A presynaptic endosomal trafficking pathway controls synaptic growth signaling

Structural remodeling of synapses in response to growth signals leads to long-lasting alterations in neuronal function in many systems. Synaptic growth factor receptors alter their signaling properties during transit through the endocytic pathway, but the mechanisms controlling cargo traffic between endocytic compartments remain unclear. Nwk (Nervous Wreck) is a presynaptic F-BAR/SH3 protein that regulates synaptic growth signaling in Drosophila melanogaster. In this paper, we show that Nwk acts through a physical interaction with sorting nexin 16 (SNX16). SNX16 promotes synaptic growth signaling by activated bone morphogenic protein receptors, and live imaging in neurons reveals that SNX16-positive early endosomes undergo transient interactions with Nwk-containing recycling endosomes. We identify an alternative signal termination pathway in the absence of Snx16 that is controlled by endosomal sorting complex required for transport (ESCRT)-mediated internalization of receptors into the endosomal lumen. Our results define a presynaptic trafficking pathway mediated by SNX16, NWK, and the ESCRT complex that functions to control synaptic growth signaling at the interface between endosomal compartments.     

Syndecan-2 induces filopodia by active cdc42Hs

The syndecans, a family of transmembrane heparan sulfate proteoglycans, are ubiquitous molecules whose intracellular function is still unknown. To examine the function of syndecan-2, one of the most abundant heparan sulfate proteoglycan in fibroblasts, we performed transfection studies in COS-1 and Swiss 3T3 cells. Endogenous syndecan-2 colocalized with F-actin in cortical structures. Overexpression of full-length syndecan-2 induced the formation of long filopodia-like structures. These changes correlated with a rearrangement of the actin cytoskeleton, which strongly colocalized with syndecan-2. Overexpression of syndecan-2 lacking the extracellular domain increased the number of microspikes on the cell surface but failed to induce filopodia. Addition of heparin blocked the effect of full-length syndecan-2, suggesting that heparan sulfate chains in the extracellular domain are necessary to induce filopodia. Coexpression of cdc42Hs negative-dominant N17 blocked syndecan-2-induced filopodia and cdc42Hs positive-dominant V12 had a synergic effect. This indicates that active cdc42Hs is necessary for syndecan-2 induction of filopodia. These results provide a link between syndecan-2, actin cytoskeleton, and cdc42Hs.     

Neuromodulatory changes in short-term synaptic dynamics may be mediated by two distinct mechanisms of presynaptic calcium entry

Although synaptic output is known to be modulated by changes in presynaptic calcium channels, additional pathways for calcium entry into the presynaptic terminal, such as non-selective channels, could contribute to modulation of short term synaptic dynamics. We address this issue using computational modeling. The neuropeptide proctolin modulates the inhibitory synapse from the lateral pyloric (LP) to the pyloric dilator (PD) neuron, two slow-wave bursting neurons in the pyloric network of the crab Cancer borealis. Proctolin enhances the strength of this synapse and also changes its dynamics. Whereas in control saline the synapse shows depression independent of the amplitude of the presynaptic LP signal, in proctolin, with high-amplitude presynaptic LP stimulation the synapse remains depressing while low-amplitude stimulation causes facilitation. We use simple calcium-dependent release models to explore two alternative mechanisms underlying these modulatory effects. In the first model, proctolin directly targets calcium channels by changing their activation kinetics which results in gradual accumulation of calcium with low-amplitude presynaptic stimulation, leading to facilitation. The second model uses the fact that proctolin is known to activate a non-specific cation current I _MI . In this model, we assume that the MI channels have some permeability to calcium, modeled to be a result of slow conformation change after binding calcium. This generates a gradual increase in calcium influx into the presynaptic terminals through the modulatory channel similar to that described in the first model. Each of these models can explain the modulation of the synapse by proctolin but with different consequences for network activity.     

Activity-induced Notch signaling in neurons requires Arc/Arg3.1 and is essential for synaptic plasticity in hippocampal networks

Notch signaling in the nervous system has been most studied in the context of cell fate specification. However, numerous studies have suggested that Notch also regulates neuronal morphology, synaptic plasticity, learning, and memory. Here we show that Notch1 and its ligand Jagged1 are present at the synapse, and that Notch signaling in neurons occurs in response to synaptic activity. In addition, neuronal Notch signaling is positively regulated by Arc/Arg3.1, an activity-induced gene required for synaptic plasticity. In Arc/Arg3.1 mutant neurons, the proteolytic activation of Notch1 is disrupted both in vivo and in vitro. Conditional deletion of Notch1 in the postnatal hippocampus disrupted both long-term potentiation (LTP) and long-term depression (LTD), and led to deficits in learning and short-term memory. Thus, Notch signaling is dynamically regulated in response to neuronal activity, Arc/Arg3.1 is a context-dependent Notch regulator, and Notch1 is required for the synaptic plasticity that contributes to memory formation.     

Selective control of basolateral membrane protein polarity by cdc42

The rho GTPase cdc42 is implicated in several aspects of cell polarity. A recent study (Kroschewski R, Hall A, Mellman I. Nat Cell Biol 1999;1:8–13) demonstrated that a dominant negative mutant of cdc42 abolishes the polarity of basolateral membrane proteins in MDCK cells, but did not elucidate whether this effect was selective for basolateral proteins or nonselective for all secreted proteins. To answer this question, we analyzed the polarity of newly synthesized membrane and soluble proteins in MDCK cell lines previously induced to overexpress mutant forms of cdc42. GTPase-deficient and dominant negative cdc42 did not affect the apical targeting of a newly synthesized apical membrane protein, but reversed to apical the distribution of two exogenous basolateral membrane proteins. In striking contrast, GTPase-deficient cdc42 did not affect polarized exocytosis of endogenous soluble proteins, either apical or basolateral. The exquisitely selective regulation of polarized protein targeting by cdc42 may allow cells to fine-tune their membrane composition in response to extracellular signals during development, migration and in response to injury.     

A role for local calcium signaling in rapid synaptic partner selection by dendritic filopodia

Synapse elimination is an important process underlying the establishment of functional neuronal networks during development. Here, we tested the idea that neurons select among potential synaptic partners already during initial contact formation between dendritic filopodia and axons-well before mature synapses are established. We show that filopodia frequently make contact with axons, and while some contacts are selectively stabilized, many are short-lived. More specifically, we demonstrate that contacts with a certain population of GABAergic axons never get stabilized, indicating that filopodia already early on select between different types of axons. Local dendritic calcium transients that are independent of glutamate occur within seconds after contact formation, and their frequency is high where contacts become stabilized and low at short-lived contacts. Thus, filopodia are capable of choosing between potential synaptic partners well before a mature synapse is established.     

Cytoskeletal alterations in Dictyostelium induced by expression of human cdc42

The rho family of small G proteins has been shown to be involved in controlling actin filament dynamics in cells. To evaluate the functional overlap between human and Dictyostelium G proteins, we conditionally expressed constitutively active human cdc42 (V12-cdc42) in Dictyostelium cells. Upon induction, cells adopted a unique morphology: a flattened shape with wrinkles running from the cell edge toward the center. The appearance of these wrinkles is highly dynamic so that the cells cycle between the wrinkled and relatively normal morphologies. Phalloidin staining indicates that the stellate wrinkles contain dense actin structures and also that numerous filopods project vertically from the center of these cells. Consistent with the hypothesis that cdc42 induces actin polymerization in vivo, cells expressing V12-cdc42 show an increase in the amount of F-actin associated with the cytoskeleton. This is accompanied by an increase in the association of the actin-binding proteins 34-kDa bundler, ABP-120 and alpha-actinin with the cytoskeleton. In conclusion, human cdc42 has various effects on the Dictyostelium actin cytoskeleton consistent with a conserved role of small GTPases in control of the cytoskeleton.     

Associative short-term synaptic plasticity mediated by endocannabinoids

Associative learning is important on rapid timescales, but no suitable form of short-term plasticity has been identified that is both associative and synapse specific. Here, we assess whether endocannabinoids can mediate such plasticity. In the cerebellum, bursts of parallel fiber (PF) activity evoke endocannabinoid release from Purkinje cell dendrites that results in retrograde synaptic inhibition lasting seconds. We find that the powerful climbing fiber (CF) to Purkinje cell synapse regulates this inhibition. Compared to PF stimulation alone, coactivation of PF and CF synapses greatly enhanced endocannabinoid-mediated inhibition of PF synapses. Retrograde inhibition was restricted to PFs activated within several hundred milliseconds of CF activation. This associative plasticity reflects two aspects of calcium-dependent endocannabinoid release. First, PF-mediated activation of metabotropic glutamate receptors locally reduced the dendritic calcium levels required for endocannabinoid release. Second, CF and PF coactivation evoked localized supralinear dendritic calcium signals. Thus, endocannabinoids mediate transient associative synaptic plasticity.     

Promyogenic function of Integrin/FAK signaling is mediated by Cdo, Cdc42 and MyoD

The Integrin-mediated cell adhesion to the extracellular matrix is implicated in the control of proliferation, survival, migration and differentiation of myoblasts. Focal adhesion kinase (FAK) mediates signals from Integrins and plays an essential role in myotube formation. Cdo forms a multiprotein complex that includes other cell adhesion molecules like Cadherins and Boc. Multiple signals emanate from such complexes, including Cdc42 and p38MAPK pathways to activate MyoD. Here we show that C2C12 myoblasts cultured in suspension or on Poly-L-Lysine (PLL), a well known Integrin-independent substratum, failed to express Cdo and MyoD, while the expression of Cadherins and Boc was unchanged. In addition, the activation of Akt and p38MAPK as well as the expression of Cdc42 was affected in these cells. Overexpression of FAK rescued MyoD and Cdo expression as well as myotube formation of C2C12 cells on PLL. Furthermore, reintroduction of Cdo induced enhanced myotube formation on PLL and increased the expression of myogenic markers. Inhibition of ROCK or overexpression of Cdc42-V12 in C2C12 cells upregulated Cdc42 and MyoD expression and rescued defective myoblast differentiation. Taken together, these data indicate that the Integrin/FAK signaling pathway is required for myoblast differentiation by regulating the expression of the promyogenic factors, Cdo, MyoD and Cdc42.     

Functional diversity on synaptic plasticity mediated by endocannabinoids

Endocannabinoids (eCBs) act as modulators of synaptic transmission through activation of a number of receptors, including, but not limited to, cannabinoid receptor 1 (CB1). eCBs share CB1 receptors as a common target with Δ⁹-tetrahydrocannabinol (THC), the main psychoactive ingredient in marijuana. Although THC has been used for recreational and medicinal purposes for thousands of years, little was known about its effects at the cellular level or on neuronal circuits. Identification of CB1 receptors and the subsequent development of its specific ligands has therefore enhanced our ability to study and bring together a substantial amount of knowledge regarding how marijuana and eCBs modify interneuronal communication. To date, the eCB system, composed of cannabinoid receptors, ligands and the relevant enzymes, is recognized as the best-described retrograde signalling system in the brain. Its impact on synaptic transmission is widespread and more diverse than initially thought. The aim of this review is to succinctly present the most common forms of eCB-mediated modulation of synaptic transmission, while also illustrating the multiplicity of effects resulting from specializations of this signalling system at the circuital level.     

Potentiation of glutamatergic synaptic input to supraoptic neurons by presynaptic nicotinic receptors

The release of vasopressin and oxytocin from the supraoptic nucleus (SON) neurons is tonically regulated by excitatory glutamatergic and inhibitory GABAergic synaptic inputs. Acetylcholine is known to excite SON neurons and to elicit vasopressin release. Cholinergic receptors are located pre- and postsynaptically in the SON, but their functional significance in the regulation of SON neurons is not fully understood. In this study, we determined the role of presynaptic cholinergic receptors in regulation of the excitatory glutamatergic inputs to the SON neurons. The magnocellular neurons in the rat hypothalamic slices were identified microscopically, and the spontaneous miniature excitatory postsynaptic currents (mEPSCs) were recorded using the whole cell voltage-clamp technique. The mEPSCs were abolished by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (20 microM). Acetylcholine (100 microM) significantly increased the frequency of mEPSCs of 38 SON neurons from 1.87 +/- 0.36 to 3.42 +/- 0.54 Hz but did not alter the amplitude (from 19.61 +/- 0.90 to 19.34 +/- 0.84 pA) and the decay time constant of mEPSCs. Furthermore, the nicotinic receptor antagonist mecamylamine (10 microM, n = 16), but not the muscarinic receptor antagonist atropine (100 microM, n = 12), abolished the excitatory effect of acetylcholine on the frequency of mEPSCs. These data provide new information that the excitatory effect of acetylcholine on the SON neurons is mediated, at least in part, by its effect on presynaptic glutamate release. Activation of presynaptic nicotinic, but not muscarinic, receptors located in the glutamatergic terminals increases the excitatory synaptic input to the SON neurons of the hypothalamus.     

Regulation of hyphal morphogenesis by cdc42 and rac1 homologues in Aspergillus nidulans

The ability of filamentous fungi to form hyphae requires the establishment and maintenance of a stable polarity axis. Based on studies in yeasts and animals, the GTPases Cdc42 and Rac1 are presumed to play a central role in organizing the morphogenetic machinery to enable axis formation and stabilization. Here, we report that Cdc42 (ModA) and Rac1 (RacA) share an overlapping function required for polarity establishment in Aspergillus nidulans. Nevertheless, Cdc42 appears to have a more important role in hyphal morphogenesis in that it alone is required for the timely formation of lateral branches. In addition, we provide genetic evidence suggesting that the polarisome components SepA and SpaA function downstream of Cdc42 in a pathway that may regulate microfilament formation. Finally, we show that microtubules become essential for the establishment of hyphal polarity when the function of either Cdc42 or SepA is compromised. Our results are consistent with the action of parallel Cdc42 and microtubule-based pathways in regulating the formation of a stable axis of hyphal polarity in A. nidulans.     

Calcium signaling and synaptic modulation: regulation of endocannabinoid-mediated synaptic modulation by calcium

Postsynaptic Ca2+ signal influences synaptic transmission through multiple mechanisms. Some of them involve retrograde messengers that are released from postsynaptic neurons in a Ca2+-dependent manner and modulate transmitter release through activation of presynaptic receptors. Recent studies have revealed essential roles of endocannabinoids in retrograde modulation of synaptic transmission. Endocannabinoid release is induced by either postsynaptic Ca2+ elevation alone or activation of postsynaptic Gq/11-coupled receptors with or without Ca2+ elevation. The former pathway is independent of phospholipase Cbeta (PLCbeta) and requires a large Ca2+ elevation to a micromolar range. The latter pathway requires PLCbeta and is facilitated by a moderate Ca2+ elevation to a submicromolar range. This facilitation is caused by Ca2+-dependency of receptor-driven PLCbeta activation. The released endocannabinoids then activate presynaptic cannabinoid receptor type 1 (CB1), and suppress transmitter release from presynaptic terminals. Both CB1 receptors and Gq/11-coupled receptors are widely distributed in the brain. Thus, the endocannabinoid-mediated retrograde modulation may be an important and widespread mechanism in the brain, by which postsynaptic events including Gq/11-coupled receptor activation and Ca2+ elevation can retrogradely influence presynaptic function.     

Metabolic stress signaling mediated by mixed-lineage kinases

Saturated free fatty acid (FFA) is a major source of metabolic stress that activates the c-Jun NH(2)-terminal kinase (JNK). This FFA-stimulated JNK pathway is relevant to hallmarks of metabolic syndrome, including insulin resistance. Here we used gene ablation studies in mice to demonstrate a central role for mixed-lineage protein kinases (MLK) in this signaling pathway. Saturated FFA causes protein kinase C (PKC)-dependent activation of MLK3 that subsequently causes increased JNK activity by a mechanism that requires the MAP kinase kinases MKK4 and MKK7. Loss of PKC, MLK3, MKK4, or MKK7 expression prevents FFA-stimulated JNK activation. Together, these data establish a signaling pathway that mediates effects of metabolic stress on insulin resistance.     

Redox signaling mediated by the gut microbiota

Abstract

The microbiota that occupies the mammalian intestine can modulate a range of physiological functions, including control over immune responses, epithelial barrier function, and cellular proliferation. While commensal prokaryotic organisms are well known to stimulate inflammatory signaling networks, less is known about control over homeostatic pathways. Recent work has shown that gut epithelia contacted by enteric commensal bacteria rapidly generate reactive oxygen species (ROS). While the induced production of ROS in professional phagocytes via stimulation of formyl peptide receptors (FPRs) and activation of NADPH oxidase 2 (Nox2) is a well-studied process, ROS are also similarly elicited in other cell types, including intestinal epithelia, in response to microbial signals via FPRs and the epithelial NADPH oxidase 1 (Nox1). ROS generated by Nox enzymes have been shown to function as critical second messengers in multiple signal transduction pathways via the rapid and transient oxidative inactivation of a distinct class of sensor proteins bearing oxidant-sensitive thiol groups. These redox-sensitive proteins include tyrosine phosphatases that serve as regulators of MAP kinase pathways, focal adhesion kinase, as well as components involved in NF-κB activation. As microbe-elicited ROS has been shown to stimulate cellular proliferation and motility, and to modulate innate immune signaling, we hypothesize that many of the established effects of the normal microbiota on intestinal physiology may be at least partially mediated by this ROS-dependent mechanism.