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.

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.

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.

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 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.

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.

Mitochondrial and Neuronal Dysfunctions in L1 Mutant Mice.

Adhesion molecules regulate cell proliferation, migration, survival, neuritogenesis, synapse formation and synaptic plasticity during the nervous system's development and in the adult. Among such molecules, the neural cell adhesion molecule L1 contributes to these functions during development, and in synapse formation, synaptic plasticity and regeneration after trauma. Proteolytic cleavage of L1 by different proteases is essential for these functions. A proteolytic fragment of 70 kDa (abbreviated L1-70) comprising part of the extracellular domain and the transmembrane and intracellular domains was shown to interact with mitochondrial proteins and is suggested to be involved in mitochondrial functions. To further determine the role of L1-70 in mitochondria, we generated two lines of gene-edited mice expressing full-length L1, but no or only low levels of L1-70. We showed that in the absence of L1-70, mitochondria in cultured cerebellar neurons move more retrogradely and exhibit reduced mitochondrial membrane potential, impaired Complex I activity and lower ATP levels compared to wild-type littermates. Neither neuronal migration, neuronal survival nor neuritogenesis in these mutants were stimulated with a function-triggering L1 antibody or with small agonistic L1 mimetics. These results suggest that L1-70 is important for mitochondrial homeostasis and that its absence contributes to the L1 syndrome phenotypes.

The Cell Adhesion Molecule L1 Interacts with Methyl CpG Binding Protein 2 via Its Intracellular Domain.

Cell adhesion molecule L1 regulates multiple cell functions, and L1 deficiency is linked to several neural diseases. Recently, we have identified methyl CpG binding protein 2 (MeCP2) as a potential binding partner of the intracellular L1 domain. By ELISA we show here that L1's intracellular domain binds directly to MeCP2 via the sequence motif KDET. Proximity ligation assay with cultured cerebellar and cortical neurons suggests a close association between L1 and MeCP2 in nuclei of neurons. Immunoprecipitation using MeCP2 antibodies and nuclear mouse brain extracts indicates that MeCP2 interacts with an L1 fragment of ~55 kDa (L1-55). Proximity ligation assay indicates that metalloproteases, ß-site of amyloid precursor protein cleaving enzyme (BACE1) and É£-secretase, are involved in the generation of L1-55. Reduction in MeCP2 expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons as well as the migration of L1-expressing HEK293 cells. Moreover, L1 siRNA, MeCP2 siRNA, or a cell-penetrating KDET-containing L1 peptide leads to reduced levels of myocyte enhancer factor 2C (Mef2c) mRNA and protein in cortical neurons, suggesting that the MeCP2/L1 interaction regulates Mef2c expression. Altogether, the present findings indicate that the interaction of the novel fragment L1-55 with MeCP2 affects L1-dependent functions, such as neurite outgrowth and neuronal migration.

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.

Cell adhesion molecule L1 interacts with the chromo shadow domain of heterochromatin protein 1 isoforms α, ß, and É£ via its intracellular domain.

Cell adhesion molecule L1 regulates multiple cell functions and L1 deficiency is linked to several neural diseases. Proteolytic processing generates functionally decisive L1 fragments, which are imported into the nucleus. By computational analysis, we found at L1's C-terminal end the chromo shadow domain-binding motif PxVxL, which directs the binding of nuclear proteins to the heterochromatin protein 1 (HP1) isoforms α, ß, and É£. By enzyme-linked immunosorbent assay, we show that the intracellular L1 domain binds to all HP1 isoforms. These interactions involve the HP1 chromo shadow domain and are mediated via the sequence 1158 KDET1161 in the intracellular domain of murine L1, but not by L1's C-terminal PxVxL motif. Immunoprecipitation using nuclear extracts from the brain and from cultured cerebellar and cortical neurons indicates that HP1 isoforms interact with a yet unknown nuclear L1 fragment of approximately 55 kDa (L1-55), which carries ubiquitin residues. Proximity ligation indicates a close association between L1-55 and the HP1 isoforms in neuronal nuclei. This association is reduced after the treatment of neurons with inhibitors of metalloproteases, ß-site of amyloid precursor protein cleaving enzyme (BACE1), or É£-secretase, suggesting that cleavage of full-length L1 by these proteases generates L1-55. Reduction of HP1α, -ß, or -É£ expression by siRNA decreases L1-dependent neurite outgrowth from cultured cortical neurons and decreases the L1-dependent migration of L1-transfected HEK293 cells in a scratch assay. These findings indicate that the interaction of the novel fragment L1-55 with HP1 isoforms in nuclei affects L1-dependent functions, such as neurite outgrowth and neuronal migration.

Proteins Binding to the Carbohydrate HNK-1: Common Origins?

The human natural killer (HNK-1) carbohydrate plays important roles during nervous system development, regeneration after trauma and synaptic plasticity. Four proteins have been identified as receptors for HNK-1: the laminin adhesion molecule, high-mobility group box 1 and 2 (also called amphoterin) and cadherin 2 (also called N-cadherin). Because of HNK-1's importance, we asked whether additional receptors for HNK-1 exist and whether the four identified proteins share any similarity in their primary structures. A set of 40,000 sequences homologous to the known HNK-1 receptors was selected and used for large-scale sequence alignments and motif searches. Although there are conserved regions and highly conserved sites within each of these protein families, there was no sequence similarity or conserved sequence motifs found to be shared by all families. Since HNK-1 receptors have not been compared regarding binding constants and since it is not known whether the sulfated or non-sulfated part of HKN-1 represents the structurally crucial ligand, the receptors are more heterogeneous in primary structure than anticipated, possibly involving different receptor or ligand regions. We thus conclude that the primary protein structure may not be the sole determinant for a bona fide HNK-1 receptor, rendering receptor structure more complex than originally assumed.

Amelioration of the abnormal phenotype of a new L1 syndrome mouse mutation with L1 mimetics.

L1 syndrome is a rare developmental disorder characterized by hydrocephalus of varying severity, intellectual deficits, spasticity of the legs, and adducted thumbs. Therapy is limited to symptomatic relief. Numerous gene mutations in the L1 cell adhesion molecule (L1CAM, hereafter abbreviated L1) were identified in L1 syndrome patients, and those affecting the extracellular domain of this transmembrane type 1 glycoprotein show the most severe phenotypes. Previously analyzed rodent models of the L1 syndrome focused on L1-deficient animals or mouse mutants with abrogated cell surface expression of L1, making it difficult to test L1 function-triggering mimetic compounds with potential therapeutic value. To overcome this impasse, we generated a novel L1 syndrome mouse with a mutation of aspartic acid at position 201 in the extracellular part of L1 (p.D201N, hereafter termed L1-201) that displays a cell surface-exposed L1 accessible to the L1 mimetics. Behavioral assessment revealed an increased neurological deficit score and increased locomotor activity in male L1-201 mice carrying the mutation on the X-chromosome. Histological analyses of L1-201 mice showed features of the L1 syndrome, including enlarged ventricles and reduced size of the corpus callosum. Expression levels of L1-201 protein as well as extent of cell surface biotinylation and immunofluorescence labelling of cultured cerebellar neurons were normal. Importantly, treatment of these cultures with the L1 mimetic compounds duloxetine, crotamiton, and trimebutine rescued impaired cell migration and survival as well as neuritogenesis. Altogether, the novel L1 syndrome mouse model provides a first experimental proof-of-principle for the potential therapeutic value of L1 mimetic compounds.

Revisiting the proteolytic processing of cell adhesion molecule L1.

The important functions of cell adhesion molecule L1 in the nervous system depend on diverse proteolytic enzymes which generate different L1 fragments. It has been reported that cleavage in the third fibronectin type III (FNIII) homologous domain generates the fragments L1-80 and L1-140, while cleavage in the first FNIII domain yields the fragments L1-70 and L1-135. These results raised questions concerning the L1 cleavage sites. We thus generated gene-edited mice expressing L1 with mutations of the cleavage sites either in the first or third FNIII domain. By immunoprecipitations and immunoblot analyses using brain homogenates and different L1 antibodies, we show that L1-70 and L1-135 are generated in wild-type mice, but not or only to a low extent in L1 mutant mice. L1-80 and L1-140 were not detected in wild-type or mutant mice. Mass spectrometry confirmed the results from immunoprecipitations and immunoblot analyses. Based on these observations, we propose that L1-70 and L1-135 are the predominant fragments in the mouse nervous system and that the third FNIII domain is decisive for generating these fragments. Treatment of cultured cerebellar neurons with trypsin or plasmin, which were both proposed to generate L1-80 and L1-140 by cleaving in the third FNIII domain, showed by immunoprecipitations and immunoblot analyses that both proteases lead to the generation of L1-70 and L1-135, but not L1-80 and L1-140. We discuss previous observations on the basis of our new results and propose a novel view on the molecular features that render previous and present observations compatible.

The cell adhesion molecule L1 interacts with nuclear proteins via its intracellular domain.

Proteolytic cleavage of the cell adhesion molecule L1 (L1) in brain tissue and in cultured cerebellar neurons results in the generation and nuclear import of a 30 kDa fragment comprising most of L1's C-terminal, intracellular domain. In search of molecules that interact with this domain, we performed affinity chromatography with the recombinant intracellular L1 domain and a nuclear extract from mouse brains, and identified potential nuclear L1 binding partners involved in transcriptional regulation, RNA processing and transport, DNA repair, chromatin remodeling, and nucleocytoplasmic transport. By co-immunoprecipitation and enzyme-linked immunosorbent assay using recombinant proteins, we verified the direct interaction between L1 and the nuclear binding partners non-POU domain containing octamer-binding protein and splicing factor proline/glutamine-rich. The proximity ligation assay confirmed this close interaction in cultures of cerebellar granule cells. Our findings suggest that L1 fragments regulate multiple nuclear functions in the nervous system. We discuss possible physiological and pathological roles of these interactions in regulation of chromatin structure, gene expression, RNA processing, and DNA repair.

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 32 kDa (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.

Histone H1 improves regeneration after mouse spinal cord injury and changes shape and gene expression of cultured astrocytes.

BACKGROUND: We have shown that histone H1 is a binding partner for polysialic acid (PSA) and that it improves functional recovery, axon regrowth/sprouting, and target reinnervation after mouse femoral nerve injury. OBJECTIVE: Here, we analyzed whether histone H1 affects functional recovery, axon regrowth/sprouting, and target reinnervation after spinal cord injury of adult mice. Furthermore, we tested in vitro histone H1's effect on astrocytic gene expression, cell shape and migration as well as on cell survival of cultured motoneurons. METHODS: We applied histone H1 to compressed spinal cord and determined functional recovery and number of fibrillary acidic protein (GFAP)- and neuron-glial antigen 2 (NG2)- positive glial cells, which contribute to glial scarring. Histone H1's effect on migration of astrocytes, astrocytic gene expression and motoneuronal survival was determined using scratch-wounded astroglial monolayer cultures, astrocyte cultures for microarray analysis, and motoneuron cell culture under oxidative stress conditions, respectively. RESULTS: Histone H1 application improves locomotor functions and enhances monoaminergic and cholinergic reinnervation of the spinal cord. Expression levels of GFAP and NG2 around the lesion site were decreased in histone H1-treated mice relative to vehicle-treated mice six weeks after injury. Histone H1 reduced astrocytic migration, changed the shape of GFAP- and NG2-positive glial cells and altered gene expression. Gene ontology enrichment analysis indicated that in particular genes coding for proteins involved in proliferation, differentiation, migration and apoptosis are dysregulated. The up- and down-regulation of distinct genes was confirmed by qPCR and Western blot analysis. Moreover, histone H1 reduced hydrogen peroxide-induced cell death of cultured motoneurons. CONCLUSIONS: The combined observations indicate that histone H1 locally applied to the lesion site, improves regeneration after spinal cord injury. Some of these beneficial functions of histone H1 in vivo and in vitro can be attributed to its interaction with PSA-carrying neural cell adhesion molecule.

A fragment of adhesion molecule L1 is imported into mitochondria, and regulates mitochondrial metabolism and trafficking.

The cell adhesion molecule L1 (also known as L1CAM) plays important roles in the mammalian nervous system under physiological and pathological conditions. We have previously reported that proteolytic cleavage of L1 by myelin basic protein leads to the generation of a 70 kDa transmembrane L1 fragment (L1-70) that promotes neuronal migration and neuritogenesis. Here, we provide evidence that L1-70 is imported from the cytoplasm into mitochondria. Genetic ablation of L1, inhibition of mitochondrial import of L1-70 or prevention of myelin basic protein-mediated generation of L1-70 all lead to reduced mitochondrial complex I activity, and impaired mitochondrial membrane potential, fusion, fission and motility, as well as increased retrograde transport. We identified NADH dehydrogenase ubiquinone flavoprotein 2 as a binding partner for L1, suggesting that L1-70 interacts with this complex I subunit to regulate complex I activity. The results of our study provide insights into novel functions of L1 in mitochondrial metabolism and cellular dynamics. These functions are likely to ameliorate the consequences of acute nervous system injuries and chronic neurodegenerative diseases.

A Fragment of Adhesion Molecule L1 Binds to Nuclear Receptors to Regulate Synaptic Plasticity and Motor Coordination.

Proteolytic cleavage of the neuronal isoform of the murine cell adhesion molecule L1, triggered by stimulation of the cognate L1-dependent signaling pathways, results in the generation and nuclear import of an L1 fragment that contains the intracellular domain, the transmembrane domain, and part of the extracellular domain. Here, we show that the LXXLL and FXXLF motifs in the extracellular and transmembrane domain of this L1 fragment mediate the interaction with the nuclear estrogen receptors α (ERα) and ß (ERß), peroxisome proliferator-activated receptor γ (PPARγ), and retinoid X receptor ß (RXRß). Mutations of the LXXLL motif in the transmembrane domain and of the FXXLF motif in the extracellular domain disturb the interaction of the L1 fragment with these nuclear receptors and, when introduced by viral transduction into mouse embryos in utero, result in impaired motor coordination, learning and memory, as well as synaptic connectivity in the cerebellum, in adulthood. These impairments are similar to those observed in the L1-deficient mouse. Our findings suggest that the interplay of nuclear L1 and distinct nuclear receptors is associated with synaptic contact formation and plasticity.

Proteolytic cleavage of transmembrane cell adhesion molecule L1 by extracellular matrix molecule Reelin is important for mouse brain development.

The cell adhesion molecule L1 and the extracellular matrix protein Reelin play crucial roles in the developing nervous system. Reelin is known to activate signalling cascades regulating neuronal migration by binding to lipoprotein receptors. However, the interaction of Reelin with adhesion molecules, such as L1, has remained poorly explored. Here, we report that full-length Reelin and its N-terminal fragments N-R2 and N-R6 bind to L1 and that full-length Reelin and its N-terminal fragment N-R6 proteolytically cleave L1 to generate an L1 fragment with a molecular mass of 80 kDa (L1-80). Expression of N-R6 and generation of L1-80 coincide in time at early developmental stages of the cerebral cortex. Reelin-mediated generation of L1-80 is involved in neurite outgrowth and in stimulation of migration of cultured cortical and cerebellar neurons. Morphological abnormalities in layer formation of the cerebral cortex of L1-deficient mice partially overlap with those of Reelin-deficient reeler mice. In utero electroporation of L1-80 into reeler embryos normalised the migration of cortical neurons in reeler embryos. The combined results indicate that the direct interaction between L1 and Reelin as well as the Reelin-mediated generation of L1-80 contribute to brain development at early developmental stages.