Ultramicronized N-palmitoylethanolamine Contributes to Morphine Efficacy Against Neuropathic Pain: Implication of Mast Cells and Glia.

BACKGROUND: In the current management of neuropathic pain, in addition to antidepressants and anticonvulsants, the use of opioids is wide, despite their related and well-known issues. OBJECTIVE: N-palmitoylethanolamine (PEA), a natural fatty-acid ethanolamide whose anti-inflammatory, neuroprotective, immune-modulating and anti-hyperalgesic activities are known, represents a promising candidate to modulate and/or potentiate the action of opioids. METHODS: This study was designed to evaluate if the preemptive and morphine concomitant administration of ultramicronized PEA, according to fixed or increasing doses of both compounds, delays the onset of morphine tolerance and improves its analgesic efficacy in the chronic constriction injury (CCI) model of neuropathic pain in rats. RESULTS: Behavioral experiments showed that the preemptive and co-administration of ultramicronized PEA significantly decreased the effective dose of morphine and delayed the onset of morphine tolerance. The activation of spinal microglia and astrocytes, commonly occurring both on opioid treatment and neuropathic pain, was investigated through GFAP and Iba-1 immunofluorescence. Both biomarkers were found to be increased in CCI untreated or morphine treated animals in a PEA-sensitive manner. The increased density of endoneural mast cells within the sciatic nerve of morphine-treated and untreated CCI rats was significantly reduced by ultramicronized PEA. The decrease of mast cell degranulation, evaluated in terms of reduced plasma levels of histamine and N-methyl-histamine metabolite, was mainly observed at intermediate-high doses of ultramicronized PEA, with or without morphine. CONCLUSION: Overall, these results show that the administration of ultramicronized PEA in CCI rats according to the study design fully fulfilled the hypotheses of this study.

Protective and Pain-Killer Effects of AMC3, a Novel N-Formyl Peptide Receptors (FPRs) Modulator, in Experimental Models of Rheumatoid Arthritis.

Rheumatoid arthritis is an autoimmune disorder that causes chronic joint pain, swelling, and movement impairment, resulting from prolonged inflammation-induced cartilage and bone degradation. The pathogenesis of RA, which is still unclear, makes diagnosis and treatment difficult and calls for new therapeutic strategies to cure the disease. Recent research has identified FPRs as a promising druggable target, with AMC3, a novel agonist, showing preclinical efficacy in vitro and in vivo. In vitro, AMC3 (1-30 µM) exhibited significant antioxidant effects in IL-1ß (10 ng/mL)-treated chondrocytes for 24 h. AMC3 displayed a protective effect by downregulating the mRNA expression of several pro-inflammatory and pro-algic genes (iNOS, COX-2, and VEGF-A), while upregulating genes essential for structural integrity (MMP-13, ADAMTS-4, and COLIAI). In vivo, AMC3 (10 mg kg-1) prevented hypersensitivity and restored postural balance in CFA-injected rats after 14 days. AMC3 attenuated joint alterations, reduced joint inflammatory infiltrate, pannus formation, and cartilage erosion. Chronic AMC3 administration reduced transcriptional changes of genes causing excitotoxicity and pain (EAATs and CCL2) and prevented morphological changes in astrocytes, including cell body hypertrophy, processes length, and thickness, caused by CFA in the spinal cord. This study demonstrates the usefulness of AMC3 and establishes the groundwork for further research.

Ellagitannins and triterpenoids extracts of Anogeissus leiocarpus stem bark extracts: Protective effects against osteoarthritis.

Osteoarthritis (OA) is a complex joint disease characterized by persistent pain. Unfortunately, current pharmacological therapies are unsatisfactory and characterized by side effects, reason why new strategies are needed. We tested the efficacy of different classes of compounds, ellagitannins and olean-type triterpenoids, contained in Anogeissus leiocarpus extract (Combretaceae family) in comparison to ellagitannins of Castanea sativa extract in a rat model of osteoarthritis induced by the intra-articular injection of sodium monoiodoacetate (MIA). The decoction of stem bark of A. leiocarpus AL-DEC-TOT (300 mg/kg; 4.8% triterpenoids; 11.0% tannins), the butanol extract AL-BuOH-EXT (120 mg/kg; triterpenoids 20.9%; tannins 6.4%) and its correlated aqueous residue AL-Res-H2 O (300 mg/kg; triterpenoids 0.7%; tannins 8.7%) and the decoction of C. sativa, CS-DEC-TOT, (240 mg/kg; triterpenoids 0.65%; tannins 10.8%) were orally administered for two weeks starting from the day of the damage. Behavioural tests highlighted that all stem bark extracts of A. leiocarpus counteracted hypersensitivity development, reduced spontaneous pain, and improved motor skills. Histologically, AL-DEC-TOT, AL-BuOH-EXT and AL-Res-H2 O were effective in preventing joint alterations. In conclusion, all the extracts were effective demonstrating that both olean-type triterpenoid and ellagitannin fractions have anti-hypersensitivity and restorative properties running the stem bark extracts of A. leiocarpus as a candidate in the treatment of OA.

Ultramicronized N-Palmitoylethanolamine Regulates Mast Cell-Astrocyte Crosstalk: A New Potential Mechanism Underlying the Inhibition of Morphine Tolerance.

Persistent pain can be managed with opioids, but their use is limited by the onset of tolerance. Ultramicronized N-palmitoylethanolamine (PEA) in vivo delays morphine tolerance with mechanisms that are still unclear. Since glial cells are involved in opioid tolerance and mast cells (MCs) are pivotal targets of PEA, we hypothesized that a potential mechanism by which PEA delays opioid tolerance might depend on the control of the crosstalk between these cells. Morphine treatment (30 µM, 30 min) significantly increased MC degranulation of RBL-2H3 cells, which was prevented by pre-treatment with PEA (100 µM, 18 h), as evaluated by ß-hexosaminidase assay and histamine quantification. The impact of RBL-2H3 secretome on glial cells was studied. Six-hour incubation of astrocytes with control RBL-2H3-conditioned medium, and even more so co-incubation with morphine, enhanced CCL2, IL-1ß, IL-6, Serpina3n, EAAT2 and GFAP mRNA levels. The response was significantly prevented by the secretome from PEA pre-treated RBL-2H3, except for GFAP, which was further upregulated, suggesting a selective modulation of glial signaling. In conclusion, ultramicronized PEA down-modulated both morphine-induced MC degranulation and the expression of inflammatory and pain-related genes from astrocytes challenged with RBL-2H3 medium, suggesting that PEA may delay morphine tolerance, regulating MC-astrocyte crosstalk.

Efficacy of a vegetal mixture composed of Zingiber officinale, Echinacea purpurea, and Centella asiatica in a mouse model of neuroinflammation: In vivo and ex vivo analysis.

Experimental evidence suggests that neuroinflammation is a key pathological event of many diseases affecting the nervous system. It has been well recognized that these devastating illnesses (e.g., Alzheimer's, Parkinson's, depression, and chronic pain) are multifactorial, involving many pathogenic mechanisms, reason why pharmacological treatments are unsatisfactory. The purpose of this study was to evaluate the efficacy of a vegetal mixture capable of offering a multiple approach required to manage the multifactoriality of neuroinflammation. A mixture composed of Zingiber officinale (150 mg kg-1), Echinacea purpurea (20 mg kg-1), and Centella asiatica (200 mg kg-1) was tested in a mouse model of systemic neuroinflammation induced by lipopolysaccharide (LPS, 1 mg kg-1). Repeated treatment with the vegetal mixture was able to completely counteract thermal and mechanical allodynia as reported by the Cold plate and von Frey tests, respectively, and to reduce the motor impairments as demonstrated by the Rota rod test. Moreover, the mixture was capable of neutralizing the memory loss in the Passive avoidance test and reducing depressive-like behavior in the Porsolt test, while no efficacy was shown in decreasing anhedonia as demonstrated by the Sucrose preference test. Finally, LPS stimulation caused a significant increase in the activation of glial cells, of the central complement proteins and of inflammatory cytokines in selected regions of the central nervous system (CNS), which were rebalanced in animals treated with the vegetal mixture. In conclusion, the vegetal mixture tested thwarted the plethora of symptoms evoked by LPS, thus being a potential candidate for future investigations in the context of neuroinflammation.

Inhibition of Monoacylglycerol Lipase by NSD1819 as an Effective Strategy for the Endocannabinoid System Modulation against Neuroinflammation-Related Disorders.

Neuroinflammation is a key pathological event shared by different diseases affecting the nervous system. Since the underlying mechanism of neuroinflammation is a complex and multifaceted process, current pharmacological treatments are unsatisfactory-a reason why new therapeutic approaches are mandatory. In this context, the endocannabinoid system has proven to possess neuroprotective and immunomodulatory actions under neuroinflammatory status, and its modulation could represent a valuable approach to address different inflammatory processes. To this aim, we evaluated the efficacy of a repeated treatment with NSD1819, a potent ß-lactam-based monoacylglycerol lipase inhibitor in a mouse model of neuroinflammation induced by lipopolysaccharide (LPS) injection. Mice were intraperitoneally injected with LPS 1 mg/kg for five consecutive days to induce systemic inflammation. Concurrently, NSD1819 (3 mg/kg) was daily per os administered from day 1 until the end of the experiment (day 11). Starting from day 8, behavioral measurements were performed to evaluate the effect of the treatment on cognitive impairments, allodynia, motor alterations, anhedonia, and depressive-like behaviors evoked by LPS. Histologically, glial analysis of the spinal cord was also performed. The administration of NSD1819 was able to completely counteract thermal and mechanical allodynia as highlighted by the Cold plate and von Frey tests, respectively, and to reduce motor impairments as demonstrated by the Rota rod test. Moreover, the compound was capable of neutralizing the memory loss in the Passive avoidance test, and reducing depressive-like behavior in the Porsolt test. Finally, LPS stimulation caused a significant glial cells activation in the dorsal horn of the lumbar spinal cord that was significantly recovered by NSD1819 repeated treatment. In conclusion, NSD1819 was able to thwart the plethora of symptoms evoked by LPS, thus representing a promising candidate for future applications in the context of neuroinflammation and related diseases.

The Efficacy of Camelina sativa Defatted Seed Meal against Colitis-Induced Persistent Visceral Hypersensitivity: The Relevance of PPAR α Receptor Activation in Pain Relief.

Brassicaceae are natural sources of bioactive compounds able to promote gut health. Belonging to this plant family, Camelina sativa is an ancient oil crop rich in glucosinolates, polyunsaturated fatty acids, and antioxidants that is attracting renewed attention for its nutraceutical potential. This work aimed at investigating the therapeutic effects of a defatted seed meal (DSM) of Camelina sativa on the colon damage and the persistent visceral hypersensitivity associated with colitis in rats. Inflammation was induced by the intrarectal injection of 2,4-dinitrobenzenesulfonic acid (DNBS). The acute administration of Camelina sativa DSM (0.1-1 g kg-1) showed a dose-dependent pain-relieving effect in DNBS-treated rats. The efficacy of the meal was slightly enhanced after bioactivation with myrosinase, which increased isothiocyanate availability, and drastically decreased by pre-treating the animals with the selective peroxisome proliferator-activated receptor alpha (PPAR α) receptor antagonist GW6471. Repeated treatments with Camelina sativa DSM (1 g kg-1) meal counteracted the development, as well as the persistence, of visceral hyperalgesia in DNBS-treated animals by reducing the intestinal inflammatory damage and preventing enteric neuron damage. In conclusion, Camelina sativa meal might be employed as a nutraceutical tool to manage persistent abdominal pain in patients and to promote gut healing.

Clinical presentations leading to arrhythmogenic left ventricular cardiomyopathy.

OBJECTIVES: To describe a cohort of patients with arrhythmogenic left ventricular cardiomyopathy (ALVC), focusing on the spectrum of the clinical presentations. METHODS: Patients were retrospectively evaluated between January 2012 and June 2020. Diagnosis was based on (1) ≥3 contiguous segments with subepicardial/midwall late gadolinium enhancement in the left ventricle (LV) at cardiac magnetic resonance plus a likely pathogenic/pathogenic arrhythmogenic cardiomyopathy (AC) associated genetic mutation and/or familial history of AC and/or red flags for ALVC (ie, negative T waves in V4-6/aVL, low voltages in limb leads, right bundle branch block like ventricular tachycardia) or (2) pathology examination of explanted hearts or autoptic cases suffering sudden cardiac death (SCD). Significant right ventricular involvement was an exclusion criterion. RESULTS: Fifty-two patients (63% males, age 45 years (31-53)) composed the study cohort. Twenty-one (41%) had normal echocardiogram, 13 (25%) a hypokinetic non-dilated cardiomyopathy (HNDC) and 17 (33%) a dilated cardiomyopathy (DCM). Of 47 tested patients, 29 (62%) were carriers of a pathogenic/likely pathogenic DNA variant. Clinical contexts leading to diagnosis were SCD in 3 (6%), ventricular arrhythmias in 15 (29%), chest pain in 8 (15%), heart failure in 6 (12%) and familial screening in 20 (38%). Thirty patients (57%) had previously received a diagnosis other than ALVC with a diagnostic delay of 6 years (IQR 1-7). CONCLUSIONS: ALVC is hidden in different clinical scenarios with a phenotypic spectrum ranging from normal LV to HNDC and DCM. Ventricular arrhythmias, chest pain, heart failure and SCD are the main clinical presentations, being familial screening essential for the affected relatives' identification.

[Mitral valve prolapse and mitral annulus disjunction: be aware of a potential arrhythmogenic substrate]. / Prolasso valvolare mitralico e disgiunzione mitro-anulare. La consapevolezza di un possibile substrato aritmico.

Mitral valve prolapse is a relatively common disease with a good overall prognosis. However, in specific clinical and instrumental contexts, patients at high risk of ventricular arrhythmias and sudden cardiac death can be identified. Female sex, history of palpitations or syncope, bi-leaflet myxomatous valve, ECG repolarization abnormalities in the inferior leads, complex ventricular arrhythmias, left ventricular fibrosis detected by cardiac magnetic resonance correlate with a higher risk clinical profile. Additionally, morpho-functional abnormalities of the mitral valve annulus, particularly mitral annulus disjunction, may cause a mechanical stretch at the inferior basal ventricular wall and posterior papillary muscles, predisposing to myocardial fibrosis and arrhythmias. A risk stratification strategy is needed to identify patients with mitral valve prolapse and/or mitral annulus disjunction at high risk of arrhythmias; however, few data are available. Further prospective multicenter studies are warranted, focusing on medical therapy, the role of implantable cardioverter-defibrillators for primary prevention, efficacy of targeted catheter ablation or mitral valve surgery.

Effects of Ultramicronized N-Palmitoylethanolamine Supplementation on Tramadol and Oxycodone Analgesia and Tolerance Prevention.

Chronic pain management requires increasing doses of opioids, the milestone of painkillers, which may result in the onset of tolerance with exacerbated side effects. Maintaining stable analgesia with low doses of opioids is thus imperative. N-palmitoylethanolamine (PEA) is an endogenous lipid compound endowed with pain-relieving as well as anti-inflammatory properties. The ultramicronized formulation of PEA was recently demonstrated to be able to modulate morphine's effects, delaying tolerance and improving efficacy. To evaluate the possible application to other opioids, in this study, we analysed the capacity of ultramicronized PEA to regulate analgesia and tolerance induced by oxycodone and tramadol. Pre-emptive and continuative treatment with ultramicronized PEA (30 mg kg-1, daily, per os) delayed the onset of opioid tolerance and enhanced opioid analgesia when it was acutely administered in association with tramadol (20 mg kg-1, daily, subcutaneously) or oxycodone (0.5 mg kg-1, daily, subcutaneously). Moreover, PEA exerted antinociceptive effects on tolerant rats, suggesting the use of PEA together with opioids for stable, long-lasting analgesia. To that purpose, the oxycodone dose needed to be increased from 0.3 mg kg-1 (day 1) up to 1 mg kg-1 (day 31) in the oxycodone + vehicle group; the tramadol dose was progressively enhanced from 15 mg kg-1 to 50 mg kg-1 in 31 days in the tramadol + vehicle group. Acute oral co-treatment with PEA (120 mg kg-1) achieved the same analgesia without increasing the dose of both opioids. The behavioural effects of PEA on opioid chronic treatment paralleled a decrease in astrocyte activation in the dorsal horn of the spinal cord (a marker of the development of opioid tolerance) and with a modulation of mRNA expression of IL-6 and serpin-A3. In conclusion, pre- and co-administration of ultramicronized PEA delayed the development of tramadol tolerance, potentiating either oxycodone or tramadol analgesia and allowing a long-lasting analgesic effect with a low opioid dose regimen. The use of PEA is suggested for clinical purposes to support the opioid-based management of persistent pain.

Restorative and pain-relieving effects of fibroin in preclinical models of tendinopathy.

The term tendinopathy indicates a wide spectrum of conditions characterized by alterations in tendon tissue homeostatic response and damage to the extracellular matrix. The current pharmacological approach involves the use of nonsteroidal anti-inflammatory drugs and corticosteroids often with unsatisfactory results, making essential the identification of new treatments. In this study, the pro-regenerative and protective effects of an aqueous fibroin solution (0.5-500 µg/mL) against glucose oxidase (GOx)-induced damage in rat tenocytes were investigated. Then, fibroin anti-hyperalgesic and protective actions were evaluated in two models of tendinopathy induced in rats by collagenase or carrageenan injection, respectively. In vitro, 5-10 µg/mL fibroin per se increased cell viability and reverted the morphological alterations caused by GOx (0.1 U/mL). Fibroin 10 µg/mL evoked proliferative signaling upregulating the expression of decorin, scleraxin, tenomodulin (p < 0.001), FGF-2, and tenascin-C (p < 0.01) genes. Fibroin enhanced the basal FGF-2 and MMP-9 protein concentrations and prevented their GOx-mediated decrease. Furthermore, fibroin positively modulated the production of collagen type I. In vivo, the peri-tendinous injection of fibroin (5 mg) reduced the development of spontaneous pain and hypersensitivity (p < 0.01) induced by the intra-tendinous injection of collagenase; the efficacy was comparable to that of triamcinolone. The pain-relieving action of fibroin (peri-tendinous) was confirmed in the model of tendinopathy induced by carrageenan (intra-tendinous) where this fibrous protein was also able to improve tendon matrix organization, normalizing the orientation of collagen fibers. In conclusion, the use of fibroin in tendinopathies is suggested taking advantage of its excellent mechanical properties, pain-relieving effects, and ability to promote tissue regeneration processes.

Beneficial Effects of Eruca sativa Defatted Seed Meal on Visceral Pain and Intestinal Damage Resulting from Colitis in Rats.

Most therapies used in patients affected by inflammatory bowel diseases are ineffective in preventing the development of chronic visceral hypersensitivity, mainly due to inflammation-induced enteric neuroplasticity. Glucosinolates, secondary metabolites mainly of Brassicaceae with anti-inflammatory and neuroprotective properties, are effective in treating both neuropathic and arthritis pain through H2S release and Kv7 potassium channel activation. The aim of this work was to investigate the protective and anti-hyperalgesic efficacy of a defatted seed meal from Eruca sativa Mill. (Brassicaceae), rich in glucosinolates, in a rat model of colitis induced by 2,4-dinitrobenzene sulfonic acid (DNBS). The mechanisms of action were also investigated. Visceral pain was assessed by measuring the abdominal response to colorectal distension. Fifteen days after colitis induction, the acute administration of E. sativa defatted seed meal (0.1-1 g kg-1 p.o.) dose-dependently relieved pain. This effect was hampered by co-administering an H2S scavenger or a selective Kv7 blocker. Administering E. sativa (1 g kg-1) for 14 days, starting after DNBS injection, contributed to counteracting visceral pain persistence in the post-inflammatory phase of colitis by promoting colon healing from the damage and reducing enteric gliosis. E. sativa defatted seed meal might be employed as a nutraceutical tool for supporting abdominal pain relief in patients.

Standard ECG for differential diagnosis between Anderson-Fabry disease and hypertrophic cardiomyopathy.

OBJECTIVES: To evaluate the role of the ECG in the differential diagnosis between Anderson-Fabry disease (AFD) and hypertrophic cardiomyopathy (HCM). METHODS: In this multicentre retrospective study, 111 AFD patients with left ventricular hypertrophy were compared with 111 patients with HCM, matched for sex, age and maximal wall thickness by propensity score. Independent ECG predictors of AFD were identified by multivariate analysis, and a multiparametric ECG score-based algorithm for differential diagnosis was developed. RESULTS: Short PR interval, prolonged QRS duration, right bundle branch block (RBBB), R in augmented vector left (aVL) ≥1.1 mV and inferior ST depression independently predicted AFD diagnosis. A point-by-point ECG score was then derived with the following diagnostic performances: c-statistic 0.80 (95% CI 0.74 to 0.86) for discrimination, the Hosmel-Lemeshow χ2 6.14 (p=0.189) for calibration, sensitivity 69%, specificity 84%, positive predictive value 82% and negative predictive value 72%. After bootstrap resampling, the mean optimism was 0.025, and the internal validated c-statistic for the score was 0.78. CONCLUSIONS: Standard ECG can help to differentiate AFD from HCM while investigating unexplained left ventricular hypertrophy. Short PR interval, prolonged QRS duration, RBBB, R in aVL ≥1.1 mV and inferior ST depression independently predicted AFD. Their systematic evaluation and the integration in a multiparametric ECG score can support AFD diagnosis.

Carbonic Anhydrase IV Selective Inhibitors Counteract the Development of Colitis-Associated Visceral Pain in Rats.

Persistent pain affecting patients with inflammatory bowel diseases (IBDs) is still very difficult to treat. Carbonic anhydrase (CA) represents an intriguing pharmacological target considering the anti-hyperalgesic efficacy displayed by CA inhibitors in both inflammatory and neuropathic pain models. The aim of this work was to evaluate the effect of inhibiting CA IV, particularly when expressed in the gut, on visceral pain associated with colitis induced by 2,4-di-nitrobenzene sulfonic acid (DNBS) in rats. Visceral sensitivity was assessed by measuring animals' abdominal responses to colorectal distension. Repeated treatment with the selective CA IV inhibitors AB-118 and NIK-67 effectively counteracted the development of visceral pain induced by DNBS. In addition to pain relief, AB-118 showed a protective effect against colon damage. By contrast, the anti-hyperalgesic activity of NIK-67 was independent of colon healing, suggesting a direct protective effect of NIK-67 on visceral sensitivity. The enzymatic activity and the expression of CA IV resulted significantly increased after DNBS injection. NIK-67 normalised CA IV activity in DNBS animals, while AB-118 was partially effective. None of these compounds influenced CA IV expression through the colon. Although further investigations are needed to study the underlying mechanisms, CA IV inhibitors are promising candidates in the search for therapies to relieve visceral pain in IBDs.

Pediatric Restrictive Cardiomyopathies.

Restrictive cardiomyopathy (RCM) is the least frequent phenotype among pediatric heart muscle diseases, representing only 2.5-3% of all cardiomyopathies diagnosed during childhood. Pediatric RCM has a poor prognosis, high incidence of pulmonary hypertension (PH), thromboembolic events, and sudden death, is less amenable to medical or surgical treatment with high mortality rates. In this scenario, heart transplantation remains the only successful therapeutic option. Despite a shared hemodynamic profile, characterized by severe diastolic dysfunction and restrictive ventricular filling, with normal ventricle ejection fraction and wall thickness, RCM recognizes a broad etiological spectrum, consisting of genetic/familial and acquired causes, each of which has a distinct pathophysiology and natural course. Hence, the aim of this review is to cover the causes, clinical presentation, diagnostic evaluation, treatment, and prognosis of pediatric RCM.

Nuclear mRNA retention and aberrant doppel protein expression in human astrocytic tumor cells.

Doppel (Dpl) is a paralogue of the mammalian Prion (PrP) protein. It is abundant in testis and, unlike PrP, it is expressed at low levels in the adult central nervous system (CNS). Besides, Dpl overexpression correlates with some prion-disease pathological features, such as ataxia and death of cerebellar neurons. Recently, ectopic expression of doppel was found in two different tumor types, specifically in glial and haematological cancers. In this study the doppel gene (PRND) mRNA and protein expression in PRT-HU2 and IPDDC-A2 astrocytoma-derived cell lines was investigated. Northern blot analysis revealed two equally abundant PRND mRNA isoforms, while real-time PCR, on nuclear and cytoplasmic RNA fractions, and cRNA in situ hybridization, on astrocytoma cells and bioptical specimens, showed a nuclear retention of PRND transcripts. Western blot analysis showed that the amount of protein expressed is low compared to the level of mRNA. Moreover deglycosylation studies indicated that Dpl undergoes unusual glycosylation processes. Immunohistochemistry experiments demonstrated that Dpl was mainly localised in the cytoplasm of the astrocytic tumor cells, and that it failed to be GPI-anchored to the cell membrane. This unusual cellular localization was also confirmed through EGFP-Dpl expression in astrocytomas; on the contrary, HeLa cells exhibited the expected Dpl membrane localization. Our findings suggest an aberrant doppel gene expression pattern, characterized by a substantial nuclear retention of the transcript, an altered post-translational modification of the protein and an unusual cytoplasmic localization.

Interaction between the cellular prion (PrPC) and the 2P domain K+ channel TREK-1 protein.

The cellular prion protein (PrP(C)) is a highly conserved protein throughout the evolution of mammals and therefore is thought to play important cellular functions. Despite decades of intensive researches, the physiological function of PrP(C) remains enigmatic. Differently, in particular pathological contexts, generally referred as transmissible spongiform encephalopathies, a conformational isoform of PrP(C), i.e., PrP(Sc), is considered the causative agent of these diseases. In this study, we investigated putative PrP(C) cellular functions through the identification of PrP(C) protein interactants. Using a bacterial two-hybrid approach, we identified a novel interaction between PrP(C) and a two-pore potassium channel protein, TREK-1. This interaction was further verified in transfected eukaryotic cells using co-immunoprecipitation and confocal microscopic analysis of the fluorescent transfected proteins. Importantly, in the cerebellar cortex, the endogenous PrP(C) and TREK-1 proteins exhibited co-localization signals in correspondence of the Purkinje cells. Furthermore, a deletion mapping study defined the carboxyl-terminal regions of the two proteins as the possible determinants of the PrP(C)-TREK-1 interaction. Our results indicated a novel PrP(C) interacting protein and suggested that this complex might be relevant in modulating a variety of electrophysiological-dependent cellular responses.