Synaptic changes in Alzheimer's disease and its models.

Alzheimer's disease (AD) is a highly prevalent neurodegenerative disorder characterized by a progressive loss of cognition and the presence of two hallmark lesions, senile plaques (SP) and neurofibrillary tangles (NFT), which result from the accumulation and deposition of the ß-amyloid peptide (Aß) and the aggregation of hyperphosphorylated tau protein, respectively. Initially, it was thought that Aß fibrils, which make up SP, were the root cause of the massive neurodegeneration usual found in AD brains. Over time, the longstanding emphasis on fibrillar Aß deposits and neuronal death slowly gave way to a new paradigm involving soluble oligomeric forms of Aß, which play a prominent role in triggering the cognitive deficits by specifically targeting synapses and disrupting synaptic signaling pathways. While this paradigm is widely accepted today in the AD field, the molecular details have not been fully elucidated. In this review, we address some of the important evidence, which has led to the Aß oligomer-centric hypothesis as well as some of the key findings concerning the effects of Aß oligomers on synapses at a morphological and functional level. Understanding how Aß oligomers target synapses provides an important framework for ongoing AD research, which can lead to the development of successful therapeutic strategies designed to alter or perhaps reverse the course of the disease.

Caspase-2 redux.

It has been difficult to assign caspase-2 to the effector or initiator caspase groups. It bears sequence homology to initiators (caspase-9 and CED-3), but its cleavage specificity is closer to the effectors (caspase-3 and -7). Interest in caspase-2 was dampened by the lack of a dramatic phenotype in the caspase-2 null mouse. Studies have been inhibited by the lack of knowledge about its mechanism of activation and the lack of specific methods to assay its activity. Molecular studies have defined a unique role for caspase-2 in apoptosis initiated by beta-amyloid toxicity or by trophic factor deprivation. Recently, a role for caspase-2 as an upstream initiator of mitochondrial permeabilization has been proposed. Thus, while much remains to be deciphered about caspase-2, most critically the mode of activation, it is clear that caspase-2 plays critical and singular roles in the control of programmed cell death.

Death in the balance: alternative participation of the caspase-2 and -9 pathways in neuronal death induced by nerve growth factor deprivation.

The data presented here demonstrate that sympathetic neurons have the potential to activate two alternative caspase-dependent pathways either of which is capable of mediating death induced by NGF deprivation and that these neurons have the potential to switch from one pathway to the other. The presence of these two alternative pathways to trophic factor deprivation-induced death may have implications for ensuring the correct development of the nervous system. In wild-type neurons, a caspase-2-dependent pathway is required for death, and a caspase-9-dependent pathway appears to be suppressed by endogenous inhibitors of apoptosis proteins (IAPs). In contrast, for caspase-2-null neurons, death is dependent on the caspase-9 pathway. The mechanism underlying the shift is the result of a threefold compensatory elevation of caspase-9 expression and a doubling of levels of direct IAP binding protein with low pI/(DIABLO)/second mitochondria-derived activator of caspase (Smac), an IAP inhibitor, both at the mRNA and protein levels [corrected]. These findings resolve seemingly discrepant findings regarding the roles of various caspases after NGF deprivation and raise a cautionary note regarding the interpretation of findings with caspase-null animals. The choice of the death-mediating caspase pathway in the sympathetic neurons is thus dependent on the regulated relative expression of components of the pathways including those of caspases, IAPs, and IAP inhibitors.

beta-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation.

beta-Amyloid (A beta) has been strongly implicated in the pathophysiology of Alzheimer's disease (AD), but the means by which the aggregated form of this molecule induces neuronal death have not been fully defined. Here, we examine the role of the c-Jun N-terminal kinases (JNKs) and of their substrate, c-Jun, in the death of cultured neuronal PC12 cells and sympathetic neurons evoked by exposure to aggregated A beta. The activities of JNK family members increased in neuronal PC12 cells within 2 h of A beta treatment and reached 3--4-fold elevation by 6 h. To test the role of these changes in death caused by A beta, we examined the effects of CEP-1347 (KT7515), an indolocarbazole that selectively blocks JNK activation. Inclusion of CEP-1347 (100--300 nM) in the culture medium effectively blocked the increases in cellular JNK activity caused by A beta and, at similar concentrations, protected both PC12 cells and sympathetic neurons from A beta-evoked-death. Effective protection required addition of CEP-1347 within 2 h of A beta treatment, indicating that the JNK pathway acts relatively proximally and as a trigger in the death mechanism. A dominant-negative c-Jun construct also conferred protection from A beta-evoked death, supporting a model in which JNK activation contributes to death via activation of c-Jun. Finally, CEP-1347 blocked A beta-stimulated activation of caspase-2 and -3, placing these downstream of JNK activation. These observations implicate the JNK pathway as a required element in death evoked by A beta and hence identify it as a potential therapeutic target in AD.

Identification of diverse nerve growth factor-regulated genes by serial analysis of gene expression (SAGE) profiling.

Neurotrophic factors such as nerve growth factor (NGF) promote a wide variety of responses in neurons, including differentiation, survival, plasticity, and repair. Such actions often require changes in gene expression. To identify the regulated genes and thereby to more fully understand the NGF mechanism, we carried out serial analysis of gene expression (SAGE) profiling of transcripts derived from rat PC12 cells before and after NGF-promoted neuronal differentiation. Multiple criteria supported the reliability of the profile. Approximately 157,000 SAGE tags were analyzed, representing at least 21,000 unique transcripts. Of these, nearly 800 were regulated by 6-fold or more in response to NGF. Approximately 150 of the regulated transcripts have been matched to named genes, the majority of which were not previously known to be NGF-responsive. Functional categorization of the regulated genes provides insight into the complex, integrated mechanism by which NGF promotes its multiple actions. It is anticipated that as genomic sequence information accrues the data derived here will continue to provide information about neurotrophic factor mechanisms.

Involvement of retinoblastoma family members and E2F/DP complexes in the death of neurons evoked by DNA damage.

Neuronal death evoked by DNA damage requires cyclin-dependent kinase 4 (Cdk4) and 6 activity and is accompanied by elevation of cyclin D1-associated kinase activity. Because Cdk4/6 phosphorylates retinoblastoma protein (pRb) family members that then modulate the transcriptional activity of E2F/DP1 complexes, we examined the involvement of these components in DNA damage-evoked neuronal death. Camptothecin induced rapid pRb and p107 phosphorylation at a Cdk4/6 phosphorylation site followed by selective loss of Rb and p107. The CDK inhibitor flavopiridol suppressed pRb and p107 phosphorylation and loss, implicating CDK activity in these events. Moreover, the loss of pRb and p107 appeared to be mediated by caspases because it was blocked by general caspase inhibitors. The role of phosphorylation and pRb and p107 loss in the death pathway was indicated by observations that virally mediated expression of pRb mutated at sites of phosphorylation, including the Cdk4/6 site, inhibited death. Finally, expression of dominant-negative versions of DP1, known to compromise E2F transcriptional activity, protects cortical neurons from death induced by camptothecin and sympathetic neurons from death evoked by UV treatment. Taken together, these results implicate the CDK-pRb/E2F/DP pathway as a required element in the neuronal death evoked by DNA damage.

Caspase-2 mediates neuronal cell death induced by beta-amyloid.

beta-amyloid (Abeta) has been proposed to play a role in the pathogenesis of Alzheimer's disease (AD). Deposits of insoluble Abeta are found in the brains of patients with AD and are one of the pathological hallmarks of the disease. It has been proposed that Abeta induces death by oxidative stress, possibly through the generation of peroxynitrite from superoxide and nitric oxide. In our current study, treatment with nitric oxide generators protected against Abeta-induced death, whereas inhibition of nitric oxide synthase afforded no protection, suggesting that formation of peroxynitrite is not critical for Abeta-mediated death. Previous studies have shown that aggregated Abeta can induce caspase-dependent apoptosis in cultured neurons. In all of the neuronal populations studied here (hippocampal neurons, sympathetic neurons, and PC12 cells), cell death was blocked by the broad spectrum caspase inhibitor N-benzyloxycarbonyl-val-ala-asp-fluoromethyl ketone and more specifically by the downregulation of caspase-2 with antisense oligonucleotides. In contrast, downregulation of caspase-1 or caspase-3 did not block Abeta(1-42)-induced death. Neurons from caspase-2 null mice were totally resistant to Abeta(1-42) toxicity, confirming the importance of this caspase in Abeta-induced death. The results indicate that caspase-2 is necessary for Abeta(1-42)-induced apoptosis in vitro.

Primary care outcomes in patients treated by nurse practitioners or physicians: a randomized trial.

CONTEXT: Studies have suggested that the quality of primary care delivered by nurse practitioners is equal to that of physicians. However, these studies did not measure nurse practitioner practices that had the same degree of independence as the comparison physician practices, nor did previous studies provide direct comparison of outcomes for patients with nurse practitioner or physician providers. OBJECTIVE: To compare outcomes for patients randomly assigned to nurse practitioners or physicians for primary care follow-up and ongoing care after an emergency department or urgent care visit. DESIGN: Randomized trial conducted between August 1995 and October 1997, with patient interviews at 6 months after initial appointment and health services utilization data recorded at 6 months and 1 year after initial appointment. SETTING: Four community-based primary care clinics (17 physicians) and 1 primary care clinic (7 nurse practitioners) at an urban academic medical center. PATIENTS: Of 3397 adults originally screened, 1316 patients (mean age, 45.9 years; 76.8% female; 90.3% Hispanic) who had no regular source of care and kept their initial primary care appointment were enrolled and randomized with either a nurse practitioner (n = 806) or physician (n = 510). MAIN OUTCOME MEASURES: Patient satisfaction after initial appointment (based on 15-item questionnaire); health status (Medical Outcomes Study Short-Form 36), satisfaction, and physiologic test results 6 months later; and service utilization (obtained from computer records) for 1 year after initial appointment, compared by type of provider. RESULTS: No significant differences were found in patients' health status (nurse practitioners vs physicians) at 6 months (P = .92). Physiologic test results for patients with diabetes (P = .82) or asthma (P = .77) were not different. For patients with hypertension, the diastolic value was statistically significantly lower for nurse practitioner patients (82 vs 85 mm Hg; P = .04). No significant differences were found in health services utilization after either 6 months or 1 year. There were no differences in satisfaction ratings following the initial appointment (P = .88 for overall satisfaction). Satisfaction ratings at 6 months differed for 1 of 4 dimensions measured (provider attributes), with physicians rated higher (4.2 vs 4.1 on a scale where 5 = excellent; P = .05). CONCLUSIONS: In an ambulatory care situation in which patients were randomly assigned to either nurse practitioners or physicians, and where nurse practitioners had the same authority, responsibilities, productivity and administrative requirements, and patient population as primary care physicians, patients' outcomes were comparable.

Role of cell cycle regulatory proteins in cerebellar granule neuron apoptosis.

Cerebellar granule neurons (CGNs) undergo apoptosis when deprived of depolarizing concentrations of KCl, but the underlying molecular mechanisms are not yet clear. Although caspases have been postulated to be involved in CGN cell death, inhibitors of caspases failed to prevent apoptosis under our culture conditions, suggesting an involvement of other molecules and pathways. We find that inhibitors of cyclin-dependent kinases--flavopiridol, olomoucine, and roscovitine--protect CGNs from KCl withdrawal-induced apoptosis, suggesting that cell cycle components play a significant role in the death of these neurons. Analysis of the different cell cycle regulatory elements in this model revealed that apoptosis is preceded by an increase in the level of cyclin E protein, with elevated nuclear levels of cyclin D1 and with enhanced activity of the cyclin D1- and E- associated kinases. In addition, there was a significant decrease in the level of the cyclin-dependent kinase (cdk) inhibitor p27. In agreement with these changes, analysis of a major substrate of cyclin-activated cdks, retinoblastoma protein (Rb), showed an increase in the level of phosphorylated forms within 1 hr of KCl withdrawal. Moreover, the overall levels of Rb protein were significantly reduced within 6-12 hr of KCl withdrawal and did so by a caspase-independent mechanism. All of these responses were blocked by cdk inhibitors. These findings indicate that cdks act at an early step in the pathway by which KCl withdrawal induces apoptotic death of cerebellar granule cells and suggest that additional elements of the cell cycle machinery participate in this mechanism.

Abnormal microtubule packing in processes of SF9 cells expressing the FTDP-17 V337M tau mutation.

Mutations in the gene for the microtubule associated protein, tau have been identified for fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). In vitro data have shown that FTDP-17 mutant tau proteins have a reduced ability to bind microtubules and to promote microtubule assembly. Using the baculovirus system we have examined the effect of the V337M mutation on the organization of the microtubules at the ultrastructural level. Our results show that the organization of the microtubules is disrupted in the presence of V337M tau with greater distances between the microtubules and fewer microtubules per process.

Dibutyryl cyclic AMP-induced process formation in astrocytes is associated with a decrease in tyrosine phosphorylation of focal adhesion kinase and paxillin.

Focal adhesion kinase (FAK or pp125FAK) is a cytosolic protein tyrosine kinase which plays an important role in integrin-mediated signal transduction. Adhesion of cells to the substratum correlates with an increase in tyrosine phosphorylation of FAK as well as an associated protein, paxillin. In this report we show that the tyrosine phosphorylation of FAK and paxillin are decreased during dibutyryl cyclic AMP-induced (dB-cAMP) process formation in astrocytes. When astrocytes in suspension are treated with dB-cAMP, no alteration in morphology or tyrosine phosphorylation is observed, suggesting that both phenomena are linked and adhesion dependent. Furthermore, genistein, a tyrosine kinase inhibitor, can induce process formation in such cells, underscoring the significance of protein tyrosine kinases in maintaining the morphology of adherent cells. Finally, endothelin-1, a vasopeptide which is known to inhibit process formation in astrocytes, inhibited the tyrosine dephosphorylation of proteins associated with dB-cAMP treatment. These results suggest that the formation of asymmetric processes in astrocytes results from a coordinated set of alterations in the actin cytoskeleton as well as the adhesion of the cell to the substratum. Modification of the properties of such molecules is required for process formation and the dynamic modulation of astrocytic morphology in vitro and in vivo.

Caspase-2 (Nedd-2) processing and death of trophic factor-deprived PC12 cells and sympathetic neurons occur independently of caspase-3 (CPP32)-like activity.

We have previously shown that caspase-2 (Nedd-2) is required for apoptosis induced by withdrawal of trophic support from PC12 cells and sympathetic neurons. Here, we examine the relationship of caspase-2 processing and cell death to induction of caspase-3 (CPP32)-like activity in PC12 cells. Caspase-2 processing, at a site tentatively identified as D333, led to the formation of an N-terminal 37 kDa product. This processing correlated temporally with induction of caspase-3-like activity. Agents previously shown to inhibit caspase-3-like activation, such as bcl-2 and the Cdk inhibitor flavopiridol, also acted upstream of caspase-2 processing. The general caspase inhibitors BAF and zVAD-FMK inhibited N-terminal caspase-2 processing. In contrast, the more selective caspase inhibitor DEVD-FMK inhibited the induction of caspase-3-like activity but did not affect caspase-2 processing or significantly suppress death in PC12 cells or sympathetic neurons. This indicates that caspase-3-like activity is not required for either caspase-2 processing or apoptosis in this paradigm. An antisense oligonucleotide to caspase-2 inhibited cell death but did not affect caspase-3-like activity, indicating that caspase-2 is not upstream of this activity and that activation of caspase-3-like caspases is not sufficient for death. Thus, in our paradigm, caspase-2 processing and caspase-3-like activity are induced independently of each other. Moreover, although death requires caspase-2, caspase-3-like activity is neither necessary nor sufficient for death.

Cyclin-dependent kinases participate in death of neurons evoked by DNA-damaging agents.

Previous reports have indicated that DNA-damaging treatments including certain anticancer therapeutics cause death of postmitotic nerve cells both in vitro and in vivo. Accordingly, it has become important to understand the signaling events that control this process. We recently hypothesized that certain cell cycle molecules may play an important role in neuronal death signaling evoked by DNA damage. Consequently, we examined whether cyclin-dependent kinase inhibitors (CKIs) and dominant-negative (DN) cyclin-dependent kinases (CDK) protect sympathetic and cortical neurons against DNA-damaging conditions. We show that Sindbis virus-induced expression of CKIs p16(ink4), p21(waf/cip1), and p27(kip1), as well as DN-Cdk4 and 6, but not DN-Cdk2 or 3, protect sympathetic neurons against UV irradiation- and AraC-induced death. We also demonstrate that the CKIs p16 and p27 as well as DN-Cdk4 and 6 but not DN-Cdk2 or 3 protect cortical neurons from the DNA damaging agent camptothecin. Finally, in consonance with our hypothesis and these results, cyclin D1-associated kinase activity is rapidly and highly elevated in cortical neurons upon camptothecin treatment. These results suggest that postmitotic neurons may utilize Cdk4 and 6, signals that normally control proliferation, to mediate death signaling resulting from DNA-damaging conditions.

Process formation in astrocytes: modulation of cytoskeletal proteins.

Studies on primary astrocytes cultured in vitro have shown that process formation involves changes in cytoskeletal proteins and release of tension on the substratum. Actin filament reorganization has previously been found to be the major cytoskeletal change occurring during process formation. These changes are relatively rapid with breakdown of the actin web and release of contacts occur within 15 min. of cyclic AMP treatment. The former is regulated by myosin light chain (MLC) and actin depolymerizing factor (ADF), with MLC involved in the initial release of contractile tension and ADF in both initial and longer term actin breakdown. Our results show that the dephosphorylation of MLC is due to the phosphorylation and inactivation of myosin light chain kinase (MLCK) in response to cyclic AMP. To further study the mechanisms underlying the process formation in astrocytes we used endothelin-1 (ET-1), a vasopeptide which has been shown to inhibit process formation in astrocytes and sodium fluoride which is a general phosphatase inhibitor. We observe an increase in phosphorylation of MLC on inhibition of process formation. To study the role of adhesion in process formation we used suspension cultures of astrocytes. Our results with the astrocytes in suspension suggest that the process formation in astrocytes is adhesion dependent and the changes in ADF and MLC occur only when there is process formation.

Multiple pathways of neuronal death induced by DNA-damaging agents, NGF deprivation, and oxidative stress.

Here, we compare the pathways by which DNA-damaging agents, NGF deprivation, and superoxide dismutase 1 (SOD1) depletion evoke apoptosis of sympathetic neurons. Previous work raised the hypothesis that cell cycle signaling plays a required role in neuronal apoptosis elicited by NGF deprivation and the DNA-damaging agent camptothecin. To test this hypothesis, we extended our investigation of DNA-damaging agents to cytosine arabinoside (AraC) and UV irradiation. As with NGF deprivation and camptothecin treatment, the cyclin-dependent kinase inhibitors flavopiridol and olomoucine protected neurons from apoptosis induced by AraC and UV treatment. These observations support the model that camptothecin, AraC, and UV treatment cause DNA damage, which leads to apoptosis by a mechanism that, as in the case of NGF deprivation, includes activation of cell cycle components. Flavopiridol and olomoucine, however, had no effect on death induced by SOD1 depletion, suggesting that CDKs do not play a role in this paradigm of neuronal death. To compare further the mechanisms of death evoked by NGF withdrawal, SOD1 depletion, and DNA-damaging agents, we investigated their responses to inhibitors of cysteine aspartases, elements of apoptotic pathways. The V-ICEinh and BAF, two peptide inhibitors of cysteine aspartases, protected neurons in all three death paradigms. In contrast, the cysteine aspartase inhibitory peptide zVAD-fmk conferred protection from NGF withdrawal and SOD1 depletion, but not DNA-damaging agents, whereas acYVAD-cmk protected only from SOD1 depletion. Taken together, these findings indicate that three different apoptotic stimuli activate separate pathways of death in the same neuron type.

Nedd2 is required for apoptosis after trophic factor withdrawal, but not superoxide dismutase (SOD1) downregulation, in sympathetic neurons and PC12 cells.

Activation of cysteine aspartases (caspases) seems to be a required element of apoptotic death in many paradigms. We have shown previously that general inhibitors of cysteine aspartases block apoptosis of PC12 cells and sympathetic neurons evoked by either trophic factor (nerve growth factor and/or serum) deprivation or superoxide dismutase (SOD1) downregulation. Moreover, activation of a caspase family member similar or equivalent to the interleukin-1beta-converting enzyme (ICE) was implicated for death caused by SOD1 downregulation, but not withdrawal of trophic support. The experiments presented here demonstrate that diminished expression of the cysteine aspartase Nedd2 in PC12 cells and sympathetic neurons induced by an appropriate vector peptide-linked antisense oligonucleotide rescues them from death caused by trophic factor deprivation without inhibiting apoptosis in the same cell types evoked by SOD1 downregulation. Neither the level (as revealed by Western immunoblotting) nor the cellular distribution (as revealed immunohistochemically) of Nedd2 was altered demonstrably by trophic factor deprivation. However, evidence for proteolytic processing of Nedd2 (consistent with commencement of activation) was observed in PC12 cells after withdrawal of trophic support. These findings indicate that neuronal death triggered by different initial causes may be mediated by distinct members of the cysteine aspartase family.

Induction of CPP32-like activity in PC12 cells by withdrawal of trophic support. Dissociation from apoptosis.

Inhibitors of interleukin-1beta converting enzyme (ICE) and a related group of cysteine aspartases of the ICE/ced-3 family inhibit cell death in a variety of settings, including in PC12 cells and sympathetic neurons following withdrawal of trophic support. To assess the particular member(s) of the ICE/ced-3 family that are relevant to cell death and to position their activation within the apoptotic pathway, we have used specific substrates to measure ICE-like and CPP32-like enzymatic activity in naive and neuronally differentiated PC12 cells that had been deprived of trophic support (nerve growth factor and/or serum). Rapid induction of CPP32-like, but not ICE-like, activity was observed. c-Jun kinase activation and the action of bcl-2 and other survival agents, such as cell cycle blockers, a NO generator, N-acetylcysteine, aurintricarboxylic acid, and actinomycin D occurred at a point further upstream in the apoptotic pathway compared with the aspartase activation. In living cells, zVAD-FMK, a pseudosubstrate aspartase inhibitor, blocked the activity/activation of the aspartase at concentrations about one order of magnitude lower than those required to promote survival, raising the possibility that the CPP32-like aspartase is not the main death effector in this model.

The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation.

We compare here the mechanisms of apoptotic death of PC12 cells induced by down-regulation of Cu2+,Zn2+ superoxide dismutase (SOD1) and withdrawal of trophic support (serum/nerve growth factor). Our previous results indicated that the initiating causes of death are different in each paradigm. However, bcl-2 rescues cells in either paradigm, suggesting common downstream elements to the cell death pathway. To determine whether the ICE [interleukin 1beta converting enzyme] family of proteases, which is required for apoptosis on trophic factor withdrawal, is also required for apoptosis induced by oxidative stress, we have developed a novel peptide inhibitor that mimics the common catalytic site of these enzymes and thereby blocks their access to substrates. This differs from the more usual pseudosubstrate approach to enzyme inhibition. Blockade of ICE family proteases by either this inhibitor or by a permeant competitive ICE family antagonist rescues PC12 cells from apoptotic death following apoptosis induced by down-regulation of SOD1, as well as from trophic factor/nerve growth factor deprivation. SOD1 down-regulation results in an increase in interleukin 1beta (IL- 1beta) production by the cells, and cell death under these conditions can be prevented by either blocking antibodies against IL-1beta or the IL-1 receptor antagonist (IL-1Ralpha). In contrast, trophic factor withdrawal does not increase IL-1beta secretion, and the blocking antibody failed to protect PC12 cells from trophic factor withdrawal, whereas the receptor antagonist was only partially protective at very high concentrations. There were substantial differences in the concentrations of pseudosubstrate inhibitors which rescued cells from SOD1 down-regulation and trophic factor deprivation. These results suggest the involvement of different members of the ICE family, different substrates, or both in the two different initiating causes of cell death.

Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway.

We previously showed that the downregulation of Cu/Zn superoxide dismutase (SOD1) activity in PC12 cells by exposure to an appropriate antisense oligonucleotide causes their apoptotic death. In this report, we used this model to examine the pathways by which SOD1 downregulation leads to death and to compare these pathways with those responsible for death caused by withdrawal of trophic support. To improve delivery of the SOD1 antisense oligonucleotide, we coupled it to a carrier "vector" peptide homologous to the third helix of the Drosophila Antennapedia homeodomain. This caused not only efficient cellular uptake even in the presence of serum, but also inhibition of SOD1 activity and promotion of apoptosis at 100-fold lower concentrations of oligonucleotide. Death induced by SOD1 downregulation appeared to require the reaction of superoxide with nitric oxide (NO) to form peroxynitrite. In support of this, inhibitors of NO synthase, the enzyme responsible for NO synthesis, blocked death in our experiments, whereas NO generators and donors accelerated cell death. N-Acetylcysteine and chlorophenylthiol cAMP, which rescue PC12 cells and neurons from the withdrawal of nerve growth factor and other forms of trophic support, did not protect PC12 cells from SOD1 downregulation. In contrast, overexpression of bcl-2, which also rescues these cells form loss of trophic support, was equally effective in saving the cells in the SOD1 downregulation paradigm. Taken together with past findings, such observations suggest that SOD1 downregulation and withdrawal of trophic support trigger apoptosis via distinct initial mechanisms but may utilize a common final pathway to bring about death. Our findings may be relevant to the causes and potential amelioration of neuronal degenerative disorders caused by impaired regulation of cellular levels of NO and superoxide.