ACS Chemical Neuroscience

Spreading depolarizations (SDs) are waves of mass depolarization that propagate through gray matter following Na+/K+-ATPase (NKA) failure because of stroke, traumatic brain injury or sudden cardiac arrest. SDs expand the initial site of neuronal injury and worsen clinical outcomes. The molecular events underlying SD initiation and propagation are not well understood. In this rodent study, we hypothesized that gray matter stressed by oxygen/glucose deprivation (OGD) releases a compound(s) that promotes SD, which we term a spreading depolarization activator (SDa). We used rat brain slices incubated in artificial cerebrospinal fluid (aCSF) and subjected to OGD to release a putative SDa. The aCSF was collected either prior to ("Pre-SD aCSF") or 10 min after initiation of OGD conditions ("Post-SDOGD aCSF"). These solutions were then separately superfused over a healthy, naive (non-stressed) brain slice. Post-SDOGD aCSF (with re-normalized O2 and glucose) evoked SD in 82.35% of the naive brain slices (n = 17) whereas Pre-SD aCSF evoked no SD in 10 naive slices. Then to investigate the NKA as a potential target of the SDa, we used a hemolysis assay, comparing the effects of Pre- or Post-SDOGD aCSF on red blood cell (RBC) lysis and compared it to the known hemolytic effect of the NKA-specific inhibitor, palytoxin. Post-SDOGD aCSF evoked neither swelling nor lysis of RBCs on its own. However, when a sub-threshold concentration (0.01-0.02 nM) of the specific NKA inhibitor palytoxin (PLTX) was added, a striking "priming" effect was observed, whereby Post-SDOGD aCSF evoked a highly significant increase in both RBC swelling and then hemolysis, compared to Pre-SD aCSF. High pressure liquid chromatography (HPLC) experiments show a several-fold increase in released molecules post-SD vs pre-SD. Overall, this study provides support for SDa release capable of inducing SD-associated swelling in brain slices and, when combined with a trace amount of PLTX, swelling/hemolysis of RBCs caused by NKA inhibition. A greater understanding of the molecular events underlying SD should identify novel targets to reduce recurrent SD-evoked neuronal injury under ischemic conditions.

Amyloid plaques are a hallmark of Alzheimers disease (AD) progression; however, the early stages of plaque formation and the specific amyloid-beta (A{beta}) species involved remain difficult to study. While post-mortem tissue provides insight into end-stage mature plaques, therapeutic development requires targeting the earliest A{beta} oligomers to arrest plaque formation. Furthermore, inherently toxic soluble A{beta} oligomers off-pathway from plaque formation are implicated as a driving force of AD pathology. It also remains unclear if the specific nature of key disease-relevant species can be accurately replicated in preparations of synthetic peptides.. To bridge this gap, we utilize brain organoids carrying AD mutations as a biologically authentic source for A{beta} peptides and oligomers. We demonstrate that these mutations do not disrupt organoid development and that the resulting conditioned media contains A{beta} oligomers with disease-relevant structures. Finally, we show that these oligomers can be concentrated and segregated via differential ultracentrifugation for further experimental applications.

Nicotinic acetylcholine receptors (nAChRs) belong to the pentameric ligand-gated ion channel superfamily (pLGICs). Among them, the neuronal homomeric 7 nAChR is highly permeable to calcium and plays critical roles in synaptic transmission, cell signaling, and inflammation modulation. The biogenesis of 7 nAChRs is enhanced by the chaperone proteins RIC-3 and NACHO. Previously, we reported a motif in the 5-HT3A receptor, another pLGIC, involved in RIC-3 modulation. Residues in this motif are conserved and also found within the L1-MX segment of the 7 nACh subunit. We therefore explored the regulatory roles of these conserved residues in the biogenesis of 7 nAChRs using multiple approaches, including heterologous expression in Xenopus laevis oocytes, mutagenesis, pull-down assays, cell-surface labeling, and two-electrode voltage-clamp (TEVC) recordings. We find that synthetic 7 L1-MX peptide interacts with both RIC-3 and NACHO. In particular, conserved residues W330, R332, and L336 in the L1-MX positively regulates the assembly of 7 oligomers and the biogenesis of 7nAChR. In presence of residues W330, R332, and L336, NACHO promotes an assembly of an 7 pentamer which is resistant to strong denaturing conditions. NACHO-promoted 7 pentamer is also resistant to Endo H enzyme. Sensitivity of the pentamer to moderate temperatures (37 {degrees}C, 45 {degrees}C, and 50 {degrees}C) suggests that NACHO stabilizes the pentamer via non-covalent interactions. In contrast, Ala replacements at these residues disrupt the biogenesis and abolish 7 current. NACHO and RIC-3 co-expression yields partial rescue of functional expression for some Ala replacement constructs. SUMMARYThis work identifies regulatory roles of conserved residues W330, R332, and L336 in the biogenesis of 7 nAChR. This discovery positions MX subdomain as a promising target for future drug development that can minimize adverse effects.

Muscarinic and histamine receptors are neurotransmitter-binding proteins within the large family of G protein-coupled receptors (GPCRs) and are relevant to human health and disease, including multiple sclerosis (MS), a chronic immune-mediated inflammatory demyelinating disease of the central nervous system (CNS) with neurodegenerative components. MS affects approximately 1 in 333 people, and women are affected at roughly threefold higher rates than men. A major pathological feature of MS is demyelination with incomplete remyelination of axons in the CNS. Because oligodendrocyte progenitor cells (OPCs) can differentiate into mature oligodendrocytes that restore myelin, small molecules that promote OPC differentiation represent a potential therapeutic strategy. High-throughput screening identified 18 hit compounds with EC50 values below 0.2 M, including the lead compound CN045, which showed an EC50 of 40 nM in vitro. Cheminformatic and experimental target-identification studies implicated the M1 muscarinic receptor and the H3 histamine receptor as candidate targets. To interpret these findings, we performed docking, molecular dynamics simulations, and binding free-energy analyses on complexes involving CN045 and clemastine, a known antihistamine with antimuscarinic activity. The simulations support weaker and less stable binding of CN045 to H3 than to M1 and identify residue-level interactions that contribute to stability within the M1 binding pocket. Comparisons between CN045 and clemastine at M1 further suggest that the two ligands sample different local conformational ensembles, including differences in conserved microswitch behavior associated with active-like versus inactive-like receptor states. Together, these results provide a structural framework for understanding ligand-specific M1 engagement and may help guide future optimization of remyelination-promoting compounds.

GPR52 is an orphan G protein-coupled receptor implicated in psychiatric and neurodegenerative disorders, but its endogenous ligand remains unidentified, limiting the exploration of its physiological functions and therapeutic potential. We pioneered a novel methodology for orphan GPCR ligand discovery utilizing the GPCR-activation-based (GRAB) strategy by developing GPR52-1.0, a genetically encoded fluorescent sensor. GPR52-1.0 exhibits excellent membrane trafficking and high sensitivity in HEK293T cells, cultured neurons, and acute mouse brain slices. Notably, it detects neuronal activity-dependent endogenous ligand release in the striatum, with responses abolished by a specific antagonist. This sensor provides a powerful tool for identifying GPR52s endogenous ligand(s) and enables real-time monitoring of its activation. Our work lays the foundation for uncovering GPR52s physiological roles and supports future efforts to develop GPR52-targeted therapeutics.

Serotonergic psychedelics have attracted considerable interest as promising therapeutic agents. However, the molecular mechanisms linking their acute hallucinogenic-like effects to longer-lasting neuroplastic responses remain incompletely understood, partly because of the scarcity of native neural models suitable for mechanistic studies. Here, we developed a neural stem cell-derived in vitro model capable of differentiating into neuronal and glial lineages and, after characterization, used it to investigate the molecular pharmacology of serotonergic psychedelics. A panel comprising tryptamines, phenethylamines and ergolines, including psychedelic compounds and selected non-psychedelic analogues, was evaluated alongside ketamine and TrkB agonists. Endpoints included dendritogenesis, synaptogenesis, immediate-early gene induction, BDNF expression and lactate production. TrkB silencing abolished dendritogenic responses to serotonergic psychedelics, ketamine and TrkB agonists, whereas 5-HT2A receptor silencing selectively impaired serotonergic psychedelic-induced plasticity and altered TrkB-dependent responses. Most serotonergic compounds also increased synaptogenesis and induced c-Fos and Egr-2 expression, although ligand-specific differences were evident, particularly for psilocin and the phenethylamines DOI and Ariadne. Uncoupling of Gq/11 or Gi/o protein-dependent signaling differentially modified neuroplastic and transcriptional responses, indicating a ligand and endpoint dependent contribution of both pathways. Serotonergic psychedelics further induced a 5-HT2A receptor dependent lactate response that was generally sensitive to disruption of either Gq/11 or Gi/o protein coupling. Taken together, these findings support a model in which serotonergic psychedelics recruit an integrated 5-HT2A-TrkB signaling network with distinct structural, transcriptional and metabolic outputs, and establish this neural stem cell-derived system as a valuable platform for screening and dissecting the signaling basis of psychedelic action.

The HIV-1 Vpu protein aids viral adaptation by influencing host cell pathways via protein interactions. While Vpu is mainly found in plasma and endomembranes, we recently discovered a soluble form that forms a stable, equimolar complex with Ca2+-bound calmodulin (Ca2+-CaM), potentially affecting Vpus cellular trafficking. Here, to determine the binding affinity and identify regions of soluble Vpu involved in CaM binding, we used ensemble Forster Resonance Energy Transfer (eFRET). We tested Cy3-labeled full-length (FL) Vpu, a C-terminal fragment (helices 2 and 3), and a Cy3-labeled FL Vpu V22A/W23Y mutant with substitutions in Vpus helix 1. All Vpus variants were labeled at residue L42C, and Ca2+-CaM was tagged with Cy5 at residue S39C. eFRET analysis of 100 nM Cy3-Vpu variants mixed with Cy5-Ca2+-CaM (in the range 100-600 nM) revealed dissociation constants (Kd) and binding energies ({Delta}G) for heterocomplexes. FL Vpu-Ca2+-CaM showed high stability (Kd [~]40 nM,{Delta} G [~]10.1 kcal/mol), while the truncated C-terminal region and V22A/W23Y mutant formed less stable complexes with Ca2+-CaM (Kd[~]200 nM and 800 nM,{Delta} G [~]9 kcal/mol and [~]8.3 kcal/mol). This, a binding hot spot in Vpus CaM-binding motif in helix 1 was identified, which may control the stability of Vpu-Ca2+-CaM complex and Vpus insertion in the membrane: We hypothesize that upon delivery to the membrane, the hydrophobic helix 1 of Vpu dissociates from Ca2+-CaM and inserts in the lipid bilayer; thereafter, CaM dissociates from Vpu facilitated by the reduced Vpu-Ca2+-CaM complex stability. The findings from this study advance our understanding of HIV-1 Vpu interactions with cellular components and may aid the development of antivirals.

Although a differential vulnerability of dopaminergic neurons to degeneration based on their specific location within the dorsal and ventral tiers of the substantia nigra pars compacta (SNcD and SNcV, respectively) has long been postulated, the underlying mechanisms sustaining these tier-specific differences remain poorly understood. Here, upon inducing a viral-mediated enhancement of neuromelanin (NMel) accumulation within dopaminergic neurons in non-human primates, the distribution of Lewy body-like inclusions (LBs) was analyzed within identified SNcD and SNcV neurons, together with their intracellular NMel levels. Results showed that the vast majority of intracytoplasmic inclusions were found in SNcV neurons, and indeed correlated to higher pigmentation levels. By contrast, only very few LBs were found in calbindin-positive neurons of the SNcD, which in parallel exhibited very low levels of NMel accumulation. These results postulate an additive effect made of a tier-specific location of LB burden together with high pigmentation levels as synergistic drivers sustaining the preferential vulnerability of SNcV dopaminergic neurons. Moreover, the evidence obtained here supported that NMel accumulation beyond a given threshold triggers the aggregation of endogenous -Syn in the form of LBs; therefore, approaches intended to reduce pigmentation levels in SNcV neurons would likely induce a neuroprotective effect by preventing the subsequent aggregation of -Syn.

CAPON (NOS1AP) is an adaptor protein involved in neuronal nitric oxide synthase (nNOS) signaling and has been implicated in Alzheimers disease (AD), excitotoxicity, and tau-associated neurodegeneration. Here, we report the identification of cyclic peptide ligands targeting CAPON using phage display screening of a disulfide-constrained peptide library. Phage enrichment, ELISA validation, microscale thermophoresis (MST), and biolayer interferometry (BLI) identified CAP1 as the lead peptide, exhibiting low micromolar binding affinity toward CAPON. Computational studies further supported stable CAPON-CAP1 interactions through complementary hydrophobic and electrostatic contacts. Functionally, CAP1 attenuated A{beta}42-induced neuronal toxicity, suppressed NMDA-driven nitric oxide production, and reduced pathological tau phosphorylation in neuronal models under AD-relevant stress conditions. In addition, CAP1 demonstrated favorable preliminary pharmacokinetic properties, including good aqueous solubility, plasma stability, and measurable membrane permeability. Collectively, these findings establish the first cyclic peptide ligands targeting CAPON and identify CAP1 as a promising scaffold for modulation of CAPON-dependent neurodegenerative signaling.

Fv-HSP72 is a rapid cell-penetrating human heat shock protein for the treatment of traumatic organ injuries. We have shown this re-engineered protein (HSP72) is capable of crossing the blood brain barrier (BBB) of rats suffering a controlled cortical impact (CCI) and remains in brain tissue for up to 12 hours; long after clearance from the cortex of uninjured rats. Peptide sequences unique to Fv-HSP72 allow for its differential detection from endogenous HSP72. Male Sprague-Dawley rats were divided into 10 groups of n=10 with those animals receiving a CCI subjected to a unilateral cortical contusion simulating a moderate to severe brain injury using an electronically controlled pneumatic impact device. Control groups were either uninjured (Sham), injured (TBI Only), or injured and given buffer (TBI+Vehicle). Rats treated with one of three Fv-HSP72 variants were dosed at 10 or 30mg/kg 15m post-impact, then sacrificed 48 hours later. Cortical tissues were extracted from the ipsilateral and contralateral hemispheres for biomarker analysis. Here we report results of our drug inhibiting neurodegeneration based on five biomarkers (NF-L, pNF-H, pTau [T181, T231, S396]). These results were statistically significant, especially for one of the Fv-HSP72 variants, when comparing differences both between treatment groups and within groups (i.e. when comparing ipsi-vs. contralateral hemispheres). Significant inhibition of oxidative stress (3-NT) and inflammatory (IL-6) biomarkers were also observed (both p<0.0001). With similar results obtained for a blast injury model being published elsewhere, the analyses suggest Fv-HSP72 is neuroprotective following a direct impact brain injury. One sentence summaryThis study describes the effectiveness of a biologic agent, Fv-HSP72, in significantly inhibiting neuronal tissue damage in the brain when administered after a direct cortical impact.

Formation of the {beta}-amyloid (A{beta}) plaques is a pathological hallmark of Alzheimers disease (AD), and is believed to be a primary cause of dementia in elderly individuals. In the present work, we simulated the conformational evolution of A{beta}42 dimers in solution and in membrane-like environment to explore the folding of A{beta}42 along fibrillation. The molecular dynamics (MD) simulation was steered by experimental internuclear distance restraints obtained using solid-state nuclear magnetic resonance (ssNMR) spectroscopy. Our results revealed that several hydrophobic and polar motifs within the A{beta}42 sequence played key roles in the early-stage nucleation process of fibrillation and those motifs are also the stabilizing agents in the mature fibrils judged by the energy contribution. Our results also indicated that the peptide association with membrane bilayers could modulate the structural evolution pathways towards fibrillation. These findings contributed to a better understanding of the molecular level structural polymorphisms inherent to A{beta}42 fibrils. Further, the current work demonstrated that the combination of MD simulations with ssNMR-based experimental restraints provided a reliable method for studying structural changes of A{beta}. HighlightO_LIUsing solid-state NMR restraints guided molecular dynamic simulation, {beta}-amyloid dimers displayed consistent {beta}-strand-prone regions, which are major stabilizing segments for mature fibrils. C_LIO_LI{beta}-amyloid dimers evolved differently with or without interacting with the lipid bilayers. C_LIO_LIExperimental restraints guided simulation provided molecular level insights about early-stage interactions along the progress of {beta}-amyloid fibrillation C_LI

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial immune receptor genetically and functionally linked to Alzheimers disease (AD). VG-3927, the first clinical-stage small-molecule TREM2 agonist, has been proposed to function as a transmembrane molecular glue and positive allosteric modulator (PAM). Whether it directly engages the extracellular ligand-recognition surface of TREM2 remains unknown. Here, we used a deep learning-based blind docking algorithm to map potential VG-3927 binding sites across TREM2 and identified a binding site within the ectodomain hydrophobic groove, a ligand-recognition surface previously implicated in A{beta} and apoE binding. Microscale thermophoresis (MST) confirmed direct interaction of VG-3927 with TREM2 under optimized PEG-400 buffer conditions and independently demonstrated binding of A{beta}1-42 to the receptor. Co-incubation with A{beta} reduced the VG-3927 thermophoretic response, consistent with interference at an overlapping ectodomain binding surface. Consistently, A{beta} induced a rightward shift in the VG-3927 dose-response curve in a Jurkat TREM2-DAP12 NFAT reporter assay and attenuated VG-3927-induced phospho-SYK signaling. Together, these findings support the presence of a previously unrecognized ectodomain interaction mode for VG-3927 and suggest that amyloid-associated ligand occupancy may modulate TREM2 agonist activity within the AD microenvironment.

Parkinsons disease (PD) is the second most progressive degenerative disorder of the brain due to dopaminergic (DA) neuron degenerations and alpha-synuclein (-Syn) accumulations. At present, the disease has no effective treatment. Therefore, the current study objective is to identify a novel anti-PD formula (Zhi-Shi-Huang-Wu Formula, F-2) computed at 8:4:2:1 ratio from HSP 70 promoter activators Valeriana jatamansi (V), Acori talarinowii (A), Scutellaria baicalensis (S), Fructus Schisandrae (F). Traditionally, V is used to cure memory impairments, A treats mental disorders, and chronic mild stress, S for neuroprotection, and F showed multiple therapeutic actions to treat insomnia. This study investigated the neuroprotective potential of the V, A, S, F, formula F-2 and its underlying molecular mechanisms in transgenic Caenorhabditis elegans models. A, S, F, and F-2 successfully restored 6-hydroxydopamine intoxicated DA neuron degenerations, reduced food-sensing behavior disabilities, and attenuated -Syn aggregations. Moreover, activates the lipid deposition and proteasome expressions to confirm -Syn degradations at the cellular level. Reactive oxygen species (ROS) cause oxidative stress, and A, S, F, and F-2 repressed ROS and raised SOD-3 expressions. Overall, these data indicate that V, A, S, F combined into F-2 (22.3%) are more effective against PD progression-like symptom than individual drugs V (0.7%), A (11.4%), S (9.6%), and F (12.6%). These improved neuroprotective actions of F-2 possibly due to following the antioxidative pathway. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/709540v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1a6f1f7org.highwire.dtl.DTLVardef@157a270org.highwire.dtl.DTLVardef@69a238org.highwire.dtl.DTLVardef@1194b5e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Glycoproteins lining the luminal endothelial surface form the glycocalyx, composing the tripartite blood brain barrier. We explored the glycocalyx as a source of danger signals for complement lectin pathway after ischemic stroke. Our data indicate that hypoxic microvascular cells increased -D-mannosyl and N-acetylglucosaminyl exposure after re-oxygenation, favoring mannose binding lectin (MBL) pathogenic deposition, and overexpression of inflammatory genes (ICAM-1 and MMP-2). The hypoxia-conditioned medium induced neuronal damage (reduced MAP-2), microglia and astrocytic reactivity (increased/thickened ramifications) when applied to induced pluripotent stem cell-derived neurons, astrocytes and microglia co-cultures. All these effects were counteracted by mannose-capped gold nanoparticles (Man-GNPs), shown to bind and sequester MBL from the medium. We then tested the Man-GNPs in vivo, in an ischemic stroke model using humanized mice, knocked-in for human MBL. The ischemic mice (males:females 1:1) treated with Man-GNPs (3h after the ischemic onset) exhibited less anxiety at the elevated plus maze and reduced neuronal loss at 8d after ischemia compared to vehicle-treated. Thus, multivalent Man-GNPs represent a promising approach to take MBL away from its glycoproteic targets on the ischemic endothelium, hence preventing downstream pathogenesis. Moreover, these data support circulating MBL as a druggable pharmacological target to prevent the thrombo-inflammatory events following acute brain injury.

Alzheimers disease (AD) is a common neurodegenerative disorder primarily caused by Amyloid-beta (A{beta}) toxicity. Therefore, there is an urgent need to develop novel, effective, and safe drugs to treat AD. Traditional Chinese Medicine (TCM) has a long history of use in protecting against memory impairments. Recently, TCM has attracted growing attention from researchers as a source of potent neuroprotective compounds. In this study, we focus on four TCM herbs with multiple therapeutic properties: Valeriana jatamansi (V; 20 mg/mL), Acori tatarinowii (A; 10 mg/mL), Fructus Schisandrae (F; 5 mg/mL), and Scutellaria baicalensis (S; 2.5 mg/mL). The aim is to develop a neuroprotective anti-AD formulation, named "Zhi-Shi-Wu-Huang" derived from V, A, F, and S, and evaluate its efficacy in transgenic Caenorhabditis elegans models of AD. These four TCM herbs are among the most potent activators of the HSP-70 promoter, promoting the expression of heat shock protein 70 (HSP-70), which helps prevent protein misfolding and aggregation. Additionally, V, A, F, S, and the Zhi-Shi-Wu-Huang formula were found to reduce reactive oxygen species (ROS) production and enhance the expression of superoxide dismutase-3 (sod-3) and chymotrypsin-like proteasomes. Our findings demonstrate that both the individual extracts (V, A, F, S) and the Zhi-Shi-Wu-Huang formulation significantly reduce A{beta}-induced toxicity in transgenic worms by activating the insulin/DAF-16 signaling pathway.

Colony stimulating factor 1 receptor (CSF1R) is a tyrosine kinase receptor that is expressed exclusively in microglia within the CNS. Its endogenous ligands, colony stimulating factor-1 (CSF1) and interleukin-34 (IL-34), are released from neurons, positioning CSF1R as a key mediator receptor of neuron-glia communication. CSF1R is considered not only a potential drug target, but also a biomarker of neuroinflammation. From that perspective, selective radioligands for neuroimaging are of great interest for imaging neuroinflammation and determining drug occupancy. In this study, we have validated the binding characteristics of a CSF1R inhibitor, 4-((5-MethOxy-6-((5-methoxypyridin-2-yl)methoxy)pyridin-3-yl)methyl)-2-(1-methyl-1H-pyrazol-4-yl)pyrimidine (5-MOP) as a novel CSF1R radioligand, by performing in vitro saturation binding experiments in human and murine tissues. 5-MOP was found to be selective for CSF1R among a broad range of kinases. Autoradiography revealed that [3H]5-MOP binds with high affinity (KD = 9.8 nM) to a single saturable binding site in human meningioma tissues, and this binding was displaced with known CSF1R inhibitors, including CPPC, sCSF1inh and GW-2580. In contrast, CPPC, which has been extensively used as a CSF1R radioligand showed substantial cross-reactivity to other brain kinases, including Trk A/B/C, and [3H]CPPC could only be displaced with CPPC itself, not by other ligands, including 5-MOP. These results identify [3H]5-MOP as the most selective radioligand currently available, enabling accurate detection of drug occupancy and activated microglia. Significance of the studyThis study identifies and validates a novel selective radioligand that binds CSF1R with high selectivity and low nanomolar affinity. Because CSF1R is selectively expressed in activated microglia, this radioligand could be useful for detecting neuroinflammatory activity.

The sigma-1 receptor (S1R) is an endoplasmic reticulum transmembrane protein implicated in a wide range of physiological and pathological processes, including neurodegeneration, cancer, and pain modulation. Although X-ray crystallography has revealed S1R as a trimeric assembly with a distinctive triangular architecture, the dynamic behavior of this oligomeric state and its modulation by ligands and membrane composition remain poorly understood. In particular, agonists and antagonists have been experimentally proved to differentially regulate S1R oligomerization although the underlying molecular mechanisms are still obscure. Here, we present the first atomistic molecular dynamics study of trimeric S1R embedded in a physiologically relevant lipid environment. Using a total of 12 {micro}s of simulation time, we investigate the impact of membrane composition, with a specific focus on cholesterol, as well as the conformational response of S1R to pharmacologically distinct ligands: the agonist (+)-pentazocine and the antagonist haloperidol. Our simulations reveal how ligands can alter S1R interprotomer interaction through a mechanism involving the {beta}6-strand of the protein and in particular W136, data that correlate with experimentally observed differences in S1R oligomerization. These findings provide new molecular-level insights into S1R regulation and establish a framework for rationalizing the distinct functional outcomes induced by agonists and antagonists.

This study investigates the interaction between the cationic antimicrobial peptide protamine and bacterial porin OmpF (E. coli) at the single-molecule level. Using high-resolution conductance measurements in planar lipid bilayers, strong voltage- and concentration-dependent ion current blockages with OmpF, indicating significant protamine binding were observed. Further analysis revealed that peptide length influences binding kinetics, with longer peptides showing reduced affinity and slower exchange rates. These findings demonstrate that OmpF is a tractable model for studying cationic peptide-channel interactions and translocation mechanisms relevant to antimicrobial action.

TRPA1 is a polymodal ion channel receptor known for its role in nociception. TRPA1 can be activated by local mechanical perturbations in the surrounding plasma membrane (PM) by molecules that insert in the lipid bilayer. Here, we tested whether TRPA1 function can be modulated by lipid nanoparticles (LNPs) while interacting with the target cell plasma membrane. We found that LNP induce irregular Ca2+ transients in heterologous and native TRPA1-expressing cells, which may reflect stochastic LNP-PM interactions. By using different cell types and applying selective and non-selective TRPA1 inhibitors, we revealed that the cytosolic [Ca2+] is elevated transients arise as a result through multiple mechanisms: TRPA1-dependent Ca2+ influx, TRPA1-independent Ca2+ influx, and Ca2+ mobilization from the endoplasmic reticulum. Our results describe a novel, non-canonical TRPA1 activation mechanism by LNPs, that may be relevant in the context of the development of cancer and nasal vaccines.

Amyloid {beta} peptides (A{beta}) trigger Alzheimers disease (AD) but how has remained elusive. A{beta} stimulates the {beta}2 adrenergic receptor ({beta}2AR), which forms a unique signaling complex with the L-type Ca2+ channel (LTCC) CaV1.2. LTCCs have been implicated in the etiology of dementia and AD. We show that A{beta} acutely potentiates CaV1.2 via the {beta}2AR, which triggers postsynaptic recruitment of Ca2+ permeable (CP) AMPARs in hippocampal cultures and impairs LTP in hippocampal slices within minutes. The long-term consequence is a loss of postsynaptic structure of glutamatergic synapses and neurotoxicity. Disrupting this signaling cascade with highly specific tools prevented all of these effects, unifying a number of currently divergent findings on A{beta} synaptotoxicity including dysregulation of AMPARs and synaptic plasticity. TEASERAmyloid {beta} peptide is the primary pathological agent in Alzheimers disease. It affects the nanoscale structure and function of glutamatergic synapses. The molecular mechanisms are largely unknown except for identification of several binding proteins including the {beta}2 adrenergic receptor. We show that this binding potently (EC50<100 nM) augments Ca2+ influx through the L-type Ca channel CaV1.2. This effect leads to improper recruitment of Ca2+-permeable glutamate receptors to postsynaptic sites (EC50<100 nM), synaptic dysfunction and ultimately neuronal death. This work identifies an essential mechanism in amyloid {beta} neurotoxicity and explains many of the observed postsynaptic alterations. HighlightsImmediate effects of A{beta}-induced stimulation of {beta}2AR on Cav1.2: O_LIA{beta} induces phosphorylation of Cav1.2 on S1928 by PKA C_LIO_LIA{beta} augments Cav1.2 activity via {beta}2AR-induced S1928 phosphorylation within seconds C_LI A{beta}-induced {beta}2AR - Cav1.2 signaling has the following synaptotoxic effects. O_LIA{beta} induces postsynaptic accumulation of Ca-permeable AMPARs via {beta}2AR - Cav1.2 signaling within 20 min C_LIO_LIA{beta} impairs long-term potentiation (LTP) via {beta}2AR - Cav1.2 signaling C_LIO_LIA{beta} impairs postsynaptic structure and neuronal viability over 24 h C_LIO_LIPotency of A{beta} in all the above effects is very high (100 nM A{beta} is saturating!) C_LIO_LIAll effects are prevented in S1928A KI mice and acute displaces {beta}2AR from Cav1.2 with tat-Pep1923 C_LI