Cultures Of Hippocampal Neurons Research Articles

A Method for Bioluminescence-Based RNA Monitoring Using Split-Luciferase Reconstitution Techniques.

A bioluminescence-based RNA monitoring method in living cells was developed using a split NanoLuc (NLuc) reconstitution technique. For specific recognition of a target RNA sequence, a mutant PUM-HD (mPUM) was used. The method was applied to β-actin mRNA in various cells, including primary cultures of rat hippocampal neurons. It allowed for continuous observation of intracellular localization distribution of the target mRNA in living cells, providing insights into various biological phenomena involving intracellular RNA localization and dynamics.

Perovskite Quantum Dot-Based Photovoltaic Biointerface for Photostimulation of Neurons.

A promising avenue for vision restoration against retinal degeneration is the use of semiconductor-based photovoltaic biointerfaces to substitute natural photoreceptors. Instead of silicon, perovskite has emerged as an exciting material for solar energy harvesting, and its nanocrystalline forms generally offer better stability than their bulk counterparts in addition to the distinct synthesis and fabrication steps. Herein, we synthesize tetramethylammonium lead iodide (TMAPbI3) perovskite quantum dots (QDs) as a novel photoactive material for photovoltaic biointerfaces. While the TMAPbI3 quantum dots and electrolyte interface induces Faradaic photocurrent under light illumination, the heterojunction with P3HT converts the charge-transfer process to a safe capacitive photocurrent with an improved ionic responsivity of 17.4 mA/W. The integration of the 18-nm quantum dot thickness shows good biocompatibility with primary cultures of hippocampal neurons and the photoresponse of the biointerface triggered photostimulation of the neurons. The rise of perovskite materials can stimulate novel forms of photovoltaic retina implants.

Dynamic Expression of Genes Encoding Ubiquitin Conjugating Enzymes (E2s) During Neuronal Differentiation and Maturation: Implications for Neurodevelopmental Disorders and Neurodegenerative Diseases

Background: The ubiquitination process plays a crucial role in neuronal differentiation and function. Numerous studies have focused on the expression and functions of E3 ligases during these different stages, far fewer on E2 conjugating enzymes. In mice, as in humans, these E2s belong to 17 conjugating enzyme families. Objectives: We analyzed by real-time PCR the expression dynamics of all known E2 genes during an in vitro differentiation of mouse hippocampal neuronal cultures, and after, we analyzed their stimulation with N-methyl-D-aspartate (NMDA). Results: We found that 36 of the 38 E2 genes were expressed in hippocampal neurons. Many were up-regulated during neuritogenesis and/or synaptogenesis stages, such as Ube2h, Ube2b, and Aktip. Rapid and delayed responses to NMDA stimulation were associated with the increased expression of several E2 genes, such as Ube2i, the SUMO-conjugating E2 enzyme. We also observed similar expression profiles within the same E2 gene family, consistent with the presence of similar transcription factor binding sites in their respective promoter sequences. Conclusions: Our study indicates that specific expression profiles of E2 genes are correlated with changes in neuronal differentiation and activity. A better understanding of the regulation and function of E2s is needed to better understand the role played by the ubiquitination process in physiological mechanisms and pathophysiological alterations involved in neurodevelopmental or neurodegenerative diseases.

A Nanobody-Based Proximity Ligation Assay Detects Constitutive and Stimulus-Regulated Native Arc/Arg3.1 Oligomers in Hippocampal Neuronal Dendrites.

Activity-regulated cytoskeleton-associatedprotein (Arc), the product of an immediate early gene, plays critical roles in synaptic plasticity and memory. Evidence suggests that Arc function is determined by its oligomeric state; however, methods for localization of native Arc oligomers are lacking. Here, we developed a nanobody-based proximity ligation assay (PLA) for detection, localization, and quantification of Arc-Arc complexes in primary rat hippocampal neuronal cultures. We used nanobodies with single, structurally defined epitopes in the bilobar Arc capsid domain. Nanobody H11 binds inside the N-lobe ligand pocket, while nanobody C11 binds to the C-lobe surface. For each nanobody, ALFA- and FLAG-epitope tags created a platform for antibody binding and PLA. Surprisingly, PLA puncta in neuronal dendrites revealed widespread constitutive Arc-Arc complexes. Treatment of cultures with tetrodotoxin or cycloheximide had no effect, suggesting stable complexes that are independent of recent neuronal activity and protein synthesis. To assess detection of oligomers, cultures were exposed to a cell-penetrating peptide inhibitor of the Arc oligomerization motif (OligoOFF). Arc-Arc complexes detected by H11 PLA were inhibited by OligoOff but not by control peptide. Notably, Arc complexes detected by C11 were unaffected by OligoOFF. Furthermore, we evaluated Arc complex formation after chemical stimuli that increase Arc synthesis. Brain-derived neurotrophic factor increased Arc-Arc signal detected by C11, but not H11. Conversely, dihydroxyphenylglycine (DHPG) treatment selectively enhanced H11 PLA signals. In sum, nanobody-based PLA reveals constitutive and stimulus-regulated Arc oligomers in hippocampal neuronal dendrites. A model is proposed based on detection of Arc dimer by C11 and higher-order oligomer by H11 nanobody.

Gene therapy for epilepsy targeting neuropeptide Y and its Y2 receptor to dentate gyrus granule cells

Gene therapy is emerging as an alternative option for individuals with drug-resistant focal epilepsy. Here, we explore the potential of a novel gene therapy based on Neuropeptide Y (NPY), a well-known endogenous anticonvulsant. We develop a lentiviral vector co-expressing NPY with its inhibitory receptor Y2 in which, for the first time, both transgenes are placed under the control of the minimal CamKIIa(0.4) promoter, biasing expression toward excitatory neurons and allowing autoregulation of neuronal excitability by Y2 receptor-mediated inhibition. Vector-induced NPY and Y2 expression and safety are first assessed in cultures of hippocampal neurons. In vivo experiments demonstrate efficient and nearly selective overexpression of both genes in granule cell mossy fiber terminals following vector administration in the dentate gyrus. Telemetry video-EEG monitoring reveals a reduction in the frequency and duration of seizures in the synapsin triple KO model. This study shows that targeting a small subset of neurons (hippocampal granule cells) with a combined overexpression of NPY and Y2 receptor is sufficient to reduce the occurrence of spontaneous seizures.

Elastic Properties of the Cell Surface and Metabolic Profile of an Embryonic Primary Mixed Culture of Hippocampal Neurons under Conditions of P2X3 Receptor Blockade

Elastic Properties of the Cell Surface and Metabolic Profile of an Embryonic Primary Mixed Culture of Hippocampal Neurons under Conditions of P2X3 Receptor Blockade

A biallelic mutation in CACNA2D2 associated with developmental and epileptic encephalopathy affects calcium channel-dependent as well as synaptic functions of α2δ-2.

α2δ proteins serve as auxiliary subunits of voltage-gated calcium channels and regulate channel membrane expression and current properties. Besides their channel function, α2δ proteins regulate synapse formation, differentiation, and synaptic wiring. Considering these important functions, it is not surprising that CACNA2D1-4, the genes encoding for α2δ-1 to -4 isoforms, have been implicated in neurological, neurodevelopmental, and neuropsychiatric disorders. Mutations in CACNA2D2 have been associated with developmental and epileptic encephalopathy (DEE) and cerebellar atrophy. In our present study, we performed a detailed functional characterization of the p.R593P mutation in α2δ-2, a homozygous mutation previously identified in two siblings with DEE. Importantly, we analyzed both calcium channel-dependent as well as synaptic functions of α2δ-2. Our data show that the corresponding p.R596P mutation in mouse α2δ-2 drastically decreases membrane expression and synaptic targeting of α2δ-2. This defect correlates with altered biophysical properties of postsynaptic CaV1.3 channel but has no effect on presynaptic CaV2.1 channels upon heterologous expression in tsA201 cells. However, homologous expression of α2δ-2_R596P in primary cultures of hippocampal neurons affects the ability of α2δ-2 to induce a statistically significant increase in the presynaptic abundance of endogenous CaV2.1 channels and presynaptic calcium transients. Moreover, our data demonstrate that in addition to lowering membrane expression, the p.R596P mutation reduces the trans-synaptic recruitment of GABAA receptors and presynaptic synapsin clustering in glutamatergic synapses. Lastly, the α2δ-2_R596P reduces the amplitudes of glutamatergic miniature postsynaptic currents in transduced hippocampal neurons. Taken together, our data strongly link the human biallelic p.R593P mutation to the underlying severe neurodevelopmental disorder and highlight the importance of studying α2δ mutations not only in the context of channelopathies but also synaptopathies.

Inhibition of histone methyltransferase promotes cognition and mitochondrial function in vascular dementia model

Vascular dementia (VD) is one of the most common forms of dementia worldwide, characterized by problems with reasoning, planning, judgment, and memory. This study investigated the effect of a histone methyltransferase inhibitor on cognition and mitochondrial function in a rat model of VD, as well as its impact on H2O2-induced neurotoxicity in hippocampal neuronal cultures. In the in vivo experiments, VD was induced by bilateral occlusion of the common carotid artery (CCA) for one month. The histone methyltransferase inhibitor, BIX01294, was administered intracerebroventricularly for one month (22.5 µg.kg−1 three times/week). On day 30, behavioral tests, including the novel object recognition test and elevated plus maze test, were conducted. Mitochondrial enzyme activities, including aconitase, α-ketoglutarate dehydrogenase (α-KG), complex I, and complex IV, were evaluated in the hippocampus of rats following CCA ligation. In the in vitro experiments, the effect of BIX01294 (50–600 μM) on H2O2 (400 µM)-induced cytotoxicity in hippocampal neuronal cells was assessed using the MTT assay. Flow cytometry was performed to evaluate apoptosis. Our findings revealed that BIX01294 effectively improved memory function, Krebs cycle enzyme activity, and mitochondrial function in the rat model of VD. Moreover, in vitro results showed that BIX01294 at a concentration of 100 µM significantly reversed the cytotoxicity and apoptosis induced by H2O2 in neuronal cells. These findings suggest that BIX01294 may have the potential to improve VD complications by reducing oxidative stress and inhibiting histone methylation.

Delayed recruitment of activity-dependent bulk endocytosis in Fmr1 knockout neurons.

The presynapse performs an essential role in brain communication via the activity-dependent release of neurotransmitters. However, the sequence of events through which a presynapse acquires functionality is relatively poorly understood, which is surprising, since mutations in genes essential for its operation are heavily implicated in neurodevelopmental disorders. We addressed this gap in knowledge by determining the developmental trajectory of synaptic vesicle (SV) recycling pathways in primary cultures of rat hippocampal neurons. Exploiting a series of optical and morphological assays, we revealed that the majority of nerve terminals displayed activity-dependent calcium influx from 3 days invitro (DIV), immediately followed by functional evoked exocytosis and endocytosis, although the number of responsive nerve terminals continued to increase until the second week invitro. However, the most intriguing discovery was that activity-dependent bulk endocytosis (ADBE) was only observed from DIV 14 onwards. Importantly, optimal ADBE recruitment was delayed until DIV 21 in Fmr1 knockout neurons, which model Fragile X Syndrome (FXS). This implicates the delayed recruitment of ADBE as a potential contributing factor in the development of circuit dysfunction in FXS, and potentially other neurodevelopmental disorders.

Aberrant activation of hippocampal astrocytes causes neuroinflammation and cognitive decline in mice.

Reactive astrocytes are associated with neuroinflammation and cognitive decline in diverse neuropathologies; however, the underlying mechanisms are unclear. We used optogenetic and chemogenetic tools to identify the crucial roles of the hippocampal CA1 astrocytes in cognitive decline. Our results showed that repeated optogenetic stimulation of the hippocampal CA1 astrocytes induced cognitive impairment in mice and decreased synaptic long-term potentiation (LTP), which was accompanied by the appearance of inflammatory astrocytes. Mechanistic studies conducted using knockout animal models and hippocampal neuronal cultures showed that lipocalin-2 (LCN2), derived from reactive astrocytes, mediated neuroinflammation and induced cognitive impairment by decreasing the LTP through the reduction of neuronal NMDA receptors. Sustained chemogenetic stimulation of hippocampal astrocytes provided similar results. Conversely, these phenomena were attenuated by a metabolic inhibitor of astrocytes. Fiber photometry using GCaMP revealed a high level of hippocampal astrocyte activation in the neuroinflammation model. Our findings suggest that reactive astrocytes in the hippocampus are sufficient and required to induce cognitive decline through LCN2 release and synaptic modulation. This abnormal glial-neuron interaction may contribute to the pathogenesis of cognitive disturbances in neuroinflammation-associated brain conditions.

Cytosolic Escape of Mitochondrial DNA Triggers cGAS-STING Pathway-Dependent Neuronal PANoptosis in Response to Intermittent Hypoxia.

Intermittent hypoxia (IH) is the predominant pathophysiological disturbance in obstructive sleep apnea (OSA), characterized by neuronal cell death and neurocognitive impairment. We focus on the accumulated mitochondrial DNA (mtDNA) in the cytosol, which acts as a damage-associated molecular pattern (DAMP) and activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, a known trigger for immune responses and neuronal death in degenerative diseases. However, the specific role and mechanism of the mtDNA-cGAS-STING axis in IH-induced neural damage remain largely unexplored. Here, we investigated the involvement of PANoptosis, a novel type of programmed cell death linked to cytosolic mtDNA accumulation and the cGAS-STING pathway activation, in neuronal cell death induced by IH. Our study found that PANoptosis occurred in primary cultures of hippocampal neurons and HT22 cell lines exposed to IH. In addition, we discovered that during IH, mtDNA released into the cytoplasm via the mitochondrial permeability transition pore (mPTP) activates the cGAS-STING pathway, exacerbating PANoptosis-associated neuronal death. Pharmacologically inhibiting mPTP opening or depleting mtDNA significantly reduced cGAS-STING pathway activation and PANoptosis in HT22 cells under IH. Moreover, our findings indicated that the cGAS-STING pathway primarily promotes PANoptosis by modulating endoplasmic reticulum (ER) stress. Inhibiting or silencing the cGAS-STING pathway substantially reduced ER stress-mediated neuronal death and PANoptosis, while lentivirus-mediated STING overexpression exacerbated these effects. In summary, our study elucidates that cytosolic escape of mtDNA triggers cGAS-STING pathway-dependent neuronal PANoptosis in response to IH, mainly through regulating ER stress. The discovery of the novel mechanism provides theoretical support for the prevention and treatment of neuronal damage and cognitive impairment in patients with OSA.

The mitochondrial calcium uniporter inhibitor Ru265 increases neuronal excitability and reduces neurotransmission via off-target effects.

Excitotoxicity due to mitochondrial calcium (Ca2+) overloading can trigger neuronal cell death in a variety of pathologies. Inhibiting the mitochondrial calcium uniporter (MCU) has been proposed as a therapeutic avenue to prevent calcium overloading. Ru265 (ClRu(NH3)4(μ-N)Ru(NH3)4Cl]Cl3) is a cell-permeable inhibitor of the mitochondrial calcium uniporter (MCU) with nanomolar affinity. Ru265 reduces sensorimotor deficits and neuronal death in models of ischemic stroke. However, the therapeutic use of Ru265 is limited by the induction of seizure-like behaviours. We examined the effect of Ru265 on synaptic and neuronal function in acute brain slices and hippocampal neuron cultures derived from mice, in control and where MCU expression was genetically abrogated. Ru265 decreased evoked responses from calyx terminals and induced spontaneous action potential firing of both the terminal and postsynaptic principal cell. Recordings of presynaptic Ca2+ currents suggested that Ru265 blocks the P/Q type channel, confirmed by the inhibition of currents in cells exogenously expressing the P/Q type channel. Measurements of presynaptic K+ currents further revealed that Ru265 blocked a KCNQ current, leading to increased membrane excitability, underlying spontaneous spiking. Ca2+ imaging of hippocampal neurons showed that Ru265 increased synchronized, high-amplitude events, recapitulating seizure-like activity seen in vivo. Importantly, MCU ablation did not suppress Ru265-induced increases in neuronal activity and seizures. Our findings provide a mechanistic explanation for the pro-convulsant effects of Ru265 and suggest counter screening assays based on the measurement of P/Q and KCNQ channel currents to identify safe MCU inhibitors.

Autism risk gene Cul3 alters neuronal morphology via caspase-3 activity in mouse hippocampal neurons.

Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders (NDDs) in which children display differences in social interaction/communication and repetitive stereotyped behaviors along with variable associated features. Cul3, a gene linked to ASD, encodes CUL3 (CULLIN-3), a protein that serves as a key component of a ubiquitin ligase complex with unclear function in neurons. Cul3 homozygous deletion in mice is embryonic lethal; thus, we examine the role of Cul3 deletion in early synapse development and neuronal morphology in hippocampal primary neuronal cultures. Homozygous deletion of Cul3 significantly decreased dendritic complexity and dendritic length, as well as axon formation. Synaptic spine density significantly increased, mainly in thin and stubby spines along with decreased average spine volume in Cul3 knockouts. Both heterozygous and homozygous knockout of Cul3 caused significant reductions in the density and colocalization of gephyrin/vGAT puncta, providing evidence of decreased inhibitory synapse number, while excitatory synaptic puncta vGulT1/PSD95 density remained unchanged. Based on previous studies implicating elevated caspase-3 after Cul3 deletion, we demonstrated increased caspase-3 in our neuronal cultures and decreased neuronal cell viability. We then examined the efficacy of the caspase-3 inhibitor Z-DEVD-FMK to rescue the decrease in neuronal cell viability, demonstrating reversal of the cell viability phenotype with caspase-3 inhibition. Studies have also implicated caspase-3 in neuronal morphological changes. We found that caspase-3 inhibition largely reversed the dendrite, axon, and spine morphological changes along with the inhibitory synaptic puncta changes. Overall, these data provide additional evidence that Cul3 regulates the formation or maintenance of cell morphology, GABAergic synaptic puncta, and neuronal viability in developing hippocampal neurons in culture.

Engineering 3D microenvironments with femtosecond laser-fabricated microtubes to direct neuronal network formation

The regulation of cell growth characteristics through physical structures on material surfaces plays a crucial role in advancing our understanding of cell behavior. Three-dimensional (3D) microstructures, offering multidimensional growth space and a richer nutrient environment compared to their two-dimensional counterparts, have attracted significant attention from researchers. However, the fabrication of high-precision 3D microstructures, especially those with a high aspect ratio (H-AR), remains challenging. In this study, we employ femtosecond laser to achieve exceptional precision and patterning capabilities in fabricating 3D microstructures. To enhance the manufacturing efficiency of axial 3D structures, we introduce a novel single pulse multi-photon polymerization (SP-MPP) method for efficient fabrication of H-AR micropillar and microtube structures. Our innovative approach involves a dual-aperture optical modulation method that effectively modulates the annular energy distribution, enabling the creation of H-AR microstructures. We can achieve a maximum processing height of up to 130 μm with an aspect ratio of 16 within single pulse exposure of 217 fs. Compared to the layer-by-layer scanning method, our proposed approach enhances the axial manufacturing efficiency of 3D structures by 1–2 orders of magnitude. Through experimentation, we validate the applicability of microtube structures for guiding neuronal orientation by utilizing microtube arrays in hippocampal neuronal cell culture. Our results demonstrate that neurons not only grow along the patterned microtubes but also exhibit preliminary synaptic differentiation. Furthermore, we effectively regulate the 3D patterning of neuronal growth by implementing diverse patterned microtube arrays. This proposed method enables the rapid construction of a large-scale 3D cell growth environment, offering significant advantages in inducing neuronal orientation. Consequently, this technique holds immense potential for diverse applications in biomedicine and tissue engineering, including the establishment of isolated neural networks.

Mitochondrial respiration of a primary mixed culture of neurons from hippocampus at various stages of differentiation

BACKGROUND: Primary mixed culture of neurons is often used to evaluate the molecular mechanisms underlying neurodegenerative disorders. However, isolating cells from the brain is accompanied by profound changes in cell morphology, behavior, and metabolic rate due to enzymatic disintegration and microenvironmental changes in cells. In this regard, the mitochondrial basal respiration of neurons should be considered as an indicator of the formation of functional activity of the cell.
 AIM: To evaluate mitochondrial respiration in a primary mixed culture of hippocampal neurons at various stages of differentiation.
 METHODS: This study included mice of line CD-1 and was conducted in two series: the first studied the primary mixed embryonic culture on the 18th day of gestation (E18) and the second used the postnatal culture on the 2nd day after birth (P2). A Seahorse XF HS Mini (Agilent, USA) was used to measure the functional parameters of cell metabolism. The oxygen consumption rate was calculated based on the results of the mitochondrial respiration profile of the primary mixed culture built during its differentiation.
 RESULTS: On the 5th day of neuronal differentiation in the culture, maximum mitochondrial respiration was established both in the hippocampal culture obtained from embryos on the 18th day of gestation and in the culture taken from mice on the 2nd day after birth. As cells differentiated in culture from days 2 to 11, the substrate oxidation rate increased almost twofold in the culture of hippocampal neurons obtained from embryos, which increased the metabolic potential.
 CONCLUSION: The study results prove that the hippocampus can be used to study the role of mitochondria in neurogenesis.

The role of glia in the dysregulation of neuronal spinogenesis in Ube3a-dependent ASD

Overexpression of the Ube3a gene and the resulting increase in Ube3a protein are linked to autism spectrum disorder (ASD). However, the cellular and molecular processes underlying Ube3a-dependent ASD remain unclear. Using both male and female mice, we find that neurons in the somatosensory cortex of the Ube3a 2× Tg ASD mouse model display reduced dendritic spine density and increased immature filopodia density. Importantly, the increased gene dosage of Ube3a in astrocytes alone is sufficient to confer alterations in neurons as immature dendritic protrusions, as observed in primary hippocampal neuron cultures. We show that Ube3a overexpression in astrocytes leads to a loss of astrocyte-derived spinogenic protein, thrombospondin-2 (TSP2), due to a suppression of TSP2 gene transcription. By neonatal intraventricular injection of astrocyte-specific virus, we demonstrate that Ube3a overexpression in astrocytes in vivo results in a reduction in dendritic spine maturation in prelimbic cortical neurons, accompanied with autistic-like behaviors in mice. These findings reveal an astrocytic dominance in initiating ASD pathobiology at the neuronal and behavior levels. Significance statementIncreased gene dosage of Ube3a is tied to autism spectrum disorders (ASDs), yet cellular and molecular alterations underlying autistic phenotypes remain unclear. We show that Ube3a overexpression leads to impaired dendritic spine maturation, resulting in reduced spine density and increased filopodia density. We find that dysregulation of spine development is not neuron autonomous, rather, it is mediated by an astrocytic mechanism. Increased gene dosage of Ube3a in astrocytes leads to reduced production of the spinogenic glycoprotein thrombospondin-2 (TSP2), leading to abnormalities in spines. Astrocyte-specific Ube3a overexpression in the brain in vivo confers dysregulated spine maturation concomitant with autistic-like behaviors in mice. These findings indicate the importance of astrocytes in aberrant neurodevelopment and brain function in Ube3a-depdendent ASD.

Antiretroviral drugs from multiple classes induce loss of excitatory synapses between hippocampal neurons in culture.

Introduction: Antiretroviral (ARV) drugs have improved prognoses for people living with HIV. However, HIV-associated neurocognitive disorders (HAND) persist despite undetectable viral loads. Some ARVs have been linked to neuropsychiatric effects that may contribute to HAND. Synapse loss correlates with cognitive decline in HAND and synaptic deficits may contribute to the neuropsychiatric effects of ARV drugs. Methods: Using an automated high content assay, rat hippocampal neurons in culture expressing PSD95-eGFP to label glutamatergic synapses and mCherry to fill neuronal structures were imaged before and after treatment with 25 clinically used ARVs. Results and Discussion: At a concentration of 10μM the protease inhibitors nelfinavir and saquinavir, the non-nucleoside reverse transcriptase inhibitors etravirine and the 8-OH metabolite of efavirenz, the integrase inhibitor bictegravir, and the capsid inhibitor lenacapavir produced synaptic toxicity. Only lenacapavir produced synapse loss at the nanomolar concentrations estimated free in the plasma, although all 4 ARV drugs induced synapse loss at Cmax. Evaluation of combination therapies did not reveal synergistic synaptic toxicity. Synapse loss developed fully by 24h and persisted for at least 3days. Bictegravir-induced synapse loss required activation of voltage-gated Ca2+ channels and bictegravir, etravirine, and lenacapavir produced synapse loss by an excitotoxic mechanism. These results indicate that select ARV drugs might contribute to neuropsychiatric effects in combination with drugs that bind serum proteins or in disease states in which synaptic function is altered. The high content imaging assay used here provides an efficient means to evaluate new drugs and drug combinations for potential CNS toxicity.

Human mutations in SLITRK3 implicated in GABAergic synapse development in mice.

This study reports on biallelic homozygous and monoallelic de novo variants in SLITRK3 in three unrelated families presenting with epileptic encephalopathy associated with a broad neurological involvement characterized by microcephaly, intellectual disability, seizures, and global developmental delay. SLITRK3 encodes for a transmembrane protein that is involved in controlling neurite outgrowth and inhibitory synapse development and that has an important role in brain function and neurological diseases. Using primary cultures of hippocampal neurons carrying patients' SLITRK3 variants and in combination with electrophysiology, we demonstrate that recessive variants are loss-of-function alleles. Immunostaining experiments in HEK-293 cells showed that human variants C566R and E606X change SLITRK3 protein expression patterns on the cell surface, resulting in highly accumulating defective proteins in the Golgi apparatus. By analyzing the development and phenotype of SLITRK3 KO (SLITRK3-/-) mice, the study shows evidence of enhanced susceptibility to pentylenetetrazole-induced seizure with the appearance of spontaneous epileptiform EEG as well as developmental deficits such as higher motor activities and reduced parvalbumin interneurons. Taken together, the results exhibit impaired development of the peripheral and central nervous system and support a conserved role of this transmembrane protein in neurological function. The study delineates an emerging spectrum of human core synaptopathies caused by variants in genes that encode SLITRK proteins and essential regulatory components of the synaptic machinery. The hallmark of these disorders is impaired postsynaptic neurotransmission at nerve terminals; an impaired neurotransmission resulting in a wide array of (often overlapping) clinical features, including neurodevelopmental impairment, weakness, seizures, and abnormal movements. The genetic synaptopathy caused by SLITRK3 mutations highlights the key roles of this gene in human brain development and function.

Downregulation of Ribosomal Protein Genes Is Revealed in a Model of Rat Hippocampal Neuronal Culture Activation with GABA(A)R/GlyRa2 Antagonist Picrotoxin.

Long-read transcriptome sequencing provides us with a convenient tool for the thorough study of biological processes such as neuronal plasticity. Here, we aimed to perform transcriptional profiling of rat hippocampal primary neuron cultures after stimulation with picrotoxin (PTX) to further understand molecular mechanisms of neuronal activation. To overcome the limitations of short-read RNA-Seq approaches, we performed an Oxford Nanopore Technologies MinION-based long-read sequencing and transcriptome assembly of rat primary hippocampal culture mRNA at three time points after the PTX activation. We used a specific approach to exclude uncapped mRNAs during sample preparation. Overall, we found 23,652 novel transcripts in comparison to reference annotations, out of which ~6000 were entirely novel and mostly transposon-derived loci. Analysis of differentially expressed genes (DEG) showed that 3046 genes were differentially expressed, of which 2037 were upregulated and 1009 were downregulated at 30 min after the PTX application, with only 446 and 13 genes differentially expressed at 1 h and 5 h time points, respectively. Most notably, multiple genes encoding ribosomal proteins, with a high basal expression level, were downregulated after 30 min incubation with PTX; we suggest that this indicates redistribution of transcriptional resources towards activity-induced genes. Novel loci and isoforms observed in this study may help us further understand the functional mRNA repertoire in neuronal plasticity processes. Together with other NGS techniques, differential gene expression analysis of sequencing data obtained using MinION platform might provide a simple method to optimize further study of neuronal plasticity.

Puromycin reveals a distinct conformation of neuronal ribosomes

Puromycin is covalently added to the nascent chain of proteins by the peptidyl transferase activity of the ribosome and the dissociation of the puromycylated peptide typically follows this event. It was postulated that blocking the translocation of the ribosome with emetine could retain the puromycylated peptide on the ribosome, but evidence against this has recently been published [Hobson et al., Elife 9, e60048 (2020); and Enam et al., Elife 9, e60303 (2020)]. In neurons, puromycylated nascent chains remain in the ribosome even in the absence of emetine, yet direct evidence for this has been lacking. Using biochemistry and cryoelectron microscopy, we show that the puromycylated peptides remain in the ribosome exit channel in the large subunit in a subset of neuronal ribosomes stalled in the hybrid state. These results validate previous experiments to localize stalled polysomes in neurons and provide insight into how neuronal ribosomes are stalled. Moreover, in these hybrid-state neuronal ribosomes, anisomycin, which usually blocks puromycylation, competes poorly with puromycin in the puromycylation reaction, allowing a simple assay to determine the proportion of nascent chains that are stalled in this state. In early hippocampal neuronal cultures, over 50% of all nascent peptides are found in these stalled polysomes. These results provide insights into the stalling mechanisms of neuronal ribosomes and suggest that puromycylated peptides can be used to reveal subcellular sites of hybrid-state stalled ribosomes in neurons.