The BH3-only protein Puma is both necessary and sufficient for neuronal apoptosis induced by DNA damage in sympathetic neurons

DNA damage activates apoptosis in several neuronal populations and is an important component of neuropathological conditions. While it is well established that neuronal apoptosis, induced by DNA damage, is dependent on the key cell death regulators p53 and Bax, it is unknown which proteins link the p53 signal to Bax. Using rat sympathetic neurons as an in vitro model of neuronal apoptosis, we show that cytosine arabinoside is a DNA damaging drug that induces the expression of the BH3-only pro-apoptotic genes Noxa, Puma and Bim. Increased expression occurred after p53 activation, measured by its phosphorylation at serine 15, but prior to the conformational change of Bax at the mitochondria, cytochrome c (cyt c) release and apoptosis. Hence Noxa, Puma and Bim could potentially link p53 to Bax. We directly tested this hypothesis by the use of nullizygous mice. We show that Puma, but not Bim or Noxa, is a crucial mediator of DNA damage-induced neuronal apoptosis. Despite the powerful pro-apoptotic effects of overexpressed Puma in Bax-expressing neurons, Bax nullizygous neurons were resistant to Puma-induced death. Therefore, Puma provides the critical link between p53 and Bax, and is both necessary and sufficient to mediate DNA damage-induced apoptosis of sympathetic neurons.     

RAIDD is required for apoptosis of PC12 cells and sympathetic neurons induced by trophic factor withdrawal

Caspase 2 has been implicated in trophic deprivation-induced neuronal death. We have shown that overexpression of the caspase 2-binding protein RAIDD induces neuronal apoptosis, acting synergistically with trophic deprivation. Currently, we examine the role of endogenous RAIDD in apoptosis of PC12 cells and sympathetic neurons. Expression of a truncated caspase recruitment domain-only form of caspase 2, which presumably disrupts the RAIDD interaction with endogenous caspase 2, attenuated trophic deprivation-induced apoptosis. Furthermore, downregulation of RAIDD by small interfering RNA led to inhibition of trophic deprivation-induced death, whereas death induced by DNA damage, which is not caspase 2-mediated, was not inhibited. Therefore, RAIDD, likely through interaction with caspase 2, is involved in trophic deprivation-induced neuronal apoptosis. This is the first demonstration of the involvement of RAIDD in apoptosis, and provides further support for the idea that apoptotic pathways in the same system may differ depending on the initiating stimulus.     

Differential p53 phosphorylation and activation of apoptosis-promoting genes Bax and Fas/APO-1 by irradiation and ara-C treatment

In this study, we examined the effects of radiation and ara-C on induction of apoptosis and on the apoptosis-promoting genes p53, Bax and Fas/APO-1, in BV173 human leukemia cells, which harbor the wild-type p53 gene. It has been reported that p53 upregulates Fas/APO-1 and Bax expression. Both irradiation and ara-C treatment resulted in apoptosis and induction of p53 proteins within hours. The Bax gene was activated in irradiated and ara-C-treated BV173 cells, but Fas/APO-1 was induced only in irradiated BV173 cells. Radiation and ara-C treatment did not induce Bax or Fas/APO-1 protein expression in p53-null HL60 cells. Radiation weakly induced Fas/APO-1 expression in KBM-7 cells, which harbor a partially defective p53 gene. Both HL60 and KBM-7 cells are more resistant to radiation- and ara-C-induced apoptosis than BV173 cells. These results suggest that functional p53 is necessary for the activation of Bax and Fas/APO-1 expression. However, elevated p53 protein is not sufficient to activate Fas/APO-1 gene expression in ara-C-treated cells. Using two-dimensional gel electrophoresis, we found that the p53 proteins in irradiated and ara-C-treated BV173 cells have different isoelectric points; they converged to a single isoelectric point after in vitro treatment with phosphatase. These results suggest that different genotoxic treatments cause different phosphorylations of p53, which may account for the different levels of activation of Fas/APO-1 expression.     

Neural precursor cells are protected from apoptosis induced by trophic factor withdrawal or genotoxic stress by inhibitors of glycogen synthase kinase 3

Mechanisms controlling the survival of neural precursor cells (NPCs) are critical during brain development, in adults for neuron replenishment, and after transplantation for neuron replacement. This investigation found that glycogen synthase kinase 3 (GSK3) promotes apoptotic signaling in cultured NPCs derived from embryonic mouse brain subjected to two common apoptotic conditions, trophic factor withdrawal and genotoxic stress. Trophic factor withdrawal activated GSK3 and the key apoptosis mediators Bax and caspase-3. Pharmacological inhibition of GSK3 activity produced dramatic reductions in the activation of Bax and caspase-3 and NPC death after trophic factor withdrawal. Trophic factor withdrawal-induced apoptosis was delayed in Bax knock-out NPCs, but GSK3 inhibitors provided additional protection. Genotoxic stress induced by camptothecin treatment of NPCs stabilized p53, which formed a complex with GSK3beta and activated Bax and caspase-3. Camptothecin-induced activation of caspase-3 was reduced by GSK3 inhibitors in both bax(+)(/)(+) and bax(-/-) NPCs. Thus, NPCs are sensitive to loss of trophic factors and genotoxic stress, and inhibitors of GSK3 are capable of enhancing NPC survival.     

Distinct requirements for p38alpha and c-Jun N-terminal kinase stress-activated protein kinases in different forms of apoptotic neuronal death

The stress-activated protein kinases c-Jun-activated kinase (JNK) and p38 are implicated in neuronal apoptosis. Early studies in cell lines suggested a requirement for both in the apoptosis induced by withdrawal of nerve growth factor. However, studies in neuronal cells typically implicate JNK but not p38 in apoptosis. In some cases, p38 is implicated, but the role of JNK is undefined. It remains unclear whether p38 and JNK have differing roles dependent on cell type, apoptotic stimulus, or mechanism of cell death or whether they are redundant and each sufficient to induce identical forms of cell death. We investigate the relative roles of these protein kinases in different death mechanisms in a single system, cultured cerebellar granule neurons. Apoptosis induced by withdrawal of trophic support and glutamate are mechanistically different in terms of caspase activation, DNA fragmentation profile, chromatin morphology, and dependence on de novo gene expression. Caspase-independent apoptosis induced by glutamate is accompanied by strong activation of p38, and dominant negatives and inhibitors of the p38 pathway prevent this apoptosis. In contrast, withdrawal of trophic support induces caspase-dependent death accompanied by JNK-dependent phosphorylation of c-Jun, and inhibition of JNK is sufficient to prevent the death induced by withdrawal of trophic support. Inhibition of p38 does not block withdrawal of trophic support-induced death, nor does inhibition of JNK block glutamate-induced death. We propose that mechanistically different forms of apoptosis have differing requirements for p38 and JNK activities in neurons and demonstrate that only inhibition of the appropriate kinase will prevent neurons from undergoing apoptosis.     

P53 Is Essential for Developmental Neuron Death as Regulated by the TrkA and p75 Neurotrophin Receptors

Naturally occurring sympathetic neuron death is the result of two apoptotic signaling events: one normally suppressed by NGF/TrkA survival signals, and a second activated by the p75 neurotrophin receptor. Here we demonstrate that the p53 tumor suppressor protein, likely as induced by the MEKK-JNK pathway, is an essential component of both of these apoptotic signaling cascades. In cultured neonatal sympathetic neurons, p53 protein levels are elevated in response to both NGF withdrawal and p75NTR activation. NGF withdrawal also results in elevation of a known p53 target, the apoptotic protein Bax. Functional ablation of p53 using the adenovirus E1B55K protein inhibits neuronal apoptosis as induced by either NGF withdrawal or p75 activation. Direct stimulation of the MEKK-JNK pathway using activated MEKK1 has similar effects; p53 and Bax are increased and the subsequent neuronal apoptosis can be rescued by E1B55K. Expression of p53 in sympathetic neurons indicates that p53 functions downstream of JNK and upstream of Bax. Finally, when p53 levels are reduced or absent in p53+/− or p53−/− mice, naturally occurring sympathetic neuron death is inhibited. Thus, p53 is an essential common component of two receptor-mediated signal transduction cascades that converge on the MEKK-JNK pathway to regulate the developmental death of sympathetic neurons.     

Injury‐induced spinal motor neuron apoptosis is preceded by DNA single‐strand breaks and is p53‐ and Bax‐dependent

The mechanisms of injury‐induced apoptosis of neurons within the spinal cord are not understood. We used a model of peripheral nerve‐spinal cord injury in the rat and mouse to induce motor neuron degeneration. In this animal model, unilateral avulsion of the sciatic nerve causes apoptosis of motor neurons. We tested the hypothesis that p53 and Bax regulate this neuronal apoptosis, and that DNA damage is an early upstream signal. Adult mice and rats received unilateral avulsions causing lumbar motor neurons to achieve endstage apoptosis at 7–14 days postlesion. This motor neuron apoptosis is blocked in bax^?/? and p53^?/? mice. Single‐cell gel electrophoresis (comet assay), immunocytochemistry, and quantitative immunogold electron microscopy were used to measure molecular changes in motor neurons during the progression of apoptosis. Injured motor neurons accumulate single‐strand breaks in DNA by 5 days. p53 accumulates in nuclei of motor neurons destined to undergo apoptosis. p53 is functionally activated by 4–5 days postlesion, as revealed by immunodetection of phosphorylated p53. Preapoptotically, Bax translocates to mitochondria, cytochrome c accumulates in the cytoplasm, and caspase‐3 is activated. These results demonstrate that motor neuron apoptosis in the adult spinal cord is controlled by upstream mechanisms involving DNA damage and activation of p53 and downstream mechanisms involving upregulated Bax and cytochrome c and their translocation, accumulation of mitochondria, and activation of caspase‐3. We conclude that adult motor neuron death after nerve avulsion is DNA damage‐induced, p53‐ and Bax‐dependent apoptosis. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 181–197, 2002; DOI 10.1002/neu.10026     

mtCLIC/CLIC4, an organellular chloride channel protein, is increased by DNA damage and participates in the apoptotic response to p53

mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53- and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.     

DNA damage-induced neural precursor cell apoptosis requires p53 and caspase 9 but neither Bax nor caspase 3

Programmed cell death (apoptosis) is critical for normal brain morphogenesis and may be triggered by neurotrophic factor deprivation or irreparable DNA damage. Members of the Bcl2 and caspase families regulate neuronal responsiveness to trophic factor withdrawal; however, their involvement in DNA damage-induced neuronal apoptosis is less clear. To define the molecular pathway regulating DNA damage-induced neural precursor cell apoptosis, we have examined the effects of drug and gamma-irradiation-induced DNA damage on telencephalic neural precursor cells derived from wild-type embryos and mice with targeted disruptions of apoptosis-associated genes. We found that DNA damage-induced neural precursor cell apoptosis, both in vitro and in vivo, was critically dependent on p53 and caspase 9, but neither Bax nor caspase 3 expression. Neural precursor cell apoptosis was also unaffected by targeted disruptions of Bclx and Bcl2, and unlike neurotrophic factor-deprivation-induced neuronal apoptosis, was not associated with a detectable loss of cytochrome c from mitochondria. The apoptotic pathway regulating DNA damage-induced neural precursor cell death is different from that required for normal brain morphogenesis, which involves both caspase 9 and caspase 3 but not p53, indicating that additional apoptotic stimuli regulate neural precursor cell numbers during telencephalic development.     

Injury-induced spinal motor neuron apoptosis is preceded by DNA single-strand breaks and is p53- and Bax-dependent.

The mechanisms of injury-induced apoptosis of neurons within the spinal cord are not understood. We used a model of peripheral nerve-spinal cord injury in the rat and mouse to induce motor neuron degeneration. In this animal model, unilateral avulsion of the sciatic nerve causes apoptosis of motor neurons. We tested the hypothesis that p53 and Bax regulate this neuronal apoptosis, and that DNA damage is an early upstream signal. Adult mice and rats received unilateral avulsions causing lumbar motor neurons to achieve endstage apoptosis at 7-14 days postlesion. This motor neuron apoptosis is blocked in bax(-/-) and p53(-/-) mice. Single-cell gel electrophoresis (comet assay), immunocytochemistry, and quantitative immunogold electron microscopy were used to measure molecular changes in motor neurons during the progression of apoptosis. Injured motor neurons accumulate single-strand breaks in DNA by 5 days. p53 accumulates in nuclei of motor neurons destined to undergo apoptosis. p53 is functionally activated by 4-5 days postlesion, as revealed by immunodetection of phosphorylated p53. Preapoptotically, Bax translocates to mitochondria, cytochrome c accumulates in the cytoplasm, and caspase-3 is activated. These results demonstrate that motor neuron apoptosis in the adult spinal cord is controlled by upstream mechanisms involving DNA damage and activation of p53 and downstream mechanisms involving upregulated Bax and cytochrome c and their translocation, accumulation of mitochondria, and activation of caspase-3. We conclude that adult motor neuron death after nerve avulsion is DNA damage-induced, p53- and Bax-dependent apoptosis.     

Temporal analysis of events associated with programmed cell death (apoptosis) of sympathetic neurons deprived of nerve growth factor

The time course of molecular events that accompany degeneration and death after nerve growth factor (NGF) deprivation and neuroprotection by NGF and other agents was examined in cultures of NGF-dependent neonatal rat sympathetic neurons and compared to death by apoptosis. Within 12 h after onset of NGF deprivation, glucose uptake, protein synthesis, and RNA synthesis fell precipitously followed by a moderate decrease of mitochondrial function. The molecular mechanisms underlying the NGF deprivation-induced decrease of protein synthesis and neuronal death were compared and found to be different, demonstrating that this decrease of protein synthesis is insufficient to cause death subsequently. After these early changes and during the onset of neuronal atrophy, inhibition of protein synthesis ceased to halt neuronal degeneration while readdition of NGF or a cAMP analogue remained neuroprotective for 6 h. This suggests a model in which a putative killer protein reaches lethal levels several hours before the neurons cease to respond to readdition of NGF with survival and become committed to die. Preceding loss of viability by 5 h and concurrent with commitment to die, the neuronal DNA fragmented into oligonucleosomes. The temporal and pharmacological characteristics of DNA fragmentation is consistent with DNA fragmentation being part of the mechanism that commits the neuron to die. The antimitotic and neurotoxin cytosine arabinoside induced DNA fragmentation in the presence of NGF, supporting previous evidence that it mimicked NGF deprivation-induced death closely. Thus trophic factor deprivation- induced death occurs by apoptosis and is an example of programmed cell death.     

Maneb potentiates paraquat neurotoxicity by inducing key Bcl-2 family members

An important feature of Parkinson's disease is the degeneration of dopaminergic neurons in the Substantia Nigra pars compacta . Paraquat (PQ) and MPTP cause the selective degeneration of these neurons in vivo , and combining PQ with maneb exacerbates that pathology. Elucidation of the cell death mechanisms involved is important to understand how multiple environmental toxins may contribute to sporadic Parkinson's disease. We recently reported that PQ induces neuronal apoptosis through Bak activation, in contrast to MPP⁺, the toxic metabolite of MPTP, which relies on Bax and p53. Here we show that individually PQ and maneb activate Bak, but together they trigger Bax-dependent cell death. Focusing on mechanisms responsible for this synergy, we found that maneb+PQ increased the expression of three strong Bak inhibitors, Bfl-1, Bcl-xL and Mcl-1, and also induced Bax activators that included Bik and Bim. Those responses favor Bax-dependent MOMP and apoptosis. SiRNA knockdown of Bax and Bak confirmed that individually PQ and maneb induce Bak-dependent cell death, but together they block the Bak pathway and activate apoptosis through Bax.     

Nerve growth factor withdrawal-mediated apoptosis in naive and differentiated PC12 cells through p53/caspase-3-dependent and -independent pathways

Programmed cell death is regulated in response to a variety of stimuli, including the tumor suppressor protein p53, that can mediate cell cycle arrest through p21/Waf1 and apoptosis through the Bcl-2/Bax equilibrium and caspases. Neuronal cell apoptosis has been reported to require p53, whereas other data suggest that neuronal cell death may be independent of p53. Comparison of wild type PC12 to a temperature-sensitive PC12 cell line that depresses the normal function of p53 has permitted investigation of the importance of p53 in a variety of cell functions. This study examined the role of p53 in trophic factor withdrawal-mediated apoptosis in both na?ve and differentiated PC12 cells. Our data show that as PC12 cells differentiate they are more poised to undergo apoptosis than their undifferentiated counterparts. Survival assays with XTT (sodium 3'-1-(phenylaminocarbonyl)-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene sulfonic acid) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) demonstrated that lack of p53 is initially protective against apoptosis. The window of protection is about 20 h for na?ve and 36 h for differentiated cells. Apoptosis involved caspases 3, 6, and 9. However, caspase 3 activation was absent in cells lacking p53, concomitant with the delayed apoptosis. When the expression of caspase 3 was silenced with interference RNA, wild type PC12 cells revealed a morphology and biochemistry similar to PC12[p53ts] cells, indicating that caspase 3 accounts for the observed delay in apoptosis in p53 dysfunction. These results suggest that p53 is important, but not essential, in factor withdrawal-mediated apoptosis. Parallel pathways of caspase-mediated apoptosis are activated later in the absence of functional p53.