Posted 23 October 2004

Two Hits and You're Out? A Novel Mechanistic Hypothesis of Alzheimer Disease
By Xiongwei Zhu, George Perry, Mark A. Smith, Institute of Pathology, Case Western Reserve University, Cleveland, Ohio, USA

Abstract

It is now firmly established that both oxidative stress and cell cycle mitogenic alterations are prominent and proximal features in the pathogenesis of Alzheimer disease. However, while both abnormalities are early events, occurring prior to any cytopathology, the relationship between these two events, and their role in pathophysiology was, until recently, unclear. Based on the study of mitogenic and oxidative stress signalling pathways in Alzheimer disease, we proposed a "Two Hit hypothesis" that states while either oxidative stress or abnormalities in mitotic signalling can independently serve as initiators, both processes are necessary to propagate disease pathogenesis and progression.

Background

The pathological presentation of Alzheimer disease (AD), the leading cause of senile dementia, involves regionalized neuronal death and an accumulation of intraneuronal and extracellular filaments termed neurofibrillary tangles and senile plaques, respectively [reviewed in (1)]. A clearer understanding of the mechanisms responsible for neuronal death and dysfunction should lead to a greater understanding not only of the underlying pathophysiology of the disease, but also unveil potential therapeutic opportunities. To date, despite intensive efforts, the mechanism(s) responsible for AD remain elusive, and this incomplete understanding of disease pathogenesis had greatly impacted the development of accurate animal and cellular models, and thereby retarded the development of therapeutic modalities. Even though several independent hypotheses have been proposed to link the pathological lesions and neuronal cytopathology with, among others, apolipoprotein E genotype (2,3), hyperphosphorylation of cytoskeletal proteins (4), and amyloid-β metabolism (5), not one of these theories alone is sufficient to explain the diversity of biochemical and pathological abnormalities of AD, which involves a multitude of cellular and biochemical changes. Furthermore, attempts to mimic the disease by a perturbation of one of these elements using cell or animal models, including transgenic animals, do not result in the same spectrum of pathological alterations. The most striking case is that while amyloid-β plaques are deposited in some transgenic rodent models overexpressing amyloid-β precursor protein (6), there is no neuronal loss, a seminal feature of AD, and behavioral changes poorly mimic human disease.

What many of these theories have failed to incorporate is that AD is a disease of aging (7). Importantly, this holds true even in individuals with a genetic predisposition, i.e., those individuals with an autosomal dominant inheritance of AD or in individuals with Down syndrome who develop the pathology of AD. Therefore, age is a clear contributor in 100 percent of AD cases, whatever the genetic background. The free radical theory of aging (8) posits that the aging process is associated with (i) an increase in the adventitious production of oxygen-derived radicals, i.e., reactive oxygen species (ROS), together with (ii) a concurrent decrease in the ability to defend against such ROS that leads to the accumulation of oxidatively-modified macromolecules. The decrease in ROS buffering capacity also leads to a compromised ability to deal with abnormal sources of ROS such as those associated with genetic predisposition and/or disease status. Studies over the past 10 years have established oxidative stress and damage not only in the lesions of AD, but also in neurons at risk of death (9-22). Researchers are now establishing how oxidative stress is related to other possible causes of AD, as well as whether oxidative stress is an initiator or is instead a result of the disease process. Notably, while oxidative stress is not unique to AD, it does represent one of the earliest pathological events in the disease. Therefore, while oxidative stress is a fundamental aspect of the disease, other factors, likely in synergy, also impinge on disease initiation and progression.

Given the postmitotic nature of adult neurons, it is somewhat surprising that, in AD, susceptible cortical neurons display an activated cell cycle phenotype normally only seen during developmental neurogenesis, in mitotically active cells, and in neoplastic cells [reviewed in (23)]. In neoplasia, such ectopic mitogenicity is, by definition, due to a successful dysregulated cell cycle, while in the vulnerable neurons of AD it is due to an emergence out of terminal differentiation and attempted reentry into the cell cycle (23). However, as yet, there is no evidence suggesting a successful nuclear division in AD, implying that the neurons do not complete mitosis (M-phase) [reviewed in (24)]. In fact, terminally differentiated neurons may lack the ability to complete the cell cycle such that the mitotic alterations (i.e., reactivation of cell cycle machinery) may contribute to neuronal death (24). Like oxidative stress, cell cycle alterations are extremely early events in disease pathogenesis and likely act in synergy to initiate and propagate disease.

Here, we propose a "Two Hit" hypothesis of AD stating that susceptible neurons are subject to two independent insults, oxidative and mitotic, that are both necessary and sufficient to lead to AD.

Oxidative Stress, Oxidative Stress Signalling, and Alzheimer Disease

Free radical production occurs as a ubiquitous byproduct of both oxidative phosphorylation and the myriad of oxidases necessary to support aerobic metabolism. In addition to this background level of ROS, there are a number of additional contributory sources in AD that are thought to play an important role in the disease process (Figure 1). These include, but are not limited to: i) Iron, in a redox-active state, is increased in neurofibrillary tangles as well as in amyloid-β deposits and involved in ROS production (17,25). Iron catalyzes the formation of oOH from H2O2 as well as the formation of advanced glycation end products. Furthermore, iron-induced lipid peroxidation is potentiated by aluminum (26), which also accumulates in neurofibrillary tangle-containing neurons (27); ii) Activated microglia, such as those that surround most senile plaques (28), are a source of NO and O2- (29) which can react to form peroxynitrite, leaving nitrotyrosine as an identifiable marker (16,30); iii) Amyloid-β itself has been directly implicated in ROS formation through peptidyl radicals (31-34); iv) Advanced glycation end products (9) in the presence of transition metals (17) can undergo redox cycling with consequent ROS production (35-38). Additionally, advanced glycation end products and amyloid-β activate specific receptors such as the receptor for advanced glycation end products (RAGE) and the class A scavenger-receptor to increase ROS production (39,40); v) Abnormalities in mitochondrial metabolism, such as deficiencies in key enzyme function, resulting in part from detection of the mitochondrial genome, may be a major initiating source of ROS (41-46).

An exact determination of the contribution of each source of oxidative stress is complicated if for no other reason than that most sources have positive feedback. Nonetheless, the overall result is oxidative damage including advanced glycation end products (9), nitration (16,30,47,48), lipid peroxidation adduction products (18,49-54), as well as carbonyl-modified neurofilament protein and free carbonyls (9,11,14,18,37,55-60). It is notable and of mechanistic importance that such oxidative modifications extend beyond the lesions to neurons that do not display obvious signs of degenerative change. Indeed, since oxidative crosslinking makes proteins not only insoluble [reviewed in (12,15)] but also resistant to proteolytic removal (61) by competitively inhibiting the proteosome (62), oxidative crosslinking may be a significant and initiating factor in the formation of neurofibrillary tangles (63) in the face of numerous proteolytic activities which are highly active against abnormal proteins (64). In fact, it may not be coincidental that similar fibrillary inclusions, found in neurodegenerative diseases other than AD, are also extensively ubiquitinated, e.g., Lewy/Pick bodies and Rosenthal fibers (65,66) and are also oxidatively modified (67-69). Moreover, the induction of antioxidant enzymes such as heme oxygenase-1, Cu/Zn superoxide dismutase, catalase, GSHPx, GSSG-R, peroxiredoxins and several heat shock proteins and their association with intracellular pathology (10,60,70-72) provide more credence that the vulnerable neuronal cells are mobilizing antioxidant defense in the face of increased oxidative stress.

As eluded to above, there is increasing evidence that the very earliest neuronal and pathological changes characteristic of AD show evidence of oxidative damage, and such a notion has considerable experimental support (20,21,73-75). An early and contributing role for oxidative stress and damage is borne out by clinical management of oxidative stress which appears to reduce the incidence and severity of AD (76,77). Indeed, increased levels of isoprostane, a product of polyunsaturated fatty acid oxidation, in living patients with MCI and probable AD suggest that lipid peroxidation is present at the very earliest stages of the disease (78-80). That oxidative damage, marked by lipid peroxidation, nitration, reactive carbonyls or nucleic acid oxidation, is increased in vulnerable neurons whether or not they contain neurofibrillary tangles suggests that increases in neuronal oxidative damage must precede neurofibrillary pathology formation (19,21). Moreover, a marked accumulation of active oxidative modification products, such as 8OHG and nitrotyrosine, temporally precedes Aβ deposition by decades in the cytoplasm of cerebral neurons from Down syndrome patients, who invariably develop AD symptoms in their teens and twenties (20,81). That oxidative damage is the earliest event preceding the formation of tau and amyloid-β-containing pathologies is also confirmed in AD brains (21,74) and, compellingly, in APP transgenic mice models where oxidative stress precedes amyloid-β deposition (75,82).

In sum, oxidative stress appears to play an early and chronic role in both the initiation and progression of AD.

Mitotic Abnormalities, Mitotic Signalling, and Alzheimer Disease

The cell cycle is a highly regulated process with numerous checks and balances that ensures a homeostatic balance between cell proliferation and cell death in the presence of appropriate environmental signals (Figure 2). The cell cycle is typically divided into four phases: the S phase of DNA replication and the M phase of mitosis, separated by two gap phases called G1 and G2. It is the sequential expression and activation of cyclin/cyclin-dependent kinase (Cdk) complexes, the main regulators of cell cycle progression, that orchestrate the transition from one phase to another (83). Cells can exit the cell cycle to stay at resting (G0) phase, which is the case in terminally differentiated cells. Triggered by the presence of mitotic growth factors, the resting G0 cells may reenter G1 phase due to the expression/activation of cyclin D/Cdk 4,6 complex. Thereafter, the G1/S transition is controlled by the activation of the cyclin E/Cdk2 complex (84) such that the absence of cyclin E and/or the inhibition of the cyclin E/Cdk2 complex by p21, p27, and p53 will cause the cell cycle to be arrested at the G1 checkpoint. The subsequent fate of the G1-arrested cells depends on the presence or absence of cyclin A (83) such that in the absence of cyclin A, the cells return to G0 and redifferentiate. However, in the presence of cyclin A, the cells become committed to division, lack the ability to redifferentiate and, if unable to complete the cell cycle, die via an apoptotic pathway (85). Therefore, once beyond late G1, any arrest in the cell cycle will lead to cell death. The DNA replication in the S phase and the transition to the G2 phase is regulated by the activation of cyclin A/Cdk2 complex and proliferating cell nuclear antigen (PCNA). The G2/M phase transition is controlled by cyclin B/Cdc2 complex. Any perturbation of these regulators will result in the arrest of the cell cycle at G2/M transition point and cell death.

Although the scheme depicted above is sufficient to describe the behavior of a continuously dividing cell, it fails to provide a mechanism for cells that remain at a steady-state population as is the case for terminally differentiated neurons. However, in recent years, emerging evidence shows that vulnerable neurons in AD exhibit phenotypic changes characteristic of mitotic cells, suggesting that these neurons, while not necessarily capable of completing the cell cycle, are capable of reentering the cell cycle (86-94). In support of this notion, various components of the cell cycle machinery are activated in vulnerable neurons in AD [reviewed in (23,95)]. For example, the presence of cyclin D, Cdk4 and Ki67 in diseased neurons suggests that vulnerable neurons in AD are no longer in a quiescent (G0) phase (86-89). Moreover, the presence of cyclin E/Cdk2 complex indicates that neurons have passed G1 (88) and are therefore committed to division or death without the possibility of dedifferentiation. In support of this assertion, the presence of coordinated DNA replication suggests that the susceptible neurons may complete a nearly full S phase (91). Moreover, the aberrant expression of cyclin B1/Cdc2 complex indicates that degenerating neurons in AD may even, in some cases, reach G2 phase (89,90,94,96). However, the highly unorganized nature of the cell cycle in AD neurons (24) is evident by: i) the concurrent expression and aberrant localization of PCNA and cyclin B (97); ii) the concurrent appearance of Cdk4 and p16 (87); and iii) the presence of cyclin E and cyclin B but absence of cyclin D and cyclin A (89). These abnormalities point to an inadequate or a failed control of cell cycle in these neurons that may contribute to their eventual death in AD (24). Notably, like oxidative stress, mitotic abnormalities are among the very earliest neuronal changes to occur in the disease (87,88,97,98) and not end-stage epiphenomena of neuropathology. Indeed, cell cycle markers occur prior to the appearance of gross cytopathological changes (99), and the proximal nature of mitotic events in the disease process is evident in pre-AD patients with mild cognitive impairment (100).

In sum, like oxidative stress, there is accumulating evidence that cell cycle alterations represent a very early and, thereafter, chronic contributor to disease initiation and progression.

The Two Hit Hypothesis

As detailed above, oxidative stress and aberrant mitotic signalling both play early roles in the pathogenesis of AD. However, the temporal relationship between these two events was, until recently, unclear. However, studies of oxidative stress signalling and mitotic signalling pathways reveal that oxidative stress and aberrant mitotic stimuli are both necessary to initiate and propagate AD (101). In other words, "two hits" are necessary for the development of AD whereas individuals subject to only "one hit" remain free of disease (Figure 1). To illustrate this concept, while it is clear that oxidative stress is a pervasive feature in AD at all stages, it is apparent that few neurons (less than 1/10,000 at any given time) exhibit signs of apoptosis (102,103) as would be expected under conditions of acute and high level of oxidative stress. Therefore, AD is associated with lower, but chronic, levels of oxidative stress that, in other situations, induce an adaptive response rather than cell death (104-107). Therefore, we suspect that, a uniquely chronic, tolerable exposure of neurons to oxidative stress provides an explanation for the low levels of neuronal apoptosis in AD as well as the abnormally sustained activation of SAPK pathways (108,109). Tolerable levels of oxidative stress provoke compensatory changes that lead to a shift in neuronal homeostasis and, while initially reversible, become permanent adaptive changes under chronic oxidative stress. In this new steady state, "oxidative steady state," neurons still function relatively normally, perhaps for decades (108), and individuals remain relatively cognitively intact (Figure 3). In fact, since oxidative stress is much higher in pre-AD and AD than that in normal aging, it is likely that neurons at oxidative steady state devote much of their compensatory potential to fight against oxidative stress. Unfortunately, such compensations make the neurons uniquely vulnerable to secondary insults that require other types of compensatory changes in other pathways such as those that regulate cell size and growth. Normally, neurotrophic factors such as BDNF and NGF promote the survival, growth, and/or synaptogenesis of neurons (110); however, the ectopic expression of, or increased sensitivity to, neurotrophic factors in response to cellular stress in an "oxidative steady state" may serve as the second hit and trigger a catastrophe in these neurons, leading to AD-type changes (111-113). Conversely, neurons that have reentered into what will become a futile attempt at division (i.e., mitotic steady state) are more vulnerable to changes in oxidative stress that require further adaptation. In other words, the onset of AD, at least in the absence of genetic factors, is a stochastic process that, given the nature of the detrimental "hits," is age-related in penetrance (Figure 3).

Genetic Factors and the Two Hit Hypothesis

Mutations in at least three genes, the amyloid-β precursor protein (AβPP) and the two homologous presenilin genes, PS1 and PS2, are associated with early-onset AD (114). Although all these mutations inevitably lead to increased Aβ production, the exact mechanism(s) by which mutations in these genes are involved in AD pathogenesis remains elusive. However, it is notable that these proteins share a common function, namely, a role in cell cycle control which may be key to the "two hit hypothesis."

AβPP is a single-pass membrane protein expressed at the cell surface, whose cytoplasmic C-terminus interacts with several adaptor proteins, including Fe65 and AβPP-BP1, that function as regulators of the cell cycle (115-117). For example, AβPP-BP1 is a cell cycle protein that normally negatively regulates the progression of cells into the S phase and positively regulates progression into mitosis (115,116). The other adaptor, Fe65, is a nuclear protein and also regulates negatively G1 to S phase cell cycle progression by inhibiting the key S phase enzymes (117). It is therefore conceivable that AβPP may act as a cell surface receptor to relay cell cycle-related signals. Moreover, AβPP and its proteolytic fragments (i.e., Aβ peptide and sAβPP) are mitogenic (91,118-122), and Aβ itself can promote the activation of the mitotic cycle in cultured differentiated neurons which enter the S phase and start the replication of DNA (120). sAβPP has been shown to have epithelial growth factor activity, inducing two- to threefold increases in the rate of cell proliferation and cell migration (121,122). It therefore follows that overexpression or mutation of AβPP may push neurons into an aberrant cell cycle and in support of this hypothesis, FAD mutants of AβPP have a greater capacity to drive DNA synthesis than expression of wild AβPP (115,116). The compensatory changes in response to such genetic stress that serves as a first hit may leave neurons very vulnerable to an additional hit. In this regard, it has been demonstrated that neuronal cells bearing AβPPSw mutants have significantly enhanced vulnerability to oxidative stress (123,124), reduction of trophic factors (125), UV irradiation, and staurosporine (126). AβPP transgenic mice also show increased vulnerability to oxidative stress-related conditions such as ischemia (127) and traumatic brain injury (128).

The two hit hypothesis. An initial insult, whether oxidative or mitotic, that is chronic and above threshold limits leads to a new steady state (either oxidative steady state or mitotic steady state). It is in this new steady state where neurons are vulnerable to the subsequent "second hit," which causes the AD phenotype. Reprinted with permission from Elsevier (The Lancet, 2004, 3, 219-226).

Presenilin (PS) 1 and 2 proteins also play a role in cell cycle control. For example, the overexpression of both PS1 and PS2 proteins resulted in G1 phase arrest of the cell cycle (129,130), which may be due to the decrease in Cdk4 activity and phosphorylation of the retinoblastoma tumor suppressor protein (131). Overexpression of FAD PS1/2 mutants further increase cell cycle arrest compared to wild-type PS1/2, and the degree to which the different FAD PS1 mutants inhibits cell cycle progression correlates somewhat with the age of AD onset induced by the mutations (130). Conversely, PS1 deficiency results in accelerated entry into the S phase and prolonged S phase of the cell cycle (132,133). Therefore, the disruption of PS1/2 function caused by FAD mutants could affect the regulation of the cell cycle. Neurons under such mitotic stress, which we term the "mitotic steady state," must devote much of their compensatory potential to fight against it and would be extremely vulnerable to an additional "hit." Indeed, multiple lines of evidence demonstrate that although the expression of pathogenic PS mutants is not toxic, it does enhance the susceptibility to apoptotic and necrotic insults both in vitro and in vivo.

In summary, both AβPP and PS1/2 play an important role in cell cycle control; therefore, it is conceivable that the disruption caused by FAD mutants may impair the cell cycle control of susceptible neurons. Given the fact that massive neuronal loss only occurs relatively later in life, it is conceivable that the compensatory changes to such genetic stress lead to a steady state that we call "mitotic steady state," where susceptible neurons still function normally but are very vulnerable to a second oxidative hit as evidenced by their enhanced vulnerability to additional insults.

Conclusions

That AD, like cancer, is a disease of "two hits" not only explains why current therapeutic strategies are often found wanting with respect to efficacy, but also why current models of disease pathogenesis fail to replicate the human condition. Models utilizing a two hit strategy are currently in development and should allow the development of pharmacological modalities for AD.

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