Oligomeric and fibrillar amyloid beta 42 induce platelet aggregation partially through GPVI.

The effects of the Alzheimer's disease (AD)-associated Amyloid-ß (Aß) peptides on platelet aggregation have been previously assessed, but most of these studies focused on Aß40 species. It also remains to be determined which distinct forms of Aß peptides exert differential effects on platelets. In AD, oligomeric Aß42 species is widely thought to be a major contributor to the disease pathogenesis. We, therefore, examine the ability of oligomeric and fibrillary Aß42 to affect platelet aggregation. We show that both forms of Aß42 induced significant platelet aggregation and that it is a novel ligand for the platelet receptor GPVI. Furthermore, a novel binding peptide that reduces the formation of soluble Aß42 oligomers was effective at preventing Aß42-dependent platelet aggregation. These results support a role for Aß42 oligomers in platelet hyperactivity.

Examining the potential clinical value of curcumin in the prevention and diagnosis of Alzheimer's disease.

Curcumin derived from turmeric is well documented for its anti-carcinogenic, antioxidant and anti-inflammatory properties. Recent studies show that curcumin also possesses neuroprotective and cognitive-enhancing properties that may help delay or prevent neurodegenerative diseases, including Alzheimer's disease (AD). Currently, clinical diagnosis of AD is onerous, and it is primarily based on the exclusion of other causes of dementia. In addition, phase III clinical trials of potential treatments have mostly failed, leaving disease-modifying interventions elusive. AD can be characterised neuropathologically by the deposition of extracellular ß amyloid (Aß) plaques and intracellular accumulation of tau-containing neurofibrillary tangles. Disruptions in Aß metabolism/clearance contribute to AD pathogenesis. In vitro studies have shown that Aß metabolism is altered by curcumin, and animal studies report that curcumin may influence brain function and the development of dementia, because of its antioxidant and anti-inflammatory properties, as well as its ability to influence Aß metabolism. However, clinical studies of curcumin have revealed limited effects to date, most likely because of curcumin's relatively low solubility and bioavailability, and because of selection of cohorts with diagnosed AD, in whom there is already major neuropathology. However, the fresh approach of targeting early AD pathology (by treating healthy, pre-clinical and mild cognitive impairment-stage cohorts) combined with new curcumin formulations that increase bioavailability is renewing optimism concerning curcumin-based therapy. The aim of this paper is to review the current evidence supporting an association between curcumin and modulation of AD pathology, including in vitro and in vivo studies. We also review the use of curcumin in emerging retinal imaging technology, as a fluorochrome for AD diagnostics.

A combination of physical activity and computerized brain training improves verbal memory and increases cerebral glucose metabolism in the elderly.

Physical exercise interventions and cognitive training programs have individually been reported to improve cognition in the healthy elderly population; however, the clinical significance of using a combined approach is currently lacking. This study evaluated whether physical activity (PA), computerized cognitive training and/or a combination of both could improve cognition. In this nonrandomized study, 224 healthy community-dwelling older adults (60-85 years) were assigned to 16 weeks home-based PA (n=64), computerized cognitive stimulation (n=62), a combination of both (combined, n=51) or a control group (n=47). Cognition was assessed using the Rey Auditory Verbal Learning Test, Controlled Oral Word Association Test and the CogState computerized battery at baseline, 8 and 16 weeks post intervention. Physical fitness assessments were performed at all time points. A subset (total n=45) of participants underwent [(18)F] fluorodeoxyglucose positron emission tomography scans at 16 weeks (post-intervention). One hundred and ninety-one participants completed the study and the data of 172 participants were included in the final analysis. Compared with the control group, the combined group showed improved verbal episodic memory and significantly higher brain glucose metabolism in the left sensorimotor cortex after controlling for age, sex, premorbid IQ, apolipoprotein E (APOE) status and history of head injury. The higher cerebral glucose metabolism in this brain region was positively associated with improved verbal memory seen in the combined group only. Our study provides evidence that a specific combination of physical and mental exercises for 16 weeks can improve cognition and increase cerebral glucose metabolism in cognitively intact healthy older adults.

Associations between gonadotropins, testosterone and ß amyloid in men at risk of Alzheimer's disease.

Testosterone and gonadotropins have been associated with cognitive decline in men and the modulation of ß amyloid (Aß) metabolism. The relatively few studies that have investigated whether changes in one or a combination of these hormones influence Aß levels have focused primarily on plasma Aß(1-40) and not on the more pathogenic Aß(1-42). Currently, no study has investigated whether these hormones are associated with an increase in brain amyloid deposition, ante mortem. Through the highly characterised Australian imaging, biomarkers and lifestyle study, we have determined the impact of these hormones on plasma Aß levels and brain amyloid burden (Pittsburgh compound B (PiB) retention). Spearman's rank correlation and linear regression analysis was carried out across the cohort and within subclassifications. Luteinizing hormone (LH) was the only variable shown, in the total cohort, to have a significant impact on plasma Aß(1-40) and Aß(1-42) levels (beta=0.163, P<0.001; beta=0.446, P<0.001). This held in subjective memory complainers (SMC) (Aß(1-40); beta=0.208, P=0.017; Aß(1-42); beta=0.215, P=0.017) but was absent in mild cognitive impairment (MCI) and Alzheimer's disease (AD) groups. In SMC, increased frequency of the APOE-ɛ4 allele (beta=0.536, P<0.001) and increasing serum LH levels (beta=0.421, P=0.004) had a significant impact on PiB retention. Whereas in MCI, PiB retention was associated with increased APOE-ɛ4 allele copy number (beta=0.674, P<0.001) and decreasing calculated free testosterone (beta=-0.303, P=0.043). These findings suggest a potential progressive involvement of LH and testosterone in the early preclinical stages of AD. Furthermore, these hormones should be considered while attempting to predict AD at these earliest stages of the disease.

Latrepirdine stimulates autophagy and reduces accumulation of α-synuclein in cells and in mouse brain.

Latrepirdine (Dimebon; dimebolin) is a neuroactive compound that was associated with enhanced cognition, neuroprotection and neurogenesis in laboratory animals, and has entered phase II clinical trials for both Alzheimer's disease and Huntington's disease (HD). Based on recent indications that latrepirdine protects cells against cytotoxicity associated with expression of aggregatable neurodegeneration-related proteins, including Aß42 and γ-synuclein, we sought to determine whether latrepirdine offers protection to Saccharomyces cerevisiae. We utilized separate and parallel expression in yeast of several neurodegeneration-related proteins, including α-synuclein (α-syn), the amyotrophic lateral sclerosis-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine expansion (HTT gene; htt-103Q). Latrepirdine effects on α-syn clearance and toxicity were also measured following treatment of SH-SY5Y cells or chronic treatment of wild-type mice. Latrepirdine only protected yeast against the cytotoxicity associated with α-syn, and this appeared to occur via induction of autophagy. We further report that latrepirdine stimulated the degradation of α-syn in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers of autophagic activity. Ongoing experiments will determine the utility of latrepirdine to abrogate α-syn accumulation in transgenic mouse models of α-syn neuropathology. We propose that latrepirdine may represent a novel scaffold for discovery of robust pro-autophagic/anti-neurodegeneration compounds, which might yield clinical benefit for synucleinopathies including Parkinson's disease, Lewy body dementia, rapid eye movement (REM) sleep disorder and/or multiple system atrophy, following optimization of its pro-autophagic and pro-neurogenic activities.

Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model.

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aß42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.

Effect of chronic hCG administration on Alzheimer's-related cognition and A beta accumulation in PS1KI mice.

Age-associated changes in the reproductive hormones-the gonadal steroid hormones and the gonadotropins-have been identified as potential risk factors for Alzheimer's disease (AD). However, levels of gonadotropins and estrogens are closely linked in vivo, and it has proven difficult to separate the effects of gonadotropins from the well-documented estrogenic effects on AD-related neuropathology in experimental models of menopause. To assess the effects of gonadotropins on cognition and AD biochemical markers independent of estrogenic effects, a potent analog of luteinizing hormone [human chorionic gonadotropin (hCG)] was administered to ovariectomized presenilin1 knock-in mice (PS1KI). Gonadotropin administration was found to induce hyperactivity and anxiety (Open Field Maze and Taste Neophobia Task) and working memory dysfunction, without altering reference memory (Morris Water Maze). Although gonadotropin administration modestly altered ß amyloid (Aß40) levels, levels of the longer more toxic form (Aß42) were unaffected. Furthermore, altered Aß40 levels were not associated with observed behavioral and cognitive impairments. These findings provide proof, in principle, that the gonadotropin hormones play a role in the modulation of AD-related behavior, cognition, and neuropathology.

Novel phage peptides attenuate beta amyloid-42 catalysed hydrogen peroxide production and associated neurotoxicity.

Amyloid-beta (Abeta) peptides play a central role in the pathogenesis of Alzheimer's disease. There is accumulating evidence that supports the notion that the toxicity associated with human Abeta (both 40 and 42) is dependent on its superoxide dismutase (SOD)-like activity. We developed a novel screening method involving phage display technology to identify novel peptides capable of inhibiting Abeta's neurotoxicity. Two random peptide libraries containing 6-mer and 15-mer peptide inserts were used and resulted in the identification of 25 peptides that bound human Abeta (40 or 42). Here, we show that two of the three most enriched peptides obtained significantly reduced Abeta42's SOD-like activity. A 15-mer peptide reduced Abeta42 neurotoxicity in a dose-dependent manner as evidenced by a reduction in LDH release. These findings were confirmed in the independent MTT assay. Furthermore, comparative analysis of the 15-mer peptide with Clioquinol, a known inhibitor of Abeta's metal-mediated redox activity, showed the 15-mer peptide to be equipotent to this metal chelator, under the same experimental conditions. These agents represent novel peptides that selectively target and neutralise Abeta-induced neurotoxicity and thus provide promising leads for rational drug development.

Ovariectomy and 17beta-estradiol replacement do not alter beta-amyloid levels in sheep brain.

The benefits of estrogen replacement as a preventative treatment for Alzheimer's disease (AD) are subject to debate. Because the effects of estrogen depletion and replacement on accumulation of the neurotoxic beta-amyloid (A beta) peptide in transgenic animal models of AD have been variable, we examined A beta levels and oxidative stress in a nontransgenic animal model. Sheep have traditionally been used as a model for human reproduction; however because they share 100% sequence homology with the human form of A beta, they may also have potential as a nontransgenic model for A beta biology. The effect of ovariectomy and estrogen replacement administered for 6 months via slow-release implant was examined in the brain of 4.5-yr-old sheep. A beta levels were measured by ELISA, and protein levels of the amyloid precursor protein (APP), APP C-terminal fragments (C100), and presenilin-1 were examined semiquantitatively by Western blot as markers of APP processing. Markers of oxidative stress were examined semiquantitatively by Western blot [4-hydroxy-2(E)-nonenal] and oxyblot (protein carbonyls). We found no effects of estrogen depletion and supplementation in terms of AD-related biochemical markers, including A beta levels, APP processing, and oxidative stress levels. Evidence of a trend toward increased P450 side-chain cleavage enzyme levels in the hippocampus of ovariectomized and estrogen supplemented sheep suggests that neurosteroidogenesis may compensate for gonadal estrogen depletion; however, these findings cannot explain the lack of effect of estrogen supplementation on APP processing. It is possible that supraphysiological doses of estrogen are necessary to yield antiamyloidogenic and antioxidative benefits in ovariectomized sheep.

Immunization in Alzheimer's disease: naïve hope or realistic clinical potential?

There has been considerable recent interest in vaccination of patients by immunotherapy as a potentially clinically useful methodology for combating histopathological changes in Alzheimer's disease (AD). The focus of the majority of this research has been on (1) active immunotherapy using the pre-aggregated synthetic beta-amyloid (Abeta) 42 preparation AN1792 vaccine (QS-21), or (2) passive immunization using injections of already prepared polyclonal anti-Abeta antibodies (intravenous immunoglobulin). These two clinical approaches to the treatment of patients with AD represent the focus of this review. We conclude here that, with certain caveats, immunization offers further potential as a technique for the treatment (and possible prevention) of AD. New studies are seeking to develop and apply safer vaccines that do not result in toxicity and neuroinflammation. Nevertheless, caution is warranted, and future clinical investigations are required to tackle key outstanding issues. These include the need to demonstrate efficacy in humans as well as animal models (especially with respect to the potentially toxic side effects of immunotherapy), and fine-tuning in safely guiding the immune response. The issue of defining necessary and sufficient criteria for determining clinical efficacy remains an additional important issue for future immunization trials. The vaccination methodology appears to offer substantial current promise for clearing both soluble and aggregated amyloid in AD. However, it remains to be determined whether this approach will help to repair already damaged neural systems in the disease, and the extent to which vaccination-driven amyloid clearance will impact beneficially on patients' neurocognitive capacity and their functional status. The outcomes of future studies will be important both clinically and scientifically: an important further test of the validity of the amyloid hypothesis of AD is to evaluate the impact of an effective anti-amyloid strategy on the functional status of patients with this disease.

Clearance mechanisms of Alzheimer's amyloid-beta peptide: implications for therapeutic design and diagnostic tests.

Currently, the 'amyloid hypothesis' is the most widely accepted explanation for the pathogenesis of Alzheimer's disease (AD). According to this hypothesis, altered metabolism of the amyloid-beta (Abeta) peptide is central to the pathological cascade involved in the pathogenesis of AD. Although Abeta is produced by almost every cell in the body, a physiological function for the peptide has not been determined, and the pathways by which Abeta leads to cognitive dysfunction and cell death are unclear. Numerous therapeutic approaches that target the production, toxicity and removal of Abeta are being developed worldwide. Although therapeutic treatment for AD may be imminent, the value and effectiveness of such treatment are largely dependent on early diagnosis of the disease. This review summarizes current knowledge of Abeta clearance, transport and degradation, and evaluates the use of such information in the development of diagnostic tools. The conflicting results of plasma Abeta ELISAs are discussed, as are the more promising results of Abeta imaging by positron emission tomography. Current knowledge of Abeta-binding proteins and Abeta-degrading enzymes is analysed in the context of a potential therapy for AD. Transport across the blood-brain barrier by the receptor for advanced glycation end products and efflux via the multi-ligand lipoprotein receptor LRP-1 is also reviewed. Enhancing clearance and degradation of Abeta remains an attractive therapeutic strategy, and improved understanding of Abeta clearance may lead to advances in diagnostics and interventions designed to prevent or delay the onset of AD.

Protein markers for Alzheimer disease in the frontal cortex and cerebellum.

OBJECTIVE: To compare proteins related to Alzheimer disease (AD) in the frontal cortex and cerebellum of subjects with early-onset AD (EOAD) with or without presenilin 1 (PS1) mutations with sporadic late-onset AD (LOAD) and nondemented control subjects. METHODS: Immunohistochemistry, immunoblot analysis, and ELISA were used to detect and assess protein levels in brain. RESULTS: In EOAD and to a lesser extent in LOAD, there was increased amyloid beta (Abeta) deposition (by immunohistochemistry), increased soluble Abeta (by immunoblot analysis), and specific increases in Abeta40 and Abeta42 (by ELISA) in the frontal cortex and, in some cases, in the cerebellum. Surprisingly, immunoblot analysis revealed reduced levels of PS1 in many of the subjects with EOAD with or without PS1 mutations. In those PS1 mutation-bearing subjects with the highest Abeta, PS1 was barely, if at all, detectable. This decrease in PS1 was specific and not attributable solely to neuronal loss because amyloid precursor protein (APP) and the PS1-interacting protein beta-catenin levels were unchanged. CONCLUSIONS: This study shows that in the frontal cortex and cerebellum from Alzheimer disease patients harboring certain presenilin 1 mutations, high levels of amyloid beta are associated with low levels of presenilin 1. The study provides the premise for further investigation of mechanisms underlying the downregulation of presenilin 1, which may have considerable pathogenic and therapeutic relevance.

Baculoviruses expressing the human familial Alzheimer's disease presenilin 1 mutation lacking exon 9 increase levels of an amyloid beta-like protein in Sf9 cells.

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-beta protein (Abeta) that is central to the pathogenesis of Alzheimer's disease. PS1 regulates the intramembranous proteolysis of a 99-amino-acid C-terminal fragment of the amyloid precursor protein (APP-C99), a cleavage event that releases Abeta following a reaction catalyzed by an enzyme termed 'gamma-secretase'. The molecular mechanism of PS1-mediated, gamma-secretase cleavage remains largely unresolved. In particular, controversy surrounds whether PS1 includes the catalytic site of the gamma-secretase protease or whether instead PS1 mediates gamma-secretase activity indirectly, perhaps by regulating the trafficking or presentation of substrates to the 'authentic' protease, which may be a molecule distinct from PS1. To address this issue, the baculovirus expression system was used to co-express: (i) APP-C99; (ii) a pathogenic, constitutively active mutant form of PS1 lacking exon 9 (PS1DeltaE9); (iii) nicastrin and (iv) tropomyosin in Spodoptera frugiperda (Sf9) cells. Cells infected with APP-C99 alone produced an Abeta-like species, and levels of this species were enhanced by the addition of baculoviruses bearing the PS1DeltaE9 mutation. The addition to APP-C99-infected cells of baculoviruses bearing nicastrin, also a transmembrane protein, had a neutral or inhibitory effect on the reaction; tropomyosin viruses had the same effect as nicastrin viruses. These results suggest that PS1DeltaE9 molecules expressed in Sf9 cells retain the ability to modulate Abeta levels. Baculoviral-expressed PS1DeltaE9 provides a source of microgram quantities of bioactive molecules for use as starting material for purifying and reconstituting gamma-secretase activity from its individual purified component parts.

Inhibiting amyloid precursor protein C-terminal cleavage promotes an interaction with presenilin 1.

Presenilin 1 (PS1) plays a pivotal role in the production of the amyloid-beta protein, which is central to the pathogenesis of Alzheimer's disease. It has been demonstrated that PS1 regulates the gamma-secretase proteolysis of the amyloid precursor protein (APP) C-terminal fragment (APP-C100), which is the final step in amyloid-beta protein production. The mechanism and detailed pathway of this PS1 activity has yet to be fully resolved, but it may be due to a presenilin-controlled trafficking of the APP fragment or possibly an inherent PS1 proteolytic activity. We have investigated the possibility of a direct interaction of PS1 and the APP-C100 within the high molecular mass presenilin complex. However, the APP-C100 is rapidly degraded, and if it forms, then any PS1.APP complex is likely to be very transitory. To circumvent this problem, we have utilized the protease inhibitor N-acetyl-leucyl-norleucinal (LLnL) and the lysosomotropic agent NH(4)Cl, which inhibits the turnover of the APP-C100. Under these conditions, levels of the fragment increased appreciably, and as shown by glycerol gradient analysis, the APP-C100 shifted to a higher molecular mass complex that overlapped with PS1. Immunoprecipitation studies demonstrated that a significant population of the APP-C100 co-precipitated with PS1. These findings suggest that PS1 may mediate the shuttling of APP fragments and/or facilitate their presentation for gamma-secretase cleavage through a direct interaction.