Modulation of Amyloid ß-Induced Microglia Activation and Neuronal Cell Death by Curcumin and Analogues.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is not restricted to the neuronal compartment but includes important interactions with immune cells, including microglia. Protein aggregates, common pathological hallmarks of AD, bind to pattern recognition receptors on microglia and trigger an inflammatory response, which contributes to disease progression and severity. In this context, curcumin is emerging as a potential drug candidate able to affect multiple key pathways implicated in AD, including neuroinflammation. Therefore, we studied the effect of curcumin and its structurally related analogues cur6 and cur16 on amyloid-ß (Aß)-induced microglia activation and neuronal cell death, as well as their effect on the modulation of Aß aggregation. Primary cortical microglia and neurons were exposed to two different populations of Aß42 oligomers (Aß42Os) where the oligomeric state had been assigned by capillary electrophoresis and ultrafiltration. When stimulated with high molecular weight Aß42Os, microglia released proinflammatory cytokines that led to early neuronal cell death. The studied compounds exerted an anti-inflammatory effect on high molecular weight Aß42O-stimulated microglia and possibly inhibited microglia-mediated neuronal cell toxicity. Furthermore, the tested compounds demonstrated antioligomeric activity during the process of in vitro Aß42 aggregation. These findings could be investigated further and used for the optimization of multipotent candidate molecules for AD treatment.

Oligomerization and Conformational Change Turn Monomeric ß-Amyloid and Tau Proteins Toxic: Their Role in Alzheimer's Pathogenesis.

The structural polymorphism and the physiological and pathophysiological roles of two important proteins, ß-amyloid (Aß) and tau, that play a key role in Alzheimer's disease (AD) are reviewed. Recent results demonstrate that monomeric Aß has important physiological functions. Toxic oligomeric Aß assemblies (AßOs) may play a decisive role in AD pathogenesis. The polymorph fibrillar Aß (fAß) form has a very ordered cross-ß structure and is assumed to be non-toxic. Tau monomers also have several important physiological actions; however, their oligomerization leads to toxic oligomers (TauOs). Further polymerization results in probably non-toxic fibrillar structures, among others neurofibrillary tangles (NFTs). Their structure was determined by cryo-electron microscopy at atomic level. Both AßOs and TauOs may initiate neurodegenerative processes, and their interactions and crosstalk determine the pathophysiological changes in AD. TauOs (perhaps also AßO) have prionoid character, and they may be responsible for cell-to-cell spreading of the disease. Both extra- and intracellular AßOs and TauOs (and not the previously hypothesized amyloid plaques and NFTs) may represent the novel targets of AD drug research.

Interaction of amyloid-ß (Aß) oligomers with neurexin 2α and neuroligin 1 mediates synapse damage and memory loss in mice.

Brain accumulation of the amyloid-ß protein (Aß) and synapse loss are neuropathological hallmarks of Alzheimer disease (AD). Aß oligomers (AßOs) are synaptotoxins that build up in the brains of patients and are thought to contribute to memory impairment in AD. Thus, identification of novel synaptic components that are targeted by AßOs may contribute to the elucidation of disease-relevant mechanisms. Trans-synaptic interactions between neurexins (Nrxs) and neuroligins (NLs) are essential for synapse structure, stability, and function, and reduced NL levels have been associated recently with AD. Here we investigated whether the interaction of AßOs with Nrxs or NLs mediates synapse damage and cognitive impairment in AD models. We found that AßOs interact with different isoforms of Nrx and NL, including Nrx2α and NL1. Anti-Nrx2α and anti-NL1 antibodies reduced AßO binding to hippocampal neurons and prevented AßO-induced neuronal oxidative stress and synapse loss. Anti-Nrx2α and anti-NL1 antibodies further blocked memory impairment induced by AßOs in mice. The results indicate that Nrx2α and NL1 are targets of AßOs and that prevention of this interaction reduces the deleterious impact of AßOs on synapses and cognition. Identification of Nrx2α and NL1 as synaptic components that interact with AßOs may pave the way for development of novel approaches aimed at halting synapse failure and cognitive loss in AD.

Peptides as Potential Therapeutics for Alzheimer's Disease.

Intracellular synthesis, folding, trafficking and degradation of proteins are controlled and integrated by proteostasis. The frequency of protein misfolding disorders in the human population, e.g., in Alzheimer's disease (AD), is increasing due to the aging population. AD treatment options are limited to symptomatic interventions that at best slow-down disease progression. The key biochemical change in AD is the excessive accumulation of per-se non-toxic and soluble amyloid peptides (Aß(1-37/44), in the intracellular and extracellular space, that alters proteostasis and triggers Aß modification (e.g., by reactive oxygen species (ROS)) into toxic intermediate, misfolded soluble Aß peptides, Aß dimers and Aß oligomers. The toxic intermediate Aß products aggregate into progressively less toxic and less soluble protofibrils, fibrils and senile plaques. This review focuses on peptides that inhibit toxic Aß oligomerization, Aß aggregation into fibrils, or stabilize Aß peptides in non-toxic oligomers, and discusses their potential for AD treatment.

APP Regulates Microglial Phenotype in a Mouse Model of Alzheimer's Disease.

UNLABELLED: Prior work suggests that amyloid precursor protein (APP) can function as a proinflammatory receptor on immune cells, such as monocytes and microglia. Therefore, we hypothesized that APP serves this function in microglia during Alzheimer's disease. Although fibrillar amyloid ß (Aß)-stimulated cytokine secretion from both wild-type and APP knock-out (mAPP(-/-)) microglial cultures, oligomeric Aß was unable to stimulate increased secretion from mAPP(-/-) cells. This was consistent with an ability of oligomeric Aß to bind APP. Similarly, intracerebroventricular infusions of oligomeric Aß produced less microgliosis in mAPP(-/-) mice compared with wild-type mice. The mAPP(-/-) mice crossed to an APP/PS1 transgenic mouse line demonstrated reduced microgliosis and cytokine levels and improved memory compared with wild-type mice despite robust fibrillar Aß plaque deposition. These data define a novel function for microglial APP in regulating their ability to acquire a proinflammatory phenotype during disease. SIGNIFICANCE STATEMENT: A hallmark of Alzheimer's disease (AD) brains is the accumulation of amyloid ß (Aß) peptide within plaques robustly invested with reactive microglia. This supports the notion that Aß stimulation of microglial activation is one source of brain inflammatory changes during disease. Aß is a cleavage product of the ubiquitously expressed amyloid precursor protein (APP) and is able to self-associate into a wide variety of differently sized and structurally distinct multimers. In this study, we demonstrate both in vitro and in vivo that nonfibrillar, oligomeric forms of Aß are able to interact with the parent APP protein to stimulate microglial activation. This provides a mechanism by which metabolism of APP results in possible autocrine or paracrine Aß production to drive the microgliosis associated with AD brains.

A human scFv antibody that targets and neutralizes high molecular weight pathogenic amyloid-ß oligomers.

Brain accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) is increasingly considered a key early event in the pathogenesis of Alzheimer's disease (AD). A variety of AßO species have been identified, both in vitro and in vivo, ranging from dimers to 24mers and higher order oligomers. However, there is no consensus in the literature regarding which AßO species are most germane to AD pathogenesis. Antibodies capable of specifically recognizing defined subpopulations of AßOs would be a valuable asset in the identification, isolation, and characterization of AD-relevant AßO species. Here, we report the characterization of a human single chain antibody fragment (scFv) denoted NUsc1, one of a number of scFvs we have identified that stringently distinguish AßOs from both monomeric and fibrillar Aß. NUsc1 readily detected AßOs previously bound to dendrites in cultured hippocampal neurons. In addition, NUsc1 blocked AßO binding and reduced AßO-induced neuronal oxidative stress and tau hyperphosphorylation in cultured neurons. NUsc1 further distinguished brain extracts from AD-transgenic mice from wild type (WT) mice, and detected endogenous AßOs in fixed AD brain tissue and AD brain extracts. Biochemical analyses indicated that NUsc1 targets a subpopulation of AßOs with apparent molecular mass greater than 50 kDa. Results indicate that NUsc1 targets a particular AßO species relevant to AD pathogenesis, and suggest that NUsc1 may constitute an effective tool for AD diagnostics and therapeutics.

Aging and amyloid ß oligomers enhance TLR4 expression, LPS-induced Ca2+ responses, and neuron cell death in cultured rat hippocampal neurons.

BACKGROUND: Toll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer's disease (AD). AD is associated to oligomers of the amyloid ß peptide (Aßo) that cause intracellular Ca2+ dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aßo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence. METHODS: Ca2+ imaging and immunofluorescence against annexin V and TLR4 were applied in short- and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and Aßo on cytosolic [Ca2+] and on apoptosis as well as on expression of TLR4. RESULTS: LPS increases cytosolic [Ca2+] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with Aßo increases TLR4 expression and enhances LPS-induced Ca2+ responses and neuron cell death. CONCLUSIONS: Aging and amyloid ß oligomers, the neurotoxin involved in Alzheimer's disease, enhance TLR4 expression as well as LPS-induced Ca2+ responses and neuron cell death in rat hippocampal neurons aged in vitro.

Assessing the Effects of Acute Amyloid ß Oligomer Exposure in the Rat.

Alzheimer's disease (AD) is the most common form of dementia, yet there are no therapeutic treatments that can either cure or delay its onset. Currently, the pathogenesis of AD is still uncertain, especially with respect to how the disease develops from a normal healthy brain. Amyloid ß oligomers (AßO) are highly neurotoxic proteins and are considered potential initiators to the pathogenesis of AD. Rat brains were exposed to AßO via bilateral intracerebroventricular injections. Rats were then euthanized at either 1, 3, 7 or 21-days post surgery. Rat behavioural testing was performed using the Morris water maze and open field tests. Post-mortem brain tissue was immunolabelled for Aß, microglia, and cholinergic neurons. Rats exposed to AßO showed deficits in spatial learning and anxiety-like behaviour. Acute positive staining for Aß was only observed in the corpus callosum surrounding the lateral ventricles. AßO exposed rat brains also showed a delayed increase in activated microglia within the corpus callosum and a decreased number of cholinergic neurons within the basal forebrain. Acute exposure to AßO resulted in mild learning and memory impairments with co-concomitant white matter pathology within the corpus callosum and cholinergic cell loss within the basal forebrain. Results suggest that acute exposure to AßO in the rat may be a useful tool in assessing the early phases for the pathogenesis of AD.

The Effect of Ultrasonication on the Fibrillar/ Oligomeric Structures of Aß1-42 at Different Concentrations.

The number of disease states linked the aberrant regular protein conformations to oligomers and amyloid fibrils. Amyloid beta 1-42 (Aß1-42) peptide is very hydrophobic and quickly forms the ß-rich structure and fibrillar protein aggregates in some solutions and buffer conditions. Ultrasonication pulses can disrupt amyloid fibrils to smaller fragments and produce Aß1-42 peptides of different sizes and oligomers. Herein, we investigated the effects of buffer and ultrasonication on Aß1-42 structure at low and high concentrations. After ultrasonication, the Western blot results showed that Aß1-42 fibrils were disaggregated into different sizes. The transmission electron microscopy results indicated Aß1-42 at low concentration (25 µM) in Ham's/F12 phenol red-free culture medium formed short-size fragments and oligomers. In comparison, Aß1-42 at higher concentration (100 µM) formed fibrils that break down into smaller fragments after ultrasonication. However, after regrowth, it formed mature fibrils again. Cell viability assay indicated that Aß1-42 oligomers formed at a low concentration (25 µM) were more toxic to PC12 cells than other forms. In conclusion, by applying ultrasonication pulses and controlling peptide concentration and buffer condition, we can rich Aß1-42 aggregates with a particular size and molecular structure.

Amyloid ß oligomers disrupt piriform cortical output via a serotonergic pathway.

Although olfactory deficits have been found in patients with early-stage Alzheimer's disease (AD), the underlying mechanisms remain unclear. Here we investigated whether and how human amyloid ß (Aß) oligomers affect neural activity in the piriform cortex (PC) slices of adult mice. We found that oligomeric Aß1-42 decreased the excitability of pyramidal neurons in the anterior PC. The effect was not blocked by glutamate or GABAA receptor antagonists, suggesting that Aß1-42-induced hypoactivity is independent of glutamatergic and GABAergic transmission. Interestingly, the hypoexcitability was occluded by serotonin (5-HT) and blocked by antagonists of 5-HT2C receptors, phospholipase C (PLC), and calcium-activated potassium (BK) channels. Furthermore, Aß1-42 oligomers failed to increase K+-channel currents in the presence of a BK channel blocker. Finally, 5-HT2C receptor antagonist improved olfactory memory and odor discrimination in APP/PS1 mice. The above data indicate that Aß disrupts olfactory information output from the PC via the 5-HT-5-HT2C receptor-PLC-BK channel pathway. This study reveals that serotonergic modulation is a potential novel therapeutic target for olfactory damage in AD.

Amyloid Beta Oligomers-Induced Ca2+ Entry Pathways: Role of Neuronal Networks, NMDA Receptors and Amyloid Channel Formation.

The molecular basis of amyloid toxicity in Alzheimer's disease (AD) remains controversial. Amyloid ß (Aß) oligomers promote Ca2+ influx, mitochondrial Ca2+ overload and apoptosis in hippocampal neurons in vivo and in vitro, but the primary Ca2+ entry pathways are unclear. We studied Ca2+ entry pathways induced by Aß oligomers in rat hippocampal and cerebellar neurons. Aß oligomers induce Ca2+ entry in neurons. Ca2+ responses to Aß oligomers are large after synaptic networking and prevented by blockers of synaptic transmission. In contrast, in neurons devoid of synaptic connections, Ca2+ responses to Aß oligomers are small and prevented only by blockers of amyloid channels (NA7) and NMDA receptors (MK801). A combination of NA7 and MK801 nearly abolished Ca2+ responses. Non-neuronal cells bearing NMDA receptors showed Ca2+ responses to oligomers, whereas cells without NMDA receptors did not exhibit Ca2+ responses. The expression of subunits of the NMDA receptor NR1/ NR2A and NR1/NR2B in HEK293 cells lacking endogenous NMDA receptors restored Ca2+ responses to NMDA but not to Aß oligomers. We conclude that Aß oligomers promote Ca2+ entry via amyloid channels and NMDA receptors. This may recruit distant neurons intertwisted by synaptic connections, spreading excitation and recruiting further NMDA receptors and voltage-gated Ca2+ channels, leading to excitotoxicity and neuron degeneration in AD.

Cholesterol in Membranes Facilitates Aggregation of Amyloid ß Protein at Physiologically Relevant Concentrations.

The formation of amyloid ß (1-42) (Aß42) oligomers is considered to be a critical step in the development of Alzheimer's disease (AD). However, the mechanism underlying this process at physiologically low concentrations of Aß42 remains unclear. We have previously shown that oligomers assemble at such low Aß42 monomer concentrations in vitro on phospholipid membranes. We hypothesized that membrane composition is the factor controlling the aggregation process. Accumulation of cholesterol in membranes is associated with AD development, suggesting that insertion of cholesterol into membranes may initiate the Aß42 aggregation, regardless of a low monomer concentration. We used atomic force microscopy (AFM) to test the hypothesis and directly visualize the aggregation process of Aß42 on the surface of a lipid bilayer depending on the cholesterol presence. Time-lapse AFM imaging unambiguously demonstrates that cholesterol in the lipid bilayer significantly enhances the aggregation process of Aß42 at nanomolar monomer concentration. Quantitative analysis of the AFM data shows that both the number of Aß42 oligomers and their sizes grow when cholesterol is present. Importantly, the aggregation process is dynamic, so the aggregates assembled on the membrane can dissociate from the bilayer surface into the bulk solution. Computational modeling demonstrated that the lipid bilayer containing cholesterol had an elevated affinity to Aß42. Moreover, monomers adopted the aggregation-prone conformations present in amyloid fibrils. The results lead to the model for the on-surface aggregation process in which the self-assembly of Aß oligomers is controlled by the lipid composition of cellular membranes.

Highly sensitive aptasensor based on interferometric reflectance spectroscopy for the determination of amyloid ß as an Alzheimer's disease biomarkers using nanoporous anodic alumina.

It is well known that Alzheimer's disease is one of the global challenges for the 21st century. Therefore, it is urgent to develop a reliable biosensor for the detection of this disease. Here in, we have developed for the first time, an aptasensor based on interferometric reflectance spectroscopy (IRS) for the determination of amyloid ß (Aß) oligomers that is an Alzheimer's disease biomarker. For this purpose, the nanoporous anodic alumina (NAA) was first fabricated. After that, the pore walls of the NAA were modified with (3-aminopropyl) trimethoxysilane (NAA-NH2). The amino-terminal aptamers probe were then attached to the pore walls of the NAA-NH2 by using glutaraldehyde (GA) as the cross-linking agent. Subsequently, methylene blue (MB) was immobilized into the aptamer as the photo-probe, generating the MB/G-quadruplex complex. Since MB has a high absorption coefficient, the intensity of the reflected white light to the charge-coupled device (CCD) detector decreased. In the presence of the Aß oligomers that have high affinity to the immobilized aptamer, the MB/quadruplex complex broke and MB washed away from the aptasensor. Therefore, the intensity of the reflected white light to the CCD detector increased. The increased signal intensity of the aptasensor has a logarithmic relationship with the concentration of Aß oligomers. The proposed aptasensor exhibited a good response to the concentration of Aß oligomers in the range of 0.5-50.0 µg × mL-1. The experimental detection limit was of 0.02 µg × mL-1 (at 3σ/S). The proposed optical aptasensor exhibited good selectivity, linear range, and stability.

Neurotoxic Ca2+ Signaling Induced by Amyloid-ß Oligomers in Aged Hippocampal Neurons In Vitro.

Alzheimer's disease (AD), the most prevalent dementia linked to aging, involves neurotoxic effects of amyloid ß species and dishomeostasis of intracellular Ca2+. To investigate mechanisms of AD, the effects of soluble species of amyloid ß oligomers (Aßo) prepared in medium devoid of glutamate receptor agonists can be tested on intracellular Ca2+ in long-term cultures of rat hippocampal neurons that reflect aging neurons. Furthermore, changes in expression of proteins involved in oligomer responses and AD can be tested in the same neurons using quantitative immunofluorescence. Detailed procedures for the preparation of Aß species in defined medium, long-term culture of rat hippocampal neurons mimicking aged neurons, calcium imaging and quantitative immunofluorescence in these cultures are described in this chapter.

Rapid amyloid-ß oligomer and protofibril accumulation in traumatic brain injury.

Deposition of amyloid-ß (Aß) is central to Alzheimer's disease (AD) pathogenesis and associated with progressive neurodegeneration in traumatic brain injury (TBI). We analyzed predisposing factors for Aß deposition including monomeric Aß40, Aß42 and Aß oligomers/protofibrils, Aß species with pronounced neurotoxic properties, following human TBI. Highly selective ELISAs were used to analyze N-terminally intact and truncated Aß40 and Aß42, as well as Aß oligomers/protofibrils, in human brain tissue, surgically resected from severe TBI patients (n = 12; mean age 49.5 ± 19 years) due to life-threatening brain swelling/hemorrhage within one week post-injury. The TBI tissues were compared to post-mortem AD brains (n = 5), to post-mortem tissue of neurologically intact (NI) subjects (n = 4) and to cortical biopsies obtained at surgery for idiopathic normal pressure hydrocephalus patients (iNPH; n = 4). The levels of Aß40 and Aß42 were not elevated by TBI. The levels of Aß oligomers/protofibrils in TBI were similar to those in the significantly older AD patients and increased compared to NI and iNPH controls (P < 0.05). Moreover, TBI patients carrying the AD risk genotype Apolipoprotein E epsilon3/4 (APOE ε3/4; n = 4) had increased levels of Aß oligomers/protofibrils (P < 0.05) and of both N-terminally intact and truncated Aß42 (P < 0.05) compared to APOE ε3/4-negative TBI patients (n = 8). Neuropathological analysis showed insoluble Aß aggregates (commonly referred to as Aß plaques) in three TBI patients, all of whom were APOE ε3/4 carriers. We conclude that soluble intermediary Aß aggregates form rapidly after TBI, especially among APOE ε3/4 carriers. Further research is needed to determine whether these aggregates aggravate the clinical short- and long-term outcome in TBI.

Advancements of the sFIDA method for oligomer-based diagnostics of neurodegenerative diseases.

Early diagnosis of Alzheimer's disease (AD) is of great importance for the development of therapeutics and their application in the clinical environment. Amyloid ß (Aß) oligomers are crucial for the onset and progression of AD and represent a popular drug target, being presumably the most direct biomarker. Efforts to measure Aß oligomers in body fluids are hampered by the low analyte concentration and presence of Aß monomers. The surface-based fluorescence intensity distribution analysis (sFIDA) features both highly specific and sensitive oligomer quantitation as well as total insensitivity towards monomers. In this Review, we highlight structural features of oligomeric and fibrillar Aß. Recent advancements in sFIDA assay development have been the successful automation, adaption for additional biomarkers such as α-synuclein oligomers, and significant improvement of essential assay parameters.

Is it All Said for NSAIDs in Alzheimer's Disease? Role of Mitochondrial Calcium Uptake.

OBJECTIVES: Epidemiological data suggest that non-steroidal anti-inflammatory drugs (NSAIDs) may protect against Alzheimer's disease (AD). Unfortunately, recent trials have failed in providing compelling evidence of neuroprotection. Discussion as to why NSAIDs effectivity is uncertain is ongoing. Possible explanations include the view that NSAIDs and other possible disease-modifying drugs should be provided before the patients develop symptoms of AD or cognitive decline. In addition, NSAID targets for neuroprotection are unclear. Both COX-dependent and independent mechanisms have been proposed, including γ-secretase that cleaves the amyloid precursor protein (APP) and yields amyloid ß peptide (Aß). METHODS: We have proposed a neuroprotection mechanism for NSAIDs based on inhibition of mitochondrial Ca2+ overload. Aß oligomers promote Ca2+ influx and mitochondrial Ca2+ overload leading to neuron cell death. Several non-specific NSAIDs including ibuprofen, sulindac, indomethacin and Rflurbiprofen depolarize mitochondria in the low µM range and prevent mitochondrial Ca2+ overload induced by Aß oligomers and/or N-methyl-D-aspartate (NMDA). However, at larger concentrations, NSAIDs may collapse mitochondrial potential (ΔΨ) leading to cell death. RESULTS: Accordingly, this mechanism may explain neuroprotection at low concentrations and damage at larger doses, thus providing clues on the failure of promising trials. Perhaps lower NSAID concentrations and/or alternative compounds with larger dynamic ranges should be considered for future trials to provide definitive evidence of neuroprotection against AD.

Soluble oligomeric amyloid-ß induces calcium dyshomeostasis that precedes synapse loss in the living mouse brain.

BACKGROUND: Amyloid-ß oligomers (oAß) are thought to mediate neurotoxicity in Alzheimer's disease (AD), and previous studies in AD transgenic mice suggest that calcium dysregulation may contribute to these pathological effects. Even though AD mouse models remain a valuable resource to investigate amyloid neurotoxicity, the concomitant presence of soluble Aß species, fibrillar Aß, and fragments of amyloid precursor protein (APP) complicate the interpretation of the phenotypes. METHOD: To explore the specific contribution of soluble oligomeric Aß (oAß) to calcium dyshomeostasis and synaptic morphological changes, we acutely exposed the healthy mouse brain, at 3 to 6 months of age, to naturally occurring soluble oligomers and investigated their effect on calcium levels using in vivo multiphoton imaging. RESULTS: We observed a dramatic increase in the levels of neuronal resting calcium, which was dependent upon extracellular calcium influx and activation of NMDA receptors. Ryanodine receptors, previously implicated in AD models, did not appear to be primarily involved using this experimental setting. We used the high resolution cortical volumes acquired in-vivo to measure the effect on synaptic densities and observed that, while spine density remained stable within the first hour of oAß exposure, a significant decrease in the number of dendritic spines was observed 24 h post treatment, despite restoration of intraneuronal calcium levels at this time point. CONCLUSIONS: These observations demonstrate a specific effect of oAß on NMDA-mediated calcium influx, which triggers synaptic collapse in vivo. Moreover, this work leverages a method to quantitatively measure calcium concentration at the level of neuronal processes, cell bodies and single synaptic elements repeatedly and thus can be applicable to testing putative drugs and/or other intervention methodologies.

Surface Assay for Specific Detection of Soluble Amyloid Oligomers Utilizing Pronucleon Peptides Instead of Antibodies.

Immunoassays such as enzyme-linked immunosorbent assays (ELISAs) are widely used for diagnostics; however, antibodies as detection reagents may be insufficiently selective and have other shortcomings. We present a novel non-antibody-based detection method based on binding target molecules to peptides (used as recognition molecules): a surface assay for A-ß oligomers employing a peptide comprising amino acid residues of the human ß-amyloid protein (Pronucleon peptide) as the capture agent. For the sake of convenience, we term this the "Pronucleon peptide-linked immunosorbent assay", or PLISA. Pronucleon peptides are amino acid sequences matched to target amyloids of interest, in particular soluble Aß-1-42 amyloid protein oligomers, which are widely considered as an early biomarker for Alzheimer's disease in body fluids. The Pronucleon peptide in a PLISA is immobilized on the surface and substitutes for the capture antibody used in an ELISA for binding the Aß-1-42 oligomers present in the sample. We present data comparing synthetic oligomer PLISAs in spiked buffer and body fluids (such as cerebrospinal fluid, brain extracts, or whole blood) to those from an ELISA and demonstrate better selectivity of the PLISA for amyloid ß-42 oligomers versus monomers and fibrils. The detection limit, calculated as the mean (blank) plus three standard deviations, was in the range of 0.35-1.5 pM (32-135 ng/L) (oligomers contained approximately 20 monomers on average).

Early intervention in the 3xTg-AD mice with an amyloid ß-antibody fragment ameliorates first hallmarks of Alzheimer disease.

The single-chain variable fragment, scFv-h3D6, has been shown to prevent in vitro toxicity induced by the amyloid ß (Aß) peptide in neuroblastoma cell cultures by withdrawing Aß oligomers from the amyloid pathway. Present study examined the in vivo effects of scFv-h3D6 in the triple-transgenic 3xTg-AD mouse model of Alzheimer disease. Prior to the treatment, five-month-old female animals, corresponding to early stages of the disease, showed the first behavioral and psychological symptoms of dementia -like behaviors. Cognitive deficits included long- and short-term learning and memory deficits and high swimming navigation speed. After a single intraperitoneal dose of scFv-h3D6, the swimming speed was reversed to normal levels and the learning and memory deficits were ameliorated. Brain tissues of these animals revealed a global decrease of Aß oligomers in the cortex and olfactory bulb after treatment, but this was not seen in the hippocampus and cerebellum. In the untreated 3xTg-AD animals, we observed an increase of both apoJ and apoE concentrations in the cortex, as well as an increase of apoE in the hippocampus. Treatment significantly recovered the non-pathological levels of these apolipoproteins. Our results suggest that the benefit of scFv-h3D6 occurs at both behavioral and molecular levels.