Induction of alpha-synuclein aggregation by intracellular nitrative insult

Abstract

Brain lesions containing filamentous and aggregated alpha-synuclein are hallmarks of neurodegenerative synucleinopathies. Oxidative stress has been implicated in the formation of these lesions. Using HEK 293 cells stably transfected with wild-type and mutant alpha-synuclein, we demonstrated that intracellular generation of nitrating agents results in the formation of alpha-synuclein aggregates. Cells were exposed simultaneously to nitric oxide- and superoxide-generating compounds, and the intracellular formation of peroxynitrite was demonstrated by monitoring the oxidation of dihydrorhodamine 123 and the nitration of alpha-synuclein. Light microscopy using antibodies against alpha-synuclein and electron microscopy revealed the presence of perinuclear aggregates under conditions in which peroxynitrite was generated but not when cells were exposed to nitric oxide- or superoxide-generating compounds separately. alpha-Synuclein aggregates were observed in 20-30% of cells expressing wild-type or A53T mutant alpha-synuclein and in 5% of cells expressing A30P mutant alpha-synuclein. No evidence of synuclein aggregation was observed in untransfected cells or cells expressing beta-synuclein. In contrast, selective inhibition of the proteasome resulted in the formation of aggregates detected with antibodies to ubiquitin in the majority of the untransfected cells and cells expressing alpha-synuclein. However, alpha-synuclein did not colocalize with these aggregates, indicating that inhibition of the proteasome does not promote alpha-synuclein aggregation. In addition, proteasome inhibition did not alter the steady-state levels of alpha-synuclein, but addition of the lysosomotropic agent ammonium chloride significantly increased the amount of alpha-synuclein, indicating that lysosomes are involved in degradation of alpha-synuclein. Our data indicate that nitrative and oxidative insult may initiate pathogenesis of alpha-synuclein aggregates.

Fig. 1.

Expression of synucleins in stably transfected HEK 293 cell lines. Expression of human wild-type α-syn (lane 4), A53T mutant α-syn (lane 5), A30P mutant α-syn (lane 6), and human β-synuclein (lane 7) in stably transfected HEK 293 cells is shown, as demonstrated by Western blot analysis using the anti-synuclein antibody Syn102, which reacts equally to both syn proteins (Giasson et al., 2000b). Expression of α- and β-syn is not detected in untransfected HEK 293 cells (lane 3).Lanes 1 and 2 were loaded with 10 ng of recombinant human α- and β-syn, respectively. Lanes 3–7 were loaded with 10 μg of total cell lysates.

Fig. 2.

Formation of rhodamine 123 from the oxidation of DHR 123 in HEK 293/α-syn. A, HEK 293/α-syn cells were incubated for 2 hr at 37°C with 5 μm DHR 123. After extensive washing, rhodamine 123 fluorescence was monitored over time in untreated cells (triangles) and cells treated with 1 mm PAPA/NO alone (diamonds), DMNQ alone (circles), or 1 mm PAPA/NO plus 10 μm DMNQ (squares). B, Composite fluorescence histograms obtained by flow cytometric evaluation of HEK 293/α-syn cells after exposure to PAPA/NO plus DMNQ (broken trace) and control cells (solid trace). The mean fluorescence intensity value for each histogram, indicating the percentage of cells positive for rhodamine 123 fluorescence, was 6% for control and 29% for treated cells.C, Representative epifluorescence images (magnification, 17×) of cells after exposure to nitric oxide- and superoxide-generating compounds. D, Immunoprecipitation of α-syn using the anti-α-syn monoclonal antibody SYN-1 followed by Western blotting with SYN-1 or a polyclonal anti-3-nitrotyrosine antibody (Anti-3-NT). Cells were exposed to 1 mm PAPA/NO (first lane), 5 mm paraquat (second lane), or both 1 mm PAPA/NO and 5 mm paraquat (third lane).

Fig. 3.

Formation of α-syn intracellular aggregates on exposure to nitric oxide- and superoxide-generating compounds. HEK 293/α-syn cells were fixed and stained with monoclonal anti-α-syn antibodies Syn208 and Syn202. A–C, α-Syn inclusions were readily visible in cells exposed to 1 mm PAPA/NO plus 5 μm dopamine (A), 1 mmPAPA/NO plus 100 nm rotenone (B), and 1 mm PAPA/NO plus 5 mm paraquat (C). Only diffuse background staining was noted in untransfected (D) and β-syn-transfected (E) cells exposed to 1 mm PAPA/NO plus 5 mm paraquat. Magnification: A, B, 125×; C–E, 65×.

Fig. 4.

Nitration of α-syn intracellular aggregates on exposure to nitric oxide- and superoxide-generating compounds.A, HEK 293/α-syn exposed to 1 mm PAPA/NO plus 100 nm rotenone. B, HEK 293/A30P mutant α-syn exposed to 1 mm PAPA/NO plus 5 μmdopamine. C, HEK 293/A53T mutant α-syn exposed to 1 mm PAPA/NO plus 100 nm rotenone. Column 1, Cells were fixed and stained with monoclonal anti-α-syn antibody Syn 208. Column 2, Stained with a polyclonal anti-3-nitrotyrosine antibody. Column 3, Superimposed image. Magnification, 125×.

Fig. 5.

Intracellular aggregates of α-syn on exposure to nitric oxide- and superoxide-generating compounds are nitrated at Tyr³⁹ as well as Tyr¹²⁵ and Tyr¹³⁶. A, HEK 293/α-syn exposed to 1 mm PAPA/NO plus 100 nm rotenone.B, HEK 293/α-syn exposed to 1 mm PAPA/NO plus 5 μm dopamine. Column 1, Cells were fixed and stained with monoclonal anti-nitrated α-syn antibody nSyn14, which recognizes nitrated tyrosine residue Tyr³⁹ in the N terminus of α-syn. Column 2, Stained with monoclonal anti-nitrated α-syn antibody nSyn12, which recognizes nitrated tyrosine residues Tyr¹²⁵ and Tyr¹³⁶ in the C terminus of α-syn (Giasson et al., 2000a). Magnification, 98×.

Fig. 6.

Ultrastructure of inclusions induced by nitrative damage in HEK 293/α-syn cells. Electron microscopic examination of cytoplasmic inclusions in HEK 293/α-syn cells exposed to nitric oxide- and superoxide-generating compounds was performed. InC, The sample was treated with formic acid to enhance the appearance of fibrils (arrows) in the inclusion.

Fig. 7.

Paucity of ubiquitin immunoreactivity in α-syn aggregates. Cells were stained with the mouse anti-α-syn monoclonal antibodies Syn202 and Syn208 (column 1) or a rabbit anti-ubiquitin polyclonal antibody (column 2), and inclusions were visualized by immunofluorescence. Column 3, Overlay composite of images. Cells expressing wild-type α-syn (A, B, D) or A53T mutant α-syn (C) were exposed to PAPA/NO and paraquat (A), PAPA/NO and rotenone (B, C), or paraquat alone (D). Magnification, 125×.

Fig. 8.

Formation of protein aggregates after selective inhibition of the proteasome. Untransfected HEK 293 cells (A, B) or HEK 293/α-syn cells (C, D) were treated with 10 μm lactacystin-β-lactone for 1 hr and stained with anti-α-syn monoclonal antibody Syn202 (A, C) or a rabbit anti-ubiquitin antibody (B, D). Magnification, 40×.

Fig. 9.

Formation of protein aggregates after inhibition of the proteasome. HEK 293/α-syn cells were treated with 10 μm lactacystin-β-lactone for 1 hr and stained with the mouse anti-α-syn monoclonal antibody Syn202 (A) or with a rabbit anti-ubiquitin antibody (B). InC, the fluorescence fields in A andB were merged. Note that inhibition of the proteasome with lactacystin-β-lactone results in ubiquitin aggregates but not α-syn aggregates. Magnification, 65×.

Fig. 10.

Degradation of α-syn in HEK 293 cells.A, Pulse–chase analysis of wild-type (diamonds), A30P (triangles), and A53T (squares) α-syn turnover in HEK 293 stable transfectants. The residual [³⁵S]Met is after chasing over 48 hr (n = 3). A, inset, Representative chase profile of [³⁵S]Met-labeled wild-type α-syn.UT, Immunoprecipitation from untransfected HEK 293 cells. B, Western blot analysis using the anti-α-syn antibody LB509 showing three independent experiments. HEK 293/α-syn cells were untreated (Ct) or challenged with 25 mm NH₄Cl, 10 μmlactacystin-β-lactone (Lact), or 2 μmMG132 for 24 hr. Equal amounts of protein (5 μg) were loaded in each separate lane of the gels as confirmed by the levels of β-tubulin (β-tub).