Mapping the Subcellular Distribution of α-Synuclein in Neurons using Genetically Encoded Probes for Correlated Light and Electron Microscopy: Implications for Parkinson's Disease Pathogenesis (original) (raw)
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Journal of Neuroscience, 2013
Modifications to the gene encoding human ␣-synuclein have been linked to the development of Parkinson's disease. The highly conserved structure of ␣-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human ␣-synuclein, including new genetic tags developed for correlated light microscopy and electron microscopy (the tetracysteine-biarsenical labeling system or the new fluorescent protein for electron microscopy, MiniSOG), we determined the distribution of ␣-synuclein when overexpressed in primary neurons at supramolecular and cellular scales in three dimensions (3D). We observed specific association of ␣-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, ␣-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice overexpressing human ␣-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. Three-dimensional electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulovesicular structures similar to what we observed in vitro. We propose that ␣-synuclein overexpression is associated with hypertrophy of membrane systems of the presynaptic terminalpreviouslyshowntohavearoleinvesiclerecycling.Ourdatasupporttheconclusionthat␣-synucleinisinvolvedinprocessesassociated with the sorting, channeling, packaging, and transport of synaptic material destined for degradation.
F1000 - Post-publication peer review of the biomedical literature, 2013
Modifications to the gene encoding human ␣-synuclein have been linked to the development of Parkinson's disease. The highly conserved structure of ␣-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human ␣-synuclein, including new genetic tags developed for correlated light microscopy and electron microscopy (the tetracysteine-biarsenical labeling system or the new fluorescent protein for electron microscopy, MiniSOG), we determined the distribution of ␣-synuclein when overexpressed in primary neurons at supramolecular and cellular scales in three dimensions (3D). We observed specific association of ␣-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, ␣-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice overexpressing human ␣-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. Three-dimensional electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulovesicular structures similar to what we observed in vitro. We propose that ␣-synuclein overexpression is associated with hypertrophy of membrane systems of the presynaptic terminalpreviouslyshowntohavearoleinvesiclerecycling.Ourdatasupporttheconclusionthat␣-synucleinisinvolvedinprocessesassociated with the sorting, channeling, packaging, and transport of synaptic material destined for degradation.
A pathologic cascade causing synaptic dysfunction in α-synuclein-induced neurodegeneration
Alzheimers & Dementia, 2010
Several neurodegenerative diseases are typified by intraneuronal ␣-synuclein deposits, synaptic dysfunction, and dementia. While even modest ␣-synuclein elevations can be pathologic, the precise cascade of events induced by excessive ␣-synuclein and eventually culminating in synaptotoxicity is unclear. To elucidate this, we developed a quantitative model system to evaluate evolving ␣-synucleininduced pathologic events with high spatial and temporal resolution, using cultured neurons from brains of transgenic mice overexpressing fluorescent-human-␣-synuclein. Transgenic ␣-synuclein was pathologically altered over time and overexpressing neurons showed striking neurotransmitter release deficits and enlarged synaptic vesicles; a phenotype reminiscent of previous animal models lacking critical presynaptic proteins. Indeed, several endogenous presynaptic proteins involved in exocytosis and endocytosis were undetectable in a subset of transgenic boutons ("vacant synapses") with diminished levels in the remainder, suggesting that such diminutions were triggering the overall synaptic pathology. Similar synaptic protein alterations were also retrospectively seen in human pathologic brains, highlighting potential relevance to human disease. Collectively the data suggest a previously unknown cascade of events where pathologic ␣-synuclein leads to a loss of a number of critical presynaptic proteins, thereby inducing functional synaptic deficits.
Synucleins Regulate the Kinetics of Synaptic Vesicle Endocytosis
Journal of Neuroscience, 2014
Genetic and pathological studies link ␣-synuclein to the etiology of Parkinson's disease (PD), but the normal function of this presynaptic protein remains unknown. ␣-Synuclein, an acidic lipid binding protein, shares high sequence identity with and ␥-synuclein. Previous studies have implicated synucleins in synaptic vesicle (SV) trafficking, although the precise site of synuclein action continues to be unclear. Here we show, using optical imaging, electron microscopy, and slice electrophysiology, that synucleins are required for the fast kinetics of SV endocytosis. Slowed endocytosis observed in synuclein null cultures can be rescued by individually expressing mouse ␣-, -, or ␥-synuclein, indicating they are functionally redundant. Through comparisons to dynamin knockout synapses and biochemical experiments, we suggest that synucleins act at early steps of SV endocytosis. Our results categorize ␣-synuclein with other familial PD genes known to regulate SV endocytosis, implicating this pathway in PD.
Synucleins Have Multiple Effects on Presynaptic Architecture
Cell reports, 2017
Synucleins (α, β, γ-synuclein) are a family of abundant presynaptic proteins. α-Synuclein is causally linked to the pathogenesis of Parkinson's disease (PD). In an effort to define their physiological and pathological function or functions, we investigated the effects of deleting synucleins and overexpressing α-synuclein PD mutations, in mice, on synapse architecture using electron microscopy (EM) and cryoelectron tomography (cryo-ET). We show that synucleins are regulators of presynapse size and synaptic vesicle (SV) pool organization. Using cryo-ET, we observed that deletion of synucleins increases SV tethering to the active zone but decreases the inter-linking of SVs by short connectors. These ultrastructural changes were correlated with discrete protein phosphorylation changes in αβγ-synuclein-/- neurons. We also determined that α-synuclein PD mutants (PARK1/hA30P and PARK4/hα-syn) primarily affected presynaptic cytomatrix proximal to the active zone, congruent with previous...
Acute increase of α-synuclein inhibits synaptic vesicle recycling evoked during intense stimulation
Molecular biology of the cell, 2014
Parkinson's disease is associated with multiplication of the α-synuclein gene and abnormal accumulation of the protein. In animal models, α-synuclein overexpression broadly impairs synaptic vesicle trafficking. However, the exact steps of the vesicle trafficking pathway affected by excess α-synuclein and the underlying molecular mechanisms remain unknown. Therefore we acutely increased synuclein levels at a vertebrate synapse and performed a detailed ultrastructural analysis of the effects on presynaptic membranes. At stimulated synapses (20 Hz), excess synuclein caused a loss of synaptic vesicles and an expansion of the plasma membrane, indicating an impairment of vesicle recycling. The N-terminal domain (NTD) of synuclein, which folds into an α-helix, was sufficient to reproduce these effects. In contrast, α-synuclein mutants with a disrupted N-terminal α-helix (T6K and A30P) had little effect under identical conditions. Further supporting this model, another α-synuclein mutan...
Journal of Cell Biology, 2004
Presenilin-1 null mutation (PS1 −/−) in mice is associated with morphological alterations and defects in cleavage of transmembrane proteins. Here, we demonstrate that PS1 deficiency also leads to the formation of degradative vacuoles and to the aberrant translocation of presynaptic α- and β-synuclein proteins to these organelles in the perikarya of primary neurons, concomitant with significant increases in the levels of both synucleins. Stimulation of autophagy in control neurons produced a similar mislocalization of synucleins as genetic ablation of PS1. These effects were not the result of the loss of PS1 γ-secretase activity; however, dysregulation of calcium channels in PS1 −/− cells may be involved. Finally, colocalization of α-synuclein and degradative organelles was observed in brains from patients with the Lewy body variant of AD. Thus, aberrant accumulation of α- and β-synuclein in degradative organelles are novel features of PS1 −/− neurons, and similar events may promote ...
Neuron, 2010
The protein a-synuclein accumulates in the brain of patients with sporadic Parkinson's disease (PD), and increased gene dosage causes a severe, dominantly inherited form of PD, but we know little about the effects of synuclein that precede degeneration. a-Synuclein localizes to the nerve terminal, but the knockout has little if any effect on synaptic transmission. In contrast, we now find that the modest overexpression of a-synuclein, in the range predicted for gene multiplication and in the absence of overt toxicity, markedly inhibits neurotransmitter release. The mechanism, elucidated by direct imaging of the synaptic vesicle cycle, involves a specific reduction in size of the synaptic vesicle recycling pool. Ultrastructural analysis demonstrates reduced synaptic vesicle density at the active zone, and imaging further reveals a defect in the reclustering of synaptic vesicles after endocytosis. Increased levels of a-synuclein thus produce a specific, physiological defect in synaptic vesicle recycling that precedes detectable neuropathology.
Differential Expression of Alpha-Synuclein in Hippocampal Neurons
PLoS ONE, 2014
a-Synuclein is the major pathological component of synucleinopathies including Parkinson's disease and dementia with Lewy bodies. Recent studies have demonstrated that a-synuclein also plays important roles in the release of synaptic vesicles and synaptic membrane recycling in healthy neurons. However, the precise relationship between the pathogenicity and physiological functions of a-synuclein remains to be elucidated. To address this issue, we investigated the subcellular localization of a-synuclein in normal and pathological conditions using primary mouse hippocampal neuronal cultures. While some neurons expressed high levels of a-synuclein in presynaptic boutons and cell bodies, other neurons either did not or only very weakly expressed the protein. These a-synuclein-negative cells were identified as inhibitory neurons by immunostaining with specific antibodies against glutamic acid decarboxylase (GAD), parvalbumin, and somatostatin. In contrast, a-synuclein-positive synapses were colocalized with the excitatory synapse marker vesicular glutamate transporter-1. This expression profile of a-synuclein was conserved in the hippocampus in vivo. In addition, we found that while presynaptic a-synuclein colocalizes with synapsin, a marker of presynaptic vesicles, it is not essential for activitydependent membrane recycling induced by high potassium treatment. Exogenous supply of preformed fibrils generated by recombinant a-synuclein was shown to promote the formation of Lewy body (LB) -like intracellular aggregates involving endogenous a-synuclein. GAD-positive neurons did not form LB-like aggregates following treatment with preformed fibrils, however, exogenous expression of human a-synuclein allowed intracellular aggregate formation in these cells. These results suggest the presence of a different mechanism for regulation of the expression of a-synuclein between excitatory and inhibitory neurons. Furthermore, a-synuclein expression levels may determine the efficiency of intracellular aggregate formation in different neuronal subtypes.
Presynaptic accumulation of α-synuclein causes synaptopathy and progressive neurodegeneration
2020
Alpha-synuclein (α-syn) mislocalisation and accumulation in intracellular inclusions is the major pathological hallmark of degenerative synucleinopathies, including Parkinson’s disease, Parkinson’s disease with Dementia and Dementia with Lewy Bodies. Typical symptoms are behavioural abnormalities including motor deficits that mark disease progression, while non-motor symptoms and synaptic deficits are already apparent during the early stages of disease. Synucleinopathies have therefore been considered synaptopathies that exhibit synaptic dysfunction prior to neurodegeneration. However, the mechanisms and events underlying synaptopathy are largely unknown. Here we investigated the cascade of pathological events underlying α-syn accumulation and toxicity in aDrosophilamodel of synucleinopathy by employing a combination of histological, biochemical, behavioural and electrophysiological assays. Our findings demonstrate that targeted expression of human α-syn leads to its accumulation in...