X11α haploinsufficiency enhances Aβ amyloid deposition in Alzheimer's disease transgenic mice

The neuronal adaptor protein X11α/mint-1/APBA-1 binds to the cytoplasmic domain of the amyloid precursor protein (APP) to modulate its trafficking and metabolism. We investigated the consequences of reducing X11α in a mouse model of Alzheimer's disease (AD). We crossed hAPPswe/PS-1ΔE9 transgenic (AD tg) mice with X11α heterozygous knockout mice in which X11α expression is reduced by approximately 50%. The APP C-terminal fragments C99 and C83, as well as soluble Aβ40 and Aβ42, were increased significantly in brain of X11α haploinsufficient mice. Aβ/amyloid plaque burden also increased significantly in the hippocampus and cortex of one year old AD tg/X11α (+/−) mice compared to AD tg mice. In contrast, the levels of sAPPα and sAPPβ were not altered significantly in AD tg/X11α (+/−) mice. The increased neuropathological indices of AD in mice expressing reduced X11α suggest a normal suppressor role for X11α on CNS Aβ/amyloid deposition.     

Costimulatory Effects of Interferon-γ and Interleukin-1β or Tumor Necrosis Factor α on the Synthesis of Aβ1-40 and Aβ1-42 by Human Astrocytes

Chronic inflammation and astrocytosis are characteristic histopathological features of Alzheimer's Disease (AD). Astrocytes are one of the predominant cell types in the brain. In AD they are activated and produce inflammatory components such as complement components, acute phase proteins, and cytokines. In this study we analyzed the effect of cytokines on the production of amyloid β (Aβ) in the astrocytoma cell line U373 and in primary human astrocytes isolated postmortem from healthy aged persons as well as from patients with AD. Astrocytes did not produce Aβ in the absence of stimuli or following stimulation with IL-1β, TNFα, IL-6, and TGF-β1. Neither did combinations of TNFα and IL-1β, IL-6 or TGF-β1, or the coadministration of IFNγ and IL-6 or TGF-β1 induce Aβ production. In contrast, pronounced production of Aβ1-40 and Aβ1-42 was observed when primary astrocytes or astrocytoma cells were stimulated with combinations of IFNγ and TNFα or IFNγ and IL-1β. Induction of Aβ production was accompanied by decreased glycosylation of APP as well as by increased secretion of APPsβ. Our results suggest that astrocytes may be an important source of Aβ in the presence of certain combinations of inflammatory cytokines. IFNγ in combination with TNFα or IL-1β seems to trigger Aβ production by supporting β-secretase cleavage of the immature APP molecule.     

Identification of a Novel Aspartic Protease (Asp 2) as β-Secretase

The Alzheimer's disease β-amyloid peptide (Aβ) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of β- and then γ-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of β-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal β-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the β-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Aβ. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.     

Presenilin/γ-Secretase Activity Is Located in Acidic Compartments of Live Neurons

Presenilin (PSEN)/γ-secretase is a protease complex responsible for the proteolytic processing of numerous substrates. These substrates include the amyloid precursor protein (APP), the cleavage of which by γ-secretase results in the production of β-amyloid (Aβ) peptides. However, exactly where within the neuron γ-secretase processes APP C99 to generate Aβ and APP intracellular domain (AICD) is still not fully understood. Here, we employ novel Förster resonance energy transfer (FRET)-based multiplexed imaging assays to directly “visualize” the subcellular compartment(s) in which γ-secretase primarily cleaves C99 in mouse cortex primary neurons (from both male and female embryos). Our results demonstrate that γ-secretase processes C99 mainly in LysoTracker-positive low-pH compartments. Using a new immunostaining protocol which distinguishes Aβ from C99, we also show that intracellular Aβ is significantly accumulated in the same subcellular loci. Furthermore, we found functional correlation between the endo-lysosomal pH and cellular γ-secretase activity. Taken together, our findings are consistent with Aβ being produced from C99 by γ-secretase within acidic compartments such as lysosomes and late endosomes in living neurons.SIGNIFICANCE STATEMENT Alzheimer''s disease (AD) genetics and histopathology highlight the importance of amyloid precursor protein (APP) processing by γ-secretase in pathogenesis. For the first time, this study has enabled us to directly “visualize” that γ-secretase processes C99 mainly in acidic compartments such as late endosomes and lysosomes in live neurons. Furthermore, we uncovered that intracellular β-amyloid (Aβ) is significantly accumulated in the same subcellular loci. Emerging evidence proposes the great importance of the endo-lysosomal pathway in mechanisms of misfolded proteins propagation (e.g., Tau, α-Syn). Therefore, the predominant processing of C99 and enrichment of Aβ in late endosomes and lysosomes may be critical events in the molecular cascade leading to AD.     

Distribution of β-amyloid and amyloid precursor protein in the brain of spawning (senescent) salmon: a natural, brain-aging model

Brain amyloid precursor protein (APP), a normal constituent of neurons, glial cells and cerebrospinal fluid, has several proposed functions (e.g., in neuronal growth and survival). It appears, however, that altered processing of APP is an initial or downstream step in the neuropathology of brain aging, Alzheimer's disease (AD), and Down's syndrome (DS). Some studies suggest that proteolytic cleavage of APP, producing β-amyloid (Aβ_1–42), could have neurotoxic or neuroprotective effects. In this study, we utilized antibodies to human APP₆₉₅ and Aβ_1–42, and Congo red staining, to search for amyloid deposition in the brain of semelparous spawning kokanee salmon (Oncorhynchus nerka kennerlyi). Intracellular APP₆₉₅ immunoreactivity (APP-ir) was observed in brain regions involved in gustation (glomerulosus complex), olfaction (putative hippocampus, olfactory bulb), vision (optic tectum), the stress response (nucleus preopticus and nucleus lateralis tuberis), reproductive behavior (nucleus preopticus magnocellularis, nucleus preopticus periventricularis, ventral telencephalon), and coordination (cerebellum). Intra- and extra-neuronal Aβ_1–42 immunoreactivity (Aβ-ir) were present in all APP-ir regions except the nucleus lateralis tuberis and Purkinje cells of the cerebellum (coordination). Thus, the relationship between APP and Aβ deposition during brain aging could shed light on the processing of APP into Aβ, neurodegeneration, and possible protection of neurons that are functioning in spawning but senescent salmon. Pacific salmon, with their predictable and synchronized life history, could provide research options not available with the existing models for studies of brain aging and amyloidosis.     

Residues at P2-P1 positions of - and ζ-cleavage sites are important in formation of β-amyloid peptide

Most of the Alzheimer's disease (AD)-linked mutations in amyloid precursor protein (APP), which cause abnormal production of β-amyloid (Aβ), are localized at the major β-secretase-and γ-secretase cleavage sites. In this study, using an APP-knockout mouse neuronal cell line, our data demonstrated that at the P2-P1 positions of the -cleavage site at Aβ49 and the ζ-cleavage site at Aβ46, aromatic amino acids caused a strong reduction in total Aβ. On the other hand, residues with a long side chain caused a decrease in Aβ₄₀ and a concomitant increase in Aβ₄₂ and Aβ₃₈. These findings indicate that the structures of the substituting residues at these key positions strongly determine the efficiency and preference of γ-secretase-mediated APP processing, which determines the ratio of different secreted Aβ species, a crucial factor in the disease development. Our findings provide new insight into the mechanisms of γ-secretase-mediated APP processing and, specifically, into why most AD-linked APP mutations are localized at major γ-secretase cleavage sites. This information may contribute to the development of methods of prevention and treatment of Alzheimer's disease aimed at modulating γ-secretase activity.     

Presenilin Is Essential for ApoE Secretion,a Novel Role of Presenilin Involved in Alzheimer's Disease Pathogenesis

Alzheimer''s disease (AD) is a debilitating dementia characterized by progressive memory loss and aggregation of amyloid-β (Aβ) protein into amyloid plaques in patient brains. Mutations in presenilin (PS) lead to abnormal generation of Aβ, which is the major cause of familial AD (FAD), and apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) onset. However, whether dysfunction of PS is involved in the pathogenesis of SAD is largely unknown. We found that ApoE secretion was completely abolished in PS-deficient cells and markedly decreased by inhibition of γ-secretase activity. Blockade of γ-secretase activity by a γ-secretase inhibitor, DAPT, decreased ApoE secretion, suggesting an important role of γ-secretase activity in ApoE secretion. Reduced ApoE secretion is also observed in nicastrin-deficient cells with reduced γ-secretase activity. PS deficiency enhanced nuclear translocation of ApoE and binding of ApoE to importin α4, a nuclear transport receptor. Moreover, the expression of PS mutants in PS-deficient cells suppressed the restoration effects on ApoE secretion compared with the expression of wild-type PS. Plasma ApoE levels were lower in FAD patients carrying PS1 mutations compared with normal control subjects. Our findings suggest a novel role of PS contributing to the pathogenesis of SAD by regulating ApoE secretion.SIGNIFICANCE STATEMENT Familial AD (FAD) typically results from mutations in the genes encoding amyloid precursor protein, presenilin 1 (PS1), or PS2. Many PS mutants have been found to exert impaired γ-secretase activity and increased amyloid-β 42 (Aβ42)/Aβ40 ratio, which induce early amyloid deposition and FAD. On the other hand, apolipoprotein E4 (ApoE4) is the major genetic risk factor for sporadic AD (SAD) and contributes to AD pathogenesis because it has reduced Aβ clearance capability compared with ApoE3 and ApoE2. FAD and SAD have long been considered to be caused by these two independent mechanisms; however, for the first time, we demonstrated that PS is essential for ApoE secretion and PS mutants affected ApoE secretion in vitro and in human samples, suggesting a novel mechanism by which PS is also involved in SAD pathogenesis.     

Cellular signaling roles of TGFβ, TNFα and βAPP in brain injury responses and Alzheimer's disease

β-Amyloid precursor protein (βAPP), transforming growth factor β (TGFβ), and tumor necrosis factor-α (TNFα) are remarkably pleiotropic neural cytokines/neurotrophic factors that orchestrate intricate injury-related cellular and molecular interactions. The links between these three factors include: their responses to injury; their interactive effects on astrocytes, microglia and neurons; their ability to induce cytoprotective responses in neurons; and their association with cytopathological alterations in Alzheimer's disease. Astrocytes and microglia each produce and respond to TGFβ and TNFα in characteristic ways when the brain is injured. TGFβ, TNFα and secreted forms of βAPP (sAPP) can protect neurons against excitotoxic, metabolic and oxidative insults and may thereby serve neuroprotective roles. On the other hand, under certain conditions TNFα and the fibrillogenic amyloid β-peptide (Aβ) derivative of βAPP can promote damage of neuronal and glial cells, and may play roles in neurodegenerative disorders. Studies of genetically manipulated mice in which TGFβ, TNFα or βAPP ligand or receptor levels are altered suggest important roles for each factor in cellular responses to brain injury and indicate that mediators of neural injury responses also have the potential to enhance amyloidogenesis and/or to interfere with neuroregeneration if expressed at abnormal levels or modified by strategic point mutations. Recent studies have elucidated signal transduction pathways of TGFβ (serine/threonine kinase cascades), TNFα (p55 receptor linked to a sphingomyelin-ceramide-NFκB pathway), and secreted forms of βAPP (sAPP; receptor guanylate cyclase-cGMP-cGMP-dependent kinase-K⁺ channel activation). Knowledge of these signaling pathways is revealing novel molecular targets on which to focus neuroprotective therapeutic strategies in disorders ranging from stroke to Alzheimer's disease.     

Apolipoprotein E and β-amyloid levels in the hippocampus and frontal cortex of Alzheimer's disease subjects are disease-related and apolipoprotein E genotype dependent

The ε4 allele of apolipoprotein E (apoE) is associated with increased risk for the development of Alzheimer's disease (AD), possibly due to interactions with the β-amyloid (Aβ) protein. The mechanism by which these two proteins are linked to AD is still unclear. To further assess their potential relationship with the disease, we have determined levels of apoE and Aβ isoforms from three brain regions of neuropathologically confirmed AD and non-AD tissue. In two brain regions affected by AD neuropathology, the hippocampus and frontal cortex, apoE levels were found to be decreased while Aβ_1–40 levels were increased. Levels of apoE were unchanged in AD cerebellum. Furthermore, levels of apoE and Aβ_1–40 were found to be apoE genotype dependent, with lowest levels of apoE and highest levels of Aβ_1–40 occurring in ε4 allele carriers. These results suggest that reduction in apoE levels may give rise to increased deposition of amyloid peptides in AD brain.     

S-Adenosylhomocysteine increases β-amyloid formation in BV-2 microglial cells by increased expressions of β-amyloid precursor protein and presenilin 1 and by hypomethylation of these gene promoters

S-Adenosylhomocysteine (SAH) has been implicated as a risk factor for neurodegenerative diseases such as Alzheimer's disease. As SAH is a potent inhibitor of all cellular methyltransferases, we herein examined the hypothesis that SAH may increase the formation of amyloid β-peptide (Aβ) in BV-2 mouse microglial cells through hypomethylation of presenilin 1 protein (PS1) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1), both of which cleave Aβ precursor protein (APP) to form Aβ. The results showed that SAH increased Aβ protein formation in a concentration-dependent manner (10–500 nM), and this effect of SAH was accompanied by significantly increased expression of APP and PS1 proteins, although SAH only significantly increased the expression of BACE1 at the highest concentration used (500 nM). SAH (500 nM) markedly induced hypomethylation of APP and PS1 gene promoters. Incubation of cells with 5′-azc (20 μM), also an inhibitor of DNA methyltransferases enhanced Aβ protein expression and APP and PS1 gene promoters hypomethylation. By contrast, pre-incubation of cells with betaine (1.0 mM), 30 min followed by incubation with SAH (500 nM) or 5′-azc (20 μM) for 24 h markedly prevented the expression of Aβ protein (by 50%, P < 0.05) and the gene promoter hypomethylation of APP and PS1. Taken together, this study demonstrates that SAH increases the production of Aβ in BV-2 cells possibly by increased expression of APP and induction of hypomethylation of APP and PS1 gene promoters.     

Application of quantitative LC–MS surrogate peptide methodology in the analysis of the amyloid beta peptide (Aβ) biosynthetic intermediate protein APP–βCTF

An area of current research in Alzheimer's disease (AD) is the biosynthetic pathway of amyloid beta peptides (Aβ) via consecutive proteolytic cleavages of the amyloid beta precursor protein (APP) by BACE and γ-secretase enzymes. APP is first cleaved by BACE to form a C-terminal fragment APP–βCTF, or also called C99, which then undergoes further cleavage by γ-secretase to form Aβ. Inhibitors of γ-secretase have been observed to yield a so-called ‘Aβ rise’ phenomenon whereby low inhibitor concentrations result in an increase in Aβ levels while high inhibitor concentrations result in lower Aβ levels. A previous report from our labs indicated that this phenomenon was related to ratios of APP–βCTF substrate relative to γ-secretase enzyme. A quantitative Western blot analysis was used with a recombinant C100 protein as calibration standards to assess the relationship of APP–βCTF, γ-secretase enzyme and various inhibitors resulting in the ‘Aβ rise’. An on-line liquid chromatography mass spectrometry (LC–MS) method employing the ‘surrogate peptide’ methodology was developed to accurately quantify the recombinant C100 used in the Western blot analyses. The surrogate peptide approach utilizes tryptic digestion of the protein to stoichiometrically yield a unique peptide fragment, in this case ^C100Aβ17–28 (LVFFAEDVGSNK) that can be readily detected by LC–MS. The absolute quantitative assessment of C100 was accomplished using synthetic Aβ17–28 to generate calibration curves over a 0.001–1 μM range and 15N isotopically labeled Aβ1–40 as the internal standard for enzymatic digestion and its proteolytic peptide [15N]-Aβ17–28 for the analysis.     

Overexpression of wild-type human APP in mice causes cognitive deficits and pathological features unrelated to Aβ levels

Transgenic mice expressing mutant human amyloid precursor protein (APP) develop an age-dependent amyloid pathology and memory deficits, but no overt neuronal loss. Here, in mice overexpressing wild-type human APP (hAPP_wt) we found an early memory impairment, particularly in the water maze and to a lesser extent in the object recognition task, but β-amyloid peptide (Aβ₄₂) was barely detectable in the hippocampus. In these mice, hAPP processing was basically non-amyloidogenic, with high levels of APP carboxy-terminal fragments, C83 and APP intracellular domain. A tau pathology with an early increase in the levels of phosphorylated tau in the hippocampus, a likely consequence of enhanced ERK1/2 activation, was also observed. Furthermore, these mice presented a loss of synapse-associated proteins: PSD95, AMPA and NMDA receptor subunits and phosphorylated CaMKII. Importantly, signs of neurodegeneration were found in the hippocampal CA1 subfield and in the entorhinal cortex that were associated to a marked loss of MAP2 immunoreactivity. Conversely, in mice expressing mutant hAPP, high levels of Aβ₄₂ were found in the hippocampus, but no signs of neurodegeneration were apparent. The results support the notion of Aβ-independent pathogenic pathways in Alzheimer's disease.     

A novel GSK-3β inhibitor reduces Alzheimer's pathology and rescues neuronal loss in vivo

Amyloid deposits, neurofibrillary tangles, and neuronal cell death in selectively vulnerable brain regions are the chief hallmarks in Alzheimer's (AD) brains. Glycogen synthase kinase-3 (GSK-3) is one of the key kinases required for AD-type abnormal hyperphosphorylation of tau, which is believed to be a critical event in neurofibrillary tangle formation. GSK-3 has also been recently implicated in amyloid precursor protein (APP) processing/Aβ production, apoptotic cell death, and learning and memory. Thus, GSK-3 inhibition represents a very attractive drug target in AD and other neurodegenerative disorders. To investigate whether GSK-3 inhibition can reduce amyloid and tau pathologies, neuronal cell death and memory deficits in vivo, double transgenic mice coexpressing human mutant APP and tau were treated with a novel non-ATP competitive GSK-3β inhibitor, NP12. Treatment with this thiadiazolidinone compound resulted in lower levels of tau phosphorylation, decreased amyloid deposition and plaque-associated astrocytic proliferation, protection of neurons in the entorhinal cortex and CA1 hippocampal subfield against cell death, and prevention of memory deficits in this transgenic mouse model. These results show that this novel GSK-3 inhibitor has a dual impact on amyloid and tau alterations and, perhaps even more important, on neuronal survival in vivo further suggesting that GSK-3 is a relevant therapeutic target in AD.     

LRP1 modulates APP trafficking along early compartments of the secretory pathway

The amyloid beta peptide (Aβ) is a central player in Alzheimer's disease (AD) pathology. Aβ liberation depends on APP cleavage by β- and γ-secretases. The low density lipoprotein receptor related protein 1 (LRP1) was shown to mediate APP processing at multiple steps. Newly synthesized LRP1 can interact with APP, implying an interaction between these two proteins early in the secretory pathway. We wanted to investigate whether LRP1 mediates APP trafficking along the secretory pathway, and, if so, whether it affects APP processing. Indeed, the early trafficking of APP within the secretory pathway is strongly influenced by its interaction with the C-terminal domain of LRP1. The LRP1-construct expressing an ER-retention motif, LRP-CT KKAA, had the capacity to retard APP traffic to early secretory compartments. In addition, we provide evidence that APP metabolism occurs in close conjunction with LRP1 trafficking, highlighting a new role of lipoprotein receptors in neurodegenerative diseases.     

Cytoplasmic gelsolin increases mitochondrial activity and reduces Aβ burden in a mouse model of Alzheimer's disease

Accumulation of amyloid-β (Aβ) peptides is thought to be a critical event in the pathology of Alzheimer's disease (AD), because they induce multiple neurotoxic effects, including mitochondrial dysfunction and apoptotic cell death. Therefore the reduction of Aβ is considered a primary therapeutic target. Gelsolin, an Aβ binding protein, has been shown to inhibit apoptosis, although the underlying mechanism is unclear. To clarify these effects, we manipulated cytoplasmic gelsolin levels through viral-directed overexpression in the brain of APP/Ps1 transgenic mice. We observed that gelsolin reduces brain Aβ burden in the APP/Ps1 mice, possibly by enhancing Aβ clearance via megalin. The reduction in brain Aβ levels was accompanied by an inhibition of nitric oxide production and cell death, not only in the choroid plexus but also in the cerebral cortex. Notably, overexpressed gelsolin restored the impaired mitochondrial activity in the APP/Ps1 mice, resulting in the increase of cytochrome c oxidase activity. By contrast, RNA interference to block gelsolin expression, confirmed that cytoplasmic gelsolin acts as a modulator of brain Aβ levels and its neurotoxic effects. We conclude that gelsolin might prevent brain amyloidosis and Aβ-induced apoptotic mitochondrial changes. These findings make cytoplasmic gelsolin a potential therapeutic strategy in AD.     

JNK regulates APP cleavage and degradation in a model of Alzheimer's disease

Secretion of Amyloid-beta peptide (Aβ) circulating oligomers and their aggregate forms derived by processing of beta-amyloid precursor protein (APP) are a key event in Alzheimer's disease (AD).We show that phosphorylation of APP on threonine 668 may play a role in APP metabolism in H4-APP^sw cell line, a degenerative AD model. We proved that JNK plays a fundamental role in this phosphorylation since its specific inhibition, with the JNK inhibitor peptide (D-JNKI1), induced APP degradation and prevented APP phosphorylation at T668. This results in a significant drop of βAPPs, Aβ fragments and Aβ circulating oligomers. Moreover the D-JNKI1 treatment produced a switch in the APP metabolism, since the peptide reduced the rate of the amyloidogenic processing in favour of the non-amyloidogenic one. All together our results suggest an important link between APP metabolism and the JNK pathway and contribute to shed light on the molecular signalling pathway of this disease indicating JNK as an innovative target for AD therapy.     

Acidic pH promotes the formation of toxic fibrils from β-amyloid peptide

Beta-amyloid (Aβ) peptides, the major component of senile plaques in brains of patients with Alzheimer’s disease (AD), were found in the low pH organelles (i.e. endosome/lysosome) of cultured neuronal cells. Since acidic pH values have been shown to promote the self-assembly of Aβs, which seems to be a prerequisite for their neuropathogenicity, elucidating the aggregation behavior of Aβs in acidic environments and their subsequent effects on neuronal cells may be crucial for understanding the neurodegenerative process of AD. In this study, the extent and rate of aggregation of Aβ_1–42 peptides at pH values of 5.8 and 7.4, as well as the structure and neurotoxic effects of these aggregates, were examined. We showed that Aβ_1–42 peptides formed large and complex fibrils much more efficiently at acidic rather than neutral pH. Furthermore, only the pH 5.8 Aβ aggregates induced significant apoptotic death of PC12 cells, as indicated by a decrease in 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) reduction and an increase in phosphatidylserine externalization. Taken together, our results suggest that the Aβs present in the acidic organelles may form neurotoxic fibrils more easily than those in the neutral cellular compartments.     

Interactions between Alzheimer's disease and cerebral ischemia—focus on inflammation

Progressive memory impairment, β-amyloid (Aβ) plaques associated with local inflammation, neurofibrillary tangles, and loss of neurons in selective brain areas are hallmarks of Alzheimer's disease (AD). Although β-amyloid precursor protein (APP) and Aβ have a central role in the etiology of AD, it is not clear which forms of APP or Aβ are responsible for the neuronal vulnerability in AD brain. Brain ischemia, another cause of dementia in the elderly, has recently been recognized to contribute to the pathogenesis of AD and individuals with severe cognitive decline and possibly underlying AD are at increased risk for ischemic events in the brain. Moreover, the ε4 allele of apolipoprotein E (ApoE) is a risk factor for both AD and poor outcome following brain ischemia and hemorrhage. Several factors and molecular mechanisms that lower the threshold of neuronal death in models of AD have recently been described. Among these neuroinflammation seems to play an important role. The development and maturation of both AD neuropathology and ischemic lesions in the central nervous system are characterized by activation of glial cells and upregulation of inflammatory mediators. Indeed, anti-inflammatory approaches have proven to be beneficial in the prevention and treatment of AD-like neuropathology and ischemic injuries in vivo. This review summarizes some of the findings suggesting that neuronal overexpression of human APP renders the brain more vulnerable to ischemic injury and describes the factors that are involved in increased neuronal susceptibility to ischemic stroke.     

Vascular endothelial growth factor (VEGF) affects processing of amyloid precursor protein and β-amyloidogenesis in brain slice cultures derived from transgenic Tg2576 mouse brain

The up-regulation of the angiogenic vascular endothelial growth factor (VEGF) in brains of Alzheimer patients in close relationship to β-amyloid (Aβ) plaques, suggests a link of VEGF action and processing of the amyloid precursor protein (APP). To reveal whether VEGF may affect APP processing, brain slices derived from 17-month-old transgenic Tg2576 mice were exposed with 1 ng/ml VEGF for 6, 24, and 72 h, followed by assessing cytosolic and membrane-bound APP expression, level of both soluble and fibrillar Aβ-peptides, as well as activities of α- and β-secretases in brain slice tissue preparations.Treatment of brain slices with VEGF did not significantly affect the expression level of APP, regardless of the exposure time studied. In contrast, VEGF exposure of brain slices for 6 h reduced the formation of soluble, SDS extractable Aβ(1–40) and Aβ(1–42) as compared to brain slice cultures incubated in the absence of any drug, while the fibrillar Aβ peptides did not change significantly. This effect was less pronounced 24 h after VEGF exposure, but was no longer detectable when brain slices were exposed by VEGF for 72 h, which indicates an adaptive response to chronic VEGF exposure. The VEGF-mediated reduction in Aβ formation was accompanied by a transient decrease in β-secretase activity peaking 6 h after VEGF exposure. To reveal whether the VEGF-induced changes in soluble Aβ-level may be due to actions of VEGF on Aβ fibrillogenesis, the fibrillar status of Aβ was examined using the thioflavin-T binding assay. Incubation of Aβ preparations obtained from Tg2576 mouse brain cortex, in the presence of VEGF slightly decreased the fibrillar content with increasing incubation time up to 72 h. The data demonstrate that VEGF may affect APP processing, at least in vitro, suggesting a role of VEGF in the pathogenesis of Alzheimer's disease.     

Thioflavins released from nanoparticles target fibrillar amyloid β in the hippocampus of APP/PS1 transgenic mice

For the delivery of drugs into the brain, the use of nanoparticles as carriers has been described as a promising approach. Here, we prepared nanoparticles as carriers for the model drugs thioflavin T and thioflavin S that bind fibrillar amyloid β peptides (Aβ). These polymer colloids are composed of a polystyrene core and a degradable PBCA [poly(butyl-2-cyanoacrylate)] shell with a diameter of 90–100 nm as shown by dynamic light scattering. Fluorescence spectrophotometric analysis revealed that encapsulated thioflavin T exhibited significantly stronger fluorescence than the free fluorophore. The enzymatic degradation of core-shell nanoparticles, as required in vivo, was shown after their treatment with porcine liver esterase, a non-specific esterase, in vitro. Shells of nanoparticles were dose-dependently degraded while their polystyrene cores remained intact. In the cortices of 7–14 months old APP/PS1 mice with age-dependent β-amyloidosis, thioflavins selectively targeted fibrillar Aβ after biodegradation-induced release from their nanoparticulate carriers upon intracerebral injection. Collectively, our data suggest that core-shell nanoparticles with controlled degradation in vivo can become versatile tools to trace and clear Aβ in the brain.