Molecular characterization and temporal expression profiling of presenilins in the developing porcine brain
© Madsen et al; licensee BioMed Central Ltd. 2007
Received: 17 November 2006
Accepted: 13 September 2007
Published: 13 September 2007
The transmembrane presenilin (PSEN) proteins, PSEN1 and PSEN2, have been proposed to be the catalytic components of the γ-secretase protein complex, which is an intramembranous multimeric protease involved in development, cell regulatory processes, and neurodegeneration in Alzheimer's disease. Here we describe the sequencing, chromosomal mapping, and polymorphism analysis of PSEN1 and PSEN2 in the domestic pig (Sus scrofa domesticus).
The porcine presenilin proteins showed a high degree of homology over their entire sequences to the PSENs from mouse, bovine, and human. PSEN1 and PSEN2 transcription was examined during prenatal development of the brain stem, hippocampus, cortex, basal ganglia, and cerebellum at embryonic days 60, 80, 100, and 114, which revealed distinct temporal- and tissue-specific expression profiles. Furthermore, immunohistochemical analysis of PSEN1 and PSEN2 showed similar localization of the proteins predominantly in neuronal cells in all examined brain areas.
The data provide evidence for structural and functional conservation of PSENs in mammalian lineages, and may suggest that the high sequence similarity and colocalization of PSEN1 and PSEN2 in brain tissue reflect a certain degree of functional redundancy. The data show that pigs may provide a new animal model for detailed analysis of the developmental functions of the PSENs.
The transmembrane presenilin (PSEN) 1 and 2 proteins are the catalytic subunits of the γ-secretase complex, which mediates intramembranous proteolytic cleavage of a number of transmembrane proteins [1, 2]. In addition to the PSEN proteins the γ-secretase complex includes PEN-2, nicastrin and APH-1. Examples of γ-secretase processed proteins are Notch, Jagged, CD44, LRP, NGFRd, ERBB4, and APP. A common theme in γ-secretase mediated proteolytic processing is the generation of intracellular C-terminal domains, which migrates to the nuclear compartment to regulate expression of target genes. γ-secretase-independent functions of the PSEN proteins have also been described, including regulation of protein localization, apoptosis, calcium homoeostasis, and Wnt signal transduction [1, 3]. Human PSEN1 and PSEN2 share more than 60% amino acid identity and both PSENs are processed proteolytically to generate amino- and carboxyl-fragments, which associate in the bioactive γ-secretase complex .
The PSENs are multifunctional and have important roles in embryonic development through regulation of cellular signal transduction pathways. Thus, studies of PSEN1-deficient mice showed that homozygous null mutants die perinatally and that PSEN1 is required for formation of the axial skeleton, normal neurogenesis, and neuronal survival [5, 6]. The PSEN1 mutant phenotype can in part be explained from experiments in mouse , Caenorhabditis elegans , and Drosophila melanogaster , which have demonstrated an essential role for PSENs in Notch signalling [2, 10]. PSEN2 homozygous null mutant mice are viable, presumable due to a compensation effect of the more abundantly expressed PSEN1. Interestingly, complete loss of both PSENs causes more widespread developmental defects than observed for the PSEN1 null mutant, which suggest overlapping but not identical cellular roles of PSEN1 and PSEN2 [11, 12]. Importantly, genetic and biochemical evidence have demonstrated that mutations in human PSEN1 and PSEN2 play a role in the development of familiar Alzheimer's disease by altering APP processing to increase the ratio between the more aggregation-prone Aβ42 peptide and the Aβ40 variant 
Although studies of the PSENs in Caenorhabditis elegans, Drosophila melanogaster and mice have yielded important insight into the biological role of the PSENs, several essential questions still remain concerning the developmental functions of PSEN1 and PSEN2. Strong anatomical, physiological and biochemical similarities between man and pig suggest that this animal may constitute a suitable alternative model for research related to Alzheimer's disease, although Alzheimer-like neuropathology including Aβ deposits in the brains of aged pigs to our knowledge has not yet been described. To begin to address this possibility, we here present the first molecular characterization of the porcine PSENs by sequence analysis, determination of chromosomal localization and screening for polymorphisms. Moreover, the expression profiles of PSEN1 and PSEN2 were studied both at the mRNA and protein levels in several areas of the embryonic pig brain. The results show a high degree of evolutionary conservation of both the porcine primary sequences and the expression patterns compared to those observed in human and rodents.
PSEN1 and PSEN2 cDNA and protein sequence
The amino acid sequence for porcine PSEN1 shows 64% identity to porcine PSEN2 and particularly amino acids in the transmembrane domains and the C-terminus are conserved (data not shown). Also, the two aspartic acid residues located in transmembrane domain 6 (D257 in PSEN1 and D263 in PSEN2) and the transmembrane domain 7 (D385 in PSEN1 and D366 in PSEN2), as well as the "PAL" sequence, (P433, A434, L435 in PSEN1 and P414, A415, L416 in PSEN2) are conserved in both porcine PSEN1 and PSEN2, consistent with the essential role of these residues for the protease catalytic function of the presenilins [15, 20, 21].
Mapping of porcine PSEN1 and PSEN2
A porcine-rodent somatic cell hybrid panel was used for the chromosomal mapping of porcine PSEN1 and PSEN2 genes (data not shown) . Statistical evaluation by applying the "Interpreting PCR data" program  resulted in a chromosomal assignment of the PSEN1 gene to chromosome 7q12-q26 with a probability of 0.4494 and a correlation of 1. This conclusion gains support from the facts that the specified region of porcine chromosome 7 shares homology with the human chromosome 14, and that the human PSEN1 gene has been mapped to HSA14q24.3 . However, the relatively low probability value suggests that the chromosomal position of the porcine PSEN1 gene should be considered preliminary. The PSEN2 gene was assigned to chromosome 10p11-p16 with a probability of 0.9959 and a correlation of 0.7255, which is in agreement with the syntenic relationship between porcine chromosome 10 and human chromosome 1, and the known position of PSEN2 on HSA1q31-q42 .
Single nucleotide polymorphism screening of porcine PSEN1
Genotype-frequencies of a C/T SNP in position 1163 (DQ86246) in PSEN1 intron 8 in a pig breed-panel
No. of animals
SNP position 1163
Genotype-frequencies for three SNPs in PSEN1 intron 10 (DQ86246) in a pig breed-panel
No. of animals
SNP position 1535/1575
SNP position 1600
PSEN expression in the developing porcine brain
PSEN1 and PSEN2 have been shown to be widely expressed during embryonic development and especially the expression profile in the CNS is well characterized [25–27]. Here, we measured the mRNA expression levels of PSEN1 and PSEN2 in hippocampus, cerebellum, frontal cortex, basal ganglia, and brain stem from dissected porcine foetus brains at days 60, 80, 100 and 114 of gestation using three biological samples for each of the time points. Day E115 corresponds to the normal day of birth. The PCR analyses were performed in triplicates. The requirement for a proper internal control gene was met by normalization to the GAPDH expression level to compensate for inter-PCR variation with respect to RNA integrity and sample loading. We did not find any significant variation in expression of GAPDH within the 5 different porcine brain tissues at the various developmental stages. The standard curve for the control GAPDH (R2 = 0.98), PSEN1 (R2 = 0.98), and PSEN2 (R2 = 0.98) were generated by plotting Ct values versus log μL of cDNA. The slope of the regression line was used to calculate the amount of cDNA and thus mRNA in each sample. All GAPDH cDNA's generated almost identical Ct values within each type of tissue (data not shown) and accordingly the mRNA expression levels of PSEN1 and PSEN2 were normalized to the GAPDH expression level. Ethidium bromide-staining after real time PCR confirmed specific amplification of the relevant PCR products (data not shown).
Here we describe the isolation and primary characterization of PSEN1 and PSEN2 from pigs. Thus, chromosomal mapping assigned the porcine PSEN1 and PSEN2 to SSC7q12-q23 and SSC10p11-p16, respectively, which is in accordance with the known syntenic relationships with human chromosomes 14 and 1 . Pig PSEN1 and PSEN2 have protein sequences that are highly homologous to the human counterparts. Conservation included the two catalytic aspartic acid residues located in adjacent transmembrane domains, the C-terminal "PAL" sequence involved in defining the catalytic active site conformation, all of the nine transmembrane domains, and the C-terminal part. The evolutionary conservation of these amino acid segments strongly advocate that they are essential for PSEN functions . For instance, the aspartate residues are critical for PSEN1 endoproteolysis and γ-secretase activity, leading to the hypothesis that the proteolytic cleavage generating the N- and C-terminal PSEN fragments is an autocatalytic event necessary for activation of PSEN, and that PSENs are the intrinsic catalytic subunits for γ-secretase aspartyl protease activity . Furthermore, non-conservative mutations within the invariant PAL sequence abolish PSEN1 endoproteolysis and γ-secretase activity, indicating that this sequence is crucial for the enzymatic activity . Approximately, 150 mutations in human PSEN1 and ten mutations in human PSEN2 have been associated with early onset Alzheimer's disease . We observed complete identity between pig and human PSENs of amino acid residues, which have been mutated in Alzheimer's patients. This identity extends to the mouse and cow PSENs, which underscores the functional importance of these residues, and further supports the notion that the pig PSENs share the biological functions of other mammalian PSENs.
We did not find any SNP's in exons 5, 7, 8 or 9, which are hotspots for mutations causing familiar Alzheimer's disease. However, one SNP was found in intron 8 and three others in intron 9. The allele frequencies of these SNPs differed markedly between Hampshire and the other porcine breeds, possibly reflecting the different origins of these breeds. SNPs are amenable to high-throughput analysis and, therefore, an attractive type of DNA marker for animal identification, paternity testing, and genome scans for QTL and disease genes.
Expression profiling showed a distinct spatiotemporal regulation of PSEN transcription in various brain regions at embryonic days 60, 80, 100, and 114. Thus, variation in gene expression was observed for PSEN1 in frontal cortex, cerebellum and hippocampus, whereas PSEN1 expression in basal ganglia and brainstem did not vary significantly between the different embryonic stages. The variation pointed towards a reduction in PSEN1 expression between day 60 and day 114 of gestation. Also, PSEN2 showed differential expression during the gestation period in some of the tested tissues. The variation in expression levels suggests that both PSEN1 and PSEN2 play specific functions during embryonic brain development. In this context, it is noteworthy that mice lacking PSEN1 die shortly after birth and show a thinner ventricular zone, and substantial degeneration of the subcortical region of the temporal lobe at embryonic day 14.5 and 16.5 . In comparison, PSEN2 deficient mice show no alterations in brain anatomy, and only with increasing age developed mild pulmonary fibrosis . However, mice deficient in both PSEN1 and PSEN2 display early embryonic patterning defects resulting in lethality (before E9.5) . This suggests that PSEN2 is capable of performing some PSEN1 functions in early developmental stages, and that PSEN1 largely can substitute for PSEN2 functions during development. Our immunohistochemical analysis showed that at the protein level PSEN2 are expressed in the same cells in the CNS as PSEN1 and that the PSENs localize mainly in neuronal cell types but also in astrocytes. Such localizations were in accordance with human, mouse and, rat brain studies [25, 26]. Taken together, the expression data support the notion that redundancy of PSEN1 and PSEN2 functions during development might be attributed to both the high degree of sequence similarity and the consistent expression of PSEN2 in cells also expressing PSEN1.
Our data show that the pig PSENS are conserved both at the primary sequence level and in patterns of gene expression during embryonic brain development. Since pigs share many physiologic and anatomic characteristics with humans, this makes them interesting and attractive models for developmental studies . The here presented initial characterization of the porcine PSENs could be the first step towards a future inclusion of the pig as a model animal to study and elucidate the biological functions of the PSENs.
PSEN1 and PSEN2 Isolation and sequencing
Pig brain, lymphocyte, and liver RNA was isolated with the TRI-reagent (Sigma). For RT-PCR of PSEN1 the following primers were used (PSEN1forward, 5'-TGGAGGAGAACACATGAAAGAAAG-3'; PSEN1-forward-EcoR1 5'-GGGGAATTCTGGAGGAGAACACATGAAAGAAAG-3'; PSEN1reverseEcoR1, 5'-GGGGAATTCCCTGACTTTGTTAGATGTGGACAC-3'). The RT-PCR reaction was incubated at 50°C for 60 min with the reverse primer followed by PCR with the PSEN1forward-EcoR1 and PSEN1reverse-EcoR1 primers at conditions (94°C for 3 min, 35 cycles of; 94°C, 45 sec; 62°C, 30 sec; 68°C, 2 min, followed by a final elongation step at 68°C for 7 min). Amplified DNA fragments were purified from agarose gels and either directly sequenced or EcoR1 cloned into pCDNA3 followed by DNA purification and sequencing. For RT-PCR of PSEN2 the following primers were used (PSEN2-forward, 5'-GCCATGCTCACTTTCATGGC-3'; PSEN2-reverse, 5'-CACGACTGCGTCCAGTGACC-3'). The reverse transcription reaction was accomplished using the Invitrogen reverse transcription system (Invitrogen) and 5 μg of total-RNA according to the manufacturer's instructions. Subsequently, the PCR reaction was carried out at the following conditions: (94°C for 3 min, 35 cycles of; 94°C, 45 sec; 60°C, 30 sec; 68°C, 2 min, followed by a final elongation step at 68°C for 7 min). Amplified DNA fragments were purified from agarose gels and either directly sequenced or cloned into pCR® 2.1-TOPO® Vector (Invitrogen) followed by DNA purification and sequencing. The porcine pSEN1 and pSEN2 cDNA sequences were submitted to GenBank (Accession numbers DQ853416, and DQ853415, respectively)
Radioactive probes were generated employing the nick translation kit from Invitrogen which incorporated [α-32P]dCTP into the PCR generated PSEN1-exon8 fragment. High-density colony BAC filters (a generous gift from Dr. P. D. Jong) of the porcine genome were screened with the PSEN1-exon8 probe. The filters were pre-hybridized, hybridized, washed and autoradiographed according to standard methods. Positive spots were localised and BAC DNA of positive clones was isolated using the alkaline lysis method described by Zhang et al. (1996). BAC clone 388G9 contained the PSEN1 genomic sequence and was used for intronic sequence generation.
Generation of intron sequence information
Sequences of primers and real time PCR probes
Primer and probes
PS1 Exon 5 forward primer1
PS1 Exon 5 reverse primer1
PS1 Exon 7 forward primer1
PS1 exon 7 reverse primer1
PS1 Exon 8 forward primer1
PS1 Exon 8 reverse primer1
PS1 Exon 9 forward primer1
PS1 Exon 9 reverse primer1
PS1 Exon 5 forward primer2
PS1 Exon 5 reverse primer2
PS1 Exon 7 forward primer2
PS1 exon 7 reverse primer2
PS1 Exon 8 forward primer2
PS1 Exon 8 reverse primer2
PS1 Exon 9 forward primer2
PS1 Exon 9 reverse primer2
PS1 forward primer
Real Time PCR
PS1 reverse primer
Real Time PCR
PS1 MGB probe
Real Time PCR
PS2 forward primer
Real Time PCR
PS2 reverse primer
Real Time PCR
PS2 MGB probe
Real Time PCR
GAPDH forward primer
Real Time PCR
GAPDH reverse primer
Real Time PCR
GAPDH MGB probe
Real Time PCR
Exons 5, 7, 8 and 9 and flanking intron sequences were amplified by PCR (primers listed in table 3 under SNP-screening application). Exon 5 and flanking intron sequences were amplified at conditions 50 ng DNA; 94°C for 3 min and 35 cycles; 94°C, 30 sec; 60°C, 20 sec; 72°C, 1 min. Exon 7 and flanking intron sequences were amplified at conditions 50 ng DNA; 94°C for 3 min and 35 cycles; 94°C, 20 sec; 58°C, 20 sec; 72°C, 1 min. Exon 8 and flanking intron sequences were amplified at conditions 50 ng DNA; 94°C for 3 min and 35 cycles; 94°C, 45 sec; 64°C, 30 sec; 72°C, 1 min. Exon 9 and the flanking intron sequences were amplified at conditions 50 ng DNA; 94°C for 3 min and 35 cycles; 94°C, 20 sec; 58°C, 20 sec; 72°C, 1 min. All PCR products were incubated with exozap at 37°C for 1 hour and sequenced with the forward amplification primer. The sequences were analyzed using PolyBace and checked manually in Consed.
Hybrid cell mapping
A porcine-rodent somatic cell hybrid panel was used for physical mapping (Yerle et al., 1996) of both PSEN1 and PSEN2. For PSEN1 the exon 9 forward and reverse primers 2 were used for amplification of the probe fragment. For PSEN2 the PCR primers (PSEN2exon12F; 5'-GTTTGTGTCTGACCCTCCTGCTGC-3' and PSEN2exon12R; 5'-CAGATGTAGAGCTGGTGGGGAGG-3') were used for amplification of the probe fragment. PCR's were performed in a total volume of 10 μL containing 10 ng DNA, 1 × PCR buffer, 2.5 mM of each dNTP, 5 pmol of each primer, and 0.5 U of Taq polymerase (Bioline) under the following conditions: 94°C for 3 min; 35 cycles of 94°C for 20 s, 65°C for 20 s and 72°C for 20 s, and a final elongation step for 5 min at 72°C.
Fetal pig brains were immersion fixed in formalin and paraffin-embedded tissue blocks were produced from various brain regions. 10 μm coronal sections were then obtained on coated glass slides. The sections were deparaffinized and pretreated with proteinase K for 6 min. The slides were blocked with BSA (1 mg/ml) for 10 min. Immunohistochemical demonstration of PSEN1 and PSEN2 was performed using the EnVision+ System-HRP-DAB (DAKO). The anti-PSEN1 antibody derived from human was a rabbit polyclonal antiserum 520 (a generous gift from Dr. Paul Fraser, Toronto, Canada) used in 1:100 dilution with 2 hours incubation time. The anti-PSEN2 antibody derived from human was the mouse monoclonal antibody, APS 26, used in 1:33 dilution with 2 hours incubation time (abcam). Nuclei were counterstained in haematoxylen solution. The slides were finally coverslipped with Faramount Aqueous Mounting Medium (DAKO).
200 mg frontal cortex from embryonic day 60, 80, 100, and 114 of gestation were homogenized mechanically in 1 mL extraction buffer, pH 8.3 (50 mM Tris, 10 mM EDTA, and complete protease inhibitor (Roche, Penzberg, Germany)). Subsequently, 1 mL extraction buffer was further added and 500 μL treatment buffer, pH 6.8 (0.125 M Tris, 4 % SDS, 20 % glycerol) was added to 500 μL of the cleared tissue lysate. The samples were incubated at 50°C for 25 min following protein quantification by BCA protein assay kit (Pierce, Rockford, IL). 20 μg total protein from each sample were heated to 95°C for 10 min in the treatment buffer before being separated on a 15 % Tris-HCl gel (Bio-Rad, Hercules, CA). The membranes were blocked with 5% skim milk powder in 0.05% TBST buffer and incubated overnight with anti-PSEN1 rabbit polyclonal antiserum diluted 1:250 and mouse monoclonal anti-PSEN2, APS 26, diluted 1:1000. Bound primary antibodies were detected by binding with horseradish peroxidase-conjugated anti-rabbit IgG or polyclonal anti-mouse secondary antibodies, both diluted 1:2000 (Dako, Glostrup, Denmark). The blots were visualized with BM Chemiluminescence blotting substrate according to manufactures instructions (Roche, Penzberg, Germany).
Real-time quantitative PCR assay
Total RNA was isolated from cerebellum, frontal cortex, hippocampus, brainstem, and basal ganglia from 60, 80, 100, and 114 days old porcine fetuses using the TRI Reagent™ (Sigma) in compliance with the manufacturer's instructions. Three separate tissues were applied for each type of tissue and time in gestation, yielding a total of 60 samples. The reverse transcription reaction was accomplished using an Invitrogen reverse transcription system (Invitrogen) and 5 μg of RNA according to the manufacturer's instructions. Quantitative real time PCR was performed using the TaqMan® assay and PCR amplification in an ABI-PE prism 7900 sequence detection system (PE Applied Biosystems). Primers and MGB probes were designed using the Primer Express Software 2.0 (PE Applied Biosystems), so that both forward and reverse primer spanned an exon-exon junction. The MGB probe was synthesized with VIC as a reporter dye. After an initial screening with different control genes GAPDH was chosen as the endogenous control and the MGB-probe was synthesized with VIC as a reporter dye. The primers and probes are detailed in table 3. Separate mixtures for PSEN1, PSEN2, and GAPDH were prepared and consisted of 5 μL 2× TaqMan® Universal PCR Master Mix, 0.3 μL of each primer (10 μM), 0.25 μL probe (5 μM), 2 μL of a 5-fold diluted cDNA template, and H2O to a final volume of 10 μL. Real-time PCR was done under the following conditions: 2 min at 50°C, 10 min at 95°C, 40 cycles of 95°C for 15 sec and 60°C for 1 min. For both PSEN1, PSEN2, and GAPDH PCRs were performed in triplicate. The cycle threshold (Ct) values corresponding to the PCR cycle number at which fluorescence emission in real time reaches a threshold above baseline emission were determined in SDS 2.2 (PE Applied Biosystems). To compare expression patterns in the various brain tissues at different developmental stages mRNA template concentrations for GAPDH, PSEN1, and PSEN2 were calculated using the standard curve method. Standard curves were constructed using 8 fold dilution of day 114 frontal cortex cDNA (4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μL). The mRNA quantity of each amplicon was calculated for each standard and experimental sample.
The equality of PSEN1 and PSEN2 expression levels between different time of gestation within the 5 sampled tissues were tested for statistical significance using the standalone software REST© . The statistical model applied was the Pair Wise Fixed Reallocation Randomisation Test. The assumption regarding normal distribution of the data was avoided, and differences in expression between groups were assessed using the means for statistical significance by randomization. The level of probability was set at P < 0.05 as statistically significant and 50000 randomization steps were implemented in each comparison.
We gratefully acknowledge Dr. Martine Yerle, INRA, Toulouse, France for providing the pig-rodent hybrid panel. The authors wish to thank Connie Jakobsen Juhl, Hanne Jørgensen, Birgitte Busk Christensen, and Marianne Johansen for excellent technical assistance.
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