1 Mapping of Italian research excellence in Neurodegenerative Diseases
2 1. MECHANISMS OF NEURODEGENERATION A. Macromolecular interactions and Neurodegeneration 1. Public health. Therapeutic strategies for the prevention and tratment of neurodegenerative disease. Molecular basis of amyloid aggregation 2. Protein conformational transitions that trigger the aggregation processes associated to neurodegenerative diseases : a nanotechnological approach Pier Luigi San Biagio Bruno Samorì 3. Myelin proteins and demyelinating deseases Eugenia Polverini 4. Conformational transitions of proteins from native Maria Grazia Bridelli to amyloid form 5. Role of altered composition of plasma membrane Sandro Sonnino complex lipids in lipid-protein interactions, membrane organization and neurodegeneration 6. Alterations of proteolytic pathways in neurodegenerative diseases" 7. Optical and biomolecular methods for the functional investigation of neural circuits in vivo 8. Molecular mechanisms in neurotransmission: effects in neuron and glia phenotypes in synaptic efficiency and synapropathy 9. Presynaptic molecular machinery regulationg glutamate release an pathophysiological mechanism in amyotrophic lateral sclerosis 10. Toward exploiting the screening potential of mirnome for the identification of genes and pathways involved in neurodegenerative disorders 11. Identification of signaling pathways in neurodegenerative proteinopathies 12. Neurotensin and neurotensin antagonists in an animal model of Parkinson disease: therapeutic perspectives and role of NMDA/neurotensin interaction. B. Neurotoxic stimuli 1. CSF Glycomics in AD and other neurodegenerative disease 2. Intracellular signalling during neurodegenerative events triggered upon APP overexpression Angelo Poletti Antonio Malgaroli Gian Giacomo Consalez Flavia Valtorta Maurizio Popoli Daniela Tardito Maria Penuto Tanganelli Sergio Domenico Garozzo Maurizio Taglialatela Claudio Russo 3. Effect of Age and neurodegenerative disorders in central neurotrasmission: focus on neurotrasmitter relase from neuron and glia Mario Marchi
3 4. Developing competitive in vitro and vivo models to study Alzheimer s disease 5. Evaluation of mesenchymal stell cells (MSCs) effect in Alzheimer s disease rat models competitive in vitro and vivo studies 6. New Tratment strategies (Neurodegeneration and Neuroprotection) 7. Pathways leading to tau pathology Role of nuclear tau in neurodegeneration in fronto-temporal dementia 8. Cholesterol and amyloid-beta: is there a relation? - Specific type 4 phosphodiestrerase inhibitors and their impact on the production of amyloid-beta - Cholesterol and amyloid-beta: is there a relation? - Role of non muscle myosin IIB in the processing of the amyloid precursor protein (APP) Francesca Ruberti Mariarosaria Miloso Patrizia Hrelia Fabrizio Tagliavini Roberta Ricciarelli 9. Basic research - Genetic susceptibility to Alzheimer disease and genome wide association studies - Biobanking Dario Finazzi 10. Neurotoxic stimuli, synaptic dysfunction Calro Sala 11. Neuroprotection by chemokines Cristina Limatola 12. Phatogenesis and therapeutic targets for SBMA Fabio Benfenati Mara Pennuto Chiara Scaramuzzino 13. Malattia di Alzheimer e trasduzione del segnale legata al calcio 14. Mechanisms of neurodegeneration and enhancement of adult neurogenesis to improve cognitive functions in down syndrome Armando Genazzani Andrea Contestabile 15. Pathological mechanisms of neurodegeneration Laura Gasparini triggered by tau and beta-amyloid 16. Oxidative stress and neurodegeneration Michele Mazzanti C. Neuro inflammation 1. Role of vascular inflammation and leukocyte Gabriela Constantin trafficking in neurodegenerative diseases 2. Regional vulmerability of the brain to Marina Bentivoglio neurodegeneration and immune regulatory mechanisms 3. Neurotoxic stimuli: role of astrocytes and microglia Michela Matteoli 4. Gender and hormonal/endocrine signals in Elisabetta Vegeto neuroinflammation role in alzheimer s disease and
4 brain ageing 5. Role of pro-and anti- inflammatory cytokines in the prognosis and progression of neurodegenerative diseases 6. Study of the role of pro-inflammatory chemokines in neuroinflammatory disorders and analysis of the therapeutic potential of chemokine synthesis inhibitors 7. Role of inflammation and microglial activation in neurodegenerative diseases 8. Role of inflammation in Alzheimer s disease: study of pro-inflammatory cytokines for the identification of new pathogenic and diagnostic targets Barbara Viviani Claudio Milanese Luisa Minghetti Paola Bossù D. Oxidative Stress and neurodegeneration 1. Dopamine and oxidative stress in the pathogenesis of Parkinson's disease 2. Mitochondria, apoptosis and neurodegeneration: characterization of mitochondrial dysfunction in neurodegenerative diseases - Characterization of the molecular mechanisms underlying aging, cell senescence and neurodegenerative diseases 3. Structural organization of the mitochondrial respiratory chain, generation of reactive oxygen species and neurodegeneration 4. Post-genomic biochemestry of neurodegenerative diseases Marco Bisaglia Antonella Bobba Giorgio Lenaz Annalisa Santucci E. Trophic factors involvement in the neurodegeneration process F. Endocrine and Metabolic Factors 1. mirna as sensors of neuronal stress and modulators of synaptic plasticità and memory deficits 2. Developing competitive animal models to study Alzheimer s disease 3. Genetic susceptibility to Alzheimer s disease (AD) in women: AD as a gender disease 4. Genetic susceptibility to Alzheimer s disease (Epigenetic maks of susceptibility to Alzheimer disese) 5. Role of altered lipid and cholesterol metabolism in neurodegenerations Paola Fragapane Roberto Rimondini Rosa Maria Corbo Lucia Migliore Laura Colombaioni
5 6. Role of neuroactive steroids in the neurodegeneration process 7. Selective Alzheimer Disease Indicator -1(Seladin- ò1): a liker between cholesterol, oxidative stress and Alzheimer s disease 8. Nuclear receptors, coregulators, neuroactive steroids and epigenetics in neurodegenerative disease associated to metabolic disorders Roberto Cosimo Melcangi Roberto Maggi Donatella Caruso G. Cell interactions 1. The Astrocyte, not just a Bystander in Alzheimer s disease 2. Synapse and Amyloid-beta: effects on astrocyte processes, nerve terminals and network synaptic activity Ubaldo Armato Manuela Marcoli 3. Alzheimer s disease, Ageing Luciano Domenici 4. Synaptic deficits in neurodegenerative disease Evelina Chieregatti H. Intracellular Traffiking 1. Membrane traffic and neurodegenerative diseases: Cecilia Bucci role of Rab proteins and their effectors 2. Basic research. Neurodegenerative diseases Paolo Macchi I. Animal models 1. Developing competitive animal models to study Adalberto Merighi Alzheimer s disease 2. Synaptic dysfunction in neurodegeneration: Monica DI Luca characterization of gene product in functional synaptic networks under physiological and patological conditions 3. Study of early onset Alzheimer disease in a model Renata Bartesaghi for Down syndrome, the Ts65Dn mouse 4. Iron and Neurodegeneration Sonia Levi 5. Non neurotoxic activity of Beta amyloid in animal Stefano Govoni models 6. Neurochemical and morphological in vivo and in Tiziana Antonelli vitro models in neurodegenerative diseases.
6 2. PHARMACOLOGY A. Drug Design 1. Elementary mechanisms of neurodegenerative diseases 2. Innovative drug discovery strategies for Alzheimer s disease 3. Development of ligands specifically targeting the Translocator Protein 18 kda in Alzheimer disease 4. Structural studies of acetylcholinesterases and their inibitors: implicantions for the design of new antialzheimer drugs Nino Russo Carlo Melchiorre Federico Da Settimo Alberto Cassetta B. Drug screening 1. Developemt of competitive animal models for AD- GLP grade animal facility and behavioural testing 2. Yeast model to seek for molecules which can reduce mitochondrial mutability 3. Innovative technologies for the therapy of the neurodegenerative diseases. C. Drug Delivery 1. Nanotechnology for health: application in the brain diseases 2. Hybrid drug containing reservoirs for long term release in brain diseases 3. New nanodevices targeting beta-amyloid and overcoming the blood-brain barrier for the therapy of Alzheimer disease Laura Calzà Iliana Ferrero Fortunati Luca Ferraro MA Vandelli Giovanni Tosi Angelo Montenero Massimo Masserini D. Novel Drugs 1. Lamberto Maffei 2. Synthesis and screening of new AChE/BuChE Aldo Andreani inhibitors 3. Drug design and synthesis of therapeutic agent for Olga Bruno neurodegenerative disorders, particularly for Alzheimer disease 4. Development of a novel therapeutic strategy for AD based on a natural variant of amyloid beta that Fabrizio Tagliavini
7 hinders amyloidogenesis 5. Second generation recombinant Abeta peptide immunogens as prototype vaccines for AD treatment Simone Ottonello 6. Pediatric neurodegenerative diseases Maurizio Scarpa 7. Ocular NGF administration as a novel non invasive Paola Tirassa approach to protect brain-ngf target neurons that degenerative in Alzheimer s disease 8. New symptomatic and neuroprotective terapie for Micaela Morelli parkinsons s disease 9. Innovative neuro-reparative strategies via the implementation of endogenous neurogenesis and gliogenesis: focus on the new P2Y-like GPR17 receptor Maria Pia Abbracchio 10. Pharmacological modulation of adult neurogenesis Maria Grazia Grilli 11. Study of the relationship between the Fabrizio Chiti structure/morphology of amyloid beta peptide aggregates and their toxicity in cellular, neuronal and animal models" E. Non-Pharmacological Therapies 1. Psychoeducational intervention for patients and care givers in ParKinson s Disease and other neurodegenerative diseases. Pio Enrico Ricci Bitti 2. Physical excercise and cognitive decline Maurizio Taglialatela Alfonso Di Costanzo 3. New tratment strategies (non pharmacologic therapeutic strategies in neurodegenerative diseases) 4. Non invasive brain stimulation an integrated approach to neurorehabilitation in Alzheimer disease 5. Early cognitive and electroencephalographic markers of normal and pathological ageing 6. Transplantation of ips-derived dopaminergic neurons for cell replacement in Parkinson's disease 7. Neural re generation in the cerebellum: development of cell replacement strategies for the management of spinocerebellar ataxias 8. Physical activity programs for elderly subjects with different grades of cognitive impairment 9. Promotion of Adapted Motor Activity among patients with Parkinson s disease Andrea Stracciari Orazio Zanetti Patrizia Bisiacchi Vania Broccoli Gian Giacomo Consalez Ferdinando Rossi Federico Schena Enrico Granieri
8 10. New treatment strategies in Neurodegenerative diseases with Deep brain Stimulation F. Drug Pharmacogenetics 1. Role of pharmacogenetics to identify the personalized treatment of dementia and depression. Mariachiara Sensi Alberto Pilotto G. Clinical studies 1. Neurocognition in Alzheimer Disease Carlo Caltagirone 3. HUMAN/CLINICAL RESEARCH A. Human epidemiology/risk factors 1. Pathogenic mechanisms, early diagnosis and prevention of neurodegenerative diseases following pesticide intake in animal model 2. Effects of traffic-related particulate matter on transcription of genes implicated in Gabbianelli Rosita Cinzia Nasuti Micaela Caserta neurodegeneration 3. Cronobiology and neurodegeneration Valerio Carelli 4. Gene-metal interaction as determinant of neurodegenerative diseases 5. Using prevention to reduce the burden of Alzheimer s disease 6. Construction and development of "Alzheimer-Web- GIS". Geographical correlation between Alzheimer's disease and environmental parameters. Method for mapping population-based case-control studies. 7. Prospective evaluation of biomarkers of inflammation in Mild Cognitive Impairment subtypes, and their role in the conversion from cognitive impairment to dementia and Alzheimer's disease 8. Epidemiology and risk factors for Mild Cognitive Impairment, Alzheimer's disease and dementia. Population-based 7-year follow-up study. Roberto Lucchini Stefano Mattioli Giuseppe Mele Calogero Caruso Roberto Monastero 9. [moved to B.5 Epidemiology genetics] Emilio Di Maria 10. Preclinical Diagnosis Carlo Caltagirone 11. Interactions between the products of the Herpes Manservigi Roberto
9 simplex genome and Alzheimer's disease susceptibility genes B. Epidemiology genetics 1. Genetic susceptibility to Alzheimer s disease and genome wide association studies 2. Genetic susceptibility to Alzheimer s disease: biobanking Luisa Benussi Maria Del Zompo C. Biomarkers and early diagnosis and imaging 1. Plasma biomarkers of oxidative stress and inflammation in early detection and follow-up of dementia. 2. Vitamins and antioxidants in aging and neurodegenerative diseases. 3. Phenotypic and genotypic markers of early Alzheimer s disease 4. Prioritization and annotation of AD genes, their variants and their interaction networks 5. Cerebrospinal fluid cytokine and chemokine levels as potential biomarkers for early diagnosis of Alzheimer s disease 6. Individual evaluation of cognitive decline: definition of low cost non- invasive examination battery and implementation of a predictive algorithm to identify Mild cognitive impairment 7. Impiego dei markers biologici e neuro radiologici nella diagnosi differenziale precoce 8. Biomarkers in Mild cognitive Impairment: genetics and biochemical and neuroimaging studies for early diagnosis. Patrizia Mecocci Salvatore Monaco Rita Casadio Elio Scarpini Paolo Maria Rossini Orazio Zanetti Sandro Sorbi 9. A multimodality index for early diagnosis of Flavio Nobili Alzheimer s disease 10. Lexical semantic skills, APOE and neuroanatomical Paolo Caffarra substrate of semantic memory in early diagnosis of Alzheimer's disease 11. Cognitive neuroscience of dementia: clinical and Stefano F. Cappa theoretical aspect 12. Mapping structural and functional brain network Massimo Filippi damage in neurodegenerative diseases 13. Studies are aimed at investigating whit PET the Daniela Perani brain functional parameters and neurotransmission systems in neurological and psychiatric diseases 14. Global proteomics of human fibroblast for the Maurizio Popoli identification of early biomarkers for dementia and Alzheimer s disease 15. Parkinson disease and parkinsonisms Gianfranco Spalletta 16. Mild cognitive impairment Gianfranco Spalletta
10 17. Electrophysiological study of striatal physiology in normal conditions and experimental parkinsonism: use of genetic and pathogenetic animal models 18. Celebrospinal fluid biomarkers in alzheimer s disease and other neurodegenerative disease Paolo Calabresi Lucilla Parnetti D. Diagnosis: biomarkers 1. Proteomics of cognitive and movement disorders: Identification of molecular mechanisms associated with disease onset and phenotypic variability of frontotemporal lobar degeneration 2. Peripheral markers of oxidative stress, excitotoxicity and abnormal protein metabolism in neurodegenerative disorders. 3. Validation and search of CSF biomarkers for the early differential diagnosis of cognitive impairment. Development of new diagnostic approaches for prion diseases in humans. Roberta Ghidoni Carlo Ferrarese Piero Parchi 4. Analisi Biochimica di parametri di stress ossidativo Gabriele Siciliano 5. Cognitive decline in multiple sclerosis Laura Calzà 6. Search of predictive markers of AD based on amplification of disease-specific amyloid-beta oligomers Fabrizio Tagliavini 7. Mild cognitive impairment and dementias Gianfranco Spalletta E. Diagnosis: neuropsychology 1. Handwriting analysis as a novel diagnostic tool for Alzheimer s disease in the clinical and forensic settings 2. Amnesic Mild Cognitive Impairment: neuropsychological features and new diagnostic criteria (Dubois et Al., 2007) Maurizio Balestrino Angiola Maria Fasanaro 3. Predictors of conversion from MCI to dementia Roberto Gallassi 4. Individual evaluation of cognitive decline; validation Ildebrando Appollonio of ultrafast screening tools. F. Biobanking Bioinformatics G. Ambient assisted living and socioeconomics 1. Home-based Empowered Living for people with dementia and Parkinson s disease Roberto Monastero
11 2. Neuro-rehabilitation and brain functional imaging: rehabilitation customization driven by best exploitation of celebral plasticity and health system sustability. 3. Role of information and communication technology (ICT) systems to improve quality of life, quality of care and safety of older patients with cognitive decline 4. A transdisciplinary orented proposal from a social sciences perspective Alessandra Pedrocchi Alberto Pilotto Vincenzo Maria Bruno Giorgino
12 1. MECHANISMS OF NEURODEGENERATION A- Macromolecular interactions and Neurodegeneration
13 Nome Contatti Istituto/Dipartimento Proposta di ricerca Dr. Pier Luigi San Biagio phone: cell: Istituto di Biofisica - CNR, Via Ugo La Malfa, 153, Palermo (Italy) Many untreatable neurodegenerative human disorders are associated with the aggregation of misfolded or natively unfolded proteins into amyloid fibrils. Our research activity concerns Alzheimer s Parkinson s and Albers-Schönberg diseases with the shared perspective of relating the understanding of the pathway leading to the fibrils formation to the design of appropriate drugs capable on preventing the protein aggregation. Alzheimer s disease. Recent results suggested that the main neurotoxic species in Alzheimer s Disease (AD) would not be the insoluble protein aggregates, made of extracellular deposits of Beta-amyloid peptide (Ab) fibrils, but rather the soluble oligomeric species, including small globular structures, 2.7 to 6.0 nm in diameter, and protofibrils. Conformational constrains imposed by small organic molecules could play a key role in modulating the fibrillogenesis process, so providing effective therapeutic tools to target both oligomeric and fibrillar species. In this perspective, polycyclic aromatic molecules could be of special interest as they might disrupt the molecular architectures precursors of fibrils by means of aromatic interactions, like stacking interactions with tyrosine and phenylalanine residues of Ab. In particular, natural polyphenols, composed of one or more aromatic phenolic rings, are a class of phytochemicals found in high concentrations in wine, tea, nuts, berries, fruits, cocoa, and an ample assortment of other plants. Some of them have been demonstrated to have anti-oxidant and anti-amyloidogenic effect and it has been speculated that they could prevent the development of Alzheimer's disease, not only through scavenging of reactive oxygen species, but also through directly inhibiting the deposition of fibrillar beta-amyloid in the brain. In the present project, we ll test some natural molecules, such as ferulic acid (FA), a phenolic anti-oxidant present in fruit cell walls, hypericin (Hyp), a natural pigment extracted from Hypericum perforatum, widely used as a mild antidepressant and epigallocatechin gallate (EGCG), one of the major flavonoids of green tea. The aggregation process of Ab in the presence of these natural molecules will be studied by light scattering, steady-state and time-resolved fluorescence, FTIR and CD techniques. The analysis of the correlations between the effects of the studied compounds on the various stages of amyloid fibril formation, and their known physicochemical properties, will provide novel insights on the specific role of hydrophobic and aromatic interactions. Encouraging results have already been obtained using hypericin which has been shown to affect and to interfere with the early stages of polymerization process. The furtherance of this type of study, extended to other small molecules like FA and EGCG, hopefully will meet the goal of designing effective therapeutic tools to ideally target both oligomeric and fibrillar species, thus contributing to devise AD prevention strategies.
14 Parkinson s disease. Alpha-synuclein (as) is the main constituent of Lewy bodies, intraneuronal fibrillar inclusions which are present in all patients affected by Parkinson s disease (PD). From genetic and biochemical studies it is believed that as is involved in the disease. AS is a natively unfolded protein with the ability to acquire secondary structure upon interaction with membranes or with itself. It is linked to PD by two evidences: the accumulation of amyloid fibrils of the protein and the autosomal dominant forms of the disease (A53T, A30P and E46K mutants). Both the biological role of this protein and the mechanisms involved in the ethiopathogenesis of PD are still unknown. The protein is located at the presynaptic terminal of neurons in all the CNS, where it exists free in the citosol or bound to synaptic vesicles. The membrane binding causes the formation of an amphipatic alpha-helix in the first part of the protein, which lies at the membrane surface without crossing the bilayer. Recent evidences show that as is able to bind and permeabilize cell membrane. Our investigation will be devoted (i) to the characterization, at the single molecule level, of the channel like ability of as and its architecture. The pore-forming ability of monomers and aggregates of different sizes will be investigated. Secondly, the lipid specificity will be also investigated by means of high resolution NMR and MS (both maldi-tof and esi-ms). The ability of as to provoke or protect unsaturated acyl chains of membrane lipids from oxidation will also be investigated, by studying with MS the products of oxidation. As possible therapeutic perspectives, the organic or peptidic inhibitors of proteinprotein interaction or able to clog the already formed pores will be investigated. Furthermore, if indications of any lipid specificity will be obtained, we will develop lipid formulates suitable for sequestering as lowering its level in solution. Albers-Schönberg disease. Mutations in the gene coding for the lysosomal Cl-/H+ exchanger CLC-7 lead to recessive osteopetrosis and Albers-Schönberg disease. Apart from the osteopetrotic phenotype the recessive disease is characterized by severe generalized neurodegeneration. Also knock-out of the plasma membrane localized CLC-2 lead to neurodegeneration (retinal degeneration and leukoencephalopathy). The mechanisms underlying these phenotypes are unclear. We are studying the functional properties of these two proteins using electrophysiological and optical methods, combined with mutagenesis, in order understand how their dysfunction may lead to neurodegeneration. In particular, we consider these proteins as potential risk factors for more generalized forms of neurodegeneration. Area di interesse identificata Finanziamenti ricevuti Titolo progetto Ente finanziatore Public health. Therapeutic strategies for the prevention and treatment of neurodegenerative diseases. Molecular basis of amyloid aggregation Progetto strategico di ricerca finalizzata sulla malattia di Alzheimer (Art. 12/bis D.Lgs 229/99), Beta-amyloid deposit on the cell membrane: role of metal ions and free radicals Italian 'Ministero della Salute Durata progetto 24 months, started 2001
15 Abstract del progetto Titolo progetto Ente finanziatore Characterization of some key molecular mechanisms for the neuronal damage connected to AD. Specific objectives: (i) to identify of the key steps responsible for the amyloid depost formation on membrane model systems in vitro; (ii) to evaluate the role of the membrane lipid composition on the deposit; (iii) to understand the role of metal ions and oxidative stress on the fibril growth both in solution and on membranes; (iv) to establish an in vitro test for evaluating the ability of drugs to inhibit ba aggregation. Animal neuropathies: molecular and functional analysis of the prionic proteins in Sicilian bovine race Italian 'Ministero della Salute Durata progetto 24 months, started 2003 Abstract del progetto Titolo progetto Ente finanziatore The main objective of the present proposal is to understand the mechanisms by which wild type and mutant prion proteins become misfolded in such a way as to provoke neurodegenerative diseases becoming transmissible agents. An extensive study of the aggregation process will be done by using static and dynamic light scattering and fluorescence techniques. Prin 2005 project: The effects of metal ions on the protein aggregation processes Italian 'Ministero Università e Ricerca' Durata progetto 24 months, started 2005 Abstract del progetto Titolo progetto Ente finanziatore The project concerns the study of the effects of metal ions in solution on the protein aggregation processes. Main goals are (i) to understand the molecular mechanisms relevant for amyloid aggregation and to establish the relevance of metals in modulating solvent mediated protein-protein interactions. Electrophysiological characterization of the alpha synuclein channels, a protein involved in Parkinson s disease Local Bank Foundation CARITRO Durata progetto 24 months, started 2008 Abstract del progetto Titolo progetto Ente finanziatore The aim of the project is: i) to characterize the channel like activity of alpha synuclein (as) and its role in the disease; ii) to distinguish the most active as form; iii) to verify the ability of as to bind to membranes and lipid requirements; iv) to investigate the role of as in the prevention of the oxidation of unsaturated fatty acid chains Prin 2008 project: Inhibition of beta-amyloid peptide aggregation by some natural compounds Italian 'Ministero Università e Ricerca Durata progetto 24 months, started 2010 Abstract del progetto Titolo progetto Ente finanziatore The aim of this project is to provide a molecular approach for preventing Abeta aggregation and toxicity through the use of some natural molecules potentially able of interact with Abeta by stabilizing or disrupt aggregates of different sizes. Membrane transport of chloride and protons: mechanism of function of CLC family proteins involved in epilepsy and neurodegeneration Compagnia San Paolo Durata progetto 36 months, started 2009 Abstract del progetto CLC-2 is a plasma membrane localized Cl- channel whereas CLC-7 is a lysosomal Cl-/H+ exchanger. Knock-out of CLC-2 in mice leads to retinal degeneration and leukoencephalopathy and knock-out of CLC-7 and human mutations in the CLCN7 gene
16 lead to generalized neurodegeneration. The aim of the project is to understand better the transport function of these two proteins in order to get insight into their causative role in these forms of neurodegeneration.
17 Nome Bruno Samorì Contatti E mail: Istituto/Dipartimento Laboratory of Nanoscience and Nanobiotechnology, Department of Biochemistry, University of Bologna (Italy) (http://biocfarm.unibo.it/samori/) Proposta di ricerca Area di interesse identificata Basic research Abstract According to an emerging view, the aggregation-prone proteins associated to neurodegenerative, Parkinson s, Alzheimer s, and Creutzfeldt-Jakob diseases, can switch back and forth between functional and amyloidogenic/prionogenic conformations. Their conformational polymorphism poses tremendous challenges to standard methods in structural biology, which mask the complexity of their behaviour by recording observables as time and ensemble averages. Single-molecule methodologies can instead follow the time trajectories of individual molecules or molecular assemblies, can map their energy landscapes, and obtain distributions of molecular properties. These capabilities can provide information about the monomeric state of an amyloidogenic protein and about the very early stages of its amyloid cascade. This information is crucially needed for the design of new drugs against Parkinson s or Alzheimer s disease that must be capable to target upstream of the formation of the most neurotoxic oligomeric species. In fact concerns are now growing over the possibility that more harm than cure can be created whenever the deposits in the central nervous system, are targeted instead: in this way their dynamic equilibrium with the oligomeric forms can be shifted back to the latter. We have recently shown that Atomic Force Microscopy-based Single Molecule Force Spectroscopy (AFM-SMFS) can single out the different monomeric conformers of α-synuclein (the intrinsically unstructured protein associated to Parkinson s disease) and of its mutants associated to genetic PD. (1,2). So far AFM-SMFS has been the only methodology that made it possible to achieve this goal. Ongoing projects: a) The integration of AFM-SMFS with Optical tweezers (OT) (in coll. with Carlos Bustamante (UC Berkeley). b) The determination of alpha synuclein aggregation step targeted by different potential drugs or chaperons, by using an integrated approach based on AFM- and OT-SMFS, AFM-imaging, Fluorescence Polarization Spectroscopy ( in collaboration with
18 Luigi Bubacco, University of Padova) c) The conformational equilibria or prion proteins (in coll. with G. Legname SISSA Trieste, and Motomasa Tanaka, Riken, Japan) (1) Sandal, M., et al (2008). PLoS Biol 6, (2) Brucale, et al. (2009). Chembiochem 10, Finanziamenti ricevuti Titolo progetto Nanomanipolazione di singole molecole delle proteine coinvolte in malattie neurodegenerative Ente finanziatore FIRB Nanobiotecnologie per dispositivi e sensori innovativi (G.U n. 175) Durata progetto ; FIRB Metodologie e tecnologie innovative per la farmaceutica. CINECA RBNE03PX83 Abstract del progetto Titolo progetto Sviluppo metodologie di nanomanipolazione di proteine intrinsecamente non foldate o disordinate Meccanismi Neurodegenerativi nelle Malattie da Prioni (coordin. Catia Sorgato) Ente finanziatore PRIN 2009 Durata progetto
19 Nome Contatti Istituto/Dipartimento Dr. Eugenia Polverini Department of Physics University of Parma Proposta di ricerca Our studies are - and will be - focused on myelin proteins, to the aim of clarify their structure to function relationship and the mechanisms involved in demyelinating diseases, including multiple sclerosis. Two main proteins are under investigation: the Myelin Basic Protein (MBP) and the P2 myelin protein. MBP is a highly post-translationally modified structural component of central nervous system myelin, the multilamellar membrane wrapped around nerve axons. MBP has a high net positive charge and its principal role seems to be the adhesion of the cytosolic surfaces of the compact myelin sheath, whose structural integrity determines the speed of transmission of action potentials along the axon. However, MBP seems to be a multifunctional protein, since it also interacts with other cytoskeletal and signalling proteins, tethering some of them to the oligodendrocyte plasma membrane. The three-dimensional structure of MBP in situ is still unknown, due to its intrinsic flexibility and dependence of conformation on environment. Therefore a neighbourhood-dependent structural charcterization of fragtments is attempted. In the classic 18.5 kda MBP sequence, there is a highly conserved fragment, constituting two regions with different putative functionality. The first region seems to associate with the membrane, and comprises the primary immunodominant epitope in multiple sclerosis; the second one is a proline-rich segment, that has been predicted to be a ligand for protein kinase SH3-domains. Due to the functional relevance of this fragment of MBP, we re trying a structural characterization - in the absence and in the presence of protein s post-translational modifications - by means of molecular dynamics simulations in membrane and of spectroscopic techniques (in collaboration with the University of Guelph, Ontario, Canada). P2 myelin protein is a basic peripheral nervous system protein localized to the cytoplasmic spaces in the myelin lamellae. It was recognized as the factor inducing the Experimental Allergic Neuritis, an animal model for the study of the Guillain-Barrè syndrome, a demyelinating disease of the PNS in humans. The interest for the putative autoantigenic role of the P2 protein and the need to understand its function in the myelin sheath has led to the study of its structural characteristics and behaviour, basing on the solved crystal structures. By investigating its interaction with different kinds of lipids, on the one hand we want to clarify its transport role and on the other hand we want to verify its putative involvement in the compactness of PNS myelin sheat. Different approaches are carrying out: molecular docking simulations, spectroscopical investigations and, in collaboration with the University of Oulu, Finland, crystal X-ray diffraction and small-angle X-ray scattering. Area di interesse identificata myelin proteins and demyelinating deseases Finanziamenti ricevuti Titolo progetto Ente finanziatore University local research funds (FIL) Durata progetto Abstract del progetto
20 Nome Contatti Istituto/Dipartimento Proposta di ricerca Prof. ROBERTO DE RENZI - Dr. MARIA GRAZIA BRIDELLI Physics department University of Parma Parco Area delle Scienze, 7/A, Parma ITALY The goal of our research is threefold: 1) study of the nucleation rate and the growth rate of the amyloidogenic process by controlling and modulating the solution parameters such as temperature, ph, solvent composition, and peptide concentration, not only influencing the final fibril state but also the formation kinetics. The analysis of the aggregation process will be performed in solution by employing CD and Fluorescence spectroscopies by following the change of fluorescence emission of small hydrophobic molecules, called amyloid ligands, which do not bind to specific proteins but are selective for protein aggregates with a cross β-sheet conformation. Dynamic Light Scattering measurements will allow to monitor size and shapes of the fibrils during the aggregation process. 2) study the conformational state of proteins in the native and amyloid form at very low hydration level by investigating the structural properties of the first hydration shell. It is well known that proteins are surrounded in the cell by a hydration shell formed by interacting water molecules that form patches locally distributed in dependence on the heterogeneous surface chemistry or occluded in the internal cavities. This bound water in the crowded cytoplasm may not be enough to fully cover the protein surface with a monolayer aqueous sheath, moreover about 0.4 g of water/g of protein are enough to assure protein normal functionality. Two experimental techniques can be applied to investigate this structured water to gain information on the geometry of the protein surface and secondary structure. Thermally Stimulated Depolarization Currents (TSDC) technique, applied in the past to a variety of natural and synthetic biopolymers has recently been applied to the study of lysozyme. It has allowed recognizing the main features of the hydration as a function of secondary structure, revealing a clear dominance of the β- structure in the amyloid form. Fourier Transform Infrared Spectrometry as well has proven to be highly versatile in the analysis of protein conformation by monitoring the alteration in protein secondary structure as changes in the Amide bands. 3) by means of the molecular dynamics simulation techniques, study the conformational changes that proteins undergo under the critical environmental conditions that in vitro cause the fibrils formation. This approach could be useful to investigate the pathway(s) of the transition and its molecular features. Area di interesse identificata Conformational transitions of proteins from native to amyloid form. Finanziamenti ricevuti University local research funds FIL 2008 Titolo progetto Ente finanziatore Durata progetto Abstract del progetto Hydration structure analysis of Lysozyme amyloid fibrils Ministero dell Istruzione, dell Università e della Ricerca 2 y Combined Thermally stimulated depolarization currents (TSDC) and FTIR techniques have been employed to investigate the conformation of hen egg white lysozyme in native and amyloid form, in the state of powder at very low hydration level. The spectra reveal specific features that can be attributed to water texture around the secondary structure adopted by the macromolecule.