Defining new mechanistic roles for αII spectrin in cardiac function.

Spectrins are cytoskeletal proteins essential for membrane biogenesis and regulation and serve critical roles in protein targeting and cellular signaling. αII spectrin (SPTAN1) is one of two α spectrin genes and αII spectrin dysfunction is linked to alterations in axon initial segment formation, cortical lamination, and neuronal excitability. Furthermore, human αII spectrin loss-of-function variants cause neurological disease. As global αII spectrin knockout mice are embryonic lethal, the in vivo roles of αII spectrin in adult heart are unknown and untested. Here, based on pronounced alterations in αII spectrin regulation in human heart failure we tested the in vivo roles of αII spectrin in the vertebrate heart. We created a mouse model of cardiomyocyte-selective αII spectrin-deficiency (cKO) and used this model to define the roles of αII spectrin in cardiac function. αII spectrin cKO mice displayed significant structural, cellular, and electrical phenotypes that resulted in accelerated structural remodeling, fibrosis, arrhythmia, and mortality in response to stress. At the molecular level, we demonstrate that αII spectrin plays a nodal role for global cardiac spectrin regulation, as αII spectrin cKO hearts exhibited remodeling of αI spectrin and altered ß-spectrin expression and localization. At the cellular level, αII spectrin deficiency resulted in altered expression, targeting, and regulation of cardiac ion channels NaV1.5 and KV4.3. In summary, our findings define critical and unexpected roles for the multifunctional αII spectrin protein in the heart. Furthermore, our work provides a new in vivo animal model to study the roles of αII spectrin in the cardiomyocyte.

Glial ßII Spectrin Contributes to Paranode Formation and Maintenance.

Action potential conduction along myelinated axons depends on high densities of voltage-gated Na+ channels at the nodes of Ranvier. Flanking each node, paranodal junctions (paranodes) are formed between axons and Schwann cells in the peripheral nervous system (PNS) or oligodendrocytes in the CNS. Paranodal junctions contribute to both node assembly and maintenance. Despite their importance, the molecular mechanisms responsible for paranode assembly and maintenance remain poorly understood. ßII spectrin is expressed in diverse cells and is an essential part of the submembranous cytoskeleton. Here, we show that Schwann cell ßII spectrin is highly enriched at paranodes. To elucidate the roles of glial ßII spectrin, we generated mutant mice lacking ßII spectrin in myelinating glial cells by crossing mice with a floxed allele of Sptbn1 with Cnp-Cre mice, and analyzed both male and female mice. Juvenile (4 weeks) and middle-aged (60 weeks) mutant mice showed reduced grip strength and sciatic nerve conduction slowing, whereas no phenotype was observed between 8 and 24 weeks of age. Consistent with these findings, immunofluorescence microscopy revealed disorganized paranodes in the PNS and CNS of both postnatal day 13 and middle-aged mutant mice, but not in young adult mutant mice. Electron microscopy confirmed partial loss of transverse bands at the paranodal axoglial junction in the middle-aged mutant mice in both the PNS and CNS. These findings demonstrate that a spectrin-based cytoskeleton in myelinating glia contributes to formation and maintenance of paranodal junctions.SIGNIFICANCE STATEMENT Myelinating glia form paranodal axoglial junctions that flank both sides of the nodes of Ranvier. These junctions contribute to node formation and maintenance and are essential for proper nervous system function. We found that a submembranous spectrin cytoskeleton is highly enriched at paranodes in Schwann cells. Ablation of ßII spectrin in myelinating glial cells disrupted the paranodal cell adhesion complex in both peripheral and CNSs, resulting in muscle weakness and sciatic nerve conduction slowing in juvenile and middle-aged mice. Our data show that a spectrin-based submembranous cytoskeleton in myelinating glia plays important roles in paranode formation and maintenance.

Axon initial segments: structure, function, and disease.

The axon initial segment (AIS) is located at the proximal axon and is the site of action potential initiation. This reflects the high density of ion channels found at the AIS. Adaptive changes to the location and length of the AIS can fine-tune the excitability of neurons and modulate plasticity in response to activity. The AIS plays an important role in maintaining neuronal polarity by regulating the trafficking and distribution of proteins that function in somatodendritic or axonal compartments of the neuron. In this review, we provide an overview of the AIS cytoarchitecture, mechanism of assembly, and recent studies revealing mechanisms of differential transport at the AIS that maintain axon and dendrite identities. We further discuss how genetic mutations in AIS components (i.e., ankyrins, ion channels, and spectrins) and injuries may cause neurological disorders.

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

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 ß 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 ßIV spectrin interact and form a periodic AIS cytoskeleton. To determine the role of spectrins in the nervous system, we generated Sptan1f/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 ß 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 ßIV 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.

An αII Spectrin-Based Cytoskeleton Protects Large-Diameter Myelinated Axons from Degeneration.

Axons must withstand mechanical forces, including tension, torsion, and compression. Spectrins and actin form a periodic cytoskeleton proposed to protect axons against these forces. However, because spectrins also participate in assembly of axon initial segments (AISs) and nodes of Ranvier, it is difficult to uncouple their roles in maintaining axon integrity from their functions at AIS and nodes. To overcome this problem and to determine the importance of spectrin cytoskeletons for axon integrity, we generated mice with αII spectrin-deficient peripheral sensory neurons. The axons of these neurons are very long and exposed to the mechanical forces associated with limb movement; most lack an AIS, and some are unmyelinated and have no nodes. We analyzed αII spectrin-deficient mice of both sexes and found that, in myelinated axons, αII spectrin forms a periodic cytoskeleton with ßIV and ßII spectrin at nodes of Ranvier and paranodes, respectively, but that loss of αII spectrin disrupts this organization. Avil-cre;Sptan1f/f mice have reduced numbers of nodes, disrupted paranodal junctions, and mislocalized Kv1 K+ channels. We show that the density of nodal ßIV spectrin is constant among axons, but the density of nodal αII spectrin increases with axon diameter. Remarkably, Avil-cre;Sptan1f/f mice have intact nociception and small-diameter axons, but severe ataxia due to preferential degeneration of large-diameter myelinated axons. Our results suggest that nodal αII spectrin helps resist the mechanical forces experienced by large-diameter axons, and that αII spectrin-dependent cytoskeletons are also required for assembly of nodes of Ranvier.SIGNIFICANCE STATEMENT A periodic axonal cytoskeleton consisting of actin and spectrin has been proposed to help axons resist the mechanical forces to which they are exposed (e.g., compression, torsion, and stretch). However, until now, no vertebrate animal model has tested the requirement of the spectrin cytoskeleton in maintenance of axon integrity. We demonstrate the role of the periodic spectrin-dependent cytoskeleton in axons and show that loss of αII spectrin from PNS axons causes preferential degeneration of large-diameter myelinated axons. We show that nodal αII spectrin is found at greater densities in large-diameter myelinated axons, suggesting that nodes are particularly vulnerable domains requiring a specialized cytoskeleton to protect against axon degeneration.