Adrian Robert Walmsley

Versican V2 and the central inhibitory domain of Nogo-A inhibit neurite growth via p75NTR/NgR-independent pathways that converge at RhoA.

Myelin is a major obstacle for regenerating nerve fibers of the adult mammalian central nervous system (CNS). Several proteins including Nogo-A, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and the chondroitin-sulfate proteoglycan (CSPG) Versican V2 have been identified as inhibitory components present in CNS myelin. MAG, OMgp as well as the Nogo specific domain Nogo-66 exert their inhibitory activity by binding to a neuronal receptor complex containing the Nogo-66 receptor NgR and the neurotrophin receptor p75(NTR). While this suggests a converging role of the p75(NTR)/NgR receptor complex for myelin-derived neurite growth inhibitors, we show here that NgR/p75(NTR) is not required for mediating the inhibitory activity of the two myelin components NiG, unlike Nogo-66 a distinct domain of Nogo-A, and Versican V2. Primary neurons derived from a complete null mutant of p75(NTR) are still sensitive to NiG and Versican V2. In line with this result, neurite growth of p75(NTR) deficient neurons is still significantly blocked on total bovine CNS myelin. Furthermore, modulation of RhoA and Rac1 in p75(NTR)-/- neurons persists with NiG and Versican V2. Finally, we demonstrate that neither NiG nor Versican V2 interact with the p75(NTR)/NgR receptor complex and provide evidence that the binding sites of NiG and Nogo-66 are physically distinct from each other on neural tissue. These results indicate not only the existence of neuronal receptors for myelin inhibitors independent from the p75(NTR)/NgR receptor complex but also establish Rho GTPases as a common point of signal convergence of diverse myelin-induced regeneration inhibitory pathways.

Zinc metalloproteinase-mediated cleavage of the human Nogo-66 receptor.

The central nervous system myelin components oligodendrocyte-myelin glycoprotein, myelin-associated glycoprotein and the Nogo-66 domain of Nogo-A inhibit neurite outgrowth by binding the neuronal glycosyl-phosphatidylinositol-anchored Nogo-66 receptor (NgR) that transduces the inhibitory signal to the cell interior via a transmembrane co-receptor, p75NTR. Here, we demonstrate that human NgR expressed in human neuroblastoma cells is constitutively cleaved in a post-ER compartment to generate a lipid-raft associated C-terminal fragment that is present on the cell surface and a soluble N-terminal fragment that is released into the medium. Mass spectrometric analysis demonstrated that the N-terminal fragment terminated just after the C-terminus of the ligand-binding domain of NgR. In common with other shedding mechanisms, the release of this fragment was blocked by a hydroxamate-based inhibitor of zinc metalloproteinases, but not by inhibitors of other protease classes and up-regulated by treatment with the cellular cholesterol depleting agent methyl-β-cyclodextrin. The N-terminal fragment bound Nogo-66 and blocked Nogo-66 binding to cell surface NgR but failed to associate with p75NTR, indicative of a role as a Nogo-66 antagonist. Furthermore, the N- and C-terminal fragments of NgR were detectable in human brain cortex and the N-terminal fragment was also present in human cerebrospinal fluid, demonstrating that NgR proteolysis occurs within the human nervous system. Our findings thus identify a potential cellular mechanism for the regulation of NgR function at the level of the receptor.

Neutralization of LINGO-1 during In Vitro Differentiation of Neural Stem Cells Results in Proliferation of Immature Neurons

Identifying external factors that can be used to control neural stem cells division and their differentiation to neurons, astrocytes and oligodendrocytes is of high scientific and clinical interest. Here we show that the Nogo-66 receptor interacting protein LINGO-1 is a potent regulator of neural stem cell maturation to neurons. LINGO-1 is expressed by cortical neural stem cells from E14 mouse embryos and inhibition of LINGO-1 during the first days of neural stem cell differentiation results in decreased neuronal maturation. Compared to neurons in control cultures, which after 6 days of differentiation have long extending neurites, neurons in cultures treated with anti-LINGO-1 antibodies retain an immature, round phenotype with only very short processes. Furthermore, neutralization of LINGO-1 results in a threefold increase in βIII tubulin-positive cells compared to untreated control cultures. By using BrdU incorporation assays we show that the immature neurons in LINGO-1 neutralized cultures are dividing neuroblasts. In contrast to control cultures, in which no cells were double positive for βIII tubulin and BrdU, 36% of the neurons in cultures treated with anti-LINGO-1 antibodies were proliferating after three days of differentiation. TUNEL assays revealed that the amount of cells going through apoptosis during the early phase of differentiation was significantly decreased in cultures treated with anti-LINGO-1 antibodies compared to untreated control cultures. Taken together, our results demonstrate a novel role for LINGO-1 in neural stem cell differentiation to neurons and suggest a possibility to use LINGO-1 inhibitors to compensate for neuronal cell loss in the injured brain.

LINGO-1-mediated inhibition of oligodendrocyte differentiation does not require the leucine-rich repeats and is reversed by p75NTR antagonists

LINGO-1 is a potent negative regulator of oligodendrocyte differentiation and hence may play a pivotal restrictive role during remyelination in demyelinating diseases such as multiple sclerosis. However, little is known as to which stages of oligodendrocyte differentiation are inhibited by LINGO-1, which domains of the protein are involved and whether accessory proteins are required. Here, we show that LINGO-1 expression in the human oligodendroglial cell line MO3.13 inhibited process extension and this was reversed by an anti-LINGO-1 antibody or the antagonist LINGO-1-Fc. LINGO-1 expression was also found to inhibit myelin basic protein transcription in the rat oligodendroglial cell line CG4. Both of these inhibitory actions of LINGO-1 were abrogated by deletion of the entire ectodomain or cytoplasmic domains but, surprisingly, were unaffected by deletion of the leucine-rich repeats (LRRs). As in neurons, LINGO-1 physically associated with endogenous p75(NTR) in MO3.13 cells and, correspondingly, its inhibition of process extension was reversed by antagonists of p75(NTR). Thus, LINGO-1 inhibits multiple aspects of oligodendrocyte differentiation independently of the LRRs via a process that requires p75(NTR) signalling.

The leucine-rich repeats of LINGO-1 are not required for self-interaction or interaction with the amyloid precursor protein.

LINGO-1 (leucine rich repeat and Ig domain containing Nogo receptor interacting protein-1) is a central nervous system transmembrane protein which simultaneously interacts with the Nogo-66 receptor and p75(NTR) or TROY on neurons to form a receptor complex responsible for myelin-mediated neurite outgrowth inhibition. On oligodendroglial cells, LINGO-1 interacts with p75(NTR) to constitutively inhibit multiple aspects of oligodendrocyte differentiation. Recently, LINGO-1 was identified as an in vivo interacting partner of the amyloid precursor protein (APP) and, correspondingly, cellular LINGO-1 expression was found to augment the release of the Abeta peptide, the potential causative agent of Alzheimers disease. In addition, the recombinant LINGO-1 ectodomain has been shown to self-interact in solution and after crystallisation. Here, we have used deletional mutagenesis to identify the regions on LINGO-1 that are involved in homo- and heterotypic interactions. We have found that the N-terminal region containing the leucine-rich repeats along with the transmembrane and cytoplasmic domains of LINGO-1 are not required for self-interaction or interaction with APP.

MYELIN NEUTRALIZATION FOR SPINAL CORD INJURY AND STROKE

Injuries to the central nervous system (CNS) in humans are associated with a low degree of neurological recovery and, in most cases, life-long debilitation. This, however, is not due to an intrinsic inability of CNS axons to regenerate; rather the environment of the CNS is highly inhibitory to axonal regeneration and plasticity. In recent years, great advances have been made in our understanding of the molecular components contributing to the inhibitory environment of the CNS. The majority of these have been identified as membrane proteins present in CNS myelin, such as myelin-associated glycoprotein, Nogo-A and oligodendrocyte-myelin glycoprotein, that inhibit neurite outgrowth by binding to the multimeric Nogo-66 receptor (NgR1) complex on the neuron and activating the small GTPase RhoA. Accordingly, blockade of either Nogo-A, the NgR receptor complex or RhoA using biological-based inhibitors has been shown to promote neurite outgrowth on CNS myelin substrates in vitro and, more importantly, enhance axonal regeneration and plasticity in vivo leading to improved functional recovery in animal models of acute CNS injury. Based on these promising findings, clinical trials have now been instigated in acute spinal cord injury using a function-blocking anti-Nogo-A antibody and a RhoA antagonist, respectively.