Detection, Analysis, and Quantification of GPCR Homo- and Heteroreceptor Complexes in Specific Neuronal Cell Populations Using the In Situ Proximity Ligation Assay

GPCR’s receptosome operates via coordinated changes between the receptor expression, their modifications and interactions between each other. Perturbation in specific heteroreceptor complexes and/or their balance/equilibrium with other heteroreceptor complexes and corresponding homoreceptor complexes is considered to have a role in pathogenic mechanisms. Such mechanisms lead to mental and neurological diseases, including drug addiction, depression, Parkinson’s disease, and schizophrenia. To understand the associations of GPCRs and to unravel the global picture of their receptor–receptor interactions in the brain, different experimental detection techniques for receptor–receptor interactions have been established (e.g., co-immunoprecipitation based approach). However, they have been criticized for not reflecting the cellular situation or the dynamic nature of receptor–receptor interactions. Therefore, the detection and visualization of GPCR homo- and eteroreceptor complexes in the brain remained largely unknown until recent years, when a well-characterized in situ proximity ligation assay (in situ PLA) was adapted to validate the receptor complexes in their native environment. The in situ PLA protocol presented here can be used to visualize GPCR receptor–receptor interactions in cells and tissues in a highly sensitive and specific manner. We have developed a combined method using immunohistochemistry and PLA, particularly aimed to monitor interactions between GPCRs in specific neuronal cell populations. This allows the analysis of homo- and heteroreceptor complexes at a cellular and subcellular level. The method has the advantage that it can be used in clinical specimens, providing localized, quantifiable homo- and heteroreceptor complexes detected in single cells. We compare the advantages and limitations of the methods, underlining recent progress and the growing importance of these techniques in basic research. We discuss also their potential as tools for drug development and diagnostics

Detection, Analysis, and Quantification of GPCR Homo and Heteroreceptor Complexes in Specific Neuronal Cell Populations Using the In Situ Proximity Ligation Assay

GPCR’s receptosome operates via coordinated changes between the receptor expression, their modifications and interactions between each other. Perturbation in specific heteroreceptor complexes and/or their balance/equilibrium with other heteroreceptor complexes and corresponding homoreceptor complexes is considered to have a role in pathogenic mechanisms. Such mechanisms lead to mental and neurological diseases, including drug addiction, depression, Parkinson’s disease, and schizophrenia. To understand the associations of GPCRs and to unravel the global picture of their receptor–receptor interactions in the brain, different experimental detection techniques for receptor–receptor interactions have been established (e.g., co-immunoprecipitation based approach). However, they have been criticized for not reflecting the cellular situation or the dynamic nature of receptor–receptor interactions. Therefore, the detection and visualization of GPCR homo- and heteroreceptor complexes in the brain remained largely unknown until recent years, when a well-characterized in situ proximity ligation assay (in situ PLA) was adapted to validate the receptor complexes in their native environment. The in situ PLA protocol presented here can be used to visualize GPCR receptor–receptor interactions in cells and tissues in a highly sensitive and specific manner. We have developed a combined method using immunohistochemistry and PLA, particularly aimed to monitor interactions between GPCRs in specific neuronal cell populations. This allows the analysis of homo- and heteroreceptor complexes at a cellular and subcellular level. The method has the advantage that it can be used in clinical specimens, providing localized, quantifiable homo- and heteroreceptor complexes detected in single cells. We compare the advantages and limitations of the methods, underlining recent progress and the growing importance of these techniques in basic research. We discuss also their potential as tools for drug development and diagnostics

Dopamine heteroreceptor complexes as therapeutic targets in Parkinson's disease.

NTRODUCTION: Several types of D2R and D1R heteroreceptor complexes were discovered in the indirect and direct pathways of the striatum, respectively. The hypothesis is given that changes in the function of the dopamine heteroreceptor complexes may help us understand the molecular mechanisms underlying the motor complications of long-term therapy in Parkinson's disease (PD) with l-DOPA and dopamine receptor agonists. AREAS COVERED: In the indirect pathway, the potential role of the A2AR-D2R, A2AR-D2R-mGluR5 and D2R-NMDAR heteroreceptor complexes in PD are covered and in the direct pathway, the D1R-D3R, A1R-D1R, D1R-NMDAR and putative A1R-D1R-D3R heteroreceptor complexes. EXPERT OPINION: One explanation for the more powerful ability of l-DOPA treatment versus treatment with the partial dopamine receptor agonist/antagonist activity to induce dyskinesias, may be that dopamine formed from l-DOPA acts as a full agonist. The field of D1R and D2R heteroreceptor complexes in the CNS opens up a new understanding of the wearing off of the antiparkinson actions of l-DOPA and dopamine receptor agonists and the production of l-DOPA-induced dyskinesias. It can involve a reorganization of the D1R and D2R heteroreceptor complexes and a disbalance of the D1R and D2R homomers versus non-dopamine receptor homomers in the direct and indirect pathways

The balance and integration of different forms of volume and wiring transmission in the CNS. Relevance for schizophrenia

There is the existence of two main modes of intercellular communication in the Central Nervous System (CNS): the wiring transmission (WT), the prototype being synaptic transmission and the volume transmission (VT). A novel type of WT, the tunneling nanotube type may exist. VT uses the extracellular space (ECS) and the ventricular system as important channels for chemical transmission in the CNS complementary toWTwith diffusion and flow of transmitters, ions, trophic factors, etc. in the extracellular fluid (ECF) and cerebrospinal fluid (CSF). A short distance type (extrasynaptic transmission) and a long distance type of VT including the CSF type are found together with an extracellular vesicle mediated VT, called a roamer type of VT. Via the latter subtype several messages are sent via extracellular vesicles, mainly exosomes, containing selected mRNAs and miRNAs, proteins and lipids. They are released into the ECS and diffuse and flow until the proper targets are reached. The intrinsic features of exosomes allow their specific interaction with the appropriate target cells. They roam from cell to cell to disseminate important information but can also transfer pathologically altered proteins (e.g. toxic oligomers of amyloid beta and of alphasynuclein) contributing to spread of neurodegeneration. The balance and integration of synaptic and VT signals are achieved to a substantial degree via allosteric receptor\u2013 receptor interactions in GPCR-GPCR heteroreceptor complexes and especially ion-channel receptor-GPCR heteroreceptor complexes in the plasma membrane of synaptic and extrasynaptic regions. Disturbances in these receptor\u2013receptor interactions by mild enkephalitis may contribute to development of schizophrenia, since many NMDA, GABA, D2 and 5-HT2A receptors are protomers in heteroreceptor complexes and are also known targets for antipsychotic drugs. The hypothesis is that in neuroinflammation glial microvesicles (extracellular vesicles) may be released which contain chemokine and cytokine receptors. The extracellular vesicles can be internalized into surrounding neurons via cell adhesion receptors. In view of the bioinformatic results obtained based on the triplet puzzle theory, immune receptors CXCR4, CCR2 and IL1R2 may form heteroreceptor complexes with neuronal NMDA, GABAA and GABAB receptors which involves the triplet homologies ITL, SVS, VST, GLL, LYS, and YSG. They appear to be part of the interface of these human heteroreceptor complexes built up of neural and immune receptor protomers. Through the allosteric receptor\u2013receptor interactions in such postulated pathological heteroreceptor complexes the neuronal NMDA, GABAA and GABAB protomers may change their function in neuronal networks of the brain. This process can then contribute to positive, negative and/or cognitive symptoms of schizophrenia in line with the mild encephalitis hypothesis of schizophrenia. The glially derived kynurenines via VT participate in an important way in this integration of signals especially in the glutamate synapses and are altered in schizophrenia potentially related to mild neuroinflammation

Detection of fibroblast growth factor receptor 1 (FGFR1) transactivation by muscarinic acetylcholine receptors (mAChRs) in primary neuronal hippocampal cultures through use of biochemical and morphological approaches

In addition to their canonical intracellular signals involved in the regulation of neuronal plasticity, G-protein coupled receptors can also rapidly transactivate tyrosine kinase receptors and their downstream intracellular signaling in absence of specific ligands. Here we describe our protocol for dissociating and maintaining hippocampal primary neurons in high- and low-density culture, followed by a description of methods employed to evaluate neurite outgrowth and protein phosphorylation associated with fibroblast growth factor receptor 1 transactivation by muscarinic acetylcholine receptors. Our goal was to provide the reader with detailed protocols of the abovementioned techniques and to highlight advantages and limitations of the used approaches as compared to other valid alternatives

Role of D2-like Heteroreceptor Complexes in the Effects of Cocaine, Morphine, and Hallucinogens

One emerging concept is that direct physical receptor-receptor interactions in heteroreceptor complexes or altered balance with their homoreceptor complexes population can contribute to disease progression. Antagonistic A2AR-D2R-like receptor-receptor interactions in heteroreceptor complexes in the ventral striatum play a role in cocaine addictive behaviors. A2AR-D2R heteroreceptor complexes are hypothesized to be critically involved in cocaine reward and reinstatement of seeking, and more specifically that the activation of the A2AR protomer of this heteroreceptor complex inhibits the development and maintenance of cocaine addictive behavior. D2R-5-HT2AR heteroreceptor complexes in the brain represent a new target for hallucinogenic drugs. The psychotic-like actions of the 5-HT2AR hallucinogens can involve an enhancement of D2R protomer signaling via an abnormal facilitatory allosteric receptor-receptor interaction in the D2R-5-HT2AR heteroreceptor complex in the striatum. This gives novel insights into the molecular mechanism for the therapeutic actions of atypical antipsychotic drugs with a high affinity for the 5-HT2AR receptors. D4R-MOR heteroreceptor complexes appear to exist in the GABA striosome-nigral pathway, which plays a role in habit learning and the transition from impulsive to compulsive drug use. Antagonistic allosteric D4R-MOR interactions in these complexes block the effect of morphine on MOR protomer recognition and Gi/o coupling. D4R agonists targeting the D4R protomer of this receptor complex can be a novel strategy for counteracting morphine addiction development

The receptor\u2013receptor interactions within the cytokine receptor superfamily. Role in neuroinflammation and beyond

The mild encephalitis (ME) hypothesis of schizophrenia characterized a subgroup of severe psychiatric disorders in which low level neuroinflammation casually underlies the disorder as the core pathogenic mechanism. This low level neuroinflammation prevails and is important during a critical time period of diseases and it thought to be especially relevant in affective and schizophrenic disorders. The aetiologies involved in low level neuroinflammation vary, including infections, autoimmunity, toxicity and trauma. The pathological mechanisms generally are linked to the interactions between brain cells, immune cells and solutes resulting in an effective inflammatory immune response (IIR). The IIR first requires the recruitment of cells to the site of inflammation and then their appropriate activation and regulation. Chemokines and cytokines are critical in these responses since they are both chemotactic and immunoregulatory molecules. Cytokines are immunomodulating agents such as interleukins, interferons and chemokines which regulate the response to infection, inflammation and trauma. Each cytokine has a matching cell-surface receptor which varies in their threedimensional structure and cell type location. For instance, the cytokine receptor family comprises the Immunoglobulin (Ig) receptor superfamily, the Hemopoietic growth factor (type 1) receptor family, the interferon (type 2) receptor family, the tumor necrosis factor (TNF) (type 3) receptor family, the interleukin-17 and -12 (IL-17 and IL-12) receptor family and the class A G protein-coupled receptor chemokine receptor family. All these receptor-ligand pairs have a fundamental role in providing directional cues for the immune cell trafficking and signaling, both in the context of homeostasis and disease. However, immunomodulatory agents and their receptors are not isolated entities, but instead function in complex networks involving homo- and heteroreceptor complexes formation as well as crosstalk with other signaling cascades. For example, the arrest and chemotaxis of leukocytes during homeostasis and inflammation is an orchestrated phenomenon by a multitude of cytokines which are particularly adept at adjusting rapidly to changes within the environment. The confrontation of leukocytes with different combinations of chemokines that are concomitantly produced under physiological or pathological conditions in vivo is a complex challenge and receptor\u2013receptor interaction in heteroreceptor complexes could be the molecular mechanisms behind this phenomenon. In viral-induced disease receptor\u2013receptor interactions between viral and host-coded receptors can have a special relevance. Growing evidence indicates that the homo/heterodimeric form is the basic functional structure of cytokine receptors. These receptor families are not isolated entities that are activated following ligand binding. Rather, they exist as heteroreceptor complexes and/or higher order oligomers at the cell surface, even in the absence of ligands. These heteroreceptor complexes form organized receptor networks that can be modified by changes in receptor expression and ligand levels, indicating their dynamic properties. The way in which these receptor complexes are altered into new states determine their ligand binding, their pharmacological properties and the signal. These conformations thus represent a fundamental mechanism which increases the broad diversity of cytokine receptor functions. Understanding these heteroreceptor complexes and their dynamics at the cell surface is thus critical for influencing cytokine/chemokine functions. Modulation of cytokine including chemokine receptor activities through these molecular mechanisms has significant implications for physiological and pathological processes in the immune system. It could open up new possibilities for drug discovery and drug efficacy. For instance, small interfering synthetic peptides of TM regions of cytokine/chemokinereceptorsmayinterferewiththeformation of oligomeric receptor complexes and inhibit functional activity of the receptors. Therefore, small interfering TM peptides and possibly compounds that target cytokine heteroreceptor complexes are proposed to have therapeutic applications

5HT1AR-FGFR1 Heteroreceptor Complexes Differently Modulate GIRK Currents in the Dorsal Hippocampus and the Dorsal Raphe Serotonin Nucleus of Control Rats and of a Genetic Rat Model of Depression

The midbrain raphe serotonin (5HT) neurons provide the main ascending serotonergic projection to the forebrain, including hippocampus, which has a role in the pathophysiology of depressive disorder. Serotonin 5HT1A receptor (R) activation at the soma-dendritic level of serotonergic raphe neurons and glutamatergic hippocampal pyramidal neurons leads to a decrease in neuronal firing by activation of G protein-coupled inwardly-rectifying potassium (GIRK) channels. In this raphe-hippocampal serotonin neuron system, the existence of 5HT1AR-FGFR1 heteroreceptor complexes has been proven, but the functional receptor–receptor interactions in the heterocomplexes have only been investigated in CA1 pyramidal neurons of control Sprague Dawley (SD) rats. In the current study, considering the impact of the receptor interplay in developing new antidepressant drugs, the effects of 5HT1AR-FGFR1 complex activation were investigated in hippocampal pyramidal neurons and in midbrain dorsal raphe serotonergic neurons of SD rats and of a genetic rat model of depression (the Flinders Sensitive Line (FSL) rats of SD origin) using an electrophysiological approach. The results showed that in the raphe-hippocampal 5HT system of SD rats, 5HT1AR-FGFR1 heteroreceptor activation by specific agonists reduced the ability of the 5HT1AR protomer to open the GIRK channels through the allosteric inhibitory interplay produced by the activation of the FGFR1 protomer, leading to increased neuronal firing. On the contrary, in FSL rats, FGFR1 agonist-induced inhibitory allosteric action at the 5HT1AR protomer was not able to induce this effect on GIRK channels, except in CA2 neurons where we demonstrated that the functional receptor–receptor interaction is needed for producing the effect on GIRK. In keeping with this evidence, hippocampal plasticity, evaluated as long-term potentiation induction ability in the CA1 field, was impaired by 5HT1AR activation both in SD and in FSL rats, which did not develop after combined 5HT1AR-FGFR1 heterocomplex activation in SD rats. It is therefore proposed that in the genetic FSL model of depression, there is a significant reduction in the allosteric inhibition exerted by the FGFR1 protomer on the 5HT1A protomer-mediated opening of the GIRK channels in the 5HT1AR-FGFR1 heterocomplex located in the raphe-hippocampal serotonin system. This may result in an enhanced inhibition of the dorsal raphe 5HT nerve cell and glutamatergic hippocampal CA1 pyramidal nerve cell firing, which we propose may have a role in depression