NKCC1 and KCC2 Chloride Transporters Have Different Membrane Dynamics on the Surface of Hippocampal Neurons.

Na-K-2Cl cotransporter 1 (NKCC1) regulates chloride influx in neurons and thereby GABAA receptor activity in normal and pathological conditions. Here, we characterized in hippocampal neurons the membrane expression, distribution and dynamics of exogenous NKCC1a and NKCC1b isoforms and compared them to those of the chloride extruder K-Cl cotransporter 2 (KCC2). We found that NKCC1a and NKCC1b behave quite similarly. NKCC1a/1b but not KCC2 are present along the axon initial segment where they are confined. Moreover, NKCC1a/1b are detected in the somato-dendritic compartment at a lower level than KCC2, where they form fewer, smaller and less compact clusters at perisynaptic and extrasynaptic sites. Interestingly, ~60% of dendritic clusters of NKCC1a/1b are colocalized with KCC2. They are larger and brighter than those devoid of KCC2, suggesting a particular NKCC1a/1b-KCC2 relationship. In agreement with the reduced dendritic clustering of NKCC1a/1b compared with that of KCC2, NKCC1a/1b are more mobile on the dendrite than KCC2, suggesting weaker cytoskeletal interaction. NKCC1a/b are confined to endocytic zones, where they spend more time than KCC2. However, they spend less time in these compartments than at the synapses, suggesting that they can rapidly leave endocytic zones to increase the membrane pool, which can happen in pathological conditions. Thus, NKCC1a/b have different membrane dynamics and clustering from KCC2, which helps to explain their low level in the neuronal membrane, while allowing a rapid increase in the membrane pool under pathological conditions.

5-HT1A and 5-HT2B receptor interaction and co-clustering regulate serotonergic neuron excitability.

Many psychiatric diseases have been associated with serotonin (5-HT) neuron dysfunction. The firing of 5-HT neurons is known to be under 5-HT1A receptor-mediated autoinhibition, but functional consequences of coexpressed receptors are unknown. Using co-immunoprecipitation, BRET, confocal, and super-resolution microscopy in hippocampal and 5-HT neurons, we present evidence that 5-HT1A and 5-HT2B receptors can form heterodimers and co-cluster at the plasma membrane of dendrites. Selective agonist stimulation of coexpressed 5-HT1A and 5-HT2B receptors prevents 5-HT1A receptor internalization and increases 5-HT2B receptor membrane clustering. Current clamp recordings of 5-HT neurons revealed that 5-HT1A receptor stimulation of acute slices from mice lacking 5-HT2B receptors in 5-HT neurons increased their firing activity trough Ca2+-activated potassium channel inhibition compared to 5-HT neurons from control mice. This work supports the hypothesis that the relative expression of 5-HT1A and 5-HT2B receptors tunes the neuronal excitability of serotonergic neurons through potassium channel regulation.

Lateral Diffusion of NKCC1 Contributes to Chloride Homeostasis in Neurons and Is Rapidly Regulated by the WNK Signaling Pathway.

An upregulation of the Na+-K+-2Cl- cotransporter NKCC1, the main chloride importer in mature neurons, can lead to depolarizing/excitatory responses mediated by GABA type A receptors (GABAARs) and, thus, to hyperactivity. Understanding the regulatory mechanisms of NKCC1 would help prevent intra-neuronal chloride accumulation that occurs in pathologies with defective inhibition. The cell mechanisms regulating NKCC1 are poorly understood. Here, we report in mature hippocampal neurons that GABAergic activity controls the membrane diffusion and clustering of NKCC1 via the chloride-sensitive WNK lysine deficient protein kinase 1 (WNK1) and the downstream Ste20 Pro-line Asparagine Rich Kinase (SPAK) kinase that directly phosphorylates NKCC1 on key threonine residues. At rest, this signaling pathway has little effect on intracellular Cl- concentration, but it participates in the elevation of intraneuronal Cl- concentration in hyperactivity conditions associated with an up-regulation of NKCC1. The fact that the main chloride exporter, the K+-Cl- cotransporter KCC2, is also regulated in mature neurons by the WNK1 pathway indicates that this pathway will be a target of choice in the pathology.

Introducing Diinamic, a flexible and robust method for clustering analysis in single-molecule localization microscopy.

Super-resolution microscopy allowed major improvements in our capacity to describe and explain biological organization at the nanoscale. Single-molecule localization microscopy (SMLM) uses the positions of molecules to create super-resolved images, but it can also provide new insights into the organization of molecules through appropriate pointillistic analyses that fully exploit the sparse nature of SMLM data. However, the main drawback of SMLM is the lack of analytical tools easily applicable to the diverse types of data that can arise from biological samples. Typically, a cloud of detections may be a cluster of molecules or not depending on the local density of detections, but also on the size of molecules themselves, the labeling technique, the photo-physics of the fluorophore, and the imaging conditions. We aimed to set an easy-to-use clustering analysis protocol adaptable to different types of data. Here, we introduce Diinamic, which combines different density-based analyses and optional thresholding to facilitate the detection of clusters. On simulated or real SMLM data, Diinamic correctly identified clusters of different sizes and densities, being performant even in noisy datasets with multiple detections per fluorophore. It also detected subdomains ("nanodomains") in clusters with non-homogeneous distribution of detections.

Conformational state-dependent regulation of GABAA receptor diffusion and subsynaptic domains.

The efficacy of GABAergic synapses relies on the number of postsynaptic GABAA receptors (GABAARs), which is regulated by a diffusion capture mechanism. Here, we report that the conformational state of GABAARs influences their membrane dynamics. Indeed, pharmacological and mutational manipulations of receptor favoring active or desensitized states altered GABAAR diffusion leading to the disorganization of GABAAR subsynaptic domains and gephyrin scaffold, as detected by super-resolution microscopy. Active and desensitized receptors were confined to perisynaptic endocytic zones, and some of them were further internalized. We propose that following their activation or desensitization, synaptic receptors rapidly diffuse at the periphery of the synapse where they remain confined until they switch back to a resting state or are internalized. We speculate that this allows a renewal of activatable receptors at the synapse, contributing to maintain the efficacy of the synaptic transmission, in particular on sustained GABA transmission.

Gephyrin Interacts with the K-Cl Cotransporter KCC2 to Regulate Its Surface Expression and Function in Cortical Neurons.

The K+-Cl- cotransporter KCC2, encoded by the Slc12a5 gene, is a neuron-specific chloride extruder that tunes the strength and polarity of GABAA receptor-mediated transmission. In addition to its canonical ion transport function, KCC2 also regulates spinogenesis and excitatory synaptic function through interaction with a variety of molecular partners. KCC2 is enriched in the vicinity of both glutamatergic and GABAergic synapses, the activity of which in turn regulates its membrane stability and function. KCC2 interaction with the submembrane actin cytoskeleton via 4.1N is known to control its anchoring near glutamatergic synapses on dendritic spines. However, the molecular determinants of KCC2 clustering near GABAergic synapses remain unknown. Here, we used proteomics to identify novel KCC2 interacting proteins in the adult rat neocortex. We identified both known and novel candidate KCC2 partners, including some involved in neuronal development and synaptic transmission. These include gephyrin, the main scaffolding molecule at GABAergic synapses. Gephyrin interaction with endogenous KCC2 was confirmed by immunoprecipitation from rat neocortical extracts. We showed that gephyrin stabilizes plasmalemmal KCC2 and promotes its clustering in hippocampal neurons, mostly but not exclusively near GABAergic synapses, thereby controlling KCC2-mediated chloride extrusion. This study identifies gephyrin as a novel KCC2 anchoring molecule that regulates its membrane expression and function in cortical neurons.SIGNIFICANCE STATEMENT Fast synaptic inhibition in the brain is mediated by chloride-permeable GABAA receptors (GABAARs) and therefore relies on transmembrane chloride gradients. In neurons, these gradients are primarily maintained by the K/Cl cotransporter KCC2. Therefore, understanding the mechanisms controlling KCC2 expression and function is crucial to understand its physiological regulation and rescue its function in the pathology. KCC2 function depends on its membrane expression and clustering, but the underlying mechanisms remain unknown. We describe the interaction between KCC2 and gephyrin, the main scaffolding protein at inhibitory synapses. We show that gephyrin controls plasmalemmal KCC2 clustering and that loss of gephyrin compromises KCC2 function. Our data suggest functional units comprising GABAARs, gephyrin, and KCC2 act to regulate synaptic GABA signaling.

Convergence of adenosine and GABA signaling for synapse stabilization during development.

During development, neural circuit formation requires the stabilization of active γ-aminobutyric acid­mediated (GABAergic) synapses and the elimination of inactive ones. Here, we demonstrate that, although the activation of postsynaptic GABA type A receptors (GABAARs) and adenosine A2A receptors (A2ARs) stabilizes GABAergic synapses, only A2AR activation is sufficient. Both GABAAR- and A2AR-dependent signaling pathways act synergistically to produce adenosine 3',5'-monophosphate through the recruitment of the calcium­calmodulin­adenylyl cyclase pathway. Protein kinase A, thus activated, phosphorylates gephyrin on serine residue 303, which is required for GABAAR stabilization. Finally, the stabilization of pre- and postsynaptic GABAergic elements involves the interaction between gephyrin and the synaptogenic membrane protein Slitrk3. We propose that A2ARs act as detectors of active GABAergic synapses releasing GABA, adenosine triphosphate, and adenosine to regulate their fate toward stabilization or elimination.

Serotonin 2B Receptor by Interacting with NMDA Receptor and CIPP Protein Complex May Control Structural Plasticity at Glutamatergic Synapses.

The serotonin 2B (5-HT2B) receptor coupled to Gq-protein contributes to the control of neuronal excitability and is implicated in various psychiatric disorders. The mechanisms underlying its brain function are not fully described. Using peptide affinity chromatography combined with mass spectrometry, we found that the PDZ binding motif of the 5-HT2B receptor located at its C-terminal end interacts with the scaffolding protein channel interacting PDZ protein (CIPP). We then showed, in COS-7 cells, that the association of the 5-HT2B receptor to CIPP enhanced receptor-operated inositol phosphate (IP) production without affecting its cell surface and intracellular levels. Co-immunoprecipitation experiments revealed that CIPP, the 5-HT2B receptor, and the NR1 subunit of the NMDA receptor form a macromolecular complex. CIPP increased 5-HT2B receptor clustering at the surface of primary cultured hippocampal neurons and prevented receptor dispersion following agonist stimulation, thus potentiating IP production and intracellular calcium mobilization in dendrites. CIPP or 5-HT2B receptor stimulation in turn dispersed NR1 clusters colocalized with 5-HT2B receptors and increased the density and maturation of dendritic spines. Collectively, our results suggest that the 5-HT2B receptor, the NMDA receptor, and CIPP may form a signaling platform by which serotonin can influence structural plasticity of excitatory glutamatergic synapses.

Increased surface P2X4 receptor regulates anxiety and memory in P2X4 internalization-defective knock-in mice.

ATP signaling and surface P2X4 receptors are upregulated selectively in neurons and/or glia in various CNS disorders including anxiety, chronic pain, epilepsy, ischemia, and neurodegenerative diseases. However, the cell-specific functions of P2X4 in pathological contexts remain elusive. To elucidate P2X4 functions, we created a conditional transgenic knock-in P2X4 mouse line (Floxed P2X4mCherryIN) allowing the Cre activity-dependent genetic swapping of the internalization motif of P2X4 by the fluorescent mCherry protein to prevent constitutive endocytosis of P2X4. By combining molecular, cellular, electrophysiological, and behavioral approaches, we characterized two distinct knock-in mouse lines expressing noninternalized P2X4mCherryIN either exclusively in excitatory forebrain neurons or in all cells natively expressing P2X4. The genetic substitution of wild-type P2X4 by noninternalized P2X4mCherryIN in both knock-in mouse models did not alter the sparse distribution and subcellular localization of P2X4 but increased the number of P2X4 receptors at the surface of the targeted cells mimicking the pathological increased surface P2X4 state. Increased surface P2X4 density in the hippocampus of knock-in mice altered LTP and LTD plasticity phenomena at CA1 synapses without affecting basal excitatory transmission. Moreover, these cellular events translated into anxiolytic effects and deficits in spatial memory. Our results show that increased surface density of neuronal P2X4 contributes to synaptic deficits and alterations in anxiety and memory functions consistent with the implication of P2X4 in neuropsychiatric and neurodegenerative disorders. Furthermore, these conditional P2X4mCherryIN knock-in mice will allow exploring the cell-specific roles of P2X4 in various physiological and pathological contexts.

ATP6AP2 variant impairs CNS development and neuronal survival to cause fulminant neurodegeneration.

Vacuolar H+-ATPase-dependent (V-ATPase-dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell-derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase-dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase-dependent signaling and protein degradation in the developing human central nervous system.

Activity-Dependent Inhibitory Synapse Scaling Is Determined by Gephyrin Phosphorylation and Subsequent Regulation of GABAA Receptor Diffusion.

Synaptic plasticity relies on the rapid changes in neurotransmitter receptor number at postsynaptic sites. Using superresolution photoactivatable localization microscopy imaging and quantum dot-based single-particle tracking in rat hippocampal cultured neurons, we investigated whether the phosphorylation status of the main scaffolding protein gephyrin influenced the organization of the gephyrin scaffold and GABAA receptor (GABAAR) membrane dynamics. We found that gephyrin phosphorylation regulates gephyrin microdomain compaction. Extracellular signal-regulated kinase 1/2 and glycogen synthase kinase 3ß (GSK3ß) signaling alter the gephyrin scaffold mesh differentially. Differences in scaffold organization similarly affected the diffusion of synaptic GABAARs, suggesting reduced gephyrin receptor-binding properties. In the context of synaptic scaling, our results identify a novel role of the GSK3ß signaling pathway in the activity-dependent regulation of extrasynaptic receptor surface trafficking and GSK3ß, protein kinase A, and calcium/calmodulin-dependent protein kinase IIα pathways in facilitating adaptations of synaptic receptors.

GABAA receptor dependent synaptic inhibition rapidly tunes KCC2 activity via the Cl--sensitive WNK1 kinase.

The K+-Cl- co-transporter KCC2 (SLC12A5) tunes the efficacy of GABAA receptor-mediated transmission by regulating the intraneuronal chloride concentration [Cl-]i. KCC2 undergoes activity-dependent regulation in both physiological and pathological conditions. The regulation of KCC2 by synaptic excitation is well documented; however, whether the transporter is regulated by synaptic inhibition is unknown. Here we report a mechanism of KCC2 regulation by GABAA receptor (GABAAR)-mediated transmission in mature hippocampal neurons. Enhancing GABAAR-mediated inhibition confines KCC2 to the plasma membrane, while antagonizing inhibition reduces KCC2 surface expression by increasing the lateral diffusion and endocytosis of the transporter. This mechanism utilizes Cl- as an intracellular secondary messenger and is dependent on phosphorylation of KCC2 at threonines 906 and 1007 by the Cl--sensing kinase WNK1. We propose this mechanism contributes to the homeostasis of synaptic inhibition by rapidly adjusting neuronal [Cl-]i to GABAAR activity.

Sperm-inherited organelle clearance in C. elegans relies on LC3-dependent autophagosome targeting to the pericentrosomal area.

Macroautophagic degradation of sperm-inherited organelles prevents paternal mitochondrial DNA transmission in C. elegans. The recruitment of autophagy markers around sperm mitochondria has also been observed in mouse and fly embryos but their role in degradation is debated. Both worm Atg8 ubiquitin-like proteins, LGG-1/GABARAP and LGG-2/LC3, are recruited around sperm organelles after fertilization. Whereas LGG-1 depletion affects autophagosome function, stabilizes the substrates and is lethal, we demonstrate that LGG-2 is dispensable for autophagosome formation but participates in their microtubule-dependent transport toward the pericentrosomal area prior to acidification. In the absence of LGG-2, autophagosomes and their substrates remain clustered at the cell cortex, away from the centrosomes and their associated lysosomes. Thus, the clearance of sperm organelles is delayed and their segregation between blastomeres prevented. This allowed us to reveal a role of the RAB-5/RAB-7 GTPases in autophagosome formation. In conclusion, the major contribution of LGG-2 in sperm-inherited organelle clearance resides in its capacity to mediate the retrograde transport of autophagosomes rather than their fusion with acidic compartments: a potential key function of LC3 in controlling the fate of sperm mitochondria in other species.