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Prokineticin 2 (PK2) binds to prokineticin receptor 1 and prokineticin receptor 2 (PKR1 and PKR2, respectively), two G protein-coupled receptors (GPCRs) that can mediate multiple signalling pathways by promoting the elevation of intracellular calcium and cAMP levels, phosphorylation of Akt and activation of ERK and STAT3. This work aims to evidence the conservation of protein sequence and the mechanism of PK2 binding to PKR1 to use the zebrafish model for the identification of new drugs as targets of prokineticin receptors. To this end, we first demonstrated that the zebrafish genes pk2 and pkr1 are phylogenetically related to orthologous mammalian genes by constructing evolutionary trees and performing syntenic analyses. Subsequently, by comparing the amino acid sequences, we showed that the interaction sites with PK2 are conserved in the zPKR1. Using GST pull-down and cross-linking experiments, we demonstrated the crucial role of the N-terminal region of zPKR1 for binding to the PK2. Finally, by expressing zPKR1 in CHO cells, we demonstrated the ability of zPKR1 to induce the activation of ERK and STAT3.
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The prokineticin system plays a role in hypothalamic neurons in the control of energy homeostasis. Prokineticin receptors (PKR1 and PKR2), like other G-protein-coupled receptors (GPCRs) are involved in the regulation of energy intake and expenditure and are modulated by the accessory membrane protein 2 of the melanocortin receptor (MRAP2). The aim of this work is to characterise the interaction and regulation of the non-melanocortin receptor PKR1 by MRAP2a in zebrafish (zMRAP2a) in order to use zebrafish as a model for the development of drugs targeting accessory proteins that can alter the localisation and activity of GPCRs. To this end, we first showed that zebrafish PKR1 (zPKR1) is able to interact with both zMRAP2a and human MRAP2 (hMRAP2). This interaction occurs between the N-terminal region of zPKR1 and the C-terminal domain of zMRAP2a, which shows high sequence identity with hMRAP2 and a similar propensity for dimer formation. Moreover, we demonstrated that in Chinese hamster ovary (CHO) cells, zMRAP2a or hMRAP2 are able to modulate zPKR1 activation induced by zebrafish PK2 (zPK2) resulting in an impaired ERK and STAT3 activation.
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Receptores Acoplados a Proteínas G , Proteínas de Pez Cebra , Pez Cebra , Animales , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Células CHO , Cricetulus , Unión Proteica , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Péptidos/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
The biological substrate of persistent post-COVID-19 hyposmia is still unclear. However, as many neurodegenerative diseases present with smell impairment at onset, it may theoretically reflect degeneration within the central olfactory circuits. However, no data still exist regarding the post-COVID-19 patients. As the olfactory neurons (ONs) mirror pathological changes in the brain, allowing for tracking the underlying molecular events, here, we performed a broad analysis of ONs from patients with persistent post-COVID-19 OD to identify traces of potential neurodegeneration. ONs were collected through the non-invasive brushing of the olfactory mucosa from ten patients with persistent post-COVID-19 hyposmia (lasting > 6 months after infection) and ten age/sex-matched controls. Immunofluorescence staining for protein quantification and RT-PCR for gene expression levels were combined to measure ONs markers of α-synuclein, amyloid-ß, and tau pathology, axonal injury, and mitochondrial network. Patients and controls had similar ONs levels of oligomeric α-synuclein, amyloid-ß peptide, tau protein, neurofilament light chain (NfL), cytochrome C oxidase subunit 3 (COX3), and the heat shock protein 60 (HSP60). Our findings thus did not provide evidence for synucleinopathy and amyloid-ß mismetabolism or gross traces of neuronal injury and mitochondrial dysfunction within the olfactory system in the early phase of persistent post-COVID-19 hyposmia.
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Melanocortin receptor accessory protein 2 (MRAP2) is a membrane protein that binds multiple G protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, including prokineticin receptors. These GPCRs are expressed both centrally and peripherally, and their endogenous ligands are prokineticin 1 (PK1) and prokineticin 2 (PK2). PKRs couple all G-protein subtypes, such as Gαq/11, Gαs, and Gαi, and recruit ß-arrestins upon PK2 stimulation, although the interaction between PKR2 and ß-arrestins does not trigger receptor internalisation. MRAP2 inhibits the anorexigenic effect of PK2 by binding PKR1 and PKR2. The aim of this work was to elucidate the role of MRAP2 in modulating PKR2-induced ß-arrestin-2 recruitment and ß-arrestin-mediated signalling. This study could allow the identification of new specific targets for potential new drugs useful for the treatment of the various pathologies correlated with prokineticin, in particular, obesity.
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Laryngeal cancer accounts for one-third of all head and neck tumors, with squamous cell carcinoma (SCC) being the most predominant type, followed by neuroendocrine tumors. Chromogranins, are commonly used as biomarkers for neuroendocrine tumors, including laryngeal cancer. It has been reported that secretogranin VGF, a member of the chromogranin family, can be also used as a significant biomarker for neuroendocrine tumors. However, the expression and role of VGF in laryngeal carcinomas have not been previously investigated. Therefore, the present study aimed to determine the expression levels of VGF in laryngeal SCC (LSCC). The present study collected tumor tissues, as well as serum samples, from a cohort of 15 patients with LSCC. The results of reverse transcription-quantitative PCR, western blot analysis and immunofluorescence assays showed that the selective VGF precursor was downregulated in patients with LSCC. Notably, in tumor tissue, the immunoreactivity for VGF was found in vimentin-positive cells, probably corresponding to T lymphocytes. The current preliminary study suggested that the reduced expression levels of VGF observed in tumor tissue and at the systemic level could sustain LSCC phenotype. Overall, VGF could be a potential biomarker for detecting neoplastic lesions with a higher risk of tumor invasiveness, even in non-neuroendocrine tumors.
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Rheumatoid arthritis (RA) is a chronic inflammatory disease mediated by an interdependent network of proinflammatory molecules such as chemokines. Prokineticin 2 (PK2) is a chemokine-like peptide that modulates nociceptive threshold and immuno-inflammatory processes via two G-protein-linked receptors, prokineticin receptor 1 and 2 (PKR1 and PKR2). In the present study, we investigated the effects of the prokineticin receptor antagonist PC1 on arthritic pain and the inflammatory response in type II collagen-induced arthritis (CIA) in mice. We demonstrated that PC1, administered subcutaneously from day 25 to day 35 after CIA, improved clinical signs of arthritis such as paw edema, pain, and impaired locomotor activity. In CIA mice, PC1 was also able to lower plasma malondialdehyde (MDA) levels, suggesting a role in reducing oxidative damage, as well as joint expression levels of PK2, PKRs, TNFα, IL-1ß, CD4, CD8, and NF-kB. These results suggest that blocking PKRs may be a successful strategy to control arthritic pain and pathology development. KEY MESSAGES: PK2/PKRs expression levels strongly increase in the synovium of RA mice. PC1 treatment shows anti-arthritic activity and reduces arthritis-induced pain. PC1 treatment significantly lowers synovial PK2/PKRs levels. PC1 treatment lowers plasma MDA levels and synovial levels of TNFα and IL -1ß PC1 treatment is a viable therapeutic option for RA.
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Artritis Experimental , Artritis Reumatoide , Sinovitis , Ratones , Animales , Artritis Experimental/patología , Factor de Necrosis Tumoral alfa/metabolismo , Dolor , Membrana Sinovial , Sinovitis/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismoRESUMEN
Persistent olfactory dysfunction (OD) is one of the most complaining and worrying complications of long COVID-19 because of the potential long-term neurological consequences. While causes of OD in the acute phases of the SARS-CoV-2 infection have been figured out, reasons for persistent OD are still unclear. Here we investigated the activity of two inflammatory pathways tightly linked with olfaction pathophysiology, namely Substance P (SP) and Prokineticin-2 (PK2), directly within the olfactory neurons (ONs) of patients to understand mechanisms of persistent post-COVID-19 OD. ONs were collected by non-invasive brushing from ten patients with persistent post-COVID-19 OD and ten healthy controls. Gene expression levels of SP, Neurokinin receptor 1, Interleukin-1ß (IL-1ß), PK2, PK2 receptors type 1 and 2, and Prokineticin-2-long peptide were measured in ONs by Real Time-PCR in both the groups, and correlated with residual olfaction. Immunofluorescence staining was also performed to quantify SP and PK2 proteins. OD patients, compared to controls, exhibited increased levels of both SP and PK2 in ONs, the latter proportional to residual olfaction. This work provided unprecedented, preliminary evidence that both SP and PK2 pathways may have a role in persistent post-COVID-19 OD. Namely, if the sustained activation of SP, lasting months after infection's resolution, might foster chronic inflammation and contribute to hyposmia, the PK2 expression could instead support the smell recovery.
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COVID-19 , Trastornos del Olfato , Humanos , Neuronas , Síndrome Post Agudo de COVID-19 , SARS-CoV-2 , Olfato , Sustancia PRESUMEN
OBJECTIVE: The objective of this study was to outline the dynamics of prokineticin-2 pathway in relation to clinical-pathological features of Parkinson's disease by examining olfactory neurons of patients. METHODS: Thirty-eight patients (26 de novo, newly diagnosed) and 31 sex/age-matched healthy controls underwent noninvasive mucosa brushing for olfactory neurons collection, and standard clinical assessment. Gene expression levels of prokineticin-2, prokineticin-2 receptors type 1 and 2, and prokineticin-2-long peptide were measured in olfactory neurons by real-time polymerase chain reaction (PCR); moreover, the prokineticin-2 protein and α-synuclein species (total and oligomeric) were quantified by immunofluorescence staining. RESULTS: Prokineticin-2 expression was significantly increased in Parkinson's disease. De novo patients had higher prokineticin-2 levels, directly correlated with Movement Disorder Society-Sponsored Revision of the Unified Parkinson Disease Rating Scale (MDS-UPDRS) part III motor score. In addition, oligomeric α-synuclein was higher in Parkinson's disease and directly correlated with prokineticin-2 protein levels. Total α-synuclein did not differ between patients and controls. INTERPRETATION: Prokineticin-2 is a chemokine showing neuroprotective effects in experimental models of Parkinson's disease, but translational proof of its role in patients is still lacking. Here, we used olfactory neurons as the ideal tissue to analyze molecular stages of neurodegeneration in vivo, providing unprecedented evidence that the prokineticin-2 pathway is activated in patients with Parkinson's disease. Specifically, prokineticin-2 expression in olfactory neurons was higher at early disease stages, proportional to motor severity, and associated with oligomeric α-synuclein accumulation. These data, consistently with preclinical findings, support prokineticin-2 as a candidate target in Parkinson's disease, and validate reliability of olfactory neurons to reflect pathological changes of the disease. ANN NEUROL 2023;93:196-204.
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Enfermedad de Parkinson , Humanos , alfa-Sinucleína/genética , Pruebas de Estado Mental y Demencia , Neuronas/metabolismo , Enfermedad de Parkinson/genética , Reproducibilidad de los ResultadosRESUMEN
MRAP2 is a small simple transmembrane protein arranged in a double antiparallel topology on the plasma membrane. It is expressed in the paraventricular nucleus of the hypothalamus, where it interacts with various G protein-coupled receptors, such as the prokineticin receptors, and regulates energy expenditure and appetite. The aim of this work was to analyze the functional role of the specific arginine residue at position 125 of MRAP2, which affects protein conformation, dimer formation, and PKR2 binding. Results obtained with the MRAP2 mutants R125H and R125C, which are found in human patients with extreme obesity, and mouse MRAP2, in which arginine 125 is normally replaced by histidine, were compared with those obtained with human MRAP2. Understanding the mechanism by which MRAP2 regulates G protein-coupled receptors helps in elucidating the metabolic pathways involved in metabolic dysfunction and in developing new drugs as specific targets of the MRAP2-PKR2 complex.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Arginina , Animales , Arginina/metabolismo , Humanos , Hipotálamo/metabolismo , Ratones , Núcleo Hipotalámico Paraventricular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
The role of the immune system in neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) has become clear in recent decades, as evidenced by the presence of activated microglia and astrocytes and numerous soluble mediators in the brain and peripheral tissues of affected patients. Among inflammatory mediators, chemokines play a central role in neuroinflammation due to their dual function as chemoattractants for immune cells and molecular messengers in crosstalk among CNS-resident cells. The chemokine Bv8/Prokineticin 2 (PK2) has recently emerged as an important player in many age-related and chronic diseases that are either neurodegenerative or systemic. In this perspective paper, we briefly discuss the role that PK2 and its cognate receptors play in AD and PD animal models and in patients. Given the apparent changes in PK2 blood levels in both AD and PD patients, the potential clinical value of PK2 either as a disease biomarker or as a therapeutic target for these disorders is discussed.
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Enfermedad de Alzheimer , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Animales , Encéfalo , Modelos Animales de EnfermedadRESUMEN
Melanocortin Receptor Accessory Protein 2 (MRAP2) modulates the trafficking and signal transduction of several G-protein-coupled receptors (GPCRs) involved in the control of energy homeostasis, such as Prokineticin receptors (PKRs). They bind the endogenous ligand prokineticin 2 (PK2), a novel adipokine that has an anorexic effect and modulates thermoregulation and energy homeostasis. In the present work, we used biochemical techniques to analyze the mechanism of interaction of MRAP2 with PKR2 and we identified the specific amino acid regions involved in the complex formation. Our results indicate that MRAP2 likely binds to the N-terminal region of PKR2, preventing glycosylation and consequently the correct receptor localization. We also identified a C-terminal region of MRAP2 that is critical for the interaction with PKR2. Consequently, we analyzed the role of the prokineticin transduction system in the regulation of MRAP2 expression in tissues involved in the control of food intake: at the central level, in hypothalamic explants, and at the peripheral level, in adipocytes. We demonstrated the modulation of MRAP2 expression by the prokineticin transduction system.
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Proteínas Adaptadoras Transductoras de Señales , Melanocortinas , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Portadoras/metabolismo , Melanocortinas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Melanocortina/metabolismo , Transducción de SeñalRESUMEN
Neuropathic pain (NP) originates from an injury or disease of the somatosensory nervous system. This heterogeneous origin and the possible association with other pathologies make the management of NP a real challenge. To date, there are no satisfactory treatments for this type of chronic pain. Even strong opioids, the gold-standard analgesics for nociceptive and cancer pain, display low efficacy and the paradoxical ability to exacerbate pain sensitivity in NP patients. Mounting evidence suggests that chemokine upregulation may be a common mechanism driving NP pathophysiology and chronic opioid use-related consequences (analgesic tolerance and hyperalgesia). Here, we first review preclinical studies on the role of chemokines and chemokine receptors in the development and maintenance of NP. Second, we examine the change in chemokine expression following chronic opioid use and the crosstalk between chemokine and opioid receptors. Then, we examine the effects of inhibiting specific chemokines or chemokine receptors as a strategy to increase opioid efficacy in NP. We conclude that strong opioids, along with drugs that block specific chemokine/chemokine receptor axis, might be the right compromise for a favorable risk/benefit ratio in NP management.
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Prokineticin 2 (PROK2) is a secreted bioactive peptide that regulates a variety of biological responses via two GPCRs, the prokineticin receptors (PROKRs). The aim of this study was to characterize a new alternatively spliced product of the prok2 gene consisting of four exons. The 40-amino acid peptide, designated PROK2C, is encoded by exon 1 and exon 4, and its expression was detected in the hippocampus and spinal cord of mice. PROK2C was expressed in a heterologous system, Pichia pastoris, and its binding specificity to the amino-terminal regions of PROKR1 and PROKR2 was investigated by GST pull-down experiments. In addition, the introduction of the unnatural amino acid p-benzoyl-L-phenylalanine using amber codon suppression technology demonstrated the role of tryptophan at position 212 of PROKR2 for PROK2C binding by photoactivatable cross-linking. The functional significance of this new isoform was determined in vivo by nociceptive experiments, which showed that PROK2C elicits strong sensitization of peripheral nociceptors to painful stimuli. In order to analyze the induction of PROK2C signal transduction, STAT3 and ERK phosphorylation levels were determined in mammalian CHO cells expressing PROKR1 and PROKR2. Our data show by in vivo and in vitro experiments that PROK2C can bind and activate both prokineticin receptors.
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The prokineticin-2 (PROK2) is a small peptide belonging to the prokineticin family. In humans and rodents this chemokine is primarily involved in the control of central and peripheral reproductive processes. Klinefelter's syndrome (KS) is the first cause of male genetic infertility, due to an extra X chromosome, which may occur with a classical karyotype (47, XXY) or mosaic forms (46, XY/47, XXY). In affected subjects, pubertal maturation usually begins at an adequate chronological age, but when development is almost complete, they display a primary gonadal failure, with early spermatogenesis damage, and later onset of testosterone insufficiency. Thus, the main aim of the present study was to investigate the serum levels of PROK2 in prepubertal and adult KS patients, comparing them with healthy subjects. We showed for the first time the presence of PROK2 in the children serum but with significant changes in KS individuals. Indeed, compared with healthy subjects characterized by PROK2 serum elevation during the growth, KS individuals showed constant serum levels during the sexual maturation phase (higher during the prepubertal phase but lower during the adult age). In conclusion, these data indicate that in KS individuals PROK2 may be considered a biomarker for investigating the SK infertility process.
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Hormonas Gastrointestinales/sangre , Infertilidad Masculina/diagnóstico , Síndrome de Klinefelter/sangre , Neuropéptidos/sangre , Adolescente , Adulto , Biomarcadores/sangre , Niño , Humanos , Infertilidad Masculina/etiología , Cariotipo , Síndrome de Klinefelter/complicaciones , Masculino , Persona de Mediana Edad , Maduración Sexual , Espermatogénesis , Testosterona/deficiencia , Adulto JovenRESUMEN
Acute inflammation is particularly relevant in the pathogenesis of visceral hypersensitivity associated with inflammatory bowel diseases. Glia within the enteric nervous system, as well as within the central nervous system, contributes to neuroplasticity during inflammation, but whether enteric glia has the potential to modify visceral sensitivity following colitis is still unknown. This work aimed to investigate the occurrence of changes in the neuron-glial networks controlling visceral perception along the gut-brain axis during colitis, and to assess the effects of peripheral glial manipulation. Enteric glia activity was altered by the poison fluorocitrate (FC; 10 µmol kg-1 i.p.) before inducing colitis in animals (2,4-dinitrobenzenesulfonic acid, DNBS; 30 mg in 0.25 mL EtOH 50%), and visceral sensitivity, colon damage, and glia activation along the pain pathway were studied. FC injection significantly reduced the visceral hyperalgesia, the histological damage, and the immune activation caused by DNBS. Intestinal inflammation is associated with a parallel overexpression of TRPV1 and S100ß along the gut-brain axis (colonic myenteric plexuses, dorsal root ganglion, and periaqueductal grey area). This effect was prevented by FC. Peripheral glia activity modulation emerges as a promising strategy for counteracting visceral pain induced by colitis.
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BACKGROUND: Mood and metabolic disorders are interrelated and may share common pathological processes. Autonomic neurons link the brain with the gastrointestinal tract and constitute a likely pathway for peripheral metabolic challenges to affect behaviors controlled by the brain. The activities of neurons along these pathways are regulated by glia, which exhibit phenotypic shifts in response to changes in their microenvironment. How glial changes might contribute to the behavioral effects of consuming a high-fat diet (HFD) is uncertain. Here, we tested the hypothesis that anxiogenic and depressive-like behaviors driven by consuming a HFD involve compromised duodenal barrier integrity and subsequent phenotypic changes to glia and neurons along the gut-brain axis. METHODS: C57Bl/6 male mice were exposed to a standard diet or HFD for 20 weeks. Bodyweight was monitored weekly and correlated with mucosa histological damage and duodenal expression of tight junction proteins ZO-1 and occludin at 0, 6, and 20 weeks. The expression of GFAP, TLR-4, BDNF, and DCX were investigated in duodenal myenteric plexus, nodose ganglia, and dentate gyrus of the hippocampus at the same time points. Dendritic spine number was measured in cultured neurons isolated from duodenal myenteric plexuses and hippocampi at weeks 0, 6, and 20. Depressive and anxiety behaviors were also assessed by tail suspension, forced swimming, and open field tests. RESULTS: HFD mice exhibited duodenal mucosa damage with marked infiltration of immune cells and decreased expression of ZO-1 and occludin that coincided with increasing body weight. Glial expression of GFAP and TLR4 increased in parallel in the duodenal myenteric plexuses, nodose ganglia, and hippocampus in a time-dependent manner. Glial changes were associated with a progressive decrease in BDNF, and DCX expression, fewer neuronal dendritic spines, and anxiogenic/depressive symptoms in HFD-treated mice. Fluorocitrate (FC), a glial metabolic poison, abolished these effects both in the enteric and central nervous systems and prevented behavioral alterations at week 20. CONCLUSIONS: HFD impairs duodenal barrier integrity and produces behavioral changes consistent with depressive and anxiety phenotypes. HFD-driven changes in both peripheral and central nervous systems are glial-dependent, suggesting a potential glial role in the alteration of the gut-brain signaling that occurs during metabolic disorders and psychiatric co-morbidity.
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Encéfalo/metabolismo , Encéfalo/patología , Depresión/etiología , Dieta Alta en Grasa/efectos adversos , Duodeno/patología , Trastornos Mentales/etiología , Neuroglía/metabolismo , Animales , Peso Corporal , Duodeno/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Plexo Mientérico/metabolismo , Plexo Mientérico/patología , Neuroglía/patología , Neuronas/metabolismo , Neuronas/patología , Ganglio Nudoso/metabolismo , Ganglio Nudoso/patologíaRESUMEN
The prokineticin (PK) family, prokineticin 1 and Bv8/prokineticin 2 (PROK2), initially discovered as regulators of gastrointestinal motility, interacts with two G protein-coupled receptors, PKR1 and PKR2, regulating important biological functions such as circadian rhythms, metabolism, angiogenesis, neurogenesis, muscle contractility, hematopoiesis, immune response, reproduction and pain perception. PROK2 and PK receptors, in particular PKR2, are widespread distributed in the central nervous system, in both neurons and glial cells. The PROK2 expression levels can be increased by a series of pathological insults, such as hypoxia, reactive oxygen species, beta amyloid and excitotoxic glutamate. This suggests that the PK system, participating in different cellular processes that cause neuronal death, can be a key mediator in neurological/neurodegenerative diseases. While many PROK2/PKRs effects in physiological processes have been documented, their role in neuropathological conditions is not fully clarified, since PROK2 can have a double function in the mechanisms underlying to neurodegeneration or neuroprotection. Here, we briefly outline the latest findings on the modulation of PROK2 and its cognate receptors following different pathological insults, providing information about their opposite neurotoxic and neuroprotective role in different pathological conditions.