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.

Culture Conditions for Human Induced Pluripotent Stem Cell-Derived Schwann Cells: A Two-Centre Study.

Adult human Schwann cells represent a relevant tool for studying peripheral neuropathies and developing regenerative therapies to treat nerve damage. Primary adult human Schwann cells are, however, difficult to obtain and challenging to propagate in culture. One potential solution is to generate Schwann cells from human induced pluripotent stem cells (hiPSCs). Previously published protocols, however, in our hands did not deliver sufficient viable cell numbers of hiPSC-derived Schwann cells (hiPSC-SCs). We present here, two modified protocols from two collaborating laboratories that overcome these challenges. With this, we also identified the relevant parameters to be specifically considered in any proposed differentiation protocol. Furthermore, we are, to our knowledge, the first to directly compare hiPSC-SCs to primary adult human Schwann cells using immunocytochemistry and RT-qPCR. We conclude the type of coating to be important during the differentiation process from Schwann cell precursor cells or immature Schwann cells to definitive Schwann cells, as well as the amounts of glucose in the specific differentiation medium to be crucial for increasing its efficiency and the final yield of viable hiPSC-SCs. Our hiPSC-SCs further displayed high similarity to primary adult human Schwann cells.

Impact of Gut Microbiota on the Peripheral Nervous System in Physiological, Regenerative and Pathological Conditions.

It has been widely demonstrated that the gut microbiota is responsible for essential functions in human health and that its perturbation is implicated in the development and progression of a growing list of diseases. The number of studies evaluating how the gut microbiota interacts with and influences other organs and systems in the body and vice versa is constantly increasing and several 'gut-organ axes' have already been defined. Recently, the view on the link between the gut microbiota (GM) and the peripheral nervous system (PNS) has become broader by exceeding the fact that the PNS can serve as a systemic carrier of GM-derived metabolites and products to other organs. The PNS as the communication network between the central nervous system and the periphery of the body and internal organs can rather be affected itself by GM perturbation. In this review, we summarize the current knowledge about the impact of gut microbiota on the PNS, with regard to its somatic and autonomic divisions, in physiological, regenerative and pathological conditions.

Neuropathic pain: Spotlighting anatomy, experimental models, mechanisms, and therapeutic aspects.

The International Association for the Study of Pain defines neuropathic pain as "pain arising as a direct consequence of a lesion or disease affecting the somatosensory system". The associated changes can be observed in the peripheral as well as the central nervous system. The available literature discusses a wide variety of causes as predisposing for the development and amplification of neuropathic pain. Further, key interactions within sensory pathways have been discovered, but no common molecular mechanism leading to neuropathic pain has been identified until now. In the first part of this review, the pain mediating lateral spinothalamic tract is described. Different in vivo models are presented that allow studying trauma-, chemotherapy-, virus-, and diabetes-induced neuropathic pain in rodents. We furthermore discuss approaches to assess neuropathic pain in these models. Second, the current knowledge about cellular and molecular mechanisms suggested to underlie the development of neuropathic pain is presented and discussed. A summary of established therapies that are already applied in the clinic and novel, promising approaches closes the paper. In conclusion, the established animal models are able to emulate the diversity of neuropathic pain observed in the clinics. However, the assessment of neuropathic pain in the presented in vivo models should be improved. The determination of common molecular markers with suitable in vitro models would simplify the assessment of neuropathic pain in vivo. This would furthermore provide insights into common molecular mechanisms of the disease and establish a basis to search for satisfying therapeutic approaches.

Modified Hyaluronic Acid-Laminin-Hydrogel as Luminal Filler for Clinically Approved Hollow Nerve Guides in a Rat Critical Defect Size Model.

Hollow nerve guidance conduits are approved for clinical use for defect lengths of up to 3 cm. This is because also in pre-clinical evaluation they are less effective in the support of nerve regeneration over critical defect lengths. Hydrogel luminal fillers are thought to improve the regeneration outcome by providing an optimized matrix inside bioartificial nerve grafts. We evaluated here a modified hyaluronic acid-laminin-hydrogel (M-HAL) as luminal filler for two clinically approved hollow nerve guides. Collagen-based and chitosan-based nerve guides were filled with M-HAL in two different concentrations and the regeneration outcome comprehensively studied in the acute repair rat sciatic nerve 15 mm critical defect size model. Autologous nerve graft (ANG) repair served as gold-standard control. At 120 days post-surgery, all ANG rats demonstrated electrodiagnostically detectable motor recovery. Both concentrations of the hydrogel luminal filler induced improved regeneration outcome over empty nerve guides. However, neither combination with collagen- nor chitosan-based nerve guides resulted in functional recovery comparable to the ANG repair. In contrast to our previous studies, we demonstrate here that M-HAL slightly improved the overall performance of either empty nerve guide type in the critical defect size model.

Gut microbiota depletion delays somatic peripheral nerve development and impairs neuromuscular junction maturation.

Gut microbiota is responsible for essential functions in human health. Several communication axes between gut microbiota and other organs via neural, endocrine, and immune pathways have been described, and perturbation of gut microbiota composition has been implicated in the onset and progression of an emerging number of diseases. Here, we analyzed peripheral nerves, dorsal root ganglia (DRG), and skeletal muscles of neonatal and young adult mice with the following gut microbiota status: a) germ-free (GF), b) gnotobiotic, selectively colonized with 12 specific gut bacterial strains (Oligo-Mouse-Microbiota, OMM12), or c) natural complex gut microbiota (CGM). Stereological and morphometric analyses revealed that the absence of gut microbiota impairs the development of somatic median nerves, resulting in smaller diameter and hypermyelinated axons, as well as in smaller unmyelinated fibers. Accordingly, DRG and sciatic nerve transcriptomic analyses highlighted a panel of differentially expressed developmental and myelination genes. Interestingly, the type III isoform of Neuregulin1 (NRG1), known to be a neuronal signal essential for Schwann cell myelination, was overexpressed in young adult GF mice, with consequent overexpression of the transcription factor Early Growth Response 2 (Egr2), a fundamental gene expressed by Schwann cells at the onset of myelination. Finally, GF status resulted in histologically atrophic skeletal muscles, impaired formation of neuromuscular junctions, and deregulated expression of related genes. In conclusion, we demonstrate for the first time a gut microbiota regulatory impact on proper development of the somatic peripheral nervous system and its functional connection to skeletal muscles, thus suggesting the existence of a novel 'Gut Microbiota-Peripheral Nervous System-axis.'

iPSC-Derived Striatal Medium Spiny Neurons from Patients with Multiple System Atrophy Show Hypoexcitability and Elevated α-Synuclein Release.

Multiple system atrophy of the parkinsonian type (MSA-P) is a rare, fatal neurodegenerative disease with sporadic onset. It is still unknown if MSA-P is a primary oligodendropathy or caused by neuronal pathophysiology leading to severe, α-synuclein-associated neurodegeneration, mainly in the striatum. In this study, we generated and differentiated induced pluripotent stem cells (iPSCs) from patients with the clinical diagnosis of probable MSA-P (n = 3) and from three matched healthy controls into GABAergic striatal medium spiny neurons (MSNs). We found a significantly elevated release and neuronal distribution for α-synuclein, as well as hypoexcitability in the MSNs derived from the MSA-P patients compared to the healthy controls. These data suggest that the striatal hypoexcitable neurons of MSA-P patients contribute to a pathological α-synuclein burden which is likely to spread to neighboring cells and projection targets, facilitating disease progression.

In Vivo and In Vitro Evaluation of a Novel Hyaluronic Acid-Laminin Hydrogel as Luminal Filler and Carrier System for Genetically Engineered Schwann Cells in Critical Gap Length Tubular Peripheral Nerve Graft in Rats.

In the current study we investigated the suitability of a novel hyaluronic acid-laminin hydrogel (HAL) as luminal filler and carrier system for co-transplanted cells within a composite chitosan-based nerve graft (CNG) in a rat critical nerve defect model. The HAL was meant to improve the performance of our artificial nerve guides by giving additional structural and molecular support to regrowing axons. We filled hollow CNGs or two-chambered nerve guides with an inserted longitudinal chitosan film (CNG[F]s), with cell-free HAL or cell-free HA or additionally suspended either naïve Schwann cells (SCs) or fibroblast growth factor 2-overexpressing Schwann cells (FGF2-SCs) within the gels. We subjected female Lewis rats to immediate 15 mm sciatic nerve gap reconstruction and comprehensively compared axonal and functional regeneration parameters with the gold standard autologous nerve graft (ANG) repair. Motor recovery was surveyed by means of electrodiagnostic measurements at 60, 90, and 120 days post-reconstruction. Upon explantation after 120 days, lower limb target muscles were harvested for calculation of muscle-weight ratios. Semi-thin cross-sections of nerve segments distal to the grafts were evaluated histomorphometrically. After 120 days of recovery, only ANG treatment led to full motor recovery. Surprisingly, regeneration outcomes revealed no regeneration-supportive effect of HAL alone and even an impairment of peripheral nerve regeneration when combined with SCs and FGF2-SCs. Furthermore, complementary in vitro studies, conducted to elucidate the reason for this unexpected negative result, revealed that SCs and FGF2-SCs suspended within the hydrogel relatively downregulated gene expression of regeneration-supporting neurotrophic factors. In conclusion, cell-free HAL in its current formulation did not qualify for optimizing regeneration outcome through CNG[F]s. In addition, we demonstrate that our HAL, when used as a carrier system for co-transplanted SCs, changed their gene expression profile and deteriorated the pro-regenerative milieu within the nerve guides.

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.

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.].

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).

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.

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.

RNA Editing-Systemic Relevance and Clue to Disease Mechanisms?

Recent advances in sequencing technologies led to the identification of a plethora of different genes and several hundreds of amino acid recoding edited positions. Changes in editing rates of some of these positions were associated with diseases such as atherosclerosis, myopathy, epilepsy, major depression disorder, schizophrenia and other mental disorders as well as cancer and brain tumors. This review article summarizes our current knowledge on that front and presents glycine receptor C-to-U RNA editing as a first example of disease-associated increased RNA editing that includes assessment of disease mechanisms of the corresponding gene product in an animal model.