Search

Background: The deposition of neurotoxic amyloid-β (Aβ) peptides in plaques in the brain parenchyma and in cerebral blood vessels is considered to be a key event in Alzheimer's disease (AD) pathogenesis. Although the presence and impact of full-length Aβ peptides such as Aβ1-40 and Aβ1-42 have been analyzed extensively, the deposition of N-terminally truncated Aβ peptide species has received much less attention, largely because of the lack of specific antibodies. Methods: This paper describes the generation and characterization of novel antibodies selective for Aβ4-x peptides and provides immunohistochemical evidence of Aβ4-x in the human brain and its distribution in the APP/PS1KI and 5XFAD transgenic mouse models. Results: The Aβ4-x staining pattern was restricted mainly to amyloid plaque cores and cerebral amyloid angiopathy in AD and Down syndrome cases and in both AD mouse models. In contrast, diffuse amyloid deposits were largely negative for Aβ4-x immunoreactivity. No overt intraneuronal staining was observed. Conclusions: The findings of this study are consistent with previous reports demonstrating a high aggregation propensity of Aβ4-x peptides and suggest an important role of these N-truncated Aβ species in the process of amyloidogenesis and plaque core formation. [ABSTRACT FROM AUTHOR]

According to the modified amyloid hypothesis, the key event in the pathogenesis of Alzheimer's disease (AD) is the deposition of neurotoxic amyloid β-peptides (Aβs) in plaques and cerebral blood vessels. Additionally to full-length peptides, a great diversity of N-truncated Aβ variants is derived from the larger amyloid-β protein precursor (AβPP). Vast evidence suggests that Aβx-42 isoforms play an important role in triggering neurodegeneration due to their high abundance, amyloidogenic propensity and toxicity. Although N-truncated Aβ peptides and Aβx-42 species appear to be the crucial players in AD etiology, the Aβ2-X isoforms did not receive much attention yet. The present study is the first to show immunohistochemical evidence of Aβ2-X in cases of AD and its distribution in AβPP/PS1KI and 5XFAD transgenic mouse models using a novel antibody pAB77 that has been developed using Aβ2-14 as antigen. Positive plaques and congophilic amyloid angiopathy (CAA) were observed in AD cases and in both mouse models. While in AD cases, abundant CAA and less pronounced plaque pathology was evident, the two mouse models showed predominantly extracellular Aβ deposits and minor CAA staining. Western blotting and a capillary isoelectric focusing immunoassay demonstrated the high specificity of the antibody pAb77 against Aβ-variants starting with the N-terminal Alanine-2. [ABSTRACT FROM AUTHOR]

Solanezumab and Crenezumab are two humanized antibodies targeting Amyloid-β (Aβ) which are currently tested in multiple clinical trials for the prevention of Alzheimer's disease. However, there is a scientific discussion ongoing about the target engagement of these antibodies. Here, we report the immunohistochemical staining profiles of biosimilar antibodies of Solanezumab, Crenezumab and Bapineuzumab in human formalin-fixed, paraffin-embedded tissue and human fresh frozen tissue. Furthermore, we performed a direct comparative immunohistochemistry analysis of the biosimilar versions of the humanized antibodies in different mouse models including 5XFAD, Tg4-42, TBA42, APP/PS1KI, 3xTg. The staining pattern with these humanized antibodies revealed a surprisingly similar profile. All three antibodies detected plaques, cerebral amyloid angiopathy and intraneuronal Aβ in a similar fashion. Remarkably, Solanezumab showed a strong binding affinity to plaques. We also reaffirmed that Bapineuzumab does not recognize N-truncated or modified Aβ, while Solanezumab and Crenezumab do detect N-terminally modified Aβ peptides Aβ4-42 and pyroglutamate Aβ3-42. In addition, we compared the results with the staining pattern of the mouse NT4X antibody that recognizes specifically Aβ4-42 and pyroglutamate Aβ3-42, but not full-length Aβ1-42. In contrast to the biosimilar antibodies of Solanezumab, Crenezumab and Bapineuzumab, the murine NT4X antibody shows a unique target engagement. NT4X does barely cross-react with amyloid plaques in human tissue. It does, however, detect cerebral amyloid angiopathy in human tissue. In Alzheimer mouse models, NT4X detects intraneuronal Aβ and plaques comparable to the humanized antibodies. In conclusion, the biosimilar antibodies Solanezumab, Crenezumab and Bapineuzumab strongly react with amyloid plaques, which are in contrast to the NT4X antibody that hardly recognizes plaques in human tissue. Therefore, NT4X is the first of a new class of therapeutic antibodies. [ABSTRACT FROM AUTHOR]

Background According to the modified amyloid hypothesis the main event in the pathogenesis of Alzheimer's disease (AD) is the deposition of neurotoxic amyloid β-peptide (Aβ) within neurons. Additionally to full-length peptides, a great diversity of N-truncated Aβ variants is derived from the larger amyloid precursor protein (APP). Vast evidence suggests that Aβx-42 isoforms play an important role triggering neurodegeneration due to its high abundance, amyloidogenic propensity and toxicity. Although N-truncated and Aβx-42 species have been pointed as crucial players in AD etiology, the Aβ5-x isoforms have not received much attention. Results The present study is the first to show immunohistochemical evidence of Aβ5-x in familial cases of AD (FAD) and its distribution in APP/PS1KI, 5XFAD and 3xTG transgenic mouse models. In order to probe Aβ5-x peptides we generated the AB5-3 antibody. Positive plaques and congophilic amyloid angiopathy (CAA) were observed among all the FAD cases tested carrying either APP or presenilin 1 (PS1) mutations and most of the sporadic cases of AD (SAD). Different patterns of Aβ5-x distribution were found in the mouse models carrying different combinations of autosomal mutations in the APP, PS1 and Tau genes. All of them showed extracellular Aβ deposits but none CAA. Additionally, they were all affected by a severe amyloid pathology in the hippocampus among other areas. Interestingly, neither 5XFAD nor APP/PS1KI showed any evidence for intraneuronal Aβ5-x. Conclusions Different degrees of Aβ5-x accumulations can be found in the transgenic AD mouse models and human cases expressing the sporadic or the familial form of the disease. Due to the lack of intracellular Aβ5-x, these isoforms might not be contributing to early mechanisms in the cascade of events triggering AD pathology. Brain sections obtained from SAD cases showed higher Aβ5-x-immunoreactivity in vascular deposits than in extracellular plaques, while both are equally important in the FAD cases. The difference may rely on alternative mechanisms involving Aβ5-x peptides and operating in a divergent way in the late and early onset forms of the disease. [ABSTRACT FROM AUTHOR]

AβpE3-42 (N-terminal truncated amyloid-β peptide starting with pyroglutamate at the third position) is abundant in Alzheimer's disease (AD) brain and has high aggregation propensity and cellular toxicity. Transgenic TBA42 mice expressing AβpE3-42 exhibit a neurological phenotype evident at the age of 12 months. As AD has a long presymptomatic period, early detection of imminent neurodegeneration is highly desirable. In the present work we used four-month-old presymptomatic TBA42 mice and performed a whole-brain proteome analysis in order to elucidate early AD-related pathological changes and the molecular networks involved. At least three proteins were found to be moderately (by 17% to 28%) but statistically significantly upregulated, including: nectin-like molecule 1 involved in cell-cell adhesion; Homer proteins involved in scaffolding, organizing proteins at synapse and regulating intracellular calcium within neurons; and inositol-trisphosphate 3-kinase A, which is important for InsP3 induced calcium signaling in the brain. Analysis of key nodes (regulatory molecules found on pathway intersections) identified Rho-kinase (ROCK), a serine/threonine kinase and one of the major downstream effectors of the small GTPase Rho, as well as three key nodes of the mTOR/p70S6K signaling pathway previously implicated in multiple fundamental biological processes including synaptic plasticity, and upregulated in AD. These data confirm that AD-typical molecular pathways can be detected by whole-brain shotgun proteomics in young presymptomatic mice long before the onset of behavioral changes. [ABSTRACT FROM AUTHOR]

N-truncated Aβ is highly abundant in Alzheimer disease (AD) brain and was the first Aβ peptide discovered in AD plaques. However, a possible role in AD aetiology has largely been neglected. In the present report, we demonstrate that Aβ rapidly forms aggregates possessing a high aggregation propensity in terms of monomer consumption and oligomer formation. Short-term treatment of primary cortical neurons indicated that Aβ is as toxic as pyroglutamate Aβ and Aβ. In line with these findings, treatment of wildtype mice using intraventricular Aβ injection induced significant working memory deficits with Aβ, pyroglutamate Aβ and Aβ. Transgenic mice expressing Aβ (Tg4-42 transgenic line) developed a massive CA1 pyramidal neuron loss in the hippocampus. The hippocampus-specific expression of Aβ correlates well with age-dependent spatial reference memory deficits assessed by the Morris water maze test. Our findings indicate that N-truncated Aβ triggers acute and long-lasting behavioral deficits comparable to AD typical memory dysfunction. [ABSTRACT FROM AUTHOR]

N-terminally truncated pyroglutamate amyloid-β (Aβ) starting at position 3 (AβpE3) represents a major fraction of Aβ peptides in Alzheimer's disease (AD). Recently, we have identified low molecular weight AβpE3 oligomers, which can be detected by 9D5, a novel mouse monoclonal antibody. In the present study, we analyzed the immunohistochemical staining profile in the brain of patients with AD and in the APP/PS1KI mouse model, as well as in aged rhesus monkeys. 9D5-positive microglia and blood vessels were found in many AD cases, in the transgenic mouse model, and in an aged macaque. The presence of 9D5-immunoreactivity in microglia indicates that low molecular weight AβpE3 oligomers may be phagocytosed, since in the APP/PS1KI model, Aβ is exclusively produced in neurons due to neuronal expression of transgenic AβPP. [ABSTRACT FROM AUTHOR]

Abstract: The staining protocols so far applied to study intracellular Aβ accumulation in human tissue have been inconsistent with varying use of heat and formic acid (FA) for antigen retrieval. Microwave heat treatment has been reported to enhance the staining of intraneuronal Aβ as compared to no or enzymatic pretreatment. FA is widely used to increase the staining of plaque pathology in AD, yet the effect of FA on intraneuronal Aβ staining has been reported to be low and similar to the effect of heat or even to counteract the enhancing effect of heat pretreatment on intraneuronal Aβ immunohistochemical detection. To overcome these inconsistencies, there is a need for optimization of the staining protocol for intraneuronal Aβ detection and more knowledge is required concerning the effects of the different antigen retrieval methods. In the present work, we optimized the staining protocol for intraneuronal Aβ in paraffin-embedded sections in relation to heat and FA using four different mouse models known to accumulate intraneuronal Aβ peptides. It was found that FA is essential for the staining of highly aggregated intraneuronal Aβ peptides in AD transgenic mouse tissue. [Copyright &y& Elsevier]

Accumulating evidence points to an important role of intraneuronal Aβ as a trigger of the pathological cascade of events leading to neurodegeneration and eventually to Alzheimer's disease (AD) with its typical clinical symptoms, like memory impairment and change in personality. In the present article, we review recent findings on intracellular monomeric and oligomericβ-amyloid (Aβ) generation and its pathological function in cell culture, transgenic AD mouse models and post mortem brain tissue of AD and Down syndrome patients, as well as its interaction with oxidative stress and its relevance in apoptotic cell death. Based on these results, a modified Aβ hypothesis is formulated, that integrates biochemical, neuropathological and genetic observations with AD-typical neuron loss and plaque formation. [ABSTRACT FROM AUTHOR]

The cu-binding &beta-amyloid precursor protein (APP), and the amyloid Aβ peptide have been proposed to play a role in physiological metal regulation. There is accumulating evidenceo f an unbalance Cu homeostasis with a causative or diagnostic link to Alzheimer's disease. Whereas elevated Cu levels are observed in APP knockout mice, APP overexpression results in reduced Cu in transgenic mouse brain. Moreover, Cu induces a decrease in Aβ levels in APP-transfected cells in vitro. To investigate the influence of bioavailable Cu, transgenic APP23 mice received an oral treatment with Cu-supplemented sucrose-sweetened drinking water (1). Chronic APP overexpression per se reduced superoxide dismutase 1 activity in transgenic mouse brain, which could be restored to normal levels after Cu treatment (2). A significant increase of brain Cu indicated its bioavailability on Cu treatment in APP23 mice, whereas Cu levels remained unaffected in littermate controls (3). Cu treatment lowered endogenous CNS Aβ before a detectable reduction of amyloid plaques. Thus, AOPP23 mice reveal APP-induced alterations linked to Cu homeostasis, which can be reversed by addition of dietary Cu. [ABSTRACT FROM AUTHOR]

α-Synuclein is a presynaptic protein that is implicated in the pathogenesis of various neurodegenerative diseases. Missense mutations in the α-synuclein gene are linked to familial cases of Parkinson''s disease (PD), and it has further been shown that α-synuclein is a major constituent of the Lewy bodies in sporadic PD and dementia with Lewy body (DLB). The contribution of α-synuclein to the pathological changes in Alzheimer''s disease (AD) has been currently a matter of scientific debate. Some reports hypothesized that α-synuclein may play a role in amyloid β/A4 protein (Aβ) aggregation in senile plaques, whereas recent reports challenged this finding by showing a lack of α-synuclein-immunoreactivity in Aβ plaques. In this review, we report on recent findings on the physiological and pathological role of α-synuclein and try to elucidate its possible contribution to AD pathology. [Copyright &y& Elsevier]

Abstract: There is pivotal evidence that tau pathology can be triggered by amyloid-β (Aβ) pathology in experimental systems. On the other side, studies on human brain specimen have elucidated that tau pathology may occur before amyloid pathology is present indicating that in principle tau pathology could also trigger Aβ aggregation. To address this question, we have crossed 5XFAD mice coexpressing human mutant APP695 with the Swedish, Florida, and London mutations and human mutant presenilin-1 (PS1) with the M146L and L286V mutations with the PS19 model overexpressing human mutant tau with the P301S mutation. The resulting triple transgenic model 5XFAD/PS19 has been characterized at 3 and 9 months of age. A dramatic aggravation of hyperphosphorylated tau pathology together with a dramatically increased inflammatory response and a loss of synapses and hippocampal CA1 neurons in aged 5XFAD/PS19 mice were observed. Extracellular amyloid deposits were unaltered. These data support the assumption of tau pathology being downstream of amyloid pathology, suggesting that both pathologies together trigger the severe neuron loss in the hippocampus in the 5XFAD/PS19 mouse model. [Copyright &y& Elsevier]

Pyroglutamate-modified amyloid-β (AβpE3) peptides are gaining considerable attention as potential key participants in the pathology of Alzheimer disease (AD) due to their abundance in AD brain, high aggregation propensity, stability, and cellular toxicity. Transgenic mice that produce high levels of AβpE3-42 show severe neuron loss. Recent in vitro and in vivo experiments have proven that the enzyme glutaminyl cyclase catalyzes the formation of AβpE3. In this minireview, we summarize the current knowledge on AβpE3, discussing its discovery, biochemical properties, molecular events determining formation, prevalence in the brains of AD patients, Alzheimer mouse models, and potential as a target for therapy and as a diagnostic marker. [ABSTRACT FROM AUTHOR]

According to the neurovascular hypothesis, impairment of low-density lipoprotein receptor-related protein-1 (LRP1) in brain capillaries of the blood-brain barrier (BBB) contributes to neurotoxic amyloid-β (Aβ) brain accumulation and drives Alzheimer's disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in Aβ transport and the expression of LRP1 in brain endothelium, the role of LRP1 at the BBB is uncertain. As global Lrp1 deletion in mice is lethal, appropriate models to study the function of LRP1 are lacking. Moreover, the relevance of systemic Aβ clearance to AD pathology remains unclear, as no BBB-specific knockout models have been available. Here, we developed transgenic mouse strains that allow for tamoxifen-inducible deletion of Lrp1 specifically within brain endothelial cells (Slco1c1-CreER(T2) Lrp1(fl/fl) mice) and used these mice to accurately evaluate LRP1-mediated Aβ BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [125I] Aβ(1-42). Additionally, in the 5xFAD mouse model of AD, brain endothelial-specific Lrp1 deletion reduced plasma Aβ levels and elevated soluble brain Aβ, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic Aβ elimination via the BBB. Together, our results suggest that receptor-mediated Aβ BBB clearance may be a potential target for treatment and prevention of Aβ brain accumulation in AD. [ABSTRACT FROM AUTHOR]

Abstract: The disturbed metabolism of β-amyloid peptides generated from amyloid precursor protein is widely considered as a main factor during the pathogenesis of Alzheimer''s disease. A neuropathological hallmark in the brains from cases with Alzheimer''s disease are senile plaques mainly composed of hardly soluble β-amyloid peptides comprising up to 43 amino acids. Age-dependent cortical β-amyloidosis was also shown in several transgenic mice and old individuals from various mammalian species, e.g., non-human primates. β-Amyloid1–42 is believed to be the main component in the core of senile plaques, whereas less hydrophobic β-amyloid1–40 predominantly occurs in the outer rim of plaques. Amino-terminally truncated pyroglutamyl-β-amyloidpE3–x was recently found to be a β-amyloid species of high relevance to the progression of the disease. While a few biochemical studies provided data on the co-occurrence of several β-amyloid forms, their concomitant histochemical detection is still lacking. Here, we present a novel triple immunofluorescence labelling of amino- and differently carboxy-terminally truncated β-amyloid peptides in cortical plaques from a case with Alzheimer''s disease, senile macaques and baboons, and triple transgenic mice with age-dependent β-amyloidosis and tau hyperphosphorylation. Additionally, β-amyloidpE3–x and total β-amyloid were concomitantly detected with β-amyloid peptides ending with amino acid 40 or 42, respectively. Simultaneous staining of several β-amyloid species reveals for instance vascular amyloid containing β-amyloidpE3–x in Alzheimer''s disease and monkeys, and may contribute to the further elucidation of β-amyloidosis in neurodegenerative disorders and animal models. [Copyright &y& Elsevier]

We reported previously that stabilized β-amyloid peptide dimers were derived from mutant amyloid precursor protein with a single cysteine in the ectodomain juxtamembrane position. In vivo studies revealed that two forms of SDS-stable Aβ homodimers exist, species ending at Aβ40 and Aβ42. The phenomenon of the transformation of the initially deposited 42-residue β-amyloid peptide into the amyloid fibrils of Alzheimer's disease plaques remains to be explained in physical terms, i.e. energetically and structurally. We therefore performed spectroscopic analyses revealing that engineered dimeric peptides ending at residue 42 displayed a much more pronounced β-structural transition than corresponding monomers. Specifically, the single chemically induced dimerization of Aβ peptides significantly increased the β-sheet content by a factor of 2. The Cterminal residues Ile-41 and Ala-42 of dimeric forms further increased the β-sheet content by roughly one-third. In contrast to Aβ42, the β-sheet content of the α- and γ-secretase-generated p3 fragments did not necessarily correlate with the tendency to form fibrils, although p3/17-42 had a pronounced thread forming character with fibril lengths of up to 2.5 µm. Electron microscopic images show that forms of p3/17-42 generated smaller granular particles than forms ending at residue 40. We discuss these findings in terms of Aβ1-42 dimers representing paranuclei, which self-aggregate into ribbonlike ordered fibrils by elongation. Based on Aβ42 dimerspecific titers of a polyclonal antiserum we propose that the Aβ homodimer represents a nidus for plaque formation and a well defined novel therapeutic target. [ABSTRACT FROM AUTHOR]