Pcd

Programmed cell death (PCD) is also believed to be one of the contributing factors to neuronal injury and is considered to be an active, directed process that can rapidly lead to the destruction of a cell. In contrast to necrosis, PCD is characterized by the preservation of membrane integrity and internal organellae structure, chromatin condensation with nuclear fragmentation, and the budding of cellular fragments known as "apoptotic bodies.'' In most cellular systems, the end result of PCD is termed apoptosis. The protein- or gene-mediated mechanisms of PCD form the basis of several organic brain disorders. In some clinical investigations of global cerebral ischemia, the ischemic vulnerability of cerebellar granular cells has been attributed to early apoptotic DNA fragmentation. In vitro studies also support a role for PCD during neurodegeneration. Glutamate toxicity in primary neuronal cultures may lead to double-strand DNA breaks and to early single-strand DNA breaks that are consistent with apoptotic neuronal death. The detailed understanding of the cellular mechanisms that modulate PCD may provide the basis for novel therapeutic strategies to prevent or reverse neuronal loss.

Neuronal and vascular PCD is believed to proceed through two dynamic but distinct pathways that involve both DNA fragmentation and the loss of membrane asymmetry with the exposure of membrane phosphatidylserine (PS) residues (Fig. 1). These processes are considered to be functionally independent determinants of neuronal PCD. The internucleosomal cleavage of genomic DNA into fragments may be a late event during PCD and ultimately commit a cell to its demise (Fig. 2). In contrast, the redistribution of membrane PS residues can be an early event during PCD that usually precedes DNA fragmentation and may serve to later "tag" injured cells for phagocytosis (Fig. 3).

However, one of the current central issues surrounding PCD focuses on whether this process, once initiated, is committed in nature to lead to cellular death or is reversible to the extent of preventing further neuronal injury. Experimental studies have illustrated that at least the onset of PCD can be significantly limited during therapeutic modulation, such as during neuronal ischemia, excitotoxicity, and free radical exposure. In addition, these models have demonstrated a prominent link between the generation of the free radical NO and the induction of neuronal PCD during brain injury since NO can result in the rapid induction of PCD.

DNA Degradation

Immediate Survival

PS Exposure

Chronic Survival

Figure 1 Programmed cell death (PCD) consists of two independent pathways. The potential sequence of events following cellular PCD induction is illustrated. Neuronal and vascular PCD is believed to proceed through two distinct pathways that involve both DNA degradation and the loss of membrane asymmetry with the exposure of membrane phosphatidylserine (PS) residues. The internucleoso-mal cleavage of genomic DNA into fragments may be a late event during PCD and ultimately commit a cell to its demise. In contrast, the redistribution of membrane PS residues can be an early event during PCD that usually precedes DNA fragmentation and may serve to later identify injured cells for phagocytosis.

Current techniques employed to assess PCD following NO exposure, such as terminal deoxy-UTP nick end labeling (Fig. 2) or transmission electron microscopy, are useful for identifying the extent of PCD induction. However, these procedures lack the ability to assess dynamic changes in PCD in individual cells. As an alternative, a recently developed technique can monitor the induction and change in PCD in individual living cells over a period of time. The method employs the reversible labeling of annexin V to exposed PS residues of cells undergoing PCD, an early primary event during PCD induction (Fig. 3). By exploiting the dependence of annexin V on cellular calcium to bind to exposed membrane PS residues, one can reversibly label individual neurons over time. During the induction of PCD, such as following NO exposure, progressive externalization of membrane PS residues occurs that is independent of the loss of neuronal membrane integrity.

Given the ability to continually monitor changes in the course of PCD, it is now conceivable to assess whether neuronal brain injury during PCD is, in fact, reversible in nature. Studies employing neuroprotec-tive regiments, such as the application of either trophic factors or metabotropic glutamate receptor agonists, have provided evidence supporting the concept of reversible injury during PCD. For example, since cytosolic and nuclear changes associated with PCD are evident within the first hour of NO exposure, the signal

Figure 2 Prevention of neuronal DNA fragmentation following NO exposure. A representative figure is shown of neuronal DNA fragmentation that was assessed using the terminal deoxy-UTP nick end labeling (TUNEL) assay. (A) Untreated neurons (control) are without significant TUNEL labelling. (B) Neurons were exposed to NO and DNA fragmentation was assessed 24 hr later. In the presence of NO, DNA fragmentation was present in approximately 71% of the neurons examined. (C) Treatment with the growth factor, basic fibroblast growth factor, significantly prevented neuronal injury and genomic DNA fragmentation induced by NO exposure.

Figure 2 Prevention of neuronal DNA fragmentation following NO exposure. A representative figure is shown of neuronal DNA fragmentation that was assessed using the terminal deoxy-UTP nick end labeling (TUNEL) assay. (A) Untreated neurons (control) are without significant TUNEL labelling. (B) Neurons were exposed to NO and DNA fragmentation was assessed 24 hr later. In the presence of NO, DNA fragmentation was present in approximately 71% of the neurons examined. (C) Treatment with the growth factor, basic fibroblast growth factor, significantly prevented neuronal injury and genomic DNA fragmentation induced by NO exposure.

transduction mechanisms of the metabotropic glutamate system may reverse early steps in PCD.

The ability to prevent genomic DNA degradation and maintain membrane PS asymmetry may be closely linked to the modulation of cysteine protease activity. NO is believed to be one of the signal transduction systems that can elicit cysteine protease activity and can directly stimulate caspase 1- and caspase 3-like activities. Caspase 1 has been linked to the modulation of membrane PS residues through cytoskeletal proteins such as fodrin. Caspase 3 can lead to the direct degradation of DNA through the enhancement of DNase activity. Application of some neuroprotective agents, such as trophic factors or metabotropic glutamate agonists, can directly prevent the activation of caspase 1- and caspase 3-like activities following NO exposure, suggesting that these agents may maintain both genomic DNA integrity and membrane PS asymmetry through the modulation of cysteine protease activity. In addition to directly downregulating cysteine protease activity, specific neuronal enzymes responsible for the destruction of genomic DNA and

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