The NOGO receptor NgR2, a novel αVß3 integrin effector, induces neuroendocrine differentiation in prostate cancer.

Androgen deprivation therapies aimed to target prostate cancer (PrCa) are only partially successful given the occurrence of neuroendocrine PrCa (NEPrCa), a highly aggressive and highly metastatic form of PrCa, for which there is no effective therapeutic approach. Our group has demonstrated that while absent in prostate adenocarcinoma, the αVß3 integrin expression is increased during PrCa progression toward NEPrCa. Here, we show a novel pathway activated by αVß3 that promotes NE differentiation (NED). This novel pathway requires the expression of a GPI-linked surface molecule, NgR2, also known as Nogo-66 receptor homolog 1. We show here that NgR2 is upregulated by αVß3, to which it associates; we also show that it promotes NED and anchorage-independent growth, as well as a motile phenotype of PrCa cells. Given our observations that high levels of αVß3 and, as shown here, of NgR2 are detected in human and mouse NEPrCa, our findings appear to be highly relevant to this aggressive and metastatic subtype of PrCa. This study is novel because NgR2 role has only minimally been investigated in cancer and has instead predominantly been analyzed in neurons. These data thus pave new avenues toward a comprehensive mechanistic understanding of integrin-directed signaling during PrCa progression toward a NE phenotype.

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.

The Prenylflavonoid ENDF1 Overrules Central Nervous System Growth Inhibitors and Facilitates Regeneration of DRG Neurons.

Restoration of neuronal connectivity after lesion of the central nervous system, such as spinal cord injury, is one of the biggest challenges in modern medicine. In particular, the accumulation of axon growth inhibitory factors at the site of injury constitutes a major obstacle to structural and thus functional repair. We previously investigated a group of prenylflavonoids derived from hops for their capacity to promote neuroregeneration. We identified a molecule called ENDF1 that was very potent to enhance regrowth and branching of neurites from dorsal root ganglion neurons in culture on growth promoting substrates. In the present study, we investigated ENDF1's capacity to promote regeneration of rat dorsal root ganglion neurons in vitro in the presence of three main components of the extracellular matrix acting as axon growth inhibitors: Semaphorin 3A, Ephrin A4 and mixed chondroitin sulfate proteoglycans. We report that ENDF1 application significantly promoted the percentages of sensory neurons able to regrow their neurites regardless of the presence of those inhibitors, and this to an extent similar to the one obtained after NGF treatment. Moreover, ENDF1 strongly enhanced the total neurite length and the complexity of neurites extending from neurons challenged with axon growth inhibitors. Although the impact of NGF and ENDF1 on the regeneration of neurons was similar, the activity of ENDF1 was not mediated by signaling through the TrkA receptor, indicating that each molecule act through different signaling pathways. In addition, ENDF1 did not decrease the phosphorylation of cofilin, a downstream effector of the regeneration-associated RhoA/ROCK signaling pathway. Hence, ENDF1 is a potent pro-neuroregenerative factors that could help in identifying new efficient targets for regenerative therapies of the nervous system.

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.

Chroman-like cyclic prenylflavonoids promote neuronal differentiation and neurite outgrowth and are neuroprotective.

Flavonoids target a variety of pathophysiological mechanisms and are therefore increasingly considered as compounds encompassed with therapeutic potentials in diseases such as cancer, diabetes, arteriosclerosis, and neurodegenerative diseases and mood disorders. Hops (Humulus lupulus L.) is rich in flavonoids such as the flavanone 8-prenylnaringenin, which is the most potent phytoestrogen identified so far, and the prenylchalcone xanthohumol, which has potent tumor-preventive, anti-inflammatory and antiviral activities. In the present study, we questioned whether hops-derived prenylflavonoids and synthetic derivatives thereof act on neuronal precursor cells and neuronal cell lines to induce neuronal differentiation, neurite outgrowth and neuroprotection. Therefore, mouse embryonic forebrain-derived neural precursors and Neuro2a neuroblastoma-derived cells were stimulated with the prenylflavonoids of interest, and their potential to activate the promoter of the neuronal fate-specific doublecortin gene and to stimulate neuronal differentiation and neurite outgrowth was analyzed. In this screening, we identified highly "neuroactive" compounds, which we termed "enhancement of neuronal differentiation factors" (ENDFs). The most potent molecule, ENDF1, was demonstrated to promote neuronal differentiation of neural stem cells and neurite outgrowth of cultured dorsal root ganglion neurons and protected neuronal PC12 cells from cobalt chloride-induced as well as cholinergic neurons of the nucleus basalis of Meynert from deafferentation-induced cell death. The results indicate that hops-derived prenylflavonoids such as ENDFs might be powerful molecules to promote neurogenesis, neuroregeneration and neuroprotection in cases of chronic neurodegenerative diseases, acute brain and spinal cord lesion and age-associated cognitive impairments.

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.

Rho-independent stimulation of axon outgrowth and activation of the ERK and Akt signaling pathways by C3 transferase in sensory neurons.

Peripheral nerve injury triggers the activation of RhoA in spinal motor and peripheral sensory neurons. RhoA activates a number of effector proteins including the Rho-associated kinase, ROCK, which targets the cytoskeleton and leads to inhibition of neurite outgrowth. Blockade of the Rho/ROCK pathway by pharmacological means improves axon regeneration after experimental injury. C3(bot) transferase, an exoenzyme produced by Clostridium botulinum, inactivates RhoA by ADP-ribosylation. It has been successfully applied in experimental CNS lesions to facilitate axon regeneration. Up to now it was not investigated thoroughly whether C3(bot) exerts positive effects on peripheral axon regeneration as well. In the present study, recombinant membrane permeable C3(bot) produced a small, but significant, axon outgrowth effect on peripheral sensory neurons dissociated from adult dorsal root ganglia (DRG) of the rat. Neuronal overexpression of C3, however, did not enhance axonal growth. Moreover, transfection of plasmids encoding dominant negative RhoA or RhoA specific shRNAs failed to increase axonal growth. Furthermore, we show that the C3(bot) mutant, C3(E174Q), which lacks RhoA inhibitory activity, still stimulates axonal growth. When analyzing possible signaling mechanisms we found that extracellular signal-regulated kinase (ERK) and Akt are activated by C3(bot) and ERK is induced by the C3(E174Q) mutant. Upregulation of kinase activities by C3(bot) occurs significantly faster than inactivation of RhoA indicating a RhoA-independent pathway of action by C3(bot). The induction of ERK signaling by C3(bot) was detected in embryonic hippocampal neurons, too. Taken together, although RhoA plays a central role for inhibition of axon outgrowth by myelin-derived inhibitors, it does not interfere with axonal growth of sensory neurons on a permissive substrate in vitro. C3(bot) blocks neuronal RhoA activity, but its positive effects on axon elongation and branching appear to be mediated by Rho independent mechanisms involving activation of axon growth promoting ERK and Akt kinases.

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-receptors NgR1 and NgR2 do not mediate regulation of CD4 T helper responses and CNS repair in experimental autoimmune encephalomyelitis.

Myelin-associated inhibition of axonal regrowth after injury is considered one important factor that contributes to regeneration failure in the adult central nervous system (CNS). Blocking strategies targeting this pathway have been successfully applied in several nerve injury models, including experimental autoimmune encephalomyelitis (EAE), suggesting myelin-associated inhibitors (MAIs) and functionally related molecules as targets to enhance regeneration in multiple sclerosis. NgR1 and NgR2 were identified as interaction partners for the myelin proteins Nogo-A, MAG and OMgp and are probably mediating their growth-inhibitory effects on axons, although the in vivo relevance of this pathway is currently under debate. Recently, alternative functions of MAIs and NgRs in the regulation of immune cell migration and T cell differentiation have been described. Whether and to what extent NgR1 and NgR2 are contributing to Nogo and MAG-related inhibition of neuroregeneration or immunomodulation during EAE is currently unknown. Here we show that genetic deletion of both receptors does not promote functional recovery during EAE and that NgR1 and NgR2-mediated signals play a minor role in the development of CNS inflammation. Induction of EAE in Ngr1/2-double mutant mice resulted in indifferent disease course and tissue damage when compared to WT controls. Further, the development of encephalitogenic CD4(+) Th1 and Th17 responses was unchanged. However, we observed a slightly increased leukocyte infiltration into the CNS in the absence of NgR1 and NgR2, indicating that NgRs might be involved in the regulation of immune cell migration in the CNS. Our study demonstrates the urgent need for a more detailed knowledge on the multifunctional roles of ligands and receptors involved in CNS regeneration failure.

Nogo-A expression in the brain of mice with cerebral malaria.

Cerebral malaria (CM) is associated with a high rate of transient or persistent neurological sequelae. Nogo-A, a protein that is highly expressed in the endoplasmic reticulum (ER) of the mammalian central nervous system (CNS), is involved in neuronal regeneration and synaptic plasticity in the injured CNS. The current study investigates the role of Nogo-A in the course of experimental CM. C57BL/6J mice were infected with Plasmodium berghei ANKA blood stages. Brain homogenates of mice with different clinical severity levels of CM, infected animals without CM and control animals were analyzed for Nogo-A up-regulation by Western blotting and immunohistochemistry. Brain regions with Nogo-A upregulation were evaluated by transmission electron microscopy. Densitometric analysis of Western blots yielded a statistically significant upregulation of Nogo-A in mice showing moderate to severe CM. The number of neurons and oligodendrocytes positive for Nogo-A did not differ significantly between the studied groups. However, mice with severe CM showed a significantly higher number of cells with intense Nogo-A staining in the brain stem. In this region ultrastructural alterations of the ER were regularly observed. Nogo-A is upregulated during the early course of experimental CM. In the brain stem of severely affected animals increased Nogo-A expression and ultrastructural changes of the ER were observed. These data indicate a role of Nogo-A in neuronal stress response during experimental CM.

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-B is associated with cytoskeletal structures in human monocyte-derived macrophages.

BACKGROUND: The reticulon Nogo-B participates in cellular and immunological processes in murine macrophages. Since leukocytes are an essential part of the immune system in health and disease, we decided to investigate the expression of Nogo-A, Nogo-B and Nogo-C in different human immune cell subpopulations. Furthermore, we analyzed the localization of Nogo-B in human monocyte-derived macrophages by indirect immunofluorescence stainings to gain further insight into its possible function. FINDINGS: We describe an association of Nogo-B with cytoskeletal structures and the base of filopodia, but not with focal or podosomal adhesion sites of monocyte-derived macrophages. Nogo-B positive structures are partially co-localized with RhoA staining and Rac1 positive membrane ruffles. Furthermore, Nogo-B is associated with the tubulin network, but not accumulated in the Golgi region. Although Nogo-B is present in the endoplasmic reticulum, it can also be translocated to large cell protrusions or the trailing end of migratory cells, where it is homogenously distributed. CONCLUSIONS: Two different Nogo-B staining patterns can be distinguished in macrophages: firstly we observed ER-independent Nogo-B localization in cell protrusions and at the trailing end of migrating cells. Secondly, the localization of Nogo-B in actin/RhoA/Rac1 positive regions supports an influence on cytoskeletal organization. To our knowledge this is the first report on Nogo-B expression at the base of filopodia, thus providing further insight into the distribution of this protein.

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.