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Originally published In Press as doi:10.1074/jbc.M605081200 on July 24, 2006

J. Biol. Chem., Vol. 281, Issue 38, 28079-28089, September 22, 2006
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beta-Amyloid-induced Dynamin 1 Degradation Is Mediated by N-Methyl-D-Aspartate Receptors in Hippocampal Neurons*Formula

Brent L. Kelly{ddagger}1 and Adriana Ferreira{ddagger}§2

From the {ddagger}Department of Cell and Molecular Biology, Feinberg School of Medicine and the §Institute for Neuroscience, Northwestern University, Chicago, Illinois 60611

Received for publication, May 26, 2006 , and in revised form, July 17, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer disease (AD) is a progressive, neurodegenerative disorder that leads to debilitating cognitive deficits. Although little is known about the early functional or ultrastructural changes associated with AD, it has been proposed that a stage of synaptic dysfunction might precede neurodegeneration in the development of this disease. Unfortunately, the molecular mechanisms that underlie such synaptic dysfunction remain largely unknown. Recently we have shown that beta-amyloid (Abeta), the main component of senile plaques, induced a significant decrease in dynamin 1, a protein that plays a critical role in synaptic vesicle recycling, and hence, in the signaling properties of the synapse. We report here that this dynamin 1 degradation was the result of calpain activation induced by the sustained calcium influx mediated by N-methyl-D-aspartate receptors in hippocampal neurons. In addition, our results showed that soluble oligomeric Abeta, and not fibrillar Abeta, was responsible for this sustained calcium influx, calpain activation, and dynamin 1 degradation. Considering the importance of dynamin 1 to synaptic function, these data suggest that Abeta soluble oligomers might catalyze a stage of synaptic dysfunction that precedes synapse loss and neurodegeneration. These data also highlight the calpain system as a novel therapeutic target for early stage AD intervention.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Alzheimer disease (AD)3 patients display measurable cognitive deficits before significant synapse loss becomes apparent (1, 2). Disruptions in synaptic plasticity preceding synapse loss, neurodegeneration, and plaque accumulation have been also reported in AD animal models (3–6). Together, these findings suggested that synaptic dysfunction could account for cognitive symptoms during the earliest stages of this disease (reviewed in Refs. 7 and 8). However, the molecular mechanisms that contribute to this synaptic dysfunction are not well understood. It has been postulated that the accumulation of some toxic form of beta-amyloid (Abeta) might be responsible for these changes in neuronal activity in AD (9–14). Abeta, a fragment of 40 or 42 amino acids, is the product of the sequential cleavage of the amyloid precursor protein (APP) mediated by the beta- and {gamma}-secretase enzymatic complexes (reviewed in Ref. 15). These fragments are presumably broken down and present little harm to the neurons they come in contact with in healthy individuals (16). On the other hand, under the pathologic conditions leading to AD, these soluble monomeric Abeta fragments aggregate, and eventually deposit as large fibrils in the extracellular space forming amyloid plaques (17).

Soluble oligomeric species of Abeta have been shown to be particularly toxic to synapses by disrupting long term potentiation (LTP) both in vivo and in culture systems (18, 19). Recently, a series of studies have begun to address the mechanisms underlying Abeta-induced synaptic toxicity. Data obtained using brains of AD patients showed that the levels of synaptic proteins like SNAP-25, syntaxin, and synaptotagmin were decreased (reviewed in Ref. 20). In addition, dynamin 1, a GTPase protein, which pinches off synaptic vesicles from the plasma membrane following fusion and exocytotic release of neurotransmitter, was also depleted in the brain of AD patients (21). A similar decrease in dynamin 1 content has been observed when soluble Abeta oligomers were added to cultured hippocampal neurons and in the hippocampus of AD animal model systems (22). This decreased dynamin 1 could play a critical role in synaptic dysfunction in view of results obtained in loss-of-function studies. These studies showed that synapses lost their ability to successively release neurotransmitter as synaptic vesicles became trapped at the plasma membrane and the synaptic vesicle pool was depleted in neurons in which dynamin 1 had been down-regulated (23–26). Collectively, these data suggest that Abeta-induced dynamin 1 depletion might contribute to synaptic dysfunction in AD.

In the present study, we analyzed the molecular mechanisms leading to Abeta-induced dynamin 1 depletion in hippocampal neurons. Our results showed that Abeta-induced decrease in dynamin 1 involved calpain-mediated degradation. Furthermore, these findings suggested that Abeta-induced calpain activation was dependent on the presence of Abeta soluble oligomers and on external Ca2+ influx mediated by N-methyl-D-aspartate (NMDA) receptors.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Hippocampal Cultures—Embryonic day (E) 18 rat embryos were used to prepare primary hippocampal cultures as previously described (27). Briefly, hippocampi were dissected and freed of meninges. The cells were dissociated by trypsinization followed by trituration with a fire-polished Pasteur pipette. For biochemical experiments, hippocampal neurons were plated at high density (500,000 cells/60-mm dish) in MEM with 10% horse serum (MEM10). After 2 h, the medium was changed to glia-conditioned MEM containing N2 supplements plus ovalbumin (0.1%) and sodium pyruvate (0.1 mM) (N2 medium) (28). For immunocytochemistry and Ca2+ imaging experiments, hippocampal neurons were plated onto poly-L-lysine-coated coverslips in MEM10. After 2 h, the coverslips were transferred to dishes containing an astroglial monolayer and maintained in MEM containing N2 supplements plus ovalbumin (0.1%) and sodium pyruvate (0.1 mM). These cultures contain ~95% pyramidal neurons and 5% glial cells.

Abeta Aggregation and Treatment—Synthetic Abeta1–40 (Bachem, Torrance, CA) was dissolved in N2 medium (0.1 mg/ml) and incubated for 3 days at 37 °C to preaggregate the peptide (29). This preaggregated Abeta was added to the culture medium at a final concentration of 2 µM, a concentration that has been shown to cause early events of neurotoxicity without causing overt neuronal death in cultured hippocampal neurons (22). For some experiments, preaggregated Abeta was centrifuged at 100,000 x g for 1 h to separate the soluble oligomeric (supernatant) from the insoluble fibrillar (pellet) forms of the peptide. The oligomeric fraction was obtained by removing the supernatant. The fibrillar fraction was obtained by resuspending the pellet in a volume of N2 medium equal to the supernatant. These fractions were added directly to cultured hippocampal neurons at final concentrations calculated using the initial concentration of the monomeric form of the peptide. Hippocampal neurons were grown in the presence of different Abeta forms for up to 24 h. For experiments using fura-2, synthetic Abeta1–40 was dissolved in MilliQ H2O and preaggregated as described above.

Electrophoresis and Immunoblotting—Whole cell extracts were prepared from hippocampal neurons that developed in culture. To prepare these fractions, hippocampal neurons kept in culture for 3 weeks were rinsed in PBS, scraped into Laemmli buffer, and homogenized in a boiling water bath for 10 min. Samples were run on 7.5% SDS-polyacrylamide gels and transferred to Immobilon membranes (Millipore) (30). Transfer of protein to Immobilon membranes and immunodetection were performed according to Towbin et al. (31) as modified by Ferreira and co-workers (22). Immunodetection was performed using the following antibodies: anti-{alpha}-tubulin (1:200,000; clone DM1A; Sigma), anti-dynamin 1 (1:5,000; Affinity BioReagents, Golden, CO), anti-calpain (1:200, Sigma), anti-calpastatin (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-spectrin (1:1,000; Chemicon, Temecula, CA). Secondary antibodies conjugated to horseradish peroxidase (Promega, Madison, WI) followed by enhanced chemiluminescence reagents (Amersham Biosciences) were used for the detection of proteins. Immunoreactive bands were imaged using a ChemiDoc XRS system (Bio-Rad). Densitometry of these bands was performed using Quantity One software (Bio-Rad). Unless stated differently, densitometry values were normalized using tubulin as internal controls. At least three independent experiments for each experimental condition were used for the quantitative Western blots. Data were expressed as means ± S.E. Statistical significance was analyzed by Student's t test.

Determination of Calpain Activity—Calpain activity was determined by assessing spectrin cleavage as previously described (32). Briefly, whole cell extracts were prepared from hippocampal neurons cultured in the presence or absence of preaggregated Abeta and used for quantitative Western blot analysis with a spectrin antibody as described above. Because calpain cleavage produces characteristic spectrin fragments of 145 and 150 kDa (33), we determined the 150/240-kDa ratio from treated neurons and compared it to the one obtained from untreated controls. Values obtained from these untreated controls were considered 100%. Calpain activity was also assessed by quantitative analysis of the 58-kDa active calpain fragment. Densitometry of immunoreactive bands corresponding to this 58-kDa fragment was performed as described above.

Reagents—A series of calcium channel blockers including xestospongin C (1 µM; Promega), dantrolene (10 µM; Santa Cruz Biotechnology), and nimodipine (10 µM; Calbiochem), NMDA receptor blockers including MK801 (10 µM; Calbiochem) and memantine (10 µM; Sigma), and 1,2-bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA, 20 µM; Calbiochem), were added to the medium of 3 weeks in culture hippocampal neurons 1 h prior to, and for the duration of, the preaggregated Abeta treatment (34–39). Hippocampal neurons treated as described above were used for immunoblotting and immunocytochemistry. For Ca2+ imaging, MK801 (10 µM), memantine (10 µM), or tetrodotoxin (TTX; 0.1 µM; Sigma) (40) were added to the Krebs buffer during the loading, washing, and imaging phases.

Immunocytochemistry—Hippocampal neurons cultured for 3 weeks were fixed for 20 min with 4% paraformaldehyde in PBS containing 0.12 M sucrose. They were then permeabilized in 0.3% Triton X-100 in PBS for 5 min and rinsed twice in PBS. The cells were preincubated in 10% bovine serum albumin in PBS for 1 h at 37°C and exposed to the primary antibodies overnight at 4 °C. The neurons were then rinsed in PBS and incubated with secondary antibodies for 1 h at 37°C. The following primary antibodies were used: polyclonal anti-dynamin 1 (1:1,000; Affinity BioReagents) and monoclonal anti-{alpha}-tubulin (1:600; clone DM1A). The following secondary antibodies were used: anti-mouse IgG fluorescein-conjugated and anti-rabbit IgG rhodamine-conjugated (1:1,000; Chemicon). Images were taken using Metamorph Image analysis software (Universal Imaging Corporation, Fryer Company Inc., Huntley, IL).

Calcium Imaging—Hippocampal neurons were cultured for 2 weeks on coverslips as described above. Loading with the Ca2+ indicator fura-2 was accomplished by incubating with 3 µM fura-2-AM (Molecular Probes, Eugene, OR) in Krebs-HEPES buffer (100 mM NaCl, 180 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM Na3PO4, 4.2 mM Na2CO3, 10 mM glucose, 12.5 mM HEPES, pH 7.4) for 30 min at 37 °C. The coverslips were then washed three times with Krebs-HEPES buffer, and maintained at 37 °C for 30 min to ensure complete de-esterification (41, 42). Coverslips with loaded cells were mounted in a chamber on an inverted microscope (Nikon Diaphot, Melville, NY). Neurons were then exposed to preaggregated Abeta in the absence or presence of the TTX and NMDA receptor blockers as described above. Preaggregated Abeta was added directly to the chamber and readings were taken from the soma of the hippocampal neurons. Fura-2 was excited by a xenon light source at 340 and 380 nm. The emitted fluorescence was filtered through a 520-nm filter, captured with an intensified CCD camera (Hamamatsu, Hamamatsu City, Japan) coupled to a microscope (Nikon Diaphot, Melville, NY) and analyzed with MetaFluor software (Universal Imaging Corporation). [Ca2+]i was calibrated with a range of known Ca2+ concentrations in solution with fura-2. Quantitative analysis using fura-2 imaging was performed on three independent experiments (~60 cells). Each experimental condition reflects an average of these trials and was compared with the [Ca2+]i of untreated neurons.


Figure 1
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  		FIGURE 1.
Abeta oligomers induced calpain activation in cultured hippocampal neurons. A, Western blot analysis of dynamin 1 and spectrin content in whole cell extracts prepared from hippocampal neurons (21 days in culture) treated with preaggregated Abeta (2µM) for 24 h in the absence or presence of caspase (VAD) or calpain inhibitors (ALLN). B, Western blot analysis of dynamin 1 and spectrin content in whole cell extracts prepared from hippocampal neurons (21 days in culture) treated with 2 µM mixed (Mix), insoluble fibrillar (Fib), or soluble oligomeric (Olig) Abeta for 24 h. The numbers on the right indicate the apparent molecular masses (kDa). C and D, quantitative analysis of spectrin degradation (150/240 kDa ratio) in hippocampal neurons cultured under the experimental conditions described above. {alpha}-Tubulin was used as a loading control. The values obtained in untreated controls were considered 100%. Values represent the mean ± S.E. *, differs from untreated controls, p < 0.01.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Soluble Abeta Oligomers Activated Calpain in Cultured Hippocampal Neurons—We have previously shown that preaggregated Abeta induced dynamin 1 depletion in cultured hippocampal neurons (22). Our results also suggested that calpain activation might be responsible for this Abeta effect. To gain further insights into Abeta-induced dynamin 1 degradation, as well as into the toxic form of this peptide, we performed a series of experiments using mature cultured hippocampal neurons. When 3 weeks in culture hippocampal neurons were incubated with 2 µM of preaggregated Abeta for 24 h there was a significant decrease in full-length dynamin 1 levels (100 kDa) and an increase of a ~90-kDa fragment (Fig. 1A, see also Ref. 22). We determined next whether this decrease in dynamin 1 levels was due to calpain-mediated degradation. For these experiments, we assessed calpain activation in hippocampal neurons cultured under the experimental conditions described above by determining spectrin degradation. Spectrin degradation is highly sensitive to calpain activation and considered an excellent marker for this protease activity (32). Western blot analysis of whole cells extracts reacted with a specific spectrin antibody showed a significant decrease in full-length spectrin (240 kDa) and a concomitant increase in the 150-kDa degradation fragment (Fig. 1A). Quantitative analysis of immunoreactive bands showed a significant increase in the 150/240-kDa spectrin ratio in hippocampal neurons treated with preaggregated Abeta when compared with untreated controls (Fig. 1C). These findings suggested an Abeta-induced increase in calpain activity in treated neurons. These results were confirmed by means of quantitative Western blot analysis of immunoreactive bands corresponding to the 58-kDa active form of calpain (data not shown). Since spectrin degradation could also be mediated by caspase-3, we incubated cultured hippocampal neurons with a cell-permeable, general caspase inhibitor (VAD) or a cell-permeable calpain inhibitor (ALLN) 1 h prior to the addition of preaggregated Abeta. Western blot analysis showed no changes in full-length dynamin 1 levels in hippocampal neurons incubated with ALLN and preaggregated Abeta for 24 h when compared with untreated controls. In addition, the ~90-kDa dynamin 1 fragment was not detected in cultured hippocampal neurons treated with both ALLN and preaggregated Abeta (Fig. 1A). Spectrin degradation was also attenuated in hippocampal neurons cultured in the presence of ALLN and preaggregated Abeta (Fig. 1A). Thus, no changes in full-length spectrin and/or the 150/240-kDa spectrin ratio were detected in ALLN- and preaggregated Abeta-treated hippocampal neurons when compared with untreated controls (Fig. 1C). On the other hand, the decrease in full-length dynamin 1 and the appearance of the ~90-kDa fragment in hippocampal neurons incubated with VAD and preaggregated Abeta for 24 h were similar to the ones observed in hippocampal neurons incubated with preaggregated Abeta alone (Fig. 1A). This caspase inhibitor had no effect either on Abeta-induced spectrin degradation as assessed by the significant increase in the 150/240-kDa spectrin ratio (Fig. 1C). These results ruled out the effect of caspases on Abeta-induced dynamin 1 degradation under our experimental conditions (see also Ref. 22).

We then determined whether a particular species of Abeta was responsible for calpain activation and dynamin 1 degradation in cultured hippocampal neurons. For these experiments, preaggregated Abeta was centrifuged to separate the soluble oligomeric forms of the peptide from the larger, insoluble fibrillar aggregates as previously described (22). The preaggregated Abeta (referred to as mixed because of its likelihood to contain both insoluble fibrillar Abeta along with soluble oligomeric Abeta) was added to the cultured medium of 3 weeks in culture hippocampal neurons at a final concentration of 2 µM. Twenty-four hours later, whole cell lysates were prepared and dynamin 1 levels and calpain activation were determined. Western blot analysis of immunoreactive bands showed that mixed Abeta caused a decrease in full-length dynamin 1 and an increase in its ~90-kDa proteolytic fragment (Fig. 1B). Degradation of dynamin 1 was also accompanied by the activation of calpain, as shown by the increase in the 150/240-kDa spectrin ratio (Fig. 1D). Conversely, no changes in dynamin 1 levels, or the appearance of the degradation product, were detected when 3 week in culture hippocampal neurons were incubated with fibrillar Abeta (2 µM) for 24 h (Fig. 1B). Fibrillar Abeta also failed to induce calpain activation in these neurons as assessed by spectrin degradation (Fig. 1D). On the other hand, the addition of 2 µM oligomeric Abeta induced a decrease in full-length dynamin 1, the appearance of the dynamin 1 fragment, and a significant calpain activation in cultured hippocampal neurons. Quantitative analysis showed that these changes were similar to the ones induced by the mixed Abeta in these neurons (Fig. 1D).

Abeta-induced Activation of Calpain Was Likely Due to a Disruption in Ca2+ Homeostasis—Calpastatin, an endogenous calpain inhibitor, and changes in Ca2+ levels are among the most well-studied regulators of calpain activity (reviewed in Ref. 43). To determine how calpain was activated in Abeta-treated neurons, we first examined the levels of calpastatin by means of Western blot analysis using a specific calpastatin antibody. For these experiments, we incubated cultured hippocampal neurons with preaggregated Abeta (2 µM) for 8 and 24 h. Quantitative analysis of calpastatin immunoreactive bands showed no significant changes in this calpain inhibitor levels after an 8-h exposure to preaggregated Abeta as compared with untreated controls (Fig. 2A). On the other hand, a significant increase (~40%) in calpastatin levels were detected in hippocampal neurons exposed to preaggregated Abeta for 24 h as compared with untreated ones (Fig. 2, A and B). These data suggested calpastatin levels increased in a time-dependent manner in response to Abeta. In addition, they suggested that the calpain activation observed in preaggregated Abeta-treated hippocampal neurons was not caused by a decrease in the level of calpastatin. Therefore, we studied next whether an abnormal increase in intraneuronal Ca2+ could contribute to the activation of calpain in these Abeta-treated neurons. For these experiments, we added preaggregated (mixed, soluble oligomeric, and fibrillar) Abeta (2 µM) to the hippocampal neurons that were previously loaded with fura-2. Fura-2-based calcium imaging is commonly used to measure real-time intracellular Ca2+ levels (41, 42). The addition of mixed Abeta (2 µM) produced a significant spike in Ca2+ levels (~7-fold increase above basal intraneuronal Ca2+ levels) in these cultured hippocampal neurons (Fig. 3A). Over the next 12 min, Ca2+ levels declined and eventually plateau. However, these plateau levels were significantly higher than the Ca2+ levels detected before the addition of the peptide (Fig. 3A). We next tested whether Abeta oligomers or Abeta fibrils were responsible for this disruption of Ca2+ homeostasis in hippocampal neurons. The addition of oligomeric Abeta (2 µM) induced a significant and instantaneous rise in Ca2+ (~5-fold increase above the basal intraneuronal Ca2+ levels) in hippocampal neurons (Fig. 3B). These Ca2+ levels decreased thereafter and reached plateau levels that were similar to the ones detected when hippocampal neurons were incubated in the presence of mixed Abeta (Fig. 3B). Fibrillar Abeta also induced changes in intracellular Ca2+ levels. However, these changes were more subtle and transient than the ones observed when hippocampal neurons were incubated in the presence of either mixed or soluble oligomeric Abeta. Thus, Ca2+ levels significantly increased (~2-fold increase above the basal intraneuronal Ca2+ levels) in the presence of fibrillar Abeta (Fig. 3C). These Ca2+ levels decreased to baseline levels (~80 nM) ~15 min after the addition of fibrillar Abeta (Fig. 3D). To determine whether there was a significant elevation of intraneuronal Ca2+ at the end point of our experimental period, we quantified the Ca2+ measurements 23 min after the addition of the different forms of Abeta (Fig. 3D). This quantitative analysis showed that Ca2+ levels were significantly higher in hippocampal neurons treated with either mixed or oligomeric Abeta when compared with basal levels obtained in the absence of these peptides. In contrast, hippocampal neurons that were exposed to fibrillar Abeta had recovered to baseline Ca2+ levels (Fig. 3D).


Figure 2
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  		FIGURE 2.
Abeta induced an increase in calpastatin levels in cultured hippocampal neurons. A, Western blot analysis of calpastatin in whole cell extracts prepared from hippocampal neurons (21 days in culture) treated with preaggregated Abeta (2 mM) for 8 and 24 h. The number on the right indicates the apparent molecular mass (kDa). B, quantitative analysis of the effects of Abeta on calpastatin levels in hippocampal neurons cultured under the experimental conditions described above. Results were normalized using {alpha}-tubulin as an internal control. The values obtained in untreated controls were considered 100%. Values represent the mean ± S.E. *, differs from untreated controls, p < 0.01.

 


Figure 3
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  		FIGURE 3.
Abeta oligomers elicited an elevation in intracellular Ca2+ in cultured hippocampal neurons. A–C, traces of intracellular Ca2+ levels in hippocampal neurons loaded with fura-2 and treated with 2 µM of mixed (A), oligomeric (B), or fibrillar (C) Abeta. Traces represent an average of 3 trials totaling 64, 70, and 51 neurons for mixed, oligomeric, and fibrillar Abeta treatments, respectively. D, quantitative analysis of intraneuronal Ca2+ levels at the end point of Abeta exposure. Values represent the mean ± S.E. *, differs from untreated controls, p < 0.01.

 
Abeta-induced Calpain Activation and Dynamin 1 Degradation Were Dependent on the Extracellular Influx of Ca2+—Collectively, the data described above suggested that soluble oligomeric Abeta was responsible for a sustained disruption of Ca2+ homeostasis in hippocampal neurons. However, they did not provide information regarding the Ca2+ source mobilized under our experimental conditions. To determine whether this Abeta-induced Ca2+ increase was due to intracellular release or extracellular influx, we performed a series of experiments to selectively block each one of these mechanisms. The first set of experiments was directed to block Ca2+ release from the endoplasmic reticulum (ER), the major source of intracellular Ca2+ release. Since this Ca2+ release is regulated through inositol 1,4,5,-trisphosphate receptors (IP3R) or ryanodine-mediated Ca2+ channels (RyR), we incubated 3 weeks in culture hippocampal neurons with xestospongin C (1 µM) and dantrolene (10 µM), inhibitors of IP3Rs and RyRs, respectively (36, 44, 45). Cultured hippocampal neurons treated with these inhibitors for 1 h were then incubated with preaggregated Abeta (2 µM) for an additional 24 h. Quantitative analysis of immunoreactive bands in whole cell extracts showed a significant decrease (~60%) in full-length dynamin 1 and the appearance of the dynamin 1 fragment in hippocampal neurons treated with preaggregated Abeta when compared with untreated controls (Fig. 4, A and D). Blockage of Ca2+ release from the ER with xestospongin C and dantrolene had no effect on the Abeta-induced decrease in full-length dynamin 1 levels, the appearance of the dynamin 1 fragment (Fig. 4, A and D), or calpain activation (data not shown). These data suggested that Abeta-induced degradation of dynamin 1 and calpain activation was not due to intracellular release of Ca2+ from the ER.

We next tested whether Abeta-induced dynamin 1 degradation and calpain activation was caused by the extracellular influx of Ca2+. For these experiments, 3 weeks in culture hippocampal neurons were incubated with BAPTA, a chelator of extracellular Ca2+, for 1 h prior to the addition of preaggregated Abeta (38). Western blot analysis of immunoreactive bands in whole cell extracts prepared 8 h after the addition of the peptide to BAPTA-treated cultures showed no changes in full-length dynamin 1 levels when compared with untreated controls. In addition, BAPTA prevented the appearance of the dynamin 1 fragment observed in preaggregated Abeta-treated hippocampal neurons (Fig. 4, B and E). The addition of BAPTA to the culture medium also blocked Abeta-induced calpain activation as assessed by spectrin cleavage. Thus, no changes in the 150/240-kDa spectrin ratio were detected in BAPTA-treated hippocampal neurons incubated with Abeta for 8 h as compared with untreated controls (Fig. 4, C and F). Similar results were observed when BAPTA-treated cultures were incubated with preaggregated Abeta for 24 h (Fig. 4, B, C, E, and F). The changes in dynamin 1 levels in cultured hippocampal neurons incubated in the presence or absence of preaggregated Abeta and/or BAPTA were also detected at the light microscopy level (Fig. 5, A–C). Dynamin 1 immunoreactivity was highly enriched in the cell bodies and throughout the neurites extended by untreated hippocampal neurons (Fig. 5A, see also Kelly et al., Ref. 22). This dynamin 1 staining was greatly reduced when hippocampal neurons were incubated in the presence of preaggregated Abeta (2 µM) for 24 h (Fig. 5B). In contrast, no apparent decrease in dynamin 1 immunoreactivity was detected when BAPTA was added to the culture media prior to the incubation with preaggregated Abeta (Fig. 5C). BAPTA also prevented the signs of early neuronal degeneration (varicosities along the neurites) induced by preaggregated Abeta treatment (Fig. 5, E and F). Therefore, the processes extended by preaggregated Abeta and BAPTA-treated neurons were undistinguishable from the ones extended from the cell bodies of untreated controls (Fig. 5D).


Figure 4
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  		FIGURE 4.
Abeta-induced dynamin 1 degradation and calpain activation were dependent on extracellular Ca2+ influx. A, Western blot analysis of dynamin 1 content in whole cell extracts prepared from hippocampal neurons (21 days in culture) treated with preaggregated Abeta (2 µM) in the absence or presence of RyR/IP3R blockers (X/D) for 24 h. B and C, Western blot analysis of dynamin 1 (B) and spectrin (C) content in whole cell extracts prepared from hippocampal neurons (21 days in culture) treated with preaggregated Abeta (2 µM) in the absence or presence of 20 µM BAPTA for 8 and 24 h. D and E, quantitative analysis of dynamin 1 levels in hippocampal neurons cultured under the experimental conditions described above. Full-length dynamin 1 levels were normalized using {alpha}-tubulin as an internal control. F, quantitative analysis of calpain activation in hippocampal neurons treated with Abeta and BAPTA. Calpain activation was assessed by determining spectrin degradation (150/240 kDa ratio). Values represent the mean ± S.E. *, differs from untreated controls, p < 0.01.

 


Figure 5
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  		FIGURE 5.
BAPTA blocked Abeta-induced dynamin 1 depletion in cultured hippocampal neurons. Hippocampal neurons grown in culture for 21 days were incubated with preaggregated Abeta (2 µM) in the absence (B and E) or presence (C and F) of 20 µM BAPTA for 24 h and compared with untreated controls (A and D). Neurons were co-stained using dynamin 1 (A–C) and {alpha}-tubulin antibodies (D–F). Note that the decrease in dynamin 1 immunoreactivity induced by Abeta (B) was blocked when the neurons were treated with BAPTA and Abeta (C). Scale bar, 20 µm.

 
Abeta-induced Calpain Activation and Dynamin 1 Degradation Were Dependent on the Activation of NMDA Receptors—The experiments described above suggested that preaggregated Abeta induced an extracellular Ca2+ influx. Ca2+ influx can be regulated both by NMDA receptors and/or voltage-gated calcium channels (VGCC), mechanisms that have been implicated in the neurotoxic effects of Abeta in central neurons (46–48). To test whether NMDA receptors were implicated in Abeta-induced Ca2+ influx leading to calpain activation and subsequent dynamin 1 degradation, we took advantage of specific NMDA receptor inhibitors. For these experiments, 3 weeks in culture hippocampal neurons were treated with MK801 for 1 h and then incubated in the presence of preaggregated Abeta for 24 additional hours (34). Quantitative Western blot analysis showed that MK801 fully prevented the decrease of full-length dynamin 1 and the appearance of the dynamin 1 fragment observed in Abeta-treated neurons (Fig. 6, A and C). MK801 also blocked calpain activation as assessed by spectrin cleavage as described above (Fig. 6, B and D). We also tested whether the FDA approved NMDA receptor antagonist memantine had similar effects to those of MK801 (49). Indeed, memantine completely blocked both Abeta-induced degradation of dynamin 1 and calpain activation in cultured hippocampal neurons (Fig. 6, A–D).

We next tested whether the blockage of L-type VGCCs also prevented calpain activation and dynamin 1 degradation. For these experiments, we preincubated hippocampal neurons with nimodipine, an L-type VGCCs blocker (35). No changes in dynamin 1 or in the appearance of the dynamin 1 fragment where detected from whole cell extracts of nimodipine- and Abeta-treated hippocampal neurons when compared with the ones observed in neurons treated with Abeta alone (Fig. 6, A and C). Furthermore, nimodipine did not decrease the Abeta-induced activation of calpain, as determined by quantitative analysis of the 150/240-kDa spectrin ratio (Fig. 6, B and D).

The changes in dynamin 1 levels described above were also detected when hippocampal neurons cultured under these different experimental conditions were stained using dynamin 1 and tubulin antibodies. Thus, the addition of MK801 blocked the reduction of dynamin 1 immunoreactivity and the appearance of morphological signs of early neurodegeneration induced by preaggregated Abeta (Fig. 7). These neurons showed very similar dynamin 1 distribution throughout the neuronal network as compared with untreated controls (Fig. 7C). Similar results were obtained when dynamin 1 immunoreactivity was assessed in hippocampal neurons treated with memantine prior to the addition of preaggregated Abeta-treated neurons (Fig. 7, E and J). On the other hand, nimodipine had no effect on the Abeta-induced reduction of dynamin 1 staining in cultured hippocampal neurons. Additionally, nimodipine did not prevent the morphological signs of early neurodegeneration normally induced by Abeta (Fig. 7I). Collectively, these data indicated that the blockage of NMDA receptors, but not L-type VGCCs, was able to attenuate dynamin 1 degradation in Abeta-treated hippocampal neurons.

Sustained Abeta-induced Ca2+ Increase Was Dependent on NMDA Receptors in Cultured Hippocampal Neurons—The data described above suggested that Abeta-induced calpain activation and dynamin 1 degradation was dependent on Ca2+ influx through NMDA receptors. We next ruled out that the increase in Ca2+ influx observed in hippocampal neurons treated with preaggregated Abeta was due to action potential-driven network excitation. For these experiments, we added TTX, a Na+-channel blocker, 1 h prior to the incubation of cultured hippocampal neurons in the presence of preaggregated Abeta. Fura-2 Ca2+ imaging assays showed that the addition of preaggregated Abeta to hippocampal neurons treated with TTX did not prevent the sustained activation of Ca2+ observed when these neurons were treated with preaggregated Abeta (Fig. 8, A and B). These data suggested that preaggregated Abeta caused an increase in Ca2+ that was independent from the depolarization mediated by Na+ channels. We next tested whether the addition of the NMDA receptor antagonist MK801 could affect Abeta-induced Ca2+ increase in these neurons. Indeed, the addition of MK801 significantly attenuated the initial Abeta-induced increase of Ca2+ in these neurons (170 nM versus 330 nM, respectively) (Fig. 8C). Similar results were obtained when hippocampal neurons were pretreated with memantine (Fig. 8D). Thus, memantine significantly attenuated the initial Abeta-induced increase of Ca2+ (155 nM versus 330 nM, respectively), and these values returned to baselines levels 20 min after the Abeta treatment. Quantitative analysis of Ca2+ levels at the end point showed that preaggregated Abeta-treated neurons still had significantly increased Ca2+ levels (171 nM) (Fig. 8E). Treatment with TTX did not attenuate this sustained increase in Ca2+. Conversely, treatment with the NMDA receptor antagonists MK801 or memantine allowed these neurons to return to normal baseline levels (97 and 93 nM, respectively). These data suggested that NMDA receptors played an important role in mediating the sustained Ca2+ influx induced by preaggregated Abeta.


Figure 6
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  		FIGURE 6.
NMDA receptor antagonists blocked Abeta-induced calpain activation and dynamin 1 degradation in cultured hippocampal neurons. A and B, Western blot analysis of dynamin 1 (A) and spectrin (B) content in whole cell extracts prepared from hippocampal neurons (21 days in culture) treated with preaggregated Abeta (2 µM) in the absence or presence of MK801 (MK), nimodipine (Nim), or memantine (Mem). C, quantitative analysis of dynamin 1 levels in whole cell extracts obtained from hippocampal neurons treated as described above. Full-length dynamin 1 levels were normalized using {alpha}-tubulin as an internal control. D, quantitative analysis of calpain activation as assessed by spectrin degradation (150/240-kDa ratio) in whole cell extracts obtained from hippocampal neurons treated as described above. Values represent the mean ± S.E. *, differs from untreated controls, p < 0.01.

 


Figure 7
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  		FIGURE 7.
NMDA receptor antagonists blocked Abeta-induced dynamin 1 depletion in cultured hippocampal neurons. Hippocampal neurons grown in culture for 21 days were incubated with preaggregated Abeta (2 µM) in the absence (B and G) or presence of MK801 (C and H), nimodipine (D and I), or memantine (E and J) for 24 h and compared with untreated controls (A and F). Neurons were co-stained using dynamin 1 (A–E) and {alpha}-tubulin antibodies (F–J). Note that the decrease of dynamin 1 immunoreactivity induced by Abeta (B) was blocked when the neurons were treated with MK801 (C) or memantine (E), but not nimodipine (D). Scale bar, 20 µm.

 


Figure 8
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  		FIGURE 8.
NMDA receptor antagonists blocked the sustained Ca2+ increase induced by Abeta in cultured hippocampal neurons. A–D, traces of intracellular Ca2+ levels in hippocampal neurons loaded with fura-2 and treated with preaggregated Abeta (2 µM). Intracellular Ca2+ levels in Abeta-treated hippocampal neurons were measured for 25 min in the absence (A) or presence of TTX (B), MK801 (C), or memantine (D). Traces represent an average of 3 trials totaling 64, 61, 52, and 56 neurons for Abeta, Abeta/TTX, Abeta/memantine, and Abeta/MK801 treatment, respectively. E, intraneuronal Ca2+ levels at the end point of Abeta exposure. Values represent the mean ± S.E. *, differs from untreated controls, p < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented herein indicate that oligomeric species of Abeta induce the degradation of dynamin 1 through calpain activation in hippocampal neurons. In addition, our findings suggest that Abeta-induced calpain activation is due to a disruption of extracellular Ca2+ influx mediated by NMDA receptors. Taken collectively, our data identify a potential mechanism leading to synaptic dysfunction in AD.

Synaptic activity seems to be highly susceptible to the early pathological changes that characterize AD. Thus, a stage in which synaptic function becomes defective precedes synapse loss, neurite degeneration, and neuronal death in this disease (7, 8, 14, 50). However, the molecular mechanisms that contribute to synaptic dysfunction in AD are poorly understood. A growing body of evidence suggests that several presynaptic proteins could play a role in changes in synaptic activity both in AD patients and in AD animal models (21). Building upon these previous findings, the results presented in this report suggest that dynamin 1 might be involved in synaptic dysfunction before signs of degeneration become apparent. The importance of dynamin 1 to the normal functioning of a synapse has been well documented (23–26). These studies showed that in the absence of dynamin 1, synapses lost their ability to release neurotransmitter due to ineffective synaptic vesicle recycling. In addition, the analysis of the ultrastructure of presynaptic terminals in dynamin 1-depleted neurons showed the accumulation of synaptic vesicles at the plasma membranes and a decrease in the releasable synaptic vesicle pool (25, 51). Our data identified soluble oligomers, and not fibrils, as the toxic Abeta forms capable of inducing the decrease in dynamin 1 in cultured hippocampal neurons. These data are in agreement with previous reports showing that the addition of soluble Abeta into the brain of normal rats caused a disruption in LTP, a common measure of synaptic plasticity (18). Conversely, the removal of excessive Abeta from the brain of AD model mice was able to rescue the cognitive deficits normally displayed by these mice (46, 52, 53). It is worth noting that the Abeta oligomer preparation used in this study produced mainly a soluble Abeta species of ~50–75 kDa of apparent molecular mass (supplemental Fig. S3). Soluble oligomers of a similar molecular weight have been found responsible for soluble Abeta toxicity and memory impairment in mice (54, 55). Because the aggregation of Abeta follows a progression from soluble monomers, to a range of soluble oligomers, and finally to insoluble fibrillar aggregates, it is tempting to speculate that the decrease in dynamin 1 induced by soluble Abeta oligomers could be an early step in the pathophysiology of AD.

Abeta-Induced dynamin 1 degradation correlated closely with the activation of calpain in hippocampal neurons (see also Ref. 22). A potential role for this protease in AD has begun to be addressed. Thus, numerous studies have shown an abnormal activation of the calpain system in the brain of AD patients (56–58). In addition, calpain activation has been implicated in the cleavage of a number of other proteins relevant to AD including APP, p35, and microtubule-associated proteins (59–62). These data suggested that calpain might be a key player in AD by catalyzing a linear progression from synaptic dysfunction to neuronal degeneration. As such, this protease could become a potential therapeutic target in AD. It is worth mentioning that initial studies using calpain inhibitors in a mouse model of AD showed encouraging recovery of cognitive function when animals were treated with a calpain inhibitor at an early age (63).

Our results also provided insights into specific mechanisms leading to the Abeta-induced calpain activation in central neurons. Two main mechanisms of calpain regulation have been described under different experimental conditions (reviewed in Ref. 43). One of these mechanisms involves a decrease in calpastatin, the endogenous calpain inhibitor. The decrease in this inhibitor could allow calpain activity to go unchecked and rise above basal levels. This seems not to be the case in Abeta-treated neurons. On the contrary, preaggregated Abeta induced a significant increase in calpastatin levels in cultured hippocampal neurons exposed to this peptide for at least 24 h. The analysis of the time course of this increase in calpastatin content showed that calpain activation preceded the changes in this inhibitor. These data suggested that the increased levels of calpastatin under the experimental conditions analyzed in this study might be an attempt by the neurons to dampen or compensate an abnormally active calpain system and not the cause of this activation. An alternative mechanism of calpain activation might involve Abeta-induced rise in intracellular Ca2+. Recently, it has been shown that soluble Abeta oligomers have the ability to alter the homeostasis of Ca2+ in neurons (64). We showed here that soluble Abeta oligomers induced both a significant and a sustained elevation of intraneuronal Ca2+ in cultured hippocampal neurons. On the other hand, fibrillar Abeta did not elicit such a sustained Ca2+ elevation in these neurons. Similar results have been reported when neuroblastoma cells were incubated in the presence of soluble oligomeric and fibrillar Abeta (64). Our data also indicated that the sustained Ca2+ levels observed in the presence of preaggregated Abeta were in the nanomolar range. It has been shown that in vitro, calpain 1 activation requires µM Ca2+ levels, and therefore, the Ca2+ levels observed in Abeta-treated hippocampal neurons might not be sufficient to activate calpain. However, the calcium requirements for calpain activation in a cellular environment could be significantly lower due to the presence of phospholipids (43, 65, 66). We cannot rule out either that the local Ca2+ concentration at specific subcellular compartments could be much higher than the one detected in the cell bodies of these hippocampal neurons. It is worth noting that the addition of Abeta caused a rapid Ca2+ influx, while calpain activation was not observed until 8 h after Abeta addition. These data suggest that calpain might be activated by the chronic, sustained exposure to Ca2+ and not by the initial increase in intraneuronal Ca2+ in cultured hippocampal neurons.

Our results also provided insights into the source of Ca2+ mobilized in hippocampal neurons cultured in the presence of soluble Abeta oligomers. It has been shown that the disruption of Ca2+ homeostasis from the ER may be an important event in AD. Thus, mutations in the presenilins, proteins that form the secretase complexes that cleave APP into Abeta, lead to reliable disruptions in ER Ca2+ signaling (67–70). However, Abeta-induced Ca2+ release from the ER was not the major source of disruption of Ca2+ homeostasis leading to calpain activation and dynamin 1 degradation under the experimental conditions used in this study. On the other hand, by using BAPTA to eliminate free extracellular Ca2+ we were able to completely block the Abeta-induced activation of calpain and degradation of dynamin 1. These results are in agreement with previous reports showing that excessive Ca2+ influx into neurons cause neurodegeneration (71–74). Although BAPTA effects on Ca2+ levels have been extensively characterized, we cannot completely rule out a direct effect of BAPTA on the function or stability of the NMDA receptors or a potential interaction between this Ca2+ chelator and Abeta.

Soluble Abeta oligomers could induce an extracellular influx of Ca2+ into these neurons through different pathways including the activation of VGCCs and NMDA receptors. Both mechanisms have been proposed to mediate Abeta-induced Ca2+ influx (46–48). Our results using nimodipine suggested that Abeta-induced Ca2+ influx through L-type VGCCs was not necessary for calpain activation and dynamin 1 degradation under these conditions. Conversely, NMDA receptor antagonists MK801 and memantine completely blocked both calpain activation and dynamin 1 degradation. These data strongly suggest that Abeta-induced Ca2+ influx is mediated by NMDA receptors in hippocampal neurons. Surprisingly, a recent study elegantly showed that Abeta oligomers decreased the level of available NMDA receptors at the membrane surface of hippocampal neurons (50). This might be a natural response by the neurons in order to dampen the excessive Ca2+ influx induced by Abeta. One potential mechanism by which Abeta induces increased calcium influx might involve the direct or indirect binding of this peptide to the NMDA receptor. Alternatively, Abeta could induce Ca2+ influx through the activation of AMPA receptors. However, this seems not to be the case in light of our results showing that an AMPA antagonist did not prevent the increase in Ca2+ levels in Abeta-treated hippocampal neurons (supplemental Fig. S4). Further studies will be required to fully understand this Abeta toxic effect.

Collectively, our results show that the activation of calpain by soluble Abeta oligomers could lead to dynamin 1 depletion, and hence, to a stage of synaptic dysfunction in hippocampal neurons. They also suggest that antagonists of the NMDA receptors could have protective effects in central neurons. Furthermore, they provide insights into a novel mechanism by which the FDA-approved NMDA receptor antagonist memantine could significantly improve cognitive functions not only in moderate-to-severe stage of AD but also in the earliest stages of this disease.


   FOOTNOTES
 
* This study was supported by Grant NS39080 from the National Institutes of Health (to A. F.). 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4. Back

1 Supported in part by NIA/AG20506 Training Grant and an American Foundation for Aging Research Fellowship. Back

2 To whom correspondence should be addressed: Northwestern Institute for Neuroscience, Northwestern University, Chicago, IL 60611. Tel.: 312-503-0597; Fax: 312-503-7345; E-mail: a-ferreira{at}northwestern.edu.

3 The abbreviations used are: AD, Alzheimer disease; Abeta, beta-amyloid; APP, amyloid precursor protein; LTP, long term potentiation; NMDA, N-methyl-D-aspartate; MEM, minimum essential medium; MK801, dizocilpine maleate; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; TTX, tetrodotoxin; VAD, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; ALLN, N-acetyl-Leu-Leu-Nle-CHO; IP3R, inositol 1,4,5-trisphosphate receptor; RyR, ryanodine-mediated Ca2+ channel; VGCC, voltage-gated calcium channel; AMPA, {alpha}-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; PBS, phosphate-buffered saline; ER, endoplasmic reticulum. Back


   ACKNOWLEDGMENTS
 
We thank Rachel Bergstrom, Roxanna Sinjuanu, and Peter Toth for excellent technical support and advice. We also thank Dr. Richard Miller (Northwestern University) for generously sharing his calcium imaging equipment with us.



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