Chromatin-Bound Oxidized α-Synuclein Causes Strand Breaks in Neuronal Genomes in in vitro Models of Parkinson’s Disease

Abstract

Alpha-synuclein (α-Syn) overexpression and misfolding/aggregation in degenerating dopaminergic neurons have long been implicated in Parkinson’s disease (PD). The neurotoxicity of α-Syn is enhanced by iron (Fe) and other pro-oxidant metals, leading to generation of reactive oxygen species in PD brain. Although α-Syn is predominantly localized in presynaptic nerve terminals, a small fraction exists in neuronal nuclei. However, the functional and/or pathological role of nuclear α-Syn is unclear. Following up on our earlier report that α-Syn directly binds DNA in vitro, here we confirm the nuclear localization and chromatin association of α-Syn in neurons using proximity ligation and chromatin immunoprecipitation analysis. Moderate (~2-fold) increase in α-Syn expression in neural lineage progenitor cells (NPC) derived from induced pluripotent human stem cells (iPSCs) or differentiated SHSY-5Y cells caused DNA strand breaks in the nuclear genome, which was further enhanced synergistically by Fe salts. Furthermore, α-Syn required nuclear localization for inducing genome damage as revealed by the effect of nucleus versus cytosol-specific mutants. Enhanced DNA damage by oxidized and misfolded/oligomeric α-Syn suggests that DNA nicking activity is mediated by the chemical nuclease activity of an oxidized peptide segment in the misfolded α-Syn. Consistent with this finding, a marked increase in Fe-dependent DNA breaks was observed in NPCs from a PD patient-derived iPSC line harboring triplication of the SNCA gene. Finally, α-Syn combined with Fe significantly promoted neuronal cell death. Together, these findings provide a novel molecular insight into the direct role of α-Syn in inducing neuronal genome damage, which could possibly contribute to neurodegeneration in PD.

Heterocyclic Analogs of Sulforaphane Trigger DNA Damage and Impede DNA Repair in Colon Cancer Cells: Interplay of HATs and HDACs

“…Inhibition of DNA repair by SFN analogs. A) A schematic representation of the SeeSaw reporter SSR 2.0, as published . B) Images of HCT116 cells transfected with SSR 2.0 for 24 h ( left panel ), followed by transfection with I‐SceI for 6 h ( right panel ) to induce DNA damage response, as indicated by HR‐mediated (red) or NHEJ‐mediated repair (green) fluorescence.…”

“…HCT116 cells were grown overnight in 8‐well chamber slides. After treatment for 6 h with test agents, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X‐100, and incubated with primary antibodies for CtIP (#61142, mouse monoclonal; Active Motif), PCAF (#A301‐666A, rabbit polyclonal; Bethyl Labs), or GCN5 (#3305, rabbit polyclonal, cell signaling), as per reported methodologies . Proximity ligation assays (PLA) were carried out using the Duolink PLA kit (Sigma‐Aldrich, St. Louis, MO), according to the manufacturer's recommendations.…”

Alpha‐Synuclein Modulates the Physical Properties of DNA

“…[10] aS-induced changes in transcription of PGC1alpha negatively affected mitochondrial morphology and function. Moreover, chromatin-bound oxidized aS was found to cause double-strand breaks in neuronal DNA that resulted in cell death [11] and DNA has been reported to stimulate aS amyloid formation in vitro. [12] These studies clearly emphasize that aS may have additional roles in PD that are related to nuclear gene regulation.…”

An Inducible Alpha-Synuclein Expressing Neuronal Cell Line Model for Parkinson’s Disease1

“…1C, D). Cell viability was moderately higher in differentiated cells compared to undifferentiated cells overexpressing α-synuclein (Supplementary Figure 1G), possibly due to the replication stress activated upon accumulation of DNA strand breaks induced by the presence of α-synuclein in the chromatin [21]. …”

“…Preparation of whole cell extracts and protein quantitation was performed as previously described [21]. Following electrophoresis separation in a 4–16% gradient gel, proteins were transferred onto a PVDF membrane fixed with 0.4% paraformaldehyde (PFA) in 1X PBS for 30 min at room temperature (RT), and blocked with 5% skim milk in 1X TBS-T.…”