In Alzheimer disease, ␤-amyloid peptide accumulates in the brain as insoluble amyloid plaques. Amyloid filaments, similar to those found in amyloid plaques, can be assembled in vitro from chemically synthesized ␤-peptides. In this study, we report that antibodies raised against the Nterminal region (1-28) of the ␤-amyloid peptide bind to the in vitro-formed ␤-amyloid assemblies, leading to disaggregation of the fibrils and partial restoration of the peptide's solubility. The concomitant addition of fibrillar ␤-amyloid with these antibodies to PC 12 cells leads to the inhibition of the neurotoxic effects of ␤-amyloid. Some of the mAbs raised against soluble ␤-peptide (1-28) have been found to prevent in vitro fibrillar aggregation of ␤-amyloid peptide. These experimental data suggest that site-directed mAbs interfere with the aggregation of ␤-amyloid and trigger reversal to its nontoxic, normal components. The above findings give hints on how to convert in vivo senile plaques into nontoxic, diffuse components and may have therapeutic interest for those studying Alzheimer disease and other human diseases related to amyloidogenic properties of physiological peptides and proteins.

The f3-amyloid peptide, the hallmark of Alzheimer disease, forms fibrillar toxic aggregates in brain tissue that can be dissolved only by strong denaturing agents. To study 3-amyloid formation and its inhibition, we prepared immune complexes with two monoclonal antibodies (mAbs), AMY-33 and 6F/3D, raised against f3-amyloid fragments spanning amino acid residues 1-28 and 8-17 of the f3-amyloid peptide chain, respectively. In vitro aggregation of ,3-amyloid peptide was induced by incubation for 3 h at 37°C and monitored by ELISA, negative staining electron microscopy, and fluorimetric studies. We found that the mAbs prevent the aggregation of ,B-amyloid peptide and that the inhibitory effect appears to be related to the localization of the antibodybinding sites and the nature of the aggregating agents. Preparation of mAbs against "aggregating epitopes," defined as sequences related to the sites where protein aggregation is initiated, may lead to the understanding and prevention of protein aggregation. The results of this study may provide a foundation for using mAbs in vivo to prevent the g3-amyloid peptide aggregation that is associated with Alzheimer disease.Experimental evidence that ,B-amyloid peptide (,BA4), the hallmark of Alzheimer disease (1, 2), has opposing neuritepromoting and neurotoxic properties that are related to peptide aggregation forms (3-5) has focused the development of appropriate therapeutic approaches toward reducing or eliminating the extent of amyloid fibrillar deposition in the brain (6)(7)(8). Amyloid insolubility has been one of the most insurmountable problems in the initial characterization of the constituent proteins of the isolated plaque cores from brains affected by Alzheimer disease. Strong denaturing conditions, such as high concentrations of urea, guanidine HCl, or extreme pH, are required to break and dissolve such aggregates (2, 9).Under physiological conditions, the synthetic f3A4 adopts an aggregated form and also shows a change from a neuritepromoting to a neurotoxic effect on hippocampal neurons (3-5, 10). Aggregation of fA4 has been shown to depend on pH, peptide concentration, temperature, and time of incubation (11). The so-called pathological chaperones (12) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.In this study, we investigated the ability of mAbs against ,BA4 to suppress the in vitro aggregation of f3A4 via immunocomplexation. We used a sandwich ELISA method and fluorescence monitoring, as well as electron microscope studies, to characterize the effect of two different mAbs on ,B-amyloid formation. MATERIALS AND METHODSAntibodies. Rabbit polyclonal antibodies raised against synthetic P3A4-(1-40) were obtained from Boehringer Mannheim.Aggregation of ,3A4-(1-40)-peptide was monitored by using two commercially available mAbs raised against the respective peptides 8-17 and 1-28 of 13A4 anti-human...

Early diagnosis of Alzheimer's disease is prevented by lack of means to visualize and target ␤ amyloid plaques in the brains of affected people. There are many methods of detecting amyloid plaques by staining postmortem brain tissue, but none are available for monitoring in living patients. We propose anti-␤ amyloid antibodies as a highly specific probe to monitor amyloid plaque formation in living patients. Intranasal administration of filamentous phage as delivery vector of anti-␤ amyloid antibody fragment into Alzheimer's APP transgenic mice enables in vivo targeting of ␤ amyloid plaques. The plaques were covisualized both by thioflavin-S and fluorescent-labeled antiphage antibodies in the olfactory bulb and the hippocampus region. The genetically engineered filamentous bacteriophage proved to be an efficient and nontoxic viral delivery vector to the brain, offering an obvious advantage over other mammalian vectors. The ability to image A␤ deposits in vivo would arguably provide the most useful diagnostic and monitoring test for early diagnosis of Alzheimer's disease.T he amyloid cascade hypothesis proposes that the onset and progression of Alzheimer's disease (AD) result from increased production of ␤ amyloid peptides (A␤P) and their progressive accumulation in senile plaques (reviewed in ref. 1). The clinical trials of novel techniques to decrease the burden of amyloid plaques (A␤) (2, 3) required reliable and sensitive methods to monitor plaque burden in the brains of living AD patients. Accurate targeting of A␤ in brain regions could allow the preventative treatment and monitoring of drug efficacy during and after those trials.There are different methods to detect plaques by staining postmortem brain tissue; however, none are yet available for monitoring living patients. Attempts to develop such probes have focused on Chrysamine G (CG), a derivative of Congo red that also binds A␤ in vitro and could be a potential probe for detecting plaques in vivo because it crosses the blood-brain barrier (BBB) of mice (4). Styrylbenzene derivative (BSB) is another probe recently designed to penetrate the BBB (5) and bind brain A␤ deposits, enabling radiological imaging of plaques in the brains of living animals. However, as aggregated fibrils with a ␤ pleated structure are a common neuropathological feature of several neurodegenerative diseases, amyloid-binding probes such as BSB or CG are not specific only for AD. Injection by Wengenack et al. (6) of 125 I-PUT-A␤ 1-40 into the femoral veins of 6-mo-old transgenic mice resulted in in vivo labeling of some neuritic plaques.Here we propose A␤P antibodies displayed on filamentous bacteriophage as a highly specific probe to scan brain A␤. The phage maintains the inert properties of the delivery vector and the ability to carry and preserve the biological activity of the antibodies. We demonstrated the usefulness of this A␤-specific antibody for in vivo targeting of ␤ amyloid deposition in live transgenic mice. Two complementary model systems are used to show how phages pen...

The epitope EFRH, corresponding to amino acids 3-6 within the human ␤-amyloid peptide (A␤P), acts as a regulatory site controlling both the formation and disaggregation process of the ␤-amyloid fibrils (A␤). Locking of this epitope by highly specific antibodies affects the dynamics of the entire A␤P molecule, preventing selfaggregation as well as enabling resolubilization of already formed aggregates. Production of such antibodies by repeated injections of toxic human A␤ fibrils into transgenic mice suggests the feasibility of vaccination against Alzheimer's disease. Here, we report the development of an immunization procedure for the production of effective anti-aggregating ␤-amyloid antibodies based on filamentous phages displaying the EFRH peptide as specific and nontoxic antigen. Effective autoimmune antibodies were obtained by EFRH phage administration in guinea pigs, which exhibit A␤P identical to the human A␤P region. Moreover, because of the high antigenicity of the phage, no adjuvant is required to obtain high affinity anti-aggregating IgG antibodies after a short immunization period of 3 weeks. Availability of such antibodies opens up possibilities for the development of an efficient and long-lasting vaccination for the prevention and treatment of Alzheimer's disease.Alzheimer's disease ͉ ␤-amyloid ͉ vaccine ͉ EFRH phage ͉ autoantibodies

Amyloid-␤ peptide (A␤P) that accumulates in the Alzheimer's diseased brain is derived from proteolytic processing of the amyloid precursor protein (APP) by means of ␤-and ␥-secretases. The ␤-secretase APP cleaving enzyme (BACE), which generates the N terminus of A␤P, has become a target of intense research aimed at blocking the enzyme activity, thus reducing A␤P and, subsequently, plaque formation. The search for specific inhibitors of ␤-secretase activity as a possible treatment for Alzheimer's disease intensified with the discovery that BACE may be involved in processing other non-APP substrates. The presence of the APP-BACE complex in early endosomes highlights the cell surface as a potential therapeutic target, suggesting that interference in APP-BACE interaction at the cell surface may affect amyloid-␤ production. We present here a unique approach to inhibit A␤P production by means of antibodies against the ␤-secretase cleavage site of APP. These antibodies were found to bind human APP overexpressed by CHO cells, and the formed immunocomplex was visualized in the early endosomes. Indeed, blocking of the ␤-secretase site by these antibodies interfered with BACE activity and inhibited both intracellular and extracellular A␤P formation in these cells.Alzheimer's disease ͉ ␤-secretase site ͉ monoclonal antibodies ͉ amyloid ␤-peptide production ͉ endocytic pathway A lzheimer's disease (AD) is characterized by the accumulation of senile plaques in the brain extracellular space and by intraneuronal accumulation of neurofibrillary tangles. The senile plaques are composed of deposited amyloid-␤ peptides (A␤Ps), which are derived from the enzymatic processing of a type I transmembrane protein called amyloid precursor protein (APP) (1). The ␤-secretase APP cleaving enzyme (BACE) generates the N terminus of the A␤P peptide and produces a membrane-bound C-terminal fragment (CTF), C99. This membrane-bound product serves as a substrate for ␥-secretase complex processing, which releases amyloid peptides of 40 or 42 aa. Pharmacologic and cell biology studies demonstrated that the three major enzymatic activities involved in APP processing, ␣-␤-and ␥-secretases, are distinct in their subcellular localization and in their respective cleavage products (2, 3). It was shown that ␤-secretase activity must reside both in the endosomes (4) and in the secretory pathway (5). Antibody uptake and biotinylation studies showed that most cell surface-located BACE is reinternalized into the early endosomal compartments, from where it can recycle back to the cell surface or can later be retrieved to endosomal͞lysosomal compartments and͞or to the trans-Golgi network (6, 7). The endocytic pathway, responsible for internalization and initial processing of cell surface APP in endosomes, is well established (4,(8)(9)(10). Indeed, the mutagenesis of the APP internalization signal (11) and expression of the dominantnegative dynamin mutant that prevents endocytosis in the transfected cells (10) reduced both A␤P 40 and A␤P 42 secretion levels. Recen...

Intravenous immunoglobulin (IVIg), a purified immunoglobulin fraction manufactured from the blood of healthy humans, is an FDA-approved treatment for many immune and inflammatory diseases. Recent studies have demonstrated that IVIg therapy has several positive effects on patients with Alzheimer's disease (AD). These include improving cognitive functions and lowering the level of soluble amyloid-beta peptide (AbetaP) in the brain. Nonetheless, the mechanism by which IVIg mediates the clearance of AbetaP from the AD brain currently remains unknown. In this study we investigated the molecular basis for the direct and indirect effects of IVIg on AbetaP clearance using the BV-2 cellular microglia line. Specifically, we show that IVIg dissolves preformed AbetaP fibrils in vitro. Moreover, IVIg increases cellular tolerance to AbetaP, enhances microglial migration toward AbetaP deposits, and mediates phagocytosis of AbetaP. Thus, several mechanisms can be considered when examining the effects of IVIg. Our work supports the hypothesis that IVIg interferes by more than one mechanism in clearing AbetaP from the brains of Alzheimer's patients.

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