Information

Why use embryonic neurons to study protein knockouts/mutants in long term potentiation?

Why use embryonic neurons to study protein knockouts/mutants in long term potentiation?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

Just wondering if anyone had some ideas about the question in the title. I'm just wondering why some papers use embryonic cultures of specific brain regions for neurones to test the effects of knockouts/mutations on synaptic plasticity (for example, long term potentiation, LTP) instead of adult cultures?

Many thanks


Some suggestions, there may be more:

1) The knockout may not be viable to adulthood (the animals die). Perhaps heterozygotes are viable, but to test the full knockout you need a homozygote.

2) Even if the knockout is viable to adulthood, the brain may develop adaptations to the knockout that aren't specific to the knockout itself: up- or down-regulation of certain channels, for example.

3) Plasticity is upregulated in developing brains compared to adult brains, so the effects may be more pronounced in the young tissue.

4) Younger tissue cultures better - you can keep cells alive longer, more easily.

Also good to note is that undifferentiated stem cells tend to be "neuron-default" - that is, if you just culture embryonic stem cells and you don't do anything special to them, they will tend to generate neuron-like cells. In the adult, neurons are always going to be alongside other types of cells: glia, vascular tissue, etc.


Targeted Genomic Disruption of H-ras and N-ras, Individually or in Combination, Reveals the Dispensability of Both Loci for Mouse Growth and Development

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5

Carlos Vicario-Abejón

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5

Pedro Fernández-Salguero

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5

Alberto Fernández-Medarde

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5

Nalini Swaminathan

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5

Kate Yienger

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5

Eva Lopez

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5

Marcos Malumbres

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5

Ron McKay

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5

Jerrold M. Ward

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5

Angel Pellicer

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5

Eugenio Santos

Centro de Investigación del Cáncer, IBMCC, CSIC-USAL, University of Salamanca, Salamanca, 1 and Departamento Bioquímica y Biolog໚ Molecular, Universidad de Extremadura, Badajoz, 4 Spain Laboratory of Cellular and Molecular Biology, National Cancer Institute, 2 and Laboratory of Molecular Biology, National Institute of Neurological Disorders and Stroke, 3 Bethesda, and Veterinary and Tumor Pathology Section, National Cancer Institute, Frederick, 6 Maryland and Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, New York 5


1. Introduction

L1cam is an X-linked gene that encodes the neural cell adhesion molecule L1. L1cam is a member of the transmembrane immunoglobulin gene superfamily and one of four genes in the L1cam subfamily (Chl1, Nrcam, Nfasc, and L1cam itself). L1cam and its other subfamily members are widely expressed in the developing and adult nervous system (Allen Brain Atlas). L1 in particular is known to play essential roles during neurodevelopment (Kenwrick et al., 2000 Hortsch et al., 2014 Sytnyk et al., 2017).

The extracellular domain of L1 can interact with many other cell surface ligands and receptors and its intracellular domain can activate signaling cascades. In humans, engineered loss of L1CAM expression in ES-derived cultured neurons leads to deficits in axonal and dendritic arborization (Patzke et al., 2016). Over 350 inherited and spontaneous mutations in L1CAM, predicted to lead to loss of L1 function, have been identified in the human population (NIH, U.S. National Library of Medicine, Genetics Home Reference). These mutations often lead to “L1 syndrome”, almost exclusively in males due to the X-linked nature of L1CAM. Affected individuals exhibit a range of nervous system defects with the most common manifestations including hydrocephalus due to stenosis, spastic paraplegia type 1, abnormal gait, corpus callosum agenesis, aphasia, and intellectual disabilities (including autism and schizophrenia) (Wong et al., 1995 Christaller et al., 2017).

This spectrum of human nervous system defects overlaps with the spectrum observed in L1cam knockout mice and rats. L1cam knockout rodents exhibit hydrocephalus and enlarged ventricles, impaired hind limb motor control, reduced corpus callosum and corticospinal tracts, increased perinatal lethality, smaller body size, cerebellar defects, and other neurological deficits (Dahme et al., 1997 Cohen et al., 1997 Fransen et al., 1998 Emmert et al., 2019). The importance of L1 for nervous system development is conserved beyond vertebrates. In the worm C. elegans, for example, the L1 homolog SAX-7 is required for neuron positioning and neurite branching (Dong et al., 2013 Salzberg et al., 2013 Diaz-Balzac et al., 2015 Zou et al., 2016 Yang et al., 2019).

In worms and fruit flies, the growth and branching of axons and dendrites are in some cases mediated by an interaction between L1 and the homologs of Fibroblast Growth Factor Receptors (FGFRs) (Diaz-Balzac et al., 2015 Forni et al., 2004). An L1-FGFR interaction to promote neuron maturation extends to mammalian cells, as shown in several defined culture assays (Doherty and Walsh, 1996 Saffell et al., 1997) and in the proliferation and mobilization of glioblastoma cells (Anderson and Galileo, 2016). L1 can act as a ligand that activates not only FGFRs, but also neurotrophin receptors (Colombo et al., 2014). FGFRs and neurotrophin receptors play multiple roles throughout neurodevelopment (Guillemot and Zimmer, 2011 Hrt, 2011 Park and Poo, 2013). In addition, FGF and neurotrophin receptors are important in promoting multiple steps of hippocampal neurogenesis in the adult dentate gyrus (DG) where FGFR activity is necessary and sufficient to promote both neurogenesis and dendritogenesis and Tyrosine receptor kinase B (TRKB) activity promotes dendrite maturation (Kang and Hrt, 2015 de Vincenti et al., 2019).

However, the extent to which L1 plays a role coincident with that of FGFRs and TRKs in the adult DG remains unknown. Based on L1′s essential roles during neurodevelopment across species and in some cases its direct action through tyrosine kinase receptors, we hypothesized that L1 is required for one or more steps in the differentiation lineage of newborn neurons in the adult hippocampal DG. Using several Cre-driver mouse lines to conditionally delete L1cam in different cell types of the neurogenic lineage and surrounding neurons, we find defects in neuron production, dendritogenesis, and behavior, suggesting that L1 is critical not only during developmental neurogenesis, but also during adult hippocampal neurogenesis.


Chapter 184 - Effects of cGMP-Dependent Protein Kinase Knockouts

Cyclic GMP-dependent protein kinases (cGKs) are serine/threonine kinases which are activated by the second messenger cGMP. The amino terminus of cGKI is encoded by two alternatively used exons, resulting in the production of two cGKI isoforms, cGKIα and cGKIβ. Nitric oxide (NO) and atrial-natriuretic peptide (ANP) stimulate cGMP synthesis and relax small arteries and arterioles, resulting in a decreased blood pressure. Targeted inactivation of the NO-synthase III (NOS III), ANP, or ANP receptor gene caused hypertension. NO is of major importance for the homeostasis of platelet-endothelium and platelet–platelet interactions by inhibiting the adhesion of platelets to injured endothelium, and platelet activation and aggregation. Platelet cGKI is essential to prevent intravascular adhesion and aggregation of platelets after ischemia, probably by inhibiting the activation of the platelet fibrinogen receptor, glycoprotein IIb–IIIa. These effects of cGKIβ are most likely mediated by IRAG and a reduced release of Ca 2+ from intracellular stores. cGKI plays an important role for cardiovascular and gastrointestinal homeostasis, and has discrete functions in the central and peripheral nervous system. Deletion of the cGKI gene impairs the NO/cGMP-induced relaxation of penile smooth muscle, leading to erectile dysfunction, but does not affect the motility and fertility of sperm. Furthermore, the NO/cGMP-dependent relaxation of urinary tract smooth muscle is abolished in cGKI-null mutants. cGKII regulates longitudinal bone growth, intestinal ion transport, and renin release in the kidney, and may be involved in the generation of complex behaviors like anxiety and addiction.


Results

Expression and characterization of mutant isoforms of ephrinB2

In a previous communication (Makinen et al., 2005), we had shown that mutant ephrinB2 5Y and ephrinB2 ΔV isoforms were expressed at equal levels in adult brain lysates, were tyrosine phosphorylated to similar degrees in embryo protein lysates, and were expressed on the cell surface of transfected cells at similar levels compared with wild-type ephrinB2. Although this was strong evidence for normal subcellular targeting of mutant ephrinB2 protein, we now investigated surface expression and clustering behavior of endogenous ephrinB2 in cultured neurons. Hippocampal neuron cultures were established from control homozygous knock-in embryos (at E16.5) expressing wild-type ephrinB2 (ephrinB2 WT/WT ), or mutant ephrinB2 isoforms (ephrinB2 5Y/5Y and ephrinB2 ΔV/ΔV ). After 2 DIV, we used soluble EphB4-Fc fusion protein to specifically induce ephrinB2 protein clusters. In the presence of control Fc protein, no ephrinB2 clusters were induced (data not shown). Endogenous ephrinB2 protein clusters were visualized by staining against the Fc portion of EphB4-Fc and quantified using MetaMorph software. We did not detect any marked differences in the densities and sizes of ephrinB2 clusters in neurons expressing the mutant ephrinB2 isoforms compared with wild-type knock-in ephrinB2 ( Fig. 1 A𠄾). Staining of ephrinB2 conditional knock-outs [ephrinB2-Nestincre (Grunwald et al., 2004)] with EphB4-Fc did not reveal fluorescent clusters, indicating specific binding to ephrinB2 (supplemental Fig. S1, available at www.jneurosci.org as supplemental material). These findings suggest that the majority of ephrinB2 mutant proteins were transported to the appropriate subcellular locations in hippocampal neurites. To directly visualize endogenous ephrinB2 at the CA3� synapse, we applied immunoelectron microscopic analysis using a specific ephrinB2 antibody ( Fig. 1 F–H). Using this technique, we had previously shown that ephrinB2 labeling at the postsynaptic side dropped nearly fourfold in ephrinB2 conditional knock-outs (ephrinB2-CamKIIcre) (Grunwald et al., 2004). Here, we found that the numbers of gold particles at ephrinB2-positive synapses and the number of labeled synapses were lower in the mutants than in the controls ( Fig. 1 I). Together with the results from previous work (Makinen et al., 2005), these data suggest that targeting of ephrinB2 to subcellular sites was mainly unaffected in the ephrinB2 5Y/5Y and ephrinB2 ΔV/ΔV mice (see Discussion).

Expression and biochemical characterization of ephrinB2 mutant proteins. A𠄼, Representative images of dendrites of cultured hippocampal neurons derived from the indicated ephrinB2 knock-in mice. Cells were incubated with EphB4-Fc, and ephrinB2-positive clusters were visualized by anti-Fc immunostaining. D, Quantification of the density of anti-Fc-positive clusters per micrometer of dendrite. E, Quantification of the sizes of anti-Fc-positive clusters. F–H, CA1-region synapses immunolabeled with ephrinB2 antibody and analyzed by electron microscopy. Asterisks indicate the presynaptic side of each synapse shown. Arrowheads point to the postsynaptic densities. Immunogold particles appear as small black dots. I, Quantification of ephrinB2 immunoelectron microscopy. Numbers of gold grains per synapse are shown versus numbers of labeled synapses in a pool of 300 synapses. A higher proportion of synapses are not labeled in the two mutant mice. Numbers of gold particles in ephrinB2-positive synapses are similar between mutants and controls (n = 2 mice per group *p < 0.05, t test). J, Coimmunoprecipitation (IP) of ephrinB2 with GRIP1. HeLa cells transiently transfected with constructs encoding GFP-tagged ephrinB2 wild type (wt), PDZ site deficient (ΔV), or tyrosine mutant (5Y) were lysed and immunoprecipitated using anti-GFP antibody. The proteins were immunoblotted with either anti-GFP (middle) or anti-GRIP (top) antibodies. A small amount of the total lysate was probed with anti-GRIP antibodies to visualize the levels of GRIP in the cells (bottom). Note that GRIP is not coimmunoprecipitated with ephrinB2 ΔV . K, Quantification of ephrinB2 (eB2) coimmunoprecipitation with GRIP1 [ratio between GRIP and eB2 optical density (OD)]. L, Tyrosine phosphorylation of mutant ephrinB2 proteins in adult hippocampal slices. Slices derived from ephrinB2 WT/WT control, ephrinB2 5Y/5Y , and ephrinB2 ΔV/ΔV mice were either left unstimulated or were stimulated with the general protein tyrosine phosphatase inhibitor orthovanadate. Slices were lysed, and ephrinB2 was immunoprecipitated using anti-ephrinB2 antibodies. The proteins were immunoblotted with either anti-ephrinB2 (bottom) or anti-phosphotyrosine (4G10 top) antibodies. The arrow points to ephrinB2. Error bars indicate SEM.

To evaluate PDZ binding by mutant ephrinB2 proteins, we investigated the interaction with GRIP1, a seven-PDZ domain-containing protein enriched at excitatory synapses (Dong et al., 1997) and likely to interact with ephrinB2 (Bruckner et al., 1999 Lin et al., 1999). We expressed ephrinB2 isoforms in HeLa cells and compared relative protein levels by Western blot analysis against GFP, which was attached to their extreme N terminus, a modification that does not interfere with surface expression and receptor binding (Zimmer et al., 2003 Makinen et al., 2005) ( Fig. 1 J). EphrinB2 WT and ephrinB2 5Y isoforms coimmunoprecipitated with GRIP1, demonstrating that PDZ domain-dependent binding of GRIP1 was unaffected by the removal of tyrosine residues in ephrinB2. In contrast, GRIP1 was not detected in ephrinB2 ΔV immunoprecipitates, indicating that PDZ binding of GRIP1 was destroyed [as shown previously for syntenin (Makinen et al., 2005)]. Quantification of six separate experiments revealed that the ability of ephrinB2 ΔV to coimmunoprecipitate GRIP was 10-fold lower compared with ephrinB2 WT and ephrinB2 5Y isoforms ( Fig. 1 K). To assess tyrosine phosphorylation of endogenous ephrinB2 isoforms in acute hippocampal slices of adult mice, we treated the slices with the tyrosine phosphatase inhibitor vanadate to allow maximum tyrosine kinase activity. In the presence of vanadate, the ephrinB2 ΔV protein was tyrosine phosphorylated to a similar extent as wild-type ephrinB2, suggesting that it can be a substrate for tyrosine kinases in adult hippocampus ( Fig. 1 L). As expected, the mutant ephrinB2 5Y isoform failed to get tyrosine phosphorylated under these conditions ( Fig. 1 L). These results also confirmed that the overall levels of ephrinB2 proteins in the adult mutant hippocampus were normal in the knock-in mutants. Together, these findings indicate that mutant ephrinB2 proteins are targeted to correct subcellular sites and behave as expected with respect to PDZ domain-dependent binding and susceptibility to tyrosine phosphorylation.

Normal ultrastructural morphology of ephrinB2 5Y/5Y mice

Previous reports had indicated that the neuronal receptors for ephrinB2, namely EphB1-B3 and EphA4, have (partially redundant) functions in the formation of synapses and spines (Dalva et al., 2000 Henkemeyer et al., 2003 Murai et al., 2003 Kayser et al., 2006). In EM analyses of ephrinB2 conditional knock-outs, we had previously not found differences in CA1 synapse numbers compared with littermate controls (Grunwald et al., 2004). Because the ephrinB2 isoform knock-ins are nonconditional and mutant ephrinB2 proteins are present throughout development, it is possible that effects on synapse number occur that are not seen in conditional knock-outs. We therefore quantified the numbers of synapses in the CA1 region and assessed the morphology of PSDs. We found a modest, yet significant, reduction in CA1 synapse numbers in both ephrinB2 knock-in mutants compared with ephrinB2 WT/WT controls (20% for ephrinB2 ΔV/ΔV and 15% for ephrinB2 5Y/5Y at 6 weeks of age) ( Fig. 2 A𠄽). This reduction in synapse numbers was milder at postnatal day 15 (P15 14% for ephrinB2 ΔV/ΔV and 10% for ephrinB2 5Y/5Y ) (supplemental Fig. S2, available at www.jneurosci.org as supplemental material). A previous report had indicated a modest reduction of PSD length in CA1 and CA3 regions of triple EphB knock-out mice (EphB1 −/− B2 −/− B3 −/− ) (Henkemeyer et al., 2003). We found a modest (10%) decrease of PSD length in ephrinB2 ΔV/ΔV but not in ephrinB2 5Y/5Y mice, compared with ephrinB2 WT/WT controls ( Fig. 2 E). In addition we noticed a significant increase in PSD width in ephrinB2 ΔV/ΔV but not in ephrinB2 5Y/5Y mice, compared with ephrinB2 WT/WT controls (32 and 7%, respectively) ( Fig. 2 F). The pattern of hippocampal dendrites in the stratum radiatum of the CA1 was similar based on MAP2 staining in all three lines (supplemental Fig. S3, available at www.jneurosci.org as supplemental material).

Morphologies of synapses derived from ephrinB2 mutant mice. A𠄼, Electron micrographs showing the morphology of asymmetric synapses in the CA1 region of the hippocampus of the indicated mice. PSDs are indicated by arrows. D, Numbers of synapses per 100 μm 2 in the CA1 region (n = 3 mice per genotype � synapses per animal counted) of 2-month-old mice. Note a small but significant reduction in ephrinB2 5Y/5Y and ephrinB2 ΔV/ΔV mice compared with ephrinB2 WT/WT controls (*p < 0.01). E, Quantification of PSD length. Note a small but significant reduction in ephrinB2 ΔV/ΔV mice compared with ephrinB2 WT/WT controls (*p < 0.01) and no changes in ephrinB2 5Y/5Y mice. F, Quantification of PSD width. Note a small but significant increase in ephrinB2 ΔV/ΔV mice compared with ephrinB2 WT/WT controls (*p < 0.01) and no changes in ephrinB2 5Y/5Y mice. Error bars indicate SEM.

Because ephrinB2 is predominantly localized postsynaptically at CA3� synapses, we next asked whether the modification of the cytoplasmic domain of ephrinB2 would alter the number of postsynaptic specializations in vitro. We cultured hippocampal neurons from ephrinB2 mutants (as described for Fig. 1 ) and visualized postsynaptic PSD-95-immunoreactive puncta by immunofluorescence after 20 DIV. We found no significant differences in the numbers of PSD-95-positive puncta between ephrinB2 mutants and ephrinB2 WT/WT controls ( Fig. 3 ). Together, these results suggest that the mutant ephrinB2 protein lacking all tyrosine residues (ephrinB2 5Y ) mediates normal synapse morphology in vivo and postsynaptic specializations in cultured neurons. Expression of the ephrinB2 ΔV protein leads to a slightly altered synapse morphology but normal postsynaptic specializations in cultured neurons.

Fluorescence analysis of PSD-95 puncta in cultured hippocampal neurons. A𠄼, Representative images of cells were derived from E16.5 embryos of the indicated mouse lines, cultured for 20 DIV, and immunostained using anti-PSD-95 antibodies. D, Quantification of PSD-95 puncta per 100 μm 2 . No significant differences were observed (n = 3 embryos and 90� neurons per genotype p > 0.05). Error bars indicate SEM.

Basal synaptic parameters are unchanged in ephrinB2 cytoplasmic mutants

We next examined the functional consequences of the mutations in the ephrinB2 cytoplasmic domain. We performed a number of electrophysiological tests comparing ephrinB2 ΔV/ΔV and ephrinB2 5Y/5Y mice to ephrinB2 WT/WT controls, which did not show any significant differences to wild-type mice (data not shown). First, we compared basal synaptic transmission between ephrinB2 WT/WT and mutant mice. fEPSPs were evoked with increasing stimulus intensities at CA1 synapses by stimulating Schaffer collaterals, one of the three main excitatory pathways. The sizes of the presynaptic FVs, which are proportional to the number of presynaptic axons recruited by the stimulation, were compared with the slope of the fEPSP response, to establish input–output relationships ( Fig. 4 A). We observed no differences between ephrinB2 WT/WT and ephrinB2 ΔV/ΔV or ephrinB2 5Y/5Y mice, respectively (ephrinB2 ΔV/ΔV mice: slope and percentage of the control, 93 ± 5.6% ephrinB2 5Y/5Y mice: slope and percentage of the control, 115 ± 13.2% p > 0.2, t test).

Basal synaptic parameters are unchanged in ephrinB2 cytoplasmic mutants. A, fEPSP slope at various stimulus intensities (FV). In the range of 0.1𠄰.6 mV, the three groups of mice showed similar values (ephrinB2 WT/WT , n = 17 slices ephrinB2 ΔV/ΔV , n = 15 slices ephrinB2 5Y/5Y , n = 19 slices p > 0.05, t test). B, NMDA/AMPA ratio. AMPA receptor EPSCs were evoked at a membrane potential of � mV, and NMDA receptor EPSCs were evoked at +40 mV. For control neurons, the NMDA/AMPA ratio was 1.6 ± 0.1 (n = 10 slices). The ratio of NMDA to AMPA was not significantly changed in the mutant mice (ephrinB2 ΔV/ΔV : 1.7 ± 0.3, n = 11 slices ephrinB2 5Y/5Y : 1.8 ± 0.2, n = 9 slices p > 0.05, t test). C, PPF of the EPSP at an interstimulus interval (ISI) of 40 ms from the three groups of mice was not significantly different (ephrinB2 WT/WT : 1.5 ± 0.1, n = 9 slices ephrinB2 ΔV/ΔV : 1.6 ± 0.2, n = 9 slices ephrinB2 5Y/5Y : 1.6 ± 0.1, n = 8 slices p > 0.05, t test). D, Cumulative probability plot of the mEPSC amplitudes (D1, D2) and frequencies (D3, D4) in the control and mutant mice. Neither mEPSC amplitudes nor mEPSC frequencies changed significantly in the mutant mice (AV50 amplitude for mEPSCs: ephrinB2 WT/WT , �.3 ± 0.9 pA, n = 19 slices ephrinB2 5Y/5Y , �.4 ± 0.8 pA, n = 19 slices ephrinB2 ΔV/ΔV , �.2 ± 4 pA, n = 18 slices p > 0.05, t test frequency of events: ephrinB2 WT/WT , 0.9 ± 0.2 Hz, n = 19 slices ephrinB2 5Y/5Y , 1.0 ± 0.2 Hz, n = 19 slices ephrinB2 ΔV/ΔV , 1.0 ± 0.2 Hz, n = 18 slices p > 0.05, t test). Error bars indicate SEM.

To address whether the numbers of synaptic AMPA and/or NMDA receptors were different in mutant mice, we compared the sizes of AMPA receptor EPSCs with those of NMDA receptor EPSCs by whole-cell recordings of CA1 neurons in hippocampal slices. AMPA receptor EPSCs were evoked at a membrane potential of � mV, and NMDA receptor EPSCs were evoked at +40 mV to relieve the magnesium block. The magnitude of the NMDA receptor EPSC was determined by measuring the amplitude of the EPSCs 70 ms after stimulus. For control neurons, the NMDA/AMPA ratio was 1.6 ± 0.1 (n = 10) ( Fig. 4 B). The ratio of NMDA to AMPA was not significantly changed in the mutant mice (ephrinB2 ΔV/ΔV : 1.7 ± 0.3, n = 11 ephrinB2 5Y/5Y : 1.8 ± 0.2, n = 9 p > 0.05, t test).

Next, we measured PPF, a sensitive measure of changes in the probability of transmitter release (Pr). To evaluate the effects of ephrinB2 cytoplasmic domain mutations on the Pr, we measured PPF using the ratios of the second and the first EPSP slopes at an interpulse interval of 40 ms. As shown in Figure 4 C, we found that synapses in wild-type and mutant mice exhibited very similar PPFs during basal synaptic transmission. These results indicate that presynaptic functions in these mice were normal.

Finally, we designed experiments to look for changes in functional synapse numbers. The amplitude of mEPSCs is a measure for the AMPA receptor number per synapse, whereas a change in mEPSC frequency reflects a change in the number of functional synapses. Comparison of the cumulative EPSC amplitudes and frequencies ( Fig. 4 D) for the wild-type and the mutant mice revealed no significant differences. mEPSC frequencies were measured in 2- to 3-week-old mice, at a time when electron microscopy revealed mild reductions in synapse numbers (14% for ephrinB2 ΔV/ΔV and 10% for ephrinB2 5Y/5Y mice). Because various parameters of mEPSC (such as probability of spontaneous vesicle fusion, detection level, attenuation in the dendrite) are heterogeneous from synapse to synapse, it is not surprising that small changes in synapse number are not detected as a change in mEPSC frequency. Together, these findings indicate that the ephrinB2 cytoplasmic domain mutations caused no apparent changes in excitatory circuitry.

EphrinB2–PDZ interaction and tyrosine phosphorylation sites participate in CA3� hippocampal LTP

Using two different protocols to induce LTP (tetanic and theta burst see Materials and Methods), we investigated the consequences of ephrinB2 cytoplasmic domain mutations in CA3� hippocampal LTP. Using acute slices from adult mice, either tetanic stimulation ( Fig. 5 A) or TBS ( Fig. 5 B) were applied to fibers of the CA3 presynaptic neurons, and LTP was recorded from CA1 neurons for up to 1 h after stimulation. In both stimulation protocols, the magnitude of the potentiation was different between controls and mutants. After tetanic stimulation ( Fig. 5 A), the mean EPSP slope as a percentage of baseline 55� min after stimulation was 156.4 ± 6.9% in the ephrinB2 WT/WT controls (n = 13 slices), whereas ephrinB2 ΔV/ΔV and ephrinB2 5Y/5Y mice showed significantly lower normalized slopes (117.7 ± 3.8 and 120.6 ± 8.9%, respectively p < 0.01, t test). Similar differences were observed after TBS ( Fig. 5 B), indicating that the impairments in LTP were not specific to a single induction protocol. We next investigated slices taken from P14–P20 animals. As shown in Figure 5 C, both ephrinB2 ΔV/ΔV and ephrinB2 5Y/5Y mice displayed reduced LTP compared with ephrinB2 WT/WT controls, although the difference was less dramatic than in adult slices (ephrinB2 WT/WT , 141.5 ± 5.7% ephrinB2 ΔV/ΔV , 120.8 ± 5.5% ephrinB2 5Y/5Y , 120.9 ± 4.6% p < 0.01, t test). These data indicate that hippocampal LTP requires both intact PDZ target and tyrosine phosphorylation sites in the ephrinB2 cytoplasmic domain.

EphrinB2 tyrosine phosphorylation sites are not required for LTD and depotentiation

Because LTD at CA3� hippocampal synapses requires the presence of ephrinB2 (Grunwald et al., 2004), we analyzed hippocampal LTD in slices taken from ephrinB2 WT/WT control knock-in mice and ephrinB2 signaling mutants. After recording stable baseline responses (see Materials and Methods), LTD was induced by low-frequency stimulation (LFS) of 900 stimuli at 1 Hz (15 min). As shown in Figure 6 A, ephrinB2 WT/WT controls showed a persistent decrease in fEPSPs, which lasted for at least 60 min. EphrinB2 ΔV/ΔV slices were defective in LTD and returned to baseline at 40 min after LTD induction (ephrinB2 ΔV/ΔV slices, 97.9 ± 2.2% vs ephrinB2 WT/WT slices, 84.1 ± 1.7% p < 0.01, t test). Unexpectedly, ephrinB2 5Y/5Y slices showed LTD nearly indistinguishable from ephrinB2 WT/WT controls (ephrinB2 5Y/5Y , 82.3 ± 3.4% vs ephrinB2 WT/WT slices, 84.1 ± 1.7% p = 0.56, t test).

EphrinB2 tyrosine phosphorylation sites are not required for LTD and depotentiation. A, EphrinB2–PDZ interaction but not tyrosine phosphorylation sites are required for LTD in young hippocampal slices (P14–P20). LFS induced a significant long-lasting decrease in fEPSPs in ephrinB2 WT/WT control and ephrinB2 ΔV/ΔV slices but not in ephrinB2 5Y/5Y slices (97.9 ± 2.2% in ephrinB2 ΔV/ΔV mutants compared with 82.3 ± 3.4% in ephrinB2 5Y/5Y mutants and 84.1 ± 1.7% in ephrinB2 WT/WT controls at 55� min after LFS p < 0.05). B, Depotentiation is impaired in ephrinB2 ΔV/ΔV but not ephrinB2 5Y/5Y mutants. Ten minutes after tetanus (arrow) to produce the initial phase of LTP, slices were subjected to a 15 min LFS train (horizontal line 1 Hz). The fEPSPs from ephrinB2 WT/WT control and ephrinB2 5Y/5Y slices returned to baseline (ephrinB2 WT/WT slices, 104.0 ± 7.6% ephrinB2 5Y/5Y slices, 98.9 ± 6.2%), whereas ephrinB2 ΔV/ΔV slices remained potentiated (126.9 ± 4.6% p < 0.05, compared with ephrinB2 WT/WT controls).

We additionally analyzed another form of long-term plasticity known as depotentiation, the depression of synaptic efficacy at synapses that have recently undergone LTP (O'Dell and Kandel, 1994). Although the induction protocol for depotentiation is identical to LTD, they may represent distinct processes (Montgomery and Madison, 2002, and references within). After recording a stable baseline, we applied a tetanic stimulation to induce LTP, followed (after 10 min) by a depotentiation stimulus (LFS of 900 stimuli at 1 Hz for 15 min). Slices derived from ephrinB2 WT/WT controls showed a normal initiation phase of LTP (first 10 min after tetanus application) and complete depotentiation after 15 min of LFS ( Fig. 6 B). As expected from the LTP results, both ephrinB2 ΔV/ΔV and ephrinB2 5Y/5Y slices displayed reduced early LTP, but their reaction to LFS varied: ephrinB2 ΔV/ΔV slices were defective in depotentiation and remained potentiated 55� min after LFS application (fEPSP slope size: ephrinB2 ΔV/ΔV slices, 126.9 ± 4.6% vs ephrinB2 WT/WT slices, 104.0% ± 7.7% p < 0.05, t test). In contrast, ephrinB2 5Y/5Y slices displayed complete depotentiation 1 h after the application of the LFS protocol (98.9 ± 6.2% fEPSP slope) ( Fig. 6 B), following a time course indistinguishable from controls. These findings suggest that the required function of ephrinB2 in LTD as well as in depotentiation is independent of phosphotyrosine signaling.

EphrinB2 reverse signaling in cultured neurons induces phosphorylation of NMDA receptors

In a number of different cell types, including excitatory neurons, ephrinB reverse signaling involves the rapid recruitment and activation of SFKs to ephrinB clusters (Palmer et al., 2002 Georgakopoulos et al., 2006). SFKs bind to the ephrinB cytoplasmic domain and phosphorylate ephrinB on tyrosine residues. SFKs are also the major protein tyrosine kinases that upregulate the activity of NMDA receptors (reviewed in (Salter and Kalia, 2004 Lee, 2006). SFK-mediated tyrosine phosphorylation of NMDA receptors is critical for induction of NMDA receptor-dependent LTP (Lu et al., 1998). Tyrosine phosphorylation of NMDA receptor subunits regulates their membrane trafficking. To begin addressing the biochemical events mediated by ephrinB2 reverse signaling, we investigated the degree of tyrosine phosphorylation of the NR2A subunit of NMDA receptors in cultured forebrain neurons. Stimulation of neurons derived from ephrinB2 WT/WT mice with EphB4-Fc led to a robust increase in NR2A tyrosine phosphorylation compared with control Fc-treated cultures ( Fig. 7 A𠄼). Interestingly, NR2A tyrosine phosphorylation was abnormal in mutant neurons: in the presence of the ephrinB2 ΔV isoform, basal NR2A tyrosine phosphorylation was elevated and was not further enhanced by EphB4-Fc stimulation ( Fig. 7 B). In the presence of the ephrinB2 5Y isoform, basal NR2A tyrosine phosphorylation was comparable to wild-type neurons, but EphB4-Fc stimulation did not lead to an increase in NR2A tyrosine phosphorylation. The changes in NR2A tyrosine phosphorylation correlated with similar changes in Src phosphorylation as revealed by a phospho-Src ELISA ( Fig. 7 D). These results demonstrate NR2A tyrosine phosphorylation as a consequence of ephrinB activation in cultured neurons and that both ephrinB2 ΔV and ephrinB2 5Y isoforms fail to regulate NR2A tyrosine phosphorylation.

EphrinB2 activation causes NR2A and Src tyrosine phosphorylation. Dissociated cultures of embryonic forebrain neurons (8 DIV) derived from wild-type (A) or from the indicated mutant mice (B𠄽) were stimulated with control Fc or EphB4-Fc for the indicated times. A, Note the EphB4-Fc-induced increase in NR2A tyrosine phosphorylation in wild-type neurons. B, Independent experiment using ephrinB2 WT/WT and ephrinB2 ΔV/ΔV neurons. Note the elevated basal and lack of stimulated NR2A tyrosine phosphorylation in ephrinB2 ΔV/ΔV neurons. C, Independent experiment using ephrinB2 WT/WT and ephrinB2 5Y/5Y neurons. There is no EphB4-Fc-induced increase in NR2A tyrosine phosphorylation in ephrinB2 5Y/5Y neurons. D, Cell lysates were subjected to a phospho-Src ELISA. EphB4-Fc-stimulation of ephrinB2 WT/WT neurons led to a significant induction of Src phosphorylation at position 418, which is required for Src activation (*p < 0.05, t test). Changes in Src phosphorylation observed in ephrinB2 ΔV/ΔV and ephrinB2 5Y/5Y neurons were not significant. p-NR2A, Phosphorylated NR2A.


Brain Extracellular Matrix in Health and Disease

Oleg Senkov , . Alexander Dityatev , in Progress in Brain Research , 2014

7 Reelin

Reelin is a large secreted ECM glycoprotein (400 kDa, gene RELN) that controls neuronal migration and brain development ( Cooper, 2008 Dɺrcangelo et al., 1995 Rice and Curran, 2001 Soriano and Del Rio, 2005 ). Reelin binds to the lipoprotein family receptors apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR) ( Dɺrcangelo et al., 1999 Hiesberger et al., 1999 ) and induces the phosphorylation of the adaptor protein Dab1 ( Howell et al., 1997 Howell et al., 1999 ). The downstream Reelin cascade includes several signaling pathways, including distinct members of the Src kinase family ( Arnaud et al., 2003 ), Erk1/2 ( Simo et al., 2007 ), AKT/GSK3 ( Beffert et al., 2002 ), and ubiquitination/degradation of phosphorylated mDab1 triggered by Cul5 ( Simo and Cooper, 2013 Simo et al., 2010 ).

In addition to developmental stages, Reelin is expressed in the adult cerebral cortex mainly by γ-amino-butyric acid (GABA)-positive interneurons ( Alcantara et al., 1998 ) where it has been proposed to regulate plasticity processes ( Dityatev et al., 2010a Herz and Chen, 2006 ). For instance, it has been shown that Reelin potentiates glutamatergic neurotransmission, LTP, and synaptic maturation increases the expression of AMPA and NMDA receptor subunits and favors the trafficking and substitution of NR2B subunits by NR2A subunits ( Beffert et al., 2005 Chen et al., 2005 Groc et al., 2007 Qiu and Weeber, 2007 Qiu et al., 2006b ). Moreover, Reelin has been suggested to regulate the density and stabilization of dendritic spines ( Niu et al., 2008 Ventruti et al., 2011 ). Two models have been used to address specifically the role of Reelin in the adult brain. Local in vivo injections of Reelin increase spine density, modify spine morphology, and enhance LTP ( Rogers et al., 2011 Rogers et al., 2013 ). Similarly, transgenic mice overexpressing Reelin in the adult forebrain (Reelin-OE mice) show hypertrophy of dendritic spines and enhanced glutamatergic neurotransmission and LTP ( Pujadas et al., 2010 Teixeira et al., 2011 ). Moreover, both acute administration of Reelin in wild-type mice and studies in Reelin-OE mice show enhanced associative and spatial learning and memory ( Pujadas et al., 2010 Pujadas et al., 2014 Rogers et al., 2011 Rogers et al., 2013 ). Conversely, administration of recombinant receptor-associated protein (as a Reelin signaling blocking tool) is associated with impaired performance in a hippocampus-dependent MWM test ( Stranahan et al., 2011 ).

Reelin is also highly expressed in neurogenic niches, the subventricular zone (SVZ) and the dentate gyrus, and in rostral migratory stream and olfactory bulb ( Courtes et al., 2011 Dityatev et al., 2010b ). In the SVZ, Reelin was shown to control the behavior of SVZ-derived migrating neurons, triggering them to leave prematurely the rostral migratory stream leading to ectopic neurons both along the rostral migratory pathway and in the olfactory bulb ( Courtes et al., 2011 Pujadas et al., 2010 ). In the dentate gyrus, Reelin overexpression results in increased neurogenesis and accelerated dendritic maturation, likely reflecting increased functional integration into adult circuits. Conversely, inactivation of the Reelin signaling pathway specifically in adult neuroprogenitor cells resulted in aberrant migration, decreased dendrite development, and formation of ectopic dendrites in the hilus and in the establishment of aberrant circuits ( Teixeira et al., 2012 ). These studies support a critical role for the Reelin pathway in regulating adult neurogenesis and dendritic development of adult-generated neurons. Taken together with the above data, Reelin emerges as a key regulator of adult plasticity processes important for learning and memory including synaptic plasticity and remodeling and adult neurogenesis.

Reelin is also known to be involved in a spectrum of cognitive pathological conditions, for example, its expression is compromised in the brain of schizophrenic patients and in autism, bipolar disorder, major depression, AD, and several polymorphisms, and rare variants in the RELN gene have been associated with these disorders ( Botella-Lopez et al., 2006 Chin et al., 2007 Fatemi et al., 2000 Kramer et al., 2011 Liu et al., 2010 ). However, experimental evidence in heterozygous reeler mice, expressing a half of normal Reelin protein levels, has been contradictory. While some studies show that heterozygous reeler mice have cognitive ( Brigman et al., 2006 Qiu et al., 2006a ) and sensorimotor deficits as measured in the prepulse inhibition test (PPI) ( Barr et al., 2008 ), other studies have failed to find differences ( Krueger et al., 2006 Podhorna and Didriksen, 2004 Teixeira et al., 2011 ). Moreover, additional tests associated to psychiatric-related behavioral dysfunctions (e.g., OF, BW, and FST tests) also failed to demonstrate clear differences between heterozygous reeler and wt mice ( Podhorna and Didriksen, 2004 Teixeira et al., 2011 ). Finally, relatively mild behavioral deficits have been found in VLDLR or ApoER2 mutant mice ( Barr et al., 2007 ). Taking advantage of an experimentally different approach, Teixeira et al. (2011) investigated whether Reelin overexpression may modify psychiatric-related phenotypes. While increased Reelin expression does not alter mood-related behaviors under basal conditions, Reelin overexpression was found to be protective against PPI deficits induced by NMDA antagonists, cocaine sensitization (as a model of maniac disorder), and chronic stress-induced depression phenotypes.

In relation to AD pathology, it is noteworthy that Reelin is present in amyloid plaques ( Doehner et al., 2010 ), controls APP processing ( Hoe et al., 2006 ), and reduces tau phosphorylation by inhibiting glycogen synthase kinase 3 (GSK3 Ohkubo et al., 2003 ). Importantly, Reelin has been consistently found to counteract Aβ42-induced synaptic dysfunction including LTP ( Durakoglugil et al., 2009 ). In addition, AD brain samples show altered levels of Reelin and RELN polymorphisms have been associated with this disease ( Botella-Lopez et al., 2006 Botella-Lopez et al., 2010 Chin et al., 2007 Kramer et al., 2011 ). These studies suggest that dysfunction of the Reelin pathway may be at the root of the neuropathologic mechanisms leading to sporadic, late-onset AD ( Krstic and Knuesel, 2013 Krstic et al., 2012 ). This hypothesis has been experimentally addressed. Indeed, the reduction of Reelin in an AD mouse model crossed with heterozygous reeler accelerates the onset of plaque formation and tau pathology ( Kocherhans et al., 2010 ). Conversely, Reelin overexpression in hAPPSwe/ind (J20) mice reduces amyloid plaque load, rescues dendritic spine loss in J20 mice, and enhances cognitive performance in both aged wild-type and J20 mice. At the molecular level, Reelin delays Aβ42 fibril formation—by interacting with Aβ42 soluble species including oligomers—until it is sequestered into amyloid fibrils. Importantly, Reelin overcomes the toxicity of Aβ42 oligomers in neuronal cultures ( Pujadas et al., 2014 ). Taken together with Reelin's role in plasticity, these data support a model in which the Reelin pathway may exert beneficial effects on both AD pathology and cognition by at least two complementary mechanisms: in addition to extracellular Reelin delaying amyloid fibril formation and reducing neurotoxicity by interacting with Aβ42, the activation of the Reelin cascade itself would potentiate adult plasticity events, including synaptic plasticity and adult neurogenesis, and lead to decreased GSK3 activity and tau phosphorylation. It is thus likely that activation of the Reelin pathway might represent a therapeutic strategy for ameliorating the cognitive decline and neuropathologic hallmarks associated with AD.


Comments

This is a very interesting study that provides several important advances and insights for the field. It is particularly intriguing to see that BACE1 inhibition not only reduces plaque growth, but may even shrink existing plaques. If it also happens in patients this would be fantastic news for the clinical trials with BACE inhibitors. Because AD mouse models, including the 5xFAD model used in this study, typically mimic presymptomatic AD, it would appear possible that secondary prevention trials with BACE inhibitors may not only yield reduced growth or number of plaques, but even a shrinking of pre-existing plaques and a reduction of neuroinflammation—provided that the drugs are given early enough before the symptoms start. This should and will be tested in the trials’ participants using PET imaging. These new findings contradict a recent study that demonstrated that plaques remain stable in size when BACE1 is inhibited pharmacologically. I am sure that this discrepancy will be resolved with future experiments and may depend on the mouse line used, or the level or duration of BACE1 inhibition.

We also need to consider that the amazing effects of the mouse study were accompanied by an intriguingly gradual reduction of BACE1 protein levels. It is difficult to predict exactly which inhibition level of BACE1 would be needed in humans to achieve the same results. Potentially, the levels currently used in trials are already sufficient.

Another take-home message of the study is that memory and LTP deficits were improved in the AD mice as BACE1 was suppressed. While this is good news, the study also demonstrated that LTP did not fully recover. This indicates that the therapeutically desired BACE inhibition in adult mice may interfere with physiological BACE1 functions. Besides LTP, this includes muscle spindle formation/maintenance and dendritic spine densities in adult mice. Translated to humans, this could indicate a larger number of falls or psychiatric symptoms in individuals treated with BACE inhibitors. Whether this is a realistic concern will be seen from the results of the currently ongoing and recently terminated BACE inhibitor trials.

Constitutive BACE1-deficient mice show a number of different phenotypes. Another major insight of the new study is that adult deletion of BACE1 overcomes at least one of the symptoms—the hypomyelination. The new conditional BACE1-deficient mice are an excellent tool to analyze whether the other BACE1-deficient mouse phenotypes are also of developmental origin and which functions of BACE1 are relevant in adult mice. Taken together, the new study is an excellent basis both for basic and translational BACE1 research.

I am interested to know why, under BACE1 cKO, there is, in addition to an expected decrease in C99, a decrease in C83 as well. The authors suggest that a cKO of BACE1 improves autophagy, increasing turnover of C-terminal fragments. However, there is no change in C83 in the original KO (Cai, 2001). Have the authors looked at APP trafficking in their model? I would be interested to know if the amount of sAPPα is the same, i.e. is APP still being trafficked to the cell surface?


Jin-Yu Lee and Li-Jen Lee: These authors contributed equally to this work.

Affiliations

Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan

Jin-Yu Lee, Hsin-An Shih & Mau-Sun Chang

Graduate Institute of Anatomy and Cell Biology, National Taiwan University, Taipei, Taiwan

Institute of Brain and Mind Sciences National Taiwan University, Taipei, Taiwan

Neurobiology and Cognitive Science Center, National Taiwan University, Taipei, Taiwan

Department of Superintendent Office, Mackay Memorial Hospital, Taipei, Taiwan

Department of Medical Laboratory Science and Biotechnology, Yuanpei University, Hsinchu, Taiwan

Department of Medical Technology, Jen-Teh Junior College of Medicine, Nursing and Management, Miaoli, Taiwan

Department of Life Science, College of Life Science, National Taiwan University, Taipei, Taiwan


Results

SynGAP mutant mice die shortly after birth

To explore the possible role of SynGAP in vivo, the SynGAP gene was disrupted in mice. The genomic DNA containing the 5′ end of the mouse SynGAP gene was isolated and analyzed for targeting vector construction. SynGAP is extensively spliced at the 5′ end, leading to splice variants: SynGAP-a, -b, -c, and -d (Chen et al., 1998 Kim et al., 1998 Li et al., 2001) (Fig.1A). Therefore, the exon cassette containing the first common methionine present in the shortest splice variant, SynGAP-c, was chosen for deletion, along with an adjacent exon encoding a portion of the C2 domain by replacing it with a neo R gene cassette in the reverse orientation (Fig. 1B). The normal splicing events in the targeted SynGAP gene are predicted to yield transcripts with premature stop codons. The targeted and the wild-type alleles can be differentiated by performing Southern blotting after theKpnI digestion using the inner and outer probes (Fig.1B), and the genotyping result can also be confirmed by PCR using the primers shown (Fig. 1C,right).

SynGAP splice variants and domain structure and gene targeting strategy. A, N-terminal splicing leads to different start sites and sequences in SynGAP-a, -b, -c, and -d. SynGAP protein contains a pleckstrin homology (PH) domain, a phospholipid-dependent Ca 2+ binding motif (C2) domain, a Ras GTPase-activation protein (RasGAP) domain, and a C-terminal sequence PSD-95/discs large/zona occludens-1 domain binding motif (QTRV). Alternative splicing occurs also at the C terminal with C-terminal sequences other than −QTRV.B, The SynGAP gene structure is shown (not drawn to scale) of the region analyzed for gene targeting. Targeting of the SynGAP gene was performed by replacing the SacII andEcoRI fragments of the SynGAP gene containing two exons with the neo R cassette. The targeting construct spanning the XhoI and HindIII fragment of the SynGAP gene is 11.5 kb long. Outer and inner probes were used for Southern blotting. The PCR primers used for genotyping and the predicted amplified sizes are shown. TK, Thymidine kinase. C, Genotype analyses of tail DNA of second filial generation (F2) mice by PCR and Southern blotting. The wild-type allele is detected by Southern blotting after digestion with the KpnI restriction enzyme (left). The size of the detected wild-type band is 1.8 kb longer than that of the targeted allele. Two alleles can be distinguished using PCR primer sets (right).

The chimeric male mice from two independent ES clones (17.28 and 18.8) were mated with C57BL/6 female mice to generate heterozygotes, and sibling mating of heterozygotes produced homozygotes. The F2 mice genotypes exhibited a Mendelian ratio of 1:2:1, indicating that there is no embryonic lethality caused by the null mutation of the SynGAP protein or abnormal segregation of the gene. A database search revealed that the SynGAP gene is on mouse chromosome 17. A sample Southern blotting analysis of the F2 mice is shown in Figure 1C,left, where the mobility of the targeted allele differs from that of the wild type after the KpnI digestion because the neo R gene does not contain the restriction site. The heterozygotes are indistinguishable from the wild types in their size and activity, and they breed normally. The homozygotes are indistinguishable from the wild types and the heterozygotes for the first 2 d after birth. By the third day the mutant mice begin to show less movement and do not feed from the mother mice. Between P4 and P6, the pups stay small in size, and they die between P5 and P7. These observations were confirmed in the two independent mutant mouse lines.

SynGAP gene targeting abolishes the expression of the wild-type SynGAP protein

Immunoblot analysis of mouse brain homogenates at P5 with the anti-GAP SynGAP antibody showed that expression of the 130 kDa SynGAP protein is abolished in the mutant mice (Fig.2A). However, overexposure of the immunoblot showed that a very low level of a smear of smaller proteins (∼120 kDa) could be detectable using the anti-GAP antibody. These are most likely protein products of the SynGAP gene from cryptic start sites downstream of the deleted exons in the targeted gene. These protein products are present at <2% the level of the wild-type SynGAP protein. To analyze whether deletion of SynGAP affected the expression of other neuronal proteins, various synaptic proteins were surveyed in the SynGAP mice at P5 (Fig.2B). NMDA receptor subunits, NR1 and NR2B, and associated proteins, PSD-95 and SAP102, were similar in expression level in all genotypes. Also, the level of the AMPA receptor subunit GluR1 and the AMPA receptor-associated proteins GRIP1 and GRASP1 were indistinguishable in the wild types, heterozygotes, and homozygotes.

A proper expression of SynGAP protein is abolished in the mutant mice, whereas other synaptic proteins are not affected at P5. A, Mouse brain homogenates were prepared and immunoblotted with the anti-GAP SynGAP antibodies at P5 and compared with that of rat brain homogenate at P4. With an equal amount of protein loaded in each lane, SynGAP protein expression is absent in the sample from a homozygous (Homoz) mouse.Heteroz, Heterozygous. B, The expression of proteins at synapses was examined in the SynGAP mice using the antibodies to the proteins indicated, and no detectable change in the expression was seen. C, Tissue distribution of SynGAP protein in the mutant and the wild-type mice at P5 was examined using the α-GAP domain antibody. In wild-type mice, a prominent band of ∼130 kDa was detected in the cortex and the cerebellum. In contrast, the ∼130 kDa protein was not detected in the homozygotes or in non-neuronal tissues.

Brain development in the SynGAP mutant mice

Examination of the SynGAP mutant mice at a gross anatomical level reveals typical development of tissues and organs, similar to the wild-type mice. Because SynGAP protein expression is selectively expressed in the brain (Fig. 2C), it seems likely that the prenatal development of non-neuronal tissues is not affected by the absence of SynGAP protein. Brain development at the gross anatomical level also seems normal in the mutant mice. The formation and organization of the forebrain appears to be similar in mice of all three genotypes at P5, as revealed by Nissl staining (data not shown). However, the size of the mutant mouse brain is significantly smaller, indicating that SynGAP may be crucial to the proliferation and development of neuronal tissues after birth, especially around P3. It is not clear why the SynGAP mutant mice die (see Discussion).

Decreased number of silent synapses in neurons cultured from homozygous mice

To investigate the role of SynGAP during synaptogenesis, cortical neuronal cultures were prepared from SynGAP mice and analyzed after 18–20 d in vitro (DIV). The neurons were fixed, and immunocytochemistry was performed using an anti-synaptophysin antibody to identify synapses, anti-GluR1 and anti-GluR2/3 antibodies to identify AMPA receptors, and anti-NR1 antibody for NMDA receptors. In heterozygote and homozygous neurons, synapses, identified by the anti-synaptophysin antibody, were present in similar numbers [heterozygote mice, 94.1 ± 4.7 (SD) of wild-type mice,p < 0.68 homozygote mice 109.4 ± 4.9%,p < 0.49 of wild-type mice] and pattern to those in wild-type neurons. Interestingly, AMPA receptor clusters, identified by the anti-GluR1 antibody, were present in a greater number in the homozygotes than in the heterozygotes and the wild-types (Fig.3A). A similar result was obtained using the anti-GluR2/3 antibody. Quantitation of the number of AMPA receptor puncta is shown in Figure 3B. The number of GluR1-positive clusters was increased in the homozygotes by 32.1 ± 9.0% (p < 0.05 ANOVA) compared with the wild types (Fig. 3) and was also higher in the heterozygotes (21.4 ± 8.6%). There was a slight increase in the number of NMDA receptor puncta, although this was not statistically significant (p > 0.05 ANOVA data not shown). Because the number of AMPA receptor clusters increased more than the number of NMDA receptor clusters, we determined whether the number of morphological silent synapses (synapses that contain NMDA receptors but not AMPA receptors) (Liao et al., 1999, 2001) was increased in the mutant mouse. The cultures from the SynGAP mutant mice had significantly fewer morphological silent synapses than their wild-type littermates (Fig.3C).

The number of AMPA receptor clusters in the SynGAP mutant mice is increased. A, Primary cortical cultures from the SynGAP mutant mice and their wild-type and heterozygous littermates were immunostained with anti-GluR1 antibodies after 18–20 DIV. There was an increase in the number of GluR1-positive clusters in the cultures prepared from the homozygous pups. B, Quantitation of GluR1-positive puncta in SynGAP mouse neuronal cultures at 18–20 DIV (n = 14, n = 19, and n = 13, respectively p < 0.05 ANOVA F = 3.52). C, The number of morphological silent synapses in the cultures was quantitated by comparing the number of AMPA receptor cluster/NMDA receptor cluster puncta (n = 9, n = 13, andn = 7, respectively) at 18–20 DIV.

Synaptic plasticity in SynGAP knock-out mice

We tested the role of SynGAP in hippocampal synaptic plasticity by comparing the magnitude of LTP and LTD in the CA1 region of adult wild-type and heterozygous mice. LTP induced by TBS was significantly decreased in slices from heterozygous mice (140 ± 6% of baseline at 1 hr after TBS n = 20 slices from five animals) compared with their wild-type littermates (174 ± 9% of baseline n = 16 slices from five animalsp < 0.01 Student's t test) (Fig.4A). Next we tested whether LTD is affected in the SynGAP heterozygotes. To examine LTD, we used paired pulses at an interstimulus interval of 50 msec repeated at 1 Hz for 15 min (PP-1 Hz), which has been used previously to induce LTD in hippocampal slices from adult rats (Kemp et al., 2000). As shown in Figure 4B, there was no difference in the magnitude of LTD in the heterozygous animals (80 ± 3% of baseline measured 1 hr after the start of PP-1 Hz n = 21 slices from four animals) compared with wild-type littermates (82 ± 4% of baseline n = 18 slices from four animalsp > 0.4 Student's t test). In mice, LTD induced by the PP-1 Hz protocol is completely blocked by bath application of the NMDA receptor antagonist APV (data not shown).

A, Schaffer collateral to CA1 LTP in adult SynGAP heterozygotes (● n = 20 slices from 5 animals) are significantly reduced compared with wild-type littermates (○ n = 16 slices from 5 animals). FP traces taken just before and 1 hr after TBS for wild types and heterozygotes are shown to the right. B, No significant difference in PP-LTD (PP-1Hz) in SynGAP heterozygotes (● n = 21 slices from 4 animals) and wild types (○ n = 18 slices from 4 animals). FP traces taken just before and 1 hr after the initiation of PP-1 Hz are shown to the right. C, AMPA receptor-mediated synaptic transmission measured as the initial FP slope plotted against fiber volley amplitude. Plots of both wild types (○ n = 32 slices from 8 animals) and heterozygotes (● n = 33 slices from 8 animals) essentially overlap, suggesting that synaptic transmission is normal in heterozygotes. D, No difference was observed in presynaptic function as monitored by paired-pulse facilitation between wild types (○ n = 14 slices from 5 animals) and heterozygotes (● n = 14 slices from 5 animals). Paired pulses were given at interstimulus intervals of 25, 50 100, 200, 400, 800, and 1600 msec at baseline stimulus intensity.E, Pharmacologically isolated NMDA receptor-mediated synaptic transmission does not differ much between SynGAP heterozygous and wild-type littermates. NMDA receptor-mediated synaptic responses were pharmacologically isolated by bath application of ACSF with 0 m m Mg 2+ and 10 μ m NBQX. An input–output curve was generated by plotting the amplitude of NMDA receptor (NR)-mediated FP against fiber volley amplitude. At the end of each experiment, 100 μ m d,l -APV was added to the bath, completely abolishing the responses (data not shown). Dashed lines indicate normalized FP and paired-pulse facilitation ratio.

The phenotype seen in heterozygotes was not attributable to changes in AMPA receptor-mediated synaptic transmission, because there were no detectable differences in the input–output curve (Fig. 4C). Presynaptic function measured by the paired-pulse facilitation ratio at interstimulus intervals ranging from 25 to 1600 msec were also normal in the heterozygotes (Fig. 4D). To rule out the possibility that the reduced LTP in the SynGAP heterozygotes is attributable to alterations in NMDA receptor-mediated synaptic responses, we pharmacologically isolated NMDA receptor-mediated components of synaptic transmission by recording in ACSF with 0 m m Mg 2+ and 10 μ m NBQX. The magnitude of the NMDA receptor-mediated response was measured by generating an input–output curve. We plotted NMDA receptor-mediated FP amplitude against the fiber volley amplitude to correct for variability in recruiting presynaptic fibers. As shown in Figure 4E, there is no significant effect on NMDA receptor-mediated responses in the heterozygotes.


Conclusions

The functioning of the CPEB family proteins is essential at all stages of ontogeny. CPEBs play an important role in the formation and maintenance of cell polarity, participating in mRNA transport and localization, translational repression or activation of target mRNAs [30, 81,82,83]. In the nervous system, this function is manifested in the participation of the CPEB proteins in neurogenesis and the functioning of neurons. Much attention is devoted now to the role that the prion-like conformation of these proteins plays in the formation of long-term memory.

The CPEB proteins participate in the translational control of a wide range of mRNAs and, therefore, are involved in pathologies of the nervous system. Moreover, disturbances in the functioning of the CPEB proteins cause other pathological processes, including carcinogenesis, tumor invasion, and angiogenesis. In the case of rectal cancer, breast cancer, and gliomas, the expression levels of several CPEB proteins change simultaneously, which is indicative of interactions between them in the oncological process [67, 84,85,86]. The role of the CPEB proteins in certain liver diseases and metabolic disorders (e.g., hepatosteatosis) was also revealed [87].

Thus, investigation of the role of the CPEB proteins is an extremely important fundamental task that opens up prospects for understanding the molecular mechanisms of the formation and functioning of the nervous system and other body systems, as well as for finding ways to treat a wide range of diseases.


Watch the video: Πώς μεταφέρονται τα μηνύματα κατά μήκος του νευρώνα; (September 2022).


Comments:

  1. Maucage

    Willingly I accept. In my opinion, it is an interesting question, I will take part in discussion. I know, that together we can come to a right answer.

  2. Samubei

    Wonderful, good idea

  3. Nigul

    Interesting topic, Thank you!

  4. Akinoshura

    It is a pity, that now I can not express - there is no free time. I will be released - I will necessarily express the opinion on this question.

  5. Slaed

    I am sorry that I can not help you with anything, but I am sure that they will help you find the right solution.



Write a message