48 research outputs found
Existence of Brain 5-HT1A–5-HT2A Isoreceptor Complexes with Antagonistic Allosteric Receptor–Receptor Interactions Regulating 5-HT1A Receptor Recognition
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