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• W2080455250 abstract "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 β-amyloid (Aβ), 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 Aβ-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 Aβ-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. 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 β-amyloid (Aβ), 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 Aβ-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 Aβ-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. Senile plaques, neurofibrillary tangles, synapse loss, and gross neurodegeneration are common findings in the brain of AD 1The abbreviations used are: AD, Alzheimer disease; Aβ, β-amyloid; MEM, minimum essential medium; MDL-28170, benzyloxycarbonyl-Val-Phe-CHO; PBS, phosphate-buffered saline; RT, reverse transcription; WT, wild-type. patients (1Glenner G.G. Wong C.W. Biochem. Biophys. Res. Commun. 1984; 122: 1131-1135Crossref PubMed Scopus (1261) Google Scholar, 2Grundke-Iqbal I. Iqbal K. Quinlan M. Tung Y.C. Zaidi M.S. Wisniewski H.M. J. Biol. Chem. 1986; 261: 6084-6089Abstract Full Text PDF PubMed Google Scholar, 3Terry R.D. Masliah E. Salmon D.P. Butters N. DeTeresa R. Hill R. Hansen L.A. Katzman R. Ann. Neurol. 1991; 30: 572-580Crossref PubMed Scopus (3423) Google Scholar, 4DeKosky S.T. Scheff S.W. Ann. Neurol. 1990; 27: 457-464Crossref PubMed Scopus (1701) Google Scholar). Numerous genetic, biochemical, and animal model studies have implicated the gradual buildup of Aβ, the main component of senile plaques, as the catalyst for AD. However, the mechanistic link between Aβ 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 (3Terry R.D. Masliah E. Salmon D.P. Butters N. DeTeresa R. Hill R. Hansen L.A. Katzman R. Ann. Neurol. 1991; 30: 572-580Crossref PubMed Scopus (3423) Google Scholar, 4DeKosky S.T. Scheff S.W. Ann. Neurol. 1990; 27: 457-464Crossref PubMed Scopus (1701) Google Scholar). In contrast, patients in the earliest stages of the disease show no significant decline in synapse number (5Tiraboschi P. Hansen L.A. Alford M. Masliah E. Thal L.J. Corey-Bloom J. Neurology. 2000; 55: 1278-1283Crossref PubMed Scopus (118) Google Scholar). 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 (6Selkoe D.J. Science. 2002; 298: 789-791Crossref PubMed Scopus (3438) Google Scholar, 7Yao P.J. Trends Neurosci. 2004; 27: 24-29Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). 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 (8Honer W.G. Neurobiol. Aging. 2003; 24: 1047-1062Crossref PubMed Scopus (116) Google Scholar). More recently, dynamin 1, a protein highly enriched in presynaptic terminals, has been shown to be significantly reduced in AD brains (9Yao P.J. Zhu M. Pyun E.I. Brooks A.I. Therianos S. Meyers V.E. Coleman P.D. Neurobiol. Dis. 2003; 12: 97-109Crossref PubMed Scopus (193) Google Scholar). 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 (10Clark S.G. Shurland D.L. Meyerowitz E.M. Bargmann C.I. van der Bliek A.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10438-10443Crossref PubMed Scopus (100) Google Scholar, 11Damke H. Baba T. Warnock D.E. Schmid S.L. J. Cell Biol. 1994; 127: 915-934Crossref PubMed Scopus (1046) Google Scholar). 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 (12Koenig J.H. Ikeda K. J. Neurosci. 1989; 9: 3844-3860Crossref PubMed Google Scholar, 13van der Bliek A.M. Meyerowitz E.M. Nature. 1991; 351: 411-414Crossref PubMed Scopus (596) Google Scholar). 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 Aβ was one of such stressors using hippocampal neurons that develop in culture and in an AD animal model system. Our results showed that Aβ induced a significant reduction in dynamin 1 levels that preceded synapse loss in both model systems. In cultured neurons, our data suggested that the Aβ-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. Preparation of Hippocampal Cultures—Embryonic day 18 rat embryos were used to prepare primary hippocampal cultures as described previously (14Ferreira A. Li L. Chin L.S. Greengard P. Kosik K.S. Mol. Cell. Neurosci. 1996; 8: 286-299Crossref PubMed Scopus (22) Google Scholar, 15Dawson H.N. Ferreira A. Eyster M.V. Ghoshal N. Binder L.I. Vitek M.P. J. Cell Sci. 2001; 114: 1179-1187PubMed Google Scholar). 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) (16Bottenstein J.E. Sato G.H. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 514-517Crossref PubMed Scopus (2015) Google Scholar). 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. Aβ Treatment—Synthetic Aβ-(1-40) or Aβ-(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 (17Ferreira A. Lu Q. Orecchio L. Kosik K.S. Mol. Cell. Neurosci. 1997; 9: 220-234Crossref PubMed Scopus (171) Google Scholar). This preaggregated Aβ was added to the culture medium at final concentrations ranging from 0.02 to 20 μm. For some experiments, the aggregated Aβ was centrifuged at 100,000 × 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 Aβ concentrations calculated using the initial concentration of the monomeric form of the peptide. Monomeric Aβ 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. 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 Aβ 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-α-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 β-tubulin (1:500; clone TUJ1) (18Ferreira A. Caceres A. J. Neurosci. Res. 1992; 32: 516-529Crossref PubMed Scopus (96) Google Scholar); 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 Aβ-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 Aβ 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-Aβ (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. 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 l0× 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 Aβ-treated cultured hippocampal neurons versus untreated cultured hippocampal neurons and Tg2576 hippocampi versus WT hippocampi using the ABI 7900HT Detection System (Applied Biosystems). Aβ Induced a Dynamin 1 Reduction in Cultured Hippocampal Neurons—To test whether the deposition of Aβ could cause a depletion of dynamin 1, we analyzed dynamin 1 protein levels in cultured hippocampal neurons treated with Aβ. 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 (17Ferreira A. Lu Q. Orecchio L. Kosik K.S. Mol. Cell. Neurosci. 1997; 9: 220-234Crossref PubMed Scopus (171) Google Scholar, 19Bartlett W.P. Banker G.A. J. Neurosci. 1984; 4: 1954-1965Crossref PubMed Google Scholar, 20Bartlett W.P. Banker G.A. J. Neurosci. 1984; 4: 1944-1953Crossref PubMed Google Scholar). The addition of Aβ-(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 Aβ-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 (21Wiedenmann B. Franke W.W. Cell. 1985; 41: 1017-1028Abstract Full Text PDF PubMed Scopus (1228) Google Scholar). Synaptophysin levels correlate closely with synapse numbers and are commonly used to assay for loss of synapses (5Tiraboschi P. Hansen L.A. Alford M. Masliah E. Thal L.J. Corey-Bloom J. Neurology. 2000; 55: 1278-1283Crossref PubMed Scopus (118) Google Scholar, 22Sabbagh M.N. Corey-Bloom J. Tiraboschi P. Thomas R. Masliah E. Thal L.J. Arch. Neurol. 1999; 56: 1458-1461Crossref PubMed Scopus (27) Google Scholar). On the other hand, no changes in the levels of dynamin 3, a dynamin isoform also expressed in the nervous system, were detected in Aβ-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 Aβ-treated neurons (Fig. 1A). Similar results were obtained when cultured hippocampal neurons were incubated with preaggregated Aβ-(1-42) (Fig. 1B). All experiments described below were performed with Aβ-(1-40). To determine whether Aβ induced the decrease of dynamin levels in a dose-dependent manner, mature hippocampal neurons were incubated with Aβ 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 Aβ-treated neurons as compared with untreated controls. However, synaptophysin levels were unchanged when cultured hippocampal neurons were incubated with Aβ 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 Aβ at all of the concentrations analyzed (Fig. 1C). Next, we determined whether Aβ affects dynamin levels in a time-dependent manner. For these experiments, hippocampal neurons kept in culture for 3 weeks were incubated with Aβ (2 μm) for up to 36 h (Fig. 1, E and F). The effect of Aβ 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 Aβ has been proposed to play a critical role in its neurotoxic effects, we next determined which form of the Aβ peptide was causing these effects on dynamin 1. The preaggregated Aβ preparation likely contained both large insoluble fibrillar forms and smaller soluble oligomeric forms of the peptide. Therefore, we separated these two forms of Aβ by centrifugation. To determine whether we had successfully separated the fibrillar Aβ from the oligomeric Aβ, we immunostained each fraction with an Aβ 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 Aβ had different effects on dynamin 1, we incubated cultured hippocampal neurons with the monomeric, mixed, oligomeric, and fibrillar forms of Aβ (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 Aβ (Fig. 2, B and C). We also observed the appearance of the ∼90-kDa band in the mixed and oligomeric preparations only (Fig. 2B). Treatment with Aβ 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 Aβ (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 Aβ-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 Aβ-treated neurons. These data suggested that Aβ severely depleted levels of dynamin 1 in the synapses present along the distal portion of the neurites extended by hippocampal neurons. Aβ 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 Aβ-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 Aβ-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 (23Saito K. Elce J.S. Hamos J.E. Nixon R.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2628-2632Crossref PubMed Scopus (534) Google Scholar, 24Tsuji T. Shimohama S. Kimura J. Shimizu K. Neurosci. Lett. 1998; 248: 109-112Crossref PubMed Scopus (83) Google Scholar, 25Gervais F.G. Xu D. Robertson G.S. Vaillancourt J.P. Zhu Y. Huang J. LeBlanc A. Smith D. Rigby M. Shearman M.S. Clarke E.E. Zheng H. Van Der Ploeg L.H. Ruffolo S.C. Thornberry N.A. Xanthoudakis S. Zamboni R.J. Roy S. Nicholson D.W. Cell. 1999; 97: 395-406Abstract Full Text Full Text PDF PubMed Scopus (719) Google Scholar, 26Gastard M.C. Troncoso J.C. Koliatsos V.E. Ann. Neurol. 2003; 54: 393-398Crossref PubMed Scopus (81) Google Scholar, 27Veeranna Kaji T. Boland B. Odrljin T. Mohan P. Basavarajappa B.S. Peterhoff C. Cataldo A. Rudnicki A. Amin N. Li B.S. Pant H.C. Hungund B.L. Arancio O. Nixon R.A. Am. J. Pathol. 2004; 165: 795-805Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). 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 Aβ (Fig. 4A). To further determine whether Aβ 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 Aβ incubation (Fig. 4B). The broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone did not prevent the dynamin 1 decrease induced by Aβ. 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 Aβ treatment on dynamin 1 expression in cultured hippocampal neurons. Neurons were incubated with Aβ (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 Aβ 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 Aβ for 8 and 24 h as compared with untreated controls (∼50 and ∼48% decrease, respectively) (Fig. 4D). Taken together, these data suggested that Aβ 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 Aβ 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 Aβ peptide (28Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-102Crossref PubMed Scopus (3711) Google Scholar). These mice recapitulate many characteristics of AD pathology, including Aβ plaque formation, beginning at 10-12 months of age (28Hsiao K. Chapman P. Nilsen S. Eckman C. Harigaya Y. Younkin S. Yang F. Cole G. Science. 1996; 274: 99-102Crossref PubMed Scopus (3711) Google Scholar). On the other hand, cognitive deficits were detected as early as 4 months after birth in Tg2576 mice (29Ohno M. Sametsky E.A. Younkin L.H. Oakley H. Younkin S.G. Citron M. Vassar R. Disterhoft J.F. Neuron. 2004; 41: 27-33Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). Surprisingly, changes in the number of synapses were not detected even in 1-year-old Tg2576 mice (30King D.L. Arendash G.W. Brain Res. 2002; 926: 58-68Crossref PubMed Scopus (82) Google Scholar, 31Savage M.J. Lin Y.G. Ciallella J.R. Flood D.G. Scott R.W. J. Neurosci. 2002; 22: 3376-3385Crossref PubMed Google Scholar). 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 β-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—Aβ-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 (32Siman R. Baudry M. Lynch G. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 3572-3576Crossref PubMed Scopus (255) Google Scholar). Activation of calpain results in the" @default.
• W2080455250 created "2016-06-24" @default.
• W2080455250 creator A5001214157 @default.
• W2080455250 creator A5028697711 @default.
• W2080455250 creator A5081561753 @default.
• W2080455250 date "2005-09-01" @default.
• W2080455250 modified "2023-09-29" @default.
• W2080455250 title "β-Amyloid-induced Dynamin 1 Depletion in Hippocampal Neurons" @default.
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