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.
View: Text | PDF
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

×

Figure 6

Pathogenic SPTAN1 mutations produce phenotypes similar to Sptan1 deletion.

Options: View larger image (or click on image) Download as PowerPoint
Pathogenic SPTAN1 mutations produce phenotypes similar to Sptan1 deletio...
(A) All reported αII spectrin mutations reside at the C-terminal, a critical region required for spectrin heterodimer formation. (B) Neurons cotransfected with WT SPTAN1 and GFP show normal morphology compared with GFP alone except for decreased soma size. Neurons transfected with p.R2308_M2309dup or p.E2207del are smaller and have shortened or absent apical dendrites and abnormal basal dendrites. Arrowheads indicate well-formed apical dendrites in GFP and WT SPTAN1 groups, and arrows indicate the apical tufts in layer I cortex (seen best in the GFP group). Apical dendrites and tufts in the mutant groups are minimal. (C) Higher-magnification images reveal the severely altered neuronal morphology in the mutant overexpression groups. Arrows indicate the lack of apical dendrites, and arrowheads denote impaired basal dendritic trees. Scale bars: 100 μm in B, 50 μm in C. (D) Most neurons transfected with GFP (76.2%) or WT SPTAN1 (63.9%) display intact apical dendrites. Neurons transfected with mutant SPTAN1 constructs typically lack apical dendrites (20.3% in p.R2308_M2309dup; 19.0% in p.E2207del overexpression group). Percentage data are transformed to arcsine values for statistical comparisons. (E) The soma size of neurons transfected with WT or mutant SPTAN1 is significantly smaller than that of GFP controls, and both mutants show significantly reduced soma size compared with WT SPTAN1. For apical dendrite analysis, n = 32 neurons from 3 brains for GFP control; n = 20 neurons from 3 brains with WT SPTAN1; n = 43 neurons from 4 brains with SPTAN1 p.R2308_M2309dup; n = 37 neurons from 3 brains with SPTAN1 deletion were used. For soma size analysis, 25 neurons from 3 GFP brains, 53 neurons from 3 brains with WT SPTAN1, 47 neurons from 3 brains with SPTAN1 p.R2308_M2309dup, and 50 neurons from 4 brains with SPTAN1 p.E2207del were used. ANOVA test: *P < 0.05; **P < 0.01; ***P < 0.001.