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Systematic comparison of the molecular mechanisms underlying the spreading of pathology in different neurodegenerative diseases

dc.contributor.advisorOuteiro, Tiago Fleming Prof. Dr.
dc.contributor.authorBrás, Inês
dc.titleSystematic comparison of the molecular mechanisms underlying the spreading of pathology in different neurodegenerative diseasesde
dc.contributor.refereeBraus, Gerhard Prof. Dr.
dc.description.abstractengMisfolding and accumulation of disease-related proteins are common hallmarks among several neurodegenerative diseases. This phenomenon causes the progressive loss of cognitive and motor functions correlated with specific cell loss in the brain. Despite the different clinical manifestations, these disorders share common features and molecular mechanisms, such as aggregation and accumulation of disease-related proteins in specific regions of the brain. These include alpha-synuclein aggregates in Parkinson’s disease and other synucleinopathies, inclusions of hyperphosphorylated microtubule-binding Tau in tauopathies, and extended polyglutamine protein aggregates (huntingtin) in Huntington’s disease. The association between the progression of clinical symptoms and the topographical distribution of pathology in neurodegenerative diseases has led to the hypothesis that specific pathological disease-related proteins can be transferred intercellularly in a prion-like manner. This hypothesis was created after the observation Lewy body pathology, a characteristic hallmark in synucleinopathies, within fetal dopaminergic neurons grafts in Parkinson’s disease patients. Later, injection of Tau aggregates into animal models was shown to induce pathology. More recently, similar mechanisms were proposed to occur in monogenic forms of neurodegenerative diseases after the observation of mutant huntingtin aggregates within fetal striatal allografts in the brain of Huntington’s disease patients. Furthermore, the presence of co-pathology in the brain of patients with distinct neurodegenerative diseases has implied that several proteins may overlap to contribute to the neuropathophysiology and can share common molecular mechanisms in neurodegeneration. Several mechanisms have been suggested to contribute to the spreading of alpha-synuclein, Tau and huntingtin pathology. These include diffusion through the plasma membrane, release via extracellular vesicles (as ectosomes and exosomes), misfolded-associated protein secretion pathway, membrane carriers, membrane pores, tunnelling nanotubes, endocytosis, and receptor-mediated endocytosis. To date, it is unclear if alpha-synuclein, Tau and huntingtin release to the extracellular milieu occurs through similar cellular mechanisms and their effect in receptor cells. Also, the role of protein secretion and their involvement in neuronal dysfunction and disease progression remains elusive. Further elucidation of these questions will permit a better understanding of protein propagation on disease pathogenesis in neurodegenerative diseases. In recent years, extracellular vesicles have emerged as central mediators in intercellular communication under physiological and pathological conditions. Their heterogeneity and presence in several human biofluids have led to extensive research regarding their content and functional properties as relevant biomarkers in neurodegenerative diseases. In particular, ectosomes and exosomes are considered important carriers of misfolded proteins between cells in disease. These can be internalized in diverse cell types, although their effect in neuronal activity is unclear. Discernment between ectosomes and exosomes has been difficult due to the moderate differences in their physical properties and absence of reliable markers. While exosomes have been extensively studied in the field, the role of ectosomes in the pathogenesis of neurodegenerative diseases has not been explored. Additional studies focusing in the role of ectosomes and other types of extracellular vesicles in neurodegenerative diseases will open new avenues to uncover potential disease biomarkers and therapeutic targets. In our first study, we developed a detailed differential ultracentrifugation protocol to efficiently purify ectosomes and exosomes, and provided a proteomic and functional characterization of these vesicles subtypes. Comprehensive proteomic analysis revealed specific protein composition and pathways enrichment for ectosomes and exosomes. Interestingly, ectosomes isolated from human cerebrospinal fluid and from cell media displayed enrichment in annexin-A2, suggesting this protein as an important marker for ectosomes characterization. Furthermore, treatment of neuronal cortical cultures with ectosomes and exosomes resulted in their internalization at similar ratios. Using multi-electrode array technology, we further demonstrated that extracellular vesicles internalization affects differently the spontaneous activity of neuronal cells. In our second study, we demonstrated that common cellular mechanisms are used for the transfer of alpha-synuclein, Tau and huntingtin exon-1 fragments between cells. Interestingly, we observed that these proteins are handled in different ways depending on the receptor cells. Our results reveal the release of the different disease-related proteins to the cell media at different levels in a free form and in extracellular vesicles. Overall, alpha-synuclein, Tau and normal huntingtin exon-1 were found in higher levels in the cell media in contrast to mutant huntingtin exon-1. We further observed discernible alterations in the spontaneous firing activity in primary cortical neurons after treatment with the different recombinant proteins, suggesting that the effects of alpha-synuclein, Tau and huntingtin in the extracellular space and on neuronal activity are dependent of the protein properties and not only correlated with their secretion levels. Interestingly, alpha-synuclein, Tau and huntingtin exon-1 were present in higher levels in ectosomes than in exosomes. We revealed that these vesicles could be internalized in microglial and astrocytic cells, and resulted in the production of pro-inflammatory cytokines and autophagy activation. Neuronal cells also internalized ectosomes and exosomes enriched with alpha-synuclein, Tau or huntingtin, and exhibited irregularity in the cell bursting properties that overall was correlated with the vesicles subtype. Overall, our work indicates that extracellular vesicles cargoes likely reflect core pathophysiological intracellular processes in their origin cells. A clear understanding of the specific functional properties of different vesicles subtypes might represent a step forward in the search of novel biomarkers. Furthermore, our results propose that common cellular mechanisms are used for the transfer of alpha-synuclein, Tau and huntingtin between cells. These similarities could suggest common therapeutic targets for neurodegenerative diseases, and the need to target several cellular mechanisms to halt the detrimental effects of protein transmission and pathology
dc.contributor.coRefereeZweckstetter, Markus Prof. Dr.
dc.contributor.thirdRefereeUrlaub, Henning Prof. Dr.
dc.contributor.thirdRefereeBrose, Nils Prof. Dr.
dc.contributor.thirdRefereeRizzoli, Silvio Prof. Dr.
dc.subject.engneurodegenerative diseasesde
dc.subject.engextracellular vesiclesde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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