Glioblastoma multiforme (GBM) is a diffuse brain tumor characterized by high infiltration in the brain parenchyma rendering the tumor difficult to eradicate by neurosurgery. Efforts to identify molecular targets involved in the invasive behavior of GBM suggested ion channel inhibition as a promising therapeutic approach. To determine if the Ca2+-dependent K+ channel KCa3.1 could represent a key element for GBM brain infiltration, human GL-15 cells were xenografted into the brain of SCID mice that were then treated with the specific KCa3.1 blocker TRAM-34 (1-((2-chlorophenyl) (diphenyl)methyl)-1H-pyrazole). After 5 weeks of treatment, immunofluorescence analyses of cerebral slices revealed reduced tumor infiltration and astrogliosis surrounding the tumor, compared with untreated mice. Significant reduction of tumor infiltration was also observed in the brain of mice transplanted with KCa3.1-silenced GL-15 cells, indicating a direct effect of TRAM-34 on GBM-expressed KCa3.1 channels. As KCa3.1 channels are also expressed on microglia, we investigated the effects of TRAM-34 on microglia activation in GL-15 transplanted mice and found a reduction of CD68 staining in treated mice. Similar results were observed in vitro where TRAM-34 reduced both phagocytosis and chemotactic activity of primary microglia exposed to GBM-conditioned medium. Taken together, these results indicate that KCa3.1 activity has an important role in GBM invasiveness in vivo and that its inhibition directly affects glioma cell migration and reduces astrocytosis and microglia activation in response to tumor-released factors. KCa3.1 channel inhibition therefore constitutes a potential novel therapeutic approach to reduce GBM spreading into the surrounding tissue.

The activation of ion channels is crucial during cell movement, including glioblastoma cell invasion in the brain parenchyma. In this context, we describe for the first time the contribution of intermediate conductance Ca(2+)-activated K (IK(Ca)) channel activity in the chemotactic response of human glioblastoma cell lines, primary cultures, and freshly dissociated tissues to CXC chemokine ligand 12 (CXCL12), a chemokine whose expression in glioblastoma has been correlated with its invasive capacity. We show that blockade of the IK(Ca) channel with its specific inhibitor 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) or IK(Ca) channel silencing by short hairpin RNA (shRNA) completely abolished CXCL12-induced cell migration. We further demonstrate that this is not a general mechanism in glioblastoma cell migration since epidermal growth factor (EGF), which also activates IK(Ca) channels in the glioblastoma-derived cell line GL15, stimulate cell chemotaxis even if the IK(Ca) channels have been blocked or silenced. Furthermore, we demonstrate that both CXCL12 and EGF induce Ca(2+) mobilization and IK(Ca) channel activation but only CXCL12 induces a long-term upregulation of the IK(Ca) channel activity. Furthermore, the Ca(2+)-chelating agent BAPTA-AM abolished the CXCL12-induced, but not the EGF-induced, glioblastoma cell chemotaxis. In addition, we demonstrate that the extracellular signal-regulated kinase (ERK)1/2 pathway is only partially implicated in the modulation of CXCL12-induced glioblastoma cell movement, whereas the phosphoinositol-3 kinase (PI3K) pathway is not involved. In contrast, EGF-induced glioblastoma migration requires both ERK1/2 and PI3K activity. All together these findings suggest that the efficacy of glioblastoma invasiveness might be related to an array of nonoverlapping mechanisms activated by different chemotactic agents.

Muscarinic receptors, expressed in several primary and metastatic tumours, appear to be implicated in their growth and propagation. In this work we have demonstrated that M2 muscarinic receptors are expressed in glioblastoma human specimens and in glioblastoma cell lines. Moreover, we have characterized the effects of the M2 agonist arecaidine on cell growth and survival both in two different glioblastoma cell lines (U251MG and U87MG) and in primary cultures obtained from different human biopsies. Cell growth analysis has demonstrated that the M2 agonist arecaidine strongly decreased cell proliferation in both glioma cell lines and primary cultures. This effect was dose and time dependent. FACS analysis has confirmed cell cycle arrest at G1/S and at G2/M phase in U87 cells and U251 respectively. Cell viability analysis has also shown that arecaidine induced severe apoptosis, especially in U251 cells. Chemosensitivity assays have, moreover, shown arecaidine and temozolomide similar effects on glioma cell lines, although IC50 value for arecaidine was significantly lower than temozolomide. In conclusion, we report for the first time that M2 receptor activation has a relevant role in the inhibition of glioma cell growth and survival, suggesting that M2 may be a new interesting therapeutic target to investigate for glioblastoma therapy.

We report here the expression and properties of the intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channel in the GL-15 human glioblastoma cell line. Macroscopic IK_Ca currents on GL-15 cells displayed a mean amplitude of 7.2±0.8 pA/pF at 0 mV, at day 1 after plating. The current was inhibited by clotrimazole (CTL, IC₅₀=257 nM), TRAM-34 (IC₅₀=55 nM), and charybdotoxin (CTX, IC₅₀=10.3 nM). RT-PCR analysis demonstrated the expression of mRNA encoding the IK_Ca channel in GL-15 cells. Unitary currents recorded using the inside-out configuration had a conductance of 25 pS, a K_D for Ca²⁺ of 188 nM at -100 mV, and no voltage dependence. We tested whether the IKCa channel expression in GL-15 cells could be the result of an increased ERK activity. Inhibition of the ERK pathway with the MEK antagonist PD98059 (25 µM, for 5 days) virtually suppressed the IK_Ca current in GL-15 cells. PD98059 treatment also increased the length of cellular processes and up-regulated the astrocytic differentiative marker GFAP. A significant reduction of the IKCa current amplitude was also observed with time in culture, with mean currents of 7.17±0.75 pA/pF at 1-2 days, and 3.11±1.35 pA/pF at 5-6 days after plating. This time-dependent downregulation of the IK_Ca current was not accompanied by changes in the ERK activity, as assessed by immunoblot analysis. Semiquantitative RT-PCR analysis demonstrated a ~35% reduction of the IK_Ca channel mRNA resulting from ERK inhibition and a ~50% reduction with time in culture.

Misfolding and aggregation of α-synuclein are specific features of Parkinson’s disease and other neurodegenerative diseases defined as synucleinopathies. Parkinson’s disease progression has been correlated with the formation and the extracellular release of α-synuclein aggregates, as well as with their spreading from neuron to neuron. Therapeutic interventions in the initial stages of Parkinson’s disease require a clear understanding of the mechanisms by which α-synuclein disrupts the physiological synaptic and plastic activity of the basal ganglia. For this reason, here we have identified two early time points to clarify how the intrastriatal injection of α-synuclein preformed fibrils in rodents, via retrograde transmission, induces time-dependent electrophysiological and behavioral alterations. We found that intrastriatal α-synuclein preformed fibrils perturb the firing rate of dopaminergic neurons of the substantia nigra pars compacta while the discharge of putative GABAergic cells of the substantia nigra pars reticulata is unchanged. The α-synuclein-induced dysregulation of nigrostriatal function also impairs, in a time-dependent manner, the two main forms of striatal synaptic plasticity, long-term potentiation and long-term depression. We also observed an increased glutamatergic transmission measured as an augmented frequency of spontaneous excitatory synaptic currents. These changes in neuronal function in the substantia nigra pars compacta and striatum were observed before overt neuronal death occurred. In an additional set of experiments, we were able to rescue α-synuclein-induced alterations of motor function, striatal synaptic plasticity, and increased spontaneous excitatory synaptic currents by a sub-chronic treatment with L-Dopa, a precursor of dopamine widely used in the therapy of Parkinson’s disease, clearly demonstrating that a dysfunctional dopamine system plays a critical role in the early phases of the disease.

Neuronal nicotinic acetylcholine receptors (nAChRs) containing the a5 subunit modulate nicotine consumption, and the human CHRNA5 rs16969968 polymorphism, causing the replacement of the aspartic acid residue at position 398 with an asparagine (a5DN), has recently been associated with increased use of tobacco and higher incidence of lung cancer. We show that in ventral midbrain neurons, the a5 subunit is essential for heteromeric nAChR-induced intracellular-free Ca 2+ concentration elevations and that in a5 2/2 mice, a class of large-amplitude nicotine-evoked currents is lost. Furthermore, the expression of the a5DN subunit is not able to restore nicotinic responses, indicating a loss of function by this subunit in native neurons. To understand how a5DN impairs heteromeric nAChR functions, we coexpressed a4, a5, or a5DN subunits with a dimeric concatemer (b2a4) in a heterologous system, to obtain nAChRs with fixed stoichiometry. Both a5(b2a4) 2 and a5DN(b2a4) 2 nAChRs yielded similar levels of functional expression and Ca 2+ permeability, measured as fractional Ca 2+ currents (8.2 6 0.7% and 8.0 6 1.9%, respectively), 2-fold higher than a4 (b2a4) 2 . Our results indicate that the loss of function of nicotinic responses observed in a5DN-expressing ventral midbrain neurons is neither due to an intrinsic inability of this subunit to form functional nAChRs nor to an altered Ca 2+ permeability but likely to intracellular modulation.-Sciaccaluga, M., Moriconi, C., Martinello, K., Catalano, M., Bermudez, I., Stitzel, J. A., Maskos, U., Fucile, S. Crucial role of nicotinic a5 subunit variants for Ca 2+ fluxes in ventral midbrain neurons. FASEB J. 29, 3389-3398 (2015 , and it confers specific activation, desensitization, and ion selectivity to these receptors (2, 4-8). Recently, the a5 subunit containing the nonsynonymous variant rs16969968, a single nucleotide polymorphism in which the aspartic acid residue at position 398 is replaced with an asparagine (a5DN), has been associated with increased use of tobacco (9, 10) and with a higher incidence of lung cancer (11,12). The a5 subunit is important for nicotine aversion (13,14), and in its absence (i.e., a5 2/2 mice), self-administration of nicotine increases (13, 15), which suggests that a5DN-induced increase in nicotine consumption in humans may be due to partial loss of a5* nAChR function (15,16). This subunit has been shown to increase Ca 2+ permeability of heteromeric nAChRs (4, 6). The ability of nAChRs to allow significant Ca 2+ entry in presynaptic terminals represents a key step in cholinergic modulation of neurotransmitter release (17), and the expression of a5 subunit in neurons of the reward-related mesocorticolimbic pathways (18,19) and in structures linked to aversion and withdrawal response, such as medial habenula and interpeduncular nucleus (20-22), strongly suggests that Ca 2+ fluxes mediated by a5* nAChRs are highly relevant for drug-dependence mechanisms. To clarify the role of a5 and a5DN subunits in modulating neuronal intracellular free Ca 2+...

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The swelling-activated chloride current (I Cl,Vol) is abundantly expressed in glioblastoma (GBM) cells, where it controls cell volume and invasive migration. The transduction pathway mediating I Cl,Vol activation in GBM cells is, however, poorly understood. By means of pharmacological and electrophysiological approaches, on GL-15 human GBM cells we found that I Cl,Vol activation by hypotonic swelling required the activity of a U73122-sensitive phospholipase C (PLC). I Cl,Vol activation could also be induced by the membrane-permeable diacylglycerol (DAG) analog OAG. In contrast, neither calcium (Ca(2+)) chelation by BAPTA-AM nor changes in PKC activity were able to affect I Cl,Vol activation by hypotonic swelling. We further found that R59022, an inhibitor of diacylglycerol kinase (DGK), reverted I Cl,Vol activation, suggesting the involvement of phosphatidic acid. In addition, I Cl,Vol activation required the activity of a EHT1864-sensitive Rac1 small GTPase and the resulting actin polymerization, as I Cl,Vol activation was prevented by cytochalasin B. We finally show that I Cl,Vol can be activated by the promigratory fetal calf serum in a PLC- and DGK-dependent manner. This observation is potentially relevant because blood serum can likely come in contact with glioblastoma cells in vivo as a result of the tumor-related partial breakdown of the blood-brain barrier. Given the relevance of I Cl,Vol in GBM cell volume regulation and invasiveness, the several key signaling molecules found in this study to be involved in the activation of the I Cl,Vol may represent potential therapeutic targets against this lethal cancer.