The Nogo-66 Receptors NgR1 and NgR3 Are Required for Commissural Axon Pathfinding.

Nogo-66 receptors (NgR1-3) are glycosylphosphatidyl inositol-linked proteins that belong to the leucine-rich repeat superfamily. Through binding to myelin-associated inhibitors, NgRs contribute to the inhibition of axonal regeneration after spinal cord injury. Their role in limiting synaptic plasticity and axonal outgrowth in the adult CNS has been described previously, but not much is known about their role during the development of the nervous system. Here, we show that NgR1 and NgR3 mRNAs are expressed during spinal cord development of the chicken embryo. In particular, they are expressed in the dI1 subpopulation of commissural neurons during the time when their axons navigate toward and across the floorplate, the ventral midline of the spinal cord. To assess a potential role of NgR1 and NgR3 in axon guidance, we downregulated them using in ovo RNAi and analyzed the trajectory of commissural axons by tracing them in open-book preparations of spinal cords. Our results show that loss of either NgR1 or NgR3 causes axons to stall in the midline area and to interfere with the rostral turn of postcrossing axons. In addition, we also show that NgR1, but not NgR3, requires neuronal PlexinA2 for the regulation of commissural axon guidance.SIGNIFICANCE STATEMENT Over the last decades, many studies have focused on the role of NgRs, particularly NgR1, in axonal regeneration in the injured adult CNS. Here, we show a physiological role of NgRs in guiding commissural axons during early development of the chicken spinal cord in vivo Both NgR1 and NgR3 are required for midline crossing and subsequent turning of postcrossing axons into the longitudinal axis of the spinal cord. NgR1, but not NgR3, forms a receptor complex with PlexinA2 during axon guidance. Overall, these findings provide a link between neural regenerative mechanisms and developmental processes.

Sphingosine-1-Phosphate and the S1P3 Receptor Initiate Neuronal Retraction via RhoA/ROCK Associated with CRMP2 Phosphorylation.

The bioactive lipid sphingosine-1-phosphate (S1P) is an important regulator in the nervous system. Here, we explored the role of S1P and its receptors in vitro and in preclinical models of peripheral nerve regeneration. Adult sensory neurons and motor neuron-like cells were exposed to S1P in an in vitro assay, and virtually all neurons responded with a rapid retraction of neurites and growth cone collapse which were associated with RhoA and ROCK activation. The S1P1 receptor agonist SEW2871 neither activated RhoA or neurite retraction, nor was S1P-induced neurite retraction mitigated in S1P1-deficient neurons. Depletion of S1P3 receptors however resulted in a dramatic inhibition of S1P-induced neurite retraction and was on the contrary associated with a significant elongation of neuronal processes in response to S1P. Opposing responses to S1P could be observed in the same neuron population, where S1P could activate S1P1 receptors to stimulate elongation or S1P3 receptors and retraction. S1P was, for the first time in sensory neurons, linked to the phosphorylation of collapsin response-mediated protein-2 (CRMP2), which was inhibited by ROCK inhibition. The improved sensory recovery after crush injury further supported the relevance of a critical role for S1P and receptors in fine-tuning axonal outgrowth in peripheral neurons.

Peripheral nerve regeneration and NGF-dependent neurite outgrowth of adult sensory neurons converge on STAT3 phosphorylation downstream of neuropoietic cytokine receptor gp130.

After nerve injury, adult sensory neurons can regenerate peripheral axons and reconnect with their target tissue. Initiation of outgrowth, as well as elongation of neurites over long distances, depends on the signaling of receptors for neurotrophic growth factors. Here, we investigated the importance of gp130, the signaling subunit of neuropoietic cytokine receptors in peripheral nerve regeneration. After sciatic nerve crush, functional recovery in vivo was retarded in SNS-gp130(-/-) mice, which specifically lack gp130 in sensory neurons. Correspondingly, a significantly reduced number of free nerve endings was detected in glabrous skin from SNS-gp130(-/-) compared with control mice after nerve crush. Neurite outgrowth and STAT3 activation in vitro were severely reduced in cultures in gp130-deficient cultured neurons. Surprisingly, in neurons obtained from SNS-gp130(-/-) mice the increase in neurite length was reduced not only in response to neuropoietic cytokine ligands of gp130 but also to nerve growth factor (NGF), which does not bind to gp130-containing receptors. Neurite outgrowth in the absence of neurotrophic factors was partially rescued in gp130-deficient neurons by leptin, which activates STAT3 downstream of leptic receptor and independent of gp130. The neurite outgrowth response of gp130-deficient neurons to NGF was fully restored in the presence of leptin. Based on these findings, gp130 signaling via STAT3 activation is suggested not only to be an important regulator of peripheral nerve regeneration in vitro and in vivo, but as determining factor for the growth promoting action of NGF in adult sensory neurons.

Loss of Nogo receptor homolog NgR2 alters spine morphology of CA1 neurons and emotionality in adult mice.

Molecular mechanisms which stabilize dendrites and dendritic spines are essential for regulation of neuronal plasticity in development and adulthood. The class of Nogo receptor proteins, which are critical for restricting neurite outgrowth inhibition signaling, have been shown to have roles in developmental, experience and activity induced plasticity. Here we investigated the role of the Nogo receptor homolog NgR2 in structural plasticity in a transgenic null mutant for NgR2. Using Golgi-Cox staining to analyze morphology, we show that loss of NgR2 alters spine morphology in adult CA1 pyramidal neurons of the hippocampus, significantly increasing mushroom-type spines, without altering dendritic tree complexity. Furthermore, this shift is specific to apical dendrites in distal CA1 stratum radiatum (SR). Behavioral alterations in NgR2(-/-) mice were investigated using a battery of standardized tests and showed that whilst there were no alterations in learning and memory in NgR2(-/-) mice compared to littermate controls, NgR2(-/-) displayed reduced fear expression in the contextual conditioned fear test, and exhibited reduced anxiety- and depression-related behaviors. This suggests that the loss of NgR2 results in a specific phenotype of reduced emotionality. We conclude that NgR2 has role in maintenance of mature spines and may also regulate fear and anxiety-like behaviors.

Nogo receptor homolog NgR2 expressed in sensory DRG neurons controls epidermal innervation by interaction with Versican.

Primary sensory afferents of the dorsal root ganglion (DRG) that innervate the skin detect a wide range of stimuli, such as touch, temperature, pain, and itch. Different functional classes of nociceptors project their axons to distinct target zones within the developing skin, but the molecular mechanisms that regulate target innervation are less clear. Here we report that the Nogo66 receptor homolog NgR2 is essential for proper cutaneous innervation. NgR2(-/-) mice display increased density of nonpeptidergic nociceptors in the footpad and exhibit enhanced sensitivity to mechanical force and innocuous cold temperatures. These sensory deficits are not associated with any abnormality in morphology or density of DRG neurons. However, deletion of NgR2 renders nociceptive nonpeptidergic sensory neurons insensitive to the outgrowth repulsive activity of skin-derived Versican. Biochemical evidence shows that NgR2 specifically interacts with the G3 domain of Versican. The data suggest that Versican/NgR2 signaling at the dermo-epidermal junction acts in vivo as a local suppressor of axonal plasticity to control proper density of epidermal sensory fiber innervation. Our findings not only reveal the existence of a novel and unsuspected mechanism regulating epidermal target innervation, but also provide the first evidence for a physiological role of NgR2 in the peripheral nervous system.

Nogo-A couples with Apg-1 through interaction and co-ordinate expression under hypoxic and oxidative stress.

Nogo-A is the largest isoform of the Nogo/RTN4 (reticulon 4) proteins and has been characterized as a major myelin-associated inhibitor of regenerative nerve growth in the adult CNS (central nervous system). Apart from the myelin sheath, Nogo-A is expressed at high levels in principal neurons of the CNS. The specificity of Nogo-A resides in its central domain, NiG. We identified Apg-1, a member of the stress-induced Hsp110 (heat-shock protein of 110 kDa) family, as a novel interactor of NiG/Nogo-A. The interaction is selective because Apg-1 interacts with Nogo-A/RTN4-A, but not with RTN1-A, the closest paralogue of Nogo-A. Conversely, Nogo-A binds to Apg-1, but not to Apg-2 or Hsp105, two other members of the Hsp110 family. We characterized the Nogo-A-Apg-1 interaction by affinity precipitation, co-immunoprecipitation and proximity ligation assay, using primary hippocampal neurons derived from Nogo-deficient mice. Under conditions of hypoxic and oxidative stress we found that Nogo-A and Apg-1 were tightly co-regulated in hippocampal neurons. Although both proteins were up-regulated under hypoxic conditions, their expression levels were reduced upon the addition of hydrogen peroxide. Taken together, we suggest that Nogo-A is closely involved in the neuronal response to hypoxic and oxidative stress, an observation that may be of relevance not only in stroke-induced ischaemia, but also in neuroblastoma formation.

Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons.

RTN1A is a reticulon protein with predominant localization in the endoplasmic reticulum (ER). It was previously shown that RTN1A is expressed in neurons of the mammalian central nervous system but functional information remains sparse. To elucidate the neuronal function of RTN1A, we chose to focus our investigation on identifying possible novel binding partners specifically interacting with the unique N-terminus of RTN1A. Using a nonbiased approach involving GST pull-downs and MS analysis, we identified the intracellular calcium release channel ryanodine receptor 2 (RyR2) as a direct binding partner of RTN1A. The RyR2 binding site was localized to a highly conserved 150-amino acid residue region. RTN1A displays high preference for RyR2 binding in vitro and in vivo and both proteins colocalize in hippocampal neurons and Purkinje cells. Moreover, we demonstrate the precise subcellular localization of RTN1A in Purkinje cells and show that RTN1A inhibits RyR channels in [(3)H]ryanodine binding studies on brain synaptosomes. In a functional assay, RTN1A significantly reduced RyR2-mediated Ca(2+) oscillations. Thus, RTN1A and RyR2 might act as functional partners in the regulation of cytosolic Ca(2+) dynamics the in neurons.

The Nogo-66 receptor family in the intact and diseased CNS.

The Nogo-66 receptor family (NgR) consists in three glycophosphatidylinositol (GPI)-anchored receptors (NgR1, NgR2 and NgR3), which are primarily expressed by neurons in the central and peripheral mammalian nervous system. NgR1 was identified as serving as a high affinity binding protein for the three classical myelin-associated inhibitors (MAIs) Nogo-A, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp), which limit axon regeneration and sprouting in the injured brain. Recent studies suggest that NgR signaling may also play an essential role in the intact adult CNS in restricting axonal and synaptic plasticity and are involved in neurodegenerative diseases, particularly in Alzheimer's disease pathology through modulation of ß-secretase cleavage. Here, we outline the biochemical properties of NgRs and their functional roles in the intact and diseased CNS.

Nogo receptor is involved in the adhesion of dendritic cells to myelin.

BACKGROUND: Nogo-66 receptor NgR1 and its structural homologue NgR2 are binding proteins for a number of myelin-associated inhibitory factors. After neuronal injury, these inhibitory factors are responsible for preventing axonal outgrowth via their interactions with NgR1 and NgR2 expressed on neurons. In vitro, cells expressing NgR1/2 are inhibited from adhering to and spreading on a myelin substrate. Neuronal injury also results in the presence of dendritic cells (DCs) in the central nervous system, where they can come into contact with myelin debris. The exact mechanisms of interaction of immune cells with CNS myelin are, however, poorly understood. METHODS: Human DCs were differentiated from peripheral blood monocytes and mouse DCs were differentiated from wild type and NgR1/NgR2 double knockout bone marrow precursors. NgR1 and NgR2 expression were determined with quantitative real time PCR and immunoblot, and adhesion of cells to myelin was quantified. RESULTS: We demonstrate that human immature myeloid DCs express NgR1 and NgR2, which are then down-regulated upon maturation. Human mature DCs also adhere to a much higher extent to a myelin substrate than immature DCs. We observe the same effect when the cells are plated on Nogo-66-His (binding peptide for NgR1), but not on control proteins. Mature DCs taken from Ngr1/2 knockout mice adhere to a much higher extent to myelin compared to wild type mouse DCs. In addition, Ngr1/2 knockout had no effect on in vitro DC differentiation or phenotype. CONCLUSIONS: These results indicate that a lack of NgR1/2 expression promotes the adhesion of DCs to myelin. This interaction could be important in neuroinflammatory disorders such as multiple sclerosis in which peripheral immune cells come into contact with myelin debris.

Nogo-A stabilizes the architecture of hippocampal neurons.

Although the role of myelin-derived Nogo-A as an inhibitor of axonal regeneration after CNS injury has been thoroughly described, its physiological function in the adult, uninjured CNS is less well known. We address this question in the hippocampus, where Nogo-A is expressed by neurons as well as oligodendrocytes. We used 21 d in vitro slice cultures of neonatal hippocampus where we applied different approaches to interfere with Nogo-A signaling and expression and analyze their effects on the dendritic and axonal architecture of pyramidal cells. Neutralization of Nogo-A by function-blocking antibodies induced a major alteration in the dendrite structure of hippocampal pyramidal neurons. Although spine density was not influenced by Nogo-A neutralization, spine type distribution was shifted toward a more immature phenotype. Axonal complexity and length were greatly increased. Nogo-A KO mice revealed a weak dendritic phenotype resembling the effect of the antibody treatment. To discriminate a possible cell-autonomous role of Nogo-A from an environmental, receptor-mediated function, we studied the effects of short hairpin RNA-induced knockdown of Nogo-A or NgR1, a prominent Nogo-A receptor, within individual neurons. Knockdown of Nogo-A reproduced part of the dendritic and none of the spine or axon alterations. However, downregulation of NgR1 replicated the dendritic, the axonal, and the spine alterations observed after Nogo-A neutralization. Together, our results demonstrate that Nogo-A plays a major role in stabilizing and maintaining the architecture of hippocampal pyramidal neurons. Mechanistically, although the majority of the activity of Nogo-A relies on a receptor-mediated mechanism involving NgR1, its cell-autonomous function plays a minor role.

Inhibitory activity of myelin-associated glycoprotein on sensory neurons is largely independent of NgR1 and NgR2 and resides within Ig-Like domains 4 and 5.

Myelin-associated glycoprotein (MAG) is a sialic acid binding Ig-like lectin (Siglec) which has been characterized as potent myelin-derived inhibitor of neurite outgrowth. Two members of the Nogo-receptor (NgR) family, NgR1 and NgR2, have been identified as neuronal binding proteins of MAG. In addition, gangliosides have been proposed to bind to and confer the inhibitory activity of MAG on neurons. In this study, we investigated the individual contribution of NgRs and gangliosides to MAG-mediated inhibition of sensory neurons derived from dorsal root ganglia (DRG) of ngr1, ngr2 or ngr1/ngr2 deletion mutants. We found no disinhibition of neurite growth in the absence of either NgR1 or NgR2. Sensory neurons deficient for both NgR proteins displayed only a moderate reduction of MAG-mediated inhibition of neurite growth. If treated with Vibrio cholerae neuraminidase (VCN), inhibition by MAG is further attenuated but still not annulled. Thus, disrupting all known protein and ganglioside receptors for MAG in sensory neurons does not fully abolish its inhibitory activity pointing to the existence of as yet unidentified receptors for MAG. Moreover, by employing a variety of protein mutants, we identified the Ig-like domains 4 or 5 of MAG as necessary and sufficient for growth arrest, whereas abolishing MAG's ability to bind to sialic acid did not interfere with its inhibitory activity. These findings provide new insights into the inhibitory function of MAG and suggest similarities but also major differences in MAG inhibition between sensory and central nervous system (CNS) neurons.

Phosphorylation-regulated cleavage of the reticulon protein Nogo-B by caspase-7 at a noncanonical recognition site.

Reticulons (RTNs) are a large family of transmembrane proteins present throughout the eukaryotic domain in virtually every cell type. Despite their wide distribution, their function is still mostly unknown. RTN4, also termed Nogo, comes in three isoforms, Nogo-A, -B, and -C. While Nogo-A has been described as potent inhibitor of nerve growth, Nogo-B has been implicated in vascular remodeling and regulation of apoptosis. We show here that Nogo-B gets cleaved by caspase-7, but not caspase-3, during apoptosis at a caspase nonconsensus site. By a combination of MS and site-directed mutagenesis we demonstrate that proteolytic processing of Nogo-B is regulated by phosphorylation of Ser(16) within the cleavage site. We present cyclin-dependent kinase (Cdk)1 and Cdk2 as kinases that phosphorylate Nogo-B at Ser(16) in vitro. In vivo, cleavage of Nogo-B is markedly increased in Schwann cells in a lesion model of the rat sciatic nerve. Taken together, we identified an RTN protein as one out of a selected number of caspase targets during apoptosis and as a novel substrate for Cdk1 and 2. Furthermore, our data support a functionality of caspase-7 that is distinct from closely related caspase-3.

Nogo in the injured spinal cord.

Myelin of the adult mammalian central nervous system (CNS) has been attributed to suppress structural plasticity and to impede regenerating nerve fibers. Nogo-A is possibly the best characterized of a variety of neurite growth inhibitors present in CNS myelin. Neutralizing its activity results in improved axon regrowth and functional recovery in experimental CNS lesion models of adult rodents and primates. While Nogo-A has become a major target for therapeutic intervention to promote axon regeneration in the CNS, it is realized that such an approach will likely have to be combined with other therapeutic strategies to maximize functional recovery after spinal cord injury (SCI).

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.

Increased expression of Nogo-A in hippocampal neurons of patients with temporal lobe epilepsy.

Mesial temporal lobe epilepsy (TLE) is associated with pronounced anatomical and biochemical changes in the hippocampal formation including extensive neurodegeneration, reorganization of mossy fibres and sprouting of interneurons. Although the anatomical features and some of the physiological consequences of hippocampal remodeling have been well documented, the molecular mechanisms underlying the profound and orientated outgrowth of hippocampal neurons in TLE are not yet understood. The reticulon protein Nogo-A has been associated with an inhibitory action on axon growth and plasticity. Using immunohistochemistry and in situ hybridization, we investigated the expression of Nogo-A in specimens obtained at surgery from patients with TLE compared with those obtained from autopsy controls. In control specimens, Nogo-A immunoreactivity and mRNA were mainly confined to oligodendrocytes. Only approximately 40% of the specimens revealed low expression of Nogo-A mRNA in neurons. In contrast, in TLE patients with and without Ammon's horn sclerosis, Nogo-A mRNA and immunoreactivity were markedly up-regulated in most neurons (3.6- and 4.4-fold increases in Nogo-A mRNA in granule cells of sclerotic and nonsclerotic specimens) and their processes throughout the hippocampal formation. Similar elevations in Nogo-A mRNA and protein levels were determined by quantitative RT-PCR and Western blotting. Since Nogo-A expression was also up-regulated in specimens without hippocampal sclerosis, it may be induced by seizures prior to progressing neurodegeneration.

Serum and cerebrospinal fluid antibodies to Nogo-A in patients with multiple sclerosis and acute neurological disorders.

Nogo-A is a protein associated with central nervous system (CNS) myelin thought to impair regenerative responses and to suppress sprouting and plastic changes of synaptic terminals. In this study, we report that serum IgM autoantibodies to the recombinant large N-terminal inhibitory domain of Nogo-A are a frequent finding in multiple sclerosis (MS) and acute inflammatory (IND) and non-inflammatory neurological diseases (OND), but not in neurodegenerative diseases (ND), systemic inflammatory disease and healthy controls. Furthermore, we demonstrate intrathecal production of anti-Nogo-A antibodies measured by increased IgG indices. Intrathecal anti-Nogo antibodies were significantly more frequent in patients with relapsing-remitting as compared to chronic progressive (CP) MS. We also found a highly significant negative correlation of these antibody responses with age indicating that they are more frequent in younger patients. We finally demonstrate that human anti-Nogo-A antibodies recognize native Nogo-A in brain extracts, oligodendrocytes and cells expressing human Nogo-A.

Nogo-A inhibits neurite outgrowth and cell spreading with three discrete regions.

Nogo-A is a potent neurite growth inhibitor in vitro and plays a role both in the restriction of axonal regeneration after injury and in structural plasticity in the CNS of higher vertebrates. The regions that mediate inhibition and the topology of the molecule in the plasma membrane have to be defined. Here we demonstrate the presence of three different active sites: (1) an N-terminal region involved in the inhibition of fibroblast spreading, (2) a stretch encoded by the Nogo-A-specific exon that restricts neurite outgrowth and cell spreading and induces growth cone collapse, and (3) a C-terminal region (Nogo-66) with growth cone collapsing function. We show that Nogo-A-specific active fragments bind to the cell surface of responsive cells and to rat brain cortical membranes, suggesting the existence of specific binding partners or receptors. Several antibodies against different epitopes on the Nogo-A-specific part of the protein as well as antisera against the 66 aa loop in the C-terminus stain the cell surface of living cultured oligodendrocytes. Nogo-A is also labeled by nonmembrane-permeable biotin derivatives applied to living oligodendrocyte cultures. Immunofluorescent staining of intracellular, endoplasmic reticulum-associated Nogo-A in cells after selective permeabilization of the plasma membrane reveals that the epitopes of Nogo-A, shown to be accessible at the cell surface, are exposed to the cytoplasm. This suggests that Nogo-A could have a second membrane topology. The two proposed topological variants may have different intracellular as well as extracellular functions.

Regeneration in the central nervous system.

Unlike neonatal axons, mammalian adult axons of the CNS do not regenerate after injury. This developmental loss of regenerative capacity, is correlated with the onset of myelination. Likewise, myelin, or myelin-associated components such as Nogo-A and myelin-associated glycoprotein (MAG) inhibit regeneration from older but not younger neurons. Identification of the molecular events responsible for this developmental loss of regenerative capacity is central to devise strategies to encourage regeneration in adults after injury. Endogenous levels of the cyclic nucleotides cAMP and cGMP have been suggested to determine the neuronal responsiveness to various axonal guidance factors. Elevating cAMP concentrations block Nogo-A or MAG induced inhibition of neurite outgrowth in older neurons, whereas suppressing cAMP levels in young neurons renders them susceptible to Nogo-A and MAG. Interestingly, elevated cAMP levels abrogated the Nogo-A and MAG mediated activation of RhoA and down regulation of Rac1 in adult neurons. In contrast, elevation of cAMP leads to the inactivation of RhoA and prevents activation of downstream effector proteins, while Rac is activated. We therefore conclude that the endogenous neuronal cAMP levels determine the neuronal responsiveness to myelin-associated neurite growth inhibitors by regulating rho GTPase activities.