Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplas... more Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of...
The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in mela... more The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression.
Mibefradil and NNC-55-0396, tetralol derivatives with a proven -ability to block T-type calcium c... more Mibefradil and NNC-55-0396, tetralol derivatives with a proven -ability to block T-type calcium channels in excitable cells, reduce cancer cell viability in vitro, causing cell death. Furthermore, they reduce tumor growth in preclinical models of Glioblastoma multiforme (GBM), a brain tumor of poor prognosis. Here we found that GBM cells treated with cytotoxic concentrations of NNC-55-0396 paradoxically increased cytosolic calcium levels through the activation of inositol triphosphate receptors (IP3R) and ER stress. We used pharmacological inhibitors and gene silencing to dissect the cell death pathway stimulated by NNC-55-0396 in GBM cell lines and biopsy-derived cultures. Calcium chelation or IP3R inhibition prevented NNC-55-0396-mediated cytotoxicity, indicating that ER calcium efflux is the cause of cell death. Upstream of calcium mobilization, NNC-55-0396 activated the IRE1α arm of the Unfolded Protein Response (UPR) resulting in the nuclear translocation of pro-apoptotic CHOP. Consistent with these findings, silencing IRE1α or JNK1 rescued the cell death elicited by NNC-55-0396. Therefore, we demonstrate that activation of IRE1α and calcium signaling accounts for the cytotoxicity of NNC-55-0396 in GBM cells. The delineation of the signaling pathway that mediates the abrupt cell death triggered by this compound can help the development of new therapies for GBM.
BACKGROUND AND OBJECTIVES: Cutaneous malignant melanoma arises from transformed melanocytes de no... more BACKGROUND AND OBJECTIVES: Cutaneous malignant melanoma arises from transformed melanocytes de novo or from congenital or acquired melanocytic nevi. We have recently reported that T-type Ca 2+ channels (TTCs) are upregulated in human melanoma and play an important role on cell proliferation. The aim of this study was to describe for the first time in formalin-fixed-paraffin-embedded tissue the immunoexpression of TT-Cs in biopsies of normal skin, acquired melanocytic nevi and melanoma, in order to evaluate their role in melanomagenesis and/or tumor progression, their utility as prognostic markers and their possible use in targeted therapies. METHODS: Tissue samples from normal skin, melanocytic nevi and melanoma were subjected to immunohistochemistry for two TT-Cs (Cav3.1, Cav3.2), markers of proliferation (Ki67), cell cycle (Cyclin D1), hypoxia (Glut1), vascularization (CD31) and autophagy (LC3), V600E/BRAF mutation (VE-1) and PTEN. Immunostaining was evaluated by histoscore. In silico analysis was used to assess the prognostic value of TT-Cs over-expression. RESULTS: TT-Cs immunoexpression increased gradually from normal skin to common nevi, dysplastic nevi and melanoma samples, but with differences in distribution of both isoforms. Particularly, Cav3.2 expression was significantly higher in metastatic melanoma than in primary melanoma. Statistical correlation showed a lineal interaction between PTEN-loss/ V600E-BRAF/ Cav3.1/ LC3/ Ki67/ Cyclin D1/ Cav3.2 /Glut1. Disease-free survival (DFS) and global survival (OS) correlated inversely with over-expression of Cav3.2. DFS also correlated inversely with over-expresion of Cav3.1. DISCUSSION: TT-Cs immunoexpression on melanocytic neoplasms 1) is consistent with our previous in vitro studies, 2) appears related to tumor progression, and 3) TT-Cs upregulation can be considered as a prognostic marker using TCGA database. The high expression of Cav3.2 in metastatic melanoma encourages the investigation of the use of TT-Cs blockers in targeted therapies.
Tetanus toxin decreased the: frequency of spontaneous events at the electric organ of Torpedo mar... more Tetanus toxin decreased the: frequency of spontaneous events at the electric organ of Torpedo marmorata. This reduction was up to 70% in poisoned electric organ. According to distribution analysis of miniature end plate currents, only a subpopulation of events which have small amplitudes were recorded after poisoning. Furthermore, isolated cholinergic nerve terminals showed a decrease in VAMP/synaptobrevin when poisoned with tetanus toxin under similar conditions. The relationship between the two effects of the toxin, i.e. inhibition of vesicle exocylEosis and peptidase activity on synaptobrevin, is discussed.
Clostridial neurotoxins (tetanus and botulinum toxins) are potent blockers of neurotransmitter re... more Clostridial neurotoxins (tetanus and botulinum toxins) are potent blockers of neurotransmitter release. These toxins act specifically on the nervous system by interacting with still non-identified protein receptors together with gangliosides. Whereas many biochemical data are available on their binding properties to neuronal membranes in vitro, there is poor morphological evidence of their binding to mammalian central nervous system. In the present study, the binding of tetanus and botulinum neurotoxin type A to rat brain sections is reported. Both toxins bound to nerve terminals with a broad distribution in brain. Tetanus toxin additionally bound to nerve fibres. The staining patterns were clearly shown to be due to the interaction of the heavy chains, which contain the binding moiety, with the tissue. In an attempt to investigate the nature of the acceptors present in the tissue, some sections were pre-incubated with periodic acid. This treatment resulted in the additional binding of botulinum neurotoxin type A to nerve fibres. Since the extended staining of nerve terminals was not modified by this pretreatment, it is suggested that protein receptors of clostridial neurotoxins are located at the nerve terminals, which may be common constituents of the synapses.
Synaptobrevin, SNAP-25 and syntaxin (SNAP receptor proteins) are molecular components that play a... more Synaptobrevin, SNAP-25 and syntaxin (SNAP receptor proteins) are molecular components that play a key role in the exocytotic machinery of synaptic vesicles. Their presence, distribution and interactions are reported in central and peripheral nervous systems of the electric fish Torpedo marmorata. These three proteins form a protein complex in all the nervous system regions tested, including the electric lobe and the electric organ which is innervated by pure cholinergic nerve terminals. Immunoblot analysis revealed a double protein pattern of SNAP-25 in the anterior brain and cerebellum, although a single protein band corresponding to SNAP-25 was observed in the electromotor system. Moreover, SNAP-25 showed a differential distribution in the electromotor system. It was present along nerve fibres and terminals that innervated the electric organ but it was not detected in nerve terminals at the electric lobe. Immunoisolation experiments using anti-synaptobrevin antibodies showed a tissue-specific co-existence of SNAP-25 and syntaxin with synaptobrevin in the immunoisolated organelles. In conclusion, the molecular components of the exocytotic machinery are shown to be conserved in Torpedo, although some differences mainly on SNAP-25, suggest a potential diversity in the regulation of neurosecretion.
WNT-CTNN1B signaling promotes cancer cell proliferation and stemness. Furthermore, recent evidenc... more WNT-CTNN1B signaling promotes cancer cell proliferation and stemness. Furthermore, recent evidence indicates that macroautophagy/autophagy regulates WNT signaling. Here we investigated the impact of inhibiting WNT signaling on autophagy in glioblastoma (GBM), a devastating brain tumor. Inhibiting TCF, or silencing TCF4 or CTNNB1/β-catenin upregulated SQSTM1/p62 in GBM at transcriptional and protein levels and, in turn, autophagy. DKK1/Dickkopf1, a canonical WNT receptor antagonist, also induced autophagic flux. Importantly, TCF inhibition regulated autophagy through MTOR inhibition and dephosphorylation, and nuclear translocation of TFEB, a master regulator of lysosomal biogenesis and autophagy. TCF inhibition or silencing additionally affected GBM cell proliferation and migration. Autophagy induction followed by its blockade can promote cancer cell death. In agreement with this notion, halting both TCF-CTNNB1 and autophagy pathways decreased cell viability and induced apoptosis of GBM cells through a SQSTM1-dependent mechanism involving CASP8 (caspase 8). In vivo experiments further underline the therapeutic potential of such dual targeting in GBM.
Synaptotagmins constitute a large family of membrane proteins characterized by their distinct dis... more Synaptotagmins constitute a large family of membrane proteins characterized by their distinct distributions and different biochemical features. Genetic evidence suggests that members of this protein family are likely to function as calcium sensors in calcium-regulated events in neurons, although the precise molecular mechanism remains ill defined. Here we demonstrate that different synaptotagmin isoforms (Syt I, II, and IV) are present in the same synaptic vesicle population from rat brain cortex. In addition, Syt I and II co-localize on the same small synaptic vesicle (SSV), and they heterodimerize in the presence of calcium with a concentration dependence resembling that of the starting phase of SSV exocytosis (EC50 5 6 6 4 mM). The association between Syt I and Syt II was demonstrated by immunoprecipitation of the native proteins and the recombinant cytoplasmic domains and by using fluorescence resonance energy transfer (FRET). Although a subpopulation of SSV containing Syt I and...
... Shona L. OSBORNE and Claire L. THOMAS Molecular NeuroPathobiology Laboratory: Imperial Cancer... more ... Shona L. OSBORNE and Claire L. THOMAS Molecular NeuroPathobiology Laboratory: Imperial Cancer Research Fund, 44 LincoIn's Inn Fields, London WC2A 3PX ... progress to the exocytic phase in the presence of a suitable concentration of Ca2'1up to 200 jiM1, generated by ...
dorsal root ganglia (DRG) to specific target neurons in Imperial College of Science, Technology t... more dorsal root ganglia (DRG) to specific target neurons in Imperial College of Science, Technology the spinal cord (Windle and Baxter, 1936; Mirnics and and Medicine Koerber, 1995; Ozaki and Snider, 1997). Each class of London SW7 AY sensory neurons has a characteristic dorso-ventral pro-2 MRC Centre for Developmental Neurobiology jection. Nerve growth factor (NGF)-dependent skin ther-King's College London moreceptive/nociceptive sensory neurons make central London SE1 1UL connections with cells of the dorsal horn (Figure 1B) United Kingdom (Crowley et al., 1994; Ruit et al., 1992; Smeyne et al., 1994). Slit2 regulates axonal extension and collateralization of NGF-dependent DRG sensory neurons (Wang et Summary al., 1999). On the other hand, proprioceptive sensory neurons projecting to the ventral spinal cord (VSC) are Sensory axons from dorsal root ganglia neurons are of two subtypes: group Ia afferents that carry signals guided to spinal targets by molecules differentially exfrom muscle spindles to motoneurons (MNs; Figure 1B) pressed along the dorso-ventral axis of the neural tube. and group Ib afferents that connect Golgi tendon organs NT-3-responsive muscle afferents project ventrally, to interneurons in the ventral horn (Light and Perl, 1979; cease extending, and branch upon contact with moto-Brown, 1981; Eide and Glover, 1997; Ozaki and Snider, neurons (MNs), their synaptic partners. We have iden-1997). Thermoreceptive and nociceptive axons, those tified WNT-3 as a candidate molecule that regulates that terminate in the dorsal-most layers of the spinal this process. Wnt-3 is expressed by MNs of the lateral cord, are repelled by members of the collapsin/semamotor column at the time when MNs form synapses phorin family expressed in the VSC (Fan and Raper, with sensory neurons. WNT-3 increases branching and 1995; Giger et al., 1996; Luo et al., 1995; Messersmith growth cone size while inhibiting axonal extension in et al., 1995; Puschel et al., 1995; Wright et al., 1995). NT-3-but not NGF-responsive axons. Ventral spinal However, muscle afferents that are neurotrophin-3 (NTcord secretes factors with axonal remodeling activity 3)-dependent (Ernfors et al., 1994; Hory-Lee et al., 1993; for NT-3-responsive neurons. This activity is present Klein et al., 1994; Tessarollo et al., 1994) are not inhibited at limb levels and is blocked by a WNT antagonist. We (Shepherd et al., 1997) and enter the ventral territory. The propose that WNT-3, expressed by MNs, acts as a stereotypic pattern of projections of muscle afferents retrograde signal that controls terminal arborization suggests that VSC factors, such as F11 (Perrin et al., of muscle afferents. 2001), regulate their pathfinding. Muscle Ia afferents branch as they enter the ventral horn and, upon contact Introduction with MNs, cease extending and form synaptic boutons (Kudo and Yamada, 1987; Chen and Frank, 1999). Ex-The formation of neuronal connections requires neurons plant experiments suggest that VSC provides stop and to project to their appropriate synaptic partners and to branching signals for Ia afferents (Sharma and Frank, make functional synapses. This process is initiated 1998). However, the molecular identities of ventral horn when neurons begin to send axons in search of their signals that control the terminal arborization of Ia affertargets. Upon reaching the target field, axons branch, ents remain largely unknown. cease extending, and their growth cones differentiate Recent studies suggest that members of the WNT into presynaptic terminals. A number of attractive and family of signaling proteins play a role in the formation repulsive signals have been shown to regulate the beof neuronal connections. WNT-7A, expressed by cerehavior of axons in transit to their targets (Brose and bellar granule cells, induces growth cone enlargement,
β-catenin is a central component of adherent junctions and a key effector of canonical Wnt signal... more β-catenin is a central component of adherent junctions and a key effector of canonical Wnt signalling, in which dephosphorylated Ser/Thr β-catenin regulates gene transcription. β-catenin phosphorylation at Tyr142 (PTyr142 β-catenin), which is induced by receptor and Src family Tyr kinases, represents a previously described βcatenin switch from adhesive to migratory roles. In addition to classical β-catenin roles, phosphorylated Ser/Thr β-catenin and total β-catenin were involved in centrosomal functions, including mitotic spindle formation and centrosome separation. Here we find that PTyr142 β-catenin is present in centrosomes in non-transformed and glioblastoma cells and that, in contrast to the Ser/Thr phosphorylated β-catenin, PTyr142 β-catenin centrosomal levels drop in mitosis. Furthermore, we show that the inhibitor of Spleen Tyrosine Kinase (Syk) piceatannol decreases centrosomal PTyr142 β-catenin levels, indicating that Syk regulates centrosome PTyr142 β-catenin. Our findings suggest that PTyr142 β-catenin and Syk may regulate centrosomal cohesion. This study highlights 2 the contribution of different phosphorylated β-catenin forms to the cell and centrosome cycles.
Focal adhesion kinase (FAK) is a central component of focal adhesions that regulate cancer cell p... more Focal adhesion kinase (FAK) is a central component of focal adhesions that regulate cancer cell proliferation and migration. Here, we studied the effects of FAK inhibition in glioblastoma (GBM), a fast growing brain tumor that has a poor prognosis. Treating GBM cells with the FAK inhibitor PF-573228 induced a proliferative arrest and increased cell size. PF-573228 also reduced the growth of GBM neurospheres. These effects were associated with increased p27/CDKN1B levels and β-galactosidase activity, compatible with acquisition of senescence. Interestingly, FAK inhibition repressed the expression of the autophagy cargo receptor p62/SQSTM-1. Moreover, depleting p62 in GBM cells also induced a senescent-like phenotype through transcriptional upregulation of p27. Our results indicate that FAK inhibition arrests GBM cell proliferation, resulting in cell senescence, and pinpoint p62 as being key to this process. These findings highlight the possible therapeutic value of targeting FAK in GBM.
Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplas... more Glioblastoma (GBM) is the most common tumor in the central nervous system in adults. This neoplasia shows a high capacity of growth and spreading to the surrounding brain tissue, hindering its complete surgical resection. Therefore, the finding of new antitumor therapies for GBM treatment is a priority. We have previously described that cyclin D1-CDK4 promotes GBM dissemination through the activation of the small GTPases RalA and RalB. In this paper, we show that RalB GTPase is upregulated in primary GBM cells. We found that the downregulation of Ral GTPases, mainly RalB, prevents the proliferation of primary GBM cells and triggers a senescence-like response. Moreover, downregulation of RalA and RalB reduces the viability of GBM cells growing as tumorspheres, suggesting a possible role of these GTPases in the survival of GBM stem cells. By using mouse subcutaneous xenografts, we have corroborated the role of RalB in GBM growth in vivo. Finally, we have observed that the knockdown of...
The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in mela... more The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression.
Mibefradil and NNC-55-0396, tetralol derivatives with a proven -ability to block T-type calcium c... more Mibefradil and NNC-55-0396, tetralol derivatives with a proven -ability to block T-type calcium channels in excitable cells, reduce cancer cell viability in vitro, causing cell death. Furthermore, they reduce tumor growth in preclinical models of Glioblastoma multiforme (GBM), a brain tumor of poor prognosis. Here we found that GBM cells treated with cytotoxic concentrations of NNC-55-0396 paradoxically increased cytosolic calcium levels through the activation of inositol triphosphate receptors (IP3R) and ER stress. We used pharmacological inhibitors and gene silencing to dissect the cell death pathway stimulated by NNC-55-0396 in GBM cell lines and biopsy-derived cultures. Calcium chelation or IP3R inhibition prevented NNC-55-0396-mediated cytotoxicity, indicating that ER calcium efflux is the cause of cell death. Upstream of calcium mobilization, NNC-55-0396 activated the IRE1α arm of the Unfolded Protein Response (UPR) resulting in the nuclear translocation of pro-apoptotic CHOP. Consistent with these findings, silencing IRE1α or JNK1 rescued the cell death elicited by NNC-55-0396. Therefore, we demonstrate that activation of IRE1α and calcium signaling accounts for the cytotoxicity of NNC-55-0396 in GBM cells. The delineation of the signaling pathway that mediates the abrupt cell death triggered by this compound can help the development of new therapies for GBM.
BACKGROUND AND OBJECTIVES: Cutaneous malignant melanoma arises from transformed melanocytes de no... more BACKGROUND AND OBJECTIVES: Cutaneous malignant melanoma arises from transformed melanocytes de novo or from congenital or acquired melanocytic nevi. We have recently reported that T-type Ca 2+ channels (TTCs) are upregulated in human melanoma and play an important role on cell proliferation. The aim of this study was to describe for the first time in formalin-fixed-paraffin-embedded tissue the immunoexpression of TT-Cs in biopsies of normal skin, acquired melanocytic nevi and melanoma, in order to evaluate their role in melanomagenesis and/or tumor progression, their utility as prognostic markers and their possible use in targeted therapies. METHODS: Tissue samples from normal skin, melanocytic nevi and melanoma were subjected to immunohistochemistry for two TT-Cs (Cav3.1, Cav3.2), markers of proliferation (Ki67), cell cycle (Cyclin D1), hypoxia (Glut1), vascularization (CD31) and autophagy (LC3), V600E/BRAF mutation (VE-1) and PTEN. Immunostaining was evaluated by histoscore. In silico analysis was used to assess the prognostic value of TT-Cs over-expression. RESULTS: TT-Cs immunoexpression increased gradually from normal skin to common nevi, dysplastic nevi and melanoma samples, but with differences in distribution of both isoforms. Particularly, Cav3.2 expression was significantly higher in metastatic melanoma than in primary melanoma. Statistical correlation showed a lineal interaction between PTEN-loss/ V600E-BRAF/ Cav3.1/ LC3/ Ki67/ Cyclin D1/ Cav3.2 /Glut1. Disease-free survival (DFS) and global survival (OS) correlated inversely with over-expression of Cav3.2. DFS also correlated inversely with over-expresion of Cav3.1. DISCUSSION: TT-Cs immunoexpression on melanocytic neoplasms 1) is consistent with our previous in vitro studies, 2) appears related to tumor progression, and 3) TT-Cs upregulation can be considered as a prognostic marker using TCGA database. The high expression of Cav3.2 in metastatic melanoma encourages the investigation of the use of TT-Cs blockers in targeted therapies.
Tetanus toxin decreased the: frequency of spontaneous events at the electric organ of Torpedo mar... more Tetanus toxin decreased the: frequency of spontaneous events at the electric organ of Torpedo marmorata. This reduction was up to 70% in poisoned electric organ. According to distribution analysis of miniature end plate currents, only a subpopulation of events which have small amplitudes were recorded after poisoning. Furthermore, isolated cholinergic nerve terminals showed a decrease in VAMP/synaptobrevin when poisoned with tetanus toxin under similar conditions. The relationship between the two effects of the toxin, i.e. inhibition of vesicle exocylEosis and peptidase activity on synaptobrevin, is discussed.
Clostridial neurotoxins (tetanus and botulinum toxins) are potent blockers of neurotransmitter re... more Clostridial neurotoxins (tetanus and botulinum toxins) are potent blockers of neurotransmitter release. These toxins act specifically on the nervous system by interacting with still non-identified protein receptors together with gangliosides. Whereas many biochemical data are available on their binding properties to neuronal membranes in vitro, there is poor morphological evidence of their binding to mammalian central nervous system. In the present study, the binding of tetanus and botulinum neurotoxin type A to rat brain sections is reported. Both toxins bound to nerve terminals with a broad distribution in brain. Tetanus toxin additionally bound to nerve fibres. The staining patterns were clearly shown to be due to the interaction of the heavy chains, which contain the binding moiety, with the tissue. In an attempt to investigate the nature of the acceptors present in the tissue, some sections were pre-incubated with periodic acid. This treatment resulted in the additional binding of botulinum neurotoxin type A to nerve fibres. Since the extended staining of nerve terminals was not modified by this pretreatment, it is suggested that protein receptors of clostridial neurotoxins are located at the nerve terminals, which may be common constituents of the synapses.
Synaptobrevin, SNAP-25 and syntaxin (SNAP receptor proteins) are molecular components that play a... more Synaptobrevin, SNAP-25 and syntaxin (SNAP receptor proteins) are molecular components that play a key role in the exocytotic machinery of synaptic vesicles. Their presence, distribution and interactions are reported in central and peripheral nervous systems of the electric fish Torpedo marmorata. These three proteins form a protein complex in all the nervous system regions tested, including the electric lobe and the electric organ which is innervated by pure cholinergic nerve terminals. Immunoblot analysis revealed a double protein pattern of SNAP-25 in the anterior brain and cerebellum, although a single protein band corresponding to SNAP-25 was observed in the electromotor system. Moreover, SNAP-25 showed a differential distribution in the electromotor system. It was present along nerve fibres and terminals that innervated the electric organ but it was not detected in nerve terminals at the electric lobe. Immunoisolation experiments using anti-synaptobrevin antibodies showed a tissue-specific co-existence of SNAP-25 and syntaxin with synaptobrevin in the immunoisolated organelles. In conclusion, the molecular components of the exocytotic machinery are shown to be conserved in Torpedo, although some differences mainly on SNAP-25, suggest a potential diversity in the regulation of neurosecretion.
WNT-CTNN1B signaling promotes cancer cell proliferation and stemness. Furthermore, recent evidenc... more WNT-CTNN1B signaling promotes cancer cell proliferation and stemness. Furthermore, recent evidence indicates that macroautophagy/autophagy regulates WNT signaling. Here we investigated the impact of inhibiting WNT signaling on autophagy in glioblastoma (GBM), a devastating brain tumor. Inhibiting TCF, or silencing TCF4 or CTNNB1/β-catenin upregulated SQSTM1/p62 in GBM at transcriptional and protein levels and, in turn, autophagy. DKK1/Dickkopf1, a canonical WNT receptor antagonist, also induced autophagic flux. Importantly, TCF inhibition regulated autophagy through MTOR inhibition and dephosphorylation, and nuclear translocation of TFEB, a master regulator of lysosomal biogenesis and autophagy. TCF inhibition or silencing additionally affected GBM cell proliferation and migration. Autophagy induction followed by its blockade can promote cancer cell death. In agreement with this notion, halting both TCF-CTNNB1 and autophagy pathways decreased cell viability and induced apoptosis of GBM cells through a SQSTM1-dependent mechanism involving CASP8 (caspase 8). In vivo experiments further underline the therapeutic potential of such dual targeting in GBM.
Synaptotagmins constitute a large family of membrane proteins characterized by their distinct dis... more Synaptotagmins constitute a large family of membrane proteins characterized by their distinct distributions and different biochemical features. Genetic evidence suggests that members of this protein family are likely to function as calcium sensors in calcium-regulated events in neurons, although the precise molecular mechanism remains ill defined. Here we demonstrate that different synaptotagmin isoforms (Syt I, II, and IV) are present in the same synaptic vesicle population from rat brain cortex. In addition, Syt I and II co-localize on the same small synaptic vesicle (SSV), and they heterodimerize in the presence of calcium with a concentration dependence resembling that of the starting phase of SSV exocytosis (EC50 5 6 6 4 mM). The association between Syt I and Syt II was demonstrated by immunoprecipitation of the native proteins and the recombinant cytoplasmic domains and by using fluorescence resonance energy transfer (FRET). Although a subpopulation of SSV containing Syt I and...
... Shona L. OSBORNE and Claire L. THOMAS Molecular NeuroPathobiology Laboratory: Imperial Cancer... more ... Shona L. OSBORNE and Claire L. THOMAS Molecular NeuroPathobiology Laboratory: Imperial Cancer Research Fund, 44 LincoIn's Inn Fields, London WC2A 3PX ... progress to the exocytic phase in the presence of a suitable concentration of Ca2'1up to 200 jiM1, generated by ...
dorsal root ganglia (DRG) to specific target neurons in Imperial College of Science, Technology t... more dorsal root ganglia (DRG) to specific target neurons in Imperial College of Science, Technology the spinal cord (Windle and Baxter, 1936; Mirnics and and Medicine Koerber, 1995; Ozaki and Snider, 1997). Each class of London SW7 AY sensory neurons has a characteristic dorso-ventral pro-2 MRC Centre for Developmental Neurobiology jection. Nerve growth factor (NGF)-dependent skin ther-King's College London moreceptive/nociceptive sensory neurons make central London SE1 1UL connections with cells of the dorsal horn (Figure 1B) United Kingdom (Crowley et al., 1994; Ruit et al., 1992; Smeyne et al., 1994). Slit2 regulates axonal extension and collateralization of NGF-dependent DRG sensory neurons (Wang et Summary al., 1999). On the other hand, proprioceptive sensory neurons projecting to the ventral spinal cord (VSC) are Sensory axons from dorsal root ganglia neurons are of two subtypes: group Ia afferents that carry signals guided to spinal targets by molecules differentially exfrom muscle spindles to motoneurons (MNs; Figure 1B) pressed along the dorso-ventral axis of the neural tube. and group Ib afferents that connect Golgi tendon organs NT-3-responsive muscle afferents project ventrally, to interneurons in the ventral horn (Light and Perl, 1979; cease extending, and branch upon contact with moto-Brown, 1981; Eide and Glover, 1997; Ozaki and Snider, neurons (MNs), their synaptic partners. We have iden-1997). Thermoreceptive and nociceptive axons, those tified WNT-3 as a candidate molecule that regulates that terminate in the dorsal-most layers of the spinal this process. Wnt-3 is expressed by MNs of the lateral cord, are repelled by members of the collapsin/semamotor column at the time when MNs form synapses phorin family expressed in the VSC (Fan and Raper, with sensory neurons. WNT-3 increases branching and 1995; Giger et al., 1996; Luo et al., 1995; Messersmith growth cone size while inhibiting axonal extension in et al., 1995; Puschel et al., 1995; Wright et al., 1995). NT-3-but not NGF-responsive axons. Ventral spinal However, muscle afferents that are neurotrophin-3 (NTcord secretes factors with axonal remodeling activity 3)-dependent (Ernfors et al., 1994; Hory-Lee et al., 1993; for NT-3-responsive neurons. This activity is present Klein et al., 1994; Tessarollo et al., 1994) are not inhibited at limb levels and is blocked by a WNT antagonist. We (Shepherd et al., 1997) and enter the ventral territory. The propose that WNT-3, expressed by MNs, acts as a stereotypic pattern of projections of muscle afferents retrograde signal that controls terminal arborization suggests that VSC factors, such as F11 (Perrin et al., of muscle afferents. 2001), regulate their pathfinding. Muscle Ia afferents branch as they enter the ventral horn and, upon contact Introduction with MNs, cease extending and form synaptic boutons (Kudo and Yamada, 1987; Chen and Frank, 1999). Ex-The formation of neuronal connections requires neurons plant experiments suggest that VSC provides stop and to project to their appropriate synaptic partners and to branching signals for Ia afferents (Sharma and Frank, make functional synapses. This process is initiated 1998). However, the molecular identities of ventral horn when neurons begin to send axons in search of their signals that control the terminal arborization of Ia affertargets. Upon reaching the target field, axons branch, ents remain largely unknown. cease extending, and their growth cones differentiate Recent studies suggest that members of the WNT into presynaptic terminals. A number of attractive and family of signaling proteins play a role in the formation repulsive signals have been shown to regulate the beof neuronal connections. WNT-7A, expressed by cerehavior of axons in transit to their targets (Brose and bellar granule cells, induces growth cone enlargement,
β-catenin is a central component of adherent junctions and a key effector of canonical Wnt signal... more β-catenin is a central component of adherent junctions and a key effector of canonical Wnt signalling, in which dephosphorylated Ser/Thr β-catenin regulates gene transcription. β-catenin phosphorylation at Tyr142 (PTyr142 β-catenin), which is induced by receptor and Src family Tyr kinases, represents a previously described βcatenin switch from adhesive to migratory roles. In addition to classical β-catenin roles, phosphorylated Ser/Thr β-catenin and total β-catenin were involved in centrosomal functions, including mitotic spindle formation and centrosome separation. Here we find that PTyr142 β-catenin is present in centrosomes in non-transformed and glioblastoma cells and that, in contrast to the Ser/Thr phosphorylated β-catenin, PTyr142 β-catenin centrosomal levels drop in mitosis. Furthermore, we show that the inhibitor of Spleen Tyrosine Kinase (Syk) piceatannol decreases centrosomal PTyr142 β-catenin levels, indicating that Syk regulates centrosome PTyr142 β-catenin. Our findings suggest that PTyr142 β-catenin and Syk may regulate centrosomal cohesion. This study highlights 2 the contribution of different phosphorylated β-catenin forms to the cell and centrosome cycles.
Focal adhesion kinase (FAK) is a central component of focal adhesions that regulate cancer cell p... more Focal adhesion kinase (FAK) is a central component of focal adhesions that regulate cancer cell proliferation and migration. Here, we studied the effects of FAK inhibition in glioblastoma (GBM), a fast growing brain tumor that has a poor prognosis. Treating GBM cells with the FAK inhibitor PF-573228 induced a proliferative arrest and increased cell size. PF-573228 also reduced the growth of GBM neurospheres. These effects were associated with increased p27/CDKN1B levels and β-galactosidase activity, compatible with acquisition of senescence. Interestingly, FAK inhibition repressed the expression of the autophagy cargo receptor p62/SQSTM-1. Moreover, depleting p62 in GBM cells also induced a senescent-like phenotype through transcriptional upregulation of p27. Our results indicate that FAK inhibition arrests GBM cell proliferation, resulting in cell senescence, and pinpoint p62 as being key to this process. These findings highlight the possible therapeutic value of targeting FAK in GBM.
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Papers by Judit Herreros