Tiernan T. O'Malley

New ELISAs with high specificity for soluble oligomers of amyloid β-protein detect natural Aβ oligomers in human brain but not CSF

Soluble oligomers of amyloid ß‐protein (Aß) have been increasingly linked to synaptic dysfunction, tau alteration, and neuritic dystrophy in Alzheimers disease (AD) and mouse models. There is a great need for assays that quantify Aß oligomers with high specificity and sensitivity.

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 β-protein (Aβ)-induced neuronal compromise significantly increased the quantity of all forms of extracellular tau, but the presence of Aβ 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.

A beta dimers differ from monomers in structural propensity, aggregation paths and population of synaptotoxic assemblies

Dimers of Aβ (amyloid β-protein) are believed to play an important role in Alzheimers disease. In the absence of sufficient brain-derived dimers, we studied one of the only possible dimers that could be produced in vivo, [Aβ](DiY) (dityrosine cross-linked Aβ). For comparison, we used the Aβ monomer and a design dimer cross-linked by replacement of Ser²⁶ with cystine [AβS26C]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 [Aβ](DiY) and [AβS26C]₂ have distinct aggregation pathways, they both populate bioactive soluble assemblies for longer durations than Aβ monomers. Our results indicate that the link between Aβ dimers and Alzheimers disease results from the ability of dimers to further assemble and form synaptotoxic assemblies that persist for long periods of time.

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

Epileptic activity may be more prevalent in early stage Alzheimers disease (AD) than previously believed. Several studies report spontaneous seizures and interictal discharges in mouse models of AD undergoing age-related Aβ accumulation. The mechanism by which Aβ-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 Aβ oligomers (oAβ) 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, oAβ elevated glutamate levels in the hippocampal slice medium. Recording the PS revealed that oAβ increased PS frequency and reduced LTP, and this LTP deficit was occluded by pretreatment with the GABAA antagonist picrotoxin. Whole-cell recordings showed that oAβ significantly increased spontaneous EPSC frequency. Decreasing neuronal activity by increasing GABA tone or partially blocking NMDAR activity prevented oAβ impairment of hippocampal LTP. Finally, treating slices with two antiepileptic drugs rescued the LTP inhibition induced by oAβ. We conclude that soluble Aβ oligomers at the low nanomolar levels present in AD brain increase neuronal excitability by disrupting glutamatergic/GABAergic balance, thereby impairing synaptic plasticity.

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

Much knowledge about amyloid β (Aβ) aggregation and toxicity has been acquired using synthetic peptides and mouse models, whereas less is known about soluble Aβ in human brain.

Targeted Magnetic Nanoparticles for Remote Magnetothermal Disruption of Amyloid‐β 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-β (Aβ) protein, which forms dense, insoluble plaques characteristic of Alzheimers disease. Specific targeting of dilute MNPs to Aβ aggregates is confirmed via transmission electron microscopy (TEM) and is found to be consistent with a statistical model of MNP distribution on the Aβ 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 Aβ deposits decorated with targeted MNPs. Experiments in primary hippocampal neuronal cultures indicate that the magnetothermal disruption of aggregates reduces Aβ cytotoxicity, which may enable future applications of this approach for studies of protein disaggregation in physiological environments.

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 conformers 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) pAbs 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 Aβ and transthyretin (TTR) than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated IVIg IgGs. Human pAb conformers 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 pAbs activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aβ 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.

Lessons from the Study of Covalent Dimers of the Alzheimer’s Disease-Associate Amyloid β-Protein

Alzheimer’s disease (AD) is a brain disorder that first manifests in the form of intermittent memory problems and then progresses to dementia and ultimately death. Although the precise cause of AD remains obscure evidence from multiple sources indicate that the amyloid β-protein (Aβ) plays a key role [1]. Aβ comprises a family of proteins with a common core of ~30 amino acids. These peptides are amphipatic in nature and are prone to self-associate and certain aggregates of Aβ are toxic to nerve cells. Aβ of various sequences, but most particularly those that extend C-terminal to alanine 42, are found in the tell-tale amyloid plaques which litter the brains of individuals who die with AD. Several mutations within the Aβ sequence cause early onset AD and are believed to increase the formation of toxic Aβ assemblies. However, such mutations are very rare and most cases of AD occur in individuals with the normal Aβ sequence

A NOVEL PARADIGM FOR THE IDENTIFICATION AND TARGETING OF BIOACTIVE ABETA SPECIES FROM ALZHEIMER BRAIN

Background:The amyloid beta-protein (Abeta) is believed to play an initiating in role in Alzheimer’s disease (AD), but the forms and activity of Abeta involved in pathogenesis are not well understood. Most prior studies on Abeta activity and structure have utilized synthetic or recombinant peptides and crude measures of cell death or low throughput measures of synaptic plasticity. Here we describe a high throughput paradigm which combines the use of aqueous extracts of AD brain as a source of Abeta, and a livecell imaging platform to monitor the effects of brain-derived Abeta on neurite length. Methods: Temporal cortices from 3 mild AD cases (MMSEs less than or equal to 23 within 2 years of death) were homogenized in artificial cerebrospinal fluid (w10 g in 50 ml). The homogenates were centrifuged at 150,000 g for 78 minutes, supernatants removed and a portion of each treated with the pan anti-Abeta antiserum, AW7. Rat primary hippocampal neurons were grown in 96 well plates at 5000 cells per well. AD brain extracts (+/immunodepletion) were added at 3 dilutions (1:4, 1:8 and 1:16) to DIV14 neurons and their effects on neurite number and density monitored every 2 hours for 4 days using the IncuCyte live-cell imaging system. Results: AD brain extracts contained a mixture of Abeta monomers and oligomers the majority of which were removed by AW7 immunodepletion. Addition of Abeta-containing extracts to neurons caused a timeand dose-dependent decrease in neurite length, whereas extracts immunodepleted of Abeta had no effect. The interval at which neurite length first demonstrated a statistically significant decrease was distinct for extracts of the 3 different brains, but in each case the 1:4 diluted samples caused a significant effect afterw50-60 hours post-treatment (p<0.05). Conclusions:These results indicate that soluble forms of brain-derived Abeta cause a disease-relevant change that could be exploited to identify toxic forms of Abeta from AD brain. The relatively short time frame over which decreases in neurite length occur and the use of a multi-well plate format recommend this paradigm as a tool for the discovery and optimization of agents that target toxic forms of Abeta.