Transcriptional mutagenesis of α-synuclein caused by DNA oxidation in Parkinson's disease pathogenesis

Abstract

Oxidative stress plays an essential role in the development of Parkinson's disease (PD). 8-oxo-7,8-dihydroguanine (8-oxodG, oxidized guanine) is the most abundant oxidative stress-mediated DNA lesion. However, its contributing role in underlying PD pathogenesis remains unknown. In this study, we hypothesized that 8-oxodG can generate novel α-synuclein (α-SYN) mutants with altered pathologic aggregation through a phenomenon called transcriptional mutagenesis (TM). We observed a significantly higher accumulation of 8-oxodG in the midbrain genomic DNA from PD patients compared to age-matched controls, both globally and region specifically to α-SYN. In-silico analysis predicted that forty-three amino acid positions can contribute to TM-derived α-SYN mutation. Here, we report a significantly higher load of TM-derived α-SYN mutants from the midbrain of PD patients compared to controls using a sensitive PCR-based technique. We found a novel Serine42Tyrosine (S42Y) α-SYN as the most frequently detected TM mutant, which incidentally had the highest predicted aggregation score amongst all TM variants. Immunohistochemistry of midbrain sections from PD patients using a newly characterized antibody for S42Y identified S42Y-laden Lewy bodies (LB). We further demonstrated that the S42Y TM variant significantly accelerates WT α-SYN aggregation by cell and recombinant protein-based assays. Cryo-electron tomography revealed that S42Y exhibits considerable conformational heterogeneity compared to WT fibrils. Moreover, S42Y exhibited higher neurotoxicity compared to WT α-SYN as shown in mouse primary cortical cultures and AAV-mediated overexpression in the substantia nigra of C57BL/6 J mice. To our knowledge, this is the first report describing the possible contribution of TM-generated mutations of α-SYN to LB formation and PD pathogenesis.

Keywords: 8-oxodG; Alpha-synuclein aggregation; Lewy bodies; Parkinson’s disease; Transcriptional mutagenesis.

Fig. 1

8-oxodG is significantly accumulated in the midbrain genomic DNA of Parkinson’s disease patients compared to age-matched controls. a Analysis of genomic DNA from the substantia nigra of PD postmortem brain samples (n = 8) showed a significant increase in 8-oxodG levels compared to age-matched control samples (n = 9). b Representative photomicrographs of 8-oxodG immunostaining in controls and PD midbrain samples, demonstrating increased 8-oxodG in PD compared to controls. To identify cell types, tissues were counter-stained with cresyl violet. 8-oxodG immunoreactivity is shown in dark grey after cresyl violet staining. Red arrows, dopaminergic neurons containing neuromelanin; yellow arrows, 8-oxodG stainings. c Representative confocal fluorescence images of double immunofluorescence stainings of 8-oxodG (red) with cell-type-specific markers (green) in PD midbrain samples. Dopaminergic neurons, oligodendrocytes, astrocytes, and microglia were visualized (green) with TH, Olig2, GFAP, and Iba1, respectively, together with 8-oxodG (red). Representative images of each immunofluorescence stainings from 4 PD midbrain samples containing the SN region. Scale bar = 20 µm. d Percentage of cells expressing nuclear 8-oxodG. n = 10 fields per staining. e Representative gel images of α-SYN Oxo-DIP analysis of PD and control midbrain samples. f Quantitative analysis of α-SYN Oxo-DIP, showing a significant increase in 8-oxodG levels on the exon 5 of SNCA of PD compared to controls. g Semi-quantitative RT-PCR showed no significant changes in OGG1 mRNA levels between the PD (n = 9) and control subjects (n = 9). h Similarly, analysis of OGG1 activity to cleave 8-oxodG containing oligonucleotide showed no significant difference in activity between the PD (n = 7) and control subjects (n = 9). Data represent mean ± SEM. † p < 0.01, n.s. non-significant

Fig. 2

Detection of α-SYN TM variants both in mRNA and protein. α-SYN TM variants are increased in the midbrain of PD patients compared to controls and detected in some LBs. a Hypothetical α-SYN mutations caused by TM. All the possible mutant amino acid positions of α-SYN generated by 8-oxodG-driven TM are shown in red (Bottom). Mutations associated with familial PD are shown in blue (Middle). Blue circles indicate WT α-SYN amino acids mutated in familial PD. Green circles indicate serine residues that are subject to phosphorylation. b In silico analysis of the aggregation propensity of the α-SYN TM variants using TANGO. β-aggregation propensity score for the WT and some of the α-SYN TM variants are calculated. c Overall α-SYN TM mutant levels were significantly higher in PD than in control midbrain samples. χ² = 14.186, ‡p < 0.001. d Frequency of each α-SYN TM variant varied between control and PD. (Control, n = 16; PD, n = 20) e Representative photomicrographs showing an LB immuno-positive for S42Y α-SYN. LBs were visualized using HE staining and anti-ubiquitin antibody together with anti-S42Y antibody in 6 µm-apart serial sections of PD midbrain. Black arrows, co-labeled LB. Lower panels, magnified boxed areas in the upper panels. f Double immunostaining of LBs for total α-SYN and S42Y variant. All LBs were visualized by α-SYN antibody (pink), and S42Y was detected using a specific antibody (brown). Black arrows, neuromelanin. The left column shows LBs negative for S42Y; the right column shows LBs co-stained with S42Y. g,h The total number of cells having LBs and S42Y-positive LBs for each PD individual (g) and together (h)

Fig. 3

S42Y α-SYN exhibits stronger aggregation over WT and accelerates aggregation of WT α-SYN protein. a Western blot showing increased aggregation of S42Y α-SYN over WT. Triton X-100-soluble supernatant or –insoluble pellet prepared from SNCA KO HEK293 cells transiently transfected with either backbone vector (Empty), WT or S42Y α-SYN were analyzed using anti-α-SYN antibody. The insoluble fraction was further analyzed using anti-pS129 α-SYN antibody, confirming stronger aggregation of S42Y. β-actin in the soluble fraction was used as an internal control. b Overexpression of S42Y in SNCA KO HEK293 cells showed significant increases in cells containing PK-resistant aggregates compared to the WT overexpression. White arrow heads, perinuclear punctate α-SYN aggregates detected with α-SYN immunofluorescence staining. Data represent mean ± SEM. ‡p < 0.001 (Non-parametric t-test with Mann–Whitney post-hoc corrections, two-tailed p-values). c Western blot showing that overexpression of S42Y accelerates WT α-SYN aggregation. SNCA KO HEK293 cells were transfected with WT and S42Y α-SYN plasmids with various ratios, and Triton X-100 insoluble fractions were analyzed using anti-α-SYN or anti-S42Y antibodies. β-actin in the soluble fraction was used as an internal control. d Split luciferase complementation assay exhibits a small amount of S42Y accelerates WT α-SYN aggregation. SNCA KO HEK293 cells were transfected with split luciferase tagged with WT-α-SYN (S1, S2) together with various α-SYN constructs including WT, S42Y, A53T and A53E for 24, 48, and 72 h. α-SYN aggregations were assessed by luciferase activity. Data represent mean ± SEM. * p < 0.05, †p < 0.01, ‡p < 0.001, §p < 0.0001 (One-way ANOVA with Tukey’s multiple comparison test for each time point). e ThT fluorescence traces for α-SYN fibril formation for WT, S42Y, 1:99 S42Y:WT and 10:90 S42Y:WT. The mixed monomer sample ratios represent molar equivalents. For clarity, the inset plot shows the early ThT fluorescence traces up to 20 h. The total protein concentration for each experiment was 70 μM and was conducted at a pH = 7.4 at 37 °C with shaking. Traces shown are representative of at least 3 replicates each, and error bars represent the standard error of the mean (SEM). f BCA assay results showing the amount of monomer remaining at the endpoint of the 70 μM ThT assay. Residual monomer concentrations are shown as the percentage of the starting monomer concentration (14.99% for WT; 9.63% for S42Y). Error bars represent standard deviation

Fig. 4

S42Y α-SYN fibrils show increased neurotoxicity compared to WT. Primary mouse cortical neuron cultures were incubated with either WT or S42Y fibrils over time. a Time-course neuronal degeneration was assessed using serial phase-contrast images for 7 days. Yellow arrowheads indicate intact cell bodies. b S42Y α-SYN fibrils resulted in significant neuronal death over a period of 7 days compared to WT fibrils or PBS. Data represent mean ± SEM. (Two-way repeated measures ANOVA to analyze the cell survival (%) for 7 days (F7, 14 = 4.163, p = 0.007) followed by Tukey’s post hoc tests. S42Y reduced the cell survival (%) compared to PBS treatment (p = 0.010) and WT treatment (p = 0.024) c–f S42Y α-SYN fibrils increased neuritic degeneration, double-strand DNA damage, and autophagic cell death. Representative confocal fluorescence micrographs of MAP2, γH2AX, and p62 in primary mouse cortical neurons treated with PBS, WT, or S42Y α-SYN fibrils for 7 days or 9 days (c). The number of MAP2-positive neurons having “beaded” neurites (d), γH2A.X-positive (e), and p62-positive neurons (f) was counted. Data represent mean ± SEM. *p < 0.05, †p < 0.01, ‡p < 0.001, n.s. non-significant (One-way ANOVA with Tukey’s multiple comparisons test for each time point of MAP2, rH2A.X, and P62 between three groups)

Fig. 5

Overexpression of S42Y α-SYN in the mouse SNpc shows accelerated degeneration of dopaminergic neurons compared to WT. AAV containing WT or S42T was unilaterally injected into the SN, and mice were sacrificed after 14 days. a Representative photomicrographs of immunostainings for TH, pS129, and γH2A.X in the ipsilateral and contralateral SN. b–d The number of TH-positive (b), pS129-positive (c), and γH2A.X-positive neurons (d) were counted in the WT and S42Y α-SYN overexpressed SN. e–i Microglia are highly activated by S42Y compared to WT α-SYN. Morphometric assessment of microglia was performed after immunolabeling with Iba-1 (red) and nuclear DAPI staining (blue) (e). The number of branches (f), volume (g), the maximum length of branches (h), and total length of branches (i) were measured in the WT and S42Y α-SYN overexpressed SN. Data represent mean ± SEM. *p < 0.05, †p < 0.01, ‡p < 0.001, n.s. non-significant (Non-parametric t-test with Mann–Whitney post-hoc corrections, two-tailed p-values)