Lateral olfactory tract usher substance (LOTUS), an endogenous Nogo receptor antagonist, ameliorates disease progression in amyotrophic lateral sclerosis model mice.

Nogo-Nogo receptor 1 (NgR1) signaling is significantly implicated in neurodegeneration in amyotrophic lateral sclerosis (ALS). We previously showed that lateral olfactory tract usher substance (LOTUS) is an endogenous antagonist of NgR1 that prevents all myelin-associated inhibitors (MAIs), including Nogo, from binding to NgR1. Here we investigated the role of LOTUS in ALS pathogenesis by analyzing G93A-mutated human superoxide dismutase 1 (SOD1) transgenic (Tg) mice, as an ALS model, as well as newly generated LOTUS-overexpressing SOD1 Tg mice. We examined expression profiles of LOTUS and MAIs and compared motor functions and survival periods in these mice. We also investigated motor neuron survival, glial proliferation in the lumbar spinal cord, and neuromuscular junction (NMJ) morphology. We analyzed downstream molecules of NgR1 signaling such as ROCK2, LIMK1, cofilin, and ataxin-2, and also neurotrophins. In addition, we investigated LOTUS protein levels in the ventral horn of ALS patients. We found significantly decreased LOTUS expression in both SOD1 Tg mice and ALS patients. LOTUS overexpression in SOD1 Tg mice increased lifespan and improved motor function, in association with prevention of motor neuron loss, reduced gliosis, increased NMJ innervation, maintenance of cofilin phosphorylation dynamics, decreased levels of ataxin-2, and increased levels of brain-derived neurotrophic factor (BDNF). Reduced LOTUS expression may enhance neurodegeneration in SOD1 Tg mice and ALS patients by activating NgR1 signaling, and in this study LOTUS overexpression significantly ameliorated ALS pathogenesis. LOTUS might serve as a promising therapeutic target for ALS.

Brain-derived neurotrophic factor (BDNF) induces antagonistic action to Nogo signaling by the upregulation of lateral olfactory tract usher substance (LOTUS) expression.

Neurons in the central nervous system (CNS) have limited capacity for axonal regeneration after trauma and neurological disorders due to an endogenous nonpermissive environment for axon regrowth in the CNS. Lateral olfactory tract usher substance (LOTUS) contributes to axonal tract formation in the developing brain and axonal regeneration in the adult brain as an endogenous Nogo receptor-1 (NgR1) antagonist. However, how LOTUS expression is regulated remains unclarified. This study examined molecular mechanism of regulation in LOTUS expression and found that brain-derived neurotrophic factor (BDNF) increased LOTUS expression in cultured hippocampal neurons. Exogenous application of BDNF increased LOTUS expression at both mRNA and protein levels in a dose-dependent manner. We also found that pharmacological inhibition with K252a and gene knockdown by siRNA of tropomyosin-related kinase B (TrkB), BDNF receptor suppressed BDNF-induced increase in LOTUS expression. Further pharmacological analysis of the TrkB signaling pathway revealed that BDNF increased LOTUS expression through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades, but not phospholipase C-γ (PLCγ) cascade. Additionally, treatment with c-AMP response element binding protein (CREB) inhibitor partially suppressed BDNF-induced LOTUS expression. Finally, neurite outgrowth assay in cultured hippocampal neurons revealed that BDNF treatment-induced antagonism for NgR1 by up-regulating LOTUS expression. These findings suggest that BDNF may acts as a positive regulator of LOTUS expression through the TrkB signaling, thereby inducing an antagonistic action for NgR1 function by up-regulating LOTUS expression. Also, BDNF may synergistically affect axon regrowth through the upregulation of LOTUS expression.

LOTUS suppresses amyloid ß-induced dendritic spine elimination through the blockade of amyloid ß binding to PirB.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide but has no effective treatment. Amyloid beta (Aß) protein, a primary risk factor for AD, accumulates and aggregates in the brain of patients with AD. Paired immunoglobulin-like receptor B (PirB) has been identified as a receptor of Aß and Aß-PirB molecular interactions that cause synapse elimination and synaptic dysfunction. PirB deletion has been shown to suppress Aß-induced synaptic dysfunction and behavioral deficits in AD model mice, implying that PirB mediates Aß-induced AD pathology. Therefore, inhibiting the Aß-PirB molecular interaction could be a successful approach for combating AD pathology. We previously showed that lateral olfactory tract usher substance (LOTUS) is an endogenous antagonist of type1 Nogo receptor and PirB and that LOTUS overexpression promotes neuronal regeneration following damage to the central nervous system, including spinal cord injury and ischemic stroke. Therefore, in this study, we investigated whether LOTUS inhibits Aß-PirB interaction and Aß-induced dendritic spine elimination. METHODS: The inhibitory role of LOTUS against Aß-PirB (or leukocyte immunoglobulin-like receptor subfamily B member 2: LilrB2) binding was assessed using a ligand-receptor binding assay in Cos7 cells overexpressing PirB and/or LOTUS. We assessed whether LOTUS inhibits Aß-induced intracellular alterations and synaptotoxicity using immunoblots and spine imaging in a primary cultured hippocampal neuron. RESULTS: We found that LOTUS inhibits the binding of Aß to PirB overexpressed in Cos7 cells. In addition, we found that Aß-induced dephosphorylation of cofilin and Aß-induced decrease in post-synaptic density-95 expression were suppressed in cultured hippocampal neurons from LOTUS-overexpressing transgenic (LOTUS-tg) mice compared with that in wild-type mice. Moreover, primary cultured hippocampal neurons from LOTUS-tg mice improved the Aß-induced decrease in dendritic spine density. Finally, we studied whether human LOTUS protein inhibits Aß binding to LilrB2, a human homolog of PirB, and found that human LOTUS inhibited the binding of Aß to LilrB2 in a similar manner. CONCLUSIONS: This study implied that LOTUS improved Aß-induced synapse elimination by suppressing Aß-PirB interaction in rodents and inhibited Aß-LilrB2 interaction in humans. Our findings revealed that LOTUS may be a promising therapeutic agent in counteracting Aß-induced AD pathologies.

LOTUS overexpression via ex vivo gene transduction further promotes recovery of motor function following human iPSC-NS/PC transplantation for contusive spinal cord injury.

Functional recovery is still limited mainly due to several mechanisms, such as the activation of Nogo receptor-1 (NgR1) signaling, when human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PC) are transplanted for subacute spinal cord injury (SCI). We previously reported the neuroprotective and regenerative benefits of overexpression of lateral olfactory tract usher substance (LOTUS), an endogenous NgR1 antagonist, in the injured spinal cord using transgenic mice. Here, we evaluate the effects of lentiviral transduction of LOTUS gene into hiPSC-NS/PCs before transplantation in a mouse model of subacute SCI. The transduced LOTUS contributes to neurite extension, suppression of apoptosis, and secretion of neurotrophic factors in vitro. In vivo, the hiPSC-NS/PCs enhance the survival of grafted cells and enhance axonal extension of the transplanted cells, resulting in significant restoration of motor function following SCI. Therefore, the gene transduction of LOTUS in hiPSC-NS/PCs could be a promising adjunct for transplantation therapy for SCI.

LOTUS, an endogenous Nogo receptor antagonist, is involved in synapse and memory formation.

The Nogo signal is involved in impairment of memory formation. We previously reported the lateral olfactory tract usher substance (LOTUS) as an endogenous antagonist of the Nogo receptor 1 that mediates the inhibition of axon growth and synapse formation. Moreover, we found that LOTUS plays an essential role in neural circuit formation and nerve regeneration. However, the effects of LOTUS on synapse formation and memory function have not been elucidated. Here, we clearly showed the involvement of LOTUS in synapse formation and memory function. The cultured hippocampal neurons derived from lotus gene knockout (LOTUS-KO) mice exhibited a decrease in synaptic density compared with those from wild-type mice. We also found decrease of dendritic spine formation in the adult hippocampus of LOTUS-KO mice. Finally, we demonstrated that LOTUS deficiency impairs memory formation in the social recognition test and the Morris water maze test, indicating that LOTUS is involved in functions of social and spatial learning and memory. These findings suggest that LOTUS affects synapse formation and memory function.

Nogo receptor antagonist LOTUS exerts suppression on axonal growth-inhibiting receptor PIR-B.

Damaged axons in the adult mammalian central nervous system have a restricted regenerative capacity mainly because of Nogo protein, which is a major myelin-associated axonal growth inhibitor with binding to both receptors of Nogo receptor-1 (NgR1) and paired immunoglobulin-like receptor (PIR)-B. Lateral olfactory tract usher substance (LOTUS) exerts complete suppression of NgR1-mediated axonal growth inhibition by antagonizing NgR1. However, the regulation of PIR-B functions in neurons remains unknown. In this study, protein-protein interactions analyses found that LOTUS binds to PIR-B and abolishes Nogo-binding to PIR-B completely. Reverse transcription-polymerase chain reaction and immunocytochemistry revealed that PIR-B is expressed in dorsal root ganglions (DRGs) from wild-type and Ngr1-deficient mice (male and female). In these DRG neurons, Nogo induced growth cone collapse and neurite outgrowth inhibition, but treatment with the soluble form of LOTUS completely suppressed them. Moreover, Nogo-induced growth cone collapse and neurite outgrowth inhibition in Ngr1-deficient DRG neurons were neutralized by PIR-B function-blocking antibodies, indicating that these Nogo-induced phenomena were mediated by PIR-B. Our data show that LOTUS negatively regulates a PIR-B function. LOTUS thus exerts an antagonistic action on both receptors of NgR1 and PIR-B. This may lead to an improvement in the defective regeneration of axons following injury.

LOTUS as an endogenous protein converting the adult central nervous system environment from nonpermissive to permissive for axonal regrowth after brain injury.

Central nervous system (CNS) injury, such as spinal cord injury (SCI), results in severe sensory and motor deficits due to the poor regenerative capacity of the adult CNS primarily caused by a damaged CNS environment containing a large amount of axonal growth inhibitors, such as Nogo receptor-1 (NgR1), which inhibits axonal regrowth strongly after SCI, and its five ligands. Lateral olfactory tract usher substance (LOTUS), identified in the developing brain, completely antagonizes NgR1 function, promoting neuronal regeneration and functional recovery after SCI. Therefore, we hypothesized that LOTUS might be a useful natural agent for the clinical treatment of SCI in order to increase functional recovery by converting the CNS environment from nonpermissive to permissive for neuronal regeneration. Currently, we are attempting to administer LOTUS after SCI by protein injection or gene transfection. In this report, I discuss the probability of clinical application of LOTUS for future therapy of brain injury.

Axonal regeneration and functional recovery driven by endogenous Nogo receptor antagonist LOTUS in a rat model of unilateral pyramidotomy.

The adult mammalian central nervous system (CNS) rarely recovers from injury. Myelin fragments contain axonal growth inhibitors that limit axonal regeneration, thus playing a major role in determining neural recovery. Nogo receptor-1 (NgR1) and its ligands are among the inhibitors that limit axonal regeneration. It has been previously shown that the endogenous protein, lateral olfactory tract usher substance (LOTUS), antagonizes NgR1-mediated signaling and accelerates neuronal plasticity after spinal cord injury and cerebral ischemia in mice. However, it remained unclear whether LOTUS-mediated reorganization of descending motor pathways in the adult brain is physiologically functional and contributes to functional recovery. Here, we generated LOTUS-overexpressing transgenic (LOTUS-Tg) rats to investigate the role of LOTUS in neuronal function after damage. After unilateral pyramidotomy, motor function in LOTUS-Tg rats recovered significantly compared to that in wild-type animals. In a retrograde tracing study, labeled axons spanning from the impaired side of the cervical spinal cord to the unlesioned hemisphere of the red nucleus and sensorimotor cortex were increased in LOTUS-Tg rats. Anterograde tracing from the unlesioned cortex also revealed enhanced ipsilateral connectivity to the impaired side of the cervical spinal cord in LOTUS-Tg rats. Moreover, electrophysiological analysis showed that contralesional cortex stimulation significantly increased ipsilateral forelimb movement in LOTUS-Tg rats, which was consistent with the histological findings. According to these data, LOTUS overexpression accelerates ipsilateral projection from the unlesioned cortex and promotes functional recovery after unilateral pyramidotomy. LOTUS could be a future therapeutic option for CNS injury.

LOTUS Inhibits Neuronal Apoptosis and Promotes Tract Regeneration in Contusive Spinal Cord Injury Model Mice.

Nogo receptor-1 (NgR1) signaling is involved in the limitation of axonal regeneration following spinal cord injury (SCI) through collapsing the growth cone and inhibiting neurite outgrowth. Lateral olfactory tract usher substance (LOTUS), a NgR antagonist, suppresses these pathological conditions. A previous report demonstrated the positive effects of LOTUS expression on motor function through raphespinal tract regeneration using pan-neuronally LOTUS-overexpressing transgenic mice. However, this report used a hemi-section model, which does not represent the majority of clinical SCI cases, and lacked a detailed histological analysis of other descending tracts. To determine the true therapeutic effects of LOTUS, we used a more clinically relevant contusive SCI model in female transgenic mice. Definitive tracing analyses revealed that LOTUS promoted the extensive regeneration of the reticulospinal tract across the lesion site and suppressed axonal dieback of corticospinal tract (CST). A significant increase in raphespinal tract fibers was seen from the subacute to the chronic phase after the injury, strongly suggesting that LOTUS promoted translesional elongation of this tract. Furthermore, histological analyses revealed that LOTUS had a neuroprotective effect on the injured spinal cord through suppressing cellular apoptosis during the acute phase. These neuroprotective and regenerative effects contributed to significant motor functional recovery and restoration of the motor evoked potential (MEP). Therefore, LOTUS application could prove beneficial in the treatment of SCI by promoting axonal regeneration of some descending fibers, reducing axonal dieback of CST fibers and encouraging motor function recovery.

The soluble form of LOTUS inhibits Nogo receptor type 1-mediated signaling induced by B lymphocyte stimulator and chondroitin sulfate proteoglycans.

There are global efforts in developing therapeutic strategies for central nervous system (CNS) injuries using multimodal approaches. Nogo receptor type 1 (NgR1) has been known as a primary molecule limiting neuronal regeneration in the adult CNS. We identified lateral olfactory tract usher substance (LOTUS) as an endogenous NgR1 antagonist. Membrane-bound LOTUS interacts with NgR1 and inhibits its function by blocking its ligand binding. Five molecules including Nogo, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), B lymphocyte stimulator (BLyS) and chondroitin sulfate proteoglycans (CSPGs) have been identified as NgR1 ligands. These ligands bind to NgR1 and activate NgR1 signaling, leading to axon growth inhibition such as growth cone collapse and neurite outgrowth inhibition. We have recently reported that the soluble form of LOTUS (s-LOTUS) also suppressed NgR1-mediated signaling induced by myelin axonal inhibitors (MAIs) including Nogo, MAG and OMgp by binding with both NgR1 and its co-receptor p75 neurotrophin receptor (p75NTR). Though s-LOTUS has been reported to suppress MAIs, whether s-LOTUS also suppresses NgR1 signaling induced by BLyS and CSPGs remains to be elucidated. Here, we show that s-LOTUS inhibits NgR1-mediated signaling induced by BLyS and CSPGs. Although treatment with s-LOTUS did not suppress BLyS-NgR1 interaction, s-LOTUS inhibited growth cone collapse and neurite outgrowth inhibition induced by BLyS and CSPGs in chick dorsal root ganglion (DRG) neurons. Furthermore, s-LOTUS compensated for the suppressive function of endogenous LOTUS in NgR1-mediated signaling in olfactory bulb neurons of lotus-knockout mice. These findings suggest that s-LOTUS is a potent therapeutic agent for neuronal regeneration in the CNS injuries.

The Soluble Form of LOTUS inhibits Nogo Receptor-Mediated Signaling by Interfering with the Interaction Between Nogo Receptor Type 1 and p75 Neurotrophin Receptor.

Nogo receptor type 1 (NgR1) is known to inhibit neuronal regeneration in the CNS. Previously, we have shown that lateral olfactory tract usher substance (LOTUS) interacts with NgR1 and inhibits its function by blocking its ligand binding. Therefore, LOTUS is expected to have therapeutic potential for the promotion of neuronal regeneration. However, it remains unknown whether the soluble form of LOTUS (s-LOTUS) also has an inhibitory action on NgR1 function as a candidate for therapeutic agents. Here, we show that s-LOTUS inhibits NgR1-mediated signaling by inhibiting the molecular interaction between NgR1 and its coreceptor, p75 neurotrophin receptor (p75NTR). In contrast to the membrane-bound form of LOTUS, s-LOTUS did not block ligand binding to NgR1. However, we identified p75NTR as a novel LOTUS binding partner and found that s-LOTUS suppressed the interaction between p75NTR and NgR1. s-LOTUS inhibited myelin-associated inhibitor (MAI)-induced RhoA activation in murine cortical neurons. Functional analyses revealed that s-LOTUS inhibited MAI-induced growth cone collapse and neurite outgrowth inhibition in chick DRG neurons. In addition, whereas olfactory bulb neurons of lotus-KO mice are sensitive to MAI due to a lack of LOTUS expression, treatment with s-LOTUS inhibited MAI-induced growth cone collapse in these neurons. Finally, we observed that s-LOTUS promoted axonal regeneration in optic nerve crush injury of mice (either sex). These findings suggest that s-LOTUS inhibits NgR1-mediated signaling, possibly by interfering with the interaction between NgR1 and p75NTR Therefore, s-LOTUS may have potential as a therapeutic agent for neuronal regeneration in the damaged CNS.SIGNIFICANCE STATEMENT Nogo receptor type 1 (NgR1) is a receptor well known to inhibit neuronal regeneration in the CNS. Because the membrane-bound form of lateral olfactory tract usher substance (LOTUS) antagonizes NgR1 through a cis-type molecular interaction between LOTUS and NgR1, the soluble form of LOTUS (s-LOTUS) is expected to be a therapeutic agent for neuronal regeneration. In our present study, we show that s-LOTUS inhibits the interaction between NgR1 and p75NTR, NgR1 ligand-induced RhoA activation, growth cone collapse, and neurite outgrowth inhibition and promotes axonal regeneration. Our results indicate that s-LOTUS inhibits NgR1-mediated signaling through a trans-type molecular interaction between LOTUS and NgR1 and, therefore, s-LOTUS may have therapeutic potential for neuronal regeneration.

Cerebrospinal fluid level of Nogo receptor 1 antagonist lateral olfactory tract usher substance (LOTUS) correlates inversely with the extent of neuroinflammation.

BACKGROUND: Although inflammation in the central nervous system is responsible for multiple neurological diseases, the lack of appropriate biomarkers makes it difficult to evaluate inflammatory activities in these diseases. Therefore, a new biomarker reflecting neuroinflammation is required for accurate diagnosis, appropriate therapy, and comprehension of pathogenesis of these neurological disorders. We previously reported that the cerebrospinal fluid (CSF) concentration of lateral olfactory tract usher substance (LOTUS), which promotes axonal growth as a Nogo receptor 1 antagonist, negatively correlates with disease activity in multiple sclerosis, suggesting that variation in LOTUS reflects the inflammatory activities and is a useful biomarker to evaluate the disease activity. To extend this observation, we analyzed the variation of LOTUS in the CSF of patients with bacterial and viral meningitis, which are the most common neuroinflammatory diseases. METHODS: CSF samples were retrospectively obtained from patients with meningitis (n = 40), who were followed up by CSF study at least twice, and from healthy controls (n = 27). Patients were divided into bacterial (n = 14) and viral meningitis (n = 18) after exclusion of eight patients according to the criteria of this study. LOTUS concentrations, total protein levels, and CSF cell counts in the acute and recovery phases were analyzed chronologically. We also used lipopolysaccharide-injected mice as a model of neuroinflammation to evaluate LOTUS mRNA and protein expression in the brain. RESULTS: Regardless of whether meningitis was viral or bacterial, LOTUS concentrations in the CSF of patients in acute phase were lower than those of healthy controls. As the patients recovered from meningitis, LOTUS levels in the CSF returned to the normal range. Lipopolysaccharide-injected mice also exhibited reduced LOTUS mRNA and protein expression in the brain. CONCLUSIONS: CSF levels of LOTUS correlated inversely with disease activity in both bacterial and viral meningitis, as well as in multiple sclerosis, because neuroinflammation downregulated LOTUS expression. Our data strongly suggest that variation of CSF LOTUS is associated with neuroinflammation and is useful as a biomarker for a broader range of neuroinflammatory diseases.

LOTUS overexpression accelerates neuronal plasticity after focal brain ischemia in mice.

Nogo receptor-1 (NgR1) and its ligands inhibit neuronal plasticity and limit functional recovery after brain damage such as ischemic stroke. We have previously shown that lateral olfactory tract usher substance (LOTUS) antagonizes NgR1-mediated signaling. Here, we investigated whether LOTUS enhances neuronal plasticity and functional recovery after brain focal ischemia in adult mice. Focal ischemic infarcts were induced in wild-type and LOTUS-overexpressing transgenic mice via middle cerebral artery occlusion. Endogenous LOTUS expression was increased in brain and cervical spinal cord of the contralateral side of ischemia in the chronic phase after brain ischemia. LOTUS overexpression accelerated midline-crossing axonal sprouting from the contralateral side to the ipsilateral side of ischemia in the medullar reticular formation and gray matter of denervated cervical spinal cord. Importantly, LOTUS overexpression improved neurological score highly correlated with laterality ratio of corticoreticular fibers of the medulla oblongata, indicating that LOTUS overexpression may overcome the inhibitory environment induced by NgR1 signaling for damaged motor pathway reconstruction after ischemic stroke. Thus, our data suggest that LOTUS overexpression accelerates neuronal plasticity in the brainstem and cervical spinal cord after stroke and LOTUS administration is useful for future therapeutic strategies.

Regulation of axonal regeneration by the level of function of the endogenous Nogo receptor antagonist LOTUS.

Axonal regeneration in the adult mammalian central nervous system is limited in part by the non-permissive environment, including axonal growth inhibitors such as the Nogo-A protein. How the functions of these inhibitors can be blocked remains unclear. Here, we examined the role of LOTUS, an endogenous Nogo receptor antagonist, in promoting functional recovery and neural repair after spinal cord injury (SCI), as well as axonal regeneration after optic nerve crush. Wild-type untreated mice show incomplete but substantial intrinsic motor recovery after SCI. The genetic deletion of LOTUS delays and decreases the extent of motor recovery, suggesting that LOTUS is required for spontaneous neural repair. The neuronal overexpression of LOTUS in transgenic mice promotes motor recovery after SCI, and recombinant viral overexpression of LOTUS enhances retinal ganglion cell axonal regeneration after optic nerve crush. Thus, the level of LOTUS function titrates axonal regeneration.

Blockade of chondroitin sulfate proteoglycans-induced axonal growth inhibition by LOTUS.

Chondroitin sulfate proteoglycans (CSPGs) are axon growth inhibitors in the glial scar, and restrict axon regeneration following damage to the adult mammalian central nervous system. CSPGs have recently been identified as functional ligands for Nogo receptor-1 (NgR1), which is the common receptor for Nogo proteins, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and B lymphocyte stimulator (BLyS). We have previously reported that through its binding to NgR1, lateral olfactory tract usher substance (LOTUS) suppresses Nogo, MAG, OMgp, and BLyS-induced axon growth inhibition. However, it remains unknown whether LOTUS also exerts this suppressive action on CSPG-induced axon growth inhibition. LOTUS overexpression rescued CSPG-induced growth cone collapse and neurite outgrowth inhibition in cultured dorsal root ganglion neurons, which only weakly express endogenous LOTUS. In cultured olfactory bulb neurons, which endogenously express LOTUS, the growth cone was insensitive to CSPG-induced collapse, but was sensitive to collapse induced by CSPGs in lotus-deficient mice. Our data demonstrate that LOTUS suppresses CSPG-induced axon growth inhibition, suggesting that LOTUS may represent a promising therapeutic agent for promoting axon regeneration.

Micro-CALI to Study Localized Roles of the Semaphorin Signaling Component CRMP in Axon Growth.

Elucidating the local function of proteins is essential for understanding not only the individual proteins but also the organization of the cell or even tissue as a whole. However, until now, few attempts have been made to understand local proteins function in cells because of a lack of acute inactivation technique of local proteins with high versatility. Here we describe the application of the chromophore-assisted light inactivation (CALI) method to elucidate the role of the semaphorin signaling component CRMP located within the growth cone area in axon growth and growth cone turning.

Axonal branching in lateral olfactory tract is promoted by Nogo signaling.

Mitral cells are major projection neurons of the olfactory bulb (OB) that form an axonal bundle known as the lateral olfactory tract (LOT). After axonal bundle formation, collateral branches sprout from primary axons of the LOT. Recently, we identified LOT usher substance (LOTUS) as an endogenous Nogo receptor-1 (NgR1) antagonist and demonstrated that LOTUS contributes to the formation of the LOT axonal bundle. Immunoblots revealed that the expression level of Nogo-A in the OB developmentally increased during axonal collateral formation. Next, we found that the axonal collateral branches were increased in cultured OB neurons from LOTUS-knockout (KO) mice, whereas they were decreased in cultured OB neurons from NgR1-KO mice. Knockdown of Nogo-A in cultured OB neurons reduced the number of axonal collateral branches, suggesting that endogenous Nogo-A induces axonal branching. Finally, the collateral branches of the LOT were increased in LOTUS-KO mice, whereas those in NgR1-KO mice were decreased. Moreover, the abnormal increase of axonal branching observed in LOTUS-KO mice was rescued in the double mutant of LOTUS- and NgR1-KO mice. These findings suggest that Nogo-A and NgR1 interactions may contribute to axonal branching in LOT development.

Sex-Specific Protection of Osteoarthritis by Deleting Cartilage Acid Protein 1.

Cartilage acidic protein 1 (CRTAC1) was recently identified as an elevated protein in the synovial fluid of patients with osteoarthritis (OA) by a proteomic analysis. This gene is also upregulated in both human and mouse OA by transcriptomic analysis. The objective of this study was to characterize the expression and function of CRTAC1 in OA. Here, we first confirm the increase of CRTAC1 in cartilage biopsies from OA patients undergoing joint replacement by real-time PCR and immunohistochemistry. Furthermore, we report that proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha upregulate CRTAC1 expression in primary human articular chondrocytes and synovial fibroblasts. Genetic deletion of Crtac1 in mice significantly inhibited cartilage degradation, osteophyte formation and gait abnormalities of post-traumatic OA in female, but not male, animals undergoing the destabilization of medial meniscus (DMM) surgery. Taken together, CRTAC1 is upregulated in the osteoarthritic joint and directly induced in chondrocytes and synovial fibroblasts by pro-inflammatory cytokines. This molecule is necessary for the progression of OA in female mice after DMM surgery and thus represents a potential therapy for this prevalent disease, especially for women who demonstrate higher rates and more severe OA.

[Axonal Regeneration-related Molecules as Biomarkers for Multiple Sclerosis].

Abnormalities in both the nervous and immune systems are thought to be relevant to the pathogenesis of multiple sclerosis (MS). Several functional molecules closely related to axonal regeneration play an important role in not only the nervous system, but also the immune system. Many recent studies revealed that these molecules are associated with the neurological and immunological aspects of the pathogenesis of MS. Therefore, we focused on these molecules as promising biomarkers for MS. Nogo protein and its receptor (Nogo receptor-1; NgR1) are well known representative molecules that prevent axonal regeneration, and we identified lateral olfactory tract usher substance (LOTUS) as an endogenous antagonist of NgR1. We found that LOTUS expression was decreased in the spinal cord in an experimental autoimmune encephalomyelitis mouse model and that variations in LOTUS concentration in the cerebrospinal fluid correlated well with disease activity in MS patients. On the other hand, previous studies have shown that repulsive guidance molecule-a and semaphorins, known to be involved in axonal guidance and regeneration, play a role in MS pathogenesis. We review the association of these molecules with the neurological and immunological aspects of MS pathogenesis, and we show that they are promising, clinically-relevant biomarkers for MS.