Ultrastructural development and regeneration of nodes of ranvier

The National Eye Institute awarded the University of California, Berkeley $153,560 on February 18, 2027, under the Vision Research program (CFDA 93.867) to investigate the ultrastructural development and regeneration of nodes of Ranvier in the mouse optic nerve.

The project applies cryo-electron tomography (cryo-ET) with laser phase plate enhancement to visualize nodal protein complexes in situ and understand how ion channel scaffolding proteins establish and maintain rapid neural signaling. Aim 1 advances cryo-ET methodology by deploying the laser phase plate for high-resolution imaging of node proteins, establishing a benchmark for in situ resolution. Aim 2 characterizes how nodal molecular architecture develops during early postnatal growth of the mouse optic nerve, using laser phase plate cryo-ET and conventional cryo-ET with immunogold labeling to elucidate protein clustering initiation, spatial organization of ion channel types by their tethers, cytoskeleton formation, and node-myelin boundary maintenance. Aim 3 compares degenerative and regenerative processes following demyelinating optic nerve injury to understand how node organization is lost and restored. Understanding nodal protein organization has direct relevance to human developmental signaling disorders caused by mutations in node scaffolding proteins.

Work is performed at Berkeley, California. The period of performance extends from the award date through August 31, 2029.

Abstract

Rapid sensory processing in higher vertebrates is achieved through a form of neuronal signaling known as saltatory conduction. Saltatory conduction relies on specialized segments (nodes of ranvier) of densely clustered ION channels that are spaced apart by insulating myelin sheaths. Proper tethering of ION channels within the node is critical for proper neural communication, and mutations in node scaffolding proteins cause developmental signaling disorders in humans. This project will investigate the molecular architecture (ultrastructure) of the node of ranvier as IT develops in the mouse optic nerve and regenerates after demyelinating injury. To understand how nodal protein complexes are established and organized in space, node proteins will be visualized in situ with cryo-electron tomography (cryo-ET). Aim 1 seeks to advance methods in cryo-ET by applying the laser phase plate-a microscope component that increases the contrast of electron imaging and allows for the collection of high-resolution images-to in situ tissue imaging of the node. This technique is expected to resolve the in situ conformations and binding interactions of small node proteins and serve as a benchmark for resolution obtained by in situ cryo-ET. Aim 2 seeks to characterize how the molecular architecture of the node is established during early postnatal development of the mouse optic nerve. This aim will use a combination of LPP-cryo-ET and conventional cryo-ET with immunogold labeling to elucidate how protein clustering at the node is initiated, how ION channel types are spatially organized by their protein tethers, how the unique cytoskeleton of the node is formed, and how node-myelin boundaries are maintained. Aim 3 seeks to characterize how node organization is lost and regenerated after demyelinating injury of the optic nerve. This aim will specifically compare degenerative and regenerative timecourses of nodes in young and old mice to further knowledge of how regenerative potential in the visual system is lost with age. Long-term impact to human vision research: results from aim 2 will elucidate the basic biology of node formation in the vertebrate optic nerve, furthering our mechanistic understanding of how the eye communicates with the brain. Aim 3 will set the foundation for studying ultrastructural and age-dependent node regeneration during demyelinating diseases that effect vision loss, including multiple sclerosis and neuromyelitis optica. The development of LPP-cryo-ET in aim 1 will be broadly useful for the study of tissue ultrastructures, including the investigation of altered protein organizations arising from diseases and genetic disorders.