Brain cell-specific origin of circulating microRNA biomarkers in experimental temporal lobe epilepsy.

The diagnosis of epilepsy is complex and challenging and would benefit from the availability of molecular biomarkers, ideally measurable in a biofluid such as blood. Experimental and human epilepsy are associated with altered brain and blood levels of various microRNAs (miRNAs). Evidence is lacking, however, as to whether any of the circulating pool of miRNAs originates from the brain. To explore the link between circulating miRNAs and the pathophysiology of epilepsy, we first sequenced argonaute 2 (Ago2)-bound miRNAs in plasma samples collected from mice subject to status epilepticus induced by intraamygdala microinjection of kainic acid. This identified time-dependent changes in plasma levels of miRNAs with known neuronal and microglial-cell origins. To explore whether the circulating miRNAs had originated from the brain, we generated mice expressing FLAG-Ago2 in neurons or microglia using tamoxifen-inducible Thy1 or Cx3cr1 promoters, respectively. FLAG immunoprecipitates from the plasma of these mice after seizures contained miRNAs, including let-7i-5p and miR-19b-3p. Taken together, these studies confirm that a portion of the circulating pool of miRNAs in experimental epilepsy originates from the brain, increasing support for miRNAs as mechanistic biomarkers of epilepsy.

Human neutrophils communicate remotely via calcium-dependent glutamate-induced glutamate release.

Neutrophils are white blood cells that are critical to acute inflammatory and adaptive immune responses. Their swarming-pattern behavior is controlled by multiple cellular cascades involving calcium-dependent release of various signaling molecules. Previous studies have reported that neutrophils express glutamate receptors and can release glutamate but evidence of direct neutrophil-neutrophil communication has been elusive. Here, we hold semi-suspended cultured human neutrophils in patch-clamp whole-cell mode to find that calcium mobilization induced by stimulating one neutrophil can trigger an N-methyl-D-aspartate (NMDA) receptor-driven membrane current and calcium signal in neighboring neutrophils. We employ an enzymatic-based imaging assay to image, in real time, glutamate release from neutrophils induced by glutamate released from their neighbors. These observations provide direct evidence for a positive-feedback inter-neutrophil communication that could contribute to mechanisms regulating communal neutrophil behavior.

Anti-seizure effects of JNJ-54175446 in the intra-amygdala kainic acid model of drug-resistant temporal lobe epilepsy in mice.

There remains a need for new drug targets for treatment-resistant temporal lobe epilepsy. The ATP-gated P2X7 receptor coordinates neuroinflammatory responses to tissue injury. Previous studies in mice reported that the P2X7 receptor antagonist JNJ-47965567 suppressed spontaneous seizures in the intraamygdala kainic acid model of epilepsy and reduced attendant gliosis in the hippocampus. The drug-resistance profile of this model is not fully characterised, however, and newer P2X7 receptor antagonists with superior pharmacokinetic profiles have recently entered clinical trials. Using telemetry-based continuous EEG recordings in mice, we demonstrate that spontaneous recurrent seizures in the intraamygdala kainic acid model are refractory to the common anti-seizure medicine levetiracetam. In contrast, once-daily dosing of JNJ-54175446 (30 mg/kg, intraperitoneal) resulted in a significant reduction in spontaneous recurrent seizures which lasted several days after the end of drug administration. Using a combination of immunohistochemistry and ex vivo radiotracer assay, we find that JNJ-54175446-treated mice at the end of recordings display a reduction in astrogliosis and altered microglia process morphology within the ipsilateral CA3 subfield of the hippocampus, but no difference in P2X7 receptor surface expression. The present study extends the characterisation of the drug-resistance profile of the intraamygdala kainic acid model in mice and provides further evidence that targeting the P2X7 receptor may have therapeutic applications in the treatment of temporal lobe epilepsy.

Super-resolution imaging to reveal the nanostructure of tripartite synapses.

Even though neurons are the main drivers of information processing in the brain and spinal cord, other cell types are important to mediate adequate flow of information. These include electrically passive glial cells such as microglia and astrocytes, which recently emerged as active partners facilitating proper signal transduction. In disease, these cells undergo pathophysiological changes that propel disease progression and change synaptic connections and signal transmission. In the healthy brain, astrocytic processes contact pre- and postsynaptic structures. These processes can be nanoscopic, and therefore only electron microscopy has been able to reveal their structure and morphology. However, electron microscopy is not suitable in revealing dynamic changes, and it is labour- and time-intensive. The dawn of super-resolution microscopy, techniques that 'break' the diffraction limit of conventional light microscopy, over the last decades has enabled researchers to reveal the nanoscopic synaptic environment. In this review, we highlight and discuss recent advances in our understanding of the nano-world of the so-called tripartite synapses, the relationship between pre- and postsynapse as well as astrocytic processes. Overall, novel super-resolution microscopy methods are needed to fully illuminate the intimate relationship between glia and neuronal cells that underlies signal transduction in the brain and that might be affected in diseases such as Alzheimer's disease and epilepsy.

Antagomir-mediated suppression of microRNA-134 reduces kainic acid-induced seizures in immature mice.

MicroRNAs are short non-coding RNAs that negatively regulate protein levels and perform important roles in establishing and maintaining neuronal network function. Previous studies in adult rodents have detected upregulation of microRNA-134 after prolonged seizures (status epilepticus) and demonstrated that silencing microRNA-134 using antisense oligonucleotides, termed antagomirs, has potent and long-lasting seizure-suppressive effects. Here we investigated whether targeting microRNA-134 can reduce or delay acute seizures in the immature brain. Status epilepticus was induced in 21 day-old (P21) male mice by systemic injection of 5 mg/kg kainic acid. This triggered prolonged electrographic seizures and select bilateral neuronal death within the CA3 subfield of the hippocampus. Expression of microRNA-134 and functional loading to Argonaute-2 was not significantly changed in the hippocampus after seizures in the model. Nevertheless, when levels of microRNA-134 were reduced by prior intracerebroventricular injection of an antagomir, kainic acid-induced seizures were delayed and less severe and mice displayed reduced neuronal death in the hippocampus. These studies demonstrate targeting microRNA-134 may have therapeutic applications for the treatment of seizures in children.

LTP Induction Boosts Glutamate Spillover by Driving Withdrawal of Perisynaptic Astroglia.

Extrasynaptic actions of glutamate are limited by high-affinity transporters expressed by perisynaptic astroglial processes (PAPs): this helps maintain point-to-point transmission in excitatory circuits. Memory formation in the brain is associated with synaptic remodeling, but how this affects PAPs and therefore extrasynaptic glutamate actions is poorly understood. Here, we used advanced imaging methods, in situ and in vivo, to find that a classical synaptic memory mechanism, long-term potentiation (LTP), triggers withdrawal of PAPs from potentiated synapses. Optical glutamate sensors combined with patch-clamp and 3D molecular localization reveal that LTP induction thus prompts spatial retreat of astroglial glutamate transporters, boosting glutamate spillover and NMDA-receptor-mediated inter-synaptic cross-talk. The LTP-triggered PAP withdrawal involves NKCC1 transporters and the actin-controlling protein cofilin but does not depend on major Ca2+-dependent cascades in astrocytes. We have therefore uncovered a mechanism by which a memory trace at one synapse could alter signal handling by multiple neighboring connections.

A systems approach delivers a functional microRNA catalog and expanded targets for seizure suppression in temporal lobe epilepsy.

Temporal lobe epilepsy is the most common drug-resistant form of epilepsy in adults. The reorganization of neural networks and the gene expression landscape underlying pathophysiologic network behavior in brain structures such as the hippocampus has been suggested to be controlled, in part, by microRNAs. To systematically assess their significance, we sequenced Argonaute-loaded microRNAs to define functionally engaged microRNAs in the hippocampus of three different animal models in two species and at six time points between the initial precipitating insult through to the establishment of chronic epilepsy. We then selected commonly up-regulated microRNAs for a functional in vivo therapeutic screen using oligonucleotide inhibitors. Argonaute sequencing generated 1.44 billion small RNA reads of which up to 82% were microRNAs, with over 400 unique microRNAs detected per model. Approximately half of the detected microRNAs were dysregulated in each epilepsy model. We prioritized commonly up-regulated microRNAs that were fully conserved in humans and designed custom antisense oligonucleotides for these candidate targets. Antiseizure phenotypes were observed upon knockdown of miR-10a-5p, miR-21a-5p, and miR-142a-5p and electrophysiological analyses indicated broad safety of this approach. Combined inhibition of these three microRNAs reduced spontaneous seizures in epileptic mice. Proteomic data, RNA sequencing, and pathway analysis on predicted and validated targets of these microRNAs implicated derepressed TGF-ß signaling as a shared seizure-modifying mechanism. Correspondingly, inhibition of TGF-ß signaling occluded the antiseizure effects of the antagomirs. Together, these results identify shared, dysregulated, and functionally active microRNAs during the pathogenesis of epilepsy which represent therapeutic antiseizure targets.

Correction: Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons.

[This corrects the article DOI: 10.1371/journal.pbio.3000414.].

Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons.

Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retinal degeneration, obesity, kidney failure, and cognitive impairment. In spite of progress made in our general understanding of BBS aetiology, the molecular and cellular mechanisms underlying cognitive impairment in BBS remain elusive. Here, we report that the loss of BBS proteins causes synaptic dysfunction in principal neurons, providing a possible explanation for the cognitive impairment phenotype observed in BBS patients. Using synaptosomal proteomics and immunocytochemistry, we demonstrate the presence of Bbs proteins in the postsynaptic density (PSD) of hippocampal neurons. Loss of Bbs results in a significant reduction of dendritic spines in principal neurons of Bbs mouse models. Furthermore, we show that spine deficiency correlates with events that destabilise spine architecture, such as impaired spine membrane receptor signalling, known to be involved in the maintenance of dendritic spines. Our findings suggest a role for BBS proteins in dendritic spine homeostasis that may be linked to the cognitive phenotype observed in BBS.

A Method to Visualize the Nanoscopic Morphology of Astrocytes In Vitro and In Situ.

In recent years it has become apparent that astroglia are not only essential players in brain development, homeostasis, and metabolic support but are also important for the formation and regulation of synaptic circuits. Fine astrocytic processes that can be found in the vicinity of synapses undergo considerable structural plasticity associated with age- and use-dependent changes in neural circuitries. However, due to the extraordinary complex, essentially nanoscopic morphology of astroglia, the underlying cellular mechanisms remain poorly understood.Here we detail a super-resolution microscopy approach, based on the single-molecule localisation microscopy (SMLM) technique direct stochastic optical reconstruction microscopy (dSTORM) to visualize astroglial morphology on the nanoscale. This approach enables visualization of key morphological changes that occur in nanoscopic astrocyte processes, whose characteristic size falls below the diffraction limit of conventional optical microscopy.

Biochemical autoregulatory gene therapy for focal epilepsy.

Despite the introduction of more than one dozen new antiepileptic drugs in the past 20 years, approximately one-third of people who develop epilepsy continue to have seizures on mono- or polytherapy1. Viral-vector-mediated gene transfer offers the opportunity to design a rational treatment that builds on mechanistic understanding of seizure generation and that can be targeted to specific neuronal populations in epileptogenic foci2. Several such strategies have shown encouraging results in different animal models, although clinical translation is limited by possible effects on circuits underlying cognitive, mnemonic, sensory or motor function. Here, we describe an autoregulatory antiepileptic gene therapy, which relies on neuronal inhibition in response to elevations in extracellular glutamate. It is effective in a rodent model of focal epilepsy and is well tolerated, thus lowering the barrier to clinical translation.

The Nanoworld of the Tripartite Synapse: Insights from Super-Resolution Microscopy.

Synaptic connections between individual nerve cells are fundamental to the process of information transfer and storage in the brain. Over the past decades a third key partner of the synaptic machinery has been unveiled: ultrathin processes of electrically passive astroglia which often surround pre- and postsynaptic structures. The recent advent of super-resolution (SR) microscopy has begun to uncover the dynamic nanoworld of synapses and their astroglial environment. Here we overview and discuss the current progress in our understanding of the synaptic nanoenvironment, as gleaned from the imaging methods that go beyond the diffraction limit of conventional light microscopy. We argue that such methods are essential to achieve a new level of comprehension pertinent to the principles of signal integration in the brain.

Probing nano-organization of astroglia with multi-color super-resolution microscopy.

Astroglia are essential for brain development, homeostasis, and metabolic support. They also contribute actively to the formation and regulation of synaptic circuits, by successfully handling, integrating, and propagating physiological signals of neural networks. The latter occurs mainly by engaging a versatile mechanism of internal Ca2+ fluctuations and regenerative waves prompting targeted release of signaling molecules into the extracellular space. Astroglia also show substantial structural plasticity associated with age- and use-dependent changes in neural circuitry. However, the underlying cellular mechanisms are poorly understood, mainly because of the extraordinary complex morphology of astroglial compartments on the nanoscopic scale. This complexity largely prevents direct experimental access to astroglial processes, most of which are beyond the diffraction limit of optical microscopy. Here we employed super-resolution microscopy (direct stochastic optical reconstruction microscopy; dSTORM), to visualize astroglial organization on the nanoscale, in culture and in thin brain slices, as an initial step to understand the structural basis of astrocytic nano-physiology. We were able to follow nanoscopic morphology of GFAP-enriched astrocytes, which adapt a flattened shape in culture and a sponge-like structure in situ, with GFAP fibers of varied diameters. We also visualized nanoscopic astrocytic processes using the ubiquitous cytosolic astrocyte marker proteins S100ß and glutamine synthetase. Finally, we overexpressed and imaged membrane-targeted pHluorin and lymphocyte-specific protein tyrosine kinase (N-terminal domain) -green fluorescent protein (lck-GFP), to better understand the molecular cascades underlying some common astroglia-targeted fluorescence imaging techniques. The results provide novel, albeit initial, insights into the cellular organization of astroglia on the nanoscale, paving the way for function-specific studies. © 2017 Wiley Periodicals, Inc.

A Method for the Isolation and Culture of Adult Rat Retinal Pigment Epithelial (RPE) Cells to Study Retinal Diseases.

Diseases such as age-related macular degeneration (AMD) affect the retinal pigment epithelium (RPE) and lead to the death of the epithelial cells and ultimately blindness. RPE transplantation is currently a major focus of eye research and clinical trials using human stem cell-derived RPE cells are ongoing. However, it remains to be established to which extent the source of RPE cells for transplantation affects their therapeutic efficacy and this needs to be explored in animal models. Autotransplantation of RPE cells has attractions as a therapy, but existing protocols to isolate adult RPE cells from rodents are technically difficult, time-consuming, have a low yield and are not optimized for long-term cell culturing. Here, we report a newly devised protocol which facilitates reliable and simple isolation and culture of RPE cells from adult rats. Incubation of a whole rat eyeball in 20 U/ml papain solution for 50 min yielded 4 × 10(4) viable RPE cells. These cells were hexagonal and pigmented upon culture. Using immunostaining, we demonstrated that the cells expressed RPE cell-specific marker proteins including cytokeratin 18 and RPE65, similar to RPE cells in vivo. Additionally, the cells were able to produce and secrete Bruch's membrane matrix components similar to in vivo situation. Similarly, the cultured RPE cells adhered to isolated Bruch's membrane as has previously been reported. Therefore, the protocol described in this article provides an efficient method for the rapid and easy isolation of high quantities of adult rat RPE cells. This provides a reliable platform for studying the therapeutic targets, testing the effects of drugs in a preclinical setup and to perform in vitro and in vivo transplantation experiments to study retinal diseases.

Influence of extracellular matrix components on the expression of integrins and regeneration of adult retinal ganglion cells.

PURPOSE: Retinal ganglion cells (RGCs) are exposed to injury in a variety of optic nerve diseases including glaucoma. However, not all cells respond in the same way to damage and the capacity of individual RGCs to survive or regenerate is variable. In order to elucidate factors that may be important for RGC survival and regeneration we have focussed on the extracellular matrix (ECM) and RGC integrin expression. Our specific questions were: (1) Do adult RGCs express particular sets of integrins in vitro and in vivo? (2) Can the nature of the ECM influence the expression of different integrins? (3) Can the nature of the ECM affect the survival of the cells and the length or branching complexity of their neurites? METHODS: Primary RGC cultures from adult rat retina were placed on glass coverslips treated with different substrates: Poly-L-Lysine (PL), or PL plus laminin (L), collagen I (CI), collagen IV (CIV) or fibronectin (F). After 10 days in culture, we performed double immunostaining with an antibody against ßIII-Tubulin to identify the RGCs, and antibodies against the integrin subunits: αV, α1, α3, α5, ß1 or ß3. The number of adhering and surviving cells, the number and length of the neurites and the expression of the integrin subunits on the different substrates were analysed. RESULTS: PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions. The type of substrate affected the number and length of neurites. L stimulated the longest growth. We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites. The different combinations of integrins expressed by the cells growing on different substrata suggest that RGCs expressed predominantly α1ß1 or α3ß1 on L, α1ß1 on CI and CIV, and α5ß3 on F. The activity of the integrins was demonstrated by the phosphorylation of focal adhesion kinase (FAK). CONCLUSIONS: Adult rat RGCs can survive and grow in the presence of different ECM tested. Further studies should be done to elucidate the different molecular characteristics of the RGCs subtypes in order to understand the possible different sensitivity of different RGCs to damage in diseases like glaucoma in which not all RGCs die at the same time.

Morphological plasticity of astroglia: Understanding synaptic microenvironment.

Memory formation in the brain is thought to rely on the remodeling of synaptic connections which eventually results in neural network rewiring. This remodeling is likely to involve ultrathin astroglial protrusions which often occur in the immediate vicinity of excitatory synapses. The phenomenology, cellular mechanisms, and causal relationships of such astroglial restructuring remain, however, poorly understood. This is in large part because monitoring and probing of the underpinning molecular machinery on the scale of nanoscopic astroglial compartments remains a challenge. Here we briefly summarize the current knowledge regarding the cellular organisation of astroglia in the synaptic microenvironment and discuss molecular mechanisms potentially involved in use-dependent astroglial morphogenesis. We also discuss recent observations concerning morphological astroglial plasticity, the respective monitoring methods, and some of the newly emerging techniques that might help with conceptual advances in the area.

Full length talin stimulates integrin activation and axon regeneration.

Integrin function is regulated by activation involving conformational changes that modulate ligand-binding affinity and downstream signaling. Activation is regulated through inside-out signaling which is controlled by many signaling pathways via a final common pathway through kindlin and talin, which bind to the intracellular tail of beta integrins. Previous studies have shown that the axon growth inhibitory molecules NogoA and chondroitin sulfate proteoglycans (CSPGs) inactivate integrins. Overexpressing kindlin-1 in dorsal root ganglion (DRG) neurons activates integrins, enabling their axons to overcome inhibitory molecules in the environment, and promoting regeneration in vivo following dorsal root crush. Other studies have indicated that expression of the talin head alone or with kindlin can enhance integrin activation. Here, using adult rat DRG neurons, we investigate the effects of overexpressing various forms of talin on axon growth and integrin signaling. We found that overexpression of the talin head activated axonal integrins but inhibited downstream signaling via FAK, and did not promote axon growth. Similarly, co-expression of the talin head and kindlin-1 prevented the growth-promoting effect of kindlin-1, suggesting that the talin head acts as a form of dominant negative for integrin function. Using full-length talin constructs in PC12 cells we observed that neurite growth was enhanced by the expression of wild-type talin and more so by two 'activated' forms of talin produced by point mutation (on laminin and aggrecan-laminin substrates). Nevertheless, co-expression of full-length talin with kindlin did not promote neurite growth more than either molecule alone. In vivo, we find that talin is present in PNS axons (sciatic nerve), and also in CNS axons of the corticospinal tract.

Retinal ganglion cell neuroprotection by an angiotensin II blocker in an ex vivo retinal explant model.

PURPOSE: An ex vivo organotypic retinal explant model was developed to examine retinal survival mechanisms relevant to glaucoma mediated by the renin angiotensin system in the rodent eye. METHODS: Eyes from adult Sprague Dawley rats were enucleated immediately post-mortem and used to make four retinal explants per eye. Explants were treated either with irbesartan (10 µM), vehicle or angiotensin II (2 µM) for four days. Retinal ganglion cell density was estimated by ßIII tubulin immunohistochemistry. Live imaging of superoxide formation with dihydroethidium (DHE) was performed. Protein expression was determined by Western blotting, and mRNA expression was determined by RT-PCR. RESULTS: Irbesartan (10 µM) almost doubled ganglion cell survival after four days. Angiotensin II (2 µM) reduced cell survival by 40%. Sholl analysis suggested that irbesartan improved ganglion cell dendritic arborisation compared to control and angiotensin II reduced it. Angiotensin-treated explants showed an intense DHE fluorescence not seen in irbesartan-treated explants. Analysis of protein and mRNA expression determined that the angiotensin II receptor At1R was implicated in modulation of the NADPH-dependent pathway of superoxide generation. CONCLUSION: Angiotensin II blockers protect retinal ganglion cells in this model and may be worth further investigation as a neuroprotective treatment in models of eye disease.

Kinesin KIF4A transports integrin ß1 in developing axons of cortical neurons.

CNS axons have poor regenerative ability compared to PNS axons, and mature axons regenerate less well than immature embryonic axons. The loss of regenerative ability with maturity is accompanied by the setting up of a selective transport filter in axons, restricting the types of molecule that are present. We confirm that integrins (represented by subunits ß1 and α5) are present in early cortical axons in vitro but are excluded from mature axons. Ribosomal protein and L1 show selective axonal transport through association with kinesin kif4A; we have therefore examined the hypothesis that integrin transport might also be in association with kif4A. Kif4A is present in all processes of immature cortical neurons cultured at E18, then downregulated by 14days in vitro, coinciding with the exclusion of integrin from axons. Kif4a co-localises with ß1 integrin in vesicles in neurons and non-neuronal cells, and the two molecules co-immunoprecipitate. Knockdown of KIF4A expression with shRNA reduced the level of integrin ß1 in axons of developing neurons and reduced neurite elongation on laminin, an integrin-dependent substrate. Overexpression of kif4A triggered apoptosis in neuronal and non-neuronal cells. In mature neurons expression of kif4A-GFP at a modest level did not kill the cells, and the kif4A was detectable in their axons. However this was not accompanied by an increase in integrin ß1 axonal transport, suggesting that kif4A is not the only integrin transporter, and that integrin exclusion from axons is controlled by factors other than the kif4A level.

Enhancing RPE Cell-Based Therapy Outcomes for AMD: The Role of Bruch's Membrane.

Age-related macular degeneration (AMD) is the leading cause of legal blindness in older people in the developed world. The disease involves damage to the part of the retina responsible for central vision. Degeneration of retinal pigment epithelial (RPE) cells, photoreceptors, and choriocapillaris may contribute to visual loss. Over the past decades, scientists and clinicians have tried to replace lost RPE cells in patients with AMD using cells from different sources. In recent years, advances in generating RPE cells from stem cells have been made and clinical trials are currently evaluating the safety and efficiency of replacing the degenerated RPE cell layer with stem cell-derived RPE cells. However, the therapeutic success of transplantation of stem cell-derived RPE cells may be limited unless the transplanted cells can adhere and survive in the long term in the diseased eye. One hallmark of AMD is the altered extracellular environment of Bruch's membrane to which the grafted cells have to adhere. Here, we discuss recent approaches to overcome the inhibitory environment of the diseased eye and to enhance the survival rate of transplanted RPE cells. Our aim is to highlight novel approaches that may have the potential to improve the efficacy of RPE transplantation for AMD in the future.