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Effects of rapamycin and 3-MA on caspase-3 activity and Bax expression in ganglionic eminences from E15.5 Gad67–GFP mice. (A–C) Photographs visualizing the LC3 immunoreactivity and the fluorescein isothiocyanate N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (FITC-VAD-FMK) signal in ganglionic eminences after a 6-h culture in control, 200 nM rapamycin, and 30 mM 3-MA conditions. Note that the diffuse pattern of LC3 immunolabeling is associated with a low caspase activity. (D) Quantification by fluorimetry of the effects of 200 nM rapamycin and 30 mM 3-MA on caspase-3 activity. (E) Quantification by Western blot of the effects of 200 nM rapamycin and 30 mM 3-MA on B-cell CLL/lymphoma 2 (BCL2)-associated X protein Bax expression. Each value represents the mean (±SEM) of three independent experiments. **p lt; 0.01 versus control with the unpaired t test.
Source publication
In human neonates, immature GABAergic interneurons are markedly affected by an excitotoxic insult. While in adults the interest of cell transplantation has been demonstrated in several neurological disorders, few data are available regarding the immature brain. The low survival rate
constitutes a strong limitation in the capacity of transplanted ne...
Citations
... Electron microscopy ultrastructural studies were carried out according to standardized protocols (Roux et al., 2014). Briefly, cortices obtained from P15 corpus callosum of neonates previously in utero-exposed to NaCl or ethanol were fixed in a 2% glutaraldehyde solution, postfixed with 1% osmium tetroxide, and embedded in epoxy resin. ...
Le syndrome d’alcoolisation fœtale (SAF) est provoqué par la consommation d’alcool au cours de la grossesse, et constitue l’atteinte la plus sévère des troubles causés par l’alcoolisation fœtale (TCAF). Première cause de retard mental acquis, le SAF se traduit par de multiples manifestations cliniques, et la présence de dysmorphies faciales caractéristiques permet un diagnostic périnatal précoce. Cependant, alors qu’ils pourront développer des atteintes neurodéveloppementales et comportementales, la plupart des enfants exposés in utero à l’alcool ne présentent pas ces anomalies faciales et échappent, de ce fait, à un diagnostic précoce. Les troubles engendrés par l’alcoolisation maternelle seront alors décelés tardivement, lors de la scolarisation. Bien que de multiples études aient démontré une action délétère d’une exposition in utero à l’alcool sur de nombreuses cellules nerveuses, ce n’est que plus récemment qu’un effet sur l’angiogenèse cérébrale a été caractérisé. Ainsi, des travaux réalisés au sein du laboratoire, puis confirmés par d’autres groupes, ont mis en évidence le rôle néfaste d’une alcoolisation maternelle sur l’angiogenèse cérébrale fœtale, engendrant notamment une désorganisation du réseau vasculaire cortical. Par ailleurs, d’autres groupes de recherche ont démontré l’existence d’une subtile interaction entre les vaisseaux et deux types cellulaires, les interneurones GABAergiques et les oligodendrocytes ; les vaisseaux servant de support de migration tangentielle. Ainsi, mon projet de thèse a consisté à rechercher l’impact de l’alcoolisation in utero sur la mise en place vasculo-associée des oligodendrocytes au cours du développement. Ces travaux ont pu être menés par des approches précliniques et, en partie, chez l’humain grâce à une interaction étroite avec plusieurs Services du CHU de Rouen. Les données obtenues chez la Souris confirment la désorganisation vasculaire cérébrale induite par l’alcoolisation maternelle. Toutefois, cette désorganisation observée chez le fœtus et le nouveau-né s’estompe avec l’âge. Au niveau cortical, une interaction entre oligodendrocytes et vaisseaux radiaux a été caractérisée. Alors que les vaisseaux sont désorganisés, l’association oligo-vasculaire n’est pas modifiée par l’alcool. En revanche, l’alcoolisation in utero entraîne un défaut de positionnement des cellules Olig2-positives renforçant l’hypothèse qu’une altération des routes migratoires vasculaires induirait des défauts ou un retard de positionnement cortical. De plus, des études protéiques et immunohistochimiques démontrent des altérations des processus de maturation. Une perturbation du processus de myélinisation est également retrouvée après exposition prénatale à l’alcool au sein du corps calleux et du striatum, deux régions de projection des fibres corticales. Enfin, des atteintes comportementales ont été constatées, traduisant notamment des altérations motrices à long terme. Parallèlement, des études réalisées chez l’homme sur une cohorte de fœtus exposés ou non in utero à l’alcool, démontrent la présence d’interactions oligo vasculaires sur l’ensemble de la surface corticale. De manière comparable aux études réalisées chez la Souris, un défaut de différenciation oligodendrogliale majeur a été observé chez les individus exposés in utero à l’alcool. En conclusion, ces travaux de thèse ont révélé que les oligodendrocytes intégrant le néocortex utilisent les microvaisseaux radiaux. L’alcoolisation in utero désorganise l’orientation radiale des vaisseaux et induit une altération des profils migratoires et de maturation des oligodendrocytes corticaux, associés à un défaut de myélinisation et des troubles moteurs. Ils suggèrent une dysfonction oligovasculaire induite par l’alcool et ouvrent de nouvelles pistes de recherche. En particulier, il serait intéressant de déterminer si une correction des atteintes vasculaires pourrait être en mesure de corriger les atteintes développementales du lignage oligodendrocytaire.
... In other studies, transplanted GABAergic interneuron precursors exhibit marked sensitivity to autophagy within the first 24 h following transplantation. Inhibition of autophagy increases the survival and fraction of neurite-bearing GABAergic interneurons within two weeks post-implantation [41]. While our in vitro analyses focused on the initial post-differentiation phase (3-6 days), our in vivo analyses suggest that the pro-neurogenic effects of basal MKP-1 activity in the dentate gyrus persist into adulthood. ...
Inherited and acquired defects in neurogenesis contribute to neurodevelopmental disorders, dysfunctional neural plasticity, and may underlie pathology in a range of neurodegenerative conditions. Mitogen-activated protein kinases (MAPKs) regulate the proliferation, survival, and differentiation of neural stem cells. While the balance between MAPKs and the family of MAPK dual-specificity phosphatases (DUSPs) regulates axon branching and synaptic plasticity, the specific role that DUSPs play in neurogenesis remains unexplored. In the current study, we asked whether the canonical DUSP, MAP Kinase Phosphatase-1 (MKP-1), influences neural stem cell differentiation and the extent to which DUSP-dependent autophagy is operational in this context. Under basal conditions, Mkp-1 knockout mice generated fewer doublecortin (DCX) positive neurons within the dentate gyrus (DG) characterized by the accumulation of LC3 puncta. Analyses of wild-type neural stem cell (NSC) differentiation in vitro revealed increased Mkp-1 mRNA expression during the initial 24-h period. Notably, Mkp-1 KO NSC differentiation produced fewer Tuj1-positive neurons and was associated with increased expression of the BCL2/adenovirus E1B 19-kD protein-interacting protein 3 (BNIP3) and levels of autophagy. Conversely, Bnip3 knockdown in differentiated Mkp-1 KO NSCs reduced levels of autophagy and increased neuronal yields. These results indicate that MKP-1 exerts a pro-neurogenic bias during a critical window in NSC differentiation by regulating BNIP3 and basal autophagy levels.
... As a PI3K inhibitor, 3-methyladenine (3-MA) has been reported to inhibit the activity of PI3K and block the formation of autophagosomes and autophagic vacuoles (Zhao et al. 2019). Christian et al. (2014) reported that autophagy is inhibited in the HTC11 human colon cancer cell line after 5 mM 3-MA exposure for 48 h. Autophagy is inhibited in the nerve cells of mouse embryos after treatment with 30 mM 3-MA for 6 h (Hou et al. 2016). ...
... Therefore, this study aimed to determine whether autophagy was inhibited in planarians after 3-MA exposure for different concentrations and for different lengths of time; to this end TEM was performed. On the basis of the literature (Christian et al. 2014;Hou et al. 2016;Pang et al. 2019), 5 mM and 10 mM concentrations were tested. Several different exposure times (6 and 18 h) were also selected. ...
As an important PI3K (VPS34) inhibitor, 3-methyladenine (3-MA) can block the formation of autophagic vesicles in animals. Most toxicological studies using 3-MA have shown that 3-MA leads to serious disorders via autophagy suppression in mammals. However, no toxicological research on 3-MA has been performed on individuals undergoing regeneration. The freshwater planarian has powerful regenerative capability, and it can regenerate a new brain in 5 days and undergo complete adult individual remodelling in approximately 14 days. Moreover, it is also an excellent model organism for studies on environmental toxicology due to its high chemical sensitivity and extensive distribution. Here, Dugesia japonica planarians were treated with 3-MA, and the results showed that autophagy was inhibited and Djvps34 expression levels were down-regulated. After exposure to 10 mM 3-MA for 18 h, all the controls showed normal phenotypes, while one-half of the planarians treated with 3-MA showed morphological defects. In most cases, an ulcer appeared in the middle of the body, and a normal phenotype was restored 7 days following 3-MA exposure. During regeneration, disproportionate blastemas with tissue regression were observed. Furthermore, 3-MA treatment suppressed stem cell proliferation in intact and regenerating worms. These findings demonstrate that autophagy is indispensable for tissue homeostasis and regeneration in planarians and that 3-MA treatment is detrimental to planarian regeneration via its effect on the autophagy pathway.
... Morphometric analysis was then validated by the investigator to correct possible artefacts (for example, two fused cells counted as a single object or the presence of fluorescent debris). The analysis was repeated to cover the superficial layers I-IV from the somatosensory cortex in both hemispheres and in three slices per animal [29]. ...
During cortex development, fine interactions between pyramidal cells and migrating GABA neurons are required to orchestrate correct positioning of interneurons, but cellular and molecular mechanisms are not yet clearly understood. Functional and age-specific expression of NMDA receptors by neonate endothelial cells suggests a vascular contribution to the trophic role of glutamate during cortical development. Associating functional and loss-of-function approaches, we found that glutamate stimulates activity of the endothelial proteases MMP-9 and t-PA along the pial migratory route (PMR) and radial cortical microvessels. Activation of MMP-9 was NMDAR-dependent and abrogated in t-PA−/− mice. Time-lapse recordings revealed that glutamate stimulated migration of GABA interneurons along vessels through an NMDAR-dependent mechanism. In Gad67-GFP mice, t-PA invalidation and in vivo administration of an MMP inhibitor impaired positioning of GABA interneurons in superficial cortical layers, whereas Grin1 endothelial invalidation resulted in a strong reduction of the thickness of the pial migratory route, a marked decrease of the glutamate-induced MMP-9-like activity along the PMR and a depopulation of interneurons in superficial cortical layers. This study supports that glutamate controls the vessel-associated migration of GABA interneurons by regulating the activity of endothelial proteases. This effect requires endothelial NMDAR and is t-PA-dependent. These neurodevelopmental data reinforce the debate regarding safety of molecules with NMDA-antagonist properties administered to preterm and term neonates.
... The results indicated that the downregulation of HOTTIP induced RCC cell autophagy, while the upregulation of HOTTIP inhibited RCC cell autophagy. Therefore, we added 3-MA (a common autophagy inhibitor) (Roux et al. 2014) or RAPA (a common autophagy inducer) (Kroemer et al. 2010) to regulate autophagy to further verify our results, and we again found that RCC cell proliferation, migration and invasion were significantly reversed. Finally, to explore the specific mechanism through which HOTTIP regulates RCC cell autophagy, we measured the expression of several critical proteins and found that HOTTIP could regulate RCC cell autophagy through the PI3K/Akt/Atg13 signaling pathway. ...
Objective
Renal cell carcinoma (RCC) is the most common malignancy of the urinary system, and it is a serious threat to human health. HOXA transcript at the distal tip (HOTTIP), located at the 5′ end of the HOXA locus, is a long non-coding RNA that has been newly discovered in recent years. It has been reported to promote the development of several types of tumors. Moreover, accumulating evidence has indicated that autophagy plays an important role in tumor cell survival or death. However, whether HOTTIP affects RCC development by regulating autophagy remains unknown.
Methods
In this study, we first measured HOTTIP expression in 42 paired RCC and adjacent non-tumor tissues, as well as in 4 RCC cell lines and 1 normal renal tubular epithelial cell line. Then, we selected 2 RCC cell lines to silence HOTTIP expression and 1 RCC cell line to overexpress HOTTIP, and we measured their proliferation, migration and invasion, as well as autophagy, after pretreatment with an autophagy inhibitor or inducer. In addition, we assessed the growth, metastasis and autophagy of tumors in nude mice and explored the mechanism involved.
Results
The results showed that HOTTIP expression was significantly upregulated in the RCC tissues and cell lines, and it was closely associated with TNM stage, histological grade, lymph node metastasis and patient prognosis. The in vitro and in vivo assays indicated that HOTTIP silencing inhibited RCC cell proliferation, migration and invasion and induced autophagy, and 3-MA (an autophagy inhibitor) reversed these effects. In contrast, HOTTIP overexpression and rapamycin (an autophagy inducer) yielded the opposite results. Further research revealed that HOTTIP modification could affect RCC cell autophagy via the PI3K/Akt/Atg13 signaling pathway.
Conclusions
Our study will help in finding a potential marker for RCC diagnosis and supply a target molecule for RCC treatment.
Prenatal alcohol exposure (PAE) is a major cause of nongenetic mental retardation and can lead to fetal alcohol syndrome (FAS), the most severe manifestation of fetal alcohol spectrum disorder (FASD). FASD infants present behavioral disabilities resulting from neurodevelopmental defects. Both grey and white matter lesions have been characterized and are associated with apoptotic death and/or ectopic migration profiles. In the last decade, it was shown that PAE impairs brain angiogenesis, and the radial organization of cortical microvessels is lost. Concurrently, several studies have reported that tangential migration of oligodendrocyte precursors (OPCs) originating from ganglionic eminences is vascular associated. Because numerous migrating oligodendrocytes enter the developing neocortex, the present study aimed to determine whether migrating OPCs interacted with radial cortical microvessels and whether alcohol-induced vascular impairments were associated with altered positioning and differentiation of cortical oligodendrocytes. Using a 3D morphometric analysis, the results revealed that in both human and mouse cortices, 15 to 40% of Olig2-positive cells were in close association with radial cortical microvessels, respectively. Despite perinatal vascular disorganization, PAE did not modify the vessel association of Olig2-positive cells but impaired their positioning between deep and superficial cortical layers. At the molecular level, PAE markedly but transiently reduced the expression of CNPase and MBP, two differentiation markers of immature and mature oligodendrocytes. In particular, PAE inverted their distribution profiles in cortical layers V and VI and reduced the thickness of the myelin sheath of efferent axons. These perinatal oligo-vascular defects were associated with motor disabilities that persisted in adults. Altogether, the present study provides the first evidence that Olig2-positive cells entering the neocortex are associated with radial microvessels. PAE disorganized the cortical microvasculature and delayed the positioning and differentiation of oligodendrocytes. Although most of these oligovascular defects occurred in perinatal life, the offspring developed long-term motor troubles. Altogether, these data suggest that alcohol-induced oligo-vascular impairments contribute to the neurodevelopmental issues described in FASD.
More than 75 000 man-made chemicals contaminate the environment; many of these have not been tested for toxicities. These chemicals demand quantitative high-throughput screening assays to assess them for causative roles in neurotoxicities, including Parkinson's disease and other neurodegenerative disorders. To facilitate high throughput screening for cytotoxicity to neurons, three human neuronal cellular models were compared: SH-SY5Y neuroblastoma cells, LUHMES conditionally-immortalized dopaminergic neurons, and Neural Stem Cells (NSC) derived from human fetal brain. These three cell lines were evaluated for rapidity and degree of differentiation, and sensitivity to 32 known or candidate neurotoxicants. First, expression of neural differentiation genes was assayed during a 7-day differentiation period. Of the three cell lines, LUHMES showed the highest gene expression of neuronal markers after differentiation. Both in the undifferentiated state and after 7 days of neuronal differentiation, LUHMES cells exhibited greater cytotoxic sensitivity to most of 32 suspected or known neurotoxicants than SH-SY5Y or NSCs. LUHMES cells were also unique in being more susceptible to several compounds in the differentiating state than in the undifferentiated state; including known neurotoxicants colchicine, methyl-mercury (II), and vincristine. Gene expression results suggest that differentiating LUHMES cells may be susceptible to apoptosis because they express low levels of anti-apoptotic genes BCL2 and BIRC5/survivin, whereas SH-SY5Y cells may be resistant to apoptosis because they express high levels of BCL2, BIRC5/survivin, and BIRC3 genes. Thus, LUHMES cells exhibited favorable characteristics for neuro-cytotoxicity screening: rapid differentiation into neurons that exhibit high level expression neuronal marker genes, and marked sensitivity of LUHMES cells to known neurotoxicants. Copyright © 2016 John Wiley & Sons, Ltd.
In neonates, excitotoxicity is a major process involved in hypoxic-ischemic brain lesions, and several research groups have suggested the use of NMDA antagonists for neuroprotection. However, despite their clinical interest, there is more and more evidence suggesting that, in the immature brain, these molecules exert deleterious actions on migrating GABAergic interneurons by suppressing glutamatergic trophic inputs. Consequently, preventing the side effects of NMDA antagonists would be therapeutically useful. Because macroautophagy is involved in the adaptive response to trophic deprivation, the aim of the present study was to investigate the impact of autophagy modulators on the MK801-induced death of immature GABAergic interneurons and to characterize the crosstalk between autophagic and apoptotic mechanisms in this cell type. Ex vivo, using cortical slices from NMRI and Gad67-GFP mice, we show that blockade of the NMDA receptor results in an accumulation of autophagosomes due to the disruption of the autophagic flux. This effect precedes the activation of the mitochondrial apoptotic pathway, and the degeneration of immature GABAergic neurons present in developing cortical layers II-IV and is prevented by 3-MA, an autophagy inhibitor. In contrast, modulators of autophagy (3-MA, rapamycin) do not interfere with the anti-excitotoxic and neuroprotective effect of MK801 observed in deep layers V and VI. In vivo, 3-MA blocks the rapid increase in caspase-3 cleavage induced by the blockade of NMDA receptors and prevents the resulting long-term decrease in Gad67-GFP neurons in layers II-IV. Together, these data suggest that, in the developing cortex, the suppression of glutamatergic inputs through NMDA receptor inhibition results in the impairment of the autophagic flux and the subsequent switch to apoptotic death of immature GABAergic interneurons. The concomitant inhibition of autophagy prevents this pro-apoptotic action of the NMDA blocker and favors the long-term rescue of GABAergic interneurons without interfering with its neuroprotective actions. The use of autophagy modulators in the developing brain would create new opportunities to prevent the side effects of NMDA antagonists used for neuroprotection or anesthesia.
Copyright © 2015. Published by Elsevier Inc.
