Tau and Amyloid ß Protein in Patient-Derived Aqueous Brain Extracts Act Concomitantly to Disrupt Long-Term Potentiation in Vivo.

Amyloid ß protein (Aß) and tau, the two main proteins implicated in causing Alzheimer's disease (AD), are posited to trigger synaptic dysfunction long before significant synaptic loss occurs in vulnerable circuits. Whereas soluble Aß aggregates from AD brain are well recognized potent synaptotoxins, less is known about the synaptotoxicity of soluble tau from AD or other tauopathy patient brains. Minimally manipulated patient-derived aqueous brain extracts contain the more diffusible native forms of these proteins. Here, we explore how intracerebral injection of Aß and tau present in such aqueous extracts of patient brain contribute to disruption of synaptic plasticity in the CA1 area of the male rat hippocampus. Aqueous extracts of certain AD brains acutely inhibited long-term potentiation (LTP) of synaptic transmission in a manner that required both Aß and tau. Tau-containing aqueous extracts of a brain from a patient with Pick's disease (PiD) also impaired LTP, and diffusible tau from either AD or PiD brain lowered the threshold for AD brain Aß to inhibit LTP. Remarkably, the disruption of LTP persisted for at least 2 weeks after a single injection. These findings support a critical role for diffusible tau in causing rapid onset, persistent synaptic plasticity deficits, and promoting Aß-mediated synaptic dysfunction.SIGNIFICANCE STATEMENT The microtubule-associated protein tau forms relatively insoluble fibrillar deposits in the brains of people with neurodegenerative diseases including Alzheimer's and Pick's diseases. More soluble aggregates of disease-associated tau may diffuse between cells and could cause damage to synapses in vulnerable circuits. We prepared aqueous extracts of diseased cerebral cortex and tested their ability to interfere with synaptic function in the brains of live rats. Tau in these extracts rapidly and persistently disrupted synaptic plasticity and facilitated impairments caused by amyloid ß protein, the other major pathologic protein in Alzheimer's disease. These findings show that certain diffusible forms of tau can mediate synaptic dysfunction and may be a target for therapy.

Identification of neurotoxic cross-linked amyloid-ß dimers in the Alzheimer's brain.

The primary structure of canonical amyloid-ß-protein was elucidated more than 30 years ago, yet the forms of amyloid-ß that play a role in Alzheimer's disease pathogenesis remain poorly defined. Studies of Alzheimer's disease brain extracts suggest that amyloid-ß, which migrates on sodium dodecyl sulphate polyacrylamide gel electrophoresis with a molecular weight of ∼7 kDa (7kDa-Aß), is particularly toxic; however, the nature of this species has been controversial. Using sophisticated mass spectrometry and sensitive assays of disease-relevant toxicity we show that brain-derived bioactive 7kDa-Aß contains a heterogeneous mixture of covalently cross-linked dimers in the absence of any other detectable proteins. The identification of amyloid-ß dimers may open a new phase of Alzheimer's research and allow a better understanding of Alzheimer's disease, and how to monitor and treat this devastating disorder. Future studies investigating the bioactivity of individual dimers cross-linked at known sites will be critical to this effort.

Diffusible, highly bioactive oligomers represent a critical minority of soluble Aß in Alzheimer's disease brain.

Significant data suggest that soluble Aß oligomers play an important role in Alzheimer's disease (AD), but there is great confusion over what exactly constitutes an Aß oligomer and which oligomers are toxic. Most studies have utilized synthetic Aß peptides, but the relevance of these test tube experiments to the conditions that prevail in AD is uncertain. A few groups have studied Aß extracted from human brain, but they employed vigorous tissue homogenization which is likely to release insoluble Aß that was sequestered in plaques during life. Several studies have found such extracts to possess disease-relevant activity and considerable efforts are being made to purify and better understand the forms of Aß therein. Here, we compared the abundance of Aß in AD extracts prepared by traditional homogenization versus using a far gentler extraction, and assessed their bioactivity via real-time imaging of iPSC-derived human neurons plus the sensitive functional assay of long-term potentiation. Surprisingly, the amount of Aß retrieved by gentle extraction constituted only a small portion of that released by traditional homogenization, but this readily diffusible fraction retained all of the Aß-dependent neurotoxic activity. Thus, the bulk of Aß extractable from AD brain was innocuous, and only the small portion that was aqueously diffusible caused toxicity. This unexpected finding predicts that generic anti-oligomer therapies, including Aß antibodies now in trials, may be bound up by the large pool of inactive oligomers, whereas agents that specifically target the small pool of diffusible, bioactive Aß would be more useful. Furthermore, our results indicate that efforts to purify and target toxic Aß must employ assays of disease-relevant activity. The approaches described here should enable these efforts, and may assist the study of other disease-associated aggregation-prone proteins.

C-Terminally Truncated Forms of Tau, But Not Full-Length Tau or Its C-Terminal Fragments, Are Released from Neurons Independently of Cell Death.

Recent evidence suggests that tau aggregation may spread via extracellular release and subsequent uptake by synaptically connected neurons, but little is known about the processes by which tau is released or the molecular forms of extracellular tau. To gain insight into the nature of extracellular tau, we used highly sensitive ELISAs, which, when used in tandem, are capable of differentiating between full-length (FL) tau, mid-region-bearing fragments, and C-terminal (CT) fragments. We applied these assays to the systematic study of the conditioned media of N2a cells, induced pluripotent stem cell-derived human cortical neurons, and primary rat cortical neurons, each of which was carefully assessed for viability. In all three neuronal models, the bulk of extracellular tau was free-floating and unaggregated and <0.2% was encapsulated in exosomes. Although most intracellular tau was FL, the majority of extracellular tau was CT truncated and appeared to be released both actively by living neurons and passively by dead cells. In contrast, only a small amount of extracellular tau was aggregation-competent tau (i.e., contained the microtubule-binding regions) and this material appears to be released solely due to a low level of cell death that occurs in all cell culture systems. Importantly, amyloid ß-protein (Aß)-induced neuronal compromise significantly increased the quantity of all forms of extracellular tau, but the presence of Aß before detectable cell compromise did not increase extracellular tau. Collectively, these results suggest that factors that induce neuronal death are likely to be necessary to initiate the extracellular spread of tau aggregation. SIGNIFICANCE STATEMENT: Recent studies suggest that the transfer of tau between neurons underlies the characteristic spatiotemporal progression of neurofibrillary pathology. We searched for tau in the conditioned medium of N2a cells, induced pluripotent stem cell-derived human cortical neurons, and primary rat cortical neurons and analyzed the material present using four different tau ELISAs. We demonstrate that the majority of tau released from healthy neurons is C-terminally truncated and lacks the microtubule-binding region (MTBR) thought necessary for self-aggregation. A small amount of MTBR-containing tau is present outside of cells, but this appears to be solely due to cell death. Therefore, if propagation of tau aggregation is mediated by extracellular tau, our findings suggest that neuronal compromise is required to facilitate this process.

The Aggregation Paths and Products of Aß42 Dimers Are Distinct from Those of the Aß42 Monomer.

Extracts of Alzheimer's disease (AD) brain that contain what appear to be sodium dodecyl sulfate-stable amyloid ß-protein (Aß) dimers potently block LTP and impair memory consolidation. Brain-derived dimers can be physically separated the Aß monomer, consist primarily of Aß42, and resist denaturation by chaotropic agents. In nature, covalently cross-linked Aß dimers could be generated in two ways: by the formation of a dityrosine (DiY) or an isopeptide ε-(γ-glutamyl)-lysine (Q-K) bond. We enzymatically cross-linked recombinant Aß42 monomer to produce DiY and Q-K dimers and then used a range of biophysical methods to study their aggregation. Both Q-K and DiY dimers aggregate to form soluble assemblies distinct from the fibrillar aggregates formed by the Aß monomer. The results suggest that the cross-links disfavor fibril formation from Aß dimers, thereby enhancing the concentration of soluble aggregates akin to those in aqueous extracts of AD brain. Thus, it seems that Aß dimers may play an important role in determining the formation of soluble rather than insoluble aggregates.

Soluble Aß oligomers impair hippocampal LTP by disrupting glutamatergic/GABAergic balance.

Epileptic activity may be more prevalent in early stage Alzheimer's disease (AD) than previously believed. Several studies report spontaneous seizures and interictal discharges in mouse models of AD undergoing age-related Aß accumulation. The mechanism by which Aß-induced neuronal excitability can trigger epileptiform activity remains unknown. Here, we systematically examined field excitatory postsynaptic potentials (fEPSP) in stratum radiatum and population spikes (PSs) in the adjacent stratum pyramidale of CA1 in wild-type mouse hippocampal slices. Soluble Aß oligomers (oAß) blocked hippocampal LTP and EPSP-spike (E-S) potentiation, and these effects were occluded by prior treatment with the glutamate uptake inhibitor TBOA. In accord, oAß elevated glutamate levels in the hippocampal slice medium. Recording the PS revealed that oAß increased PS frequency and reduced LTP, and this LTP deficit was occluded by pretreatment with the GABAA antagonist picrotoxin. Whole-cell recordings showed that oAß significantly increased spontaneous EPSC frequency. Decreasing neuronal activity by increasing GABA tone or partially blocking NMDAR activity prevented oAß impairment of hippocampal LTP. Finally, treating slices with two antiepileptic drugs rescued the LTP inhibition induced by oAß. We conclude that soluble Aß oligomers at the low nanomolar levels present in AD brain increase neuronal excitability by disrupting glutamatergic/GABAergic balance, thereby impairing synaptic plasticity.

Aß dimers differ from monomers in structural propensity, aggregation paths and population of synaptotoxic assemblies.

Dimers of Aß (amyloid ß-protein) are believed to play an important role in Alzheimer's disease. In the absence of sufficient brain-derived dimers, we studied one of the only possible dimers that could be produced in vivo, [Aß](DiY) (dityrosine cross-linked Aß). For comparison, we used the Aß monomer and a design dimer cross-linked by replacement of Ser²6 with cystine [AßS26C]2. We showed that similar to monomers, unaggregated dimers lack appreciable structure and fail to alter long-term potentiation. Importantly, dimers exhibit subtly different structural propensities from monomers and each other, and can self-associate to form larger assemblies. Although [Aß](DiY) and [AßS26C]2 have distinct aggregation pathways, they both populate bioactive soluble assemblies for longer durations than Aß monomers. Our results indicate that the link between Aß dimers and Alzheimer's disease results from the ability of dimers to further assemble and form synaptotoxic assemblies that persist for long periods of time.

The aqueous phase of Alzheimer's disease brain contains assemblies built from ∼4 and ∼7 kDa Aß species.

INTRODUCTION: Much knowledge about amyloid ß (Aß) aggregation and toxicity has been acquired using synthetic peptides and mouse models, whereas less is known about soluble Aß in human brain. METHODS: We analyzed aqueous extracts from multiple AD brains using an array of techniques. RESULTS: Brains can contain at least four different Aß assembly forms including: (i) monomers, (ii) a ∼7 kDa Aß species, and larger species (iii) from ∼30-150 kDa, and (iv) >160 kDa. High molecular weight species are by far the most prevalent and appear to be built from ∼7 kDa Aß species. The ∼7 kDa Aß species resist denaturation by chaotropic agents and have a higher Aß42/Aß40 ratio than monomers, and are unreactive with antibodies to Asp1 of Ab or APP residues N-terminal of Asp1. DISCUSSION: Further analysis of brain-derived ∼7 kDa Aß species, the mechanism by which they assemble and the structures they form should reveal therapeutic and diagnostic opportunities.

Identification and Prioritization of PET Neuroimaging Targets for Microglial Phenotypes Associated with Microglial Activity in Alzheimer's Disease.

Activation of microglial cells accompanies the progression of many neurodegenerative disorders, including Alzheimer's disease (AD). Development of molecular imaging tools specific to microglia can help elucidate the mechanism through which microglia contribute to the pathogenesis and progression of neurodegenerative disorders. Through analysis of published genetic, transcriptomic, and proteomic data sets, we identified 19 genes with microglia-specific expression that we then ranked based on association with the AD characteristics, change in expression, and potential druggability of the target. We believe that the process we used to identify and rank microglia-specific genes is broadly applicable to the identification and evaluation of targets in other disease areas and for applications beyond molecular imaging.

Production and Use of Recombinant Aß for Aggregation Studies.

The amyloid ß-protein (Aß) is believed to play a central role in Alzheimer's disease (AD) pathogenesis and there is great interest in understanding the process of Aß aggregation, its underlying mechanism and the species generated during aggregation and their biological activity. Although Aß has been studied for more than 30 years, analysis of its aggregation has been hampered by structural and chemical impurities. Here we provide a detailed protocol for the expression and purification of chemically and structurally homogeneous Aß monomer. We also describe a method to produce covalent Aß dimers linked by phenolic coupling of tyrosine residues.

Extracellular Forms of Aß and Tau from iPSC Models of Alzheimer's Disease Disrupt Synaptic Plasticity.

The early stages of Alzheimer's disease are associated with synaptic dysfunction prior to overt loss of neurons. To identify extracellular molecules that impair synaptic plasticity in the brain, we studied the secretomes of human iPSC-derived neuronal models of Alzheimer's disease. When introduced into the rat brain, secretomes from human neurons with either a presenilin-1 mutation, amyloid precursor protein duplication, or trisomy of chromosome 21 all strongly inhibit hippocampal long-term potentiation. Synaptic dysfunction caused by presenilin-1 mutant and amyloid precusor protein duplication secretomes is mediated by Aß peptides, whereas trisomy of chromosome 21 (trisomy 21) neuronal secretomes induce dysfunction through extracellular tau. In all cases, synaptotoxicity is relieved by antibody blockade of cellular prion protein. These data indicate that human models of Alzheimer's disease generate distinct proteins that converge at the level of cellular prion protein to induce synaptic dysfunction in vivo.

An in vitro paradigm to assess potential anti-Aß antibodies for Alzheimer's disease.

Although the amyloid ß-protein (Aß) is believed to play an initiating role in Alzheimer's disease (AD), the molecular characteristics of the key pathogenic Aß forms are not well understood. As a result, it has proved difficult to identify optimal agents that target disease-relevant forms of Aß. Here, we combined the use of Aß-rich aqueous extracts of brain samples from AD patients as a source of human Aß and live-cell imaging of iPSC-derived human neurons to develop a bioassay capable of quantifying the relative protective effects of multiple anti-Aß antibodies. We report the characterization of 1C22, an aggregate-preferring murine anti-Aß antibody, which better protects against forms of Aß oligomers that are toxic to neurites than do the murine precursors of the clinical immunotherapeutics, bapineuzumab and solanezumab. These results suggest further examination of 1C22 is warranted, and that this bioassay maybe useful as a primary screen to identify yet more potent anti-Aß therapeutics.

IgG Conformer's Binding to Amyloidogenic Aggregates.

Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs) have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1) pAb aggregates have greater activity than monomers (HMW species > dimers > monomers), 2) pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR) interactions of F(ab) regions, and 3) pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg), had up to ~200- and ~7-fold stronger binding to aggregates of Aß and transthyretin (TTR) than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aß- and Cibacron blue-isolated IVIg IgGs. Human pAb conformer's binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR) component to pAb's activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aß and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs.

Targeted Magnetic Nanoparticles for Remote Magnetothermal Disruption of Amyloid-ß Aggregates.

Remotely triggered hysteretic heat dissipation by magnetic nanoparticles (MNPs) selectively attached to targeted proteins can be used to break up self-assembled aggregates. This magnetothermal approach is applied to the amyloid-ß (Aß) protein, which forms dense, insoluble plaques characteristic of Alzheimer's disease. Specific targeting of dilute MNPs to Aß aggregates is confirmed via transmission electron microscopy (TEM) and is found to be consistent with a statistical model of MNP distribution on the Aß substrates. MNP composition and size are selected to achieve efficient hysteretic power dissipation at physiologically safe alternating magnetic field (AMF) conditions. Dynamic light scattering, fluorescence spectroscopy, and TEM are used to characterize the morphology and size distribution of aggregates before and after exposure to AMF. A dramatic reduction in aggregate size from microns to tens of nanometers is observed, suggesting that exposure to an AMF effectively destabilizes Aß deposits decorated with targeted MNPs. Experiments in primary hippocampal neuronal cultures indicate that the magnetothermal disruption of aggregates reduces Aß cytotoxicity, which may enable future applications of this approach for studies of protein disaggregation in physiological environments.

A highly sensitive novel immunoassay specifically detects low levels of soluble Aß oligomers in human cerebrospinal fluid.

INTRODUCTION: Amyloid ß-protein oligomers play a key role in Alzheimer's disease (AD), but well-validated assays that routinely detect them in cerebrospinal fluid (CSF) are just emerging. We sought to confirm and extend a recent study using the Singulex Erenna platform that reported increased mean CSF oligomer levels in AD. METHODS: We tested four antibody pairs and chose one pair that was particularly sensitive, using 1C22, our new oligomer-selective monoclonal antibody, for capture. We applied this new assay to extracts of human brain and CSF. RESULTS: A combination of 1C22 for capture and 3D6 for detection yielded an Erenna immunoassay with a lower limit of quantification of approximately 0.15 pg/ml that was highly selective for oligomers over monomers and detected a wide size-range of oligomers. Most CSFs we tested had detectable oligomer levels but with a large overlap between AD and controls and a trend for higher mean levels in mild cognitive impairment (MCI) than controls. CONCLUSION: Aß oligomers are detectable in most human CSFs, but AD and controls overlap. MCI CSFs may have a modest elevation in mean value by this assay.

Soluble Aß oligomers are rapidly sequestered from brain ISF in vivo and bind GM1 ganglioside on cellular membranes.

Soluble Aß oligomers contribute importantly to synaptotoxicity in Alzheimer's disease, but their dynamics in vivo remain unclear. Here, we found that soluble Aß oligomers were sequestered from brain interstitial fluid onto brain membranes much more rapidly than nontoxic monomers and were recovered in part as bound to GM1 ganglioside on membranes. Aß oligomers bound strongly to GM1 ganglioside, and blocking the sialic acid residue on GM1 decreased oligomer-mediated LTP impairment in mouse hippocampal slices. In a hAPP transgenic mouse model, substantial levels of GM1-bound Aß42 were recovered from brain membrane fractions. We also detected GM1-bound Aß in human CSF, and its levels correlated with Aß42, suggesting its potential as a biomarker of Aß-related membrane dysfunction. Together, these findings highlight a mechanism whereby hydrophobic Aß oligomers become sequestered onto GM1 ganglioside and presumably other lipids on neuronal membranes, where they may induce progressive functional and structural changes.