Article

Systematic Mutagenesis of -Synuclein Reveals Distinct Sequence Requirements for Physiological and Pathological Activities

Department of Molecular and Cellular Physiology, Stanford University, Stanford, California 94305-5453, and Howard Hughes Medical Institute, Stanford University, Stanford, California 94305-5453.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 10/2012; 32(43):15227-42. DOI: 10.1523/JNEUROSCI.3545-12.2012
Source: PubMed

ABSTRACT

α-Synuclein is an abundant presynaptic protein that binds to phospholipids and synaptic vesicles. Physiologically, α-synuclein functions as a SNARE-protein chaperone that promotes SNARE-complex assembly for neurotransmitter release. Pathologically, α-synuclein mutations and α-synuclein overexpression cause Parkinson's disease, and aggregates of α-synuclein are found as Lewy bodies in multiple neurodegenerative disorders ("synucleinopathies"). The relation of the physiological functions to the pathological effects of α-synuclein remains unclear. As an initial avenue of addressing this question, we here systematically examined the effect of α-synuclein mutations on its physiological and pathological activities. We generated 26 α-synuclein mutants spanning the entire molecule, and analyzed them compared with wild-type α-synuclein in seven assays that range from biochemical studies with purified α-synuclein, to analyses of α-synuclein expression in cultured neurons, to examinations of the effects of virally expressed α-synuclein introduced into the mouse substantia nigra by stereotactic injections. We found that both the N-terminal and C-terminal sequences of α-synuclein were required for its physiological function as SNARE-complex chaperone, but that these sequences were not essential for its neuropathological effects. In contrast, point mutations in the central region of α-synuclein, referred to as nonamyloid β component (residues 61-95), as well as point mutations linked to Parkinson's disease (A30P, E46K, and A53T) increased the neurotoxicity of α-synuclein but did not affect its physiological function in SNARE-complex assembly. Thus, our data show that the physiological function of α-synuclein, although protective of neurodegeneration in some contexts, is fundamentally distinct from its neuropathological effects, thereby dissociating the two activities of α-synuclein.

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    • "Assessing the normal function of -synuclein has been challenging, because: (i) -Synuclein is an intrinsically unstructured protein that cycles between a natively unfolded state in cytosol, and a helical multimeric state on membranes [71, 92–95, 100]; (ii) Overexpression of -synuclein triggers toxic effects in humans [152] [153] and in animal models [154] [155] [156], that are much worse than the effects caused by loss of -synuclein [157] [158]. This disconnection of the pathogenic activity of -synuclein from its physiological function [159] complicates findings in overexpression models; (iii) Potential compensation of -synuclein function by its isoforms -and -synuclein complicate findings in knockout animals and necessitate simultaneous knockout of all isoforms or acute manipulation, such as done via viral injections. However, -synuclein's presynaptic localization and its interaction with highly curved membranes and synaptic proteins strongly suggests a regulatory function associated with the synapse, such as synaptic activity, synaptic plasticity, learning, neurotransmitter release, dopamine metabolism, synaptic vesicle pool maintenance, and/or vesicle trafficking (Fig. 3). "
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    ABSTRACT: α-Synuclein is an abundant neuronal protein which localizes predominantly to presynaptic terminals, and is strongly linked genetically and pathologically to Parkinson's disease and other neurodegenerative diseases. While the accumulation of α-synuclein in the form of misfolded oligomers and large aggregates defines multiple neurodegenerative diseases called "synucleinopathies", its cellular function has remained largely unclear, and is the subject of intense investigation. In this review, I focus on the structural characteristics of α-synuclein, its cellular and subcellular localization, and discuss how this relates to its function in neurons, in particular at the neuronal synapse.
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    • "This hydrophobic region of 35 amino acids is indispensable for the aggregation of alpha-synuclein (Crowther et al., 1998; Giasson et al., 2001) and it has a degree of sequence similarity with other amyloidogenic peptides such as ␤-amyloid (El-Agnaf and Irvine, 2002). The third region is the C-terminal part, a proline rich area, negatively charged and highly unstructured (Fig. 1A and B) where several proteins have been reported to bind (Burré et al., 2012; Dev et al., 2003). The alpha-synuclein protein undergoes a number of posttranslational modifications mostly pathology associated. "
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    ABSTRACT: Parkinson's disease is one of several neurodegenerative diseases associated with a misfolded, aggregated and pathological protein. In Parkinson's disease this protein is alpha-synuclein and its neuronal deposits in the form of Lewy bodies are considered a hallmark of the disease. In this review we describe the clinical and experimental data that have led to think of alpha-synuclein as a prion-like protein and we summarize data from in vitro, cellular and animal models supporting this view. (C) 2014 Published by Elsevier B.V.
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    • "SybII interacts with α-synuclein on SVs and unlike synaptophysin remains bound to sybII even after its incorporation into the SNARE complex 58. The C-terminal acidic domain of α-synuclein binds to the extreme N-terminal 28 amino acids of sybII, which are not involved in SNARE complex formation 58,59 (Figure 1). It has been proposed that α-synuclein acts as a non-classical chaperone, by catalyzing the formation of SNARE complexes 58,59. "
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