Grazia Pietrini

PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells

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.

Mammalian LIN‐7 PDZ proteins associate with β‐catenin at the cell–cell junctions of epithelia and neurons

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 β‐catenin in epithelia and neurons. The association of LIN‐7 with cadherin and β‐catenin is Ca2+ dependent and is mediated by the direct binding of LIN‐7 to the C‐terminal PDZ target sequence of β‐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 β‐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 β‐catenin dependent and that one functional role of the binding is to localize LIN‐7. Moreover, when LIN‐7 is present at the β‐catenin‐containing junctions, it determines the accumulation of binding partners, thus suggesting the mechanism by which β‐catenin mediates the organization of the junctional domain.

The Proinflammatory Action of Microglial P2 Receptors Is Enhanced in SOD1 Models for Amyotrophic Lateral Sclerosis

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.

Sorting of two polytopic proteins, the gamma-aminobutyric acid and betaine transporters, in polarized epithelial cells

The γ-aminobutyric acid transporter (GAT-1) isoform of the γ-aminobutyric acid and the betaine (BGT) transporters exhibit distinct apical and basolateral distributions when introduced into Madin-Darby canine kidney cells (Pietrini, G., Suh, Y. J., Edelman, L., Rudnick, G., and Caplan, M. J. (1994) J. Biol. Chem. 269, 4668-4674). We have investigated the presence of sorting signals in their COOH-terminal cytosolic domains by expression in Madin-Darby canine kidney cells of mutated and chimeric transporters. Whereas truncated GAT-1 (ΔC-GAT) maintained the original functional activity and apical localization, either the removal (ΔC-myc BGT) or the substitution (BGS chimera) of the cytosolic tail of BGT generated proteins that accumulated in the endoplasmic reticulum. Moreover, we have found that the cytosolic tail of BGT redirected apical proteins, the polytopic GAT-1 (GBS chimera) and the monotopic human nerve growth factor receptor, to the basolateral surface. These results suggest the presence of basolateral sorting information in the cytosolic tail of BGT. We have further shown that information necessary for the exit of BGT from the endoplasmic reticulum and for the basolateral localization of the GBS chimera is contained in a short segment, rich in basic residues, within the cytosolic tail of BGT.

Cell culture models to investigate the selective vulnerability of motoneuronal mitochondria to familial ALS-linked G93ASOD1

Mitochondrial damage induced by superoxide dismutase (SOD1) mutants has been proposed to have a causative role in the selective degeneration of motoneurons in amyotrophic lateral sclerosis (ALS). In order to investigate the basis of the tissue specificity of mutant SOD1 we compared the effect of the continuous expression of wild‐type or mutant (G93A) human SOD1 on mitochondrial morphology in the NSC‐34 motoneuronal‐like, the N18TG2 neuroblastoma and the non‐neuronal Madin–Darby Canine Kidney (MDCK) cell lines. Morphological alterations of mitochondria were observed in NSC‐34 expressing the G93A mutant (NSC‐G93A) but not the wild‐type SOD1, whereas a ten‐fold greater level of total expression of the mutant had no effect on mitochondria of non‐motoneuronal cell lines. Fragmented network, swelling and cristae remodelling but not vacuolization of mitochondria or other intracellular organelles were observed only in NSC‐G93A cells. The mitochondrial alterations were not explained by a preferential localization of the mutant within NSC‐G93A mitochondria, as a higher amount of the mutant SOD1 was found in mitochondria of MDCK‐G93A cells. Our results suggest that mitochondrial vulnerability of motoneurons to G93ASOD1 is recapitulated in NSC‐34 cells, and that peculiar features in network dynamics may account for the selective alterations of motoneuronal mitochondria.

Cell type-specific recruitment of Drosophila Lin-7 to distinct MAGUK-based protein complexes defines novel roles for Sdt and Dlg-S97.

Stardust (Sdt) and Discs-Large (Dlg) are membrane-associated guanylate kinases (MAGUKs) involved in the organization of supramolecular protein complexes at distinct epithelial membrane compartments in Drosophila. Loss of either Sdt or Dlg affects epithelial development with severe effects on apico-basal polarity. Moreover, Dlg is required for the structural and functional integrity of synaptic junctions. Recent biochemical and cell culture studies have revealed that various mammalian MAGUKs can interact with mLin-7/Veli/MALS, a small PDZ-domain protein. To substantiate these findings for their in vivo significance with regard to Sdt- and Dlg-based protein complexes, we analyzed the subcellular distribution of Drosophila Lin-7 (DLin-7) and performed genetic and biochemical assays to characterize its interaction with either of the two MAGUKs. In epithelia, Sdt mediates the recruitment of DLin-7 to the subapical region, while at larval neuromuscular junctions, a particular isoform of Dlg, Dlg-S97, is required for postsynaptic localization of DLin-7. Ectopic expression of Dlg-S97 in epithelia, however, was not sufficient to induce a redistribution of DLin-7. These results imply that the recruitment of DLin-7 to MAGUK-based protein complexes is defined by cell-type specific mechanisms and that DLin-7 acts downstream of Sdt in epithelia and downstream of Dlg at synapses.

Increased internalisation and degradation of GLT-1 glial glutamate transporter in a cell model for familial amyotrophic lateral sclerosis (ALS)

It has been suggested that glutamate-induced excitotoxicity plays a central role in the development of motor neuron diseases such as amyotrophic lateral sclerosis (ALS). The GLT-1 isoform of the glutamate transporter gene family is the most important transporter involved in keeping extracellular glutamate concentration below neurotoxic levels. Its loss and an increase in extracellular glutamate has been documented in cases of sporadic and familial ALS, as well as in animal models expressing ALS-linked Cu2+-Zn2+ superoxide dismutase (SOD1) mutations, but the underlying molecular mechanisms are still unclear. We developed and characterised a cell model consisting of polarised epithelial Madin-Darby Canine Kidney (MDCK) cell lines stably expressing wild-type SOD1 or the ALS-linked SOD1 G93A mutant, and analysed the expression of glutamate transporters after transient transfection of the corresponding cDNAs. Like ALS patients and animal models of ALS, the G93A-expressing MDCK cell system showed reduced total glial GLT-1 expression, with no change in the expression of the neuronal EAAC1 glutamate transporter isoform. Morphological analysis revealed the intracellular redistribution of GLT-1 to acidic compartments, whereas the surface distribution of other glutamate transporters (neuronal EAAC1 and glial GLAST) was not affected. Moreover, mutant SOD1 affected the cytosolic tail of GLT-1 because reduced protein expression of EAAC-GLT but not GLT-EAAC chimeras was found in G93A-expressing cell lines. GLT-1 downregulation was greatly induced by inhibition of protein synthesis, and prevented by treatment with chloroquine aimed at inhibiting the activity of acidic degradative compartments. Negligible effect on the protein level or distribution of GLT-1 was observed in cells overexpressing wild-type SOD1. The specific decrease in the GLT-1 isoform of glutamate transporters is therefore recapitulated in G93A-expressing MDCK cell lines, thus suggesting an autonomous cell mechanism underlying the loss of GLT-1 in ALS. Our data indicate that the continuous expression of mutant SOD1 causes the downregulation of GLT-1 by increasing the internalisation and degradation of the surface transporter, and suggest that the cytosolic tail of GLT-1 is required to target the transporter to degradation.

NADH-cytochrome b5 reductase and cytochrome b5 isoforms as models for the study of post-translational targeting to the endoplasmic reticulum

Cytochrome b 5 and NADH‐cytochrome b 5, reductase are integral membrane proteins with cytosolic active domains and short membrane anchors, which are inserted post‐translationally into their target membranes. Both are produced as different isoforms, with different localizations, in mammalian cells. In the rat, the reductase gene generates two transcripts by an alternative promoter mechanism: a ubiquitous mRNA coding for the myristylated membrane‐bound form, and an erythroid mRNA which generates both the soluble form and a nonmyristylated membrane‐binding form. The available evidence indicates that the ubiquitous myristylated form binds to the cytosolic face of both outer mitochondrial membranes and ER. In contrast, two genes code for two homologous forms of cytochrome b 5, one of which is found on outer mitochondrial membranes, the other on the ER. The gene specifying the ER form probably also generates an erythroid‐specific mRNA by alternative splicing, which codes for soluble cytochrome b 5. Possible molecular mechanisms responsible for the observed localizations of these different enzyme isoforms are discussed.

Protein Kinase C-mediated Phosphorylation of the BGT1 Epithelial γ-Aminobutyric Acid Transporter Regulates Its Association with LIN7 PDZ Proteins A POST-TRANSLATIONAL MECHANISM REGULATING TRANSPORTER SURFACE DENSITY

The Na/Cl-dependent BGT1 transporter has osmoprotective functions by importing the small osmolyte betaine into the cytosol of renal medullary epithelial cells. We have demonstrated previously that the surface localization of the transporter in Madin-Darby canine kidney cells depends on its association with the LIN7 PDZ protein through a PDZ target sequence in the last 5 residues of the transporter (-KETHL). Here we describe a protein kinase C (PKC)-mediated mechanism regulating the association between BGT1 and LIN7. Reduced transport activity paralleled by the intracellular relocalization of the transporter was observed in response to the PKC activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. This activation caused clathrin-dependent internalization of the transporter and its targeting to a recycling compartment that contains the truncated transporter lacking the LIN7 binding motif (BGTΔ5) but not the LIN7 partner of BGT1. The decreased association between BGT1 and LIN7 was demonstrated further by coimmunoprecipitation studies and in vitro binding to recombinant LIN7 fusion protein. The TPA treatment induced phosphorylation of surface BGT1 on serine and threonine residues. However, a greater increase in phosphothreonines than phosphoserines was measured in the wild type transporter, whereas the opposite was true in the BGTSer mutant in which a serine replaced the threonine 612 in the LIN7 association motif (-KESHL). No similar increase in relative phosphoserines or phosphothreonines was found in the BGTΔ5 transporter. Moreover, phosphorylation of threonine 612 in a BGT COOH-terminal peptide impaired its association with recombinant LIN7. Taken together, these data demonstrate that the post-translational regulation of BGT1 surface density is a result of transporter phosphorylation and that threonine 612 is an essential residue in this PKC-mediated regulation.

Analysis of neuromuscular junctions and effects of anabolic steroid administration in the SOD1G93A mouse model of ALS

Several lines of evidence indicate that neuromuscular junction (NMJ) destruction and disassembly is an early phenomenon in amyotrophic lateral sclerosis (ALS). Here we analyzed by confocal and electron microscopy the NMJ structure in the diaphragm of SOD1G93A mice at symptom onset. In these mice, which provide a model for familial ALS, diaphragm denervation (~50%) as well as gastrocnemius denervation (~40%) was found. In addition, the size of the synaptic vesicle pool was reduced and alterations of mitochondria were observed in approximately 40% of the remaining presynaptic terminals. Chronic treatment of SOD1G93A mice with the anabolic steroid nandrolone during the presymptomatic stage preserved the diaphragm muscle mass and features indicative of synaptic activity. These features were represented by the number of vesicles docked within 200 nm from the presynaptic membrane and area of acetylcholine receptor clusters. Structural preservation of mitochondria was documented in presynaptic terminals. However, innervation of diaphragm muscle fibers was only slightly increased in nandrolone-treated SOD1-mutant mice. Altogether the results point out and define fine structural alterations of diaphragm NMJs in the murine model of familial ALS at symptom onset, and indicate that nandrolone may prevent or delay structural alterations in NMJ mitochondria and stimulate presynaptic activity but does not prevent muscle denervation during the disease.