A systematic investigation of the protein kinases involved in NMDA receptor-dependent LTD: evidence for a role of GSK-3 but not other serine/threonine kinases

Background: The signalling mechanisms involved in the induction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term depression (LTD) in the hippocampus are poorly understood. Numerous studies have presented evidence both for and against a variety of second messengers systems being involved in LTD induction. Here we provide the first systematic investigation of the involvement of serine/threonine (ser/thr) protein kinases in NMDAR-LTD, using whole-cell recordings from CA1 pyramidal neurons. Results: Using a panel of 23 inhibitors individually loaded into the recorded neurons, we can discount the involvement of at least 57 kinases, including PKA, PKC, CaMKII, p38 MAPK and DYRK1A. However, we have been able to confirm a role for the ser/thr protein kinase, glycogen synthase kinase 3 (GSK-3). Conclusion: The present study is the first to investigate the role of 58 ser/thr protein kinases in LTD in the same study. Of these 58 protein kinases, we have found evidence for the involvement of only one, GSK-3, in LTD

The Somatostatin 2A Receptor Is Enriched in Migrating Neurons during Rat and Human Brain Development and Stimulates Migration and Axonal Outgrowth

The neuropeptide somatostatin has been suggested to play an important role during neuronal development in addition to its established modulatory impact on neuroendocrine, motor and cognitive functions in adults. Although six somatostatin G protein-coupled receptors have been discovered, little is known about their distribution and function in the developing mammalian brain. In this study, we have first characterized the developmental expression of the somatostatin receptor sst2A, the subtype found most prominently in the adult rat and human nervous system. In the rat, the sst2A receptor expression appears as early as E12 and is restricted to post-mitotic neuronal populations leaving the ventricular zone. From E12 on, migrating neuronal populations immunopositive for the receptor were observed in numerous developing regions including the cerebral cortex, hippocampus and ganglionic eminences. Intense but transient immunoreactive signals were detected in the deep part of the external granular layer of the cerebellum, the rostral migratory stream and in tyrosine hydroxylase- and serotonin- positive neurons and axons. Activation of the sst2A receptor in vitro in rat cerebellar microexplants and primary hippocampal neurons revealed stimulatory effects on neuronal migration and axonal growth, respectively. In the human cortex, receptor immunoreactivity was located in the preplate at early development stages (8 gestational weeks) and was enriched to the outer part of the germinal zone at later stages. In the cerebellum, the deep part of the external granular layer was strongly immunoreactive at 19 gestational weeks, similar to the finding in rodents. In addition, migrating granule cells in the internal granular layer were also receptor-positive. Together, theses results strongly suggest that the somatostatin sst2A receptor participates in the development and maturation of specific neuronal populations during rat and human brain ontogenesis

Rabconnectin-3α is required for the morphological maturation of GnRH neurons and kisspeptin responsiveness

A few hundred hypothalamic neurons form a complex network that controls reproduction in mammals by secreting gonadotropin-releasing hormone (GnRH). Timely postnatal changes in GnRH secretion are essential for pubertal onset. During the juvenile period, GnRH neurons undergo morphological remodeling, concomitantly achieving an increased responsiveness to kisspeptin, the main secretagogue of GnRH. However, the link between GnRH neuron activity and their morphology remains unknown. Here, we show that brain expression levels of Dmxl2, which encodes the vesicular protein rabconnectin-3α, determine the capacity of GnRH neurons to be activated by kisspeptin and estradiol. We also demonstrate that Dmxl2 expression levels control the pruning of GnRH dendrites, highlighting an unexpected role for a vesicular protein in the maturation of GnRH neuronal network. This effect is mediated by rabconnectin-3α in neurons or glial cells afferent to GnRH neurons. The widespread expression of Dmxl2 in several brain areas raises the intriguing hypothesis that rabconnectin-3α could be involved in the maturation of other neuronal populations

Ionotropic glutamate receptors in GtoPdb v.2021.3

The ionotropic glutamate receptors comprise members of the NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) and kainate receptor classes, named originally according to their preferred, synthetic, agonist [35, 92, 155]. Receptor heterogeneity within each class arises from the homo-oligomeric, or hetero-oligomeric, assembly of distinct subunits into cation-selective tetramers. Each subunit of the tetrameric complex comprises an extracellular amino terminal domain (ATD), an extracellular ligand binding domain (LBD), 3 TM domains (M1, M3 and M4), a channel lining re-entrant 'p-loop' (M2) located between M1 and M3 and an intracellular carboxy- terminal domain (CTD) [99, 68, 107, 155, 82]. The X-ray structure of a homomeric ionotropic glutamate receptor (GluA2- see below) has recently been solved at 3.6Å resolution [143] and although providing the most complete structural information current available may not representative of the subunit arrangement of, for example, the heteromeric NMDA receptors [71]. It is beyond the scope of this supplement to discuss the pharmacology of individual ionotropic glutamate receptor isoforms in detail; such information can be gleaned from [35, 66, 31, 77, 42, 114, 24, 65, 155, 112, 113, 162]. Agents that discriminate between subunit isoforms are, where appropriate, noted in the tables and additional compounds that distinguish between receptor isoforms are indicated in the text below.The classification of glutamate receptor subunits has been re-addressed by NC-IUPHAR [28]. The scheme developed recommends a nomenclature for ionotropic glutamate receptor subunits that is adopted here.NMDA receptorsNMDA receptors assemble as obligate heteromers that may be drawn from GluN1, GluN2A, GluN2B, GluN2C, GluN2D, GluN3A and GluN3B subunits. Alternative splicing can generate eight isoforms of GluN1 with differing pharmacological properties. Various splice variants of GluN2B, 2C, 2D and GluN3A have also been reported. Activation of NMDA receptors containing GluN1 and GluN2 subunits requires the binding of two agonists, glutamate to the S1 and S2 regions of the GluN2 subunit and glycine to S1 and S2 regions of the GluN1 subunit [41, 25]. The minimal requirement for efficient functional expression of NMDA receptors in vitro is a di-heteromeric assembly of GluN1 and at least one GluN2 subunit variant, as a dimer of heterodimers arrangement in the extracellular domain [48, 99, 71]. However, more complex tri-heteromeric assemblies, incorporating multiple subtypes of GluN2 subunit, or GluN3 subunits, can be generated in vitro and occur in vivo. The NMDA receptor channel commonly has a high relative permeability to Ca2+ and is blocked, in a voltage-dependent manner, by Mg2+ such that at resting potentials the response is substantially inhibited.AMPA and Kainate receptorsAMPA receptors assemble as homomers, or heteromers, that may be drawn from GluA1, GluA2, GluA3 and GluA4 subunits. Transmembrane AMPA receptor regulatory proteins (TARPs) of class I (i.e. γ2, γ3, γ4 and γ8) act, with variable stoichiometry, as auxiliary subunits to AMPA receptors and influence their trafficking, single channel conductance gating and pharmacology (reviewed in [43, 103, 153, 64]). Functional kainate receptors can be expressed as homomers of GluK1, GluK2 or GluK3 subunits. GluK1-3 subunits are also capable of assembling into heterotetramers (e.g. GluK1/K2; [87, 119, 118]). Two additional kainate receptor subunits, GluK4 and GluK5, when expressed individually, form high affinity binding sites for kainate, but lack function, but can form heteromers when expressed with GluK1-3 subunits (e.g. GluK2/K5; reviewed in [119, 65, 118]). Kainate receptors may also exhibit 'metabotropic' functions [87, 131]. As found for AMPA receptors, kainate receptors are modulated by auxiliary subunits (Neto proteins, [118, 88]). An important function difference between AMPA and kainate receptors is that the latter require extracellular Na+ and Cl- for their activation [11, 120]. RNA encoding the GluA2 subunit undergoes extensive RNA editing in which the codon encoding a p-loop glutamine residue (Q) is converted to one encoding arginine (R). This Q/R site strongly influences the biophysical properties of the receptor. Recombinant AMPA receptors lacking RNA edited GluA2 subunits are: (1) permeable to Ca2+; (2) blocked by intracellular polyamines at depolarized potentials causing inward rectification (the latter being reduced by TARPs); (3) blocked by extracellular argiotoxin and joro spider toxins and (4) demonstrate higher channel conductances than receptors containing the edited form of GluA2 [139, 63]. GluK1 and GluK2, but not other kainate receptor subunits, are similarly edited and broadly similar functional characteristics apply to kainate receptors lacking either an RNA edited GluK1, or GluK2, subunit [87, 118]. Native AMPA and kainate receptors displaying differential channel conductances, Ca2+ permeabilites and sensitivity to block by intracellular polyamines have been identified [30, 63, 91]. GluA1-4 can exist as two variants generated by alternative splicing (termed ‘flip’ and ‘flop’) that differ in their desensitization kinetics and their desensitization in the presence of cyclothiazide which stabilises the nondesensitized state. TARPs also stabilise the non-desensitized conformation of AMPA receptors and facilitate the action of cyclothiazide [103]. Splice variants of GluK1-3 also exist which affects their trafficking [87, 118]

Ionotropic glutamate receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The ionotropic glutamate receptors comprise members of the NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) and kainate receptor classes, named originally according to their preferred, synthetic, agonist [34, 87, 147]. Receptor heterogeneity within each class arises from the homo-oligomeric, or hetero-oligomeric, assembly of distinct subunits into cation-selective tetramers. Each subunit of the tetrameric complex comprises an extracellular amino terminal domain (ATD), an extracellular ligand binding domain (LBD), three transmembrane domains composed of three membrane spans (M1, M3 and M4), a channel lining re-entrant ‘p-loop’ (M2) located between M1 and M3 and an intracellular carboxy- terminal domain (CTD) [94, 66, 102, 147, 77]. The X-ray structure of a homomeric ionotropic glutamate receptor (GluA2 – see below) has recently been solved at 3.6Å resolution [135] and although providing the most complete structural information current available may not representative of the subunit arrangement of, for example, the heteromeric NMDA receptors [69]. It is beyond the scope of this supplement to discuss the pharmacology of individual ionotropic glutamate receptor isoforms in detail; such information can be gleaned from [34, 65, 30, 73, 41, 108, 23, 64, 147, 106, 107, 152]. Agents that discriminate between subunit isoforms are, where appropriate, noted in the tables and additional compounds that distinguish between receptor isoforms are indicated in the text below.The classification of glutamate receptor subunits has been re-addressed by NC-IUPHAR [27]. The scheme developed recommends a nomenclature for ionotropic glutamate receptor subunits that is adopted here.NMDA receptorsNMDA receptors assemble as obligate heteromers that may be drawn from GluN1, GluN2A, GluN2B, GluN2C, GluN2D, GluN3A and GluN3B subunits. Alternative splicing can generate eight isoforms of GluN1 with differing pharmacological properties. Various splice variants of GluN2B, 2C, 2D and GluN3A have also been reported. Activation of NMDA receptors containing GluN1 and GluN2 subunits requires the binding of two agonists, glutamate to the S1 and S2 regions of the GluN2 subunit and glycine to S1 and S2 regions of the GluN1 subunit [40, 24]. The minimal requirement for efficient functional expression of NMDA receptors in vitro is a di-heteromeric assembly of GluN1 and at least one GluN2 subunit variant, as a dimer of heterodimers arrangement in the extracellular domain [47, 94, 69]. However, more complex tri-heteromeric assemblies, incorporating multiple subtypes of GluN2 subunit, or GluN3 subunits, can be generated in vitro and occur in vivo. The NMDA receptor channel commonly has a high relative permeability to Ca2+ and is blocked, in a voltage-dependent manner, by Mg2+ such that at resting potentials the response is substantially inhibited.AMPA and Kainate receptorsAMPA receptors assemble as homomers, or heteromers, that may be drawn from GluA1, GluA2, GluA3 and GluA4 subunits. Transmembrane AMPA receptor regulatory proteins (TARPs) of class I (i.e. γ2, γ3, γ4 and γ8) act, with variable stoichiometry, as auxiliary subunits to AMPA receptors and influence their trafficking, single channel conductance gating and pharmacology (reviewed in [42, 98, 145, 63]). Functional kainate receptors can be expressed as homomers of GluK1, GluK2 or GluK3 subunits. GluK1-3 subunits are also capable of assembling into heterotetramers (e.g. GluK1/K2; [82, 113, 112]). Two additional kainate receptor subunits, GluK4 and GluK5, when expressed individually, form high affinity binding sites for kainate, but lack function, but can form heteromers when expressed with GluK1-3 subunits (e.g. GluK2/K5; reviewed in [113, 64, 112]). Kainate receptors may also exhibit ‘metabotropic’ functions [82, 123]. As found for AMPA receptors, kainate receptors are modulated by auxiliary subunits (Neto proteins, [112, 83]). An important function difference between AMPA and kainate receptors is that the latter require extracellular Na+ and Cl- for their activation [11, 114]. RNA encoding the GluA2 subunit undergoes extensive RNA editing in which the codon encoding a p-loop glutamine residue (Q) is converted to one encoding arginine (R). This Q/R site strongly influences the biophysical properties of the receptor. Recombinant AMPA receptors lacking RNA edited GluA2 subunits are: (1) permeable to Ca2+; (2) blocked by intracellular polyamines at depolarized potentials causing inward rectification (the latter being reduced by TARPs); (3) blocked by extracellular argiotoxin and Joro spider toxins and (4) demonstrate higher channel conductances than receptors containing the edited form of GluA2 [131, 62]. GluK1 and GluK2, but not other kainate receptor subunits, are similarly edited and broadly similar functional characteristics apply to kainate receptors lacking either an RNA edited GluK1, or GluK2, subunit [82, 112]. Native AMPA and kainate receptors displaying differential channel conductances, Ca2+ permeabilites and sensitivity to block by intracellular polyamines have been identified [29, 62, 86]. GluA1-4 can exist as two variants generated by alternative splicing (termed ‘flip’ and ‘flop’) that differ in their desensitization kinetics and their desensitization in the presence of cyclothiazide which stabilises the nondesensitized state. TARPs also stabilise the non-desensitized conformation of AMPA receptors and facilitate the action of cyclothiazide [98]. Splice variants of GluK1-3 also exist which affects their trafficking [82, 112]

Ionotropic glutamate receptors in GtoPdb v.2023.1

The ionotropic glutamate receptors comprise members of the NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid) and kainate receptor classes, named originally according to their preferred, synthetic, agonist [36, 94, 157]. Receptor heterogeneity within each class arises from the homo-oligomeric, or hetero-oligomeric, assembly of distinct subunits into cation-selective tetramers. Each subunit of the tetrameric complex comprises an extracellular amino terminal domain (ATD), an extracellular ligand binding domain (LBD), 3 TM domains (M1, M3 and M4), a channel lining re-entrant 'p-loop' (M2) located between M1 and M3 and an intracellular carboxy- terminal domain (CTD) [101, 70, 109, 157, 84]. The X-ray structure of a homomeric ionotropic glutamate receptor (GluA2- see below) has recently been solved at 3.6Å resolution [145] and although providing the most complete structural information current available may not representative of the subunit arrangement of, for example, the heteromeric NMDA receptors [73]. It is beyond the scope of this supplement to discuss the pharmacology of individual ionotropic glutamate receptor isoforms in detail; such information can be gleaned from [36, 68, 32, 79, 43, 116, 25, 67, 157, 114, 115, 165]. Agents that discriminate between subunit isoforms are, where appropriate, noted in the tables and additional compounds that distinguish between receptor isoforms are indicated in the text below.The classification of glutamate receptor subunits has been re-addressed by NC-IUPHAR [29]. The scheme developed recommends a nomenclature for ionotropic glutamate receptor subunits that is adopted here.NMDA receptorsNMDA receptors assemble as obligate heteromers that may be drawn from GluN1, GluN2A, GluN2B, GluN2C, GluN2D, GluN3A and GluN3B subunits. Alternative splicing can generate eight isoforms of GluN1 with differing pharmacological properties. Various splice variants of GluN2B, 2C, 2D and GluN3A have also been reported. Activation of NMDA receptors containing GluN1 and GluN2 subunits requires the binding of two agonists, glutamate to the S1 and S2 regions of the GluN2 subunit and glycine to S1 and S2 regions of the GluN1 subunit [42, 26]. The minimal requirement for efficient functional expression of NMDA receptors in vitro is a di-heteromeric assembly of GluN1 and at least one GluN2 subunit variant, as a dimer of heterodimers arrangement in the extracellular domain [49, 101, 73]. However, more complex tri-heteromeric assemblies, incorporating multiple subtypes of GluN2 subunit, or GluN3 subunits, can be generated in vitro and occur in vivo. The NMDA receptor channel commonly has a high relative permeability to Ca2+ and is blocked, in a voltage-dependent manner, by Mg2+ such that at resting potentials the response is substantially inhibited.AMPA and Kainate receptorsAMPA receptors assemble as homomers, or heteromers, that may be drawn from GluA1, GluA2, GluA3 and GluA4 subunits. Transmembrane AMPA receptor regulatory proteins (TARPs) of class I (i.e. γ2, γ3, γ4 and γ8) act, with variable stoichiometry, as auxiliary subunits to AMPA receptors and influence their trafficking, single channel conductance gating and pharmacology (reviewed in [44, 105, 155, 66]). Functional kainate receptors can be expressed as homomers of GluK1, GluK2 or GluK3 subunits. GluK1-3 subunits are also capable of assembling into heterotetramers (e.g. GluK1/K2; [89, 121, 120]). Two additional kainate receptor subunits, GluK4 and GluK5, when expressed individually, form high affinity binding sites for kainate, but lack function, but can form heteromers when expressed with GluK1-3 subunits (e.g. GluK2/K5; reviewed in [121, 67, 120]). Kainate receptors may also exhibit 'metabotropic' functions [89, 133]. As found for AMPA receptors, kainate receptors are modulated by auxiliary subunits (Neto proteins, [120, 90]). An important function difference between AMPA and kainate receptors is that the latter require extracellular Na+ and Cl- for their activation [12, 122]. RNA encoding the GluA2 subunit undergoes extensive RNA editing in which the codon encoding a p-loop glutamine residue (Q) is converted to one encoding arginine (R). This Q/R site strongly influences the biophysical properties of the receptor. Recombinant AMPA receptors lacking RNA edited GluA2 subunits are: (1) permeable to Ca2+; (2) blocked by intracellular polyamines at depolarized potentials causing inward rectification (the latter being reduced by TARPs); (3) blocked by extracellular argiotoxin and joro spider toxins and (4) demonstrate higher channel conductances than receptors containing the edited form of GluA2 [141, 65]. GluK1 and GluK2, but not other kainate receptor subunits, are similarly edited and broadly similar functional characteristics apply to kainate receptors lacking either an RNA edited GluK1, or GluK2, subunit [89, 120]. Native AMPA and kainate receptors displaying differential channel conductances, Ca2+ permeabilites and sensitivity to block by intracellular polyamines have been identified [31, 65, 93]. GluA1-4 can exist as two variants generated by alternative splicing (termed ‘flip’ and ‘flop’) that differ in their desensitization kinetics and their desensitization in the presence of cyclothiazide which stabilises the nondesensitized state. TARPs also stabilise the non-desensitized conformation of AMPA receptors and facilitate the action of cyclothiazide [105]. Splice variants of GluK1-3 also exist which affects their trafficking [89, 120]

Synaptoimmunology - Roles in health and disease

Abstract Mounting evidence suggests that the nervous and immune systems are intricately linked. Many proteins first identified in the immune system have since been detected at synapses, playing different roles in normal and pathological situations. In addition, novel immunological functions are emerging for proteins typically expressed at synapses. Under normal conditions, release of inflammatory mediators generally represents an adaptive and regulated response of the brain to immune signals. On the other hand, when immune challenge becomes prolonged and/or uncontrolled, the consequent inflammatory response leads to maladaptive synaptic plasticity and brain disorders. In this review, we will first provide a summary of the cell signaling pathways in neurons and immune cells. We will then examine how immunological mechanisms might influence synaptic function, and in particular synaptic plasticity, in the healthy and pathological CNS. A better understanding of neuro-immune system interactions in brain circuitries relevant to neuropsychiatric and neurological disorders should provide specific biomarkers to measure the status of the neuroimmunological response and help design novel neuroimmune-targeted therapeutics

Dynamin I phosphorylation by GSK3 controls activity-dependent bulk endocytosis of synaptic vesicles.

Glycogen synthase kinase 3 (GSK3) is a critical enzyme in neuronal physiology; however, it is not yet known whether it has any specific role in presynaptic function. We found that GSK3 phosphorylates a residue on the large GTPase dynamin I (Ser-774) both in vitro and in primary rat neuronal cultures. This was dependent on prior phosphorylation of Ser-778 by cyclin-dependent kinase 5. Using both acute inhibition with pharmacological antagonists and silencing of expression with short hairpin RNA, we found that GSK3 was specifically required for activity-dependent bulk endocytosis (ADBE) but not clathrin-mediated endocytosis. Moreover we found that the specific phosphorylation of Ser-774 on dynamin I by GSK3 was both necessary and sufficient for ADBE. These results demonstrate a presynaptic role for GSK3 and they indicate that a protein kinase signaling cascade prepares synaptic vesicles for retrieval during elevated neuronal activity

Reversal of Synapse Degeneration by Restoring Wnt Signaling in the Adult Hippocampus

Synapse degeneration occurs early in neurodegenerative diseases and correlates strongly with cognitive decline in Alzheimer's disease (AD). The molecular mechanisms that trigger synapse vulnerability and those that promote synapse regeneration after substantial synaptic failure remain poorly understood. Increasing evidence suggests a link between a deficiency in Wnt signaling and AD. The secreted Wnt antagonist Dickkopf-1 (Dkk1), which is elevated in AD, contributes to amyloid-β-mediated synaptic failure. However, the impact of Dkk1 at the circuit level and the mechanism by which synapses disassemble have not yet been explored. Using a transgenic mouse model that inducibly expresses Dkk1 in the hippocampus, we demonstrate that Dkk1 triggers synapse loss, impairs long-term potentiation, enhances long-term depression, and induces learning and memory deficits. We decipher the mechanism involved in synapse loss induced by Dkk1 as it can be prevented by combined inhibition of the Gsk3 and RhoA-Rock pathways. Notably, after loss of synaptic connectivity, reactivation of the Wnt pathway by cessation of Dkk1 expression completely restores synapse number, synaptic plasticity, and long-term memory. These findings demonstrate the remarkable capacity of adult neurons to regenerate functional circuits and highlight Wnt signaling as a targetable pathway for neuronal circuit recovery after synapse degeneration

Control of Cerebellar Long-Term Potentiation by P-Rex-Family Guanine-Nucleotide Exchange Factors and Phosphoinositide 3-Kinase

Long-term potentiation (LTP) at the parallel fibre-Purkinje cell synapse in the cerebellum is a recently described and poorly characterized form of synaptic plasticity. The induction mechanism for LTP at this synapse is considered reciprocal to "classical" LTP at hippocampal CA1 pyramidal neurons: kinases promote increased trafficking of AMPA receptors into the postsynaptic density in the hippocampus, whereas phosphatases decrease internalization of AMPA receptors in the cerebellum. In the hippocampus, LTP occurs in overlapping phases, with the transition from early to late phases requiring the consolidation of initial induction processes by structural re-arrangements at the synapse. Many signalling pathways have been implicated in this process, including PI3 kinases and Rho GTPases.We hypothesized that analogous phases are present in cerebellar LTP, and took as the starting point for investigation our recent discovery that P-Rex--a Rac guanine nucleotide exchange factor which is activated by PtdIns(3,4,5)P(3)--is highly expressed in mouse cerebellar Purkinje neurons and plays a role in motor coordination. We found that LTP evoked at parallel fibre synapses by 1 Hz stimulation or by NO donors was not sustained beyond 30 min when P-Rex was eliminated or Rac inhibited, suggesting that cerebellar LTP exhibits a late phase analogous to hippocampal LTP. In contrast, inhibition of PI3 kinase activity eliminated LTP at the induction stage.Our data suggest that a PI3K/P-Rex/Rac pathway is required for late phase LTP in the mouse cerebellum, and that other PI3K targets, which remain to be discovered, control LTP induction