Dendritic spine morphogenesis during development and plasticity

In the rat hippocampus, first synapses are formed as early as postnatal day 1 (P1) (Fiala et al., 1998) and the density of synapses gradually increases during the following weeks – doubling from P7 to P15 and from P15 to adulthood (Harris and Stevens, 1989; Harris et al., 1992). In dissociated hippocampal neuronal cultures, synapse development follows roughly in a sim- ilar time course: during the second week the synapse number doubles or triples and keeps increasing until the third week, after which it tends to fall (Papa et al., 1995; Boyer et al., 1998). Synapses are frequently found on filopodia (long thin protrusions), on stubby spines and on dendritic shafts in the brain (Miller and Peters, 1981; Fiala et al., 1998); they are later gradu- ally replaced by or converted to, synapses on mushroom-like spines that have a well-defined head (Harris, 1999). Filopodia are remarkably more motile than mushroom-shaped spines and with a mean lifetime of onlyv10 minutes versus hours and days. These thin protrusions are more transient than mature dendritic spines in culture (Dailey and Smith, 1996; Ziv and Smith, 1996). Filopodial motility might help to contact the “right” axon and can therefore be related to synaptogenesis (Ziv and Smith, 1996; Bonhoeffer and Yuste, 2002). Filopodia

4.5. Eph receptors in the adult brain

have been proposed to be the precursors of mature mushroom-shaped spines (Ziv and Smith, 1996; Harris, 1999; Bonhoeffer and Yuste, 2002) and once formed, synapses are not rigid. Dur- ing development, synaptic connections undergo an activity-dependent remodelling (Katz and Shatz, 1996; Shatz, 1996) and also in the adult brain synapses undergo long-term modifica- tions (synaptic plasticity) that are believed to be the basis of information storage (learning and memory) (Bonhoeffer and Yuste, 2002; Li and Sheng, 2003).

The polymerization of actin, the main cytoskeletal element of the spine, is necessary for the shape and motility of the spine (Matus et al., 2000; Bonhoeffer and Yuste, 2002). As described in more detail before, many signalling proteins downstream of Eph receptors are known reg- ulators of actin dynamics and therefore the activation of these receptors is required for spine development and maintenance.

Kalirin & Intersectin

EphB2 also promotes dendritic spine development through the activation of the GEFs Kalirin and Intersectin, which activate the two Rho family GTPases, Rac1 and Cdc42, respectively (Irie and Yamaguchi, 2002; Penzes et al., 2003). In the first pathway, long-term activation of EphB2 by ephrinB1-Fc induces clustering, phosphorylation and synaptic translocation of the endoge- nous Rho-GEF Kalirin to the postsynaptic site in immature hippocampal neurons and pro- motes spine morphogenesis. Unlike Intersectin and Ephexin, Kalirin binds only to activated EphB2 receptors. EphB2 activation also upregulates the activation of the serine/threonine kinase p21 activated kinase (Pak), a Rac1 downstream effector that promotes actin cytoskele- ton rearrangement. Transfection of dominant negative forms of Kalirin, Rac1 and the Pak inhibitory domain all prevent spine morphogenesis in response to ephrinB1 stimulation, sug- gesting that Kalirin regulates Rac1 activation downstream of activated EphB2 receptors (Pen- zes et al., 2003). The second downstream pathway that link EphB2 to spine morphogenesis in- volves Intersectin and Cdc42. Intersectin, an exchange factor for Cdc42, is predominantly ex- pressed in neurons and associated in an activity-independent manner with the kinase-domain of EphB2, but not EphA4. EphB2 cooperates synergistically with neural Wiskott-Aldrich syn- drome protein (N-WASP) to activate Intersectin, which in turn activates Cdc42. N-WASP bind to the actin-related protein 2/3 (Arp2/3) complex in the presence of activated Cdc42 and activate Arp2/3-mediated polymerization of branched actin filaments (Irie and Yamaguchi, 2002). Transfection of dominant-negative forms of Cdc42, Intersectin and N-WASP inhibit spine morphogenesis (Figure 4.10). Thus, this pathway provides a direct link between EphB receptor activation and actin dynamics and has been proposed to promote branching of actin

Figure 4.10. EphB receptors drive dendritic spine morphogenesis. Phosphorylation of the proteoglycan syndecan-2 downstream of activated EphB2 receptors is critical for promoting dendritic spine formation. Ac- tivated EphB2 receptors also activate the Rho-GEF proteins, Intersectin and Kalirin, which in turn activate the Rho family GTPases, Cdc42 and Rac1, respectively. The activated GTPases interact with downstream effector proteins which induce actin rearrangements, thereby promoting the EphB2-induced spine morphogenesis. The phosphorylation of syndecan-2 downstream of EphB2 is critical to induce association with EphB2 and its cluster- ing. Clustered syndecan-2 recruits its cytoplasmic ligands toward subsynaptic localization, thereby promoting the morphological maturation of spines.

filaments, leading to the enlargement of the spine head.

Syndecan-2

Ethell et al. found that clustering of the cell surface proteoglycan syndecan-2 promotes den- dritic spine maturation (Ethell and Yamaguchi, 1999). In a follow-up study they could demon- strate that tyrosine phosphorylation of syndecan-2 downstream of EphB2 receptor activation, but not EphAs, induces syndecan clustering and transforms immature filopodia-like dendritic protrusions into mature, mushroom-shaped dendritic spines in cultured hippocampal neu-

4.5. Eph receptors in the adult brain

rons. EphB2 phosphorylates syndecan-2 on two cytoplasmic tyrosines and this phosphoryla- tion is necessary for the association of both proteins (Ethell et al., 2001). Clustered syndecan-2 recruits cytoplasmic proteins, such as CASK, syntenin and Synbindin through the interaction of the C-terminal EFYA sequence, leading to the morphological maturation of spines (Figure 4.10)(Ethell and Yamaguchi, 1999).

NMDA receptor

Four recent studies demonstrate the involvement of Eph receptors forward signalling in the assembly of postsynaptic complexes at the excitatory synapses (Dalva et al., 2000; Ethell et al., 2001; Takasu et al., 2002; Penzes et al., 2003). The activation of the EphB receptor by solu- ble preclustered ephrinB1-Fc induces the direct association of EphB2 receptors with the NR1 subunit of the NMDA glutamate receptor in large raft-like patches within one hour in young immature cultured neurons. This interaction is mediated by the extracellular regions of the two receptors. Although this interaction is independent from the kinase activity of EphB re- ceptors, the authors showed that forward signalling via EphB2 is required for further steps in synapse formation. This is all very important because the NMDA receptor plays a central role in synaptic plasticity and is known to be the first recruited glutamate-gated ion channel to the immature synapse. Ca2+/Calmodulin-dependent kinase II (CamKII) and Grb10 were also found to colocalize with NR1 and EphB2 following ephrinB stimulation (Dalva et al., 2000). More recent data showed that EphB2 recruits and activates the cytoplasmic tyrosine kinase Src, which in turn phosphorylates subunits of the NMDA receptor (Grunwald et al., 2001; Takasu et al., 2002). Phosphorylation of the NR2B subunit enhances Ca2+ influx through the NMDA receptor in response to glutamate and increases phosphorylation of the transcription factor Ca2+/cAMP-responsive element binding protein (CREB) and CREB-dependent tran- scriptional events that may affect synapse formation, maturation and plasticity (Grunwald et al., 2001; Takasu et al., 2002).

EphB-knockout mice

Consistent with thesein vitrofindings, EphB2 null mice show reduced levels of NR1-containing NMDA receptors in hippocampal synapses (Henderson et al., 2001) and show deficits in activity-dependent synaptic plasticity (LTP and LTD). Targeted expression of a truncated kinase-deficient form of EphB2 rescued the EphB2-knockout phenotype, suggesting that ephrinB reverse signalling might play a role at the synapse. However,EphB2-knockout mice have normal hippocampal synapse morphology and density, which indicates that EphB2 is

Figure 4.11. NMDA and EphB2 receptor and their possible roles during synapse formation, maturation and synaptic plasticity. EphB2 receptor activation leads to the recruitment of SFKs, direct interaction with the NR1 subunit and clustering of the NMDA receptor (in young cultures). The activated SFKs phosphorylate the NMDA receptor subunits NR2B (grey spheres), which enhances Ca2+influx through the ion channel after glutamate (pink spheres) stimulation and potentiates phosphorylation (grey spheres) of the transcription factor CREB. CREB- dependent transcriptional events and other EphB2 downstream signalling cascades may influence synapse for- mation, maturation and synaptic plasticity. The site of ephrin localization has not yet been addressed (question marks).

not critically required for most aspects of synapse development (Grunwald et al., 2001; Hen- derson et al., 2001). In contrast, overexpression of kinase-deficient EphB2 in dissociated hip- pocampal neurons (one week old) that presumably block phosphorylation of multiple EphB family members (EphB1-B3), results in a decrease of synapse density (Dalva et al., 2000) and dendritic protrusions remain filopodial in old cultures (three weeks old)(Ethell et al., 2001). Hippocampal neurons from triple EphB1/EphB2/EphB3-knockout mice fail to form spinesin vitroand develop abnormal spinesin vivo, indicating that multiple EphB receptors cooperate to promote spine morphogenesis and synapse formation in the hippocampus (Henkemeyer et al., 2003). Thus, EphB receptor signalling pathways are required for spine morphogenesis.