ACS Chemical Neuroscience

Insulin amyloid aggregation is a key pathological and pharmaceutical concern, particularly in the context of Type-2 Diabetes (T2D), where amyloid deposition of protein can impair therapeutic efficacy and contribute to cell death leading to local tissue damage. Although gangliosides--glycosphingolipids containing sialic acid residues--are known to modulate amyloid formation in neurodegenerative disorders, their influence on insulin aggregation remains largely unexplored. In this study, we investigate the effects of gangliosides GM3 and GD3 on insulin aggregation. Using Thioflavin-T (ThT) based fluorescence kinetics, Fourier Transform Infrared (FTIR) spectroscopy, Circular Dichroism (CD) spectroscopy, Small Angle X-ray Scattering (SAXS), Nuclear Magnetic Resonance (NMR) spectroscopy, and Transmission Electron Microscopy (TEM), the aggregation pathway, changes in the secondary structure and morphology of insulin aggregates have been characterized. Our results show that both GM3 and GD3 lipids accelerated insulin aggregation in a concentration-dependent manner while steering the pathway away from classical fibril formation, producing short, beaded structures distinct from the extended fibrils observed under lipid-free conditions. CD and FTIR data analyses revealed that insulin in the presence of gangliosides formed non-fibrillar intermediates with distinct secondary structures: {beta}-sheet-rich globular clusters in presence of GD3 and -helical intermediates in GM3-treated samples. Cytotoxicity assays further demonstrated that ganglioside-induced aggregates are significantly less toxic to cells when compared to insulin-only aggregates. Furthermore, ganglioside-bound insulin oligomers retain seeding capacity, suggesting that they can nucleate further aggregation despite their non-fibrillar morphology. These findings underscore the role of gangliosides in modulating insulin amyloid polymorphism and toxicity, offering new insights into their potential impact on the pathology of T2D and treatment strategies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/703542v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@5bf40eorg.highwire.dtl.DTLVardef@f400ddorg.highwire.dtl.DTLVardef@164dcd8org.highwire.dtl.DTLVardef@def4e7_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIGangliosides GD3 and GM3 accelerate insulin aggregation, forming non-fibrillar assemblies. C_LIO_LIGanglioside-bound insulin aggregates are less cytotoxic than fibrillar aggregates. C_LIO_LIDespite altered morphology, ganglioside-bound aggregates retain seeding ability. C_LI

Mucopolysaccharidosis III (MPS III or Sanfilippo disease) is a spectrum of 4 genetic disorders (MPS IIIA-D), caused by defects in the genes SGSH, NAGLU, HGSNAT and GNS encoding enzymes involved in degradation of heparan sulfate (HS). HS accumulates in brain tissues and causes neuronal dysfunction and neurodegeneration leading to neuropsychiatric problems, developmental delays, childhood dementia, blindness and death during the second decade of life. Previously, we demonstrated that pathophysiological mechanisms, underlying MPS IIIC in mouse models, involves functional pathological changes, affecting synaptogenesis and synaptic transmission and leading to learning and memory deficits. These results suggested that a treatment for MPS III could be developed by using compounds inducing synaptogenesis. In the current study, we tested the efficacy of a synthetic peptide ACTH(4-7)PGP, an analog of adrenocorticotropic hormone fragment, previously used as a neuroprotective and anti-inflammatory medication for treatment of acute neurological conditions, including stroke. We show that intranasal administration of ACTH(4-7)PGP restores defective synaptic transmission in CA1 pyramidal neurons of MPS IIIA and MPS IIIC mouse models and rescues the decrease in synaptic proteins in cultured MPS IIIC mouse hippocampal neurons and iPSC-derived neurons of human MPS IIIA, MPS IIIB and MPS IIIC patients. Furthermore, daily intranasal administration of ACTH(4-7)PGP to MPS IIIC and MPS IIIA mice reduces hyperactivity and rescues defects in working and spatial memory, delays progression of CNS pathology including neuroinflammation and axonal demyelination, and increases the lifespan. Together with the absence of any adverse reactions to ACTH(4-7)PGP in the MPS III and WT mice, our results justify testing the drugs efficacy in clinical settings.

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.

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.

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.

Macromolecular crowding modulates enzyme behavior in crowder- and protein-specific ways, yet its impact on viral proteases, which are often key therapeutic targets, remains unclear. Here, we investigated the hepatitis C virus NS3/4A protease under increasing concentrations of polyethylene glycols (PEGs), ficoll, dextran, and lysozyme using a fluorescence-based activity assay and intrinsic tryptophan fluorescence. PEGs reduced catalytic activity while leaving substrate binding largely unaffected or moderately enhanced. These effects were accompanied by a moderate tryptophan fluorescence spectral narrowing, consistent with reduced heterogeneity in local conformational environments. In contrast, ficoll enhanced catalytic efficiency despite stronger fluorescence quenching, indicating local structural changes that favored catalysis. Dextran and lysozyme inhibited protease activity through distinct kinetic patterns, likely reflecting differences in their size, shape, and chemical properties. Thermal analysis revealed crowder-specific local structural changes in NS3/4A without global unfolding up to 65{degrees}C, with differences in local stability and flexibility corroborating the observed kinetic effects. These findings demonstrate that macromolecular crowding modulates NS3/4A catalysis through crowder-specific effects on local structure.

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.

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.

Benzodiazepines represent a major class of drugs prescribed as treatments for anxiety, insomnia and seizures. They act by engaging GABAA receptors in the central nervous system to depress neuronal excitability. Here, the polypharmacology of benzodiazepines was explored by studying their engagement of human TRP channels revealing clonazepam acts as a robust and selective activator of the TRPM8 ion channel. Clonazepam-evoked Ca2+ signals were observed in cells expressing human TRPM8 channels, and in mouse trigeminal neurons. These responses were completely blocked by pharmacological or genetic inhibition of TRPM8 function. This discovery likely explains why clonazepam is an effective treatment for the painful oral condition known as burning mouth disorder, where local activation of TRPM8 channels in sensory neurons mitigates the painful symptoms of this disease.

The brain, though less than 10% of body mass, contains about 25% of total cholesterol (CHL), emphasizing CHLs key role in neuronal function. Many CHL actions are stereospecific, as shown by differences from its 3-hydroxy epimer, epicholesterol (epiCHL). How this minor structural change alters membrane properties and sterol transport remains unclear. Here, we compare fluorescent analogs of CHL (cholestatrienol, CTL) and epiCHL (epicholestatrienol, epiCTL), which closely mimic their natural counterparts. Biophysical membrane properties, such as flip-flop, acyl-chain ordering, and interbilayer transfer, depend on the orientation of the 3-hydroxy group. Similarly, transport by sterol transport proteins (STPs) and intracellular trafficking of the sterols in human astrocytes are stereospecific. Treatment with 25-hydroxycholesterol increases uptake of both epimers, but only CTL shows enhanced esterification and lipid droplet storage. These findings demonstrate that subtle cholesterol structural changes affect cellular homeostasis and establish epiCTL as a useful probe of sterol stereospecificity and trafficking.

Globally, about 264 million individuals across all age groups are impacted by depression, a prevalent central nervous system (CNS) condition. Chronic and enduring depression might result in significant health consequences. Numerous pharmaceutical antidepressants exist for the management of mild to severe depression, largely functioning by modifying neurotransmitter levels in the brain. Nevertheless, these drugs frequently induce a variety of side effects, such as insomnia, constipation, exhaustion, drowsiness, and anxiety. Saffron (Crocus sativus L.) is widely acknowledged as a natural antidepressant with little adverse effects. This study investigated the potential antidepressant mechanisms of saffrons principal bioactive compounds safranal, crocin, and picrocrocin via molecular docking against critical target proteins associated with depression, namely the dopamine transporter (DAT), serotonin transporter (SERT), and monoamine oxidase B (MAO-B). Molecular docking was conducted with AutoDock 4.2 to assess the binding affinity and interaction energy of these drugs with the target proteins. Furthermore, Discovery Studio facilitated the viewing and study of both interacting and non-interacting residues at the docking sites, juxtaposing these interactions with those of established inhibitors in crystal structures. The permeability of the blood-brain barrier (BBB), pharmacokinetic characteristics, and toxicity profiles of saffron components were evaluated using SWISS ADME, DataWarrior, and Osiris Molecular Property Explorer. Among the evaluated elements, safranal had the greatest potential as a competitive inhibitor of the dopamine transporter, according to its notable blood-brain barrier permeability, robust binding affinity, and analogous interaction residues in comparison to nortriptyline, a recognized inhibitor. Our findings indicate that safranal may be a viable natural alternative to traditional antidepressants, with minimized adverse effects.

There is a pressing need for effective alternatives to opioid analgesics, the development of which requires the identification of novel anti-nociceptive drug targets. Here, we have further investigated the anti-nociceptive properties of a GPR35 agonist, cromolyn, in an in vitro model of inflammatory sensitisation. We used ratiometric Ca2+ imaging of cultured sensory neurons to examine the effect of cromolyn on prostaglandin E2 (PGE2)-mediated sensitisation of the pro-nociceptive ion channel, transient receptor potential cation channel, subfamily V, member 1 (TRPV1). The sensitisation of TRPV1 by PGE2 was inhibited by cromolyn in a GPR35-dependent manner. These observations provide further evidence in support of an anti-nociceptive role for GPR35, highlighting the potential use of GPR35 agonists as analgesics.

CAPON (also known as NOS1AP) is an adaptor protein of neuronal nitric oxide synthase (nNOS) that has been implicated in the progression of multiple neurodegenerative diseases, making it an attractive but largely unexplored therapeutic target. To identify small molecule CAPON modulators, we screened a library of 10,000 compounds for CAPON binding using affinity selection-mass spectrometry (AS-MS), which led to the identification of compound MA48 as a potential CAPON binder. Subsequent biophysical validation using microscale thermophoresis (MST) confirmed direct binding, with MA48 exhibiting a dissociation constant (Kd) of 11.9 {micro}M. Structure-activity relationship (SAR) analysis combined with molecular docking was performed to elucidate key pharmacophoric features underlying the MA48/CAPON interaction. To determine whether MA48 disrupts the CAPON-nNOS interaction in a cellular context, we conducted a NanoBRET assay, which demonstrated that MA48 significantly inhibited this interaction in living cells. Collectively, these findings suggest that MA48 represents the first reported small molecule inhibitor of CAPON and provides a foundation for further development of CAPON-targeted therapeutics.

Soluble oligomers and transmembrane channels formed by the 42-residue variant of amyloid beta (A{beta}42) play key roles in Alzheimers disease. Unfortunately, detailed structures of these assemblies have not been determined. Our group addresses this problem by developing atomic scale models. Previously we proposed that both soluble A{beta}42 oligomers and transmembrane channels have symmetric concentric {beta}-barrel structures. Here we expand this hypothesis to include GM1 gangliosides and sometimes cholesterol and lattice models of channel assemblies. The presence of GM1 gangliosides increases the toxicity of A{beta}42, enhances its ability to penetrate liposome membranes, and facilitates interactions between adjacent liposomes. Although the conformations of numerous model assemblies vary, in these models the carboxyl group of GM1 always binds to side-chains of histidine 13 and/or histidine 14. Our soluble oligomer models are consistent with electron microscopy images of beaded annular protofibrils. Our models of membrane-bound assemblies are consistent with the following: freeze-fracture and atomic force microscopy images of A{beta}42 in lipid bilayers, secondary structure results, the calcium hypothesis of Alzheimers Disease, effects of lithium depletion on AD, established {beta}-barrel theory, and energetic criteria.