[Show abstract][Hide abstract] ABSTRACT: Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer’s dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)—a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer’s model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug discovery for membrane protein targets.
PLoS ONE 04/2015; 10(4):e0125263. DOI:10.1371/journal.pone.0125263 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein aggregation is common to dozens of diseases including prionoses, diabetes, Parkinson’s and Alzheimer’s. Over the past 15 years, there has been a paradigm shift in understanding the structural basis for these proteinopathies. Precedent for this shift has come from investigation of soluble Aβ oligomers (AβOs), toxins now widely regarded as instigating neuron damage leading to Alzheimer’s dementia. Toxic AβOs accumulate in AD brain and constitute long-lived alternatives to the disease-defining Aβ fibrils deposited in amyloid plaques. Key experiments using fibril-free AβO solutions demonstrated that while Aβ is essential for memory loss, the fibrillar Aβ in amyloid deposits is not the agent. The AD-like cellular pathologies induced by AβOs suggest their impact provides a unifying mechanism for AD pathogenesis, explaining why early stage disease is specific for memory and accounting for major facets of AD neuropathology. Alternative ideas for triggering mechanisms are being actively investigated. Some research favors insertion of AβOs into membrane, while other evidence supports ligand-like accumulation at particular synapses. Over a dozen candidate toxin receptors have been proposed. AβO binding triggers a redistribution of critical synaptic proteins and induces hyperactivity in metabotropic and ionotropic glutamate receptors. This leads to Ca2+ overload and instigates major facets of AD neuropathology, including tau hyperphosphorylation, insulin resistance, oxidative stress, and synapse loss. Because different species of AβOs have been identified, a remaining question is which oligomer is the major pathogenic culprit. The possibility has been raised that more than one species plays a role. Despite some key unknowns, the clinical relevance of AβOs has been established, and new studies are beginning to point to co-morbidities such as diabetes and hypercholesterolemia as etiological factors. Because pathogenic AβOs appear early in the disease, they offer appealing targets for therapeutics and diagnostics. Promising therapeutic strategies include use of CNS insulin signaling enhancers to protect against the presence of toxins and elimination of the toxins through use of highly specific AβO antibodies. An AD-dependent accumulation of AβOs in CSF suggests their potential use as biomarkers and new AβO probes are opening the door to brain imaging. Overall, current evidence indicates that Aβ oligomers provide a substantive molecular basis for the cause, treatment and diagnosis of Alzheimer’s disease.
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is associated with peripheral metabolic disorders. Clinical/epidemiological data indicate increased risk of diabetes in AD patients. Here, we show that intracerebroventricular infusion of AD-associated Aβ oligomers (AβOs) in mice triggered peripheral glucose intolerance, a phenomenon further verified in two transgenic mouse models of AD. Systemically injected AβOs failed to induce glucose intolerance, suggesting AβOs target brain regions involved in peripheral metabolic control. Accordingly, we show that AβOs affected hypothalamic neurons in culture, inducing eukaryotic translation initiation factor 2α phosphorylation (eIF2α-P). AβOs further induced eIF2α-P and activated pro-inflammatory IKKβ/NF-κB signaling in the hypothalamus of mice and macaques. AβOs failed to trigger peripheral glucose intolerance in tumor necrosis factor-α (TNF-α) receptor 1 knockout mice. Pharmacological inhibition of brain inflammation and endoplasmic reticulum stress prevented glucose intolerance in mice, indicating that AβOs act via a central route to affect peripheral glucose homeostasis. While the hypothalamus has been largely ignored in the AD field, our findings indicate that AβOs affect this brain region and reveal novel shared molecular mechanisms between hypothalamic dysfunction in metabolic disorders and AD.
EMBO Molecular Medicine 01/2015; 7(2). DOI:10.15252/emmm.201404183 · 8.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: One way to image the molecular pathology in Alzheimer's disease is by positron emission tomography using probes that target amyloid fibrils. However, these fibrils are not closely linked to the development of the disease. It is now thought that early-stage biomarkers that instigate memory loss are composed of Aβ oligomers. Here, we report a sensitive molecular magnetic resonance imaging contrast probe that is specific for Aβ oligomers. We attach oligomer-specific antibodies onto magnetic nanostructures and show that the complex is stable and binds to Aβ oligomers on cells and brain tissues to give a magnetic resonance imaging signal. When intranasally administered to an Alzheimer's disease mouse model, the probe readily reached hippocampal Aβ oligomers. In isolated samples of human brain tissue, we observed a magnetic resonance imaging signal that distinguished Alzheimer's disease from controls. Such nanostructures that target neurotoxic Aβ oligomers are potentially useful for evaluating the efficacy of new drugs and ultimately for early-stage Alzheimer's disease diagnosis and disease management.
[Show abstract][Hide abstract] ABSTRACT: Alzheimer disease (AD), the most prevalent type of dementia, has been associated with the accumulation of amyloid β oligomers (AβOs) in the central nervous system. AβOs vary widely in size, ranging from dimers to larger than 100 kDa. Evidence indicates that not all oligomers are toxic, and there is yet no consensus on the size of the actual toxic oligomer. Here we used NU4, a conformation-dependent anti-AβO monoclonal antibody, to investigate size and shape of a toxic AβO assembly. By using size-exclusion chromatography and immuno-based detection we isolated an AßO-NU4 complex amenable for biochemical and morphological studies. The apparent molecular mass of the NU4-targeted oligomer was 80 kDa. AFM imaging of the AβO-NU4 complex showed a size distribution centered at 5.37 nm, an increment of 1.5 nm compared to the size of AβOs (3.85 nm). This increment was compatible with the size of NU4 (1.3 nm), suggesting a 1:1 oligomer to NU4 ratio. NU4-reactive oligomers extracted from AD human brain concentrated in a molecular mass range similar to that found for in vitro-prepared oligomers, supporting the relevance of the species herein studied. These results represent an important step towards understanding the connection between AβO size and toxicity.
ACS Chemical Neuroscience 10/2014; 5(12). DOI:10.1021/cn500156r · 4.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is a devastating neurodegenerative disorder and a major medical problem. Here, we have investigated the impact of amyloid-␤ (A␤) oligomers, AD-related neurotoxins, in the brains of rats and adult nonhuman primates (cynomolgus ma-caques). Soluble A␤ oligomers are known to accumulate in the brains of AD patients and correlate with disease-associated cognitive dysfunction. When injected into the lateral ventricle of rats and macaques, A␤ oligomers diffused into the brain and accumulated in several regions associated with memory and cognitive functions. Cardinal features of AD pathology, including synapse loss, tau hyper-phosphorylation, astrocyte and microglial activation, were observed in regions of the macaque brain where A␤ oligomers were abun-dantly detected. Most importantly, oligomer injections induced AD-type neurofibrillary tangle formation in the macaque brain. These outcomes were specifically associated with A␤ oligomers, as fibrillar amyloid deposits were not detected in oligomer-injected brains. Human and macaque brains share significant similarities in terms of overall architecture and functional networks. Thus, generation of a macaque model of AD that links A␤ oligomers to tau and synaptic pathology has the potential to greatly advance our understanding of mechanisms centrally implicated in AD pathogenesis. Furthermore, development of disease-modifying therapeutics for AD has been hampered by the difficulty in translating therapies that work in rodents to humans. This new approach may be a highly relevant nonhuman primate model for testing therapeutic interventions for AD.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 10/2014; 34(41):13643. DOI:10.1523/JNEUROSCI.1353-14.2014 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oligomers of the Aβ42 peptide are significant neurotoxins linked to Alzheimer's disease (AD). Histidine (His) residues present at the N terminus of Aβ42 are believed to influence toxicity by either serving as metal–ion binding sites (which promote oligomerization and oxidative damage) or facilitating synaptic binding. Transition metal complexes that bind to these residues and modulate Aβ toxicity have emerged as therapeutic candidates. Cobalt(III) Schiff base complexes (Co–sb) were evaluated for their ability to interact with Aβ peptides. HPLC-MS, NMR, fluorescence, and DFT studies demonstrated that Co–sb complexes could interact with the His residues in a truncated Aβ16 peptide representing the Aβ42 N terminus. Coordination of Co–sb complexes altered the structure of Aβ42 peptides and promoted the formation of large soluble oligomers. Interestingly, this structural perturbation of Aβ correlated to reduced synaptic binding to hippocampal neurons. These results demonstrate the promise of Co–sb complexes in anti-AD therapeutic approaches.
[Show abstract][Hide abstract] ABSTRACT: Numerous studies have implicated the abnormal accumulation of intraneuronal amyloid-beta (Abeta) as an important contributor to Alzheimer's disease (AD) pathology, capable of triggering neuroinflammation, tau hyperphosphorylation and cognitive deficits. However, the occurrence and pathological relevance of intracellular Abeta remain a matter of controversial debate. In this study, we have used a multidimensional approach including high-magnification and super-resolution microscopy, cerebro-spinal fluid (CSF) mass spectrometry analysis and ELISA to investigate the Abeta pathology and its associated cognitive impairments, in a novel transgenic rat model overexpressing human APP. Our microscopy studies with quantitative co-localization analysis revealed the presence of intraneuronal Abeta in transgenic rats, with an immunological signal that was clearly distinguished from that of the amyloid precursor protein (APP) and its C-terminal fragments (CTFs). The early intraneuronal pathology was accompanied by a significant elevation of soluble Abeta42 peptides that paralleled the presence and progression of early cognitive deficits, several months prior to amyloid plaque deposition. Abeta38, Abeta39, Abeta40 and Abeta42 peptides were detected in the rat CSF by MALDI-MS analysis even at the plaque-free stages; suggesting that a combination of intracellular and soluble extracellular Abeta may be responsible for impairing cognition at early time points. Taken together, our results demonstrate that the intraneuronal development of AD-like amyloid pathology includes a mixture of molecular species (Abeta, APP and CTFs) of which a considerable component is Abeta; and that the early presence of these species within neurons has deleterious effects in the CNS, even before the development of full-blown AD-like pathology.
[Show abstract][Hide abstract] ABSTRACT: The AβO hypothesis for AD was introduced in 1998. It proposed that neuron damage leading to dementia was instigated by diffusible oligomeric Aβ toxins. The proposed mechanism was disruption of neuronal signal transduction triggered by ligand-like binding of AβOs to unknown toxin receptors. The hypothesis is supported by experimental findings that AβOs can target specific synapses, disrupt plasticity and memory mechanisms, and induce AD-type pathology; it is strengthened, moreover, by data showing AD-dependent accumulation of toxic AβOs in humans and animal models. Surprisingly, molecular interactions between AβOs and neuron surfaces are still a conundrum, although key pieces of the puzzle have been found. The extracellular presence of AβOs is indicated by detection of AβOs in AD CSF, albeit at low levels. The involvement of surface-bound AβOs is indicated by the ability of AβO antibodies delivered intra-nasally to target the hippocampal neuropil of AD Tg mice (suggesting intranasal antibody delivery could be of value for therapeutics and for early diagnostic PET imaging). Interactions between extracellular AβOs and brain cell surfaces appear to depend on multiple cell surface proteins. Although these proteins often are proposed as AβO receptors, the important interactions may be less direct. Emblematic of this complexity is the PrP protein, whose involvement in AβO toxicity is extensively documented. Nonetheless, we find that when PrP is virtually eliminated from differentiated hippocampal neurons by siRNA (delivered with high efficiency by nanoparticles) or by enzymatic surface removal of PrP (using PIPLC), there is no loss of synaptic AβO accumulation. Analogously, nano-scale artificial membranes (Nanodiscs) containing solubilized synaptic membrane proteins show no loss of AβO specific binding after PrP removal. At present, a full understanding of AβO binding sites and transduction mechanisms is still not available. Synaptic Nanodiscs, however, with a full complement of membrane proteins including unidentified AβO binding sites, provide an unbiased platform to screen small molecule libraries for antagonist useful as therapeutic lead compounds.
Neurobiology of Aging 03/2014; 35:S12. DOI:10.1016/j.neurobiolaging.2014.01.076 · 5.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) and type 2 diabetes appear to share similar pathogenic mechanisms. dsRNA-dependent protein kinase (PKR) underlies peripheral insulin resistance in metabolic disorders. PKR phosphorylates eukaryotic translation initiation factor 2α (eIF2α-P), and AD brains exhibit elevated phospho-PKR and eIF2α-P levels. Whether and how PKR and eIF2α-P participate in defective brain insulin signaling and cognitive impairment in AD are unknown. We report that β-amyloid oligomers, AD-associated toxins, activate PKR in a tumor necrosis factor α (TNF-α)-dependent manner, resulting in eIF2α-P, neuronal insulin receptor substrate (IRS-1) inhibition, synapse loss, and memory impairment. Brain phospho-PKR and eIF2α-P were elevated in AD animal models, including monkeys given intracerebroventricular oligomer infusions. Oligomers failed to trigger eIF2α-P and cognitive impairment in PKR(-/-) and TNFR1(-/-) mice. Bolstering insulin signaling rescued phospho-PKR and eIF2α-P. Results reveal pathogenic mechanisms shared by AD and diabetes and establish that proinflammatory signaling mediates oligomer-induced IRS-1 inhibition and PKR-dependent synapse and memory loss.
[Show abstract][Hide abstract] ABSTRACT: Brain accumulation of soluble amyloid-β oligomers (AβOs) has been implicated in synapse failure and cognitive impairment in Alzheimer's disease (AD). However, whether and how oligomers of different sizes induce synapse dysfunction is a matter of controversy. Here, we report that low-molecular-weight (LMW) and high-molecular-weight (HMW) Aβ oligomers differentially impact synapses and memory. A single intracerebroventricular injection of LMW AβOs (10 pmol) induced rapid and persistent cognitive impairment in mice. On the other hand, memory deficit induced by HMW AβOs (10 pmol) was found to be reversible. While memory impairment in LMW oligomer-injected mice was associated with decreased hippocampal synaptophysin and GluN2B immunoreactivities, synaptic pathology was not detected in the hippocampi of HMW oligomer-injected mice. On the other hand, HMW oligomers, but not LMW oligomers, induced oxidative stress in hippocampal neurons. Memantine rescued both neuronal oxidative stress and the transient memory impairment caused by HMW oligomers, but did not prevent the persistent cognitive deficit induced by LMW oligomers. Results establish that different Aβ oligomer assemblies act in an orchestrated manner, inducing different pathologies and leading to synapse dysfunction. Furthermore, results suggest a mechanistic explanation for the limited efficacy of memantine in preventing memory loss in AD.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 06/2013; 33(23):9626-34. DOI:10.1523/JNEUROSCI.0482-13.2013 · 6.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amyloid-β peptide (Aβ) fragment misfolding may play a crucial role in the progression of Alzheimer's disease (AD) pathophysiology as well as epigenetic mechanisms at the DNA and histone level. We hypothesized that histone H3 homeostasis is disrupted in association with the appearance of soluble Aβ at an early stage in AD progression. We identified, localized, and compared histone H3 modifications in multiple model systems (neural-like SH-SY5Y, primary neurons, Tg2576 mice, and AD neocortex), and narrowed our focus to investigate 3 key motifs associated with regulating transcriptional activation and inhibition: acetylated lysine 14, phosphorylated serine 10 and dimethylated lysine 9. Our results in vitro and in vivo indicate that multimeric soluble Aβ may be a potent signaling molecule indirectly modulating the transcriptional activity of DNA by modulating histone H3 homeostasis. These findings reveal potential loci of transcriptional disruption relevant to AD. Identifying genes that undergo significant epigenetic alterations in response to Aβ could aid in the understanding of the pathogenesis of AD, as well as suggesting possible new treatment strategies.
Neurobiology of aging 04/2013; 34(9). DOI:10.1016/j.neurobiolaging.2012.12.028 · 5.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oligomeric forms of amyloid-β peptide (Aβ) are thought to play a pivotal role in the pathogenesis of Alzheimers disease (AD), but the mechanism involved is still unclear. Here, we generated induced pluripotent stem cells (iPSCs) from familial and sporadic AD patients and differentiated them into neural cells. Aβ oligomers accumulated in iPSC-derived neurons and astrocytes in cells from patients with a familial amyloid precursor protein (APP)-E693 mutation and sporadic AD, leading to endoplasmic reticulum (ER) and oxidative stress. The accumulated Aβ oligomers were not proteolytically resistant, and docosahexaenoic acid (DHA) treatment alleviated the stress responses in the AD neural cells. Differential manifestation of ER stress and DHA responsiveness may help explain variable clinical results obtained with the use of DHA treatment and suggests that DHA may in fact be effective for a subset of patients. It also illustrates how patient-specific iPSCs can be useful for analyzing AD pathogenesis and evaluating drugs.
[Show abstract][Hide abstract] ABSTRACT: Alzheimer's disease (AD) is a global health crisis with limited treatment options. Despite major advances in neurotherapeutics, poor brain penetration due to the blood-brain barrier continues to pose a big challenge in overcoming the access of therapeutics to the central nervous system. In that regard, the non-invasive intranasal route of brain targeting is gaining considerable attention. The nasal mucosa offers a large surface area, rapid absorption, and avoidance of first-pass metabolism increasing drug bioavailability with less systemic side effects. Intranasal delivery is known to utilize olfactory, rostral migratory stream, and trigeminal routes to reach the brain. This investigation confirmed that intranasal delivery of oligomeric amyloid-β antibody (NU4) utilized all three routes to enter the brain with a resident time of 96 hours post single bolus intranasal administration, and showed evidence of perikaryal and parenchymal uptake of NU4 in 5XFAD mouse brain, confirming the intranasal route as a non-invasive and efficient way of delivering therapeutics to the brain. In addition, this study demonstrated that intranasal delivery of NU4 antibody lowered cerebral amyloid-β and improved spatial learning in 5XFAD mice.
[Show abstract][Hide abstract] ABSTRACT: Oligomeric forms of amyloid-β peptide (Aβ) are thought to play a pivotal role in the pathogenesis of Alzheimer's disease (AD), but the mechanism involved is still unclear. Here, we generated induced pluripotent stem cells (iPSCs) from familial and sporadic AD patients and differentiated them into neural cells. Aβ oligomers accumulated in iPSC-derived neurons and astrocytes in cells from patients with a familial amyloid precursor protein (APP)-E693Δ mutation and sporadic AD, leading to endoplasmic reticulum (ER) and oxidative stress. The accumulated Aβ oligomers were not proteolytically resistant, and docosahexaenoic acid (DHA) treatment alleviated the stress responses in the AD neural cells. Differential manifestation of ER stress and DHA responsiveness may help explain variable clinical results obtained with the use of DHA treatment and suggests that DHA may in fact be effective for a subset of patients. It also illustrates how patient-specific iPSCs can be useful for analyzing AD pathogenesis and evaluating drugs.