Molecular cloning and characterization of presenilin gene in Bombyx mori

Abstract

Presenilin (PS), the catalytic core of the γ-secretase complex, is considered to be a causative protein of the early‑onset familial form of Alzheimer's disease. Aging is a risk factor for Alzheimer's disease and a number of genetic studies have utilized Bombyx mori (B. mori) as a model, making it possible to use B. mori to investigate Alzheimer's disease. However, the homologous gene of human PS in B. mori has remained to be elucidated. In the present study, the PS homologue gene in B. mori was identified and characterized, and six B. mori presenilin (BmPS) mRNA transcripts were generated by selecting multiple transcription start sites and/or alternative splice sites. The longest mRNA of BmPS (termed BmPS1) contains a 153 nt 5' untranslated region (UTR), a 1,440 nt open reading frame and a 1,063 nt 3' UTR. The predicted protein of BmPS1 consists of 479 amino acid residues and has two highly‑conserved aspartate residues, which form the catalytic core of aspartic proteases. It exhibits a sequence identity of ~44 and 51% with homologues in Homo sapiens and Drosophila melanogaster, respectively. However, the amino acid sequence of the BmPS loop region does not completely match between the two B. mori strains R13Q and Dazao. Genomic analysis revealed that B. mori had a single copy of the BmPS gene, which was composed of 14 exons. A total of four isoforms of BmPS (BmPS‑A, ‑B, ‑C and ‑D) owing to multiple transcriptional start sites and alternative splice sites were identified. The alternative splicing events occurring in the loop region improved the diversity of the BmPS protein and were detectable in all tissues, as determined using reverse transcription quantitative polymerase chain reaction (RT‑qPCR). Furthermore, the expression levels of BmPS in the brain at different developmental stages were detected using RT‑qPCR, and significantly higher expression levels of BmPS were found in the adult stage compared with those in the larval and pupal stages. The present study on BmPS provided insight into the pathogenesis of Alzheimer's disease and mechanisms of silkworm developmental regulation.

Amyloid Precursor Proteins Are Dynamically Trafficked and Processed during Neuronal Development

“…As with other APP family proteins, insect APPL is subject to cleavage by a combination of α, β, and γ-secretases (Figures 1G–J), consistent with the identification of all three secretase classes in multiple insect systems (Luo et al, 1990; Ramabhadran et al, 1993; Fossgreen et al, 1998; Greeve et al, 2004; Groth et al, 2010; Bolkan et al, 2012; Prussing et al, 2013; Pace et al, 2014; Bourdet et al, 2015; Lavore et al, 2015; Zheng et al, 2015). Likewise, our studies showed that Manduca APPL is preferentially cleaved by α-secretases under normal circumstances, as previously reported for both Drosophila APPL and human APP 695 (Haass et al, 2012; Wentzell et al, 2012; Zhang et al, 2012).…”

Presenilin is involved in larval‐pupal metamorphosis development of Bombyx mori

“…In a previous study, we found that Bmps was expressed in the brain (Zheng et al, 2015). To further investigated the spatial and temporal expressional pattern, the mRNA level of Bmps were quantified at three representative developmental stages: wandering stage (W), day 1 pupal stage (P1) and day 5 pupal stage (P5).…”

“…As a model animal, the silkworm has numerous advantages including short generation time and clear genetic background. The ps homologous gene in Bombyx mori has been identified, and six Bmps messenger RNA (mRNA) transcripts were generated by selecting multiple transcription start sites and/or alternative splice sites (Zheng et al, 2015). In Drosophila melanogaster , PS plays vital roles during metamorphosis and absence of Dps leads to early pupal‐lethal (Guo et al, 1999).…”

Aluminum toxicity related to SOD and expression of presenilin and CREB in Bombyx mori

“…Extracted proteins were separated by 10% SDS/PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). Polyclonal rabbit anti‐BmPS primary antibody recognizing the RERRDEGAAPRR peptide sequence of BmPS (Zheng et al., ) was prepared by ABclonal (Hubei, China) and used to detect BmPS protein (1:500 dilution). CREB and p‐CREB were detected using polyclonal rabbit anti‐CREB primary antibody (1:1,000 dilution; Signalway Antibody LLC, College Park, MD, USA) and monoclonal mouse anti‐p‐CREB primary antibody (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), respectively.…”

“…In Al‐treated animals, it has been demonstrated that Al enter the brain and disrupt learning and memory functions by enhancing the production and aggregation of amyloid‐β (Aβ) in vitro and in vivo, with a marked increase in the expression of enzymes participating in Aβ metabolism such as presenilin (Kawahara, Kato, & Kuroda, ; Matsuzaki et al., ; Walton & Wang, ; Wang et al., ). Our previous study cloned silkworm ( Bombyx mori ) presenilin (BmPS) (Zheng, Chao, Fan, Zhao, & Song, ), leading us to hypothesize that Al would have similar effects on BmPS. cAMP response element‐binding protein (CREB) is a molecular switch that implicates in controlling long‐term synaptic plasticity and long‐term memory (Lonze & Ginty, ; Vitolo et al., ).…”