Search Results (27)

Prostate stem cell antigen (PSCA) is a Ly6/uPAR protein that targets neuronal nicotinic acetylcholine receptors (nAChRs). It exists in membrane-tethered and soluble forms, with the latter upregulated in Alzheimer’s disease. We hypothesize that PSCA may be linked to a wider spectrum of neurological diseases and could induce neuroinflammation. Indeed, PSCA expression is significantly upregulated in the brain of patients with multiple sclerosis, Huntington’s disease, Down syndrome, bipolar disorder, and HIV-associated dementia. To investigate PSCA’s structure, pharmacology, and inflammatory function, we produced a correctly folded water-soluble recombinant analog (ws-PSCA). In primary hippocampal neurons and astrocytes, ws-PSCA differently regulates secretion of inflammatory factors and adhesion molecules and induces pro-inflammatory responses by increasing TNFβ secretion. Heteronuclear NMR and ¹⁵N relaxation measurements reveal a classical β-structural three-finger fold with conformationally disordered loops II and III. Positive charge clustering on the molecular surface suggests the functional importance of ionic interactions by these loops. Electrophysiological studies in Xenopus oocytes point on ws-PSCA inhibition of α3β2-, high-, and low-sensitive variants of α4β2- (IC₅₀ ~50, 27, and 15 μM, respectively) but not α4β4-nAChRs, suggesting targeting of the β2 subunit. Ensemble docking and molecular dynamics simulations predict PSCA binding to high-sensitive α4β2-nAChR at α4/β2 and β2/β2 interfaces. Complexes are stabilized by ionic and hydrogen bonds between PSCA’s loops II and III and the primary and complementary receptor subunits, including glycosyl groups. This study gives new structural and functional insights into PSCA’s interaction with molecular targets and provides clues to understand its role in the brain function and mental disorders. Full article

Voltage-gated Ca²⁺ channels (VGCCs) are regulated by four CaVβ subunits (CaVβ1–CaVβ4), each showing specific expression patterns in excitable cells. While primarily known for regulating VGCC function, CaVβ proteins also have channel-independent roles, including gene expression modulation. Among these, CaVβ1 is expressed in skeletal muscle as multiple isoforms. The adult isoform, CaVβ1D, localizes at the triad and modulates CaV1 activity during Excitation–Contraction Coupling (ECC). In this study, we investigated the lesser-known embryonic/perinatal CaVβ1 isoforms and their roles in neuromuscular junction (NMJ) formation, maturation, and maintenance. We found that CaVβ1 isoform expression is developmentally regulated through differential promoter activation. Specifically, CaVβ1A is expressed in embryonic muscle and reactivated in denervated adult muscle, alongside the known CaVβ1E isoform. Nerve injury in adult muscle triggers a shift in promoter usage, resulting in re-expression of embryonic/perinatal Cacnb1A and Cacnb1E transcripts. Functional analyses using aneural agrin-induced AChR clustering on primary myotubes demonstrated that these isoforms contribute to NMJ formation. Additionally, their expression during early post-natal development is essential for NMJ maturation and long-term maintenance. These findings reveal previously unrecognized roles of CaVβ1 isoforms beyond VGCC regulation, highlighting their significance in neuromuscular system development and homeostasis. Full article

Sitophilus zeamais, a major pest of stored grains, causes significant post-harvest losses and challenges effective control. While synthetic insecticides pose risks of resistance and toxicity, essential oils (EOs) offer a safer alternative. However, the insecticidal potential of their individual volatile constituents (VCs) remains largely unexplored. This study evaluated the insecticidal activity of 51 EO-derived volatile compounds (VCs) against S. zeamais, identifying the most toxic ones, optimizing 15 synergistic mixtures, and assessing their effects on key insect enzymes. A structure–activity relationship (SAR) analysis determined functional groups associated with insecticidal activity, while a cluster analysis pre-selected 29 ternary mixtures, later refined using response surface methodology (RSM). Additionally, enzymatic assays explored their impact on detoxification and nervous system enzymes, providing insights into potential mechanisms of action. Among the 51 VCs tested, 37 exhibited significant toxicity, with 11 acting as fumigants and 13 displaying contact toxicity. Monocyclic monoterpenoids with ketone or alcohol functional groups and exocyclic unsaturation demonstrated the highest insecticidal activity via both exposure routes. Notably, pulegone enantiomers were particularly effective (LC₅₀ < 0.1 mg/L, LD₅₀ < 7.5 µg/adult). Among the optimized mixtures, 10 displayed strong insecticidal effects, 8 were active through both routes, and 5 exhibited synergistic fumigant interactions. The most effective formulations were M2 (R-pulegone + S-pulegone + S-carvone, LC₅₀ 0.48 mg/L) and M20 (isopulegone + δ-3-carene, LC₅₀ 2.06 mg/L), showing the strongest fumigant and synergistic effects, respectively. Enzymatic assays revealed that while some compounds mildly inhibited GST and CAT, others, such as δ-3-carene (IC₅₀ 0.19 mg/L), significantly inhibited AChE. Five mixtures exhibited synergistic neurotoxicity, with M20 (IC₅₀ 0.61 mg/L) and M12 (IC₅₀ 0.81 mg/L) emerging as the most potent AChE inhibitors. These findings highlight the potential of plant-derived volatile compounds as bioinsecticides, leveraging synergistic interactions to enhance efficacy, disrupt enzymatic pathways, and mitigate resistance. Full article

Advanced paternal age increases the risk of transmitting de novo germline mutations, particularly missense mutations activating the receptor tyrosine kinase (RTK) signalling pathway, as exemplified by the FGFR3 mutation, which is linked to achondroplasia (ACH). This risk is attributed to the expansion of spermatogonial stem cells carrying the mutation, forming sub-clonal clusters in the ageing testis, thereby increasing the frequency of mutant sperm and the number of affected offspring from older fathers. While prior studies proposed a correlation between sub-clonal cluster expansion in the testis and elevated mutant sperm production in older donors, limited data exist on the universality of this phenomenon. Our study addresses this gap by examining the testis-expansion patterns, as well as the increases in mutations in sperm for two FGFR3 variants—c.1138G>A (p.G380R) and c.1948A>G (p.K650E)—which are associated with ACH or thanatophoric dysplasia (TDII), respectively. Unlike the ACH mutation, which showed sub-clonal expansion events in an aged testis and a significant increase in mutant sperm with the donor’s age, as also reported in other studies, the TDII mutation showed focal mutation pockets in the testis but exhibited reduced transmission into sperm and no significant age-related increase. The mechanism behind this divergence remains unclear, suggesting potential pleiotropic effects of aberrant RTK signalling in the male germline, possibly hindering differentiation requiring meiosis. This study provides further insights into the transmission risks of micro-mosaics associated with advanced paternal age in the male germline. Full article

Simufilam is a novel oral drug candidate in Phase 3 clinical trials for Alzheimer’s disease (AD) dementia. This small molecule binds an altered form of filamin A (FLNA) that occurs in AD. This drug action disrupts FLNA’s aberrant linkage to the α7 nicotinic acetylcholine receptor (α7nAChR), thereby blocking soluble amyloid beta_1–42 (Aβ₄₂)’s signaling via α7nAChR that hyperphosphorylates tau. Here, we aimed to clarify simufilam’s mechanism. We now show that simufilam reduced Aβ₄₂ binding to α7nAChR with a 10-picomolar IC₅₀ using time-resolved fluorescence resonance energy transfer (TR-FRET), a robust technology to detect highly sensitive molecular interactions. We also show that FLNA links to multiple inflammatory receptors in addition to Toll-like receptor 4 (TLR4) in postmortem human AD brains and in AD transgenic mice: TLR2, C-X-C chemokine receptor type 4 (CXCR4), C-C chemokine receptor type 5 (CCR5), and T-cell co-receptor cluster of differentiation 4 (CD4). These aberrant FLNA linkages, which can be induced in a healthy control brain by Aβ₄₂ incubation, were disrupted by simufilam. Simufilam reduced inflammatory cytokine release from Aβ₄₂-stimulated human astrocytes. In the AD transgenic mice, CCR5–G protein coupling was elevated, indicating persistent activation. Oral simufilam reduced both the FLNA–CCR5 linkage and the CCR5–G protein coupling in these mice, while restoring CCR5′s responsivity to C-C chemokine ligand 3 (CCL3). By disrupting aberrant FLNA–receptor interactions critical to AD pathogenic pathways, simufilam may promote brain health. Full article

Although skeletal muscle (hSKM) has been proven to be actively involved in Amyotrophic Lateral Sclerosis (ALS) neuromuscular junction (NMJ) dysfunction, it is rarely considered as a pharmacological target in preclinical drug discovery. This project investigated how improving ALS hSKM viability and function effects NMJ integrity. Phenotypic ALS NMJ human-on-a-chip models developed from patient-derived induced pluripotent stem cells (iPSCs) were used to study the effect of hSKM-specific creatine treatment on clinically relevant functional ALS NMJ parameters, such as NMJ numbers, fidelity, stability, and fatigue index. Results indicated comparatively enhanced NMJ numbers, fidelity, and stability, as well as reduced fatigue index, across all hSKM-specific creatine-treated systems. Immunocytochemical analysis of the NMJs also revealed improved post-synaptic nicotinic Acetylcholine receptor (AChR) clustering and cluster size in systems supplemented with creatine relative to the un-dosed control. This work strongly suggests hSKM as a therapeutic target in ALS drug discovery. It also demonstrates the need to consider all tissues involved in multi-systemic diseases, such as ALS, in drug discovery efforts. Finally, this work further establishes the BioMEMs NMJ platform as an effective means of performing mutation-specific drug screening, which is a step towards personalized medicine for rare diseases. Full article

Accelerated postsynaptic remodelling and disturbance of neuromuscular transmission are common features of autoimmune neurodegenerative diseases. Homer protein isoform expression, crosslinking activity and neuromuscular subcellular localisation are studied in mouse hind limb muscles of an experimentally induced autoimmune model of Myasthenia Gravis (EAMG) and correlated to motor end plate integrity. Soleus (SOL), extensor digitorum longus (EDL) and gastrocnemius (GAS) skeletal muscles are investigated. nAChR membrane clusters were studied to monitor neuromuscular junction (NMJ) integrity. Fibre-type cross-sectional area (CSA) analysis is carried out in order to determine the extent of muscle atrophy. Our findings clearly showed that crosslinking activity of Homer long forms (Homer 1b/c and Homer2a/b) are decreased in slow-twitch and increased in fast-twitch muscle of EAMG whereas the short form of Homer that disrupts Homer crosslinking (Homer1a) is upregulated in slow-twitch muscle only. Densitometry analysis showed a 125% increase in Homer protein expression in EDL, and a 45% decrease in SOL of EAMG mice. In contrast, nAChR fluorescence pixel intensity decreased in endplates of EAMG mice, more distinct in type-I dominant SOL muscle. Morphometric CSA of EAMG vs. control (CTR) revealed a significant reduction in EDL but not in GAS and SOL. Taken together, these results indicate that postsynaptic Homer signalling is impaired in slow-twitch SOL muscle from EAMG mice and provide compelling evidence suggesting a functional coupling between Homer and nAChR, underscoring the key role of Homer in skeletal muscle neurophysiology. Full article

In vitro neuromuscular junction (NMJ) models are powerful tools for studying neuromuscular disorders. Although linearly patterned culture surfaces have been reported to be useful for the formation of in vitro NMJ models using mouse motor neuron (MNs) and skeletal muscle (SkM) myotubes, it is unclear how the linearly patterned culture surface increases acetylcholine receptor (AChR) clustering, one of the steps in the process of NMJ formation, and whether this increases the in vitro NMJ formation efficiency of co-cultured human MNs and SkM myotubes. In this study, we investigated the effects of a linearly patterned culture surface on AChR clustering in myotubes and examined the possible mechanism of the increase in AChR clustering using gene expression analysis, as well as the effects of the patterned surface on the efficiency of NMJ formation between co-cultured human SkM myotubes and human iPSC-derived MNs. Our results suggest that better differentiation of myotubes on the patterned surface, compared to the flat surface, induced gene expression of integrin α7 and AChR ε-subunit, thereby increasing AChR clustering. Furthermore, we found that the number of NMJs between human SkM cells and MNs increased upon co-culture on the linearly patterned surface, suggesting the usefulness of the patterned surface for creating in vitro human NMJ models. Full article

Dystrophin is a cytoskeletal protein contributing to the organization of the neuromuscular junction. In Duchenne muscular dystrophy, due to dystrophin absence, the distribution of endplate acetylcholine receptors (AChRs) becomes disorganized. It is still debated whether this is due to the absence of dystrophin or to the repeated damage/regeneration cycles typical of dystrophic muscle. We addressed this controversy studying the endplate in the first 3 postnatal weeks, when muscle damage in dystrophic (mdx) mice is minimal. By synaptic and extra-synaptic patch-clamp recordings in acutely dissociated mdx and wt muscle fibers, we recorded unitary events due to openings of AChR-channels containing the γ and ε subunit. We also examined AChR distribution at the endplate by immunofluorescence assays. No differences between wt and mdx fibers were found in the γ/ε switch, nor in the AChR organization at the endplates up to 21 postnatal days. Conversely, we detected a delayed appearance and disappearance of patches with high channel opening frequency in mdx fibers. Our data emphasize that the innervation-dependent γ/ε switch and AChR organization in the endplate are not affected by the absence of dystrophin, while extra-synaptic AChR cluster formation and disassembly could be differentially regulated in mdx mice. Full article

The cell-surface topography and density of nicotinic acetylcholine receptors (nAChRs) play a key functional role in the synapse. Here we employ in parallel two labeling and two super-resolution microscopy strategies to characterize the distribution of this receptor at the plasma membrane of the mammalian clonal cell line CHO-K1/A5. Cells were interrogated with two targeted techniques (confocal microscopy and stimulated emission depletion (STED) nanoscopy) and single-molecule nanoscopy (stochastic optical reconstruction microscopy, STORM) using the same fluorophore, Alexa Fluor 647, tagged onto either α-bungarotoxin (BTX) or the monoclonal antibody mAb35. Analysis of the topography of nanometer-sized aggregates (“nanoclusters”) was carried out using STORMGraph, a quantitative clustering analysis for single-molecule localization microscopy based on graph theory and community detection, and ASTRICS, an inter-cluster similarity algorithm based on computational geometry. Antibody-induced crosslinking of receptors resulted in nanoclusters with a larger number of receptor molecules and higher densities than those observed in BTX-labeled samples. STORM and STED provided complementary information, STED rendering a direct map of the mesoscale nAChR distribution at distances ~10-times larger than the nanocluster centroid distances measured in STORM samples. By applying photon threshold filtering analysis, we show that it is also possible to detect the mesoscale organization in STORM images. Full article

The heteromeric assembly of α3 and β4 subunits of acetylcholine nicotinic receptors (nAChRs) seems to mediate the secretory response in bovine chromaffin cells. However, there is no information about the localization of these nAChRs in relationship with the secretory active zones in this cellular model. The present work presents the first evidence that, in fact, a population of these receptors is associated through the F-actin cytoskeleton with exocytotic machinery components, as detected by SNAP-25 labeling. Furthermore, we also prove that, upon stimulation, the probability to find α3β4 nAChRs very close to exocytotic events increases with randomized distributions, thus substantiating the clear dynamic behavior of these receptors during the secretory process. Modeling on secretory dynamics and secretory component distributions supports the idea that α3β4 nAChR cluster mobility could help with improving the efficiency of the secretory response of chromaffin cells. Our study is limited by the use of conventional confocal microscopy; in this sense, a strengthening to our conclusions could come from the use of super-resolution microscopy techniques in the near future. Full article

The perturbation of nicotinic cholinergic receptors is thought to underlie many neurodegenerative and neuropsychiatric disorders, such as Alzheimer’s and schizophrenia. We previously identified that the tumor suppressor gene, MEN1, regulates both the expression and synaptic targeting of α7 nAChRs in the mouse hippocampal neurons in vitro. Here we sought to determine whether the α7 nAChRs gene expression reciprocally regulates the expression of menin, the protein encoded by the MEN1 gene, and if this interplay impacts learning and memory. We demonstrate here that α7 nAChRs knockdown (KD) both in in vitro and in vivo, initially upregulated and then subsequently downregulated menin expression. Exogenous expression of menin using an AAV transduction approach rescued α7 nAChRs KD mediated functional and behavioral deficits specifically in hippocampal (CA1) neurons. These effects involved the modulation of the α7 nAChR subunit expression and functional clustering at the synaptic sites. Our data thus demonstrates a novel and important interplay between the MEN1 gene and the α7 nAChRs in regulating hippocampal-dependent learning and memory. Full article

Currently, the etiology of many neuromuscular disorders remains unknown. Many of them are characterized by aberrations in the maturation of the neuromuscular junction (NMJ) postsynaptic machinery. Unfortunately, the molecular factors involved in this process are still largely unknown, which poses a great challenge for identifying potential therapeutic targets. Here, we identified Tks5 as a novel interactor of αdystrobrevin-1, which is a crucial component of the NMJ postsynaptic machinery. Tks5 has been previously shown in cancer cells to be an important regulator of actin-rich structures known as invadosomes. However, a role of this scaffold protein at a synapse has never been studied. We show that Tks5 is crucial for remodeling of the NMJ postsynaptic machinery by regulating the organization of structures similar to the invadosomes, known as synaptic podosomes. Additionally, it is involved in the maintenance of the integrity of acetylcholine receptor (AChR) clusters and regulation of their turnover. Lastly, our data indicate that these Tks5 functions may be mediated by its involvement in recruitment of actin filaments to the postsynaptic machinery. Collectively, we show for the first time that the Tks5 protein is involved in regulation of the postsynaptic machinery. Full article

Proper muscle function depends on the neuromuscular junctions (NMJs), which mature postnatally to complex “pretzel-like” structures, allowing for effective synaptic transmission. Postsynaptic acetylcholine receptors (AChRs) at NMJs are anchored in the actin cytoskeleton and clustered by the scaffold protein rapsyn, recruiting various actin-organizing proteins. Mechanisms driving the maturation of the postsynaptic machinery and regulating rapsyn interactions with the cytoskeleton are still poorly understood. Drebrin is an actin and microtubule cross-linker essential for the functioning of the synapses in the brain, but its role at NMJs remains elusive. We used immunohistochemistry, RNA interference, drebrin inhibitor 3,5-bis-trifluoromethyl pyrazole (BTP2) and co-immunopreciptation to explore the role of this protein at the postsynaptic machinery. We identify drebrin as a postsynaptic protein colocalizing with the AChRs both in vitro and in vivo. We also show that drebrin is enriched at synaptic podosomes. Downregulation of drebrin or blocking its interaction with actin in cultured myotubes impairs the organization of AChR clusters and the cluster-associated microtubule network. Finally, we demonstrate that drebrin interacts with rapsyn and a drebrin interactor, plus-end-tracking protein EB3. Our results reveal an interplay between drebrin and cluster-stabilizing machinery involving rapsyn, actin cytoskeleton, and microtubules. Full article

Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients. Full article