Analysis of Gpr126 function defines distinct mechanisms controlling the initiation and maturation of myelin

Abstract

SUMMARYIn peripheral nerves, Schwann cells form the myelin sheath, which allows the efficient propagation of action potentials along axons. The transcription factor Krox20 regulates the initiation of myelination in Schwann cells and is also required to maintain mature myelin. The adhesion G protein-coupled receptor (GPCR) Gpr126 is essential for Schwann cells to initiate myelination, but previous studies have not addressed the role of Gpr126 signaling in myelin maturation and maintenance. Through analysis of Gpr126 in zebrafish, we define two distinct mechanisms controlling the initiation and maturation of myelin. We show that gpr126 mutant Schwann cells elaborate mature myelin sheaths and maintain krox20 expression for months, provided that the early signaling defect is bypassed by transient elevation of cAMP. At the onset of myelination, Gpr126 and protein kinase A (PKA) function as a switch that allows Schwann cells to initiate krox20 expression and myelination. After myelination is initiated, krox20 expression is maintained and myelin maturation proceeds independently of Gpr126 signaling. Transgenic analysis indicates that the Krox20 cis-regulatory myelinating Schwann cell element (MSE) becomes active at the onset of myelination and that this activity is dependent on Gpr126 signaling. Activity of the MSE declines after initiation, suggesting that other elements are responsible for maintaining krox20 expression in mature nerves. We also show that elevated cAMP does not initiate myelination in the absence of functional Neuregulin 1 (Nrg1) signaling. These results indicate that the mechanisms regulating the initiation of myelination are distinct from those mediating the maturation and maintenance of myelin.

Glial Cell Development and Function in Zebrafish

“…1B). Expression of a constitutively active PKA in Schwann cells rescued mbp expression in gpr126 mutant zebrafish (Glenn and Talbot 2013a), and 8-CPT-cAMP, a selective agonist of PKA, rescued myelination in cultures of murine Schwann cells lacking Gpr126 . The key substrates of PKA in myelinating Schwann cells are not yet known, but cAMPresponse element-binding protein (CREB) is a strong candidate (Mandemakers et al 2000;Jagalur et al 2011;Glenn and Talbot 2013b).…”

“…Interestingly, Gpr126 is not required after myelination initiates, because a short pulse of forskolin not only promotes the onset of myelination in gpr126 mutants, but also allows the mutants to form compact myelin that matures and persists for months after the treatment (Glenn and Talbot 2013a). Thus, the current data indicate that Gpr126 activates a transiently required signaling pathway that includes G s , adenylyl cyclase 6, and PKA, culminating with the transcriptional activation of oct6 and perhaps other key downstream regulators of myelination (Fig.…”

G Protein-Coupled Receptors in Myelinating Glia

“…Many adhesion GPCRs undergo autoproteolysis at the GPS motif, resulting in a protein that is separated into an N-terminal fragment (NTF) and a C-terminal fragment (CTF) [10,14]. Gpr126/Adgrg6 was discovered to be essential for Schwann cell myelination by a forward genetic screen in zebrafish [15,16], and subsequent work has firmly demonstrated that the CTF of Gpr126/Adgrg6 couples to Gs proteins, elevates 3', 5'-cyclic adenosine monophosphate (cAMP) levels, activates protein kinase A (PKA), and initiates transcription of key regulators (e.g., Oct6/ Pou3f1) of terminal Schwann cell differentiation in zebrafish as well as mammals [16][17][18][19][20][21][22]. In mammals, Gpr126 mutants also present with limb contracture defects [17]; importantly, human mutations in GPR126/ADGRG6 as well as Adenylate cyclase gene 6 (ADCY6, which encodes a cAMP-synthesizing protein) cause limb and myelination defects, highlighting the conserved and critical function of this pathway in the human PNS [23,24].…”

A zinc finger protein that regulates oligodendrocyte specification, migration, and myelination in zebrafish

“…Tissue-specific expression plasmids were made by LR recombination between this plasmid, pTol2, p3′E-polyA plasmid and the following tissue-specific promoter-containing p5′E plasmids: huC promoter (neurons; Shiau et al, 2013), mpeg1 (macrophages and microglia; Shiau et al, 2013) and claudin k (oligodendrocytes; Münzel et al, 2012). sox10 promoter flanked by Tol2 sequences was previously described by Glenn and Talbot (2013). Briefly, znf16l with adapter sequences were amplified with the following primers: 5′-GTGGCCGCAGAACGAGTGGACCGGCCGCCACCatgagccgaaaaaggaa-3′ and 5′-CATGTCTGGATCATCATCGATTttacccagtttgcaccttgtc-3′ (upper case indicates homology to the sox10 promoter vector and lower case indicates gene-specific sequence) and cloned into the sox10-promoter vector with CloneEZ PCR-Cloning kit (GenScript).…”