(J-382) An Engineered Human Brain Microphysiological Model to Study Propagation of Tau Pathology

Introduction:
Many neurodegenerative conditions, including Alzheimer’s Disease (AD) and Frontotemporal Dementia (FTD), are characterized by dense neurofibrillary tangles, which are formed by pathological aggregates of the microtubule-associated protein tau. Each disease is believed to be caused by a different conformation of neurofibrillary tangle that spreads among neurons and contributes to cell death. However, after decades of research, basic questions about the underlying mechanisms of these so-called tauopathies remain unanswered. Most studies of tauopathies have been performed in mice, which are limited in only providing information about endpoint pathology, have limited relevance to human physiology, and are inherently a low throughput model system. Hence, an in vitro model in a human genetic background would allow for broad exploration and characterization of tauopathies and a better understanding of disease progression. To address this challenge, our lab has developed a triple-chamber microfluidic device that supports 3D culture of induced pluripotent stem cell-derived neurons with flanking perfusion chambers. Using a peptide-functionalized gelatin hydrogel previously developed by our lab, we can generate synaptically connected networks of neurons. Then, the device is used to inoculate the central cell culture zone with tau seeds on one side through one of the perfusion channels, and in combination with a genetically encoded FRET biosensor that reports tau aggregation in neurons, we can monitor spread of tau pathology in live cells using recombinant or patient-derived tau seeds. This model provides a novel strategy to study human tauopathies.

Materials and Methods: Microfluidic devices were fabricated by generating a 3D-printed mold coated in parylene to prevent cytotoxicity for production of PDMS devices plasma-bonded to glass slides. GelMA-Cad hydrogel was synthesized by conjugating methacrylate and neural-cadherin adhesion peptide to the backbone of gelatin. Neurons were resuspended in 10% GelMA-Cad with 0.05% LAP, embedded into central chamber of the device and UV-crosslinked. Patient-derived lysates containing tau were isolated by sonicating frontal lobe tissue suspended in 10% TBS with protease inhibitor cocktail and collecting supernatant. Fluorescence microscopy was used to validate tau diffusion into device and propagation of human brain-derived tau seeds with FRET biosensor cells.

Results, Conclusions, and Discussions: We have successfully designed and fabricated a multi-chamber device for inoculating synaptically connected neurons with patient tau seeds. We have previously demonstrated the presence of phosphorylated tau (pTau), a marker of tau aggregation and precursor to tau pathology, in neurons on the opposite side of the device and observed high pTau accumulation in central bodies of neurons, demonstrating that aggregated tau was propagated through synaptically connected neurons in the hydrogel. We have found that dosing biosensor cells with human AD and FTD brain lysates increases the number of aggregates detected compared to non-diseased human control lysates. These preliminary results demonstrate that we can build a model system that characterizes tau propagation across different disease types.

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