Evidence for α-synuclein prions causing multiple system atrophy in humans with parkinsonism

Abstract

Prions are proteins that adopt alternative conformations that become self-propagating; the PrP(Sc) prion causes the rare human disorder Creutzfeldt-Jakob disease (CJD). We report here that multiple system atrophy (MSA) is caused by a different human prion composed of the α-synuclein protein. MSA is a slowly evolving disorder characterized by progressive loss of autonomic nervous system function and often signs of parkinsonism; the neuropathological hallmark of MSA is glial cytoplasmic inclusions consisting of filaments of α-synuclein. To determine whether human α-synuclein forms prions, we examined 14 human brain homogenates for transmission to cultured human embryonic kidney (HEK) cells expressing full-length, mutant human α-synuclein fused to yellow fluorescent protein (α-syn140*A53T-YFP) and TgM83(+/-) mice expressing α-synuclein (A53T). The TgM83(+/-) mice that were hemizygous for the mutant transgene did not develop spontaneous illness; in contrast, the TgM83(+/+) mice that were homozygous developed neurological dysfunction. Brain extracts from 14 MSA cases all transmitted neurodegeneration to TgM83(+/-) mice after incubation periods of ∼120 d, which was accompanied by deposition of α-synuclein within neuronal cell bodies and axons. All of the MSA extracts also induced aggregation of α-syn*A53T-YFP in cultured cells, whereas none of six Parkinson's disease (PD) extracts or a control sample did so. Our findings argue that MSA is caused by a unique strain of α-synuclein prions, which is different from the putative prions causing PD and from those causing spontaneous neurodegeneration in TgM83(+/+) mice. Remarkably, α-synuclein is the first new human prion to be identified, to our knowledge, since the discovery a half century ago that CJD was transmissible.

Keywords: Parkinson's disease; neurodegeneration; strains; synucleinopathies.

Fig. 1.

Neuropathological and biochemical analysis of synucleinopathy cases. (A) Gross pathology of one representative sample (MSA10) demonstrating depigmentation of the substantia nigra compared with nondiseased control. (B) Immunohistochemical detection of α-synuclein deposits in patient samples. Two representative PD samples and four representative MSA samples were stained for α-synuclein using the antibodies clone 42 (BD Biosciences; PD1, PD6, MSA1, MSA7, MSA10) and LB509 (MSA12). Arrowheads point to Lewy bodies, arrows to GCIs. (Scale bar, 50 µm for all samples.) (C) Immunoblots of brain homogenates of human control (C1), PD, and MSA cases show total human α-synuclein (Top) and detergent-insoluble phosphorylated α-synuclein (Middle). The brain region used for each case is noted: FC (frontal cortex), SN (substantia nigra), BG (basal ganglia), and P (pons). Blots were probed for actin as a loading control (Bottom). Total human α-synuclein was probed using the monoclonal antibody Syn211, and S129-specific, phosphorylated α-synuclein was probed with antibody EP1536Y. Molecular weight markers of migrated protein standards are shown in kilodaltons.

Fig. 2.

A cell infectivity assay can quantify infectivity in synucleinopathy tissue samples. (A) Representative images of α-syn140*A53T–YFP–expressing cells infected with PTA-precipitated brain homogenate from control (C1), PD, or MSA patients. YFP is shown in green. (Scale bars, 100 µm.) (B) Box and whisker plot of cell infectivity from PD and MSA samples shows a significant difference between the two groups (P < 0.001). Whiskers indicate maximum and minimum values. (C) For each of the MSA samples tested in both cell assay and mouse bioassay, cell infectivity and incubation time were significantly inversely correlated (R² = 0.27, P = 0.026).

Fig. 3.

Inoculation of α-synuclein aggregates from MSA but not PD cases induced deposition of phosphorylated α-synuclein and reactive astrogliosis. (A) Brain homogenates were prepared from control (C1), PD, and MSA patients and IC inoculated into TgM83^+/− mice. Mice inoculated with MSA homogenates, but not PD or control homogenates, showed deposition of phosphorylated α-synuclein in the brainstem (top panels, staining with EP1536Y antibody); Insets show a 4× magnification relative to the main image. These mice also showed prominent reactive astrogliosis, as indicated by GFAP staining (bottom panels). (Scale bars, 50 μm.) (B) Representative immunoblot shows total α-synuclein (Top) and detergent-insoluble phosphorylated α-synuclein (Middle) in the brains of TgM83^+/− mice inoculated with homogenates from C1, PD, or MSA patients. The brain region for each inoculum is noted: FC (frontal cortex), P (pons), and SN (substantia nigra). Total α-synuclein was detected from the crude brain homogenates using the Syn211 antibody; phosphorylated α-synuclein was probed with the EP1536Y antibody. Actin is shown as a loading control (Bottom). Molecular weight markers of migrated protein standards are shown in kilodaltons. (C) Phosphorylated α-synuclein in the brains of TgM83^+/− mice inoculated with PD or MSA was quantified by densitometry and expressed as an x-fold difference compared with mice inoculated with nondiseased control brain (C1). The brain-region origin for each inoculum is noted: BG (basal ganglia), FC (frontal cortex), P (pons), and SN (substantia nigra). For each inoculum, data from two animals are shown, except for the MSA11 inoculum, for which only one animal was available for biochemical analysis.

Fig. S1.

Distribution of phosphorylated α-synuclein deposition in the brains of TgM83^+/− mice inoculated with control, PD, and MSA cases. Immunostaining for phosphorylated α-synuclein using antibody EP1536Y was performed in the brains of TgM83^+/− mice inoculated with homogenate prepared from a nondiseased control brain (360 d after inoculation), a PD case (360 d after inoculation), and two MSA cases (90 and 109 d after inoculation). Several brain regions were assessed for the deposition of phosphorylated α-synuclein, which was only found following inoculation with MSA homogenates. (Scale bars, 50 µm.)