Clustering of Tau fibrils impairs the synaptic composition of α3-Na+/K+-ATPase and AMPA receptors.

Tau assemblies have prion-like properties: they propagate from one neuron to another and amplify by seeding the aggregation of endogenous Tau. Although key in prion-like propagation, the binding of exogenous Tau assemblies to the plasma membrane of naïve neurons is not understood. We report that fibrillar Tau forms clusters at the plasma membrane following lateral diffusion. We found that the fibrils interact with the Na+/K+-ATPase (NKA) and AMPA receptors. The consequence of the clustering is a reduction in the amount of α3-NKA and an increase in the amount of GluA2-AMPA receptor at synapses. Furthermore, fibrillar Tau destabilizes functional NKA complexes. Tau and α-synuclein aggregates often co-exist in patients' brains. We now show evidences for cross-talk between these pathogenic aggregates with α-synuclein fibrils dramatically enhancing fibrillar Tau clustering and synaptic localization. Our results suggest that fibrillar α-synuclein and Tau cross-talk at the plasma membrane imbalance neuronal homeostasis.

Differential Membrane Binding and Seeding of Distinct α-Synuclein Fibrillar Polymorphs.

The aggregation of the protein α-synuclein (α-Syn) leads to different synucleinopathies. We recently showed that structurally distinct fibrillar α-Syn polymorphs trigger either Parkinson's disease or multiple system atrophy hallmarks in vivo. Here, we establish a structural-molecular basis for these observations. We show that distinct fibrillar α-Syn polymorphs bind to and cluster differentially at the plasma membrane in both primary neuronal cultures and organotypic hippocampal slice cultures from wild-type mice. We demonstrate a polymorph-dependent and concentration-dependent seeding. We show a polymorph-dependent differential synaptic redistribution of α3-Na+/K+-ATPase, GluA2 subunit containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and GluN2B-subunit containing N-methyl-D-aspartate receptors, but not GluA1 subunit containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and metabotropic glutamate receptor 5 receptors. We also demonstrate polymorph-dependent alteration in neuronal network activity upon seeded aggregation of α-Syn. Our findings bring new, to our knowledge, insight into how distinct α-Syn polymorphs differentially bind to and seed monomeric α-Syn aggregation within neurons, thus affecting neuronal homeostasis through the redistribution of synaptic proteins.

α-synuclein assemblies sequester neuronal α3-Na+/K+-ATPase and impair Na+ gradient.

Extracellular α-synuclein (α-syn) assemblies can be up-taken by neurons; however, their interaction with the plasma membrane and proteins has not been studied specifically. Here we demonstrate that α-syn assemblies form clusters within the plasma membrane of neurons. Using a proteomic-based approach, we identify the α3-subunit of Na+/K+-ATPase (NKA) as a cell surface partner of α-syn assemblies. The interaction strength depended on the state of α-syn, fibrils being the strongest, oligomers weak, and monomers none. Mutations within the neuron-specific α3-subunit are linked to rapid-onset dystonia Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). We show that freely diffusing α3-NKA are trapped within α-syn clusters resulting in α3-NKA redistribution and formation of larger nanoclusters. This creates regions within the plasma membrane with reduced local densities of α3-NKA, thereby decreasing the efficiency of Na+ extrusion following stimulus. Thus, interactions of α3-NKA with extracellular α-syn assemblies reduce its pumping activity as its mutations in RDP/AHC.

A Simple and Powerful Analysis of Lateral Subdiffusion Using Single Particle Tracking.

In biological membranes, many factors such as cytoskeleton, lipid composition, crowding, and molecular interactions deviate lateral diffusion from the expected random walks. These factors have different effects on diffusion but act simultaneously, so the observed diffusion is a complex mixture of diffusive behaviors (directed, Brownian, anomalous, or confined). Therefore, commonly used approaches to quantify diffusion based on averaging of the displacements such as the mean square displacement, are not adapted to the analysis of this heterogeneity. We introduce a parameter-the packing coefficient Pc, which gives an estimate of the degree of free movement that a molecule displays in a period of time independently of its global diffusivity. Applying this approach to two different situations (diffusion of a lipid probe and trapping of receptors at synapses), we show that Pc detected and localized temporary changes of diffusive behavior both in time and in space. More importantly, it allowed the detection of periods with very high confinement as well as their frequency and duration, and thus it can be used to calculate the effective kon and koff of scaffolding interactions such as those that immobilize receptors at synapses.

Shape matters in protein mobility within membranes.

The lateral mobility of proteins within cell membranes is usually thought to be dependent on their size and modulated by local heterogeneities of the membrane. Experiments using single-particle tracking on reconstituted membranes demonstrate that protein diffusion is significantly influenced by the interplay of membrane curvature, membrane tension, and protein shape. We find that the curvature-coupled voltage-gated potassium channel (KvAP) undergoes a significant increase in protein mobility under tension, whereas the mobility of the curvature-neutral water channel aquaporin 0 (AQP0) is insensitive to it. Such observations are well explained in terms of an effective friction coefficient of the protein induced by the local membrane deformation.

The Catalytic Efficiency of Lipin 1ß Increases by Physically Interacting with the Proto-oncoprotein c-Fos.

Phosphatidic acid (PA) is a central precursor for membrane phospholipid biosynthesis. The lipin family is a magnesium-dependent type I PA phosphatase involved in de novo synthesis of neutral lipids and phospholipids. The regulation of lipin activity may govern the pathways by which these lipids are synthesized and control the cellular levels of important signaling lipids. Moreover, the proto-oncoprotein c-Fos has an emerging role in glycerolipid synthesis regulation; by interacting with key synthesizing enzymes it is able to increase overall phospho- and glycolipid synthesis. We studied the lipin 1ß enzyme activity in a cell-free system using PA/Triton X-100 mixed micelles as substrate, analyzing it in the presence/absence of c-Fos. We found that lipin 1ß kcat value increases around 40% in the presence of c-Fos, with no change in the lipin 1ß affinity for the PA/Triton X-100 mixed micelles. We also probed a physical interaction between both proteins. Although the c-Fos domain involved in lipin activation is its basic domain, the interaction domain is mapped to the N-terminal c-Fos. In conclusion, we provide evidence for a novel positive regulator of lipin 1ß PA phosphatase activity that is not achieved via altering its subcellular localization or affinity for membranes but rather through directly increasing its catalytic efficiency.

c-Fos-activated synthesis of nuclear phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] promotes global transcriptional changes.

c-Fos is a well-recognized member of the AP-1 (activator protein-1) family of transcription factors. In addition to this canonical activity, we previously showed that cytoplasmic c-Fos activates phospholipid synthesis through a mechanism independent of its genomic AP-1 activity. c-Fos associates with particular enzymes of the lipid synthesis pathway at the endoplasmic reticulum and increases the Vmax of the reactions without modifying the Km values. This lipid synthesis activation is associated with events of differentiation and proliferation that require high rates of membrane biogenesis. Since lipid synthesis also occurs in the nucleus, and different phospholipids have been assigned transcription regulatory functions, in the present study we examine if c-Fos also acts as a regulator of phospholipid synthesis in the nucleus. Furthermore, we examine if c-Fos modulates transcription through its phospholipid synthesis activator capacity. We show that nuclear-localized c-Fos associates with and activates PI4P5K (phosphatidylinositol-4-monophosphate 5-kinase), but not with PI4KIIIß (type IIIß phosphatidylinositol 4-kinase) thus promoting PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) formation, which, in turn, promotes transcriptional changes. We propose c-Fos as a key regulator of nuclear PtdIns(4,5)P2 synthesis in response to growth signals that results in c-Fos-dependent transcriptional changes promoted by the newly synthesized lipids.

Mapping the energy and diffusion landscapes of membrane proteins at the cell surface using high-density single-molecule imaging and Bayesian inference: application to the multiscale dynamics of glycine receptors in the neuronal membrane.

Protein mobility is conventionally analyzed in terms of an effective diffusion. Yet, this description often fails to properly distinguish and evaluate the physical parameters (such as the membrane friction) and the biochemical interactions governing the motion. Here, we present a method combining high-density single-molecule imaging and statistical inference to separately map the diffusion and energy landscapes of membrane proteins across the cell surface at ~100 nm resolution (with acquisition of a few minutes). Upon applying these analytical tools to glycine neurotransmitter receptors at inhibitory synapses, we find that gephyrin scaffolds act as shallow energy traps (~3 kBT) for glycine neurotransmitter receptors, with a depth modulated by the biochemical properties of the receptor-gephyrin interaction loop. In turn, the inferred maps can be used to simulate the dynamics of proteins in the membrane, from the level of individual receptors to that of the population, and thereby, to model the stochastic fluctuations of physiological parameters (such as the number of receptors at synapses). Overall, our approach provides a powerful and comprehensive framework with which to analyze biochemical interactions in living cells and to decipher the multiscale dynamics of biomolecules in complex cellular environments.

Differential control of thrombospondin over synaptic glycine and AMPA receptors in spinal cord neurons.

Thrombospondin-1 (TSP-1) is a large extracellular matrix protein secreted by astrocytes during development and inflammation. In the developing CNS, TSP-1 is involved in neuronal migration and adhesion, neurite outgrowth, and synaptogenesis. We investigated the effects of TSP-1 on neurons with mature synapses using immunocytochemistry, single-particle tracking, surface biotinylation, and calcium imaging. We show that in cultured rat spinal cord neurons TSP-1 decreased neuronal excitability by reducing the accumulation of excitatory AMPA receptors (AMPARs) and increasing that of inhibitory glycine receptors (GlyRs) in synapses. The effects of TSP-1 on GlyRs were dependent on the activation of excitatory receptors. These changes were abolished by blocking ß1-integrins and mimicked by blocking ß3-integrins. In the presence of TSP-1, AMPARs were less stabilized at synapses, increasing their lateral diffusion and endocytosis. Interestingly, TSP-1 counteracted the increased neuronal excitability and neuronal death induced by TNFα. These results suggest a role of TSP-1 in controlling the balance between excitation and inhibition which could help the recovery of normal synaptic activity after injury responses.

Activity-dependent regulation of the K/Cl transporter KCC2 membrane diffusion, clustering, and function in hippocampal neurons.

The neuronal K/Cl transporter KCC2 exports chloride ions and thereby influences the efficacy and polarity of GABA signaling in the brain. KCC2 is also critical for dendritic spine morphogenesis and the maintenance of glutamatergic transmission in cortical neurons. Because KCC2 plays a pivotal role in the function of central synapses, it is of particular importance to understand the cellular and molecular mechanisms underlying its regulation. Here, we studied the impact of membrane diffusion and clustering on KCC2 function. KCC2 forms clusters in the vicinity of both excitatory and inhibitory synapses. Using quantum-dot-based single-particle tracking on rat primary hippocampal neurons, we show that KCC2 is slowed down and confined at excitatory and inhibitory synapses compared with extrasynaptic regions. However, KCC2 escapes inhibitory synapses faster than excitatory synapses, reflecting stronger molecular constraints at the latter. Interfering with KCC2-actin interactions or inhibiting F-actin polymerization releases diffusion constraints on KCC2 at excitatory but not inhibitory synapses. Thus, F-actin constrains KCC2 diffusion at excitatory synapses, whereas KCC2 is confined at inhibitory synapses by a distinct mechanism. Finally, increased neuronal activity rapidly increases the diffusion coefficient and decreases the dwell time of KCC2 at excitatory synapses. This effect involves NMDAR activation, Ca(2+) influx, KCC2 S940 dephosphorylation and calpain protease cleavage of KCC2 and is accompanied by reduced KCC2 clustering and ion transport function. Thus, activity-dependent regulation of KCC2 lateral diffusion and clustering allows for a rapid regulation of chloride homeostasis in neurons.

Learning, AMPA receptor mobility and synaptic plasticity depend on n-cofilin-mediated actin dynamics.

Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin-binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n-cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long-term potentiation and long-term depression. Loss of n-cofilin-mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n-cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability.

Mobility in geometrically confined membranes.

Lipid and protein lateral mobility is essential for biological function. Our theoretical understanding of this mobility can be traced to the seminal work of Saffman and Delbrück, who predicted a logarithmic dependence of the protein diffusion coefficient (i) on the inverse of the size of the protein and (ii) on the "membrane size" for membranes of finite size [Saffman P, Delbrück M (1975) Proc Natl Acad Sci USA 72:3111-3113]. Although the experimental proof of the first prediction is a matter of debate, the second has not previously been thought to be experimentally accessible. Here, we construct just such a geometrically confined membrane by forming lipid bilayer nanotubes of controlled radii connected to giant liposomes. We followed the diffusion of individual molecules in the tubular membrane using single particle tracking of quantum dots coupled to lipids or voltage-gated potassium channels KvAP, while changing the membrane tube radius from approximately 250 to 10 nm. We found that both lipid and protein diffusion was slower in tubular membranes with smaller radii. The protein diffusion coefficient decreased as much as 5-fold compared to diffusion on the effectively flat membrane of the giant liposomes. Both lipid and protein diffusion data are consistent with the predictions of a hydrodynamic theory that extends the work of Saffman and Delbrück to cylindrical geometries. This study therefore provides strong experimental support for the ubiquitous Saffman-Delbrück theory and elucidates the role of membrane geometry and size in regulating lateral diffusion.

ß-amyloid and ATP-induced diffusional trapping of astrocyte and neuronal metabotropic glutamate type-5 receptors.

ß-Amyloid (Aß) oligomers initiate synaptotoxicity following their interaction with the plasma membrane. Several proteins including metabotropic glutamate type 5 receptors (mGluR5s) contribute to this process. We observed an overexpression of mGluR5s in reactive astrocytes surrounding Aß plaques in brain sections from an Alzheimer's disease mouse model. In a simplified cell culture system, using immunocytochemistry and single molecule imaging, we demonstrated a rapid binding of Aß oligomers on the plasma membrane of astrocytes. The resulting aggregates of Aß oligomers led to the diffusional trapping and clustering of mGluR5s. Further, Aß oligomers induced an increase in ATP release following activation of astroglial mGluR5s by its agonist. ATP slowed mGluR5s diffusion in astrocytes as well as in neurons co-cultured with astrocytes. This effect, which is purinergic receptor-dependent, was not observed in pure neuronal cultures. Thus, Aß oligomer- and mGluR5-dependent ATP release by astrocytes may contribute to the overall deleterious effect of mGluR5s in Alzheimer's disease. GLIA 2013;61:1673-1686.

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.

Heat shock cognate protein 70 regulates gephyrin clustering.

Formation and stabilization of postsynaptic glycine receptor (GlyR) clusters result from their association with the polymerized scaffold protein gephyrin. At the cell surface, lateral diffusion and local trapping of GlyR by synaptic gephyrin clusters is one of the main factors controlling their number. However, the mechanisms regulating gephyrin/GlyR cluster sizes are not fully understood. To identify molecular binding partners able to control gephyrin cluster stability, we performed pull-down assays with full-length or truncated gephyrin forms incubated in a rat spinal cord extract, combined with mass spectrometric analysis. We found that heat shock cognate protein 70 (Hsc70), a constitutive member of the heat shock protein 70 (Hsp70) family, selectively binds to the gephyrin G-domain. Immunoelectron microscopy of mouse spinal cord sections showed that Hsc70 could be colocalized with gephyrin at inhibitory synapses. Furthermore, ternary Hsc70-gephyrin-GlyR coclusters were formed following transfection of COS-7 cells. Upon overexpression of Hsc70 in mouse spinal cord neurons, synaptic accumulation of gephyrin was significantly decreased, but GlyR amounts were unaffected. In the same way, Hsc70 inhibition increased gephyrin accumulation at inhibitory synapses without modifying GlyR clustering. Single particle tracking experiments revealed that the increase of gephyrin molecules reduced GlyR diffusion rates without altering GlyR residency at synapses. Our findings demonstrate that Hsc70 regulates gephyrin polymerization independently of its interaction with GlyR. Therefore, gephyrin polymerization and synaptic clustering of GlyR are uncoupled events.

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.

Control of the postsynaptic membrane viscosity.

The physical properties of the postsynaptic membrane (PSM), including its viscosity, determine its capacity to regulate the net flux of synaptic membrane proteins such as neurotransmitter receptors. To address these properties, we studied the lateral diffusion of glycophosphatidylinositol-anchored green fluorescent protein and cholera toxin bound to the external leaflet of the plasma membrane. Relative to extrasynaptic regions, their mobility was reduced at synapses and even more at inhibitory than at excitatory ones. This indicates a higher density of obstacles and/or higher membrane viscosity at inhibitory contacts. Actin depolymerization reduced the confinement and accelerated a population of fast, mobile molecules. The compaction of obstacles thus depends on actin cytoskeleton integrity. Cholesterol depletion increased the mobility of the slow diffusing molecules, allowing them to diffuse more rapidly through the crowded PSM. Thus, the PSM has lipid-raft properties, and the density of obstacles to diffusion depends on filamentous actin. Therefore, lipid composition and actin-dependent protein compaction regulate viscosity of the PSM and, consequently, the molecular flow in and out of synapses.

Molecular Crowding and Diffusion-Capture in Synapses.

Cell membranes often contain domains with important physiological functions. A typical example are neuronal synapses, whose capacity to capture receptors for neurotransmitters is central to neuronal functions. Receptors diffuse in the membrane until they are stabilized by interactions with stable elements, the scaffold. Single particle tracking experiments demonstrated that these interactions are rather weak and that lateral diffusion is strongly impaired in the post-synaptic membrane due to molecular crowding. We investigated how the distribution of scaffolding molecules and molecular crowding affect the capture of receptors. In particle-based Monte Carlo simulations, based on experimental data of molecular diffusion and organization, crowding enhanced the receptor-scaffold interaction but reduced the capture of new molecules. The distribution of scaffolding sites in several clusters reduced crowding and fostered the exchange of molecules accelerating synaptic plasticity. Synapses could switch between two regimes, becoming more stable or more plastic depending on the internal distribution of molecules.

The excitatory postsynaptic density is a size exclusion diffusion environment.

Receptors are concentrated in the postsynaptic membrane but can enter and exit synapses rapidly during both basal turnover and processes of synaptic plasticity. How the exchange of receptors by lateral diffusion between synaptic and extrasynaptic areas is regulated remains largely unknown. We investigated the structural properties of the postsynaptic membrane that allow these movements by addressing the diffusion behaviors of AMPA receptors (AMPARs) and different lipids. Using single molecule tracking we found that not only AMPARs but also lipids, which are not synaptically enriched, display confined diffusion at synapses. Each molecule type displays a different average confinement area, smaller molecules being confined to smaller areas. Glutamate application increases the mobility of all molecules. The structure of the synaptic membrane is thus probably organized as a size exclusion matrix and this controls the rate of exchange of molecules with the extrasynaptic membrane.

Topographical cues control the morphology and dynamics of migrating cortical interneurons.

In mammalian embryos, cortical interneurons travel long distances among complex three-dimensional tissues before integrating into cortical circuits. Several molecular guiding cues involved in this migration process have been identified, but the influence of physical parameters remains poorly understood. In the present study, we have investigated in vitro the influence of the topography of the microenvironment on the migration of primary cortical interneurons released from mouse embryonic explants. We found that arrays of PDMS micro-pillars of 10 µm size and spacing, either round or square, influenced both the morphology and the migratory behavior of interneurons. Strikingly, most interneurons exhibited a single and long leading process oriented along the diagonals of the square pillared array, whereas leading processes of interneurons migrating in-between round pillars were shorter, often branched and oriented in all available directions. Accordingly, dynamic studies revealed that growth cone divisions were twice more frequent in round than in square pillars. Both soma and leading process tips presented forward directed movements within square pillars, contrasting with the erratic trajectories and more dynamic movements observed among round pillars. In support of these observations, long interneurons migrating in square pillars displayed tight bundles of stable microtubules aligned in the direction of migration. Overall, our results show that micron-sized topography provides global spatial constraints promoting the establishment of different morphological and migratory states. Remarkably, these different states belong to the natural range of migratory behaviors of cortical interneurons, highlighting the potential importance of topographical cues in the guidance of these embryonic neurons, and more generally in brain development.