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Address correspondence to Andréa LeBlanc, The Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, The Sir Mortimer B. Davis Jewish General Hospital, 3755 Côte Ste-Catherine Rd., Montréal, Québec H3T 1E2, Canada. Tel.: (514) 340-8260 Fax: (514) 340-8295. E-mail: andrea.leblanc{at}mcgill.ca
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Abstract |
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Key Words: Alzheimer's disease; amylois ß peptide; p53; Bax; neurotoxicity
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Introduction |
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Results |
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p53 is involved in intracellular Aß142-mediated neurotoxicity
Bax is transcriptionally regulated by p53; therefore, we tested whether p53 activation was involved in intracellular Aß142-mediated neurotoxicity. We chose to use the p53R273H dominant negative (p53DN) mutant because it effectively inhibits p53 transcriptional activation of Bax (Aurelio et al., 2000). Whereas the expression of p53 wild-type (WT) or p53DN does not induce neuronal apoptosis in absence or presence of Aß140, the p53DN, but not the p53WT, effectively inhibits Aß142-mediated neurotoxicity (Fig. 6). The inability of the p53DN to inhibit the toxicity of Bax when expressed from the CMV promoter of a cDNA construct or to inhibit cell death by recombinant active caspase-6 attests to the specificity of the p53DN against Aß142-mediated cell death. Together with the inhibition of Aß142-mediated neurotoxicity using Bax antibodies, these results suggest that Aß142 activates p53 which regulates Bax expression to induce cell death.
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Discussion |
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The in vivo cytotoxicity of intracellular Aß142 is supported by the results of LaFerla and colleagues (1995) who showed that neurofilament L promoter-directed expression of cytosolic Aß142 results in neuronal cell death in mice (LaFerla et al., 1996). Additionally, our experiments show that levels of Aß142 likely to be more physiological can rapidly induce cell death and that this toxicity is restricted to a nonfibrillar form of Aß142 peptide.
The extreme toxicity of intracellular Aß142 raises some important questions. First, can Aß142, which is made through the secretory pathway, access the cytosol? Normally, it should not. However, there are three possible modes of entry of Aß142 into the cytosol. Insoluble Aß42 in the ER could access the cytosol through the ER quality control system where misfolded proteins are reverse translocated to the cytosol, ubiquitinated, and degraded through the proteosome system (Lippincott-Schwartz et al., 1988; Bonifacino et al., 1990; Bonifacino and Lippincott-Schwartz, 1991; Greenfield et al., 1999). In aging, the degradation of the Aß142 could be compromised by a reduction of proteosomal activity thus resulting in cytosolic Aß142 neurotoxicity (Merker et al., 2001). Second, newly synthesized Aß142 could be directed to endosomes/lysosomes either from the trans-Golgi or by endocytosis of secreted peptide. In this situation, the Aß142 would have to be released in the cytosol through breakdown of membrane or passive diffusion of the peptide from endosomes/lysosomes into the cytosol. Interestingly, it has been shown that Aß142, but not Aß140, increases lysosomal membrane permeability possibly resulting in leakage of the Aß142 in the cytosol (Yang et al., 1998). Third, the secreted Aß142 could passively diffuse back into the cytosol through the plasma membrane. Many have reported the neuronal toxicity of the extracellular Aß peptides on primary and cell line systems (for review see Klein et al., 2001). In contrast to these findings, we were unable to find toxicity of the extracellular Aß142 or Aß140 in our cultures of human neurons despite high concentrations of the peptides. However, we have previously shown that the amyloid peptides render neurons vulnerable to a secondary insult (Paradis et al., 1996). Others have also observed this effect in human neuronal cultures (Mattson et al., 1992). Possibly, the secondary stress increases Aß receptors or endocytosis or alters the plasma membrane permeability resulting in the diffusion of the extracellular Aß142 peptide inside the cell. However, this latter possibility is unlikely to be a primary cause of AD, as intracellular Aß142 accumulation precedes senile plaques but nevertheless could contribute to a secondary round of cell death after the deposition of extracellular amyloid.
The selective toxicity of Aß142 compared with Aß140 poses another interesting problem: the extracellular Aß peptide toxicity has been attributed to the fibrillar, protofibrillar, or aggregating properties of Aß (for review see Klein et al., 2001). For intracellular Aß142 toxicity, there exists three possibilities. First, it could be that the extra two amino acids on Aß142 are responsible for inducing toxicity. Second, as has previously been suggested, amyloid fibrils could be the toxic molecules. However, both less toxic Aß140 and extremely toxic Aß142 form significant amounts of fibrils and nonfibrillized Aß142 is still very toxic. Third, the oligomers observed by Western blot analysis in the nonfibrillized and fibrillized Aß142 could be responsible for cell death (Walsh et al., 1999). Furthermore, it remains a possibility that the forms observed by electron microscopy and by Western blot analysis change when microinjected in neurons due to association with certain cytosolic factors (Walsh et al., 1999; Nilsberth et al., 2001). Therefore, much work will be required to elucidate the toxic structure of the Aß142 peptides.
We further show that Aß142 mediates neurotoxicity through the known p53 and Bax cell death pathway. p53 expression increases in the transgenic neurons of cytosolically expressed Aß142 and in AD neurons (LaFerla et al., 1996; de la Monte et al., 1997, 1998; Kitamura et al., 1997; Seidl et al., 1999). In addition, synthetic p53 inhibitors can prevent extracellular Aß mediated toxicity of hippocampal neuron cultures (Culmsee et al., 2001). Extracellular Aß can upregulate Bax expression or require Bax to mediate cytotoxicity (Paradis et al., 1996; Selznick et al., 2000; Culmsee et al., 2001). Furthermore, Bax protein levels increase in AD (MacGibbon et al., 1997; Nagy and Esiri, 1997; Su et al., 1997; Tortosa et al., 1998; Giannakopoulos et al., 1999), although this was not confirmed by all studies (Engidawork et al., 2001). Therefore, the identification of the role of the p53Bax cell death pathway in intracellular Aß142-mediated neurotoxicity reveals additional therapeutic targets that could be used against AD. Because p53 is activated through phosphorylation, the results suggest that Aß142 induces a kinase or inhibits a phosphatase responsible for this phosphorylation. Given the known importance of tau hyperphosphorylation in AD, one wonders whether the activation of p53 could be linked to the phosphorylation of tau through intracellular Aß142 induction of kinase activity.
The mechanism by which p53 is activated by intracellular Aß142 remains to be elucidated. It is interesting to note that Aß142 interacts with ERAB, an intracellular ER and mitochondria-localized member of the alcohol dehydrogenase family (Yan et al., 1997). In transfected Cos cells, ERAB facilitates the toxicity of extracellular Aß142 and APPV717G mutant (Yan et al., 1999). Whether ERAB is involved in cytosolic Aß142 toxicity remains to be determined but since ERAB is localized to mitochondria and ER, it is unlikely to have access to the injected Aß142 unless mitochondrial ERAB is released into the cytosol during apoptosis as are many other mitochondrial factors.
It is well known that Bax activates caspases by promoting the release of mitochondrial cytochrome c, which forms an apoptosome with a number of other factors including caspases (Adrain and Martin, 2001). The caspase-9 has specifically been shown to be activated by cytochrome c and further activate caspase-3. Our result showing that the caspase-6 and -8, but not the caspase-3 inhibitors, prevent Aß142 toxicity is surprising. The lack of involvement of caspase-3 is likely due to the fact that these neurons contain high levels of inhibitor of apoptosis proteins known to inhibit caspase-3 (Zhang and LeBlanc; unpublished data). These results are consistent with our previous observation that caspase-6, but not caspase-3, is activated in serum-deprived primary human neurons and that only recombinant active caspase-6 induces apoptosis of human primary neurons (LeBlanc et al., 1999; Zhang et al., 2000). Because little is known of caspase-6 regulation, much more work will be required to understand how caspase-6 is activated in the presence of intracellular Aß142.
Uncovering the acute human neuronal toxicity of intracellular Aß142 questions the validity of the currently developed therapies against extracellular amyloid in AD. If intracellular Aß142 precedes extracellular amyloid deposits, then anti-amyloid therapies need to be cell permeable. Otherwise, they may have little effect on the prevention or early treatment of AD but could be beneficial to prevent further damage incurred by the extracellular amyloid. However, if the intracellular Aß142 is a consequence of extracellular amyloid deposits, then these therapies would presumably be advantageous to AD patients. Attempts directed toward the inhibition of the secretases responsible for the production of Aß142 should also have a favorable impact on the disease assuming that it is only Aß142 that is the problem in AD and not an underlying general problem with the secretory pathway resulting in the misfolding and cytosolic accumulation of other insoluble proteins. Our study indicates that in addition to the antiamyloid strategies, antineuronal cell death therapies against p53, Bax, or caspases could be extremely valuable in preventing neuronal loss in AD.
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Materials and methods |
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Primers were designed to amplify secreted and cytosolic Aß140 and Aß142 from APP695. These primers amplify the entire Aß sequence with an additional methionine ATG codon at the 5' end and a stop codon at the 3' end to ensure translation. Cytosolic Aß was amplified with the Aß140/42 forward primer 5'-TCA CTC GAG AAT GGA TGC AGA ATT CCG ACA T-3' (contains a built-in 5' XhoI site) and Aß142 reverse primer 5'-ATG GAT CCT TAC GCT ATG ACA ACA CCG AA-3' (has a 3' BamH1 site) or Aß140 reverse primer 5'-TCG ATC CTT AGA CAA CAC CGC CCA CCA TG-3' (has a 3' BamH1 site). Secreted Aß was made by linking the APP signal peptide (SP) to the Aß sequence. The SP was amplified with APP-SP1 forward primer, 5'-TTA CTC GAG ATG CTG CCC GGT TTG GCA-3' (contains a 5' XhoI site) and APP-SP2 reverse primer, 5'-GGA ATT CTG CAT CCA TCG CCC GAG CCG TCC AGG C-3' (contains a 3' EcoR1 site). A ligation between EcoR1- cleaved PCR-amplified Aß coding sequence and the EcoR1-cleaved SP sequence was reamplified with the Aß140/42 forward primer and Aß140 or Aß142 reverse primers. The PCR-amplified SP and Aß sequence were cloned into the prokaryotic pBSKII and eukaryotic episomal pCep4ß vectors through the XhoI/BamHI restriction sites. All clones were restriction mapped and sequenced before use.
Neutralizing Bax antibodies
Monoclonal anti-Bax 6A7 (amino acids 1224; PharMingen) and 2D2 (amino acids 316; Trevigen), polyclonal anti-Bax N-20 (Santa Cruz Biotechnology, Inc.), monoclonal anti-APP 22C11 (Roche), mouse IgG, or rabbit sera were diluted at 50 µg/ml (for 6A7, 2D2, mouse IgG, and 22C11) or 25 µg/ml (for polyclonal anti-Bax and rabbit sera) in PBS before use. A toxicity curve was done to determine these concentrations as the highest nontoxic concentrations that can be injected in neurons.
Cell cultures
Primary cultures of human neurons and astrocytes were prepared from 1117-wk-old fetal brains as described previously (LeBlanc, 1995). The McGill University Institutional Review Board (Montréal, Québec, Canada) has approved this procedure following Canadian Institutes of Health and National Institutes of Health ethical guidelines. Brain tissues were dissociated with 0.25% trypsin (GIBCO BRL) in PBS at 37°C for 15 min. The trypsin was inactivated with 10% decomplemented FBS (HyClone). The dissociated cells were triturated in 0.1 mg/ml DNaseI (GIBCO BRL), filtered successively through 130 µm nylon mesh (Sefar Canada, Inc.) and Falcon 70-µm cell strainers (Becton Dickinson), and centrifuged at 5,000 g for 10 min at 10°C to pellet the cells. The cell pellet was washed once with PBS and once with MEM (GIBCO BRL) in Earle's balanced salt solution containing 0.225% sodium bicarbonate, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1% dextrose, antibiotic Pen-Strep (all from GIBCO BRL), and 5% decomplemented FBS. The cells were plated at a density of 3 x 106 cells/ml on poly-L-lysinecoated ACLARTM (Cat. No., 33C; thickness, 0.5 mm; Allied Chemical) coverslips. Neuron cultures were treated successively three times with 1 mM fluorodeoxyuridine (GIBCO BRL) at feeding, and subsequently every week to prevent proliferation of dividing cells. In general, the neurons attach to the coverslips within 24 h and develop dense neuritic networks within 3 d. The cultures contain 9095% neurons and 510% astrocytes (LeBlanc, 1995). Microinjections or treatments were performed 10 d after plating for neurons and astrocytes.
Human neuroblastoma M17 cells were obtained from Dr. J. Biedler (Cellular Biochemistry and Genetics, New York, NY) and cultured on ACLARTM coverslips at 106 cells/ml in OPTI-MEM (GIBCO BRL) containing 5% FBS. Human teratocarcinoma NT2 (Stratagene) and neuroblastoma La-N-1 cells, a gift from Dr. L. Culp (Case Western Reserve University, Cleveland, OH), were cultured on ACLARTM coverslips at 106 cells/ml in DME (GIBCO BRL) containing 10% FBS. BHK cells, a gift from William Bowers (University of Rochester, Rochester, NY), and the mice NIH3T3 fibroblasts, a gift from Dr. Stephane Richard (McGill University, Montréal, Québec, Canada) were grown in DME and 10% FBS.
Aß peptides
Initially (for Fig. 1), Aß peptides (Bachem) were dissolved in sterile distilled water at 25 µM and incubated at 37°C for 5 d. The peptides' stock solutions were frozen and diluted in PBS immediately before microinjection. Thereafter, nonfibrillar Aß peptides (American Peptide Co.) were disaggregated at 25 µM in 5 mM Tris buffer pH 7.4, an aliquot diluted to 0.25 µM and immediately frozen at -20°C in aliquots of 50 µl. The remaining 25-µM solution was incubated at 37°C in Eppendorf tubes with continuous mixing by inversion to fibrillize the peptides. After incubation, the samples were removed, vortexed, sonicated twice for 1 min in a bath type sonicator (ELMA GmbH & Co. KG), and frozen at -20°C in 50 µl aliquots. Each aliquot was used once to avoid possible effects of freeze and thaw cycles.
Electron microscopy
A 3-µl aliquot of Aß peptide was placed on freshly cleaved mica plates (BioForce Laboratory, Inc.). The specimens were air dried and subsequently transferred to a Balzers High-Vacuum Freeze-Etch Unit (model 301) under a 1.3 x 10-4 Pa vacuum. The specimens were shadowed with platinum (BAL-TEC EM-Technology and Application, NH) at a 30° angle and coated with a carbon film platinum (BAL-TEC EM-Technology and Application). The replicas were detached from the mica by flotation in deionized water and transferred onto a 300-mesh grid (Canemco, Inc.). The grids were examined with a Joel 200FX transmission electron microscope (Joel) at 21,000x magnification.
Western blot analysis of Aß peptides
Nonfibrillar and fibrillar forms of Aß140 and Aß142 (5 µg) were added to sample buffer and electrophoresed on a triple layer (4%, 10%, 16.5%) Tris-Tricine gel at 50 V for 1 h followed by 70 V for 16 h (Schagger and Von Jagow, 1987). The proteins were transferred to Immobilon-P PVDF Membrane (Millipore) at 200 milliamps for 2 h. The membrane was blocked by 5% nonfat milk in Tris buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h, incubated with a 1/100 dilution of the anti-Aß117 antibody 6E10 (Signet) and detected by chemiluminescence.
Microinjection
Thin-walled Borosilicate glass capillaries (OD 1.0 mm, ID 0.5 mm) with microfilament (MTW100F-4; World Precision Instrument) were pulled with a Flaming/Brown Micropipette Puller (P-87; Sutter) to obtain injection needles with a tip diameter of 0.5 µm. Microinjections were performed in the cytosol of each cell using the Eppendorf Microinjector 5246 and Burleigh Micromanipulator MIS-5000. Human neurons were injected with 25 pl/shot at an injection pressure of 100 hPa, a compensation pressure of 50 hPa, and an injection time of 0.1 s. Human astrocytes, M17, NT2, La-N-1, BHK, and NIH 3T3 cells were injected with 8 pl/shot at an injection pressure of 50 hPa, a compensation pressure of 30 hPa and an injection time of 0.1 s. The diluted peptides were injected at the indicated concentrations with 100 µg/ml DTR (MW: 3000; Molecular Probes) as a fluorescent marker to recognize the injected cells. Approximately 90% neurons and NT2 cells, and 50% astrocytes, M17, La-N-1, BHK, and NIH 3T3 cells survive the injections for at least 16 d.
Measurement of neuronal apoptosis
Cells were fixed in freshly prepared 4% paraformaldehyde/4% sucrose for 20 min at room temperature and permeabilized in 0.1% Triton X-100, 0.1% sodium citrate on ice for 2 min. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was performed using the In situ Cell Death Detection Kit I as described by the manufacturer (Roche). The percentage of cell death was determined as the ratio of the number of DTR-TUNELdouble positive cells over the total number of DTR-positive cells.
Hoechst staining was used to recognize cell nuclei and detect apoptotic nuclear condensation and fragmentation. Hoechst dye (Intergen) was dissolved in sterile distilled water at 200 µg/ml and diluted 500 times in PBS immediately before staining. After the incubation for TUNEL staining, cells were washed three times for 10 min each in PBS, treated with the diluted Hoechst dye for 15 min at room temperature (in the dark), washed three times for 10 min each in PBS, washed once in water for 5 min, and mounted with ImmunonTM mounting medium (Shandon) onto glass slides to be observed under the fluorescence microscope.
Treatment with caspase inhibitors, cycloheximide, and actinomycin D
Caspase pan inhibitor, Z-Valine-Alanine-Aspartic acid-fluoromethylketone (Z-VAD-fmk) (Biomol), caspase-1 inhibitor, Z-Tyrosine-Valine-Alanine-Aspartic acid-fmk (Z-YVAD-fmk), caspase-6 inhibitor, Z-Valine-Glutamic acid-Isoleucine-Aspartic acid-fmk (Z-VEID-fmk), caspase-3 inhibitor, Z-Aspartic acid-Glutamic acid-Valine-Aspartic acid-fmk (Z-DEVD-fmk), and caspase-8 inhibitor, Z-Isoleucine-Glutamic acid-Threomine-Aspartic acid-fmk (Z-IETD-fmk) (Sigma-Aldrich), were dissolved in 100% DMSO (Sigma-Aldrich) at 20 mM and were diluted at 5 µM into culture media immediately before use. Stock solutions of 5 mg/ml cycloheximide (Sigma-Aldrich) and 200 µM actinomycin D (Sigma-Aldrich) were made in sterile distilled water and diluted to 5 µg/ml for cycloheximide and 5 µM for actinomycin D in the culture media immediately before use. The media was changed every 48 h.
Statistical evaluation
One-way or two-way analyses of variance (ANOVAs) with post hoc tests (Statview 5.01) determined the statistical significance of the difference between treatments. The Dunnett's test was used when comparing several groups with one certain group (control), for example, comparison between different treatment groups vs. untreated group. The Sheffé's test was applied when comparing between every other group, for example, comparison between each treatment group. A P value of <0.05 was taken as the criteria for statistical significance.
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Footnotes |
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Acknowledgments |
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Submitted: 24 October 2001
Revised: 18 December 2001
Accepted: 21 December 2001
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References |
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