Fabio Benfenati

Istituto Italiano di Tecnologia, Genova, Liguria, Italy

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Publications (382)1723.74 Total impact


  • No preview · Article · Jan 2016 · Parkinsonism & Related Disorders
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    ABSTRACT: PRoline-Rich Transmembrane protein 2 (PRRT2) has been identified as single causative gene for a group of paroxysmal syndromes of infancy including epilepsy, paroxysmal movement disorders and migraine. Based on topology predictions, PRRT2 has been assigned to the recently characterized family of Dispanins, whose members share the 2-transmembrane domain topology with a large N- and short C-termini oriented towards the outside of the cell. Since PRRT2 plays a role at synapse, it is important to confirm the exact orientation of its N- and C-termini with respect to the plasma membrane, in order to get clues on its possible function. Using a combination of different experimental approaches, including live immunolabeling, immunogold electron microscopy, surface biotinylation and computational modeling, we demonstrate a novel topology for this protein. PRRT2 is a type II transmembrane protein, whereby only the second hydrophobic segment spans the plasma membrane while the first one is associated with the internal surface of the membrane and forms a helix-loop-helix structure without crossing it. Most importantly, the large proline-rich N-terminal domain is not exposed to the extracellular space, but is rather localized intracellularly and only the short C terminus is extracellular (Ncyt/Cexo topology). Accordingly, we showed that PRRT2 interacts with the SH3 domain bearing protein Intersectin 1, an intracellular protein involved in synaptic vesicle cycling. These findings will contribute to clarify the PRRT2 role at synapse and the understanding of pathogenic mechanisms on the basis of PRRT2 related neurological disorders.
    Preview · Article · Jan 2016 · Journal of Biological Chemistry
  • Antonella Marte · Mirko Messa · Fabio Benfenati · Franco Onofri
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    ABSTRACT: Synapsins (Syns) are synaptic vesicle-associated phosphoproteins involved in neuronal development and neurotransmitter release. While Syns are implicated in the regulation of brain-derived neurotrophic factor (BDNF)-induced neurotransmitter release, their role in the BDNF developmental effects has not been fully elucidated. By using primary cortical neurons from Syn I knockout (KO) and Syn I/II/III KO mice, we studied the effects of BDNF and nerve growth factor (NGF) on axonal growth. While NGF had similar effects in all genotypes, BDNF induced significant differences in Syn KO axonal outgrowth compared to wild type (WT), an effect that was rescued by the re-expression of Syn I. Moreover, the significant increase of axonal branching induced by BDNF in WT neurons was not detectable in Syn KO neurons. The expression analysis of BDNF receptors in Syn KO neurons revealed a significant decrease of the full length TrkB receptor and an increase in the levels of the truncated TrkB.t1 isoform and p75(NTR) associated with a marked reduction of the BDNF-induced MAPK/Erk activation. By using the Trk inhibitor K252a, we demonstrated that these differences in BDNF effects were dependent on a TrkB/p75(NTR) imbalance. The data indicate that Syn I plays a pivotal role in the BDNF signal transduction during axonal growth.
    No preview · Article · Jan 2016 · Molecular Neurobiology
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    ABSTRACT: Recycling of synaptic vesicles (SVs) is a fundamental step in the process of neurotransmission. Endocytosed SV can travel directly into the recycling pool or recycle through endosomes but little is known about the molecular actors regulating the switch between these SV recycling routes. ADP ribosylation factor 6 (Arf6) is a small GTPase known to participate in constitutive trafficking between plasma membrane and early endosomes. Here we have morphologically and functionally investigated Arf6-silenced hippocampal synapses and found an activity dependent accumulation of synaptic endosome-like organelles and increased release-competent docked SVs. These features were phenocopied by pharmacological blockage of Arf6 activation. The data reveal an unexpected role for this small GTPase in reducing the size of the readily releasable pool of SVs and in channeling retrieved SVs toward direct recycling rather than endosomal sorting. We propose that Arf6 acts at the presynapse to define the fate of an endocytosed SV.
    Preview · Article · Jan 2016 · eLife Sciences
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    ABSTRACT: Optogenetics provides new ways to activate gene transcription; however, no attempts have been made as yet to modulate mammalian transcription factors. We report the light-mediated regulation of the repressor element 1 (RE1)-silencing transcription factor (REST), a master regulator of neural genes. To tune REST activity, we selected two protein domains that impair REST-DNA binding or recruitment of the cofactor mSin3a. Computational modeling guided the fusion of the inhibitory domains to the light-sensitive Avena sativa light-oxygen-voltage-sensing (LOV) 2-phototrophin 1 (AsLOV2). By expressing AsLOV2 chimeras in Neuro2a cells, we achieved light-dependent modulation of REST target genes that was associated with an improved neural differentiation. In primary neurons, light-mediated REST inhibition increased Na(+)-channel 1.2 and brain-derived neurotrophic factor transcription and boosted Na(+) currents and neuronal firing. This optogenetic approach allows the coordinated expression of a cluster of genes impinging on neuronal activity, providing a tool for studying neuronal physiology and correcting gene expression changes taking place in brain diseases.
    Full-text · Article · Dec 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Objective: To report on the identification of intrathecally synthesized immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies to synapsin, a synaptic vesicle-associated protein, in a patient with limbic encephalitis. Methods: Methods included clinical characterization, indirect immunofluorescence, immunoprecipitation, mass spectrometry, immunoblots of wild-type and synapsin I/II/III knockout mice, and cell-based assays with synapsin Ia, Ib, IIa, and IIb plasmids. Results: A 69-year-old man presented with confusion, disorientation, seizures, and left hippocampal hyperintensities on MRI. CSF examinations revealed an intrathecal IgA and IgG synthesis. Except for IgG antibodies to voltage-gated potassium channels in CSF, screening for known neuronal autoantibodies in serum and CSF was negative. However, indirect immunofluorescence using the patient's CSF showed binding of IgA to mouse hippocampus, amygdala, and cerebellum. Immunoprecipitation with CSF IgA followed by mass spectrometry identified synapsin as autoantigenic target. Knockout tissues and cell-based assays confirmed that IgA and IgG in the patient's CSF and serum reacted with synapsin Ia, Ib, and IIa. Calculation of antibody indices proved intrathecal synthesis of anti-synapsin IgA and IgG. The patient responded clinically to immunotherapy but developed left hippocampal atrophy. CSF IgA or IgG of the patient did not bind to live, unfixed, and nonpermeabilized mouse hippocampal neurons, compatible with synapsin being an intracellular antigen. Conclusions: This report identifies isoforms of the synaptic vesicle-associated protein synapsin as targets of intrathecally produced IgA and IgG antibodies in a patient with limbic encephalitis. Future studies should clarify the prevalence and pathogenic relevance of anti-synapsin antibodies in limbic encephalitis.
    Full-text · Article · Nov 2015
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    ABSTRACT: Transplantation of GABAergic interneurons (INs) can provide long-term functional benefits in animal models of epilepsy and other neurological disorders. Whereas GABAergic INs can be differentiated from embryonic stem cells, alternative sources of GABAergic INs may be more tractable for disease modeling and transplantation. We identified five factors (Foxg1, Sox2, Ascl1, Dlx5, and Lhx6) that convert mouse fibroblasts into induced GABAergic INs (iGABA-INs) possessing molecular signatures of telencephalic INs. Factor overexpression activates transcriptional networks required for GABAergic fate specification. iGABA-INs display progressively maturing firing patterns comparable to cortical INs, form functional synapses, and release GABA. Importantly, iGABA-INs survive and mature upon being grafted into mouse hippocampus. Optogenetic stimulation demonstrated functional integration of grafted iGABA-INs into host circuitry, triggering inhibition of host granule neuron activity. These five factors also converted human cells into functional GABAergic INs. These properties suggest that iGABA-INs have potential for disease modeling and cell-based therapeutic approaches to neurological disorders.
    Full-text · Article · Nov 2015 · Cell stem cell
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    ABSTRACT: Synapsin III (SynIII) is a neuron-specific phosphoprotein that plays a unique role in neuronal development. SynIII is phosphorylated by cAMP-dependent protein kinase (PKA) at a highly conserved phosphorylation site and by cyclin-dependent kinase-5 (Cdk5) at a newly described site. Although SynIII is known to be involved in axon elongation in vitro, the role of its phosphorylation byPKAand Cdk5 in the modulation of this process is unknown.Weexpressed either wild-type (WT) or phosphorylation-site mutants of SynIII in primary SynIII knock-out (KO) mouse neurons at early stages of in vitro development. Whereas the neurite elongation phenotype of SynIII KO neurons was fully rescued by the expression ofWTSynIII, the expression of nonphosphorylatable and pseudo-phosphorylated PKA mutants was ineffective. Also, the nonphosphorylatable Cdk5 mutant was unable to rescue the neurite elongation phenotype of SynIII KO neurons. By contrast, the pseudo-phosphorylated mutant rescued the delay in neuronal maturation and axonal elongation, revealing a Cdk5- dependent regulation of SynIII function. Interestingly, SynIII KO neurons also exhibited decreased survival that was fully rescued by the expression of WT SynIII, but not by its phosphorylation mutants, and was associated with increased activated caspase3 and altered tropomyosin receptor kinase B isoform expression. These results indicate that PKA and Cdk5 phosphorylation is required for the physiological action of SynIII on axon specification and neurite outgrowth and that the expression of a functional SynIII is crucial for cell survival.
    No preview · Article · Sep 2015 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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    ABSTRACT: The long term stability of the interface between neural tissue and implanted microelectrodes is very important both for research and clinical purposes. To address this challenge we developed a probe composed by an ultrathin and ultra-flexible wire as tether and a low- impedance gold sphere microelectrode as recording/stimulation site. We further improved the microsphere performance using PEDOT-CNT composite coating and we mimic the biological composition of the host tissue by coating the probe with fibroblast cells.
    No preview · Article · Jul 2015
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    G. Lanzani · M.R. Antognazza · N. Martino · D. Ghezzi · F. Benfenati
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    ABSTRACT: The capability to selectively affect vital functions in cell networks and sub-cell compartments in vitro or in vivo is a mission critical tool in neuroscience and medicine. Optical excitation is one of the main strategies used to achieve high spatial and temporal resolution. In the following we describe recent results and future approaches of cell photostimulation mediated by organic semiconducting polymers.
    Full-text · Article · Jul 2015
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    ABSTRACT: We recently reported a Q555X mutation of synapsin 1 (SYN1) on chromosome Xp11-q21 in a family segregating partial epilepsy and autistic spectrum disorder. Herein, we provide a detailed description of the epileptic syndrome in the original family. A total of 34 members from a large French-Canadian family were evaluated. Family members with seizures or epilepsy underwent (when possible) clinical, neuropsychological, electrophysiologic, and neuroimaging assessments. Epilepsy was diagnosed in 10 family members (4 deceased, 6 living). In addition to occasional spontaneous complex partial seizures, seven family members clearly had reflex seizures triggered by bathing or showering. Hippocampal atrophy was found in two of five epileptic family members family members who underwent magnetic resonance (MR) imaging. Video-electroencephalography (EEG) recordings of three triggered seizures in two affected members showed rhythmic theta activity over temporal head regions. Ictal single-photon emission computed tomography (SPECT) showed temporoinsular perfusion changes. Detailed neuropsychological assessments revealed that SYN1 Q555X male mutation carriers showed specific language impairment and mild autistic spectrum disorder. Female carriers also exhibited reading impairments and febrile seizures but no chronic epilepsy. Available evidence suggests that impaired SYN1 function is associated with hyperexcitability of the temporoinsular network and disturbance of high mental functions such as language and social interaction. The presence of reflex bathing seizures, a most peculiar clinical feature, could be helpful in identifying other patients with this syndrome. Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.
    No preview · Article · Jun 2015 · Epilepsia
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    ABSTRACT: Kidins220/ ARMS acts as a signaling platform at the plasma membrane and is implicated in a multitude of neuronal functions, including the control of neuronal activity. Here, we used the Kidins220(-/-) mouse model to study the effects of Kidins220 ablation on neuronal excitability. Multi-electrode array recordings showed reduced evoked spiking activity in Kidins220(-/-) hippocampal networks, which was compatible with the increased excitability of GABAergic neurons determined by current-clamp recordings. Spike waveform analysis further indicated an increased sodium conductance in this neuronal subpopulation. Kidins220 association with brain voltage-gated sodium channels was shown by co-immunoprecipitation experiments and Na(+) current recordings in transfected HEK293 cells, which revealed dramatic alterations of kinetics and voltage-dependence. Finally, an in silico interneuronal model incorporating the Kidins220-induced Na(+) current alterations reproduced the firing phenotype observed in Kidins220(-/-) neurons. These results identify Kidins220 as a novel modulator of Nav channel activity, broadening our understanding of the molecular mechanisms regulating network excitability. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Full-text · Article · Jun 2015 · Journal of Biological Chemistry
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    ABSTRACT: The main neuropathological features of Parkinson's disease (PD) are dopaminergic nigrostriatal neuron degeneration and intraneuronal and intraneuritic proteinaceous inclusions named Lewy bodies and Lewy neurites, mainly composed of α-synuclein (α-syn). The neuronal phosphoprotein synapsin III, is a pivotal regulator of dopamine (DA) neuron synaptic function. Here, we show that α-syn interacts with and modulates synapsin III. The absence of α-syn causes a selective increase and redistribution of synapsin III and changes the organization of synaptic vesicle pools in DA neurons. In α-syn null mice, the alterations of synapsin III induce an increased locomotor response to the stimulation of synapsin-dependent DA overflow, despite these mice show decreased basal and depolarization-dependent striatal DA release. Of note, synapsin III seems to be involved in α-syn aggregation, that also coaxes its increase and redistribution. Furthermore, synapsin III is accumulated in the caudate/putamen of PD patients. These findings support a reciprocal modulatory interaction of α-syn and synapsin III in the regulation of DA neuron synaptic function. © 2015. Published by The Company of Biologists Ltd.
    Full-text · Article · May 2015 · Journal of Cell Science
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    ABSTRACT: Synapsin III (SynIII) is a phosphoprotein that is highly expressed at early stages of neuronal development. Whereas in vitro evidence suggests a role for SynIII in neuronal differentiation, in vivo evidence is lacking. Here, we demonstrate that in vivo downregulation of SynIII expression affects neuronal migration and orientation. By contrast, SynIII overexpression affects neuronal migration, but not orientation. We identify a cyclin-dependent kinase-5 (CDK5) phosphorylation site on SynIII and use phosphomutant rescue experiments to demonstrate its role in SynIII function. Finally, we show that SynIII phosphorylation at the CDK5 site is induced by activation of the semaphorin-3A (Sema3A) pathway, which is implicated in migration and orientation of cortical pyramidal neurons (PNs) and is known to activate CDK5. Thus, fine-tuning of SynIII expression and phosphorylation by CDK5 activation through Sema3A activity is essential for proper neuronal migration and orientation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Apr 2015 · Cell Reports
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    ABSTRACT: Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications, bearing a huge potential, from basic researches to clinical applications. In particular, light sensitive conjugated polymers can be exploited as a new approach for optical modulation of cellular activity. In this work we focus on light-induced changes in the membrane potential of Human Embryonic Kidney (HEK-293) cells grown on top of a poly(3-hexylthiophene) (P3HT) thin film. On top of a capacitive charging of the polymer interface, we identify and fully characterize two concomitant mechanisms, leading to membrane depolarization and hyperpolarisation, both mediated by a thermal effect. Our results can be usefully exploited in the creation of a new platform for light-controlled cell manipulation, with possible applications in neuroscience and medicine.
    Full-text · Article · Mar 2015 · Scientific Reports
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    ABSTRACT: Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications, bearing a huge potential, from basic researches to clinical applications. In particular, light sensitive conjugated polymers can be exploited as a new approach for optical modulation of cellular activity. In this work we focus on light-induced changes in the membrane potential of Human Embryonic Kidney (HEK-293) cells grown on top of a poly(3-hexylthiophene) (P3HT) thin film. On top of a capacitive charging of the polymer interface, we identify and fully characterize two concomitant mechanisms, leading to membrane depolarization and hyperpolarisation, both mediated by a thermal effect. Our results can be usefully exploited in the creation of a new platform for light-controlled cell manipulation, with possible applications in neuroscience and medicine.
    Preview · Article · Mar 2015
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    ABSTRACT: Trace Amine-Associated Receptor 1 (TAAR1) is a G protein-coupled receptor expressed in the mammalian brain and known to influence subcortical monoaminergic transmission. Monoamines, such as dopamine, play also an important role within the prefrontal cortex (PFC) circuitry, which is critically involved in high order cognitive processes. TAAR1 selective ligands have shown potential antipsychotic, antidepressant and pro-cognitive effects in experimental animal models; however, it remains unclear if TAAR1 can affect PFC-related processes and functions. In this study, we document distinct pattern of expression of TAAR1 in the PFC, as well as altered subunit composition and deficient functionality of the glutamate N-methyl-D-aspartate (NMDA) receptors in the pyramidal neurons of layer V of PFC in mice lacking TAAR1. The dysregulated cortical glutamate transmission in TAAR1-KO mice was associated with aberrant behaviors in several tests, indicating a perseverative and impulsive phenotype of mutants. Conversely, pharmacological activation of TAAR1 with selective agonists reduced premature impulsive responses observed in the fixed-interval conditioning schedule in normal mice. Our study indicates that TAAR1 plays an important role in the modulation of NMDA receptor-mediated glutamate transmission in the PFC and related functions. Furthermore, these data suggest that development of TAAR1-based drugs could provide a novel therapeutic approach for the treatment of disorders related to aberrant cortical functions.Neuropsychopharmacology accepted article preview online, 09 March 2015. doi:10.1038/npp.2015.65.
    Full-text · Article · Mar 2015 · Neuropsychopharmacology: official publication of the American College of Neuropsychopharmacology
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    Dataset: bhu301supp

    Full-text · Dataset · Jan 2015
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    ABSTRACT: The functional consequence of γ-aminobutyric acid (GABA) release at mossy fiber terminals is still a debated topic. Here, we provide multiple evidence of GABA release in cultured autaptic hippocampal granule cells. In ∼50% of the excitatory autaptic neurons, GABA, VGAT, or GAD67 colocalized with vesicular glutamate transporter 1-positive puncta, where both GABAB and GABAA receptors (Rs) were present. Patch-clamp recordings showed a clear enhancement of autaptic excitatory postsynaptic currents in response to the application of the GABABR antagonist CGP58845 only in neurons positive to the selective granule cell marker Prox1, and expressing low levels of GAD67. Indeed, GCP non-responsive excitatory autaptic neurons were both Prox1- and GAD67-negative. Although the amount of released GABA was not sufficient to activate functional postsynaptic GABAARs, it effectively activated presynaptic GABABRs that maintain a tonic "brake" on the probability of release and on the size of the readily releasable pool and contributed to resting potential hyperpolarization possibly through extrasynaptic GABAAR activation. The autocrine inhibition exerted by GABABRs on glutamate release enhanced both paired-pulse facilitation and post-tetanic potentiation. Such GABABR-mediated changes in short-term plasticity confer to immature granule cells the capability to modulate their filtering properties in an activity-dependent fashion, with remarkable consequences on the dynamic behavior of neural circuits. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
    Full-text · Article · Jan 2015 · Cerebral Cortex
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    ABSTRACT: The synapsins constitute a family of evolutionarily conserved neuronal phosphoproteins associated with the cytosolic surface of synaptic vesicles. In mammals, the family comprises three members encoded by distinct genes that give rise to various splicing isoforms. In the central nervous system, the vast majority of neurons expresses at least one synapsin isoform. However, the functions of these proteins are not fully understood to date. Given their ability to bind both the vesicular membrane and actin filaments in a phosphorylation-dependent manner, the classical role attributed to synapsins is the reversible anchorage of synaptic vesicles to the cytoskeletal matrix present in the presynaptic terminal. However, recent evidences suggest the implication of synapsins in other aspects of the synaptic vesicle life cycle, such as docking, fusion and recycling. Genetic manipulation of synapsins in various in vitro and in vivo models has proved that they are dispensable for the proper development of functional neuronal networks but are essential modulators of synaptic neurotransmission and play differential roles at excitatory and inhibitory synapses. Indeed, mice lacking synapsins are viable and do not display gross brain abnormalities but exhibit generalised epileptic seizures as well as autism-related behavioural abnormalities. Consistently, several mutations have been identified in SYN1 and SYN2 genes in patients affected by epilepsy and/or autism spectrum disorders. This chapter overviews the current knowledge about synapsin structure and function in the modulation of synaptic vesicle release, as well as the mechanisms leading to synaptic pathology when their properties are altered.
    No preview · Chapter · Jan 2015

Publication Stats

14k Citations
1,723.74 Total Impact Points

Institutions

  • 2007-2016
    • Istituto Italiano di Tecnologia
      • Department of Neuroscience and Brain Technologies
      Genova, Liguria, Italy
  • 2001-2015
    • Università degli Studi di Genova
      • Dipartimento di Medicina sperimentale (DIMES)
      Genova, Liguria, Italy
  • 2012
    • Harvard University
      Cambridge, Massachusetts, United States
  • 2002-2007
    • Università Vita-Salute San Raffaele
      • Faculty of Psychology
      Milano, Lombardy, Italy
  • 1993-2006
    • University of Rome Tor Vergata
      Roma, Latium, Italy
    • Università Degli Studi Roma Tre
      Roma, Latium, Italy
  • 1988-2005
    • The Rockefeller University
      • Laboratory of Molecular and Cellular Neuroscience
      New York, New York, United States
  • 1995-2000
    • San Raffaele Scientific Institute
      Milano, Lombardy, Italy
  • 1992-1993
    • University of Milan
      • Center of Cytopharmacology CNR
      Milano, Lombardy, Italy
  • 1981-1993
    • Università degli Studi di Modena e Reggio Emilia
      • Department of Biomedical, Metabolical and Neurosciences
      Modène, Emilia-Romagna, Italy
  • 1991
    • Stockholm University
      Tukholma, Stockholm, Sweden
  • 1982-1990
    • Karolinska Institutet
      • Institutionen för neurovetenskap
      Solna, Stockholm, Sweden
  • 1982-1986
    • Mario Negri Institute for Pharmacological Research
      Milano, Lombardy, Italy
  • 1978-1980
    • University of Bologna
      Bolonia, Emilia-Romagna, Italy