Dr Zsuzsanna Nagy
email: z.nagy@bham.ac.uk

The dysregulation of the cell cycle and the diagnosis of Alzheimer's disease

Keywords: Alzheimer's disease, cell cycle, cyclin dependent kinase, cyclin dependent kinase inhibitor

The 'silent epidemic' of Alzheimer's disease is becoming a considerable social and economical problem in the developed countries. Especially so, because we still cannot diagnose the disease early enough, and there is no disease-modifying treatment.

. It is imperative therefore that we find early biomarkers for sporadic AD, which could identify patients before substantial pathology develops.

who are in the pre-clinical stages of the disease and reduce heterogeneity in clinical trials. If the marker also reflects the progression of the disease it can be used as target for clinical trials [START_REF] Mayeux | Biomarkers: potential uses and limitations[END_REF].

The diagnosis of Alzheimer's disease

At present the diagnosis of 'definite' Alzheimer's disease can only be made by histopathological assessment after autopsy. While the patient is alive the clinical diagnosis carries varying amounts of uncertainty allowing the patients to fall into two categories: probable or possible Alzheimer's disease (NINCDS-ARDRA). Either category requires the patient to show clinical signs of dementia. Additionally the clinical diagnosis relies mainly on the exclusion of other neurological disorders, which could cause dementia, rather than the identification of an Alzheimer-specific clinical profile. While the positive predictive value of clinical diagnosis of 'probable AD' is high (89-100%) the diagnostic categories described above have a very high false negative rate, excluding patients suffering from early/mid stages of the disease [START_REF] Nagy | Accuracy of clinical operational diagnostic criteria for Alzheimer's disease in relation to different pathological diagnostic protocols[END_REF].

Existing biomarkers for Alzheimer's disease

Because amyloid deposition in the brain is a defining feature of AD the measurement of markers related to APP and β-amyloid metabolism appeared attractive candidates as biomarkers for the disease. Despite much initial enthusiasm it transpired that measurements of β-amyloid from plasma (or serum) might relate to age or renal function but does not allow the discrimination between healthy elderly controls and AD patients (reviewed in [START_REF] Irizarry | Biomarkers of Alzheimer disease in plasma[END_REF]). Furthermore the plasma β-amyloid levels do not correlate to dementia severity and did not reflect the effectiveness of different

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types of therapeutic interventions targeted to reduce β-amyloid accumulation in the brain (reviewed in [START_REF] Irizarry | Biomarkers of Alzheimer disease in plasma[END_REF]). The possibility that the ratio of different APP isoforms from platelets could be used for diagnosis and monitoring disease progression and therapeutic effects was also raised. However, technical limitations make this marker difficult to standardise.

Cardiovascular risk factors, such as high cholesterol ApoE4 genotype hypertension and elevated plasma homocysteine levels were also identified as risk factors for sporadic AD. While they appear to be good antecedent biomarkers they do not fulfil the role of biomarkers of 'disease'. The same is true for markers of oxidative stress (antioxidant levels and lipid peroxidation products) (reviewed in [START_REF] Irizarry | Biomarkers of Alzheimer disease in plasma[END_REF]).

As the CSF is regarded as a mirror of the biochemical changes occurring in the brain, considerable effort has been put into identifying biomarkers for AD in the CSF.

In turn total tau and phospho-tau have been found to be elevated in AD patients relative to controls while β-amyloid is reduced in the CSF of the patient population.

Although some studies claim good specificity and sensitivity for these markers in differentiating AD patients from controls, they do not differentiate AD from other types of dementia (reviewed in [START_REF] Blennow | CSF biomarkers for Alzheimer's disease: use in early diagnosis and evaluation of drug treatment[END_REF]). The main shortcoming of these markers is that they appear only once the disease has caused considerable accumulation of AD-type pathology, and as such they are relatively late markers.

A substantial number of imaging markers of the anatomy, chemistry, cognitive performance or behavioural assessments. As such, neuroimaging has the potential to provide more sensitive and reliable markers of therapeutic efficacy, which could reduce the length of clinical trials for disease modifying therapies (reviewed in [START_REF] Dickersonand | Neuroimaging biomarkers for clinical trials of disease-modifying therapies in Alzheimer's disease[END_REF]). The main caveat of these techniques however, is that imaging reflects the accumulation of pathology (whether it is tissue atrophy or the accumulation of ADtype pathology) and subsequent functional deficit. Furthermore, due to large personal variability, most of these imaging techniques allow patient identification based only on the rate of change rather than on one baseline observation. This makes the use of imaging techniques for early diagnosis and trial stratification rather difficult.

In summary, so far there is no consistent, reproducible, sensitive, or specific marker for AD diagnosis, risk, progression, or treatment effects. If we aspire to identify disease-modifying therapies, which could delay the clinical symptoms of AD in cognitively intact individuals who are at high risk, we need markers, which reflect the pathophysiology of the disease.

Cell cycle and AD

The cell cycle hypothesis for the pathogenesis of Alzheimer's disease was originally based on the observation that neurones in the brain of elderly demented patients express cell division-related proteins [START_REF] Nagy | The cell division cycle and the pathophysiology of Alzheimer's disease[END_REF][START_REF] Gartner | Induction of p21ras in Alzheimer pathology[END_REF][START_REF] Arendt | Neuronal expression of cycline dependent kinase inhibitors of the INK4 family in Alzheimer's disease[END_REF]. The differential expression of these cell cycle markers suggested that the neurones in the adult nervous system are able to re-enter the cell division cycle [START_REF] Nagy | Cell cycle markers in the hippocampus in Alzheimer's disease[END_REF][START_REF] Nagyand | Neuronal cyclin expression in the hippocampus in temporal lobe epilepsy[END_REF][START_REF] Vincent | Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer's disease brain[END_REF]. In normal circumstances the cell cycle re-entry (from G0 to G1) is followed by cell cycle arrest in an early stage (before the G1/S transition point), when re-differentiation is still possible. It has also been suggested that in the healthy brain the transient re-entry into the cell cycle might be part of the synaptic remodelling process [START_REF] Arendt | Synaptic plasticity and cell cycle activation in neurons are alternative effector pathways: the 'Dr. Jekyll and Mr. Hyde concept' of Alzheimer's disease or the yin and yang of neuroplasticity[END_REF].

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In Alzheimer's disease the G1/S control mechanisms fail. Neurones are allowed to replicate their DNA [START_REF] Yang | DNA replication precedes neuronal cell death in Alzheimer's disease[END_REF] and progress into the late, G2 phase of the cycle [START_REF] Nagy | The cell division cycle and the pathophysiology of Alzheimer's disease[END_REF]. Since the neurones are not able to divide, they remain in this very late stage of the cell cycle. In turn the cellular mechanisms activated in the G2 phase of the cycle drive the formation of neurofibrillary tangles and amyloid plaques [START_REF] Suzuki | Cell cycle-dependent regulation of the phosphorylation and metabolism of the Alzheimer amyloid precursor protein[END_REF][START_REF] Baumann | Abnormal Alzheimer-like phosphorylation of tau-protein by cyclindependent kinases cdk2 and cdk5[END_REF] The mitogenic stimulation of neurones is a necessary to trigger the cell cycle.

Many of the identified risk factors for Alzheimer's disease, such as elevated plasma homocysteine levels [START_REF] Nagy | Hyperhomocysteinemia in Alzheimer's disease and expression of cell cycle markers in brain[END_REF], ageing [START_REF] Arendt | Synaptic plasticity and cell cycle activation in neurons are alternative effector pathways: the 'Dr. Jekyll and Mr. Hyde concept' of Alzheimer's disease or the yin and yang of neuroplasticity[END_REF], menopause [START_REF] Casadesus | The estrogen myth: potential use of gonadotropinreleasing hormone agonists for the treatment of Alzheimer's disease[END_REF][START_REF] Casadesus | Evidence for the role of gonadotropin hormones in the development of Alzheimer disease[END_REF], low thyroid levels [START_REF] Lustig | Sex hormone modulation of neural development in vitro[END_REF][START_REF] Mussa | Influence of thyroid in nervous system growth[END_REF], low level prolonged oxidative stress [START_REF] Langleyand | Oxidative stress-induced death in the nervous system: cell cycle dependent or independent?[END_REF][START_REF] Kleinand | Oxidative stress, cell cycle, and neurodegeneration[END_REF], head injury or low education, can either represent mitogenic signalling for neurones or facilitate cell cycle re-entry in vulnerable neuronal populations (reviewed by Arendt [START_REF] Arendt | Synaptic plasticity and cell cycle activation in neurons are alternative effector pathways: the 'Dr. Jekyll and Mr. Hyde concept' of Alzheimer's disease or the yin and yang of neuroplasticity[END_REF]). The link between synaptic plasticity and cell cycle reactivation also explains how ApoE4 genotype, which is associated with reduced plastic capacity of neurones [START_REF] Arendt | Plastic neuronal remodeling is impaired in patients with Alzheimer's disease carrying apolipoprotein epsilon 4 allele[END_REF] is a risk factor for Alzheimer's disease.

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The G1/S regulatory failure While cell cycle re-entry of neurones appears to be a prerequisite of Alzheimer's disease, on its own is not sufficient to cause the disease [START_REF] Zhu | Alzheimer's disease: the two-hit hypothesis[END_REF][START_REF] Nagy | Cell cycle regulatory failure in neurones: causes and consequences[END_REF]. As discussed above, the cell cycle re-entry may just represent a short transient part of synaptic remodelling in the healthy elderly [START_REF] Arendt | Synaptic plasticity and cell cycle activation in neurons are alternative effector pathways: the 'Dr. Jekyll and Mr. Hyde concept' of Alzheimer's disease or the yin and yang of neuroplasticity[END_REF]. The difference between the healthy aging and AD seems to be the extent of cell cycle progression rather than cell cycle re-entry. This suggests that the transition from healthy ageing to AD is the point when neurones bypass the G1/S restriction point and replicate their DNA. Another member of the family, p27 kip1 (CDKN1B, 600778), was mapped to 12p13. In quiescent cells relatively high levels of the protein are expressed, but protein expression rapidly declines upon cell cycle induction. The CDK inhibitory activity of the protein is linked to its nuclear localisation, while the cytoplasmic accumulation of the protein is associated with high proliferative activity of certain cancers. The phosphorylation and ubiquitination of the protein leads to the segregation of p27 kip1 in the cytoplasm followed by its degradation. The truncation of p27 kip1 by caspase 3 in apoptotic cells leads to the deactivation of this CDKI similar to p21 cip1 . Germline variants of the gene have been found associated with increased risk of certain cancers (http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=600778 ).

The third member of this CDKI protein family is p57 kip2 mapped to 11p15.5 (CDKN1C; 600856). The CDK inhibitor region of the protein is highly homologous with the p21 cip1 protein. However, unlike the previous two members of the family the p57 kip2 is partially imprinted, the maternal allele being preferentially expressed. Loss of function (mutations on the gene, loss of maternal allele) was found to be associated with sporadic cancers as well as hereditary syndromes such as Simpson-Golabi-Behmel syndrome and Beckwith-Wiedemann syndrome (http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=600856 ).

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All three proteins have been found to be expressed in the cytoplasm of neurones in AD patients [START_REF] Nagy | The last neuronal division: a unifying hypothesis for the pathogenesis of Alzheimer's disease[END_REF][START_REF] Chang | Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases[END_REF][START_REF] Ogawa | Increased p27, an essential component of cell cycle control, in Alzheimer's disease[END_REF][START_REF] Zhu | Elevated expression of a regulator of the G2/M phase of the cell cycle, neuronal CIP-1-associated regulator of cyclin B, in Alzheimer's disease[END_REF], often in tangle bearing neurones and distorted neuritic processes of plaques. The association with tangles and sequestration in the in apoptotic cells is a tempting possibility. However, neurones appear to avoid the classical apoptotic process exactly due to the fact that they do not amplify their downstream cascade of caspases, including caspases 3 [START_REF] Raina | Abortive apoptosis in Alzheimer's disease[END_REF].

In summary, it appears likely that the inactivation of CDKIs plays a major role in the G1/S regulatory failure in neurones responsible for the development of AD-type pathology. However, the causes of this inactivation remain elusive.

The causes of the G1/S regulatory failure in AD.

There is evidence in the literature suggesting that the cell cycle regulatory failure in AD is not restricted to neurones. It has been shown that there is an association between solid tumours and the incidence of AD [START_REF] Burke | Occurrence of cancer in Alzheimer and elderly control patients: an epidemiologic necropsy study[END_REF]. Additionally peripheral cell, such as lymphocytes from AD patients showed altered response to mitogenic stimulation relative to control subjects [START_REF] Stieler | Impairment of mitogenic activation of peripheral blood lymphocytes in Alzheimer's disease[END_REF][START_REF] Urcelay | Enhanced proliferation of lymphoblasts from patients with Alzheimer dementia associated with calmodulin-dependent activation of the na+/H+ exchanger[END_REF]. Fibroblasts collected from AD patients also shown an aberrant cell cycle-dependent Ca response [START_REF] Tatebayashi | Cell-cycle-dependent abnormal calcium response in fibroblasts from patients with familial Alzheimer's disease[END_REF]. A more recent study has found that the differential phosphorylation of Erk1 and Erk2 in

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response to bradykinin in fibroblasts can distinguish AD patients from healthy elderly and patients suffering from non-AD dementias [START_REF] Khanand | An internally controlled peripheral biomarker for Alzheimer's disease: Erk1 and Erk2 responses to the inflammatory signal bradykinin[END_REF].

We have also found that this specific failure of the G1/S transition checkpoint is not be restricted to neurons alone. Lymphocytes of AD sufferers also show signs of G1 regulatory failure [START_REF] Nagy | Cell cycle kinesis in lymphocytes in the diagnosis of Alzheimer's disease[END_REF]. Additionally, in subjects showing neuropsychological signs of pre-clinical AD, the lymphocyte response was similar to that seen in AD patients [START_REF] Nagy | Cell cycle kinesis in lymphocytes in the diagnosis of Alzheimer's disease[END_REF].

These findings also imply that the AD-related G1/S regulatory failure is probably due to genomic variations of the cyclin dependent kinase inhibitors (CDKIs) involved in the regulation of the G1/S transition.

In order to investigate this possibility we genotyped a relatively small but unselected group of AD patients and control subject (which included both healthy elderly people and patients suffering from other non-AD dementias). We have concentrated on known, mainly cancer-related SNPs on the p21 cip1 p27 kip1 and p57 kip2 genes.

There are two variants on the p21 cip1 gene associated with increased tumour frequency [START_REF] Mousses | Two variants of the CIP1/WAF1 gene occur together and are associated with human cancer[END_REF][START_REF] Chedid | A single nucleotide substitution at codon 31 (Ser/Arg) defines a polymorphism in a highly conserved region of the p53-inducible gene WAF1/CIP1[END_REF][START_REF] Sjalander | Association between the p21 codon 31 A1 (arg) allele and lung cancer[END_REF]. One of the variants is a Ser-to-Arg substitution in codon 31 while the other was a C-to-T change in the 3-prime untranslated region of the p21 cip1 gene 20 bp following the stop codon. The amino acid substitution is within the CDKI region of the protein, and probably affects the binding of the inhibitor to the cyclin CDK complex. The role of the intronic SNP is less clear, but it has been suggested that it might affect the stability of the mRNA and alter protein levels in the cytoplasm.

We have genotyped the patients using the methods described previously [START_REF] Mousses | Two variants of the CIP1/WAF1 gene occur together and are associated with human cancer[END_REF].

No homozygous patient occurred in our cohort. The simultaneous presence of both variants increased the risk of developing AD slightly, albeit significantly (Table 1).

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Interestingly however, the effect of the genotype was gender dependent (Table 1), similar to IGF-1R [START_REF] Garcia | Association of Insulin-Like Growth Factor-1 Receptor Polymorphism in Dementia[END_REF] ABCA1 [START_REF] Sundar | Gender-specific association of ATP-binding cassette transporter 1 (ABCA1) polymorphisms with the risk of late-onset Alzheimer's disease[END_REF] and ESR2 [START_REF] Pirskanen | Estrogen receptor beta gene variants are associated with increased risk of Alzheimer's disease in women[END_REF]. We have also found that the presence of the two SNPs on p21 cip1 was associated with a reduced age of onset (p=0.002) (Figure 1) in AD patients (both males and females).

In late (neocortical) stage AD patients (n=59, of which 9 had variant p21 cip1 ) the number of Cyclin B positive (G2 phase) neurones was significantly higher in patients with the p21 cip1 variant (Figure 2), indicating that the variant is associated with the progression of the cell cycle in neurones. In early stage (entorhinal) patients (n=40, of which 5 had variant p21 cip1 ) however, the p21 cip1 variant was associated with significantly lower levels of synaptophysin (Figure 3).

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The p21 cip1 protein is also responsible for the cell cycle arrest in response to oxidative stress induced DNA damage downstream of p38. Thus the functional loss of p21 cip1 may lead to deficiencies in the neuronal stress-response. In this context it is interesting that earlier studies found that both mitogenic stimulation and oxidative stress are required for the progression of AD [START_REF] Zhu | Alzheimer's disease: the two-hit hypothesis[END_REF].

For p27 kip1 we analysed the effect of the Gly-to-Val polymorphism on codon 109. The VV genotype has been found to be associated with increased risk of cancer [START_REF] Kibel | CDKN1A and CDKN1B polymorphisms and risk of advanced prostate carcinoma[END_REF]. The polymorphism lies in the p38 jab1 binding domain of the protein and probably reduces the CDKI activity of the protein by altering its degradation. We found that the VV genotype also increases the risk of AD (Odds Ratio 1.72, p=0.049) (Table 2).

However, this SNP had no effect on the age of onset of dementia in our AD patients (data not shown). There are no SNPs on the p57kip2 gene known to be associated with cancer and the known SNPs are not characterised in terms of population diversity. We have found that the SNP on codon 159, which leads to an Ala-to-Val substitution, is associated with an increased risk (Odds Ratio=1.66, no statistical significance due to small sample) of AD in a small group of patients (Table 3). The presence of the SNP

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was also associated with a significantly (p=0.04) reduced age of onset in our AD patients (Figure 4). The polymorphism does not lie within the CDKI domain of p57 kip2 . The function of the region affected is not known. The interpretation of these, in terms of their functional significance, is made more complex by the fact that p57 kip2 is imprinted.

New early diagnostic possibilities for Alzheimer's disease

The discovery of the pathogenic pathway responsible for the development of Alzheimer's disease holds the promise of early diagnostic protocols, novel biomarkers and disease-modifying therapies for Alzheimer's disease.

As mentioned earlier, there are two types of biomarkers.

The antecedent biomarkers measure the exposure to environmental risk and genetic risk factors, allowing a more accurate risk prediction for an individual patient.

In this sense all the factors which contribute to the mitogenic stimulation of mature neurones can be regarded as antecedent biomarkers. Elevated plasma Recent studies suggest that the identification of disease biomarkers, based on detecting the AD-type cell cycle deregulation, is possible.

The proliferative capacity and survival of cultured lymphocytes is altered in AD patients relative to age-matched controls [START_REF] Stieler | Impairment of mitogenic activation of peripheral blood lymphocytes in Alzheimer's disease[END_REF][START_REF] De Las Cuevas | Altered transcriptional regulators in response to serum in immortalized lymphocytes from Alzheimer's disease patients[END_REF]. The apparent discrepancy between the two reports may have several reasons. Besides the possible differences in the selection/inclusion criteria for AD patients and controls, the experimental design and measures of lymphocyte proliferative capacity in the two studies were different. It is also known that the proliferative potential of peripheral lymphocytes is affected by age, therefore even slight differences in the age of AD patients and controls will affect the outcome of these studies. In addition, in an individual patient, the use of COX-2

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inhibitor NSAIDS, or a simple cold might alter the response of the lymphocytes to mitogenic stimulation in vitro. Larger controlled studies would be required to clarify the cause of the apparent discrepancy.

The specific G1/S regulatory failure, typical to neurones in AD, is also detectable from lymphocytes [START_REF] Nagy | Cell cycle kinesis in lymphocytes in the diagnosis of Alzheimer's disease[END_REF]. The study was aimed specifically at the detection of any defects of the G1/S transition control. We found that in response to cell cycle inhibitors the relative lengthening of the G1 phase in exponentially proliferating peripheral lymphocytes was significantly reduced in AD patients even when the clinical picture was atypical due to other co-existing disease processes. The test also identified the early, pr-clinical stage AD patients. It is reassuring that the results of patients suffering from non-AD dementias were similar to the control group. The effect of the cell cycle inhibitor rapamicin used to induce G1/S arrest depends on the integrity and expression of the p27 kip1 CDKI. The presence of the polymorphism on p27 kip1 described earlier could explain the lack of response to rapamicin. The other finding of the study was that in lymphocytes from AD patients sub-lethal oxidative stress did not induce cell cycle arrest to allow for DNA-repair. This finding was later replicated in fibroblasts from AD patients [START_REF] Uberti | Selective impairment of p53-mediated cell death in fibroblasts from sporadic Alzheimer's disease patients[END_REF]. At least in a subset of patients this phenomenon might be due to the p21 cip1 polymorphism associated with increased risk of AD and an earlier onset. Since oxidative stress is one of the factors believed to be necessary for the development of AD [START_REF] Zhu | Alzheimer's disease: the two-hit hypothesis[END_REF], the lack of the repair mechanisms could certainly explain the earlier appearance of the pathology.

The recent finding of differential Erk1 and Erk2 phosphorylation in response to bradykinin [START_REF] Dixonand | Regulation of mitogenesis by kinins in arterial smooth muscle cells[END_REF][START_REF] Dixon | Bradykinin B1 receptor blocks PDGF-induced mitogenesis by prolonging ERK activation and increasing p27Kip1[END_REF] (inflammatory agonist and G1 inhibitor) in fibroblasts from AD patients [START_REF] Khanand | An internally controlled peripheral biomarker for Alzheimer's disease: Erk1 and Erk2 responses to the inflammatory signal bradykinin[END_REF] further strengthens the hypothesis that that p27 kip1 mediated cell cycle

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responses are aberrant in AD patients and could be used as an early diagnostic marker for the disease.

The early peripheral markers of the G1/S regulatory failure appear to perform well as disease biomarkers and would be equally useful in clinical practice and drug trial design. However, more controlled studies are necessary to validate and standardise the protocols designed to identify patients with AD-type cell cycle regulatory deficits. The main caveat of course of these markers is that, although they identify AD patients accurately and early, they do not reflect the progression of the disease and ca not be used as outcome measures in clinical trials.

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A

  C C E P T E D M A N U S C R I P T Introduction By definition biomarkers fall into two categories: Biomarkers of 'exposure' and biomarkers of 'disease'. The Biomarkers of exposure (antecedent biomarkers) include measure of exposure to environmental risk factors and genetic risk factors. They are particularly useful for risk prediction. The biomarkers of 'disease' are indicators of biological factors which represent either a prodromal manifestation of the disease or a surrogate manifestation of the disease. These markers allow the identification of individuals A C C E P T E D M A N U S C R I P T

  physiology and pathology of AD became available in recent years. Many of these have also been validated against clinical and pathologic measured of the disease. Most of these markers indicate a more rapid change over time in AD patients than in agematched controls. It also appears that some changes identified by neuroimaging are more sensitive measures of disease progression than the standard measures of A C C E P T E D M A N U S C R I P T

  in apoptosis incompetent neurones. Based on this hypothesis the development of Alzheimer's disease is a 'two stage' process: cell cycle re-entry followed by regulatory failure at the G1/S transition point. The hypothesis poses several questions. What are the mitogenic stimuli that are able to drive mature differentiated neurones into the cell division cycle? What are the consequences of DNA replication in neurones that are 50-60 years old? What are the causes of the cell cycle regulatory failure at the G1/S transition point in some people but not others? Mitogenic stimuli in the brain.

  The cell cycle is driven by the sequential expression and activation of cyclin/cyclin dependent kinase (CDK) complexes and inhibited by the cyclin dependent kinase inhibitors (CDKIs). At each checkpoint different groups of cyclins kinases and CDKIs ensure that the cell responds adequately to the extra-and intracellular factors, which support either cell division or differentiation. When the intracellular environment is not capable of supporting the events initiated by extracellular signals cell death is initiated.At the G1/S transition point the cell cycle is driven by the activity of the cyclin E/CDK2 complex and inhibited by the members of the kip-cip protein family. There are three identified proteins in this family of CDKIs.The p21 cip1 (p21waf1, CDKN1A; 116899) gene for the protein has been mapped to 6p21.2. It is a short 21 kd protein, which inhibits the activity of CDK2 activated by cyclins D1, E and A, therefore inhibiting several phases of the cell cycle. Its expression is regulated at transcriptional level by p53, thus making this CDKI one of the effectors of DNA-damage induced cell cycle arrest at the G1/S and G2/M checkpoints. The successful cell cycle arrest, mediated by p21 cip1 will protect cells against DNA damage-induced apoptosis and conversely the inactivation of the protein A C C E P T E D M A N U S C R I P T will favour the induction of the p53-dependent apoptosis. Additionally the truncation of the C terminus of the protein by caspases 3 (and possibly other caspases) in apoptotic cells leads to a loss of the nuclear localisation signal, exit from the nucleus and loss of cell cycle inhibitor activity. As an inhibitor of the G1/S transition point it plays a part in the cell cycle exit and the activation of the differentiation programme in many cell types. The allelic variants of the gene are associated with elevated risk of tumours (http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=116899 ).

  cytoplasm imply inactive CDKIs, which would explain the cell cycle regulatory failure at the G1/S transition point and uninhibited progression of neurones through DNA replication. However, the inconsistent expression pattern of the three proteins and large variations from one patient to another imply that AD is a heterogeneous disorder associated with the deactivation of different CDKIs in different patients. The question is, though what inactivates the CDKIs in neurones? Truncation by caspases

A

  C C E P T E D M A N U S C R I P Thomocysteine levels, low thyroid and oestrogen levels can all be included in this category.Since the G1/S regulatory failure is important for disease development the genetic polymorphisms on the CDKI genes described above also constitute antecedent biomarkers. However, since neither the potential mitogenic stimuli nor the G1/S regulatory failure lead to AD-type changes in neurones on their own, the real risk of developing AD is only present in individuals where both types of risk factors are present. In this context risk assessment in any individual patient would require multiple tests, including genotyping for several genes, and the determination of complex risk-profiles.The disease biomarkers indicate the presence of phenomena or factors which are either a pre-clinical or a surrogate manifestation of the disease. These markers allow early diagnosis of individual patients but they are also important for the stratification of clinical trials.

Figure 1 .Figure 2 .

 12 Figure 1. The age of onset in Alzheimer's patients is reduced in the presence of the variant p21 cip1 gene. Y axis indicates the age of onset in years, error bars indicate the

Table 1 .

 1 The relative frequency of the variant p21 cip1 gene in AD patients and age matched control subjects. Odds ratio calculations indicate an association between the variant and Alzheimer's disease. This association is stronger in men.

	Diagnosis	P21 cip1 Variant P21 cip1	Relative	Odds Ratio
			Normal	Frequency
	AD	21	184	10%	1.64
	Men	10	84	11%	1.97
	Women	11	100	10%	1.42
	Control	20	288	6%
	Men	8	133	6%
	Women	12	155	7%

Table 2 .

 2 The relative frequency of the variant p27 kip1 gene in AD patients and age

	matched control subjects. Patient numbers and the relative frequency values
	(expressed in %).
	Diagnosis	AA109 G/G	AA109 V/G AA109 V/V
	AD	3 (4.05%)	34 (45.94%) 37 (50%)
	Control	9 (11.39%)	41 (51.89%) 29 (39.18%)

Table 3 .

 3 The relative frequency of the variant p57 kip2 gene in AD patients and age matched control subjects. Patient numbers and the relative frequency values (expressed in %).

	Diagnosis	AA159 A/A	AA159 A/V AA159 V/V
	AD	29 (53.70%)	21 (38.89%) 4 (7.41%)
	Control	73 (65.77%)	31 (27.93%) 7 (6.31%)

Acknowledgements

I would like to thank all members of Oxford Project to Investigate Memory and Ageing (OPTIMA). Without their help and the multiple collaborations this work would not have been possible. I am also grateful to all the patients, their families, carers and clinicians involved in this study. I am also grateful to former and present members of the group involved in sample preparation and some of the genotyping work, especially Dr. Anila Syed, Mrs. Margaret Reading and Miss Sharon Timms.

Ethical approval was granted by the Central Oxfordshire Research Ethics Committee.

The lymphocyte studies were supported by Research into Ageing, while the genotyping work was funded by the Jules Thorn Charitable Trust.


J Rashidian

G O Iyirhiaro

Ph.D David S Park
email: dpark@uottawa.ca

Cell cycle machinery and stroke

Keywords: Stroke, CDKs, cyclins, cell cycle, apoptosis

Stroke results from a transient or permanent reduction in blood flow to the brain. The mechanisms involving neuronal death following ischemic insult are complex and not fully understood. One signal which may control ischemic neuronal death is the inappropriate activation of cell cycle regulators including cyclins, cyclin dependent kinases (CDKs) and endogenous cyclin dependent kinase inhibitors (CDKIs). In dividing cells, activation of cell cycle machinery induces cell proliferation. In the context of terminally differentiated-neurons, however, aberrant activation of these elements triggers neuronal death. Indeed, there are several lines of correlative and functional evidence supporting this "cell cycle/neuronal death hypothesis". The objective of this review is to summarize the findings implicating cell cycle machinery in ischemic neuronal death from in vitro and in vivo studies. Importantly, determining and blocking the signaling pathway(s) by which these molecules act to mediate ischemic neuronal death, in conjunction with other targets may provide a viable therapeutic strategy for stroke damage.

Introduction

Stroke occurs primarily as a result of a transient or permanent interruption of blood supply to the brain. This condition can stem from an occluded or ruptured blood vessels and in some cases cardiac arrest. Consequently, neurons in the affected brain region, or the whole brain in the case of a cardiac arrest, are deprived of oxygen and glucose. This sets in motion a cascade of cellular activities that ultimately culminate in neuronal cell death [START_REF] Dirnagl | Pathobiology of ischaemic stroke: An integrated view[END_REF][START_REF] Endres | Ischemia and stroke[END_REF][START_REF] Nedergaard | Role of glial cells in cerebral ischemia[END_REF] .

Presently, stroke is a leading cause of death and permanent disability in industrialized nations.

Stroke occurs on average every 45 seconds in the USA (www.strokecenter.org). The American Heart Association (AHA) estimates the cost of treating stroke related injury and disability at $58 billion for 2006 alone (www.americanheart.org). Currently the treatment of stroke is mainly reliant on the use of thrombolytics such as tissue plasminogen activator (TPA), which themselves can pose an inherent risk of intracerebral hemorrhage. This limits the use of TPA to only certain cases of stroke.

Furthermore, the efficacy of TPA depends on timely presentation of <3 hours which excludes 95% of stroke patients [START_REF] Barber | Why are stroke patients excluded from tpa therapy? An analysis of patient eligibility[END_REF][START_REF] O'hare | Cyclin-dependent kinases as potential targets to improve stroke outcome[END_REF] . Thus there is a need to develop new and efficacious neuroprotective strategies for the treatment of stroke.

The development of new strategies for stroke hinges on better understanding of the complex cellular and molecular interplay that ensue following stroke. A maelstrom of dyregulated molecules and potential perpetrators of ischemic neuronal death have been recently suggested. One of these exciting new developments involves the role of cell cycle molecules such as the cyclin dependent kinases. The notion that cell cycle machinery may mediate ischemic neuronal death is not unique to stroke. Indeed research evidence from numerous labs have demonstrated correlative relationship between the dysregulation of cell cycle machinery and neuronal death models of neurodegenerative diseases such as Parkinson's disease (PD) [START_REF] El-Khodor | Ectopic expression of cell cycle markers in models of induced programmed cell death in dopamine neurons of the rat substantia nigra pars compacta[END_REF][START_REF] Jordan-Sciutto | Expression patterns of retinoblastoma protein in parkinson disease[END_REF] , Alzheimer's disease (AD) [START_REF] Milton | The amyloid-beta peptide binds to cyclin b1 and increases human cyclindependent kinase-1 activity[END_REF][START_REF] Nagy | Expression of cell division markers in the hippocampus in alzheimer's disease and other neurodegenerative conditions[END_REF][START_REF] Mcshea | Abnormal expression of the cell cycle regulators p16 and cdk4 in alzheimer's disease[END_REF], Amyotrophic

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Lateral Sclerosis (ALS) [START_REF] Nguyen | Cell cycle regulators in the neuronal death pathway of amyotrophic lateral sclerosis caused by mutant superoxide dismutase 1[END_REF][START_REF] Ranganathan | Hyperphosphorylation of the retinoblastoma gene product and altered subcellular distribution of e2f-1 during alzheimer's disease and amyotrophic lateral sclerosis[END_REF], and Niemann-Pick type C disease [START_REF] Husseman | Mitotic activation: A convergent mechanism for a cohort of neurodegenerative diseases[END_REF][START_REF] Zhang | Cyclin-dependent kinase inhibitors attenuate protein hyperphosphorylation, cytoskeletal lesion formation, and motor defects in niemann-pick type c mice[END_REF] . Whether or not cell cycle mechanism is similar in neurodegenerative diseases and acute conditions such stroke is unknown. This review will focus only on evidence implicating cell cycle molecules in ischemic neuronal death. We will also briefly discuss the potential involvement of other members of the CDK family not directly involved in cell cycle control, but only as potential activators of cell cycle machinery in stroke.

Ischemic Neuronal Death

The mechanism(s) of ischemic neuronal death is complex and may be determined by factors such as the location, severity and duration of insult. For example, neuronal death in the ischemic core, the region most severely affected by the lack of blood flow, occurs within minutes to a few hours and is marked predominantly by necrotic and excitotoxic cell death . Neuronal death in the ischemic penumbra, the region less severely affected by ischemia, is marked mainly by delayed apoptotic-like death that can progress over a period of days.

The precise sequence of events leading to neuronal death following stroke is at present not fully defined. However, a core picture is developing. Following the disruption of blood flow, the affected brain region(s) undergo a period of hypoxia resulting in a decrease in cellular ATP, due primarily to impaired mitochondrial oxidative respiration, a process that generates majority of the cellular energy In addition to the events describe above other stressors such as oxidative stress, and DNA damage have been demonstrated to mediate ischemic neuronal death. Furthermore, extrinsic stressors such as the activation of glial cells and inflammation may impinge on ischemic neurons to mediate their demise. These later findings are particularly relevant when it comes to therapeutic interventions. In fact, cell cycle regulation may also play a critical role with these non neuronal cell types in brain injury [START_REF] Cernak | Role of the cell cycle in the pathobiology of central nervous system trauma[END_REF][START_REF] Di Giovanni | Cell cycle inhibition provides neuroprotection and reduces glial proliferation and scar formation after traumatic brain injury[END_REF] . However, we will not mention this further in this review, but will instead focus on how the cell cycle machinery may impact neurons more directly.

Cell cycle regulation

Cell cycle is a highly regulated process. Timing progression of cell cycle through different phases, G0, G1 S, G2, and M requires an orchestrated functions of several elements, including cyclins, cyclin-dependent kinases (CDKs), retinoblastoma protein (Rb; pocket proteins) and E2F

complex proteins [START_REF] Pines | Cyclins and their associated cyclin-dependent kinases in the human cell cycle[END_REF]. Different complexes of cyclin-CDK drive each phase of cell cycle [START_REF] Nurse | A long twentieth century of the cell cycle and beyond[END_REF]. In this regard, the current model is that cyclin D-cdk4/6 and cyclin E-cdk2 complexes regulate G1/S progression, cyclin A-cdk2 complexes mediate S/G2 transitions, and cyclin B-cdc2 complexes mediate M-phase progression [START_REF] Ekholm | Regulation of g(1) cyclin-dependent kinases in the mammalian cell cycle[END_REF][START_REF] Pines | Cyclins and cyclin-dependent kinases: A biochemical view[END_REF] . In addition, a recent report has suggested that cyclin C-cdk3 complexes also regulate G0/G1 transition [START_REF] Ren | Cyclin c/cdk3 promotes rb-dependent g0 exit[END_REF] (Figure1). CDKs activity is regulated by binding to activating cyclin partners [START_REF] Lees | Cyclin dependent kinase regulation[END_REF] as well as endogenous CDK inhibitors such as members of the INK4 (p15, p16, p18, p19) and Cip/Kip (p21, p27, p57) families [START_REF] Morgan | Principles of cdk regulation[END_REF][START_REF] Sherr | Cdk inhibitors: Positive and negative regulators of g1phase progression[END_REF] . Finally, phosphorylation also plays a critical role in CDK regulation. For example, cyclin H-cdk7-Mat1 (Cdk Activating Kinase;

CAK complex) by phosphorylation of a central threonine [START_REF] Fisher | A novel cyclin associates with mo15/cdk7 to form the cdk-activating kinase[END_REF] and cdc25 by dephosphorylation of thr14 (on cdc2) or tyr15 (on cdk2) modulate CDKs conformation to a fully active structure so they recognize their substrates more efficiently [START_REF] Morgan | Principles of cdk regulation[END_REF] . CDKs mediate cell cycle progression by phosphorylating downstream targets [START_REF] Morgan | Cyclin-dependent kinases: Engines, clocks, and microprocessors[END_REF] . For example, an important target of G1 CDKs is the

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tumor suppressor, Rb [START_REF] Weinberg | The retinoblastoma protein and cell cycle control[END_REF]. In mid-to late-G1, Rb is sequentially phosphorylated by cyclin D1-cdk4/6

and cyclin E-cdk2 complexes. Once hyperphosphorylated, Rb repression of E2F containing complexes is alleviated. E2F, along with a required binding partner DP activate genes required for S phase progression [START_REF] Chellappan | The e2f transcription factor is a cellular target for the rb protein[END_REF][START_REF] Dyson | The regulation of e2f by prb-family proteins[END_REF][START_REF] Harbour | Cdk phosphorylation triggers sequential intramolecular interactions that progressively block rb functions as cells move through g1[END_REF].

A growing body of exciting evidence indicates that CDKs may have a function beyond its canonical role in cell cycle regulation. Indeed, it has been suggested that neuronal death may be controlled by the inappropriate activation of some of cell cycle elements [START_REF] Becker | Beyond proliferation--cell cycle control of neuronal survival and differentiation in the developing mammalian brain[END_REF]. The hypothesis, compatible with the general cell cycle/neuronal death notion, is that activation of these regulatory elements, in the context of post-mitotic neurons, signals for death rather than cell cycle progression.

Several lines of in vitro and in vivo evidence support this hypothesis that aberrant activation of cell cycle involves in different pathological conditions such as stroke [START_REF] Osuga | Cyclin-dependent kinases as a therapeutic target for stroke[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][START_REF] Rashidian | Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo[END_REF] , AD [START_REF] Herrup | Re-expression of cell cycle proteins induces neuronal cell death during alzheimer's disease[END_REF], PD [START_REF] El-Khodor | Ectopic expression of cell cycle markers in models of induced programmed cell death in dopamine neurons of the rat substantia nigra pars compacta[END_REF][START_REF] Jordan-Sciutto | Expression patterns of retinoblastoma protein in parkinson disease[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] .

In this review, we will first start our discussion with deregulation/upregulation of cell cycle regulators in in vitro paradigms of ischemic neuronal death and then will provide in vivo evidence suggesting that cell cycle might control death following ischemic neuronal injury in the adult animal.

Finally, we will touch upon the role of other CDKs, in particular the neuronal cdk5 which has important roles in neuronal development and death and which might also link to cell cycle signals.

In vitro evidence for involvement of cell cycle elements in stroke

Generally, terminally differentiated cells such as neuron do not divide. Indeed as neurons undertake the process of terminal differentiation they irreversibly withdraw from the cell cycle. The levels and activity of key cell cycle regulators are downregulated in differentiated neurons [START_REF] Kranenburg | Inhibition of cyclindependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells[END_REF][START_REF] Sumrejkanchanakij | Role of cyclin d1 cytoplasmic sequestration in the survival of postmitotic neurons[END_REF] .

For example, Cdk4, a regulator of G1/S transition becomes increasingly associated with the CDK inhibitor p27 and its activity declines in parts due to loss of phosphorylation by its activator CAK

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7 [START_REF] Kranenburg | Inhibition of cyclindependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells[END_REF]. Similarly, the levels and activity of cdk2 another important regulator of the G1/S is downregulated in differentiated neurons. Consequently, the levels of hypophosphorylated Rb increases resulting in greater E2F1 sequestration [START_REF] Kranenburg | Inhibition of cyclindependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells[END_REF]. Silencing E2F1 in neurons is particularly important since its overexpression promotes aberrant S-phase entry in neurons [START_REF] Azuma-Hara | Regulation and deregulation of e2f1 in postmitotic neurons differentiated from embryonal carcinoma p19 cells[END_REF] . Furthermore, overexpression of E2F1 alone in the context of a post-mitotic neuron and in the absence of insult induces neuronal death [START_REF] Azuma-Hara | Regulation and deregulation of e2f1 in postmitotic neurons differentiated from embryonal carcinoma p19 cells[END_REF][START_REF] Giovanni | E2f1 mediates death of b-amyloid-treated cortical neurons in a manner independent of p53 and dependent on bax and caspase 3[END_REF] . Downregulation of cell cycle machinery and inhibition of E2F1 activity through its sequestration by Rb is thus an important feature of terminally differentiated neurons. This is underscored by observations that overexpression of Rb or the CDK inhibitor p27 is sufficient to induce neuronal differentiation [START_REF] Kranenburg | Inhibition of cyclindependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells[END_REF]. In addition to downregulation of CDK levels and activity, observations by Sumrejkanchanakij et al. [START_REF] Sumrejkanchanakij | Cytoplasmic sequestration of cyclin d1 associated with cell cycle withdrawal of neuroblastoma cells[END_REF] suggests that the cell cycle may be held in check in post-mitotic neurons through cellular redistribution of cell cycle components. For example, in undifferentiated neurons cyclin D1 appears both cytoplasmic and nuclear but becomes predominantly cytoplasmic in differentiated neuroblastoma and cortical neurons [START_REF] Sumrejkanchanakij | Role of cyclin d1 cytoplasmic sequestration in the survival of postmitotic neurons[END_REF][START_REF] Sumrejkanchanakij | Cytoplasmic sequestration of cyclin d1 associated with cell cycle withdrawal of neuroblastoma cells[END_REF] . Ectopic expression of cyclin D1 in differentiated neurons leads to cytoplasmic sequestration whereas forced nuclear expression by fusing to a nuclear localization signal induces apoptosis [START_REF] Sumrejkanchanakij | Role of cyclin d1 cytoplasmic sequestration in the survival of postmitotic neurons[END_REF] . This suggests that cellular redistribution of cell cycle machinery is an important aspect in the maintenance of quiescent state in adult neurons. While the above evidence indicates what happens under normal developmental conditions, accumulating research evidence also suggests that cell cycle machinery are activated in adult neurons in response to numerous stressors such as DNA damage, NGF deprivation, proteosome inhibition and ß-amyloid toxicity and models of ischemic neuronal death [START_REF] Giovanni | E2f1 mediates death of b-amyloid-treated cortical neurons in a manner independent of p53 and dependent on bax and caspase 3[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][START_REF] Katchanov | Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death[END_REF][START_REF] Park | Inhibitors of cyclin-dependent kinases promote survival of post-mitotic neuronally differentiated pc12 cells and sympathetic neurons[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][START_REF] Park | G1/s cell cycle blockers and inhibitors of cyclin-dependent kinases suppress camptothecin-induced neuronal apoptosis[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] Verdaguer | Inhibition of cdks: A strategy for preventing kainic acid-induced apoptosis in neurons[END_REF] .

In regards to the latter type of injury, multiple lines of evidence in vitro suggest a role for cell cycle regulators as mediators of ischemic injury. Firstly, Katchanov and colleagues [START_REF] Katchanov | Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death[END_REF] reported lose of the CDK inhibitor p27 following oxygen glucose deprivation (OGD) of neocortical neurons. This

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8 group also reported increases in cyclin D1 protein levels and activation of cdk2 following OGD [START_REF] Katchanov | Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death[END_REF] .

Similar increase in cyclin D1, cyclin E, and cdk2 is observed following kainic acid (KA) induced death in cerebellar granule neurons (CGNs) [START_REF] Verdaguer | Kainic acid-induced apoptosis in cerebellar granule neurons: An attempt at cell cycle re-entry[END_REF][START_REF] Giardina | Kainate-induced apoptosis in cultured murine cerebellar granule cells elevates expression of the cell cycle gene cyclin d1[END_REF]. Secondly, Rb, a downstream target of cyclinD/Cdk4 is increasingly phosphorylated following hypoxia/reoxygenation and KA induced death [START_REF] Rashidian | Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo[END_REF][START_REF] Smith | Involvement of the transcription factor e2f1/rb in kainic acid-induced death of murine cerebellar granule cells[END_REF] suggesting inactivation of Rb and activation of the cell cycle in these neurons.

Additionally, increase in E2F1 (downstream effector of Cdk4) protein levels and proliferating cell nulear antigen (PCNA) another S-phase marker (required for completion of DNA synthesis and repair) is observed following KA induced death [START_REF] Verdaguer | Kainic acid-induced apoptosis in cerebellar granule neurons: An attempt at cell cycle re-entry[END_REF][START_REF] Verdaguer | Inhibition of cell cycle pathway by flavopiridol promotes survival of cerebellar granule cells after an excitotoxic treatment[END_REF] . Finally, increase in E2F1 mRNA transcript is also observed following OGD [START_REF] Gendron | Attenuation of neurotoxicity in cortical cultures and hippocampal slices from e2f1 knockout mice[END_REF]. These observations discussed above seem to implicate the reactivation of cell cycle machinery in ischemic neuronal death and have lead to the hypothesis that inappropriate activation of the cell cycle in the context of differentiated neurons may underlie ischemic neuronal death. However, this evidence only showed the activation of cell cycle components and does not address the issue of whether this signal is required for death. In other words, does the activation of the cell cycle in adult neurons underlie neuronal death in stroke or are these observations simply an artifact of the cell death process? Studies using pharmacological inhibitors of cell cycle regulators seem to lend support to the former. For example, treatment of neurons with the CDK inhibitors olomoucine, roscovitine, 3-amino thioacridone (3-ATA) and flavopiridol has been shown to protect cortical neurons and CGNs from OGD and KA induced cell death [START_REF] Katchanov | Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death[END_REF][START_REF] Verdaguer | Inhibition of cdks: A strategy for preventing kainic acid-induced apoptosis in neurons[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] Giardina | Kainate receptor-mediated apoptosis in primary cultures of cerebellar granule cells is attenuated by mitogen-activated protein and cyclindependent kinase inhibitors[END_REF] . While these pharmacological studies supports the involvement of cell cycle components in ischemic neuronal death, the caveat to these studies is the relatively non-specific nature of inhibitors such as flavopiridol which in addition to the cell cycle CDKs can potently inhibit non-mitotic CDKs such as cdk5 [START_REF] De Azevedo | Structural basis for specificity and potency of a flavonoid inhibitor of human cdk2, a cell cycle kinase[END_REF] , cdk7 [START_REF] Fisher | A novel cyclin associates with mo15/cdk7 to form the cdk-activating kinase[END_REF] as well as non-CDK-related kinases such as GSK-3ß [START_REF] Leclerc | Indirubins inhibit glycogen synthase kinase-3 beta and cdk5/p25, two protein kinases involved in abnormal tau phosphorylation in alzheimer's disease. A property common to most cyclin-dependent kinase inhibitors?[END_REF]. To address this, the functional relevance of cell cycle machinery in ischemic neuronal death was demonstrated by utilizing genetic manipulations of components of the cell cycle. Firstly,

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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] showed that adenoviral delivered kinase dead cdk4 protects CGNs from hypoxia mediated delayed death. Secondly, they showed that neurons derived from mice expressing kinase dead cdk4 or null for its regulator cyclin D1 are resistant to hypoxia mediated ischemic death [START_REF] Rashidian | Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo[END_REF] .

Further support for the functional role of cell cycle pathway in ischemic neuronal death is provided by observations that cortical neurons and CGNs derived from E2F1 null mice are less susceptible to death mediated by OGD and KA induced death respectively [START_REF] Smith | Involvement of the transcription factor e2f1/rb in kainic acid-induced death of murine cerebellar granule cells[END_REF][START_REF] Gendron | Attenuation of neurotoxicity in cortical cultures and hippocampal slices from e2f1 knockout mice[END_REF] . Furthermore, E2F1 deficiency improves the recovering of CA1 neurons from loss of synaptic transmission following anoxic insult of hippocampal slices in vitro [START_REF] Gendron | Attenuation of neurotoxicity in cortical cultures and hippocampal slices from e2f1 knockout mice[END_REF] . The above in vitro evidence is summarized in tables 1 and 2.

Taken together these in vitro evidence strongly implicate the reactivation of cell cycle components in ischemic neuronal death. What evidence is there to implicate the involvement of cell cycle machinery in vivo? Are these observations relevant in intact physiological systems?

In vivo evidence for involvement of cell cycle elements in stroke

The above evidence indicates that neurons grown in culture can re-activate cell cycle signals upon an exogenous stress leading to death. This suggests that terminally differentiated, post mitotic neurons from embryonic or early post natal sources have retained their capacity to reactivate cell cycle CDK mediated signals. However, these neurons have only recently become post-mitotic and might represent a unique situation where the cell cycle machinery is still present. Indeed, reports have shown that while downregulated, multiple CDKs, cyclins as well as there regulators exist in cultured neurons [START_REF] Kranenburg | Inhibition of cyclindependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells[END_REF][START_REF] Sumrejkanchanakij | Role of cyclin d1 cytoplasmic sequestration in the survival of postmitotic neurons[END_REF] . Questions remain about the more relevant situation where ischemic injury occurs in fully matured adult neurons where there is likely very little if any cell cycle machinery present under basal conditions. Here too, there is accumulating evidence that upregulation/activation of cell cycle machinery occurs and this leads to death in neurons following stroke insult.

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The first body of work linking cell cycle to ischemic injury in the adult context is the correlative data demonstrating that CDKs are changed following stroke injury in vivo. In these situations, a central player may again be the cdk4-cyclin D complex. For example, Cdk4/6 kinase activity/levels along with its activator cyclin D1 are upregulated in different models of ischemia in vivo. It has been shown that cyclin D1 is expressed in infarct region following MCAO model of focal ischemia [START_REF] Osuga | Cyclin-dependent kinases as a therapeutic target for stroke[END_REF][START_REF] Katchanov | Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death[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 | Neuronal damage and plasticity identified by microtubule-associated protein 2, growth-associated protein 43, and cyclin d1 immunoreactivity after focal cerebral ischemia in rats[END_REF][START_REF] Wen | Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats[END_REF] and in vulnerable regions of the hippocampus following transient global ischemia [START_REF] Wiessner | Cyclin d1 messenger rna is induced in microglia rather than neurons following transient forebrain ischaemia[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] 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] Small | Characterization of cyclin d1 expression in a rat global model of cerebral ischemia[END_REF][START_REF] Kato | Cell cycle protein expression in proliferating microglia and astrocytes following transient global cerebral ischemia in the rat[END_REF] . Consistent with these observations, increased levels of cyclin D1 has been demonstrated in response to an excitotoxic death induced by systemic injection of KA [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] Liu | Increased expression of cyclin d1 in the adult rat brain following kainic acid treatment[END_REF][START_REF] Park | Cell cycle regulators in neuronal death evoked by excitotoxic stress: Implications for neurodegeneration and its treatment[END_REF][START_REF] Ino | Cyclin-dependent kinase 4 and cyclin d1 are required for excitotoxin-induced neuronal cell death in vivo[END_REF] and transient spinal cord ischemia [START_REF] Sakurai | Cyclin d1 and cdk4 protein induction in motor neurons after transient spinal cord ischemia in rabbits[END_REF] . Cdk4 itself is also changed during ischemic insult. Aberrant expression of cdk4 has been shown in MCAO [START_REF] Hayashi | DNA fragmentation precedes aberrant expression of cell cycle-related protein in rat brain after mca occlusion[END_REF] and clip models of focal ischemia [START_REF] Osuga | Cyclin-dependent kinases as a therapeutic target for stroke[END_REF].

Furthermore, other groups have reported induction of cdk4 expression in other models such as transient global ischemia [START_REF] Kato | Cell cycle protein expression in proliferating microglia and astrocytes following transient global cerebral ischemia in the rat[END_REF] , KA-mediated cortical death [START_REF] Ino | Cyclin-dependent kinase 4 and cyclin d1 are required for excitotoxin-induced neuronal cell death in vivo[END_REF] and transient spinal cord ischemia [START_REF] Sakurai | Cyclin d1 and cdk4 protein induction in motor neurons after transient spinal cord ischemia in rabbits[END_REF].

Although the above evidence implicates cyclinD1-cdk4 complexes in ischemic injury, it must be kept in mind that other components of G1 phase might have potential roles in vivo as well. For instance, cdk6 is also activated by cyclin D [START_REF] Pines | Cyclins and their associated cyclin-dependent kinases in the human cell cycle[END_REF]. In addition, there are some lines of data suggesting deregulation of cdk2 after MCAO and a hypoxic cerebral ischemia [START_REF] Katchanov | Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death[END_REF][START_REF] Kuan | Hypoxiaischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain[END_REF] . Consistent with this, Li et al. [START_REF] Li | Apoptosis and protein expression after focal cerebral ischemia in rat[END_REF] provided evidence that cdk2, as well as its partner, cyclin A, are deregulated following focal ischemia.

In line with the upregulation/activation of cell cycle CDK complexes, evidence also point to a modulation of endogenous regulators of CDKs, the CDKIs. For example, Katchanov et al. [START_REF] Katchanov | Mild cerebral ischemia induces loss of cyclin-dependent kinase inhibitors and activation of cell cycle machinery before delayed neuronal cell death[END_REF] demonstrated that p16 is downregulated very early in dying neurons, followed by upregulation of cyclin D1 after a mild cerebral ischemia. Additional intriguing observations suggesting that CDKIs might have a functional role in ischemic death were provided by Van Lookeren et al. [START_REF] Van Lookeren Campagne | Cell cycle-related gene expression in the adult rat brain: Selective induction of cyclin g1 and p21waf1/cip1 in neurons following focal cerebral ischemia[END_REF][START_REF] Van Lookeren Campagne | Increased expression of cyclin g1 and p21waf1/cip1 in neurons following transient forebrain ischemia: Comparison with early DNA damage[END_REF]. They

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11 indicated that p21 expression is enhanced in surviving neurons surrounding dead area in both focal and transient forebrain ischemia. They suggested that elevation of p21 is actually a response to the ischemic insult to protect the neurons by arresting them in first steps of the cell cycle. In line with these findings, p21 mRNA was elevated in perifocal ischemia regions following focal ischemia, while no significant change was observed in ischemic area [START_REF] Schmidt-Kastner | Differential changes of bax, caspase-3 and p21 mrna expression after transient focal brain ischemia in the rat[END_REF]. More importantly, Kuan et al. [START_REF] Kuan | Hypoxiaischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain[END_REF] demonstrated that p16 and p27 are depleted in dying hippocampal neurons and this may promote G1/S phase transition following hypoxia-ischemia insult.

The above mentioned in vivo evidence does not indicate whether these signals actually play a role in ischemic damage. To address this, several labs, including ours, have provided more direct data by carrying out additional functional studies. In support of a require role, inhibition of the cell cycle regulating proteins is protective in some of the ischemic models. For instance, administration of a synthetic cdk4 inhibitor, or alternatively, cyclin D1 and cdk4 antisense oligonucleotides has been shown to be neuroprotective in MCAO and KA-inducing excitotoxicity models, respectively [START_REF] Wen | Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats[END_REF][START_REF] Ino | Cyclin-dependent kinase 4 and cyclin d1 are required for excitotoxin-induced neuronal cell death in vivo[END_REF].

We have recently shown that flavopiridol, a CDK inhibitor, administrated before or after the insult, blocks the delayed death evoked by focal or global ischemia [START_REF] Osuga | Cyclin-dependent kinases as a therapeutic target for stroke[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]. Importantly, flavopiridol blocked activation of the cdk-Rb pathway, reduced E2F1 levels and improved behavioral performance [START_REF] Osuga | Cyclin-dependent kinases as a therapeutic target for stroke[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] . More critically, we also investigated involvement of specific CDKs by expression of kinase dead dominant negative forms of several cell cycle CDKs, as well as cdk5, in delayed and excitotoxic types of ischemia. In this research for the first time we provided functional evidence that in adult system, cyclin D1-cdk4 complex has a key role in inducing delayed neuronal death [START_REF] Rashidian | Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo[END_REF] . We also showed that phosphorylation of Rb, mediated by this complex, occurs very early following global ischemia and that this activation is inhibited by DN cdk4 expression [START_REF] Rashidian | Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo[END_REF].

Expression of DN cdk4 also improves behavioral performance in rats suggesting that brains are not

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12 only neuroprotected, but that this leads to improved function [START_REF] Rashidian | Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo[END_REF] . This final point is particularly relevant to the goal of human therapy.

How do cyclin-CDK complexes mediate a potential ischemic death signal, in vivo? In other words, what is (are) the downstream target(s) for these complexes in adult systems? Recent findings suggest that inactivation of Rb leads to neuronal apoptosis, in vivo. Supporting this, overexpression of large T antigen in cerebellar Purkinje cells initially resulted in DNA synthesis but eventually caused neuronal degeneration in transgenic mice [START_REF] Feddersen | Disrupted cerebellar cortical development and progressive degeneration of purkinje cells in sv40 t antigen transgenic mice[END_REF]. Rb deficiency is also embryonically lethal because of widespread apoptosis in CNS and PNS [START_REF] Jacks | Effects of an rb mutation in the mouse[END_REF][START_REF] Lee | Mice deficient for rb are nonviable and show defects in neurogenesis and haematopoiesis[END_REF]. However, in this case, much of the death may be due to non neuron autonomous events and not directly caused by Rb deficiency [START_REF] Ferguson | Telencephalon-specific rb knockouts reveal enhanced neurogenesis, survival and abnormal cortical development[END_REF].

Observations in different in vivo models of ischemia have suggested that CDKs activity induced by apoptotic insults lead to phosphorylation and inactivation of Rb. For example, we have shown that Rb phosphorylation (on ser-795, a consensus site for CDKs) is an early that event happens a few hours after focal ischemia [START_REF] Osuga | Cyclin-dependent kinases as a therapeutic target for stroke[END_REF], global ischemia [START_REF] Wang | Inhibition of cyclin-dependent kinases improves ca1 neuronal survival and behavioral performance after global ischemia in the rat[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 KA injection [START_REF] Park | Cell cycle regulators in neuronal death evoked by excitotoxic stress: Implications for neurodegeneration and its treatment[END_REF]. Moreover, other groups have also shown that Rb is progressively phosphorylated at CDKs consensus sites after focal ischemia induced by MCAO and hypoxic cerebral ischemia and that this might trigger apoptotic neuronal death pathways [START_REF] Wen | Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats[END_REF][START_REF] Kuan | Hypoxiaischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain[END_REF][START_REF] Hayashi | Phosphorylation of retinoblastoma protein in rat brain after transient middle cerebral artery occlusion[END_REF].

To investigate the mechanism by which Rb inactivation may promote death, E2F family proteins, as a group of proteins regulated by Rb, have been studied in vivo. In this regard, it has been suggested that E2F may play an important role in neuronal death in select conditions such as stroke.

For example, E2F1 deficient mice has been shown to exhibit smaller brain injury and improved behavior after focal ischemia [START_REF] Macmanus | Decreased brain infarct following focal ischemia in mice lacking the transcription factor e2f1[END_REF][START_REF] Macmanus | Absence of the transcription factor e2f1 attenuates brain injury and improves behavior after focal ischemia in mice[END_REF] . In agreement with this, upregulation of E2F1 has been implicated in animal models of ischemia. For instance, we have shown that focal ischemia induces E2F1 expression which occurs following Rb phosphorylation and administration of CDK inhibitor diminishes this effect and protects the neurons [START_REF] Osuga | Cyclin-dependent kinases as a therapeutic target for stroke[END_REF]. Microarray analysis also has also revealed that

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E2F1 is among the upregulated genes after global ischemia [START_REF] Jin | Microarray analysis of hippocampal gene expression in global cerebral ischemia[END_REF]. Finally, a very recent study demonstrated that E2F1 and its target gene, c-myb, is upregulated in hippocampus following global ischemia [START_REF] Hwang | The pattern of e2f1 and c-myb immunoreactivities in the ca1 region is different from those in the ca2/3 region of the gerbil hippocampus induced by transient ischemia[END_REF]. What factor(s) determine which responsive genes, whether cell cycle or apoptotic, be regulated? It is not clear yet, but there is an idea suggesting that there is a pool of transcriptional activity for E2F. When this activity gets to a specific threshold the genes regulating cell cycle progression are induced. But when this activity reaches a second higher threshold, apoptotic genes are induced and death mechanisms start [START_REF] Trimarchi | Sibling rivalry in the e2f family[END_REF]. Almost nothing is known about the targets of E2F1 in ischemic death, in vivo, and this area needs to be intensively investigated.

Although what has been discussed so far suggests that cell cycle is aborted mostly at G1/S checkpoint in ischemic death, some reports have shown S phase entry. Indeed, neurons start to replicate DNA before they die in some systems. To investigate whether DNA synthesis is induced in dying neurons, induction of PCNA and incorporation of Brdu have been used as markers for S phase.

In this regard, induction of PCNA has been demonstrated in several models of focal and global ischemia [START_REF] Wen | Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats[END_REF][START_REF] Kato | Cell cycle protein expression in proliferating microglia and astrocytes following transient global cerebral ischemia in the rat[END_REF][START_REF] Li | Apoptosis and protein expression after focal cerebral ischemia in rat[END_REF][START_REF] Tomasevic | Changes in proliferating cell nuclear antigen, a protein involved in DNA repair, in vulnerable hippocampal neurons following global cerebral ischemia[END_REF]. In other reports, incorporation of Brdu in apoptotic neurons is indicative of tendency for DNA synthesis in these cells [START_REF] Wen | Cell-cycle regulators are involved in transient cerebral ischemia induced neuronal apoptosis in female rats[END_REF][START_REF] Kuan | Hypoxiaischemia induces DNA synthesis without cell proliferation in dying neurons in adult rodent brain[END_REF] . Interestingly, these injured, "S phase positive" neurons contained elevated G1 cyclin-CDK complexes and co-localized with apoptotic markers (TUNEL positive); suggesting that cell cycle is already triggered and they have transited to S phase to resume DNA synthesis but for some reasons they undergo apoptosis and do not replicate.

Therefore, there is no in vivo evidence of G2 entry in ischemic neurons, to our knowledge.

Nevertheless, expression of cdc2, as E2F target and a G2 marker has been investigated in other neuronal death systems [START_REF] Konishi | The e2f-cdc2 cell-cycle pathway specifically mediates activity deprivation-induced apoptosis of postmitotic neurons[END_REF]. This might be an important area of future research in the stroke field.

In spite of the accumulating in vivo experimental studies demonstrating aberrant deregulation of cell cycle in stroke, there is little known about how/whether these processes occur in humans.

Recently, a group investigated cell cycle elements in brains of postmortem patients who died after

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14 cardiac arrest or focal brain infarction. They reported elevated levels of cyclin D1, cdk2 and cdk4 [START_REF] Love | Neuronal expression of cell cycle-related proteins after brain ischaemia in man[END_REF] . This study shows that results obtained from experimental studies in animals may be relevant to the human condition.

The in vivo evidence discussed above is summarized in tables 1 and 2.

Role of other CDK members including cdk5 in stroke

In addition to the core cell cycle components discussed above, other cyclins and CDK members which are not exclusively or fully linked with cell cycle regulation may also participate in ischemic response. For example, cyclin H, a component of the CAK complex is increased in CA3, dentate gyrus, and cortex after global ischemia [START_REF] Jin | Molecular cloning of a cell cycle regulation gene cyclin h from ischemic rat brain: Expression in neurons after global cerebral ischemia[END_REF]. Since these areas are resistant to this type of ischemia, it has been suggested that cyclin H might have a role in survival through its DNA repair function in ischemic injury. Cyclin G is another member of the cyclins family but its role in cell cycle is controversial. While some evidence shows its stimulating role in growth [START_REF] Smith | The p53-regulated cyclin g gene promotes cell growth: P53 downstream effectors cyclin g and gadd45 exert different effects on cisplatin chemosensitivity[END_REF][START_REF] Skotzko | Retroviral vectormediated gene transfer of antisense cyclin g1 (cycg1) inhibits proliferation of human osteogenic sarcoma cells[END_REF], others suggesting it inhibits growth [START_REF] Bates | Characterisation of human cyclin g1 and g2: DNA damage inducible genes[END_REF][START_REF] Okamoto | A role of cyclin g in the process of apoptosis[END_REF] in proliferating cells. The function of cyclin G in neurons is also unclear.

Nevertheless, it has been shown that cyclin G1 is increased following MCAO and global ischemia [START_REF] Van Lookeren Campagne | Cell cycle-related gene expression in the adult rat brain: Selective induction of cyclin g1 and p21waf1/cip1 in neurons following focal cerebral ischemia[END_REF][START_REF] Van Lookeren Campagne | Increased expression of cyclin g1 and p21waf1/cip1 in neurons following transient forebrain ischemia: Comparison with early DNA damage[END_REF], as well as spinal cord injury [START_REF] Di Giovanni | Gene profiling in spinal cord injury shows role of cell cycle in neuronal death[END_REF]. Furthermore, Maeda et al. [START_REF] Maeda | The p53-independent nuclear translocation of cyclin g1 in degenerating neurons by ischemic and traumatic insults[END_REF] demonstrated that cyclin G1 is translocated into the nucleus of degenerating neurons following transient common carotid artery occlusion (CCAO) and MCAO. Therefore, they suggested that the cytoplasmic cyclin G1 might be associated with cell survival while nuclear cyclin G1 might signal death.

Perhaps the strongest evidence for the involvement of a CDK member not classically associated with the cell cycle involves cdk5. Cdk5 is a unique member of cyclin-dependent kinases family. Unlike the other CDKs, it does not require a cyclin to be activated. P35 and p39, instead, are activators of cdk5 and are present mainly in neurons [START_REF] Tsai | P35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5[END_REF][START_REF] Tang | An isoform of the neuronal cyclin-dependent kinase 5 (cdk5) activator[END_REF]. For this reason, although is ubiquitously expressed, cdk5 activity is mainly restricted to nervous system [START_REF] Tsai | Activity and expression pattern of cyclin-dependent kinase 5 in the embryonic mouse nervous system[END_REF] . Even though there

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15 is no functional evidence for involvement of cdk5 directly in the core cell cycle machinery, some recent observations have led to the idea that it might be associated with cell cycle components and, therefore, affects its transition. Initially it was shown that cdk5 was able to bind to cyclins D1, D3

and E [START_REF] Xiong | D type cyclins associate with multiple protein kinases and the DNA replication and repair factor pcna[END_REF][START_REF] Miyajima | Cyclin e is expressed in neurons and forms complexes with cdk5[END_REF][START_REF] Zhang | Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes[END_REF]. However, this binding did not lead to increased CDK activity. Further studies showed that cdk5 could phosphorylate Rb in vitro [START_REF] Hamdane | P25/cdk5-mediated retinoblastoma phosphorylation is an early event in neuronal cell death[END_REF]. In this study, overexpression of p25 in inducible-neuroblastoma cells led to deregulation of cell cycle elements such as elevation of cyclins A and B1 and cdc2, downregulation of p27 and finally apoptosis. Interestingly, Rb phosphorylation on ser807/811 sites was an early event in this study and was mediated by cdk5 activity since it was abolished by a cdk5 inhibitor [START_REF] Hamdane | P25/cdk5-mediated retinoblastoma phosphorylation is an early event in neuronal cell death[END_REF]. Finally, a very recent report revealed that cdk5 is necessary for neuronal cell cycle arrest [START_REF] Cicero | Cyclin-dependent kinase 5 is essential for neuronal cell cycle arrest and differentiation[END_REF] . This group suggested that cdk5 connects and inhibits cell cycle possibly by competing with cdk4/6 for binding to cyclins or through phosphorylation and inhibition of Rb or, alternatively, by phosphorylation of RNA polymerase.

Cdk5 has been implicated in different neurodegenerative disorders including brain ischemia.

The generally accepted concept about cdk5 is that cdk5/p35 complexes are regulators of normal biological functions of neuron. But under some neuropathological conditions p35 is cleaved to a more potent activator, p25 [START_REF] Patrick | Conversion of p35 to p25 deregulates cdk5 activity and promotes neurodegeneration[END_REF]. Production of p25 unmasks pathological face of cdk5 since it has prolonged half life, over activates, dislocates and might change cdk5 substrate specificity. There is increasing evidence, though, that argue this model. Several studies have debated role of p25 in pathogenecity of cdk5 in some circumstances [START_REF] Tandon | Brain levels of cdk5 activator p25 are not increased in alzheimer's or other neurodegenerative diseases with neurofibrillary tangles[END_REF][START_REF] Yoo | P25 protein in neurodegeneration[END_REF][START_REF] Takashima | Involvement of cyclin dependent kinase5 activator p25 on tau phosphorylation in mouse brain[END_REF][START_REF] Takahashi | Mutant superoxide dismutase 1 causes motor neuron degeneration independent of cyclin-dependent kinase 5 activation by p35 or p25[END_REF][START_REF] Hallows | P35/p25 is not essential for tau and cytoskeletal pathology or neuronal loss in niemann-pick type c disease[END_REF] . While an exhaustive review of this issue is not possible here, we will suggest that, depending upon different factors; both p35 and p25 are able to drive "good-normal" cdk5 to "bad-pathogenic" cdk5.

Contribution of cdk5 in stroke has not been intensively studied and our knowledge about desregulated cdk5 in stroke comes from very recent research. One of the initial reports came from Hayashi et al. study [START_REF] Hayashi | Expression of cyclin-dependent kinase 5 and its activator p35 in rat brain after middle cerebral artery occlusion[END_REF] . They showed that immunoreactivity of cdk5 and p35/p25 is enhanced in

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MCAO model. Likewise, we [START_REF] Rashidian | Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo[END_REF] as well as Wang et al. [START_REF] Wang | Cdk5 activation induces hippocampal ca1 cell death by directly phosphorylating nmda receptors[END_REF] , have recently provided evidence that cdk5 is a mediator of excitotixc neuronal death in global and focal ischemia.

Similar to classic cell cycle CDKs, the important question of the mechanism by which aberrant cdk5 causes neuronal damage remains. Several possibilities may exist. For example, it has been reported that cdk5 can phosphorylate the NMDA receptors regulating calcium influx [START_REF] Wang | Cdk5 activation induces hippocampal ca1 cell death by directly phosphorylating nmda receptors[END_REF].

Interestingly tau, a microtubule-associated protein which is connected to Alzheimer's disease pathology, has also been recently shown to be as a substrate for cdk5 following ischemia [START_REF] Morioka | Hyperphosphorylation at serine 199/202 of tau factor in the gerbil hippocampus after transient forebrain ischemia[END_REF]. What this means is unknown, however. There might also be other candidates such as MEF2 (Myocyte Enhance Factor-2) or p53 transcription factors. Both have been shown to be regulated by cdk5 and both have been implicated in ischemic/excitotoxic death [START_REF] Gong | Cdk5-mediated inhibition of the protective effects of transcription factor mef2 in neurotoxicity-induced apoptosis[END_REF][START_REF] Smith | Calpain-regulated p35/cdk5 plays a central role in dopaminergic neuron death through modulation of the transcription factor myocyte enhancer factor 2[END_REF][START_REF] Zhang | Cdk5 phosphorylates p53 and regulates its activity[END_REF]. However, the link between cdk5 and these factors have not been yet addressed in in vivo stroke studies.

Conclusion:

As summarized above, there is increasing evidence of the involvement of multiple cell cycle inhibitors appear to at least meet some of these criteria. For example, the CDK inhibition has been shown to be neuroprotective in multiple models and lead to improved behavior/function. However, the CDK inhibitor flavopiridol, at least, seems to lack long term efficacy. This may be due to the non selective nature of flavopiridol or and indication that CDK inhibition by itself can not completely normalize neuron function. These issues will have to be carefully addressed before CDK inhibitors can be contemplated for future clinical trials.

  required to maintain proper cell functions. The reduction of cellular energy results in the impairment of vital cellular functions such as the maintenance of the Na+/K+ pump, massive depolarization and excessive release of glutamate. Over activation of the glutamate channel particularly the N-methyl-D-aspartate receptor (NMDAR)-type channels mediates massive influx of Ca2+ resulting in cellular excitotoxicity and neuronal death. Increase in intracellular free calcium results in the activation of Ca2+ sensitive enzymes such as calpains proteases which then act to activate other molecules and A C C E P T E D M A N U S C R I P T

  regulatory signals in ischemic injury. Several important questions remain however, and include: 1) What are the mechanism(s) by which cell cycle signals are actually activated following stroke injury; 2) Are there other mechanisms by which cell cycle CDKs may impact death other than through modulation of Rb; 3) Does cdk5 regulate cell cycle signals in models of stroke 4) Are cell cycle targets in and of themselves a viable therapeutic option for stroke. The latter question is of particular importance since most pharmacotherapeutic strategies tested in the clinic for treatment of stroke have failed. One reason for this is likely that preclinical testing in animal models has not been sufficiently stringent prior to clinical trials. In this regard, vigorous benchmarks of a) efficacy of any strategy in multiple animal models, b) behavioral and functional improvements beyond neuroprotection and c) long lasting efficacy have been established. Cell cycle

Figure 1 :

 1 Figure 1: Schematic representation of our current understanding of the mammalian cell cycle. The cell

Acknowledgements:

This review was supported by funds from Heart and Stroke of Ontario, Canadian Institute for Health Research, The Centre for Brain Recovery, and Canadian Stroke Network.

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