Identification and characterization of prokineticin receptor 2 splicing variant and its modulation in an animal model of alzheimer's disease

Prokineticin 2 is a peptide that is widely distributed in the nervous system and influences a variety of brain functions, such as pain, food intake and circadian rhythms. We previously demonstrated that, in the animal model of Alzheimer’s disease, induced by the intracerebroventricular administration of Aβ1-42, there is a modulation of the prokineticin system in rat hippocampus. Prokineticin 2 is a able to mediate its signaling through two different G-protein coupled receptors, designated PKR1 and PKR2, belonging to the neuropeptide Y receptor class. These two receptors have different tissue distributions: PKR1 is expressed in diverse peripheral organs with relatively high levels in the small intestines and lung, whereas PKR2 is predominantly expressed in the central nervous system. The PKRs activate multiple intracellular signal-transduction pathways: they are Gαq-coupled receptors and promoting intracellular calcium mobilization but they also couple to Gαi (especially PKR2) and Gαs proteins. In rat hippocampus we identified a mRNA encoding for a PKR2 splice variant, that lacking the second exon, gives rise to a four-transmembrane protein denominated TM 4-7. Expression of this splicing variant in yeast, allowed us to demonstrate that TM 4-7 dimerizes with PKR2 long form and that this heterodimer binds to G protein subtypes with different specificity respect to PKR2 wild-type. Moreover we evidenced that, following Aβ1-42 intracerebroventricular injection in rat, the PKR2 hippocampal levels slightly increased respect to control animals whereas there was a strong up-regulation of the PKR2 splicing variant, TM 4-7. We showed that the increased levels of TM 4-7 determined a modulation of PKR2 signal transduction hindering STAT3 activation

The role of prokineticin 2 in oxidative stress and in neuropathological processes

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

Antagonism of the prokineticin system prevents and reverses allodynia and inflammation in a mouse model of diabetes

Neuropathic pain is a severe diabetes complication and its treatment is not satisfactory. It is associated with neuroinflammation-related events that participate in pain generation and chronicization. Prokineticins are a new family of chemokines that has emerged as critical players in immune system, inflammation and pain. We investigated the role of prokineticins and their receptors as modulators of neuropathic pain and inflammatory responses in experimental diabetes. In streptozotocin-induced-diabetes in mice, the time course expression of prokineticin and its receptors was evaluated in spinal cord and sciatic nerves, and correlated with mechanical allodynia. Spinal cord and sciatic nerve pro- and anti-inflammatory cytokines were measured as protein and mRNA, and spinal cord GluR subunits expression studied. The effect of preventive and therapeutic treatment with the prokineticin receptor antagonist PC1 on behavioural and biochemical parameters was evaluated. Peripheral immune activation was assessed measuring macrophage and T-helper cytokine production. An up-regulation of the Prokineticin system was present in spinal cord and nerves of diabetic mice, and correlated with allodynia. Therapeutic PC1 reversed allodynia while preventive treatment blocked its development. PC1 normalized prokineticin levels and prevented the up-regulation of GluN2B subunits in the spinal cord. The antagonist restored the pro-/anti-inflammatory cytokine balance altered in spinal cord and nerves and also reduced peripheral immune system activation in diabetic mice, decreasing macrophage proinflammatory cytokines and the T-helper 1 phenotype. The prokineticin system contributes to altered sensitivity in diabetic neuropathy and its inhibition blocked both allodynia and inflammatory events underlying disease

High-fat diet impairs duodenal barrier function and elicits glia-dependent changes along the gut-brain axis that are required for anxiogenic and depressive-like behaviors

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

Investigation of hospital discharge cases and SARS-CoV-2 introduction into Lothian care homes

Background The first epidemic wave of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Scotland resulted in high case numbers and mortality in care homes. In Lothian, over one-third of care homes reported an outbreak, while there was limited testing of hospital patients discharged to care homes. Aim To investigate patients discharged from hospitals as a source of SARS-CoV-2 introduction into care homes during the first epidemic wave. Methods A clinical review was performed for all patients discharges from hospitals to care homes from 1st March 2020 to 31st May 2020. Episodes were ruled out based on coronavirus disease 2019 (COVID-19) test history, clinical assessment at discharge, whole-genome sequencing (WGS) data and an infectious period of 14 days. Clinical samples were processed for WGS, and consensus genomes generated were used for analysis using Cluster Investigation and Virus Epidemiological Tool software. Patient timelines were obtained using electronic hospital records. Findings In total, 787 patients discharged from hospitals to care homes were identified. Of these, 776 (99%) were ruled out for subsequent introduction of SARS-CoV-2 into care homes. However, for 10 episodes, the results were inconclusive as there was low genomic diversity in consensus genomes or no sequencing data were available. Only one discharge episode had a genomic, time and location link to positive cases during hospital admission, leading to 10 positive cases in their care home. Conclusion The majority of patients discharged from hospitals were ruled out for introduction of SARS-CoV-2 into care homes, highlighting the importance of screening all new admissions when faced with a novel emerging virus and no available vaccine

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant

Targeting the prokineticin system to control chronic pain and inflammation

Prokineticin1 and prokineticin2 belong to a new family of chemokines identified in several species including mammals and characterized by the presence of five disulfide bridges. These proteins signal through two G-coupled receptors (prokineticin-receptor1 and prokineticin-receptor2) widely expressed in all tissues and involved in a large spectrum of biological activities, including angiogenesis, hematopoiesis, immune processes, inflammation and nociceptive transmission. Prokineticin2 is overexpressed in inflamed tissues and has a crucial role in neutrophil dependent inflammation and hypernociception. Following tissue inflammation, peripheral nerve injury, cancer, bone metastasis the expression of prokineticin2 and of the prokineticin-receptor2 is increased also within dorsal root ganglia and spinal cord. Prokineticin receptors, highly expressed in nociceptor endings and dorsal root ganglia, exert a tonic activation of TRPV1 and TRPA1 contributing to peripheral sensitization. Prokineticin2-induced activation of the prokineticin receptors in the spinal dorsal horn and in activated astrocytes contributes to central sensitization and maintains chronic and neuropathic pain. Prokineticin2, acting on prokineticin receptors on monocytes, macrophages and dendritic cells, induces chemotaxis and release of inflammatory and pronociceptive cytokines. Hence, the prokineticin system represents a novel therapeutic target in chronic pain conditions. Evaluation of the mechanism of action of prokineticin2 and the potential effectiveness of its inhibition is discussed

PRELIMINARY DATA OF THE IN VITRO CULTURE RESPONSE OF ROSA CANINA L. SPECIES

The in vitro cultures of Rosa canina L. were initiated starting from shoot tips harvested during various periods of the vegetative season. The first observations ascertained that the offshoots cut off at the beginning of July offred a best in vitro response, and the MS medium supplemented with BAP (1.0 mg/l) and IBA (0.5 mg/l) induced both the multiple shooting and the formation of callus surrounding the shoots’ base. Some of the shoots provided roots on this medium formula (sporadically). The hormone-free MS medium stimulated the rooting of the shoots, and scarcely the production of callus at the stem base. The future tests aim to fiind out the best medium variants to enhance the direct caulogenesis, the rooting of the in vitro shoots, and the survival of the regenerants during the ex vitro environment

CYTOGENETIC STUDIES ON SEVERAL HIPPOPHAƠ RHAMNOIDES L. GENOTYPES

Sea-buckthorn (Hippophaë rhamnoides (2n=24)) is a dioecious plant with a very obvious morpho-physiological polymorphism. This species is one of the most valuable fruit-bearing shrubs of the spontaneous and also of the cultivated flora, due to its content of biologically active substances of its leaves, fruit and shoots. The research has been accomplished on root apical meristems from germinated seeds that belong to four genotypes characteristic for Bacău county - Dospineúti, ùerpeni 11, ùerbăneúti 4, Sfiútofca 18, and to one genotype of the Danube Delta - Sfântul Gheorghe 5. Cytogenetical studies evinced that the mitotic index (MI) was high, and it varied with the genotype. The highest MI was evinced in the ùerbăneúti 4 genotype (53.13), and the lowest – in ùerpeni 11 (40.08). Cell distribution per mitotic phases is approximately the same, the highest percentage was represented by cells in prophase, followed by metaphases, telophases and anaphases. There is a rather high frequency and a quite large spectrum of  chromosomal aberrations identified in this species (with variations due to the 5 different genotypes). Of all the chromosomal aberrations evinced during the ana-telophases of mitotic root apical meristems (bridges, delayed chromosomes, expelled chromosomes, fragments, micronuclei), the highest frequency was represented by ana-telophases with bridges. There has been noticed the presence of chromosomal abnormalities in metaphasic and prophasic cells

Chemokines in alzheimer’s disease: new insights into prokineticins, chemokine-like proteins

Alzheimer’s disease is the most common neurodegenerative disorder characterized by the presence of β-amyloid aggregates deposited as senile plaques and by the presence of neurofibrillary tangles of tau protein. To date, there is a broad consensus on the idea that neuroinflammation is one of the most important component in Alzheimer’s disease pathogenesis. Chemokines and their receptors, beside the well-known role in the immune system, are widely expressed in the nervous system, where they play a significant role in the neuroinflammatory processes. Prokineticins are a new family of chemokine-like molecules involved in numerous physiological and pathological processes including immunity, pain, inflammation, and neuroinflammation. Prokineticin 2 (PROK2) and its receptors PKR1 and PKR2 are widely expressed in the central nervous system in both neuronal and glial cells. In Alzheimer’s disease, PROK2 sustains the neuroinflammatory condition and contributes to neurotoxicity, since its expression is strongly upregulated by amyloid-β peptide and reversed by the PKR antagonist PC1. This review aims to summarize the current knowledge on the neurotoxic and/or neuroprotective function of chemokines in Alzheimer’s disease, focusing on the prokineticin system: it represents a new field of investigation that can stimulate the research of innovative pharmacotherapeutic strategies