Parkinson’s disease (PD) is a progressive neurodegenerative disorder marked by dopaminergic neuron loss in the substantia nigra and misfolded alpha-synuclein accumulation in Lewy bodies. These pathological changes disrupt motor and non-motor circuits, leading to hallmark motor symptoms (bradykinesia, rigidity, tremor, postural instability) and non-motor symptoms such as REM sleep behavior disorder (RBD), depression, anosmia, and autonomic dysfunction. RBD, characterized by loss of atonia during REM sleep and vivid, often violent dreams, is increasingly recognized as a prodromal phase of Parkinson's. Despite advances in early symptom recognition, PD diagnosis remains reliant on clinical identification of motor signs. Imaging techniques, such as dopamine transporter scans or magnetic resonance, support diagnosis but are typically employed after significant neurodegeneration. This underscores the need for tools to detect PD in its prodromal phase, when interventions could potentially alter the progression. Mitochondrial dysfunction and oxidative stress are central mechanisms in Parkinson's pathogenesis. Impaired mitochondrial energy production and elevated reactive oxygen species contribute to neuronal damage and alpha-synuclein aggregation, creating a cycle of neurodegeneration. These processes are linked to the prodromal phase, particularly in individuals with RBD. This project investigates the relationship between iRBD and PD, focusing on mitochondrial dysfunction, oxidative stress, and alpha-synuclein accumulation as potential biomarkers and therapeutic targets. The study analyzed mitochondrial proteins, antioxidant enzymes, oxidative markers, and alpha-synuclein in PBMCs, plasma, and sEVs from 58 patients divided into four groups: 20 controls, 18 iRBD, 10 RBD-PD (REM sleep disorders before PD), and 10 PD-RBD (PD symptoms before RBD onset). PBMCs and plasma were isolated via density gradient centrifugation, and sEVs were obtained through ultracentrifugation and characterized biochemically. Protein quantification was performed using BCA assays, while alpha-synuclein levels were measured via ELISA and Western Blot. Oxidative stress markers and enzymatic activities were assessed using TBARS, SOD, and catalase activity assays. Results revealed significant increases in TBARS (lipid peroxidation) in iRBD compared to controls, with slightly lower levels in RBD-PD, suggesting oxidative damage peaks in early stages of neurodegeneration. This reduction in RBD-PD could be attributed to compensatory mechanisms or to the fact that oxidative stress may act as an early trigger and then decreases as the pathology progresses. In PBMCs, increased Complex II and decreased Complex IV expression in iRBD compared to controls and PD indicate mitochondrial dysfunction and imbalances in the electron transport chain driving elevated ROS production. Analysis of sEV-contained alpha-synuclein showed progressively higher levels from controls to iRBD and RBD-PD, supporting its potential as a biomarker for monitoring disease progression. In conclusion, mitochondrial and oxidative alterations in PBMCs, plasma, and sEVs of iRBD patients may serve as predictive biomarkers for neurodegeneration and phenoconversion to PD. These findings highlight the potential of mitochondrial function and oxidative stress assessments in identifying individuals at higher risk of synucleinopathy. Early intervention with neuroprotective strategies may benefit from such insights, although further studies with larger, longitudinal cohorts are needed to confirm these findings and establish clinical relevance.
La malattia di Parkinson (MP) è un disturbo neurodegenerativo progressivo caratterizzato dalla perdita di neuroni dopaminergici nella substantia nigra e dall'accumulo di alfa-sinucleina soggetta a fenomeni di misfolding nei corpi di Lewy. Questi cambiamenti patologici alterano i circuiti motori e non motori, causando sintomi caratteristici come bradicinesia, rigidità, tremore e instabilità posturale, oltre a sintomi non motori quali il disturbo del comportamento del sonno REM (RBD), la depressione, l’anosmia e la disfunzione autonomica. Tra questi, l’RBD, una parasomnia caratterizzata dalla perdita dell’atonia muscolare durante il sonno REM e da sogni vividi, spesso violenti, è considerato una fase prodromica cruciale della MP. Nonostante i progressi nel riconoscimento dei sintomi precoci, la diagnosi della MP si basa principalmente sull’osservazione clinica dei segni motori. Tecniche di imaging, come la scansione dei trasportatori di dopamina o la risonanza magnetica, possono supportare il processo diagnostico ma sono impiegate solo in stadi avanzati, quando la neurodegenerazione è già significativa. Pertanto, è essenziale sviluppare strumenti diagnostici che consentano di identificare la MP nella fase prodromica, massimizzando il potenziale degli interventi terapeutici. La disfunzione mitocondriale e lo stress ossidativo sono meccanismi chiave nella patogenesi della MP. La ridotta produzione di energia mitocondriale e l'aumento di specie reattive dell’ossigeno (ROS) contribuiscono al danno neuronale e favoriscono l’aggregazione di alfa-sinucleina, innescando un ciclo di neurodegenerazione. Questi processi sono particolarmente rilevanti nei soggetti con RBD, che presentano un rischio elevato di conversione verso la MP. Questo studio ha analizzato le alterazioni mitocondriali e ossidative in pazienti con iRBD, focalizzandosi su proteine mitocondriali, enzimi antiossidanti, marcatori ossidativi e alfa-sinucleina come potenziali biomarcatori. Sono stati inclusi 58 soggetti, suddivisi in quattro gruppi: 20 controlli, 18 con iRBD, 10 con RBD-PD (disturbi del sonno REM che precedono la MP) e 10 con PD-RBD (sintomi motori precedenti l’RBD). I PBMCs e il plasma sono stati isolati mediante centrifugazione, mentre le sEVs sono state ottenute tramite ultracentrifugazione. Le proteine sono state quantificate con il saggio BCA e i livelli di alfa-sinucleina sono stati misurati con ELISA e Western Blot. I marcatori di stress ossidativo, inclusi TBARS, SOD e catalasi, sono stati analizzati per valutare lo stato redox. I risultati hanno evidenziato un aumento significativo dei livelli di TBARS nei pazienti iRBD rispetto ai controlli, con una diminuzione nei RBD-PD, suggerendo che lo stress ossidativo sia più marcato nelle fasi iniziali della neurodegenerazione, Questa riduzione potrebbe essere attribuita a meccanismi compensatori che emergono nello stato patologico (diversi da SOD e catalasi, che sono invariati) o al fatto che lo stress ossidativo possa agire come un innesco precoce per poi diminuire man mano che la patologia progredisce. Nei PBMCs, è stato osservato un aumento dell’espressione del Complesso II e una riduzione del Complesso IV nei soggetti iRBD, indicando uno squilibrio nella catena di trasporto degli elettroni che potrebbe favorire la produzione eccessiva di ROS. Inoltre, i livelli di alfa-sinucleina nelle sEVs aumentano progressivamente dai controlli agli iRBD fino ai RBD-PD, rafforzando il suo potenziale come biomarcatore. Questi risultati suggeriscono che le alterazioni mitocondriali e ossidative in iRBD possano rappresentare biomarcatori utili per identificare individui a rischio di MP e guidare interventi neuroprotettivi. Studi futuri con coorti più ampie sono necessari per validare queste osservazioni.
Valutazione della disfunzione mitocondriale, dello stress ossidativo e dell’alfa-sinucleina nel disturbo comportamentale del sonno REM idiopatico come marcatori predittivi della fenoconversione verso la malattia di Parkinson
ABATI, REBECCA
2023/2024
Abstract
Parkinson’s disease (PD) is a progressive neurodegenerative disorder marked by dopaminergic neuron loss in the substantia nigra and misfolded alpha-synuclein accumulation in Lewy bodies. These pathological changes disrupt motor and non-motor circuits, leading to hallmark motor symptoms (bradykinesia, rigidity, tremor, postural instability) and non-motor symptoms such as REM sleep behavior disorder (RBD), depression, anosmia, and autonomic dysfunction. RBD, characterized by loss of atonia during REM sleep and vivid, often violent dreams, is increasingly recognized as a prodromal phase of Parkinson's. Despite advances in early symptom recognition, PD diagnosis remains reliant on clinical identification of motor signs. Imaging techniques, such as dopamine transporter scans or magnetic resonance, support diagnosis but are typically employed after significant neurodegeneration. This underscores the need for tools to detect PD in its prodromal phase, when interventions could potentially alter the progression. Mitochondrial dysfunction and oxidative stress are central mechanisms in Parkinson's pathogenesis. Impaired mitochondrial energy production and elevated reactive oxygen species contribute to neuronal damage and alpha-synuclein aggregation, creating a cycle of neurodegeneration. These processes are linked to the prodromal phase, particularly in individuals with RBD. This project investigates the relationship between iRBD and PD, focusing on mitochondrial dysfunction, oxidative stress, and alpha-synuclein accumulation as potential biomarkers and therapeutic targets. The study analyzed mitochondrial proteins, antioxidant enzymes, oxidative markers, and alpha-synuclein in PBMCs, plasma, and sEVs from 58 patients divided into four groups: 20 controls, 18 iRBD, 10 RBD-PD (REM sleep disorders before PD), and 10 PD-RBD (PD symptoms before RBD onset). PBMCs and plasma were isolated via density gradient centrifugation, and sEVs were obtained through ultracentrifugation and characterized biochemically. Protein quantification was performed using BCA assays, while alpha-synuclein levels were measured via ELISA and Western Blot. Oxidative stress markers and enzymatic activities were assessed using TBARS, SOD, and catalase activity assays. Results revealed significant increases in TBARS (lipid peroxidation) in iRBD compared to controls, with slightly lower levels in RBD-PD, suggesting oxidative damage peaks in early stages of neurodegeneration. This reduction in RBD-PD could be attributed to compensatory mechanisms or to the fact that oxidative stress may act as an early trigger and then decreases as the pathology progresses. In PBMCs, increased Complex II and decreased Complex IV expression in iRBD compared to controls and PD indicate mitochondrial dysfunction and imbalances in the electron transport chain driving elevated ROS production. Analysis of sEV-contained alpha-synuclein showed progressively higher levels from controls to iRBD and RBD-PD, supporting its potential as a biomarker for monitoring disease progression. In conclusion, mitochondrial and oxidative alterations in PBMCs, plasma, and sEVs of iRBD patients may serve as predictive biomarkers for neurodegeneration and phenoconversion to PD. These findings highlight the potential of mitochondrial function and oxidative stress assessments in identifying individuals at higher risk of synucleinopathy. Early intervention with neuroprotective strategies may benefit from such insights, although further studies with larger, longitudinal cohorts are needed to confirm these findings and establish clinical relevance.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14239/28473