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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - NEUROTUNN (Mechanisms of Î±-synuclein spreading, implications for synucleinopathies)

Teaser

Summary

Parkinsonâ€™s Disease (PD) is a major health problem particularly for European aging populations. Since the etiology of PD remains unknown and an effective therapeutic intervention is not yet available, the recent observations that prion-like mechanisms underlie the pathological spreading of misfolded Î±-synuclein (Î±-syn) and other proteins represent a ground-breaking discovery with extensive implications. PD is a neurodegenerative disorder related to aging, characterized by intracellular deposits of aggregated Î±-synuclein (Î±-syn) known as Lewy bodies and Lewy neurites. In PD patients, at late stages of disease, deposits of Î±-syn aggregates are widely spread in the central nervous system (CNS). Several studies have shown that Lewy bodies may propagate within the brain in a prion-like manner, which means that the disease may be transmissible by self-propagation of the protein misfolding process in a similar way as prions transmit prion diseases. However, the exact underlying route(s) allowing the physical movement of the protein aggregates from one cell to another and the cellular players involved in this process are not yet fully understood. Thus, considerable amount of research has focused in clarifying this possibility with the aim of translating the knowledge of the basic disease mechanisms into development of novel strategies for early diagnosis and efficient treatment. Accordingly, by using novel systems of primary cultures combined with state-of-the-art imaging approaches, the general aims of the project were to 1) unveil the mechanisms of propagation of Î±-syn protein assemblies, 2) assess the possible involvement of tunneling nanotubes (TNTs), which are thin actin-rich membrane bridges that allow exchange of cellular components between cells, in the transfer process and, 3) evaluate if non-cell autonomous processes, namely neuron-glial interactions, contribute to either clearance or dissemination of Î±-syn aggregated species between the CNS cells. In summary, this project addresses three open questions in the field; the unequivocal identification of the mechanism of Î±-syn transfer in neurons, the possible involvement of TNTs in Î±-syn transfer and the impact of glial cells to the pathology. Providing answers to these and other related research questions is of paramount importance mainly from a therapeutic point of view.

Work performed

One of the most interesting discoveries originated from my research work that is directly related to this proposal, was the identification of one mechanism of -syn propagation using quantitative fluorescence microscopy of co-cultured neuronal cells. In that work, I demonstrated that -syn fibrils efficiently transfer from one cell to the other through tunneling nanotubes (TNTs) inside lysosomal vesicles. Furthermore, I also showed for the first time the formation of TNTs between neuronal cells and primary neurons and determined and quantified that the propagation of -syn fibrils between primary neurons is mainly dependent on cell-to-cell contact, which is of vital importance for understanding how PD progress. Another important contribution of my work to the field of synucleinopathies is the establishment of several in vitro and ex vivo models that allowed the quantification of a selective intercellular communication between astrocytes and neurons. In that work, I demonstrated the role of astrocytes in the intercellular transfer and fate of Î±-syn fibrils. Besides, I showed that Î±-syn fibrils are transferred in a non-cell autonomous manner, but the transfer efficiency changes depending on the cell types; Î±-syn is efficiently transferred from astrocytes to astrocytes and from neurons to astrocytes, but less efficiently from astrocytes to neurons. However, differently from neurons, which are unable to degrade the fibrils, astrocytes efficiently degrade fibrillar Î±-syn, suggesting an active role for these cells in clearing Î±-syn deposits. In addition, I successfully developed a novel co-culture system where primary mouse astrocytes were cultured on top of organotypic hippocampal slices, where they integrated and interacted with the cells of the slice. This new model has proven to be important for studying Î±-syn trafficking in the disease state, given that it represents the first model to look at cellular transfer of Î±-syn in the presence of all glial cell types.

The results derived from this project have been disseminated in the following peer-reviewed articles, meetings and conferences:

Loria F, et al, 2017, DOI: 10.1007/s00401-017-1746-2

Abounit S et al, 2016, DOI: 10.15252/embj.201593411

In vitro and ex vivo assessment of the role of astrocytes in the spreading of alpha-synuclein aggregates. The Society of Neuroscience Annual Meeting. Washington DC, USA. 2017.

In vitro and ex vivo assessment of Î±-synuclein spreading in neurons and astrocytes. Seminar, Institut Pasteur, Paris, France. 2017.

Identification of the cellular players involved in the spreading of Î±-synuclein. The 13th International Conference on Alzheimerâ€™s & Parkinsonâ€™s Diseases. Vienna, Austria. 2017.

Identification of cellular players involved in the spreading of Î±-synuclein. BCI Annual Retreat. Institut Pasteur, Paris, France. 2016.

In vitro and ex vivo assessment and quantification of Î±-synuclein intercellular spreading. Institut Pasteur, Quantitative Biology Meeting. Paris, France. 2016.

Mechanisms of alpha-synuclein spreading in neurons, role of tunneling nanotubes. EMBO Workshop on Membrane fusion in health and disease. Paris, France. 2016.

Mechanisms of Î±-synuclein intercellular spreading involved in Parkinsonâ€™s disease progression. EMBO Workshop on Membrane fusion in health and disease. Paris, France. 2016. Poster.

Mechanisms of Î±-synuclein intercellular spreading involved in Parkinsonâ€™s disease progression. StaPa-ADIC Joint Retreat Normandie, France. 2016.

Final results

Overall, the results derived from this project have i) extended the knowledge concerning Î±-syn dynamics in neurons and glial cells, ii) filled fundamental gaps in basic knowledge of TNTs, iii) identified novel mechanisms of intercellular communication, iv) brought advances to the understanding of neurodegenerative pathologies and v) contributed to the possible development of novel therapies for treating PD and other neurodegenerative diseases that take into account glial cells and TNTs. Ultimately, in the short-term, the researcher had the greatest chance to contribute to the advancement in the understanding of a disease with a high incidence in Europe for which searching of solutions in the form of basic knowledge or treatments is a priority.