The PDZ target motifs located in the C-terminal end of many receptors and ion channels mediate protein-protein interactions by binding to specific PDZ-containing proteins. These interactions are involved in the localization of surface proteins on specialized membrane domains of neuronal and epithelial cells. However, the molecular mechanism responsible for this PDZ protein-dependent polarized localization is still unclear. This study first demonstrated that the epithelial γ-aminobutyric acid (GABA) transporter (BGT-1) contains a PDZ target motif that mediates the interaction with the PDZ protein LIN-7 in Madin-Darby canine kidney (MDCK) cells, and then investigated the role of this interaction in the basolateral localization of the transporter. It was found that although the transporters from which the PDZ target motif was deleted were still targeted to the basolateral surface, they were not retained but internalized in an endosomal recycling compartment. Furthermore, an interfering BGT peptide determined the intracellular relocation of the native transporter. These data indicate that interactions with PDZ proteins determine the polarized surface localization of target proteins by means of retention and not targeting mechanisms. PDZ proteins may, therefore, act as a sort of membrane protein sorting machinery which, by recognizing retention signals (the PDZ target sequences), prevents protein internalization.

Abstract:The GLT-1 and GLAST astroglial transporters are the glutamate transporters mainly involved in maintaining physiological extracellular glutamate concentrations. Defects in neurotransmitter glutamate transport may represent an important component of glutamateinduced neurodegenerative disorders (such as amyotrophic lateral sclerosis) and CNS insults (ischemia and epilepsy). We characterized the protein expression of GLT-1 and GLAST in primary astrocyte-neuron cocultures derived from rat hippocampal tissues during neuron differentiation/maturation. GLT-1 and GLAST are expressed by morphologically distinct glial fibrillary acidic proteinpositive astrocytes, and their expression correlates with the status of neuron differentiation/maturation and activity. Up-regulation of the transporters paralleled the content of the synaptophysin synaptic vesicle marker p38, and down-regulation was a consequence of glutamateinduced neuronal death or the reduction of synaptic activity. Finally, soluble factors in neuronal-conditioned media prevented the down-regulation of the GLT-1 and GLAST proteins. Although other mechanisms may participate in regulating GLT-1 and GLAST in the CNS, our data indicate that soluble factors dependent on neuronal activity play a major regulating role in hippocampal cocultures.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the selective loss of lower and upper motoneurons. The pathology is imputable in ∼2% of cases to mutations in the ubiquitous enzyme Cu, Zn superoxide dismutase (SOD1). Common theories to explain the pathogenic mechanisms of ALS include activation of microglia, responsible for the release of proinflammatory factors. However, how mutant SOD1 affects microglial activation and subsequently injures neurons is still unclear. Considering that extracellular ATP, through purinergic P2 receptors, constitutes a well recognized neuron-to-microglia alarm signal, the aim of this study was to investigate how the expression of mutant SOD1 affects P2 receptor-mediated proinflammatory microglial properties. We used primary and immortalized microglial cells from mutant SOD1 mice to explore several aspects of activation by purinergic ligands and to analyze the overall effect of such stimulation on the viability of NSC-34 and SH-SY5Y neuronal cell lines. We observed up-regulation of P2X4, P2X7, and P2Y6 receptors and down-regulation of ATP-hydrolyzing activities in mutant SOD1 microglia. This potentiation of the purinergic machinery reflected into enhanced sensitivity mainly to 2′-3′-O-(benzoyl-benzoyl) ATP, a P2X7 receptor preferential agonist, and translated into deeper morphological changes, enhancement of TNF-α and cyclooxygenase-2 content, and finally into toxic effects exerted on neuronal cell lines by microglia expressing mutant SOD1. All these parameters were prevented by the antagonist Brilliant Blue G. The purinergic activation of microglia may thus constitute a new route involved in the progression of ALS to be exploited to potentially halt the disease.

The heterotrimeric PDZ complex containing LIN-2, LIN-7 and LIN-10 is known to be involved in the organization of epithelial and neuronal junctions in Caenorhabditis elegans and mammals. We report here that mammalian LIN-7 PDZ proteins form a complex with cadherin and b-catenin in epithelia and neurons. The association of LIN-7 with cadherin and b-catenin is Ca 2+ dependent and is mediated by the direct binding of LIN-7 to the C-terminal PDZ target sequence of b-catenin, as demonstrated by means of co-immunoprecipitation experiments and in vitro binding assays with the recombinant glutathione S-transferase:LIN-7A. The presence of b-catenin at the junction is required in order to relocate LIN-7 from the cytosol to cadherin-mediated adhesions, thus indicating that LIN-7 junctional recruitment is b-catenin dependent and that one functional role of the binding is to localize LIN-7. Moreover, when LIN-7 is present at the b-catenin-containing junctions, it determines the accumulation of binding partners, thus suggesting the mechanism by which b-catenin mediates the organization of the junctional domain.

The distributions of isoforms of the Na,KATPase a subunit were determined in mature cultured hippocampal neurons and in a polarized epithelial cefl line. We find that hippocampal neurons express the al and a3 isoforms in the membranes of both axons and dendrites. In contrast the al and a3 proteins are exclusively basolateral when expressed endogenously or by stable transfection in renal epithelial cells. These data suggest that epithelial cells and hippocampal neurons localize these proteins by different mechanisms. These observations contrast with those made for the vesicular stomatitis virus and the influenza glycoproteins, which are polarized in both epithelial and neuronal cells. Na,K-ATPase is an integral membrane protein complex responsible for establishing the electrochemical gradients of Na+ and K+ ions across the plasma membranes of mammalian cells. The enzyme is composed of two subunits, a and P, both of which exist in multiple isoforms (1). The a polypeptide is thought to be the catalytically active subunit, and the (3 polypeptide seems to be necessary for the assembly and transport of the sodium pump to the plasma membrane (2). Three a-subunit isoforms (al, a2, and a3), encoded by three genes, have been cloned from the rat (3). The three a isoforms manifest ""90% identity at the amino acid level and exhibit a tissue-specific and developmentally regulated pattern of expression. Polarized epithelial cells express the al isoform and, in most native and cultured epithelial cells, the enzyme is localized exclusively to the basolateral membrane domain (4, 5). In brain, al and a2 are expressed both in neuronal and in nonneuronal cells, whereas a3 is the neuron-specific isoform (6).Although epithelial cells and neurons differ markedly in function, both cell types share the need to polarize their surface membranes. Recent studies that made use of enveloped RNA viruses to examine sorting phenomena in hippocampal neurons in culture (7) have suggested that epithelial cells and neurons may share common sorting mechanisms for membrane proteins. These studies revealed that hippocampal neurons infected with influenza virus target the hemagglutinin glycoprotein to the axons, whereas vesicular stomatitis virus (VSV)-infected neurons sort the VSV glycoprotein to their dendrites. Moreover, Thy-i, aglycosylphosphatidylinositol-linked protein endogenously expressed in hippocampal neurons, is exclusively localized on the axonal surface (8). It is known that glycosylphosphatidylinositol-linked proteins are sorted to the apical surface ofpolarized epithelial cells (9). These results suggest that the neuronal axon is the counterpart ofthe epithelial apical domain whereas the dendrite is the counterpart of the basolateral membrane.In light of the basolateral localization of al in epithelia, we were interested to determine whether this protein is restricted in its distribution to the dendrites of polarized hippocampal neurons. Support for this hypothesis can be found in a recent immunocytochemical analysis of rat hippoc...