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Neurofibrillary tangles are one of the pathologic hallmarks of Alzheimer's disease (AD). They are composed of paired helical filaments (PHF) containing hyperphosphorylated forms of tau. Hyperphosphorylation of certain tau sites favors its dissociation from the microtubules (MT), interfering with axonal transport and compromising the function and viability of neurons. Reappearance of cell cycle proteins have been reported in neurons exhibiting tau aggregation, suggesting that an aberrant cell cycle occurs before neurons die. Cell cycle suppression in neurons is crucial to survival, thus prevention of progression through the cell cycle may offer a therapeutic approach. Using a neuroblastoma cell line overexpressing 3-repeat (3R) tau, we investigated the effects of cell cycle inhibitors on tau phosphorylation. G2/M phase inhibitors did not alter phosphorylation of tau at Ser-202 and Ser-396/404 at the lower doses, but did at higher doses. Ser-202 and Ser-396/404 are phosphorylation sites of early and late neurofibrillary tangles, respectively, in AD. Cisplatin, a G1 phase inhibitor, did not phosphorylate tau. Cyclophosphamide and phosphoramide mustard, DNA cross-linking agents, decreased tau phosphorylation at Ser-396/404 site, but increased phosphorylation at Ser-202. These studies demonstrate that the G2/M blockers have a dose-dependent effect on tau phosphorylation. This seems to be a consequence of both the disruption of MT-organization and MT-dynamics when doses are higher, but only a disruption of MT-dynamics with lower doses. These results are also in agreement with the lack of phosphorylation seen for cisplatin, another inhibitor that produces disruption of the MT-dynamics.
Alzheimer's disease (AD) is the leading cause of dementia worldwide. Over 5 million Americans are afflicted with AD in 2007. Neurofibrillary tangles are one of the pathologic hallmarks of AD. Neurofibrillary tangles are composed of paired helical filaments (PHF) containing hyperphosphorylated forms of the microtubule associated protein, tau. Hyperphosphorylation of certain Ser or Thr phosphorylation tau sites favors its dissociation from the microtubules (MT), leading to destabilization of the neuronal cytoskeleton (Billingsley and Kincaid 1997). Reappearance of cell cycle proteins has been reported in neurons exhibiting tau aggregation, suggesting that an aberrant cell cycle occurs before neurons die (Andorfer et al. 2005). Understanding the mechanisms underlying tau phosphorylation by targeting the cell cycle with compounds that produce cell cycle suppression could provide molecular targets for future therapeutic interventions.
G2/M blockers of the cell cycle such as paclitaxel and Vinca alkaloids (e.g., vinblastine, vincristine) are commonly used as antimitotic drugs (Bhalla 2003; Mollinedo and Gajate 2003; Jordan and Wilson 2004). At lower doses, these drugs have a common mechanism of action; they interfere with polymerization dynamics of the microtubule by hyperstabilization or by destabilization of microtubules and activate the mitotic spindle checkpoint (Blagosklonny and Fojo 1999; Gorbsky 2001; Jordan 2002; Cleveland et al. 2003; Bharadwaj and Yu 2004). The spindle checkpoint causes extended mitotic arrest. During mitosis, the dynamic instability of microtubules has been observed to rise approximately tenfold (Kinoshita et al. 2002). Paclitaxel and the Vinca alkaloids act by suppressing dynamic instability of mitotic spindle microtubules and thus halt cell division at the metaphase/anaphase transition (Jordan 2002). After prolonged periods of mitotic arrest, the cells eventually undergo apoptosis and die either during the mitotic arrest or after cells exit mitosis without normal chromosome segregation (Blagosklonny and Fojo 1999; Weaver and Cleveland 2005).
Cyclophosphamide and phosphoramide mustard (PM), both DNA cross-linking agents, are capable of inducing apoptosis (Colvin 1999). The main effect of cyclophosphamide is due to its active metabolite PM. This metabolite forms DNA crosslinks between (interstrand) and within (intrastrand) DNA strands at guanine N7 positions. Cisplatin is also a DNA cross-linking agent that is most effective in G1 of the cell cycle. It preferentially binds at N7 of the purines adenine and guanine, and at N3 of the pyrimidines cytosine and uracil forming DNA crosslinks (90% intrastrand). These DNA cross-linking agents produce alterations of the DNA structure that prevent replication. They also activate cellular repair mechanisms in an attempt to remove by excision the defective section of DNA. Cell cycle checkpoints detect the presence of defective DNA, and trigger a series of responses that lead to apoptotic cell death.
Using a neuroblastoma cell line overexpressing 3-repeat (3R) tau we investigated the effects of cell cycle inhibitor drugs on tau phosphorylation at low and high doses. In these studies, drugs that produce arrest at G2/M phase, like paclitaxel, vincristine (VC), and vinblastine (VBT) increased phosphorylation of tau at Ser-202 and Ser-396/404 at higher doses, but not at lower doses. Cyclophosphamide and PM, both DNA cross-linking agents, decreased tau phosphorylation at Ser-396/404 site, but increased tau phosphorylation at Ser-202. Cisplatin, another DNA cross-linking agent most effective in G1 phase of the cell cycle, did not affect phosphorylation of tau at these sites.
Mouse neuroblastoma N2A cells (provided courtesy of Dr. Philippe Marambaud) were maintained at 37°C and 5% CO2, in Dulbecco's modified minimal essential medium (DMEM; Life Technologies, Burlington, ON, Canada) supplemented with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) penicillin and streptomycin (Life Technologies, Burlington, ON). N2A cells were transient transfected with a 3R human tau construct using GeneCarrier reagent (Epoch Biolabs, TX, USA) and Lipofectamine (Invitrogen, CA, USA) and stable lines were selected with 1 mg/ml medium Geneticin (Invitrogen, CA, USA). These stably transfected N2A cells expressing the 3R human tau were then used in this study and will be referred to as N2aTau3R cells. To differentiate the cells, N2aTau3R cells were treated with retinoic acid (1 μM) for 24 h in culture medium containing 0.1% FBS. Paclitaxel, vincristine, vinblastine, cisplatin, and cyclophosphamide monohydrate were obtained from Sigma (St. Louis, MO, USA). PM (as the cyclohexylammonium salt) was acquired from the National Cancer Institute.
After incubation with drugs or Dulbecco's modified Eagle medium (DMEM), cells (undifferentiated and differentiated N2a stable-transfected cell cultures) were washed twice with PBS and scraped off the plates in lysis buffer. The lysis buffer composition was: ELB buffer (50 mM HEPES [pH 7.5], 250 mM NaCl, 0.1% Nonidet P-40 [NP-40], 1 mM ethylenediaminetetraacetic acid (EDTA) including protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride [PMSF], 100 μg of aprotinin, 10 μg of leupeptin, and 10 μg of pepstatin per ml, and 1 mM benzamidine). The lysates were centrifuged at 13,500 rpm for 15 min at 4°C, and supernatants were collected. Protein concentration of the cell lysate was determined in each case using the Pierce protein assay kit. Cell lysates were then stored at −20°C. Protein samples (20 μg protein per lane) was first boiled for 7 min, and then subjected by SDS-PAGE using 8–15% polyacrylamide gels and transferred to nitrocellulose membranes. The nitrocellulose sheets were probed with different antibodies: PHF-1 (Ser-396/404), CP13 (Ser-202) and CP27 (recognizes all human tau isoforms). Polyclonal anti-β-Actin antibody (Santa Cruz Biotechnology, CA, USA) and monoclonal Anti-α-tubulin antibody (Sigma, MO, USA) were used as controls. All nitrocellulose sheets were then labeled with anti-mouse or anti-rabbit antibodies conjugated with peroxidase; visualization was performed using a standard ECL detection procedure.
Paclitaxel, a microtubule-stabilizing agent, is an anti-mitotic drug that induces apoptosis in cancer cells by causing cell cycle arrest at the G2/M transition (Blagosklonny and Fojo 1999; Jordan et al. 1993), activating JNK (Amato et al. 1998; Wang et al. 1998) and inducing phosphorylation of bcl-2 (Blagosklonny et al. 1997; Eisenhauer and Vermorken 1998). The binding of paclitaxel to the β-tubulin subunits in microtubules promotes polymerization of tubulin and disruption of microtubule dynamics, leading to a sustained mitotic arrest and ultimately to apoptotic cell death (Jordan and Wilson 1998).
Paclitaxel interferes with the normal function of microtubule growth by hyperstabilization of their structure. The resulting microtubule/paclitaxel complex does not have the ability to disassemble. This adversely affects cell function because the shortening and lengthening of microtubules (dynamic instability) is necessary for their function as a mechanism to transport other cellular components (e.g., the mitosis where microtubules position the chromosomes during their replication and subsequent separation into the two daughter-cell nuclei). Despite these adverse effects, low doses of paclitaxel may have a therapeutic benefit in human tauopathies by offsetting the loss of normal tau functions that result from its hyperphosphorylation and sequestration into tangles (Zhang et al. 2005).
To further assess the effect of paclitaxel treatment on tau phosphorylation at low and high doses in N2aTau3R cells, we used immunoblot analysis and the CP13, PHF-1 and CP27 tau specific antibodies. CP13 recognizes an epitope phosphorylated at Ser202 and PHF-1 recognizes tau phosphorylated on Ser396/404 (Busciglio et al. 1995). The CP27 antibody recognizes total tau levels independent of phosphorylation state. The N2aTau3R cells under both undifferentiated and differentiated conditions were treated with 10 nM or 1 μM paclitaxel for 8 h before examining tau phosphorylation.
Phosphorylation of tau protein at both epitopes Ser202 and Ser396/404 was increased at 8 h following incubation with 1 μM paclitaxel (higher dose). At the lower dose (10 nM), there was no apparent difference in phosphorylation at either Ser202 or Ser396/404 epitope compared to the untreated cells; regardless of the differentiation status of the cells (Fig. 1a,b). In the high-dose (1 μM) paclitaxel-treated cells, the electrophoretic profile of tau was also modified. This shift in apparent molecular weight associates with hyperphosphorylation of tau. Moreover, the CP27 antibody also recognizes phosphorylated tau species as indicated by the presence of a mobility shift in the tau band from cells treated with paclitaxel. Densitometric quantitation of the CP-13, PHF1, and CP27 immunoreactive bands is shown in (Fig. 1c,d). These results demonstrate that paclitaxel at low dose does not phosphorylate tau, thus opening a window for potential therapeutic treatment at low doses.
Vincristine and VBT block the cell cycle in G2/M phase by depolymerizing microtubules and/or inhibiting their dynamics, eventually leading to cell death (Jordan 2002; Weaver and Cleveland 2005). To assess the effect of VC and VBT treatments on tau phosphorylation in N2aTau3R cells, we used the CP13, PHF-1 and CP27 antibodies. N2aTau3R cells were treated with 1 nM or 1 μM VC for 24 h for the undifferentiated condition and with 1 nM (data not shown), 1 μM or 10 μM VC for the differentiated condition (Fig. 2a,b).
Phosphorylation of tau protein was increased at 24 h after incubation with 1 μM or 10 μM VC (higher doses) for both epitopes Ser202 and Ser396/404. At lower dose (1 nM), there was no apparent difference in phosphorylation for either epitope compared to the untreated cells for both conditions, undifferentiated and differentiated. In VC-treated cells with the higher doses (1 μM or 10 μM), the electrophoretic profile of tau was modified (Fig. 2a,b). The shift in apparent molecular weight indicates hyperphosphorylation of tau. The CP27 antibody also recognizes phosphorylated tau species as indicated by the presence of a mobility shift in the tau band from cells treated with VC.
Undifferentiated and differentiated N2aTau3R cells were also treated with 1 nM or 1 μM VBT for 24 h. Phosphorylation of tau protein was clearly increased at 24 h after incubation with 1 μM VBT for both epitopes Ser202 and Ser396/404 (Fig. 3a,b). However, at lower dose (1 nM) there was no apparent difference in phosphorylation at either epitope when compared to the untreated cells. In the high-dose (1 μM) VBT-treated cells, the electrophoretic profile of tau was also modified in favor of more tau species (Fig. 3a,b). Densitometric quantitation of the immunoreactive bands for both treatments, VC and VBT, is shown in Figs. 2c,d, and 3c,d, respectively.
Cisplatin is a DNA cross-linking agent (90% intrastrand crosslinks). Although it is generally known as a phase-nonspecific agent, it may be most effective in G1 of the cell cycle. Cell cycle checkpoints detect the presence of defective DNA, and trigger a series of responses that lead to cellular apoptosis by preventing transcription and upregulation of P53, P21, and Bax.
When either undifferentiated or differentiated N2aTau3R cells were treated with cisplatin (1 nM or 1 μM) for 24 h, phosphorylation of tau protein at epitopes Ser202 and Ser396/404 was unchanged compared to the untreated cells (Fig. 4a,b). CP27 antibody, which recognizes total tau, showed no differences in the electrophoretic profile between different doses of cisplatin and untreated cells. Densitometry measurements are shown in Fig. 4c and d.
Cyclophosphamide and PM are both DNA cross-linking agents that are capable of inducing apoptosis. Oxidative activation of cyclophosphamide by hepatic cytochromes to 4-hydroxycyclophosphamide (4-OH-CPA) leads to spontaneous (non-enzymatic) reactions, ultimately resulting in the production of PM, which is the reactive alkylating agent of therapeutic consequences (Ludeman 1999). The half-life of PM at physiological pH has been reported to be very short and complete loss of free PM should occur within a few hours (Ludeman 1999).
The N2aTau3R cells were treated with 1 mM cyclophosphamide or 1 mM PM for 3 h under undifferentiated and differentiated conditions. For the undifferentiated condition, phosphorylation of tau protein was clearly increased for the Ser202 epitope at 3-h incubation with both cyclophosphamide and PM. For the Ser396/404 epitope, phosphorylation of tau protein was decreased at 3-h incubation with the same treatments. CP27 antibody showed no differences between treated and untreated cells (Fig. 5a). For the differentiated condition, there were no differences between treated and untreated cells in phosphorylation of tau protein as shown by the different antibodies CP13, PHF-1, and CP27 (Fig. 5b). Densitometric quantitation of the CP-13, PHF1, and CP27 immunoreactive bands for these blots is shown in Fig. 5c and d.
Tau protein promotes microtubule assembly, but the phosphorylation of certain tau epitopes reduces microtubule binding leading to destabilization of the neuronal cytoskeleton (Billingsley and Kincaid 1997). Reappearance of cell cycle markers have been reported in neurons exhibiting tau aggregation, suggesting that an aberrant cell cycle occurs before neurons die. Prevention of progression through the cell cycle may offer a therapeutic approach if drugs do not induce additional phosphorylation of tau. In the present study, we show that G2/M cell cycle blocker treatments do not induce hyperphosphorylation of tau at lower doses independent of being MT-stabilizing/destabilizing drugs, but induce hyperphosphorylation of tau on Ser 202 and Ser-396/404 consistently at higher doses.
Lower doses of the MT-stabilizing drug paclitaxel could have a therapeutic benefit in human tauopathies by offsetting the loss of normal tau functions that result from its hyperphosphorylation and sequestration into tangles (Zhang et al. 2005). Treatment with paclitaxel restores fast axonal transport in spinal axons, increases axonal MTs, and ameliorates motor impairments in tau transgenic mice that developed filamentous tau inclusions (mostly in the spinal cord) (Ishihara et al. 1999, 2001; Zhang et al. 2005). Paclitaxel also blocks Aβ-induced phosphorylation of tau by preventing the cleavage of p35 by calpain (Li et al. 2003). Nanomolar concentrations of paclitaxel can protect neurons against various toxic insults and enhance survival by maintaining Ca2+ homeostasis (Burke et al. 1994; Furukawa and Mattson 1995; Furukawa et al. 2003; Sponne et al. 2003; Michaelis et al. 2005) without evidence of toxicity (Trushina et al. 2003). Our study demonstrates that tau is not phosphorylated by MT-stabilizing/destabilizing drugs at lower doses, but it is phosphorylated at higher doses. Lower doses of these agents do not alter MT organization; meanwhile, higher doses produce disruption of the MT organization (Becvarova et al. 2006). Consequently, MT organization disruption, but not MT-dynamics disruption, is required to promote the hyperphosphorylation of tau. Adding a layer of complexity, tau phosphorylation may have different effects, depending on site. A recent study analyzed site-specific effects of tau phosphorylation on its microtubule assembly activity and self-aggregation (Liu et al. 2007). Tau phosphorylation at the c-terminal region by glycogen synthase kinase-3β (GSK-3β) increased its microtubule assembly activity although promoted its self-aggregation markedly.
Cisplatin, a DNA cross-linking agent, produces alterations of the DNA structure that prevents replication. In addition, it is also able to produce microtubule disassembly by direct tubulin modification (e.g., when platinated, tubulin does not assemble into microtubules, consequently altering MT dynamics) (Boekelheide et al. 1992; Tulub and Stefanov 2001). In our studies, we did not observe tau hyperphosphorylation at the low/high doses used in the study for the treatments with cisplatin. This is also consistent with our results with VC, VBT, and paclitaxel treatments in which tau protein is hyperphosphorylated only when there is alteration of the MT organization but not of the MT-dynamics.
Cyclophosphamide and PM are also DNA cross-linking agents that prevent replication. In our study, both compounds decreased tau phosphorylation at Ser-396/404 when cells were undifferentiated, but increased tau phosphorylation at Ser-202. These contradictory effects may be due to the binding/interfering of the compound with the Ser-396/404 site, so that consequently there is no phosphorylation of tau at that particular site. Another interpretation of these opposing effects on tau phosphorylation between the Ser396/404 and Ser-202 sites could be that cyclophosphamide and its metabolite may have pleiotrophic effects that lead to differentially activation of tau kinases. Clearly, further work is needed to clarify the relationship between DNA cross-linking agents and their effects on microtubule stability/dynamics and how these two phenomena modulate tau phosphorylation. In conclusion, understanding the mechanisms underlying tau phosphorylation by targeting the cell cycle with compounds that produce cell cycle suppression may help to understand why reappearance of cell cycle proteins plays such a critical role in vulnerable neurons. Further studies are needed to provide more answers to the changing of the course of early events of this illness.
Phosphoramide mustard was a gift from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute.
Concepcion Conejero-Goldberg, The Litwin-Zucker Research Center for Study of Alzheimer's Disease, The Feinstein Institute for Medical Research, North Shore University Hospital, 350 Community Drive, Manhasset, NY 11030, USA.
Kirk Townsend, The Litwin-Zucker Research Center for Study of Alzheimer's Disease, The Feinstein Institute for Medical Research, North Shore University Hospital, 350 Community Drive, Manhasset, NY 11030, USA.
Peter Davies, The Litwin-Zucker Research Center for Study of Alzheimer's Disease, The Feinstein Institute for Medical Research, North Shore University Hospital, 350 Community Drive, Manhasset, NY 11030, USA; Department of Pathology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.