Lloyd A Greene

David X Liu

Carol M Troy

Subhas C Biswas

Cell Cycle Molecules Define A Pathway Required For Neuron Death In Development And Disease

Keywords: Neuron death, apoptosis, cell cycle, Cdk4, Myb, Bim

We review here evidence defining a molecular pathway that includes cell cyclerelated molecules and that appears to play a required role in neuron death during normal development as well as in disease and trauma. The pathway starts with inappropriate activation of cyclin dependent kinase 4 (Cdk4) in neurons which leads to hyper-phosphorylation of the pRb family member p130. This in turn results in dissociation of p130 and its associated chromatin modifiers Suv39H1 and HDAC1 from the transcription factor E2F4. Dissociation of this complex results in derepression of genes with E2F binding sites including those encoding the transcription factors B-and C-Myb. Once elevated in neurons, B-and C-Myb proteins bind to the promoter for the pro-apoptotic BH3-only protein Bim and promote its induction. Bim then interacts with the core cellular apoptotic machinery, leading to c aspase activation and apoptotic death. This pathway is supported by a variety of observations and experimental findings that implicate it as a required element for neuron loss in development and in many nervous system traumas and disorders. The components of this pathway appear to represent potential therapeutic targets for prevention of disease-associated neuron death.

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Introduction and overview.

As recounted by reviews in this special issue and elsewhere [START_REF] Arendt | Aberrancies in signal transduction and cell cycle related events in Alzheimer's disease[END_REF][START_REF] Becker | Cell cycle regulation of neuronal apoptosis in development and disease[END_REF][START_REF] Copani | Alzheimer's disease research enters a "new cycle": how significant?[END_REF][START_REF] Greene | Cell cycle molecules and vertebrate neuron death: E2F at the hub[END_REF][START_REF] Herrup | Divide and die: cell cycle events as triggers of nerve cell death[END_REF][START_REF] Liu | Neuronal apoptosis at the G1/S cell cycle checkpoint[END_REF][START_REF] Nagy | The last neuronal division: a unifying hypothesis for the pathogenesis of Alzheimer's disease[END_REF][START_REF] Neve | The cell cycle as a therapeutic target for Alzheimer's disease[END_REF][START_REF] Smith | Emerging pathogenic role for cyclin dependent kinases in neurodegeneration[END_REF][START_REF] Ueberham | The expression of cell cycle proteins in neurons and its relevance for Alzheimer's disease[END_REF][START_REF] Vincent | The cell cycle and human neurodegenerative disease[END_REF][START_REF] Webber | The cell cycle in Alzheimer disease: a unique target for neuropharmacology[END_REF], cell cycle-related molecules appear to play key roles in neuron death associated with development and disease. Our aim here will be to describe the components of a molecular pathway in which cell cycle elements participate in neuron death. We will relate how activation of a cyclin-dependent kinase in neurons triggers a chain of molecular events that ultimately engages the core apoptotic machinery and culminates in cellular demise.

There are six overarching themes of this article. The first is that there are striking and mutually informative similarities between the molecular mechanisms that govern neuron death during normal development and in neurological disorders. The second is that neuron death in both instances involves similar changes in the activities and expression of molecules otherwise associated with the cell cycle. The third is that the pathway includes the sequential activation of cyclin-dependent kinase 4 (Cdk4); hyper-phosphorylation of the Rb family member p130; dissociation of p130 and associated chromatin modifiers from the transcription factor E2F4, resulting in gene de-repression; de-repression of the genes encoding the transcription factors B-and C-Myb; enhanced occupancy by B-and C-Myb of the promoter for the pro-apoptotic BH3-only protein Bim; and activation by Bim of the core apoptotic machinery. The fourth theme is that neuron death mediated by this pathway is not simply due to an aborted attempt to traverse the cell cycle, but rather reflects alternative actions of molecules that are also involved in cell proliferation. Fifth, although this pathway is necessary for neuron death under many conditions, it is not sufficient and works in conjunction with additional signaling pathways to mediate apoptosis. Lastly, our understanding of the specific molecules that contribute to neuron death in disease provides targets for novel therapeutic opportunities.

Cell cycle molecules and developmental neuron death.

During normal vertebrate development, approximately 50% of all post-mitotic neurons that are born undergo cell death [START_REF] Oppenheim | Cell death during development of the nervous system[END_REF]. An abundance of evidence supports a model in which the decision of which neurons live or die is regulated by their competition for limiting amounts of target-derived trophic factors [START_REF] Davies | The neurotrophic hypothesis: where does it stand?[END_REF][START_REF] Bennet | Neuronal cell death, nerve growth factor and neurotrophic models: 50 years on[END_REF][START_REF] Levi-Montalcini | The saga of the nerve growth factor[END_REF][START_REF] Cowan | Viktor Hamburger and Rita Levi-Montalcini: the path to the discovery of nerve growth factor[END_REF]. The best studied of these trophic factors with respect to neuronal survival and death is nerve growth factor (NGF). In vitro studies, largely (but by no means exclusively) carried out with rodent sympathetic neurons and PC12 pheochromocytoma cells have revealed a wealth of information about what happens in nerve cells when they are deprived of NGF and how this results in their apoptotic death. This has been strongly complemented by in vivo and in vitro studies with other trophic factor systems and models.

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Among early observations relevant to our theme was that NGF deprivation in sympathetic neuron cultures was followed by elevation of transcripts encoding cyclin D1 (CycD1), a regulator of cyclin dependent kinases 4 and 6 (Cdk4/6) [START_REF] Freeman | Analysis of cell cycle-related gene expression in postmitotic neurons: selective induction of Cyclin D1 during programmed cell death[END_REF]. In proliferation-competent cells, an increase in CycD1 protein levels triggers movement from the G0 or G1 phase of the cycle into G1/S [START_REF] Stevaux | A revised picture of the E2F transcriptional network and RB function[END_REF][START_REF] Cobrinik | Pocket proteins and cell cycle control[END_REF]. It was also reported that the apoptotic stimulus of serum deprivation in cultures of neuronally differentiated murine neuroblastoma cells resulted in activation of CycD1-dependent cdk activity [START_REF] Kranenburg | Cyclin D1 is an essential mediator of apoptotic neuronal cell death[END_REF]. Moreover, blockade of this activity by over-expression of the cdk inhibitory protein p16INK4A1, was protective. Additional evidence linking loss of trophic support to dysregulation of G1/S markers and other cell cycle proteins came from a genetic model in which post-mitotic cerebellar granule cells die due to loss of their synaptic targets and presumptive source of trophic support [START_REF] Herrup | The induction of multiple cell cycle e vents precedes target-related neuronal death[END_REF]. In this model, neuron death was also associated with elevation of cyclin D and other cell cyclerelated proteins.

If G1/S-related events are required for developmental neuron death, it follows that agents that interfere with the G1/S transition should be protective. Indeed, blockade of this transition with a dominant-negative form of Ras or with a variety of small chemical G1/S inhibitors effectively rescued neuronal cells from NGF deprivation [START_REF] Ferrari | Proliferative inhibition by dominant-negative Ras rescues naive and neuronally differentiated PC12 cells from apoptotic death[END_REF][START_REF] Farinelli | Cell cycle blockers mimosine, ciclopirox, and deferoxamine prevent the death of PC12 cells and postmitotic sympathetic neurons after removal of trophic support[END_REF].

The potential involvement of cyclin D1 in developmental neuron death implicates its partners cdks 4 and 6. These play important roles in permitting replicationcompetent cells to undergo the G1/S transition [START_REF] Stevaux | A revised picture of the E2F transcriptional network and RB function[END_REF][START_REF] Cobrinik | Pocket proteins and cell cycle control[END_REF]. One test of the potential importance of these proteins in developmental cell death has been the employment of small chemical cdk inhibitors such as olomoucine, roscovitine and flavopiridol. These have proved to be remarkably effective in blocking neuronal apoptosis evoked by trophic factor deprivation [START_REF] Park | Inhibitors of cyclin-dependent kinases promote survival of post-mitotic neuronally differentiated PC12 cells and sympathetic neurons[END_REF][START_REF] Markus | Survival-promoting activity of inhibitors of cyclin-dependent kinases on primary neurons correlates with inhibition of c-Jun kinase-1[END_REF][START_REF] Maas | Apoptosis of central and peripheral neurons can be prevented with cyclin-dependent kinase/mitogen-activated protein kinase inhibitors[END_REF][START_REF] Padmanabhan | Role of cell cycle regulatory proteins in cerebellar granule neuron apoptosis[END_REF][START_REF] Appert-Collin | Cyclin dependent kinase inhibitors prevent apoptosis of postmitotic mouse motoneurons[END_REF]. Although widely employed, one weakness of such drugs is that they lack specificity both within the cdk family and with respect to other kinases [START_REF] Bain | The specificities of protein kinase inhibitors: an update[END_REF][START_REF] Blagden | Drugging cell cycle kinases in cancer therapy[END_REF][START_REF] Sausville | Cyclin-dependent kinase modulators studied at the NCI: preclinical and clinical studies[END_REF][START_REF] Sridhar | Selectivity and potency of cyclindependent kinase inhibitors[END_REF] and this has complicated the mechanistic interpretation of their actions in neuron death. An alternative approach has been the delivery of constructs encoding naturally occurring cdk inhibitor proteins such as p21, p16(INK4A1), and p27(kip1). Over-expression of these proteins protects neurons from trophic factor deprivation, reinforcing a role for cdks in neuronal developmental cell death [START_REF] Kranenburg | Cyclin D1 is an essential mediator of apoptotic neuronal cell death[END_REF][START_REF] Park | Cyclin dependent kinase inhibitors and dominant negative cyclin dependent kinase 4 and 6 promote survival of NGF-deprived sympathetic neurons[END_REF]. To test the roles of specific cdks in neuron death, dominant-negative forms of these proteins have been expressed in neurons. It was observed that virally-delivered dominant-negative forms of Cdk4 and 6, but not of Cdk2 and Cdk3 protected sympathetic neurons from NGF deprivation [START_REF] Park | Cyclin dependent kinase inhibitors and dominant negative cyclin dependent kinase 4 and 6 promote survival of NGF-deprived sympathetic neurons[END_REF]. Because of the potential capacity of dominant negative constructs to interact with and inhibit shared targets and regulators, we have more recently used shRNAs to specifically knock down Cdk4 [START_REF] Biswas | Bim is a direct target of a neuronal E2F-dependent apoptotic pathway[END_REF]. This too has protected against NGF deprivation, further implicating this partner of CycD1 as a required element in the mechanism by which loss of trophic support triggers apoptotic death.

Although Cdk4 has multiple potential targets, its major substrates during cell proliferation are members of the Rb family of "pocket" proteins, namely pRb, p107 and p130 [START_REF] Stevaux | A revised picture of the E2F transcriptional network and RB function[END_REF][START_REF] Cobrinik | Pocket proteins and cell cycle control[END_REF]. (In this review, we will use Rb to refer to the entire family and pRb to indicate this specific family member). In proliferating cells, hyper-phosphorylation of Rb by Cdk4 is an important event in the G1/S transition. In accordance with the involvement of Cdk4 activation in developmental neuron death, all three Rb family members rapidly undergo hyper-phosphorylation in response to trophic factor deprivation [START_REF] Padmanabhan | Role of cell cycle regulatory proteins in cerebellar granule neuron apoptosis[END_REF][START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF].

Although initial studies focused on pRb and its role in survival and death of postmitotic neurons [START_REF] Jacks | Effects of an Rb mutation in the mouse[END_REF][START_REF] Feddersen | In vivo viability of postmitotic Purkinje neurons requires pRb family member function[END_REF], more recent findings suggest that this protein may be more involved in regulating proliferation of neuronal precursor cells than in maintaining neuron survival [START_REF] Ferguson | Telencephalon-specific Rb knockouts reveal enhanced neurogenesis, survival and abnormal cortical development[END_REF][START_REF] Macpherson | Conditional mutation of Rb causes cell cycle defects without apoptosis in the central nervous system[END_REF]. Our findings suggest that the family member that is most important for regulating neuron survival and death in p130 [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF]. In support of this, electrophoretic mobility shift assays (EMSA) indicate that relevant E2F-binding DNA complexes formed in post-mitotic neurons contain mainly p130 rather than pRb or p107. Moreover, the importance of p130 in neuron survival is underscored by the observation that knockdown of p130, but not of pRb or p107 induces neuron death [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF].

How does p130 maintain neuron survival and how does its hyper-phosphorylation by Cdk4 result in developmental neuron death? An important (but not the only) activity of Rb family proteins is to associate with members of the E2F transcription factor family [START_REF] Stevaux | A revised picture of the E2F transcriptional network and RB function[END_REF][START_REF] Cobrinik | Pocket proteins and cell cycle control[END_REF]. Such complexes have two roles. One is to suppress E2F's transactivating activity and the other is to repress genes to which they are bound.

Hyper-phosphorylation of Rb by Cdk4 causes such complexes to dissociate. This has two potential consequences: release of E2F so that it can now transactivate suitable targets and de-repression and induction of genes otherwise silenced by E2F-Rb complexes [START_REF] Stevaux | A revised picture of the E2F transcriptional network and RB function[END_REF][START_REF] Cobrinik | Pocket proteins and cell cycle control[END_REF].

In response to trophic factor deprivation, as described above, p130 undergoes hyper-phosphorylation. It appears that a major effect of this is translocation of p130 out of the nucleus [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF]. This presumably further reduces its capacity to participate in gene repression. EMSA, chromatin immunoprecipitation (ChIP) and luciferase reporter assays all confirm that the associations of p130 with its DNA targets are lost in response to NGF deprivation and that this requires cdk activity [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF].

As noted above, loss of p130-E2F complexes due to hyper-phosphorylation by activated Cdk4 permits both elevated E2F transactivational activity and derepression of genes with E2F binding sites. Of these, multiple lines of evidence point to the latter as the most relevant to developmental neuronal death [START_REF] Liu | Regulation of neuronal survival and death by E2Fdependent gene repression and derepression[END_REF]. For instance, a decoy DNA construct with multiple E2F binding sites that effectively soaks up endogenous E2F is sufficient to trigger neuron death. This is consistent with the de-repression mechanism of death in that sequestration of E2F should result in loss of E2F-p130 complexes from DNA. On the other hand, if death were due to E2F-driven transactivation, such a construct would be (but is not) protective.

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Another supportive finding is that constructs encoding phosphorylation-resistant Rb mutants or fusion proteins between E2F and Rb, which should selectively bind to and silence genes with E2F binding sites, effectively protect neurons from NGF deprivation [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF][START_REF] Liu | Regulation of neuronal survival and death by E2Fdependent gene repression and derepression[END_REF]. As discussed below, additional experiments show de-repression of specific death-associated genes when NGF is withdrawn.

There is partial insight as to how p130 represses genes that lead to developmental neuron death.

In addition to associating with E2F, Rb family members simultaneously bind molecules that remodel chromatin [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF]. Thus, within complexes with E2F, Rb family members serve as tethers for proteins that modify chromatin and that promote gene silencing. Two such p130-associated proteins have been identified in neurons, histone deacetylase (HDAC1) and Suv39H1 [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF]. Both are associated with p130 in healthy neurons and both are lost from complexes with p130 and E2F on DNA under conditions of NGF withdrawal [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF]. Suv39H1 (suppressor of variegation 3-9 homolog 1) is a histone methyltransferase that transfers methyl groups to lysine 9 on histone H3 [START_REF] Schotta | SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing[END_REF] while HDAC1 deacetylates histones [START_REF] Marks | Histone deacetylases[END_REF]. Both modifications promote gene repression. As evidence of its role in maintaining neuron survival, loss of cellular Suv39H1 or transfection with an Suv39H1 mutant that binds p130 but lacks methyltransferase activity causes both gene de-repression and neuron death [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF]. In addition, inhibition of HDAC activity also leads to gene derepression and to neuron death [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF][START_REF] Salminen | Neuronal apoptosis induced by histone deacetylase inhibitors[END_REF][START_REF] Boutillier | Selective E2F-dependent gene transcription is controlled by histone deacetylase activity during neuronal apoptosis[END_REF]. Finally, although E2F-p130 fusion constructs protect neurons from NGF deprivation, such constructs that are defective in binding HDAC and Suv39H1 lack protective activity [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF]. There are other chromatin modifiers in addition to HDAC1 and Suv39H1 that could conceivably play roles in p130-dependent neuron survival [cf. 46] and it will be of interest to identify these in future studies.

Like the Rb family, the E2F family consists of multiple members and there appears to be a selectivity for the association between specific Rb and E2F proteins [START_REF] Stevaux | A revised picture of the E2F transcriptional network and RB function[END_REF][START_REF] Cobrinik | Pocket proteins and cell cycle control[END_REF][START_REF] Crosby | Opposing roles of E2Fs in cell proliferation and death[END_REF][START_REF] Dimova | The E2F transcriptional network: old acquaintances with new faces[END_REF]. In particular, the preferred partners for p130 are E2F4 and E2F5. Our finding indicate the most relevant partner for p130 in maintaining neuron survival is E2F4 [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF]. E2F4, but not other E2F family members were found to occupy the promoter of the E2F-repressed pro-apoptotic gene B-Myb in healthy neurons (see below for further discussion) and was bound there to p130, but not pRb or p107. In addition, knock-down of E2F4,as in the case of p130, is sufficient to trigger apoptotic neuronal death. Taken together, such findings support the view that intact E2F4-p130 complexes and their associated chromatin modifiers are required to maintain neuron survival and that in trophic factor deprivation, such complexes are lost, resulting in de-repression of genes that promote death.

A key issue regards the identities of the death-associated genes that are repressed by p130-E2F4 complexes to maintain neuron survival. One important family of such molecules has been identified to date, the B-and C-members of the Myb family of transcription factors. B-and C-Myb are classic examples of genes that possess binding sites for E2F and that are repressed by E2F-Rb family member complexes [START_REF] Joaquin | Cell cycle regulation by the B-Myb transcription factor[END_REF][START_REF] Sala | B-MYB, a transcription factor implicated in regulating cell cycle, apoptosis and cancer[END_REF]. In response to cdk-dependent hyper-phosphorylation of Rb family members in proliferating cells, B-and C-Myb are induced and play major roles in promoting cell cycle progression [START_REF] Joaquin | Cell cycle regulation by the B-Myb transcription factor[END_REF][START_REF] Sala | B-MYB, a transcription factor implicated in regulating cell cycle, apoptosis and cancer[END_REF]. B-and C-Myb are also induced in neurons after NGF deprivation [START_REF] Liu | Regulation of neuronal survival and death by E2Fdependent gene repression and derepression[END_REF], but in this case, they promote apoptotic death. In healthy neurons, the promoters for B-and C-myb are occupied by p130-E2F4 along with Suv39H1 and HDAC1, and B-and C-Myb expression is suppressed. In response to NGF deprivation, E2F4-p130-Suv39H1/HDAC1 complexes are lost from at least the Bmyb promoter and expression of Myb transcripts and protein is elevated [START_REF] Liu | Regulation of neuron survival and death by p130 and associated chromatin modifiers[END_REF]. This induction promotes death in that over-expression of B-or C-myb triggers neuronal apoptosis while suppression of B -or C -myb induction by siRNA or antisense constructs protects neurons from NGF deprivation [START_REF] Liu | Regulation of neuronal survival and death by E2Fdependent gene repression and derepression[END_REF][START_REF] Liu | B-myb and C-myb play required roles in neuronal apoptosis evoked by nerve growth factor deprivation and DNA damage[END_REF]. These findings thus establish that B-and C-Myb are among the genes that are de-repressed in neurons when p130 is hyper-phosphorylated after NGF deprivation and that their expression is required for apoptotic death under such conditions.

Because Mybs are transcription factors, their role in neuron death most likely results from regulation of other genes. To date, we have identified one important and relevant target of Mybs in cells deprived of trophic support and this is the apoptosis facilitator molecule Bim/Bcl2-like 11. Bim is a pro-apoptotic member of the Bcl2 family that belongs to the subgroup of BH3-only proteins [START_REF] Strasser | The role of bim, a proapoptotic BH3only member of the Bcl-2 family in cell-death control[END_REF][START_REF] Willis | Life in the balance: how BH3-only proteins induce apoptosis[END_REF]. Current evidence indicates that Bim binds to and neutralizes the function of pro-survival proteins such as Bclx and Bcl2 and by this means promotes caspase activation and apoptotic death. Bim is induced in response to NGF deprivation and its knockdown partially protects neurons from withdrawal of trophic support [START_REF] Biswas | Bim is a direct target of a neuronal E2F-dependent apoptotic pathway[END_REF][START_REF] Putcha | Induction of BIM, a proapoptotic BH3-only BCL-2 family member, is critical for neuronal apoptosis[END_REF][START_REF] Whitfield | Dominantnegative c -Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release[END_REF][START_REF] Biswas | Nerve growth factor (NGF) down-regulates the Bcl-2 homology 3 (BH3) domain-only protein Bim and suppresses its proapoptotic activity by phosphorylation[END_REF]. Therefore, it appears to be among the regulated genes that are involved in promoting developmental neuron death. Two binding sites for Mybs have been found in the Bim promoter. Significantly, we found that mutation of these sites abolishes the induction of a Bim reporter construct that occurs in response to NGF deprivation [START_REF] Biswas | Bim is a direct target of a neuronal E2F-dependent apoptotic pathway[END_REF]. Moreover, occupancy of these sites by at least C-myb is elevated by NGF withdrawal. Additional evidence that Bim is a target of Mybs comes from findings that induction of Bim by NGF deprivation is blocked by Myb shRNAs [START_REF] Biswas | Bim is a direct target of a neuronal E2F-dependent apoptotic pathway[END_REF]. In line with the pathway described above, Bim induction via Mybs during NGF deprivation is dependent on Cdk4 and E2F-mediated gene de-repression [START_REF] Biswas | Bim is a direct target of a neuronal E2F-dependent apoptotic pathway[END_REF]. Thus, Bim induction after NGF withdrawal is blocked by cdk inhibitors and Cdk4 shRNA and is suppressed by the E2F-Rb fusion construct described above that silences genes with E2F binding sites.

Considered together, present findings indicate that cell-cycle-related events that are triggered by loss of trophic support in neurons constitute a pathway that culminates in induction of at least one protein, Bim, with clear pro-apoptotic activity and in apoptotic death (see the scheme in Figure 1). To summarize, loss of trophic support in models of developmental neuron death leads sequentially to 1) activation of Cdk4; 2) hyper-phosphorylation of p130; 3) dissociation of complexes containing E2F4, p130, Suv39H1 and HDAC1; 4) de-repression of genes occupied by the latter complexes, including those encoding B-and C-myb; 5) accumulation of elevated levels of cellular B-and C-myb proteins; 6) binding of B-and C-myb to the promoter for the gene encoding the pro-apoptotic protein Bim; 7) induction of Bim protein; 8) engagement and activation of the core apoptotic machinery by Bim via its neutralization of pro-survival Bcl2 proteins; 9) caspase activation and death.

Cell cycle molecules in neuron death associated with diverse apoptotic stimuli. Although many of the mechanistic studies described above have focused on neuron death associated with NGF deprivation, it is important to emphasize that at least several aspects of this scheme appear to pertain to a number of additional apoptotic paradigms. For instance, many of the cell cycle pathway elements that characterize neuron death evoked by NGF deprivation also mediate death of both peripheral and CNS neurons caused by DNA damaging agents [START_REF] Liu | B-myb and C-myb play required roles in neuronal apoptosis evoked by nerve growth factor deprivation and DNA damage[END_REF][START_REF] Park | Cyclin-dependent kinases participate in death of neurons evoked by DNAdamaging agents[END_REF][START_REF] Park | Involvement of retinoblastoma family members and E2F/DP complexes in the death of neurons evoked by DNA damage[END_REF]. Components of this pathway have also been implicated death of various neuron types brought about by various insults including hypoxia [START_REF] Osuga | Cyclin-dependent kinases as a therapeutic target for stroke[END_REF][START_REF] Wen | Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats[END_REF], excitotoxic injury [START_REF] Giardina | Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin D1[END_REF][START_REF] Ino | Cyclin-dependent kinase 4 and cyclin D1 are required for excitotoxin-induced neuronal cell death in vivo[END_REF][START_REF] Park | Cell cycle regulators in neuronal death evoked by excitotoxic stress: implications for neurodegeneration and its treatment[END_REF], physical trauma [START_REF] Di Giovanni | Gene profiling in spinal cord injury shows role of cell cycle in neuronal death[END_REF], proteasomal inhibitors [START_REF] Boutillier | Depolarization regulates cyclin D1 degradation and neuronal apoptosis: a hypothesis about the role of the ubiquitin/proteasome signalling pathway[END_REF][START_REF] Rideout | Cyclin-dependent kinase activity is required for apoptotic death but not inclusion formation in cortical neurons after proteasomal inhibition[END_REF][START_REF] Staropoli | The ubiquitin-proteasome pathway is necessary for maintenance of the postmitotic status of neurons[END_REF], and withdrawal of support by hyperpolarizing levels of K+ [START_REF] Padmanabhan | Role of cell cycle regulatory proteins in cerebellar granule neuron apoptosis[END_REF]. Such considerations by no means imply that the p athway described here or variations that employ alternative sets of cycle-related proteins are universally involved in neuron death. Nevertheless, present data encourage us consider the apoptotic cell-cycle pathway in paradigms well beyond just the case of NGF deprivation. In this context, we next review evidence for the role of the apoptotic cell cycle pathway in neurological disorders.

Cell cycle molecules in neuron death associated with neurological disorders: parallels with developmental neuron death.

Several types of evidence have linked cell cycle molecules to neuron death in disease. The first are correlative observations that cell cycle-related proteins are elevated in neurons that are at risk to die in various neurodegenerative disorders, traumatic injuries and ischemia/stroke, either in human disease or models thereof. These findings have been extensively reviewed in this volume and elsewhere and so need not be recounted here in depth. However, of particular relevance is that these changes include the same molecules involved in developmental neuron death. For instance neuronal cyclin D is elevated in models of ischemia/stroke [START_REF] Park | Involvement of retinoblastoma family members and E2F/DP complexes in the death of neurons evoked by DNA damage[END_REF][START_REF] Osuga | Cyclin-dependent kinases as a therapeutic target for stroke[END_REF][START_REF] Guegan | c-Jun and cyclin D1 proteins as mediators of neuronal death after a focal ischaemic insult[END_REF][START_REF] Li | Granule cell apoptosis and protein expression in hippocampal dentate gyrus after forebrain ischemia in the rat[END_REF][START_REF] Kuroiwa | Expression of cell cycle-related genes in dying cells in retinal ischemic injury[END_REF][START_REF] Timsit | Increased cyclin D1 in vulnerable neurons in the hippocampus after ischaemia and epilepsy: a modulator of in vivo programmed cell death?[END_REF][START_REF] Chen | Cyclooxygenase-2 activity contributes to neuronal expression of cyclin D1 after anoxia/ischemia in vitro and in vivo[END_REF][START_REF] Sakurai | Cyclin D1 and Cdk4 protein induction in motor neurons after transient spinal cord ischemia in rabbits[END_REF], ALS [START_REF] Nguyen | Cell cycle regulators in the neuronal death pathway of amyotrophic lateral sclerosis caused by mutant superoxide dismutase 1[END_REF], Alzheimer disease (AD) [START_REF] Copani | Mitotic signaling by beta-amyloid causes neuronal death[END_REF], excitotoxic stress [START_REF] Staropoli | The ubiquitin-proteasome pathway is necessary for maintenance of the postmitotic status of neurons[END_REF][START_REF] Guegan | c-Jun and cyclin D1 proteins as mediators of neuronal death after a focal ischaemic insult[END_REF][START_REF] Li | Granule cell apoptosis and protein expression in hippocampal dentate gyrus after forebrain ischemia in the rat[END_REF], and spinal cord injury [START_REF] Di Giovanni | Gene profiling in spinal cord injury shows role of cell cycle in neuronal death[END_REF] as well as in affected neurons in human patients with AD [START_REF] Busser | Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer's disease brain[END_REF][START_REF] Yang | DNA replication precedes neuronal cell death in Alzheimer's disease[END_REF][START_REF] Yang | Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer's disease[END_REF][START_REF] Hoozemans | Cyclin D1 and cyclin E are co-localized with cyclooxygenase 2 (COX-2) in pyramidal neurons in Alzheimer disease temporal cortex[END_REF], ischemia [START_REF] Love | Neuronal expression of cell cycle-related proteins after brain ischaemia in man[END_REF] and sporadic ALS [START_REF] Ranganathan | Alterations in G(1) to S phase cell-cycle regulators during amyotrophic lateral sclerosis[END_REF]. Likewise, neuronal Cdk4 expression is reported to be up in models of ischemia/stroke [START_REF] Park | Involvement of retinoblastoma family members and E2F/DP complexes in the death of neurons evoked by DNA damage[END_REF][START_REF] Sakurai | Cyclin D1 and Cdk4 protein induction in motor neurons after transient spinal cord ischemia in rabbits[END_REF], excitotoxicity [START_REF] Ino | Cyclin-dependent kinase 4 and cyclin D1 are required for excitotoxin-induced neuronal cell death in vivo[END_REF], ALS [START_REF] Nguyen | Cell cycle regulators in the neuronal death pathway of amyotrophic lateral sclerosis caused by mutant superoxide dismutase 1[END_REF] as well as in neurons from patients with AD [START_REF] Busser | Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer's disease brain[END_REF][START_REF] Mcshea | Abnormal expression of the cell cycle regulators P16 and CDK4 in Alzheimer's disease[END_REF] and stroke [START_REF] Love | Neuronal expression of cell cycle-related proteins after brain ischaemia in man[END_REF]. Consistent with elevated cyclin D and Cdk4, phospho-pRb immunostaining in relevant neurons is increased in models of ischemia/stroke [START_REF] Osuga | Cyclin-dependent kinases as a therapeutic target for stroke[END_REF][START_REF] Hayashi | Phosphorylation of retinoblastoma protein in rat brain after transient middle cerebral artery occlusion[END_REF][START_REF] Rashidian | Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo[END_REF] and AD [START_REF] Xiang | Cyclooxygenase (COX)-2 and cell cycle activity in a transgenic mouse model of Alzheimer's disease neuropathology[END_REF][START_REF] Giovanni | Involvement of cell cycle elements, cyclin-dependent kinases, pRb, and E2F x DP, in B-amyloid-induced neuronal death[END_REF] and in patients with Parkinson's Disease (PD) [START_REF] Jordan-Sciutto | Expression patterns of retinoblastoma protein in Parkinson disease[END_REF], AD [START_REF] Hoozemans | Cyclin D1 and cyclin E are co-localized with cyclooxygenase 2 (COX-2) in pyramidal neurons in Alzheimer disease temporal cortex[END_REF][START_REF] Jordan-Sciutto | Altered distribution of cell cycle transcriptional regulators during Alzheimer disease[END_REF] and sporadic ALS [START_REF] Ranganathan | Alterations in G(1) to S phase cell-cycle regulators during amyotrophic lateral sclerosis[END_REF]. With respect to Bim induction, this has been found for experimental optic nerve transection [START_REF] Napankangas | Rat retinal ganglion cells upregulate the pro-apoptotic BH3-only protein Bim after optic nerve transection[END_REF], seizure [START_REF] Shinoda | Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy[END_REF] and neonatal hypoxia/ischemia [START_REF] Ness | Selective involvement of BH3-only Bcl-2 family members Bim and Bad in neonatal hypoxia-ischemia[END_REF] and in human seizure [START_REF] Shinoda | Bim regulation may determine hippocampal vulnerability after injurious seizures and in temporal lobe epilepsy[END_REF]. Western immunoblotting of AD and control frontal cortex also revealed elevation of Bim protein in the former [START_REF] Engidawork | Expression of apoptosis related proteins in brains of patients with Alzheimer's disease[END_REF].

A C C E P T E D M A N U S C R I P T

Although there has been little work on Mybs in neurodegenerative diseases, there is evidence for their de-repression in at least one human disorder, autoimmune paraneoplastic cerebellar degeneration (PCD). In this malady, the target antigen of auto-antibodies is pcd-17, a protein that blocks B-myb de-repression [START_REF] Sakai | Effect of a paraneoplastic cerebellar degeneration-associated neural protein on B -myb promoter activity[END_REF]. The pcd-17 auto-antibodies appear to enter Purkinje cells and block pcd-17 function and by this means, permit B-Myb de-repression and consequent death.

The second type of evidence that cell-cycle related molecules play roles in nervous system disease is based on experimental manipulation of their activity or expression in cell culture and animal models. Among the most widely used approaches is the application of small chemical Cdk inhibitors (generally olomoucine, roscovitine or flavopiridol, though others have also been employed) to disease models and the demonstration that these have ameliorative actions on behavior and/or neuron survival. Examples in which this approach has been successful include models of AD [START_REF] Di Giovanni | Gene profiling in spinal cord injury shows role of cell cycle in neuronal death[END_REF], ischemia/stroke [START_REF] Park | Involvement of retinoblastoma family members and E2F/DP complexes in the death of neurons evoked by DNA damage[END_REF][START_REF] Wang | Inhibition of cyclin-dependent kinases improves CA1 neuronal survival and behavioral performance after global ischemia in the rat[END_REF], excitotoxic stress [START_REF] Park | Cell cycle regulators in neuronal death evoked by excitotoxic stress: implications for neurodegeneration and its treatment[END_REF][START_REF] Verdaguer | Inhibition of cell cycle pathway by flavopiridol promotes survival of cerebellar granule cells after an excitotoxic treatment[END_REF][START_REF] Verdaguer | 3-Amino thioacridone, a selective cyclin-dependent kinase 4 inhibitor, attenuates kainic acidinduced apoptosis in neurons[END_REF], Niemann-Pick Disease [START_REF] Zhang | Cyclin-dependent kinase inhibitors attenuate protein hyperphosphorylation, cytoskeletal lesion formation, and motor defects in Niemann-Pick Type C mice[END_REF], traumatic brain injury [START_REF] Di Giovanni | Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury[END_REF], optic nerve transection [START_REF] Lefevre | Involvement of cyclindependent kinases in axotomy-induced retinal ganglion cell death[END_REF], and spinal cord injury [START_REF] Tian | Suppression of astroglial scar formation and enhanced axonal regeneration associated with functional recovery in a spinal cord injury rat model by the cell cycle inhibitor olomoucine[END_REF].

While the experimental use of such chemical inhibitors has supported the involvement of a cdk-dependent pathway in disease-related neuron death and may well point the way to clinically useful drugs, there are several serious reservations about this interpretation. One issue regards the specificity of such agents for cdks. As in the case of most drugs, one cannot rule out actions on other classes of enzymes, especially kinases, and indeed such activities have been described for flavopiridol, olomoucine and roscovitine [cf 30 -33]. However, the fact that all three inhibitors seem to have similar actions in protecting neurons does argue that noncdk actions are less likely.

A related concern is the selectivity of these agents for various cdk family members. Each of these drugs targets different cdks with variable levels of specificity. For instance, flavopiridol has been described as a "pan-cdk" inhibitor [START_REF] Sausville | Cyclin-dependent kinase modulators studied at the NCI: preclinical and clinical studies[END_REF] and there are no published reports in which the actions of flavopiridol or other such inhibitors have been systematically evaluated on all known cdks. To compound the issue, accumulating evidence has implicated Cdk5, which is a target of many cdk inhibitors, in neuron death associated with neurological disorders [101][START_REF] O'hare | Differential roles of nuclear and cytoplasmic cyclin-dependent kinase 5 in apoptotic and excitotoxic neuronal death[END_REF][START_REF] Shelton | Cyclin-dependent kinase-5 in neurodegeneration[END_REF][START_REF] Weishaupt | Cyclin-dependent kinase 5 (CDK5) and neuronal cell death[END_REF][START_REF] Lim | Cyclin-dependent kinases in neural development and degeneration[END_REF]. This raises several possibilities. One is that cdks other than, or in addition to, Cdk4 are involved in disease-related neuron death. In this light, Rashidian et al. [START_REF] Rashidian | Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo[END_REF] have suggested that both Cdk4 and Cdk5 play complementary roles in neuron death in hypoxia/ischemia with Cdk4 regulating apoptotic-like death and Cdk5 promoting excitotoxic death. Another possibility is that cdks in addition to Cdk4 drive hyperphosphorylation of Rb family members. It has been recently reported that activated Cdk5 can directly hyper-phosphorylate Rb in a neuronal cell death model [START_REF] Hamdane | p25/Cdk5-mediated retinoblastoma phosphorylation is an early event in neuronal cell death[END_REF]. If this finding holds for additional death paradigms, it may be that disease-related Cdk5-dependent neuron death is, at least in part, mediated via the phospho-p130/Myb/Bim pathway described here.

Owing to the potential limitations of present small chemical cdk inhibitors, alternative strategies are required to test the involvement of specific cdks in disease-associated neuron death. One approach has been the use of dominant-negative cdks. Studies with such reagents showed that dominant-negative forms of Cdk4 and 6 protected cultured neurons from death evoked by β-amyloid [START_REF] Giovanni | Involvement of cell cycle elements, cyclin-dependent kinases, pRb, and E2F x DP, in B-amyloid-induced neuronal death[END_REF]. Because dominant-negative proteins may have non-selective actions such as interactions with substrates common for other cdks, we have employed a knockdown strategy with shRNAs. We find that an shRNA targeted to Cdk4 protects cultured neurons from β-amyloid [START_REF] Biwas | Bim is elevated in alzheimer disease neurons and is required for beta-amyloid induced neuronal apoptosis[END_REF], thus reinforcing the conclusion that at least in this model, Cdk4 is required for neuron death. This observation also supports the idea that Cdk4 mediates neuron death in neurodegenerative disorders such as AD in which it appears to be elevated and/or activated.

Our finding that knockdown of Cdk4 suppresses death evoked by β-amyloid has led us to further pursue the hypothesis that the same pathway identified in developmental cell death is also activated in AD. We found that neuron death in the cellular β-amyloid model was associated with induction of Bim message and protein and that shRNA targeted to Bim was highly protective [START_REF] Biwas | Bim is elevated in alzheimer disease neurons and is required for beta-amyloid induced neuronal apoptosis[END_REF]. Moreover, Bim induction as well as death were blocked in this system by knocking down B-Myb. Thus, in this cellular AD model, many of the same cycle-related mechanisms that characterize the death induced by NGF deprivation are present, including the requirement for Cdk4, hyper-phosphorylation of Rb proteins and the essential induction of B-Myb and Bim.

Indications that the above pathway mediates neuron death by β-amyloid in culture have led us to explore whether this might also occur in AD itself. As reviewed above, susceptible neurons in AD show elevated expression of cyclin D, Cdk4 and hyper-phosphorylated Rb. We find that a large proportion of such neurons also coexpress elevated levels of Bim [START_REF] Biwas | Bim is elevated in alzheimer disease neurons and is required for beta-amyloid induced neuronal apoptosis[END_REF]. These findings thus establish that affected neurons in AD brains express both the distal and proximal elements of the death pathway we have defined in developmental neuron death and in cellular models of AD. We would anticipate comparable findings in additional neurological disorders.

Other cell cycle elements in neurological disease.

The molecules that constitute the pathway described here (cyclin D, Cdk4, phospho-Rb and Mybs) are normally associated with the G1/S transition. However, a potential complication is the somewhat bewildering observation that neurons at risk in a variety of disorders show simultaneous elevation of proteins that are considered to be markers of additional phases of the cycle in proliferating cells. How do these additional cell cycle proteins fit into the scheme presented here? A variety of approaches have revealed that E2F-Rb complexes, including E2F4-p130, bind to and regulate numerous cell-cycle associated genes [START_REF] Dimova | The E2F transcriptional network: old acquaintances with new faces[END_REF][START_REF] Balciunaite | Pocket protein complexes are recruited to distinct targets in quiescent and proliferating cells[END_REF]. In proliferating cells, hyper-phosphorylation of Rb and loss of such complexes permits induction of genes that promote DNA synthesis and an orderly progression to the next stages of the cycle. In neurons, by contrast, hyper-phophorylation and loss of E2F-Rb complexes will also lead to induction of multiple cell-cycle associated proteins, but in this case, leads to cell death. Although our suspicion is that many of the cell-cycle-related proteins that are induced as a result of neuron disease or injury are bystanders of p130 hyper-phosphorylation and play little if any role in death, others may contribute. For instance, up-regulation of cyclins B and E has been noted in AD [START_REF] Timsit | Increased cyclin D1 in vulnerable neurons in the hippocampus after ischaemia and epilepsy: a modulator of in vivo programmed cell death?[END_REF][START_REF] Husseman | Mitotic activation: a convergent mechanism for a cohort of neurodegenerative diseases[END_REF][START_REF] Nagy | Cell cycle markers in the hippocampus in Alzheimer's disease[END_REF]. These in turn activate Cdk1 and Cdk2, respectively both of which can target Rb family members [START_REF] Lin | Retinoblastoma cancer suppressor gene product is a substrate of the cell cycle regulator cdc2 kinase[END_REF][START_REF] Berthet | Combined loss of Cdk2 and Cdk4 results in embryonic lethality and Rb hypophosphorylation[END_REF]. Activation of these kinases may thus mimic or augment the death-promoting actions of Cdk4 in neurons. In addition such kinase may have other relevant targets. For instance, Cdk1 can target and activate the pro-apoptotic BH3-domain molecule BAD [START_REF] Becker | Cell cycle regulation of neuronal apoptosis in development and disease[END_REF]. Another potential player is Cdk7 which phosphorylates and activates other cdks including Cdks 1,2 and 4. Cdk7 levels are reported to be elevated in susceptible neurons in AD [START_REF] Zhu | Neuronal CDK7 in hippocampus is related to aging and Alzheimer disease[END_REF].

The presence of markers for various stages of the cycle in disease-affected neurons has led to suggestions that such cells undergo an "aborted" or "dysregulated" cell cycle and that this is what is responsible for their demise, perhaps by triggering death-promoting check point controls. In our view, the critical event is not the attempt to synthesize DNA or to traverse the cycle, but rather the de-repression of genes such as B -and C -Myb that can have multiple functions in addition to promoting proliferation. For example, depending on the cellular context, B-Myb can induce either proliferation or cell survival [START_REF] Joaquin | Cell cycle regulation by the B-Myb transcription factor[END_REF][START_REF] Sala | B-MYB, a transcription factor implicated in regulating cell cycle, apoptosis and cancer[END_REF] or, as reviewed here, neuron death [START_REF] Liu | Regulation of neuronal survival and death by E2Fdependent gene repression and derepression[END_REF][START_REF] Liu | B-myb and C-myb play required roles in neuronal apoptosis evoked by nerve growth factor deprivation and DNA damage[END_REF]. As we argue below, the choice between these actions and the reason that Myb is fatal in a neuronal context, may depend on which additional transcriptional regulators are present.

Another point to consider regarding the relationship between cell cycle and neuron death in disease is that in our model, the key E2F and Rb family proteins involved are E2F4 and p130. Our culture experiments indicate that these are the major family members complexed with DNA and the Myb promoter in healthy post-mitotic neurons and that such associations are lost upon activation of Cdk4 and result in death. Present findings indicate that E2F4 and p130 are not generally involved in regulating cell proliferation (in contrast to E2F1-3 and pRb and p107) and that they appear at high levels in the nervous system when neurons become post-mitotic [START_REF] Corbeil | Characterization of transcription factor E2F complexes during muscle and neuronal differentiation[END_REF][START_REF] Baldi | Differential expression of Rb2/p130 and p107 in normal human tissues and in primary lung cancer[END_REF]. In neurological diseases, the consequence of losing E2F4-p130 complexes due to activation of Cdk4 may therefore be to trigger death rather than replication [see 47].

Neurological disorders and trophic factor deprivation.

We have drawn parallels here between the apoptotic pathways involved in neuron death associated with development and with disease. Observations that treatments such as exposure to β-amyloid can rapidly activate this mechanism suggest that it can occur independently of loss of trophic signaling. On the other hand, one consequence of a number of neurodegenerative disorders and of nervous system trauma is impairment or loss of access to and/or retrograde transport of trophic factors [START_REF] Chao | Neurotrophin signalling in health and disease[END_REF][START_REF] Salehi | Increased App Expression in a Mouse Model of Down's Syndrome Disrupts NGF Transport and Causes Cholinergic Neuron Degeneration[END_REF]. Thus, the mechanism described here by which cell cycle molecules contribute to neuron death in neurological disorders may be driven both by direct apoptotic stimuli as well as by loss of trophic support.

The potential role of aggregated proteins and proteasomal dysfunction in activation of the apoptotic cell cycle pathway in neurodegenerative disorders.

Dysfunction of the ubiquitin-proteasomal system is an additional potential, and not necessarily mutually exclusive contributor to activation of the neuronal apoptotic cell cycle pathway. There is accumulation of aggregated proteins in a number of degenerative disorders and this may cause or be the result of reduced neuronal ubiquitin-proteasomal activity [reviewed in [START_REF] Petrucelli | Mechanism of neurodegenerative disease: role of the ubiquitin proteasome system[END_REF][START_REF] De Vrij | Protein quality control in Alzheimer's disease by the ubiquitin proteasome system[END_REF]. It has been further suggested that failure ubiquitin-proteasomal triggers neuron death and degeneration in such diseases [START_REF] Petrucelli | Mechanism of neurodegenerative disease: role of the ubiquitin proteasome system[END_REF][START_REF] De Vrij | Protein quality control in Alzheimer's disease by the ubiquitin proteasome system[END_REF]. The ubiquitin-proteasomal system plays a major role in regulation of cell cycle by degrading key proteins such as cyclins that promote transition from one phase of the cycle to another [reviewed in 120]. A relevant example is CycD which is degraded by the proteasomal system to prevent activation of cdks such as cdk4 and movement of cells into G1/S. Thus, one could imagine that a neuron with compromised proteasomal function might accumulate CycD as well as other potential activators of the apoptotic cell cycle pathway. Evidence to support this concept has come from experiments with cultured cerebellar neurons induced to die by removal of supporting levels of K+ or by treatment with proteasomal inhibitors [START_REF] Boutillier | Depolarization regulates cyclin D1 degradation and neuronal apoptosis: a hypothesis about the role of the ubiquitin/proteasome signalling pathway[END_REF]. Several additional studies have shown that neuron death induced by proteasomal inhibitors induce and require activation of the cell cycle machinery, including cdk4 [START_REF] Rideout | Cyclin-dependent kinase activity is required for apoptotic death but not inclusion formation in cortical neurons after proteasomal inhibition[END_REF][START_REF] Staropoli | The ubiquitin-proteasome pathway is necessary for maintenance of the postmitotic status of neurons[END_REF]. Thus, irrespective of the initial cause, the reduction of ubiquitin-proteasomal function that occurs in neurodegenerative disorders could contribute to activation of the apoptotic pathway described here.

Cell cycle molecules are necessary, but not sufficient triggers of neuron death.

We have argued that activation of cell cycle molecules is necessary for neuron death in development and disease. However, they are unlikely to be sufficient in this regard. Even in systems in which there is strong evidence for an essential role of cell cycle molecules in neuron death, additional required pathways have been uncovered. A good example is the pathway dependent on activation of the c-Jun Nterminal protein kinases (JNKs). The JNK pathway has been implicated as required for neuron death associated with diverse causes including AD [START_REF] Troy | beta-Amyloid-induced neuronal apoptosis requires c -Jun Nterminal kinase activation[END_REF][START_REF] Okazawa | The JNK/c-Jun cascade and Alzheimer's disease[END_REF], PD [START_REF] Silva | Mixed lineage kinase-c-jun N-terminal kinase signaling pathway: a new therapeutic target in Parkinson's disease[END_REF], hypoxia/ischemia [START_REF] Irving | Role of mitogen-and stress-activated kinases in ischemic injury[END_REF] and trophic factor deprivation [START_REF] Maroney | CEP-1347 (KT7515), an inhibitor of JNK activation, rescues sympathetic neurons and neuronally differentiated PC12 cells from death evoked by three distinct insults[END_REF][START_REF] Ham | c-Jun and the transcriptional control of neuronal apoptosis[END_REF]. At least part of the pro-apoptotic activity of JNKs appears to be due to their capacity to phosphorylate and activate the transcription factor c-Jun [START_REF] Ham | c-Jun and the transcriptional control of neuronal apoptosis[END_REF]. One aspect that the JNK pathway shares in common with the apoptotic cell cycle pathway described here is that it too can lead to activation of the pro-apoptotic protein Bim in neurons. This has been observed thus far for NGF deprivation [START_REF] Whitfield | Dominantnegative c -Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release[END_REF][START_REF] Harris | BH3-only Bcl-2 family members are coordinately regulated by the JNK pathway and require Bax to induce apoptosis in neurons[END_REF], ischemia [START_REF] Okuno | The c-Jun N-terminal protein kinase signaling pathway mediates Bax activation and subsequent neuronal apoptosis through interaction with Bim after transient focal cerebral ischemia[END_REF] and β-amyloid exposure [S.C. Biswas and L.A. Greene, unpublished]. Moreover, induction of Bim by apoptotic stimuli in neurons is suppressed by inhibition of either the cell cycle or JNK pathways [START_REF] Biswas | Bim is a direct target of a neuronal E2F-dependent apoptotic pathway[END_REF]. Thus, it appears that both pathways must be activated simultaneously to promote maximum Bim induction and death. Yet a third transcriptional pathway can also regulate Bim expression in neurons, and that is one involving the FOXO subfamily of Forkhead transcription factors [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF]. FOXO appears to be involved in both Bim induction and in death evoked by NGFdeprivation [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF]. In the presence of NGF, FOXO proteins are phosphorylated by a PI3K/AKT-dependent pathway and are excluded from the nucleus; with NGF deprivation, de-phosphorylation permits FOXO movement to the nucleus and regulation of death-promoting genes including Bim [START_REF] Gan | Nuclear/cytoplasmic shuttling of the transcription factor FoxO1 is regulated by neurotrophic factors[END_REF].

If at least three independent transcriptional pathways appear to regulate Bim expression in neurons and if each plays a required role in death, how is this coordinated? Examination of the Bim promoter reveals conserved binding sites for Myb [START_REF] Biswas | Bim is a direct target of a neuronal E2F-dependent apoptotic pathway[END_REF] and FOXO [START_REF] Gilley | FOXO transcription factors directly activate bim gene expression and promote apoptosis in sympathetic neurons[END_REF] proteins. In addition, we find that the promoter also possesses a binding site for c-Jun [S.C. Biswas and L.A. Greene, unpublished]. Thus, the Bim promoter contains regulatory binding sites for products of all three pathways (i.e. the cell cycle, JNK and FOXO pathway). We have further observed that inhibition of any one of the three pathways is sufficient to block NGFdeprivation-induced activation of a reporter driven by the Bim promoter [S.C. Biswas and L.A. Greene, unpublished]. These findings point to and support a model in which neuron death requires the convergence of multiple independent pathways to induce the pro-apoptotic protein Bim. That is, under physiologic conditions, each of the pathways appears to be required for Bim induction and for neuron death, but none is sufficient. For full Bim induction and for neuron death, all three pathways need to be activated simultaneously. One possible reason for the evolution of such a mechanism is that it acts as a "failsafe" to protect neurons from dying if only a single pathway is activated inappropriately. This consideration may also explain why Myb induction might be lead to death in a neuronal context, but not in proliferating cells. That is, death would only occur if the complementary transcription factors were activated and this would not be the case for proliferating cells in which FOXO proteins are inactivated by growth-factor-dependent PI3K/AKT signaling. This convergence model is also consistent with the progressive loss of neurons that characterizes many neurodegenerative disorders. If neuron death in such diseases requires a threshold level of activation of pro-apoptotic proteins such as Bim, this would not occur until all three pathways were sufficiently activated.

Therapeutic perspectives

The characterization of a pathway that is dependent on defined cell cycle molecules such as Cdk4 and that is required for neuron death in disease opens the door to specific therapeutic intervention. One obvious approach that appears promising thus far in models of spinal cord injury [START_REF] Tian | Suppression of astroglial scar formation and enhanced axonal regeneration associated with functional recovery in a spinal cord injury rat model by the cell cycle inhibitor olomoucine[END_REF] and ischemia/stroke [START_REF] Wang | Inhibition of cyclin-dependent kinases improves CA1 neuronal survival and behavioral performance after global ischemia in the rat[END_REF] is the use of small molecule cdk inhibitors. Issues such as specificity, side effects on proliferating tissues, and access to the nervous system must be considered before moving forward. Fortunately, mainly due to the potential use of such agents for treatment of cancers, efforts are underway to develop a variety of drugs that are selective for specific cdks [START_REF] Sridhar | Selectivity and potency of cyclindependent kinase inhibitors[END_REF][START_REF] Hirai | Recent advances in the development of selective small molecule inhibitors for cyclin-dependent kinases[END_REF]. However, a major, though presumably soluble, roadblock in evaluating such drugs for their neuroprotective actions in suitable cell and animal models has been their limited accessibility to the overall research community. Another issue is whether such agents might be useful for inhibition of neuron death in progressive neurodegenerative disorders. It remains to be seen whether even the most specific cdk inhibitors could be tolerated for prolonged periods of time and whether blockade of neuron death in these disorders is sufficient to yield therapeutic effects.

The model we have described here includes molecules in addition to cdks. This raises the possibility that these too might be considered as potential therapeutic targets. One advantage of blocking the death pathway at such levels is that they are upstream of activation of the core apoptotic machinery and thus of cellular degeneration due to mitochondrial dysfunction. Bim is attractive in this regard in that, based on the viability of Bim null mice, it appears to be dispensable for survival of the organism. Moreover, basal Bim levels in normal brain appear to be very low. Various strategies such as targeted siRNAs or antisense oligonucleotides or sitespecific drugs that interact with Bim and block its interaction with anti-apoptotic Bcl2 family members could thus prove useful in sustaining neuron survival in disease.

It is not unlikely that the cell-cycle-related molecular pathway recounted here is more complex than presently understood and that it possesses additional components and regulatory features.

Although this would further challenge our capacity to understand the mechanisms that govern neuron death in development and disease, identifying additional pathway components may also provide new opportunities and targets for therapeutic intervention.

A

  C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T A C C E P T E D M A N U S C R I P T

Figure 1 :

 1 Figure1: Scheme depicting a molecular pathway by which cell-cycle-related molecules promote cell death in neurons in development and disease. A. Survival conditions. Under conditions of neuron survival, p130 forms complexes with E2F4 on the promoters of genes including those encoding B-and C-Myb. p130 tethers chromatin-modifying proteins such as Suv39H1 and HDAC1 to the promoter and represses their transcription. Cdk4 is inactive and consequently p130 is hypophosphorylated and binds tightly to E2F4. B. Apoptotic conditions. Under apoptotic conditions, Cdk4 associates with Cyclin D and is active. This leads to hyperphosphorylation of p130 which in turn causes it and its bound chromatin modifiers to dissociate from E2F4 and from promoters of genes including B-and C-Myb. This results in gene de-repression and elevation of cellular levels of B-and C-Myb. The Mybs bind to the promoter of the gene encoding the pro-apoptotic BH3-only protein Bim. The levels of intracellular Bim rise and it engages the core cell death machinery, promoting caspase activation and apoptosis.

Acknowledgements:

Supported in part by grants from the National Institutes of Health-NINDS and Parkinson's Disease Foundation.