Chronic Lithium Treatment Increases Telomere Length in Parietal Cortex and Hippocampus of Triple-Transgenic Alzheimer’s Disease Mice

Abstract

Telomere length (TL) is a biomarker of cell aging, and its shortening has been linked to several age-related diseases. In Alzheimer’s disease (AD), telomere shortening has been associated with neuroinflammation and oxidative stress. The majority of studies on TL in AD were based on leucocyte DNA, with little information about its status in the central nervous system. In addition to other neuroprotective effects, lithium has been implicated in the maintenance of TL. The present study aims to determine the effect of chronic lithium treatment on TL in different regions of the mouse brain, using a triple-transgenic mouse model (3xTg-AD). Eighteen transgenic and 22 wild-type (Wt) male mice were treated for eight months with chow containing 1.0 g (Li1) or 2.0 g (Li2) of lithium carbonate/kg, or standard chow (Li0). DNA was extracted from parietal cortex, hippocampus and olfactory epithelium and TL was quantified by real-time PCR. Chronic lithium treatment was associated with longer telomeres in the hippocampus (Li2, p = 0.0159) and in the parietal cortex (Li1, p = 0.0375) of 3xTg-AD compared to Wt. Our findings suggest that chronic lithium treatment does affect telomere maintenance, but the magnitude and nature of this effect depend on the working concentrations of lithium and characteristics of the tissue. This effect was observed when comparing 3xTg-AD with Wt mice, suggesting that the presence of AD pathology was required for the lithium modulation of TL.

Keywords

Alzheimer’s disease hippocampus lithium olfactory epithelium parietal cortex telomere transgenic mice

INTRODUCTION

Telomeres are DNA-protein complexes present on chromosomes extremities. In vertebrates, they consist of tandem repeats of the hexanucleotide TTAGGG. Telomeres shorten at each cell division due to the end replication problem [1]. When they are at the maximum of their shortening, the cell enters in senescence, extinguishing its replication potential and irreversibly losing its proliferative capacities [2]. This process is followed by differential protein synthesis and morphofunctional changes [3, 4], altering the tissue homeostasis and leading to the process known as aging. In fact, several age-related diseases have been related to altered telomere length (TL), such as cardiovascular diseases [5, 6], age-related diabetes [7, 8] and neurodegenerative disorders [9 –11] including Alzheimer’s disease (AD) [12 –15].

AD is the most common cause of dementia in elderly and is characterized by the presence of extracellular aggregates of amyloid-β peptides (senile plaques) and intracellular deposits of hyperphosphorylated tau protein (neurofibrillary tangles) in the brain. These alterations correlate with progressive neuronal dysfunction and degeneration, resulting in severe brain atrophy and cognitive impairments [16]. AD has a complex etiology, relying on interaction of several genetic and environmental factors, such as oxidative stress and inflammation [17], both of which can accelerate telomere shortening in peripheral blood leucocytes [14, 18].

Neurodegeneration in AD is established with different intensities in CA1 region of hippocampus [19, 20], association neocortex (parieto-temporal, inferolateral temporal and prefrontal cortices) [21, 22], and olfactory epithelium [23, 24]. However, as the vast majority of studies use peripheral DNA as matrix, little is known about how progressive neurodegeneration affects telomeres in different brain regions. On this matter, Lukens et al. [25] showed reduced TL in the cerebellum of AD patients when compared to healthy controls. On the other hand, Thomas et al. [26] found longer telomeres in the hippocampus of AD subjects.

Lithium is an important mood stabilizer and has long been used to treat bipolar disorder [27]. It seems to stabilize neural activities and promote neural plasticity and neuroprotection [28], though the mechanisms underlying these effects are poorly understood. Lithium has been proposed as a possible treatment for patients with cognitive impairment [29]. It is expected that its neuroprotective and neurotrophic modulation effects [30, 31] may prevent AD progression [32, 33]. The therapeutic window to warrant such effect is probably distinct from that used in mood disorders. Studies have shown relevant clinical and biological effects at sub therapeutic or even lower (micromolar) working concentrations of lithium [29, 31 , 35]. Martinsson et al. [36] observed longer telomeres in leukocytes of bipolar patients treated with lithium for 2 years, and a positive correlation between TL and treatment response. Additionally, lithium seems to have an important role on modulation of telomerase activity via glycogen synthase kinase 3 beta inhibition [37, 38].

Studies regarding TL in brain tissue are few and controversial, and may not correlate with findings in peripheral blood [12, 25]. Moreover, the effects of lithium on TL are equally scarce [36, 39]. In the present study we addressed TL in different brain structures in a transgenic mouse model of AD, and determined the effect of chronic lithium treatment on TL.

MATERIALS AND METHODS

Animal treatment

All animal experiments were approved by the local Ethics Committee (CAPPesqn° 1293/09) in accordance with Directive 2010/63/EU. Fifty-three young-adult male mice were purchased from Jackson Laboratory: of these, 27 were triple-transgenic AD mice, B6;129-Psen1^tm1MpmTg (APP_Swe,tau_P301L) 1Lfa/Mmjax (3xTg-AD; Tg), and 26 were wild-type (B6129SF2/J; Wt). Three-month old mice were kept under controlled environment at the Central Animal Facility at the Faculty of Medicine of the University of Sao Paulo (FMUSP), housed in standard cages (four or five animals per cage) sized 41×34×16 cm (length×width×height) at controlled temperature (22±1°C), relative humidity (50–60%) and 12 h light/dark cycle. 3xTg-AD and Wt mice were randomly divided into six subgroups: 1) Li0, 3xTg-AD (n = 9) or Wt (n = 9) mice fed with regular chow (treatment control groups); 2) Li1, 3xTg-AD (n = 9) or Wt (n = 9) mice fed with 1.0 g Li₂CO₃/kg chow; 3) Li2, 3xTg-AD (n = 9) or Wt (n = 8) mice fed with 2.0 Li₂CO₃/kg chow. Lithium-supplemented pellets were weekly prepared by Nutri Experimental^® (Brazil), by mixing lithium carbonate (Li₂CO₃; Merck) to regular chow. All groups had access to food and water ad libitum. Lithium-treated animals also received a bottle with 0.9% NaCl. Treatments lasted for 34 weeks, until animals reached 11 months of age (middle aged). Animal weight was measured monthly throughout the experiment. Forty animals reached the endpoint of the intervention, being 18 3xTg-AD and 22 Wt. Thirteen mice (24.5% of the initial sample) died during the intervention phase; of these, 9 were transgenic and 4 Wt mice (30% and 15% of each initial group respectively). Surviving animals were beheaded, parietal cortex, hippocampus and olfactory epithelium were dissected and immediately frozen at –80°C until use.

Measurement of serum lithium concentration

Serum lithium concentrations (mM) were determined two days before the end of treatment and animal sacrifice. Blood was collected from tail veins into heparin-coated tubes and the plasma was separated by centrifugation. Lithium concentration was determined by an ion analyzer (Electrolyte Analyzer 9180, Roche). Mean serum concentrations of lithium in each group are shown in Table 1. Both lithium diets yielded sub-therapeutic concentrations ranging from 0.11 mM (Li1) to 0.57 mM (Li2), whereas in mice fed with standard chow lithium levels were undetectable. Although serum concentrations of lithium tended to be higher among Li2 compared to Li1 mice, statistically significant differences were only observed among Wt animals (Table 1).

Table 1

Characterization of groups according to genetic characteristics of mice and intervention

Group	N (T0/T1)	Lithemia (mmol/L)	T0 weight (g)	T1 weight (g)	Weight change (g)
WtLi0	9/8	–	27.30±2.54	42.18±5.67	14.88±4.43
WtLi1	9/8	0.16±0.04	24.33±1.66	37.79±3.71	13.46±4.39
WtLi2	8/6	0.26±0.07	27.57±2.70	37.07±4.73	9.50±6.74
TgLi0	9/6	–	26.97±3.52	38.87±5.13	11.90±5,03
TgLi1	9/5	0.21±0.07	23.68±2.47	33.36±3.78	9.68±3.45
TgLi2	9/7	0.35±0.20	22.94±3.42	34.71±4.27	11.77±4.55

Wild type (Wt) and triple transgenic (3xTg-AD; Tg) were allocated into experimental (lithium intervention) and comparison groups, i.e., 1 g of Li₂CO₃/Kg of chow (Li1); 2 g of Li₂CO₃/Kg of chow (Li2); standard chow (untreated controls; Li0). Animal mortality (second column) is depicted by the number (N) of experimental animals at baseline (T0) and endpoint (T1). All values are presented as mean±standard deviations (SD). n, number of mice in each group; g, grams. Statistical analysis of the number of mice (baseline/endpoint) and weight change per group (Wt versus Tg): WtLi0/Li1/Li2 versus TgLi0/Li1/Li2, N.S.; or exposure to lithium treatment (lithium versus non-lithium): WtLi0 versus WtLi1/Li2, N.S.; TgLi0 versus TgLi1/Li2, N.S.

DNA extraction

DNA was extracted using AllPrep DNA/RNA mini kit (QIAGEN), a spin column-based nucleic acid purification method. Tissue was disrupted and homogenized in Buffer RLT Plus (supplied by the kit) with addition of 2-mercaptoethanol, to neutralize released RNAses. Post-homogenization extraction process was automatized using QIACube (QIAGEN), according to manufacturer’s instructions. By the end of the process, DNA yield is ready for downstream use. DNA samples were quantified with NanoDrop (Thermo) and evaluated.

Genomic DNA integrity

High DNA integrity is needed for optimal TL measurement [40]. These evaluations were performed by a capillary electrophoresis equipment (Fragment Analyzer, Advanced Analytical) along with DNF-488 High Sensitivity Genomic DNA Analysis kit (Advanced Analytical). The results were analyzed in PROSize 2.0 software (Advanced Analytical), which generates a virtual electrophoresis gel, fragments graphs and a Genomic Quality Number (GQN), varying from 0 to 10, that indicates DNA integrity. Only DNA samples with GQN values higher than 8.5 proceeded to qPCR. Nine samples were excluded from the analysis due to low genomic DNA integrity, being 3 from parietal cortex tissue (1 from Tg-Li2; 1 from Wt-Li0; 1 from Wt-Li1), and 6 from olfactory epithelium (1 from each group).

To measure TL, a qPCR analysis was performed according to the methodology previously described by Callicott and Womack [41]. The following primers were used: telomere (5’ CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT 3’, forward; 5’ GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT 3’, reverse; Exxtend) [41]; single copy gene 36B4, which codes a highly conserved ribosomal subunit used as reference for quantification (5’CAG CAA GTG GGA AGG TGT AAT CC 3’, forward; 5’ CCC ATT CTA TCA TCA ACG GGT ACA A 3’, reverse; Exxtend) [42]. qPCR was performed in the LineGene 9600 (Bioer) using Go Taq^®qPCRMasterMix (Promega), 20 ng of DNA, 300 nM of both telomere primers (for the telomere reaction), 300 nM and 500 nM for 36B4 forward and reverse primers, respectively (for the 36B4 reaction) in a final volume of 25 μL. All samples were analyzed in triplicates.

Standard curve for absolute TL quantification

Absolute quantification of TL was obtained by establishing two standard curves (telomere and 36B4) with HPLC-purified synthetized oligomers, as described by O’Callaghan and Fenech [43]. Designed oligomers were: (5’ TTA GGG 3’) x14 for telomeres (Exxtend); 5’ ACT GGT CTA GGA CCC GAG AAG ACC TCC TTC TTC CAG GCT TTG GGC ATC ACC ACG AAA ATC TCC AGA GGC ACC ATT GA 3’ for 36B4 (Exxtend). Six serial 1:4 dilutions were performed for each oligomer, ranging from 600 pg to 0.58 pg for telomere, and 9.37 pg to 0.0075 pg for 36B4. Results were shown as T/S ratio (where T stands for telomere and S for single copy gene 36B4).

Statistical analysis

Statistical analysis was conducted using R (version 3.2.0). Differences in TL between Tg and Wt mice within each brain region was accessed with one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. The R function used (TukeyHSD) already incorporated adjustment for multiple comparisons. Statistical differences were considered significant if adjusted p < 0.05.

RESULTS

Characterization of the sample at study endpoint

Of the initial sample of 53 mice, 40 reached the endpoint after 8 months of treatment. The attrition rate was higher among 3xTg-AD mice (30%) compared to Wt animals (15%), but the statistical analysis of these differences did not prove significant (OR 2.75, CI_95 % 0.7–10.4), probably due to the small sample size. Also, the chronic exposure to lithium was not statistically associated with a higher attrition rate (26% and 22% in Li1/Li2 versus Li0 groups, respectively). Therefore, based on the analysis of the attrition rate, we understand that animal mortality was predominantly related to the underlying AD pathology in 3xTg-AD mice rather than to the chronic exposure to lithium treatment. Animals that reached the endpoint of the intervention after 8 months had 148% increase in body weight, as expected, but without significant differences between groups. No statistically significant differences were observed in body weight changes, or in the comparison of baseline versus endpoint weight of Wt and Tg mice within the same treatment group, nor in lithium-treated versus control mice (Table 1).

Telomere length in parietal cortex

Figure 1 shows T/S ratio in the parietal cortex according to the exposure to lithium treatment in Wt and 3xTg-AD mice. In the absence of lithium treatment (Li0), we observed similar TL in Wt and 3xTg-AD (basal status) (p = 1.00). No significant differences were found comparing Wt animals treated with lithium-enriched chow (Li1 and Li2) to standard chow (Li0), indicating no effect of lithium on TL in non-transgenic animals (WtLi0 versus WtLi1, p = 1.00; WtLi0 versus WtLi2, p = 1.00). In 3xTg-AD mice, there was a 98% increase in telomere length in the Li1 group, and a 55% increase in the Li2 group, compared to controls (Li0, 3xTg-AD), but these differences were not statistically significant (TgLi0 versus TgLi1, p = 0.07; TgLi0 versus TgLi2, p = 0.54). Likewise, lithium dose regimens (Li1 versus Li2) were not associated with statistically significant effects on TL, considering Wt and 3xTg-AD groups separately (WtLi1 versus WtLi2, p = 1.00; TgLi1 versus TgLi2, p = 0.79). One way ANOVA with Tukey’s post hoc test indicated a significant difference between Wt and 3xTg-AD chronically treated with 1.0 g of Li₂CO₃/Kg of chow (Li1) (p = 0.04), with telomeres 109% longer in the transgenic group. This effect was not observed in mice treated with the higher dose of lithium (Li2) (p = 0.26).

Fig.1

Comparison of telomere length in parietal cortex of animals. Graphs show mean telomere length in Kb (T/S ratio) of each group and standard deviations. Tg, triple transgenic mice (3xTg-AD); Wt, wild type mice; Li0, treatment with standard chow; Li1, treatment with 1 g of of Li₂CO₃/Kg of chow; Li2, treatment with 2 g of Li₂CO₃/Kg of chow. Data show longer telomere length (^*p = 0.0375) in parietal cortex of TgLi1 mice when compared with WtLi1.

Telomere length in hippocampus

T/S ratio data in hippocampus of Wt and 3xTg-AD animals treated with lithium-enriched or regular chow are shown in Fig. 2. Like the findings in parietal cortex, TL in the hippocampal tissue was statistically similar in untreated (Li0) Wt and 3xTg-AD mice (basal status) (p = 0.86). The comparison of lithium-treated mice (Li1 and Li2) to controls (Li0) indicated no statistically significant differences in TL when the two groups of animals (Wt and 3xTg-AD) were analyzed separately (WtLi0 versus WtLi1, p = 1.00; WtLi0 versus WtLi2, p = 0.98; TgLi0 versus TgL1, p = 0.94; TgLi0 versus TgLi2, p = 0.56). Among transgenic mice, we observed a bi-directional effect on TL in lithium-treated mice compared to controls (Li0): lower lithium doses (Li1) were associated with a 19% reduction, and higher doses (Li2) with a 32% increase in TL in the hippocampus; however, these differences were not statistically significant (TgLi0 versus TgLi1, p = 0.94; TgLi0 versus TgLi2, p = 0.56). One way ANOVA with Tukey’s post hoc test indicated a statistically significant, two-fold increase (105%) in TL in 3xTg-AD mice chronically treated with 2.0 g of Li₂CO₃/Kg of chow (Li2) compared to Wt mice receiving the same treatment (p = 0.02).

Fig.2

Comparison of telomere length in hippocampus of animals. Graphs show mean telomere length in Kb (T/S ratio) of each group and standard deviations. Tg, triple transgenic mice (3xTg-AD); Wt, wild type mice; Li0, treatment with standard chow; Li1, treatment with 1 g of of Li₂CO₃/Kg of chow; Li2, treatment with 2 g of Li₂CO₃/Kg of chow. Data show longer telomere length (^*p = 0.0159) in hippocampus of TgLi2 mice when compared with WtLi2.

Telomere length in olfactory epithelium

Using the same statistical approach to address the effects of lithium on TL in the olfactory epithelium of 3xTg-AD and Wt mice, we did not find any statistically significant differences (p > 0.05) that might be attributed to the effect of treatment or to the genetic differences of the two animal groups. We observed a high dispersion of data, indicating inconsistency of this determination in this tissue. Figure 3 depicts T/S ratio data in olfactory epithelium in Wt and 3xTg-AD animals.

Fig.3

Comparison of telomere length in olfactory epithelium of animals. Graphs show mean telomere length in Kb (T/S ratio) of each group and standard deviations. Tg, triple transgenic mice (3xTg-AD); Wt, wild type mice; Li0, treatment with standard chow; Li1, treatment with 1 g of of Li₂CO₃/Kg of chow; Li2, treatment with 2 g of Li₂CO₃/Kg of chow.

DISCUSSION

Our data provide evidence that chronic lithium treatment is associated with longer telomeres in the brain tissue of triple transgenic mice as compared to wild type animals. Distinct effects were observed according to lithium doses and brain regions, i.e., at lower doses (1.0 g of Li₂CO₃/Kg of chow) in the parietal cortex, and at higher doses (2.0 g of Li₂CO₃/Kg of chow) in the hippocampus. No such effect was observed in the olfactory epithelium. These results indicate that the effect of chronic lithium treatment on cerebral telomere length seems to be restricted to transgenic mice when compared to Wt. The present set of data suggests that the presence of AD pathology (which is admitted to occur as a consequence of the triple genetic modification of these mice) is required for the biological effect of lithium on telomeres.

Lithium may have an important role on the modulation of telomerase, a ribonucleoprotein enzyme responsible for telomere maintenance [38 , 45]. Telomerase reverse transcriptase (TERT) is the catalytic subunit that regulates and limits telomerase activity [44, 46]. The regulation of the hTERT gene, which encodes for TERT in humans, occurs primarily at the transcription level and is dependent of β-catenin activity. β-catenin is degraded by glycogen synthase kinase-3 beta (GSK-3β), an important and highly expressed kinase in neurons that is inhibited by lithium. Therefore, the availability of β-catenin is determined by the activation state of GSK-3β, i.e., lithium inhibits GSK-3β leading to β-catenin retention (preventing its degradation), which yields the activation of hTERT transcription and further effect on the elongation of telomeres [37]. GSK-3β is known to be overactive in AD, being relevant to major pathological process such as of amyloid-β (Aβ) deposition [47] and tau hyperphosphorylation [48, 49]. Therefore, we acknowledge as a limitation of our study the fact that we did not determine GSK-3β activity along with telomere length in the present samples; another relevant control that may be relevant in future experiments would be the use of other GSK3 inhibitors in addition to lithium.

Moreover, lithium is known to modulate and up-regulate several neurotrophic factors in the brain, such as brain-derived neurotrophic factor (BDNF) [34]. BDNF is an important neurotrophin involved in synaptic plasticity, neuronal survival and dendritic branching [50, 51]. BDNF increases telomerase activity, and the suppression of TERT synthesis impaired the neuronal protection promoted by BDNF against glutamate-induced death in embryonic brain neurons [52]. The effects of BDNF are mainly mediated by the tyrosine kinase receptor-B, which activates several downstream signaling pathways [53]. Among these pathways, the PI₃K/Akt and MAPK/ERK₁/₂ cascades seem to be mediators of the neuroprotective effects of BDNF [54, 55]. Studies show that activation of both pathways can regulate telomerase at transcriptional and post-transcriptional levels [56 –59].

Lithium regulation of other important homeostatic factors (e.g., interleukins, phospholipase A2), is distinctly affected by its concentration [31, 60]. In a recent study conducted in our group, De-Paula et al. [34] reported increased BDNF secretion in primary cultures of hippocampal and cortical neurons chronically treated with subtherapeutic (0.02 and 0.2 mM) and therapeutic (2 mM) concentrations of lithium; this effect was more prominent in cortical neurons. Distinct brain regions may display tissue specificities and, therefore, differences in the capacity to respond to pharmacological stimuli [61]. This notion may provide support to our findings showing that lithium distinctly modulates TL according to dose and tissue involved.

Additionally, our data do not support the notion that diseased (AD-transgenic) mice might bear shorter telomeres when compared to healthy mice. In humans, as suggested by studies conducted in samples of AD patients and reinforced by a recent meta-analysis [12], the AD-pathology is associated with reduction in TL. It is noticeable, though, that most such studies were based on leucocytes drawn from peripheral blood. Results from studies that assessed the post mortem brain yielded inconsistent findings. According to Lukens et al. [25], no differences in TL were observed in the cerebellum of AD patients as compared to age-matched healthy controls. Thomas et al. [26] evidenced longer telomeres in the hippocampus of AD patients compared to healthy controls. In addition, it was shown that rats exposed to stress during development exhibit shortened telomeres in the hippocampus, but not on the pre-frontal cortex [62]. These studies suggest that TL findings in the peripheral blood may not be representative of the effects in the central nervous system, although the reasons for these discrepancies are poorly understood. Also, recent stereological studies in mature (eleven month old) 3xTg-AD mice indicated that, in spite of the occurrence of cerebral atrophy and reduced hippocampal volume, there was preservation of the total number of CA1 pyramidal neurons [63, 64], suggesting a self-preservation mechanism that may reflect in enhanced TL maintenance. Neuronal death is apparent in older 3xTg-AD mice (18 to 23 months old), indicating that the progressive severity of the disease overcomes the survival strategies that are engaged in earlier stages of the aging process [65]. In the present study, we used 11 month old mice, and it is possible that the AD pathology is not fully established, though it is stated that these animals already display progressive extracellular deposition of Aβ within the brain [66, 67] and present signs of cognitive impairment [68]. Taken together, these pieces of evidence suggest that aging itself may play a much more important role in telomere shortening than the inflammation subsequent to Aβ deposition in this animal model [69].

Finally, our data showed high dispersion of TL in olfactory bulb among the two animal groups. This tissue is the final destination of neuroblasts (neural stem cells) that originated from the subventricular zone and then migrate through the rostral migratory stream [70, 71]. The neuroblasts integrate into the network of the olfactory bulb, providing a high level of plasticity, important for rodent adaptation to new olfactory cues in the habitat. As TL is a cell marker of aging, and is affected by number of cell divisions, it is plausible that a tissue that accounts for both old and newborn cells may present with a larger variability in results. We speculate that TL analysis through a lysate of this tissue may not be the best approach.

To the best of our knowledge, this is the first study to determine TL in the brain tissue of AD triple transgenic mice. Our findings show a regulatory effect of lithium in maintenance and elongation of TL on the AD-injured brain, with regional specificities regarding the therapeutic window for this effect. In addition, we show no telomere shortening in the brain tissue of 11-month-old triple transgenic mice. These effects may be modulated by several cellular pathways (e.g., telomerase activity and TERT expression, BDNF signaling, GSK-β phosphorylation) and, therefore, future studies should encompass these mechanisms to further elucidate the protective properties of lithium on telomere homeostasis. For the time being, the present indications of the lithium effect on telomeres as a potential neuroprotective mechanism in AD should be regarded as preliminary.

Footnotes

ACKNOWLEDGMENTS

This study was supported by the State of Sao Paulo Research Support Foundation (FAPESP; Projects 09/52825-8, 2014/14211-6, 2016/01302-9) and National Council for Scientific and Technological Development (CNPq; Project 442795/2014-9). The Laboratory of Neuroscience (LIM-27), University of Sao Paulo, receives financial support from the Alzira Denise Hertzog Silva Benevolent Association (ABADHS).

Authors’ disclosures available online ().

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