Soluble Amyloid Beta Research Articles

A hydrogel biosensor for high selective and sensitive detection of amyloid-beta oligomers.

BackgroundAlzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive and memory impairment. It is the most common neurological disease that causes dementia. Soluble amyloid-beta oligomers (AβO) in blood or cerebrospinal fluid (CSF) are the pathogenic biomarker correlated with AD.MethodsA simple electrochemical biosensor using graphene oxide/gold nanoparticles (GNPs) hydrogel electrode was developed in this study. Thiolated cellular prion protein (PrPC) peptide probe was immobilized on GNPs of the hydrogel electrode to construct an AβO biosensor. Electrochemical impedance spectroscopy was utilized for AβO analysis.ResultsThe specific binding between AβO and PrPC probes on the hydrogel electrode resulted in an increase in the electron-transfer resistance. The biosensor showed high specificity and sensitivity for AβO detection. It could selectively differentiate AβO from amyloid-beta (Aβ) monomers or fibrils. Meanwhile, it was highly sensitive to detect as low as 0.1 pM AβO in artificial CSF or blood plasma. The linear range for AβO detection is from 0.1 pM to 10 nM.ConclusionThis biosensor could be used as a cost-effective tool for early diagnosis of AD due to its high electrochemical performance and bionic structure.

Brain tissue Aβ42 levels are linked to shunt response in idiopathic normal pressure hydrocephalus.

The authors conducted a study to test if the cortical brain tissue levels of soluble amyloid beta (Aβ) reflect the propensity of cortical Aβ aggregate formation and may be an additional factor predicting surgical outcome following idiopathic normal pressure hydrocephalus (iNPH) treatment. Highly selective ELISAs (enzyme-linked immunosorbent assays) were used to quantify soluble Aβ40, Aβ42, and neurotoxic Aβ oligomers/protofibrils, associated with Aβ aggregation, in cortical biopsy samples obtained in patients with iNPH (n = 20), sampled during ventriculoperitoneal (VP) shunt surgery. Patients underwent pre- and postoperative (3-month) clinical assessment with a modified iNPH scale. The preoperative CSF biomarkers and the levels of soluble and insoluble Aβ species in cortical biopsy samples were analyzed for their association with a favorable outcome following the VP shunt procedure, defined as a ≥ 5-point increase in the iNPH scale. The brain tissue levels of Aβ42 were negatively correlated with CSF Aβ42 (Spearman’s r = -0.53, p < 0.05). The Aβ40, Aβ42, and Aβ oligomer/protofibril levels in cortical biopsy samples were higher in patients with insoluble cortical Aβ aggregates (p < 0.05). The preoperative CSF Aβ42 levels were similar in patients responding (n = 11) and not responding (n = 9) to VP shunt treatment at 3 months postsurgery. In contrast, the presence of cortical Aβ aggregates and high brain tissue Aβ42 levels were associated with a poor outcome following VP shunt treatment (p < 0.05). Brain tissue measurements of soluble Aβ species are feasible. Since high Aβ42 levels in cortical biopsy samples obtained in patients with iNPH indicated a poor surgical outcome, tissue levels of Aβ species may be associated with the clinical response to shunt treatment.

Induced dural lymphangiogenesis facilities soluble amyloid-beta clearance from brain in a transgenic mouse model of Alzheimer's disease.

Impaired amyloid-β clearance from the brain is a core pathological event in Alzheimer's disease. The therapeutic effect of current pharmacotherapies is unsatisfactory, and some treatments cause severe side effects. The meningeal lymphatic vessels might be a new route for amyloid-β clearance. This study investigated whether promoting dural lymphangiogenesis facilitated the clearance of amyloid-β from the brain. First, human lymphatic endothelial cells were treated with 100 ng/mL recombinant human vascular endothelial growth factor-C (rhVEGF-C) protein. Light microscopy verified that rhVEGF-C, a specific ligand for vascular endothelial growth factor receptor-3 (VEGFR-3), significantly promoted tube formation of human lymphatic endothelial cells in vitro. In an in vivo study, 200 μg/mL rhVEGF-C was injected into the cisterna magna of APP/PS1 transgenic mice, once every 2 days, four times in total. Immunofluorescence staining demonstrated high levels of dural lymphangiogenesis in Alzheimer's disease mice. One week after rhVEGF-C administration, enzyme-linked immunosorbent assay results showed that levels of soluble amyloid-β were decreased in cerebrospinal fluid and brain. The Morris water maze test demonstrated that spatial cognition was restored. These results indicate that the upregulation of dural lymphangiogenesis facilities amyloid-β clearance from the brain of APP/PS1 mice, suggesting the potential of the VEGF-C/VEGFR-3 signaling pathway as a therapeutic target for Alzheimer's disease.

Early changes in hyppocampal neurogenesis induced by soluble Ab1-42 oligomers

Alzheimer's disease is characterized by the loss of neurons, the accumulation of intracellular neurofibrillary tangles and extracellular amyloid plaques in the brain. However, there are contradicting data on differences in neurogenesis at the onset of the disease or before the formation of amyloid plaques. As awareness of the importance of the pre-symptom phase in neurodegenerative diseases grows in the context of early diagnosis and pathogenesis, we analyzed the critical periods of adult hippocampal neurogenesis at an early stage under the action of soluble Ab1-42 beta-amyloid. The proliferation, migration and neuronal cells survival were evaluated in mice with an injection of soluble amyloid beta-oligomers. It was found that the injection of Ab1-42 oligomers causes a decrease in cell proliferation in the mouse hippocampus. Despite the preservation of the neuroblast pool in animals after beta-amyloid injection, the process of radial migration is disrupted, and an increase in apoptosis in the neurogenic niche was revealed. Thus, our results demonstrate damage of neurogenesis critical stages: the progenitor cells, neuroblast migration, the integration of immature neurons, and the survival of neurons under application of soluble beta-amyloid oligomers. The obtained data indicate decline in proliferation rate in the subgranular zone, that is accompanied by ectopic differentiation and disturbed migration, producing, apparently, abnormal neurons that have lower survival rates. That could lead to a decrease in mature neurons numbers and the number of cells in the granular layer of the dentate gyrus.

Emerging role of amyloid beta in stress response: Implication for depression and diabetes

Chronic stress is considered a widely accepted risk factor for the development of neuropsychiatric and neurological disorders. Indeed, high cortisol levels, and, thus, hypothalamic pituitary adrenal (HPA)-axis dysregulation, have been indicated as the most frequent alteration in patients affected by depression, as well as by Alzheimer's disease (AD). Furthermore, depressive state has been pointed as an early manifestation of AD, advocating an overlap between these neuropathological events. We have previously demonstrated that central soluble beta amyloid 1-42 (Aβ) administration peptide induces a depressive like-behavior in rats, with altered HPA axis activation, reduced cortical serotonin and neurotrophin levels. The crucial role of Aβ in stress response is becoming more and more evident, indeed many reports indicate that its release is increased in stressful conditions and stress-based paradigm. Furthermore, it has been reported that stress controls Aβ production and/or clearance. Chronic stress is responsible of inducing neuroinflammation processes and reduced serotoninergic tone, both pathophysiological mechanisms proposed in the association of depression with another chronic disease, such as diabetes. Likewise, AD has also been indicated as type 3 diabetes, considering the large body of literature that suggests common biological bases. Thus, the main aim of the present review is to evaluate the most recent literature findings in humans and animal models in regard to the role of Aβ in stress response and in relation to the biological substrates and pathological pathways common to AD and comorbid diseases, such as depression and diabetes.

CROSSBETA STABLE Aβ OLIGOMERS: DISEASE-RELEVANT DRUG TARGETS AND VERSATILE REFERENCE STANDARDS IN BIOMARKER ASSAYS IN AD

There is accumulating evidence that soluble amyloid beta (Aβ) oligomers, rather than the amyloid fibrils, are the principal pathogenic species in Alzheimer disease (AD). Because they exhibit acute synaptic toxicity and induce neurodegenerative processes, and because the Aβ oligomer levels in cerebrospinal fluid appear to correlate with disease severity and progression, these species constitute an optimal drug target and possible useful diagnostic marker in early AD. Unfortunately, the instability and limited characterization of oligomer preparations have seriously impeded research and development in this field. Crossbeta has addressed this problem by developing a technology to reproducibly generate stable soluble Aß1–42 oligomers that are devoid of monomeric or fibrillar Aß and show biophysical properties indistinguishable from the native species. Furthermore, Crossbeta oligomers demonstrate typical functionality of pathological Aβ species such as neurotoxicity, in vivo LTP depression and induction of inflammatory response. The availability of the stable oligomers opens previously inaccessible avenues for R&D. For example, it enabled high throughput screening of drug-like compound libraries. Derived from a 100k compound screening effort, small molecule CBB68 was identified and shown to neutralize oligomer-induced neurotoxicity in rat primary neurons and to rescue the synaptic deficit induced by Aβ oligomers in vivo as well as in vitro. In particular, the in vivo LTP assay demonstrated the efficacy and brain penetration of CBB68 after intravenous administration. In another application, Crossbeta's oligomers have also been used to select shark antibodies that specifically recognize a conformational oligomeric epitope and bind to Aß1–42 oligomers with high affinity (sub-nM), but not to the monomers or fibrils. This antibody provides potential to selectively quantify Aß1–42 oligomers in patient material as part of a high sensitivity biomarker assay together with the stable Crossbeta oligomers as calibrator. Furthermore, the oligomers are excellent research tools, e.g. in disease-relevant bioassays. We show and conclude that the stable oligomers enable multiple applications and constitute optimum drug targets.

SOLUBLE AMYLOID-BETA AGGREGATES FROM HUMAN ALZHEIMER’S DISEASE BRAINS

Soluble amyloid-beta (Aβ) aggregates likely contribute substantially to the dementia that characterizes Alzheimer's disease. However, despite intensive study of in vitro preparations and animal models, little is known about the characteristics of soluble Aβ aggregates in the human Alzheimer's disease brain. Here we present a new method for extracting soluble Aβ aggregates from human brains, separating them from insoluble aggregates and Aβ monomers using differential ultracentrifugation, and purifying them >6000 fold by dual antibody immunoprecipitation. Our approach to isolating soluble Aβ aggregates from human brain involved using 2–3 grams of frontal or parietal cortex from pathologically confirmed CDR3 (severe) AD cases. Tissue was homogenized in 1xPBS containing protease inhibitors and subjected to differential ultracentrifugation, and dual antibody immunoprecipitation. We examined the effect of multiple extractions, the use of detergents during extraction, and chromatographic methods to improve enrichment of the Aβ aggregates prior to immunopurification. The stability of the aggregates was characterized during purification using size-exclusion chromatography, sucrose density gradient centrifugation, immunoelectron microscopy, and quantitative tracking of the Aβ aggregates at each step. We developed a method that results in <40% loss of starting material and purifying them >6000 fold with no detectible ex vivo aggregation of monomeric Aβ, and no apparent ex vivo alterations in soluble aggregate sizes. Loss of Aβ was greatly reduced by universal blocking of materials with albumin. By immunoelectron microscopy, soluble Aβ aggregates typically appear as clusters of 10–20 nanometer diameter ovoid structures with 2–3 amino-terminal Aβ antibody binding sites, distinct from previously characterized structures. A major challenge has been that the specific forms of soluble Aβ aggregates most relevant to human disease have not been determined. Aβ can potentially aggregate into a vast number of forms, consisting of different numbers of Aβ peptides, various size forms of Aβ, multiple Aβ post-translational modifications and alternative structural configurations of Aβ. Here we present a method to purify soluble Aβ aggregates directly from frozen human AD brain tissue, reasoning this would be the most relevant approach to facilitate investigation into the characteristics of native soluble Aβ aggregates, and deepen our understanding of Alzheimer's dementia.

SAFETY OF SOLANEZUMAB IN PHASE 3 STUDIES IN INDIVIDUALS WITH MILD ALZHEIMER’S DISEASE DEMENTIA

Solanezumab, a humanized monoclonal antibody that binds to the mid-domain of soluble amyloid-beta peptide was developed for treatment of Alzheimer's disease (AD). EXPEDITION, EXPEDITION2 and EXPEDITION3 were Phase 3, 18-month, placebo-controlled studies investigating solanezumab (400mg IV/4 weeks) in individuals with mild (EXPEDITION3) or mild-to-moderate (EXPEDITION and EXPEDITION2) AD dementia at baseline. We present treatment-emergent adverse event (AE) findings from the 18-month double-blind periods of EXPEDITION3 and from the pooled data from EXPEDITION and EXPEDITION2 (subjects with mild AD dementia at baseline in either study) and EXPEDITION3. Mild AD dementia was defined as meeting National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer's Disease and Related Disorders Association criteria for probable AD and having a Mini-Mental State Examination 20–26 inclusive at baseline. For analyses of EXPEDITION3 data, p-values for treatment group comparisons of percentages were obtained using Fisher's exact test. For analyses of the pooled data, p-values were obtained from the Cochran-Mantel-Haenszel (CMH) test of general association stratified by study. EXPEDITION3 included 2129 subjects, aged 55–91 years; the pooled database included 3435 subjects, aged 55–94 years. Deaths, serious AEs, discontinuations due to an AE, treatment-emergent AEs were evenly distributed across study treatment groups (Table 1). In EXPEDITION3, there were no statistically significant treatment group differences at the individual Preferred Term level for any serious AE. For the pooled data, among serious AEs, Aortic Aneurysm was significantly more common in the solanezumab group while Seizure, Cerebrovascular Accident, Hyponatraemia and Sepsis were more common in the placebo group. There were also no significant differences between treatment groups in changes in amyloid-related imaging abnormality microhemorrhage/hemosiderin (ARIA-H) deposition, presence of ARIA-edema/effusion (ARIA-E, all asymptomatic), or incidence of treatment-emergent anti-solanezumab antibodies or incidence of cardiac ischemic or arrhythmia AEs (Table 1). The incidence of several treatment-emergent AEs was significantly different between treatment groups (Table 2 and 3). EXPEDITION3 safety findings from the mild AD dementia population are mostly consistent with previously published safety findings from the EXPEDITION and EXPEDITION2, with no new safety signals detected. Any suggestion of an increase incidence of cardiovascular events seen in EXPEDITION and EXPEDITION2 was not evident in EXPEDITION3.

Is testosterone important for women as they age?

In women, is testosterone an obligatory precursor for extra-gonadal estradiol production after menopause, and has important direct androgenic genomic and nongenomic effects. Testosterone levels in women decline with age from the 4th decade, reaching a nadir in the 7th decade of life. Testosterone levels are positively associated with sexual function in women, and multiple randomised placebo-controlled trials have shown that testosterone therapy can be effective for the treatment of female sexual dysfunction in postmenopausal women. However, low testosterone in ageing women has multiple potential undesirable consequences that have been somewhat ignored. In the brain, testosterone exhibits neuroprotection against oxidative stress, serum deprivation-induced apoptosis and soluble amyloid beta toxicity, and clinical trials suggest exogenous testosterone enhances cognitive performance in postmenopausal women. Lower free testosterone in older women is associated with a decline in bone mineral density and a greater risk of hip fracture. Observational studies implicate testosterone as having favourable cardiovascular effects measured by surrogate outcomes, however associations between endogenous testosterone and CVD risk, and total mortality, particularly in older women are yet to be established.

Antidepressant drugs for beta amyloid-induced depression: A new standpoint?

Mounting evidence suggests that depression represents a risk factor and an early manifestation of Alzheimer's disease (AD). Neuropsychiatric symptoms may derive from neurobiological changes in specific brain areas and may be considered prodromal of dementia. We have previously reported the depressive-like profile in rats receiving a single intracerebroventricular injection of soluble amyloid beta protein (ßA). Here, we verified the effect of different classes of antidepressants on the ßA-induced depressive behavior and on cortical monoamine levels. To these purposes, the forced swimming test was performed and cortical levels of serotonin (5-HT) and noradrenaline (NA) were quantified by high performance liquid chromatography (HPLC). We found that acute fluoxetine (20mg/kg, s.c.), reboxetine (10mg/kg, s.c.), and ketamine (15mg/kg, i.p.) significantly reduced the immobility in ßA-treated rats compared to controls. Fluoxetine and reboxetine reversed 5-HT reduction, while βA-induced NA increase was further enhanced by all treatments. Treatments with fluoxetine, reboxetine and ketamine were able to revert soluble ßA-induced decrease of cortical BDNF levels, while only fluoxetine and ketamine, but not reboxetine, had the same effects on cortical NGF expression. Moreover, plasma soluble ßA-levels were lowered by fluoxetine, but not reboxetine and ketamine, treatments.Our data suggest that different classes of antidepressants yield a short-acting effect on rat soluble ßA-induced depressive profile. Thus, we hypothesize a novel common mechanism of action of these drugs also based upon a “ßA lowering” effect. Although further investigations are still needed, our study might open a new scenario for unravelling the molecular antidepressant mechanisms of these drugs.

A sensitive and selective electrochemical biosensor for the determination of beta-amyloid oligomer by inhibiting the peptide-triggered in situ assembly of silver nanoparticles.

Soluble beta-amyloid (Aβ) oligomer is believed to be the most important toxic species in the brain of Alzheimer’s disease (AD) patients. Thus, it is critical to develop a simple method for the selective detection of Aβ oligomer with low cost and high sensitivity. In this paper, we report an electrochemical method for the detection of Aβ oligomer with a peptide as the bioreceptor and silver nanoparticle (AgNP) aggregates as the redox reporters. This strategy is based on the conversion of AgNP-based colorimetric assay into electrochemical analysis. Specifically, the peptide immobilized on the electrode surface and presented in solution triggered together the in situ formation of AgNP aggregates, which produced a well-defined electrochemical signal. However, the specific binding of Aβ oligomer to the immobilized peptide prevented the in situ assembly of AgNPs. As a result, a poor electrochemical signal was observed. The detection limit of the method was found to be 6 pM. Furthermore, the amenability of this method for the analysis of Aβ oligomer in serum and artificial cerebrospinal fluid (aCSF) samples was demonstrated.

In silico investigation on the inhibition of Aβ42 aggregation by Aβ40 peptide by potential of mean force study

Recent experimental data revealed that small, soluble Amyloid beta (Aβ42) oligomers, especially dimers impair synaptic plasticity and memory leading to Alzheimer’s disease. Here, we have studied dimerization of Aβ42/Aβ42 homo-dimer and Aβ40/Aβ42 hetero-dimer in terms of free energy profile by all-atom simulations using the ff99SB force field. We have found that in the presence of Aβ40 peptide, there exists a strong tendency to form a hetero-dimer with Aβ42 peptide, suggesting that a possible co-oligomerization. Furthermore, we have investigated the effects of Aβ40 on the Aβ42 peptide. Our study also shows that in presence of Aβ40, the beta-content of Aβ42 monomer is reduced. Additionally, certain residues important for bending in Aβ42 peptide attained an increased flexibility in the presence of Aβ40. The salt-bridge destabilization also manifested the impact of Aβ40 on Aβ42 peptide as a whole. Based on this, one may expect that Aβ40 inhibits the aggregation propensity of Aβ42. Moreover, the binding free energy obtained by the molecular mechanics–Poisson–Boltzmann surface area method also revealed a strong affinity between the two isoforms thereby suggests that Aβ40 binding induces conformational change in Aβ42. Our results suggest that co-oligomerization of Aβ isoforms may play a substantial role in Alzheimer’s disease.

Overexpression of EphB2 in hippocampus rescues impaired NMDA receptors trafficking and cognitive dysfunction in Alzheimer model

Alzheimer’s disease (AD) is a progressive neurodegenerative disease, which affects more and more people. But there is still no effective treatment for preventing or reversing the progression of the disease. Soluble amyloid-beta (Aβ) oligomers, also known as Aβ-derived diffusible ligands (ADDLs) play an important role in AD. Synaptic activity and cognition critically depend on the function of glutamate receptors. Targeting N-methyl-D-aspartic acid (NMDA) receptors trafficking and its regulation is a new strategy for AD early treatment. EphB2 is a key regulator of synaptic localization of NMDA receptors. Aβ oligomers could bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation in the proteasome. Here we identified that overexpression of EphB2 with lentiviral vectors in dorsal hippocampus improved impaired memory deficits and anxiety or depression-like behaviors in APPswe/PS1-dE9 (APP/PS1) transgenic mice. Phosphorylation and surface expression of GluN2B-containing NMDA receptors were also improved. Overexpression of EphB2 also rescued the ADDLs-induced depletion of the expression of EphB2 and GluN2B-containing NMDA receptors trafficking in cultured hippocampal neurons. These results suggest that improving the decreased expression of EphB2 and subsequent GluN2B-containing NMDA receptors trafficking in hippocampus may be a promising strategy for AD treatment.

A bispecific Tribody PET radioligand for visualization of amyloid-beta protofibrils – a new concept for neuroimaging

Antibodies are highly specific for their target molecules, but their poor brain penetrance has restricted their use as PET ligands for imaging of targets within the CNS. The aim of this study was to develop an antibody-based radioligand, using the TribodyTM format, for PET imaging of soluble amyloid-beta (Aβ) protofibrils, which are suggested to cause neurodegeneration in Alzheimer's disease. Antibodies, even when expressed in smaller engineered formats, are large molecules that do not enter the brain in sufficient amounts for imaging purposes. Hence, their transport across the blood-brain barrier (BBB) needs to be facilitated, for example through interaction with the transferrin receptor (TfR). Thus, a Fab fragment of the TfR antibody 8D3 was fused with two single chain variable fragments (scFv) of the Aβ protofibril selective antibody mAb158. Five TribodyTM proteins (A1-A5) were generated with different linkers between the Fab-8D3 and scFv-158. All proteins bound to TfR and Aβ protofibrils in vitro. Three of the proteins (A1-A3) were radiolabeled with iodine-125 and studied ex vivo in wild-type (wt) and transgenic mice overexpressing human Aβ. The systemic pharmacokinetics were similar with half-lives in blood of around 9h for all three ligands. Brain concentrations at 2h were around 1% of the injected dose per gram brain tissue, which is similar to what is observed for small molecular radioligands and at least 10-fold higher than antibodies in general. At 72h, transgenic mice showed higher concentrations of radioactivity in the brain than wt mice (12, 15- and 16-fold for A1, A2 and A3 respectively), except in the cerebellum, an area largely devoid of Aβ pathology. A3 was then labelled with iodine-124 for in vivo positron emission tomography (PET) imaging. Brain concentrations were quantified in six different regions showing a clear distinction both quantitatively and visually between wt and transgenic mice and a good correlation with Aβ pathology. We have thus produced a recombinant, bispecific protein, actively transported into the brain, for PET imaging within the CNS. In a longer perspective, this technique may enable imaging of other proteins involved in neurodegenerative diseases for which imaging agents are completely lacking today.

Management of Alzheimers Disease: Role of Existing Therapies, Traditional Medicines and New Treatment Targets

Dementia, a common problem in elderly, is rising worldwide including India. Alzheimer’s disease is the main etiology for dementia. Currently anticholinesterases, N-methyl-D-aspartate receptor antagonists, disease modifying agents form the mainstay of treatment of Alzheimer’s disease. However, recent breakthroughs in Alzheimer’s disease research have given multiple new treatment targets. This review summarizes the role of existing therapies and their limitations, role of traditional medicines and new treatment targets. The newer agents include molecules targeting tau protein like modulators of τ-kinases or phosphatases, kinase inhibitors, τ-aggregation inhibitors. It also includes molecules which targets amyloid plaques like, inhibitors or modulators of the secretases, amyloid-β aggregation inhibitors and amyloid-plaque degradation enhancers. Role of nootropics, statins and antipsychotics in the management of Alzheimer’s disease is limited and lacks conclusive evidence. Studies with vaccines have shown reduced amyloid beta and τ-pathology, neutralized soluble amyloid beta oligomers. But search for the appropriate vaccine for Alzheimer’s disease is still on. Antioxidants, metal chelators, Metal-protein attenuating compounds, metal complexes have also shown some potential. Traditional medicines like huperzine A and galantamine have been used for management of Alzheimer’s disease. Traditional medicinal system in India particularly Ayurveda mentions neuroprotective actions of plant drugs like Brahmi, Shankhapushpi, Amla, Guduchi, Tulsi, Ashwagandha and Haritaki, which can help in improving the treatment armamentarium for Alzheimer’s disease.The understanding of Alzheimer’s disease neurobiology is improving now and the search for better treatment options is still on. This provides an opportunity for more research on integrated approach using existing therapies, traditional and alternative medicines with newer treatments and potential candidates for management of Alzheimer’s disease.

Homocysteine metabolism is associated with cerebrospinal fluid levels of soluble amyloid precursor protein and amyloid beta.

Disturbed homocysteine metabolism may contribute to amyloidogenesis by modulating the amyloid precursor protein (APP) production and processing. The objective of this study was to investigate the relationships between cerebral amyloid production and both blood and cerebrospinal fluid (CSF) markers of the homocysteine metabolism. We assessed CSF concentrations of soluble APPα, soluble APPβ, and amyloid β1-42 (Aβ1-42), as well as plasma levels of homocysteine (Hcys), total vitamin B12, and folate, and CSF concentrations of homocysteine (Hcys-CSF), 5-methyltetrahydrofolate (5-MTHF), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) in 59 subjects with normal cognition. Linear regression analyses were performed to assess associations between homocysteine metabolism parameters and amyloid production. The study was approved by the Ethical Committee of the University of Bonn. After controlling for age, gender, APOEe4 status, and albumin ratio (Qalb), higher Aβ1-42 CSF levels were associated with high Hcys and low vitamin B12 plasma levels as well as with high Hcys, high SAH, and low 5-MTHF CSF levels. Higher CSF concentrations of sAPPα and sAPPβ were associated with high SAH levels. The results suggest that disturbed homocysteine metabolism is related to increased CSF levels of sAPP forms and Aβ1-42, and may contribute to the accumulation of amyloid pathology in the brain. Disturbed homocysteine metabolism may contribute to amyloidogenesis by modulating the amyloid precursor protein (APP) production and processing. We found associations between CSF levels of soluble APP forms and Aβ1-42, and markers of the homocysteine metabolism in both plasma and CSF in adults with normal cognition. Disturbed homocysteine metabolism may represent a target for preventive and early disease-modifying interventions in Alzheimer's disease.

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps.

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer's disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (Aβo) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble Aβo correlate with memory deficits in AD models and humans. The Aβo-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated Aβo infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning Aβo-induced memory deficits at the onset of AD. Since soluble Aβo aggregate relatively fast into insoluble Aβ fibrils that correlate poorly with the clinical state of patients, soluble Aβo are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of Aβo and continuous infusion of Aβo antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of Aβo in pre-dementia patients.

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer's disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (Aβo) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble Aβo correlate with memory deficits in AD models and humans. The Aβo-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated Aβo infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning Aβo-induced memory deficits at the onset of AD. Since soluble Aβo aggregate relatively fast into insoluble Aβ fibrils that correlate poorly with the clinical state of patients, soluble Aβo are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of Aβo and continuous infusion of Aβo antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of Aβo in pre-dementia patients.

Early effects of Aβ1-42 oligomers injection in mice: Involvement of PI3K/Akt/GSK3 and MAPK/ERK1/2 pathways

Neuronal and synaptic loss are the best pathological correlates for memory decline in Alzheimer's disease (AD). Soluble beta-amyloid oligomers (AβO) are considered to putatively play a crucial role in the early synapse loss and cognitive impairment observed in AD. Evidence suggests that oxidative stress and apoptosis are involved in the mechanism of Aβ-induced neurotoxicity and AD pathogenesis. This study aimed to explore the molecular mechanisms that contribute to the early memory deficits induced by intracerebroventricular injection of AβO in mice. Ten days after a single AβO injection memory impairments were observed, as measured by Morris water maze and novel object recognition tests. Cognitive decline was associated with increased oxidative stress, caspase-9 activation, and decreased hippocampal synaptophysin immunoreactivity. Furthermore, GSH levels were significantly higher in AβO-injected mice than in sham mice, showing that a protective mechanism might develop due to oxidative stress. Additionally, AβO-induced toxicity was aligned with an increment of the activation of Akt and ERK1/2, and reduced activity of GSK3. These findings suggest that AβO injection triggers a cascade of events that mimic the key neuropathological hallmarks of AD. Aβ acute injection helps to better understand how this peptide impairs specific signaling pathways leading to synaptic and memory dysfunctions. Thus, this model is a valid tool for investigating AD and may suggest a new way to develop neuroprotective therapies at such early stages of the disease.

Small molecule NPT-440-1 inhibits ionic flux through Aβ1 -42 pores: Implications for Alzheimer's disease therapeutics

Increased levels of soluble amyloid-beta (Aβ) oligomers are suspected to underlie Alzheimer's disease (AD) pathophysiology. These oligomers have been shown to form multi-subunit Aβ pores in bilayers and induce uncontrolled, neurotoxic, ion flux, particularly calcium ions, across cellular membranes that might underlie cognitive impairment in AD. Small molecule interventions that modulate pore activity could effectively prevent or ameliorate their toxic activity. Here we examined the efficacy of a small molecule, NPT-440-1, on modulating amyloid pore permeability. Co-incubation of B103 rat neuronal cells with NPT-440-1 and Aβ1-42 prevented calcium influx. In purified lipid bilayers, we show that a 10-15min preincubation, prior to membrane introduction, was required to prevent conductance. Thioflavin-T and circular dichroism both suggested a reduction in Aβ1-42 β-sheet content during this incubation period. Combined with previous studies on site-specific amino acid substitutions, these results suggest that pharmacological modulation of Aβ1-42 could prevent amyloid pore-mediated AD pathogenesis.