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The development of distinct cellular layers and precise synaptic circuits is essential for the formation of well functioning cortical structures in the mammalian brain. The extracellular protein Reelin through the activation of a core signaling pathway including the ApoER2 and VLDLR receptors and the adapter protein Dab1, controls the positioning of radially migrating principal neurons, promotes the extension of dendritic processes in immature forebrain neurons, and affects synaptic transmission. Here we report for the first time that the Reelin signaling pathway promotes the development of postsynaptic structures such as dendritic spines in hippocampal pyramidal neurons. Our data underscore the importance of Reelin as a factor that promotes the maturation of target neuronal populations and the development of excitatory circuits in the postnatal hippocampus. These findings may have implications for understanding the origin of cognitive disorders associated with Reelin deficiency.
A late step in neuronal maturation, which occurs mostly at postnatal ages, consists of synapse formation and the establishment of connectivity. Synaptogenesis is the result of a complex series of events that includes the acquisition of synaptic competence and the apposition of pre-and post-synaptic anatomical structures (Craig et al., 2006). A variety of secreted or cell adhesion proteins have been implicated in the genesis of excitatory or inhibitory synapses, but much remains to be understood about this important process at the molecular level.
In recent years the extracellular protein Reelin has emerged as an important factor that affects several steps in brain development, from neuronal migration to dendritogenesis and synaptic transmission. Homozygous reeler mice lacking Reelin are severely ataxic and exhibit disrupted cellular layers and dendritic trees in neocortical, hippocampal and cerebellar structures (reviewed by (Lambert de Rouvroit and Goffinet, 1998; D’Arcangelo, 2005b)). Heterozygous reeler mice on the other hand appear normal, but dendrite development is delayed (Niu et al., 2004) and the mice are impaired in at least some tests of cognitive function (Tueting et al., 1999; Niu et al., 2004; Brigman et al., 2006; Krueger et al., 2006). The activity of Reelin in layer formation and dendritogenesis is mediated by the same basic signaling pathway, consisting of two Reelin receptors (ApoER2 and VLDLR), src-family kinases (SFKs) Src and Fyn, and the adapter protein Disabled-1 (Dab1) (reviewed by (D’Arcangelo, 2005b)). Homozygous mice lacking ApoER2 and VLDLR (Trommsdorff et al., 1999), Src and Fyn (Kuo et al., 2005) or Dab1 (Howell et al., 1997; Sheldon et al., 1997; Ware et al., 1997), like reeler, exhibit ataxia, disruption of cortical cellular layers, and impaired dendrite development (Tabata and Nakajima, 2002; Niu et al., 2004; Olson et al., 2006; Maclaurin et al., 2007).
Previous anatomical studies of mutant mice revealed that Reelin is important for the development of precise synaptic connectivity in the cerebellum (Mariani, 1982), hippocampus (Borrell et al., 1999) and retina (Rice et al., 2001), However, these defects appeared to arise from improper pruning or branching of presynaptic axonal fibers, or from an indirect deficit in neuron survival. A more direct role for Reelin in the modulation of synaptic function in the brain has been appreciated in recent years (reviewed by (D’Arcangelo, 2005a; Herz and Chen, 2006)). These studies demonstrate that Reelin affects synaptic strength and plasticity. However, it is not yet known whether the Reelin pathway directly affects the formation or the stabilization of anatomical synapses during development.
In this study we investigated the effect of the Reelin pathway on the development of postsynaptic structures in hippocampal glutamatergic synapses. Dendritic spines in apical dendrites of fluorescently labeled hippocampal pyramidal neurons were analyzed in vivo and in organotypic cultures by confocal microscopy. The expression and the recruitment of postsynaptic proteins to hippocampal synaptosomes were also examined by biochemical assays. We demonstrate for the first time a direct role of the canonical Reelin pathway in the formation or stabilization of dendritic spines in the postnatal hippocampus.
Cell culture medium and reagents were purchased from Invitrogen. G10, a mouse monoclonal antibody against Reelin, was purified from hybridoma cell culture supernatants using Hi-Trap protein G columns (Amersham Biosciences). GST-RAP was prepared as previously described (Niu et al., 2004). PP2 and PP3 were from Calbiochem. Antibodies used were: rabbit anti-Dab1 Rockland Immunochemicals), rabbit anti-Synaptophysin (Synaptic Systems), mouse anti-Actin (Chemicon), mouse anti-PSD-95 (Chemicon), rabbit anti-NR2A (Chemicon), and mouse anti-Synapsin IIA (BD Transduction Laboratories). Reelin was obtained as the conditioned medium of the stable cell line CER (Niu et al., 2004). Mock medium was prepared from the parental 293-EBNA cell line. Both media were concentrated approximately 30 fold by centrifugation using Amicon Ultra filters (Millipore) at 2,680x g for 20 minutes prior to addition to neuronal cultures.
All the experiments were performed in accordance with procedures approved by the Animal Protocol Review Committee of Baylor College of Medicine and Rutgers according to National and Institutional Guidelines for animal care established by the National Institutes of Health and approved by the competent Animal Ethics Committee. reeler mice (B6C3Fe-a/a-Relnrl/+) were obtained from The Jackson Laboratories and genotyped by PCR using the following primers: forward, TAATCTGTCCTCACTCTGCC; reverse wild-type, ACAGTTGACATACCTTAATC; and reverse reeler, TGCATTAATGTGCAGTGTTG. PCR conditions were as follows: 1 cycle 5 min at 94°C; 30 cycles 1 min at 94°C, 2 min at 55°C, 3 min at 72°C; 1 cycle 10 min at 72°C. Thy1-YFP transgenic mice (B6.Cg-TgN(Thy1-YFPH)2Jrs) were obtained from The Jackson Laboratories and genotyped by PCR as suggested by the distributor. Dab1 knockout mice were obtained from J.A. Cooper and genotyped as described (Howell et al., 1997). Heterozygous reeler or heterozygous Dab1 knock out mice were crossed with Thy1-YFP transgenic mice to generate the reeler-YFP or Dab1 knock out-YFP mouse colonies, respectively.
Hippocampal cultures were prepared essentially as described previously (Stoppini et al., 1991) from wild type, heterozygous and homozygous reeler littermates or wild type, heterozygous and homozygous Dab1 knock out littermates expressing the YFP transgene. The hippocampi were dissected at postnatal day (P) 4 and placed in Leibovitz’s L-15 media. Meningeal membranes were pealed off and the tissues were placed on the stage of a custom-built tissue chopper. Transverse slices (375 μm thick) were cut and placed on Millicell (Millipore) membranes soaked in culture medium containing 98% Neurobasal-A, 2% B-27 supplement and 0.5 mM glutamine. Typically 6–7 slices were cultured on one membrane and maintained 37°C in 5% CO2 in water-jacked incubator. Culture medium was changed every other day. Sister cultures from the same animal were treated with various agents or left untreated for 11 days in vitro. Chronic treatment of slices was started on the day following dissection and was maintained by adding 30x concentrated mock or Reelin conditioned medium (containing approximately 100 ng/ml Reelin), 50 μg/ml GST-RAP, 50 μg/ml GST, 5 μM PP2 or 5 μM Experiments were terminated by fixation in fresh 4% paraformaldehyde. A blind code was assigned to each culture dish by a different experimenter prior to imaging acquisition and analysis.
Fixed organotypic cultures (375 μm thick) were lifted from membrane inserts with a fine paintbrush and kept free-floating in PBS. Free-floating sections (40 μm thick) were also prepared from the brain of reeler-YFP and Dab1KO-YFP mice perfused at P21 or P32. All sections were blocked in PBS containing 0.25% Triton X-100 and 5% normal goat serum for h, incubated overnight at 4°C with primary antibodies and then incubated for one hour with secondary antibodies Alexa 594 Goat Anti-Rabbit or Alexa 594 Goat Anti-Mouse IgG (Invitrogen) for immunofluorescence. To visualize YFP fluorescence the sections were directly imaged by confocal microscopy using a FluoView FV300 (Olympus) or an LSM 510 Meta (Zeiss) confocal laser scanning microscope. Some brain sections were also processed for anti-GFP immunohistochemistry using the Vectastain Elite ABC kit (Vector Laboratories) and the signal was detected by 3,3′-diaminobenzidine (DAB) substrate (Vector Laboratories). To measure the extension of apical dendrites, YFP-labeled pyramidal neurons in the CA1 region of hippocampal tissue sections obtained from wild type and heterozygous reeler mice were imaged using an Olympus IX50 fluorescence microscope. The length of apical projections from the pyramidal layer to the bottom of the stratum lacunosum moleculare (SLM) was measured using the NIH Image J Software. A total of 5 sections were analyzed for each genotype, and three measurements were obtained from each section. The Student’s t-Test was used for statistical analysis.
YFP-labeled dendrites in 375 μm-thick organotypic cultures or 40 μm-thick brain sections were imaged using confocal microscopy. For quantitative analysis, apical dendritic segments 20–40 μm long in the stratum radiatum (SR) and SLM were imaged using the FluoView FV300 confocal microscope and a 60x UPlanApo objective (numerical aperture (NA) =0.65–1.25 (Olympus) followed by 3 x digital zoom. Kalman accumulation averaging of 2 was used to reduce the noise. Maximum projection images were generated from 0.15 μm incremental steps in the z axis. Apical dendrites could be readily identified in wild type and heterozygous reeler or Dab1 knock out mice based on their stereotypical projections. For homozygous mutant sections where pyramidal neurons appeared disorganized and disoriented, apical dendrites were tentatively identified based on their relative longer extension compared to basal branches. Dendritic branches were classified as 2nd–4th order according to their topological centrifugal order, where branch order starts at 1 for branches connected to the soma and increases after each branch point. Primary dendrites were not included in the analysis. Terminal branches that could not be traced to the soma but that exhibited a terminal tip were analyzed separately from 2nd–4th order branches. These may include cut ends and do not necessarily correspond to the most distal branches. Dendritic spines were scored only if they exhibited a distinct morphology defined as the presence of a neck and mushroom-shaped head or thin-shaped head. These types of spine were by far the most abundant protrusions observed in P21/P32 mice brain sections or 11 DIV culture slices. Stubby protrusions, where neck and head structures could not be distinguished, or filopodia were not included in the analysis. Spines were quantified from confocal image projections of dendritic segments traced with a digital Neurolucida using the NeuroExplorer software (MBF Bioscience). The data in each experimental or control sample were obtained from 25–35 dendritic segments of multiple neurons from multiple sections or slices. All dendritic segments were analyzed in blind with respect to the genotype or the treatment received in each experimental group. Each type of experiment was repeated three times and the results were combined for final analysis. The Student’s t-Test was used to determine statistical significance (p<0.05).
The hippocampi of P21 or P32 wild type, heterozygous and mutant littermates were processed for synaptosome fractionation at the same time. The tissue was dissected in 0.9% NaCl and placed in 500 μL of ice-cold homogenization buffer containing 0.32 M Sucrose, 1 mM EDTA, 5 mM HEPES (pH 7.4), and protease inhibitor Complete Mini tablet (Roche) for homogenization (Glas-Col Homogenizer, Thomas Scientific). Total lysates were centrifuged at 800 x g for 10 minutes at 4°C. The supernatant was collected and centrifuged again at 800 x g for 10 minutes at 4°C. The supernatant was collected and subjected to a third centrifugation at 7200 x g for 15 minutes at 4°C. The pellet, containing the synaptosome fraction, was resuspended in 100 μL of homogenization buffer. The protein concentration of the total lysates as well as the synaptosome fractions were measured and normalized with homogenization buffer.
10 μg of total lysate or synaptosome fractions were loaded in 4–12% Tris-Glycine SDS-PAGE gels (Invitrogen), separated at 120 V for 2 hours, and transferred to 0.22 μm nitrocellulose membrane (Invitrogen) at 200 mAmps for 2 hours. The membranes were blocked with 3% milk /1X TBS-T (Tris-Buffered Saline with 0.1% Tween-20) for 1 hour at room temperature followed by incubation with primary antibodies and then secondary antibodies diluted in 0.3% milk/1X TBS-T for 1 hour at room temperature. Membranes were washed 3–4 times in 1X TBS-T for 1 hour, incubated with ECL-Plus Western Blotting Detection System (Amersham) for 5 minutes, and exposed to autoradiographic film (Denville). X-ray films were scanned for densitometry analysis using NIH Image J software. The percent change of the protein levels in heterozygous or mutants from wild type synaptosome fractions was measured. The Student’s t-Test was used for statistical analysis.
In the embryonic and early postnatal hippocampus Reelin is expressed at high levels by Cajal-Retzius cells, which secrete large amounts of the protein in the stratum lacunosum moleculare (SLM) of the hippocampus proper and in the outer marginal layer (OML) of the dentate gyrus (D’Arcangelo et al., 1995; Alcantara et al., 1998). GABAergic interneurons in all layers of the hippocampus also begin to express Reelin at early postnatal ages but persist throughout adult life while Cajal-Retzius cells slowly disappear. Components of the Reelin signaling pathway such as ApoER2, VLDLR and Dab1 are expressed throughout development in hippocampal pyramidal and dentate gyrus neurons (Trommsdorff et al., 1999; Niu et al., 2004).
To investigate the development of dendritic spines in Reelin target cells we took advantage of a transgenic mouse line expressing yellow fluorescent protein (YFP) in selected neuronal populations under the control of the Thy1 promoter (line H) (Feng et al., 2000). In this line, high levels of YFP expression are observed in the hippocampus starting from the second postnatal week and are maintained throughout adulthood in some pyramidal neurons in areas CA1 and CA3, and in many dentate gyrus neurons. We bred these transgenic mice with reeler heterozygous mice to generate a reeler-YFP colony and then crossed heterozygous mice to obtain YFP-positive wild type, heterozygous and homozygous reeler mice for our study. When hippocampal sections from postnatal day (P) 21 mice were examined by fluorescence confocal microscopy, YFP-positive neurons appeared to be normally placed in the pyramidal layer of wild type and heterozygous reeler mice, but they were clearly ectopic in homozygous reeler mutants, as expected (Figure 1A–C). To study spine development we focused on apical dendrites of CA1 pyramidal neurons because the limited number of YFP-positive processes facilitates their analysis, and also because they project toward the Reelin-rich SLM and thus are more likely to be affected by this protein. The overall length and orientation of the apical tree of YFP-positive CA1 pyramidal neurons appeared normal in wild type (Figure 1D) and heterozygous reeler mice (Figure 1E), whereas these projections were obviously misoriented and stunted in homozygous reeler mutants (Figure 1F). To examine apical dendrites in more detail we collected composite, high-resolution confocal stack images of YFP-labeled projections in comparable CA1 regions of wild type, heterozygous and homozygous reeler mice (Figure 1G–I). These images confirmed that apical dendrites appear morphologically normal in heterozygous mice, whereas they are severely reduced in length and complexity in homozygous reeler mice. Quantitative analysis of the thickness of the apical projection, defined as the distance between the pyramidal layer and the hippocampal fissure, further demonstrated that the extension of apical dendrites is similar in wild type and heterozygous mice (Figure 1J). We then analyzed 2nd–4th order and terminal apical branches of YFP-positive pyramidal neurons in the stratum radiatum (SR) and SLM to determine whether they bear dendritic spines. When dendritic segments from P21 mice were imaged at high magnification by confocal microscopy, mature dendritic spines with distinct neck and head morphology were readily observed in all genotypes (Figure 1K–M and Suppl. Figure 1A–C). Double labeling with antibodies against the presynaptic marker synaptophysin revealed that axon terminals were juxtaposed to at least some YFP-labeled spines in both, wild type or mutant dendrites, indicating that these latter structures can participate in the formation of synapses (Figure 1N and O). However, in this study we focused on the effect of Reelin on the development of postsynaptic components of the synapse defined as YFP-labeled mushroom shaped spine protrusions, and not that of the synapse as a whole. Quantitative analysis indicated that the density of spines was dramatically and significantly reduced in heterozygous and homozygous reeler mutants compared to wild type mice (p <0.001). Values in the 2nd–4th order branches were: wild type 0.94 ± 0.02 spines/μm, heterozygous 0.46 ± 0.03 spines/μm, and mutant 0.36 ± 0.02 spines/μm (Figure 1P). Values in the terminal segments were: wild type 0.83 ± 0.04 spines/μm, heterozygous 0.38 ± 0.03 spines/μm, and mutant 0.31 ± 0.02 spines/μm (Suppl. Figure 1D). This deficit cannot be attributed to neuronal ectopia since neurons were normally positioned in reeler heterozygous mice. We also analyzed apical dendrites in older (P32) mice. The data indicate that spine density is also drastically reduced in both heterozygous and homozygous reeler mice at this age in 2nd–4th order apical branches (Figure 1Q) as well as terminals (Suppl Figure 1E). These data demonstrate that normal levels of Reelin are required in the adult brain for the development or the maintenance of hippocampal dendritic spines.
To gain biochemical evidence of spine abnormalities, we also analyzed the expression of proteins normally associated with these structures in the reeler background. We first prepared total hippocampal protein extracts and crude synaptosomal fractions from adult wild type mice and verified by Western blot analysis that the synaptosomal fractions were enriched, as expected, for presynaptic proteins such as Synapsin IIA, and postsynaptic markers such as PSD-95 and the NMDA receptor subunit NR2A (Figure 2A). These latter proteins have been previously shown to associate with the Reelin receptor ApoER2 in postsynaptic density fractions (Beffert et al., 2005). We then prepared lysates and crude synaptosomal fractions from the hippocampi of wild type, heterozygous and homozygous reeler littermates. Western blot analysis of total lysates revealed that the levels of NR2A and PSD-95 were similar in all genotypes (Figure 2B and C). However, when synaptosomal fractions were analyzed, we found that the levels of NR2A and PSD-95 were significantly decreased in heterozygous and homozygous reeler mice compared to wild type littermates (Figure 2B and D). Levels of NR2A were: 22 ± 17% in heterozygous (78% reduction from wild type) and 16 ± 8.3% in homozygous reeler mice (84% reduction from wild type). Levels of PSD-95 were: 30.7 ± 1.3% in heterozygous (69.3% reduction from wild type) and 16.3 ± 1.5% in homozygous reeler mice (83.7% reduction from wild type). These observations further indicate that Reelin deficiency leads to the failed development or maintenance of dendritic spines.
To further investigate the role of Reelin in spine formation in an in vitro system that could be experimentally manipulated, we generated hippocampal organotypic cultures from littermates of the reeler-YFP mouse line. The cultures were routinely established at postnatal day 4 and maintained for 11 days in vitro (DIV) to allow neuronal maturation to take place. When cultures were fixed and imaged, YFP-positive pyramidal neurons appeared arranged in a diffuse cellular layer in slice cultures obtained from wild type (Figure 3A) or heterozygous reeler mice (not shown), whereas they appeared clustered and disorganized in slices derived from homozygous reeler mutants (Figure 3C). Immunofluorescence analysis confirmed that Reelin was abundantly expressed in Cajal-Retzius cells located in the SLM at the border between the hippocampus proper and the dentate gyrus (Figure 3B), as it is normally observed in vivo. Confocal analysis of dendritic branches (Figure 3D) and terminals (Suppl. Figure 1F) indicated that mature spines were present in explants of all genotypes, but that spine density was strongly affected by the Reelin mutation. Quantitative analysis (Figure 3E) demonstrated a dramatic reduction in spines density on apical dendrites of heterozygous and homozygous reeler mice, similar to the in vivo results described above. Values in apical branches were: wild type 0.867 ± 0.03 spines/μm, heterozygous 0.507 ± 0.034 spines/μm (42% reduction compared to wild type), and homozygous reeler mice 0.272 ± 0.026 spines/μm (~69% reduction compared to wild type). Values in apical terminals were: wild type 0.738 ± 0.032 spines/μm, heterozygous 0.352 ± 0.018 spines/μm (52% reduction compared to wild type), and homozygous reeler mice 0.286 ± 0.018 spines/μm (~61% reduction compared to wild type).
To determine whether Reelin directly promotes the formation of dendritic spines, cultured slices obtained from mutant reeler hippocampus were incubated continuously with a conditioned medium containing recombinant Reelin or with a mock medium for 10 DIV. The data show that Reelin treatment rescued the reeler phenotype and lead to a significant increase in the density of dendritic spines compared to untreated explants or to cultures treated with mock medium (Figure 3D and E). Densities in apical branches were 0.75 ± 0.04 spines/μm in Reelin-treated explants and 0.22 ± 0.03 spines/μm in mock-treated explants. Densities in terminal branches (Suppl. Figure 1G) were 0.69 ± 0.05 spines/μm in Reelin-treated explants and 0.24 ± 0.02 spines/sm in mock-treated explants. The difference between Reelin and mock values was statistically significant (p < 0.001) in both, branches and terminals. The density value achieved in reeler explants cultured in the presence of Reelin was similar to that of wild type cultures. These findings suggest that the observed reduced density of dendritic spines in heterozygous and homozygous mutant reeler mice is directly caused by Reelin deficiency in vivo and in vitro.
High affinity Reelin receptors of the lipoprotein receptor superfamily, ApoER2 and VLDLR, are expressed in hippocampal pyramidal neurons (Niu et al., 2004). To determine if their binding activity is necessary for Reelin-induced spine development we prepared sister hippocampal organotypic cultures from mutant reeler mice and incubated them with Reelin in the presence or absence of the competitive antagonist Receptor Associated Protein (RAP) fused to Glutathione S-Transferase (GST). This protein is well known to inhibit Reelin binding to ApoER2 and VLDLR, and to prevent Dab1 phosphorylation (Hiesberger et al., 1999). As a control we used GST alone or a mock conditioned medium. As above, Reelin treatment alone increased spine density in apical branches (Figures 3F) as well as terminal dendrites (Suppl. Figure 1G) by approximately 3-fold compared to untreated control or mock treatment. Spine densities in apical branches of reeler explants were as follows: 0.63 ± 0.03 spines/μm in Reelin-treated explants, 0.25 ± 0.02 spines/μm in mock-treated explants, and 0.21 ± 0.01 spines/μm in untreated explants. Densities in terminal branches of reeler explants were as follows: 0.68 ± 0.02 spines/μm in Reelin-treated explants, 0.18 ± 0.02 spines/sm in mock-treated explants, and 0.26 ± 0.02 spines/μm in untreated explants. However, the addition of RAP completely prevented Reelin induction and resulted in spine density values similar to those observed in control explants, whereas GST had no effect (Figure 3D and F). Apical branches of Reelin+RAP-treated explants had a density of 0.25 ± 0.02 spines/μm whereas Reelin+GST-treated explants had a density of 0.70 ± 0.03 spines/μm. Terminal branches (Suppl. Figure 1G) of Reelin+RAP-treated explants had a density of 0.26 ± 0.02 spines/μm, whereas Reelin+GST-treated explants had a density of 0.68 ± 0.03 spines/μm. The difference between Reelin+RAP and Reelin alone or Reelin+GST was statistically significant (p< 0.001). These results strongly suggest that Reelin binding to VLDLR and ApoER2 is necessary to promote the formation of dendritic spines.
Dab1 is a cytoplasmic adapter protein that binds to VLDLR and ApoER2 and mediates Reelin signaling (Hiesberger et al., 1999; Howell et al., 1999). The phenotype of Dab1 homozygous null mutants is virtually indistinguishable from reeler, and is characterized by extensive neuronal ectopia (Howell et al., 1997). Like heterozygous reeler, heterozygous Dab1 mutants exhibit no dyslamination of cellular layers. To investigate the role of Dab1 in spine formation, we generated a Dab1 knockout-YFP mouse colony mice as described above for reeler mice, and obtained YFP-positive mice of all possible Dab1 genotypes. Immunofluorescence analysis of hippocampal sections revealed that Dab1 is detectable in hippocampal pyramidal neurons, including YFP-positive neurons, in wild type, but not in knock out mutant mice (Figures 4A–E). As expected, YFP-positive neurons were properly positioned in wild type and heterozygous Dab1 mice, and appeared ectopic in homozygous knock out mutants (Figures 4F–H). Next, we examined dendritic spines present on YFP-labeled apical branches (Figures 4I–K) and terminals (Suppl. Figure 2A–C) of CA1 pyramidal neurons. As in reeler, quantitative analysis revealed that the density of dendritic spines is significantly reduced in heterozygous as well as homozygous Dab1 mutants compared to wild type littermates (Figures 4L and Suppl. Figure 2D). Densities in apical dendritic branches were as follows: wild type 0.75 ± 0.06 spines/μm, heterozygous 0.47 ± 0.03 spines/μm, and mutant 0.34 ± 0.03 spines/μm. Densities in terminal branches were as follows: wild type 0.76 ± 0.03 spines/μm, heterozygous 0.35 ± 0.02 spines/μm, and mutant 0.41 ± 0.03 spines/μm. The difference between heterozygous or mutant and wild type mice was statistically significant for both branches and terminals (p < 0.001).
To determine whether the expression and the recruitment of postsynaptic proteins to the spines are also affected by the Dab1 genotype we carried out Western blot analysis of total hippocampal lysates and crude synaptosomal fractions. The data indicate that, like in reeler mice, the total levels of NR2A and PSD-95 in hippocampal extracts did not significantly change in heterozygous or homozygous Dab1 mutants compared to wild type littermates (Figure 5A). However, as in reeler, levels of NR2A and PSD-95 in synaptosomal preparations were significantly reduced in both, heterozygous as well as homozygous Dab1 mutants compared to wild type (Figure 5B). Noticeably however, the levels of NR2A in heterozygous Dab1 knock out mice were intermediate between those of wild type and homozygous mice, whereas the levels of PSD-95 were similarly reduced in both, heterozygous and homozygous Dab1 mutants. Levels of NR2A were: 66 ± 5.7% in heterozygous (34% reduction from wild type) and 15 ± 3.6% in homozygous Dab1 mutant mice (85% reduction from wild type). Levels of PSD-95 were: 33 ± 4.8% in heterozygous (67% reduction from wild type) and 44 ± 8.6% in homozygous Dab1 knock out mice (56% reduction from wild type). These data suggests that Dab1 activity primarily affects the intracellular localization of postsynaptic proteins.
To confirm our in vivo observation that spine density is reduced in Dab1 mutant mice, we also established hippocampal organotypic cultures from YFP-positive wild type, heterozygous and homozygous Dab1 knock out mice (Figures 5A–C) and examined dendritic spines in apical branches (Figures 6D) and terminals (Suppl. Figure 2E). Again, we observed a significant reduction in spine density in heterozygous and homozygous Dab1 mutant cultures compared to wild type (p < 0.001). Densities in the apical branches were: wild type 0.72 ± 0.04 spines/μm, heterozygous 0.26 ± 0.03 spines/μm, and mutant 0.24 ± 0.03 spines/μm. Values in terminal branches were: wild type 0.57 ± 0.05 spines/μm, heterozygous 0.19 ± 0.02 spines/μm, and mutant 0.08 ± 0.02 spines/μm. Thus, both in vivo and in vitro data indicate that Dab1 plays a role in dendritic spine formation.
Previous studies demonstrated that in order for Dab1 to transduce Reelin signaling, src-family kinases (SFKs) must phosphorylate it on specific tyrosine residues (Howell et al., 2000; Keshvara et al., 2001; Ballif et al., 2003). Addition of the specific SFK inhibitor PP2, but not the inert control compound PP3, blocks Reelin-induced Dab1 phosphorylation as well as the activation of downstream kinases such as Akt (Arnaud et al., 2003; Bock and Herz, 2003). We previously showed that PP2 also blocks Reelin-induced dendrite elongation in dissociated hippocampal neurons (Niu et al., 2004). Here we used this pharmacological inhibitor to test the role of SFKs in dendritic spine formation using wild type YFP-positive hippocampal cultures. Addition of PP2 at doses known to effectively block Dab1 phosphorylation resulted in a significant reduction of spine density in apical branches and terminals of pyramidal neurons compared to controls (p < 0.001), whereas PP3 had no effect (Figures 6E and Supp. Figure 2F). Densities in the apical branches of PP2-treated explants were 0.21 ± 0.01 spines//μm, PP3-treated explants 0.64 ± 0.03 spines/μm), and untreated explants 0.60 ± 0.03 spines//μm. Densities in the terminal branches were: PP2-treated explants, 0.17 ± 0.01 spines//μm, PP3-treated explants, 0.57 ± 0.03 spines//μm, and untreated explants 0.53 ± 0.03 spines//μm. Together with the findings described above these data indicate that Reelin-induced and SFK-mediated Dab1 phosphorylation is important for the formation or maintenance of dendritic spines.
We investigated the role of Reelin signaling on dendritic spine formation, a prerequisite for synaptogenesis and the establishment of neuronal connectivity, and thus a crucial aspect of postnatal hippocampal development. We demonstrated that loss or reduction of Reelin signaling results in a dramatic defect in the density of dendritic spines in hippocampal pyramidal neurons in vivo and in vitro. Reduced spine density was observed in apical dendrites of heterozygous and homozygous mutant reeler mice (Figures 1 and and3).3). This phenotype is caused by a deficit in native Reelin, since it can be rescued in vitro by the addition of recombinant Reelin to the medium of reeler hippocampal cultures (Figure 3). A similar phenotype consisting of reduced spine density was also seen in the hippocampus of Dab1 heterozygous and homozygous knock out mice in vivo and in vitro (Figures 4 and and6).6). Through the use of specific inhibitors we further provided evidence that ApoER2 and VLDLR receptors (Figure 3) and SFKs (Figure 6) are also crucial for the formation or maintenance of dendritic spines. It will be interesting in the future to investigate whether the PI3K/Akt/mTOR pathway, a downstream branch of the Reelin pathway that is important for dendrite growth (Jossin and Goffinet, 2007), also mediates its effect on spine development. Overall our data highlight a biological function of the Reelin pathway in the postnatal brain, which affects neuronal maturation and enables the formation of normal circuitry in the mammalian brain.
We generated colonies of reeler-YFP or Dab1 knock out-YFP mice to better visualize hippocampal dendrites and spines. To expand on our in vivo observations we established hippocampal cultures from these transgenic lines. YFP was detectable in many pyramidal and dentate neurons beginning at 2 DIV and remained cell type-specific for the entire time in culture. Mature spines were observed starting at 5 DIV and continued to develop until at least 15 DIV. We selected 11 DIV as the most reliable time point when explants appeared healthy and mature spines could be observed and compared across different mutant genotypes. Our in vitro data are strikingly similar to those obtained in vivo, demonstrating a deficit in spine density in heterozygous or homozygous reeler and Dab1 mutants. We also verified that organotypic cultures appropriately expressed Reelin mostly in Cajal-Retzius cells located at the boundary between the hippocampus proper and the dentate gyrus. These observations further indicate that the hippocampal organotypic culture is a valid model system to study Reelin-induced spine development.
Our findings are consistent with previous electron microscopy studies indicating that heterozygous reeler mice have fewer spines than normal in the cortical gray matter (Liu et al., 2001), and that secreted Reelin accumulates in the extracellular matrix around dendrites and spines of cortical pyramidal neurons (Rodriguez et al., 2000). Given that at least some ApoER2 has also been localized to postsynaptic structures (Beffert et al, 2005), our data suggest that Reelin may act locally near synaptic contacts to promote spine development. However, it is also conceivable that Reelin affects spine development by acting remotely, in the cell body or in cellular processes to regulate the trafficking and localization of postsynaptic proteins at the synapse. Since postsynaptic proteins such as PSD-95 and the NMDA receptor interact with the ApoER2 (Beffert et al., 2005; Hoe et al., 2006), and Dab1 promotes membrane localization of Reelin receptors (Morimura et al., 2005), it is possible that changes in receptor trafficking in mutant mice may contribute to their reduction at the synapse. Alternatively, a specific defect in the trafficking of synaptic proteins and/or their assembly at the dendritic spines may underlie their deficit in Reelin signaling mutants. Further investigation will be necessary to elucidate the exact cause of the observed decrease of postsynaptic proteins in heterozygous and mutant synaptosomal fractions. Noticeably, the decrease in synaptosomal levels of postsynaptic proteins in either heterozygous or homozygous mice was more dramatic compared to the decrease in spine density. This suggests that postsynaptic structures can form even in the presence of reduced levels of NR2A and PSD-95, however, it is possible that some of these spines may not be stable or may be unable to form active synapses. The static imaging technique utilized in this study does not allow us to determine whether the activity of Reelin induces the formation of new spines, promotes the maturation of immature spines or favors the maintenance of mature spines at the sites of synaptic contact. Further studies using dynamic imaging techniques are necessary to address this issue.
The observed reduced density of spines seen in conditions where the Reelin signaling pathway is suppressed may be viewed as the long-term result of poor dendrite development. Previous studies have in fact shown that dendrite development is impaired in young (3–5DIV) hippocampal cultures of heterozygous or homozygous reeler mice. However, dendrite elongation in long-term cultures of Reelin signaling mutants appears normal (Maclaurin et al., 2007). In the present study we also found that the extension of apical dendrites is normal in heterozygous reeler mice examined at postnatal day 21 or 32, suggesting that the dendritic growth defect may be transient in mice with reduced Reelin activity, whereas the spine deficit may persist into adult ages.
In the past few years a number of studies focused on the role of Reelin in the postnatal and adult brain, particularly emphasizing its effect on synaptic transmission, plasticity, learning and memory (reviewed by (Herz and Chen, 2006)). Addition of recombinant Reelin promotes hippocampal long term potentiation (LTP) through the activity of lipoprotein receptors (Weeber et al., 2002). A splicing variant of ApoER2 capable of interacting with PSD-95 and the JNK interacting protein (JIP) is important for Reelin-induced LTP and the formation of spatial memory (Beffert et al., 2005). The role of Reelin in synaptic function is mediated in part through interactions between ApoER2 and the NMDA receptor (Beffert et al., 2005). These proteins form a synaptic complex that controls Ca++ entry and thus regulate synaptic plasticity. In addition, Reelin signaling is also important for the regulation of NMDA receptor subunit composition during hippocampal maturation (Sinagra et al., 2005; Groc et al., 2007) and the NMDA receptor-mediated activity in cortical neurons (Chen et al., 2005). Recent studies further revealed that Reelin also enhances glutamatergic transmission through AMPA receptors (Qiu and Weeber, 2007). We further demonstrated here that Reelin, through the activation of its signaling pathway, impacts the recruitment of postsynaptic proteins to the spines thus favoring the development of anatomical synaptic structures, in addition to impacting the physiological activity of the synapse.
Complete or partial loss of Reelin is associated with a variety of developmental brain disorders. Absence of Reelin due to genetic mutations results in lissencephaly with cerebellar hypoplasia (LCH), a phenotype similar to reeler characterized by severe and widespread neuronal migration defects (Hong et al., 2000). LCH patients also exhibit seizures, indicative of abnormal synaptic activity. Reduced RELN expression due to epigenetic mechanisms is associated with schizophrenia and psychotic bipolar disorder (Impagnatiello et al., 1998; Guidotti et al., 2000; Eastwood and Harrison, 2003; Grayson et al., 2005; Herz and Chen, 2006). Genetic association studies also supported an association of RELN gene variants with treatment-resistant schizophrenia (Goldberger et al., 2005), particularly in women (Shifman et al., 2008). A recent study further suggested that allelic variants of RELN contribute to specific endophenotypes of schizophrenia such as working memory and executive functioning (Wedenoja et al., 2007). Reduced Reelin expression was also reported in autistic patients (Fatemi et al., 2001). Genetic studies further supported a role for the RELN gene in the susceptibility to autism, at least in some populations (Persico et al., 2001; Zhang et al., 2002; Skaar et al., 2004; Serajee et al., 2006). Interestingly, we found that the spine density deficit is prominent in heterozygous reeler mice, where reduced Reelin levels are present, and it is quite similar to that of homozygous mutants. This unexpected result is further corroborated by explant and Dab1 mutant anatomical data, and by biochemical data. Overall, the results suggest that the control of spine development by Reelin is independent of the regulation of neuronal positioning and further validate the use of heterozygous reeler mice as models of cognitive dysfunctions.
In addition to developmental brain disorders, Reelin has also been implicated in neurodegenerative diseases, suggesting a convergence of these two apparently distinct types of disorders (Bothwell and Giniger, 2000). Independent studies reported altered Reelin expression and glycosylation patterns (Botella-Lopez et al., 2006), and reductions of Reelin-expressing pyramidal neurons in the entorhinal cortex of Alzheimer’s disease brains (Chin et al., 2007). Whether developmental or degenerative in nature, cognitive disorders likely arise from abnormalities in synaptic connectivity or function. Our findings provide a plausible mechanism by which Reelin deficiency may be a potential risk factor for cognitive dysfunctions.
We thank T. Curran for J. Herz for plasmid constructs, A. Goffinet for Reelin antibodies, J. Cooper for Dab1 knock out mice, J. Swann and H-C. Lu for technical advice and helpful discussion, and the Neuroscience Imaging Facility in the W.M. Keck Center for Collaborative Neuroscience at Rutgers for assistance with confocal microscopy. Supported by NIH R01 NS042616 from NINDS (G.D.) and a Research Supplement to Promote Diversity in Health-Related Research (O.Y.).
Senior Editor: Dr. Marie T. Filbin