023 + CO-1-Microcine | Staurosporine treatment in hiPSC-derived motor neurons produce gaps in the spectrin lattice of axons

Cellular and Molecular Neurobiology

Author: Nahir Guadalupe Gazal | Email: nggazal@immf.uncor.edu

Nahir Gazal , María Castellanos-Montiel , Guillermina Bruno , Lale Gursu , Ghazal Haghi , Gilles Maussion , Thomas Durcan , Nicolás Unsain

1° Instituto de Investigación Médica Mercedes y Martín Ferreyra (INIMEC), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Universidad Nacional de Córdoba, Córdoba, Argentina
2° Early Drug Discovery Unit, Montreal Neurologial Institute-Hospital, Canadá

The membrane-associated periodic skeleton (MPS) is a protein structure of actin “rings” located transversely to the axon and separated every 190 nm by “spacers” of ?/?-spectrin tetramers. In mature neurons, the MPS is organized along almost the entire axonal shaft that correlates with ?II-spectrin’s homogenous distribution. During the maturation of human induced Pluripotent Stem Cells (hiPSCs) in culture, we observed an intriguing interruption in the otherwise uniform distribution of ?II-spectrin along axons, referred to as ?II-spectrin gaps (?IIs-gaps). These appear as stretches devoid of ?II-spectrin. To determine if ?IIs-gaps are associated with axonal constriction or loss, phase contrast and co-immunofluorescence analysis against various cytoskeletal and membrane proteins demonstrated that the lack of ?II-spectrin is specific and that the axon shows no changes in those areas. Consequently, we subjected 2-week-old cultures to acute stress using sub-lethal doses of staurosporine, arsenite and L-glutamate. Remarkably, a significant increase in the occurrence of axons with ?IIs-gaps under staurosporine treatment was observed. STED microscopy of ?II-spectrin showed that the MPS is unaffected outside of the ?IIs-gaps. Staurosporine was also found to induce ?IIs-gaps in dorsal root ganglion neurons, but not in hippocampal neurons, derived from mouse embryos. We believe the study of ?IIs-gaps will provide valuable insights into the formation and dynamics of the MPS in axons.