Specific contribution of Reelin expressed by Cajal-Retzius cells or GABAergic interneurons to cortical lamination.

The extracellular protein Reelin, expressed by Cajal-Retzius (CR) cells at early stages of cortical development and at late stages by GABAergic interneurons, regulates radial migration and the "inside-out" pattern of positioning. Current models of Reelin functions in corticogenesis focus on early CR cell-derived Reelin in layer I. However, developmental disorders linked to Reelin deficits, such as schizophrenia and autism, are related to GABAergic interneuron-derived Reelin, although its role in migration has not been established. Here we selectively inactivated the Reln gene in CR cells or GABAergic interneurons. We show that CR cells have a major role in the inside-out order of migration, while CR and GABAergic cells sequentially cooperate to prevent invasion of cortical neurons into layer I. Furthermore, GABAergic cell-derived Reelin compensates some features of the reeler phenotype and is needed for the fine tuning of the layer-specific distribution of cortical neurons. In the hippocampus, the inactivation of Reelin in CR cells causes dramatic alterations in the dentate gyrus and mild defects in the hippocampus proper. These findings lead to a model in which both CR and GABAergic cell-derived Reelin cooperate to build the inside-out order of corticogenesis, which might provide a better understanding of the mechanisms involved in the pathogenesis of neuropsychiatric disorders linked to abnormal migration and Reelin deficits.

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.

Functional protection in J20/VLW mice: a model of non-demented with Alzheimer's disease neuropathology.

Alzheimer's disease comprises amyloid-ß and hyperphosphorylated Tau accumulation, imbalanced neuronal activity, aberrant oscillatory rhythms and cognitive deficits. Non-demented with Alzheimer's disease neuropathology defines a novel clinical entity with amyloid-ß and Tau pathologies but preserved cognition. The mechanisms underlying such neuroprotection remain undetermined and animal models of non-demented with Alzheimer's disease neuropathology are currently unavailable. We demonstrate that J20/VLW mice (accumulating amyloid-ß and hyperphosphorylated Tau) exhibit preserved hippocampal rhythmic activity and cognition, as opposed to J20 and VLW animals, which show significant alterations. Furthermore, we show that the overexpression of mutant human Tau in coexistence with amyloid-ß accumulation renders a particular hyperphosphorylated Tau signature in hippocampal interneurons. The GABAergic septohippocampal pathway, responsible for hippocampal rhythmic activity, is preserved in J20/VLW mice, in contrast to single mutants. Our data highlight J20/VLW mice as a suitable animal model in which to explore the mechanisms driving cognitive preservation in non-demented with Alzheimer's disease neuropathology. Moreover, they suggest that a differential Tau phosphorylation pattern in hippocampal interneurons prevents the loss of GABAergic septohippocampal innervation and alterations in local field potentials, thereby avoiding cognitive deficits.

The armadillo-repeat containing X-linked protein 3, ARMCX3, is a negative regulator of the browning of adipose tissue associated with obesity.

OBJECTIVES: To determine the role of armadillo repeat-containing X-linked protein 3 (ARMCX3) in the thermogenic plasticity of adipose tissue. METHODS: Adipose tissues were characterized in Armcx3-KO male mice. Armcx3 gene expression was analyzed in adipose tissue from mice exposed to thermogenic inducers (cold, ß3-adenergic stimulus) and in differentiating brown and beige cells in culture. Analyses encompassed circulating metabolite and hormonal profiling, tissue characterization, histology, gene expression patterns, and immunoblot assays. Armcx3 gene expression was assessed in subcutaneous adipose tissue from lean individuals and individuals with obesity and was correlated with expression of marker genes of adipose browning. The effects of adenoviral-mediated overexpression of ARMCX3 on differentiating brown adipocyte gene expression and respiratory activity were determined. RESULTS: Male mice lacking ARMCX3 showed significant induction of white adipose tissue browning. In humans, ARMCX3 expression in subcutaneous adipose tissue was inversely correlated with the expression of marker genes of thermogenic activity, including CIDEA, mitochondrial transcripts, and creatine kinase-B. Armcx3 expression in adipose tissues was repressed by thermogenic activation (cold or ß3-adrenergic stimulation) and was upregulated by obesity in mice and humans. Experimentally-induced increases in Armcx3 caused down-regulation of thermogenesis-related genes and reduced mitochondrial oxidative activity of adipocytes in culture, whereas siRNA-mediated Armcx3 knocking-down enhanced expression of thermogenesis-related genes. CONCLUSION: ARMCX3 is a novel player in the control of thermogenic adipose tissue plasticity that acts to repress acquisition of the browning phenotype and shows a direct association with indicators of obesity in mice and humans.

Growth cone repulsion to Netrin-1 depends on lipid raft microdomains enriched in UNC5 receptors.

During brain development, Uncoordinated locomotion 5 (UNC5) receptors control axonal extension through their sensing of the guidance molecule Netrin-1. The correct positioning of receptors into cholesterol-enriched membrane raft microdomains is crucial for the efficient transduction of the recognized signals. However, whether such microdomains are required for the appropriate axonal guidance mediated by UNC5 receptors remains unknown. Here, we combine the use of confocal microscopy, live-cell FRAP analysis and single-particle tracking PALM to characterize the distribution of UNC5 receptors into raft microdomains, revealing differences in their membrane mobility properties. Using pharmacological and genetic approaches in primary neuronal cultures and brain cerebellar explants we further demonstrate that disrupting raft microdomains inhibits the chemorepulsive response of growth cones and axons against Netrin-1. Together, our findings indicate that the distribution of all UNC5 receptors into cholesterol-enriched raft microdomains is heterogeneous and that the specific localization has functional consequences for the axonal chemorepulsion against Netrin-1.

Reversible silencing of endogenous receptors in intact brain tissue using 2-photon pharmacology.

The physiological activity of proteins is often studied with loss-of-function genetic approaches, but the corresponding phenotypes develop slowly and can be confounding. Photopharmacology allows direct, fast, and reversible control of endogenous protein activity, with spatiotemporal resolution set by the illumination method. Here, we combine a photoswitchable allosteric modulator (alloswitch) and 2-photon excitation using pulsed near-infrared lasers to reversibly silence metabotropic glutamate 5 (mGlu5) receptor activity in intact brain tissue. Endogenous receptors can be photoactivated in neurons and astrocytes with pharmacological selectivity and with an axial resolution between 5 and 10 µm. Thus, 2-photon pharmacology using alloswitch allows investigating mGlu5-dependent processes in wild-type animals, including synaptic formation and plasticity, and signaling pathways from intracellular organelles.

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.

A conserved role for Syntaxin-1 in pre- and post-commissural midline axonal guidance in fly, chick, and mouse.

Axonal growth and guidance rely on correct growth cone responses to guidance cues. Unlike the signaling cascades that link axonal growth to cytoskeletal dynamics, little is known about the crosstalk mechanisms between guidance and membrane dynamics and turnover. Recent studies indicate that whereas axonal attraction requires exocytosis, chemorepulsion relies on endocytosis. Indeed, our own studies have shown that Netrin-1/Deleted in Colorectal Cancer (DCC) signaling triggers exocytosis through the SNARE Syntaxin-1 (STX1). However, limited in vivo evidence is available about the role of SNARE proteins in axonal guidance. To address this issue, here we systematically deleted SNARE genes in three species. We show that loss-of-function of STX1 results in pre- and post-commissural axonal guidance defects in the midline of fly, chick, and mouse embryos. Inactivation of VAMP2, Ti-VAMP, and SNAP25 led to additional abnormalities in axonal guidance. We also confirmed that STX1 loss-of-function results in reduced sensitivity of commissural axons to Slit-2 and Netrin-1. Finally, genetic interaction studies in Drosophila show that STX1 interacts with both the Netrin-1/DCC and Robo/Slit pathways. Our data provide evidence of an evolutionarily conserved role of STX1 and SNARE proteins in midline axonal guidance in vivo, by regulating both pre- and post-commissural guidance mechanisms.

One Raft to Guide Them All, and in Axon Regeneration Inhibit Them.

Central nervous system damage caused by traumatic injuries, iatrogenicity due to surgical interventions, stroke and neurodegenerative diseases is one of the most prevalent reasons for physical disability worldwide. During development, axons must elongate from the neuronal cell body to contact their precise target cell and establish functional connections. However, the capacity of the adult nervous system to restore its functionality after injury is limited. Given the inefficacy of the nervous system to heal and regenerate after damage, new therapies are under investigation to enhance axonal regeneration. Axon guidance cues and receptors, as well as the molecular machinery activated after nervous system damage, are organized into lipid raft microdomains, a term typically used to describe nanoscale membrane domains enriched in cholesterol and glycosphingolipids that act as signaling platforms for certain transmembrane proteins. Here, we systematically review the most recent findings that link the stability of lipid rafts and their composition with the capacity of axons to regenerate and rebuild functional neural circuits after damage.

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.

Differential accumulation of Tau phosphorylated at residues Thr231, Ser262 and Thr205 in hippocampal interneurons and its modulation by Tau mutations (VLW) and amyloid-ß peptide.

Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß peptide (Aß) and hyperphosphorylated Tau protein (P-Tau). Our recent data showed a differential accumulation of Tau protein phosphorylated at residue Thr231 (pThr231) in distinct hippocampal neurons in VLW mice-a model that overexpresses mutated human Tau. Here we demonstrate that, in VLW mice, the accumulation of human P-Tau in pyramidal cells induces the phosphorylation of murine Tau at residue Thr231 in hippocampal interneurons. In addition, we show that pSer262 and pThr205 Tau are present specifically in the soma of some hippocampal interneurons in control mice. Analysis of J20 mice-a model that accumulates Aß-and of VLW animals showed that the density of hippocampal interneurons accumulating pThr205 Tau is lower in VLW mice than in controls. In contrast, the density of interneurons accumulating pThr205 Tau in J20 mice was increased compared to controls in hippocampal regions with a higher Aß plaque load, thereby suggesting that pThr205 Tau is induced by Aß. No significant differences were found between the density of hippocampal interneurons positive for pSer262 Tau in VLW or J20 mice compared to control animals. We also show that pSer262 and pThr205 Tau are present in the soma of some hippocampal interneurons containing Parvalbumin, Calbindin or Calretinin in control, VLW, and J20 mice. Moreover, our results reveal that some interneurons in human hippocampi of cases of AD and control cases accumulate pSer262 and pThr205 Tau. Taken together, these data point to a specific role of pSer262 and pThr205 Tau in the soma of hippocampal interneurons in control and pathological conditions.

New partners and phosphorylation sites of focal adhesion kinase identified by mass spectrometry.

The regulation of focal adhesion kinase (FAK) involves phosphorylation and multiple interactions with other signaling proteins. Some of these pathways are relevant for nervous system functions such as branching, axonal guidance, and plasticity. In this study, we screened mouse brain to identify FAK-interactive proteins and phosphorylatable residues as a first step to address the neuronal functions of this kinase. Using mass spectrometry analysis, we identified new phosphorylated sites (Thr 952, Thr 1048, and Ser 1049), which lie in the FAT domain; and putative new partners for FAK, which include cytoskeletal proteins such as drebrin and MAP 6, adhesion regulators such as neurabin-2 and plakophilin 1, and synapse-associated proteins such as SynGAP and a NMDA receptor subunit. Our findings support the participation of brain-localized FAK in neuronal plasticity.

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.

Regulation of patterned dynamics of local exocytosis in growth cones by netrin-1.

Axonal guidance and synaptic specification depends on specific signaling mechanisms that occur in growth cones. While several signaling pathways implicated in cone navigation have been identified, membrane dynamics in growth cones remains largely unknown. We took advantage of SynaptopHluorin and high-speed optical recordings to monitor the patterns of membrane dynamics in rat hippocampal growth cones. We show that exocytosis occurs both at the peripheral and central domains, including filopodia, and that SynaptopHluorin signals occur as spontaneous patterned peaks. Such transients average approximately two per minute and last ∼30 s. We also demonstrate that the chemoattractant Netrin-1 elicits increases in the frequency and slopes of these transients, with peaks averaging up to six per minute in the peripheral domain. Netrin-1-dependent regulation of exocytotic events requires the activation of the Erk1/2 and SFK pathways. Furthermore, we show that domains with high SynaptopHluorin signals correlate with high local calcium concentrations and that local, spontaneous calcium increases are associated with higher SynaptopHluorin signals. These findings demonstrate highly stereotyped, spontaneous transients of local exocytosis in growth cones and that these transients are positively regulated by chemoattractant molecules such as Netrin-1.

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.

The GABAergic septohippocampal pathway is directly involved in internal processes related to operant reward learning.

We studied the role of γ-aminobutyric acid (GABA)ergic septohippocampal projections in medial septum (MS) self-stimulation of behaving mice. Self-stimulation was evoked in wild-type (WT) mice using instrumental conditioning procedures and in J20 mutant mice, a type of mouse with a significant deficit in GABAergic septohippocampal projections. J20 mice showed a significant modification in hippocampal activities, including a different response for input/output curves and the paired-pulse test, a larger long-term potentiation (LTP), and a delayed acquisition and lower performance in the MS self-stimulation task. LTP evoked at the CA3-CA1 synapse further decreased self-stimulation performance in J20, but not in WT, mice. MS self-stimulation evoked a decrease in the amplitude of field excitatory postsynaptic potentials (fEPSPs) at the CA3-CA1 synapse in WT, but not in J20, mice. This self-stimulation-dependent decrease in the amplitude of fEPSPs was also observed in the presence of another positive reinforcer (food collected during an operant task) and was canceled by the local administration of an antibody-inhibiting glutamate decarboxylase 65 (GAD65). LTP evoked in the GAD65Ab-treated group was also larger than in controls. The hippocampus has a different susceptibility to septal GABAergic inputs depending on ongoing cognitive processes, and the GABAergic septohippocampal pathway is involved in consummatory processes related to operant rewards.

Benz[c,d]indolium-containing Monomethine Cyanine Dyes: Synthesis and Photophysical Properties.

Asymmetric monomethine cyanines have been extensively used as probes for nucleic acids among other biological systems. Herein we report the synthesis of seven monomethine cyanine dyes that have been successfully prepared with various heterocyclic moieties such as quinoline, benzoxazole, benzothiazole, dimethyl indole, and benz[e]indole adjoining benz[c,d]indol-1-ium, which was found to directly influence their optical and energy profiles. In this study the optical properties vs. structural changes were investigated using nuclear magnetic resonance and computational approaches. The twisted conformation unique to monomethine cyanines was exploited in DNA binding studies where the newly designed sensor displayed an increase in fluorescence when bound in the DNA grooves compared to the unbound form.

The diverse roles and multiple forms of focal adhesion kinase in brain.

Although it was originally characterized as a constituent of focal adhesions in fibroblasts, focal adhesion kinase (FAK) is now considered to be not only a mediator of adhesion processes but also a crucial regulator of guidance and a modulator of gene expression. FAK is the main transducer of the integrin signaling required to stabilize the actin cytoskeleton. However, additional activities have been described over the years. In the brain, FAK deserves particular attention as it is found in various alternatively spliced forms - these distributed in multiple subcellular compartments or bound to multiple partners. Moreover, its signaling involves not only phosphorylation but also ubiquitination and proteolysis. Several experimental cell models demonstrate that FAK increases or decreases migration, participates in differentiation and contributes to plasticity events. In addition, this kinase is linked to cell survival in cancer and apoptosis. This review focuses on the diversity of events involving brain-located forms of FAK.