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Cell membranes isolated from brain tissues, obtained surgically from six patients afflicted with drug-resistant temporal lobe epilepsy and from one nonepileptic patient afflicted with a cerebral oligodendroglioma, were injected into frog oocytes. By using this approach, the oocytes acquire human GAB[A.sub.A] receptors, and we have shown previously that the 'epileptic receptors' (receptors transplanted from epileptic brains) display a marked run-down during repetitive applications of GABA. It was found that exposure to the neurotrophin BDNF increased the amplitude of the 'GABA currents' (currents elicited by GABA) generated by the epileptic receptors and decreased their run-down; both events being blocked by K252A, a neurotrophin tyrosine kinase receptor B inhibitor. These effects of BDNF were not mimicked by nerve growth factor. In contrast, the GAB[A.SUB.A] receptors transplanted from the nonepileptic human hippocampal uncus (obtained during surgical resection as part of the nontumoral tissue from the oligodendroglioma margins) or receptors expressed by injecting rat recombinant [alpha]1[beta]2[gamma]2 GAB[A.SUB.A] receptor subunit cDNAs generated GABA currents whose time-course and run-down were not altered by BDNF. Loading the oocytes with the [Ca.sup.2+] chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate-acetoxymethyl ester (BAPTA-AM), or treating them with Rp-8-Br-cAMP, an inhibitor of the cAMP-dependent PKA, did not alter the GABA currents. However, staurosporine (a broad spectrum PK inhibitor), bisindolylmaleimide I (a PKC inhibitor), and U73122 (a phospholipase C inhibitor) blocked the BDNF-induced effects on the epileptic GABA currents. Our results indicate that BDNF potentiates the epileptic GAB[A.SUB.A] currents and antagonizes their use-dependent run-down, thus strengthening GABAergic inhibition, probably by means of activation of tyrosine kinase receptor B receptors and of both PLC and PKC. microtransplantation into Xenopus oocytes | temporal lobe epilepsy

The properties of [gamma]-aminobutyric acid (GABA) type A receptors (GAB[A.sub.A] receptors) microtransplanted from the human epileptic brain to the plasma membrane of Xenopus oocytes were compared with those recorded directly from neurons, or glial cells, in human brains slices. Cell membranes isolated from brain specimens, surgically obtained from six patients afflicted with drug-resistant temporal lobe epilepsy (TLE) were injected into frog oocytes. Within a few hours, these oocytes acquired GAB[A.sub.A] receptors that generated GABA currents with an unusual run-down, which was inhibited by orthovanadate and okadaic acid. In contrast, receptors derived from membranes of a nonepileptic hippocampal uncus, membranes from mouse brain, or recombinant rat [alpha]1[beta]2[gamma]2-GABA receptors exhibited a much less pronounced GABA-current rundown. Moreover, the GAB[A.sub.A] receptors of pyramidal neurons in temporal neocortex slices from the same six epileptic patients exhibited a stronger run-down than the receptors of rat pyramidal neurons. Interestingly, the GAB[A.sub.A] receptors of neighboring glial cells remained substantially stable after repetitive activation. Therefore, the excessive GABA-current run-down observed in the membrane-injected oocytes recapitulates essentially what occurs in neurons, rather than in glial cells. Quantitative RT-PCR analyses from the same TLE neocortex specimens revealed that GAB[A.sub.A]receptor [beta]1, [beta]2, [beta]3, and [gamma]2 subunit mRNAs were significantly overexpressed (8- to 33-fold) compared with control autopsy tissues. Our results suggest that an abnormal GABA-receptor subunit transcription in the TLE brain leads to the expression of run-down-enhanced GAB[A.sub.A] receptors. Blockage of phospbatases stabilizes the TLE GAB[A.sub.A] receptors and strengthens GABAergic inhibition. It may be that this process can be targeted to develop new treatments for intractable epilepsy. temporal lobe epilepsy | microtransplantation into Xenopus oocyte | okadaic acid | [gamma]-aminobutyric acid-current run-down | human tissue slices

Cell membranes isolated from brain tissues, obtained surgically from six patients afflicted with drug-resistant temporal lobe epilepsy and from one nonepileptic patient afflicted with a cerebral oligodendroglioma, were injected into frog oocytes. By using this approach, the oocytes acquire human GABAA receptors, and we have shown previously that the "epileptic receptors" (receptors transplanted from epileptic brains) display a marked run-down during repetitive applications of GABA. It was found that exposure to the neurotrophin BDNF increased the amplitude of the "GABA currents" (currents elicited by GABA) generated by the epileptic receptors and decreased their run-down; both events being blocked by K252A, a neurotrophin tyrosine kinase receptor B inhibitor. These effects of BDNF were not mimicked by nerve growth factor. In contrast, the GABAA receptors transplanted from the nonepileptic human hippocampal uncus (obtained during surgical resection as part of the nontumoral tissue from the oligo- dendroglioma margins) or receptors expressed by injecting rat recombinant αβ27gamma;2 GABAA receptor subunit cDNAs generated GABA currents whose time-course and run-down were not altered by BDNF. Loading the oocytes with the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N' -tetraacetate-acetoxymethyl ester (BAPTA-AM), or treating them with Rp-8-Br-cAMP, an inhibitor of the cAMP-dependent PKA, did not alter the GABA currents. However, staurosporine (a broad spectrum PK inhibitor), bisindolylmaleimide I (a PKC inhibitor), and U73122 (a phospholipase C inhibitor) blocked the BDNF-induced effects on the epileptic GABA currents. Our results indicate that BDNF potentiates the epileptic GABAA currents and antagonizes their use-dependent run-down, thus strengthening GABAergic inhibition, probably by means of activation of tyrosine kinase receptor B receptors and of both PLC and PKC. [ABSTRACT FROM AUTHOR]

In recent years, several studies described the close relationship between the composition of gut microbiota and brain functions, highlighting the importance of gut-derived metabolites in mediating neuronal and glial cells cross-talk in physiological and pathological condition. Gut dysbiosis may affects cerebral tumors growth and progression, but the specific metabolites involved in this modulation have not been identified yet. Using a syngeneic mouse model of glioma, we have investigated the role of dysbiosis induced by the administration of non-absorbable antibiotics on mouse metabolome and on tumor microenvironment. We report that antibiotics treatment induced: (1) alteration of the gut and brain metabolome profiles; (2) modeling of tumor microenvironment toward a pro-angiogenic phenotype in which microglia and glioma cells are actively involved; (3) increased glioma stemness; (4) trans-differentiation of glioma cells into endothelial precursor cells, thus increasing vasculogenesis. We propose glycine as a metabolite that, in ABX-induced dysbiosis, shapes brain microenvironment and contributes to glioma growth and progression., (© 2024. The Author(s).)

Astrocytes are highly plastic cells whose activity is essential to maintain the cerebral homeostasis, regulating synaptogenesis and synaptic transmission, vascular and metabolic functions, ions, neuro- and gliotransmitters concentrations. In pathological conditions, astrocytes may undergo transient or long-lasting molecular and functional changes that contribute to disease resolution or exacerbation. In recent years, many studies demonstrated that non-neoplastic astrocytes are key cells of the tumor microenvironment that contribute to the pathogenesis of glioblastoma, the most common primary malignant brain tumor and of secondary metastatic brain tumors. This Mini Review covers the recent development of research on non-neoplastic astrocytes as tumor-modulators. Their double-edged capability to promote cancer progression or to represent potential tools to counteract brain tumors will be discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Catalano, Limatola and Trettel.)

Gut microorganisms and the products of their metabolism thoroughly affect host brain development, function and behavior. Since alterations of brain plasticity and cognition have been demonstrated upon motor, sensorial and social enrichment of the housing conditions, we hypothesized that gut microbiota and metabolome could be altered by environmental stimuli, providing part of the missing link among environmental signals and brain effects. In this preliminary study, metagenomic and metabolomic analyses of mice housed in different environmental conditions, standard and enriched, identify environment-specific microbial communities and metabolic profiles. We show that mice housed in an enriched environment have distinctive microbiota composition with a reduction in gut bacterial richness and biodiversity and are characterized by a metabolomic fingerprint with the increase of formate and acetate and the decrease of bile salts. We demonstrate that mice treated with a mixture of formate and acetate recapitulate some of the brain plasticity effects modulated by environmental enrichment, such as hippocampal neurogenesis, neurotrophin production, short-term plasticity and cognitive behaviors, that can be further exploited to decipher the mechanisms involved in experience-dependent brain plasticity., (© 2022. The Author(s).)

Glioblastoma (GBM) is the most aggressive form of glioma tumor in adult brain. Among the numerous factors responsible for GBM cell proliferation and invasion, neurotransmitters such as dopamine, serotonin and glutamate can play key roles. Studies performed in mice housed in germ-free (GF) conditions demonstrated the relevance of the gut-brain axis in a number of physiological and pathological conditions. The gut-brain communication is made possible by vagal/nervous and blood/lymphatic routes and pave the way for reciprocal modulation of functions. The gut microbiota produces and consumes a wide range of molecules, including neurotransmitters (dopamine, norepinephrine, serotonin, gamma-aminobutyric acid [GABA], and glutamate) that reach their cellular targets through the bloodstream. Growing evidence in animals suggests that modulation of these neurotransmitters by the microbiota impacts host neurophysiology and behavior, and affects neural cell progenitors and glial cells, along with having effects on tumor cell growth. In this review we propose a new perspective connecting neurotransmitter modulation by gut microbiota to glioma progression.

In the CNS, chemokines and chemokine receptors are involved in pleiotropic physiological and pathological activities. Several evidences demonstrated that chemokine signaling in the CNS plays key homeostatic roles and, being expressed on neurons, glia and endothelial cells, chemokines mediate the bidirectional cross-talk among parenchymal cells. An efficient communication between neurons and glia is crucial to establish and maintain a healthy brain environment which ensures normal functionality. Glial cells behave as active sensors of environmental changes induced by neuronal activity or detrimental insults, supporting and exerting neuroprotective activities. In this review we summarize the evidence that chemokines (CXCL12, CX3CL1, CXCL16 and CCL2) modulate neuroprotective processes upon different noxious stimuli and participate to orchestrate neurons-microglia-astrocytes action to preserve and limit brain damage. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries., (Copyright © 2019 IBRO. Published by Elsevier Ltd. All rights reserved.)

Objective: To investigate the molecular cause(s) underlying a severe form of infantile-onset parkinsonism and characterize functionally the identified variants., Methods: A trio-based whole exome sequencing (WES) approach was used to identify the candidate variants underlying the disorder. In silico modeling, and in vitro and in vivo studies were performed to explore the impact of these variants on protein function and relevant cellular processes., Results: WES analysis identified biallelic variants in WARS2, encoding the mitochondrial tryptophanyl tRNA synthetase (mtTrpRS), a gene whose mutations have recently been associated with multiple neurological phenotypes, including childhood-onset, levodopa-responsive or unresponsive parkinsonism in a few patients. A substantial reduction of mtTrpRS levels in mitochondria and reduced OXPHOS function was demonstrated, supporting their pathogenicity. Based on the infantile-onset and severity of the phenotype, additional variants were considered as possible genetic modifiers. Functional assessment of a selected panel of candidates pointed to a de novo missense mutation in CHRNA6, encoding the α6 subunit of neuronal nicotinic receptors, which are involved in the cholinergic modulation of dopamine release in the striatum, as a second event likely contributing to the phenotype. In silico, in vitro (Xenopus oocytes and GH4C1 cells) and in vivo (C. elegans) analyses demonstrated the disruptive effects of the mutation on acetylcholine receptor structure and function., Conclusion: Our findings consolidate the association between biallelic WARS2 mutations and movement disorders, and suggest CHRNA6 as a genetic modifier of the phenotype., Competing Interests: Declarations of competing interest The authors report no conflicts of interest relevant to the manuscript., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In this chapter we describe the state of the art knowledge of the role played by myeloid cells in promoting and supporting the growth and the invasive properties of a deadly brain tumor, glioblastoma. We provide a review of the works describing the intercellular communication among glioma and associated microglia/macrophage cells (GAMs) using in vitro cellular models derived from mice, rats and human patients and in vivo animal models using syngeneic or xenogeneic experimental systems. Special emphasis will be given to 1) the timing alteration of brain microenvironment under the influence of glioma, 2) the bidirectional communication among tumor and GAMs, 3) possible approaches to interfere with or to guide these interactions, with the aim to identify molecular and cellular targets which could revert or delay the vicious cycle that favors tumor biology.

Microglia are patrolling cells that sense changes in the brain microenvironment and respond acquiring distinct phenotypes that can be either beneficial or detrimental for brain homeostasis. Anti-inflammatory microglia release soluble factors that might promote brain repair; however, in glioma, anti-inflammatory microglia dampen immune response and promote a brain microenvironment that foster tumor growth and invasion. The chemokine CXCL16 is expressed in the brain, where it is neuroprotective against brain ischemia, and it has been found to be over-expressed in glioblastoma (GBM). Considering that CXCL16 specific receptor CXCR6 is diffusely expressed in the brain including in microglia cells, we wanted to investigate the role of CXCL16 in the modulation of microglia cell activity and phenotype, and in the progression of glioma. Here we report that CXCL16 drives microglia polarization toward an anti-inflammatory phenotype, also restraining microglia polarization toward an inflammatory phenotype upon LPS and IFNγ stimulation. In the context of glioma, we demonstrate that CXCL16 released by tumor cells is determinant in promoting glioma associated microglia/macrophages (GAMs) modulation toward an anti-inflammatory/pro-tumor phenotype, and that cxcr6ko mice, orthotopically implanted into the brain with GL261 glioma cells,survive longer compared to wild-type mice. We also describe that CXCL16/CXCR6 signaling acts directly on mouse glioma cells, as well as human primary GBM cells, promoting tumor cell growth, migration and invasion. All together these data suggest that CXCL16 signaling could represent a good target to modulate microglia phenotype in order to restrain inflammation or to limit glioma progression.

Oleandrin is a glycoside that inhibits the ubiquitous enzyme Na + /K + -ATPase. In addition to its known effects on cardiac muscle, recent in vitro and in vivo evidence highlighted its potential for anticancer properties. Here, we evaluated for the first time the effect of oleandrin on brain tumors. To this aim, mice were transplanted with human or murine glioma and analyzed for tumor progression upon oleandrin treatment. In both systems, oleandrin impaired glioma development, reduced tumor size, and inhibited cell proliferation. We demonstrated that oleandrin does the following: (1) enhances the brain-derived neurotrophic factor (BDNF) level in the brain; (2) reduces both microglia/macrophage infiltration and CD68 immunoreactivity in the tumor mass; (3) decreases astrogliosis in peritumoral area; and (4) reduces glioma cell infiltration in healthy parenchyma. In BDNF-deficient mice (bdnftm1Jae/J) and in glioma cells silenced for TrkB receptor expression, oleandrin was not effective, indicating a crucial role for BDNF in oleandrin's protective and antitumor functions. In addition, we found that oleandrin increases survival of temozolomide-treated mice. These results encourage the development of oleandrin as possible coadjuvant agent in clinical trials of glioma treatment. SIGNIFICANCE STATEMENT In this work, we paved the road for a new therapeutic approach for the treatment of brain tumors, demonstrating the potential of using the cardioactive glycoside oleandrin as a coadjuvant drug to standard chemotherapeutics such as temozolomide. In murine models of glioma, we demonstrated that oleandrin significantly increased mouse survival and reduced tumor growth both directly on tumor cells and indirectly by promoting an antitumor brain microenvironment with a key protective role played by the neurotrophin brain-derived neurotrophic factor., (Copyright © 2017 the authors 0270-6474/17/373926-14$15.00/0.)

Chemokines have several physio-pathological roles in the brain. Among them, the modulation of synaptic contacts and neurotransmission recently emerged as crucial activities during brain development, in adulthood, upon neuroinflammation and neurodegenerative diseases. CXCL16 is a chemokine normally expressed in the brain, where it exerts neuroprotective activity against glutamate-induced damages through cross communication with astrocytes and the involvement of the adenosine receptor type 3 (A3R) and the chemokine CCL2. Here we demonstrated for the first time that CXCL16 exerts a modulatory activity on inhibitory and excitatory synaptic transmission in CA1 area. We found that CXCL16 increases the frequency of the miniature inhibitory synaptic currents (mIPSCs) and the paired-pulse ratio (PPR) of evoked IPSCs (eIPSCs), suggesting a presynaptic modulation of the probability of GABA release. In addition, CXCL16 increases the frequency of the miniature excitatory synaptic currents (mEPSCs) and reduces the PPR of evoked excitatory transmission, indicating that the chemokine also modulates and enhances the release of glutamate. These effects were not present in the A3RKO mice and in WT slices treated with minocycline, confirming the involvement of A3 receptors and introducing microglial cells as key mediators of the modulatory activity of CXCL16 on neurons.

Adenosine is a widespread neuromodulator within the CNS and its extracellular level is increased during hypoxia or intense synaptic activity, modulating pre- and postsynaptic sites. We studied the neuromodulatory action of adenosine on glutamatergic currents in the hippocampus, showing that activation of multiple adenosine receptors (ARs) by basal adenosine impacts postsynaptic site. Specifically, the stimulation of both A1R and A3R reduces AMPA currents, while A2AR has an opposite potentiating effect. The effect of ARs stimulation on glutamatergic currents in hippocampal cultures was investigated using pharmacological and genetic approaches. A3R inhibition by MRS1523 increased GluR1-Ser845 phosphorylation and potentiated AMPA current amplitude, increasing the apparent affinity for the agonist. A similar effect was observed blocking A1R with DPCPX or by genetic deletion of either A3R or A1R. Conversely, impairment of A2AR reduced AMPA currents, and decreased agonist sensitivity. Consistently, in hippocampal slices, ARs activation by AR agonist NECA modulated glutamatergic current amplitude evoked by AMPA application or afferent fiber stimulation. Opposite effects of AR subtypes stimulation are likely associated to changes in GluR1 phosphorylation and represent a novel mechanism of physiological modulation of glutamatergic transmission by adenosine, likely acting in normal conditions in the brain, depending on the level of extracellular adenosine and the distribution of AR subtypes.

Fractalkine (CX3CL1) is an intriguing chemokine that plays a central role in the nervous system. The expression of CX3CL1 on neurons and of its receptor CX3CR1 on microglia facilitates a privileged interaction, playing important roles in regulating the function and maturation of these cells. CX3CL1 is reported to have neuroprotective and anti-inflammatory activities in several experimental systems and animal models of disease, and its expression correlates with positive outcomes in human neuropathologies. However, a comparable amount of evidence shows that CX3CL1 sustains neuroinflammatory conditions and contributes to neurotoxicity. This review discusses the evidence in favor of the CX3CL1/CX3CR1 pair being neuroprotective and other evidence that it is neurotoxic. Our aim is to stimulate future research examining the molecular and cellular determinants responsible for this unique functional switch, which could be important for several neuropathologies., (© 2015 New York Academy of Sciences.)

Neuronal death induced by overactivation of N-methyl-d-aspartate receptors (NMDARs) is implicated in the pathophysiology of many neurodegenerative diseases such as stroke, epilepsy and traumatic brain injury. This toxic effect is mainly mediated by NR2B-containing extrasynaptic NMDARs, while NR2A-containing synaptic NMDARs contribute to cell survival, suggesting the possibility of therapeutic approaches targeting specific receptor subunits. We report that fractalkine/CX3CL1 protects hippocampal neurons from NMDA-induced cell death with a mechanism requiring the adenosine receptors type 2A (A2AR). This is different from CX3CL1-induced protection from glutamate (Glu)-induced cell death, that fully depends on A1R and requires in part A3R. We show that CX3CL1 neuroprotection against NMDA excitotoxicity involves D-serine, a co-agonist of NR2A/NMDAR, resulting in cyclic AMP-dependent transcription factor cyclic-AMP response element-binding protein (CREB) phosphorylation.

Upon noxious insults, cells of the brain parenchyma activate endogenous self-protective mechanisms to counteract brain damage. Interplay between microglia and astrocytes can be determinant to build a physiological response to noxious stimuli arisen from injury or stress, thus understanding the cross talk between microglia and astrocytes would be helpful to elucidate the role of glial cells in endogenous protective mechanisms and might contribute to the development of new strategy to mobilize such program and reduce brain cell death. Here we demonstrate that chemokines CX3CL1 and CXCL16 are molecular players that synergistically drive cross-talk between neurons, microglia and astrocytes to promote physiological neuroprotective mechanisms that counteract neuronal cell death due to ischemic and excitotoxic insults. In an in vivo model of permanent middle cerebral artery occlusion (pMCAO) we found that exogenous administration of soluble CXCL16 reduces ischemic volume and that, upon pMCAO, endogenous CXCL16 signaling restrains brain damage, being ischemic volume reduced in mice that lack CXCL16 receptor. We demonstrated that CX3CL1, acting on microglia, elicits CXCL16 release from glia and this is important to induce neroprotection since lack of CXCL16 signaling impairs CX3CL1 neuroprotection against both in vitro Glu-excitotoxic insult and pMCAO. Moreover the activity of adenosine receptor A3R and the astrocytic release of CCL2 play also a role in trasmembrane chemokine neuroprotective effect, since their inactivation reduces CX3CL1- and CXCL16 induced neuroprotection.

A role for chemokines as molecules mediating neuron-glia cross talk has emerged in recent years, both in physiological and pathological conditions. We demonstrate here for the first time that the chemokine CXCL16 and its unique receptor CXCR6 are functionally expressed in the CNS, and induce neuroprotection against excitotoxic damage due to excessive glutamate (Glu) exposure and oxygen glucose deprivation (OGD). In mice and rats we found that, to exert neuroprotection, CXCL16 requires the presence of extracellular adenosine (ADO), and that pharmacological or genetic inactivation of the ADO A(3) receptor, A(3)R, prevents CXCL16 effect. In experiments with astrocytes cocultured with cxcr6(gfp/gfp) hippocampal cells, we demonstrate that CXCL16 acts directly on astrocytes to release soluble factors that are essential to mediate neuroprotection. In particular, we report that (1) upon stimulation with CXCL16 astrocytes release monocyte chemoattractant protein-1/CCL2 and (2) the neuroprotective effect of CXCL16 is reduced in the presence of neutralizing CCL2 antibody. In conclusion, we found that chemokine CXCL16 is able to mediate cross talk between astrocytes and neighboring neurons and, in pathological conditions such as excessive Glu or OGD exposure, is able to counteract neuronal cell death through an ADO-dependent chemokine-induced chemokine-release mechanism.

Adenosine modulates the function of nicotinic ACh receptors (nAChRs) in a variety of preparations, possibly through pathways involving protein kinase A (PKA), but these phenomena have not yet been investigated in detail. In this work we studied, using the patch clamp technique, the functional modulation of recombinant human α3β4 nAChR by the A2A adenosine receptor, co-expressed in HEK cells. Tonic activation of A2A receptor slowed current decay during prolonged applications of nicotine and accelerated receptor recovery from desensitization. Together, these changes resulted into a more sustained current response upon multiple nicotine or ACh applications. These findings were confirmed in cultured mouse superior cervical ganglion neurones, which express nAChR containing the α3 subunit together with β2 and/or β4 and A2A receptor. Expression of the A2A receptor in HEK cells also increased the apparent potency of nAChR for nicotine, further supporting a general A2A-induced gain of function for nAChR. These effects were dependent on PKA since the direct activation of PKA mimicked, and its inhibition prevented almost completely, the effects of the A2A receptor. Mutations of R385 and S388 in the cytoplasmic loop of the α3 subunit abolished the functional modulation of nAChR induced by activation of A2A receptor, PKA and other Ser/Thr kinases, suggesting that this region constitutes a putative consensus site for these kinases. These data provide conclusive evidence that activation of the A2A receptor determines functional changes

Recently identified mutations in the genes encoding the neuronal nicotinic ACh receptor (nAChR) subunits in patients affected by sporadic amyotrophic lateral sclerosis (sALS) may represent a factor which enhances disease susceptibility, in particular in association with ambient causes such as cigarette smoking. In this work, we characterize the functional properties of nAChRs containing the β4R349C subunit, the mutation most frequently encountered in sALS patients. The mutation was coexpressed with wild-type α3 or α4 subunits or with mutant α4R487Q subunit, which has been detected in one patient together with β4R349C mutation. None of the functional parameters examined showed differences between α4β4 and α4R487Qβ4 nAChRs. By contrast, β4R349C mutation, independent of the companion α subunit, caused the reduction in potency of both ACh and nicotine, decreased the density of whole-cell current evoked by maximal transmitter concentrations, and altered the kinetics of ACh-evoked whole-cell currents. These data confirm that sALS-associated mutations in nicotinic subunits may markedly perturb cholinergic transmission in individuals bearing the mutations.

Fractalkine/CX3CL1 is a neuron-associated chemokine, which modulates microglia-induced neurotoxicity activating the specific and unique receptor CX3CR1. CX3CL1/CX3CR1 interaction modulates the release of cytokines from microglia, reducing the level of tumor necrosis factor-alpha, interleukin-1-beta, and nitric oxide and induces the production of neurotrophic substances, both in vivo and in vitro. We have recently shown that blocking adenosine A(1) receptors (A(1)R) with the specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) abolishes CX3CL1-mediated rescue of neuronal excitotoxic death and that CX3CL1 induces the release of adenosine from microglia. In this study, we show that the presence of extracellular adenosine is mandatory for the neurotrophic effect of CX3CL1 as reducing adenosine levels in hippocampal cultures, by adenosine deaminase treatment, strongly impairs CX3CL1-mediated neuroprotection. Furthermore, we confirm the predominant role of microglia in mediating the neuronal effects of CX3CL1, because the selective depletion of microglia from hippocampal cultures treated with clodronate-filled liposomes causes the complete loss of effect of CX3CL1. We also show that hippocampal neurons obtained from A(1)R(-/-) mice are not protected by CX3CL1 whereas A(2A)R(-/-) neurons are. The requirement of functional A(1)R for neuroprotection is not unique for CX3CL1 as A(1)R(-/-) hippocampal neurons are not rescued from Glu-induced cell death by other neurotrophins such as brain-derived neurotrophic factor and erythropoietin, which are fully active on wt neurons.

We have examined how the chemokine fractalkine/CX(3)CL1 influences long-term potentiation (LTP) in CA1 mouse hippocampal slices. Field potentials (fEPSPs) were recorded upon electrical stimulation of Schaffer collaterals. It was found that application of CX(3)CL1 inhibits LTP when present during the critical induction period. LTP impairment (i) failed to occur in CX(3)CR1 deficient mice (CX(3)CR1(GFP/GFP)) and in the presence of okadaic acid (OA); (ii) required the activation of adenosine receptor 3 (A(3)R), since it was prevented in A(3)R-deficient mice or by MRS1523, a selective A(3)R antagonist. Together, these findings indicate that CX(3)CL1 inhibits hippocampal LTP through A(3)R activity.

The chemokine interleukin 8/CXCL8 induces the phosphorylation of the GluR1 subunit of the AMPA-type glutamate receptor in neurons and transfected HEK cells, on both serine 845 (S845) and 831 (S831) residues. We previously described that CXCL8 receptor CXCR2 and GluR1 co-precipitate and that GluR1/CXCR2 co-expression both in HEK cells and neurons impairs CXCL8-induced cell migration. Here we show that replacement of S845 with Ala (A), but not with Glu (E), strongly reduces GluR1/CXCR2 interaction and abolishes the impairment of CXCL8-induced cell migration. Considered together our findings point to the phosphorylated state of S845GluR1 as a determinant of GluR1-CXCR2 physical coupling.

Fractalkine/CX3CL1 and its specific receptor CX3CR1 are constitutively expressed in several regions of the CNS and are reported to mediate neuron-microglial interaction, synaptic transmission, and neuronal protection from toxic insults. CX3CL1 is released both by neuronal and astrocytic cells, whereas CX3CR1 is mainly expressed by microglial cells and neurons. Microglial cells efficiently migrate in response to CX3CL1, whereas no evidence is reported to date on CX3CL1-induced neuronal migration. For this reason, we have investigated in vitro the effects of CX3CL1 on basal migration of neurons and of the microglial and astrocytic populations, all these cells being obtained from the hippocampus and the cerebellum of newborn rats. We report that CX3CL1 stimulates microglial cell migration but efficiently reduces basal neuronal movement, regardless of the brain source. The effect of CX3CL1 is pertussis toxin (PTX) sensitive and PI3K dependent on hippocampal neurons, while it is PTX sensitive, PI3K dependent, and ERK dependent on cerebellar granules. Interestingly, CX3CL1 also increases neuron adhesion to the extracellular matrix component laminin, with mechanisms dependent on PTX-sensitive G proteins, and on the ERK and PI3K pathways. Both the reduction of migration and the increase of neuron adhesion require the activation of the beta(1) and alpha(6) integrin subunits with the exception of cerebellar neuron migration, which is only dependent on the beta(1) subunit. More importantly, in neurons, CX3CL1/CXCL12 cotreatment abolished the effect mediated by a single chemokine on chemotaxis and adhesion. In conclusion, our findings indicate that CX3CL1 reduces neuronal migration by increasing cell adhesion through integrin-dependent mechanisms in hippocampal and cerebellar neurons.

We examined the role of cysteine (Cys) residues present in chemokine receptor CXCR2 for proper surface expression, dimerization, signaling, and chemotaxis. To address this issue, serine or leucine residues were substituted for Cys, generating nine CXCR2 mutants transiently expressed in HEK cells. Single substitution of Cys residues present in the three extracellular loops (C119L, C196L, C286S) or in the seventh-transmembrane (TM) domain (C308L) abolished CXCL8 agonist binding, while no Cys substitution abolished surface receptor expression. We have previously demonstrated that CXCR2 dimerizes under reducing conditions, due to hydrophobic interactions that involve TM3 regions, and here we show that the dimer/monomer CXCR2 ratio drastically increases when analyzed under non-reducing conditions. We report that none of the Cys-deficient CXCR2 mutants abolishes receptor dimerization, demonstrating that Cys-Cys bonds are not the exclusive determinant of CXCR2 dimerization. Furthermore, both wt- and Cys-mutated CXCR2 dimers are expressed at the cell surface, indicating that receptor dimers are efficiently transferred at the plasma membrane. We also show that every Cys substitution in CXCR2, including those that still bind CXCL8, results in an impairment of receptor activity, analyzed as cell chemotaxis and intracellular signaling, suggesting that some structural requirement is likely fulfilled by Cys presence.

Homo- and hetero-oligomerization have been reported for several G protein-coupled receptors (GPCRs). The CXCR2 is a GPCR that is activated, among the others, by the chemokines CXCL8 (interleukin-8) and CXCL2 (growth-related gene product beta) to induce cell chemotaxis. We have investigated the oligomerization of CXCR2 receptors expressed in human embryonic kidney cells and generated a series of truncated mutants to determine whether they could negatively regulate the wild-type (wt) receptor functions. CXCR2 receptor oligomerization was also studied by coimmunoprecipitation of green fluorescent protein- and V5-tagged CXCR2. Truncated CXCR2 receptors retained their ability to form oligomers only if the region between the amino acids Ala-106 and Lys-163 was present. In contrast, all of the deletion mutants analyzed were able to form heterodimers with the wt CXCR2 receptor, albeit with different efficiency, competing for wt/wt dimer formation. The truncated CXCR2 mutants were not functional and, when coexpressed with wt CXCR2, interfered with receptor functions, impairing cell signaling and chemotaxis. When CXCR2 was expressed with the AMPA-type glutamate receptor GluR1, CXCR2 dimerization was again impaired in a dose-dependent way, and receptor functions were prejudiced. In contrast, CXCR1, a chemokine receptor that shares many similarities with CXCR2, did not dimerize alone or with CXCR2 and when coexpressed with CXCR2 did not impair receptor signaling and chemotaxis. The formation of CXCR2 dimers was also confirmed in cerebellar neuron cells. Taken together, we conclude from these studies that CXCR2 functions as a dimer and that truncated receptors negatively modulate receptor activities competing for the formation of wt/wt dimers.

The Xenopus oocyte is used as a convenient cell expression system to study the structure and function of heterogenic transmitter receptors and ion channels. Recently, we introduced a method to microtransplant already assembled neurotransmitter receptors from the human brain to the plasma membrane of Xenopus oocytes. The same approach was used here to transplant neurotransmitter receptors expressed from cultured cells to the oocytes. Membrane vesicles prepared from a human embryonic kidney cell line (HEK293) stably expressing the rat glutamate receptor 1 were injected into oocytes, and, within a few hours, the oocyte plasma membrane acquired alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptors, which had the same properties as those expressed in the original HEK cells. Analogously, oocytes injected with membranes prepared from rat pituitary GH(4)C1 cells, stably expressing homomeric human neuronal alpha 7 nicotinic acetylcholine receptors (alpha 7-AcChoRs), incorporated in their plasma membrane AcChoRs that behaved as those expressed in GH(4)C1 cells. Similar results were obtained with HEK cells stably expressing heteromeric human neuronal alpha 4 beta 2-AcChoRs. All this makes the Xenopus oocyte a powerful tool for detailed investigations of receptors and other proteins expressed in the membrane of cultured cells.

Defects in gene transcription and mitochondrial function have been implicated in the dominant disease process that leads to the loss of striatal neurons in Huntington's disease (HD). Here we have used precise genetic HD mouse and striatal cell models to investigate the hypothesis that decreased cAMP responsive element (CRE)-mediated gene transcription may reflect impaired energy metabolism. We found that reduced CRE-signaling in Hdh(Q111) striatum, monitored by brain derived neurotrophic factor and phospho-CRE binding protein (CREB), predated inclusion formation. Furthermore, cAMP levels in Hdh(Q111) striatum declined from an early age (10 weeks), and cAMP was significantly decreased in HD postmortem brain and lymphoblastoid cells, attesting to a chronic deficit in man. Reduced CRE-signaling in cultured STHdh(Q111) striatal cells was associated with cytosolic CREB binding protein that mirrored diminished cAMP synthesis. Moreover, mutant cells exhibited mitochondrial respiratory chain impairment, evidenced by decreased ATP and ATP/ADP ratio, impaired MTT conversion and heightened sensitivity to 3-nitropropionic acid. Thus, our findings strongly suggest that impaired ATP synthesis and diminished cAMP levels amplify the early HD disease cascade by decreasing CRE-regulated gene transcription and altering energy dependent processes essential to neuronal cell survival.

We compared the signal transduction pathways activated by stromal cell-derived factor-1 (CXCL12) chemokine in two different cell systems: primary cultures of rat cerebellar granule neurons (CGN) and human neuroepithelioma CHP100 cells. Both cell types express functional CXC chemokine receptor 4 (CXCR4), which is coupled both to extracellular signal-regulated kinase (ERK) and Akt phosphorylation pathways. The activation of ERK shows different dependency on the phosphatidylinositol 3-kinase (PI3-K) pathway and different sensitivity to pertussis toxin (PTX) treatment, indicative of coupling to different G proteins in the two cell systems considered. We demonstrate that the inhibition of either the ERK kinase or the PI3-K pathways blocks the CXCL12 induced-chemotaxis in CHP100 cells; while only PI3-K activity is stringently necessary for CGN migration.