JOURNAL OF NEUROCHEMISTRY
| 2014 | 130 | 469–471
,
doi: 10.1111/jnc.12723
,
,
*Division of Fundamental Neurobiology, University Health Network, Toronto, Ontario, Canada †Department of Physiology, University of Toronto, Toronto, Ontario, Canada Read the full article ‘HPC-1/syntaxin 1A and syntaxin 1B play distinct roles in neuronal survival’ on page 514.
Syntaxin-1 is a central component of neuronal soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex (S€ ollner et al. 1993) and believed to play an essential role in neurotransmitter exocytosis. The lack of syntaxin-1 results in an entire loss of both evoked and spontaneous neurotransmitter release in Drosophila (Schulze et al. 1995) and a near complete paralysis in C. elegans (Saifee et al. 1998), causing pre- or perinatal death of the organisms. However, a crucial in vivo function of syntaxin-1 has not been demonstrated in mammals. Dr Akagawa, the discoverer of syntaxin-1A as a marker of early amacrine cell development in rat retina in 1985 (Barnstable et al. 1985), and his colleagues previously generated syntaxin-1A knockout (KO) mice (Fujiwara et al. 2006). The mice surprisingly retained normal life span and basic neurotransmission, whereas their synaptic plasticity such as long-term potentiation was partially impaired (Fujiwara et al. 2006; Mishima et al. 2012). This modest phenotype was considered to be because of the functional redundancy of syntaxin-1A and -1B (Bennett et al. 1992). To address the role of syntaxin-1B in vivo, Dr S€ udhof’s group generated mice (Gerber et al. 2008) harboring syntaxin-1B ‘open’ conformation mutations (L165A/E166A) (Dulubova et al. 1999). The knock-in mice developed lethal seizures after 2 weeks of age regardless of the presence or absence of syntaxin-1A, indicating the importance of syntaxin-1B. Nonetheless, neurons from syntaxin-1A null mice carrying the knock-in ‘open’ conformation mutations of syntaxin-1B exhibited relatively normal neurotransmission with an increased size of readily releasable pool (Gerber et al. 2008), making in vivo function of syntaxin-1B unclear. To answer this question, Kofuji et al. (2014) from Dr Akagawa’s group have now generated syntaxin-1B null mice and analyzed their phenotypes in an article published in this issue of Journal of Neurochemistry. Kofuji et al. (2014) found that almost all syntaxin-1B KO mice died by post-natal day 14 and exhibited motor function
impairments tested by righting reflex, presumably owing to the reduced neurotransmitter release (data not shown). Importantly, the mice displayed several abnormal brain morphologies. These include a thinner molecular layer and a reduced branching of Purkinje cell dendrites in the cerebellum, as well as a smaller number of neurons in the hippocampus. The authors also found that syntaxin-1B KO neurons exhibited reduced viability in culture beginning at 9 DIV, while the morphology of surviving neurons seems no different from other genotypes, suggesting that the surviving ones develop normally. Interestingly, this reduced viability of syntaxin-1B KO neurons was found to be partially rescued by culturing them with wild-type (WT) or syntaxin-1A KO glial feeder layer. Subsequently, similar rescue effects were observed when the authors cultured them with glial conditioned medium from the WT or syntaxin-1A KO glial cells. However, neither glial feeder layer nor glial conditioned medium from syntaxin-1B KO mice ensured neuronal survival, suggesting a lack of support from the glial cells in these mice. In addition, the application of neurotropic factors brain-derived neurotrophic factor (BDNF) and NT-3 to neuronal culture partially restored the survival of syntaxin1B KO neurons. The authors also found that the syntaxin1B-deficient glial cells exhibited perinuclear localization of BDNF-positive puncta with reduced numbers at the cell periphery compared to WT glial cells. At the protein level, Munc18-1, an essential regulator of syntaxin-1 in exocytosis (Han et al. 2010), was decreased in syntaxin-1B-deficient glial cells. Moreover, the release of BDNF was significantly reduced in syntaxin-1B KO glial cells under high potassium stimulation, while BDNF mRNA levels were not altered Received February 26, 2014; accepted March 6, 2014. Address correspondence and reprint requests to Shuzo Sugita, Division of Fundamental Neurobiology, University Health Network, Krembil Discovery Tower, 7KD-419, 60 Leonard Street, Toronto, Ontario, M5T 2S8, Canada. E-mail: [email protected] Abbreviations used: KO, knockout; WT, wild type.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 469--471
469
470
Review
Fig. 1 A schematic model illustrates dual roles of syntaxin-1B in releasing neurotropic factors and neurotransmitters from glial cells and neurons, respectively. Syntaxin-1B, which is a predominant isoform of syntaxin1 in glial cells, forms a non-neuronal SNARE complex with SNAP-23 and VAMP-3 (Trang et al. 2011). The SNARE complex is responsible for the release of neurotropic factors such as BDNF from glial cells to promote neuronal survival. When such release is inhibited in the absence of syntaxin-1B, neurodegeneration occurs. At the pre-synaptic terminal of a neuron, either syntaxin-1A or -1B forms a SNARE complex with SNAP25 and VAMP-2 for neurotransmitter release. Perturbation of neuronal exocytosis or other processes in syntaxin-1B KO could possibly cause neurodegeneration in cell-autonomous manner.
compared to other genotypes. Based on these results, Kofuji et al. (2014) propose that the syntaxin-1B plays a crucial role in neuronal survival by supporting the release of neurotrophic factors from glial cells (Fig. 1). The restoration of syntaxin-1B KO neuronal survival by the glial feeder layer was clearly evident, but these neurons still exhibited reduced survival even in the presence of glial feeder layer and neurotrophic factors when compared with the WT neurons. Therefore, syntaxin-1B seems to play an additional role in neuroprotection in a cell-autonomous manner. An important insight into the requirement of target (t)-SNARE proteins, syntaxin-1 and SNAP-25, in neuroprotection comes from recent analyses of CSPa KO mice by Dr S€udhof’s group. They reported that loss of CSPa in mice causes severe neurodegeneration (Fernandez-Chac on et al. 2004). Later, they discovered that CSPa functions as a molecular chaperone toward SNAP-25 and that in the absence of CSPa, the level of SNAP-25 and the formation of the SNARE complex are severely compromised (Sharma et al. 2011, 2012). Interestingly, it was previously shown that SNAP-25 KO neurons also exhibit a neurodegenerative phenotype (Washbourne et al. 2002). More recently, cleavages of syntaxin-1 and SNAP-25 by botulinum neurotoxins have been demonstrated to cause neurodegeneration without affecting neurotransmitter release (Peng et al. 2013). These results as well as the present study by Kofuji et al. (2014) suggest that appropriate regulations of syntaxin-1 and SNAP-25 by their respective chaperoning proteins may be necessary for the survival of neurons. The underlying mechanism of the cell-autonomous neuroprotective function of syntaxin-1 and SNAP-25 awaits future study.
Many questions about the in vivo function of syntaxin-1 remain unanswered. These include: (i) How severely impaired is the neurotransmitter exocytosis in syntaxin-1B KO neurons? (ii) Are the impairment of neurotransmitter release and neurodegeneration phenotypes seen in syntaxin-1B KO neurons further augmented by the additional syntaxin-1A KO? (iii) Do syntaxin-1A and -1B null mice survive to birth? (iv) Why do syntain-1A and -1B have different roles in neuroprotection and exocytosis? The availability of syntaxin-1A and -1B null mice generated by Dr Akagawa’s group will definitely contribute to further elucidation of the in vivo function of these important synaptic proteins in neuroprotection and exocytosis. Acknowledgments and conflict of interest disclosure The authors have no conflict of interest to declare.
References Barnstable C., Hofstein R. and Akagawa K. (1985) A marker of early amacrine cell development in rat retina. Brain Res. 352, 286–290. Bennett M., Calakos N. and Scheller R. (1992) Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257, 255–259. Dulubova I., Sugita S., Hill S., Hosaka M., Fernandez I., S€udhof T. C. and Rizo J. (1999) A conformational switch in syntaxin during exocytosis: role of munc18. EMBO J. 18, 4372–4382. Fernandez-Chacon R., W€olfel M., Nishimune H. et al. (2004) The synaptic vesicle protein CSP alpha prevents presynaptic degeneration. Neuron 42, 237–251.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 469--471
Review
Fujiwara T., Mishima T., Kofuji T., Chiba T., Tanaka K., Yamamoto A. and Akagawa K. (2006) Analysis of knock-out mice to determine the role of HPC-1/syntaxin 1A in expressing synaptic plasticity. J. Neurosci. 26, 5767–5776. Gerber S., Rah J., Min S. et al. (2008) Conformational switch of syntaxin-1 controls synaptic vesicle fusion. Science 321, 1507–1510. Han G. A., Malintan N. T., Collins B. M., Meunier F. A. and Sugita S. (2010) Munc18-1 as a key regulator of neurosecretion. J. Neurochem. 115, 1–10. Kofuji T., Fujiwara T., Sanada M., Mishima T. and Akagawa K. (2014) HPC-1/syntaxin 1A and syntaxin 1B play distinct roles in neuronal survival. J. Neurochem. 130, 514–525. Mishima T., Fujiwara T., Kofuji T. and Akagawa K. (2012) Impairment of catecholamine systems during induction of long-term potentiation at hippocampal CA1 synapses in HPC-1/syntaxin 1A knock-out mice. J. Neurosci. 32, 381–389. Peng L., Liu H., Ruan H. et al. (2013) Cytotoxicity of botulinum neurotoxins reveals a direct role of syntaxin 1 and SNAP-25 in neuron survival. Nat. Commun. 4, 1472. Saifee O., Wei L. and Nonet M. L. (1998) The Caenorhabditis elegans unc-64 locus encodes a syntaxin that interacts genetically with synaptobrevin. Mol. Biol. Cell 9, 1235–1252.
471
Schulze K., Broadie K., Perin M. and Bellen H. (1995) Genetic and electrophysiological studies of Drosophila syntaxin-1A demonstrate its role in nonneuronal secretion and neurotransmission. Cell 80, 311–320. Sharma M., Burre J. and S€udhof T. C. (2011) CSPa promotes SNAREcomplex assembly by chaperoning SNAP-25 during synaptic activity. Nat. Cell Biol. 13, 30–39. Sharma M., Burre J., Bronk P., Zhang Y., Xu W. and S€udhof T. C. (2012) CSPa knockout causes neurodegeneration by impairing SNAP-25 function. EMBO J. 31, 829–841. S€ollner T., Bennett M., Whiteheart S., Scheller R. and Rothman J. (1993) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75, 409–418. Trang T., Beggs S. and Salter M. W. (2011) Brain-derived neurotrophic factor from microglia: a molecular substrate for neuropathic pain. Neuron Glia Biol. 7, 99–108. Washbourne P., Thompson P., Carta M. et al. (2002) Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis. Nat. Neurosci. 5, 19–26.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 469--471
| 2014 | 130 | 469–471
,
doi: 10.1111/jnc.12723
,
,
*Division of Fundamental Neurobiology, University Health Network, Toronto, Ontario, Canada †Department of Physiology, University of Toronto, Toronto, Ontario, Canada Read the full article ‘HPC-1/syntaxin 1A and syntaxin 1B play distinct roles in neuronal survival’ on page 514.
Syntaxin-1 is a central component of neuronal soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex (S€ ollner et al. 1993) and believed to play an essential role in neurotransmitter exocytosis. The lack of syntaxin-1 results in an entire loss of both evoked and spontaneous neurotransmitter release in Drosophila (Schulze et al. 1995) and a near complete paralysis in C. elegans (Saifee et al. 1998), causing pre- or perinatal death of the organisms. However, a crucial in vivo function of syntaxin-1 has not been demonstrated in mammals. Dr Akagawa, the discoverer of syntaxin-1A as a marker of early amacrine cell development in rat retina in 1985 (Barnstable et al. 1985), and his colleagues previously generated syntaxin-1A knockout (KO) mice (Fujiwara et al. 2006). The mice surprisingly retained normal life span and basic neurotransmission, whereas their synaptic plasticity such as long-term potentiation was partially impaired (Fujiwara et al. 2006; Mishima et al. 2012). This modest phenotype was considered to be because of the functional redundancy of syntaxin-1A and -1B (Bennett et al. 1992). To address the role of syntaxin-1B in vivo, Dr S€ udhof’s group generated mice (Gerber et al. 2008) harboring syntaxin-1B ‘open’ conformation mutations (L165A/E166A) (Dulubova et al. 1999). The knock-in mice developed lethal seizures after 2 weeks of age regardless of the presence or absence of syntaxin-1A, indicating the importance of syntaxin-1B. Nonetheless, neurons from syntaxin-1A null mice carrying the knock-in ‘open’ conformation mutations of syntaxin-1B exhibited relatively normal neurotransmission with an increased size of readily releasable pool (Gerber et al. 2008), making in vivo function of syntaxin-1B unclear. To answer this question, Kofuji et al. (2014) from Dr Akagawa’s group have now generated syntaxin-1B null mice and analyzed their phenotypes in an article published in this issue of Journal of Neurochemistry. Kofuji et al. (2014) found that almost all syntaxin-1B KO mice died by post-natal day 14 and exhibited motor function
impairments tested by righting reflex, presumably owing to the reduced neurotransmitter release (data not shown). Importantly, the mice displayed several abnormal brain morphologies. These include a thinner molecular layer and a reduced branching of Purkinje cell dendrites in the cerebellum, as well as a smaller number of neurons in the hippocampus. The authors also found that syntaxin-1B KO neurons exhibited reduced viability in culture beginning at 9 DIV, while the morphology of surviving neurons seems no different from other genotypes, suggesting that the surviving ones develop normally. Interestingly, this reduced viability of syntaxin-1B KO neurons was found to be partially rescued by culturing them with wild-type (WT) or syntaxin-1A KO glial feeder layer. Subsequently, similar rescue effects were observed when the authors cultured them with glial conditioned medium from the WT or syntaxin-1A KO glial cells. However, neither glial feeder layer nor glial conditioned medium from syntaxin-1B KO mice ensured neuronal survival, suggesting a lack of support from the glial cells in these mice. In addition, the application of neurotropic factors brain-derived neurotrophic factor (BDNF) and NT-3 to neuronal culture partially restored the survival of syntaxin1B KO neurons. The authors also found that the syntaxin1B-deficient glial cells exhibited perinuclear localization of BDNF-positive puncta with reduced numbers at the cell periphery compared to WT glial cells. At the protein level, Munc18-1, an essential regulator of syntaxin-1 in exocytosis (Han et al. 2010), was decreased in syntaxin-1B-deficient glial cells. Moreover, the release of BDNF was significantly reduced in syntaxin-1B KO glial cells under high potassium stimulation, while BDNF mRNA levels were not altered Received February 26, 2014; accepted March 6, 2014. Address correspondence and reprint requests to Shuzo Sugita, Division of Fundamental Neurobiology, University Health Network, Krembil Discovery Tower, 7KD-419, 60 Leonard Street, Toronto, Ontario, M5T 2S8, Canada. E-mail: [email protected] Abbreviations used: KO, knockout; WT, wild type.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 469--471
469
470
Review
Fig. 1 A schematic model illustrates dual roles of syntaxin-1B in releasing neurotropic factors and neurotransmitters from glial cells and neurons, respectively. Syntaxin-1B, which is a predominant isoform of syntaxin1 in glial cells, forms a non-neuronal SNARE complex with SNAP-23 and VAMP-3 (Trang et al. 2011). The SNARE complex is responsible for the release of neurotropic factors such as BDNF from glial cells to promote neuronal survival. When such release is inhibited in the absence of syntaxin-1B, neurodegeneration occurs. At the pre-synaptic terminal of a neuron, either syntaxin-1A or -1B forms a SNARE complex with SNAP25 and VAMP-2 for neurotransmitter release. Perturbation of neuronal exocytosis or other processes in syntaxin-1B KO could possibly cause neurodegeneration in cell-autonomous manner.
compared to other genotypes. Based on these results, Kofuji et al. (2014) propose that the syntaxin-1B plays a crucial role in neuronal survival by supporting the release of neurotrophic factors from glial cells (Fig. 1). The restoration of syntaxin-1B KO neuronal survival by the glial feeder layer was clearly evident, but these neurons still exhibited reduced survival even in the presence of glial feeder layer and neurotrophic factors when compared with the WT neurons. Therefore, syntaxin-1B seems to play an additional role in neuroprotection in a cell-autonomous manner. An important insight into the requirement of target (t)-SNARE proteins, syntaxin-1 and SNAP-25, in neuroprotection comes from recent analyses of CSPa KO mice by Dr S€udhof’s group. They reported that loss of CSPa in mice causes severe neurodegeneration (Fernandez-Chac on et al. 2004). Later, they discovered that CSPa functions as a molecular chaperone toward SNAP-25 and that in the absence of CSPa, the level of SNAP-25 and the formation of the SNARE complex are severely compromised (Sharma et al. 2011, 2012). Interestingly, it was previously shown that SNAP-25 KO neurons also exhibit a neurodegenerative phenotype (Washbourne et al. 2002). More recently, cleavages of syntaxin-1 and SNAP-25 by botulinum neurotoxins have been demonstrated to cause neurodegeneration without affecting neurotransmitter release (Peng et al. 2013). These results as well as the present study by Kofuji et al. (2014) suggest that appropriate regulations of syntaxin-1 and SNAP-25 by their respective chaperoning proteins may be necessary for the survival of neurons. The underlying mechanism of the cell-autonomous neuroprotective function of syntaxin-1 and SNAP-25 awaits future study.
Many questions about the in vivo function of syntaxin-1 remain unanswered. These include: (i) How severely impaired is the neurotransmitter exocytosis in syntaxin-1B KO neurons? (ii) Are the impairment of neurotransmitter release and neurodegeneration phenotypes seen in syntaxin-1B KO neurons further augmented by the additional syntaxin-1A KO? (iii) Do syntaxin-1A and -1B null mice survive to birth? (iv) Why do syntain-1A and -1B have different roles in neuroprotection and exocytosis? The availability of syntaxin-1A and -1B null mice generated by Dr Akagawa’s group will definitely contribute to further elucidation of the in vivo function of these important synaptic proteins in neuroprotection and exocytosis. Acknowledgments and conflict of interest disclosure The authors have no conflict of interest to declare.
References Barnstable C., Hofstein R. and Akagawa K. (1985) A marker of early amacrine cell development in rat retina. Brain Res. 352, 286–290. Bennett M., Calakos N. and Scheller R. (1992) Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science 257, 255–259. Dulubova I., Sugita S., Hill S., Hosaka M., Fernandez I., S€udhof T. C. and Rizo J. (1999) A conformational switch in syntaxin during exocytosis: role of munc18. EMBO J. 18, 4372–4382. Fernandez-Chacon R., W€olfel M., Nishimune H. et al. (2004) The synaptic vesicle protein CSP alpha prevents presynaptic degeneration. Neuron 42, 237–251.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 469--471
Review
Fujiwara T., Mishima T., Kofuji T., Chiba T., Tanaka K., Yamamoto A. and Akagawa K. (2006) Analysis of knock-out mice to determine the role of HPC-1/syntaxin 1A in expressing synaptic plasticity. J. Neurosci. 26, 5767–5776. Gerber S., Rah J., Min S. et al. (2008) Conformational switch of syntaxin-1 controls synaptic vesicle fusion. Science 321, 1507–1510. Han G. A., Malintan N. T., Collins B. M., Meunier F. A. and Sugita S. (2010) Munc18-1 as a key regulator of neurosecretion. J. Neurochem. 115, 1–10. Kofuji T., Fujiwara T., Sanada M., Mishima T. and Akagawa K. (2014) HPC-1/syntaxin 1A and syntaxin 1B play distinct roles in neuronal survival. J. Neurochem. 130, 514–525. Mishima T., Fujiwara T., Kofuji T. and Akagawa K. (2012) Impairment of catecholamine systems during induction of long-term potentiation at hippocampal CA1 synapses in HPC-1/syntaxin 1A knock-out mice. J. Neurosci. 32, 381–389. Peng L., Liu H., Ruan H. et al. (2013) Cytotoxicity of botulinum neurotoxins reveals a direct role of syntaxin 1 and SNAP-25 in neuron survival. Nat. Commun. 4, 1472. Saifee O., Wei L. and Nonet M. L. (1998) The Caenorhabditis elegans unc-64 locus encodes a syntaxin that interacts genetically with synaptobrevin. Mol. Biol. Cell 9, 1235–1252.
471
Schulze K., Broadie K., Perin M. and Bellen H. (1995) Genetic and electrophysiological studies of Drosophila syntaxin-1A demonstrate its role in nonneuronal secretion and neurotransmission. Cell 80, 311–320. Sharma M., Burre J. and S€udhof T. C. (2011) CSPa promotes SNAREcomplex assembly by chaperoning SNAP-25 during synaptic activity. Nat. Cell Biol. 13, 30–39. Sharma M., Burre J., Bronk P., Zhang Y., Xu W. and S€udhof T. C. (2012) CSPa knockout causes neurodegeneration by impairing SNAP-25 function. EMBO J. 31, 829–841. S€ollner T., Bennett M., Whiteheart S., Scheller R. and Rothman J. (1993) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75, 409–418. Trang T., Beggs S. and Salter M. W. (2011) Brain-derived neurotrophic factor from microglia: a molecular substrate for neuropathic pain. Neuron Glia Biol. 7, 99–108. Washbourne P., Thompson P., Carta M. et al. (2002) Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis. Nat. Neurosci. 5, 19–26.
© 2014 International Society for Neurochemistry, J. Neurochem. (2014) 130, 469--471
