Human-Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Showed Neuronal Differentiation, Neurite Extension, and Formation of Synaptic Structures in Rodent Ischemic Stroke Brains.

Ischemic stroke is a major cerebrovascular disease with high morbidity and mortality rates; however, effective treatments for ischemic stroke-related neurological dysfunction have yet to be developed. In this study, we generated neural progenitor cells from human leukocyte antigen major loci gene-homozygous-induced pluripotent stem cells (hiPSC-NPCs) and evaluated their therapeutic effects against ischemic stroke. hiPSC-NPCs were intracerebrally transplanted into rat ischemic brains produced by transient middle cerebral artery occlusion at either the subacute or acute stage, and their in vivo survival, differentiation, and efficacy for functional improvement in neurological dysfunction were evaluated. hiPSC-NPCs were histologically identified in host brain tissues and showed neuronal differentiation into vGLUT-positive glutamatergic neurons, extended neurites into both the ipsilateral infarct and contralateral healthy hemispheres, and synaptic structures formed 12 weeks after both acute and subacute stage transplantation. They also improved neurological function when transplanted at the subacute stage with γ-secretase inhibitor pretreatment. However, their effects were modest and not significant and showed a possible risk of cells remaining in their undifferentiated and immature status in acute-stage transplantation. These results suggest that hiPSC-NPCs show cell replacement effects in ischemic stroke-damaged neural tissues, but their efficacy is insufficient for neurological functional improvement after acute or subacute transplantation. Further optimization of cell preparation methods and the timing of transplantation is required to balance the efficacy and safety of hiPSC-NPC transplantation.

Secondary Publication: Proposal for Points of Consideration for Pluripotent Stem Cell Culture.

Human pluripotent stem cells, such as human embryonic stem cells and human induced pluripotent stem cells, are used in basic research and various applied fields, including drug discovery and regenerative medicine. Stem cell technologies have developed rapidly in recent years, and the supply of culture materials has improved. This has facilitated the culture of human pluripotent stem cells and has enabled an increasing number of researchers and bioengineers to access this technology. At the same time, it is a challenge to share the basic concepts and techniques of this technology among researchers and technicians to ensure the reproducibility of research results. Human pluripotent stem cells differ from conventional somatic cells in many aspects, and many points need to be considered in their handling, even for those experienced in cell culture. Therefore, we have prepared this proposal, "Points of Consideration for Pluripotent Stem Cell Culture," to promote the effective use of human pluripotent stem cells. This proposal includes seven items to be considered and practices to be confirmed before using human pluripotent stem cells. These are laws/guidelines and consent/material transfer agreements, diversity of pluripotent stem cells, culture materials, thawing procedure, media exchange and cell passaging, freezing procedure, and culture management. We aim for the concept of these points of consideration to be shared by researchers and technicians involved in the cell culture of pluripotent stem cells. In this way, we hope the reliability of research using pluripotent stem cells can be improved, and cell culture technology will advance.

Super-resolution imaging reveals the relationship between CaMKIIß and drebrin within dendritic spines.

Dendritic spines are unique postsynaptic structures that emerge from the dendrites of neurons. They undergo activity-dependent morphological changes known as structural plasticity. The changes involve actin cytoskeletal remodeling, which is regulated by actin-binding proteins. CaMKII is a crucial molecule in synaptic plasticity. Notably, CaMKIIß subtype is known to bind to filamentous-actin and is closely involved in structural plasticity. We have shown that CaMKIIß binds to drebrin, and is localized in spines as both drebrin-dependent and drebrin-independent pools. However, the nanoscale relationship between drebrin and CaMKIIß within dendritic spines has not been clarified. In this study, we used stochastic optical reconstruction microscopy (STORM) to examine the detailed localization of these proteins. STORM imaging showed that CaMKIIß co-localized with drebrin in the core region of spines, and localized in the submembrane region of spines without drebrin. Interestingly, the dissociation of CaMKIIß and drebrin in the core region was induced by NMDA receptor activation. In drebrin knockdown neurons, CaMKIIß was decreased in the core region but not in the submembrane region. Together it indicates that the clustering of CaMKIIß in the spine core region is dependent on drebrin. These findings suggest that drebrin-dependent CaMKIIß is in a standby state before its activation.

Erratum: Dendritic spine formation and synapse maturation in transcription factor-induced human iPSC-derived neurons.

[This corrects the article DOI: 10.1016/j.isci.2023.106285.].

Dendritic spine formation and synapse maturation in transcription factor-induced human iPSC-derived neurons.

Synaptic maturation is reportedly limited in human induced pluripotent stem cell (iPSC)-derived neurons. Notably, their ability to reach postnatal-like stages and form dendritic spines has been difficult to demonstrate unless using long-term cultured organoids. Recent transcription factor (TF)-based induction methods allow the accelerated generation of differentiated neurons, which offers an unprecedented opportunity to address further progression into late developmental stages. Herein, we report on a comprehensive time-course study of TF-induced iPSC neurons cultured in vitro through an intrinsic maturation program following neurogenesis. Moreover, we determined the transcriptional and morphological sequences of key developmental events associated with spinogenesis, including the conversion of drebrin to its brain-specific isoform A and the N-methyl-D-aspartate (NMDA) receptor subunit switch. TF-induced iPSC neurons successfully acquired structural and functional synaptic maturity, which will critically expand their utility in modeling higher brain functions and disorders.

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons.

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

Adenosine A1 receptor antagonist-induced facilitation of postsynaptic AMPA currents in pyramidal neurons of the rat hippocampal CA2 area.

Adenosine A1 receptors (A1R) are widely expressed in hippocampal pyramidal neurons and their presynaptic terminals. It is well known that endogenous adenosine regulates hippocampal function through the activation of A1R in hippocampal pyramidal neurons and has been reported that blockade of A1R induces stronger potentiation of excitatory synaptic transmission in CA2 pyramidal neurons than in CA1 pyramidal neurons. This strong potentiation of CA2 neurons is thought to be caused by the specific modulation of excitatory synaptic transmission through postsynaptic A1R. However, the direct effects of A1R on postsynaptic AMPA channels remain unknown because of the technical difficulties of patch-clamp recording from mature hippocampal CA2 neurons. We recorded synaptic currents from pyramidal neurons in CA1 and CA2 and analyzed the effects of an A1R antagonist on stimulation-evoked synaptic transmission and local application-induced postsynaptic AMPA currents. The antagonist increased the amplitude of evoked synaptic transmission in neurons in both CA1 and CA2. This facilitation was larger in pyramidal neurons in CA2 than in CA1. The antagonist also increased postsynaptic AMPA currents in neurons in CA2 but not in CA1. This facilitation of CA2 AMPA currents was occluded by the intracellular application of a G-protein blocker. Even with the blockade of postsynaptic G-protein signaling, the A1R antagonist increased evoked synaptic transmission in neurons in CA2. These results suggest that synaptic transmission in pyramidal neurons in CA2 is regulated by both presynaptic and postsynaptic A1R. Moreover, A1R regulate excitatory synaptic transmission in pyramidal neurons in CA2 through the characteristic postsynaptic modulation of AMPA currents.

Impairment of ciliary dynamics in an APP knock-in mouse model of Alzheimer's disease.

The primary cilium is a specialized microtubule-based sensory organelle that extends from the cell body of nearly all cell types. Neuronal primary cilia, which have their own unique signaling repertoire, are crucial for neuronal integrity and the maintenance of neuronal connectivity throughout adulthood. Dysfunction of cilia structure and ciliary signaling is associated with a variety of genetic syndromes, termed ciliopathies. One of the characteristic features of human ciliopathies is impairment of memory and cognition, which is also observed in Alzheimer's disease (AD). Amyloid ß peptide (Aß) is produced through the proteolytic processing of amyloid precursor protein (APP), and Aß accumulation in the brain is proposed to be an early toxic event in the pathogenesis of AD. To evaluate the effect of increased Aß level on primary cilia, we assessed ciliary dynamics in hippocampal neurons in an APP knock-in AD model (AppNL-G-F mice) compared to that in wild-type mice. Neuronal cilia length in the CA1, CA3, and dentate gyrus (DG) of wild-type mice increased significantly with age. In AppNL-G-F mice, such elongation was detected in the DG but not in the CA1 and CA3, where more Aß accumulation was observed. We further demonstrated that Aß1-42 treatment decreased cilia length both in hTERT-RPE1 cells and dissociated rat hippocampal neurons. There is growing evidence that reduced cilia length is associated with perturbations of synaptic connectivity and dendrite complexity. Thus, our observations raise the important possibility that structural alterations in neuronal cilia might have a role in AD development.

Neurons Induce Tiled Astrocytes with Branches That Avoid Each Other.

Neurons induce astrocyte branches that approach synapses. Each astrocyte tiles by expanding branches in an exclusive territory, with limited entries for the neighboring astrocyte branches. However, how astrocytes form exclusive territories is not known. For example, the extensive branching of astrocytes may sterically interfere with the penetration of other astrocyte branches. Alternatively, astrocyte branches may actively avoid each other or remove overlapped branches to establish a territory. Here, we show time-lapse imaging of the multi-order branching process of GFP-labeled astrocytes. Astrocyte branches grow in the direction where other astrocyte branches do not exist. Neurons that had just started to grow dendrites were able to induce astrocyte branching and tiling. Upon neuronal loss by glutamate excitotoxicity, astrocytes' terminal processes retracted and more branches went over other branches. Our results indicate that neurons induce astrocyte branches and make them avoid each other.

Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro.

The generation of mature synaptic structures using neurons differentiated from human-induced pluripotent stem cells (hiPSC-neurons) is expected to be applied to physiological studies of synapses in human cells and to pathological studies of diseases that cause abnormal synaptic function. Although it has been reported that synapses themselves change from an immature to a mature state as neurons mature, there are few reports that clearly show when and how human stem cell-derived neurons change to mature synaptic structures. This study was designed to elucidate the synapse formation process of hiPSC-neurons. We propagated hiPSC-derived neural progenitor cells (hiPSC-NPCs) that expressed localized markers of the ventral hindbrain as neurospheres by dual SMAD inhibition and then differentiated them into hiPSC-neurons in vitro. After 49 days of in vitro differentiation, hiPSC-neurons significantly expressed pre- and postsynaptic markers at both the transcript and protein levels. However, the expression of postsynaptic markers was lower than in normal human or normal rat brain tissues, and immunostaining analysis showed that it was relatively modest and was lower than that of presynaptic markers and that its localization in synaptic structures was insufficient. Neurophysiological analysis using a microelectrode array also revealed that no synaptic activity was generated on hiPSC-neurons at 49 days of differentiation. Analysis of subtype markers by immunostaining revealed that most hiPSC-neurons expressed vesicular glutamate transporter 2 (VGLUT2). The presence or absence of NGF, which is required for the survival of cholinergic neurons, had no effect on their cell fractionation. These results suggest that during the synaptogenesis of hiPSC-neurons, the formation of presynaptic structures is not the only requirement for the formation of postsynaptic structures and that the mRNA expression of postsynaptic markers does not correlate with the formation of their mature structures. Technically, we also confirmed a certain level of robustness and reproducibility of our neuronal differentiation method in a multicenter setting, which will be helpful for future research. Synapse formation with mature postsynaptic structures will remain an interesting issue for stem cell-derived neurons, and the present method can be used to obtain early and stable quality neuronal cultures from hiPSC-NPCs.

Ciliary GPCR-based transcriptome as a key regulator of cilia length control.

The primary cilium is a plasma membrane-protruding sensory organelle that efficiently conveys signaling cascades in a highly ordered microenvironment. Its signaling is mediated, in part, by a limited set of GPCRs preferentially enriched in the cilium membrane. This includes melanin-concentrating hormone (MCH) receptor 1 (MCHR1), which plays a role in feeding and mood. In addition to its receptor composition, the length of the cilium is a characteristic parameter that is implicated in its function. We previously found that MCH can dynamically shorten cilia length via the Gi/o and Akt pathways in both MCHR1-expressing hTERT-RPE1 cells (hRPE1 cells) and rat hippocampal neurons. However, the detailed mechanisms by which MCH regulates cilia length through ciliary MCHR1 remains unclear. In this study, we aimed to determine the transcriptome changes in MCHR1-expressing hRPE1 cells in response to MCH to identify the target molecules involved in cilia length regulation via MCHR1 activation. RNA sequencing analysis of ciliated cells subjected to MCH treatment showed upregulation of 424 genes and downregulation of 112 genes compared with static control cells. Validation by quantitative real-time PCR, knocking down, and CRISPR/Cas9-mediated knockout technology identified a molecule, PDZ and LIM domain-containing protein 5 (PDLIM5). Thus, it was considered as the most significant key factor for MCHR1-mediated shortening of cilia length. Additional analyses revealed that the actin-binding protein alpha-actinin 1/4 is a crucial downstream target of the PDLIM5 signaling pathway that exerts an effect on MCHR1-induced cilia shortening. In the endogenous MCHR1-expressing hippocampus, transcriptional upregulation of PDLIM5 and actinin 1/4, following the application of MCH, was detected when the MCHR1-positive cilia were shortened. Together, our transcriptome study based on ciliary MCHR1 function uncovered a novel and important regulatory step underlying cilia length control. These results will potentially serve as a basis for understanding the mechanism underlying the development of obesity and mood disorders.

NMDA receptor-dependent and -independent effects of natural compounds and crude drugs on synaptic states as revealed by drebrin imaging analysis.

Effective drugs that can cure cognitive impairments remain elusive. Because synaptic dysfunction has been correlated with cognitive impairments, drug development to target synaptic dysfunction is important. Recently, natural compounds and crude drugs have emerged as potential therapeutic agents for cognitive disorders. However, their effects on synaptic function remain unclear, because of lack of evaluation system with high reproducibility. We have recently developed highly reproducible in vitro high-content imaging analysis system for evaluation of synaptic function using drebrin as a marker for synaptic states. Therefore, we aimed to examine the direct effects of well-known natural compounds and crude drugs on synaptic states using this system. Rat hippocampal neurons were treated using natural compounds (nobiletin, diosgenin and tenuifolin) and crude drugs (Uncaria Hook [UH], Bezoar Bovis [BB], Coptis Rhizome [CR], Phellodendron Bark [PB] and Polygala Root [PR]). Immunocytochemical analysis was performed, and dendrite lengths and drebrin cluster densities were automatically quantified. We found that diosgenin, tenuifolin, CR, PB and PR decreased drebrin cluster densities, and the effects of PB and PR were partially dependent on N-methyl-D-aspartic acid-type glutamate receptors (NMDARs). Nobiletin and UH did not show any effects, whereas low-dose BB treatment increased drebrin cluster densities. Our results showed that diosgenin, tenuifolin, BB, CR, PB and PR appeared to directly change synaptic states. Particularly, the NMDAR dependency of PB and PR appears to affect synaptic plasticity.

Phosphorylation of GAP-43 T172 is a molecular marker of growing axons in a wide range of mammals including primates.

GAP-43 is a vertebrate neuron-specific protein and that is strongly related to axon growth and regeneration; thus, this protein has been utilized as a classical molecular marker of these events and growth cones. Although GAP-43 was biochemically characterized more than a quarter century ago, how this protein is related to these events is still not clear. Recently, we identified many phosphorylation sites in the growth cone membrane proteins of rodent brains. Two phosphorylation sites of GAP-43, S96 and T172, were found within the top 10 hit sites among all proteins. S96 has already been characterized (Kawasaki et al., 2018), and here, phosphorylation of T172 was characterized. In vitro (cultured neurons) and in vivo, an antibody specific to phosphorylated T172 (pT172 antibody) specifically recognized cultured growth cones and growing axons in developing mouse neurons, respectively. Immunoblotting showed that pT172 antigens were more rapidly downregulated throughout development than those of pS96 antibody. From the primary structure, this phosphorylation site was predicted to be conserved in a wide range of animals including primates. In the developing marmoset brainstem and in differentiated neurons derived from human induced pluripotent stem cells, immunoreactivity with pT172 antibody revealed patterns similar to those in mice. pT172 antibody also labeled regenerating axons following sciatic nerve injury. Taken together, the T172 residue is widely conserved in a wide range of mammals including primates, and pT172 is a new candidate molecular marker for growing axons.

Properties of primary cilia in melanin-concentrating hormone receptor 1-bearing hippocampal neurons in vivo and in vitro.

The primary cilium is a solitary organelle that organizes a sensitive signaling hub in a highly ordered microenvironment. Cilia are plastic structures, changing their length in response to bioactive substances, and ciliary length may be regulated to ensure efficient signaling capacity. Mammalian brain neurons possess primary cilia that are enriched in a set of G protein-coupled receptors (GPCRs), including the feeding-related melanin-concentrating hormone (MCH) receptor 1 (MCHR1). We previously demonstrated a novel biological phenomenon, ciliary MCHR1-mediated cilia length shortening through Gi/o and Akt signaling, using a simple cell culture model of human retinal pigmented epithelial RPE1 cells exogenously expressing MCHR1. In the present study, we characterized the properties of endogenous MCHR1-expressing primary cilia in hippocampal neurons in rodents. Using cultured dissociated rat hippocampal neurons in vitro, we showed that MCH triggered cilia length reduction involved in MCHR1-Gi/o and -Akt signaling. In rat hippocampal slice cultures with preservation of the cytoarchitecture and cell populations, ciliary MCHR1 was abundantly located in the CA1 and CA3 regions, but not in the dentate gyrus. Notably, treatment of slice cultures with MCH induced Gi/o- and Akt-dependent cilia shortening in the CA1 region without influencing cilia length in the CA3 region. Regarding the in vivo mouse brain, we observed higher levels of ciliary MCHR1 in the CA1 and CA3 regions as well as in slice cultures. In the starved state mice, a marked increase in MCH mRNA expression was detected in the lateral hypothalamus. Furthermore, MCHR1-positive cilia length in the hippocampal CA1 region was significantly shortened in fasted mice compared with fed mice. The present findings focused on the hippocampus provide a potential approach to investigate how MCHR1-driven cilia shortening regulates neuronal activity and physiological function toward feeding and memory tasks.

Specific induction and long-term maintenance of high purity ventricular cardiomyocytes from human induced pluripotent stem cells.

Currently, cardiomyocyte (CM) differentiation methods require a purification step after CM induction to ensure the high purity of the cell population. Here we show an improved human CM differentiation protocol with which high-purity ventricular-type CMs can be obtained and maintained without any CM purification process. We induced and collected a mesodermal cell population (platelet-derived growth factor receptor-α (PDGFRα)-positive cells) that can respond to CM differentiation cues, and then stimulated CM differentiation by means of Wnt inhibition. This method reproducibly generated CMs with purities above 95% in several human pluripotent stem cell lines. Furthermore, these CM populations were maintained in culture at such high purity without any further CM purification step for over 200 days. The majority of these CMs (>95%) exhibited a ventricular-like phenotype with a tendency to structural and electrophysiological maturation, including T-tubule-like structure formation and the ability to respond to QT prolongation drugs. This is a simple and valuable method to stably generate CM populations suitable for cardiac toxicology testing, disease modeling and regenerative medicine.

Systematic expression analysis of genes related to generation of action potentials in human iPS cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a valuable tool to characterize the pharmacology and toxic effects of drugs on heart cells. In particular, hiPSC-CMs can be used to identify drugs that generate arrhythmias. However, it is unclear whether the expression of genes related to generation of CM action potentials differs between hiPSC-CM cell lines and the mature human heart. To address this, we obtained accurate gene expression profiles of commercially available hiPSC-CM cell lines with quantitative real time RT-PCR analysis. Expression analysis of ten cardiac proteins important for generation of action potentials and three cardiac proteins important for muscle contractility was performed using GAPDH for normalization. Comparison revealed large variations in expression levels among hiPSC-CM cell lines and between hiPSC-CMs and normal human heart. In general, gene expression in hiPSC-CM cell lines was more similar to an immature, stem-like cell than a mature cardiomyocyte from human heart samples. These results provide quantitative information about differences in gene expression between hiPSC-CM cell lines, essential for interpreting pharmacology experiments. Our approach can be used as an experimental guideline for future research on gene expression in hiPSC-CMs.

High-content imaging analysis for detecting the loss of drebrin clusters along dendrites in cultured hippocampal neurons.

INTRODUCTION: Detection of drug effects on neuronal synapses is important for predicting their adverse effects. We have used drebrin as a marker to detect the synaptic changes in cultured neurons. High concentration of glutamate decreases the amount of drebrin in synapses. To increase the availability of this method for high throughput analysis, we applied the drebrin-based evaluation of synapses to high-content imaging analysis using microplates. METHODS: Three weeks old cultured neurons were fixed and processed for immunocytochemistry to visualize drebrin clusters, dendrites and neuronal cell bodies. After automated image acquisition, total number of drebrin clusters per fields, linear density of drebrin cluster along dendrites, dendrite length and neuron number were automatically measured by a custom-designed protocol. RESULTS: Automated image acquisition and analysis showed that dendrite length and drebrin cluster density along dendrites are measured consistently and reproducibly. In addition, application of 10-100 µM glutamate for 10 min or 0.5-50 µM latrunculin A for 5 min significantly decreased drebrin cluster density without affecting neuron number. These results were consistent with our previous results using manual image acquisition and analysis with regular fluorescence microscope and image analysis software. Furthermore, 0.3 or 1.0 µM staurosporine for 24 h significantly decreased neuron number. DISCUSSION: The present study demonstrates that this high-throughput imaging analysis of drebrin cluster density along dendrites for detecting the effects of substances on synapses is sensitive enough to detect the effects of glutamate receptor activation and latrunculin A treatment, and indicates that this analysis will be useful for safety pharmacology study.

Hyaluronan synthesis supports glutamate transporter activity.

Hyaluronan is synthesized, secreted, and anchored by hyaluronan synthases (HAS) at the plasma membrane and comprises the backbone of perineuronal nets around neuronal soma and dendrites. However, the molecular targets of hyaluronan to regulate synaptic transmission in the central nervous system have not been fully identified. Here, we report that hyaluronan is a negative regulator of excitatory signals. At excitatory synapses, glutamate is removed by glutamate transporters to turn off the signal and prevent excitotoxicity. Hyaluronan synthesized by HAS supports the activity of glial glutamate transporter 1 (GLT1). GLT1 also retracted from cellular processes of cultured astrocytes after hyaluronidase treatment and hyaluronan synthesis inhibition. A serial knockout study showed that all three HAS subtypes recruit GLT1 to cellular processes. Furthermore, hyaluronidase treatment activated neurons in a dissociated rat hippocampal culture and caused neuronal damage due to excitotoxicity. Our findings reveal that hyaluronan helps to turn off excitatory signals by supporting glutamate clearance. Cover Image for this issue: doi: 10.1111/jnc.14516.

Characterization of Functional Primary Cilia in Human Induced Pluripotent Stem Cell-Derived Neurons.

Recent advances in human induced pluripotent stem cells (hiPSCs) offer new possibilities for biomedical research and clinical applications. Neurons differentiated from hiPSCs may be promising tools to develop novel treatment methods for various neurological diseases. However, the detailed process underlying functional maturation of hiPSC-derived neurons remains poorly understood. Here, we analyze the developmental architecture of hiPSC-derived cortical neurons, iCell GlutaNeurons, focusing on the primary cilium, a single sensory organelle that protrudes from the surface of most growth-arrested vertebrate cells. To characterize the neuronal cilia, cells were cultured for various periods and evaluated immunohistochemically by co-staining with antibodies against ciliary markers Arl13b and MAP2. Primary cilia were detected in neurons within days, and their prevalence and length increased with increasing days in culture. Treatment with the mood stabilizer lithium led to primary cilia length elongation, while treatment with the orexigenic neuropeptide melanin-concentrating hormone caused cilia length shortening in iCell GlutaNeurons. The present findings suggest that iCell GlutaNeurons develop neuronal primary cilia together with the signaling machinery for regulation of cilia length. Our approach to the primary cilium as a cellular antenna can be useful for both assessment of neuronal maturation and validation of pharmaceutical agents in hiPSC-derived neurons.

Assessment of NMDA receptor inhibition of phencyclidine analogues using a high-throughput drebrin immunocytochemical assay.

INTRODUCTION: In recent years, new psychoactive substances (NPS) have been widely distributed for abuse purposes. Effective measures to counter the spread of NPS are to promptly legislate them through the risk assessment. Phencyclidine analogues having inhibitory effects toward NMDA receptor (NMDAR) have recently emerged in Japan. Therefore, it is important to establish a high-throughput system for efficiently detecting NPS that can inhibit NMDAR activity. METHODS: Hippocampal neurons prepared from embryonic rats were incubated in 96-well microplates. After 3 weeks in vitro, cultured neurons were preincubated with phencyclidine (PCP) or PCP-analogues, including 3-methoxyphencyclidine (3-MeO-PCP) and 4-[1-(3-methoxyphenyl)cyclohexyl]morpholine (3-MeO-PCMo), and then treated with 100 µM glutamate for 10 min. After fixation, cultured neurons were immunostained with anti-drebrin and anti-MAP2 antibodies. The linear cluster density of drebrin along the dendrites was automatically quantified using a protocol that was originally developed by us. RESULTS: The high-throughput immunocytochemical assay, measuring drebrin cluster density of cultured neurons, demonstrated that glutamate-induced reduction of drebrin cluster density in 96-well plates is competitively inhibited by NMDAR antagonist, APV. The reduction was also antagonized by PCP, 3-MeO-PCP and 3-MeO-PCMo. The inhibitory activity of 3-MeO-PCMo was lower than that of PCP or 3-MeO-PCP, with IC50 values of 26.67 µM (3-MeO-PCMo), 2.02 µM (PCP) and 1.51 µM (3-MeO-PCP). DISCUSSION: The relative efficacy among PCP, 3-MeO-PCP and 3-MeO-PCMo calculated from IC50 are similar to those from Ki values. This suggests that the high-throughput imaging analysis is useful to speculate the Ki values of new PCP analogues without performing the kinetic studies.