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 February 1, 2018; First 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 4

Sptan1 deletion disrupts AIS formation and neuronal polarity in vivo.

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Sptan1 deletion disrupts AIS formation and neuronal polarity in vivo. (...
(A) Confocal images from a P21 control after prior transfection at E14–15 showing GFP+ cortical neurons (green) with typical pyramidal morphology and ankyrin-G immunolabeling (red) of their proximal axon (arrows). (B) In contrast, Sptan1 CRISPR–transfected cells lack ankyrin-G staining. (C and D) Adenylyl cyclase III (ACIII, red), a marker for primary neuronal cilia, is seen in the typical apical location of a control GFP-labeled neuron (C, arrowhead) but is mislocalized (D, arrowheads) to the basal pole in GFP+ neurons after αII spectrin deletion, indicating disrupted neuronal polarity. Blue is bisbenzimide (BB) nuclear stain. Scale bars: 10 μm in B (for A and B), 10 μm in D (for C and D). (E) Quantification of the percentages of cells with ACIII staining in the upper quadrants of the soma in control (92.9%) and Sptan1 CRISPR groups (22.0%). n = 42 neurons from 4 brains with Sptan1 CRIPSR A transfection, n = 39 from 3 brains with control CRISPR transfection. The percentage data are transformed to arcsine value for statistical analysis with a 1-tailed t test; **P < 0.01.