The Transcriptionally Active Amyloid Precursor Protein (APP) Intracellular Domain Is Preferentially Produced from the 695 Isoform of APP in a β-Secretase-dependent Pathway

Abstract

Amyloidogenic processing of the amyloid precursor protein (APP) by ␤-and ␥-secretases generates several biologically active products, including amyloid-␤ (A␤) and the APP intracellular domain (AICD). AICD regulates transcription of several neuronal genes, especially the A␤-degrading enzyme, neprilysin (NEP). APP exists in several alternatively spliced isoforms, APP 695 , APP 751 , and APP 770 . We have examined whether each isoform can contribute to AICD generation and hence up-regulation of NEP expression. Using SH-SY5Y neuronal cells stably expressing each of the APP isoforms, we observed that only APP 695 up-regulated nuclear AICD levels (9-fold) and NEP expression (6-fold). A characteristic feature of Alzheimer disease (AD) 5 is the presence in the brain of extracellular amyloid plaques composed of the amyloid ␤-peptide (principally A␤ 1-40 and A␤ 1-42 ), which is derived from the transmembrane amyloid precursor protein (APP). Hence, for almost two decades, the amyloid cascade hypothesis (1, 2) has driven much AD research with a focus on the prevention of A␤ accumulation or the enhancement of its clearance as primary therapeutic strategies. In the amyloidogenic pathway of APP metabolism, A␤ is formed through the sequential actions of ␤-and ␥-secretases, whereas the non-amyloidogenic ␣-secretase pathway precludes A␤ formation. Enzymic clearance of A␤ is mediated by several enzymes, of which the metallopeptidase neprilysin (NEP) is a key contributor, and up-regulation of A␤-degrading enzymes is a potential therapeutic strategy (3, 4).Three major isoforms of APP are produced due to the alternative splicing of exons 7 and 8, which encode a 56-amino acid Kunitz-type proteinase inhibitor (KPI) domain and a 19-amino acid domain that shares sequence identity with the OX-2 antigen of thymus-derived lymphoid cells, respectively (5). The longest isoform, APP 770 , contains both the KPI and the OX-2 domains, whereas APP 751 contains only the KPI domain. The shortest isoform, APP 695 , lacks both domains. In the brain, APP 695 is expressed at high levels, and the APP 751/770 isoforms are expressed at significantly lower levels, although there are regional differences, and it has been suggested that the balance between the KPI-and non-KPI-containing isoforms may be an important factor influencing A␤ deposition (6). In the AD brain (7-9) and in response to N-methyl-Daspartate (NMDA) receptor stimulation (10, 11), there is an increase in the proportion of KPI-to non-KPI-containing isoforms of APP. This has led to the suggestion that the KPIcontaining isoforms of APP can exert important neuroprotective functions, and thus their up-regulation in the AD brain or in response to excitotoxic insult may be to protect against further neuronal loss (12, 13).A major unmet scientific need in the AD field is still to understand the normal function of APP (14). An added complexity is whether the different APP isoforms have similar or distinct localizations, metabolism, and roles (15). A long standing enigma in APP biology has additi...

Gamma-Secretase Inhibitor Activity of a <b><i>Pterocarpus erinaceus</i></b> Extract

“…In the crude membrane extract assay (fig. 4a), AICD production was concomitant to a decrease in β-CTF but not α-CTF, which is also in line with the recent hypothesis that AICD is preferentially produced from APP in a β-secretase dependent manner [32]. It is of note that inhibition of DAPT in in vitro assays was observed at concentrations higher than those used to achieve inhibition of Aβ production in cultured cells (10 µ M vs. 250 n M ).…”

The Amyloid Precursor Protein Represses Expression of Acetylcholinesterase in Neuronal Cell Lines

“…The SN56 cell line is a neuronal cell line derived from murine septal neurons of the basal forebrain (53). A stable SH-SY5Y (APP 695 ) cell line has been generated and characterized previously (37). We also generated a stable SN56 (APP 695 ) cell line that showed substantial increases in APP 695 protein (mature and immature) and also in mRNA transcripts as compared with mock-transfected cells (Fig.…”

“…Protein samples (20 -50 g) were run for 90 min (30 mA and 300 V) using a Bio-Rad gel rig and Invitrogen PowerEase 500 power supply. Western blotting was performed as described previously (37). Primary antibodies used were for AChE (AChE (C-16) catalog no.…”

“…1A) (36). Functional AICD is mostly formed in the amyloidogenic pathway, predominantly from the APP 695 isoform (37)(38)(39). Much of the transcriptional regulation by AICD is dependent on binding of the Fe65 protein, which may facilitate its nuclear translocation (40,41).…”

A Greek Tragedy: The Growing Complexity of Alzheimer Amyloid Precursor Protein Proteolysis

“…Although the AICD produced via either the amyloidogenic or non-amyloidogenic pathways have the same peptide sequence, they appear to be functionally distinct. The AICD produced following ␤-secretase cleavage is transported to the nucleus and binds to the neprilysin gene promoter, whereas that produced following ␣-secretase cleavage is rapidly degraded in the cytosol by insulin-degrading enzyme (6).…”