Debra Yager

Familial Alzheimer's Disease–Linked Presenilin 1 Variants Elevate Aβ1–42/1–40 Ratio In Vitro and In Vivo

Mutations in the presenilin 1 (PS1) and presenilin 2 genes cosegregate with the majority of early-onset familial Alzheimers disease (FAD) pedigrees. We now document that the Abeta1-42(43)/Abeta1-40 ratio in the conditioned media of independent N2a cell lines expressing three FAD-linked PS1 variants is uniformly elevated relative to cells expressing similar levels of wild-type PS1. Similarly, the Abeta1-42(43)/Abeta1-40 ratio is elevated in the brains of young transgenic animals coexpressing a chimeric amyloid precursor protein (APP) and an FAD-linked PS1 variant compared with brains of transgenic mice expressing APP alone or transgenic mice coexpressing wild-type human PS1 and APP. These studies provide compelling support for the view that one mechanism by which these mutant PS1 cause AD is by increasing the extracellular concentration of Abeta peptides terminating at 42(43), species that foster Abeta deposition.

Substrate-targeting γ-secretase modulators

Selective lowering of Aβ42 levels (the 42-residue isoform of the amyloid-β peptide) with small-molecule γ-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 γ-secretase complex, but instead labelled the β-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-β peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP γ-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28–36 of amyloid-β, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-β act as GSMs, and some GSMs alter the production of cell-derived amyloid-β 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 Aβ42 production and inhibition of amyloid-β aggregation, which may synergistically reduce amyloid-β 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.

Flemish and Dutch Mutations in Amyloid β Precursor Protein Have Different Effects on Amyloid β Secretion

Mutations in the amyloid beta precursor protein (APP) gene cosegregate with autosomal dominant Alzheimer disease (AD). Brain pathology of AD is characterized by amyloid deposition in senile plaques and by neurofibrillary tangles. Amyloid deposits in AD brains consist of amyloid beta (A beta), a 4-kDa proteolytic product of APP. In contrast, two other mutations in APP, the Flemish APP692 and Dutch APP693 mutations, are associated with autosomal dominant cerebral hemorrhages due to congophilic amyloid angiopathy (CAA) in the presence or absence of AD pathology, respectively. Both mutations are located within A beta near the constitutive cleavage site. While a common effect of AD-linked mutations is to elevate A beta 42 extracellular concentrations, not much is known about the effect of APP692 and APP693. Here we provide evidence that APP692 and APP693 have a different effect on A beta secretion as determined by cDNA transfection experiments. While APP692 upregulates both A beta 40 and A beta 42 secretion, APP693 does not. These data corroborate with previous findings that increased A beta secretion and particularly of A beta 42, is specific for AD pathology.

Plasma amyloid β protein is elevated in late-onset Alzheimer disease families

Objective: Plasma Aβ levels are elevated in early-onset Alzheimer disease (AD) caused by autosomal dominant mutations. Our objective was to determine whether similar genetic elevations exist in late-onset AD (LOAD). Methods: We measured plasma Aβ in first-degree relatives of patients with LOAD in a cross-sectional series and in extended LOAD families. We screened these subjects for pathogenic mutations in early-onset AD genes and determined their ApoE genotypes. Results: Plasma Aβ is significantly elevated in the LOAD first-degree relatives in comparison to unrelated controls and married-in spouses. These elevations are not due to ApoE ε4 or pathogenic coding mutations in the known early-onset AD genes. Conclusions: The findings provide strong evidence for the existence of novel, as yet unknown genetic factors that affect late-onset Alzheimer disease by increasing Aβ.

β-Secretase cleavage of the amyloid precursor protein mediates neuronal apoptosis caused by familial Alzheimer’s disease mutations ☆

The amyloid precursor protein (APP) is cleaved by two enzymes, beta-secretase and gamma-secretase, to generate the pathological amyloid beta (Abeta) peptide. Expression of familial Alzheimers disease (FAD) mutants of APP in primary neurons causes both intracellular accumulation of the C-terminal beta-secretase cleavage product of APP and increased secretion of Abeta, and eventually results in apoptotic death of the cells. To determine whether either of these two processing products of APP is involved in this apoptotic pathway, we first modeled experimentally the accumulation of the beta-secretase cleavage product in neurons. The C-terminal 100 amino acids (C100) of APP, with and without a signal peptide, was expressed in cells via recombinant herpes simplex virus (HSV) vectors. Both transgene products were targeted to the membrane, and both caused apoptosis in the neurons, implicating the beta-secretase cleavage product of APP in apoptosis caused by FAD APPs. Expression in neurons of a mutant of FAD APP that inhibited beta-secretase cleavage inhibited its ability to cause apoptosis. However, expression in neurons of a mutant of FAD APP that inhibited gamma-secretase cleavage did not inhibit the ability of this mutant to cause apoptosis. These data suggested that the C-terminal beta-secretase cleavage product of APP, but not Abeta, mediates the apoptosis caused by FAD mutants of APP. Consistent with this hypothesis, C31, which is generated from the beta-secretase cleavage product, itself caused neuronal apoptosis. Inhibitors of caspases 3, 6 and 8, but not of caspase 9, inhibited the apoptosis caused by FAD mutants of APP. It may be inferred from these data that beta-secretase cleavage of FAD mutants of APP allows the appropriate caspase access to its site of action to produce C31, which directly causes neuronal apoptosis.

Reduction of Aβ accumulation in the Tg2576 animal model of Alzheimer’s disease after oral administration of the phosphatidyl-inositol kinase inhibitor wortmannin 1

The abnormal accumulation of the amyloid β protein (Aβ) has been implicated as an early and critical event in the etiology and pathogenesis of Alzheimers disease (AD). Compounds that reduce Aβ accumulation may therefore be useful therapeutically. In cell‐based screens we detected a significant reduction in Aβ concentration after treatment with the phosphatidylinositol kinase inhibitors wortmannin and LY294002. To determine the effect of this class of compounds on in vivo Aβ accumulation, we administered wortmannin to the Tg2576 mouse model of AD. Oral administration of wortmannin over four months resulted in a significant, non‐overlapping 40%–50% reduction in the number of senile plaques, one of the pathological hallmarks of AD. Sandwich ELISA analysis of formic acid extractable Aβ in the brain of treated animals indicates that both Aβ40 and the longer, more amyloidogenic form of the peptide, Aβ42, were significantly reduced. These data provide the first direct evidence that compounds identified by their ability to reduce Aβ concentration in vitro can reduce Aβ accumulation and deposition in the brain, thus establishing a basic paradigm for the identification and evaluation of additional compounds that lower Aβ accumulation.

Glycosylphosphatidylinositol-anchored Proteins Play an Important Role in the Biogenesis of the Alzheimer’s Amyloid β-Protein

The Alzheimer’s amyloid protein (Aβ) is released from the larger amyloid β-protein precursor (APP) by unidentified enzymes referred to as β- and γ-secretase. β-Secretase cleaves APP on the amino side of Aβ producing a large secreted derivative (sAPPβ) and an Aβ-bearing C-terminal derivative that is subsequently cleaved by γ-secretase to release Aβ. Alternative cleavage of the APP by α-secretase at Aβ16/17 releases the secreted derivative sAPPα. In yeast, α-secretase activity has been attributed to glycosylphosphatidylinositol (GPI)-anchored aspartyl proteases. To examine the role of GPI-anchored proteins, we specifically removed these proteins from the surface of mammalian cells using phosphatidylinositol-specific phospholipase C (PI-PLC). PI-PLC treatment of fetal guinea pig brain cultures substantially reduced the amount of Aβ40 and Aβ42 in the medium but had no effect on sAPPα. A mutant CHO cell line (gpi85), which lacks GPI-anchored proteins, secreted lower levels of Aβ40, Aβ42, and sAPPβ than its parental line (GPI+). When this parental line was treated with PI-PLC, Aβ40, Aβ42, and sAPPβ decreased to levels similar to those observed in the mutant line, and the mutant line was resistant to these effects of PI-PLC. These findings provide strong evidence that one or more GPI-anchored proteins play an important role in β-secretase activity and Aβ secretion in mammalian cells. The cell-surface GPI-anchored protein(s) involved in Aβ biogenesis may be excellent therapeutic target(s) in Alzheimer’s disease.

High throughput screens for the identification of compounds that alter the accumulation of the Alzheimer's amyloid β peptide (Aβ)

Evidence gathered over the last two decades suggests that β amyloid (Aβ), the predominant proteinaceous component of senile plaques, plays an early and critical role in the etiology and pathogenesis of Alzheimers disease (AD). Thus, it is reasonable to hypothesize that compounds capable of reducing the accumulation of Aβ may be of value therapeutically. Additionally, compounds that influence Aβ accumulation may be useful as tools to further dissect the cellular pathways that regulate Aβ production and accumulation. To screen for compounds that affect Aβ levels, we have established high throughput, cell-based assays capable of the sensitive and selective detection of Aβ40 in parallel with the more amyloidogenic form of the peptide, Aβ42. To validate the approach, we examined the effects of several compounds previously identified to influence Aβ accumulation. Analysis of peptide accumulation following treatment with these compounds showed results similar to those previously published. Currently, we are using this assay to screen drugs that have already received FDA approval for the treatment of other diseases and over-the-counter natural product extracts. If compounds such as these can be identified that lower Aβ in the brain, they may represent one of the fastest and most cost effective methods to therapy.

Natural product extracts that reduce accumulation of the Alzheimer's amyloid beta peptide: selective reduction in A beta42.

Alzheimer’s disease (AD) is the most common form of dementia in the elderly. Without a treatment that significantly delays the progression of the disease over 14 million Americans are likely to be affected with AD by the middle of the 21st Century, presenting an enormous economic and social burden. Evidence gathered over the last two decades has implicated the abnormal accumulation of Aβ, in particular the longer more amyloidogenic form Aβ42, as a potential causative agent in the disease. To screen for compounds that reduce Aβ accumulation we have established several high throughput, cell based screens capable of the sensitive and selective detection of Aβ40 and Aβ42. Using these screens we have analyzed a proprietary library of natural product extracts for their ability to influence Aβ accumulation. Using this approach, we have identified several agents capable of influencing total Aβ concentration. In addition, we have identified one extract that selectively reduces Aβ42. Intracerebroventricular administration of this agent to mice results in a selective reduction in Aβ42 in the brain.