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J. Biol. Chem., Vol. 281, Issue 1, 229-240, January 6, 2006

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Microtubule-associated Protein MAP1A, MAP1B, and MAP2 Proteolysis during Soluble Amyloid -Peptide-induced Neuronal Apoptosis

SYNERGISTIC INVOLVEMENT OF CALPAIN AND CASPASE-3^*

From the Lipidomix, JeuneÉquipe 2482, Laboratoire Médecine et Thérapeutique Moléculaire, Institut National Polytechnique de Lorraine, 54500 Vandoeuvre-lès-Nancy, France

Received for publication, July 7, 2005 , and in revised form, October 12, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
A growing body of evidence supports the notion that soluble oligomeric forms of the amyloid -peptide (A) may be the proximate effectors of neuronal injuries and death in the early stages of Alzheimer disease. However, the molecular mechanisms associated with neuronal apoptosis induced by soluble A remain to be elucidated. We recently demonstrated the involvement of an early reactive oxygen species-dependent perturbation of the microtubule network (Sponne, I., Fifre, A., Drouet, B., Klein, C., Koziel, V., Pincon-Raymond, M., Olivier, J.-L., Chambaz, J., and Pillot, T. (2003) J. Biol. Chem. 278, 3437–3445). Because microtubule-associated proteins (MAPs) are responsible for the polymerization, stabilization, and dynamics of the microtubule network, we investigated whether MAPs might represent the intracellular targets that would enable us to explain the microtubule perturbation involved in soluble A-mediated neuronal apoptosis. The data presented here show that soluble A oligomers induce a time-dependent degradation of MAP1A, MAP1B, and MAP2 involving a perturbation of Ca²⁺ homeostasis with subsequent calpain activation that, on its own, is sufficient to induce the proteolysis of isoforms MAP2a, MAP2b, and MAP2c. In contrast, MAP1A and MAP1B sequential proteolysis results from the A-mediated activation of caspase-3 and calpain. The prevention of MAP1A, MAP1B, and MAP2 proteolysis by antioxidants highlights the early reactive oxygen species generation in the perturbation of the microtubule network induced by soluble A. These data clearly demonstrate the impact of cytoskeletal perturbations on soluble A-mediated cell death and support the notion of microtubule-stabilizing agents as effective Alzheimer disease drugs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Microtubules are polymers of - and -tubulin dimers that mediate many functions in neurons, including organelle transport and cell shape establishment and maintenance as well as axonal elongation and growth cone steering in neurons. The polymerization, stabilization, and dynamic properties of microtubules are influenced by interactions with microtubule-associated proteins (MAPs).³ Members of this protein family are classified by size: high molecular mass proteins (MAP1A, MAP1B, MAP2a, and MAP2b) and intermediate molecular mass proteins (MAP2c, MAP2d, and tau) (1–3). Numerous studies have shown that neuronal apoptotic cell death involves alterations of the microtubule network consequent to calpain and effector caspase activation (4, 5). These calcium-dependent proteases are responsible for the degradation and turnover of a broad repertoire of MAP substrates, some of which they share, such as II-spectrin and tau, and some of which are specific, as is the case of calpain-degraded MAP1B and MAP2.

Intraneuronal neurofibrillary tangles (NFTs) are one of the histopathological hallmarks in brains of patients diagnosed with Alzheimer disease (AD), a progressive dementia that manifests primarily as a profound inability to form new memories. These NFTs are composed of hyperphosphorylated tau organized into paired helical filaments (PHFs) (6). In addition, AD is also associated with the presence of extracellular senile plaques (7) formed as a consequence of the gradual accumulation and aggregation of the amyloid -peptide (A) into fibrils (8). Despite evidence that A represents a key factor in AD (9), the nature of the toxic form of A involved early in AD pathology remains to be determined. The issue of which pool (soluble or aggregated) of A in brain is more deleterious in the early stages of AD is still controversial (10). However, clinicopathological hallmarks of AD correlate far better with the soluble pool of A (11, 12). Moreover, several studies in transgenic mice have indicated that specific cognitive deficits, neurodegeneration, and synaptic loss might occur before any histologically detectable formation of senile plaques (13, 14). So, in reports from our group (15–18) and others (19, 20), attention has been paid to the soluble oligomeric forms of A as the principal mediators of neurodegeneration in the early stages of AD development (10, 19–23).

Increasing evidence suggests that the selective neuronal cell death in AD involves activation of caspases, which critically participate in apoptosis through the initiation of intracellular pathways and the proteolytic cleavage of several target proteins responsible for the typical morphological changes of apoptosis (24, 25). Indeed, some experimental studies report the presence of activated forms of caspases and the accumulation of caspase-derived products in post-mortem AD brain tissue, similar to what has been observed in in vitro models of A neurotoxicity (26–28). In addition, there is considerable evidence that an increased activity of calpain associated with impaired calcium homeostasis (29, 30) may be involved in AD development (31, 32). A region-specific increase in calpain activity has been demonstrated in the early stages of AD development, even before synaptic loss, neuronal degeneration, and tau hyperphosphorylation and aggregation (33). Moreover, in AD brain, the active form of calpain co-localizes with NFTs, senile plaques, and neuropil threads (34), just like MAP1B and MAP2 (35, 36), which supports the capacity of MAP2 to form structures resembling PHFs via its microtubule-binding region (37). Moreover, MAP1 and MAP2 or their proteolytic products have been suggested to act as effectors of cell death in AD (1, 38). These observations strongly suggest the early involvement of caspases and calpains in the cytoskeletal disorganization and degeneration of neurons in AD (39).

We recently demonstrated that soluble A oligomers induce neuronal apoptosis by biphasic modification of plasma membrane fluidity, leading to perturbation of the microtubule network that is dependent on the induction of early oxidative stress (17). We therefore tested the hypothesis that MAP modifications could be involved in the microtubule perturbation induced by soluble A oligomers. The data presented here establish for the first time that calpain- and/or caspase-3-dependent proteolysis of MAP1A, MAP1B, and MAP2 could be a critical event involved in the microtubule disorganization and subsequent apoptosis induced by soluble A in cortical neurons.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—A-(1–40), A-(1–42), A-(40–1), caspase substrates (Ac-DEVD-7-amido-4-methylcoumarin (AMC) and Ac-LEHD-AMC), and inhibitor peptides (benzyloxycarbonyl-VAD-fluoromethyl ketone (fmk), Ac-DEVD-fmk, and Ac-LEHD-chloromethyl ketone) were purchased from Bachem. A-(1–40) and A-(1–42) used in this study are synthetic peptides identical to the most predominant A forms found in AD brains. Indeed, A is a heterogeneous proteolytic fragment derived from sequential cleavage of the amyloid precursor protein by a variety of enzymes classically termed "secretases." Initial cleavage of the amyloid precursor protein by -secretase is followed by -secretase processing to release either the predominant 40-amino acid-long species (referred to here as A-(1–40)) or the slightly larger 42-amino acid-long variant (referred to here as A-(1–42)) according to the cleavage site along the amyloid precursor protein primary sequence. A-(40–1) is a synthetic peptide with the same amino acid composition and the reverse sequence of A-(1–40). A-(40–1) is widely used as a non-neurotoxic negative control. The LIVE/DEAD viability/cytotoxicity kit (catalog no. L-3224) and the calcium indicator Fluo-3/AM (catalog no. F-1242) were purchased from Molecular Probes. The calpain substrate (succinyl-LY-AMC) and inhibitors (MDL28170, N-acetyl-Leu-Leu-norleucinal, and N-acetyl-Leu-Leu-Met) were purchased from Calbiochem. All polyclonal antibodies were obtained from Santa Cruz Biotechnology, Inc., except mouse anti--tubulin monoclonal antibody (MAB380, Chemicon International, Inc.) used in immunofluorescence studies and Alexa Fluor 488-conjugated donkey anti-goat antibody (Molecular Probes catalog no. A-11055). Unless otherwise indicated, materials used for cell culture were obtained from Invitrogen. All other chemicals were of high purity and were from Sigma.

Neuronal Cell Culture Studies—Primary cultures of cerebral cortical neurons were prepared from embryonic day 15 C57BL/6J mouse fetuses as described previously (16). Briefly, dissociated cells were plated at 10⁵ cells/cm² in plastic dishes or on glass coverslips precoated overnight with poly-DL-ornithine (1.5 or 15 µg/ml, respectively). The cells were cultured in chemically defined Dulbecco's modified Eagle's medium/nutrient mixture F-12 serum-free medium and supplemented with 500 nM insulin, 60 µM putrescine, 30 nM sodium selenite, 100 µM transferrin, 100 nM progesterone, and 0.1% (w/v) ovalbumin. Cultures were maintained at 35 °C in a humidified 6% CO₂ atmosphere. After 6–7 days in vitro, the cell population was determined to be at least 95% neuronal by immunostaining as described previously (16). Cortical neurons were treated at 6–7 days in vitro for the indicated times with soluble oligomers of A-(1–40) prepared as described (16). Alternatively, neurons were preincubated with different inhibitors or Ca²⁺-chelating agent 2 h before and throughout the treatment with soluble A oligomers.

Neuronal Viability and Apoptosis—Cell viability was initially determined by morphological observation. A neurotoxicity was also assessed quantitatively by monitoring the mitochondrial reduction activity using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously (16). The simultaneous determination of live and dead cells was achieved with the LIVE/DEAD viability/cytotoxicity kit according to the manufacturer's instructions.

To monitor apoptosis, cell nuclei were visualized using 4',6-diamidino-2-phenylindole (DAPI). The neuronal cells (grown on glass coverslips) were washed with phosphate-buffered saline (PBS), fixed in PBS containing 4% paraformaldehyde for 30 min at room temperature, incubated for 10 min at room temperature with DAPI (0.1 µg/ml), washed with PBS, mounted in Fluoprep (bioMérieux S. A.), and visualized using a Leitz Aristoplan microscope equipped for fluorescence with a Fluotar x40/1.3 objective. Photographs were taken using a Nikon DXM1200 digital camera. To evaluate the percentage of apoptotic cells, five independent fields of the microscope were counted (200 cells) in three separate experiments, with three determinations in each experiment.

Measurement of Caspase-like Proteolytic Activities—The caspase activities were measured by cleavage of the substrates Ac-DEVD-AMC and Ac-LEHD-AMC as described previously (17). Briefly, at the indicated time points after A treatment, the cells were washed three times with ice-cold PBS and incubated for 20 min on ice in buffer containing 25 mM Hepes (pH 7.5), 1% (v/v) Triton X-100, 5 mM EDTA, 1 mM EGTA, 5 mM MgCl₂, 5 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml each pepstatin and leupeptin, and 5 µg/ml aprotinin. After homogenization, the cells collected were then lysed by three cycles of freezing/thawing and centrifuged at 4 °C for 10 min at 10,000 x g, and the protein concentration was assayed using the BCA protein assay kit (Pierce). Fifty µg of proteins were incubated for 2 h with substrates (100 µM) initially dissolved at 10 mM in Me₂SO. The cleavage of the caspase substrates was monitored by fluorescence emission at 460 nm after excitation at 360 nm using a Fluostar microplate reader (BMG LABTECH GmbH).

Measurement of Calpain-like Proteolytic Activity—The µ/m-calpain activity was measured by cleavage of the cyclic fluorogenic substrate succinyl-Leu-Tyr-AMC. Briefly, at the indicated time points following peptide treatment, cortical neurons were incubated for 1 h at 35°C in a humidified 6% CO₂ atmosphere with 50 µM succinyl-LY-AMC. (The stock solution was 50 mM in Me₂SO.) Following this, cells were washed with Hanks' balanced saline solution, and fluorescence was directly recorded on culture dishes using the Fluostar microplate reader with 360-nm excitation and 460-nm emission filters.

Western Blot Analysis—Primary cortical neurons cultured under different conditions were washed with ice-cold PBS. Cells were then solubilized in 25 mM Tris-HCl (pH 7.4) lysis buffer containing 150 mM NaCl, 1 mM EDTA, 1% (w/v) sodium deoxycholate, 1% (v/v) Nonidet P-40, 0.1% (w/v) SDS, and protease inhibitors (Complete, Roche Applied Science). After homogenization, the cells collected were lysed by three cycles of freezing/thawing and finally centrifuged at 4 °C for 10 min at 10,000 x g. The protein concentration in the supernatant was determined using the BCA protein assay kit. Samples (10 µg) were mixed with an equal volume of 2x Laemmli buffer, denatured by heating the mixture for 5 min at 100 °C, and then resolved by 6% SDS-PAGE. The proteins separated were transferred using a Mini Trans-Blot cell onto polyvinylidene difluoride membranes (Immobilon-P, Millipore Corp.), and the membranes were blocked for 2 h with Tris-buffered saline (pH 7.4) and 0.1% (v/v) Tween 20 (TBST buffer) containing 5% (w/v) bovine serum albumin. The polyvinylidene difluoride membranes were then incubated overnight at 4 °C with primary antibodies diluted in TBST buffer containing 5% bovine serum albumin as follows: goat polyclonal antibodies against MAP1A (Santa Cruz Biotechnology catalog no. sc-8969; 1:500 dilution), MAP1B (catalog no. sc-8970; 1:500 dilution), the four isoforms MAP2a/MAP2b/MAP2c/MAP2d (catalog no. sc-12012; 1:500 dilution), II-spectrin (catalog no. sc-7465; 1:500 dilution), the calpain regulatory subunit (catalog no. sc-7528; 1:500 dilution), and CPP32 (caspase-3; catalog no. sc-1225; 1:500 dilution) and rabbit polyclonal antibody against -tubulin (catalog no. sc-12012; 1:1000 dilution). After being washed with TBST buffer, the membranes were incubated for 1 h at room temperature with the corresponding horseradish peroxidase-conjugated preadsorbed secondary antibodies (catalog numbers sc-2054, sc-2055, and sc-2056; 1:5000 dilution). Subsequently, immunoreactive proteins were visualized using the enhanced chemiluminescence protocol (ECL kit, Amersham Biosciences). Quantity One software, associated with the VersaDoc imaging system (Model 1000, Bio-Rad), was used to quantify signals.

View larger version (41K): [in this window] [in a new window]   FIGURE 1. Time- and dose-dependent neuronal apoptosis induced by soluble A oligomers. Primary cortical neurons were cultured for 24 h in the absence or presence of 5 µM soluble A oligomers. Neuronal apoptosis is illustrated here with representative microscopic fields of phase-contrast micrographs (A), of the two-color fluorescence cell viability assay (LIVE/DEAD viability/cytotoxicity kit) (B), and of cell nuclei staining with DAPI (C). A neurotoxicity was next determined by monitoring the mitochondrial reduction activity using the MTT assay (D). Cortical neurons were exposed to increasing concentrations of soluble A (0.05–5 µM) for 24 or 48 h. Data are the means ± S.E. of three different experiments, with four determinations in each experiment, and are normalized to the effect of vehicle (designated as 100%). Differences between control and treated groups were analyzed using Student's t test. ***, p < 0.001.

For cytosolic Ca²⁺ imaging, the neuronal cells were grown on glass coverslips and subjected to the same protocol as described above. Cells were washed with PBS, fixed in PBS containing 4% paraformaldehyde for 30 min at room temperature, washed again with PBS, mounted in Fluoprep, and visualized under fluorescence microscopy.

Immunofluorescence Studies—Following treatment, neurons were fixed in PBS containing 4% (w/v) paraformaldehyde for 30 min at room temperature. The cells were permeabilized with PBS containing 3% bovine serum albumin and 0.1% (v/v) Triton X-100 for 30 min at room temperature and then incubated for 1 h at room temperature under constant shaking with monoclonal anti--tubulin antibody (1:500 dilution) or with goat polyclonal antibody against MAP1A (1:500 dilution), MAP1B (1:500 dilution), or MAP2 (1:500 dilution). After several washes with PBS, the cells were incubated for 1 h with TRITC-conjugated donkey anti-mouse IgG (Santa Cruz Biotechnology catalog number sc-2300; 1:300 dilution) or Alexa Fluor 488-conjugated donkey anti-goat antibody (1:1000 dilution), washed with PBS, labeled with DAPI as described above, and mounted in Fluoprep. The microtubules were visualized by fluorescence microscopy as described above.

Electron Microscopy—Cortical neurons were fixed for 2 h at 4°C in 2.5% (w/v) glutaraldehyde and 0.5% (w/v) tannic acid in 0.1 M cacodylate buffer (pH 7.4). The neurons were then post-fixed for 2 h at 4 °C in 2% (w/v) osmic acid in phosphate buffer. After being dehydrated in a graded alcohol series, the samples were embedded in Epon resin (Poly/Bed 812, Polysciences, Inc.), and ultrathin sections (70 nm) were obtained using a Reichert Ultracut E ultramicrotome. Thin sections were counterstained with uranyl acetate and lead citrate prior to examination with a Jeol 100CX microscope.

Statistical Analyses—StatView computer software was used for statistical analysis. Data are from three separate experiments, with four determinations in each experiment, and are normalized to the effect of vehicle (designated as 100%). Values are expressed as the means ± S.E. Differences between control and treated groups were analyzed using Student's t test. Multiple pairwise comparisons among the groups of data were performed using analysis of variance, followed by Scheffe's post hoc test. Statistical differences were determined at p < 0.05. The Western blot analysis results and fluorescent images shown are representative of at least three separate experiments.

View larger version (30K): [in this window] [in a new window]   FIGURE 2. Increase in cytosolic Ca2+ levels induced by soluble A oligomers. Primary cortical neurons were cultured in the absence or presence of 5 µM A for 12 h or 5 µM ionomycin for 5 min. Cytosolic Ca2+ levels are visualized here by representative microscopic fields of photomicrographs of Fluo-3/AM-stained neurons (A). The perturbation of Ca2+ homeostasis was next quantified using cortical neurons incubated in the absence or presence of 5 µM A for 3, 6, or 12 h (B). The influence of Ca2+-chelating agent was investigated by incubating cortical neurons with 1 mM EGTA before and throughout the treatment with soluble A. No significant differences were found between control cells and cells treated with EGTA only (data not shown). Data are the means ± S.E. of three different experiments, with four determinations in each experiment, and are normalized to the effect of vehicle (designated as 100%). **, p < 0.01; ***, p < 0.001. Statistical differences among the subgroups for each condition were performed by analysis of variance, followed by Scheffe's post hoc test. #, p < 0.05. No significant difference was found between EGTA-treated and control cells.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Soluble Oligomers of A Induce Perturbation of Cellular Ca²⁺ Homeostasis—Cytosolic Ca²⁺ levels were first visualized using the fluorescent probe Fluo-3/AM. Labeling of control cells revealed weak cytoplasmic labeling, whereas cortical neurons exposed to 5 µM A for 12 h exhibited increased fluorescence intensity, reflecting enhanced intracellular Ca²⁺ levels (Fig. 2A). As a positive control, a dramatic rise in cytosolic Ca²⁺ levels was noted after a 5-min treatment of cortical neurons with the Ca²⁺ ionophore ionomycin at 5 µM (Fig. 2A). The cytosolic Ca²⁺ content of cortical neurons was then quantified after treatment with 5 µM A for increasing incubation times. The results revealed a time-dependent elevation of the cytosolic Ca²⁺ concentration (17.3 ± 2.7, 45.7 ± 3.5, and 77.5 ± 3.8% relative to controls at 3, 6, and 12 h, respectively; p < 0.001). Interestingly, A-induced cytosolic Ca²⁺ influx was almost completely inhibited by addition of the Ca²⁺-chelating agent EGTA to the culture medium (increase of 77.5 ± 3.8% compared with 19.3 ± 4.1% relative to controls at 12 h; p < 0.05) (Fig. 2B). The positive control of the deregulation of Ca²⁺ homeostasis induced by 5 µM ionomycin for 5 min caused an increase of 610 ± 35% (p < 0.001) (data not shown). It is interesting to note that nifedipine, an inhibitor of cell membrane L-type Ca²⁺ channels, protected cortical neurons from soluble A-mediated apoptosis.⁴ Altogether, these data suggest that a perturbation of cellular Ca²⁺ homeostasis is involved in the soluble A-mediated neuronal death.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Calpain, caspase-9, and caspase-3 activation induced by soluble A oligomers. Cortical neurons were incubated in the absence or presence of 5 µM A for 12 h. Calpain, caspase-9, and caspase-3 activation was first monitored by measuring the proteolytic cleavage of the µ/m-calpain, caspase-9, and caspase-3 fluorogenic substrates (succinyl (Suc)-LY-AMC, Ac-LEHD-AMC, and Ac-DEVD-AMC, respectively) (A). Data are the means ± S.E. of three different experiments, with four determinations in each experiment, and are normalized to the effect of vehicle (designated as 100%). Differences between control and treated groups were analyzed using Student's t test. ***, p < 0.001. Calpain and caspase-3 activation was next investigated by immunoblot analysis of II-spectrin breakdown product (SBDP) appearance, calpain regulatory subunit autolysis, and procaspase-3 processing of cortical neurons incubated in the absence (control (Ctl)) or presence of 5 µM A for 12 h (B and C). Blots were stripped and reprobed with goat anti--tubulin polyclonal antibody.

View larger version (131K): [in this window] [in a new window]   FIGURE 4. Early cytoskeletal perturbations induced by soluble A oligomers. Cortical neurons were cultured in the absence or presence of 5 µM A for 6 h. The microtubule network was visualized by immunofluorescence using anti--tubulin (A) or anti--tubulin (B) monoclonal antibody and by electron microscopy (C). The total magnification for all transmission electron micrographs was x20,000. Scale bars = 0.5 µm.

View larger version (70K): [in this window] [in a new window]   FIGURE 5. Time-dependent degradation of microtubule-associated proteins MAP1A and MAP1B and three isoforms of MAP2 induced by soluble A oligomers. The degradation of MAP1A, MAP1B, and three isoforms of MAP2 was first visualized by representative microscopic fields of immunofluorescence (A). Cortical neurons were cultured for 12 h in the absence or presence of 5 µM A. The observed degradation was next monitored by immunoblot analysis of MAP1A, MAP1B, and MAP2 isoform levels in primary cortical neurons incubated in the absence (control (Ctl)) or presence of 5 µM A for 12 and 24 h (B). Blots were stripped and reprobed with goat anti--tubulin polyclonal antibody. Apparent molecular mass markers are indicated on the left.

View larger version (42K): [in this window] [in a new window]   FIGURE 6. Specificity of the degradation of MAP1A, MAP1B, and three MAP2 isoforms involved in neuronal apoptosis induced by soluble A oligomers. The degradation of MAP1A, MAP1B, and three MAP2 isoforms induced by the two most common forms of the A peptide and the reverse A peptide was studied by immunoblot analysis of cortical neurons incubated in the absence or presence of A-(1–40), A-(1–42), or A-(40–1) at 5 µM for 24 h. Alternatively, we also monitored the effects of the microtubule-destabilizing agent nocodazole incubated for 1 h at 0.5 µM. Blots were stripped and reprobed with goat anti--tubulin polyclonal antibody.

Calpain and/or Caspase-3 Mediates Soluble A-dependent Proteolysis of MAP1A, MAP1B, and MAP2—We further tested the hypothesis that calpain and/or caspase-3 might be involved in the soluble A-induced degradation of MAP1A, MAP1B, and three MAP2 isoforms (MAP2a, MAP2b, and MAP2c) by monitoring the sensitivity of their degradation to different specific and nonspecific inhibitors. Immunoblot analysis showed that 1 mM EGTA as well as 10 µM MDL28170 (two inhibitors of calpain activity) prevented the degradation of MAP1A, MAP1B, MAP2a, MAP2b, and MAP2c induced by the treatment of neurons with 5 µM A for 24 h (Fig. 7, A and B). In the case of MAP1A and MAP1B, the triplet of degradation products at 75 kDa disappeared completely without fully recovering the amount of native proteins compared with untreated cells. Indeed, inhibition of calpain by EGTA or MDL28170 resulted in the production of an intermediate proteolytic fragment of 250 kDa (Fig. 7A) that probably resulted from the cleavage of MAP1A and MAP1B by other protease(s). Interestingly, the formation of this intermediate cleavage fragment was completely inhibited in the presence of DEVD-fmk, an irreversible specific inhibitor of caspase-3. The treatment of neurons with 100 µM DEVD-fmk prevented MAP1A and MAP1B from being degraded, with a nearly complete recovery of the quantity of native proteins (Fig. 7A). However, the final proteolytic products were still faintly present, probably due to incomplete inhibition of caspase-3, associated with a preserved calpain activity (Fig. 7A). Finally, when calpain and caspase-3 inhibitors were used simultaneously, MAP1A and MAP1B degradation was almost completely inhibited, with the combined effects being greater than the individual effect of either MDL28170 or DEVD-fmk. This is further illustrated by the full recovery of the quantity of native proteins and the absence of intermediate and final proteolytic fragments. Similarly, having demonstrated that calpain and caspase-3 are responsible for MAP1A and MAP1B proteolysis, we also examined the involvement of these proteases in the degradation of the three MAP2 isoforms upon soluble A treatment. The presence of EGTA and MDL28170 significantly abolished the proteolysis of MAP2a, MAP2b, and MAP2c (Fig. 7B). In contrast, the specific inhibition of caspase-3 by 100 µM DEVD-fmk had no effect on the A-induced degradation of MAP2 (data not shown). Interestingly, exposure of cortical neurons for 24 h to the Ca²⁺ ionophore ionomycin (0.5 µM), which activates calpain, resulted in the degradation of the three MAP2 isoforms, similar to cells incubated with 5 µM A (Fig. 7B). This effect of ionomycin on MAP2 was inhibited by the calpain-specific inhibitor MDL28170 (Fig. 7B). Similar results were obtained using another family of calpain inhibitors (N-protected tripeptide aldehyde) such as calpain inhibitors I and II (N-acetyl-Leu-Leu-norleucinal and N-acetyl-Leu-Leu-Met, respectively) instead of MDL28170 and a pan-caspase inhibitor (benzyloxycarbonyl-VAD-fmk) instead of the caspase-3 inhibitor (data not shown).

Soluble A-induced Proteolysis of MAP1A, MAP1B, and MAP2 Is Oxidative Stress-dependent—In a previous report, we demonstrated that soluble A oligomers induce neuronal apoptosis via an early increase in the formation of reactive oxygen species preceding the A-mediated microtubule perturbation (17). To characterize the involvement of oxidative stress in A-induced MAP degradation, cortical neurons were preincubated with Trolox, a cell-permeable and water-soluble derivative of -tocopherol. Preincubation of neurons with 1 mM Trolox prior to soluble A exposure induced a persistent increase in cell survival as monitored by the MTT assay (61.1 ± 2.3% compared with 28.2 ± 3.2% reduction relative to controls; p < 0.05) (Fig. 8A). We thus investigated the effect of Trolox on the soluble A-induced degradation of MAP1A, MAP1B, and MAP2. Immunoblot analysis showed that 1 mM Trolox partially prevented the degradation of MAP1A, MAP1B, MAP2a, MAP2b, and MAP2c induced by treatment of cortical neurons with 5 µM A for 24 h (Fig. 8B). These data were assessed by densitometric analysis, and the results show that Trolox incubation significantly reduced A-induced MAP cleavage as well as generation of degradation products (Table 1). Taken together, these results indicate that soluble A-(1–40) oligomers induce a reactive oxygen species-dependent disorganization of the cytoskeleton that involves MAP degradation.

View this table: [in this window] [in a new window]   TABLE 1 Densitometric analysis of Trolox inhibitory effect on MAP protecolysis induced by soluble A The protective effect of Trolox on MAP protecolysis was monitored by densitometric analysis of the signals revealed after immunoblot examination of the MAP1A, MAP1B, and MAP2 levels in cortical primary neurons incubated in the absence (Control) or presence of 5 µM A for 24 h. Alternatively, neurons were incubated with 1 mM Trolox before and throughout the treatment with soluble A. Data are means ± S.E. of three different experiments and are normalized to the effect of vehicle (Control; designated as 100%) (see Footnotes a, c, and d). Statistical differences among the two A-treated subgroups were identified by performing analysis of variance, followed by Scheffe's post hoc test (see Footnote b). No significant difference was found between Trolox-treated and control cells.

View larger version (61K): [in this window] [in a new window]   FIGURE 7. Synergistic involvement of calpain and caspase-3 in A-dependent MAP1A, MAP1B, and MAP2 degradation. The involvement of calpain and caspase-3 activation in MAP degradation was monitored by immunoblot analysis of cortical neurons incubated in the absence or presence of 5 µM A or 0.5 µM ionomycin for 24 h. The influence of calpain and caspase-3 inhibitors and Ca2+-chelating agent was investigated, in the case of MAP1A and MAP1B proteolysis (A), by incubating cortical neurons with 10 µM calpain inhibitor III (MDL28170), 100 µM DEVD-fmk, or 1 mM EGTA. Alternatively, in the case of proteolysis of MAP2a, MAP2b, and MAP2c (B), this involvement was investigated by incubating cortical neurons with 10 µM MDL28170 or 1 mM EGTA before and throughout the treatment with soluble A. Blots were stripped and reprobed with goat anti--tubulin polyclonal antibody. We have verified that cells treated with the inhibitors only exhibited the same MAP profiles as control cells (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 8. Partial inhibition of A-dependent MAP1A, MAP1B, and MAP2 proteolysis by Trolox. Cortical neurons were exposed to 5 µM A for 24 h. The protective effect of Trolox on A neurotoxicity was first determined by monitoring the mitochondrial reduction activity using the MTT assay (A). Data are the means ± S.E. of three different experiments, with four determinations in each experiment, and are normalized to the effect of vehicle (designated as 100%). ***, p < 0.001. Statistical differences among the subgroups for each condition were performed by analysis of variance, followed by Scheffe's post hoc test. #, p < 0.05. No significant differences were found between Trolox-treated and control cells. The effect of Trolox on MAP proteolysis was next monitored by immunoblot analysis of MAP1A, MAP1B, and three MAP2 isoform levels in primary cortical neurons incubated in the absence (control (Ctl)) or presence of 5 µM A for 24 h (B). Cortical neurons were incubated with 1 mM Trolox before and throughout the treatment with soluble A prior to immunoblot analysis. Blots were stripped and reprobed with goat anti--tubulin polyclonal antibody. We have verified that Trolox-treated and control cells exhibited the same MAP profiles (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The results presented in this study provide evidence that the proteolysis of several structural MAPs is directly involved in the disruption of the microtubule network involved in the neuronal apoptosis induced by soluble oligomers of both A-(1–40) and A-(1–42). Indeed, soluble A-mediated apoptosis involved an early perturbation of Ca²⁺ homeostasis with subsequent calpain activation. Calpain activation alone was required for the proteolysis of the three isoforms MAP2a, MAP2b, and MAP2c, whereas the A-dependent activation of caspase-3 and calpain was responsible in a sequential manner for MAP1A and MAP1B proteolysis. By demonstrating that the proteolysis of MAP1A, MAP1B, and the three MAP2 isoforms was prevented by the presence of the antioxidant Trolox, we have confirmed the early involvement of reactive oxygen species generation and activation of several caspases (caspase-3 and caspase-9) preceding and inducing the perturbation of the microtubule network induced by non-fibrillar A (17).

The "amyloid cascade hypothesis" has been challenged in favor of an alternative hypothesis involving the more recently described soluble A assemblies (10, 21–23), which could clarify many observations that cannot be fully explained, including the poor correlation between senile plaques and clinical symptoms of AD (13, 14). However, the molecular events associated with the cell death induced by soluble oligomers of A remain only partially defined. It is of particular interest to note that the levels of soluble A have been reported to directly correlate with NFT density (11). Furthermore, the severity of dementia and the extensive neuronal loss correlated far better with the number of NFTs than with the extent of A deposition in the early stages of AD development (48). This would support the notion that modifications of the cytoskeleton of neurons might be involved early in the progression of AD pathology (49). Here, we have provided evidence that neurons exposed to soluble A display a disruption of the microtubule architecture even before the typical morphological and biochemical alterations of apoptosis are detectable (Figs. 4 and 5A). A previous study has indicated that an obvious microtubule deficit can be met in the absence of formation of PHFs in the early stages of AD development (50). Together with our data, these results therefore suggest that, besides the alteration of the structure and properties of tau (the most studied member of the MAP family), modifications of other members of this family (such as MAP1A, MAP1B, and MAP2) can contribute to the perturbation of the microtubule network in AD and ultimately lead to neuronal degeneration without accumulation of amyloid deposits.

Increasing evidence highlights the critical outcome of MAP modification in cytoskeletal disorganization associated with the early stages of AD development. A decreased content of MAP1B and tau associated with cytoskeletal breakdown was found in the brains of AD patients compared with those of control individuals, suggesting a decreased capacity of microtubule assembly and stability (51). These results are consistent with those of Iqbal et al. (52) describing a decreased capacity in the in vitro microtubule assembly from brain extracts of AD patients. Moreover, the formation of neuropil threads involves a series of dendritic changes, including not only PHF accumulation, but also dramatic structural alterations such as microtubule perturbation and modification and/or aggregation of several MAPs. One study has shown an early decrease in MAP2 labeling within dendrites from AD brain (53). In addition, accumulation of soluble A oligomers in a transgenic mouse model of AD results in a decrease in the density of presynaptic terminals and in MAP2 labeling well before these mice develop plaques (13). Other studies have demonstrated that MAP1B and MAP2 co-localize with NFTs (35, 36). Despite the still unclear role of MAP1B and MAP2 in AD-associated neurodegeneration, several studies have highlighted the possible involvement of aberrant phosphorylation and proteolysis in several fragments (35, 54, 55), leading to MAP incorporation into PHFs as effectors of neurodegeneration in AD. Moreover, it has been demonstrated that small MAP2a and MAP2b fragments and MAP2c (via their microtubule-binding region) readily polymerize into structures resembling PHFs (37).

In this study, we have provided the first biochemical evidence that depletion of functional MAP1A, MAP1B, and MAP2 induced by soluble A might in part explain the disruption of the microtubule network during neuronal apoptosis (Figs. 5, 6, 7). We propose, as a mechanism of proteolysis, that the degradation of MAP1A and MAP1B is initiated by a caspase-3-dependent proteolytic cleavage to yield the 250-kDa fragments, whose accumulation might modify their properties and susceptibility to calpain proteolysis. The changes might include an altered conformation as well as abnormal interactions with other proteins or aggregation of MAP fragments. After being cleaved by caspase-3, these large MAP1A and MAP1B fragments can be further degraded by calpain into multiple smaller fragments. The pattern of limited proteolysis of MAP1A and MAP1B is in keeping with the fact that caspase-3 and calpain are known to generate fragments of limited size during the proteolysis of their substrates without further and complete degradation into very small peptides and amino acids (5, 40). However, it is possible that other caspases with shared specificity for caspase-3-sensitive sites in MAP1A and MAP1B contribute to their proteolysis and generate the intermediate fragment. Indeed, caspase-2 shares similarities with caspase-3 in their substrate preference and cleaves at the same DXXD consensus sequence (56). We have eliminated this possibility by showing that a caspase-2 inhibitor (N-acetyl-Val-Asp-Val-Ala-aspartatal) used under the same conditions did not have any effect on MAP1A and MAP1B proteolysis (data not shown). Moreover, we have determined that calpain is not able to degrade full-length MAP1A and MAP1B if they have not been previously and independently cleaved by caspase-3. Indeed, treatment of cortical neurons with ionomycin increased the intracellular Ca²⁺ levels and subsequently activated calpain, but did not cause MAP1A and MAP1B degradation (data not shown). Calpain and caspase-3 interact in different ways to mediate limited proteolysis of a large number of proteins (4, 5, 40). This process can occur on the adjacent (57, 58) or separate (57–60) calpain and caspase-3 cleavage sites along the primary sequence according to the stimulus (61) and depending on their cell type (62). It is also interesting to note that the degradation of tau (63) and huntingtin (64) occurs according to the same sequential process as described for MAP1A and MAP1B in this study, i.e. an initial degradation by caspase-3, followed by calpain proteolysis.

In this study, we have demonstrated that soluble A induces a perturbation of Ca²⁺ homeostasis (Fig. 2). Several lines of evidence that strongly support a role for alteration of cellular Ca²⁺ homeostasis in the pathogenesis of AD come from studies of the effects of exogenously applied A on cultured neurons (30) and from analysis of brain tissues from AD patients in association with neurodegenerative process (29). Interestingly, some investigators have suggested that Ca²⁺ dysfunction occurs during the initial phases of the disease, even before the development of overt symptoms and before any obvious extracellular A deposits (65, 66). Consequently, destabilization of neuronal Ca²⁺ homeostasis might also be an important trigger of apoptosis via microtubule modifications (67) and activation of an array of cellular enzymes, including the calpain system (31). We have demonstrated here that exposure to soluble A results in calpain-dependent proteolysis of the three isoforms MAP2a, MAP2b, and MAP2c (Fig. 7B). It has already been shown that MAP2a, MAP2b, and MAP2c are substrates for activated calpain (2). The calpain-dependent degradation of high molecular mass MAP2 is known to occur as an early intracellular structural event in response to traumatic (68), seizure-related (69), and focal ischemic (70, 71) brain injuries and to glutamate excitotoxicity (72). Similar MAP2 degradation is also associated with the proteolysis of MAP1A and MAP1B involving calpain activation in response to focal ischemic injuries (70) as well as in different in vivo and in vitro experimental models of neurotoxicity using various neurotoxic substances (73–76), in bipolar disorder (77), and in other neurodegenerative disease such as amyotrophic lateral sclerosis (78). However, it is noteworthy that the degradation of MAP2 is not associated with the proteolysis of MAP1B in the striatum or hippocampus after ischemia, whereas more extensive proteolysis of MAP2 and MAP1B occurs in other vulnerable brain areas (71). This discrepancy in the proteolysis of MAP2 and MAP1B depending on the brain area and/or the stimulus highlights that activated caspase-3 is necessary for the proteolysis of MAP1 family members.

Unlike caspase-3, which requires a specific amino acid sequence at cleavage sites (DXXD), calpain does not seem to have a strict sequence requirement for substrate cleavage, but rather recognizes secondary hydrophilic sequences called PEST sequences. These PEST sequences represent protein regions rich in proline, glutamate, aspartate, serine, and threonine. In addition to being recognition sites for calpain, such negatively charged clusters may bind Ca²⁺ and consequently locally activate calpain. It is even believed that the substrate specificity of calpain could be governed by the tertiary and quaternary structures of the polypeptide chain. The presence of accessible PEST sequences all along MAP1B, MAP2a, MAP2b, and MAP2c (as described by Friedrich and Aszodi (79)) has been established by several in vitro studies that described rapid proteolysis by the two typical purified µ- and m-calpains (80–83). Protein sequence analysis led us to identify a number of putative DXXD cleavage sites for caspase-3 along the entire primary sequence of the structurally related proteins MAP1A and MAP1B. Furthermore, using the computer program PESTfind (84), we have observed that MAP1A and MAP1B contain a large number of PEST sequences.

Not only could MAP proteolysis result in loss of ability to promote the assembly and stabilization of microtubules, but also the MAP fragments could be toxic. Indeed, the cleavage affecting the tubulin-binding domain and the remaining two-thirds of MAP1A, MAP1B, and MAP2 representing the projection domain is likely to influence their association with microtubules, the spacing between microtubules, and their interactions with other cytoskeletal proteins or the plasma membrane (1, 2). It may also lead to negative regulation of MAP functions, known to provoke microtubule destabilization, modification of vesicle trafficking, and perturbation of signal transduction pathways. The hypothesis of loss of function may explain the association reported between estrogen-induced protection against A-(1–40) neurotoxicity and increased expression of MAP1B and MAP2c (85). Caspase-3- and/or calpain-mediated MAP degradation and generation of several fragments may also contribute to the progression of soluble A-induced neuronal cell death by release of proteolytic fragments that may exert a cytoskeletal disassembling function and/or pro-apoptotic effects. Indeed, caspase-3 cleavage products of tau were observed to induce apoptosis, turning tau itself into an effector of apoptosis (86, 87). Also, overexpression of N-terminal fragments of MAP1B has been shown to promote neuronal apoptosis (38).

We demonstrated previously that caspase-3 inhibition prevents neuronal apoptosis induced by soluble A (17). In contrast, inhibition of calpain by several families of inhibitors (MDL28170, N-acetyl-Leu-Leu-norleucinal, and N-acetyl-Leu-Leu-Met) had no effect on A-induced apoptosis (data not shown). This suggests that caspase-3 activation is predominant and sufficient in MAP1A and MAP1B degradation and subsequent functional perturbations. Indeed, the sequential involvement of caspase-3 and calpain in the proteolysis of these MAPs would explain the complete recovery of the quantity of native proteins by inhibiting caspase-3, but not calpain (Fig. 7A). However, determining the relative roles of calpain and caspases in neuronal apoptosis is complicated by a growing body of evidence delineating a cross-talk between the two proteolytic systems (88). In this study, we demonstrated that A-induced apoptosis required the activation of caspase-3 and caspase-9 by directly measuring caspase-like activities (Fig. 3A). Interestingly, calpain inhibitors further enhanced these proteolytic activities significantly even though A-induced calpain activation was efficiently prevented (supplemental figure). The calpain-dependent cleavage of procaspase-3 has been shown to generate the active form and to lead to cell apoptosis (89). In contrast, activation of calpain may also prevent entry of cells into a caspase-dependent cell death program by inhibiting the activities of caspase-3 and caspase-9. Interestingly, the calpain-mediated truncation of procaspase-9 produces an inactive peptide that is unable to participate in cytochrome c- and dATP-induced caspase-3 activation (90–92). Another possibility is that the calpain-mediated proteolysis of procaspase-3 directly inhibits caspase-3 activation (91, 92). Additionally, the calpain-mediated proteolysis of active forms of caspase-3 and caspase-9 can be equally responsible for the down-regulation of activation (93). Moreover, in addition to direct cleavage of caspases, calpain has been reported to degrade several apoptotic regulatory proteins, including the apoptosome components apoptotic protease-activating factor-1 and Bax (94), indirectly inhibiting subsequent caspase-3 activation. Taken together, our results suggest that calpain may play a dual role during the time course of soluble A-mediated neuronal apoptosis, negatively regulating caspase-9 and caspase-3 activation on the one hand and taking part in MAP degradation involved in the apoptotic process on the other.

In summary, this work identifies a novel mechanism associated with soluble A-induced neuronal apoptosis. Our results reveal a direct relationship between A exposure and the proteolysis of several MAPs, appearing to involve a caspase- and calpain-dependent pathway. Identification of this pathway may lead to the development of more effective therapeutic strategies for AD aimed at preventing soluble A-mediated cytoskeletal breakdown and subsequent apoptosis.

	FOOTNOTES

 
^* This work was supported in part by INSERM and by a grant from the Aventis French Network on Molecular Mechanisms in Alzheimer Disease. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.

¹ Recipient of a fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche and a grant from the Fondation pour la Recherche Médicale.

² To whom correspondence should be addressed: Lab. Médecine et Thérapeutique Moléculaire, Inst. National Polytechnique de Lorraine, 15 rue du Bois de la Champelle, 54500 Vandoeuvre-lès-Nancy, France. Tel.: 33-3-8367-8211; Fax: 33-3-8367-8999; E-mail: Thierry.Pillot{at}mtm.nancy.inserm.fr.

³ The abbreviations used are: MAP, microtubule-associated protein; NFTs, neurofibrillary tangles; AD, Alzheimer disease; PHFs, paired helical filaments; A, amyloid -peptide; AMC, 7-amido-4-methylcoumarin; fmk, fluoromethyl ketone; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI, 4',6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine isothiocyanate.

⁴ A. Fifre, I. Sponne, V. Koziel, B. Kriem, F. T. Yen-Potin, B. E. Bihain, J.-L. Olivier, T. Oster, and T. Pillot, unpublished data.