Eric Boué-Grabot

Expression of GABA receptor Ρ subunits in rat brain

Abstract: The GABA receptor ρ1, ρ2, and ρ3 subunits are expressed in the retina where they form bicuculline‐insensitive GABAC receptors. We used northern blot, in situ hybridization, and RT‐PCR analysis to study the expression of ρ subunits in rat brains. In situ hybridization allowed us to detect ρ‐subunit expression in the superficial gray layer of the superior colliculus and in the cerebellar Purkinje cells. RT‐PCR experiments indicated that (a) in retina and in domains that may contain functional GABAC receptors, ρ2 and ρ1 subunits are expressed at similar levels; and (b) in domains and in tissues that are unlikely to contain GABAC receptors, ρ2 mRNA is enriched relative to ρ1 mRNA. These results suggest that both ρ1 and ρ2 subunits are necessary to form a functional GABAC receptor. The use of RT‐PCR also showed that, except in the superior colliculus, ρ3 is expressed along with ρ1 and ρ2 subunits. We also raised an antibody against a peptide sequence unique to the ρ1 subunit. The use of this antibody on cerebellum revealed the rat ρ1 subunit in the soma and dendrites of Purkinje neurons. The allocation of GABAC receptor subunits to identified neurons paves the way for future electrophysiological studies.

Functional and biochemical evidence for heteromeric ATP-gated channels composed of P2X1 and P2X5 subunits.

The mammalian P2X receptor gene family encodes two-transmembrane domain nonselective cation channels gated by extracellular ATP. Anatomical localization data obtained by in situ hybridization and immunocytochemistry have shown that neuronal P2X subunits are expressed in specific but overlapping distribution patterns. Therefore, the native ionotropic ATP receptors diversity most likely arises from interactions between different P2X subunits that generate hetero-multimers phenotypically distinct from homomeric channels. Rat P2X1 and P2X5 mRNAs are localized within common subsets of peripheral and central sensory neurons as well as spinal motoneurons. The present study demonstrates a functional association between P2X1 and P2X5subunits giving rise to hybrid ATP-gated channels endowed with the pharmacology of P2X1 and the kinetics of P2X5. When expressed in Xenopus oocytes, hetero-oligomeric P2X1+5 ATP receptors were characterized by slowly desensitizing currents highly sensitive to the agonist α,β-methylene ATP (EC50 = 1.1 μm) and to the antagonist trinitrophenyl ATP (IC50 = 64 nm), observed with neither P2X1 nor P2X5 alone. Direct physical evidence for P2X1+5co-assembly was provided by reciprocal subunit-specific co-purifications between epitope-tagged P2X1 and P2X5 subunits transfected in HEK-293A cells.

PSD-95 expression controls l-DOPA dyskinesia through dopamine D1 receptor trafficking

L-DOPA-induced dyskinesia (LID), a detrimental consequence of dopamine replacement therapy for Parkinsons disease, is associated with an alteration in dopamine D1 receptor (D1R) and glutamate receptor interactions. We hypothesized that the synaptic scaffolding protein PSD-95 plays a pivotal role in this process, as it interacts with D1R, regulates its trafficking and function, and is overexpressed in LID. Here, we demonstrate in rat and macaque models that disrupting the interaction between D1R and PSD-95 in the striatum reduces LID development and severity. Single quantum dot imaging revealed that this benefit was achieved primarily by destabilizing D1R localization, via increased lateral diffusion followed by increased internalization and diminished surface expression. These findings indicate that altering D1R trafficking via synapse-associated scaffolding proteins may be useful in the treatment of dyskinesia in Parkinsons patients.

Phosphoinositides Regulate P2X4 ATP-Gated Channels through Direct Interactions

P2X receptors are ATP-gated nonselective cation channels highly permeable to calcium that contribute to nociception and inflammatory responses. The P2X4 subtype, upregulated in activated microglia, is thought to play a critical role in the development of tactile allodynia following peripheral nerve injury. Posttranslational regulation of P2X4 function is crucial to the cellular mechanisms of neuropathic pain, however it remains poorly understood. Here, we show that the phosphoinositides PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3), products of phosphorylation by wortmannin-sensitive phosphatidylinositol 4-kinases and phosphatidylinositol 3-kinases, can modulate the function of native and recombinant P2X4 receptor channels. In BV-2 microglial cells, depleting the intracellular levels of PIP2 and PIP3 with wortmannin significantly decreased P2X4 current amplitude and P2X4-mediated calcium entry measured in patch clamp recordings and ratiometric ion imaging, respectively. Wortmannin-induced depletion of phosphoinositides in Xenopus oocytes decreased the current amplitude of P2X4 responses by converting ATP into a partial agonist. It also decreased their recovery from desensitization and affected their kinetics. Injection of phosphoinositides in wortmannin-treated oocytes reversed these effects and application of PIP2 on excised inside-out macropatches rescued P2X4 currents from rundown. Moreover, we report the direct interaction of phospholipids with the proximal C-terminal domain of P2X4 subunit (Cys360–Val375) using an in vitro binding assay. These results demonstrate novel regulatory roles of the major signaling phosphoinositides PIP2 and PIP3 on P2X4 function through direct channel–lipid interactions.

A γ2(R43Q) Mutation, Linked to Epilepsy in Humans, Alters GABAA Receptor Assembly and Modifies Subunit Composition on the Cell Surface

Genetic defects leading to epilepsy have been identified in γ2 GABAA receptor subunit. A γ2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a γ2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABAA receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant γ2 constructs and showed that the γ 2(R43Q) mutation prevented surface expression of the subunit, unlike γ2(K289M) substitution. Several γ2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with α3- and β3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the γ2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant γ2(R43Q) was retained within intracellular compartments, αβ complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that γ2(R43Q) was able to associate with α3- or β3-subunits, although the stoichiometry of the complex with α3 was altered. Our data show that γ2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABAA receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the γ2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous γ2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.

Unique functional properties of a sensory neuronal P2X ATP-gated channel from zebrafish.

Abstract: We report here the structural and functional characterization of an ionotropic P2X ATP receptor from the lower vertebrate zebrafish (Danio rerio). The full‐length cDNA encodes a 410‐amino acid‐long channel subunit zP2X3, which shares only 54% identity with closest mammalian P2X subunits. When expressed in Xenopus oocytes in homomeric form, ATP‐gated zP2X3 channels evoked a unique nonselective cationic current with faster rise time, faster kinetics of desensitization, and slower recovery than any other known P2X channel. Interestingly, the order of agonist potency for this P2X receptor was found similar to that of distantly related P2X7 receptors, with benzoylbenzoyl ATP (EC50 = 5 μM) ≫ ATP (EC50 = 350 μM) = ADP > α,β‐methylene ATP (EC50 = 480 μM). zP2X3 receptors are highly sensitive to blockade by the antagonist trinitrophenyl ATP (IC50 < 5 nM) but are weakly sensitive to the noncompetitive antagonist pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulfonic acid. zP2X3 subunit mRNA is exclusively expressed at high levels in trigeminal neurons and Rohon‐Beard cells during embryonic development, suggesting that neuronal P2X receptors mediating fast ATP responses were selected early in the vertebrate phylogeny to play an important role in sensory pathways.

Molecular and electrophysiological evidence for a GABAc receptor in thyrotropin-secreting cells.

In the pituitary, GABA regulates the release of several hormones via different receptors. GABAC receptors are heterooligomers that differ from GABAA receptors in that they containρ -subunits and are insensitive to bicuculline. However, molecular and functional evidence for the presence of GABAC receptors outside the retina has yet to be established. The present work was performed on guinea pig and rat pituitaries. Both Northern blot and RT-PCR analysis showed that, although ρ1- and ρ2-subunits were expressed at similar levels in the rat retina, ρ1 messenger RNA (mRNA) was enriched, relative to ρ2 mRNA in the rat pituitary. Northern blot experiments also showed that, in the pituitary, ρ1 andρ 2 mRNAs are shorter in size than those expressed in the retina. The use of a subunit-specific antibody revealed colocalization ofρ 1-subunit and anti-TSH labeling on rat pituitary sections. TSH guinea pig pituitary cells were also labeled with a ρ-subunit antiserum. Moreover, whole-cell patch clamp on single guinea pi...

Selective Inactivation of Striatal FosB/ΔFosB-Expressing Neurons Alleviates L-DOPA–Induced Dyskinesia

BACKGROUND ΔFosB is a surrogate marker of L-DOPA-induced dyskinesia (LID), the unavoidable disabling consequence of Parkinsons disease L-DOPA long-term treatment. However, the relationship between the electrical activity of FosB/ΔFosB-expressing neurons and LID manifestation is unknown. METHODS We used the Daun02 prodrug-inactivation method associated with lentiviral expression of β-galactosidase under the control of the FosB promoter to investigate a causal link between the activity of FosB/ΔFosB-expressing neurons and dyskinesia severity in both rat and monkey models of Parkinsons disease and LID. Whole-cell recordings of medium spiny neurons (MSNs) were performed to assess the effects of Daun02 and daunorubicin on neuronal excitability. RESULTS We first show that daunorubicin, the active product of Daun02 metabolism by β-galactosidase, decreases the activity of MSNs in rat brain slices and that Daun02 strongly decreases the excitability of rat MSN primary cultures expressing β-galactosidase upon D1 dopamine receptor stimulation. We then demonstrate that the selective, and reversible, inhibition of FosB/ΔFosB-expressing striatal neurons with Daun02 decreases the severity of LID while improving the beneficial effect of L-DOPA. CONCLUSIONS These results establish that FosB/ΔFosB accumulation ultimately results in altered neuronal electrical properties sustaining maladaptive circuits leading not only to LID but also to a blunted response to L-DOPA. These findings further reveal that targeting dyskinesia can be achieved without reducing the antiparkinsonian properties of L-DOPA when specifically inhibiting FosB/ΔFosB-accumulating neurons.

ATP P2X Receptors Downregulate AMPA Receptor Trafficking and Postsynaptic Efficacy in Hippocampal Neurons.

P2X receptors (P2XRs) are ATP-gated cation channels widely expressed in the brain where they mediate action of extracellular ATP released by neurons or glia. Although purinergic signaling has multiple effects on synaptic transmission and plasticity, P2XR function at brain synapses remains to be established. Here, we show that activation of postsynaptic P2XRs by exogenous ATP or noradrenaline-dependent glial release of endogenous ATP decreases the amplitude of miniature excitatory postsynaptic currents and AMPA-evoked currents in cultured hippocampal neurons. We also observed a P2X-mediated depression of field potentials recorded in CA1 region from brain slices. P2X2Rs trigger dynamin-dependent internalization of AMPA receptors (AMPARs), leading to reduced surface AMPARs in dendrites and at synapses. AMPAR alteration required calcium influx through opened ATP-gated channels and phosphatase or CamKII activities. These findings indicate that postsynaptic P2XRs play a critical role in regulating the surface expression of AMPARs and thereby regulate the synaptic strength.

An intracellular motif of P2X(3) receptors is required for functional cross-talk with GABA(A) receptors in nociceptive DRG neurons.

Functional cross‐talk between structurally unrelated P2X ATP receptors and members of the ‘cys‐loop’ receptor‐channel superfamily represents a recently‐discovered mechanism for rapid modulation of information processing. The extent and the mechanism of the inhibitory cross‐talks between these two classes of ionotropic receptors remain poorly understood, however. Both ionic and molecular coupling were proposed to explain cross‐inhibition between P2X subtypes and GABAA receptors, suggesting a P2X subunit‐dependent mechanism. We show here that cross‐inhibition between neuronal P2X3 or P2X2+3 and GABAA receptors does not depend on chloride and calcium ions. We identified an intracellular QST386–388 motif in P2X3 subunits which is required for the functional coupling with GABAA receptors. Moreover the cross‐inhibition between native P2X3 and GABA receptors in cultured rat dorsal root ganglia (DRG) neurons is abolished by infusion of a peptide containing the QST motif as well as by viral expression of the main intracellular loop of GABAAβ3 subunits. We provide evidence that P2X3 and GABAA receptors are colocalized in the soma and central processes of nociceptive DRG neurons, suggesting that specific intracellular P2X3‐GABAA subunit interactions underlie a pre‐synaptic cross‐talk that might contribute to the regulation of sensory synaptic transmission in the spinal cord.