Analysis of gene expression during aging of CGNs in culture: implication of SLIT2 and NPY in senescence.

Senescence is the major key factor that leads to the loss of neurons throughout aging. Cellular senescence is not the consequence of single cause, but there are multiple aspects which may induce senescence in a cell. Various causes such as gene expression, molecular interactions and protein processing and chromatin organization are described as causal factor for senescence. It is well known that the damage to the nuclear or mitochondrial DNA contributes to the aging either directly by inducing the apoptosis/cellular senescence or indirectly by altering cellular functions. The significant nuclear DNA damage with the age is directly associated with the continuous declining in DNA repair. The continuous decline in expression of topoisomerase 2 beta (Topo IIß) in cultured cerebellar granule neurons over time indicated the decline in the repair of damage DNA. DNA Topo IIß is an enzyme that is crucial for solving topological problems of DNA and thus has an important role in DNA repair. The enzyme is predominantly present in non-proliferating cells such as neurons. In this paper, we have studied the genes which were differentially expressed over time in cultured cerebellar granule neurons (CGNs) and identified potential genes associated with the senescence. Our results showed that the two genes neuropeptide Y (Npy) and Slit homolog 2 (Drosophila) (Slit2) gradually increase during aging, and upon suppression of these two genes, there was gradual increase in cell viability along with restoration of the expression of Topo IIß and potential repair proteins.

Topoisomerase IIß regulates base excision repair capacity of neurons.

Topoisomerase IIß (TopoIIß), an enzyme involved in DNA rearrangements, is predominantly present in brain and its levels are shown to decrease with age. This study characterizes the function of TopoIIß in regulating BER (base excision repair) activity. TopoIIß deficient granule neurons (CGNT⁻) show greater sensitivity to N-ethyl N-nitroso urea (ENU)-mediated DNA damage. The cell-free extracts of TopoIIß knockdown cells (ECGNT⁻) show a significant decrease in G-U BER activity during ENU-treatment as well as during recovery, suggesting that TopoIIß promotes G-U BER activity. Since G-U BER activity is not affected in the presence of ICRF-193, catalytic inhibitor of TopoIIß, the activity of enzyme per se may not be participating in BER activity. Further characterization of the activities of BER enzymes present in ECGNT⁻ shows that uracil DNA-glycosylase (UDG) and ligase (LIG) activities decrease significantly in both ENU treatment and recovery. Supplementation of TopoIIß to ECGNT⁻ does not restore ligation activity and ICRF-193 does not influence the LIG activity. These results suggest a role, at least an indirect one, of TopoIIß in the repair of ENU-mediated strand breaks via BER pathway including the activities of UDG and LIG.