αII Spectrin Forms a Periodic Cytoskeleton at the Axon Initial Segment and Is Required for Nervous System Function

Abstract

Spectrins form a submembranous cytoskeleton proposed to confer strength and flexibility to neurons and to participate in ion channel clustering at axon initial segments (AIS) and nodes of Ranvier. Neuronal spectrin cytoskeletons consist of diverse β subunits and αII spectrin. Although αII spectrin is found in neurons in both axonal and somatodendritic domains, using proteomics, biochemistry, and superresolution microscopy, we show that αII and βIV spectrin interact and form a periodic AIS cytoskeleton. To determine the role of spectrins in the nervous system, we generated Sptan1^f/f mice for deletion of CNS αII spectrin. We analyzed αII spectrin-deficient mice of both sexes and found that loss of αII spectrin causes profound reductions in all β spectrins. αII spectrin-deficient mice die before 1 month of age and have disrupted AIS and many other neurological impairments including seizures, disrupted cortical lamination, and widespread neurodegeneration. These results demonstrate the importance of the spectrin cytoskeleton both at the AIS and throughout the nervous system.SIGNIFICANCE STATEMENT Spectrin cytoskeletons play diverse roles in neurons, including assembly of excitable domains such as the axon initial segment (AIS) and nodes of Ranvier. However, the molecular composition and structure of these cytoskeletons remain poorly understood. Here, we show that αII spectrin partners with βIV spectrin to form a periodic cytoskeleton at the AIS. Using a new αII spectrin conditional knock-out mouse, we show that αII spectrin is required for AIS assembly, neuronal excitability, cortical lamination, and to protect against neurodegeneration. These results demonstrate the broad importance of spectrin cytoskeletons for nervous system function and development and have important implications for nervous system injuries and diseases because disruption of the spectrin cytoskeleton is a common molecular pathology.

Keywords: ankyrin; axon; axon initial segment; cytoskeleton; node of Ranvier; spectrin.

Figure 1.

αII spectrin is found at the AIS. a, Immunoblot of neurofascin (Nfasc) purified from rat brain using an anti-Nfasc affinity column. Fractions 5–9 are shown. b, Proteins purified from the Nfasc affinity column identified by mass spectrometry. c, Immunostaining of 12 DIV rat hippocampal neurons using antibodies against αII spectrin (red), βIV spectrin (green), and MAP2 (blue). d, Detergent-extracted 12 DIV rat hippocampal neurons immunostained using antibodies against αII spectrin (red), βIV spectrin (green), and MAP2 (blue). Scale bar, 20 μm.

Figure 2.

αII spectrin forms a periodic cytoskeleton with βIV spectrin at AIS. a–d, Conventional fluorescence (a) and STORM (b, c) imaging of 12 DIV cultured hippocampal neurons labeled with antibodies against αII spectrin. The box in a corresponds to the STORM image shown in b. The box in b corresponds to the STORM image shown in c. The region between the lines in c was used to generate an αII spectrin intensity profile d. Scale bars: a, 10 μm; b, 2 μm; c, 1 μm. e–h, Conventional fluorescence (e) and STORM (f, g) imaging of detergent-extracted 12 DIV cultured hippocampal neurons labeled with antibodies against αII spectrin. The box in e corresponds to the STORM image shown in f. The box in f corresponds to the STORM image shown in g. The region between the lines in g was used to generate an αII spectrin intensity profile (h). Scale bars: e, 10 μm; f, 2 μm; g, 1 μm. i–l, Conventional fluorescence (i) and STORM (j, k) imaging of detergent-extracted 12 DIV cultured hippocampal neurons labeled with antibodies against βIV spectrin. The box in i corresponds to the STORM image shown in j. The box in j corresponds to the STORM image shown in k. The region between the lines in k was used to generate a βIV spectrin intensity profile (l). Scale bars: i, 10 μm; j, 2 μm; k, 1 μm. m–p, Conventional fluorescence (m) obtained by projecting frames from the PAINT acquisition sequence and DNA-PAINT (n, o) imaging of 12 DIV cultured hippocampal neurons labeled with antibodies against αII spectrin and βIV spectrin. The box in m corresponds to the PAINT image shown in n. The box in n corresponds to the PAINT image shown in o. The region between the lines in o was used to generate αII spectrin (green) and βIV spectrin (magenta) intensity profiles (p). Scale bars: m, 10 μm; n, 2 μm; o, 1 μm.

Figure 3.

αII spectrin and βIV spectrin splice variants can interact through SR1-2 and SR14-15. a, Diagram of βIV spectrin splice variants and their domains. ABD, actin-binding domain; SD, specific domain; PH, pleckstrin homology domain. b, Cotransfection of COS-7 cells with GFP-tagged αII spectrin and myc-tagged βIVΣ1 or βIVΣ6. Immunoprecipitation of αII spectrin-GFP coimmunoprecipitates both myc-βIVΣ1 and myc-βIVΣ6. Immunoblots were performed using antibodies against myc and GFP. Molecular weights are shown in kDa. c, Transfection of GFP-tagged αII spectrin in COS-7 cells. Immunoprecipitation of αII spectrin-GFP coimmunoprecipitates endogenous βII spectrin. d, Cotransfection of COS-7 cells with GFP-tagged αII spectrin and myc-tagged SR1, SR1-2, or SR1-3. Immunoprecipitation using anti-GFP antibodies coimmunoprecipitates myc-SR1-2 and myc-SR1-3. Molecular weights are shown in kilodaltons (kDa). Coimmunoprecipitation between αII spectrin-GFP and myc-βIVΣ1 is included as a positive control. e, Cotransfection of COS-7 cells with αII spectrin-GFP and myc-tagged SR10-11, SR10-13 or SR14-15. αII spectrin-GFP coimmunoprecipitates myc-SR14-15. Molecular weights are shown in kilodaltons (kDa). Coimmunoprecipitation between AnkG-GFP and myc-SR14-15 is included as a positive control.

Figure 4.

Mice lacking nervous system αII spectrin. a, αII spectrin immunostaining in cerebella of P12 Sptan1^f/f and Nestin-cre;Sptan1^f/f mice. b, Immunoblots of brain membrane homogenates from P7 Sptan1^f/f and Nestin-cre;Sptan1^f/f mice using antibodies against the indicated spectrins. Blood was used as the positive control for αI spectrin. Neurofilament-M (NF-M) served as the loading control. Protein levels were calculated as the percentage of Nestin-cre;Sptan1^f/f mice compared with Sptan1^f/f mice. n = 3 mice per genotype. Mean ± SEM αII: p = 0.0009; βI: p = 0.0234; βII: p = 0.0042; βIII: p = 0.032; βIV-Σ1: p = 0.0112; βIV-Σ6: p = 0.0174. αII: t₍₄₎ = 21.47; βI: t₍₄₎ = 3.57; βII: t₍₄₎ = 5.89; βIII: t₍₄₎ = 3.228; βIV-Σ1: t₍₄₎ = 4.46; βIV-Σ6: t₍₄₎ = 3.912. c, Survival curve for Sptan1^f/f (n = 26) and Nestin-cre;Sptan1^f/f mice (n = 18). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5.

Loss of αII spectrin disrupts the AIS cytoskeleton. a, AIS immunostained for βIV spectrin in P18 Sptan1^f/f and Nestin-cre;Sptan1^f/f cerebral cortex. Scale bar, 20 μm. b–e, Conventional fluorescence (b) and STORM (c, d) imaging of GFP⁻ 12 DIV cultured hippocampal neurons labeled with antibodies against GFP (blue), ankG (red), and βIV spectrin (green). The box in b corresponds to the STORM image shown in c. The box in c corresponds to the STORM image shown in d. The region between the lines in d was used to generate a βIV spectrin intensity profile (e). Scale bars: b, 10 μm; c, 2 μm; d, 1 μm. f–i, Conventional fluorescence (f) and STORM (g, h) imaging of GFP⁺ 12 DIV cultured hippocampal neurons labeled with antibodies against GFP (blue), ankG (red), and βIV spectrin (green). The box in f corresponds to the STORM image shown in g. The box in g corresponds to the STORM image shown in h. The region between the lines in h was used to generate a βIV spectrin intensity profile (i). Scale bars: f, 10 μm; g, 2 μm; h, 1 μm. j–m, Conventional fluorescence (j) and STORM (k, l) imaging of GFP⁻ 12 DIV cultured hippocampal neurons labeled with antibodies against GFP (blue), βII spectrin (red), and βIV spectrin (green). The box in j corresponds to the STORM image shown in k. The box in k corresponds to the STORM image shown in l. The region between the lines in l was used to generate a βII spectrin intensity profile (m). Scale bars: j, 10 μm; k, 2 μm; l, 1 μm. n–q, Conventional fluorescence (n) and STORM (o, p) imaging of GFP⁺ 12 DIV cultured hippocampal neurons labeled with antibodies against GFP (blue), βII spectrin (red), and βIV spectrin (green). The box in n corresponds to the STORM image shown in o. The box in o corresponds to the STORM image shown in p. The region between the lines in p was used to generate a βII spectrin intensity profile (q). Scale bars: n, 10 μm; o, 2 μm; p, 1 μm.

Figure 6.

αII spectrin-deficient mice have seizures. a, Video EEG monitoring of awake and behaving P12–P14 mice revealed generalized seizure discharges in Nestin-cre;Sptan1^f/f mice that were not detected in Sptan1^f/f littermates. b, Quantification of spikes/h recorded in Sptan1^f/f and Nestin-cre;Sptan1^f/f mice. *p = 0.0159.

Figure 7.

αII spectrin-deficient mice have disrupted cortical lamination and cerebellar organization. a, b, Immunostaining of cerebral cortex using antibodies against Reelin (layer I), Cux1 (layers II–IV), Ctip2 (layer V), FoxP2 (layer VI), and ctgf (subplate) at P7 (a) and P12 (b). Scale bar, 50 μm. c, d, Quantification of percentage of labeled cells (Reelin, red; Cux1, blue; Ctip2, green; Foxp2, magenta; ctgf, cyan) in their respective cortical layers (I, II-IV, V, VI, VII) in P7 (c) or P12 (d) control (close column) or αII spectrin-deficient (open column) cerebral cortex. e, f, GFP immunoreactivity in P7 rats after in utero electroporation using GFP alone (e) or GFP and αII spectrin shRNA (f) plasmids. Scale bar, 50 μm. g, Sholl analysis of dendritic complexity in control or αII spectrin-deficient hippocampal neurons in vitro (n = 3). Error bars indicate ± SEM. h, Immunostaining of P18 Sptan1^f/f and Nestin-cre;Sptan1^f/f cerebella using antibodies against Kv1.2 (green), βIV spectrin (red), and calbindin (blue). Scale bar, 50 μm. i, Orientation and length of Purkinje neuron AIS in Sptan1^f/f and Nestin-cre;Sptan1^f/f mice.

Figure 8.

Nestin-cre;Sptan1^f/f mice have widespread axon degeneration. a–c, Immunostaining of cerebellum (a), thalamus (b), and corpus callosum (c) in Sptan1^f/f and Nestin-cre;Sptan1^f/f mice using antibodies against β-APP (green) and NeuN (red). Scale bar, 50 μm.