Rat nicastrin gene: cDNA isolation, mRNA variants and expression pattern analysis

Molecular Brain Research 136 (2005) 12 – 22 www.elsevier.com/locate/molbrainres

Research report

Rat nicastrin gene: cDNA isolation, mRNA variants and expression $ pattern analysis Annamaria Confalonia,T, Alessio Crestinia, Diego Albanib, Paola Piscopoa, Lorenzo Malvezzi Campeggia, Liana Terrenib, Marco Tartagliaa, Gianluigi Forlonib a b

Department of Cellular Biology and Neuroscience, Istituto Superiore di Sanita`, 299 Viale Regina Elena, 00161 Rome, Italy Biology of Neurodegenerative Disorders Laboratory, Istituto di Ricerche, Farmacologiche, Mario Negri, 20157 Milano, Italy Accepted 20 December 2004 Available online 16 March 2005

Abstract Nicastrin is a type 1 transmembrane glycoprotein that interacts with presenilin, Aph-1, and Pen-2 proteins to form a high molecular complex with gamma secretase activity. Then, nicastrin has a central role in presenilin-mediated processing of beta-amyloid precursor protein and in some aspects of Notch/glp-1 signaling in vivo. Here, we isolated a rat nicastrin cDNA and investigated gene expression in embryonic and adult rat tissues. The predicted amino acid sequence is comprised of 708 residues and showed a high degree of identity with other vertebrate orthologs. Besides full-length nicastrin mRNA, we identified an alternative spliced variant lacking the whole exon 3 and predicted to encode a 62-residue-long truncated protein. Full-length nicastrin mRNA was observed to be ubiquitously expressed, while the spliced variant was preferentially transcribed in the nervous system, whether in embryonic or adult neural tissues. Studies performed on primary cell cultures demonstrated that the short isoform was expressed in neurons, but not in astrocyte and microglial cells. Further experiments performed to verify the presence of the variant in neuroblastoma culture failed to show any truncated protein. Treatments by cyclohexamide showed the involvement of a quality control-based surveillance mechanism, which selectively degrades the exon 3-skipped isoform. In summary, this is the first report describing a novel skipped isoform of nicastrin which may suggest a new possible control mechanism based on the alternative splicing and nonsense-mediated mRNA decay to regulate brain protein expression and provide newer insights into potential implication in Alzheimer’s disease. D 2005 Elsevier B.V. All rights reserved. Theme: Cellular and molecular biology Topic: Gene structure and function: general Keywords: Nicastrin; Alternative splicing; Tissue expression; Culture expression; Rattus norvegicus; Nonsense-mediated mRNA decay

1. Introduction

Abbreviations: Ah, beta amyloid; AD, Alzheimer’s disease; APP, h-amyloid precursor protein; BACE, beta-site APP cleaving enzyme; FAD, familial Alzheimer’s disease; Ncstn, nicastrin; NMD, nonsense-mediated mRNA decay; Presenilin 1, PS1; Presenilin 2, PS2; PSs, presenilins $ Nucleotide sequence data reported in this paper are available in the GenBankk/NCBI databases under accession numbers NM174864 and AF510722. T Corresponding author. Fax: +39 6 49387143. E-mail address: [email protected] (A. Confaloni). 0169-328X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.12.022

g-Secretase is a membrane protein complex regulating the transmembranous proteolysis of h-amyloid precursor protein (APP) [28] to release the Alzheimer’s disease (AD)associated amyloid h-peptide (Ah) [18]. This multimolecular protein complex requires the presenilin 1 and 2 heterodimers (PS1/PS2) [9,32,44] which apparently build up the active site, beyond three cofactors: Anterior pharynx defective-1 (Aph-1) [15], Presenilin enhancer-2 (Pen-2) [14], and a highly glycosylated form of nicastrin (Ncstn) [23]. Recently, g-secretase activity has been reconstituted by

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the co-expression of these proteins in the yeast Saccharomyces cerevisiae, showing that presenilins (PSs), Aph-1, Pen-2, and Ncstn form the minimal set of components required for catalytic activity [10]. Ncstn is a type 1 transmembrane glycoprotein that is also involved in the presenilin-dependent proteolytic processing of Notch, another substrate of g-secretase [6,46]. Human Ncstn gene maps to a region of chromosome 1 (1q22–q23) that, in two independent genome-wide surveys, has generated evidence for genetic linkage and/or allelic association with Alzheimer’s disease [22,47]. The gene encodes a 709residue-long protein characterized by a putative signal peptide, a long amino-terminal hydrophilic domain containing glycosylation, N-myristoylation, and phosphorylation motifs, a 20-residue putative hydrophobic transmembrane domain, and a short hydrophilic carboxyl terminus of 20 residues [46]. Ncstn does not exhibit a significant amino acid sequence homology or motif similarity to other functionally characterized proteins, with the exception of an aminopeptidase/transferrin receptor superfamily domain [11,30]. Molecular cloning experiments showed that deletion of a conserved hydrophobic domain of the protein (DYIGS) results in a decrease of Ah peptide production. Moreover, induced site-specific missense mutation of the DYIGS motif to AAIGS leads to a significant increase of Ah and especially in Ah-42 secretion [46]. So far, all Ncstn partners involved in g-secretase complex with the exception of PEN-2 are characterized by the occurrence of an alternative splicing, which produces isoforms with uncertain function but specific distribution in systemic and cerebral districts [31,37,42]. Alternative splicing is a critical post-transcriptional event leading to an increase in transcriptome diversity and contributing to a higher functional diversification of genomes during evolution [2]. This process, which affects over a third of all human genes, is able to allow a single genetic locus to generate multiple mRNA isoforms [3,21,25]. This mechanism produces diversity and specificity at the cell, tissue, or developmental levels in an economical way. Furthermore, it seems to allow a trial/error approach for the evolution of the gene structure by decreasing, together with the nonsensemediated mRNA decay (NMD), the selective pressure on genes. NMD is an eukaryotic mRNA surveillance mechanism that reduces the abundance of transcripts containing a premature termination codon (PTC) [4,26,33]. Lewis et al. [25] observe that alternative splicing and NMD are widely coupled. One-third of alternative splicing variants that they examined contained PTC, making them targets for the surveillance mechanism. To the best of our knowledge, no splice variant of Ncstn was reported. In this regard, we tried to elucidate the expression of Ncstn using in vitro and in vivo models. To clarify Ncstn mRNA expression in rat, we first cloned Ncstn cDNA to obtain the complete sequence and examined the presence of splice variants in systemic and neural tissues. We identified a specific skipped isoform of the

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Ncstn mRNA in neuronal cells both in embryonic and adult tissues and analyzed some elements of the splice site. This spliced variant was also identified in a human neuroblastoma line. Experimental treatments allowed us to assess that both Ncstn mRNAs are constitutively expressed in neuroblastoma cells but we failed to identify any C-terminal truncated protein. We have also shown that the skipped variant transcript was a substrate to NMD that should be considered as one of the processes regulating Ncstn expression in neuronal cells.

2. Materials and methods 2.1. Cell culture Cortical neuron cultures were obtained from fetal rat cortices on embryonic day 17 as reported elsewhere [12]. Briefly, cortical cells were dissociated by trituration with Pasteur pipet and plated (1.5  106 cells per well) in 12-well dishes (Falcon) precoated with poly-d-lysine (50 mg/ml; Sigma). Cells were cultured in Dulbecco’s modified minimal essential medium (DMEM; GIBCO) supplemented with 10% fetal calf serum (FCS; Hyclone), 2 mM lglutamine at 37 8C, 5% CO2. After 4 days in vitro, nonneuronal cell division was halted by exposure to 10 mM cytosine arabinoside. Glial cell cultures were prepared from newborn rat pups as described previously [13] and grown in poly-d-lysinecoated Primaria dishes (Falcon), containing DMEM supplemented with 10% FCS, 2 mM l-glutamine at 37 8C, 5% CO2. Microglial cells were harvested from these primary mixed cultures by the method of Yao et al. [45]. After 10 days, flasks containing mixed glial cultures were added with fresh media and placed on a shaker at 37 8C for 12–16 h. Media containing microglia were then placed in new flasks. After 30–60 min, the medium was aspirated and replaced with fresh DMEM. Adherent cells (astrocytes) were then exposed for 5 min to 0.25% trypsin (Difco), followed by the addition of DMEM/FCS 10%. The suspension was centrifuged and the pellet was suspended again in medium containing 10% FCS. Cells were plated at a density of 5  10 4 cells/ml. Human neuroblastoma LAN-5 cells were grown at 80– 90% confluency on 50 ml tissue culture flask (Falcon) in RPMI 1640 medium (Euro CloneR) supplemented with 10% fetal bovine serum (FBS; Euro Clone) and 2 mM lGlutamine (Life Technologies). Cells were maintained at 37 8C, 5% CO2 until 7 days in vitro and then detached by 1 mM EDTA (Biorad) in phosphate-buffered saline (PBS, pH 7.4) and harvested for the experimental procedure. For cyclohexamide treatment, LAN-5 cells were seeded at 2  105 cells in 12-well plates and grown o.n. The day after, cyclohexamide (Sigma) was added at the final concentration of 5 mg/ml. Cells were then incubated for 0.5–3 h and lysed in RNA lysis buffer for total RNA extraction [34].

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2.2. RNA extraction from rat tissue Systemic (liver, spleen, lung, heart) and neural (cortex, cerebellum, hippocampus, olfactory bulbs) fresh tissue samples were prepared on ice, in semisterile conditions, from Sprague–Dawley embryos (E18) and adult rats, collected in RNA later (AmbionR) and stored at 20 8C until use. Total RNA was extracted from tissues (10 mg) according to manufacturer’s protocol (RNAqueousk 4PCR, AmbionR) and quantified by GeneQuantk RNA/DNA Calculator (Amersham Pharmacia Biotech). 2.3. Rat Ncstn cDNA isolation For first-strand cDNA synthesis, we performed a reverse transcription of 2 Ag of total RNA from adult rat cortex with 200 units of SuperScript IIk reverse transcriptase (Life Technologies) and 2.5 AM of oligo (dT)16 in a total reaction volume of 20 Al and incubated sequentially at 42 8C for 1 h and at 95 8C for 15 min. A volume of 0.5 Al of cDNA was amplified using the Elongase Kit (Invitrogen), according to the manufacturer’s protocol. PCR reactions, using primers NCS5A (5V-TTC CGC TCA GCA GAG AGG CAA-3V) and NCS3B (5V-TCC AGT GAC AAA TTA GTG GTT-3V), were carried out in 50 Al containing 1 U elongase enzyme mix, 200 AM each dNTP, 1.8 mM Mg2+ by a MJ Thermal Cycler. PCR conditions were as follows: 94 8C, 30 s; 94 8C, 30 s; 55 8C, 30 s; 68 8C, 3 min (32 cycles); 68 8C, 10 min. The products were then resolved on 2% of agarose gel (SeaplaqueR GTG Agarose-BMA) and the bands of interest were excised, purified (NucleoSpin Extract, MachereyNagel), and cloned in a pCRII-TOPO vector (Invitrogen). A preliminary treatment was performed to add a 3V-terminal deoxyadenosine to blunt-ended PCR products (Qiagen AAddition Kit). Two independent clones obtained from independent PCR reactions were sequenced using an ABI PRISM 377 DNA Sequencer (Applied Biosystems) and the ABI BigDye Terminator Sequencing Kit (Applied Biosystems). DNA sequences were analyzed by the Sequencing Analysis and AutoAssembler software packages. DNA and protein sequence alignments were performed by using the MacVector v.7.0 package. The variability of rat Ncstn splice site of exon 3-skipped (D3) isoform was tested by cloning and sequencing the gelpurified DNA of the specific band. PCR amplification reactions were performed in a total volume of 50 Al using 2 U of AmpliTaq Gold DNA polymerase (Applied Biosystems) in 1 Gold buffer supplemented with 50 AM of each dNTP, 1.5 mM MgCl2, DMSO 5%, 0.5 AM Nic5 (5V-GCT CAA CGC CAC TCA TCA GAT TG-3V) and Nic3 (5V-TGG GCA CTG TAC ACT AGG AGA GA-3V) primers, and 0.5 Al of adult rat cortex first-strand cDNA. PCR conditions were as follows: 8 min at 94 8C, thirty five cycles of 45 s at 94 8C, 30 s at 62 8C, and 45 s at 72 8C; 10 min at 72 8C. The amplified products of D3-isoform (141 bp) were cloned by Qiagen PCR cloning kit. Twenty different clones were

sequenced as described above and splice site variability was individually checked out. 2.4. Expression of Ncstn mRNAs in rat tissues To investigate the expression levels of full-length and D3-isoform Ncstn mRNAs in embryonic and adult tissues, 2 Ag of total RNA was converted into first-strand cDNA, as described previously, except for using 2.5 AM random hexamers (Applied Biosystems) as primers. Primer annealing was performed at 25 8C, 10 min. The forward primer NCS5A and the reverse primer NCS3F (5V-TCT GGG TTG ATG CTG AAG GTG-3V) were used to amplify cDNA portion encompassing exon 3 with expected size of 758 and 634 bp in the full-length and D3-isoform cDNAs, respectively. Such primer pair was designed to minimize the possibility of an amplification of different length products and to avoid the amplification of excessively long PCR products. Briefly, 0.5 Al of cDNA was amplified with 0.5 AM of each primer, 50 AM of each dNTP, 1.5 mM MgCl2, DMSO 5% (SIGMA), 1 Reaction Buffer, and 2.0 U of Taq Gold (Applied Biosystems) in a final volume of 50 Al. PCR cycles were as follows: 8 min at 94 8C; thirty five cycles of 45 s at 94 8C, 30 s at 65 8C; 1 min at 72 8C; 10 min at 72 8C. PCR reaction aliquots (1 Al) were analyzed by a Bioanalyzer with LIF detection (Agilent Technologies, Waldbronn, Germany) [27]. Microchips used in these analyses were prepared with the dsDNA 1000 LabChip kit (Agilent Technologies) for qualitative and quantitative evaluation of dsDNA fragments in the range 25–1000 bp, according to the manufacturer’s instructions. 2.5. Expression of Ncstn mRNAs in rat primary cultures Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) and reverse-transcribed according to the same procedure described for tissue expression. First-strand cDNA pools were used to amplify Ncstn (NCS5A and NCS3F as described above). The reaction products were separated, detected, and determined with the Agilent 2100 bioanalyzer using the DNA 1000 LabChip kit (Agilent Technologies). 2.6. Ncstn mRNA expression in human neuroblastoma cell lines Total RNA from neuroblastoma LAN-5 cells (2  106) was extracted by RNAqueous-4PCR (Ambion) and reversetranscribed as previously described. To investigate expression levels of full-length and D3-isoform Ncstn mRNAs, forward primer Nic5 and reverse primer Nic3 were used. These primers are respectively located in exon 2 and exon 4, according to the genomic organization of the human Ncstn gene (AF240468). Expected sizes of PCR products from full-length and D3-isoform Ncstn cDNAs were 265 bp and 141 bp, respectively. PCR products were obtained after forty

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cycles for a better visualization of transcription secondary products of neuroblastoma Ncstn gene. Thirty PCR cycles were chosen for semiquantitative assay on the base of a comparative analysis of PCR reactions to maintain the reaction linearity in both transcripts. h-Actin was used as housekeeping gene (NM031144), forward primer 5V-GTC GAC AAC GGC TCC GGC ATG-3V, reverse primer 5VCTC TTG CTC TGG GCC TCG TCG C-3V, product length 158 bp. The products were separated on a 2% agarose gel (Biorad) and revealed by ethidium bromide fluorescence. Pictures of gels were taken and the signal intensities of bands quantified by Quantity One Software of the FX Imager Apparatus (Biorad). 2.7. Exonic splicing enhancer consensus analysis The search for exonic splicing enhancers (ESEs) consensus sequence was performed by ESEfinder (release 2.0: http://exon.cshl.edu/ESE) [5]. The program uses weight matrices, obtained by functional SELEX experiment, to identify the binding motifs for human Ser/Arg-rich proteins

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(SR): SF2/ASF, SC35, SRp40, and SRp55. Default threshold values were selected for each matrix but only scores higher than 4.0 and with consensus set in proximity to donor (exon 2)/acceptor (exon3) sites were considered. Rat sequence analysis was qualified by the extent of homology between human and rat SR proteins. In fact, evolutive differences could introduce changes in protein consensus recognition, which are not evaluated by the program. 2.8. Western blot LAN-5 cells were plated in 12-well plates (2  105 cells/well) and grown o.n. The day after, the proteasome inhibitor MG132 (Sigma) was added (final concentration 10 mM) for a further 24 h. Then cells were harvested in hot Laemli buffer, separated on 15% SDS–PAGE, and finally transferred to a nitrocellulose filter (Biorad). The filter was then incubated o.n. in a 1:500 dilution of goat antibody (N-19), to the N-terminus of Ncstn (Santa Cruz Biotechnologies), and then in a secondary antibody HRPconjugated (1:5000) for 1 h. Immunoreactive bands were

Fig. 1. Nucleotide and putative amino acid sequences of rat Ncstn cDNA. (A) Rat Ncstn cDNA is shown by single triplets with corresponding putative aa. Underline represents the amino acid sequence encoded by the skipping variant identified in rat cortex tissue. (B) Schematic representation of full-length and

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revealed by ECL detection system (Amersham Pharmacia Biotech). Alternatively, if deglycosylation was required, at the end of MG132 incubation, cells were harvested and protein extracts preincubated with PNGase (New England Biolabs) according to the manufacturer’s instructions. Deglycosylated samples were then subjected to SDS– PAGE and blotted as described above. Preabsorption of the antiserum with the peptide used for immunization (Santa Cruz Biotechnologies) in a 1:5 molar relationship was done as control for binding specificity.

3. Results 3.1. Isolation of rat Ncstn cDNAs Two rat Ncstn cDNA species were isolated by RT-PCR from adult cortex total RNAs. Cloning and sequencing of the predominant cDNA isoform revealed an open reading

frame of 2127 nucleotides that encoded for a protein of 708 aa residues, corresponding to full-length Ncstn product (Fig. 1A). Sequence comparison with human (AF240468), mouse (AF240469), and Drosophila (AF240470) orthologous proteins indicated an identity of 88%, 95%, and 28%, respectively (Fig. 2). We next analyzed sequence conservation of rat Ncstn mRNA finding that the hydrophobic DIYGS motif (336– 341 residues of the rat sequence) did not show any variations. The comparison of human, murine, and rat aa sequences showed a high homology in the C-terminal section of the protein in which has been identified the transmembrane domain (669–698 residues) [46]. Also the putative catalytic residue of the aminopeptidase/transferrin receptor superfamily domain, localized near the DYIGS motif, was equally conserved (333 residue) [30]. As previously observed for the mouse, rat Ncstn lacks the codon codifying the arginine19 amino acid in comparison to human protein (Fig. 2).

Fig. 2. Homology analysis of amino acid sequence of Ncstn in different species. Predicted aa sequence of R. norvegicus (708 aa), Mus musculus (708 aa), Homo sapiens (709 aa), and Drosophila melanogaster (695 aa) orthologues. Dark and grey shading denote, respectively, an amino acidic identity and a conservative substitution; the absence of background indicates a non-conservative event, dash indicates a residue deletion. The rat sequence shares an 88% and 95% of identity with human and murine Ncstn and the 28% with D. melanogaster.

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In addition to the major Ncstn transcript, a second shorter mRNA isoform identical to the full-length cDNA, but lacking 124 nucleotides, was isolated. The skipped mRNA sequence corresponded to the whole exon 3 of human Ncstn sequence (Fig. 1B). The skipping event is predicted to result in a frame-shift of the open reading frame with the introduction of multiple stop codons and in a protein of 62 residues identical to the amino-terminal portion of the full-length protein encoded by exons 1 and 2. The analysis of the splice site, regarding the skipped isoform, did not demonstrate any sequence variability in the acceptor site pointing out, on the contrary, a conserved alternative splicing pattern in neuronal cells. The complete nucleotide sequences of full-length and D3-isoform mRNA variants were deposited with GenBank accession NM174864 and AF510722, respectively.

were observed at the exon 2 donor site GUGUAAGU (consensus 2AGAGUPuAGU+6) in which GU nucleotides (bold) mismatch the consensus sequence. Moreover, the exon 3 acceptor site GCAGCU (consensus 4NPyA+2 NPyAGAPuN ) showed a pyrimidine (bold) instead of the consensus purine. The polypyrimidine tract at the 3V region of the intron 2 is characterized by the presence of the purines (GAUCCAUUCUAUCCUGGC), which interrupt the pyrimydine-rich binding site. Rat exon 3 scanning revealed possible binding sites for SRp40 (UUAGUGG score 4.04, AUACAGG score 4.96) and SRp55 (CACGUA, score 4.33) proteins. In exon 2, we identified a consensus for SC35 (GGCUGCCA, score 4.22) protein in tight proximity (2 nucleotides) with the donor site, beyond a UGUGGG consensus for a possible exonic splicing silencer.

3.2. Analysis of cis-acting elements

3.3. Ncstn mRNA expression in rat tissues and primary cultures

To investigate the skipping event mechanism, we analyzed the rat Ncstn sequence and characterized the elements able to influence the alternative splicing. We assessed the classic cis-acting elements (branch point, pyrymidine-rich tract, donor/acceptor sites) involved in alternative splicing in rat genomic sequence (NW_047399) between exon 2 and 3. Variations of consensus sequences

RT-PCR assay was used to investigate the expression profiles of both full-length and D3-isoform Ncstn mRNA variants in rat tissues of neural (cortex, cerebellum, hippocampus, olfactory bulbs) and systemic (liver, spleen, lung, heart) origin (Fig. 3). Full-length mRNA was observed to be ubiquitously expressed both in embryonic and adult tissues,

Fig. 3. Expression of a differentially spliced Ncstn mRNA in embryonic and adult rat tissues. Shown is the microchip high-voltage electrophoretic run representing the Ncstn rat splicing variant in neural (olfactory bulbs, cortex, hippocampus, cerebellum) (lanes 2–8) and systemic tissues (heart, liver, spleen, lung) (lanes 9–16). The 758-bp band corresponding to the expected size for the PCR product of the first six exons of the full-length Ncstn is ubiquitous, while the skipping variant is preferentially expressed in the nervous system, whether in embryonic (lanes 2–8) or adult (lanes 9–16) neural tissues. Data are representative of five independent experiments. The bands at 15 and 1500 bp correspond to the standards for instrumental lining up, while lane 1 shows DNA 1000 LabChip ladder (Agilent Technologies).

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and it was the predominant Ncstn mRNA. D3-Isoform mRNA was expressed in neural tissues, where Ncstn protein is believed to carry out a physiological role, cooperating in the assembly of g-secretase protein complex. We analyzed the relative expression of full-length and D3-isoform mRNA variants in rat neuron and glial primary cell cultures (Fig. 4). Significantly, full-length mRNA variant was expressed in both cortical neurons and glial cells, while D3-isoform was detected in cortical neurons, but not in microglia and astrocytes. 3.4. Ncstn expression in human neuroblastoma LAN-5 cells In order to further clarify the occurrence of D3-isoform mRNA variant, we analyzed Ncstn transcript expression in human LAN-5 neuroblastoma by RT-PCR. Two products of 265 and 141 bp, corresponding to full-length and exon 3skipped mRNA variants, were observed (Fig. 5). Direct sequencing of the shorter product confirmed the skipping event, with loss of the entire exon 3. In homeostatic cell culture conditions, full-length Ncstn mRNA was the prominent transcript. Further, the presence of a 62-aa peptide was investigated by Western blot on cell lysate and culture medium in control

Fig. 5. Ncstn full-length and skipped forms in LAN-5 neuroblastoma cells. The expression of the alternatively spliced short form was examined in neuroblastoma cells using a product obtained by PCR reaction of forty cycles and by two oligos in the 3V region of exon 2 and 5V region of exon 4. The electrophoretic run of the Ncstn cDNA shows two amplification products of 265 bp and 141 bp corresponding to the full-length and the D3-isoform, respectively. DNA ladder size (100 bp) marker is indicated on the left.

conditions and after treatment with proteasome inhibitor and N-deglycosylase. If rat Ncstn isoform is translated, these treatments would evidence a putative 6-kDa peptide detectable by N-terminal anti-nicastrin antibody. In all tested conditions, the presence of the peptide was undetectable (Fig. 6) while we were able to reveal both native and immature glycosylated forms of Ncstn. Although more accurate investigations will be necessary to definitely exclude the production of the small Ncstn peptide, our results indicate that the alternative splicing is unlikely devoted to the synthesis of a functional peptide. 3.5. Effect of the cyclohexamide treatment on mRNA levels in skipped and full-length forms of Ncstn To investigate whether the mRNA Ncstn is targeted to NMD, according to Brenner and coworkers [17], we treated Lan-5 neuroblastoma with the translational inhibitor cyclohexamide. Then, a semiquantitative competitive RT-PCR assay was used to study the effects of this treatment on mRNA levels of skipped and full-length Ncstn forms in Lan-5 neuroblastoma. The skipping variant levels increased 1.5 and 2 times after 0.5 and 3 h of treatment, respectively, compared to control (0.5 h cyclohexamide treatment vs. control: 1.47 F 0.07 vs. 1 F 0.11, P = 0.013; 3 h cyclohexamide treatment vs. control 2.08 F 0.09 vs. 1 F 0.11, P = 0.005 Student’s t test) (Fig. 7), while the fulllength transcript remains unchanged.

Fig. 4. Identification of Ncstn transcripts in rat primary cell cultures by microchip high-voltage electrophoresis. Total RNA from different primary cell cultures was reverse-transcribed and amplified by PCR. The full-length Ncstn transcript is expressed in cortical neurons (lanes 2–5), microglia (lanes 6–9), and astrocytes (lanes 10–13). In contrast, the 634-bp band produced by the exon 3 skipping is present exclusively in cortical neurons and undetectable both in microglia and astrocytes. Bands at 1500 bp and 20 bp represent standards for the instrumental lining up.

4. Discussion Ncstn is a transmembrane glycoprotein of type 1 whose individual role as a cofactor in g-secretase complex is still unclear. Accumulating evidence indicates that the protein

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Fig. 6. Western blot of Ncstn on LAN-5 cells. (a) LAN-5 cells were treated as described in Materials and methods. Equal amounts of the proteic extracts were separated on 15% SDS–PAGE and transferred to nitrocellulose filter. The filter was then incubated o.n. in a 1:500 dilution of goat antibody (N-19) to the Nterminus of Ncstn. The double band corresponds to the glycosylated (upper) and deglycosylated (lower) forms of the protein. A single band was observed when the proteic extracts were preincubated with PNGase. MW, molecular weights. (b) The same nitrocellulose filter described in (a) was stripped and reprobed with N-terminal anti-Ncstn antibody. Preabsorption of the antiserum with the peptide used for immunization was done as control for binding specificity as described in Materials and methods.

interacts with PS1 and PS2 and has a central role in both presenilin-mediated processing of APP and Notch/glp-1 signaling [10,46]. Although human Ncstn gene is localized in a region of chromosome 1 previously associated to AD [1,22,47], recent studies failed to identify pathogenic mutations able to produce a hereditary form of AD [7,8]. However, the presence of a modified risk factor for familial early onset AD associated to a particular haplotype of Ncstn gene has been suggested [8,20].

In this study, we reported the isolation of rat Ncstn transcript and the occurrence of an alternative mRNA variant. The predicted rat Ncstn amino acid sequence shows a high homology with known orthologous sequences. Notably, the previously identified regions with a presumed physiologic significance [30,46] are completely conserved. The single missense N417Y change identified by Dermaut et al. in AD patients [8] is not conserved among species. Ncstn expression in some human tissues was described in the original paper of Yu et al. [46] but their Northern blot,

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Fig. 7. Effect of cyclohexamide treatment on mRNA levels in skipped and full-length forms of Ncstn. Lan-5 neuroblastoma cell medium was added with cyclohexamide for 0, 0.5, and 3 h. mRNAs extracted from LAN-5 cells were tested by a semiquantitative competitive RT-PCR assay. The figure shows Ncstn and h-actin transcripts (thirty PCR cycles were used to maintain the reaction linearity in both transcripts) before and after cyclohexamide treatment (upper panel). Ratios of skipped and full-length Ncstn and h-actin mRNAs are statistically analyzed (lower panel). Each value represents the mean F SE of three separate experiments. *P b 0.05, **P b 0.01 vs. control group (Student’s t test).

concerning adult tissues, showed no evidence of a second transcript. This could be due to the small exon 3 size or to the relatively faint quantitative expression of spliced mRNA compared with full-length Ncstn. Furthermore, a recent article described a constitutive expression of Ncstn in human neural culture lines without finding any secondary expression product [36]. Here, we investigated Ncstn mRNA expression in several neural tissues and systemic organs, both in embryo and adult rat. Ncstn appears to be expressed in each examined tissue, whether in embryonic or adult stages. This distribution of expression matches the similar ubiquitous pattern of presenilins and APP, confirming that the expression of g-secretase-linked proteins is not restricted to the central nervous system [40,41,43]. Additionally, to the widespread expression of full-length mRNA, we detected a spliced isoform, which appeared to be specific of the embryonic and adult central nervous system. Ncstn short variant is produced by skipping of the entire exon 3, while the open reading frame encodes an initial amino acid sequence identical to full-length form up to the splice site, which inserts a premature stop codon after 62 aa. To find out more on the mechanism regulating alternative splicing, we tested the variability of the acceptor sequence at the splice site in Ncstn-skipped isoform without finding any

heterogeneity. Our results indicate that, in case of alternative splicing of the pre-messenger RNA, there is no lack of precision in the recognition of 3V splice site at the exon 4 by neuronal cell splicing machinery, suggesting that D3-isoform is a bona fide product of the Ncstn pre-mRNA processing derived from a perfect skipping event. It is well known that changes in splicing pattern derive from changes in the definition of splice sites at the ends of intron/exon boundaries [16]. The analysis of the rat sequences to assess the cis-acting elements of the exons involved in the D3-skipping revealed some mismatches in the donor/acceptor sites at exons 2 and 3 as compared to the consensus sequences. The presence of purines inside the polypyrimidine tract at the 3V end of the intron 2 was assessed as well. These consensus variations in Ncstn cisacting elements could reduce the binding affinity of the spliceosome complex and make it dependent on the presence of tissue-specific factors. The control of alternative splice site recognition is mediated by members of the SR (serine/ arginine rich) protein family of splicing factors, which bind to the splicing enhancer and inhibitor elements [29]. So, the intrinsic weakness of the cis-acting elements associated with the cooperative action of trans-acting factors could be an important aspect of the Ncstn alternative splicing mechanism in neurons. At this regard, we also identified some exonic cis-elements distinct from the classical splicing signals. In fact, we found some ESE consenses for a class of transacting factors (SR proteins) by computational analysis of exon sequence. These ESE elements could affect the specific skipping event, being situated at the Ncstn exon 2 and 3 boundaries, in proximity to the sites recognized by the spliceosome machinery. Further studies, however, will be necessary to understand the role of these putative binding sites on the exon 3 exclusion in neuronal Ncstn. The mRNA expression, both of normal and alternative spliced isoforms, was evaluated in neuronal and glial rat primary cell cultures. The distinct patterns of Ncstn transcript expression among cell types led us to consider the possibility that an alternative isoform might have distinct cellular function as compared to full-length Ncstn messenger. The spliced form was also expressed in the human neuroblastoma LAN-5 cell line suggesting that it is not a peculiar feature of Rattus norvegicus but an evolutionary conserved isoform. The identified short Ncstn isoform belongs to a relatively large class of prematurely terminated transcripts, which are not translated and then, presumably incapable of generating functional products. Nevertheless, an analysis of human mRNA sequences assesses that 35% of alternative isoforms show that this feature could be potentially harmful [17,25], because truncated versions of the protein might exert dominant-negative effects on phenotypes, so that their expression must be strictly regulated. In this regard, all eukaryotes distinguish normal and premature termination codons (PTCs) from each other, considering their position to that of the final intron. A conserved surveillance system

A. Confaloni et al. / Molecular Brain Research 136 (2005) 12–22

termed nonsense-mediated mRNA decay (NMD) works by assessing the quality of mRNAs to ensure that they are suitable for translation. The memory of gene structure in RNA processing is saved by marking the exon–exon boundary with junction complex (EJC) deposited by the spliceosome [24]. The presence of EJC or factors associated with it downstream to a stop codon points out a transcript as premature and activates an accelerated degradation [38]. This pathway selectively eliminates dangerous transcripts also giving a recessive mode of inheritance to genetic disorders due to a nonsense mutation or mistakes in RNA processing. Vice versa it is well known that the escape from accelerated turnover of NMD-candidate messengers can trigger the display of a dominant form of the diseases as brachydactyly type B [39] or h-thalassaemias [19]. Our experimental data indicate that Ncstn D3-isoform mRNA is targeted to NMD and then submitted to a surveillance mechanism that recognizes and degrades the isoform containing the premature termination codons. The NMD pathway could also be involved in AD by a regulated unproductive splicing and translation (RUST) [17] due to the presence of an NMD candidate isoform among the presenilin 1 transcripts [35]. In light of the analogue isoform now identified in Ncstn, this fact suggests that a mechanism as the RUST may be a pervasive, underestimated means of regulating protein expression of g-secretase cofactors. Either nonsense presenilin [35] or Ncstn transcripts, according to our results, seem to have tissue-specific distribution if compared to the widespread expression of a full-length form. This observation raises the possibility that a premature transcriptional termination could have physiological relevance for the above genes, modulating a more complex expression pathway than was originally thought. The establishment of the temporal and spatial distribution of eventual secondary transcription products of g-secretase complex could provide a necessary preamble to explain potential control mechanisms that confer susceptibility to AD.

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The authors thank Dr. V. Cesi and Dr. G. Raschella`, Ente Nuove tecnologie Energia Ambiente (ENEA), Rome, Italy, for the LAN-5 neuroblastoma cells. We would like to thank Nora Perrone-Bizzozero for stimulating discussions. The study was partially supported by the Ministry of Health of Italy (Project bRuolo della Nicastrina nell’etiopatogenesi dell’Alzheimer familiareQ; Alz.8) and by Istituto Superiore di Sanita` (Ricerca Intramurale, grant no.1149/RI). References [1] D. Blacker, L. Bertram, A.J. Saunders, T.J. Moscarillo, M.S. Albert, H. Wiener, R.T. Perry, J.S. Collins, L.E. Harrell, R.C. Go, A. Mahoney, T. Beaty, M.D. Fallin, D. Avramopoulos, G.A. Chase, M.F. Folstein, M.G. Mc Innis, S.S. Bassett, K.J. Doheny, E.W. Pugh, R.E.

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