Myelination is a complex process requiring coordination of directional motility and an increase in glial cell size to generate a multilamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin-1 (NRG1) expressed on the axon surface. Here we identify cofilin1, an actin depolymerizing and severing protein, as a downstream target of NRG1 signaling in rat Schwann cells (SCs). In isolated SCs, NRG1 promotes dephosphorylation of cofilin1 and its upstream regulators, LIM kinase (LIMK) and Slingshot-1 phosphatase (SSH1), leading to cofilin1 activation and recruitment to the leading edge of the plasma membrane. These changes are associated with rapid membrane expansion yielding a 35–50% increase in SC size within 30 min. Cofilin1-deficient SCs increase phosphorylation of ErbB2, ERK, focal adhesion kinase, and paxillin in response to NRG1, but fail to increase in size possibly due to stabilization of unusually long focal adhesions. Cofilin1-deficient SCs cocultured with sensory neurons do not myelinate. Ultrastructural analysis reveals that they unsuccessfully segregate or engage axons and form only patchy basal lamina. After 48 h of coculturing with neurons, cofilin1-deficient SCs do not align or elongate on axons and often form adhesions with the underlying substrate. This study identifies cofilin1 and its upstream regulators, LIMK and SSH1, as end targets of a NRG1 signaling pathway and demonstrates that cofilin1 is necessary for dynamic changes in the cytoskeleton needed for axon engagement and myelination by SCs.

One of the most significant interactions between Schwann cells and neurons is myelin sheath formation. Myelination is a vertebrate adaptation that enables rapid conduction of action potentials without a commensurate increase in axon diameter. In vitro neuronal systems provide a unique modality to study both factors influencing myelination and diseases associated with myelination. Currently, no in vitro system for motoneuron myelination by Schwann cells has been demonstrated. This work details the myelination of motoneuron axons by Schwann cells, with complete node of Ranvier formation, in a defined in vitro culture system. This defined system utilizes a novel serum-free medium in combination with the non-biological substrate, N-1[3 (trimethoxysilyl) propyl] diethylenetriamine (DETA). The myelinated segments and nodal proteins were visualized and quantified using confocal microscopy. This defined system provides a highly controlled, reproducible model for studying Schwann cell interactions with motoneurons as well as the myelination process and its effect on neuronal plasticity. Furthermore, an in vitro system that would allow studies of motoneuron myelination would be beneficial for understanding peripheral demyelinating neuropathies such as diabetes induced peripheral neuropathy and could lead to a better understanding of CNS demyelinating diseases like multiple sclerosis, as well as neuromuscular junction maturation and maintenance.

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Mutations in the neurofibromatosis type 2 (NF2) gene cause formation of schwannomas and other tumors in the nervous system. The NF2 protein, Schwannomin/Merlin, is a cytoskeleton-associated tumor suppressor regulated by phosphorylation at serine 518 (S518). Unphosphorylated Schwannomin restricts cell proliferation in part by inhibiting Rac-and p21-activated kinase (Pak). In a negative-feedback loop, Pak phosphorylates Schwannomin inactivating its ability to inhibit Pak. Little is known about receptor mechanisms that promote Pak activity and Schwannomin phosphorylation. Here we demonstrate in primary Schwann cells (SCs) that Schwannomin is rapidly phosphorylated on S518 by Pak following laminin-1 binding to b1 integrin, and by protein kinase A following neuregulin-1b (NRG1b) binding to ErbB2/ErbB3 receptors. These receptors, together with phosphorylated Schwannomin, P-Pak, Cdc42 and paxillin are enriched at the distal tips of SC processes, and can be isolated as a complex using b1 integrin antibody. Dual stimulation with laminin-1 and NRG1b does not synergistically increase Schwannomin phosphorylation because ErbB2 kinase partially antagonizes integrin-dependent activation of Pak. These results identify two parallel, but interactive pathways that inactivate the tumor suppressor activity of Schwannomin to allow proliferation of subconfluent SCs. Moreover, they identify ErbB2, ErbB3 and b1 integrins as potential therapeutic targets for NF2.

The Neurofibromatosis type 2 tumor suppressor, schwannomin (Sch) is a plasma membrane-cytoskeleton linking protein that regulates receptor signaling and actin dynamics. We examined Sch’s role in specifying morphological changes needed for Schwann cell (SC) function in vitro. Isolated Sch-GFP-expressing SCs extended bipolar processes 82% longer than those formed by GFP-expressing cells. In contrast, SCs expressing dominant negative Sch-BBA-GFP extended bipolar processes 16% shorter than controls and 64% shorter than Sch-GFP-expressing SCs. nf2 gene inactivation caused isolated mouse SCs to transition from bipolar to multipolar cells. Live imaging revealed that SCs co-expressing Sch-GFP and dominant negative RacN17 behaved similarly in dorsal root ganglion explant cultures; they quickly aligned on axons and slowly elongated bipolar processes. In contrast, SCs expressing constitutively active RacV12 underwent continuous transitions in morphology that interfered with axon alignment. When co-cultured with neurons under myelin-promoting conditions, Sch-GFP-expressing SCs elaborated longer myelin segments than GFP-expressing SCs. In contrast, Sch-BBA-GFP-expressing SCs failed to align on or myelinate axons. Together, these results demonstrate that Sch plays an essential role in inducing and/or maintaining the SC’s spindle shape and suggest that the mechanism involves Sch-dependent inhibition of Rac activity. By stabilizing the bipolar morphology, Sch promotes alignment of SCs with axons and ultimately influences myelin segment length.