Enzymatic digestion was halted by the addition of complete culture media; DMEM:F12 (Invitrogen) made up of 10% fetal bovine serum (FBS; Gibco), 1% Penicillin/Streptomycin (Gibco), and GlutaMAX (Invitrogen). role in -syn transfer and further highlight the importance that non-neuronal cells, such as pericytes play in disease progression. Parkinsons disease (PD) is the second most common neurodegenerative disease after Alzheimers disease1. Pathologically, PD is usually characterized by the presence of intracellular inclusions called Lewy bodies. The main protein component of Lewy bodies is usually -synuclein (-syn), a synaptic protein that has a conformational plasticity allowing various structural conformations. In PD, -syn misfolds and subsequently forms aggregates2. Landmark studies designed to investigate early origin and progressive spread of -syn throughout the human brain exhibited that this olfactory bulb and locus coeruleus are the regions that are affected Rabbit polyclonal to Cyclin B1.a member of the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle.Cyclins function as regulators of CDK kinases. first in the body. It is only in later stages of the disease that Lewy bodies are seen in the neocortex and substantia nigra, and this is also when the main motor symptoms of PD become apparent3. Therefore, it appears that -syn precipitates spread from early affected brain regions, to more central areas of the brain. Other studies support this -syn spread theory. Intrastriatal grafts from healthy embryonic dopaminergic neurons, given to PD patients, contained -syn-positive Lewy bodies when the brain was autopsied more than 10 years later4,5. -syn is not restricted to the central nervous system and is able to cross the blood-brain barrier in both directions6, with -syn and its phosphorylated form being present in human blood plasma7. Pericytes are uniquely positioned within the neurovascular unit between endothelial cells of brain capillaries, astrocytes and neurons8. Pericytes regulate the key neurovascular functions including bloodCbrain barrier Cilengitide formation and maintenance. In Alzheimers disease pericyte deficiency directly leads to the development of tau pathology and an early neuronal loss that is normally absent in A-precursor protein transgenic mice9. Because of this transport and a dysfunctional blood-brain barrier influencing pathogenesis and progression in PD, it is important to look at the role that pericytes play in the spread of -syn in PD8. Tunneling nanotubes (TNTs) have been shown to act as a conduit for -syn transfer in mouse neuron-like CAD cells10, but there is no published evidence of this in human non-neuronal cells such as pericytes. TNTs are long, thin, F-actin-based membranous channels that connect cells and allow transfer of materials11,12. TNT diameter typically ranges from 50C200? nm and their length can vary dramatically and reach up to several cell diameters12. TNTs are very dynamic Cilengitide structures and can connect cells for several seconds up to multiple hours. Currently, there are two proposed models for TNT formation. The actin-driven protrusion mechanism involves one or two protrusive events that connect the membrane Cilengitide of the two cells. The cell-dislodgement Cilengitide mechanism involves two cells in close contact that allow their membranes to fuse, and as the cells migrate away from each other, TNTs are formed, composed of membrane originating from either one or both cells13,14. There are no known specific TNT markers, making them hard to study. To avoid this confusion with similar-looking structures, it is pivotal to clearly define TNTs. The following TNT definition was recently proposed: TNTs contain actin, attach two cells and are not attached to the substrate11. In addition, the transfer of a signal or cargo needs to be added as an essential distinguishing characteristic of TNTs. Only this criterion allows for the differentiation of TNTs from other similar structures that function in movement and adherence as opposed to communication. In this study, we decided that SH-SY5Y cells and human primary brain pericytes use TNTs as a mechanism for intercellular -syn transfer and show that TNTs provide for the transport of -syn pathology. Our results suggest the possibility that TNTs assist in the spread of -syn throughout the brain and emphasize the role that non-neuronal cells such as pericytes play in -syn progression in PD. Results -synuclein transfers through tunneling nanotubes in SH-SY5Y cells To understand whether -syn can be intercellularly transferred, we set up co-cultures of SH-SY5Y cells.