ABSTRACT
Aggregation and spreading of α-Synuclein (αSyn) are hallmarks of several neurodegenerative diseases, thus monitoring human αSyn (hαSyn) in animal models or cell cultures is vital for the field. However, the detection of native hαSyn in such systems is challenging. We show that the nanobody NbSyn87, previously-described to bind hαSyn, also shows cross-reactivity for the proteasomal subunit Rpn10. As such, when the NbSyn87 is expressed in the absence of hαSyn, it is continuously degraded by the proteasome, while it is stabilized when it binds to hαSyn. Here, we exploit this feature to design a new Fluorescent Reporter for hαSyn (FluoReSyn) by fusing NbSyn87 to fluorescent proteins, which results in fluorescence signal fluctuations depending on the presence and amounts of intracellular hαSyn. We characterize this biosensor in cells and tissues to finally reveal the presence of transmittable αSyn in human cerebrospinal fluid, demonstrating the potential of FluoReSyn for clinical research and diagnostics.
Subject(s)
Cytosol/metabolism , Luminescent Proteins/metabolism , Single-Domain Antibodies/metabolism , alpha-Synuclein/metabolism , Adult , Aged , Aged, 80 and over , Animals , Cells, Cultured , Cytosol/chemistry , Female , Fluorescence , HEK293 Cells , Humans , Luminescent Proteins/chemistry , Luminescent Proteins/genetics , Male , Microscopy, Fluorescence, Multiphoton , Middle Aged , Neurons/cytology , Neurons/metabolism , Rats, Wistar , Single-Domain Antibodies/genetics , alpha-Synuclein/cerebrospinal fluid , alpha-Synuclein/genetics , Red Fluorescent ProteinABSTRACT
Aggregation of α-synuclein (αS) is involved in the pathogenesis of Parkinson's disease (PD) and a variety of related neurodegenerative disorders. The physiological function of αS is largely unknown. We demonstrate with in vitro vesicle fusion experiments that αS has an inhibitory function on membrane fusion. Upon increased expression in cultured cells and in Caenorhabditis elegans, αS binds to mitochondria and leads to mitochondrial fragmentation. In C. elegans age-dependent fragmentation of mitochondria is enhanced and shifted to an earlier time point upon expression of exogenous αS. In contrast, siRNA-mediated downregulation of αS results in elongated mitochondria in cell culture. αS can act independently of mitochondrial fusion and fission proteins in shifting the dynamic morphologic equilibrium of mitochondria towards reduced fusion. Upon cellular fusion, αS prevents fusion of differently labelled mitochondrial populations. Thus, αS inhibits fusion due to its unique membrane interaction. Finally, mitochondrial fragmentation induced by expression of αS is rescued by coexpression of PINK1, parkin or DJ-1 but not the PD-associated mutations PINK1 G309D and parkin Δ1-79 or by DJ-1 C106A.
Subject(s)
Intracellular Signaling Peptides and Proteins/metabolism , Membrane Fusion/physiology , Mitochondria/metabolism , Oncogene Proteins/metabolism , Protein Kinases/metabolism , Ubiquitin-Protein Ligases/metabolism , alpha-Synuclein/metabolism , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Cell Line , Humans , Immunohistochemistry , Intracellular Signaling Peptides and Proteins/genetics , Mitochondria/ultrastructure , Oncogene Proteins/genetics , Parkinson Disease/metabolism , Parkinson Disease/pathology , Protein Deglycase DJ-1 , Protein Kinases/genetics , Ubiquitin-Protein Ligases/genetics , alpha-Synuclein/geneticsABSTRACT
The relation of alpha-synuclein (alphaS) aggregation to Parkinson's disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated alphaS species have in neurotoxicity in vivo, we generated alphaS variants by a structure-based rational design. Biophysical analysis revealed that the alphaS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre-fibrillar alphaS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between alphaS aggregates with impaired beta-structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric alphaS species and their in vivo function.
