Eg where synapse genes (esp. glutamatergic) may be affecting B and T cell receptors —
The Scaffolding Protein Synapse-Associated Protein 97 Is Required for Enhanced Signaling Through Isotype-Switched IgG Memory B Cell Receptors (2012, Science Signaling)
After their first encounter with a foreign antigen, naive B cells that have immunoglobulin M (IgM) B cell receptors (BCRs) trigger the primary antibody response and the generation of memory B cells with IgG BCRs. When these memory B cells reencounter the same antigen, the cell surface IgG BCRs stimulate their rapid differentiation into plasma cells that release large amounts of IgG antibodies.
We showed that the conserved cytoplasmic tail of the IgG BCR, which contains a putative PDZ (postsynaptic density 95/disc large/zona occludens 1)–binding motif, associated with synapse-associated protein 97 (SAP97), a PDZ domain–containing scaffolding molecule that is involved in controlling receptor density and signal strength at neuronal synapses. SAP97 accumulated and bound to IgG BCRs in the immunological synapses that formed in response to B cell engagement with antigen. Knocking down SAP97 in IgG+ B cells or mutating the putative PDZ-binding motif in the BCR tail impaired formation of the immunological synapse, initiation of IgG BCR signaling, and downstream activation of the mitogen-activated protein kinase p38.
Thus, heightened B cell memory responses are encoded, in part, by a mechanism that involves SAP97 serving as a scaffolding protein in the IgG BCR immunological synapse.
We showed that the conserved cytoplasmic tail of the IgG BCR, which contains a putative PDZ (postsynaptic density 95/disc large/zona occludens 1)–binding motif, associated with synapse-associated protein 97 (SAP97), a PDZ domain–containing scaffolding molecule that is involved in controlling receptor density and signal strength at neuronal synapses. SAP97 accumulated and bound to IgG BCRs in the immunological synapses that formed in response to B cell engagement with antigen. Knocking down SAP97 in IgG+ B cells or mutating the putative PDZ-binding motif in the BCR tail impaired formation of the immunological synapse, initiation of IgG BCR signaling, and downstream activation of the mitogen-activated protein kinase p38.
Thus, heightened B cell memory responses are encoded, in part, by a mechanism that involves SAP97 serving as a scaffolding protein in the IgG BCR immunological synapse.
The role of PSD-95 in the rearrangement of Kv1.3 channels to the immunological synapse (2013, Pflügers Archiv - European Journal of Physiology)
Establishment of the immunological synapse (IS) between T lymphocytes and antigen-presenting cells is a key step in the adaptive immune response. Several proteins accumulate in the IS, such as the Kv1.3 potassium channel; however, the mechanism of this translocation is unknown.
PSD-95 and SAP97 are adaptor proteins that regulate the polarized cell surface expression and localization of Kv1 channels in neurons. We investigated whether these proteins affect the redistribution of Kv1.3 into the IS in non-excitable human T cells. We show here that PSD-95 and SAP97 are expressed in Jurkat and interact with the C terminus of Kv1.3.
Disruption of the interaction between PSD-95 or SAP97 and Kv1.3 in Jurkat was realized by the expression of a C-terminal truncated Kv1.3, which lacks the binding domain for these proteins, or by the knockdown of the expression of PSD-95 or SAP97 using specific shRNA. Expression of the truncated Kv1.3 or knockdown of PSD-95, but not the knockdown of SAP97, inhibited the recruitment of Kv1.3 into the IS; the fraction of cells showing polarized Kv1.3 expression upon engagement in an IS was significantly lower than in control cells expressing the full-length Kv1.3, and the rearrangement of Kv1.3 did not show time dependence. In contrast, Jurkat cells expressing the full-length channel showed marked time dependence in the recruitment into the IS peaking at 1 min after the conjugation of the cells.
These results demonstrate that PSD-95 participates in the targeting of Kv1.3 into the IS, implying its important role in human T-cell activation.
PSD-95 and SAP97 are adaptor proteins that regulate the polarized cell surface expression and localization of Kv1 channels in neurons. We investigated whether these proteins affect the redistribution of Kv1.3 into the IS in non-excitable human T cells. We show here that PSD-95 and SAP97 are expressed in Jurkat and interact with the C terminus of Kv1.3.
Disruption of the interaction between PSD-95 or SAP97 and Kv1.3 in Jurkat was realized by the expression of a C-terminal truncated Kv1.3, which lacks the binding domain for these proteins, or by the knockdown of the expression of PSD-95 or SAP97 using specific shRNA. Expression of the truncated Kv1.3 or knockdown of PSD-95, but not the knockdown of SAP97, inhibited the recruitment of Kv1.3 into the IS; the fraction of cells showing polarized Kv1.3 expression upon engagement in an IS was significantly lower than in control cells expressing the full-length Kv1.3, and the rearrangement of Kv1.3 did not show time dependence. In contrast, Jurkat cells expressing the full-length channel showed marked time dependence in the recruitment into the IS peaking at 1 min after the conjugation of the cells.
These results demonstrate that PSD-95 participates in the targeting of Kv1.3 into the IS, implying its important role in human T-cell activation.
The neurotransmitter glutamate and human T cells: glutamate receptors and glutamate-induced direct and potent effects on normal human T cells, cancerous human leukemia and lymphoma T cells, and autoimmune human T cells (2014, Journal of Neural Transmission)
Glutamate is the most important excitatory neurotransmitter of the nervous system, critically needed for the brain’s development and function. Glutamate has also a signaling role in peripheral organs.
Herein, we discuss glutamate receptors (GluRs) and glutamate-induced direct effects on human T cells. T cells are the most important cells of the adaptive immune system, crucially needed for eradication of all infectious organisms and cancer. Normal, cancer and autoimmune human T cells express functional ionotropic and metabotropic GluRs. Different GluR subtypes are expressed in different T cell subtypes, and in resting vs. activated T cells.
Glutamate by itself, at low physiological 10−8M to 10−5M concentrations and via its several types of GluRs, activates many key T cell functions in normal human T cells, among them adhesion, migration, proliferation, intracellular Ca2+ fluxes, outward K+ currents and more. Glutamate also protects activated T cells from antigen-induced apoptotic cell death. By doing all that, glutamate can improve substantially the function and survival of resting and activated human T cells. Yet, glutamate’s direct effects on T cells depend dramatically on its concentration and might be inhibitory at excess pathological 10−3M glutamate concentrations.
The effects of glutamate on T cells also depend on the specific GluRs types expressed on the target T cells, the T cell’s type and subtype, the T cell’s resting or activated state, and the presence or absence of other simultaneous stimuli besides glutamate. Glutamate also seems to play an active role in T cell diseases. For example, glutamate at several concentrations induces or enhances significantly very important functions of human T-leukemia and T-lymphoma cells, among them adhesion to the extracellular matrix, migration, in vivo engraftment into solid organs, and the production and secretion of the cancer-associated matrix metalloproteinase MMP-9 and its inducer CD147. Glutamate induces all these effects via activation of GluRs highly expressed in human T-leukemia and T-lymphoma cells. Glutamate also affects T cell-mediated autoimmune diseases.
With regards to multiple sclerosis (MS), GluR3 is highly expressed in T cells of MS patients, and upregulated significantly during relapse and when there is neurological evidence of disease activity. Moreover, glutamate or AMPA (10−8M to 10−5M) enhances the proliferation of autoreactive T cells of MS patients in response to myelin proteins. Thus, glutamate may play an active role in MS. Glutamate and its receptors also seem to be involved in autoimmune rheumatoid arthritis and systemic lupus erythematosus. Finally, T cells can produce and release glutamate that in turn affects other cells, and during the contact between T cells and dendritic cells, the latter cells release glutamate that has potent effects on the T cells.
Together, these evidences show that glutamate has very potent effects on normal, and also on cancer and autoimmune pathological T cells. Moreover, these evidences suggest that glutamate and glutamate-receptor agonists might be used for inducing and boosting beneficial T cell functions, for example, T cell activity against cancer and infectious organisms, and that glutamate-receptor antagonists might be used for preventing glutamate-induced activating effects on detrimental autoimmune and cancerous T cells.
Herein, we discuss glutamate receptors (GluRs) and glutamate-induced direct effects on human T cells. T cells are the most important cells of the adaptive immune system, crucially needed for eradication of all infectious organisms and cancer. Normal, cancer and autoimmune human T cells express functional ionotropic and metabotropic GluRs. Different GluR subtypes are expressed in different T cell subtypes, and in resting vs. activated T cells.
Glutamate by itself, at low physiological 10−8M to 10−5M concentrations and via its several types of GluRs, activates many key T cell functions in normal human T cells, among them adhesion, migration, proliferation, intracellular Ca2+ fluxes, outward K+ currents and more. Glutamate also protects activated T cells from antigen-induced apoptotic cell death. By doing all that, glutamate can improve substantially the function and survival of resting and activated human T cells. Yet, glutamate’s direct effects on T cells depend dramatically on its concentration and might be inhibitory at excess pathological 10−3M glutamate concentrations.
The effects of glutamate on T cells also depend on the specific GluRs types expressed on the target T cells, the T cell’s type and subtype, the T cell’s resting or activated state, and the presence or absence of other simultaneous stimuli besides glutamate. Glutamate also seems to play an active role in T cell diseases. For example, glutamate at several concentrations induces or enhances significantly very important functions of human T-leukemia and T-lymphoma cells, among them adhesion to the extracellular matrix, migration, in vivo engraftment into solid organs, and the production and secretion of the cancer-associated matrix metalloproteinase MMP-9 and its inducer CD147. Glutamate induces all these effects via activation of GluRs highly expressed in human T-leukemia and T-lymphoma cells. Glutamate also affects T cell-mediated autoimmune diseases.
With regards to multiple sclerosis (MS), GluR3 is highly expressed in T cells of MS patients, and upregulated significantly during relapse and when there is neurological evidence of disease activity. Moreover, glutamate or AMPA (10−8M to 10−5M) enhances the proliferation of autoreactive T cells of MS patients in response to myelin proteins. Thus, glutamate may play an active role in MS. Glutamate and its receptors also seem to be involved in autoimmune rheumatoid arthritis and systemic lupus erythematosus. Finally, T cells can produce and release glutamate that in turn affects other cells, and during the contact between T cells and dendritic cells, the latter cells release glutamate that has potent effects on the T cells.
Together, these evidences show that glutamate has very potent effects on normal, and also on cancer and autoimmune pathological T cells. Moreover, these evidences suggest that glutamate and glutamate-receptor agonists might be used for inducing and boosting beneficial T cell functions, for example, T cell activity against cancer and infectious organisms, and that glutamate-receptor antagonists might be used for preventing glutamate-induced activating effects on detrimental autoimmune and cancerous T cells.
Glutamate receptor–T cell receptor signaling potentiates full CD8+ T cell activation and effector function in tumor immunity (2025, iScience)
Glutamate is best known as an excitatory neurotransmitter. However, its roles in T cell immunity remain underrecognized. We investigated the interplay between glutamate receptors (GluRs) and T cell receptors (TCRs) during CD8+ T cell activation. Our findings reveal that GluR expression in CD8+ T lymphocytes strongly correlates with the activation of TCR-CD28 signaling, enhancing their antitumor effector responses.
Conversely, pharmacologic antagonism of GluRs in activated CD8+ T cells disrupts the colocalization of GluR with TCRVβ8.1, reduces the phosphorylation of TCR-signaling intermediates, alters calcium flux, and impairs the metabolic switch to glycolysis essential for T cell activation. Moreover, these disruptions blunt clonal proliferation and compromise the tumor-cytolytic capacity of CD8+ T cells.
Thus, the glutamatergic system—via the GluR−TCR signaling complex—plays a critical amplifier role in activating CD8+ T cells and eliciting their full antitumor activity. This mechanistic insight reveals a previously underappreciated axis in T cell biology and opens avenues for immunotherapy regimens targeting GluR−TCR interactions to augment T cell–mediated responses in cancer and potentially other immunopathologies.
HIGHLIGHTS
• Expression of GluR, CD69 and CD25 is critical for the activation of CD8+ T lymphocytes
• Inhibiting GluR signaling blunts CD8+ T cell proliferation, glycolysis and effector function
• Activation of the GluR−TCR−CD28 axis elicits full CD8+ T cell antitumor effector responses
• Engagement of mGluR1 shapes T cell immunity in the solid tumor microenvironment
Conversely, pharmacologic antagonism of GluRs in activated CD8+ T cells disrupts the colocalization of GluR with TCRVβ8.1, reduces the phosphorylation of TCR-signaling intermediates, alters calcium flux, and impairs the metabolic switch to glycolysis essential for T cell activation. Moreover, these disruptions blunt clonal proliferation and compromise the tumor-cytolytic capacity of CD8+ T cells.
Thus, the glutamatergic system—via the GluR−TCR signaling complex—plays a critical amplifier role in activating CD8+ T cells and eliciting their full antitumor activity. This mechanistic insight reveals a previously underappreciated axis in T cell biology and opens avenues for immunotherapy regimens targeting GluR−TCR interactions to augment T cell–mediated responses in cancer and potentially other immunopathologies.
HIGHLIGHTS
• Expression of GluR, CD69 and CD25 is critical for the activation of CD8+ T lymphocytes
• Inhibiting GluR signaling blunts CD8+ T cell proliferation, glycolysis and effector function
• Activation of the GluR−TCR−CD28 axis elicits full CD8+ T cell antitumor effector responses
• Engagement of mGluR1 shapes T cell immunity in the solid tumor microenvironment
Or vascular function —
The synaptic proteins neurexins and neuroligins are widely expressed in the vascular system and contribute to its functions (2009, Proceedings of the National Academy of Sciences)
Unlike other neuronal counterparts, primary synaptic proteins are not known to be involved in vascular physiology. Here, we demonstrate that neurexins and neuroligins, which constitute large and complex families of fundamental players in synaptic activity, are produced and processed by endothelial and vascular smooth muscle cells throughout the vasculature. Moreover, they are dynamically regulated during vessel remodeling and form endogenous complexes in large vessels as well as in the brain.
We used the chicken chorioallantoic membrane as a system to pursue functional studies and demonstrate that a monoclonal recombinant antibody against β-neurexin inhibits angiogenesis, whereas exogenous neuroligin has a role in promoting angiogenesis. Finally, as an insight into the mechanism of action of β-neurexin, we show that the anti-β-neurexin antibody influences vessel tone in isolated chicken arteries.
Our finding strongly supports the idea that even the most complex and plastic events taking place in the nervous system (i.e., synaptic activity) share molecular cues with the vascular system.
We used the chicken chorioallantoic membrane as a system to pursue functional studies and demonstrate that a monoclonal recombinant antibody against β-neurexin inhibits angiogenesis, whereas exogenous neuroligin has a role in promoting angiogenesis. Finally, as an insight into the mechanism of action of β-neurexin, we show that the anti-β-neurexin antibody influences vessel tone in isolated chicken arteries.
Our finding strongly supports the idea that even the most complex and plastic events taking place in the nervous system (i.e., synaptic activity) share molecular cues with the vascular system.
Neurexins and neuroligins: synapses look out of the nervous system (2011, Cellular and Molecular Life Sciences)
The scientific interest in the family of the so-called nervous vascular parallels has been growing steadily for the past 15 years, either by addition of new members to the group or, lately, by deepening the analysis of established concepts and mediators. Proteins governing both neurons and vascular cells are known to be involved in events such as cell fate determination and migration/guidance but not in the last and apparently most complex step of nervous system development, the formation and maturation of synapses.
Hence, the recent addition to this family of the specific synaptic proteins, Neurexin and Neuroligin, is a double innovation. The two proteins, which were thought to be “simple” adhesive links between the pre- and post-synaptic sides of chemical synapses, are in fact extremely complex and modulate the most subtle synaptic activities. We will discuss the relevant data and the intriguing challenge of transferring synaptic activities to vascular functions.
Hence, the recent addition to this family of the specific synaptic proteins, Neurexin and Neuroligin, is a double innovation. The two proteins, which were thought to be “simple” adhesive links between the pre- and post-synaptic sides of chemical synapses, are in fact extremely complex and modulate the most subtle synaptic activities. We will discuss the relevant data and the intriguing challenge of transferring synaptic activities to vascular functions.
NMDA-Type Glutamate Receptor Activation Promotes Vascular Remodeling and Pulmonary Arterial Hypertension (2018, Circulation)
BACKGROUND
Excessive proliferation and apoptosis resistance in pulmonary vascular cells underlie vascular remodeling in pulmonary arterial hypertension (PAH). Specific treatments for PAH exist, mostly targeting endothelial dysfunction, but high pulmonary arterial pressure still causes heart failure and death. Pulmonary vascular remodeling may be driven by metabolic reprogramming of vascular cells to increase glutaminolysis and glutamate production. The N-methyl-d-aspartate receptor (NMDAR), a major neuronal glutamate receptor, is also expressed on vascular cells, but its role in PAH is unknown.
METHODS
We assessed the status of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and controls through mass spectrometry imaging, Western blotting, and immunohistochemistry. We measured the glutamate release from cultured pulmonary vascular cells using enzymatic assays and analyzed NMDAR regulation/phosphorylation through Western blot experiments. The effect of NMDAR blockade on human pulmonary arterial smooth muscle cell proliferation was determined using a BrdU incorporation assay. We assessed the role of NMDARs in vascular remodeling associated to pulmonary hypertension, in both smooth muscle-specific NMDAR knockout mice exposed to chronic hypoxia and the monocrotaline rat model of pulmonary hypertension using NMDAR blockers.
RESULTS
We report glutamate accumulation, upregulation of the NMDAR, and NMDAR engagement reflected by increases in GluN1-subunit phosphorylation in the pulmonary arteries of human patients with PAH. Kv channel inhibition and type A-selective endothelin receptor activation amplified calcium-dependent glutamate release from human pulmonary arterial smooth muscle cell, and type A-selective endothelin receptor and platelet-derived growth factor receptor activation led to NMDAR engagement, highlighting crosstalk between the glutamate-NMDAR axis and major PAH-associated pathways. The platelet-derived growth factor-BB-induced proliferation of human pulmonary arterial smooth muscle cells involved NMDAR activation and phosphorylated GluN1 subunit localization to cell-cell contacts, consistent with glutamatergic communication between proliferating human pulmonary arterial smooth muscle cells via NMDARs. Smooth-muscle NMDAR deficiency in mice attenuated the vascular remodeling triggered by chronic hypoxia, highlighting the role of vascular NMDARs in pulmonary hypertension. Pharmacological NMDAR blockade in the monocrotaline rat model of pulmonary hypertension had beneficial effects on cardiac and vascular remodeling, decreasing endothelial dysfunction, cell proliferation, and apoptosis resistance while disrupting the glutamate-NMDAR pathway in pulmonary arteries.
CONCLUSIONS
These results reveal a dysregulation of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and identify vascular NMDARs as targets for antiremodeling treatments in PAH.
Excessive proliferation and apoptosis resistance in pulmonary vascular cells underlie vascular remodeling in pulmonary arterial hypertension (PAH). Specific treatments for PAH exist, mostly targeting endothelial dysfunction, but high pulmonary arterial pressure still causes heart failure and death. Pulmonary vascular remodeling may be driven by metabolic reprogramming of vascular cells to increase glutaminolysis and glutamate production. The N-methyl-d-aspartate receptor (NMDAR), a major neuronal glutamate receptor, is also expressed on vascular cells, but its role in PAH is unknown.
METHODS
We assessed the status of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and controls through mass spectrometry imaging, Western blotting, and immunohistochemistry. We measured the glutamate release from cultured pulmonary vascular cells using enzymatic assays and analyzed NMDAR regulation/phosphorylation through Western blot experiments. The effect of NMDAR blockade on human pulmonary arterial smooth muscle cell proliferation was determined using a BrdU incorporation assay. We assessed the role of NMDARs in vascular remodeling associated to pulmonary hypertension, in both smooth muscle-specific NMDAR knockout mice exposed to chronic hypoxia and the monocrotaline rat model of pulmonary hypertension using NMDAR blockers.
RESULTS
We report glutamate accumulation, upregulation of the NMDAR, and NMDAR engagement reflected by increases in GluN1-subunit phosphorylation in the pulmonary arteries of human patients with PAH. Kv channel inhibition and type A-selective endothelin receptor activation amplified calcium-dependent glutamate release from human pulmonary arterial smooth muscle cell, and type A-selective endothelin receptor and platelet-derived growth factor receptor activation led to NMDAR engagement, highlighting crosstalk between the glutamate-NMDAR axis and major PAH-associated pathways. The platelet-derived growth factor-BB-induced proliferation of human pulmonary arterial smooth muscle cells involved NMDAR activation and phosphorylated GluN1 subunit localization to cell-cell contacts, consistent with glutamatergic communication between proliferating human pulmonary arterial smooth muscle cells via NMDARs. Smooth-muscle NMDAR deficiency in mice attenuated the vascular remodeling triggered by chronic hypoxia, highlighting the role of vascular NMDARs in pulmonary hypertension. Pharmacological NMDAR blockade in the monocrotaline rat model of pulmonary hypertension had beneficial effects on cardiac and vascular remodeling, decreasing endothelial dysfunction, cell proliferation, and apoptosis resistance while disrupting the glutamate-NMDAR pathway in pulmonary arteries.
CONCLUSIONS
These results reveal a dysregulation of the glutamate-NMDAR axis in the pulmonary arteries of patients with PAH and identify vascular NMDARs as targets for antiremodeling treatments in PAH.
