Functional Relationships between L1CAM, LC3, ATG12, and Aß.

Abnormal protein accumulations in the brain are linked to aging and the pathogenesis of dementia of various types, including Alzheimer's disease. These accumulations can be reduced by cell indigenous mechanisms. Among these is autophagy, whereby proteins are transferred to lysosomes for degradation. Autophagic dysfunction hampers the elimination of pathogenic protein aggregations that contribute to cell death. We had observed that the adhesion molecule L1 interacts with microtubule-associated protein 1 light-chain 3 (LC3), which is needed for autophagy substrate selection. L1 increases cell survival in an LC3-dependent manner via its extracellular LC3 interacting region (LIR). L1 also interacts with Aß and reduces the Aß plaque load in an AD model mouse. Based on these results, we investigated whether L1 could contribute to autophagy of aggregated Aß and its clearance. We here show that L1 interacts with autophagy-related protein 12 (ATG12) via its LIR domain, whereas interaction with ubiquitin-binding protein p62/SQSTM1 does not depend on LIR. Aß, bound to L1, is carried to the autophagosome leading to Aß elimination. Showing that the mitophagy-related L1-70 fragment is ubiquitinated, we expect that the p62/SQSTM1 pathway also contributes to Aß elimination. We propose that enhancing L1 functions may contribute to therapy in humans.

GPR50 regulates neuronal development as a mitophagy receptor.

Neurons rely heavily on high mitochondrial metabolism to provide sufficient energy for proper development. However, it remains unclear how neurons maintain high oxidative phosphorylation (OXPHOS) during development. Mitophagy plays a pivotal role in maintaining mitochondrial quality and quantity. We herein describe that G protein-coupled receptor 50 (GPR50) is a novel mitophagy receptor, which harbors the LC3-interacting region (LIR) and is required in mitophagy under stress conditions. Although it does not localize in mitochondria under normal culturing conditions, GPR50 is recruited to the depolarized mitochondrial membrane upon mitophagy stress, which marks the mitochondrial portion and recruits the assembling autophagosomes, eventually facilitating the mitochondrial fragments to be engulfed by the autophagosomes. Mutations Δ502-505 and T532A attenuate GPR50-mediated mitophagy by disrupting the binding of GPR50 to LC3 and the mitochondrial recruitment of GPR50. Deficiency of GPR50 causes the accumulation of damaged mitochondria and disrupts OXPHOS, resulting in insufficient ATP production and excessive ROS generation, eventually impairing neuronal development. GPR50-deficient mice exhibit impaired social recognition, which is rescued by prenatal treatment with mitoQ, a mitochondrially antioxidant. The present study identifies GPR50 as a novel mitophagy receptor that is required to maintain mitochondrial OXPHOS in developing neurons.

Self-Assembled Fibroblast Growth Factor Nanoparticles as a Therapeutic for Oxidant-Induced Neuronal and Skin Cell Injury.

Traumatic brain injury (TBI) and spinal cord injury (SCI) are neurological conditions that result from immediate mechanical injury, as well as delayed injury caused by local inflammation. Furthermore, TBI and SCI often lead to secondary complications, including pressure wounds of the skin, which can heal slowly and are prone to infection. Pressure wounds are localized areas of damaged tissue caused by prolonged pressure on the skin due to immobility and loss of neurological sensation. With the aim to ameliorate these symptoms, we investigated whether fibroblast growth factors 2 (FGF-2) could contribute to recovery. FGF-2 plays a significant role in both neurogenesis and skin wound healing. We developed a recombinant fusion protein containing FGF-2 linked to elastin-like polypeptides (FGF-ELP) that spontaneously self-assembles into nanoparticles at around 33 °C. The nanoparticle's size was ranging between 220 and 250 nm in diameter at 2 µM. We tested this construct for its ability to address neuronal and skin cell injuries. Hydrogen peroxide was used to induce oxidant-mediated injury on cultured neuronal cells to mimic the impact of reactive oxidants released during the inflammatory response in vivo. We found that FGF-ELP nanoparticles protected against hydrogen peroxide-mediated injury and promoted neurite outgrowth. In the skin cell models, cells were depleted from serum to mimic the reduced levels of nutrients and growth factors in chronic skin wounds. FGF-ELP increased the proliferation and migration of human keratinocytes, fibroblasts, and endothelial cells. FGF-ELP is, therefore, a potentially useful agent to provide both neuroprotection and promotion of cellular processes involved in skin wound healing.

In Vitro and In Vivo Studies of Melanoma Cell Migration by Antagonistic Mimetics of Adhesion Molecule L1CAM.

Melanoma, the deadliest type of skin cancer, has a high propensity to metastasize to other organs, including the brain, lymph nodes, lungs, and bones. While progress has been made in managing melanoma with targeted and immune therapies, many patients do not benefit from these current treatment modalities. Tumor cell migration is the initial step for invasion and metastasis. A better understanding of the molecular mechanisms underlying metastasis is crucial for developing therapeutic strategies for metastatic diseases, including melanoma. The cell adhesion molecule L1CAM (CD171, in short L1) is upregulated in many human cancers, enhancing tumor cell migration. Earlier studies showed that the small-molecule antagonistic mimetics of L1 suppress glioblastoma cell migration in vitro. This study aims to evaluate if L1 mimetic antagonists can inhibit melanoma cell migration in vitro and in vivo. We showed that two antagonistic mimetics of L1, anagrelide and 2-hydroxy-5-fluoropyrimidine (2H5F), reduced melanoma cell migration in vitro. In in vivo allograft studies, only 2H5F-treated female mice showed a decrease in tumor volume.

Mice Mutated in the First Fibronectin Domain of Adhesion Molecule L1 Show Brain Malformations and Behavioral Abnormalities.

The X-chromosome-linked cell adhesion molecule L1 (L1CAM), a glycoprotein mainly expressed by neurons in the central and peripheral nervous systems, has been implicated in many neural processes, including neuronal migration and survival, neuritogenesis, synapse formation, synaptic plasticity and regeneration. L1 consists of extracellular, transmembrane and cytoplasmic domains. Proteolytic cleavage of L1's extracellular and transmembrane domains by different proteases generates several L1 fragments with different functions. We found that myelin basic protein (MBP) cleaves L1's extracellular domain, leading to enhanced neuritogenesis and neuronal survival in vitro. To investigate in vivo the importance of the MBP-generated 70 kDa fragment (L1-70), we generated mice with an arginine to alanine substitution at position 687 (L1/687), thereby disrupting L1's MBP cleavage site and obliterating L1-70. Young adult L1/687 males showed normal anxiety and circadian rhythm activities but enhanced locomotion, while females showed altered social interactions. Older L1/687 males were impaired in motor coordination. Furthermore, L1/687 male and female mice had a larger hippocampus, with more neurons in the dentate gyrus and more proliferating cells in the subgranular layer, while the thickness of the corpus callosum and the size of lateral ventricles were normal. In summary, subtle mutant morphological changes result in subtle behavioral changes.

Chicoric Acid Ameliorated Beta-Amyloid Pathology and Enhanced Expression of Synaptic-Function-Related Markers via L1CAM in Alzheimer's Disease Models.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disease. The accumulation of amyloid-beta (Aß) plaques is a distinctive pathological feature of AD patients. The aims of this study were to evaluate the therapeutic effect of chicoric acid (CA) on AD models and to explore its underlying mechanisms. APPswe/Ind SH-SY5Y cells and 5xFAD mice were treated with CA. Soluble Aß1-42 and Aß plaque levels were analyzed by ELISA and immunohistochemistry, respectively. Transcriptome sequencing was used to compare the changes in hippocampal gene expression profiles among the 5xFAD mouse groups. The specific gene expression levels were quantified by qRT-PCR and Western blot analysis. It was found that CA treatment reduced the Aß1-42 levels in the APPswe/Ind cells and 5xFAD mice. It also reduced the Aß plaque levels as well as the APP and BACE1 levels. Transcriptome analysis showed that CA affected the synaptic-plasticity-related genes in the 5xFAD mice. The levels of L1CAM, PSD-95 and synaptophysin were increased in the APPswe/Ind SH-SY5Y cells and 5xFAD mice treated with CA, which could be inhibited by administering siRNA-L1CAM to the CA-treated APPswe/Ind SH-SY5Y cells. In summary, CA reduced Aß levels and increased the expression levels of synaptic-function-related markers via L1CAM in AD models.

Single Nucleotide Polymorphism in Cell Adhesion Molecule L1 Affects Learning and Memory in a Mouse Model of Traumatic Brain Injury.

The L1 cell adhesion molecule (L1) has demonstrated a range of beneficial effects in animal models of spinal cord injury, neurodegenerative disease, and ischemia; however, the role of L1 in TBI has not been fully examined. Mutations in the L1 gene affecting the extracellular domain of this type 1 transmembrane glycoprotein have been identified in patients with L1 syndrome. These patients suffer from hydrocephalus, MASA (mental retardation, adducted thumbs, shuffling gait, aphasia) symptoms, and corpus callosum agenesis. Clinicians have observed that recovery post-traumatic brain injury (TBI) varies among the population. This variability may be explained by the genetic differences present in the general population. In this study, we utilized a novel mouse model of L1 syndrome with a mutation at aspartic acid position 201 in the extracellular domain of L1 (L1-201). We assessed the impact of this specific single nucleotide polymorphism (SNP) localized to the X-chromosome L1 gene on recovery outcomes following TBI by comparing the L1-201 mouse mutants with their wild-type littermates. We demonstrate that male L1-201 mice exhibit significantly worse learning and memory outcomes in the Morris water maze after lateral fluid percussion (LFP) injury compared to male wild-type mice and a trend to worse motor function on the rotarod. However, no significant changes were observed in markers for inflammatory responses or apoptosis after TBI.

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

Introduction: 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. Methods: 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. Results: 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. Discussion: 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.

Small Organic Compounds Mimicking the Effector Domain of Myristoylated Alanine-Rich C-Kinase Substrate Stimulate Female-Specific Neurite Outgrowth.

Myristoylated alanine-rich C-kinase substrate (MARCKS) is a critical member of a signaling cascade that influences disease-relevant neural functions such as neural growth and plasticity. The effector domain (ED) of MARCKS interacts with the extracellular glycan polysialic acid (PSA) through the cell membrane to stimulate neurite outgrowth in cell culture. We have shown that a synthetic ED peptide improves functional recovery after spinal cord injury in female but not male mice. However, peptides themselves are unstable in therapeutic applications, so we investigated more pharmacologically relevant small organic compounds that mimic the ED peptide to maximize therapeutic potential. Using competition ELISAs, we screened small organic compound libraries to identify molecules that structurally and functionally mimic the ED peptide of MARCKS. Since we had shown sex-specific effects of MARCKS on spinal cord injury recovery, we assayed neuronal viability as well as neurite outgrowth from cultured cerebellar granule cells of female and male mice separately. We found that epigallocatechin, amiodarone, sertraline, tegaserod, and nonyloxytryptamine bind to a monoclonal antibody against the ED peptide, and compounds stimulate neurite outgrowth in cultured cerebellar granule cells of female mice only. Therefore, a search for compounds that act in males appears warranted.

Interaction of L1CAM with LC3 Is Required for L1-Dependent Neurite Outgrowth and Neuronal Survival.

The neural cell adhesion molecule L1 (also called L1CAM or CD171) functions not only in cell migration, but also in cell survival, differentiation, myelination, neurite outgrowth, and signaling during nervous system development and in adults. The proteolytic cleavage of L1 in its extracellular domain generates soluble fragments which are shed into the extracellular space and transmembrane fragments that are internalized into the cell and transported to various organelles to regulate cellular functions. To identify novel intracellular interaction partners of L1, we searched for protein-protein interaction motifs and found two potential microtubule-associated protein 1 light-chain 3 (LC3)-interacting region (LIR) motifs within L1, one in its extracellular domain and one in its intracellular domain. By ELISA, immunoprecipitation, and proximity ligation assay using L1 mutant mice lacking the 70 kDa L1 fragment (L1-70), we showed that L1-70 interacts with LC3 via the extracellular LIR motif in the fourth fibronectin type III domain, but not by the motif in the intracellular domain. The disruption of the L1-LC3 interaction reduces L1-mediated neurite outgrowth and neuronal survival.

Deficiency in the neural cell adhesion molecule 2 (NCAM2) reduces axonal levels of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), affects axonal organization in the hippocampus, and leads to behavioral deficits.

The neural cell adhesion molecule 2 (NCAM2) regulates axonal organization in the central nervous system via mechanisms that have remained poorly understood. We now show that NCAM2 increases axonal levels of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), a protease that regulates axonal guidance. In brains of NCAM2-deficient mice, BACE1 levels are reduced in hippocampal mossy fiber projections, and the infrapyramidal bundle of these projections is shortened. This abnormal axonal organization correlates with impaired short-term spatial memory and cognitive flexibility in NCAM2-deficient male and female mice. Self-grooming, rearing, digging and olfactory acuity are increased in NCAM2-deficient male mice, when compared with littermate wild-type mice of the same sex. NCAM2-deficient female mice also show increased self-grooming, but are reduced in rearing, and do not differ from female wild-type mice in olfactory acuity and digging behavior. Our results indicate that errors in axonal guidance and organization caused by impaired BACE1 function can underlie the manifestation of neurodevelopmental disorders, including autism as found in humans with deletions of the NCAM2 gene.

Mice Mutated in the Third Fibronectin Domain of L1 Show Enhanced Hippocampal Neuronal Cell Death, Astrogliosis and Alterations in Behavior.

Adhesion molecules play major roles in cell proliferation, migration, survival, neurite outgrowth and synapse formation during nervous system development and in adulthood. The neural cell adhesion molecule L1 contributes to these functions during development and in synapse formation and synaptic plasticity after trauma in adulthood. Mutations of L1 in humans result in L1 syndrome, which is associated with mild-to-severe brain malformations and mental disabilities. Furthermore, mutations in the extracellular domain were shown to cause a severe phenotype more often than mutations in the intracellular domain. To explore the outcome of a mutation in the extracellular domain, we generated mice with disruption of the dibasic sequences RK and KR that localize to position 858RKHSKR863 in the third fibronectin type III domain of murine L1. These mice exhibit alterations in exploratory behavior and enhanced marble burying activity. Mutant mice display higher numbers of caspase 3-positive neurons, a reduced number of principle neurons in the hippocampus, and an enhanced number of glial cells. Experiments suggest that disruption of the dibasic sequence in L1 results in subtle impairments in brain structure and functions leading to obsessive-like behavior in males and reduced anxiety in females.

The L1 cell adhesion molecule constrains dendritic spine density in pyramidal neurons of the mouse cerebral cortex.

A novel function for the L1 cell adhesion molecule, which binds the actin adaptor protein Ankyrin was identified in constraining dendritic spine density on pyramidal neurons in the mouse neocortex. In an L1-null mouse mutant increased spine density was observed on apical but not basal dendrites of pyramidal neurons in diverse cortical areas (prefrontal cortex layer 2/3, motor cortex layer 5, visual cortex layer 4. The Ankyrin binding motif (FIGQY) in the L1 cytoplasmic domain was critical for spine regulation, as demonstrated by increased spine density and altered spine morphology in the prefrontal cortex of a mouse knock-in mutant (L1YH) harboring a tyrosine (Y) to histidine (H) mutation in the FIGQY motif, which disrupted L1-Ankyrin association. This mutation is a known variant in the human L1 syndrome of intellectual disability. L1 was localized by immunofluorescence staining to spine heads and dendrites of cortical pyramidal neurons. L1 coimmunoprecipitated with Ankyrin B (220 kDa isoform) from lysates of wild type but not L1YH forebrain. This study provides insight into the molecular mechanism of spine regulation and underscores the potential for this adhesion molecule to regulate cognitive and other L1-related functions that are abnormal in the L1 syndrome.

X-ray structure and function of fibronectin domains two and three of the neural cell adhesion molecule L1.

The cell adhesion molecule L1 (L1CAM, L1 in short) plays crucial roles during neural development, regeneration after injury, synapse formation, synaptic plasticity and tumor cell migration. L1 belongs to the immunoglobulin superfamily and comprises in its extracellular part six immunoglobulin (Ig)-like domains and five fibronectin type III homologous repeats (FNs). The second Ig-like domain has been validated for self- (so-called homophilic) binding between cells. Antibodies against this domain inhibit neuronal migration in vitro and in vivo. The fibronectin type III homologous repeats FN2 and FN3 bind small molecule agonistic L1 mimetics and contribute to signal transduction. FN3 has a stretch of 25 amino acids that can be triggered with a monoclonal antibody, or the L1 mimetics, to enhance neurite outgrowth and neuronal cell migration in vitro and in vivo. To correlate the structural features of these FNs with function, we determined a high-resolution crystal structure of a FN2FN3 fragment, which is functionally active in cerebellar granule cells and binds several mimetics. The structure illustrates that both domains are connected by a short linker sequence allowing a flexible and largely independent organization of both domains. This becomes further evident by comparing the X-ray crystal structure with models derived from Small-Angle X-ray Scattering (SAXS) data for FN2FN3 in solution. Based on the X-ray crystal structure, we identified five glycosylation sites which we believe are crucial for folding and stability of these domains. Our study signifies an advance in the understanding of structure-functional relationships of L1.

The Interactions of the 70 kDa Fragment of Cell Adhesion Molecule L1 with Topoisomerase 1, Peroxisome Proliferator-Activated Receptor γ and NADH Dehydrogenase (Ubiquinone) Flavoprotein 2 Are Involved in Gene Expression and Neuronal L1-Dependent Functions.

The cell adhesion molecule L1 is essential not only for neural development, but also for synaptic functions and regeneration after trauma in adulthood. Abnormalities in L1 functions cause developmental and degenerative disorders. L1's functions critically depend on proteolysis which underlies dynamic cell interactions and signal transduction. We showed that a 70 kDa fragment (L1-70) supports mitochondrial functions and gene transcription. To gain further insights into L1-70's functions, we investigated several binding partners. Here we show that L1-70 interacts with topoisomerase 1 (TOP1), peroxisome proliferator-activated receptor γ (PPARγ) and NADH dehydrogenase (ubiquinone) flavoprotein 2 (NDUFV2). TOP1, PPARγ and NDUFV2 siRNAs reduced L1-dependent neurite outgrowth, and the topoisomerase inhibitors topotecan and irinotecan inhibited L1-dependent neurite outgrowth, neuronal survival and migration. In cultured neurons, L1 siRNA reduces the expression levels of the long autism genes neurexin-1 (Nrxn1) and neuroligin-1 (Nlgn1) and of the mitochondrially encoded gene NADH:ubiquinone oxidoreductase core subunit 2 (ND2). In mutant mice lacking L1-70, Nrxn1 and Nlgn1, but not ND2, mRNA levels are reduced. Since L1-70's interactions with TOP1, PPARγ and NDUFV2 contribute to the expression of two essential long autism genes and regulate important neuronal functions, we propose that L1 may not only ameliorate neurological problems, but also psychiatric dysfunctions.

The KDET Motif in the Intracellular Domain of the Cell Adhesion Molecule L1 Interacts with Several Nuclear, Cytoplasmic, and Mitochondrial Proteins Essential for Neuronal Functions.

Abnormal functions of the cell adhesion molecule L1 are linked to several neural diseases. Proteolytic L1 fragments were reported to interact with nuclear and mitochondrial proteins to regulate events in the developing and the adult nervous system. Recently, we identified a 55 kDa L1 fragment (L1-55) that interacts with methyl CpG binding protein 2 (MeCP2) and heterochromatin protein 1 (HP1) via the KDET motif. We now show that L1-55 also interacts with histone H1.4 (HistH1e) via this motif. Moreover, we show that this motif binds to NADH dehydrogenase ubiquinone flavoprotein 2 (NDUFV2), splicing factor proline/glutamine-rich (SFPQ), the non-POU domain containing octamer-binding protein (NonO), paraspeckle component 1 (PSPC1), WD-repeat protein 5 (WDR5), heat shock cognate protein 71 kDa (Hsc70), and synaptotagmin 1 (SYT1). Furthermore, applications of HistH1e, NDUFV2, SFPQ, NonO, PSPC1, WDR5, Hsc70, or SYT1 siRNAs or a cell-penetrating KDET-carrying peptide decrease L1-dependent neurite outgrowth and the survival of cultured neurons. These findings indicate that L1's KDET motif binds to an unexpectedly large number of molecules that are essential for nervous system-related functions, such as neurite outgrowth and neuronal survival. In summary, L1 interacts with cytoplasmic, nuclear and mitochondrial proteins to regulate development and, in adults, the formation, maintenance, and flexibility of neural functions.

Effects of L1 adhesion molecule agonistic mimetics on signal transduction in neuronal functions.

The L1 cell adhesion molecule plays an essential role in neural development and repair. It is not only a 'lock and key' recognition molecule, but an important signal transducer that stimulates regenerative-beneficial cellular functions such as neurite outgrowth, neuronal cell migration, survival, myelination, and synapse formation. Triggering L1 functions after neurotrauma improves functional recovery. In addition, loss-of-function mutations in the L1 gene lead to the L1 syndrome, a rare, X-linked neurodevelopmental disorder with an incidence of approximately 1:30,000 in newborn males. To use L1 for beneficial functions, we screened small compound libraries for L1 agonistic mimetics that trigger L1 functions and improve conditions in animal models of neurotrauma and the L1 syndrome. To understand the mechanisms underlying these functions, it is important to gain a better understanding of L1-dependent cellular signaling that is triggered by the L1 agonistic mimetics. We tested the cell signaling features of L1 agonistic mimetics that contribute to neurite outgrowth and neuronal migration. Our findings indicates that L1 agonistic mimetics trigger the same cell signaling pathways underlying neurite outgrowth, but only the L1 mimetics tacrine, polydatin, trimebutine and honokiol trigger neuronal migration. In contrast, the mimetics crotamiton and duloxetine did not affect neuronal migration, thus limiting their use in increasing neuronal migration, leaving open the question of whether this is a desired or not desired feature in the adult.

CHL1-Deficient and Wild-Type Male Mice do Not Differ in Locomotor Recovery from Spinal Cord Injury.

CHL1 is a close homolog of L1, a cell adhesion molecule that plays major roles in neural and tumor cell functions. We had found that young adult female mice deficient in CHL1 recovered better than their wild-type female littermates after thoracic Spinal Cord Injury (SCI). This observation was surprising, because CHL1 increases neurite outgrowth in vitro. Injury of adult mouse central and peripheral nervous systems upregulate CHL1 expression in neurons and astrocytes, consistent with CHL1's pro-active, homophilic interaction between CHL1 surface molecules in wild-type mice. After SCI, CHL1 expression was observed to increase in the glial scar, areas of axonal regrowth and remodeling of neural circuits. These observations were made only in females, and we therefore sought to analyze SCI in CHL1-deficient male mice. We now show that CHL1-deficient males did not recover better or worse than their male wild-type littermates. Primary and secondary lesion volumes were similar in the two genotypes, as seen in female mice which were studied in parallel with male mice. Assessment of peripheral leukocytes showed a significant increase in numbers of blood neutrophils at 24 h after SCI in males, but not in females. Lymphocyte numbers in mutant males increased slightly, but numbers of lymphocytes or monocytes did not differ significantly between males or females. These results indicate that CHL1-deficient males and females differ in the number of neutrophils but not lymphocytes or monocytes, suggesting that the difference between males and females is unlikely due to differences in leukocytes.

The BACE1-generated C-terminal fragment of the neural cell adhesion molecule 2 (NCAM2) promotes BACE1 targeting to Rab11-positive endosomes.

Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), also known as ß-secretase, is an aspartic protease. The sorting of this enzyme into Rab11-positive recycling endosomes regulates the BACE1-mediated cleavage of its substrates, however, the mechanisms underlying this targeting remain poorly understood. The neural cell adhesion molecule 2 (NCAM2) is a substrate of BACE1. We show that BACE1 cleaves NCAM2 in cultured hippocampal neurons and NCAM2-transfected CHO cells. The C-terminal fragment of NCAM2 that comprises the intracellular domain and a small portion of NCAM2's extracellular domain, associates with BACE1. This association is not affected in cells with inhibited endocytosis, indicating that the interaction of NCAM2 and BACE1 precedes the targeting of BACE1 from the cell surface to endosomes. In neurons and CHO cells, this fragment and BACE1 co-localize in Rab11-positive endosomes. Overexpression of full-length NCAM2 or a recombinant NCAM2 fragment containing the transmembrane and intracellular domains but lacking the extracellular domain leads to an increase in BACE1 levels in these organelles. In NCAM2-deficient neurons, the levels of BACE1 are increased at the cell surface and reduced in intracellular organelles. These effects are correlated with increased levels of the soluble extracellular domain of BACE1 in the brains of NCAM2-deficient mice, suggesting increased shedding of BACE1 from the cell surface. Of note, shedding of the extracellular domain of Sez6, a protein cleaved exclusively by BACE1, is reduced in NCAM2-deficient animals. These results indicate that the BACE1-generated fragment of NCAM2 regulates BACE1 activity by promoting the targeting of BACE1 to Rab11-positive endosomes.

Interactions between the Polysialylated Neural Cell Adhesion Molecule and the Transient Receptor Potential Canonical Channels 1, 4, and 5 Induce Entry of Ca2+ into Neurons.

The neural cell adhesion molecule (NCAM) plays important functional roles in the developing and mature nervous systems. Here, we show that the transient receptor potential canonical (TRPC) ion channels TRPC1, -4, and -5 not only interact with the intracellular domains of the transmembrane isoforms NCAM140 and NCAM180, but also with the glycan polysialic acid (PSA) covalently attached to the NCAM protein backbone. NCAM antibody treatment leads to the opening of TRPC1, -4, and -5 hetero- or homomers at the plasma membrane and to the influx of Ca2+ into cultured cortical neurons and CHO cells expressing NCAM, PSA, and TRPC1 and -4 or TRPC1 and -5. NCAM-stimulated Ca2+ entry was blocked by the TRPC inhibitor Pico145 or the bacterial PSA homolog colominic acid. NCAM-stimulated Ca2+ influx was detectable neither in NCAM-deficient cortical neurons nor in TRPC1/4- or TRPC1/5-expressing CHO cells that express NCAM, but not PSA. NCAM-induced neurite outgrowth was reduced by TRPC inhibitors and a function-blocking TRPC1 antibody. A characteristic signaling feature was that extracellular signal-regulated kinase 1/2 phosphorylation was also reduced by TRPC inhibitors. Our findings indicate that the interaction of NCAM with TRPC1, -4, and -5 contributes to the NCAM-stimulated and PSA-dependent Ca2+ entry into neurons thereby influencing essential neural functions.