Sergio Fucile

Ca2+ permeability of mouse and chick nicotinic acetylcholine receptors expressed in transiently transfected human cells

1 Combinations of cDNAs encoding mouse and chick nicotinic acetylcholine receptor (nAChR) subunits were transiently transfected into human BOSC 23 cells, and the expressed receptors were studied by simultaneously recording transmembrane currents and fluorescence transients using the whole‐cell patch‐clamp technique, and confocal microscopy with the Ca2+ indicator dye fluo‐3. 2 The fractional Ca2+ current, Pf, of nAChRs was evaluated as the normalized ratio of nicotine‐evoked fluorescence transient over total charge entering the cell (F/Q ratio). Mouse fetal muscle nAChR channels had a Pf,αβγδ value of 2.1 %. The substitution of the γ subunit with the ɛ subunit resulted in a 2‐fold increase in Pf (4.2 %). The difference in Ca2+ permeability was confirmed by determination of Ca2+/Cs+ permeability ratios. 3 Among the chick neuronal nAChRs tested, Pf,α3β4 was 4.6 %, while Pf,α4β4 and Pf,α4β2 were 3.0 % and 2.9 %, respectively. 4 The amplitude of the current elicited by the activation of α3β4 nAChRs increased as the external Ca2+ concentration was raised from 2 to 110 mM, whereas currents flowing through all other nAChRs tested were reduced to various extents. 5 Our findings indicate that the adult‐type muscle nAChR (αβɛδ) is more permeable to Ca2+ than the fetal‐type (αβγδ), while ganglionic‐like α3β4 nAChR is more permeable to Ca2+ than the examined α4‐containing nAChRs. The functional significance is discussed.

Fractional Ca2+ current through human neuronal α7 nicotinic acetylcholine receptors

The neuronal alpha7 nicotinic acetylcholine (ACh) receptor is believed to be a highly Ca(2+) permeable ligand-gated receptor-channel. However, the contribution of Ca(2+) to cationic current generated by ACh has not yet been directly measured to date. Simultaneous fluorescence and whole-cell current measurements using the Ca(2+) indicator dye fura-2 were made in GH4C1 pituitary cells stably expressing human alpha7 receptors and the fractional Ca(2+) current (the proportion of whole-cell current carried by Ca(2+); P(f)) was determined. We report that the P(f) value was 11.4+/-1.3%. This value was significantly larger than P(f) of human L248Talpha7 receptor mutant (P(f)=6.3+/-1.0%) and of rat alpha7 receptor (P(f)=8.8+/-1.5%) both determined in transiently transfected GH4C1 cells. In our knowledge, the findings here reported indicate the human alpha7 receptors are the most Ca(2+) conductive homomeric ligand-gated receptor-channels expressed in a heterologous cell system.

Ca2+ permeability of nicotinic acetylcholine receptors from rat dorsal root ganglion neurones

Ca2+ entry through neuronal nicotinic ACh receptors (nAChRs) modulates many biological processes in nervous tissue. In order to study the functional role of nAChRs in peripheral sensory signalling, we measured their Ca2+ permeability in rat dorsal root ganglion (DRG) neurones, and analysed the effects of nAChR‐mediated Ca2+ influx on the function of the vanilloid receptor TRPV1. The fractional Ca2+ current (Pf, i.e. the percentage of current carried by Ca2+ ions) flowing through nAChR channels was measured by Ca2+ imaging fluorescence microscopy in combination with the patch‐clamp technique. Functional nAChRs were expressed in a subset of adult DRG neurones (about 24% of the cells), typically with small to medium size as measured by their capacitance (40 ± 3 pF). In most cells, ACh evoked slowly desensitizing currents, insensitive to methyllycaconitine (MLA, 10 nm), a potent antagonist of homomeric nAChRs. Fast decaying currents, probably mediated by α7*‐nAChRs (i.e. native α7‐containing nAChRs), were observed in 15% of ACh‐responsive cells, in which slowly decaying currents, mediated by heteromeric nAChRs, were simultaneously present. The nAChRs of adult DRG neurones exhibited a Pf value of 2.2 ± 0.6% in the presence of MLA and 1.9 ± 0.6% (P > 0.1) in the absence of MLA, indicating that homomeric MLA‐sensitive nAChRs do not contribute to Ca2+ entry into adult DRG neurones. Conversely, 10% of neonatal DRG neurones showed ACh‐evoked currents completely blocked by MLA. In these neurones, nAChRs showed a larger Pf value (9.5 ± 1.5%), indicating the expression of bona fide α7*‐nAChRs. Finally, we report that Ca2+ influx through nAChRs in adult DRG neurones negatively modulated the TRPV1‐mediated responses, representing a possible mechanism underlying the analgesic properties of nicotinic agonists on sensory neurones.

FTY720 (fingolimod) is a neuroprotective and disease-modifying agent in cellular and mouse models of Huntington disease

Huntington disease (HD) is a genetic neurodegenerative disorder for which there is currently no cure and no way to stop or even slow the brain changes it causes. In the present study, we aimed to investigate whether FTY720, the first approved oral therapy for multiple sclerosis, may be effective in HD models and eventually constitute an alternative therapeutic approach for the treatment of the disease. Here, we utilized preclinical target validation paradigms and examined the in vivo efficacy of chronic administration of FTY720 in R6/2 HD mouse model. Our findings indicate that FTY720 improved motor function, prolonged survival and reduced brain atrophy in R6/2 mice. The beneficial effect of FTY720 administration was associated with a significant strengthening of neuronal activity and connectivity and, with reduction of mutant huntingtin aggregates, and it was also paralleled by increased phosphorylation of mutant huntingtin at serine 13/16 residues that are predicted to attenuate protein toxicity.

The human adult subtype ACh receptor channel has high Ca2+ permeability and predisposes to endplate Ca2+ overloading

Slow‐channel congenital myasthenic syndrome, caused by mutations in subunits of the endplate ACh receptor (AChR), results in prolonged synaptic currents and excitotoxic injury of the postsynaptic region by Ca2+ overloading. The Ca2+ overloading could be due entirely to the prolonged openings of the AChR channel or could be abetted by enhanced Ca2+ permeability of the mutant channels. We therefore measured the fractional Ca2+ current, defined as the percentage of the total ACh‐evoked current carried by Ca2+ ions (Pf), for AChRs harbouring the αG153S or the αV249F slow‐channel mutation, and for wild‐type human AChRs in which Pf has not yet been determined. Experiments were performed in transiently transfected GH4C1 cells and human myotubes with simultaneous recording of ACh‐evoked whole‐cell currents and fura‐2 fluorescence signals. We found that the Pf of the wild‐type human endplate AChR was unexpectedly high (Pf∼7%), but neither the αV249F nor the αG153S mutation altered Pf. Fetal human AChRs containing either the wild‐type or the mutated α subunit had a much lower Pf (2–3%). We conclude that the Ca2+ permeability of human endplate AChRs is higher than that reported for any other human nicotinic AChR, with the exception of α7‐containing AChRs (Pf > 10%); and that neither the αG153S nor the αV249F mutations affect the Pf of fetal or adult endplate AChRs. However, the intrinsically high Ca2+ permeability of human AChRs probably predisposes to development of the endplate myopathy when opening events of the AChR channel are prolonged by altered AChR‐channel kinetics.

The single-channel properties of human acetylcholine α7 receptors are altered by fusing α7 to the green fluorescent protein

Neuronal nicotinic acetylcholine (AcCho) receptors composed of α7-subunits (α7-AcChoRs) are involved in many physiological activities. Nevertheless, very little is known about their single-channel characteristics. By using outside-out patch-clamp recordings from Xenopus oocytes expressing wild-type (wt) α7-AcChoRs, we identified two classes of channel conductance: a low conductance (γL) of 72 pS and a high one (γH) of 87 pS, with mean open-times (τop) of 0.6 ms. The same classes of conductances, but longer τop (3 ms), were seen in experiments with chimeric α7 receptors in which the wtα7 extracellular C terminus was fused to the green fluorescent protein (wtα7-GFP AcChoRs). In contrast, channels with three different conductances were gated by AcCho in oocytes expressing α7 receptors carrying a Leu-to-Thr 248 mutation (mutα7) or oocytes expressing chimeric mutα7-GFP receptors. These conductance levels were significantly smaller, and their mean open-times were larger, than those of wtα7-AcChoRs. Interestingly, in the absence of AcCho, these oocytes showed single-channel openings of the same conductances, but shorter τop, than those activated by AcCho. Accordingly, human homomeric wtα7 receptors open channels of high conductance and brief lifetime, and fusion to GFP lengthens their lifetime. In contrast, mutα7 receptors open channels of lower conductance and longer lifetime than those gated by wtα7-AcChoRs, and these parameters are not greatly altered by fusing the mutα7 to GFP. All this evidence shows that GFP-tagging can alter importantly receptor kinetics, a fact that has to be taken into account whenever tagged proteins are used to study their function.

Fractalkine/CX3CL1 modulates GABAA currents in human temporal lobe epilepsy.

The chemokine fractalkine/CX3CL1 and its receptor CX3CR1 are widely expressed in the central nervous system (CNS). Recent evidence showed that CX3CL1 participates in inflammatory responses that are common features of CNS disorders, such as epilepsy. Mesial temporal lobe epilepsy (MTLE) is the prevalent form of focal epilepsy in adults, and hippocampal sclerosis (HS) represents the most common underlying pathologic abnormality, as demonstrated at autopsy and postresection studies. Relevant features of MTLE are a characteristic pattern of neuronal loss, as are astrogliosis and microglia activation. Several factors affect epileptogenesis in patients with MTLE, including a lack of γ‐aminobutyric acid (GABA)ergic inhibitory efficacy. Therefore, experiments were designed to investigate whether, in MTLE brain tissues, CX3CL1 may influence GABAA receptor (GABAAR) mediatedtransmission, with a particular focus on the action of CX3CL1 on the use‐dependent decrease (rundown) of the GABA‐evoked currents (IGABA), a feature underlying the reduction of GABAergic function in epileptic tissue.

GABAA-current rundown of temporal lobe epilepsy is associated with repetitive activation of GABAA phasic receptors

A study was made of the “rundown” of GABAA receptors, microtransplanted to Xenopus oocytes from surgically resected brain tissues of patients afflicted with drug-resistant human mesial temporal lobe epilepsy (mTLE). Cell membranes, isolated from mTLE neocortex specimens, were injected into frog oocytes that rapidly incorporated functional GABAA receptors. Upon repetitive activation with GABA (1 mM), “epileptic” GABAA receptors exhibited a GABAA-current (IGABA) rundown that was significantly enhanced by Zn2+ (≤250 μM), and practically abolished by the high-affinity GABAA receptor inverse agonist SR95531 (gabazine; 2.5–25 μM). Conversely, IGABA generated by “control” GABAA receptors microtransplanted from nonepileptic temporal lobe, lesional TLE, or authoptic disease-free tissues remained stable during repetitive stimulation, even in oocytes treated with Zn2+. We conclude that rundown of mTLE epileptic receptors depends on the presence of “phasic GABAA receptors” that have low sensitivity to antagonism by Zn2+. Additionally, we found that GABAA receptors, microtransplanted from the cerebral cortex of adult rats exhibiting recurrent seizures, caused by pilocarpine-induced status epilepticus, showed greater rundown than control tissue, an event also occurring in patch-clamped rat pyramidal neurons. Rundown of epileptic rat receptors resembled that of human mTLE receptors, being enhanced by Zn2+ (40 μM) and sensitive to the antiepileptic agent levetiracetam, the neurotrophin brain-derived neurotrophic factor, and the phosphatase blocker okadaic acid. Our findings point to the rundown of GABAA receptors as a hallmark of TLE and suggest that modulating tonic and phasic mTLE GABAA receptor activity may represent a useful therapeutic approach to the disease.

Rare Missense Variants of Neuronal Nicotinic Acetylcholine Receptor Altering Receptor Function Are Associated with Sporadic Amyotrophic Lateral Sclerosis

Sporadic amyotrophic lateral sclerosis (SALS) is a motor neuron degenerative disease of unknown etiology. Current thinking on SALS is that multiple genetic and environmental factors contribute to disease liability. Since neuronal acetylcholine receptors (nAChRs) are part of the glutamatergic pathway, we searched for sequence variants in CHRNA3, CHRNA4 and CHRNB4 genes, encoding neuronal nicotinic AChR subunits, in 245 SALS patients and in 450 controls. We characterized missense variants by in vitro mutagenesis, cell transfection and electrophysiology. Sequencing the regions encoding the intracellular loop of AChRs subunits disclosed 15 missense variants (6.1%) in 14 patients compared with only six variants (1.3%) in controls (P = 0.001; OR 4.48, 95% CI 1.7-11.8). The frequency of variants in exons encoding extracellular and transmembrane domains and in intronic regions did not differ. NAChRs formed by mutant alpha3 and alpha4 and wild-type (WT) beta4 subunits exhibited altered affinity for nicotine (Nic), reduced use-dependent rundown of Nic-activated currents (I(Nic)) and reduced desensitization leading to sustained intracellular Ca(2+) concentration, in comparison with WT-nAChR. The cellular loop has a crucial importance for receptor trafficking and regulating ion channel properties. Missense variants in this domain are significantly over-represented in SALS patients and alter functional properties of nAChR in vitro, resulting in increased Ca(2+) entry into the cells. We suggest that these gain-of-function variants might contribute to disease liability in a subset of SALS because Ca(2+) signals mediate nAChRs neuromodulatory effects, including regulation of glutamate release and control of cell survival.

Cyclin D1 degradation enhances endothelial cell survival upon oxidative stress

The understanding of endothelial cell responses to oxidative stress may provide insights into aging mechanisms and into the pathogenesis of numerous cardiovascular diseases. In this study, we examined the regulation and the functional role of cyclin D1, a crucial player in cell proliferation and survival. On H2O2 treatment, endothelial cells showed a rapid down‐modulation of cyclin D1. Other D‐cyclins were similarly regulated, and this decrease was also observed after exposure to other oxidative stress‐inducing stimuli, namely 1,3‐bis (2 chloroethyl)‐1 nitrosourea treatment and ischemia. H2O2 treatment induced cyclin D1 ubiquitination followed by proteasome degradation. Phospholipase C inhibition prevented cyclin D1 degradation, and its activation triggered cyclin D1 down‐modulation in the absence of oxidative stress. Activated phospholipase C generates inositol‐1,4,5‐trisphosphate (IP3) and Ca2+ release from internal stores. We found that both IP3‐receptor inhibition and intracellular Ca2+ chelation prevented cyclin D1 degradation induced by oxidative stress. Furthermore, Ca2+ increase was transduced by Ca2+/calmodulin‐dependent protein kinase (CaMK). In fact, H2O2 stimulated CaMK activity, CaMK inhibitors prevented H2O2‐induced cyclin D1 down‐modulation, and CaMK overexpression induced cyclin D1 degradation. Finally, overriding of cyclin D1 down‐modulation via its forced overexpression or via CaMK inhibition increased cell sensitivity to H2O2‐induced apoptotic cell death. Thus, cyclin D1 degradation enhances endothelial cell survival on oxidative stress.—Fasanaro, P., Magenta, A., Zaccagnini, G., Cicchillitti, L., Fucile, S., Eusebi, F., Biglioli, P., Capogrossi, M. C., Martelli, F. Cyclin D1 degradation enhances endothelial cell survival upon oxidative stress. FASEB J. 20, E503–E515 (2006)