The neurotransmitter receptor for glycine (GlyR) is a ligand-gated chloride-permeable ion channel in the central nervous system, and dysregulation of this channel is associated with diverse neurological disorders. Cesium (Cs+) is an agonist of GlyRs, and recent atomistic molecular dynamic simulations suggested amino acids D141, E192, and D194 with negatively charged side chains as possible binding sites for Cs+. To test this hypothesis, we mutated these positions to code for alanine with a neutral hydrophobic side chain or lysine with a positively charged side chain. The correspondingly mutated GlyR channels were expressed in HEK293T cells and analyzed using whole-cell patch clamp electrophysiology. The results show that D141 mutations had no considerable effect on GlyR currents, E192K prolonged desensitization and D194 turned out to be critical for general (glycine- and Cs+-dependent) GlyR activation, which was not due to differentially reduced cell surface expression of all the investigated GlyR mutants. Thus, E192 and D194 are critically involved in cell surface GlyR activation by Cs+ and glycine.

Zebrafish have an impressive capacity to regenerate neurons in the central nervous system. However, regeneration of the principal neuron of the evolutionary conserved cerebellum, the Purkinje cell (PC), is believed to be limited to developmental stages based on invasive lesions. In contrast, non-invasive cell type-specific ablation by induced apoptosis closely represents a process of neurodegeneration. We demonstrate that the ablated larval PC population entirely recovers in number, quickly reestablishes electrophysiological properties, and properly integrates into circuits to regulate cerebellum-controlled behavior. PC progenitors are present in larvae and adults, and PC ablation in adult cerebelli results in an impressive PC regeneration of different PC subtypes able to restore behavioral impairments. Interestingly, caudal PCs are more resistant to ablation and regenerate more efficiently, suggesting a rostro-caudal pattern of de- and regeneration properties. These findings demonstrate that the zebrafish cerebellum is able to regenerate functional PCs during all stages of the animal's life.

The chemical biology of native nucleic acid modifications has seen an intense upswing, first concerning DNA modifications in the field of epigenetics and then concerning RNA modifications in a field that was correspondingly rebaptized epitranscriptomics by analogy. The German Research Foundation (DFG) has funded several consortia with a scientific focus in these fields, strengthening the traditionally well-developed nucleic acid chemistry community and inciting it to team up with colleagues from the life sciences and data science to tackle interdisciplinary challenges. This Perspective focuses on the genesis, scientific outcome, and downstream impact of the DFG priority program SPP1784 and offers insight into how it fecundated further consortia in the field. Pertinent research was funded from mid-2015 to 2022, including an extension related to the coronavirus pandemic. Despite being a detriment to research activity in general, the pandemic has resulted in tremendously boosted interest in the field of RNA and RNA modifications as a consequence of their widespread and successful use in vaccination campaigns against SARS-CoV-2. Funded principal investigators published over 250 pertinent papers with a very substantial impact on the field. The program also helped to redirect numerous laboratories toward this dynamic field. Finally, SPP1784 spawned initiatives for several funded consortia that continue to drive the fields of nucleic acid modification.

The monovalent cations sodium and potassium are crucial for the proper functioning of excitable cells, but, in addition, other monovalent alkali metal ions such as cesium and lithium can also affect neuronal physiology. For instance, there have been recent reports of adverse effects resulting from self-administered high concentrations of cesium in disease conditions, prompting the Food and Drug Administration (FDA) to issue an alert concerning cesium chloride. As we recently found that the monovalent cation NH4+ activates glycine receptors (GlyRs), we investigated the effects of alkali metal ions on the function of the GlyR, which belongs to one of the most widely distributed neurotransmitter receptors in the peripheral and central nervous systems. Whole-cell voltage clamp electrophysiology was performed with HEK293T cells transiently expressing different splice and RNA-edited variants of GlyR α2 and α3 homopentameric channels. By examining the influence of various milli- and sub-millimolar concentrations of lithium, sodium, potassium, and cesium on these GlyRs in comparison to its natural ligand glycine (0.1 mM), we could show that cesium activates GlyRs in a concentration- and post-transcriptional-dependent way. Additionally, we conducted atomistic molecular dynamic simulations on GlyR α3 embedded in a membrane bilayer with potassium and cesium, respectively. The simulations revealed slightly different GlyR-ion binding profiles for potassium and cesium, identifying interactions near the glycine binding pocket (potassium and cesium) and close to the RNA-edited site (cesium) in the extracellular GlyR domain. Together, these findings show that cesium acts as an agonist of GlyRs.

IntroductionThe cerebellum is organized into functional regions each dedicated to process different motor or sensory inputs for controlling different locomotor behaviors. This functional regionalization is prominent in the evolutionary conserved single-cell layered Purkinje cell (PC) population. Fragmented gene expression domains suggest a genetic organization of PC layer regionalization during cerebellum development. However, the establishment of such functionally specific domains during PC differentiation remained elusive. Methods and resultsWe show the progressive emergence of functional regionalization of PCs from broad responses to spatially restricted regions in zebrafish by means of in vivo Ca2+-imaging during stereotypic locomotive behavior. Moreover, we reveal that formation of new dendritic spines during cerebellar development using in vivo imaging parallels the time course of functional domain development. Pharmacological as well as cell-type specific optogenetic inhibition of PC neuronal activity results in reduced PC dendritic spine density and an altered stagnant pattern of functional domain formation in the PC layer. DiscussionHence, our study suggests that functional regionalization of the PC layer is driven by physiological activity of maturing PCs themselves.

Synaptic inhibition is essential for shaping the dynamics of neuronal networks, and aberrant inhibition is linked to epilepsy. Gephyrin (Geph) is the principal scaffolding protein at inhibitory synapses and is essential for postsynaptic clustering of glycine (GlyRs) and GABA type A receptors. Consequently, gephyrin is crucial for maintaining the relationship between excitation and inhibition in normal brain function and mutations in the gephyrin gene (GPHN) are associated with neurodevelopmental disorders and epilepsy. We identified bi-allelic variants in the GPHN gene, namely the missense mutation c.1264G > A and splice acceptor variant c.1315-2A > G, in a patient with developmental and epileptic encephalopathy. We demonstrate that the splice acceptor variant leads to nonsense-mediated mRNA decay. Furthermore, the missense variant (D422N) alters gephyrin structure, as examined by analytical size exclusion chromatography and circular dichroism-spectroscopy, thus leading to reduced receptor clustering and sensitivity towards calpain-mediated cleavage. In addition, both alterations contribute to an observed reduction of inhibitory signal transmission in neurons, which likely contributes to the pathological encephalopathy.

Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-α3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-α3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-α3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson’s and dual-color spatiotemporal correlation analysis to prove that GlyR-α3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via cell-attached single-channel electrophysiology we show that heteropentamers exhibit currents in between those of K and L variants. Our data are compatible with a model where α3 heteropentamerization fine-tunes mobility and activity of GlyR-α3 channels, which is important to understand and tackle α3 related diseases.

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore. Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

The human body is composed of many different cell types which communicate with each other. In particular, the brain consists of billions of neurons and non-neuronal cells which are interconnected and require tight and precise regulation of cellular processes. RNA editing is a cellular process that diversifies gene function by enzymatic deamination of cytidine or adenine. This can result in changes of protein structure and function. Altered RNA editing is becoming increasingly associated with all kind of disease, but most approaches use advanced sequencing technologies to analyze bulk material. However, it is also becoming progressively evident that changes in RNA editing have to be analyzed and considered in a cell type specific way. We present here a triple fluorescence reporter system that discriminates between Apobec1- and Apobec3-dependent C-to-U RNA editing at the single cell level. In particular, the Apobec3 reporter enables C-to-U RNA editing inducible protein expression through generation of a RNA splice donor site. We used the new system here to analyze Apobec1- and Apobec3-dependent RNA editing in primary neuron culture. The results reveal a large heterogeneity of C-to-U RNA editing in neurons and glia cells, and they show that GABAergic neurons are not able to perform Apobec1-dependent RNA editing, but Apobec3-dependent editing. Altogether, the new system can be the foundation of therapeutic application systems that counteract changes in Apobec3-dependent RNA editing in disease while simultaneously monitoring Apobec1-dependent RNA editing at the single cell level.

Glycine receptors (GlyRs) are ligand-gated pentameric chloride channels in the central nervous system. GlyR-α3 is a possible target for chronic pain treatment and temporal lobe epilepsy. Alternative splicing into K or L variants determines the subcellular fate and function of GlyR-α3, yet it remains to be shown whether its different splice variants can functionally co-assemble, and what the properties of such heteropentamers would be. Here, we subjected GlyR-α3 to a combined fluorescence microscopy and electrophysiology analysis. We employ masked Pearson’s and dual-color spatiotemporal correlation analysis to prove that GlyR-α3 splice variants heteropentamerize, adopting the mobility of the K variant. Fluorescence-based single-subunit counting experiments revealed a variable and concentration ratio dependent hetero-stoichiometry. Via single-channel on-cell patch clamp we show heteropentameric conductances resemble those of the α3K splice variant. Our data are compatible with a model where α3 heteropentamerization fine-tunes mobility and activity of GlyR α3 channels, which is important to understand and tackle α3 related diseases. Summary The glycine receptor α3 is key to the central nervous system’s physiology and involved in chronic pain and epilepsy. In this paper, Lemmens et al. reveal and functionally characterize α3 splice variant heteropentamerization via advanced single-molecule fluorescence image analysis. Declarations Funding We acknowledge the UHasselt Advanced Optical Microscopy Centre (AOMC). Prof. Em. Marcel Ameloot, the Research Foundation Flanders (FWO, project G0H3716N) and the province of Limburg (Belgium) (tUL Impuls II) are acknowledged for funding the microscopy hardware. V. Lemmens is grateful for a doctoral scholarship from the UHasselt (17DOC11BOF) and KU Leuven (C14/16/053) Special Research Funds (BOF). Conflicts of interest / competing interests No conflicts of interest apply. Ethics approval Not applicable Availability of data and material All data and material are available upon request. Code availability Fluctuation imaging and co-localization analyses were performed in the software package PAM [71]. The software is available as source code, requiring MATLAB to run, or as pre-compiled standalone distributions for Windows or MacOS at http://www.cup.uni-muenchen.de/pc/lamb/software/pam.html or hosted in Git repositories under http://www.gitlab.com/PAM-PIE/PAM and http://www.gitlab.com/PAM-PIE/PAMcompiled . A detailed user manual is available at http://pam.readthedocs.io . Author contributions Conceptualization Meier J.C., Brône B. and Hendrix J.; Investigation and formal analysis Lemmens V. and Thevelein B.; Software development Hendrix J.; Writing the original draft Lemmens V., Thevelein B and Hendrix, J.; Review and editing by all authors.

EDITORIAL article Front. Mol. Neurosci., 12 October 2021 | https://doi.org/10.3389/fnmol.2021.773308

Unambiguous imaging of C → U edited mRNA calls for a method that distinguishes a locally high concentration of unbound probe or single nucleotide mismatched target from a locally low concentration of matched mRNA target. To address this issue, we combine FIT probes as a "chemical" detection system with the "biological" MS2 technique. Ratio measurements provide a convenient parameter to discriminate the edited from the unedited state of mRNA encoding for GlyR α2 in HEK cells.

Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy with about 30% of patients developing pharmacoresistance. These patients continue to suffer from seizures despite polytherapy with antiepileptic drugs (AEDs) and have an increased risk for premature death, thus requiring further efforts for the development of new antiepileptic therapies. The molecule dimethylethanolamine (DMEA) has been tested as a potential treatment in various neurological diseases, albeit the functional mechanism of action was never fully understood. In this study, we investigated the effects of DMEA on neuronal activity in single-cell recordings of primary neuronal cultures. DMEA decreased the frequency of spontaneous synaptic events in a concentration-dependent manner with no apparent effect on resting membrane potential (RMP) or action potential (AP) threshold. We further tested whether DMEA can exert antiepileptic effects in human brain tissue ex vivo. We analyzed the effect of DMEA on epileptiform activity in the CA1 region of the resected hippocampus of TLE patients in vitro by recording extracellular field potentials in the pyramidal cell layer. Epileptiform burst activity in resected hippocampal tissue from TLE patients remained stable over several hours and was pharmacologically suppressed by lacosamide, demonstrating the applicability of our platform to test antiepileptic efficacy. Similar to lacosamide, DMEA also suppressed epileptiform activity in the majority of samples, albeit with variable interindividual effects. In conclusion, DMEA might present a new approach for treatment in pharmacoresistant TLE and further studies will be required to identify its exact mechanism of action and the involved molecular targets.

Three states in one blow: a FRET relay system distinguishes between (i) the absence of RNA target, (ii) the presence of C → U edited RNA or (iii) unedited RNA target by means of ratio measurements.

C-to-U RNA editing of glycine receptors (GlyR) can play an important role in disease progression of temporal lobe epilepsy (TLE) as it may contribute in a neuron type-specific way to neuropsychiatric symptoms of the disease. It is therefore necessary to develop tools that allow identification of neuron types that express RNA-edited GlyR protein. In this study, we identify NH4 as agonist of C-to-U RNA edited GlyRs. Furthermore, we generated a new molecular C-to-U RNA editing sensor tool that detects Apobec-1- dependent RNA editing in HEPG2 cells and rat primary hippocampal neurons. Using this sensor combined with NH4 application, we were able to identify C-to-U RNA editing-competent neurons and expression of C-to-U RNA-edited GlyR protein in neurons. Bioinformatic analysis of 1,000 Genome Project Phase 3 allele frequencies coding for human Apobec-1 80M and 80I variants showed differences between populations, and the results revealed a preference of the 80I variant to generate RNA-edited GlyR protein. Finally, we established a new PCR-based restriction fragment length polymorphism (RFLP) approach to profile mRNA expression with regard to the genetic APOBEC1 dimorphism of patients with intractable temporal lobe epilepsy (iTLE) and found that the patients fall into two groups. Patients with expression of the Apobec-1 80I variant mostly suffered from simple or complex partial seizures, whereas patients with 80M expression exhibited secondarily generalized seizure activity. Thus, our method allows the characterization of Apobec-1 80M and 80l variants in the brain and provides a new way to epidemiologically and semiologically classify iTLE according to the two different APOBEC1 alleles. Together, these results demonstrate Apobec-1-dependent expression of RNA-edited GlyR protein in neurons and identify the APOBEC1 80I/M-coding alleles as new genetic risk factors for iTLE patients.

BACKGROUND:Insights into human brain diseases may emerge from tissue obtained after operations on patients. However techniques requiring transduction of transgenes carried by viral vectors cannot be applied to acute human tissue. NEW METHOD:We show that organotypic culture techniques can be used to maintain tissue from patients with three different neurological syndromes for several weeks in vitro. Optimized viral vector techniques and promoters for transgene expression are described. RESULTS:Region-specific differences in neuronal form, firing pattern and organization as well as pathological activities were maintained over 40-50 days in culture. Both adeno-associated virus and lentivirus based vectors were persistently expressed from ∼10 days after application, providing 30-40 days to exploit genetically expressed constructs. Different promoters, including hSyn, e/hSyn, CMV and CaMKII, provided cell-type specific transgene expression. The Ca probe GCaMP let us explore epileptogenic synchrony and a FRET-based probe was used to follow activity of the kinase mTORC1. COMPARISON WITH EXISTING METHODS:The use of a defined culture medium, with low concentrations of amino acids and no growth factors, permitted organotypic culture of tissue from humans aged 3-62 years. Epileptic activity was maintained and excitability changed relatively little until ∼6 weeks in culture. CONCLUSIONS:Characteristic morphology and region-specific neuronal activities are maintained in organotypic culture of tissue from patients diagnosed with mesial temporal lobe epilepsy, cortical dysplasia and cortical glioblastoma. Viral vector techniques permit expression of probes for long-term measurements of multi-cellular activity and intra-cellular signaling.

Compared to sites in protein-coding sequences many more targets undergoing adenosine to inosine (A-to-I) RNA editing were discovered in non-coding regions of human cerebral transcripts, particularly in genetic transposable elements called retrotransposons. We review here the interaction mechanisms of RNA editing and retrotransposons and their impact on normal function and human neurological diseases. Exemplarily, A-to-I editing of retrotransposons embedded in protein-coding mRNAs can contribute to protein abundance and function via circular RNA formation, alternative splicing, and exonization or silencing of retrotransposons. Interactions leading to disease are not very well understood. We describe human diseases with involvement of the central nervous system including inborn errors of metabolism, neurodevelopmental disorders, neuroinflammatory and neurodegenerative and paroxysmal diseases, in which retrotransposons (Alu and/or L1 elements) appear to be causally involved in genetic rearrangements. Sole binding of single-stranded retrotransposon transcripts by RNA editing enzymes rather than enzymatic deamination may have a homeostatic effect on retrotransposon turnover. We also review evidence in support of the emerging pathophysiological function of A-to-I editing of retrotransposons in inflammation and its implication for different neurological diseases including amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's and Parkinson's disease, and epilepsy.

Self-propagating amyloid-β (Aβ) aggregates or seeds possibly drive pathogenesis of Alzheimer's disease (AD). Small molecules targeting such structures might act therapeutically in vivo. Here, a fluorescence polarization assay was established that enables the detection of compound effects on both seeded and spontaneous Aβ42 aggregation. In a focused screen of anti-amyloid compounds, we identified Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]), a small molecule that potently delays both seeded and non-seeded Aβ42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aβ42 peptides and decreases the seeding activity of Aβ aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aβ plaques and decreased neuroinflammation-related gene expression changes in brains of 5xFAD transgenic mice. Finally, improved nesting behavior was observed upon treatment with the compound. Together, our evidence supports targeting of self-propagating Aβ structures with small molecules as a valid therapeutic strategy.

Compared to sites in protein-coding sequences many more targets undergoing adenosine to inosine (A-to-I) RNA editing were discovered in non-coding regions of human cerebral transcripts, particularly in genetic transposable elements called retrotransposons. We review here the interaction mechanisms of RNA editing and retrotransposons and their impact on normal function and human neurological diseases. Exemplarily, A-to-I editing of retrotransposons embedded in protein-coding mRNAs can contribute to protein abundance and function via circular RNA formation, alternative splicing, and exonization or silencing of retrotransposons. Interactions leading to disease are not very well understood. We describe human diseases with involvement of the central nervous system including inborn errors of metabolism, neurodevelopmental disorders, neuroinflammatory and neurodegenerative and paroxysmal diseases, in which retrotransposons (Alu and/or L1 elements) appear to be causally involved in genetic rearrangements. Sole binding of single-stranded retrotransposon transcripts by RNA editing enzymes rather than enzymatic deamination may have a homeostatic effect on retrotransposon turnover. We also review evidence in support of the emerging pathophysiological function of A-to-I editing of retrotransposons in inflammation and its implication for different neurological diseases including amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's and Parkinson's disease, and epilepsy.

Department of Chemistry, Humboldt-Unive D-12489 Berlin, Germany. E-mail: oliver.sei Institute of Physical Science and Informat Anhui 230601, China Zoological Institute, Technical University B Braunschweig, Germany Max Delbrück Centrum für Molekulare M Berlin, Germany Technische Universität Darmstadt, Depart 64287 Darmstadt, Germany † Electronic supplementary information synthesis, uorescence spectra, absorban are available online. See DOI: 10.1039/c8s Cite this: Chem. Sci., 2018, 9, 4794