Critical roles of αII spectrin in brain development and epileptic encephalopathy
Yu Wang, … , Paul M. Jenkins, Jack M. Parent
Yu Wang, … , Paul M. Jenkins, Jack M. Parent
Published January 16, 2018
Citation Information: J Clin Invest. 2018;128(2):760-773. https://doi.org/10.1172/JCI95743.
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Research Article Development Neuroscience

Critical roles of αII spectrin in brain development and epileptic encephalopathy

Abstract

The nonerythrocytic α-spectrin-1 (SPTAN1) gene encodes the cytoskeletal protein αII spectrin. Mutations in SPTAN1 cause early infantile epileptic encephalopathy type 5 (EIEE5); however, the role of αII spectrin in neurodevelopment and EIEE5 pathogenesis is unknown. Prior work suggests that αII spectrin is absent in the axon initial segment (AIS) and contributes to a diffusion barrier in the distal axon. Here, we have shown that αII spectrin is expressed ubiquitously in rodent and human somatodendritic and axonal domains. CRISPR-mediated deletion of Sptan1 in embryonic rat forebrain by in utero electroporation caused altered dendritic and axonal development, loss of the AIS, and decreased inhibitory innervation. Overexpression of human EIEE5 mutant SPTAN1 in embryonic rat forebrain and mouse hippocampal neurons led to similar developmental defects that were also observed in EIEE5 patient-derived neurons. Additionally, patient-derived neurons displayed aggregation of spectrin complexes. Taken together, these findings implicate αII spectrin in critical aspects of dendritic and axonal development and synaptogenesis, and support a dominant-negative mechanism of SPTAN1 mutations in EIEE5.

Authors

Yu Wang, Tuo Ji, Andrew D. Nelson, Katarzyna Glanowska, Geoffrey G. Murphy, Paul M. Jenkins, Jack M. Parent

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Figure 7

Patient iPSC-derived neurons show aberrant neurite development and spectrin aggregation.

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Patient iPSC-derived neurons show aberrant neurite development and spect...
(A) Patient and control iPSCs express the pluripotency markers SOX2, SSEA4, and OCT3/4. After differentiation into neural progenitors, both control and EIEE5 patient cells form similarly appearing neural rosettes that express Musashi and Pax6. (B) Cells were transduced with GFP-expressing lentivirus (CBh-GFP, green), and neurons were identified by Tuj1 immunoreactivity (red). Total process length per cell was measured in GFP+ cells and was significantly shorter in patient neurons. Two-tailed t test: *P < 0.05. n = 44 neurons per group from 4 biological replicates per group. (C) Confocal images showing coexpression of the neuronal marker MAP2 (green), αII spectrin (red), and βIII spectrin (magenta) in control (top row) and patient (bottom row) iPSC-derived neurons. Note the overlap of GFP and spectrins in control processes (arrows in top panels), but aggregation of spectrins in patient cells, with lack of spectrin immunoreactivity in GFP+ processes (arrows in bottom panels). Two different iPSC lines derived from patient and control fibroblasts were used. Scale bars: 20 μm.