In the adult mammalian CNS, chondroitin sulfate proteoglycans (CSPGs) and myelin–associated inhibitors (MAIs) stabilize neuronal structure and restrict compensatory sprouting following injury. The Nogo receptor family members NgR1 and NgR2 bind to MAIs and have been implicated in neuronal inhibition. Here we show that NgR1 and NgR3 bind with high–affinity to the glycosaminoglycan moiety of proteoglycans and participate in CSPG inhibition in cultured neurons. Nogo receptor triple mutants (NgR123−/−), but not single mutants, show enhanced axonal regeneration following retro–orbital optic nerve crush injury. The combined loss of NgR1 and NgR3 (NgR13−/−), but not NgR1 and NgR2 (NgR12−/−), is sufficient to mimic the NgR123−/− regeneration phenotype. Regeneration in NgR13−/− mice is further enhanced by simultaneous ablation of RPTPσ, a known CSPG receptor. Collectively, these results identify NgR1 and NgR3 as novel CSPG receptors, demonstrate functional redundancy among CSPG receptors, and provide unexpected evidence for shared mechanisms of MAI and CSPG inhibition.

Mature myeloid cells (macrophages andCD11b IntroductionMyeloid cells, such as macrophages and dendritic cells (DCs), are a prominent constituent of inflammatory infiltrates in the central nervous system (CNS) during multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). 1,2 These cells not only serve as antigen-presenting cells for the reactivation of infiltrating myelin-reactive CD4 ϩ T cells but are thought to directly inflict tissue damage through secretion of toxic factors, such as reactive oxygen species, proteases, and tumor necrosis factor-␣ (TNF-␣). 3,4 They might also recruit naive myelin-reactive T cells into the effector pool in the context of epitope spreading. 5 We and others have demonstrated that bone marrow-derived CD11c ϩ major histocompatibility complex (MHC) class II ϩ DCs accumulate in the CNS during EAE and have the capacity to polarize naive T cells along encephalitogenic Th1 and Th17 lineages. 2,6 However, the circulating cell that gives rise to CNS-infiltrating DCs and macrophages has yet to be defined. The specific chemokine pathways and adhesion molecule interactions required for infiltration of the CNS by myeloid cells will depend on whether they cross the blood-brain barrier as immature monocytes or as macrophages and DCs. Therefore, identification of the differentiation status of the migrating cell holds implications regarding candidate therapeutic targets in neuroinflammatory diseases, such as multiple sclerosis (MS).Under steady-state conditions, mature myeloid lineages are maintained within lymphoid and peripheral tissues through controlled release of bone marrow progenitors/precursors into the peripheral circulation. 7 In the setting of infection or injury, myeloid cell mobilization is accelerated to meet the demands imposed by the increased turnover of macrophages and DCs at the site of inflammation. 8,9 The pathways underlying expansion of peripheral myeloid cell pools under stress are thought to serve an adaptive role by reinforcing host protection against infectious agents and by promoting wound healing. 8 Conversely, leukocyte-mobilizing pathways might be subverted to sustain target organ inflammation during relapsing or chronic autoimmune disease. For example, the number of macrophages and DCs in the CNS contracts during remissions and rebounds during exacerbations of EAE, suggesting that myeloid precursors might be released at a heightened rate before, or in concert with, clinical disease activity. 10,11 Recently, it was shown that CCL2 expression by nonhematopoetic (likely glial) cells is important for the accumulation of proinflammatory DCs in the CNS during acute EAE. 12 Furthermore, transgenic animals that simultaneously express CCL2 in the CNS and Fms-like tyrosine kinase 3 ligand in the periphery spontaneously develop meningeal and perivascular inflammation in association with an ascending paralysis. The neuroinflammation in this model appears to be primarily driven by myeloid cells and occurs independent of T and B lymphocytes. 13 The receptor ...

Summary Touch perception begins with activation of low-threshold mechanoreceptors (LTMRs) in the periphery. LTMR terminals exhibit tremendous morphological heterogeneity that specifies their mechanical receptivity. In a survey of mammalian skin, we found a preponderance of neurofilament-heavy chain+ circumferential endings associated with hair follicles, prompting us to develop a genetic strategy to interrogate these neurons. Targeted in vivo recordings revealed them to be Aβ Field-LTMRs, identified 50 years ago but largely elusive thereafter. Remarkably, while Aβ Field-LTMRs are highly sensitive to gentle stroking of the skin, they are unresponsive to hair deflection, and they encode skin indentation in the noxious range across large, spotty receptive fields. Individual Aβ Field-LTMRs form up to 180 circumferential endings, making them the most anatomically expansive LTMR identified to date. Thus, Aβ Field-LTMRs are a major mammalian LTMR subtype that forms circumferential endings in hairy skin, and their sensitivity to gentle skin stroking arises through integration across many low-sensitivity circumferential endings.

Voltage-gated Naϩ channel ␤1 subunits are multifunctional, participating in channel modulation and cell adhesion in vitro. We previously demonstrated that ␤1 promotes neurite outgrowth of cultured cerebellar granule neurons (CGNs) via homophilic adhesion. Both lipid raft-associated kinases and nonraft fibroblast growth factor (FGF) receptors are implicated in cell adhesion molecule-mediated neurite extension. In the present study, we reveal that ␤1-mediated neurite outgrowth is abrogated in Fyn and contactin (Cntn) null CGNs. ␤1 protein levels are unchanged in Fyn null brains, whereas levels are significantly reduced in Cntn null brain lysates. FGF or EGF (epidermal growth factor) receptor kinase inhibitors have no effect on ␤1-mediated neurite extension. These results suggest that ␤1-mediated neurite outgrowth occurs through a lipid raft signaling mechanism that requires the presence of both fyn kinase and contactin. In vivo, Scn1b null mice show defective CGN axon extension and fasciculation indicating that ␤1 plays a role in cerebellar microorganization. In addition, we find that axonal pathfinding and fasciculation are abnormal in corticospinal tracts of Scn1b null mice consistent with the suggestion that ␤1 may have widespread effects on postnatal neuronal development. These data are the first to demonstrate a cell-adhesive role for ␤1 in vivo. We conclude that voltage-gated Na ϩ channel ␤1 subunits signal via multiple pathways on multiple timescales and play important roles in the postnatal development of the CNS.

SummaryIn the injured adult mammalian central nervous system (CNS), products are generated that inhibit neuronal sprouting and regeneration. In recent years, most attention has focused on the myelin-associated inhibitory proteins (MAIs) Nogo-A, OMgp, and myelin-associated glycoprotein (MAG). Binding of MAIs to neuronal cell-surface receptors leads to activation of RhoA, growth cone collapse, and neurite outgrowth inhibition. In the present study, we identify low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) as a high-affinity, endocytic receptor for MAG. In contrast with previously identified MAG receptors, binding of MAG to LRP1 occurs independently of terminal sialic acids. In primary neurons, functional inactivation of LRP1 with receptor-associated protein, depletion by RNA interference (RNAi) knock-down, or LRP1 gene deletion is sufficient to significantly reverse MAG and myelin-mediated inhibition of neurite outgrowth. Similar results are observed when LRP1 is antagonized in PC12 and N2a cells. By contrast, inhibiting LRP1 does not attenuate inhibition of neurite outgrowth caused by chondroitin sulfate proteoglycans. Mechanistic studies in N2a cells showed that LRP1 and p75NTR associate in a MAG-dependent manner and that MAG-mediated activation of RhoA may involve both LRP1 and p75NTR. LRP1 derivatives that include the complement-like repeat clusters CII and CIV bind MAG and other MAIs. When CII and CIV were expressed as Fc-fusion proteins, these proteins, purified full-length LRP1 and shed LRP1 all attenuated the inhibition of neurite outgrowth caused by MAG and CNS myelin in primary neurons. Collectively, our studies identify LRP1 as a novel MAG receptor that functions in neurite outgrowth inhibition.

During development of the peripheral nervous system, excess neurons are generated, most of which will be lost by programmed cell death due to a limited supply of neurotrophic factors from their targets. Other environmental factors, such as ‘competition factors' produced by neurons themselves, and axon guidance molecules have also been implicated in developmental cell death. Semaphorin 3A (Sema3A), in addition to its function as a chemorepulsive guidance cue, can also induce death of sensory neurons in vitro. The extent to which Sema3A regulates developmental cell death in vivo, however, is debated. We show that in compartmentalized cultures of rat sympathetic neurons, a Sema3A-initiated apoptosis signal is retrogradely transported from axon terminals to cell bodies to induce cell death. Sema3A-mediated apoptosis utilizes the extrinsic pathway and requires both neuropilin 1 and plexin A3. Sema3A is not retrogradely transported in older, survival factor-independent sympathetic neurons, and is much less effective at inducing apoptosis in these neurons. Importantly, deletion of either neuropilin 1 or plexin A3 significantly reduces developmental cell death in the superior cervical ganglia. Taken together, a Sema3A-initiated apoptotic signaling complex regulates the apoptosis of sympathetic neurons during the period of naturally occurring cell death.

The voltage-gated sodium channel gene Scn1b encodes the auxiliary subunit beta1, which is widely distributed in neurons and glia of the central and peripheral nervous systems, cardiac myocytes, skeletal muscle myocytes, and neuroendocrine cells. We showed previously that the Scn1b null mutation results in a complex and severe phenotype that includes retarded growth, seizures, ataxia, and death by postnatal day 21. We generated a floxed allele of Scn1b by inserting loxP sites surrounding the second coding exon. Ubiquitous deletion of the floxed exon by Cre recombinase using CMV-Cre-transgenic mice produced the Scn1b(del) allele. The null phenotype of Scn1b(del) homozygotes is indistinguishable from that of Scn1b nulls and confirms the invivo inactivation of Scn1b. Conditional inactivation ofthe floxed allele will make it possible to circumvent the lethality that results from complete loss of this gene, such that the physiological role of Scn1b in specific cell types and/or specific developmental time points can be investigated.

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