Clonally expanded B cells in multiple sclerosis bind EBV EBNA1 and GlialCAM.

Multiple sclerosis (MS) is a heterogenous autoimmune disease in which autoreactive lymphocytes attack the myelin sheath of the central nervous system. B lymphocytes in the cerebrospinal fluid (CSF) of patients with MS contribute to inflammation and secrete oligoclonal immunoglobulins1,2. Epstein-Barr virus (EBV) infection has been epidemiologically linked to MS, but its pathological role remains unclear3. Here we demonstrate high-affinity molecular mimicry between the EBV transcription factor EBV nuclear antigen 1 (EBNA1) and the central nervous system protein glial cell adhesion molecule (GlialCAM) and provide structural and in vivo functional evidence for its relevance. A cross-reactive CSF-derived antibody was initially identified by single-cell sequencing of the paired-chain B cell repertoire of MS blood and CSF, followed by protein microarray-based testing of recombinantly expressed CSF-derived antibodies against MS-associated viruses. Sequence analysis, affinity measurements and the crystal structure of the EBNA1-peptide epitope in complex with the autoreactive Fab fragment enabled tracking of the development of the naive EBNA1-restricted antibody to a mature EBNA1-GlialCAM cross-reactive antibody. Molecular mimicry is facilitated by a post-translational modification of GlialCAM. EBNA1 immunization exacerbates disease in a mouse model of MS, and anti-EBNA1 and anti-GlialCAM antibodies are prevalent in patients with MS. Our results provide a mechanistic link for the association between MS and EBV and could guide the development of new MS therapies.

NgCAM and VAMP2 reveal that direct delivery and dendritic degradation maintain axonal polarity.

Neurons are polarized cells of extreme scale and compartmentalization. To fulfill their role in electrochemical signaling, axons must maintain a specific complement of membrane proteins. Despite being the subject of considerable attention, the trafficking pathway of axonal membrane proteins is not well understood. Two pathways, direct delivery and transcytosis, have been proposed. Previous studies reached contradictory conclusions about which of these mediates delivery of axonal membrane proteins to their destination, in part because they evaluated long-term distribution changes and not vesicle transport. We developed a novel strategy to selectively label vesicles in different trafficking pathways and determined the trafficking of two canonical axonal membrane proteins, neuron-glia cell adhesion molecule and vesicle-associated membrane protein-2. Results from detailed quantitative analyses of transporting vesicles differed substantially from previous studies and found that axonal membrane proteins overwhelmingly undergo direct delivery. Transcytosis plays only a minor role in axonal delivery of these proteins. In addition, we identified a novel pathway by which wayward axonal proteins that reach the dendritic plasma membrane are targeted to lysosomes. These results redefine how axonal proteins achieve their polarized distribution, a crucial requirement for elucidating the underlying molecular mechanisms.

Megalencephalic leukoencephalopathy with subcortical cysts is a developmental disorder of the gliovascular unit.

Absence of the astrocyte-specific membrane protein MLC1 is responsible for megalencephalic leukoencephalopathy with subcortical cysts (MLC), a rare type of leukodystrophy characterized by early-onset macrocephaly and progressive white matter vacuolation that lead to ataxia, spasticity, and cognitive decline. During postnatal development (from P5 to P15 in the mouse), MLC1 forms a membrane complex with GlialCAM (another astrocytic transmembrane protein) at the junctions between perivascular astrocytic processes. Perivascular astrocytic processes along with blood vessels form the gliovascular unit. It was not previously known how MLC1 influences the physiology of the gliovascular unit. Here, using the Mlc1 knock-out mouse model of MLC, we demonstrated that MLC1 controls the postnatal development and organization of perivascular astrocytic processes, vascular smooth muscle cell contractility, neurovascular coupling, and intraparenchymal interstitial fluid clearance. Our data suggest that MLC is a developmental disorder of the gliovascular unit, and perivascular astrocytic processes and vascular smooth muscle cell maturation defects are primary events in the pathogenesis of MLC and therapeutic targets for this disease.

HepaCAM controls astrocyte self-organization and coupling.

Astrocytes extensively infiltrate the neuropil to regulate critical aspects of synaptic development and function. This process is regulated by transcellular interactions between astrocytes and neurons via cell adhesion molecules. How astrocytes coordinate developmental processes among one another to parse out the synaptic neuropil and form non-overlapping territories is unknown. Here we identify a molecular mechanism regulating astrocyte-astrocyte interactions during development to coordinate astrocyte morphogenesis and gap junction coupling. We show that hepaCAM, a disease-linked, astrocyte-enriched cell adhesion molecule, regulates astrocyte competition for territory and morphological complexity in the developing mouse cortex. Furthermore, conditional deletion of Hepacam from developing astrocytes significantly impairs gap junction coupling between astrocytes and disrupts the balance between synaptic excitation and inhibition. Mutations in HEPACAM cause megalencephalic leukoencephalopathy with subcortical cysts in humans. Therefore, our findings suggest that disruption of astrocyte self-organization mechanisms could be an underlying cause of neural pathology.

Identification of the GlialCAM interactome: the G protein-coupled receptors GPRC5B and GPR37L1 modulate megalencephalic leukoencephalopathy proteins.

Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy, which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related to adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to an impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.

Cellular basis of ClC-2 Cl- channel-related brain and testis pathologies.

The ClC-2 chloride channel is expressed in the plasma membrane of almost all mammalian cells. Mutations that cause the loss of ClC-2 function lead to retinal and testicular degeneration and leukodystrophy, whereas gain-of-function mutations cause hyperaldosteronism. Leukodystrophy is also observed with a loss of GlialCAM, a cell adhesion molecule that binds to ClC-2 in glia. GlialCAM changes the localization of ClC-2 and opens the channel by altering its gating. We now used cell type-specific deletion of ClC-2 in mice to show that retinal and testicular degeneration depend on a loss of ClC-2 in retinal pigment epithelial cells and Sertoli cells, respectively, whereas leukodystrophy was fully developed only when ClC-2 was disrupted in both astrocytes and oligodendrocytes. The leukodystrophy of Glialcam-/- mice could not be rescued by crosses with Clcn2op/op mice in which a mutation mimics the "opening" of ClC-2 by GlialCAM. These data indicate that GlialCAM-induced changes in biophysical properties of ClC-2 are irrelevant for GLIALCAM-related leukodystrophy. Taken together, our findings suggest that the pathology caused by Clcn2 disruption results from disturbed extracellular ion homeostasis and identifies the cells involved in this process.

Matrix metalloprotease-mediated cleavage of neural glial-related cell adhesion molecules activates quiescent olfactory stem cells via EGFR.

Quiescent stem cells have been found in multiple adult organs, and activation of these stem cells is critical to the restoration of damaged tissues in response to injury or stress. Existing evidence suggests that extrinsic cues from the extracellular matrix or supporting cells of various stem cell niches may interact with intrinsic components to initiate stem cell differentiation, but the molecular and cellular mechanisms regulating their activation are not fully understood. In the present study, we find that olfactory horizontal basal cells (HBCs) are stimulated by neural glial-related cell adhesion molecules (NrCAMs). NrCAM activation requires matrix metalloproteases (MMPs) and epidermal growth factor receptors (EGFRs). Inhibiting MMP activity or EGFR activation not only blocks HBC proliferation in the cultured olfactory organoids, but also severely suppresses HBC proliferation in the olfactory epithelium following methimazole-induced injury, resulting in a delay of olfactory mucosa reconstitution and functional recovery of the injured mice. Both NrCAMs and EGFR are expressed by the HBCs and their expression increases upon injury. Our data indicate that MMP-mediated cleavage of NrCAMs serves as an autocrine or paracrine signal that activates EGFRs on HBCs to trigger HBC proliferation and differentiation to reconstruct the entire olfactory epithelium following injury.

Comparison of zebrafish and mice knockouts for Megalencephalic Leukoencephalopathy proteins indicates that GlialCAM/MLC1 forms a functional unit.

BACKGROUND: Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a rare type of leukodystrophy characterized by astrocyte and myelin vacuolization, epilepsy and early-onset macrocephaly. MLC is caused by mutations in MLC1 or GLIALCAM, coding for two membrane proteins with an unknown function that form a complex specifically expressed in astrocytes at cell-cell junctions. Recent studies in Mlc1-/- or Glialcam-/- mice and mlc1-/- zebrafish have shown that MLC1 regulates glial surface levels of GlialCAM in vivo and that GlialCAM is also required for MLC1 expression and localization at cell-cell junctions. METHODS: We have generated and analysed glialcama-/- zebrafish. We also generated zebrafish glialcama-/- mlc1-/- and mice double KO for both genes and performed magnetic resonance imaging, histological studies and biochemical analyses. RESULTS: glialcama-/- shows megalencephaly and increased fluid accumulation. In both zebrafish and mice, this phenotype is not aggravated by additional elimination of mlc1. Unlike mice, mlc1 protein expression and localization are unaltered in glialcama-/- zebrafish, possibly because there is an up-regulation of mlc1 mRNA. In line with these results, MLC1 overexpressed in Glialcam-/- mouse primary astrocytes is located at cell-cell junctions. CONCLUSIONS: This work indicates that the two proteins involved in the pathogenesis of MLC, GlialCAM and MLC1, form a functional unit, and thus, that loss-of-function mutations in these genes cause leukodystrophy through a common pathway.

Identification in Chinese patients with GLIALCAM mutations of megalencephalic leukoencephalopathy with subcortical cysts and brain pathological study on Glialcam knock-in mouse models.

BACKGROUND: Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare neurological degenerative disorder caused by the mutations of MLC1 or GLIALCAM with autosomal recessive or autosomal dominant inheritance and a different prognosis, characterized by macrocephaly, delayed motor and cognitive development, and bilateral abnormal signals in cerebral white matter (WM) with or without cysts on magnetic resonance imaging (MRI). This study aimed to reveal the clinical and genetic features of MLC patients with GLIALCAM mutations and to explore the brain pathological characteristics and prognosis of mouse models with different modes of inheritance. METHODS: Clinical information and peripheral venous blood were collected from six families. Genetic analysis was performed by Sanger sequencing of GLIALCAM. GlialcamArg92Trp/+ and GlialcamLys68Met/Thr132Asn mouse models were generated based on mutations from patients (c.274C>T(p.Arg92Trp) (c.203A>T(p.Lys68Met), and c.395C>A (p.Thr132Asn))). Brain pathologies of the mouse models at different time points were analyzed. RESULTS: Six patients were clinically diagnosed with MLC. Of the six patients, five (Pt1-Pt5) presented with a heterozygous mutation in GLIALCAM (c.274C>T(p.Arg92Trp) or c.275G>C(p.Arg92Pro)) and were diagnosed with MLC2B; the remaining patient (Pt6) with two compound heterozygous mutations in GLIALCAM (c.203A>T (p.Lys68Met) and c.395C>A (p.Thr132Asn)) was diagnosed with MLC2A. The mutation c.275C>G (p.Arg92Pro) has not been reported before. Clinical manifestations of the patient with MLC2A (Pt6) progressed with regression, whereas the course of the five MLC2B patients remained stable or improved. The GlialcamArg92Trp/+ and GlialcamLys68Met/ Thr132Asn mouse models showed vacuolization in the anterior commissural WM at 1 month of age and vacuolization in the cerebellar WM at 3 and 6 months, respectively. At 9 months, the vacuolization of the GlialcamLys68Met/ Thr132Asn mouse model was heavier than that of the GlialcamArg92Trp/+ mouse model. Decreased expression of Glialcam in GlialcamArg92Trp/+ and GlialcamLys68Met/ Thr132Asn mice may contribute to the vacuolization. CONCLUSIONS: Clinical and genetic characterization of patients with MLC and GLIALCAM mutations revealed a novel mutation, expanding the spectrum of GLIALCAM mutations. The first Glialcam mouse model with autosomal recessive inheritance and a new Glialcam mouse model with autosomal dominant inheritance were generated. The two mouse models with different modes of inheritance showed different degrees of brain pathological features, which were consistent with the patients' phenotype and further confirmed the pathogenicity of the corresponding mutations.

Postnatal development of the astrocyte perivascular MLC1/GlialCAM complex defines a temporal window for the gliovascular unit maturation.

Astrocytes, the most abundant glial cells of the central nervous system are morphologically complex. They display numerous processes interacting with synapses and blood vessels. At the vascular interface, astrocyte endfeet-terminated processes almost entirely cover the blood vessel surface and participate to the gliovascular unit where important vascular properties of the brain are set such as the blood-brain barrier (BBB) integrity. How specific morphological and functional interactions between astrocytes and the vascular compartment develop has not been fully investigated. Here, we elaborated an original experimental strategy to study the postnatal development of astrocyte perivascular endfeet. Using purified gliovascular units, we focused on the postnatal expression of MLC1 and GlialCAM, two transmembrane proteins forming a complex enriched at the junction between mature astrocyte perivascular endfeet. We showed that MLC1 and GlialCAM were enriched and assembled into mature complexes in astrocyte perivascular endfeet between postnatal days 10 and 15, after the formation of astrocyte perivascular Aquaporin 4 water channels. These events correlated with the increased expression of Claudin-5 and P-gP, two endothelial-specific BBB components. These results illustrate for the first time that astrocyte perivascular endfeet differentiation is a complex and progressive process which correlates with BBB maturation. Moreover, our results suggest that maturation of the astrocyte endfeet MLC1/GlialCAM complex between postnatal days 10 and 15 might be a key event in the gliovascular unit maturation.

Depolarization causes the formation of a ternary complex between GlialCAM, MLC1 and ClC-2 in astrocytes: implications in megalencephalic leukoencephalopathy.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of leukodystrophy caused by mutations in either MLC1 or GLIALCAM. GlialCAM is necessary for the correct targeting of MLC1, but also for the targeting of the Cl- channel ClC-2. Furthermore, GlialCAM modifies ClC-2 functional properties in vitro. However, in vivo studies in GlialCAM-/- mice have shown that the modification of ClC-2 activity only occurs in oligodendrocytes, despite GlialCAM and ClC-2 being expressed in astrocytes. Thus, the relationship between GlialCAM, MLC1 and ClC-2 in astrocytes is unknown. Here, we show that GlialCAM, ClC-2 and MLC1 can form a ternary complex in cultured astrocytes, but only under depolarizing conditions. We also provide biochemical evidences that this ternary complex exists in vivo. The formation of this complex changes ClC-2 localization in the membrane and its functional properties. ClC-2 association with GlialCAM/MLC1 depends on calcium flux through L-type calcium channels and activation of calcium-dependent calpain proteases. Based on these studies, we propose that the chloride influx mediated by GlialCAM/MLC1/ClC-2 in astrocytes may be needed to compensate an excess of potassium, as occurs in conditions of high neuronal activity. We suggest that a defect in this compensation may contribute to the pathogenesis of MLC disease.

Cumulative effects of the ApoE genotype and gender on the synaptic proteome and oxidative stress in the mouse brain.

Elderly females, particularly those carrying the apolipoprotein E (ApoE)-ε4 allele, have a higher risk of developing Alzheimer's disease (AD). However, the underlying mechanism for this increased susceptibility remains unclear. In this study, we investigated the effects of the ApoE genotype and gender on the proteome of synaptosomes. We isolated synaptosomes and used label-free quantitative proteomics, to report, for the first time, that the synaptosomal proteomic profiles in the cortex of female human-ApoE4 mice exhibited significantly reduced expression of proteins related to energy metabolism, which was accompanied by increased levels of oxidative stress. In addition, we also first demonstrated that the proteomic response in synaptic termini was more susceptible than that in the soma to the adverse effects induced by genders and genotypes. This suggests that synaptic mitochondria might be 'older' than mitochondria in the soma of neurons; therefore, they might contain increased cumulative damage from oxidative stress. Furthermore, female human-ApoE4 mice had much lower oestrogen levels in the cortex and treatment with oestrogen protected ApoE3 stable transfected C6 neurons from oxidative stress. Overall, this study reveals complex ApoE- and gender-dependent effects on synaptic function and also provides a basis for future studies of candidates based on specific pathways involved in the pathogenesis of AD. The lack of oestrogen-mediated protection regulated by the ApoE genotype led to synaptic mitochondrial dysfunction and increased oxidative stress, which might make older females more susceptible to AD.

Disrupting MLC1 and GlialCAM and ClC-2 interactions in leukodystrophy entails glial chloride channel dysfunction.

Defects in the astrocytic membrane protein MLC1, the adhesion molecule GlialCAM or the chloride channel ClC-2 underlie human leukoencephalopathies. Whereas GlialCAM binds ClC-2 and MLC1, and modifies ClC-2 currents in vitro, no functional connections between MLC1 and ClC-2 are known. Here we investigate this by generating loss-of-function Glialcam and Mlc1 mouse models manifesting myelin vacuolization. We find that ClC-2 is unnecessary for MLC1 and GlialCAM localization in brain, whereas GlialCAM is important for targeting MLC1 and ClC-2 to specialized glial domains in vivo and for modifying ClC-2's biophysical properties specifically in oligodendrocytes (OLs), the cells chiefly affected by vacuolization. Unexpectedly, MLC1 is crucial for proper localization of GlialCAM and ClC-2, and for changing ClC-2 currents. Our data unmask an unforeseen functional relationship between MLC1 and ClC-2 in vivo, which is probably mediated by GlialCAM, and suggest that ClC-2 participates in the pathogenesis of megalencephalic leukoencephalopathy with subcortical cysts.

Early postnatal expression and localization of matrix metalloproteinases-2 and -9 during establishment of rat hippocampal synaptic circuitry.

Matrix metalloproteinases (MMPs) are extracellular proteolytic enzymes that contribute to pericellular remodeling in a variety of tissues, including brain, where they function in adult hippocampal synaptic structural and functional plasticity. Synaptic plasticity and remodeling are also important for development of connectivity, but it is unclear whether MMPs--particularly MMP-2 and -9, the major MMPs operative in brain--contribute at these stages. Here, we use a combination of biochemical and anatomical methods to characterize expression and localization of MMP-2 and MMP-9 in early postnatal and adult rat hippocampus. Gene and protein expression of these MMPs were evident throughout hippocampus at all ages examined, but expression levels were highest during the first postnatal week. MMP-2 and MMP-9 immunolocalized to punctate structures within the neuropil that codistributed with foci of proteolytic activity, as well as with markers of growing axons and synapses. Taken together, discrete foci of MMP proteolysis are likely important for actively shaping and remodeling cellular and connectional architecture as hippocampal circuitry is becoming established during early postnatal life.

Trypsin causes platelet activation independently of known protease-activated receptors.

To identify a physiological agonist of PAR3, we used PAR4 null murine platelets, which were known to express only PAR3. In this study, we tested several proteases and found that trypsin, but not heat-inactivated trypsin, activated PAR4 null murine platelets. Even at high concentrations, trypsin caused shape change without increasing intracellular calcium levels in PAR4 null murine platelets. Consistent with this result, the Gq inhibitor YM-254890 had no effect on trypsin-induced shape change. However, trypsin-induced platelet shape change was abolished by either p160ROCK inhibitor, Y27632 or H1152. Furthermore, trypsin caused phosphorylation of myosin light chain (Thr18), but not Akt or Erk. Surprisingly, trypsin caused a similar shape change in PAR4-desensitised PAR3 null murine platelets as in PAR4null murine platelets, indicating that trypsin did not activate PAR3 to cause shape change. More interestingly, the Src family kinase (SFK) inhibitor PP2 abolished trypsin-induced, but not AYPGKF-induced, shape change. Hence, trypsin activated a novel signalling pathway through RhoA/p160ROCK and was regulated by SFKs. In conclusion, our study demonstrates a novel protease signalling pathway in platelets that is independent of PARs. This protease-induced novel signalling pathway regulates platelet shape change through SFKs and p160ROCK.

Neurodegeneración con acúmulo cerebral de hierro / Neurodegeneration with brain iron accumulation

Introducción. Las alteraciones en el metabolismo del hierro se han relacionado con diversas enfermedades neurodegenerativas, en algunas de ellas se trata del principal elemento de la enfermedad mientras que en otras, como la enfermedad de Alzheimer o Parkinson, se ha descrito su alteración pero no se conoce su significado fisiopatológico exacto. Objetivo. Revisión bibliográfica sobre el conocimiento actual de las enfermedades que cursan con depósitos encefálicos de hierro. Desarrollo. Se denomina neurodegeneración asociada a depósito de hierro (NAEH), a un grupo heterogéneo de enfermedades hereditarias (la mayor parte de ellas autosómicas recesivas) que cursan con depósitos de hierro en determinadas áreas cerebrales. La causa más frecuente es la mutación en el gen PANK2, recibiendo el nombre de Neurodegeneración asociada a Pantotenatocinasa (NAPC). La descripción inicial corresponde al síndrome de Hallervorden-Spatz. El cuadro clínico incluye manifestaciones motoras y deterioro psicomotor con una evolución progresiva hasta la incapacidad y fallecimiento del paciente. La resonancia magnética muestra alteraciones características afectando fundamentalmente al globo pálido. También se incluyen dentro de la NAEH la Neuroferritinopatia, la aceruloplasminemia y algunas formas asociadas a mutaciones en el gen PLA2G6, enfermedades que comparten algunas características clínicas y la presencia de alteraciones en la RM aunque con diferencias entre ellas. Conclusiones. El conocimiento de las distintas alteraciones genéticas ha permitido una mejor clasificación nosológica de la NAEH. Tanto la clínica como el patrón de alteraciones en RM pueden resultar de utilidad en el diagnóstico. Por el momento no se dispone de tratamientos que modifiquen el curso de la enfermedad (AU)

Introduction. Alterations in iron metabolism have been related with several neurodegenerative diseases. In some cases it is the main element of the disease while in others, such as Alzheimer's or Parkinson’s disease, its alteration has been reported but its exact pathophysiological significance remains unknown. Aims. The aim of the study was to carry out a review of the literature on the current knowledge about diseases that are accompanied by deposits of iron in the brain. Development. The term neurodegeneration with brain iron accumulation (NBIA) is used to refer to a heterogeneous group of hereditary (mostly autosomal recessive) diseases that are accompanied by deposits of iron in certain areas of the brain. The most frequent cause is a mutation in the PANK2 gene, which is called Pantothenate Kinase-Associated Neurodegeneration (PKAN). The initial description corresponds to Hallervorden-Spatz syndrome. The clinical picture includes motor manifestations and psychomotor deterioration with a progressive development until disability and the death of the patient. Magnetic resonance imaging shows characteristic alterations mainly involving the globus pallidus. NBIA also includes neuroferritinopathy, aceruloplasminemia and some forms associated to mutations in the PLA2G6 gene, diseases that share certain clinical characteristics, and the presence of alterations in the MRI scan, although there are differences from one to another. Conclusions. Understanding the different genetic alterations has made it possible to achieve a better nosological classification of NBIA. Both the clinical features and the pattern of alterations in MRI can be useful in the diagnosis. For the time being there are no treatments that modify the course of the disease (AU)

Endocytosis and endosomes at the crossroads of regulating trafficking of axon outgrowth-modifying receptors.

In neurons, many receptors must be localized correctly to axons or dendrites for proper function. During development, receptors for nerve growth and guidance are targeted to axons and localized to growth cones where receptor activation by ligands results in promotion or inhibition of axon growth. Signaling outcomes downstream of ligand binding are determined by the location, levels and residence times of receptors on the neuronal plasma membrane. Therefore, the mechanisms controlling the trafficking of these receptors are crucial to the proper wiring of circuits. Membrane proteins accumulate on the axonal surface by multiple routes, including polarized sorting in the trans Golgi network, sorting in endosomes and removal by endocytosis. Endosomes also play important roles in the signaling pathways for both growth-promoting and -inhibiting molecules: signaling endosomes derived from endocytosis are important for signaling from growth cones to cell bodies. Growth-promoting neurotrophins and growth-inhibiting Nogo-A can use EHD4/Pincher-dependent endocytosis at the growth cone for their respective retrograde signaling. In addition to retrograde transport of endosomes, anterograde transport to axons in endosomes also occurs for several receptors, including the axon outgrowth-promoting cell adhesion molecule L1/NgCAM and TrkA. L1/NgCAM also depends on EHD4/Pincher-dependent endocytosis for its axonal polarization. In this review, we will focus on receptors whose trafficking has been reported to be modulated by the EHD4/Pincher family of endosomal regulators, namely L1/NgCAM, Trk and Nogo-A. We will first summarize the pathways underlying the axonal transport of these proteins and then discuss the potential roles of EHD4/Pincher in mediating their endocytosis.

Increased expression of the close homolog of the adhesion molecule L1 in different cell types over time after rat spinal cord contusion.

The close homolog of the adhesion molecule L1 (CHL1) is important during CNS development, but a study with CHL1 knockout mice showed greater functional recovery after spinal cord injury (SCI) in its absence. We investigated CHL1 expression from 1 to 28 days after clinically relevant contusive SCI in Sprague-Dawley rats. Western blot analysis showed that CHL1 expression was significantly up-regulated at day 1 and further increased over 4 weeks after SCI. Immunohistochemistry of tissue sections showed that CHL1 in the intact spinal cord was expressed at low levels. By 1 day and through 4 weeks after SCI, CHL1 became highly expressed in NG2(+) cells. Hypertrophic GFAP(+) astrocytes also expressed CHL1 by 1 week after injury. The increase in CHL1 protein paralleled that of NG2 in the first week and GFAP between 1 and 4 weeks after injury. At 4 weeks, NG2(+) /CHL1(+) cells and GFAP(+) /CHL1(+) astrocytes were concentrated at the boundary between residual spinal cord tissue and the central lesion. NF200(+) spinal cord axons approached but did not penetrate this boundary. In contrast, CHL1(+) cells in the central lesion at 1 week and later colabeled with p75 and NG2 and were chronically associated with many NF200(+) axons, presumably axons that had sprouted in association with CHL1(+) Schwann cells infiltrating the cord after contusion. Thus, our study demonstrates up-regulation of CHL1 in multiple cell types and locations in a rat model of contusion injury and suggests that this molecule may be involved both in inhibition of axonal regeneration and in recovery processes after SCI.

Neuronal early endosomes require EHD1 for L1/NgCAM trafficking.

In neurons, the endosomal system is essential for membrane receptor trafficking to dendrites and axons and thereby participates in various neuronal functions, such as neurite outgrowth and synaptic plasticity. A multitude of regulators coordinates trafficking through endosomes, but most of them have not been studied in detail in neurons. In non-neuronal cells, EHD1 (Eps15 homology-domain containing protein 1) functions in the recycling endosome and is required for endosome-to-plasma membrane transport of multiple cargos. In this study, we analyze the role of EHD1 in neurons. In particular, we investigate whether EHD1 is required for polarized trafficking of the dendritically targeted transferrin and the axonal adhesion molecule L1/NgCAM (neuron-glia cell adhesion molecule) and, if so, in what compartment it is required. We find that endosomal recycling of both L1/NgCAM and transferrin is impaired when EHD1 is downregulated. We show that EHD1 colocalizes with L1/NgCAM and transferrin mostly in EEA1 (early endosome antigen 1)-positive early endosomes and less extensively with recycling endosomes. Using live imaging, we observe that EHD1 is stably associated with endosomal membranes during their maturation into EEA1-positive compartments and often persists on them longer than EEA1. Finally, we show that downregulation of EHD1 causes a delay of L1/NgCAM in exiting EEA1-positive endosomes, resulting in impaired targeting of L1/NgCAM to the axonal membrane. We conclude that, in neurons, EHD1 functions in early endosomes rather than (or possibly in addition to) recycling endosomes. These findings point to the existence of neuronal adaptations of the endosomal system.

Células gliales y actividad sináptica: control traduccional del acople metabólico / Glial cells and synaptic activity: translational control of metabolic coupling

Introducción. Consideradas tradicionalmente como células de soporte, las células gliales constituyen la inmensa mayoría de las células cerebrales al superar en número a las neuronas por un factor de diez. Poco se conoce de su participación en la fisiología cerebral, a pesar de su ubicación privilegiada, envolviendo las sinapsis. Las células de la estirpe glial participan en la formación de la denominada barrera hematoencefálica y representan una conexión entre la concentración de metabolitos en el compartimiento sistémico y el líquido cefalorraquídeo. Desarrollo. En este artículo analizamos los fenómenos moleculares desencadenados por el ácido glutámico en las células gliales y su participación en el acople metabólico establecido entre estas células y las neuronas. Conclusiones. El control de la traducción selectiva de ARN mensajero constituye la base molecular del acople entre la liberación sostenida de glutamato, la captura de este neurotransmisor y la producción y liberación de glutamina por las células gliales (AU)

Introduction. Traditionally regarded as supportive cells, glial cells have been barely studied in the context of brain physiology. No attention has been paid to the fact that these cells outcome neurons by an estimated factor of ten, and more importantly that they surround synapses. Moreover, cells of glial linage influence the formation of the so-called brain blood barrier representing a link between the concentration of metabolites in the systemic compartment and thecerebrospinal fluid. Development. Using as a model system the cerebellar glutamatergic synapses, in this contribution, we analyze the molecular transactions triggered by glutamate within glial cells that are involved neuronal-glia metabolic coupling. Conclusions. A tight coupling between sustained neuronal glutamate release, glial glutamate uptake, glial glutamine production and release is based on the control of the translation of selective mRNAs (AU)