Final Report Summary - ELANSCI (Role of Lgi and Adam proteins in nerve development and function)
Project context and objectives
The myelin sheath is a lamellar structure that surrounds nerve fibres in the vertebrate nervous system and is produced by Schwann cells (SC) in the peripheral nervous system (PNS). The importance of the myelin sheath for the nervous system's normal function is underscored by the large range of neurological diseases associated with its dysfunction.
The myelination of axons in the developing PNS results from interactions between SC and axons. Our previous studies identified the Lgi4 gene (Leucine-rich-glioma inactivated4) as a key regulator of myelination in the PNS. Lgi4 is a secreted protein and predominantly expressed in the nervous system. Its mechanism of action is unknown but may involve binding to members of the Adam (A disintegrin and metalloprotease) family of transmembrane proteins. In addition, we recently found that Lgi4 and Adam22 are expressed in SC and sensory neurons, and that Lgi4 binds directly to Adam22 without a requirement for additional membrane associated factors.
Work performed
We wanted to determine whether Lgi4-Adam22 function involves a paracrine and/or an autocrine mechanism of action. In a first approach, we generated primary sensory neuron cultures from Adam22+/neo and Adam22neo/neo embryos and seeded these cultures with wild-type rat SC. Immunostaining and western Blot analysis of myelin formation showed that rat SC readily ensheath and myelinate Adam22+/neo neurons, whereas Adam22neo/neo neurons are not myelinated. These results indicate that Adam22 function is required in the neuron. In a second parallel approach, we generated a conditional Adam22 null allele using the Wnt1Cre driver, thereby deleting Adam22 in all neural crest derivatives (sensory neurons, endoneurial fibroblasts and SC). The sciatic nerve is a mixed nerve containing both primary sensory and motor fibres. Histological analysis of myelin formation in the sciatic nerve revealed a mosaic pattern of normally myelinated fibres and promyelin arrested fibres. Examination of the ventral motor and dorsal sensory roots revealed normal myelinated motor fibres and severely hypomyelinated sensory fibres respectively. This data demonstrate that Adam22 is not required in SC for myelin formation. Thus, axonally expressed Adam22 serves as the major receptor for Schwann cell-derived Lgi4 in PNS myelination.
In contrast to previously identified ligand-receptor pairs, Lgi4 is secreted from SC and binds to axonally expressed Adam22. Hence, we wanted to elucidate how axonal Adam22 signals back to the SC and promotes myelination. In order to establish the relevance of the different domains of Adam22 we started by transfecting DRG neurons in culture with the two main adam22 isoforms detected in the PNS that differ in the presence/absence of axon 27. Immunohistochemistry analysis of myelin formation showed that both isoforms of adam22 rescue myelination on Adam22D1/D1 DRG cocultures. This result indicates that the information required for driving PNS myelination is not contained in axon 27 of adam22 and validates this system as an excellent system to dissect the different domains of adam22 involved in PNS myelination. We are now testing the role of the different domains of adam22, the extracellular domain, transmembrane domain and cytosolic tail by making chimera adam22-adam23 proteins using the homologue adam23 domain. In addition, we are also examining the role of the PDZ-binding and SH3-binding motifs located in the cytosolic tail by using the corresponding deleted adam22 version.
Main results
Identifying the mechanisms by which axonal Adam22 governs SC myelination during development is an important question, the answer to which may provide novel approaches for treating myelin diseases. As such, our results are of particular interest to those in the biomedical research community who are actively pursuing novel strategies to modulate myelin formation and maintenance in hereditary and acquired peripheral neuropathies.
The myelin sheath is a lamellar structure that surrounds nerve fibres in the vertebrate nervous system and is produced by Schwann cells (SC) in the peripheral nervous system (PNS). The importance of the myelin sheath for the nervous system's normal function is underscored by the large range of neurological diseases associated with its dysfunction.
The myelination of axons in the developing PNS results from interactions between SC and axons. Our previous studies identified the Lgi4 gene (Leucine-rich-glioma inactivated4) as a key regulator of myelination in the PNS. Lgi4 is a secreted protein and predominantly expressed in the nervous system. Its mechanism of action is unknown but may involve binding to members of the Adam (A disintegrin and metalloprotease) family of transmembrane proteins. In addition, we recently found that Lgi4 and Adam22 are expressed in SC and sensory neurons, and that Lgi4 binds directly to Adam22 without a requirement for additional membrane associated factors.
Work performed
We wanted to determine whether Lgi4-Adam22 function involves a paracrine and/or an autocrine mechanism of action. In a first approach, we generated primary sensory neuron cultures from Adam22+/neo and Adam22neo/neo embryos and seeded these cultures with wild-type rat SC. Immunostaining and western Blot analysis of myelin formation showed that rat SC readily ensheath and myelinate Adam22+/neo neurons, whereas Adam22neo/neo neurons are not myelinated. These results indicate that Adam22 function is required in the neuron. In a second parallel approach, we generated a conditional Adam22 null allele using the Wnt1Cre driver, thereby deleting Adam22 in all neural crest derivatives (sensory neurons, endoneurial fibroblasts and SC). The sciatic nerve is a mixed nerve containing both primary sensory and motor fibres. Histological analysis of myelin formation in the sciatic nerve revealed a mosaic pattern of normally myelinated fibres and promyelin arrested fibres. Examination of the ventral motor and dorsal sensory roots revealed normal myelinated motor fibres and severely hypomyelinated sensory fibres respectively. This data demonstrate that Adam22 is not required in SC for myelin formation. Thus, axonally expressed Adam22 serves as the major receptor for Schwann cell-derived Lgi4 in PNS myelination.
In contrast to previously identified ligand-receptor pairs, Lgi4 is secreted from SC and binds to axonally expressed Adam22. Hence, we wanted to elucidate how axonal Adam22 signals back to the SC and promotes myelination. In order to establish the relevance of the different domains of Adam22 we started by transfecting DRG neurons in culture with the two main adam22 isoforms detected in the PNS that differ in the presence/absence of axon 27. Immunohistochemistry analysis of myelin formation showed that both isoforms of adam22 rescue myelination on Adam22D1/D1 DRG cocultures. This result indicates that the information required for driving PNS myelination is not contained in axon 27 of adam22 and validates this system as an excellent system to dissect the different domains of adam22 involved in PNS myelination. We are now testing the role of the different domains of adam22, the extracellular domain, transmembrane domain and cytosolic tail by making chimera adam22-adam23 proteins using the homologue adam23 domain. In addition, we are also examining the role of the PDZ-binding and SH3-binding motifs located in the cytosolic tail by using the corresponding deleted adam22 version.
Main results
Identifying the mechanisms by which axonal Adam22 governs SC myelination during development is an important question, the answer to which may provide novel approaches for treating myelin diseases. As such, our results are of particular interest to those in the biomedical research community who are actively pursuing novel strategies to modulate myelin formation and maintenance in hereditary and acquired peripheral neuropathies.