beta-Secretase cleavage is not required for generation of the intracellular C-terminal domain of the amyloid precursor family of proteins.

The amyloid precursor family of proteins are of considerable interest, both because of their role in Alzheimer's disease pathogenesis and because of their normal physiological functions. In mammals, the amyloid precursor protein (APP) has two homologs, amyloid precursor-like protein (APLP) 1 and APLP2. All three proteins undergo ectodomain shedding and regulated intramembrane proteolysis, and important functions have been attributed to the full-length proteins, shed ectodomains, C-terminal fragments and intracellular domains (ICDs). One of the proteases that is known to cleave APP and that is essential for generation of the amyloid beta-protein is the beta-site APP-cleaving enzyme 1 (BACE1). Here, we investigated the effects of genetic manipulation of BACE1 on the processing of the APP family of proteins. BACE1 expression regulated the levels and species of full-length APLP1, APP and APLP2, of their shed ectodomains, and of their membrane-bound C-terminal fragments. In particular, APP processing appears to be tightly regulated, with changes in beta-cleaved APPs (APPsbeta) being compensated for by changes in alpha-cleaved APPs (APPsalpha). In contrast, the total levels of soluble cleaved APLP1 and APLP2 species were less tightly regulated, and fluctuated with BACE1 expression. Importantly, the production of ICDs for all three proteins was not decreased by loss of BACE1 activity. These results indicate that BACE1 is involved in regulating ectodomain shedding, maturation and trafficking of the APP family of proteins. Consequently, whereas inhibition of BACE1 is unlikely to adversely affect potential ICD-mediated signaling, it may alter other important facets of amyloid precursor-like protein/APP biology.

gamma-secretase processing of APLP1 leads to the production of a p3-like peptide that does not aggregate and is not toxic to neurons.

The amyloid precursor-like protein-1 (APLP1) is a member of a protein family that includes the Alzheimer's disease-associated amyloid precursor protein (APP). While much is known about the proteolytic processing of APP, fewer details are available about APLP1. Using Chinese hamster ovarian cells stably transfected with human APLP1 and a novel juxtamembrane anti-APLP1 antibody, we demonstrate the detection of a secreted approximately 3.5 kDa APLP1-derived peptide (ALP-1). The production of this peptide is abolished by inhibition of gamma-secretase, but not beta-secretase, suggesting that ALP-1 is analogous to the p3 fragment produced from APP. However, unlike p3 or Abeta, ALP-1 shows no obvious propensity for aggregation and is not toxic to neuronal cells. Moreover, using two distinct experimental paradigms, we demonstrate that neither cell-derived nor chemically synthesized ALP-1 influences the oligomerization or aggregation of Abeta.

Certain inhibitors of synthetic amyloid beta-peptide (Abeta) fibrillogenesis block oligomerization of natural Abeta and thereby rescue long-term potentiation.

Recent studies support the hypothesis that soluble oligomers of amyloid beta-peptide (Abeta) rather than mature amyloid fibrils are the earliest effectors of synaptic compromise in Alzheimer's disease. We took advantage of an amyloid precursor protein-overexpressing cell line that secretes SDS-stable Abeta oligomers to search for inhibitors of the pathobiological effects of natural human Abeta oligomers. Here, we identify small molecules that inhibit formation of soluble Abeta oligomers and thus abrogate their block of long-term potentiation (LTP). Furthermore, we show that cell-derived Abeta oligomers can be separated from monomers by size exclusion chromatography under nondenaturing conditions and that the isolated, soluble oligomers, but not monomers, block LTP. The identification of small molecules that inhibit early Abeta oligomer formation and rescue LTP inhibition offers a rational approach for therapeutic intervention in Alzheimer's disease and highlights the utility of our cell-culture paradigm as a useful secondary screen for compounds designed to inhibit early steps in Abeta oligomerization under biologically relevant conditions.

Soluble Arctic amyloid beta protein inhibits hippocampal long-term potentiation in vivo.

Mutations in the amyloid precursor protein that result in substitutions of glutamic acid at residue 22 of the amyloid beta protein (A beta) with glutamine (Q22, Dutch) or glycine (G22, Arctic) cause aggressive familial neurological diseases characterized by cerebrovascular haemorrhages or Alzheimer's-type dementia, respectively. The present study compared the ability of these peptides to block long-term potentiation (LTP) of glutamatergic transmission in the hippocampus in vivo. The effects of intracerebroventricular injection of wild-type, Q22 and G22 A beta(1-40) peptides were examined in the CA1 area of urethane-anaesthetized rats. Both mutant peptides were approximately 100-fold more potent than wild-type A beta at inhibiting LTP induced by high-frequency stimulation when solutions of A beta were freshly prepared. Fibrillar material, as determined by electron microscopy, was obvious in all these peptide solutions and exhibited appreciable Congo Red binding, particularly for A beta(1-40)G22 and A beta(1-40)Q22. A soluble fraction of A beta(1-40)G22, obtained following high-speed centrifugation, retained full activity of the peptide solution to inhibit LTP, providing strong evidence that in the case of the Arctic disease a soluble nonfibrillar form of A beta may represent the primary mediator of A beta-related cognitive deficits, particularly early in the disease. In contrast, nonfibrillar soluble A beta(1-40)Q22 supernatant solution was approximately 10-fold less potent at inhibiting LTP than A beta(1-40)G22, a finding consistent with fibrillar A beta contributing to the inhibition of LTP by the Dutch peptide.

gamma-Secretase cleavage and binding to FE65 regulate the nuclear translocation of the intracellular C-terminal domain (ICD) of the APP family of proteins.

Regulated intramembrane proteolysis (RIP) of the amyloid precursor protein (APP) produces amyloid beta-protein (Abeta), the probable causative agent of Alzheimer's disease (AD), and is therefore an important target for therapeutic intervention. However, there is a burgeoning consensus that gamma-secretase, one of the proteases that generates Abeta, is also critical for the signal transduction of APP and a growing list of other receptors. APP is a member of a gene family that includes two amyloid precursor-like proteins, APLP1 and APLP2. Although APP and the APLPs undergo similar proteolytic processing, there is little information about the role of their gamma-secretase-generated intracellular domains (ICDs). Here, we show that APLP1 and 2 undergo presenilin-dependent RIP similar to APP, resulting in the release of a approximately 6 kDa ICD for each protein. Each of the ICDs are degraded by an insulin degrading enzyme-like activity, but they can be stabilized by members of the FE65 family and translocate to the nucleus. Given that modulation of APP processing is a therapeutic target and that the APLPs are processed in a manner similar to APP, any strategy aimed at altering APP proteolysis will have to take into account possible effects on signaling by APLP 1 and 2.

Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo.

Although extensive data support a central pathogenic role for amyloid beta protein (Abeta) in Alzheimer's disease, the amyloid hypothesis remains controversial, in part because a specific neurotoxic species of Abeta and the nature of its effects on synaptic function have not been defined in vivo. Here we report that natural oligomers of human Abeta are formed soon after generation of the peptide within specific intracellular vesicles and are subsequently secreted from the cell. Cerebral microinjection of cell medium containing these oligomers and abundant Abeta monomers but no amyloid fibrils markedly inhibited hippocampal long-term potentiation (LTP) in rats in vivo. Immunodepletion from the medium of all Abeta species completely abrogated this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Abeta monomers but not oligomers, did not prevent the inhibition of LTP. Therefore, Abeta oligomers, in the absence of monomers and amyloid fibrils, disrupted synaptic plasticity in vivo at concentrations found in human brain and cerebrospinal fluid. Finally, treatment of cells with gamma-secretase inhibitors prevented oligomer formation at doses that allowed appreciable monomer production, and such medium no longer disrupted LTP, indicating that synaptotoxic Abeta oligomers can be targeted therapeutically.