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.

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.

Amyloid precursor protein (APP) and amyloid ß (Aß) interact with cell adhesion molecules: Implications in Alzheimer's disease and normal physiology.

Alzheimer's disease (AD) is an incurable neurodegenerative disorder in which dysfunction and loss of synapses and neurons lead to cognitive impairment and death. Accumulation and aggregation of neurotoxic amyloid-ß (Aß) peptides generated via amyloidogenic processing of amyloid precursor protein (APP) is considered to play a central role in the disease etiology. APP interacts with cell adhesion molecules, which influence the normal physiological functions of APP, its amyloidogenic and non-amyloidogenic processing, and formation of Aß aggregates. These cell surface glycoproteins also mediate attachment of Aß to the neuronal cell surface and induce intracellular signaling contributing to Aß toxicity. In this review, we discuss the current knowledge surrounding the interactions of cell adhesion molecules with APP and Aß and analyze the evidence of the critical role these proteins play in regulating the processing and physiological function of APP as well as Aß toxicity. This is a necessary piece of the complex AD puzzle, which we should understand in order to develop safe and effective therapeutic interventions for AD.

RNA polyadenylation patterns in the human transcriptome.

The eukaryotic transcriptome undergoes various post-transcriptional modifications which assists gene expression. Polyadenylation is a molecular process occurring at the 3'-end of the RNA molecule which involves the poly(A) polymerase attaching adenine monophosphate molecules in a chain-like fashion to assemble a poly(A) tail. Multiple RNA isoforms are produced with differing 3'-UTR and exonic compositions through alternative polyadenylation (APA) which enhances the diversification of alternatively spliced mRNA transcripts. To study polyadenylation patterns, novel methods have been developed using short-read and long-read sequencing technologies to analyse the 3'-ends of the transcript. Recent studies have identified unique polyadenylation patterns in different cellular functions, including oncogenic activity, which could prove valuable in the understanding of medical genetics, particularly in the discovery of biomarkers in diseased states. We present a review of current literature reporting on polyadenylation and the biological relevance in the mammalian transcriptome, with a focus on the human transcriptome. Additionally, we have explored the various methods available to detect polyadenylation patterns using second and third generation sequencing technologies.

Neural glycomics: the sweet side of nervous system functions.

The success of investigations on the structure and function of the genome (genomics) has been paralleled by an equally awesome progress in the analysis of protein structure and function (proteomics). We propose that the investigation of carbohydrate structures that go beyond a cell's metabolism is a rapidly developing frontier in our expanding knowledge on the structure and function of carbohydrates (glycomics). No other functional system appears to be suited as well as the nervous system to study the functions of glycans, which had been originally characterized outside the nervous system. In this review, we describe the multiple studies on the functions of LewisX, the human natural killer cell antigen-1 (HNK-1), as well as oligomannosidic and sialic (neuraminic) acids. We attempt to show the sophistication of these structures in ontogenetic development, synaptic function and plasticity, and recovery from trauma, with a view on neurodegeneration and possibilities to ameliorate deterioration. In view of clinical applications, we emphasize the need for glycomimetic small organic compounds which surpass the usefulness of natural glycans in that they are metabolically more stable, more parsimonious to synthesize or isolate, and more advantageous for therapy, since many of them pass the blood brain barrier and are drug-approved for treatments other than those in the nervous system, thus allowing a more ready access for application in neurological diseases. We describe the isolation of such mimetic compounds using not only Western NIH, but also traditional Chinese medical libraries. With this review, we hope to deepen the interests in this exciting field.

Cell Adhesion Molecules and Protein Synthesis Regulation in Neurons.

Cell adhesion molecules (CAMs) mediate interactions of neurons with the extracellular environment by forming adhesive bonds with CAMs on adjacent membranes or via binding to proteins of the extracellular matrix. Binding of CAMs to their extracellular ligands results in the activation of intracellular signaling cascades, leading to changes in neuronal structure and the molecular composition and function of neuronal contacts. Ultimately, many of these changes depend on the synthesis of new proteins. In this review, we summarize the evidence showing that CAMs regulate protein synthesis by modulating the activity of transcription factors, gene expression, protein translation, and the structure and distribution of organelles involved in protein synthesis and transport.

Trafficking and Activity of Glutamate and GABA Receptors: Regulation by Cell Adhesion Molecules.

The efficient targeting of ionotropic receptors to postsynaptic sites is essential for the function of chemical excitatory and inhibitory synapses, constituting the majority of synapses in the brain. A growing body of evidence indicates that cell adhesion molecules (CAMs), which accumulate at synapses at the earliest stages of synaptogenesis, are critical for this process. A diverse variety of CAMs assemble into complexes with glutamate and GABA receptors and regulate the targeting of these receptors to the cell surface and synapses. Presynaptically localized CAMs provide an additional level of regulation, sending a trans-synaptic signal that can regulate synaptic strength at the level of receptor trafficking. Apart from controlling the numbers of receptors present at postsynaptic sites, CAMs can also influence synaptic strength by modulating the conductivity of single receptor channels. CAMs thus act to maintain basal synaptic transmission and are essential for many forms of activity dependent synaptic plasticity. These activities of CAMs may underlie the association between CAM gene mutations and synaptic pathology and represent fundamental mechanisms by which synaptic strength is dynamically tuned at both excitatory and inhibitory synapses.

NCAM regulates temporal specification of neural progenitor cells via profilin2 during corticogenesis.

The development of cerebral cortex requires spatially and temporally orchestrated proliferation, migration, and differentiation of neural progenitor cells (NPCs). The molecular mechanisms underlying cortical development are, however, not fully understood. The neural cell adhesion molecule (NCAM) has been suggested to play a role in corticogenesis. Here we show that NCAM is dynamically expressed in the developing cortex. NCAM expression in NPCs is highest in the neurogenic period and declines during the gliogenic period. In mice bearing an NPC-specific NCAM deletion, proliferation of NPCs is reduced, and production of cortical neurons is delayed, while formation of cortical glia is advanced. Mechanistically, NCAM enhances actin polymerization in NPCs by interacting with actin-associated protein profilin2. NCAM-dependent regulation of NPCs is blocked by mutations in the profilin2 binding site. Thus, NCAM plays an essential role in NPC proliferation and fate decision during cortical development by regulating profilin2-dependent actin polymerization.

Early transcriptome changes in response to chemical long-term potentiation induced via activation of synaptic NMDA receptors in mouse hippocampal neurons.

Long term potentiation (LTP) is a form of synaptic plasticity. In the present study LTP was induced via activation of synaptic NMDA receptors in primary hippocampal neuron cultures from neonate mice and RNA was isolated for RNA sequencing at 20 min following LTP induction. RNA sequencing and differential expression testing was performed to determine the identity and abundance of protein-coding and non-coding RNAs in control and LTP induced neuron cultures. We show that expression levels of a small group of transcripts encoding proteins involved in negative regulation of gene expression (Adcyap1, Id3), protein translation (Rpl22L1), extracellular structure organization (Bgn), intracellular signalling (Ppm1H, Ntsr2, Cldn10) and protein citrullination (PAD2) are downregulated in the stimulated neurons. Our results suggest that the early stages of LTP are accompanied by the remodelling of the biosynthetic machinery, interactions with the extracellular matrix and intracellular signalling pathways at the transcriptional level.

Neural Cell Adhesion Molecule 2 (NCAM2)-Induced c-Src-Dependent Propagation of Submembrane Ca2+ Spikes Along Dendrites Inhibits Synapse Maturation.

The neural cell adhesion molecule 2 (NCAM2) is encoded by a gene on chromosome 21 in humans. NCAM2 accumulates in synapses, but its role in regulation of synapse formation remains poorly understood. We demonstrate that an increase in NCAM2 levels results in increased instability of dendritic protrusions and reduced conversion of protrusions to dendritic spines in mouse cortical neurons. NCAM2 overexpression induces an increase in the frequency of submembrane Ca2+ spikes localized in individual dendritic protrusions and promotes propagation of submembrane Ca2+ spikes over segments of dendrites or the whole dendritic tree. NCAM2-dependent submembrane Ca2+ spikes are L-type voltage-gated Ca2+ channel-dependent, and their propagation but not initiation depends on the c-Src protein tyrosine kinase. Inhibition of initiation or propagation of NCAM2-dependent submembrane Ca2+ spikes reduces the NCAM2-dependent instability of dendritic protrusions. Synaptic boutons formed on dendrites of neurons with elevated NCAM2 expression are enriched in the protein marker of immature synapses GAP43, and the number of boutons with mature activity-dependent synaptic vesicle recycling is reduced. Our results indicate that synapse maturation is inhibited in NCAM2-overexpressing neurons and suggest that changes in NCAM2 levels and altered submembrane Ca2+ dynamics can cause defects in synapse maturation in Down syndrome and other brain disorders associated with abnormal NCAM2 expression.

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.

Glycosylphosphatidylinositol-Anchored Immunoglobulin Superfamily Cell Adhesion Molecules and Their Role in Neuronal Development and Synapse Regulation.

Immunoglobulin superfamily (IgSF) cell adhesion molecules (CAMs) are cell surface glycoproteins that not only mediate interactions between neurons but also between neurons and other cells in the nervous system. While typical IgSF CAMs are transmembrane molecules, this superfamily also includes CAMs, which do not possess transmembrane and intracellular domains and are instead attached to the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. In this review, we focus on the role GPI-anchored IgSF CAMs have as signal transducers and ligands in neurons, and discuss their functions in regulation of neuronal development, synapse formation, synaptic plasticity, learning, and behavior. We also review the links between GPI-anchored IgSF CAMs and brain disorders.

Replicable Expansion and Differentiation of Neural Precursors from Adult Canine Skin.

Repopulation of brain circuits by neural precursors is a potential therapeutic strategy for neurodegenerative disorders; however, choice of cell is critical. Previously, we introduced a two-step culture system that generates a high yield of neural precursors from small samples of adult canine skin. Here, we probe their gene and protein expression profiles in comparison with dermal fibroblasts and brain-derived neural stem cells and characterize their neuronal potential. To date, we have produced >50 skin-derived neural precursor (SKN) lines. SKNs can be cultured in a highly replicable fashion and uniformly express a panel of identifying markers. Upon differentiation, they self-upregulate neural specification genes, generating neurons with basic electrophysiological functionality. This unique population of neural precursors, derived from mature skin, overcomes many of the practical issues that have limited clinical translation of alternative cell types. Easily accessible, neuronally committed, and patient specific, SKNs may have potential for the treatment of brain disorders.

Neural Cell Adhesion Molecules of the Immunoglobulin Superfamily Regulate Synapse Formation, Maintenance, and Function.

Immunoglobulin superfamily adhesion molecules are among the most abundant proteins in vertebrate and invertebrate nervous systems. Prominent family members are the neural cell adhesion molecules NCAM and L1, which were the first to be shown to be essential not only in development but also in synaptic function and as key regulators of synapse formation, synaptic activity, plasticity, and synaptic vesicle recycling at distinct developmental and activity stages. In addition to interacting with each other, adhesion molecules interact with ion channels and cytokine and neurotransmitter receptors. Mutations in their genes are linked to neurological disorders associated with abnormal development and synaptic functioning. This review presents an overview of recent studies on these molecules and their crucial impact on neurological disorders.

Synaptic Cell Adhesion Molecules in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative brain disorder associated with the loss of synapses between neurons in the brain. Synaptic cell adhesion molecules are cell surface glycoproteins which are expressed at the synaptic plasma membranes of neurons. These proteins play key roles in formation and maintenance of synapses and regulation of synaptic plasticity. Genetic studies and biochemical analysis of the human brain tissue, cerebrospinal fluid, and sera from AD patients indicate that levels and function of synaptic cell adhesion molecules are affected in AD. Synaptic cell adhesion molecules interact with Aß, a peptide accumulating in AD brains, which affects their expression and synaptic localization. Synaptic cell adhesion molecules also regulate the production of Aß via interaction with the key enzymes involved in Aß formation. Aß-dependent changes in synaptic adhesion affect the function and integrity of synapses suggesting that alterations in synaptic adhesion play key roles in the disruption of neuronal networks in AD.

Transcriptional regulation of long-term potentiation.

Long-term potentiation (LTP), the persistent strengthening of synapses following high levels of stimulation, is a form of synaptic plasticity that has been studied extensively as a possible mechanism for learning and memory formation. The strengthening of the synapse that occurs during LTP requires cascades of complex molecular processes and the coordinated remodeling of pre-synaptic and post-synaptic neurons. Despite over four decades of research, our understanding of the transcriptional mechanisms and molecular processes underlying LTP remains incomplete. Identification of all the proteins and non-coding RNA transcripts expressed during LTP may provide greater insight into the molecular mechanisms involved in learning and memory formation.

KCa1.1, a calcium-activated potassium channel subunit alpha 1, is targeted by miR-17-5p and modulates cell migration in malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is an aggressive, locally invasive, cancer elicited by asbestos exposure and almost invariably a fatal diagnosis. To date, we are one of the leading laboratory that compared microRNA expression profiles in MPM and normal mesothelium samples in order to identify dysregulated microRNAs with functional roles in mesothelioma. We interrogated a significant collection of MPM tumors and normal pleural samples in our biobank in search for novel therapeutic targets. METHODS: Utilizing mRNA-microRNA correlations based on differential gene expression using Gene Set Enrichment Analysis (GSEA), we systematically combined publicly available gene expression datasets with our own MPM data in order to identify candidate targets for MPM therapy. RESULTS: We identified enrichment of target binding sites for the miR-17 and miR-30 families in both MPM tumors and cell lines. RT-qPCR revealed that members of both families were significantly downregulated in MPM tumors and cell lines. Interestingly, lower expression of miR-17-5p (P = 0.022) and miR-20a-5p (P = 0.026) was clearly associated with epithelioid histology. We interrogated the predicted targets of these differentially expressed microRNA families in MPM cell lines, and identified KCa1.1, a calcium-activated potassium channel subunit alpha 1 encoded by the KCNMA1 gene, as a target of miR-17-5p. KCa1.1 was overexpressed in MPM cells compared to the (normal) mesothelial line MeT-5A, and was also upregulated in patient tumor samples compared to normal mesothelium. Transfection of MPM cells with a miR-17-5p mimic or KCNMA1-specific siRNAs reduced mRNA expression of KCa1.1 and inhibited MPM cell migration. Similarly, treatment with paxilline, a small molecule inhibitor of KCa1.1, resulted in suppression of MPM cell migration. CONCLUSION: These functional data implicating KCa1.1 in MPM cell migration support our integrative approach using MPM gene expression datasets to identify novel and potentially druggable targets.

Reciprocal Interactions between Cell Adhesion Molecules of the Immunoglobulin Superfamily and the Cytoskeleton in Neurons.

Cell adhesion molecules of the immunoglobulin superfamily (IgSF) including the neural cell adhesion molecule (NCAM) and members of the L1 family of neuronal cell adhesion molecules play important functions in the developing nervous system by regulating formation, growth and branching of neurites, and establishment of the synaptic contacts between neurons. In the mature brain, members of IgSF regulate synapse composition, function, and plasticity required for learning and memory. The intracellular domains of IgSF cell adhesion molecules interact with the components of the cytoskeleton including the submembrane actin-spectrin meshwork, actin microfilaments, and microtubules. In this review, we summarize current data indicating that interactions between IgSF cell adhesion molecules and the cytoskeleton are reciprocal, and that while IgSF cell adhesion molecules regulate the assembly of the cytoskeleton, the cytoskeleton plays an important role in regulation of the functions of IgSF cell adhesion molecules. Reciprocal interactions between NCAM and L1 family members and the cytoskeleton and their role in neuronal differentiation and synapse formation are discussed in detail.