Cellular senescence is a state of permanent cell cycle arrest that results in tissue dysfunction and is associated with aging and age-related diseases . The types of senescence by cause are generally addressed in two: replicative senescence, where cells stop dividing due to a DNA damage response (DDR) induced by critical telomere shortening; and premature senescence, where any of several stressors, including mutations in oncogenes or tumor-suppressor genes, DNA damage, endoplasmic reticulum (ER) stress, and oxidative stress causes the cell cycle arrest . Since DNA damage, which is a defining attribute of replicative senescence, can also appear in premature senescence, definitively identifying the type of senescence a cell has undergone can, in some cases, be challenging . Besides the standard features of arrested cell cycle and function loss, it has been reported that some senescent cells acquire an inflammatory phenotype termed as senescent-associated secretory phenotype (SASP), where they engage in paracrine inflammatory signaling, driving chronic inflammatory diseases . Since one function of senescence is oncogenesis prevention, tumor-suppressor proteins' expression or activity tends to be elevated in senescent cells . Tumor-suppressor proteins most commonly used in identifying senescent cells are p53, p21, and p16/INK4A . The causes of senescence are multifactorial, as stated above and often overlap. The two nearly universal senescence factors are DDR, whether caused by telomere attrition, mitochondrial dysfunction/reactive oxygen species (ROS), or ionizing radiation; and elevated activity of the tumor-suppressor proteins p53 or p16/INK4a [6, 7]. Since its discovery, multiple markers of senescence have been identified. A typical senescent cell marker is a change in morphology, where cells become enlarged and flattened . Though depending on the type of cells or senescence trigger, the morphological change can manifest differently; for example, a morphological alteration that appears with ER stress is increased vacuolization in the cytoplasm . Historically, the first biochemical marker of senescence that is still commonly used is increased activity of the β-galactosidase enzyme, usually termed "senescence-associated β-galactosidase (or SA-β-gal)" . While this was initially shown only in cultured cells' replicative senescence, later in vivo studies have reported β-gal activity in senescent-like cells in animals [10, 11].
The senescence of neurons and other central nervous system cells contributes to neurodegenerative diseases [12, 13]. Although senescence in postmitotic cells like neurons may sound counterintuitive, several in vivo and in vitro studies have reported senescent-like states identified by other markers of senescence in neurons and glia [12, 11, 13]. Premature senescence, especially SASP, has been implicated in various age-related diseases, including diabetes, osteoarthritis, atherosclerosis, cancer, Alzheimer's disease, and Parkinson's disease [12, 1]. As poor health brought about by aging creates a substantial economic burden , it is advantageous to reduce the rate of degenerative aging and increase global health.
Lithium, the first-line treatment against bipolar disorder, has been shown to have neuroprotective effects in clinical, pre-clinical, and in vitro studies . Mechanisms of lithium that might help exert its protective role include reducing pro-apoptotic/pro-senescence p53 and increasing anti-apoptotic Bcl-2 and brain-derived neurotrophic factor (BDNF) . The effect of lithium in senescence induction seems to depend on cell type, the senescence trigger, and the strength of pro-apoptotic vs. pro-survival signals . The primary anti-senescence action of lithium is the inhibition of glycogen synthase kinase 3 (GSK3) , a protein that helps p53 to exert its role . A study of replicative senescence in human fibroblasts shows that lithium, by its anti-GSK3 action, lets late-passage cells enter a reversible quiescence state instead of senescence . Lithium also reverses the rise in p53 and p21 protein expression and β-gal activity in late passage fibroblasts . In endothelial cells, lithium has a pro-senescence effect that is independent of its GSK3 inhibition, where it upregulates the expression of matrix metalloproteinase 1 (MMP1) , an enzyme often secreted by SASP cells . Similar results have also been observed in rat nucleus pulposus cells, where lithium again induced senescence and a rise in matrix metalloproteinases . A recent study on human iPSC-derived astrocytes (in a model without a senescence trigger) shows that low-dose lithium can prevent senescence and SASP markers, including β-gal activity and mRNA expression of p16, p21, and IL-1β .
MicroRNAs (miRNAs) are 22-nt members of short non-coding RNA species that regulate expression by binding to 3’-UTR of their target genes. Naturally, miRNAs that target pathways suppressing senescence tend to be upregulated in senescent cells [24, 25]. A well-studied senescence-associated miRNA is miR-34a , which suppresses telomerase activity and expression of SIRT1, a histone deacetylase that promotes cell survival . Another biomarker of senescence –primarily observed in oncogene-induced senescence– is the condensation of repressive epigenetic marks in chromatin into multiple focal points, termed senescence-associated heterochromatic foci (SAHF), which can be visually detected by DAPI staining or antibodies for methylations common in senescence such as H3K9me3 . As none of the senescence markers have been specific enough to identify the senescent state exclusively, most studies use multiple markers in combination .
In this study, to gain insight into how lithium can prevent the occurrence or ameliorate effects of premature senescence in neurons, we have used an H2O2-induced (i.e., ROS-mediated) premature senescence model, together with lithium treatment on the human neuroblastoma cell line SH-SY5Y. We show via Sudan Black B staining, β-Gal activity assay, and by detecting SAHFs, that lithium protects against senescence in SH-SY5Y cells. Moreover, we have observed that lithium has a modulatory effect on miR-34a-5p, a miRNA that is associated with aging [29, 30] and targets –among others- the mRNA of SIRT1 , a longevity associated protein that boosts mitochondrial function . Therefore, this study aims to examine Li's effects on oxidative stress-induced neuronal senescence and unravel the related signaling mechanisms.