The prolyl isomerase Pin1 acts as a novel molecular switch for TNF-alpha-induced priming of the NADPH oxidase in human neutrophils.

Neutrophils play a key role in host defense by releasing reactive oxygen species (ROS). However, excessive ROS production by neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can damage bystander tissues, thereby contributing to inflammatory diseases. Tumor necrosis factor-α (TNF-α), a major mediator of inflammation, does not activate NADPH oxidase but induces a state of hyperresponsiveness to subsequent stimuli, an action known as priming. The molecular mechanisms by which TNF-α primes the NADPH oxidase are unknown. Here we show that Pin1, a unique cis-trans prolyl isomerase, is a previously unrecognized regulator of TNF-α-induced NADPH oxidase hyperactivation. We first showed that Pin1 is expressed in neutrophil cytosol and that its activity is markedly enhanced by TNF-α. Inhibition of Pin1 activity with juglone or with a specific peptide inhibitor abrogated TNF-α-induced priming of neutrophil ROS production induced by N-formyl-methionyl-leucyl-phenylalanine peptide (fMLF). TNF-α enhanced fMLF-induced Pin1 and p47phox translocation to the membranes and juglone inhibited this process. Pin1 binds to p47phox via phosphorylated Ser345, thereby inducing conformational changes that facilitate p47phox phosphorylation on other sites by protein kinase C. These findings indicate that Pin1 is critical for TNF-α-induced priming of NADPH oxidase and for excessive ROS production. Pin1 inhibition could potentially represent a novel anti-inflammatory strategy.

Aquaporin-4 Surface Trafficking Regulates Astrocytic Process Motility and Synaptic Activity in Health and Autoimmune Disease.

Astrocytes constantly adapt their ramified morphology in order to support brain cell assemblies. Such plasticity is partly mediated by ion and water fluxes, which rely on the water channel aquaporin-4 (AQP4). The mechanism by which this channel locally contributes to process dynamics has remained elusive. Using a combination of single-molecule and calcium imaging approaches, we here investigated in hippocampal astrocytes the dynamic distribution of the AQP4 isoforms M1 and M23. Surface AQP4-M1 formed small aggregates that contrast with the large AQP4-M23 clusters that are enriched near glutamatergic synapses. Strikingly, stabilizing surface AQP4-M23 tuned the motility of astrocyte processes and favors glutamate synapse activity. Furthermore, human autoantibodies directed against AQP4 from neuromyelitis optica (NMO) patients impaired AQP4-M23 dynamic distribution and, consequently, astrocyte process and synaptic activity. Collectively, it emerges that the membrane dynamics of AQP4 isoform regulate brain cell assemblies in health and autoimmune brain disease targeting AQP4.

Dynamics of surface neurotransmitter receptors and transporters in glial cells: Single molecule insights.

The surface dynamics of neurotransmitter receptors and transporters, as well as ion channels, has been well-documented in neurons, revealing complex molecular behaviour and key physiological functions. However, our understanding of the membrane trafficking and dynamics of the signalling molecules located at the plasma membrane of glial cells is still in its infancy. Yet, recent breakthroughs in the field of glial cells have been obtained using combination of superresolution microscopy, single molecule imaging, and electrophysiological recordings. Here, we review our current knowledge on the surface dynamics of neurotransmitter receptors, transporters and ion channels, in glial cells. It has emerged that the brain cell network activity, synaptic activity, and calcium signalling, regulate the surface distribution and dynamics of these molecules. Remarkably, the dynamics of a given neurotransmitter receptor/transporter at the plasma membrane of a glial cell or neuron is unique, revealing the existence of cell-type specific regulatory pathways. Thus, investigating the dynamics of signalling proteins at the surface of glial cells will likely shed new light on our understanding of glial cell physiology and pathology.

A quantitative analysis of cellular and synaptic localization of GAT-1 and GAT-3 in rat neocortex.

High-affinity plasma membrane GABA transporters GAT-1 and GAT-3 contribute to the modulation of GABA-mediated inhibition in adult mammalian cerebral cortex. How GATs regulate inhibition in neocortical circuits remains however poorly understood for the lack of information on key localizational features. In this study, we used quantitative pre- and post-embedding electron microscopy to define the distribution of GAT-1 and GAT-3 in elements contributing to synapses and to unveil their ultrastructural organization at adult cortical GABAergic synapses. GAT-1 and GAT-3 were found in both neuronal and astrocytic processes: GAT-1 was prevalently segregated in neuronal elements and in profiles contributing to synapses, whereas GAT-3 was mostly expressed in astrocytes and did not exhibit a preferential distribution in elements contributing to synapses. Analysis of the ultrastructural distribution of GAT-1 and GAT-3 in the plasma membrane of axon terminals and perisynaptic astrocytic processes of symmetric synapses in relation to the active zone revealed that GAT-1 was more concentrated in restricted perisynaptic and extrasynaptic regions, whereas GAT-3 was prominent in extrasynaptic areas. These studies provide a basis for understanding the role GAT-1 and GAT-3 play in the modulation of GABA-mediated phasic and tonic inhibition in cerebral cortex.

Plasma membrane transporters GAT-1 and GAT-3 contribute to heterogeneity of GABAergic synapses in neocortex.

Cortical GABAergic synapses exhibit a high degree of molecular, anatomical and functional heterogeneity of their neurons of origins, presynaptic mechanisms, receptors, and scaffolding proteins. GABA transporters (GATs) have an important role in regulating GABA levels; among them, GAT-1 and GAT-3 play a prominent role in modulating tonic and phasic GABAAR-mediated inhibition. We asked whether GAT-1 and GAT-3 contribute to generating heterogeneity by studying their ultrastructural localization at cortical symmetric synapses using pre- and post-embedding electron microcopy. GAT-1 and GAT-3 staining at symmetric synapses showed that in some cases the transporters were localized exclusively over axon terminals; in others they were in both axon terminals and perisynaptic astrocytic processes; and in some others GAT-1 and GAT-3 were in perisynaptic astrocytic processes only. Moreover, we showed that the organizational pattern of GAT-1, but not of GAT-3, exhibits a certain degree of specificity related to the post-synaptic target of GABAergic synapses. These findings show that symmetric synapses expressing GAT-1 or GAT-3 are heterogeneous, and indicate that plasma membrane transporters can contribute to synaptic heterogeneity.

A Role for GAT-1 in Presynaptic GABA Homeostasis?

In monoamine-releasing terminals, neurotransmitter transporters - in addition to terminating synaptic transmission by clearing released transmitters from the extracellular space - are the primary mechanism for replenishing transmitter stores and thus regulate presynaptic homeostasis. Here, we analyze whether GAT-1, the main plasma membrane GABA transporter, plays a similar role in GABAergic terminals. Re-examination of existing literature and recent data gathered in our laboratory show that GABA homeostasis in GABAergic terminals is dominated by the activity of the GABA synthesizing enzyme and that GAT-1-mediated GABA transport contributes to cytosolic GABA levels. However, analysis of GAT-1 KO, besides demonstrating the effects of reduced clearance, reveals the existence of changes compatible with an impaired presynaptic function, as miniature IPSCs frequency is reduced by one-third and glutamic acid decarboxylases and phosphate-activated glutaminase levels are significantly up-regulated. Although the changes observed are less robust than those reported in mice with impaired dopamine, noradrenaline, and serotonin plasma membrane transporters, they suggest that in GABAergic terminals GAT-1 impacts on presynaptic GABA homeostasis, and may contribute to the activity-dependent regulation of inhibitory efficacy.