Cooperative dimerization of the POU domain protein Brn-2 on a new motif activates the neuronal promoter of the human aromatic L-amino acid decarboxylase gene

The neuronal promoter of the human aromatic L-amino acid decarboxylase (AADC) gene contains a perfectly palindromic element (TB) that conforms to the structure of a POU domain protein binding site of the MORE+2 type. The TB motif (located at nts -900/-872 relative to the neuronal cap site) bears striking similarities with the dimeric Pit-1 binding site from growth hormone gene promoter (GH-1), and it enhanced the activity of the minimal tk promoter in transfected SK-N-BE neuroblastoma cells. In transfected COS-7 cells, the expression of a 3xTB-tk-luc was stimulated up to 11-fold by the overexpressed Brn-2 protein. In AADC gene neuronal promoter, we previously characterized a bipartite regulatory element (ONF for octamer-like/NF-Y, nts -86/-57) that binds Brn-2 and NF-Y proteins in a cooperative manner. We now show that both TB and ONF sites participate in the activation of the neuronal promoter by Brn-2. EMSA experiments showed that the recombinant Brn-2 POU domain dimerized on the TB element in a cooperative manner. By site directed mutagenesis of the POU domain of Brn-2, the dimerization interface on the TB element was localized to the hydrophobic pocket of the POU specific domain and the C-terminal part of the POU homeodomain.     

Gliopodia extend the range of direct glia-neuron communication during the CNS development in Drosophila

Midline glia are a source of cues for neuronal navigation and differentiation in the Drosophila CNS. Despite their importance, how glia and neurons communicate during the development is not fully understood. Here, we examined dynamic morphology of midline glia and assessed their direct cellular interactions with neurons within the embryonic CNS. Midline glia extend filopodia-like "gliopodia" from the onset of axogenesis through the near completion of embryonic neural development. The most abundant and stable within the commissures, gliopodia frequently contact neurites extending from the neuropil on either side of the midline. Misexpression of Rac1N17 in midline glia not only reduces the number of gliopodia but also shifts the position of neuropils towards the midline. Midline-secreted signaling protein Slit accumulates along the surface of gliopodia. Mutant analysis supports the idea that gliopodia contribute to its presentation on neuronal surfaces at both the commissures and neuropils. We propose that gliopodia extend the range of direct glia-neuron communication during CNS development.     

The actin-binding protein Canoe/AF-6 forms a complex with Robo and is required for Slit-Robo signaling during axon pathfinding at the CNS midline

Axon guidance is a key process during nervous system development and regeneration. One of the best established paradigms to study the mechanisms underlying this process is the axon decision of whether or not to cross the midline in the Drosophila CNS. An essential regulator of that decision is the well conserved Slit-Robo signaling pathway. Slit guidance cues act through Robo receptors to repel axons from the midline. Despite good progress in our knowledge about these proteins, the intracellular mechanisms associated with Robo function remain poorly defined. In this work, we found that the scaffolding protein Canoe (Cno), the Drosophila orthologue of AF-6/Afadin, is essential for Slit-Robo signaling. Cno is expressed along longitudinal axonal pioneer tracts, and longitudinal Robo/Fasciclin2-positive axons aberrantly cross the midline in cno mutant embryos. cno mutant primary neurons show a significant reduction of Robo localized in growth cone filopodia and Cno forms a complex with Robo in vivo. Moreover, the commissureless (comm) phenotype (i.e., lack of commissures due to constitutive surface presentation of Robo in all neurons) is suppressed in comm, cno double-mutant embryos. Specific genetic interactions between cno, slit, robo, and genes encoding other components of the Robo pathway, such as Neurexin-IV, Syndecan, and Rac GTPases, further confirm that Cno functionally interacts with the Slit-Robo pathway. Our data argue that Cno is a novel regulator of the Slit-Robo signaling pathway, crucial for regulating the subcellular localization of Robo and for transducing its signaling to the actin cytoskeleton during axon guidance at the midline.     

Nestin enhancer requirements for expression in normal and injured adult CNS

The nestin gene is expressed in many CNS stem/progenitor cells, both in the embryo and the adult, and nestin is used commonly as a marker for these cells. In this report we analyze nestin enhancer requirements in the adult CNS, using transgenic mice carrying reporter genes linked to three different nestin enhancer constructs: the genomic rat nestin gene and 5 kb of upstream nestin sequence (NesPlacZ/3), 636 bp of the rat nestin second intron (E/nestin:EGFP), and a corresponding 714 bp region from the human second intron (Nes714tk/lacZ). NesPlacZ/3 and E/nestin:EGFP mice showed reporter gene expression in stem cell-containing regions of brain and spinal cord during normal conditions. NesPlacZ/3 and E/nestin:EGFP mice showed increased expression in spinal cord after injury and NesPlacZ/3 mice displayed elevated expression in the periventricular area of the brain after injury, which was not the case for the E/nestin:EGFP mice. In contrast, no expression in adult CNS in vivo was seen in the Nes714tk/lacZ mice carrying the human enhancer, neither during normal conditions nor after injury. The Nes714 tk/lacZ mice, however, expressed the reporter gene in reactive astrocytes and CNS stem cells cultured ex vivo. Collectively, this suggests a species difference for the nestin enhancer function in adult CNS and that elements outside the second intron enhancer are required for the full injury response in vivo.     

Nogos and the Nogo-66 receptor: factors inhibiting CNS neuron regeneration

The recently cloned gene Nogo, whose alternative splice products correspond to the antigenic target of the central nervous system (CNS) regeneration enhancing monoclonal antibody IN-1, codes for membrane proteins enriched in brain, particularly in oligodendrocytes. The 66-amino acid extracellular domain of Nogo (Nogo-66) interacts with a high-affinity receptor (NgR), a glycosylphosphatidylinositol (GPI)-linked protein with multiple leucine-rich repeats. The amino terminal cytoplasmic domain of Nogo appears to have a general cellular growth inhibitory effect. Nogo-66, on the other hand, specifically retards neurite outgrowth and induces growth cone collapse, possibly through its interaction with NgR and as yet unidentified transmembrane coreceptors. Recent results also suggest that Nogo expression may induce apoptosis in tumor cells. Together, these proteins provide new molecular handles for the design of therapeutic interventions for CNS injuries and neurodegenerative diseases, as well as possible leads to anticancer strategies.     

A novel scaffold protein, TANC, possibly a rat homolog of Drosophila rolling pebbles (rols), forms a multiprotein complex with various postsynaptic density proteins

We cloned from the rat brain a novel gene, tanc (GenBank Accession No. AB098072), which encoded a protein containing three tetratricopeptide repeats (TPRs), ten ankyrin repeats and a coiled-coil region, and is possibly a rat homolog of Drosophila rolling pebbles (rols). The tanc gene was expressed widely in the adult rat brain. Subcellular distribution, immunohistochemical study of the brain and immunocytochemical studies of cultured neuronal cells indicated the postsynaptic localization of TANC protein of 200 kDa. Pull-down experiments showed that TANC protein bound PSD-95, SAP97, and Homer via its C-terminal PDZ-binding motif, -ESNV, and fodrin via both its ankyrin repeats and the TPRs together with the coiled-coil domain. TANC also bound the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. An immunoprecipitation study showed TANC association with various postsynaptic proteins, including guanylate kinase-associated protein (GKAP), alpha-internexin, and N-methyl-D-aspartate (NMDA)-type glutamate receptor 2B and AMPA-type glutamate receptor (GluR1) subunits. These results suggest that TANC protein may work as a postsynaptic scaffold component by forming a multiprotein complex with various postsynaptic density proteins.