N-truncated Aβ Research Articles

The effect of histidine, histamine, and imidazole on electrochemical properties of Cu(II) complexes of Aβ peptides containing His-2 and His-3 motifs.

The N-truncation of amyloid beta (Aβ) peptides could lead to peptide sequences with the histidine residue at the second and third positions, creating His-2 and His-3 motifs, known as high-affinity Cu(II) binding sites. In such complexes, the Cu(II) ion is arrested in a rigid structure of a square-planar arrangement of nitrogen donors, which highly limits its susceptibility to Cu(II) reduction. Cu(II) reduction fuels the Cu(II)/Cu(I) redox cycle, which is engaged in the production of reactive oxygen species (ROS). Employing electrochemical techniques, cyclic voltammetry (CV) and differential pulse voltammetry (DPV), together with UV-vis spectroscopy, we showed that low-molecular-weight (LMW) substances, such as imidazole, histamine, and histidine, could enhance the redox activity of Cu(II) complexes of three models of N-truncated Aβ peptides, Aβ4-9, Aβ5-9, and Aβ12-16, identifying three main mechanisms. LMW compounds could effectively compete with Aβ peptides for Cu(II) ions, forming Cu(II)/LMW species, which are more prone to Cu(II) reduction. LMW substances could also shift the equilibrium between the Cu(II)/Aβ species towards the species with higher susceptibility to Cu(II) reduction. Finally, the presence of LMW molecules could promote Cu(I) reoxidation in ternary Cu(II)/Aβ/LMW systems. The obtained results raise further questions regarding the Cu(II) redox activity in Alzheimer's disease.

Enhanced ion mobility resolution of Abeta isomers from human brain using high-resolution demultiplexing software.

Isomerization of aspartic acid (Asp) residues in long-lived proteins is a key feature associated with neurodegenerative proteinopathies such as Alzheimer's disease (AD). Recently, using ultra high-performance liquid chromatography (UHPLC) coupled with drift tube ion mobility mass spectrometry (DTIMS-MS), we documented the extensive Asp isomerization in amyloid-beta (Aβ) peptides depositing in the extracellular cortical plaques (senile plaques) of the AD brain. Aβ1-15 was estimated to be ~ 85% isomerized, while Aβ4-15 another major constituent of these senile plaques was ~ 50% isomerized in AD brain. Low resolution on the standard demultiplexed ion mobility resulted in poor separation of these N-truncated Aβ isomers in the ion mobility domain. Here, using the same ion multiplexed dataset, we applied new post-acquisition data reconstruction technique, high-resolution demultiplexing (HRdm), to improve the resolution of these Aβ isomers in the ion mobility dimension. We demonstrate that for the complex proteomic AD brain digests, HRdm could successfully resolve three out of four major Asp isomers of Aβ1-15. For Aβ2-15 and Aβ4-15, the significant resolution enhancement in the HRdm data resulted in baseline peak separation of the respective Asp isomers. An analysis of two-peak resolution (Rpp) and peak-to-peak separation (ΔP) indicated twofold enhancement for the Asp-isomerized Aβ species. HRdm performed with an effective resolving power (Rp) of between 150 and 160 for the highest deconvolution settings in comparison to ~ 40 to 65 in the standard settings. These major resolution improvements in the ion mobility domain for the endogenous Aβ isomers demonstrate the feasibility of in situ measurement of peptide isomers and their role in the mechanism of amyloid plaque formation in AD.

Tuning Receptor Properties of Metal-Amyloid Beta Complexes. Studies on the Interaction between Ni(II)-Aβ5-9 and Phosphates/Nucleotides.

Amyloid-beta (Aβ) peptides, potentially relevant in the pathology of Alzheimer’s disease, possess distinctive coordination properties, enabling an effective binding of transition-metal ions, with a preference for Cu(II). In this work, we found that a N-truncated Aβ analogue bearing a His-2 motif, Aβ5–9, forms a stable Ni(II) high-spin octahedral complex at a physiological pH of 7.4 with labile coordination sites and facilitates ternary interactions with phosphates and nucleotides. As the pH increased above 9, a spin transition from a high-spin to a low-spin square-planar Ni(II) complex was observed. Employing electrochemical techniques, we showed that interactions between the binary Ni(II)–Aβ5–9 complex and phosphate species result in significant changes in the Ni(II) oxidation signal. Thus, the Ni(II)–Aβ5–9 complex could potentially serve as a receptor in electrochemical biosensors for phosphate species. The obtained results could also be important for nickel toxicology.

Discovery of a novel pseudo β-hairpin structure of N-truncated amyloid-β for use as a vaccine against Alzheimer's disease.

One of the hallmarks of Alzheimer's disease (AD) are deposits of amyloid-beta (Aβ) protein in amyloid plaques in the brain. The Aβ peptide exists in several forms, including full-length Aβ1-42 and Aβ1-40 - and the N-truncated species, pyroglutamate Aβ3-42 and Aβ4-42, which appear to play a major role in neurodegeneration. We previously identified a murine antibody (TAP01), which binds specifically to soluble, non-plaque N-truncated Aβ species. By solving crystal structures for TAP01 family antibodies bound to pyroglutamate Aβ3-14, we identified a novel pseudo β-hairpin structure in the N-terminal region of Aβ and show that this underpins its unique binding properties. We engineered a stabilised cyclic form of Aβ1-14 (N-Truncated Amyloid Peptide AntibodieS; the 'TAPAS' vaccine) and showed that this adopts the same 3-dimensional conformation as the native sequence when bound to TAP01. Active immunisation of two mouse models of AD with the TAPAS vaccine led to a striking reduction in amyloid-plaque formation, a rescue of brain glucose metabolism, a stabilisation in neuron loss, and a rescue of memory deficiencies. Treating both models with the humanised version of the TAP01 antibody had similar positive effects. Here we report the discovery of a unique conformational epitope in the N-terminal region of Aβ, which offers new routes for active and passive immunisation against AD.

N-Truncated Aβ Starting at Position Four-Biochemical Features, Preclinical Models, and Potential as Drug Target in Alzheimer's Disease.

The discussion of whether amyloid plaque Aβ is a valid drug target to fight Alzheimer’s disease (AD) has been a matter of scientific dispute for decades. This question can only be settled by successful clinical trials and the approval of disease-modifying drugs. However, many clinical trials with antibodies against different regions of the amyloid Aβ peptide have been discontinued, as they did not meet the clinical endpoints required. Recently, passive immunization of AD patients with Donanemab, an antibody directed against the N-terminus of pyroglutamate Aβ, showed beneficial effects in a phase II trial, supporting the concept that N-truncated Aβ is a relevant target for AD therapy. There is long-standing evidence that N-truncated Aβ variants are the main variants found in amyloid plaques besides full-length Aβ1–42, t, therefore their role in triggering AD pathology and as targets for drug development are of interest. While the contribution of pyroglutamate Aβ3–42 to AD pathology has been well studied in the past, the potential role of Aβ4–42 has been largely neglected. The present review will therefore focus on Aβ4–42 as a possible drug target based on human and mouse pathology, in vitro and in vivo toxicity, and anti-Aβ4-X therapeutic effects in preclinical models.

Oxidase Reactivity of CuII Bound to N-Truncated Aβ Peptides Promoted by Dopamine.

The redox chemistry of copper(II) is strongly modulated by the coordination to amyloid-β peptides and by the stability of the resulting complexes. Amino-terminal copper and nickel binding motifs (ATCUN) identified in truncated Aβ sequences starting with Phe4 show very high affinity for copper(II) ions. Herein, we study the oxidase activity of [Cu–Aβ4−x] and [Cu–Aβ1−x] complexes toward dopamine and other catechols. The results show that the CuII–ATCUN site is not redox-inert; the reduction of the metal is induced by coordination of catechol to the metal and occurs through an inner sphere reaction. The generation of a ternary [CuII–Aβ–catechol] species determines the efficiency of the oxidation, although the reaction rate is ruled by reoxidation of the CuI complex. In addition to the N-terminal coordination site, the two vicinal histidines, His13 and His14, provide a second Cu-binding motif. Catechol oxidation studies together with structural insight from the mixed dinuclear complexes Ni/Cu–Aβ4−x reveal that the His-tandem is able to bind CuII ions independently of the ATCUN site, but the N-terminal metal complexation reduces the conformational mobility of the peptide chain, preventing the binding and oxidative reactivity toward catechol of CuII bound to the secondary site.

Ion channel formation by N-terminally truncated Aβ (4–42): relevance for the pathogenesis of Alzheimer's disease

Aβ deposition is a pathological hallmark of Alzheimer's disease (AD). Besides the full-length amyloid forming peptides (Aβ1–40 and Aβ1–42), biochemical analyses of brain deposits have identified a variety of N- and C-terminally truncated Aβ variants in sporadic and familial AD patients. However, their relevance for AD pathogenesis remains largely understudied. We demonstrate that Aβ4–42 exhibits a high tendency to form β-sheet structures leading to fast self-aggregation and formation of oligomeric assemblies. Atomic force microscopy and electrophysiological studies reveal that Aβ4–42 forms highly stable ion channels in lipid membranes. These channels that are blocked by monoclonal antibodies specifically recognizing the N-terminus of Aβ4–42. An Aβ variant with a double truncation at phenylalanine-4 and leucine 34, (Aβ4–34), exhibits unstable channel formation capability. Taken together the results presented herein highlight the potential benefit of C-terminal proteolytic cleavage and further support an important pathogenic role for N-truncated Aβ species in AD pathophysiology.

CuII Binding Properties of N-Truncated Aβ Peptides: In Search of Biological Function.

As life expectancy increases, the number of people affected by progressive and irreversible dementia, Alzheimer's Disease (AD), is predicted to grow. No drug designs seem to be working in humans, apparently because the origins of AD have not been identified. Invoking amyloid cascade, metal ions, and ROS production hypothesis of AD, herein we share our point of view on Cu(II) binding properties of Aβ4-x, the most prevalent N-truncated Aβ peptide, currently known as the main constituent of amyloid plaques. The capability of Aβ4-x to rapidly take over copper from previously tested Aβ1-x peptides and form highly stable complexes, redox unreactive and resistant to copper exchange reactions, prompted us to propose physiological roles for these peptides. We discuss the new findings on the reactivity of Cu(II)Aβ4-x with coexisting biomolecules in the context of synaptic cleft; we suggest that the role of Aβ4-x peptides is to quench Cu(II) toxicity in the brain and maintain neurotransmission.

Dipeptidyl-Peptidase Activity of Meprin β Links N-truncation of Aβ with Glutaminyl Cyclase-Catalyzed pGlu-Aβ Formation.

The formation of amyloid-β (Aβ) peptides is causally involved in the development of Alzheimer's disease (AD). A significant proportion of deposited Aβ is N-terminally truncated and modified at the N-terminus by a pGlu-residue (pGlu-Aβ). These forms show enhanced neurotoxicity compared to full-length Aβ. Although the truncation may occur by aminopeptidases after formation of Aβ, recently discovered processing pathways of amyloid-β protein precursor (AβPP) by proteases such as meprin β may also be involved. Here, we assessed a role of meprin β in forming Aβ3-40/42, which is the precursor of pGlu-Aβ3-40/42 generated by glutaminyl cyclase (QC). Similar to QC, meprin β mRNA is significantly upregulated in postmortem brain from AD patients. A histochemical analysis supports the presence of meprin β in neurons and astrocytes in the vicinity of pGlu-Aβ containing deposits. Cleavage of AβPP-derived peptides by meprin β in vitro results in peptides Aβ1-x, Aβ2-x, and Aβ3-x. The formation of N-truncated Aβ by meprin β was also corroborated in cell culture. A subset of the generated peptides was converted into pGlu-Aβ3-40 by an addition of glutaminyl cyclase, supporting the preceding formation of Aβ3-40. Further analysis of the meprin β cleavage revealed a yet unknown dipeptidyl-peptidase-like activity specific for the N-terminus of Aβ1-x. Thus, our data suggest that meprin β contributes to the formation of N-truncated Aβ by endopeptidase and exopeptidase activity to generate the substrate for QC-catalyzed pGlu-Aβ formation.

Interplay between Copper, Neprilysin, and N-Truncation of β-Amyloid.

Sporadic Alzheimer's disease (AD) is associated with an inefficient clearance of the β-amyloid (Aβ) peptide from the central nervous system. The protein levels and activity of the Zn2+-dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aβ levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aβ fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition. Divalent copper noncompetitively inhibited NEP ( Ki = 1.0 μM), while proteolysis of Aβ yielded the soluble, Aβ4-9 fragment that can bind Cu2+ with femtomolar affinity at pH 7.4. This provides Aβ4-9 with the potential to act as a Cu2+ carrier and to mediate its own production by preventing NEP inhibition. Enzyme inhibition at high Zn2+ concentrations ( Ki = 20 μM) further suggests a mechanism for modulating NEP activity, Aβ4-9 production, and Cu2+ homeostasis.

Synergistic Effect on Neurodegeneration by N-Truncated Aβ4−42 and Pyroglutamate Aβ3−42 in a Mouse Model of Alzheimer's Disease

The N-terminally truncated pyroglutamate Aβ3−42 (AβpE3−42) and Aβ4−42 peptides are known to be highly abundant in the brain of Alzheimer's disease (AD) patients. Both peptides show enhanced aggregation and neurotoxicity in comparison to full-length Aβ, suggesting that these amyloid peptides may play an important role in the pathogenesis of AD. The aim of the present work was to study the direct effect of the combination of AβpE3−42 and Aβ4−42 on ongoing AD-related neuron loss, pathology, and neurological deficits in transgenic mice. Bigenic mice were generated by crossing the established TBA42 and Tg4-42 mouse models expressing the N-truncated Aβ peptides AβpE3−42 and Aβ4−42, respectively. After generation of the bigenic mice, detailed phenotypical characterization was performed using either immunostainings to evaluate amyloid pathology or quantification of neuron numbers using design-based stereology. The elevated plus maze was used to study anxiety levels. In order to evaluate sensori-motor deficits, the inverted grid, the balance beam and the string suspension tasks were applied. We could demonstrate that co-expression of AβpE3−42 and Aβ4−42 accelerates neuron loss in the CA1 pyramidal layer of young bigenic mice as seen by reduced neuron numbers in comparison to single transgenic homozygous mice expressing either AβpE3−42 or Aβ4−42. This observation coincides with the robust intraneuronal Aβ accumulation observed in the bigenic mice. In addition, loss of anxiety and motor deficits were enhanced in an age-dependent manner. The sensori-motor deficits correlate with the abundant spinal cord pathology, as demonstrated by robust intracellular Aβ accumulation within motor neurons and extracellular Aβ deposition. Our observations demonstrate that a combination of AβpE3−42 and Aβ4−42 has a stronger effect on ongoing AD pathology than the peptides alone. Therefore, AβpE3−42 and Aβ4−42 might represent excellent potential therapeutic targets and diagnostic markers for AD.

CENTRAL CATHEPSIN B INHIBITION BY CA074 DOES NOT ALTER THE LEVELS OF NEUROTOXIC Aβ SPECIES IN THE BRAIN OF HAPP-LON MICE

The cysteine protease cathepsin B (CatB) was previously reported as a potential drug target for disease modification in Alzheimer’s disease (AD). The direction in which CatB activity needs to be regulated to provide beneficial effects has been a matter of debate. While a study by Mueller-Steiner et al. (2006) pointed towards a neuroprotective role of CatB, Hook et al. (2008, 2014) demonstrated that CatB inhibition or CatB knockout improved memory deficits, reduced amyloid plaques, and brain Aβ40/42 levels. Notably, pyroglutamate (pGlu) Aβ, an N-truncated and neurotoxic species, was also decreased suggesting CatB inhibitors might have potential for slowing down AD progression. The aim of this study was to validate the role of CatB in the production of neurotoxic Aβ species using a selective CatB inhibitor to increase confidence in CatB as a putative drug target. Transgenic hAPP-Lon (14 months old) and hAPP-Lon/PS1 (13 months old) mice were treated i.c.v. with 6μg/day CA074Me prodrug (n=16) or vehicle (n=16) for 4 weeks using ALZET minipumps. The animals were sacrificed and one hemisphere was analyzed for pharmacokinetics by mass spectrometry (MS). The other hemisphere was homogenized with Guanidine-HCl and analyzed for Aβ38/40/42 by MSD immunoassay and ELISA, for Aβ pGlu3-40/42 by ELISA, and for 11 different N-truncated and pGlu Aβ species by immunoaffinity MS. The pharmacokinetic analysis revealed that the free concentration of the active drug CA074 in the brain of both transgenic mouse strains was three times higher than IC50 suggesting efficient inhibition of Cat B in vivo. The results of our Aβ analyses with different immunoassays and immunoaffinity MS were very consistent. Brain levels of all Aβ species (Aβ40/42, Aβ pGlu3-40/42, N-truncated Aβs) after CA074 treatment did not differ from vehicle controls, both in hAPP-Lon and hAPP-Lon/PS1 mice. In a well-designed study and using various analytical methods, we obtained consistent results but were not able to confirm the published effects of CatB inhibition on brain levels of Aβ40/42 and Aβ pGlu3-40/42. We conclude that inhibition of CatB with the aim to lower the production of neurotoxic Aβ species is not an attractive therapeutic approach for AD.

STRUCTURAL BASIS OF UNIQUE SELECTIVITY OF ADUCANUMAB FOR OLIGOMERIC FORMS OF AMYLOID-β

There has been a renewed interest in immunotherapy targeting Aβ peptide for treatment of Alzheimer’s disease (AD) with the intriguing Phase 1b findings showing aducanumab significantly reduces amyloid in patients’ brains. Hypotheses being investigated for positive clinical data are the preferred epitope and selectivity for toxic Aβ aggregates. Aducanumab (BIIB037) is a human monoclonal antibody derived from a healthy donor cohort of elderly subjects lacking signs of cognitive impairment, which is in advanced clinical testing for treatment of AD. Aducanumab is unique among amyloid-targeted antibodies for its exquisite selectivity for both soluble oligomeric and insoluble fibrillar aggregates of Aβ found in brain β-amyloid plaques. To gain further insight into the epitope conformation and selectivity for Aβ aggregates, we investigated the structural details of amino-terminal epitope in Aβ using X-ray crystallography of aducanumab Fab:Aβ complex. Biochemical validation of critical interface residues was confirmed using standard ELISA and PepSpot binding assays. Here we report the crystal structure of a recombinant Fab fragment of aducanumab in complex with Aβ1-11 peptide solved at 2.5Å resolution. The N-terminus of Aβ is bound to aducanumab in an extended conformation with the binding interface composed primarily of Phe 4 and His 6 of Aβ. Comparison with other N-terminal Aβ targeting antibodies reveals that aducanumab has an exceptionally compact epitope, which contributes to its selectivity and high avidity for Aβ oligomers and fibrils, including aggregates composed of N-truncated Aβ variants, without targeting physiologic Aβ monomers. In silico Rosetta-based interface and antibody–flexible Aβ docking analysis further highlight aducanumab’s distinct biophysical interaction with the Aβ monomer that further corroborates its selectivity for Aβ oligomers. Aducanumab selectively targets pathologic Aβ conformations displayed by oligomeric and fibrillary aggregates. Our results provide the structural basis for this unique binding profile that differentiates aducanumab from other N-terminal specific antibodies, both in the conformation and orientation of the bound Aβ peptide, as well as the molecular details that comprise the antibody-peptide interface.

Tuning the Redox Properties of Copper(II) Complexes with Amyloid-β Peptides

Copper complexes of metal binding domains of synthesized amyloid-β peptides – Aβ(1-16) and N-truncated Aβ(4-16) containing a novel N-terminal FRH sequence, as well as its shorter mutants were characterized by cyclic voltammetry. The influence of the peptide sequence and peptide to copper molar ratio on the electrochemical properties of the obtained structures were studied and discussed. The reversibility of the studied redox processes in copper complexes with Aβ(4-x) derivatives was also investigated. The results indicate the crucial role of Tyr10 in the redox process of the Aβ(4-x) complex, including the removal of reversibility of the Cu(II)/Cu(III) redox couple.

Alzheimer therapy with an antibody against N-terminal Abeta 4-X and pyroglutamate Abeta 3-X.

Full-length Aβ1-42 and Aβ1-40, N-truncated pyroglutamate Aβ3-42 and Aβ4-42 are major variants in the Alzheimer brain. Aβ4-42 has not been considered as a therapeutic target yet. We demonstrate that the antibody NT4X and its Fab fragment reacting with both the free N-terminus of Aβ4-x and pyroglutamate Aβ3-X mitigated neuron loss in Tg4-42 mice expressing Aβ4-42 and completely rescued spatial reference memory deficits after passive immunization. NT4X and its Fab fragment also rescued working memory deficits in wild type mice induced by intraventricular injection of Aβ4-42. NT4X reduced pyroglutamate Aβ3-x, Aβx-40 and Thioflavin-S positive plaque load after passive immunization of 5XFAD mice. Aβ1-x and Aβx-42 plaque deposits were unchanged. Importantly, for the first time, we demonstrate that passive immunization using the antibody NT4X is therapeutically beneficial in Alzheimer mouse models showing that N-truncated Aβ starting with position four in addition to pyroglutamate Aβ3-x is a relevant target to fight Alzheimer’s disease.

N-truncated Aβ2-X starting with position two in sporadic Alzheimer's disease cases and two Alzheimer mouse models.

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.

NT4X-167; Novel N-terminal Aβ specific antibody, prevents in vitro and in vivo toxicity of highly toxic Aβ4-42 species

Objectives: The novel monoclonal antibody (NT4X-167) specifically reacts with N-truncated Aβ at position 4 of Aβ. It binds N-truncated Aβ under native and denaturing conditions but only rescues in vitro toxicity of Aβ4-42 and not that of pyroglutamate AβpE3–42. One objective was to determine whether the NT4X-167 antibody and its respective Fab fragment could prevent behavioral deficits (Y-Maze), caused by Aβ4-42 injection in wildtype mice. Another was to determine if the antibody and its respective Fab could rescue the in vivo toxicity of Aβ4-42 in the Tg4-42 mouse model, by using passive immunization. Methods: Cell toxicity assay, in vivo toxicity, Y-Maze, mAB antibody production, immunostaining, passive immunization. Results: The novel Aβ4-x immunoreactive antibody NT4X-167 detected high molecular weight aggregates derived from N-truncated Aβ species. Phenylalanine at position four of Aβ was imperative for antibody binding. Both full-length and the Fab fragment of the antibody were able to prevent the in vitro toxicity caused by Aβ4-42 in rat primary cortical neuron cultures. Aβ4-42 IntraCerebroVentricular injection into wildtype mice induced a behavioral deficit, shown as a reduction in alteration rate in a Y-Maze, which was prevented using the NT4X-167. The Fab fragment of the antibody, at a higher dosage, was also able to prevent the behavioral deficit in a replicate experiment. The passive immunization was able to rescue both spatial memory deficits and significantly mitigate neuron loss. Conclusions: NT4X-167 and Fab demonstrate binding and prevention of Aβ4-42 toxicity in vitro and in vivo. NT4X full length antibody and Fab prevent Aβ4-42 induced behavioral deficits in wildtype mice. Functionally, the full length antibody and its respective Fab fragment exhibit the same profile. It would thus be of great interest to study the structural binding characteristics of the NT4X-167, represented by its Fab fragment, to the highly toxic N-truncated Aβ4-x species.

Focusing the amyloid cascade hypothesis on N-truncated Abeta peptides as drug targets against Alzheimer's disease.

Although N-truncated Aβ variants are known to be the main constituent of amyloid plaques in the brains of patients with Alzheimer’s disease, their potential as targets for pharmacological intervention has only recently been investigated. In the last few years, the Alzheimer field has experienced a paradigm shift with the ever increasing understanding that targeting amyloid plaques has not led to a successful immunotherapy. On the other hand, there can be no doubt that the amyloid cascade hypothesis is central to the etiology of Alzheimer’s disease, raising the question as to why it is apparently failing to translate into the clinic. In this review, we aim to refocus the amyloid hypothesis integrating N-truncated Aβ peptides based on mounting evidence that they may represent better targets than full-length Aβ. In addition to Aβ peptides starting with an Asp at position 1, a variety of different N-truncated Aβ peptides have been identified starting with amino residue Ala-2, pyroglutamylated Glu-3, Phe-4, Arg-5, His-6, Asp-7, Ser-8, Gly-9, Tyr-10 and pyroglutamylated Glu-11. Certain forms of N-truncated species are better correlates for early pathological changes found pre-symptomatically more often than others. There is also evidence that, together with full-length Aβ, they might be physiologically detectable and are naturally secreted by neurons. Others are known to form soluble aggregates, which have neurotoxic properties in transgenic mouse models. It has been clearly demonstrated by several groups that some N-truncated Aβs dominate full-length Aβ in the brains of Alzheimer’s patients. We try to address which of the N-truncated variants may be promising therapeutic targets and which enzymes might be involved in the generation of these peptides

Abundance of Aβ5-xlike immunoreactivity in transgenic 5XFAD, APP/PS1KI and 3xTG mice, sporadic and familial Alzheimer’s disease

BackgroundAccording 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.ResultsThe 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.ConclusionsDifferent 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.

The link between intraneuronal N-truncated amyloid-β peptide and oxidatively modified lipids in idiopathic autism and dup(15q11.2-q13)/autism

BackgroundAutism is a neurodevelopmental disorder of unknown etiopathogenesis associated with structural and functional abnormalities of neurons and increased formation of reactive oxygen species. Our previous study revealed enhanced accumulation of amino-terminally truncated amyloid-β (Aβ) in brain neurons and glia in children and adults with autism. Verification of the hypothesis that intraneuronal Aβ may cause oxidative stress was the aim of this study.ResultsThe relationships between neuronal Aβ and oxidative stress markers—4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA)—were examined in the frontal cortex from individuals aged 7–32 years with idiopathic autism or with chromosome 15q11.2-q13 duplications (dup(15)) with autism, and age-matched controls. Quantification of confocal microscopy images revealed significantly higher levels of neuronal N-truncated Aβ and HNE and MDA in idiopathic autism and dup(15)/autism than in controls. Lipid peroxidation products were detected in all mitochondria and lipofuscin deposits, in numerous autophagic vacuoles and lysosomes, and in less than 5% of synapses. Neuronal Aβ was co-localized with HNE and MDA, and increased Aβ levels correlated with higher levels of HNE and MDA.ConclusionsThe results suggest a self-enhancing pathological process in autism that is initiated by intraneuronal deposition of N-truncated Aβ in childhood. The cascade of events includes altered APP metabolism and abnormal intracellular accumulation of N-terminally truncated Aβ which is a source of reactive oxygen species, which in turn increase the formation of lipid peroxidation products. The latter enhance Aβ deposition and sustain the cascade of changes contributing to metabolic and functional impairments of neurons in autism of an unknown etiology and caused by chromosome 15q11.2-q13 duplication.