Salta ai contenuti. | Salta alla navigazione

Strumenti personali

sito non più aggiornato
 

Sezioni

Tu sei qui: Home / Ricerca / 2008

Responsabile Ciro Isidoro
Settore di ricerca Patologia Generale
Personale docente    
Personale non-docente Roberta Castino  Carlo Follo  Nicol Trincheri  Natascia Bellio  Claudia Peracchio  Ilaria Fiorentiono  Matteo Ozzano   
Obiettivi della ricerca 1) Regolazione e ruolo dell`autofagia nelle malattie neurodegenerative
Risultati ottenuti 1)  1. In neuroblastoma cells over-expressing a truncated C98X vasopressin (VP) precursor autophagy, a macromolecular degradation process, was shown to be essential for assuring cell survival. Ectopic expression of either the wild-type or the mutated C98X AVP altered neither the expression nor the phosphorylation of the pro-survival signalling molecule Akt. Strikingly, the ectopic adenoviral-directed expression of a constitutively active Akt, instead of preserving cell survival, resulted in the suppression of autophagy, and precipitated Bax-mediated cell death.
2. Methamphetamine abuse is toxic to dopaminergic neurons, causing nigrostriatal denervation and striatal dopamine loss. We found that autophagy is rapidly up-regulated in response to methamphetamine. Confocal fluorescence microscopy and immuno-electron microscopy studies demonstrated the presence of alpha-synuclein aggregates in autophagy-lysosomal structures in cells exposed to methamphetamine, a condition compatible with cell survival. Inhibition of autophagy either by pharmacologic or genetic manipulation of the class III Phosphatidylinositol-3 kinase-mediated signaling prevented the removal of alpha-synuclein aggregates and precipitated a bax-mediated mitochondrial apoptosis pathway.
3. The intracellular assembly of tau aggregates is a pathological hallmark shared by Alzheimer`s disease and other neurodegenerative disorders known collectively as tauopathies. To model how tau fibrillogenesis evolves in tauopathies, we established transfectant M1C cultures from human neuroblastoma BE(2)-M17D cells that inducibly express human tau. By using the lysosomotropic agent, chloroquine (CQ), and the PI3 kinase inhibitor 3-methyadenine we could demonstrate that the autophagic-lysosomal system plays a role in the clearance of tau, and that dysfunction of this system results in the formation of tau oligomers and insoluble aggregates.
4. In a pilot clinical study that we recently published we found that lithium administration slows the progression of Amyotrophic Lateral Sclerosis (ALS) in human patients. This clinical study was published in addition with basic (in vitro) and pre-clinical (in vivo) data demonstrating a defect of autophagy as a final common pathway in the genesis of ALS. In fact, lithium was used as an autophagy inducer. In detailing the protective effects of lithium we found for the first time that this drug stimulates the biogenesis of mitochondria in the central nervous system and, uniquely in the spinal cord, it induces neuronogenesis and neuronal differentiation. In particular, the effects induced by lithium can be summarized as follows: (i) the removal of altered mitochondria and protein aggregates; (ii) the biogenesis of well-structured mitochondria; (iii) the suppression of glial proliferation; (iv) the differentiation of newly formed neurons in the spinal cord towards a specific phenotype.


 
Obiettivi della ricerca 2) Meccanismi di citotossicità e chemioresistenza nei tumori: ruolo del sistema autofagico-lisosomico
Risultati ottenuti 2) In two human ovary carcinoma cell lines, A2780 and NIHOVCAR3 respectively chemosensitive and chemoresistant to taxol and etoposide, we have demonstrated that: 1. cathepsin D, not cathepsin B or L, mediates the activation of the mitochondrial intrinsic death pathway in A2780 cells; 2. lysosome leakage and cytosolic relocation of cathepsin D occurs in the chemosensitive A2780 cells, not in the chemoresistant NIHOVCAR3 cells; 3. bax is essential for the permeabilization of both lysosomes and mitochondria in A2780 cells exposed to chemotherapy drugs; 4. cathepsin D activity is mandatory for the oligomerization of bax on both mitochondrial and lysosomal membranes.
In human colorectal DLD1 cancer cells, the dietary bioflavonoid resveratrol (RV) rapidly induced autophagy. This effect was reversible (on removal of the drug) and was associated with increased expression and cytosolic redistribution of the proteins Beclin1 and LC3 II. Supplementing the cells with asparagine (Asn) abrogated the Beclin-dependent autophagy. When applied acutely (2 h), RV was not toxic; however, reiterate chronic (48 h) exposure to RV eventually led to annexin V- and terminal deoxinucleotidyl transferase-mediated dUTP-biotin nick end labeling-positive cell death. This toxic effect was autophagy dependent, as it was prevented either by Asn, by expressing a dominant-negative lipid kinase-deficient class III phosphoinositide 3-phosphate kinase, or by RNA interference knockdown of Beclin1. Lamp2b silencing abolished the fusion of autophagosomes with lysosomes and preserved cell viability despite the ongoing formation of autophagosomes in cells chronically exposed to RV. The pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone inhibited RV-induced cell death, but not autophagy. These results uncover a novel pathway of RV cytotoxicity in which the autophagy-lysosomal system acts as a primary contributor.

 
Obiettivi della ricerca 3) Ruolo della catepsina D nella funzione della ghiandola mammaria
Risultati ottenuti 3)  Cathepsin D (CD), a lysosomal aspartic protease present in mammary tissue and milk in various molecular forms, is also found in the incubation medium of mammary acini in molecular forms that are proteolytically active on prolactin at a physiological pH. Because prolactin controls the vesicular traffic in mammary cells, we studied, in vivo and in vitro, its effects on the polarized transport and secretion of various forms of CD in the rat mammary gland. CD accumulated in vesicles not involved in endocytosis in the basal region of cells. Prolactin increased this accumulation and the release of endosomal active single-chain CD at the basal side of acini. The CD-mediated proteolysis of prolactin, leading to the antiangiogenic 16-kDa form, at a physiological pH, was observed only in conditioned medium but not milk. These data support the novel concept that an active molecular form of CD, secreted at the basal side of the mammary epithelium, participates in processing blood-borne prolactin outside the cell, this polarized secretion being controlled by prolactin itself.
 
Collaborazioni in atto Collaborazioni internazionali e nazionali in atto:
1. Bonnie F. Sloane, Department of Pharmacology della Wayne State University di Detroit (USA). (Biogenesi e funzione delle catepsine)
2. David Murphy, Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol (UK) (autofagia nelle malattie neurodegenerative)
3. Andrej Hasilik, Institute fuer Physiologische Chemie, Klinikum der Philipps-Universitaet Marburg (D). (Biogenesi e funzione delle catepsine)
4. Tamara Lah, National Institute of Biology, Ljubljana (Slovenia) (Citotossicità e autofagia nel glioblastoma)
5. Guido kroemer, INSERM di Villejuif, Università Parigi IX, (mitofagia e mitocondriogenesi)
6. Francesco Fornai, Università di Pisa (autofagia e neurodegenerazione)
7. Eugenia Dogliotti, Istituto Superiore di Sanità, (autofagia nel danno e riparo del DNA)
8. Salvatore Coluccia, Dipartimento di Chimica, Università di Torino (funzionalizzazione di nanoparticelle mesoporose a scopo diagnostico-terapeutico)
9. Emanuele Albano – Rita Carini, Dipartimento di Scienze Mediche, Università del Piemonte Orientale di Novara (ruolo dei lisosomi nella morte cellulare e nel precondizionamento ischemico di epatociti)
10. Guido Valente, Laboratorio di Anatomia Patologica, Università del Piemonte Orientale di Novara (Nuovi marcatori molecolari prognostici nel cancro).
11.  Michelle Ollivier, INRA Unité Génomique et Physiologie de la Lactation, Jouy-en-Josas, CEDEX (F). (Ruolo della catepsina D nella funzione della ghiandola mammaria)
 
Comunicazioni a congressi   
Pubblicazioni
Akt induces apoptosis in neuroblastoma cells expressing a C98X vasopressin mutant following autophagy suppression.
Suppression of autophagy precipitates neuronal cell death following low doses of methamphetamine.
Prolactin promotes the secretion of active cathepsin D at the basal side of rat mammary acini.
Autophagic-lysosomal perturbation enhances tau aggregation in transfectants with induced wild-type tau expression.
Lithium delays progression of amyotrophic lateral sclerosis.
Resveratrol-induced apoptosis depends on the lipid kinase activity of Vps34 and on the formation of autophagolysosomes.
A fast and simple method for simultaneous mixed site-specific mutagenesis of a wide coding sequence.
Autophagy and amyotrophic lateral sclerosis: The multiple roles of lithium.
Role of autophagy during methamphetamine neurotoxicity.
The transport of soluble lysosomal hydrolases from the Golgi complex to lysosomes