John Chua - Academia.edu (original) (raw)

Papers by John Chua

Proceedings of the National Academy of Sciences, 2012

Presynaptic nerve terminals are formed from preassembled vesicles that are delivered to the prosp... more Presynaptic nerve terminals are formed from preassembled vesicles that are delivered to the prospective synapse by kinesin-mediated axonal transport. However, precisely how the various cargoes are linked to the motor proteins remains unclear. Here, we report a transport complex linking syntaxin 1a (Stx) and Munc18, two proteins functioning in synaptic vesicle exocytosis at the presynaptic plasma membrane, to the motor protein Kinesin-1 via the kinesin adaptor FEZ1. Mutation of the FEZ1 ortholog UNC-76 in Caenorhabditis elegans causes defects in the axonal transport of Stx. We also show that binding of FEZ1 to Kinesin-1 and Munc18 is regulated by phosphorylation, with a conserved site (serine 58) being essential for binding. When expressed in C. elegans , wild-type but not phosphorylation-deficient FEZ1 (S58A) restored axonal transport of Stx. We conclude that FEZ1 operates as a kinesin adaptor for the transport of Stx, with cargo loading and unloading being regulated by protein kina...

Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also... more Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradat...

Autophagy, 2008

Research in autophagy continues to accelerate, 1 and as a result many new scientists are entering... more Research in autophagy continues to accelerate, 1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have ...

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 13, 2018

Elevated iron deposition has been reported in Parkinson's disease. However, the route of iron... more Elevated iron deposition has been reported in Parkinson's disease. However, the route of iron uptake leading to high deposition in the substantia nigra is unresolved. Here, we show a mechanism in enhanced Fe uptake via S-nitrosylation of DMT1. While DMT1 could be S-nitrosylated by exogenous nitric oxide donors, in human PD brains, endogenously S-nitrosylated DMT1 was detected in post-mortem substantia nigra. Patch-clamp electrophysiological recordings and iron uptake assays confirmed increased Mn or Fe uptake through S-nitrosylated DMT1. We identified two major S-nitrosylation sites, C23 and C540, by mass spectrometry and DMT1 C23A or C540A substitutions abolished NO-mediated DMT1 current increase. To evaluate significance, lipopolysaccharide was stereotaxically injected into the substantia nigra of female and male mice to induce inflammation and production of NO. The intranigral lipopolysaccharide injection resulted in corresponding increase in Fe deposition, JNK activation, do...

Molecular neurobiology, Jan 17, 2017

Sphingomyelinases are a family of enzymes that hydrolyze sphingomyelin to generate phosphocholine... more Sphingomyelinases are a family of enzymes that hydrolyze sphingomyelin to generate phosphocholine and ceramide. The brain distribution and function of neutral sphingomyelinase 2 (nSMase2) were elucidated in this study. nSMase2 mRNA expression was greatest in the striatum, followed by the prefrontal cortex, hippocampus, cerebellum, thalamus, brainstem, and olfactory bulb. The striatum had the highest level of nSMase2 protein expression, followed by the prefrontal cortex, thalamus, hippocampus, brainstem, and cerebellum. Dense immunolabeling was observed in the striatum, including the caudate-putamen, while moderately dense staining was found in the olfactory bulb and cerebral neocortex. Electron microscopy of the caudate-putamen showed nSMase2 immunoreaction product was present in small diameter dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled axon terminals containing small round vesicles; and characteristics of glutamatergic axons. Lipidomic analysis ...

Scientific reports, Jun 1, 2016

Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated ... more Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer's disease, a neurodegenerative disorder exhibiting impaired axonal transport a...

Scientific reports, Jun 1, 2016

Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated ... more Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer's disease, a neurodegenerative disorder exhibiting impaired axonal transport a...

Frontiers in synaptic neuroscience, 2016

Targeted intracellular movement of presynaptic proteins plays important roles during synapse form... more Targeted intracellular movement of presynaptic proteins plays important roles during synapse formation and, later, in the homeostatic maintenance of mature synapses. Movement of these proteins, often as vesicular packages, is mediated by motor complexes travelling along intracellular cytoskeletal networks. Presynaptic protein transport by kinesin motors in particular plays important roles during synaptogenesis to bring newly synthesized proteins to establish nascent synaptic sites. Conversely, movement of proteins away from presynaptic sites by Dynein motors enables synapse-nuclear signaling and allows for synaptic renewal through degradation of unwanted or damaged proteins. Remarkably, recent data has indicated that synaptic and protein trafficking machineries can modulate each other's functions. Here, we survey the mechanisms involved in moving presynaptic components to and away from synapses and how this process supports presynaptic function.

Cells, 2016

Presynaptic neurotransmitter release is dominated by the synaptic vesicle (SV) cycle and entails ... more Presynaptic neurotransmitter release is dominated by the synaptic vesicle (SV) cycle and entails the biogenesis, fusion, recycling, reformation or turnover of synaptic vesicles-a process involving bulk movement of membrane and proteins. As key mediators of membrane trafficking, small GTPases from the Rab family of proteins play critical roles in this process by acting as molecular switches that dynamically interact with and regulate the functions of different sets of macromolecular complexes involved in each stage of the cycle. Importantly, mutations affecting Rabs, and their regulators or effectors have now been identified that are implicated in severe neurological and neurodevelopmental disorders. Here, we summarize the roles and functions of presynaptic Rabs and discuss their involvement in the regulation of presynaptic function.

Cellular and Molecular Life Sciences, 2014

The release of neurotransmitters from synaptic vesicles exocytosing at presynaptic nerve terminal... more The release of neurotransmitters from synaptic vesicles exocytosing at presynaptic nerve terminals is a critical event in the initiation of synaptic transmission. This event occurs at specialized sites known as active zones. The task of faithfully executing various steps in the process is undertaken by careful orchestration of overlapping sets of molecular nano-machineries upon a core macromolecular scaffold situated at active zones. However, their composition remains incompletely elucidated. This review provides an overview of the role of the active zone in mediating neurotransmitter release and summarizes the recent progress using neuroproteomic approaches to decipher their composition. Key proteins of these nano-machineries are highlighted.

Neuron, 2013

Neurotransmission involves calcium-triggered fusion of docked synaptic vesicles at specialized pr... more Neurotransmission involves calcium-triggered fusion of docked synaptic vesicles at specialized presynaptic release sites. While many of the participating proteins have been identified, the molecular composition of these sites has not been characterized comprehensively. Here, we report a procedure to biochemically isolate fractions highly enriched in docked synaptic vesicles. The fraction is largely free of postsynaptic proteins and most other organelles while containing most known synaptic vesicle and active zone proteins. Numerous presynaptic transmembrane proteins were also identified, together with over 30 uncharacterized proteins, many of which are evolutionarily conserved. Quantitative proteomic comparison of glutamate- and GABA-specific docking complexes revealed that, except of neurotransmitter-specific enzymes and transporters, only few proteins were selectively enriched in either fraction. We conclude that the core machinery involved in vesicle docking and exocytosis does not show compositional differences between the two types of synapses.

eLife, Jan 26, 2016

Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles w... more Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes as...

The Journal of Neuroscience the Official Journal of the Society For Neuroscience, Jun 10, 2010

Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reac... more Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reactions. In synapses, Rab3 and Rab5 proteins have been implicated in the exo-endocytic cycling of synaptic vesicles (SVs), but an involvement of additional Rabs cannot be excluded. Here, combining high-resolution mass spectrometry and chemical labeling (iTRAQ) together with quantitative immunoblotting and fluorescence microscopy, we have determined the exocytotic (Rab3a, Rab3b, Rab3c, and Rab27b) and endocytic (Rab4b, Rab5a/b, Rab10, Rab11b, and Rab14) Rab machinery of SVs. Analysis of two closely related proteins, Rab3a and Rab27b, revealed colocalization in synaptic nerve terminals, where they reside on distinct but overlapping SV pools. Moreover, whereas Rab3a readily dissociates from SVs during Ca 2ϩ -triggered exocytosis, and is susceptible to membrane extraction by Rab-GDI, Rab27b persists on SV membranes upon stimulation and is resistant to GDI-coupled Rab retrieval. Finally, we demonstrate that selective modulation of the GTP/GDP switch mechanism of Rab27b impairs SV recycling, suggesting that Rab27b, probably in concert with Rab3s, is involved in SV exocytosis.

Proceedings of the National Academy of Sciences, 2012

Presynaptic nerve terminals are formed from preassembled vesicles that are delivered to the prosp... more Presynaptic nerve terminals are formed from preassembled vesicles that are delivered to the prospective synapse by kinesin-mediated axonal transport. However, precisely how the various cargoes are linked to the motor proteins remains unclear. Here, we report a transport complex linking syntaxin 1a (Stx) and Munc18, two proteins functioning in synaptic vesicle exocytosis at the presynaptic plasma membrane, to the motor protein Kinesin-1 via the kinesin adaptor FEZ1. Mutation of the FEZ1 ortholog UNC-76 in Caenorhabditis elegans causes defects in the axonal transport of Stx. We also show that binding of FEZ1 to Kinesin-1 and Munc18 is regulated by phosphorylation, with a conserved site (serine 58) being essential for binding. When expressed in C. elegans , wild-type but not phosphorylation-deficient FEZ1 (S58A) restored axonal transport of Stx. We conclude that FEZ1 operates as a kinesin adaptor for the transport of Stx, with cargo loading and unloading being regulated by protein kina...

Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also... more Small GTPases of the Rab family not only regulate target recognition in membrane traffic but also control other cellular functions such as cytoskeletal transport and autophagy. Here we show that Rab26 is specifically associated with clusters of synaptic vesicles in neurites. Overexpression of active but not of GDP-preferring Rab26 enhances vesicle clustering, which is particularly conspicuous for the EGFP-tagged variant, resulting in a massive accumulation of synaptic vesicles in neuronal somata without altering the distribution of other organelles. Both endogenous and induced clusters co-localize with autophagy-related proteins such as Atg16L1, LC3B and Rab33B but not with other organelles. Furthermore, Atg16L1 appears to be a direct effector of Rab26 and binds Rab26 in its GTP-bound form, albeit only with low affinity. We propose that Rab26 selectively directs synaptic and secretory vesicles into preautophagosomal structures, suggesting the presence of a novel pathway for degradat...

Autophagy, 2008

Research in autophagy continues to accelerate, 1 and as a result many new scientists are entering... more Research in autophagy continues to accelerate, 1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have ...

The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 13, 2018

Elevated iron deposition has been reported in Parkinson's disease. However, the route of iron... more Elevated iron deposition has been reported in Parkinson's disease. However, the route of iron uptake leading to high deposition in the substantia nigra is unresolved. Here, we show a mechanism in enhanced Fe uptake via S-nitrosylation of DMT1. While DMT1 could be S-nitrosylated by exogenous nitric oxide donors, in human PD brains, endogenously S-nitrosylated DMT1 was detected in post-mortem substantia nigra. Patch-clamp electrophysiological recordings and iron uptake assays confirmed increased Mn or Fe uptake through S-nitrosylated DMT1. We identified two major S-nitrosylation sites, C23 and C540, by mass spectrometry and DMT1 C23A or C540A substitutions abolished NO-mediated DMT1 current increase. To evaluate significance, lipopolysaccharide was stereotaxically injected into the substantia nigra of female and male mice to induce inflammation and production of NO. The intranigral lipopolysaccharide injection resulted in corresponding increase in Fe deposition, JNK activation, do...

Molecular neurobiology, Jan 17, 2017

Sphingomyelinases are a family of enzymes that hydrolyze sphingomyelin to generate phosphocholine... more Sphingomyelinases are a family of enzymes that hydrolyze sphingomyelin to generate phosphocholine and ceramide. The brain distribution and function of neutral sphingomyelinase 2 (nSMase2) were elucidated in this study. nSMase2 mRNA expression was greatest in the striatum, followed by the prefrontal cortex, hippocampus, cerebellum, thalamus, brainstem, and olfactory bulb. The striatum had the highest level of nSMase2 protein expression, followed by the prefrontal cortex, thalamus, hippocampus, brainstem, and cerebellum. Dense immunolabeling was observed in the striatum, including the caudate-putamen, while moderately dense staining was found in the olfactory bulb and cerebral neocortex. Electron microscopy of the caudate-putamen showed nSMase2 immunoreaction product was present in small diameter dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled axon terminals containing small round vesicles; and characteristics of glutamatergic axons. Lipidomic analysis ...

Scientific reports, Jun 1, 2016

Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated ... more Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer's disease, a neurodegenerative disorder exhibiting impaired axonal transport a...

Scientific reports, Jun 1, 2016

Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated ... more Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer's disease, a neurodegenerative disorder exhibiting impaired axonal transport a...

Frontiers in synaptic neuroscience, 2016

Targeted intracellular movement of presynaptic proteins plays important roles during synapse form... more Targeted intracellular movement of presynaptic proteins plays important roles during synapse formation and, later, in the homeostatic maintenance of mature synapses. Movement of these proteins, often as vesicular packages, is mediated by motor complexes travelling along intracellular cytoskeletal networks. Presynaptic protein transport by kinesin motors in particular plays important roles during synaptogenesis to bring newly synthesized proteins to establish nascent synaptic sites. Conversely, movement of proteins away from presynaptic sites by Dynein motors enables synapse-nuclear signaling and allows for synaptic renewal through degradation of unwanted or damaged proteins. Remarkably, recent data has indicated that synaptic and protein trafficking machineries can modulate each other's functions. Here, we survey the mechanisms involved in moving presynaptic components to and away from synapses and how this process supports presynaptic function.

Cells, 2016

Presynaptic neurotransmitter release is dominated by the synaptic vesicle (SV) cycle and entails ... more Presynaptic neurotransmitter release is dominated by the synaptic vesicle (SV) cycle and entails the biogenesis, fusion, recycling, reformation or turnover of synaptic vesicles-a process involving bulk movement of membrane and proteins. As key mediators of membrane trafficking, small GTPases from the Rab family of proteins play critical roles in this process by acting as molecular switches that dynamically interact with and regulate the functions of different sets of macromolecular complexes involved in each stage of the cycle. Importantly, mutations affecting Rabs, and their regulators or effectors have now been identified that are implicated in severe neurological and neurodevelopmental disorders. Here, we summarize the roles and functions of presynaptic Rabs and discuss their involvement in the regulation of presynaptic function.

Cellular and Molecular Life Sciences, 2014

The release of neurotransmitters from synaptic vesicles exocytosing at presynaptic nerve terminal... more The release of neurotransmitters from synaptic vesicles exocytosing at presynaptic nerve terminals is a critical event in the initiation of synaptic transmission. This event occurs at specialized sites known as active zones. The task of faithfully executing various steps in the process is undertaken by careful orchestration of overlapping sets of molecular nano-machineries upon a core macromolecular scaffold situated at active zones. However, their composition remains incompletely elucidated. This review provides an overview of the role of the active zone in mediating neurotransmitter release and summarizes the recent progress using neuroproteomic approaches to decipher their composition. Key proteins of these nano-machineries are highlighted.

Neuron, 2013

Neurotransmission involves calcium-triggered fusion of docked synaptic vesicles at specialized pr... more Neurotransmission involves calcium-triggered fusion of docked synaptic vesicles at specialized presynaptic release sites. While many of the participating proteins have been identified, the molecular composition of these sites has not been characterized comprehensively. Here, we report a procedure to biochemically isolate fractions highly enriched in docked synaptic vesicles. The fraction is largely free of postsynaptic proteins and most other organelles while containing most known synaptic vesicle and active zone proteins. Numerous presynaptic transmembrane proteins were also identified, together with over 30 uncharacterized proteins, many of which are evolutionarily conserved. Quantitative proteomic comparison of glutamate- and GABA-specific docking complexes revealed that, except of neurotransmitter-specific enzymes and transporters, only few proteins were selectively enriched in either fraction. We conclude that the core machinery involved in vesicle docking and exocytosis does not show compositional differences between the two types of synapses.

eLife, Jan 26, 2016

Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles w... more Neurotransmitter release is mediated by the fast, calcium-triggered fusion of synaptic vesicles with the presynaptic plasma membrane, followed by endocytosis and recycling of the membrane of synaptic vesicles. While many of the proteins governing these processes are known, their regulation is only beginning to be understood. Here we have applied quantitative phosphoproteomics to identify changes in phosphorylation status of presynaptic proteins in resting and stimulated nerve terminals isolated from the brains of Wistar rats. Using rigorous quantification, we identified 252 phosphosites that are either up- or downregulated upon triggering calcium-dependent exocytosis. Particularly pronounced were regulated changes of phosphosites within protein constituents of the presynaptic active zone, including bassoon, piccolo, and RIM1. Additionally, we have mapped kinases and phosphatases that are activated upon stimulation. Overall, our study provides a snapshot of phosphorylation changes as...

The Journal of Neuroscience the Official Journal of the Society For Neuroscience, Jun 10, 2010

Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reac... more Rab GTPases are molecular switches that orchestrate protein complexes before membrane fusion reactions. In synapses, Rab3 and Rab5 proteins have been implicated in the exo-endocytic cycling of synaptic vesicles (SVs), but an involvement of additional Rabs cannot be excluded. Here, combining high-resolution mass spectrometry and chemical labeling (iTRAQ) together with quantitative immunoblotting and fluorescence microscopy, we have determined the exocytotic (Rab3a, Rab3b, Rab3c, and Rab27b) and endocytic (Rab4b, Rab5a/b, Rab10, Rab11b, and Rab14) Rab machinery of SVs. Analysis of two closely related proteins, Rab3a and Rab27b, revealed colocalization in synaptic nerve terminals, where they reside on distinct but overlapping SV pools. Moreover, whereas Rab3a readily dissociates from SVs during Ca 2ϩ -triggered exocytosis, and is susceptible to membrane extraction by Rab-GDI, Rab27b persists on SV membranes upon stimulation and is resistant to GDI-coupled Rab retrieval. Finally, we demonstrate that selective modulation of the GTP/GDP switch mechanism of Rab27b impairs SV recycling, suggesting that Rab27b, probably in concert with Rab3s, is involved in SV exocytosis.