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Human Molecular Genetics Advance Access originally published online on November 2, 2007
Human Molecular Genetics 2008 17(3):402-412; doi:10.1093/hmg/ddm317
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Interference with splicing of Presenilin transcripts has potent dominant negative effects on Presenilin activity

Svanhild Nornes1, Morgan Newman1, Giuseppe Verdile2,3,4, Simon Wells1, Cristi L. Stoick-Cooper5,6, Ben Tucker1, Inna Frederich-Sleptsova2,3, Ralph Martins2,3,4 and Michael Lardelli1,*

1 Discipline of Genetics, School of Molecular and Biomedical Science, The University of Adelaide, SA 5005, Australia 2 Centre of Excellence for Alzheimer’s Disease Research and Care, School of Exercise, Biomedical and Health Sciences, Edith Cowan University, Joondalup, WA 6027, Australia 3 Sir James McCusker Alzheimer’s Disease Research Unit, Hollywood Private Hospital, Nedlands, WA 6009, Australia 4 School of Psychiatry and Clinical Neurosciences, University of Western Australia, Crawley, WA 6009, Australia 5 Department of Pharmacology, Howard Hughes Medical Institute, Institute for Stem Cell and Regenerative Medicine 6 Graduate Program in Neurobiology and Behavior, University of Washington School of Medicine, Seattle, WA 98195, USA

* To whom correspondence should be addressed. Tel: +61 883033212; Fax: +61 883034362; Email: michael.lardelli{at}adelaide.edu.au

Received August 19, 2007; Revised October 8, 2007; Accepted October 25, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY
 FUNDING
 REFERENCES
 
Missense mutations in the PRESENILIN1 (PSEN1) gene frequently underlie familial Alzheimer’s disease (FAD). Nonsense and most splicing mutations result in the synthesis of truncated peptides, and it has been assumed that truncated PSEN1 protein is functionless so that heterozygotes for these mutations are unaffected. Some FAD mutations affecting PSEN1 mRNA splicing cause loss of exon 8 or 9 sequences while maintaining the reading frame. We attempted to model these exon-loss mutations in zebrafish embryos by injecting morpholino antisense oligonucleotides (morpholinos) directed against splice acceptor sites in zebrafish psen1 transcripts. However, this produced cryptic changes in splicing potentially forming mRNAs encoding truncated presenilin proteins. Aberrant splicing in the region between exons 6 and 8 produces potent dominant negative effects on Psen1 protein activity, including Notch signalling, and causes a hydrocephalus phenotype. Reductions in Psen1 activity feedback positively to increase psen1 transcription through a mechanism apparently independent of {gamma}-secretase. We present evidence that the dominant negative effects are mediated through production of truncated Psen1 peptides that interfere with the normal activity of both Psen1 and Psen2. Mutations causing such truncations would be dominant lethal in embryo development. Somatic cellular changes in ageing cells that interfere with PSEN1 splicing, or otherwise cause protein truncation, might contribute to sporadic Alzheimer’s disease, cancer and other diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY
 FUNDING
 REFERENCES
 
Alzheimer’s disease (AD) is the most prevalent form of degenerative dementia and is characterized by generalized brain atrophy beginning in the medial temporal lobes and affecting memory and cognition. At the histological level, extracellular condensations of Amyoid β peptide (Aβ plaques) and intraneuronal accumulations of tau protein as neurofibrillary tangles (NFTs) are observed. The neural degeneration observed in the related condition, frontotemporal dementia (FTD) is more localized to frontal and temporal brain regions and affects, primarily, behaviour and language. Pick’s disease is a familial form of FTD characterized by ovoid, intraneuronal accumulations of tau protein named Pick bodies. Mutations in the gene PSEN1 have been linked to familial forms of AD and FTD including Pick’s disease (reviewed in 1).

Of the 165 known dominant PSEN1 mutations causing familial AD (FAD) or FTD, almost all are missense mutations that do not truncate the protein. One mutation, PSEN1 delta 4, affects the exon 4 splice donor site producing mRNAs encoding inactive 7 kDa peptides. However, this mutation also produces mRNAs encoding full-length protein with an additional amino acid inserted after amino acid 113 (2). A mutation of the splice donor site of PSEN1 exon 6 causing FTD shows similar characteristics—exon 6 is lost from some transcripts that consequently encode truncated, putatively inactive proteins but full-length transcript is also produced encoding PSEN1 protein with an altered amino acid sequence (G183V). It is uncertain whether the FTD is due to production of truncated or altered full-length proteins (3). The PSEN1 L271V mutation increases production of transcripts lacking exon 8 (a naturally occurring isoform). However, the reading frame is maintained and, when translated, forms a protein lacking {gamma}-secretase activity but which interacts with normal PSEN1 protein to influence Aβ1–42 production (4). PSEN1 exon 9 can also be lost from transcripts by mutation of the exon 9 splice acceptor site or exon deletion. This does not cause a frameshift and produces a protein showing {gamma}-secretase activity without endoproteolysis and that raises the Aβ42:Aβ40 ratio (4,5). All other known presenilin mutations are missense. Therefore, in almost all PSEN1 (and PSEN2) mutations, the C-terminal sequence of the protein is retained.

There is evidence for aberrant splicing of presenilins in sporadic forms of AD (SAD). Loss of PSEN2 exon 5, generating a truncated form of PSEN2 protein, has been observed in SAD (6,7). Furthermore, aberrant splicing of PSEN1 has been reported in cases of FTD (8). There is also evidence from conditional deletion of Psen1 in mouse epidermis that presenilin activity is required for tumour suppression (9).

The failure to recover nonsense mutations truncating PSEN1 proteins in FAD or FTD led to the suggestion that such mutations represent null alleles without dominant activity (1). However, this hypothesis has not been tested experimentally.

Zebrafish possess orthologues of the human PSEN1 and PSEN2 genes denoted psen1 and psen2, respectively (10,11). Here we use zebrafish embryos to analyse the effects of interference with normal presenilin splicing. We find that low-level aberrant splicing of psen1 transcripts in the region between exons 6 and 8 produces potent dominant negative effects that increase psen1 transcription, suppress Notch signalling and cause a dramatic hydrocephalus phenotype. These effects appear to be mediated by production of truncated forms of Psen1 protein that interfere with both Psen1 and Psen2 function. The potency of the dominant negative effects and the occurrence of aberrant splicing in some forms of dementia and cancer suggest that somatic changes in presenilin splicing may play a role in the pathogenesis of SAD, FTD and other diseases.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY
 FUNDING
 REFERENCES
 
Inhibition of normal psen1 splicing or translation stimulates psen1 transcription through a {gamma}-secretase-independent mechanism
We attempted to alter splicing of psen1 transcripts in zebrafish embryos to model human PSEN1 mutations causing loss of exon 8 sequences but found that a morpholino blocking the splice acceptor site for exon 8 on psen1 transcripts (Mo8Ac) was inefficient and caused a low level of inclusion of intron 7 (Fig. 1B and C). To quantify the effect of this morpholino on the level of normally spliced psen1 mRNA, we used quantitative RT–PCR (QRT–PCR). This showed, unexpectedly, almost a 2-fold increase in psen1 mRNA when splicing to exon 8 was partially inhibited (Fig. 1D). Similarly, a 2- and 3-fold increase, respectively, was observed when translation of Psen1 protein was blocked using a morpholino binding over the start codon of psen1 mRNA (MoTln) or the splice acceptor site of exon 7 (Mo7Ac, Fig. 1D). The QRT–PCR on MoTln-injected embryos was conducted on sequence within intron 11 (Fig. 1B) supporting that the observed increases in mRNA abundance are not due to increased mRNA stability. Together, these results indicate that aberrant splicing of psen1 transcripts after exon 6 or loss of Psen1 protein apparently increase psen1 transcription.


Figure 1
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  		Figure 1. Effects of morpholinos on psen1 transcription. Morpholinos are indicated by names without their ‘Mo’ prefix except for MoCont which is indicated by ‘Co’. (A) Diagram approximately indicating the areas of Psen1 protein encoded by psen1 exons. The N-terminus (N) lies in the cytosol and the C-terminus (C) may be luminal or extracellular. Alternating thick and thin lines and accompanying numbers indicate which exons contribute to which areas of the protein. The approximate positions of the 200th codon (200), endonuclease cleavage site (arrowhead), aspartate residues critical for substrate proteolysis (D) and C-terminal polyclonal antibody binding domain (line adjacent to exon 10-encoded sequence) are indicated. (B) Diagram (not to scale) showing an area of psen1 exon/intron structure. Boxes enclosing numbers indicate numbered exons. Thin connecting lines indicate introns. Morpholino binding sites are indicated by thick black lines. Arrowheads indicate sites of PCR primer binding with primer names italicized. A ‘Q’ prefix indicates primers used in QRT–PCR. Thin black lines connect paired PCR primers. Thin boxes with numbers indicate areas sequenced from cloned PCR fragments and clone designations respectively (see Supplementary Material, Data S1 for sequence data). (C) RT–PCR on embryos injected with morpholinos (at 0.5 mM as per Fig. 3). Primers used in PCRs are indicated below electrophoresis images. Black arrowheads indicate PCR product size from normally spliced psen1 mRNA. Numbers and lines beside images indicate DNA fragment sizes (in kb). White letters indicate products of aberrant splicing. (D) Histogram of QRT–PCR analysis quantifying psen1 transcription after morpholino injection (at 0.5 mM as per Fig. 3) relative to control only (MoCont-injected) embryos. ef-1{alpha} used as a reference standard. Statistical significance is indicated above the columns.

 
To determine whether the apparent increase in psen1 transcription after loss of Psen1 protein might be due to a feedback mechanism dependent upon {gamma}-secretase activity, we used QRT–PCR to determine the levels of psen1 mRNA in embryos at 48 h post-fertilisation (hpf) that had been treated for 42 h with the {gamma}-secretase inhibitor DAPT (12,13). No consistently significant changes in the abundance of psen1 mRNA were observed (as there was considerable variability between replicates). Similar results were obtained for QRT–PCR analysis of human PSEN1 mRNA in DAPT-treated HEK-293 cells (14) (see Materials and Methods and Supplementary Material, Data S2). Thus, the apparent stimulation of psen1 transcription by loss of Psen1 protein may occur through a mechanism independent of {gamma}-secretase activity.

Interference with psen1 splicing causes hydrocephalus by a potent dominant negative mechanism
The limited interference with splicing to exon 8 caused by Mo8Ac causes a range of developmental phenotypes indicative of changes in Notch signalling and including a dramatic hydrocephalus phenotype in zebrafish embryos at 48 hpf (Fig. 2 and Table 1). Injection of a negative control morpholino (MoCont) has no effect while injection of Mo7Ac also produced hydrocephalus (Fig. 2 and Table 1). The sequences of MoTln, Mo7Ac and Mo8Ac are completely unrelated indicating that this phenotype does not result from non-specific activities of these oligonucleotides. The specificity of these morpholino effects is supported further by our observations of similar phenotypic and splicing effects after injection of morpholinos interfering with the splice acceptor sites of exons 7 and 8 of the psen2 gene and the fact that Mo8Ac injection does not alter splicing of the unrelated gene cyclin G1 (ccng1, data not shown).


Figure 2
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  		Figure 2. Phenotypic effects of loss of Psen1 activity. (A) Wild-type embryo (upper) and embryo with strong Psen1-loss phenotype (lower) at 48 hpf. Arrowheads indicate expanded hindbrain and midbrain ventricular spaces. The yolk extension (indicated by bar labelled ye on normal embryo) is absent and the yolk ball (y) is increased in size. Melanocytes are fewer in number and smaller in size such that overall pigmentation appears decreased. The ‘head angle’ (the intersection of the eye-to-otic vesicle axis with the notochord axis) is increased and the spinal cord (sc) is thinner. Overall, the Psen1-loss embryo is smaller than wild type. (B and C) Lateral views of wild type and Psen1-loss embryos respectively at 28 hpf in the region of the yolk extension (rostral to left). Embryos have been subjected to in situ transcript hybridisation with a neurog1 probe. More darker staining cells are seen in the Psen1-loss embryo and the yolk extension (ye in wild-type embryo) is absent. The position of the cloaca is marked with an asterisk. (D and E) Melanocytes on the lateral yolk ball at 48 hpf in wild type and Psen1-loss embryos, respectively. The single melanocyte in (D) is much larger than the two melanocytes in focus in (E). Size bars indicate 100 µm.

 


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  		Table 1. Results of morpholino and mRNA injections (Supplementary Data S3 and 4)

 
RT–PCR using primers spanning exons 6–10 revealed only a low level of aberrant splicing due to Mo8Ac and none due to Mo7Ac (Fig. 1B and C). However, intron 6 is over 4 kb in length and, if Mo7Ac results in inclusion of this intron into mRNA, it would be difficult to detect by RT–PCR using primers in flanking exons. We subsequently performed RT–PCR using a primer pair binding in exon 6 and within intron 6 (primers 1F and 4R, respectively; Fig. 1B). This revealed relatively high levels of RNA incorporating intron 6 sequence in embryos injected with Mo7Ac compared with those injected with MoCont alone (Supplementary Material, Data S5).

Inhibition of psen1 mRNA translation by MoTln produces a decrease in Notch signalling [as shown by increased neurogenin1 (neurog1) expression], reduction in the width of the yolk extension and hydrocephalus indicating that these are loss-of-function phenotypes (Fig. 2 and Table 1). This also indicates that Mo7Ac and Mo8Ac significantly reduce Psen1 function despite not reducing the levels of normal psen1 mRNA. Thus, the apparently low levels of aberrant splicing caused by Mo7Ac and Mo8Ac must act in a dominant negative fashion to reduce Psen1 activity (Table 1). The loss of function produced by Mo7Ac and Mo8Ac is also significantly more severe than that seen after translation blockage at least in terms of the easily quantifiable yolk-extension phenotype (Supplementary Material, Data S6). To confirm this dominant negative action, we titrated the effective concentrations of MoTln, Mo7Ac and Mo8Ac and observed the effects on Psen1 protein by western blotting (Fig. 3A and C). Injection of MoTln results in dose-dependent loss of the C-terminal fragment (CTF) of Psen1 that correlates with the hydrocephalus phenotype. In contrast, injection of Mo7Ac or Mo8Ac produces no visible effect on Psen1 CTF levels, even at concentrations 5- and 2.5-fold above their minimum effective concentrations, respectively (Fig. 3A and C). Western blots using our antibody against the CTF of Psen1 failed to reveal Psen1 holoprotein at 48 hpf, nor did they reveal any changes in the higher molecular weight complexes incorporating CTF protein sequence in embryos injected with Mo7Ac, Mo8Ac or MoCont (data not shown). We have not been able to observe the effects of these morpholinos on the N-terminal fragment (NTF) of zebrafish Psen1 since there is currently no antibody available.


Figure 3
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  		Figure 3. Analysis of Psen1 protein expression in morpholino-injected embryos. Embryos were injected with negative control morpholino MoCont alone (Cont) or in a mixture with experimental morpholinos affecting psen1 transcript splicing or translation (indicated by names lacking the ‘Mo’ prefix) at a constant total concentration of 1 mM. (numbers indicate mM of experimental morpholino in the mixture.) Embryos were harvested at 48 hpf and lysate from 10 embryos per gel lane was subjected to SDS–PAGE and blotted before probing with an antibody against the CTF of Psen1. Gels were stripped and re-probed with an antibody against β-Tubulin as a loading control. Asterisks indicate the lowest morpholino concentrations at which a Psen1-loss phenotype was seen. (A) Nearly, all Psen1 CTF must be lost before a phenotype is seen when translation is blocked with MoTln but no Psen1 CTF loss is apparent at concentrations of Mo7Ac five times greater than that required to produce a phenotype. (B) Visible loss of Psen1 CTF is seen after injection at 0.5 mM with MoTln, Mo6Do and Mo9Ac but not after injection with Mo7Ac and Mo8Ac (or 1 mM MoCont). The Psen1 CTF loss after injection of Mo9Ac is insufficient to produce a Psen1-loss phenotype. (C) Titration to find the lowest concentrations of Mo6Do and Mo8Ac that can produce a Psen1-loss phenotype. Mo6Do must cause a near total loss of Psen1 CTF before a phenotype is seen. Injection of Mo8Ac at 0.5 mM (a concentration 2.5x that required to produce a phenotype) has no discernible effect on Psen1 CTF levels compared with MoCont-injected embryos.

 
A morpholino modelling Pick’s disease mutation G183V has no apparent dominant negative effect
Since interference with splicing after exon 6 potently inhibits normal Psen1 function and causes hydrocephalus we predict that mutations causing these effects in humans would be dominant lethal. This would explain why such mutations have never been recovered in human pedigrees. However, the viable G183V mutation of human PSEN1 affects the splice donor site of exon 6 causing loss of exon 6 from some transcripts (3). This implies that interference with splicing before exon 6 may not have dominant negative/lethal effects. We attempted to model the Pick’s disease mutation in zebrafish by injection of a morpholino, Mo6Do, binding to the splice donor site of exon 6 in psen1 transcripts. RT–PCR analysis showed that this produces significant interference with splicing out of introns 5 and 6 (Fig. 1C). Mo6Do injection can reduce Psen1 activity sufficiently to cause hydrocephalus (Table 1). However, titration of Mo6Do and western blotting indicates that the loss of Psen1 activity occurs through simple loss of Psen1 protein rather than a dominant negative effect (Table 1 and Fig. 3C). Similar results were obtained using a morpholino, Mo9Ac, to block the splice acceptor site of exon 9. Mo9Ac causes failure to splice out intron 8 in a large proportion of transcripts (Fig. 1B and C). However, in contrast to Mo6Do, injection of Mo9Ac does not reduce Psen1 protein levels sufficiently to cause hydrocephalus and there is no observable dominant negative effect (Table 1 and Fig. 3B). Thus, the dominant negative effects produced by interference with splicing of psen1 transcripts appear to require the inclusion of exon 6 sequence and the exclusion of exon 8 sequence.

The dominant negative effects of splicing interference are due to formation of truncated Psen1 peptides
Most aberrant splicing of psen1 transcripts would be expected to produce mRNAs coding for truncated peptides. In analyses of protein structure, it is commonly observed that truncated proteins can bind to a subset of the normal interacting partners and so dominantly suppress normal protein function. Therefore, we tested whether truncation of Psen1 proteins might produce dominant negatively acting peptides. Interference with splicing to the acceptor site of exon7 is expected to produce Psen1 peptides truncated after exon 6 sequence. We synthesized mRNA in vitro corresponding to Psen1 truncated after exon 6 (mRNA{Delta}exon7+) and injected this into zebrafish embryos at the 1-cell stage. We then observed the effects on embryos at 28 hpf (neurog1 expression), 28–30 hpf (yolk extension) and 48 hpf (brain ventricles, Table 1).

Injection of mRNA{Delta}exon7+ did not consistently produce hydrocephalus. However, mRNAs injected into zebrafish embryos are far less stable than morpholinos so this might be due to premature mRNA breakdown. We were able to observe a highly significant increase in neurog1 expression after injection of mRNA{Delta}exon7+ (relative to injection of the functionless mRNA, mRNAcont, P << 0.001, Table 1). This supports that the dominant negative effect of aberrant splicing is due to the production of truncated peptides. Consistent with these observations, injection of an mRNA coding for full-length Psen1 protein (mRNAfulllength, engineered so as not to bind MoTln) can rescue the effects of Psen1 translation blockage on neurog1 expression at 28 hpf but not hydrocephalus at 48 hpf (Table 1). However, injection of mRNAfulllength is unable to rescue the effects of Mo8Ac on neurog1 expression (Table 1). This supports the dominant negative character of the psen1 loss of function caused by Mo8Ac.

Effects of aberrant psen1 splicing on dorsal longitudinal ascending neuron number support dominant negative interference with Psen2 function
Interference with the splice acceptor sites of psen1 exons 7 and 8 produces a higher proportion of embryos with a severe loss-of-function phenotype than simple blockage of Psen1 translation (Table 1 and Supplementary Material, Data S6). In mice, Psen1 and Psen2 have partially redundant activities (15) and their similar structures imply the possibility of interaction. Thus, the severe phenotypes produced by Mo7Ac and Mo8Ac injection suggest that Psen1 protein truncated in the region between exons 6 and 8 may interfere with normal function of both Psen1 and Psen2. Suppression of Psen2 protein translation, but not Psen1 translation, produces a highly significant increase in the number of dorsal longitudinal ascending (DoLA) interneurons (manuscript in preparation). Therefore, we asked whether the dominant negative effects of aberrant psen1 splicing could alter DoLA neuron number. DoLA number was assessed at 30 hpf after injection of Mo7Ac, Mo8Ac, MoTln, or a morpholino blocking Psen2 translation, MoPS2Tln (Fig. 4 and Supplementary Material, Data S7 and 8). As expected, inhibition of Psen1 translation with MoTln (denoted ‘PS1’ in Fig. 4) did not significantly change DoLA number (a slight decrease was observed). However, Mo7Ac and Mo8Ac produced highly significant increases in DoLA number similar to blockage of Psen2 translation. This supports that Psen1 protein truncated in the region between exons 6 and 8 can interfere with, and inhibit, Psen2 activity.


Figure 4
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  		Figure 4. Effects on DoLA neuron number of blockage of Psen1 and Psen2 activity. Mean DoLA numbers in embryos (n ≥ 52) injected with 0.5 mM morpholinos (+0.5 mM MoCont as per Fig. Fig. 3) and then identified by in situ transcript hybridization at 30 hpf with a probe against tbx16 transcripts. Cont, PS2, PS1, 7Ac and 8Ac indicate embryos injected with MoCont, MoPS2Tln, MoTln, Mo7Ac and Mo8Ac, respectively. The significance of differences in mean DoLA number between embryos injected with MoCont and the other morpholinos (P-values from a two-tailed t-test) is given above each column. ns, not significant. Blockage of Psen1 translation has no significant effect on DoLA number but interference with psen1 transcript splicing by Mo7Ac or Mo8Ac increases DoLA number in a similar manner to blockage of Psen2 translation. See also Supplementary Material, Data S7.

 
Loss of Psen1 function does not appear to disturb formation or behaviour of ventricular cilia
Hydrocephalus in zebrafish and mouse embryos has been reported as resulting from interference in the structure and function of ventricular cilia. Ventricular cilia are clearly visible in the central canal of the spinal cord of living zebrafish embryos at 48 hpf (16). We examined these cilia in embryos injected with Mo7Ac or with negative control MoCont. We did not observe any obvious effect on the length, density or motion of cilia in this region (n = 4 for each treatment; Supplementary Material, Data S9 and 10). Thus, the hydrocephalus effect produced by loss of Psen1 function may be by a mechanism other than disturbance of cilial function. Interestingly, blockage of {gamma}-secretase activity by treatment of embryos with the drug DAPT does not produce hydrocephalus (data not shown) implying that the hydrocephalus phenotype is caused by loss of a presenilin activity independent of its role in {gamma}-secretase activity. This is consistent with the failure of DAPT to affect the cilia of Kuppfer’s vesicle, a structure involved in left–right patterning in zebrafish (17). Work in mammalian cells has shown that the influence of Psen1 on β-catenin-mediated transcription is independent of its role in {gamma}-secretase (18) and β-catenin is known to play a role in the function of some cilia (19,20). Zebrafish bearing the TOPdGFP transgene can be used to assay changes in β-catenin/Lef1-mediated transcription, e.g. following β-catenin phosphorylation (21). To date, we have not observed any consistent changes in β-catenin/Lef1-mediated transcription following injection of morpholinos altering psen1 activity at the limits of resolution available with the TOPdGFP system (data not shown). TOPdGFP reports β-catenin-mediated transcription through the Lef1 promoter, but not via derepression of TCF, so it is possible that our psen1 morpholinos alter β-catenin-mediated transcription in a manner not detectable with this system. This is consistent with observations in mammalian cells where Presenilin1 has been shown to negatively regulate β-catenin/TCF-mediated transcription through its interaction with plakoglobin ({gamma}-catenin) (22).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY
 FUNDING
 REFERENCES
 
PSEN1 is the major locus for mutations causing FAD. Although a great number of dominant, missense mutations have been characterized widely spread throughout the open-reading frame of this gene, over 70% of AD mutations in PSEN1 and PSEN2 fall in exons 5–8 (encoding the second to fifth transmembrane domains) indicating a critical role of this region in presenilin function. Mann et al. (23) showed that AD caused by mutations in PSEN1 before codon 200 is frequently characterized by the existence of few densely cored amyloid plaques and mild to moderate vascular amyloid deposition (amyloid angiopathy), whereas mutations after codon 200 produce increased numbers of these plaques that are larger in size and are associated with vessels showing severe amyloid angiopathy. Codon 200 is found early in exon 7 supporting that disruption of Psen1 protein structure after exon 6 has more severe effects on presenilin function.

The failure to find mutations in PSEN1 causing aberrant splicing or protein truncation after exon 6 in human AD or FTD pedigrees has previously been assumed to be due to these mutations causing loss of function with no effect on {gamma}-secretase activity. Indeed, our experiments blocking Psen1 function show that this might lead to dosage compensation by upregulation of psen1 transcription. However, we have also shown that interference with psen1 splicing in the region between exons 6 and 8 causes a potent dominant negative effect that results in apparent suppression of Notch signalling indicating a reduction in presenilin activity. This may occur through production of low levels of truncated proteins that potently interfere with normal presenilin function. De novo mutations causing such changes would be expected to be dominant lethal during human embryogenesis since changes in presenilin function and Notch signalling disturb cell differentiation and embryo development and are lethal in mice (24–26). The dramatic hydrocephalus we observed in zebrafish also suggests that de novo mutations causing truncation of PSEN1 protein might account for a proportion of human congenital hydrocephalus.

The potently dominant negative effects of interference in presenilin splicing suggest that this might be important in sporadic forms of disease. Evin et al. (8) noted aberrant splicing of PSEN1 in apparently sporadic cases of FTD. These authors did not observe aberrant splicing of human PSEN1 in SAD. However, the potency of apparently slight changes in splicing in the region between exons 6 and 8 of zebrafish psen1 suggests that even low-level changes in splicing in these regions might have an effect. Also, our experiments show that the levels of aberrant presenilin gene splicing required for dominant negative action may be difficult to detect using RT–PCR for a number of technical reasons (such as failure to splice out large introns from transcripts). PSEN1 has also been identified as a tumour suppressor in mammalian skin, possibly via its action in phosphorylating and destabilizing β-catenin (9,27), although Deng et al. (28) presented evidence that this was probably due, instead, to decreased Notch signalling. Aberrant splicing is commonly found in cancer (29). Intriguingly, Sato et al. (30) showed that β-catenin can interact with proteins regulating splicing and suggested that activation of β-catenin might contribute to the aberrant splicing seen in human cancers. Since loss of presenilin activity can reduce phosphorylation of β-catenin and increase its transcription activation activity (27), dual positive feedback loops are possible whereby aberrant splicing generates dominant suppression of PSEN1 activity that subsequently increases both β-catenin activity and PSEN1 transcription to further increase aberrant splicing and the dominant negative effect. We believe it likely that somatic changes altering PSEN1 transcript splicing will be found to contribute to the progression of some tumour types.

Interference with splicing of presenilin transcripts affects multiple presenilin functions
The presenilin proteins have multiple molecular functions, at least some of which are independent of their role in {gamma}-secretase activity. For example, N-terminally truncated forms of PSEN2 have been shown to inhibit Fas- and tumor necrosis factor alpha-induced apoptosis (31,32). Also, presenilins form calcium channels in the endoplasmic reticulum and AD mutations in PSEN1 apparently increase calcium flux (reviewed in 33). As described above, presenilins can also affect phosphorylation of β-catenin. The increase in neurog1 expression observed by interference in presenilin splicing or injection of mRNA encoding truncated presenilin proteins is most likely due to a decrease in Notch signalling through decreased {gamma}-secretase activity (34–36). The observed decrease in pigmentation is partially explained by reduced formation of melanocytes due to reduced differentiation of trunk neural crest cells. This is also probably an effect of loss of Notch signalling (Nornes et al., manuscript in preparation) (37).

While blockage of presenilin function by morpholino injection produces hydrocephalus, treatment of embryos with DAPT to block {gamma}-secretase activity does not produce this phenotype. This suggests that the hydrocephalus effect of DAPT treatment may not result from decreased {gamma}-secretase activity but, rather, a decrease in another of presenilin’s molecular activities. One possibility is that decreased presenilin activity may decrease the association between cadherin and PI3K giving decreased PI3K/Akt signalling, and thus reducing repression of GSK3β activity (38). These authors found that promotion of PI3K/Akt signalling by PSEN1 is not sensitive to inhibitors of {gamma}-secretase. Interestingly, reduced PI3K/Akt signalling would also reduce the PI3K/Akt cell survival signal and might contribute to neural cell death (although no large increase in cell death has been observed upon loss of presenilin activity in zebrafish) (36).

Are changes in presenilin activity the common molecular link between SAD and FAD?
While the great majority of AD cases are sporadic, mutations in PSEN1 account for the majority of FAD cases in which the mutant gene has been identified (39). Sporadic AD and FAD show similar histological pathologies (e.g. amyloid plaques and NFTs). However unlike FAD, SAD is associated with a number of genetic, hormonal and environmental risk factors (reviewed in 40). The genetic predisposition to FAD has allowed more insight into molecular mechanisms underlying the disease pathogenesis.

The wide dispersal of dominant FAD mutations throughout the PSEN1 protein has long represented an enigma for understanding the function of PSEN1 in {gamma}-secretase activity. Recently, it has been proposed that FAD-associated mutations may result in a reduction in presenilin activity (‘loss of function’) resulting in a change of the proportion of cleavage events occurring at alternative sites in APP to favour production of Aβ42 over Aβ40 (reviewed in 41,42). Loss of one copy of the Psen1 gene in a mouse model of AD does not promote AD pathology (43) implying that the heterozygous missense mutations in PSEN1 that promote FAD reduce PSEN1 activity by a dominant negative mechanism (i.e. they interfere with the activity of normal PSEN1 protein). Our results support this idea in a number of ways. First, Mo7Ac and Mo8Ac appear to reduce Psen1 activity without affecting the normal concentration of Psen1 CTF implying a dominant negative mechanism. Second, the dominant negative effects of Mo7Ac and Mo8Ac injection appear to extend to suppression of psen2 activity, raising the possibility that FAD mutations in human PSEN1 might also interfere with PSEN2 activity. Third, Mo9Ac produces dramatic effects on psen1 transcript splicing (Fig. 1C) but only mild effects on Psen1 CTF levels (Fig. 3B) and no obvious Psen1-loss phenotype. This indicates that mechanisms exist to maintain, robustly, stable Psen1 protein levels in the face of variable gene expression. Indeed, severe disruption of Psen1 protein levels by MoTln or Mo6Do is required before a phenotype becomes evident (Fig. 3A and C).

While the protein products of the various PSEN1 FAD mutant alleles might dominantly suppress the activity of normal PSEN1 proteins, they do not lack PSEN1 activity completely since transgenic mice expressing only the human PSEN1 FAD mutant A246E (and not endogenous mouse Psen1) possess sufficient {gamma}-secretase activity for embryo development (44,45) while those lacking Psen1 activity die (24). However missense mutants of PSEN1 lacking all {gamma}-secretase activity have been generated. These mutations were generated in the two aspartate residues, D257 and D385, that are thought to form part of the catalytic site for substrate proteolysis and are coded within exons 8 and 10 (Fig. 1A) (46). Expression of a Presenilin protein in which either of these two aspartate residues is mutated to alanine (i.e. D257A and/or D385A) decreases {gamma}-secretase activity in cultured cells (46) by inhibiting the endoproteolysis of endogenous Presenilin holoproteins and the incorporation of NTF and CTF into higher molecular weight complexes (47). The potent dominant negative activity of Psen1 when truncated in the region between that coded by exons 6 and 8 may be due to formation of what is, essentially, a truncated NTF. This may be competent to incorporate into higher molecular weight complexes to disrupt the NTF:CTF heterodimers of active {gamma}-secretase. A truncation of Psen1 after exon 8 sequence would create an NTF lacking as few as two amino acid residues of the normal NTF sequence (48) and might permit {gamma}-secretase activity. Confirmation of this model will require development of an antibody (or tagged protein) to allow detection, specifically, of the NTF of zebrafish Psen1.

The idea that reduction in PSEN1 activity might underlie FAD, raises the possibility that reduction in PSEN1 activity through somatic cellular changes might also result in pathological changes in APP cleavage and promote the development of SAD. Our research provides insight into a mechanism through which reductions in PSEN1 activity may occur in otherwise genetically normal somatic cells. Aberrant splicing of presenilin transcripts in the region between exons 6 and 8 or the production of truncated presenilin proteins by other means have the potential to affect PSEN1 activity, raising the question of whether such events might play a role in SAD. Indeed, changes in splicing are frequently observed in aged cells (reviewed in 49) and in cancer (reviewed in 50). Alterations in gene splicing are also observed in myotonic dystrophy due to sequestration of splicing regulators by transcripts of expanded trinucleotide repeat sequences (reviewed in 51). Since Notch signalling is required for maintenance of the muscle cell progenitor pool (52), we can speculate that changes in PSEN1 transcript splicing in the cells of myotonic dystrophy patients might reduce {gamma}-secretase activity and Notch signalling and contribute to the diverse symptoms of this disease. Future work should validate that truncation of human PSEN1 protein can have potent dominant negative effects and should examine whether such truncations occur in these diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY
 FUNDING
 REFERENCES
 
This work was conducted under the auspices of animal ethics committees of The University of Adelaide, Edith Cowan University and the University of Washington School of Medicine.

Morpholino and mRNA injection
Morpholinos were synthesized by Gene Tools LLC (Corvallis, OR, USA) and are listed in Table 2. cDNAs for mRNA production were engineered using PCR on a psen1 cDNA clone containing the entire open-reading frame. These were designed to include an optimal Kozac’s sequence (5'-GCCACCATG-3') at the upstream end and were ligated into vector pXT7 (53) between the Xho I and Spe I sites. Construct mRNAfulllength includes the entire psen1 open-reading frame. mRNA{Delta}exon7+ includes all codons to the end of exon 6 sequence followed by a stop codon. These constructs were restricted with XbaI before transcription using the mMessage mMachine T7 Ultra kit (Ambion Inc., Austin, TX, USA) to generate capped mRNA. The negative control mRNA construct, mRNAcont, consists of the full-length of the psen2 open-reading frame mutated such that the fourth codon is altered to a stop codon; see Nornes et al. (54) for details. All mRNAs were precipitated with LiCl and then redissolved in water for injection.


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  		Table 2. PCR primer and morpholino sequences

 
Fertilized zebrafish eggs were rinsed in embryo medium (55) and injected before cleavage. To ensure consistency of morpholino injection, eggs were always injected with solutions at 1 mM total concentration. Dilutions of experimental morpholinos were carried out by mixing these with standard negative control morpholino, MoCont. This ensured that phenotypic variation observed was not due to change in the total injection concentration. The maximum concentration of experimental morpholino injected was always 0.5 mM (i.e. a mixture of 0.5 mM MoCont + 0.5 mM experimental morpholino). Titration of morpholinos to find effective concentrations is shown in Supplementary Material, Data S3. Messenger RNAs were injected at 200 ng/µl. When an embryo was injected with both morpholino and mRNA, the morpholino was injected first followed by mRNA injection before first cleavage.

Western analysis
Procedures for western blotting of zebrafish embryos using polyclonal antibodies against Psen1 CTF, Psen2 NTF and β-tubulin have been described previously (54).

Isolation of RNA, reverse transcription and RT–PCR
RNA was extracted from morpholino-injected embryos at 48 hpf using the ‘QIAGEN RNAeasy Mini Kit’ (QIAGEN, Hilden, Germany) and precipitated with LiCl (Ambion). Quality and concentration were checked by electrophoresis on a 1% agarose/Tris–borate–EDTA gel prior to reverse transcription.

For cDNA production, whole cell RNA at up to 1 µg/µl was reverse transcribed with SuperScript III Reverse Transcriptase (Invitrogen Corporation, Carlsbad, CA, USA) according to manufacturer’s instructions.

To detect aberrant splicing products after injection of MoTln, Mo7Ac, Mo8Ac and Mo9Ac, primers spanned exons 6–10, and for Mo6Do, primers spanned exons 5 and 6 and 6 and 7. Primers were synthesized by GeneWorks Pty Ltd (Adelaide, Australia) and Proligo (Boulder, Colorado, USA). A 25 µl PCR reaction including 2 µl of cDNA, 1 µl of each gene-specific primer (10 µM), 2 µl 10 mM dNTPs, 1 µl 2 U/µl Dynazyme DNA polymerase (Finnzymes Oy, Espoo, Finland) was performed for 30–35 cycles with a temperature of 94°C for 30 s, 55–60°C for 1 min and 72°C for 2 min on a PTC-200, Peltier Thermal cycler (MJ Research Inc., Waltham, MA, USA). Products were resolved by electrophoresis on 1–2% agarose/Tris–Acetate–EDTA gels. Products of interest were excised from the gel and purified using the ‘QIAGEN Minelute gel extraction kit’ (QIAGEN).

Sequencing analysis of splicing isoforms
Purified amplified cDNA products were cloned in the pGEMT easy vector system (Promega Corp., Madison, WI, USA) for Cycle Sequencing using M13 primers and Big Dye Terminator Mix (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s instructions. The products were analysed on an ABI DNA sequencer (Applied Biosystems).

Quantitative PCR
The relative standard curve method for quantification was used in this study to determine the expression of experimental samples compared with a basis sample. For experimental samples, target quantity was determined from the standard curve and then compared with the basis sample to determine the fold change in expression. Gene specific primers were designed using Primer Express software (Applied Biosystems). For zebrafish embryos injected with Mo7Ac or Mo8Ac primers amplified products spanning exon 6–7 or exons 7–8 respectively. For embryos injected with MoTln, primers were designed to amplify a region in intron 11 since translation blockage might stabilize mRNAs (Fig. 1B and Table 2). For DAPT-treated embryos, primers were positioned in exon 5 (Qzpsen1F) and spanning the exon 5/exon 6 boundary (Qzpsen1R). Primers were also designed for the ubiquitously expressed control gene ef-1{alpha} (Table 2). The starting cDNA quantity was from a pool of 60–80 injected embryos for each treatment. For QRT–PCR on HEK-293 cells, PSEN1 gene specific primers were QhPSEN1F and QhPSEN1R and control gene primers were QACTBF and QACTBR amplifying cDNA from the gene ACTB (Table 2). The reaction mixture consisted of 200 ng of sample cDNA, 18 µM of forward and reverse primers and SYBR green master mix PCR solution (Applied Biosystems). For generation of the standard curve, cDNA from embryos injected with MoCont (or, for DAPT-treatment experiments, from embryos or HEK-293 cells treated with DMSO only) was diluted to 25, 50, 100 and 200 ng. Each sample and standard curve reaction was repeated five times separately for zebrafish psen1 and ef-1{alpha} except for zebrafish and HEK-293 cell DAPT treatment experiments where three repeats were performed. Amplification conditions were 2 min at 50°C followed by 10 min at 95°C, and then 40 cycles of 15 s at 95°C and 1 min at 60°C. Amplification was performed on an ABI 7000 Sequence Detection System (Applied Biosystems) using 96 well plates. Cycle thresholds obtained from morpholino-injected or DAPT-treated embryos were averaged and normalized against the expression of ef-1{alpha}. Cycle thresholds obtained from DAPT-treated HEK-293 cells were averaged and normalized against the expression of ACTB. When five repeats were conducted, the highest and lowest repeats were eliminated from the final analysis and each treated sample was compared with the control sample resulting in fold changes of expression.

Treatment of zebrafish embryos and HEK-293 cells with DAPT
Zebrafish embryos were collected at fertilization and groups of 20–25 embryos were placed in individual wells of a microtitre tray with 1 ml per group of E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4). At 6 hpf, one group was left untreated, one group was treated with 1% DMSO in E3 medium and a third group was treated with 100 µM DAPT in 1% DMSO in E3 medium. Embryos were harvested for RNA purification and QRT–PCR at 48 hpf.

HEK-293 cells were maintained in DMEM containing 5% (v/v) fetal calf serum, 2 mM L-glutamine, 10 mM D-glucose, 50 IU/ml penicillin, 50 µg/ml streptomycin. Cells were subcultured into six-well plates, 24 h before treatment, at a density of 1 x 105 cells/well. The cells were either left untreated, treated with vehicle (DMSO), or the {gamma}-secretase inhibitor, DAPT (0.5 or 5 µM) for 18 h at 37°C. Following treatment, cells underwent purification of RNA which was converted to cDNA for QRT–PCR using the FastLane Cell cDNA kit (QIAGEN) according to the manufacturer’s instructions.

In situ transcript hybridization and statistical analysis
Embryos were fixed in 4% formaldehyde in embryo medium before manual dechorionation. Whole mount in situ transcript hybridization was then performed essentially as described by (56) using probes against tbx16 (57) and neurog1 (58). Where neurog1 expression was to be compared in embryos injected with different morpholinos, the tips of tails were cut off fixed embryos from one treatment (to aid later identification) and then the embryos from the two treatments were pooled prior to the start of the whole mount in situ hybridization procedure. To compare the intensity of neurog1 staining between embryo treatments, an embryo from one treatment judged to be of moderate staining was used in side-by-side comparisons with all other embryos from both treatments. These were judged to fall into two groups: equal + less intense staining or more intense staining (or, for rescue experiments, equal + more intense staining or less intense staining). Since the results of each experiment were measured against an internal standard, data from replicates could be pooled before statistical analysis using a contingency chi-square test (Supplementary Material, Data S4). For counting of DoLA neurons (labelled by tbx16 expression) in embryos subject to injection of different morpholinos, embryos were mounted on slides in glycerol under raised cover slips and then examined using differential interference contrast optics (which were also used for all photomicrography). The number of cells per embryo occurring in each treatment was compared with the number found in embryos injected with MoCont using a two-tailed t-test (Supplementary Material, Data S7).

Observation of cilia
Living embryos at 48 hpf were treated with 0.17 mg/ml tricaine before mounting in a mixture of tricaine, embryo medium and methyl cellulose. Cilia were observed in the caudal extremity of the central canal of the spinal cord under 630x magnification using a Leica SP5 spectral scanning confocal microscope with associated software (Leica Microsystems GmbH, Wetzlar, Germany).


   SUPPLEMENTARY
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY
FUNDING
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY
 FUNDING
REFERENCES
 
M.L., R.M., S.N. and G.V. are supported by Project Grant 453622 from the Australian National Health and Medical Research Council. M.L. and S.N. were supported by funds from the Cancer Council of SA, the ARC Centre for the Molecular Genetics of Development and the late Douglas Anders. R.M. is supported by grants from the McCusker Foundation for Alzheimer’s Disease Research, Department of Veterans Affairs, and Hollywood Private Hospital, G.V. is generously supported by a grant from Mr Warren Milner (Milner English College—Perth, Western Australia) and Ms Helen Sewell, I.S. is supported by a grant from the Centre of Excellence for Alzheimer’s disease Research and Care. C.S.-C. is a recipient of an NIH-funded Cardiovascular Pathology Training Grant.


   ACKNOWLEDGEMENT
 
We thank Vladimir Korzh for the neurog1 cDNA clone.

Conflict of Interest statement. The authors declare no conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY
 FUNDING
 REFERENCES
 

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