While numerous studies have examined the existence of increased reactive oxygen species (ROS) in later-onset neurodegenerative disorders, the mechanism by which neurons die under conditions of oxidative stress remains largely unknown.
Fairly recent evidence has suggested that one mechanism linked to the death of terminally differentiated neurons is aberrant reentry into the cell cycle.
This phenomenon has been reported in Alzheimer disease (AD) patients, Down syndrome patients, and several mouse neurodegenerative models.
Here it will be overviewed recent findings regarding the influence of oxidative stress on neurodegeneration and possible connections between oxidative stress and unscheduled cell cycle reentry, the understanding of which could lead to new strategies in the development of therapeutic agents for neurodegenerative disorders.
Oxidative stress and neuronal death
Under normal physiological conditions, it is estimated that up to 1% of the mitochondrial electron flow leads to the formation of superoxide (O2•–), the primary oxygen free radical produced by mitochondria; and interference with electron transport can dramatically increase O2•– production.
While these partially reduced oxygen species can attack iron sulfur centers in a variety of enzymes, O2•– is rapidly converted within the cell to hydrogen peroxide (H2O2) by the superoxide dismutases (SOD1, SOD2, and SOD3).
However, H2O2 can react with reduced transition metals, via the Fenton reaction, to produce the highly reactive hydroxyl radical (•OH), a far more damaging molecule to the cell.
In addition to forming H2O2, O2•– radicals can rapidly react with nitric oxide (NO) to generate cytotoxic peroxynitrite anions (ONOO–).
Peroxynitrite can react with carbon dioxide, leading to protein damage via the formation of nitrotyrosine and lipid oxidation.
The generation of ROS in normal cells, including neurons, is under tight homeostatic control.
To help detoxify ROS, biological antioxidants, including glutathione, α-tocopherol (vitamin E), carotenoids, and ascorbic acid, will react with most oxidants.
In addition, the antioxidant enzymes catalase and glutathione peroxidase detoxify H2O2 by converting it to O2 and H2O.
However, when ROS levels exceed the antioxidant capacity of a cell, a deleterious condition known as oxidative stress occurs.
Unchecked, excessive ROS can lead to the destruction of cellular components including lipids, protein, and DNA, and ultimately cell death via apoptosis or necrosis.
Although numerous in vitro studies have implicated ROS in neuronal death, the relative lack of in vivo evidence has contributed to some controversy surrounding the role of ROS in the pathophysiology of later-onset neurodegenerative disorders.
Markers of oxidative stress are found in postmortem examination of brains from patients with many neurodegenerative disorders.
However, whether oxidative stress is involved in the development and/or progression of these disorders, or is merely associated with end-stage disease, is in dispute.
DNA oxidation, protein oxidation, and lipid peroxidation have been reported in regions containing neurofibrillary tangles and senile plaques of brains from AD patients.
As an apparent compensatory response, increased levels of catalase, glutathione peroxidase, and glutathione reductase were observed in the hippocampus and inferior parietal lobe of brains of patients with AD.
Biochemical assays for salicylate hydroxylation using β-amyloid (Aβ) fragments, the primary constituent of senile plaques, suggest that these peptides can generate free radicals that may underlie some of the molecular alterations observed in AD brains.
In agreement, increases in Aβ deposition resulted in the induction of oxidative stress in transgenic mice overexpressing the mutant amyloid precursor protein and presenilin 1, two proteins implicated in the progression of AD.
These results suggest that Aβ may be involved in free radical generation.
However, other studies suggest that Aβ may be a cellular response to oxidative stress or an antioxidant and implicate other processes as primary in generating free radical damage.
Dopaminergic neurons in the substantia nigra (SNc) of brains of Parkinson disease (PD) patients also exhibit hallmarks of oxidative stress, including lipid peroxidation, nucleic acid and protein oxidation, and changes in some antioxidants.
Furthermore, α-synuclein, the main deposition product in inclusions in the SNc of PD patients, is a specific target of nitration, suggestive of the role of oxidative damage in the formation of these inclusions.
In agreement, in vitro studies have shown that these aggregates are stabilized by oxidation.
These findings and the intrinsic potential for the oxidative metabolism of dopamine to generate ROS, have suggested that oxidative stress may be involved in the death of neurons in the SNc of these patients.
In addition, compounds that can generate ROS and/or disrupt the electron transport chain, have been found to induce symptoms of PD in animal models, including, in some cases, the deposition of α-synuclein aggregates.
These substances also represent environmental risk factors for PD.
Oxidative damage has also been reported in several other age-related neurodegenerative diseases, including Huntington disease, progressive supranuclear palsy, amyotrophic lateral sclerosis, and prion disorders, all of which have abnormal protein aggregation as a major component of their pathology.
Although recent research had questioned the relationship between protein fibril formation and disease, it is also clear that some sort of protein aggregation plays an important role in the pathologies of these various diseases and that oxidative stress and protein inclusions may cooperate in neuronal death in several neurodegenerative disorders.
Intrinsic oxidative stresses, and presumably cell damage, increase with age due to either diminished antioxidant defenses or the increase in mitochondrial dysfunction.
These age-related effects, compounded with genetic and environmental risk factors, and the high-energy dependence but relatively low level of antioxidants in the brain, may provide a unifying mechanism for the high incidence of neurodegenerative disorders in the aged population.
Cell cycle and neuron death
The misregulation of the cell cycle in many cell types can lead to unchecked proliferation and neoplastic disease.
As in other cell types, the cell cycle in the CNS is tightly regulated.
Neuronal precursors proceed through the cell cycle to produce larger numbers of neurons than are needed, and excess neurons are eliminated by selective apoptosis.
However, once neurons are terminally differentiated, they are maintained in a quiescent G0 state and no longer cycle.
A decade ago it was hypothesized that cell cycle abnormalities may be intimately connected to the death of terminally differentiated neurons.
The bases for this hypothesis included the observation that tumors arising from terminally differentiated neurons are extremely rare, indicating that these cells are resistant to neoplastic transformation.
Experimental evidence for this hypothesis included the demonstration that forced expression of oncogenes in terminally differentiated cells, including neurons, can cause cell death rather than cell proliferation.
In vitro studies have examined the link between aberrant cell cycle reentry and neuronal cell death in an attempt to better understand the mechanisms by which neurons can be forced back into the cell cycle.
These results demonstrate that although at least some postmitotic neurons retain the capacity to respond to growth factors by reentering into the cell cycle, such stimulation causes apoptosis rather than proliferation.
Unscheduled cell cycle reentry can also be induced by neurotoxic insults.
Several animal models directly or indirectly support the hypothesis that aberrant cell cycle reentry leads to neuronal death.
Recent evidence suggests that cell cycle reentry precedes neuronal apoptosis in human neurodegenerative diseases as well.
Data from these studies and others support the idea that mitogenic signals may lead to an increase in cell cycle proteins, thus driving neurons into aberrant cell cycle reentry.
Furthermore, it is clear that when terminally differentiated neurons are forced into the cell cycle, they do not proliferate – they die.
Although the mechanisms by which cell cycle reentry causes cell death are unknown, a direct link between cell cycle and neuronal death was recently made with the observation that CDC2, a cell cycle regulator, induces the phosphorylation and activation of BAD, a trigger of apoptosis (Fig.1.).
Connections between oxidative stress and cell cycle
The multiple demonstrations of biomarkers of oxidative stress in many age-related neurodegenerative disorders, combined with the more recent reports of cell cycle abnormalities in neurons from these patients, suggest that these processes may be intertwined at the molecular level.
At first, the idea of oxidative stress and cell cycle reentry seems counterintuitive.
In fact, examination of the current literature on the effect of oxidative stress on the cell cycle reveals that increases in ROS-induced DNA damage are correlated with cell cycle arrest.
However, whether ROS-exposed cells undergo growth arrest or apoptosis may depend in part on where the cell resides in the cell cycle when insulted.
For example, human fibroblasts treated with H2O2 underwent either cell cycle arrest or apoptosis.
The majority of the apoptotic fibroblasts were in the S phase of the cell cycle, whereas growth-arrested cells were predominantly in the G1 or the G2/M phase.
This apoptotic death of fibroblasts in the S phase is consistent with the death of neurons that have aberrantly reentered the cell cycle.
Oxidative stress and cell cycle reentry lead to cell death in the harlequin mouse.
An 80% reduction of flavoprotein expression in the Hq mutant mouse is associated with increased activity of total glutathione (GSH) and catalase, presumably through increases in hydrogen peroxide (H2O2).
Surviving neurons may express additional antioxidant pathways.
Increases in H2O2 could lead to cellular damage via formation of hydroxyl radicals (•OH) by the Fenton reaction.
In addition, oxidative stress may trigger cell cycle reentry in some terminally differentiated neurons.
These neurons undergo apoptosis after DNA replication.
The studies clearly indicate that various types of neurons respond differently to the down regulation of flavoprotein (and presumably the increase in free radicals).
Some neurons undergo apoptosis correlated with cell cycle reentry, whereas others die without signs of either process.
Furthermore, those neurons that survive may be more resistant to oxidative stress.
The sensitivity to oxidative stress differs between neuron types.
Cerebellar granule cells exposed to a 15-minute pulse of 100 μM H2O2 displayed only a 25% survival rate after 15 hours in culture.
In contrast, a similar number of cortical neurons survived when exposed to 100 μM H2O2 for 24 hours, suggesting that granule cells are more sensitive to oxidative stress than are cortical neurons.
Therefore, Hq mutant mice provide a model to examine both the influence of oxidative stress on cell cycle reentry and the mechanisms underlying the differential response of different types of neurons to oxidative stress.
Although both oxidative stress and cell cycle reentry have been implicated in the onset of later-onset neurodegenerative diseases and clearly occur together at the cellular level in Hq mutant mice, the mechanism by which oxidative stress may lead to cell cycle abnormalities remains unknown.
Cumulative DNA damage caused by endogenous free radicals has been suggested to underlie cancer and other age-related disorders, including neurodegeneration.
Free radical attack that has been shown to alter the base-pairing properties of guanine in vitro assays is elevated in nuclear and mitochondrial DNA in neurons in diseased brain regions of patients with neurodegenerative disorders.
Furthermore, studies have shown that increases in this modified base are correlated with increased incidence of cancer (and therefore cell cycle abnormalities).
In addition to base modifications, oxidative stress can cause other potentially mutational events including strand breaks, discontinuous loss of heterozygosity, and large deletions.
Thus, if the oxidation of DNA surpasses the DNA-repair capacity of the cell, mutations could accumulate, leading to the loss of genome stability.
Like malignant transformation, cell cycle reentry in neurons could require somatic mutation of a complex group of genes.
Furthermore, like cancer, the genetic alterations required to cause cell cycle reentry may be specific to particular cell types.
Another potential indirect mechanism for oxidative stress in the induction of aberrant cell cycle reentry is through histone deacetylation.
Other evidence suggests that oxidative stress may more directly trigger unscheduled cell cycle reentry.
While exposure of cells to moderate concentrations of hydrogen peroxide induces growth arrest, and exposure to high concentrations induces apoptosis and/or necrosis, low amounts of H2O2 have been shown to stimulate cell proliferation.
One potential mechanism by which oxidative stress could induce cell proliferation is through oxidative stimulation of mitogenic pathways.
Indeed, ROS have been implicated in cell signaling, specifically through mitogens.
In non-neuronal systems, oxidative stress has been shown to upregulate growth factors.
The transactivation of growth factor receptors appears to signal through several pathways, including the extracellular signal-regulated kinases (ERKs; a subset of the MAPKs), PI3K/AKT, and phospholipase C-γ1.
The activation of each of these signaling pathways has been shown to induce either apoptosis or cell survival depending on the cell type and oxidative insult.
ROS can also activate the transcription factor and tumor suppressor protein p53.
Although this protein plays an important role as a sensor of genotoxic stress and regulator of genes necessary for growth arrest and cell death, recent evidence suggests that p53 activation can in turn activate genes, including antioxidants and heparin-binding EGF-like factor that function in compensatory survival pathways.
ROS and/or oxidative damage can activate gene transcription.
However, as discussed previously, transcribed genes may be implicated in either cell survival or cell death.
Lastly, oxidative stress is well known for causing protein damage via nitration or oxidation.
Such protein modifications have been repeatedly shown in postmortem brain tissue from patients with neurodegenerative disorders, and these reactions can occur generally or have protein specificity.
Oxidation could gradually disable proteins necessary for cell cycle repression.
In summary, although unscheduled cell cycle reentry may be mediated by many different insults, evidence is mounting that both cell cycle and oxidative stress may be team players in neurodegenerative disorders.
Here it was discussed some of the potential pathways by which the repression of the cell cycle in neurons may be modulated by ROS.
Given the distinctive pathology of different neurodegenerative diseases, it is likely that specific types of neurons may respond to different signaling pathways, or need activation of multiple signaling pathways.
The testing of the roles of these and other potential pathways in neurodegeneration will require the development and analysis of additional animal models, in addition to further correlative human studies.
However, the careful dissection of the interplay between oxidative stress and the cell cycle will greatly advance our understanding of several debilitating neurodegenerative disorders.