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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 5

Loss of αII spectrin alters inhibitory innervation.

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Loss of αII spectrin alters inhibitory innervation.
(A) Confocal image o...
(A) Confocal image of a neuron in P21 rat cortex that displays typical pyramidal cell morphology after co-electroporation of gephyrin-FingR-GFP (green) and RFP (red) by IUE at E13–14. Arrows indicate the AIS where GFP+ puncta (green) and ankyrin-G (purple) cluster and colocalize; arrowheads indicate punctate GFP labeling along the dendrites. The star indicates excess nuclear gephyrin-FingR-GFP, caused by saturation of endogenous gephyrin binding that leads to a feedback mechanism within the FingR system. Bisbenzimide nuclear stain in blue. (B) Neurons cotransfected with control CRISPR, RFP, and gephyrin-FingR-GFP show abundant GFP+ puncta along axonal and somatodendritic domains. (C) Sptan1 CRISPR/Cas9–transfected neurons, in contrast, show dramatically decreased GFP+ puncta. Insets in middle panels of B and C are higher-magnification images. Scale bars: 10 μm in A, 10 μm in C (for B and C), 5 μm in C inset for insets in B and C. (D) Quantification of GFP+ puncta density on proximal somatodendritic domains and axons (AIS excluded) shows a 45.8% reduction in Sptan1-deleted neurons. One-tailed t test. (E) Frequency (left) and amplitude (right) of miniature inhibitory postsynaptic currents (mIPSCs) are altered in the Sptan1-deleted neurons. Two-tailed t test. (F) Examples of voltage-clamp mIPSC recordings from control (upper trace) and Sptan1-deleted (lower trace) neurons. (G) mIPSC rise times for GFP+ cells from control and Sptan1 CRISPR–transfected cells. The mean rise time is significantly shorter for Sptan1 CRISPR cells. Two-tailed t test performed in G. One-tailed t test: *P < 0.05; ***P < 0.001. In FingR-gephyrin experiments, n = 21 neurons (3 brains) with Sptan1 CRISPR A transfection and n = 19 neurons (3 brains) with control CRISPR transfection were analyzed. In patch-clamp experiments, n = 19 Sptan1-deleted and n = 16 control neurons (from ≥3 brains and 3 neurons per brain per condition) were analyzed.

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ISSN: 0021-9738 (print), 1558-8238 (online)

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