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Originally published In Press as doi:10.1074/jbc.M503259200 on July 15, 2005 Originally published In Press as doi:10.1074/jbc.M503259200 on July 8, 2005 Originally published In Press as doi:10.1074/jbc.M503259200 on July 7, 2005

J. Biol. Chem., Vol. 280, Issue 36, 31746-31753, September 9, 2005
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{beta}-Amyloid-induced Dynamin 1 Depletion in Hippocampal Neurons

A POTENTIAL MECHANISM FOR EARLY COGNITIVE DECLINE IN ALZHEIMER DISEASE*

Brent L. Kelly{ddagger}§, Robert Vassar{ddagger}, and Adriana Ferreira{ddagger}||

From the {ddagger}Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University and the Institute for Neuroscience, Northwestern University, Chicago, Illinois 6061

Received for publication, March 24, 2005 , and in revised form, June 17, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Synaptic dysfunction is one of the earliest events in the pathogenesis of Alzheimer disease (AD). However, the molecular mechanisms underlying synaptic defects in AD are largely unknown. We report here that {beta}-amyloid (A{beta}), the main component of senile plaques, induced a significant decrease in dynamin 1, a protein that is essential for synaptic vesicle recycling and, hence, for memory formation and information processing. The A{beta}-induced dynamin 1 decrease occurred in the absence of overt synaptic loss and was also observed in the Tg2576 mouse model of AD. In addition, our results provided evidence that the A{beta}-induced decrease in dynamin 1 was likely the result of a calpain-mediated cleavage of dynamin 1 protein and possibly the down-regulation of dynamin 1 gene expression. These data suggest a mechanism to explain the early cognitive loss without a major decline in synapse number observed in AD and propose a novel therapeutic target for AD intervention.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Senile plaques, neurofibrillary tangles, synapse loss, and gross neurodegeneration are common findings in the brain of AD1 patients (1-4). Numerous genetic, biochemical, and animal model studies have implicated the gradual buildup of A{beta}, the main component of senile plaques, as the catalyst for AD. However, the mechanistic link between A{beta} accumulation and the progressive cognitive impairment associated with this disease has not been elucidated. Synapse loss seems to be the best morphological correlate of the functional deficits observed in the mid-to-late stages of AD (3, 4). In contrast, patients in the earliest stages of the disease show no significant decline in synapse number (5). Based on these findings, it has been hypothesized that a stage of synaptic dysfunction might precede frank synapse loss, plaque accumulation, and tangle formation in AD (6, 7). The mechanisms underlying such synaptic dysfunction remain unknown. It is tempting to speculate that proteins involved in synaptic vesicle biogenesis and/or recycling might play a critical role in AD. Data obtained recently seem to support this view. Thus, changes in the levels of a number of presynaptic proteins, including SNAP-25, syntaxin, and synaptotagmin, have been reported in AD (8). More recently, dynamin 1, a protein highly enriched in presynaptic terminals, has been shown to be significantly reduced in AD brains (9). Dynamin 1, a well studied neuron-specific mechanochemical GTPase, pinches off synaptic vesicles, freeing them from the membrane and allowing them to re-enter the synaptic vesicle pool to be refilled for future release (10, 11). The essential role for dynamin 1 in vesicle scission and synaptic function has been best supported by studies of the Drosophila model shibire, a temperature-sensitive mutant of a dynamin ortholog (12, 13). At restrictive temperatures these flies displayed a paralysis phenotype. This functional deficit was accompanied by the depletion of synaptic vesicles and the accumulation of invaginated pits at pre-synaptic membranes adjacent to the synaptic clefts. Collectively, these data suggested that the stressors that induce dynamin 1 loss-of-function could result in the diminution of available synaptic vesicles leading to synaptic dysfunction.

In the present study we analyzed whether A{beta} was one of such stressors using hippocampal neurons that develop in culture and in an AD animal model system. Our results showed that A{beta} induced a significant reduction in dynamin 1 levels that preceded synapse loss in both model systems. In cultured neurons, our data suggested that the A{beta}-induced decrease in dynamin 1 was likely the result of a bimodal mechanism that involved calpain-mediated proteolysis and the down-regulation of dynamin 1 gene expression. On the other hand, in the AD mouse model Tg2576 the decrease in dynamin 1 was mainly the result of calpain activation. These mechanisms identify novel therapeutic targets to address the synaptic dysfunction preceding synapse loss and neurodegeneration in the context of AD.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of Hippocampal Cultures—Embryonic day 18 rat embryos were used to prepare primary hippocampal cultures as described previously (14, 15). 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 per 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) (16). For immunocytochemistry studies the 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.

A{beta} Treatment—Synthetic A{beta}-(1-40) or A{beta}-(1-42) (Sigma-Aldrich) was dissolved in N2 medium at 0.1 mg/ml and incubated for 4 days at 37 °C to preaggregate the peptide (17). This preaggregated A{beta} was added to the culture medium at final concentrations ranging from 0.02 to 20 µM. For some experiments, the aggregated A{beta} was centrifuged at 100,000 x g for 1 h to separate the oligomeric (supernatant) from the 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 the neurons at final A{beta} concentrations calculated using the initial concentration of the monomeric form of the peptide. Monomeric A{beta} preparations were prepared by dissolving the peptide in N2 medium immediately before use. Hippocampal neurons were grown in the presence of the peptide for up to 36 h.



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  		FIG. 1.
A{beta} induced a decrease in dynamin levels in a dose- and time-dependent manner in cultured hippocampal neurons. A and B, Western blot analysis of dynamin 1 (Dyn 1), dynamin 2 (Dyn 2), dynamin 3 (Dyn 3), and synaptophysin (Syn) content in whole cell extracts prepared from hippocampal neurons (21-days in vitro) cultured in the absence (column C) or presence of A{beta}-(1-40) (A{beta}) or A{beta}-(1-42) (A{beta}42) (20 µM) for 36 h. Note the decrease in dynamin 1 (100 kDa) and dynamin 2 immunoreactive bands and the appearance of a second dynamin 1 (~90 kDa) immunoreactive band in A{beta}-treated neurons. C and E, Western blot analysis of dynamin (Dyn) and synaptophysin (Syn) content in whole cell extracts prepared from hippocampal neurons (21 days in vitro) cultured with increasing concentrations of A{beta} for 36 h (C) or cultured in the presence of A{beta} (2 µM) for up to 36 h (E). D and F, quantification analysis of the dose-response (panel C) and time course (panel E) effects of A{beta} in cultured hippocampal neurons. The results were normalized using tubulin as the internal control. The values obtained in untreated controls were considered 100%. Values represent the mean ± S.E. obtained from 6-8 independent experiments. *, p < 0.05; **, p < 0.01 (differs from untreated controls).

 
Protease Inhibitors and in Vitro Cleavage—A series of cell-permeable inhibitors, including the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (5-50 µM; Promega, Madison, WI) and the calpain inhibitors N-acetyl-Leu-Leu-norleucine-CHO (5-50 µM; Santa Cruz Biotechnology, Santa Cruz, CA), calpeptin (0.1-10 µM; Calbiochem, San Diego, CA), and MDL-28170 (0.1-10 µM; Calbiochem), were added to the medium of hippocampal neurons (which had been kept in culture 3 weeks) 1 h prior to and for the duration of the A{beta} treatment. For experiments using purified caspase-3 (Sigma-Aldrich) and calpain (Calbiochem), whole cell lysates prepared from cultured hippocampal neurons or tissue from the hippocampal region were incubated with caspase-3 or calpain for 1 h at 37 °C. Both proteases were used at final concentrations ranging from 0.002 to 200 units.

Electrophoresis and Immunoblotting—Whole cell extracts were prepared from hippocampal neurons that developed either in culture or in vivo. 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. Hippocampi were also dissected from SwAPP695 transgenic (Tg2576) mice and strain-matched, wild-type (WT) mice. The mice used for these experiments were 2-month-old WT (n = 3), 2-month-old Tg2576 (n = 3), 5-month-old WT (n = 4), 5-month-old Tg2576 (n = 6), 8-month-old WT (n = 3), and 8-month-old Tg2576 (n = 5) mice. Hippocampi were mechanically homogenized in Laemmli buffer and boiled for 10 min. Samples were run on 7.5% SDS-polyacrylamide gels and transferred to Immobilon membranes (Millipore, Billerica, MA). Immunodetection was performed using the following antibodies: anti-{alpha}-tubulin (1:200,000; clone DM1A; Sigma-Aldrich); anti-dynamin 1 raised against an immunogen corresponding to amino acids 633-647 (1:5,000; Affinity BioReagents, Golden, CO); anti-dynamin 1 (1:1,000; gift from Dr. Mark McNiven, Mayo Clinic, Rochester, MN); anti-dynamin 2 (1:1,000; Affinity BioReagents); anti-dynamin 3 (1:1,000; Affinity BioReagents); anti-synaptophysin (1:100,000; Santa Cruz Biotechnology); anti-class III {beta}-tubulin (1:500; clone TUJ1) (18); and anti-spectrin (1:1,000; Chemicon, Temecula, CA). Secondary antibodies conjugated to horseradish peroxidase (Promega) followed by enhanced chemiluminescence reagents (Amersham Biosciences) were used for the detection of proteins. Immunoreactive bands were detected and imaged using a ChemiDoc XRS (Bio-Rad). Densitometry of the images was performed using Quantity One software (Bio-Rad). Densitometric values were normalized using tubulin as an internal control.

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 primary antibodies used for neuronal staining were polyclonal anti-dynamin 1 (1:1,000; Affinity BioReagents) and monoclonal anti-synaptophysin (1:1,000; Santa Cruz Biotechnology). The secondary antibodies used were the anti-mouse IgG fluorescein-conjugated antibody and anti-rabbit IgG rhodamine-conjugated antibody (1:1,000; Chemicon). To quantify immunoreactive spots, untreated and A{beta}-treated cultured hippocampal neurons were stained using dynamin 1 and synaptophysin antibodies as described above. Five random fields were selected for the quantification of dynamin 1 and synaptophysin immunoreactive spots, which was done at a set intensity using MetaMorph Image analysis software (Universal Imaging Corporation, Fryer Company Inc., Huntley, IL). For the staining of the A{beta} peptide, the aggregated, fibrillar, and oligomeric fractions were dried on a slide, fixed with 4% paraformaldehyde, and rinsed twice in PBS. The fractions were preincubated in 10% bovine serum albumin in PBS for 1 h at 37 °C and exposed to anti-A{beta} (1:500; clone 6e10; Sigma-Aldrich) overnight at 4 °C. The fractions were then rinsed in PBS and incubated with anti-mouse IgG fluorescein-conjugated antibody for 1 h at 37 °C.



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  		FIG. 2.
Oligomeric forms of A{beta} depleted dynamin 1 in cultured hippocampal neurons. A, A{beta} peptide aggregated for 4 days at 37 °C (Mix) was centrifuged (100,000 x g) to separate the oligomers (Olig) from the fibrils (Fib) and stained with an anti-A{beta} antibody. B, Western blot analysis of dynamin 1 (Dyn 1) and synaptophysin (Syn) in whole cell extracts prepared from hippocampal neurons (21 days in vitro) cultured with 2 µM monomeric (Mon), mixed (Mix), fibrillar (Fib), or oligomeric (Olig) A{beta} for 24 h. {alpha}-tub, {alpha}-tubulin. C, quantification analysis of dynamin 1 (Dyn 1) and synaptophysin (Syn) protein levels in cultured hippocampal neurons treated with these different forms of A{beta}. The results were normalized using {alpha}-tubulin as an internal control (column C). The values obtained in untreated controls were considered 100%. Values represent the mean ± S.E. obtained from three independent experiments. *, p < 0.05; **, p < 0.01 (differs from untreated controls).

 



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  		FIG. 3.
Fibrillar A{beta} decreased dynamin 1 at synaptic sites in cultured hippocampal neurons. Hippocampal neurons kept in culture for 21 days were incubated in the absence (A-D) or presence (E-H) of A{beta} (2 µM) for 24 h, fixed, and co-stained with dynamin 1 (A and E) and synaptophysin (B and F) antibodies. Note the decrease of dynamin 1 staining in the distal portions of processes extended by A{beta}-treated neurons (G and H) as compared with untreated controls (C and D). The scale bar for low magnification pictures (A-C and E-G) is shown in panel G, whereas the scale bar for the high magnification pictures (D and H) is shown in panel H. Scale bars, 20 µm

 
RT-PCR—To obtain total mRNA, cultured hippocampal neurons and hippocampi obtained from WT and Tg2576 mice were homogenized in TRIzol® Reagent (Invitrogen). RNA was extracted by phenol/chloroform according to the TRIzol® Reagent manufacturer's protocol. Reverse transcription was performed in 20-µl reactions containing 1 µg of sample RNA, 2.5 units of murine leukemia virus reverse transcriptase, 2.5 µM random hexamers, 1 unit of RNase inhibitor, 1 mM dATP, 1 mM dCTP, 1 mM dTTP, 1 mM dGTP, 5 mM MgCl2 solution, and 2 µl of l0x buffer II (PerkinElmer Life Sciences GeneAmp RNA PCR core kit, catalog number N808-0143). Tubes were incubated at 42 °C for 15 min at 99 °C for 5 min and then at 5 °C for 5 min. Real time RT-PCR was performed using 18 S ribosomal subunit primers (Applied Biosystems, Foster City, CA) as endogenous controls and dynamin 1-specific primers (Applied Biosystems) as the target genes. Real-time RT-PCR reactions were performed in 20-µl reactions using TaqMan universal PCR master mix (Applied Biosystems), 135 ng of cDNA, and TaqMan® primers, which included probes conjugated with fluorescein as the reporter dyes. All real time RT-PCR reactions were performed in triplicate and analyzed as relative quantifications of dynamin 1 in A{beta}-treated cultured hippocampal neurons versus untreated cultured hippocampal neurons and Tg2576 hippocampi versus WT hippocampi using the ABI 7900HT Detection System (Applied Biosystems).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
A{beta} Induced a Dynamin 1 Reduction in Cultured Hippocampal Neurons—To test whether the deposition of A{beta} could cause a depletion of dynamin 1, we analyzed dynamin 1 protein levels in cultured hippocampal neurons treated with A{beta}. We have chosen this model system because the hippocampus is one of the brain regions most affected in AD. In addition, synaptic integrity in the hippocampus is crucial to memory formation. Furthermore, we and others have shown previously that, when kept in culture for >3 weeks, hippocampal neurons reproduce the molecular composition and functional characteristics of mature neurons in vivo (17, 19, 20). The addition of A{beta}-(1-40) (20 µM) to the culture medium of these mature hippocampal neurons induced a significant decrease in dynamin 1 (100 kDa) as determined by Western blot analysis. Dynamin 2, a ubiquitous dynamin isoform, as well as synaptophysin, a pre-synaptic protein, were also decreased in A{beta}-treated neurons (Fig. 1A). However, the loss of dynamin 1 and 2 was more extensive than that of synaptophysin. Synaptophysin is a synaptic vesicle protein with four transmembrane domains that anchor it to synaptic vesicles in the presynaptic compartment of nerve terminals (21). Synaptophysin levels correlate closely with synapse numbers and are commonly used to assay for loss of synapses (5, 22). On the other hand, no changes in the levels of dynamin 3, a dynamin isoform also expressed in the nervous system, were detected in A{beta}-treated neurons when compared with untreated controls. Interestingly, in addition to the changes in dynamin 1 levels described above, a second smaller dynamin 1 immunoreactive band (~90 kDa) was detected in the A{beta}-treated neurons (Fig. 1A). Similar results were obtained when cultured hippocampal neurons were incubated with preaggregated A{beta}-(1-42) (Fig. 1B). All experiments described below were performed with A{beta}-(1-40).

To determine whether A{beta} induced the decrease of dynamin levels in a dose-dependent manner, mature hippocampal neurons were incubated with A{beta} at final concentrations ranging from 0.02 to 20 µM for 36 h. The content of dynamin 1, 2, and 3, as well as synaptophysin in whole cell lysates was determined by means of Western blot analysis (Fig. 1, C and D). Our results showed a dose-dependent decrease in the levels of dynamin 1 and 2, but not of dynamin 3, in A{beta}-treated neurons as compared with untreated controls. However, synaptophysin levels were unchanged when cultured hippocampal neurons were incubated with A{beta} at concentrations below 20 µM. These results suggested that synapse numbers were not affected at these lower concentrations. On the other hand, the appearance of the ~90-kDa dynamin 1 immunoreactive band was evident in cell extracts prepared from hippocampal neurons cultured in the presence of A{beta} at all of the concentrations analyzed (Fig. 1C). Next, we determined whether A{beta} affects dynamin levels in a time-dependent manner. For these experiments, hippocampal neurons kept in culture for 3 weeks were incubated with A{beta} (2 µM) for up to 36 h (Fig. 1, E and F). The effect of A{beta} on dynamin 1 and 2 levels was evident as early as 8 h after the addition of the peptide. In contrast, no changes in dynamin 3 and synaptophysin levels were detected throughout the time period analyzed (Fig. 1, C and E).

Because the aggregation state of A{beta} has been proposed to play a critical role in its neurotoxic effects, we next determined which form of the A{beta} peptide was causing these effects on dynamin 1. The preaggregated A{beta} preparation likely contained both large insoluble fibrillar forms and smaller soluble oligomeric forms of the peptide. Therefore, we separated these two forms of A{beta} by centrifugation. To determine whether we had successfully separated the fibrillar A{beta} from the oligomeric A{beta}, we immunostained each fraction with an A{beta} antibody. The aggregated, pre-centrifuged preparation, termed "mixed," showed large globular immunoreactive aggregates along with smaller species (Fig. 2A). Following centrifugation, the oligomeric fraction showed numerous small spherical immunoreactive species, while the fibrillar fraction showed mainly large globular aggregates (Fig. 2A). To test whether these separate forms of A{beta} had different effects on dynamin 1, we incubated cultured hippocampal neurons with the monomeric, mixed, oligomeric, and fibrillar forms of A{beta} (2 µM each) for 24 h. Western blot analysis showed a decrease in dynamin 1 levels only in whole cell extracts obtained from hippocampal neurons incubated with the mixed and oligomeric forms of A{beta} (Fig. 2, B and C). We also observed the appearance of the ~90-kDa band in the mixed and oligomeric preparations only (Fig. 2B).



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  		FIG. 4.
A{beta} induced the reduction of dynamin 1 both at transcriptional and post-transcriptional levels in cultured hippocampal neurons. A, Western blot analysis of dynamin 1 content in whole cell extracts obtained from hippocampal neurons (21 days in vivo) incubated in the presence of increasing concentrations of recombinant caspase-3 or calpain. Note that calpain incubation produced a lower molecular mass dynamin 1 immunoreactive band (~90 kDa) similar to the one obtained when cultured hippocampal neurons were treated with A{beta} (far right column). Far left column, control. B, Western blot analysis of dynamin 1 content in whole cell extracts prepared from hippocampal neurons (21 days in vivo) treated with increasing concentrations of various protease inhibitors for 1 h prior to the addition of A{beta} (2 µM). Inhibitors included a general caspase inhibitor (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK)), a calpain/proteosome inhibitor (N-acetyl-Leu-Leu-norleucine-CHO (ALLN)), and calpain inhibitors (calpeptin and MDL-28170 (MDL)). Only inhibitors blocking calpain activity prevented cleavage of dynamin 1. Columns C, control. C, conventional RT-PCR analysis of dynamin 1 (Dyn 1) mRNA bands in untreated and A{beta}-treated (2 µM) cultured hippocampal neurons. {beta}-actin ({beta}-act) was used as an internal control. Column C, control. D, real time RT-PCR of dynamin 1 mRNA in untreated and A{beta}-treated (2 µM) cultured hippocampal neurons. The dynamin 1 signal was normalized using the 18 S ribosomal gene as an endogenous control. Column C, control. Values obtained in untreated controls were set at 100%. Values represent the mean ± S.E. obtained from six independent experiments. *, p < 0.05; **, p < 0.01 (differs from untreated controls).

 
Treatment with A{beta} also altered the distribution of dynamin 1 in mature hippocampal neurons. In untreated neurons, dynamin 1 was highly enriched in the cell bodies. In addition, robust dynamin 1 punctate immunostaining was detected along the processes extended by these pyramidal neurons (Fig. 3A). Synaptophysin staining appeared in a typical punctuate pattern distributed along the processes (Fig. 3B). Most dynamin 1 immunoreactive spots co-localized with synaptophysin at synaptic sites (Fig. 3, C and D). However, dynamin 1 immunoreactivity was also detected in extrasynaptic areas. The incubation of hippocampal neurons with A{beta} (2 µM) for 24 h affected neither the morphology nor the distribution of synaptophysin staining (Fig. 3F). In contrast, dynamin 1 immunoreactivity was greatly decreased throughout the neuritic network and was mainly restricted to the cell body and adjacent regions of the processes extended by A{beta}-treated hippocampal neurons (Fig. 3E). As a consequence, synaptophysin immunoreactivity did not co-localize with that of dynamin 1 in distal neurites (Fig. 3, G and H). Quantitative analysis of the dynamin 1 and synaptophysin immunoreactive puncta showed a similar number of spots detected by each antibody in untreated controls (726 ± 127 dynamin 1 and 651 ± 52 synaptophysin). On the other hand, a significant decrease in the number of dynamin 1 (204 ± 146) puncta, but not synaptophysin (706 ± 165) puncta, was detected in A{beta}-treated neurons. These data suggested that A{beta} severely depleted levels of dynamin 1 in the synapses present along the distal portion of the neurites extended by hippocampal neurons.



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  		FIG. 5.
Dynamin 1 was decreased in the hippocampi of Tg2576 mice. A, Western blot analysis of dynamin 1 (Dyn 1) and class III {beta}-tubulin in whole cell extracts prepared from the hippocampi of 2-, 5-, and 8-month-old Tg2576 (TG) mice and strain- and age-matched WT mice. B, Western blot analysis of synaptophysin (Syn) and class III {beta}-tubulin in whole cell extracts prepared from the hippocampi of 2-, 5-, and 8-month-old Tg2576 mice and strain-, and age-matched WT mice. C and D, quantification of dynamin 1 from panel A and synaptophysin from panel B, respectively. The results were normalized using class III {beta}-tubulin as internal control. Values represent the mean ± S.E. *, p < 0.05 (differs from WT, which is set at 100%).

 
A{beta} Induced Dynamin 1 Reduction by a Bimodal Mechanism—We then determined to what extent post-translational degradation and/or down-regulation of the expression of dynamin 1 contributed to the A{beta}-induced decrease in its protein levels observed in cultured hippocampal neurons. The results described previously showing the appearance of a second lower molecular mass immunoreactive band in A{beta}-treated neurons strongly suggested that a post-translational proteolytic event was involved in the decrease of dynamin 1 levels. Two proteases proposed to play a role in the pathogenesis of AD are caspase-3 and calpain (23-27). Therefore, we first tested the ability of these proteases to cleave dynamin 1 in vitro (Fig. 4A). Whole cell lysates prepared from untreated cultured hippocampal neurons were incubated with increasing amounts of recombinant caspase-3 or calpain. Caspase-3 failed to cleave dynamin 1 at any of the concentrations used in our in vitro assays. On the other hand, Western blot analysis showed a dose-dependent decrease of full-length dynamin 1 when incubated with calpain. In addition, recombinant calpain cleavage generated a second dynamin 1 band similar to the one detected when hippocampal neurons were incubated with A{beta} (Fig. 4A). To further determine whether A{beta} could induce the proteolysis of dynamin 1 in hippocampal neurons, we treated the neurons with several cell-permeable inhibitors of caspase-3 or calpain prior to A{beta} incubation (Fig. 4B). The broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone did not prevent the dynamin 1 decrease induced by A{beta}. Conversely, three different calpain inhibitors, N-acetyl-Leu-Leu-norleucine-CHO, calpeptin, and MDL-28170 did block the cleavage of dynamin 1 in a dose-dependent manner (Fig. 4B).

Next, we investigated the effect of A{beta} treatment on dynamin 1 expression in cultured hippocampal neurons. Neurons were incubated with A{beta} (2 µM) for 8 and 24 h, and their RNA was harvested for RT-PCR. Conventional RT-PCR bands showed a qualitative decrease in dynamin 1 mRNA from cultured hippocampal neurons that were incubated with A{beta} for 8 and 24 h (Fig. 4C). Real time RT-PCR showed a significant decrease of dynamin 1 mRNA from hippocampal neurons incubated in the presence of A{beta} for 8 and 24 h as compared with untreated controls (~50 and ~48% decrease, respectively) (Fig. 4D). Taken together, these data suggested that A{beta} induced the decrease of dynamin 1 in cultured hippocampal neurons by a bimodal process involving both calpain-mediated proteolysis and a decrease in dynamin 1 expression.

Decreased Dynamin 1 Levels Were Also Detected in the Hippocampus of Tg2576 Mice—Finally, we studied whether endogenous A{beta} had similar affects on dynamin 1 in the AD animal model Tg2576. Tg2576 transgenic mice harbor a double mutation in the amyloid precursor protein that enhances production of the A{beta} peptide (28). These mice recapitulate many characteristics of AD pathology, including A{beta} plaque formation, beginning at 10-12 months of age (28). On the other hand, cognitive deficits were detected as early as 4 months after birth in Tg2576 mice (29). Surprisingly, changes in the number of synapses were not detected even in 1-year-old Tg2576 mice (30, 31). The cognitive impairments observed in these mice in the absence of pathology suggested a discrete mechanism affecting neuronal function. Dynamin 1 might play a key role in such a mechanism. To examine this possibility, we compared dynamin 1 and synaptophysin protein levels in whole cell extracts prepared from the hippocampi of 2-, 5-, and 8-month-old Tg2576 mice with those of strain- and age-matched WT mice (Fig. 5). Dynamin 1 and synaptophysin levels were normalized using neuron-specific {beta}-tubulin as an internal control. No differences in dynamin 1 protein levels were detected in the Tg2576 mice as compared with WT mice 2 months after birth (Fig. 5, A and C). On the other hand, beginning at 5 months of age, Tg2576 mice showed a significant decline (~22%) in dynamin 1 levels. This declining trend in dynamin 1 levels continued at 8 months of age when Tg2576 mice show an even greater (~36%) decrease in dynamin 1 levels when compared with WT mice. Importantly, no changes in synaptophysin protein levels were detected in the Tg2576 mice when compared with WT mice throughout the whole period analyzed (Fig. 5, B and D). These data indicated that a decrease in dynamin 1 protein occurred independently from synapse loss both in neurons that developed in culture and in situ.

Decrease of Dynamin 1 in Tg2576 Mice Was Likely Due to Calpain Activation—A{beta}-induced calpain activation resulted in the cleavage of dynamin 1 and the appearance of a fragment of ~90 kDa in cultured hippocampal neurons. If calpain was abnormally activated in the hippocampus of Tg2576 mice, we would expect to see a dynamin 1 fragment band at approximately the same molecular mass (~90 kDa). No dynamin 1 cleaved bands were detected in the WT or 2-month-old Tg2576 mice (Fig. 6A). By contrast, cleaved products were detected directly below full-length dynamin 1 in 5- and 8-month-old Tg2576 mice. To test whether calpain was responsible for generating this faster migrating dynamin 1 band, we incubated hippocampal lysates obtained from 2-month-old Tg2576 mice with calpain for 1 h. Western blot analysis showed the presence of a faster migrating dynamin 1 band in 2-month-old lysates incubated with calpain and untreated lysates from 8-month-old Tg2576 mice. On the other hand, no such cleaved fragment was detected in untreated lysates obtained from 2-month-old mice (Fig. 6B). To further assess calpain activation in the hippocampus of these mice, we analyzed the degradation of spectrin, a common calpain substrate (32). Activation of calpain results in the cleavage of full-length spectrin (240 kDa) to break down products of ~145- and 150-kDa apparent molecular masses (33). An increase in the ratio of 150/240-kDa spectrin bands was detected in Tg2576 mice when compared with strain- and age-matched WT mice at 5 and 8 months after birth (Fig. 6C). Interestingly, calpain activation in these mice seems to correlate with the decrease in dynamin 1 and the appearance of the dynamin 1 fragment.



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  		FIG. 6.
Decrease of dynamin 1 in Tg2576 mice was consistent with calpain activation. A, Western blot analysis of dynamin 1 (Dyn 1) in whole cell extracts prepared from the hippocampi of 2-, 5-, and 8-month-old Tg2576 (TG) mice and strain- and age-matched WT mice. Note the appearance of a lower molecular mass dynamin 1 band (~90 kDa) in the 5- and 8-month-old Tg2576 mice. B, Western blot analysis of dynamin 1 in whole cell extracts prepared from the hippocampi of 2- and 8-month-old Tg2576 mice. The 2-month-old samples were either untreated (-) or incubated with 0.2 units of calpain (+) and run next to an untreated sample from an 8-month-old Tg2576 mouse. Note that calpain treatment in the 2-month-old sample produced a similar fast migrating band as that in the 8-month-old untreated sample. C, Western blot analysis of spectrin in whole cell extracts prepared from the hippocampi of 2-, 5-, and 8-month-old Tg2576 mice and strain- and age-matched WT mice. The antibody used recognized both full-length spectrin (240 kDa) and calpain-mediated cleavage products (150 and 145 kDa). Spectrin degradation was assessed by determining the 150/240-kDa ratio. {alpha}-Tubulin was used as a loading control. D, real time RT-PCR of dynamin 1 mRNA obtained from the hippocampi of 2-, 5-, and 8-month-old Tg2576 mice and strain- and age-matched WT mice. The dynamin 1 signal was normalized using the 18 S ribosomal gene as an endogenous control. Values represent the mean ± S.E. *, p < 0.05; **, p < 0.01 (differs from WT, which is set at 100%).

 
We next studied dynamin 1 mRNA levels in the hippocampi of these transgenic mice. Relative quantification real time PCR showed that at 2 and 5 months, Tg2576 mice have no significant change in dynamin 1 mRNA levels compared with strain- and age-matched WT (Fig. 6D). A significant increase (~84%) in dynamin 1 mRNA expression was detected in 8-month-old Tg2576 mice when compared with WT mice.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented herein indicated that A{beta} induced a decrease of dynamin 1 in hippocampal neurons. In addition, our findings suggested that this A{beta}-induced dynamin 1 decrease was the result of a bimodal mechanism that involved calpain-mediated proteolysis and down-regulation of dynamin 1 gene expression in cultured hippocampal neurons. In Tg2576 mice, on the other hand, the decrease in the dynamin 1 protein was likely due to calpain-mediated proteolysis. Given the importance of dynamin 1 to synaptic function, these results provide insights into the molecular mechanisms underlying the cognitive decline observed in the early stages of AD. Furthermore, they help to bridge an extensive gap between our knowledge of the mechanisms leading to A{beta} accumulation in different brain areas and synaptic deficits in AD patients.

Synaptic dysfunction has been a tempting explanation for the mild cognitive deficits observed at early stages of the disease when no significant decline in the synapse number could be detected (5). However, the mechanisms underlying these functional deficits are not known. These results indicated that changes in dynamin 1 could mediate, at least in part, these deficits. Thus, low concentrations of A{beta} (~10-fold lower than insoluble A{beta} levels in AD brains) (34) significantly reduced dynamin 1 protein levels in a time- and dose-dependent manner in cultured hippocampal neurons. Importantly, when used at these concentrations A{beta} induced the reduction in dynamin 1 prior to synapse loss and/or neurite degeneration in cultured hippocampal neurons. Similar results were obtained when the levels of dynamin 1 were analyzed in the hippocampal region of Tg2576 mice, the most commonly used AD animal model. These mice, which have enhanced production of A{beta}-(1-40) (5-fold) and A{beta}-(1-42) (14-fold) in their hippocampi, showed a significant reduction in dynamin 1 protein levels in the absence of synapse loss as determined using synaptophysin as a synaptic marker.

Our results suggested that decreased dynamin 1 levels might lead to changes in synaptic vesicle recycling and the accumulation of fused vesicles at the membrane, thus affecting synaptic function in the complex neuronal network of the hippocampus. This network depends on flawless synaptic vesicle turnover in millions of synapses for memory acquisition following long term potentiation (35, 36). Our data support previous studies showing that A{beta}-induced cognitive decline is a reversible or transient phenomenon in animal models (37, 38). These studies have suggested that cognition decline due to A{beta} is not because of frank synapse loss or neurodegeneration but rather a stage of synaptic dysfunction that does not cause permanent disruptions in neuronal networks important to learning and memory. It is tempting to speculate that this stage of synaptic dysfunction is mediated by a decrease in dynamin 1, leading to the depletion of releasable synaptic vesicles due to the inability to endocytose from the presynaptic membrane. Interestingly, it has been documented that the average synaptic area in the dentate gyrus and frontal cortex was significantly larger in AD brains than in age-matched controls (39, 40). This particular ultrastructural abnormality could be a result of the accumulation of fused synaptic vesicles at the synapse. Similar ultrastructural features have been described in temperature-sensitive mutants of a dynamin ortholog in Drosophila (12, 13).

This study also provided insights into the mechanisms leading to A{beta}-induced decrease in dynamin 1 levels. Our in vitro and in vivo experiments suggested that calpain mediated dynamin 1 proteolysis in A{beta}-treated hippocampal neurons. The activation of this protease in the presence of A{beta} is in agreement with previous reports showing that AD patients had increased levels of active calpain in their central nervous system (23, 24, 27). Calpain has also been linked to the dysregulation of Cdk5 leading to aberrant phosphorylation of tau, another salient feature of AD (41). Furthermore, initial studies using calpain inhibitors in a mouse model of AD showed an encouraging recovery of cognitive function in transgenic mice that were treated with a calpain inhibitor at an early age (42). The mechanisms by which A{beta} induces the activation of this protease have not been completely elucidated. Calpain is a cytosolic cysteine protease physiologically activated by intracellular increases in Ca2+ (43). Thus, one potential mechanism of activation could involve the disruption of Ca2+ homeostasis. The molecular mechanism by which A{beta} causes calcium dyshomeostasis is yet to be fully identified. To this point, cases have been made for increased calcium influx through glutamate receptors, receptor-independent membrane permeabilization, and intracellular calcium leakage (44-46).

Our results obtained using cultured hippocampal neurons support the idea that A{beta} also reduces dynamin 1 expression. This A{beta}-induced decrease in dynamin 1 might involve the regulation of its transcription factor. Expression of the dynamin 1 gene is partly controlled by, and dependent on, the transcription factor Sp1 (47). Sp1 is activated by cAMP-dependent protein kinase A-mediated phosphorylation (48). Therefore, disruptions in the cAMP/protein kinase A/Sp1 pathway could have significant effects on the level of dynamin 1 gene expression and synaptic function. Support for this hypothesis has been provided by a recent study showing that A{beta} blocked protein kinase A activity and long term potentiation in cultured hippocampal neurons (49).

Surprisingly, dynamin 1 mRNA levels were increased in the hippocampi of 8-month-old Tg2576 mice. The results obtained using this AD animal model system are in agreement with a previous report showing an increase in dynamin 1 expression from brains of these mice (9). This apparent discrepancy between the effects of A{beta} on the expression of dynamin 1 mRNA in neurons that develop either in culture or in situ could be due to several factors. First, compensatory mechanisms leading to an increase in dynamin 1 mRNA could be triggered by the chronic cleavage of this protein in Tg2576 mice. These mechanisms might not get into play under the acute conditions leading to dynamin 1 cleavage like the ones observed in A{beta}-treated cultured neurons. Alternatively, the increase in dynamin 1 mRNA observed in the hippocampal region of the Tg2576 mice could be the result of different responses of specific neuronal populations to A{beta}. It is possible that granular cells from the dentate gyrus, absent from our cultures prepared from embryonic tissue, increase the transcription of dynamin 1 in response to the overexpression of the double-mutated amyloid precursor proteins present in these transgenic animals. Regardless, it is worth noting that our results obtained using cultured hippocampal neurons closely resembled the decrease in dynamin 1 expression observed in the brains of AD patients (9).

Taken collectively, our results suggest that A{beta} induces a decrease of dynamin 1 in hippocampal neurons. This effect on dynamin 1 is an early event that precedes synapse loss both in A{beta}-treated cultured hippocampal neurons and in hippocampal neurons obtained from Tg2576 mice. These findings support a previous study that showed the decrease of dynamin 1 in human AD brains (9). In addition, these results present a molecular explanation for the cognitive decline in absence of synapse loss seen in the early stages of AD. The idea that synapses become dysfunctional before they are lost presents an attractive window for therapeutic intervention before irreversible neuronal damage is done. Thus, drugs that inhibit calpain and/or stimulate protein kinase A, two attractive targets to prevent synaptic dysfunction, could become useful therapeutic tools for treating AD.


   FOOTNOTES
 
* This study was supported in part by NIA, National Institutes of Health Grant R01A G022560 (to R. V.), National Institutes of Health Grant NS39080, and an Alzheimer's Association Investigator-initiated Research Grant (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

§ Supported by NIA, National Institutes of Health Training Grant AG20506 and a fellowship from the American Foundation for Aging Research. Back

|| To whom correspondence should be addressed. Tel.: 312-503-0597; Fax: 312-503-7345; E-mail: a-ferreira{at}northwestern.edu.

1 The abbreviations used are: AD, Alzheimer disease; A{beta}, {beta}-amyloid; MEM, minimum essential medium; MDL-28170, benzyloxycarbonyl-Val-Phe-CHO; PBS, phosphate-buffered saline; RT, reverse transcription; WT, wild-type. Back


   ACKNOWLEDGMENTS
 
We thank Caryn Tournell, Kelsi Anderson, and Holly Oakley for excellent technical support. We thank Dr. Mark McNiven (Mayo Clinic, Rochester, MN) for the generous gift of a dynamin 1 antibody. We also thank Tracy O'Connor for help with real time PCR.



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 DISCUSSION
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