Transmembrane AMPA Receptor Regulatory Proteins Research Articles

Preclinical Evaluation of Azabenzimidazole-Based PET Radioligands for γ-8 Dependent Transmembrane AMPA Receptor Regulatory Protein Imaging.

AMPA glutamate receptors (AMPARs) play a pivotal role in excitatory neurotransmission, particularly in the hippocampus where the TARP γ-8 subunit is enriched and serves as a target for emerging anti-epileptic drugs. To enable in vivo visualization of TARP γ-8 distribution and expression by positron emission tomography (PET), this study focuses on the development of novel 18 F-labeled TARP γ-8 inhibitors and their corresponding precursors, stemming from the azabenzimidazole scaffold. The resulting radioligands [18 F]TARP-2204 and [18 F]TARP-2205 were successfully synthesized with acceptable radiochemical yield, high molar activity, and excellent radiochemical purity. In vitro autoradiography demonstrates high level of specific binding of [18 F]TARP-2205 to TARP γ-8 in both rat and nonhuman primate brain tissues. However, unexpected radiodefluorination in PET imaging studies of rodents emphasizes the need for further structural refinement. This work serves as an excellent starting point for the development of future 18 F-labeled TARP γ-8 PET tracers, offering valuable insights into medicinal chemistry design, radiosynthesis and subsequent PET evaluation.

Transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor regulatory protein expression during the development of absence seizures in genetic absence epilepsy rats from Strasbourg.

The transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) regulatory proteins (TARPs), γ2 (stargazin), γ3, γ4, γ5, γ7, and γ8, are a family of proteins that regulate AMPAR trafficking, expression, and biophysical properties that could have a role in the development of absence seizures. Here, we evaluated the expression of TARPs and AMPARs across the development of epilepsy in the genetic absence epilepsy rats from Strasbourg (GAERS) model of idiopathic generalized epilepsy (IGE) with absence seizures. Pre-epileptic (7-day-old), early epileptic (6-week-old), and chronically epileptic (16-week-old) GAERS, and age-matched male nonepileptic control rats (NEC) were used. Electroencephalographic (EEG) recordings were acquired from the 6- and 16-week-old animals to quantify seizure expression. Somatosensory cortex (SCx) and whole thalamus were collected from all the animals to evaluate TARP and AMPAR mRNA expression. Analysis of the EEG demonstrated a gradual increase in the number and duration of seizures across GAERS development. mRNA expression of the TARPs γ2, γ3, γ4, γ5, and γ8 in the SCx, and γ4 and γ5 in the thalamus, increased as the seizures started and progressed in the GAERS compared to NEC. There was a temporal association between increased TARP expression and seizures in GAERS, highlighting TARPs as potential targets for developing novel treatments for IGE with absence seizures.

P-023 - Development of a novel Azabenzimidazoles-Based PET radioligands for γ-8 dependent transmembrane AMPA receptor regulatory protein imaging

P-023 - Development of a novel Azabenzimidazoles-Based PET radioligands for γ-8 dependent transmembrane AMPA receptor regulatory protein imaging

GSG1L-containing AMPA receptor complexes are defined by their spatiotemporal expression, native interactome and allosteric sites

Transmembrane AMPA receptor regulatory proteins (TARPs) and germ cell-specific gene 1-like protein (GSG1L) are claudin-type AMPA receptor (AMPAR) auxiliary subunits that profoundly regulate glutamatergic synapse strength and plasticity. While AMPAR-TARP complexes have been extensively studied, less is known about GSG1L-containing AMPARs. Here, we show that GSG1L’s spatiotemporal expression, native interactome and allosteric sites are distinct. GSG1L generally expresses late during brain development in a region-specific manner, constituting about 5% of all AMPAR complexes in adulthood. While GSG1L can co-assemble with TARPs or cornichons (CNIHs), it also assembles as the sole auxiliary subunit. Unexpectedly, GSG1L acts through two discrete evolutionarily-conserved sites on the agonist-binding domain with a weak allosteric interaction at the TARP/KGK site to slow desensitization, and a stronger interaction at a different site that slows recovery from desensitization. Together, these distinctions help explain GSG1L’s evolutionary past and how it fulfills a unique signaling role within glutamatergic synapses.

Differential regulation of tetramerization of the AMPA receptor glutamate–gated ion channel by auxiliary subunits

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) auxiliary subunits are specialized, nontransient binding partners of AMPARs that modulate AMPAR channel gating properties and pharmacology, as well as their biogenesis and trafficking. The most well-characterized families of auxiliary subunits are transmembrane AMPAR regulatory proteins (TARPs), cornichon homologs (CNIHs), and the more recently discovered GSG1-L. These auxiliary subunits can promote or reduce surface expression of AMPARs (composed of GluA1-4 subunits) in neurons, thereby impacting their functional role in membrane signaling. Here, we show that CNIH-2 enhances the tetramerization of WT and mutant AMPARs, presumably by increasing the overall stability of the tetrameric complex, an effect that is mainly mediated by interactions with the transmembrane domain of the receptor. We also find CNIH-2 and CNIH-3 show receptor subunit-specific actions in this regard with CNIH-2 enhancing both GluA1 and GluA2 tetramerization, whereas CNIH-3 only weakly enhances GluA1 tetramerization. These results are consistent with the proposed role of CNIHs as endoplasmic reticulum cargo transporters for AMPARs. In contrast, TARP γ-2, TARP γ-8, and GSG1-L have no or negligible effect on AMPAR tetramerization. On the other hand, TARP γ-2 can enhance receptor tetramerization but only when directly fused with the receptor at a maximal stoichiometry. Notably, surface expression of functional AMPARs was enhanced by CNIH-2 to a greater extent than TARP γ-2, suggesting that this distinction aids in maturation and membrane expression. These experiments define a functional distinction between CNIHs and other auxiliary subunits in the regulation of AMPAR biogenesis.

TARPγ2 Is Required for Normal AMPA Receptor Expression and Function in Direction-Selective Circuits of the Mammalian Retina.

AMPA receptors (AMPARs) are the major mediators of fast excitatory neurotransmission in the retina as in other parts of the brain. In most neurons, the synaptic targeting, pharmacology, and function of AMPARs are influenced by auxiliary subunits including the transmembrane AMPA receptor regulatory proteins (TARPs). However, it is unclear which TARP subunits are present at retinal synapses and how they influence receptor localization and function. Here, we show that TARPɣ2 (stargazin) is associated with AMPARs in the synaptic layers of the mouse, rabbit, macaque, and human retina. In most species, TARPɣ2 expression was high where starburst amacrine cells (SACs) ramify and transcriptomic analyses suggest correspondingly high gene expression in mouse and human SACs. Synaptic expression of GluA2, GluA3, and GluA4 was significantly reduced in a mouse mutant lacking TARPɣ2 expression (stargazer mouse; stg), whereas GluA1 levels were unaffected. AMPAR-mediated light-evoked EPSCs in ON-SACs from stg mice were ∼30% smaller compared with heterozygous littermates. There was also loss of a transient ON pathway-driven GABAergic input to ON-SACs in stg mutants. Direction-selective ganglion cells in the stg mouse showed normal directional tuning, but their surround inhibition and thus spatial tuning was reduced. Our results indicate that TARPɣ2 is required for normal synaptic expression of GluA2, GluA3, and GluA4 in the inner retina. The presence of residual AMPAR expression in the stargazer mutant suggests that other TARP subunits may compensate in the absence of TARPɣ2.

Synaptic memory and CaMKII: a review.

CaMKII and long-term potentiation (LTP) were discovered within a decade of each other and have been inextricably intertwined ever since. However, like many marriages, it has had its up and downs. Based on the unique biochemical properties of CaMKII, it was proposed as a memory molecule before any direct physiological linkage was made to LTP. This review will assess the state of this marriage 40 years on. How well does the physiological evidence support the role of CaMKII in synaptic memory and what are the remaining outstanding issues?

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].

Negative modulation of AMPA receptors bound to transmembrane AMPA receptor regulatory protein γ-8 blunts the positive reinforcing properties of alcohol and sucrose in a brain region-dependent manner in male mice.

The development and progression of alcohol use disorder (AUD) are widely viewed as maladaptive neuroplasticity. The transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory protein γ8 (TARP γ-8) is a molecular mechanism of neuroplasticity that has not been evaluated in AUD or other addictions. To address this gap in knowledge, we evaluated the mechanistic role of TARP γ-8 bound AMPAR activity in the basolateral amygdala (BLA) and ventral hippocampus (vHPC) in the positive reinforcing effects of alcohol, which drive repetitive alcohol use throughout the course of AUD, in male C57BL/6J mice. These brain regions were selected because they exhibit high levels of TARP γ-8 expression and send glutamate projections to the nucleus accumbens (NAc), which is a key nucleus in the brain reward pathway. Site-specific pharmacological inhibition of AMPARs bound to TARP γ-8 in the BLA via bilateral infusion of the selective negative modulator JNJ-55511118 (0-2µg/µl/side) significantly decreased operant alcohol self-administration with no effect on sucrose self-administration in behavior-matched controls. Temporal analysis showed that reductions in alcohol-reinforced response rate occurred > 25min after the onset of responding, consistent with a blunting of the positive reinforcing effects of alcohol in the absence of nonspecific behavioral effects. In contrast, inhibition of TARP γ-8 bound AMPARs in the vHPC selectively decreased sucrose self-administration with no effect on alcohol. This study reveals a novel brain region-specific role of TARP γ-8 bound AMPARs as a molecular mechanism of the positive reinforcing effects of alcohol and non-drug rewards.

Enhanced functional detection of synaptic calcium-permeable AMPA receptors using intracellular NASPM.

Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic plasticity and pathology. They can be distinguished from GluA2-containing calcium-impermeable AMPARs by the inward rectification of their currents, which reflects voltage-dependent channel block by intracellular spermine. However, the efficacy of this weakly permeant blocker is differentially altered by the presence of AMPAR auxiliary subunits - including transmembrane AMPAR regulatory proteins, cornichons, and GSG1L - which are widely expressed in neurons and glia. This complicates the interpretation of rectification as a measure of CP-AMPAR expression. Here, we show that the inclusion of the spider toxin analog 1-naphthylacetyl spermine (NASPM) in the intracellular solution results in a complete block of GluA1-mediated outward currents irrespective of the type of associated auxiliary subunit. In neurons from GluA2-knockout mice expressing only CP-AMPARs, intracellular NASPM, unlike spermine, completely blocks outward synaptic currents. Thus, our results identify a functional measure of CP-AMPARs, that is unaffected by their auxiliary subunit content.

Alternative splicing of the flip/flop cassette and TARP auxiliary subunits engage in a privileged relationship that fine-tunes AMPA receptor gating.

Alternative splicing of AMPA-type glutamate receptors (AMPARs) and allosteric modulation by auxiliary subunits, such as transmembrane AMPAR regulatory proteins (TARPs), are two important mechanisms that regulate the time course of glutamatergic neurotransmission. Prior work has shown that alternative splicing of the flip/flop cassette profoundly regulates TARP γ2 modulation, where flip receptor gating exhibits robust sensitivity to TARPs while flop isoforms are relatively insensitive to TARP modulation. Whether this splice variant-specific regulation extends to other auxiliary subunit families, such as cornichons (CNIHs), GSG1L, or CKAMPs, remains unknown. Here, we demonstrate that CNIH-3 modulation is unaffected by AMPAR alternative splicing due to inherent differences in how CNIH-3 and TARP γ2 modify channel gating. CNIH-3 slows receptor deactivation from the outset of current decay, consistent with structural evidence showing its point of contact at the level of the pore. In contrast, TARP γ2 acts through the KGK site of the ligand-binding domain (LBD) to slow the onset of desensitization. Although GSG1L and CKAMP44 primarily slow recovery from desensitization, their effects on channel gating are unaffected by alternative splicing, further underlining that structural events leading to the onset and recovery from desensitization are separable. Together, this work establishes that alternative splicing and TARP auxiliary subunits form a unique partnership that governs fast glutamatergic signaling at central synapses. Since proteomic studies suggest that all native AMPARs co-assemble with at least two TARPs, allosteric coupling between the flip/flop cassette and TARPs may represent a common design element in all AMPAR complexes of the mammalian brain.SIGNIFICANCE STATEMENT:All fast excitatory neurotransmission in the mammalian brain is mediated by AMPA-type glutamate receptors (AMPARs). The time course of AMPAR gating can be regulated by two distinct mechanisms: alternative splicing of the flip/flop cassette and association with auxiliary subunits. Although these regulatory mechanisms have been well studied individually, it is not clear whether alternative splicing impacts auxiliary protein modulation of AMPARs. Here, we compare the four main families of AMPAR auxiliary subunits, TARPs (γ2), Cornichons (CNIH-3), GSG1L and CKAMPs (CKAMP44), and find a privileged relationship between TARPs and the flip/flop cassette that is not shared by others. The flop cassette acts as a master switch to override TARP action, and this coupling represents a way to fine-tune AMPAR signaling.

Inhibition mechanism of AMPA receptors by the pore blocker memantine.

Fast excitatory synaptic transmission in the mammalian central nervous system is mediated by AMPA-type ionotropic glutamate receptors. AMPA receptors co-assemble with auxiliary proteins, such as transmembrane AMPA receptor regulatory proteins (TARPs), cornichons, cysteine knot proteins and GSG1; which regulate AMPAR assembly, trafficking, gating, and pharmacology. TARPs are the most studied AMPAR auxiliary proteins for their role in the synaptic plasticity. The coupling between AMPA and TARPs change their kinetic and pharmacology properties. Here we show that in the presence of TARPs GluA2 receptors are inhibited by memantine. Memantine inhibition is dependent on charge at site 607, glutamine to arginine mutation showing a larger inhibition by memantine relative to the that GluA2(R) and the GluA2(Q). The inhibition by memantine is activity- and voltage-dependent, suggesting a pore block mechanism.

Small-animal PET study for noninvasive quantification of transmembrane AMPA receptor regulatory protein γ-8 (TARP γ-8) in the brain

Transmembrane AMPA receptor regulatory protein γ-8 (TARP γ-8) mediates various AMPA receptor functions. Recently, [11C]TARP-2105 was developed as a PET ligand for TARP γ-8 imaging. We performed a full kinetic analysis of [11C]TARP-2105 using PET with [11C]TARP-2105 for the first time. The distribution volume (VT), which is a macro parameter consisting of the K1–k4 rate constants in the two-tissue compartment model analysis, exhibited the following rank order: hippocampus (1.4 ± 0.3) > amygdala (1.0 ± 0.2) > frontal cortex (0.9 ± 0.2) > striatum (0.8 ± 0.2) ≫ cerebellum (0.5 ± 0.1) ≈ thalamus (0.5 ± 0.1) > pons (0.4 ± 0.1 mL/cm3). These heterogenous VT values corresponded with the order of biological distribution of TARP γ-8 in the brain. To validate the reference tissue model, the binding potential (BPND) of [11C]TARP-2105 for TARP γ-8 was estimated using general methods (SRTM, MRTM0, Logan reference model, and ratio method). These BPNDs based on reference models indicated excellent correlation (R2 > 0.9) to the indirect BPNDs based on 2TCM with moderate reproducibility (%variability ≈ 10). PET with [11C]TARP-2105 enabled noninvasive BPND estimation and visual mapping of TARP γ-8 in the living rat brain.

Expression and Interaction Proteomics of GluA1- and GluA3-Subunit-Containing AMPARs Reveal Distinct Protein Composition.

The AMPA glutamate receptor (AMPAR) is the major type of synaptic excitatory ionotropic receptor in the brain. AMPARs have four different subunits, GluA1-4 (each encoded by different genes, Gria1, Gria2, Gria3 and Gria4), that can form distinct tetrameric assemblies. The most abundant AMPAR subtypes in the hippocampus are GluA1/2 and GluA2/3 heterotetramers. Each subtype contributes differentially to mechanisms of synaptic plasticity, which may be in part caused by how these receptors are regulated by specific associated proteins. A broad range of AMPAR interacting proteins have been identified, including the well-studied transmembrane AMPA receptor regulatory proteins TARP-γ2 (also known as Stargazin) and TARP-γ8, Cornichon homolog 2 (CNIH-2) and many others. Several interactors were shown to affect biogenesis, AMPAR trafficking, and channel properties, alone or in distinct assemblies, and several revealed preferred binding to specific AMPAR subunits. To date, a systematic specific interactome analysis of the major GluA1/2 and GluA2/3 AMPAR subtypes separately is lacking. To reveal interactors belonging to specific AMPAR subcomplexes, we performed both expression and interaction proteomics on hippocampi of wildtype and Gria1- or Gria3 knock-out mice. Whereas GluA1/2 receptors co-purified TARP-γ8, synapse differentiation-induced protein 4 (SynDIG4, also known as Prrt1) and CNIH-2 with highest abundances, GluA2/3 receptors revealed strongest co-purification of CNIH-2, TARP-γ2, and Noelin1 (or Olfactomedin-1). Further analysis revealed that TARP-γ8-SynDIG4 interact directly and co-assemble into an AMPAR subcomplex especially at synaptic sites. Together, these data provide a framework for further functional analysis into AMPAR subtype specific pathways in health and disease.

Imaging of Transmembrane AMPA Receptor Regulatory Proteins by Positron Emission Tomography.

The transmembrane α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein γ-8 (TARP γ-8) constitutes an auxiliary subunit of AMPA receptors, which mediates various brain functions including learning and memory. TARP γ-8 has emerged as a promising therapeutic target for central nervous system disorders. Despite considerable efforts, previously reported TARP γ-8 PET radioligands, such as [11C]TARP-1903 and [11C]TARP-1811 series, were plagued by limited brain uptake and/or high nonspecific binding in vivo. Herein, we developed two novel 11C-labeled probes, [11C]8 and [11C]15 (also named as [11C]TARP-2105), of which the latter exhibited a reasonable brain uptake as well as specific binding toward TARP γ-8 both in vitro and in vivo, as confirmed by blocking experiments with the commercially available TARP γ-8 inhibitor, JNJ-55511118 in the TARP γ-8-rich hippocampus. Overall, [11C]15 exhibited promising tracer characteristics and proved to be a lead positron-emission tomography ligand for the non-invasive quantification of TARP γ-8 in the mammalian brain.

P-088 - Radiosynthesis of 6-(2-cyclobutyl-5-(methyl-11C)-3H-imidazo[4,5-b]pyridin-3-yl)benzo[d]thiazol-2(3H)-one and (2-cyclobutyl-3-(1H-indazol-5-yl)-5-[11C]methyl-3H-imidazo[4,5-b]pyridine for imaging γ-8 dependent transmembrane AMPA receptor regulatory protein

P-088 - Radiosynthesis of 6-(2-cyclobutyl-5-(methyl-11C)-3H-imidazo[4,5-b]pyridin-3-yl)benzo[d]thiazol-2(3H)-one and (2-cyclobutyl-3-(1H-indazol-5-yl)-5-[11C]methyl-3H-imidazo[4,5-b]pyridine for imaging γ-8 dependent transmembrane AMPA receptor regulatory protein

Aberrant hippocampal transmission and behavior in mice with a stargazin mutation linked to intellectual disability

Mutations linked to neurodevelopmental disorders, such as intellectual disability (ID), are frequently found in genes that encode for proteins of the excitatory synapse. Transmembrane AMPA receptor regulatory proteins (TARPs) are AMPA receptor auxiliary proteins that regulate crucial aspects of receptor function. Here, we investigate a mutant form of the TARP family member stargazin, described in an ID patient. Molecular dynamics analyses predicted that the ID-associated stargazin variant, V143L, weakens the overall interface of the AMPAR:stargazin complex and impairs the stability of the complex. Knock-in mice harboring the V143L stargazin mutation manifest cognitive and social deficits and hippocampal synaptic transmission defects, resembling phenotypes displayed by ID patients. In the hippocampus of stargazin V143L mice, CA1 neurons show impaired spine maturation, abnormal synaptic transmission and long-term potentiation specifically in basal dendrites, and synaptic ultrastructural alterations. These data suggest a causal role for mutated stargazin in the pathogenesis of ID and unveil a new role for stargazin in the development and function of hippocampal synapses.

Mechanisms underlying TARP modulation of the GluA1/2-\u03b38 AMPA receptor

AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor’s ligand-binding domains (LBDs) leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs) through currently incompletely resolved mechanisms. Here, electron cryo-microscopy structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states (at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop (‘β1’), elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the gate, and forms the major cation binding site in the conduction path. Our results provide a mechanistic framework of how TARPs modulate AMPAR gating and conductance.

Bidirectional protein–protein interactions control liquid–liquid phase separation of PSD-95 and its interaction partners

SummaryThe organization of the postsynaptic density (PSD), a protein-dense semi-membraneless organelle, is mediated by numerous specific protein–protein interactions (PPIs) which constitute a functional postsynapse. The PSD protein 95 (PSD-95) interacts with a manifold of proteins, including the C-terminal of transmembrane AMPA receptor (AMPAR) regulatory proteins (TARPs). Here, we uncover the minimal essential peptide responsible for the Stargazin (TARP-γ2)-mediated liquid–liquid phase separation (LLPS) formation of PSD-95 and other key protein constituents of the PSD. Furthermore, we find that pharmacological inhibitors of PSD-95 can facilitate the formation of LLPS. We found that in some cases LLPS formation is dependent on multivalent interactions, while in other cases short, highly charged peptides are sufficient to promote LLPS in complex systems. This study offers a new perspective on PSD-95 interactions and their role in LLPS formation, while also considering the role of affinity over multivalency in LLPS systems.

Deficiency of transmembrane AMPA receptor regulatory protein γ-8 leads to attention-deficit hyperactivity disorder-like behavior in mice.

Attention-deficit hyperactivity disorder (ADHD) is a neurodevelopmental disorder prevalent in school-age children. At present, however, its etiologies and risk factors are unknown. Transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein γ-8 (TARP γ-8, also known as calcium voltage-gated channel auxiliary subunit gamma 8 (CACNG8)) is an auxiliary AMPA receptor (AMPAR) subunit. Here, we report an association between TARP γ-8 and ADHD, whereby adolescent TARP γ-8 knockout (KO) mice exhibited ADHD-like behaviors, including hyperactivity, impulsivity, anxiety, impaired cognition, and memory deficits. Human single-nucleotide polymorphism (SNP) analysis also revealed strong associations between intronic alleles in CACNG8 genes and ADHD susceptibility. In addition, synaptosomal proteomic analysis revealed dysfunction of the AMPA glutamate receptor complex in the hippocampi of TARP γ-8 KO mice. Proteomic analysis also revealed dysregulation of dopaminergic and glutamatergic transmissions in the prefrontal cortices of TARP γ-8 KO mice. Methylphenidate (MPH), which is commonly used to treat ADHD, significantly rescued the major behavioral deficits and abnormal synaptosomal proteins in TARP γ-8 KO mice. Notably, MPH significantly reversed the up-regulation of Grik2 and Slc6a3 in the prefrontal cortex. MPH also significantly improved synaptic AMPAR complex function by up-regulating other AMPAR auxiliary proteins in hippocampal synaptosomes. Taken together, our results suggest that TARP γ-8 is involved in the development of ADHD in humans. This study provides a useful alternative animal model with ADHD-like phenotypes related to TARP γ-8 deficiency, which has great potential for the development of new therapies.