PA700, the regulatory complex of the 26S proteasome, interferes with α-synuclein assembly

Laboratoire de Génétique Moléculaire de la Neurotransmission et des Processus Neurodégénératifs, Centre National de la Recherche Scientifique, Hôpital de la Pitié Salpêtrière, Paris, France.
FEBS Journal (Impact Factor: 4). 09/2005; 272(16):4023-33. DOI: 10.1111/j.1742-4658.2005.04776.x
Source: PubMed


Parkinson's disease is characterized by the loss of dopaminergic neurons in the nigrostriatal pathway accompanied by the presence of intracellular cytoplasmic inclusions, termed Lewy bodies. Fibrillized alpha-synuclein forms the major component of Lewy bodies. We reported a specific interaction between rat alpha-synuclein and tat binding protein 1, a subunit of PA700, the regulatory complex of the 26S proteasome. It has been demonstrated that PA700 prevents the aggregation of misfolded, nonubiquinated substrates. In this study, we examine the effect of PA700 on the aggregation of wild-type and A53T mutant alpha-synuclein. PA700 inhibits both wild-type and A53T alpha-synuclein fibril formation as measured by Thioflavin T fluorescence. Using size exclusion chromatography, we present evidence for a stable PA700-alpha-synuclein complex. Sedimentation analyses reveal that PA700 sequesters alpha-synuclein in an assembly incompetent form. Analysis of the morphology of wild-type and A53T alpha-synuclein aggregates during the course of fibrillization by electron microscopy demonstrate the formation of amyloid-like fibrils. Secondary structure analyses of wild-type and A53T alpha-synuclein assembled in the presence of PA700 revealed a decrease in the overall amount of assembled alpha-synuclein with no significant change in protein conformation. Thus, PA700 acts on alpha-synuclein assembly and not on the structure of fibrils. We hypothesize that PA700 sequesters alpha-synuclein oligomeric species that are the precursors of the fibrillar form of the protein, thus preventing its assembly into fibrils.

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    • "Recombinant human A53T αS was expressed and purified as described previously [33]. Soluble A53T αS was incubated in buffer A (50 mM Tris–HCl, pH 7.5, 150 mM KCl) at 37°C under continuous shaking in an Eppendorf Thermomixer set at 600 r.p.m. "
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    ABSTRACT: The accumulation of misfolded proteins appears as a fundamental pathogenic process in human neurodegenerative diseases. In the case of synucleinopathies such as Parkinson's disease (PD) or dementia with Lewy bodies (DLB), the intraneuronal deposition of aggregated alpha-synuclein (alphaS) is a major characteristic of the disease, but the molecular basis distinguishing the disease-associated protein (alphaSD) from its normal counterpart remains poorly understood. However, recent research suggests that a prion-like mechanism could be involved in the inter-cellular and inter-molecular propagation of aggregation of the protein within the nervous system. Our data confirm our previous observations of disease acceleration in a transgenic mouse line (M83) overexpressing a mutated (A53T) form of human alphaS, following inoculation of either brain extracts from sick M83 mice or fibrillar recombinant alphaS. A similar phenomenon is observed following a "second passage" in the M83 mouse model, including after stereotactic inoculations into the hippocampus or cerebellum. For further molecular analyses of alphaSD, we designed an ELISA test that identifies alphaSD specifically in sick mice and in the brain regions targeted by the pathological process in this mouse model. alphaSD distribution, mainly in the caudal brain regions and spinal cord, overall appears remarkably uniform, whatever the conditions of experimental challenge. In addition to specific detection of alphaSD immunoreactivity using an antibody against Ser129 phosphorylated alphaS, similar results were observed in ELISA with several other antibodies against the C-terminal part of alphaS, including an antibody against non phosphorylated alphaS. This also indicated consistent immunoreactivity of the murine alphaS protein specifically in the affected brain regions of sick mice. Prion-like behaviour in propagation of the disease-associated alphaS was confirmed with the M83 transgenic mouse model, that could be followed by an ELISA test. The ELISA data question their possible relationship with the conformational differences between the disease-associated alphaS and its normal counterpart.
    03/2014; 2(1):29. DOI:10.1186/2051-5960-2-29
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    • "We expressed recombinant wild-type a-syn in Escherichia coli strain BL21(DE3) (Stratagene) and we purified it as previously described (Ghee et al., 2005). We then determined the concentration of a-syn spectrophotometrically using an extinction coefficient of 5,960 M 21 cm 21 at 280 nm. "
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    ABSTRACT: The origin of α-synuclein (α-syn)-positive glial cytoplasmic inclusions found in oligodendrocytes in multiple system atrophy (MSA) is enigmatic, given the fact that oligodendrocytes do not express α-syn mRNA. Recently, neuron-to-neuron transfer of α-syn was suggested to contribute to the pathogenesis of Parkinson's disease. In this study, we explored whether a similar transfer of α-syn might occur from neurons to oligodendrocytes, which conceivably could explain how glial cytoplasmic inclusions are formed. We studied oligodendrocytes in vitro and in vivo and examined their ability to take up different α-syn assemblies. First, we treated oligodendrocytes with monomeric, oligomeric, and fibrillar forms of α-syn proteins and investigated whether α-syn uptake is dynamin-dependent. Second, we injected the same α-syn species into the mouse cortex to assess their uptake in vivo. Finally, we monitored the presence of human α-syn within rat oligodendroglial cells grafted in the striatum of hosts displaying Adeno-Associated Virus-mediated overexpression of human α-syn in the nigro-striatal pathway. Here, we show that oligodendrocytes take up recombinant α-syn monomers, oligomers and, to a lesser extent, fibrils in vitro in a concentration and time-dependent manner, and that this process is inhibited by dynasore. Further, we demonstrate in our injection model that oligodendrocytes also internalize α-syn in vivo. Finally, we provide the first direct evidence that α-syn can transfer to grafted oligodendroglial cells from host rat brain neurons overexpressing human α-syn. Our findings support the hypothesis of a neuron-to-oligodendrocyte transfer of α-syn, a mechanism that may play a crucial role in the progression and pathogenesis of MSA. GLIA 2013;
    Glia 03/2014; 62(3). DOI:10.1002/glia.22611 · 6.03 Impact Factor
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    • "We expressed recombinant S-tagged wild-type (WT) human α-syn in Escherichia coli strain BL21 (DE3) (Stratagene) and purified it as described previously [26]. We determined α-syn concentration by spectrophotometry using an extinction coefficient of 5,960/M/cm at 280 nm. "
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    ABSTRACT: α-Synuclein (α-syn) is a protein prevalent in neural tissue and known to undergo axonal transport. Intracellular α-syn aggregates are a hallmark of Parkinson’s disease (PD). Braak and collaborators have suggested that in people who are destined to eventually develop PD, α-syn aggregate pathology progresses following a stereotypic pattern, starting in the olfactory bulb (OB) and the gut. α-Synuclein aggregates are postulated to spread to interconnected brain regions over several years. Thus, propagation of the pathology via neural pathways can potentially explain how α-syn aggregates spread in PD. We have now studied if α-syn can transfer from the OB to other brain structures through neural connections, by injecting different molecular species of human α-syn (monomers, oligomers, fibrils) into the OB of wild-type mice. We found that non-fibrillar human α-syn is taken up very quickly by OB neurons. Within minutes to hours, it is also found in neurons in structures connected to the OB. Conversely, when we injected bovine serum albumin used as a control protein, we found that it does not diffuse beyond the OB, is rarely taken up by OB cells, and does not transfer to other structures. Taken together, our results show that OB cells readily take up α-syn, and that monomeric and oligomeric, but not fibrillar, forms of α-syn are rapidly transferred to interconnected structures within the timeframe we explored. Our results support the idea that α-syn can transfer along neural pathways and thereby contribute to the progression of the α-syn-related pathology. Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1160-3) contains supplementary material, which is available to authorized users.
    Acta Neuropathologica 08/2013; 126(4). DOI:10.1007/s00401-013-1160-3 · 10.76 Impact Factor
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