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

View Transcript of Live Discussion — Posted 6 September 2006

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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