Emerging Pathogenic Role for Cyclin Dependent Kinases in Neurodegeneration

While the requirement of CDKs in cell cycle control is well established, the participation of CDK family members in other important biological processes are now beginning to be uncovered. Paramount in these newly defined roles is the surprising involvement of CDKs in neuronal development and death. These discoveries are somewhat of a paradox considering the terminally differentiated state of neurons. This brief perspective will focus on the role of CDKs in neuronal death and neurodegeneration. In this regard, we will primarily explore two (of potentially many) ways by which CDKs may enable neuronal death. The first involves the effects of ectopic activation of cell cycle related CDKs in a terminal post mitotic environment. The second explores how cdk5, a neuron specific cdk required for normal neuronal function, can be usurped to signal death.     

Cyclin D1, cdk4, and Bim are involved in thrombin-induced apoptosis in cultured cortical neurons

Thrombin, a multifunctional serine protease, is neurotoxic in vitro and in vivo. Thrombin has been shown to be increased in Alzheimer's disease (AD) and other neuropathological conditions and could be a mediator of pathological neuronal cell death in the brain. The mechanisms of thrombin-induced neuronal cell death are incompletely understood. The objective of this study is to explore mechanisms that contribute to thrombin-induced neuronal apoptosis focusing on the role of cell cycle regulators and the pro-apoptotic protein Bim (Bcl-2-interacting mediator of cell death) in this process. Our data show that thrombin treatment of primary cerebral cortical cultures results in dose-dependent apoptotic cell death. Exposure of neuronal cultures to thrombin leads to induction of cell cycle proteins cyclin D1 and cyclin E, at both mRNA and protein levels. In addition, thrombin treatment causes the appearance of cyclin-dependent kinase 4 (cdk4) and expression of the pro-apoptotic protein Bim. Inhibition of cdk4 prevents both induction of Bim expression and thrombin-induced neuronal apoptosis. These data demonstrate that thrombin-induced apoptosis proceeds via cell cycle activation involving cdk4 resulting in induction of Bim. Thus, cell cycle proteins could be therapeutic targets in diseases such as AD where thrombin has been implicated.     

Cell cycle machinery and stroke

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.     

Cyclin-dependent kinase 5 (CDK5) and neuronal cell death

Many neurological disorders like Parkinson's and Alzheimer's disease, amyotrophic lateral sclerosis (ALS) or stroke have in common a definite loss of CNS neurons due to apoptotic or necrotic neuronal cell death. Previous studies suggested that proapoptotic stimuli may trigger an abortive and, therefore, eventually fatal cell cycle reentry in postmitotic neurons. Neuroprotective effects of small molecule inhibitors of cyclin-dependent kinases (CDKs), which are key regulators of cell cycle progression, support the cell cycle theory of neuronal apoptosis. However, growing evidence suggests that deregulated CDK5, which is not involved in cell cycle control, rather than cell cycle relevant members of the CDK family, promotes neuronal cell death. Here we summarize the current knowledge about the involvement of CDK5 in neuronal cell death and discuss possible up- or downstream partners of CDK5. Moreover, we discuss potential therapeutic options that might arise from the identification of CDK5 as an important upstream element of neuronal cell death cascades.     

Identification of an hexapeptide that binds to a surface pocket in cyclin A and inhibits the catalytic activity of the complex cyclin-dependent kinase 2-cyclin A

The protein-protein complexes formed between different cyclins and cyclin-dependent kinases (CDKs) are central to cell cycle regulation. These complexes represent interesting points of chemical intervention for the development of antineoplastic molecules. Here we describe the identification of an all d-amino acid hexapeptide, termed NBI1, that inhibits the kinase activity of the cyclin-dependent kinase 2 (cdk2)-cyclin A complex through selective binding to cyclin A. The mechanism of inhibition is non-competitive for ATP and non-competitive for protein substrates. In contrast to the existing CDKs peptide inhibitors, the hexapeptide NBI1 interferes with the formation of the cdk2-cyclin A complex. Furthermore, a cell-permeable derivative of NBI1 induces apoptosis and inhibits proliferation of tumor cell lines. Thus, the NBI1-binding site on cyclin A may represent a new target site for the selective inhibition of activity cdk2-cyclin A complex.     

Redundancy or specificity? The role of the CDK Pho85 in cell cycle control

It is generally accepted that progression through the eukaryotic cell cycle is driven by cyclin-dependent kinases (CDKs), which are regulated by interaction with oscillatory expressed proteins called cyclins. CDKs may be separated into 2 categories: essential and non-essential. Understandably, more attention has been focused on essential CDKs because they are shown to control cell cycle progression to a greater degree. After clearly determining the basic and “core” mechanisms of essential CDKs, several questions arise. What role do non-essential CDKs play? Are these CDKs functionally redundant and do they serve as a mere backup? Or might they be responsible for some accessory tasks in cell cycle progression or control? In the present review we will try to answer these questions based on recent findings on the involvement of non-essential CDKs in cell cycle progression. We will analyse the most recent information with regard to these questions in the yeast Saccharomyces cerevisiae, a well-established eukaryotic model, and in its unique non-essential CDK involved in the cell cycle, Pho85. We will also briefly extend our discussion to higher eukaryotic systems.     

Interplay between MEK-ERK signaling, cyclin D1, and cyclin-dependent kinase 5 regulates cell cycle reentry and apoptosis of neurons

In response to neurotoxic signals, postmitotic neurons make attempts to reenter the cell cycle, which results in their death. Although several cell cycle proteins have been implicated in cell cycle-related neuronal apoptosis (CRNA), the molecular mechanisms that underlie this important event are poorly understood. Here, we demonstrate that neurotoxic agents such as β-amyloid peptide cause aberrant activation of mitogen-activated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling, which promotes the entry of neurons into the cell cycle, resulting in their apoptosis. The MEK-ERK pathway regulates CRNA by elevating the levels of cyclin D1. The increase in cyclin D1 attenuates the activation of cyclin-dependent kinase 5 (cdk5) by its neuronal activator p35. The inhibition of p35-cdk5 activity results in enhanced MEK-ERK signaling, leading to CRNA. These studies highlight how neurotoxic signals reprogram and alter the neuronal signaling machinery to promote their entry into the cell cycle, which eventually leads to neuronal cell death.     

PACAP38 protects rat cortical neurons against the neurotoxicity evoked by sodium nitroprusside and thrombin

Pituitary adenylate cyclase-activating polypeptide (PACAP) 38 is a multifunctional anti-inflammatory and anti-apoptotic neuropeptide widely distributed in the nervous system. The objective of this study is to determine whether PACAP38 is neuroprotective against sodium nitroprusside (SNP) and thrombin, two mechanistically distinct neurotoxic agents. Treatment of primary cortical neuronal cultures with 1 mM SNP for 4 h causes neuronal cell death that is significantly reduced by 100 nM PACAP38. PACAP38 down-regulates SNP-induced cell cycle protein (cyclin E) expression and up-regulates p57(KIP2), a cyclin-dependent kinase inhibitor as well as the anti-apoptotic protein Bcl-2. Similarly, neuronal death induced by 100 nM thrombin or the thrombin receptor activating peptide (TRAP 6) is reduced by PACAP38 treatment. Thrombin-stimulated cell cycle protein (cdk4) expression is decreased by PACAP38 while PACAP38 inhibits thrombin-mediated reduction of p57(KIP2). However, the decrease in Bcl-2 evoked by thrombin is not affected by PACAP38. Finally, both SNP and thrombin (or TRAP) increase caspase 3 activity, an effect that is decreased by PACAP38. These data show that PACAP38 supports neuronal survival in vitro suppressing cell cycle progression and enhancing anti-apoptotic proteins. Our results support the possibility that PACAP could be a useful therapeutic agent for reducing neuronal cell death in neurodegenerative diseases.     

Neuroprotective effect of TNFalpha against the beta-amyloid neurotoxicity mediated by CDK5 kinase

The tumor necrosis factor alpha (TNFalpha) plays a dual role in producing either neurodegeneration or neuroprotection in the central nervous system. Despite that TNFalpha was initially described as a cell death inductor, neuroprotective effects against cell death induced by several neurotoxic insults have been reported. Tau hyperphosphorylation and neuronal death found in Alzheimer disease is mediated by deregulation of the cdk5/p35 complex induced by Abeta treatments. Since TNFalpha affects cdk5 activity, we investigated its possible protective role against the Abeta-induced neurodegeneration, as mediated by cdk5. TNFalpha pretreatments significantly reduced the hippocampal neuronal cell death induced by the effects of Abeta(42) peptide. In addition, this pretreatment reduced the increase in the activity of cdk5 induced by Abeta(42) in primary neurons. Next, we investigated the Alzheimer type phosphorylation of tau protein induced by Abeta(42). We observed that the pretreatment of neurons with TNFalpha reduces tau hyperphosphorylation. Taken together, these results define a novel neuroprotective effect of TNFalpha in preventing neuronal cell death and cdk5-dependent tau hyperphosphorylation. This phenomenon, taken together with other previous findings, suggests that the inflammatory response due to Abeta peptide plays a key role in the development of Alzheimer etiopathogenesis.     

Cell cycle S phase markers are expressed in cerebral neuron nuclei of cats infected by the Feline Panleukopenia Virus

The cell cycle-associated neuronal death hypothesis, which has been proposed as a common mechanism for most neurodegenerative diseases, is notably supported by evidencing cell cycle effectors in neurons. However, in naturally occurring nervous system diseases, these markers are not expressed in neuron nuclei but in cytoplasmic compartments. In other respects, the Feline Panleukopenia Virus (FPV) is able to complete its cycle in mature brain neurons in the feline species. As a parvovirus, the FPV is strictly dependent on its host cell reaching the cell cycle S phase to start its multiplication. In this retrospective study on the whole brain of 12 cats with naturally-occurring, FPV-associated cerebellar atrophy, VP2 capsid protein expression was detected by immunostaining not only in some brain neuronal nuclei but also in neuronal cytoplasm in 2 cats, suggesting that viral mRNA translation was still occurring. In these cats, double immunostainings demonstrated the expression of cell cycle S phase markers cyclin A, cdk2 and PCNA in neuronal nuclei. Parvoviruses are able to maintain their host cells in S phase by triggering the DNA damage response. S139 phospho H2A1, a key player in the cell cycle arrest, was detected in some neuronal nuclei, supporting that infected neurons were also blocked into the S phase. PCR studies did not support a co-infection with an adeno or herpes virus. ERK1/2 nuclear accumulation was observed in some neurons suggesting that the ERK signaling pathway might be involved as a mechanism driving these neurons far into the cell cycle.     

Cyclin dependent kinase inhibitors prevent apoptosis of postmitotic mouse motoneurons

Recent evidence suggests that apoptosis in post-mitotic neurons involves an aborted attempt of cells to re-enter the cell cycle which is characterized by increased expression of cyclins, such as cyclin D1, prior to death. However, such cyclins activation prior to apoptotic cell death remains controversial. Many neurological disorders are characterized by neuronal loss, particularly amyotrophic lateral sclerosis (ALS). ALS is a motoneuronal degenerative condition in which motoneuron loss could be due to an inappropriate return of these cells in the cell cycle. In the present study, we observed that deprivation of neurotrophic factor in purified motoneuron cultures induces an apoptotic pathway. After neurotrophic factor withdrawal, DAPI (4,6-diamidin-2-phenylindol dichlorohydrate) staining revealed the presence of nuclear condensation, DNA fragmentation, and perinuclear apoptotic body. Similarly, release of apoptotic microparticles and activation of caspases-3 and -9 were observed within the first hours following neurotrophic factor withdrawal. Next, we tested whether inhibition of cell cycle-related cyclin-dependent kinases (cdks) can prevent motoneuronal cell death. We showed that three cdk inhibitors, olomoucine, roscovitine and flavopiridol, suppress the death of motoneurons. Finally, we observed early increases in cyclin D1 and cyclin E expression after withdrawal of neurotrophic factors. These findings support the hypothesis that after removal of trophic support, post-mitotic neuronal cells die due to an attempt to re-enter the cell cycle in an uncoordinated and inappropriate manner.     

Species-specific and conserved epitopes on mouse and human E-selectin important for leukocyte adhesion

The cdk5 and its activator p35 constitute one of the main tau-phosphorylating systems in neuronal cells. Under normal conditions for neurons, its activity is required for modulating tau involvement in neuronal polarity and in development of the mammalian central nervous system. Recently, we reported that the treatment of rat hippocampal cells in culture with fibrillary beta-amyloid (Abeta) results in deregulation of the protein kinase cdk5. The neurotoxic effects of Abeta fibrils were prevented by inhibition of cdk5 activity by butyrolactone I or by using antisense oligonucleotides that control the expression of this kinase. Here, we show that the Abeta-promoted increase of cdk5 activity is associated with changes in tau phosphorylation patterns and in the intraneuronal distribution of tau. In addition to hippocampal cells, deregulation of cdk5 was observed in other cell types. However, butyrolactone I prevented Abeta-induced cell death only in neuronal cells in which cdk5 activation was sensitive to Abeta fibrils. This lost of cdk5 regulation in hippocampal cells exposed to Abeta fibrils appears to be associated with an increase in the cdk5-p35 complex stability. Complex stabilization was sensitive to phosphorylation of cdk5. However, no changes in cdk5 and p35 mRNAs were observed, suggesting that the main effects on cdk5 occur at the posttranslational level. These studies indicate that cdk5 phosphorylation and the formation of an abnormally active cdk5-p35 complex are directly involved in the molecular paths leading to the neurodegenerative process of rat hippocampal neurons triggered by Abeta fibrils.     

Inhibition of tau phosphorylating protein kinase cdk5 prevents beta-amyloid-induced neuronal death.

The key target of this study was the tau protein kinase II system (TPK II) involving the catalytic subunit cdk5 and the regulatory component p35. TPK II is one of the tau phosphorylating systems in neuronal cells, thus regulating its functions in the cytoskeletal dynamics and the extension of neuronal processes. This research led to demonstration that the treatment of rat hippocampal cells in culture with fibrillary beta-amyloid (Abeta) results in a significant increase of the cdk5 enzymatic activity. Interestingly, the data also showed that the neurotoxic effect of 1-20 microM Abeta on primary cultures markedly diminished with co-incubation of hippocampal cells with the amyloid fibers plus the cdk5 inhibitor butyrolactone I. This inhibitor protected brain cells against Abeta-induced cell death in a concentration dependent fashion. Moreover, death was also prevented by a cdk5 antisense probe, but not by an oligonucleotide with a random sequence. The cdk5 antisense also reduced neuronal expression of cdk5 compared with the random oligonucleotide. The studies indicate that cdk5 plays a major role in the molecular path leading to the neurodegenerative process triggered by the amyloid fibers in primary cultures of rat hippocampal neurons. These findings are of interest in the context of the pathogenesis of Alzheimer's disease.     

Fas-induced changes in cdc2 and cdk2 kinase activity are not sufficient for triggering apoptosis in HUT-78 cells

Recent evidence suggested a role for the cell cycle dependent kinases cdc2 and cdk2 in apoptosis. An important mechanism by which many cell types could undergo apoptosis is through the activation of the Fas molecule on the cell membrane. To investigate whether Fas-induced cell death activated cdc2 and cdk2 kinases inappropriately, the human T lymphoma cells HUT-78, which express a high copy number of Fas, and two other previously characterized subclones of the same cell line which express mutant, cell death-deficient dominant-negative forms of Fas, were Fas-challenged and the changes in cdc2 and cdk2 kinase activity monitored. In both wild-type and Fas-mutated HUT-78 cells, apoptosis was associated simultaneously with decreased cdc2 and increased cdk2 activity. This association suggested that changes in cdc2 and cdk2 kinase activity are secondary events in cell death mediated by Fas. J. Cell. Biochem. 64:579–585. © 1997 Wiley-Liss, Inc.     

P16Ink4a tumor suppressor function in lung cancer cells involves cyclin-dependent kinase 2 inhibition by Cip/Kip protein redistribution.

As cell cycle regulators whose activity is frequently altered in human cancers, cyclin-dependent kinases (cdks) are novel targets for therapeutic intervention. cdk inhibition is an emerging strategy for the treatment of non-small cell lung carcinomas (NSCLCs) because most derived cell lines express functional retinoblastoma protein (Rb) but appear to bypass its function with inappropriate cdk activity. Elevated cdk4/cdk6 activity in NSCLC cells is often due to inactivation of the p16Ink4a cdk inhibitor. To model the effects of cdk4/cdk6 inhibition, we have expressed p16Ink4a in a Rb-positive NSCLC cell line that lacks endogenous p16Ink4a expression. Whereas cdk4/cdk6 inhibition and Rb dephosphorylation are expected on p16Ink4a expression, we have also observed indirect cdk2 inhibition. cdk2 inactivation by the redistribution of other cdk inhibitors may be required for p16Ink4a-mediated growth suppression of Rb-positive cells. The implications of such a requirement on the use of chemical cdk inhibitors to treat human cancers will be discussed.