Apoptosis Of Neural Precursor Cells Research Articles

P53 upregulated mediator of apoptosis (Puma) deficiency increases survival of adult neural stem cells generated physiologically in the hippocampus, but does not protect stem cells generated in surplus after an excitotoxic lesion.

Neurogenesis occurs in the mammalian brain throughout adulthood and increases in response to metabolic, toxic or traumatic insults. To remove potentially superfluous or unwanted neural stem cells/neuronal progenitors, their rate of proliferation and differentiation is fine-tuned against their rate of apoptosis. Apoptosis requires the transcriptional and posttranslational activation of Bcl-2-homolgy domain 3 (BH3)-only proteins. Previously, we demonstrated that the BH3-only protein p53-upregulated mediator of apoptosis (Puma) controls the physiological rate of apoptosis of neural precursor cells in the adult mouse hippocampus. Puma's role in controlling a lesion-induced increase in neural stem cells is currently not known. We employed a model of local, N-methyl-D-asparte (NMDA)-induced excitotoxic injury to the CA1 hippocampal subfield and immunofluorescence labelling to produce increased neural stem cell proliferation/ neurogenesis in the dentate gyrus at two survival times following the excitotoxic lesion. Deletion of puma failed to rescue any NMDA-induced increase in adult born cells as assessed by BrdU or Doublecortin labelling in the long-term. No difference in the proportion of BrdU/NeuN-positive cells comparing the different genotypes and treatments suggested that the phenotypic fate of the cells was preserved regardless of the genotype and the treatment. While neurogenesis is up-regulated in puma-deficient animals following NMDA-induced excitotoxicity to the hippocampal CA1 subfield, puma deficiency could not protect this surplus of newly generated cells from apoptotic cell death.

Microglia-derived TNFα induces apoptosis in neural precursor cells via transcriptional activation of the Bcl-2 family member Puma

Neuroinflammation is a common feature of acute neurological conditions such as stroke and spinal cord injury, as well as neurodegenerative conditions such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis. Previous studies have demonstrated that acute neuroinflammation can adversely affect the survival of neural precursor cells (NPCs) and thereby limit the capacity for regeneration and repair. However, the mechanisms by which neuroinflammatory processes induce NPC death remain unclear. Microglia are key mediators of neuroinflammation and when activated to induce a pro-inflammatory state produce a number of factors that could affect NPC survival. Importantly, in the present study we demonstrate that tumor necrosis factor α (TNFα) produced by lipopolysaccharide-activated microglia is necessary and sufficient to trigger apoptosis in mouse NPCs in vitro. Furthermore, we demonstrate that microglia-derived TNFα induces NPC apoptosis via a mitochondrial pathway regulated by the Bcl-2 family protein Bax. BH3-only proteins are known to play a key role in regulating Bax activation and we demonstrate that microglia-derived TNFα induces the expression of the BH3-only family member Puma in NPCs via an NF-κB-dependent mechanism. Specifically, we show that NF-κB is activated in NPCs treated with conditioned media from activated microglia and that Puma induction and NPC apoptosis is blocked by the NF-κB inhibitor BAY-117082. Importantly, we have determined that NPC apoptosis induced by activated microglia-derived TNFα is attenuated in Puma-deficient NPCs, indicating that Puma induction is required for NPC death. Consistent with this, we demonstrate that Puma-deficient NPCs exhibit an ∼13-fold increase in survival as compared with wild-type NPCs following transplantation into the inflammatory environment of the injured spinal cord in vivo. In summary, we have identified a key signaling pathway that regulates neuroinflammation induced apoptosis in NPCs in vitro and in vivo that could be targeted to promote regeneration and repair in diverse neurological conditions.

Mcl-1 regulates the survival of adult neural precursor cells

Since the discovery of neural precursor cells (NPCs) in the adult mammalian brain, there has been a lot of excitement surrounding the potential for regeneration in the adult brain. For instance, many studies have shown that a significant number of NPCs will migrate to a site of injury and differentiate into all of the neural lineages. However, one of the main challenges affecting endogenous neural regeneration is that many of the NPCs that migrate to the injury site ultimately undergo apoptosis. Therefore, we sought to determine whether myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic Bcl-2 protein, would promote the survival of adult NPCs by impeding apoptosis. To do this, we first confirmed that Mcl-1 is endogenously expressed within the adult NPC population using BrdU labeling assays. Next, we conditionally deleted Mcl-1 in adult NPCs using cre/lox technology and expressed Cre from the NPC-specific promoter Nestin. In vitro, cells that had Mcl-1 conditionally deleted had a 2-fold increase in apoptosis when compared to controls. In vivo, we used electroporation to conditionally delete Mcl-1 in adult NPCs and assessed apoptosis at 72h. after electroporation. As in our in vitro results, there was a 2-fold increase in apoptosis when Mcl-1 was conditionally deleted. Finally, we found that Mcl-1 over-expression reduced the endogenous rate of adult NPC apoptosis 2-fold in vitro. Collectively, these results demonstrate that Mcl-1 is crucial for the survival of adult NPCs and may be a promising target for future neural regeneration therapies.

Lysosome Dysfunction Triggers Atg7-dependent Neural Apoptosis

Macroautophagy (autophagy) is a process wherein bulk cytosolic proteins and damaged organelles are sequestered and degraded via the lysosome. Alterations in autophagy-associated proteins have been shown to cause neural tube closure defects, neurodegeneration, and tumor formation. Normal lysosome function is critical for autophagy completion and when altered may lead to an accumulation of autophagic vacuoles (AVs) and caspase activation. The tumor suppressor p53 is highly expressed in neural precursor cells (NPCs) and has an important role in the regulation of both autophagy and apoptosis. We hypothesized that altered lysosome function would lead to NPC death via an interaction between autophagy- and apoptosis-associated proteins. To test our hypothesis, we utilized FGF2-expanded NPCs and the neural stem cell line, C17.2, in combination with the lysosomotropic agent chloroquine (CQ) and the vacuolar ATPase inhibitor bafilomycin A1 (Baf A1). Both CQ and Baf A1 caused concentration- and time-dependent AV accumulation, p53 phosphorylation, increased damage regulator autophagy modulator levels, caspase-3 activation, and cell death. Short hairpin RNA knockdown of Atg7, but not Beclin1, expression significantly inhibited CQ- and Baf A1-induced cell death, indicating that Atg7 is an upstream mediator of lysosome dysfunction-induced cell death. Cell death and/or caspase-3 activation was also attenuated by protein synthesis inhibition, p53 deficiency, or Bax deficiency, indicating involvement of the intrinsic apoptotic death pathway. In contrast to lysosome dysfunction, starvation-induced AV accumulation was inhibited by either Atg7 or Beclin1 knockdown, and Atg7 knockdown had no effect on starvation-induced death. These findings indicate that Atg7- and Beclin1-induced autophagy plays a cytoprotective role during starvation but that Atg7 has a unique pro-apoptotic function in response to lysosome dysfunction.

Interleukin-1β mediates proliferation and differentiation of multipotent neural precursor cells through the activation of SAPK/JNK pathway

Neural precursor cells (NPCs) have been experimentally used to repair the damaged nervous system either by exogenous transplantation or by endogenous activation. In post-injury inflammation, an array of cytokines including interleukin-1β (IL-1β) are released by host as well as invading immune cells and increased markedly. In the present study, we investigated the effects of IL-1β on the survival, proliferation, differentiation and migration of NPCs as well as underlying intracellular signaling pathways. NPCs derived from the E16 rat brain were expanded in neurospheres that were found to express IL-1β, IL-1RI and IL-1RII, but not IL-1α and IL-1ra. IL-1β inhibited the proliferation of NPCs in a dose-dependent manner, an effect that can be reversed by IL-1ra, an antagonist for IL-1 receptor. This inhibitory effect of IL-1β on NPCs proliferation resulted in part from its effect on increased apoptosis of NPCs. Moreover, IL-1ra did not affect NPCs lineage fate but rather inhibited GFAP expression in differentiated astrocytes. We also found that IL-1ra had no effect on the transmigration of NPCs in vitro. Finally, we showed that the effect of IL-1β on NPCs proliferation and differentiation appeared to be mediated by SAPK/JNK, but not ERK, P38MAPK nor NF-κB pathways. These findings collectively suggest that the inflammatory environment following CNS injuries may influence the ability of NPCs to repair the damage.

BH3-Only Proapoptotic Bcl-2 Family Members Noxa and Puma Mediate Neural Precursor Cell Death

Neural precursor cells (NPCs) are highly sensitive to genotoxic injury, which triggers activation of the intrinsic mitochondria-dependent apoptotic pathway. This pathway is typically initiated by members of the BH3 (Bcl-2 homology 3)-only subgroup of the Bcl-2 (B-cell CLL/lymphoma 2) protein family, which are positioned upstream in the apoptotic pathway to respond to specific death stimuli. We have shown previously that NPCs deficient in the tumor suppressor protein p53 show significantly less death after exposure to genotoxic injury or to staurosporine (STS), a broad kinase inhibitor and potent apoptosis inducer. p53 has been shown to regulate the expression of both Noxa and Puma, two BH3-only proteins, although their involvement in p53-dependent cell death appears to be cell-type and stimulus specific. A systematic comparison of the relative contributions of Noxa and Puma to NPC apoptosis has not yet been performed. We hypothesized that p53-dependent transcription of Noxa and Puma leads to death in telencephalic NPCs exposed to genotoxic stress. We found that genotoxic injury induces a rapid p53-dependent increase in expression of Noxa and Puma mRNA in telencephalic NPCs. Furthermore, deficiency of either Noxa or Puma inhibited DNA damage-induced caspase-3 activation and cell death in telencephalic NPCs in vitro. However, only Puma deficiency protected telencephalic ventricular zone NPCs from death in vivo. In contrast to genotoxic injury, STS produced a p53-independent increase in Noxa and Puma expression, but neither Noxa nor Puma was required for STS-induced NPC death. Together, these experiments identify Noxa and Puma as important regulators of genotoxin-induced telencephalic NPC death.

Reactive species and apoptosis of neural precursor cells after γ-irradiation

This study addresses the participation of radiation-induced free radicals, mainly nitric oxide (NO), in modulating the apoptotic response in an in vitro model of neural cortical precursor cells exposed to γ-radiation. Cortical cells obtained from rats at 17 gestational day (GD) were irradiated with a dose of 2 Gy. The percentage of apoptotic cells was significantly increased 4 h post-irradiation (pi). NO content showed a significant increase after 30 min pi and the rate of generation reached a maximum 1 h pi. Luminol-dependent chemiluminescence (CL) was significantly higher in cells after 2 h pi as compared to control cells and this profile was maintained up to 4 h pi. Supplementation with l-NAME significantly increased light emission. Administration of superoxide dismutase (SOD) following l-NAME addition prevented the observed changes due to l-NAME administration. The caspase inhibitor zDEVD-fmk significantly reduced the radical generation. Moreover, the cellular decrease in NO content occurred coincidentally with the rise in oxygen radical generation and the activation of caspase-3. In vitro irradiation of neural precursor cells allowed us to suggest that an early radiation-induced generation of NO could exert a neuroprotective role. However, despite this NO initial protective effect and its role modulating the response against γ-radiation, NO generation was not able of fully preventing radiation-induced apoptosis.

TNF-α-induced chemokine production and apoptosis in human neural precursor cells

Recent studies have shown that proinflammatory cytokines damage rodent neural precursor cells (NPCs), a source of self-renewing, multipotent cells that play an important role in the developing as well as adult brain. In this study, the effects of tumor necrosis factor alpha (TNF-alpha) on cytokine and chemokine production by human NPCs (>98% nestin- and >90% A2B5-positive), obtained from 6- to 8-week-old fetal brain specimens, were evaluated. NPCs stimulated with this proinflammatory cytokine were found to produce abundant amounts of the chemokines monocyte chemoattractant protein 1 (MCP-1)/CC chemokine ligand 2 (CCL2) and interferon-inducible protein 10 (IP-10)/CXC chemokine ligand 10 (CXCL10) in a time- and concentration-dependent manner. TNF-alpha treatment also induced NPC apoptosis. Receptors for TNF [TNFRI (p55) and TNFRII (p75)] mRNA were constitutively expressed on NPCs. However, only TNFRI was involved in TNF-alpha-induced chemokine production and apoptosis by NPCs, as anti-TNFRI but not anti-TNFRII antibodies blocked the stimulatory effect. TNF-alpha treatment induced p38 mitogen-activated protein kinase (MAPK) phosphorylation in NPCs, and SB202190, an inhibitor of p38 MAPK, blocked TNF-alpha-induced chemokine production. Thus, this study demonstrated that NPCs constitutively express receptors for TNF-alpha, which when activated, trigger via a p38 MAPK signaling pathway production of two chemokines, MCP-1/CCL2 and IP-10/CXCL10, which are involved in infectious and inflammatory diseases of the brain.

Mechanisms of radiation-induced hippocampal neuron cytotoxicity and implications on memory and learning disorders

Mechanisms of radiation-induced hippocampal neuron cytotoxicity and implications on memory and learning disorders

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.