Expression of the rat connexin 39 (rCx39) gene in myoblasts and myotubes in developing and regenerating skeletal muscles: an in situ hybridization study.

We report a detailed analysis of the expression pattern of the recently identified rat connexin gene, named rat connexin 39 (rCx39), both during embryonic development and in adult life. Qualitative and quantitative reverse transcription/polymerase chain reaction analysis showed intense expression of rCx39 restricted to differentiating skeletal muscles, with a peak of expression detected at 18 days of embryonic life, followed by a rapid decline to undetectable levels within the first week of postnatal life. A combination of the in situ hybridization technique for the detection of rCx39 mRNA and immunohistochemistry for myogenin, a myoblast-specific marker, allowed us to establish that the mR…

Central nicotinic receptors, neurotrophic factors and neuroprotection

The multiple combinations of nAChR subunits identified in central nervous structures possess distinct pharmacological and physiological properties. A growing number of data have shown that compounds interacting with neuronal nAChRs have, both in vivo and in vitro, the potential to be neuroprotective and that treatment with nAChR agonists elicit long-lasting improving of cognitive performance in a variety of behavioural tests in rats, monkeys and humans. Epidemiological and clinical studies suggested also a potential neuroprotective/trophic role of (-)-nicotine in neurodegenerative disease, such as Alzheimer's and Parkinson's disease. Taken together experimental and clinical data largely ind…

Involvement of estrogen receptors in the resveratrol-mediated increase in dopamine transporter in human dopaminergic neurons and in striatum of female mice.

Treatment with resveratrol (RSV) has been shown to protect vulnerable neurons after various brain injuries and in neurodegenerative diseases. The mechanisms for the effects of RSV in brain are not fully understood, but RSV may affect the expression of various gene products. RSV is structurally related to the synthetic estrogen, diethylstilbestrol so the effects of RSV may be gender-specific. Here we studied the role of RSV in the regulation of dopamine transporter (DAT) in the striatum using male and female mice. The basic levels of DAT in the striatum showed no sex difference, but the levels increased significantly by RSV (20 mg/kg i.p.) in female but not in male mice. Pretreatment of mice…

Fibroblast Growth Factor Receptor 1– 5-Hydroxytryptamine 1A Heteroreceptor Complexes and Their Enhancement of Hippocampal Plasticity

Background The hippocampus and its 5-hydroxytryptamine transmission plays an important role in depression related to its involvement in limbic circuit plasticity. Methods The analysis was made with bioluminescence resonance energy transfer, co-immunoprecipitation, in situ proximity ligation assay, binding assay, in cell western and the forced swim test. Results Using bioluminescence resonance energy transfer analysis, fibroblast growth factor receptor 1 (FGFR1)-5-hydroxytryptamine 1A (5-HT1A) receptor complexes have been demonstrated and their specificity and agonist modulation characterized. Their presence based on co-immunoprecipitation and proximity ligation assay has also been indicated…

FGF-2/FGFR1 neurotrophic system expression level and its basal activation do not account for the age-dependent decline of precursor cell proliferation in the subventricular zone of rat brain

It is largely accepted that neurogenesis in the adult brain decreases with age and reduced levels of local neurotrophic support is speculated to be a contributing factor. Among neurotrophic factors involved on neurogenesis, we focused our attention on the neurotrophic system fibroblast growth factor-2 (FGF-2) and its receptor FGFR1, a potent modulator of precursor cell proliferation. In the present work, we aimed to analyse if potential age-dependent changes of the FGF-2/FGFR1 neurotrophic system may give account for the age-dependent decline of precursor cell proliferation in the neurogenic region of the subventricular zone (SVZ) in the rat brain. Using in situ hybridization and western bl…

Fibroblast growth factor-21 enhances mitochondrial functions and increases the activity of PGC-1α in human dopaminergic neurons via Sirtuin-1

Abstract Mitochondrial dysfunctions accompany several neurodegenerative disorders and contribute to disease pathogenesis among others in Parkinson’s disease (PD). Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a major regulator of mitochondrial functions and biogenesis, and was suggested as a therapeutic target in PD. PGC-1α is regulated by both transcriptional and posttranslational events involving also the action of growth factors. Fibroblast growth factor-21 (FGF21) is a regulator of glucose and fatty acid metabolism in the body but little is known about its action in the brain. We show here that FGF21 increased the levels and activity of PGC-1α and elevated mito…

Protective, antioxidant and antiproliferative activity of grapefruit integropectin on sh-sy5y cells

Tested in vitro on SH-SY5Y neuroblastoma cells, grapefruit IntegroPectin is a powerful protective, antioxidant and antiproliferative agent. The strong antioxidant properties of this new citrus pectin, and its ability to preserve mitochondrial membrane potential and morphology, severely impaired in neurodegenerative disorders, make it an attractive therapeutic and preventive agent for the treatment of oxidative stress-associated brain disorders. Similarly, the ability of this pectic polymer rich in RG-I regions, as well as in naringin, linalool, linalool oxide and limonene adsorbed at the outer surface, to inhibit cell proliferation or even kill, at high doses, neoplastic cells may have open…

Corrigendum to “Antiabsence effects of carbenoxolone in two genetic animal models of absence epilepsy (WAG/Rij rats and lh/lh mice)”

Corrigendum to ‘‘Antiabsence effects of carbenoxolone in two genetic animal models of absence epilepsy (WAG/Rij rats and lh/lh mice)’’ Pietro Gareri , Daniele Condorelli , Natale Belluardo , Rita Citraro , Vincenza Barresi , Angela Trovato-Salinaro , Giuseppa Mudo‘ , Guido Ferreri Ibbadu , Emilio Russo , Giovambattista De Sarro a,* a Section of Pharmacology, Department of Experimental and Clinical Medicine, Faculty of Medicine and Surgery, University of Catanzaro, Catanzaro, Italy b Section of Biochemistry and Molecular Biology, Department of Chemical Sciences, University of Catania, Catania, Italy Department of Experimental Medicine, University of Palermo, Palermo, Italy

Beneficial Role of Exercise in the Modulation of

Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive lethal disorder caused by the lack of dystrophin, which determines myofibers mechanical instability, oxidative stress, inflammation, and susceptibility to contraction-induced injuries. Unfortunately, at present, there is no efficient therapy for DMD. Beyond several promising gene- and stem cells-based strategies under investigation, physical activity may represent a valid noninvasive therapeutic approach to slow down the progression of the pathology. However, ethical issues, the limited number of studies in humans and the lack of consistency of the investigated training interventions generate loss of consensus regarding …

Downregulation of the Astroglial Connexin Expression and Neurodegeneration after Pilocarpine-Induced Status Epilepticus

Astrocytic networks and gap junctional communication mediated by connexins (Cxs) have been repeatedly implicated in seizures, epileptogenesis, and epilepsy. However, the effect of seizures on Cx expression is controversial. The present study focused on the response of Cxs to status epilepticus (SE), which is in turn an epileptogenic insult. The expression of neuronal Cx36 and astrocytic Cx30 and Cx43 mRNAs was investigated in the brain of rats in the first day after pilocarpine-induced SE. In situ hybridization revealed a progressive decrease in Cx43 and Cx30 mRNA levels, significantly marked 24 h after SE onset in neocortical areas and the hippocampus, and in most thalamic domains, whereas…

Nicotine-induced FGF-2 mRNA in rat brain is preserved during aging

Indirect trophic actions of nicotine on brain during aging are suggested from observations describing nicotine as a cognitive enhancer, increasing vigilance and improving learning and memory, and both in vitro and in vivo models have demonstrated neuroprotective effects of nAChR agonists. Previously, we have reported that an acute intermittent (-)nicotine treatment significantly increases fibroblast growth factor-2 (FGF-2) mRNA and protein in several brain regions of rat brain. The present study was designed to analyse if nicotine-induced FGF-2 expression in the rat brain was preserved during aging. Using in situ hybridization and quantitative RNase protection assay the present paper report…

Serotonin Heteroreceptor Complexes and Their Integration of Signals in Neurons and Astroglia—Relevance for Mental Diseases

The heteroreceptor complexes present a novel biological principle for signal integration. These complexes and their allosteric receptor–receptor interactions are bidirectional and novel targets for treatment of CNS diseases including mental diseases. The existence of D2R-5-HT2AR heterocomplexes can help explain the anti-schizophrenic effects of atypical antipsychotic drugs not only based on blockade of 5-HT2AR and of D2R in higher doses but also based on blocking the allosteric enhancement of D2R protomer signaling by 5-HT2AR protomer activation. This research opens a new understanding of the integration of DA and 5-HT signals released from DA and 5-HT nerve terminal networks. The biologica…

Increase of the FGFR1 signaling in the FGFR1-5-HT1A heteroreceptor complex in midbrain raphe 5-HT neuron systems via allosteric receptor-receptor interaction

The ascending midbrain 5-HT neurons known to contain 5-HT1A autoreceptors may be dysregulated in depression due to a reduced trophic support. New findings show existence of FGFR1-5-HT1A heteroreceptor complexes in the rat hippocampus with a partial characterization of their interface and in midbrain raphe 5-HT nerve cells. With in situ Proximity Ligation Assay (PLA) and supported by co-location of the FGFR1 and 5-HT1A immunoreactivities in midbrain raphe 5-HT cells, evidence for the existence of FGFR1-5-HT1A heteroreceptor complexes were obtained in the dorsal and median raphe nuclei of the Sprague–Dawley rat. Their existence in the rat medullary raphe RN33B cell cultures was also establish…

Neuronal expression and regulation of rat inhibitor of apoptosis protein-2 by kainic acid in the rat brain

Inhibitors of apoptosis proteins (IAPs) define a protein family with the ability to counteract cell death by the inhibition of different caspases activated during apoptosis. These proteins are present in different cells, however, the function and roles of IAPs in brain tissue are not fully understood. We report here that RIAP-2, the rat homologue of human cIAP-1/HIAP-2, is expressed in different areas of rat brain as shown by in situ hybridization and immunohistochemistry. Brain regions with relatively high expression of RIAP-2 mRNA included cortex, cerebellum and different subregions of rat hippocampus. Double labelling using a specific anti-RIAP antibody and markers for neurons and glial …

Time-course of GDNF and its receptor expression after brain injury in the rat

Abstract The aim of the present work was to perform, by in situ hybridization, a time-course analysis of the glial cell line-derived neurotrophic factor (GDNF) and its receptor mRNA expression in two models of brain injury in the rat: (a) excitotoxic lesion by ibotenic acid injection in the hippocampal formation; (b) mechanical lesion by needle insertion through the cerebral cortex including the white matter of the corpus callosum. The time-course analysis, ranging from 6 h to 8 days, showed that the GDNF and its receptor (RET, GFRα-1 and GFRα-2) mRNA expressions were differentially up-regulated in both models of lesion. This in vivo regulation of the GDNF and its receptor mRNA expression i…

Increase in Bcl-2 phosphorylation and reduced levels of BH3-only Bcl-2 family proteins in kainic acid-mediated neuronal death in the rat brain.

Kainic acid induces excitotoxicity and nerve cell degeneration in vulnerable regions of rat brain, most markedly in hippocampus and amygdala. Part of the cell death following kainic acid is apoptotic as shown by caspase 3 activation and chromatin condensation. Here we have studied the regulation of pro- and anti-apoptotic proteins belonging to the Bcl-2 family in rat hippocampus and amygdala by kainic acid in relationship to ensuing neuronal death. The pro-apoptotic protein Bax was up-regulated in hippocampus 6 h after kainic acid administration. The increase in Bax was followed by the appearance of TdT-mediated dUTP nick end labelling-positive cells which were prominent at 24 h. Immunohist…

Reduction of mdx mouse muscle degeneration by low-intensity endurance exercise: a proteomic analysis in quadriceps muscle of exercised versus sedentary mdx mice

By proteomic analysis we found an up-regulation of four carbonic anhydrase-3 (CA3) isoforms and a down-regulation of superoxide dismutase [Cu-Zn] (SODC) in quadriceps of sedentary X-linked muscular dystrophy (mdx) mice as compared with wild–type (WT) mice and the levels were significantly restored to WT values following low-intensity endurance exercise.

Neuroprotective and Mitoprotective Effects of Lemon IntegroPectin on SH-SY5Y Cells

AbstractLemon IntegroPectin obtained via hydrodynamic cavitation of organic lemon processing waste in water shows significant neuroprotective activity in vitro, as first reported in this study investigating the effects of both lemon IntegroPectin and commercial citrus pectin on cell viability, cell morphology, reactive oxygen species (ROS) production and mitochondria perturbation induced by treatment of neuronal SH-SY5Y human cells with H2O2. Mediated by ROS including H2O2 and its derivatives, oxidative stress alters numerous cellular processes, including mitochondrial regulation and cell signaling, propagating cellular injury that leads to incurable neurodegenerative diseases. These result…

Enhancement of the FGFR1 signaling in the FGFR1-5-HT1A heteroreceptor complex in midbrain raphe 5-HT neuron systems. Relevance for neuroplasticity and depression

New findings show existence of FGFR1-5-HT1A heteroreceptor complexes in 5-HT nerve cells of the dorsal and median raphe nuclei of the rat midbrain and hippocampus. Synergistic receptor-receptor interactions in these receptor complexes indicated their enhancing role in hippocampal plasticity. The existence of FGFR1-5-HT1A heteroreceptor complexes also in midbrain raphe 5-HT nerve cells open up the possibility that antidepressant drugs by increasing extracellular 5-HT levels can cause an activation of the FGF-2/FGFR1 mechanism in these nerve cells as well. Therefore, the agonist modulation of the FGFR1-5-HT1A heteroreceptor complexes and their specific role is now determined in rat medullary …

Adipose Stromal/Stem Cell-Derived Extracellular Vesicles: Potential Next-Generation Anti-Obesity Agents

Over the last decade, several compounds have been identified for the treatment of obesity. However, due to the complexity of the disease, many pharmacological interventions have raised concerns about their efficacy and safety. Therefore, it is important to discover new factors involved in the induction/progression of obesity. Adipose stromal/stem cells (ASCs), which are mostly isolated from subcutaneous adipose tissue, are the primary cells contributing to the expansion of fat mass. Like other cells, ASCs release nanoparticles known as extracellular vesicles (EVs), which are being actively studied for their potential applications in a variety of diseases. Here, we focused on the importance …

Peroxisome proliferator-activated receptor-γ coactivator-1α mediates neuroprotection against excitotoxic brain injury in transgenic mice: role of mitochondria and X-linked inhibitor of apoptosis protein

Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) is a transcriptional coactivator involved in the regulation of mitochondrial biogenesis and cell defense. The functions of PGC-1α in physiology of brain mitochondria are, however, not fully understood. To address this we have studied wild-type and transgenic mice with a two-fold overexpression of PGC-1α in brain neurons. Data showed that the relative number and basal respiration of brain mitochondria were increased in PGC-1α transgenic mice compared with wild-type mitochondria. These changes occurred concomitantly with altered levels of proteins involved in oxidative phosphorylation (OXPHOS) as studied by proteomi…

Current disease modifying approaches to treat Parkinson's disease

Parkinson's disease (PD is a progressive neurological disorder characterized by the degeneration and death of midbrain dopamine and non-dopamine neurons in the brain leading to motor dysfunctions and other symptoms, which seriously influence the quality of life of PD patients. The drug L-dopa can alleviate the motor symptoms in PD, but so far there are no rational therapies targeting the underlying neurodegenerative processes. Despite intensive research, the molecular mechanisms causing neuronal loss are not fully understood which has hampered the development of new drugs and disease-modifying therapies. Neurotrophic factors are by virtue of their survival promoting activities attract candi…

Anti-inflammatory and cognitive effects of interferon-β1a (IFNβ1a) in a rat model of Alzheimer’s disease

Background: Aβ 1-42 peptide abnormal production is associated with the development and maintenance of neuroinflammation and oxidative stress in brains from Alzheimer disease (AD) patients. Suppression of neuroinflammation may then represent a suitable therapeutic target in AD. We evaluated the efficacy of IFNβ1a in attenuating cognitive impairment and inflammation in an animal model of AD. Methods: A rat model of AD was obtained by intra-hippocampal injection of Aβ 1-42 peptide (23 μg/2 μl). After 6 days, 3.6 μg of IFNβ1a was given subcutaneously (s.c.) for 12 days. Using the novel object recognition (NOR) test, we evaluated changes in cognitive function. Measurement of pro-inflammatory or …

Acute intermittent nicotine treatment induces fibroblast growth factor-2 in the subventricular zone of the adult rat brain and enhances neuronal precursor cell proliferation

Abstract Over the past years, evidence has accumulated that stem cells are present in the adult brain, and generate neurons and/or glia from two active germinal zones: the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus. This study shows that acute intermittent nicotine treatment significantly enhances neuronal precursor cell proliferation in the SVZ of adult rat brain, but not in the SGZ of the hippocampus, and pre-treatment with mecamylamine, a nonselective nAChR antagonist, blocks the enhanced precursor proliferation by nicotine. This effect is supported by up-regulation of fibroblast growth factor-2 (FGF-2) mRNA …

Bidirectional Control between Cholesterol Shuttle and Purine Signal at the Central Nervous System.

Recent studies have highlighted the mechanisms controlling the formation of cerebral cholesterol, which is synthesized in situ primarily by astrocytes, where it is loaded onto apolipoproteins and delivered to neurons and oligodendrocytes through interactions with specific lipoprotein receptors. The “cholesterol shuttle” is influenced by numerous proteins or carbohydrates, which mainly modulate the lipoprotein receptor activity, function and signaling. These molecules, provided with enzymatic/proteolytic activity leading to the formation of peptide fragments of different sizes and specific sequences, could be also responsible for machinery malfunctions, which are associated with neurological…

Connexin36 (Cx36) expression and protein detection in the mouse carotid body and myenteric plexus

AbstractAlthough connexin36 (Cx36) has been studied in several tissues, it is notable that no data are available on Cx36 expression in the carotid body and the intestine. The present study was undertaken to evaluate using immunohistochemistry, PCR and Western blotting procedures, whether Cx36 was expressed in the mouse carotid body and in the intestine at ileum and colon level. In the carotid body, Cx36 was detected as diffuse punctate immunostaining and as protein by Western blotting and mRNA by RT-PCR. Cx36 punctate immunostaining was also evident in the intestine with localization restricted to the myenteric plexus of both the ileum and the colon, and this detection was also confirmed by…

New neuroprotective effect of lemon integropectin on neuronal cellular model

Lemon IntegroPectin obtained via hydrodynamic cavitation of organic lemon processing waste in water shows significant neuroprotective activity in vitro, as first reported in this study investigating the effects of both lemon IntegroPectin and commercial citrus pectin on cell viability, cell morphology, reactive oxygen species (ROS) production, and mitochondria perturbation induced by treatment of neuronal SH-SY5Y human cells with H2O2. Mediated by ROS, including H2O2 and its derivatives, oxidative stress alters numerous cellular processes, such as mitochondrial regulation and cell signaling, propagating cellular injury that leads to incurable neurodegenerative diseases. These results, and t…

Regulation of connexin gene expression during skeletal muscle regeneration in the adult rat

In the adult skeletal muscle, various kinds of trauma promote proliferation of satellite cells that differentiate into myoblasts forming new myofibers or to repair the damaged one. The aim of present work was to perform a comparative spatial and temporal analysis of connexin (Cx) 37, Cx39, Cx40, Cx43, and Cx45 expression in the adult regenerating skeletal muscle in response to crush injury. Within 24 h from injury, Cx37 expression was upregulated in the endothelial cells of blood vessels, and, 5 days after injury, Cx37-expressing cells were found inside the area of lesion and formed clusters generating new blood vessels with endothelial cells expressing Cx37. Three days after injury, Cx39 m…

The nicotinic acetylcholine receptor agonist (±)-epibatidine increases FGF-2 mRNA and protein levels in the rat brain

Abstract In a previous work, we showed that acute intermittent nicotine treatment up-regulates the level of fibroblast growth factor-2 (FGF-2) mRNA in brain regions of tel- and mesencephalon of rats suggesting that neuroprotective effect of (−)nicotine may, at least in part, involve an activation of the neuronal FGF-2 signalling. The present experiments were designed to extend the study on the nicotinic receptor mediated up-regulation of FGF-2 mRNA levels to the use of the potent nicotinic acetylcholine receptor (nAChR) agonist (±)-epibatidine. The (±)-epibatidine treatment led to a strong and long lasting up-regulation of FGF-2 mRNA expression in the cerebral cortex, in the hippocampal for…

Modulation of the TGF-β1-induced epithelial to mesenchymal transition (EMT) mediated by P1 and P2 purine receptors in MDCK cells

Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-β1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-β1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-β1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation o…

Speaker 4: Dasiel Borroto – Escuela, Sweden

Neurotrophic effects of central nicotinic receptor activation

A growing number of data have shown that compounds interacting with neuronal nicotinic acetylcholine receptors (nAChRs) have, both in vivo and in vitro, the potential to be neuroprotective and that treatment with nAChR agonists elicit long-lasting improvement of cognitive performance in a variety of behavioural tests in rats, monkeys and humans. Epidemiological and clinical studies suggested also a potential neuroprotective/trophic role of (-)-nicotine in neurodegenerative disease, such as Alzheimer's disease and Parkinson's disease. This neuroprotective/trophic role of nAChR activation has been mainly mediated by alpha7 and alpha4beta2 nAChR subtypes, as evidenced using selective nAChR ant…

Transplantation of prodrug-converting neural progenitor cells for brain tumor therapy

Since neural progenitor cells can engraft stably into brain tumors and differentiate along the neuronal and glial line, we tested the hypothesis that transplanted cytosine deaminase (CD)-expressing ST14A cells (an immortalized neural progenitor cell line) can convert locally 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) and produce a regression of glioma tumors. ST14A, retrovirally transduced with the E. coli CD gene, showed a strong bystander effect on glioma cells as assessed by in vitro assay. Intracerebral injection of C6 glioma cells generated a rapidly growing tumoral mass. DiI prelabeled ST14A, coinjected into the rat brain with C6 glioma cells, survived in the tumoral mass up t…

Fibroblast growth factor-2 and its receptor expression in proliferating precursor cells of the subventricular zone in the adult rat brain

Several findings have suggested the existence in the subventricular zone (SVZ) from sagittal sections of adult rat brain of a trophic mechanism, mediated by fibroblast growth factor-2 (FGF-2) and its multiple high-affinity FGF receptors (FGFRs), regulating neurogenesis mainly by controlling precursor cell proliferation. However, no clear data are available on the expression of FGF-2 and FGFRs in proliferating precursor cells of the SVZ. To address these questions we examined FGF-2 mRNA and its FGFR mRNA expression in proliferating precursor cells of the SVZ by using a double labeling procedure, combining in situ hybridization for FGF-2 and its FGFR mRNA with immunohistochemistry for bromode…

Distribution and Function of Gap Junction Coupling in Cortical GABAergic Neurons.

Although gap junctions have been observed in GABAergic interneurons of several brain regions, this chapter focuses on the distribution and functions of gap junctions and connexins in inhibitory interneurons of the cerebral cortex and hippocampus. Evidence for interconnections mediated by electrical synapses is reported for at least eight cerebral cortex interneuron types, classified on the basis of morphology, electrophysiology and molecular markers. The main differences in the organization of these interneuronal networks are summarized in terms of homologous and heterologous electrical coupling and mutual chemical inhibition. The role of connexin36 (Cx36) in forming neuronal electrical syn…

The Guanine-Based Purinergic System: The Tale of An Orphan Neuromodulation.

Guanine-based purines (GBPs) have been recently proposed to be not only metabolic agents but also extracellular signaling molecules that regulate important functions in the central nervous system. In such way, GBPs-mediated neuroprotection, behavioral responses and neuronal plasticity have been broadly described in the literature. However, while a number of these functions (i.e., GBPs neurothophic effects) have been well-established, the molecular mechanisms behind these GBPs-dependent effects are still unknown. Furthermore, no plasma membrane receptors for GBPs have been described so far, thus GBPs are still considered orphan neuromodulators. Interestingly, an intricate and controversial f…

Guanine inhibits the growth of human glioma and melanoma cell lines by interacting with GPR23

Guanine-based purines (GBPs) exert numerous biological effects at the central nervous system through putative membrane receptors, the existence of which is still elusive. To shed light on this question, we screened orphan and poorly characterized G protein-coupled receptors (GPRs), selecting those that showed a high purinoreceptor similarity and were expressed in glioma cells, where GBPs exerted a powerful antiproliferative effect. Of the GPRs chosen, only the silencing of GPR23, also known as lysophosphatidic acid (LPA) 4 receptor, counteracted GBP-induced growth inhibition in U87 cells. Guanine (GUA) was the most potent compound behind the GPR23-mediated effect, acting as the endpoint eff…

Anti-inflammatory and antioxidant effects of muscarinic acetylcholine receptor (mAChR) activation in the rat hippocampus

AbstractRecently we found that acute treatment with Oxotremorine (Oxo), a non-selective mAChRs agonist, up-regulates heat shock proteins and activates their transcription factor heat shock factor 1 in the rat hippocampus. Here we aimed to investigate: a) if acute treatment with Oxo may regulate pro-inflammatory or anti-inflammatory cytokines and oxidative stress in the rat hippocampus; b) if chronic restraint stress (CRS) induces inflammatory or oxidative alterations in the hippocampus and whether such alterations may be affected by chronic treatment with Oxo. In the acute experiment, rats were injected with single dose of Oxo (0.4 mg/kg) and sacrificed at 24 h, 48 h and 72 h. In the CRS ex…

The existence of FGFR1-5-HT1A receptor heterocomplexes in midbrain 5-HT neurons of the rat: relevance for neuroplasticity.

The ascending midbrain 5-HT neurons to the forebrain may be dysregulated in depression and have a reduced trophic support. Within situproximity ligation assay (PLA) and supported by coimmunoprecipitation and colocation of the FGFR1 and 5-HT1A immunoreactivities in the midbrain raphe cells, evidence for the existence of FGFR1–5-HT1A receptor heterocomplexes in the dorsal and median raphe nuclei of the Sprague Dawley rat as well as in the rat medullary raphe RN33B cells has been obtained. Especially after combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA clusters was found in the RN33B cells. Similar results were reached with the FRET technique in HEK293T cells,…

Lack of Dystrophin Affects Bronchial Epithelium inmdxMice

Mild exercise training may positively affect the course of Duchenne Muscular Dystrophy (DMD). Training causes mild bronchial epithelial injury in both humans and mice, but no study assessed the effects of exercise in mdx mice, a well known model of DMD. The airway epithelium was examined in mdx (C57BL/10ScSn-Dmdmdx) mice, and in wild type (WT, C57BL/10ScSc) mice either under sedentary conditions (mdx-SD, WT-SD) or during mild exercise training (mdx-EX, WT-EX). At baseline, and after 30 and 45 days of training (5 d/wk for 6 weeks), epithelial morphology and markers of regeneration, apoptosis, and cellular stress were assessed. The number of goblet cells in bronchial epithelium was much lower…

Identification and functional expression of HCx31.9, a novel gap junction gene

By combining in silico and bench molecular biology methods we have identified a novel human gap junction gene that encodes a protein designated HCx31.9. We have determined its human chromosomal location and gene structure, and we have identified a putative mouse ortholog, mCx30.2. We have observed the presence of HCx31.9 in human cerebral cortex, liver, heart, spleen, lung, and kidney and the presence of mCx30.2 in mouse cerebral cortex, liver and lung. Moreover, preliminary data on the electrophysiological properties of HCx31.9 have been obtained by functional expression in paired Xenopus oocytes and in transfected N2A cells.

Altered gastrointestinal motility in an animal model of Lesch-Nyhan disease.

Mutations in the HGPRT1 gene, which encodes hypoxanthine-guanine phosphoribosyltransferase (HGprt), housekeeping enzyme responsible for recycling purines, lead to Lesch-Nyhan disease (LND). Clinical expression of LND indicates that HGprt deficiency has adverse effects on gastrointestinal motility. Therefore, we aimed to evaluate intestinal motility in HGprt knockout mice (HGprt(−)). Spontaneous and neurally evoked mechanical activity was recorded in vitro as changes in isometric tension in circular muscle strips of distal colon. HGprt(−) tissues showed a lower in amplitude spontaneous activity and atropine-sensitivity neural contraction compared to control mice. The responses to carbachol a…

The FGF-2/FGFRs neurotrophic system promotes neurogenesis in the adult brain.

Neurogenesis occurs in two regions of the adult brain, namely, the subventricular zone (SVZ) throughout the wall of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG) in hippocampal formation. Adult neurogenesis requires several neurotrophic factors to sustain and regulate the proliferation and differentiation of the adult stem cell population. In the present review, we examine the cellular and functional aspects of a trophic system mediated by fibroblast growth factor-2 (FGF-2) and its receptors (FGFRs) related to neurogenesis in the SVZ and SGZ of the adult rat brain. In the SVZ, FGF-2 is expressed in GFAP-positive cells of SVZ but is not present in proliferati…

Resveratrol reduces oxidative stress and cell death and increases mitochondrial antioxidants and XIAP in PC6.3-cells.

Resveratrol, a polyphenol derived e.g. from red grapes, has been shown to mediate several positive biological actions such as protection of cells against oxidative stress. It can also influence cell signaling, but the mechanisms behind its antioxidant properties are largely unknown. Here we show that RSV reduces oxidative stress and enhances cell survival in PC6.3 cells depending on the concentration. In these cells, RSV increased the levels of antioxidants, SOD2 and TRX2, and of X chromosome-linked inhibitor of apoptosis protein. RSV also activated NFκB signaling as shown using luciferase reporter constructs. These findings show that RSV regulates oxidative stress and mitochondrial antioxi…

Interactions between cholinergic and fibroblast growth factor receptors in brain trophism and plasticity

Acetylcholine, acting on both nicotinic receptors (nAChRs) and muscarinic receptors (mAChRs), plays a role in the regulation of synaptic plasticity, being involved in the regulation of cellular processes and cognitive functions, such as learning, memory and attention. Recently, G protein coupled receptors (GPCRs), including mAChRs, have been reported to transactivate tyrosine-kinase receptors (RTK), such as epidermal growth factor receptor (EGFR), and initiate their intracellular signaling. In this minireview we have first analysed the RTK transactivation mechanisms, involving cholinergic receptors, and thereafter the interplay between AChR and neurotrophic factor systems built up by FGF2 a…

Guanosine negatively modulates the gastric motor function in mouse

The aim of the present study was to evaluate if guanine-based purines may affect the gastric motor function in mouse. Thus, the influence of guanosine on the gastric emptying rate in vivo was determined and its effects on spontaneous gastric mechanical activity, detected as changes of the intraluminal pressure, were analyzed in vitro before and after different treatments. Gastric gavage of guanosine (1.75-10 mg/kg) delayed the gastric emptying. Guanosine (30 μM-1 mM) induced a concentration-dependent relaxation of isolated stomach, which was not affected by the inhibition of the purine nucleoside phosphorylase enzyme by 4'-deaza-1'-aza-2'-deoxy-1'-(9-methylene)-immucillin-H. The inhibitory …

Manipulation of HSP70-SOD1 Expression Modulates SH-SY5Y Differentiation and Susceptibility to Oxidative Stress-Dependent Cell Damage: Involvement in Oxotremorine-M-Mediated Neuroprotective Effects

The differentiation of neural progenitors is a complex process that integrates different signals to drive transcriptional changes, which mediate metabolic, electrophysiological, and morphological cellular specializations. Understanding these adjustments is essential within the framework of stem cell and cancer research and therapy. Human neuroblastoma SH-SY5Y cells, widely used in neurobiology research, can be differentiated into neuronal-like cells through serum deprivation and retinoic acid (RA) supplementation. In our study, we observed that the differentiation process triggers the expression of Heat Shock Protein 70 (HSP70). Notably, inhibition of HSP70 expression by KNK437 causes a dra…

The nicotinic acetylcholine receptor agonist ABT-594 increases FGF-2 expression in various rat brain regions

The present experiments were designed to extend previous work showing that acute intermittent (-)nicotine treatment upregulates the level of fibroblast growth factor-2 (FGF2) mRNA in several rat brain regions, by the use of the nicotinic acetylcholine receptor (nAChR) agonist ABT-594 with preferential selectivity for the alpha4beta2 nAChR subtype. ABT594 treatment led to a well-defined temporal and regional upregulation of FGF-2 mRNA. A double labelling analysis showed that the up-regulation of FGF-2 mRNA involves both neuronal and non-neuronal cells. The effects of ABT-594 on FGF-2 expression were antagonized by the preferential alpha4beta2 antagonist dihydrobetaerythroidine (DHbetaE), but…

Endoplasmic Reticulum Stress Inhibition Protects against Excitotoxic Neuronal Injury in the Rat Brain

Elevated brain glutamate with activation of neuronal glutamate receptors accompanies neurological disorders, such as epilepsy and brain trauma. However, the mechanisms by which excitotoxicity triggers neuronal injury are not fully understood. We have studied the glutamate receptor agonist kainic acid (KA) inducing seizures and excitotoxic cell death. KA caused the disintegration of the endoplasmic reticulum (ER) membrane in hippocampal neurons and ER stress with the activation of the ER proteins Bip, Chop, and caspase-12. Salubrinal, inhibiting eIF2α (eukaryotic translation initiation factor 2 subunit α) dephosphorylation, significantly reduced KA-induced ER stress and neuronal deathin vivo…

Nicotinic receptor agonists as neuroprotective/neurotrophic drugs. Progress in molecular mechanisms

In the present work we reviewed recent advances concerning neuroprotective/neurotrophic effects of acute or chronic nicotine exposure, and the signalling pathways mediating these effects, including mechanisms implicated in nicotine addiction and nAChR desensitization. Experimental and clinical data largely indicate long-lasting effects of nicotine and nicotinic agonists that imply a neuroprotective/neurotrophic role of nAChR activation, involving mainly alpha 7 and alpha 4 beta 2 nAChR subtypes, as evidenced using selective nAChR agonists. Compounds interacting with neuronal nAChRs have the potential to be neuroprotective and treatment with nAChR agonists elicits long-lasting neurotrophic e…

Uncovering the Signaling Pathway behind Extracellular Guanine-Induced Activation of NO System: New Perspectives in Memory-Related Disorders

Mounting evidence suggests that the guanine-based purines stand out as key player in cell metabolism and in several models of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Guanosine (GUO) and guanine (GUA) are extracellular signaling molecules derived from the breakdown of the correspondent nucleotide, GTP, and their intracellular and extracellular levels are regulated by the fine-tuned activity of two major enzymes, purine nucleoside phosphorylase (PNP) and guanine deaminase (GDA). Noteworthy, GUO and GUA, seem to play opposite roles in the modulation of cognitive functions, such as learning and memory. Indeed GUO, despite exerting neuroprotective, anti-apoptot…

Evidence for the existence of FGFR1-5-HT1A heteroreceptor complexes in the midbrain raphe 5-HT system.

The ascending midbrain 5-HT neurons known to contain 5-HT1A autoreceptors may be dysregulated in depression due to a reduced trophic support. With in situ proximity ligation assay (PLA) and supported by co-location of the FGFR1 and 5-HT1A immunoreactivities in midbrain raphe 5-HT cells, evidence for the existence of FGFR1–5-HT1A heteroreceptor complexes were obtained in the dorsal and median raphe nuclei of the Sprague–Dawley rat. Their existence in the rat medullary raphe RN33B cell cultures was also established. After combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA positive clusters was found in the RN33B cells. Similar results were reached upon coactivati…

Existence of muscarinic acetylcholine receptor (mAChR) and fibroblast growth factor receptor (FGFR) heteroreceptor complexes and their enhancement of neurite outgrowth in neural hippocampal cultures

Abstract Background Recently, it was demonstrated that G-protein-coupled receptors (GPCRs) can transactivate tyrosine kinase receptors in absence of their ligands. In this work, driven by the observation that mAChRs and fibroblast growth factor receptors (FGFRs) share signalling pathways and regulation of brain functions, it was decided to explore whether mAChRs activation may transactivate FGFRs and, if so, to characterize the related trophic effects in cultured hippocampal neurons. Methods Oxotremorine-M transactivation of FGFRs and related trophic effects were tested in primary hippocampal neurons. Western blotting and in situ proximity ligation assay (PLA) were used to detect FGFR phosp…

Identification of calcium sensing receptor (CaSR) mRNA-expressing cells in normal and injured rat brain

Calcium sensing receptor (CaSR), isolated for the first time from bovine and human parathyroid, is a G-protein-coupled receptors that has been involved in diverse physiological functions. At present a complete in vivo work on the identification of CaSR mRNA-expressing cells in the adult brain lacks and this investigation was undertaken in order to acquire more information on cell type expressing CaSR mRNA in the rat brain and to analyse for the first time its expression in different experimental models of brain injury. The expression of CaSR mRNAs was found mainly in scattered cells throughout almost all the brain regions. A double labeling analysis showed a colocalization of CaSR mRNA expr…

mGluR2/3 agonist LY379268, by enhancing the production of GDNF, induces a time-related phosphorylation of RET receptor and intracellular signaling Erk1/2 in mouse striatum.

In the present study we aimed to verify if the enhancement of glial cell line-derived neurotrophic factor (GDNF) production in mouse striatum following treatment with LY379268 may also induce in the nigrostriatal system a time-related activation of RET receptor and its specific intracellular signaling. For this purpose, we have investigated the effects of LY379268 treatment on RET phosphorylation at the Tyr1062 and on downstream signaling Erk1/2, Akt and PLCγ1 pathway activation. The results showed that treatment with LY379268 (3 mg/kg) induces a significant increase of GDNF levels and time-related RET and Erk1/2 phosphorylation in the striatum. These increases were detected at 24 h and 48 …

Parkinson's disease: towards better preclinical models and personalized treatments.

Non peer reviewed

Connexin-30 mRNA Is Up-Regulated in Astrocytes and Expressed in Apoptotic Neuronal Cells of Rat Brain Following Kainate-Induced Seizures

Glial connexins (Cxs) make an extensively interconnected functional syncytium created by a network of gap junctions between astrocytes and oligodendrocytes. Among Cxs expressed in the brain, Cx30 is expressed in grey matter astrocytes, as shown at the protein level by immunoistochemistry. In the present study we aimed to perform a detailed study of the regional distribution of Cx30 mRNA in the adult and postnatal developing rat brain, analyzing its expression by in situ hybridization, and determining its cell type localization by double labeling. Recently, it has been suggested that neuronal activity may control the level of intercellular communication between astrocytes through gap junctio…

Expression of connexin36 in the adult and developing rat brain.

The distribution of connexin36 (Cx36) in the adult rat brain and retina has been analysed at the protein (immunofluorescence) and mRNA (in situ hybridization) level. Cx36 immunoreactivity, consisting primarily of round or elongated puncta, is highly enriched in specific brain regions (inferior olive and the olfactory bulb), in the retina, in the anterior pituitary and in the pineal gland, in agreement with the high levels of Cx36 mRNA in the same regions. A lower density of immunoreactive puncta can be observed in several brain regions, where only scattered subpopulations of cells express Cx36 mRNA. By combining in situ hybridization for Cx36 mRNA with immunohistochemistry for a general neu…

Structure, chromosomal localization, and brain expression of human Cx36 gene

Rat connexin-36 (Cx36) is the first gap junction protein shown to be expressed predominantly in neuronal cells of the mammalian central nervous system. As a prerequisite for studies devoted to the investigation of the possible role of this connexin in human neurological diseases, we report the cloning and sequencing of the human Cx36 gene, its chromosomal localization, and its pattern of expression in the human brain analyzed by radioactive in situ hybridization. The determination of the human gene sequence revealed that the coding sequence of Cx36 is highly conserved (98% identity at the protein level with the mouse and rat Cx36 and 80% with the ortholog perch and skate Cx35), and that the…

Recovery of Damaged Skeletal Muscle in mdx Mice through Low-intensity Endurance Exercise

The lack of dystrophin in mdx mice leads to cycles of muscle degeneration and regeneration processes. Various strategies have been proposed in order to reduce the muscle-wasting component of muscular dystrophy, including implementation of an exercise programme. The aim of this study was to examine how low-intensity endurance exercise affects the degeneration-regeneration process in dystrophic muscle of male mdx mice. Mice were subjected to low-intensity endurance exercise by running on a motorized Rota-Rod for 5 days/week for 6 weeks. Histomorphological analysis showed a signifi cant reduction of measured inflammatory-necrotic areas in both gastrocnemius and quadriceps muscle of exercised m…

Transcription factor gene expression profiling after acute intermittent nicotine treatment in the rat cerebral cortex

Several studies in different in vitro and in vivo models have demonstrated neuroprotective effects of nicotinic receptor agonists and indirect trophic actions of nicotine on brain are suggested from observations describing nicotine as a cognitive enhancer by increasing vigilance and improving learning and memory. While an increasing number of studies have given evidence of neuroprotective and neurotrophic effects of nicotine treatment, the molecular mechanism mediating the neurotrophic effects of nicotine are not fully understood. Previously in an analysis of several neurotrophic factors as possible mediators of nicotine-induced neuroprotection and/or neurotrophic effects we could reveal th…

Group II metabotropic glutamate receptor activation by agonist LY379268 treatment increases the expression of brain derived neurotrophic factor in the mouse brain

A number of in vitro and in vivo studies using selective agonists have indicated a neuroprotective role for group-II metabotropic glutamate (mGlu2/3) receptors in various models of neuronal injury. Although an interplay among neurotrophic factors and mGlu2/3 receptors signalling system has been suggested as possible mechanism involved on neuroprotection, at present poor information are available concerning the in vivo regulation by mGlu2/3 receptors activation of specific neurotrophic factors. By using in situ hybridization and western blotting methods the aim of present study was to analyse the potential regulatory role of selective mGluR2/3 agonist LY379268 treatment on brain derived neur…

Guanosine modulates K+ membrane currents in SH-SY5Y cells: involvement of adenosine receptors

AbstractGuanosine (GUO), widely considered a key signaling mediator, is implicated in the regulation of several cellular processes. While its interaction with neural membranes has been described, GUO still is an orphan neuromodulator. It has been postulated that GUO may eventually interact with potassium channels and adenosine (ADO) receptors (ARs), both particularly important for the control of cellular excitability. Accordingly, here, we investigated the effects of GUO on the bioelectric activity of human neuroblastoma SH-SY5Y cells by whole-cell patch-clamp recordings. We first explored the contribution of voltage-dependent K+ channels and, besides this, the role of ARs in the regulation…

Nicotinic receptors and neurotrophic factors

Expression of neurotrophins, GDNF, and their receptors in rat thyroid tissue

Levels of mRNA for neurotrophins (brain-derived neurotrophic factor, BDNF; neurotrophin 3, NT-3; neurotrophin 4, NT-4) and their receptors (trkA, trkB, trkC) and for glial cell line-derived neurotrophic factor (GDNF) and its receptors (ret, GDNFR-alpha) were measured in rat thyroid tissue by ribonuclease protection assays. In thyroid tissue the NT-3 mRNA level was threefold lower and the NT-4 mRNA level sixfold higher than those detected in adult rat hippocampus, while BDNF mRNA was undetectable. Very low levels of mRNA for truncated trkB and trkC receptors and no catalytic trkA, trkB or trkC were found. In conclusion NT-3 and NT-4, but not the corresponding functional receptors, are expres…

Investigating the Role of Guanosine on Human Neuroblastoma Cell Differentiation and the Underlying Molecular Mechanisms

Neuroblastoma arises from neural crest cell precursors failing to complete the process of differentiation. Thus, agents helping tumor cells to differentiate into normal cells can represent a valid therapeutic strategy. Here, we evaluated whether guanosine (GUO), a natural purine nucleoside, which is able to induce differentiation of many cell types, may cause the differentiation of human neuroblastoma SH-SY5Y cells and the molecular mechanisms involved. We found that GUO, added to the cell culture medium, promoted neuron-like cell differentiation in a time- and concentration-dependent manner. This effect was mainly due to an extracellular GUO action since nucleoside transporter inhibitors r…

Mild Aerobic Exercise Training Hardly Affects the Diaphragm ofmdxMice

In the mdx mice model of Duchenne Muscular Dystrophy (DMD), mild endurance exercise training positively affected limb skeletal muscles, whereas few and controversial data exist on the effects of training on the diaphragm. The diaphragm was examined in mdx (C57BL/10ScSn-Dmdmdx) and wild-type (WT, C57BL/10ScSc) mice under sedentary conditions (mdx-SD, WT-SD) and during mild exercise training (mdx-EX, WT-EX). At baseline, and after 30 and 45 days (training: 5 d/wk for 6 weeks), diaphragm muscle morphology and Cx39 protein were assessed. In addition, tissue levels of the chaperonins Hsp60 and Hsp70 and the p65 subunit of nuclear factor-kB (NF-kB) were measured in diaphragm, gastrocnemius, and q…

Nicotine-induced fibroblast growth factor-2 restores the age-related decline of precursor cell proliferation in the subventricular zone of rat brain.

Precursor cell proliferation is present in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus of adult rat and persists during aging although at reduced levels. Previous studies have shown that acute intermittent nicotine treatment significantly increases fibroblast growth factor-2 (FGF-2) expression in several brain regions of aged rats. The aim of the present investigation was to test the hypothesis that nicotine-induced expression of FGF-2 may restore the age-related decline of precursor cell proliferation. It was first demonstrated that nicotine treatment increases both mRNA and protein FGF-2 in the SVZ of aged …

Cellular expression of connexins in the rat brain: neuronal localization, effects of kainate-induced seizures and expression in apoptotic neuronal cells.

The identification of connexins (Cxs) expressed in neuronal cells represents a crucial step for understanding the direct communication between neurons and between neuron and glia. In the present work, using a double-labelling method combining in situ hybridization for Cx mRNAs with immunohistochemical detection for neuronal markers, we provide evidence that, among cerebral connexins (Cx26, Cx32, Cx36, Cx37, Cx40, Cx43, Cx45 and Cx47), only Cx45 and Cx36 mRNAs are localized in neuronal cells in both developing and adult rat brain. In order to establish whether connexin expression is influenced in vivo by abnormal neuronal activity, we examined the short-term effects of kainate-induced seizur…

Expression of Cx36 in mammalian neurons

Cx36 is the first mammalian member of a novel subgroup of the connexin family, characterized by a long cytoplasmic loop, a peculiar gene structure and a preferential expression in cell types of neural origin. In the present review we summarize the evidence in favour of its predominant expression in neuronal cells in the mammalian central nervous system, such as results from experiments with specific neurotoxins and co-localization of Cx36 mRNA and a neuronal marker. We also report a detailed description of Cx36 mRNA distribution in the rat and human central nervous system by in situ hybridization and, for each brain region, we correlate the novel findings with previous morphological or func…

Crosstalk between receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCR) in the brain: Focus on heteroreceptor complexes and related functional neurotrophic effects

Neuronal events are regulated by the integration of several complex signaling networks in which G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) are considered key players of an intense bidirectional cross-communication in the cell, generating signaling mechanisms that, at the same time, connect and diversify the traditional signal transduction pathways activated by the single receptor. For this receptor-receptor crosstalk, the two classes of receptors form heteroreceptor complexes resulting in RTKs transactivation and in growth-promoting signals. In this review, we describe heteroreceptor complexes between GPCR and RTKs in the central nervous system (CNS) and their …

Detection of Fibroblast Growth Factor Receptor 1 (FGFR1) Transactivation by Muscarinic Acetylcholine Receptors (mAChRs) in Primary Neuronal Hippocampal Cultures Through Use of Biochemical and Morphological Approaches

In addition to their canonical intracellular signals involved in the regulation of neuronal plasticity, G-protein coupled receptors can also rapidly transactivate tyrosine kinase receptors and their downstream intracellular signaling in absence of specific ligands. Here we describe our protocol for dissociating and maintaining hippocampal primary neurons in high- and low-density culture, followed by a description of methods employed to evaluate neurite outgrowth and protein phosphorylation associated with fibroblast growth factor receptor 1 transactivation by muscarinic acetylcholine receptors. Our goal was to provide the reader with detailed protocols of the abovementioned techniques and t…

Heat shock protein (Hsp) regulation by muscarinic acetylcholine receptor (mAChR) activation in the rat hippocampus.

The cholinergic system plays a crucial role in modulating in the central nervous system physiological responses such as neurogenesis, neuronal differentiation, synaptic plasticity, and neuroprotection. In a recent study, we showed that Oxotremorine-M, a non-selective muscarinic acetylcholine receptor agonist, is able to transactivate the fibroblast growth factor receptor and to produce a significant increase in the hippocampal primary neurite outgrowth. In the present study we aimed to explore in the rat hippocampus the possible effect of acute or chronic treatment with Oxotremorine-M on some heat shock proteins (Hsp60, Hsp70, Hsp90) and on activation of related transcription factor heat sh…

Transgenic expression and activation of PGC-1α protect dopaminergic neurons in the MPTP mouse model of Parkinson’s disease

Mitochondrial dysfunction and oxidative stress occur in Parkinson’s disease (PD), but little is known about the molecular mechanisms controlling these events. Peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α) is a transcriptional coactivator that is a master regulator of oxidative stress and mitochondrial metabolism. We show here that transgenic mice overexpressing PGC-1α in dopaminergic neurons are resistant against cell degeneration induced by the neurotoxin MPTP. The increase in neuronal viability was accompanied by elevated levels of mitochondrial antioxidants SOD2 and Trx2 in the substantia nigra of transgenic mice. PGC-1α overexpression also protected against MP…

Cellular localization of mGluR3 and mGluR5 mRNAs in normal and injured rat brain

Abstract In order to understand the role of metabotropic glutamate receptors (mGluRs) in the brain, it is important to know how the mGluRs are differentially expressed among the different cell types. At present, the cellular expression of mGluR3 and mGluR5 has been mostly studied in terms of proteins with observations suggesting the expression of both mGluR3 and mGluR5 in neuronal and in glial cells. In order to verify the brain cell type-expressing mGluR3 and mGluR5 mRNAs, both in normal and injured brain, we performed a double labeling analysis, by in situ hybridization for mGluR3 or mGluR5 mRNA and immunohistochemistry for specific cellular markers. This approach allowed us to find mGluR…

Neuroprotective, antioxidant and antiproliferative activity of grapefruit IntegroPectin on SH-SY5Y cells

AbstractTested in vitro on SH-SY5Y neuroblastoma cells, grapefruit IntegroPectin is a powerful neuroprotective, antioxidant and antiproliferative agent. The strong antioxidant properties of grapefruit IntegroPectin, and its ability to preserve mitochondrial membrane potential and morphology, severely impaired in neurodegenerative disorders, make this new biopolymer highly soluble in water an attractive therapeutic agent for oxidative stress-associated brain disorders. Similarly, the ability of this new citrus pectin rich in naringin, linalool, linalool oxide and limonene adsorbed at the outer surface to inhibit cell proliferation or even kill, at high doses, neoplastic cells, coupled to its…

Agonist-induced formation of FGFR1 homodimers and signaling differ among members of the FGF family

Fibroblast growth factor receptor 1 (FGFR1) is known to be activated by homodimerization in the presence of both the FGF agonist ligand and heparan sulfate glycosaminoglycan. FGFR1 homodimers in turn trigger a variety of downstream signaling cascades via autophosphorylation of tyrosine residues in the cytoplasmic domain of FGFR1. By means of Bioluminescence Energy Resonance Transfer (BRET) as a sign of FGFR1 homodimerization, we evaluated in HEK293T cells the effects of all known FGF agonist ligands on homodimer formation. A significant correlation between BRET(2) signaling and ERK1/2 phosphorylation was observed, leading to a further characterization of the binding and signaling properties…

Guanosine-Mediated Anxiolytic-Like Effect: Interplay with Adenosine A1 and A2A Receptors

Acute or chronic administration of guanosine (GUO) induces anxiolytic-like effects, for which the adenosine (ADO) system involvement has been postulated yet without a direct experimental evidence. Thus, we aimed to investigate whether adenosine receptors (ARs) are involved in the GUO-mediated anxiolytic-like effect, evaluated by three anxiety-related paradigms in rats. First, we confirmed that acute treatment with GUO exerts an anxiolytic-like effect. Subsequently, we investigated the effects of pretreatment with ADO or A1R (CPA, CCPA) or A2AR (CGS21680) agonists 10 min prior to GUO on a GUO-induced anxiolytic-like effect. All the combined treatments blocked the GUO anxiolytic-like effect, …

Neuroprotective and Antioxidant Role of Oxotremorine-M, a Non-selective Muscarinic Acetylcholine Receptors Agonist, in a Cellular Model of Alzheimer Disease.

AbstractAlzheimer disease (AD) is a multifactorial and age-dependent neurodegenerative disorder, whose pathogenesis, classically associated with the formation of senile plaques and neurofibrillary tangles, is also dependent on oxidative stress and neuroinflammation chronicization. Currently, the standard symptomatic therapy, based on acetylcholinesterase inhibitors, showed a limited therapeutic potential, whereas disease-modifying treatment strategies are still under extensive research. Previous studies have demonstrated that Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptors agonist, exerts neurotrophic functions in primary neurons, and modulates oxidative stress and …

Detection, Analysis, and Quantification of GPCR Homo- and Heteroreceptor Complexes in Specific Neuronal Cell Populations Using the In Situ Proximity Ligation Assay

GPCR’s receptosome operates via coordinated changes between the receptor expression, their modifications and interactions between each other. Perturbation in specific heteroreceptor complexes and/or their balance/equilibrium with other heteroreceptor complexes and corresponding homoreceptor complexes is considered to have a role in pathogenic mechanisms. Such mechanisms lead to mental and neurological diseases, including drug addiction, depression, Parkinson’s disease, and schizophrenia. To understand the associations of GPCRs and to unravel the global picture of their receptor–receptor interactions in the brain, different experimental detection techniques for receptor–receptor interactions…

Anxiolytic effects of muscarinic acetylcholine receptors agonist oxotremorine in chronically stressed rats and related changes in BDNF and FGF2 levels in the hippocampus and prefrontal cortex.

Rationale: In depressive disorders, one of the mechanisms proposed for antidepressant drugs is the enhancement of synaptic plasticity in the hippocampus and cerebral cortex. Previously, we showed that the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine (Oxo) increases neuronal plasticity in hippocampal neurons via FGFR1 transactivation. Objectives: Here, we aimed to explore (a) whether Oxo exerts anxiolytic effect in the rat model of anxiety-depression-like behavior induced by chronic restraint stress (CRS), and (b) if the anxiolytic effect of Oxo is associated with the modulation of neurotrophic factors, brain-derived neurotrophic factor (BDNF) and fibroblast growth factor-2…

Dendritic localization of mammalian neuralized mRNA encoding a protein with transcription repression activities.

Drosophila neurogenic gene neuralized (neu) is required for the maintenance of neuroblast cell fate and differentiation. In the present study we have characterized a mouse and a rat homologue of Drosophila neu. Mammalian neu1 encodes several C-terminal RING zinc finger proteins with one or two neuralized homology repeat (NHR) domains. Mammalian neu1 mRNAs are predominantly expressed in the nervous system and in the skeletal muscle with the highest levels in the adult. In the nervous system neu1 mRNAs are expressed in neurons and dendritically localized in several brain regions, suggesting a role of neu1 in the regulation of synaptic function. Mammalian neu1 isoforms exhibit transcription re…

Small airways in in sedentary and endurance-trained dystrophic (mdx) mice

The effects of mild endurance exercise training on the small airways in mdx mice are unknown. We compared epithelial thickness and turnover, apoptosis, and stress marker expression in small airways of mdx mice and wild-type (WT) controls, at rest and during exercise training. Mdx and WT mice were randomly assigned to sedentary (mdx-S, n=17; WT-S, n=19) or trained (mdx-EX, n=14; WT-EX, n=16) groups. Low-intensity endurance training (running on a wheel) was done 5 d/wk for 6 wk at progressively increasing speed (rpm from 16 to 24) and time (15 min to 1 h). Lungs were processed for light microscopy and periodic acid Schiff (PAS) staining. Hsp60 and PCNA were quantified by immunohistochemistry.…