Wu, H.-F. | bioRxiv Science

bioRxiv2025

A 3D Human Neuron-on-Chip Platform to Monitor Neuronal Injury Responses

Traumatic brain injury (TBI) is a major cause of neurological dysfunction and long-term neurodegeneration, yet the intrinsic neuronal contributions to TBI pathophysiology remain incompletely defined. Here, we present a novel Neuron-on-Chip microfluidic platform that can be used to mechanically injure mature human prefrontal cortex neurons (hPFCs) embedded in three-dimensional (3D) hydrogels, enabling the study of injury responses in pure neuronal cultures. We assessed real-time calcium dynamics across 13 metrics of single-cell and network activity, revealing a biphasic injury response: an early phase (0.5-24 hr) characterized by excitotoxicity, hyper-synchronized bursting, and network collapse; and a late phase (8 d) marked by sustained depolarization and structural remodeling. Secretome profiling uncovered progressive elevations in extracellular pT181 and total Tau from days 1 to 5 post-injury. Cytokine analyses identified early (24 hr) elevations in IP-10, IL-10, IFN2, and NCAM, and late increases (8 d) in CXCL9 and MPO, linking neuronal activity changes to stage-specific inflammatory signaling. Immunocytochemistry and immunoblotting confirmed temporally ordered upregulation of calpain-1 and active caspase-3 (days 1-3), phosphorylated Tau (AT8+, days 5-8), and neurofibrillary tangle-like Tau aggregates (NFT+, day 8). These findings establish our platform as a scalable microphysiological model for probing the dynamic cellular and molecular sequelae of neuronal response to injury, offering insights into neurodegeneration and opportunities for therapeutic discovery.

neuroscience↗

bioRxiv2023

Genipin Crosslinks the Extracellular Matrix to Rescue Developmental and Degenerative Defects, and Accelerates Regeneration of Peripheral Neurons

The peripheral nervous system (PNS) is essential for proper body function. A high percentage of the population suffer nerve degeneration or peripheral damage. For example, over 40% of patients with diabetes or undergoing chemotherapy develop peripheral neuropathies. Despite this, there are major gaps in the knowledge of human PNS development and therefore, there are no available treatments. Familial Dysautonomia (FD) is a devastating disorder that specifically affects the PNS making it an ideal model to study PNS dysfunction. FD is caused by a homozygous point mutation in ELP1 leading to developmental and degenerative defects in the sensory and autonomic lineages. We previously employed human pluripotent stem cells (hPSCs) to show that peripheral sensory neurons (SNs) are not generated efficiently and degenerate over time in FD. Here, we conducted a chemical screen to identify compounds able to rescue this SN differentiation inefficiency. We identified that genipin, a compound prescribed in Traditional Chinese Medicine for neurodegenerative disorders, restores neural crest and SN development in FD, both in the hPSC model and in a FD mouse model. Additionally, genipin prevented FD neuronal degeneration, suggesting that it could be offered to patients suffering from PNS neurodegenerative disorders. We found that genipin crosslinks the extracellular matrix, increases the stiffness of the ECM, reorganizes the actin cytoskeleton, and promotes transcription of YAP-dependent genes. Finally, we show that genipin enhances axon regeneration in an in vitro axotomy model in healthy sensory and sympathetic neurons (part of the PNS) and in prefrontal cortical neurons (part of the central nervous system, CNS). Our results suggest genipin can be used as a promising drug candidate for treatment of neurodevelopmental and neurodegenerative diseases, and as a enhancer of neuronal regeneration. One sentence summaryGenipin rescues the developmental and degenerative phenotypes of the peripheral neuropathy familial dysautonomia and enhances neuron regeneration after injury.

neuroscience↗