Andrea M. Vincent

Prevention of nitric oxide-induced neuronal injury through the modulation of independent pathways of programmed cell death.

Neuronal injury may be dependent upon the generation of the free radical nitric oxide (NO) and the subsequent induction of programed cell death (PCD). Although the nature of this injury may be both preventable and reversible, the underlying mechanisms that mediate PCD are not well understood. Using the agent nicotinamide as an investigative tool in primary rat hippocampal neurons, the authors examined the ability to modulate two independent components of PCD, namely the degradation of genomic DNA and the early exposure of membrane phosphatidylserine (PS) residues. Neuronal injury was determined through trypan blue dye exclusion, DNA fragmentation, externalization of membrane PS residues, cysteine protease activation, and the measurement of intracellular pH (pHi). Exposure to the NO donors SIN-1 and NOC-9 (300 μmol/L) alone rapidly increased genomic DNA fragmentation from 20 ± 4% to 71 ± 5% and membrane PS exposure from 14 ± 3% to 76 ± 9% over a 24-hour period. Administration of a neuroprotective concentration of nicotinamide (12.5 mmol/L) consistently maintained DNA integrity and prevented the progression of membrane PS exposure. Posttreatment paradigms with nicotinamide at 2, 4, and 6 hours after NO exposure further demonstrated the ability of this agent to prevent and reverse neuronal PCD. Although not dependent upon pHi, neuroprotection by nicotinamide was linked to the modulation of two independent components of neuronal PCD through the regulation of caspase 1 and caspase 3-like activities and the DNA repair enzyme poly(ADP-ribose) polymerase. The current work lays the foundation for the development of therapeutic strategies that may not only prevent the course of PCD, but may also offer the ability for the repair of neurons that have been identified through the loss of membrane asymmetry for subsequent destruction.

Membrane asymmetry and DNA degradation: functionally distinct determinants of neuronal programmed cell death.

The ability to elucidate the molecular mechanisms that modulate programmed cell death (PCD) may provide the crucial clues to unravel the cellular basis of neurodegenerative disorders. Employing both a novel assay to follow serially PCD in individual living neurons and the neuroprotective agent lubeluzole as an investigative tool, we examined the development of nitric oxide (NO)‐induced PCD over time through the reversible annexin V labelling of membrane phosphatidylserine (PS) exposure and the electron microscopy of genomic DNA in primary rat hippocampal neurons. Exposure to the NO generators SNP (300 μM) or NOC‐9 (300 μM) alone increased annexin V‐positive neurons in the population from 7% ± 4% in untreated cultures to 13% ± 4% at 1 hr and to 61% ± 5% at 24 hr. Administration of a neuroprotective concentration of lubeluzole (750 nM) at the time of NO exposure initially prevented the exposure of PS residues, but consistently maintained DNA integrity over a 24 hr period. During posttreatment paradigms of lubeluzole (750 nM) at 2, 4, and 6 hr following NO exposure, progression of membrane PS inversion was reversed and subsequently suppressed over a 24 hr course. Our work illustrates that neuronal PCD is composed of at least two physiologically distinct and separate pathways that consist of the externalization of membrane PS residues and the independent maintenance of genomic DNA integrity. In addition, neuronal injury is fluid and reversible in nature, suggesting a “window of opportunity” for the repair and reversal of neurons yet to be committed to PCD. J. Neurosci. Res. 59:568–580, 2000

Group I and Group III metabotropic glutamate receptor subtypes provide enhanced neuroprotection

Neuroprotection by the metabotropic glutamate receptor (mGluR) system has been linked to the modulation of both the free radical nitric oxide (NO) and programmed cell death (PCD). Because the cellular mechanisms that ultimately determine neuronal PCD rely upon the independent pathways of genomic DNA degradation, externalization of membrane phosphatidylserine (PS) residues, and the activation of associated cysteine proteases, we investigated the ability of the individual mGluR subtypes to modulate the distinct pathways of NO‐induced PCD in primary rat hippocampal neurons. Membrane PS residue externalization occurred within the initial 3 hr after exposure to the NO donors (300 μM SNP or 300 μM NOC‐9), preceded genomic DNA fragmentation, and was present in 80 ± 2% of the neurons within a 24‐hr period. NO exposure also led to the rapid induction of both caspase 1‐like and caspase 3‐like activities that were determined to be necessary, at least in part, for the generation of NO‐induced genomic DNA degradation, but distinct from the detrimental effects of intracellular acidification. Yet, only caspase 1‐like activity was necessary for the modulation of PS residue externalization. Activation of group I mGluR subtypes utilized an effective, “upstream” mechanism for the inhibition of cysteine protease activity that offered an enhanced level of neuroprotection through both the preservation of genomic DNA integrity and the maintenance of PS membrane asymmetry. Group II and Group III mGluR subtypes maintained DNA integrity and group III mGluR subtypes additionally prevented PS residue externalization through mechanisms that were targeted below the level of caspase activation. Our work elucidates the independent nature of the mGluR subtypes to not only provide discrete levels of protection against neuronal PCD, but also offer robust therapeutic strategies for neurodegenerative disease. J. Neurosci. Res. 62:257–272, 2000.

Direct Temporal Analysis of Apoptosis Induction in Living Adherent Neurons

Destruction of neurons through the genetically directed process of programmed cell death (PCD) is an area of intense interest because this is the underlying mechanism in a variety of developmental and neurodegenerative diseases. The ability to identify and track viable neurons subjected to PCD could be invaluable in development of strategies to prevent or reverse the downstream mechanisms of neuronal PCD. We have developed a novel assay for PCD in viable, adherent cells using annexin V labeling. Annexin V binds to the highly negatively charged plasma membrane phosphatidylserine residues that undergo membrane translocation during PCD. Current annexin V techniques are almost exclusively restricted to flow cytometric analysis. Our unique technique permits repeated examination of individual viable neurons without altering their survival. Correlation with electron microscopy and dye exclusion assays demonstrate both sensitivity and specificity for our method to detect PCD. To our knowledge, this is the first account of a technique that positively identifies PCD in viable, adherent cells.

NEURONAL INTRACELLULAR PH DIRECTLY MEDIATES NITRIC OXIDE-INDUCED PROGRAMMED CELL DEATH

Neuronal injury is intricately linked to the activation of three distinct neuronal endonucleases. Since these endonucleases are exquisitely pH dependent, we investigated in primary rat hippocampal neurons the role of intracellular pH (pH(i)) regulation during nitric oxide (NO)-induced toxicity. Neuronal injury was assessed by both a 0.4% Trypan blue dye exclusion survival assay and programmed cell death (PCD) with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) 24 h following treatment with the NO generators sodium nitroprusside (300 microM), 3-morpholinosydnonimine (300 microM), or 6-(2-hyrdroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hex anamine (300 microM). The pH(i) was measured using the fluorescent probe BCECF. NO exposure yielded a rapid intracellular acidification during the initial 30 min from pH(i) 7.36 +/- 0.01 to approximately 7.00 (p <.0001). Within 45 min, a biphasic alkaline response was evident, with pH(i) reaching 7.40 +/- 0.02, that was persistent for a 6-h period. To mimic the effect of NO-induced acidification, neurons were acid-loaded with ammonium ions to yield a pH(i) of 7.09 +/- 0.02 for 30 min. Similar to NO toxicity, neuronal survival decreased to 45 +/- 2% (24 h) and DNA fragmentation increased to 58 +/- 8% (24 h) (p <.0001). Although neuronal caspases did not play a dominant role, neuronal injury and the induction of PCD during intracellular acidification were dependent upon enhanced endonuclease activity. Furthermore, maintenance of an alkaline pH(i) of 7.60 +/- 0.02 during the initial 30 min of NO exposure prevented neuronal injury, suggesting the necessity for the rapid but transient induction of intracellular acidification during NO toxicity. Through the identification of the critical role of both NO-induced intracellular acidification and the induction of the neuronal endonuclease activity, our work suggests a potential regulatory trigger for the prevention of neuronal degeneration.

METABOTROPIC GLUTAMATE RECEPTORS PREVENT NITRIC OXIDE-INDUCED PROGRAMMED CELL DEATH

Activation of metabotropic glutamate receptor (mGluR) subtypes can prevent neuronal injury through the signal transduction pathways of nitric oxide (NO). It is this link to NO free radical injury and subsequent DNA damage that is the most intriguing. We therefore examined whether neuronal protection through mGluR activation was dependent on the molecular mechanisms of programmed cell death (PCD). The NO generators sodium nitroprusside and 3‐morpholino‐sydnonimine were administered to induce NO toxicity in primary hippocampal neurons. PCD was documented by hematoxylin and eosin nuclear staining, DNA gel electrophoresis, transmission electron microscopy, and protein synthesis assays. Following NO exposure, PCD induction was rapid and robust in approximately 70% of the neuronal population. Activation of specific mGluR subtypes with 1S,3R‐ACPD and L‐AP4, agents that are neuroprotective against NO, significantly limited the progression of PCD. In contrast, antagonism of mGluRs with L‐AP3 did not prevent the development of PCD. Induction of new protein synthesis, a common requisite for PCD, was evident following NO exposure, but did not appear to represent a principal pathway of modulation by the mGluR agonists. Our studies suggest that mGluR modulation of NO‐induced PCD represents a primary molecular pathway responsible for neuronal survival. Further elucidation of the molecular mGluR signaling pathways may yield new insight into specific genetic regulatory mechanisms responsible for neuronal injury. J. Neurosci. Res. 50: 549–564, 1997.

Group I metabotropic receptors down-regulate nitric oxide induced caspase-3 activity in rat hippocampal neurons

Invoking the modulation of parallel cellular pathways, the G-protein metabotropic glutamate receptors (mGluRs) and nitric oxide (NO) have been shown to require a host of signal transduction pathways to modulate neuronal programmed cell death (PCD). Since the cysteine protease caspase-3 (CPP32) is one of the principal mediators of PCD in several nonneuronal cell systems, we investigated whether CPP32 activity was linked to both NO induced PCD and mGluR neuroprotection. We demonstrate that NO directly increases the activity of CPP32 by approximately 400% over a 6 h period that is necessary, at least in part, for the generation of neuronal PCD. Activation of only Group I mGluRs completely ameliorates the induction of CPP32 activity by NO and prevents the induction of PCD.

Critical temporal modulation of neuronal programmed cell injury.

Abstract1. As a free radical, nitric oxide (NO) may be toxic to neurons through mechanisms that directly involve DNA damage. Lubeluzole, a novel benzothiazole compound, has recently been demonstrated to be neuroprotective through the signal transduction pathways of NO. We therefore examined whether neuroprotection by lubeluzole was dependent upon the molecular pathways of programmed cell death (PCD).2. In primary hippocampal neurons, evidence of PCD was determined by hematoxylin and eosin (H&E) stain, transmission electron microscopy, and annexin-V binding. NO administration with the NO generators sodium nitroprusside (300 μM) or SIN-1 (300 μM) directly induced PCD.3. Neurons positive for PCD increased from 22 ± 3% (untreated) to 72 ± 3% (NO) over a 24-hr period. Coadministration of NO and lubeluzole (750 nM), a neuroprotective concentration, actively decreased PCD expression on H&E stain from 72 ± 3% (NO only) to 25 ± 3% (NO and lubeluzole). Significant reduction in DNA fragmentation by lubeluzole also was evident on electron microscopy. Application of lubeluzole in concentrations that were not neuroprotective or administration of the biologically inactive R-isomer did not significantly alter NO-induced PCD, suggesting that neuroprotection by lubeluzole was intimately linked to the modulation of PCD. Lubeluzole also was able to prevent the initial stages of cellular membrane inversion labeled with annexin-V binding, an early and sensitive indicator of PCD. Interestingly, the critical period for lubeluzole to reverse PCD induction appeared to be within the first 4 hr following NO exposure.4. Further investigation into the neuroprotective pathways that alter PCD may provide greater insight into the molecular mechanisms that ultimately determine neuronal injury.

Dual Pathways of Free Radical Injury in Vascular Endothelial Cells: Loss of Nuclear DNA Integrity and Membrane Asymmetry During Enhanced Cysteine Protease Activity

Cerebral ischemic vascular flow has been linked to endothelial release of the free radical nitric oxide (NO) and downstream activation of programmed cell death (PCD). The molecular mechanisms that mediate endothelial cell (EC) PCD are diverse in nature and require further definition. We therefore investigated the role of NO-mediated PCD and caspase-3-like activity during EC injury. We demonstrate that free radical-induced EC injury involves two distinct pathways that lead to PCD: one that modulates membrane PS exposure and another that regulates genomic DNA integrity. We also illustrate that externalization of membrane PS residues is a distinct event that occurs independent from the degradation of genomic DNA. In addition, the induction of EC PCD is robust and dependent, at least in part, on the generation of caspase-3-like activity. Further dissection of the role of the molecular mechanisms that mediate cerebral vascular injury is crucial to identify therapy that can govern both EC and neuronal cell survival.