β-Amyloid-induced Dynamin 1 Degradation Is Mediated by N-Methyl-D-Aspartate Receptors in Hippocampal Neurons*

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 β-amyloid (Aβ), 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 Aβ, and not fibrillar Aβ, 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 Aβ 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.

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 ␤-amyloid (A␤), 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 A␤, and not fibrillar A␤, 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 A␤ 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.
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)(4)(5)(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 ␤-amyloid (A␤) might be responsible for these changes in neuronal activity in AD (9 -14). A␤, a fragment of 40 or 42 amino acids, is the product of the sequential cleavage of the amyloid precursor protein (APP) mediated by the ␤and ␥-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 A␤ fragments aggregate, and eventually deposit as large fibrils in the extracellular space forming amyloid plaques (17).
Soluble oligomeric species of A␤ 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 A␤-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 A␤ 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)(24)(25)(26). Collectively, these data suggest that A␤-induced dynamin 1 depletion might contribute to synaptic dysfunction in AD.
In the present study, we analyzed the molecular mechanisms leading to A␤-induced dynamin 1 depletion in hippocampal neurons. Our results showed that A␤-induced decrease in dynamin 1 involved calpain-mediated degradation. Furthermore, these findings suggested that A␤-induced calpain activation was dependent on the presence of A␤ soluble oligomers and on external Ca 2ϩ influx mediated by N-methyl-D-aspartate (NMDA) receptors.

EXPERIMENTAL PROCEDURES
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 Ca 2ϩ 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 ϳ95% pyramidal neurons and 5% glial cells.
A␤ Aggregation and Treatment-Synthetic A␤ 1-40 (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 A␤ was added to the culture medium at a final concentration of 2 M, 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 A␤ was centrifuged at 100,000 ϫ 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 A␤ forms for up to 24 h. For experiments using fura-2, synthetic A␤ 1-40 was dissolved in MilliQ H 2 O 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-␣-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 antispectrin (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 pro-teins. 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 Ϯ 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 A␤ 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.
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-␣-tubulin (1:600; clone DM1A). The following secondary antibodies were used: anti-mouse IgG fluorescein-conjugated and antirabbit 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 Ca 2ϩ indicator fura-2 was accomplished by incubating with 3 M fura-2-AM (Molecular Probes, Eugene, OR) in Krebs-HEPES buffer (100 mM NaCl, 180 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 1 mM Na 3 PO 4 , 4.2 mM Na 2 CO 3 , 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 A␤ in the absence or presence of the TTX and NMDA receptor blockers as described above. Preaggregated A␤ 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). [Ca 2ϩ ] i was calibrated with a range of known Ca 2ϩ concentrations in solution with fura-2. Quantitative analysis using fura-2 imaging was performed on three independent experiments (ϳ60 cells). Each experimental condition reflects an average of these trials and was compared with the [Ca 2ϩ ] i of untreated neurons.

Soluble A␤ Oligomers Activated Calpain in Cultured
Hippocampal Neurons-We have previously shown that preaggregated A␤ induced dynamin 1 depletion in cultured hippocampal neurons (22). Our results also suggested that calpain activation might be responsible for this A␤ effect. To gain further insights into A␤-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 M of preaggregated A␤ for 24 h there was a significant decrease in full-length dynamin 1 levels (100 kDa) and an increase of a ϳ90-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 A␤ when compared with untreated controls (Fig. 1C). These findings suggested an A␤-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 A␤. Western blot analysis showed no changes in full-length dynamin 1 levels in hippocampal neurons incubated with ALLN and preaggregated A␤ for 24 h when compared with untreated controls. In addition, the ϳ90-kDa dynamin 1 fragment was not detected in cultured hippocampal neurons treated with both ALLN and preaggregated A␤ (Fig. 1A). Spectrin degradation was also attenuated in hippocampal neurons cultured in the presence of ALLN and preaggregated A␤ (Fig.  1A). Thus, no changes in full-length spectrin and/or the 150/ 240-kDa spectrin ratio were detected in ALLN-and preaggregated A␤-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 ϳ90-kDa fragment in hippocampal neurons incubated with VAD and preaggregated A␤ for 24 h were similar to the ones observed in hippocampal neurons incubated with preaggregated A␤ alone (Fig. 1A). This caspase inhibitor had no effect either on A␤-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 A␤-induced dynamin 1 degradation under our experimental conditions (see also Ref. 22). We then determined whether a particular species of A␤ was responsible for calpain activation and dynamin 1 degradation in cultured hippocampal neurons. For these experiments, preaggregated A␤ was centrifuged to separate the soluble oligomeric forms of the peptide from the larger, insoluble fibrillar aggregates as previously described (22). The preaggregated A␤ (referred to as mixed because of its likelihood to contain both insoluble fibrillar A␤ along with soluble oligomeric A␤) was added to the cultured medium of 3 weeks in culture hippocampal neurons at a final concentration of 2 M. 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 A␤ caused a decrease in full-length dynamin 1 and an increase in its ϳ90-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 A␤ (2 M) for 24 h (Fig. 1B). Fibrillar A␤ also failed to induce calpain activation in these neurons as assessed by spectrin degradation (Fig. 1D). On the other hand, the addition of 2 M oligomeric A␤ 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 A␤ in these neurons (Fig. 1D).

A␤-induced Activation of Calpain Was Likely Due to a Disruption in Ca 2ϩ
Homeostasis-Calpastatin, an endogenous calpain inhibitor, and changes in Ca 2ϩ levels are among the most well-studied regulators of calpain activity (reviewed in Ref. 43).
To determine how calpain was activated in A␤-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 A␤ (2 M) 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 A␤ as compared with untreated controls ( Fig. 2A). On the other hand, a significant increase (ϳ40%) in calpastatin levels were detected in hippocampal neurons exposed to preaggregated A␤ 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 A␤. In addition, they suggested that the calpain activation observed in preaggregated A␤-treated hippocampal neurons was not caused by a decrease in the level of calpastatin. Therefore, we studied next whether an abnormal increase in intraneuronal Ca 2ϩ could contribute to the activation of calpain in these A␤-treated neurons. For these experiments, we added preaggregated (mixed, soluble oligomeric, and fibrillar) A␤ (2 M) to the hippocampal neurons that were previously loaded with fura-2. Fura-2-based calcium imaging is commonly used to measure real-time intracellular Ca 2ϩ levels (41,42). The addition of mixed A␤ (2 M) produced a significant spike in Ca 2ϩ levels (ϳ7-fold increase above basal intraneuronal Ca 2ϩ levels) in these cultured hippocampal neurons (Fig. 3A). Over the next 12 min, Ca 2ϩ levels declined and eventually plateau. However, these plateau levels were significantly higher than the Ca 2ϩ levels detected before the addition of the peptide (Fig.  3A). We next tested whether A␤ oligomers or A␤ fibrils were responsible for this disruption of Ca 2ϩ homeostasis in hippocampal neurons. The addition of oligomeric A␤ (2 M) induced a significant and instantaneous rise in Ca 2ϩ (ϳ5-fold increase above the basal intraneuronal Ca 2ϩ levels) in hippocampal neurons (Fig. 3B). These Ca 2ϩ levels decreased thereafter and reached plateau levels that were similar to the ones detected when hippocampal neurons were incubated in the presence of mixed A␤ (Fig. 3B). Fibrillar A␤ also induced changes in intracellular Ca 2ϩ 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 A␤. Thus, Ca 2ϩ levels significantly increased (ϳ2-fold increase above the basal intraneuronal Ca 2ϩ levels) in the presence of fibrillar A␤ (Fig. 3C). These Ca 2ϩ levels decreased to baseline levels (ϳ80 nM) ϳ15 min after the addition of fibrillar A␤ (Fig. 3D). To determine whether there was a significant elevation of intraneuronal Ca 2ϩ at the end point of our experimental period, we quantified the Ca 2ϩ measurements 23 min after the addition of the different forms of A␤ (Fig. 3D). This quantitative analysis showed that Ca 2ϩ levels were significantly higher in hippocampal neurons treated with either mixed or oligomeric A␤ when compared with basal levels obtained in the absence of these peptides. In contrast, hippocampal neurons that were exposed to fibrillar A␤ had recovered to baseline Ca 2ϩ levels (Fig. 3D).

A␤-induced Calpain Activation and Dynamin 1 Degradation
Were Dependent on the Extracellular Influx of Ca 2ϩ -Collectively, the data described above suggested that soluble oligomeric A␤ was responsible for a sustained disruption of Ca 2ϩ homeostasis in hippocampal neurons. However, they did not provide information regarding the Ca 2ϩ source mobilized under our experimental conditions. To determine whether this A␤-induced Ca 2ϩ 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 Ca 2ϩ release from the endoplasmic reticulum (ER), the major source of intracellular Ca 2ϩ release. Since this Ca 2ϩ release is regulated through inositol 1,4,5,-trisphosphate receptors (IP 3 R) or ryanodine-mediated Ca 2ϩ channels (RyR), we incubated 3 weeks in culture hippocampal neurons with xestospongin C (1 M) and dantrolene (10 M), inhibitors of IP 3 Rs and RyRs, respectively (36,44,45). Cultured hippocampal neurons treated with these inhibitors for 1 h were then incubated with preaggregated A␤ (2 M) for an additional 24 h. Quantitative analysis of immunoreactive bands in whole cell extracts showed a significant decrease (ϳ60%) in fulllength dynamin 1 and the appearance of the dynamin 1 fragment in hippocampal neurons treated with preaggregated A␤ when compared with untreated controls (Fig. 4, A and D). Blockage of Ca 2ϩ release from the ER with xestospongin C and dantrolene had no effect on the A␤-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 A␤-induced degradation of dynamin 1 and calpain activation was not due to intracellular release of Ca 2ϩ from the ER.
We next tested whether A␤-induced dynamin 1 degradation and calpain activation was caused by the extracellular influx of Ca 2ϩ . For these experiments, 3 weeks in culture hippocampal neurons were incubated with BAPTA, a chelator of extracellular Ca 2ϩ , for 1 h prior to the addition of preaggregated A␤ (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 A␤-treated hippocampal neurons (Fig. 4, B and E). The addition of BAPTA to the culture medium also blocked A␤-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 A␤ 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 A␤ 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 A␤ 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 A␤ (2 M) 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 A␤ (Fig. 5C). BAPTA also prevented the signs of early neuronal degeneration (varicosities along the neurites) induced by preaggregated A␤ treatment (Fig. 5, E and F). Therefore, the processes extended by preaggregated A␤ and BAPTA-treated neurons were undistinguishable from the ones extended from the cell bodies of untreated controls (Fig. 5D).
A␤-induced Calpain Activation and Dynamin 1 Degradation Were Dependent on the Activation of NMDA Receptors-The experiments described above suggested that preaggregated A␤ induced an extracellular Ca 2ϩ influx. Ca 2ϩ 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 A␤ in central neurons (46 -48). To test whether NMDA receptors were implicated in A␤-induced Ca 2ϩ 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 A␤ for 24 additional hours (34). Quantitative Western blot analysis showed that MK801 fully prevented the decrease of fulllength dynamin 1 and the appearance of the dynamin 1 fragment observed in A␤-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 A␤-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 A␤-treated hippocampal neurons when compared with the ones observed in neurons treated with A␤ alone (Fig. 6, A and C). Furthermore, nimodipine did not decrease the A␤-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 A␤ (Fig. 7). These neurons showed very similar dynamin 1 distribution throughout the neuronal network as compared with untreated controls (Fig. 7C). Similar  Neurons were co-stained using dynamin 1 (A-C ) and ␣-tubulin antibodies (D-F ). Note that the decrease in dynamin 1 immunoreactivity induced by A␤ (B) was blocked when the neurons were treated with BAPTA and A␤ (C ). Scale bar, 20 m. results were obtained when dynamin 1 immunoreactivity was assessed in hippocampal neurons treated with memantine prior to the addition of preaggregated A␤-treated neurons (Fig.  7, E and J). On the other hand, nimodipine had no effect on the A␤-induced reduction of dynamin 1 staining in cultured hippocampal neurons. Additionally, nimodipine did not prevent the morphological signs of early neurodegeneration normally induced by A␤ (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 A␤-treated hippocampal neurons.
Sustained A␤-induced Ca 2ϩ Increase Was Dependent on NMDA Receptors in Cultured Hippocampal Neurons-The data described above suggested that A␤-induced calpain activation and dynamin 1 degradation was dependent on Ca 2ϩ influx through NMDA receptors. We next ruled out that the increase in Ca 2ϩ influx observed in hippocampal neurons treated with preaggregated A␤ was due to action potentialdriven network excitation. For these experiments, we added TTX, a Na ϩ -channel blocker, 1 h prior to the incubation of cultured hippocampal neurons in the presence of preaggregated A␤. Fura-2 Ca 2ϩ imaging assays showed that the addition of preaggregated A␤ to hippocampal neurons treated with TTX did not prevent the sustained activation of Ca 2ϩ observed when these neurons were treated with preaggregated A␤ (Fig. 8, A  and B). These data suggested that preaggregated A␤ caused an increase in Ca 2ϩ that was independent from the depolarization mediated by Na ϩ channels. We next tested whether the addition of the NMDA receptor antagonist MK801 could affect A␤-induced Ca 2ϩ increase in these neurons. Indeed, the addition of MK801 significantly attenuated the initial A␤-induced increase of Ca 2ϩ 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 A␤-induced increase of Ca 2ϩ (155 nM versus 330 nM, respectively), and these values returned to baselines levels 20 min after the A␤ treatment. Quantitative analysis of Ca 2ϩ levels at the end point showed that preaggregated A␤-treated neurons still had significantly increased Ca 2ϩ levels (171 nM) (Fig. 8E). Treatment with TTX did not attenuate this sustained increase in Ca 2ϩ . 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 Ca 2ϩ influx induced by preaggregated A␤.

DISCUSSION
The results presented herein indicate that oligomeric species of A␤ induce the degradation of dynamin 1 through calpain activation in hippocampal neurons. In addition, our findings suggest that A␤-induced calpain activation is due to a disruption of extracellular Ca 2ϩ 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)(24)(25)(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 A␤ 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 A␤ into the brain of normal rats caused a disruption in LTP, a common measure of synaptic plasticity (18). Conversely, the removal of excessive A␤ 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 A␤ oligomer preparation used in this study produced mainly a soluble A␤ species of ϳ50 -75 kDa of apparent molecular mass (supplemental Fig. S3). Soluble oligomers of a similar molecular weight have been found responsible for soluble A␤ toxicity and memory impairment in mice (54,55). Because the aggregation of A␤ 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 A␤ oligomers could be an early step in the pathophysiology of AD.
A␤-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 A␤-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 A␤-treated neurons. On the contrary, preaggregated A␤ 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 A␤-induced rise in intracellular Ca 2ϩ . Recently, it has been shown that soluble A␤ oligomers have the ability to alter the homeostasis of Ca 2ϩ in neurons (64). We showed here that soluble A␤ oligomers induced both a significant and a sustained elevation of intraneuronal Ca 2ϩ in cultured hippocampal neurons. On the other hand, fibrillar A␤ did not elicit such a sustained Ca 2ϩ elevation in these neurons. Similar results have been reported when neuroblastoma cells were incubated in the presence of soluble oligomeric and fibrillar A␤ (64). Our data also indicated that the sustained Ca 2ϩ levels observed in the presence of preaggregated A␤ were in the nanomolar range. It has been shown that in vitro, calpain 1 activation requires M Ca 2ϩ levels, and therefore, the Ca 2ϩ levels observed in A␤-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 Ca 2ϩ 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 A␤ caused a rapid Ca 2ϩ influx, while calpain activation was not observed until 8 h after A␤ addition. These data suggest that calpain might be activated by the chronic, sustained exposure to Ca 2ϩ and not by the initial increase in intraneuronal Ca 2ϩ in cultured hippocampal neurons.
Our results also provided insights into the source of Ca 2ϩ mobilized in hippocampal neurons cultured in the presence of soluble A␤ oligomers. It has been shown that the disruption of Ca 2ϩ 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 A␤, lead to reliable disruptions in ER Ca 2ϩ signaling (67)(68)(69)(70). However, A␤-induced Ca 2ϩ release from the ER was not the major source of disruption of Ca 2ϩ 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 Ca 2ϩ we were able to completely block the A␤-induced activation of calpain and degradation of dynamin 1. These results are in agreement with previous reports showing that excessive Ca 2ϩ influx into neurons cause neurodegeneration (71)(72)(73)(74). Although BAPTA effects on Ca 2ϩ 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 Ca 2ϩ chelator and A␤.
Soluble A␤ oligomers could induce an extracellular influx of Ca 2ϩ into these neurons through different pathways including the activation of VGCCs and NMDA receptors. Both mechanisms have been proposed to mediate A␤-induced Ca 2ϩ influx (46 -48). Our results using nimodipine suggested that A␤-induced Ca 2ϩ 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 A␤-induced Ca 2ϩ influx is mediated by NMDA receptors in hippocampal neurons. Surprisingly, a recent study elegantly showed that A␤ 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 Ca 2ϩ influx induced by A␤. One potential mechanism by which A␤ induces increased calcium influx might involve the direct or indirect binding of this peptide to the NMDA receptor. Alternatively, A␤ could induce Ca 2ϩ 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 Ca 2ϩ levels in A␤-treated hippocampal neurons (supplemental Fig.  S4). Further studies will be required to fully understand this A␤ toxic effect.
Collectively, our results show that the activation of calpain by soluble A␤ 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.