Neuronal and astrocytic tetraploidy is increased in drug-resistant epilepsy.

AIMS: Epilepsy is one of the most prevalent neurological diseases. A third of patients with epilepsy remain drug-resistant. The exact aetiology of drug-resistant epilepsy (DRE) is still unknown. Neuronal tetraploidy has been associated with neuropathology. The aim of this study was to assess the presence of tetraploid neurons and astrocytes in DRE. METHODS: For that purpose, cortex, hippocampus and amygdala samples were obtained from patients subjected to surgical resection of the epileptogenic zone. Post-mortem brain tissue of subjects without previous records of neurological, neurodegenerative or psychiatric diseases was used as control. RESULTS: The percentage of tetraploid cells was measured by immunostaining of neurons (NeuN) or astrocytes (S100ß) followed by flow cytometry analysis. The results were confirmed by image cytometry (ImageStream X Amnis System Cytometer) and with an alternative astrocyte biomarker (NDRG2). Statistical comparison was performed using univariate tests. A total of 22 patients and 10 controls were included. Tetraploid neurons and astrocytes were found both in healthy individuals and DRE patients in the three brain areas analysed: cortex, hippocampus and amygdala. DRE patients presented a higher number of tetraploid neurons (p = 0.020) and astrocytes (p = 0.002) in the hippocampus than controls. These results were validated by image cytometry. CONCLUSIONS: We demonstrated the presence of both tetraploid neurons and astrocytes in healthy subjects as well as increased levels of both cell populations in DRE patients. Herein, we describe for the first time the presence of tetraploid astrocytes in healthy subjects. Furthermore, these results provide new insights into epilepsy, opening new avenues for future treatment.

Serum Amyloid A1/Toll-Like Receptor-4 Axis, an Important Link between Inflammation and Outcome of TBI Patients.

Traumatic brain injury (TBI) is one of the leading causes of mortality and disability worldwide without any validated biomarker or set of biomarkers to help the diagnosis and evaluation of the evolution/prognosis of TBI patients. To achieve this aim, a deeper knowledge of the biochemical and pathophysiological processes triggered after the trauma is essential. Here, we identified the serum amyloid A1 protein-Toll-like receptor 4 (SAA1-TLR4) axis as an important link between inflammation and the outcome of TBI patients. Using serum and mRNA from white blood cells (WBC) of TBI patients, we found a positive correlation between serum SAA1 levels and injury severity, as well as with the 6-month outcome of TBI patients. SAA1 levels also correlate with the presence of TLR4 mRNA in WBC. In vitro, we found that SAA1 contributes to inflammation via TLR4 activation that releases inflammatory cytokines, which in turn increases SAA1 levels, establishing a positive proinflammatory loop. In vivo, post-TBI treatment with the TLR4-antagonist TAK242 reduces SAA1 levels, improves neurobehavioral outcome, and prevents blood-brain barrier disruption. Our data support further evaluation of (i) post-TBI treatment in the presence of TLR4 inhibition for limiting TBI-induced damage and (ii) SAA1-TLR4 as a biomarker of injury progression in TBI patients.

The use of transcranial motor-evoked potentials, somatosensory-evoked potentials and free-run electromyography for proper placement of paddle leads in chronic pain.

Introduction: As an alternative to those patients who cannot be performed an awake spinal cord stimulation (SCS) or had been percutaneously implanted with poor pain relief outcomes, neurophysiological monitoring through transcranial motor evoked potentials (MEPs), somatosensory-evoked potentials (SSEPs) and free-run electromyography (EMG) under general anesthesia allows the correct placement of surgical leads and provide objective responses.Methods: An initial series of 15 patients undergoing SCS implantation for chronic pain. Physiologic midline was determined with 32-channel NIM-Eclipse System equipment. During neurophysiological monitoring, MEPs, SSEPs, EMG and CMAPs were recorded.Results: MEPs, SSEPs, and EMG were able to target spinal cord physiological midline during SCS to all patients. Physiologic midline was deviated in 53% patients. No warning events in SSEPs, MEPs, or EMG were recorded in any patient.Conclusions: Bilateral CMAPs recording allows placement of paddle leads in physiological midline, obtaining an accurate coverage, pain relief and avoid unpleasant or ineffective stimulation postoperatively. While these neurophysiological techniques are generally used to provide information on the state of the nervous system and prevent neurological injury risks during SCS, our work has shown that can accurate direct lead placement.

Axonal and Myelin Neuroprotection by the Peptoid BN201 in Brain Inflammation.

The development of neuroprotective therapies is a sought-after goal. By screening combinatorial chemical libraries using in vitro assays, we identified the small molecule BN201 that promotes the survival of cultured neural cells when subjected to oxidative stress or when deprived of trophic factors. Moreover, BN201 promotes neuronal differentiation, the differentiation of precursor cells to mature oligodendrocytes in vitro, and the myelination of new axons. BN201 modulates several kinases participating in the insulin growth factor 1 pathway including serum-glucocorticoid kinase and midkine, inducing the phosphorylation of NDRG1 and the translocation of the transcription factor Foxo3 to the cytoplasm. In vivo, BN201 prevents axonal and neuronal loss, and it promotes remyelination in models of multiple sclerosis, chemically induced demyelination, and glaucoma. In summary, we provide a new promising strategy to promote neuroaxonal survival and remyelination, potentially preventing disability in brain diseases.

Introduction of macarpine as a novel cell-permeant DNA dye for live cell imaging and flow cytometry sorting.

BACKGROUND INFORMATION: Macarpine (MA) is a quaternary benzophenanthridine plant alkaloid isolated from Macleaya microcarpa or Stylophorum lasiocarpum. Benzophenanthridine alkaloids are interesting natural products that display antiproliferative, antimicrobial, antifungal and anti-inflammatory activities, and also fluorescence properties. In a previous study, we demonstrated that thanks to its ability to interact with DNA and its spectral properties MA could be used as a supravital DNA probe for fluorescence microscopy and flow cytometry including analyses of the cell cycle. In this study, we evaluated the suitability of MA as a DNA dye for time-lapse microscopy and flow-cytometric cell sorting. RESULTS: Living A-375 and MEF cells stained with MA were monitored by time-lapse microscopy for 24 h. Mitoses were observed at MA concentrations up to 0.5 µg/ml during the first 2-3 h. After this period of time, cells treated with MA at concentrations of 0.75 and 0.5 µg/ml underwent apoptosis. Cells cultivated with MA at concentration of 0.25 µg/ml or lower survived throughout the 24 h period. Toxicity of MA was dependent on light wavelength and frequency of image capturing. The intensity of MA fluorescence decreased during the incubation. MA concentration of 0.1 µg/ml was identified as the most suitable for live cell imaging with respect to fluorescence intensity and toxicity. MA at the concentration 10 µg/ml was used for sorting of enhanced green fluorescent protein (EGFP)-labelled neurons and fibroblasts yielding profiles similar to those obtained with DRAQ5. Contrary to DRAQ5, MA-stained cells survived in culture, and the sorted cells lost the MA signal suggesting reversible binding of the dye to the DNA. CONCLUSION: The results proved that MA may readily be used for chromosomes depicting and mitosis monitoring by time-lapse microscopy. In addition, MA has shown to be a suitable probe for sorting of EGFP-labelled cells, including neurons, that survived the labelling process. SIGNIFICANCE: In consideration of the results, we highly anticipate an onward use of MA in a broad range of applications based on live cell sorting and imaging, for example, cell synchronisation and monitoring of proliferation as an important experimental and/or diagnostic utility.

Neuronal injury external to the retina rapidly activates retinal glia, followed by elevation of markers for cell cycle re-entry and death in retinal ganglion cells.

Retinal ganglion cells (RGCs) are neurons that relay visual signals from the retina to the brain. The RGC cell bodies reside in the retina and their fibers form the optic nerve. Full transection (axotomy) of the optic nerve is an extra-retinal injury model of RGC degeneration. Optic nerve transection permits time-kinetic studies of neurodegenerative mechanisms in neurons and resident glia of the retina, the early events of which are reported here. One day after injury, and before atrophy of RGC cell bodies was apparent, glia had increased levels of phospho-Akt, phospho-S6, and phospho-ERK1/2; however, these signals were not detected in injured RGCs. Three days after injury there were increased levels of phospho-Rb and cyclin A proteins detected in RGCs, whereas these signals were not detected in glia. DNA hyperploidy was also detected in RGCs, indicative of cell cycle re-entry by these post-mitotic neurons. These events culminated in RGC death, which is delayed by pharmacological inhibition of the MAPK/ERK pathway. Our data show that a remote injury to RGC axons rapidly conveys a signal that activates retinal glia, followed by RGC cell cycle re-entry, DNA hyperploidy, and neuronal death that is delayed by preventing glial MAPK/ERK activation. These results demonstrate that complex and variable neuro-glia interactions regulate healthy and injured states in the adult mammalian retina.

Lateral inhibition and neurogenesis: novel aspects in motion.

Neuronal production in metazoans is tightly controlled by Delta/Notch-dependent signals regulating lateral inhibition. It is currently thought that lateral inhibition takes place in clusters of precursors with equal capacity to trigger and receive Notch-dependent inhibitory signals. However, this view neglects crucial dynamical aspects of the process. In this review, we discuss two of these dynamic factors, whose alterations yield dysfunctions in neurogenesis. First, precursors show variable neurogenic capacity as they go through the cell cycle. Second, differentiating precursors are in direct contact with non-neurogenic cells at the wavefront of expanding neurogenic domains. We discuss the mechanisms adopted by Metazoa to prevent these dysfunctions in the lateral inhibitory process, which include cell cycle synchronization occurring in the invertebrate neural epithelium and during primary neurogenesis in anamniotes, interkinetic nuclear movement in the vertebrate neuroepithelium and generalized Delta expression ahead of the neurogenic wavefront. The emerging concept is that lateral inhibition during neurogenesis occurs in dynamic clusters of precursors and requires specific mechanisms to avoid distortions resulting from the interaction between neurogenic and non-neurogenic precursors. The advance in visualizing Notch dynamics with real-time imaging at cellular and subcellular levels will notably contribute to our understanding of these novel "aspects of motion" in neurogenesis.

Regulation of neuronal differentiation at the neurogenic wavefront.

Signaling mediated by the Delta/Notch system controls the process of lateral inhibition, known to regulate neurogenesis in metazoans. Lateral inhibition takes place in equivalence groups formed by cells having equal capacity to differentiate, and it results in the singling out of precursors, which subsequently become neurons. During normal development, areas of active neurogenesis spread through non-neurogenic regions in response to specific morphogens, giving rise to neurogenic wavefronts. Close contact of these wavefronts with non-neurogenic cells is expected to affect lateral inhibition. Therefore, a mechanism should exist in these regions to prevent disturbances of the lateral inhibitory process. Focusing on the developing chick retina, we show that Dll1 is widely expressed by non-neurogenic precursors located at the periphery of this tissue, a region lacking Notch1, lFng, and differentiation-related gene expression. We investigated the role of this Dll1 expression through mathematical modeling. Our analysis predicts that the absence of Dll1 ahead of the neurogenic wavefront results in reduced robustness of the lateral inhibition process, often linked to enhanced neurogenesis and the presence of morphological alterations of the wavefront itself. These predictions are consistent with previous observations in the retina of mice in which Dll1 is conditionally mutated. The predictive capacity of our mathematical model was confirmed further by mimicking published results on the perturbation of morphogenetic furrow progression in the eye imaginal disc of Drosophila. Altogether, we propose that Notch-independent Delta expression ahead of the neurogenic wavefront is required to avoid perturbations in lateral inhibition and wavefront progression, thus optimizing the neurogenic process.

Neurotrophin receptors TrkA and TrkC cause neuronal death whereas TrkB does not.

Neurons of the peripheral nervous system have long been known to require survival factors to prevent their death during development. But why they selectively become dependent on secretory molecules has remained a mystery, as is the observation that in the central nervous system, most neurons do not show this dependency. Using engineered embryonic stem cells, we show here that the neurotrophin receptors TrkA and TrkC (tropomyosin receptor kinase A and C, also known as Ntrk1 and Ntrk3, respectively) instruct developing neurons to die, both in vitro and in vivo. By contrast, TrkB (also known as Ntrk2), a closely related receptor primarily expressed in the central nervous system, does not. These results indicate that TrkA and TrkC behave as dependence receptors, explaining why developing sympathetic and sensory neurons become trophic-factor-dependent for survival. We suggest that the expansion of the Trk gene family that accompanied the segregation of the peripheral from the central nervous system generated a novel mechanism of cell number control.

Somatic tetraploidy in vertebrate neurons: Implications in physiology and pathology.

The presence of polyploid neurons in the vertebrate nervous system has been a subject of debate since the 1960s. At that time, Purkinje cells were proposed to be tetraploid, but technical limitations impeded to reach a clear conclusion, and the current belief is that most vertebrate neurons are diploid. By using up-to-date approaches we have recently demonstrated the existence of a subpopulation of tetraploid retinal ganglion cells (RGCs) in the vertebrate retina. In the chick, these neurons show large somas and extensive dendritic trees and most of them express a marker specific for RGCs innervating a specific lamina of the optic tectum. We have also demonstrated that these neurons are generated in response to nerve growth factor (NGF) acting through the neurotrophin receptor p75 (p75(NTR)), which induces E2F1 activity and cell cycle re-entry in migrating RGC neuroblasts lacking retinoblastoma (Rb) protein. We have also showed that brain-derived neurotrophic factor (BDNF) prevents G(2)/M transition in the tetraploid RGCs, thus being crucial for the maintenance of the tetraploid status as well as the survival of these neurons. The realization that tetraploid neurons can be readily observed in the vertebrate nervous system has important physiological consequences, which are discussed in this commentary.

Cell cycle control of Notch signaling and the functional regionalization of the neuroepithelium during vertebrate neurogenesis.

A critical feature of vertebrate neural precursors is the to-and-fro displacement of their nuclei as cell cycle progresses, thus giving rise to a pseudostratified epithelium. This nuclear behavior, referred to as interkinetic nuclear migration (INM), is translated into the disposition of the cell somas at different orthogonal levels depending on the cell cycle stage they are. The finding that important regulators of neurogenesis, such as the proneural and neurogenic genes, undergo cyclic changes of expression and function in coordination with the cell cycle and the INM, and that the neurogenic process correlates with a particular window of the cell cycle, in coincidence with the apical localization in the neuroepithelium of neural precursors, is a novel concept that facilitates our understanding of the neurogenic process in vertebrates. As such, recent data support the notion that the three-dimensional structure of the neuroepithelium is crucial for proper neuronal production. In this review, we describe current knowledge of the molecular mechanisms involved in the differential expression and function of the proneural and neurogenic gene products along the cell cycle, and we discuss important consequences for vertebrate neurogenesis derived from this observation.

Instability of Notch1 and Delta1 mRNAs and reduced Notch activity in vertebrate neuroepithelial cells undergoing S-phase.

Vertebrate neurogenesis is controlled through lateral inhibitory signals triggered by the Notch receptor and its ligand Delta. In the E4 chick embryo, the capacity of neural precursors to express the neurogenic genes Notch1 and Delta1 becomes reduced during S-phase, suggesting that their competence to trigger lateral inhibitory signals varies at different stages of the cell cycle. Here we show that the reduction of neurogenic gene expression during S-phase is extensive to later developmental stages and to other species; and it correlates with lower expression of lunatic Fringe and diminished capability to induce the expression of cHairy1/Hes1 and Hes5-1. We also show that the cell cycle-dependence of Notch1 and Delta1 expression is due to a remarkable decrease of mRNA stability during S-phase. These results provide evidence that the capacity of vertebrate neural precursors to express neurogenic genes and trigger lateral inhibitory signals is functionally coordinated with the cell cycle.

Single mage gene in the chicken genome encodes CMage, a protein with functional similarities to mammalian type II Mage proteins.

In mammals, the type II melanoma antigen (Mage) protein family is constituted by at least 10 closely related members that are expressed in different tissues, including the nervous system. These proteins are believed to regulate cell cycle withdrawal, neuronal differentiation, and apoptosis. However, the analysis of their specific function has been complicated by functional redundancy. In accordance with previous studies in teleosts and Drosophila, we present evidence that only one mage gene exists in genomes from protists, fungi, plants, nematodes, insects, and nonmammalian vertebrates. We have identified the chicken mage gene and cloned the cDNA encoding the chick Mage protein (CMage). CMage shares close homology with the type II Mage protein family, and, as previously shown for the type II Mage proteins Necdin and Mage-G1, it can interact with the transcription factor E2F-1. CMage is expressed in specific regions of the developing nervous system including the retinal ganglion cell layer, the ventral horn of the spinal cord, and the dorsal root ganglia, coinciding with the expression of the neurotrophin receptor p75 (p75(NTR)) in these regions. We show that the intracellular domain of p75(NTR) can interact with both CMage and Necdin, thus preventing the binding of the latter proteins to the transcription factor E2F-1, and facilitating the proapoptotic activity of E2F-1 in N1E-115 differentiating neurons. The presence of a single mage gene in the chicken genome, together with the close functional resemblance between CMage and Necdin, makes this species ideal to further analyze signal transduction through type II Mage proteins.

Mitochondrial c-Jun NH2-terminal kinase prevents the accumulation of reactive oxygen species and reduces necrotic damage in neural tumor cells that lack trophic support.

In response to different stress signals, the c-Jun NH(2)-terminal kinase (JNK) can trigger cell death. However, JNK also facilitates the survival and cell cycle progression of tumor cells by mechanisms that are poorly defined. Here, we show that schwannoma RN22 cells can survive and proliferate under serum-free conditions although serum withdrawal rapidly induces mitochondrial fission and swelling. Although the morphologic changes observed in the mitochondria did not trigger cytochrome c release, they were accompanied by an increase in the mitochondrial membrane potential (DeltaPsi(M)) and of immunoreactivity for active JNK in these organelles. Pharmacologic inhibition of JNK provoked a further increase of the DeltaPsi(M), an increase in reactive oxygen species (ROS) production, and a sustained decrease in cell viability due to necrosis. This increase in necrosis was prevented by the presence of ROS scavengers. Immunoreactivity for active JNK was also observed in the mitochondria of neuroblastoma 1E-115 and neuroblastoma 2a neuroblastoma cell lines on serum withdrawal, whereas active JNK was barely detected in serum-deprived fibroblasts. Accordingly, the reduction in neural tumor cell viability induced by JNK inhibition was largely attenuated in serum-deprived fibroblasts. These data indicate that local activation of JNK in the mitochondria can protect against necrotic cell death associated with ROS production, facilitating the growth of neural tumor cells subjected to serum deprivation.

Interkinetic nuclear movement may provide spatial clues to the regulation of neurogenesis.

During the transition from S phase to mitosis, vertebrate neuroepithelial cells displace their nuclei and subsequently migrate from the basal membrane to the apical surface of the neuroepithelium, a phenomenon termed interkinetic nuclear movement (INM). Here we provide evidence that cycling neuroepithelial cells pass through a neurogenic state in which they are situated apically, as defined by the capacity to express Notch1, Delta1, and Neurogenin2 (Ngn2). Based on this scenario, we have developed a mathematical model to analyze the influence of INM on neurogenesis. In the absence of INM, the model predicted an increase in the rate of neurogenesis due to the reduction in the influence of inhibitory signals on cells in the neurogenic state. This exacerbation in neurogenesis led to the diminished growth of the neuroepithelium and a reduction in the later production of neurons. Pharmacological perturbation of the stereotypical distribution of precursors along the orthogonal axis of the neuroepithelium resulted in an excess of neurogenesis, as seen by the expression of Ngn2, and of the neuronal marker RA4 in the retina. These findings suggest that INM might be important for the efficient and continued production of neurons in G0, since it is involved in defining a proneural cluster in the ventricular part of the neuroepithelium that contains precursors at stages of the mitotic cycle compatible with neuronal differentiation.

Interkinetic nuclear movement in the vertebrate neuroepithelium: encounters with an old acquaintance.

The vertebrate neuroepithelium is a highly efficient structure with respect to the process of neurogenesis. The orthogonal arrangement of the nuclei with respect to the surface of the epithelium facilitates the dramatic increase in cell density necessary to produce a high number of neurons. Moreover, the spatial organization of the neuroepithelium reflects the segregation of cells that are transiently embarked upon distinct functions, thereby avoiding any interference between these populations in terms of their physiological activities. Two main regions can be distinguished: an apical neurogenetic zone, where lateral inhibitory signals and neurogenic gene expression can be observed, and a basally located pre-neurogenetic zone, which contains cells replicating their DNA and able to receive the signals that will modify their fate.