Developmental synaptic regulator, TWEAK/Fn14 signaling, is a determinant of synaptic function in models of stroke and neurodegeneration.

Identifying molecular mediators of neural circuit development and/or function that contribute to circuit dysfunction when aberrantly reengaged in neurological disorders is of high importance. The role of the TWEAK/Fn14 pathway, which was recently reported to be a microglial/neuronal axis mediating synaptic refinement in experience-dependent visual development, has not been explored in synaptic function within the mature central nervous system. By combining electrophysiological and phosphoproteomic approaches, we show that TWEAK acutely dampens basal synaptic transmission and plasticity through neuronal Fn14 and impacts the phosphorylation state of pre- and postsynaptic proteins in adult mouse hippocampal slices. Importantly, this is relevant in two models featuring synaptic deficits. Blocking TWEAK/Fn14 signaling augments synaptic function in hippocampal slices from amyloid-beta-overexpressing mice. After stroke, genetic or pharmacological inhibition of TWEAK/Fn14 signaling augments basal synaptic transmission and normalizes plasticity. Our data support a glial/neuronal axis that critically modifies synaptic physiology and pathophysiology in different contexts in the mature brain and may be a therapeutic target for improving neurophysiological outcomes.

Functional activity of anti-LINGO-1 antibody opicinumab requires target engagement at a secondary binding site.

LINGO-1 is a membrane protein of the central nervous system (CNS) that suppresses myelination of axons. Preclinical studies have revealed that blockade of LINGO-1 function leads to CNS repair in demyelinating animal models. The anti-LINGO-1 antibody Li81 (opicinumab), which blocks LINGO-1 function and shows robust remyelinating activity in animal models, is currently being investigated in a Phase 2 clinical trial as a potential treatment for individuals with relapsing forms of multiple sclerosis (AFFINITY: clinical trial.gov number NCT03222973). Li81 has the unusual feature that it contains two LINGO-1 binding sites: a classical site utilizing its complementarity-determining regions and a cryptic secondary site involving Li81 light chain framework residues that recruits a second LINGO-1 molecule only after engagement of the primary binding site. Concurrent binding at both sites leads to formation of a 2:2 complex of LINGO-1 with the Li81 antigen-binding fragment, and higher order complexes with intact Li81 antibody. To elucidate the role of the secondary binding site, we designed a series of Li81 variant constructs that eliminate it while retaining the classic site contacts. These Li81 mutants retained the high affinity binding to LINGO-1, but lost the antibody-induced oligodendrocyte progenitor cell (OPC) differentiation activity and myelination activity in OPC- dorsal root ganglion neuron cocultures seen with Li81. The mutations also attenuate antibody-induced internalization of LINGO-1 on cultured cortical neurons, OPCs, and cells over-expressing LINGO-1. Together these studies reveal that engagement at both LINGO-1 binding sites of Li81 is critical for robust functional activity of the antibody.

Remodeling of the intestinal brush border underlies adhesion and virulence of an enteric pathogen.

UNLABELLED: Intestinal colonization by Vibrio parahaemolyticus-the most common cause of seafood-borne bacterial enteritis worldwide-induces extensive disruption of intestinal microvilli. In orogastrically infected infant rabbits, reorganization of the apical brush border membrane includes effacement of some microvilli and marked elongation of others. All diarrhea, inflammation, and intestinal pathology associated with V. parahaemolyticus infection are dependent upon one of its type 3 secretion systems (T3SS2); however, translocated effectors that directly mediate brush border restructuring and bacterial adhesion are not known. Here, we demonstrate that the effector VopV is essential for V. parahaemolyticus intestinal colonization and therefore its pathogenicity, that it induces effacement of brush border microvilli, and that this effacement is required for adhesion of V. parahaemolyticus to enterocytes. VopV contains multiple functionally independent and mechanistically distinct domains through which it disrupts microvilli. We show that interaction between VopV and filamin, as well as VopV's previously noted interaction with actin, mediates enterocyte cytoskeletal reorganization. VopV's multipronged approach to epithelial restructuring, coupled with its impact on colonization, suggests that remodeling of the epithelial brush border is a critical step in pathogenesis. IMPORTANCE: Colonization of the small bowel by Vibrio parahaemolyticus, the most common bacterial agent of seafood-borne enteric disease, induces extensive structural changes in the intestinal epithelium. Here, we show that this diarrheal pathogen's colonization and virulence depend upon VopV, a bacterial protein that is transferred into host epithelial cells. VopV induces marked rearrangement of the apical epithelial cell membrane, including elimination of microvilli, by two means: through interaction with actin and through a previously unrecognized interaction with the actin-cross-linking protein filamin. VopV-mediated "effacement" of microvilli enables V. parahaemolyticus to adhere to host cells, although VopV may not directly mediate adhesion. VopV's effects on microvillus structure and bacterial adhesion likely account for its essential role in V. parahaemolyticus intestinal pathogenesis. Our findings suggest a new role for filamin in brush border maintenance and raise the possibility that microvillus effacement is a common strategy among enteric pathogens for enhancing adhesion to host cells.

Multivalent immune complexes divert FcRn to lysosomes by exclusion from recycling sorting tubules.

The neonatal receptor for immunoglobulin G (IgG; FcRn) prevents IgG degradation by efficiently sorting IgG into recycling endosomes and away from lysosomes. When bound to IgG-opsonized antigen complexes, however, FcRn traffics cargo into lysosomes, where antigen processing can occur. Here we address the mechanism of sorting when FcRn is bound to multivalent IgG-opsonized antigens. We find that only the unbound receptor or FcRn bound to monomeric IgG is sorted into recycling tubules emerging from early endosomes. Cross-linked FcRn is never visualized in tubules containing the unbound receptor. Similar results are found for transferrin receptor, suggesting a general mechanism of action. Deletion or replacement of the FcRn cytoplasmic tail does not prevent diversion of trafficking to lysosomes upon cross-linking. Thus physical properties of the lumenal ligand-receptor complex appear to act as key determinants for sorting between the recycling and lysosomal pathways by regulating FcRn entry into recycling tubules.

Release of cellular tension signals self-restorative ventral lamellipodia to heal barrier micro-wounds.

Basic mechanisms by which cellular barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust barrier function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique ventral lamellipodia that propagate via integrins toward and across these "micro-wounds" to close them. This novel actin remodeling activity progressively healed multiple micro-wounds in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that ventral lamellipodia formed as a response to force imbalance and specifically loss of isometric tension. Ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that barrier disruptions are detected as local release of isometric tension/force unloading, which is directly coupled to reactive oxygen species-dependent self-restorative actin remodeling dynamics.

Similar uptake but different trafficking and escape routes of reovirus virions and infectious subvirion particles imaged in polarized Madin-Darby canine kidney cells.

Polarized epithelial cells that line the digestive, respiratory, and genitourinary tracts form a barrier that many viruses must breach to infect their hosts. Current understanding of cell entry by mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs), generated from MRV virions by extracellular proteolysis in the digestive tract, are mostly derived from in vitro studies with nonpolarized cells. Recent live-cell imaging advances allow us for the first time to visualize events at the apical surface of polarized cells. In this study, we used spinning-disk confocal fluorescence microscopy with high temporal and spatial resolution to follow the uptake and trafficking dynamics of single MRV virions and ISVPs at the apical surface of live polarized Madin-Darby canine kidney cells. Both types of particles were internalized by clathrin-mediated endocytosis, but virions and ISVPs exhibited strikingly different trafficking after uptake. While virions reached early and late endosomes, ISVPs did not and instead escaped the endocytic pathway from an earlier location. This study highlights the broad advantages of using live-cell imaging combined with single-particle tracking for identifying key steps in cell entry by viruses.

Lipid sorting by ceramide structure from plasma membrane to ER for the cholera toxin receptor ganglioside GM1.

The glycosphingolipid GM1 binds cholera toxin (CT) on host cells and carries it retrograde from the plasma membrane (PM) through endosomes, the trans-Golgi (TGN), and the endoplasmic reticulum (ER) to induce toxicity. To elucidate how a membrane lipid can specify trafficking in these pathways, we synthesized GM1 isoforms with alternate ceramide domains and imaged their trafficking in live cells. Only GM1 with unsaturated acyl chains sorted efficiently from PM to TGN and ER. Toxin binding, which effectively crosslinks GM1 lipids, was dispensable, but membrane cholesterol and the lipid raft-associated proteins actin and flotillin were required. The results implicate a protein-dependent mechanism of lipid sorting by ceramide structure and provide a molecular explanation for the diversity and specificity of retrograde trafficking by CT in host cells.

Insights on the trafficking and retro-translocation of glycosphingolipid-binding bacterial toxins.

Some bacterial toxins and viruses have evolved the capacity to bind mammalian glycosphingolipids to gain access to the cell interior, where they can co-opt the endogenous mechanisms of cellular trafficking and protein translocation machinery to cause toxicity. Cholera toxin (CT) is one of the best-studied examples, and is the virulence factor responsible for massive secretory diarrhea seen in cholera. CT enters host cells by binding to monosialotetrahexosylganglioside (GM1 gangliosides) at the plasma membrane where it is transported retrograde through the trans-Golgi network (TGN) into the endoplasmic reticulum (ER). In the ER, a portion of CT, the CT-A1 polypeptide, is unfolded and then "retro-translocated" to the cytosol by hijacking components of the ER associated degradation pathway (ERAD) for misfolded proteins. CT-A1 rapidly refolds in the cytosol, thus avoiding degradation by the proteasome and inducing toxicity. Here, we highlight recent advances in our understanding of how the bacterial AB(5) toxins induce disease. We highlight the molecular mechanisms by which these toxins use glycosphingolipid to traffic within cells, with special attention to how the cell senses and sorts the lipid receptors. We also discuss several new studies that address the mechanisms of toxin unfolding in the ER and the mechanisms of CT A1-chain retro-translocation to the cytosol.

Intoxication of zebrafish and mammalian cells by cholera toxin depends on the flotillin/reggie proteins but not Derlin-1 or -2.

Cholera toxin (CT) causes the massive secretory diarrhea associated with epidemic cholera. To induce disease, CT enters the cytosol of host cells by co-opting a lipid-based sorting pathway from the plasma membrane, through the trans-Golgi network (TGN), and into the endoplasmic reticulum (ER). In the ER, a portion of the toxin is unfolded and retro- translocated to the cytosol. Here, we established zebrafish as a genetic model of intoxication and examined the Derlin and flotillin proteins, which are thought to be usurped by CT for retro-translocation and lipid sorting, respectively. Using antisense morpholino oligomers and siRNA, we found that depletion of Derlin-1, a component of the Hrd-1 retro-translocation complex, was dispensable for CT-induced toxicity. In contrast, the lipid raft-associated proteins flotillin-1 and -2 were required. We found that in mammalian cells, CT intoxication was dependent on the flotillins for trafficking between plasma membrane/endosomes and two pathways into the ER, only one of which appears to intersect the TGN. These results revise current models for CT intoxication and implicate protein scaffolding of lipid rafts in the endo-somal sorting of the toxin-GM1 complex.

The length of vesicular stomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis.

Microbial pathogens exploit the clathrin endocytic machinery to enter host cells. Vesicular stomatitis virus (VSV), an enveloped virus with bullet-shaped virions that measure 70 x 200 nm, enters cells by clathrin-dependent endocytosis. We showed previously that VSV particles exceed the capacity of typical clathrin-coated vesicles and instead enter through endocytic carriers that acquire a partial clathrin coat and require local actin filament assembly to complete vesicle budding and internalization. To understand why the actin system is required for VSV uptake, we compared the internalization mechanisms of VSV and its shorter (75 nm long) defective interfering particle, DI-T. By imaging the uptake of individual particles into live cells, we found that, as with parental virions, DI-T enters via the clathrin endocytic pathway. Unlike VSV, DI-T internalization occurs through complete clathrin-coated vesicles and does not require actin polymerization. Since VSV and DI-T particles display similar surface densities of the same attachment glycoprotein, we conclude that the physical properties of the particle dictate whether a virus-containing clathrin pit engages the actin system. We suggest that the elongated shape of a VSV particle prevents full enclosure by the clathrin coat and that stalling of coat assembly triggers recruitment of the actin machinery to finish the internalization process. Since some enveloped viruses have pleomorphic particle shapes and sizes, our work suggests that they may use altered modes of endocytic uptake. More generally, our findings show the importance of cargo geometry for specifying cellular entry modes, even when the receptor recognition properties of a ligand are maintained.

Shiga toxin increases formation of clathrin-coated pits through Syk kinase.

Clathrin-dependent endocytosis is a main entry mechanism for the glycolipid-binding Shiga toxin (Stx), although clathrin-independent pathways are also involved. Binding of Stx to its receptor Gb3 not only is essential for Stx retrograde transport to the endoplasmic reticulum and toxicity but also activates signaling through the tyrosine kinase Syk. We previously described that Syk activity is important for Stx entry, but it remained unclear how this kinase modulates endocytosis of Stx. Here we characterized the effects of Stx and Syk on clathrin-coated pit formation. We found that acute treatment with Stx results in an increase in the number of clathrin-coated profiles as determined by electron microscopy and on the number of structures containing the endocytic AP-2 adaptor at the plasma membrane determined by live-cell spinning disk confocal imaging. These responses to Stx require functional Syk activity. We propose that a signaling pathway mediated by Syk and modulated by Stx leads to an increased number of endocytic clathrin-coated structures, thus providing a possible mechanism by which Stx enhances its own endocytosis.

The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity.

The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.

Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization.

Many viruses that enter cells by clathrin-dependent endocytosis are significantly larger than the dimensions of a typical clathrin-coated vesicle. The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain. Here we examined how clathrin-coated vesicles accommodate vesicular stomatitis virus (VSV) during its entry into cells. Using high-resolution imaging of the internalization of single viral particles into cells expressing fluorescent clathrin and adaptor molecules, we show that VSV enters cells through partially clathrin-coated vesicles. We found that on average, virus-containing vesicles contain more clathrin and clathrin adaptor molecules than conventional vesicles, but this increase is insufficient to permit full coating of the vesicle. We further show that virus-containing vesicles depend upon the actin machinery for their internalization. Specifically, we found that components of the actin machinery are recruited to virus-containing vesicles, and chemical inhibition of actin polymerization trapped viral particles in vesicles at the plasma membrane. By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit. This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery.

Nicotinic acetylcholine receptor is internalized via a Rac-dependent, dynamin-independent endocytic pathway.

Endocytosis of the nicotinic acetylcholine receptor (AChR) is a proposed major mechanism of neuromodulation at neuromuscular junctions and in the pathology of synapses in the central nervous system. We show that binding of the competitive antagonist alpha-bungarotoxin (alphaBTX) or antibody-mediated cross-linking induces the internalization of cell surface AChR to late endosomes when expressed heterologously in Chinese hamster ovary cells or endogenously in C2C12 myocytes. Internalization occurs via sequestration of AChR-alphaBTX complexes in narrow, tubular, surface-connected compartments, which are indicated by differential surface accessibility of fluorescently tagged alphaBTX-AChR complexes to small and large molecules and real-time total internal reflection fluorescence imaging. Internalization occurs in the absence of clathrin, caveolin, or dynamin but requires actin polymerization. alphaBTX binding triggers c-Src phosphorylation and subsequently activates the Rho guanosine triphosphatase Rac1. Consequently, inhibition of c-Src kinase activity, Rac1 activity, or actin polymerization inhibits internalization via this unusual endocytic mechanism. This pathway may regulate AChR levels at ligand-gated synapses and in pathological conditions such as the autoimmune disease myasthenia gravis.

Role of lipids and actin in the formation of clathrin-coated pits.

Assembly of clathrin-coated pits and their maturation into coated vesicles requires coordinated interactions between specific lipids and several structural and regulatory proteins. In the presence of primary alcohols, phospholipase D generates phosphatidylalcohols instead of PA, reducing stimulation of phosphatidyl inositol 5-kinase (PI5K) and hence decreasing formation of phosphoinositide-4,5-biphosphate (PIP(2)). Using live-cell imaging, we have shown that acute treatment of cells with 1-butanol or other small primary alcohols induces rapid disassembly of coated pits at the plasma membrane and blocks appearance of new ones. Addition of exogenous PIP(2) reverses this effect. Coated pits and vesicles reappear synchronously upon removal of 1-butanol; we have used this synchrony to assess the role of actin in coated vesicle assembly. Prolonged inhibition of actin polymerization by latrunculin A or cytochalasin D reduced by approximately 50% the frequency of coated pit formation without affecting maturation into coated vesicles. As in control cells, removal of 1-butanol in the continued presence of an actin depolymerizer led to synchronous appearance of new pits, which matured normally. Thus, remodeling of the actin cytoskeleton is not essential for clathrin-coated vesicle assembly but may indirectly affect the nucleation of clathrin-coated pits.

A burst of auxilin recruitment determines the onset of clathrin-coated vesicle uncoating.

Clathrin-coated pits assemble on a membrane and pinch off as coated vesicles. The released vesicles then rapidly lose their clathrin coats in a process mediated by the ATPase Hsc70, recruited by auxilin, a J-domain-containing cofactor. How is the uncoating process regulated? We find that during coat assembly small and variable amounts of auxilin are recruited transiently but that a much larger burst of association occurs after the peak of dynamin signal, during the transition between membrane constriction and vesicle budding. We show that the auxilin burst depends on domains of the protein likely to interact with lipid head groups. We conclude that the timing of auxilin recruitment determines the onset of uncoating. We propose that, when a diffusion barrier is established at the constricting neck of a fully formed coated pit and immediately after vesicle budding, accumulation of a specific lipid can recruit sufficient auxilin molecules to trigger uncoating.

Dynasore, a cell-permeable inhibitor of dynamin.

Dynamin is essential for clathrin-dependent coated vesicle formation. It is required for membrane budding at a late stage during the transition from a fully formed pit to a pinched-off vesicle. Dynamin may also fulfill other roles during earlier stages of vesicle formation. We have screened about 16,000 small molecules and have identified 1, named here dynasore, that interferes in vitro with the GTPase activity of dynamin1, dynamin2, and Drp1, the mitochondrial dynamin, but not of other small GTPases. Dynasore acts as a potent inhibitor of endocytic pathways known to depend on dynamin by rapidly blocking coated vesicle formation within seconds of dynasore addition. Two types of coated pit intermediates accumulate during dynasore treatment, U-shaped, half formed pits and O-shaped, fully formed pits, captured while pinching off. Thus, dynamin acts at two steps during clathrin coat formation; GTP hydrolysis is probably needed at both steps.

Perforin triggers a plasma membrane-repair response that facilitates CTL induction of apoptosis.

Perforin delivers granzymes to induce target-cell apoptosis. At high concentrations, perforin multimerizes in the plasma membrane to form pores. However, whether granzymes enter target cells via membrane pores is uncertain. Here we find that perforin at physiologically relevant concentrations and during cell-mediated lysis creates pores in the target-cell membrane, transiently allowing Ca(2+) and small dyes into the cell. The Ca(2+) flux triggers a wounded membrane-repair response in which internal vesicles, including lysosomes and endosomes, donate their membranes to reseal the damaged membrane. Perforin also triggers the rapid endocytosis of granzymes into large EEA-1-staining vesicles. The restoration of target-cell membrane integrity by triggering the repair response is necessary for target cells subjected to cytotoxic T lymphocyte attack to avoid necrosis and undergo the slower process of programmed cell death. Thus, the target cell actively participates in determining its own fate during cell-mediated death.

Possible role of deep tubular invaginations of the plasma membrane in MHC-I trafficking.

Tubules and vesicles are membrane carriers involved in traffic along the endocytic and secretory routes. The small GTPase Arf6 regulates a recycling branch of short dynamic tubular intermediates used by major histocompatibility class I (MHC-I) molecules to traffic through vesicles between endosomes and the plasma membrane. We observed that Arf6 also affects a second network of very long and stable tubules containing MHC-I, many of which correspond to deep invaginations of the plasma membrane. Treatment with wortmannin, an inhibitor of phosphatidylinositol-3-phosphate kinase, prevents formation of the short dynamic tubules while increasing the number of the long and very stable ones. Expression of NefAAAA, a mutant form of HIV Nef, increases the number of cells containing the stable tubules, and is used here as a tool to facilitate their study. Photoactivation of NefAAAA-PA-GFP demonstrates that this molecule traffics from endosomes to the tubules. Finally, live-cell imaging also shows internalization of MHC-I molecules into these tubules, suggesting that this is an additional route for MHC-I traffic.

The transcription factor NFAT3 mediates neuronal survival.

Neuronal apoptosis is critical for normal development of the mammalian nervous system and also contributes to the pathogenesis of ischemic and degenerative diseases of the brain. Apoptosis of neurons is tightly regulated by extrinsic signals including growth factors and neuronal activity, but the intracellular mechanisms by which these signals promote neuronal survival are incompletely understood. We report that the transcription factor NFAT3 plays a critical role in mediating survival of granule neurons of the developing cerebellum. NFAT3 accumulated in the nucleus of primary granule neurons under survival conditions of serum growth factors and neuronal activity that was elicited by depolarization with high K(+). In contrast, deprivation of serum and K(+), which leads to neuronal apoptosis, triggered NFAT3 nuclear export. Treatment of granule neurons with Li(+), an inhibitor of the NFAT export kinase GSK3, prevented the nuclear export of NFAT3 and increased granule cell survival even under pro-apoptotic conditions. Thus, the nuclear localization of NFAT3 correlated tightly with granule neuron survival. Consistent with a pro-survival function for NFAT3, genetic knockdown of NFAT3 by RNA interference in primary granule neurons led to increased apoptosis even in neurons cultured under survival conditions. Conversely, expression of a constitutively active form of NFAT protected neurons against apoptosis induced by serum withdrawal and low K(+). Taken together, these results reveal an essential function for NFAT3-mediated transcription in neuronal survival that may play important roles in the developing and mature brain.