Engineering a Human Glial-Neuronal Microphysiological System to Study Myelination and Axonal-Synaptic Pathology in Alzheimer's Disease

Axonopathy, defined as morphological and functional axonal dysfunction, has emerged as a key factor in the pathophysiology of Alzheimer’s disease (AD). Axonal transport (AT) is required to sustain neuronal integrity, with AT dysfunction potentially leading to axonal and synaptic degeneration. Abnormal axons consistent with impaired AT are found in AD brains, and elevated levels of axonal damage biomarkers have been detected in preclinical AD. Moreover, AT deficits have been linked to aberrant amyloid beta (Aβ) production, indicating that axonopathy may represent an early pathogenic event in AD. However, it remains unclear whether axonopathy is a primary cause, facilitator, or consequence of AD. In the central nervous system, oligodendrocytes enable rapid conduction and provide metabolic and structural support through axonal myelination. Aging is associated with myelin degradation, with demyelination contributing to neurological disabilities in AD and other neurodegenerative conditions. Despite the recognition of axonopathy as an early hallmark of AD, most existing in vitro models exclude oligodendrocytes and fail to replicate the structure and interactions needed for myelination. To overcome these challenges, the proposed work aims to establish a human tri-culture microphysiological system that integrates multiple brain cell types and promotes the maturation of axonal and synaptic connectivity, enabling mechanistic studies of brain aging diseases, particularly AD, with unprecedented spatiotemporal resolution. In Aim 1, this project will develop and validate a tri-culture platform that enables axonal maturation, myelination, and synaptic connectivity under physiological conditions. Here, neurons, astrocytes, and oligodendrocytes will be co-cultured on a microfluidic platform designed to promote directional axonal growth, myelin formation, and presynaptic, synaptic, and postsynaptic compartmentalization. The axonal structure, transport, nodal architecture, synaptic structure, and electrophysiological status will be assessed via live imaging, confocal microscopy, electron microscopy, and microelectrode-array recordings. In Aim 2, this project will determine the roles of oligodendrocytes and myelination in AD-like axonopathy and synaptic phenotypes. The tri-culture system developed in Aim 1 will be exposed to exogenous Aβ to induce an AD-like axonopathy, revealing the impact of myelin and glial cells on axonopathy based on axonal swelling, intracellular calcium levels, and the release of axonal damage biomarkers. Through these efforts, the proposed work will establish a highly manipulable, reductionist microfluidic platform that enables the dissection of temporality and causality in axonal and synaptic dysfunction within a glia-relevant context. In addition to filling a major technical gap in the field, this model will provide a foundation for future mechanistic studies of white matter vulnerability and circuit dysfunction and will facilitate the identification of early biomarkers and potential drug targets, informing intervention strategies for preclinical stages of AD as well as a broad range of neurodegenerative and aging disorders. Project Number: 1R21AG101690-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: Valentina Fossati (+1 co-PI) | Institution: JACKSON LABORATORY, BAR HARBOR, ME | Award Amount: $486,750 | Activity Code: R21 | Study Section: Special Emphasis Panel[ZRG1 AN-G (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11354973