Combined Exposure to Simulated Microgravity and Acute or Chronic Radiation Reduces Neuronal Network Integrity and Survival.

During orbital or interplanetary space flights, astronauts are exposed to cosmic radiations and microgravity. However, most earth-based studies on the potential health risks of space conditions have investigated the effects of these two conditions separately. This study aimed at assessing the combined effect of radiation exposure and microgravity on neuronal morphology and survival in vitro. In particular, we investigated the effects of simulated microgravity after acute (X-rays) or during chronic (Californium-252) exposure to ionizing radiation using mouse mature neuron cultures. Acute exposure to low (0.1 Gy) doses of X-rays caused a delay in neurite outgrowth and a reduction in soma size, while only the high dose impaired neuronal survival. Of interest, the strongest effect on neuronal morphology and survival was evident in cells exposed to microgravity and in particular in cells exposed to both microgravity and radiation. Removal of neurons from simulated microgravity for a period of 24 h was not sufficient to recover neurite length, whereas the soma size showed a clear re-adaptation to normal ground conditions. Genome-wide gene expression analysis confirmed a modulation of genes involved in neurite extension, cell survival and synaptic communication, suggesting that these changes might be responsible for the observed morphological effects. In general, the observed synergistic changes in neuronal network integrity and cell survival induced by simulated space conditions might help to better evaluate the astronaut's health risks and underline the importance of investigating the central nervous system and long-term cognition during and after a space flight.

Identification of novel radiation-induced p53-dependent transcripts extensively regulated during mouse brain development.

Ionizing radiation is a potent activator of the tumor suppressor gene p53, which itself regulates the transcription of genes involved in canonical pathways such as the cell cycle, DNA repair and apoptosis as well as other biological processes like metabolism, autophagy, differentiation and development. In this study, we performed a meta-analysis on gene expression data from different in vivo and in vitro experiments to identify a signature of early radiation-responsive genes which were predicted to be predominantly regulated by p53. Moreover, we found that several genes expressed different transcript isoforms after irradiation in a p53-dependent manner. Among this gene signature, we identified novel p53 targets, some of which have not yet been functionally characterized. Surprisingly, in contrast to genes from the canonical p53-regulated pathways, our gene signature was found to be highly enriched during embryonic and post-natal brain development and during in vitro neuronal differentiation. Furthermore, we could show that for a number of genes, radiation-responsive transcript variants were upregulated during development and differentiation, while radiation non-responsive variants were not. This suggests that radiation exposure of the developing brain and immature cortical neurons results in the p53-mediated activation of a neuronal differentiation program. Overall, our results further increase the knowledge of the radiation-induced p53 network of the embryonic brain and provide more evidence concerning the importance of p53 and its transcriptional targets during mouse brain development.

MorphoNeuroNet: an automated method for dense neurite network analysis.

High content cell-based screens are rapidly gaining popularity in the context of neuronal regeneration studies. To analyze neuronal morphology, automatic image analysis pipelines have been conceived, which accurately quantify the shape changes of neurons in cell cultures with non-dense neurite networks. However, most existing methods show poor performance for well-connected and differentiated neuronal networks, which may serve as valuable models for inter alia synaptogenesis. Here, we present a fully automated method for quantifying the morphology of neurons and the density of neurite networks, in dense neuronal cultures, which are grown for more than 10 days. MorphoNeuroNet, written as a script for ImageJ, Java based freeware, automatically determines various morphological parameters of the soma and the neurites (size, shape, starting points, and fractional occupation). The image analysis pipeline consists of a multi-tier approach in which the somas are segmented by adaptive region growing using nuclei as seeds, and the neurites are delineated by a combination of various intensity and edge detection algorithms. Quantitative comparison showed a superior performance of MorphoNeuroNet to existing analysis tools, especially for revealing subtle changes in thin neurites, which have weak fluorescence intensity compared to the rest of the network. The proposed method will help determining the effects of compounds on cultures with dense neurite networks, thereby boosting physiological relevance of cell-based assays in the context of neuronal diseases.

Morphological and physiological changes in mature in vitro neuronal networks towards exposure to short-, middle- or long-term simulated microgravity.

One of the objectives of the current international space programmes is to investigate the possible effects of the space environment on the crew health. The aim of this work was to assess the particular effects of simulated microgravity on mature primary neuronal networks and specially their plasticity and connectivity. For this purpose, primary mouse neurons were first grown for 10 days as a dense network before being placed in the Random Positioning Machine (RPM), simulating microgravity. These cultures were then used to investigate the impact of short- (1 h), middle- (24 h) and long-term (10 days) exposure to microgravity at the level of neurite network density, cell morphology and motility as well as cytoskeleton properties in established two-dimensional mature neuronal networks. Image processing analysis of dense neuronal networks exposed to simulated microgravity and their subsequent recovery under ground conditions revealed different neuronal responses depending on the duration period of exposure. After short- and middle-term exposures to simulated microgravity, changes in neurite network, neuron morphology and viability were observed with significant alterations followed by fast recovery processes. Long exposure to simulated microgravity revealed a high adaptation of single neurons to the new gravity conditions as well as a partial adaptation of neuronal networks. This latter was concomitant to an increase of apoptosis. However, neurons and neuronal networks exposed for long-term to simulated microgravity required longer recovery time to re-adapt to the ground gravity. In conclusion, a clear modulation in neuronal plasticity was evidenced through morphological and physiological changes in primary neuronal cultures during and after simulated microgravity exposure. These changes were dependent on the duration of exposure to microgravity.

Non-conventional apoptotic response to ionising radiation mediated by N-methyl D-aspartate receptors in immature neuronal cells.

During cortical development, N-methyl D-aspartate (NMDA) receptors are highly involved in neuronal maturation and synapse establishment. Their implication in the phenomenon of excitotoxicity has been extensively described in several neurodegenerative diseases due to the permissive entry of Ca2+ ions and massive accumulation in the intracellular compartment, which is highly toxic to cells. Ionising radiation is also a source of stress to the cells, particularly immature neurons. Their capacity to induce cell death has been described for various cell types either by directly damaging the DNA or indirectly through the generation of reactive oxygen species responsible for the activation of a battery of stress response effectors leading in certain cases, to cell death. In this study, in order to determine whether a link exists between NMDA receptors-mediated excitotoxicity and radiation-induced cell death, we evaluated radiation-induced cell death in vitro and in vivo in maturing neurons during the fetal period. Cell death induction was assessed by TUNEL, caspase-3 activity and DNA ladder assays, with or without the administration of dizocilpine (MK-801), a non-competitive NMDA receptor antagonist which blocks neuronal Ca2+ influx. To further investigate the possible involvement of Ca2+-dependent enzyme activation, known to occur at high Ca2+ concentrations, we examined the protective effect of a calpain inhibitor on cell death induced by radiation. Doses ranging from 0.2 to 0.6 Gy of X-rays elicited a clear apoptotic response that was prevented by the injection of dizocilpine (MK-801) or calpain inhibitor. These data demonstrate the involvement of NMDA receptors in radiation-induced neuronal death by the activation of downstream effectors, including calpain-related pathways. An increased apoptotic process elicited by radiation, occurring independently of the normal developmental scheme, may eliminate post-mitotic but immature neuronal cells and deeply impair the establishment of the neuronal network, which in the case of cortical development is critical for cognitive capacities.

V-ATPase membrane sector associates with synaptobrevin to modulate neurotransmitter release.

Acidification of synaptic vesicles by the vacuolar proton ATPase is essential for loading with neurotransmitter. Debated findings have suggested that V-ATPase membrane domain (V0) also contributes to Ca(2+)-dependent transmitter release via a direct role in vesicle membrane fusion, but the underlying mechanisms remain obscure. We now report a direct interaction between V0 c-subunit and the v-SNARE synaptobrevin, constituting a molecular link between the V-ATPase and SNARE-mediated fusion. Interaction domains were mapped to the membrane-proximal domain of VAMP2 and the cytosolic 3.4 loop of c-subunit. Acute perturbation of this interaction with c-subunit 3.4 loop peptides did not affect synaptic vesicle proton pump activity, but induced a substantial decrease in neurotransmitter release probability, inhibiting glutamatergic as well as cholinergic transmission in cortical slices and cultured sympathetic neurons, respectively. Thus, V-ATPase may ensure two independent functions: proton transport by a fully assembled V-ATPase and a role in SNARE-dependent exocytosis by the V0 sector.