Research ArticleIndependent and cooperative action of Psen2 with Psen1 in zebrafish embryos
Introduction
The presenilin genes, PSEN1 and PSEN2 were discovered as loci mutated in a large proportion of human pedigrees showing inherited early onset Alzheimer's disease. These two genes with closely related structures encode components of “γ-secretase” complexes that cleave transmembrane proteins within lipid bilayers including the Notch receptor, amyloid precursor protein, E-cadherin, Nectin1 and others [1], [2]. Presenilins are also known to act in phosphorylation of β-catenin [3], as calcium channels that allow calcium ions to flow from the ER into the cytoplasm [4] and in processing and trafficking of tyrosinase required for conversion of tyrosine into the pigment melanin [5]. In addition, presenilin1 and presenilin2 may have other undiscovered functions since they have been observed in interphase kinetochores in the nucleus [6].
All non-human vertebrates studied to date appear to have single orthologues of the two presenilin genes. Knockout of mouse Psen1 showed that this gene has essential functions during embryo development [7]. The phenotype of mice lacking Psen1 activity is very similar to that of loss of Notch1 function implying that one of the major activities controlled by Psen1 during development is Notch signalling. In contrast, mice lacking Psen2 activity are viable and fertile and show only subtle changes in lung tissue and lung haemorrhage [8], [9]. Psen2 has previously been identified as regulating apoptosis and a truncated form of Psen2 protein was shown to inhibit apoptosis in a cultured cell survival assay [10]. However, progress in understanding the function of Psen2 has been hampered by lack of an assay for its non-apoptotic activities.
Presenilin proteins exist in a variety of isoforms. The longest forms of these proteins appear to have nine transmembrane domains although the conformation has been disputed [11], [12]. The form of presenilin protein that is active in the γ-secretase complex (together with cofactors nicastrin, PEN2 and APH1) is cleaved endoproteolytically within the “cytoplasmic loop” domain to form N- and C-terminal fragments (NTF and CTF). In previous work we have identified the zebrafish orthologue of human PSEN2 [13] and we have examined protein expression and function of the zebrafish orthologue of human PSEN1 in embryogenesis [14], [15]. By injection of antisense morpholino oligonucleotides (“morpholinos”) which bind to zebrafish psen1 mRNA to inhibit translation, we were able to show that psen1 performs similar roles to that of its mouse orthologue during embryogenesis.
The function of Psen2 in zebrafish embryos has been analysed by Campbell et al. [16] in the context of its participation in the γ-secretase complex with the protein product of the zebrafish orthologue of human PEN2, psenen. These authors noted that inhibition of Psen2 translation by the morpholino “Psen2MO1” reduced expression of the gene her6 that is thought to be a target of Notch signalling in the developing retina [17]. We have previously shown that inhibition of Psen2 translation increases the number of Dorsal Longitudinal Ascending (DoLA) interneurons that differentiate in the developing spinal cord of zebrafish embryos. Since these cells are unaffected by decreased translation of Psen1 we used this phenomenon to demonstrate that truncated forms of Psen1 protein appear to have the ability to inhibit the function of Psen2 in a dominant negative manner [15].
In this paper we compare the activities of Psen1 and Psen2 in the differentiation of two cell types in zebrafish embryos, melanocytes and DoLAs. Inhibition of Psen2 (or Psen1) translation causes loss of trunk and tail (but not cranial) melanocytes and a reciprocal increase in Rohon-Beard neuron number apparently due to diminished Notch signalling. Interestingly, differentiation of another spinal cord neuron type, Dorsal Longitudinal Ascending (DoLA) interneurons, is affected in an unequal fashion by loss of Psen2 or Psen1. Psen2 loss increases DoLA number, while Psen1 loss has no effect. However, the effect on DoLA number of Psen2 loss is ameliorated by loss of Psen1. The effects of Psen2 loss on DoLA and melanocyte cell numbers provide sensitive bioassays for analysis of the non-apoptotic activity of Psen2.
Section snippets
Ethics
This work was conducted under the auspices of The Animal Ethics Committee of The University of Adelaide and in accordance with EC Directive 86/609/EEC for animal experiments and the Uniform Requirements for manuscripts submitted to Biomedical journals.
Morpholinos and RNA injection
Morpholinos were synthesised by Gene Tools (LLC, Corvallis, OR, USA) and are listed in Table 1. They were dissolved in distilled water at a concentration of 2 mM. All morpholino injections were preformed at 1 mM total morpholino concentration.
Morpholinos inhibiting Psen2 protein translation
Two morpholino antisense oligonucleotides, “Psen2MO1” [16] and “MoPS2Tln” [15], have previously been used successfully to inhibit translation of Psen2 protein in zebrafish embryos but description of their phenotypic effects has been very limited. Psen2MO1 binds over the translation start codon of psen2 mRNA while MoPS2Tln binds in a non-overlapping region of the 5′ untranslated region (5′UTR). Injection of either of these morpholinos into fertilised eggs results in similar developmental defects
Psen2 has non-redundant roles in zebrafish embryogenesis
In this paper we have analysed the effects of changes in Psen1 and Psen2 activity on the development of three zebrafish cell types, melanocytes, Rohon-Beard dorsal sensory neurons and DoLA interneurons. This revealed that Psen2 has important and non-redundant roles in development of zebrafish embryos in contrast to the results published for mice [9] (and see below). Decreased Psen2 activity causes decreased melanocyte formation and increased neuron number, apparently through changes in Notch
Acknowledgments
This work was supported by a project grant from the National Health and Medical Research Council of Australia (453622), the Cancer Council of South Australia (S 24/04), the ARC Special Research Centre for the Molecular Genetics of Development (S00001541) and the School of Molecular and Biomedical Sciences of The University of Adelaide. Professor Martins is supported by grants from the McCusker Foundation for Alzheimer's Disease Research, Department of Veterans Affairs, National Health and
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