Regulation of young-adult neurogenesis and neuronal differentiation by neural cell adhesion molecule 2 (NCAM2).

Adult neurogenesis persists in mammals in the neurogenic zones, where newborn neurons are incorporated into preexisting circuits to preserve and improve learning and memory tasks. Relevant structural elements of the neurogenic niches include the family of cell adhesion molecules (CAMs), which participate in signal transduction and regulate the survival, division, and differentiation of radial glial progenitors (RGPs). Here we analyzed the functions of neural cell adhesion molecule 2 (NCAM2) in the regulation of RGPs in adult neurogenesis and during corticogenesis. We characterized the presence of NCAM2 across the main cell types of the neurogenic process in the dentate gyrus, revealing different levels of NCAM2 amid the progression of RGPs and the formation of neurons. We showed that Ncam2 overexpression in adult mice arrested progenitors in an RGP-like state, affecting the normal course of young-adult neurogenesis. Furthermore, changes in Ncam2 levels during corticogenesis led to transient migratory deficits but did not affect the survival and proliferation of RGPs, suggesting a differential role of NCAM2 in adult and embryonic stages. Our data reinforce the relevance of CAMs in the neurogenic process by revealing a significant role of Ncam2 levels in the regulation of RGPs during young-adult neurogenesis in the hippocampus.

NCAM2 Regulates Dendritic and Axonal Differentiation through the Cytoskeletal Proteins MAP2 and 14-3-3.

Neural cell adhesion molecule 2 (NCAM2) is involved in the development and plasticity of the olfactory system. Genetic data have implicated the NCAM2 gene in neurodevelopmental disorders including Down syndrome and autism, although its role in cortical development is unknown. Here, we show that while overexpression of NCAM2 in hippocampal neurons leads to minor alterations, its downregulation severely compromises dendritic architecture, leading to an aberrant phenotype including shorter dendritic trees, retraction of dendrites, and emergence of numerous somatic neurites. Further, our data reveal alterations in the axonal tree and deficits in neuronal polarization. In vivo studies confirm the phenotype and reveal an unexpected role for NCAM2 in cortical migration. Proteomic and cell biology experiments show that NCAM2 molecules exert their functions through a protein complex with the cytoskeletal-associated proteins MAP2 and 14-3-3γ and ζ. We provide evidence that NCAM2 depletion results in destabilization of the microtubular network and reduced MAP2 signal. Our results demonstrate a role for NCAM2 in dendritic formation and maintenance, and in neural polarization and migration, through interaction of NCAM2 with microtubule-associated proteins.

The Hidden Side of NCAM Family: NCAM2, a Key Cytoskeleton Organization Molecule Regulating Multiple Neural Functions.

Although it has been over 20 years since Neural Cell Adhesion Molecule 2 (NCAM2) was identified as the second member of the NCAM family with a high expression in the nervous system, the knowledge of NCAM2 is still eclipsed by NCAM1. The first studies with NCAM2 focused on the olfactory bulb, where this protein has a key role in axonal projection and axonal/dendritic compartmentalization. In contrast to NCAM1, NCAM2's functions and partners in the brain during development and adulthood have remained largely unknown until not long ago. Recent studies have revealed the importance of NCAM2 in nervous system development. NCAM2 governs neuronal morphogenesis and axodendritic architecture, and controls important neuron-specific processes such as neuronal differentiation, synaptogenesis and memory formation. In the adult brain, NCAM2 is highly expressed in dendritic spines, and it regulates synaptic plasticity and learning processes. NCAM2's functions are related to its ability to adapt to the external inputs of the cell and to modify the cytoskeleton accordingly. Different studies show that NCAM2 interacts with proteins involved in cytoskeleton stability and proteins that regulate calcium influx, which could also modify the cytoskeleton. In this review, we examine the evidence that points to NCAM2 as a crucial cytoskeleton regulation protein during brain development and adulthood. This key function of NCAM2 may offer promising new therapeutic approaches for the treatment of neurodevelopmental diseases and neurodegenerative disorders.

New Partners Identified by Mass Spectrometry Assay Reveal Functions of NCAM2 in Neural Cytoskeleton Organization.

Neuronal cell adhesion molecule 2 (NCAM2) is a membrane protein with an important role in the morphological development of neurons. In the cortex and the hippocampus, NCAM2 is essential for proper neuronal differentiation, dendritic and axonal outgrowth and synapse formation. However, little is known about NCAM2 functional mechanisms and its interactive partners during brain development. Here we used mass spectrometry to study the molecular interactome of NCAM2 in the second postnatal week of the mouse cerebral cortex. We found that NCAM2 interacts with >100 proteins involved in numerous processes, including neuronal morphogenesis and synaptogenesis. We validated the most relevant interactors, including Neurofilaments (NEFs), Microtubule-associated protein 2 (MAP2), Calcium/calmodulin kinase II alpha (CaMKIIα), Actin and Nogo. An in silico analysis of the cytosolic tail of the NCAM2.1 isoform revealed specific phosphorylation site motifs with a putative affinity for some of these interactors. Our results expand the knowledge of NCAM2 interactome and confirm the key role of NCAM2 in cytoskeleton organization, neuronal morphogenesis and synaptogenesis. These findings are of interest in explaining the phenotypes observed in different pathologies with alterations in the NCAM2 gene.

Reelin Regulates the Maturation of Dendritic Spines, Synaptogenesis and Glial Ensheathment of Newborn Granule Cells.

Significance Statement: The extracellular protein Reelin has an important role in neurological diseases, including epilepsy, Alzheimer's disease and psychiatric diseases, targeting hippocampal circuits. Here we address the role of Reelin in the development of synaptic contacts in adult-generated granule cells (GCs), a neuronal population that is crucial for learning and memory and implicated in neurological and psychiatric diseases. We found that the Reelin pathway controls the shapes, sizes, and types of dendritic spines, the complexity of multisynaptic innervations and the degree of the perisynaptic astroglial ensheathment that controls synaptic homeostasis. These findings show a pivotal role of Reelin in GC synaptogenesis and provide a foundation for structural circuit alterations caused by Reelin deregulation that may occur in neurological and psychiatric disorders.

Unconventional EGF-induced ERK1/2-mediated Kv1.3 endocytosis.

The potassium channel Kv1.3 plays roles in immunity, neuronal development and sensory discrimination. Regulation of Kv1.3 by kinase signaling has been studied. In this context, EGF binds to specific receptors (EGFR) and triggers tyrosine kinase-dependent signaling, which down-regulates Kv1.3 currents. We show that Kv1.3 undergoes EGF-dependent endocytosis. This EGF-mediated mechanism is relevant because is involved in adult neural stem cell fate determination. We demonstrated that changes in Kv1.3 subcellular distribution upon EGFR activation were due to Kv1.3 clathrin-dependent endocytosis, which targets the Kv1.3 channels to the lysosomal degradative pathway. Interestingly, our results further revealed that relevant tyrosines and other interacting motifs, such as PDZ and SH3 domains, were not involved in the EGF-dependent Kv1.3 internalization. However, a new, and yet undescribed mechanism, of ERK1/2-mediated threonine phosphorylation is crucial for the EGF-mediated Kv1.3 endocytosis. Our results demonstrate that EGF triggers the down-regulation of Kv1.3 activity and its expression at the cell surface, which is important for the development and migration of adult neural progenitors.

ApoER2 processing by presenilin-1 modulates reelin expression.

The reelin signaling protein and its downstream components have been associated with synaptic plasticity and neurotransmission. The reelin signaling pathway begins with the binding of reelin to the transmembrane lipoprotein receptor apolipoprotein E receptor 2 (ApoER2), which in turns induces the sequential cleavage of ApoER2 by the sequential action of α- and γ-secretases. Using conditional-knockout mice of the catalytic component of the γ-secretase complex, presenilin 1 (PS1), we demonstrated increased brain ApoER2 and reelin protein and transcript levels, with no changes in the number of reelin-positive cells. Using the human SH-SY5Y neuroblastoma cell line, we showed that ApoER2 processing occurs in the presence of PS1, producing an intracellular ApoER2 C-terminal fragment. In addition, the pharmacologic inhibition of γ-secretase in SH-SY5Y cells led to increased reelin levels. Overexpression of ApoER2 decreased reelin mRNA levels in these cells. A luciferase reporter gene assay and nuclear fractionation confirmed that increased amounts of intracellular fragment of ApoER2 suppressed reelin expression at a transcriptional level. Chromatin immunoprecipitation experiments corroborated that the intracellular fragment of ApoER2 bound to the RELN promoter region. Our study suggests that PS1/γ-secretase-dependent processing of the reelin receptor ApoER2 inhibits reelin expression and may regulate its signaling.

Cell-autonomous inactivation of the reelin pathway impairs adult neurogenesis in the hippocampus.

Adult hippocampal neurogenesis is thought to be essential for learning and memory, and has been implicated in the pathogenesis of several disorders. Although recent studies have identified key factors regulating neuroprogenitor proliferation in the adult hippocampus, the mechanisms that control the migration and integration of adult-born neurons into circuits are largely unknown. Reelin is an extracellular matrix protein that is vital for neuronal development. Activation of the Reelin cascade leads to phosphorylation of Disabled-1, an adaptor protein required for Reelin signaling. Here we used transgenic mouse and retroviral reporters along with Reelin signaling gain-of-function and loss-of-function studies to show that the Reelin pathway regulates migration and dendritic development of adult-generated hippocampal neurons. Whereas overexpression of Reelin accelerated dendritic maturation, inactivation of the Reelin signaling pathway specifically in adult neuroprogenitor cells resulted in aberrant migration, decreased dendrite development, formation of ectopic dendrites in the hilus, and the establishment of aberrant circuits. Our findings support a cell-autonomous and critical role for the Reelin pathway in regulating dendritic development and the integration of adult-generated granule cells and point to this pathway as a key regulator of adult neurogenesis. Moreover, our data reveal a novel role of the Reelin cascade in adult brain function with potential implications for the pathogenesis of several neurological and psychiatric disorders.

Adult-specific Reelin expression alters striatal neuronal organization: implications for neuropsychiatric disorders.

In addition to neuronal migration, brain development, and adult plasticity, the extracellular matrix protein Reelin has been extensively implicated in human psychiatric disorders such as schizophrenia, bipolar disorder, and autism spectrum disorder. Moreover, heterozygous reeler mice exhibit features reminiscent of these disorders, while overexpression of Reelin protects against its manifestation. However, how Reelin influences the structure and circuits of the striatal complex, a key region for the above-mentioned disorders, is far from being understood, especially when altered Reelin expression levels are found at adult stages. In the present study, we took advantage of complementary conditional gain- and loss-of-function mouse models to investigate how Reelin levels may modify adult brain striatal structure and neuronal composition. Using immunohistochemical techniques, we determined that Reelin does not seem to influence the striatal patch and matrix organization (studied by µ-opioid receptor immunohistochemistry) nor the density of medium spiny neurons (MSNs, studied with DARPP-32). We show that overexpression of Reelin leads to increased numbers of striatal parvalbumin- and cholinergic-interneurons, and to a slight increase in tyrosine hydroxylase-positive projections. We conclude that increased Reelin levels might modulate the numbers of striatal interneurons and the density of the nigrostriatal dopaminergic projections, suggesting that these changes may be involved in the protection of Reelin against neuropsychiatric disorders.

Reelin regulates postnatal neurogenesis and enhances spine hypertrophy and long-term potentiation.

Reelin, an extracellular protein essential for neural migration and lamination, is also expressed in the adult brain. To unravel the function of this protein in the adult forebrain, we generated transgenic mice that overexpress Reelin under the control of the CaMKIIalpha promoter. Overexpression of Reelin increased adult neurogenesis and impaired the migration and positioning of adult-generated neurons. In the hippocampus, the overexpression of Reelin resulted in an increase in synaptic contacts and hypertrophy of dendritic spines. Induction of long-term potentiation (LTP) in alert-behaving mice showed that Reelin overexpression evokes a dramatic increase in LTP responses. Hippocampal field EPSP during a classical conditioning paradigm was also increased in these mice. Our results indicate that Reelin levels in the adult brain regulate neurogenesis and migration, as well as the structural and functional properties of synapses. These observations suggest that Reelin controls developmental processes that remain active in the adult brain.

Mechanism suppressing glycogen synthesis in neurons and its demise in progressive myoclonus epilepsy.

Glycogen synthesis is normally absent in neurons. However, inclusion bodies resembling abnormal glycogen accumulate in several neurological diseases, particularly in progressive myoclonus epilepsy or Lafora disease. We show here that mouse neurons have the enzymatic machinery for synthesizing glycogen, but that it is suppressed by retention of muscle glycogen synthase (MGS) in the phosphorylated, inactive state. This suppression was further ensured by a complex of laforin and malin, which are the two proteins whose mutations cause Lafora disease. The laforin-malin complex caused proteasome-dependent degradation both of the adaptor protein targeting to glycogen, PTG, which brings protein phosphatase 1 to MGS for activation, and of MGS itself. Enforced expression of PTG led to glycogen deposition in neurons and caused apoptosis. Therefore, the malin-laforin complex ensures a blockade of neuronal glycogen synthesis even under intense glycogenic conditions. Here we explain the formation of polyglucosan inclusions in Lafora disease by demonstrating a crucial role for laforin and malin in glycogen synthesis.

Alzheimer's disease modifies progenitor cell expression of monoamine oxidase B in the subventricular zone.

In the adult brain, progenitor cells remaining in the subventricular zone (SVZ) are frequently identified as glial fibrillary acidic protein (GFAP)-positive cells that retain attributes reminiscent of radial glia. Because the very high expression of monoamine oxidase B (MAO-B) in the subventricular area has been related to epithelial and astroglial expression, we sought to ascertain whether it was also expressed by progenitor cells of human control and Alzheimer's disease (AD) patients. In the SVZ, epithelial cells and astrocyte-like cells presented rich MAO-B activity and immunolabeling. Nestin-positive cells were found in the same area, showing a radial glia-like morphology. When coimmunostaining and confocal microscopy were performed, most nestin-positive cells showed MAO-B activity and labeling. The increased progenitor activity in SVZ proposed for AD patients was confirmed by the positive correlation between the SVZ nestin/MAO-B ratio and the progression of the disease. Nestin/GFAP-positive cells, devoid of MAO-B, can represent a distinct subpopulation of an earlier phase of maturation. This would indicate that MAO-B expression takes place in a further step of nestin/GFAP-positive cell differentiation. In the early AD stages, the discrete MAO-B reduction, different from the severe GFAP decrease, would reflect the capacity of this population of MAO-B-positive progenitor cells to adapt to the neurodegenerative process.

Reelin reverts biochemical, physiological and cognitive alterations in mouse models of Tauopathy.

Reelin is an extracellular protein crucial for adult brain plasticity. Moreover, Reelin is protective against amyloid-ß (Aß) pathology in Alzheimer's Disease (AD), reducing plaque deposition, synaptic loss and cognitive decline. Given that Tau protein plays a key role in AD pathogenesis, and that the Reelin pathway modulates Tau phosphorylation, here we explored the involvement of Reelin in AD-related Tau pathology. We found that Reelin overexpression modulates the levels of Tau phosphorylation in AD-related epitopes in VLW mice expressing human mutant Tau. in vitro, Reelin reduced the Aß-induced missorting of axonal Tau and neurofilament proteins to dendrites. Reelin also reverted in vivo the toxic somatodendritic localization of phosphorylated Tau. Finally, overexpression of Reelin in VLW mice improved long-term potentiation and long-term memory cognitive performance thus masking the cognitive and physiological deficits in VLW mice. These data suggest that the Reelin pathway, which is also protective against Aß pathology, modulates fundamental traits of Tau pathology, strengthening the potential of Reelin as a therapeutic target in AD.

MAP1B is required for Netrin 1 signaling in neuronal migration and axonal guidance.

BACKGROUND: The signaling cascades governing neuronal migration and axonal guidance link extracellular signals to cytoskeletal components. MAP1B is a neuron-specific microtubule-associated protein implicated in the crosstalk between microtubules and actin filaments. RESULTS: Here we show that Netrin 1 regulates, both in vivo and in vitro, mode I MAP1B phosphorylation, which controls MAP1B activity, in a signaling pathway that depends essentially on the kinases GSK3 and CDK5. We also show that map1B-deficient neurons from the lower rhombic lip and other brain regions have reduced chemoattractive responses to Netrin 1 in vitro. Furthermore, map1B mutant mice have severe abnormalities, similar to those described in netrin 1-deficient mice, in axonal tracts and in the pontine nuclei. CONCLUSIONS: These data indicate that MAP1B phosphorylation is controlled by Netrin 1 and that the lack of MAP1B impairs Netrin 1-mediated chemoattraction in vitro and in vivo. Thus, MAP1B may be a downstream effector in the Netrin 1-signaling pathway.

Reelin Exerts Structural, Biochemical and Transcriptional Regulation Over Presynaptic and Postsynaptic Elements in the Adult Hippocampus.

Reelin regulates neuronal positioning and synaptogenesis in the developing brain, and adult brain plasticity. Here we used transgenic mice overexpressing Reelin (Reelin-OE mice) to perform a comprehensive dissection of the effects of this protein on the structural and biochemical features of dendritic spines and axon terminals in the adult hippocampus. Electron microscopy (EM) revealed both higher density of synapses and structural complexity of both pre- and postsynaptic elements in transgenic mice than in WT mice. Dendritic spines had larger spine apparatuses, which correlated with a redistribution of Synaptopodin. Most of the changes observed in Reelin-OE mice were reversible after blockade of transgene expression, thus supporting the specificity of the observed phenotypes. Western blot and transcriptional analyses did not show major changes in the expression of pre- or postsynaptic proteins, including SNARE proteins, glutamate receptors, and scaffolding and signaling proteins. However, EM immunogold assays revealed that the NMDA receptor subunits NR2a and NR2b, and p-Cofilin showed a redistribution from synaptic to extrasynaptic pools. Taken together with previous studies, the present results suggest that Reelin regulates the structural and biochemical properties of adult hippocampal synapses by increasing their density and morphological complexity and by modifying the distribution and trafficking of major glutamatergic components.

Hydrogen/deuterium exchange-protected oligomers populated during Aß fibril formation correlate with neuronal cell death.

The aggregation of the amyloid-ß peptide (Aß) to form fibrils and plaques is strongly associated with Alzheimer's disease (AD). Although it is well established that this process generates neurotoxicity, it is also heterogeneous with a variety of species being formed during the conversion process. This heterogeneity makes it difficult to detect and characterize each of the aggregates formed, which precludes establishing the specific features responsible for the neurotoxicity observed. Here we use pulse-labeling hydrogen-deuterium exchange experiments analyzed by electrospray ionization mass spectrometry (PL-HDX-ESI-MS) to distinguish three ensembles populated during the aggregation of the 40 and 42 residue forms of the Aß peptide, Aß40 and Aß42, on the basis of differences in their persistent structure. Noticeably, two of them are more abundant at the beginning and at the end of the lag phase and are therefore not detectable by conventional assays such as Thioflavin T (ThT). The ensembles populated at different stages of the aggregation process have a surprisingly consistent average degree of exchange, indicating that there are definite structural transitions between the different stages of aggregation. To determine whether an ensemble of species with a given hydrogen exchange pattern correlates with neurotoxicity, we combined PL-HDX-ESI-MS experiments with parallel measurements of the neurotoxicity of the samples under study. The results of this dual approach show that the maximum toxicity correlates with the ensemble comprising HDX protected oligomers, indicating that development of persistent structure within Aß oligomers is a determinant of neurotoxicity.

Reelin delays amyloid-beta fibril formation and rescues cognitive deficits in a model of Alzheimer's disease.

Reelin is an extracellular matrix protein that is crucial for neural development and adult brain plasticity. While the Reelin signalling cascade has been reported to be associated with Alzheimer's disease (AD), the role of Reelin in this pathology is not understood. Here we use an in vitro approach to show that Reelin interacts with amyloid-ß (Aß42) soluble species, delays Aß42 fibril formation and is recruited into amyloid fibrils. Furthermore, Reelin protects against both the neuronal death and dendritic spine loss induced by Aß42 oligomers. In mice carrying the APP(Swe/Ind) mutation (J20 mice), Reelin overexpression delays amyloid plaque formation and rescues the recognition memory deficits. Our results indicate that by interacting with Aß42 soluble species, delaying Aß plaque formation, protecting against neuronal death and dendritic spine loss and preventing AD cognitive deficits, the Reelin pathway deserves consideration as a therapeutic target for the treatment of AD pathogenesis.

Somatic signature of brain-specific single nucleotide variations in sporadic Alzheimer's disease.

BACKGROUND: Although genome-wide association studies have shown that genetic factors increase the risk of suffering late-onset, sporadic Alzheimer's disease (SAD), the molecular mechanisms responsible remain largely unknown. OBJECTIVE: The aim of the study was to investigate the presence of somatic, brain-specific single nucleotide variations (SNV) in the hippocampus of SAD samples. METHODS: By using bioinformatic tools, we compared whole exome sequences in paired blood and hippocampal genomic DNAs from 17 SAD patients and from 2 controls and 2 vascular dementia patients. RESULTS: We found a remarkable number of SNVs in SAD brains (~575 per patient) that were not detected in blood. Loci with hippocampus-specific (hs)-SNVs were common to several patients, with 38 genes being present in 6 or more patients out of the 17. While some of these SNVs were in genes previously related to SAD (e.g., CSMD1, LRP2), most hs-SNVs occurred in loci previously unrelated to SAD. The most frequent genes with hs-SNVs were associated with neurotransmission, DNA metabolism, neuronal transport, and muscular function. Interestingly, 19 recurrent hs-SNVs were common to 3 SAD patients. Repetitive loci or hs-SNVs were underrepresented in the hippocampus of control or vascular dementia donors, or in the cerebellum of SAD patients. CONCLUSION: Our data suggest that adult blood and brain have different DNA genomic variations, and that somatic genetic mosaicism and brain-specific genome reshaping may contribute to SAD pathogenesis and cognitive differences between individuals.

Reelin controls progenitor cell migration in the healthy and pathological adult mouse brain.

Understanding the signals that control migration of neural progenitor cells in the adult brain may provide new therapeutic opportunities. Reelin is best known for its role in regulating cell migration during brain development, but we now demonstrate a novel function for reelin in the injured adult brain. First, we show that Reelin is upregulated around lesions. Second, experimentally increasing Reelin expression levels in healthy mouse brain leads to a change in the migratory behavior of subventricular zone-derived progenitors, triggering them to leave the rostral migratory stream (RMS) to which they are normally restricted during their migration to the olfactory bulb. Third, we reveal that Reelin increases endogenous progenitor cell dispersal in periventricular structures independently of any chemoattraction but via cell detachment and chemokinetic action, and thereby potentiates spontaneous cell recruitment to demyelination lesions in the corpus callosum. Conversely, animals lacking Reelin signaling exhibit reduced endogenous progenitor recruitment at the lesion site. Altogether, these results demonstrate that beyond its known role during brain development, Reelin is a key player in post-lesional cell migration in the adult brain. Finally our findings provide proof of concept that allowing progenitors to escape from the RMS is a potential therapeutic approach to promote myelin repair.

Overexpression of Reelin prevents the manifestation of behavioral phenotypes related to schizophrenia and bipolar disorder.

Despite the impact of schizophrenia and mood disorders, which in extreme cases can lead to death, recent decades have brought little progress in the development of new treatments. Recent studies have shown that Reelin, an extracellular protein that is critical for neuronal development, is reduced in schizophrenia and bipolar disorder patients. However, data on a causal or protective role of Reelin in psychiatric diseases is scarce. In order to study the direct influence of Reelin's levels on behavior, we subjected two mouse lines, in which Reelin levels are either reduced (Reelin heterozygous mice) or increased (Reelin overexpressing mice), to a battery of behavioral tests: open-field, black-white box, novelty-suppressed-feeding, forced-swim-test, chronic corticosterone treatment followed by forced-swim-test, cocaine sensitization and pre-pulse inhibition (PPI) deficits induced by N-methyl-D-aspartate (NMDA) antagonists. These tests were designed to model some aspects of psychiatric disorders such as schizophrenia, mood, and anxiety disorders. We found no differences between Reeler heterozygous mice and their wild-type littermates. However, Reelin overexpression in the mouse forebrain reduced the time spent floating in the forced-swim-test in mice subjected to chronic corticosterone treatment, reduced behavioral sensitization to cocaine, and reduced PPI deficits induced by a NMDA antagonist. In addition, we demonstrate that while stress increased NMDA NR2B-mediated synaptic transmission, known to be implicated in depression, Reelin overexpression significantly reduced it. Together, these results point to the Reelin signaling pathway as a relevant drug target for the treatment of a range of psychiatric disorders.