Optimized Protocol for Proportionate CNS Cell Retrieval as a Versatile Platform for Cellular and Molecular Phenomapping in Aging and Neurodegeneration

Efficient purification of viable neural cells from the mature CNS has been historically challenging due to the heterogeneity of the inherent cell populations. Moreover, changes in cellular interconnections, membrane lipid and cholesterol compositions, compartment-specific biophysical properties, and intercellular space constituents demand technical adjustments for cell isolation at different stages of maturation and aging. Though such obstacles are addressed and partially overcome for embryonic premature and mature CNS tissues, procedural adaptations to an aged, progeroid, and degenerative CNS environment are underrepresented. Here, we describe a practical workflow for the acquisition and phenomapping of CNS neural cells at states of health, physiological and precocious aging, and genetically provoked neurodegeneration. Following recent, unprecedented evidence of post-mitotic cellular senescence (PoMiCS), the protocol appears suitable for such de novo characterization and phenotypic opposition to classical senescence. Technically, the protocol is rapid, efficient as for cellular yield and well preserves physiological cell proportions. It is suitable for a variety of downstream applications aiming at cell type-specific interrogations, including cell culture systems, Flow-FISH, flow cytometry/FACS, senescence studies, and retrieval of omic-scale DNA, RNA, and protein profiles. We expect suitability for transfer to other CNS targets and to a broad spectrum of engineered systems addressing aging, neurodegeneration, progeria, and senescence

Amitosenescence and Pseudomitosenescence: Putative New Players in the Aging Process

Replicative senescence has initially been defined as a stress reaction of replication-competent cultured cells in vitro, resulting in an ultimate cell cycle arrest at preserved growth and viability. Classically, it has been linked to critical telomere curtailment following repetitive cell divisions, and later described as a response to oncogenes and other stressors. Currently, there are compelling new directions indicating that a comparable state of cellular senescence might be adopted also by postmitotic cell entities, including terminally differentiated neurons. However, the cellular upstream inducers and molecular downstream cues mediating a senescence-like state in neurons (amitosenescence) are ill-defined. Here, we address the phenomenon of abortive atypical cell cycle activity in light of amitosenescence, and discuss why such replicative reprogramming might provide a yet unconsidered source to explain senescence in maturated neurons. We also hypothesize the existence of a G0 subphase as a priming factor for cell cycle re-entry, in analogy to discoveries in quiescent muscle stem cells. In conclusion, we propose a revision of our current view on the process and definition of senescence by encompassing a primarily replication-incompetent state (amitosenescence), which might be expanded by events of atypical cell cycle activity (pseudomitosenescence)