Alpha-synuclein regulates nucleolar DNA double-strand break repair in melanoma

Abstract

Although an increased risk of the skin cancer melanoma in people with Parkinson's Disease (PD) has been shown in multiple studies, the mechanisms involved are poorly understood, but increased expression of the PD-associated protein alpha-synuclein (αSyn) in melanoma cells may be important. Our previous work suggests that αSyn can facilitate DNA double-strand break (DSB) repair, promoting genomic stability. We now show that αSyn is preferentially enriched within the nucleolus in the SK-MEL28 melanoma cell line, where it colocalizes with DNA damage markers and DSBs. Inducing DSBs specifically within nucleolar ribosomal DNA (rDNA) increases αSyn levels near sites of damage. αSyn knockout increases DNA damage within the nucleolus at baseline, after specific rDNA DSB induction, and prolongs the rate of recovery from this induced damage. αSyn is important downstream of ATM signaling to facilitate 53BP1 recruitment to DSBs, reducing micronuclei formation and promoting cellular proliferation, migration, and invasion.

Figure 1.. Alpha-synuclein localizes to the granular component of the nucleolus.

A, B) SK-Mel28 cells were seeded on PLL-coated coverslips and then fixed and stained for alpha-synuclein (Syn1), nucleolar markers (nucleophosmin, treacle, and nucleostemin), and DAPI. Cells were imaged on the Zeiss 980 confocal microscope with Airyscan and colocalization was analyzed in Imaris software. Error bars represent Standard Error of the Mean (SEM) with quantification from 3 biological replicates. ****p<0.0001 by T-test. C). SK-Mel28 cells were seeded on PLL-coated coverslips and then fixed and stained for alpha-synuclein (Syn1), fibrillar center marker (RPA194), dense fibrillar component marker (fibrillarin), granular component marker (nucleophosmin), and DAPI. Cells were imaged on the Zeiss 980 confocal microscope with Airyscan oversampling and Joint Deconvolution and Channel Alignment post-processing. 3D renderings were produced using Imaris software. D) SK-Mel28 control and KO cell pellets were fixed with 0.1M sodium cacodylate buffer (pH 7.2) containing 0.05% glutaraldehyde, 4% paraformaldehyde, and 0.1% picric acid for 2 hours at RT. Thin sections were cut on an ultramicrotome (EM UC7) using a diamond knife. Sections were permeabilized and stained for alpha-synuclein (MJFR1, 1:75) (12nm colloidal gold particles). The stained sections were observed by a JEOL 1400 transmission electron microscope. Red labelling denotes nuclear membrane. Blue labelling denotes the outline of the nucleolus. FC=Fibrillar Center. Arrows point to representative MJFR1 staining. Quantification of 3 biological replicates. ****p<0.0001 by T-test. Error bars denote SEM.

Figure 2.. Alpha-synuclein interacts with γH2AX in the nucleolus and knocking out alpha-synuclein leads to increased ATM/ATR-driven H2AX phosphorylation.

A, C) SK-Mel28 cells were seeded on PLL-coated coverslips and then fixed and stained for alpha-synuclein (Syn1), DSB marker (γH2AX), nucleolar mask (nucleostemin), and DAPI. Cells were imaged on the Zeiss 980 confocal microscope with airyscan and data was analyzed using FIJI (intensity) or Imaris (colocalization). Quantification represents quantification from 3 biological replicates. * p<0.05, *** p<0.001, **** p<0.0001 by T-test or ANOVA. Error bars denote SEM for all graphs. N=nucleus, Nu=nucleolus. Same γH2AX quantification of control cells between A and C. B) SK-Mel28 control and KO cells were seeded on PDL-coated coverslips and then fixed in 4% paraformaldehyde. Proximity Ligation Assay (Duolink) was completed using antibodies against Syn1 and γH2AX. Cells were imaged on the Zeiss 980 confocal microscope and number of foci per nucleus was measured using CellProfiler while masking for the nucleus using DAPI. Each figure shows representative images and quantification from 3 biological replicates. ****p<0.0001 by T-test. Error bars denote SEM. D) SK-Mel28 cells (control/KO/KI) were lysed and a nuclear fractionation was performed. Nuclear protein was run out on SDS-PAGE and probed for γH2AX and total protein. Western blots were imaged on Licor CLx imager. *** p<0.001 by ANOVA. Error bars denote SEM. Quantification from 4 biological replicates. E, F) SK-Mel28 cells were seeded on PLL-coated coverslips and treated with DMSO, KU-60019 (10μM), VE-822 (0.1μM), or NU-7441 (1μM) for 24 hours. Cells were fixed and stained for Syn1, γH2AX, nucleostemin and DAPI. Mean intensity of γH2AX signal within DAPI and nucleostemin masks analyzed using FIJI. ** p<0.01, *** p<0.001, **** p<0.0001 by ANOVA. Error bars denote SEM. Quantification from 3 biological replicates.

Figure 3.. Alpha-synuclein is important in global DNA damage response pathways.

A) SK-Mel28 cells (control/KO/KI) were seeded on PLL-coated coverslips and treated with DMSO or 100μg/ml bleomycin for 1 hour. Cells were fixed and stained for Syn1, γH2AX, and DAPI. Mean intensity of γH2AX signal within DAPI masks were analyzed using FIJI. ** p<0.01, **** p<0.0001 by ANOVA. Error bars denote SEM. Quantification from 3 biological replicates. Same γH2AX quantification between graphs with control cells. B) SK-Mel28 cells (control/KO/KI) were treated with DMSO or 100μg/ml bleomycin for 1 hour and lysed and a nuclear fractionation was performed. Nuclear protein was run out on SDS-PAGE and probed for γH2AX and total protein. Western blots were imaged on Licor CLx imager. * p<0.05, **** p<0.0001. Error bars denote SEM. Quantification from 4 biological replicates. C) SK-Mel28 cells were seeded in a black-welled PDL-coated 96 well plate and treated with DMSO or 100μg/ml bleomycin for 1 hour. Cells were processed according to the In-Cell Western manufacturer instructions and stained for γH2AX (800) and CellTag (700). Plates were imaged on the Licor CLx. ** p<0.01, *** p<0.001, **** p<0.0001 by ANOVA. Error bars denote SEM. Quantification from 3 biological replicates. D) SK-Mel28 cells (control/KO/KI) were seeded on PLL-coated coverslips and treated with DMSO or 100μg/ml bleomycin for 1 hour. Cells were fixed and stained for Syn1, γH2AX, nucleostemin, and DAPI. Mean intensity of γH2AX signal within nucleostemin and DAPI masks were analyzed using FIJI. * p<0.05, ** p<0.01, ***p<0.001, **** p<0.0001 by ANOVA. Error bars denote SEM. N=nucleus, Nu=nucleolus. Quantification from 3 biological replicates. E) SK-Mel28 cells were seeded on PDL-coated coverslips, treated with DMSO or 100μg/ml bleomycin for 1 hour, and fixed in 4% paraformaldehyde. DNA Damage In Situ Ligation Followed by Proximity Ligation Assay (Duolink) was completed as previously described (Galbiati and Fagagna, 2019) using a DNA linker and Syn1. Cells were imaged on the Zeiss 980 confocal microscope and number of foci per nucleus was measured using CellProfiler within nuclear masking with DAPI. Each figure shows representative images and quantification from 3 biological replicates. ****p<0.0001 by ANOVA. Error bars denote SEM.

Figure 4.. Inducing rDNA DSBs increases alpha-synuclein levels and localization to sites of damage and alpha-synuclein knockout significantly increases damage signature.

A) SK-Mel28 cells (control/KO/KI) were seeded on PLL-coated coverslips and then transfected with WT and H98A I-PpoI mRNA. 6-hours post transfection, cells were fixed and stained for Syn1, γH2AX, nucleostemin, and DAPI. Cells were imaged on the Zeiss 980 confocal microscope and data was analyzed using FIJI. Quantification from 3 biological replicates. * p<0.05, ****p<0.0001 by ANOVA. Error bars denote SEM. B) SK-Mel28 cells (control/KO/KI) were transfected with WT and H98A I-PpoI mRNA. 6-hours post transfection, cells were lysed and a nuclear fractionation was performed. Nuclear protein was run out on SDS-PAGE and probed for γH2AX and total protein. Quantification from 4–6 biological replicates. *p<0.05, **p<0.01, ****p<0.0001 by ANOVA. Error bars denote SEM. C) SK-Mel28 cells (control/KO/KI) were seeded on PLL-coated coverslips and then transfected with Cas9 (TrueCut Cas9 Protein v2, Invitrogen) and guideRNAs that target portions of the 28S unit of rDNA or non-targeting control (NT vs. Guide 1+3). Twenty-four hours after transfection, cells were fixed and stained for γH2AX, treacle, RNA polymerase II, and DAPI. Cells were imaged on the Zeiss 980 confocal microscope and analyzed using RNA polymerase II anti-masking. γH2AX intensity was measured within a radius of treacle-identified nucleolar cap. Quantification from 3 biological replicates. ** p<0.01, ****p<0.0001 by ANOVA. Error bars denote SEM. D) SK-Mel28 cells (control/KO) were seeded on PDL-coated coverslips and then transfected with WT and H98A I-PpoI mRNA. 6-hours post transfection, cells were fixed and DNA Damage In Situ Ligation Followed by Proximity Ligation Assay (Duolink) was completed as previously described (Galbiati and Fagagna, 2019) using a DNA linker and Syn1. A treacle co-stain was included to identify nucleolar cap formation. Cells were imaged on the Zeiss 980 confocal microscope and number of foci per nucleus was measured using CellProfiler within nuclear masking with DAPI. Each figure shows representative images and quantification from 3 biological replicates. ****p<0.0001 by ANOVA. Error bars denote SEM. E) SK-Mel29 control cells expressing Synuclein-GFP or Empty Vector-GFP (EV). Yellow arrows show targeting of laser-induced damage (LID) pulse in the nucleolus. Baseline (t=−6s) and after LID (t=6 and 23s) images show accumulation of Synuclein-GFP at DNA damage site. Data from graph shows calculated enrichment ratio at LID site (compared to an adjacent site in the nucleolus). Quantification from >20 cells over 2 biological replicates. ****p<0.0001 by t-test. Error bars denote SEM.

Figure 5.. Alpha-synuclein knockout significantly impairs recovery of rDNA damage in an ATM-dependent manner.

A) SK-Mel28 cells were seeded in a black-welled PDL-coated 96 well plate and treated with DMSO, KU-60019 (10μM), VE-822 (0.1μM), or NU-7441 (1μM) for 24 hours. Cells were then treated with WT and H98A I-PpoI mRNA for 6 hours in the presence of the inhibitors. Cells were processed according to the In-Cell Western manufacturer instructions and stained for γH2AX (800) and CellTag (700). Plates were imaged on the Licor CLx. *p<0.05, ** p<0.01, *** p<0.001 by ANOVA. Error bars denote SEM. Quantification from 6 biological replicates. Normalization to control cells transfected with I-PpoI H98A and treated with DMSO. B) SK-Mel28 cells (control/KO/KI) were seeded on PLL-coated coverslips and then transfected with WT and H98A I-PpoI mRNA. At the indicated time point post transfection, cells were fixed and stained for Syn1, γH2AX, nucleostemin, and DAPI. Cells were imaged on the Zeiss 980 confocal microscope and data was analyzed using FIJI. Quantification from 3 biological replicates. Statistical labeling denotes significance between control and KO cell lines, ****p<0.0001 by ANOVA. C) SK-Mel28 cells (control/KO/KI) were transfected with WT and H98A I-PpoI mRNA. After 24 hours, cells were lysed and a nuclear fractionation was performed. Nuclear protein was run out on SDS-PAGE and probed for γH2AX and total protein. Quantification from 5 biological replicates. *p<0.05, **p<0.01, ***p<0.001, by ANOVA. Error bars denote SEM.

Figure 6.. Alpha-synuclein localizes to DSB-induced nucleolar caps and is important for nucleolar cap recovery.

A) SK-Mel28 cells were seeded on PLL-coated coverslips and then treated with WT I-PpoI mRNA for 6 hours or 100ng/ml Actinomycin D for 1 hour prior to fixation. Cells were stained for Syn1, nucleolar caps marker (UBF), nucleostemin, and DAPI. Cells were imaged on the Zeiss 980 confocal microscope with Airyscan oversampling and Joint Deconvolution and Channel Alignment post-processing. 3D renderings and distance from cap analysis were produced using Imaris software. Quantification from 5 biological replicates. Error bars denote SEM. Statistical significance was calculated via nonlinear regression (99% confidence interval). B) SK-Mel28 cells (control/KO/KI) were seeded on PDL-coated 8-well Ibidi plates. Cells were transfected with 800ng GFP-Treacle using Lipofectamine 3000. Twenty-four hours post-transfection, cells were treated with WT I-PpoI mRNA. Four hours after treatment, live-cell imaging was performed using the Zeiss Celldiscoverer 7 and imaged for 15 hours. Quantification from 5 biological replicates. *p<0.05, ****p<0.0001 by two-way ANOVA or Mantel-Cox test.

Figure 7.. Alpha-synuclein knockout leads to dysregulated recruitment of 53BP1 to nucleolar caps and significantly increases occurrences of micronuclei formation with impaired cellular growth phenotypes.

A) SK-Mel28 cells (control/KO/KI) were seeded on PLL-coated coverslips and then transfected with WT I-PpoI mRNA. 6-hours post transfection, cells were fixed and stained for Syn1, UBF, 53BP1, and DAPI. Cells were imaged on the Zeiss 980 confocal microscope and data was analyzed using FIJI. Quantification from 4 biological replicates. * p<0.05, ****p<0.0001 by ANOVA. Error bars denote SEM. B) SK-Mel28 cells were seeded on PLL-coated coverslips and then transfected with WT I-PpoI mRNA. 0, 6, and 24-hours post transfection, cells were fixed and stained for DAPI. Cells were imaged on the Zeiss 980 confocal microscope. Micronuclei were hand counted with the experimenter blinded to cell condition. Quantification from 3 biological replicates. *p<0.05, **p<0.01 by ANOVA. Error bars denote SEM. C) SK-Mel28 cells were plated at 6,000 cells per well on 80μg/ml Matrigel coated ImageLock plates. Graphs represent temporal progression of total confluence (proliferation), or wound closure using Relative Wound Density as the metric to measure migration or invasion. Data represents time-course of means of each cell line among 3 biological replicates. *95%CI, ***99%CI by nonlinear regression (migration) or simple linear regression (proliferation and invasion) analysis.

Figure 8.. Transcriptomic analysis reveals alpha-synuclein involvement in nucleolar DNA damage pathways.

A) Total RNA was extracted from SK-Mel28 control and KO cells and sent for RNA-sequencing analysis at Indiana University. Differentially expressed gene transcripts in KO cells compared to control were identified. These were cross-referenced to over 500 nucleolar-specific genes. 64 genes were identified and plotted on a volcano plot using p-value and fold change. All transcripts, greater than 1 standard deviation and less than −1 standard deviation were plotted. Red labelling indicated RT-PCR validated genes. B) All significant upregulated and downregulated transcripts underwent gene ontology analysis, which included pathways associated with molecular function, cellular component, and biological process. Gene sets found to be significant after Benjamini-Hochberg multiple testing correction (P.adj < 0.05) were marked with an asterisk. C) Graphic outlining mechanistic function of alpha-synuclein in the nucleolar DNA damage response pathway.