IgG Conformer's Binding to Amyloidogenic Aggregates.

Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs) have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1) pAb aggregates have greater activity than monomers (HMW species > dimers > monomers), 2) pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR) interactions of F(ab) regions, and 3) pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg), had up to ~200- and ~7-fold stronger binding to aggregates of Aß and transthyretin (TTR) than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aß- and Cibacron blue-isolated IVIg IgGs. Human pAb conformer's binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR) component to pAb's activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aß and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs.

Secreted amyloid ß-proteins in a cell culture model include N-terminally extended peptides that impair synaptic plasticity.

Evidence for a central role of amyloid ß-protein (Aß) in the genesis of Alzheimer's disease (AD) has led to advanced human trials of Aß-lowering agents. The "amyloid hypothesis" of AD postulates deleterious effects of small, soluble forms of Aß on synaptic form and function. Because selectively targeting synaptotoxic forms of soluble Aß could be therapeutically advantageous, it is important to understand the full range of soluble Aß derivatives. We previously described a Chinese hamster ovary (CHO) cell line (7PA2 cells) that stably expresses mutant human amyloid precursor protein (APP). Here, we extend this work by purifying an sodium dodecyl sulfate (SDS)-stable, ∼8 kDa Aß species from the 7PA2 medium. Mass spectrometry confirmed its identity as a noncovalently bonded Aß40 homodimer that impaired hippocampal long-term potentiation (LTP) in vivo. We further report the detection of Aß-containing fragments of APP in the 7PA2 medium that extend N-terminal from Asp1 of Aß. These N-terminally extended Aß-containing monomeric fragments are distinct from soluble Aß oligomers formed from Aß1-40/42 monomers and are bioactive synaptotoxins secreted by 7PA2 cells. Importantly, decreasing ß-secretase processing of APP elevated these alternative synaptotoxic APP fragments. We conclude that certain synaptotoxic Aß-containing species can arise from APP processing events N-terminal to the classical ß-secretase cleavage site.

Human anti-Aß IgGs target conformational epitopes on synthetic dimer assemblies and the AD brain-derived peptide.

Soluble non-fibrillar assemblies of amyloid-beta (Aß) and aggregated tau protein are the proximate synaptotoxic species associated with Alzheimer's disease (AD). Anti-Aß immunotherapy is a promising and advanced therapeutic strategy, but the precise Aß species to target is not yet known. Previously, we and others have shown that natural human IgGs (NAbs) target diverse Aß conformers and have therapeutic potential. We now demonstrate that these antibodies bound with nM avidity to conformational epitopes on plate-immobilized synthetic Aß dimer assemblies, including synaptotoxic protofibrils, and targeted these conformers in solution. Importantly, NAbs also recognized Aß extracted from the water-soluble phase of human AD brain, including species that migrated on denaturing PAGE as SDS-stable dimers. The critical reliance on Aß's conformational state for NAb binding, and not a linear sequence epitope, was confirmed by the antibody's nM reactivity with plate-immobilized protofibrills, and weak uM binding to synthetic Aß monomers and peptide fragments. The antibody's lack of reactivity against a linear sequence epitope was confirmed by our ability to isolate anti-Aß NAbs from intravenous immunoglobulin using affinity matrices, immunoglobulin light chain fibrils and Cibacron blue, which had no sequence similarity with the peptide. These findings suggest that further investigations on the molecular basis and the therapeutic/diagnostic potential of anti-Aß NAbs are warranted.

Isolation of low-n amyloid ß-protein oligomers from cultured cells, CSF, and brain.

Recent data suggest that soluble, non-fibrillar assemblies of the amyloid ß-protein (Aß) may mediate the synaptic deficits that characterize the early stages of Alzheimer's disease. Consequently, much effort has been expended in isolating and studying a variety of different Aß assemblies. Here, we describe the use of immunoprecipitation/western blotting and size exclusion chromatography/western blotting to characterize Aß present in conditioned medium from cultured cells, human cerebrospinal fluid, and human cortex extracted with aqueous buffer, detergent, and formic acid.

Cognitive effects of cell-derived and synthetically derived Aß oligomers.

Soluble forms of amyloid-ß peptide (Aß) are a molecular focus in Alzheimer's disease research. Soluble Aß dimers (≈8 kDa), trimers (≈12 kDa), tetramers (≈16 kDa) and Aß*56 (≈56 kDa) have shown biological activity. These Aß molecules have been derived from diverse sources, including chemical synthesis, transfected cells, and mouse and human brain, leading to uncertainty about toxicity and potency. Herein, synthetic Aß peptide-derived oligomers, cell- and brain-derived low-n oligomers, and Aß*56, were injected intracerebroventricularly (icv) into rats assayed under the Alternating Lever Cyclic Ratio (ALCR) cognitive assay. Cognitive deficits were detected at 1.3 µM of synthetic Aß oligomers and at low nanomolar concentrations of cell-secreted Aß oligomers. Trimers, from transgenic mouse brain (Tg2576), did not cause cognitive impairment at any dose tested, whereas Aß*56 induced concentration-dependent cognitive impairment at 0.9 and 1.3µM. Thus, while multiple forms of Aß have cognition impairing activity, there are significant differences in effective concentration and potency.

The presence of sodium dodecyl sulphate-stable Abeta dimers is strongly associated with Alzheimer-type dementia.

The molecular pathways leading to Alzheimer-type dementia are not well understood, but the amyloid beta-protein is believed to be centrally involved. The quantity of amyloid beta-protein containing plaques does not correlate well with clinical status, suggesting that if amyloid beta-protein is pathogenic it involves soluble non-plaque material. Using 43 brains from the Newcastle cohort of the population-representative Medical Research Council Cognitive Function and Ageing Study, we examined the relationship between biochemically distinct forms of amyloid beta-protein and the presence of Alzheimer-type dementia. Cortical samples were serially extracted with Tris-buffered saline, Tris-buffered saline containing 1% TX-100 and with 88% formic acid and extracts analysed for amyloid beta-protein by immunoprecipitation/western blotting. The cohort was divisible into those with dementia at death with (n = 14) or without (n = 10) significant Alzheimer-type pathology, and those who were not demented (n = 19). Amyloid beta-protein monomer in extracts produced using Tris-buffered saline and Tris-buffered saline containing 1% TX-100 were strongly associated with Alzheimer type dementia (P < 0.001) and sodium dodecyl sulphate-stable amyloid beta-protein dimer was detected specifically and sensitively in Tris-buffered saline, Tris-buffered saline containing 1% TX-100 and formic acid extracts of Alzheimer brain. Amyloid beta-protein monomer in the formic acid fraction closely correlated with diffuse and neuritic plaque burden, but was not specific for dementia. These findings support the hypothesis that soluble amyloid beta-protein is a major correlate of dementia associated with Alzheimer-type pathology and is likely to be intimately involved in the pathogenesis of cognitive failure.

Substrate-targeting gamma-secretase modulators.

Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.

Amyloid beta protein dimer-containing human CSF disrupts synaptic plasticity: prevention by systemic passive immunization.

The current development of immunotherapy for Alzheimer's disease is based on the assumption that human-derived amyloid beta protein (Abeta) can be targeted in a similar manner to animal cell-derived or synthetic Abeta. Because the structure of Abeta depends on its source and the presence of cofactors, it is of great interest to determine whether human-derived oligomeric Abeta species impair brain function and, if so, whether or not their disruptive effects can be prevented using antibodies. We report that untreated ex vivo human CSF that contains Abeta dimers rapidly inhibits hippocampal long-term potentiation in vivo and that acute systemic infusion of an anti-Abeta monoclonal antibody can prevent this disruption of synaptic plasticity. Abeta monomer isolated from human CSF did not affect long-term potentiation. These results strongly support a strategy of passive immunization against soluble Abeta oligomers in early Alzheimer's disease.