Julia V. Fadeeva

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 β protein (Aβ) in Alzheimers disease, the amyloid hypothesis remains controversial, in part because a specific neurotoxic species of Aβ and the nature of its effects on synaptic function have not been defined in vivo. Here we report that natural oligomers of human Aβ 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 Aβ monomers but no amyloid fibrils markedly inhibited hippocampal long-term potentiation (LTP) in rats in vivo. Immunodepletion from the medium of all Aβ species completely abrogated this effect. Pretreatment of the medium with insulin-degrading enzyme, which degrades Aβ monomers but not oligomers, did not prevent the inhibition of LTP. Therefore, Aβ 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 γ-secretase inhibitors prevented oligomer formation at doses that allowed appreciable monomer production, and such medium no longer disrupted LTP, indicating that synaptotoxic Aβ oligomers can be targeted therapeutically.

Certain Inhibitors of Synthetic Amyloid β-Peptide (Aβ) Fibrillogenesis Block Oligomerization of Natural Aβ and Thereby Rescue Long-Term Potentiation

Recent studies support the hypothesis that soluble oligomers of amyloid β-peptide (Aβ) rather than mature amyloid fibrils are the earliest effectors of synaptic compromise in Alzheimers disease. We took advantage of an amyloid precursor protein-overexpressing cell line that secretes SDS-stable Aβ oligomers to search for inhibitors of the pathobiological effects of natural human Aβ oligomers. Here, we identify small molecules that inhibit formation of soluble Aβ oligomers and thus abrogate their block of long-term potentiation (LTP). Furthermore, we show that cell-derived Aβ 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 Aβ oligomer formation and rescue LTP inhibition offers a rational approach for therapeutic intervention in Alzheimers disease and highlights the utility of our cell-culture paradigm as a useful secondary screen for compounds designed to inhibit early steps in Aβ oligomerization under biologically relevant conditions.

The role of cell-derived oligomers of Aβ in Alzheimer's disease and avenues for therapeutic intervention

Burgeoning evidence suggests that soluble oligomers of Abeta (amyloid beta-protein) are the earliest effectors of synaptic compromise in Alzheimers disease. Whereas most other investigators have employed synthetic Abeta peptides, we have taken advantage of a beta-amyloid precursor protein-overexpressing cell line (referred to as 7PA2) that secretes sub-nanomolar levels of low-n oligomers of Abeta. These are composed of heterogeneous Abeta peptides that migrate on SDS/PAGE as dimers, trimers and tetramers. When injected into the lateral ventricle of rats in vivo, these soluble oligomers inhibit hippocampal long-term potentiation and alter the memory of a complex learned behaviour. Biochemical manipulation of 7PA2 medium including immunodepletion with Abeta-specific antibodies and fractionation by size-exclusion chromatography allowed us to unambiguously attribute these effects to low-n oligomers. Using this paradigm we have tested compounds directed at three prominent amyloid-based therapeutic targets: inhibition of the secretases responsible for Abeta production, inhibition of Abeta aggregation and immunization against Abeta. In each case, compounds capable of reducing oligomer production or antibodies that avidly bind Abeta oligomers also ameliorate the synaptotoxic effects of these natural, cell-derived oligomers.

Soluble Arctic amyloid β 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 β protein (Aβ) with glutamine (Q22, Dutch) or glycine (G22, Arctic) cause aggressive familial neurological diseases characterized by cerebrovascular haemorrhages or Alzheimers‐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 Aβ(1–40) peptides were examined in the CA1 area of urethane‐anaesthetized rats. Both mutant peptides were ≈ 100‐fold more potent than wild‐type Aβ at inhibiting LTP induced by high‐frequency stimulation when solutions of Aβ 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 Aβ(1–40)G22 and Aβ(1–40)Q22. A soluble fraction of Aβ(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 Aβ may represent the primary mediator of Aβ‐related cognitive deficits, particularly early in the disease. In contrast, nonfibrillar soluble Aβ(1–40)Q22 supernatant solution was ≈ 10‐fold less potent at inhibiting LTP than Aβ(1–40)G22, a finding consistent with fibrillar Aβ contributing to the inhibition of LTP by the Dutch peptide.

The APP family of proteins: similarities and differences.

Overwhelming evidence indicates that the Abeta (amyloid beta-peptide) plays a critical role in the pathogenesis of Alzheimers disease. Abeta is derived from the APP (amyloid precursor protein) by the action of two aspartyl proteases (beta- and gamma-secretases) that are leading candidates for therapeutic intervention. APP is a member of a multigene family that includes APLP1 (amyloid precursor-like protein 1) and APLP2. Both APLPs are processed in a manner analogous to APP, with all three proteins subject to ectodomain shedding and subsequent cleavage by gamma-secretase. Careful study of the APP family of proteins has already revealed important insights about APP. Here, we will review how knowledge of the similarities and differences between APP and the APLPs may prove useful for the development of novel disease-modifying therapeutics.

β-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 β‐protein is the β‐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 β‐cleaved APPs (APPsβ) being compensated for by changes in α‐cleaved APPs (APPsα). 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.

γ-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 Alzheimers 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.