Ginetta Collo

Tissue distribution of the P2X7 receptor.

The P2X7 receptor is a bifunctional molecule. The binding of ATP induces within milliseconds the opening of a channel selective for small cations, and within seconds a larger pore opens which allows permeation by molecules as large as propidium dyes (629 Da). In situ hybridization using a digoxigenin-labelled riboprobe, and immunohistochemistry using an antibody raised against a C-terminal peptide sequence, were used to determine the distribution of the P2X7 receptor mRNA and protein in rat and mouse tissues and cell lines. The brain of newborn rats showed a 6 kb RNA by Northern blotting, but this was not detectable in adult brain. By in situ hybridization and immunohistochemistry, there was heavy labelling of ependymal cells in both newborn and adult brain, but the brain parenchyma showed no labelling. However, P2X7 receptor-immunoreactive cells appeared in the penumbral region around an area of necrosis evoked by prior occlusion of the middle cerebral artery, suggesting expression of the receptor by activated microglia. NTW8 cells, a mouse microglial cell line, strongly expressed the P2X7 receptor mRNA and protein. The P2X7 receptor mRNA and protein were also observed in the majority of bone marrow cells, including those separately identified by their expression of other antigens as granulocytes, monocyte/macrophages and B lymphocytes. The expression of P2X7 receptor by brain macrophages rather than neurons would be consistent with a role in brain repair following inflammation, infarction or immune insult.

P2X Receptors: An Emerging Channel Family

P2X receptors are ligand-gated ion channels activated by ATP. Early evidence for the role of extracellular ATP in signalling was based on the ability of ATP and its analogues to contract smooth muscle; this work has been reviewed by Burnstock (1980). Subsequently, ATP was shown to gate channels on a subset of sensory neurons (Jahr and Jessell, 1983). These and other observations have led to the identification of distinct P2 purinergic receptors which are divided into metabotropic (P2Y) and ionotropic families (P2X and P2Z) (Abbracchio and Bumstock, 1994; Fredholm et al., 1994). Several P2Y receptors have been cloned (Barnard et al., 1994); they display seven transmembrane structures and signal via a variety of second messenger systems, including inositol triphosphate production and the inhibition of CAMP (Harden et al., 1995). P2Z receptors are channel-forming proteins present on the surface of macrophages (Nuttle et al., 1993; Wiley et al., 1994; Bretschneider et al., 1995). Since the activation of P2Z receptors induces the formation of large pores, which results in cell lysis, the physiological role of these receptors has remained unresolved. Cloning of cDNA has recently shown that P2X receptors constitute a new class of channel-forming proteins which are structurally distinct from other ligand-gated channels, such as those for glutamate or acetylcholine (Brake et al., 1994; Valera et al., 1994). Cloned P2X receptors form rapidly activated, non-selective cationic channels which are activated by micromolar concentrations of extracellular ATP. The extent of P2X receptor diversity in structure, distribution and phenotype is being currently addressed. This review looks at answers that have been obtained with the molecularly cloned receptors and at the relationship between P2X and P2Z.

The NMDA/D1 Receptor Complex as a New Target in Drug Development

Dopamine and glutamate have been shown to extensively interact in the striatum, nucleus accumbens, hippocampus and prefrontal cortex, to regulate different physiological functions, including locomotor activity, positive reinforcement, attention and working memory. Although dysfunctions of dopamine transmission have long been identified as critical determinants of neurological and neuropsychiatric disorders, such as Parkinsons disease and schizophrenia, there is now increasing evidence that concurrent alterations of dopamine and glutamate function may play a central role in the pathophysiology of these diseases. Thus, defining the characteristics of dopamine-glutamate interactions may be crucial to identify alternative molecular targets for the development of novel pharmacological tools. At the postsynaptic level, interactions between the dopamine D1 and the glutamate NMDA receptors appear to be particularly relevant. Different mechanisms are involved in this interactions: 1) D1R-dependent, second messenger-mediated phosphorylation of NMDAR subunits; 2) coordinated regulation of receptor trafficking at synaptic sites; 3) formation of an heteromeric D1/NMDA receptor complex. In this paper we review the molecular mechanisms, functional implications and pharmacological significance of D1R/NMDAR interaction via direct protein-protein oligomerization.

Alpha‐synuclein aggregation and cell death triggered by energy deprivation and dopamine overload are counteracted by D2/D3 receptor activation

Progressive degeneration and intraneuronal Lewy bodies made of filamentous α‐synuclein (α‐syn) in dopaminergic cells of the nigrostriatal system are characteristics of Parkinson’s disease (PD). Glucose uptake is reduced in some of the brain regions affected by PD neurodegenerative changes. Defects in mitochondrial activity in the substantia nigra have been observed in the brain of patients affected by PD and substantia nigra lesions can induce the onset of a secondary parkinsonism. Thus, energy starvation and consequently metabolic impairment to dopaminergic neurons may be related to the onset of PD. On this line, we evaluated the effect of nutrient starvation, reproduced ‘in vitro’ by glucose deprivation (GD), in primary mesecephalic neuronal cultures and dopaminergic‐differentiated SH‐SY5Y cells, to evaluate if decreased glucose support to dopaminergic cells can lead to mitochondrial damage, neurodegeneration and α‐syn misfolding. Furthermore, we investigated the effect of dopamine (DA) treatment in the presence of a DA‐uptake inhibitor or of the D2/D3 receptor (D2R/D3R) agonist quinpirole on GD‐treated cells, to evaluate the efficacy of these therapeutic compounds. We found that GD induced the formation of fibrillary aggregated α‐syn inclusions containing the DA transporter in dopaminergic cells. These alterations were accompanied by dopaminergic cell death and were exacerbated by DA overload. Conversely, the block of DA uptake and D2R/D3R agonist treatment exerted neuroprotective effects. These data indicate that glucose starvation is likely involved in the induction of PD‐related pathological changes in dopaminergic neurons. These changes may be counteracted by the block of DA uptake and by dopaminergic agonist treatment.

Redistribution of DAT/α-Synuclein Complexes Visualized by “In Situ” Proximity Ligation Assay in Transgenic Mice Modelling Early Parkinson's Disease

Alpha-synuclein, the major component of Lewy bodies, is thought to play a central role in the onset of synaptic dysfunctions in Parkinsons disease (PD). In particular, α-synuclein may affect dopaminergic neuron function as it interacts with a key protein modulating dopamine (DA) content at the synapse: the DA transporter (DAT). Indeed, recent evidence from our “in vitro” studies showed that α-synuclein aggregation decreases the expression and membrane trafficking of the DAT as the DAT is retained into α-synuclein-immunopositive inclusions. This notwithstanding, “in vivo” studies on PD animal models investigating whether DAT distribution is altered by the pathological overexpression and aggregation of α-synuclein are missing. By using the proximity ligation assay, a technique which allows the “in situ” visualization of protein-protein interactions, we studied the occurrence of alterations in the distribution of DAT/α-synuclein complexes in the SYN120 transgenic mouse model, showing insoluble α-synuclein aggregates into dopaminergic neurons of the nigrostriatal system, reduced striatal DA levels and an altered distribution of synaptic proteins in the striatum. We found that DAT/α-synuclein complexes were markedly redistributed in the striatum and substantia nigra of SYN120 mice. These alterations were accompanied by a significant increase of DAT striatal levels in transgenic animals when compared to wild type littermates. Our data indicate that, in the early pathogenesis of PD, α-synuclein acts as a fine modulator of the dopaminergic synapse by regulating the subcellular distribution of key proteins such as the DAT.

Dopamine D3 receptor-preferring agonists increase dendrite arborization of mesencephalic dopaminergic neurons via extracellular signal-regulated kinase phosphorylation.

Clinical improvements in Parkinson’s disease produced by dopamine D3 receptor‐preferring agonists have been related to their neuroprotective actions and, more recently, to their neuroregenerative properties. However, it is unclear whether dopamine agonists produce their neurotrophic effects by acting directly on receptors expressed by the mesencephalic dopaminergic neurons or indirectly on receptors expressed by astrocytes, via release of neurotrophic factors. In this study, we investigated the effects of the dopamine D3 receptor‐preferring agonists quinpirole and 7‐hydroxy‐N,N‐di‐propyl‐2‐aminotetralin (7‐OH‐DPAT), as well as of the indirect agonist amphetamine, on dopaminergic neurons identified by tyrosine hydroxylase immunoreactivity (TH‐IR). Experiments were performed on neuronal‐enriched primary cultures containing less than 0.5% of astrocytes prepared from the mouse embryo mesencephalon. After 3 days of incubation, both quinpirole (1–10 μm) and 7‐OH‐DPAT (5–500 nm) dose‐dependently increased the maximal dendrite length (P < 0.001), number of primary dendrites (P < 0.01) and [3H]dopamine uptake (P < 0.01) of TH‐IR‐positive mesencephalic neurons. Similar effects were observed with 10 μm amphetamine. All neurotrophic effects were blocked by the unselective D2/D3 receptor antagonist sulpiride (5 μm) and by the selective D3 receptor antagonist SB‐277011‐A at a low dose (50 nm). Quinpirole and 7‐OH‐DPAT also increased the phosphorylation of extracellular signal‐regulated kinase (ERK) within minutes, an effect blocked by pretreatment with SB‐277011‐A. Inhibition of the D2/D3 receptor signalling pathway to ERK was obtained with PD98059, GF109203 or LY294002, resulting in blockade of neurotrophic effects. These data suggest that dopamine agonists increase dendritic arborizations of mesencephalic dopaminergic neurons via a direct effect on D2/D3 receptors, preferentially involving D3 receptor‐dependent neurotransmission.

Nicotine-Induced Structural Plasticity in Mesencephalic Dopaminergic Neurons Is Mediated by Dopamine D3 Receptors and Akt-mTORC1 Signaling

Although long-term exposure to nicotine is highly addictive, one beneficial consequence of chronic tobacco use is a reduced risk for Parkinson’s disease. Of interest, these effects both reflect structural and functional plasticity of brain circuits controlling reward and motor behavior and, specifically, recruitment of nicotinic acetylcholine receptors (nAChR) in mesencephalic dopaminergic neurons. Because the underlying cellular mechanisms are poorly understood, we addressed this issue with use of primary cultures of mouse mesencephalic dopaminergic neurons. Exposure to nicotine (1–10 μM) for 72 hours in vitro increased dendritic arborization and soma size in primary cultures. These effects were blocked by mecamylamine and dihydro-β-erythroidine, but not methyllycaconitine. The involvement of α4β2 nAChR was supported by the lack of nicotine-induced structural remodeling in neurons from α4 null mutant mice (KO). Challenge with nicotine triggered phosphorylation of the extracellular signal-regulated kinase (ERK) and the thymoma viral proto-oncogene (Akt), followed by activation of the mammalian target of rapamycin complex 1 (mTORC1)-dependent p70 ribosomal S6 protein kinase. Upstream pathway blockade using the phosphatidylinositol 3-kinase inhibitor LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride] resulted in suppression of nicotine-induced phosphorylations and structural plasticity. These effects were dependent on functional DA D3 receptor (D3R), because nicotine was inactive both in cultures from D3R KO mice and after pharmacologic blockade with D3R antagonist trans-N-4-2-(6-cyano-1,2,3, 4-tetrahydroisoquinolin-2-yl)ethylcyclohexyl-4-quinolinecarboxamide (SB-277011-A) (50 nM). Finally, exposure to nicotine in utero (5 mg/kg/day for 5 days) resulted in increased soma area of DAergic neurons of newborn mice, effects not observed in D3 receptor null mutant mice mice. These findings indicate that nicotine-induced structural plasticity at mesencephalic dopaminergic neurons involves α4β2 nAChRs together with dopamine D3R-mediated recruitment of ERK/Akt-mTORC1 signaling.

Pre-synaptic dopamine D(3) receptor mediates cocaine-induced structural plasticity in mesencephalic dopaminergic neurons via ERK and Akt pathways.

J. Neurochem. (2012) 120, 765–778.

Dopamine D3 receptor ligands for drug addiction treatment: update on recent findings.

The dopamine D3 receptor is located in the limbic area and apparently mediates selective effects on motivation to take drugs and drug-seeking behaviors, so that there has been considerable interest on the possible use of D3 receptor ligands to treat drug addiction. However, only recently selective tools allowing studying this receptor have been developed. This chapter presents an overview of findings that were presented at a symposium on the conference Dopamine 2013 in Sardinia in May 2013. Novel neurobiological findings indicate that drugs of abuse can lead to significant structural plasticity in rodent brain and that this is dependent on the availability of functional dopamine D3 autoreceptor, whose activation increased phosphorylation in the ERK pathway and in the Akt/mTORC1 pathway indicating the parallel engagement of a series of intracellular signaling pathways all involved in cell growth and survival. Preclinical findings using animal models of drug-seeking behaviors confirm that D3 antagonists have a promising profile to treat drug addiction across drugs of abuse type. Imaging the D3 is now feasible in human subjects. Notably, the development of (+)-4-propyl-9-hydroxynaphthoxazine ligand used in positron emission tomography (PET) studies in humans allows to measure D3 and D2 receptors based on the area of the brain under study. This PET ligand has been used to confirm up-regulation of D3 sites in psychostimulant users and to reveal that tobacco smoking produces elevation of dopamine at the level of D3 sites. There are now novel antagonists being developed, but also old drugs such as buspirone, that are available to test the D3 hypothesis in humans. The first results of clinical investigations are now being provided. Overall, those recent findings support further exploration of D3 ligands to treat drug addiction.

The neurobiology of dopamine receptors: evolution from the dual concept to heterodimer complexes

Context: G protein-coupled receptors (GPCRs) have been classically thought to work as monomeric entities. The current view of their organization, however, assumes that they are part of highly organized molecular complexes, where different receptors and interacting proteins are clustered. These heteromers have peculiar pharmacological, signaling, and trafficking properties. GPCR heteromerization, raising different combinatorial possibilities, thus underlies an unexpected level of diversity within this receptor family. Methods: In this paper, we summarize recent data, reported by different research groups, suggesting that the dopamine (DA) D1 receptor forms heteromers with receptors of the same family and with structurally and functionally divergent receptors. Results and discussion: DA D1 and D3 receptors and glutamate NMDA receptors regulate rewarding mechanisms and motivated behavior, modulate emotional and cognitive processes and regulate locomotor activity by extensive cross-talk mechanisms. Co-localization of D1 and D3 receptors and D1 and NMDA receptors in specific neuronal populations in the striatum and nucleus accumbens, moreover, suggested that their cross-talk may involve direct interactions. By using different experimental approaches various groups have, in fact, demonstrated the existence of D1-NMDA and D1-D3 heteromers, in both transfected cell systems and in the straitum, with peculiar pharmacological, signaling, and functional properties. The putative role of the D1-D3 and D1-NMDA heteromers in the physiological regulation of striatal function and in the development of motor dysfunctions will be discussed.