SOX2 and SOX9 Expression in Developing Postnatal Opossum (Monodelphis domestica) Cortex.

(1) Background: Central nervous system (CNS) development is characterized by dynamic changes in cell proliferation and differentiation. Key regulators of these transitions are the transcription factors such as SOX2 and SOX9. SOX2 is involved in the maintenance of progenitor cell state and neural stem cell multipotency, while SOX9, expressed in neurogenic niches, plays an important role in neuron/glia switch with predominant expression in astrocytes in the adult brain. (2) Methods: To validate SOX2 and SOX9 expression patterns in developing opossum (Monodelphis domestica) cortex, we used immunohistochemistry (IHC) and the isotropic fractionator method on fixed cortical tissue from comparable postnatal ages, as well as dissociated primary neuronal cultures. (3) Results: Neurons positive for both neuronal (TUJ1 or NeuN) and stem cell (SOX2) markers were identified, and their presence was confirmed with all methods and postnatal age groups (P4-6, P6-18, and P30) analyzed. SOX9 showed exclusive staining in non-neuronal cells, and it was coexpressed with SOX2. (4) Conclusions: The persistence of SOX2 expression in developing cortical neurons of M. domestica during the first postnatal month implies the functional role of SOX2 during neuronal differentiation and maturation, which was not previously reported in opossums.

Label-Free Long-Term Methods for Live Cell Imaging of Neurons: New Opportunities.

Time-lapse light microscopy combined with in vitro neuronal cultures has provided a significant contribution to the field of Developmental Neuroscience. The establishment of the neuronal polarity, i.e., formation of axons and dendrites, key structures responsible for inter-neuronal signaling, was described in 1988 by Dotti, Sullivan and Banker in a milestone paper that continues to be cited 30 years later. In the following decades, numerous fluorescently labeled tags and dyes were developed for live cell imaging, providing tremendous advancements in terms of resolution, acquisition speed and the ability to track specific cell structures. However, long-term recordings with fluorescence-based approaches remain challenging because of light-induced phototoxicity and/or interference of tags with cell physiology (e.g., perturbed cytoskeletal dynamics) resulting in compromised cell viability leading to cell death. Therefore, a label-free approach remains the most desirable method in long-term imaging of living neurons. In this paper we will focus on label-free high-resolution methods that can be successfully used over a prolonged period. We propose novel tools such as scanning ion conductance microscopy (SICM) or digital holography microscopy (DHM) that could provide new insights into live cell dynamics during neuronal development and regeneration after injury.

An update to pain management after spinal cord injury: from pharmacology to circRNAs.

Neuropathic pain (NP) following a spinal cord injury (SCI) is often hard to control and therapies should be focused on the physical, psychological, behavioral, social, and environmental factors that may contribute to chronic sensory symptoms. Novel therapeutic treatments for NP management should be based on the combination of pharmacological and nonpharmacological options. Some of them are addressed in this review with a focus on mechanisms and novel treatments. Several reports demonstrated an aberrant expression of non-coding RNAs (ncRNAs) that may represent key regulatory factors with a crucial role in the pathophysiology of NP and as potential diagnostic biomarkers. This review analyses the latest evidence for cellular and molecular mechanisms associated with the role of circular RNAs (circRNAs) in the management of pain after SCI. Advantages in the use of circRNA are their stability (up to 48 h), and specificity as sponges of different miRNAs related to SCI and nerve injury. The present review discusses novel data about deregulated circRNAs (up or downregulated) that sponge miRNAs, and promote cellular and molecular interactions with mRNAs and proteins. This data support the concept that circRNAs could be considered as novel potential therapeutic targets for NP management especially after spinal cord injuries.

The Potential Connection between Molecular Changes and Biomarkers Related to ALS and the Development and Regeneration of CNS.

Neurodegenerative diseases are one of the greatest medical burdens of the modern age, being mostly incurable and with limited prognostic and diagnostic tools. Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the loss of motoneurons, with a complex etiology, combining genetic, epigenetic, and environmental causes. The neuroprotective therapeutic approaches are very limited, while the diagnostics rely on clinical examination and the exclusion of other diseases. The recent advancement in the discovery of molecular pathways and gene mutations involved in ALS has deepened the understanding of the disease pathology and opened the possibility for new treatments and diagnostic procedures. Recently, 15 risk loci with distinct genetic architectures and neuron-specific biology were identified as linked to ALS through common and rare variant association analyses. Interestingly, the quantity of related proteins to these genes has been found to change during early postnatal development in mammalian spinal cord tissue (opossum Monodelphis domestica) at the particular time when neuroregeneration stops being possible. Here, we discuss the possibility that the ALS-related genes/proteins could be connected to neuroregeneration and development. Moreover, since the regulation of gene expression in developmental checkpoints is frequently regulated by non-coding RNAs, we propose that studying the changes in the composition and quantity of non-coding RNA molecules, both in ALS patients and in the developing central nervous (CNS) system of the opossum at the time when neuroregeneration ceases, could reveal potential biomarkers useful in ALS prognosis and diagnosis.

The Role of ATF3 in Neuronal Differentiation and Development of Neuronal Networks in Opossum Postnatal Cortical Cultures.

Activating transcription factor 3 (ATF3), a member of the ATF/cAMP response element-binding (CREB) family, is upregulated by various intracellular and extracellular signals such as injury and signals related to cell proliferation. ATF3 also belongs to the regeneration-associated genes (RAG) group of transcription factors. RAG and ATF/CREB transcription factors that play an important role in embryonic neuronal development and PNS regeneration may also be involved in postnatal neuronal differentiation and development, as well as in the regeneration of the injured CNS. Here we investigated the effect of ATF3 in differentiation, neural outgrowth, network formation, and regeneration after injury using postnatal dissociated cortical neurons derived from neonatal opossums (Monodelphis domestica). Our results show that RAG and ATF genes are differentially expressed in early differentiated neurons versus undifferentiated neurospheres and that many members of those families, ATF3 in particular, are upregulated in cortical cultures obtained from younger animals that have the ability to fully functionally regenerate spinal cord after injury. In addition, we observed different intracellular localization of ATF3 that shifts from nuclear (in neuronal progenitors) to cytoplasmic (in more mature neurons) during neuronal differentiation. The ATF3 inhibition, pharmacological or by specific antibody, reduced the neurite outgrowth and differentiation and caused increased cell death in early differentiating cortical neuronal cultures, suggesting the importance of ATF3 in the CNS development of neonatal opossums. Finally, we investigated the regeneration capacity of primary cortical cultures after mechanical injury using the scratch assay. Remarkably, neonatal opossum-derived cultures retain their capacity to regenerate for up to 1 month in vitro. Inhibition of ATF3 correlates with reduced neurite outgrowth and regeneration after injury. These results indicate that ATF3, and possibly other members of RAG and ATF/CREB family of transcription factors, have an important role both during cortical postnatal development and in response after injury.

Proteomic analysis of opossum Monodelphis domestica spinal cord reveals the changes of proteins related to neurodegenerative diseases during developmental period when neuroregeneration stops being possible.

One of the major challenges of modern neurobiology concerns the inability of the adult mammalian central nervous system (CNS) to regenerate and repair itself after injury. It is still unclear why the ability to regenerate CNS is lost during evolution and development and why it becomes very limited in adult mammals. A convenient model to study cellular and molecular basis of this loss is neonatal opossum (Monodelphis domestica). Opossums are marsupials that are born very immature with the unique possibility to successfully regenerate postnatal spinal cord after injury in the first two weeks of their life, after which this ability abbruptly stops. Using comparative proteomic approach we identified the proteins that are differentially distributed in opossum spinal tissue that can and cannot regenerate after injury, among which stand out the proteins related to neurodegenerative diseases (NDD), such as Huntington, Parkinson and Alzheimer's disease, previously detected by comparative transcriptomics on the analog tissue. The different distribution of the selected proteins detected by comparative proteomics was further confirmed by Western blot (WB), and the changes in the expression of related genes were analysed by quantitative reverse transcription PCR (qRT-PCR). Furthermore, we explored the cellular localization of the selected proteins using immunofluorescent microscopy. To our knowledge, this is the first report on proteins differentially present in developing, non-injured mammalian spinal cord tissue with different regenerative capacities. The results of this study indicate that the proteins known to have an important role in the pathophysiology of neurodegeneration in aged CNS, could also have an important phyisological role during CNS postnatal development and in neuroregeneration process.

Circular RNAs: The Novel Actors in Pathophysiology of Spinal Cord Injury.

Spinal Cord Injury (SCI) can elicit a progressive loss of nerve cells promoting disability, morbidity, and even mortality. Despite different triggering mechanisms, a cascade of molecular events involving complex gene alterations and activation of the neuroimmune system influence either cell damage or repair. Effective therapies to avoid secondary mechanisms underlying SCI are still lacking. The recent progression in circular RNAs (circRNAs) research has drawn increasing attention and opened a new insight on SCI pathology. circRNAs differ from traditional linear RNAs and have emerged as the active elements to regulate gene expression as well as to facilitate the immune response involved in pathophysiology-related conditions. In this review, we focus on the impact and possible close relationship of circRNAs with pathophysiological mechanisms following SCI, where circRNAs could be the key transcriptional regulatory molecules to define neuronal death or survival. Advances in circRNAs research provide new insight on potential biomarkers and effective therapeutic targets for SCI patients.

Establishment of Long-Term Primary Cortical Neuronal Cultures From Neonatal Opossum Monodelphis domestica.

Primary dissociated neuronal cultures have become a standard model for studying central nervous system (CNS) development. Such cultures are predominantly prepared from the hippocampus or cortex of rodents (mice and rats), while other mammals are less used. Here, we describe the establishment and extensive characterization of the primary dissociated neuronal cultures derived from the cortex of the gray South American short-tailed opossums, Monodelphis domestica. Opossums are unique in their ability to fully regenerate their CNS after an injury during their early postnatal development. Thus, we used cortex of postnatal day (P) 3-5 opossum to establish long-surviving and nearly pure neuronal cultures, as well as mixed cultures composed of radial glia cells (RGCs) in which their neurogenic and gliogenic potential was confirmed. Both types of cultures can survive for more than 1 month in vitro. We also prepared neuronal cultures from the P16-18 opossum cortex, which were composed of astrocytes and microglia, in addition to neurons. The long-surviving opossum primary dissociated neuronal cultures represent a novel mammalian in vitro platform particularly useful to study CNS development and regeneration.

Glia in amyotrophic lateral sclerosis and spinal cord injury: common therapeutic targets.

The toolkit for repairing damaged neurons in amyotrophic lateral sclerosis (ALS) and spinal cord injury (SCI) is extremely limited. Here, we reviewed the in vitro and in vivo studies and clinical trials on nonneuronal cells in the neurodegenerative processes common to both these conditions. Special focus was directed to microglia and astrocytes, because their activation and proliferation, also known as neuroinflammation, is a key driver of neurodegeneration. Neuroinflammation is a multifaceted process that evolves during the disease course, and can be either beneficial or toxic to neurons. Given the fundamental regulatory functions of glia, pathogenic mechanisms in neuroinflammation represent promising therapeutic targets. We also discussed neuroprotective, immunosuppressive, and stem-cell based approaches applicable to both ALS and SCI.

Pharmacological induction of Heat Shock Protein 70 by celastrol protects motoneurons from excitotoxicity in rat spinal cord in vitro.

The secondary phase of spinal cord injury arising after the primary lesion largely extends the damage severity with delayed negative consequences for sensory-motor pathways. It is, therefore, important to find out if enhancing intrinsic mechanisms of neuroprotection can spare motoneurons that are very vulnerable cells. This issue was investigated with an in vitro model of rat spinal cord excitotoxicity monitored for up to 24 hr after the primary injury evoked by kainate. This study sought to pharmacologically boost the expression of heat shock proteins (HSP) to protect spinal motoneurons using celastrol to investigate if the rat spinal cord can upregulate HSP as neuroprotective mechanism. Despite its narrow range of drug safety in vitro, celastrol was not toxic to the rat spinal cord at 0.75 µM concentration and enhanced the expression of HSP70 by motoneurons. When celastrol was applied either before or after kainate, the number of dead motoneurons was significantly decreased and the nuclear localization of the cell death biomarker AIF strongly inhibited. Nevertheless, electrophysiological recording showed that protection of lumbar motor networks by celastrol was rather limited as reflex activity was impaired and fictive locomotion largely depressed, suggesting that functional deficit persisted, though the networks could express slow rhythmic oscillations. While our data do not exclude further recovery at later times beyond the experimental observations, the present results indicate that the upregulated expression of HSP in the aftermath of acute injury may be an interesting avenue for early protection of spinal motoneurons.

Loss of inhibitory synapses causes locomotor network dysfunction of the rat spinal cord during prolonged maintenance in vitro.

The isolated spinal cord of the neonatal rat is widely employed to clarify the basic mechanisms of network development or the early phase of degeneration after injury. Nevertheless, this preparation survives in Krebs solution up to 24 h only, making it desirable to explore approaches to extend its survival for longitudinal studies. The present report shows that culturing the spinal cord in oxygenated enriched Basal Medium Eagle (BME) provided excellent preservation of neurons (including motoneurons), glia and primary afferents (including dorsal root ganglia) for up to 72 h. Using DMEM medium was unsuccessful. Novel characteristics of spinal networks emerged with strong spontaneous activity, and deficit in fictive locomotion patterns with stereotypically slow cycles. Staining with markers for synaptic proteins synapsin 1 and synaptophysin showed thoroughly weaker signal after 3 days in vitro. Immunohistochemical staining of markers for glutamatergic and glycinergic neurons indicated significant reduction of the latter. Likewise, there was lower expression of the GABA-synthesizing enzyme GAD65. Thus, malfunction of locomotor networks appeared related to loss of inhibitory synapses. This phenomenon did not occur in analogous opossum preparations of the spinal cord kept in vitro. In conclusion, despite histological data suggesting that cultured spinal cords were undamaged (except for inhibitory biomarkers), electrophysiological data revealed important functional impairment. Thus, the downregulation of inhibitory synapses may account for the progressive hyperexcitability of rat spinal networks despite apparently normal histological appearance. Our observations may help to understand the basis of certain delayed effects of spinal injury like chronic pain and spasticity.

Role of HSP70 in motoneuron survival after excitotoxic stress in a rat spinal cord injury model in vitro.

The outcome for gait recovery from paralysis due to spinal lesion remains uncertain even when damage is limited. One critical factor is the survival of motoneurons, which are very vulnerable cells. To clarify the early pathophysiological mechanisms of spinal damage, an in vitro injury model of the rat spinal cord caused by moderate excitotoxicity was used. With this preparation we investigated whether motoneuron survival was dependent on the expression of the neuroprotective protein HSP70. In the present study excitotoxicity evoked by kainate induced delayed (24 h) loss (35%) of motoneurons, which became pyknotic with translocation of the cell death biomarker apoptosis-inducing factor (AIF) to the nucleus. This process was concomitant with suppression of locomotor network electrical activity. Surviving cells showed strong expression of HSP70 without nuclear AIF. The HSP70 inhibitor VER155008 per se induced neurotoxicity similar to that of kainate, while the HSP90 inhibitor geldanamycin did not damage spinal tissue. Electrophysiological recording following kainate or VER155008 indicated depression of motoneuron field potentials, with decreased excitability and impaired synaptic transmission. When these two drugs were applied together, more intense neurotoxicity emerged. Our data indicate that HSP70 was one important contributor to motoneuron survival and suggest that enhancing HSP70 activity is a potential future strategy for neuroprotecting these cells.

ATF3 is a novel nuclear marker for migrating ependymal stem cells in the rat spinal cord.

The present study identified ATF3 as a novel dynamic marker for ependymal stem/progenitor cells (nestin, vimentin and SOX2 positive) around the central canal of the neonatal or adult rat spinal cord. While quiescent ependymal cells showed cytoplasmic ATF3 expression, during 6-24h in vitro these cells mobilized and acquired intense nuclear ATF3 staining. Their migratory pattern followed a centrifugal pathway toward the dorsal and ventral funiculi, reminiscent of the rostral migratory stream of the brain subventricular stem cells. Thus, the chain cell formation was, by analogy, termed funicular migratory stream (FMS). The FMS process preceded the strong proliferation of ependymal cells occurring only after 24h in vitro. Pharmacological inhibition of MAPK-p38 and JNK/c-Jun (upstream effectors of ATF3 activation) prevented the FMS mobilization of ATF3 nuclear-positive cells. Excitotoxicity or ischemia-like conditions, reported to evoke neuronal and glial injury, did not further enhance migration of ependymal cells at 24h, suggesting that, at this early stage of damage, the FMS phenomenon had peaked and that more extensive repair processes are delayed beyond this time point. ATF3 is, therefore, useful to identify activation and migration of endogenous stem cells of the rat spinal cord in vitro.

Microelectrode arrays in combination with in vitro models of spinal cord injury as tools to investigate pathological changes in network activity: facts and promises.

Microelectrode arrays (MEAs) represent an important tool to study the basic characteristics of spinal networks that control locomotion in physiological conditions. Fundamental properties of this neuronal rhythmicity like burst origin, propagation, coordination, and resilience can, thus, be investigated at multiple sites within a certain spinal topography and neighboring circuits. A novel challenge will be to apply this technology to unveil the mechanisms underlying pathological processes evoked by spinal cord injury (SCI). To achieve this goal, it is necessary to fully identify spinal networks that make up the locomotor central pattern generator (CPG) and to understand their operational rules. In this review, the use of isolated spinal cord preparations from rodents, or organotypic spinal slice cultures is discussed to study rhythmic activity. In particular, this review surveys our recently developed in vitro models of SCI by evoking excitotoxic (or even hypoxic/dysmetabolic) damage to spinal networks and assessing the impact on rhythmic activity and cell survival. These pathological processes which evolve via different cell death mechanisms are discussed as a paradigm to apply MEA recording for detailed mapping of the functional damage and its time-dependent evolution.

Postnatal developmental profile of neurons and glia in motor nuclei of the brainstem and spinal cord, and its comparison with organotypic slice cultures.

In vitro preparations of the neonatal rat spinal cord or brainstem are useful to investigate the organization of motor networks and their dysfunction in neurological disease models. Long-term spinal cord organotypic cultures can extend our understanding of such pathophysiological processes over longer times. It is, however, surprising that detailed descriptions of the type (and number) of neurons and glia in such preparations are currently unavailable to evaluate cell-selectivity of experimental damage. The focus of the present immunohistochemical study is the novel characterization of the cell population in the lumbar locomotor region of the rat spinal cord and in the brainstem motor nucleus hypoglossus at 0-4 postnatal days, and its comparison with spinal organotypic cultures at 2-22 days in vitro. In the nucleus hypoglossus, neurons were 40% of all cells and 80% of these were motoneurons. Astrocytes (35% of total cells) were the main glial cells, while microglia was <10%. In the spinal gray matter, the highest neuronal density was in the dorsal horn (>80%) and the lowest in the ventral horn (≤57%) with inverse astroglia numbers and few microglia. The number of neurons (including motoneurons) and astrocytes was stable after birth. Like in the spinal cord, motoneurons in organotypic spinal culture were <10% of ventral horn cells, with neurons <40%, and the rest made up by glia. The present report indicates a comparable degree of neuronal and glial maturation in brainstem and spinal motor nuclei, and that this condition is also observed in 3-week-old organotypic cultures.

Molecular Mechanisms Underlying Cell Death in Spinal Networks in Relation to Locomotor Activity After Acute Injury in vitro.

Understanding the pathophysiological changes triggered by an acute spinal cord injury is a primary goal to prevent and treat chronic disability with a mechanism-based approach. After the primary phase of rapid cell death at the injury site, secondary damage occurs via autodestruction of unscathed tissue through complex cell-death mechanisms that comprise caspase-dependent and caspase-independent pathways. To devise novel neuroprotective strategies to restore locomotion, it is, therefore, necessary to focus on the death mechanisms of neurons and glia within spinal locomotor networks. To this end, the availability of in vitro preparations of the rodent spinal cord capable of expressing locomotor-like oscillatory patterns recorded electrophysiologically from motoneuron pools offers the novel opportunity to correlate locomotor network function with molecular and histological changes long after an acute experimental lesion. Distinct forms of damage to the in vitro spinal cord, namely excitotoxic stimulation or severe metabolic perturbation (with oxidative stress, hypoxia/aglycemia), can be applied with differential outcome in terms of cell types and functional loss. In either case, cell death is a delayed phenomenon developing over several hours. Neurons are more vulnerable to excitotoxicity and more resistant to metabolic perturbation, while the opposite holds true for glia. Neurons mainly die because of hyperactivation of poly(ADP-ribose) polymerase-1 (PARP-1) with subsequent DNA damage and mitochondrial energy collapse. Conversely, glial cells die predominantly by apoptosis. It is likely that early neuroprotection against acute spinal injury may require tailor-made drugs targeted to specific cell-death processes of certain cell types within the locomotor circuitry. Furthermore, comparison of network size and function before and after graded injury provides an estimate of the minimal network membership to express the locomotor program.

Effects of 6(5H)-phenanthridinone, an inhibitor of poly(ADP-ribose)polymerase-1 activity (PARP-1), on locomotor networks of the rat isolated spinal cord.

Excitotoxicity is considered to be a major pathophysiological mechanism responsible for extensive neuronal death after acute spinal injury. The chief effector of such a neuronal death is thought to be the hyperactivation of intracellular PARP-1 that leads to cell energy depletion and DNA damage with the manifestation of non-apoptotic cell death termed parthanatos. An in vitro lesion model using the neonatal rat spinal cord has recently shown PARP-1 overactivity to be closely related to neuronal losses after an excitotoxic challenge by kainate: in this system the PARP-1 inhibitor 6(5H)-phenanthridinone (PHE) appeared to be a moderate histological neuroprotector. This article investigated whether PHE could actually preserve the function of locomotor networks in vitro from excitotoxicity. Bath-applied PHE (after a 60 min kainate application) failed to recover locomotor network function 24 h later. When the PHE administration was advanced by 30 min (during the administration of kainate), locomotor function could still not be recovered, while basic network rhythmicity persisted. Histochemical analysis showed that, even if the number of surviving neurons was improved with this protocol, it had failed to reach the threshold of minimal network membership necessary for expressing locomotor patterns. These results suggest that PARP-1 hyperactivity was a rapid onset mechanism of neuronal loss after an excitotoxic challenge and that more selective and faster-acting PARP-1 inhibitors are warranted to explore their potential neuroprotective role.

Developmental changes of gene expression after spinal cord injury in neonatal opossums.

Changes in gene expression have been measured 24h after injury to mammalian spinal cords that can and cannot regenerate. In opossums there is a critical period of development when regeneration stops being possible: at 9 days postnatal cervical spinal cords regenerate, at 12 days they do not. By the use of marsupial cDNA microarrays, we detected 158 genes that respond differentially to injury at the two ages critical for regeneration. For selected candidates additional measurements were made by real-time PCR and sites of their expression were shown by immunostaining. Candidate genes have been classified so as to select those that promote or prevent regeneration. Up-regulated by injury at 8 days and/or down-regulated by injury at 13 days were genes known to promote growth, such as Mitogen-activated protein kinase kinase 1 or transcription factor TCF7L2. By contrast, at 13 days, up-regulation occurred of inhibitory molecules, including annexins, ephrins, and genes related to apoptosis and neurodegenerative diseases. Certain genes such as calmodulin 1 and NOGO, changed expression similarly in animals that could and could not regenerate without any additional changes in response to injury. These findings confirmed and extended changes of gene expression found in earlier screens on 9 and 12 ay preparations without lesions and provide a comprehensive list of genes that serve as a basis for testing how identified molecules, singly or in combination, promote and prevent central nervous system regeneration.