Ubiquitination of alpha-synuclein is not required for formation of pathological inclusions in alpha-synucleinopathies

Abstract

alpha-Synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are neurodegenerative disorders in which abnormal inclusions containing alpha-synuclein accumulate in selectively vulnerable neurons and glia. In this report, immunohistochemistry demonstrates ubiquitin in subsets of alpha-synuclein inclusions in dementia with Lewy bodies and multiple system atrophy. Biochemistry demonstrates that alpha-synuclein in the sodium dodecyl sulfate-soluble fractions of diseased brains is ubiquitinated, with mono- and di-ubiquitinated species predominating over polyubiquitinated forms. Similar immunohistochemical and biochemical characteristics were observed in an A53T mutant human alpha-synuclein transgenic mouse model of neurodegenerative alpha-synucleinopathies. Furthermore, in vitro ubiquitination of alpha-synuclein fibrils recapitulated the pattern of alpha-synuclein ubiquitination observed in human disease and the A53T alpha-synuclein mouse model. These results suggest that ubiquitination of alpha-synuclein is not required for inclusion formation and follows the fibrillization of alpha-synuclein.

Figure 1.

Ubiquitin immunostaining of α-syn pathological inclusions in DLB and MSA. Immunohistochemistry of cingulate cortex from a DLB patient (DLB-2) (A and B) and cerebellum from a MSA patient (MSA-6) (C and D) stained using monoclonal anti-α-syn antibodies Syn303 (A and C) and monoclonal anti-ubiquitin antibody mAb 1510 (B and D). In A and B, arrows highlight immunoreactive cortical LBs, whereas in C and D arrows indicate stained GCIs. Double-label immunofluorescence of cortical LBs in the cingulate cortex of a patient with DLB (DLB-5) (E–G) and GCIs in the cerebellum of a patient with MSA (MSA-8) (H–J) with rabbit anti-α-syn antibody SNL-4 (green, E and H) and murine anti-ubiquitin antibody mAb 1510 (red, F and I). The overlays of staining with both antibodies are shown in G and J. In H to J, arrows indicate inclusions stained with antibodies to both α-syn and ubiquitin, whereas arrowheads depict inclusions stained only with antibodies to α-syn. Scale bars: 80 μm (A, B, E–J), 40 μm (C, D).

Figure 2.

Insoluble α-syn in diseased brain is ubiquitinated. A: Western blot analysis of biochemically fractionated cingulate cortex from a patient with DLB (DLB-3). Immunoblots were developed with anti-α-syn antibodies LB509 and Syn208 as well as anti-ubiquitin antibody mAb 1510. Twenty μl of LS fraction (lane 1), TX fraction (lane 2), sarkosyl-soluble fraction (lane 3), and SDS-soluble fraction (lane 4) were loaded in separate lanes of 15% SDS-polyacrylamide gels. Note that the SDS-soluble fraction is four times as concentrated as each of the other fractions in that 2.5 ml/g of SDS buffer was used for tissue extraction versus 10 ml/g for each of the other fractions. Arrowhead indicates α-syn monomer (αS) and bracket indicates ubiquitin monomer (Ub). Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. B: Western blot analysis (with antibodies Syn208 and mAb 1510) of the SDS-soluble fraction from the cingulate cortex of normal brains (NL-1, NL-2) and DLB brains (DLB-1, DLB-2, and DLB-3). Twenty μl of SDS-soluble fraction was loaded in each lane of a 15% gel. One hundred ng of recombinant human α-syn was loaded in the indicated lanes. C: Western blot analysis of the SDS-soluble fraction from the cingulate cortex of case DLB-1 using various anti-α-syn (LB509, Syn102, Syn211) and anti-ubiquitin (mAb 1510, Conj8) antibodies. Arrows depict mono-, di-, and tri-ubiquitinated forms of α-syn. D: Immunoprecipitation followed by Western blot analysis. α-Syn in the SDS-soluble fraction of the cingulate cortex of DLB-1 was isolated by immunoprecipitation with anti-α-syn antibodies. The sample was analyzed by Western blot analysis using anti-α-syn antibody LB509 and anti-ubiquitin antibody mAb 1510 (arrows, mono- and di-ubiquitinated forms of α-syn; * and **, possible ubiquitinated forms of α-syn in which ubiquitin moieties may be masking the LB509 epitope; ***, modified form of α-syn, possibly dimerized α-syn). E: Ubiquitinated α-syn from DLB brain can be deubiquitinated by UCH-L1 in vitro. SDS-soluble fraction from the cingulate cortex of NL-1 or DLB-1 was untreated or reacted with 50 nmol/L of UCH-L1. The samples were analyzed by Western blot analysis using LB509. R represents a lane loaded with 100 ng of recombinant human α-syn. The mobility of molecular mass markers (kd) is depicted on the left of each panel.

Figure 3.

Ubiquitin immunostaining of α-syn inclusions in mice expressing A53T human α-syn. Double-label immunofluorescence of α-syn inclusions in the spinal cord (A–C) and pons (D–F) of M83 homozygous A53T human α-syn transgenic mice with rabbit anti-α-syn antibody SNL-4 (green, A and D) and mouse anti-ubiquitin antibody mAb 1510 (red, B and E). The overlays are shown in C and F. Arrows indicate α-syn inclusions that are not ubiquitin-positive. Scale bar, 80 μm.

Figure 4.

α-Syn is ubiquitinated in inclusions in A53T α-syn transgenic mice. A: Western blot analysis of the SDS-soluble fractions of cortex (C) and spinal cord (S) from 12-month-old nontransgenic (nTg) mice, homozygous transgenic mice expressing wild-type human α-syn (line M7), and homozygous transgenic mice expressing A53T human α-syn (line M83) using the anti-α-syn antibody LB509. Note the accumulation of α-syn and higher molecular mass species in the SDS-soluble fraction of M83 transgenic mice. B: Accumulation of higher molecular mass species of α-synuclein in the spinal cord of M83 and M91 transgenic mice coincides with presence of ubiquitin immunoreactive bands. Ten μl of each SDS-soluble fraction was loaded in separate lanes of a 15% polyacrylamide gel. Arrows indicate ubiquitinated forms of α-syn in the M91 mouse. The bracket indicates ubiquitinated proteins that are unlikely to be α-syn because they are not labeled by the anti-α-syn antibody LB509. The mobility of molecular mass markers (kd) is indicated on the left of each panel.

Figure 5.

In vitro ubiquitination of monomeric and fibrillar α-syn. Left: Unassembled or fibrillar α-syn (0.5 mg/ml) was subjected to in vitro ubiquitination as described in Materials and Methods. Equal volumes of unassembled α-syn, ubiquitination reaction without α-syn, ubiquitination reaction with unassembled α-syn, and ubiquitination reaction with fibrillar α-syn were loaded in separate lanes of 15% SDS-polyacrylamide gels that were transferred electrophoretically onto nitrocellulose and analyzed by Western blotting with LB509. Right: Western blot analysis (using LB509) of the SDS-soluble fraction of cingulate cortex from three DLB cases. Arrows indicate ubiquitinated forms of α-syn. The mobility of molecular mass markers (kd) is depicted to the left of the panel.