A brain-enriched circular RNA controls excitatory neurotransmission and restricts sensitivity to aversive stimuli.

Circular RNAs (circRNAs) are a large class of noncoding RNAs. Despite the identification of thousands of circular transcripts, the biological significance of most of them remains unexplored, partly because of the lack of effective methods for generating loss-of-function animal models. In this study, we focused on circTulp4, an abundant circRNA derived from the Tulp4 gene that is enriched in the brain and synaptic compartments. By creating a circTulp4-deficient mouse model, in which we mutated the splice acceptor site responsible for generating circTulp4 without affecting the linear mRNA or protein levels, we were able to conduct a comprehensive phenotypic analysis. Our results demonstrate that circTulp4 is critical in regulating neuronal and brain physiology, modulating the strength of excitatory neurotransmission and sensitivity to aversive stimuli. This study provides evidence that circRNAs can regulate biologically relevant functions in neurons, with modulatory effects at multiple levels of the phenotype, establishing a proof of principle for the regulatory role of circRNAs in neural processes.

Influence of RNA circularity on Target RNA-Directed MicroRNA Degradation.

A subset of circular RNAs (circRNAs) and linear RNAs have been proposed to 'sponge' or block microRNA activity. Additionally, certain RNAs induce microRNA destruction through the process of Target RNA-Directed MicroRNA Degradation (TDMD), but whether both linear and circular transcripts are equivalent in driving TDMD is unknown. Here, we studied whether circular/linear topology of endogenous and artificial RNA targets affects TDMD. Consistent with previous knowledge that Cdr1as (ciRS-7) circular RNA protects miR-7 from Cyrano-mediated TDMD, we demonstrate that depletion of Cdr1as reduces miR-7 abundance. In contrast, overexpression of an artificial linear version of Cdr1as drives miR-7 degradation. Using plasmids that express a circRNA with minimal co-expressed cognate linear RNA, we show differential effects on TDMD that cannot be attributed to the nucleotide sequence, as the TDMD properties of a sequence often differ when in a circular versus linear form. By analysing RNA sequencing data of a neuron differentiation system, we further detect potential effects of circRNAs on microRNA stability. Our results support the view that RNA circularity influences TDMD, either enhancing or inhibiting it on specific microRNAs.

Super-resolution Imaging of Energy Transfer by Intensity-Based STED-FRET.

Förster resonance energy transfer (FRET) imaging methods provide unique insight into the spatial distribution of energy transfer and (bio)molecular interaction events, though they deliver average information for an ensemble of events included in a diffraction-limited volume. Coupling super-resolution fluorescence microscopy and FRET has been a challenging and elusive task. Here, we present STED-FRET, a method of general applicability to obtain super-resolved energy transfer images. In addition to higher spatial resolution, STED-FRET provides a more accurate quantification of interaction and has the capacity of suppressing contributions of noninteracting partners, which are otherwise masked by averaging in conventional imaging. The method capabilities were first demonstrated on DNA-origami model systems, verified on uniformly double-labeled microtubules, and then utilized to image biomolecular interactions in the membrane-associated periodic skeleton (MPS) of neurons.

Three-dimensional total-internal reflection fluorescence nanoscopy with nanometric axial resolution by photometric localization of single molecules.

Single-molecule localization microscopy enables far-field imaging with lateral resolution in the range of 10 to 20 nanometres, exploiting the fact that the centre position of a single-molecule's image can be determined with much higher accuracy than the size of that image itself. However, attaining the same level of resolution in the axial (third) dimension remains challenging. Here, we present Supercritical Illumination Microscopy Photometric z-Localization with Enhanced Resolution (SIMPLER), a photometric method to decode the axial position of single molecules in a total internal reflection fluorescence microscope. SIMPLER requires no hardware modification whatsoever to a conventional total internal reflection fluorescence microscope and complements any 2D single-molecule localization microscopy method to deliver 3D images with nearly isotropic nanometric resolution. Performance examples include SIMPLER-direct stochastic optical reconstruction microscopy images of the nuclear pore complex with sub-20 nm axial localization precision and visualization of microtubule cross-sections through SIMPLER-DNA points accumulation for imaging in nanoscale topography with sub-10 nm axial localization precision.

Detrimental Effects of HMGB-1 Require Microglial-Astroglial Interaction: Implications for the Status Epilepticus -Induced Neuroinflammation.

Temporal Lobe Epilepsy (TLE) is the most common form of human epilepsy and available treatments with antiepileptic drugs are not disease-modifying therapies. The neuroinflammation, neuronal death and exacerbated plasticity that occur during the silent period, following the initial precipitating event (IPE), seem to be crucial for epileptogenesis. Damage Associated Molecular Patterns (DAMP) such as HMGB-1, are released early during this period concomitantly with a phenomenon of reactive gliosis and neurodegeneration. Here, using a combination of primary neuronal and glial cell cultures, we show that exposure to HMGB-1 induces dendrite loss and neurodegeneration in a glial-dependent manner. In glial cells, loss of function studies showed that HMGB-1 exposure induces NF-κB activation by engaging a signaling pathway that involves TLR2, TLR4, and RAGE. In the absence of glial cells, HMGB-1 failed to induce neurodegeneration of primary cultured cortical neurons. Moreover, purified astrocytes were unable to fully respond to HMGB-1 with NF-κB activation and required microglial cooperation. In agreement, in vivo HMGB-1 blockage with glycyrrhizin, immediately after pilocarpine-induced status epilepticus (SE), reduced neuronal degeneration, reactive astrogliosis and microgliosis in the long term. We conclude that microglial-astroglial cooperation is required for astrocytes to respond to HMGB-1 and to induce neurodegeneration. Disruption of this HMGB-1 mediated signaling pathway shows beneficial effects by reducing neuroinflammation and neurodegeneration after SE. Thus, early treatment strategies during the latency period aimed at blocking downstream signaling pathways activated by HMGB-1 are likely to have a significant effect in the neuroinflammation and neurodegeneration that are proposed as key factors in epileptogenesis.

Toll-Like Receptor 4 (TLR4) and Triggering Receptor Expressed on Myeloid Cells-2 (TREM-2) Activation Balance Astrocyte Polarization into a Proinflammatory Phenotype.

Astrocytes react to brain injury with a generic response known as reactive gliosis, which involves activation of multiple intracellular pathways including several that may be beneficial for neuronal survival. However, by unknown mechanisms, reactive astrocytes can polarize into a proinflammatory phenotype that induces neurodegeneration. In order to study reactive gliosis and astroglial polarization into a proinflammatory phenotype, we used cortical devascularization-induced brain ischemia in Wistar rats and primary astroglial cell cultures exposed to oxygen-glucose deprivation (OGD). We analyzed the profile of TLR4 expression and the consequences of its activation by gain- and loss-of-function studies, and the effects produced by the activation of triggering receptor expressed on myeloid cells-2 (TREM-2), a negative regulator of TLR4 signaling. Both OGD exposure on primary astroglial cell cultures and cortical devascularization brain ischemia in rats induced TLR4 expression in astrocytes. In vivo, astroglial TLR4 expression was specifically observed in the ischemic penumbra surrounding necrotic core. Functional studies showed that OGD increased the astroglial response to the TLR4 agonist lipopolysaccharide (LPS), and conversely, TLR4 knockout primary astrocytes had impaired nuclear factor kappa-B (NF-κB) activation when exposed to LPS. In gain-of-function studies, plasmid-mediated TLR4 over-expression exacerbated astroglial response to LPS as shown by sustained NF-κB activation and increased expression of proinflammatory cytokines IL-1ß and TNFα. TREM-2 expression, although present in naïve primary astrocytes, was induced by OGD, LPS, or high-mobility group box 1 protein (HMGB-1) exposure. TREM-2 activation by antibody cross-linking or the overexpression of TREM-2 intracellular adaptor, DAP12, partially suppressed LPS-induced NF-κB activation in purified astrocytic cultures. In vivo, TREM-2 expression was observed in macrophages and astrocytes located in the ischemic penumbra. While TREM-2+ macrophages were abundant at 3 days post-lesion (DPL) in the ischemic core, TREM-2+ astrocytes persisted in the penumbra until 14DPL. This study demonstrates that TLR4 expression increases astroglial sensitivity to ligands facilitating astrocyte conversion towards a proinflammatory phenotype, and that astroglial TREM-2 modulates this response reducing the downstream NF-κB activation. Therefore, the availability of TLR4 and TREM-2 ligands in the ischemic environment may control proinflammatory astroglial conversion to the neurodegenerative phenotype.

The proinflammatory RAGE/NF-κB pathway is involved in neuronal damage and reactive gliosis in a model of sleep apnea by intermittent hypoxia.

Sleep apnea (SA) causes long-lasting changes in neuronal circuitry, which persist even in patients successfully treated for the acute effects of the disease. Evidence obtained from the intermittent hypoxia (IH) experimental model of SA has shown neuronal death, impairment in learning and memory and reactive gliosis that may account for cognitive and structural alterations observed in human patients. However, little is known about the mechanism controlling these deleterious effects that may be useful as therapeutic targets in SA. The Receptor for Advanced Glycation End products (RAGE) and its downstream effector Nuclear Factor Kappa B (NF-κB) have been related to neuronal death and astroglial conversion to the pro-inflammatory neurodegenerative phenotype. RAGE expression and its ligand S100B were shown to be increased in experimental models of SA. We here used dissociated mixed hippocampal cell cultures and male Wistar rats exposed to IH cycles and observed that NF-κB is activated in glial cells and neurons after IH. To disclose the relative contribution of the S100B/RAGE/NF-κB pathway to neuronal damage and reactive gliosis after IH we performed sequential loss of function studies using RAGE or S100B neutralizing antibodies, a herpes simplex virus (HSV)-derived amplicon vector that induces the expression of RAGEΔcyto (dominant negative RAGE) and a chemical blocker of NF-κB. Our results show that NF-κB activation peaks 3 days after IH exposure, and that RAGE or NF-κB blockage during this critical period significantly improves neuronal survival and reduces reactive gliosis. Both in vitro and in vivo, S100B blockage altered reactive gliosis but did not have significant effects on neuronal survival. We conclude that both RAGE and downstream NF-κB signaling are centrally involved in the neuronal alterations found in SA models, and that blockage of these pathways is a tempting strategy for preventing neuronal degeneration and reactive gliosis in SA.

Gabapentin administration reduces reactive gliosis and neurodegeneration after pilocarpine-induced status epilepticus.

The lithium-pilocarpine model of epilepsy reproduces in rodents several features of human temporal lobe epilepsy, by inducing an acute status epilepticus (SE) followed by a latency period. It has been proposed that the neuronal network reorganization that occurs during latency determines the subsequent appearance of spontaneous recurrent seizures. The aim of this study was to evaluate neuronal and glial responses during the latency period that follows SE. Given the potential role of astrocytes in the post-SE network reorganization, through the secretion of synaptogenic molecules such as thrombospondins, we also studied the effect of treatment with the α2δ1 thrombospondin receptor antagonist gabapentin. Adult male Wistar rats received 3 mEq/kg LiCl, and 20 h later 30 mg/kg pilocarpine. Once SE was achieved, seizures were stopped with 20 mg/kg diazepam. Animals then received 400 mg/kg/day gabapentin or saline for either 4 or 14 days. In vitro experiments were performed in dissociated mixed hippocampal cell culture exposed to glutamate, and subsequently treated with gabapentin or vehicle. During the latency period, the hippocampus and pyriform cortex of SE-animals presented a profuse reactive astrogliosis, with increased GFAP and nestin expression. Gliosis intensity was dependent on the Racine stage attained by the animals and peaked 15 days after SE. Microglia was also reactive after SE, and followed the same pattern. Neuronal degeneration was present in SE-animals, and also depended on the Racine stage and the SE duration. Polysialic-acid NCAM (PSA-NCAM) expression was increased in hippocampal CA-1 and dentate gyrus of SE-animals. Gabapentin treatment was able to reduce reactive gliosis, decrease neuronal loss and normalize PSA-NCAM staining in hippocampal CA-1. In vitro, gabapentin treatment partially prevented the dendritic loss and reactive gliosis caused by glutamate excitotoxicity. Our results show that gabapentin treatment during the latency period after SE protects neurons and normalizes PSA-NCAM probably by direct interaction with neurons and glia.