Search

IntroductionNeurophysiological manifestations selectively associated with amyloid beta and tau depositions in Alzheimer's disease (AD) are useful network biomarkers to identify peptide specific pathological processes. The objective of this study was to validate the associations between reduced neuronal synchrony within alpha oscillations and neurofibrillary tangle (NFT) density in autopsy examination, in patients with AD.MethodsIn a well-characterized clinicopathological cohort of AD patients (n=13), we quantified neuronal synchrony within alpha (8-12Hz) and delta-theta (2-8Hz) oscillations, using magnetoencephalography during the disease course, within six selected neocortical and hippocampal regions, including angular gyrus, superior temporal gurus, middle frontal gyrus, primary motor cortex, CA1, and subiculum, and correlated these with regional NFT density quantified at histopathological examination.ResultsAbnormal synchrony in alpha, but not in delta-theta, significantly predicted the NFT density at post mortem neuropathological examination.DiscussionReduced alpha synchrony is a sensitive neurophysiological index associated with pathological tau, and a potential network biomarker for clinical trials, to gauge the extent of network dysfunction and the degree of rescue in treatments targeting tau pathways in AD.

DNQX and TTX were bioactive. (A–C) Experiments illustrating effects of DNQX (A) and TTX (B, C). (A) A single experiment was performed to confirm that DNQX blocks AMPA-induced neurotoxicity in primary neuronal cultures. Data represent means ± SEM of technical replicates (4–6 wells per condition). WT neurons were treated with vehicle or the competitive AMPAR antagonist DNQX (20 μM) 1 h before and throughout exposure to different concentrations of AMPA. Raw AlamarBlue fluorescence measurements are shown in arbitrary units. (B, C) TTX blocked spontaneous and induced neuronal activity in acute brain slices. Slices of somatosensory cortex were prepared and pyramidal neurons were patch-clamped as described [9]. Whole-cell membrane voltage recordings were made in artificial cerebrospinal fluid (aCSF) in the absence or presence of 3 μM TTX (from the same batch used for cell culture experiments). (B) TTX prevented spontaneous neuronal activity as well as activity induced by 100 pA/200 ms current stimulations (arrowheads) or by a membrane voltage ramp (between asterisks). Scale bar indicates 20 s of recording time. (C) Representative membrane voltage responses elicited by 100 pA/200 ms current stimulations in control aCSF and after addition of 3 μM TTX. (PDF 4818 kb)

Glutamate-induced neurotoxicity depends on NMDARs and extracellular Ca2+. (A, B) Experiments illustrating Ca2+ dependence of glutamate-induced neurotoxicity (A) and neurotoxicity caused by different glutamate receptor agonists (B). (A) One experiment was performed to confirm that reduction of extracellular Ca2+ counteracts glutamate-induced neurotoxicity. WT neurons were treated with Ca2+-containing isotonic solution or 0 mM Ca2+ solution 1 h prior to and throughout exposure to different concentrations of glutamate. Raw AlamarBlue fluorescence measurements are shown in arbitrary units. (B) Neurotoxicity was assessed as above in WT neuronal cultures treated with the indicated glutamate receptor agonists for 15 min at DIV 13. Data represent means ± SEM of technical replicates (8–16 wells per condition) from 1 to 2 experiments. (PDF 4803 kb)

Proximity ligation assay (PLA) to quantify association between tau and Fyn in neurons. (A) Diagram illustrating key features of the PLA assay. Neuronal cultures from Mapt −/− mice were transfected on DIV7 with a plasmid encoding GFP-P2A or GFP-P2A-mTau and fixed for PLA and immunostaining on DIV8. The GFP-P2A-mTau fusion protein is posttranslationally cleaved at the C-terminal side of the P2A peptide, resulting in the production of GFP-P2A and mTau at a 1:1 molar ratio. The mTau expressed was either WT or mutant (Fig. 5a). GFP signals were used to identify transfected neurons and PLA signals to measure the association between exogenous mTau and endogenous Fyn (see Methods for details). mTau immunofluorescence was used to normalize PLA signals, because mTau expression levels varied among neurons and correlated with PLA signals, as indicated in (C). (B, C) Neurons were transfected with plasmids encoding GFP-P2A, GFP-P2A-mTauWT, or GFP-P2A-mTauAxxA7 on DIV7 and analyzed by PLA and tau immunostaining on DIV8. (B) Representative photomicrographs showing levels of GFP signals (left), PLA signals (middle), and tau immunoreactivity (right) in three neurons from different culture wells. Scale bar: 50 μm. (C) Correlation between PLA signals and Tau5 immunofluorescence across individual neurons in cultures transfected with GFP-P2A-mTauWT (black: R2 = 0.6436, P

Variability of glutamate-induced neurotoxicity measurements across independent experiments. In each of seven independent experiments (carried out on different days), primary neurons pooled from multiple WT mice were cultured on a single 96-well plate and transduced with lentiviral vectors encoding shSCR or shTau (44–48 wells per shRNA) at the time of plating (DIV0). On DIV 13, neurons were exposed to glutamate (4–8 wells per concentration) for 15 min, followed by alamarBlue assay of neurotoxicity 24 h later. Different from the other figures, the box plots shown here represent the distribution of the raw fluorescence signals (in arbitrary units) measured in each set of wells (technical replicates) per glutamate concentration for each experimental condition (shSCR and shTau) and experiment (1–7). The lower and upper ends of the boxes represent the 25th and 75th quartile of the respective distributions. The horizontal line in each box represents the median. The ends of the whiskers terminate at the farthest points that are within 1.5 times the inter-quartile range (difference between upper and lower ends of the box). Individual dots shown in some of the panels represent outliers that fell outside the range defined by the whiskers. Note that, across experiments, neurons of the same genotype that were exposed to similar experimental conditions showed systematic variations in the mean and variance of their responses at given doses as well as in the shapes of their dose-response curves. Therefore, accounting for experiment-to-experiment variability by mean models of intensity (e.g., linear mixed effect models) and minimizing the number of dose-specific parameters to be estimated by assuming dose-response models (e.g., linear, cubic, or Hill) were not suitable for the analysis of this data. (PDF 4937 kb)

ObjectiveSeizures are more frequent in patients with Alzheimer's disease (AD) and can hasten cognitive decline. However, the incidence of subclinical epileptiform activity in AD and its consequences are unknown. Motivated by results from animal studies, we hypothesized higher than expected rates of subclinical epileptiform activity in AD with deleterious effects on cognition.MethodsWe prospectively enrolled 33 patients (mean age, 62 years) who met criteria for AD, but had no history of seizures, and 19 age-matched, cognitively normal controls. Subclinical epileptiform activity was assessed, blinded to diagnosis, by overnight long-term video-electroencephalography (EEG) and a 1-hour resting magnetoencephalography exam with simultaneous EEG. Patients also had comprehensive clinical and cognitive evaluations, assessed longitudinally over an average period of 3.3 years.ResultsSubclinical epileptiform activity was detected in 42.4% of AD patients and 10.5% of controls (p = 0.02). At the time of monitoring, AD patients with epileptiform activity did not differ clinically from those without such activity. However, patients with subclinical epileptiform activity showed faster declines in global cognition, determined by the Mini-Mental State Examination (3.9 points/year in patients with epileptiform activity vs 1.6 points/year in patients without; p = 0.006), and in executive function (p = 0.01).InterpretationExtended monitoring detects subclinical epileptiform activity in a substantial proportion of patients with AD. Patients with this indicator of network hyperexcitability are at risk for accelerated cognitive decline and might benefit from antiepileptic therapies. These data call for more sensitive and comprehensive neurophysiological assessments in AD patient evaluations and impending clinical trials. Ann Neurol 2016;80:858-870.