Experience with German Research Consortia in the Field of Chemical Biology of Native Nucleic Acid Modifications.

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.

Cesium activates the neurotransmitter receptor for glycine.

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.

Functional regionalization of the differentiating cerebellar Purkinje cell population occurs in an activity-dependent manner.

Introduction: The 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 results: We 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. Discussion: Hence, our study suggests that functional regionalization of the PC layer is driven by physiological activity of maturing PCs themselves.

Lifelong regeneration of cerebellar Purkinje cells after induced cell ablation in zebrafish.

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.

Hetero-pentamerization determines mobility and conductance of Glycine receptor α3 splice variants.

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.

Biallelic gephyrin variants lead to impaired GABAergic inhibition in a patient with developmental and epileptic encephalopathy.

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.

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal.

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.

Chemo-biological mRNA imaging with single nucleotide specificity.

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.

Dimethylethanolamine Decreases Epileptiform Activity in Acute Human Hippocampal Slices in vitro.

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.

Corrigendum: A Novel RNA Editing Sensor Tool and a Specific Agonist Determine Neuronal Protein Expression of RNA-Edited Glycine Receptors and Identify a Genomic APOBEC1 Dimorphism as a New Genetic Risk Factor of Epilepsy.

[This corrects the article DOI: 10.3389/fnmol.2017.00439.].

The Anti-amyloid Compound DO1 Decreases Plaque Pathology and Neuroinflammation-Related Expression Changes in 5xFAD Transgenic Mice.

Self-propagating amyloid-ß (Aß) 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 Aß42 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 Aß42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aß42 peptides and decreases the seeding activity of Aß aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aß 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 Aß structures with small molecules as a valid therapeutic strategy.

RNA Editing and Retrotransposons in Neurology.

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.

A bright FIT-PNA hybridization probe for the hybridization state specific analysis of a C → U RNA edit via FRET in a binary system.

Oligonucleotide probes that show enhanced fluorescence upon nucleic acid hybridization enable the detection and visualization of specific mRNA molecules, in vitro and in cellulo. A challenging problem is the analysis of single nucleotide alterations that occur, for example, when cellular mRNA is subject to C → U editing. Given the length required for uniqueness of the targeted segment, the commonly used probes do not provide the level of sequence specificity needed to discriminate single base mismatched hybridization. Herein we introduce a binary probe system based on fluorescence resonance energy transfer (FRET) that distinguishes three possible states i.e. (i) absence of target, (ii) presence of edited (matched) and (iii) unedited (single base mismatched) target. To address the shortcomings of read-out via FRET, we designed donor probes that avoid bleed through into the acceptor channel and nevertheless provide a high intensity of FRET signaling. We show the combined use of thiazole orange (TO) and an oxazolopyridine analogue (JO), linked as base surrogates in modified PNA FIT-probes that serve as FRET donor for a second, near-infrared (NIR)-labeled strand. In absence of target, donor emission is low and FRET cannot occur in lieu of the lacking co-alignment of probes. Hybridization of the TO/JO-PNA FIT-probe with the (unedited RNA) target leads to high brightness of emission at 540 nm. Co-alignment of the NIR-acceptor strand ensues from recognition of edited RNA inducing emission at 690 nm. We show imaging of mRNA in fixed and live cells and discuss the homogeneous detection and intracellular imaging of a single nucleotide mRNA edit used by nature to post-transcriptionally modify the function of the Glycine Receptor (GlyR).

Imaging pathological activities of human brain tissue in organotypic culture.

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.

S-sulfocysteine/NMDA receptor-dependent signaling underlies neurodegeneration in molybdenum cofactor deficiency.

Molybdenum cofactor deficiency (MoCD) is an autosomal recessive inborn error of metabolism characterized by neurodegeneration and death in early childhood. The rapid and progressive neurodegeneration in MoCD presents a major clinical challenge and may relate to the poor understanding of the molecular mechanisms involved. Recently, we reported that treating patients with cyclic pyranopterin monophosphate (cPMP) is a successful therapy for a subset of infants with MoCD and prevents irreversible brain damage. Here, we studied S-sulfocysteine (SSC), a structural analog of glutamate that accumulates in the plasma and urine of patients with MoCD, and demonstrated that it acts as an N-methyl D-aspartate receptor (NMDA-R) agonist, leading to calcium influx and downstream cell signaling events and neurotoxicity. SSC treatment activated the protease calpain, and calpain-dependent degradation of the inhibitory synaptic protein gephyrin subsequently exacerbated SSC-mediated excitotoxicity and promoted loss of GABAergic synapses. Pharmacological blockade of NMDA-R, calcium influx, or calpain activity abolished SSC and glutamate neurotoxicity in primary murine neurons. Finally, the NMDA-R antagonist memantine was protective against the manifestation of symptoms in a tungstate-induced MoCD mouse model. These findings demonstrate that SSC drives excitotoxic neurodegeneration in MoCD and introduce NMDA-R antagonists as potential therapeutics for this fatal disease.

Human iPSC-Derived Neural Progenitors Are an Effective Drug Discovery Model for Neurological mtDNA Disorders.

Mitochondrial DNA (mtDNA) mutations frequently cause neurological diseases. Modeling of these defects has been difficult because of the challenges associated with engineering mtDNA. We show here that neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) retain the parental mtDNA profile and exhibit a metabolic switch toward oxidative phosphorylation. NPCs derived in this way from patients carrying a deleterious homoplasmic mutation in the mitochondrial gene MT-ATP6 (m.9185T>C) showed defective ATP production and abnormally high mitochondrial membrane potential (MMP), plus altered calcium homeostasis, which represents a potential cause of neural impairment. High-content screening of FDA-approved drugs using the MMP phenotype highlighted avanafil, which we found was able to partially rescue the calcium defect in patient NPCs and differentiated neurons. Overall, our results show that iPSC-derived NPCs provide an effective model for drug screening to target mtDNA disorders that affect the nervous system.

Step-by-step guide to building an inexpensive 3D printed motorized positioning stage for automated high-content screening microscopy.

High-content screening microscopy relies on automation infrastructure that is typically proprietary, non-customizable, costly and requires a high level of skill to use and maintain. The increasing availability of rapid prototyping technology makes it possible to quickly engineer alternatives to conventional automation infrastructure that are low-cost and user-friendly. Here, we describe a 3D printed inexpensive open source and scalable motorized positioning stage for automated high-content screening microscopy and provide detailed step-by-step instructions to re-building the device, including a comprehensive parts list, 3D design files in STEP (Standard for the Exchange of Product model data) and STL (Standard Tessellation Language) format, electronic circuits and wiring diagrams as well as software code. System assembly including 3D printing requires approx. 30h. The fully assembled device is light-weight (1.1kg), small (33×20×8cm) and extremely low-cost (approx. EUR 250). We describe positioning characteristics of the stage, including spatial resolution, accuracy and repeatability, compare imaging data generated with our device to data obtained using a commercially available microplate reader, demonstrate its suitability to high-content microscopy in 96-well high-throughput screening format and validate its applicability to automated functional Cl-- and Ca2+-imaging with recombinant HEK293 cells as a model system. A time-lapse video of the stage during operation and as part of a custom assembled screening robot can be found at https://vimeo.com/158813199.