Método Inductivo

Since both neuronal viability and synaptic transmission are highly dependent on the architecture of the microtubule and actin cytoskeletons, their dynamics will be discussed in more detail here. Importantly, cytoskeleton dynamics largely determine the morphological constraints within which cells and synapses may function physiologically. Members of the doublecortin family are known to induce microtubule polymerization and stabilization and to interact with F-actin (Francis et al., 1999; Lin et al., 2000; Edelman et al., 2005; Coquelle et al., 2006; Vreugdenhil et al., 2007). CARP is highly homologous to the S/P-rich C-terminal parts of both DCX and DCLK, a domain that is implicated in protein interactions (Friocourt et al., 2001; Moores et al., 2004). As mentioned above, CARP was found to interact with Grb2, most likely through SH-3 domain binding. As Grb2 has been implicated in regulation of the actin cytoskeleton (Buday et al., 2002), this then suggests CARP indirectly plays a role in regulation of the actin cytoskeleton.

Trk receptor Grb2 Membrane Nucleus Neuronal Viability Neuronal Plasticity BDNF-LTP DCLK-short induction ERK1/2 DCLK-short- CARP SOS Cytoplasm

et al., 2007; Yan et al., 2007). Importantly, the viability of neuronal cells is linked to a change in the availability of neurotrophic factors and their receptors (Nichols et al., 2005; Schaaf et al., 2000). Thus, through interaction with Grb2, CARP may interfere with this cascade, thereby affecting the viability and morphology of neurons.

As described above, CARP is highly induced by kainate-induced seizures (Vreugdenhil et al., 1999) and this is also a time during which several neurotropic factors are induced (Gall and Lauterborn, 1992; Lindvall et al., 1994). Interestingly, another neurotrophic factor, BDNF, is a major regulator of LTP induced by high- frequency stimulation of excitatory synapses (Bramham and Messaoudi, 2005). BDNF is released from glutamatergic synapses following high-frequency stimulation and performs its intracellular signaling via TrkB receptors (Nawa and Takei, 2001; Balkowiec and Katz, 2002). In fact, BDNF stimulation itself induces LTP and this phenomenon is accompanied by a robust increase of CARP expression (Wibrand et al., 2006). Strikingly, mice with brain specific over- expression of TrkC, which is capable of binding both Neurotrophin (NT)-3 and BDNF, develop a phenotype that is similar to the electrophysiological phenotype of high-CARP mice: highly increased evoked extracellular fEPSPs at the CA3/CA1 synapse (Schenk et al., 2010; Sahún et al, 2007). Collectively, these data underscore CARPs potential to intervene with neurotrophic factor signalling. Evidence for involvement in neurotrophic factor signalling not only exists for CARP, but also for DCLK-short. Recently, nerve growth factor (NGF) stimulation of Neuroscreen-1 PC12 cells has been shown to highly induce the expression of

DCLK-short (Dijkmans et al., 2008; 2009). Moreover, activation of DCLK-short in

vitro depends critically on ERK1/2 activation, leading to phosphorylation of a specific serine residue situated within DCLK-short’s S/P-rich domain (Dijkmans et al., 2009). Given the abundant examples of processes in which concomitant activity and regulation of DCLK gene expression and neurotrophic factors is evident, we propose that CARP and DCLK-short exert their functions, at least partly, through interference with neurotrophic signal transduction and that interaction with Grb2 is highly probable (Figure 4).

Figure 4. Cartoon showing DCLK-short and CARP in relation to neurotrophic factor signaling. For details see section 2.3.

2.4 The DCLK gene and cytoskeleton dynamics

Since both neuronal viability and synaptic transmission are highly dependent on the architecture of the microtubule and actin cytoskeletons, their dynamics will be discussed in more detail here. Importantly, cytoskeleton dynamics largely determine the morphological constraints within which cells and synapses may function physiologically. Members of the doublecortin family are known to induce microtubule polymerization and stabilization and to interact with F-actin (Francis et al., 1999; Lin et al., 2000; Edelman et al., 2005; Coquelle et al., 2006; Vreugdenhil et al., 2007). CARP is highly homologous to the S/P-rich C-terminal parts of both DCX and DCLK, a domain that is implicated in protein interactions (Friocourt et al., 2001; Moores et al., 2004). As mentioned above, CARP was found to interact with Grb2, most likely through SH-3 domain binding. As Grb2 has been implicated in regulation of the actin cytoskeleton (Buday et al., 2002), this then suggests CARP indirectly plays a role in regulation of the actin cytoskeleton.

Trk receptor Grb2 Membrane Nucleus Neuronal Viability Neuronal Plasticity BDNF-LTP DCLK-short induction ERK1/2 DCLK-short- CARP SOS Cytoplasm

Chapter 6

Previously, the DCX domain containing isoforms of the DCLK gene have been held responsible for the role of DCLK in dynamic rearrangements of the microtubule cytoskeleton. In fact, DCL plays a pivotal role in determining neuronal fate by regulating mitotic spindle positioning and stability (Shu et al., 2006; Vreugdenhil et al., 2007; Boekhoorn et al., 2008). Moreover, DCL over-expression results in robust formation of microtubule bundles. (Vreugdenhil et al., 2007; Fitzsimons et al., 2008). Using a tubulin polymerization assay I demonstrated that CARP increases DCL-induced polymerization of tubulin in a dose-dependent fashion (Schenk et al., 2007). Through a similar mechanism, CARP may exert its pro-apoptotic properties through arrest of the microtubule skeleton.

CARP up-regulation has been found following kainate-induced seizures (Vreugdenhil et al., 1999), BDNF-LTP (Wibrand et al., 2006), D1-receptor stimulation (Berke et al., 1998; Glavan et al., 2002), and in apoptotic cells (Schenk et al., 2007). A common feature of these processes is the requirement of cytoskeleton rearrangements underlying the plasticity of specific neuronal circuits (Reviewed in: Cai and Sheng, 2009; Conde and Cáceres, 2009; Kueh and Mitchison, 2009). Another observation that makes a potential role for the CARP domain in cytoskeleton regulation possible is that several of the co-regulated genes that accompany the induction of CARP following BDNF-LTP have known functions in excitatory synaptogenesis and axon guidance (Wibrand et al., 2006). More specifically, BDNF-LTP is associated with the induction and dendritic transport of transcripts of the immediate early gene activity-regulated cytoskeleton associated protein Arc (Lyford et al., 1995; Ying et al., 2002). BDNF activates a process of synaptic consolidation that dependents critically on Arc transcription and translation (Bramham and Messaoudi, 2005). During this process formation of stable LTP is associated with insertion of glutamate receptors at cell membranes and structural remodeling of spines (Geinisman, 2000; Harris et al., 2003). Importantly, these changes are closely related to regulation of actin dynamics (Okamoto et al., 2004; Zito et al., 2004; Oertner and Matus, 2005). These findings, in combination with the structural overlap between DCX and DCLK on the one hand and CARP on the other hand, raises the possibility that CARP affects cytoskeleton stability. Thus by affecting cytoskeleton stability, CARP may, together

with other players, influence neuronal viability.

Evidence suggests that DCLK-short also interacts with the cytoskeleton as it co- localizes with F-actin in growth cones of neurites and may regulate neuritogenesis in a phophorylation dependent fashion (Dijkmans et al., 2009). CaMKs are highly homologous to DCLK-short and from this perspective it is of interest to note that CaMKI and CaMKII also play regulatory roles in actin dynamics, thereby affecting neuronal morphology (Suizu et al., 2002; Okamoto et al., 2007; Penzes et al., 2008). CaMKI regulates actin rearrangements in growth cones and affects neurite outgrowth of granule cells in the hippocampus (Wayman et al., 2004; 2008), whereas CaMKII interacts with F-actin directly and regulates synaptic strength and dendritic morphology in hippocampal neurons (Shen and Meyer, 1999; Okamoto et al., 2007). Interestingly, putative substrates of CaMKs that are implicated in actin dynamics include guanine exchange factors (Gefs; Wayman et al., 2004). Deregulation of Rap1Gef was observed in the hippocampus of high-CARP mice, underscoring the possible involvement of the CARP domain in cytoskeleton rearrangements and its regulatory role in processes that are similar to those where CaMKs are of importance.

Our observations and those of others strongly suggest a role for the DCLK gene in cytoskeleton dynamics, potentially through interference with neurotrophic factor signalling. Altogether, this suggests that members of the DCLK gene family lacking DCX domains, i.e. CARP and DCLK-short, also have relevant biological functions related to the state of the cytoskeleton.

Previously, the DCX domain containing isoforms of the DCLK gene have been held responsible for the role of DCLK in dynamic rearrangements of the microtubule cytoskeleton. In fact, DCL plays a pivotal role in determining neuronal fate by regulating mitotic spindle positioning and stability (Shu et al., 2006; Vreugdenhil et al., 2007; Boekhoorn et al., 2008). Moreover, DCL over-expression results in robust formation of microtubule bundles. (Vreugdenhil et al., 2007; Fitzsimons et al., 2008). Using a tubulin polymerization assay I demonstrated that CARP increases DCL-induced polymerization of tubulin in a dose-dependent fashion (Schenk et al., 2007). Through a similar mechanism, CARP may exert its pro-apoptotic properties through arrest of the microtubule skeleton.

CARP up-regulation has been found following kainate-induced seizures (Vreugdenhil et al., 1999), BDNF-LTP (Wibrand et al., 2006), D1-receptor stimulation (Berke et al., 1998; Glavan et al., 2002), and in apoptotic cells (Schenk et al., 2007). A common feature of these processes is the requirement of cytoskeleton rearrangements underlying the plasticity of specific neuronal circuits (Reviewed in: Cai and Sheng, 2009; Conde and Cáceres, 2009; Kueh and Mitchison, 2009). Another observation that makes a potential role for the CARP domain in cytoskeleton regulation possible is that several of the co-regulated genes that accompany the induction of CARP following BDNF-LTP have known functions in excitatory synaptogenesis and axon guidance (Wibrand et al., 2006). More specifically, BDNF-LTP is associated with the induction and dendritic transport of transcripts of the immediate early gene activity-regulated cytoskeleton associated protein Arc (Lyford et al., 1995; Ying et al., 2002). BDNF activates a process of synaptic consolidation that dependents critically on Arc transcription and translation (Bramham and Messaoudi, 2005). During this process formation of stable LTP is associated with insertion of glutamate receptors at cell membranes and structural remodeling of spines (Geinisman, 2000; Harris et al., 2003). Importantly, these changes are closely related to regulation of actin dynamics (Okamoto et al., 2004; Zito et al., 2004; Oertner and Matus, 2005). These findings, in combination with the structural overlap between DCX and DCLK on the one hand and CARP on the other hand, raises the possibility that CARP affects cytoskeleton stability. Thus by affecting cytoskeleton stability, CARP may, together

with other players, influence neuronal viability.

Evidence suggests that DCLK-short also interacts with the cytoskeleton as it co- localizes with F-actin in growth cones of neurites and may regulate neuritogenesis in a phophorylation dependent fashion (Dijkmans et al., 2009). CaMKs are highly homologous to DCLK-short and from this perspective it is of interest to note that CaMKI and CaMKII also play regulatory roles in actin dynamics, thereby affecting neuronal morphology (Suizu et al., 2002; Okamoto et al., 2007; Penzes et al., 2008). CaMKI regulates actin rearrangements in growth cones and affects neurite outgrowth of granule cells in the hippocampus (Wayman et al., 2004; 2008), whereas CaMKII interacts with F-actin directly and regulates synaptic strength and dendritic morphology in hippocampal neurons (Shen and Meyer, 1999; Okamoto et al., 2007). Interestingly, putative substrates of CaMKs that are implicated in actin dynamics include guanine exchange factors (Gefs; Wayman et al., 2004). Deregulation of Rap1Gef was observed in the hippocampus of high-CARP mice, underscoring the possible involvement of the CARP domain in cytoskeleton rearrangements and its regulatory role in processes that are similar to those where CaMKs are of importance.

Our observations and those of others strongly suggest a role for the DCLK gene in cytoskeleton dynamics, potentially through interference with neurotrophic factor signalling. Altogether, this suggests that members of the DCLK gene family lacking DCX domains, i.e. CARP and DCLK-short, also have relevant biological functions related to the state of the cytoskeleton.

Figure 5. Neuronal viability, neuronal plasticity, neurotrophic factor signalling and regulation of cytoskeleton dynamics are processes which are of crucial importance for DCLK-gene function. Induction of DCLK-short and CARP and their reinforcing (+) and inhibiting effects (-) on neuronal viability and plasticity are indicated.