Lithium Prevents Telomere Shortening in Cortical Neurons in Amyloid-Beta Induced Toxicity

doi:10.21203/rs.3.rs-260558/v1

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Lithium Prevents Telomere Shortening in Cortical Neurons in Amyloid-Beta Induced Toxicity

https://doi.org/10.21203/rs.3.rs-260558/v1

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posted 03 Mar, 2021

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Background

There is consistent evidence of the potential benefits of lithium attenuating mechanisms of neurodegeneration, including those related to the pathophysiology of Alzheimer’s disease (AD), and facilitating neurotrophic and protective responses, including maintenance of telomere length. The aim was to investigate the protective effect of the pre-treatment with lithium on amyloid-beta (Aβ)-induced toxicity and telomere length in neurons.

Methods

Cortical neurons were treated with lithium chloride at therapeutic and subtherapeutic concentrations (2mM, 0.2mM and 0.02mM) for seven days. Amyloid toxicity was induced 24 hours before the end of lithium treatment.

Results

Lithium resulted in 120% (2mM), 180% (0.2mM) and 140% (0.02mM) increments in telomere length as compared to untreated controls. Incubation with Aβ_1-42 was associated with significant reductions in MTT uptake (33%) and telomere length (83%) as compared to controls.

Conclusions

Lithium prevented loss of culture viability and telomere shortening in neuronal cultures challenged with Aβ fibrils.

Developmental Neuroscience

telomere

lithium

cortical neurons

amyloid-beta

Alzheimer’s disease

Telomeres are terminal DNA regions that lose 50-150pb fragments at each cycle of cell division. As a consequence, telomeres work as a ‘biological clock’, avoiding the endless proliferation of cells and establishing the replicative senescence (Alberts et al. 2014).Telomere shortening may also occur as a consequence of oxidative stress in the brain, along with protein oxidation and lipid peroxidation (Spilsbury et al. 2015; HUANG et al. 2016). Evidence of telomere shortening has been reported in age-associated diseases, including Alzheimer’s (AD) and related neurodegenerative disorders (Boccardi et al. 2015).

Pharmacological compounds that hinder or attenuate telomere shortening by oxidative stress and other mechanisms, therefore preserving cell function and survival of neuronal cells, may be helpful in the treatment or prevention of age-related neurodegenerative disorders. There is a substantial body of evidence from experimental and clinical models suggesting that lithium, has additional neurotrophic and protective properties(Forlenza et al. 2014; De-Paula et al. 2016c); these properties may be relevant to disease modification in certain neuropsychiatric and neurodegenerative diseases (Nunes et al. 2013), and AD (Forlenza et al. 2011; De-Paula et al. 2016c).

Therefore, the overall effect of lithium may have specific disease-modifying properties in AD by tackling core pathophysiologic mechanisms(Forlenza et al. 2011, 2019), and also deliver a myriad of unspecific yet potentially relevant clinical benefits by its multiple effects on cellular function, including the regulation of telomere homeostasis. In a recent study conducted in our group, using a triple-transgenic mouse model of AD, we were able to provide evidence of a dose-dependent, tissue-specific effect of long-term lithium treatment on the maintenance of telomeres in the presence of AD pathology (Cardillo et al. 2018a). In the present study, we address the protective effect of chronic lithium treatment on Aβ-induced telomere shortening in cultured cortical neurons.

Compliance with Ethical Standards

All animal experiments were approved by the local Ethics Committee (CAPPesq nº1293/09) in accordance with Directive 2018/63/EU, committee of University of Sao Paulo Medical School, Brazil, under the protocol number nº1293/09. All national guidelines were taken into consideration.

Primary cultures of cortical neurons:

Primary neuronal cultures were prepared according to the (De-Paula et al. 2015, 2016c, a) method. Treatment with beta-amyloid peptide (Aβ_1-42) was done for 24 hours from the 9th to the 10th DIC, as described elsewhere (Apel et al. 2009). To assess the effect of lithium on telomere length in amyloid-challenged cultures, we used working concentration of 5μM of the Aβ_1-42 peptide, previously incubated to turn into fibrillary state (Apel et al. 2009). After ten days in culture, neuronal viability was microscopically ascertained prior to harvesting the cultured cells upon completion of the incubations.

Assessment of cell viability:

The viability of neuronal cultures was quantitatively assessed by the MTT method [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], which estimates the percentage of living cells in a given substrate compared to an experimental control. Brieﬂy, 50μl of MTT solution (5 mg/ml in PBS) was added to each well (1x10⁵ cells/ml) and multi-well plates were incubated for 3h at 37ºC and 5% CO₂. Then, 500μl of 10% sodium-dodecyl sulfate (SDS) in 0.01N HCl was added. After overnight incubation, the absorbance was measured by spectrophotometry at 570 nm.

DNA extraction:

We used the AllPrepDNA/RNA mini kit (QIAGEN) following the manufacturer’s guidelines. The samples were quantified by spectrophotometry with the NanoDrop apparatus (ThermoFisher Scientific), and the samples were diluted to reach the final concentration of 25ng/μL of DNA. Amplification primers:The complete protocol of primer sequence (36B4, control gene, and telomere region), dilutions and reaction time are described in the Supplementary material 1.Statistical analysis:An independent‐sample Student's t‐test was used to determine the difference between MTT mean values in treatment conditions compared to respective controls. The analysis of telomere size was done according to the 2-ΔΔCT method adapted (Jendrzejewski et al. 2011), comparing (triplicates samples) the target region (telomeres) with the control region (36B4). Finally, we obtained the 2-ΔΔCT defining the control condition ratio as 100% to compare the telomere size with other experimental conditions.

Lithium treatment was associated with increased viability of cultured cortical neurons, as indicated by MTT assays. This effect was dose-dependent, although statistical significance was only achieved with the midle dose of lithium chloride (0.2mM), in which case there was a 34% increase in MTT uptake (Figure 1). Treatment with Aβ_1-42 (5μM) significantly impaired neuronal viability. This effect was partially (0.02mM, increased 32%) or totally (0.2mM and 2mM increased 57% and 65% respectively) reverted by the pre-treatment of cultures with lithium chloride for seven days, all such effects reaching statistical significance compared to the effect of Aβ_1-42 alone.

Figure 2 illustrates the analysis of telomere length in primary cultures of cortical neurons treated with lithium chloride (0.02mM; 0.2mM and 2mM) for seven days (DIC 4 through 11) and challenged with fibrillary Aβ_1-42, for 24 hours at the 11^th DIC, compared to controls. Telomere length was significantly increased as a consequence of lithium treatment alone, with increments of 140%, 180% and 120% (respectively), as compared to the untreated control. The incubation of neurons with 5μM Aβ_1-42resulted in a statistically significant, 46% reduction in telomere length (compared to untreated control). This effect was totally reverted by pre-treatment with lithium chloride, with final telomere lengths of greater magnitude that the one registered in non-disturbed neurons (controls).

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 Aβ_1-42, 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 Aβ_1-42. 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 Aβ_1-42, 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.

Funding:

This work was supported by: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Grant No. 466625/2014-6; National Institute for Biomarker Research in Neuropsychiatry (INBION), Grants Nos. 14/50873-3, 2016/01302-9, 2017/14418-8, 2018/08621-8 (FAPESP), 88887.463672/2019-00 PNPD/CAPES (Programa Nacional de Pós-Doutorado/Capes) and 465412/2014-9 (CNPq); Associação Beneficente Alzira Denise Hertzog da Silva (ABADHS).

Acknowledgments and FundingThis work was supported by: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Grant No. 466625/2014-6; National Institute for Biomarker Research in Neuropsychiatry (INBION), Grants Nos. 14/50873-3, 2016/01302-9 (FAPESP) and 465412/2014-9 (CNPq); Associação Beneficente Alzira Denise Hertzog da Silva (ABADHS).Conflict of Interest

All authors have no conflict of interest and are in accordance with the publication

Availability of data and material

The raw data for the realization of the average will be made available if requested by the authors.

Author's Contribution:

Rafael Martins Themoteo: Performed all cell cultures, data analysis and preparation of the manuscript.

Vanessa de Jesus Rodrigues de Paula: Performed all cell cultures, data analysis and preparation of the manuscript.

Nicole Kemberly Ribeiro Rocha: Performed all cell viability tests, data analysis and preparation of manuscript.

Helena Brentani: Data analysis and preparation of the manuscript.

Orestes Vicente Forlenza: Data analysis and preparation of the manuscript

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Lithium Prevents Telomere Shortening in Cortical Neurons in Amyloid-Beta Induced Toxicity

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Abstract

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Introduction

Materials And Methods

Results

Discussion

Declarations

References

Supplementary Files

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