The tissue homogenate was centrifuged at 4C for 2 min at 1000 rpm (Beckman, Allegra 6R) as well as the supernatant was stored on ice
April 15, 2022
The tissue homogenate was centrifuged at 4C for 2 min at 1000 rpm (Beckman, Allegra 6R) as well as the supernatant was stored on ice. is usually deleted and followed by a 13 aa MAG-binding motif of the NgR2 stalk, shows superior binding of OMgp, Nogo-66, and MAG compared with wild-type NgR1 or NgR2. Soluble NgROMNI (NgROMNI-Fc) binds strongly to membrane-bound inhibitors and promotes neurite outgrowth on both MAG and CNS myelin substrates. Thus, NgROMNI-Fc may offer therapeutic opportunities following nervous system injury or disease where myelin inhibits neuronal regeneration. Introduction Adult mammalian CNS myelin contains growth inhibitory factors that contribute to the regenerative failure of severed axons following injury (Schwab, 1993). Several myelin inhibitors have been recognized and most of the attention has focused on MAG, Nogo-A, and OMgp (Filbin, 2003; Xie and Zheng, 2008). The Nogo receptors NgR1 and NgR2 show overlapping, yet unique, binding preferences for myelin inhibitors. Nogo-A and OMgp bind selectively and with high affinity to NgR1 (Liu et al., 2006), while MAG binds preferentially to NgR2 but also interacts with NgR1 (Giger et al., 2008). is necessary for growth cone collapse in response to acutely offered myelin inhibitors (Kim et al., 2004; Chivatakarn et al., 2007), but is usually dispensable for neurite outgrowth inhibition on substrate-bound Nogo-66 (Zheng et al., 2005) or MAG or OMgp (Chivatakarn et al., 2007; Venkatesh et al., 2007; Williams et al., 2008). Mechanistically, this apparent dichotomy of the role of NgR1 in neuronal growth inhibitory responses is usually poorly comprehended. Physiological signaling limits experience-dependent plasticity in the visual cortex (McGee et al., 2005), and in the adult hippocampus, regulates activity-dependent synaptic strength and dendritic spine morphology (Lee et al., 2008). Following CNS GW0742 injury, limits axon collateral sprouting but not long-distance regenerative growth of severed corticospinal tract fibers (Kim et al., 2004; Zheng et al., 2005; Cafferty and Strittmatter, 2006). MAG is usually a member of the siglec family of sialic acid-binding Ig lectins and uses neuronal cell-type-specific mechanisms to mediate growth inhibition. Cerebellar granule neurons (CGNs) but not dorsal root ganglion (DRG) neurons deficient for complex gangliosides are Rabbit Polyclonal to Cytochrome P450 39A1 more resistant to MAG inhibition. In retinal ganglion cells (RGCs), hippocampal and DRG neurons, functional depletion of gangliosides or NgR1 alone is not sufficient to attenuate MAG inhibition. Simultaneous loss of terminal sialic acids and NgR1, however, significantly attenuates MAG inhibition (Mehta et al., GW0742 2007; Venkatesh et al., 2007). A receptor complex comprised of NgR1, Lingo-1, and p75 or TROY has been implicated in signaling Nogo-66, OMgp, and MAG inhibition of neurite outgrowth (Yiu and He, 2006). nor is necessary for MAG inhibition of CGNs or RGCs (Zheng et al., 2005; Venkatesh et al., 2007). MAG-induced repulsive growth cone steering requires the presence of an arginine-glycine-aspartate (RGD)-dependent conversation with neuronal 1-integrin (Goh et al., 2008). The ligand-binding domain name (LBD) of NgR1 is composed of 8.5 canonical LRRs flanked by cysteine-rich LRR-NT and LRR-CT cap GW0742 domains. The LBD harbors overlapping, yet distinct, binding pouches for Nogo, OMgp, and MAG (Schimmele and Plckthun, 2005; Laurn et al., 2007). In soluble form, the NgR1 LBD [NgR1(310)] has CNS myelin inhibitor antagonistic properties (Fournier et al., 2002; Liu et al., 2002; He et al., 2003; Zheng et al., 2005). Following spinal cord injury, NgR1(310)-Fc promotes sprouting and regenerative growth of severed corticospinal and raphespinal fibers (Li et al., 2004; Wang et al., 2006). Here, we define the structural basis of the MAG association with NgR1 and NgR2 and develop a soluble chimeric Nogo receptor variant with potent CNS myelin antagonistic properties. Materials and Methods Recombinant DNA constructs. Chimeric receptors were generated by PCR using rat NgR1, NgR2, or NgR3 cDNA themes and put together in the expression vector pMT21 (Venkatesh et al., 2005). To fuse PCR-amplified receptor fragments, either endogenous restriction enzyme sites or newly introduced restriction sites were used that resulted in either no amino acid substitution or conservative substitutions. None of the conserved leucine.