Cell cycle regulators in neuronal death evoked by excitotoxic stress: implications for neurodegeneration and its treatment
Introduction
Regulation of selective neuronal death in neurological disorders such as Parkinson’s (PD), Huntingtons (HD), Alzheimers (AD) disease and stroke is complex and dependent upon severity and type of death stimuli. While several death initiators have been proposed, excitotoxic stress is likely to be one important proximal regulator of neuronal injury in these conditions [1], [11], [16], [19]. For example, glutamate and its related excitatory amino acids (EAA) cause death of CNS neurons when administered both in vivo [16], [67]and in vitro [14], [31]. Administration of NMDA antagonists ameliorate damage due to stroke [19], [41] and the dopamine neuron specific toxin MPTP [10], [79]. While the mechanisms that regulate excitotoxic damage are not fully understood, growing evidence suggests that excitotoxicity may initiate both necrotic and apoptotic death mechanisms. In this article, we will discuss the potential role of cell cycle molecules in the delayed apoptotic death evoked by excitotoxicity. Our findings both support a role of cell cycle molecules in such death and suggest a potential therapeutic target.
The effects of EAAs appear to be transduced primarily, although not exclusively, through ionotropic receptors of the NMDA and AMPA/KA subtypes [16] and possess both acute and delayed characteristics. The immediate neurotoxic/necrotic effects are most likely due to deregulation of ionic flux and consequent cell swelling [19]. Delayed death may be associated with Ca2+ influx through NMDA, and in some cases, kainate type channels [14], [31], [65]. Elevated levels of free intracellular Ca2+ affect a wide range of signal transduction pathways such as calpains [2], [3] and nitric oxide synthase [17], [18]. While the effects of Ca2+ influx are not fully delineated, one important consequence is an increase of radical oxygen species (ROS) and oxidative stress. For example, increased NO production may lead to an increase in highly reactive peroxynitrite species [6], [7], [29]. Increased ROS also may result from activation of Ca2+ dependent proteases like calpains which catalyze conversion of xanthine dehydrogenase to xanthine oxidase yielding superoxide radicals [42]. It must be noted that excitotoxic damage may not necessarily be a primary initiator of neuronal death. In some cases of neurodegeneration and/or in certain neurodegenerative models, mitochondrial damage may lead to loss of ATP, partial membrane depolarization, activation of NMDA receptors and to death by a secondary excitotoxic related mechanism [1], [11].
The relative contributions of necrotic and delayed death after excitotoxicity are variable and likely dependent upon a various factors such as strength of insult and level of ATP within a cell [52], [64]. Moreover, some evidence suggests that the delayed death pathway may predominate when necrotic pathways of death are inhibited [52], [64]. Thus, in any given excitotoxic death paradigm, it is important to find means to dissect the two death mechanisms.
While it is likely that multiple proximal signaling events contribute to delayed neuronal death, recent evidence has suggested that signals normally associated with cell cycle control may play an important role in the death signal. Quite simply put, neuronal injury, in some circumstances, may inappropriately upregulate cell cycle related signals. This response, in the context of terminally differentiated neurons leads to death rather than cell cycle progression.
The cell cycle, like apoptosis, is a tightly regulated process. A few salient points of cell cycle regulation will be touched on here. The timing of cell cycle progression is in large part due to the actions of cyclin dependent kinases (CDKs) [61], [62]. Activation of the CDKs requires binding of their respective partners, the cyclins. On a simplistic level, Cdk4/6/cyclinD, Cdk2/cyclinE and cdk3 (with an unknown partner) complexes control regulation of the G1/S phase of the cell cycle, and Cdk2/cyclinA controls S phase progression, while, the Cdk1/cyclinB kinases mediate G2/M progression [61], [62]. The complexity of CDK regulation is highlighted by the fact that activation is also mediated through activating and inactivating phosphorylations and endogenous cell molecules which include members of the cip/kip family, p21, p27, p57, and INK family members, p15, p16, and p19 [45]. While the focus of this article is on the potential involvement in the death mechanism of molecules that normally regulate the G1/S phase of the cell cycle, the involvement of M phase regulators such as Cdc2 has also been suggested [23], [73].
One set of important targets of the CDKs which regulate G1/S phase progression are members of the Rb tumor suppressor family (p105, p107, p130) [13], [75]. Hypophosphorylated Rb family members bind to E2F transcription family members, inhibiting their function [13], [69], [75]. Once phosphorylated by the CDKs, Rb family members are released from E2F members that can then transactivate and regulate cell cycle progression. Rb family-E2F complexes also mediate gene repression and this repression is lost upon phosphorylation by CDKs. Such a loss of repressive activity may also contribute to cell cycle control. While the roles of Rb family members as negative regulators of cell cycle progression are best characterized, it must be pointed out that the Rb family likely has other functions, including differentiation [38]. For example, there are reports that Rb interacts with numerous (at least 70 to date) proteins in addition to E2F family members (for example) [76].
The evidence for cell cycle involvement in neuronal death can be summarized in two major points. First, in vitro [22], [23], [24], [26] and in vivo [12], [37], [43], [47], [70], [73] evidence has indicated that cell cycle regulators are upregulated during some circumstances of neuronal death. Second, inhibition of cell cycle elements is protective in certain paradigms of neuronal death. For example, cyclinD1 transcripts and Cdk4/Cdc2 protein levels are upregulated during death of sympathetic neurons and neuronal PC12 cells deprived of NGF [22], [23]. In addition, cyclinD1, cyclinB, and Cdk4 levels are upregulated in brains of AD patients and during stroke/excitotoxic damage [12], [37], [39], [43], [47], [70], [73]. Multiple lines of evidence suggest that these presumed CDK complexes are active. For example, we have shown that cyclin D1 associated kinase activity increases during death of cultured cortical neurons evoked by DNA damage [57]. In addition, increases in cyclin B associated kinases activity have been observed in brains of AD patients. [73]. Numerous reports have indicated that an important target of the CDKs, Rb, is phosphorylated on a CDK consensus site during neuronal injury evoked by stroke [50], DNA damage [55], B-amyloid [15], [26], cisplatinum [24] or K+ deprivation [51]. Finally, an increase in E2F1 transcripts and/or protein, a possible consequence of Rb deregulation have been described in models of neuronal death induced by B-amyloid toxicity [26], K+ deprivation [49], and ischemia [50]. These studies suggest not only an increase in cell cycle associated regulatory proteins with injury, but also an increase in their activities.
Is enhanced cell cycle molecule activity a death signal in neurons or merely a protective response or epiphenomenon? In a number of the cases that we have tested, cell cycle molecules appear to play an active role in induction of death. For example, inhibition of CDK activity by pharmacological agents or by expression of dominant negative forms of CDKs 4 and 6 protect against neuronal death evoked by beta amyloid [15], [26], DNA damage [56], [57], [58], loss of trophic support [53], [54] and loss of depolarization [51].
While cell cycle regulators have been implicated in a wide variety of death paradigms, little attention has been given to the issue of whether they play an important role in death initiated by excitotoxic/oxidative stress. In fact, death of sympathetic neurons evoked by superoxide 1 dismutase depletion, a paradigm of oxidative stress, was not inhibitable by CDK inhibitors [58]. However, growing evidence, including the work that follows, suggests that delayed death initiated by excitotoxic stress may be regulated through cell cycle components as well. Such observations have potentially relevant implications for amelioration of neural damage associated with aging.
Section snippets
Kainic acid treatment of mice
CD-1 mice (8–10 weeks of age; Charles River) were administered intraperitoneal (i.p.) injections of saline or KA (35 mg/kg in saline; Sigma). At the appropriate times indicated in the text, the animals were sacrificed and prepared for histologic and immunohistochemical analyses.
Histology and immunohistochemistry
Mice were perfused with 50 ml saline followed by 50 ml of 4% paraformaldehyde in phosphate-buffered saline (PBS). The brains were removed, postfixed in 10% formalin for 5 days, and then paraffin embedded. Brain sections
Cell cycle markers are induced in vulnerable neurons after KA treatment in vivo
KA, a potent excitotoxin, activates the AMPA/kainate class of glutamate receptors and is known to cause death of defined regions of the brain including the CA3/CA1 neurons of the hippocampus [67]. We examined whether the cell cycle pathway was activated in these neurons. As previously described, i.p. injection of KA (35 mg/kg) produced rigid posture within 10 min after injection. Automation, such as head bobbing and repetitive scratching occurred shortly after and seizures were detected within
Discussion
The results presented here support the intriguing possibility that cell cycle related death pathways may be activated in response to some forms of excitotoxic stress. First, cell cycle pathways which may include cyclin D1 associated kinase activity and pRb phosphorylation are activated in vulnerable regions of the hippocampus after KA administration. Second, the CDK inhibitor flavopiridol, when coadministered with MK-801, protects cultured neurons from 3-NP induced death. While these findings
Acknowledgements
This work was supported by grants from the Heart and Stroke foundation of Canada (DSP) and National Institutes of Health and Blanchette Rockefeller Foundation (LAG). DSP is a recipient of a Glaxo Wellcome Research Chair.
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