ßOHB Protective Pathways in Aralar-KO Neurons and Brain: An Alternative to Ketogenic Diet.

Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier expressed in neurons, is the regulatory component of the NADH malate-aspartate shuttle. AGC1 deficiency is a neuropediatric rare disease characterized by hypomyelination, hypotonia, developmental arrest, and epilepsy. We have investigated whether ß-hydroxybutyrate (ßOHB), the main ketone body (KB) produced in ketogenic diet (KD), is neuroprotective in aralar-knock-out (KO) neurons and mice. We report that ßOHB efficiently recovers aralar-KO neurons from deficits in basal-stimulated and glutamate-stimulated respiration, effects requiring ßOHB entry into the neuron, and protects from glutamate excitotoxicity. Aralar-deficient mice were fed a KD to investigate its therapeutic potential early in development, but this approach was unfeasible. Therefore, aralar-KO pups were treated without distinction of gender with daily intraperitoneal injections of ßOHB during 5 d. This treatment resulted in a recovery of striatal markers of the dopaminergic system including dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio, and vesicular monoamine transporter 2 (VMAT2) protein. Regarding postnatal myelination, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) myelin proteins were markedly increased in the cortices of ßOHB-treated aralar-KO mice. Although brain Asp and NAA levels did not change by ßOHB administration, a 4-d ßOHB treatment to aralar-KO, but not to control, neurons led to a substantial increase in Asp (3-fold) and NAA (4-fold) levels. These results suggest that the lack of increase in brain Asp and NAA is possibly because of its active utilization by the aralar-KO brain and the likely involvement of neuronal NAA in postnatal myelination in these mice. The effectiveness of ßOHB as a therapeutic treatment in AGC1 deficiency deserves further investigation.SIGNIFICANCE STATEMENTAralar deficiency induces a fatal phenotype in humans and mice and is associated with impaired neurodevelopment, epilepsy, and hypomyelination. In neurons, highly expressing aralar, its deficiency causes a metabolic blockade hampering mitochondrial energetics and respiration. Here, we find that ßOHB, the main metabolic product in KD, recovers defective mitochondrial respiration bypassing the metabolic failure in aralar-deficient neurons. ßOHB oxidation in mitochondria boosts the synthesis of cytosolic aspartate (Asp) and NAA, which is impeded by aralar deficiency, presumably through citrate-malate shuttle. In aralar-knock-out (KO) mice, ßOHB recovers from the drastic drop in specific dopaminergic and myelin markers. The ßOHB-induced myelin synthesis occurring together with the marked increment in neuronal NAA synthesis supports the role of NAA as a lipid precursor during postnatal myelination.

Siglecs in Brain Function and Neurological Disorders.

Siglecs (Sialic acid-binding immunoglobulin-type lectins) are a I-type lectin that typically binds sialic acid. Siglecs are predominantly expressed in immune cells and generate activating or inhibitory signals. They are also shown to be expressed on the surface of cells in the nervous system and have been shown to play central roles in neuroinflammation. There has been a plethora of reviews outlining the studies pertaining to Siglecs in immune cells. However, this review aims to compile the articles on the role of Siglecs in brain function and neurological disorders. In humans, the most abundant Siglecs are CD33 (Siglec-3), Siglec-4 (myelin-associated glycoprotein/MAG), and Siglec-11, Whereas in mice the most abundant are Siglec-1 (sialoadhesin), Siglec-2 (CD22), Siglec-E, Siglec-F, and Siglec-H. This review is divided into three parts. Firstly, we discuss the general biological aspects of Siglecs that are expressed in nervous tissue. Secondly, we discuss about the role of Siglecs in brain function and molecular mechanism for their function. Finally, we collate the available information on Siglecs and neurological disorders. It is intriguing to study this family of proteins in neurological disorders because they carry immunoinhibitory and immunoactivating motifs that can be vital in neuroinflammation.

Glial Sulfatides and Neuronal Complex Gangliosides Are Functionally Interdependent in Maintaining Myelinating Axon Integrity.

Sulfatides and gangliosides are raft-associated glycolipids essential for maintaining myelinated nerve integrity. Mice deficient in sulfatide (cerebroside sulfotransferase knock-out, CST-/-) or complex gangliosides (ß-1,4-N-acetylegalactosaminyltransferase1 knock-out, GalNAc-T-/-) display prominent disorganization of proteins at the node of Ranvier (NoR) in early life and age-dependent neurodegeneration. Loss of neuronal rather than glial complex gangliosides underpins the GalNAc-T-/- phenotype, as shown by neuron- or glial-specific rescue, whereas sulfatide is principally expressed and functional in glial membranes. The similarities in NoR phenotype of CST-/-, GalNAc-T-/-, and axo-glial protein-deficient mice suggests that these glycolipids stabilize membrane proteins including neurofascin155 (NF155) and myelin-associated glycoprotein (MAG) at axo-glial junctions. To assess the functional interactions between sulfatide and gangliosides, CST-/- and GalNAc-T-/- genotypes were interbred. CST-/-× GalNAc-T-/- mice develop normally to postnatal day 10 (P10), but all die between P20 and P25, coinciding with peak myelination. Ultrastructural, immunohistological, and biochemical analysis of either sex revealed widespread axonal degeneration and disruption to the axo-glial junction at the NoR. In addition to sulfatide-dependent loss of NF155, CST-/- × GalNAc-T-/- mice exhibited a major reduction in MAG protein levels in CNS myelin compared with WT and single-lipid-deficient mice. The CST-/- × GalNAc-T-/- phenotype was fully restored to that of CST-/- mice by neuron-specific expression of complex gangliosides, but not by their glial-specific expression nor by the global expression of a-series gangliosides. These data indicate that sulfatide and complex b-series gangliosides on the glial and neuronal membranes, respectively, act in concert to promote NF155 and MAG in maintaining the stable axo-glial interactions essential for normal nerve function.SIGNIFICANCE STATEMENT Sulfatides and complex gangliosides are membrane glycolipids with important roles in maintaining nervous system integrity. Node of Ranvier maintenance in particular requires stable compartmentalization of multiple membrane proteins. The axo-glial adhesion molecules neurofascin155 (NF155) and myelin-associated glycoprotein (MAG) require membrane microdomains containing either sulfatides or complex gangliosides to localize and function effectively. The cooperative roles of these microdomains and associated proteins are unknown. Here, we show vital interdependent roles for sulfatides and complex gangliosides because double (but not single) deficiency causes a rapidly lethal phenotype at an early age. These findings suggest that sulfatides and complex gangliosides on opposing axo-glial membranes are responsible for essential tethering of the axo-glial junction proteins NF155 and MAG, which interact to maintain the nodal complex.

Expression and function of myelin-associated proteins and their common receptor NgR on oligodendrocyte progenitor cells.

Nogo-A, oligodendrocyte myelin glycoprotein (OMgp) and myelin-associated glycoprotein (MAG) are known as myelin-associated proteins that inhibit axon growth by binding a common receptor, the Nogo66 receptor (NgR). In the CNS, Nogo-A, OMgp and MAG are predominantly expressed by oligodendrocytes. As our previous study revealed that oligodendrocyte progenitor cells (OPCs) did not inhibit neurite outgrowth, it is not clear whether these myelin-associated proteins are expressed in OPCs, and what functions they perform if they are expressed in OPCs. In the present study, with OPCs induced from neural precursor cells (NPCs) derived from rat embryonic spinal cord, and oligodendrocytes differentiated from OPCs, we have observed the expression patterns of Nogo-A, OMgp, MAG and NgR in NPCs, OPCs and oligodendrocytes by immunostaining and western blot assay. We found that Nogo-A could be detected in all tested cells; OMgp could be detected in OPCs and oligodendrocytes, but not in NPCs; MAG was only detected in oligodendrocytes; while NgR could be detected in NPCs and OPCs, but not in oligodendrocytes. These results indicated that the expression pattern of MAG and NgR in OPCs was totally different from that of oligodendrocytes, which might be one of the factors that led to the discrepancy between the two cells in promoting neurite outgrowth. By respectively blocking Nogo-A, OMgp and NgR expressed on OPCs with their corresponding antibodies, we further investigated their roles in the proliferation and differentiation of OPCs, as well as the possible signal pathways involved in. Our results showed that when OPCs were cultured under proliferation condition, blocking Nogo-A, OMgp or NgR did not affect the proliferation of OPCs, but could all significantly prolong their processes. And this effect on OPC processes might involve the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. When OPCs were cultured under differentiation condition (containing tri-iodothyronine, T3), blocking Nogo-A, OMgp or NgR could all inhibit the differentiation of OPCs, and this effect might involve the extracellular signal-regulated kinases1/2 (Erk1/2) signaling pathway. These results suggested that under proliferation environment, the functions of Nogo-A, OMgp and NgR expressed in OPCs might be to control the length of processes, thus maintaining the morphology of OPCs. While in differentiation environment, the functions of Nogo-A, OMgp and NgR expressed in OPCs turned to promote the differentiation of OPCs, thus facilitating the maturation of oligodendrocytes. And NgR, as the common receptor for Nogo-A and OMgp, might be the main molecule that mediated these functions in OPCs.

Oligodendrocyte myelin glycoprotein does not influence node of ranvier structure or assembly.

Oligodendrocyte myelin glycoprotein (OMgp) is expressed by both neurons and oligodendrocytes in the CNS. It has been implicated in growth cone collapse and neurite outgrowth inhibition by signaling through the Nogo receptor and paired Ig-like receptor B (PirB). OMgp was also reported to be an extracellular matrix (ECM) protein surrounding CNS nodes of Ranvier and proposed to function as (1) an inhibitor of nodal collateral sprouting and (2) an important contributor to proper nodal and paranodal architecture. However, we show here that the anti-OMgp antiserum used in previous studies to define the functions of OMgp at nodes is not specific. Among all reported nodal ECM components, the antiserum exhibited strong cross-reactivity against versican V2 isoform, a chondroitin sulfate proteoglycan. Furthermore, the OMgp antiserum labeled OMgp-null nodes, but not nodes from versican V2-deficient mice, and preadsorption of the OMgp antiserum with recombinant versican V2 blocked nodal labeling. Analysis of CNS nodes in OMgp-null mice failed to reveal any nodal or paranodal defects, or increased nodal collateral sprouting, indicating that OMgp does not participate in CNS node of Ranvier assembly or maintenance. We successfully identified a highly specific anti-OMgp antibody and observed OMgp staining in white matter only after initiation of myelination. OMgp immunoreactivity decorated the surface of mature myelinated axons, but was excluded from compact myelin and nodes. Together, our results strongly argue against the nodal localization of OMgp and its proposed functions at nodes, and reveal OMgp's authentic localization relative to nodes and myelin.

Oligodendrocyte-myelin glycoprotein and Nogo negatively regulate activity-dependent synaptic plasticity.

In the adult mammalian CNS, the growth inhibitors oligodendrocyte-myelin glycoprotein (OMgp) and the reticulon RTN4 (Nogo) are broadly expressed in oligodendrocytes and neurons. Nogo and OMgp complex with the neuronal cell surface receptors Nogo receptor-1 (NgR1) and paired Ig-like receptor-B (PirB) to regulate neuronal morphology. In the healthy CNS, NgR1 regulates dendritic spine shape and attenuates activity-driven synaptic plasticity at Schaffer collateral-CA1 synapses. Here, we examine whether Nogo and OMgp influence functional synaptic plasticity, the efficacy by which synaptic transmission occurs. In acute hippocampal slices of adult mice, Nogo-66 and OMgp suppress NMDA receptor-dependent long-term potentiation (LTP) when locally applied to Schaffer collateral-CA1 synapses. Neither Nogo-66 nor OMgp influences basal synaptic transmission or paired-pulse facilitation, a form of short-term synaptic plasticity. PirB(-/-) and NgR1(-/-) single mutants and NgR1(-/-);PirB(-/-) double mutants show normal LTP, indistinguishable from wild-type controls. In juvenile mice, LTD in NgR1(-/-), but not PirB(-/-), slices is absent. Mechanistic studies revealed that Nogo-66 and OMgp suppress LTP in an NgR1-dependent manner. OMgp inhibits LTP in part through PirB but independently of p75. This suggests that NgR1 and PirB participate in ligand-dependent inhibition of synaptic plasticity. Loss of NgR1 leads to increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), signaling intermediates known to regulate neuronal growth and synaptic function. In primary cortical neurons, BDNF elicited phosphorylation of AKT and p70S6 kinase is attenuated in the presence of myelin inhibitors. Collectively, we provide evidence that mechanisms of neuronal growth inhibition and inhibition of synaptic strength are related. Thus, myelin inhibitors and their receptors may coordinate structural and functional neuronal plasticity in CNS health and disease.

Bex1 is involved in the regeneration of axons after injury.

Successful axonal regeneration is a complex process determined by both axonal environment and endogenous neural capability of the regenerating axons in the central and the peripheral nervous systems. Numerous external inhibitory factors inhibit axonal regeneration after injury. In response, neurons express various regeneration-associated genes to overcome this inhibition and increase the intrinsic growth capacity. In the present study, we show that the brain-expressed X-linked (Bex1) protein was over-expressed as a result of peripheral axonal damage. Bex1 antagonized the axon outgrowth inhibitory effect of myelin-associated glycoprotein. The involvement of Bex1 in axon regeneration was further confirmed in vivo. We have demonstrated that Bex1 knock-out mice showed lower capability for regeneration after peripheral nerve injury than wild-type animals. Wild-type mice could recover from sciatic nerve injury much faster than Bex1 knock-out mice. Our findings suggest that Bex1 could be considered as regeneration-associated gene.

Asymmetric endocytosis and remodeling of beta1-integrin adhesions during growth cone chemorepulsion by MAG.

Gradients of chemorepellent factors released from myelin may impair axon pathfinding and neuroregeneration after injury. We found that, analogously to the process of chemotaxis in invasive tumor cells, axonal growth cones of Xenopus spinal neurons modulate the functional distribution of integrin receptors during chemorepulsion induced by myelin-associated glycoprotein (MAG). A focal MAG gradient induced polarized endocytosis and concomitant asymmetric loss of beta(1)-integrin and vinculin-containing adhesions on the repellent side during repulsive turning. Loss of symmetrical beta(1)-integrin function was both necessary and sufficient for chemorepulsion, which required internalization by clathrin-mediated endocytosis. Induction of repulsive Ca(2+) signals was necessary and sufficient for the stimulated rapid endocytosis of beta(1)-integrin. Altogether, these findings identify beta(1)-integrin as an important functional cargo during Ca(2+)-dependent rapid endocytosis stimulated by a diffusible guidance cue. Such dynamic redistribution allows the growth cone to rapidly adjust adhesiveness across its axis, an essential feature for initiating chemotactic turning.

Assessing spinal axon regeneration and sprouting in Nogo-, MAG-, and OMgp-deficient mice.

A central hypothesis for the limited capacity for adult central nervous system (CNS) axons to regenerate is the presence of myelin-derived axon growth inhibitors, the role of which, however, remains poorly understood. We have conducted a comprehensive genetic analysis of the three major myelin inhibitors, Nogo, MAG, and OMgp, in injury-induced axonal growth, including compensatory sprouting of uninjured axons and regeneration of injured axons. While deleting any one inhibitor in mice enhanced sprouting of corticospinal or raphespinal serotonergic axons, there was neither associated behavioral improvement nor a synergistic effect of deleting all three inhibitors. Furthermore, triple-mutant mice failed to exhibit enhanced regeneration of either axonal tract after spinal cord injury. Our data indicate that while Nogo, MAG, and OMgp may modulate axon sprouting, they do not play a central role in CNS axon regeneration failure.

Myelin-associated glycoprotein mediates membrane fusion and entry of neurotropic herpesviruses.

Varicella-zoster virus (VZV) and herpes simplex virus (HSV) are prevalent neurotropic herpesviruses that cause various nervous system diseases. Similar to other enveloped viruses, membrane fusion is an essential process for viral entry. Therefore, identification of host molecules that mediate membrane fusion is important to understand the mechanism of viral infection. Here, we demonstrate that myelin-associated glycoprotein (MAG), mainly distributed in neural tissues, associates with VZV glycoprotein B (gB) and promotes cell-cell fusion when coexpressed with VZV gB and gH/gL. VZV preferentially infected MAG-transfected oligodendroglial cells. MAG also associated with HSV-1 gB and enhanced HSV-1 infection of promyelocytes. These findings suggested that MAG is involved in VZV and HSV infection of neural tissues.

Axonal protective effects of the myelin-associated glycoprotein.

Progressive axonal degeneration follows demyelination in many neurological diseases, including multiple sclerosis and inherited demyelinating neuropathies, such as Charcot-Marie-Tooth disease. One glial molecule, the myelin-associated glycoprotein (MAG), located in the adaxonal plasmalemma of myelin-producing cells, is known to signal to the axon and to modulate axonal caliber through phosphorylation of axonal neurofilament proteins. This report establishes for the first time that MAG also promotes resistance to axonal injury and prevents axonal degeneration both in cell culture and in vivo. This effect on axonal stability depends on the RGD domain around arginine 118 in the extracellular portion of MAG, but it is independent of Nogo signaling in the axon. Exploiting this pathway may lead to therapeutic strategies for neurological diseases characterized by axonal loss.

A hypothesis about the relationship of myelin-associated glycoprotein's function in myelinated axons to its capacity to inhibit neurite outgrowth.

The myelin-associated glycoprotein (MAG) is selectively localized in periaxonal Schwann cell and oligodendroglial membranes of myelin sheaths suggesting that it functions in glia-axon interactions in the PNS and CNS, and this is supported by much experimental evidence. In addition, MAG is now well known as one of several white matter inhibitors of neurite outgrowth in vitro and axonal regeneration in vivo, and this latter area of research has provided a substantial amount of information about neuronal receptors or receptor complexes for MAG. This article makes the hypothesis that the capacity of MAG to inhibit outgrowth of immature developing or regenerating neurites is an aberration of its normal physiological function to promote the maturation, maintenance, and survival of myelinated axons. The overview summarizes the literature on the function of MAG in PNS and CNS myelin sheaths and its role as an inhibitor of neurite outgrowth to put this hypothesis into perspective. Additional research is needed to determine if receptors and signaling systems similar to those responsible for MAG inhibition of neurite outgrowth also promote the maturation, maintenance, and survival of myelinated axons as hypothesized here, or if substantially different MAG-mediated signaling mechanisms are operative at the glia-axon junction.

Nogo-A and myelin-associated glycoprotein differently regulate oligodendrocyte maturation and myelin formation.

Nogo-A is one of the most potent oligodendrocyte-derived inhibitors for axonal regrowth in the injured adult CNS. However, the physiological function of Nogo-A in development and in healthy oligodendrocytes is still unknown. In the present study, we investigated the role of Nogo-A for myelin formation in the developing optic nerve. By quantitative real-time PCR, we found that the expression of Nogo-A increased faster in differentiating oligodendrocytes than that of the major myelin proteins MBP (myelin basic protein), PLP (proteolipid protein)/DM20, and CNP (2',3'-cyclic nucleotide 3'-phosphodiesterase). The analysis of optic nerves and cerebella of mice deficient for Nogo-A (Nogo-A(-/-)) revealed a marked delay of oligodendrocyte differentiation, myelin sheath formation, and axonal caliber growth within the first postnatal month. The combined deletion of Nogo-A and MAG caused a more severe transient hypomyelination. In contrast to MAG(-/-) mice, Nogo-A(-/-) mutants did not present abnormalities in the structure of myelin sheaths and Ranvier nodes. The common binding protein for Nogo-A and MAG, NgR1, was exclusively upregulated in MAG(-/-) animals, whereas the level of Lingo-1, a coreceptor, remained unchanged. Together, our results demonstrate that Nogo-A and MAG are differently involved in oligodendrocyte maturation in vivo, and suggest that Nogo-A may influence also remyelination in pathological conditions such as multiple sclerosis.

BDNF activates CaMKIV and PKA in parallel to block MAG-mediated inhibition of neurite outgrowth.

The environment of the adult CNS prevents axonal regeneration after injury. This inhibition of axonal regeneration can be blocked by elevating cAMP. Previously, we showed that the cAMP pathway can be activated via pre-treatment with neurotrophins and requires activation of several signaling pathways which converge at activation of the transcription factor, CREB. Here, we show that calcium/calmodulin-dependent kinase IV (CaMKIV) is necessary for the neurotrophin-induced phosphorylation of CREB and the block of myelin-mediated inhibition of axonal growth. Pharmacological inhibition of CaMKIV or over-expression of a dominant-negative mutant form of CaMKIV blocks the neurotrophin effect. Interestingly, CaMKIV activation is not necessary if cAMP levels is already elevated. Finally, calcium flux from intracellular stores is necessary for this CaMKIV signaling. These results demonstrate that CaMKIV is another player in the neurotrophin-induced signaling which leads to axonal regeneration and therefore, is a potential target for therapeutic intervention following injury to the adult CNS.

Transient 5-(4-phenylbutoxy)psoralen (PAP-1) treatment dissociates developing pathologies in autoimmune optic neuritis into two distinct pathology profiles.

Discovery of treatments to protect axonal function of neurons and prevent permanent disability associated with progressive multiple sclerosis (MS) has faced the uphill challenge of assessing relatively small changes in accumulated axon damage within a background environment that is disorganized by CNS inflammation. We hypothesized that transient immunosuppression after initiation of MS-like autoimmune mechanisms would disassociate development of MS-like myelinated axon pathology from development of CNS inflammation in a rat model of autoimmune optic neuritis (AON). A rat model of myelin oligodendrocyte glycoprotein peptide-induced AON was transiently treated (on days 3-7 after antigen exposure) with 5-(4-phenylbutoxy)psoralen (PAP-1), an immunomodulatory drug previously shown specifically to suppress proliferation of effector memory T-cells and immunoglobulin class-switched B-cells. Thirteen days after antigen exposure, optic nerves were harvested for quantitative assessment of 12 MS-associated pathologies using microfluorimetry. With one exception, the immunoreactivities (-ir) for eight markers of MS-like neuroinflammation and immune infiltration were significantly reduced (P < 0.05) by transient PAP-1 treatment, often to levels significantly below those detected in normal control rat optic nerves. With one exception, four immunoreactive markers of MS-like myelinated axon pathology were detected at levels indicating increased axon/myelin pathology compared with vehicle-treated rats with AON (P < 0.05). These data suggest the conclusion that early causative mechanisms in CNS autoimmunity initiate signaling mechanisms that diverge into two separate pathways, one that is strongly associated with inflammatory responses and one that is associated predominantly with disturbed axon-myelin interactions and impaired fast axonal transport.

Rap1 is involved in the signal transduction of myelin-associated glycoprotein.

Myelin-associated glycoprotein (MAG) is a well-characterized axon growth inhibitor in the adult vertebrate nervous system. Several signals that play roles in inhibiting axon growth have been identified. Here, we report that soluble MAG induces activation of Rap1 in postnatal cerebellar granule neurons (CGNs) and dorsal root ganglion (DRG) neurons. The p75 receptor associates with activated Rap1 and is internalized in response to MAG. After MAG is applied to the distal axons of the sciatic nerves, the activated Rap1, internalized p75 receptor, and MAG are retrogradely trafficked via axons to the cell bodies of the DRG neurons. Rap1 activity is required for survival of the DRG neurons as well as CGNs when treated with MAG. The transport of the signaling complex containing the p75 receptor and Rap1 may play a role in the effect of MAG.

[Molecular mechanism and regulation of axon growth inhibition].

In the adult mammalian central nervous system (CNS), it is well known that injured axons exhibit very limited regeneration ability. Due to this lack of appropriate axonal regeneration, a traumatic damage to the adult brain and spinal cord frequently causes permanent neuronal deficits such as paralysis. Several axon growth inhibitors, including myelin-associated glycoprotein, Nogo, and oligodensrocyte myelin glycoprotein, in the CNS have been identified in the myelin. Receptor complex comprising of the Nogo receptor, the p75 receptor, and LINGO-1 transduces the signals from all of these inhibitors in vitro. Downstream of these inhibitors, activation of small GTPase RhoA and its effector Rho-kinase has been shown to be a key element for neurite growth inhibition and growth cone collapse elicited by these inhibitors. Consistent with these findings in vitro, inhibition of RhoA or Rho-kinase in vivo promotes axon growth and functional recovery after spinal cord injury. Recently, several developmental guidance proteins, including repulsive guidance molecules, semaphorin, and ephrin are suggested to be involved in axon growth inhibition after injury to the CNS. Thus, multiple axon growth inhibitors seem to contribute to inability of the injured axons to regenerate, and therapeutic strategy to block the multiple axon growth inhibitors may provide efficient tools that produce functional regeneration following injuries to the CNS. In addition, it is noted that synaptic plasticity in pre-existing pathways and the formation of new circuits through collateral sprouting of lesioned and unlesioned fibers are important components of the spontaneous recovery process. The molecular mechanism of this phenomenon is poorly understood, and elucidation of this will contribute to enhancement of functional recovery after incomplete injury to the CNS. I will summarize recent findings regarding these issues.

MUC1 is a counter-receptor for myelin-associated glycoprotein (Siglec-4a) and their interaction contributes to adhesion in pancreatic cancer perineural invasion.

Perineural invasion in pancreatic adenocarcinoma, a common pathologic phenomenon whereby cancer cells invade and intimately contact the endoneurium of pancreatic nerves, is thought to contribute to both pain and local disease recurrence. MUC1, a type I transmembrane mucin that can affect the adhesive properties of cells, contains a large extracellular tandem repeat domain, which is heavily glycosylated in normal epithelia, but is overexpressed and differentially glycosylated in pancreatic cancer. This altered glycosylation includes the shortened core I O-glycans for monosialyl and disialyl T antigens. Myelin-associated glycoprotein (MAG), a membrane-bound protein expressed on oligodendrocytes and Schwann cells, binds myelin to neurons. MAG's preferred ligands are derivatives of the monosialyl and disialyl T antigen. We investigated whether MUC1 is a counter-receptor for MAG and if their interaction contributed to pancreatic perineural invasion. Results showed that MAG binds pancreatic cells expressing MUC1, that this binding is sialidase-sensitive, and that MAG physically associates with MUC1. Heterotypic adhesion assays between pancreatic cancer cells and Schwann cells revealed that increased expression of MUC1 or MAG enhanced adhesion. Conversely, specific inhibition of MAG or sialyl-T MUC1 partially blocked adhesion. Immunohistochemical analysis of pancreatic perineural invasion showed the expression of both MUC1 and MAG. These results support the hypothesis that the adhesive interactions between MUC1 and MAG are of biological significance in pancreatic cancer perineural invasion.

The Nogo-66 receptor NgR1 is required only for the acute growth cone-collapsing but not the chronic growth-inhibitory actions of myelin inhibitors.

Neuronal Nogo-66 receptor 1 (NgR1) has been proposed to function as an obligatory coreceptor for the myelin-derived ligands Nogo-A, oligodendrocyte myelin glycoprotein (OMgp), and myelin-associated glycoprotein (MAG) to mediate neurite outgrowth inhibition by these ligands. To examine the contribution of neuronal NgR1 to outgrowth inhibition, we used two different strategies, genetic ablation of NgR1 through the germline and transient short hairpin RNA interference (shRNAi)-mediated knock-down. To monitor growth inhibition, two different paradigms were used, chronic presentation of substrate-bound inhibitor to measure neurite extension and acute application of soluble inhibitor to assay growth cone collapse. We find that regardless of the NgR1 genotype, membrane-bound MAG strongly inhibits neurite outgrowth of primary cerebellar, sensory, and cortical neurons. Similarly, substrate-bound OMgp strongly inhibits neurite outgrowth of NgR1 wild-type and mutant sensory neurons. Consistent with these results, shRNAi-mediated knock-down of neuronal NgR1 does not result in a substantial release of L-MAG (large MAG) inhibition. When applied acutely, however, MAG-Fc and OMgp-Fc induce a modest degree of growth cone collapse that is significantly attenuated in NgR1-null neurons compared with wild-type controls. Based on our findings and previous studies with Nogo-66, we propose that neuronal NgR1 has a circumscribed role in regulating cytoskeletal dynamics after acute exposure to soluble MAG, OMgp, or Nogo-66, but is not required for these ligands to mediate their growth-inhibitory properties in chronic outgrowth experiments. Our results thus provide unexpected evidence that the growth cone-collapsing activities and substrate growth-inhibitory activities of inhibitory ligands can be dissociated. We also conclude that chronic axon growth inhibition by myelin is mediated by NgR1-independent mechanisms.