Copolymeric NiPAM:BAM nanoparticles of varying hydrophobicity were found to retard fibrillation of the Alzheimer's disease-associated amyloid beta protein (Abeta). We found that these nanoparticles affect mainly the nucleation step of Abeta fibrillation. The elongation step is largely unaffected by the particles, and once the Abeta is nucleated, the fibrillation process occurs with the same rate as in the absence of nanoparticles. The extension of the lag phase for fibrillation of Abeta is strongly dependent on both the amount and surface character of the nanoparticles. Surface plasmon resonance studies show that Abeta binds to the nanoparticles and provide rate and equilibrium constants for the interaction. Numerical analysis of the kinetic data for fibrillation suggests that binding of monomeric Abeta and prefibrillar oligomers to the nanoparticles prevents fibrillation. Moreover, we find that fibrillation of Abeta initiated in the absence of nanoparticles can be reversed by addition of nanoparticles up to a particular time point before mature fibrils appear.

We report the development of a high-level bacterial expression system for the Alzheimer’s disease-associated amyloid β-peptide (Aβ), together with a scaleable and inexpensive purification procedure. Aβ(1–40) and Aβ(1–42) coding sequences together with added ATG codons were cloned directly into a Pet vector to facilitate production of Met-Aβ(1–40) and Met-Aβ(1–42), referred to as Aβ(Μ1–40) and Aβ(Μ1–42), respectively. The expression sequences were designed using codons preferred by Escherichia coli, and the two peptides were expressed in this host in inclusion bodies. Peptides were purified from inclusion bodies using a combination of anion-exchange chromatography and centrifugal filtration. The method described requires little specialized equipment and provides a facile and inexpensive procedure for production of large amounts of very pure Aβ peptides. Recombinant peptides generated using this protocol produced amyloid fibrils that were indistinguishable from those formed by chemically synthesized Aβ1–40 and Aβ1–42. Formation of fibrils by all peptides was concentration-dependent, and exhibited kinetics typical of a nucleation-dependent polymerization reaction. Recombinant and synthetic peptides exhibited a similar toxic effect on hippocampal neurons, with acute treatment causing inhibition of MTT reduction, and chronic treatment resulting in neuritic degeneration and cell loss.

Overwhelming evidence indicates that the Abeta (amyloid beta-peptide) plays a critical role in the pathogenesis of Alzheimer's disease. Abeta is derived from the APP (amyloid precursor protein) by the action of two aspartyl proteases (beta- and gamma-secretases) that are leading candidates for therapeutic intervention. APP is a member of a multigene family that includes APLP1 (amyloid precursor-like protein 1) and APLP2. Both APLPs are processed in a manner analogous to APP, with all three proteins subject to ectodomain shedding and subsequent cleavage by gamma-secretase. Careful study of the APP family of proteins has already revealed important insights about APP. Here, we will review how knowledge of the similarities and differences between APP and the APLPs may prove useful for the development of novel disease-modifying therapeutics.

Among the changes that typify Alzheimer's disease (AD) are neuroinflammation and microglial activation, amyloid deposition perhaps resulting from compromised microglial function and iron accumulation. Data from Genome Wide Association Studies (GWAS) identified a number of gene variants that endow a significant risk of developing AD and several of these encode proteins expressed in microglia and proteins that are implicated in the immune response. This suggests that neuroinflammation and the accompanying microglial activation are likely to contribute to the pathogenesis of the disease. The trigger(s) leading to these changes remain to be identified. In this study, we set out to examine the link between the inflammatory, metabolic and iron-retentive signature of microglia in vitro and in transgenic mice that overexpress the amyloid precursor protein (APP) and presenilin 1 (PS1; APP/ PS1 mice), a commonly used animal model of AD. Stimulation of cultured microglia with interferon (IFN)γ and amyloid-β (Aβ) induced an inflammatory phenotype and switched the metabolic profile and iron handling of microglia so that the cells became glycolytic and iron retentive, and the phagocytic and chemotactic function of the cells was reduced. Analysis of APP/PS1 mice by magnetic resonance imaging (MRI) revealed genotype-related hypointense areas in the hippocampus consistent with iron deposition, and immunohistochemical analysis indicated that the iron accumulated in microglia, particularly in microglia that decorated Aβ deposits. Isolated microglia prepared from APP/PS1 mice were characterized by a switch to a glycolytic and iron-retentive phenotype and phagocytosis of Aβ was reduced in these cells. This evidence suggests that the switch to glycolysis in microglia may kick-start a cascade of events that ultimately leads to microglial dysfunction and Aβ accumulation.Brain Pathology 29 (2019) 606-621

Amyloid-␤ (A␤) is a major constituent of the neuritic plaque found in the brain of Alzheimer's disease patients, and a great deal of evidence suggests that the neuronal loss that is associated with the disease is a consequence of the actions of A␤. In the past few years, it has become apparent that activation of c-Jun N-terminal kinase (JNK) mediates some of the effects of A␤ on cultured cells; in particular, the evidence suggests that A␤-triggered JNK activation leads to cell death. In this study, we investigated the effect of intracerebroventricular injection of A␤ (1-40) on signaling events in the hippocampus and on long term potentiation in Schaffer collateral CA1 pyramidal cell synapses in vivo. We report that A␤ One of the pathological hallmarks of Alzheimer's disease (AD) 1 is an accumulation of plaques consisting predominately of amyloid-␤ (A␤) peptide, which is processed from amyloid precursor protein by the action of ␤-and ␥-secretase (1). Neuronal cell loss is one feature of AD, and evidence from analysis of changes in cultured cells suggests that A␤ acts as the executioner. Thus, neuronal cultures exposed to A␤ demonstrate signs of apoptosis (2-4), and previous evidence from this laboratory has revealed that cultured cortical neurons exposed to A␤ exhibited increased expression of the tumor suppressor p53; increased activation of caspase-3, a marker of apoptotic cell death; and increased TUNEL reactivity (5). The evidence is consistent with the idea that activation of the stress-activated protein kinase, c-Jun N-terminal kinase (JNK) played a significant role, because depletion of JNK1 following exposure to antisense oligonucleotide prevented the effects of A␤ (5). Similarly, Morishima et al. (6) reported that A␤ increased phosphorylation of JNK and c-Jun in cultured cortical neurons and that these changes were associated with expression of the death inducer Fas ligand (FasL). Others have reported findings that support a role for JNK activation in mediating at least certain effects of A␤. For instance, A␤-induced parallel increases in JNK activation and TUNEL reactivity in PC12 cells (7), whereas activation of JNK was shown to be localized to amyloid deposits in 7-and 12-month-old mice that overexpress amyloid precursor protein (8).It has emerged in several experimental models that increased JNK phosphorylation is associated with deficits in synaptic function; for instance, increased activation of JNK has been reported in the hippocampi of aged rats (9, 10), rats exposed to whole body irradiation (11), and rats injected with the proinflammatory cytokine, interleukin (IL)-1␤ (12) or lipopolysaccharide (13), and in all cases glutamate release was decreased. In each of these experimental conditions, long term potentiation (LTP), a model of synaptic plasticity, was markedly impaired, and this impairment was coupled with an increased hippocampal concentration of IL-1␤.A number of groups have reported that A␤ administration exerts an inhibitory effect on LTP. For instance, A␤ peptides (14 -16) and natu...

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Astrocytes, the most numerous glial cell in the brain, have multiple functions and are key to maintenance of homeostasis in the central nervous system. Microglia are the resident immunocompetent cells in the brain and share several functions with macrophages, including their phagocytic ability. Indeed microglia are the resident phagocytes in the brain and express numerous cell surface proteins which act to enable receptor-mediated phagocytosis. However recent evidence suggests that astrocytes express some genes which permit phagocytosis of phosphatidylserine-decorated cells and this probably explains sporadic reports in the literature which suggest that astrocytes become phagocytic following brain trauma. Here we examined the potential of astrocytes to phagocytose fluorescently-labelled latex beads and amyloid-β (Aβ) and report that they competently engulf both in a manner that relies on actin polymerization since it was inhibited by cytochalasin D. The data indicate that incubation of cultured astrocytes or microglia with Aβ increased phagocytosis and markers of activation of both cell types. Aβ was found to markedly increase expression of the putative Aβ-binding receptors CD36 and CD47 in astrocytes, while it decreased expression of the receptor for advanced glycation endproducts (RAGE). It is demonstrated that blocking these receptors using a neutralizing antibody attenuated Aβ-induced phagocytosis of latex beads by astrocytes. Interestingly blocking these receptors also decreased uptake of beads even in the absence of Aβ. Here we demonstrate that astrocytes are competent phagocytes and are capable of engulfing Aβ.