Usenovic M1, Niroomand S1, Drolet RE1, Yao L1, Gaspar RC1, Hatcher NG1, Schachter J1, Renger JJ1, and Parmentier-Batteur S1
- Neuroscience, Merck Research Laboratories, West Point, Pennsylvania 19486
Neuronal inclusions of hyperphosphorylated and aggregated tau protein are a pathological hallmark of several neurodegenerative tauopathies, including Alzheimer’s disease (AD). The hypothesis of tau transmission in AD has emerged from histopathological studies of the spatial and temporal progression of tau pathology in postmortem patient brains. Increasing evidence in cellular and animal models supports the phenomenon of intercellular spreading of tau. However, the molecular and cellular mechanisms of pathogenic tau transmission remain unknown. The studies described herein investigate tau pathology propagation using human neurons derived from induced pluripotent stem cells. Neurons were seeded with full-length human tau monomers and oligomers and chronic effects on neuronal viability and function were examined over time. Tau oligomer-treated neurons exhibited an increase in aggregated and phosphorylated pathological tau. These effects were associated with neurite retraction, loss of synapses, aberrant calcium homeostasis, and imbalanced neurotransmitter release. In contrast, tau monomer treatment did not produce any measureable changes. This work supports the hypothesis that tau oligomers are toxic species that can drive the spread of tau pathology and neurodegeneration.
CovalX Technology Used (Click each option to learn more)
Tau oligomer and monomer sample sets were analysed by MALDI-TOF mass spectrometry. These sample sets contained 5 μM each of tau oligomer preparations, tau monomer preparations and heparin. Prior to analysis, a single sample set was crosslinked using the CovalX K200 Stabilization Kit. The Tau and heparin solutions were incubated for 3 hours at 4 °C with the K200 and then all samples (untreated and treated) underwent methanol-chloroform liquid-liquid extraction. The tau and heparin precipitates were centrifuged at 10,000 x g for 5 minutes before the supernatants were removed and placed in a vacuum to dry. The dried sample pellets were reconstituted using 5 μl of formic acid and then diluted in a 1:3 saturated solution of sinapic acid that was used as the matrix (10 mg/ml mixed in a 50% solution of acetonitrile and 0.1% trifluoroacetic acid). 1 μl of each sample/matrix solutions were spotted onto a MALDI target and crystallized at room temperature. Analysis of the samples was performed using a mass spectrometer that had been modified with a CovalX HM2 detection system. The data was then analyzed using the CovalX Complex Tracker software. Using the data gathered from performing MALDI-TOF MS analysis, the researchers were able to observe the presence of tau monomers, dimers, trimers and tetramers within the crosslinked tau oligomers.