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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

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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 3

Altered neuronal morphology after Sptan1 deletion.

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Altered neuronal morphology after Sptan1 deletion.
(A) Confocal images o...
(A) Confocal images of P21 brain sections after control CRISPR transfection at E14–15 show normal-appearing GFP-labeled cells in cortical layer II/III (left). A higher-magnification image of the boxed region (right) reveals typical pyramidal neuron morphology with a single apical dendrite and multiple basal dendrites. (B) Neurons transfected with Sptan1 CRISPR A reach the appropriate cortical layers but appear disorganized, lack apical dendrites, and have abnormal basal dendritic architecture that typically consists of a single short, thickened but exuberant process. (C) Reconstructed neurons transfected with control CRISPR or Sptan1 CRISPR A or B. Note that neurons transfected in vivo with Sptan1 CRISPR A or B are morphologically similar, indicating the specificity of the gene editing. (D and E) Although Sptan1 deletion severely alters neuronal morphology, the GFP+ cells coexpress the neuronal marker NeuN (red in D) and the superficial cortical layer marker Cux1 (red in E), suggesting preserved neuronal differentiation. Scale bars: 25 μm in B (for A and B), 10 μm in E (for D and E). (F) Quantification of percentages of GFP-labeled cells with apical dendrites and numbers of basal dendrites per GFP+ cell in neurons transfected with control plasmid or Sptan1 CRISPR A. A total of 39 neurons from 3 brains with Sptan1 CRISPR A transfection and 43 neurons from 3 brains from the CRISPR control group were quantified. Percentage data are transformed to arcsine value for statistical analysis. One-tailed t test: ***P < 0.001.
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