amyloid-beta-peptides and Glioma

The neuropathological hallmarks of Alzheimer's disease (AD) are extracellular plaques built up by the accumulation of the amyloid-β protein precursor (AβPP)-derived peptide β (Aβ), and intracellular tangles of hyperphosphorylated tau protein. Sirtuin 2 (SIRT2) is a member of the sirtuin family, featuring conserved enzymes with deacetylase activity and involved in several cell molecular pathways. We investigated the importance of SIRT2 inhibition in AD. We inhibited SIRT2 by small molecules (AGK-2, AK-7) and examined AβPP metabolism in H4-SW neuroglioma cells overexpressing AβPP and two AD transgenic mouse models (3xTg-AD and APP23). The in vitro studies suggested that the inhibition of SIRT2 reduced Aβ production; in vivo data showed an improvement of cognitive performance in the novel object recognition test, and an effect on AβPP proteolytic processing leading to a reduction of soluble β-AβPP and an increase of soluble α-AβPP protein. In 3xTg-AD mice, we noticed that total tau protein level rose. Overall, our pre-clinical data support a role for SIRT2 inhibition in the improvement of cognitive performance and the modulation of molecular mechanisms relevant for AD, thus deserving attention as possible therapeutic strategy.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Benzamides; Brain; Calcium-Binding Proteins; Cell Line, Tumor; Cognition Disorders; Disease Models, Animal; Enzyme Inhibitors; Furans; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Glioma; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microfilament Proteins; Peptide Fragments; Phosphorylation; Quinolines; Sirtuin 2; Sulfonamides

Alzheimer disease (AD) is characterized by chronic neuroinflammation, which may lead to dysfunction in neuronal circuits. Although reactive microglia are found in association with accumulation of beta amyloid (Aβ) plaques in the AD brain, their contribution to neuronal cell loss remains speculative. A major genetic risk factor for sporadic AD is inheritance of the apolipoprotein (apo) E4 allele, which has been shown to contribute significantly to neurodegeneration in AD. Many studies have documented the ability of Aβ fibrils in vitro to induce microglia to undergo phenotypic activation, which results in the secretion and/or expression of a plethora of free radicals and pro-inflammatory mediators. These mediators, such as reactive nitrogen/oxygen species and IL-1β as well as cyclooxygenase-2 (COX-2) with associated prostaglandin E2 (PGE(2)), are believed to be neurotoxic and to contribute to the underlying cause of AD. We have used the human H4 neuroglioma cells as a model astroglial system to examine the interactions between IL-1β and nitric oxide (NO) as co-stimulators of Aβ(1-40) in enhancing the expression of COX-2 and production of PGE(2) in the presence of recombinant human apolipoprotein E4 (apoE4). Neither Aβ(1-40) nor its reverse sequence analog Aβ(40-1) alone had a significant effect on COX-2 expression and PGE(2) production in the cells. In contrast, after co-incubation with apoE4, Aβ(1-40) increased IL-1β-induced COX-2 expression and PGE(2) production. Aβ(12-28), which binds with high affinity to apoE4, blocked apoE4-mediated effects on Aβ(1-40). Furthermore, (±)-S-Nitroso-N-acetylpenicillamine (SNAP), an agent that releases nitric oxide (NO) in situ, alone did not affect IL-1β-induced COX-2 expression, but increased PGE(2) production only. Addition of Aβ(1-40) preincubated with apoE4 to H4 cells in the presence of SNAP led to an additive IL-1β-induced COX-2 expression and PGE(2) production. These observations indicate that increased PGE(2) resulted from increased nitrosative stress, which is enhanced by apoE4. Thus a molecular understanding of the interactions of apoE4 with Aβ, NO and IL-1β on the regulation of the COX-2/prostaglandin pathway may open new avenues in understanding the mechanism of development of neurodegenerative disease such as AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Apolipoprotein E4; Brain Neoplasms; Cell Line, Tumor; Cell Survival; Cyclooxygenase 2; Glioma; Humans; Interleukin-1beta; Nerve Degeneration; Nitric Oxide; Peptide Fragments

Accumulation and deposition of β-amyloid protein (Aβ) are the hallmark features of Alzheimer's disease. The inhalation anesthetic isoflurane has been shown to induce caspase activation and increase Aβ accumulation. In addition, recent studies suggest that isoflurane may directly promote the formation of cytotoxic soluble Aβ oligomers, which are thought to be the key pathological species in AD. In contrast, propofol, the most commonly used intravenous anesthetic, has been reported to have neuroprotective effects. We therefore set out to compare the effects of isoflurane and propofol alone and in combination on caspase-3 activation and Aβ oligomerization in vitro and in vivo. Naïve and stably-transfected H4 human neuroglioma cells that express human amyloid precursor protein, the precursor for Aβ; neonatal mice; and conditioned cell culture media containing secreted human Aβ40 or Aβ42 were treated with isoflurane and/or propofol. Here we show for the first time that propofol can attenuate isoflurane-induced caspase-3 activation in cultured cells and in the brain tissues of neonatal mice. Furthermore, propofol-mediated caspase inhibition occurred when there were elevated levels of Aβ. Finally, isoflurane alone induces Aβ42, but not Aβ40, oligomerization, and propofol can inhibit the isoflurane-mediated oligomerization of Aβ42. These data suggest that propofol may mitigate the caspase-3 activation by attenuating the isoflurane-induced Aβ42 oligomerization. Our findings provide novel insights into the possible mechanisms of isoflurane-induced neurotoxicity that may aid in the development of strategies to minimize potential adverse effects associated with the administration of anesthetics to patients.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Anesthetics, Inhalation; Anesthetics, Intravenous; Animals; Apoptosis; Blotting, Western; Brain; Caspase 3; Enzyme Activation; Glioma; Humans; Isoflurane; Mice; Mice, Inbred C57BL; Mice, Transgenic; Peptide Fragments; Propofol; Protein Multimerization; Tumor Cells, Cultured

A disrupted blood-brain barrier (BBB) might have major effects on the progression of Alzheimer's disease (AD). This supports the theory of blood as a chronic source of exogenous amyloid-beta (Abeta) peptide as well as other neurotoxic substances in the brain, which would normally be excluded by an intact BBB. In addition to Abeta, neuroinflammation is suggested to contribute to the pathological conditions in AD and is a known disruptor of BBB integrity. Consequently, new therapeutic approaches to stabilize the BBB should be developed. Serum derived immunoglobulins, also called intravenous immunoglobulins (IVIG), are gained from plasma of healthy individuals and were found to induce positive effects in some patients with AD, but mechanisms of action are unclear by now. Moreover, there are no data on how IVIG affects the BBB itself. Therefore, we examined the potency of Abeta peptides as well as neuroinflammatory mediators (TNF-alpha and LPS) either alone or after simultaneous application of IVIG to disintegrate the BBB by evaluating the transport rate of carboxyfluorescein, a marker of paracellular leakage. Our results showed beneficial effects of IVIG on a disrupted BBB, which could positively influence diseases outcome in AD. With a stabilized BBB the brain is prevented from systemic Abeta entry, as well as from entry of other toxic substances from the blood.

Topics: Amyloid beta-Peptides; Animals; Biological Transport; Blood-Brain Barrier; Cell Line, Tumor; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Drug Interactions; Fluoresceins; Fluorometry; Glioma; Immunoglobulins, Intravenous; Lipopolysaccharides; Peptide Fragments; Rats; Time Factors; Tumor Necrosis Factor-alpha

The detection of cell cycle proteins in Alzheimer's disease (AD) brains may represent an early event leading to neurodegeneration. To identify cell cycle modifiers with anti-Abeta properties, we assessed the effect of Differentiation-Inducing Factor-1 (DIF-1), a unique, small-molecule from Dictyostelium discoideum, on the proteolysis of the amyloid beta-protein precursor (APP) in a variety of different cell types. We show that DIF-1 slows cell cycle progression through G0/G1 that correlates with a reduction in cyclin D1 protein levels. Western blot analysis of DIF-treated cells and conditioned medium revealed decreases in the levels of secreted APP, mature APP, and C-terminal fragments. Assessment of conditioned media by sandwich ELISA showed reduced levels of Abeta40 and Abeta42, also demonstrating that treatment with DIF-1 effectively decreases the ratio of Abeta42 to Abeta40. In addition, DIF-1 significantly diminished APP phosphorylation at residue T668. Interestingly, site-directed mutagenesis of APP residue Thr668 to alanine or glutamic acid abolished the effect of DIF-1 on APP proteolysis and restored secreted levels of Abeta. Finally, DIF-1 prevented the accumulation of APP C-terminal fragments induced by the proteasome inhibitor lactacystin, and calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal (ALLN). Our findings suggest that DIF-1 affects G0/G1-associated amyloidogenic processing of APP by a gamma-secretase-, proteasome- and calpain-insensitive pathway, and that this effect requires the presence of residue Thr668.

Topics: Acetylcysteine; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Benzazepines; Cell Line; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; Cyclin D1; Fibroblasts; Glioma; Hexanones; Humans; Hydrocarbons, Chlorinated; Indoles; Leupeptins; Mice; Peptide Fragments; Proteasome Inhibitors; Purines; Recombinant Fusion Proteins; Roscovitine; Threonine

The cytoplasmic tail of amyloid precursor protein (APP) possesses the NPTY motif to which several phosphotyrosine-binding domain-containing proteins bind, including X11alpha and mDab1. X11alpha has been shown to slow cellular APP processing and reduce secretion of Abeta peptides. However, the effect of mDab1 on APP processing has not been determined. Here, we show that mDab1 increases the levels of cellular mature APP and promotes its processing by the secretases in both transiently transfected HEK 293 cells and in neuroglioma U251 cells. These effects derive specifically from the interaction of APP with mDab1 since they are not observed in APP deletion mutants lacking the interaction module NPTY. We further demonstrate that mDab1 enhances cell surface expression of APP, possibly by interfering with its endocytosis. Interestingly, X11alpha and mDab1 exert opposing effects on APP processing. However, when both proteins are co-expressed the effect of X11alpha overrides that of mDab1. Taken together, these results suggest that the relative stoichiometry and binding affinity of the adaptor proteins determines the final outcome on APP metabolism.

Topics: Age Factors; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Biotinylation; Blotting, Western; Brain; Cadherins; Carrier Proteins; Cell Line; Endocytosis; Enzyme-Linked Immunosorbent Assay; Gene Expression; Glioma; Humans; Immunoprecipitation; Mice; Mice, Transgenic; Nerve Tissue Proteins; Peptide Fragments; Protein Transport; Sequence Deletion; Transfection