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Activation of the Notch pathway in Down syndrome: cross-talk of Notch and APP David F. Fischer,*,†,1,2 Renske van Dijk,*,2 Jacqueline A. Sluijs,* Suresh M. Nair,* Marco Racchi,‡ Christiaan N. Levelt,§ Fred W. van Leeuwen,* and Elly M. Hol* *Netherlands Institute for Brain Research, Amsterdam, The Netherlands; †Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; ‡Department of Experimental and Applied Pharmacology, University of Pavia, Italy; and §The Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands Down syndrome (DS) patients suffer from mental retardation, but also display enhanced -APP production and develop cortical amyloid plaques at an early age. As -APP and Notch are both processed by ␥-secretase, we analyzed expression of the Notch signaling pathway in the adult DS brain and in a model system for DS, human trisomy 21 fibroblasts by quantitative PCR. In adult DS cortex we found that Notch1, Dll1 and Hes1 expression is up-regulated. Moreover, DS fibroblasts and Alzheimer disease cortex also show overexpression of Notch1 and Dll1, indicating that enhanced -APP processing found in both DS and AD could be instrumental in these changes. Using pull-down studies we could demonstrate interaction of APP with Notch1, suggesting that these transmembrane proteins form heterodimers, but independent of ␥-secretase. We could demonstrate binding of the intracellular domain of Notch1 to the APP adaptor protein Fe65. Furthermore, activated Notch1 can transactivate an APP target gene, Kai1, and vice versa, activated APP can trans-activate the classical Notch target gene Hes1. These data suggest that Notch expression is activated in Down syndrome, possibly through cross-talk with APP signaling. This interaction might affect brain development, since the Notch pathway plays a pivotal role in neuron-glia differentiation.— Activation of the Notch pathway in Down syndrome: cross-talk of Notch and APP. Fischer, D. F., van Dijk, R., Sluijs, J. A., Nair, S. M., Racchi, M., Levelt, C. N., van Leeuwen, F. W., Hol, E. M. FASEB J. 19, 1451–1458 (2005)
ABSTRACT
Key Words: Alzheimer disease 䡠 ␥-secretase complex 䡠 APP 䡠 Notch intracellular domain
Recently, studies of Notch signaling and amyloid  precursor protein (APP) processing have converged (1, 2). Notch signaling involves cleavage by ␥-secretase (3, 4), the same proteolytic activity that is required for processing of APP (5, 6). Notch has been discovered as a component of a signaling network that regulates neuronal and glial differentiation (7, 8), whereas APP 0892-6638/05/0019-1451 © FASEB
was discovered as a gene involved in sporadic and familial Alzheimer disease and Down syndrome (9). Upon binding of a ligand such as delta-like protein 1 (Dll1) to the single-pass transmembrane receptor Notch, an extracellular cleavage of Notch is followed by intra-membrane cleavage by ␥-secretase and release of the Notch intracellular domain (NICD). NICD translocates to the nucleus and acts as a transcriptional activator (4). The intra-membrane processing of APP by ␥-secretase is highly similar, resulting in generation of AICD (10, 11). Only recently has F-spondin been identified as a ligand for APP (12). The four components of the ␥-secretase complex have been elucidated as presenilin 1 (PS1) or 2 (PS2), nicastrin, aph-1, and pen2 (13, 14). NICD interacts with CSL transcription factors (4) to activate target genes such as Hes1 and Hes5 (15). Only Kai1 has been identified as a target gene for the transcription factor complex of AICD, Fe65, and tip60 (10). It has been shown in vitro that the Swedish mutant of APP and Notch are competitive substrates of ␥-secretase (16), and we reasoned that enhanced processing of either APP or Notch would affect signaling of the other. The early accumulation of -amyloid as plaques in trisomy 21 Down syndrome (DS) brain is thought to result from the overexpression and enhanced processing of APP (9, 17). In this report we have examined the result of trisomy 21 on the expression of genes in the APP and Notch signaling pathways to investigate potential cross-talk between APP and Notch such as through competition for ␥-secretase. Surprisingly, we find that components of the Notch pathway are activated in the DS brain. Furthermore, we show that APP and Notch interact directly, potentially as heterodimers, and can activate reciprocal target genes.
1
Correspondence: Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, De Boelelaan 1085, Amsterdam 1081 HV, The Netherlands. E-mail:
[email protected] 2 These authors contributed equally to this work. doi: 10.1096/fj.04-3395.com 1451
MATERIALS AND METHODS Gene expression studies Human brain material was obtained from the Netherlands Brain Bank and kept at – 80°C after being snap-frozen in liquid nitrogen (for clinical and pathological data, see supplementary Table 1). Approximately 100 mg pieces of tissue (gray matter) were cut in a frozen state and immediately processed with the Trizol RNA purification kit (Invitrogen, San Diego, CA, USA). RNA was concentrated to a concentration of 1 mg/mL by ethanol precipitation and stored at –20°C until use. cDNA was prepared from 2 g of RNA with 250 ng of random hexamers (Amersham Pharmacia, Arlington Heights, IL, USA) and Superscript II reverse transcriptase (Invitrogen) in a total volume of 20 L. Real-time quantitative PCR was performed as described earlier (18). Three reference genes were used to correct for experimental variations; elongation factor 1␣, E2 ubiquitinconjugating enzyme Ube2d2 and ribosomal protein S27a. These three reference genes are highly correlated in expression in the human brain (r⫽0.737 for EF1␣ to Ube2d2, r⫽0.619 for Ube2d2 to rS27a, r⫽0.873 for EF1␣ to rS27a with a 2-tailed Spearman’s correlation). Statistical analysis was performed with a Mann-Whitney U test for pair wise comparisons (nonparametric), Spearman’s test for correlations using SPSS 11 for Mac. DNA cloning and constructs Reporter plasmids Hes1-pGL3 and Kai1-pGL3 were constructed by cloning the mouse Hes1 promoter from –200 to ⫹150 or the mouse Kai1 promoter from –1200 to ⫹20 (Celera mouse genome sequence) in pGL3-basic (Promega, Madison, WI, USA). Dominantly negative Notch (DN-notch) contained the mouse Notch1 sequence (GenBank CAA77941) from aa 1 to 1760, fused to EGFP; CA-Notch contained the mouse Notch1 sequence (GenBank CAA77941) from aa 1741 onward similar to construct FCDN1 (19) with a bicistronic EGFP; APP695 was kindly provided by T. Hartmann, Heidelberg and cloned in pcDNA3; CA-APP contained the Cterminal 63 amino acids of human -APP in pcDNA3; see also Fig. 2C. Fe65 expression plasmid was kindly provided by T. Russo, University of Naples, Italy; the trkB construct contains the rat trkB-T1 sequence in pEGFP-N1 (Clontech, Palo Alto, CA, USA); EGFP-F is from Clontech; APP⫹1 constructs have been described in ref 20. All constructs were verified by sequencing.
immunoprecipitated by rotating the suspension head-overhead at 4°C overnight. The antibodies used for immunoprecipitation were a polyclonal antibody 80750 (22) or a monoclonal 22C11 (23) against APP695 (kindly provided by T. Hartmann, ZMBH, Heidelberg, Germany dilution 1:1000), a polyclonal antibody against Fe65 (24) (kindly provided by L. Mercken, Aventis Pharma, Neurodegenerative Disease Group, Vitry-sur-Seine, France, 1:2500), a monoclonal antibody against Notch1 (MAB5352, Chemicon, Temecula, CA, USA, 1:200) or a polyclonal antibody against GFP to detect DN-Notch, trkB or EGFP-F (A-11122, Molecular Probes, Eugene, OR, USA, 1:1000). Beads were washed 3 times with buffer AA (50 mM Tris-HCl pH 8.0, 0.5 M NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.5% deoxycholic acid sodium salt, 0.1% SDS), transferred to a new Eppendorf tube, and washed once more in buffer AA. Then the beads were resuspended in loading buffer (50 mM Tris-HCl pH 6.8, 5% -mercaptoethanol, 2% SDS, 0.1% BFB, 10% glycerol) and proteins were separated on a 7.5% SDS-PAGE gel and transferred by semi-dry blot onto nitro-cellulose. Blots were probed with a monoclonal antibody against Notch1 (MAB5352, Chemicon, 1:200), a polyclonal antibody against Fe65 (1:5000) or a monoclonal antibody 22C11 against APP (1:200). All antibodies for Western blot were diluted in supermix (0.05 M Tris, 0.15 M NaCl, 0.25% gelatin, and 0.5% Triton X-100, pH 7.4). After overnight incubation with the primary antibody at 4°C, the blots were washed with Tris-buffered saline-Tween (65 mM Tris-HCl pH 7.5, 0.15 M NaCl, 0.05% Tween 20) and incubated with secondary rabbit or mouse polyclonal antibodies conjugated to horseradish peroxidase in supermix (DAKO, Glostrup, Denmark, dilution 1:1000). Labeled proteins were visualized using the Western Lightning Chemiluminescence Reagent Plus kit (Perkin Elmer Life Sciences, Boston, MA, USA). Luciferase assays Cells were harvested 46 h after transfection and luciferase activity was measured by using the Luciferase Assay System (Promega, Madison, WI, USA). Total amount of proteins was determined by the DC protein assay (Bio-Rad, Herculus, CA, USA) or according to Bradford methods (25). Luciferase measurements were normalized for protein content and the transfection efficiency was checked on a Western blot. Data are plotted as the means ⫾ se from three different experiments and the statistical significance was assessed by a paired Student t test (P⬍0.05). Within each experiment, transfections were performed in duplicate, with minor variations between these samples.
Cell culture and transient transfections Primary human shoulder skin fibroblasts were cultured as described (21); RNA was isolated from confluent dishes at passage number 4 to 9. HEK 293T cell lines were cultured and transfected as described previously (20). Immunoprecipitation and Western blot analysis Approximately 24 h after transfection cells were washed in phosphate-buffered saline (PBS, pH 7.4) and scraped in 1 mL of buffer A (10 mM Tris-HCl pH 8.0, 0.15 M NaCl, 0.1% Nonidet P-40, 0.1% Triton X-100, 20 mM EDTA pH 8.0). After addition of protease inhibitors (100 M phenylmethylsulfonyl fluoride and 10 g/mL leupeptin) cells were sonicated twice for 10 s and centrifuged for 1 min at 10,000 g. To 600 L of the supernatant 500 L buffer A, primary antibody and protein-A Sepharose beads were added and proteins were 1452
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RESULTS We analyzed gene expression of components of the Notch signaling pathway in cortex from 10 DS patients (age 64.6⫾6.7 years) and 14 controls (age 62.4⫾16.5 years) by real-time quantitative RT-PCR (see supplementary tables for clinical details and raw data). In DS cortex, we found significant up-regulation of Notch1, Notch2, and Dll1 expression (Fig. 1A). The Notch target gene Hes1 (26) was also up-regulated (1.5-fold, P⫽0.019); Hes5 is not statistically significant increased due to large inter-patient variation (Fig. 1A). Also in the DS fibroblast cultures are Notch1 and Dll1 significantly up-regulated (Fig. 1B). Because APP processing and -amyloid generation have been shown to be
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enhanced in DS brain (27) and DS fibroblasts (21), the observed Notch1 up-regulation correlates with enhanced APP processing. Indeed, sporadic Alzheimer patients who suffer from enhanced -amyloid formation due to enhanced APP processing (28), have upregulated Notch1 (2.2-fold, P⫽0.005) and Dll1 (3.2fold, P⫽0.001, Fig. 1C). As Notch1, Notch2, Dll1 and APP are all processed by ␥-secretase (3, 29 –31), Notch signaling could be affected by enhanced expression of the ␥-secretase complex (13, 14). In neither Down syndrome nor the fibroblast model of DS could we detect significant up-regulation of the five constituents of the ␥-secretase complex: presenilin 1 and 2 (PS1 and PS2), nicastrin, pen-2, and aph-1 (supplementary tables). These data suggest that expression of Notch is affected by other aspects of APP processing. Cross-talk between APP and Notch signaling
Figure 1. Gene expression analysis. A) Gene expression of NOTCH1, NOTCH2, DLL1, HES1, and HES5 in adult control cortex (n⫽14, open bars) and DS cortex (n⫽10, gray bars). B) Gene expression of NOTCH1, NOTCH2, DLL1, HES1, and HES5 in adult human fibroblasts (n⫽3, open bars) and DS fibroblasts (n⫽5, gray bars). C) Gene expression of NOTCH1 and DLL1 in adult control cortex (n⫽8, open bars) and Alzheimer cortex (n⫽6, gray bars). The box contains 50% of the data points with the median indicated by the hairline, the whiskers represent the maximum and minimum expression levels in the group, with outliers (points whose 〉-APP AND NOTCH INTERACT
To examine the potential cross-talk between APP signaling and Notch signaling, we used the well-studied regulation of the Hes1 promoter by activated Notch (26, 32). For APP signaling, we cloned the Kai1 promoter, which has been shown to be regulated by APP intracellular domain (AICD) (33) in a luciferase reporter plasmid. As expected, a constitutively active form of Notch1 (CA-Notch) (19) induced the Hes1 reporter in transient transfections in HEK293 cells, whereas a dominant negative form of Notch1 (DN-Notch) repressed the Hes1 reporter (Fig. 2A). Strikingly, DNNotch repressed basal expression of Kai1 (2-fold) and CA-Notch induced Kai1 (1.5- to 2-fold), suggesting that this gene is under regulation of the Notch pathway (Fig. 2B). CA-APP, a mutant of APP similar to CA-Notch in structure, and encoding the expected AICD peptide (34) was able to significantly induce the Hes1 construct (2-fold), although this required the presence of Fe65 (Fig. 2A), a known cofactor of AICD (33, 34). Coexpression of CA-APP and CA-Notch did not result in higher induction levels of the reporter constructs (Fig. 2), perhaps due to limiting amounts of unknown signaling components. Indeed, the effect of CA-APP was relatively small, compared with that of CA-Notch. Fe65 did not influence the activity of the DN-Notch construct, suggesting that Fe65 requires the intracellular domain of Notch1 to activate transcription, a mechanism that resembles the coactivating properties of Fe65 and the APP intracellular domain (33). Very similar data were obtained in neuronally differentiated neuroblastoma cells (supplementary figure).
value is either greater than Upper ⫹ 1.5 * Inner Quartile Distance or less than Lower Quartile Distance-1.5 * Inner Quartile Distance) indicated by an open circle. Asterisks indicate statistically significant (P⬍0.05 Mann-Whitney) changes. 1453
Figure 2. Transactivation of Hes1luciferase (A) and Kai1-luciferase (B) constructs in HEK293 cells. Luciferase reporter plasmids were cotransfected with expression plasmids for dominant-negative Notch1 (DN-Notch), constitutively active Notch1 (CA-Notch), constitutively active APP or AICD (CA-APP), or both active proteins in the presence or absence of a Fe65 expression plasmid. Induction levels are compared with empty expression plasmids in 5 independent experiments. Asterisks indicate statistically significant (P⬍0.05 paired t test) changes. C) Schematic representation of the APP and Notch1 expression vectors used. Domains are indicated by symbols, numbers indicate amino acid number.
Figure 3. Interaction of APP695 with CA-Notch (and Fe65). HEK293T cells were transfected with CA-Notch, and/or an empty construct (mock), and/or APP695 , and/or Fe65 (see ⫹ and – above lanes 1 to 16). Cell lysates were immunoprecipitated with an antibody against APP (80750) (A), Fe65 (B, D), and Notch (C), followed by Western blot with an antibody against Notch1 (A, B), Fe65 (C), and APP (22C11) (D). Below each immunoprecipitation is a Western blot showing the input of each protein in the cell lysate. A) When APP695 was immunoprecipitated from cell lysates transfected with APP695 , CA-Notch with or without Fe65 transfection, CA-Notch could be detected by Western blot with an antibody against Notch1 (lanes 2– 4). B) When Fe65 was pulled down from cell lysates transfected with CA-Notch and APP695 with or without Fe65 transfection, Notch could be detected in lysates containing Fe65, CA-Notch with or without APP695 (lanes 6, 8). C) Immunoprecipitation of Notch from lysates expressing CA-Notch, Fe65 with or without APP695 , followed by Western blot with an antibody against Fe65, resulted in detection of Fe65 (lanes 10, 12). D) When Fe65 was immunoprecipitated from cell lysates expressing Fe65, APP695 , with or without CA-Notch, APP695 could be detected by Western blot with 22C11 (lanes 13, 14, 16). Expression of transfected constructs in total lysates (INPUT, lanes 1 to 16) was analyzed by Western blot with antibodies against Notch1, APP695 (22C11), and Fe65. 1454
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Figure 4. Interaction of APP695 with DN-Notch. HEK293T cells were transfected with DN-Notch and APP695 , in the absence (A) or presence (B) of Fe65 (see ⫹ and – above lanes 1 to 8). Cell lysates were immunoprecipitated with an antibody against GFP, which recognizes DN-Notch, followed by Western blot with 22C11, recognizing APP695 or with an antibody against Fe65. When DN-Notch was immunoprecipitated from cell lysates transfected with DN-Notch and APP695 with or without Fe65 transfection, APP695 was detected by Western blot with 22C11 (lanes 2, 4). C) Immunoprecipitation of cell lysates with an antibody against GFP did not pull down Fe65 together with DN-Notch (lanes 5– 8). Expression of transfected constructs in total lysates was analyzed by Western blot with antibodies against GFP, APP695 (22C11), and Fe65 (INPUT, lanes 1– 8).
binding to APP (33, 35), we examined whether Fe65 bound Notch1. Indeed, the intracellular domain of Notch1 bound Fe65 in immunoprecipitation experiments from transfected cell lines (Fig. 3B, C). In the same experiment we could find the known interaction between APP and Fe65 (Fig. 3D) but, remarkably, the intracellular domain of Notch1 and APP also had a direct interaction independent of Fe65 (Fig. 3A, compare lanes 1 and 2). DN-Notch, which lacks the intracellular domain of Notch1, interacted with APP independent of transfected Fe65 (Fig. 4A, B, compare lanes 1 and 2 or 3 and 4 for the reverse experiment). We could not detect binding of Fe65 to the extracellular domain of Notch1 (DN-Notch) (Fig. 4C), which is in accordance with Fe65 being present intracellularly and in accordance with our promoter activation experiments (Fig. 2). We could further map the extracellular interaction domain of APP by using a mutant of APP, APP⫹1 (20), which is processed similar to APP but lacks the transmembrane domain, and is therefore secreted efficiently. Significantly, APP⫹1 did not bind CA-Notch (Fig. 5A), but interacted strongly with DN-Notch (Fig. 5B), suggesting that the extracellular domains of APP and Notch1 have a direct interaction independent of the ␥-secretase site. The reverse pull-down experiment gave the same data (Fig. 5C, D). The specificity of interaction of APP and APP⫹1 with DN-Notch was further demonstrated by showing that both APP proteins do not interact with another overexpressed singlepass transmembrane receptor, trkB (Fig. 6, lanes 4, 5), or with membrane-associated EGFP (Fig. 6, lanes 6 and 7).
Binding of Notch to Fe65 and APP DISCUSSION The promoter activation experiments indicated that there may be cross-talk between APP and Notch downstream signaling, and that Fe65 may stimulate Notch signaling. As Fe65 acts in the APP signaling pathway by
We have presented data showing cross-talk between APP and Notch signaling pathways. Biochemically, APP and Notch1 bind through two nonoverlapping do-
Figure 5. Interaction of APP⫹1 with DN-Notch and CA-Notch. HEK293T cells were transfected with APP⫹1 and CA-Notch (A) or DN-Notch (B). Cell lysates were immunoprecipitated with an antibody against APP⫹1 (Amy6), followed by Western blot with an antibody against Notch1 (A), or with an antibody against GFP (B). In the reverse experiment (C, D), CA-Notch (C), or DN-Notch (D) were immunoprecipitated, after which APP⫹1 was detected with Amy6. Expression of transfected constructs in total lysates was analyzed by Western blot with antibodies against GFP, APP⫹1 (Amy6), and Notch (INPUT, lanes 1 to 8).
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38). However, because the intracellular domain of Notch1 (CA-Notch) lacks the ␥-secretase processing site, it is unlikely that the interaction of APP and Notch1 we describe involves ␥-secretase. We did not observe an increase in mRNA levels of the components of the ␥-secretase complex, but cannot rule out that the activity of ␥-secretase is post-transcriptionally activated. Cross-talk of the Notch signaling pathway with other signaling pathways is not unprecedented, as recently the Notch1 intracellular domain has been shown to bind to Smad3, which results in cross-talk with TGF- signaling (39). Homodimerization of APP (40) and heterodimerization of APP and APP⫹1 (20) leads to activation of APP processing. Heterodimerization of APP and Notch1 could affect ligand binding and processing of both proteins. Indeed, the interaction between APP and Notch1 has recently been confirmed in an open screen approach (41) Implications for Down syndrome
Figure 6. The interaction of APP695 or APP⫹1 is specific for Notch1. HEK293 cells were transfected with APP695 (lanes 2, 4, 6) or APP⫹1 (lanes 3, 5, 7) and DN-Notch (lanes 1–3), intracellularly GFP-tagged trkB (lanes 4, 5), or farnesylated EGFP-F (lanes 6 and 7). Immunoprecipitation was performed with an antibody against GFP, recognizing DN-Notch, trkB, and EGFP-F, Western blot detection was performed with 22C11, recognizing APP695 and APP⫹1. Expression of transfected constructs in total lysates was analyzed by Western blot with antibodies against GFP or APP (22C11) (INPUT, lanes 1 to 7).
mains. The extracellular domain of Notch1 (DN-Notch construct) could bind APP, but not a mutant of APP lacking the signal-peptide (data not shown). Thus, the extracellular interaction is dependent on processing of APP and Notch1. The intracellular interaction of Notch1 and APP appears to be independent of Fe65, although this adaptor protein binds to both CA-Notch and CA-APP. The binding of Notch1 to Fe65 has not been described previously, but similar to APP, low density lipoprotein receptor-related protein binds Fe65 (36) and is processed through ␥-secretase (37). The interaction of Fe65 with these proteins has been shown to occur via an NPxY motif (35, 36), but the intracellular domain of Notch1 lacks such a motif, indicating that Notch1-Fe65 complex formation is mechanistically distinct. It has been suggested that APP and Notch1 interact through competition for the ␥-secretase complex (2, 6, 1456
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In the adult DS cortex we found up-regulation of Notch family members and ligands. In a microarray profile of DS fetal astrocytes, Notch4 was shown to be up-regulated 2.79-fold, P ⫽ 0.01 (42). The observed Notch1 and Dll1 overexpression in DS fibroblasts indicates that activation of the Notch pathway is directly caused by the genetic disequilibrium of genes on chromosome 21, and not by a change in neuronal or glial differentiation. We could not identify any gene known to be involved in the Notch signaling pathway on human chromosome 21 (43), suggesting that the interaction with APP we describe here could cause Notch upregulation.
CONCLUSIONS We propose that in the DS brain, enhanced processing of APP induces disequilibrium of Notch signaling by direct APP-Notch1 interaction, as demonstrated by coimmunoprecipitation, or by cross-talk of the signaling pathways as demonstrated by the luciferase reporter studies. This disequilibrium can be measured in peripheral tissues such as cultured fibroblasts. It has been well established that activation of Notch promotes gliagenesis but suppresses neurogenesis (8, 44, 45). Indeed, a reduced neuronal density has been reported in Down syndrome (46). We would like to thank K. Schuurman for contribution to experiments and A. Salehi (Stanford Universtity) for fruitful discussions. We thank the Netherlands Brain Bank (coordinator R. Ravid, Amsterdam, The Netherlands) for providing the postmortem brain material; T. Hartmann (ZMBH, Heidelberg, Germany) for providing the 22C11 antibody and APP695 construct; T. Russo, University of Naples, Italy for the Fe65 expression plasmid. This research was supported by Platform Alternatieven voor Dierproeven (PAD98.19), EU 5th framework (QLRT-02238), Jan Dekkerstichting en dr. Ludgardine Bouwmanstichting (99-17), HFSP (RG0148/
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1999-B), NWO memory processes and dementia (970-10-029 and 970-10-002), and Hersenstichting Nederland (H00.06).
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The FASEB Journal
Received for publication November 9, 2004. Accepted for publication April 27, 2005.
FISCHER, ET AL.
