Erasure of fear memories is prevented by Nogo Receptor 1 in adulthood.

Critical periods are temporary windows of heightened neural plasticity early in development. For example, fear memories in juvenile rodents are subject to erasure following extinction training, while after closure of this critical period, extinction training only temporarily and weakly suppresses fear memories. Persistence of fear memories is important for survival, but the inability to effectively adapt to the trauma is a characteristic of post-traumatic stress disorder (PTSD). We examined whether Nogo Receptor 1 (NgR1) regulates the plasticity associated with fear extinction. The loss of NgR1 function in adulthood eliminates spontaneous fear recovery and fear renewal, with a restoration of fear reacquisition rate equal to that of naive mice; thus, mimicking the phenotype observed in juvenile rodents. Regional gene disruption demonstrates that NgR1 expression is required in both the basolateral amygdala (BLA) and infralimbic (IL) cortex to prevent fear erasure. NgR1 expression by parvalbumin expressing interneurons is essential for limiting extinction-dependent plasticity. NgR1 gene deletion enhances anatomical changes of inhibitory synapse markers after extinction training. Thus, NgR1 robustly inhibits elimination of fear expression in the adult brain and could serve as a therapeutic target for anxiety disorders, such as PTSD.

The current state-of-the-art of spinal cord imaging: methods.

A first-ever spinal cord imaging meeting was sponsored by the International Spinal Research Trust and the Wings for Life Foundation with the aim of identifying the current state-of-the-art of spinal cord imaging, the current greatest challenges, and greatest needs for future development. This meeting was attended by a small group of invited experts spanning all aspects of spinal cord imaging from basic research to clinical practice. The greatest current challenges for spinal cord imaging were identified as arising from the imaging environment itself; difficult imaging environment created by the bone surrounding the spinal canal, physiological motion of the cord and adjacent tissues, and small cross-sectional dimensions of the spinal cord, exacerbated by metallic implants often present in injured patients. Challenges were also identified as a result of a lack of "critical mass" of researchers taking on the development of spinal cord imaging, affecting both the rate of progress in the field, and the demand for equipment and software to manufacturers to produce the necessary tools. Here we define the current state-of-the-art of spinal cord imaging, discuss the underlying theory and challenges, and present the evidence for the current and potential power of these methods. In two review papers (part I and part II), we propose that the challenges can be overcome with advances in methods, improving availability and effectiveness of methods, and linking existing researchers to create the necessary scientific and clinical network to advance the rate of progress and impact of the research.

The current state-of-the-art of spinal cord imaging: applications.

A first-ever spinal cord imaging meeting was sponsored by the International Spinal Research Trust and the Wings for Life Foundation with the aim of identifying the current state-of-the-art of spinal cord imaging, the current greatest challenges, and greatest needs for future development. This meeting was attended by a small group of invited experts spanning all aspects of spinal cord imaging from basic research to clinical practice. The greatest current challenges for spinal cord imaging were identified as arising from the imaging environment itself; difficult imaging environment created by the bone surrounding the spinal canal, physiological motion of the cord and adjacent tissues, and small crosssectional dimensions of the spinal cord, exacerbated by metallic implants often present in injured patients. Challenges were also identified as a result of a lack of "critical mass" of researchers taking on the development of spinal cord imaging, affecting both the rate of progress in the field, and the demand for equipment and software to manufacturers to produce the necessary tools. Here we define the current state-of-the-art of spinal cord imaging, discuss the underlying theory and challenges, and present the evidence for the current and potential power of these methods. In two review papers (part I and part II), we propose that the challenges can be overcome with advances in methods, improving availability and effectiveness of methods, and linking existing researchers to create the necessary scientific and clinical network to advance the rate of progress and impact of the research.

Small-molecule-induced Rho-inhibition: NSAIDs after spinal cord injury.

Limited axonal plasticity within the central nervous system (CNS) is a major restriction for functional recovery after CNS injury. The small GTPase RhoA is a key molecule of the converging downstream cascade that leads to the inhibition of axonal re-growth. The Rho-pathway integrates growth inhibitory signals derived from extracellular cues, such as chondroitin sulfate proteoglycans, Nogo-A, myelin-associated glycoprotein, oligodendrocyte-myelin glycoprotein, Ephrins and repulsive guidance molecule-A, into the damaged axon. Consequently, the activation of RhoA results in growth cone collapse and finally outgrowth failure. In turn, the inhibition of RhoA-activation blinds the injured axon to its growth inhibitory environment resulting in enhanced axonal sprouting and plasticity. This has been demonstrated in various CNS-injury models for direct RhoA-inhibition and for downstream/upstream blockade of the RhoA-associated pathway. In addition, RhoA-inhibition reduces apoptotic cell death and secondary damage and improves locomotor recovery in clinically relevant models after experimental spinal cord injury (SCI). Unexpectedly, a subset of "small molecules" from the group of non-steroid anti-inflammatory drugs, particularly the FDA-approved ibuprofen, has recently been identified as (1) inhibiting RhoA-activation, (2) enhancing axonal sprouting/regeneration, (3) protecting "tissue at risk" (neuroprotection) and (4) improving motor recovery confined to realistic therapeutical time-frames in clinically relevant SCI models. Here, we survey the effect of small-molecule-induced RhoA-inhibition on axonal plasticity and neurofunctional outcome in CNS injury paradigms. Furthermore, we discuss the body of preclinical evidence for a possible clinical translation with a focus on ibuprofen and illustrate putative risks and benefits for the treatment of acute SCI.

IFITM3 restricts virus-induced inflammatory cytokine production by limiting Nogo-B mediated TLR responses.

Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that limits viral pathogenesis and exerts poorly understood immunoregulatory functions. Here, using human and mouse models, we demonstrate that IFITM3 promotes MyD88-dependent, TLR-mediated IL-6 production following exposure to cytomegalovirus (CMV). IFITM3 also restricts IL-6 production in response to influenza and SARS-CoV-2. In dendritic cells, IFITM3 binds to the reticulon 4 isoform Nogo-B and promotes its proteasomal degradation. We reveal that Nogo-B mediates TLR-dependent pro-inflammatory cytokine production and promotes viral pathogenesis in vivo, and in the case of TLR2 responses, this process involves alteration of TLR2 cellular localization. Nogo-B deletion abrogates inflammatory cytokine responses and associated disease in virus-infected IFITM3-deficient mice. Thus, we uncover Nogo-B as a driver of viral pathogenesis and highlight an immunoregulatory pathway in which IFITM3 fine-tunes the responsiveness of myeloid cells to viral stimulation.

Semaphorin-mediated axonal guidance via Rho-related G proteins.

For many growing axons, interaction with an extracelluar Semaphorin signal leads to growth cone collapse and axon repulsion. Semaphorin receptors composed of Neuropilins and Plexins transduce extracellular cues into changes in the growth cone actin cytoskeleton. The data implicating Rho family G proteins in Semaphorin signaling and in other axon guidance events are considered here. Recent work makes it clear that Rac1 is required for this process. In particular, there is intriguing new evidence that the Plexin receptors communicate directly with members of the Rho family GTPases, although uncertainties remain concerning how Plexins alter Rac1 function. The CRMP (collapsin response mediator protein) family is also required for Plexin-based Semaphorin signaling, and new data demonstrate direct links to Rho and Rac1-based signaling.

Semaphorin3A enhances endocytosis at sites of receptor-F-actin colocalization during growth cone collapse.

Axonal growth cone collapse is accompanied by a reduction in filopodial F-actin. We demonstrate here that semaphorin 3A (Sema3A) induces a coordinated rearrangement of Sema3A receptors and F-actin during growth cone collapse. Differential interference contrast microscopy reveals that some sites of Sema3A-induced F-actin reorganization correlate with discrete vacuoles, structures involved in endocytosis. Endocytosis of FITC-dextran by the growth cone is enhanced during Sema3A treatment, and sites of dextran accumulation colocalize with actin-rich vacuoles and ridges of membrane. Furthermore, the Sema3A receptor proteins, neuropilin-1 and plexin, and the Sema3A signaling molecule, rac1, also reorganize to vacuoles and membrane ridges after Sema3A treatment. These data support a model whereby Sema3A stimulates endocytosis by focal and coordinated rearrangement of receptor and cytoskeletal elements. Dextran accumulation is also increased in retinal ganglion cell (RGC) growth cones, in response to ephrin A5, and in RGC and DRG growth cones, in response to myelin and phorbol-ester. Therefore, enhanced endocytosis may be a general principle of physiologic growth cone collapse. We suggest that growth cone collapse is mediated by both actin filament rearrangements and alterations in membrane dynamics.

Mediation by G proteins of signals that cause collapse of growth cones.

During development, motion of nerve growth cones ceases on contact with particular targets. The signaling mechanism is unknown. In culture, growth cone collapse can be caused by solubilized embryonic brain membranes, central nervous system myelin, a 35-kilodalton protein isolated from myelin, and mastoparan. Collapse induced by each of these is blocked by pertussis toxin. Thus, collapse of growth cones is mediated by G protein-coupled receptors, which may be activated by proteins associated with the cell surface as well as by soluble ligands.

Plexina1 autoinhibition by the plexin sema domain.

Semaphorin 3A (Sema3A) binds to neuropilin-1 (NP1) and activates the transmembrane Plexin to transduce a repulsive axon guidance signal. Here, we show that Sema3 signals are transduced equally effectively by PlexinA1 or PlexinA2, but not by PlexinA3. Deletion analysis of the PlexinA1 ectodomain demonstrates that the sema domain prevents PlexinA1 activation in the basal state. Sema-deleted PlexinA1 is constitutively active, producing cell contraction, growth cone collapse, and inhibition of neurite outgrowth. The sema domain of PlexinA1 physically associates with the remainder of the PlexinA1 ectodomain and can reverse constitutive activation. Both the sema portion and the remainder of the ectodomain of PlexinA1 associate with NP1 in a Sema3A-independent fashion. Plexin A1 is autoinhibited by its sema domain, and Sema3A/NP1 releases this inhibition.

Functional expression of sodium channel mutations identified in families with periodic paralysis.

Two mutations in the sodium channel alpha subunit that have been implicated as the cause of periodic paralysis were studied by functional expression in a mammalian cell line. Both mutations disrupted inactivation without affecting the time course of the onset of the sodium current or the single-channel conductance. This is the same functional defect that was observed in myotubes cultured from affected patients and proves that these mutations are not benign polymorphisms. Unlike the currents in the myotubes, however, there was no consistent potassium dependence for the noninactivating component. These mutations also define new regions of the sodium channel alpha subunit that are involved in the process of inactivation.

Neuropilin-1 extracellular domains mediate semaphorin D/III-induced growth cone collapse.

Somatosensory axon outgrowth is repulsed when soluble semaphorin D (semD) binds to growth cone neuropilin-1 (Npn-1). Here, semD ligand binding studies of Npn-1 mutants demonstrate that the sema domain binds to the amino-terminal quarter, or complement-binding (CUB) domain, of Npn-1. By herpes simplex virus- (HSV-) mediated expression of Npn-1 mutants in chick retinal ganglion cells, we show that semD-induced growth cone collapse requires two segments of the ectodomain of Npn-1, the CUB domain and the juxtamembrane portion, or MAM (meprin, A5, mu) domain. In contrast, the transmembrane segment and cytoplasmic tail of Npn-1 are not required for biologic activity. These data imply that the CUB and MAM ectodomains of Npn-1 interact with another transmembrane growth cone protein that in turn transduces a semD signal into axon repulsion.

Semaphorins A and E act as antagonists of neuropilin-1 and agonists of neuropilin-2 receptors.

Neuropilin-1 (NP-1) has been identified as a necessary component of a semaphorin D (SemD) receptor that repulses dorsal root ganglion (DRG) axons during development. SemA and SemE are related to SemD and bind to NP-1, but do not repulse DRG axons. By expressing NP-1 in retinal neurons and NP-2 in DRG neurons, we demonstrate that neuropilins are sufficient to determine the functional specificity of semaphorin responsiveness. SemA and SemE block SemD binding to NP-1 and abolish SemD repulsion in axons expressing NP-1. SemA and SemE seem to have a newly discovered protein antagonist capacity at NP-1 receptors, whereas they act as agonists at receptors containing NP-2.

Repulsive factors and axon regeneration in the CNS.

During the past year, a major advance in the study of axon regeneration was the molecular cloning of Nogo. The expression of Nogo protein by CNS myelin may be a major factor in the failure of CNS axon regeneration. The effect of disrupting Nogo-dependent axon inhibition can now be studied conclusively. In related work, immunization with a Nogo-containing CNS myelin preparation was shown to promote regeneration and dramatic functional recovery after spinal cord trauma.

Angiotensin-converting enzyme in the male rat reproductive system: autoradiographic visualization with [3H]captopril.

[3H]Captopril autoradiography visualizes angiotensin-converting enzyme (ACE; EC 3.14.5.1) in the reproductive tract of male rats. [3H]Captopril binds to testicular slices with high affinity (Kd = 4.4 nM) and displays a pharmacological profile similar to that of ACE activity. High densities of [3H] captopril autoradiographic silver grains are found over spermatid heads and in the lumen of seminiferous tubules in stages I-VIII and XII-XIV. Tubules in stages IX-XI exhibit only one fifth the level of binding. The basal epithelium and interstitial tissue are not labeled. The initial segment of the epididymis contains very low levels of grains. The head of the epididymis demonstrates intense grain density at the luminal surface of the epithelium, with little luminal labeling. In a progression to the tail of the epididymis, epithelium labeling declines, and luminal grains increase. The lumen of the vas deferens is also labeled. ACE from testis and several regions of the epididymis has been categorized with respect to its particulate vs. soluble nature and its ability to be precipitated by an antirat lung ACE monoclonal antibody. Testicular ACE is particulate and not immunoprecipitable. The distribution of immunoprecipitable particulate ACE in the epididymis is similar to that of autoradiographic silver grains over the epithelium. The concentration of particulate but nonprecipitable ACE gradually rises from the initial segment to the tail of the epididymis, similar to the distribution of luminal [3H]captopril-associated grains. Soluble ACE activity, present in equal concentration throughout the epididymis and not immunoprecipitable, may not be detected by autoradiography of [3H]captopril.

Angiotensin-converting enzyme localized in the rat pituitary and adrenal glands by [3H]captopril autoradiography.

We have localized angiotensin-converting enzyme (EC 3.14.5.1) in the rat pituitary and adrenal glands by [3H]captopril autoradiography. The maximal numbers of [3H] captopril-binding sites are: posterior pituitary, 4500 fmol/mg protein; anterior pituitary, 1950 fmol/mg; intermediate pituitary, less than 100 fmol/mg; adrenal medulla, 480 fmol/mg; and adrenal cortex, less than 25 fmol/mg. The distribution within the posterior pituitary and adrenal medulla is homogeneous, whereas that in the anterior pituitary is patchy. Subcellular fractionation of the bovine adrenal medulla reveals enrichment of angiotension-converting enzyme in plasma membrane fractions, but not in chromaffin granules. [3H]Captopril autoradiography in the rat pituitary gland is unaltered by dehydration, adrenalectomy, or reserpine treatment and in Brattleboro rats. [3H]Captopril binding in the adrenal medulla is increased by 75% 3 weeks after hypophysectomy and is elevated by 80% after reserpine treatment. The change after hypophysectomy is not reversed by dexamethasone treatment.

Enkephalin convertase in the gastrointestinal tract an associated organs characterized and localized with [3H]guanidinoethylmercaptosuccinic acid.

Enkephalin convertase (carboxypeptidase EH; EC 3.4.17.10), a carboxypeptidase B-like enzyme which processes hormone and neuropeptide precursors, has been characterized in the gastrointestinal tract, submandibular gland, and pancreas using a binding assay with [3H]guanidinoethylmercaptosuccinic acid. Binding to homogenates of the membrane and soluble fractions of stomach, small intestine, colon, and submandibular gland is saturable, with Kd values of about 2 nM. Partial purification of the membrane fractions reveals a Co+2-stimulated carboxypeptidase B activity which is not detectable in crude homogenates. In vitro autoradiography with [3H]guanidinoethylmercaptosuccinic acid localizes enkephalin convertase to the epithelial cells of the stomach, colon, and intestine, the islet cells of the pancreas, and the acinar cells of the submandibular gland. This localization contrasts to the distribution of enkephalins and other neuropeptides in the gastrointestinal tract and associated organs, suggesting that enkephalin convertase may serve functions other than neuropeptide and prohormone processing.

Enkephalin convertase demonstrated in the pituitary and adrenal gland by [3H]guanidinoethylmercaptosuccinic acid autoradiography: dehydration decreases neurohypophyseal levels.

[3H]Guanidinoethylmercaptosuccinic acid (GEMSA) autoradiography demonstrates the particulate form of a carboxypeptidase B-like peptide processing enzyme, enkephalin convertase (EC 3.4.17.10), in the rat pituitary and adrenal glands. The maximal number of binding sites (Bmax) for [3H]GEMSA is 20 pmol/mg protein in the intermediate lobe of the pituitary, 12.0 pmol/mg protein in the posterior pituitary lobe, 15 pmol/mg protein in the anterior pituitary lobe, 5.8 pmol/mg protein in the adrenal medulla, and less than 0.3 pmol/mg protein in the adrenal cortex. The labeling pattern is homogeneous within each of these regions. Subcellular fractionation of the bovine adrenal medulla demonstrates that [3H] GEMSA-binding sites are localized to chromaffin granules. In Brattleboro rats and dehydrated rats, the level of posterior pituitary [3H]GEMSA binding is less than 25% of that in control animals. This decrease is abolished by arginine vasopressin treatment of Brattleboro rats or rehydration of dehydrated rats. There are no changes in [3H] GEMSA binding in the supraoptic nucleus or magnocellular portion of the paraventricular nucleus of the hypothalamus under any of these conditions, suggesting that the alterations observed in the neurohypophysis result from an increased rate of loss of enkephalin convertase. The level of anterior pituitary enkephalin convertase is unchanged by dehydration, adrenalectomy, or dexamethasone or in Brattleboro rats. [3H]GEMSA labeling in the intermediate pituitary lobe is unaffected by dehydration and haloperidol treatment and in Brattleboro rats. The adrenal medullary enzyme is not altered by reserpine, hypophysectomy, or splanchnic denervation or in Brattleboro rats.

Angiotensin-converting enzyme in the testis and epididymis: differential development and pituitary regulation of isozymes.

Angiotensin-converting enzyme (ACE, EC 3.14.5.1) is found in particulate fractions of the epididymis but not in soluble epididymal fractions or in the testis of 4-week-old rats. [3H]Captopril autoradiography of testis and epididymis from 4-week-old rats confirms the association of ACE with epididymal ducts but not the testis. ACE appears in the testis between 4 and 6 weeks of age. Soluble ACE is not detectable in the epididymis until 6-7 weeks of age. Within the epididymis, regions closest to the testis develop soluble ACE activity about 1 week before those nearest to the vas deferens. Hypophysectomy of 10 week-old-rats depletes greater than 95% of ACE activity from the testis and soluble fractions of the epididymis, with little change in ACE levels from particulate fractions of the epididymis. [3H]Captopril autoradiography after hypophysectomy reveals luminal and epithelial ACE in the epididymis. The presence of particulate ACE in the epididymis under conditions where there is no testicular ACE indicates that the two forms are synthesized separately. However, soluble ACE from the epididymis might be derived from the membrane-associated ACE of the testis. Such a relationship is supported by the lag of 1 week between the development of ACE in the initial segment of the epididymis and the tail of the epididymis, and by the occurrence of soluble epididymis ACE only in those animals with testicular ACE activity.

Nogo: a molecular determinant of axonal growth and regeneration.

Following injury, axons of the adult mammalian central nervous system (CNS) fail to regenerate. As a result, CNS trauma generally results in severe and persistent functional deficits. The inability of CNS axons to regenerate is largely associated with nonneuronal aspects of the CNS environment that are inhibitory to axonal elongation. This inhibition is mediated by the glial scar, including reactive astrocytes, and by the myelin-associated neurite outgrowth inhibitors chondroitin sulfate proteoglycans, myelin-associated glycoprotein, and Nogo. Nogo is an integral membrane protein that localizes to CNS, but not peripheral nervous system, myelin. In vitro characterization of Nogo has demonstrated its function as a potent inhibitor of axon elongation. In vivo neutralization of Nogo activity results in enhanced axonal regeneration and functional recovery following CNS injury as well as increased plasticity in uninjured CNS fibers. These findings suggest that Nogo may be a major contributor to the nonpermissive nature of the CNS environment.

Enkephalin convertase: characterization and localization using [3H]guanidinoethylmercaptosuccinic acid.

Enkephalin convertase (carboxypeptidase E,H; EC 3.4.17.10) is a carboxypeptidase B-like enzyme which appears to be physiologically associated with the biosynthesis of the enkephalins and certain other peptides. We have localized enkephalin convertase in the brain and other tissues autoradiographically by labeling studies with [3H]guanidinoethylmercaptosuccinic acid ([3H]GEMSA). In the brain, [3H]GEMSA localizations parallel enkephalin distribution but with certain exceptions, suggesting a role in relation to other peptides. In the pancreas, [3H]GEMSA binding sites are localized to the islets suggesting an involvement in insulin, glucagon, or somatostatin formation. The selective concentration of [3H]GEMSA grains in cardiac atria suggests a link to atrial natriuretic factor.