α-Synuclein–enhanced green fluorescent protein fusion proteins form proteasome sensitive inclusions in primary neurons

doi:10.1016/S0306-4522(01)00113-0

Neuroscience

Volume 104, Issue 3, 14 June 2001, Pages 901-912

https://doi.org/10.1016/S0306-4522(01)00113-0 Get rights and content

Abstract

α-Synuclein accumulates in the brains of sporadic Parkinson’s disease patients as a major component of Lewy bodies, and mutations in α-synuclein are associated with familial forms of Parkinson’s disease. The pathogenic mechanisms that precede and promote the aggregation of α-synuclein into Lewy bodies in neurons remain to be determined. Here, we constructed a series of α-synuclein–enhanced green fluorescent protein (α-synucleinEGFP, SynEGFP) fusion proteins to address whether the Parkinson’s disease-associated mutations alter the subcellular distribution of α-synuclein, and to use as a tool for experimental manipulations to induce aggregate formation. When transfected into mouse cultured primary neurons, the 49-kDa α-synucleinEGFP fusion proteins are partially truncated to a ∼27-kDa form. This non-fluorescent carboxy-terminally modified fusion protein spontaneously forms inclusions in the neuronal cytoplasm. A marked increase in the accumulation of inclusions is detected following treatment with each of three proteasome inhibitors, n-acetyl-leu-leu-norleucinal, lactacystin and MG132. Interestingly, Ala30Pro α-synucleinEGFP does not form the cytoplasmic inclusions that are characteristic of wild-type and Ala53Thr α-synucleinEGFP, supporting the idea that the Ala30Pro α-synuclein protein conformation differs from wild-type α-synuclein. Similar inclusions are formed if α-synuclein carboxy-terminus is modified by the addition of a V5/6×Histidine epitope tag. By contrast, overexpression of unmodified α-synuclein does not lead to aggregate formation. Furthermore, synphilin-1, an α-synuclein interacting protein also found in Lewy bodies, colocalizes with the carboxy-terminally truncated α-synuclein fusion protein in discrete cytoplasmic inclusions.

Our finding that manipulations of the carboxy-terminus of α-synuclein lead to inclusion formation may provide a model for studies of the pathogenic mechanisms of α-synuclein aggregation in Lewy bodies.

Section snippets

Isolation of α-synuclein cDNA, generation of mutant α-synuclein and fusion proteins

α-Synuclein cDNA was amplified by polymerase chain reaction (PCR) from random primed brain cDNAs (as described previously) (McLean et al., 2000) and cloned into either pSI (Promega, Madison, WI, USA) to create wild-type α-synuclein (WTSyn) expression construct or pEGFP-N3 (Clontech, Palo Alto, CA, USA), minus the stop codon, to create WTSynEGFP fusion construct. Alternatively, the α-synuclein cDNA was cloned into pcDNA3.1/V5-His-TOPO expression vector (Invitrogen, Carlsbad, CA, USA), minus the

Results

To facilitate our ongoing studies into the subcellular localization of α-synuclein, we created α-synucleinEGFP fusion constructs by fusing EGFP to the C-terminus of α-synuclein, and performed transient transfection of primary cortical neurons with a WTSynEGFP fusion construct. Confocal scanning microscopy reveals a widespread, diffuse distribution of the fusion protein throughout the cell body, neurites and nucleus, essentially identical to that of overexpressed α-synuclein (Fig. 1) or

Discussion

Our studies of α-synucleinEGFP fusion proteins have led to five novel observations. (i) In general, the cellular distribution of the α-synucleinEGFP fusion proteins is identical to that seen (by immunocytochemistry) for overexpressed, unmodified α-synuclein (Fig. 1) and for endogenous α-synuclein (McLean et al., 2000). (ii) However, we found that several C-terminally modified forms of α-synuclein, including a truncated form of the EGFP fusion protein, have a propensity to form inclusions in

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Hybrids of polyphenolic/quinone acids, the potential preventive and therapeutic drugs for PD: Disaggregate α-Syn fibrils, inhibit inclusions, and repair damaged neurons in mice
2023, European Journal of Medicinal Chemistry
Neurotoxic α-Syn fibers, the main components of Lewy bodies, play a key role in the development of PD characterized by a progressive loss of dopaminergic neurons. Here, we designed and synthesized the hybrids of polyphenolic/quinone acids. The candidate compounds showed high α-Syn aggregation inhibitory activities in vitro with IC₅₀ down to 1.6 μM. The inhibition went through the aggregation process by stabilizing the conformation of α-Syn proteostasis and preventing β-sheets aggregation, especially in the lag phase. Furthermore, the candidate drugs could disintegrate the preformed varisized aggregates into pony-size aggregates and functional monomers and continually inhibit the re-aggregation. The activities of anti-aggregation and aggregates depolymerization result in the reduction of inclusions in neuron cells. The candidate drugs also show high anti-oxidation and low cytotoxicity. They finally repair the damaged neurons in 6-OHDA-lesioned C57 mice and significantly improve PD-like symptoms of the PD model mice. The hybrids are promising molecules for PD prevention and therapy.© 2022 Elsevier Masson SAS. All rights reserved.
Monitoring α-synuclein aggregation
2023, Neurobiology of Disease
Synucleinopathies, including Parkinson's disease (PD), dementia with Lewy Bodies (DLB), and multiple system atrophy (MSA), are characterized by the misfolding and subsequent aggregation of alpha-synuclein (α-syn) that accumulates in cytoplasmic inclusions bodies in the cells of affected brain regions. Since the seminal report of likely-aggregated α-syn presence within the Lewy bodies by Spillantini et al. in 1997, the keyword “synuclein aggregation” has appeared in over 6000 papers (Source: PubMed October 2022). Studying, observing, describing, and quantifying α-syn aggregation is therefore of paramount importance, whether it happens in tubo, in vitro, in post-mortem samples, or in vivo. The past few years have witnessed tremendous progress in understanding aggregation mechanisms and identifying various polymorphs. In this context of growing complexity, it is of utmost importance to understand what tools we possess, what exact information they provide, and in what context they may be applied. Nonetheless, it is also crucial to rationalize the relevance of the information and the limitations of these methods for gauging the final result. In this review, we present the main techniques that have shaped the current views about α-syn structure and dynamics, with particular emphasis on the recent breakthroughs that may change our understanding of synucleinopathies.
A water-soluble manganese(II) octanediaoate/phenanthroline complex acts as an antioxidant and attenuates alpha-synuclein toxicity
2022, Biochimica et Biophysica Acta - Molecular Basis of Disease
The overproduction of reactive oxygen species (ROS) induces oxidative stress, a well-known process associated with aging and several human pathologies, such as cancer and neurodegenerative diseases. A large number of synthetic compounds have been described as antioxidant enzyme mimics, capable of eliminating ROS and/or reducing oxidative damage. In this study, we investigated the antioxidant activity of a water-soluble 1,10-phenantroline-octanediaoate Mn²⁺-complex on cells under oxidative stress, and assessed its capacity to attenuate alpha-synuclein (aSyn) toxicity and aggregation, a process associated with increased oxidative stress. This Mn²⁺-complex exhibited a significant antioxidant potential, reducing intracelular oxidation and increasing oxidative stress resistance in S. cerevisiae cells and in vivo, in G. mellonella, increasing the activity of the intracellular antioxidant enzymes superoxide dismutase and catalase. Strikingly, the Mn²⁺-complex reduced both aSyn oligomerization and aggregation in human cell cultures and, using NMR and DFT/molecular docking we confirmed its interaction with the C-terminal region of aSyn. In conclusion, the Mn²⁺-complex appears as an excellent lead for the design of new phenanthroline derivatives as alternative compounds for preventing oxidative damages and oxidative stress - related diseases.
Buffering capacity is determinant for restoring early α-synuclein aggregation
2022, Biophysical Chemistry
For disordered proteins, including α-synuclein (Syn), the aggregation of which is implicated in Parkinson's disease, it is known that at mild acidic and at the pI solution conditions the use of either strong or weak electrolytes minimized Syn aggregation. The mechanism is driven by electrostatic forces but remains, however, poorly understood. To address this issue, we used two biological buffers as weak electrolytes, at a low concentration (10 mM) and monitored the aggregation of Syn solutions from pH 7 to pH 2, by means of light scattering techniques. When the citrate buffer was used, in which there is buffering capacity in the pH range studied, the maximum of Syn aggregation was very close to the isoelectric point (pI = 4.7). When using tris-HCl, in which there is almost no buffering capacity in the pH range studied, it was for the first time observed a slow transition of the pI (of ca. 1 h) from 4.7 to 4–3, for a 33.5 μM protein concentration, as an example. We also observed in the protein solutions (in tris-HCl) the very early formation of large Syn aggregates. When there is buffering capacity, such as pH 7, these early large Syn aggregates dissociate, followed by association/aggregation. When there is no buffering capacity, such as pH 3, the referred early large Syn aggregates only dissociate. Overall, early large Syn aggregates dissociation can cause entropy in the protein solutions and Syn aggregation is only restored by the altered electrostatic forces due to the existing buffering capacity. Finally, by using an innovative strategy based in the ANS dye fluorescence intensity variation, we determined of the occurrence of the liquid-liquid phase separation process at pH 7 Syn solutions.
Modeling the cellular fate of alpha-synuclein aggregates: A pathway to pathology
2022, Current Opinion in Neurobiology
Parkinson's disease is a progressive neurodegenerative disorder that is characterized by pathological protein inclusions that form in the brains of patients, leading to neuron loss and the observed clinical symptoms. These inclusions, containing aggregates of the protein α-Synuclein, spread throughout the brain as the disease progresses. This spreading follows patterns that inform our understanding of the disease. One way to further our understanding of disease progression is to model the discrete steps from when a cell first encounters an aggregate to when those aggregates propagate to new cells. This review will serve to highlight the recent progress made in the effort to better understand the mechanistic steps that determine how this propagation happens at the cellular level.
An integrated genomic approach to dissect the genetic landscape regulating the cell-to-cell transfer of α-synuclein
2021, Cell Reports
Citation Excerpt :
We added an RFP tag to αSyn to enable its detection through imaging, without extra processing steps for antibody staining. Addition of a tag to αSyn modifies its ability to form aggregates (McLean et al., 2001), therefore possibly also modifying its ability to spread (Gustafsson et al., 2018). However, we have controlled for that limitation by including a negative control, GFP-2A-RFP.
Neuropathological and experimental evidence suggests that the cell-to-cell transfer of α-synuclein has an important role in the pathogenesis of Parkinson’s disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook a small interfering RNA (siRNA), genome-wide screen to identify genes regulating the cell-to-cell transfer of α-synuclein. A genetically encoded reporter, GFP-2A-αSynuclein-RFP, suitable for separating donor and recipient cells, was transiently transfected into HEK cells stably overexpressing α-synuclein. We find that 38 genes regulate the transfer of α-synuclein-RFP, one of which is ITGA8, a candidate gene identified through a recent PD genome-wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) show that those hits cluster in networks that include known PD genes more frequently than expected by random chance. The findings expand our understanding of the mechanism of α-synuclein spread.

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Neuroscience

Abstract

Section snippets

Isolation of α-synuclein cDNA, generation of mutant α-synuclein and fusion proteins

Results

Discussion

References (23)

α-Synuclein and the Parkinson’s disease-related mutant Ala53Thr-α-synuclein do not undergo proteasomal degradation in HEK293 and neuronal cells

Neurosci. Lett.

Degradation of α-synuclein by the proteasome

J. Biol. Chem.

Synthetic filaments assembled from C-terminally truncated α-synuclein

FEBS Lett.

Mutant and wild-type human α-synucleins assemble into elongated filaments with distinct morphologies in vitro

J. Biol. Chem.

Role of cytochrome c as a stimulator of α-synuclein aggregation in Lewy body disease

J. Biol. Chem.

Binding of α-synuclein to brain vesicles is abolished by familial Parkinson’s disease mutation

J. Biol. Chem.

Proteasome inhibitors: valuable new tools for cell biologists

Trends Cell Biol.

Membrane association and protein conformation of α-synuclein in intact neurons: Effect of Parkinson’s disease linked mutations

J. Biol. Chem.

α-Synuclein fibrillogenesis is nucleation-dependent: implications for the pathogenesis of Parkinson’s disease

J. Biol. Chem.

Aggregation of α-synuclein in Lewy bodies of sporadic Parkinson’s disease and dementia with Lewy bodies

Am. J. Pathol.

Accelerated in vitro fibril formation by a mutant α-synuclein linked to early-onset Parkinson disease

Nat. Med.

Cited by (194)

Hybrids of polyphenolic/quinone acids, the potential preventive and therapeutic drugs for PD: Disaggregate α-Syn fibrils, inhibit inclusions, and repair damaged neurons in mice

Monitoring α-synuclein aggregation

A water-soluble manganese(II) octanediaoate/phenanthroline complex acts as an antioxidant and attenuates alpha-synuclein toxicity

Buffering capacity is determinant for restoring early α-synuclein aggregation

Modeling the cellular fate of alpha-synuclein aggregates: A pathway to pathology

An integrated genomic approach to dissect the genetic landscape regulating the cell-to-cell transfer of α-synuclein