Morphological and functional effects of altered cysteine string protein at the Drosophila larval neuromuscular junction

The synaptic vesicle-associated cysteine string protein (CSP) is critical for neurotransmitter release at the neuromuscular junction (NMJ) of Drosophila, where the approximately 4% of mutant flies lacking CSP that survive to adulthood exhibit spastic jumping and shaking, temperature-sensitive paralysis, and premature death. Previously, it has been shown that CSP is also required for nerve terminal growth and the prevention of neurodegeneration in Drosophila and mice. At larval csp null mutant NMJs of Drosophila, intracellular recordings from the muscle showed that evoked release is significantly reduced at room temperature. However, it remained unclear whether the reduction in evoked release might be due to a loss of synaptic boutons, loss of synapses, and alterations in trafficking of vesicles to synapses. To resolve these issues, we have examined synaptic structure and function of csp null mutant NMJs at the level of single boutons. csp null mutations proportionally reduce the number of synaptic boutons of both motor neurons (1s and 1b) innervating larval muscles 6 and 7, while the number of synapses per bouton remains normal. However, focal recordings from individual synaptic boutons show that nerve-evoked neurotransmitter release is also impaired in both 1s and 1b boutons. Further, our ultrastructural analyses show that the reduction in evoked release at low stimulation frequencies is not due to a loss of synapses or to alterations in docked vesicles at synapses. Together, these data suggest that CSP promotes synaptic growth and evoked neurotransmitter release by mechanistically independent signaling pathways.     

Individual differences in response to novelty predict prefrontal cortex dopamine transporter function and cell surface expression

Previous research has shown that individual differences in response to novelty predict self-administration and the locomotor response to psychostimulant drugs of abuse. The aim of the present study was to determine if individual differences in response to novelty based on inescapable or free-choice novelty tests predict dopamine transporter (DAT) function and trafficking as well as nicotine-induced modulation of DAT. Results show that the maximal velocity (Vmax) of [3H]dopamine uptake into prefrontal cortex (PFC) synaptosomes correlated negatively with the locomotor response to inescapable novelty. In contrast, Vmax correlated positively with novelty place preference in the free-choice novelty test. The divergent correlations between DAT and the two behavioral phenotypes suggest a differential contribution of DAT in these phenotypes, which are known not to be isomorphic. Furthermore, rats categorized as high responders to inescapable novelty had lower Vmax values, which were accompanied by less DAT expression at the cell surface in PFC compared with low responders, suggesting that inherent individual differences in DAT cellular localization may underlie the differential response to novelty. Compared with the saline control, nicotine increased Vmax and cell surface DAT immunoreactivity in PFC from high responders but not from low responders. Similarly, nicotine increased Vmax and cell surface DAT in PFC in rats classified as low in novelty place preference but not in rats classified as high in novelty place preference. Thus, despite the different behavioral phenotypes, the pharmacological effect of nicotine to increase DAT function and cell surface expression was apparent, such that rats with inherently lower DAT function show a greater sensitivity to the neurochemical effect of nicotine.     

A specific role of AGS3 in the surface expression of plasma membrane proteins

Activator of G protein signaling 3 (AGS3), originally identified in a functional screen for mammalian proteins that activate heterotrimeric G protein signaling, is known to be involved in drug-seeking behavior and is up-regulated during cocaine withdrawal in animal models. These observations indicate a potential role for AGS3 in the formation or maintenance of neural plasticity. We have found that the overexpression of AGS3 alters the surface-to-total ratios of a subset of heterologously expressed plasma membrane receptors and channels. Further analysis of the endocytic trafficking of one such protein by a biotin-based internalization assay suggests that overexpression of AGS3 moderately affects the internalization or recycling of surface proteins. Moreover, AGS3 overexpression and siRNA-mediated knockdown of AGS3 both result in the dispersal of two endogenously expressed trans-Golgi network (TGN)-associated cargo proteins without influencing those in the cis- or medial-Golgi compartments. Finally, adding a TGN-localization signal to a CD4-derived reporter renders the trafficking of fusion protein sensitive to AGS3. Taken together, our data support a model wherein AGS3 modulates the protein trafficking along the TGN/plasma membrane/endosome loop.     

Pathomechanism of leukoaraiosis

Ischemic demyelination in the white matter of the brain is a frequent clinical entity. In neuroimaging terms, it is referred to as leukoaraiosis (LA). LA can reflect a broad public health problem, which is caused by a cognitive impairment ranging from mild slowness of thinking to full-blown subcortical dementia. One-quarter of subjects aged 65 yr or over are affected by some degree of white matter changes. There are a number of genetic factors that can be associated with circulatory disturbances of the white matter of the brain. A slight chronic hypoperfusion or an endothelial dysfunction associated with unfavorable genetic variations such as methylenete-trahydrofolate reductase C677T variation and angiotensin-converting enzyme I/D polymorphism then may lead indirectly to a malfunction of the molecular cross-talk between the nucleus and the mitochondria. This results in a decrease in the production of energy in the glia cells and thereby the beginning of demyelination. From another aspect, the presence of either the apolipoprotein E2 or 4 alleles may cause an increased vulnerability to a slight chronic hypoperfusion of the white matter by reducing the range of mechanical and chemical flexibility of the glial cytoskeleton. In consequence of the chronic hypoperfusion, the functionally damaged kinesin protein gives rise also to the disturbances of the trafficking of the myelin basic protein mRNAs in the oligoden-drocytes. On the basis of the current knowledge on LA, this article suggests a hypothetical molecular bridge between the genetic, biochemical, and clinical processes.     

Molecular determinants of kainate receptor trafficking

Glutamate receptors of the kainate subtype are ionotropic receptors that play a key role in the modulation of neuronal network activity. The role of kainate receptors depends on their precise membrane and subcellular localization in presynaptic, extrasynaptic and postsynaptic domains. These receptors are composed of the combination of five subunits, three of them having several splice variants. The subunits and splice variants show great divergence in their C-terminal cytoplasmic tail domains, which have been implicated in intracellular trafficking of homomeric and heteromeric receptors. Differential trafficking of kainate receptors to specific neuronal compartments likely relies on interactions between the different kainate receptor subunits with distinct subsets of protein partners that interact with C-terminal domains. These C-terminal domains have also been implicated in the degradation of kainate receptors. Finally, the phosphorylation of the C-terminal domain regulates receptor trafficking and function. This review summarizes our knowledge on the regulation of membrane delivery and trafficking of kainate receptors implicating C-terminal domains of the different isoforms and focuses on the identification and characterization of the function of interacting partners.     

Protein kinase C–dependent trafficking of glutamate transporters excitatory amino acid carrier 1 and glutamate transporter 1b in cultured cerebellar granule cells

Previous data showed that cell surface expression of the glutamate transporters GLT1a and excitatory amino acid carrier 1 (EAAC1), localized in glia and neurons of the CNS, can be regulated by protein kinase C (PKC). Regulation and physiological importance of GLT1b, a splice variant of GLT1a, is not understood. In the present study we used cultured cerebellar granule cells (CGCs) from mice to investigate PKC dependent trafficking of GLT1b in comparison to GLT1a and EAAC1 using immunohistochemistry and subcellular fractionation followed by Western blotting. In neurites of CGCs, GLT1b and EAAC1 were localized to different aggregates of vesicles that were different from vesicle aggregates containing vesicular glutamate transporters. In CGCs cultured with low-potassium medium, stimulation of PKC by phorbol ester enhanced the formation of large varicosities in neurites that exhibited immunoreactivity for GLT1a, GLT1b, and EAAC1. Stimulation of PKC leads to a significant increase of GLT1b and EAAC1 in the plasma membrane whereas GLT1a in the plasma membrane was decreased. Following PKC stimulation, also a significant increase of transporter-mediated glutamate uptake representing sodium dependent glutamate uptake, was observed. Similarly, the fraction of glutamate uptake, that was sensitive to the inhibitor WAY-213613 and represents uptake by GLT1a and GLT1b, was increased after stimulation by PKC. The findings suggest that PKC is similarly involved in regulation of surface trafficking of GLT1b and EAAC1 and that PKC stimulated increase in surface location of GLT1b and EAAC1 in glutamatergic CGCs.     

PSGL-1 function in immunity and steady state homeostasis

Summary: The substantial importance of P-selectin glycoprotein ligand 1 (PSGL-1) in leukocyte trafficking has continued to emerge beyond its initial identification as a selectin ligand. PSGL-1 seemed to be a relatively simple molecule with an extracellular mucin domain extended as a flexible rod, teleologically consistent with its primary role in tethering leukocytes to endothelial selectins. The rolling interaction between leukocyte and endothelium mediated by this selectin-PSGL-1 interaction requires branched O-glycan extensions on specific PSGL-1 amino acid residues. In some cells, such as neutrophils, the glycosyltransferases involved in formation of the O-glycans are constitutively expressed, while in other cells, such as T cells, they are expressed only after appropriate activation. Thus, PSGL-1 supports leukocyte recruitment in both innate and adaptive arms of the immune response. A complex array of amino acids within the selectins engage multiple sugar residues of the branched O-glycans on PSGL-1 and provide the molecular interactions responsible for the velcro-like catch bonds that support leukocyte rolling. Such binding of PSGL-1 can also induce signaling events that influence cell phenotype and function. Scrutiny of PSGL-1 has revealed a better understanding of how it performs as a selectin ligand and yielded unexpected insights that extend its scope from supporting leukocyte rolling in inflammatory settings to homeostasis including stem cell homing to the thymus and mature T-cell homing to secondary lymphoid organs. PSGL-1 has been found to bind homeostatic chemokines CCL19 and CCL21 and to support the chemotactic response to these chemokines. Surprisingly, the O-glycan modifications of PSGL-1 that support rolling mediated by selectins in inflammatory conditions interfere with PSGL-1 binding to homeostatic chemokines and thereby limit responsiveness to the chemotactic cues used in steady state T-cell traffic. The multi-level influence of PSGL-1 on cell traffic in both inflammatory and steady state settings is therefore substantially determined by the orchestrated addition of O-glycans. However, central as specific O-glycosylation is to PSGL-1 function, in vivo regulation of PSGL-1 glycosylation in T cells remains poorly understood. It is our purpose herein to review what is known, and not known, of PSGL-1 glycosylation and to update understanding of PSGL-1 functional scope.     

Modulation of dendritic spine development and plasticity by BDNF and vesicular trafficking: fundamental roles in neurodevelopmental disorders associated with mental retardation and autism

The process of axonal and dendritic development establishes the synaptic circuitry of the central nervous system (CNS) and is the result of interactions between intrinsic molecular factors and the external environment. One growth factor that has a compelling function in neuronal development is the neurotrophin brain-derived neurotrophic factor (BDNF). BDNF participates in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development. Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Several genes that are either mutated or deregulated in neurodevelopmental disorders associated with mental retardation have now been identified, and several mouse models of these disorders have been generated and characterized. Interestingly, abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, and in mouse models of these disorders as well. Abnormalities in dendritic and synaptic differentiation are thought to underlie altered synaptic function and network connectivity, thus contributing to the clinical outcome. Here, we review the roles of BDNF and vesicular trafficking in axonal and dendritic differentiation in the context of dendritic and axonal morphological impairments commonly observed in neurodevelopmental disorders associated with mental retardation.     

Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3ζ in the endosomal compartment

Phosphorylation of the T-cell receptor complex (TcR/CD3) mediates the survival and antigen-induced activation of T cells. TcR/CD3 phosphorylation is usually monitored using phospho-specific antibodies, which precludes dynamic measurements. Here, we have developed genetically encoded, live-cell reporters that enable simultaneous monitoring of the phosphorylation state and intracellular trafficking of CD3ζ, the major signal-transducing subunit of the TcR/CD3. We show that these reporters provide accurate readouts of TcR/CD3 phosphorylation and are sensitive to the local balance of kinase and phosphatase activities acting upon TcR/CD3. Using these reporters, we demonstrate that, in addition to the expected activation-dependent phosphorylation at the plasma membrane, tyrosine-phosphorylated CD3ζ accumulates on endosomal vesicles distinct from lysosomes. These results suggest that an intracellular pool of phosphorylated CD3ζ may help to sustain TcR/CD3 signaling after the receptor internalization.     

Genetic reduction of striatal-enriched tyrosine phosphatase (STEP) reverses cognitive and cellular deficits in an Alzheimer's disease mouse model

Alzheimer's disease (AD) is a progressive and incurable neurodegenerative disorder. Early in the pathophysiology of AD, synaptic function is disrupted by soluble Aβ oligomers, possibly through Aβ-mediated internalization of NMDA receptors. Striatal-enriched phosphatase (STEP) is a tyrosine phosphatase that regulates the internalization of NMDA receptors. Recent work shows that STEP is elevated in the prefrontal cortex of human AD patients and in animal models of AD. Here, we use genetic manipulations to reduce STEP activity in a triple transgenic AD mouse model and show that a decrease in STEP levels reverses cognitive and cellular deficits observed in these mice. Our results suggest that STEP inhibitors may prove therapeutic for this devastating disorder.