Olanzapine, risperidone and ziprasidone differently affect lysosomal function and autophagy, reflecting their different metabolic risk in patients.

The metabolic effects induced by antipsychotics in vitro depend on their action on the trafficking and biosynthesis of sterols and lipids. Previous research showed that antipsychotics with different adverse effects in patients cause similar alterations in vitro, suggesting the low clinical usefulness of cellular studies. Moreover, the inhibition of peripheral AMPK was suggested as potential aetiopathogenic mechanisms of olanzapine, and different effects on autophagy were reported for several antipsychotics. We thus assessed, in clinically-relevant culture conditions, the aetiopathogenic mechanisms of olanzapine, risperidone and ziprasidone, antipsychotics with respectively high, medium, low metabolic risk in patients, finding relevant differences among them. We highlighted that: olanzapine impairs lysosomal function affecting autophagy and autophagosome clearance, and increasing intracellular lipids and sterols; ziprasidone activates AMPK increasing the autophagic flux and reducing intracellular lipids; risperidone increases lipid accumulation, while it does not affect lysosomal function. These in vitro differences align with their different impact on patients. We also provided evidence that metformin add-on improved autophagy in olanzapine-treated cells and reduced lipid accumulation induced by both risperidone and olanzapine in an AMPK-dependent way; metformin also increased the production of bile acids to eliminate cholesterol accumulations caused by olanzapine. These results have different clinical implications. We demonstrated that antipsychotics with different metabolic impacts on patients actually have different mechanisms of action, thus supporting the possibility of a personalised antipsychotic treatment. Moreover, we found that metformin can fully revert the phenotype caused by risperidone but not the one caused by olanzapine, that still activates SREBP2.

Targeting the multifaceted neurotoxicity of Alzheimer's disease by tailored functionalisation of the curcumin scaffold.

Simultaneous modulation of multifaceted toxicity arising from neuroinflammation, oxidative stress, and mitochondrial dysfunction represents a valuable therapeutic strategy to tackle Alzheimer's disease. Among the significant hallmarks of the disorder, Aß protein and its aggregation products are well-recognised triggers of the neurotoxic cascade. In this study, by tailored modification of the curcumin-based lead compound 1, we aimed at developing a small library of hybrid compounds targeting Aß protein oligomerisation and the consequent neurotoxic events. Interestingly, from in vitro studies, analogues 3 and 4, bearing a substituted triazole moiety, emerged as multifunctional agents able to counteract Aß aggregation, neuroinflammation and oxidative stress. In vivo proof-of-concept evaluations, performed in a Drosophila oxidative stress model, allowed us to identify compound 4 as a promising lead candidate.

Rescue of lysosomal function as therapeutic strategy for SPG15 hereditary spastic paraplegia.

SPG15 is a hereditary spastic paraplegia subtype caused by mutations in Spastizin, a protein encoded by the ZFYVE26 gene. Spastizin is involved in autophagosome maturation and autophagic lysosome reformation and SPG15-related mutations lead to autophagic lysosome reformation defects with lysosome enlargement, free lysosome depletion and autophagosome accumulation. Symptomatic and rehabilitative treatments are the only therapy currently available for patients. Here, we targeted autophagy and lysosomes in SPG15 patient-derived cells by using a library of autophagy-modulating compounds. We identified a rose of compounds affecting intracellular calcium levels, the calcium-calpain pathway or lysosomal functions, which reduced autophagosome accumulation. The six most effective compounds were tested in vivo in a new SPG15 loss of function Drosophila model that mimicked the reported SPG15 phenotype, with autophagosome accumulation, enlarged lysosomes, reduced free lysosomes, autophagic lysosome reformation defects and locomotor deficit. These compounds, namely verapamil, Bay K8644, 2',5'-dideoxyadenosine, trehalose, Small-Molecule Enhancer of Rapamycin 28 and trifluoperazine, improved lysosome biogenesis and function in vivo, demonstrating that lysosomes are a key pharmacological target to rescue SPG15 phenotype. Among the others, the Small-Molecule Enhancer of Rapamycin 28 was the most effective, rescuing both autophagic lysosome reformation defects and locomotor deficit, and could be considered as a potential therapeutic compound for this hereditary spastic paraplegia subtype.

Dysbindin-1A modulation of astrocytic dopamine and basal ganglia dependent behaviors relevant to schizophrenia.

The mechanisms underlying the dichotomic cortical/basal ganglia dopaminergic abnormalities in schizophrenia are unclear. Astrocytes are important non-neuronal modulators of brain circuits, but their role in dopaminergic system remains poorly explored. Microarray analyses, immunohistochemistry, and two-photon laser scanning microscopy revealed that Dys1 hypofunction increases the reactivity of astrocytes, which express only the Dys1A isoform. Notably, behavioral and electrochemical assessments in mice selectively lacking the Dys1A isoform unraveled a more prominent impact of Dys1A in behavioral and dopaminergic/D2 alterations related to basal ganglia, but not cortical functioning. Ex vivo electron microscopy and protein expression analyses indicated that selective Dys1A disruption might alter intracellular trafficking in astrocytes, but not in neurons. In agreement, Dys1A disruption only in astrocytes resulted in decreased motivation and sensorimotor gating deficits, increased astrocytic dopamine D2 receptors and decreased dopaminergic tone within basal ganglia. These processes might have clinical relevance because the caudate, but not the cortex, of patients with schizophrenia shows a reduction of the Dys1A isoform. Therefore, we started to show a hitherto unknown role for the Dys1A isoform in astrocytic-related modulation of basal ganglia behavioral and dopaminergic phenotypes, with relevance to schizophrenia.

Nitrobenzoxadiazole derivatives of the rat selective toxicant norbormide as fluorescent probes for live cell imaging.

Norbormide [5-(α-hydroxy-α-2-pyridylbenzyl)-7-(α-2-pyridylbenzylidene)-5-norbornene-2,3-dicarboximide] (NRB, 1), an existing but infrequently used rodenticide, is known to be uniquely toxic to rats, but relatively harmless to other rodents and mammals. As a vasoactive agent, NRB induces a species-specific vasocontractile effect that is restricted to the peripheral arteries of the rat. Despite the precise mechanisms behind this phenomenon having yet to be fully clarified, it is postulated that the molecular target of NRB could be located within the plasma membrane of rat peripheral artery myocytes (e.g. rat caudal artery myocytes). As such, the primary objective of this study was to develop a fluorescently labelled derivative of NRB to investigate its subcellular distribution/localization in both NRB-sensitive (freshly isolated rat caudal artery myocytes, FIRCAMs) and NRB-insensitive (human hepatic stellate, LX2) cells. Of the examples prepared, lead structure endo-NRB-NBD-bPA subsequently demonstrated retention of the parent toxicant's pharmacological profile (in terms of its ability to induce both a vasocontractile response in rat caudal artery rings in vitro, and a lethal end-point in rats in vivo). Endo-NRB-NBD-bPA was also shown to be significantly less permeable (an integral feature in the design of fluorescent probes targeting cell-surface receptors) to both LX2 cells and FIRCAMs. Disappointingly, no fluorescence could be observed on the plasma membrane of FIRCAMs stained with endo-NRB-NBD-bPA.

Cholesterol Metabolic Reprogramming in Cancer and Its Pharmacological Modulation as Therapeutic Strategy.

Cholesterol is a ubiquitous sterol with many biological functions, which are crucial for proper cellular signaling and physiology. Indeed, cholesterol is essential in maintaining membrane physical properties, while its metabolism is involved in bile acid production and steroid hormone biosynthesis. Additionally, isoprenoids metabolites of the mevalonate pathway support protein-prenylation and dolichol, ubiquinone and the heme a biosynthesis. Cancer cells rely on cholesterol to satisfy their increased nutrient demands and to support their uncontrolled growth, thus promoting tumor development and progression. Indeed, transformed cells reprogram cholesterol metabolism either by increasing its uptake and de novo biosynthesis, or deregulating the efflux. Alternatively, tumor can efficiently accumulate cholesterol into lipid droplets and deeply modify the activity of key cholesterol homeostasis regulators. In light of these considerations, altered pathways of cholesterol metabolism might represent intriguing pharmacological targets for the development of exploitable strategies in the context of cancer therapy. Thus, this work aims to discuss the emerging evidence of in vitro and in vivo studies, as well as clinical trials, on the role of cholesterol pathways in the treatment of cancer, starting from already available cholesterol-lowering drugs (statins or fibrates), and moving towards novel potential pharmacological inhibitors or selective target modulators.

Norbormide-Based Probes and Their Application for Mitochondrial Imaging in Drosophila Melanogaster.

Fluorescent live imaging on Drosophila melanogaster is a microscopy technique in rapid expansion. The growing number of probes available to detect cellular components and the relatively easy genetic manipulation of fruit fly make this model one of the most used for in vivo analysis of several physiological and/or pathological processes. Here we describe the chemical synthesis of two norbormide-derived BODIPY-conjugated fluorescent probes (NRBMC009 and NRBZLW0047). Moreover, we describe the larval dissection method, and subsequent live imaging acquisition. Both probes are able to label mitochondria in different Drosophila larval tissues, which allows for the characterization of mitochondrial morphological alterations by using a simple and quick method that avoids the fixation artefacts that often occur in immunofluorescence studies.

Hereditary Spastic Paraplegia and Future Therapeutic Directions: Beneficial Effects of Small Compounds Acting on Cellular Stress.

Hereditary spastic paraplegia (HSP) is a group of inherited neurodegenerative conditions that share a characteristic feature of degeneration of the longest axons within the corticospinal tract, which leads to progressive spasticity and weakness of the lower limbs. Mutations of over 70 genes produce defects in various biological pathways: axonal transport, lipid metabolism, endoplasmic reticulum (ER) shaping, mitochondrial function, and endosomal trafficking. HSPs suffer from an adequate therapeutic plan. Currently the treatments foreseen for patients affected by this pathology are physiotherapy, to maintain the outgoing tone, and muscle relaxant therapies for spasticity. Very few clinical studies have been conducted, and it's urgent to implement preclinical animal studies devoted to pharmacological test and screening, to expand the rose of compounds potentially attractive for clinical trials. Small animal models, such as Drosophila melanogaster and zebrafish, have been generated, analyzed, and used as preclinical model for screening of compounds and their effects. In this work, we briefly described the role of HSP-linked proteins in the organization of ER endomembrane system and in the regulation of ER homeostasis and stress as a common pathological mechanism for these HSP forms. We then focused our attention on the pharmacodynamic and pharmacokinetic features of some recently identified molecules with antioxidant property, such as salubrinal, guanabenz, N-acetyl cysteine, methylene blue, rapamycin, and naringenin, and on their potential use in future clinical studies. Expanding the models and the pharmacological screening for HSP disease is necessary to give an opportunity to patients and clinicians to test new molecules.

Exploiting the Potential of Drosophila Models in Lysosomal Storage Disorders: Pathological Mechanisms and Drug Discovery.

Lysosomal storage disorders (LSDs) represent a complex and heterogeneous group of rare genetic diseases due to mutations in genes coding for lysosomal enzymes, membrane proteins or transporters. This leads to the accumulation of undegraded materials within lysosomes and a broad range of severe clinical features, often including the impairment of central nervous system (CNS). When available, enzyme replacement therapy slows the disease progression although it is not curative; also, most recombinant enzymes cannot cross the blood-brain barrier, leaving the CNS untreated. The inefficient degradative capability of the lysosomes has a negative impact on the flux through the endolysosomal and autophagic pathways; therefore, dysregulation of these pathways is increasingly emerging as a relevant disease mechanism in LSDs. In the last twenty years, different LSD Drosophila models have been generated, mainly for diseases presenting with neurological involvement. The fruit fly provides a large selection of tools to investigate lysosomes, autophagy and endocytic pathways in vivo, as well as to analyse neuronal and glial cells. The possibility to use Drosophila in drug repurposing and discovery makes it an attractive model for LSDs lacking effective therapies. Here, ee describe the major cellular pathways implicated in LSDs pathogenesis, the approaches available for their study and the Drosophila models developed for these diseases. Finally, we highlight a possible use of LSDs Drosophila models for drug screening studies.

Further assessment of Salvia haenkei as an innovative strategy to counteract skin photo-aging and restore the barrier integrity.

Skin is the essential barrier of the human body which performs multiple functions. Endogenous factors, in concert with external assaults, continuously affect skin integrity, leading to distinct structural changes that influence not only the skin appearance but also its various physiological functions. Alterations of the barrier functions lead to an increased risk of developing disease and side reactions, thus the importance of maintaining the integrity of the epidermal barrier and slowing down the skin aging process is evident. Salvia haenkei (SH) has been recently identified as a potential anti-senescence agent; its extract is able to decrease the level of senescent cells by affecting the IL1α release and reducing reactive oxygen species (ROS) generation. In this study, SH extract was tested on human keratinocyte cell line (HaCaT) exposed to stress factors related to premature aging of cells such as free radicals and ultraviolet B radiation. We confirmed that SH acts as scavenger of ROS and found its ability to restore the skin barrier integrity by reinforcing the cytoskeleton structure, sealing the tight junctions and increasing the migration rate of cells. Given these results, this work becomes relevant, identifying Salvia haenkei as a compound useful for anti-aging skin treatment in clinical performance.

Drosophila D-idua Reduction Mimics Mucopolysaccharidosis Type I Disease-Related Phenotypes.

Deficit of the IDUA (α-L-iduronidase) enzyme causes the lysosomal storage disorder mucopolysaccharidosis type I (MPS I), a rare pediatric neurometabolic disease, due to pathological variants in the IDUA gene and is characterized by the accumulation of the undegraded mucopolysaccharides heparan sulfate and dermatan sulfate into lysosomes, with secondary cellular consequences that are still mostly unclarified. Here, we report a new fruit fly RNAi-mediated knockdown model of a IDUA homolog (D-idua) displaying a phenotype mimicking some typical molecular features of Lysosomal Storage Disorders (LSD). In this study, we showed that D-idua is a vital gene in Drosophila and that ubiquitous reduction of its expression leads to lethality during the pupal stage, when the precise degradation/synthesis of macromolecules, together with a functional autophagic pathway, are indispensable for the correct development to the adult stage. Tissue-specific analysis of the D-idua model showed an increase in the number and size of lysosomes in the brain and muscle. Moreover, the incorrect acidification of lysosomes led to dysfunctional lysosome-autophagosome fusion and the consequent block of autophagy flux. A concomitant metabolic drift of glycolysis and lipogenesis pathways was observed. After starvation, D-idua larvae showed a quite complete rescue of both autophagy/lysosome phenotypes and metabolic alterations. Metabolism and autophagy are strictly interconnected vital processes that contribute to maintain homeostatic control of energy balance, and little is known about this regulation in LSDs. Our results provide new starting points for future investigations on the disease's pathogenic mechanisms and possible pharmacological manipulations.

Serenoa repens and Urtica dioica Fixed Combination: In-Vitro Validation of a Therapy for Benign Prostatic Hyperplasia (BPH).

Benign prostatic hyperplasia (BPH) is an age-related chronic disorder, characterized by the hyperproliferation of prostatic epithelial and stromal cells, which drives prostate enlargement. Since BPH aetiology and progression have been associated with the persistence of an inflammatory stimulus, induced both by Nuclear Factor-kappa B (NF-κB) activation and reactive oxygen species (ROS) production, the inhibition of these pathways could result in a good tool for its clinical treatment. This study aimed to evaluate the antioxidant and anti-inflammatory activity of a combined formulation of Serenoa repens and Urtica dioica (SR/UD) in an in vitro human model of BPH. The results confirmed both the antioxidant and the anti-inflammatory effects of SR/UD. In fact, SR/UD simultaneously reduced ROS production, NF-κB translocation inside the nucleus, and, consequently, interleukin 6 (IL-6) and interleukin 8 (IL-8) production. Furthermore, the effect of SR/UD was also tested in a human androgen-independent prostate cell model, PC3. SR/UD did not show any significant antioxidant and anti-inflammatory effect, but was able to reduce NF-κB translocation. Taken together, these results suggested a promising role of SR/UD in BPH and BPH-linked disorder prevention.

Naringenin Ameliorates Drosophila ReepA Hereditary Spastic Paraplegia-Linked Phenotypes.

Defects in the endoplasmic reticulum (ER) membrane shaping and interaction with other organelles seem to be a crucial mechanism underlying Hereditary Spastic Paraplegia (HSP) neurodegeneration. REEP1, a transmembrane protein belonging to TB2/HVA22 family, is implicated in SPG31, an autosomal dominant form of HSP, and its interaction with Atlastin/SPG3A and Spastin/SPG4, the other two major HSP linked proteins, has been demonstrated to play a crucial role in modifying ER architecture. In addition, the Drosophila ortholog of REEP1, named ReepA, has been found to regulate the response to ER neuronal stress. Herein we investigated the role of ReepA in ER morphology and stress response. ReepA is upregulated under stress conditions and aging. Our data show that ReepA triggers a selective activation of Ire1 and Atf6 branches of Unfolded Protein Response (UPR) and modifies ER morphology. Drosophila lacking ReepA showed Atf6 and Ire1 activation, expansion of ER sheet-like structures, locomotor dysfunction and shortened lifespan. Furthermore, we found that naringenin, a flavonoid that possesses strong antioxidant and neuroprotective activity, can rescue the cellular phenotypes, the lifespan and locomotor disability associated with ReepA loss of function. Our data highlight the importance of ER homeostasis in nervous system functionality and HSP neurodegenerative mechanisms, opening new opportunities for HSP treatment.

Western Diet-Induced Metabolic Alterations Affect Circulating Markers of Liver Function before the Development of Steatosis.

Since nutrition might have a significant impact on liver function, we analyzed the early effect of Western-type diet on hepatic tissue and lipid and drug metabolism in Wistar-Kyoto rats (n = 8); eight rats fed with a standard diet were used as controls. Histological analysis of liver tissue was performed, and plasma biochemical parameters were measured. Plasma concentration of six bile acids was determined by ultra-liquid chromatography-tandem mass spectrometry UHPLC-MS/MS. Hepatic gene expressions of enzymes involved in drug and lipid metabolism were assessed by means of real-time reverse transcription (qRT)-PCR. Liver of rats fed with a Western diet did not show macroscopic histological alterations, but number and diameter of lipid droplets increased, as well as DGAT1, GPAT4, SCD, FASN and SREBP2 expression. Furthermore, Western diet-fed animals showed an increase in the activation of hepatic stellate cells and macrophage number in liver tissue, as well as a significant increase in AST and bilirubin levels (p < 0.01), and in the LDL:HDL cholesterol ratio (p < 0.001). Plasma chenodeoxycholic acid concentration increased significantly, whereas cholic acid decreased (p < 0.05), and cytochrome P450 genes were generally downregulated. Significant changes in hepatic lipid and drug metabolism are early induced by the Western diet, prior to steatosis development. Such changes are associated with a peculiar alteration in circulating bile acids, which could represent an early marker of non-alcoholic fatty liver disease (NAFLD) development.

The Fine Tuning of Drp1-Dependent Mitochondrial Remodeling and Autophagy Controls Neuronal Differentiation.

Mitochondria play a critical role in neuronal function and neurodegenerative disorders, including Alzheimer's, Parkinson's and Huntington diseases and amyotrophic lateral sclerosis, that show mitochondrial dysfunctions associated with excessive fission and increased levels of the fission protein dynamin-related protein 1 (Drp1). Our data demonstrate that Drp1 regulates the transcriptional program induced by retinoic acid (RA), leading to neuronal differentiation. When Drp1 was overexpressed, mitochondria underwent remodeling but failed to elongate and this enhanced autophagy and apoptosis. When Drp1 was blocked during differentiation by overexpressing the dominant negative form or was silenced, mitochondria maintained the same elongated shape, without remodeling and this increased cell death. The enhanced apoptosis, observed with both fragmented or elongated mitochondria, was associated with increased induction of unfolded protein response (UPR) and ER-associated degradation (ERAD) processes that finally affect neuronal differentiation. These findings suggest that physiological fission and mitochondrial remodeling, associated with early autophagy induction are essential for neuronal differentiation. We thus reveal the importance of mitochondrial changes to generate viable neurons and highlight that, rather than multiple parallel events, mitochondrial changes, autophagy and apoptosis proceed in a stepwise fashion during neuronal differentiation affecting the nuclear transcriptional program.

Live applications of norbormide-based fluorescent probes in Drosophila melanogaster.

In this study we investigated the performance of two norbormide (NRB)-derived fluorescent probes, NRBMC009 (green) and NRBZLW0047 (red), on dissected, living larvae of Drosophila, to verify their potential application in live cell imaging confocal microscopy. To this end, larval tissues were exposed to NRB probes alone or in combination with other commercial dyes or GFP-tagged protein markers. Both probes were rapidly internalized by most tissues (except the central nervous system) allowing each organ in the microscope field to be readily distinguished at low magnification. At the cellular level, the probes showed a very similar distribution (except for fat bodies), defined by loss of signal in the nucleus and plasma membrane, and a preferential localization to endoplasmic reticulum (ER) and mitochondria. They also recognized ER and mitochondrial phenotypes in the skeletal muscles of fruit fly models that had loss of function mutations in the atlastin and mitofusin genes, suggesting NRBMC009 and NRBZLW0047 as potentially useful screening tools for characterizing ER and mitochondria morphological alterations. Feeding of larvae and adult Drosophilae with the NRB-derived dyes led to staining of the gut and its epithelial cells, revealing a potential role in food intake assays. In addition, when flies were exposed to either dye over their entire life cycle no apparent functional or morphological abnormalities were detected. Rapid internalization, a bright signal, a compatibility with other available fluorescent probes and GFP-tagged protein markers, and a lack of toxicity make NRBZLW0047 and, particularly, NRBMC009 highly performing fluorescent probes for live cell microscopy studies and food intake assays in Drosophila.

Antipsychotics Promote Metabolic Disorders Disrupting Cellular Lipid Metabolism and Trafficking.

Antipsychotics frequently cause obesity and related metabolic disorders that current psychopharmacological/endocrinological theories do not explain consistently. An integrative/alternative theory implies metabolic alterations happening at the cellular level. Many observations in vitro and in vivo, and pivotal observations in humans, point towards chemical properties of antipsychotics, independent of receptor binding characteristics. Being amphiphilic weak bases, antipsychotics can disrupt lysosomal function, affecting cholesterol trafficking; moreover, by chemical mimicry, antipsychotics can inhibit cholesterol biosynthesis. These two molecular adverse effects may trigger a cascade of transcriptional and biochemical events, ultimately reducing available cholesterol while increasing cholesterol precursors and fatty acids. The macroscopic manifestation of these molecular alterations includes decreased high-density lipoprotein and increased very low-density lipoprotein and triglycerides that may translate into obesity and related metabolic disorders.

Interplay between colonic inflammation and tachykininergic pathways in the onset of colonic dysmotility in a mouse model of diet-induced obesity.

BACKGROUND: The murine model of high fat diet (HFD)-induced obesity is characterized by an increment of intestinal permeability, secondary to an impairment of mucosal epithelial barrier and enteric inflammation, followed by morphofunctional rearrangement of the enteric nervous system. The present study investigated the involvement of abdominal macrophages in the mechanisms underlying the development of enteric dysmotility associated with obesity. METHODS: Wild type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD, 18% kcal from fat) for 8 weeks. Groups of mice fed with NCD or HFD were treated with clodronate encapsulated into liposomes to deplete abdominal macrophages. Tachykininergic contractions, elicited by electrical stimulation or exogenous substance P (SP), were recorded in vitro from longitudinal muscle colonic preparations. Substance P distribution was examined by confocal immunohistochemistry. The density of macrophages in the colonic wall was examined by immunohistochemical analysis. Malondialdehyde (MDA, colorimetric assay) and IL-1ß (ELISA assay) levels were also evaluated. RESULTS: MDA and IL-1ß levels were increased in colonic tissues from HFD-treated animals. In colonic preparations, electrically evoked tachykininergic contractions were enhanced in HFD mice. Immunohistochemistry displayed an increase in substance P immunoreactivity in myenteric ganglia, as well as in the muscular layers of colonic cryosections from obese mice. Macrophage depletion in HFD mice was associated with a significant reduction of colonic inflammation. In addition, the decrease in macrophage density attenuated the morphofunctional alterations of tachykininergic pathways observed in obese mice. CONCLUSION: Obesity elicited by HFD determines a condition of colonic inflammation, followed by a marked rearrangement of motor excitatory tachykininergic enteric nerves. Macrophage depletion counteracted the morphofunctional changes of colonic neuromuscular compartment, suggesting a critical role for these immune cells in the onset of enteric dysmotility associated with obesity.

The ecto-enzymes CD73 and adenosine deaminase modulate 5'-AMP-derived adenosine in myofibroblasts of the rat small intestine.

Adenosine is a versatile signaling molecule recognized to physiologically influence gut motor functions. Both the duration and magnitude of adenosine signaling in enteric neuromuscular function depend on its availability, which is regulated by the ecto-enzymes ecto-5'-nucleotidase (CD73), alkaline phosphatase (AP), and ecto-adenosine deaminase (ADA) and by dipyridamole-sensitive equilibrative transporters (ENTs). Our purpose was to assess the involvement of CD73, APs, ecto-ADA in the formation of AMP-derived adenosine in primary cultures of ileal myofibroblasts (IMFs). IMFs were isolated from rat ileum longitudinal muscle segments by means of primary explant technique and identified by immunofluorescence staining for vimentin and α-smooth muscle actin. IMFs confluent monolayers were exposed to exogenous 5'-AMP in the presence or absence of CD73, APs, ecto-ADA, or ENTs inhibitors. The formation of adenosine and its metabolites in the IMFs medium was monitored by high-performance liquid chromatography. The distribution of CD73 and ADA in IMFs was detected by confocal immunocytochemistry and qRT-PCR. Exogenous 5'-AMP was rapidly cleared being almost undetectable after 60-min incubation, while adenosine levels significantly increased. Treatment of IMFs with CD73 inhibitors markedly reduced 5'-AMP clearance whereas ADA blockade or inhibition of both ADA and ENTs prevented adenosine catabolism. By contrast, inhibition of APs did not affect 5'-AMP metabolism. Immunofluorescence staining and qRT-PCR analysis confirmed the expression of CD73 and ADA in IMFs. Overall, our data show that in IMFs an extracellular AMP-adenosine pathway is functionally active and among the different enzymatic pathways regulating extracellular adenosine levels, CD73 and ecto-ADA represent the critical catabolic pathway.

Synthesis and Biological Characterization of a New Norbormide Derived Bodipy FL-Conjugated Fluorescent Probe for Cell Imaging.

Background: Norbormide (NRB) is a selective rat toxicant endowed with vasoconstrictor activity confined to the rat peripheral arteries. In a recent work we used a fluorescent derivative of NRB (NRB-AF12), obtained by coupling the NBD fluorophore to the parent molecule via a linker, in order to gain information about the possible site of action of the unlabeled compound. We found that NRB-AF12 labeled intracellular organelles in both NRB-sensitive and -insensitive cells and we accordingly proposed its use as a scaffold for the development of a new class of fluorescent probes. In this study, we examined the fluorescent properties of a BODIPY FL-conjugated NRB probe (MC009) developed: (A) to verify if NRB distribution could be influenced by the attached fluorophore; (B) to improve the fluorescent performance of NRB-AF12. Methods: MC009 characteristics were investigated by confocal fluorescence microscopy, in freshly isolated rat caudal artery myocytes (FIRCAM) and in LX2 cells, representative of NRB-sensitive and insensitive cells, respectively. Main results: In both FIRCAM and LX2 cells MC009 stained endoplasmic reticulum, mitochondria, Golgi apparatus and lipid droplets, revealing the same intracellular distribution as NRB-AF12, and, at the same time, had both improved photostability and gave a more intense fluorescent signal at lower concentrations than was possible with NRB-AF12, which resulted in a better and finer visualization of intracellular structures. Furthermore, MC009 was effective in cellular labeling in both living and fixed cells. At the concentration used to stain the cells, MC009 did not show any cytotoxic effect and did not affect the regular progression of cell cycle and division. Conclusions: This study demonstrates that the distribution of fluorescently labeled NRB is not affected by the type of fluorophore attached to the parent compound, supporting the idea that the localization of the fluorescent derivatives may reasonably reflect that of the parent compound. In addition, we observed a marked improvement in the fluorescent properties of BODIPY FL-conjugated NRB (MC009) over its NBD-derived counterpart (NRB-AF12), confirming NRB as a scaffold for the development of new, high performance, non-toxic fluorescent probes for the labeling of intracellular structures in both living and fixed cells.