Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Compared to men, women display lower PD incidence and prevalence, later onset, and milder symptoms. Key pathological features of PD are the loss of dopaminergic neurons in the substantia nigra and the accumulation of α-synuclein. This protein, normally expressed as component of the central and enteric nervous systems (CNS and ENS, respectively), in pathological conditions aggregates to form proteinaceous inclusions observed in the brain of PD patients, but also in their intestines. In addition to the well-known motor deficits, PD is also characterized by a number of non-motor symptoms, including constipation, hyposmia, anxiety and depression. Among these, constipation is one of the most common and one of the earliest, appearing even more than 20 years before disease diagnosis. On the basis of the early appearance of intestinal dysfunction, together with the abnormal α-synuclein deposition in the ENS observed before CNS neurodegeneration, Braak and colleagues suggested that α-synuclein pathology may start in the gut and then propagate to the brain. We previously showed that male mice lacking the NF-kB/c-Rel subunit (c-rel-/- mice) reproduce a Braak-like pattern of ascending α-synuclein deposition in the olfactory bulbs and brainstem nuclei. The pathology progression is accompanied by the appearance of both non-motor and motor symptoms, including hyposmia at 2 months, anxiety- and depressive-like behaviour at 12 months, and motor deficits and apathy at 18 months. The aim of this thesis work is to investigate whether the pathology in the c-rel-/- mouse model of PD starts at gut level. This PhD thesis consists of three parts. In Part I, I characterized the progression of intestinal dysfunction and pathology in c-rel-/- male mice by using a combination of behavioral tests, as well as microscopy and biochemistry methods. From 2 months of age, c-rel-/- mice displayed intestinal constipation. At 2 months, c-rel-/- mice exhibited a mild α-synuclein accumulation in the distal colon. Similarly, accumulation of phosphorylated α-synuclein was already present in proximal colon at 2 months, and increased at 10 months. Gut α-synucleinopathy was paralleled by increasing inflammation, as pointed out by the augmented levels of infiltrating leukocytes in the proximal colon of 10-month-old c-rel-/- mice and a progressive colon shortening. Finally, 10-month-old c-rel-/- mice displayed higher levels of oxidative stress in the gut. Taken together, these results indicate that the c-rel-/- male mice reproduce progressive pathology and dysfunction also at gut levels. In Part II, I determined whether gut dysfunction and pathology in c-rel-/- male mice were related to an altered microbiome composition. Stool pellets collected from wt and c-rel-/- mice at 0.5, 6-9 and 18-20 months were subjected to culturomics and Next Generation Sequencing analyses. The complementary culturomics and metagenomic approaches indicated a remarkable difference between wt and c-rel-/- mice at 0.5 months. Indeed, young c-rel-/- male mice displayed a pro-inflammatory microbiome characterized by the reduction of Verrucomicrobiacae and Enterobacteriaceae bacteria families, and the increase of Prevotellacae. In Part III, I investigated sexual dimorphism in c-rel-/- mice, with emphasis on motor and intestinal symptoms, gut pathology and microbiome composition. The comparison of aged wt and c-rel-/- male and female mice revealed the absence of significant motor deficits in c-rel-/- females. Also constipation was milder in c-rel-/- females when compared with c-rel-/- males. Lastly, the observation that aged c-rel-/- females did not present colon shortening, suggested a reduced intestinal inflammation. Despite the milder gut dysfunction and pathology observed in c-rel-/- females, males and females lacking c-Rel share a similar alteration in microbiome composition.
La malattia di Parkinson (MP) è la più comune malattia neurodegenerativa del movimento. Rispetto agli uomini, le donne mostrano incidenza e prevalenza più basse, esordio tardivo, e sintomi più blandi. Caratteristiche principali della MP sono la perdita di neuroni dopaminergici nella sostanza nera e l’accumulo di α-sinucleina. Questa proteina, normalmente espressa nel sistema nervoso centrale ed enterico (SNC e SNE), in condizioni patologiche aggrega formando inclusioni proteinacee osservabili nei cervelli e negli intestini dei pazienti affetti da MP. Oltre ai noti deficit motori, la MP è caratterizzata da sintomi non motori che spesso precedono l’insorgenza dei disturbi del movimento, tra cui stipsi, iposmia, ansia e depressione, disturbi del sonno. Tra i sintomi non motori, la stipsi è uno dei più comuni e dei più precoci, potendo insorgere anche più di 20 anni prima della diagnosi della malattia. Sulla base della comparsa precoce della disfunzione intestinale e dell’accumulo anormale di α-sinucleina nel SNE prima della neurodegenerazione del SNC, Braak ha proposto che l’accumulo di α-sinucleina possa iniziare nell’intestino per poi propagarsi al cervello. Abbiamo precedentemente dimostrato che topi maschi mancanti della subunità NF-kB/c-Rel (topi c-rel-/-) riproducono un profilo di accumulo ascendente di α-sinucleina nei bulbi olfattori e nei nuclei del tronco encefalico simile a quello proposto da Braak. La progressione della patologia è accompagnata dalla comparsa di sintomi non motori e motori, quali l’iposmia a 2 mesi, ansia e comportamento simil-depressivo a 12, deficit motori ed apatia a 18. Lo scopo di questo lavoro è di investigare se nel modello di MP rappresentato dal topo c-rel-/- la patologia abbia inizio a livello intestinale. Questa tesi di dottorato è divisa in tre parti. Nella Parte I ho caratterizzato la progressione della patologia intestinale nei topi c-rel-/- maschi utilizzando una combinazione di test comportamentali, metodi microscopici e biochimici. A partire dall’età di 2 mesi, i topi c-rel-/- mostrano un moderato accumulo di α-sinucleina nel colon distale. In maniera simile, l’accumulo di α-sinucleina fosforilata è presente nel colon prossimale a 2 mesi, e peggiora a 10. In parallelo è osservabile una progressiva infiammazione, come indicato dai leucociti infiltrati nel colon prossimale dei topi c-rel-/- di 10 mesi, e da un accorciamento del colon. Infine, i topi c-rel-/- di 10 mesi mostrano alti livelli di stress ossidativo nell’intestino. Nel loro insieme, questi risultati mostrano come i topi c-rel-/- maschi riproducano una patologia progressiva anche a livello intestinale. Nella Parte II ho determinato se la patologia intestinale nei topi c-rel-/- maschi fosse legata ad una composizione alterata del microbioma. Campioni di feci sono stati raccolti dai topi wt e c-rel-/- a 0.5, 6-9 e 18-20 mesi e processati mediante analisi culturomica e Next Generation Sequencing. Tale approccio complementare ha indicato importanti differenze tra i topi wt e c-rel-/- a 0.5 mesi. I giovani topi c-rel-/- presentavano un microbioma pro-infiammatorio caratterizzato da una riduzione delle famiglie batteriche delle Verrucomicrobiacae e Enterobacteriaceae, ed un aumento di Prevotellacae. Nella Parte III ho studiato il dimorfismo sessuale nei topi c-rel-/-, con enfasi sui sintomi motori ed intestinali, sulla patologia intestinale e sulla composizione del microbioma. Il confronto tra i topi wt e c-rel-/- maschi e femmine ha rivelato un’assenza di deficit motori significativi ed una stipsi ridotta nelle femmine c-rel-/-. Inoltre, l’osservazione che le femmine c-rel-/- anziane non presentano accorciamento del colon ha suggerito una ridotta infiammazione intestinale. A fronte delle ridotte disfunzioni intestinali osservate nelle femmine c-rel-/-, i topi maschi e femmine c-rel-/- condividevano una simile alterazione nella composizione del microbioma.
From gut to brain: sexually dimorphic pathology in the c-rel-/- mouse mimicking Parkinson’s disease progression / Parrella, Edoardo. - (2022 Jun 10).
From gut to brain: sexually dimorphic pathology in the c-rel-/- mouse mimicking Parkinson’s disease progression.
PARRELLA, Edoardo
2022-06-10
Abstract
Parkinson’s disease (PD) is the most common neurodegenerative movement disorder. Compared to men, women display lower PD incidence and prevalence, later onset, and milder symptoms. Key pathological features of PD are the loss of dopaminergic neurons in the substantia nigra and the accumulation of α-synuclein. This protein, normally expressed as component of the central and enteric nervous systems (CNS and ENS, respectively), in pathological conditions aggregates to form proteinaceous inclusions observed in the brain of PD patients, but also in their intestines. In addition to the well-known motor deficits, PD is also characterized by a number of non-motor symptoms, including constipation, hyposmia, anxiety and depression. Among these, constipation is one of the most common and one of the earliest, appearing even more than 20 years before disease diagnosis. On the basis of the early appearance of intestinal dysfunction, together with the abnormal α-synuclein deposition in the ENS observed before CNS neurodegeneration, Braak and colleagues suggested that α-synuclein pathology may start in the gut and then propagate to the brain. We previously showed that male mice lacking the NF-kB/c-Rel subunit (c-rel-/- mice) reproduce a Braak-like pattern of ascending α-synuclein deposition in the olfactory bulbs and brainstem nuclei. The pathology progression is accompanied by the appearance of both non-motor and motor symptoms, including hyposmia at 2 months, anxiety- and depressive-like behaviour at 12 months, and motor deficits and apathy at 18 months. The aim of this thesis work is to investigate whether the pathology in the c-rel-/- mouse model of PD starts at gut level. This PhD thesis consists of three parts. In Part I, I characterized the progression of intestinal dysfunction and pathology in c-rel-/- male mice by using a combination of behavioral tests, as well as microscopy and biochemistry methods. From 2 months of age, c-rel-/- mice displayed intestinal constipation. At 2 months, c-rel-/- mice exhibited a mild α-synuclein accumulation in the distal colon. Similarly, accumulation of phosphorylated α-synuclein was already present in proximal colon at 2 months, and increased at 10 months. Gut α-synucleinopathy was paralleled by increasing inflammation, as pointed out by the augmented levels of infiltrating leukocytes in the proximal colon of 10-month-old c-rel-/- mice and a progressive colon shortening. Finally, 10-month-old c-rel-/- mice displayed higher levels of oxidative stress in the gut. Taken together, these results indicate that the c-rel-/- male mice reproduce progressive pathology and dysfunction also at gut levels. In Part II, I determined whether gut dysfunction and pathology in c-rel-/- male mice were related to an altered microbiome composition. Stool pellets collected from wt and c-rel-/- mice at 0.5, 6-9 and 18-20 months were subjected to culturomics and Next Generation Sequencing analyses. The complementary culturomics and metagenomic approaches indicated a remarkable difference between wt and c-rel-/- mice at 0.5 months. Indeed, young c-rel-/- male mice displayed a pro-inflammatory microbiome characterized by the reduction of Verrucomicrobiacae and Enterobacteriaceae bacteria families, and the increase of Prevotellacae. In Part III, I investigated sexual dimorphism in c-rel-/- mice, with emphasis on motor and intestinal symptoms, gut pathology and microbiome composition. The comparison of aged wt and c-rel-/- male and female mice revealed the absence of significant motor deficits in c-rel-/- females. Also constipation was milder in c-rel-/- females when compared with c-rel-/- males. Lastly, the observation that aged c-rel-/- females did not present colon shortening, suggested a reduced intestinal inflammation. Despite the milder gut dysfunction and pathology observed in c-rel-/- females, males and females lacking c-Rel share a similar alteration in microbiome composition.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
