Muscle-specific ankyrins 1 (sAnk1) are a group of small ankyrin 1 isoforms, of which sAnk1.5 is the most abundant. sAnk1 are localized in the sarcoplasmic reticulum (SR) membrane from where they interact with obscurin, a myofibrillar protein. This interaction appears to contribute to stabilize the SR close to the myofibrils. Here we report the structural and functional characterization of skeletal muscles from sAnk1 knockout mice (KO). Deletion of sAnk1 did not change the expression and localization of SR proteins in 4-6 month old sAnk1 KO mice. Structurally, the main modification observed in skeletal muscles of adult sAnk1 KO mice (4-6 months of age) was the reduction of SR volume at the sarcomere A band level. With increasing age (at 12-15 months) EDL skeletal muscles of sAnk1 KO mice develop prematurely large tubular aggregates, whereas diaphragm undergoes significant structural damage. Parallel functional studies revealed specific changes in the contractile performance of muscles from sAnk1 KO mice and a reduced exercise tolerance in an endurance test on treadmill compared to control mice. Moreover, reduced Q? charge and L-type Ca(2+)current, that are indexes of affected e-c coupling, were observed in diaphragm fibers from 12-15 month old mice, but not in other skeletal muscles from sAnk1 KO mice. Altogether, these findings show that the ablation of sAnk1, by altering the organization of the SR, renders skeletal muscles susceptible to undergo structural and functional alterations more evident with age, and point to an important contribution of sAnk1 to the maintenance of the longitudinal SR architecture.
The aggregation of the amyloid-? peptide (A?) to form fibrils and plaques is strongly associated with Alzheimer's disease (AD). Although it is well established that this process generates neurotoxicity, it is also heterogeneous with a variety of species being formed during the conversion process. This heterogeneity makes it difficult to detect and characterize each of the aggregates formed, which precludes establishing the specific features responsible for the neurotoxicity observed. Here we use pulse-labeling hydrogen-deuterium exchange experiments analyzed by electrospray ionization mass spectrometry (PL-HDX-ESI-MS) to distinguish three ensembles populated during the aggregation of the 40 and 42 residue forms of the A? peptide, A?40 and A?42, on the basis of differences in their persistent structure. Noticeably, two of them are more abundant at the beginning and at the end of the lag phase and are therefore not detectable by conventional assays such as Thioflavin T (ThT). The ensembles populated at different stages of the aggregation process have a surprisingly consistent average degree of exchange, indicating that there are definite structural transitions between the different stages of aggregation. To determine whether an ensemble of species with a given hydrogen exchange pattern correlates with neurotoxicity, we combined PL-HDX-ESI-MS experiments with parallel measurements of the neurotoxicity of the samples under study. The results of this dual approach show that the maximum toxicity correlates with the ensemble comprising HDX protected oligomers, indicating that development of persistent structure within A? oligomers is a determinant of neurotoxicity.
A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high-affinity Ca(2+) -binding sites of CASQ1 and alters the kinetics of Ca(2+) release in muscle fibers. Expression of the mutated CASQ1 protein in COS-7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron-dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca(2+) release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.
The mechanism of endoplasmic reticulum (ER)-Golgi complex (GC) traffic is conserved from yeast to higher animals, but the architectures and the dynamics of vesicles' traffic between ER and GC vary across cell types and species. Skeletal muscle is a unique tissue in which ER and GC undergo a structural reorganization during differentiation that completely remodels the secretory pathway. In mature skeletal muscle, the ER is turned into sarcoplasmic reticulum, which is composed of junctional and longitudinal regions specialized, respectively, in calcium release and uptake during contraction. During skeletal muscle differentiation, GC acquires a particular fragmented organization as it appears as spots both at the nuclear poles and along the fibers. The ubiquitary-expressed Yip1A isoform has been proposed to be involved in anterograde trafficking from the ER exit sites to the cis-side of the GC and in ER and GC architecture organization. We investigated the role of Yip1 in skeletal muscle. Here we report that, following skeletal muscle development, the expression of the Yip1A decreases and is replaced by the muscle-specific Yip1B isoform. Confocal microscope analysis revealed that in adult skeletal muscle the Yip1B isoform is localized in the ER-Golgi intermediate and cis-Golgi compartments. Finally, skeletal muscle knockdown experiments in vitro and in vivo suggested that Yip1B is not involved in GC structure maintenance.
Although genome-wide association studies have shown that genetic factors increase the risk of suffering late-onset, sporadic Alzheimer's disease (SAD), the molecular mechanisms responsible remain largely unknown.
This study aims to evaluate the effect of combined use of the racemic flavanone Naringenin (NRG) and the protein sericin as TNF-? blockers. Sericin (SMs) and (R/S) NRG-loaded Sericin (SNRGMs) microparticles were prepared by spray-drying, characterized in terms of morphology and particle size distribution, and encapsulation efficiency was determined. Concerning morphology and particle size distribution of microparticles, results indicated that they were not affected by the presence of NRG. The encapsulation efficiency was almost quantitative (93%), thus proving that sericin can be advantageously loaded with (R/S) NRG. Biological evaluation of (R/S) NRG, SMs and SNRGMs was then performed in lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (hPBMC). SNRGMs resulted cytotoxic at the higher dose used (200 ?g/mL) and the effect was greater than (R/S) NRG alone. Moreover, even if sericin alone was not effective in suppressing LPS-induced serum TNF-? levels, SNRGMs loaded with 9.3% of (R/S) NRG were significantly more potent than (R/S) NRG alone. In summary, this study provides the proof of concept that sericin-based microspheres loaded with TNF-?-blockers could contribute to the down regulation of the cytokine and represents the starting point for the development of new topical formulations for the treatment of middle-stage psoriasis.
Reelin is an extracellular matrix protein that is crucial for neural development and adult brain plasticity. While the Reelin signalling cascade has been reported to be associated with Alzheimer's disease (AD), the role of Reelin in this pathology is not understood. Here we use an in vitro approach to show that Reelin interacts with amyloid-? (A?42) soluble species, delays A?42 fibril formation and is recruited into amyloid fibrils. Furthermore, Reelin protects against both the neuronal death and dendritic spine loss induced by A?42 oligomers. In mice carrying the APP(Swe/Ind) mutation (J20 mice), Reelin overexpression delays amyloid plaque formation and rescues the recognition memory deficits. Our results indicate that by interacting with A?42 soluble species, delaying A? plaque formation, protecting against neuronal death and dendritic spine loss and preventing AD cognitive deficits, the Reelin pathway deserves consideration as a therapeutic target for the treatment of AD pathogenesis.
(RS)-Naringenin is a flavanone well-known for its beneficial health-related properties, such as its anti-inflammatory activity. The preparative enantioselective chromatographic resolution of commercial (RS)-naringenin was performed on a Chiralpak AD-H column (500×50 mm i.d., dp 20 ?m) using MeOH as eluent. The developed method is in accordance with the principles of green chemistry, since the environmental impact was lowered by recycling of the eluent, and allowed the production of gram amounts of each enantiomer with high purity (chemical purity >99%, enantiomeric excess (ee) >94%). Racemic and enantiomeric naringenin were subjected to an exhaustive in vitro investigation of anti-inflammatory activity, aimed at evaluating the relevance of chirality. The assay with cultured human peripheral blood mononuclear cells (hPBMC) activated by phytohemagglutinin A revealed that (R)-naringenin was more effective in inhibiting T-cell proliferation than the (S)-enantiomer and the racemate. Moreover, (R)-naringenin significantly reduced proinflammatory cytokine levels such as those of TNF-? and, with less potency, IL-6. These results evidenced the anti-inflammatory potential of naringenin and the higher capacity of (R)-naringenin to inhibit both in vitro hPBMC proliferation and cytokine secretion at non toxic doses. Thus, (R)-naringenin is a promising candidate for in vivo investigation.
The identification of novel molecular targets crucially involved in motor neuron degeneration/survival is a necessary step for the development of hopefully more effective therapeutic strategies for amyotrophic lateral sclerosis (ALS) patients. In this view, S1R, an endoplasmic reticulum (ER)-resident receptor with chaperone-like activity, has recently attracted great interest. S1R is involved in several processes leading to acute and chronic neurodegeneration, including ALS pathology. Treatment with the S1R agonist PRE-084 improves locomotor function and motor neuron survival in presymptomatic and early symptomatic mutant SOD1-G93A ALS mice. Here, we tested the efficacy of PRE-084 in a model of spontaneous motor neuron degeneration, the wobbler mouse (wr) as a proof of concept that S1R may be regarded as a key therapeutic target also for ALS cases not linked to SOD1 mutation. Increased staining for S1R was detectable in morphologically spared cervical spinal cord motor neurons of wr mice both at early (6th week) and late (12th week) phases of clinical progression. S1R signal was also detectable in hypertrophic astrocytes and reactive microglia of wr mice. Chronic treatment with PRE-084 (three times a week, for 8weeks), starting at symptom onset, significantly increased the levels of BDNF in the gray matter, improved motor neuron survival and ameliorated paw abnormality and grip strength performance. In addition, the treatment significantly reduced the number of reactive astrocytes whereas, that of CD11b+ microglial cells was increased. A deeper evaluation of microglial markers revealed significant increased number of cells positive for the pan-macrophage marker CD68 and of CD206+ cells, involved in tissue restoration, in the white matter of PRE-084-treated mice. The mRNA levels of TNF-? and IL-1? were not affected by PRE-084 treatment. Thus, our results support pharmacological manipulation of S1R as a promising strategy to cure ALS and point to increased availability of growth factors and modulation of astrocytosis and of macrophage/microglia as part of the mechanisms involved in S1R-mediated neuroprotection.
In this study we addressed the role of chirality in the biological activity of RC-33, recently studied by us in its racemic form. An asymmetric synthesis procedure was the first experiment, leading to the desired enantioenriched RC-33 but with an enantiomeric excess (ee) not good enough for supporting the in vitro investigation. An enantioselective high-performance liquid chromatography (HPLC) procedure was then successfully carried out, yielding both RC-33 enantiomers in amounts and optical purity suitable for the pharmacological study. The absolute configuration of pure enantiomers was easily assigned exploiting the asymmetric synthesis previously devised. As emerged in the preliminary in vitro biological investigation, (S)- and (R)-RC-33 possess a comparable affinity towards the ?1 receptor and a very a similar behavior in the calcium influx assay, resulting in an equally effective ?1 receptor agonist. Overall, the results obtained so far suggest that the interaction with the biological target is nonstereoselective and leads us to hypothesize that there is a lack of stereoselectivity in the biological activity of RC-33.
Our recent research efforts identified racemic RC-33 as a potent and metabolically stable ?? receptor agonist. Herein we describe the isolation of pure RC-33 enantiomers by chiral chromatography, assignment of their absolute configuration, and in vitro biological studies in order to address the role of chirality in the biological activity of these compounds and their metabolic processing. The binding of enantiopure RC-33 to the ?? receptor was also investigated in silico by molecular dynamics simulations. Both RC-33 enantiomers showed similar affinities for the ?? receptor and appeared to be almost equally effective as ?? receptor agonists. However, the R-configured enantiomer showed higher in vitro hepatic metabolic stability in the presence of NADPH than the S enantiomer. Overall, the results presented herein led us to select (R)-RC-33 as the optimal candidate for further in vivo studies in an animal model of amyotrophic lateral sclerosis.
Despite indisputable progress in the molecular and genetic aspects of amyotrophic lateral sclerosis (ALS), a mechanistic comprehension of the neurodegenerative processes typical of this disorder is still missing and no effective cures to halt the progression of this pathology have yet been developed. Therefore, it seems that a substantial improvement of the outcome of ALS treatments may depend on a better understanding of the molecular mechanisms underlying neuronal pathology and survival as well as on the establishment of novel etiological therapeutic strategies. Noteworthy, a convergence of recent data from multiple studies suggests that, in cellular and animal models of ALS, a complex pathological interplay subsists between motor neurons and their non-neuronal neighbours, particularly glial cells. These observations not only have drawn attention to the physiopathological changes glial cells undergo during ALS progression, but they have moved the focus of the investigations from intrinsic defects and weakening of motor neurons to glia-neuron interactions. In this review, we summarize the growing body of evidence supporting the concept that different glial populations are critically involved in the dreadful chain of events leading to motor neuron sufferance and death in various forms of ALS. The outlined observations strongly suggest that glial cells can be the targets for novel therapeutic interventions in ALS.
Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20?M), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10?M) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKC? activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKC? accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that activation of the PKC? and ERK1/2 pathways participate in regulating neuronal differentiation. Furthermore, since PKC has been classified as one of the cognitive kinases, and activation of PKC is considered a potential therapeutic strategy for the treatment of cognitive disorders, our findings suggest that HMI-1b11 represents a promising lead compound in research aimed to prevent or counteract memory impairment.
Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and ?-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of ?-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity.
Sigma receptors are involved in several central nervous system (CNS) disorders, including mood disorders (depression and anxiety), psychosis, schizophrenia, movement disorders (i.e., Parkinsons disease) and memory deficits (i.e., Alzheimers disease). Recently, the involvement of sigma receptors in neuropathic pain and cancer has also been observed.
Embryonic lethal abnormal vision (ELAV) proteins are RNA-binding proteins that bind specific adenine and uridine-rich elements mainly located in the 3-untranslated region of target mRNAs, preventing their otherwise rapid degradation and thus increasing gene expression. Starting from our previous discovery and applying an integrated in vitro/in silico approach, herein we report a deeper understanding of the mRNA-stabilizing activity of four peptides derived from the ELAV proteins structure. The stabilizing effect on the VEGF transcript (mRNAVEGF ) exerted by each peptide, tested individually, was initially evaluated, and no effects were evidenced. Hence, the biological effects of all peptides couples were investigated. Interestingly, in accordance with preliminary molecular dynamics results, only one of all possible peptide couples resulted highly effective in stabilizing mRNAVEGF . These two peptides were thus identified as valuable starting point for designing small molecules with ELAV-mimicking properties.
Strong pharmacological evidences indicate that ?1 receptors are implicated in the pathophysiology of all major CNS disorders. In the last years our research group has conducted extensive studies aimed at discovering novel ?1 ligands and we recently selected (R/S)-RC-33 as a novel potent and selective ?1 receptor agonist. As continuation of our work in this field, here we report our efforts in the development of this new ?1 receptor agonist. Initially, we investigated the binding of (R) and (S) enantiomers of RC-33 to the ?1 receptor by in silico experiments. The close values of the predicted affinity of (R)-RC-33 and (S)-RC-33 for the protein evidenced the non-stereoselective binding of RC-33 to the ?1 receptor; this, in turn, supported further development and characterization of RC-33 in its racemic form. Subsequently, we set-up a scaled-up, optimized synthesis of (R/S)-RC-33 along with some compound characterization data (e.g., solubility in different media and solid state characterization by thermal analysis techniques). Finally, metabolic studies of RC-33 in different biological matrices (e.g., plasma, blood, and hepatic S9 fraction) of different species (e.g., rat, mouse, dog, and human) were performed. (R/S)-RC-33 is generally stable in all examined biological matrices, with the only exception of rat and human liver S9 fractions in the presence of NADPH. In such conditions, the compound is subjected to a relevant oxidative metabolism, with a degradation of approximately 65% in rat and 69% in human. Taken together, our results demonstrated that (R/S)-RC-33 is a highly potent, selective, metabolically stable ?1 agonist, a promising novel neuroprotective drug candidate.
Proteolipid protein 1 (PLP1) gene-related disorders due to mutations in the PLP1 include a wide spectrum of X-linked disorders ranging from severe connatal Pelizaeus-Merzbacher disease (PMD) to spastic paraplegia 2 (SPG2). Duplications, deletions or point mutations in coding and noncoding regions of the PLP1 gene may occur. We report the clinical, neuroradiologic and molecular findings in six patients from two unrelated families. The affected males showed severe mental retardation, spastic tetraparesis, inability of walking and pes cavus at onset in early infancy. Brain magnetic resonance imaging (MRI) showed hypomyelination and brain atrophy. Nystagmus was never observed. The affected females showed adult-onset progressive spastic paraparesis leading to wheel-chair dependency and subtle white matter changes on brain MRI. Molecular studies in the two families identified two different intronic mutations, the novel c.622+2T>C and the known c.622+1G>A, leading to the skipping of PLP1-exon 4. The clinical presentation of the affected males did not consistently fit in any of the PLP1-related disorder subtypes (i.e., connatal or classic PMD, SPG2 and PLP1 null syndrome), and in addition, the carrier females were symptomatic despite the severe clinical picture of their respective probands. This study provides new insight into the genotype-phenotype correlations of patients with PLP1 splice-site mutations.
Abstract Extracts of Crataegus monogyna Jacq. (hawthorn) show an interesting free radical scavenging (FRS) effect, related to their flavonoids content. Unfortunately, their oral administration is affected by their low bioavailability. The aim of this work is to obtain a multiparticulate drug delivery system for hawthorn extracts for oral administration. The extracts from flowering tops (FL) or fruits (FR) of hawthorn were obtained with maceration, using ethanol as an extraction solvent, and their antioxidant activity was evaluated. FL extract showed the highest FRS activity (EC50 3.72?±?1.21?µg/ml), so it was selected to prepare microparticulate systems by a spray-drying technique, which were characterized by granulometric analysis, scanning electron microscopy-energy dispersive X-ray spectroscopy, confocal fluorescence microscopy and hyperoside content. Antioxidant activity was evaluated before and after gastrointestinal transit in vitro simulation. Results indicate that the microparticulate systems maintained the antioxidant activity of hawthorn also after gastrointestinal transit in vitro simulation, exhibiting properties suitable for oral administration.
Collective evidence indicates that motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is non-cell-autonomous and requires the interaction with the neighboring astrocytes. Recently, we reported that a subpopulation of spinal cord astrocytes degenerates in the microenvironment of motor neurons in the hSOD1(G93A) mouse model of ALS. Mechanistic studies in vitro identified a role for the excitatory amino acid glutamate in the gliodegenerative process via the activation of its inositol 1,4,5-triphosphate (IP(3))-generating metabotropic receptor 5 (mGluR5). Since non-physiological formation of IP(3) can prompt IP(3) receptor (IP(3)R)-mediated Ca(2+) release from the intracellular stores and trigger various forms of cell death, here we investigated the intracellular Ca(2+) signaling that occurs downstream of mGluR5 in hSOD1(G93A)-expressing astrocytes. Contrary to wild-type cells, stimulation of mGluR5 causes aberrant and persistent elevations of intracellular Ca(2+) concentrations ([Ca(2+)](i)) in the absence of spontaneous oscillations. The interaction of IP(3)Rs with the anti-apoptotic protein Bcl-X(L) was previously described to prevent cell death by modulating intracellular Ca(2+) signals. In mutant SOD1-expressing astrocytes, we found that the sole BH4 domain of Bcl-X(L), fused to the protein transduction domain of the HIV-1 TAT protein (TAT-BH4), is sufficient to restore sustained Ca(2+) oscillations and cell death resistance. Furthermore, chronic treatment of hSOD1(G93A) mice with the TAT-BH4 peptide reduces focal degeneration of astrocytes, slightly delays the onset of the disease and improves both motor performance and animal lifespan. Our results point at TAT-BH4 as a novel glioprotective agent with a therapeutic potential for ALS.
Docking of 3-amino-1H-indazoles complexed with glycogen synthase kinase 3 beta (GSK3?) was performed to gain insight into the structural requirements and preferred conformations of these inhibitors. The study was conducted on a selected set of 57 compounds with variation in structure and activity. We found that the most active compounds established three hydrogen bonds with the residues of the hinge region of GSK3?, but some of the less active compounds have other binding modes. In addition, models able to predict GSK3? inhibitory activities (IC(50) ) of the studied compounds were obtained by 3D-QSAR methods CoMFA and CoMSIA. Ligand-based and receptor-guided alignment methods were utilized. Adequate R(2) and Q(2) values were obtained by each method, although some striking differences existed between the obtained contour maps. Each of the predictive models exhibited a similar ability to predict the activity of a test set. The application of docking and quantitative structure-activity relationship together allowed conclusions to be drawn for the choice of suitable GSK3? inhibitors.
Despite the impact of schizophrenia and mood disorders, which in extreme cases can lead to death, recent decades have brought little progress in the development of new treatments. Recent studies have shown that Reelin, an extracellular protein that is critical for neuronal development, is reduced in schizophrenia and bipolar disorder patients. However, data on a causal or protective role of Reelin in psychiatric diseases is scarce. In order to study the direct influence of Reelins levels on behavior, we subjected two mouse lines, in which Reelin levels are either reduced (Reelin heterozygous mice) or increased (Reelin overexpressing mice), to a battery of behavioral tests: open-field, black-white box, novelty-suppressed-feeding, forced-swim-test, chronic corticosterone treatment followed by forced-swim-test, cocaine sensitization and pre-pulse inhibition (PPI) deficits induced by N-methyl-D-aspartate (NMDA) antagonists. These tests were designed to model some aspects of psychiatric disorders such as schizophrenia, mood, and anxiety disorders. We found no differences between Reeler heterozygous mice and their wild-type littermates. However, Reelin overexpression in the mouse forebrain reduced the time spent floating in the forced-swim-test in mice subjected to chronic corticosterone treatment, reduced behavioral sensitization to cocaine, and reduced PPI deficits induced by a NMDA antagonist. In addition, we demonstrate that while stress increased NMDA NR2B-mediated synaptic transmission, known to be implicated in depression, Reelin overexpression significantly reduced it. Together, these results point to the Reelin signaling pathway as a relevant drug target for the treatment of a range of psychiatric disorders.
The seminal discovery that glial cells, particularly astrocytes, can release a number of gliotransmitters that serve as signalling molecules for the cross-talk with neighbouring cellular populations has recently changed our perception of brain functioning, as well as our view of the pathogenesis of several disorders of the CNS. Since glutamate was one of the first gliotransmitters to be identified and characterized, we tackle the mechanisms that underlie its release from astrocytes, including the Ca2+ signals underlying its efflux from astroglia, and we discuss the involvement of these events in a number of relevant physiological processes, from the modulatory control of neighbouring synapses to the regulation of blood supply to cerebral tissues. The relevance of these mechanisms strongly indicates that the contribution of glial cells and gliotransmission to the activities of the brain cannot be overlooked, and any study of CNS physiopathology needs to consider glial biology to have a comprehensive overview of brain function and dysfunction. Abnormalites in the signalling that controls the astrocytic release of glutamate are described in several experimental models of neurological disorders, for example, AIDS dementia complex, Alzheimers disease and cerebral ischaemia. While the modalities of glutamate release from astrocytes remain poorly understood, and this represents a major impediment to the definition of novel therapeutic strategies targeting this process at the molecular level, some key mediators deputed to the control of the glial release of this excitatory amino acid have been identified. Among these, we can mention, for instance, proinflammatory cytokines, such as tumour necrosis factor-?, and prostaglandins. Agents that are able to block the major steps of tumour necrosis factor-? and prostaglandin production and/or signalling can be proposed as novel therapeutic targets for the treatment of these disorders.
Pantothenate kinase-associated neurodegeneration (PKAN) is a neurodegenerative disorder caused by pantothenate kinase (PANK2) gene mutations. Brain magnetic resonance imaging (MRI) typically shows the "eye-of-the-tiger" sign, i.e. bilateral pallidal T2 hypointensity with a small central region of T2-hyperintensity.
Herein we report the synthesis, drug-likeness evaluation, and in vitro studies of new sigma (?) ligands based on arylalkenylaminic scaffold. For the most active olefin the corresponding arylalkylamine was studied. Novel arylalkenylamines generally possess high ?(1) receptor affinity (K(i) values <25 nM) and good ?(1)/?(2) selectivity (K(i)?(2) >100). Particularly, the piperidine derivative (E)-17 and its arylalkylamine analog (R,S)-33 were observed to be excellent ?(1) receptor ligands (K(i)=0.70 and 0.86 nM, respectively) and to display significantly high selectivity over ?(2), ?-, and ?-opioid receptors and phencyclidine (PCP) binding site of the N-methyl-d-aspartate (NMDA) receptors. Moreover in PC12 cells (R,S)-33 promoted the nerve growth factor (NGF)-induced neurite outgrowth and elongation. Co-administration of the selective ?(1) receptor antagonist BD-1063 totally counteracted this effect, confirming that ?(1) receptors are involved in the (R,S)-33 modulation of the NGF effect in PC12 cells and suggesting a ?(1) agonist profile. As a part of our work, a threedimensional ?(1) pharmacophore model was also developed employing GALAHAD methodology. Only active compounds were used for deriving this model. The model included two hydrophobes and a positive nitrogen as relevant features and it was able to discriminate between molecules with and without affinity toward ?(1) receptor subtype.
Current therapies have changed systemic vasculitis from a disease with a high rate of mortality to a chronic curable condition. A limited percentage of patients either remains refractory to conventional treatment or experiences dose-limiting side effects.
B cells play a central role in systemic lupus erythematosus (SLE). Rituximab is expected to induce apoptosis of all the CD20-positive B cells. A proportion of patients are refractory or intolerant to standard immunosuppression. These are candidate to new therapeutic options.
The HPLC enantioselective separation of (R/S)-Naringenin, a chiral flavonoid found in several fruits juices and well-known for its beneficial health-related properties, including antioxidant, anti-inflammatory, cancer chemopreventive, immunomodulating and antimicrobial activities, has been performed on both analytical and (semi)-preparative scale using an amylose derived Chiralpak AD chiral stationary phase (CSP). A standard screening protocol for cellulose and amylose based CSPs was firstly applied to analytical Chiralcel OD-H and Chiralpak AD-H, as well as to Lux Cellulose-1, Lux Cellulose-2 and Lux Amylose-2 in order to identify the best experimental condition for the subsequent scaling-up. Using Chiralpak AD-H and eluting with pure methanol (without acidic or basic additives) relatively short retention times, high enantioselectivity and good resolution (?=1.49, R(s)=3.48) were observed. Therefore, these experimental conditions were properly scaled-up to (semi)-preparative scale using both a pre-packed Regispack column and a Chiralpak AD column packed in house with bulk CSP. The developed preparative method proved to be superior to previously published methods in terms of elution times, separation and resolution and is suitable for obtaining a quick access to the desired enantiomers with high enantiomeric excess and amounts sufficient for biological investigations. Future scale-up options (enantioselective supercritical fluid chromatography or HPLC in the Simulated Moving Bed mode) were also evaluated. It could be shown that both methodologies have a high potential for future production of Naringenin enantiomers by enantioselective chromatography.
This is research of a qualitative nature that sought to analyze the perceptions of both the health team and users of the Pediatric Unit of the University Hospital of Santa Catarina Federal University with respect to the participation of dental surgeons in the healthcare of hospitalized children. Data were collected through interviews based on a form with semi-structured questions and analyzed by the content analysis technique and the analysis-reflection-synthesis process. Eight professionals from the health team, as well as seven caregivers and five hospitalized children took part in the interviews. The dental care provided to these children was given by students of the Undergraduate and Graduate Program in Dentistry, via an extension project of a preventive educational nature. Results revealed broad acceptance and perception of the importance of the participation of dentists in the context of the Unit as team members implementing the concept of comprehensive healthcare, and as support professionals for the health team in the care, streamlining and optimization of interdisciplinary work. Participants perceive the effective participation of dentists in the healthcare of hospitalized children as being very important and even essential.
Mesenchymal stem cells (MSCs) from human adult adipose tissue (A-MSCs) have a better differentiative ability than MSCs derived from the derma (D-MSCs). To test whether this difference is associated with differences in the content of multi-potent progenitors in A-MSCs, the number and the differentiative properties of multi-potent progenitors have been analyzed in various preparations of A-MSCs and D-MSCs. Adipogenic and osteogenic differentiation performed on colony-forming units have revealed that adipogenic and osteogenic progenitors are similar in the two populations, with only a slighty better performance of A-MSCs over D-MSCs from passages p0 to p15. An analysis of the presence of tri-, bi-, uni- and nulli-potent progenitors isolated immediately after isolation from tissues (p0) has shown comparable numbers of tri-potent and bi-potent progenitors in MSCs from the two tissues, whereas a higher content in uni-potent cells committed to adipocytes and a lower content in nulli-potent cells has been observed in A-MSCs. Furthermore, we have characterized the progenitors present in A-MSCs after six passages in vitro to verify the way in which in vitro culture can affect content in progenitor cells. We have observed that the percentage of tri-potent cells in A-MSCs at p6 remains similar to that observed at p0, although bi-potent and uni-potent progenitors committed to osteogenic differentiation increase at p6, whereas nulli-potent cells decrease at p6. These data indicate that the greater differentiative ability of A-MSC populations does not correlate directly with the number of multi-potent progenitors, suggesting that other factors influence the differentiation of bulk populations of A-MSCs.
The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca(2+) stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca(2+) uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+)-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca(2+) stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca(2+) signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca(2+) concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca(2+) transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca(2+) transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca(2+) stores by bafilomycin. By contrast, NAADP failed to evoke a Ca(2+) transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca(2+) transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca(2+) release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.
The sophistication of intracellular Ca ( 2+) signalling reflects the necessity for the many different types of cells to fine tuning their specific activities. This can, at least in part, be explained by the molecular complexity of the Ca ( 2+) signalling machinery, consisting of different intracellular Ca ( 2+) release channel types, each including multiple isoforms and alternative splicing variants. This commentary will go over the main points concerning expression and functional characterization of alternative splicing variants of inositol 1,4,5-trisphosphate and ryanodine receptor isoforms. Many of these variants display specific activation or regulatory features. In addition, dominant negative effects of non-functional alternative splicing variants have been also described for both InsP3Rs and RyRs channels. Recently, a novel RyR2 transcript has been identified by Takasawa and co-workers in pancreatic islets. This novel RyR2 transcript has been proposed to act as an intracellular target for cADPR signalling, which has been demonstrated to be important for insulin secretion. Future characterization of this RyR2 transcript may represent a significant advancement in understanding the mechanisms underlying regulation of Ca ( 2+) release by cADPR.
A microwave-assisted HWE olefination process of readily accessible aryl-alkyl ketones has been developed to provide a rapid access to (Z)-3,3-trisubstituted-alpha,beta-unsaturated methyl esters, key building blocks for the synthesis of biologically active compounds.
Reelin, an extracellular protein essential for neural migration and lamination, is also expressed in the adult brain. To unravel the function of this protein in the adult forebrain, we generated transgenic mice that overexpress Reelin under the control of the CaMKIIalpha promoter. Overexpression of Reelin increased adult neurogenesis and impaired the migration and positioning of adult-generated neurons. In the hippocampus, the overexpression of Reelin resulted in an increase in synaptic contacts and hypertrophy of dendritic spines. Induction of long-term potentiation (LTP) in alert-behaving mice showed that Reelin overexpression evokes a dramatic increase in LTP responses. Hippocampal field EPSP during a classical conditioning paradigm was also increased in these mice. Our results indicate that Reelin levels in the adult brain regulate neurogenesis and migration, as well as the structural and functional properties of synapses. These observations suggest that Reelin controls developmental processes that remain active in the adult brain.
Immune and inflammatory response activation is a common feature of connective tissue diseases and systemic vasculitis. The aim of our study was to evaluate the possible involvement of TNFalpha c.-308A > G, IL-10 c.-1082A > G, uteroglobin c.38A > G, TGFbeta 1 c.869C > T and NFkappaB2 c.-1837T > C gene polymorphisms in susceptibility to connective tissue diseases. Our study cohort included 68 unrelated patients affected by rheumatoid arthritis (RA) (37 patients) and ANCA-positive [micropolyangiitis (mPA) 17 patients] or ANCA-negative systemic vasculitis [including 8 patients with Henoch-Schönlein purpura (HSP) and 6 patients with mixed cryoglobulinaemia (MC)] as well as 98 control subjects. Allele frequency analysis of uteroglobin c.38G > A polymorphism showed a significant increase in the c.38A allele in patients (p= 0.002). Genotype frequency analysis of uteroglobin and NF-kappaB2 gene polymorphisms in patients showed an increase in c.38GA and c.38AA genotypes in the uteroglobin gene (p=0.02) coupled with an increase in homozygous c.-1837CC in the NF-kappaB2 gene (p=0.02). Our data suggest that genetic variation in UG and NF-kappaB2 pathways could have effects in connective tissue disease susceptibility.
In order to investigate the molecular features involved in sigma receptors (sigma-Rs) binding, new compounds based on arylalkylaminoalcoholic, arylalkenyl- and arylalkylaminic scaffolds were synthesized and their affinity towards sigma(1)- and sigma(2)-Rs subtypes was evaluated. The most promising compounds were also screened for their affinity at micro-opioid, delta-opioid and kappa-opioid receptors. Biological results are herein presented and discussed.
Four peptides corresponding to highly conserved motives within the first two RNA recognition motif-type domains of ELAV proteins were prepared, and their effect on the stability of NOVA-1 and VEGF ELAV-target mRNAs was evaluated. Biological results show that in the presence of phorbol 12-myristate 13-acetate (a PKC activator triggering the ELAV pathway), an equimolar mixture of peptides induces a statistically significant stabilization of the selected transcripts, suggesting a synergic effect of the two stimuli.
For decades, astrocytes have been regarded as passive partners of neurons in central nervous system (CNS) function. Studies of the last 20 years, however, challenged this view by demonstrating that astrocytes possess functional receptors for neurotransmitters and respond to their stimulation via release of gliotransmitters, including glutamate. Notably, astrocytes react to synaptically released neurotransmitters with intracellular calcium ([Ca(2+)]) elevations, which result in the release of glutamate via regulated exocytosis and, possibly, other mechanisms. These findings have led to a new concept of neuron-glia intercommunication where astrocytes play an unsuspected dynamic role by integrating neuronal inputs and modulating synaptic activity. The additional observation that glutamate release from astrocytes is controlled by molecules linked to inflammatory reactions, such as the cytokine tumor necrosis factor alpha (TNFalpha) and prostaglandins (PGs), suggests that glia-to-neuron signalling may be sensitive to changes in the production of these mediators occurring in pathological conditions. Indeed, a local, parenchymal brain inflammatory reaction (neuroinflammation) characterized by astrocytic and microglial activation has been reported in several neurodegenerative disorders, including AIDS dementia complex, Alzheimers disease and amyotrophic lateral sclerosis. This transition may be accompanied by functional de-regulation and even degeneration of the astrocytes with the consequent disruption of the cross-talk normally occurring between these cells and neurons. Incorrect neuron-astrocyte interactions may be involved in neuronal derangement and contribute to disease development. The findings reported in this review suggest that a better comprehension of the glutamatergic interplay between neurons and astrocytes may provide information about normal brain function and also highlight potential molecular targets for therapeutic interventions in pathology.
We describe here the synthesis and the binding interaction with sigma(1) and sigma(2) receptors of a series of new benzo[d]oxazol-2(3H)-one derivatives variously substituted on the N-benzyl moiety. The results of binding studies confirm the notion that the benzoxazolone moiety confers preference towards sigma(1) sites and establish that the ability to bind to sigma(1), but not to sigma(2) receptors, is strongly affected by the kind and the position of the substituents introduced in the N-benzyl ring. In fact, compounds with substitutions in para-position with atoms of Cl, H or F or with a CH(3) group exhibit a higher affinity for sigma(1) receptors than the corresponding ortho-substituted compounds. The highest affinity and selectivity, with K(i) values of 0.1 and 427 nM for sigma(1) and sigma(2) receptors, respectively, and a corresponding K(i)sigma(2)/K(i)sigma(1) selectivity ratio of 4270 were found for the Cl-substituted compound. These results indicate that benzo[d]oxazol-2(3H)-one derivatives are among the most selective and sigma(1) receptor-preferring ligands currently available.
The cellular prion protein (PrP(C)) is a highly conserved glycoprotein of unknown biological function. To gain insight into the physiological role of PrP(C), we generated a novel PrP knockout cell line, named PrP(o/o) ML, by immortalization of neuroepithelial precursor cells derived from the cerebellum of PrP-knockout mice using the temperature-sensitive simian virus 40 (SV40) large T antigen. We demonstrated that the PrP(o/o) ML cell line is a unipotent precursor line with glutamatergic properties, which can acquire neuronal features when cultivated under specific conditions. The role of the prion protein in the process of neuronal differentiation was then analyzed in the PrP(o/o) ML cells reconstituted with either the full-length or an amino-terminally deleted form of the prion protein. We show that the expression of PrP(C) facilitates the processes of neuronal differentiation and neuritogenesis and that the deletion of its amino-terminal domain reduces the efficiency, but does not suppress this activity. This cell line represents a useful tool for studying PrP-dependent signal transduction pathways during differentiation of neuronal stem/precursor cells.
We performed the asymmetric synthesis of four enantiopure benzo[d] isothiazo-3-or 5-yloxypropanolamine derivatives, previously described as competitive antagonists at beta-adrenoceptors. The chemical characterization of each enantiomer was accomplished by (1)H NMR and HPLC/DAD/CD. The direct chromatographic separation of the enantiomers via chiral HPLC was investigated. The best resolutions were achieved using cellulose tris (3,5-dimethylphenyl carbamate) (Chiralcel OD-H) and amylose tris (3,5-dimethylphenyl carbamate) (Chiralpak AD). The enantiomers obtained had enantiomeric purities suitable for biological assays. Tested in isolated rat cardiac and intestinal tissues to evaluate their effects at beta(1)- and beta(3)-adrenoceptors, the (S)-enantiomers revealed a higher degree of antagonism than (R)-enantiomers at both subtypes, even though their activity was greater at the cardiac beta(1)-subtype. The potent and cardiospecific antagonistic effect exerted by the compounds tested suggests that the benzisothiazole moiety could be an interesting scaffold for discovering new chiral beta-blocking drugs.
The feasibility and outcomes of homeopathic therapy in a group of type-2 diabetes mellitus patients with diabetic neuropathy were studied in a prospective observational study. Patients were followed from baseline (T0) for 6 months (T1) and for 12 months (T2), treatment was adjusted as necessary. Primary outcome was diabetic neuropathy symptom (DNS) score, secondary outcomes were clinical evolution and short-form-36 (SF-36)-evaluated quality of life (QOL).
Raynauds phenomenon (RP) is a clinical sign of precocious abnormal microcirculation and can be considered a major risk factor for the development of connective tissue disease, especially systemic sclerosis (SSc). Nailfold videocapillaroscopy is the most valuable tool for the early diagnosis of SSc and related disorders. It allows classification of capillary abnormalities. Scoring capillaroscopic alterations, which change significantly during patient follow-up, should be systematically used in order to monitor microangiopathy. The effectiveness of the nailfold videocapillaroscopy in allowing an early diagnosis of SSc and monitoring the progression of the disease, and its predictive value of clinical complications make it a powerful tool for clinical evaluation and research.
Glucocorticoids have never been studied in a placebo-controlled manner in giant cell arteritis (GCA), but their effectiveness is well established. However, evidence for the efficacy of immunosuppressant drugs as steroid-sparing agents in this disease is highly desirable, especially in elderly patients. We report the use of mycophenolate mofetil (MMF) as a steroid-sparing agent in three patients (mean age 78 years) with GCA, at high risk of longterm high dose glucocorticoids because of type II diabetes mellitus, obesity, hypertension or osteoporosis.
Growing evidence indicates that astrocytes cannot be just considered as passive supportive cells deputed to preserve neuronal activity and survival, but rather they are involved in a striking number of active functions that are critical to the performance of the central nervous system (CNS). As a consequence, it is becoming more and more evident that the peculiar properties of these cells can actively contribute to the extraordinary functional complexity of the brain and spinal cord. This new perception of the functioning of the CNS opens up a wide range of new possibilities to interpret various physiological and pathological events, and moves the focus beyond the neuronal compartment toward astrocyte-neuron interactions. With this in mind, here we provide a synopsis of the activities astrocytes perform in normal conditions, and we try to discuss what goes wrong with these cells in specific pathological conditions, such as Alzheimer Disease, prion diseases and amyotrophic lateral sclerosis.
Recent evidence highlights the peroxisome proliferator-activated receptors (PPARs) as critical neuroprotective factors in several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). To gain new mechanistic insights into the role of these receptors in the context of ALS, here we investigated how PPAR transcriptional activity varies in hSOD1(G93A) ALS transgenic mice. We demonstrate that PPAR?-driven transcription selectively increases in the spinal cord of symptomatic hSOD1(G93A) mice. This phenomenon correlates with the up-regulation of target genes, such as lipoprotein lipase and glutathione S-transferase ?-2, which are implicated in scavenging lipid peroxidation by-products. Such events are associated with enhanced PPAR? immunoreactivity within motor neuronal nuclei. This observation, and the fact that PPAR? displays increased responsiveness in cultured hSOD1(G93A) motor neurons, points to a role for this receptor in neutralizing deleterious lipoperoxidation derivatives within the motor cells. Consistently, in both motor neuron-like cultures and animal models, we report that PPAR? is activated by lipid peroxidation end products, such as 4-hydroxynonenal, whose levels are elevated in the cerebrospinal fluid and spinal cord from ALS patients. We propose that the accumulation of critical concentrations of lipid peroxidation adducts during ALS progression leads to the activation of PPAR? in motor neurons. This in turn triggers self-protective mechanisms that involve the up-regulation of lipid detoxification enzymes, such as lipoprotein lipase and glutathione S-transferase ?-2. Our findings indicate that anticipating natural protective reactions by pharmacologically modulating PPAR? transcriptional activity may attenuate neurodegeneration by limiting the damage induced by lipid peroxidation derivatives.
Phytochemical investigation on the Amygdalus lycioides Spach branchelets resulted in the isolation of four chiral flavanones: (2R,3R)-Taxifolin, (2R,3R)-aromadendrin, (S)-5,7,3,5-tetrahydroxyflavanone and (S)-naringenin. The flavanones were isolated by semi-preparative HPLC, their structures elucidated based on spectroscopic data and their absolute configuration assigned. As a part of our ethnobotanical-directed search for novel TNF? inhibitors, the bioassay-guided fractionation of the n-hexane-acetone (n-Hex-Ac, 1:1 v/v) Amygdalus lycioides Spach branchelets extract was performed. In this way, (S)-naringenin was identified as the constituent responsible for the TNF? blocking effect, being effective in vitro and in vivo after oral administration. This is the first investigation on bioactive secondary metabolites of Amygdalus lycioides Spach branchelets.
Ca2+ release necessary for muscle contraction occurs at the junctional domain of the sarcoplasmic reticulum (j-SR). It requires the assembly of a large multi-protein complex containing the ryanodine receptor (RyR) and additional proteins, including triadin and calsequestrin. The signals which drive these proteins to the j-SR and how they assemble to form this multi-protein complex are poorly understood. To address aspects of these questions we studied the localization, dynamic properties and molecular interactions of triadin. We identified three regions, named TR1, TR2 and TR3, that contribute to the localization of triadin at the j-SR. Fluorescence recovery after photobleaching (FRAP) experiments showed that triadin is stably associated with the j-SR and that this association is mediated by TR3. Protein pull-down experiments indicated that TR3 contains binding sites for calsequestrin-1 and that triadin clustering can be enhanced by binding to calsequestrin-1. These findings were confirmed by FRET experiments. Interestingly, the stable association of triadin to the j-SR was significantly decreased in myotubes from calsequestrin-1 knockout mice. Altogether, these results identify three regions in triadin that mediate targeting to the j-SR and reveal a role for calsequestrin-1 in promoting the stable association of triadin to the multi-protein complex associated with RyR.
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