Publications by authors named "Andrea Becchetti"

63 Publications

An early Sox2-dependent gene expression programme required for hippocampal dentate gyrus development.

Open Biol 2021 Feb 24;11(2):200339. Epub 2021 Feb 24.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy.

The hippocampus is a brain area central for cognition. Mutations in the human SOX2 transcription factor cause neurodevelopmental defects, leading to intellectual disability and seizures, together with hippocampal dysplasia. We generated an allelic series of Sox2 conditional mutations in mouse, deleting Sox2 at different developmental stages. Late Sox2 deletion (from E11.5, via Nestin-Cre) affects only postnatal hippocampal development; earlier deletion (from E10.5, Emx1-Cre) significantly reduces the dentate gyrus (DG), and the earliest deletion (from E9.5, FoxG1-Cre) causes drastic abnormalities, with almost complete absence of the DG. We identify a set of functionally interconnected genes (Gli3, Wnt3a, Cxcr4, p73 and Tbr2), known to play essential roles in hippocampal embryogenesis, which are downregulated in early Sox2 mutants, and (Gli3 and Cxcr4) directly controlled by SOX2; their downregulation provides plausible molecular mechanisms contributing to the defect. Electrophysiological studies of the Emx1-Cre mouse model reveal altered excitatory transmission in CA1 and CA3 regions.
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http://dx.doi.org/10.1098/rsob.200339DOI Listing
February 2021

Data describing the effects of potassium channels modulators on outward currents measured in human lymphoma cell lines.

Data Brief 2021 Feb 21;34:106668. Epub 2020 Dec 21.

Department of Experimental and Clinical Medicine, University of Florence, I‑50134 Florence, Italy.

In the present work, applying the whole-cell patch-clamp technique in voltage clamp mode, we have investigated the effects of different drugs, such as riluzole, Psora-4 and Tram-34, on the potassium currents in four human lymphoma cell lines. We focused on outward currents mediated by two potassium channels (Kv1.3 and KCa3.1), which are known to play a key physiological role in lymphoid cells. The currents were evoked by voltage ramps ranging from -120 mV to +40 mV and the conductance of the two potassium channels was measured between +20 mV and +40 mV, both in the absence and in the presence of the specific blockers Psora-4 (Kv1.3; 1 µM) and Tram-34 (KCa3.1; 1 µM). The effect of the latter was tested after KCa3.1 channels were activated by riluzole 10 µM. Taken together, these data could be useful as an indication of the functional characteristics of the potassium channels in human lymphomas and represent a starting point for the study of potassium conductance in cellular models of these tumors.
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http://dx.doi.org/10.1016/j.dib.2020.106668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7772536PMC
February 2021

Nicotinic Receptors in Sleep-Related Hypermotor Epilepsy: Pathophysiology and Pharmacology.

Brain Sci 2020 Nov 25;10(12). Epub 2020 Nov 25.

Department of Biosciences, University of Milano, 20133 Milano, Italy.

Sleep-related hypermotor epilepsy (SHE) is characterized by hyperkinetic focal seizures, mainly arising in the neocortex during non-rapid eye movements (NREM) sleep. The familial form is autosomal dominant SHE (ADSHE), which can be caused by mutations in genes encoding subunits of the neuronal nicotinic acetylcholine receptor (nAChR), Na-gated K channels, as well as non-channel signaling proteins, such as components of the gap activity toward rags 1 (GATOR1) macromolecular complex. The causative genes may have different roles in developing and mature brains. Under this respect, nicotinic receptors are paradigmatic, as different pathophysiological roles are exerted by distinct nAChR subunits in adult and developing brains. The widest evidence concerns α4 and β2 subunits. These participate in heteromeric nAChRs that are major modulators of excitability in mature neocortical circuits as well as regulate postnatal synaptogenesis. However, growing evidence implicates mutant α2 subunits in ADSHE, which poses interpretive difficulties as very little is known about the function of α2-containing (α2*) nAChRs in the human brain. Planning rational therapy must consider that pharmacological treatment could have different effects on synaptic maturation and adult excitability. We discuss recent attempts towards precision medicine in the mature brain and possible approaches to target developmental stages. These issues have general relevance in epilepsy treatment, as the pathogenesis of genetic epilepsies is increasingly recognized to involve developmental alterations.
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http://dx.doi.org/10.3390/brainsci10120907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761363PMC
November 2020

Impaired flickering of the permeability transition pore causes SPG7 spastic paraplegia.

EBioMedicine 2020 Nov 9;61:103050. Epub 2020 Oct 9.

Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli-Naples, Italy; Vita-Salute San Raffaele University, Milan, Italy. Electronic address:

Background: Mutations of the mitochondrial protein paraplegin cause hereditary spastic paraplegia type 7 (SPG7), a so-far untreatable degenerative disease of the upper motoneuron with still undefined pathomechanism. The intermittent mitochondrial permeability transition pore (mPTP) opening, called flickering, is an essential process that operates to maintain mitochondrial homeostasis by reducing intra-matrix Ca and reactive oxygen species (ROS) concentration, and is critical for efficient synaptic function.

Methods: We use a fluorescence-based approach to measure mPTP flickering in living cells and biochemical and molecular biology techniques to dissect the pathogenic mechanism of SPG7. In the SPG7 animal model we evaluate the potential improvement of the motor defect, neuroinflammation and neurodegeneration by means of an mPTP inducer, the benzodiazepine Bz-423.

Findings: We demonstrate that paraplegin is required for efficient transient opening of the mPTP, that is impaired in both SPG7 patients-derived fibroblasts and primary neurons from Spg7 mice. We show that dysregulation of mPTP opening at the pre-synaptic terminal impairs neurotransmitter release leading to ineffective synaptic transmission. Lack of paraplegin impairs mPTP flickering by a mechanism involving increased expression and activity of sirtuin3, which promotes deacetylation of cyclophilin D, thus hampering mPTP opening. Pharmacological treatment with Bz-423, which bypasses the activity of CypD, normalizes synaptic transmission and rescues the motor impairment of the SPG7 mouse model.

Interpretation: mPTP targeting opens a new avenue for the potential therapy of this form of spastic paraplegia.

Funding: Telethon Foundation grant (TGMGCSBX16TT); Dept. of Defense, US Army, grant W81XWH-18-1-0001.
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http://dx.doi.org/10.1016/j.ebiom.2020.103050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553352PMC
November 2020

K11.1 Potassium Channel and the Na/H Antiporter NHE1 Modulate Adhesion-Dependent Intracellular pH in Colorectal Cancer Cells.

Front Pharmacol 2020 10;11:848. Epub 2020 Jun 10.

Section of Internal Medicine, Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

Increasing evidence indicates that ion channels and transporters cooperate in regulating different aspects of tumor pathophysiology. In cancer cells, H/HCO transporters usually invert the transmembrane pH gradient typically observed in non-neoplastic cells, which is thought to contribute to cancer malignancy. To what extent the pH-regulating transporters are functionally linked to K channels, which are central regulators of cell membrane potential (V), is unclear. We thus investigated in colorectal cancer cells the implication of the pH-regulating transporters and K11.1 (also known as hERG1) in the pH modifications stimulated by integrin-dependent cell adhesion. Colorectal cancer cell lines (HCT 116 and HT 29) were seeded onto β1 integrin-dependent substrates, collagen I and fibronectin. This led to a transient cytoplasmic alkalinization, which peaked at 90 min of incubation, lasted approximately 180 min, and was inhibited by antibodies blocking the β1 integrin. The effect was sensitive to amiloride (10 µM) and cariporide (5 µM), suggesting that it was mainly caused by the activity of the Na/H antiporter NHE1. Blocking K11.1 with E4031 shows that channel activity contributed to modulate the β1 integrin-dependent pH increase. Interestingly, both NHE1 and K11.1 modulated the colorectal cancer cell motility triggered by β1 integrin-dependent adhesion. Finally, the β1 integrin subunit, K11.1 and NHE1 co-immunoprecipitated in colorectal cancer cells seeded onto Collagen I, suggesting the formation of a macromolecular complex following integrin-mediated adhesion. We conclude that the interaction between K11.1, NHE1, and β1 integrin contributes to regulate colorectal cancer intracellular pH in relation to the tumor microenvironment, suggesting novel pharmacological targets to counteract pro-invasive and, hence, pro-metastatic behavior in colorectal cancer.
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http://dx.doi.org/10.3389/fphar.2020.00848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7297984PMC
June 2020

Variants in and Identified in Patients with Insular Epilepsy.

Can J Neurol Sci 2020 11 15;47(6):800-809. Epub 2020 Jun 15.

Neurology Division, Centre Hospitalier de l'Université de Montréal, Montreal, Canada.

Purpose: Our purpose was to determine the role of CHRNA4 and CHRNB2 in insular epilepsy.

Method: We identified two patients with drug-resistant predominantly sleep-related hypermotor seizures, one harboring a heterozygous missense variant (c.77C>T; p. Thr26Met) in the CHRNB2 gene and the other a heterozygous missense variant (c.1079G>A; p. Arg360Gln) in the CHRNA4 gene. The patients underwent electrophysiological and neuroimaging studies, and we performed functional characterization of the p. Thr26Met (c.77C>T) in the CHRNB2 gene.

Results: We localized the epileptic foci to the left insula in the first case (now seizure-free following epilepsy surgery) and to both insulae in the second case. Based on tools predicting the possible impact of amino acid substitutions on the structure and function of proteins (sorting intolerant from tolerant and PolyPhen-2), variants identified in this report could be deleterious. Functional expression in human cell lines of α4β2 (wild-type), α4β2-Thr26Met (homozygote), and α4β2/β2-Thr26Met (heterozygote) nicotinic acetylcholine receptors revealed that the mutant subunit led to significantly higher whole-cell nicotinic currents. This feature was observed in both homo- and heterozygous conditions and was not accompanied by major alterations of the current reversal potential or the shape of the concentration-response relation.

Conclusions: This study suggests that variants in CHRNB2 and CHRNA4, initially linked to autosomal dominant nocturnal frontal lobe epilepsy, are also found in patients with predominantly sleep-related insular epilepsy. Although the reported variants should be considered of unknown clinical significance for the moment, identification of additional similar cases and further functional studies could eventually strengthen this association.
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http://dx.doi.org/10.1017/cjn.2020.126DOI Listing
November 2020

Correction: Clarithromycin inhibits autophagy in colorectal cancer by regulating the hERG1 potassium channel interaction with PI3K.

Cell Death Dis 2020 Mar 30;11(3):209. Epub 2020 Mar 30.

Department of Experimental and Clinical Medicine, University of Firenze, Viale GB Morgagni 50, 50134, Firenze, Italy.

The financial support for this Article was not fully acknowledged. The acknowledgements should have included the following: We thank M. Lulli (University of Florence, Italy) for acquiring images of immunofluorescence-labeled cells. This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro (#15627, #21510 and #19766 to A.A.); PAR FAS-Linea di Azione 1.1-Azione 1.1.2-Bando FAS Salute. 2014 (DD 4042/ 2014) Project OMITERC to A.A.; FAR 2018 to A.B.
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http://dx.doi.org/10.1038/s41419-020-2407-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105481PMC
March 2020

Clarithromycin inhibits autophagy in colorectal cancer by regulating the hERG1 potassium channel interaction with PI3K.

Cell Death Dis 2020 03 2;11(3):161. Epub 2020 Mar 2.

Department of Experimental and Clinical Medicine, University of Firenze, Viale GB Morgagni 50, 50134, Firenze, Italy.

We have studied how the macrolide antibiotic Clarithromycin (Cla) regulates autophagy, which sustains cell survival and resistance to chemotherapy in cancer. We found Cla to inhibit the growth of human colorectal cancer (CRC) cells, by modulating the autophagic flux and triggering apoptosis. The accumulation of cytosolic autophagosomes accompanied by the modulation of autophagic markers LC3-II and p62/SQSTM1, points to autophagy exhaustion. Because Cla is known to bind human Ether-à-go-go Related Gene 1 (hERG1) K channels, we studied if its effects depended on hERG1 and its conformational states. By availing of hERG1 mutants with different gating properties, we found that fluorescently labelled Cla preferentially bound to the closed channels. Furthermore, by sequestering the channel in the closed conformation, Cla inhibited the formation of a macromolecular complex between hERG1 and the p85 subunit of PI3K. This strongly reduced Akt phosphorylation, and stimulated the p53-dependent cell apoptosis, as witnessed by late caspase activation. Finally, Cla enhanced the cytotoxic effect of 5-fluorouracil (5-FU), the main chemotherapeutic agent in CRC, in vitro and in a xenograft CRC model. We conclude that Cla affects the autophagic flux by impairing the signaling pathway linking hERG1 and PI3K. Combining Cla with 5-FU might be a novel therapeutic option in CRC.
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http://dx.doi.org/10.1038/s41419-020-2349-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052256PMC
March 2020

Niosomal Formulation of a Lipoyl-Carnosine Derivative Targeting TRPA1 Channels in Brain.

Pharmaceutics 2019 Dec 10;11(12). Epub 2019 Dec 10.

Department of Chemistry "Ugo Schiff", University of Florence, 50019 Florence, Italy.

The transient receptor potential akyrin type-1 (TRPA1) is a non-selective cation channel playing a pivotal role in pain sensation and neurogenic inflammation. TRPA1 channels expressed in the central nervous system (CNS) have a critical role in the modulation of cortical spreading depression (CSD), which is a key pathophysiological basis of migraine pain. ADM_09 is a recently developed lipoic acid-based TRPA1 antagonist that is able to revert oxaliplatin-induced neuropathic pain and inflammatory trigeminal allodynia. In this context, aiming at developing drugs that are able to target TRPA1 channels in the CNS and promote an antioxidant effect, permeability across the blood-brain barrier (BBB) represents a central issue. Niosomes are nanovesicles that can be functionalized with specific ligands selectively recognized by transporters expressed on the BBB. In this work, the activity of ADM_09 on neocortex cultures was studied, and an efficient formulation to cross the BBB was developed with the aim of increasing the concentration of ADM_09 into the brain and selectively delivering it to the CNS rapidly after parenteral administration.
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http://dx.doi.org/10.3390/pharmaceutics11120669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956366PMC
December 2019

Data describing the effects of the Macrolide Antibiotic Clarithromycin on preclinical mouse models of Colorectal Cancer.

Data Brief 2019 Oct 22;26:104406. Epub 2019 Aug 22.

Department of Experimental and Clinical Medicine, University of Firenze, Viale GB Morgagni 50, 50134 Firenze, Italy.

Macrolide antibiotics, such as Clarithromycin (Cla), have been proven to exert anti-tumour activity in several preclinical models of different types of cancer. Cla can exert its anti-tumour effects through different mechanisms, e.g. by blocking the autophagic flux, inducing apoptosis or inhibiting tumour-induced angiogenesis. The clinical benefit of Cla in treating various tumours in combination with conventional treatment was confirmed in extensive clinical studies in patients suffering from non-small cell lung cancer, breast cancer, multiple myeloma and other haematological malignancies. Data regarding the anti-cancer effect of Cla on Colorectal Cancer (CRC) are still lacking. This article shares data on the efficacy of Cla in two xenograft models of CRC. Our results show that Cla treatment reduces tumour growth and increases the overall survival in CRC mouse xenograft models. Moreover, the Western blot analysis of autophagic and apoptotic markers suggests that the anti-tumour effects of Cla are related to a modulation of both cellular processes. The data suggest that it will worth consider Cla as treatment option for CRC patients.
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http://dx.doi.org/10.1016/j.dib.2019.104406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727004PMC
October 2019

and Nocturnal Frontal Lobe Epilepsy: Identification and Characterization of a Novel Loss of Function Mutation.

Front Mol Neurosci 2019 12;12:17. Epub 2019 Feb 12.

School of Medicine and Surgery, University of Milano - Bicocca, Monza, Italy.

Mutations in genes coding for subunits of the neuronal nicotinic acetylcholine receptor (nAChR) have been involved in familial sleep-related hypermotor epilepsy (also named autosomal dominant nocturnal frontal lobe epilepsy, ADNFLE). Most of these mutations reside in and genes, coding for the α4 and β2 nAChR subunits, respectively. Two mutations with contrasting functional effects were also identified in the gene coding for the α2 subunit. Here, we report the third mutation in the , found in a patient showing ADNFLE. The patient was examined by scalp EEG, contrast-enhanced brain magnetic resonance imaging (MRI), and nocturnal video-polysomnographic recording. All exons and the exon-intron boundaries of , , , , were amplified and Sanger sequenced. In the proband, we found a c.754T>C (p.Tyr252His) missense mutation located in the N-terminal ligand-binding domain and inherited from the mother. Functional studies were performed by transient co-expression of α2 and α2 , with either β2 or β4, in human embryonic kidney (HEK293) cells. Equimolar amounts of subunits expression were obtained by using F2A-based multi-cistronic constructs encoding for the genes relative to the nAChR subunits of interest and for the enhanced green fluorescent protein. The mutation reduced the maximal currents by approximately 80% in response to saturating concentrations of nicotine in homo- and heterozygous form, in both the α2β4 and α2β2 nAChR subtypes. The effect was accompanied by a strong right-shift of the concentration-response to nicotine. Similar effects were observed using ACh. Negligible effects were produced by α2 on the current reversal potential. Moreover, binding of (±)-[H]Epibatidine revealed an approximately 10-fold decrease of both K and B (bound ligand in saturating conditions), in cells expressing α2. The reduced B and whole-cell currents were not caused by a decrease in mutant receptor expression, as minor effects were produced by α2 on the level of transcripts and the membrane expression of α2β4 nAChR. Overall, these results suggest that α2 strongly reduced the number of channels bound to the agonist, without significantly altering the overall channel expression. We conclude that mutations in are more commonly linked to ADNFLE than previously thought, and may cause a loss-of-function phenotype.
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http://dx.doi.org/10.3389/fnmol.2019.00017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379349PMC
February 2019

The Activity of Kv 11.1 Potassium Channel Modulates F-Actin Organization During Cell Migration of Pancreatic Ductal Adenocarcinoma Cells.

Cancers (Basel) 2019 Jan 23;11(2). Epub 2019 Jan 23.

Department of Experimental and Clinical Medicine, University of Florence, Viale GB Morgagni 50, 50134 Florence, Italy.

Cell migration exerts a pivotal role in tumor progression, underlying cell invasion and metastatic spread. The cell migratory program requires f-actin re-organization, generally coordinated with the assembly of focal adhesions. Ion channels are emerging actors in regulating cell migration, through different mechanisms. We studied the role of the voltage dependent potassium channel K 11.1 on cell migration of pancreatic ductal adenocarcinoma (PDAC) cells, focusing on its effects on f-actin organization and dynamics. Cells were cultured either on fibronectin (FN) or on a desmoplastic matrix (DM) with the addition of a conditioned medium produced by pancreatic stellate cells (PSC) maintained in hypoxia (Hypo-PSC-CM), to better mimic the PDAC microenvironment. K11.1 was essential to maintain stress fibers in a less organized arrangement in cells cultured on FN. When PDAC cells were cultured on DM plus Hypo-PSC-CM, K11.1 activity determined the organization of cortical f-actin into sparse and long filopodia, and allowed f-actin polymerization at a high speed. In both conditions, blocking K11.1 impaired PDAC cell migration, and, on cells cultured onto FN, the effect was accompanied by a decrease of basal intracellular Ca concentration. We conclude that K11.1 is implicated in sustaining pro-metastatic signals in pancreatic cancer, through a reorganization of f-actin in stress fibers and a modulation of filopodia formation and dynamics.
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http://dx.doi.org/10.3390/cancers11020135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6406627PMC
January 2019

Ion Channel Conformations Regulate Integrin-Dependent Signaling.

Trends Cell Biol 2019 04 8;29(4):298-307. Epub 2019 Jan 8.

Department of Experimental and Clinical Medicine, University of Florence, 50134 Firenze, Italy.

Cell-matrix adhesion determines the choice between different cell fates and is accompanied by substantial changes in ion transport. The greatest evidence is the bidirectional interplay occurring between integrin receptors and K channels. These proteins can form signaling hubs that regulate cell proliferation, differentiation, and migration in normal and neoplastic tissue. Recent results show that the physical interaction with integrins determines the balance of the open and closed K channel states, and individual channel conformations regulate distinct downstream pathways. We propose a model of how these mechanisms regulate proliferation and metastasis in cancer cells. In particular, we suggest that the neoplastic progression could be modulated by targeting specific ion channel conformations.
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http://dx.doi.org/10.1016/j.tcb.2018.12.005DOI Listing
April 2019

Postnatal Changes in K/Cl Cotransporter-2 Expression in the Forebrain of Mice Bearing a Mutant Nicotinic Subunit Linked to Sleep-Related Epilepsy.

Neuroscience 2018 08 24;386:91-107. Epub 2018 Jun 24.

Department of Biotechnology and Biosciences, and NeuroMI-Milan Center of Neuroscience, University of Milano-Bicocca, Piazza della Scienza, 2, 20126 Milano, Italy. Electronic address:

The Na/K/Cl cotransporter-1 (NKCC1) and the K/Cl cotransporter-2 (KCC2) set the transmembrane Cl gradient in the brain, and are implicated in epileptogenesis. We studied the postnatal distribution of NKCC1 and KCC2 in wild-type (WT) mice, and in a mouse model of sleep-related epilepsy, carrying the mutant β2-V287L subunit of the nicotinic acetylcholine receptor (nAChR). In WT neocortex, immunohistochemistry showed a wide distribution of NKCC1 in neurons and astrocytes. At birth, KCC2 was localized in neuronal somata, whereas at subsequent stages it was mainly found in the somatodendritic compartment. The cotransporters' expression was quantified by densitometry in the transgenic strain. KCC2 expression increased during the first postnatal weeks, while the NKCC1 amount remained stable, after birth. In mice expressing β2-V287L, the KCC2 amount in layer V of prefrontal cortex (PFC) was lower than in the control littermates at postnatal day 8 (P8), with no concomitant change in NKCC1. Consistently, the GABAergic excitatory to inhibitory switch was delayed in PFC layer V of mice carrying β2-V287L. At P60, the amount of KCC2 was instead higher in mice bearing the transgene. Irrespective of genotype, NKCC1 and KCC2 were abundantly expressed in the neuropil of most thalamic nuclei since birth. However, KCC2 expression decreased by P60 in the reticular nucleus, and more so in mice expressing β2-V287L. Therefore, a complex regulatory interplay occurs between heteromeric nAChRs and KCC2 in postnatal forebrain. The pathogenetic effect of β2-V287L may depend on altered KCC2 amounts in PFC during synaptogenesis, as well as in mature thalamocortical circuits.
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http://dx.doi.org/10.1016/j.neuroscience.2018.06.030DOI Listing
August 2018

The combined activation of K3.1 and inhibition of K11.1/hERG1 currents contribute to overcome Cisplatin resistance in colorectal cancer cells.

Br J Cancer 2018 01 21;118(2):200-212. Epub 2017 Nov 21.

Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence, Viale G.B. Morgagni 50, 50134 Firenze, Italy.

Background: Platinum-based drugs such as Cisplatin are commonly employed for cancer treatment. Despite an initial therapeutic response, Cisplatin treatment often results in the development of chemoresistance. To identify novel approaches to overcome Cisplatin resistance, we tested Cisplatin in combination with K channel modulators on colorectal cancer (CRC) cells.

Methods: The functional expression of Ca-activated (K3.1, also known as KCNN4) and voltage-dependent (K11.1, also known as KCNH2 or hERG1) K channels was determined in two CRC cell lines (HCT-116 and HCT-8) by molecular and electrophysiological techniques. Cisplatin and several K channel modulators were tested in vitro for their action on K currents, cell vitality, apoptosis, cell cycle, proliferation, intracellular signalling and Platinum uptake. These effects were also analysed in a mouse model mimicking Cisplatin resistance.

Results: Cisplatin-resistant CRC cells expressed higher levels of K3.1 and K11.1 channels, compared with Cisplatin-sensitive CRC cells. In resistant cells, K3.1 activators (SKA-31) and K11.1 inhibitors (E4031) had a synergistic action with Cisplatin in triggering apoptosis and inhibiting proliferation. The effect was maximal when K3.1 activation and K11.1 inhibition were combined. In fact, similar results were produced by Riluzole, which is able to both activate K3.1 and inhibit K11.1. Cisplatin uptake into resistant cells depended on K3.1 channel activity, as it was potentiated by K3.1 activators. K11.1 blockade led to increased K3.1 expression and thereby stimulated Cisplatin uptake. Finally, the combined administration of a K3.1 activator and a K11.1 inhibitor also overcame Cisplatin resistance in vivo.

Conclusions: As Riluzole, an activator of K3.1 and inhibitor of K11.1 channels, is in clinical use, our results suggest that this compound may be useful in the clinic to improve Cisplatin efficacy and overcome Cisplatin resistance in CRC.
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http://dx.doi.org/10.1038/bjc.2017.392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5785745PMC
January 2018

Mesoporous silica nanoparticles trigger mitophagy in endothelial cells and perturb neuronal network activity in a size- and time-dependent manner.

Int J Nanomedicine 2017 8;12:3547-3559. Epub 2017 May 8.

Nanomedicine Center, School of Medicine and Surgery, University of Milano-Bicocca, Monza.

Purpose: Mesoporous silica nanoparticles (MSNPs) are excellent candidates for biomedical applications and drug delivery to different human body areas, the brain included. Although toxicity at cellular level has been investigated, we are still far from using MSNPs in the clinic, because the mechanisms involved in the cellular responses activated by MSNPs have not yet been elucidated.

Materials And Methods: This study used an in vitro multiparametric approach to clarify relationships among size, dose, and time of exposure of MSNPs (0.05-1 mg/mL dose range), and cellular responses by analyzing the morphology, viability, and functionality of human vascular endothelial cells and neurons.

Results: The results showed that 24 hours of exposure of endothelial cells to 250 nm MSNPs exerted higher toxicity in terms of mitochondrial activity and membrane integrity than 30 nm MSN at the same dose. This was due to induced cell autophagy (in particular mitophagy), probably consequent to MSNP cellular uptake (>20%). Interestingly, after 24 hours of treatment with 30 nm MSNPs, very low MSNP uptake (<1%) and an increase in nitric oxide production (30%, <0.01) were measured. This suggests that MSNPs were able to affect endothelial functionality from outside the cells. These differences could be attributed to the different protein-corona composition of the MSNPs used, as suggested by sodium dodecyl sulfate polyacrylamide-gel electrophoresis analysis of the plasma proteins covering the MSNP surface. Moreover, doses of MSNPs up to 0.25 mg/mL perturbed network activity by increasing excitability, as detected by multielectrode-array technology, without affecting neuronal cell viability.

Conclusion: These results suggest that MSNPs may be low-risk if prepared with a diameter <30 nm and if they reach human tissues at doses <0.25 mg/mL. These important advances could help the rational design of NPs intended for biomedical uses, demonstrating that careful toxicity evaluation is necessary before using MSNPs in patients.
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http://dx.doi.org/10.2147/IJN.S127663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5428814PMC
August 2017

The conformational state of hERG1 channels determines integrin association, downstream signaling, and cancer progression.

Sci Signal 2017 Apr 4;10(473). Epub 2017 Apr 4.

Department of Experimental and Clinical Medicine, University of Firenze, Viale G.B. Morgagni 50, 50134 Firenze, Italy.

Ion channels regulate cell proliferation, differentiation, and migration in normal and neoplastic cells through cell-cell and cell-extracellular matrix (ECM) transmembrane receptors called integrins. K flux through the human ether-à-go-go-related gene 1 (hERG1) channel shapes action potential firing in excitable cells such as cardiomyocytes. Its abundance is often aberrantly high in tumors, where it modulates integrin-mediated signaling. We found that hERG1 interacted with the β integrin subunit at the plasma membrane of human cancer cells. This interaction was not detected in cardiomyocytes because of the presence of the hERG1 auxiliary subunit KCNE1 (potassium voltage-gated channel subfamily E regulatory subunit 1), which blocked the β integrin-hERG1 interaction. Although open hERG1 channels did not interact as strongly with β integrins as did closed channels, current flow through hERG1 channels was necessary to activate the integrin-dependent phosphorylation of Tyr in focal adhesion kinase (FAK) in both normal and cancer cells. In immunodeficient mice, proliferation was inhibited in breast cancer cells expressing forms of hERG1 with impaired K flow, whereas metastasis of breast cancer cells was reduced when the hERG1/β integrin interaction was disrupted. We conclude that the interaction of β integrins with hERG1 channels in cancer cells stimulated distinct signaling pathways that depended on the conformational state of hERG1 and affected different aspects of tumor progression.
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http://dx.doi.org/10.1126/scisignal.aaf3236DOI Listing
April 2017

hERG Channels: From Antitargets to Novel Targets for Cancer Therapy.

Clin Cancer Res 2017 Jan 30;23(1):3-5. Epub 2016 Nov 30.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.

In this issue of Clinical Cancer Research, evidence is provided on how to avoid cardiotoxicity when targeting hERG K channel for cancer therapy. hERG regulates different aspects of neoplastic progression. Although its blockade has effective anticancer effects in experimental models, it may lead to fatal arrhythmias in humans. Clin Cancer Res; 23(1); 3-5. ©2016 AACRSee related article by Pointer et al., p. 73.
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http://dx.doi.org/10.1158/1078-0432.CCR-16-2322DOI Listing
January 2017

α4β2 nicotinic receptors stimulate GABA release onto fast-spiking cells in layer V of mouse prefrontal (Fr2) cortex.

Neuroscience 2017 01 26;340:48-61. Epub 2016 Oct 26.

Department of Biotechnology and Biosciences, and NeuroMI (Milan Center of Neuroscience), University of Milano-Bicocca, piazza della Scienza 2, Milano 20126, Italy. Electronic address:

Nicotinic acetylcholine receptors (nAChRs) produce widespread and complex effects on neocortex excitability. We studied how heteromeric nAChRs regulate inhibitory post-synaptic currents (IPSCs), in fast-spiking (FS) layer V neurons of the mouse frontal area 2 (Fr2). In the presence of blockers of ionotropic glutamate receptors, tonic application of 10μM nicotine augmented the spontaneous IPSC frequency, with minor alterations of amplitudes and kinetics. These effects were studied since the 3rd postnatal week, and persisted throughout the first two months of postnatal life. The action of nicotine was blocked by 1μM dihydro-β-erythroidine (DHβE; specific for α4 nAChRs), but not 10nM methyllycaconitine (MLA; specific for α7 nAChRs). It was mimicked by 10nM 5-iodo-3-[2(S)-azetidinylmethoxy]pyridine (5-IA; which activates β2 nAChRs). Similar results were obtained on miniature IPSCs (mIPSCs). Moreover, during the first five postnatal weeks, approximately 50% of FS cells displayed DHβE-sensitive whole-cell nicotinic currents. This percentage decreased to ∼5% in mice older than P45. By confocal microscopy, the α4 nAChR subunit was immunocytochemically identified on interneurons expressing either parvalbumin (PV), which mainly labels FS cells, or somatostatin (SOM), which labels the other major interneuron population in layer V. GABAergic terminals expressing α4 were observed to be juxtaposed to PV-positive (PV+) cells. A fraction of these terminals displayed PV immunoreactivity. We conclude that α4β2 nAChRs can produce sustained regulation of FS cells in Fr2 layer V. The effect presents a presynaptic component, whereas the somatic regulation decreases with age. These mechanisms may contribute to the nAChR-dependent stimulation of excitability during cognitive tasks as well as to the hyperexcitability caused by hyperfunctional heteromeric nAChRs in sleep-related epilepsy.
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http://dx.doi.org/10.1016/j.neuroscience.2016.10.045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5231322PMC
January 2017

Neuron-glia cross talk revealed in reverberating networks by simultaneous extracellular recording of spikes and astrocytes' glutamate transporter and K+ currents.

J Neurophysiol 2016 12 28;116(6):2706-2719. Epub 2016 Sep 28.

Department of Biotechnologies and Biosciences and Milan Center For Neuroscience (NeuroMI), University of Milano-Bicocca, Milan, Italy; and

Astrocytes uptake synaptically released glutamate with electrogenic transporters (GluT) and buffer the spike-dependent extracellular K excess with background K channels. We studied neuronal spikes and the slower astrocytic signals on reverberating neocortical cultures and organotypic slices from mouse brains. Spike trains and glial responses were simultaneously captured from individual sites of multielectrode arrays (MEA) by splitting the recorded traces into appropriate filters and reconstructing the original signal by deconvolution. GluT currents were identified by using dl-threo-β-benzyloxyaspartate (TBOA). K currents were blocked by 30 μM Ba, suggesting a major contribution of inwardly rectifying K currents. Both types of current were tightly correlated with the spike rate, and their astrocytic origin was tested in primary cultures by blocking glial proliferation with cytosine β-d-arabinofuranoside (AraC). The spike-related, time-locked inward and outward K currents in different regions of the astrocyte syncytium were consistent with the assumptions of the spatial K buffering model. In organotypic slices from ventral tegmental area and prefrontal cortex, the GluT current amplitudes exceeded those observed in primary cultures by several orders of magnitude, which allowed to directly measure transporter currents with a single electrode. Simultaneously measuring cell signals displaying widely different amplitudes and kinetics will help clarify the neuron-glia interplay and make it possible to follow the cross talk between different cell types in excitable as well as nonexcitable tissue.
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http://dx.doi.org/10.1152/jn.00509.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133298PMC
December 2016

Why we forget our dreams: Acetylcholine and norepinephrine in wakefulness and REM sleep.

Behav Brain Sci 2016 Jan;39:e202

Department of Biosciences,University of Milano,20133

The ascending fibers releasing norepinephrine and acetylcholine are highly active during wakefulness. In contrast, during rapid-eye-movement sleep, the neocortical tone is sustained mainly by acetylcholine. By comparing the different physiological features of the norepinephrine and acetylcholine systems in the light of the GANE (glutamate amplifies noradrenergic effects) model, we suggest how to interpret some functional differences between waking and rapid-eye-movement sleep.
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http://dx.doi.org/10.1017/S0140525X15001739DOI Listing
January 2016

Agonist and antagonist effects of tobacco-related nitrosamines on human α4β2 nicotinic acetylcholine receptors.

Front Pharmacol 2015 22;6:201. Epub 2015 Sep 22.

Department of Biotechnology and Biosciences, University of Milano-Bicocca Milano, Italy.

Regulation of the "neuronal" nicotinic acetylcholine receptors (nAChRs) is implicated in both tobacco addiction and smoking-dependent tumor promotion. Some of these effects are caused by the tobacco-derived N-nitrosamines, which are carcinogenic compounds that avidly bind to nAChRs. However, the functional effects of these drugs on specific nAChR subtypes are largely unknown. By using patch-clamp methods, we tested 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) on human α4β2 nAChRs. These latter are widely distributed in the mammalian brain and are also frequently expressed outside the nervous system. NNK behaved as a partial agonist, with an apparent EC50 of 16.7 μM. At 100 μM, it activated 16% of the maximal current activated by nicotine. When NNK was co-applied with nicotine, it potentiated the currents elicited by nicotine concentrations ≤ 100 nM. At higher concentrations of nicotine, NNK always inhibited the α4β2 nAChR. In contrast, NNN was a pure inhibitor of this nAChR subtype, with IC50 of approximately 1 nM in the presence of 10 μM nicotine. The effects of both NNK and NNN were mainly competitive and largely independent of Vm. The different actions of NNN and NNK must be taken into account when interpreting their biological effects in vitro and in vivo.
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http://dx.doi.org/10.3389/fphar.2015.00201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585029PMC
October 2015

Novel perspectives in cancer therapy: Targeting ion channels.

Drug Resist Updat 2015 Jul-Aug;21-22:11-9. Epub 2015 Jul 6.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, 20126, Italy.

By controlling ion fluxes at multiple time scales, ion channels shape rapid cell signals, such as action potential and synaptic transmission, as well as much slower processes, such as mitosis and cell migration. As is currently increasingly recognized, a variety of channel types are involved in cancer hallmarks, and regulate specific stages of neoplastic progression. Long-term in vitro work has established that inhibition of these ion channels impairs the growth of cancer cells. Recently, these studies have been followed up in vivo, hence revealing that ion channels constitute promising pharmacological targets in oncology. The channel proteins can be often accessed from the extracellular milieu, which allows use of lower drug doses and decrease untoward toxicity. However, because of the central physiological roles exerted by ion channels in excitable cells, other types of side effects may arise, the gravest of which is cardiac arrhythmia. A paradigmatic case is offered by Kv11.1 (hERG1) channels. HERG1 blockers attenuate the progression of both hematologic malignancies and solid tumors, but may also lead to the lengthening of the electrocardiographic QT interval, thus predisposing the patient to ventricular arrhythmias. These side effects can be avoided by specifically inhibiting the channel isoforms which are highly expressed in certain tumors, such as Kv11.1B and the neonatal forms of voltage-gated Na(+) channels. Preclinical studies are also being explored in breast and prostate cancer (targeting voltage-gated Na(+) channels), and gliomas (targeting CLC-3). Overall, the possible approaches to improve the efficacy and safety of ion channel targeting in oncology include: (1) the development of specific inhibitors for the channel subtypes expressed in specific tumors; (2) drug delivery into the tumor by using antibodies or nanotechnology-based approaches; (3) combination regimen therapy and (4) blocking specific conformational states of the ion channel. We believe that expanding this relatively neglected field of oncology research might lead to unforeseen therapeutic benefits for cancer patients.
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http://dx.doi.org/10.1016/j.drup.2015.06.002DOI Listing
June 2016

Commentary: "Comparison of spike parameters from optically identified GABAergic and glutamatergic neurons in sparse cortical cultures".

Front Cell Neurosci 2015 22;9:157. Epub 2015 Apr 22.

Department of Biotechnology and Biosciences, University of Milano-Bicocca Milan, Italy.

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http://dx.doi.org/10.3389/fncel.2015.00157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406083PMC
May 2015

Nocturnal frontal lobe epilepsy with paroxysmal arousals due to CHRNA2 loss of function.

Neurology 2015 Apr 13;84(15):1520-8. Epub 2015 Mar 13.

From the Pediatric Neurology and Neurogenetics Unit and Laboratories (V.C., L.C., F.M., C.M., R.G.), A. Meyer Children's Hospital-University of Florence; Department of Biotechnology and Biosciences and Center of Neuroscience (P.A., S.B., A.B.), Università di Milano-Bicocca, Milan; Neurophysiopathology Unit (M.A., A.M., C.L., F.P., A.R.), Sleep and Epilepsy Center, Department of Systems Medicine, University of Rome Tor Vergata General Hospital, Rome; IRCCS Neuromed (A.R.), Pozzilli, Isernia; and IRCCS Stella Maris Foundation (R.G.), Calambrone, Pisa, Italy.

Objective: We assessed the mutation frequency in nicotinic acetylcholine receptor (nAChR) subunits CHRNA4, CHRNB2, and CHRNA2 in a cohort including autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and sporadic nocturnal frontal lobe epilepsy (NFLE). Upon finding a novel mutation in CHRNA2 in a large family, we tested in vitro its functional effects.

Methods: We sequenced all the coding exons and their flanking intronic regions in 150 probands (73 NFLE, 77 ADNFLE), in most of whom diagnosis had been validated by EEG recording of seizures. Upon finding a missense mutation in CHRNA2, we measured whole-cell currents in human embryonic kidney cells in both wild-type and mutant α2β4 and α2β2 nAChR subtypes stimulated with nicotine.

Results: We found a c.889A>T (p.Ile297Phe) mutation in the proband (≈0.6% of the whole cohort) of a large ADNFLE family (1.2% of familial cases) and confirmed its segregation in all 6 living affected individuals. Video-EEG studies demonstrated sleep-related paroxysmal epileptic arousals in all mutation carriers. Oxcarbazepine treatment was effective in all. Whole-cell current density was reduced to about 40% in heterozygosity and to 0% in homozygosity, with minor effects on channel permeability and sensitivity to nicotine.

Conclusion: ADNFLE had previously been associated with CHRNA2 dysfunction in one family, in which a gain of function mutation was demonstrated. We confirm the causative role of CHRNA2 mutations in ADNFLE and demonstrate that also loss of function of α2 nAChRs may have pathogenic effects. CHRNA2 mutations are a rare cause of ADNFLE but this gene should be included in mutation screening.
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http://dx.doi.org/10.1212/WNL.0000000000001471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408286PMC
April 2015

The role of nicotinic acetylcholine receptors in autosomal dominant nocturnal frontal lobe epilepsy.

Front Physiol 2015 11;6:22. Epub 2015 Feb 11.

Department of Biosciences, University of Milano Milano, Italy.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a focal epilepsy with attacks typically arising in the frontal lobe during non-rapid eye movement (NREM) sleep. It is characterized by clusters of complex and stereotyped hypermotor seizures, frequently accompanied by sudden arousals. Cognitive and psychiatric symptoms may be also observed. Approximately 12% of the ADNFLE families carry mutations on genes coding for subunits of the heteromeric neuronal nicotinic receptors (nAChRs). This is consistent with the widespread expression of these receptors, particularly the α4β2(*) subtype, in the neocortex and thalamus. However, understanding how mutant nAChRs lead to partial frontal epilepsy is far from being straightforward because of the complexity of the cholinergic regulation in both developing and mature brains. The relation with the sleep-waking cycle must be also explained. We discuss some possible pathogenetic mechanisms in the light of recent advances about the nAChR role in prefrontal regions as well as the studies carried out in murine models of ADNFLE. Functional evidence points to alterations in prefrontal GABA release, and the synaptic unbalance probably arises during the cortical circuit maturation. Although most of the available functional evidence concerns mutations on nAChR subunit genes, other genes have been recently implicated in the disease, such as KCNT1 (coding for a Na(+)-dependent K(+) channel), DEPD5 (Disheveled, Egl-10 and Pleckstrin Domain-containing protein 5), and CRH (Corticotropin-Releasing Hormone). Overall, the uncertainties about both the etiology and the pathogenesis of ADNFLE point to the current gaps in our knowledge the regulation of neuronal networks in the cerebral cortex.
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http://dx.doi.org/10.3389/fphys.2015.00022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4324070PMC
February 2015

New pyrimido-indole compound CD-160130 preferentially inhibits the KV11.1B isoform and produces antileukemic effects without cardiotoxicity.

Mol Pharmacol 2015 Feb 19;87(2):183-96. Epub 2014 Nov 19.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy (L.G., S.P., A.A.); Department of Chemistry "Ugo Schiff," University of Florence, Florence, Italy (M.M., A.P.); DI.V.A.L. Toscana srl, Sesto Fiorentino, Italy (M.D.A., M.M.); Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom (R.C., R.K., J.S.M.); BlackSwan Pharma GmbH, Leipzig, Germany (W.T., K.M.); Oncohematology Laboratory, Department of Woman and Child Health, University of Padova, Padova, Italy (G.B.); and Department of Biotechnologies and Biosciences, University of Milano-Bicocca, Milan, Italy (A.B.)

KV11.1 (hERG1) channels are often overexpressed in human cancers. In leukemias, KV11.1 regulates pro-survival signals that promote resistance to chemotherapy, raising the possibility that inhibitors of KV11.1 could be therapeutically beneficial. However, because of the role of KV11.1 in cardiac repolarization, blocking these channels may cause cardiac arrhythmias. We show that CD-160130, a novel pyrimido-indole compound, blocks KV11.1 channels with a higher efficacy for the KV11.1 isoform B, in which the IC50 (1.8 μM) was approximately 10-fold lower than observed in KV11.1 isoform A. At this concentration, CD-160130 also had minor effects on Kir2.1, KV 1.3, Kv1.5, and KCa3.1. In vitro, CD-160130 induced leukemia cell apoptosis, and could overcome bone marrow mesenchymal stromal cell (MSC)-induced chemoresistance. This effect was caused by interference with the survival signaling pathways triggered by MSCs. In vivo, CD-160130 produced an antileukemic activity, stronger than that caused by cytarabine. Consistent with its atypical target specificity, CD-160130 did not bind to the main binding site of the arrhythmogenic KV11.1 blockers (the Phe656 pore residue). Importantly, in guinea pigs CD-160130 produced neither alteration of the cardiac action potential shape in dissociated cardiomyocytes nor any lengthening of the QT interval in vivo. Moreover, CD-160130 had no myelotoxicity on human bone marrow-derived cells. Therefore, CD-160130 is a promising first-in-class compound to attempt oncologic therapy without cardiotoxicity, based on targeting KV11.1. Because leukemia and cardiac cells tend to express different ratios of the A and B KV11.1 isoforms, the pharmacological properties of CD-160130 may depend, at least in part, on isoform specificity.
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http://dx.doi.org/10.1124/mol.114.094920DOI Listing
February 2015

Empirically founded genotype-phenotype maps from mammalian cyclic nucleotide-gated ion channels.

Authors:
Andrea Becchetti

J Theor Biol 2014 Dec 27;363:205-15. Epub 2014 Aug 27.

Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milano, Italy. Electronic address:

A major barrier between evolutionary and functional biology is the difficulty of determining appropriate genotype-phenotype-fitness maps, particularly in metazoans. Concrete perspectives towards unifying these approaches are offered by studies on the physiological systems that depend on ion channel dynamics. I focus on the cyclic nucleotide-gated (CNG) channels implicated in the photoreceptor's response to light. From an evolutionary standpoint, sensory systems offers interpretative advantages, as the relation between the sensory response and environment is relatively straightforward. For CNG and other ion channels, extensive data are available about the physiological consequences of scanning mutagenesis on sensitive protein domains, such as the conduction pore. Mutant ion channels can be easily studied in living cells, so that the relation between genotypes and phenotypes is less speculative than usual. By relying on relatively simple theoretical frameworks, I used these data to relate the sequence space with phenotypes at increasing hierarchical levels. These empirical genotype-phenotype and phenotype-phenotype landscapes became smoother at higher integration levels, especially in heterozygous condition. The epistatic interaction between sites was analyzed from double mutant constructs. Magnitude epistasis was common. Moreover, evidence of reciprocal sign epistasis and the presence of permissive mutations were also observed, which suggest how adaptive regions can be connected across maladaptive valleys. The approach I describe suggests a way to better relate the evolutionary dynamics with the underlying physiology.
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http://dx.doi.org/10.1016/j.jtbi.2014.08.038DOI Listing
December 2014

Multi-electrode array study of neuronal cultures expressing nicotinic β2-V287L subunits, linked to autosomal dominant nocturnal frontal lobe epilepsy. An in vitro model of spontaneous epilepsy.

Front Neural Circuits 2014 24;8:87. Epub 2014 Jul 24.

Department of Biotechnology and Biosciences, University of Milano-Bicocca Milano, Italy.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is a partial sleep-related epilepsy which can be caused by mutant neuronal nicotinic acetylcholine receptors (nAChR). We applied multi-electrode array (MEA) recording methods to study the spontaneous firing activity of neocortical cultures obtained from mice expressing or not (WT) an ADNFLE-linked nAChR subunit (β2-V287L). More than 100,000 up-states were recorded during experiments sampling from several thousand neurons. Data were analyzed by using a fast sliding-window procedure which computes histograms of the up-state durations. Differently from the WT, cultures expressing β2-V287L displayed long (10-32 s) synaptic-induced up-state firing events. The occurrence of such long up-states was prevented by both negative (gabazine, penicillin G) and positive (benzodiazepines) modulators of GABAA receptors. Carbamazepine (CBZ), a drug of choice in ADNFLE patients, also inhibited the long up-states at micromolar concentrations. In cultures expressing β2-V287L, no significant effect was observed on the action potential waveform either in the absence or in the presence of pharmacological treatment. Our results show that some aspects of the spontaneous hyperexcitability displayed by a murine model of a human channelopathy can be reproduced in neuronal cultures. In particular, our cultures represent an in vitro chronic model of spontaneous epileptiform activity, i.e., not requiring pre-treatment with convulsants. This opens the way to the study in vitro of the role of β2-V287L on synaptic formation. Moreover, our neocortical cultures on MEA platforms allow to determine the effects of prolonged pharmacological treatment on spontaneous network hyperexcitability (which is impossible in the short-living brain slices). Methods such as the one we illustrate in the present paper should also considerably facilitate the preliminary screening of antiepileptic drugs (AEDs), thereby reducing the number of in vivo experiments.
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http://dx.doi.org/10.3389/fncir.2014.00087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4109561PMC
March 2015

NAMI-A is highly cytotoxic toward leukaemia cell lines: evidence of inhibition of KCa 3.1 channels.

Dalton Trans 2014 Aug;43(32):12150-5

Department of Experimental and Clinical Medicine, University of Florence, Viale GB Morgagni 50, 50134 Firenze, Italy.

We report here that the established anticancer ruthenium(iii) complex NAMI-A induces potent and selective cytotoxic effects in a few leukaemia cell lines. These results sound very surprising after 20 years of intense studies on NAMI-A, commonly considered as a "non-cytotoxic" antimetastatic agent. In addition, evidence is given for selective inhibition of KCa 3.1 channels. The implications of these findings are discussed.
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http://dx.doi.org/10.1039/c4dt01356eDOI Listing
August 2014