!

Paul Coleman: Fred, to try to rephrase the question, I am thinking about where the frameshift editing you describe may fit into the pathological cascade of AD.

Fred van Leeuwen: Whether it is cause or consequence is still under investigation. However it must be a very early event as in Down syndrome patients it precedes neuropathology. More data will be presented at the SFN meeting in LA.

Asalehi: In DS, There is very clear overexpression of APP. which we think is related to the frequency of occurrence of +1.

Fred van Leeuwen: You can fire the next question.

Paul Coleman: I wonder whether it could be an early event in view of the IR being limited to a subset of Alz-50, Bodian, Mc1 positive neurons.

Fred van Leeuwen: Paul, from the cover of Science you can see that more neurons are stained in Vibratome sections. Paraffin is not ideal. But we feel that it starts in a few neurons as seen in non-demented controls.

Paul Coleman: Hello Jill.

Rachel: In the Brattleboro rat, and for the wt rats, frameshifted RNAs appear in the neurons. Is this dependent primarily on age? i.e. have stronger promoter constructs been tested in transgenic animals?

Fred van Leeuwen: Rachel, in rats it is dependent on age. That relation was not found in humans for vasopressin, but the sample might have been too small.

Howard Federoff: Are there any data from transgenic as of yet?

Elly Hol: We have a few transgenic lines and we are currently analyzing them. Chrisw: I was interested if the +1 proteins led to altered distribution of the WT proteins in transfected cells (maybe aggregates).

Elly Hol: About the transfected cells: we do not have any indications that +1 proteins lead to an altered distribution of the WT proteins.

Howard Federoff: Have other post-mitotic tissues from AD patients been examined for deletional editing?

Elly Hol: No, we didn't check other post-mitotic tissues from AD patients.

Rachel: But how highly expressed is vasopressin in rats relative to humans?

Fred van Leeuwen: We do not know, Rachel.

Paul Coleman: I have looked at your web site. Actually, the few neurons that are positive in non-demented controls raises another question in my mind, which is, what constitutes a control in AD studies. We have found that a significant proportion of our so-called control cases (based on clinical studies) are actually Braak Stage I-III on neuropath exam. In the latest consensus statement on the neuropathological diagnosis of AD, the statement is that any evidence of NFT or SP in normals is not normal. So, I guess I am raising the question of whether the +1 IR you see in controls represents the same kind of co-localization you see in AD brain.

Asalehi: Paul, it could be the case. But all the brains here diagnosed for AD are stage IV of Braak.

Fred van Leeuwen: Yes Paul. If the first tangles appear there is +1 immunoreactivity as well. Once again this work was based upon paraffin sections. Using Vibratome sections it might even be seen earlier.

Paul Coleman: Ahmad - that's good. But really my question relates to the presence of NFT in control brains, and whether from a neuropath point of view, these control brains are really early AD, with some cells, the ones with NFT, representing more advanced disease.

Rperez: This is Ruth Perez in Pittsburgh. Hello to all. Do you have an idea why the +1 APP was found in only 50% of the AD patients? Were they sporadic cases or FAD?

Elly Hol: Hi Ruth, All the cases we studied were sporadic cases. The 50% is not correct, if you accumulate all brain areas we looked at is 70%. This is based on immunostainings on paraffin sections. We now prefer to study Vibratome sections, and we are convinced that we will find APP+1 immunoreactivity in almost all AD cases. The Vibratome method is more sensitive.

Howard Federoff: Federoff will return to the mundane. Thanks to all.

Paul Coleman: Fred, I think you have a very important point about Vibratome sections, in that I believe it would be of great interest to the issue of whether the +1 IR is an early event in the pathology of single cells or a later event.

Asalehi: Indeed the controls with some IR +1, were the same with higher numbers of NFTs or plaques, However, they did not reach to that point to be diagnosed as AD.

Chrisw: Is there any evidence that in addition to the increase in +1 proteins that there is an increase in undegraded and perhaps untranslated aberrant mRNA in AD patients suggesting the phenomenon is related aberrant transcription (and the accumulation of junk RNA) and not the +1 proteins?

Elly Hol: Chris, you might be quite right that there is an accumulation of junk RNA or mutated RNA. One of the possibilities is that a system like the 'RNA surveillance' system in yeast is not functioning in AD patients. If this is true than mutated RNA is not degraded anymore and can be translated in aberrant proteins: such as the +1 proteins.

Rperez: Could the accumulation of the +1 proteins be toxic to neurons? Or could the loss of the trophic APP secretion products diminish neuronal survival?

Fred van Leeuwen: +1 proteins could indeed be toxic but it is possibly not acute. See our discussion about for ubiquitin(slow accumulation of aberrant proteins). AD lasts 5-10 years.

Rachel: How does the presenilin story fit with +1 products?

Fred van Leeuwen: The presenilin story will be checked quite soon in brain material from different presenilin families.

Paul Coleman: Fred et al, the reason I am pursuing the line of inquiry that I am following relates to the data from Brad Hyman's lab, as well as data we have, suggesting that the formation of NFT is not necessary to the loss of neurons in AD. If your +1 IR is largely in NFT or PHF + cells, then it might not be a primary factor in neuron loss. It could, of course have other great significance.

Asalehi: Paul, you are right that NFTs may not induce cell death, However, only NFTs and not NPs correlate with the severity of dementia.

Fred van Leeuwen: Paul, Heiko Braak has reported that tangles correlate only with dementia.

Marc_paradis: Hello all - my question concerns the specificity of your finding. Why should a brain (i.e. a large assemblage of anatomically distinct neurons and/or astrocytes) suddenly start producing post-transcriptional +1 mutations only in APP and ubiquitin B? Especially if a similar post-transcriptional mechanism is postulated for ALS?

Elly Hol: Marc, I believe that most certainly not only APP and ubiquitin-B are affected. But we started out with these two.

Chrisw: Do you think that the phenomenon of +1 proteins is a generalized pathology of several neurodegenerative diseases. Have you looked at animal models of AD (APP mice)?

Marc_paradis: Precisely, Chrisw, if +1 proteins are a general phenomenon for neurodegeneration, some preceding disease-specific insult must also exist (as with people who argue a central role for apoptosis). What might this AD-specific +1 insult be?

Chrisw: Marc that is why I was curious about transgenic models have the same phenomenon of +1 IR.

Fred van Leeuwen: Marc, it is probably the common denominator but we don't know what that may be.

Marc_paradis: Speaking of apoptosis, is there evidence that apoptotic cells generate +1 or any other kind of post-transcriptional mutations?

Paul Coleman: Ahmad, I think the bulk of evidence does support that NFT correlate with dementia. However, see a fairly recent paper by Carl Cotman and B. Cummings that indicates that NFT and plaque burden correlate equally well with dementia, depending on the IHC procedures used.

Asalehi: Paul, that is fine, but still it does not rule out that the process of neurofibrillary degeneration plays a major role in AD.

Paul Coleman: Ahmad, I agree. I see synapse loss as really the underlying best correlate presently known, and we have evidence that NFT neurons contribute significantly to synapse loss, not non-NFT neurons. The proportion of synapse loss due to still live NFT neurons and to death of neurons is undoubtedly different in different projection systems, but remains to be determined for any system.

Asalehi: Paul, I agree. But you can add decreased neuronal metabolism as another hallmark of AD.

Msmith: Sorry, I'm late...damn traffic.

{JUNE KINOSHITA} Rachel tells Fred van Leeuwen I am sorry not to be saying much, but this is interesting!

Elly Hol: About the transgenic lines. We tested several transgenic lines (from Karen Hsiao, Staufenbiel, Fred van Leeuwen, Bruce Lamb) with different mutations in the APP molecule and wt APP. None of them detectably produced APP+1.

Chrisw: Elly Hol, that seems contrary since you speculate that high APP levels result in +1 APP reactivity.

Elly Hol: Chris, these mice are healthy and may be able to cope with aberrant RNA. Thus if mutations do occur the RNA, this RNA is efficiently degraded.

Marc_paradis: But then for each of those lines, strong arguments could be made that they are poor models of AD.

Rperez: Since the transgenics did not generate +1 APP, does this suggest that aging plays a role?

Chrisw: or perhaps environmental factors (in regard to the transgenic data)?

Fred van Leeuwen: The APP transgenic mice may bear incomplete constructs (without introns, except those of Bruce Lamb). Thus the environment of the transgene may be important.

Rachel: Did these mutants affect the dinucleotide repeat regions?

Fred van Leeuwen: Rachel, the APP transgenic mice do not have a dinucleotide deletion.

Kieran_breen: Have you looked for +1 APP in any of the PS mutant mice?

Fred van Leeuwen: Kieran, no we did not look in the PS transgenic mice.

Paul Coleman: Marc wants to know what role apoptosis may play in generating +1 or any other post-transcriptional modifications.

Asalehi: Paul, I don't know about the role of apoptosis in generating +1. However, there are not much data supporting the notion that apoptosis may play a major role in AD.

Fred van Leeuwen: Marc's question is a good one. It may be that an inefficiently working ubiquitin system may somehow induce apoptosis. But cell death in AD is only clear in the hippocampus(CA1 and subiculum) , locus coeruleus and the entorhinal cortex. It is not a clear phenomenon for every brain area in AD.

Paul Coleman: Breen asks whether there is anything known about the role of free radicals in generating post-transcriptional modifications.

Fred van Leeuwen: Not known as yet. SOD models may interesting.

Rachel: I wonder if there is an AD transgenic model that everyone agrees on. Does it make APP+1??

Rachel: Also, isn't the hippocampus problem the most important in terms of memory and Alzheimer's?

Paul Coleman: Rachel, I regard your question as one open to all. My opinion is that at present transgenics model very well selected aspects of AD, but I know of no tg that models all currently known aspects, or even all the major pathologies.

Elly Hol: Rachel, the transgenics we tested did not make APP+1. Maybe crossbreeding of several lines, can lead to an improved AD transgenic model. I think Ubiquitin +1 overexpression can lead to problems in the transgenic lines, as it probably will interfere with the proteasomal protein breakdown. Although it remains to be seen if AD-like neuropathology will appear in these mice (plaques and tangles).

Marc_paradis: The other half of the apoptosis question is necrosis, including sick cells which do not commit to the apoptotic pathway. What role might such necrotic cells play in generating post-transcriptional mutations?

Fred van Leeuwen: We have addressed that question in multiple sclerosis patients and did not find any +1 immunoreactivity.

Chrisw: Is there any evidence that mutant transcriptional machinery might be involved and resulting in +1 proteins. Have you looked at RNA polymerase etc? Maybe these proteins are susceptible to environmental factors (free radicals)?

Elly Hol: Chrisw, no we did not look at RNA polymerases. It might be well possible that the transcriptional machinery is involved.

Paul Coleman: Your Science paper refers to data from PD. Have you examined other dementing disorders?

Fred van Leeuwen: Yes we are currently studying Pick's disease, ALS. Multiple system atrophy and Lewy Body Disease. No definite results as yet.

Rperez: Have the Athena PD-APP mice been analyzed?

Fred van Leeuwen: We have asked Karen Chen for these mice but so far we did not receive them.

Msmith: Obvious Q but here goes anyway...any effect of ApoE? Or, have you looked at the ApoE Tg animals yet?

Fred van Leeuwen: As yet we did not look to ApoE Tg animals.

Asalehi: No clear effect of ApoE was found on IR of +1.

Chrisw: Do you speculate a causal link between environmental factors resulting in + IR?

Elly Hol: Everything that increases the speed of aging and the transcriptional activity might result in +1 IR.

Msmith: Tissues aside from brain?

Fred van Leeuwen: We did not yet study tissues outside the brain but see our ref.30 in the Science paper. What about progeria?

Elly Hol: I am sorry...Progeria?????

Msmith: I forget the other names.....disease of premature aging...12 year old kids look like grandfathers/mothers!

June Kinoshita: We've now gone past one hour. Let's let the authors wrap up the remaining questions. Any further questions can be submitted to the news group. Rperez: Thanks to all.

Elly Hol: Oh progeria, Might be very interesting to study. Do you know somebody who can provide us with material of these patients?

Paul Coleman: Sorry I have to leave. This has been most enjoyable. Thank you June for organizing this, and thanks to Fred, Ahmad, Elly and all participants.

Fred van Leeuwen: Thanks for all the interesting questions and feel free to send e-mails.

June Kinoshita: Thank you very much Paul!

Elly Hol: Thanks to you too Paul.

Asalehi: Thanks very much Paul.

Chrisw: Thank you for an excellent discussion.

June Kinoshita: Thank you for the excellent discussion text, Chris! Thank you Fred, Elly and Ahmad. I have to leave now. See you in Amsterdam.

Msmith: Toodle-pip.

Chrisw: Thank you again.

Asalehi: Thank you June. Hope to see you here in Amsterdam.

Background

Background Text
By Chris Weihl

Familial Alzheimer's Disease (FAD) has been associated with a variety of point mutations in three genes, APP (amyloid precursor protein), PS1 (presenilin 1) and PS2 (presenilin 2). These point mutations are single base pair changes at the genomic DNA level and are inherited from generation to generation. The mutated gene thus encodes a mutant protein that results in FAD. Conventional genetic analysis of AD patients has primarily focused upon the identification of these types of genomic mutations.

Recently mutations have been discovered in several proteins exclusively associated with neurodegenerative diseases, that are not found at the genomic level, suggesting that neurons are capable of altering genetic information post transcriptionally. It has been known that mammalian neurons are unique in their ability to "edit" their mRNA transcripts in order to increase phenotypic variability (O'Connell, 1997). In particular, glutamate and serotonin receptors are edited at specific bases (adenine to guanine) creating amino acid changes not found at the DNA level (ibid). While this form of RNA editing has not been associated with disease, two other novel forms of post transcriptional RNA editing have recently been associated with sporadic amyotrophic lateral sclerosis (ALS) and AD.

Rothstein and colleagues identified multiple splice variants in the mRNA of EAAT2 (excitatory amino acid transporter type 2 abundant in glia) resulting from aberrant splicing events exclusively in sporadic ALS patients (Lin et al., 1998). The splice variants were not the result of changes at the genomic DNA level but were created post-transcriptionally. In the paper under discussion van Leeuwen and colleagues identified a novel form of RNA editing resulting in a frameshift mutation of transcribed mRNAs that was not found at the genomic DNA level and was highly specific for AD and Down's syndrome patients (van Leeuwen et al., 1998).

In order to understand the potential mechanisms involved in these pathogenic RNA editing events, it is necessary to review the steps involved in cellular protein synthesis. In brief, genomic DNA is transcribed into pre-mRNA which contains exons (protein coding sequences) and introns (non coding sequences) in the nucleus of a cell. Introns are then spliced from the pre-mRNA via a complex mechanism mediated through a spliceosome into mRNA. mRNA is then shuttled to the cytoplasm and translated via ribosomes into functional proteins. Hence alterations that result in mutant protein can be introduced at any of the aforementioned steps. In addition, all of the events involved in protein synthesis are mediated by multiple protein complexes, and mutations in any of these proteins may result in a mutant protein. In fact two other neurodegenerative diseases, spinal muscular atrophy (SMA) and fragile X syndrome (FMRP), are the result of mutations in the proteins SMN (associated with splicing machinery) and FRAXA (associated with RNA trafficking from the nucleus to the cytoplasm) respectively, suggesting that post-transcriptional mRNA changes may be a general mechanism in the pathogenesis of disease (Feng et al., 1997; Liu et al., 1997; Siomi et al., 1993).

Discussion of paper
Previous studies have alluded to a novel mechanism of mutant protein reversion in vasopressin deficient rats (VP-/-) (Evans et al., 1994). Genetic analysis revealed that VP-/- rats contain a GA deletion in a GAGAG motif in the vasopressin allele resulting in an out of frame nonfunctional vasopressin protein. However, a small number of magnocellular neurons (the neurons that secrete vasopressin) express immunoreactivity for both the mutant and normal vasopressin protein in VP-/- rats. Further characterization revealed that magnocellular neurons in VP-/- rats are capable of reverting this phenotype to VP-/+ resulting in an in-frame vasopressin mRNA transcript and functional protein. Furthermore wild-type rats also alter their vasopressin transcripts in the opposite manner creating mutant vasopressin suggesting that this phenomenon was not induced by the diseased state of VP-/- rats.

The paper under discussion investigates sporadic AD patients' mRNA for frameshift mutations not found at the genomic level. Van Leeuwen and colleagues surmised that two proteins associated with AD, APP and ubiquitin-B, that contained multiple GAGAG motifs, might exhibit frameshift mutants in AD patients. In order to examine the presence of putative +1 proteins (frameshifted proteins), they generated antibodies to mutant proteins APP+1 and ubiquitin-B+1 and immunostained tissue sections from AD, Down's and normal brains. Increased immunoreactivity of the frontal cortex, temporal cortex and hippocampus were strongly correlated with AD and Down's syndrome brain tissue. In addition, this correlation was not present in other neurodegenerative diseases or in unaffected brain regions (i.e. striatum) suggesting specificity to AD and Down's syndrome. Co-analysis of +1 protein immunoreactivity with neuropathology suggested that APP+1 and ubiquitin+1 co-existed in the same neurons and were strongly immunoreactive in dystrophic neurites, neuropil threads and neurofibrillary tangles. Western analysis of the immunodetected proteins demonstrated a 38 kDa APP+1 protein and 11 kDa ubiquitin-B+1 protein from AD brain homogenates corresponding to their expected molecular masses.

In an effort to identify the frequency and the specific mRNA mutations involved, van Leeuwen performed RT-PCR and sequencing of APP and ubiquitin-B transcripts in normal and AD patients. As expected, GA deletions, as well as some GT and CT deletions were found more often in AD and Down's syndrome brain homogenates than in normal control brains. However, the frequency of these mutant transcripts was low (1/2000 sequenced transcripts).

It is possible that the mutations found in the mRNA transcripts are due to mutations at the genomic level in single neurons or mRNA editing during or after transcription. In order to determine if the GA deletions were present in neuronal DNA, van Leeuwen performed two rigorous PCR control experiments. One strategy involved PCR amplification and subsequent sequencing of APP and ubiquitin-B genes from genomic DNA and the second strategy used mutation specific PCR primers to detect the GA deletion in APP and ubiquitin-B. Both strategies failed to detect genomic alterations, supporting the hypothesis that frameshift deletions in APP and ubiquitin-B occur during or after transcription.

Mechanisms of RNA editing are slowly emerging as potential mediators of neurodegenerative disease. In particular, the current paper addresses a novel finding of frameshift deletions present in two proteins associated with AD. Further investigation into the mechanisms of RNA modification as well as similar phenomena in other degenerative diseases will provide clues to the pathogenesis of sporadic AD.

Experimental Questions
1) Was the crossreactivity of the antibodies tested? Perhaps AD brains (and dystrophic neurites, etc.) contain non-specific immunoreactive proteins that were detected by the APP+1 and ubiquitin-B+1 antibodies.

Reply from van Leeuwen: Yes, we extensively tested the crossreactivity of the antibodies. APP+1 antibody revealed a band of 38 kDa on a Western blot, as expected by translation of the mRNA sequence into the protein sequence. Although this might be a degradation product of the APP+1 protein, as due to the acidic domain in APP one could also expect a MW of 60-70 kDA. However APP+1 produced in stably transfected cells has also an apparent MW of 38 kDA, as shown with the APP+1 antibody on a Western blot. Ubi B+1 stained an 11 kD protein on the Western blot and in addition, HPLC fractionation in combination with a radioimmunoassay, showed that recombinant Ubi B+1 co-eluted with Ubi-B+1 immunoreactivity from a temporal cortex homogenate of an AD patient. We tried immunoprecipitation with the APP+1 antibody but failed. Therefore we consider trying to immunoprecipitate with 22C11 antibody, which recognizes the the N-terminal part of APP+1. Moreover one must realize that a band at a certain position does not automatically mean that the antiserum is specific(see our letter to the Editor in the J.Histochem.Cytochem.25, 388-391,1977).

2) Was there a correlation between +1 immunoreactivity and age, considering that aged nondemented patients contain some AD-like pathology?

Reply from van Leeuwen: The next important question is: what is first? neuropathology or +1 proteins? At the next meeting of the Society for Neuroscience in Los Angeles, we will present new data on this issue. A correlation between +1 immunoreactivity and age exists. As soon as UbiB+1 immunoreactivity is apparent, also some neuropathology can be seen. You can find these details on our website.

3) Have you investigated the presence of other mutant transcripts not associated with AD that might suggest a more generalized phenomenon in these patients?

Reply from van Leeuwen: Yes, we investigated the presence of tubulin immunoreactivity in AD pathology. No staining was obtained. However, it might still be that we are dealing with a general phenomenon, and that also in these proteins dinucleotide deletions occur. In that case our choice of the epitope (to make the antibody) might not have been optimal.

Questions for the authors
1) In your previously published paper "Frameshift mutations at two hotspots in vasopressin transcripts in post-mitotic neurons" which addresses the novel mechanism of GA deletions in a GAGAG motif as a reversion phenomenon in vasopressin-null Brattleboro rats, you argue against an RNA editing mechanism and suggest that the mutations have occurred in the genome of solitary neurons. However in strong contrast, the current report on frameshift mutations associated with AD suggests that these mutations are generated via a novel RNA editing mechanism. What evidence supports either a genomic mutation in individual neurons or RNA edited transcripts?

Reply from van Leeuwen: Initially we thought that the mutations are introduced at the genomic level (see Mutation Research 338 (1995) 173-182, p. 178). RNA-editing was supposed to take place in all cells and not only in a few cells. Up till now we have no evidence which is in favor of the hypothesis of a genomic somatic mutation. We extensively studied the genomic DNA of Brattleboro rats and of AD and DS patients, but we couldn't find any mutation in the genomic sequence. In addition, we neither have evidence that the mutation is taking place at the transcript level. However, circumstantial evidence led us to conclude that we were dealing with a mutational event that occurs at the transcript level. First of all, APP+1 and Ubi+1 are colocalized and it is highly unlikely that 2 somatic mutations in 2 different transcripts occur in the same neuron. Secondly, in the Brattleboro rat we found out that substitution with vasopressin resulted in a decrease in the number of cells expressing vasopressin +1 immunoreactivity. This is most probably caused by a down-regulation in the transcription of the vasopressin gene.

2) A recent paper by Rothstein has shown that aberrant splice mutants of EAAT2 generated post-transcriptionally result in a change in the normal proteins localization and function when overexpressed in mammalian cells. Considering that neurons may express both wild type and +1 frameshift mutants, is there evidence suggesting that APP+1 or ubiquitin-B+1 may elicit a dominant negative effect on the wild-type proteins? Or perhaps a decrease in the functional APP and ubiquitin-B protein results in a recessive defect in the neurons, as you suggest in the paper?

Reply from van Leeuwen: We have no evidence for a dominant negative effect of APP+1 and Ubi-B+1. For Ubiquitin-B+1 one can imagine that the +1 protein interferes with the function of the wild-type protein. This is currently under investigation. In case of APP+1 it is more complex, as the function of the protein is not known. We started transgenesis studies and we hope that these studies will provide us with data that can give an answer on the question of the dominance of the mutation.

3) +1 protein immunoreactivity in non-AD patients as well as one non-neuropathological Down's syndrome patient demonstrated little presence of mutant protein. However patients with pathology showed abundant immunoreactivity. Could the presence of +1 proteins be a downstream effect or consequence of AD pathology? Do you speculate that a mechanism of RNA frameshift mutations is causally related to sporadic AD?

Reply from van Leeuwen: As +1 immunoreactivity coincides with neuropathology (see our website), it could as well be a cause or consequence of the neuropathology. Studies with transgenic mice might give the answer to this question. The mechanism of the frameshift mutations might be causally related to neuropathology, but it might also be possible that a failure in the mechanisms that normally degrade aberrant RNA and protein is causally related to AD. In both cases, ALS and AD a similar failure in the RNA degradation mechanism might lead to the presence of mutant RNA and might be be the cause of neurodegeneration.

4) Your previous study in Brattleboro rats suggested that wild-type rats as well as VP-/- rats exhibited the same phenomenon (albeit under opposite circumstances) of frameshift mutations. Why in the present study do normal and non-demented controls not show immunoreactivity to APP+1 or ubiquitin+1 antibodies since the phenomenon is not related to disease state as suggested by the Brattleboro rats.

Reply from van Leeuwen: Note that aged non-demented controls (>72 years) do show ubiquitinB+1 immunoreactivity (see our website). The phenomenon is related to overexpression of genes as is seen in homozygous Brattleboro rats and Down syndrome patients (see also next question).

5) You speculate that the increase in frameshifted proteins in AD and Down's syndrome brains may be a result of increased transcription of these proteins. If this is true why does your data not suggest an increase in APP+1 protein in Down's syndrome (containing trisomy of chromosome 21 and APP) versus AD patient brains?

Reply from van Leeuwen: If you look to table I of the Science paper you will notice that the percentage of DS patients showing immunoreactivity for APP+1 in the frontal and temporal cortex and the hippocampus, is much higher compared to AD patients. In fact the difference is larger if we exclude the non-demented Down syndrome patient.

6) RNA editing via single base pair changes is mediated by a family of enzymes known as dsRNA-specific editases. What proposed mechanism is involved in this novel form of frameshifted RNA editing found in AD patients? Are flanking RNA sequences important considering that exon 9 GA deletions were more prevalent than exon 10 GA deletions from APP mRNA?

Reply from van Leeuwen: The mechanism by which the mutation occurs is not clear to us. We searched for consensus sequences in the RNA flanking GAGAG-sequence, but we could not find a clear motif that occurred in APP, UbiquitinB and vasopressin. Instead of RNA-editing the dinucleotide deletion can be explained by the occurrence of RNA-polymerase slippage or stuttering. The rate of this process (GA-deletion) can be promoted by increased transcription. In the case of APP we detected more GA-deletions in exon 9 than in exon 10, this is probably due to an extended GAGAG motif: GAGAGAGA.

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References

Webinar Citations

1. Frameshift Mutants of β Amyloid Precursor Protein and Ubiquitin-B in Alzheimer's and Down's Syndrome Patients 28 May 1998

Paper Citations

1. van Leeuwen FW, de Kleijn DP, van den Hurk HH, Neubauer A, Sonnemans MA, Sluijs JA, Köycü S, Ramdjielal RD, Salehi A, Martens GJ, Grosveld FG, Peter J, Burbach H, Hol EM. Frameshift mutants of beta amyloid precursor protein and ubiquitin-B in Alzheimer's and Down patients. Science. 1998 Jan 9;279(5348):242-7. PubMed.

External Citations

1. our website

Further Reading