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Dottorando: Renzo VANNA Matricola: R08140

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Dottorando: Renzo VANNA Matricola: R08140 Università degli Studi di Milano Scuola di Dottorato in Medicina Molecolare Dipartimento di Scienze e Tecnologie Biomediche Curriculum di Genomica, Proteomica e Tecnologie correlate Ciclo XXIV Anno Accademico 2010/2011 Dottorando: Renzo VANNA Matricola: R08140 CARATTERIZZAZIONE DELL’ORGANELLO DI NEUROMELANINA DELLA SUBSTANTIA NIGRA UMANA CON APPROCCIO PROTEOMICO Direttore della Scuola: Ch.mo Prof. Mario Clerici Tutore: Prof.ssa Cecilia GELFI Correlatore: Dott. Luigi ZECCA II SOMMARIO La substantia nigra umana è l’area del cervello principalmente colpita dalla malattia di Parkinson. La caratteristica più evidente della substantia nigra è la presenza di neuromelanina (NM), un particolare pigmento melanico che si accumula nei neuroni all’interno di organelli circondati da membrana e ricchi di lipidi (dolicoli). La singolare presenza di elevate quantità di organelli di NM all’interno dell’area cerebrale più vulnerabile alla malattia di Parkinson è fonte di molto interesse. Ad oggi, non è stato chiarito in che modo la presenza di organelli di NM possa interferire con la vulnerabilità dei neuroni durante l’invecchiamento. D’altra parte, negli ultimi anni sta emergendo una forte correlazione tra invecchiamento e neurodegenerazione, e l’accumulo di materiale intracellulare difficilmente degradabile (proteine danneggiate, corpi di inclusione, lipofuscine, ecc.). L’accumulo di materiale intracellulare può infatti essere fonte di stress cellulare e fattore di rischio per la neurodegenerazione. In questo lavoro di tesi sono stati preparati campioni di organelli di NM (integri e trattati con shock termico per la rimozione delle membrane) e di pigmento di NM, isolati da tessuto umano (substantia nigra) da soggetti sani post-mortem. I campioni sono stati analizzati in replicato per la definizione del profiling proteomico attraverso tecnologia proteomica MudPIT. Il profiling consiste in 1142 proteine totali delle quali 48 sono state definite con buona confidenza come rappresentative dei campioni. Alcune proteine sono state localizzare nelle diverse regioni dell’organello (pigmento, membrane e parte solubile) grazie al confronto tra i campioni. Dall’analisi proteomica emerge il carattere lisosomiale dell’organello di NM: tra le 48 proteine rappresentative, più di 20 sono di origine lisosomiale. Vengono identificate anche proteine coinvolte nel folding proteico (alpha-crystallin), nella degradazione di proteine (ubiquitin), nell’omeostasi del ferro (ferritin L), nei meccanismi antiossidanti e nella formazione di aggregati intracellulari. Sono state osservate anche proteine di accumulo (mitochindrial subunit c, saposin) tipiche di malattie lisosomiali. Dalle analisi non risulta però né la presenza di enzimi per la sintesi di lipidi né la presenza di proteine potenzialmente coinvolte nella biosintesi di composti melanici. L’organello di NM possiede quindi le caratteristiche di un organello di origine lisosomiale/autofagica caratterizzato anche dall’accumulo di materiale indegradabile e da una probabile perdita di efficienza degradativa. I dati ottenuti sono in accordo con recenti ipotesi secondo le quali l’organello si formerebbe attraverso il sequestro di NM e di altre molecole potenzialmente tossiche per il neurone (proteine e lipidi danneggiati, metalli). Dai dati di proteomica sembra che questo meccanismo protettivo, con il passare del tempo, porti all’accumulo di materiale difficilmente degradabili e questo implicherebbe una probabile diminuzione della capacità degradativa della cellula. L’organello di NM potrebbe quindi avere un ruolo protettivo ma allo steso tempo potrebbe aumentare la vulnerabilità del neurone durante l’invecchiamento. III ABSTRACT Substantia nigra is the most affected brain region in Parkinson's disease. Substantia nigra is characterized by strong pigmentation derived by the presence of neuromelanin (NM), a particular melanic pigment that accumulates during aging in neurons in typical membrane-bounded organelles with high levels of lipids (dolichols). The occurrence of an elevated number of NM organelles in the most vulnerable brain region during Parkinson’s disease is of special interest. It is not clear if NM organelles may interfere with the vulnerability of neurons during aging. Nevertheless, many evidences support the correlation between aging and neurodegeneration, and the accumulation of intracellular indigestible materials (damaged proteins, inclusion bodies and lipofuscins). The accumulation of indigestible materials inside the cell may increase cellular stress and neurodegeneration. In this study, samples of NM organelles (whole and without membranes) and NM pigment were isolated from human post-mortem tissue (substantia nigra) of healthy subjects. Samples were analysed in duplicate to obtaining the proteomic profile of the NM organelle using MudPIT proteomic technology. A total of 1142 proteins were identified, of which 48 were observed with high confidence as representative of samples. Comparing results from different samples, some proteins were assigned to different regions of the NM organelle (NM pigment, membrane, soluble portion). From the proteomic profile emerges the lysosomal nature of the NM organelle: among the 48 representative proteins, more than 20 are lysosomal proteins. In addition, other groups of proteins were identified: proteins involved in protein folding (alpha-crystallin) and degradation (ubiquitin), proteins important to iron homeostasis (ferritin L), antioxidant proteins and proteins involved in intracellular aggregates formation. Furthermore, typical biomarkers of lysosomal storage disease are observed (mitochondrial subunit c, saposins). On the other hand, any enzyme potentially involved in the melanogenic process and any lipids biosynthetic enzyme were identified. This study demonstrates that the NM organelle has the features of both a lysosomal/autophagic organelle and an aged and damaged lysosome accumulating indigestible material. These data are in agreement with recent hypothesis suggesting that the NM organelle formation originate by sequestration of NM and other potentially toxic molecules as damaged proteins, damaged lipids and metals. Our data suggest that this protective mechanism, during aging, may induce accumulation of indigestible material inside the NM organelles and, as consequence, a decreased degradative function of the cell. In summary, e suggest that NM organelle could confer protection to neurons and, at the same time, could increase cellular vulnerability in aging conditions. IV INDICE SOMMARIO............................................................................................................. III ABSTRACT ............................................................................................................. IV INDICE...................................................................................................................... V LISTA DELLE ABBREVIAZIONI ............................................................................ IX 1 INTRODUZIONE ........................................................................................... 1 1.1 INVECCHIAMENTO: CARATTERISTICHE E MECCANISMI COINVOLTI .................... 1 1.1.1 Caratteristiche dell’invecchiamento ...................................................... 2 1.1.2 Accumulo di proteine e meccanismi degradativi ................................. 2 1.1.2.1 Sistema ubiquitina/proteasoma e invecchiamento ....................................... 2 1.1.2.2 Lisosomi e invecchiamento........................................................................... 2 1.1.2.3 Autofagia e invecchiamento.......................................................................... 3 1.2 LA MALATTIA DI PARKINSON......................................................................... 5 1.2.1 Cenni storici............................................................................................. 5 1.2.2 Sintomatologia......................................................................................... 5 1.2.3 Neurochimica e neuropatologia del PD................................................. 5 1.2.4 Eziologia del PD....................................................................................... 9 1.2.4.1 Cause genetiche per comprendere la patogenesi del PD............................ 9 1.2.4.2 Fattori ambientali.........................................................................................13 1.2.5 Ipotesi recenti sulla patogenesi del PD ............................................... 15 1.2.5.1 Stress ossidativo.........................................................................................17 1.2.5.2 Difunzione mitocondriale.............................................................................17 1.2.5.3 Omeostasi delle proteine: proteasoma, lisosomi e autofagia.....................17 1.2.5.4 Infiammazione e microglia ..........................................................................18 1.3 MELANINE ................................................................................................. 19 1.3.1 Principali studi sulle melanine ............................................................. 19 1.3.2 Principali funzioni delle melanine ........................................................ 21 1.3.3 La biogenesi dei melanosomi............................................................... 21 1.3.4 Proteine del melanosoma ..................................................................... 22 1.4 NEUROMELANINA......................................................................................
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