Molecule Close Homolog Of L1 Research Articles

CHL1 inhibits cell proliferation, migration and invasion by regulating the NF‑κB signaling pathway in colorectal cancer.

Cell adhesion molecule close homolog of L1 (CHL1) is implicated in tumorigenesis of various malignancies. However, its role and underlying molecular mechanisms in colorectal cancer (CRC) remain unclear. The present study aimed to evaluate the specific biological functions and mechanisms of CHL1, in order to provide a theoretical basis for the use of CHL1 as a biological target in CRC. CHL1 expression was originally determined in CRC cell lines. Subsequently, CHL1 overexpression was induced by plasmid transfection in HT29 and SW480 cells, and cell proliferation, migration and invasion were evaluated using the Cell Counting Kit-8, clone formation, organoids formation and Transwell assays. Immunofluorescence and western blotting were performed to assess the protein expression of E-cadherin or N-cadherin. Differentially expressed genes (DEGs) were further evaluated using RNA-sequencing (RNA-seq) in HT29 and SW480 cells following CHL1 overexpression and functional enrichment analysis. Western blotting was performed to validate the expression of proteins related to the nuclear factor κB (NF-κB) signaling pathway. The TNMplot online database revealed the significant downregulation of CHL1 in CRC tissues. The results indicated that exogenous CHL1 overexpression significantly inhibited the proliferative, organoid-forming, migratory and invasive abilities of HT29 and SW480 cells, and increased E-cadherin protein expression. Additionally, CHL1 overexpression reduced xenograft tumor growth in vivo. RNA-seq and functional analysis revealed that DEGs in CHL1 overexpressing cells were mainly enriched in the NF-κB signaling pathway. The expression of p-p65 and p-p65/p65 ratio were significantly reduced in HT29 and SW480 cells, following CHL1 overexpression. Additionally, the inhibitory effects of CHL1 overexpression on CRC cell proliferation, organoid formation, migration and invasion were partially counteracted following the overexpression of p65 expression. Overall, the present study demonstrates that CHL1 inhibits CRC cell growth, migration and invasion through the inactivation of the NF-κB signaling pathway.

CHL1 depletion affects dopamine receptor D2-dependent modulation of mouse behavior.

The dopaminergic system plays a key role in the appropriate functioning of the central nervous system, where it is essential for emotional balance, arousal, reward, and motor control. The cell adhesion molecule close homolog of L1 (CHL1) contributes to dopaminergic system development, and CHL1 and the dopamine receptor D2 (D2R) are associated with mental disorders like schizophrenia, addiction, autism spectrum disorder and depression. Here, we investigated how the interplay between CHL1 and D2R affects the behavior of young adult male and female wild-type (CHL+/+) and CHL1-deficient (CHL1-/-) mice, when D2R agonist quinpirole and antagonist sulpiride are applied. Low doses of quinpirole (0.02 mg/kg body weight) induced hypolocomotion of CHL1+/+ and CHL1-/- males and females, but led to a delayed response in CHL1-/- mice. Sulpiride (1 mg/kg body weight) affected locomotion of CHL1-/- females and social interaction of CHL1+/+ females as well as social interactions of CHL1-/- and CHL1+/+ males. Quinpirole increased novelty-seeking behavior of CHL1-/- males compared to CHL1+/+ males. Vehicle-treated CHL1-/- males and females showed enhanced working memory and reduced stress-related behavior. We propose that CHL1 regulates D2R-dependent functions in vivo. Deficiency of CHL1 leads to abnormal locomotor activity and emotionality, and to sex-dependent behavioral differences.

Interplay in neural functions of cell adhesion molecule close homolog of L1 (CHL1) and Programmed Cell Death 6 (PDCD6).

Close homolog of L1 (CHL1) is a cell adhesion molecule of the immunoglobulin superfamily. It promotes neuritogenesis and survival of neurons in vitro. In vivo, CHL1 promotes nervous system development, regeneration after trauma, and synaptic function and plasticity. We identified programmed cell death 6 (PDCD6) as a novel binding partner of the CHL1 intracellular domain (CHL1‐ICD). Co‐immunoprecipitation, pull‐down assay with CHL1‐ICD, and proximity ligation in cerebellum and pons of 3‐day‐old and 6‐month‐old mice, as well as in cultured cerebellar granule neurons and cortical astrocytes indicate an association between PDCD6 and CHL1. The Ca2+‐chelator BAPTA‐AM inhibited the association between CHL1 and PDCD6. The treatment of cerebellar granule neurons with a cell‐penetrating peptide comprising the cell surface proximal 30 N‐terminal amino acids of CHL1‐ICD inhibited the association between CHL1 and PDCD6 and PDCD6‐ and CHL1‐triggered neuronal survival. These results suggest that PDCD6 contributes to CHL1 functions in the nervous system.

Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma.

In a previous study, we demonstrated that CHL1, the neuronal cell adhesion molecule close homolog of L1, acts as a tumor suppressor in human neuroblastoma (NB), a still highly lethal childhood malignancy, influencing its differentiation and proliferation degree. Here we found that ezrin, one of the ERM (ezrin, radixin, moesin) proteins involved in cytoskeleton organization, strongly interacts with CHL1. The low expression of EZRIN, as well as the low expression of CHL1 and of the neuronal differentiation marker MAP2, correlates with poor outcome in NB patients. Knock-down of ezrin in HTLA-230 cell line induces neurite retraction, enhances cell proliferation and migration, and triggers anchorage-independent growth, with effects very similar to those already obtained by CHL1 silencing. Furthermore, lack of ezrin inhibits the expression of MAP2 and of the oncosuppressor molecule p53, whereas it enhances MAPK activation, all typical features of tumor aggressiveness. As already described, CHL1 overexpression in IMR-32 cell line provokes an opposite trend, but the co-silencing of ezrin reduces these effects, confirming the hypothesis that CHL1 acts in close connection with ezrin. Overall, our data show that ezrin reinforces the differentiating and oncosuppressive functions of CHL1, identifying this ERM protein as a new targetable molecule for NB therapy.

Analysis of the functional sequences in the promoter region of the human adhesion molecule close homolog of L1

Background Close Homolog of L1 (CHL1) is a member of the L1 family of cell adhesion molecules. CHL1 gene is located on human chromosome 3 and has been linked to several pathologies, including 3p deletion syndrome, schizophrenia, and tumor growth and metastasis. Objective The goal of the present study was to determine which region of the CHL1 promoter is most competent in driving CHL1 gene expression. Methods: Five candidate DNA fragments in the promoter regions were selected by screening across six species for evolutionary conserved sequences. The activity of these five promoter regions was quantitatively evaluated using a GFP reporter gene in transfection experiments, performed in C6 glioma cells. Results Of the five promoter regions tested, three drove reporter GFP expression, with the conserved region 6 (CR6, Gene ID AC066595.5, 25851-26850) being the most active for transcription. Conclusion The identification of the CR6 activity provides a better understanding of the regulatory mechanisms underlying CHL1 expression. It may help future discovery of therapeutic strategies that involve influencing critical promoter regions to drive transcriptional regulation of the mammalian CHL1 gene. HIGHLIGHTS Conserved regions of CHL1 promoter sequences were identified by in-silico analysis. Five conserved regions were tested for gene regulatory activity using a reporter assay. Conserved regions CR5, CR6 and CR7 show gene regulatory function in a reporter assay. Co-transfection of CR5 and CR6 yielded the highest reporter activity. The core region of CR6 (CR6core) was identified as a cis-acting element. In-tandem promoter CR5core-CR6core was the best in a reporter assay.

Downregulation of Adhesion Molecule CHL1 in B Cells but Not T Cells of Patients with Major Depression and in the Brain of Mice with Chronic Stress.

Depression is a common serious mental disorder with unclear pathogenesis. Currently, specific diagnostic biomarkers are yet to be characterized. The close homolog of L1 (CHL1) is a L1 family cell adhesion molecule involved in the regulation of neuronal survival and growth. Although genome-wide expression profiling of human lymphoblastoid cell lines (LCLs) reported neural cell adhesion molecule (NCAM) L1 as a tentative biomarker for selective serotonin reuptake inhibitor (SSRI) antidepressant response, the involvement of CHL1 in depression is unclear. In this study, using a well-established chronic unpredictable mild stress (CUMS) depression mouse model, we examined the mRNA and protein expression of CHL1 in normal control, CUMS, vehicle (VEH), fluoxetine (FLU), and clozapine (CLO) groups. We found that in the CUMS group, both mRNA and protein expression of CHL1 were downregulated in both the hippocampus and the cortex. Treatment of CUMS mice with FLU and CLO reversed CHL1 mRNA and protein expression. In the human study, we showed that CHL1 expression was significantly downregulated in monocytes of unipolar and bipolar depressive patients compared with healthy donors (HD) at both mRNA and protein levels. Consistently, ELISA showed that CHL1 levels in the serum of patients with depression were reduced and negatively correlated with their HRSD-21 scores. Further flow cytometry studies showed that the reduced number of CHL1 positive CD19+ and CD20+ B cells of patients with depression was subsequently reversed with antidepressant treatment. Our findings suggested that downregulation of CHL1 from both immune cells and the brain may be linked to the immunopathogenesis of depression. In conclusion, CHL1 may be an important predictive marker for both diagnosis and treatment outcome of depression.

Cell adhesion molecule close homolog of L1 binds to the dopamine receptor D2 and inhibits the internalization of its short isoform.

Cell adhesion molecule close homolog of L1 (CHL1) and the dopamine receptor D2 (DRD2) are associated with psychiatric and mental disorders. We here show that DRD2 interacts with CHL1 in mouse brain, as evidenced by co-immunostaining, proximity ligation assay, co-immunoprecipitation, and pull-down assay with recombinant extracellular CHL1 domain fused to Fc (CHL1-Fc). Direct binding of CHL1-Fc to the first extracellular loop of DRD2 was shown by ELISA. Using HEK cells transfected to co-express CHL1 and the short (DRD2-S) or long (DRD2-L) DRD2 isoforms, co-localization of CHL1 and both isoforms was observed by immunostaining and proximity ligation assay. Moreover, CHL1 inhibited agonist-triggered internalization of DRD2-S. Proximity ligation assay showed close interaction between CHL1 and DRD2 in neurons expressing dopamine- and cAMP-regulated phosphoprotein of 32kDa (DARPP32) or tyrosine hydroxylase (TH) in tissue sections of adult mouse striatum. In cultures of striatum or ventral midbrain, CHL1 was also closely associated with DRD2 in DARPP32- or TH-immunopositive cells, respectively. In the dorsal striatum of CHL1-deficient mice, lower levels of DRD2 and phosphorylated TH were observed, when compared to wild-type littermates. In the ventral striatum of CHL1-deficient mice, levels of phosphorylated DARPP32 were reduced. We propose that CHL1 regulates DRD2-dependent presynaptic and postsynaptic functions.

Close Homolog of L1 Regulates Dendritic Spine Density in the Mouse Cerebral Cortex Through Semaphorin 3B.

Dendritic spines in the developing mammalian neocortex are initially overproduced and then eliminated during adolescence to achieve appropriate levels of excitation in mature networks. We show here that the L1 family cell adhesion molecule Close Homolog of L1 (CHL1) and secreted repellent ligand Semaphorin 3B (Sema3B) function together to induce dendritic spine pruning in developing cortical pyramidal neurons. Loss of CHL1 in null mutant mice in both genders resulted in increased spine density and a greater proportion of immature spines on apical dendrites in the prefrontal and visual cortex. Electron microscopy showed that excitatory spine synapses with postsynaptic densities were increased in the CHL1-null cortex, and electrophysiological recording in prefrontal slices from mutant mice revealed deficiencies in excitatory synaptic transmission. Mechanistically, Sema3B protein induced elimination of spines on apical dendrites of cortical neurons cultured from wild-type but not CHL1-null embryos. Sema3B was secreted by the cortical neuron cultures, and its levels increased when cells were treated with the GABA antagonist gabazine. In vivo CHL1 was coexpressed with Sema3B in pyramidal neuron subpopulations and formed a complex with Sema3B receptor subunits Neuropilin-2 and PlexinA4. CHL1 and NrCAM, a closely related L1 adhesion molecule, localized primarily to distinct spines and promoted spine elimination to Sema3B or Sema3F, respectively. These results support a new concept in which selective spine elimination is achieved through different secreted semaphorins and L1 family adhesion molecules to sculpt functional neural circuits during postnatal maturation.SIGNIFICANCE STATEMENT Dendritic spines in the mammalian neocortex are initially overproduced and then pruned in adolescent life through unclear mechanisms to sculpt maturing cortical circuits. Here, we show that spine and excitatory synapse density of pyramidal neurons in the developing neocortex is regulated by the L1 adhesion molecule, Close Homolog of L1 (CHL1). CHL1 mediated spine pruning in response to the secreted repellent ligand Semaphorin 3B and associated with receptor subunits Neuropilin-2 and PlexinA4. CHL1 and related L1 adhesion molecule NrCAM localized to distinct spines, and promoted spine elimination to Semaphorin 3B and -3F, respectively. These results support a new concept in which selective elimination of individual spines and nascent synapses can be achieved through the action of distinct secreted semaphorins and L1 adhesion molecules.

CHL1 gene acts as a tumor suppressor in human neuroblastoma.

Neuroblastoma is an aggressive, relapse-prone childhood tumor of the sympathetic nervous system that accounts for 15% of pediatric cancer deaths. A distal portion of human chromosome 3p is often deleted in neuroblastoma, this region may contain one or more putative tumor suppressor genes. A 2.54 Mb region at 3p26.3 encompassing the smallest region of deletion pinpointed CHL1 gene, the locus for neuronal cell adhesion molecule close homolog of L1. We found that low CHL1 expression predicted poor outcome in neuroblastoma patients. Here we have used two inducible cell models to analyze the impact of CHL1 on neuroblastoma biology. Over-expression of CHL1 induced neurite-like outgrowth and markers of neuronal differentiation in neuroblastoma cells, halted tumor progression, inhibited anchorage-independent colony formation, and suppressed the growth of human tumor xenografts. Conversely, knock-down of CHL1 induced neurite retraction and activation of Rho GTPases, enhanced cell proliferation and migration, triggered colony formation and anchorage-independent growth, accelerated growth in orthotopic xenografts mouse model. Our findings demonstrate unambiguously that CHL1 acts as a regulator of proliferation and differentiation of neuroblastoma cells through inhibition of the MAPKs and Akt pathways. CHL1 is a novel candidate tumor suppressor in neuroblastoma, and its associated pathways may represent a promising target for future therapeutic interventions.

Cell Adhesion Molecule Close Homolog of L1 (CHL1) Guides the Regrowth of Regenerating Motor Axons and Regulates Synaptic Coverage of Motor Neurons.

The close homolog of L1 (CHL1) is a cell adhesion molecule involved in regulation of neuronal differentiation and survival, neurite outgrowth and axon guidance during development. In the mature nervous system, CHL1 regulates synaptic activity and plasticity. The aim of the present study was to evaluate the influence of CHL1 on peripheral nerve regeneration after trauma. Using the established model of mouse femoral nerve regeneration, CHL1 knock-out mice were investigated in comparison to the wild type littermates. First, non-injured mice of both genotypes were compared regarding the synaptic phenotypes in the corresponding spinal cord segment. While no differences in phenotypes were detectable in the femoral nerve, corresponding segments in the spinal cord were observed to differ in that inhibitory perisomatic innervation of motor neurons was increased in CHL1-deficient mice, and numbers of perisomatic cholinergic synapses on motor neuronal somata were reduced. Regarding the femoral nerve after injury, CHL1-deficient mice demonstrated preferential motor axon regrowth into the saphenous vs. quadriceps branch after nerve transection upstream of the nerve bifurcation by 8 weeks after transection, indicating decreased preferential motor re-innervation. Furthermore, in injured wild-type mice, enhanced CHL1 expression was observed in regenerating axons in the proximal nerve stump upstream of the bifurcation at days 1, 3, 5, 7 and 14, and in the distal stump at days 7 and 14 after injury, when compared to non-injured mice. Injury-related upregulation of CHL1 expression was more pronounced in axons than in Schwann cells. Despite a more pronounced capacity for preferential motor axon regrowth in wild-type vs. mutant mice, only a tendency for difference in recovery of motor functions was observed between genotypes, without statistical significance Taken together, these results indicate that CHL1 is involved in peripheral nerve regeneration, because it guides regrowing axons into the appropriate nerve branch and regulates synaptic coverage in the spinal cord.

α-secretase ADAM10 physically interacts with β-secretase BACE1 in neurons and regulates CHL1 proteolysis

α-secretase and β-secretase are known to compete for amyloid precursor protein (APP) processing and thus play a vital role in Alzheimer's disease pathogenesis. A disintegrin and metalloproteinase 10 (ADAM10) and β-site APP cleaving enzyme 1 (BACE1) mediate the major activities of α-secretase and β-secretase in brain and share various common substrates. However, whether they function separately or together is poorly understood. Here, we show that ADAM10 and BACE1 co-localize in the neurites of mouse primary neurons. Co-immunoprecipitation and fluorescence resonance energy transfer analysis revealed that ADAM10 and BACE1 interact with each other under both endogenous and exogenous conditions. In addition, we found that ADAM10 enhances the proteolysis of neural cell adhesion molecule close homolog of L1 (CHL1) by BACE1. Further studies found that ADAM10-BACE1 interaction interfering peptide LT52 attenuates the regulation of ADAM10 on BACE1-mediated cleavage of CHL1. Our data indicate that ADAM10-BACE1 interaction regulates the proteolysis of some specific substrates and may play a potential role in brain function.

The cell adhesion molecule CHL1 interacts with patched-1 to regulate apoptosis during postnatal cerebellar development

The immunoglobulin superfamily adhesion molecule close homolog of L1 (CHL1) plays important roles during nervous system development. Here, we identified the hedgehog receptor patched-1 (PTCH1) as a novel CHL1-binding protein and showed that CHL1 interacts with the first extracellular loop of PTCH1 via its extracellular domain. Colocalization and co-immunoprecipitation of CHL1 with PTCH1 suggest an association of CHL1 with this major component of the hedgehog signaling pathway. The trans-interaction of CHL1 with PTCH1 promotes neuronal survival in cultures of dissociated cerebellar granule cells and of organotypic cerebellar slices. An inhibitor of the PTCH1-regulated hedgehog signal transducer, smoothened (SMO), and inhibitors of RhoA and Rho-associated kinase (ROCK) 1 and 2 prevent CHL1-dependent survival of cultured cerebellar granule cells and survival of cerebellar granule and Purkinje cells in organotypic cultures. In histological sections from 10- and 14-day-old CHL1-deficient mice, enhanced apoptosis of granule, but not Purkinje, cells was observed. The results of the present study indicate that CHL1 triggers PTCH1-, SMO-, RhoA- and ROCK-dependent signal transduction pathways to promote neuronal survival after cessation of the major morphogenetic events during mouse cerebellar development.

The cell adhesion molecule CHL1 interacts with patched-1 to regulate apoptosis during postnatal cerebellar development.

The immunoglobulin superfamily adhesion molecule close homolog of L1 (CHL1) plays important roles during nervous system development. Here, we identified the hedgehog receptor patched-1 (PTCH1) as a novel CHL1-binding protein and showed that CHL1 interacts with the first extracellular loop of PTCH1 via its extracellular domain. Colocalization and co-immunoprecipitation of CHL1 with PTCH1 suggest an association of CHL1 with this major component of the hedgehog signaling pathway. The trans-interaction of CHL1 with PTCH1 promotes neuronal survival in cultures of dissociated cerebellar granule cells and of organotypic cerebellar slices. An inhibitor of the PTCH1-regulated hedgehog signal transducer, smoothened (SMO), and inhibitors of RhoA and Rho-associated kinase (ROCK) 1 and 2 prevent CHL1-dependent survival of cultured cerebellar granule cells and survival of cerebellar granule and Purkinje cells in organotypic cultures. In histological sections from 10- and 14-day-old CHL1-deficient mice, enhanced apoptosis of granule, but not Purkinje, cells was observed. The results of the present study indicate that CHL1 triggers PTCH1-, SMO-, RhoA- and ROCK-dependent signal transduction pathways to promote neuronal survival after cessation of the major morphogenetic events during mouse cerebellar development.

Interaction between CHL1 and serotonin receptor 2c regulates signal transduction and behavior in mice.

The serotonergic system plays important roles in multiple functions of the nervous system and its malfunctioning leads to neurological and psychiatric disorders. Here, we show that the cell adhesion molecule close homolog of L1 (CHL1), which has been linked to mental disorders, binds to a peptide stretch in the third intracellular loop of the serotonin 2c (5-HT2c) receptor through its intracellular domain. Moreover, we provide evidence that CHL1 deficiency in mice leads to 5-HT2c-receptor-related reduction in locomotor activity and reactivity to novelty, and that CHL1 regulates signaling pathways triggered by constitutively active isoforms of the 5-HT2c receptor. Furthermore, we found that the 5-HT2c receptor and CHL1 colocalize in striatal and hippocampal GABAergic neurons, and that 5-HT2c receptor phosphorylation and its association with phosphatase and tensin homolog (PTEN) and β-arrestin 2 is regulated by CHL1. Our results demonstrate that CHL1 regulates signal transduction pathways through constitutively active 5-HT2c receptor isoforms, thereby altering 5-HT2c receptor functions and implicating CHL1 as a new modulator of the serotonergic system.

Interaction of the cell adhesion molecule CHL1 with vitronectin, integrins, and the plasminogen activator inhibitor-2 promotes CHL1-induced neurite outgrowth and neuronal migration.

The cell adhesion molecule close homolog of L1 (CHL1) plays important functional roles in the developing and adult nervous system. In search of the binding partners that mediate the diverse and sometimes opposing functions of CHL1, the extracellular matrix-associated proteins vitronectin and plasminogen activator inhibitor-2 (PAI-2) were identified as novel CHL1 interaction partners and tested for involvement in CHL1-dependent functions during mouse cerebellar development. CHL1-induced cerebellar neurite outgrowth and cell migration at postnatal days 6-8 were inhibited by a CHL1-derived peptide comprising the integrin binding RGD motif, and by antibodies against vitronectin or several integrins, indicating a vitronectin-dependent integrin-mediated pathway. A PAI-2-derived peptide, or antibodies against PAI-2, urokinase type plasminogen activator (uPA), uPA receptor, and several integrins reduced cell migration. CHL1 colocalized with vitronectin, PAI-2, and several integrins in cerebellar granule cells, suggesting an association among these proteins. Interestingly, at the slightly earlier age of 4-5 d, cerebellar neurons did not depend on CHL1 for neuritogenesis and cell migration. However, differentiation of progenitor cells into neurons at this stage was dependent on homophilic CHL1-CHL1 interactions. These observations indicate that homophilic CHL1 trans-interactions regulate differentiation of neuronal progenitor cells at early postnatal stages, while heterophilic trans-interactions of CHL1 with vitronectin, integrins, and the plasminogen activator system regulate neuritogenesis and neuronal cell migration at a later postnatal stage of cerebellar morphogenesis. Thus, within very narrow time windows in postnatal cerebellar development, distinct types of molecular interactions mediated by CHL1 underlie the diverse functions of this protein.

Lipid Raft-dependent Endocytosis of Close Homolog of Adhesion Molecule L1 (CHL1) Promotes Neuritogenesis

CHL1 plays a dual role by either promoting or inhibiting neuritogenesis. We report here that neuritogenesis-promoting ligand-dependent cell surface clustering of CHL1 induces palmitoylation and lipid raft-dependent endocytosis of CHL1. We identify βII spectrin as a binding partner of CHL1, and we show that partial disruption of the complex between CHL1 and βII spectrin accompanies CHL1 endocytosis. Inhibition of the association of CHL1 with lipid rafts by pharmacological disruption of lipid rafts or by mutation of cysteine 1102 within the intracellular domain of CHL1 reduces endocytosis of CHL1. Endocytosis of CHL1 is also reduced by nifedipine, an inhibitor of the L-type voltage-dependent Ca(2+) channels. CHL1-dependent neurite outgrowth is reduced by inhibitors of lipid raft assembly, inhibitors of voltage-dependent Ca(2+) channels, and overexpression of CHL1 with mutated cysteine Cys-1102. Our results suggest that ligand-induced and lipid raft-dependent regulation of CHL1 adhesion via Ca(2+)-dependent remodeling of the CHL1-βII spectrin complex and CHL1 endocytosis are required for CHL1-dependent neurite outgrowth.

β-Site Amyloid Precursor Protein (APP)-cleaving Enzyme 1 (BACE1)-deficient Mice Exhibit a Close Homolog of L1 (CHL1) Loss-of-function Phenotype Involving Axon Guidance Defects

BACE1 is the β-secretase enzyme that initiates production of the β-amyloid peptide involved in Alzheimer disease. However, little is known about the functions of BACE1. BACE1-deficient mice exhibit mild but complex neurological phenotypes suggesting therapeutic BACE1 inhibition may not be completely free of mechanism-based side effects. Recently, we have reported that BACE1 null mice have axon guidance defects in olfactory sensory neuron projections to glomeruli in the olfactory bulb. Here, we show that BACE1 deficiency also causes an axon guidance defect in the hippocampus, a shortened and disorganized infrapyramidal bundle of the mossy fiber projection from the dentate gyrus to CA3. Although we observed that a classical axon guidance molecule, EphA4, was cleaved by BACE1 when co-expressed with BACE1 in HEK293 cells, we could find no evidence of BACE1 processing of EphA4 in the brain. Remarkably, we discovered that the axon guidance defects of BACE1(-/-) mice were strikingly similar to those of mice deficient in a recently identified BACE1 substrate, the neural cell adhesion molecule close homolog of L1 (CHL1) that is involved in neurite outgrowth. CHL1 undergoes BACE1-dependent processing in BACE1(+/+), but not BACE1(-/-), hippocampus, and olfactory bulb, indicating that CHL1 is a BACE1 substrate in vivo. Finally, BACE1 and CHL1 co-localize in the terminals of hippocampal mossy fibers, olfactory sensory neuron axons, and growth cones of primary hippocampal neurons. We conclude that BACE1(-/-) axon guidance defects are likely the result of abrogated BACE1 processing of CHL1 and that BACE1 deficiency produces a CHL1 loss-of-function phenotype. Our results imply the possibility that axon mis-targeting may occur in adult neurogenic and/or regenerating neurons as a result of chronic BACE1 inhibition and add a note of caution to BACE1 inhibitor development.

L1 and CHL1 Cooperate in Thalamocortical Axon Targeting

Neural cell adhesion molecule close homolog of L1 (CHL1) is a regulator of topographic targeting of thalamic axons to the somatosensory cortex (S1) but little is known about its cooperation with other L1 class molecules. To investigate this, CHL1(-/-)/L1(-/y) double mutant mice were generated and analyzed for thalamocortical axon topography. Double mutants exhibited a striking posterior shift of axons from motor thalamic nuclei to the visual cortex (V1), which was not observed in single mutants. In wild-type (WT) embryos, L1 and CHL1 were coexpressed in the dorsal thalamus (DT) and on fibers along the thalamocortical projection in the ventral telencephalon and cortex. L1 and CHL1 colocalized on growth cones and neurites of cortical and thalamic neurons in culture. Growth cone collapse assays with WT and mutant neurons demonstrated a requirement for L1 and CHL1 in repellent responses to EphrinA5, a guidance factor for thalamic axons. L1 coimmunoprecipitated with the principal EphrinA5 receptors expressed in the DT (EphA3, EphA4, and EphA7), whereas CHL1 associated selectively with EphA7. These results implicate a novel mechanism in which L1 and CHL1 interact with individual EphA receptors and cooperate to guide subpopulations of thalamic axons to distinct neocortical areas essential for thalamocortical connectivity.

Better functional outcome of compression spinal cord injury in mice is associated with enhanced H-reflex responses

Alterations in spinal reflexes and functional improvements occur after incomplete spinal cord injury but the relationship between these phenomena is not understood. Here we show that spontaneous functional recovery after compression injury of the spinal cord at low-thoracic level (Th10–12) in C57BL/6J mice is associated with a progressively increasing, over 3 months, excitability of the plantar H-reflex. The stimulation rate-sensitive H-reflex depression, already strongly reduced at 1 week after injury, when compared with non-injured mice, decreased further during the observation time period. Twelve weeks after injury, the degree of motor recovery estimated by single-frame motion analysis in individual animals correlated positively with their H-reflex responses at 2-Hz stimulation. Functional recovery and reflex alterations were accompanied by an increase in glycine/GABAergic and glutamatergic terminals around motoneuron cell bodies between 6 and 12 weeks after injury. Enhanced H-reflex responses at frequencies between 0.1 and 5 Hz were also observed in mice deficient in the extracellular matrix glycoprotein tenascin-R and the adhesion molecule close homolog of L1, mice previously shown to have better motor recovery after spinal cord injury than wild-type littermates. These results indicate that better functional outcome of compression spinal cord injury in mice is associated with alterations of the monosynaptic reflex pathway which facilitate motoneuron recruitment. Our observations support the view that plasticity of spinal circuitries underlies specific aspects of motor recovery and demonstrate the usefulness of H-reflex analyses in studies on spinal cord injury in mice.

Close Homolog of L1 and Neuropilin 1 Mediate Guidance of Thalamocortical Axons at the Ventral Telencephalon

We report a cooperation between the neural adhesion molecule close homolog of L1 (CHL1) and the semaphorin 3A (Sema3A) receptor, neuropilin 1 (Npn1), important for establishment of area-specific thalamocortical projections. CHL1 deletion in mice selectively disrupted the projection of somatosensory thalamic axons from the ventrobasal (VB) nuclei, causing them to shift caudally and target the visual cortex. At the ventral telencephalon, an intermediate target with graded Sema3A expression, VB axons were caudally shifted in CHL1- embryos and in Npn1(Sema-/-) mutants, in which axons are nonresponsive to Sema3A. CHL1 colocalized with Npn1 on thalamic axons, and associated with Npn1 through a sequence in the CHL1 Ig1 domain that was required for Sema3A-induced growth cone collapse. These results identify a novel function for CHL1 in thalamic axon responsiveness to ventral telencephalic cues, and demonstrate a role for CHL1 and Npn1 in establishment of proper targeting of specific thalamocortical projections.