Staurosporine treatment in hiPSC-derived motor neurons produce gaps in the spectrin lattice of axons

NG GAZAL; M CASTELLANOS-MONTIEL; G BRUNO; L GURSU; G HAGHI; G MAUSSION; T DURCAN; N UNSAIN

San Luis

Congreso; XXXVIII Congreso Reunión Anual - Sociedad Argentina de Investigación en Neurociencia. San Luis, Argentina; 2023

Sociedad Argentina de Investigación en Neurociencia

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 α/β-spectrin tetramers. In mature neurons, the MPS is organized along almost the entire axonal shaft that correlates with βII-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 βII-spectrin along axons, referred to as βII-spectrin gaps (βIIs-gaps). These appear as stretches devoid of βII-spectrin. To determine if βIIs-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 βII-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 βIIs-gaps under staurosporine treatment was observed. STED microscopy of βII-spectrin showed that the MPS is unaffected outside of the βIIs-gaps. Staurosporine was also found to induce βIIs-gaps in dorsal root ganglion neurons, but not in hippocampal neurons, derived from mouse embryos. We believe the study of βIIs-gaps will provide valuable insights into the formation and dynamics of the MPS in axons.