Lucio Annunziato
Lucio Annunziato
affiliation: Università di Napoli Federico II
research area(s): Neuroscience, Molecular Biology
Course: Neurosciences
University/Istitution: Università di Napoli Federico II
Lucio Annunziato is Full Professor of Pharmacology, School of Medicine, "Federico II" University of Naples. He is coordinator of the Ph. D. Program in Neuroscience, School of Medicine, "Federico II" University of Naples. He is author of 188 publications in international peer-reviewed journals, including PNAS, Nature Cell Biology, J.Biol.Chem., Mol. Pharmacol., J. Neurochem., J. Pharmacol. Exp.Ther., Br. J. of Pharmacol., Stroke, Neuropharmacology, Endocrinology, J Neuroscience, J. Cerebral Blood Flow and Metabolism. He is member of the following research commissions: CEE, ISS, Ministero della Salute, CNR, MIUR, AIFA and of the ethic commission of Federico II University of Naples. He is referee for the European Commission for the Evaluation of Drugs (EMEA). He had the following research grants: Progetto Finalizzato Biotecnologie, CNR; Progetto Coordinato Agenzia 2000, CNR; Progetto Strategico Ricerca Finalizzata Alzheimer, Ministero Della Salute; Cofinanziamento MIUR; Progetto FIRB MIUR, Fondi Regionali. He was invited speaker to several World Congress of Pharmacology. He is president elected of Italian Society of Neuroscience. EDITORIAL ACTIVITY Section Editor: Current Opinion in Pharmacology Referee for Molecular Pharmacology; Journal Pharmacology Experimental Therapeutics; Molecular and Cellular Neuroscience; American Journal Physiology; Journal Biological Chemistry; Embo Reports; Journal Neuroscience; Journal of Neurophysiology; Journal of Neurochemistry; Stroke; Molecular and Cellular Neuroscience; Journal of Neuroscience Research; Neuroscience; Neuroendocrinology; Biochemical Pharmacology; Brain Research; Neuroscience Letters; Life Science; EMBO Reports, Journal of Endocrinology; Journal of Endocrinology Investigation; Pharmacology Biochemistry and Behaviour; Editor of "SIF Notizie" (1992-97), for the Italian Society of Pharmacology; Field Editor of Pharmacological Research. SOCIETY MEMBERSHIPS: Italian Society of Pharmacology; British Pharmacological Society; American Endocrine Society; American Physiological Society; American Society for Neuroscience; Executive Council of the Italian Research Association for Brain Aging; Italian Society for Neuroscience; Secretary General and Member of the Executive Council of the Italian Society of Pharmacology; Italian Delegate to the International Union of Pharmacology (IUPHAR).
The Sodium Calcium exchanger, because of its involvement in Na+ and Ca2+ homeostasis, plays a main role in the development of ischemic neuronal death. Previous studies demonstrated that, during in vitro hypoxia or in vivo ischemia, when the homeostasis of Na+ and Ca2+ is altered, the Sodium Calcium exchanger modulates the extent of neuronal damage. Moreover, compounds that activate the exchanger decrease the neuronal damage, while compounds capable to inhibit the exchanger exacerbate the lesion. Among the three different isoforms of the sodium calcium exchanger, only NCX1 and NCX3 are involved in the development of the ischemic damage and other neurodegenerative diseases.
Finally, recent evidences demonstrated that NCX1.4 e NCX1.5, two splicing forms of NCX1 are expressed in the brain and in particular in the neurons. 1) Identification of Transcriptional Factors Modulating ncx1 Gene Expression during ischemia. This research line is addressed to characterize the molecular determinants regulating ncx1 gene expression during the ischemic insult. ncx1 gene encodes for a protein ubiquitous expressed and involved in the maintenance of the ionic cellular homeostasis both physiological and pathological conditions. To address this issue the whole brain specific promoter region has been isolated and inserted in an eukaryotic expression vector in front of the luciferase gene. This construct has been transfected in the continue cell line SHSY-5Y in order to correlate the luciferase activity with promoters strength. To identify the transcriptional factors activated by the anoxic insult and modulating ncx1 expression, two sperimental approaches are routinely used: (1) site-specific mutagenesis of the promoter regions probably involved in complexes with peculiar transcriptional factors, and (2) band-shift and supershift experiments to identify these factors. 2) Functional characterization of neuronal sodium/calcium isoforms NCX1.4 and NCX1.5 in cellular and animal models of cerebral ischemia. The aim of the present research line is to study the functional role of NCX1.4 and NCX1.5 neuronal isoforms both in physiological and in phatological conditions such as cerebral ischemia by in vitro and in vivo aproaches. Cellular Models: SHSY-5Y and BHK cellsransfected with the two isoforms of NCX1. Animal models: transgenic mice e obtained in our laboratory in which NCX1.4 and NCX1.5 overexpression will be induced in neurons in specific different brain areas by administration of Tamoxifen. Functional studies: Single cell microfluorimetric assay and 45Ca2+ uptake experiments are routinely used to evaluate the functional properties of the two splice variants in experimental conditions able to activate sodium calcium exchanger in the two mode of operation, forward and reverse mode, depending on intracellular ionic homeostasis. These experiments are also conducted in the presence and in the absence of pharmacological tools able to modulate NCX activity, in order to better clarify the role of the two splicing variants. The experiments performed in the transgenic mice overexpressing NCX1.4 and NCX 1.5 are addressed to investigate the effects of the overexpression of these two splice variants of NCX1 on the extension of ischemic damage in mice exposed to permanent occlusion of middle cerebral artery (pMCAO). Finally, the subcellular localization of NCX1.4 and NCX 1.5 is also object of investigation in primary cultures of neurons derived from different brain areas of the transgenic mice overexpressing the two isoforms of NCX1. In these cells immunocytochemistry and confocal experiments are performed. 3) Study of transduction mechanisms responsible for the activity and expression of Sodium-Calcium exchanger (NCX) during differentiation and microglia activation. It is well known that Ca2+ ion plays a crucial role during cellular differentiation and microglia activation through the induction of several transduction pathways such as MAP-K, PI3"-K, CREB. NCX is a family of proteins playing a critical role in controlling intracellular Ca2+ homeostasis. The aims of the present research line are to study: a. The role of NCX isoforms in cellular differentiation and microglia activation; b. The transduction pathways involved in the modulation of NCX isoforms activity during cellular differentiation. c. To identify the cellular expression of NCX isoforms in the ischemic brain.
The experimantal approaches used to address these issues are immunocitochemistry, immunoistochemistry, Western Blotting analysis and Microfluorimetric techniques.
Molinaro P, Viggiano D, Nisticò R, Sirabella R, Secondo A, Boscia F, Pannaccione A, Scorziello A, Mehdawy B, Sokolow S, Herchuelz A, Di Renzo GF, Annunziato L (2011) Na+-Ca2+ Exchanger (NCX3) Knock-Out Mice Display an Impairment in Hippocampal Long-Term Potentiation and Spatial Learning and Memory. J Neurosci 31:7312-7321
Lignitto L, Carlucci A, Sepe M, Stefan E, Cuomo O, Nisticò R, Scorziello A, Savoia C, Garbi C, Annunziato L, Feliciello A (2011) Control of PKA stability and signalling by the RING ligase praja2. Nature Cell Biol. 13:412-22
Valsecchi V, Pignataro G, Del Prete A, Sirabella R, Matrone C, Boscia F, Scorziello A, Sisalli MJ, Esposito E, Zambrano N, Di Renzo G, Annunziato L (2011) NCX1 is a novel target gene for hypoxia-inducible factor-1 in ischemic brain preconditioning. Stroke 42:754-63
Secondo A, Molinaro P, Pannaccione A, Esposito A, Cantile M, Lippiello P, Sirabella R, Iwamoto T, Di Renzo G, Annunziato L (2011) Nitric oxide stimulates NCX1 and NCX2 but inhibits NCX3 isoform by three distinct molecular determinants. Mol Pharmacol 79:558-68
Pignataro G, Esposito E, Cuomo O, Sirabella R, Boscia F, Guida N, Di Renzo G, Annunziato L (2010) The NCX3 isoform of the Na+/Ca2+ exchanger contributes to neuroprotection elicited by ischemic postconditioning. J Cereb Blood Flow Metab 31:362-70
Esposito F, Pignataro G, Di Renzo G, Spinali A, Paccone A, Tedeschi G, Annunziato L (2010) Alcohol increases spontaneous BOLD signal fluctuations in the visual network. Neuroimage. 53:534-43
Boscia F, Gala R, Pannaccione A, Secondo A, Scorziello A, Di Renzo G, Annunziato L (2009) NCX1 expression and functional activity increase in microglia invading the infarct core. Stroke 40:3608-17
Secondo A, Pannaccione A, Molinaro P, Ambrosino P, Lippiello P, Esposito A, Cantile M, Khatri PR, Melisi D, Di Renzo G, Annunziato L (2009) Molecular pharmacology of the amiloride analog 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-n-[[(2,4-dimethylbenzyl)-amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) as a pan inhibitor of the Na+-Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in stably transfected cells. J Pharmacol Exp Ther 331:212-21
Sirabella R, Secondo A, Pannaccione A, Scorziello A, Valsecchi V, Adornetto A, Bilo L, Di Renzo G, Annunziato L (2009) Anoxia-induced NF-kappaB-dependent upregulation of NCX1 contributes to Ca2+ refilling into endoplasmic reticulum in cortical neurons. Stroke 40:922-9
Pignataro G, Scorziello A, Di Renzo G, Annunziato L (2009) Post-ischemic brain damage: effect of ischemic preconditioning and postconditioning and identification of potential candidates for stroke therapy. FEBS J. 276:46-57
Molinaro P, Cuomo O, Pignataro G, Boscia F, Sirabella R, Pannaccione A, Secondo A, Scorziello A, Adornetto A, Gala R, Viggiano D, Sokolow S, Herchuelz A, Schurmans S, Di Renzo G, Annunziato L (2008) "Target distruption of Na+-Ca2+ exchanger 3 (NCX3) gene leads to a worsening of ischemic brain damage". J Neurosc 28:1179-1184
Cuomo O, Gala R, Pignataro G, Boscia F, Secondo A, Scorziello A, Pannaccione A, Viggiano D, Adornetto A, Molinaro P, Li XF, Lytton J, Di Renzo G, Annunziato L (2008) "A critical role for the potassium-dependent sodium-calcium exchanger NCKX2 in protection against focal ischemic brain damage". J Neurosc 28:2053-63
Carlucci A, Adornetto A, Scorziello A, Viggiano D, Foca M, Cuomo O, Annunziato L, Gottesman M, Feliciello A (2008) "Proteolysis of AKAP121 regulates mitochondrial activity during cellular hypoxia and brain ischaemia". EMBO J. 27:1073-84
Molinaro P, Cuomo O, Pignataro G, Boscia F, Sirabella R, Pannaccione A, Secondo A, Scorziello A, Adornetto A, Gala R, Viggiano D, Sokolow S, Herchuelz A, Schurmans S, Di Renzo G, Annunziato L (2008) "Targeted disruption of Na+/Ca2+ exchanger 3 (NCX3) gene leads to a worsening of ischemic brain damage. J Neurosc 28:1179-1184
Formisano L, Saggese M, Secondo A, Sirabella R, Vito P, Valsecchi V, Molinaro P, Di Renzo G, Annunziato L (2008) "The two isoforms of the Na+/Ca2+ exchanger, NCX1 and NCX3, constitute novel additional targets for the prosurvival action of Akt/protein kinase B pathway". Mol Pharm 73:727-37
Scorziello A, Santillo M, Adornetto A, Dell'aversano C, Sirabella R, Damiano S, Canzoniero LM, Renzo GF, Annunziato L (2007) "NO-induced neuroprotection in ischemic preconditioning stimulates mitochondrial Mn-SOD activity and expression via RAS/ERK1/2 pathway". J Neuroch 103:1472-80
Cataldi M, Lariccia V, Marzaioli V, Cavaccini A, Curia G, Viggiano D, Canzoniero LM, Di Renzo G, Avoli M, Annunziato L (2007) "Zn2+ Slows Down Cav3.3 Gating Kinetics: Implications for Thalamocortical Activity". J Neurophysiol 98:2274-84
Pannaccione A, Boscia F, Scorziello A, Adornetto A, Castaldo P, Sirabella R, Taglialatela M, Di Renzo G, Annunziato L (2007) "Up-regulation and increased activity of KV3.4 channels and its accessory subunit MIRP2 induced by amyloid peptide is involved in apoptotic neuronal death. Mol Pharm 72:665-73
Project Title:
The aim proposed in the present project will be achieved (1) by synthesizing new compounds able to activate NCX, and (2) by characterizing their mechanism of action at the molecular level and their use as neuroprotectant in focal cerebral ischemia.
In the first part of the project, carried out in collaboration with the Department of Medicinal Chemistry, School of Pharmacy, Federico II University of Naples, we will select the most active compounds acting on NCX so far identified. The antagonist active compounds selected will be examined by algorithm methods in order to generate further molecules using Topliss scheme or bioisosteric replacement. In parallel, and depending on the results obtained using ligand binding domain, the most active candidate
will be analyzed by molecular docking (Autodock or Glide) and then modified in order to generate compounds able to activate NCX. Valuable drug candidates will be examined and, if required, modified in order to improve their physico-chemical characteristics (solubility, hydrophilic-lipophilic balance, membrane permeation) in order to optimise pharmacokinetic parameters. Collectively, these strategies will allow us to identify the chemical determinants required to act on new potential target sites on the NCX structure. Following this step, the activity of these new chemical synthesized compounds on the different NCX isoforms will be screened by FURA-2 microfluorimetric imaging, electrophysiology and 45Ca2+ radiotracer technique, in NCX stably transfected cells. These experiments will be carried out in the Department of Neuroscience. The second part of the project will allow us to identify and characterize the new potential target sites on NCX, by means of chimera and site-direct mutagenesis strategies, thus providing us instruments for the rational design of more effective and selective NCX ligands. In addition, the use of transgenic mice lacking and/or overexpressing NCX1, NCX2, NCX3 will allow us to identify the NCX isoform involved in stroke neuroprotection.
This strategy may lead to identify selective NCX activators that can target distinct isoforms of NCX. In the last part of the project we will define the therapeutic profile of compounds activating NCX in cerebral ischemia. In particular,
the therapeutic profile of newly synthesized compounds activating NCX will be investigated in in vitro and
in vivo models reproducing human brain ischemia. Newly synthesized compounds able to specifically act on a single NCX isoform will be evaluated for their possible neurobeneficial effect on primary neuronal or organotypic cultures subjected to experimental conditions mimicking cerebral ischemia. The same compounds will be then tested in wild-type mice subjected to focal cerebral ischemia. Finally, the generation of conditional tissue-specific knock-out mice for each NCX gene products will allow us to incontrovertibly validate the activity and specificity of newly synthesized drugs on NCX function under pathophysiological conditions. The experimental strategies above described are available and routinely used at the Division of Pharmacology of Department of Neuroscience.

Project Title:
Identification of vulnerability factors and pharmacological therapy of neuronal loss in Amyotrophic Lateral Sclerosis in human and transgenic animals
The main objectives of the OU NEUROPHARMA. will be: (1) To characterize the role played by the three isoforms of Na+/Ca2+ exchanger (NCX), a plasma membrane protein that plays a prominent role in controlling intracellular homeostasis of Ca2+ and Na+ ions, in motor neurons of animals transgenic for G93A SOD1 and of animals exposed to cycad neurotoxins including BMAA; (2) To correlate the dysregulation of intraneuronal Ca2+ ions with the mitochondrial dysfunction as assessed by mitochondrial membrane potential and mitochondrial Ca2+ measurements in motor neurons of animals transgenic for G93A SOD1 and of animals exposed to cycad neurotoxins including BMAA; (3) To evaluate the neuroprotective effect exerted by the growth factors GH, IGF-I and VEGF and their synthetic analogues in motor neurons of animals transgenic for G93A SOD1 and of animals exposed to cycad neurotoxins including BMAA; (4) To correlate the possible neuroprotective role exerted by the growth factors VEGF, GH, IGF-I on the expression and function of each of three NCX isoforms; (5) To generate conditional tissue-specific transgenic mice each lacking one of the three NCX isoforms and to breed these NCX knock-out mice with transgenic G93A SOD1 mice in order to obtain new transgenic mice characterized by both SOD1 mutation and heterozygosis for 1 and 3 NCX alleles. NCX knock-out mice will also be exposed to BMAA neurotoxin. These two approaches will allow to dissect the role played by the antiporter in these new animal models of ALS.
In order to characterize the phenotype and to follow longitudinally the progression of neurodegenerative processes in these new animal models of ALS, OU IBB-CNR will perform (6) MR relaxometry and diffusion imaging to monitor the progression of the disease in the above described animal models of ALS to detect the brain extension of global degeneration, including brainstem nuclei and spinal cord.
To translate the results of ALS preclinical studies obtained at the cellular and animal level, integrated by the imaging data, the OU NEUROLOGY will evaluate (7) the efficacy of GH treatment for 12 months versus placebo in reducing neuronal loss. In particular, the primary end point of this trial will be the quantification of neuronal death in the motor cortex of ALS patients, as measured by N-Acetyl-Aspartate/Creatine (NAA/Cre) reduction by MRSI, performed at OU SDN. Secondary end points will be the assessment of GH efficacy on motor function, quality of life, patient survival, and treatment safety.
In order to assess the efficacy of GH therapy and to follow longitudinally the progression of neurodegenerative processes in these patients, OU SDN, due to the limited resolution of MRS, will develop (8) a new technique for the integrated analysis of structural MRI along with MRS data to correct for the apparent decrease in the concentration of metabolites in atrophic cerebral structures in ALS patients. The development of this new analysis technique will allow the OOUU SDN/IBB-CNR (9) to correlate neuronal loss and brain morphological changes with disease progression and therapy effectiveness, allowing to clarify the respective weight of structural and metabolic alterations seen in ALS patients (e.g. if NAA loss matches atrophy severity and location, as detected by conventional MRI).