Histamine beyond its effects on allergy: Potential therapeutic benefits for the treatment of Amyotrophic Lateral Sclerosis (ALS).

ALS currently remains a challenge despite many efforts in performing successful clinical trials and formulating therapeutic solutions. By learning from current failures and striving for success, scientists and clinicians are checking every possibility to search for missing hints and efficacious treatments. Because the disease is very complex and heterogeneous and, moreover, targeting not only motor neurons but also several different cell types including muscle, glial, and immune cells, the right answer to ALS is conceivably a multidrug strategy or the use of broad-spectrum molecules. The aim of the present work is to gather evidence about novel perspectives on ALS pathogenesis and to present recent and innovative paradigms for therapy. In particular, we describe how an old molecule possessing immunomodulatory and neuroprotective functions beyond its recognized effects on allergy, histamine, might have a renewed and far-reaching momentum in ALS

Mapping P2X and P2Y receptor proteins in striatum and substantia nigra: An immunohistological study

Our work aimed to provide a topographical analysis of all known ionotropic P2X1–7 and metabotropic P2Y1,2,4,6,11–14 receptors that are present in vivo at the protein level in the basal ganglia nuclei and particularly in rat brain slices from striatum and substantia nigra. By immunohistochemistry-confocal and Western blotting techniques, we show that, with the exception of P2Y11,13 receptors, all other subtypes are specifically expressed in these areas in different amounts, with ratings of low (P2X5,6 and P2Y1,6,14 in striatum), medium (P2X3 in striatum and substantia nigra, P2X6,7 and P2Y1 in substantia nigra) and high. Moreover, we describe that P2 receptors are localized on neurons (colocalizing with neurofilament light, medium and heavy chains) with features that are either dopaminergic (colocalizing with tyrosine hydroxylase) or GABAergic (colocalizing with parvalbumin and calbindin), and they are also present on astrocytes (P2Y2,4, colocalizing with glial fibrillary acidic protein). In addition, we aimed to investigate the expression of P2 receptors after dopamine denervation, obtained by using unilateral injection of 6-hydroxydopamine as an animal model of Parkinson’s disease. This generates a rearrangement of P2 proteins: most P2X and P2Y receptors are decreased on GABAergic and dopaminergic neurons, in the lesioned striatum and substantia nigra, respectively, as a consequence of dopaminergic denervation and/or neuronal degeneration. Conversely, P2X1,3,4,6 on GABAergic neurons and P2Y4 on astrocytes augment their expression exclusively in the lesioned substantia nigra reticulata, probably as a compensatory reaction to dopamine shortage. These results disclose the presence of P2 receptors in the normal and lesioned nigro-striatal circuit, and suggest their potential participation in the mechanisms of Parkinson’s disease

P2Y1 receptor switches to neurons from glia in juvenile versus neonatal rat cerebellar cortex

<p>Abstract</p> <p>Background</p> <p>In the CNS, several P2 receptors for extracellular nucleotides are identified on neurons and glial cells to participate to neuron-neuron, glia-glia and glia-neuron communication.</p> <p>Results</p> <p>In this work, we describe the cellular and subcellular presence of metabotropic P2Y₁receptor in rat cerebellum at two distinct developmental ages, by means of immunofluorescence-confocal and electron microscopy as well as western blotting and direct membrane separation techniques. At postnatal day 21, we find that P2Y₁receptor in addition to Purkinje neurons, is abundant on neuronal specializations identified as noradrenergic by anatomical, morphological and biochemical features. P2Y₁receptor immunoreactivity colocalizes with dopamine β-hydroxylase, tyrosine hydroxylase, neurofilament light chain, synaptophysin and flotillin, but not with glial fibrillary acidic protein for astrocytes. P2Y₁receptor is found enriched in membrane microdomains such as lipid rafts, in cerebellar synaptic vesicles, and is moreover visualized on synaptic varicosities by electron microscopy analysis. When examined at postnatal day 7, P2Y₁receptor immunoreactivity is instead predominantly expressed only on Bergmann and astroglial cells, as shown by colocalization with glial fibrillary acidic protein rather then neuronal markers. At this age, we moreover identify that P2Y₁receptor-positive Bergmann fibers wrap up doublecortin-positive granule cells stretching along them, while migrating through the cerebellar layers.</p> <p>Conclusion</p> <p>Membrane components including purinergic receptors are already known to mediate cellular contact and aggregation in platelets. Our results suggesting a potential role for P2Y₁protein in cell junction/communication and development, are totally innovative for the CNS.</p

Synaptic P2X7 and oxygen/glucose deprivation in organotypic hippocampal cultures.

The P2X7 receptor for extracellular ATP is the main candidate, among P2 receptors, inducing cell death in the immune system. Here, we demonstrate the direct participation of this receptor to cell damage induced by oxygen/glucose deprivation, in the ex vivo model of organotypic hippocampal cultures. By pharmacological and immunological approaches, we show that P2X7 is rapidly and transiently up regulated in hippocampal areas eliciting metabolism impairment. Moreover, the P2 antagonists 2′,3′,-dialdehyde ATP and reactive blue 2 prevent both up regulation of this receptor and hypoxic/hypoglycemic damage. By confocal laser microscopy, we show that P2X7 is present at the synaptic level of fibers extending from the CA1–2 pyramidal cell layer throughout the strata oriens and radiatum, but absent on oligodendrocytes, astrocytes or neuronal cell bodies. Colocalization of P2X7 is obtained with neurofilament-L protein and with synaptophysin, not with myelin basic protein, glial fibrillary acidic protein or a marker for neuronal nuclei. P2X7 up regulation and diffuse cellular damage are also induced by 3′-O-(4-benzoyl) benzoyl-ATP, an agonist selective but not exclusive for P2X7. In summary, our study demonstrates that P2X7 not only directly participates to the hypoxic/hypoglycemic process, but also owns specific phenotypic localization. We do not exclude that it might serve as a sensor of dysregulated neuronal activity and ATP release, both occurring during oxygen/glucose deprivation

N-Glycans mutations rule oligomeric assembly and functional expression of P2X3 receptor for extracellular ATP

N-Glycosylation affects the function of ion channels at the level of multisubunit assembly, protein trafficking, ligand binding and channel opening. Like the majority of membrane proteins, ionotropic P2X receptors for extracellular ATP are glycosylated in their extracellular moiety. Here, we used site-directed mutagenesis to the four predicted N-glycosylation sites of P2X3 receptor (Asn139, Asn170, Asn194 and Asn290) and performed comparative analysis of the role of N-glycans on protein stability, plasma membrane delivery, trimer formation and inward currents. We have found that in transiently transfected HEK293 cells, Asn170 is apparently the most important site for receptor stability, since its mutation causes a primary loss in protein content and indirect failure in membrane expression, oligomeric association and inward current responses. Even stronger effects are obtained when mutating Thr172 in the same glycosylation consensus. Asn194 and Asn290 are the most dispensable, since even their simultaneous mutation does not affect any tested receptor feature. All double mutants containing Asn170 mutation or the Asn139/Asn290 double mutant are instead almost unable to assemble into a functional trimeric structure. The main emerging finding is that the inability to assemble into trimers might account for the impaired function in P2X3 mutants where residue Asn170 is replaced. These results improve our knowledge about the role of N-glycosylation in proper folding and oligomeric association of P2X3 recepto

Vitamin B6 rescues insulin resistance and glucose-induced DNA damage caused by reduced activity of Drosophila PI3K

: The insulin signaling pathway controls cell growth and metabolism, thus its deregulation is associated with both cancer and diabetes. Phosphatidylinositol 3-kinase (PI3K) contributes to the cascade of phosphorylation events occurring in the insulin pathway by activating the protein kinase B (PKB/AKT), which phosphorylates several substrates, including those involved in glucose uptake and storage. PI3K inactivating mutations are associated with insulin resistance while activating mutations are identified in human cancers. Here we show that RNAi-induced depletion of the Drosophila PI3K catalytic subunit (Dp110) results in diabetic phenotypes such as hyperglycemia, body size reduction, and decreased glycogen content. Interestingly, we found that hyperglycemia produces chromosome aberrations (CABs) triggered by the accumulation of advanced glycation end-products and reactive oxygen species. Rearing PI3KRNAi flies in a medium supplemented with pyridoxal 5'-phosphate (PLP; the catalytically active form of vitamin B6) rescues DNA damage while, in contrast, treating PI3KRNAi larvae with the PLP inhibitor 4-deoxypyridoxine strongly enhances CAB frequency. Interestingly, PLP supplementation rescues also diabetic phenotypes. Taken together, our results provide a strong link between impaired PI3K activity and genomic instability, a crucial relationship that needs to be monitored not only in diabetes due to impaired insulin signaling but also in cancer therapies based on PI3K inhibitors. In addition, our findings confirm the notion that vitamin B6 is a good natural remedy to counteract insulin resistance and its complications

The S100B Inhibitor Pentamidine Ameliorates Clinical Score and Neuropathology of Relapsing-Remitting Multiple Sclerosis Mouse Model

S100B is an astrocytic protein acting either as an intracellular regulator or an extracellular signaling molecule. A direct correlation between increased amount of S100B and demyelination and inflammatory processes has been demonstrated. The aim of this study is to investigate the possible role of a small molecule able to bind and inhibit S100B, pentamidine, in the modulation of disease progression in the relapsing-remitting experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. By the daily evaluation of clinical scores and neuropathologic-molecular analysis performed in the central nervous system, we observed that pentamidine is able to delay the acute phase of the disease and to inhibit remission, resulting in an amelioration of clinical score when compared with untreated relapsing-remitting experimental autoimmune encephalomyelitis mice. Moreover, we observed a significant reduction of proinflammatory cytokines expression levels in the brains of treated versus untreated mice, in addition to a reduction of nitric oxide synthase activity. Immunohistochemistry confirmed that the inhibition of S100B was able to modify the neuropathology of the disease, reducing immune infiltrates and partially protecting the brain from the damage. Overall, our results indicate that pentamidine targeting the S100B protein is a novel potential drug to be considered for multiple sclerosis treatment

A Model of Ischemia-Induced Neuroblast Activation in the Adult Subventricular Zone

We have developed a rat brain organotypic culture model, in which tissue slices contain cortex-subventricular zone-striatum regions, to model neuroblast activity in response to in vitro ischemia. Neuroblast activation has been described in terms of two main parameters, proliferation and migration from the subventricular zone into the injured cortex. We observed distinct phases of neuroblast activation as is known to occur after in vivo ischemia. Thus, immediately after oxygen/glucose deprivation (6–24 hours), neuroblasts reduce their proliferative and migratory activity, whereas, at longer time points after the insult (2 to 5 days), they start to proliferate and migrate into the damaged cortex. Antagonism of ionotropic receptors for extracellular ATP during and after the insult unmasks an early activation of neuroblasts in the subventricular zone, which responded with a rapid and intense migration of neuroblasts into the damaged cortex (within 24 hours). The process is further enhanced by elevating the production of the chemoattractant SDf-1α and may also be boosted by blocking the activation of microglia. This organotypic model which we have developed is an excellent in vitro system to study neurogenesis after ischemia and other neurodegenerative diseases. Its application has revealed a SOS response to oxygen/glucose deprivation, which is inhibited by unfavorable conditions due to the ischemic environment. Finally, experimental quantifications have allowed us to elaborate a mathematical model to describe neuroblast activation and to develop a computer simulation which should have promising applications for the screening of drug candidates for novel therapies of ischemia-related pathologies