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Nervous system development requires a sequence of processes such as neuronal migration, the development of dendrites and dendritic spines and the formation of synapses. The extracellular matrix protein Reelin plays an important role in these processes, Reelin regulates for example the migration of neurons from proliferative zones to their target positions in the brain. As a consequence, layered structures are formed in the neocortex, the hippocampus and cerebellum (Lambert de Rouvroit et al., 1999). Reelin exerts its functions by binding to two transmembrane receptors, apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR). This binding causes phosphorylation of the intracellular adapter protein Disabled-1 (Dab1) (D’Arcangelo et al., 1999) via activation of Src-family kinases (SFKs) (Bock and Herz, 2003), leading to cytoskeletal reorganization which enables cell migration and morphological changes (Lambert de Rouvroit and Goffinet, 2001). Since ApoER2 and VLDLR do not possess intrinsic kinase activity to activate SFKs, the existence of a co-receptor was suggested. EphrinBs are transmembrane ligands for Eph receptors and have signaling capabilities required for axon guidance (Cowan et al., 2004), dendritic spine maturation (Segura et al., 2007) and synaptic plasticity (Essmann et al., 2008; Grunwald et al., 2004). As stimulation of cultured cortical neurons with soluble EphB receptors causes recruitment of SFKs to ephrinB-containing membrane patches and SFK activation (Palmer et al., 2002), we investigated whether ephrinB ligands would be the missing co-receptors in the Reelin signaling pathway functioning during neuronal migration, dendritic spine maturation and synaptic plasticity. We found that the extracellular part of ephrinBs directly binds to Reelin and that ephrinBs interact with Dab1, phospho-Dab1, ApoER2 and VLDLR. EphrinB3 is localized in the same neurons as ApoER2 and Dab1 in the cortex and hippocampus, and in the cerebellum ephrinB2 is detected in neurons that express Dab1. To investigate the requirement of ephrinBs for neuronal migration, triple knockout mice lacking all ephrinB ligands were analyzed. The cortical layering of ephrinB1, B2, B3 knockout brains is inverted, showing the outside-in pattern typical for the reeler cortex. The hippocampus and cerebellum of triple knockout mice also exhibit reeler-like malformations, although less penetrant than the cortical defects. Dab1 phosphorylation is impaired in mice lacking ephrinB3 and this effect is strongly enhanced in neurons lacking all ephrin ligands. Moreover, activation of ephrinB3 reverse signaling induces Dab1phosphorylation in reeler primary neurons. In agreement with an important regulatory function of ephrinBs in Reelin signaling, activation of ephrinB3 reverse signaling is even able to rescue reeler defects in cortical layering in organotypic slice cultures. In summary, all these results identify ephrinBs as co-receptors for Reelin signaling, playing essential roles in neuronal migration during the development of cortex, hippocampus and cerebellum (Sentürk et al., 2011).
Synaptopodin is the founding member of a family of actin-associated proline-rich proteins. It is present in a subset of telencephalic dendritic spines, where it is tightly associated with the dendritic spine apparatus, a putative calcium store. Synaptopodin-deficient mice lack the spine apparatus and show deficits in long-term potentiation and spatial memory. Thus, synaptopodin appears to play a role in synaptic plasticity. In the present thesis, three major questions were addressed: (1) What is the distribution of synaptopodin and the spine apparatus in identified hippocampal neurons? (2) Is the distribution of synaptopodin affected by denervation? (3) Is synaptopodin involved in the regulation of denervation-induced spine loss? The major findings of this thesis are: (1) Immunohistochemistry in the hippocampus of wildtype and EGFP-transgenic mice revealed significant layer-specific differences in the prevalence of synaptopodin at the level of individual neurons. (2) Light and electron microscopic analysis also revealed the presence of synaptopodin in axon initial segments of cortical and hippocampal principal neurons. There, it was found to be an essential component of the cisternal organelle, a putative axonal homologue of the dendritic spine apparatus. (3) Immunohistochemistry in the rat fascia dentata before and following entorhinal deafferentation revealed changes in synaptopodin expression in denervated and non-denervated layers of the hippocampus, suggesting that the distribution of synaptopodin in hippocampal neurons is regulated by presynaptic signals. (4) The dynamics of denervation-induced spine plasticity were studied in vitro using confocal live imaging of organotypic entorhino-hippocampal slice cultures. Whereas spines were remarkably stable under control conditions, spine loss and spine formation were seen following denervation. No significant differences were observed between cultures from wildtype and synaptopodin-deficient mice, suggesting that synaptopodin is not involved in lesion-induced spine plasticity. (5) Finally, a set of transgenic mice expressing fluorescently tagged synaptopodin were generated to facilitate future experiments on the dynamics and function of synaptopodin. In summary, this thesis presents novel findings on (1) the subcellular distribution of synaptopodin in spines and the axon initial segment, (2) the molecular composition of the cisternal organelle, and (3) the dynamics of spines and the spine apparatus organelle following deafferentation in vivo and in vitro.
In the adult mammalian central nervous system, two defined neurogenic regions retain the capacity to generate new neurons throughout adulthood, namely the subependymal zone (SEZ) at the lateral ventricles and the subgranular layer of the hippocampus (SGL). Adult neurogenesis consists of a whole set of events including proliferation, fate specification, migration, survival and finally synaptic integration of newly born neurons. Each of these events is controlled by the interplay of numerous factors. In this study two signalling systems were analysed with regard to their functional role in adult neurogenesis in vivo, namely the purinergic system and the growth factor EGF. Neither short- nor long-term application of the P2Y receptor agonists UTP and ADPβS and the P2Y receptor antagonist suramin into the lateral ventricle of adult mice altered cell responses as compared to vehicle controls in vivo. In contrast, analysis of the expansion rates of cultured neural stem cells (NSCs) from knockout mice revealed a strong increase in the number of NSCs from NTPDase2-/- mice, whereas cell numbers of NSCs from P2Y1-/- and P2Y2-/- mice were significantly reduced in comparison to wildtype levels. Notably, in vivo proliferation rates were potently elevated in the SGL and the SEZ of NTPDase2-deficient mice. However, in vivo proliferation in both neurogenic niches of the single receptor knockout mice P2Y1-/- and P2Y2-/- and P2Y1-/- P2Y2-/-double-knockout mice did not differ significantly from the wildtype. In mice lacking the P2Y2 receptor the survival of newly born neurons in the hippocampal granule cell layer was significantly increased. These data provide the first line of evidence that purinergic signalling is involved in the control of neural stem cells behaviour not only in vitro but also in vivo. In order to further characterise the role of epidermal growth factor (EGF) in adult neurogenesis, transit amplifying precursors (TAPs) and type B astrocytes were identified as EGF-responsive cell populations following ventricular EGF injection, whereas ependymal cells, neuroblasts and NG2-positive cells did not or only to a minor extent respond to EGF injection. These EGF-responsive cell populations were found on both, the septal as well as striatal lateral ventricle walls. Long-term ventricular EGF infusion for 6d, 1. increased cell proliferation of both ventricle walls revealing a gradient along the rostro-caudal axis, 2. altered the balance between neuronal and macroglial cell fates to generate oligodendrocyte precursors and 3. lead to an entire remodelling of the classical architecture of the SEZ.
Active neurogenesis continuously takes place in the dentate gyrus of the adult mammalian brain. The dentate gyrus of the adult rodent hippocampus contains an astrocytelike cell population that is regarded as residual radial glia. These cells reside with their cell bodies in the subgranular layer (SGL). Radial processes traverse the granule cell layer (GCL) and form bushy ramifications in the inner molecular layer (IML). The residual radial glial cells apparently represent neuronal progenitor cells that can give rise to functionally integrated granule cells. To date the cellular and molecular events driving a subpopulation of these cells into neurogenesis as well as the cellular transition states are poorly understood. The present study shows, that in the mouse dentate gyrus, this cell type selectively expresses surfacelocated ATPhydrolyzing activity and is immunopositive for nucleoside triphosphate diphosphohydrolase 2 (NTPDase2). NTPDase2 is an ectoenzyme and hydrolyzes extracellular nucleoside triphosphates such as ATP or UTP to their respective nucleoside diphosphates. The enzyme becomes expressed in the hippocampus during late embryogenesis from E17 onwards, and is thus not involved in early brain development. Its embryonicpattern of expression mirrors dentate migration of neuroblasts and the formation of the primary and finally the tertiary dentate matrix. NTPDase2 is also expressed by a transient population of cortical radial glia from late embryonic development until postnatal day 5. NTPDase2 can be employed as a novel markerfor defining cellular transition states along the neurogenic pathway. It is associated with subpopulations of GFAP and nestinpositive cells. These intermediate filaments are typically expressed by the progenitor cells of the dentate gyrus. In addition there is a considerable overlap with doublecortinand PSANCAM positive cells. The expression of the microtubuleassociated protein doublecortin and of PSANCAM which are expressed by migrating neuroblasts is indicative of a transition of progenitors to a neural phenotype or an immature form of granule cell. NTPDase2 is no longer associated with young neurons and with maturegranule cells, as indicated by the lack of doubleimmunostaining for III tubulin and NeuN, respectively. Furthermore, β S100positive astrocytes do not express NTPDase2 validating that NTPDase2 is also not associated with later stages of gliogenesis. Experiments with the Sphase marker bromodeoxyuridine (BrdU) demonstrate that NTPDase2positive cell proliferate. Postmitotic BrdU-labeled cells preferentially acquire an NTPDase2positive phenotype. Many of these cells were also positive for GFAP. The contribution of BrdUlabeled cells positive for NTPDase2 increased with time from 2 h to 72 h, validating a strong association of NTPDase2 with proliferating cells of the dentate gyrus. The colocalization studies with various markers and the results of the experiments suggestthat NTPDase2 is associated with cell types of varying maturation states but not with mature neurons or astrocytes. Studies on the formation of neurospheres from the dentate gyrus validate previous data suggesting that the hippocampal progenitors have little capacity for self renewal in vitro. In situ hybridization results indicate the presence of one of the metabotropic purinergic receptor subtypes (the P2Y1 receptor) within the adult neurogenic regions, the dentate gyrus and the lateral walls of the lateral ventricles. A patchclamp analysis demonstrates the presence of functional ionotropic nucleotide receptor (P2X receptors) in progenitor cells expressing nestin promotordriven GFP. They suggest that the signaling pathway via extracellular nucleotides and nucleotide receptors may play a role in the control of adult hippocampal neurogenesis.
Studies in particular of the last decade showed that active neurogenesis continuously takes place in the subventricular zone (SVZ) of the lateral ventricles of the adult rodent brain. Neurogenesis in the SVZ leads to migration of neuroblasts within the rostral migratory stream (RMS) and mature neuron formation mainly in the olfactory bulb (OB). According to present understanding, glial cells with astrocytic properties represent the actual adult neural stem cells. The cell types representing the various cellular transition states leading to the formation of mature neurons as well as the mechanisms controlling adult neurogenesis and neuroblast migration are poorly understood. A previous study from this laboratory demonstrated that the ATP-hydrolyzing enzyme nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is associated with type B cells, the presumptive neural stem cells. NTPDase2 is a protein of the plasma membrane with its catalytic site facing the extracellular space. It hydrolyzes extracellular nucleoside triphosphates to their respective nucleoside diphosphates. This raises the possibility that the signaling pathway via extracellular nucleotides is involved in the control of adult neurogenesis. Neurons as well as glial cells express several subtypes of receptors (P2 receptors) that are responsive to the nucleotides ATP, ADP, UTP, or UDP. P2X receptors are ATP-gated Na+, K+ and Ca2+ permeable ion channels, P2Y receptors are coupled to trimeric G-proteins. In order to probe for a functional role of nucleotides in adult neurogenesis, the present study referred to an in vitro system (neurospheres). Neurospheres produced from isolates of the mouse SVZ and cultured in the presence of EGF and bFGF expressed the neural stem cell marker nestin and also GFAP, S100β, NTPDase2 and tissue non-specific alkaline phosphatase. Neurospheres generated from the cells of the subventricular zone were multipotenital. This was revealed by immunostaining of differentiated cells with markers for astrocytes, neurons and oligodendrocytes. The presence of ecto-nucleotidase was verified by analyzing the free phosphate released from nucleotides. The tissue non-specific form of alkaline phosphatase was the predominant enzyme. Both NTPDase2 and TNAP could be identified by immunocytochemistry and Western blotting. Hydrolysis was not observed for p-nitrophenyl thymidine monophosphate, a substrate of members of the ectonucleotide pyrophosphatase/phosphodiesterase family (NPP1 to NPP3). Since ecto-nucleotidases control the availability of extracellular nucleotide agonists, neurospheres were studied for the potential expression and functional role of nucleotide receptors. Neurospheres responded to extracellular nucleotides with a transient rise in Ca2+ (ATP = ADP > UTP). The rise in Ca2+ was due to P2Y receptors. The Ca2+ response was unaltered in the absence of extracellular Ca2+ and strongly reduced by thapsigargin, a blocker of internal Ca2+ stores. The P2Y1 antagonist MRS2179 strongly reduced the ATP- or ADP-induced increase in Ca2+, suggesting the involvement of a P2Y1 receptor. In addition, suramin and PPADS, non-selective antagonists for P2 receptors, inhibited most of the Ca2+ response. The agonistic activity of UTP and the lack of response to UDP implied the additional presence of a P2Y2 and/or a P2Y4 receptors and the absence of a functional P2Y6 receptor. RT-PCR experiments demonstrated that neurospheres expressed P2Y1 and P2Y2 receptors but not P2Y4 receptor. That the majority of the Ca2+ response to ATP was mediated via P2Y1 receptors was also confirmed by analysis of P2Y1 knockout mice and by application of the P2Y1 receptor-specific antagonist MRS2179. In addition, agonists of P2Y1 and P2Y2 receptors and low concentrations of adenosine augmented cell proliferation inspite of the presence of mitogenic growth factors. Neurosphere cell proliferation was attenuated after application of MRS2179 and in neurospheres from P2Y1 receptor knockout mice. These results infer a nucleotide receptor-mediated synergism that augments growth factor-mediated cell proliferation. Taken together these results suggest that P2Y-mediated nucleotidergic signalling is involved in neurosphere function and possibly also in adult neurogenesis in situ.
In the present study the cryo-immunogold technique was used and optimized for investigating the ultrastructure and immunolabeling of synaptic proteins. It is evidently a suitable method for the localization of membrane proteins since the antigens are not treated with any chemical denaturation before immunolabeling except for the fixation and since the antigens are directly exposed to the surface of the cryo-ultrasections. The v-SNARE VAMP II and the vesicle-associated proteins SV2 and Rab3A were detected extensively at small vesicles in the mossy fiber terminals. The t-SNARE SNAP-25, and N-type and P/Q type Ca2+ channels were allocated to the plasma membrane both at the active zone and outside the active zone. SNAP-25 and N-type Ca2+ channels appeared also at synaptic vesicles. A significantly increased immunolabeling of VAMP II, SV2, Rab3A, SNAP-25 and N-type Ca2+ channels was found at the active zones of fast synapses, indicating a concentration of these proteins at sites of exocytosis. The widespread distribution of the t-SNARE SNAP-25 at the axonal plasma membrane reveals that membrane-targeting specificity cannot be determined solely by v/t-SNARE interactions. Additional control components are required to assure the docking and exocytosis of the synaptic vesicles at active zones. The novel protein Bassoon was only found at active zones of central synapses and showed the highest specific labeling among all proteins investigated. Its labeling pattern implies an association of Bassoon with the presynaptic dense projections, the structural guide for vesicle exocytosis. The involvement of Bassoon in the organization of the neurotransmitter release site suggests that Bassoon may play an important role in determining the specificity of vesicle docking and fusion. In the neurosecretory endings of neurohypophysis the synaptic proteins VAMP II, SNAP- 25, SV2, Rab3A, and the N-type Ca2+ channels showed a preferential labeling over microvesicles. Moreover, the immunolabeling intensity of these proteins over microvesicles corresponded closely to that over synaptic vesicles. This suggests that these synaptic proteins share an identical association with synaptic vesicle and microvesicles. A significant labeling of SNAP-25, the N-type Ca2+ channels and VAMP II was also detected at the plasma membrane near the clustered microvesicles, indicating the competence of microvesicles for docking and exocytosis along the plasma membrane in the absence of active zones. No significant labeling of VAMP II, SNAP-25, SV2 and N-type Ca2+ channel was observed at the membrane of neurosecretory granules. This is in agreement with the notion that synaptic vesicles and microvesicles possess regulatory mechanisms for exocytosis different from those of granules. In contrast, a/ß-SNAP and NSF were found on the granules, and Rab3A and the P/Q-type Ca2+ channels on granules in a subset of terminals. Rab3A is associated specifically with the oxytocin-containing granule population. Interestingly, some plasma membrane proteins, such as SNAP-25 and even N-type Ca2+ channels and P/Q-type Ca2+ channels, were observed not only at the plasma membrane but also at the vesicular organelles. This suggests that these vesicular organelles may be involved in transporting newly synthesized proteins from the soma to the plasma membrane of the terminal. Furthermore, the vesicular pool of the Ca2+ channels may serve in the stimulationinduced translocation into the plasma membrane when required. Using the conventional preembedding method with Epon and the post-embedding method with LR Gold, VAMP II was localized at vesicular organelles of varying size and on horseradish peroxidase filled endocytic organelles in cultured astrocytes, with and without stimulation in the presence of the horseradish peroxidase. This indicates that VAMP II is involved in the cycle of vesicular exocytosis and endocytosis in astrocytes. U373 cells are capable of expressing all three members of the synaptic SNARE complex (v-SNARE VAMP II, t-SNARE syntaxin I and SNAP25). This indicates the competence of U373 to carry out regulated exocytosis by means of the classical SNARE mechanism. In addition, the ubiquitous v-SNARE cellubrevin and the endosome-associated small GTPbinding protein Rab5 could be expressed in U373 cells. All recombinant synaptic proteins investigated in U373 cells revealed a punctuate cellular distribution under the fluorescence microscope, suggesting that they are mainly associated with intracellular compartments. The cryo-electron microscopy provided direct evidence for the association of all expressed proteins with electron-lucent vesicular organelles. It further supports the potential of U373 MG cells to release low molecular weight messengers by a regulated exocytosis mechanism. In addition, myc-VAMP II was found on dispersed granules. Probably, VAMP II also participates in the exocytosis event of granules in U373 cells. Gold labeling for the two presumptive t-SNAREs syntaxin I and SNAP-25 in U373 cells was confined to the vesicular organelles. At the ultrastructural level no significant labeling was identified at the plasma membrane. The high level of colocalization of the two SNARE proteins VAMP II and syntaxin I in the cell body and in cell processes suggests that the two proteins are mostly sorted into identical vesicular organelles. A partial colocalization of VAMP II and cellubrevin as well as of VAMP II and Rab5 was observed under the fluorescence microscope. At the ultrastructural level, a colocalization of VAMP II and cellubrevin as well as of VAMP II and Rab5 was found on some clustered vesicles. The partial colocalization of VAMP II and cellubrevin implies that they similarly function as v-SNAREs. The partial colocalization of Rab5 with VAMP II in U373 cells suggests that the endosomal protein Rab5 is associated with VAMP II-containing organelles during some stages of their life cycle.
In the adult mammalian brain stem cells within defined neurogenic niches retain the capacity for lifelong de novo generation of neurons. The subventricular zone (SVZ) of the lateral ventricles and the subgranular layer (SGL) of the hippocampal dentate gyrus (DG) have been identified as the two major sites of adult neurogenesis. Moreover, the third ventricle in the hypothalamus is emerging as a new neurogenic niche in the adult brain. Extracellular purine and pyrimidine nucleotides are involved in the control of both embryonic and adult neuro-genesis. These nucleotides act via ionotropic P2X or metabotropic P2Y receptors and studies of the adult SVZ and the DG provide strong evidence that ATP promotes progenitor cell proliferation in this stem cell rich regions. Previous studies have shown that the extracellular nucleotide-hydrolyzing enzyme NTPDase2 is highly expressed by adult neural stem and progenitor cells of the SVZ and the rostral migratory stream (RMS), the hippocampal SGL, and the third ventricle. NTPDase2 preferentially hydrolyzes extracellular nucleoside triphosphates (NTPs) and, to a lower extent, diphosphates, thus modulating their effect on nearby nucleotide receptors. Deletion of the enzyme increases extracellular NTP concentrations, and might indicate roles of purinergic signaling in adult neurogenesis. As shown by enzyme histochemistry, genetic deletion of NTPDase2 essentially eliminates ATPase activity in neurogenic niches but does not affect protein expression levels and activity of other ectonucleotidases. Lack of NTPDase2 leads to expansion of the hippocampal stem cell pool as well as of the inter-mediate progenitor type-2 cells. Cell expansion is lost at around type-3 stage, paralleled by increased labeling for caspase-3, indicating increased apoptosis, and decreased levels in CREB phosphorylation in doublecortin-expressing cells, diminishing survival in this cell population. In line with increased cell death, P2Y12 receptor-expressing microglia is enriched at the hilus orientated side of the granule cell layer. These data strongly suggest that NTPDase2 functions as central homeostatic regulator of nucleotide-mediated neural progenitor cell proliferation and expansion in the adult brain by balancing extracellular nucleotide concentrations and activation of purinergic receptors.
In order to further characterize the role of purinergic signaling in adult neurogenesis, the ADP-sensitive P2Y13 receptor was identified as a potential candidate whose activation might inhibit neurogenesis in the hippocampal dentate gyrus and the newly identified neurogenic niche at the third ventricle. Deletion of P2ry13 increased progenitor cell proliferation and long-term progenitor survival as well as new neuron formation in the hippocampal neurogenic niche. This was further paralleled by increased thickening of the granule cell layer, CREB phosphorylation, and expression of the neuronal activity marker c-Fos. Increased progenitor cell proliferation and progenitor survival persist in aged P2ry13 knockout animals. However, in the ventral dentate gyrus proliferation and expansion levels of progenitor cells did not differ significantly from the wild type. This study strongly supports the notion that extracellular nucleotides significantly contribute to the control of adult neurogenesis in the dentate gyrus in situ. Data in this work suggest that activation of the P2Y13 receptor dampens progenitor cell proliferation, new neuron formation, and neuronal activity. In contrast to several in vitro studies and studies in the SVZ in situ, a contribution of the ATP/ADP-sensitive P2Y1 receptor could not be confirmed in the dentate gyrus in vivo.
To unravel implications of purinergic signaling and P2Y13 receptor action in the control of adult hypothalamic neurogenesis a pilot study was performed. Mice null for P2ry13 revealed increased progenitor cell proliferation at the third ventricle as well as long-term progeny survival and new neuron formation in the hypothalamus. In contrast to results obtained in the dentate gyrus expression of the neuronal activity marker c-Fos was significantly decreased in hypothalamic nuclei, indicating increased inhibition of appetite-regulating neuronal circuits by surplus neurons in knockout animals. These data provide first evidence that extracellular nucleotide signaling contributes to the control of adult hypothalamic neurogenesis in situ. Activation of the P2Y13 receptor inhibits progenitor cell proliferation, long-term survival and neuron formation and therefore controls inhibition of appetite-regulating circuits in the adult rodent hypothalamus.
Synaptic plasticity is the basis for information storage, learning and memory and is achieved by modulation of the synaptic transmission. The amount of active AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazol-propionic acid) receptors at the synapse determines the transmission properties, therefore the regulation of AMPA receptor trafficking affects the synaptic strength. The protein GRIP (glutamate receptor interacting protein) binds to AMPA receptors and is one of the important regulators of AMPA receptor stability at the synapse (Dong et al., 1997; Osten et al., 2000). Previous studies have shown that the ablation of ephrinB2 or ephrinB3 in the nervous system leads to severe defects in hippocampal LTP (long term potentiation) and LTD (long term depression) (Grunwald et al., 2004). We found that ephrinB2 ligands play an important role in the stabilization of AMPA receptors at the cellular membrane (Essmann et al., 2008). Treating cultured hippocampal neurons with AMPA resulted in a robust AMPA receptor internalization, which could be inhibited by simultaneous ephrinB2 activation with soluble EphB4-Fc fusion proteins. Conditional hippocampal ephrinB2 knock-out (KO) neurons showed enhanced constitutive internalization of AMPA receptors. Interaction and interference experiments revealed that ephrinB ligands and AMPA receptors are bridged by GRIP. This interaction is regulated by phosphorylation of a single serine residue in close proximity to the C-terminal PDZ protein target site in ephrinB ligands (Essmann et al., 2008). To investigate the in vivo relevance of this previously undescribed feature of ephrinB reverse signaling, we generated ephrinB2 S-9>A knock-in mice, where the serine at position -9 was replaced by an alanine to prevent phosphorylation. The mutated ephrinB2 of this mouse line was expressed and able to form clusters following stimulation with the preclustered receptor EphB4-Fc. Surface ephrinB2 cluster size and cluster number was slightly smaller in comparison to wild type (WT) mice. Analyzing AMPA receptor internalization, we oserved an increased basal GluR2 endocytosis in cultured hippocampal neurons of ephrinB2 S-9>A mice. Dendrite and spine morphology was similar in pyramidal CA1 neurons of brain slices from adult ephrinB2 S-9>A and WT mice, suggesting a redundancy between the different ephrinB familily members.
Apart from regulating AMPA receptor stability at the synapse, GRIP1 also has an important role in the secretory pathway to deliver cargo proteins along microtubules to dendrites and synapses (Setou et al., 2002). Proteins involved in synaptic transmission and plasticity, as well as lipids required for the outgrowth and remodeling of dendrites and axons have to be transported. We showed in our laboratory with a directed proteomic analysis using the tandem affinity purification-mass spectrometry methodology (Angrand et al., 2006) and with immunoprecipitation assays with brain lysates that the small regulatory protein 14-3-3 interacts with GRIP1. Further immunoprecipitation assays with lysates from HeLa cells transfected with various parts and sequence mutants of GRIP1 revealed that threonine 956 in the linker region L2 between PDZ6 and PDZ7 of GRIP1 is necessary for the interaction with 14-3-3. GRIP1 has been postulated to influence dendritic arborization and maintenance in hippocampal neurons in culture due to defective kinesin-dependent transport along microtubules (Hoogenraad et al 2005). In order to address the role of the association of GRIP1 and 14-3-3 in dendritogenesis, we transfected rat hippocampal neurons with GRIP1-WT and GRIP1 mutants and performed Sholl analysis to evaluate dendritic arborization defects. We could observe striking increased formation and growth of dendrites in developing neurons as well as in mature neurons overexpressing GRIP1-WT. However, overexpression of GRIP1-T956A, where the threonine 956 was replaced by an alanine to prevent phosphorylation, did not show enhanced dendritogenesis, indicating a role for threonine 956 phosphorylation in dendrite branching. To investigate the importance of the interaction between GRIP1 and 14-3-3 in vivo, we generated transgenic mouse lines with a GRIP1-T956A transgene or a GRIP1-WT transgene as control. These mice were crossed with heterozygous GRIP1 mice and by further breedings we obtained some surviver mice carrying either the wild type or the mutated GRIP1 transgene in the usually embryonic lethal GRIP1-KO background (Bladt et al., 2002; Takamiya et al., 2004). In embryonic day (E) 14.5 cultured hippocampal GRIP1-KO neurons we could observe reduced dendritic growth. We also showed reduced GluR2 staining on the dendritic surface in cultured hippocampal neurons from GRIP1-KO and GRIP1-KO neurons containing the GRIP1-T956A transgene. GRIP1-KO neurons containing the GRIP1-WT transgene showed a similar surface GluR2 signal intensity as WT neurons. Reduced surface GluR2 staining in GRIP1-KO neurons and GRIP1-KO neurons with the GRIP1-T956A transgene might be a consequence of defective kinesin-dependent transport of GluR2 to dendrites, indicating an important role of threonine 956 phosphorylation of GRIP1 for GluR2 trafficking.
Fuer die schlechte Prognose von Glioblastompatienten mit einer ueberlebenszeit von 9-15 Monaten (Norden and Wen, 2006) ist vor allem die hohe Invasivitaet dieser Tumore verantwortlich. Nach operativer Entfernung des Haupttumors entstehen aus den verbleibenden invadierten Zellen sekundaere Tumore, die sich mitunter ueber weite Bereiche des Hirns verteilen. Des Weitern sind die hochinvasiven Tumorzellen oft resistent gegen Chemo- und Strahlentherapie (Drappatz et al., 2009; Lefranc et al., 2005). In Maustumormodellen und Pateinten konnte zudem gezeigt werden, dass die neuartige antiangiogenetische Therapie zwar das Tumorwachstum verringert, jedoch die Invasivitaet stark erhoeht. (Norden et al., 2008; Ebos et al., 2009; Paez-Ribes et al., 2009). Ueber die Mechanismen die diese hohen Invasivitaet induzieren, ist bislang nur sehr wenig bekannt. Die durch Reduktion von Blutgefaessen steigende Hypoxie des Tumors foerdert die Expression von Matrix-Metalloproteinasen (MMPs). Dies fuehrt zum Abbau der extrazelluaeren Matrix des umgebenden gesunden Gewebes und beguenstigt dadurch die Tumorzellinvasion (Indelicato et al., 2010; Miyazaki et al., 2008; Shyu et al., 2007). Die Umformung des Aktinzytoskeletts und damit die Mobilitaet von Zellen wird vorwiegend durch ein akkurates Zusammenspeil der Rho GTPasen Rac, Rho und Cdc42, kontrolliert (Ridley et al., 2003). Fuer die Organisation von Axonen im Nervensystem und fuer die Blut- und Lymphgefaessbildung wurde gezeigt, dass die Interaktion der Eph-Rezeptortyrosinkinasen und Ihrer Ephrin-Liganden Signalwege induziert, die in die Regulation dieses Zusammenspiels involviert sind (Egea and Klein, 2007; Makinen et al., 2005; Palmer et al., 2002; Sawamiphak et al., 2010). Des Weiteren zeigt die Analyse der Genloci von Eph-Rezeptoren und Ephrinen in verschieden Hirntumoren eine gehaeufte Deletionen des Ephrin-B2-Gens. Die Quantifizierung von Ephrin-B2 mRNA in diesen Tumoren hat ausserdem ergeben, dass mit zunehmender Malignitaet die Expression von Ephrin-B2 sinkt. Aus diesen Gruenden wurden die Untersuchungen in dieser Arbeit auf die Rolle von Ephrin-B2 anhaengigen Signalwegen in der Glioblastomzellinvasion konzentriert. In einem modifiziertem Boyden-Chamber-Assay konnte gezeigt werden, dass das Ephrin-B2 induzierte EphB4 forward signaling und EphB4 induzierte Ephrin-B2 reverse signaling die Invasivitaet der human Glioblastomzelllinien LN-229, G55 und SNB-19 reduziert. In einem Maustumormodel konnte weiterhin gezeigt werden, dass Ephrin-B2 Knock-Out (KO) Astrozytomzellen, im Vergleich zu Wild-Typ (WT) Zellen, Tumore mit einem groesseren Volumen und einer erhoehten Invasivitaet bilden. Da die Expressionslevel fuer die Ephrin-B2 bindenden Rezeptoren EphA4, EphB1 EphB3 und EphB6 auch im adulten Hirn hoch sind (Hafner et al., 2004), weisen diese in vitro und in vivo Ergebnisse auf eine Tumorsupressorfunktion von Ephrin-B2 hin, die durch repulsive Effekte des Ephrin-B2 reverse signaling vermittelte werden koennten. Dies geht mit Erkenntnissen ueber kolorektale Tumore einher (Batlle et al., 2005). Die in einem Sphaeroid-Invasionsassay mit einer EphB-Rezeptoren freien Umgebung beobachtete verminderte Invasion von Ephrin-B2 WT deutet auf eine zusaetzliche invasionsblockierende Rolle der Ephrin-B2-Eph-Rezeptor Interaktion zwischen benachbarten Tumorzellen hin, wie sie auch in Brusttumoren gefunden wurde (Noren et al., 2006). Es scheint als sei Tumorprogression und Invasion erst moeglich, nachdem die Expression von Ephrin-B2 vermindert wurde. Es konnte weiterhin gezeigt werden, dass in hypoxischen Glioblastomzellen die Ephrin-B2 Expression durch die direkte Bindung des den Transkriptionsfaktors ZEB2 an den Ephrin-B2 Promoter reprimiert wird. In einem Weiteren Maustumormodel konnte gezeigt werden, dass die Blockierung der ZEB2 Expression mittels shRNA und die damit einhergehenden Inhibition der hypoxie induzierten Ephrin-B2 Repression das Wachstum und die Invasivitaet von Glioblastomen verringert. Zusaetzlich wurde gezeigt, dass der Verlust von ZEB2 ausreicht, die durch antiangiogenetische Therapie induzierte stark erhoehte Invasivitaet zu vermeiden. Die in dieser Arbeit gewonnen Erkenntnisse fuehren zu folgendem Modelmechanismus. In kleinen normoxischen Tumoren koennen repulsive Effekte des Ephrin-B2 reverse signalings und EphB forward signalings zwischen Tumorzellen und Zellen des umgebenden Gewebes die Ausbreitung und Invasion des Tumors unterdruecken. Zusaetzlich koennte das Ephrin-B2 induzierte EphB forward signaling zwischen benachbarten Tumorzellen die Mobilitaet der Tumorzellen wie in Brusttumoren inhibieren. Beim Erreichen einer bestimmten Tumorgroesse tritt Hypoxie auf, wodurch HIF-1alpha stabilisiert wird. Dies fuehrt dann zur ZEB2 Expression und leitet die Repression von Ephrin-B2 ein, was wiederum zur erhoehten Tumorzellemobilitaet und im Zusammenspiel mit MMPs zu Invasion fuehren kann. Gleichzeitig werden durch den HIF-induzierten VEGF-Gradienten neue Blutgefaesse rekrutiert. Damit wird der hypoxie-induzierten Invasivitaet entgegengewirkt. Wird mittels antiangiogenetischer Behandlung versucht Tumorprogression entgegenzuwirken, resultiert daraus eine erneut gesteigerte Hypoxie, die dann durch die ZEB2 vermittelte Repression von Ephrin-B2 wieder eine erhoehte Invasivitaet induzieren kann. Das Blockieren der ZEB2 Expression kann dieser durch antiangiogenetischen Behandlung induzierten Invasivitaet entgegenwirken.
Lesion of the rat entorhinal cortex denervates the outer molecular layer of the fascia dentata followed by layer-specific axonal sprouting of uninjured fibers in the denervated zone. One of the candidate molecules regulating the laminar-specific sprouting response in the outer molecular layer is the transmembrane chondroitin sulfate proteoglycan NG2. NG2 is found in glial scars and has been suggested to impede axonal regeneration following injury of the spinal cord. The present study adressed the question whether NG2 could also regulate axonal growth in denervated areas of the brain. Therefore, (1) changes in NG2 mRNA and NG2 protein levels, (2) the cellular and the extracellular localisation of the molecule, (3) the identity of NG2 expressing cells, and (4) the generation of NG2-positive cells were studied in the rat fascia dentata before and following entorhinal deafferentation. Laser microdissection was employed to selectively harvest the denervated molecular layer and combined with quantitative reverse transcription-PCR to measure changes in NG2 mRNA amount (6h, 12h, 2d, 4d, 7d post lesion). The study revealed increases of NG2 mRNA at day 2 (2.5-fold) and day 4 (2-fold) post lesion. Immunocytochemistry was used to detect changes in NG2 protein distribution (1d, 4d, 7d, 10d, 14d, 30d, 6 months post lesion). NG2 staining was increased in the denervated outer molecular layer at 1 day post lesion, reached a maximum at 10 days post lesion, and returned to control levels within 6 month. Interestingly, the accumulation of NG2 protein was strongly restricted to the denervated outer molecular layer forming a border to the unaffected inner molecular layer. Using electron microscopy, NG2-immunoprecipitate was localized not only on glial surfaces and in the extracellular matrix but also in the vicinity of neuronal profiles indicating that NG2 is secreted following denervation. Double-labelings of NG2-immunopositive cells with markers for astrocytes, microglia/macrophages, and oligodendrocytes suggested that NG2-cells are a distinct glial subpopulation before and after entorhinal deafferentation. Bromodeoxyuridine-labeling revealed that some of the NG2-positive cells are postlesional generated. Taken together, the data revealed a layer-specific upregulation of NG2 in the denervated outer molecular layer of the fascia dentata that coincides with the sprouting response of uninjured fibers. This suggests that NG2 could regulate lesion-induced axonal growth in denervated areas of the brain.