Senescence and impaired DNA damage responses in alpha-synucleinopathy models.

α-Synuclein is a crucial element in the pathogenesis of Parkinson's disease (PD) and related neurological diseases. Although numerous studies have presented potential mechanisms underlying its pathogenesis, the understanding of α-synuclein-mediated neurodegeneration remains far from complete. Here, we show that overexpression of α-synuclein leads to impaired DNA repair and cellular senescence. Transcriptome analysis showed that α-synuclein overexpression led to cellular senescence with activation of the p53 pathway and DNA damage responses (DDRs). Chromatin immunoprecipitation analyses using p53 and γH2AX, chromosomal markers of DNA damage, revealed that these proteins bind to promoters and regulate the expression of DDR and cellular senescence genes. Cellular marker analyses confirmed cellular senescence and the accumulation of DNA double-strand breaks. The non-homologous end joining (NHEJ) DNA repair pathway was activated in α-synuclein-overexpressing cells. However, the expression of MRE11, a key component of the DSB repair system, was reduced, suggesting that the repair pathway induction was incomplete. Neuropathological examination of α-synuclein transgenic mice showed increased levels of phospho-α-synuclein and DNA double-strand breaks, as well as markers of cellular senescence, at an early, presymptomatic stage. These results suggest that the accumulation of DNA double-strand breaks (DSBs) and cellular senescence are intermediaries of α-synuclein-induced pathogenesis in PD.

Alpha-Synuclein Inclusion Formation in Human Oligodendrocytes.

Multiple system atrophy (MSA) is a neurodegenerative disease characterized by presence of α-synuclein-positive inclusions in the cytoplasm of oligodendrocytes. These glial cytoplasmic inclusions (GCIs) are considered an integral part of the pathogenesis of MSA, leading to demyelination and neuronal demise. What is most puzzling in the research fields of GCIs is the origin of α-synuclein aggregates in GCIs, since adult oligodendrocytes do not express high levels of α-synuclein. The most recent leading hypothesis is that GCIs form via transfer and accumulation of α-synuclein from neurons to oligodendrocytes. However, studies regarding this subject are limited due to the absence of proper human cell models, to demonstrate the entry and accumulation of neuronal α-synuclein in human oligodendrocytes. Here, we generated mature human oligodendrocytes that can take up neuronderived α-synuclein and form GCI-like inclusions. Mature human oligodendrocytes are derived from neural stem cells via "oligosphere" formation and then into oligodendrocytes, treating the cells with the proper differentiation factors at each step. In the final cell preparations, oligodendrocytes consist of the majority population, while some astrocytes and unidentified stem cell-like cells were present as well. When these cells were exposed to α-synuclein proteins secreted from neuron-like human neuroblastoma cells, oligodendrocytes developed perinuclear inclusion bodies with α-synuclein immunoreactivity, resembling GCIs, while the stem cell-like cells showed α-synuclein-positive, scattered puncta in the cytoplasm. In conclusion, we have established a human oligodendrocyte model for the study of GCI formation, and the characterization and use of this model might pave the way for understanding the pathogenesis of MSA.

Cell-to-cell Transmission of Polyglutamine Aggregates in C. elegans.

Huntington disease (HD) is an inherited neurodegenerative disorder characterized by motor and cognitive dysfunction caused by expansion of polyglutamine (polyQ) repeat in exon 1 of huntingtin (HTT). In patients, the number of glutamine residues in polyQ tracts are over 35, and it is correlated with age of onset, severity, and disease progression. Expansion of polyQ increases the propensity for HTT protein aggregation, process known to be implicated in neurodegeneration. These pathological aggregates can be transmitted from neuron to another neuron, and this process may explain the pathological spreading of polyQ aggregates. Here, we developed an in vivo model for studying transmission of polyQ aggregates in a highly quantitative manner in real time. HTT exon 1 with expanded polyQ was fused with either N-terminal or C-terminal fragments of Venus fluorescence protein and expressed in pharyngeal muscles and associated neurons, respectively, of C. elegans. Transmission of polyQ proteins was detected using bimolecular fluorescence complementation (BiFC). Mutant polyQ (Q97) was transmitted much more efficiently than wild type polyQ (Q25) and forms numerous inclusion bodies as well. The transmission of Q97 was gradually increased with aging of animal. The animals with polyQ transmission exhibited degenerative phenotypes, such as nerve degeneration, impaired pharyngeal pumping behavior, and reduced life span. The C. elegans model presented here would be a useful in vivo model system for the study of polyQ aggregate propagation and might be applied to the screening of genetic and chemical modifiers of the propagation.