Schwann cell-secreted PGE2 promotes sensory neuron excitability during development.

Electrical excitability-the ability to fire and propagate action potentials-is a signature feature of neurons. How neurons become excitable during development and whether excitability is an intrinsic property of neurons remain unclear. Here, we demonstrate that Schwann cells, the most abundant glia in the peripheral nervous system, promote somatosensory neuron excitability during development. We find that Schwann cells secrete prostaglandin E2, which is necessary and sufficient to induce developing somatosensory neurons to express normal levels of genes required for neuronal function, including voltage-gated sodium channels, and to fire action potential trains. Inactivating this signaling pathway in Schwann cells impairs somatosensory neuron maturation, causing multimodal sensory defects that persist into adulthood. Collectively, our studies uncover a neurodevelopmental role for prostaglandin E2 distinct from its established role in inflammation, revealing a cell non-autonomous mechanism by which glia regulate neuronal excitability to enable the development of normal sensory functions.

Investigating the Relation between Anxiety Sensitivity and Substance Use: What Are the Roles of Social Anxiety and Outcome Expectancies?

Objective: Research on the association between anxiety sensitivity (AS) and substance use is mixed, with some studies showing a positive association and others showing no association. Other relevant variables, such as social anxiety and outcome expectancies, may help us understand how and for whom AS is linked to substance use. This study tested (a) the associations between AS and alcohol use, cannabis use, and drinking games and pregaming behaviors among young adults, and (b) the mediating role of social anxiety and moderating role of outcome expectancies in these associations. Method: Participants (N = 199, 69% women) were young adults (19 to 25 years) who completed a 30-minute online self-report questionnaire on their substance use. Results: Results revealed significant negative associations between AS and drinking game and pregaming participation. AS was not directly associated with other substance use outcomes. The association between AS and hazardous cannabis use was moderated by relaxation and tension reduction expectancies, but outcome expectancies did not moderate any of the other associations between AS and substance use outcomes. Social anxiety mediated the associations between AS and hazardous cannabis use and both drinking game and pregaming participation. Conclusions: Findings highlight the complex association between AS and different substance use outcomes. Outcome expectancies and social anxiety may help explain how AS is associated with hazardous cannabis use and drinking game/pregaming participation, respectively. More effective interventions can be developed by understanding the relation between AS and substance use.

Mental health and physical activity in SCI: Is anxiety sensitivity important?

Compared to research conducted with nondisabled samples, little is known about the relation between mental health and physical activity (PA) in individuals with a spinal cord injury (SCI). Despite this population being more at risk of experiencing anxiety and depression and less likely to engage in PA, few studies have investigated other factors that may impact this association in this population such as anxiety sensitivity (AS). AS is a fear of physiological arousal sensations, and importantly has been shown to be negatively associated with PA in people without disabilities. It is unknown if the changes to how one experiences physiological sensations after a SCI impacts the relation between AS and PA. OBJECTIVE: This study investigated which forms of PA are predicted by anxiety and depression and whether AS is predictive of PA in this population. RESEARCH METHOD: 98 participants with a SCI (both paraplegia and tetraplegia) completed an online questionnaire that had measures of PA, AS, and anxiety and depression. RESULTS: It was found that symptoms of anxiety were significantly associated with mild-intensity PA. Interestingly AS was positively associated with moderate-intensity PA. CONCLUSIONS: The results of this study show that the relation between mental health and PA in this sample may not mirror what has been found in people without disabilities. More research is needed to replicate these findings as well as to investigate other potential mechanisms that may be relevant for people with a SCI. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Anxiety Sensitivity in the Sexual Context: Links between Sexual Anxiety Sensitivity and Sexual Well-Being.

Anxiety sensitivity, the fear of physiological arousal sensations, has been linked to lower sexual frequency, poorer sexual function, and greater sexual anxiety. The current study assessed whether anxiety sensitivity specific to the sexual context, termed sexual anxiety sensitivity, was linked to a wide range of indicators of sexual well-being over and above associations accounted for by general anxiety sensitivity. As a first step, we developed the Sexual Anxiety Sensitivity Inventory (SASI). Participants were 484 adults aged 19 to 60 years old who completed an on-line survey. To develop the SASI, we constructed parallel items to those on the Anxiety Sensitivity Scale-3 (ASI-3; Taylor et al., 2007). The SASI demonstrated the same three-factor structure as the ASI-3 and showed high internal consistency providing evidence for its reliability. As predicted, sexual anxiety sensitivity was significantly associated with all ten of the markers of the behavioral, cognitive-affective, and functional domains of sexual well-being assessed and six of these associations remained significant after controlling for general anxiety sensitivity. The results provide evidence that sexual anxiety sensitivity is an important construct for understanding individuals' sexual well-being and provide initial evidence that the specificity of the SASI has value as a reliable and valid measure for assessing sex-related anxiety sensitivity. Implications for clinicians and researchers are discussed.

Gender Differences and the Influence of Body Composition on Land and Pool-Based Assessments of Anaerobic Power and Capacity.

Consistent differences between males and females have been shown in land-based measurements of anaerobic power and capacity. However, these differences have not been investigated for a tethered 30-s maximal swimming test (TST). The purpose of this study is to explore gender differences in land and pool-based assessments of anaerobic power (Fpeak) and capacity (Fmean), as well as the influence of body composition. Thirteen males and fifteen females completed land (Wingate (WAnT)) and pool-based (TST) measures of anaerobic power and capacity previously described in the literature. Additionally, the subjects completed assessments of body composition via air displacement plethysmography. The males produced higher force than the females for Fpeak (p < 0.001) and Fmean (p = 0.008) during the TST. However, linear regression analysis determined that lean mass significantly predicted Fpeak (p = 0.002) and Fmean (p < 0.001) during the TST, while gender was no longer significant (p = 0.694 and p = 0.136, respectively). In conclusion, increases in anaerobic power and capacity (Fpeak and Fmean) may be a function of increased lean mass in males and females, warranting future research on the impact of resistance training programs on force production and swimming performance.

Epigenome-wide association study of alcohol consumption in N = 8161 individuals and relevance to alcohol use disorder pathophysiology: identification of the cystine/glutamate transporter SLC7A11 as a top target.

Alcohol misuse is common in many societies worldwide and is associated with extensive morbidity and mortality, often leading to alcohol use disorders (AUD) and alcohol-related end-organ damage. The underlying mechanisms contributing to the development of AUD are largely unknown; however, growing evidence suggests that alcohol consumption is strongly associated with alterations in DNA methylation. Identification of alcohol-associated methylomic variation might provide novel insights into pathophysiology and novel treatment targets for AUD. Here we performed the largest single-cohort epigenome-wide association study (EWAS) of alcohol consumption to date (N = 8161) and cross-validated findings in AUD populations with relevant endophenotypes, as well as alcohol-related animal models. Results showed 2504 CpGs significantly associated with alcohol consumption (Bonferroni p value < 6.8 × 10-8) with the five leading probes located in SLC7A11 (p = 7.75 × 10-108), JDP2 (p = 1.44 × 10-56), GAS5 (p = 2.71 × 10-47), TRA2B (p = 3.54 × 10-42), and SLC43A1 (p = 1.18 × 10-40). Genes annotated to associated CpG sites are implicated in liver and brain function, the cellular response to alcohol and alcohol-associated diseases, including hypertension and Alzheimer's disease. Two-sample Mendelian randomization confirmed the causal relationship of consumption on AUD risk (inverse variance weighted (IVW) p = 5.37 × 10-09). A methylation-based predictor of alcohol consumption was able to discriminate AUD cases in two independent cohorts (p = 6.32 × 10-38 and p = 5.41 × 10-14). The top EWAS probe cg06690548, located in the cystine/glutamate transporter SLC7A11, was replicated in an independent cohort of AUD and control participants (N = 615) and showed strong hypomethylation in AUD (p < 10-17). Decreased CpG methylation at this probe was consistently associated with clinical measures including increased heavy drinking days (p < 10-4), increased liver function enzymes (GGT (p = 1.03 × 10-21), ALT (p = 1.29 × 10-6), and AST (p = 1.97 × 10-8)) in individuals with AUD. Postmortem brain analyses documented increased SLC7A11 expression in the frontal cortex of individuals with AUD and animal models showed marked increased expression in liver, suggesting a mechanism by which alcohol leads to hypomethylation-induced overexpression of SLC7A11. Taken together, our EWAS discovery sample and subsequent validation of the top probe in AUD suggest a strong role of abnormal glutamate signaling mediated by methylomic variation in SLC7A11. Our data are intriguing given the prominent role of glutamate signaling in brain and liver and might provide an important target for therapeutic intervention.

PCSK9 and the Gut-Liver-Brain Axis: A Novel Therapeutic Target for Immune Regulation in Alcohol Use Disorder.

Alcohol use disorder (AUD) is a chronic relapsing disorder characterized by an impaired ability to control or stop alcohol intake and is associated with organ damage including alcohol-associated liver disease (ALD) and progressive neurodegeneration. The etiology of AUD is complex, but organ injury due to chronic alcohol use can be partially attributed to systemic and local inflammation along the gut-liver-brain axis. Excessive alcohol use can result in translocation of bacterial products into circulation, increased expression of pro-inflammatory cytokines, and activation of immune cells, including macrophages and/or microglia in the liver and brain. One potential mediator of this alcohol-induced inflammation is proprotein convertase subtilisin/kexin type 9 (PCSK9). PCSK9 is primarily known for its regulation of plasma low-density lipoprotein cholesterol but has more recently been shown to influence inflammatory responses in the liver and brain. In rodent and post-mortem brain studies, chronic alcohol use altered methylation of the PCSK9 gene and increased expression of PCSK9 in the liver and cerebral spinal fluid. Additionally, PCSK9 inhibition in a rat model of ALD attenuated liver inflammation and steatosis. PCSK9 may play an important role in alcohol-induced pathologies along the gut-liver-brain axis and may be a novel therapeutic target for AUD-related liver and brain inflammation.

Epigenome-wide association study and multi-tissue replication of individuals with alcohol use disorder: evidence for abnormal glucocorticoid signaling pathway gene regulation.

Alcohol use disorder (AUD) is a chronic debilitating disorder with limited treatment options and poorly defined pathophysiology. There are substantial genetic and epigenetic components; however, the underlying mechanisms contributing to AUD remain largely unknown. We conducted the largest DNA methylation epigenome-wide association study (EWAS) analyses currently available for AUD (total N = 625) and employed a top hit replication (N = 4798) using a cross-tissue/cross-phenotypic approach with the goal of identifying novel epigenetic targets relevant to AUD. Results show that a network of differentially methylated regions in glucocorticoid signaling and inflammation-related genes were associated with alcohol use behaviors. A top probe consistently associated across all cohorts was located in the long non-coding RNA growth arrest specific five gene (GAS5) (p < 10-24). GAS5 has been implicated in regulating transcriptional activity of the glucocorticoid receptor and has multiple functions related to apoptosis, immune function and various cancers. Endophenotypic analyses using peripheral cortisol levels and neuroimaging paradigms showed that methylomic variation in GAS5 network-related probes were associated with stress phenotypes. Postmortem brain analyses documented increased GAS5 expression in the amygdala of individuals with AUD. Our data suggest that alcohol use is associated with differential methylation in the glucocorticoid system that might influence stress and inflammatory reactivity and subsequently risk for AUD.

Out of breath, out of time: interactions between HIF and circadian rhythms.

Humans have internal circadian clocks that ensure that important physiological functions occur at specific times of the day. These molecular clocks are regulated at the genomic level and exist in most cells of the body. Multiple circadian resetting cues have been identified, including light, temperature, and food. Recently, oxygen has been identified as a resetting cue, and emerging science indicates that this occurs through interactions at the cellular level between the circadian transcription-translation feedback loop and the hypoxia-inducible pathway (hypoxia-inducible factor; subject of the 2019 Nobel Prize in Physiology or Medicine). This review will cover recently identified relationships between HIF and proteins of the circadian clock. Interactions between the circadian clock and hypoxia could have wide-reaching implications for human diseases, and understanding the molecular mechanisms regulating these overlapping pathways may open up new strategies for drug discovery.

Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) in the Brain and Relevance for Neuropsychiatric Disorders.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has long been studied in the liver due to its regulation of plasma low-density lipoprotein cholesterol (LDL-C) and its causal role in familial hypercholesterolemia. Although PCSK9 was first discovered in cerebellar neurons undergoing apoptosis, its function in the central nervous system (CNS) is less clear. PCSK9 has been shown to be involved in neuronal differentiation, LDL receptor family metabolism, apoptosis, and inflammation in the brain, but in vitro and in vivo studies offer contradictory findings. PCSK9 expression in the adult brain is low but is highly upregulated during disease states. Cerebral spinal fluid (CSF) PCSK9 concentrations are correlated with neural tube defects and neurodegenerative diseases in human patients. Epigenetic studies reveal that chronic alcohol use may modulate methylation of the PCSK9 gene and genetic studies show that patients with gain-of-function PCSK9 variants have higher LDL-C and an increased risk of ischemic stroke. Early safety studies of the PCSK9 inhibitors evolocumab and alirocumab, used to treat hypercholesterolemia, hinted that PCSK9 inhibition may negatively impact cognition but more recent, longer-term clinical trials found no adverse neurocognitive events. The purpose of this review is to elucidate the role of PCSK9 in the brain, particularly its role in disease pathogenesis.

Synaptotagmin interaction with SNAP-25 governs vesicle docking, priming, and fusion triggering.

SNARE complex assembly constitutes a key step in exocytosis that is rendered Ca(2+)-dependent by interactions with synaptotagmin-1. Two putative sites for synaptotagmin binding have recently been identified in SNAP-25 using biochemical methods: one located around the center and another at the C-terminal end of the SNARE bundle. However, it is still unclear whether and how synaptotagmin-1 × SNARE interactions at these sites are involved in regulating fast neurotransmitter release. Here, we have used electrophysiological techniques with high time-resolution to directly investigate the mechanistic ramifications of proposed SNAP-25 × synaptotagmin-1 interaction in mouse chromaffin cells. We demonstrate that the postulated central binding domain surrounding layer zero covers both SNARE motifs of SNAP-25 and is essential for vesicle docking, priming, and fast fusion-triggering. Mutation of this site caused no further functional alterations in synaptotagmin-1-deficient cells, indicating that the central acidic patch indeed constitutes a mechanistically relevant synaptotagmin-1 interaction site. Moreover, our data show that the C-terminal binding interface only plays a subsidiary role in triggering but is required for the full size of the readily releasable pool. Intriguingly, we also found that mutation of synaptotagmin-1 interaction sites led to more pronounced phenotypes in the context of the adult neuronal isoform SNAP-25B than in the embryonic isoform SNAP-25A. Further experiments demonstrated that stronger synaptotagmin-1 × SNAP-25B interactions allow for the larger primed vesicle pool supported by SNAP-25 isoform B. Thus, synaptotagmin-1 × SNARE interactions are not only required for multiple mechanistic steps en route to fusion but also underlie the developmental control of the releasable vesicle pool.

N-terminal acetylation of the neuronal protein SNAP-25 is revealed by the SMI81 monoclonal antibody.

The monoclonal antibody SMI81 binds SNAP-25, a major player in neurotransmitter release, with high affinity and has previously been used to follow changes in the levels of this protein in neuropsychiatric disorders. We report here that the SMI81 epitope is present at the extreme N-terminus of SNAP-25 and, unusually, cannot be recognized when present as an internal sequence. Although it is known that SNAP-25 can be palmitoylated and phosphorylated in brain, we now reveal the existence of a third modification, acetylation of the N-terminus. This acetylation event greatly increases the efficiency of SMI81 antibody binding. We show that this highly specific antibody can be used for studying brain function in many vertebrate organisms.

Synaptic vesicle docking: sphingosine regulates syntaxin1 interaction with Munc18.

Consensus exists that lipids must play key functions in synaptic activity but precise mechanistic information is limited. Acid sphingomyelinase knockout mice (ASMko) are a suitable model to address the role of sphingolipids in synaptic regulation as they recapitulate a mental retardation syndrome, Niemann Pick disease type A (NPA), and their neurons have altered levels of sphingomyelin (SM) and its derivatives. Electrophysiological recordings showed that ASMko hippocampi have increased paired-pulse facilitation and post-tetanic potentiation. Consistently, electron microscopy revealed reduced number of docked vesicles. Biochemical analysis of ASMko synaptic membranes unveiled higher amounts of SM and sphingosine (Se) and enhanced interaction of the docking molecules Munc18 and syntaxin1. In vitro reconstitution assays demonstrated that Se changes syntaxin1 conformation enhancing its interaction with Munc18. Moreover, Se reduces vesicle docking in primary neurons and increases paired-pulse facilitation when added to wt hippocampal slices. These data provide with a novel mechanism for synaptic vesicle control by sphingolipids and could explain cognitive deficits of NPA patients.

DOC2B acts as a calcium switch and enhances vesicle fusion.

Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusion-competent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PM was supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release.

Cross-linking of phospholipid membranes is a conserved property of calcium-sensitive synaptotagmins.

Synaptotagmins are vesicular proteins implicated in many membrane trafficking events. They are highly conserved in evolution and the mammalian family contains 16 isoforms. We now show that the tandem C2 domains of several calcium-sensitive synaptotagmin isoforms tested, including Drosophila synaptotagmin, rapidly cross-link phospholipid membranes. In contrast to the tandem structure, individual C2 domains failed to trigger membrane cross-linking in several novel assays. Large-scale liposomal aggregation driven by tandem C2 domains in response to calcium was confirmed by the following techniques: turbidity assay, dynamic light-scattering and both confocal and negative stain electron microscopy. Firm cross-linking of membranes was evident from laser trap experiments. High-resolution cryo-electron microscopy revealed that membrane cross-linking by tandem C2 domains results in a constant distance of approximately 9 nm between the apposed membranes. Our findings show the conserved nature of this important property of synaptotagmin, demonstrate the significance of the tandem C2 domain structure and provide a plausible explanation for the accelerating effect of synaptotagmins on membrane fusion.

Real-time assay for monitoring membrane association of lipid-binding domains.

The C2 domain is a common protein module which mediates calcium-dependent phospholipid binding. Several assays have previously been developed to measure membrane association. However, these assays either have technical drawbacks or are laborious to carry out. We now present a simple solution-based turbidity method for rapidly assaying membrane association of single lipid-binding domains in real time. We used the first C2 domain of synaptotagmin 1 (C2A) as a model lipid-binding moiety. Our use of the common dimeric glutathione-S-transferase (GST) fusion tag allowed two C2A domains to be brought into close proximity. Consequently, calcium-triggered phospholipid binding by this artificially dimerized C2A resulted in liposomal aggregation, easily assayed by following absorbance of the solution at 350 nm. The assay is simple and sensitive and can be scaled up conveniently for use in a multiwell plate format, allowing high-throughput screening. In our screens, we identified nickel as a novel activator of synaptotagmin 1 C2A domain membrane association. Finally, we show that the turbidity method can be applied to the study of other GST-tagged lipid-binding proteins such as epsin, protein kinase C-beta, and synaptobrevin.

The mechanism of an exceptional case of reinitiation after translation of a long ORF reveals why such events do not generally occur in mammalian mRNA translation.

The subgenomic mRNA of feline caliciviruses is bicistronic with the two cistrons overlapping by four nucleotides, ..AUGA. The upstream cistron encodes a 75-kDa major capsid protein precursor (pre-VP1), and the downstream cistron a 10-kDa minor capsid protein. The kinetics of translation in reticulocyte lysates show that the downstream cistron is translated by a termination-reinitiation process, which is unusual in not requiring eIF4G or the eIF4F complex. Reinitiation requires the 3'-terminal 87 nucleotides (nt) of the pre-VP1 ORF, but no other viral sequences. The reinitiation site is selected by virtue of its proximity to this 87-nt element, and not its proximity to the pre-VP1 ORF stop codon, although this must be located not more than approximately 30 nt downstream from the restart codon. This 87-nt element was shown to bind 40S ribosomal subunits and initiation factor eIF3, and addition of supplementary eIF3 enhanced reinitiation efficiency. Mutants defective in reinitiation showed reduced affinity for eIF3 or defective 40S subunit binding (or both). These results suggest a mechanism in which some of the eIF3/40S complexes formed during disassembly of post-termination ribosomes bind to this 87-nt element in a position appropriate for reinitiation following acquisition of an eIF2/GTP/Met-tRNA i ternary complex.

Phospholipases and fatty acid signalling in exocytosis.

Vesicle fusion is a ubiquitous biological process involved in general membrane trafficking and a variety of specialized events, for example release of neurotransmitters and hormones, sperm acrosome exocytosis, plasma membrane repair and neurite outgrowth. Many vesicle fusion events have long been known to be activated by phospholipases and products of their activity, such as polyunsaturated arachidonic acid. Polyunsaturated fatty acids (PUFAs) have been proposed to have a number of multiple effectors, including ion channels and the cytoskeleton, but the precise mechanism of PUFA action is still unclear. It was recently reported that omega-3 and omega-6 PUFAs can act on syntaxin, a plasma membrane protein directly involved in vesicle fusion. In this review, we will discuss the role of this new mode of PUFA action in exocytosis.

Mechanism of arachidonic acid action on syntaxin-Munc18.

Syntaxin and Munc18 are, in tandem, essential for exocytosis in all eukaryotes. Recently, it was shown that Munc18 inhibition of neuronal syntaxin 1 can be overcome by arachidonic acid, indicating that this common second messenger acts to disrupt the syntaxin-Munc18 interaction. Here, we show that arachidonic acid can stimulate syntaxin 1 alone, indicating that it is syntaxin 1 that undergoes a structural change in the syntaxin 1-Munc18 complex. Arachidonic acid is incapable of dissociating Munc18 from syntaxin 1 and, crucially, Munc18 remains associated with syntaxin 1 after arachidonic-acid-induced syntaxin 1 binding to synaptosomal-associated protein 25 kDa (SNAP25). We also show that the same principle operates in the case of the ubiquitous syntaxin 3 isoform, highlighting the conserved nature of the mechanism of arachidonic acid action. Neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptors (SNAREs) can be isolated from brain membranes in a complex with endogenous Munc18, consistent with a proposed function of Munc18 in vesicle docking and fusion.