Synucleinopathies such as Parkinson's disease are characterized by the pathological deposition of misfolded a-synuclein aggregates into inclusions throughout the central and peripheral nervous system. Mounting evidence suggests that intercellular propagation of a-synuclein aggregates may contribute to the neuropathology; however, the mechanism by which spread occurs is not fully understood. By using quantitative fluorescence microscopy with co-cultured neurons, here we show that a-synuclein fibrils efficiently transfer from donor to acceptor cells through tunneling nanotubes (TNTs) inside lysosomal vesicles. Following transfer through TNTs, a-synuclein fibrils are able to seed soluble a-synuclein aggregation in the cytosol of acceptor cells. We propose that donor cells overloaded with a-synuclein aggregates in lysosomes dispose of this material by hijacking TNT-mediated intercellular trafficking. Our findings thus reveal a possible novel role of TNTs and lysosomes in the progression of synucleinopathies.

ABSTRACT. Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases caused by the misfolding of the cellular prion protein to an infectious form PrP Sc . The intercellular transfer of PrP Sc is a question of immediate interest as the cell-to-cell movement of the infectious particle causes the inexorable propagation of disease. We have previously identified tunneling nanotubes (TNTs) as one mechanism by which PrP Sc can move between cells. Here we investigate further the details of this mechanism and show that PrP Sc travels within TNTs in endolysosomal vesicles. Additionally we show that prion infection of CAD cells increases both the number of TNTs and intercellular transfer of membranous vesicles, thereby possibly playing an active role in its own intercellular transfer via TNTs.

Prion diseases are caused by misfolding of the cellular protein PrPC to an infectious conformer, PrPSc. Intercellular PrPSc transfer propagates conversion and allows infectivity to move from the periphery to the brain. However, how prions spread between cells of the central nervous system is unclear. Astrocytes are specialized non-neuronal cells within the brain that have a number of functions indispensable for brain homeostasis. Interestingly, they are one of the earliest sites of prion accumulation in the brain. A fundamental question arising from this observation is whether these cells are involved in intercellular prion transfer and thereby disease propagation. Using co-culture systems between primary infected astrocytes and granule neurons or neuronal cell lines, we provide direct evidence that prion-infected astrocytes can disseminate prion to neurons. Though astrocytes are capable of secreting PrP, this is an inefficient method of transferring prion infectivity. Efficient transfer required co-culturing and direct cell contact. Astrocytes form numerous intercellular connections including tunneling nanotubes, containing PrPSc, often colocalized with endolysosomal vesicles, which may constitute the major mechanism of transfer. Because of their role in intercellular transfer of prions astrocytes may influence progression of the disease.

Tunneling nanotubes (TNTs) are actin-enriched membranous channels enabling cells to communicate over long distances. TNT-like structures form between various cell types and mediate the exchange of different cargos, such as ions, vesicles, organelles and pathogens; thus, they may play a role in physiological conditions and diseases (e.g. cancer and infection). TNTs also allow the intercellular passage of protein aggregates related to neurodegenerative diseases, thus propagating protein misfolding. Understanding the mechanism of TNT formation is mandatory in order to reveal the mechanism of disease propagation and to uncover their physiological function. Vesicular transport controlled by the small GTPases Rab11a and Rab8a can promote the formation of different plasma membrane protrusions (filopodia, cilia and neurites). Here, we report that inhibiting membrane recycling reduces the number of TNT-connected cells and that overexpression of Rab11a and Rab8a increases the number of TNT-connected cells and the propagation of vesicles between cells in co-culture. We demonstrate that these two Rab GTPases act in a cascade in which Rab11a activation of Rab8a is independent of Rabin8. We also show that VAMP3 acts downstream of Rab8a to regulate TNT formation.

Tunneling nanotubes (TNTs) are F-actin rich structures that connect distant cells, allowing the transport of many cellular components, including vesicles, organelles and molecules. Rab GTPases are the major regulators of vesicle trafficking and also participate in actin cytoskeleton remodelling, therefore, we examined their role in TNTs. Rab35 functions with several proteins that are involved in vesicle trafficking such as ACAP2, MICAL-L1, ARF6 and EHD1, which are known to be involved in neurite outgrowth. Here we show that Rab35 promotes TNT formation and TNT-mediated vesicle transfer in a neuronal cell line. Furthermore, our data indicates that Rab35-GTP, ACAP2, ARF6-GDP and EHD1 act in a cascade mechanism to promote TNT formation. Interestingly, MICAL-L1 overexpression, shown to be necessary for the action of Rab35 on neurite outgrowth, showed no effect on TNTs, indicating that TNT formation and neurite outgrowth may be processed through similar but not identical pathways, further supporting the unique identity of these cellular protrusions.