Alpha-synuclein modulates the repair of genomic DNA double-strand breaks in a DNA-PKcs-regulated manner.

α-synuclein (αSyn) is a presynaptic and nuclear protein that aggregates in important neurodegenerative diseases such as Parkinson's Disease (PD), Parkinson's Disease Dementia (PDD) and Lewy Body Dementia (LBD). Our past work suggests that nuclear αSyn may regulate forms of DNA double-strand break (DSB) repair in HAP1 cells after DNA damage induction with the chemotherapeutic agent bleomycin1. Here, we report that genetic deletion of αSyn specifically impairs the non-homologous end-joining (NHEJ) pathway of DSB repair using an extrachromosomal plasmid-based repair assay in HAP1 cells. Notably, induction of a single DSB at a precise genomic location using a CRISPR/Cas9 lentiviral approach also showed the importance of αSyn in regulating NHEJ in HAP1 cells and primary mouse cortical neuron cultures. This modulation of DSB repair is regulated by the activity of the DNA damage response signaling kinase DNA-PKcs, since the effect of αSyn loss-of-function is reversed by DNA-PKcs inhibition. Together, these findings suggest that αSyn plays an important physiologic role in regulating DSB repair in both a transformed cell line and in primary cortical neurons. Loss of this nuclear function may contribute to the neuronal genomic instability detected in PD, PDD and LBD and points to DNA-PKcs as a potential therapeutic target.

Genetic deletion of Polo-like kinase 2 reduces alpha-synuclein serine-129 phosphorylation in presynaptic terminals but not Lewy bodies.

Phosphorylation of alpha-synuclein at serine-129 is an important marker of pathologically relevant, aggregated forms of the protein in several important human diseases, including Parkinson's disease, Dementia with Lewy bodies, and Multiple system atrophy. Although several kinases have been shown to be capable of phosphorylating alpha-synuclein in various model systems, the identity of the kinase that phosphorylates alpha-synuclein in the Lewy body remains unknown. One member of the Polo-like kinase family, PLK2, is a strong candidate for being the Lewy body kinase. To examine this possibility, we have used a combination of approaches, including biochemical, immunohistochemical, and in vivo multiphoton imaging techniques to study the consequences of PLK2 genetic deletion on alpha-synuclein phosphorylation in both the presynaptic terminal and preformed fibril-induced Lewy body pathology in mouse cortex. We find that PLK2 deletion reduces presynaptic terminal alpha-synuclein serine-129 phosphorylation, but has no effect on Lewy body phosphorylation levels. Serine-129 mutation to the phosphomimetic alanine or the unphosphorylatable analog aspartate does not change the rate of cell death of Lewy inclusion-bearing neurons in our in vivo multiphoton imaging paradigm, but PLK2 deletion does slow the rate of neuronal death. Our data indicate that inhibition of PLK2 represents a promising avenue for developing new therapeutics, but that the mechanism of neuroprotection by PLK2 inhibition is not likely due to reducing alpha-synuclein serine-129 phosphorylation and that the true Lewy body kinase still awaits discovery.

Polo-like kinase inhibition leads to neuroprotection of neurons bearing alpha-synuclein Lewy body-like inclusions in vivo.

α-synuclein (αSyn) and S129 phosphorylated αSyn (pSyn) define synucleinopathies like Parkinson's disease (PD). Targeting S129 αSyn kinases, like the Polo-like kinase (PLK) family, could provide a therapeutic strategy to limit degeneration of cells bearing aggregated αSyn inclusions. Using longitudinal in vivo multiphoton imaging in mouse cortex after αSyn inclusion induction, we find an increase in cell survival of inclusion-bearing neurons after PLK inhibition. PLK inhibition is associated with increased αSyn levels within inclusions and increased nuclear DNA damage repair markers. Overall, these findings suggest that PLK inhibition may serve as a potential therapeutic strategy for limiting neurodegeneration in PD.

Alpha-synuclein regulates the repair of genomic DNA double-strand breaks in a DNA-PKcs-dependent manner.

α-synuclein (αSyn) is a presynaptic and nuclear protein that aggregates in important neurodegenerative diseases such as Parkinson's Disease (PD), Parkinson's Disease Dementia (PDD) and Lewy Body Dementia (LBD). Our past work suggests that nuclear αSyn may regulate forms of DNA double-strand break (DSB) repair in HAP1 cells after DNA damage induction with the chemotherapeutic agent bleomycin1. Here, we report that genetic deletion of αSyn specifically impairs the non-homologous end-joining (NHEJ) pathway of DSB repair using an extrachromosomal plasmid-based repair assay in HAP1 cells. Importantly, induction of a single DSB at a precise genomic location using a CRISPR/Cas9 lentiviral approach also showed the importance of αSyn in regulating NHEJ in HAP1 cells and primary mouse cortical neuron cultures. This modulation of DSB repair is dependent on the activity of the DNA damage response signaling kinase DNA-PKcs, since the effect of αSyn loss-of-function is reversed by DNA-PKcs inhibition. Using in vivo multiphoton imaging in mouse cortex after induction of αSyn pathology, we find an increase in longitudinal cell survival of inclusion-bearing neurons after Polo-like kinase (PLK) inhibition, which is associated with an increase in the amount of aggregated αSyn within inclusions. Together, these findings suggest that αSyn plays an important physiologic role in regulating DSB repair in both a transformed cell line and in primary cortical neurons. Loss of this nuclear function may contribute to the neuronal genomic instability detected in PD, PDD and DLB and points to DNA-PKcs and PLK as potential therapeutic targets.