Brain insulin resistance accelerates Aß fibrillogenesis by inducing GM1 ganglioside clustering in the presynaptic membranes.

Type 2 diabetes mellitus is thought to be a significant risk factor for Alzheimer's disease. Insulin resistance also affects the central nervous system by regulating key processes, such as neuronal survival and longevity, learning and memory. However, the mechanisms underlying these effects remain uncertain. To investigate whether insulin resistance is associated with the assembly of amyloid ß-protein (Aß) at the cell surface of neurons, we inhibited insulin-signalling pathways of primary neurons. The treatments of insulin receptor (IR)-knockdown and a phosphatidylinositol 3-kinase inhibitor (LY294002), but not an extracellular signal-regulated kinase inhibitor, induced an increase in GM1 ganglioside (GM1) levels in detergent-resistant membrane microdomains of the neurons. The aged db/db mouse brain exhibited reduction in IR expression and phosphorylation of Akt, which later induced an increase in the high-density GM1-clusters on synaptosomes. Neurons treated with IR knockdown or LY294002, and synaptosomes of the aged db/db mouse brains markedly accelerated an assembly of Aßs. These results suggest that ageing and peripheral insulin resistance induce brain insulin resistance, which accelerates the assembly of Aßs by increasing and clustering of GM1 in detergent-resistant membrane microdomains of neuronal membranes.

Naphthoquinone-tyrptophan reduces neurotoxic Aß*56 levels and improves cognition in Alzheimer's disease animal model.

An increasing body of evidence indicates a role for oligomers of the amyloid-ß peptide (Aß) in the neurotoxicity of this peptide and the pathology of Alzheimer's disease (AD). Several neurotoxic oligomeric forms of Aß have been noted ranging from the larger Amyloid ß-Derived Diffusible Ligands (ADDLs) to smaller trimers and dimers of Aß. More recently a dodecameric form of Aß with a 56 kDa molecular weight, denoted Aß*56, was shown to cause memory impairment in AD model mice. Here, we present for the first time a potential therapeutic strategy for AD that targets the early stages in the formation of neurotoxic Aß*56 oligomers using a modified quinone-Tryptophan small molecule N-(3-chloro-1,4-dihydro-1,4-dioxo-2-naphthalenyl)-L-Tryptophan (Cl-NQTrp). Using NMR spectroscopy we show that this compound binds the aromatic recognition core of Aß and prevents the formation of oligomers. We assessed the effect of Cl-NQTrp in vivo in transgenic flies expressing Aß(1-42) in their nervous system. When these flies were fed with Cl-NQTrp a marked alleviation of their Aß-engendered reduced life span and defective locomotion was observed. Finally, intraperitoneal injection of Cl-NQTrp into an aggressive AD mouse model reduced the level of the Aß*56 species in their brain and reversed their cognitive defects. Further experiments should assess whether this is a direct effect of the drug in the brain or an indirect peripheral effect. This is the first demonstration that targeted reduction of Aß*56 results in amelioration of AD symptoms. This second generation of tryptophan-modified naphthoquinones could therefore serve as potent disease modifying therapeutic for AD.

Tau-dependent microtubule disassembly initiated by prefibrillar beta-amyloid.

Alzheimer's Disease (AD) is defined histopathologically by extracellular beta-amyloid (Abeta) fibrils plus intraneuronal tau filaments. Studies of transgenic mice and cultured cells indicate that AD is caused by a pathological cascade in which Abeta lies upstream of tau, but the steps that connect Abeta to tau have remained undefined. We demonstrate that tau confers acute hypersensitivity of microtubules to prefibrillar, extracellular Abeta in nonneuronal cells that express transfected tau and in cultured neurons that express endogenous tau. Prefibrillar Abeta42 was active at submicromolar concentrations, several-fold below those required for equivalent effects of prefibrillar Abeta40, and microtubules were insensitive to fibrillar Abeta. The active region of tau was localized to an N-terminal domain that does not bind microtubules and is not part of the region of tau that assembles into filaments. These results suggest that a seminal cell biological event in AD pathogenesis is acute, tau-dependent loss of microtubule integrity caused by exposure of neurons to readily diffusible Abeta.

Inhibition of Alzheimer's amyloid toxicity with a tricyclic pyrone molecule in vitro and in vivo.

Small beta-amyloid (Abeta) 1-42 aggregates are toxic to neurons and may be the primary toxic species in Alzheimer's disease (AD). Methods to reduce the level of Abeta, prevent Abeta aggregation, and eliminate existing Abeta aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with Abeta oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with Abeta42 oligomer. Circular dichroism spectroscopy reveals monomeric Abeta42 peptide remains as a random coil/alpha-helix structure in the presence of CP2 over 48 h. Atomic force microscopy studies show CP2 exhibits similar ability to inhibit Abeta42 aggregation as that of Congo red and curcumin. Atomic force microscopy closed-fluid cell study demonstrates that CP2 disaggregates Abeta42 oligomers and protofibrils. CP2 also blocks Abeta fibrillations using a protein quantification method. Treatment of 5x familial Alzheimer's disease mice, a robust Abeta42-producing animal model of AD, with a 2-week course of CP2 resulted in 40% and 50% decreases in non-fibrillar and fibrillar Abeta species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing Abeta aggregation and disaggregating existing Abeta oligomers and protofibrils.

Inhibition of tau fibrillization by oleocanthal via reaction with the amino groups of tau.

Tau is a microtubule-associated protein that promotes microtubule assembly and stability. In Alzheimer's disease and related tauopathies, tau fibrillizes and aggregates into neurofibrillary tangles. Recently, oleocanthal isolated from extra virgin olive oil was found to display non-steroidal anti-inflammatory activity similar to ibuprofen. As our unpublished data indicates an inhibitory effect of oleocanthal on amyloid beta peptide fibrillization, we reasoned that it might inhibit tau fibrillization as well. Herein, we demonstrate that oleocanthal abrogates fibrillization of tau by locking tau into the naturally unfolded state. Using PHF6 consisting of the amino acid residues VQIVYK, a hexapeptide within the third repeat of tau that is essential for fibrillization, we show that oleocanthal forms an adduct with the lysine via initial Schiff base formation. Structure and function studies demonstrate that the two aldehyde groups of oleocanthal are required for the inhibitory activity. These two aldehyde groups show certain specificity when titrated with free lysine and oleocanthal does not significantly affect the normal function of tau. These findings provide a potential scheme for the development of novel therapies for neurodegenerative tauopathies.

Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate.

Resveratrol, a natural phytoalexin found in grapes and red wine, increases longevity in the short-lived invertebrates Caenorhabditis elegans and Drosophila and exerts a variety of biological effects in vertebrates, including protection from ischemia and neurotoxicity. Its effects on vertebrate lifespan were not yet known. The relatively long lifespan of mice, which live at least 2.5 years, is a hurdle for life-long pharmacological trials. Here, the authors used the short-lived seasonal fish Nothobranchius furzeri with a maximum recorded lifespan of 13 weeks in captivity. Short lifespan in this species is not the result of spontaneous or targeted genetic mutations, but a natural trait correlated with the necessity to breed in an ephemeral habitat and tied with accelerated development and expression of ageing biomarkers at a cellular level. Resveratrol was added to the food starting in early adulthood and caused a dose-dependent increase of median and maximum lifespan. In addition, resveratrol delays the age-dependent decay of locomotor activity and cognitive performances and reduces the expression of neurofibrillary degeneration in the brain. These results demonstrate that food supplementation with resveratrol prolongs lifespan and retards the expression of age-dependent traits in a short-lived vertebrate.

Helical substructure of neurofilaments isolated from Myxicola and squid giant axons.

Neurofilaments purified from invertebrate giant axons have been analyzed with the electron microscope. The neurofilaments have a helical substructure which is most easily observed when the neurofilaments are partially denatured with 0.5 M KCl or 2 M urea. When the ropelike structure comprising the neurofilaments untwists, two strands 4--5.5nm in diameter can be resolved. Upon further denaturation these strands break up into rod-shaped segments and subsequently these segments roll up into amorphous globular structures. Stained, filled densities can be resolved within the strand segments, and these resemble similar structures observed within the intact neurofilaments. The strands appear to consist of protofilaments 2--2.5 nm in diameter. These observations suggest that the neurofilament is a ropelike, helical structure composed of two strands twisted tightly around each other, and they su-port the filamentous rather than the golbular model of intermediate filament structure.

Axoplasmic transport in the crayfish nerve cord. The role of fibrillar constituents of neurons.

(a) Axoplasmic transport of tritium-labeled proteins in crayfish nerve cord was confirmed at a slow rate of 1 mm/day. A second proteinaceous component which moves at a rate of 10 mm/day was also detected. Radioautography and biochemical analysis indicate that proteins migrating at these velocities have a perikaryal origin and move caudad within axons as sharply defined peaks. (b) Evidence is presented for the blockage of the slow and the fast movement of proteins by intraganglionic injection of the anti-mitotic agent vinblastine sulfate (0.1 mM). (c) Electron microscope observations of vinblastine-treated ganglia revealed a reduction in the number of axonal microtubules and the formation of intracellular aggregates presumably composed of microtubular protein. (d) These findings would be compatible with the involvement of microtubules in both slow and fast axoplasmic transport. However, the block induced by vinblastine was detected in regions of the cord (up to 10 mm away from the injection site) where the number and morphology of microtubules appeared unaltered. In addition, axons showing effects of vinblastine occasionally contained mitochondria with remarkably dense and thickened membranes. (e) In association with the surfaces of axonal microtubules are lateral filamentous elements (40-80 A in diameter) which also showed vinblastine-induced alterations. Our observations indicate that such filiform structures, associated with microtubules, may be a necessary component in the transport mechanism(s).

Preventing beta-amyloid fibrillization and deposition: beta-sheet breakers and pathological chaperone inhibitors.

Central to the pathogenesis of Alzheimer's disease (AD) is the conversion of normal, soluble beta-amyloid (sAbeta) to oligomeric, fibrillar Abeta. This process of conformational conversion can be influenced by interactions with other proteins that can stabilize the disease-associated state; these proteins have been termed 'pathological chaperones'. In a number of AD models, intervention that block soluble Abeta aggregation, including beta-sheet breakers, and compounds that block interactions with pathological chaperones, have been shown to be highly effective. When combined with early pathology detection, these therapeutic strategies hold great promise as effective and relatively toxicity free methods of preventing AD related pathology.

Inhibition of HEWL fibril formation by taxifolin: Mechanism of action.

Among therapeutic approaches for amyloid-related diseases, attention has recently turned to the use of natural products as effective anti-aggregation compounds. Although a wealth of in vitro and in vivo evidence indicates some common inhibitory activity of these compounds, they don't generally suggest the same mechanism of action. Here, we show that taxifolin, a ubiquitous bioactive constituent of foods and herbs, inhibits formation of HEWL amyloid fibrils and their related toxicity by causing formation of very large globular, chain-like aggregates. A range of amyloid-specific techniques were employed to characterize this process. We found that taxifolin exerts its effect by binding to HEWL prefibrillar species, rather than by stabilizing the molecule in its native-like state. Furthermore, it's binding results in diverting the amyloid pathway toward formation of very large globular, chain-like aggregates with low ß-sheet content and reduced solvent-exposed hydrophobic patches. ThT fluorescence measurements show that the binding capacity of taxifolin is significantly reduced, upon generation of large protofibrillar aggregates at the end of growth phase. We believe these results may help design promising inhibitors of protein aggregation for amyloid-related diseases.

Inhibition of the formation of amyloid beta-protein fibrils using biocompatible nanogels as artificial chaperones.

The formation of fibrils by amyloid beta-protein (Abeta) is considered as a key step in the pathology of Alzheimer's disease (AD). Inhibiting the aggregation of Abeta is a promising approach for AD therapy. In this study, we used biocompatible nanogels composed of a polysaccharide pullulan backbone with hydrophobic cholesterol moieties (cholesterol-bearing pullulan, CHP) as artificial chaperones to inhibit the formation of Abeta-(1-42) fibrils with marked amyloidgenic activity and cytotoxicity. The CHP-nanogels incorporated up to 6-8 Abeta-(1-42) molecules per particle and induced a change in the conformation of Abeta from a random coil to alpha-helix- or beta-sheet-rich structure. This structure was stable even after a 24-h incubation at 37 degrees C and the aggregation of Abeta-(1-42) was suppressed. Furthermore, the dissociation of the nanogels caused by the addition of methyl-beta-cyclodextrin released monomeric Abeta molecules. Nanogels composed of amino-group-modified CHP (CHPNH(2)) with positive charges under physiological conditions had a greater inhibitory effect than CHP-nanogels, suggesting the importance of electrostatic interactions between CHPNH(2) and Abeta for inhibiting the formation of fibrils. In addition, CHPNH(2) nanogels protected PC12 cells from Abeta toxicity.

Bacopa monniera extract reduces amyloid levels in PSAPP mice.

PSAPP mice expressing the "Swedish" amyloid precursor protein and M146L presenilin-1 mutations are a well-characterized model for spontaneous amyloid plaque formation. Bacopa monniera has a long history of use in India as an anti-aging and memory-enhancing ethnobotanical therapy. To evaluate the effect of Bacopa monniera extract (BME) on amyloid (Abeta) pathology in PSAPP mice, two doses of BME (40 or 160 mg/kg/day) were administered starting at 2 months of age for either 2 or 8 months. Our present data suggests that BME lowers Abeta 1-40 and 1-42 levels in cortex by as much as 60%, and reverses Y-maze performance and open field hyperlocomotion behavioral changes present in PSAPP mice. The areas encompassed by Congo Red-positive fibrillar amyloid deposits, however, were not altered by BME treatment. The data suggest that BME has potential application in Alzheimer's disease therapeutics.

Neurofilaments are nonessential to the pathogenesis of toxicant-induced axonal degeneration.

Axonal neurofilament (NF) accumulations occur before development of symptoms and before other pathological changes among idiopathic neurodegenerative diseases and toxic neuropathies, suggesting a cause-effect relationship. The dependence of symptoms and axonal degeneration on neurofilament accumulation has been tested here in a transgenic mouse model (Eyer and Peterson, 1994) lacking axonal NFs and using two prototypic toxicant models. Chronic acrylamide (ACR) or 2,5-hexanedione exposure resulted in progressive and cumulative increases in sensorimotor deficits. Neurobehavioral tests demonstrated similar expression of neurotoxicity in transgenic (T) mice and their nontransgenic (NT) littermates (containing normal numbers of axonal NFs). Axonal lesions were frequently observed after exposure to either toxicant. Quantitation of ACR-induced lesions demonstrated the distal location of pathology and equal susceptibility of T and NT axons. We conclude that axonal NFs have no effect on neurotoxicity and the pattern of pathology in these mammalian toxic neuropathies. These results also suggest that the role of neurofilament accumulation in the pathogenesis of neurodegenerative diseases requires careful evaluation.

Non-steroidal anti-inflammatory drugs have anti-amyloidogenic effects for Alzheimer's beta-amyloid fibrils in vitro.

The pathogenesis of Alzheimer's disease (AD) is characterized by cerebral deposits of amyloid beta-peptides (A beta) and neurofibrillary tangles which are surrounded by inflammatory cells. Long-term use of non-steroidal anti-inflammatory drugs (NSAIDs) reduces the risk of developing AD and delays the onset of the disease. In the present study, we used fluorescence spectroscopy with thioflavin T and electron microscopy to examine the effects of NSAIDs such as ibuprofen, aspirin, meclofenamic acid sodium salt, diclofenac sodium salt, ketoprofen, flurbiprofen, naproxen, sulindac sulfide and indomethacin on the formation, extension, and destabilization of beta-amyloid fibrils (fA beta) at pH 7.5 at 37 degrees C in vitro. All examined NSAIDs dose-dependently inhibited formation of fA beta from fresh A beta(1-40) and A beta(1-42), as well as their extension. Moreover, these NSAIDs dose-dependently destabilized preformed fA betas. The overall activity of the molecules examined was in the following order: ibuprofen approximately sulindac sulfide >or= meclofenamic acid sodium salt>aspirin approximately ketoprofen >or= flurbiprofen approximately diclofenac sodium salt>naproxen approximately indomethacin. Although the mechanisms by which these NSAIDs inhibit fA beta formation from A beta, and destabilize preformed fA beta in vitro are still unclear, NSAIDs may be promising for the prevention and treatment of AD.

Pharmacological approaches of neurofibrillary degeneration.

Alzheimer disease (AD) and related tauopathies are all characterized histopathologically by neurofibrillary degeneration. The neurofibrillary changes, whether of paired helical filaments (PHF), twisted ribbons or straight filaments (SF) are made up of abnormally hyperphosphorylated tau. Unlike normal tau which promotes assembly and maintains structure of microtubules, the abnormal tau not only lacks these functions but also sequesters normal tau, MAP1 and MAP2, and causes disassembly of microtubules. This toxic behavior of the abnormal tau is solely due to its hyperphosphorylation because dephosphorylation restores it into a normal-like protein. The abnormal hyperphosphorylation also promotes the self-assembly of tau into PHF/SF. The state of phosphorylation of a phosphoprotein is the function of the activities of protein kinases and as well as of protein phosphatases that regulate the level of phosphorylation. A cause of the abnormal hyperphosphorylation in AD brain is a decrease in the activity of protein phosphatase (PP)-2A, a major regulator of the phosphorylation of tau. A decrease in PP-2A activity results in the abnormal hyperphosphorylation of tau not only by decreased dephosphorylation of tau but also by stimulating the activities of tau kinases like CaMKII, PKA and MAP kinases which are regulated by PP-2A. Thus, the abnormal hyperphosphorylation can be inhibited both by inhibition of the activity/s of a tau protein kinase and as well as by restoration of the activity/s of a tau protein phosphatase. The development of drugs that inhibit neurofibrillary degeneration is a very promising and feasible therapeutic approach to inhibit the progression of AD and related tauopathies.

Synthesis of 6-[2-(benzoxazol-2-ylmethylamino)ethoxy]-1-alkyl-1H-indole-2-carboxylic acid and inhibitory activity on beta-amyloid aggregation.

6-[2-(benzoxazol-2-ylmethylamino)ethoxy]-1-alkyl-1H-indole-2-carboxylic acids were designed and synthesized as beta-amyloid (Abeta) fibril assembly inhibitors. Their inhibitory activity on Abeta, aggregation was evaluated by thioflavin T assay although their activities were insignificant.

Peripheral nerve changes induced by methyl n-butyl ketone and potentiation by methyl ethyl ketone.

A study of the sequential morphological changes in the peripheral nerve induced by experimental inhalation exposure of methyl n-butyl ketone (MBK) revealed that the earliest change was an increase in the number of neurofilaments in the large myelinated nerve fibers. This change occurred prior to axonal swelling or myelin thinning. As the duration of exposure lengthened the number of neurofilaments gradually increased and ultimately produced axonal swelling with secondary thinning of the myelin sheath. This appears to be the pathogenesis of the "giant axonal" neuropathy. Another change observed early in this neuropathy was the presence of inpouchings of the myelin sheath, which also increased in number in parallel to the duration of exposure. A careful study of the sequential changes in the entire motor unit did not show a predilection for early morphological changes at the axon terminal. Abnormalities at the neuromuscular junction occurred only after a full spectrum of changes were seen in the main nerve trunk, nerve roots and intramuscular nerves. An important observation was the marked potentiation of peripheral neurotoxicity observed when animals were exposed to MBK in combination with methyl ethyl ketone (MEK) at a ratio of 1:5, MBK:MEK. The latter solvent showed no neurotoxic effect alone. This might help explain a recent outbreak of a polyneuropathy affecting many workers. One further observation was that the sural nerve of a patient with prolonged exposure to MBK showed changes similar to those induced experimentally.

Chronic model of neurofibrillary changes induced in mature rabbits by metallic aluminum.

A slurry of aluminum powder injected into the brains of mature rabbits produced neurofibrillary changes in neurons of spinal cord and cerebrum similar to those produced by aluminum chloride, and with similar topography and rates of formation. The major difference observed with this preparation was that many rabbits survived several weeks or months before having any obvious seizures, compared to 2 to 3 weeks with aluminum chloride, and some survived with no obvious symptoms, apparently indefinitely (12 months being the longest time before sacrifice). This chronic animal model of neurofibrillary changes, induced in a mature nervous system, will allow better investigations of alterations in the biochemistry, pathology, behavior and cognition which may occur.

Progressing encephalomyelopathy with muscular atrophy, induced by aluminum powder.

The injection of aluminum powder into the cerebrospinal fluid of adult rabbits induced a slowly progressing encephalomyelopathy characterized at first by alteration of posture and then by myoclonic jerks and muscle weakness. Neurofibrillary degeneration was the hallmark of the disease and involved most of the gray areas. Giant axonal swellings were also numerous, particularly in the proximal axonal segment of neurons of the anterior horns. In the anterior horns the number of neurons with neurofibrillary degeneration decreased with time, while the images of neuronophagia increased in number in the rabbits killed in the second and third month after aluminum injection. In these animals there were also pathologic changes in the peripheral nerves and muscles. The peripheral nerve showed wallerian-like degeneration. Furthermore, in some animals, the presence of nodal axonal swellings and of paranodal myelin retraction were expression also of a distal axonopathy. Neurogenic muscular atrophy appeared in animals sacrificed in the second and third month after injection.

Apolipoprotein E increases the fibrillogenic potential of synthetic peptides derived from Alzheimer's, gelsolin and AA amyloids.

Apolipoprotein E (apoE) has been found in association with several different types of systemic and cerebral amyloid deposits and the presence of the epsilon 4 allele constitutes a risk factor for Alzheimer's disease. It has been shown that apoE binds and promotes the fibrillogenesis in vitro of Alzheimer's amyloid beta-peptide, suggesting an important role for apoE in the modulation of amyloidogenesis. Due to the co-localization of apoE with several biochemically distinct amyloid deposits, it has been proposed that apoE plays a general role modulating and/or participating in amyloidosis. In the present study, we show for the first time that apoE, isolated from human plasma, increases fibril formation of synthetic peptides comprising the amyloidogenic sequences of gelsolin amyloid related to familial amyloidosis Finnish type, and amyloid A found in secondary amyloidosis and familial Mediterranean fever. Our results suggest that apoE acts as a general pathological chaperone in various amyloidoses by enhancing the transition from soluble peptides into amyloid-forming, pathological molecules.