Beatriz Cubelos

Cux1 and Cux2 regulate dendritic branching, spine morphology, and synapses of the upper layer neurons of the cortex.

Dendrite branching and spine formation determines the function of morphologically distinct and specialized neuronal subclasses. However, little is known about the programs instructing specific branching patterns in vertebrate neurons and whether such programs influence dendritic spines and synapses. Using knockout and knockdown studies combined with morphological, molecular, and electrophysiological analysis, we show that the homeobox Cux1 and Cux2 are intrinsic and complementary regulators of dendrite branching, spine development, and synapse formation in layer II-III neurons of the cerebral cortex. Cux genes control the number and maturation of dendritic spines partly through direct regulation of the expression of Xlr3b and Xlr4b, chromatin remodeling genes previously implicated in cognitive defects. Accordingly, abnormal dendrites and synapses in Cux2(-/-) mice correlate with reduced synaptic function and defects in working memory. These demonstrate critical roles of Cux in dendritogenesis and highlight subclass-specific mechanisms of synapse regulation that contribute to the establishment of cognitive circuits.

Essential function for the GTPase TC21 in homeostatic antigen receptor signaling

T cell antigen receptors (TCRs) and B cell antigen receptors (BCRs) transmit low-grade signals necessary for the survival and maintenance of mature cell pools. We show here that TC21, a small GTPase encoded by Rras2, interacted constitutively with both kinds of receptors. Expression of a dominant negative TC21 mutant in T cells produced a rapid decrease in cell viability, and Rras2−/− mice were lymphopenic, possibly as a result of diminished homeostatic proliferation and impaired T cell and B cell survival. In contrast, TC21 was overexpressed in several human lymphoid malignancies. Finally, the p110δ catalytic subunit of phosphatidylinositol-3-OH kinase (PI(3)K) was recruited to the TCR and BCR in a TC21-dependent way. Consequently, we propose TC21 directly links antigen receptors to PI(3)K-mediated survival pathways.

Amino acid transporter SNAT5 localizes to glial cells in the rat brain

The SNAT5 transporter is a neutral amino acid carrier whose function remains unclear. Structural and mechanistically, SNAT5 is closely related to the SNAT3 transporter that mediates the efflux of glutamine from glial cells and that participates in the glutamate‐glutamine cycle in the brain. In this study, we have analyzed the distribution of SNAT5 in the rat central nervous system using specific antibodies. Through immunoblotting we observed that SNAT5 is ubiquitously but unevenly distributed in the CNS. It accumulates most intensely in the neocortex, the hippocampus, the striatum, and the spinal cord, whereas moderate levels were found in the thalamus, hypothalamus, and brainstem. Light microscopy revealed that the distribution of SNAT5 paralleled that of the vesicular glutamate transporter vGLUT1 in the forebrain regions, whereas in the diencephalon and brainstem, SNAT5 staining was better correlated with that of vGLUT1 and vGLUT2. However, the cellular localization differed from that of the glutamatergic markers, since SNAT5 was expressed exclusively in astrocyte cell bodies and their processes, ensheathing glutamatergic GABAergic and glycinergic terminals. The presence of SNAT5 in astrocyte processes was confirmed by electron microscopy. They were seen not only to surround different neuronal structures, but they were also found in astrocyte endfeet. Taking into consideration the higher levels of SNAT5 in the neighborhood of glutamatergic terminals and the ability of this transporter family to promote the efflux of amino acids from intracellular stores (including glutamine and perhaps glycine), this transporter is likely to be involved in glutamatergic pathways in the brain.

Immunohistochemical localization of the amino acid transporter SNAT2 in the rat brain

SNAT2 is a neutral amino acid carrier that belongs to the system A family. Since its function in the nervous system remains unclear, we have analyzed its distribution in the rat CNS using specific antisera. Although SNAT2 is expressed widely in the CNS, it is enriched in the spinal cord and the brainstem nuclei, especially those of the auditory system. At the cellular level, SNAT2 was preferentially located in neuronal cell bodies and processes, although it was also strongly expressed in the meninges and ependyma. In astrocytes, the localization of SNAT2 was more restricted since it was intensely expressed in the perivascular end-feet, glia limitans, cerebellar astrocytes and Bergmann glia, but it was less intense in astrocytes of the cerebral parenchyma. Among neurons, the primary sensory neurons of the mesencephalic trigeminal nucleus appeared to be those that most strongly express SNAT2, but many other neurons, including cortical pyramidal cells and their dendrites were also intensely stained. In several regions the transporter was detected in axons, especially in the brainstem, and its presence in both dendrites and axons was confirmed by confocal microscopy and ultrastructural studies. However, while SNAT2 was observed in the large principal dendrites and the small distal dendrites, it was only found in axonal shafts and was excluded from terminals. Some glutamatergic neurons were among the more intensely labeled cells whereas SNAT2 was not detected on GABAergic neurons. The expression of SNAT2 partially coincides with that reported for SNAT1, especially in glutamatergic neurons. Hence, both proteins could fulfill complementary roles in replenishing glutamate pools and be differentially regulated under different physiological conditions. They also seem to co-localize in non-neuronal cells probably contributing to amino acid fluxes through the blood-brain barrier.

The scaffolding protein PSD‐95 interacts with the glycine transporter GLYT1 and impairs its internalization

Recent evidence indicates that the glycine transporter‐1 (GLYT1) plays a role in regulation of NMDA receptor function through tight control of glycine concentration in its surrounding medium. Immunohistochemical studies have demonstrated that, as well as being found in glial cells, GLYT1 is also associated with the pre‐ and postsynaptic aspects of glutamatergic synapses. In this article, we describe the interaction between GLYT1 and PSD‐95 in the rat brain, PSD‐95 being a scaffolding protein that participates in the organization of glutamatergic synapses. Mutational analysis reveals that the C‐terminal sequence of GLYT1 (–SRI) is necessary for the transporter to interact with the PDZ domains I and II of PSD‐95. This C‐terminal tripeptide motif also seems to be involved in the trafficking of GLYT1 to the membrane, although this process does not involve PDZ proteins. GLYT1 is able to recruit PSD‐95 to the plasma membrane, but it does not affect its clustering. However, the interaction stabilizes this transporter at the plasma membrane, blocking its internalization and producing a significant increase in the Vmax of glycine uptake. We hypothesize that PSD‐95 might act as a scaffold for GLYT1 and NMDA receptors, allowing GLYT1 to regulate the concentrations of glycine in the micro‐environment of NMDA receptors.

Cux‐1 and Cux‐2 control the development of Reelin expressing cortical interneurons

Homeodomain transcription factors play important roles in the specification and differentiation of neuronal subpopulations. In the cerebral cortex, the expression patterns of Cux‐1 and Cux‐2 in the medial ganglionic eminence (MGE) suggest a role for these transcription factors in the development of interneurons, a heterogeneous neuronal population. In this report, we describe expression of Cux‐1 and Cux‐2 proteins in Reelin‐secreting interneurons of the cortical plate, but not in calretinin or parvalbumin subpopulations. The role of Cux genes in the development of Reelin positive neurons was studied using Cux‐1 and Cux‐2 knockout mice. These experiments demonstrate that Cux‐1−/−; Cux‐2−/− double mutation is embryonically lethal. Although this phenotype is highly penetrant, a small proportion of mice develop to birth (P0). Analysis of these animals demonstrate that expression of Reelin is completely absent in layers II–IV of Cux‐1−/−; Cux‐2−/− double mutant mice, but it is not affected in the cortex of Cux‐1−/− or Cux‐2−/− single mutants. No Cux‐1−/−; Cux‐2−/− double‐mutant were collected after P0. Since, GABA‐ergic populations mature at late postnatal stages, this did not allow us to analyze the expression of subclass specific markers and define the affected interneuron subpopulations. Our analysis of Cux‐1−/−; Cux‐2−/− double mutant thus demonstrates essential yet redundant roles for Cux‐1 and Cux‐2 in specifying Reelin expressing cortical interneurons.

CXCR4/CXCR7 Molecular Involvement in Neuronal and Neural Progenitor Migration: Focus in CNS Repair

In the adult brain, neural progenitor cells (NPCs) reside in the subventricular zone (SVZ) of the lateral ventricles, the dentate gyrus and the olfactory bulb. Following CNS insult, NPCs from the SVZ can migrate along the rostral migratory stream (RMS), a migration of NPCs that is directed by proinflammatory cytokines. Cells expressing CXCR4 follow a homing signal that ultimately leads to neuronal integration and CNS repair, although such molecules can also promote NPC quiescence. The ligand, SDF1 alpha (or CXCL12) is one of the chemokines secreted at sites of injury that it is known to attract NSC‐derived neuroblasts, cells that express CXCR4. In function of its concentration, CXCL12 can induce different responses, promoting NPC migration at low concentrations while favoring cell adhesion via EGF and the alpha 6 integrin at high CXCL12 concentrations. However, the preclinical effectiveness of chemokines and their relationship with NPC mobilization requires further study, particularly with respect to CNS repair. NPC migration may also be affected by the release of cytokines or chemokines induced by local inflammation, through autocrine or paracrine mechanisms, as well as through erythropoietin (EPO) or nitric oxide (NO) release. CXCL12 activity requires G‐coupled proteins and the availability of its ligand may be modulated by its binding to CXCR7, for which it shows a stronger affinity than for CXCR4. J. Cell. Physiol. 230: 27–42, 2015.

The glutamate transporter GLT1b interacts with the scaffold protein PSD-95

The glutamate transporter (GLT1) regulates glutamate concentrations in glutamatergic synapses and it is expressed in at least two isoforms, GLT1a and GLT1b. In this work, we show that the C‐terminus of GLT1b is able to interact with the PDZ domains of a number of proteins. Notably, one of them might be the scaffold protein post‐synaptic density (PSD‐95). GLT1b formed co‐immunoprecipitable complexes with PSD‐95 in solubilizated rat brain extracts, complexes that also contained NMDA receptors. Co‐transfection of GLT1b, PSD‐95, and NMDA receptor subunits in heterologous expression systems recapitulated in vitro the interactions among these proteins that had been observed in the rat brain extracts and revealed the importance of the GLT1b C‐terminal PDZ binding motif in tethering this transporter to PSD‐95. Significantly, co‐expression of GLT1b and PSD‐95 increased the Vmax of the transporter by decreasing the rate of GLT1b endocytosis. Moreover, GLT1b transfected into primary cultured neurons or glia formed protein clusters that co‐localized with co‐transfected PSD‐95, clusters that in these neurons accumulated preferentially in dendritic spines. We hypothesize that the GLT1b/PSD‐95 interaction, characterized here in vitro, might anchor this transporter close to the post‐synaptic glutamate receptors, thereby permitting the fine regulation of glutamate concentrations in this microenvironment. This tight association might also facilitate the regulation of GLT1b through the signaling pathways initiated by the activation of glutamate receptors.

Mechanisms of endoplasmic-reticulum export of glycine transporter-1 (GLYT1)

The GLYT1 (glycine transporter-1) regulates both glycinergic and glutamatergic neurotransmission by controlling the reuptake of glycine at synapses. Trafficking to the cell surface of GLYT1 is critical for its function. In the present paper, by using mutational analysis of the GLYT1 C-terminal domain, we identified the evolutionarily conserved motif R(575)L(576)(X(8))D(585) as being necessary for ER (endoplasmic reticulum) export. This is probably due to its capacity to bind Sec24D, a component of the COPII (coatomer coat protein II) complex. This ER export motif was active when introduced into the related GLYT2 transporter but not in the unrelated VSVG (vesicular-stomatitis virus glycoprotein)-GLYT1 protein in which this motif was mutated but was not transported to the plasma membrane, although this effect was rescued by co-expressing these mutants with wild-type GLYT1. This behaviour suggests that GLYT1 might form oligomers along the trafficking pathway. Cross-linking assays performed in rat brain synaptosomes and FRET (fluorescence resonance energy transfer) microscopy in living cells confirmed the existence of GLYT1 oligomers. In summary, we have identified a motif involved in the ER exit of GLYT1 and, in analysing the influence of this motif, we have found evidence that oligomerization is important for the trafficking of GLYT1 to the cell surface. Because this motif is conserved in the NSS (sodium- and chloride-dependent neurotransmitter transporter) family, it is possible that this finding could be extrapolated to other related transporters.

The glycine transporter GLYT1 interacts with Sec3, a component of the exocyst complex

Evidence is accumulating that the glycine transporter GLYT1 regulates NMDA receptor function by modulating the glycine concentration in glutamatergic synapses. In this article, we describe a physical and functional interaction between GLYT1 and the exocyst complex. Through a yeast two-hybrid screen to search for proteins capable of interacting with the intracellular C-terminal tail of GLYT1, we identified a protein that is highly homologous to the human and mouse Sec3 protein, a component of the exocyst complex. Pull-down and immunoprecipitation assays confirmed the physical interaction between the C-terminus of GLYT1 and Sec3. Subsequently, immunofluorescence experiments indicated that Sec3-GFP was partially recruited to the plasma membrane upon coexpression with GLYT1. The interaction of GLYT1 with exocyst components was also observed in the native rat brain since complexes immunoprecipitated from brain extracts with anti-GLYT1 antibodies contained both Sec6 and Sec8. Functional assays revealed that Sec3 increased the transporter capacity of GLYT1, suggesting that the exocyst favors insertion of GLYT1 into the plasma membrane.