A novel method for DNA delivery into bacteria using cationic copolymers.

Amphiphilic copolymers have a wide variety of medical and biotechnological applications, including DNA transfection in eukaryotic cells. Still, no polymer-primed transfection of prokaryotic cells has been described. The reversible addition-fragmentation chain transfer (RAFT) polymer synthesis technique and the reversible deactivation radical polymerization variants allow the design of polymers with well-controlled molar mass, morphology, and hydrophilicity/hydrophobicity ratios. RAFT was used to synthesize two amphiphilic copolymers containing different ratios of the amphiphilic poly[2-(dimethyl-amino) ethyl methacrylate] and the hydrophobic poly [methyl methacrylate]. These copolymers bound to pUC-19 DNA and successfully transfected non-competent Escherichia coli DH5α, with transformation efficiency in the range of 103 colony-forming units per µg of plasmid DNA. These results demonstrate prokaryote transformation using polymers with controlled amphiphilic/hydrophobic ratios.

A novel method for DNA delivery into bacteria using cationic copolymers

Amphiphilic copolymers have a wide variety of medical and biotechnological applications, including DNA transfection in eukaryotic cells. Still, no polymer-primed transfection of prokaryotic cells has been described. The reversible addition-fragmentation chain transfer (RAFT) polymer synthesis technique and the reversible deactivation radical polymerization variants allow the design of polymers with well-controlled molar mass, morphology, and hydrophilicity/hydrophobicity ratios. RAFT was used to synthesize two amphiphilic copolymers containing different ratios of the amphiphilic poly[2-(dimethyl-amino) ethyl methacrylate] and the hydrophobic poly [methyl methacrylate]. These copolymers bound to pUC-19 DNA and successfully transfected non-competent Escherichia coli DH5α, with transformation efficiency in the range of 103 colony-forming units per µg of plasmid DNA. These results demonstrate prokaryote transformation using polymers with controlled amphiphilic/hydrophobic ratios.

Nerve growth factor derivative NGF61/100 promotes outgrowth of primary sensory neurons with reduced signs of nociceptive sensitization.

Nerve Growth Factor (NGF) is being considered as a therapeutic candidate for Alzheimer's disease. However, the development of an NGF-based therapy is limited by its potent pain activity. We have developed a "painless" derivative form of human NGF (NGF61/100), characterized by identical neurotrophic properties but a reduced nociceptive sensitization activity in vivo. Here we characterized the response of rat dorsal root ganglia neurons (DRG) to the NGF derivative NGF61/100, in comparison to that of control NGF (NGF61), analyzing the expression of noxious pro-nociceptive mediators. NGF61/100 displays a neurotrophic activity on DRG neurons comparable to that of control NGF61, despite a reduced activation of PLCγ, Akt and Erk1/2. NGF61/100 does not differ from NGF61 in its ability to up-regulate Substance P (SP) and Calcitonin Gene Related Peptide (CGRP) expression. However, upon Bradykinin (BK) stimulation, NGF61/100-treated DRG neurons release a much lower amount of SP and CGRP, compared to control NGF61 pre-treated neurons. This effect of painless NGF is explained by the reduced up-regulation of BK receptor 2 (B2R), respect to control NGF61. As a consequence, BK treatment reduced phosphorylation of the transient receptor channel subfamily V member 1 (TRPV1) in NGF61/100-treated cultures and induced a significantly lower intracellular Ca2+ mobilization, responsible for the lower release of noxious mediators. Transcriptomic analysis of DRG neurons treated with NGF61/100 or control NGF allowed identifying a small number of nociceptive-related genes that constitute an "NGF pain fingerprint", whose differential regulation by NGF61/100 provides a strong mechanistic basis for its selective reduced pain sensitizing actions.

NH2-truncated human tau induces deregulated mitophagy in neurons by aberrant recruitment of Parkin and UCHL-1: implications in Alzheimer's disease.

Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20-22 kDa NH2-tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH2htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH2htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH2htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH2htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH2htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance.

Activity-dependent structural plasticity of Purkinje cell spines in cerebellar vermis and hemisphere.

The environmental enrichment (EE) paradigm is widely used to study experience-dependent brain plasticity. In spite of a long history of research, the EE influence on neuronal morphology has not yet been described in relation to the different regions of the cerebellum. Thus, aim of the present study was to characterize the EE effects on density and size of dendritic spines of Purkinje cell proximal and distal compartments in cerebellar vermian and hemispherical regions. Male Wistar rats were housed in an enriched or standard environment for 3.5 months from the 21st post-natal day onwards. The morphological features of Purkinje cell spines were visualized on calbindin immunofluorescence-stained cerebellar vermian and hemispherical sections. Density, area, length and head diameter of spines were manually (ImageJ) or automatically (Imaris) quantified. Results demonstrated that the Purkinje cell spine density was higher in enriched rats than in controls on both proximal and distal dendrite compartments in the hemisphere, while it increased only on distal compartment in the vermis. As for spine size, a significant increase of area, length and head diameter was found in the distal dendrites in both vermis and hemisphere. Thus, the exposure to a complex environment enhances synapse formation and plasticity either in the vermis involved in balance and locomotion and in the hemisphere involved in complex motor adaptations and acquisition of new motor strategies. These data highlight the importance of cerebellar activity-dependent structural plasticity underling the EE-related high-level performances.

Controlling the activation of the Bv8/prokineticin system reduces neuroinflammation and abolishes thermal and tactile hyperalgesia in neuropathic animals.

BACKGROUND AND PURPOSE: Chemokines are involved in neuroinflammation and contribute to chronic pain processing. The new chemokine prokineticin 2 (PROK2) and its receptors (PKR1 and PKR2 ) have a role in inflammatory pain and immunomodulation. In the present study, we investigated the involvement of PROK2 and its receptors in neuropathic pain. EXPERIMENTAL APPROACH: Effects of single, intrathecal, perineural and s.c. injections of the PKR antagonist PC1, or of 1 week s.c. treatment, on thermal hyperalgesia and tactile allodynia was evaluated in mice with chronic constriction of the sciatic nerve (CCI). Expression and localization of PROK2 and of its receptors at peripheral and central level was evaluated 10 days after CCI, following treatment for 1 week with saline or PC1. IL-1ß and IL-10 levels, along with glia activation, were evaluated. KEY RESULTS: Subcutaneous, intrathecal and perineural PC1 acutely abolished the CCI-induced hyperalgesia and allodynia. At 10 days after CCI, PROK2 and its receptor PKR2 were up-regulated in nociceptors, in Schwann cells and in activated astrocytes of the spinal cord. Therapeutic treatment with PC1 (s.c., 1 week) alleviated established thermal hyperalgesia and allodynia, reduced the injury-induced overexpression of PROK2, significantly blunted nerve injury-induced microgliosis and astrocyte activation in the spinal cord and restored the physiological levels of proinflammatory and anti-inflammatory cytokines in periphery and in spinal cord. CONCLUSION AND IMPLICATIONS: The prokineticin system contributes to pain modulation via neuron-glia interaction. Sustained inhibition of the prokineticin system, at peripheral or central levels, blocked both pain symptoms and some events underlying disease progression.

AD-linked, toxic NH2 human tau affects the quality control of mitochondria in neurons.

Functional as well as structural alterations in mitochondria size, shape and distribution are precipitating, early events in progression of Alzheimer's Disease (AD). We reported that a 20-22kDa NH2-tau fragment (aka NH2htau), mapping between 26 and 230 amino acids of the longest human tau isoform, is detected in cellular and animal AD models and is neurotoxic in hippocampal neurons. The NH2htau -but not the physiological full-length protein- interacts with Aß at human AD synapses and cooperates with it in inhibiting the mitochondrial ANT-1-dependent ADP/ATP exchange. Here we show that the NH2htau also adversely affects the interplay between the mitochondria dynamics and their selective autophagic clearance. Fragmentation and perinuclear mislocalization of mitochondria with smaller size and density are early found in dying NH2htau-expressing neurons. The specific effect of NH2htau on quality control of mitochondria is accompanied by (i) net reduction in their mass in correlation with a general Parkin-mediated remodeling of membrane proteome; (ii) their extensive association with LC3 and LAMP1 autophagic markers; (iii) bioenergetic deficits and (iv) in vitro synaptic pathology. These results suggest that NH2htau can compromise the mitochondrial biology thereby contributing to AD synaptic deficits not only by ANT-1 inactivation but also, indirectly, by impairing the quality control mechanism of these organelles.

ProNGF\NGF imbalance triggers learning and memory deficits, neurodegeneration and spontaneous epileptic-like discharges in transgenic mice.

ProNGF, the precursor of mature nerve growth factor (NGF), is the most abundant form of NGF in the brain. ProNGF and mature NGF differ significantly in their receptor interaction properties and in their bioactivity. ProNGF increases markedly in the cortex of Alzheimer's disease (AD) brains and proNGF\NGF imbalance has been postulated to play a role in neurodegeneration. However, a direct proof for a causal link between increased proNGF and AD neurodegeneration is lacking. In order to evaluate the consequences of increased levels of proNGF in the postnatal brain, transgenic mice expressing a furin cleavage-resistant form of proNGF, under the control of the neuron-specific mouse Thy1.2 promoter, were derived and characterized. Different transgenic lines displayed a phenotypic gradient of neurodegenerative severity features. We focused the analysis on the two lines TgproNGF#3 and TgproNGF#72, which shared learning and memory impairments in behavioral tests, cholinergic deficit and increased Aß-peptide immunoreactivity. In addition, TgproNGF#3 mice developed Aß oligomer immunoreactivity, as well as late diffuse astrocytosis. Both TgproNGF lines also display electrophysiological alterations related to spontaneous epileptic-like events. The results provide direct evidence that alterations in the proNGF/NGF balance in the adult brain can be an upstream driver of neurodegeneration, contributing to a circular loop linking alterations of proNGF/NGF equilibrium to excitatory/inhibitory synaptic imbalance and amyloid precursor protein (APP) dysmetabolism.

Nucleophosmin/B26 regulates PTEN through interaction with HAUSP in acute myeloid leukemia.

PTEN (phosphatase and tensin homolog deleted in chromosome 10) is a bona fide dual lipid and protein phosphatase with cytoplasmic (Cy) and nuclear localization. PTEN nuclear exclusion has been associated with tumorigenesis. Nucleophosmin (NPM1) is frequently mutated in acute myeloid leukemia (AML) and displays Cy localization in mutated nucleophosmin (NPMc+) AML. Here we show that NPM1 directly interacts with herpes virus-associated ubiquitin specific protease (HAUSP), which is known as a PTEN deubiquitinating enzyme. Strikingly, PTEN is aberrantly localized in AML carrying NPMc+. Mechanistically, NPM1 in the nucleus opposes HAUSP-mediated deubiquitination and this promotes the shuttle of PTEN to the cytoplasm. In the cytoplasm, NPMc+ prevents HAUSP from deubiquitinating PTEN, causing the latter to stay in the cytoplasm where it is polyubiquitinated and degraded. Our findings delineate a new NPM1-HAUSP molecular interaction controlling PTEN deubiquitination and trafficking.

BAG3 controls angiogenesis through regulation of ERK phosphorylation.

BAG3 is a co-chaperone of the heat shock protein (Hsp) 70, is expressed in many cell types upon cell stress, however, its expression is constitutive in many tumours. We and others have previously shown that in neoplastic cells BAG3 exerts an anti-apoptotic function thus favoring tumour progression. As a consequence we have proposed BAG3 as a target of antineoplastic therapies. Here we identify a novel role for BAG3 in regulation of neo-angiogenesis and show that its downregulation results in reduced angiogenesis therefore expanding the role of BAG3 as a therapeutical target. In brief we show that BAG3 is expressed in endothelial cells and is essential for the interaction between ERK and its phosphatase DUSP6, as a consequence its removal results in reduced binding of DUSP6 to ERK and sustained ERK phosphorylation that in turn determines increased levels of p21 and p15 and cell-cycle arrest in the G1 phase.

Electroacupuncture counteracts the development of thermal hyperalgesia and the alteration of nerve growth factor and sensory neuromodulators induced by streptozotocin in adult rats.

AIMS/HYPOTHESIS: Diabetes is considered the leading cause of neuropathies in developed countries. Dysfunction of nerve growth factor (NGF) production and/or utilisation may lead to the establishment of diabetic neuropathies. Electroacupuncture has been proved effective in the treatment of human neuropathic pain as well as in modulating NGF production/activity. We aimed at using electroacupuncture to correct the development of thermal hyperalgesia and the tissue alteration of NGF and sensory neuromodulators in a rat model of type 1 diabetes. METHODS: Adult rats were injected with streptozotocin to induce diabetes and subsequently treated with low-frequency electroacupuncture for 3 weeks. Variation in thermal sensitivity was studied during the experimental course. Hindpaw skin and spinal cord protein content of NGF, NGF receptor tyrosine kinase A (TrkA), substance P (SP), transient receptor potential vanilloid 1 (TRPV1) receptor and glutamic acid decarboxylase-67 (GAD-67) were measured after electroacupuncture treatments. The skin and spinal cord cellular distribution of TrkA was analysed to explore NGF signalling. RESULTS: Early after streptozotocin treatment, thermal hyperalgesia developed that was corrected by electroacupuncture. The parallel increases in NGF and TrkA in the spinal cord were counteracted by electroacupuncture. Streptozotocin also induced variation in skin/spinal TrkA phosphorylation, increases in skin SP and spinal TRPV1 and a decrease in spinal GAD-67. These changes were counteracted by electroacupuncture. CONCLUSIONS/INTERPRETATION: Our results point to the potential of electroacupuncture as a supportive therapy for the treatment of diabetic neuropathies. The efficacy of electroacupuncture might depend on its actions on spinal/peripheral NGF synthesis/utilisation and normalisation of the levels of several sensory neuromodulators.

Endogenous Aß causes cell death via early tau hyperphosphorylation.

Alzheimer's disease (AD) is characterized by Aß overproduction and tau hyperphosphorylation. We report that an early, transient and site-specific AD-like tau hyperphosphorylation at Ser262 and Thr231 epitopes is temporally and causally related with an activation of the endogenous amyloidogenic pathway that we previously reported in hippocampal neurons undergoing cell death upon NGF withdrawal [Matrone, C., Ciotti, M.T., Mercanti, D., Marolda, R., Calissano, P., 2008b. NGF and BDNF signaling control amyloidogenic route and Ab production in hippocampal neurons. Proc. Natl. Acad. Sci. 105, 13138-13143]. Such tau hyperphosphorylation, as well as apoptotic death, is (i) blocked by 4G8 and 6E10 Aß antibodies or by specific ß and/or γ-secretases inhibitors; (ii) temporally precedes tau cleavage mediated by a delayed (6-12h after NGF withdrawal) activation of caspase-3 and calpain-I; (iii) under control of Akt-GSK3ß-mediated signaling. Finally, we show that such site-specific tau hyperphosphorylation causes tau detachment from microtubules and an impairment of mitochondrial trafficking. These results depict, for the first time, a rapid interplay between endogenous Aß and tau post-translational modifications which act co-ordinately to compromise neuronal functions in the same neuronal system, under physiological conditions as seen in AD brain.

Short- but not long-lasting treadmill running reduces allodynia and improves functional recovery after peripheral nerve injury.

We analyzed the effects of different treadmill running protocols on the functional recovery after chronic constriction injury (CCI) of the sciatic nerve in mice. We found that a treadmill protocol of short-lasting running (1 h/d for 5 days after CCI) reduced the neuropathy-induced mechanical allodynia and normalized the weight bearing and the sciatic static index of the injured hindpaw. At difference, a treadmill protocol of long-lasting running (1 h/d for more than 5 days after CCI) was unfavorable both for allodynia and for functional recovery. Behavioral results were correlated with immunofluorescence assays of microglia and astrocytes activation in L4/L5 lumbar spinal cord sections. We found a differential pattern of activation characterized by: (i) reduced microglia expression, after both short- and long-lasting treadmill running; (ii) reduced astrocytes expression after short-lasting treadmill running; and, (iii) persistence of astrocytes expression after long-lasting treadmill running. Finally, in sections of injured sciatic nerves, we analyzed the expression of Cdc2 and GAP-43 proteins that are both up-regulated during peripheral regenerative processes. Compared to mice subjected to long-lasting treadmill running, mice subjected to short-lasting treadmill running showed an acceleration of the regenerative processes at the injured sciatic nerve. Our data demonstrate that short-lasting treadmill running, by reducing the neuropathic pain symptoms and facilitating the regenerative processes of the injured nerve, have beneficial rehabilitative effects on the functional recovery after peripheral nerve injury.

SP protects cerebellar granule cells against beta-amyloid-induced apoptosis by down-regulation and reduced activity of Kv4 potassium channels.

The tachykinin endecapeptide substance P (SP) has been demonstrated to exert a functional role in neurodegenerative disorders, including Alzheimer's disease (AD). Aim of the present study was to evaluate the SP neuroprotective potential against apoptosis induced by the neurotoxic beta-amyloid peptide (A beta) in cultured rat cerebellar granule cells (CGCs). We found that SP protects CGCs against both A beta(25-35)- and A beta(1-42)-induced apoptotic CGCs death as revealed by live/dead cell assay, Hoechst staining and caspase(s)-induced PARP-1 cleavage, through an Akt-dependent mechanism. Since in CGCs the fast inactivating or A-type K(+) current (I(KA)) was potentiated by A beta treatment through up-regulation of Kv4 subunits, we investigated whether I(KA) and the related potassium channel subunits could be involved in the SP anti-apoptotic activity. Patch-clamp experiments showed that the A beta-induced increase of I(KA) current amplitude was reversed by SP treatment. In addition, as revealed by Western blot analysis and immunofluorescence studies, SP prevented the up-regulation of Kv4.2 and Kv4.3 channel subunits expression. These results indicate that SP plays a role in the regulation of voltage-gated potassium channels in A beta-mediated neuronal death and may represent a new approach in the understanding and treatment of AD.

Remote cell death in the cerebellar system.

Functional impairment after focal CNS lesion is highly dependent on damage that occurs in regions that are remote but functionally connected to the primary lesion site. This pattern is particularly evident in the cerebellar system, in which functional interactions between the cerebellar cortex, deep cerebellar nuclei, and precerebellar stations are of paramount importance. Diffuse degeneration after development of a focal CNS lesion has been associated with poor outcomes in several pathologies, such as stroke, multiple sclerosis, and brain trauma. A greater understanding of the mechanisms that underlie the spread of death signals from focal lesions, however, can aid in identifying a neuroprotective approach for CNS pathologies. To this end, studies on degenerative mechanisms in the inferior olive and pontine nuclei after focal cerebellar damage have been a valuable asset in which pharmacological approaches have been tested. In this review, we focus on mechanisms of remote cell death in cerebellar circuits, analyzing the neuroprotective effects of inflammation-modulating drugs in particular.

Involvement of cannabinoid CB1- and CB2-receptors in the modulation of exocrine pancreatic secretion.

The role of the cannabinoid system in the regulation of exocrine pancreatic secretion was investigated by studying the effects of the synthetic CB1- and CB2-receptors agonist, WIN55,212, on amylase secretion in isolated lobules and acini of guinea pig and rat, and the expression of CB-receptors in rat pancreatic tissue by immuno-chemistry and Western-blot analysis in both basal and cerulein (CK)-induced pancreatitis condition. In pancreatic lobules of guinea pig and rat, WIN55,212 significantly inhibited amylase release stimulated by KCl depolarization through inhibition of presynaptic acetylcholine release, but did not modify basal, carbachol- or CK-stimulated amylase secretion. The effect of WIN55,212 was significantly reduced by pre-treatment with selective CB1- and CB2-receptor antagonists. The antagonists, when given alone, did not affect the KCl-evoked response. Conversely, WIN55,212 was unable to affect basal and CK- or carbachol-stimulated amylase release from pancreatic acini of guinea pig and rat. Immunofluorescent staining of rat pancreatic tissues showed that CB1- and CB2-receptors are expressed in lobules and in acinar cells and their presence in acinar cells was also shown by Western-blot analysis. After CK-induced pancreatitis, the expression of CB1-receptors in acinar cells was not changed, whilst a down-regulation of CB2-receptors was observed. In conclusion, the present study shows that WIN55,212 inhibits amylase release from guinea pig and rat pancreatic lobules and, for the first time, that cannabinoid receptors are expressed in lobules of the rat pancreas, suggesting an inhibitory presynaptic role of this receptor system. Finally, in rat pancreatic acinar cells, CB1- and CB2-receptors, expressed both in basal conditions and after CK-induced pancreatitis but inactive on amylase secretion, have an unknown role both in physiological and pathological conditions.

Minocycline attenuates microglial activation but fails to mitigate degeneration in inferior olive and pontine nuclei after focal cerebellar lesion.

Degenerative changes in areas remote from the primary lesion site have been linked to the clinical outcome of focal brain damage, and inflammatory mechanisms have been considered to play a key role in the pathogenesis of these remote cell death phenomena. Minocycline is a tetracycline derivative, therapeutically effective in various experimental models of central nervous system (CNS) injuries that include inflammatory and apoptotic mechanisms, although recent findings have yielded mixed results. In this study, we investigated the effectiveness of minocycline treatment in reducing remote cell death. Glial activation and neuronal loss in precerebellar stations following cerebellar lesion were investigated using immunohistochemistry and Western blot techniques. Our results show that minocycline was effective in reducing microglial activations in axotomized precerebellar nuclei, but failed to mitigate either astrocytic response or neuronal loss. This finding supports the role of minocycline in modulating inflammatory response after CNS lesion and suggests its ineffectiveness in influencing degenerative phenomena in areas remote from the primary lesion site.

Methylprednisolone treatment delays remote cell death after focal brain lesion.

Glucocorticoids have a prominent role in the treatment of CNS injuries. However, the cellular consequences of glucocorticoid treatment on remote degenerative responses after focal brain lesions have been poorly investigated. Here we examine the effectiveness of a high dose (50 mg/kg) of methylprednisolone sodium succinate (MPSS) in reducing neuronal loss, glial response and glial-derived inflammatory mediators in inferior olive and pontine nuclei after lesion of the contralateral cerebellar hemisphere using immunohistochemistry and Western blot techniques. Quantitative analysis demonstrated that MPSS treatment significantly improved the survival of neurons in remote precerebellar stations. This survival was accompanied by reduction in the postlesional activation of microglia, astrocytes and interleukin-1 beta (IL-1beta). Cell death resumed after suspension of MPSS treatment and this delayed wave of cell loss was paralleled by reactivation of the inflammatory markers analyzed. The present study confirms the importance of inflammatory events in inducing remote cell death and that this type of degeneration can be delayed by MPSS treatment. Furthermore, the sustained effect of MPSS treatment, up to 28 days postlesion, and the reactivation of the degenerative phenomena after its suspension, support the hypothesis that glucocorticoid treatment, although capable of delaying cell death mechanisms, is not effective in blocking the cascade of remote degenerative events started by the primary lesion.

Cortical and subcortical distribution of ionotropic purinergic receptor subunit type 1 (P2X(1)R) immunoreactive neurons in the rat forebrain.

Ionotropic purinergic receptors (P2XR) are ATP-gated cationic channels composed of seven known subunits (P2X(1-7)R) and involved in different functions in neural tissue. Although their presence has been demonstrated in the brain, few studies have investigated their expression pattern. In particular, ionotropic purinergic receptor subunit type 1 (P2X(1)R) has been observed in the cerebellum and in brainstem nuclei. The present study investigates the P2X(1)R expression pattern in the rat forebrain using immunohistochemistry. The specificity of the immunolabeling has been verified by Western blotting and in situ hybridization methods. P2X(1)R immunoreactivity was specifically localized in neurons, dendrites and axons throughout the forebrain. Characteristic differences in the distribution of P2X(1)R were observed in different cortical areas. In prefrontal, cingulate and perirhinal cortices, very intense labeling was present in neuronal bodies. In frontal, parietal, temporal and occipital cortices, immunostaining was lighter and mainly found in dendrites and axons. The hippocampal formation was intensely labeled. Labeling was present almost exclusively in dendrites and axons and never in neuronal bodies. The diencephalon was devoid of P2X(1)R positive neurons or fibers except for the medial habenular nucleus, which showed very intense P2X(1)R immunostaining. Furthermore, two subcortical regions, namely, the nucleus centralis of the amygdala and the bed nucleus of the stria terminalis, showed intense P2X(1)R neuronal labeling. Present data indicate that P2X(1)R are prevalent in forebrain areas involved in the integration of cognitive, limbic and autonomic functions.

Do ATP and NO interact in the CNS?

Enzymatically derived NO and extracellular ATP are receiving greater attention due to their role as messengers in the CNS during different physiological and pathological processes. Ionotropic (P2XR) and metabotropic (P2YR) purinergic receptors mediate ATP effects and are present throughout the body. Particularly P2XR are crucial for brain plasticity mechanisms, and are involved in the pathogenesis of different CNS illnesses. NO does not have a specific receptor and its actions are directly dependent on the production on demand by different nitric oxide synthase isoforms. NO synthesizing enzymes are present virtually in all tissues, and NO influences multifarious physiological and pathological functions. Interestingly, various are the tissue and organs modulated by both ATP and NO, such as the immune, brain and vascular systems. Moreover, direct interactions between purinergic and nitrergic mechanisms outside the CNS are well documented, with several studies also indicating that ATP and NO do participate to the same CNS functions. In the past few years, further experimental evidence supported the physiological and pathological relevance of ATP and NO direct interactions in the CNS. The aim of the present review is to provide an account of the available information on the interplay between purinergic and nitrergic systems, focussing on the CNS. The already established relevance of ATP and NO in different pathological processes would predict that the knowledge of ATP/NO cross-talk mechanisms would support pharmacological approaches toward the development of novel ATP/NO combined pharmacological agents.