Adult mammalian axon regeneration is generally successful in the peripheral nervous system (PNS) but is dismally poor in the central nervous system (CNS). However, many classes of CNS axons can extend for long distances in peripheral nerve grafts. A comparison of myelin from the CNS and the PNS has revealed that CNS white matter is selectively inhibitory for axonal outgrowth. Several components of CNS white matter, NI35, NI250(Nogo) and MAG, that have inhibitory activity for axon extension have been described. The IN-1 antibody, which recognizes NI35 and NI250(Nogo), allows moderate degrees of axonal regeneration and functional recovery after spinal cord injury. Here we identify Nogo as a member of the Reticulon family, Reticulon 4-A. Nogo is expressed by oligodendrocytes but not by Schwann cells, and associates primarily with the endoplasmic reticulum. A 66-residue lumenal/extracellular domain inhibits axonal extension and collapses dorsal root ganglion growth cones. In contrast to Nogo, Reticulon 1 and 3 are not expressed by oligodendrocytes, and the 66-residue lumenal/extracellular domains from Reticulon 1, 2 and 3 do not inhibit axonal regeneration. These data provide a molecular basis to assess the contribution of Nogo to the failure of axonal regeneration in the adult CNS.

Innate immunity is an evolutionarily ancient system that provides organisms with immediately available defense mechanisms through recognition of pathogen-associated molecular patterns. We show that in the CNS, specific activation of innate immunity through a Toll-like receptor 4 (TLR4)-dependent pathway leads to neurodegeneration. We identify microglia as the major lipopolysaccharide (LPS)-responsive cell in the CNS. TLR4 activation leads to extensive neuronal death in vitro that depends on the presence of microglia. LPS leads to dramatic neuronal loss in cultures prepared from wild-type mice but does not induce neuronal injury in CNS cultures derived from tlr4 mutant mice. In an in vivo model of neurodegeneration, stimulating the innate immune response with LPS converts a subthreshold hypoxic-ischemic insult from no discernable neuronal injury to severe axonal and neuronal loss. In contrast, animals bearing a loss-of-function mutation in the tlr4 gene are resistant to neuronal injury in the same model. The present study demonstrates a mechanistic link among innate immunity, TLRs, and neurodegeneration. Systemic infection is associated with sustained worsening in many diseases of the CNS, yet the molecular and cellular relationship between infection outside the CNS and potential neuronal loss within the CNS is elusive. Activation of microglia, bone marrow-derived macrophage-like cells that function as the resident immune defense system of the brain (1), is a characteristic feature of most neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, multiple sclerosis, AIDS dementia complex, and amyotrophic lateral sclerosis as well as ischemia and posttraumatic brain injury (2-4). Neurotoxicity induced by ␤-amyloid or HIV proteins in mixed CNS cultures depends on the presence and activation of microglia (5, 6). Liberatore et al. (7) demonstrated in vivo that microglial inducible nitric oxide synthase plays a crucial role in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration in the MPTP mouse model of Parkinson's disease.The evolutionarily ancient innate immune system provides the first line of host defense against a large variety of pathogens and also controls many aspects of the adaptive immune response (8). Cells of the innate immune system recognize invariant molecular structures of pathogens termed pathogen-associated molecular patterns through a series of genetically conserved and stable cell-surface receptors related to the Drosophila gene toll that thus are referred to as Toll-like receptors (TLRs) (9).TLR4 functions as the signal-transducing receptor for the endotoxin lipopolysaccharide (LPS) (10), which is a major component of the outer membrane of Gram-negative bacteria. LPS binds to the serum protein LPS-binding protein and the soluble or glycosylphosphatidylinositol-anchored CD14. This complex in turn binds to TLR4 (11) and initiates an intracellular signaling pathway that regulates gene expression through derepression of the transcriptional a...

The immediate or innate immune response is the first line of defense against diverse microbial pathogens and requires the expression of recently discovered toll-like receptors (TLRs). TLR4 serves as a specific receptor for lipopolysaccharide (LPS) and is localized on the surface of a subset of mammalian cells. Although innate immunity is a necessary host defense against microbial pathogens, the consequences of its activation in the CNS can be deleterious, as we show here in a developing neural model. We examined the major non-neuronal cell types in the CNS for expression of TLR4 and found that microglia expressed high levels, whereas astrocytes and oligodendrocytes expressed none. Consistent with TLR4 expression solely in microglia, we show that microglia are the only CNS glial cells that bind fluorescently tagged lipopolysaccharide. Lipopolysaccharide led to extensive oligodendrocyte death in culture only under conditions in which microglia were present. To determine whether TLR4 is necessary for lipopolysaccharide-induced oligodendrocyte death in mixed glial cultures, we studied cultures generated from mice bearing a loss-of-function mutation in the tlr4 gene. Lipopolysaccharide failed to induce oligodendrocyte death in such cultures, in contrast to the death induced in cultures from wild-type mice. Finally, stereotactic intracerebral injection of lipopolysaccharide into the developing pericallosal white matter of immature rodents resulted in loss of oligodendrocytes and hypomyelination and periventricular cysts. Our data provide a general mechanistic link between (1) lipopolysaccharide and similar microbial molecular motifs and (2) injury to oligodendrocytes and myelin as occurs in periventricular leukomalacia and multiple sclerosis.

Failure of remyelination is largely responsible for sustained neurologic symptoms in multiple sclerosis (MS). MS lesions contain hyaluronan deposits that inhibit oligodendrocyte precursor cell (OPC) maturation. However, the mechanism behind this inhibition is unclear. We report here that Toll-like receptor 2 (TLR2) is expressed by oligodendrocytes and is up-regulated in MS lesions. Pathogenderived TLR2 agonists, but not agonists for other TLRs, inhibit OPC maturation in vitro. Hyaluronan-mediated inhibition of OPC maturation requires TLR2 and MyD88, a TLR2 adaptor molecule. Ablated expression of TLR2 also enhances remyelination in a lysolecithin animal model. Hyaluronidases expressed by OPCs degrade hyaluronan to hyaluronan oligomers, a requirement for hyaluronan/TLR2 signaling. MS lesions contain both TLR2 + oligodendrocytes and lowmolecular-weight hyaluronan, consistent with their importance to remyelination in MS. We thus have defined a mechanism controlling remyelination failure in MS where hyaluronan is degraded by hyaluronidases into hyaluronan oligomers that block OPC maturation and remyelination through TLR2-MyD88 signaling.hyaluronidase | MyD88 | innate immunity I n multiple sclerosis (MS), destruction of CNS myelin accounts for a majority of neurologic symptoms. MS patients typically exhibit a relapsing/remitting course in which relapses result from inflammation and demyelination and remissions result from resolution of inflammation and remyelination. Secondary progressive MS, which typically begins in patients after a decade of relapsing/ remitting MS, exhibits irreversible neurologic disability.Most chronic MS lesions show little if any remyelination. The failure of remyelination in MS theoretically could be the consequence of a deficiency in the number of oligodendrocyte progenitor cells (OPCs), absence of a promyelination signal, or the presence of inhibitory influences on OPCs. It therefore is of interest that the histopathology of the MS lesion has revealed the presence of OPCs and premyelinating oligodendrocytes in chronic MS lesions. The premyelinating oligodendrocytes extend processes that contact but fail to myelinate axons (1-4), thus suggesting failure of remyelination is caused by the loss of promyelination signals or the presence of inhibitory signals.Recently, the glycosaminoglycan hyaluronan was identified within MS lesions and found to inhibit OPC maturation and remyelination in an MS animal model (5). The mechanism underlying this inhibition is unknown. Because hyaluronan can function as an endogenous mammalian ligand for Toll-like receptors (TLRs) 2 and 4 in innate immune cell activation (6-9), we reasoned that TLR stimulation also may be required for hyaluronan-mediated blocking of OPC maturation.Although TLRs and the Drosophila ortholog Toll have welldescribed functions in innate immune cells (6-12), TLRs also have potent functions outside the immune system. Toll and TLR have diverse roles in axonal pathfinding, dorsoventral patterning, and cell-fate determination (13). In particul...

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Axon regeneration in the adult CNS is limited by the presence of inhibitory proteins. An interaction of Nogo on the oligodendrocyte surface with Nogo-66 Receptor (NgR) on axons has been suggested to play an important role in limiting axonal growth. Here, we compare the localization of these two proteins immunohistochemically as a test of this hypothesis. Throughout much of the adult CNS, Nogo-A is detected on oligodendrocyte processes surrounding myelinated axons, including areas of axon-oligodendrocyte contact. The NgR protein is detected selectively in neurons and is present throughout axons, indicating that Nogo-A and its receptor are juxtaposed along the course of myelinated fibers. NgR protein expression is restricted to postnatal neurons and their axons. In contrast, Nogo-A is observed in myelinating oligodendrocytes, embryonic muscle, and neurons, suggesting that Nogo-A has additional physiologic roles unrelated to NgR binding. After spinal cord injury, Nogo-A is upregulated to a moderate degree, whereas NgR levels are maintained at constant levels. Taken together, these data confirm the apposition of Nogo ligand and NgR receptor in situations of limited axonal regeneration and support the hypothesis that this system regulates CNS axonal plasticity and recovery from injury.

Reactive microglia in the CNS have been implicated in the pathogenesis of white matter disorders, such as periventricular leukomalacia and multiple sclerosis. However, the mechanism by which activated microglia kill oligodendrocytes (OLs) remains elusive. Here we show that lipopolysaccharide (LPS)-induced death of developing OLs is caused by microglia-derived peroxynitrite, the reaction product of nitric oxide (NO) and superoxide anion. Blocking peroxynitrite formation with nitric oxide synthase inhibitors, superoxide dismutase mimics, or a decomposition catalyst abrogated the cytotoxicity. Only microglia, but not OLs, expressed inducible NO synthase (iNOS) after LPS challenge; microglia from iNOS knockout mice were not cytotoxic upon activation. The molecular source for superoxide was identified as the superoxidegenerating enzyme NADPH oxidase. The oxidase was activated upon LPS exposure, and its inhibition prevented microglial toxicity toward OLs. Furthermore, microglia isolated from mice deficient in the catalytic component of the oxidase, gp91 phox , failed to induce cell death. Our results reveal a role for NADPH oxidase in LPSinduced OL death and suggest that peroxynitrite produced by iNOS and NADPH oxidase in activated microglia may play an important role in the pathogenesis of white matter disorders.inflammation ͉ lipopolysaccharide ͉ cerebral palsy ͉ reactive nitrogen species ͉ periventricular leukomalacia M icroglia are the resident macrophage-like cells of the CNS.They play a pivotal role in the innate immune response of CNS and are the first line of defense against microorganism invasion and injury (1). Localized activation of microglia, however, has also been implicated in the pathogenesis of a number of neurological diseases and disorders, including ischemia; AIDS-associated dementia (2); chronic neurodegenerative diseases (3), such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis; and white matter disorders, such as multiple sclerosis (4) and periventricular leukomalacia (PVL) (5). Microglia are extremely responsive to environmental or immunological challenges and are the predominant cell type producing inflammation-mediated neurodegeneration. Upon activation, microglia release proinflammatory molecules, such as cytokines and chemokines, and mediators of cell injury, such as proteases and reactive oxygen species (ROS)͞reactive nitrogen species (6-8). Although necessary for normal functions, activation of microglia requires tight regulation to avoid bystander injury to other CNS cells. The identification of primary toxic factors, localization of their molecular sources, and the elucidation of signaling pathways associated with microglial overactivation are therefore of paramount importance to our understanding of cellular injury in the CNS.PVL is the lesion of cerebral white matter that underlies most cases of cerebral palsy developing in premature infants (9). It is characterized by focal necrotic lesions in the deep white matter and by diffuse injury to premyelinatin...

Toll receptors in Drosophila melanogaster function in morphogenesis and host defense. Mammalian orthologues of Toll, the Toll-like receptors (TLRs), have been studied extensively for their essential functions in controlling innate and adaptive immune responses. We report that TLR8 is dynamically expressed during mouse brain development and localizes to neurons and axons. Agonist stimulation of TLR8 in cultured cortical neurons causes inhibition of neurite outgrowth and induces apoptosis in a dissociable manner. Our evidence indicates that such TLR8-mediated neuronal responses do not involve the canonical TLR–NF-κB signaling pathway. These findings reveal novel functions for TLR8 in the mammalian nervous system that are distinct from the classical role of TLRs in immunity.