Neuroprotective role of lithium-mediated rest restoration to ameliorate oxidative stres in down syndrome cell model

Caused by an extra human chromosome 21 (HSA21), Down syndrome (DS) is the most common genetic cause of cognitive impairment and early onset of aging. DS individuals usually manifest neurological deficits with neuropsychiatric disorders. Some of the overexpressed genes on HSA21 are responsible for antioxidative events that support and maintain neurogenesis and neuroplasticity. Dysregulation of these genes in DS individuals is likely to cause chronic oxidative stress, which is associated with neuropathologies seen in DS individuals. Repressor Element-1 Silencing Transcription factor (REST), a key player in the neuronal epigenome, has been reported to be dysregulated in the DS brain. Lithium, an FDA-approved drug for bipolar disorder, recently had been re-purposed for its neuroprotective roles against neurodegenerative diseases. Encouragingly, previous studies have proven the ability of lithium to restore REST in aging neurons. However, the neuroprotective role of lithium in targeting oxidative stress in DS is not extensively investigated. Hence, this study aimed to investigate the neuroprotective potential of lithium to restore nuclear REST and ameliorate oxidative stress in DS in vitro model. In this study, 3 pairs of disomic wildtype and trisomic DS-induced pluripotent stem cell (iPSC) lines were procured. The iPSCs were differentiated into neural stem cells (NSCs), and then into neurons. Subsequently, immunocytochemistry (ICC) was performed at each developmental stage to characterize the cells. Prior to lithium treatment, cell viability assay was performed on eight different dosages of lithium (2.5, 5.0, 7.5, 10.0, 12.5, 15.0, 17.5 and 20.0 mM), to establish the range of safety dosage of lithium treatment in neurons. The neurons were then treated with 10 mM of lithium for 24 hours. To evaluate the post-treatment effect, nuclear REST expression in both control and DS untreated and lithium-treated neurons were quantified via ImageJ software. The DCFDA intracellular reactive oxygen species (ROS) assay was performed to measure the cellular ROS level in the lithium-treated and stress-induced neurons. From the ICC results, it revealed a successful characterization of cells in each development stage. Interestingly, the nuclear REST expression level which was found to be significantly lower in the trisomic DS neurons, has been successfully restored after being treated with lithium. When challenged with hydrogen peroxide (H2O2), the DS neurons exhibited significantly elevated ROS compared to the untreated ones, demonstrating inherently heightened vulnerability of DS neurons towards oxidative stress. In addition, upon H2O2 exposure, the lithium treatment has successfully reduced the ROS levels in control neurons to near-baseline level, proving that lithium can indeed combat oxidative stress. Collectively, these findings showed a positive association between lithium treatment, REST restoration and oxidative stress. It is indeed exciting to discover that the REST dysregulation in DS neurons can be rescued via lithium treatment. Hence, this study suggested that repurposing lithium could be an impactful contribution towards the discovery of therapeutic strategies in DS neuropathologies.

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