Conformational signaling required for synaptic plasticity by the NMDA receptor complex

The NMDA receptor (NMDAR) is known to transmit important information by conducting calcium ions. However, some recent studies suggest that activation of NMDARs can trigger synaptic plasticity in the absence of ion flow. Does ligand binding transmit information to signaling molecules that mediate synaptic plasticity? Using Förster resonance energy transfer (FRET) imaging of fluorescently tagged proteins expressed in neurons, conformational signaling is identified within the NMDAR complex that is essential for downstream actions. Ligand binding transiently reduces FRET between the NMDAR cytoplasmic domain (cd) and the associated protein phosphatase 1 (PP1), requiring NMDARcd movement, and persistently reduces FRET between the NMDARcd and calcium/calmodulin-dependent protein kinase II (CaMKII), a process requiring PP1 activity. These studies directly monitor agonist-driven conformational signaling at the NMDAR complex required for synaptic plasticity.Agonist binding to the NMDAR is required for two major forms of synaptic plasticity: long-term potentiation (LTP) and long-term depression (LTD) (1). Surprisingly, activation of NMDARs can produce plasticity in opposite directions, with LTP enhancing transmission and LTD reducing transmission. A model was developed (2, 3) to explain how activation of NMDAR could produce these opposing phenomena: strong stimuli during LTP induction drive a large flux of Ca²⁺ through NMDARs, leading to a large increase in intracellular calcium ion concentration ([Ca²⁺]_i) that activates one series of biochemical steps leading to synaptic potentiation; a weaker stimulus during LTD induction drives a more reduced flux of Ca²⁺ through NMDARs, producing a modest increase in [Ca²⁺]_i that activates a different series of biochemical steps, leading to synaptic depression. However, this model is not consistent with recent studies suggesting that no change in [Ca²⁺]_i is required for LTD, and instead invokes metabotropic signaling by the NMDAR (4). Studies supporting an ion-flow-independent role for NMDARs in LTD (4–7) and other processes (7–13) stand in contrast to studies proposing that flow of Ca²⁺ through NMDAR is required for LTD (14) (see ref. 15 for additional references). An important test of an ion-flow-independent model would be to measure directly signaling actions by NMDARs in the absence of ion flow.     

GluN3A expression restricts spine maturation via inhibition of GIT1/Rac1 signaling

NMDA-type glutamate receptors (NMDARs) guide the activity-dependent remodeling of excitatory synapses and associated dendritic spines during critical periods of postnatal brain development. Whereas mature NMDARs composed of GluN1 and GluN2 subunits mediate synapse plasticity and promote spine growth and stabilization, juvenile NMDARs containing GluN3A subunits are thought to inhibit these processes via yet unknown mechanisms. Here, we report that GluN3A binds G protein-coupled receptor kinase-interacting protein (GIT1), a postsynaptic scaffold that assembles actin regulatory complexes, including the Rac1 guanine nucleotide exchange factor βPIX, to promote Rac1 activation in spines. Binding to GluN3A limits the synaptic localization of GIT1 and its ability to complex βPIX, leading to decreased Rac1 activation and reduced spine density and size in primary cultured neurons. Conversely, knocking out GluN3A favors the formation of GIT1/βPIX complexes and increases the activation of Rac1 and its main effector p21-activated kinase. We further show that binding of GluN3A to GIT1 is regulated by synaptic activity, a response that might restrict the negative regulatory effects of GluN3A on actin signaling to inactive synapses. Our results identify inhibition of Rac1/p21-activated kinase actin signaling pathways as an activity-dependent mechanism mediating the inhibitory effects of GluN3A on spine morphogenesis.During the development of neural circuits, a phase of intense synaptogenesis is followed by a period of activity-dependent remodeling (or “synaptic refinement”) in which more than half of the initially formed synapses are eliminated, whereas other connections will mature and be kept (1, 2). The subunit composition of NMDA-type glutamate receptors (NMDARs) expressed by individual synapses during this critical period is a key factor influencing functional and structural synaptic plasticity and, in turn, synapse fate (3). Mature NMDARs composed of GluN1 and GluN2 subunits drive the maturation of active synapses by detecting coincident pre- and postsynaptic activity and coupling this activity to signaling pathways that trigger the enlargement and stabilization of synapses and associated dendritic spines (4–6). This structural plasticity is critical for coupling the wiring of neural circuits to experience and supporting the long-term maintenance of spines and memories. During the refinement stage, NMDARs additionally contain GluN3A subunits that serve as a brake on synapse maturation and stabilization, which might provide a counterbalance to limit synapse numbers. Supporting this idea, loss of GluN3A increases spine density and size (7) and accelerates the expression of markers of synaptic maturation (8), whereas overexpression reduces synapse and spine density and yields a higher proportion of smaller, immature spines (9). However, the downstream mechanisms by which GluN3A inhibits synapse and spine maturation remain unknown.Spines are actin-rich, and their structural remodeling relies on rearrangements of the actin cytoskeleton (10–13). Cytoskeletal rearrangements are regulated by the Rho family of small GTPases, and two members of this family, Rac1 and RhoA, are major regulators of spine remodeling (14, 15). Rho-GTPases act as molecular switches that cycle between an inactive GDP-bound conformation and an active GTP-bound conformation (16). Their activation state is controlled by guanine exchange factors (GEFs), which promote the exchange of GDP for GTP, and GTPase-activating proteins (GAPs), which catalyze GTP hydrolysis. Several Rac1-specific GEFs, including Kalirin7, Tiam1, and βPIX, are targeted to synapses via interactions with scaffolding proteins, which allows local regulation of actin remodeling in spines and its coupling to synaptic activity (12, 17–22). Although many studies have shown that NMDAR activation induces cytoskeletal and spine remodeling by activating Rac1-GEFs (23–25), much less is known about pathways that restrict excitatory synapse maturation and/or promote elimination.Here, we identify a physical association between the intracellular C-terminal domain of GluN3A subunits and G protein-coupled receptor kinase-interacting protein (GIT1), a postsynaptic scaffold that assembles a multiprotein signaling complex with Rac1 and the Rac1-GEF βPIX to regulate actin dynamics in spines (19, 26). GIT1 selectively bound juvenile NMDARs containing GluN3A but not mature NMDAR subtypes, and binding was regulated by activity because it could be enhanced or reduced, respectively, by brief episodes of synaptic inactivity or synaptic stimulation. A functional analysis demonstrates that binding to GluN3A interferes with the synaptic localization of GIT1 and its ability to recruit βPIX, leading to decreased Rac1 activation. We finally show that GluN3A-induced reductions in spine density and size critically require GIT1 binding. We propose that the coupling of GIT1/GluN3A binding to synapse use might provide an effective mechanism with which to restrict the maturation and growth of inactive synapses in a selective manner.     

Disruption of hippocampal–prefrontal cortex activity by dopamine D2R-dependent LTD of NMDAR transmission

Functional connectivity between the hippocampus and prefrontal cortex (PFC) is essential for associative recognition memory and working memory. Disruption of hippocampal–PFC synchrony occurs in schizophrenia, which is characterized by hypofunction of NMDA receptor (NMDAR)-mediated transmission. We demonstrate that activity of dopamine D2-like receptors (D2Rs) leads selectively to long-term depression (LTD) of hippocampal–PFC NMDAR-mediated synaptic transmission. We show that dopamine-dependent LTD of NMDAR-mediated transmission profoundly disrupts normal synaptic transmission between hippocampus and PFC. These results show how dopaminergic activation induces long-term hypofunction of NMDARs, which can contribute to disordered functional connectivity, a characteristic that is a hallmark of psychiatric disorders such as schizophrenia.The hippocampus to medial prefrontal cortex (PFC) projection is important for executive function and working and long-term memory (1, 2). Glutamatergic neurons of the ventral hippocampal cornu ammonis 1 (CA1) region project directly to layers 2–6 of ipsilateral PFC, and this connection synchronizes PFC and hippocampal activity during particular behavioral conditions (3–5). Disruption of hippocampal–PFC synchrony is associated with cognitive deficits that occur in disorders such as schizophrenia (6). Hippocampal–PFC uncoupling can be achieved by NMDA receptor (NMDAR) antagonism (7), and NMDAR hypofunction is a recognized feature of schizophrenia (8). However, it is unclear, first, how changes in NMDAR function at this synapse may arise, and second, how NMDAR hypofunction affects hippocampal–PFC synaptic transmission.Canonically, NMDARs are considered to contribute little to single synaptic events, but the slow kinetics of NMDARs contribute to maintaining depolarization, leading to the generation of bursts of action potentials (9–13). Furthermore, NMDARs coordinate spike timing relative to the phase of field potential oscillations (14, 15). NMDAR transmission itself undergoes synaptic plasticity (16, 17), and this can have a profound effect on sustained depolarization, burst firing, synaptic integration, and metaplasticity (9, 11, 18, 19). In PFC, NMDARs are oppositely regulated by dopamine receptors; D1-like receptors (D1Rs) potentiate and D2-like receptors (D2Rs) depress NMDAR currents (20). Interestingly, NMDAR hypofunction (8, 21) and dopamine D2 receptor activity (22) are potentially converging mechanisms contributing to schizophrenia (23).We now examine the contribution of NMDARs to transmission at the hippocampal–PFC synapse. We show that NMDAR activity provides sustained depolarization that can trigger action potentials during bursts of hippocampal input to PFC. We next demonstrate that dopamine D2 receptor-dependent long-term depression (LTD) of NMDAR transmission profoundly attenuates summation of synaptic transmission and neuronal firing at the hippocampal–PFC input. These findings allow for a mechanistic understanding of how alterations in dopamine and NMDAR function can lead to the disruption of hippocampal–PFC functional connectivity, which characterizes certain psychiatric disorders.     

Identity of the NMDA receptor coagonist is synapse specific and developmentally regulated in the hippocampus

NMDA receptors (NMDARs) require the coagonists d-serine or glycine for their activation, but whether the identity of the coagonist could be synapse specific and developmentally regulated remains elusive. We therefore investigated the contribution of d-serine and glycine by recording NMDAR-mediated responses at hippocampal Schaffer collaterals (SC)–CA1 and medial perforant path–dentate gyrus (mPP–DG) synapses in juvenile and adult rats. Selective depletion of endogenous coagonists with enzymatic scavengers as well as pharmacological inhibition of endogenous d-amino acid oxidase activity revealed that d-serine is the preferred coagonist at SC–CA1 mature synapses, whereas, unexpectedly, glycine is mainly involved at mPP–DG synapses. Nevertheless, both coagonist functions are driven by the levels of synaptic activity as inferred by recording long-term potentiation generated at both connections. This regional compartmentalization in the coagonist identity is associated to different GluN1/GluN2A to GluN1/GluN2B subunit composition of synaptic NMDARs. During postnatal development, the replacement of GluN2B- by GluN2A-containing NMDARs at SC–CA1 synapses parallels a change in the identity of the coagonist from glycine to d-serine. In contrast, NMDARs subunit composition at mPP–DG synapses is not altered and glycine remains the main coagonist throughout postnatal development. Altogether, our observations disclose an unprecedented relationship in the identity of the coagonist not only with the GluN2 subunit composition at synaptic NMDARs but also with astrocyte activity in the developing and mature hippocampus that reconciles the complementary functions of d-serine and glycine in modulating NMDARs during the maturation of tripartite glutamatergic synapses.The glutamate-gated N-methyl-d-aspartate receptors (NMDARs) play a critical role in structural and functional plasticity at synapses during postnatal brain development and in adulthood (1) and are therefore central to many cognitive functions such as learning and memory (2). Disturbances of their functions have been associated to a broad range of neurological and psychiatric disorders (3). NMDARs are heterotetramers typically composed of GluN1 and GluN2 subunits (3, 4), and the precise subunit composition determines NMDAR functional and trafficking properties (3, 4).NMDARs are unique among neurotransmitter receptors because their activation requires the binding of both glutamate and a coagonist initially thought to be glycine (5, 6). Nevertheless, subsequent studies have shown that d-serine synthesized by serine racemase (SR) (7) would be the preferred endogenous coagonist for synaptic NMDARs in many areas of the mature brain (8), raising controversies about “where, when, and how” glycine and d-serine might regulate NMDARs at synapses in the brain. This controversy is highlighted by the recent findings showing that d-serine and glycine both released by neurons come into play to regulate synaptic NMDAR-dependent functions at the hippocampal Schaffer collateral (SC)–CA1 synapses of adult brain (9), whereas others found no evidence for a function of glycine at this connection (10). Possible explanations for the relative contribution of d-serine and glycine in gating mature NMDARs were recently given by two recent studies. First, it was shown at hippocampal SC–CA1 synapses that d-serine would target GluN1/GluN2A-containing NMDARs, which are preferentially present within the synapse, whereas glycine would rather target GluN1/GluN2B-containing NMDARs located extrasynaptically (10). Second, it was proposed that the identity of the effective coagonist at synapses could depend on synaptic activity levels with tonic activation of NMDARs under low-activity conditions supported by ambient d-serine, whereas glycine will contribute in response to enhanced afferent activity (11).So far, most studies have explored the functions of d-serine vs. glycine at excitatory synapses in the adult brain or during aging (8) where GluN2A-expressing NMDARs prevail (1). Intriguingly, the respective role of the coagonists during postnatal development awaits to be addressed. Considerable evidence indicates that the NMDAR composition at excitatory synapses undergo an experience-dependent developmental switch from primarily GluN2B to GluN2A subunits during the first 2 wk of maturation and refinement of cortical circuits in the postnatal brain (1, 12–14). However, how this developmental switch is controlled is still elusive, and we do not know how it could be regulated or associated to the action of a preferred coagonist.The present study aimed at investigating the relative synapse specificity and the time window at which d-serine and glycine enter in function to drive NMDAR activity of developing and mature excitatory synapses in the hippocampus. In particular, we sought to elucidate whether the preference for one of the two coagonists could be related to any of the GluN2 subtypes of NMDARs.     

Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces

Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo–cross-linker p-azido-l-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion.Introducing light-sensitive moieties into proteins provides a powerful approach to investigate molecular mechanisms as well as biological functions with high temporal and spatial resolution (1, 2). An attractive strategy to engineer light responsiveness relies on the use of photoreactive unnatural amino acids (UAAs), allowing site-specific incorporation in a protein target. The methodology relies on the read-through of an unassigned codon (commonly the amber stop codon) in an mRNA by a suppressor tRNA aminoacylated with a desired UAA. Using this approach, UAAs with unique chemical functionalities including light-sensitivity have been successfully incorporated into ion channels and neurotransmitter receptors, significantly contributing to our understanding of receptor function (3, 4). However, the challenging synthesis of the chemically acylated tRNA has limited the general applicability of the approach. The recent development of genetically engineered suppressor tRNA/aminoacyl-tRNA synthetase pairs with altered amino acid specificity allowed for aminoacylation in the expression system in situ. This method provided a major step forward by advancing the UAA technology to the all-genetic–based level, also known as “the genetic-code expansion” (5–7).Here, we present the design of a light-sensitive ionotropic glutamate receptor (iGluR) through the genetic incorporation of a photoreactive UAA. Our approach takes advantage of the recent development of the genetic-code expansion in Xenopus oocytes (8), which is a classical vehicle for heterologous expression and functional characterization of ligand-gated ion channels (LGICs). We focused on NMDA receptors (NMDARs), which play pivotal roles in brain physiology and pathology (9). NMDARs are obligatory heterotetramers commonly composed of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. Although GluN1 is encoded by a single gene, there are four types of GluN2 subunits (GluN2A to -D) encoded by four different genes, which endow NMDARs with different properties including channel open probabilities (P_o) and sensitivities to allosteric modulators (9). The extracellular region of both GluN1 and GluN2 subunits consists of a tandem of large clamshell-like domains comprising an N-terminal domain (NTD) and an agonist-binding domain (ABD) (Fig. 1A). Besides having essential functions in receptor assembly (10, 11), recent studies of the NTDs have also revealed that the individual GluN2 (12–14) and GluN1 (15) NTDs fine-tune NMDAR gating and pharmacological properties by undergoing large-range conformational changes. The recent X-ray crystal structure of a GluN1/GluN2B NTD complex reveals a unique arrangement of the two NTD protomers with intersubunit interactions distinct from those observed in AMPA and kainate receptors (16, 17). However, the importance of these dimer interfaces in the subunit-specific receptor regulation is poorly understood. We show that encoding the photoreactive UAA p-azido-l-phenylalanine (AzF) at the NTD upper lobe dimer interface in GluN1/GluN2B receptors serves as a photoswitch, triggering irreversible decrease of channel activity upon UV exposure. We further investigated the photo-induced conformational changes at the NTD dimer interfaces, as well as the subunit-dependent regulation, identifying structural determinants that differ between GluN2A- and GluN2B-containing NMDARs. Finally, we applied our approach to mammalian cells, including cultured hippocampal neurons, providing evidence for the transferability of light-sensitive NMDARs to more native cellular environments. Our results not only prove the feasibility of designing light-controlled NMDARs by introducing a genetically encoded photoreactive UAA at a conformational sensitive site, but also reveal aspects of the NMDAR assembly as highly subtype specific.Open in a separate windowFig. 1.Light inactivation of GluN1/GluN2B NMDARs incorporating a genetically encoded photoactive UAA. (A, Left) Four plasmids encoding the GluN1 subunit with an amber stop codon at position Y109 (red dot), the wt GluN2 subunit, the suppressor tRNA (Yam), and the engineered tRNA synthetase (RS) were coinjected into Xenopus oocytes. (Center) Crystal structure of the GluA2 AMPA receptor (40). The three major domains—N-terminal domain (NTD), agonist-binding domain (ABD) and transmembrane domain (TMD)—are arranged in layers. One NTD dimer is highlighted. (Right) Crystal structure of the NMDAR GluN1/GluN2B NTD heterodimer (16); the ifenprodil molecule is omitted for clarity. LL, lower lobe; UL, upper lobe. The GluN1-Y109 site is highlighted. On UV irradiation, the azide moiety generates a biradical, which subsequently can react with a nearby residue to form a covalent adduct. (B) Current amplitudes from oocytes injected with plasmids as indicated, in the absence or presence of UAAs. For each condition, 20 oocytes were tested. Only currents >10 nA were plotted. (C) Representative current traces showing UV-induced current inhibition of GluN1-Y109AzF/GluN2B receptors but not wt GluN1/GluN2B receptors. (D) UV-induced current modifications at wt GluN1/GluN2B (1.11 ± 0.13; n = 8), GluN1-Y109AzF/GluN2Bwt with (0.28 ± 0.05; n = 16) or without (0.31 ± 0.05; n = 5) agonist, and GluN1-Y109Bpa/GluN2Bwt (1.15 ± 0.06; n = 5) receptors. Error bars, SD. (E) MK-801 inhibition kinetics of wt GluN1/GluN2B and GluN1-Y109AzF/GluN2B receptors before and after UV treatment.     

Single-molecule imaging of the functional crosstalk between surface NMDA and dopamine D1 receptors

Dopamine is a powerful modulator of glutamatergic neurotransmission and NMDA receptor-dependent synaptic plasticity. Although several intracellular cascades participating in this functional dialogue have been identified over the last few decades, the molecular crosstalk between surface dopamine and glutamate NMDA receptor (NMDAR) signaling still remains poorly understood. Using a combination of single-molecule detection imaging and electrophysiology in live hippocampal neurons, we demonstrate here that dopamine D1 receptors (D1Rs) and NMDARs form dynamic surface clusters in the vicinity of glutamate synapses. Strikingly, D1R activation or D1R/NMDAR direct interaction disruption decreases the size of these clusters, increases NMDAR synaptic content through a fast lateral redistribution of the receptors, and favors long-term synaptic potentiation. Together, these data demonstrate the presence of dynamic D1R/NMDAR perisynaptic reservoirs favoring a rapid and bidirectional surface crosstalk between receptors and set the plasma membrane as the primary stage of the dopamine–glutamate interplay.Hippocampal dopaminergic neuromodulation participates in several cognitive functions including novelty detection and long-term memory storage (1, 2). As a consequence, impairments in hippocampal neuromodulatory transmission affect synaptic plasticity at glutamatergic synapses, prevent learning and memory formation, and have been proposed to be a cellular substrate for neurodevelopmental psychiatric disorders such as schizophrenia (3). In the hippocampus and cortex, pyramidal neurons express mostly dopamine D1 and D5 receptors along their dendritic tree (4–6). Their recruitment affects the trafficking and surface expression of glutamate NMDA receptors (NMDARs), two processes that are essential for excitatory neurotransmission and synaptic plasticity. Indeed, activating dopamine D1 receptors (D1Rs) promotes the surface expression and function of NMDAR and thereby favors the long-term potentiation of excitatory glutamate synapses (7–10). Reciprocally, the activation of NMDAR modulates D1R surface expression and signaling (11). The bidirectional dialogue between dopamine and glutamate NMDAR-associated signaling thus involves changes in membrane receptor content and trafficking.Although this functional interaction is usually considered as relying on intracellular protein kinase signaling cascades (7, 10, 12), physical interactions between D1R and NMDAR at the plasma membrane were recently reported to stabilize laterally diffusing surface D1R in spines, modulate D1R- and NMDAR-mediated signaling, and influence working memory (13–16). Thus, direct interactions between these receptors could contribute to the regulation of their surface distributions and play a major role in the dopamine–glutamate interplay (15, 17). In particular, because the regulation of NMDAR synaptic content involves surface diffusion processes in and out of synaptic and extrasynaptic compartments (18), the possibility emerges that dopamine might modulate NMDAR-dependent synaptic transmission by tuning NMDAR lateral dynamics through D1R–NMDAR physical interactions. To address this question and investigate the role of the D1R/NMDAR surface crosstalk in synaptic physiology, we here assessed the surface distribution and trafficking of D1 and NMDA receptors in rat hippocampal neurons using a combination of high-resolution single-nanoparticle tracking, bulk imaging, and electrophysiology.     

Nicotinic and muscarinic agonists and acetylcholinesterase inhibitors stimulate a common pathway to enhance GluN2B-NMDAR responses

Nicotinic and muscarinic ACh receptor agonists and acetylcholinesterase inhibitors (AChEIs) can enhance cognitive function. However, it is unknown whether a common signaling pathway is involved in the effect. Here, we show that in vivo administration of nicotine, AChEIs, and an m1 muscarinic (m1) agonist increase glutamate receptor, ionotropic, N-methyl D-aspartate 2B (GluN2B)-containing NMDA receptor (NR2B-NMDAR) responses, a necessary component in memory formation, in hippocampal CA1 pyramidal cells, and that coadministration of the m1 antagonist pirenzepine prevents the effect of cholinergic drugs. These observations suggest that the effect of nicotine is secondary to increased release of ACh via the activation of nicotinic ACh receptors (nAChRs) and involves m1 receptor activation through ACh. In vitro activation of m1 receptors causes the selective enhancement of NR2B-NMDAR responses in CA1 pyramidal cells, and in vivo exposure to cholinergic drugs occludes the in vitro effect. Furthermore, in vivo exposure to cholinergic drugs suppresses the potentiating effect of Src on NMDAR responses in vitro. These results suggest that exposure to cholinergic drugs maximally stimulates the m1/guanine nucleotide-binding protein subunit alpha q/PKC/proline-rich tyrosine kinase 2/Src signaling pathway for the potentiation of NMDAR responses in vivo, occluding the in vitro effects of m1 activation and Src. Thus, our results indicate not only that nAChRs, ACh, and m1 receptors are on the same pathway involving Src signaling but also that NR2B-NMDARs are a point of convergence of cholinergic and glutamatergic pathways involved in learning and memory.Nicotinic and muscarinic agonists can produce cognitive enhancement (1, 2). Acetylcholinesterase inhibitors (AChEIs) also cause cognitive enhancement by increasing ACh levels (3, 4). However, it is largely unknown whether the effect of ACh is mediated by nicotinic ACh receptors (nAChRs), muscarinic receptors, or both. Studies involving cholinergic lesions and local administration of cholinergic antagonists indicate that both nAChRs and muscarinic receptors located in the hippocampus are of particular importance for learning and memory processes (5–8). However, the mechanisms by which these receptors mediate cognitive enhancement largely remain to be elucidated.Synaptic plasticity is thought to be a critical component underlying learning and memory (9, 10), and the NMDA receptor (NMDAR) is a key component of synaptic plasticity (9, 11). Thus, studies of the modulation of NMDAR responses and long-term potentiation (LTP) induction by cholinergic drugs (12–20) help elucidate the mechanisms of cholinergic facilitation of learning and memory. In vitro acute nicotine can potentiate NMDAR-mediated responses in CA1 pyramidal cells in hippocampal slices via at least two different mechanisms (16, 18). One of these mechanisms is absent after a selective cholinergic lesion (21) and is paradoxically blocked by the muscarinic antagonist atropine (18), suggesting not only a critical role of nicotine-induced ACh release but also the involvement of muscarinic receptor activation in the effect of nicotine. This pathway appears to be stimulated by systemic nicotine administration in rats and most likely involves Src signaling (18, 19), which is known to be initiated via acute activation of m1 muscarinic (m1) receptors in CA1 pyramidal cells (22). An implication of these observations is that there is a common signaling pathway stimulated by cognitive-enhancing cholinergic drugs, leading to the enhancement of NMDAR-mediated responses in CA1 pyramidal cells. Thus, in this study, we investigated the link between nicotine and NMDARs in rats by administrating drugs that target different cholinergic proteins.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

Molecular lock regulates binding of glycine to a primitive NMDA receptor

The earliest metazoan ancestors of humans include the ctenophore Mnemiopsis leidyi. The genome of this comb jelly encodes homologs of vertebrate ionotropic glutamate receptors (iGluRs) that are distantly related to glycine-activated NMDA receptors and that bind glycine with unusually high affinity. Using ligand-binding domain (LBD) mutants for electrophysiological analysis, we demonstrate that perturbing a ctenophore-specific interdomain Arg-Glu salt bridge that is notably absent from vertebrate AMPA, kainate, and NMDA iGluRs greatly increases the rate of recovery from desensitization, while biochemical analysis reveals a large decrease in affinity for glycine. X-ray crystallographic analysis details rearrangements in the binding pocket stemming from the mutations, and molecular dynamics simulations suggest that the interdomain salt bridge acts as a steric barrier regulating ligand binding and that the free energy required to access open conformations in the glycine-bound LBD is largely responsible for differences in ligand affinity among the LBD variants.Glutamate receptor ion channels (iGluRs) are membrane proteins that mediate excitatory synaptic transmission in the brain by detecting release of the amino acid glutamate from nerve terminals (1). In combination with GluN2 subunits, which bind glutamate, NMDA subtype iGluRs use glycine as a coagonist, which binds to GluN1, GluN3A, and GluN3B subunits (2–6). NMDA receptors play key roles in synaptic plasticity and memory formation, and mutations of NMDA receptor genes underlie a diverse set of neurological and psychiatric diseases (7). Like all iGluRs, NMDA receptors are assembled from modular subunits containing amino terminal and S1S2 ligand binding domains (LBDs), which can be genetically isolated and expressed as soluble proteins for biochemical and structural analysis (4, 8–10). The LBDs of both the glutamate and glycine binding subunits are clamshell-shaped proteins of molecular mass around 30 kDa in which two lobes are connected by a hinge formed by antiparallel β-strands; in the activated state, ligands are trapped in a cavity formed when the clamshell closes. Strikingly, the volume of the ligand binding cavity for the GluN1, GluN3A, and GluN3B subunits is just large enough to accommodate glycine, whereas iGluR glutamate binding subunits have cavities that are four to five times larger and bind both glutamate and up to six or seven water molecules (4, 10–13).We recently reported the discovery of glycine-activated iGluRs from the comb jelly Mnemiopsis leidyi and the sea gooseberry Pleurobrachia bachei, candidates for earliest lineage metazoans, for which ML032222a and PbGluR3 glycine complex crystal structures reveal a salt bridge at the perimeter of the ligand binding cleft (14). This salt bridge links the upper and lower lobes of the LBD in the closed cleft glycine-bound conformation. Ctenophore iGluR subunits bind glycine with such high affinity that the ligand cannot be removed by exhaustive dialysis, suggesting an unusually stable ligand-bound closed-cleft conformation, perhaps stabilized by the interdomain salt bridge. Prior electrophysiological and crystallographic studies on vertebrate AMPA and kainate subtype iGluRs revealed that the stability of the closed cleft conformation is determined not only by contacts of the LBD with the neurotransmitter ligand but also by contacts formed between the upper and lower lobes of the clamshell assembly that occur only in the ligand-bound closed-cleft conformation (15, 16). Comparison of crystal structures of ctenophore iGluR LBDs with those of vertebrate NMDA receptor GluN1 and GluN3 subunits that also bind glycine, but for which apo proteins can be prepared without difficulty (4, 10), reveals that the salt bridge is unique to ctenophore iGluRs, further suggesting that it might underlie the high stability of the glycine complex.To investigate this, we prepared ML032222a mutant proteins and analyzed their ligand binding properties using electrophysiological, biochemical, and crystallographic techniques. To gain further insight into how these mutants perturb large-scale LBD dynamics, we computed conformational free energy landscapes for the apo state and glycine complexes of wild-type (WT) ML032222a and the R703K and E423S mutants, which weaken and break the interdomain salt bridge, respectively. This analysis reveals that, similar to vertebrate GluN1 and GluN3 glycine binding subunits, the apo state for ML032222a can access closed cleft conformations, although it is more stable in slightly open conformations. The R703K and E423S mutants destabilize closed cleft conformations for the glycine complex. Conformational dynamics inferred from the free energy landscapes suggest that the interdomain salt bridge is positioned at the most likely point of ligand entry to (and exit from) the binding pocket and thus acts as a steric barrier regulating the binding and dissociation of glycine.     

PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95–like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.The postsynaptic density (PSD) at excitatory glutamatergic synapses, appearing in electron micrographs as a prominent electron-dense thickening lining the postsynaptic membrane (1) is a complex macromolecular machine positioned across from synaptic vesicle release sites at the presynaptic active zone. The PSD clusters and organizes neurotransmitter receptors and signaling molecules at the postsynaptic membrane, transmits and processes synaptic signals, and can undergo structural changes to encode and store information (2–5). Two types of ionotropic glutamate receptors, AMPA receptors (AMPARs) and NMDA receptors (NMDARs), present at PSDs of excitatory synapses (6–10) mediate almost all synaptic transmission in the brain (11). Biochemistry and mass spectrometry of the detergent-extracted cellular fraction of PSDs have additionally identified many proteins associated with AMPAR and NMDAR complexes (12, 13).The membrane-associated guanylate kinases (MAGUKs), a class of abundant scaffold proteins consisting of PSD-95, PSD-93, synapse-associated protein (SAP)102, and SAP97, interact directly with NMDARs (14–18). These MAGUK proteins share conserved modular structures consisting of three PDZ domains (19, 20) and one SH3-GK supermodule (21). PDZ domains of MAGUKs bind to a conserved motif at the extreme C-terminal region of GluN2 subunits of NMDARs (16, 22). PSD-95 controls the number of AMPARs at the PSD through interactions with auxiliary proteins, such as Stargazin/TARPs in complex with AMPARs (23–25). Single-particle tracking of AMPARs provides evidence that AMPAR/Stargazin complexes are stabilized by PSD-95 at the membrane (26), where PSD-95 is thought to provide hot spots for accumulating AMPARs at synapses (27, 28). Germ-line knockout of PSD-95 reduces AMPAR transmission with little effects on NMDARs (29), whereas acute loss of single members of the MAGUK family decreases primarily AMPAR-mediated synaptic transmission (30–32), and removal of multiple MAGUKs results in greater losses of transmission mediated by both AMPARs and NMDARs (30).PSD-95 and PSD-93 include N-terminal palmitoylation sites that enable PSD-95 and PSD-93 to associate with membrane lipids. N-terminal palmitoylation of PSD-95 is necessary for its synaptic localization, clustering of receptors (33–35), and stability at the PSD (36). PSD-95 palmitoylation regulates synaptic strength by controlling the accumulation of AMPARs at the PSD (35). Consistent with these results, a recent immunogold electron microscopy (immuno-EM) mapping of the positions of the two ends of the PSD-95 molecule at the PSD shows that its N terminus is located at the membrane, whereas its C terminus is farther away from the membrane in a relatively extended configuration, where it is vertically oriented with respect to the membrane (3, 4, 37). In contrast, neither SAP102 nor SAP97 has palmitoylation sites. SAP97 contains a L27 domain at the N terminus (31, 38), which might be involved in self-association, and has a role in sorting and trafficking of AMPARs and NMDARs (39) but is not required for basal synaptic transmission (40).The MAGUK family proteins interact with a host of other proteins in the PSD, such as GKAP (41, 42), which binds to the GK domain of the MAGUKs, whereas GKAPs in turn bind Shank and Homer (43–45). Both Shank and Homer can interact with actin-associated proteins, thus indirectly linking the core PSD structure to the actin system prevalent in the cytoplasm of dendritic spines (45). MAGUKs interact with signaling complexes such as AKAPs (46, 47), K channels (48), and postsynaptic adhesion molecules such as neuroligin (49, 50). With an average density of 300–400 molecules per PSD (51, 52), the MAGUKs outnumber glutamate receptors by a significant margin. With so many potential binding partners, the MAGUKs are positioned to play an important role in organizing glutamate receptors as well as other scaffolding and signaling molecules at the PSD (53).We have examined the consequences of knocking down three key MAGUKs on excitatory synaptic transmission and found an ∼80% reduction in both AMPAR and NMDAR synaptic responses (54). Interestingly, despite the rather ubiquitous distribution of MAGUKs at excitatory synapses, the reduction in synaptic AMPAR-mediated transmission appeared to be attributable primarily to an all-or-none loss of functional synapses. We present evidence that after the knockdown, there is an initial uniform decrease in AMPARs across all synapses, but over a 4-d period, a consolidation process in which a “winner-take-all” phenomenon occurs (54).Here, we have used EM tomography (3, 4) to study the structural effects of knocking down the three key MAGUKs at the PSD to develop a molecular model of the organization of the core PSD structure in intact hippocampal spine synapses. PSDs in intact synapses show numerous regularly spaced and membrane-associated vertical filaments containing PSD-95 in extended conformation connecting with NMDAR and AMPAR-type complexes. These vertical structures in turn contact horizontal elements, resulting in a molecular scaffold supporting a core PSD structure (3, 4, 37). Thus, vertical filaments appear to be of crucial importance in sustaining the core PSD structure. Here, we show that knocking down three key synaptic MAGUKs results in a profound loss of vertical filaments and the electron-dense materials manifested by the PSD. The loss of MAGUKs is accompanied by a dramatic loss of both NMDAR- and AMPAR-type structures at the PSD.     

Self-assembly of alkynylplatinum(II) terpyridine amphiphiles into nanostructures via steric control and metal–metal interactions

A series of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing the hydrophilic oligo(para-phenylene ethynylene) with two 3,6,9-trioxadec-1-yloxy chains was designed and synthesized. The mononuclear alkynylplatinum(II) terpyridine complex was found to display a very strong tendency toward the formation of supramolecular structures. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would lead to the formation of nanotubes or helical ribbons. These desirable nanostructures were found to be governed by the steric bulk on the platinum(II) terpyridine moieties, which modulates the directional metal−metal interactions and controls the formation of nanotubes or helical ribbons. Detailed analysis of temperature-dependent UV-visible absorption spectra of the nanostructured tubular aggregates also provided insights into the assembly mechanism and showed the role of metal−metal interactions in the cooperative supramolecular polymerization of the amphiphilic platinum(II) complexes.Square-planar d⁸ platinum(II) polypyridine complexes have long been known to exhibit intriguing spectroscopic and luminescence properties (1–54) as well as interesting solid-state polymorphism associated with metal−metal and π−π stacking interactions (1–14, 25). Earlier work by our group showed the first example, to our knowledge, of an alkynylplatinum(II) terpyridine system [Pt(tpy)(C ≡ CR)]⁺ that incorporates σ-donating and solubilizing alkynyl ligands together with the formation of Pt···Pt interactions to exhibit notable color changes and luminescence enhancements on solvent composition change (25) and polyelectrolyte addition (26). This approach has provided access to the alkynylplatinum(II) terpyridine and other related cyclometalated platinum(II) complexes, with functionalities that can self-assemble into metallogels (27–31), liquid crystals (32, 33), and other different molecular architectures, such as hairpin conformation (34), helices (35–38), nanostructures (39–45), and molecular tweezers (46, 47), as well as having a wide range of applications in molecular recognition (48–52), biomolecular labeling (48–52), and materials science (53, 54). Recently, metal-containing amphiphiles have also emerged as a building block for supramolecular architectures (42–44, 55–59). Their self-assembly has always been found to yield different molecular architectures with unprecedented complexity through the multiple noncovalent interactions on the introduction of external stimuli (42–44, 55–59).Helical architecture is one of the most exciting self-assembled morphologies because of the uniqueness for the functional and topological properties (60–69). Helical ribbons composed of amphiphiles, such as diacetylenic lipids, glutamates, and peptide-based amphiphiles, are often precursors for the growth of tubular structures on an increase in the width or the merging of the edges of ribbons (64, 65). Recently, the optimization of nanotube formation vs. helical nanostructures has aroused considerable interests and can be achieved through a fine interplay of the influence on the amphiphilic property of molecules (66), choice of counteranions (67, 68), or pH values of the media (69), which would govern the self-assembly of molecules into desirable aggregates of helical ribbons or nanotube scaffolds. However, a precise control of supramolecular morphology between helical ribbons and nanotubes remains challenging, particularly for the polycyclic aromatics in the field of molecular assembly (64–69). Oligo(para-phenylene ethynylene)s (OPEs) with solely π−π stacking interactions are well-recognized to self-assemble into supramolecular system of various nanostructures but rarely result in the formation of tubular scaffolds (70–73). In view of the rich photophysical properties of square-planar d⁸ platinum(II) systems and their propensity toward formation of directional Pt···Pt interactions in distinctive morphologies (27–31, 39–45), it is anticipated that such directional and noncovalent metal−metal interactions might be capable of directing or dictating molecular ordering and alignment to give desirable nanostructures of helical ribbons or nanotubes in a precise and controllable manner.Herein, we report the design and synthesis of mono- and dinuclear alkynylplatinum(II) terpyridine complexes containing hydrophilic OPEs with two 3,6,9-trioxadec-1-yloxy chains. The mononuclear alkynylplatinum(II) terpyridine complex with amphiphilic property is found to show a strong tendency toward the formation of supramolecular structures on diffusion of diethyl ether in dichloromethane or dimethyl sulfoxide (DMSO) solution. Interestingly, additional end-capping with another platinum(II) terpyridine moiety of various steric bulk at the terminal alkyne would result in nanotubes or helical ribbons in the self-assembly process. To the best of our knowledge, this finding represents the first example of the utilization of the steric bulk of the moieties, which modulates the formation of directional metal−metal interactions to precisely control the formation of nanotubes or helical ribbons in the self-assembly process. Application of the nucleation–elongation model into this assembly process by UV-visible (UV-vis) absorption spectroscopic studies has elucidated the nature of the molecular self-assembly, and more importantly, it has revealed the role of metal−metal interactions in the formation of these two types of nanostructures.     

Transition paths,diffusive processes,and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.Protein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1, 2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7–15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16–25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the free energy minimum of the unfolded state. The development of ultrafast laser spectroscopy (29, 30) and single-molecule (27, 28, 31) techniques have made it possible to establish upper bounds on the transition path time (t_TP) ranging from <200 and <10 μs by earlier (27) and more recent (28), respectively, direct single-molecule FRET to <2 μs (30) by bulk relaxation measurements. Consistent with these observations, recent extensive atomic simulations have also provided estimated t_TP values of the order of ∼1 μs (32, 33). These advances offer exciting prospects of characterizing the productive events along folding TPs.It is timely, therefore, to further the theoretical investigation of TP-related questions (19). To this end, we used coarse-grained C_α models (14) to perform extensive simulations of the folding trajectories of small proteins with 56- to 86-aa residues. These tractable models are useful, because despite significant progress, current atomic models cannot provide the same degree of sampling coverage for proteins of comparable sizes (32, 33). In addition to structural insights, this study provides previously unexplored vantage points to compare the diffusion and TST pictures of folding. Deviations of folding behaviors from TST predictions are not unexpected, because TST is mostly applicable to simple gas reactions; however, the nature and extent of the deviations have not been much explored. Our explicit-chain simulation data conform well to the diffusion picture but not as well to TST. In particular, the preexponential factors of the simulated folding rates exhibit a small but appreciable variation that depends on native topology. These findings and others reported below underscore the importance of single-molecule measurements (13, 27, 28, 31, 34, 35) in assessing the merits of proposed scenarios and organizing principles of folding (7–25, 36, 37).     

Potentiation of cytotoxic chemotherapy by growth hormone-releasing hormone agonists

The dismal prognosis of malignant brain tumors drives the development of new treatment modalities. In view of the multiple activities of growth hormone-releasing hormone (GHRH), we hypothesized that pretreatment with a GHRH agonist, JI-34, might increase the susceptibility of U-87 MG glioblastoma multiforme (GBM) cells to subsequent treatment with the cytotoxic drug, doxorubicin (DOX). This concept was corroborated by our findings, in vivo, showing that the combination of the GHRH agonist, JI-34, and DOX inhibited the growth of GBM tumors, transplanted into nude mice, more than DOX alone. In vitro, the pretreatment of GBM cells with JI-34 potentiated inhibitory effects of DOX on cell proliferation, diminished cell size and viability, and promoted apoptotic processes, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation assay, ApoLive-Glo multiplex assay, and cell volumetric assay. Proteomic studies further revealed that the pretreatment with GHRH agonist evoked differentiation decreasing the expression of the neuroectodermal stem cell antigen, nestin, and up-regulating the glial maturation marker, GFAP. The GHRH agonist also reduced the release of humoral regulators of glial growth, such as FGF basic and TGFβ. Proteomic and gene-expression (RT-PCR) studies confirmed the strong proapoptotic activity (increase in p53, decrease in v-myc and Bcl-2) and anti-invasive potential (decrease in integrin α3) of the combination of GHRH agonist and DOX. These findings indicate that the GHRH agonists can potentiate the anticancer activity of the traditional chemotherapeutic drug, DOX, by multiple mechanisms including the induction of differentiation of cancer cells.Glioblastoma multiforme (GBM) is one of the most aggressive human cancers, and the afflicted patients inevitably succumb. The dismal outcome of this malignancy demands great efforts to find improved methods of treatment (1). Many compounds have been synthesized in our laboratory in the past few years that have proven to be effective against diverse malignant tumors (2–14). These are peptide analogs of hypothalamic hormones: luteinizing hormone-releasing hormone (LHRH), growth hormone-releasing hormone (GHRH), somatostatin, and analogs of other neuropeptides such as bombesin and gastrin-releasing peptide. The receptors for these peptides have been found to be widely distributed in the human body, including in many types of cancers (2–14). The regulatory functions of these hypothalamic hormones and other neuropeptides are not confined to the hypothalamo–hypophyseal system or, even more broadly, to the central nervous system (CNS). In particular, GHRH can induce the differentiation of ovarian granulosa cells and other cells in the reproductive system and function as a growth factor in various normal tissues, benign tumors, and malignancies (2–4, 6, 11, 14–18). Previously, we also reported that antagonistic cytototoxic derivatives of some of these neuropeptides are able to inhibit the growth of several malignant cell lines (2–14).Our earlier studies showed that treatment with antagonists of LHRH or GHRH rarely effects complete regression of glioblastoma-derived tumors (5, 7, 10, 11). Previous studies also suggested that growth factors such as EGF or agonistic analogs of LHRH serving as carriers for cytotoxic analogs and functioning as growth factors may sensitize cancer cells to cytotoxic treatments (10, 19) through the activation of maturation processes. We therefore hypothesized that pretreatment with one of our GHRH agonists, such as JI-34 (20), which has shown effects on growth and differentiation in other cell lines (17, 18, 21, 22), might decrease the pluripotency and the adaptability of GBM cells and thereby increase their susceptibility to cytotoxic treatment.In vivo, tumor cells were implanted into athymic nude mice, tumor growth was recorded weekly, and final tumor mass was measured upon autopsy. In vitro, proliferation assays were used for the determination of neoplastic proliferation and cell growth. Changes in stem (nestin) and maturation (GFAP) antigen expression was evaluated with Western blot studies in vivo and with immunocytochemistry in vitro. The production of glial growth factors (FGF basic, TGFβ) was verified by ELISA. Further, using the Human Cancer Pathway Finder real-time quantitative PCR, numerous genes that play a role in the development of cancer were evaluated. We placed particular emphasis on the measurement of apoptosis, using the ApoLive-Glo Multiplex Assay kit and by detection of the expression of the proapoptotic p53 protein. This overall approach permitted the evaluation of the effect of GHRH agonist, JI-34, on the response to chemotherapy with doxorubicin.     

α-Synuclein assembles into higher-order multimers upon membrane binding to promote SNARE complex formation

Physiologically, α-synuclein chaperones soluble NSF attachment protein receptor (SNARE) complex assembly and may also perform other functions; pathologically, in contrast, α-synuclein misfolds into neurotoxic aggregates that mediate neurodegeneration and propagate between neurons. In neurons, α-synuclein exists in an equilibrium between cytosolic and membrane-bound states. Cytosolic α-synuclein appears to be natively unfolded, whereas membrane-bound α-synuclein adopts an α-helical conformation. Although the majority of studies showed that cytosolic α-synuclein is monomeric, it is unknown whether membrane-bound α-synuclein is also monomeric, and whether chaperoning of SNARE complex assembly by α-synuclein involves its cytosolic or membrane-bound state. Here, we show using chemical cross-linking and fluorescence resonance energy transfer (FRET) that α-synuclein multimerizes into large homomeric complexes upon membrane binding. The FRET experiments indicated that the multimers of membrane-bound α-synuclein exhibit defined intermolecular contacts, suggesting an ordered array. Moreover, we demonstrate that α-synuclein promotes SNARE complex assembly at the presynaptic plasma membrane in its multimeric membrane-bound state, but not in its monomeric cytosolic state. Our data delineate a folding pathway for α-synuclein that ranges from a monomeric, natively unfolded form in cytosol to a physiologically functional, multimeric form upon membrane binding, and show that only the latter but not the former acts as a SNARE complex chaperone at the presynaptic terminal, and may protect against neurodegeneration.α-Synuclein is an abundant presynaptic protein that physiologically acts to promote soluble NSF attachment protein receptor (SNARE) complex assembly in vitro and in vivo (1–3). Point mutations in α-synuclein (A30P, E46K, H50Q, G51D, and A53T) as well as α-synuclein gene duplications and triplications produce early-onset Parkinson''s disease (PD) (4–10). Moreover, α-synuclein is a major component of intracellular protein aggregates called Lewy bodies, which are pathological hallmarks of neurodegenerative disorders such as PD, Lewy body dementia, and multiple system atrophy (11–14). Strikingly, neurotoxic α-synuclein aggregates propagate between neurons during neurodegeneration, suggesting that such α-synuclein aggregates are not only intrinsically neurotoxic but also nucleate additional fibrillization (15–18).α-Synuclein is highly concentrated in presynaptic terminals where α-synuclein exists in an equilibrium between a soluble and a membrane-bound state, and is associated with synaptic vesicles (19–22). The labile association of α-synuclein with membranes (23, 24) suggests that binding of α-synuclein to synaptic vesicles, and its dissociation from these vesicles, may regulate its physiological function. Membrane-bound α-synuclein assumes an α-helical conformation (25–32), whereas cytosolic α-synuclein is natively unfolded and monomeric (refs. 25, 26, 31, and 32; however, see refs. 33 and 34 and Discussion for a divergent view). Membrane binding by α-synuclein is likely physiologically important because in in vitro experiments, α-synuclein remodels membranes (35, 36), influences lipid packing (37, 38), and induces vesicle clustering (39). Moreover, membranes were found to be important for the neuropathological effects of α-synuclein (40–44).However, the relation of membrane binding to the in vivo function of α-synuclein remains unexplored, and it is unknown whether α-synuclein binds to membranes as a monomer or oligomer. Thus, in the present study we have investigated the nature of the membrane-bound state of α-synuclein and its relation to its physiological function in SNARE complex assembly. We found that soluble monomeric α-synuclein assembles into higher-order multimers upon membrane binding and that membrane binding of α-synuclein is required for its physiological activity in promoting SNARE complex assembly at the synapse.     

The evolution of self-control

Cognition presents evolutionary research with one of its greatest challenges. Cognitive evolution has been explained at the proximate level by shifts in absolute and relative brain volume and at the ultimate level by differences in social and dietary complexity. However, no study has integrated the experimental and phylogenetic approach at the scale required to rigorously test these explanations. Instead, previous research has largely relied on various measures of brain size as proxies for cognitive abilities. We experimentally evaluated these major evolutionary explanations by quantitatively comparing the cognitive performance of 567 individuals representing 36 species on two problem-solving tasks measuring self-control. Phylogenetic analysis revealed that absolute brain volume best predicted performance across species and accounted for considerably more variance than brain volume controlling for body mass. This result corroborates recent advances in evolutionary neurobiology and illustrates the cognitive consequences of cortical reorganization through increases in brain volume. Within primates, dietary breadth but not social group size was a strong predictor of species differences in self-control. Our results implicate robust evolutionary relationships between dietary breadth, absolute brain volume, and self-control. These findings provide a significant first step toward quantifying the primate cognitive phenome and explaining the process of cognitive evolution.Since Darwin, understanding the evolution of cognition has been widely regarded as one of the greatest challenges for evolutionary research (1). Although researchers have identified surprising cognitive flexibility in a range of species (2–40) and potentially derived features of human psychology (41–61), we know much less about the major forces shaping cognitive evolution (62–71). With the notable exception of Bitterman’s landmark studies conducted several decades ago (63, 72–74), most research comparing cognition across species has been limited to small taxonomic samples (70, 75). With limited comparable experimental data on how cognition varies across species, previous research has largely relied on proxies for cognition (e.g., brain size) or metaanalyses when testing hypotheses about cognitive evolution (76–92). The lack of cognitive data collected with similar methods across large samples of species precludes meaningful species comparisons that can reveal the major forces shaping cognitive evolution across species, including humans (48, 70, 89, 93–98).To address these challenges we measured cognitive skills for self-control in 36 species of mammals and birds (Fig. 1 and Tables S1–S4) tested using the same experimental procedures, and evaluated the leading hypotheses for the neuroanatomical underpinnings and ecological drivers of variance in animal cognition. At the proximate level, both absolute (77, 99–107) and relative brain size (108–112) have been proposed as mechanisms supporting cognitive evolution. Evolutionary increases in brain size (both absolute and relative) and cortical reorganization are hallmarks of the human lineage and are believed to index commensurate changes in cognitive abilities (52, 105, 113–115). Further, given the high metabolic costs of brain tissue (116–121) and remarkable variance in brain size across species (108, 122), it is expected that the energetic costs of large brains are offset by the advantages of improved cognition. The cortical reorganization hypothesis suggests that selection for absolutely larger brains—and concomitant cortical reorganization—was the predominant mechanism supporting cognitive evolution (77, 91, 100–106, 120). In contrast, the encephalization hypothesis argues that an increase in brain volume relative to body size was of primary importance (108, 110, 111, 123). Both of these hypotheses have received support through analyses aggregating data from published studies of primate cognition and reports of “intelligent” behavior in nature—both of which correlate with measures of brain size (76, 77, 84, 92, 110, 124).Open in a separate windowFig. 1.A phylogeny of the species included in this study. Branch lengths are proportional to time except where long branches have been truncated by parallel diagonal lines (split between mammals and birds ∼292 Mya).With respect to selective pressures, both social and dietary complexities have been proposed as ultimate causes of cognitive evolution. The social intelligence hypothesis proposes that increased social complexity (frequently indexed by social group size) was the major selective pressure in primate cognitive evolution (6, 44, 48, 50, 87, 115, 120, 125–141). This hypothesis is supported by studies showing a positive correlation between a species’ typical group size and the neocortex ratio (80, 81, 85–87, 129, 142–145), cognitive differences between closely related species with different group sizes (130, 137, 146, 147), and evidence for cognitive convergence between highly social species (26, 31, 148–150). The foraging hypothesis posits that dietary complexity, indexed by field reports of dietary breadth and reliance on fruit (a spatiotemporally distributed resource), was the primary driver of primate cognitive evolution (151–154). This hypothesis is supported by studies linking diet quality and brain size in primates (79, 81, 86, 142, 155), and experimental studies documenting species differences in cognition that relate to feeding ecology (94, 156–166).Although each of these hypotheses has received empirical support, a comparison of the relative contributions of the different proximate and ultimate explanations requires (i) a cognitive dataset covering a large number of species tested using comparable experimental procedures; (ii) cognitive tasks that allow valid measurement across a range of species with differing morphology, perception, and temperament; (iii) a representative sample within each species to obtain accurate estimates of species-typical cognition; (iv) phylogenetic comparative methods appropriate for testing evolutionary hypotheses; and (v) unprecedented collaboration to collect these data from populations of animals around the world (70).Here, we present, to our knowledge, the first large-scale collaborative dataset and comparative analysis of this kind, focusing on the evolution of self-control. We chose to measure self-control—the ability to inhibit a prepotent but ultimately counterproductive behavior—because it is a crucial and well-studied component of executive function and is involved in diverse decision-making processes (167–169). For example, animals require self-control when avoiding feeding or mating in view of a higher-ranking individual, sharing food with kin, or searching for food in a new area rather than a previously rewarding foraging site. In humans, self-control has been linked to health, economic, social, and academic achievement, and is known to be heritable (170–172). In song sparrows, a study using one of the tasks reported here found a correlation between self-control and song repertoire size, a predictor of fitness in this species (173). In primates, performance on a series of nonsocial self-control control tasks was related to variability in social systems (174), illustrating the potential link between these skills and socioecology. Thus, tasks that quantify self-control are ideal for comparison across taxa given its robust behavioral correlates, heritable basis, and potential impact on reproductive success.In this study we tested subjects on two previously implemented self-control tasks. In the A-not-B task (27 species, n = 344), subjects were first familiarized with finding food in one location (container A) for three consecutive trials. In the test trial, subjects initially saw the food hidden in the same location (container A), but then moved to a new location (container B) before they were allowed to search (Movie S1). In the cylinder task (32 species, n = 439), subjects were first familiarized with finding a piece of food hidden inside an opaque cylinder. In the following 10 test trials, a transparent cylinder was substituted for the opaque cylinder. To successfully retrieve the food, subjects needed to inhibit the impulse to reach for the food directly (bumping into the cylinder) in favor of the detour response they had used during the familiarization phase (Movie S2).Thus, the test trials in both tasks required subjects to inhibit a prepotent motor response (searching in the previously rewarded location or reaching directly for the visible food), but the nature of the correct response varied between tasks. Specifically, in the A-not-B task subjects were required to inhibit the response that was previously successful (searching in location A) whereas in the cylinder task subjects were required to perform the same response as in familiarization trials (detour response), but in the context of novel task demands (visible food directly in front of the subject).     

Glycine activated ion channel subunits encoded by ctenophore glutamate receptor genes

Recent genome projects for ctenophores have revealed the presence of numerous ionotropic glutamate receptors (iGluRs) in Mnemiopsis leidyi and Pleurobrachia bachei, among our earliest metazoan ancestors. Sequence alignments and phylogenetic analysis show that these form a distinct clade from the well-characterized AMPA, kainate, and NMDA iGluR subtypes found in vertebrates. Although annotated as glutamate and kainate receptors, crystal structures of the ML032222a and PbiGluR3 ligand-binding domains (LBDs) reveal endogenous glycine in the binding pocket, whereas ligand-binding assays show that glycine binds with nanomolar affinity; biochemical assays and structural analysis establish that glutamate is occluded from the binding cavity. Further analysis reveals ctenophore-specific features, such as an interdomain Arg-Glu salt bridge, present only in subunits that bind glycine, but also a conserved disulfide in loop 1 of the LBD that is found in all vertebrate NMDA but not AMPA or kainate receptors. We hypothesize that ctenophore iGluRs are related to an early ancestor of NMDA receptors, suggesting a common evolutionary path for ctenophores and bilaterian species, and suggest that future work should consider both glycine and glutamate as candidate neurotransmitters in ctenophore species.In the nervous system and neuromuscular junction of many animal species, the amino acid l-glutamate acts as an excitatory neurotransmitter. The molecular organization of glutamate receptor ion channel (iGluR) subunits into an amino terminal domain (ATD), and a ligand binding domain (LBD) bisected by insertion of a pore loop ion channel generates a unique structural signature, distinct from that for other neurotransmitter receptors, that is easily identified by sequence analysis. Using this approach, hundreds of iGluR homologs are emerging from genome sequencing projects (1–5). Virtually all of these are glutamate receptors in name only; their functional properties, physiological function, and the ligands they bind have yet to be determined. Recent large-scale sequencing projects, which place ctenophores as candidates for the earliest metazoan lineage, reveal that iGluR homologs are abundantly represented in the genomes of the comb jelly Mnemiopsis leidyi and the sea gooseberry Pleurobrachia bachei, suggesting that glutamate was selected to act as a neurotransmitter very early in evolution (4, 5). The muscle cells of P. bachei respond to application of glutamate with action potential generation and both species have neural networks and exhibit complex predatory behaviors that might also be generated by iGluR activity (4, 5). However, as for most species studied in sequencing projects, ctenophore iGluRs have yet to be characterized.By contrast to our primitive state of knowledge for iGluRs recently discovered by genome sequencing projects, the iGluRs of vertebrate species have been extensively characterized, and based on their ligand binding properties, amino acid sequences, functional properties, and structures, have been classified into AMPA, kainate, NMDA, and delta receptors (6–14). Of these, NMDA receptors are unique in that they form heteromeric assemblies that require binding of both glycine to GluN1 or GluN3 subunits and glutamate to GluN2 subunits for activation of ion channel gating (12, 14–17), as well as depolarization to relieve ion channel block by extracellular Mg²⁺ (18, 19). The initial annotation of the M. leidyi genome identified 16 candidate iGluR genes (4), whereas in the draft genome of P. bachei, 14 iGluRs were annotated as kainate-like receptors (5). In view of growing interest in the molecular evolution of ion channels and receptors, and the pivotal role that ctenophores play in our current understanding of nervous system development (20), we initiated a structural and functional characterization of glutamate receptors expressed in both species. To our surprise, we identified a large subset of ctenophore iGluRs from both M. leidyi and P. bachei that bind glycine but not glutamate with nanomolar affinity; one of these, ML03222a, forms homomeric glycine activated ion channels when expressed in Xenopus oocytes. By contrast, a second M. leidyi iGluR subunit, ML05909a, forms homomeric ion channels that are activated by millimolar concentrations of glutamate, while by contrast glycine acts as a very weak partial agonist that inhibits responses to glutamate. Comparison of the LBD crystal structures of two ctenophore iGluRs that bind glycine with nanomolar affinity, with the crystal structures of glutamate and glycine binding subunits of classical NMDA receptors, reveals a conserved disulfide in loop 1 of the LBD found in all ctenophore iGluRs, and also in NMDA but not AMPA or kainate receptors; however, the mechanisms underlying selectivity for glycine are distinct in vertebrate and ctenophore iGluRs, suggesting that NMDA receptors evolved from an ancestral protein, further modification of which occurred after bilaterians split from ctenophores during the evolution of metazoan species.     

Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

Mechanosensitive ion channels are sensors probing membrane tension in all species; despite their importance and vital role in many cell functions, their gating mechanism remains to be elucidated. Here, we determined the conditions for releasing intact mechanosensitive channel of large conductance (MscL) proteins from their detergents in the gas phase using native ion mobility–mass spectrometry (IM-MS). By using IM-MS, we could detect the native mass of MscL from Escherichia coli, determine various global structural changes during its gating by measuring the rotationally averaged collision cross-sections, and show that it can function in the absence of a lipid bilayer. We could detect global conformational changes during MscL gating as small as 3%. Our findings will allow studying native structure of many other membrane proteins.One of the best candidates to explore the gating of mechanosensitive channels is the mechanosensitive channel of large conductance (MscL) from Escherichia coli. The crystal structure of MscL in its closed/nearly closed state from Mycobacterium tuberculosis revealed this channel as a homopentamer (1). Each subunit has a cytoplasmic N- and C-terminal domain as well as two α-helical transmembrane (TM) domains, TM1 and TM2, which are connected by a periplasmic loop. The five TM1 helices form the pore and the more peripheral TM2 helices interact with the lipid bilayer.MscL detects changes in membrane tension invoked by a hypoosmotic shock and couples the tension sensing directly to large conformational changes (1, 2). On the basis of a large body of structural and theoretical data, numerous gating models of MscL have been proposed (3–9). These models agree upon (i) the hydrophobic pore constriction of the channel and (ii) the channel opens by an iris-like rotation—i.e., a tilting and outward movement of transmembrane helices that make the channel wider and shorter (5). This mechanism is supported by patch-clamp (10), disulfide cross-linking (11), FRET spectroscopy (12), and site-directed spin labeling EPR experiments (6, 7), as well as computational studies (13–15). So far, direct experimental results have only been observed for short-range local structural changes, and no measure of the overall global structural changes during channel gating have been reported. Because there is no crystal structure available for the open MscL channel, elucidating overall global structural changes from the onset of channel activation is of utmost importance for our understanding of the gating mechanism of mechanosensitive channels. Here, we provide direct experimental evidence for the key areal changes occurring during channel gating by combining our ability to activate MscL in a controlled manner to different subopen states (16) with a native ion mobility-mass spectrometry (IM-MS) approach.