Neurons In Developing Brain Research Articles

Anesthetic neurotoxicity and long-term behavior disturbance

Background Anesthesia increases the death rate of neurons in the developing brain,which has been confirmed by a large number of animal experiments.Combined or repeated anesthesia is more likely to result in long-term impairments of brain function.Objective This article reviews the possible mechanisms of anesthetic neurotoxicity and long-term behavior disturbance,and brings together the recent evidences of the neurotoxicity to non-human primates and humans.Content The ideas generally accepted on anesthetic neurotoxicity,the possible mechanisms of anesthetic neurotoxicity and long-term impairments of brain function,and main results of recent studies on anesthetic neurotoxicity to non-human primates and humans.Trend The existing evidences suggest that the influence of anesthesia on the developing brain can not be ignored.Further studies are warranted. Key words: Anesthesia; Neurotoxicity; Long-term behavior

Temporal and spatial expression of preprotachykinin A mRNA in the developing filial mice brain after maternal administration of monosodium glutamate at a late stage of pregnancy

In the early stages of brain development, exposure of excessive monosodium glutamate (MSG) to neurons causes animal functional and behavioral disorders in adulthood. To investigate the effects of excessive MSG during pregnancy on the neurons in the developing brain, in situ hybridization was used. In mice, the expression of preprotachykinin A mRNA (PPT A mRNA) was assessed in neurons of in the brain after MSG treatment. Brain tissue sections were hybridized with specific digoxigenin-labeled RNA probes. The number of cells that expressed PPT A mRNA gradually decreased from 10-day-old (10d) to 60-day-old (60d) MSG-treated and normal animals. In the MSG-treated and normal mice, the PPT A mRNA-positive neurons almost disappeared in 90-day-old (90d) mice. The expression of PPT A mRNA significantly decreased at 10d in most of the brain regions of MSG-treated mice including the cerebral cortex (CC), hippocampal subregions of CA1, CA2 (CA1, CA2), habenula nucleus (HAB), hypothalamic periventricular nucleus (PE), hypothalamic arcuate nucleus (AR), median eminence (ME), amygdala nucleus (AMY), endopiriform nucleus (EN), and hypothalamic ventromedial nucleus (VMH) and dorsomedial nucleus (DMH). In the hippocampal CA4 subregions (CA4), paraventricular nucleus (PV) and caudate putamen (CPU), however, they were not significantly altered. Furthermore, in CC, hippocampal CA3 subregion (CA3), PE and EN regions the number of PPT A mRNA-positive neurons decreased at 20 days old (20d), but increased significantly in CA2 and CPU. At 30 days old (30d), the positive neuron number decreased in AMY, and they did not change in other regions. At 60d, the number of positive neurons significantly decreased in PV and ME, but increased in AMY. In the other observed regions, no changes were found. These results show that maternal administration of excessive MSG at a late stage of pregnancy significantly decreases PPT A mRNA expression in most of the brain regions of filial mice. This suggests that glutamate-induced excitotoxicity may affect the metabolism of precursors of substance P in developing brain neurons. The present study provides insights into the plasticity and vulnerability of neuron in different brain regions to glutamate excitotoxicity.

Role of IL-6 in the Neural Stem Cell Differentiation

In the developing brain neurons, astrocytes, and oligodendrocytes are differentiated from common multipotent progenitors called neural stem cells. We have examined the effect of cytokines on mouse neuroepithelial cells that are known to contain neural stem cells. Cytokines belonging to the interleukin (IL)-6 family and those classified into the bone morphogenetic protein (BMP) family act in synergy on neuroepithelial cells to induce astrocyte differentiation. Cooperation of these two types of cytokines is explained by the formulation of a complex between their respective downstream transcription factors (signal tranducer and activator of transcription [STAT]-3 and Smad1), bridged by a transcriptional coactivator (p300). Whereas BMPs family cytokines are involved in astrocyte differentiation, they inhibit neuronal differentiation. This appears to change the fate of neural progenitor cells from neurogenesis to astrocytogenesis. Interestingly, during brain development, neuronal differentiation starts at a very early stage and continues throughout development, whereas astrocytes appear just before term. We have shown that astrocyte differentiation is largely dependent on IL-6 family cytokine-mediated STAT3 activation and that there exists a STAT3 binding element in the promoter region of the gene for the astrocyte marker glial fibrillary acidic protein. A cytosine residue in this element is highly methylated in neuroepithelial cells in a mid-gestational stage but becomes demethylated in accordance with brain development. Because this methylation inhibits STAT3 binding, we suggest that DNA methylation is a critical determinant in the developmental stage-dependent regulation of astrocytogeneis. In conclusion, fate of neural stem cells during development is regulated by cell-intrinsic programs, such as epigenetic modification (including DNA methylation), and signaling crosstalk of cell-external mediators (including IL-6 family cytokines).

Sequential expression patterns of apoptosis- and cell cycle-related proteins in neuronal response to severe or mild transient hypoxia

Severe hypoxia was shown to induce apoptotic death in developing brain neurons, whereas mild hypoxia was demonstrated to stimulate neurogenesis. Since the apoptotic process may share common pathways with mitosis, expression profiles of proteins involved in apoptosis or the cell cycle were analyzed by immunohistochemistry and/or western blotting, in relation with cell outcome of cultured neurons from fetal rat forebrain subjected to either lethal (6 h) or non-lethal (3 h) hypoxia (95% N 2/5% CO 2). Hypoxia for 6 h led to apoptosis that was inhibited by the cell cycle blocker olomoucine. Transient overexpression of proliferating cell nuclear antigen was followed by increasing expression of p53, p21, Bax and caspases, whereas Bcl-2 and heat shock proteins were progressively repressed. Conversely, a 3-h hypoxic insult initiated neuronal mitosis, with increased thymidine incorporation. In these conditions, levels of proliferating cell nuclear antigen, Rb, Bcl-2 and heat shock proteins were persistently elevated, while expression of p53, p21, Bax and caspases gradually decreased. These data confirm that hypoxia promotes cell cycle activation, whatever the stress intensity. This process is then aborted following apoptosis-inducing hypoxia, whereas sublethal insult would trigger neurogenesis, at least in developing brain neurons in vitro, by stimulating timed expression of neurogenic and survival-associated proteins.

The protein kinase Cdk5

A set of different protein kinases have been involved in tau phosphorylations, including glycogen synthase kinase 3β (GSK3β), MARK kinase, MAP kinase, the cyclin‐dependent kinase 5 (Cdk5) system and others. The latter system include the catalytic component Cdk5 and the regulatory proteins p35, p25 and p39. Cdk5 and its neuron‐specific activator p35 are essential molecules for neuronal migration and for the laminar configuration of the cerebral cortex. Recent evidence that the Cdk5/p35 complex concentrates at the leading edge of axonal growth cones, together with the involvement of this system in the phosphorylation of neuronal microtubule‐asociated proteins (MAPs), provide further support to the role of this protein kinase in regulating axonal extension in developing brain neurons. Although the aminoacid sequence of p35 has little similarity with those of normal cyclins, studies have shown that its activation domain may adopt a conformation of the cyclin‐folded structure. The computed structure for Cdk5 is compatible with experimental data obtained from studies on the Cdk5/p35 complex, and has allowed predictions on the protein interacting domains. This enzyme exhibits a wide cell distribution, even though a regulated Cdk5 activity has been shown only in neuronal cells. Cdk5 has been characterized as a proline‐directed Ser/Thr protein kinase, that contributes to phosphorylation of human tau on Ser202, Thr205, Ser235 and Ser404. Cdk5 is active in postmitiotic neurons, and it has been implicated in cytoskeleton assembly and its organization during axonal growth. In addition to tau and other MAPs, Cdk5 phosphorylates the high molecular weight neurofilament proteins at their C‐terminal domain. Moreover, nestin, a protein that regulates cytoskeleton organization of neuronal and muscular cells during development of early embryos, and several other regulatory proteins appear to be substrates of Cdk5 and are phosphorylated by this kinase. Studies also suggest, that in addition to Cdk5 involvement in neuronal differentiation, its activity is induced during myogenesis, however, the mechanisms of how this activity is regulated during muscular differentiation has not yet been elucidated. Recent studies have shown that the β‐amyloid peptide (Aβ) induces a deregulation of Cdk5 in cultured brain cells, and raises the question on the possible roles of this tau‐phosphorylating protein kinase in the sequence of molecular events leading to neuronal death triggered by Aβ. In this context, there are evidence that Cdk5 is involved in tau hyperphosphorylation promoted by Aβ in its fibrillary form. Cdk5 inhibitors protect hippocampal neurons against both tau anomalous phosphorylations and neuronal death. The links between the studies on the Cdk5/p35 system in normal neurogenesis and its claimed participation in neurodegeneration, provide the framework to understand the regulatory relevance of this kinase system, and changes in its regulation that may be implicated in disturbances such as those occurring in Alzheimer disease.

The protein kinase Cdk5. Structural aspects, roles in neurogenesis and involvement in Alzheimer's pathology.

A set of different protein kinases have been involved in tau phosphorylations, including glycogen synthase kinase 3beta (GSK3 beta), MARK kinase, MAP kinase, the cyclin-dependent kinase 5 (Cdk5) system and others. The latter system include the catalytic component Cdk5 and the regulatory proteins p35, p25 and p39. Cdk5 and its neuron-specific activator p35 are essential molecules for neuronal migration and for the laminar configuration of the cerebral cortex. Recent evidence that the Cdk5/p35 complex concentrates at the leading edge of axonal growth cones, together with the involvement of this system in the phosphorylation of neuronal microtubule-asociated proteins (MAPs), provide further support to the role of this protein kinase in regulating axonal extension in developing brain neurons. Although the aminoacid sequence of p35 has little similarity with those of normal cyclins, studies have shown that its activation domain may adopt a conformation of the cyclin-folded structure. The computed structure for Cdk5 is compatible with experimental data obtained from studies on the Cdk5/p35 complex, and has allowed predictions on the protein interacting domains. This enzyme exhibits a wide cell distribution, even though a regulated Cdk5 activity has been shown only in neuronal cells. Cdk5 has been characterized as a proline-directed Ser/Thr protein kinase, that contributes to phosphorylation of human tau on Ser202, Thr205, Ser235 and Ser404. Cdk5 is active in postmitiotic neurons, and it has been implicated in cytoskeleton assembly and its organization during axonal growth. In addition to tau and other MAPs, Cdk5 phosphorylates the high molecular weight neurofilament proteins at their C-terminal domain. Moreover, nestin, a protein that regulates cytoskeleton organization of neuronal and muscular cells during development of early embryos, and several other regulatory proteins appear to be substrates of Cdk5 and are phosphorylated by this kinase. Studies also suggest, that in addition to Cdk5 involvement in neuronal differentiation, its activity is induced during myogenesis, however, the mechanisms of how this activity is regulated during muscular differentiation has not yet been elucidated. Recent studies have shown that the beta-amyloid peptide (A beta) induces a deregulation of Cdk5 in cultured brain cells, and raises the question on the possible roles of this tau-phosphorylating protein kinase in the sequence of molecular events leading to neuronal death triggered by A beta. In this context, there are evidence that Cdk5 is involved in tau hyperphosphorylation promoted by A beta in its fibrillary form. Cdk5 inhibitors protect hippocampal neurons against both tau anomalous phosphorylations and neuronal death. The links between the studies on the Cdk5/p35 system in normal neurogenesis and its claimed participation in neurodegeneration, provide the framework to understand the regulatory relevance of this kinase system, and changes in its regulation that may be implicated in disturbances such as those occurring in Alzheimer disease.

Does DNA damage sculpt the developing brain?

The death of differentiated neurons during their formation of synaptic connections has been widely documented and forms the basis of the trophic theory of nervous system development, in which an initial overproduction of neurons is followed by the death of neurons that do not establish functional synaptic connections. Recently, it has been shown that many neural precursor cells (NPC) die while still in a proliferative state, before having differentiated into neurons. DNA damage, including double-strand breaks, occurs in NPC during brain development and can either be successfully repaired or can trigger a form of programmed cell death called apoptosis. D'Sa-Eipper and coworkers 1xDNA damage-induced neural precursor cell apoptosis requires p53 and caspase-9 but neither Bax nor caspase-3. D'Sa-Eipper, C. et al. Development. 2001; 128: 137–146PubMedSee all References1 now provide insight into the similarities and differences between the molecular cascades that occur during natural death of NPC and death of the same cells induced by DNA-damaging chemicals and γ-radiation. They performed complementary cell culture and in vivo studies of NPC death in mice lacking proteins that play pivotal roles in cell apoptosis, including the tumor suppressor protein p53, the BCL-2 family members BAX (pro-apoptotic) and BCL-2 and BCL-x (anti-apoptotic) and members of the caspase family of apoptosis effector proteins (caspases 3 and 9). The requirements for death that is induced by the DNA-damaging agent AraC and by γ-radiation were different to those required for naturally occurring death of NPC: death triggered by chemical- and radiation-induced DNA damage required p53 and caspase-9, whereas natural cell death did not.The new findings raise several questions, including is the DNA damage that is observed in NPC undergoing natural cell death a trigger for cell death, or is it merely a consequence of the death process? Recent findings have shown that deficiency of DNA repair enzymes results in excessive apoptosis of NPC in developing mouse embryos 2xCell birth, cell death, cell diversity and DNA breaks: how do they all fit together?. Gilmore, E.C. et al. Trends Neurosci. 2000; 23: 100–105Abstract | Full Text | Full Text PDF | PubMed | Scopus (78)See all References2 suggesting that accumulation of DNA damage is sufficient to trigger developmental death in the nervous system. Does turning off a DNA protection or repair mechanism trigger a different cell-death cascade compared with DNA damage induced by exogenous factors? That DNA damage is necessary for apoptosis of NPC during brain development has been suggested by recent studies of telomerase, an enzyme that adds a six-base DNA repeat onto chromosome ends (telomeres). Levels of telomerase activity dramatically decrease in embryonic mouse cerebral cortex during the time period when death of NPC occurs 3xThe catalytic subunit of telomerase is expressed in developing brain neurons and serves a cell survival-promoting function. Fu, W. et al. J. Mol. Neurosci. 2000; 14: 3–15Crossref | PubMedSee all References3. Telomerase can protect cells against DNA damage-induced apoptosis, consistent with a similar mechanism for promoting the survival of NPC.These studies suggest that there are differences in the biochemical cascades responsible for the neural cell deaths that occur at different stages of development. The differences might be related to aspects of the cell cycle because NPC are mitotic, whereas death associated with synaptogenesis occurs in postmitotic cells. Perhaps differential expression of pro- and anti-apoptotic regulatory proteins accounts for their differential involvement in particular types of developmental cell death. Another possibility is that the stimulus for death of NPC, which is currently unknown, could be quite different from the insufficient neurotrophic factor signaling that might trigger apoptosis in differentiated neurons. Finally, it will be of interest to determine the contributions of DNA damage to neuronal apoptosis in neurodegenerative disorders such as Alzheimer's and Parkinson's diseases.

The association of MHC genes with autism.

Several immune abnormalities have been noted in autistic subjects. These associations have been extended to the Major Histocompatibility Complex (MHC), a section of DNA remarkable for the number of encoded proteins with immunological functions. The strongest MHC association identified thus far is for the null allele of C4B in the class III region. The complex allelic composition of C4 as determined by immunoelectrophoresis is discussed. Low levels of C4 resulting from the null allele may be important in disease pathogenesis especially since C4 has been identified in developing brain neurons. The DNA region just telomeric to C4 has several genes including tumor necrosis factor which encode proteins with immunological functions. These proteins may act in concert with C4 in disease contribution and the genes should be more closely examined.

Emerging roles for telomerase in neuronal development and apoptosis.

Telomerase is an enzyme consisting of a reverse transcriptase called TERT and an RNA component that adds repeats of a DNA sequence (TTAGGG) to the ends of chromosomes, thereby preventing their shortening and cell cycle arrest. Telomerase levels are high in neural progenitor cells and neurons during early development, and decrease in association with cell differentiation. A role for TERT in regulation of developmental death of neurons is suggested by a decrease in TERT expression that coincides with the period of neuronal death and by data showing that TERT promotes survival of developing brain neurons. Suppression of telomerase activity and TERT expression promotes apoptosis, whereas overexpression of TERT prevents apoptosis by suppressing cell death at a premitochondrial step in the death cascade Moreover, neurotrophic factors known to play important roles in brain development can regulate telomerase activity and TERT expression in cultured neural cells. A better understanding of the functions of telomerase and TERT in neuronal differentiation and survival may lead to novel approaches for preventing neuronal death and promoting recovery in various neurodegenerative conditions. J. Neurosci. Res. 63:1-9, 2001. Published 2001 Wiley-Liss, Inc.

Mechanisms governing the differentiation of a serotonergic phenotype in culture

In this study, we explored whether a serotonergic (5-HT) phenotype could be novelly induced in the phenotypically plastic neurons of the developing striatum. We found that the 5-HT biosynthetic enzyme tryptophan hydroxylase (TPH) was expressed in nearly 10% of neurons following treatment with an extract derived from adult raphe tissue. This effect was mimicked by co-treatment with a growth factor (aFGF, bFGF or BDNF; but not GDNF, IGF-1, EGF or TGF) and the neurotransmitter 5-HT (but not GABA, dopamine, glutamate) and/or a protein kinase activator (IBMX, forskolin, TPA). Treatment with combined factors (aFGF+5-HT+IBMX+forskolin+TPA) yielded the greatest level of TPH induction (15.6%). Moreover, TPH was enzymatically active (112.8±36 pmol/mg per h) and produced detectable levels of 5-HT (2.12±0.30 ng) and its metabolite 5-HIAA (4.24±0.11 ng) in maximally stimulated cultures. These findings demonstrate that it is possible to promote the differentiation of serotonergic phenotypic traits in developing brain neurons in culture.

Patterning defects in the primary axonal scaffolds caused by the mutations of the extradenticle and homothorax genes in the embryonic Drosophila brain.

During early brain development in Drosophila a highly stereotyped pattern of axonal scaffolds evolves by precise pioneering and selective fasciculation of neural fibers in the newly formed brain neuromeres. Using an axonal marker, Fasciclin II, we show that the activities of the extradenticle (exd) and homothorax (hth) genes are essential to this axonal patterning in the embryonic brain. Both genes are expressed in the developing brain neurons, including many of the tract founder cluster cells. Consistent with their expression profiles, mutations of exd and hth strongly perturb the primary axonal scaffolds. Furthermore, we show that mutations of exd and hth result in profound patterning defects of the developing brain at the molecular level including stimulation of the orthodenticle gene and suppression of the empty spiracles and cervical homeotic genes. In addition, expression of a Drosophila Pax6 gene, eyeless, is significantly suppressed in the mutants except for the most anterior region. These results reveal that, in addition to their homeotic regulatory functions in trunk development, exd and hth have important roles in patterning the developing brain through coordinately regulating various nuclear regulatory genes, and imply molecular commonalities between the developmental mechanisms of the brain and trunk segments, which were conventionally considered to be largely independent.

The catalytic subunit of telomerase is expressed in developing brain neurons and serves a cell survival-promoting function.

Telomerase, a specialized reverse transcriptase (RT) linked to cell immortalization and cancer, has been thought not to be expressed in postmitotic cells. We now report that telomerase activity and its essential catalytic subunit, telomerase reverse transcriptase (TERT), are expressed in neurons in the brains of rodents during embryonic and early postnatal development, and are subsequently downregulated. Suppression of TERT expression in cultured embryonic hippocampal neurons increases their vulnerability to apoptosis and excitotoxicity. Overexpression of TERT in PC12 cells suppresses apoptosis induced by trophic factor withdrawal. TERT exerts its anti-apoptotic action at an early stage of the cell death process prior to mitochondrial dysfunction and caspase activation. TERT may serve a neuron survival-promoting function in the developing brain, and downregulation of TERT in the adult brain may contribute to increased neuronal vulnerability in various age-related neurodegenerative disorders.

Involvement of caspase-1 proteases in hypoxic brain injury. Effects of their inhibitors in developing neurons

To further explore the contribution of caspase-1/interleukin-1β-converting enzyme in the consequences of hypoxia in developing brain neurons, its temporal expression profile was analysed by immunohistochemistry and western blotting in cultured neurons from the embryonic rat forebrain subjected to a hypoxic stress (95% N 2/5% CO 2 for 6 h), and proteolytic activity of caspase-1 was monitored as a function of time by measuring the degradation of a selective colorimetric substrate (N-acetyl-Tyr-Val-Ala-Asp- p-nitroanilide). In addition, the influence of pre- and posthypoxic treatments by caspase-1 inhibitors (N-acetyl-Tyr-Val-Ala-Asp-aldehyde and N-acetyl-Tyr-Val-Ala-Asp-chloromethylketone) was tested on cell outcome. Hypoxia led to delayed apoptotic neuronal death, with an elevation of the expression of both pro-caspase-1 and caspase-1 active cleavage product (ICE p20) for up to 96 h after cell reoxygenation. As reflected by cleavage of the specific substrate, caspase-1 activity progressively increased between 24 h and 96 h posthypoxia, and was blocked by inhibitors in a dose-dependent fashion. The inhibitory compounds, including when given 24 h after hypoxia, prevented neuronal death, reduced apoptosis hallmarks and also increased the number of mitotic neurons, suggesting they might promote neurogenesis. Similar observations were made when neurons were exposed to a sublethal hypoxia (i.e. 3 h). These data emphasize the participation of caspase-1 in neuronal injury consecutive to oxygen deprivation, and provide new insight into the possible cellular mechanisms by which caspase inhibitors may protect developing brain neurons.

CPP32/CASPASE-3-like proteases in hypoxia-induced apoptosis in developing brain neurons

Since caspase members have been identified as effectors of apoptosis, the role of CPP32/caspase-3 was further explored in cultured neurons from the embryonic rat forebrain submitted to a 6-h hypoxia which has previously been shown to induce apoptotic death within four days after reoxygenation, whereas a shorter aggression (i.e., for 3 h) leads by the same time to an increased number of living neurons, suggesting that sublethal hypoxia may promote neurogenesis. Neuronal expression of the active cleavage product of CPP32 (CPP32 p20) increased specifically after hypoxia for 6 h to finally reach 985% over control normoxic values at 96 h post-insult, while a 3-h hypoxia triggered the inducible stress protein HSP70 that has been shown to inhibit caspase-3. Proteolytic activity of caspase-3 was progressively stimulated by lethal hypoxia, as reflected by the degradation of two selective substrates, including poly (ADP–ribose) polymerase (PARP). Caspase-3 activity was blocked specifically and dose-dependently by the peptide inhibitor, DEVD–CHO, that reduced the number of apoptotic cells and prevented the hypoxia-induced decrease in cell viability, including when given 24 h post-insult. Interestingly, in these conditions, the inhibitory compounds enhanced the number of mitotic neurons. These data emphasize the critical role of caspase-3 in neuronal injury consecutive to hypoxia. Whereas caspase inhibitors may provide benefit over a broad therapeutic window, they might allow developing neurons to complete their cell cycle initiated in response to stress, as it is the case for sublethal hypoxia.

Murine Acid α-Glucosidase: Cell-Specific mRNA Differential Expression during Development and Maturation

Acid alpha-glucosidase (GAA) cleaves the alpha1-4 and alpha1-6 glycosidic linkages of glycogen and related alpha-glucosyl substrates within lysosomes. Its deficiency results in glycogen storage disease type II (GSDII) variants including Pompe disease. To gain insight into the tissue patterns of involvement by glycogen storage in GSDII, GAA mRNA expression in mouse tissues was evaluated by Northern blot and in situ hybridization analyses. Extensive temporal and spatial variation of GAA mRNA was observed. During preterm maturation, GAA mRNA levels of whole mice progressively increased as assessed by Northern analysis. By in situ hybridization with GAA antisense mRNA, low signals were detected in most tissues throughout gestation. However, increased expression in specific cell types of different tissues was observed beginning at 16 days post coitum in developing brain neurons, primitive inner ear cells, and seminiferous tubular epithelium. In adult mice, whole-organ GAA mRNA levels were highest in brain, moderate in heart, liver, and skeletal muscle, and lowest in the series kidney > lung > testis > spleen. By in situ hybridization, the highest-intensity signals were in neurons of the central and peripheral nervous systems whereas neuroglial cells had only low-level signal. Signals of moderate intensity were in cardiomyocytes whereas low signals were in hepatocytes and skeletal muscle myocytes and very low in cells of the lungs, thymus, pancreas, spleen, and adrenal glands. However, testicular Sertoli cells and kidney tubular epithelial cells had significant signals even though surrounding cells had very low signals. The discrete temporal and spatial variations of GAA mRNA during development indicate different physiological roles for this enzyme in various cell types and developmental stages.

Sequential activation of activator protein-1-related transcription factors and JNK protein kinases may contribute to apoptotic death induced by transient hypoxia in developing brain neurons

Previous studies have demonstrated that transient hypoxia (6 h) induces apoptotic death in cultured neurons isolated from the fetal rat forebrain. Since activation of c-Jun N-terminal kinases (JNKs) and subsequent phosphorylation of c-Jun are suspected to be involved in the apoptotic pathway in several cell types, the time course of activator protein-1 (AP-1) DNA-binding, in line with induction of the AP-1 components and JNK activation, was examined during hypoxia/reoxygenation in the same model. Gel shift analysis depicted the presence of functional AP-1 transcription factors in both control and hypoxic neurons. One hour after the onset of hypoxia, all AP-1 components were markedly overexpressed. They include c-Jun, Jun B, Jun D, c-Fos and Fos-related antigens. Whereas, only c-Jun remained elevated for up to 96 h post-reoxygenation, time at which neurons were injured, other gene products showed patterned induction/repression as hypoxia progressed and then during the post-reoxygenation period, with Fos-related antigens being finally induced at 96 h. Only JNK1 was constitutively detected in cultured neurons, and its expression was inhibited during hypoxia. Nonetheless, both JNK1 and JNK3 were markedly, but transiently, induced at 48 h post-reoxygenation, when apoptosis-related morphological features became apparent. These data support the hypothesis that transient hypoxia, independently of ischemia, may trigger apoptosis through JNK signaling pathway in developing brain neurons.

Prevention from Hypoxia-Induced Apoptosis by Preconditioning Cultured Neurons from the Developing Brain • 252

It has been previously shown that a 6-h hypoxia leads to delayed apoptotic cell death in cultured neurons isolated from fetal rat forebrain. Conversely, exposure to hypoxia for 3h increases the number of living neurons scored by the end of a 96h-period of reoxygenation, when compared to controls maintained in normoxia (Pediatr Res, 1997,41:289A). Such vital changes are associated to selective alterations in the synthesis of specific proteins. In order to test the hypothesis that a preceding period of hypoxia may induce tolerance to the effects of a long-lasting insult, cultured neurons after 6 days in vitro were submitted to hypoxia (95% N2-5% CO2) for 6h (H6 group) or to the same hypoxia preceded the day before by a transient exposure to hypoxia for 1h (P1H6 group). As a function of time following reoxygenation, cell viability was measured by the tetrazolium salt MTT, DNA synthesis was assessed by [3H]thymidine incorporation, and DNA integrity was analyzed using the fluorescent dye DAPI. Whereas changes in the synthesis of total proteins were monitored by [3H]leucine incorporation, the expression profile of specific proteins such as Bcl-2, HSP70 as well as PCNA, a cofactor for DNA polymerase, was analyzed by immunohistochemistry. At 96h post-reoxygenation, cell viability decreased by 36% (n=15) in H6 group, while it was significantly enhanced (14%, n=15) in P1H6 group as compared to normoxic controls. The use of DAPI revealed the presence of apoptotic neurons (23%, n=15) in the H6 group, while P1H6 neurons did not exhibit any sign of apoptosis but showed a significant stimulation of their mitotic activity. In parallel, thymidine incorporation increased by 20% (n=12) in the latter group, and decreased by 12% (n=12) below basal values in H6. After a biphasic increase of total protein synthesis, the final rates of leucine incorporation were significantly reduced at 96h post-reoxygenation (43%, n=12) in H6, whereas they remained in the range of basal values in P1H6. Finally, immunohistochemical analysis showed overexpression of PCNA, Bcl-2 and HSP70 starting from 24h after reoxygenation in P1H6 group, the expression of these proteins being unaffected in H6 neurons compared to normoxic controls. These data suggest that a preceding period of brief hypoxia promotes molecular events which render developing brain neurons more resistant to severe hypoxic stress. Adaptative mechanisms may involve anti-apoptotic gene products as well as related proteins which regulate the cell cycle.

Expression of the Class III β‐tubulin isotype in developing neurons in culture

The expression of the class III beta-tubulin isotype was studied in cultured brain neurons by means of a monoclonal antibody (TuJ1). The results obtained indicate that during early axonal outgrowth most of the class III beta-tubulin is not incorporated into microtubules, a phenomenon which is also observed under conditions which alter the rate and extent of the neurite outgrowth response. On the other hand, a dramatic increase in its incorporation into microtubules is observed after the neurons have differentiated their neurites as axons and dendrites. In addition, the appearance of colchicine-resistant microtubules containing this isotype, a phenomenon which occurs late in neurite development, is highly coincident with the appearance of stable microtubules containing high molecular weight microtubule-associated proteins (MAPs). This pattern is different from that of the accumulation and incorporation of other beta-tubulin isotypes into microtubules. Taken collectively, our results indicate that differences exist in the in vivo utilization of tubulin isotypes in developing brain neurons and suggest that the class III beta-tubulin isotype is not a primary factor involved in the regulation of microtubule assembly during early neurite outgrowth, but that it may be important for maintaining further neurite elongation and/or determining some unique binding property of MAPs to specific microtubule subsets.

Epidermal growth factor and transforming growth factor α induce c‐fos gene expression in retinal muller cells in vivo

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are peptides that act at a common receptor and are mitogenic for immature astrocytes and trophic for developing brain neurons in vitro. However, a role for these growth factors in the mature nervous system has not been established. To investigate the actions of EGF and TGF alpha in the adult central nervous system (CNS) in vivo, the growth factors were injected into the vitreous cavity of adult male rabbits. After varying intervals, the retinas were examined for c-fos mRNA by Northern blot hybridization or Fos (and Fos-related antigen) protein by immunocytochemistry. EGF induction of c-fos mRNA occurs within 30 min and persists more than 4 hr. Fos nuclear immunostaining is induced selectively in nuclei of Muller cells by both EGF and TGF alpha. Fos-like immunoreactivity appears within 1 hr and persists more than 9 hr after EGF injection. These observations demonstrate that mature retinal Muller cells respond to exogenously applied EGF and TGF alpha in vivo, although the effect of the growth factors is not necessarily direct. The expression of c-fos and other immediate early genes provides a short-term marker that can be used to investigate the role of growth factors in normal retinal physiology and responses to injury.

Migration of nuclei and perikaryal cytoplasm along the cytoplasmic processes of differentiated neurolastoma cells

Time-lapse microcinematography of the cultures of mouse neuroblastoma C-1300 revealed several cases of directional translocation of nucleus — containing cell body along the elongated cytoplasmic processes of differentiated neuron-like cells. This unusual type of intracellular movement observed in vitro is similar to perikaryal translocation previously described in certain types of developing brain neurons in vivo . Translocation of nuclei was often accompanied by local contractions of surrounding cytoplasm. Repeated contractions and relaxations of cytoplasm were also observed in the same cultures in the course of transformation of undifferentiated well-spread neuroblast-like into differentiated neuron-like cells. Possible mechanisms of perikaryal translocations are discussed.