Glutamate-specific gene linked to human brain evolution enhances synaptic plasticity and cognitive processes.

The human brain is characterized by the upregulation of synaptic, mainly glutamatergic, transmission, but its evolutionary origin(s) remain elusive. Here we approached this fundamental question by studying mice transgenic (Tg) for GLUD2, a human gene involved in glutamate metabolism that emerged in the hominoid and evolved concomitantly with brain expansion. We demonstrate that Tg mice express the human enzyme in hippocampal astrocytes and CA1-CA3 pyramidal neurons. LTP, evoked by theta-burst stimulation, is markedly enhanced in the CA3-CA1 synapses of Tg mice, with patch-clamp recordings from CA1 pyramidal neurons revealing increased sNMDA currents. LTP enhancement is blocked by D-lactate, implying that GLUD2 potentiates L-lactate-mediated astrocyte-neuron interaction. Dendritic spine density and synaptogenesis are increased in the hippocampus of Tg mice, which exhibit enhanced responses to sensory stimuli and improved performance on complex memory tasks. Hence, GLUD2 likely contributed to human brain evolution by enhancing synaptic plasticity and metabolic processes central to cognitive functions.

Nicotinamide enhances myelin production after demyelination through reduction of astrogliosis and microgliosis.

Caloric restriction is the chronic reduction of total caloric intake without malnutrition and has attracted a lot of attention as, among multiple other effects, it attenuates demyelination and stimulates remyelination. In this study we have evaluated the effect of nicotinamide (NAM), a well-known caloric restriction mimetic, on myelin production upon demyelinating conditions. NAM is the derivative of nicotinic acid (vitamin B3) and a precursor of nicotinamide adenine dinucleotide (NAD+), a ubiquitous metabolic cofactor. Here, we use cortical slices ex vivo subjected to demyelination or cultured upon normal conditions, a lysolecithin (LPC)-induced focal demyelination mouse model as well as primary glial cultures. Our data show that NAM enhances both myelination and remyelination ex vivo, while it also induces myelin production after LPC-induced focal demyelination ex vivo and in vivo. The increased myelin production is accompanied by reduction in both astrogliosis and microgliosis in vivo. There is no direct effect of NAM on the oligodendrocyte lineage, as no differences are observed in oligodendrocyte precursor cell proliferation or differentiation or in the number of mature oligodendrocytes. On the other hand, NAM affects both microglia and astrocytes as it decreases the population of M1-activated microglia, while reducing the pro-inflammatory phenotype of astrocytes as assayed by the reduction of TNF-α. Overall, we show that the increased myelin production that follows NAM treatment in vivo is accompanied by a decrease in both astrocyte and microglia accumulation at the lesion site. Our data indicate that NAM influences astrocytes and microglia directly, in favor of the remyelination process by promoting a less inflammatory environment.

Autophagic degradation of CNS myelin maintains axon integrity.

(Macro)autophagy is a major lysosome-dependent degradation mechanism which engulfs, removes and recycles unwanted cytoplasmic material, including damaged organelles and toxic protein aggregates. Although a few studies implicate autophagy in CNS demyelinating pathologies, its role, particularly in mature oligodendrocytes and CNS myelin, remains poorly studied. Here, using both pharmacological and genetic inhibition of the autophagic machinery, we provide evidence that autophagy is an essential mechanism for oligodendrocyte maturation in vitro. Our study reveals that two core myelin proteins, namely proteolipid protein (PLP) and myelin basic protein (MBP) are incorporated into autophagosomes in oligodendrocytes, resulting in their degradation. Furthermore, we ablated atg5, a core gene of the autophagic machinery, specifically in myelinating glial cells in vivo by tamoxifen administration (plp-Cre ERT2 ; atg5 f/f ) and showed that myelin maintenance is perturbed, leading to PLP accumulation. Significant morphological defects in myelin membrane such as decompaction accompanied with increased axonal degeneration are observed. As a result, the mice exhibit behavioral deficits. In summary, our data highlight that the maintenance of adult myelin homeostasis in the CNS requires the involvement of a fully functional autophagic machinery.

Neuronal, but not glial, Contactin 2 negatively regulates axon regeneration in the injured adult optic nerve.

Mammalian adult neurons of the central nervous system (CNS) display limited ability to regrow axons after trauma. The developmental decline in their regenerative ability has been attributed to both intrinsic and extrinsic factors, including postnatal suppression of transcription factors and non-neuronal inhibitory components, respectively. The cell adhesion molecule Contactin 2 (CNTN2) is expressed in neurons and oligodendrocytes in the CNS. Neuronal CNTN2 is highly regulated during development and plays critical roles in axon growth and guidance and neuronal migration. On the other hand, CNTN2 expressed by oligodendrocytes interferes with the myelination process, with its ablation resulting in hypomyelination. In the current study, we investigate the role of CNTN2 in neuronal survival and axon regeneration after trauma, in the murine optic nerve crush (ONC) model. We unveil distinct roles for neuronal and glial CNTN2 in regenerative responses. Surprisingly, our data show a conflicting role of neuronal and glial CNTN2 in axon regeneration. Although glial CNTN2 as well as hypomyelination are dispensable for both neuronal survival and axon regeneration following ONC, the neuronal counterpart comprises a negative regulator of regeneration. Specifically, we reveal a novel mechanism of action for neuronal CNTN2, implicating the inhibition of Akt signalling pathway. The in vitro analysis indicates a BDNF-independent mode of action and biochemical data suggest the implication of the truncated form of TrkB neurotrophin receptor. In conclusion, CNTN2 expressed in CNS neurons serves as an inhibitor of axon regeneration after trauma and its mechanism of action involves the neutralization of Akt-mediated neuroprotective effects.

Implication of Contactins in Demyelinating Pathologies.

Demyelinating pathologies comprise of a variety of conditions where either central or peripheral myelin is attacked, resulting in white matter lesions and neurodegeneration. Myelinated axons are organized into molecularly distinct domains, and this segregation is crucial for their proper function. These defined domains are differentially affected at the different stages of demyelination as well as at the lesion and perilesion sites. Among the main players in myelinated axon organization are proteins of the contactin (CNTN) group of the immunoglobulin superfamily (IgSF) of cell adhesion molecules, namely Contactin-1 and Contactin-2 (CNTN1, CNTN2). The two contactins perform their functions through intermolecular interactions, which are crucial for myelinated axon integrity and functionality. In this review, we focus on the implication of these two molecules as well as their interactors in demyelinating pathologies in humans. At first, we describe the organization and function of myelinated axons in the central (CNS) and the peripheral (PNS) nervous system, further analyzing the role of CNTN1 and CNTN2 as well as their interactors in myelination. In the last section, studies showing the correlation of the two contactins with demyelinating pathologies are reviewed, highlighting the importance of these recognition molecules in shaping the function of the nervous system in multiple ways.

UniProt-Related Documents (UniReD): assisting wet lab biologists in their quest on finding novel counterparts in a protein network.

The in-depth study of protein-protein interactions (PPIs) is of key importance for understanding how cells operate. Therefore, in the past few years, many experimental as well as computational approaches have been developed for the identification and discovery of such interactions. Here, we present UniReD, a user-friendly, computational prediction tool which analyses biomedical literature in order to extract known protein associations and suggest undocumented ones. As a proof of concept, we demonstrate its usefulness by experimentally validating six predicted interactions and by benchmarking it against public databases of experimentally validated PPIs succeeding a high coverage. We believe that UniReD can become an important and intuitive resource for experimental biologists in their quest for finding novel associations within a protein network and a useful tool to complement experimental approaches (e.g. mass spectrometry) by producing sorted lists of candidate proteins for further experimental validation. UniReD is available at http://bioinformatics.med.uoc.gr/unired/.

Ablation of CNTN2+ Pyramidal Neurons During Development Results in Defects in Neocortical Size and Axonal Tract Formation.

Corticothalamic axons express Contactin-2 (CNTN2/TAG-1), a neuronal recognition molecule of the immunoglobulin superfamily involved in neurogenesis, neurite outgrowth, and fasciculation. TAG-1, which is expressed transiently by cortical pyramidal neurons during embryonic development, has been shown to be fundamental for axonal recognition, cellular migration, and neuronal proliferation in the developing cortex. Although Tag-1 -/- mice do not exhibit any obvious defects in the corticofugal system, the role of TAG-1+ neurons during the development of the cortex remains elusive. We have generated a mouse model expressing EGFP under the Tag-1 promoter and encompassing the coding sequence of Diptheria Toxin subunit A (DTA) under quiescence with no effect on the expression of endogenous Tag-1. We show that while the line recapitulates the expression pattern of the molecule, it highlights an extended expression in the forebrain, including multiple axonal tracts and neuronal populations, both spatially and temporally. Crossing these mice to the Emx1-Cre strain, we ablated the vast majority of TAG-1+ cortical neurons. Among the observed defects were a significantly smaller cortex, a reduction of corticothalamic axons as well as callosal and commissural defects. Such defects are common in neurodevelopmental disorders, thus this mouse could serve as a useful model to study physiological and pathophysiological cortical development.

Models and treatments for traumatic optic neuropathy and demyelinating optic neuritis.

Pathologies of the optic nerve could result as primary insults in the visual tract or as secondary deficits due to inflammation, demyelination, or compressing effects of the surrounding tissue. The extent of damage may vary from mild to severe, differently affecting patient vision, with the most severe forms leading to complete uni- or bilateral visual loss. The aim of researchers and clinicians in the field is to alleviate the symptoms of these, yet uncurable pathologies, taking advantage of known and novel potential therapeutic approaches, alone or in combinations, and applying them in a limited time window after the insult. In this review, we discuss the epidemiological and clinical profile as well as the pathophysiological mechanisms of two main categories of optic nerve pathologies, namely traumatic optic neuropathy and optic neuritis, focusing on the demyelinating form of the latter. Moreover, we report on the main rodent models mimicking these pathologies or some of their clinical aspects. The current treatment options will also be reviewed and novel approaches will be discussed.

The function of contactin-2/TAG-1 in oligodendrocytes in health and demyelinating pathology.

The oligodendrocyte maturation process and the transition from the pre-myelinating to the myelinating state are extremely important during development and in pathology. In the present study, we have investigated the role of the cell adhesion molecule CNTN2/TAG-1 on oligodendrocyte proliferation, differentiation, myelination, and function during development and under pathological conditions. With the combination of in vivo, in vitro, ultrastructural, and electrophysiological methods, we have mapped the expression of CNTN2 protein in the oligodendrocyte lineage during the different stages of myelination and its involvement on oligodendrocyte maturation, branching, myelin-gene expression, myelination, and axonal function. The cuprizone model of central nervous system demyelination was further used to assess CNTN2 in pathology. During development, CNTN2 can transiently affect the expression levels of myelin and myelin-regulating genes, while its absence results in reduced oligodendrocyte branching, hypomyelination of fiber tracts and impaired axonal conduction. In pathology, CNTN2 absence does not affect the extent of de- and remyelination. However during remyelination, a novel, CNTN2-independent mechanism is revealed that is able to recluster voltage gated potassium channels (VGKCs) resulting in the improvement of fiber conduction.

The Kv1-associated molecules TAG-1 and Caspr2 are selectively targeted to the axon initial segment in hippocampal neurons.

Caspr2 and TAG-1 (also known as CNTNAP2 and CNTN2, respectively) are cell adhesion molecules (CAMs) associated with the voltage-gated potassium channels Kv1.1 and Kv1.2 (also known as KCNA1 and KCNA2, respectively) at regions controlling axonal excitability, namely, the axon initial segment (AIS) and juxtaparanodes of myelinated axons. The distribution of Kv1 at juxtaparanodes requires axo-glial contacts mediated by Caspr2 and TAG-1. In the present study, we found that TAG-1 strongly colocalizes with Kv1.2 at the AIS of cultured hippocampal neurons, whereas Caspr2 is uniformly expressed along the axolemma. Live-cell imaging revealed that Caspr2 and TAG-1 are sorted together in axonal transport vesicles. Therefore, their differential distribution may result from diffusion and trapping mechanisms induced by selective partnerships. By using deletion constructs, we identified two molecular determinants of Caspr2 that regulate its axonal positioning. First, the LNG2-EGF1 modules in the ectodomain of Caspr2, which are involved in its axonal distribution. Deletion of these modules promotes AIS localization and association with TAG-1. Second, the cytoplasmic PDZ-binding site of Caspr2, which could elicit AIS enrichment and recruitment of the membrane-associated guanylate kinase (MAGuK) protein MPP2. Hence, the selective distribution of Caspr2 and TAG-1 may be regulated, allowing them to modulate the strategic function of the Kv1 complex along axons.

Tag1 deficiency results in olfactory dysfunction through impaired migration of mitral cells.

The olfactory system provides mammals with the abilities to investigate, communicate and interact with their environment. These functions are achieved through a finely organized circuit starting from the nasal cavity, passing through the olfactory bulb and ending in various cortical areas. We show that the absence of transient axonal glycoprotein-1 (Tag1)/contactin-2 (Cntn2) in mice results in a significant and selective defect in the number of the main projection neurons in the olfactory bulb, namely the mitral cells. A subpopulation of these projection neurons is reduced in Tag1-deficient mice as a result of impaired migration. We demonstrate that the detected alterations in the number of mitral cells are well correlated with diminished odor discrimination ability and social long-term memory formation. Reduced neuronal activation in the olfactory bulb and the corresponding olfactory cortex suggest that Tag1 is crucial for the olfactory circuit formation in mice. Our results underpin the significance of a numerical defect in the mitral cell layer in the processing and integration of odorant information and subsequently in animal behavior.

Axons and myelinating glia: An intimate contact.

The coordination of the vertebrate nervous system requires high velocity signal transmission between different brain areas. High speed nerve conduction is achieved in the myelinated fibers of both the central and the peripheral nervous system where the myelin sheath acts as an insulator of the axon. The interactions between the glial cell and the adjacent axon, namely axo-glial interactions, segregate the fiber in distinct molecular and functional domains that ensure the rapid propagation of action potentials. These domains are the node of Ranvier, the paranode, the juxtaparanode and the internode and are characterized by multiprotein complexes between voltage-gated ion channels, cell adhesion molecules, members of the Neurexin family and cytoskeletal proteins. In the present review, we outline recent evidence on the key players of axo-glial interactions, depicting their importance in myelinated fiber physiology and disease.

The expression of TAG-1 in glial cells is sufficient for the formation of the juxtaparanodal complex and the phenotypic rescue of tag-1 homozygous mutants in the CNS.

Myelinated fibers are organized into specialized domains that ensure the rapid propagation of action potentials and are characterized by protein complexes underlying axoglial interactions. TAG-1 (Transient Axonal Glycoprotein-1), a cell adhesion molecule of the Ig superfamily, is expressed by neurons as well as by myelinating glia. It is essential for the molecular organization of myelinated fibers as it maintains the integrity of the juxtaparanodal region through its interactions with Caspr2 and the voltage-gated potassium channels (VGKCs) on the axolemma. Since TAG-1 is the only known component of the juxtaparanodal complex expressed by the glial cell, it is important to clarify its role in the molecular organization of juxtaparanodes. For this purpose, we generated transgenic mice that exclusively express TAG-1 in oligodendrocytes and lack endogenous gene expression (Tag-1(-/-);plp(Tg(rTag-1))). Phenotypic analysis clearly demonstrates that glial TAG-1 is sufficient for the proper organization and maintenance of the juxtaparanodal domain in the CNS. Biochemical analysis shows that glial TAG-1 physically interacts with Caspr2 and VGKCs. Ultrastructural and behavioral analysis of Tag-1(-/-);plp(Tg(rTag-1)) mice shows that the expression of glial TAG-1 is sufficient to restore the axonal and myelin deficits as well as the behavioral defects observed in Tag-1(-/-) animals. Together, these data highlight the pivotal role of myelinating glia on axonal domain differentiation and organization.

Activation of somatostatin receptor (sst 5) protects the rat retina from AMPA-induced neurotoxicity.

In a recent study, we employed an in vivo model of retinal excitotoxicity to investigate the neuroprotective effect of somatostatinergic agents. Intravitreal administration of somatostatin and sst(2) selective agonists protected the retina from (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA) induced excitotoxicity. The sst(1) and sst(4) selective ligands had no effect (Kiagiadaki and Thermos, 2008). The presence of sst(5) receptors in rat retina was only recently reported (Ke and Zhong, 2007). Synthetic agonists that activate sst(2) receptors also bind with high affinity to the sst(5) subtype. In the present study the putative neuroprotective effects of sst(5) receptor activation were investigated. Adult female and male Sprague-Dawley (250-350g) rats were employed. Groups of animals received intravitreally PBS (50mM) or AMPA (42 nmol/eye) alone or in combination with L-817,818 (sst(5), 10(-5), 10(-4)M). To exclude neuroprotective effects via the activation of sst(2) receptors, L-817,818 (10(-4)M) was coinjected with the sst(2) antagonist CYN-154806 (10(-4)M). Immunohistochemistry (IHC) studies using the anti-retinal marker choline acetyltransferase (ChAT) and TUNEL staining were employed to examine retinal cell loss and protection. IHC and Western blot analysis were also employed to assess whether the sst(5) receptors are viable in the AMPA treated tissue as compared to control retina. sst(5) receptors were not affected by AMPA. L-817,818 protected the retina from the AMPA insult in the dose of 10(-4)M, while CYN-154806 (10(-4)M) had no effect on the sst(5) neuroprotection. TUNEL staining confirmed the AMPA-induced retinal toxicity and the L-817,818 neuroprotection. These results demonstrate for the first time that sst(5) receptors are functional in the retina, and that sst(5) analogs administered intravitreally protect the retina from excitotoxicity. Further studies are essential to ascertain the therapeutic relevance of these results.

Impairment of learning and memory in TAG-1 deficient mice associated with shorter CNS internodes and disrupted juxtaparanodes.

The cell adhesion molecule TAG-1 is expressed by neurons and glial cells and plays a role in axon outgrowth, migration and fasciculation during development. TAG-1 is also required for the clustering of Kv1.1/1.2 potassium channels and Caspr2 at the juxtaparanodes of myelinated fibers. Behavioral examination of TAG-1 deficient mice (Tag-1(-/-)) showed cognitive impairments in the Morris water maze and novel object recognition tests, reduced spontaneous motor activity, abnormal gait coordination and increased response latency to noxious stimulation. Investigation at the molecular level revealed impaired juxtaparanodal clustering of Caspr2 and Kv1.1/1.2 in the hippocampus, entorhinal cortex, cerebellum and olfactory bulb, with diffusion into the internode. Caspr2 and Kv1.1 levels were reduced in the cerebellum and olfactory bulb. Moreover, Tag-1(-/-) mice had shorter internodes in the cerebral and cerebellar white matter. The detected molecular alterations may account for the behavioural deficits and hyperexcitability in these animals.

Structural requirement of TAG-1 for retinal ganglion cell axons and myelin in the mouse optic nerve.

White matter axons organize into fascicles that grow over long distances and traverse very diverse environments. The molecular mechanisms preserving this structure of white matter axonal tracts are not well known. Here, we used the optic nerve as a model and investigated the role of TAG-1, a cell adhesion molecule expressed by retinal axons. TAG-1 was first expressed in the embryonic retinal ganglion cells (RGCs) and later in the postnatal myelin-forming cells in the optic nerve. We describe the consequences of genetic loss of Tag-1 on the developing and adult retinogeniculate tract. Tag-1-null embryos display anomalies in the caliber of RGC axons, associated with an abnormal organization of the astroglial network in the optic nerve. The contralateral projections in the lateral geniculate nucleus are expanded postnatally. In the adult, Tag-1-null mice show a loss of RGC axons, with persistent abnormalities of axonal caliber and additional cytoskeleton and myelination defects. Therefore, TAG-1 is an essential regulator of the structure of RGC axons and their surrounding glial cells in the optic nerve.