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Schizophrenia and Synapse: Emerging Role of Presynaptic Fusion Machinery
COMMENTARY
Schizophrenia and Synapse: Emerging Role of Presynaptic Fusion Machinery Mikhail Khvotchev
B
illions of neurons in the brain communicate with each other via specialized junctions called synapses. Each neuron forms thousands of synaptic connections that integrate into complex neural networks responsible for information processing, memory storage, and other fundamental brain functions. Not surprisingly, the dysfunction of synapses has emerged as a major etiologic factor in many brain diseases including widespread neurodegenerative (Alzheimer’s, Parkinsonism), neurodevelopmental (autism), and psychiatric conditions (schizophrenia, depression). Synapses are morphologically and functionally distinct; they undergo rapid transient or long-lasting changes but share basic architecture and principles of function. Each synapse consists of a presynaptic and a postsynaptic terminal separated by a narrow synaptic cleft. Presynaptic terminal is filled with hundreds of synaptic vesicles loaded with a neurotransmitter. Arrival of an action potential triggers rapid fusion of synaptic vesicles within the specialized area of the presynaptic plasma membrane called the active zone and release of the neurotransmitter. Fusion of synaptic vesicles is a probabilistic, yet tightly controlled, event that is in most cases highly synchronized with calcium entry through the voltage-gated calcium channels. Released neurotransmitter is next detected by clustered neurotransmitter receptors of the postsynaptic terminal. At synapses, the minimal fusion machinery consists of three soluble N-ethylmalemide-sensitive factor attachment protein receptor (SNARE) proteins: syntaxin-1 and synaptosome-associated protein of 25 kD (SNAP-25) engaged in the binary complex on the plasma membrane and synaptobrevin-2/vesicle-associated membrane protein 2 on the synaptic vesicles. These proteins form stable core complex in a process that brings two membranes into close proximity and overcomes the energy barrier for fusion (1). Multiple additional proteins are required to control the availability of syntaxin-1 for the SNARE complex formation (Munc13s, Munc18s), to synchronize membrane fusion with transient calcium ion influx (synaptotagmins, complexins), and to provide temporal and spatial regulation (Rab proteins, active zone proteins) (2). The complex network of protein interactions involved in the membrane fusion at the synapse is highly dynamic and plastic, yet little is known about the mechanisms of their regulation. Protein phosphorylation is one established mechanism to regulate protein interactions in vivo. Most of the protein constituents of the synaptic fusion machinery are known substrates for multiple protein kinases (3). Some of the most abundant synaptic proteins undergo activity-dependent cycles of phosphorylation and dephosphorylation; however, their functional implications remain largely untested in vivo. Syntaxin-1 is one of the key hubs for the regulation of synaptic fusion machinery. It adopts an From the Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas. Address correspondence to Mikhail Khvotchev, Ph.D., Department of Neuroscience, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9111; E-mail: mkhvoc@mednet. swmed.edu. Received Dec 2, 2009; revised Dec 7, 2009; accepted Dec 11, 2009.
0006-3223/10/$36.00 doi:10.1016/j.biopsych.2009.12.007
open conformation that is compatible with the SNARE complex formation and an inhibitory closed conformation. These conformational states of syntaxin-1 are regulated by an essential element of the fusion machinery, a Sec1/Munc18 (SM) protein Munc18 that can both stabilize the closed conformation or interact with syntaxin-1 engaged in SNARE complex (4,5). Syntaxin-1 is constitutively phosphorylated by protein kinase casein kinase II (CK2) at the N-terminal serine-14 (6). This modification is of very low abundance in embryonic brain but gradually increases during postnatal development to 40% of the total protein levels in adults. Interestingly, the serine-14 phosphorylation of syntaxin-1 appears to be insensitive to acute changes in activity at the synapse. Phosphorylated syntaxin-1 has a higher propensity for binding to the plasma membrane SNARE protein SNAP-25 but not for the synaptic vesicle SNARE synaptobrevin-2. Ser-14 phosphorylated syntaxin-1 localizes predominantly to the extrasynaptic areas of axons, raising the possibility that the modified syntaxin-1 may be involved in extrasynaptic membrane fusion (Figure 1). Recent in vitro studies suggested that phosphorylation at Ser-14 interferes with permissive complex of Munc18 and syntaxin-1 but has no effect on the inhibitory complex of Munc18 and closed syntaxin-1 (7). One of the powerful molecular analyses available to study pathological processes in human brain involves protein profiling in postmortem brain tissue. This approach, however, critically depends on high-quality representative selection of tissue samples to control for differences related to individuals, postmortem handling of the tissue, effects of chronic drug treatment, etc. These considerations are especially important when labile posttranslational modifications such as phosphorylation are analyzed. Previous studies have identified multiple abnormalities in the levels and the phosphorylation status of synaptic proteins in the brains (mostly cortex) of schizophrenic patients. These include both presynaptic (SNAP-25) and postsynaptic proteins (glutamate receptors) (8,9). Syntaxin-1 levels are normal in patients with schizophrenia; however, the levels and activity of protein kinase CK2 are reduced. This prompted Castillo et al. (10) to investigate the phosphorylation status of syntaxin-1 in schizophrenic patients. Using a newly raised antibody that selectively recognizes the Ser-14 phosphorylated syntaxin-1 and variety of other antibodies, the authors confirmed that CK2 levels were decreased to 75% of the control levels in the cortices from schizophrenic patients. In turn, total levels of syntaxin-1 were not changed, but Ser-14 phosphorylated syntaxin-1 was reduced by almost 25%. Importantly, the changes in phosphorylation were not global, because the phosphorylation status of rabphilin, another abundant presynaptic phosphoprotein, was unchanged in schizophrenic patients. Phosphorylation is a highly labile protein modification that can rapidly deteriorate in postmortem tissues. The authors also showed that in rat brains the Ser-14 phosphorylation of syntaxin-1 dropped significantly during the first 8 hours after death but remained relatively stable thereafter up to 20 hours. This result suggests that human tissue samples may also be productively compared if they are collected within the similar time frame. Is the change in syntaxin-1 phosphorylation specific for BIOL PSYCHIATRY 2010;67:197–198 © 2010 Society of Biological Psychiatry
198 BIOL PSYCHIATRY 2010;67:197–198
Commentary
Presynaptic terminal
SNAP-25
N
N
Syntaxin-1 SNAP-25 N
Syntaxin-1
SV
Synaptobrevin/ VAMP
Synaptobrevin/ VAMP N
SV
Extrasynaptic axonal compartment
N
Active zone
Presynaptic neurotransmitter release
N
P
P
Munc18
N
Axonal plasma membrane
Extrasynaptic neurotransmitter release
Figure 1. A model for syntaxin-1 function in neurotransmitter release. On the left, at presynaptic terminals syntaxin-1 forms SNARE complex with SNAP-25 and synaptobrevin/VAMP in a process assisted by SM protein Munc18. Ensuing membrane fusion results in release of a neurotransmitter stored in the lumen of synaptic vesicles and activation of postsynaptic receptors. On the right, syntaxin-1 phosphorylated by CK2 at the Ser-14 is enriched in the axonal extrasynaptic plasma membrane, where it associates with SNAP-25. Modified syntaxin-1 supports membrane fusion and neurotransmitter release at extrasynaptic sites, possibly independent of Munc18. CK2, casein kinase II; Ser-14, serine-14; SM, Sec1/Munc18 like; SNARE, soluble N-ethylmalemide-sensitive factor attachment protein receptor; SNAP-25, synaptosome-associated protein of 25 kD; VAMP, vesicle-associated membrane protein.
schizophrenic brains or does it also occur in other psychiatric conditions? The detailed analysis of syntaxin-1 phosphorylation in clinically depressed patients performed by Castillo et al. (10) revealed no significant changes, suggesting at least some degree of pathophysiological specificity. The phosphorylation of syntaxin-1 can also be affected by the chronic treatment with antipsychotics and other drugs used in schizophrenic patients. The authors provide evidence that this was not the case. In brains obtained from rats chronically treated with antipsychotics, syntaxin-1 phosphorylation was significantly increased. In addition, the grouping of the tissue samples based on whether or not the patients had received the standard drug treatment revealed a trend for increased syntaxin-1 phosphorylation in treated patients. What is the functional and pathophysiological significance of changes in protein kinase CK2 mediated phosphorylation of syntaxin-1? To begin addressing this difficult question, the authors performed analysis of syntaxin-1 interacting partners: the SNARE proteins, SNAP-25 and synaptobrevin-2, and the SM protein, Munc18. Immunoprecipitation with total syntaxin-1 antibody revealed no change in interaction with synaptobrevin-2 and a marked decrease in interaction with SNAP-25 (⬃25%) in the samples from schizophrenic brains with significantly reduced levels of syntaxin-1 phosphorylation. This observation is in good agreement with a previously reported increase in binding of Ser-14 phosphorylated syntaxin-1 to SNAP-25 but not synaptobrevin-2 (6). However, a potential decrease in the total SNAP-25 levels previously seen in schizophrenic brains may have also contributed to this finding. Surprisingly, significant decrease in co-immunoprecipitation of Munc18 was also observed in the same abnormal samples. Syntaxin-1 phosphorylation at Ser-14 does not affect the closed syntaxin-1/Munc18 interaction in vitro (6). The same phosphorylation appears to interfere with the second mode of interaction, a permissive complex between syntaxin-1 and Munc18 (7). Future studies will be needed to address this discrepancy. Finally, Castillo et al. (10) observe a decrease in SNARE complexes in samples prepared from schizophrenic brains. This analysis, however, is complicated by oligomerization of native SNARE complexes and possible changes in SNARE complexes in postmortem tissue.
www.sobp.org/journal
In summary, in this study, Castillo et al. (10) provide important insight toward the molecular etiology of schizophrenia. The observed decrease in CK2 mediated phosphorylation of syntaxin-1 in schizophrenic brains may underlie malfunction at the extrasynaptic fusion sites along the axons in this widespread human neuropathology. The functional implications of these findings and the physiological importance of syntaxin-1 phosphorylation will provide an exciting avenue for future schizophrenia research.
The author reported no biomedical financial interests or potential conflict of interests. 1. Jahn R, Scheller RH (2006): SNAREs—Engines for membrane fusion. Nat Rev Mol Cell Biol 7:631– 643. 2. Sudhof TC, Rothman JE (2009): Membrane fusion: Grappling with SNARE and SM proteins. Science 323:474 – 477. 3. Snyder DA, Kelly ML, Woodbury DJ (2006): SNARE complex regulation by phosphorylation. Cell Biochem Biophys 45:111–123. 4. Dulubova I, Khvotchev M, Liu S, Huryeva I, Sudhof TC, Rizo J (2007): Munc18-1 binds directly to the neuronal SNARE complex. Proc Natl Acad Sci U S A 104:2697–2702. 5. Khvotchev M, Dulubova I, Sun J, Dai H, Rizo J, Sudhof TC (2007): Dual modes of Munc18-1/SNARE interactions are coupled by functionally critical binding to syntaxin-1 N terminus. J Neurosci 27:12147–12155. 6. Foletti DL, Lin R, Finley MA, Scheller RH (2000): Phosphorylated syntaxin 1 is localized to discrete domains along a subset of axons. J Neurosci 20:4535– 4544. 7. Rickman C, Duncan RR (2009): MUNC18/syntaxin interaction kinetics control secretory vesicle dynamics [published online ahead of print September 11]. J Biol Chem. 8. Li B, Devidze N, Barengolts D, Prostak N, Sphicas E, Apicella AJ, et al. (2009): NMDA receptor phosphorylation at a site affected in schizophrenia controls synaptic and behavioral plasticity. J Neurosci 29:11965– 11972. 9. Thompson PM, Sower AC, Perrone-Bizzozero NI (1998): Altered levels of the synaptosomal associated protein SNAP-25 in schizophrenia. Biol Psychiatry 43:239 –243. 10. Castillo MA, Ghose S, Tamminga CA, Ulery-Reynolds PG (2009): Deficits in syntaxin 1 phosphorylation in schizophrenia prefrontal cortex [published online ahead of print September 11]. Biol Psychiatry.
Schizophrenia and Synapse: Emerging Role of Presynaptic Fusion Machinery Mikhail Khvotchev
B
illions of neurons in the brain communicate with each other via specialized junctions called synapses. Each neuron forms thousands of synaptic connections that integrate into complex neural networks responsible for information processing, memory storage, and other fundamental brain functions. Not surprisingly, the dysfunction of synapses has emerged as a major etiologic factor in many brain diseases including widespread neurodegenerative (Alzheimer’s, Parkinsonism), neurodevelopmental (autism), and psychiatric conditions (schizophrenia, depression). Synapses are morphologically and functionally distinct; they undergo rapid transient or long-lasting changes but share basic architecture and principles of function. Each synapse consists of a presynaptic and a postsynaptic terminal separated by a narrow synaptic cleft. Presynaptic terminal is filled with hundreds of synaptic vesicles loaded with a neurotransmitter. Arrival of an action potential triggers rapid fusion of synaptic vesicles within the specialized area of the presynaptic plasma membrane called the active zone and release of the neurotransmitter. Fusion of synaptic vesicles is a probabilistic, yet tightly controlled, event that is in most cases highly synchronized with calcium entry through the voltage-gated calcium channels. Released neurotransmitter is next detected by clustered neurotransmitter receptors of the postsynaptic terminal. At synapses, the minimal fusion machinery consists of three soluble N-ethylmalemide-sensitive factor attachment protein receptor (SNARE) proteins: syntaxin-1 and synaptosome-associated protein of 25 kD (SNAP-25) engaged in the binary complex on the plasma membrane and synaptobrevin-2/vesicle-associated membrane protein 2 on the synaptic vesicles. These proteins form stable core complex in a process that brings two membranes into close proximity and overcomes the energy barrier for fusion (1). Multiple additional proteins are required to control the availability of syntaxin-1 for the SNARE complex formation (Munc13s, Munc18s), to synchronize membrane fusion with transient calcium ion influx (synaptotagmins, complexins), and to provide temporal and spatial regulation (Rab proteins, active zone proteins) (2). The complex network of protein interactions involved in the membrane fusion at the synapse is highly dynamic and plastic, yet little is known about the mechanisms of their regulation. Protein phosphorylation is one established mechanism to regulate protein interactions in vivo. Most of the protein constituents of the synaptic fusion machinery are known substrates for multiple protein kinases (3). Some of the most abundant synaptic proteins undergo activity-dependent cycles of phosphorylation and dephosphorylation; however, their functional implications remain largely untested in vivo. Syntaxin-1 is one of the key hubs for the regulation of synaptic fusion machinery. It adopts an From the Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, Texas. Address correspondence to Mikhail Khvotchev, Ph.D., Department of Neuroscience, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390-9111; E-mail: mkhvoc@mednet. swmed.edu. Received Dec 2, 2009; revised Dec 7, 2009; accepted Dec 11, 2009.
0006-3223/10/$36.00 doi:10.1016/j.biopsych.2009.12.007
open conformation that is compatible with the SNARE complex formation and an inhibitory closed conformation. These conformational states of syntaxin-1 are regulated by an essential element of the fusion machinery, a Sec1/Munc18 (SM) protein Munc18 that can both stabilize the closed conformation or interact with syntaxin-1 engaged in SNARE complex (4,5). Syntaxin-1 is constitutively phosphorylated by protein kinase casein kinase II (CK2) at the N-terminal serine-14 (6). This modification is of very low abundance in embryonic brain but gradually increases during postnatal development to 40% of the total protein levels in adults. Interestingly, the serine-14 phosphorylation of syntaxin-1 appears to be insensitive to acute changes in activity at the synapse. Phosphorylated syntaxin-1 has a higher propensity for binding to the plasma membrane SNARE protein SNAP-25 but not for the synaptic vesicle SNARE synaptobrevin-2. Ser-14 phosphorylated syntaxin-1 localizes predominantly to the extrasynaptic areas of axons, raising the possibility that the modified syntaxin-1 may be involved in extrasynaptic membrane fusion (Figure 1). Recent in vitro studies suggested that phosphorylation at Ser-14 interferes with permissive complex of Munc18 and syntaxin-1 but has no effect on the inhibitory complex of Munc18 and closed syntaxin-1 (7). One of the powerful molecular analyses available to study pathological processes in human brain involves protein profiling in postmortem brain tissue. This approach, however, critically depends on high-quality representative selection of tissue samples to control for differences related to individuals, postmortem handling of the tissue, effects of chronic drug treatment, etc. These considerations are especially important when labile posttranslational modifications such as phosphorylation are analyzed. Previous studies have identified multiple abnormalities in the levels and the phosphorylation status of synaptic proteins in the brains (mostly cortex) of schizophrenic patients. These include both presynaptic (SNAP-25) and postsynaptic proteins (glutamate receptors) (8,9). Syntaxin-1 levels are normal in patients with schizophrenia; however, the levels and activity of protein kinase CK2 are reduced. This prompted Castillo et al. (10) to investigate the phosphorylation status of syntaxin-1 in schizophrenic patients. Using a newly raised antibody that selectively recognizes the Ser-14 phosphorylated syntaxin-1 and variety of other antibodies, the authors confirmed that CK2 levels were decreased to 75% of the control levels in the cortices from schizophrenic patients. In turn, total levels of syntaxin-1 were not changed, but Ser-14 phosphorylated syntaxin-1 was reduced by almost 25%. Importantly, the changes in phosphorylation were not global, because the phosphorylation status of rabphilin, another abundant presynaptic phosphoprotein, was unchanged in schizophrenic patients. Phosphorylation is a highly labile protein modification that can rapidly deteriorate in postmortem tissues. The authors also showed that in rat brains the Ser-14 phosphorylation of syntaxin-1 dropped significantly during the first 8 hours after death but remained relatively stable thereafter up to 20 hours. This result suggests that human tissue samples may also be productively compared if they are collected within the similar time frame. Is the change in syntaxin-1 phosphorylation specific for BIOL PSYCHIATRY 2010;67:197–198 © 2010 Society of Biological Psychiatry
198 BIOL PSYCHIATRY 2010;67:197–198
Commentary
Presynaptic terminal
SNAP-25
N
N
Syntaxin-1 SNAP-25 N
Syntaxin-1
SV
Synaptobrevin/ VAMP
Synaptobrevin/ VAMP N
SV
Extrasynaptic axonal compartment
N
Active zone
Presynaptic neurotransmitter release
N
P
P
Munc18
N
Axonal plasma membrane
Extrasynaptic neurotransmitter release
Figure 1. A model for syntaxin-1 function in neurotransmitter release. On the left, at presynaptic terminals syntaxin-1 forms SNARE complex with SNAP-25 and synaptobrevin/VAMP in a process assisted by SM protein Munc18. Ensuing membrane fusion results in release of a neurotransmitter stored in the lumen of synaptic vesicles and activation of postsynaptic receptors. On the right, syntaxin-1 phosphorylated by CK2 at the Ser-14 is enriched in the axonal extrasynaptic plasma membrane, where it associates with SNAP-25. Modified syntaxin-1 supports membrane fusion and neurotransmitter release at extrasynaptic sites, possibly independent of Munc18. CK2, casein kinase II; Ser-14, serine-14; SM, Sec1/Munc18 like; SNARE, soluble N-ethylmalemide-sensitive factor attachment protein receptor; SNAP-25, synaptosome-associated protein of 25 kD; VAMP, vesicle-associated membrane protein.
schizophrenic brains or does it also occur in other psychiatric conditions? The detailed analysis of syntaxin-1 phosphorylation in clinically depressed patients performed by Castillo et al. (10) revealed no significant changes, suggesting at least some degree of pathophysiological specificity. The phosphorylation of syntaxin-1 can also be affected by the chronic treatment with antipsychotics and other drugs used in schizophrenic patients. The authors provide evidence that this was not the case. In brains obtained from rats chronically treated with antipsychotics, syntaxin-1 phosphorylation was significantly increased. In addition, the grouping of the tissue samples based on whether or not the patients had received the standard drug treatment revealed a trend for increased syntaxin-1 phosphorylation in treated patients. What is the functional and pathophysiological significance of changes in protein kinase CK2 mediated phosphorylation of syntaxin-1? To begin addressing this difficult question, the authors performed analysis of syntaxin-1 interacting partners: the SNARE proteins, SNAP-25 and synaptobrevin-2, and the SM protein, Munc18. Immunoprecipitation with total syntaxin-1 antibody revealed no change in interaction with synaptobrevin-2 and a marked decrease in interaction with SNAP-25 (⬃25%) in the samples from schizophrenic brains with significantly reduced levels of syntaxin-1 phosphorylation. This observation is in good agreement with a previously reported increase in binding of Ser-14 phosphorylated syntaxin-1 to SNAP-25 but not synaptobrevin-2 (6). However, a potential decrease in the total SNAP-25 levels previously seen in schizophrenic brains may have also contributed to this finding. Surprisingly, significant decrease in co-immunoprecipitation of Munc18 was also observed in the same abnormal samples. Syntaxin-1 phosphorylation at Ser-14 does not affect the closed syntaxin-1/Munc18 interaction in vitro (6). The same phosphorylation appears to interfere with the second mode of interaction, a permissive complex between syntaxin-1 and Munc18 (7). Future studies will be needed to address this discrepancy. Finally, Castillo et al. (10) observe a decrease in SNARE complexes in samples prepared from schizophrenic brains. This analysis, however, is complicated by oligomerization of native SNARE complexes and possible changes in SNARE complexes in postmortem tissue.
www.sobp.org/journal
In summary, in this study, Castillo et al. (10) provide important insight toward the molecular etiology of schizophrenia. The observed decrease in CK2 mediated phosphorylation of syntaxin-1 in schizophrenic brains may underlie malfunction at the extrasynaptic fusion sites along the axons in this widespread human neuropathology. The functional implications of these findings and the physiological importance of syntaxin-1 phosphorylation will provide an exciting avenue for future schizophrenia research.
The author reported no biomedical financial interests or potential conflict of interests. 1. Jahn R, Scheller RH (2006): SNAREs—Engines for membrane fusion. Nat Rev Mol Cell Biol 7:631– 643. 2. Sudhof TC, Rothman JE (2009): Membrane fusion: Grappling with SNARE and SM proteins. Science 323:474 – 477. 3. Snyder DA, Kelly ML, Woodbury DJ (2006): SNARE complex regulation by phosphorylation. Cell Biochem Biophys 45:111–123. 4. Dulubova I, Khvotchev M, Liu S, Huryeva I, Sudhof TC, Rizo J (2007): Munc18-1 binds directly to the neuronal SNARE complex. Proc Natl Acad Sci U S A 104:2697–2702. 5. Khvotchev M, Dulubova I, Sun J, Dai H, Rizo J, Sudhof TC (2007): Dual modes of Munc18-1/SNARE interactions are coupled by functionally critical binding to syntaxin-1 N terminus. J Neurosci 27:12147–12155. 6. Foletti DL, Lin R, Finley MA, Scheller RH (2000): Phosphorylated syntaxin 1 is localized to discrete domains along a subset of axons. J Neurosci 20:4535– 4544. 7. Rickman C, Duncan RR (2009): MUNC18/syntaxin interaction kinetics control secretory vesicle dynamics [published online ahead of print September 11]. J Biol Chem. 8. Li B, Devidze N, Barengolts D, Prostak N, Sphicas E, Apicella AJ, et al. (2009): NMDA receptor phosphorylation at a site affected in schizophrenia controls synaptic and behavioral plasticity. J Neurosci 29:11965– 11972. 9. Thompson PM, Sower AC, Perrone-Bizzozero NI (1998): Altered levels of the synaptosomal associated protein SNAP-25 in schizophrenia. Biol Psychiatry 43:239 –243. 10. Castillo MA, Ghose S, Tamminga CA, Ulery-Reynolds PG (2009): Deficits in syntaxin 1 phosphorylation in schizophrenia prefrontal cortex [published online ahead of print September 11]. Biol Psychiatry.