Timing of neurodegeneration and beta-amyloid (Abeta) peptide deposition in the brain of aging kokanee salmon

Brains of kokanee salmon (Oncorhynchus nerka kennerlyi) in one of four reproductive stages (sexually immature, maturing, sexually mature, and spawning) were stained with cresyl violet and silver stain to visualize neurodegeneration. These reproductive stages correlate with increasing somatic aging of kokanee salmon, which die after spawning. Twenty-four regions of each brain were examined. Brains of sexually immature fish exhibited low levels of neurodegeneration, whereas neurodegeneration was more marked in maturing fish and greatest in spawning fish. Neurodegeneration was present in specific regions of the telencephalon, diencephalon, mesencephalon, and rhombencephalon. Pyknotic neurons were observed in all regions previously reported to be immunopositive for A beta. Regions that did not exhibit neurodegeneration during aging included the magnocellular vestibular nucleus, the nucleus lateralis tuberis of the hypothalamus, and Purkinje cells of the cerebellum, all of which also lack A beta; perhaps these regions are neuroprotected. In 14 of 16 brain areas for which data were available on both the increase in A beta deposition and pyknosis, neurodegeneration preceded or appeared more or less simultaneously with A beta production, whereas in only two regions did A beta deposition precede neurodegeneration. This information supports the hypothesis that A beta deposition is a downstream product of neurodegeneration in most brain regions. Other conclusions are that the degree of neurodegeneration varies among brain regions, neurodegeneration begins in maturing fish and peaks in spawning fish, the timing of neurodegeneration varies among brain regions, and some regions do not exhibit accelerated neurodegeneration during aging.     

Intraneuronal amyloid precursor protein (APP) and appearance of extracellular β‐amyloid peptide (aβ) in the brain of aging kokanee salmon

Antibodies to human amyloid precursor protein (APP₆₉₅) and beta‐amyloid peptide (Aβ_1‐42) were used to determine timing of amyloidosis in the brain of kokanee salmon (Oncorhynchus nerka kennerlyi) in one of four reproductive stages: immature (IM), maturing (MA), sexually mature (SM), and spawning (SP), representing a range of aging from somatically mature but sexually immature to spawning and somatic senescence. In IM fish, immunoreactive (ir) intracellular APP occurred in 18 of 23 brain regions. During sexual maturation and aging, the number of neurons expressing APP increased in 11 of these APP‐ir regions. Aβ‐ir was absent in IM fish, present in seven regions in MA fish, moderately abundant in 15 regions in SM fish, and was most abundant in all brain regions of SP fish exhibiting Aβ‐ir. Intracellular APP‐ir was observed in brain regions involved in sensory integration, olfaction, vision, stress responses, reproduction, and coordination. Intra‐ and extracellular Aβ_1‐42 immunoreactivity (Aβ‐ir) was present in all APP‐ir regions except the nucleus lateralis tuberis (hypothalamus) and Purkinje cells (cerebellum). APP‐ir and Aβ deposition increase during aging. APP‐ir is present in IM fish; Aβ‐ir usually appears first in MA or SM fish and increases in SM fish as does APP‐ir. Extracellular Aβ deposition dramatically increases between SM and SP stages (1–2 weeks) in all fish, indicating an extremely rapid and synchronized process. Rapid senescence observed in pacific salmon could make them a useful model to investigate timing of amyloidosis and neurodegeneration during brain aging. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 11–20, 2002     

A β‐amino acid modified heptapeptide containing a designed recognition element disrupts fibrillization of the amyloid β‐peptide

We study the complex formation of a peptide βAβAKLVFF, previously developed by our group, with Aβ(1–42) in aqueous solution. Circular dichroism spectroscopy is used to probe the interactions between βAβAKLVFF and Aβ(1–42), and to study the secondary structure of the species in solution. Thioflavin T fluorescence spectroscopy shows that the population of fibers is higher in βAβAKLVFF/Aβ(1–42) mixtures compared to pure Aβ(1–42) solutions. TEM and cryo‐TEM demonstrate that co‐incubation of βAβAKLVFF with Aβ(1–42) causes the formation of extended dense networks of branched fibrils, very different from the straight fibrils observed for Aβ(1–42) alone. Neurotoxicity assays show that although βAβAKLVFF alters the fibrillization of Aβ(1–42), it does not decrease the neurotoxicity, which suggests that toxic oligomeric Aβ(1–42) species are still present in the βAβAKLVFF/Aβ(1–42) mixtures. Our results show that our designed peptide binds to Aβ(1–42) and changes the amyloid fibril morphology. This is shown to not necessarily translate into reduced toxicity. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of Aβ[1‐42] and its derivatives with improved efficiency

It has been proved that the principal component of senile plaques is aggregates of β‐amyloid peptide (Aβ) in cases of one of the most common forms of age‐related neurodegenerative disorders, Alzheimer's disease (AD). Although the synthetic methods for the synthesis of Aβ peptides have been developed since their first syntheses, Aβ[1‐42] is still problematic to prepare. The highly hydrophobic composition of Aβ[1‐42] results in aggregation between resin‐bound peptide chains or intrachain aggregation which leads to a decrease in the rates of deprotection and repetitive incomplete coupling reactions during 9‐flurenylmethoxycarbonyl (Fmoc) synthesis. In order to avoid aggregation and/or disrupt internal aggregation during stepwise Fmoc solid phase synthesis and to improve the quality of crude products, several attempts have been made. Since highly pure Aβ peptides in large quantities are used in biological experiments, we wanted to develop a method for a rational synthesis of human Aβ[1‐42] with high purity and adequate yield. This paper reports a convenient methodology with a novel solvent system for the synthesis of Aβ[1‐42], its N‐terminally truncated derivatives Aβ[4‐42] and Aβ[5‐42], and Aβ[1‐42] labeled with 7‐amino‐4‐methyl‐3‐coumarinylacetic acid (AMCA) at the N‐terminus using Fmoc strategy. The use of 10% anisole in Dimethylformamide/Dichloromethane (DMF/DCM) can substantially improve the purity and yield of crude Aβ[1‐42] and has been shown to be an optimal coupling condition for the synthesis of Aβ[1‐42]. Anisole is a cheap and simple aid in the synthesis of ‘difficult sequences’ where other solvents are less successful in the prevention of aggregation during the synthesis. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd.     

Interactions of amyloid Aβ(1–42) peptide with self‐assembled peptide nanospheres

In this work we have probed the interactions of the amyloid Aβ(1–42) peptide with self‐assembled nanospheres. The nanospheres were formed by self‐assembly of a newly developed bolaamphiphile bis(N‐alpha‐amido‐methionine)‐1,8 octane dicarboxylate under aqueous conditions. It was found that the interactions of the Aβ(1–42) peptide with the nanospheres were concentration as well as pH dependent and the peptide largely adopts a random coil structure upon interacting with the nanospheres. Further, upon incorporation with the nanospheres, we observed a relative diminution in the aggregation of Aβ(1–42) at low concentrations of Aβ(1–42). The interactions between the nanospheres and the Aβ(1–42) peptide were investigated by atomic force microscopy, transmission electron microscopy, circular dichroism, FTIR and fluorescence spectroscopy, and the degree of fibrillation in the presence and absence of nanospheres was monitored by the Thioflavine T assay. We believe that the outcome from this work will help further elucidate the binding properties of Aβ peptide as well as designing nanostructures as templates for further investigating the nucleation and fibrillation process of Aβ‐like peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Neurotoxicity of acetylcholinesterase amyloid β-peptide aggregates is dependent on the type of Aβ peptide and the AChE concentration present in the complexes

Alzheimer’s disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid β-peptide (Aβ) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Aβ_1–40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE-Aβ complexes were found to be more toxic than those formed without the enzyme, for Aβ_1–40 and Aβ_1–42, but not for amyloid fibrils formed with Aβ_Val18→Ala, a synthetic variant of the Aβ_1–40 peptide. Of all the AChE-Aβ complexes tested the one containing the Aβ_1–40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Aβ_1–40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE-Aβ_1–40 aggregates are more toxic than those of AChE-Aβ_1–42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.     

Promoting α‐secretase cleavage of beta‐amyloid with engineered proteolytic antibody fragments

Deposition of beta‐amyloid (Aβ) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of Aβ levels by various therapeutic approaches is actively being pursued. A potentially non‐inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze Aβ at its α‐secretase site. We have previously identified a light chain fragment, mk18, with α‐secretase‐like catalytic activity, producing the 1–16 and 17–40 amino acid fragments of Aβ40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking α‐secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for Aβ. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the α‐secretase site of Aβ. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3‐ and 6‐fold increase in catalytic activity (k_cat/K_M) toward the synthetic Aβ substrate compared to the original scFv primarily due to an expected decrease in K_M rather than an increase in k_cat. This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for Aβ. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble Aβ levels in vivo. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009     

Rationally designed dehydroalanine (ΔAla)‐containing peptides inhibit amyloid‐β (Aβ) peptide aggregation

Among the pathological hallmarks of Alzheimer's disease (AD) is the deposition of amyloid‐β (Aβ) peptides, primarily Aβ (1–40) and Aβ (1–42), in the brain as senile plaques. A large body of evidence suggests that cognitive decline and dementia in AD patients arise from the formation of various aggregated forms of Aβ, including oligomers, protofibrils and fibrils. Hence, there is increasing interest in designing molecular agents that can impede the aggregation process and that can lead to the development of therapeutically viable compounds. Here, we demonstrate the ability of the specifically designed α,β‐dehydroalanine (ΔAla)‐containing peptides P1 (K‐L‐V‐F‐ΔA‐I‐ΔA) and P2 (K‐F‐ΔA‐ΔA‐ΔA‐F) to inhibit Aβ (1–42) aggregation. The mechanism of interaction of the two peptides with Aβ (1–42) seemed to be different and distinct. Overall, the data reveal a novel application of ΔAla‐containing peptides as tools to disrupt Aβ aggregation that may lead to the development of anti‐amyloid therapies not only for AD but also for many other protein misfolding diseases. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 456–465, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

N‐terminal truncated pyroglutamyl β amyloid peptide Aβpy3‐42 shows a faster aggregation kinetics than the full‐length Aβ1‐42

We tested directly the differences in the aggregation kinetics of three important β amyloid peptides, the full‐length Aβ1‐42, and the two N‐terminal truncated and pyroglutamil modified Aβpy3‐42 and Aβpy11‐42 found in different relative concentrations in the brains in normal aging and in Alzheimer disease. By following the circular dichroism signal and the ThT fluorescence of the solution in phosphate buffer, we found substantially faster aggregation kinetics for Aβpy3‐42. This behavior is due to the particular sequence of this peptide, which is also responsible for the specific oligomeric aggregation states, found by TEM, during the fibrillization process, which are very different from those of Aβ1‐42, more prone to fibril formation. In addition, Aβpy3‐42 is found here to have an inhibitory effect on Aβ1‐42 fibrillogenesis, coherently with its known greater infective power. This is an indication of the important role of this peptide in the aggregation process of β‐peptides in Alzheimer disease. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 861–873, 2009. This article was originally published online as an accepted preprint. The “Published Online“ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Critical swimming velocities of juvenile sockeye salmon and kokanee, the anadromous and non-anadromous forms of Oncorhynchus nerka (Walbaum)

In streams tributary to the North Pacific, anadromous sockeye salmon and non-anadromous kokanee, Oncorhynchus nerka (Walbaum), occasionally spawn sympatrically and male kokanee may act as 'sneaks’to spawn with the larger female sockeye. Despite this interbreeding, sockeye and kokanee exhibit persistent biochemical genetic differences at several enzyme loci. Genetic differences between forms may be maintained by selection against‘hybrids’due to the different life histories of sockeye and kokanee; sockeye make extensive smolt, oceanic, and spawning migrations while kokanee reside permanently in fresh water. We tested the sustained swimming abilities of juvenile sockeye, kokanee, and sockeye (female) × kokanee (male) hybrids to see if hybrids were inferior to sockeye in a trait that is probably under stronger selection in an anadromous life history. Sockeye had significantly greater mean critical swimming velocities (U_crit) than kokanee of the same size raised under identical conditions (8.3 v. 7.3 body lengths s^?1 respectively). When tested 1 month later the mean U_crit of sockeye was only marginally greater than that for sockeye × kokanee hybrids (both c. 6.6 body lengths s^?1). Sockeye swimming performance was also less variable than that of either kokanee or hybrids. Sockeye tended to have slimmer bodies and longer caudal regions than kokanee or sockeye × kokanee hybrids of the same size. Sockeye also had significantly more vertebrae than kokanee and hybrids, while hybrids had more vertebrae than kokanee. These morphological differences may have contributed to the differences in swimming performance. We concluded: (i) that juvenile sockeye and kokanee have diverged with respect to sustained swimming performance and that reduced performance by kokanee may be due to relaxed selection for sustained swimming performance associated with their non-anadromous life history, (ii) that sockeye × kokanee hybrids appear to have modestly lower swimming capabilities than pure sockeye, and (iii) if the variability in swimming performance is associated with differences in survival in nature, then such differences may promote divergence between sympatric sockeye and kokanee.     

Rational design of amyloid β peptide–binding proteins: Pseudo‐Aβ β‐sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Aβ

Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid β‐peptide (Aβ) is thought to be a toxic agent by self‐assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo‐Aβ β‐sheet surfaces and evaluated their abilities to bind to Aβ and inhibit Aβ oligomerization. Two GFP variants P13H and AP93Q bound tightly to Aβ, K_d = 260 nM and K_d = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Aβ oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Aβ‐derived sequences was designed and bound more tightly to Aβ (K_d = 100 nM) than those having only two pseudo‐Aβ strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Aβ more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo‐Aβ β‐sheet structures onto a protein surface arranged in the β‐barrel structure is useful to construct molecules capable of binding tightly to Aβ and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Intraneuronal amyloid precursor protein (APP) and appearance of extracellular beta-amyloid peptide (abeta) in the brain of aging kokanee salmon

Antibodies to human amyloid precursor protein (APP(695)) and beta-amyloid peptide (A beta(1-42)) were used to determine timing of amyloidosis in the brain of kokanee salmon (Oncorhynchus nerka kennerlyi) in one of four reproductive stages: immature (IM), maturing (MA), sexually mature (SM), and spawning (SP), representing a range of aging from somatically mature but sexually immature to spawning and somatic senescence. In IM fish, immunoreactive (ir) intracellular APP occurred in 18 of 23 brain regions. During sexual maturation and aging, the number of neurons expressing APP increased in 11 of these APP-ir regions. A beta-ir was absent in IM fish, present in seven regions in MA fish, moderately abundant in 15 regions in SM fish, and was most abundant in all brain regions of SP fish exhibiting A beta-ir. Intracellular APP-ir was observed in brain regions involved in sensory integration, olfaction, vision, stress responses, reproduction, and coordination. Intra- and extracellular A beta(1-42) immunoreactivity (A beta-ir) was present in all APP-ir regions except the nucleus lateralis tuberis (hypothalamus) and Purkinje cells (cerebellum). APP-ir and A beta deposition increase during aging. APP-ir is present in IM fish; A beta-ir usually appears first in MA or SM fish and increases in SM fish as does APP-ir. Extracellular A beta deposition dramatically increases between SM and SP stages (1-2 weeks) in all fish, indicating an extremely rapid and synchronized process. Rapid senescence observed in pacific salmon could make them a useful model to investigate timing of amyloidosis and neurodegeneration during brain aging.     

Antibodies bound to Aβ oligomers potentiate the neurotoxicity of Aβ by activating microglia

Beta amyloid (Aβ) oligomers are thought to contribute to the pathogenesis of Alzheimer's disease. However, clinical trials using Aβ immunization were unsuccessful due to strong brain inflammation, the mechanisms of which are poorly understood. In this study we tested whether monoclonal antibodies to oligomeric Aβ would prevent the neurotoxicity of Aβ oligomers in primary neuronal‐glial cultures. However, surprisingly, the antibodies dramatically increased the neurotoxicity of Aβ. Antibodies bound to monomeric Aβ fragments were non‐toxic to cultured neurons, while antibodies to other oligomeric proteins: hamster polyomavirus major capsid protein, human metapneumovirus nucleocapsid protein, and measles virus nucleocapsid protein, strongly potentiated the neurotoxicity of their antigens. The neurotoxicity of antibody‐oligomeric antigen complexes was abolished by removal of the Fc region from the antibodies or by removal of microglia from cultures, and was accompanied by inflammatory activation and proliferation of the microglia in culture. In conclusion, we find that immune complexes formed by Aβ oligomers or other oligomeric/multimeric antigens and their specific antibodies can cause death and loss of neurons in primary neuronal‐glial cultures via Fc‐dependent microglial activation. The results suggest that therapies resulting in antibodies to oligomeric Aβ or oligomeric brain virus proteins should be used with caution or with suppression of microglial activation.

     

Antifibrillizing agents catalyze the formation of unstable intermediate aggregates of beta‐amyloid

Although Alzheimer's disease (AD) is characterized by the extracellular deposition of fibrillar aggregates of beta‐amyloid (Aβ), transient oligomeric species of Aβ are increasingly implicated in the pathogenesis of AD. Natively unfolded monomeric Aβ can misfold and progressively assemble into fibrillar aggregates, following a well‐established “on pathway” seeded‐nucleation mechanism. Here, we show that three simple saccharides, mannose, sucrose, and raffinose, alter Aβ aggregation kinetics and morphology. The saccharides inhibit formation of Aβ fibrils but promote formation of various oligomeric aggregate species through different “off pathway” aggregation mechanisms at 37°C but not at 60°C. The various oligomeric Aβ aggregates formed when coincubated with the different saccharides are morphologically distinct but all are toxic toward SH‐SY5Y human neuroblastoma cells, increasing the level of toxicity and greatly prolonging toxicity compared with Aβ alone. As a wide variety of anti‐Aβ aggregation strategies are being actively pursued as potential therapeutics for AD, these studies suggest that care must be taken to ensure that the therapeutic agents also block toxic oligomeric Aβ assembly as well as inhibit fibril formation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Neuronal localization of the TNFα converting enzyme (TACE) in brain tissue and its correlation to amyloid plaques

The tumor necrosis factor (TNF)‐α converting enzyme (TACE) can cleave the cell‐surface ectodomain of the amyloid‐β precursor protein (APP), thus decreasing the generation of amyloid‐β (Aβ) by cultured non‐neuronal cells. While the amyloidogenic processing of APP in neurons is linked to the pathogenesis of Alzheimer's disease (AD), the expression of TACE in neurons has not yet been examined. Thus, we assessed TACE expression in a series of neuronal and non‐neuronal cell types by Western blots. We found that TACE was present in neurons and was only faintly detectable in lysates of astrocytes, oligodendrocytes, and microglial cells. Immunohistochemical analysis was used to determine the cellular localization of TACE in the human brain, and its expression was detected in distinct neuronal populations, including pyramidal neurons of the cerebral cortex and granular cell layer neurons in the hippocampus. Very low levels of TACE were seen in the cerebellum, with Purkinje cells at the granular‐molecular boundary staining faintly. Because TACE was localized predominantly in areas of the brain that are affected by amyloid plaques in AD, we examined its expression in a series of AD brains. We found that AD and control brains showed similar levels of TACE staining, as well as similar patterns of TACE expression. By double labeling for Aβ plaques and TACE, we found that TACE‐positive neurons often colocalized with amyloid plaques in AD brains. These observations support a neuronal role for TACE and suggest a mechanism for its involvement in AD pathogenesis as an antagonist of Aβ formation. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 40–46, 2001     

Identification of new Presenilin‐1 phosphosites: implication for γ‐secretase activity and Aβ production

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid‐beta (Aβ) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ‐secretase is responsible for the intramembrane proteolysis of the amyloid‐β precursor protein (APP), which leads to the production of the toxic Aβ peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ‐secretase activity, to reduce Aβ42 production. Because phosphorylation of proteins is a post‐translational modification known to modulate the activity of many different enzymes, we used electrospray (LC‐MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ‐secretase. We identified 11 new single or double phosphosites in two well‐defined domains of Presenilin‐1 (PS1), the catalytic subunit of the γ‐secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ‐secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ‐secretase activity and the production of the Alzheimer's Aβ peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aβ plaque formation in AD.