Search

BackgroundMany identified mechanisms could be upstream of the prominent amyloid-β (Aβ) plaques in Alzheimer's disease (AD).ObjectiveTo profile the progression of pathology in AD.MethodsWe monitored metabolic signaling, redox stress, intraneuronal amyloid-β (iAβ) accumulation, and extracellular plaque deposition in the brains of 3xTg-AD mice across the lifespan.ResultsIntracellular accumulation of aggregated Aβ in the CA1 pyramidal cells at 9 months preceded extracellular plaques that first presented in the CA1 at 16 months of age. In biochemical assays, brain glutathione (GSH) declined with age in both 3xTg-AD and non-transgenic controls, but the decline was accelerated in 3xTg-AD brains from 2 to 4 months. The decline in GSH correlated exponentially with the rise in iAβ. Integrated metabolic signaling as the ratio of phospho-Akt (pAkt) to total Akt (tAkt) in the PI3kinase and mTOR pathway declined at 6, 9, and 12 months, before rising at 16 and 20 months. These pAkt/tAkt ratios correlated with both iAβ and GSH levels in a U-shaped relationship. Selective vulnerability of age-related AD-genotype-specific pAkt changes was greatest in the CA1 pyramidal cell layer. To demonstrate redox causation, iAβ accumulation was lowered in cultured middle-age adult 3xTg-AD neurons by treatment of the oxidized redox state in the neurons with exogenous cysteine.ConclusionThe order of pathologic progression in the 3xTg-AD mouse was loss of GSH (oxidative redox shift) followed by a pAkt/tAkt metabolic shift in CA1, iAβ accumulation in CA1, and extracellular Aβ deposition. Upstream targets may prove strategically more effective for therapy before irreversible changes.

Soluble oligomers of aggregated tau accompany the accumulation of insoluble amyloid fibrils, a histological hallmark of Alzheimer disease (AD) and two dozen related neurodegenerative diseases. Both oligomers and fibrils seed the spread of Tau pathology, and by virtue of their low molecular weight and relative solubility, oligomers may be particularly pernicious seeds. Here, we report the formation of in vitro tau oligomers formed by an ionic liquid (IL15). Using IL15-induced recombinant tau oligomers and a dot blot assay, we discovered a mAb (M204) that binds oligomeric tau, but not tau monomers or fibrils. M204 and an engineered single-chain variable fragment (scFv) inhibited seeding by IL15-induced tau oligomers and pathological extracts from donors with AD and chronic traumatic encephalopathy. This finding suggests that M204-scFv targets pathological structures that are formed by tau in neurodegenerative diseases. We found that M204-scFv itself partitions into oligomeric forms that inhibit seeding differently, and crystal structures of the M204-scFv monomer, dimer, and trimer revealed conformational differences that explain differences among these forms in binding and inhibition. The efficiency of M204-scFv antibodies to inhibit the seeding by brain tissue extracts from different donors with tauopathies varied among individuals, indicating the possible existence of distinct amyloid polymorphs. We propose that by binding to oligomers, which are hypothesized to be the earliest seeding-competent species, M204-scFv may have potential as an early-stage diagnostic for AD and tauopathies, and also could guide the development of promising therapeutic antibodies.

This work provides new insight into the age-related basis of Alzheimer's disease (AD), the composition of intraneuronal amyloid (iAβ), and the mechanism of an age-related increase in iAβ in adult AD-model mouse neurons. A new end-specific antibody for Aβ45 and another for aggregated forms of Aβ provide new insight into the composition of iAβ and the mechanism of accumulation in old adult neurons from the 3xTg-AD model mouse. iAβ levels containing aggregates of Aβ45 increased 30-50-fold in neurons from young to old age and were further stimulated upon glutamate treatment. iAβ was 8 times more abundant in 3xTg-AD than non-transgenic neurons with imaged particle sizes following the same log-log distribution, suggesting a similar snow-ball mechanism of intracellular biogenesis. Pathologically misfolded and mislocalized Alz50 tau colocalized with iAβ and rapidly increased following a brief metabolic stress with glutamate. AβPP-CTF, Aβ45, and aggregated Aβ colocalized most strongly with mitochondria and endosomes and less with lysosomes and autophagosomes. Differences in iAβ by sex were minor. These results suggest that incomplete carboxyl-terminal trimming of long Aβs by gamma-secretase produced large intracellular deposits which limited completion of autophagy in aged neurons. Understanding the mechanism of age-related changes in iAβ processing may lead to application of countermeasures to prolong dementia-free health span.

Alzheimer's disease (AD), the most common age-related neurodegenerative disorder, is currently conceptualized as a disease of synaptic failure. Synaptic impairments are robust within the AD brain and better correlate with dementia severity when compared with other pathological features of the disease. Nevertheless, the series of events that promote synaptic failure still remain under debate, as potential triggers such as β-amyloid (Aβ) can vary in size, configuration and cellular location, challenging data interpretation in causation studies. Here we present data obtained using adeno-associated viral (AAV) constructs that drive the expression of oligomeric Aβ either intra or extracellularly. We observed that expression of Aβ in both cellular compartments affect learning and memory, reduce the number of synapses and the expression of synaptic-related proteins, and disrupt chemical long-term potentiation (cLTP). Together, these findings indicate that during the progression AD the early accumulation of Aβ inside neurons is sufficient to promote morphological and functional cellular toxicity, a phenomenon that can be exacerbated by the buildup of Aβ in the brain parenchyma. Moreover, our AAV constructs represent a valuable tool in the investigation of the pathological properties of Aβ oligomers both in vivo and in vitro.

Alzheimer's disease (AD) pathology is characterized by plaques of amyloid beta (Aβ) and neurofibrillary tangles of tau. Aβ aggregation is thought to occur at early stages of the disease, and ultimately gives way to the formation of tau tangles which track with cognitive decline in humans. Here, we report the crystal structure of an Aβ core segment determined by MicroED and in it, note characteristics of both fibrillar and oligomeric structure. Using this structure, we designed peptide-based inhibitors that reduce Aβ aggregation and toxicity of already-aggregated species. Unexpectedly, we also found that these inhibitors reduce the efficiency of Aβ-mediated tau aggregation, and moreover reduce aggregation and self-seeding of tau fibrils. The ability of these inhibitors to interfere with both Aβ and tau seeds suggests these fibrils share a common epitope, and supports the hypothesis that cross-seeding is one mechanism by which amyloid is linked to tau aggregation and could promote cognitive decline.

BackgroundBesides the two main classical features of amyloid beta aggregation and tau-containing neurofibrillary tangle deposition, neuroinflammation plays an important yet unclear role in the pathophysiology of Alzheimer's disease (AD). Microglia are believed to be key mediators of neuroinflammation during AD and responsible for the regulation of brain homeostasis by balancing neurotoxicity and neuroprotective events. We have previously reported evidence that neuritic plaques are derived from dead neurons that have accumulated intraneuronal amyloid and further recruit Iba1-positive cells, which play a role in either neuronal demise or neuritic plaque maturation or both.MethodsTo study the impact of microglia on neuritic plaque development, we treated two-month-old 5XFAD mice with a selective colony stimulation factor 1 receptor (CSF1R) inhibitor, PLX3397, for a period of 3 months, resulting in a significant ablation of microglia. Directly after this treatment, we analyzed the amount of intraneuronal amyloid and neuritic plaques and performed behavioral studies including Y-maze, fear conditioning and elevated plus maze.ResultsWe found that early long-term PLX3397 administration results in a dramatic reduction of both intraneuronal amyloid as well as neuritic plaque deposition. PLX3397 treated young 5XFAD mice also displayed a significant decrease of soluble fibrillar amyloid oligomers in brain lysates, a depletion of soluble pre-fibrillar oligomers in plasma and an improvement in cognitive function measured by fear conditioning tests.ConclusionsOur findings demonstrate that CSF1R signaling, either directly on neurons or mediated by microglia, is crucial for the accumulation of intraneuronal amyloid and formation of neuritic plaques, suggesting that these two events are serially linked in a causal pathway leading to neurodegeneration and neuritic plaque formation. CSF1R inhibitors represent potential preventative or therapeutic approach that target the very earliest stages of the formation of intraneuronal amyloid and neuritic plaques.

Amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aβ and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aβ and hIAPP, termed Aβ(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aβ and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aβ(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aβ and hIAPP.

Most cases of Alzheimer's disease (AD) are sporadic, but a small percentage of AD cases, called familial AD (FAD), are associated with mutations in presenilin 1, presenilin 2, or the amyloid precursor protein. Amyloid precursor protein mutations falling within the amyloid-β (Aβ) sequence lead to a wide range of disease phenotypes. There is increasing evidence that distinct amyloid structures distinguished by amyloid conformation-dependent monoclonal antibodies have similarly distinct roles in pathology. It is possible that this phenotypic diversity of FAD associated with mutations within the Aβ sequence is due to differences in the conformations adopted by mutant Aβ peptides, but the effects of FAD mutations on aggregation kinetics and conformational and morphological changes of the Aβ peptide are poorly defined. To gain more insight into this possibility, we therefore investigated the effects of 11 FAD mutations on the aggregation kinetics of Aβ, as well as its ability to form distinct conformations recognized by a panel of amyloid conformation-specific monoclonal antibodies. We found that most FAD mutations increased the rate of aggregation of Aβ. The FAD mutations also led to the adoption of alternative amyloid conformations distinguished by monoclonal antibodies and resulted in the formation of distinct aggregate morphologies as determined by transmission electron microscopy. In addition, several of the mutant peptides displayed a large reduction in thioflavin T fluorescence, despite forming abundant fibrils indicating that thioflavin T is a probe of conformational polymorphisms rather than a reliable indicator of fibrillization. Taken together, these results indicate that FAD mutations falling within the Aβ sequence lead to dramatic changes in aggregation kinetics and influence the ability of Aβ to form immunologically and morphologically distinct amyloid structures.

hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19-29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15-25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19-29 S20G may serve as a model for the toxic spine of hIAPP.

In Alzheimer's disease, soluble Aβ oligomers are believed to play important roles in the disease pathogenesis, and their levels correlate with cognitive impairment. We have previously shown that Aβ oligomers can be categorized into multiple structural classes based on their reactivity with conformation-dependent antibodies. In this study, we analyzed the structures of Aβ40 oligomers belonging to two of these classes: fibrillar and prefibrillar oligomers. We found that fibrillar oligomers were similar in structure to fibrils but were less stable towards denaturation while prefibrillar oligomers were found to be partially disordered. These results are consistent with previously proposed structures for both oligomer classes while providing additional structural information.

Amyloid-β (Aβ) is present in humans as a 39- to 42-amino acid residue metabolic product of the amyloid precursor protein. Although the two predominant forms, Aβ(1-40) and Aβ(1-42), differ in only two residues, they display different biophysical, biological, and clinical behavior. Aβ(1-42) is the more neurotoxic species, aggregates much faster, and dominates in senile plaque of Alzheimer's disease (AD) patients. Although small Aβ oligomers are believed to be the neurotoxic species, Aβ amyloid fibrils are, because of their presence in plaques, a pathological hallmark of AD and appear to play an important role in disease progression through cell-to-cell transmissibility. Here, we solved the 3D structure of a disease-relevant Aβ(1-42) fibril polymorph, combining data from solid-state NMR spectroscopy and mass-per-length measurements from EM. The 3D structure is composed of two molecules per fibril layer, with residues 15-42 forming a double-horseshoe-like cross-β-sheet entity with maximally buried hydrophobic side chains. Residues 1-14 are partially ordered and in a β-strand conformation, but do not display unambiguous distance restraints to the remainder of the core structure.

BackgroundRecent studies have implicated specific assembly subtypes of β-amyloid (Aβ) peptide, specifically soluble oligomers (soAβ) as disease-relevant structures that may underlie memory loss in Alzheimer disease. Removing existing soluble and insoluble Aβ assemblies is thought to be essential for any attempt at stabilizing brain function and slowing cognitive decline in Alzheimer disease. IV immunoglobulin (IVIg) therapies have been shown to contain naturally occurring polyclonal antibodies that recognize conformational neoepitopes of soluble or insoluble Aβ assemblies including soAβ. These naturally occurring polyclonal antibodies have been suggested to underlie the apparent clinical benefits of IVIg. However, direct evidence linking anti-Aβ antibodies to the clinical bioactivity of IVIg has been lacking.MethodsFive-month-old female Dutch APP E693Q mice were treated for 3 months with neat IVIg or with IVIg that had been affinity-depleted over immobilized Aβ conformers in 1 of 2 assembly states. Memory was assessed in a battery of tests followed by quantification of brain soAβ levels using standard anti-soAβ antibodies.ResultsWe provide evidence that NU4-type soAβ (NU4-soAβ) assemblies accumulate in the brains of Dutch APP E693Q mice and are associated with defects in memory, even in the absence of insoluble Aβ plaques. Memory benefits were associated with depletion from APP E693Q mouse brain of NU4-soAβ and A11-soAβ but not OC-type fibrillar Aβ oligomers.ConclusionsWe propose that targeting of specific soAβ assembly subtypes may be an important consideration in the therapeutic and/or prophylactic benefit of anti-Aβ antibody drugs.

Increased dietary consumption of docosahexaenoic acid (DHA) is associated with decreased risk for Alzheimer's disease (AD). These effects have been postulated to arise from DHA's pleiotropic effects on AD pathophysiology, including its effects on β-amyloid (Aβ) production, aggregation, and toxicity. While in vitro studies suggest that DHA may inhibit and reverse the formation of toxic Aβ oligomers, it remains uncertain whether these mechanisms operate in vivo at the physiological concentrations of DHA attainable through dietary supplementation. We sought to clarify the effects of dietary DHA supplementation on Aβ indices in a transgenic APP/PS1 rat model of AD. Animals maintained on a DHA-supplemented diet exhibited reductions in hippocampal Aβ plaque density and modest improvements on behavioral testing relative to those maintained on a DHA-depleted diet. However, DHA supplementation also increased overall soluble Aβ oligomer levels in the hippocampus. Further quantification of specific conformational populations of Aβ oligomers indicated that DHA supplementation increased fibrillar (i.e. putatively less toxic) Aβ oligomers and decreased prefibrillar (i.e. putatively more toxic) Aβ oligomers. These results provide in vivo evidence suggesting that DHA can modulate Aβ aggregation by stabilizing soluble fibrillar Aβ oligomers and thus reduce the formation of both Aβ plaques and prefibrillar Aβ oligomers. However, since fibrillar Aβ oligomers still retain inherent neurotoxicity, DHA may need to be combined with other interventions that can additionally reduce fibrillar Aβ oligomer levels for more effective prevention of AD in clinical settings.

Rapid activation causes remodeling of atrial myocytes resembling that which occurs in experimental and human atrial fibrillation (AF). Using this cellular model, we previously observed transcriptional upregulation of proteins implicated in protein misfolding and amyloidosis. For organ-specific amyloidoses such as Alzheimer's disease, preamyloid oligomers (PAOs) are now recognized to be the primary cytotoxic species. In the setting of oxidative stress, highly-reactive lipid-derived mediators known as γ-ketoaldehydes (γ-KAs) have been identified that rapidly adduct proteins and cause PAO formation for amyloid β1-42 implicated in Alzheimer's. We hypothesized that rapid activation of atrial cells triggers oxidative stress with lipid peroxidation and formation of γ-KAs, which then rapidly crosslink proteins to generate PAOs. To investigate this hypothesis, rapidly-paced and control, spontaneously-beating atrial HL-1 cells were probed with a conformation-specific antibody recognizing PAOs. Rapid stimulation of atrial cells caused the generation of cytosolic PAOs along with a myocyte stress response (e.g., transcriptional upregulation of Nppa and Hspa1a), both of which were absent in control, unpaced cells. Rapid activation also caused the formation of superoxide and γ-KA adducts in atriomyocytes, while direct exposure of cells to γ-KAs resulted in PAO production. Increased cytosolic atrial natriuretic peptide (ANP), and the generation of ANP oligomers with exposure to γ-KAs and rapid atrial HL-1 cell stimulation, strongly suggest a role for ANP in PAO formation. Salicylamine (SA) is a small molecule scavenger of γ-KAs that can protect proteins from modification by these reactive compounds. PAO formation and transcriptional remodeling were inhibited when cells were stimulated in the presence of SA, but not with the antioxidant curcumin, which is incapable of scavenging γ-KAs. These results demonstrate that γ-KAs promote protein misfolding and PAO formation as a component of the atrial cell stress response to rapid activation, and they provide a potential mechanistic link between oxidative stress and atrial cell injury.

Genetic analysis of familial forms of Alzheimer's disease (AD) causally links the proteolytic processing of the amyloid precursor protein (APP) and AD. However, the specific type of amyloid and mechanisms of amyloid pathogenesis remain unclear. We conducted a detailed analysis of intracellular amyloid with an aggregation specific conformation dependent monoclonal antibody, M78, raised against fibrillar Aß42. M78 immunoreactivity colocalizes with Aß and the carboxyl terminus of APP (APP-CTF) immunoreactivities in perinuclear compartments at intermediate times in 10month 3XTg-AD mice, indicating that this represents misfolded and aggregated protein rather than normally folded APP. At 12months, M78 immunoreactivity also accumulates in the nucleus. Neuritic plaques at 12months display the same spatial organization of centrally colocalized M78, diffuse chromatin and neuronal nuclear NeuN staining surrounded by peripheral M78 and APP-CTF immunoreactivity as observed in neurons, indicating that neuritic plaques arise from degenerating neurons with intracellular amyloid immunoreactivity. The same staining pattern was observed in neuritic plaques in human AD brains, showing elevated intracellular M78 immunoreactivity at intermediate stages of amyloid pathology (Braak A and B) compared to no amyloid pathology and late stage amyloid pathology (Braak 0 and C, respectively). These results indicate that intraneuronal protein aggregation and amyloid accumulation is an early event in AD and that neuritic plaques are initiated by the degeneration and death of neurons by a mechanism that may be related to the formation of extracellular traps by neutrophils.

Type 2 diabetes (T2D) is characterized by a deficiency in β cell mass, increased β cell apoptosis, and extracellular accumulation of islet amyloid derived from islet amyloid polypeptide (IAPP), which β cells coexpress with insulin. IAPP expression is increased in the context of insulin resistance, the major risk factor for developing T2D. Human IAPP is potentially toxic, especially as membrane-permeant oligomers, which have been observed to accumulate within β cells of patients with T2D and rodents expressing human IAPP. Here, we determined that β cell IAPP content is regulated by autophagy through p62-dependent lysosomal degradation. Induction of high levels of human IAPP in mouse β cells resulted in accumulation of this amyloidogenic protein as relatively inert fibrils within cytosolic p62-positive inclusions, which temporarily averts formation of toxic oligomers. Mice hemizygous for transgenic expression of human IAPP did not develop diabetes; however, loss of β cell-specific autophagy in these animals induced diabetes, which was attributable to accumulation of toxic human IAPP oligomers and loss of β cell mass. In human IAPP-expressing mice that lack β cell autophagy, increased oxidative damage and loss of an antioxidant-protective pathway appeared to contribute to increased β cell apoptosis. These findings indicate that autophagy/lysosomal degradation defends β cells against proteotoxicity induced by oligomerization-prone human IAPP.

Preeclampsia is a pregnancy-specific disorder of unknown etiology and a leading contributor to maternal and perinatal morbidity and mortality worldwide. Because there is no cure other than delivery, preeclampsia is the leading cause of iatrogenic preterm birth. We show that preeclampsia shares pathophysiologic features with recognized protein misfolding disorders. These features include urine congophilia (affinity for the amyloidophilic dye Congo red), affinity for conformational state-dependent antibodies, and dysregulation of prototype proteolytic enzymes involved in amyloid precursor protein (APP) processing. Assessment of global protein misfolding load in pregnancy based on urine congophilia (Congo red dot test) carries diagnostic and prognostic potential for preeclampsia. We used conformational state-dependent antibodies to demonstrate the presence of generic supramolecular assemblies (prefibrillar oligomers and annular protofibrils), which vary in quantitative and qualitative representation with preeclampsia severity. In the first attempt to characterize the preeclampsia misfoldome, we report that the urine congophilic material includes proteoforms of ceruloplasmin, immunoglobulin free light chains, SERPINA1, albumin, interferon-inducible protein 6-16, and Alzheimer's β-amyloid. The human placenta abundantly expresses APP along with prototype APP-processing enzymes, of which the α-secretase ADAM10, the β-secretases BACE1 and BACE2, and the γ-secretase presenilin-1 were all up-regulated in preeclampsia. The presence of β-amyloid aggregates in placentas of women with preeclampsia and fetal growth restriction further supports the notion that this condition should join the growing list of protein conformational disorders. If these aggregates play a pathophysiologic role, our findings may lead to treatment for preeclampsia.

Abnormalities in atrial myocardium increase the likelihood of arrhythmias, including atrial fibrillation (AF). The deposition of misfolded protein, or amyloidosis, plays an important role in the pathophysiology of many diseases, including human cardiomyopathies. We have shown that genes implicated in amyloidosis are activated in a cellular model of AF, with the development of preamyloid oligomers (PAOs). PAOs are intermediates in the formation of amyloid fibrils, and they are now recognized to be the cytotoxic species during amyloidosis. To investigate the presence of PAOs in human atrium, we developed a microscopic imaging-based protocol to enable robust and reproducible quantitative analysis of PAO burden in atrial samples harvested at the time of elective cardiac surgery. Using PAO- and myocardial-specific antibodies, we found that PAO distribution was typically heterogeneous within a myocardial sample. Rigorous imaging and analysis protocols were developed to quantify the relative area of myocardium containing PAOs, termed the Green/Red ratio (G/R), for a given sample. Using these methods, reproducible G/R values were obtained when different sections of a sample were independently processed, imaged, and analyzed by different investigators. This robust technique will enable studies to investigate the role of this novel structural abnormality in the pathophysiology of and arrhythmia generation in human atrial tissue.

Abstract Background It is well established that vaccination of humans and transgenic animals against fibrillar Aβ prevents amyloid accumulation in plaques and preserves cognitive function in transgenic mouse models. However, autoimmune side effects have halted the development of vaccines based on full length human Aβ. Further development of an effective vaccine depends on overcoming these side effects while maintaining an effective immune response. Results We have previously reported that the immune response to amyloid oligomers is largely directed against generic epitopes that are common to amyloid oligomers of many different proteins and independent of a specific amino acid sequence. Here we have examined whether we can exploit this generic immune response to develop a vaccine that targets amyloid oligomers using a non-human random sequence amyloid oligomer. In order to study the effect of vaccination against generic oligomer epitopes, a random sequence oligomer (3A) was selected as it forms oligomers that react with the oligomer specific A11 antibody. Oligomer mimics from 3A peptide, Aβ, islet amyloid polypeptide (IAPP), and Aβ fibrils were used to vaccinate Tg2576 mice, which develop a progressive accumulation of plaques and cognitive impairment. Vaccination with the 3A random sequence antigen was just as effective as vaccination with the other antigens in improving cognitive function and reducing total plaque load (Aβ burden) in the Tg2576 mouse brains, but was associated with a much lower incidence of micro hemorrhage than Aβ antigens. Conclusion These results shows that the amyloid Aβ sequence is not necessary to produce a protective immune response that specifically targets generic amyloid oligomers. Using a non-human, random sequence antigen may facilitate the development of a vaccine that avoids autoimmune side effects.

The impact of synthetic amyloid β (1–42) (Aβ1–42) oligomers on biophysical properties of voltage-gated potassium channels Kv 1.3 and lipid bilayer membranes (BLMs) was quantified for protocols using hexafluoroisopropanol (HFIP) or sodium hydroxide (NaOH) as solvents prior to initiating the oligomer formation. Regardless of the solvent used Aβ1–42 samples contained oligomers that reacted with the conformation-specific antibodies A11 and OC and had similar size distributions as determined by dynamic light scattering. Patch-clamp recordings of the potassium currents showed that synthetic Aβ1–42 oligomers accelerate the activation and inactivation kinetics of Kv 1.3 current with no significant effect on current amplitude. In contrast to oligomeric samples, freshly prepared, presumably monomeric, Aβ1–42 solutions had no effect on Kv 1.3 channel properties. Aβ1–42 oligomers had no effect on the steady-state current (at −80 mV) recorded from Kv 1.3-expressing cells but increased the conductance of artificial BLMs in a dose-dependent fashion. Formation of amyloid channels, however, was not observed due to conditions of the experiments. To exclude the effects of HFIP (used to dissolve lyophilized Aβ1–42 peptide), and trifluoroacetic acid (TFA) (used during Aβ1–42 synthesis), we determined concentrations of these fluorinated compounds in the stock Aβ1–42 solutions by 19F NMR. After extensive evaporation, the concentration of HFIP in the 100× stock Aβ1–42 solutions was ∼1.7 μM. The concentration of residual TFA in the 70× stock Aβ1–42 solutions was ∼20 μM. Even at the stock concentrations neither HFIP nor TFA alone had any effect on potassium currents or BLMs. The Aβ1–42 oligomers prepared with HFIP as solvent, however, were more potent in the electrophysiological tests, suggesting that fluorinated compounds, such as HFIP or structurally-related inhalational anesthetics, may affect Aβ1–42 aggregation and potentially enhance ability of oligomers to modulate voltage-gated ion channels and biological membrane properties.

Background/aimsOligomerization of amyloid beta (Aβ) is a hypothesized step in the formation of plaques in Alzheimer's disease (AD) but has been difficult to demonstrate in vivo in humans. As persons destined to develop familial AD (FAD) due to fully penetrant autosomal dominant mutations are essentially certain to develop the disease, they provide the opportunity to identify oligomers during the presymptomatic stage of the disease.MethodsWe measured levels of Aβ(42) using a conventional immunoassay and prefibrillar, fibrillar, and annular protofibrillar oligomers using polyclonal conformation-dependent antibodies in the cerebrospinal fluid (CSF) of 7 persons at risk for inheriting FAD mutations. Levels of oligomers were compared between FAD mutation carriers and noncarriers.ResultsCompared to 2 noncarriers, annular protofibrillar oligomers were elevated, prefibrillar and fibrillar oligomers trended towards elevation and Aβ(42) monomer trended towards being decreased in 5 FAD mutation carriers.ConclusionOur data provide evidence for an identifiable elevation of CSF oligomers during the presymptomatic phase of FAD.

Background: Inflammation is associated with A beta pathology in Alzheimer's disease (AD) and transgenic AD models. Previously, it has been demonstrated that chronic stimulation of the immune response induces pro-inflammatory cytokines IL-1 beta and TNF-alpha which contribute to neurodegeneration. However, recent evidence has shown that inducing the adaptive immune response reduces A beta pathology and is neuroprotective. Low concentrations of IFN-gamma modulate the adaptive immune response by directing microglia to differentiate to antigen presenting cells. Our objective was to determine if exercise could induce a shift from the immune profile in aged (17-19 months) Tg2576 mice to a response that reduces A beta pathology. Methods: TG (n = 29) and WT (n = 27) mice were divided into sedentary (SED) and exercised (RUN) groups. RUN animals were provided an in-cage running wheel for 3 weeks. Tissue was harvested and hippocampus and cortex dissected out. Quantitative data was analyzed using 2 x 2 ANOVA and student's t-tests. Results: IL-1 beta and TNF-alpha were significantly greater in hippocampi from sedentary Tg2576 (TG(SED)) mice than in wildtype (WT(SED)) (p = 0.04, p = 0.006). Immune response proteins IFN-gamma and MIP-1 alpha are lower in TG(SED) mice than in WT(SED) (p = 0.03, p = 0.07). Following three weeks of voluntary wheel running, IL-1 beta and TNF-alpha decreased to levels indistinguishable from WT. Concurrently, IFN-gamma. and MIP-1 alpha increased in TG(RUN). Increased CD40 and MHCII, markers of antigen presentation, were observed in TG(RUN) animals compared to TG(SED), as well as CD11c staining in and around plaques and vasculature. Additional vascular reactivity observed in TG(RUN) is consistent with an alternative activation immune pathway, involving perivascular macrophages. Significant decreases in soluble A beta(40) (p = 0.01) and soluble fibrillar A beta (p = 0.01) were observed in the exercised transgenic animals. Conclusion: Exercise shifts the immune response from innate to an adaptive or alternative response. This shift in immune response coincides with a decrease in A beta in advanced pathological states.

Background: The multifunctional protein vitronectin is present within the deposits associated with Alzheimer disease (AD), age-related macular degeneration (AMD), atherosclerosis, systemic amyloidoses, and glomerulonephritis. The extent to which vitronectin contributes to amyloid formation within these plaques, which contain misfolded, amyloidogenic proteins, and the role of vitronectin in the pathophysiology of the aforementioned diseases is currently unknown. The investigation of vitronectin aggregation is significant since the formation of oligomeric and fibrillar structures are common features of amyloid proteins. Results: We observed vitronectin immunoreactivity in senile plaques of AD brain, which exhibited overlap with the amyloid fibril-specific OC antibody, suggesting that vitronectin is deposited at sites of amyloid formation. Of particular interest is the growing body of evidence indicating that soluble nonfibrillar oligomers may be responsible for the development and progression of amyloid diseases. In this study we demonstrate that both plasma-purified and recombinant human vitronectin readily form spherical oligomers and typical amyloid fibrils. Vitronectin oligomers are toxic to cultured neuroblastoma and retinal pigment epithelium (RPE) cells, possibly via a membrane-dependent mechanism, as they cause leakage of synthetic vesicles. Oligomer toxicity was attenuated in RPE cells by the anti-oligomer A11 antibody. Vitronectin fibrils contain a C-terminal protease-resistant fragment, which may approximate the core region of residues essential to amyloid formation. Conclusion: These data reveal the propensity of vitronectin to behave as an amyloid protein and put forth the possibilities that accumulation of misfolded vitronectin may contribute to aggregate formation seen in age-related amyloid diseases.

Background: Amyloid-related degenerative diseases are associated with the accumulation of misfolded proteins as amyloid fibrils in tissue. In Alzheimer disease (AD), amyloid accumulates in several distinct types of insoluble plaque deposits, intracellular A beta and as soluble oligomers and the relationships between these deposits and their pathological significance remains unclear. Conformation dependent antibodies have been reported that specifically recognize distinct assembly states of amyloids, including prefibrillar oligomers and fibrils. Results: We immunized rabbits with a morphologically homogeneous population of A beta 42 fibrils. The resulting immune serum (OC) specifically recognizes fibrils, but not random coil monomer or prefibrillar oligomers, indicating fibrils display a distinct conformation dependent epitope that is absent in prefibrillar oligomers. The fibril epitope is also displayed by fibrils of other types of amyloids, indicating that the epitope is a generic feature of the polypeptide backbone. The fibril specific antibody also recognizes 100,000 x G soluble fibrillar oligomers ranging in size from dimer to greater than 250 kDa on western blots. The fibrillar oligomers recognized by OC are immunologically distinct from prefibrillar oligomers recognized by A11, even though their sizes overlap broadly, indicating that size is not a reliable indicator of oligomer conformation. The immune response to prefibrillar oligomers and fibrils is not sequence specific and antisera of the same specificity are produced in response to immunization with islet amyloid polypeptide prefibrillar oligomer mimics and fibrils. The fibril specific antibodies stain all types of amyloid deposits in human AD brain. Diffuse amyloid deposits stain intensely with anti-fibril antibody although they are thioflavin S negative, suggesting that they are indeed fibrillar in conformation. OC also stains islet amyloid deposits in transgenic mouse models of type II diabetes, demonstrating its generic specificity for amyloid fibrils. Conclusion: Since the fibril specific antibodies are conformation dependent, sequence-independent, and recognize epitopes that are distinct from those present in prefibrillar oligomers, they may have broad utility for detecting and characterizing the accumulation of amyloid fibrils and fibrillar type oligomers in degenerative diseases.

BackgroundAlzheimer's disease, a common dementia of the elder, is characterized by accumulation of protein amyloid deposits in the brain. Immunization to prevent this accumulation has been proposed as a therapeutic possibility, although adverse inflammatory reactions in human trials indicate the need for novel vaccination strategies.MethodHere vaccination with novel amyloid peptide immunogens was assessed in a transgenic mouse model displaying age-related accumulation of fibrillar plaques.ResultsImmunization with any conformation of the amyloid peptide initiated at 12 months of age (at which time fibrillar amyloid has just begun to accumulate) showed significant decrease in total and fibrillar amyloid deposits and in glial reactivity relative to control transgenic animals. In contrast, there was no significant decrease in amyloid deposition or glial activation in mice in which vaccination was initiated at 16 months of age, despite the presence of similar levels anti-Abeta antibodies in young and old animals vaccinated with a given immunogen. Interestingly, immunization with an oligomeric conformation of Abeta was equally as effective as other amyloid peptides at reducing plaque accumulation. However, the antibodies generated by immunization with the oligomeric conformation of Abeta have more limited epitope reactivity than those generated by fAbeta, and the microglial response was significantly less robust.ConclusionThese results suggest that a more specific immunogen such as oligomeric Abeta can be designed that achieves the goal of depleting amyloid while reducing potential detrimental inflammatory reactions. In addition, the data show that active immunization of older Tg2576 mice with any amyloid conformation is not as efficient at reducing amyloid accumulation and related pathology as immunization of younger mice, and that serum anti-amyloid antibody levels are not quantitatively related to reduced amyloid-associated pathology.

Introduction: The inositol polyphosphate‐5‐phosphatase D (INPP5D) gene encodes a dual‐specificity phosphatase that can dephosphorylate both phospholipids and phosphoproteins. Single nucleotide polymorphisms in INPP5D impact risk for developing late onset sporadic Alzheimer's disease (LOAD). Methods: To assess the consequences of inducible Inpp5d knockdown in microglia of APPKM670/671NL/PSEN1Δexon9 (PSAPP) mice, we injected 3‐month‐old Inpp5dfl/fl/Cx3cr1CreER/+ and PSAPP/Inpp5dfl/fl/Cx3cr1CreER/+ mice with either tamoxifen (TAM) or corn oil (CO) to induce recombination. Results: At age 6 months, we found that the percent area of 6E10+ deposits and plaque‐associated microglia in Inpp5d knockdown mice were increased compared to controls. Spatial transcriptomics identified a plaque‐specific expression profile that was extensively altered by Inpp5d knockdown. Discussion: These results demonstrate that conditional Inpp5d downregulation in the PSAPP mouse increases plaque burden and recruitment of microglia to plaques. Spatial transcriptomics highlighted an extended gene expression signature associated with plaques and identified CST7 (cystatin F) as a novel marker of plaques. Highlights: Inpp5d knockdown increases plaque burden and plaque‐associated microglia number.Spatial transcriptomics identifies an expanded plaque‐specific gene expression profile.Plaque‐induced gene expression is altered by Inpp5d knockdown in microglia.Our plaque‐associated gene signature overlaps with human Alzheimer's disease gene networks. [ABSTRACT FROM AUTHOR]