Loss of SATB1 Induces a p21 Dependent Cellular Senescence Phenotype in Dopaminergic Neurons

Abstract

Cellular senescence is a mechanism used by mitotic cells to prevent uncontrolled cell division. As senescent cells persist in tissues, they cause local inflammation and are harmful to surrounding cells, contributing to aging. Generally, neurodegenerative diseases, such as Parkinson's, are disorders of aging. The contribution of cellular senescence to neurodegeneration is still unclear. SATB1 is a DNA binding protein associated with Parkinson's disease. We report that SATB1 prevents cellular senescence in post-mitotic dopaminergic neurons. Loss of SATB1 causes activation of a cellular senescence transcriptional program in dopamine neurons, both in human stem cell-derived dopaminergic neurons and in mice. We observed phenotypes which are central to cellular senescence in SATB1 knockout dopamine neurons in vitro and in vivo. Moreover, we found that SATB1 directly represses expression of the pro-senescence factor, p21, in dopaminergic neurons. Our data implicate senescence of dopamine neurons as a contributing factor to the pathology of Parkinson's disease. hESC shows normal appearance. Scale bar: 200 µm. (D) Immunolabeling of DA precursor markers in WT and SATB1 KO hESC derived DA neurons after 16 days of differentiation reveals no difference in marker expression. (E) Depiction of spontaneous action potentials of WT and SATB1 KO DA neurons at different time points show that both genotypes differentiate into mature DA neurons between day 35 and 40. Scale bar: 20 mV, 2 s. (F) DA neurons at day 40 show significant differences in maintenance of response to positive current injections. Scale bar: 20 mV, 200 ms. (G) Longitudinal comparison of cell survival of WT vs. SATB1 KO DA neurons revealed a significant reduction in SATB1 KO survival between day 30 and 40, reaching a plateau at ~50%. Data are represented as mean ± SEM. (H) Quantification of neurite morphology and complexity in WT and SATB1 KO DA neurons. Data are represented as mean ± SEM. (I) Triple immunolabeling of cortical shows that both WT as well as SATB1 KO neurons express the essential markers for cortical neurons. Scale bar: 50 µm. (J) SATB1 KO cortical neurons show no significant change in survival during differentiation. Data are represented as mean ± SEM. (K) Quantification of neurite morphology and complexity in WT and SATB1 KO CTX neurons. (L)Representative western blot and quantification depicts that WT DA neurons express significantly higher levels of SATB1 than WT cortical neurons, suggesting a higher demand in DA neurons. Data are represented as mean ± SEM. See also Figure S1.

Cortical neurons develop a senescence-like phenotype promoted by dysfunctional autophagy

“…Nevertheless, a neuronal SASP is supported by other observations, such as the induction of expression of pro-inflammatory cytokines like TNF-α and CCL2 [33]. Interestingly, dopaminergic neurons with senescent phenotype due to lack of expression of SATB1, a DNA binding protein reduced in the vulnerable region of Parkinson´s Disease patients, express several SASP components, including MCP-1 [34], the cytokine we found significantly secreted by senescent cortical cells. MCP-1 is also secreted by senescent human mesenchymal stem cells and induces paracrine senescence; according to our findings, MCP-1 expression is mediated by GATA4 [28], a transcription factor we found increased in senescent cortical neurons.…”

Pluripotent Stem Cell Therapies for Parkinson Disease: Present Challenges and Future Opportunities

“…We have recently reported that NURR1 (NR4A2) can serve as a reliable marker of early post-mitotic mDA neurons under floor-plate differentiation conditions. An NURR1:H2B-GFP reporter line allowed enrichment of mDA neurons in vitro that yielded nearly pure populations based on mDA markers such as FOXA2, LMX1A, NURR1 and TH including by using flow based and sc-RT-qPCR analyses (Riessland et al, 2019). While genetic engineering and FACS-based purification of neurons presents major challenges of human translation, it may be possible to adapt this approach for MACS-based or genetic selection strategies more suitable or translation.…”

SARS-CoV-2 Infection Causes Dopaminergic Neuron Senescence

“…To examine the impact of SARS-CoV-2 infection on DA neurons, Nurr1:GFP reporter hPSCs were differentiated toward a DA neuron fate using a previously established strategy 8,9 . The resulting DA neurons were validated by the expression of Nurr1:GFP, TH, and FOXA2 (Extended Data Fig.…”