Spontaneous changes in brain striatal dopamine synthesis and storage dynamics ex vivo reveal end-product feedback-inhibition of tyrosine hydroxylase.

Synaptic events are important to define treatment strategies for brain disorders. In the present paper, freshly obtained rat brain striatal minces were incubated under different times and conditions to determine dopamine biosynthesis, storage, and tyrosine hydroxylase phosphorylation. Remarkably, we found that endogenous dopamine spontaneously accumulated during tissue incubation at 37 °C ex vivo while dopamine synthesis simultaneously decreased. We analyzed whether these changes in brain dopamine biosynthesis and storage were linked to dopamine feedback inhibition of its synthesis-limiting enzyme tyrosine hydroxylase. The aromatic-l-amino-acid decarboxylase inhibitor NSD-1015 prevented both effects. As expected, dopamine accumulation was increased with l-DOPA addition or VMAT2-overexpression, and dopamine synthesis decreased further with added dopamine, the VMAT2 inhibitor tetrabenazine or D2 auto-receptor activation with quinpirole, accordingly to the known synaptic effects of these treatments. Phosphorylation activation and inhibition of tyrosine hydroxylase on Ser31 and Ser40 with okadaic acid, Sp-cAMP and PD98059 also exerted the expected effects. However, no clear-cut association was found between dopamine feedback inhibition of its own biosynthesis and changes of tyrosine hydroxylase phosphorylation, assessed by Western blot and mass spectrometry. The later technique also revealed a new Thr30 phosphorylation in rat tyrosine hydroxylase. Our methodological assessment of brain dopamine synthesis and storage dynamics ex vivo could be applied to predict the in vivo effects of pharmacological interventions in animal models of dopamine-related disorders.

Modulation of dopamine D1 receptors via histamine H3 receptors is a novel therapeutic target for Huntington's disease.

Early Huntington's disease (HD) include over-activation of dopamine D1 receptors (D1R), producing an imbalance in dopaminergic neurotransmission and cell death. To reduce D1R over-activation, we present a strategy based on targeting complexes of D1R and histamine H3 receptors (H3R). Using an HD mouse striatal cell model and HD mouse organotypic brain slices we found that D1R-induced cell death signaling and neuronal degeneration, are mitigated by an H3R antagonist. We demonstrate that the D1R-H3R heteromer is expressed in HD mice at early but not late stages of HD, correlating with HD progression. In accordance, we found this target expressed in human control subjects and low-grade HD patients. Finally, treatment of HD mice with an H3R antagonist prevented cognitive and motor learning deficits and the loss of heteromer expression. Taken together, our results indicate that D1R - H3R heteromers play a pivotal role in dopamine signaling and represent novel targets for treating HD.

D1-mGlu5 heteromers mediate noncanonical dopamine signaling in Parkinson's disease.

Dopamine receptor D1 modulates glutamatergic transmission in cortico-basal ganglia circuits and represents a major target of L-DOPA therapy in Parkinson's disease. Here we show that D1 and metabotropic glutamate type 5 (mGlu5) receptors can form previously unknown heteromeric entities with distinctive functional properties. Interacting with Gq proteins, cell-surface D1-mGlu5 heteromers exacerbated PLC signaling and intracellular calcium release in response to either glutamate or dopamine. In rodent models of Parkinson's disease, D1-mGlu5 nanocomplexes were strongly upregulated in the dopamine-denervated striatum, resulting in a synergistic activation of PLC signaling by D1 and mGlu5 receptor agonists. In turn, D1-mGlu5-dependent PLC signaling was causally linked with excessive activation of extracellular signal-regulated kinases in striatal neurons, leading to dyskinesia in animals treated with L-DOPA or D1 receptor agonists. The discovery of D1-mGlu5 functional heteromers mediating maladaptive molecular and motor responses in the dopamine-denervated striatum may prompt the development of new therapeutic principles for Parkinson's disease.

Discovery of a small molecule modulator of the Kv1.1/Kvß1 channel complex that reduces neuronal excitability and in vitro epileptiform activity.

AIMS: Kv1.1 (KCNA1) channels contribute to the control of neuronal excitability and have been associated with epilepsy. Kv1.1 channels can associate with the cytoplasmic Kvß1 subunit resulting in rapid inactivating A-type currents. We hypothesized that removal of channel inactivation, by modulating Kv1.1/Kvß1 interaction with a small molecule, would lead to decreased neuronal excitability and anticonvulsant activity. METHODS: We applied high-throughput screening to identify ligands able to modulate the Kv1.1-T1 domain/Kvß1 protein complex. We then selected a compound that was characterized on recombinant Kv1.1/Kvß1 channels by electrophysiology and further evaluated on sustained neuronal firing and on in vitro epileptiform activity using a high K+ -low Ca2+ model in hippocampal slices. RESULTS: We identified a novel compound able to modulate the interaction of the Kv1.1/Kvß1 complex and that produced a functional inhibition of Kv1.1/Kvß1 channel inactivation. We demonstrated that this compound reduced the sustained repetitive firing in hippocampal neurons and was able to abolish the development of in vitro epileptiform activity. CONCLUSIONS: This study describes a rational drug discovery approach for the identification of novel ligands that inhibit Kv1.1 channel inactivation and provides pharmacological evidence that such a mechanism translates into physiological effects by reducing in vitro epileptiform activity.

Class C G protein-coupled receptors: reviving old couples with new partners.

G protein-coupled receptors (GPCRs) are key players in cell communication and are encoded by the largest family in our genome. As such, GPCRs represent the main targets in drug development programs. Sequence analysis revealed several classes of GPCRs: the class A rhodopsin-like receptors represent the majority, the class B includes the secretin-like and adhesion GPCRs, the class F includes the frizzled receptors, and the class C includes receptors for the main neurotransmitters, glutamate and GABA, and those for sweet and umami taste and calcium receptors. Class C receptors are far more complex than other GPCRs, being mandatory dimers, with each subunit being composed of several domains. In this review, we summarize our actual knowledge regarding the activation mechanism and subunit organization of class C GPCRs, and how this brings information for many other GPCRs.

Allosteric nanobodies uncover a role of hippocampal mGlu2 receptor homodimers in contextual fear consolidation.

Antibodies have enormous therapeutic and biotechnology potential. G protein-coupled receptors (GPCRs), the main targets in drug development, are of major interest in antibody development programs. Metabotropic glutamate receptors are dimeric GPCRs that can control synaptic activity in a multitude of ways. Here we identify llama nanobodies that specifically recognize mGlu2 receptors, among the eight subtypes of mGluR subunits. Among these nanobodies, DN10 and 13 are positive allosteric modulators (PAM) on homodimeric mGlu2, while DN10 displays also a significant partial agonist activity. DN10 and DN13 have no effect on mGlu2-3 and mGlu2-4 heterodimers. These PAMs enhance the inhibitory action of the orthosteric mGlu2/mGlu3 agonist, DCG-IV, at mossy fiber terminals in the CA3 region of hippocampal slices. DN13 also impairs contextual fear memory when injected in the CA3 region of hippocampal region. These data highlight the potential of developing antibodies with allosteric actions on GPCRs to better define their roles in vivo.

Allosteric control of an asymmetric transduction in a G protein-coupled receptor heterodimer.

GPCRs play critical roles in cell communication. Although GPCRs can form heteromers, their role in signaling remains elusive. Here we used rat metabotropic glutamate (mGlu) receptors as prototypical dimers to study the functional interaction between each subunit. mGluRs can form both constitutive homo- and heterodimers. Whereas both mGlu2 and mGlu4 couple to G proteins, G protein activation is mediated by mGlu4 heptahelical domain (HD) exclusively in mGlu2-4 heterodimers. Such asymmetric transduction results from the action of both the dimeric extracellular domain, and an allosteric activation by the partially-activated non-functional mGlu2 HD. G proteins activation by mGlu2 HD occurs if either the mGlu2 HD is occupied by a positive allosteric modulator or if mGlu4 HD is inhibited by a negative modulator. These data revealed an oriented asymmetry in mGlu heterodimers that can be controlled with allosteric modulators. They provide new insight on the allosteric interaction between subunits in a GPCR dimer.

Pharmacological evidence for a metabotropic glutamate receptor heterodimer in neuronal cells.

Metabotropic glutamate receptors (mGluRs) are mandatory dimers playing important roles in regulating CNS function. Although assumed to form exclusive homodimers, 16 possible heterodimeric mGluRs have been proposed but their existence in native cells remains elusive. Here, we set up two assays to specifically identify the pharmacological properties of rat mGlu heterodimers composed of mGlu2 and 4 subunits. We used either a heterodimer-specific conformational LRET-based biosensor or a system that guarantees the cell surface targeting of the heterodimer only. We identified mGlu2-4 specific pharmacological fingerprints that were also observed in a neuronal cell line and in lateral perforant path terminals naturally expressing mGlu2 and mGlu4. These results bring strong evidence for the existence of mGlu2-4 heterodimers in native cells. In addition to reporting a general approach to characterize heterodimeric mGluRs, our study opens new avenues to understanding the pathophysiological roles of mGlu heterodimers.

HTS-compatible FRET-based conformational sensors clarify membrane receptor activation.

Cell surface receptors represent a vast majority of drug targets. Efforts have been conducted to develop biosensors reporting their conformational changes in live cells for pharmacological and functional studies. Although Förster resonance energy transfer (FRET) appears to be an ideal approach, its use is limited by the low signal-to-noise ratio. Here we report a toolbox composed of a combination of labeling technologies, specific fluorophores compatible with time-resolved FRET and a novel method to quantify signals. This approach enables the development of receptor biosensors with a large signal-to-noise ratio. We illustrate the usefulness of this toolbox through the development of biosensors for various G-protein-coupled receptors and receptor tyrosine kinases. These receptors include mGlu, GABAB, LH, PTH, EGF and insulin receptors among others. These biosensors can be used for high-throughput studies and also revealed new information on the activation process of these receptors in their cellular environment.

Heteroreceptor Complexes Formed by Dopamine D1, Histamine H3, and N-Methyl-D-Aspartate Glutamate Receptors as Targets to Prevent Neuronal Death in Alzheimer's Disease.

Alzheimer's disease (AD) is a neurodegenerative disorder causing progressive memory loss and cognitive dysfunction. Anti-AD strategies targeting cell receptors consider them as isolated units. However, many cell surface receptors cooperate and physically contact each other forming complexes having different biochemical properties than individual receptors. We here report the discovery of dopamine D1, histamine H3, and N-methyl-D-aspartate (NMDA) glutamate receptor heteromers in heterologous systems and in rodent brain cortex. Heteromers were detected by co-immunoprecipitation and in situ proximity ligation assays (PLA) in the rat cortex where H3 receptor agonists, via negative cross-talk, and H3 receptor antagonists, via cross-antagonism, decreased D1 receptor agonist signaling determined by ERK1/2 or Akt phosphorylation, and counteracted D1 receptor-mediated excitotoxic cell death. Both D1 and H3 receptor antagonists also counteracted NMDA toxicity suggesting a complex interaction between NMDA receptors and D1-H3 receptor heteromer function. Likely due to heteromerization, H3 receptors act as allosteric regulator for D1 and NMDA receptors. By bioluminescence resonance energy transfer (BRET), we demonstrated that D1 or H3 receptors form heteromers with NR1A/NR2B NMDA receptor subunits. D1-H3-NMDA receptor complexes were confirmed by BRET combined with fluorescence complementation. The endogenous expression of complexes in mouse cortex was determined by PLA and similar expression was observed in wild-type and APP/PS1 mice. Consistent with allosteric receptor-receptor interactions within the complex, H3 receptor antagonists reduced NMDA or D1 receptor-mediated excitotoxic cell death in cortical organotypic cultures. Moreover, H3 receptor antagonists reverted the toxicity induced by ß1-42-amyloid peptide. Thus, histamine H3 receptors in D1-H3-NMDA heteroreceptor complexes arise as promising targets to prevent neurodegeneration.

Orexin-corticotropin-releasing factor receptor heteromers in the ventral tegmental area as targets for cocaine.

Release of the neuropeptides corticotropin-releasing factor (CRF) and orexin-A in the ventral tegmental area (VTA) play an important role in stress-induced cocaine-seeking behavior. We provide evidence for pharmacologically significant interactions between CRF and orexin-A that depend on oligomerization of CRF1 receptor (CRF1R) and orexin OX1 receptors (OX1R). CRF1R-OX1R heteromers are the conduits of a negative crosstalk between orexin-A and CRF as demonstrated in transfected cells and rat VTA, in which they significantly modulate dendritic dopamine release. The cocaine target σ1 receptor (σ1R) also associates with the CRF1R-OX1R heteromer. Cocaine binding to the σ1R-CRF1R-OX1R complex promotes a long-term disruption of the orexin-A-CRF negative crosstalk. Through this mechanism, cocaine sensitizes VTA cells to the excitatory effects of both CRF and orexin-A, thus providing a mechanism by which stress induces cocaine seeking.

Cocaine disrupts histamine H3 receptor modulation of dopamine D1 receptor signaling: σ1-D1-H3 receptor complexes as key targets for reducing cocaine's effects.

The general effects of cocaine are not well understood at the molecular level. What is known is that the dopamine D1 receptor plays an important role. Here we show that a key mechanism may be cocaine's blockade of the histamine H3 receptor-mediated inhibition of D1 receptor function. This blockade requires the σ1 receptor and occurs upon cocaine binding to σ1-D1-H3 receptor complexes. The cocaine-mediated disruption leaves an uninhibited D1 receptor that activates Gs, freely recruits ß-arrestin, increases p-ERK 1/2 levels, and induces cell death when over activated. Using in vitro assays with transfected cells and in ex vivo experiments using both rats acutely treated or self-administered with cocaine along with mice depleted of σ1 receptor, we show that blockade of σ1 receptor by an antagonist restores the protective H3 receptor-mediated brake on D1 receptor signaling and prevents the cell death from elevated D1 receptor signaling. These findings suggest that a combination therapy of σ1R antagonists with H3 receptor agonists could serve to reduce some effects of cocaine.

Circadian-related heteromerization of adrenergic and dopamine D4 receptors modulates melatonin synthesis and release in the pineal gland.

The role of the pineal gland is to translate the rhythmic cycles of night and day encoded by the retina into hormonal signals that are transmitted to the rest of the neuronal system in the form of serotonin and melatonin synthesis and release. Here we describe that the production of both melatonin and serotonin by the pineal gland is regulated by a circadian-related heteromerization of adrenergic and dopamine D4 receptors. Through α(1B)-D4 and ß1-D4 receptor heteromers dopamine inhibits adrenergic receptor signaling and blocks the synthesis of melatonin induced by adrenergic receptor ligands. This inhibition was not observed at hours of the day when D4 was not expressed. These data provide a new perspective on dopamine function and constitute the first example of a circadian-controlled receptor heteromer. The unanticipated heteromerization between adrenergic and dopamine D4 receptors provides a feedback mechanism for the neuronal hormone system in the form of dopamine to control circadian inputs.

Cannabinoid receptors CB1 and CB2 form functional heteromers in brain.

Exploring the role of cannabinoid CB(2) receptors in the brain, we present evidence of CB(2) receptor molecular and functional interaction with cannabinoid CB(1) receptors. Using biophysical and biochemical approaches, we discovered that CB(2) receptors can form heteromers with CB(1) receptors in transfected neuronal cells and in rat brain pineal gland, nucleus accumbens, and globus pallidus. Within CB(1)-CB(2) receptor heteromers expressed in a neuronal cell model, agonist co-activation of CB(1) and CB(2) receptors resulted in a negative cross-talk in Akt phosphorylation and neurite outgrowth. Moreover, one specific characteristic of CB(1)-CB(2) receptor heteromers consists of both the ability of CB(1) receptor antagonists to block the effect of CB(2) receptor agonists and, conversely, the ability of CB(2) receptor antagonists to block the effect of CB(1) receptor agonists, showing a bidirectional cross-antagonism phenomenon. Taken together, these data illuminate the mechanism by which CB(2) receptors can negatively modulate CB(1) receptor function.

Constitutive activity of H3 autoreceptors modulates histamine synthesis in rat brain through the cAMP/PKA pathway.

We previously described that agonist-activated histamine H3 autoreceptors inhibit the stimulation of histamine synthesis mediated by calcium/calmodulin- and cAMP-dependent protein kinases (CaMKII and PKA respectively) in histaminergic nerve endings. In the absence of an agonist H3 receptors show partial constitutive activity, so we hypothesized that suppression of constitutive activity by an inverse agonist could stimulate these transduction pathways. We show here that the H3 inverse agonist thioperamide increases histamine synthesis in rat brain cortical slices independently from the amounts of extracellular histamine. Thioperamide effects were mimicked by the inverse agonists clobenpropit and A-331440, but not by the neutral antagonist VUF-5681. In contrast, coincubation with VUF-5681 suppressed thioperamide effects. The effects of thioperamide were completely blocked by the PKA inhibitor peptide myristoyl-PKI14-22, a peptide that did not block depolarization stimulation of histamine synthesis. In addition, thioperamide effects required depolarization and were impaired by blockade of N-type calcium channels (mediating depolarization), but not by CaMKII inhibition. These results indicate that constitutive activity of H3 receptors in rat brain cortex inhibits the adenylate cyclase/PKA pathway, and perhaps also the opening of N-type voltage sensitive calcium channels, but apparently not CaMKII.

H3 autoreceptors modulate histamine synthesis through calcium/calmodulin- and cAMP-dependent protein kinase pathways.

H(3) autoreceptors provide feedback control of neurotransmitter synthesis in histaminergic neurons, but the transduction pathways involved are poorly understood. In rat brain cortical slices, histamine synthesis can be stimulated by depolarization and inhibited by H(3) agonists. We show that histamine synthesis stimulation by depolarization with 30 mM K(+) requires extracellular calcium entry, mostly through N-type channels, and subsequent activation of calcium/calmodulin-dependent protein kinase type II. In vitro, this kinase phosphorylated and activated histidine decarboxylase, the histamine-synthesizing enzyme. Inhibition of depolarization-stimulated histamine synthesis by the histamine H(3) receptor agonist imetit was impaired by preincubation with pertussis toxin and by the presence of a myristoylated peptide (myristoyl-N-QEHAQEPERQYMHIGTMVE-FAYALVGK) blocking the actions of G-protein betagamma subunits. The stimulation of another G(i/o)-coupled receptor, adenosine A(1), also decreased depolarization-stimulated histamine synthesis. In contrast, protein kinase A activation, which is also repressed by H(3) receptors, elicited a depolarization- and calcium/calmodulin-independent stimulation of histamine synthesis. Protein kinase A was able also to phosphorylate and activate histidine decarboxylase in vitro. These results show how depolarization activates histamine synthesis in nerve endings and demonstrate that both pathways modulating neurotransmitter synthesis are controlled by H(3) autoreceptors.