The most common neurodegenerative diseases in the world are Alzheimer's disease (AD) and Parkison's disease (PD). According to a study carried out in 2015 by the Global Burden of Disease Study (GBD), approximately 29.8 and 6.2 million people worldwide are affected respectively by AD and PD. These neurodegenerative diseases share some common characteristics that alter cellular proteostasis, such as oxidative stress, the influence of environmental stress, protein dysfunction and aging; there-fore they are called "conformational diseases", characterized by the abundance of damaged proteins that lead to neuronal aggregation and death. AD is characterized by the formation of amyloid plaques (accumulations of beta-amyloid peptide) and tangles (neurofibrillar accumulations of tau protein) while PD is characterized by the presence of cy-toplasmic inclusions called Lewy bodies, with different aggregate proteins (such as α-synuclein). The ubiquitin proteasome system (UPS), the major intracellular protein degradation complex, and Ac-ylaminoacyl-PEptide Hydrolase (APEH), a ubiquitous bifunctional enzyme, which exhibits exopepti-dase activity on N-acyl-peptides and endoprotease activity on oxidized proteins, they are very im-portant protagonists in maintaining protein homeostasis. Recently, many studies have focused on the search for sensitive and specific molecules that can help neurodegenerative disorders diagnosis; among these, a combined action between proteasome and APEH in maintaining cellular homeostasis in patients with AD has been reported. The present work, starting from a study done on AD patients, aims to investigate the enzymatic activi-ties of UPS and APEH and the expression levels of their own genes and of others involved in their metabolic pathways, in blood samples of patients with PD compared to healthy controls (HC). The study, which involved three different operating units, consisted in the recruitment of forty-six par-ticipants, divided into two groups on the basis of clinical profiles, of which 23 PD and 23 HC; a ve-nous sampling was carried out, various cellular blood fractions (erythrocytes, granulocytes, lympho-cytes and monocytes) were then collected in order to measure, by means of spectrofluorimetric analy-sis, the APEH exopeptidase APEH and chymotyptic proteasome activities. Total RNA, extracted from the whole blood of twenty patients of which 10 PD and 10 HC, was used for the gene expression of the APEH and the proteasome by real time PCR. The expression of genes related to the UPS and APEH metabolic pathways was analyzed with the aim of investigating the molecular mechanisms underlying Parkinson's. In particular:-UBA1, coding for the first enzyme involved in the ubiquitination process (enzyme acti-vating ubiquitin like modifier); -UBE2I, encoding the second enzyme involved in the ubiquitination process (ubiquitin-like modifier activation enzyme); -PRKN, coding for parkin (ubiquitin ligase enzyme); -NAA10, encoding the enzymatic subunit of the N-alpha acetyltransferase A complex; -ACY1, coding for aminoacylase 1 - SOD2, superoxide dismutase; - PARK1, alpha synuclein. The results of this thesis work show a different trend compared to the results obtained from the study previously carried out on AD; in particular, enzymatic activities and gene expression levels increase compared to healthy controls. These results help to understand the molecular mechanisms of AD and PD and show the importance that the molecular biomarkers, object of this study, could play in the onset of these diseases. In this sense, if the results obtained are confirmed in larger prospective studies, the measurement of the activity of the systems under study, using a simple blood sample, could simplify the diagnostic ap-proaches of these diseases.
Le malattie neurodegenerative più diffuse al mondo sono il morbo di Alzheimer (AD) ed il morbo di Parkison (PD). Secondo uno studio realizzato nel 2015 dal Global Burden of Disease Study (GBD), sono affette rispettivamente da AD e PD circa 29,8 e 6,2 milioni di persone nel mondo. Queste malattie neurodegenerative condividono alcune caratteristiche comuni che alterano la proteo-stasi cellulare, come lo stress ossidativo, l'influenza dello stress ambientale, la disfunzione delle pro-teine e l’invecchiamento; pertanto sono chiamate “malattie conformazionali”, caratterizzate dall'ab-bondanza di proteine danneggiate che portano all'aggregazione e alla morte neuronale. L’AD è caratterizzato dalla formazione di placche amiloidi (accumuli di peptide beta-amiloide) e gro-vigli (accumuli neurofibrillari di proteina tau) mentre il PD è caratterizzato dalla presenza di inclusioni citoplasmatiche dette corpi di Lewy, con proteine differenti aggregati (come α-sinucleina). Il sistema ubiquitina proteasoma (UPS), il principale complesso di degradazione delle proteine intracellulari, e l'Acylaminoacyl-PEptide Hydrolase (APEH), un enzima bifunzionale onnipresente, che esibisce attività di esopeptidasi sui N-acil-peptidi e attività di endoproteasi sulle proteine ossidate, sono protagonisti molto importanti nel mantenimento dell'omeostasi proteica. Recentemente, molti studi si sono concentrati sulla ricerca di molecole sensibili e specifiche che pos-sano aiutare nella diagnosi dei disturbi neurodegenerativi; tra questi, è stata segnalata un'azione com-binata tra proteasoma e APEH nel mantenimento dell'omeostasi cellulare nei pazienti con AD. Il presente lavoro, partendo da uno studio fatto su pazienti AD,vuole indagare le attività enzimatiche del UPS e APEH ed i livelli di espressione dei loro stessi geni e di altri coinvolti nelle loro vie metabo-liche, in campioni di sangue di pazienti con PD rispetto ai controlli sani (HC). Lo studio, che ha coinvolto tre diverse unità operative, è consistito nel reclutamento di quarantasei partecipanti, divisi in due gruppi sulla base dei profili clinici, di cui 23 PD e 23 HC; è stato effettuato un prelievo venoso, sono state poi raccolte varie frazioni cellulari del sangue (eritrociti, granulociti, linfociti e monociti) allo scopo di misurare, mediante analisi spettrofluorimetrica, le attività esopepti-dasica dell’APEH e chimotriptica del proteasoma. L’RNA totale, estratto dal sangue intero di venti pazienti di cui 10 PD e 10 HC, è stato usato perl'e-spressione genica dell’APEH e del proteasoma mediante PCR real time. Si è analizzato l’espressione dei geni correlati alle vie metaboliche UPS e APEH con l’obiettivo di in-dagare i meccanismi molecolari che sono alla base del Parkinson. In particolare: - UBA1, codificante per il primo enzima coinvolto nel processo di ubiquitinazione (enzima attivante l'ubiquitina like modificatore); -UBE2I, codificante per il secondo enzima coinvolto nel processo di ubiquitinazione (enzima di atti-vazione del modificatore simile all'ubiquitina); -PRKN, codificante per parkin (enzima ubiquitina ligasi); -NAA10, codificante per la subunità enzimatica del complesso N-alfa acetiltransferasi A; -ACY1, codificante per l'aminoacilasi 1; - SOD2, superossido dismutasi; - PARK1, alfa sinucleina. I risultati di questo lavoro di tesi,mostrano una tendenza diversa rispetto ai risultati ottenuti dallo studio precedentemente realizzato sull’AD; in particolare le attività enzimatiche ed i livelli di espressione ge-nica aumentano rispetto ai controlli sani. Questi risultati aiutano a comprendere i meccanismi molecolari di AD e PD e mostrano l’importanza che i biomarcatori molecolari, oggetto di questo studio, potrebbero svolgere nell'insorgenza di tali pa-tologie. In tal senso, se i risultati ottenuti saranno confermati in studi prospettici più ampi, la misurazione dell'attività dei sistemi in studio, utilizzando un semplice prelievo di sangue, potrebbe semplificare gli approcci diagnostici di queste malattie.
Studio dell'acilpeptide idrolasi (APEH) e del proteasoma nell'insorgenza di malattie neurodegenerative e loro possibile utilizzo come marker diagnostici
FUSCO, Carmela
2022-05-04
Abstract
The most common neurodegenerative diseases in the world are Alzheimer's disease (AD) and Parkison's disease (PD). According to a study carried out in 2015 by the Global Burden of Disease Study (GBD), approximately 29.8 and 6.2 million people worldwide are affected respectively by AD and PD. These neurodegenerative diseases share some common characteristics that alter cellular proteostasis, such as oxidative stress, the influence of environmental stress, protein dysfunction and aging; there-fore they are called "conformational diseases", characterized by the abundance of damaged proteins that lead to neuronal aggregation and death. AD is characterized by the formation of amyloid plaques (accumulations of beta-amyloid peptide) and tangles (neurofibrillar accumulations of tau protein) while PD is characterized by the presence of cy-toplasmic inclusions called Lewy bodies, with different aggregate proteins (such as α-synuclein). The ubiquitin proteasome system (UPS), the major intracellular protein degradation complex, and Ac-ylaminoacyl-PEptide Hydrolase (APEH), a ubiquitous bifunctional enzyme, which exhibits exopepti-dase activity on N-acyl-peptides and endoprotease activity on oxidized proteins, they are very im-portant protagonists in maintaining protein homeostasis. Recently, many studies have focused on the search for sensitive and specific molecules that can help neurodegenerative disorders diagnosis; among these, a combined action between proteasome and APEH in maintaining cellular homeostasis in patients with AD has been reported. The present work, starting from a study done on AD patients, aims to investigate the enzymatic activi-ties of UPS and APEH and the expression levels of their own genes and of others involved in their metabolic pathways, in blood samples of patients with PD compared to healthy controls (HC). The study, which involved three different operating units, consisted in the recruitment of forty-six par-ticipants, divided into two groups on the basis of clinical profiles, of which 23 PD and 23 HC; a ve-nous sampling was carried out, various cellular blood fractions (erythrocytes, granulocytes, lympho-cytes and monocytes) were then collected in order to measure, by means of spectrofluorimetric analy-sis, the APEH exopeptidase APEH and chymotyptic proteasome activities. Total RNA, extracted from the whole blood of twenty patients of which 10 PD and 10 HC, was used for the gene expression of the APEH and the proteasome by real time PCR. The expression of genes related to the UPS and APEH metabolic pathways was analyzed with the aim of investigating the molecular mechanisms underlying Parkinson's. In particular:-UBA1, coding for the first enzyme involved in the ubiquitination process (enzyme acti-vating ubiquitin like modifier); -UBE2I, encoding the second enzyme involved in the ubiquitination process (ubiquitin-like modifier activation enzyme); -PRKN, coding for parkin (ubiquitin ligase enzyme); -NAA10, encoding the enzymatic subunit of the N-alpha acetyltransferase A complex; -ACY1, coding for aminoacylase 1 - SOD2, superoxide dismutase; - PARK1, alpha synuclein. The results of this thesis work show a different trend compared to the results obtained from the study previously carried out on AD; in particular, enzymatic activities and gene expression levels increase compared to healthy controls. These results help to understand the molecular mechanisms of AD and PD and show the importance that the molecular biomarkers, object of this study, could play in the onset of these diseases. In this sense, if the results obtained are confirmed in larger prospective studies, the measurement of the activity of the systems under study, using a simple blood sample, could simplify the diagnostic ap-proaches of these diseases.File | Dimensione | Formato | |
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