Hippocampal acetylcholine modulates stress-related behaviors independent of specific cholinergic inputs.

Acetylcholine (ACh) levels are elevated in actively depressed subjects. Conversely, antagonism of either nicotinic or muscarinic ACh receptors can have antidepressant effects in humans and decrease stress-relevant behaviors in rodents. Consistent with a role for ACh in mediating maladaptive responses to stress, brain ACh levels increase in response to stressful challenges, whereas systemically blocking acetylcholinesterase (AChE, the primary ACh degradative enzyme) elicits depression-like symptoms in human subjects, and selectively blocking AChE in the hippocampus increases relevant behaviors in rodents. We used an ACh sensor to characterize stress-evoked ACh release, then used chemogenetic, optogenetic and pharmacological approaches to determine whether cholinergic inputs from the medial septum/diagonal bands of Broca (MSDBB) or ChAT-positive neurons intrinsic to the hippocampus mediate stress-relevant behaviors in mice. Chemogenetic inhibition or activation of MSDBB cholinergic neurons did not result in significant behavioral effects, while inhibition attenuated the behavioral effects of physostigmine. In contrast, optogenetic stimulation of septohippocampal terminals or selective chemogenetic activation of ChAT-positive inputs to hippocampus increased stress-related behaviors. Finally, stimulation of sparse ChAT-positive hippocampal neurons increased stress-related behaviors in one ChAT-Cre line, which were attenuated by local infusion of cholinergic antagonists. These studies suggest that ACh signaling results in maladaptive behavioral responses to stress if the balance of signaling is shifted toward increased hippocampal engagement.

Rationally derived inhibitors of hepatitis C virus (HCV) p7 channel activity reveal prospect for bimodal antiviral therapy.

Since the 1960s, a single class of agent has been licensed targeting virus-encoded ion channels, or 'viroporins', contrasting the success of channel blocking drugs in other areas of medicine. Although resistance arose to these prototypic adamantane inhibitors of the influenza A virus (IAV) M2 proton channel, a growing number of clinically and economically important viruses are now recognised to encode essential viroporins providing potential targets for modern drug discovery. We describe the first rationally designed viroporin inhibitor with a comprehensive structure-activity relationship (SAR). This step-change in understanding not only revealed a second biological function for the p7 viroporin from hepatitis C virus (HCV) during virus entry, but also enabled the synthesis of a labelled tool compound that retained biological activity. Hence, p7 inhibitors (p7i) represent a unique class of HCV antiviral targeting both the spread and establishment of infection, as well as a precedent for future viroporin-targeted drug discovery.

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

FUS ALS-causative mutations impair FUS autoregulation and splicing factor networks through intron retention.

Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.

Cytoplasmic functions of TDP-43 and FUS and their role in ALS.

TAR DNA-binding protein of 43 kDa (TDP-43) and fused in sarcoma (FUS) are RNA binding proteins (RBPs) primarily located in the nucleus, and involved in numerous aspects of RNA metabolism. Both proteins can be found to be depleted from the nucleus and accumulated in cytoplasmic inclusions in two major neurodegenerative conditions, amyotrophic lateral sclerosis and frontotemporal dementia. Recent evidences suggest that, in addition to their nuclear functions, both TDP-43 and FUS are involved in multiple processes in the cytoplasm, including mRNA stability and transport, translation, the stress response, mitochondrial function and autophagy regulation. Here, we review the most recent advances in understanding their functions in the cytoplasm and how these are affected in disease.

Interaction between noradrenergic and cholinergic signaling in amygdala regulates anxiety- and depression-related behaviors in mice.

Medications that target the noradrenergic system are important therapeutics for depression and anxiety disorders. More recently, clinical studies have shown that the α2-noradrenergic receptor (α2AR) agonist guanfacine can decrease stress-induced smoking relapse during acute abstinence, suggesting that targeting the noradrenergic system may aid in smoking cessation through effects on stress pathways in the brain. Acetylcholine (ACh), like the nicotine in tobacco, acts at nicotinic acetylcholine receptors (nAChRs) to regulate behaviors related to anxiety and depression. We therefore investigated interactions between guanfacine and ACh signaling in tests of anxiolytic and antidepressant efficacy in female and male C57BL/6J mice, focusing on the amygdala as a potential site of noradrenergic/cholinergic interaction. The antidepressant-like effects of guanfacine were blocked by shRNA-mediated knockdown of α2AR in amygdala. Knockdown of the high-affinity ß2 nAChR subunit in amygdala also prevented antidepressant-like effects of guanfacine, suggesting that these behavioral effects require ACh signaling through ß2-containing nAChRs in this brain area. Ablation of NE terminals prevented the anxiolytic- and antidepressant-like effects of the nicotinic partial agonist cytisine, whereas administration of the cholinesterase antagonist physostigmine induced a depression-like phenotype that was not altered by knocking down α2AR in the amygdala. These studies suggest that ACh and NE have opposing actions on behaviors related to anxiety and depression and that cholinergic signaling through ß2-containing nAChRs and noradrenergic signaling through α2a receptors in neurons of the amygdala are critical for regulation of these behaviors.

Oncolytic reovirus as a combined antiviral and anti-tumour agent for the treatment of liver cancer.

OBJECTIVE: Oncolytic viruses (OVs) represent promising, proinflammatory cancer treatments. Here, we explored whether OV-induced innate immune responses could simultaneously inhibit HCV while suppressing hepatocellular carcinoma (HCC). Furthermore, we extended this exemplar to other models of virus-associated cancer. DESIGN AND RESULTS: Clinical grade oncolytic orthoreovirus (Reo) elicited innate immune activation within primary human liver tissue in the absence of cytotoxicity and independently of viral genome replication. As well as achieving therapy in preclinical models of HCC through the activation of innate degranulating immune cells, Reo-induced cytokine responses efficiently suppressed HCV replication both in vitro and in vivo. Furthermore, Reo-induced innate responses were also effective against models of HBV-associated HCC, as well as an alternative endogenous model of Epstein-Barr virus-associated lymphoma. Interestingly, Reo appeared superior to the majority of OVs in its ability to elicit innate inflammatory responses from primary liver tissue. CONCLUSIONS: We propose that Reo and other select proinflammatory OV may be used in the treatment of multiple cancers associated with oncogenic virus infections, simultaneously reducing both virus-associated oncogenic drive and tumour burden. In the case of HCV-associated HCC (HCV-HCC), Reo should be considered as an alternative agent to supplement and support current HCV-HCC therapies, particularly in those countries where access to new HCV antiviral treatments may be limited.

Release of Infectious Hepatitis C Virus from Huh7 Cells Occurs via a trans-Golgi Network-to-Endosome Pathway Independent of Very-Low-Density Lipoprotein Secretion.

UNLABELLED: The release of infectious hepatitis C virus (HCV) particles from infected cells remains poorly characterized. We previously demonstrated that virus release is dependent on the endosomal sorting complex required for transport (ESCRT). Here, we show a critical role of trans-Golgi network (TGN)-endosome trafficking during the assembly, but principally the secretion, of infectious virus. This was demonstrated by both small interfering RNA (siRNA)-mediated silencing of TGN-associated adaptor proteins and a panel of dominant negative (DN) Rab GTPases involved in TGN-endosome trafficking steps. Importantly, interfering with factors critical for HCV release did not have a concomitant effect on secretion of triglycerides, ApoB, or ApoE, indicating that particles are likely released from Huh7 cells via pathways distinct from that of very-low-density lipoprotein (VLDL). Finally, we show that HCV NS2 perturbs TGN architecture, redistributing TGN membranes to closely associate with HCV core protein residing on lipid droplets. These findings support the notion that HCV hijacks TGN-endosome trafficking to facilitate particle assembly and release. Moreover, although essential for assembly and infectivity, the trafficking of mature virions is seemingly independent of host lipoproteins. IMPORTANCE: The mechanisms by which infectious hepatitis C virus particles are assembled and released from the cell are poorly understood. We show that the virus subverts host cell trafficking pathways to effect the release of virus particles and disrupts the structure of the Golgi apparatus, a key cellular organelle involved in secretion. In addition, we demonstrate that the mechanisms used by the virus to exit the cell are distinct from those used by the cell to release lipoproteins, suggesting that the virus effects a unique modification to cellular trafficking pathways.

Antidepressant-like effects of guanfacine and sex-specific differences in effects on c-fos immunoreactivity and paired-pulse ratio in male and female mice.

RATIONALE: The a2A-noradrenergic agonist guanfacine can decreases stress-induced smoking in female, but not male, human smokers. It is not known whether these effects are due to effects on mood regulation and/or result from nicotinic-cholinergic interactions. OBJECTIVES: The objective of the study was to determine whether there are sex differences in the effect of guanfacine in tests of anxiolytic and antidepressant efficacy in mice at baseline and in a hypercholinergic model of depression induced by the acetylcholinesterase inhibitor physostigmine. METHODS: The effects of guanfacine were measured in the light/dark box, tail suspension, and the forced swim test in female and male C57BL/6J mice. In parallel, electrophysiological properties were evaluated in the prefrontal cortex, a critical brain region involved in stress responses. c-fos immunoreactivity was measured in other brain regions known to regulate mood. RESULTS: Despite a baseline sex difference in behavior in the forced swim test (female mice were more immobile), guanfacine had similar, dose-dependent, antidepressant-like effects in mice of both sexes (optimal dose, 0.15 mg/kg). An antidepressant-like effect of guanfacine was also observed following pre-treatment with physostigmine. A sex difference in the paired-pulse ratio in the prefrontal cortex (PFC) (male, 1.4; female, 2.1) was observed at baseline that was normalized by guanfacine. Other brain areas involved in cholinergic control of depression-like behaviors, including the basolateral amygdala and lateral septum, showed sex-specific changes in c-fos expression. CONCLUSIONS: Guanfacine has a robust antidepressant-like effect and can reverse a depression-like state induced by increased acetylcholine (ACh) signaling. These data suggest that different brain areas are recruited in female and male mice, despite similar behavioral responses to guanfacine.

Expression of the 5-HT1A serotonin receptor in the hippocampus is required for social stress resilience and the antidepressant-like effects induced by the nicotinic partial agonist cytisine.

Nicotinic acetylcholine receptor (nAChR) blockers potentiate the effects of selective serotonin reuptake inhibitors (SSRIs) in some treatment-resistant patients; however, it is not known whether these effects are independent, or whether the two neurotransmitter systems act synergistically. We first determined that the SSRI fluoxetine and the nicotinic partial agonist cytisine have synergistic effects in a mouse model of antidepressant efficacy, whereas serotonin depletion blocked the effects of cytisine. Using a pharmacological approach, we found that the 5-HT1A agonist 8-OH-DPAT also potentiated the antidepressant-like effects of cytisine, suggesting that this subtype might mediate the interaction between the serotonergic and cholinergic systems. The 5-HT1A receptors are located both presynaptically and postsynaptically. We therefore knocked down 5-HT1A receptors in either the dorsal raphe (presynaptic autoreceptors) or the hippocampus (a brain area with high expression of 5-HT1A heteroreceptors sensitive to cholinergic effects on affective behaviors). Knockdown of 5-HT1A receptors in hippocampus, but not dorsal raphe, significantly decreased the antidepressant-like effect of cytisine. This study suggests that serotonin signaling through postsynaptic 5-HT1A receptors in the hippocampus is critical for the antidepressant-like effects of a cholinergic drug and begins to elucidate the molecular mechanisms underlying interactions between the serotonergic and cholinergic systems related to mood disorders.

NS2 is dispensable for efficient assembly of hepatitis C virus-like particles in a bipartite trans-encapsidation system.

Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1-based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated subgenomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late-acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted, chimeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.

Structure-guided design affirms inhibitors of hepatitis C virus p7 as a viable class of antivirals targeting virion release.

UNLABELLED: Current interferon-based therapy for hepatitis C virus (HCV) infection is inadequate, prompting a shift toward combinations of direct-acting antivirals (DAA) with the first protease-targeted drugs licensed in 2012. Many compounds are in the pipeline yet primarily target only three viral proteins, namely, NS3/4A protease, NS5B polymerase, and NS5A. With concerns growing over resistance, broadening the repertoire for DAA targets is a major priority. Here we describe the complete structure of the HCV p7 protein as a monomeric hairpin, solved using a novel combination of chemical shift and nuclear Overhauser effect (NOE)-based methods. This represents atomic resolution information for a full-length virus-coded ion channel, or "viroporin," whose essential functions represent a clinically proven class of antiviral target exploited previously for influenza A virus therapy. Specific drug-protein interactions validate an allosteric site on the channel periphery and its relevance is demonstrated by the selection of novel, structurally diverse inhibitory small molecules with nanomolar potency in culture. Hit compounds represent a 10,000-fold improvement over prototypes, suppress rimantadine resistance polymorphisms at submicromolar concentrations, and show activity against other HCV genotypes. CONCLUSION: This proof-of-principle that structure-guided design can lead to drug-like molecules affirms p7 as a much-needed new target in the burgeoning era of HCV DAA.

Mutations in hepatitis C virus p7 reduce both the egress and infectivity of assembled particles via impaired proton channel function.

Hepatitis C virus (HCV) p7 protein is critical for the efficient production of infectious virions in culture. p7 undergoes genotype-specific protein-protein interactions as well as displaying channel-forming activity, making it unclear whether the phenotypes of deleterious p7 mutations result from the disruption of one or both of these functions. Here, we showed that proton channel activity alone, provided in trans by either influenza virus M2 or genotype 1b HCV p7, was both necessary and sufficient to restore infectious particle production to genotype 2a HCV (JFH-1 isolate) carrying deleterious p7 alanine substitutions within the p7 dibasic loop (R33A, R35A), and the N-terminal trans-membrane region (N15 : C16 : H17/AAA). Both mutations markedly reduced mature p7 abundance, with those in the dibasic loop also significantly reducing levels of mature E2 and NS2. Interestingly, whilst M2 and genotype 1b p7 restored the same level of intracellular infectivity as JFH-1 p7, supplementing with the isogenic protein led to a further increase in secreted infectivity, suggesting a late-acting role for genotype-specific p7 protein interactions. Finally, cells infected by viruses carrying p7 mutations contained non-infectious core-containing particles with densities equivalent to WT HCV, indicating a requirement for p7 proton channel activity in conferring an infectious phenotype to virions.

Formation of higher-order foot-and-mouth disease virus 3D(pol) complexes is dependent on elongation activity.

The replication of many viruses involves the formation of higher-order structures or replication "factories." We show that the key replication enzyme of foot-and-mouth disease virus (FMDV), the RNA-dependent RNA polymerase, forms fibrils in vitro. Although there are similarities with previously characterized poliovirus polymerase fibrils, FMDV fibrils are narrower, are composed of both protein and RNA, and, importantly, are seen only when all components of an elongation assay are present. Furthermore, an inhibitory RNA aptamer prevents fibril formation.

Resistance mutations define specific antiviral effects for inhibitors of the hepatitis C virus p7 ion channel.

UNLABELLED: The hepatitis C virus (HCV) p7 ion channel plays a critical role during infectious virus production and represents an important new therapeutic target. Its activity is blocked by structurally distinct classes of small molecules, with sensitivity varying between isolate p7 sequences. Although this is indicative of specific protein-drug interactions, a lack of high-resolution structural information has precluded the identification of inhibitor binding sites, and their modes of action remain undefined. Furthermore, a lack of clinical efficacy for existing p7 inhibitors has cast doubt over their specific antiviral effects. We identified specific resistance mutations that define the mode of action for two classes of p7 inhibitor: adamantanes and alkylated imino sugars (IS). Adamantane resistance was mediated by an L20F mutation, which has been documented in clinical trials. Molecular modeling revealed that L20 resided within a membrane-exposed binding pocket, where drug binding prevented low pH-mediated channel opening. The peripheral binding pocket was further validated by a panel of adamantane derivatives as well as a bespoke molecule designed to bind the region with high affinity. By contrast, an F25A polymorphism found in genotype 3a HCV conferred IS resistance and confirmed that these compounds intercalate between p7 protomers, preventing channel oligomerization. Neither resistance mutation significantly reduced viral fitness in culture, consistent with a low genetic barrier to resistance occurring in vivo. Furthermore, no cross-resistance was observed for the mutant phenotypes, and the two inhibitor classes showed additive effects against wild-type HCV. CONCLUSION: These observations support the notion that p7 inhibitor combinations could be a useful addition to future HCV-specific therapies.

Role of myristoylation and N-terminal basic residues in membrane association of the human immunodeficiency virus type 1 Nef protein.

Human immunodeficiency virus type 1 Nef protein is N-terminally myristoylated, a modification reported to be required for the association of Nef with cytoplasmic membranes. As myristate alone is not sufficient to anchor a protein stably into a membrane, it has been suggested that N-terminal basic residues contribute to Nef membrane association via electrostatic interactions with acidic phospholipids. Here, data are presented pertaining to the role of the myristate and basic residues in Nef membrane association, subcellular localization and function. Firstly, by using a biochemical assay for membrane association it was shown that, whereas myristoylation of Nef was not essential, mutation of a cluster of four arginines between residues 17 and 22 reduced membrane association dramatically. Mutation of two lysines at residues 4 and 7 had negligible effect alone, but when combined with the arginine substitutions, abrogated membrane association completely. By using indirect immunofluorescence, it was demonstrated that mutation of either of the two basic clusters altered the subcellular distribution of Nef dramatically. Thirdly, the requirement of the arginine and lysine clusters for Nef-mediated CD4 down modulation was shown to correlate precisely with membrane association. These data suggest that membrane localization and subcellular targeting of Nef are controlled by a complex interplay of signals at the N terminus of the protein.

The di-leucine motif in the cytoplasmic tail of CD4 is not required for binding to human immunodeficiency virus type 1 Nef, but is critical for CD4 down-modulation.

The human immunodeficiency virus type 1 (HIV-1) nef gene encodes a 205 residue, myristoylated phosphoprotein that has been shown to play a critical role in the replication and pathogenesis of the virus. One of the most studied functions of the Nef protein is the down-modulation of cell surface CD4. Nef has been reported to interact with both the cytoplasmic tail of CD4 and proteins that are components of the endocytic machinery, thereby enhancing the endocytosis of CD4 through clathrin-coated pits. A di-leucine motif in the cytoplasmic tail of CD4 (residues 413/414) was reported to be essential both for Nef mediated down-modulation and for Nef binding. In order to further characterize the involvement of this di-leucine motif in CD4 down-modulation we generated a CD4 mutant in which the leucines were substituted by alanines, termed CD4(LL-AA). We demonstrate here that, contrary to previous data obtained with the cytoplasmic tail of CD4 alone, full-length CD4(LL-AA) bound to Nef both in vivo, in recombinant baculovirus-infected Sf9 cells, and in vitro. In contrast the di-leucine motif was required for both Nef-mediated and phorbol ester-induced CD4 down-modulation, suggesting that the essential requirement for the di-leucine motif in CD4 down-modulation reflects the fact that this motif is needed for the interactions of CD4 with the endocytic machinery, not for the interaction with Nef. We have also exploited the observation that CD4(LL-AA) is refractory to Nef-mediated down-modulation to provide the first experimental evidence for a physical interaction between Nef and CD4 in intact mammalian cells.