Deorphanization of G Protein Coupled Receptors: A Historical Perspective.

Counting over 800 members, G protein coupled receptors (GPCRs) form the largest family of membrane receptors encoded in the human genome. Since the discovery of G proteins and GPCRs in the late 1970s and early 1980s, a significant portion of the GPCR research has been focused on identifying ligand/receptor pairs in parallel to studies related to their signaling properties. Despite significant advancements, about a fourth of the ∼400 nonodorant GPCRs are still considered orphan because their natural or endogenous ligands have yet to be identified. We should consider that every GPCR was once an orphan and that endogenous ligands have often been associated with biologic effects without a complete understanding of the molecular identity of their target receptors. Within this framework, this review offers a historical perspective on deorphanization processes for representative GPCRs, including the ghrelin receptor, γ aminobutyric acid B receptor, apelin receptor, cannabinoid receptors, and GPR15. It explores three main scenarios encountered in deorphanization efforts and discusses key questions and methodologies employed in elucidating ligand-receptor interactions, providing insights for future research endeavors. SIGNIFICANCE STATEMENT: Understanding how scientists have historically approached the issue of GPCR deorphanization and pairing of biologically active ligands with their cognate receptors are relevant topics in pharmacology. In fact, the biology of each GPCR, including its pathophysiological involvement, has often been uncovered only after its deorphanization, illuminating druggable targets for various diseases. Furthermore, uncovered endogenous ligands have therapeutic value as many ligands-or derivates thereof-are developed into drugs.

Gα Protein Signaling Bias at Serotonin 1A Receptor.

Serotonin 1A receptor (5-HT1AR) is a clinically relevant target because of its involvement in several central and peripheral functions, including sleep, temperature homeostasis, processing of emotions, and response to stress. As a G protein coupled receptor (GPCR) activating numerous Gα i/o/z family members, 5-HT1AR can potentially modulate multiple intracellular signaling pathways in response to different therapeutics. Here, we applied a cell-based bioluminescence resonance energy transfer assay to quantify how ten structurally diverse 5-HT1AR agonists exert biased signaling by differentially stimulating Gα i/o/z family members. Our concentration-response analysis of the activation of each Gα i/o/z protein revealed unique potency and efficacy profiles of selected agonists when compared with the reference 5-hydroxytryptamine, serotonin. Overall, our analysis of signaling bias identified groups of ligands sharing comparable G protein activation selectivity and also drugs with unique selectivity profiles. We observed, for example, a strong bias of F-15599 toward the activation of Gα i3 that was unique among the agonists tested: we found a biased factor of +2.19 when comparing the activation of Gα i3 versus Gα i2 by F-15599, while it was -0.29 for 8-hydroxy-2-(di-n-propylamino) tetralin. Similarly, vortioxetine showed a biased factor of +1.06 for Gα z versus Gα oA, while it was -1.38 for vilazodone. Considering that alternative signaling pathways are regulated downstream of each Gα protein, our data suggest that the unique pharmacological properties of the tested agonists could result in multiple unrelated cellular outcomes. Further investigation is needed to reveal how this type of ligand bias could affect cellular responses and to illuminate the molecular mechanisms underlying therapeutic profile and side effects of each drug. SIGNIFICANCE STATEMENT: Serotonin 1a receptor (5-HT1AR) activates several members of the Gi/o/z protein family. Here, we examined ten structurally diverse and clinically relevant agonists acting on 5-HT1AR and identified distinctive bias patterns among G proteins. Considering the diversity of their intracellular effectors and signaling properties, this data reveal novel mechanisms underlying both therapeutic and undesirable effects.

Death education for Palliative care: a european project for University students.

BACKGROUND: The need to spread the culture of palliative care and to train health care professionals from undergraduate courses is recognised internationally. The article presents the outcomes of a project devoted to palliative care training in university courses in four countries. AIMS: This article considered the outcomes of a course designed for university students who had the potential to work in a palliative care team. The main aim was to check the efficacy of the course and the motivation to work in palliative care settings, considering the impact of fear and representations of death. METHODS: The project presented the essential contents related to palliative care, using psychodramatic and photo-voice techniques. Longitudinal measurements were taken using a quantitative method design to detect changes among the students involved. The project involved 341 students at the first administration of the survey consisted of a protocol composed of standardized questionnaires in five countries (Austria, Israel, Italy, Poland and Romania), of whom 276 completed the pre- and post-surveys-165 of them in the experimental group and 111, in the control group. RESULTS: The experience showed that it is possible to address death-related issues seriously and competently without necessarily causing discomfort and despondency in students. The results of the changes over time in the experimental and control groups highlight how the view of death as annihilation is correlated with the fear of death and the need for avoidance of thoughts concerning dying. The main result is that competence in palliative care facilitates familiarisation with issues of death and dying, as well as the ability to work in this area, thereby enhancing interpersonal skills. CONCLUSION: The project showed that it is possible to implement death education on palliative care topics in undergraduate courses to increase motivation to work in this field.

Death education and photovoice at school: A workshop with Italian high school students.

The study shows the effects of a death education workshop with Italian high school students. Students (N = 416) from 10 high schools throughout Italy participated: 212 in the experimental group and 204 in the control group. Four weekly, two-hour workshops about the themes of death, with theoretical lessons and an experiential artistic activity of photovoice. Results show that the students in the experimental group showed significantly increased self-efficacy levels and existential anxiety levels did not increase after death education intervention. Educating youth about death could give them the skills to improve the management of death-related events and cope with negative emotions.

Identifying the prevalence of unmet supportive care needs among family caregivers of cancer patients: an Italian investigation on home palliative care setting.

PURPOSE: Family caregivers (FCs) are crucial resources in caring for cancer patients at home. The aim of this investigation was (1) to measure the prevalence of unmet needs reported by FCs of cancer patients in home palliative care, and (2) to investigate whether their needs change as their socio-demographic characteristics and the patients' functional abilities change. METHODS: FCs completed a battery of self-report questionnaires, including the Cancer Caregiving Tasks, Consequences, and Needs (CaTCoN). RESULTS: Data were collected from 251 FCs (74 men and 177 women, mean age 58.5 ± 14.2 years). Most of the participants experienced a substantial caregiving workload related to practical help (89.8%), provided some or a lot of personal care (73.1%), and psychological support (67.7%) to patients. More than half of the FCs reported that the patient's disease caused them negative physical effects (62.7%). Emotional, psychosocial, and psychological needs were referred. Some FCs reported that the patient's disease caused them a lot of stress (57.3%) and that they did not have enough time for friends/acquaintances (69.5%) and family (55.7%). The need to see a psychologist also emerged (44.0%). Age, caregiving duration, and patients' functional status correlated with FCs' unmet needs. Women reported more negative social, physical, and psychological consequences and a more frequent need to talk to a psychologist. CONCLUSION: The analysis demonstrated that cancer caregiving is burdensome. The results can guide the development and implementation of tailored programs or support policies so that FCs can provide appropriate care to patients while preserving their own well-being.

Home palliative care professionals perception of challenges during the Covid-19 outbreak: A qualitative study.

BACKGROUND: Home palliative care services have played an essential role during the first wave of the SARS-CoV-2 outbreak by providing symptom control, drug procurement, and psychological support for frail patients and their families unable to leave their homes. AIM: To understand how home palliative care professionals were affected by the outbreak, describing changes and challenges in their daily work as well as their reactions to the Covid-19 pandemic in Italy. DESIGN: Qualitative study conducted using telephone semi-structured interviews, with thematic analysis. SETTING/PARTICIPANTS: Thirty home care professionals working for an Italian non-profit organization which provides home palliative care for cancer patients and their families. RESULTS: Three main themes were identified. The first theme showed both patient-related and practice-related challenges participants faced in their daily work, requiring the implementation of different communication methods and patient and family education on risk prevention. The second theme showed the perception of increased responsibility and being the only landmark for family played a decisive role in participants' positive attitude. The third theme highlighted the participants' perception of the critical role of a home care setting in this emergency situation. CONCLUSIONS: The first wave of the Covid-19 pandemic brought many challenges and stressors for home palliative care professionals. On the other side, they reported a satisfaction with their critical role in carrying out their work with patients at risk.

Synaptic GluN2A-Containing NMDA Receptors: From Physiology to Pathological Synaptic Plasticity.

N-Methyl-d-Aspartate Receptors (NMDARs) are ionotropic glutamate-gated receptors. NMDARs are tetramers composed by several homologous subunits of GluN1-, GluN2-, or GluN3-type, leading to the existence in the central nervous system of a high variety of receptor subtypes with different pharmacological and signaling properties. NMDAR subunit composition is strictly regulated during development and by activity-dependent synaptic plasticity. Given the differences between GluN2 regulatory subunits of NMDAR in several functions, here we will focus on the synaptic pool of NMDARs containing the GluN2A subunit, addressing its role in both physiology and pathological synaptic plasticity as well as the contribution in these events of different types of GluN2A-interacting proteins.

Gz Enhanced Signal Transduction assaY (GZESTY) for GPCR deorphanization.

G protein-coupled receptors (GPCRs) are key pharmacological targets, yet many remain underutilized due to unknown activation mechanisms and ligands. Orphan GPCRs, lacking identified natural ligands, are a high priority for research, as identifying their ligands will aid in understanding their functions and potential as drug targets. Most GPCRs, including orphans, couple to Gi/o/z family members, however current assays to detect their activation are limited, hindering ligand identification efforts. We introduce GZESTY, a highly sensitive, cell-based assay developed in an easily deliverable format designed to study the pharmacology of Gi/o/z-coupled GPCRs and assist in deorphanization. We optimized assay conditions and developed an all-in-one vector employing novel cloning methods to ensure the correct expression ratio of GZESTY components. GZESTY successfully assessed activation of a library of ligand-activated GPCRs, detecting both full and partial agonism, as well as responses from endogenous GPCRs. Notably, with GZESTY we established the presence of endogenous ligands for GPR176 and GPR37 in brain extracts, validating its use in deorphanization efforts. This assay enhances the ability to find ligands for orphan GPCRs, expanding the toolkit for GPCR pharmacologists.

CYP46A1-mediated cholesterol turnover induces sex-specific changes in cognition and counteracts memory loss in ovariectomized mice.

The brain-specific enzyme CYP46A1 controls cholesterol turnover by converting cholesterol into 24S-hydroxycholesterol (24OH). Dysregulation of brain cholesterol turnover and reduced CYP46A1 levels are observed in Alzheimer's disease (AD). In this study, we report that CYP46A1 overexpression in aged female mice leads to enhanced estrogen signaling in the hippocampus and improved cognitive functions. In contrast, age-matched CYP46A1 overexpressing males show anxiety-like behavior, worsened memory, and elevated levels of 5α-dihydrotestosterone in the hippocampus. We report that, in neurons, 24OH contributes to these divergent effects by activating sex hormone signaling, including estrogen receptors. CYP46A1 overexpression in female mice protects from memory impairments induced by ovariectomy while having no effects in gonadectomized males. Last, we measured cerebrospinal fluid levels of 24OH in a clinical cohort of patients with AD and found that 24OH negatively correlates with neurodegeneration markers only in women. We suggest that CYP46A1 activation is a valuable pharmacological target for enhancing estrogen signaling in women at risk of developing neurodegenerative diseases.

Probing the orphan receptors: Tools and directions.

The endogenous ligands activating a large fraction of the G Protein Coupled Receptor (GPCR) family members have yet to be identified. These receptors are commonly labeled as orphans (oGPCRs), and because of the absence of available pharmacological tools they are currently understudied. Nonetheless, genome wide association studies, together with research using animal models identified many physiological functions regulated by oGPCRs. Similarly, mutations in some oGPCRs have been associated with rare genetic disorders or with an increased risk of developing pathologies. The once underestimated pharmacological potential of targeting oGPCRs is increasingly being exploited by the development of novel tools to understand their biology and by drug discovery endeavors aimed at identifying new modulators of their activity. Here, we summarize recent advancements in the field of oGPCRs and future directions.

Comprehensive Spatial Profile of the Orphan G Protein Coupled Receptor GPRC5B Expression in Mouse Brain.

Orphan G Protein Coupled Receptors (GPCRs) are GPCRs whose endogenous ligands are unknown or still debated. Due to the lack of pharmacological modulators, the physiological function of orphan GPCRs is understudied. However, relevant physiological roles associated with orphan GPCRs have been revealed by analysis of animal models and genome wide association studies illuminating an untapped potential for drug discovery. G Protein Coupled Receptor class C Group 5 Member B (GPRC5B) is among the most expressed GPCRs in the central nervous system. Thus, the expression profiling of GPRC5B is an essential step toward understanding GPRC5B function in health and disease. In this study, we generated new GPRC5B polyclonal antibodies and investigated the expression levels of GPRC5B across different organs and brain regions. We identified high levels of GPRC5B glycosylation both in transfected cells and in mouse brain. Moreover, in situ hybridization imaging analysis indicated that Gprc5b was expressed at the highest level in olfactory bulb, hippocampus, cerebellum, and pons. To dissect expression within various neuronal populations, we conducted a comprehensive spatial profiling of Gprc5b across excitatory and inhibitory neuronal types in medial prefrontal cortex, motor cortex, hippocampal regions, hypothalamus, and cerebellum. Overall, we discovered that GABAergic neurons displayed higher Gprc5b expression levels than glutamatergic neurons in most of the analyzed regions with the important exception of the hippocampal dentate gyrus. Overall, the expression analysis of GPRC5B in mouse brain will guide functional studies ultimately positioning GPRC5B in pathophysiological mechanisms and drug discovery.

Rabphilin-3A Drives Structural Modifications of Dendritic Spines Induced by Long-Term Potentiation.

The interaction of Rabphilin-3A (Rph3A) with the NMDA receptor (NMDAR) in hippocampal neurons plays a pivotal role in the synaptic retention of this receptor. The formation of a Rph3A/NMDAR complex is needed for the induction of long-term potentiation and NMDAR-dependent hippocampal behaviors, such as spatial learning. Moreover, Rph3A can also interact with AMPA receptors (AMPARs) through the formation of a complex with myosin Va. Here, we used a confocal imaging approach to show that Rph3A overexpression in primary hippocampal neuronal cultures is sufficient to promote increased dendritic spine density. This morphological event is correlated with an increase in GluN2A-containing NMDARs at synaptic membranes and a decrease in the surface levels of GluA1-containing AMPARs. These molecular and morphological modifications of dendritic spines are sufficient to occlude the spine formation induced by long-term potentiation, but do not prevent the spine loss induced by long-term depression. Overall, our results demonstrate a key role for Rph3A in the modulation of structural synaptic plasticity at hippocampal synapses that correlates with its interactions with both NMDARs and AMPARs.

Tackling the Pandemic a Year Later: Burnout Among Home Palliative Care Clinicians.

CONTEXT: The COVID-19 pandemic strongly challenged healthcare workers, disrupting their work routine and impacting on their professional life. A previous investigation explored levels of burnout and psychological morbidity among palliative care professionals (PCPs) during COVID-19 first wave. OBJECTIVE: To update data about burnout and psychological morbidity among PCPs after a year of COVID-19 pandemic. METHODS: The same questionnaires on burnout (Maslach Burnout Inventory, MBI) and psychological morbidity (General Health Questionnaire 12 items, GHQ-12) were administered a year after. Differences in MBI and GHQ-12 scores obtained in the two studies (COVID2020 and COVID2021), as well as distributions of PCPs showing burnout symptoms and psychological morbidity were analyzed and compared. We also explored the association between the three dimensions of burnout and socio-demographic and professional characteristics. RESULTS: The sample consisted of 145 PCPs (59% physicians and 41% nurses). Response rate (70.4%) was quite similar to the previous study (73.2%). No differences were observed in the frequency of burnout between COVID2021 and COVID2020; the PCPs in COVID2021 reported marginally higher level of EE (P = .049) and this result is confirmed in physicians (P = .010) while no difference was observed in nurses (P = .326). In addition, the percentage of cases showing psychological morbidity significantly decreased. CONCLUSION: Our findings show stable levels of burnout and decreasing levels of psychological morbidity among PCPs one year after the onset of the COVID-19 pandemic. However, more research is needed to detail the significance of emotional exhaustion dimension, a variable influenced by the survey.

Caring Advanced Cancer Patients at Home During COVID-19 Outbreak: Burnout and Psychological Morbidity Among Palliative Care Professionals in Italy.

CONTEXT: Providing palliative care (PC) at home for patients with advanced cancer has become essential during the COVID-19 emergency. Nevertheless, the home PC professionals (PCPs) faced a challenging situation because of increased number of discharged patients, reduced availability of health-care facilities, and physical/relational barriers between them and patients. OBJECTIVES: This study aimed to investigate the impact of COVID-19 pandemic on burnout and psychological morbidity among home PCPs in Italy. METHODS: One hundred and ninety-eight PC physicians and nurses working in home assistance in Italy were invited to participate. The results obtained by the investigation conducted during the COVID-19 emergency (COVID2020) were compared with data collected in 2016 in the same setting (BURNOUT2016). The questionnaires (socio-demographics, Maslach Burnout Inventory and General Health Questionnaire-12) were the same for both the surveys. The PCPs participating in COVID2020 survey (n = 145) were mostly the same (70%) who participated in the BURNOUT2016 study (n = 179). RESULTS: One hundred and forty-five PCPs participated in the study (response rate 73.2%). During the COVID-19 emergency, home PCPs presented a lower burnout frequency (P < .001) and higher level of personal accomplishment than in 2016 (P = .047). Conversely, the risk for psychological morbidity was significantly higher during the pandemic (P < .001). CONCLUSIONS: In the age of COVID-19, the awareness of being at the forefront of containing the pandemic along with the sense of responsibility toward their high-risk patients may arouse PCPs' psychological distress, but, on the other hand, this condition may improve their sense of professional satisfaction and personal accomplishment.

Students' Confidence and Interest in Palliative and Bereavement Care: A European Study.

As part of a European Erasmus Plus project entitled Death Education for Palliative Psychology, this study assessed the ways in which Master's Degree students in psychology and the creative arts therapies self-rated their confidence and interest in death education and palliative and bereavement care. In five countries (Austria, Israel, Italy, Poland, Romania), 344 students completed an online questionnaire, and 37 students were interviewed to better understand their views, interest, and confidence. The results revealed some significant differences between countries, and showed that older respondents with previous experience as formal caregivers for end-of-life clients showed greater interest in obtaining practical clinical competence in these fields. A mediation analysis indicated that students' previous care experiences and past loss experiences were related to students' current interest in death education and palliative and bereavement care through the mediation of their sense of confidence in this field. The qualitative findings identified five shared themes: life and death, learning about death, the psychological burden, personal experience and robust training, and four key training needs. Overall, students' interest in studying and working with terminal illness and death are rooted in internal resources, a preliminary sense of confidence, but also external requirements.

Burnout in Home Palliative Care: What Is the Role of Coping Strategies?

OBJECTIVE: The study examines psychophysical distress of health-care professionals providing home-based palliative care. The aim is to investigate potential correlations between dimensions of burnout and different coping strategies. METHODS: The present study is an observational cross-sectional investigation. The study involved all the home palliative care teams of an Italian nonprofit organization. Of a total of 275 practitioners working for the organization, 207 (75%) decided to participate in the study and complete questionnaires. Questionnaires employed were Maslach Burnout Inventory, General Health Questionnaire 12, Psychophysiological Questionnaire of CBA 2.0, and Coping Orientation to Problems Experienced. Professionals were physicians (50%), nurses (36%), and psychologists (14%). There were no exclusion criteria. Data were processed by SPSS 23 and analyses employed were Spearman ρ, Mann-Whitney U test, and 1-way analysis of variance on ranks. RESULTS: Among participants, a low number of professionals were emotionally exhausted (11%) or not fulfilled at work (20%), whereas most of them complained of depersonalization symptoms (67%). Emotional exhaustion and depersonalization were found to be associated with avoidance coping strategies, whereas problem-solving and positive attitude were negatively associated with emotional exhaustion and positively with personal accomplishment. Moreover, using avoidance strategies was related to a worse psychological and physical condition. CONCLUSIONS: Findings suggest the need to provide professionals training programs about coping and communication skills tailored to fit the professionals' needs according to their work experience in palliative care and aimed at improving the approach to patients and relatives.

The Synaptonuclear Messenger RNF10 Acts as an Architect of Neuronal Morphology.

The Ring Finger Protein 10 [RNF10] is a novel synapse-to-nucleus signaling protein that specifically links activation of synaptic NMDA receptors to modulation of gene expression. RNF10 dissociation from the GluN2A subunit of the NMDA receptor represents the first step of its synaptonuclear transport and it is followed by an importin-dependent translocation into the nucleus. Here, we have identified protein kinase C [PKC]-dependent phosphorylation of RNF10 Ser31 as a key step for RNF10 detachment from NMDA receptor and its subsequent trafficking to the nucleus. We show that pSer31-RNF10 plays a role both in synaptonuclear signaling and in neuronal morphology. In particular, the prevention of Ser31 RNF10 phosphorylation induces a decrease in spine density, neuronal branching, and CREB signaling, while opposite effects are obtained by mimicking a stable RNF10 phosphorylation at Ser31. Overall, these results add novel information about the functional and structural role of synaptonuclear protein messengers in shaping dendritic architecture in hippocampal neurons.

Linking NMDA Receptor Synaptic Retention to Synaptic Plasticity and Cognition.

NMDA receptor (NMDAR) subunit composition plays a pivotal role in synaptic plasticity at excitatory synapses. Still, the mechanisms responsible for the synaptic retention of NMDARs following induction of plasticity need to be fully elucidated. Rabphilin3A (Rph3A) is involved in the stabilization of NMDARs at synapses through the formation of a complex with GluN2A and PSD-95. Here we used different protocols to induce synaptic plasticity in the presence or absence of agents modulating Rph3A function. The use of Forskolin/Rolipram/Picrotoxin cocktail to induce chemical LTP led to synaptic accumulation of Rph3A and formation of synaptic GluN2A/Rph3A complex. Notably, Rph3A silencing or use of peptides interfering with the GluN2A/Rph3A complex blocked LTP induction. Moreover, in vivo disruption of GluN2A/Rph3A complex led to a profound alteration of spatial memory. Overall, our results demonstrate a molecular mechanism needed for NMDAR stabilization at synapses after plasticity induction and to trigger downstream signaling events necessary for cognitive behavior.