A Novel Presenilin‐2 Splice Variant in Human Alzheimer's Disease Brain Tissue

Abstract

Abstract:Mutations in the presenilin-1 (PS-1) and presenilin-2 (PS-2) genes account for the majority of cases of early-onset familial Alzheimer's disease (AD). Alternative splicing forms of the PS-1 and PS-2 gene products have previously been reported in fibroblast and brain tissue from both familial and sporadic AD patients, as well as from normal tissues and cell lines. We demonstrate here unusual alternative splicing of the PS-2 gene that leads to the generation of mRNA lacking exon 5 in human brain tissue. This product was more frequently detected in brain tissue from sporadic AD patients (70.0%; 21 of 30) than from normal age-matched controls (17.6%; three of 17). In cultured neuroblastoma cells, this splice variant was generated in hypoxia but not under other forms of cellular stress. Hypoxia-mediated induction of this splice variant was blocked by pretreatment of neuroblastoma cells with the protein synthesis inhibitor cycloheximide or antioxidants such as N-acetylcysteine and diphenyl iodonium, suggesting that hypoxia-mediated oxidant stress might, at least in part, underlie the alternative splicing of PS-2 mRNA through de novo protein synthesis. Furthermore, the stable transfectants of this splice variant produced the N-terminal part of PS-2 protein (15 kDa) and were more susceptible to cellular stresses than control transfectants. These results suggest the possibility that altered presenilin gene products in stress conditions may also participate in the pathogenesis of AD.

Induced HMGA1a expression causes aberrant splicing of Presenilin-2 pre-mRNA in sporadic Alzheimer's disease

“…9,10 We reported the preferential expression of a characteristic splicing variant of the PS2 gene that lacks exon 5 (isoform termed PS2V) in the sporadic AD brain, which is not caused by a mutation of the gene but possibly by a trans-acting factor. 11 We showed that PS2V protein impaired the signaling pathway of the unfolded protein response, in a manner similar to familial AD-linked PS1 mutant proteins, and caused significant increases in the production of Ab protein. 12 Intriguingly, we observed PS2V-encoded aberrant proteins in fragile pyramidal cells of the hippocampal CA1 region and in cells of the cerebral cortex of sporadic AD brains.…”

“…In contrast, only the full-length PS2 product was detected in both HeLa and HEK 293T cells by hypoxia. The shorter PS2V isoform was not detected when cells were subjected to other ADrelated stresses, including exposure to tunicamycin (TM), Ab, or H 2 O 2 ( Figure 1b; data not shown; Sato et al 11 ) Site-specific binding to exon 5 of PS2 pre-mRNA is observed by hypoxia Since the PS2V isoform was detected in SK-N-SH cells only by hypoxia, we assumed that a PS2 pre-mRNA-binding factor, which leads to skipping of exon 5 of the PS2 transcript, exists in nuclear extracts from the hypoxic SK-N-SH cells. To detect the PS2 pre-mRNA-binding factor, we performed a premRNA-binding assay by ultraviolet (UV) crosslinking to various 35 S-labeled probes derived from PS2 pre-mRNA (Figure 2a and b).…”

“…11,12 Transient transfections of various expression plasmids were carried out using LipofectAmine (Gibco BRL). The full-length cDNAs of HMGA1a, HMGA1b, and U1-70K were amplified by RT-PCR with attached BamHI and EcoRI sites, and subcloned into pcDNA3(+) vector (Invitrogen) between corresponding restriction enzyme sites.…”

“…11,12 In sporadic AD brains, a unique shorter product PS2V, which lacked exon 5, was detected along with a major RT-PCR product corresponding to the fulllength PS2 mRNA (Figure 1a). We found that SK-N-SH neuroblastoma cell line also produced a shorter PS2V isoform besides the PS2 product by hypoxia (Figure 1b).…”

“…The aberrant splicing resulting in the PS2V isoform is hypoxia-inducible in cultured neuroblastoma cells. 11 We identified a protein that binds to the exon 5 region, and demonstrated that it causes aberrant splicing of the PS2 transcript. The identified protein was a high mobility group protein A1a (HMGA1a, also known as HMG-I formerly), which is a member of the AT-hook protein (HMGA) family.…”