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


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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    • "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.
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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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    ABSTRACT: Synaptobrevin II (sybII) is a key fusogenic molecule on synaptic vesicles (SVs) therefore the active maintenance of both its conformation and location in sufficient numbers on this organelle is critical in both mediating and sustaining neurotransmitter release. Recently three proteins have been identified having key roles in the presentation, trafficking and retrieval of sybII during the fusion and endocytosis of SVs. The nerve terminal protein α-synuclein catalyses sybII entry into SNARE complexes, whereas the monomeric adaptor protein AP180 is required for sybII retrieval during SV endocytosis. Overarching these events is the tetraspan SV protein synaptophysin, which is a major sybII interaction partner on the SV. This review will evaluate recent studies to propose working models for the control of sybII traffic by synaptophysin and other sybtraps (sybII trafficking partners) and suggest how dysfunction in sybII traffic may contribute to human disease.
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    • "The latter results are more in keeping with our own studies here. Based on 2-dimensional PAGE (Native/SDS) experiments, in all likelihood brain mitochondria are impaired mainly by a soluble monomeric form and, to a lesser extent, carboxyl terminally truncated forms of overexpressed human α-synuclein [51], [52]. Perhaps released terminal peptide fragments (10–20 amino acids in length) of human α-synuclein might also be toxic to brain mitochondria at low doses. "
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    ABSTRACT: While most forms of Parkinson's Disease (PD) are sporadic in nature, a small percentage of PD have genetic causes as first described for dominant, single base pair changes as well as duplication and triplication in the α-synuclein gene. The α-synuclein gene encodes a 140 amino acid residue protein that interacts with a variety of organelles including synaptic vesicles, lysosomes, endoplasmic reticulum/Golgi vesicles and, reported more recently, mitochondria. Here we examined the structural and functional interactions of human α-synuclein with brain mitochondria obtained from an early, pre-manifest mouse model for PD over-expressing human α-synuclein (ASOTg). The membrane potential in ASOTg brain mitochondria was decreased relative to wildtype (WT) mitochondria, while reactive oxygen species (ROS) were elevated in ASOTg brain mitochondria. No selective interaction of human α-synuclein with mitochondrial electron transport complexes cI-cV was detected. Monomeric human α-synuclein plus carboxyl terminally truncated forms were the predominant isoforms detected in ASOTg brain mitochondria by 2-dimensional PAGE (Native/SDS) and immunoblotting. Oligomers or fibrils were not detected with amyloid conformational antibodies. Mass spectrometry of human α-synuclein in both ASOTg brain mitochondria and homogenates from surgically resected human cortex demonstrated that the protein was full-length and postranslationally modified by N-terminal acetylation. Overall the study showed that accumulation of full-length, N-terminally acetylated human α-synuclein was sufficient to disrupt brain mitochondrial function in adult mice.
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