Apoptotic and Oxidative Indicators in Alzheimer’s Disease

Dogma suggests that cell death mechanisms can present with either a necrotic or an apoptotic phenotype. Recent evidence, however, seems to point to a far more complex picture, in which apoptotic and necrotic phenotypes might present simultaneously. For example, TUNEL positivity in cardiomyocytes does not necessarily entail the presence of cell death that has an apoptotic phenotype.

Complicating the matter further is the promiscuous use of the same term, apoptosis, to mean different things at different times, by conflating process (a cell death program) with product (the apoptotic phenotype). Cell death can be classified into programmed cell death, which entails a global/extrinsic program of cell death, and a cell death program(s), which entails a local/intrinsic/ cellular death program.

The latter can present in a variety of phenotypes ranging from necrosis to apoptosis or as a combination phenotype, while the former is seen primarily during development and presents with an apoptotic phenotype. These ideas are useful when we come across novel phenomena or in situations where there is ambiguity as to the nature of cell death, i.e., Alzheimer’s disease (AD), where the earliest perceptible event is the presence of oxidative stress.

Indeed, the presence of oxidative stress markers in AD parallels neuronal susceptibility to cell death in AD. Here we will review methods to detect both the proximal event (oxidative stress) as well as the most distal event (cell death) in AD.

Mechanisms of Cell Death in Alzheimer’s Disease

A great deal of the research in AD over the last several decades has concentrated on the precise mechanisms responsible for the death of susceptible neurons. This is complicated by the fact that the nature and time-course of neuronal cell death in AD is controversial. The two cellular events that are a feature in AD include oxidative stress and apoptosis, with oxidative stress preceding apoptosis.

As evidence supporting an apoptotic mechanism was continually increasing, questions began to be asked about the plausibility of this claim. Although various studies point to neuronal death in AD as being the result of a cell death program that results in an apoptotic phenotype, the stereotypical manifestations that characterize the terminal phases of this apoptotic cell death program, such as detachment, chromatin condensation, nuclear segmentation, blebbing, and apoptotic bodies, are not seen either together or by themselves in AD and not for lack of trying.

Furthermore, apoptosis requires only 16–24h for completion and therefore, in a chronic disease like AD with an average duration of almost 10 yr, less than one in about 4000 cells should be undergoing apoptosis at any given time; i.e., observation of apoptotic events should be rare. Indeed, if all the neurons that are reported as having DNA cleavage were undergoing apoptosis, the brain would rapidly be stripped of neurons and the symptomatology would be abbreviated to months and not years—this is certainly not the case in AD.

Apoptosis Detection Based on Morphological Criteria

The first established criterion for cell death via an apoptotic mechanism was the characteristic cell morphology including blebbing and nuclear condensation and segmentation. The nuclear features of the apoptotic process that have been identified include condensation of chromatin around the periphery of the nucleus and fragmentation of the nucleus into multiple chromatin bodies surrounded by nuclear envelope remnants.

Ironically, enucleation does not prevent apoptosis from going forward in the cytoplasm. These changes, which can be detected by either light or electron microscopy, are the most accurate indicators for the involvement of apoptosis in the death of any given cell.

This is true in spite of the fact that the exact time-course of these nuclear characteristics remains unclear and that these changes do not seem necessary for the completion of apoptosis given that enucleated cells can complete the process. The fact that such morphological changes are considered the gold standard for the presence of apoptosis calls into question the claim that cell death in AD is apoptotic given that these hallmark end-stage signs of an apoptotic cell death program such as nuclear chromatin condensation and apoptotic bodies are not seen.

Apoptosis Detection Based on DNA Fragmentation

Apoptosis detection using DNA fragmentation is among the most widely used criteria for detection of apoptosis based on the fact that in apoptosis cation-dependent endonucleases cleave genomic DNA between nucleosomes. DNA fragmentation laddering depends on the fact that, in the case of apoptotic cell death, one expects to see mono- and oligonucleosomal-sized DNA fragments as opposed to the high-molecular weight-species normally seen with genomic DNA, creating a distinctive electrophoretic banding pattern in agrose gel systems.

While this method can be used effectively when evaluating cells in vitro and in biopsy tissue, its value in establishing apoptotic processes in AD is limited. In fact, assays based on DNA fragmentation may represent the greatest source of misunderstanding in associating AD with apoptosis, because similar mono- and oligonucleosomal patterns can be seen in a variety of conditions in which histones protect DNA from a variety of insults, including necrosis and oxidative damage.

Detection of Apoptosis Based on Effectors of Cytoplasmic Changes

Despite the extremely highly conserved nature that has been described for the apoptotic process, the upstream effectors of this mode of cell death have proven highly variable. One aspect, however, that has proven to be fairly conserved is the activation of a cascade of proteases of the caspase family, which are thought to mediate the very early stages of apoptosis.

Although caspases are known to cleave a large number of cellular substrates after aspartate residues, their main action in apoptosis is to cleave other caspase family members. All of the caspases are synthesized as proenzymes that are activated by autocleavage mediated by one of a number of extracellular stimuli or by cleavage performed by another caspase family member that lies upstream of it. Caspases are generally classified into one of two groups, the initiator as well as the executioner proteins of apoptosis.

The initiator caspases are those that lie directly downstream of extracellular or intracellular activators, while the activation of the executioner capases are thought to lead directly to the commencement of the apoptotic cell death program. Due to their role as the most important effectors of apoptosis, a great deal of effort has been focused toward using these proteases to detect apoptosis in both cells and cell populations.

Neurons are Vulnerable to Oxidative Stress

Neurons of the central nervous system are subject to a number of unique conditions that make them particularly vulnerable to oxidative stress and its sequelae that can culminate in cell death. This vulnerability is a consequence of the excessive oxygen utilization, the high unsaturated lipid content of neuronal membranes as compared to normal plasma membrane, and the postmitotic nature of primary neurons.

Due to their unique status, a balance between the production of ROS and antioxidant defenses is of particularly significance in neurons. Under normal physiological conditions, damage produced by oxygen radicals is kept in check by an efficient array of antioxidant systems that display an impressive level of redundancy, which provide multiple lines of defense.

However, in cases of age-related neurodegeneration, like that observed in AD, this balance between oxidative radicals and antioxidant defenses is altered, allowing various forms of cellular and molecular damage. This disequilibrium is intensified by the simultaneous low levels of the major neuronal antioxidant, glutathione.

The Role of In Situ Methodology

The value of using in situ methods rather than bulk analysis of tissue samples cannot be overemphasized when considering a system as structurally and cellularly complex as the nervous system. In addition to the fact that vulnerable neurons comprise only a small percentage of the affected brain region, it is essential to consider other cell types that may be compromised by the disease state, such as glia.

It is quite possible that, while neurons may display one type of damage or response, other support cells may demonstrate compensatory responses disparate from those dis- played by neurons. While at present this point is far from being established, it is enough of a theoretical concern to undertake consistent in situ studies in parallel with bulk analysis experiments. Further, the results of bulk analysis of oxidative damage are complicated by the contribution of long-lived proteins that are modified constantly by the process of normal physiological aging.

The Biochemistry of Oxidative Stress

Direct protein oxidation, mediated by diffusible hydroxyl radical (•OH) or metal-catalyzed “site-specific” oxidation at the sites of metal-ion binding, are the most obvious form of protein damage resulting from oxidative imbalance. This type of interaction can result in backbone cleavage events or in the specific oxidation of side-chain moieties, sometimes involving crosslinking reactions.

Since oxidative stress is associated with high local concentrations of both superoxide and nitric oxide, produced by the inducible isoform of nitric oxide synthase, the product of their combination, peroxynitrite, has become an important secondary oxidative stress marker. The formation of peroxynitrite can then lead to the nitration and/or oxidation of Tyr and Trp residues.


Author: Arun K. Raina, Lawrence M. Sayre, Craig S. Atwood,

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