Objective The lesions of Parkinson's disease spread through the brain in a characteristic pattern that corresponds to axonal projections. Previous observations suggest that misfolded α-synuclein could behave as a prion, moving from neuron to neuron and causing endogenous α-synuclein to misfold. Here, we characterized and quantified the axonal transport of α-synuclein fibrils and showed that fibrils could be transferred from axons to second-order neurons following anterograde transport. Methods We grew primary cortical mouse neurons in microfluidic devices to separate soma from axonal projections in fluidically isolated microenvironments. We used live-cell imaging and immunofluorescence to characterize the transport of fluorescent α-synuclein fibrils and their transfer to second-order neurons. Results Fibrillar α-synuclein was internalized by primary neurons and transported in axons with kinetics consistent with slow component-b of axonal transport (fast axonal transport with saltatory movement). Fibrillar α-synuclein was readily observed in the cell bodies of second-order neurons following anterograde axonal transport. Axon-to-soma transfer appeared not to require synaptic contacts. Interpretation These results support the hypothesis that the progression of Parkinson's disease can be caused by neuron-to-neuron spread of α-synuclein aggregates and that the anatomical pattern of progression of lesions between axonally connected areas results from the axonal transport of such aggregates. That the transfer did not appear to be transsynaptic gives hope that α-synuclein fibrils could be intercepted by drugs during the extra-cellular phase of their journey.

Select members of the Reoviridae are the only nonenveloped viruses known to induce syncytium formation. The fusogenic orthoreoviruses accomplish cell-cell fusion through a distinct class of membrane fusioninducing proteins referred to as the fusion-associated small transmembrane (FAST) proteins. The p15 membrane fusion protein of baboon reovirus is unique among the FAST proteins in that it contains two hydrophobic regions (H1 and H2) recognized as potential transmembrane (TM) domains, suggesting a polytopic topology. However, detailed topological analysis of p15 indicated only the H1 domain is membrane spanning. In the absence of an N-terminal signal peptide, the H1 TM domain serves as a reverse signal-anchor to direct p15 membrane insertion and a bitopic N exoplasmic /C cytoplasmic topology. This topology results in the translocation of the smallest ectodomain (ϳ20 residues) of any known viral fusion protein, with the majority of p15 positioned on the cytosolic side of the membrane. Mutagenic analysis indicated the unusual presence of an N-terminal myristic acid on the small p15 ectodomain is essential to the fusion process. Furthermore, the only other hydrophobic region (H2) present in p15, aside from the TM domain, is located within the endodomain. Consequently, the p15 ectodomain is devoid of a fusion peptide motif, a hallmark feature of membrane fusion proteins. The exceedingly small, myristoylated ectodomain and the unusual topological distribution of structural motifs in this nonenveloped virus membrane fusion protein necessitate alternate models of proteinmediated membrane fusion.The baboon reovirus (BRV) p15 protein is a novel member of the recently described fusion-associated small transmembrane (FAST) protein family (9,11,45). The FAST proteins are unusual membrane fusion proteins encoded by the fusogenic subgroup of orthoreoviruses, one of the few examples of nonenveloped viruses that induce cell-cell fusion and syncytium formation (15,18). At 10 to 15 kDa, the reovirus FAST proteins are the smallest known viral membrane fusion proteins and are unlikely to undergo the types of extensive structural rearrangements required for enveloped virus fusion protein activity (27,48). The FAST proteins are also the only examples of nonstructural viral proteins that induce membrane fusion (11, 45). As a result of their nonstructural nature, the FAST proteins play no role in reovirus entry. Their sole purpose appears to reflect enhanced dissemination of the infection via syncytium formation, following FAST protein expression in reovirus-infected cells (16,17). The unusual structural features of the FAST proteins and their unique role in the virus replication cycle suggest the mechanism of FAST-mediated membrane fusion is unlikely to adhere to the existing paradigm, which is derived from studies of the enveloped virus fusion proteins (5,48,52).In addition to BRV p15, FAST proteins have been recently characterized from avian reovirus (ARV), Nelson Bay reovirus (NBV), and reptilian reovirus (RRV) (9, 45). Although th...

The FAST proteins are a unique family of virus-encoded cell-cell membrane fusion proteins. In the absence of a cleavable N-terminal signal peptide, a single-pass transmembrane domain (TMD) functions as a reverse signal-anchor to direct the FAST proteins into the plasma membrane in an N exo /C cyt topology. There is little information available on the role of the FAST protein TMD in the cell-cell membrane fusion reaction. We show that in the absence of conservation in the length or primary amino acid sequence, the p14 TMD can be functionally exchanged with the TMDs of the p10 and p15 FAST proteins. This is not the case for chimeric p14 proteins containing the TMDs of two different enveloped viral fusion proteins or a cellular membrane protein; such chimeric proteins were defective for both pore formation and syncytiogenesis. TMD structural features that are conserved within members of the FAST protein family presumably play direct roles in the fusion reaction. Molecular modeling suggests that the funnel-shaped architecture of the FAST protein TMDs may represent such a conserved structural and functional motif. Interestingly, although heterologous TMDs exert diverse influences on the trafficking of the p14 FAST protein, these TMDs are capable of functioning as reverse signal-anchor sequences to direct p14 into lipid rafts in the correct membrane topology. The FAST protein TMDs are therefore not primary determinants of type III protein topology, but they do play a direct, sequence-independent role in the membrane fusion reaction.The fusion-associated small transmembrane (FAST) proteins are a unique family of membrane fusion proteins encoded by the fusogenic reoviruses (20). At 95 to 140 amino acids in size, the FAST proteins are the smallest known viral membrane fusion proteins. Rather than mediating virus-cell fusion, the FAST proteins are nonstructural viral proteins that are expressed on the surfaces of virus-infected or -transfected cells, where they induce cell-cell fusion and the formation of multinucleated syncytia. A purified FAST protein, when reconstituted into liposome membranes, induces liposome-cell and liposome-liposome fusion, indicating the FAST proteins are bona fide membrane fusion proteins (54). In their natural biological context as cell-cell fusogens, however, the FAST proteins exploit cellular adhesins and actin remodeling to maximize their cell-cell fusion potential (40). Studies further suggest that cell-cell fusion mediated by the FAST proteins may contribute to rapid localized dissemination of the infection, followed by apoptosis-induced disruption of the syncytia, resulting in a burst of infectious-progeny-virus release (19,21,41). This two-step process for virus dissemination mediated by the FAST proteins may contribute to the natural pathogenicity of the fusogenic reoviruses. How this remarkable family of virus-encoded fusogens induce membrane fusion and syncytium formation remains unclear, but several recent studies have defined specific subdomains and structural motifs likely to be in...

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WXand Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipidanchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.Biological membrane fusion is dependent on protein catalysts to mediate the lipid rearrangements required for membrane merger (1, 2). The fusion-associated small transmembrane (FAST) proteins are one such family of membrane fusion catalysts (3). The FAST proteins are a unique group of small (95-140 amino acids) integral membrane proteins encoded by the fusogenic reoviruses, an unusual group of non-enveloped viruses that induce syncytium formation (3-6). Three distinct members of the FAST protein family have been described in recent years: the homologous p10 proteins of avian reovirus and Nelson Bay reovirus and the unrelated p14 and p15 proteins of reptilian reovirus and baboon reovirus, respectively (3-5). Unlike the well characterized fusion proteins of enveloped viruses (7), the FAST proteins are nonstructural viral proteins and are therefore not involved in viral entry into cells. Following their expression inside virus-infected or -transfected cells, the FAST proteins traffic through the endoplasmic reticulum-Golgi pathway to assume a bitopic N exoplasmic /C cytoplasmic topology in the plasma membrane, where they mediate fusion of virus-infected cells to neighboring uninfected cells (8 -10). Therefore, the FAST proteins function more as "cellular" rather than viral fusion proteins, mediating cell-cell rather than viruscell membrane fusion. Furthermore, a recent study using the purified p14 FAST protein of reptilian reovirus reconstituted into artificial lipid bilayers indicated that the FAST proteins are both necessary and sufficient to mediate membrane fusion (11).In addition to their unique role in the viral replication cycle, the FAST proteins are also structurally ...

The reovirus fusion-associated small transmembrane (FAST) proteins evolved to induce cell-cell, rather than virus-cell, membrane fusion. It is unclear whether the FAST protein fusion reaction proceeds in the same manner as the enveloped virus fusion proteins. We now show that fluorescence-based cell-cell and cell-RBC hemifusion assays are unsuited for detecting lipid mixing in the absence of content mixing during FAST protein-mediated membrane fusion. Furthermore, membrane curvature agents that inhibit hemifusion or promote pore formation mediated by influenza hemagglutinin had no effect on p14-induced cell-cell fusion, even under conditions of limiting p14 concentrations. Standard assays used to detect fusion intermediates induced by enveloped virus fusion proteins are therefore not applicable to the FAST proteins. These results suggest the possibility that the nature of the fusion intermediates or the mechanisms used to transit through the various stages of the fusion reaction may differ between these distinct classes of viral fusogens.