In the present study, we show that fibrillary amyloid-beta caused a statistically significant decrease in cell viability and telomere length in primary cortical neurons; these effects were partially or totally reverted by the pre-treatment of cultures with lithium chloride for seven days (chronic treatment). Our data reinforce the evidence supporting the multi-modal neuroprotective effects of lithium against amyloid toxicity, as reported in distinct experimental models (Cuello et al. 2019), and further suggest that the overall neurotoxic effect of Aβ1-42, as depicted by a decrease in culture viability, is accompanied by impairments in telomere homeostasis. In this regard, lithium not only prevented amyloid-induced telomere shortening, but also was able to promote telomere elongation in spite of the presence of Aβ1-42. This effect was observed at all working concentrations of lithium, i.e., ranging from micromolar to therapeutic levels.
Abnormal telomere shortening leads to premature senescence, cell arrest and loss of physiological functions (Boccardi et al. 2015). Amyloid toxicity has been associated with telomere shortening mostly via mechanisms related to inflammation, oxidative stress and DNA degradation (Cai et al. 2013). However, a study by (Wang et al. 2015) showed that intracellular Aβ co-localizes in telomeres, inducing cell senescence and telomere shortening independently of the aforementioned mechanisms, supporting that the inhibition of telomerase activity may be an additional factor related to Aβ-induced cytotoxicity.
Telomerase, a key enzyme in the maintenance of telomere integrity, is regulated by its own catalytic subunit, the telomerase reverse transcriptase (TERT). Zhang et al. showed that the transcription of the hTERT gene is positively regulated by components of the Wnt/β-catenin pathway, leading to its increased expression. Whilst glycogen synthase-kinase 3-beta (GSK3β) phosohorylates β-catenin, leading to its degradation by the ubiquitin-proteasome pathway (Zhang et al. 2012a), its inhibition by lithium yields the retention of β-catenin and further downstream effects on the transcriptional activity of the hTERT gene (Cardillo et al. 2018b). Previous studies from our group and others indicate that lithium modulates multiple intracellular signaling cascades, with well-established effects such as inhibition of GSK3β (Boccardi et al. 2015); activation of Wnt/beta-catenin signaling (Zhang et al. 2012b; De-Paula et al. 2020); increase in the synthesis and release of neurotrophic factors, particularly brain-derived neurotrophic factor (BDNF) (De-Paula et al. 2016b); inhibition of apoptosis (Zhang et al. 2012b). Lithium also increases metabolic efficiency and respiratory rate in mitochondria (Jakobsson et al. 2017) and modulates inflammatory response (De-Paula et al. 2016c) and oxidative stress (Quiroz et al. 2010). Therefore, multiple biological effects of lithium may converge to the modulation of telomere homeostasis, in addition to the up-regulation of other neurotrophic/protective responses.
Our data in primary cortical neurons suggest that chronic lithium treatment alone may lead to a substantial increase in telomere length (4- to 7-fold, dose-dependent increments), although this effect failed to reach statistical significance, in view of the high variability of results. This effect would presumably reach statistical significance by increasing the number of replicates, which is unfortunately not available at the present time and represents a limitation of the study. In summary, our study shows that pre-treatment with lithium protected neurons against telomere shortening induced by Aβ1-42, restoring parameters similar to (or even higher than) baseline measures. Such effect was also observed with low working concentrations of lithium (0.02mM and 0.2mM), corroborating the notion that subtherapeutic or even lower (micromolar) concentrations of lithium may be effective in the modulation of biological responses (Forlenza et al. 2012; Cardillo et al. 2018b). In this regard, the present set of data suggests that the maintenance of telomere length is an additional mechanism by which lithium exerts neuroprotection, attenuating Aβ toxicity, with potentially relevant implications for the treatment and prevention of cognitive decline and dementia in AD.