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Keywords:

  • amyloid-β;
  • calpain;
  • cyclin-dependent kinase 5;
  • microtubules;
  • paclitaxel;
  • tau

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

One hallmark of Alzheimer's disease (AD) is the formation of neurofibrillary tangles, aggregated paired helical filaments composed of hyperphosphorylated tau. Amyloid-β (Aβ) induces tau hyperphosphorylation, decreases microtubule (MT) stability and induces neuronal death. MT stabilizing agents have been proposed as potential therapeutics that may minimize Aβ toxicity and here we report that paclitaxel (taxol) prevents cell death induced by Aβ peptides, inhibits Aβ-induced activation of cyclin-dependent kinase 5 (cdk5) and decreases tau hyperphosphorylation. Taxol did not inhibit cdk5 directly but significantly blocked Aβ-induced calpain activation and decreased formation of the cdk5 activator, p25, from p35. Taxol specifically inhibited the Aβ-induced activation of the cytosolic cdk5-p25 complex, but not the membrane-associated cdk5-p35 complex. MT-stabilization was necessary for neuroprotection and inhibition of cdk5 but was not sufficient to prevent cell death induced by overexpression of p25. As taxol is not permeable to the blood–brain barrier, we assessed the potential of taxanes to attenuate Aβ toxicity in adult animals using a succinylated taxol analog (TX67) permeable to the blood–brain barrier. TX67, but not taxol, attenuated the magnitude of both basal and Aβ-induced cdk5 activation in acutely dissociated cortical cultures prepared from drug treated adult mice. These results suggest that MT-stabilizing agents may provide a therapeutic approach to decrease Aβ toxicity and neurofibrillary pathology in AD and other tauopathies.

Abbreviations used

amyloid-beta

AD

Alzheimer's disease

cdk5

cyclin-dependent kinase 5

CNS

central nervous system

DAB

10-deacetylbaccatin III

JNK

c-jun N-terminal kinase

MTs

microtubules

NFTs

neurofibrillary tangles

PHFs

paried helical filaments

TX67

10-succinyl paclitaxel

Alzheimer's disease (AD) is characterized by the loss of specific groups of neurons and the presence of two brain lesions, amyloid plaques and neurofibrillary tangles (NFTs) (Lee et al. 2001; Selkoe 2001b). NFTs are composed of intracellular aggregates of highly insoluble paired helical filaments (PHFs) made up of fibrils of the MT-associated protein tau (Goedert 1997). Although the relationship between these two principal lesions is unclear, the discovery that multiple tau gene mutations occur in frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) has provided strong evidence that tau abnormalities are sufficient to promote neurodegeneration (Scheuner et al. 1996; Goedert et al. 1998; Spillantini et al. 1998)., Abnormal phosphorylation of tau by Aβ peptides promotes the loss of its microtubule (MT) stabilizing ability (Busciglio et al. 1995) and contributes to neurite degeneration and the formation of PHFs (Lee et al. 2001). These observations led to the hypothesis that MT-stabilizing agents, such as paclitaxel (taxol), might help overcome the inadequate binding of hyperphosphorylated tau to MTs and slow the progression of neurofibrillary pathology and cell death (Lee et al. 1994). We have reported previously that taxol can protect primary cortical neurons from Aβ-induced cell death (Michaelis et al. 1998). However, whether the neuroprotective action of taxol resulted solely from increased MT-stabilization or had contributions from other effects, such as regulating tau phosphorylation, was unclear. The cascade of events that lead to tau hyperphosphorylation may be a critical step in the pathogenesis of tauopathies (Goedert et al. 1998). Although tau is phosphorylated by numerous kinases in vivo (Billingsley and Kincaid 1997), recent attention has focused upon the role of cdk5 as an important tau kinase whose activity is enhanced in response to Aβ in cultured neurons (Alvarez et al. 1999, 2001) and in AD brain (Lee et al. 1999; Patrick et al. 1999). Cdk5 is a 33-kDa serine/threonine kinase that is active primarily in neurons (Ino et al. 1994) and can associate with MTs indirectly (Sobue et al. 2000). Cdk5 activity is regulated through association with the specific cyclin-related activator molecules, p35, p39, p25 and p29 (Dhavan et al. 2001). The calpain-directed proteolysis of p35/p39 (Kusakawa et al. 2000; Lee et al. 2000; Nath et al. 2000; Patzke and Tsai 2002a) releases p25/p29 from an N-terminal membrane tether and may delocalize cdk5/p25 (cdk5/p29) complexes from the plasma membrane and decrease phosphorylation of physiologic membrane substrates (Niethammer et al. 2000; Zukerberg et al. 2000). p35 has a short cellular half-life (Patrick et al. 1998, 1999) and its increased degradation can lead to cytoplasmic accumulation of cdk5/p25 complexes (Dhavan et al. 2001). As overexpression of cdk5/p25 in cells (Patrick et al. 1999) or transgenic mice (Ahlijanian et al. 2000) enhances tau phosphorylation, the ability of cdk5/p25 but not cdk5/p35 to serve as a tau kinase has lead to the concept that tau is a pathological substrate for cdk5 (Dhavan et al. 2001). Thus, treatments that affect the turnover and/or production of p35 should indirectly impact on cdk5 activity. In this report, we demonstrate that taxol inhibits an Aβ-induced pathway that links increased calpain activity to enhanced p25 production, cdk5 activation and tau phosphorylation. Although MT-stabilization by taxol was necessary for neuroprotection and inhibition of cdk5 activity, it was not sufficient to protect neurons from constitutive cdk5 activation following overexpression of p25 in primary cortical neurons. As the therapeutic potential of taxol in AD is limited by its bioavailability to the brain, we show that administration to adult mice of a taxol analog permeant to the blood–brain barrier inhibits Aβ-induced cdk5 activation. Collectively, our data suggest that MT-stabilization is tightly linked to the regulation of tau phosphorylation and that MT-stabilizing drugs may be potential therapeutic agents to slow the development of neurofibrillary pathologies.

Materials

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Polyclonal antibodies directed against cdk5 (C-8), the C-terminus of p35 (C-19) and the N-terminus of p35 (N-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). AT-8 antibody was purchased from Endogen and the Tau-5 and PHF-1 antibodies were provided by Dr Peter Davies (Albert Einstein College of Medicine). The pAdTrack-CMV (cytomegalovirus) shuttle vector and pAdEASY-1 adenoviral backbone vector were generous gifts from Dr T. C. He (Johns Hopkins University Medical School). Dr L-H. Tsai (Harvard Medical School) kindly provided the p35 cDNA construct. Taxol was purchased from Dabur India, Ltd. and the succinylated taxol analog (TX67) was prepared by parallel solution phase synthesis (Liu et al. 2002). Histone H1, bovine brain tau, N-succinyl-leu-tyr-7-amido-4-methylcoumarin, and Aβ1−42 peptides were purchased from Sigma/Aldrich Chemicals (St Louis, MO, USA). Aβ25−35 was synthesized by the Biochemical Research Services Laboratory at the University of Kansas. [γ-32P]ATP was from DuPont–NEN, Boston, MA, USA.

Cell culture and drug administration

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Primary cortical neurons were prepared from embryonic day 18 Sprague-Dawley rat pups as described previously (Michaelis et al. 1994). Following trituration, the cells were resuspended in Dulbecco's modified Eagle's medium/F12 (DMEM/F12) containing 15 mm KHCO3, 10% fetal calf serum and plated onto 35 or 60 mm poly d-lysine-coated dishes at a density of 6.5 × 105 or 2 × 106 cells/dish for viability and biochemical assays, respectively. Twenty-four hours after plating, the cells were washed and placed in defined medium (DMEM/F12, 0.1 g/L transferrin-APO form, 5 mg/L insulin, 0.1 mm putrescine, 10 nm progesterone, 30 nm sodium selenite, and 1 mm sodium pyruvate) for the duration of the experiment. After 4 days in vitro, the cells were treated with 10 μm Aβ peptides in the absence or presence of 100 nm taxol. Taxol was stored as a 1 mm stock solution in dimethyl sulfoxide (DMSO) and diluted to 25 μm in H2O prior to the experiment. The final concentration of DMSO in all cultures was 0.01%.

TX67 is a substituted taxol analog that replaces the C-10 acetyl group of taxol with succinic acid (Liu et al. 2002). Adult mice received intraperitoneal injections (0.1 mL) of 8 mg/kg TX67 every 2 days over 16 days. TX67 was dissolved in a (1 : 1) solution of ethanol/Cremephor EL (polyethoxylated castor oil that is used clinically for taxol injections) and diluted 1 : 6 with 133 mm sterile saline immediately prior to injection. Animals receiving taxol were treated similarly. The dosing schedule appeared to have little effect on the general health of the animals, as all gained weight and displayed no obvious behavioral or physical anomalies. All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee and in compliance with standards and regulations for care and use of laboratory rodents set by the National Institutes of Health.

Cultures of acutely dissociated neurons from the control and drug-treated animals were prepared after killing the animals with CO2. The brains were rapidly excised and placed in ice-cold isotonic buffer (5.0 mm HEPES, pH 7.4, 132 mm NaCl, 5.36 mm KCl, 0.21 mm Na2HPO4, 0.22 mm KH2PO4, 2.77 mm glucose and 58.4 mm sucrose). The cerebral cortices were dissected, the grey matter minced into small pieces and the tissue incubated with 0.25% trypsin in calcium and magnesium free Hank's solution for 1 h at 37°C. The tissue fragments were triturated to disperse the cells and placed into 20 mL of DMEM/F12 medium containing 10% fetal calf serum and 15 mm KHCO3. The cell suspension was filtered through a sterile 41 μm nylon mesh filter to remove large clumps, the cells collected by centrifugation and resuspended in pre-warmed DMEM/F12 medium containing 10% fetal calf serum. The cells were treated immediately with 20 μm25−35 for 24 h and both adherent and non-adherent cells were harvested, resuspended in lysis buffer and ex vivo cdk5 activity assessed as described below.

Immunoprecipitation of Cdk5 and in vitro kinase assay

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Cells were harvested in lysis buffer (20 mm Tris-HCl pH 7.4, 140 mm NaCl, 1 mm PMSF, 1 mm Na3NO4, 10 mm NaF, 0.1% Nonidet-40, 1 mm EDTA, 1 × complete protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA) and 26 μmN-acetyl-leu-leu-norleucinal, ALLN) and cell debris was removed by centrifugation at 10 000 g for 10 min at 4°C. Protein concentration was determined using the Bio-Rad dye and bovine serum albumin as the standard. Protein (200 μg) was incubated with 2 μg of Cdk5 antibody (C-8) or p35 antibodies (N-20 or C-19) for 2 h at 4°C. Protein A-Sepharose beads (40 μL of a 50% slurry) were added to the samples and immune complexes were formed by incubation for 1 h at 4°C. The beads were sedimented by centrifugation, washed two times with lysis buffer and one time with kinase buffer (50 mm Tris-HCl, pH, 7.4, 80 mmβ-glycero-phosphate, 20 mm EGTA, 15 mm MgCl2, and 1 mm dithiothreitol). The beads were resuspended in 30 μL of kinase buffer containing 50 μm ATP, 1.25 μCi of [γ-32P]ATP, and 1 μg of histone H1 or purified tau protein. The samples were incubated at 24°C for 30 min (histone H1) or 37°C for 30 min (tau) and the reaction was stopped by adding 10 μL of 4 × sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) loading buffer. Proteins were separated by SDS–PAGE and the radioactive bands were quantified using a phosphoimager.

In some experiments, cdk5 was immunoprecipitated from cytosolic and particulate fractions. Neurons were treated with Aβ in the absence and presence of taxol and scraped into detergent-free lysis buffer to avoid solubilizing membrane bound cdk5/p35. The cells were sonicated and debris was removed by centrifugation at 10 000 g for 5 min. An aliquot of the whole cell lysate was removed (100 μg) and 100 μg was centrifuged at 100 000 g for 1 h at 4°C in a table top Optima-Max ultracentrifuge. The supernatant (cytosol) was transferred to a fresh tube and Nonidet-40 was added to a final concentration of 0.1%. The pellet (membrane) was washed and then solubilized in lysis buffer containing 0.1% Nonidet-40. Cdk5 was immunoprecipitated from the cytosolic and membrane fractions with 2 μg of the C-19 p35 antibody and kinase activity assessed as described above.

Cell viability measurements

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

25−35 (1.3 mg/mL) and Aβ1−42 (4.5 mg/mL) were prepared by resuspending the peptides in H2O followed by dilution with 10 mm Tris-HC1, pH 7.4, to 1 mm. The peptides were then aged at 37°C for 24 h to induce aggregation. Cell viability was determined using the live/dead assay as described previously (Michaelis et al. 1998). The cells were stained with 10 μm propidium iodide (PI) and 150 nm calcein-acetoxymethylester (calcein-AM, Molecular Probes, Eugene, OR, USA) for 30 min at 37°C and imaged by fluorescence microscopy. Six images were captured from each dish on marked fields with a CCD camera and all the cells in each field were counted to determine total cell number. Calcein-AM labels the viable cells (green) and PI stains the dead cells (red). Cell viability was calculated as a percentage of viable cells to total cell number.

Protein half-life assay

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Neurons were treated with vehicle or taxol for 4 days followed by the addition of 30 μg/mL cycloheximide for 0–480 mins (Patrick et al. 1998). At the indicated time, the cells were scraped into lysis buffer, cell debris was removed by centrifugation at 10 000 g for 10 min at 4°C and an equal amount of protein from each time point was subjected to SDS–PAGE. In assessing the effect of Aβ and taxol treatment on p35/p25 levels, complete protease inhibitors and 26 μm ALLN were always added to the lysis buffer immediately before harvesting and the samples were processed directly for SDS–PAGE without freezing and thawing of the lysates. The proteins were transferred to nitrocellulose and p35/p25 detected by immunoblot analysis using a C-terminal antibody. The blot was stripped and re-probed for the presence of tau (tau-5 antibody) and cdk5. The amount of p35 expression at each time point was quantified by densitometry and expressed as per cent remaining relative to that present at time 0.

Calpain activity assay

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Neurons were harvested after 4 days of treatment in calpain lysis buffer (20 mm Tris pH 7.4, 140 mm NaCl, 0.1% Nonidet-40) and cell debris was removed by centrifugation as described above. Protein (30 μg) from each sample was loaded in quadruplicate to a 96-well plate and the reaction initiated with 130 μm substrate without the addition of exogenous calcium. The substrate for this assay, N-succinyl-leu-tyr-7-amido-4-methylcoumarin, is a non-fluorescent peptide which strongly fluoresces after cleavage by calpain (Xie and Johnson 1997). The increase in fluorescence intensity was recorded at 30°C for 3 h in a Bio-Tek FL600 microplate fluorometer and normalized to total protein. In some assays, 100 nm taxol was added directly to the reaction mix containing 0.4 units of purified calpain 1 (Calbiochem, San Diego, CA, USA) and 5 mm Ca2+ in lysis buffer.

Generation of p25 recombinant adenovirus

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

p35-pcDNA3 served as a template for amplifying p25 by PCR. The forward primer (5′-GTCGACGGTACCATGGCCCAGCCCCCACCG-3′) incorporated a KpnI site and an ATG start codon upstream of the N-terminal alanine for p25 (amino acid 98 of p35) (Tsai et al. 1994). The reverse primer (5′-CTCGAGTTACCGATCCAGGCCTAG-3′) was engineered with a XhoI site. The amplified PCR product was subcloned directly into TOPO2.1 (Invitrogen, Carslbad, CA, USA) and sequenced in both directions for errors. The p25-TOPO2.1 cDNA was digested with KpnI and XhoI, the approximate 0.6-kb p25 fragment was gel purified and subcloned into the pAdTrack-CMV shuttle vector (He et al. 1998). p25-AdTrack-CMV was linearized with PmeI and electroporated into RecA+ bacteria (BJ5183) with 1 μg of pAdEASY-1 as described (He et al. 1998). Bacterial clones containing recombinant adenoviral DNA were verified by restriction mapping and recombinant viruses were generated by transfection of HEK293 cells. Virus was amplified by four rounds of infection and purified from 20 × 15 cm plates of HEK293 cells using two rounds of centrifugation through CsCl gradients. The residual CsCl was removed by dialysis against 10 mm Tris-HCl, pH 8.0, 100 mm NaCl, 0.1% bovine serum albumin, 20% glycerol in a Slide-a-Lyzer cassette. Recombinant adenoviruses were titered based upon number of green fluorescent foci in an agar-overlay assay as described (He et al. 1998). Cortical neurons were infected with blank or p25-recombinant adenoviruses at 100–400 pfu/cell. The infection efficiency as monitored by cells expressing the green fluorescent protein ranged from 40 to 50%.

Taxol protects against cell death induced by Aβ peptides

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Primary neuronal cell cultures derived from the brains of embryonic rat pups (Michaelis et al. 1994) were used to demonstrate the protective effect of taxol against Aβ-induced toxicity. Taxol pre-treated cells were minimally distinguishable from control cells, indicating that the MT-stabilizing properties of the drug did not affect neuronal morphology (Fig. 1a). Our previous studies solely used the Aβ25−35 peptide to assess the effect of taxol on cell death (Michaelis et al. 1998), but this Aβ peptide fragment is not produced in AD. Therefore, we assessed the effect of taxol against neuronal toxicity induced by Aβ1−42, the physiologically relevant Aβ peptide that accumulates in AD (Selkoe 2001a). Whereas cells treated with Aβ1−42 were pyknotic with fragmented and degenerating neurites, taxol preserved neurite morphology and overall appearance in the presence of Aβ1−42 (Fig. 1a). Further, quantitation of cell survival indicated that 100 nm taxol afforded significant protection against cell death induced by 10 μm1−42 (Fig. 1b). As taxol rescues neurons against cell death induced by Aβ1−42 with results qualitatively similar to Aβ25−35, most remaining studies used this shorter Aβ peptide.

image

Figure 1. Cell death induced by Aβ1−42 is prevented by taxol pre-treatment. (a) Neurons were treated with buffer, 100 nm taxol or 10 μm1−42 in the absence or presence of taxol for 4 days. The cells were stained with calcein-AM and propidium iodide, photographed and cell viability determined. (b) The results from the quantitative analysis of neuronal viability from three experiments. *, p < 0.001 compared with control; #, p < 0.001 compared with Aβ only. At least 200 cells per treatment were counted in each experiment.

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To assess whether the neuroprotective effects of taxol required MT-stabilization, cells were treated with 10-deactylbaccatin III, a taxol precursor that is ineffective at stabilizing MTs (Wang et al. 1998). 10-Deactylbaccatin III provided no significant protection against Aβ25−35 toxicity (Table 1) which strongly supports that the MT-stabilizing properties of taxol are indeed necessary for neuroprotection. As neurons undergoing Aβ-induced degeneration exhibit increased phosphorylation of the MT-interacting protein tau which leads to MT destabilization (Busciglio et al. 1995), taxol may help maintain MT stability and consequently regulate the interaction of tau with Aβ-activated tau kinases. Therefore, we addressed the possibility that taxol may decrease Aβ toxicity by stabilizing MTs and interfering with mechanisms regulating Aβ-induced tau hyperphosphorylation.

Table 1.  TX67 but not 10-deacetylbaccatin III protects cortical neurons against Aβ toxicity
 Cell viabilityPer cent control
  1. Neurons were pre-treated for 2 h with 100 nm of the indicated drug and the cells were stimulated with 10 μm25−35. After 4 days, cell viability was determined with the live/dead assay. ap < 0.001 compared with vehicle minus Aβ, bp < 0.001 compared with vehicle plus Aβ.

 Minus AβPlus Aβ
Vehicle10057.6 ± 4.6a
10-deacetylbaccatin III91.3 ± 1.260.5 ± 3.2a
10-succinyl taxol (TX-67)93.7 ± 3.485.3 ± 2.9b

Taxol inhibits Aβ-induced tau phosphorylation

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

To assess the in vivo effect of Aβ25−35 and taxol treatment on tau phosphorylation in primary neurons, we used the AT-8, PHF-1 and Tau-5 antibodies. AT-8 recognizes an epitope phosphorylated at Ser202 and Thr205 while PHF-1 recognizes tau phosphorylated on Ser396 and Ser404 (Busciglio et al. 1995). The Tau-5 antibody recognizes total tau levels independent of phosphorylation state.

Neurons were pre-treated with 100 nm taxol for 2 h and challenged with 10 μmΑβ25−35. After an additional 4 days, cell lysates were prepared and the proteins present in a post-nuclear supernatant fraction were resolved by SDS–PAGE. After transferring the proteins to nitrocellulose, tau phosphorylation and total tau levels were determined by immunoblot analysis. Aβ25−35 significantly increased the amount of hyperphosphorylated tau detected by both the AT-8 and PHF-1 antibodies (Fig. 2a). Increased hyperphosphorylation was completely prevented by taxol which had no effect on total tau levels as determined using the Tau-5 antibody. Moreover, the Tau-5 antibody also recognizes phosphorylated tau species as indicated by the presence of a mobility shift in the tau band from cells treated with Aβ25−35. This species of tau was also abolished in the taxol-treated cells. The expression of cdk5 also remained the same in all the treatments and provides an additional control for protein loading. Densitometric quantitation of the PHF-1 immunoreactive bands from several experiments indicated that taxol significantly decreased both basal and Aβ-induced tau phosphorylation (Fig. 2b).

image

Figure 2. Taxol blocks Aβ-induced tau hyperphosphorylation. (a) Neurons were exposed to buffer (Con), 100 nm taxol (T) or 10 μm25−35 in the absence (Aβ) or presence of taxol (T + Aβ) for 4 days. Whole cell lysates were prepared and total protein was separated by SDS–PAGE (20 μg/lane). After transfer to nitrocellulose, the membrane was probed for phosphorylated tau (AT-8 or PHF-1 antibodies), stripped and re-probed for total tau using the Tau-5 antibody. Phosphorylated tau (pTau) is indicated by the arrows. Bottom panel shows the expression level of cdk5 from the same samples. (b) Tau phosphorylation detected with the PHF-1 antibody was quantitated by densitometry from five experiments. *, p < 0.05 compared with control; #, p < 0.001 compared with Aβ25−35 only.

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Taxol blocks Aβ-induced activation of cdk5/p25 complexes

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Tau is phosphorylated by numerous kinases in vivo including glycogen synthase kinase 3β, MT-affinity regulated kinases, protein kinase A and cdk5 (Billingsley and Kincaid 1997; Lee et al. 2001; Maccioni et al. 2001). Aβ25−35 slightly stimulated glycogen synthase kinase 3β (∼1.2-fold) under our culture conditions and taxol inhibited this activation (data not shown). However, Aβ25−35 more robustly and reproducibly activated cdk5 (Fig. 3a). Because cdk5 is active primarily in neurons (Ino et al. 1994), associates with MTs indirectly through interactions with a MT-binding repeat of non-phosphorylated tau (Sobue et al. 2000), and its phosphorylation of tau directly leads to MT destabilization (Evans et al. 2000), we primarily focused upon the potential of taxol in regulating the activity of this tau kinase.

image

Figure 3. Taxol but not 10-deacetylbaccatin III inhibits Aβ-induced cdk5 activation. Neurons were exposed for 4 days to buffer (Con), 100 nm taxol (T) or 10 μm25−35 in the absence (Aβ) or presence of taxol (T + Aβ) and cell lysates were prepared. (a) Cdk5 was immunoprecipitated from whole cell lysates and kinase activity was assessed using tau as the substrate. Inset shows representative autoradiogram for the effect of Aβ25−35 and taxol treatment on in vitro tau phosphorylation. Tau phosphorylation was quantitated from four experiments. *, p < 0.01 compared with control; #, p < 0.001 compared with Aβ25−35 only. (b) Cdk5 was immunoprecipitated from untreated neurons and kinase activity assessed in the absence or presence of 100 nm taxol. Taxol was added directly to the reaction mixture prior to the addition of histone H1 or tau, the radiolabeled products were separated by electrophoresis and the level of phosphorylation determined using a phosphoimager. Results shown are mean ± SEM from three experiments. (c) Cells were incubated for 4 days with buffer (Control), 100 nm 10-deacetylbaccatin III (DAB) or 10 μm25−35 in the absence (Aβ) or presence of 100 nm 10-deacetylbaccatin III (Aβ + DAB). Cells were harvested and cdk5 activity assessed as described above using histone H1 as the substrate. Upper panel shows histone H1 phosphorylation and bottom panel shows equivalent levels of cdk5 in each sample.

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Neurons were treated with Aβ25−35 in the absence and presence of 100 nm taxol for 4 days, whole cell lysates were prepared and cdk5 was immunoprecipitated. To assess cdk5 activity, the isolated immune-complexes were incubated in kinase buffer in the presence of [γ-32P]ATP and either histone H1 or bovine brain tau as an in vitro substrate. Aβ25−35 induced a 1.5-fold increase in cdk5-mediated tau phosphorylation that was completely prevented by taxol treatment (Fig. 3a); it is important to note that the modest increase in total cdk5 activity in whole cell lysates is likely due to Aβ25−35 preferentially activating only the cytosolic pool of cdk5/p25 complexes (see below).

To determine if taxol may decrease cdk5 activity by directly inhibiting the enzyme, cdk5 was immunoprecipitated from untreated neurons and an in vitro kinase assay was performed as above except that 100 nm taxol was added directly to the reaction mix prior to initiating the reaction by the addition of substrate. Under these conditions, taxol had absolutely no effect on the phosphorylation of histone H1 or tau by cdk5 (Fig. 3b). These data suggest that the reversal of the Aβ-induced increase in cdk5 activity by taxol is not due to a direct inhibition of the kinase by the drug and requires cell signaling events. To examine the relationship between MT stabilization and the inhibitory effect of taxol on Aβ-induced cdk5 activation, neurons were treated with 10-deacetylbaccatin III. After 4 days of treatment, the cells were harvested, cdk5 activity immunoprecipitated and kinase activity assessed using histone H1 as the substrate. In contrast to taxol treatment, 10-deacetylbaccatin III had no effect on inhibiting Aβ-induced cdk5 activation (Fig. 3c). These results suggest that MT stabilization is necessary for the inhibitory effect of taxol on cdk5 activation by Aβ25−35.

Cdk5 activity is regulated by interaction with specific activator proteins, p35 and p25, that have different subcellular localizations. Due to the presence of an N-terminal myristoylation site, p35 is essential for targeting cdk5 to the plasma membrane (Tsai et al. 1994; Dhavan et al. 2001). In contrast, a calpain-directed proteolytic degradation of p35 releases p25 from the N-terminal region of p35 that is tethered to the membrane and increases the amount of cytosolic cdk5/p25 complexes (Kusakawa et al. 2000; Lee et al. 2000; Nath et al. 2000). Because the above experiments used an antibody directed against cdk5, we could not determine whether Aβ25−35 activated cdk5/p35 or cdk5/p25 complexes nor whether one or both complexes may be inhibited by taxol.

To determine the effect of Aβ25−35 and taxol on the activity of cdk5/p35 versus cdk5/p25 complexes, two approaches were taken. In the first approach, cdk5 activity was assessed following immunoprecipitation with antibodies that recognize either the N-terminus (N-20) or C-terminus (C-19) of p35. As p35 and p25 share the same C-terminus, the C-19 p35 antibody will immunoprecipitate both p35 and p25 and provide an assessment of total cdk5 activity. However, the N-20 p35 antibody is directed against an N-terminus region that is lacking in p25 and provides a measure of kinase activity associated with only the cdk5/p35 complex. Therefore, the contribution of the cdk5/p25 complex to total cdk5 activity may be measured indirectly by subtracting the cdk5/p35-specific activity (N-20 antibody) from the total activity (cdk5/p35 + cdk5/p25) obtained following immunoprecipitation with the C-19 antibody.

Neurons were treated with Aβ25−35 in the absence or presence of taxol, the cells were harvested in lysis buffer and each whole cell lysate was divided into two × 100 μg aliquots. Each aliquot was then incubated with 2 μg of the C-19 or the N-20 p35 antibody, the immune complexes were isolated with protein A Sepharose and cdk5 activity was assessed as described above. Similar to results in Fig. 3a, Aβ25−35 treatment activated total cdk5 activity (C-19 p35 antibody) but kinase activity associated with the cdk5/p35 complex (N-20 p35 antibody) did not contribute to the overall increase (Fig. 4a). Indeed, total cdk5 was activated by Aβ25−35 about 1.5-fold. However, subtracting the p35-specific activity from total activity indicated that cdk5/p25 complexes were activated by Aβ25−35 approximately 2.1–2.3-fold (Fig. 4b). Moreover, taxol significantly inhibited kinase activity associated with the cdk5/p25 complexes but had no effect on the activity of cdk5/p35 complexes (Fig. 4b).

image

Figure 4. Taxol inhibits the activity of cdk5/p25 complexes. (a) Neurons were treated as indicated and the whole cell lysates were divided into 2 × 100 μg aliquots. Cdk5 was immunoprecipitated from each aliquot with either the C-19 p35 antibody which recovers cdk5/(p35 + p25) complexes or the N-20 p35 antibody which recovers only cdk5/p35 complexes. Cdk5 activity was assayed and phosphorylated histone H1 separated by SDS–PAGE and detected by phosphoimaging. (b) The relative density of histone H1 phosphorylation by cdk5(p35 + p25) and cdk5/(p35) complexes was quantitated from three experiments. Results shown for cdk5/(p25) were obtained by subtracting the relative density of histone H1 phosphorylation obtained for cdk5/(p35) (5.4 ± 0.8) from cdk5/(p35 + p25) (13.5 ± 1.6) and expressed as fold control. *, p < 0.01 compared to control; #, p < 0.001 compared to Aβ only. (c) Neurons were treated as indicated and an aliquot of the total lysate saved. Of the remaining whole cell lysates, 100 μg was separated into cytosolic and membrane fractions by centrifugation. Each fraction was immunoprecipitated with the C-19 p35 antibody and cdk5 activity assessed using histone H1 as the substrate. Upper panel, histone H1 phosphorylation; middle panel, immunoblot of p35 and p25 levels in membrane and cytosolic fractions; lower panel, immunoblot for the level of cdk5 in each sample.

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To provide a more direct assessment of the effect of Aβ25−35 and taxol on the activity of cdk5/p25 complexes, our second approach exploited the differential compartmentation of cdk5/p35 versus cdk5/p25 complexes. As cdk5/p35 is primarily membrane-associated whereas cdk5/p25 is cytosolic (Nikolic and Tsai 2000), soluble and particulate fractions of treated neurons were prepared as described in Experimental procedures. Prior to centrifugation, an aliquot of the whole cell lysate was removed to determine total cdk5 activity. Cdk5 present in the whole cell lysate, soluble and particulate fractions was immunoprecipitated with the C-19 p35 antibody and kinase determined as described above. Once again, Aβ25−35 activated total cdk5 activity immunoprecipitated from whole cell lysates by 1.5-fold and this activation was completely inhibited by taxol (Fig. 4c). However, cdk5/p25 activity immunoprecipitated from the cytosolic fraction was activated greater than 3-fold by Aβ25−35 and this activation was inhibited by about 55% in cells treated with taxol and Aβ25−35. Similar to our results above, Aβ25−35 and taxol had no effect on the activity of cdk5/p35 complexes immunoprecipitated from the membrane fraction. Together, these results strongly support that Aβ-induced cdk5 activity is associated primarily with cytosolic cdk5/p25 complexes and that taxol specifically decreases the Aβ-induced activation of the cdk5/p25 complex.

Taxol prevents Aβ-induced changes in the ratio of p35/25

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Because taxol was specifically inhibiting the activity of cdk5/p25 complexes, we next addressed potential mechanisms for this effect. p35 is a short-lived protein that undergoes relatively rapid degradation by calpain-mediated proteolysis (Lee et al. 2000) and via interaction with the proteasome (Patrick et al. 1998). Moreover, recent reports suggest that Aβ increases the degradation of p35 to p25 in AD brain (Lee et al. 1999; Patrick et al. 1999) and cultured primary neurons (Lee et al. 2000). Similarly, Aβ25−35 treatment induced a significant 1.5-fold increase in the production of p25 in the cortical neurons after 4 days (Fig. 5a) and co-treatment of the cells with Aβ25−35 and taxol completely prevented this increase. Thus, decreased p25 production correlated with the effect of taxol on decreasing cdk5/p25 activity.

image

Figure 5. Taxol increases the half-life of p35 and decreases the formation of p25 by Aβ25−35. (a) Neurons were exposed for 4 days to buffer (Con), 100 nm taxol (T) or 10 μm25−35 in the absence (Aβ) or presence of taxol (T + Aβ), cell lysates were prepared and p25 levels were determined by immunoblot analysis. Inset shows representative immunoblot for the effect of Aβ25−35 and taxol treatment on p25 expression. p25 expression was quantitated densitometrically from four experiments. *, p < 0.001 compared with control; #, p < 0.001 compared with Aβ only. (b) Neurons were treated with vehicle or 100 nm taxol for 4 days, 30 μg/mL cycloheximide (CHX) was added, and the cells were harvested at the indicated time after CHX addition. Whole cell lysates were subjected to SDS–PAGE (20 μg per lane) and the half-life of p35 was measured by immunoblot analysis. (c) Expression of p35 was quantitated by densitometry and the approximate half-life of p35 in control and taxol treated cells is indicated by the arrows. (d) Separate aliquots from the same neuronal lysates used above were subjected to SDS–PAGE and the effect of taxol on the half-life of tau and cdk5 was determined by immunoblot analysis.

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To determine if taxol was decreasing the turnover of p35, we measured its half-life in cortical neurons treated with vehicle or 100 nm taxol for 4 days. To inhibit protein synthesis, 30 μg/mL of cycloheximide was added to the cells (Patrick et al. 1998) and the neurons were scraped into lysis buffer at various times between 0 and 480 min following the addition of cycloheximide. Similar amounts of total protein from each time point were separated by SDS–PAGE, the proteins were transferred to nitrocellulose and the presence of p35 and p25 was determined by immunoblot analysis using the C-19 p35 antibody. In control neurons the half-life of p35 was around 140–160 min (Fig. 5b and c), substantially longer then the 20–30 mins that has been reported in cortical neurons cultured for 3 days in vitro (Patrick et al. 1998) or in transfected COS-7 cells (Patrick et al. 1999). Whether this difference is related to the age of the cultures, specific differences in culture conditions, antibodies or cell type is unknown. Nevertheless, addition of 100 nm taxol to the cortical neurons for 4 days increased the half-life of p35 to about 320–340 min (Fig. 5b and c). Importantly, using the same cell extracts, taxol had no effect on the half-life of either tau or cdk5, indicating that it was not non-specifically decreasing protein degradation (Fig. 5d). The lack of change in cdk5 and tau levels also indicates that the increased half-life of p35 in the presence of taxol is not due to differences in protein loading.

Phosphorylation of p35 by cdk5 enhances its degradation via the proteasome and direct inhibition of cdk5 with roscovitine can increase the half-life of p35 (Patrick et al. 1998). Therefore, a decrease in cdk5 activity would be expected to increase the half-life of p35. However, our results indicate that taxol does not directly inhibit cdk5, suggesting that it stabilizes p35 by another mechanism. Although we cannot rule out the possibility that taxol may increase the half-life of p35 by interfering with its accessibility to serve as a substrate for cdk5, the calpain-mediated cleavage of p35 also leads to enhanced formation of p25 (Kusakawa et al. 2000; Lee et al. 2000). Because taxol stabilized p35 and decreased p25 formation, we examined the effect of Aβ25−35 and taxol on calpain activity. Primary cortical neurons were treated with Aβ25−35 in the absence or presence of taxol for 4 days, cell lysates were prepared and calpain activity was determined fluorometrically (Xie and Johnson 1997). As anticipated, Aβ25−35 treatment induced an approximate 2-fold activation of calpain (Fig. 6). Although taxol decreased basal calpain activity, it also inhibited calpain activation induced by Aβ25−35. As a positive control, the assay was verified by incubating the cell lysate with ALLN, a calpain inhibitor that completely blocked hydrolysis of the substrate.

image

Figure 6. Inhibition of Aβ-induced calpain activation by taxol. Neuronal cultures were exposed for 4 days to 10 μm25−35 in the absence or presence of 100 nm taxol and cell lysates prepared. Calpain activity was monitored fluorometrically in quadruplicate and the results shown are from four experiments.

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The inhibition of calpain activity by taxol was not due to decreases in the level of calpain-1 (μ-calpain) or calpain-2 (m-calpain) nor to increases in the expression of the endogenous calpain inhibitor, calpastatin (data not shown). It should be noted, however, that 13 different calpains are broadly expressed (Huang and Wang 2001). Importantly, taxol had no effect on endogenous calpain activity when it was added directly to cell lysates prepared from untreated neurons and incubated with the calpain substrate. This result suggests that taxol does not inhibit calpain directly. However, to rule out the possibility that some endogenous proteins or lipids present in the cell lysate were sequestering taxol and preventing it from inhibiting calpain directly, purified calpain-1 was incubated with the peptide substrate plus 5 mm Ca2+ in the presence and absence of 100 nm taxol. Once again, taxol had no inhibitory effect on calpain-1 activity with the purified enzyme (data not shown). Collectively, these results suggest that a primary point of action of taxol is upstream of calpain activation by Aβ25−35.

Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Transgenic mice overexpressing p25 exhibit cytoskeletal disruptions and increased phosphorylation of neurofilaments and tau (Ahlijanian et al. 2000). Moreover, tau phosphorylation by cdk5 decreases MT stability (Lee et al. 2001). If MT stabilization by taxol is sufficient to protect neurons against Aβ toxicity, then it should be protective even during ectopic expression of p25 and increased tau phosphorylation. Primary cortical neurons were infected with a recombinant p25 adenovirus and protein expression and cdk5 activity determined over a period of 4 days. Overexpression of p25 was maximal 2 days after infection (Fig. 7a) and increased cdk5 activity about 2-fold using either histone H1 or tau as the substrate (Fig. 7b). Therefore, we examined the effect of p25 overexpression (200 pfu/cell) on the protection against Aβ25−35 toxicity by taxol at this time.

image

Figure 7. Overexpression of p25 reverses the neuroprotective effect of taxol. (a) Neurons were infected with recombinant adenoviruses at 0–400 pfu/cell for 2 days and cell lysates were prepared. Following SDS-PAGE (20 μg/lane), p25 was detected by immunoblot analysis. (b) Cell lysates were prepared from uninfected neurons or neurons infected with 200 pfu/cell of blank adenovirus or p25 adenovirus for 2 days. Cdk5 was immunoprecipitated and its activity was assessed using histone H1 or tau as the substrate in an in vitro kinase assay. The upper panels show the extent of histone and tau phosphorylation. Band intensity was quantitated using a phosphoimager and expressed as fold-increase relative to basal activity of uninfected cells. The results from three experiments are shown. (c) Neurons were infected with blank or p25 adenoviruses at 200 pfu/cell. After 2 h, the neurons were treated with buffer (Con), 100 nm taxol (T) or 10 μm25−35 in the absence (Aβ) or presence of taxol (T + Aβ) for 2 days. Cell lysates were prepared and p25 levels were assessed by immunoblot analysis. Cdk5 (C-8 antibody) was immunoprecipitated and its activity assessed using histone H1 as the substrate. Total cdk5 levels (lower panel) were similar between each treatment. (d) Cells infected with blank or p25 adenovirus were treated with 10 μm25−35 in the absence or presence of 100 nm taxol for 2 days and cell viability was assessed. Values presented are from four experiments. The per cent viability in uninfected neuronal cultures was 76.6 ± 2.6. *, p < 0.05 for Aβ compared to respective control; **, p < 0.05 blank virus-taxol compared with blank virus-(taxol + Aβ); #, p < 0.001 blank virus-(taxol + Aβ) compared with p25 virus-(taxol + Aβ).

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Similar to uninfected cells (Fig. 5a), taxol inhibited the Aβ-induced increase in p25 expression (Fig. 7c, upper panel) and cdk5-mediated tau phosphorylation (Fig. 7c, middle panel) in cells infected with the blank virus. In contrast, neither Aβ25−35 nor taxol changed p25 expression (Fig. 7c, upper panel) or cdk5-mediated histone H1 phosphorylation (Fig. 7c, middle panel) in neurons infected with the p25 adenovirus. As taxol did not decrease cdk5 activity in cells overexpressing p25, these results also support that the inhibitory effect of taxol on Aβ-induced cdk5 activation requires the disruption of signal transduction events and is not due to a direct inhibition of the kinase by taxol.

25−35 induced a significant level of cell death in neurons infected with blank or p25 adenoviruses (Fig. 7d). However, p25 overexpression only modestly decreased cell viability regardless of the absence or presence of Aβ25−35. The modest effect of p25 expression on increasing the extent of cell death may be due to using these cells 2 days post infection. At this time, the viability of neurons infected with blank virus (71 ± 3.1%) was similar to uninfected neurons (76.6 ± 2.6%). Although p25 expression was significantly more effective at inducing cell death 3–4 days post infection, viability of neurons treated with blank virus alone was also decreasing (data not shown). Importantly, viral infection did not non-specifically alter the response to taxol as the drug also protected against Aβ25−35 toxicity in neurons infected with blank virus (compare bars 3 and 7). In contrast, taxol did not significantly inhibit cell death in response to increased p25 expression in the absence (compare bars 2 and 6) or presence (compare bars 4 and 8) of Aβ25−35. Indeed, forced expression of p25 reversed the neuroprotective effect of taxol seen in cells infected with blank virus and treated with Aβ25−35 (compare bars 7 and 8). As ectopic expression of p25 had no effect on the stabilization of MTs by taxol (data not shown), these data suggest that MT stabilization alone is not sufficient to overcome the detrimental effect of enhanced cdk5 activity.

Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Unfortunately, identifying the most active compound in cell cultures with regards to MT stabilization, intracellular signaling and neuroprotection is usually not sufficient to overcome the difficulties of showing activity in an intact organism. In this regard, the potential for taxol as a therapeutic agent either for AD or cancers of the central nervous system (CNS) is limited by its lack of penetration across the blood–brain barrier.

The blood–brain barrier is composed of numerous tight junctions between capillary endothelial cells that create a low passive permeability across this barrier and a selective exchange of molecules from the circulation into the CNS (Thiel and Audus 2001). Additionally, the presence of the P-glycoprotein in cells forming the blood–brain barrier permits the rapid efflux of lipophilic molecules such as taxol which limits the effectiveness of this drug in treating tumors of the CNS (Heimans et al. 1994; Brouty-Boye et al. 1995). Although pharmacologic inhibition of this efflux can increase the brain level of taxol 4-fold (van Asperen et al. 1997), transport of essential macromolecules across the blood–brain barrier may also be facilitated by highly selective transporter proteins (Thiel and Audus 2001). The presence of specific small molecule transporters offers the possibility that modification of taxol may circumvent P-glycoprotein-mediated efflux and enhance its permeability across the blood–brain barrier while retaining its MT-stabilizing and neuroprotective properties.

Using combinatorial chemistry and a strategy that exploited the presence of specific transporters for basic amino acids, biotin, amines and monocarboxylic acids, a library of compounds was prepared that modified taxol with these various molecules (Liu et al. 2002). From this library, a succinylated taxol derivative (10-succinyl taxol, TX67) emerged as a candidate molecule that efficiently stabilized MTs (Liu et al. 2002), bypassed the P-glycoprotein efflux system and has a 150-fold greater permeability than taxol at therapeutically relevant concentrations in a cellular model for the blood–brain barrier (Michaelis et al. 2002). Importantly, TX67 also was neuroprotective against Aβ in cultured primary neurons (Table 1). Therefore, we assessed whether systemic administration of TX67 may mimic the effect of taxol on inhibiting Aβ-induced cdk5 activation in acutely dissociated neurons prepared from TX67-treated animals. Adult mice were injected every other day for 16 days with vehicle, 8 mg/kg of TX67 or a similar dose of taxol. To determine the in vivo effectiveness of TX67 versus taxol, cultures of acutely dissociated cortical neurons were prepared and the cells were treated ex vivo with buffer or 20 μm25−35 immediately after their preparation. We used a higher concentration of Aβ25−35 in these studies as the acutely dissociated neurons are still partially clumped and represent a mixed population of cells. After 24 h, the cells were harvested, cdk5 was immunoprecipitated and histone H1 phosphorylation was assessed using the in vitro kinase assay. Similar to the cultured embryonic cortical neurons, Aβ25−35 induced a 1.8-fold increase in cdk5 activity in acutely dissociated neurons obtained from adult animals that received the drug vehicle only (Fig. 8a). Administration of taxol to the animals was ineffective at preventing the Aβ-induced activation of cdk5, consistent with its poor ability to cross the blood–brain barrier (Cavaletti et al. 2000). In contrast, TX67 markedly decreased the magnitude of basal cdk5 activity and blocked its activation by Aβ25−35 (Fig. 8a and b). Overall, these data provide compelling evidence for proof of principle that taxol analogs may be useful for attenuating the magnitude of some aspects of Aβ signal transduction in vivo.

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Figure 8. Systemic administration of a succinylated taxol analog (TX67) blocks Aβ-induced cdk5 activation. (a) Animals were given intraperitoneal injections of the drug vehicle, 8 mg/kg TX 67 or taxol every other day for 16 days. The animals were killed, cultures of acutely dissociated cortical neurons were prepared and treated with 20 μm25−35 for 24 h. Cell lysates were prepared, cdk5 was immunoprecipitated and its activity was assessed using histone H1 as the substrate (upper panel). The amount of cdk5 in each sample was determined by immunoblot analysis (lower panel). Histone H1 phosphorylation was quantitated with a phosphoimager and normalized to the expression level of cdk5 to account for the slight differences in cdk5 levels between samples. Numbers below the upper panel indicate fold histone H1 phosphorylation relative to control neurons obtained from animals receiving the drug vehicle. (a) Quantitation of the effect of TX-67 administration on Aβ-induced histone H1 phosphorylation. Values are from three animals for each treatment. *, p < 0.01 compared with control neurons from animals receiving the drug vehicle; #, p < 0.01 compared with Aβ-treated neurons from animals receiving the drug vehicle.

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Lee and colleagues originally proposed that MT stabilizing agents may be useful therapeutics for slowing the progression of the neurofibrillary pathology that is one hallmark of advanced AD (Lee et al. 1994). In this report, we provide insight into the molecular mechanism by which taxol protects cortical neurons from toxicity by Aβ peptides. Although taxol stabilized MTs in Aβ25−35 treated cells, it also decreased Aβ-induced calpain activation, p25 production and activation of cdk5/p25 complexes. Collectively, these results suggest that taxol acts upstream of calpain in regulating the cdk5 pathway.

The cdk5/p25 complex as a therapeutic target in AD

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

The induction of tau hyperphosphorylation by cdk5/p25 complexes in cultured cells and transgenic mice suggests that this complex may contribute to the increased amount of hyperphosphorylated tau present in PHFs from AD patients (Dhavan et al. 2001). Increased cdk5 activity may also provide a mechanistic link in the etiology of motor neuron dysfunction in amyotrophic lateral sclerosis (Nguyen et al. 2001; Patzke and Tsai 2002b). Thus, the cdk5/p25 complex is emerging as an attractive pharmacological target in neurodegenerative diseases.

Direct inhibition of cdk5 with kinase inhibitors can prevent Aβ-induced cell death of primary hippocampal neurons (Alvarez et al. 1999) and our results are consistent with these data. However, taxol did not inhibit cdk5 activity directly but decreased kinase activity, presumably by regulating the formation of p25. These data suggest that the pharmacologic regulation of p25 production may provide an alternative therapeutic approach to regulating cdk5 activity. Although a calpain inhibitor would obviously be attractive for preventing this degradation, calpain is critical to regulating cytoskeletal proteins and numerous metabolic functions in cells. This broad spectrum of bioactivity contributes to the considerable lack of specificity and toxicity that current calpain inhibitors exhibit toward cells (Nakagawa and Yuan 2000; Wang 2000; Huang and Wang 2001).

A caveat to the potential of cdk5 as a therapeutic target, with respect to AD at least, hinges upon whether enhanced production of p25 and chronic activation of cdk5 activity is a consistent phenomenon in AD brain. Recent studies from post-mortem brain tissue obtained from AD patients suggest that p25 levels may be elevated in this disease (Patrick et al. 1999, 2001). Consistent with increased p25 expression, pre-frontal cortex from post-mortem brain of AD patients showed a significant increase in cdk5 activity relative to control patients, when corrected for either the level of cdk5 expression or for neuronal loss (Lee et al. 1999). In contrast, others have reported that p25 levels were decreased in post-mortem tissue from AD versus control brain (Taniguchi et al. 2001; Yoo and Lubec 2001). One complicating factor that may contribute to these discrepancies is the effect of the post-mortem interval on induction of an artifactual increase in p25 production via calpain-mediated degradation (Kusakawa et al. 2000; Taniguchi et al. 2001). Thus, a critical assessment using either pharmacologic or genetic manipulation of the cdk5 pathway in animals and examination of its role in the development of neurofibrillary pathology will be required to characterize more fully the role of this pathway in AD.

Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Increased tau phosphorylation by cdk5 is sufficient to directly decrease stabilization of MTs and contribute to Aβ toxicity (Evans et al. 2000). MT-stabilization by taxol, and presumably taxol analogs, is necessary for neuroprotection against Aβ toxicity as 10-deactylbaccatin III does not stabilize MTs and was ineffective at preventing Aβ-induced cell death. Further, MT stabilization is also necessary for the inhibition of the cdk5 pathway by taxol as 10-deacetylbacctin III did not inhibit the activation of cdk5 by Aβ25−35. However, MT stabilization alone was not sufficient to protect neurons against Aβ25−35 toxicity in neurons ectopically expressing p25. Similarly, activation of c-jun N-terminal kinase (JNK) by taxol in breast cancer cells also requires MT binding (Wang et al. 1998) and no clear evidence exists that any effect of taxol on cell signaling is necessarily independent of its MT binding properties at therapeutically relevant concentrations (Blagosklonny and Fojo 1999). Together, these data support the hypothesis that the state-of-stability or dynamic instability of axonal MTs represents a signaling pathway within neurons and that the integrity of MT structure may serve as a sensor of the normal homeostasis in cells. A schematic summary of the role of taxanes in regulating Aβ toxicity is presented in Fig. 9. We propose that taxol may minimize Aβ toxicity through distinct but interrelated contributions: (i) its ability to stabilize MTs in the presence of Aβ and (ii) an inhibition of Aβ-induced calpain activation which minimizes proteolysis of p35 to p25, leading to decreased activation of cdk5/p25 complexes and subsequent tau phosphorylation. Finally, as Aβ induces the loss of intraluminal Ca2+ pools in the endoplasmic reticulum (ER) (Siman et al. 2001), the inhibition of calpain by taxol raises the possibility that MT-stabilization may link to minimizing ER stress. We are currently examining the role of taxol and the cdk5 pathway in regulating the activity of ER-resident proteins such as presenilins and caspase 12; the later is activated by calpain cleavage and is critical to Aβ -induced apoptosis (Nakagawa and Yuan 2000; Yoneda et al. 2001).

image

Figure 9. Taxol inhibition of the cdk5 pathway occurs at the level of calpain activation by Aβ. See text for details. CalpainL-low activity; calpainH- high activity. The R group at the C10 position contains an acetyl moiety in taxol and this group is substituted with succinate in TX-67.

Download figure to PowerPoint

An important consideration in our approach of using taxol and taxol analogs as neuroprotective agents derives from the likelihood that these compounds may affect other aspects of cell signaling (Blagosklonny and Fojo 1999). Taxol is well recognized as an anti-mitotic agent that induces apoptosis in cancer cells by causing cell cycle arrest at the G2/M transition (Jordan et al. 1993; Blagosklonny and Fojo 1999), activating JNK (Wang et al. 1998; Amato et al. 1998) and inducing phosphorylation of bcl-2 (Blagosklonny et al. 1996; Blagosklonny et al. 1997). Additionally, in macrophages and monocytes, taxol exerts lipopolysaccharide-like effects at high concentrations (10–30 μm) (Ding et al. 1990; Manthey et al. 1993), which, although therapeutically unsustainable (Blagosklonny and Fojo 1999), may contribute to its apoptotic actions.

It has been reported recently that 100 nm taxol can also induce apoptosis in cortical neurons through a pathway independent of bcl-2 phosphorylation but still requiring activation of a nuclear pool of JNK and phosphorylation of the c-jun transcription factor (Figueroa-Masot et al. 2001). In contrast, addition of 1–10 μm taxol had no effect on neuronal morphology and presumably the viability of hippocampal neurons obtained from wild type or tau-deficient mice (Rapoport et al. 2002). Indeed, taxol had no significant effect on the activation of caspase 3 and significantly decreased the Aβ-induced activation of this enzymatic marker of apoptosis (Michaelis et al. 1998). Although we have not examined the effect of taxol on neuronal viability under the conditions used by Xia and colleagues (Figueroa-Masot et al. 2001), it is possible that the addition of 10% fetal calf serum to the primary cultures used in these studies may provide very strong survival signals upon which the neurons become dependent. Under these growth conditions, taxol may promote apoptosis by stabilizing MTs and decreasing the activity of phosphoinositide 3-kinase (Figueroa-Masot et al. 2001).

Similar to our results, a recent study also found that taxol did not induce death in neurons isolated from wild type or tau-deficient animals in the absence of Aβ treatment (Rapoport et al. 2002). However, taxol was not protective against Aβ treatment of neurons from wild type animals. Interestingly, neurons from tau-deficient animals were insensitive to Aβ-induced neurite degeneration unless the MTs were first stabilized with taxol. These results have led to the proposal that increasing MT stability may not permit cells to compensate for degenerative cues arising from Aβ treatment (Rapoport et al. 2002). The reason for the underlying difference between the protective effect of taxol against Aβ peptides in our study versus Rapoport et al. (2002) is unclear but both studies support that tau plays a central role in regulating cellular responses to Aβ peptides. Nevertheless, the rather dichotomous effect of taxol on post-mitotic neurons under different growth conditions raises the issue that the in vivo effects of taxol at therapeutically sustainable concentrations (5–200 nm) (Blagosklonny and Fojo 1999) may be influenced by the balance between survival and stress signals that impact on the state-of-stability or dynamic instability of axonal MTs in a given neuronal population.

The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References

Numerous lipophilic drugs, including taxol, do not penetrate the blood–brain barrier due to the presence of a P-glycoprotein efflux system, the multidrug resistant protein (MDR1) (Teraski and Tsuji 1995). We circumvented this problem by using a succinylated taxol analog that is not effluxed by P-glycoprotein present in the capillary endothelial cells that form the blood–brain barrier (Michaelis et al. 2002). Indeed, neurons obtained from TX67 but not taxol treated animals, showed decreased basal cdk5 activity and were resistant to the Aβ-induced activation of this tau kinase. This outcome suggests that, in contrast to the use of direct inhibitors of cdk5 that interact with the ATP binding domain (i.e. roscovitine), taxol analogs may permit an alternative regulation of cdk5 which may allow the enzyme to respond to cellular signals. In this regard, a basal level of cdk5 activity is associated with enhanced neuronal survival (Li et al. 2002).

In summary, our results support the premise that MT-stabilizing agents based upon the taxol backbone can be rationally designed and may have potential usefulness in treating neurofibrillary pathology if they cross the blood–brain barrier, stabilize MTs and attenuate tau kinase activity. If tau phosphorylation by cdk5 is sufficient to destabilize MTs (Evans et al. 2000), taxol analogs or other MT-stabilizing agents that are more permeable to the blood–brain barrier (i.e. epothilones) may minimize MT destabilization and prove beneficial in slowing the formation of NFTs during the progression of AD and other tauopathies.

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. Materials
  5. Cell culture and drug administration
  6. Immunoprecipitation of Cdk5 and in vitro kinase assay
  7. Cell viability measurements
  8. Protein half-life assay
  9. Calpain activity assay
  10. Generation of p25 recombinant adenovirus
  11. Statistical analysis
  12. Results
  13. Taxol protects against cell death induced by Aβ peptides
  14. Taxol inhibits Aβ-induced tau phosphorylation
  15. Taxol blocks Aβ-induced activation of cdk5/p25 complexes
  16. Taxol prevents Aβ-induced changes in the ratio of p35/25
  17. Overexpression of p25 reverses taxol-mediated neuroprotection against Aβ-induced cell death
  18. Systemic administration of a taxol analog mimics the effect of taxol on inhibiting activation of cdk5 by Aβ25−35
  19. Discussion
  20. The cdk5/p25 complex as a therapeutic target in AD
  21. Taxanes and neurodegenerative disease: the conundrum of multiple mechanisms-multiple outcomes
  22. The in vivo potential of MT-stabilizing agents to minimize neurofibrillary pathology
  23. Acknowledgements
  24. References
  • Ahlijanian M. K., Barrezueta N. X., Williams R. D., Jakowski A., Kowsz K. P., McCarthy S., Coskran T., Carlo A., Seymour P. A., Burkhardt J. E., Nelson R. B. and McNeish J. D. (2000) Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5. Proc. Natl Acad. Sci. USA 97, 29102915.
  • Alvarez A., Toro R., Caceres A. and Maccioni R. B. (1999) Inhibition of tau phosphorylating protein kinase cdk5 prevents beta-amyloid-induced neuronal death. FEBS Lett. 459, 421426.
  • Alvarez A., Munoz J. P. and Maccioni R. B. (2001) A Cdk5-p35 stable complex is involved in the beta-amyloid-induced deregulation of Cdk5 activity in hippocampal neurons. Exp. Cell Res. 264, 266274.
  • Amato S. F., Swart J. M., Berg M., Wanebo H. J., Mehta S. R. and Chiles T. C. (1998) Transient stimulation of the c-Jun – NH2-terminal kinase/activator protein 1 pathway and inhibition of extracellular signal-regulated kinase are early effects in paclitaxel-mediated apoptosis in human B lymphoblasts. Cancer Res. 58, 241247.
  • Van Asperen J., Van Tellingen O., Sparreboom A., Schinkel A. H., Borst P., Nooijen W. J. and Beijnen J. H. (1997) Enhanced oral bioavailability of paclitaxel in mice treated with the P-glycoprotein blocker SDZ PSC 833. Br. J. Cancer 76, 11811183.
  • Billingsley M. L. and Kincaid R. L. (1997) Regulated phosphorylation and dephosphorylation of tau protein: effects on microtubule interaction, intracellular trafficking and neurodegeneration. Biochem. J. 323, 577591.
  • Blagosklonny M. V. and Fojo T. (1999) Molecular effects of paclitaxel: myths and reality (a critical review). Int. J. Cancer 83, 151156.
  • Blagosklonny M. V., Schulte T., Nguyen P., Trepel J. and Neckers L. M. (1996) Taxol-induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. Cancer Res. 56, 18511854.
  • Blagosklonny M. V., Giannakakou P., El Deiry W. S., Kingston D. G., Higgs P. I., Neckers L. and Fojo T. (1997) Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death. Cancer Res. 57, 130135.
  • Brouty-Boye D., Kolonias D., Wu C. J., Savaraj N. and Lampidis T. J. (1995) Relationship of multidrug resistance to rhodamine-123 selectivity between carcinoma and normal epithelial cells: taxol and vinblastine modulate drug efflux. Cancer Res. 55, 16331638.
  • Busciglio J., Lorenzo A., Yeh J. and Yanker B. A. (1995) β-Amyloid fibrils induce tau phosphorylation and loss of microtubule binding. Neuron 14, 879888.
  • Cavaletti G., Cavalletti E., Oggioni N., Sottani C., Minoia C., D'Incalci M., Zucchetti M., Marmiroli P. and Tredici G. (2000) Distribution of paclitaxel within the nervous system of the rat after repeated intravenous administration. Neurotoxicology 21, 389393.
  • Dhavan R., Tsai L. H. and Tsai L. H. (2001) A decade of cdk5. Nat. Rev. Mol. Cell Biol. 2, 749759.
  • Ding A. H., Porteu F., Sanchez E. and Nathan C. F. (1990) Shared actions of endotoxin and taxol on TNF receptors and release. Science 248, 370372.
  • Evans D. B., Rank K. B., Bhattacharya K., Thomsen D. R., Gurney M. E. and Sharma S. K. (2000) Tau phosphorylation at serine 396 and serine 404 by human recombinant tau protein kinase II inhibits tau's ability to promote microtubule assembly. J. Biol. Chem. 275, 2497724983.
  • Figueroa-Masot X. A., Hetman M., Higgins M. J., Kokot N. and Xia Z. (2001) Taxol induces apoptosis in cortical neurons by a mechanism independent of Bcl-2 phosphorylation. J. Neurosci. 21, 46574667.
  • Goedert M. (1997) The neurofibrillary pathology of Alzheimer's disease. Neuroscientist 3, 131141.
  • Goedert M., Crowther R. A. and Spillantini M. G. (1998) Tau mutations cause frontotemporal dementias. Neuron 21, 955958.
  • He T. C., Zhou S., Da Costa L. T., Yu J., Kinzler K. W. and Vogelstein B. (1998) A simplified system for generating recombinant adenoviruses. Proc. Natl Acad. Sci. USA 95, 25092514.
  • Heimans J. J., Vermorken J. B., Wolbers J. G., Eeltink C. M., Meijer O. W., Taphoorn M. J. and Beijnen J. H. (1994) Paclitaxel (taxol) concentrations in brain tumor tissue. Ann. Oncol. 5, 951953.
  • Huang Y. and Wang K. K. (2001) The calpain family and human disease. Trends Mol. Med. 7, 355362.
  • Ino H., Ishizuka T., Chiba T. and Tatibana M. (1994) Expression of CDK5 (PSSALRE kinase), a neural cdc2-related protein kinase, in the mature and developing mouse central and peripheral nervous systems. Brain Res. 661, 196206.
  • Jordan M. A., Toso R. J., Thrower D. and Wilson L. (1993) Mechanism of mitotic block and inhibition of cell proliferation by taxol at low concentrations. Proc. Natl Acad. Sci. USA 90, 95529556.
  • Kusakawa G., Saito T., Onuki R., Ishiguro K., Kishimoto T. and Hisanaga S. (2000) Calpain-dependent proteolytic cleavage of the p35 cyclin-dependent kinase 5 activator to p25. J. Biol. Chem. 275, 1716617172.
  • Lee V. M., Daughenbaugh R. and Trojanowski J. Q. (1994) Microtubule stabilizing drugs for the treatment of Alzheimer's disease. Neurobiol. Aging 15, S87S89.
  • Lee V. M., Goedert M. and Trojanowski J. Q. (2001) Neurodegenerative tauopathies. Annu. Rev. Neurosci. 24, 11211159.
  • Lee K. Y., Clark A. W., Rosales J. L., Chapman K., Fung T. and Johnston R. N. (1999) Elevated neuronal Cdc2-like kinase activity in the Alzheimer disease brain. Neurosci. Res. 34, 2129.
  • Lee M. S., Kwon Y. T., Li M., Peng J., Friedlander R. M. and Tsai L. H. (2000) Neurotoxicity induces cleavage of p35 to p25 by calpain. Nature 405, 360364.
  • Liu Y., Ali S. M., Boge T. C., Georg G. I., Victory S., Zygmunt J., Marquez R. T. and Himes R. H. (2002) A systematic SAR study of C10 modified paclitaxel analogues using a combinatorial approach. Comb. Chem. High Throughput Screen 5, 3948.
  • Maccioni R. B., Otth C., Concha I. I. and Munoz J. P. (2001) The protein kinase Cdk5. Structural aspects, roles in neurogenesis and involvement in Alzheimer's pathology. Eur. J. Biochem. 268, 15181527.
  • Manthey C. L., Qureshi N., Stutz P. L. and Vogel S. N. (1993) Lipopolysaccharide antagonists block taxol-induced signaling in murine macrophages. J. Exp. Med. 178, 695702.
  • Michaelis M. L., Walsh J. L., Pal R., Hurlbert M., Hoel G., Bland K., Foye J. and Kwong W. H. (1994) Immunologic localization and kinetic characterization of a Na+/Ca2+ exchanger in neuronal and non-neuronal cells. Brain Res. 661, 104116.
  • Michaelis M. L., Ranciat N., Chen T., Bechtel M. R. R., Hepperle M., Liu Y. and Georg G. (1998) Protection against β-amyloid toxicity in primary neurons by paclitaxel (taxol). J. Neurochem. 70, 16231627.
  • Michaelis M. L., Chen Y., Hill S., Reiff E., Georg G., Kice A. and Audus K. (2002) Amyloid peptide toxicity and microtubule-stabilizing drugs. J. Mol. Neurosci. 19, 101105.
  • Nakagawa T. and Yuan J. (2000) Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis. J. Cell Biol. 150, 887894.
  • Nath R., Davis M., Probert A. W., Kupina N. C., Ren X., Schielke G. P. and Wang K. K. (2000) Processing of cdk5 activator p35 to its truncated form (p25) by calpain in acutely injured neuronal cells. Biochem. Biophys. Res. Commun. 274, 1621.
  • Nguyen M. D., Lariviere R. C. and Julien J. P. (2001) Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions. Neuron 30, 135147.
  • Niethammer M., Smith D. S., Ayala R., Peng J., Ko J., Lee M. S., Morabito M. and Tsai L. H. (2000) NUDEL is a novel Cdk5 substrate that associates with LIS1 and cytoplasmic dynein. Neuron 28, 697711.
  • Nikolic M. and Tsai L. H. (2000) Activity and regulation of p35/Cdk5 kinase complex. Meth Enzymol. 325, 200213.
  • Patrick G. N., Zhou P., Kwon Y. T., Howley P. M. and Tsai L. H. (1998) p35, the neuronal-specific activator of cyclin-dependent kinase 5 (Cdk5) is degraded by the ubiquitin-proteasome pathway. J. Biol. Chem. 273, 2405724064.
  • Patrick G. N., Zukerberg L., De Nikolic M., La M. S., Dikkes P. and Tsai L. H. (1999) Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration. Nature 402, 615622.
  • Patrick G. N., Zukerberg L., De Nikolic M., La M. S., Dikkes P. and Tsai L. H. (2001) reply: p25 protein in neurodegeneration. Nature 411, 764765.
  • Patzke H. and Tsai L. H. (2002a) Calpain-mediated cleavage of the cyclin-dependent kinase 5 activator p39 to p29. J. Biol. Chem. 277, 80548060.
  • Patzke H. and Tsai L. H. (2002b) Cdk5 sinks into ALS. Trends Neurosci. 25, 810.
  • Rapoport M., Dawson H. N., Lester I. B., Vitek M. P. and Ferreira A. (2002) Tau is essential to β-amyloid-induced neurotoxicity. Proc. Natl Acad. Sci. USA 99, 63646369.
  • Scheuner D., Eckman C., Jensen M., Song X., Citron M., Suzuki N., Bird T. D., Hardy J., Hutton M., Kukull W., Larson E., Levy-Lahad E., Viitanen M., Peskind E., Poorkaj P., Schellenberg G., Tanzi R., Wasco W., Lannfelt L., Selkoe D. and Younkin S. (1996) Secreted amyloid beta-protein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease. Nat. Med. 2, 864870.
  • Selkoe D. J. (2001a) Alzheimer's disease: genes, proteins, and therapy. Physiol. Rev. 81, 741766.
  • Selkoe D. J. (2001b) Clearing the brain's amyloid cobwebs. Neuron 32, 177180.
  • Siman R., Flood D. G., Thinakaran G. and Neumar R. W. (2001) Endoplasmic reticulum stress-induced cysteine protease activation in cortical neurons: effect of an Alzheimer's disease-linked presenilin-1 knock-in mutation. J. Biol. Chem. 276, 4473644743.
  • Sobue K., Agarwal-Mawal A., Li W., Sun W., Miura Y. and Paudel H. K. (2000) Interaction of neuronal Cdc2-like protein kinase with microtubule-associated protein tau. J. Biol. Chem. 275, 1667316680.
  • Spillantini M. G., Murrell J. R., Goedert M., Farlow M. R., Klug A. and Ghetti B. (1998) Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc. Natl Acad. Sci. USA 95, 77377741.
  • Taniguchi S., Fujita Y., Hayashi S., Kakita A., Takahashi H., Murayama S., Saido T. C., Hisanaga S., Iwatsubo T. and Hasegawa M. (2001) Calpain-mediated degradation of p35 to p25 in postmortem human and rat brains. FEBS Lett. 489, 4650.
  • Teraski T. and Tsuji A. (1995) Controlling transport and metabolism, in Peptide Based Drug Design (TaylorM. D. and AmidonG. L., eds) , pp. 297316. Amer. Chem. Soc., Washington, DC.
  • Thiel V. E. and Audus K. L. (2001) Nitric oxide and blood–brain barrier integrity. Antioxid. Redox Signal. 3, 273278.
  • Tsai L. H., Delalle I., Caviness V. S. Jr, Chae T. and Harlow E. (1994) p35 is a neural-specific regulatory subunit of cyclin-dependent kinase 5. Nature 371, 419423.
  • Wang K. K. (2000) Calpain and caspase: can you tell the difference? Trends Neurosci. 23, 2026.
  • Wang T. H., Wang H. S., Ichijo H., Giannakakou P., Foster J. S., Fojo T. and Wimalasena J. (1998) Microtubule-interfering agents activate c-Jun N-terminal kinase/stress-activated protein kinase through both Ras and apoptosis signal-regulating kinase pathways. J. Biol. Chem. 273, 49284936.
  • Xie H. and Johnson G. V. (1997) Ceramide selectively decreases tau levels in differentiated PC12 cells through modulation of calpain I. J. Neurochem. 69, 10201030.
  • Yoneda T., Imaizumi K., Oono K., Yui D., Gomi F., Katayama T. and Tohyama M. (2001) Activation of caspase-12, an endoplastic reticulum (ER) resident caspase, through tumor necrosis factor receptor-associated factor 2-dependent mechanism in response to the ER stress. J. Biol. Chem. 276, 1393513940.
  • Yoo B. C. and Lubec G. (2001) p25 protein in neurodegeneration. Nature 411, 763764.
  • Zukerberg L. R., Patrick G. N., Nikolic M., Humbert S., Wu C. L., Lanier L. M., Gertler F. B., Vidal M., Van Etten R. A. and Tsai L. H. (2000) Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth. Neuron 26, 633646.