The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

In-vitro Approaches to Investigate the Detrimental Effect of Light on Dopaminergic Neurons.

Our recent study revealed that fluorescent lamp light can penetrate deep into the brain of mice and rats leading to the development of typical histological characteristics associated with Parkinson's disease such as the loss of dopamine neurons in the substantia nigra. Monochromatic LED lights were thus used in this work to deepen our knowledge on the effects of the major wavelength peaks of fluorescent light on mouse and human dopaminergic cells. In particular, we exposed immortalized dopaminergic MN9D neuronal cells, primary cultures of mouse mesencephalic dopaminergic cells and human dopaminergic neurons differentiated from induced pluripotent stem cells (hiPSC) to different LED light wavelengths. We found that chronic exposure to LED light reduced overall undifferentiated MN9D cell number, with the most significant effects observed at wavelengths of 485 nm and 610 nm. Moreover, LED light especially at 610 nm was able to negatively impact on the survival of mouse mesencephalic dopaminergic cells and of human dopaminergic neurons derived from hiPSC. Notably, differentiated MN9D dopaminergic cells, which closely resemble mature dopamine neuronal phenotype, acutely exposed for 3 h at 610 nm, showed a clear increase in ROS production and cytotoxicity compared to controls undifferentiated MN9D cells. These increases were even more pronounced by the co-treatment with the oxidative agent H2O2. Collectively, these findings suggest that specific wavelengths, particularly those capable of penetrating deep into the brain, could potentially pose an environmental hazard in relation to Parkinson's disease.

Unraveling the Functional Significance of Unstructured Regions in G Protein-Coupled Receptors.

Unstructured regions in functional proteins have gained attention in recent years due to advancements in informatics tools and biophysical methods. G protein-coupled receptors (GPCRs), a large family of cell surface receptors, contain unstructured regions in the form of the i3 loop and C-terminus. This review provides an overview of the functional significance of these regions in GPCRs. GPCRs transmit signals from the extracellular environment to the cell interior, regulating various physiological processes. The i3 loop, located between the fifth and sixth transmembrane helices, and the C-terminus, connected to the seventh transmembrane helix, are determinant of interactions with G proteins and with other intracellular partners such as arrestins. Recent studies demonstrate that the i3 loop and C-terminus play critical roles in allosterically regulating GPCR activation. They can act as autoregulators, adopting conformations that, by restricting G protein access, modulate receptor coupling specificity. The length and unstructured nature of the i3 loop and C-terminus provide unique advantages in GPCR interactions with intracellular protein partners. They act as "fishing lines", expanding the radius of interaction and enabling GPCRs to tether scaffolding proteins, thus facilitating receptor stability during cell membrane movements. Additionally, the i3 loop may be involved in domain swapping between GPCRs, generating novel receptor dimers with distinct binding and coupling characteristics. Overall, the i3 loop and C-terminus are now widely recognized as crucial elements in GPCR function and regulation. Understanding their functional roles enhances our comprehension of GPCR structure and signaling complexity and holds promise for advancements in receptor pharmacology and drug development.

GPCRs in Intracellular Compartments: New Targets for Drug Discovery.

The architecture of eukaryotic cells is defined by extensive membrane-delimited compartments, which entails separate metabolic processes that would otherwise interfere with each other, leading to functional differences between cells. G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors, and their signal transduction is traditionally viewed as a chain of events initiated from the plasma membrane. Furthermore, their intracellular trafficking, internalization, and recycling were considered only to regulate receptor desensitization and cell surface expression. On the contrary, accumulating data strongly suggest that GPCRs also signal from intracellular compartments. GPCRs localize in the membranes of endosomes, nucleus, Golgi and endoplasmic reticulum apparatuses, mitochondria, and cell division compartments. Importantly, from these sites they have shown to orchestrate multiple signals that regulate different cell pathways. In this review, we summarize the current knowledge of this fascinating phenomenon, explaining how GPCRs reach the intracellular sites, are stimulated by the endogenous ligands, and their potential physiological/pathophysiological roles. Finally, we illustrate several mechanisms involved in the modulation of the compartmentalized GPCR signaling by drugs and endogenous ligands. Understanding how GPCR signaling compartmentalization is regulated will provide a unique opportunity to develop novel pharmaceutical approaches to target GPCRs and potentially lead the way towards new therapeutic approaches.

Interaction of the preferential D3 agonist (+)PHNO with dopamine D3-D2 receptor heterodimers and diverse classes of monoamine receptor: relevance for PET imaging.

(+)-4-Propyl-9-hydroxynaphthoxazine ((+)PHNO) is a high affinity, preferential dopamine D3 versus D2 agonist employed in view of its high specificity and excellent signal-to-noise ratio as a radiotracer for positron emission tomography (PET) imaging. Surprisingly, its profile at other classes of monoamine receptor remains undocumented. In addition to hD3 and hD2L receptors, (+)PHNO revealed high affinity at hD4.4 but not hD1 or hD5 receptors. It also revealed significant affinity for several other G protein-coupled monoaminergic receptors, in particular h5-HT1A and h5-HT7. (+)PHNO behaved as a full agonist at hD4.4 and h5-HT1A receptors with potencies comparable to its actions at hD3 and hD2L receptors, and with less potency at 5-HT7 receptors. In binding assays with membranes derived from cells co-expressing hD3 and hD2L receptors and labeled with [3H]Nemonapride or [3H]Spiperone, the proportion of high affinity binding sites recognized by (+)PHNO was higher than an equivalent mixture of membranes from cells expressing hD3or hD2L receptors, suggesting that (+)PHNO promotes formation of hD3-hD2L heterodimers. Further, in cells co-expressing hD3 and hD2L receptors, (+)PHNO showed higher efficacy for inhibiting forskolin stimulated adenylyl cyclase and inducing adenylyl cyclase super-sensitization than in cells transfected with only hD2L receptors. In conclusion, (+)PHNO is a potent agonist at hD4.4, h5-HT1A and h5-HT7 as well as hD3 and hD2L receptors, and it potently activates dopamine hD3-hD2L heterodimers. These interactions should be considered when interpreting PET studies with [11C](+)PHNO and may be relevant to its functional and potential clinical properties in Parkinson's disease and other disorders.

Integration and Spatial Organization of Signaling by G Protein-Coupled Receptor Homo- and Heterodimers.

Information flow from a source to a receiver becomes informative when the recipient can process the signal into a meaningful form. Information exchange and interpretation is essential in biology and understanding how cells integrate signals from a variety of information-coding molecules into complex orchestrated responses is a major challenge for modern cell biology. In complex organisms, cell to cell communication occurs mostly through neurotransmitters and hormones, and receptors are responsible for signal recognition at the membrane level and information transduction inside the cell. The G protein-coupled receptors (GPCRs) are the largest family of membrane receptors, with nearly 800 genes coding for these proteins. The recognition that GPCRs may physically interact with each other has led to the hypothesis that their dimeric state can provide the framework for temporal coincidence in signaling pathways. Furthermore, the formation of GPCRs higher order oligomers provides the structural basis for organizing distinct cell compartments along the plasma membrane where confined increases in second messengers may be perceived and discriminated. Here, we summarize evidence that supports these conjectures, fostering new ideas about the physiological role played by receptor homo- and hetero-oligomerization in cell biology.

Romidepsin (FK228) fails in counteracting the transformed phenotype of rhabdomyosarcoma cells but efficiently radiosensitizes, in vitro and in vivo, the alveolar phenotype subtype.

PURPOSE: Herein we describe the in vitro and in vivo activity of FK228 (Romidepsin), an inhibitor of class I HDACs, in counteracting and radiosensitizing embryonal (ERMS, fusion-negative) and alveolar (ARMS, fusion-positive) rhabdomyosarcoma (RMS). METHODS: RH30 (ARMS, fusion-positive) and RD (ERMS, fusion-negative) cell lines and human multipotent mesenchymal stromal cells (HMSC) were used. Flow cytometry analysis, RT-qPCR, western blotting and enzymatic assays were performed. Irradiation was delivered by using an x-6 MV photon linear accelerator. FK228 (1.2 mg/kg) in vivo activity, combined or not with radiation therapy (2 Gy), was assessed in murine xenografts. RESULTS: Compared to HMSC, RMS expressed low levels of class I HDACs. In vitro, FK228, as single agents, reversibly downregulated class I HDACs expression and activity and induced oxidative stress, DNA damage and a concomitant growth arrest associated with PARP-1-mediated transient non-apoptotic cell death. Surviving cells upregulated the expression of cyclin A, B, D1, p27, Myc and activated PI3K/Akt/mTOR and MAPK signaling, known to be differently involved in cancer chemoresistance. Interestingly, while no radiosensitizing effects were detected, in vitro or in vivo, on RD cells, FK228 markedly radiosensitized RH30 cells by impairing antioxidant and DSBs repair pathways in vitro. Further, FK228 when combined with RT in vivo significantly reduced tumor mass in mouse RH30 xenografts. CONCLUSION: FK228 did not show antitumor activity as a single agent whilst its combination with RT resulted in radiosensitization of fusion-positive RMS cells, thus representing a possible strategy for the treatment of the most aggressive RMS subtype.

Atypical Antipsychotics and Metabolic Syndrome: From Molecular Mechanisms to Clinical Differences.

Atypical antipsychotics (AAPs) are commonly prescribed medications to treat schizophrenia, bipolar disorders and other psychotic disorders. However, they might cause metabolic syndrome (MetS) in terms of weight gain, dyslipidemia, type 2 diabetes (T2D), and high blood pressure, which are responsible for reduced life expectancy and poor adherence. Importantly, there is clear evidence that early metabolic disturbances can precede weight gain, even if the latter still remains the hallmark of AAPs use. In fact, AAPs interfere profoundly with glucose and lipid homeostasis acting mostly on hypothalamus, liver, pancreatic ß-cells, adipose tissue, and skeletal muscle. Their actions on hypothalamic centers via dopamine, serotonin, acetylcholine, and histamine receptors affect neuropeptides and 5'AMP-activated protein kinase (AMPK) activity, thus producing a supraphysiological sympathetic outflow augmenting levels of glucagon and hepatic glucose production. In addition, altered insulin secretion, dyslipidemia, fat deposition in the liver and adipose tissues, and insulin resistance become aggravating factors for MetS. In clinical practice, among AAPs, olanzapine and clozapine are associated with the highest risk of MetS, whereas quetiapine, risperidone, asenapine and amisulpride cause moderate alterations. The new AAPs such as ziprasidone, lurasidone and the partial agonist aripiprazole seem more tolerable on the metabolic profile. However, these aspects must be considered together with the differences among AAPs in terms of their efficacy, where clozapine still remains the most effective. Intriguingly, there seems to be a correlation between AAP's higher clinical efficacy and increase risk of metabolic alterations. Finally, a multidisciplinary approach combining psychoeducation and therapeutic drug monitoring (TDM) is proposed as a first-line strategy to avoid the MetS. In addition, pharmacological treatments are discussed as well.

Allosteric Modulators of G Protein-Coupled Dopamine and Serotonin Receptors: A New Class of Atypical Antipsychotics.

Schizophrenia was first described by Emil Krapelin in the 19th century as one of the major mental illnesses causing disability worldwide. Since the introduction of chlorpromazine in 1952, strategies aimed at modifying the activity of dopamine receptors have played a major role for the treatment of schizophrenia. The introduction of atypical antipsychotics with clozapine broadened the range of potential targets for the treatment of this psychiatric disease, as they also modify the activity of the serotoninergic receptors. Interestingly, all marketed drugs for schizophrenia bind to the orthosteric binding pocket of the receptor as competitive antagonists or partial agonists. In recent years, a strong effort to develop allosteric modulators as potential therapeutic agents for schizophrenia was made, mainly for the several advantages in their use. In particular, the allosteric binding sites are topographically distinct from the orthosteric pockets, and thus drugs targeting these sites have a higher degree of receptor subunit specificity. Moreover, "pure" allosteric modulators maintain the temporal and spatial fidelity of native orthosteric ligand. Furthermore, allosteric modulators have a "ceiling effect", and their modulatory effect is saturated above certain concentrations. In this review, we summarize the progresses made in the identification of allosteric drugs for dopamine and serotonin receptors, which could lead to a new generation of atypical antipsychotics with a better profile, especially in terms of reduced side effects.

A New Threat to Dopamine Neurons: The Downside of Artificial Light.

Growing awareness of adverse impacts of artificial light on human health has led to recognize light pollution as a significant global environmental issue. Despite, a large number of studies in rodent and monkey models of Parkinson's disease have reported that near infrared light has neuroprotective effects on dopaminergic neurons, recent findings have shown that prolonged exposure of rodents and birds to fluorescent artificial light results in an increase of neuromelanin granules in substantia nigra and loss of dopaminergic neurons. The observed detrimental effect seems to be dependent on a direct effect of light on the substantia nigra rather than a secondary effect of the alterations of circadian rhythms. Moreover, inferences from animal models to human studies have shown a positive correlation between the prevalence of Parkinson's disease and light pollution. The present article discusses experimental evidence supporting a potentially deleterious impact of light on dopaminergic neurons and highlights the mechanisms whereby light might damage neuronal tissue. Moreover, it analyses epidemiological evidence that suggests light pollution to be an environmental risk factor for Parkinson's disease.

Antitumorigenic Effects of Inhibiting Ephrin Receptor Kinase Signaling by GLPG1790 against Colorectal Cancer Cell Lines In Vitro and In Vivo.

Erythropoietin-producing hepatocellular receptors (Eph) promote the onset and sustain the progression of cancers such as colorectal cancer (CRC), in which the A2 subtype of Eph receptor expression has been shown to correlate with a poor prognosis and has been identified as a promising therapeutic target. Herein, we investigated, in vitro and in vivo, the effects of treatment with GLPG1790, a potent pan-Eph inhibitor. The small molecule has selective activity against the EphA2 isoform in human HCT116 and HCT15 CRC cell lines expressing a constitutively active form of RAS concurrently with a wild-type or mutant form of p53, respectively. GLPG1790 reduced EPHA2 phosphorylation/activation and induced G1/S cell-cycle growth arrest by downregulating the expression of cyclin E and PCNA, while upregulating p21Waf1/Cip1 and p27Cip/Kip. The inhibition of ephrin signaling induced quiescence in HCT15 and senescence in HCT116 cells. While investigating the role of CRC-related, pro-oncogenic p53 and RAS pathways, we found that GLPG1790 upregulated p53 expression and that silencing p53 or inhibiting RAS (human rat sarcoma)/ERKs (extracellular signal-regulated kinase) signaling restrained the ability of GLPG1790 to induce senescence in HCT116 cells. On the other hand, HCT15 silencing of p53 predisposed cells to GLPG1790-induced senescence, whilst no effects of ERK inhibition were observed. Finally, GLPG1790 hindered the epithelial-mesenchymal transition, reduced the migratory capacities of CRC, and affected tumor formation in xenograft models in vivo more efficiently using HCT116 than HCT15 for xenografts. Taken together, our data suggest the therapeutic potential of GLPG1790 as a signal transduction-based therapeutic strategy in to treat CRC.

Modulating the dose-rate differently affects the responsiveness of human epithelial prostate- and mesenchymal rhabdomyosarcoma-cancer cell line to radiation.

Purpose: Radiation therapy (RT), by using ionizing radiation (IR), destroys cancer cells inducing DNA damage. Despite several studies are continuously performed to identify the best curative dose of IR, the role of dose-rate, IR delivered per unit of time, on tumor control is still largely unknown.Materials and methods: Rhabdomyosarcoma (RMS) and prostate cancer (PCa) cell lines were irradiated with 2 or 10 Gy delivered at dose-rates of 1.5, 2.5, 5.5 and 10.1 Gy/min. Cell-survival rate and cell cycle distribution were evaluated by clonogenic assays and flow cytometry, respectively. The production of reactive oxygen species (ROS) was detected by cytometry. Quantitative polymerase chain reaction assessed the expression of anti-oxidant-related factors including NRF2, SODs, CAT and GPx4 and miRNAs (miR-22, -126, -210, -375, -146a, -34a). Annexin V and caspase-8, -9 and -3 activity were assessed to characterize cell death. Senescence was determined by assessing ß-galactosidase (SA-ß-gal) activity. Immunoblotting was performed to assess the expression/activation of: i) phosphorylated H2AX (γ-H2AX), markers of DNA double strand breaks (DSBs); ii) p19Kip1/Cip1, p21Waf1/Cip1 and p27Kip1/Cip1, senescence-related-markers; iii) p62, LC3-I and LC3-II, regulators of autophagy; iv) ATM, RAD51, DNA-PKcs, Ku70 and Ku80, mediators of DSBs repair.Results: Low dose-rate (LDR) more efficiently induced apoptosis and senescence in RMS while high dose-rate (HDR) necrosis in PCa. This paralleled with a lower ability of LDR-RMS and HDR-PCa irradiated cells to activate DSBs repair. Modulating the dose rate did not differently affect the anti-oxidant ability of cancer cells.Conclusion: The present results indicate that a stronger cytotoxic effect was induced by modulating the dose-rate in a cancer cell-dependent manner, this suggesting that choose the dose-rate based on the individual patient's tumor characteristics could be strategic for effective RT exposures.

Neurotoxic and Neuroprotective Role of Exosomes in Parkinson's Disease.

Exosomes are extracellular vesicles produced by eukaryotic cells that are also found in most biological fluids and tissues. While they were initially thought to act as compartments for removal of cellular debris, they are now recognized as important tools for cell-to-cell communication and for the transfer of pathogens between the cells. They have attracted particular interest in neurodegenerative diseases for their potential role in transferring prion-like proteins between neurons, and in Parkinson's disease (PD), they have been shown to spread oligomers of α-synuclein in the brain accelerating the progression of this pathology. A potential neuroprotective role of exosomes has also been equally proposed in PD as they could limit the toxicity of α-synuclein by clearing them out of the cells. Exosomes have also attracted considerable attention for use as drug vehicles. Being nonimmunogenic in nature, they provide an unprecedented opportunity to enhance the delivery of incorporated drugs to target cells. In this review, we discuss current knowledge about the potential neurotoxic and neuroprotective role of exosomes and their potential application as drug delivery systems in PD.

Pro-differentiating and radiosensitizing effects of inhibiting HDACs by PXD-101 (Belinostat) in in vitro and in vivo models of human rhabdomyosarcoma cell lines.

This study describes the in vitro and in vivo activity of PXD-101 (Belinostat), a novel hydroxamic acid-type pan-HDACs inhibitor characterized by a larger safety and efficacy, on myogenic-derived PAX3/FOXO1 fusion protein positive (RH30) or negative (RD) expressing rhabdomyosarcoma (RMS) cell lines. PXD-101 at low doses efficiently inhibited HDACs activity and counteracted the transformed phenotype of RMS by inducing growth arrest and apoptosis, affecting cancer stem cells population and inducing differentiation in RD. Notably, PXD-101 induced oxidative stress promoting DNA damages and affected the ability of RMS to assemble mitotic spindle. PXD-101 radiosensitized by inducing G2 cell cycle growth arrest, enhancing the radiation's ability to induce ROS accumulation and compromising both the ability of RMS to detoxify from ROS and to repair DNA damage. PXD-101 transcriptionally and post-transcriptionally affected c-Myc expression, key master regulator of rhabdomyosarcomagenesis and RMS radioresistance. All in vitro data were corroborated by in vivo experiments showing the cytostatic effects of PXD-101 when used alone and at low dose and its ability to promote the RT-induced killing of RMS. Taken together, our data confirm that altered HDACs activity plays a key role in RMS genesis and suggest PXD-101 as a valid therapeutic strategy particularly in combination with RT.

Parkinson's disease and light: The bright and the Dark sides.

Light exerts a major influence on human behaviour and health, mainly owing to the importance of sight in our lives, but also due to its entrainment of daily rhythms via the suprachiasmatic nucleus, the master pacemaker. Light may also be a useful clinical medium, as in lumino-therapy for the improvement of depressed mood. Further, as discussed herein, local application of near infrared light to the substantia nigra exerts neuroprotective properties in models of Parkinson's disease. However, light also has a darker side. In general, as regards the growing problem to human health - and the natural world - of excess exposure to artificial light: both urban glow and ubiquitous screens. Moreover, over-exposure to light, in particular fluorescent light, disrupts circadian rhythms and sleep, and may damage dopaminergic neurons. Is it, then, a neglected risk factor for Parkinson's disease? The present article discusses epidemiological and experimental evidence supporting beneficial and potentially deleterious impact of light on dopaminergic neurons and highlights the mechanisms whereby light might influence neuronal tissue.

Distinctive binding properties of the negative allosteric modulator, [3H]SB269,652, at recombinant dopamine D3 receptors.

Recently, employing radioligand displacement and functional coupling studies, we demonstrated that SB269,652 (N-[(1r,4r)-4-[2-(7-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-1H-indole-2-carboxamide) interacts in an atypical manner with dopamine D3 receptor displaying a unique profile reminiscent of a negative allosteric ligand. Here, we characterized the binding of radiolabelled [3H]SB269,652 to human dopamine D3 receptor stably expressed in Chinese Hamster Ovary cells. Under saturating conditions, SB269,652 showed a KD value of ≈ 1nM. Consistent with high selectivity for human dopamine D3 receptor, [3H]SB269,652 binding was undetectable in cells expressing human dopamine D1, D2L or D4 receptors and absent in synaptosomes from dopamine D3 receptor knockout vs. wild-type mice. In contrast to saturation binding experiments, the dissociation kinetics of [3H]SB269,652 from human dopamine D3 receptors initiated with an excess of unlabelled ligand were best fitted by a bi-exponential binding model. Supporting the kinetic data, competition experiments with haloperidol, S33084 (a dopamine D3 receptor antagonist) or dopamine, were best described by a two-site model. In co-transfection experiments binding of SB269,652 to dopamine D3 receptor was able to influence the functional coupling of dopamine D2 receptor, supporting the notion that SB269,652 is a negative allosteric modulator across receptor dimers. However, because SB269,652 decreases the rate of [3H]nemonapride dissociation, the present data suggest that SB269,652 behaves as a bitopic antagonist at unoccupied dopamine D3 receptor, binding simultaneously to both orthosteric and allosteric sites, and as a pure negative allosteric modulator when receptors are occupied and it can solely bind to the allosteric site.

Dichlorodiphenyltrichloroethane (DDT) induced extracellular vesicle formation: a potential role in organochlorine increased risk of Parkinson's disease.

A number of studies have demonstrated that rural living and exposure to pesticides such as dichlorodiphenyltrichloroethane (DDT) highly increase the chances of developing Parkinson's disease. In a previous work, we have found that DDT leads to the formation of vesicular buds that are released from the cells upon fusion of an intermediate endocytic compartment with the plasma membrane. Since extracellular vesicles like exosomes have been implicated in the development of neurodegenerative diseases through the propagation of neurotoxic misfolded proteins from neuron to neuron, in this minireview we propose that organochlorine pesticides could enhance the risk of neurodegenerative diseases by increasing the formation of exosomes.

The First Negative Allosteric Modulator for Dopamine D2 and D3 Receptors, SB269652 May Lead to a New Generation of Antipsychotic Drugs.

D2 and D3 dopamine receptors belong to the largest family of cell surface proteins in eukaryotes, the G protein-coupled receptors (GPCRs). Considering their crucial physiologic functions and their relatively accessible cellular locations, GPCRs represent one of the most important classes of therapeutic targets. Until recently, the only strategy to develop drugs regulating GPCR activity was through the identification of compounds that directly acted on the orthosteric sites for endogenous ligands. However, many efforts have recently been made to identify small molecules that are able to interact with allosteric sites. These sites are less well-conserved, therefore allosteric ligands have greater selectivity on the specific receptor. Strikingly, the use of allosteric modulators can provide specific advantages, such as an increased selectivity for GPCR subunits and the ability to introduce specific beneficial therapeutic effects without disrupting the integrity of complex physiologically regulated networks. In 2010, our group unexpectedly found that N-[(1r,4r)-4-[2-(7-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-1H-indole-2-carboxamide (SB269652), a compound supposed to interact with the orthosteric binding site of dopamine receptors, was actually a negative allosteric modulator of D2- and D3-receptor dimers, thus identifying the first allosteric small molecule acting on these important therapeutic targets. This review addresses the progress in understanding the molecular mechanisms of interaction between the negative modulator SB269652 and D2 and D3 dopamine receptor monomers and dimers, and surveys the prospects for developing new dopamine receptor allosteric drugs with SB269652 as the leading compound.

Fluorescent light induces neurodegeneration in the rodent nigrostriatal system but near infrared LED light does not.

We investigated the effects of continuous artificial light exposure on the mouse substantia nigra (SN). A three month exposure of C57Bl/6J mice to white fluorescent light induced a 30% reduction in dopamine (DA) neurons in SN compared to controls, accompanied by a decrease of DA and its metabolites in the striatum. After six months of exposure, neurodegeneration progressed slightly, but the level of DA returned to the basal level, while the metabolites increased with respect to the control. Three month exposure to near infrared LED light (∼710nm) did not alter DA neurons in SN, nor did it decrease DA and its metabolites in the striatum. Furthermore mesencephalic cell viability, as tested by [3H]DA uptake, did not change. Finally, we observed that 710nm LED light, locally conveyed in the rat SN, could modulate the firing activity of extracellular-recorded DA neurons. These data suggest that light can be detrimental or beneficial to DA neurons in SN, depending on the source and wavelength.

Variants of G protein-coupled receptors: a reappraisal of their role in receptor regulation.

Truncated or shorter forms of G protein-coupled receptors (GPCRs), originating by alternative splicing, have been considered physiologically irrelevant for a rather long time. Nevertheless, it is now recognized that alternative splicing variants of GPCRs greatly increase the total number of receptor isoforms and can regulate receptor trafficking and signalling. Furthermore, dimerization of these truncated variants with other receptors concurs to expand receptor diversity. Highly truncated variants of GPCRs, typically, are retained in the endoplasmic reticulum (ER) and by heteromerization prevent the wild-type receptor to reach the plasma membrane, exerting a dominant-negative effect on its function. This can be responsible for some pathological conditions but in some other cases, it can offer protection from a disease because the expression of the receptor, that is necessary for binding an infectious agent, is attenuated. Here, we propose a possible new mechanism of creation of truncated GPCR variants through an internal ribosome entry site (IRES), a nucleotide sequence that allows cap independent translation of proteins by recruiting the ribosome in proximity of an internal initiation codon. We suggest that an IRES, situated in the third cytoplasmic loop, could be responsible for the translation of the last two transmembrane (TM) regions of the muscarinic M2receptor. IRES driven expression of this C-terminal part of the muscarinic M2receptor could represent a novel and additional mechanism of receptor regulation.