Electrophysiological Assays Research Articles

A macro-transection model of brain trauma for neuromaterial testing with functional electrophysiological readouts.

Functional recovery in penetrating neurological injury is hampered by a lack of clinical regenerative therapies. Biomaterial therapies show promise as medical materials for neural repair through immunomodulation, structural support, and delivery of therapeutic biomolecules. However, a lack of facile and pathology-mimetic models for therapeutic testing is a bottleneck in neural tissue engineering research. We have deployed a two-dimensional, high-density multicellular cortical brain sheet to develop a facile model of injury (macrotransection/scratch wound) in vitro. The model encompasses the major neural cell types involved in pathological responses post-injury. Critically, we observed hallmark pathological responses in injury foci including cell scarring, immune cell infiltration, precursor cell migration, and short-range axonal sprouting. Delivering test magnetic particles to evaluate the potential of the model for biomaterial screening shows a high uptake of introduced magnetic particles by injury-activated immune cells, mimicking in vivo findings. Finally, we proved it is feasible to create reproducible traumatic injuries in the brain sheet (in multielectrode array devices in situ) characterized by focal loss of electrical spiking in injury sites, offering the potential for longer term, electrophysiology plus histology assays. To our knowledge, this is the first in vitro simulation of transecting injury in a two-dimensional multicellular cortical brain cell sheet, that allows for combined histological and electrophysiological readouts of damage/repair. The patho-mimicry and adaptability of this simplified model of brain injury could benefit the testing of biomaterial therapeutics in regenerative neurology, with the option for functional electrophysiological readouts.

Rescue of Mutant CFTR Channel Activity by Investigational Co-Potentiator Therapy.

Background: The potentiator VX-770 (ivacaftor) has been approved as a monotherapy for over 95 cystic fibrosis (CF)-causing variants associated with gating/conductance defects of the CF transmembrane conductance regulator (CFTR) channel. However, despite its therapeutic success, VX-770 only partially restores CFTR activity for many of these variants, indicating they may benefit from the combination of potentiators exhibiting distinct mechanisms of action (i.e., co-potentiators). We previously identified LSO-24, a hydroxy-1,2,3-triazole-based compound, as a modest potentiator of p.Arg334Trp-CFTR, a variant with a conductance defect for which no modulator therapy is currently approved. Objective/Methods: We synthesized a new set of LSO-24 structure-based compounds, screened their effects on p.Arg334Trp-CFTR activity, and assessed the additivity of hit compounds to VX-770, ABBV-974, ABBV-3067, and apigenin. After validation by electrophysiological assays, the most promising hits were also assessed in cells expressing other variants with defective gating/conductance, namely p.Pro205Ser, p.Ser549Arg, p.Gly551Asp, p.Ser945Leu, and p.Gly1349Asp. Results: We found that five compounds were able to increase p.Arg334Trp-CFTR activity with similar efficacy, but slightly greater potency promoted by LSO-150 and LSO-153 (EC50: 1.01 and 1.26 μM, respectively). These two compounds also displayed a higher rescue of p.Arg334Trp-CFTR activity in combination with VX-770, ABBV-974, and ABBV-3067, but not with apigenin. When tested in cells expressing other CFTR variants, LSO-24 and its derivative LSO-150 increased CFTR activity for the variants p.Ser549Arg, p.Gly551Asp, and p.Ser945Leu with a further effect in combination with VX-770 or ABBV-3067. No potentiator was able to rescue CFTR activity in p.Pro205Ser-expressing cells, while p.Gly1349Asp-CFTR responded to VX-770 and ABBV-3067 but not to LSO-24 or LSO-150. Conclusions: Our data suggest that these new potentiators might share a common mechanism with apigenin, which is conceivably distinct from that of VX-770 and ABBV-3067. The additive rescue of p.Arg334Trp-, p.Ser549Arg-, p.Gly551Asp-, and p.Ser945Leu-CFTR also indicates that these variants could benefit from the development of a co-potentiator therapy.

Characterization of a Novel Male Pheromone Compound in Leucoptera sinuella (Lepidoptera: Lyonetiidae) and Its Role in Courtship Behavior.

The poplar moth, Leucoptera sinuella (Lepidoptera: Lyonetiidae), is widely distributed across Europe, Asia, and parts of Africa. It was first identified in Chile in 2015 and has since become a significant pest in the agricultural sector. Additionally, economic losses are further aggravated by the presence of L. sinuella pupae in nearby fruit orchards. This study investigated the presence of a male-produced sex pheromone in L. sinuella, focusing on the hairpencil (HP) glands, which are known as dissemination structures for male pheromones in lepidopterans. Male HP glands were solvent-extracted and analyzed by gas chromatography-mass spectrometry. Chemical microderivatization and comparisons of mass spectra and retention indices of natural compounds with synthetic standards led to the identification of two compounds: (Z)-3-decenyl hexanoate (the major component) and (Z)-3-decen-1-ol (a minor component). Although electrophysiological assays did not show detectable antennal responses to (Z)-3-decenyl hexanoate, behavioral bioassays demonstrated its role as a short-range courtship signal in L. sinuella. Males with ablated hairpencils exhibited significantly reduced courtship success compared to controls; however, exposure of females to synthetic (Z)-3-decenyl hexanoate or HP extract restored male courtship success. To our knowledge, (Z)-3-decenyl hexanoate has not been previously reported as a sex pheromone component in any insect species, making this discovery an intriguing addition to the diversity of chemical communication in insects.

Phlebotomus perniciosus response to volatile organic compounds of dogs and humans.

The olfactory response of insect vectors such as phlebotomine sand flies is a key facet for investigating their interactions with vertebrate hosts and associated vector-borne pathogens. Such studies are mainly performed by assessing the electrophysiological response and the olfactory behaviour of these arthropods towards volatile organic compounds (VOCs) produced by hosts. Nonetheless, few studies are available for species of the subgenera Lutzomyia and Nyssomyia in South America, leaving a void for Old World sand fly species of the genus Phlebotomus. In this study, we evaluated the olfactory responses of Phlebotomus perniciosus, one of the most important vectors of Leishmania infantum in the Old World. To test the P. perniciosus behavioural response to VOCs, 28 compounds isolated from humans and dogs were assessed using electrophysiological (i.e., electroantennogram, EAG) and behavioural assays (i.e., Y-tube olfactometer). In the EAG trials, 14 compounds (i.e., acetic acid, nonanoic acid, 2-propanol, 2-butanol, pentanal, hexanal, nonanal, trans-2-nonenal, decanal, myrcene, p-cymene, verbenone, 2-ethyl-1-hexanol, and acetonitrile) elicited high antennal responses (i.e., ≥ 0.30 mV) in female sand flies, being those VOCs selected for the behavioural assays. From the 14 compounds tested in the Y-tube olfactometer, nonanal was significantly attractive for P. perniciosus females, whereas myrcene and p-cymene were significantly repellents (p < 0.05). The attraction indexes varied from 0.53 for nonanal (i.e., most attractive) to -0.47 to p-cymene (i.e., most repellent). Overall, our results shed light on the role of olfactory cues routing host seeking behaviour in P. perniciosus, with implications to develop sustainable sand fly monitoring as well as control in leishmaniasis endemic areas.

In vitro safety evaluation of dopamine D3R antagonist, R-VK4-116, as a potential medication for the treatment of opioid use disorder.

R-VK4-116 is currently being developed as a medication to treat opioid use disorder (OUD). To characterize in vitro safety properties of R-VK4-116, metabolic stability in hepatocytes or liver microsomes, metabolite identification, metabolism/transporter-mediated drug interactions, lysosomal perturbation, mitochondrial toxicity, off-target enzyme effects, cellular and nuclear receptor functional assays, electrophysiological assays, CiPA, KINOMEscanTM, plasma protein binding, phospholipidosis and steatosis assays were performed. Overall, R-VK4-116 was metabolically stable in hepatocytes and microsomes. Four major metabolites were detected: two mono-oxidation products, one di-oxidation product, and one demethylated plus di-oxidation product. CYP2D6 and CYP3A4 were major contributors in R-VK4-116 metabolism. Further, R-VK4-116 did not induce/inhibit CYP enzymes. However, R-VK4-116 inhibited BCRP/P-gp, and showed antagonist effects on α1A(h), H1(h) and agonist effect on hGABAA∞1β2γ2 at 10 μM. R-VK4-116 inhibited hERG and Kir2.1 at a high concentration of 100 μM. KINOMEscanTM provided 5 hits (CHEK2, HPK1, MARK3, SRPK2 and TNK1) with Kds of >10 μM. Further, R-VK4-116 was bound to human, rat and dog plasma proteins (~83-93%). R-VK4-116 did not induce lysosome perturbation, mitochondrial toxicity, phospholipidosis, or steatosis at ≤10 μM. These results demonstrated that R-VK4-116 possesses favorable in vitro safety properties and supports further development as a potential medication for OUD.

Investigation of epilepsy-related genes in a Drosophila model.

Complex genetic architecture is the major cause of heterogeneity in epilepsy, which poses challenges for accurate diagnosis and precise treatment. A large number of epilepsy candidate genes have been identified from clinical studies, particularly with the widespread use of next-generation sequencing. Validating these candidate genes is emerging as a valuable yet challenging task. Drosophila serves as an ideal animal model for validating candidate genes associated with neurogenetic disorders such as epilepsy, due to its rapid reproduction rate, powerful genetic tools, and efficient use of ethological and electrophysiological assays. Here, we systematically summarize the advantageous techniques of the Drosophila model used to investigate epilepsy genes, including genetic tools for manipulating target gene expression, ethological assays for seizure-like behaviors, electrophysiological techniques, and functional imaging for recording neural activity. We then introduce several typical strategies for identifying epilepsy genes and provide new insights into gene-gene interactions in epilepsy with polygenic causes. We summarize well- established precision medicine strategies for epilepsy and discuss prospective treatment options, including drug therapy and gene therapy for genetic epilepsy based on the Drosophila model. Finally, we also address genetic counseling and assisted reproductive technology as potential approaches for the prevention of genetic epilepsy.

A new regulation mechanism for KCNN4, the Ca2+-dependent K+ channel, by molecular interactions with the Ca2+pump PMCA4b.

KCNN4, a Ca2+-activated K+ channel, is involved in various physiological and pathological processes. It is essential for epithelial transport, immune system, and other physiological mechanisms, but its activation is also involved in cancer pathophysiology as well as red blood cell (RBC) disorders. The activation of KCNN4 in RBC leads to loss of KCl and water, a mechanism known as the "Gardos effect" described 70 years ago. This Ca2+-induced dehydration is irreversible in human RBC and must be tightly controlled to prevent not only hemolysis but also alterations in RBC rheological properties. In this study, we have investigated the regulation of KCNN4 activity after changes in RBC Ca2+ concentration. Using electrophysiology, immunoprecipitation, and proximity ligation assay in human embryonic kidney 293-transfected cells, K562cells, or RBCs, we have found that KCNN4 and the Ca2+ pump PMCA4b (plasma membrane calcium-transporting ATPase 4b) interact tightly with each other, such that the C-terminal domain of PMCA4b regulates KCNN4 activity, independently of the Ca2+ extrusion activity of the pump. This regulation was not restricted to KCNN4: the small-conductance Ca2+-activated K+ channel KCNN2 was similarly regulated by the calcium pump. We propose a new mechanism that could control KCNN4 activity by a molecular inhibitory interaction with PMCA4b. It is suggested that this mechanism could attenuate erythrocyte dehydration in response to an increase in intracellular Ca2+.

An overview of drug-induced sodium channel blockade and changes in cardiac conduction: Implications for drug safety.

The human voltage-gated sodium channel Nav1.5 (hNav1.5/SCN5A) plays a critical role in the initiation and propagation of action potentials in cardiac myocytes, and its modulation by various drugs has significant implications for cardiac safety. Drug-dependent block of Nav1.5 current (INa) can lead to significant alterations in cardiac electrophysiology, potentially resulting in conduction slowing and an increased risk of proarrhythmic events. This review aims to provide a comprehensive overview of the mechanisms by which various pharmacological agents interact with Nav1.5, focusing on the molecular determinants of drug binding and the resultant electrophysiological effects. We discuss the structural features of Nav1.5 that influence drug affinity and specificity. Special attention is given to the concept of state-dependent block, where drug binding is influenced by the conformational state of the channel, and its relevance to therapeutic efficacy and safety. The review also examines the clinical implications of INa block, highlighting case studies of drugs that have been associated with adverse cardiac events, and how the Vaughan-Williams Classification system has been employed to qualify "unsafe" sodium channel block. Furthermore, we explore the methodologies currently used to assess INa block in nonclinical and clinical settings, with the hope of providing a weight of evidence approach including in silico modeling, invitro electrophysiological assays and invivo cardiac safety studies for mitigating proarrhythmic risk early in drug discovery. This review underscores the importance of understanding Nav1.5 pharmacology in the context of drug development and cardiac risk assessment.

Gain of bipolar disorder-related lncRNA AP1AR-DT in mice induces depressive and anxiety-like behaviors by reducing Negr1-mediated excitatory synaptic transmission

BackgroundBipolar disorder is a complex polygenic disorder that is characterized by recurrent episodes of depression and mania, the heterogeneity of which is likely complicated by epigenetic modifications that remain to be elucidated.MethodsWe performed transcriptomic analysis of peripheral blood RNA from monozygotic (MZ) twins discordant for bipolar disorder to identify disease-associated differentially expressed long noncoding RNAs (DE-lncRNAs), which were further validated in the PsychENCODE brain RNA-seq dataset. We then performed behavioral tests, electrophysiological assays, chromatin immunoprecipitation, and PCR to investigate the function of DE-lncRNAs in the mouse and cell models. Statistical analyses were performed using GraphPad Prism 9.0 or SPSS.ResultsWe identified a bipolar disorder-associated upregulated long non-coding RNA (lncRNA), AP1AR-DT. We observed that overexpression of AP1AR-DT in the mouse medial prefrontal cortex (mPFC) resulted in a reduction of both the total spine density and the spontaneous excitatory postsynaptic current (sEPSC) frequency of mPFC neurons as well as depressive and anxiety-like behaviors. A combination of the results of brain transcriptome analysis of AP1AR-DT overexpressing mice brains with the known genes associated with bipolar disorder revealed that NEGR1, which encodes neuronal growth regulator 1, is one of the AP1AR-DT targets and is reduced in vivo upon gain of AP1AR-DT in mice. We further demonstrated that overexpression of recombinant Negr1 in the mPFC neurons of AP1AR-DTOE mice ameliorates depressive and anxiety-like behaviors and normalizes the reduced excitatory synaptic transmission induced by the gain of AP1AR-DT. We finally identified that AP1AR-DT reduces NEGR1 expression by competing for the transcriptional activator NRF1 in the overlapping binding site of the NEGR1 promoter region.ConclusionsThe epigenetic and pathophysiological mechanism linking AP1AR-DT to the modulation of depressive and anxiety-like behaviors and excitatory synaptic function provides etiological implications for bipolar disorder.

Multiscale Responsive Kinetic Modeling: Quantifying Biomolecular Reaction Flux under Varying Electrochemical Conditions.

Attaining a complete thermodynamic and kinetic characterization for processes involving multiple interconnected rare-event transitions remains a central challenge in molecular biophysics. This challenge is amplified when the process must be understood under a range of reaction conditions. Herein, we present a novel condition-responsive kinetic modeling framework that can combine the strengths of bottom-up rate quantification from multiscale simulations with top-down solution refinement using both equilibrium and nonequilibrium experimental data. Although this framework can be applied to any process, we demonstrate its use for electrochemically driven transport through channels and transporters via the development of electrochemically responsive rates. Using the Cl-/H+ antiporter ClC-ec1 as a model system, we show how optimal and predictive kinetic solutions can be obtained when the solution space is grounded by thermodynamic constraints, seeded through multiscale rate quantification, and further refined with experimental data, such as electrophysiology assays. Turning to the Shaker K+ channel, we demonstrate that optimal solutions and biophysical insights can also be obtained with sufficient experimental data. This multi-pathway method also proves capable of identifying single-pathway dominant channel mechanisms but reveals that competing and off-pathway flux is still essential to replicate experimental findings and to describe concentration-dependent channel rectification.

The development of a novel high-throughput membrane potential assay and a solid-supported membrane (SSM)-based electrophysiological assay to study the pharmacological inhibition of GLUT9/SLC2A9 isoforms in a drug discovery program

GLUT9/SLC2A9 is a urate transporter and takes a fundamental role in the maintenance of normal serum urate levels. GLUT9 is the sole transporter of reabsorbed urate from renal epithelial cells to blood, thus making it an ideal pharmacological target for the development of urate-lowering drugs. None of the three currently available assays for studying GLUT9 pharmacological inhibition can support a high throughput drug discovery screening campaign. In this manuscript we present two novel assay technologies which can be used in a drug discovery screening cascade for GLUT9: a GLUT9 membrane potential assay for primary screening; and a solid-supported membrane (SSM)-based supported electrophysiological assay for secondary screening.

Cryo-EM structure of the heteromeric TRPC1/TRPC4 channel.

Transient receptor potential (TRP) ion channels have a crucial role as cellular sensors, mediating diverse physical and chemical stimuli. The formation of heteromeric structures expands the functionality of TRP channels; however, their molecular architecture remains largely unknown. Here we present the cryo-electron microscopy structures of the human TRPC1/TRPC4 heteromer in the apo and antagonist-bound states, both consisting of one TRPC1 subunit and three TRPC4 subunits. The heteromer structure reveals a distinct ion-conduction pathway, including an asymmetrically constricted selectivity filter and an asymmetric lower gate, primarily attributed to the incorporation of TRPC1. Through a structure-guided electrophysiological assay, we show that both the selectivity filter and the lower part of the S6 helix participate in deciding overall preference for permeating monovalent cations. Moreover, we reveal that the introduction of one lysine residue of TRPC1 into the tetrameric central cavity is enough to render one of the most important functional consequences of TRPC heteromerization: reduced calcium permeability. Our results establish a framework for addressing the structure-function relationship of the heteromeric TRP channels.

Emotional comorbidities in epilepsy result from seizure-induced corticosterone activity

People with epilepsy often have psychiatric comorbidities that can significantly impair their quality of life. We previously reported that repeated seizure activity persistently alters endocannabinoid (eCB) signaling in the amygdala which accounts for comorbid emotional dysregulation in rats, however, the mechanism by which these alterations in eCB signaling within the epileptic brain occur is unclear. Endocannabinoid signaling is influenced by corticosterone (CORT) to modulate cognitive and emotional processes and a hyperactive hypothalamic-pituitary-adrenal (HPA) axis occurs in both people with epilepsy and nonhuman animal models of epilepsy.We employed selective pharmacological tools and a variety of approaches including whole-cell patch-clamp electrophysiology, behavioural paradigms and biochemical assays in amygdala kindled adult male Long-Evans rats. We aimed to determine whether seizures induce hypersecretion of CORT and the role this plays in eCB system dysregulation, impaired fear memory, and anxiety-like behaviours associated with seizure activity.Plasma CORT levels were significantly and consistently elevated following seizures over the course of kindling. Pre-seizure administration with the CORT synthesis inhibitor metyrapone prevented this seizure-induced CORT increase, prevented amygdala anandamide downregulation, and synaptic alteration induced by seizure activity. Moreover, treatment with metyrapone or combined glucocorticoid receptor (GR)/mineralocorticoid receptor (MR) antagonists prior to each elicited seizure were equally effective in preventing chronically altered anxiety-like behaviour and fear memory responses.Inhibiting seizure-induced corticosterone synthesis, or directly blocking the effects of CORT at GR/MR prevents deleterious changes in emotional processing and could be a treatment option for emotional comorbidities in epilepsy.

Excitation-contraction coupling inhibitors potentiate the actions of botulinum neurotoxin type A at the neuromuscular junction.

Botulinum neurotoxin type A1 (BoNT/A) is one of the most potent neurotoxins known. At the same time, it is also one of the safest therapeutic agents used for the treatment of several human disorders and in aesthetic medicine. Notwithstanding great effectiveness, strategies to accelerate the onset and prolong BoNT/A action would significantly ameliorate its pharmacological effects with beneficial outcomes for clinical use. Here, we combined BoNT/A with two fast-acting inhibitors of excitation-contraction coupling inhibitors (ECCI), either the μ-conotoxin CnIIIC or dantrolene, and tested the effect of their co-injection on a model of hind-limb paralysis in rodents using behavioural, biochemical, imaging and electrophysiological assays. The BoNT/A-ECCI combinations accelerated the onset of muscle relaxation. Surprisingly, they also potentiated the peak effect and extended the duration of the three BoNT/A commercial preparations OnabotulinumtoxinA, AbobotulinumtoxinA and IncobotulinumtoxinA. ECCI co-injection increased the number of BoNT/A molecules entering motoneuron terminals, which induced a faster and greater cleavage of SNAP-25 during the onset and peak phases, and prolonged the attenuation of nerve-muscle neurotransmission during the recovery phase. We estimate that ECCI co-injection yields a threefold potentiation in BoNT/A pharmacological activity. Overall, our results show that the pharmacological activity of BoNT/A can be combined and synergized with other bioactive molecules and uncover a novel strategy to enhance the neuromuscular effects of BoNT/A without altering the neurotoxin moiety or intrinsic activity, thus maintaining its exceptional safety profile.

Emc1 is essential for vision and zebrafish photoreceptor outer segment morphogenesis.

Inherited retinal diseases (IRDs) are a rare group of eye disorders characterized by progressive dysfunction and degeneration of retinal cells. In this study, we characterized the raifteirí (raf) zebrafish, a novel model of inherited blindness, identified through an unbiased ENU mutagenesis screen. A mutation in the largest subunit of the endoplasmic reticulum membrane protein complex, emc1 was subsequently identified as the causative raf mutation. We sought to elucidate the cellular and molecular phenotypes in the emc1-/- knockout model and explore the association of emc1 with retinal degeneration. Visual behavior and retinal electrophysiology assays demonstrated that emc1-/- mutants had severe visual impairments. Retinal histology and morphometric analysis revealed extensive abnormalities, including thinning of the photoreceptor layer, in addition to large gaps surrounding the lens. Notably, photoreceptor outer segments were drastically smaller, outer segment protein expression was altered and hyaloid vasculature development was disrupted. Transcriptomic profiling identified cone and rod-specific phototransduction genes significantly downregulated by loss of emc1. These data shed light on why emc1 is a causative gene in inherited retinal disease and how outer segment morphogenesis is regulated.

N318L Blocks the Interaction of Fluralaner but Not Broflanilide or Fipronil with the Insect GABA Receptor In Vivo.

Fluralaner is a novel insecticide targeting the ionotropic GABA receptor (GABAR) subunit, RDL. A recent study revealed that N316L, a substitution of asparagine (N) with leucine (L), in the second transmembrane (M2)-spanning region reduced the antagonist action of fluralaner on the housefly Musca domestica RDL (MdRDL) in vitro. To verify the impact of N316L in vivo, the corresponding mutation (N318L) in the fruitfly Drosophila melanogaster RDL (DmRDL) was constructed using CRISPR/Cas9 genome editing. The homozygous DmRDLN318L mutant showed a 9.87-fold resistance to fluralaner compared with w1118 while still being highly sensitive to broflanilide and fipronil, which is consistent with those findings observed in the electrophysiology assays of the homomeric DmRDLWT or DmRDLN318L channel. Moreover, DmRDLN318L led to malformed ovaries, stunted eggs, and sterility in homozygous females. These results highlighted N318 as a molecular site for fluralaner in vivo and in vitro and might elucidate the resistance mechanisms of insects against fluralaner.

Structure and functional studies of Avt1, a novel peptide from the sea anemone Aulactinia veratra

Sea anemones are a rich source of peptide toxins spanning a diverse range of biological activities, typically targeting proteins such as ion channels, receptors and transporters. These peptide toxins and their analogues are usually highly stable and selective for their molecular targets, rendering them of interest as molecular tools, insecticides and therapeutics. Recent transcriptomic and proteomic analyses of the sea anemone Aulactinia veratra identified a novel 28-residue peptide, designated Avt1. Avt1 was produced using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The liquid chromatography-mass spectrometry profile of synthetic Avt1 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds. The solution structure determined by NMR revealed that Avt1 adopts an inhibitor cystine knot (ICK) fold, in which a ring is formed by two disulfide bonds with a third disulfide penetrating the ring to create the pseudo-knot. This structure provides ICK peptides with high structural, thermal and proteolytic stability. Consistent with its ICK structure, Avt1 was resistant to proteolysis by trypsin, chymotrypsin and pepsin, although it was not a trypsin inhibitor. Avt1 at 100 nM showed no activity in patch–clamp electrophysiological assays against several mammalian voltage-gated ion channels, but has structural features similar to toxins targeting insect sodium ion channels. Although sequence homologues of Avt1 are found in a number of sea anemones, this is the first representative of this family to be characterised structurally and functionally.

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids.

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulateaspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

Unveiling the Neurodegenerative Alterations through Oral Stem Cells

Parkinson’s disease (PD) is a neurodegenerative condition characterized by the progressive and selective loss of dopaminergic (DAergic) neurons in the midbrain. The replacement of neuromelanin (NM)–containing DAergic neurons in the substantia nigra and the enhancement of NM concentration could offer a promising and safe approach to treating PD symptoms. The objective of this study was to investigate and compare the potential of human periapical-cysts mesenchymal stem cells (hPCy-MSCs) and dental pulp stem cells (DPSCs) to differentiate into DAergic NM-producing neurons and to generate functional 3-dimensional (3D) midbrain-like organoids in vitro. We assessed the changes in morphology and behavior of neuron-like cells (NLCs) as well as the expression of molecular markers characterizing the DAergic neurons. Furthermore, we observed electrically active and functionally mature DAergic neurons by means of electrophysiological assays, NM dosage assays, and the quantification of dopamine release by high-performance liquid chromatography. Our results demonstrate for the first time that both hPCy-MSCs and DPSCs are capable of differentiating into NLCs, further confirmed by the increase in lactate levels in the medium of cells exposed to neurogenic conditions. Importantly, we have induced such NLCs to further differentiate into functional DAergic NM-producing neurons. Finally, 3D midbrain-like organoids have been produced from oral stem cells: they appear as neurosphere-like structures diffusely expressing the neural marker β-III tubulin and containing NM-like granules. Our findings open up a novel and fascinating opportunity to rethink oral stem cells, and the derived 3D disease models, as a strategic and reliable tool for unveiling the neurodegenerative alterations.

Ginsenoside Rg1 ameliorates stress-exacerbated Parkinson's disease in mice by eliminating RTP801 and α-synuclein autophagic degradation obstacle.

Emerging evidence shows that psychological stress promotes the progression of Parkinson's disease (PD) and the onset of dyskinesia in non-PD individuals, highlighting a potential avenue for therapeutic intervention. We previously reported that chronic restraint-induced psychological stress precipitated the onset of parkinsonism in 10-month-old transgenic mice expressing mutant human α-synuclein (αSyn) (hαSyn A53T). We refer to these as chronic stress-genetic susceptibility (CSGS) PD model mice. In this study we investigated whether ginsenoside Rg1, a principal compound in ginseng notable for soothing the mind, could alleviate PD deterioration induced by psychological stress. Ten-month-old transgenic hαSyn A53T mice were subjected to 4 weeks' restraint stress to simulate chronic stress conditions that worsen PD, meanwhile the mice were treated with Rg1 (40 mg· kg-1 ·d-1, i.g.), and followed by functional magnetic resonance imaging (fMRI) and a variety of neurobehavioral tests. We showed that treatment with Rg1 significantly alleviated both motor and non-motor symptoms associated with PD. Functional MRI revealed that Rg1 treatment enhanced connectivity between brain regions implicated in PD, and in vivo multi-channel electrophysiological assay showed improvements in dyskinesia-related electrical activity. In addition, Rg1 treatment significantly attenuated the degeneration of dopaminergic neurons and reduced the pathological aggregation of αSyn in the striatum and SNc. We revealed that Rg1 treatment selectively reduced the level of the stress-sensitive protein RTP801 in SNc under chronic stress conditions, without impacting the acute stress response. HPLC-MS/MS analysis coupled with site-directed mutation showed that Rg1 promoted the ubiquitination and subsequent degradation of RTP801 at residues K188 and K218, a process mediated by the Parkin RING2 domain. Utilizing αSyn A53T+; RTP801-/- mice, we confirmed the critical role of RTP801 in stress-aggravated PD and its necessity for Rg1's protective effects. Moreover, Rg1 alleviated obstacles in αSyn autophagic degradation by ameliorating the RTP801-TXNIP-mediated deficiency of ATP13A2. Collectively, our results suggest that ginsenoside Rg1 holds promise as a therapeutic choice for treating PD-sensitive individuals who especially experience high levels of stress and self-imposed expectations.