Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging

Arthur Antonio Ruiz Pereira; Alessandra Macedo Pinto; Helena Nascimento Malerba; Mariana Toricelli; Hudson Sousa Buck; Tania Araujo Viel

doi:10.1371/journal.pone.0299534

. 2024 Apr 4;19(4):e0299534. doi: 10.1371/journal.pone.0299534

Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging

Arthur Antonio Ruiz Pereira ^1,², Alessandra Macedo Pinto ³, Helena Nascimento Malerba ^1,², Mariana Toricelli ², Hudson Sousa Buck ^2,⁴, Tania Araujo Viel ^1,^2,^3,^*

Editor: Kyung-Wan Baek⁵

PMCID: PMC10994667 PMID: 38574297

Abstract

Alzheimer’s disease (AD) is the most common neuronal disorder that leads to the development of dementia. Until nowadays, some therapies may alleviate the symptoms, but there is no pharmacological treatment. Microdosing lithium has been used to modify the pathological characteristics of the disease, with effects in both experimental and clinical conditions. The present work aimed to analyze the effects of this treatment on spatial memory, anxiety, and molecular mechanisms related to long-term memory formation during the aging process of a mouse model of accelerated aging (SAMP-8). Female SAMP-8 showed learning and memory impairments together with disruption of memory mechanisms, neuronal loss, and increased density of senile plaques compared to their natural control strain, the senescence-accelerated mouse resistant (SAMR-1). Chronic treatment with lithium promoted memory maintenance, reduction in anxiety, and maintenance of proteins related to memory formation and neuronal density. The density of senile plaques was also reduced. An increase in the density of gamma-aminobutyric acid A (GABA_A) and α7 nicotinic cholinergic receptors was also observed and related to neuroprotection and anxiety reduction. In addition, this microdose of lithium inhibited the activation of glycogen synthase kinase-3beta (GSK-3β), the classical mechanism of lithium cell effects, which could contribute to the preservation of the memory mechanism and reduction in senile plaque formation. This work shows that lithium effects in neuroprotection along the aging process are not related to a unique cellular mechanism but produce multiple effects that slowly protect the brain along the aging process.

Introduction

Memory impairment is still the most relevant and early symptom of Alzheimer’s disease among the cognitive domains. As a way to construct experiences and personality, memory is the capacity to acquire, save, and evoke information, which demands morphological changes in synapses [1]. The increase in strengthening and quantity of neuronal connexions is referred to as neuroplasticity and is the most accepted model to explain long-term memory formation. The mechanism for neuroplasticity occurs in the hippocampus and other brain regions due to synaptic changes that last for an extended period after gene transcription, resulting in an increase in the efficacy of synaptic transmission that can also be modulated by epigenetic mechanisms [2,3]. Although the molecular and electrophysiological mechanisms of long-term memory are well known, there is no efficient pharmacological strategy capable of treating or restoring the structural and functional loss that happens in AD and other neurodegenerative diseases.

Lithium was first described to help treat manic episodes by the psychiatrist John Cade in 1949 [4]. Later, it was approved for the treatment of mania and bipolar disorders by the American Agency Food and Drug Administration in 1970. Until today, it is considered an efficient drug for the treatment of manic episodes, depression, and mixed episodes [5]. More recent works show that lithium has a modulator effect in several psychiatric, neurological, and neurodegenerative processes, such as anxiety, Alzheimer’s disease, Parkinson’s disease, and ischemic stroke, among others [6–10].

Despite the evident benefits of lithium salts to bipolar disorders and other psychiatric and neurological diseases, the main limiting factor for its prescription by psychiatrists is the side effects that this metal produces, as reported recently [5]. In this way, our group and others have used lithium salts in low-dose, and the benefits were evident in clinical trials, mouse models of Alzheimer’s disease, and human cells [11–16]. In our previous works, we used the term "microdose lithium" as the lithium dose for humans was 300 μg administered daily (1.5 mg of lithium carbonate—[15]) and the dose of 5 μg/day/animal (0,25 mg/kg/day) for mice [14]. Many groups have shown that low doses of Lithium (15–300 μg) are efficient against dementia and have little side effects in long-term protocols [10,17–19]. However, these doses are not in current usage by physicians to prevent Alzheimer’s disease or as an adjuvant strategy to mild cognitive impairment treatment. Our hypothesis for this fact is that there are not enough experimental data with biological mechanisms to support its clinical use. In this way, the aim of the present work was to verify if the chronic use of lithium in low dose could maintain the density of proteins related to memory formation. Long-term spatial memory and anxiety were verified in senescence-accelerated mice prone(SAMP-8), a mouse model of accelerated aging and Alzheimer’s disease (Takeda, 1999; Morley et al., 2012). SAMP-8 lives in approximately 50% of normal mice (roughly 12 months). In this period, they present declines in learning and memory processing, emotional alterations (like anxiety and depression-like behaviors), abnormal circadian rhythms, and brain atrophy (Takeda, 1999). SAMR-1 animals, used as controls, have regular aging development. These results obtained in the present work further support the chronic use of this metal in low doses to delay the evolution of the behavioral effects of neurodegenerative processes.

Materials and methods

Animals

The accelerated aging mouse line comes from AKR/J animals. In 1968, at Kyoto University, after brother-sister crossings, some litters showed some different characteristics such as decreased activity, periophthalmic lesions, and early death, but without any evidence of malformation. This new phenotype presented was inherited by other generations [20]. Years later, in 1975, the litters that presented the phenotypes described above were isolated as "senescence-prone" parents, and the animals that did not have an altered life course were "senescence-resistant" parents [20].

Nowadays there are several lineages of SAMP animals; the isolation of these lineages occurred due to the different characteristics they presented. SAMP-8 animals present the hallmarks of accelerated aging, early death, memory and learning deficits, presence of amyloid plaques in the hippocampus, blood-brain barrier dysfunction, loss of neurons, and cortical atrophy, as described by other groups [21,22].

Two breeding families of senescence-accelerated mice prone (SAMP-8) and senescence-accelerated mice resistant (SAMR-1) were kindly donated by Dr. Eliana Akamine (ICB-USP), and a colony was established in the animal facility of the Department of Physiological Sciences of Santa Casa de Sao Paulo School of Medical Sciences, Sao Paulo, Brazil. 24 female SAMP-8 and 11 female SAMR-1 were maintained in groups of 2–6 in ventilated cages (Alesco, Brazil) with food and water ad libitum. Controlled room temperature (24–26°C), humidity (55%), and a 12-hour light/dark cycle were maintained in the animal facility, and mice were kept until they completed months of age (the dead-point established in our protocol).

All animal protocols were performed according to the ethics principle for the use of laboratory animals of the Brazilian Society of Laboratory Animal Science (SBCAL, Brazil) and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Publication number 86–23, Bethesda, MD). The proceedings were approved by the Animal Ethics Committee of the Santa Casa de Sao Paulo School of Medical Sciences (protocol number 12/2016), and all effort was made to minimize the number of animals and their suffering, following the 3R’s principle (Replacement, Reduction, and Refinement).

Treatment with microdose lithium

Lithium, in the form of lithium carbonate (Li₂CO₃), was dissolved in water and administered ad libitum. The dose used was the same as published by our research team before for mice treatment [13,14] and proportional to that used in a previous manuscript with Alzheimer’s disease’s patients [15]. The dose used in mice corresponds to 1.5 mg of lithium carbonate/day or 0.006 mEq of lithium/kg. The dose was adjusted according to the mice’s pharmacokinetics profile [23]. The final dose was 5ug/day/animal, corresponding to 0.25 mg/kg/day.

Experimental design

For behavioral experiments, it was used 11 female SAMR-1 and 24 female SAMP-8 divided into two groups: untreated (n = 12) and treated with Li₂CO₃ ad libitum (n = 12).

The timeline of the experimental design is shown in Fig 1.

Behavioral tests

Evaluation of spatial memory with Barnes maze

Spatial memory was evaluated using the Barnes maze, following the protocol of previous work (Baraldi et al., 2013; Balthazar et al., 2018; Malerba et al., 2021). Briefly, each animal was placed in the center of a white, 100 cm diameter board with 30 holes arranged radially. Under one of the holes, a dark box was established as an escape box. A fluorescent lamp was placed above the center of the arena, and a radio "out of frequency" was played. Animals had 5 min to find the escape box. Once inside the box, the lights and radio were turned off, and the animal was left quiet for 1 minute. The learning phase was performed when animals were two months old, and sessions were repeated once a day for five consecutive days (Acquisition Session). The task was repeated when animals were 5, 8, and 10 months old to assess memory recovery. The time to enter the escape box and the number of wrong holes explored before it entered the escape box were registered.

Evaluation of anxiolytic-like effects with the elevated plus maze

The Elevated Plus Maze comprised two open opposed arms (50 x 10 cm) crossed by two closed opposed arms (50 x 10 cm and 40 cm high walls). The maze was maintained 50 cm from the floor. The percentage of the number of entries in open arms over total entries in open and closed arms was registered. Also, the percentage of time spent in open arms concerning whole time was calculated. In addition, the number of head-dippings (when the animal dipped the head to explore the environment outside of the maze) was also registered. Animals were submitted to this test when they reached ten months of age.

Biochemical and histological analysis

After behavioral tests, mice were anesthetized with 4% isoflurane, and the brain and blood were collected and immediately frozen in dimethylbutane at -50°C and stored at -80°C until used. One hemisphere was used for western blot and ELISA analysis, whereas the other was used for tissue analysis.

Analysis of the density of proteins related to neuroplasticity and long-term memory

Hippocampal tissues were isolated and homogenized in lysis buffer containing 50 mM Tris–HCl (pH 7.4), 0.1% Triton X-100, 4 mM ethylene glycol tetraacetic acid (EGTA), 10 mM ethylenediamine tetraacetic acid (EDTA) and a tablet with protease inhibitors (Complete Protease Inhibitor Cocktail Tablets, Roche, Basileia, Switzerland) and phosphatase inhibitors (PhosSTOP Phosphatase Inhibitor Cocktail, Roche, Basileia, Switzerland). Samples were centrifuged (12,000 rpm, 15 min, 4°C), the supernatant was separated, and the total protein was determined with the Bradford method (1976). After that, proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. To ensure equal protein loading (Salinovich and Montelaro, 1986) and to evaluate the loading quality, the Ponceau S Red (Sigma-Aldrich, St-Louis, Missouri, USA) method was used to stain the membranes before probing with the antibodies.

Membranes were preincubated with Tris buffer saline (Sigma-Aldrich, St-Louis, Missouri, USA) with 0.1% Tween-20 (TBST) and 1% bovine serum albumin (BSA, Sigma-Aldrich, St-Louis, Missouri, USA) for 1h in room temperature. Following that, membranes were incubated overnight at 4°C with the following primary antibodies, diluted in the same buffer: β-actin (Cell Signaling, 4970, 1:1000); synaptophysin (Abcam, ab8049,1:500); postsynaptic density protein 95 (PSD95, Cell Signaling, 2507, 1:1000—a protein that stabilizes the synaptic expression of glutamate receptors); N-methyl-D-aspartate receptor subunit R2B (NMDA-R2B, Abcam, ab65783, 1:1000—a receptor for the neurotransmitter glutamate, involved in memory formation); α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (GluA2/3/4 subunits, Cell Signaling, 2460, 1:1000 another receptor for glutamate that is also involved in memory formation), gamma aminobutyric acid, subunit A receptor (GABA_A, Abcam, ab33299, 1:5000—a receptor for the neurotransmitter GABA, involved in modulation of memory and anxiety, among other functions), α7 nicotinic receptor (Millipore, AB15332, 1:1000—a receptor involved in the control of glutamate release and modulation of memory formation and recovery); calcium/calmodulin-dependent protein kinase IV (CaMK-IV, Cell Signaling, 4032, 1:1000—a protein activated by increases in the concentration of calcium and calmodulin as a consequence of glutamate receptors activation); glycogen synthase kinase-3 beta (GSK-3β, Cell Signaling, 12456, 1:1000—one of the principal pharmacological targets for lithium in the cells); phosphorylated glycogen synthase kinase-3 beta (pGSK-3β (Ser 9), Cell Signaling, 5558P, 1:1000—the increase in phosphorylation of GSK-3β inhibits this protein, leading to cell survival and neuroprotection). The membranes were then washed and incubated with a secondary biotinylated-antibody horseradish peroxidase-conjugated anti-IgG (Millipore, AP187P, 1:4000) for 2 h at room temperature. The immune complexes were detected using chemiluminescence (Western Lightning ECL Pro, Perkin Elmer, NEL120001EA) read in Image Quant Las 500 (GE). The density of immunoblotting was quantified with Image J software 1.8.0 (National Institutes of Health, Bethesda, Maryland, EUA).

Evaluation of neurons and astrocyte densities in the hippocampus

For immunofluorescence of neuronal and astrocytic labeling, the hemispheres of 5 animals per group were separated, and 20 μm samples were obtained in a cryostat (-22°C to -20°C, Microm HM 404N, Francehville, France). Brain sections were placed in gelatin-coated slides and kept at -80°C until use.

For neuronal labeling, samples were fixed with 100% acetone for 5 min, washed with PBS (pH 7.4) for 1 min, and incubated with 0.6% H₂O₂ in methanol for 20 min. After that, slides were washed in PBS for 5 min and incubated with normal serum for 1 hour. Following that period, samples were incubated with an anti-NeuN primary antibody (Millipore, ABN78, 1:500) for 1 hour again. After that, samples were washed with PBS for 5 min and incubated with the secondary antibody Alexa Fluor 597 (goat anti-rabbit IgG, Life Technologies, 1:200) for 2 hours. At the end of this incubation, samples were washed with PBS for 5 min and, soon after, incubated with an anti-GFAP antibody (Abcam, ab16997, 1:200) for 1 hour for astrocytic labeling. Following that, samples were washed for 5 min with PBS and incubated with the secondary antibody Alexa Fluor 488 (goat anti-rabbit IgG, Life Technologies, 1:200) for 2 hours. In the end, samples were washed again with PBS for 5 min and the slides were covered with Fluoroshild with DAPI (Sigma, F6057).

Images of the hippocampal regions granular layer of the dentate gyrus (GrDG), CA1 and CA3 were acquired using the Leica DMi8 inverted microscope and analyzed in the LasX software (Leica Microsystems). The filter used for markings made with NeuN was GFP with excitation at 488 nm and emission at 510 nm. The filter used for GFAP with excitation 561 nm and 594 nm emission. To visualize the cell nucleus, filter for DAPI with 345 nm excitation and 460 nm emission.

Images of six sections of each animal’s brain were acquired with a 10x objective lens in the region between -1.70 mm to 2.46 mm, starting from bregma [24]. Quantification was performed in that region using imageJ software 1.51j8 (National Institutes of Health, Bethesda, MD, United States).

Evaluation of the density of senile plaques

For the analysis of the presence and density of senile plaques, thioflavin S was used. Slides with 20 μm brain slices were warmed for 10 min to room temperature and washed with PBS containing 0.1% Triton X-100 (PBST) for 3 min. This process was repeated three times. After that, slides were immersed in 0.1% thioflavin S (Sigma T1892) diluted in PBST for 5 min. Following that, three washes for 3 min with PBS were done followed by immersion in 70% alcohol for 5 min. After a new wash, slides were closed with Fluoroshild with DAPI (Sigma, F6057) and stored at 4°C. Positive labelling signal was differentiated from the background by visualization in higher magnification and by the experience of the operator.

Images of hippocampal areas CA1, CA3 and the granular layer of the dentate gyrus (GrDG) were obtained in an inverted microscope (Leica, DMi8) and analyzed using LasX software (Leica Microsystems).

Statistical analysis

Data were expressed as means ± SEM (standard error of means) and analyzed with the Graph Pad Prism program (GraphPad Software, San Diego, CA, version 6). For the Barnes maze test, data from SAMR-1 and SAMP-8 groups were compared. After that, SAMP-8 and lithium-treated SAMP-8 groups were compared. Results from this test were analyzed using a two-way analysis of variance (repeated measures) followed by Bonferroni’s multiple comparison test. In molecular and tissue analysis, the homogeneity of sample variance was tested using D’Agostine and Pearson omnibus variety test and Shapiro Wilk normality test. As long as one of the tests indicated no variance among groups, one-way ANOVA was performed followed by Tukey’s multiple comparison test.

In all analyses only probability values (P) less than 0.05 were considered statistically significant.

Results

Behavioral experiments

Chronic microdose lithium treatment preserved memory of SAMP-8

Before the beginning of treatments, at 2 months of age, SAMR-1 (n = 11) and SAMP-8 (n = 24) were submitted to the Barnes maze to verify their spatial memory. During the five days of the “learning period”, a significant difference in the behavior between both groups was recorded [F_(1,155) = 91.28, p < 0.0001] which was reflected in a significant interaction among data along the time of observation [F_(4,155) = 8.70, p < 0.0001]. SAMR-1 learned where the escape box was, as a significant reduction of 82.5% [F_(8,32) = 3.43, p = 0.0058] was observed in the time to find this escape box on the 5^th day when compared to the 1^st day in the maze (Fig 2A). However, SAMP-8 did not succeed in entering the escape box during the established time, having a significant increase of 1.4 fold in time to enter the escape box on the 5^th day, when compared to the 1^st day [F_(23,92) = 1.80, p = 0.0256] (Fig 2A). The number of wrong holes explored before entering the right one was also significantly different between both groups along the “learning period” [F_(1,155) = 47.34, p < 0.0001]. SAMP-8 group explored significant more holes before finding the right one along the 5 days of the "learning period" [F_(23,92) = 10.33, p < 0.0001], whereas SAMR-1 had no difficulties to learn where the escape box was [F_(8,32) = 1.01, p = 0.4507] (Fig 2D).

Fig 2 — Spatial memory was evaluated in the Barnes maze and the time to enter the escape box (A-C) and the number of wrong holes explored (D-F) were recorded. Graphs A and D show the observation during learning time of groups SAMR-1 (n = 11) and SAMP-8 (n = 24), as indicated by the legend in D. Along the mice aging process, the behaviour of non-treated SAMP-8 was compared to SAMR-1 (B and E, as indicated by the legend in E) and SAMP-8 Li⁺ (C and F, as indicated by the legend in F). Along the 8 months’ period of observations, some animals have died (for different reasons) and the final number of animals in each group was 9 animals in the SAMR-1, 9 animals in the SAMP-8 and 7 animals in the SAMP-8-Li group. Symbols and vertical bars are means ± SEM. *: p < 0.05; **: p < 0.01; #: p < 0.0001.

After the “learning period”, the SAMP-8 group was randomly divided (n = 12 in each group) and the treatments began. Female SAMP-8 were treated with microdose lithium for 8 months (from 2 months until 10 months of age). The animals were resubmitted to the test at 5, 8 and 10 months of age. The difference in time to enter the escape box between SAMR-1 and SAMP-8 was maintained along with all expositions of mice to the Barnes maze [F_(1,17) = 174.60, p < 0001] (Fig 2B). The same was observed concerning the number of holes explored before the animal gets to the escape box, as SAMP-8 explored a significantly greater number of holes until they complete 5 months of age [F_(1,68) = 36.58, p < 0.0001]. This difference between SAMR-1 and SAMP-8 became not significant in the next two evaluations when animals were 8 and 10 months old (Fig 2E).

The treatment of SAMP-8 with lithium promoted a significant difference in behaviour [F_(1,74) = 5.08, p = 0.0272] that was also significant along time [F_(3,74) = 4.29, p = 0.0076] (Fig 2C), as SAMP-8-Li showed significant less time to enter in the escape box (Fig 2C) and also explored significantly less holes to find the escape box [F_(1,70) = 5.68, p = 0.0199] along the repeated expositions [F_(3,70) = 4.17, p = 0.0089] (Fig 2F), suggesting that they kept the memory of the escape box place. In comparison with SAMR-1 group, treatment with lithium made the SAMP-8 treated group (SAMP-8 Li⁺) make as fewer errors as SAMR-1 since 8 months of age, showing the efficacy of this long-term treatment.

Chronic microdose lithium treatment reduced the anxiety of SAMP-8

Concerning the evaluation of anxiety behavior, all animals were submitted to tests when they were 10 months old. In the elevated plus-maze, SAMP-8 presented a significant decrease of 33.8% in percentage of entries in the open arms (t = 2.83, dF = 16, p = 0.0120, Fig 3A) and also a significant decrease of 77.7% in time spent in open arms (t = 9.02, dF = 16, p < 0.0001, Fig 3B) when compared to SAMR-1, suggesting an increase in anxiety-like behavior. Chronic lithium treatment made no difference in the percentage of entries but increased the time spent in open arms (t = 2.32, dF = 14, p = 0.0357, Fig 3B). Concerning the head-dippings, a significant decrease of 83.5% in the number of movements made by SAMP-8 was observed, when compared to SAMR-1 (t = 4.87, dF = 16, p = 0.0002, Fig 3C), whereas the treatment with lithium significantly increased the behaviour of SAMP-8 (t = 3.41, dF = 14, p = 0.0043, Fig 3C), suggesting a decrease in anxiety-like behavior.

Fig 3 — The behavior was evaluated in the elevated plus maze as % of entries (A) and percentage of time (B) number in the open arms and head-dippings (C). Symbols are individual points and histograms and vertical bars are means ± SEM. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001. SAMR-1 (n = 9), SAMP-8 (n = 9), SAMP-8-Li (n = 7).

Low-dose lithium preserved molecular markers related to memory and neuroplasticity

To verify if chronic low-dose lithium changed the density of proteins in the hippocampus related to long-term memory formation, western-blot analysis for LTP markers was done in samples from the three groups. Initially, it was verified that the SAMP-8 group presented a significant reduction of 73.0% in the density of NMDA receptors compared to SAMR-1 samples (p = 0.0438). Chronic treatment with lithium maintained the density of those receptors as an increased density of 3.8 fold was detected when compared to non-treated SAMP-8, which made SAMP-8-Li comparable to SAMR-1 (Fig 4A). Regarding the other glutamate receptor AMPA, although a slightly similar profile was observed, no significant difference was observed among groups (Fig 4B). The decrease in density of NMDA receptor was followed by a decrease in its anchoring protein, PSD95, as in SAMP-8 mice samples it was verified a significant reduction of 46.7% in the density of this protein when compared to SAMR-1 ones (p = 0.0265). Treatment with lithium maintained the density of PSD95 similar to that observed in SAMR-1 samples and significantly higher in 2.3 fold when compared to SAMP-8 samples (p = 0.0019 (Fig 4C). As a consequence of activation of NMDA receptors and increase in intracellular calcium concentration, there is an increase in activation of CAMK-IV. With the decrease in the density of NMDA receptors, SAMP-8 mice also showed a significant decrease of 37.7% in the density of CAMK-IV (p = 0.0087) compared to SAMR-1. However, chronic treatment with lithium did not change the density of CAMK-IV in hippocampus samples, and SAMP-8-Li animals also showed a significant reduction of 45.5% in CAMK-IV density compared to SAMR-1 samples (p = 0.0019). This means that low-dose lithium treatment did not influence the expression and density of this protein (Fig 4D).

Fig 4 — Histograms and vertical bars are means ± SEM. *: p <0.05; **: p <0.01; b: p < 0.0001. The SAMR-1 group is represented in black, the SAMP-8 group is represented in red and SAMP-8-Li group is represented in blue. For these experiments 6 to 7 animals of each group were randomly assigned.

To verify if other neurotransmitter systems that modulate memory maintenance also changed in the SAMP model, α7 nicotinic acetylcholine receptors (nAChR) and GABA_A receptors’ densities were analyzed. In SAMP-8 animals, it was verified a significant decrease of 38.1% in α7 nAChR when compared to SAMR-1 samples (p = 0.0166). Animals treated with lithium presented a non-significant increase of 1.43 fold in the density of this receptor (p = 0.1069) and this was not different from SAMR-1 animals either. (Fig 4E). With GABA_A receptors, SAMP-8 presented a non-significant decrease of 32.7% in the density of this receptor when compared to SAMR-1 samples (p = 0.1946). SAMP-8 treated with lithium presented a significant increase of 1.73 fold in the levels of GABA_A receptors compared to untreated SAMP-8 (p = 0.0448) and became comparable to SAMR-1 levels (Fig 4F).

To analyse the density of synaptic terminals in the three groups, synaptophysin was quantified. It was verified a non-significant decrease of 24.4% in the density of this protein in SAMP-8 samples, when compared to SAMR-1 group (p = 0.1009). Treatment with lithium significantly increased in 1.5-fold the density of this protein in SAMP-8-Li samples compared to untreated SAMP-8 (p = 0.0090). This density was even higher than the one detected in SAMR-1 samples (1.13 fold), but this was not statistically significant (Fig 4G).

When it comes to the classical mechanism of action of lithium in the cells, i.e., phosphorylation of GSK-3β in Ser9, it was verified a significant decrease of 67.8% in the phosphorylation of this protein in SAMP-8, when compared to SAMR-1 (p < 0.0001). Treatment with microdose lithium promoted a significant increase of 1.87 fold in GSK-3β phosphorylation when compared to untreated SAMP-8 (p = 0.0201). However, SAMP-8 Li still presented a significant decrease of 39.9% in GSK-3β phosphorylation when compared to SAMR-1 samples (p 0.0013) (Fig 4H).

Effects of chronic microdose lithium treatment on neuronal, astrocytic and senile plaques density

The density of neurons and astrocytes was analyzed in hippocampal areas CA1, CA3 and the granular layer of the dentate gyrus (GrDG). In CA1 there was no difference in neuronal density when comparing tissue samples from SAMR-1 and SAMP-8 (t = 1.69, df = 8, p = 0.1289). Chronic treatment with lithium in microdoses slightly increased the neuronal density in SAMP-8 samples (t = 2.28, df = 8, p = 0.0516, Fig 5). Nevertheless in CA3 (Fig 6) and in GrDG (Fig 7) areas the density of neurons in SAMP-8 samples was significantly lower (CA3: t = 3.02, df = 8, p = 0.0165; GrDG: t = 3.30, df = 8, p = 0.0108) when compared to the density of neurons in the same areas in SAMR-1 samples. Treatment with lithium along the aging process maintained the density of neurons in CA3 and GrDG similar to those from the SAMR-1 group, and significantly higher when compared to untreated SAMP-8 samples (CA3: t = 4.64, df = 8, p = 0.0017; GrDG: t = 5.50, df = 8, p = 0.0006, Figs 6 and 7).

Fig 5 — Labeling of NeuN (green) and GFAP (red) in the CA1 hippocampal region of SAMR-1 (A–D), SAMP-8 (E–H) and SAMP-8 Li (I–L). Cellular nuclei were labeled with DAPI (blue). Images were obtained using an inverted DMi8 microscope (Leica, Wetzlar, Germany) with a 10× objective. Scale bar: 100μm. M and N: Percentage of NeuN and GFAP labeling in the CA1 area of the three groups. Histograms and vertical bars are means ± SEM of samples from 5 animals per group. *: p < 0.05.

Fig 6 — Labeling of NeuN (green) and GFAP (red) in the CA3 hippocampal region of SAMR-1 (A–D), SAMP-8 (E–H) and SAMP-8 Li (I–L). Cellular nuclei were labeled with DAPI (blue). Images were obtained using an inverted DMi8 microscope (Leica, Wetzlar, Germany) with a 10× objective. Scale bar: 100μm. M and N: Percentage of NeuN and GFAP labeling in the CA3 area of the three groups. Histograms and vertical bars are means ± SEM of samples from 5 animals per group. *: p < 0.05.

Fig 7 — Labeling of NeuN (green) and GFAP (red) in the GrDG hippocampal region of SAMR-1 (A–D), SAMP-8 (E–H) and SAMP-8 Li (I–L). Cellular nuclei were labeled with DAPI (blue). Images were obtained using an inverted DMi8 microscope (Leica, Wetzlar, Germany) with a 10× objective. Scale bar: 100μm. M and N: Percentage of NeuN and GFAP labeling in the GrDG area of the three groups. Histograms and vertical bars are means ± SEM of samples from 5 animals per group. *: p < 0.05; **: p < 0.01.

Concerning the density of astrocytes, no difference between SAMR-1 and SAMP-8 was observed in the three hippocampal areas analyzed. In the same way, long-term treatment with microdose lithium did not change the astrocytes’ density as well (Figs 5–7).

About the senile plaque density, one of the hallmarks of Alzheimer’s disease, a significant increase of 4.19 fold was observed in SAMP-8 samples, when compared to SAMR-1 (t = 3.45, df = 8, p = 0.0087). Animals treated with lithium presented a significant reduction of 54.9% of this density (t = 2.45, df = 8, p = 0.0398, Fig 8).

Fig 8 — Labeling was done with Thioflavin-S 1% (green) in the hippocampus of SAMR-1 (A–C), SAMP-8 (D–F) and SAMP-8 Li (G–I). Cellular nuclei were labeled with DAPI (blue). Images were obtained using an inverted DMi8 microscope (Leica, Wetzlar, Germany) with a 20× objective. Scale bar: 100μm. J: Quantification of the total number of plaques in the hippocampus. Histograms and vertical bars are means ± SEM of samples from 5 animals per group. *: p < 0.05; **: p < 0.01.

Discussion

Alzheimer’s disease (AD) is still the most important cause of dementia worldwide, but until today that is no cure for this devastating disease. In the last years, our research team showed the beneficial effects of the treatment with microdose lithium for memory maintenance and neuroprotection in a transgenic mice model of AD [14], but the cellular mechanisms of this low dosage of lithium are still lacking.

Microdose lithium is being considered as a disease-modifying strategy both in human and animal observations (Nunes et al., 2013, 2015a; Relaño-Ginés et al., 2018), which turns it into a hope for treatment of these diseases, along with lifestyle changes, like the practice of physical activity and the environmental enrichment, strategies that protect the brain from the development of AD and others neurodegenerative diseases (Toricelli et al., 2020; Malerba et al., 2021). In previous works our research team showed that effects of this low dosage are seen when a physiological/pathological disturbance is present, as is observed in AD mice models [14]. In this way, there was no effect of low dosage lithium in wild-type mice, unless for an anxiety reduction (Nunes et al., 2015). For this reason, in the present work, we investigated the effects of microdose lithium in SAMP-8, a mice model of accelerated aging, that is also being accepted as a model of dementia (Tomobe and Nomura, 2009; Akiguchi et al., 2017). In the present study, we only used female mice. Many studies well documented the higher prevalence of Alzheimer’s disease in women compared to men (for examples, please verify [25–30]. The prevailing hypothesis attributes this gender-specific discrepancy to the longer life expectancy of women, when compared to men. Similar to the observation made in male SAMP-8 and transgenic mice for AD in previous works (Nunes et al., 2015a; Malerba et al., 2021), female SAMP-8 also showed a reduction in learning skills as well as in long-term memory, when compared to their natural control SAMR-1, although some molecular mechanisms for the lithium protection were different in female and male animals. In other mice strains the age-related decline in estrogen sensitivity in their tissues, has been demonstrated to relate to exacerbated age-related pathologies as memory decline [31,32].

Effects on molecular mechanisms involved in memory formation

The behavioural observation with lithium treatment was corroborated with the analysis of proteins involved with the most accepted biological model of long-term memory formation, the long-term potentiation. In the present study, it was clear that those mechanisms are disrupted in female SAMP-8, which explains the low capacity of these animals to learn and memorize tasks. Interestingly, in previous work from our research group, no difference in NMDA receptors density was observed in hippocampus homogenates of male SAMP-8 mice when compared to SAMR-1 at 10 months of age [13]. In the present work, the density of both NMDA and PSD95 in the female SAMP-8 hippocampus were lower when compared to SAMR-1 samples. Although not measured in the present work, the relevance of female hormones as estrogen to NMDA binding in rodents’ hippocampus was already described [33–35]. In SAMP-8 animals treated with lithium, the increased density of NMDA receptors and PSD95 corroborates to the action of lithium in the LTP signaling mechanism mediated by glutamate. PSD95 is an important scaffolding protein in the glutamatergic synapse, mediating interactions between signaling proteins, such as AMPA and NMDA receptors (Sheng and Kim, 2011). In addition, it plays a fundamental role in the structure of excitatory synapses, binding the receptors to cytoskeletal proteins and mediating synaptic morphology and receptor traffic (De Wilde et al., 2016). Although no difference in the density of AMPA receptors was seen, the increase in NMDA receptors and PSD95 in lithium-treated animals may indicate an increase in efficiency and synaptic strength in glutamate-mediated signaling.

Effects on neuronal and synapses survivor and senile plaques’ densities

The preservation of neuronal density is a typical characteristic of the treatment with the lithium in low-dose, as we have been observed this effect in the hippocampus of 16-months-old transgenic mice model for AD [14] and 10-months-old male SAMP-8 [13] with the chronic treatment with lithium. Also the treatment of ex-vivo hippocampus slices with lithium in low-dose also evidenced the neuroprotection observed in vivo [36]. In the present work, the same neuroprotective effect was observed when female SAMP-8 were treated with the same lithium dose. This show a consistent effect of this treatment along the aging process of mice that reflects the maintenance of memory as observed in humans treated with similar doses [15]. Interestingly no effects on astrocytes (as observed with GFAP labeling) was observed with this treatment.

Increases in senile plaques are a hallmark of AD. Although some groups have not detected senile plaques in the mouse model used in the present work, our research team and others showed previously that Thioflavin S is able to stain senile plaques in SAMP-8, as evidenced by the observed fluorescence in brain tissue from these animals [13,37]. These newer investigation, including our own, suggest that Thiflavin S is capable of staining senile plaques in SAMP-8. Besides disrupting neuronal transmission, Aβ plaques can inactivate NMDA receptors, compromising the intracellular cascade for memory consolidation (Yin et al., 2015). In our study, it was observed a decrease in the density of CAMK-IV in SAMP-8, a Ca²⁺-dependent protein kinase that is part of the long-term memory formation pathway (Ahmed et al., 2000). SAMP-8 animals treated with lithium presented a significant decrease in senile plaques with the increase in expression of NMDA receptors density without, however, a change in CAMK-IV density, which means that the neuroprotective mechanism of lithium in memory formation does not necessarily affects all the proteins in that molecular pathway.

Another synaptic protein that was maintained with the lithium treatment was the synaptophysin. This was already observed in a transgenic model of AD treated with the same lithium dose, together with the increase in the density of the brain-derived neurotrophic factor (BDNF) (Nunes et al., 2015a). Corroborating with these observations and others made with the same microdose lithium in other AD animal models (Toricelli et al., 2020; Malerba et al., 2021), lithium treatment maintained the density of neuronal cells in female SAMP-8 animals, which reinforces its role in neuroprotection as mentioned by other groups in experimental and clinical observations (Palmos et al., 2021; Puglisi-Allegra et al., 2021).

Effects on α7 nicotinic acetylcholine and GABA_A receptors

Many neurotransmitter systems contribute to memory maintenance, including the cholinergic system which has a prominent role in the induction and modulation of LTP. Most studies show the loss of nicotinic receptors in AD patients, especially the α7 subtype (α7nAChR) that contributes to the modulation of long-term memory formation (Hampel et al., 2018; Alkadhi, 2021). Upholding of this system is one of the strategies for delaying the advance of AD in the initial and moderate phases of the illness with the use of cholinesterase inhibitors (Athar et al., 2021). In presynaptic neurons, the α7 nicotinic receptor induces the synthesis and release of glutamate, an essential neurotransmitter involved in neuroplasticity (Lozada et al., 2012; Haam and Yakel, 2017). In postsynaptic neurons, α7 nicotinic receptor activation induces the activity of CaMK-IV and transcription factors such as CREB. Recently our group showed that inhibition of the α7 nicotinic receptor prevented memory retrieval in mice submitted to an experimental model of neurodegeneration (Telles-Longui et al., 2019). In the present work, SAMP-8 presented a significant decrease in the density of α7nAChR when compared to SAMR-1, which may be related to the critical observed memory loss. However, lithium-treated SAMP-8 mice presented no loss in the density of this receptor, which could be a significant fact in memory maintenance.

Another receptor that was preserved with lithium treatment in the SAMP-8 hippocampus was the GABA_A receptor. This receptor is implicated both in memory maintenance and in anxiety mechanisms. Some works already pointed out the relationship between memory loss and the development of anxiety and depression. These conditions, if not observed and treated with care, may turn into risk factors for developing AD in old age (Sinoff and Werner, 2003; Kassem et al., 2017).

Lithium carbonate has been considered a successful strategy to control humor changes in bipolar human disorder. Other recent pieces of evidence pointed to the potential role of the metal lithium as an anxiolytic agent combined or not with other anxiolytic drugs (Walia et al., 2019) with implications for the mania phase of bipolar disorder (Chaves Filho et al., 2020), as well as to neuroprotection in the neurodegenerative process (Nunes et al., 2015b; Won and Kim, 2017; Morlet et al., 2018; Habib et al., 2019; Malerba et al., 2021). In the present work, long-term treatment with microdose lithium reduced the anxiety-like behavior and increased the density of GABA_A receptors, which may also have contributed to memory maintenance along the aging process of SAMP-8.

Inhibition of the GSK-3β

One of the neuroprotective mechanisms attributed to lithium involves the direct inhibition of the enzyme glycogen synthase kinase 3β (GSK-3β) (Klein and Melton, 1996). The inhibition of GSK-3β through phosphorylation in ser9 promotes the maintenance of synaptic function and axonal transport and decreases apoptosis and inflammatory markers mediated or not by NF-κB (Llorens-Martín et al., 2014; Toricelli et al., 2020).

In the present study, it was shown that SAMP-8 animals treated with lithium had an increase in GSK-3β inhibition when compared with SAMP-8 animals without treatment, confirming the activation of the classical mechanism. Besides, lithium-treated animals presented a reduction in the density of senile plaques. A previous study using human and mouse neuroblastoma cell culture suggests that inhibition of GSK-3β by AR-A014418 decreases APP cleavage by BACE1, reducing the production of Aβ peptides. This regulatory action of GSK-3β in the production of Aβ seems to be mediated by NF-κB, where the decrease in the signaling of this pathway reduces the gene expression of BACE1 (Ly et al., 2013). Corroborating with this information, in a previous study, our group showed that microdose lithium decreased the density of NF-κB and other inflammatory markers in the organotypic hippocampal culture of SAMP-8 animals (Toricelli et al., 2020).

In conclusion, chronic microdose lithium seems to act as a long-term memory and neuronal protector, promoting functional and structural maintenance of several pathways compromised in AD. The maintenance of memory observed in this and previous studies by our research team put pieces of a puzzle together to show that there is no unique mechanism capable of explaining the beneficial effects of this treatment but rather a combination of multiple neuroprotective mechanisms. Increases in BDNF levels, observed in male SAMP-8 [13] and in transgenic mice model for Alzheimer’s disease [14], synaptic and neuronal maintenance in hippocampal areas of CA1, CA3 and GrDG, as well as inhibition of GSK-3β (all seen in the present work), reduction of neuroinflammation observed in SAMP-8 ex vivo hippocampus [36] and reduction of senile plaques (also observed in this and previous works), are some of the observed effects that may, altogether, contribute to long-term behavioral observations. Also, changes seen in this study in GABA_A and α7 nicotinic receptors suggest an essential role of lithium in maintaining and restoring the balance in the formation of memory and reduction of anxious behavior. Therefore, this work brings a necessary contribution to the understanding of the mechanisms of microdose lithium treatment along the aging process to promote neuronal protection, memory maintenance, and reduction of anxiety, as abstracted in Fig 9.

Fig 9 — Many steps in the LTP formation were disrupted in SAMP-8 mice, while chronic treatment with lithium in microdose avoided the decrease in many protein densities, allowing memory maintenance in older mice.

Supporting information

S1 File. Images of original membranes with antibody and Ponceau reagent markings.

(PDF)

pone.0299534.s001.pdf^{(845.4KB, pdf)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

TAV 2016/07115-6 and 2020/14133-6 MT 2017/21655-6 The Sao Paulo Research Foundation (FAPESP) www.fapesp.br AARP, AMP, HNM received student fellowship from: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001 https://www.gov.br/capes/pt-br The sponsors did not play any role in the present work.

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PONE-D-23-36699Multiple mechanisms of microdose lithium protect behavioral deficits and molecular mechanisms for memory formation in SAMP-8, a mouse model of accelerated aging.PLOS ONE

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Reviewer #1: Arthur Antonio Ruiz Pereira et al., in their article "Multiple mechanisms of microdose lithium protect behavioral deficits and molecular mechanisms for memory formation in SAMP-8, a mouse model of accelerated aging.," apply chronic treatment with lithium to show its effects on spatial memory, anxiety, and molecular mechanisms related to long- term memory formation during the aging process of a mouse model of accelerated aging (SAMP-8).

Critique:

I. Introduction.

1. The authors used long-term microdose lithium to treat SAMP-8 mice as a model of late-onset AD. The authors admit that the hypothesis is not novel, and lithium was suggested as an anti-AD drug by several groups. The authors should reference the papers related to the topic.

a. Christopher Baethge (2020) stated that low-dose lithium is efficient against dementia (1)

b. Robert Haussmann et al. (2021) suggested that lithium is a therapeutic option in Alzheimer’s disease (2).

c. Shanquan Chen et al. (2022) performed a retrospective cohort study to show the Association between lithium use and the incidence of dementia (3).

2. The novelty of the study is not presented clearly. The authors mention their clinical study with low-dose lithium. Of note, short-term treatment with lithium did not demonstrate promising results (4). However, long-term treatment protocol has been applied already. Orestes V. Forlenza et al. (2019) investigated the clinical and biological effects of long-term lithium treatment in older adults with amnestic mild cognitive impairment. They ran a long-term (24-month-long) randomised clinical trial to show the effects of lithium (5).

Moreover, a systematic review by Sivan Mauer et al. (2014) demonstrated that Lithium, in both standard and trace doses, appears to have biological benefits for dementia (6). Shinji Matsunaga et al. (2015) performed a systematic review and Meta-Analysis of several clinical trials to conclude that lithium treatment may have beneficial effects on cognitive performance in subjects with MCI and AD dementia.

Therefore, we know that micro-doses of lithium (15-300 µg) are efficient against dementia and have little side-effects in long-term protocols.

Consequently, the authors must clearly state the hypothesis and experimental goals while considering animal welfare for long-term experiments.

II. Methods.

1. Treatments. The authors must justify the dose applied and present the calculations. Unfortunately, the reference they provide does not have it.

2. Experimental design. The authors use only females in their study. Please clarify and justify it.

3. Behavioral tests. Barnes maze. The authors apply a very unusual protocol for the Barnes maze, which generally assesses spatial memory acquisition and recall. And here. The authors repeated the task when animals were 5, 8, and 10 months old to assess memory recovery. So, please define the term “memory recovery” and present a reference showing the method.

4. Evaluation of the density of senile plaques with Thioflavin S. The authors used a solution of thioflavin S (Sigma T1892) to stain amyloid plaques in SAMP-8 mice. The same method has been used by numerous labs in the transgenic AD animals. However, aged SAMP-8 mice, in contrast to AD mice, do not present amyloid-beta plaques detectable with thioflavin S (7). Please explain or apply another staining (IHC) for amyloid to show changes.

III. Results.

1. Fig. 2. The authors applied repeated measures ANOVA to show the effects of treatment with low-dose lithium on spatial memory, however, the presentation of the results is not correct. You cannot demonstrate statistical significance with repeated measures ANOVA by the way you did it (at the points of measurements) instead, you must analyze the entire plot and compare the curves.

2. Fig. 8. You are only displaying artifacts, no plaques. Look at reference #7 fig. 3.

IV. Discussion. There is no molecular mechanism explaining the effects of low-dose lithium.

I propose a theory that could be a good fit.

1. In 1986 Adlercreutz et al. showed that Lithium lowers renal, cardiac and splenic ornithine decarboxylase activity (ODC) in mice (8).

2. In 1992 Gad M. Gilad Gad Gilad et al. demonstrated that chronic lithium treatment suppresses ornithine decarboxylase (ODC) activity in the brain (9).

3. Kan et al. (2015) convincingly demonstrated that pharmacologic disruption of the arginine utilization pathway by an inhibitor (DMFO) of arginase and ornithine decarboxylase protects the mice from AD-like pathology (10).

4. Polis et al. (2018) showed that arginase inhibition reverses cognitive decline and synaptic loss in a murine model of Alzheimer’s Disease (11).

Accordingly, to enhance the quality of their work and deliver a unique and noteworthy research paper, the authors must employ additional techniques to investigate the hypothesis based on the findings of various groups about the influence of lithium salts on arginase and ODC expression levels and activities. It might be WB, IHC, or another method. I believe you have the lysates and the brains to run an experiment.

V. It is necessary to improve the quality of scientific English language. This includes correcting any errors in spelling, grammar, and punctuation to make the text clearer and more precise.

References:

1. Baethge, C. Low-dose lithium against dementia. Int J Bipolar Disord 8, 25 (2020). https://doi.org/10.1186/s40345-020-00188-z

2. Haussmann R, Noppes F, Brandt MD, Bauer M, Donix M. Lithium: A therapeutic option in Alzheimer's disease and its prodromal stages? Neurosci Lett. 2021 Aug 24;760:136044. doi: 10.1016/j.neulet.2021.136044. Epub 2021 Jun 10. PMID: 34119602.

3. Chen S, Underwood BR, Jones PB, Lewis JR, Cardinal RN. Association between lithium use and the incidence of dementia and its subtypes: A retrospective cohort study. PLoS Med. 2022 Mar 17;19(3):e1003941. doi: 10.1371/journal.pmed.1003941. PMID: 35298477; PMCID: PMC8929585.

4. Hampel H, Ewers M, Bürger K, Annas P, Mörtberg A, Bogstedt A, Frölich L, Schröder J, Schönknecht P, Riepe MW, Kraft I, Gasser T, Leyhe T, Möller HJ, Kurz A, Basun H. Lithium trial in Alzheimer's disease: a randomized, single-blind, placebo-controlled, multicenter 10-week study. J Clin Psychiatry. 2009 Jun;70(6):922-31. PMID: 19573486.

5. Forlenza, O., Radanovic, M., Talib, L., & Gattaz, W. (2019). Clinical and biological effects of long-term lithium treatment in older adults with amnestic mild cognitive impairment: Randomised clinical trial. The British Journal of Psychiatry, 215(5), 668-674. doi:10.1192/bjp.2019.76

6. Mauer S, Vergne D, Ghaemi SN. Standard and trace-dose lithium: a systematic review of dementia prevention and other behavioral benefits. Aust N Z J Psychiatry. 2014 Sep;48(9):809-18. doi: 10.1177/0004867414536932. Epub 2014 Jun 11. PMID: 24919696.

7. Porquet D, Andrés-Benito P, Griñán-Ferré C, Camins A, Ferrer I, Canudas AM, Del Valle J, Pallàs M. Amyloid and tau pathology of familial Alzheimer's disease APP/PS1 mouse model in a senescence phenotype background (SAMP8). Age (Dordr). 2015 Feb;37(1):9747. doi: 10.1007/s11357-015-9747-3. Epub 2015 Feb 8. PMID: 25663420; PMCID: PMC4320125.

8. Adlercreutz C, Rosengren E, Uvelius B. Lithium lowers renal, cardiac and splenic ornithine decarboxylase activity in mice. Experientia. 1986 Apr 15;42(4):409. doi: 10.1007/BF02118632. PMID: 3007201.

9. Gilad GM, Gilad VH, Wyatt RJ, Casero RA Jr. Chronic lithium treatment prevents the dexamethasone-induced increase of brain polyamine metabolizing enzymes. Life Sci. 1992;50(18):PL149-54. doi: 10.1016/0024-3205(92)90289-2. PMID: 1313939.

10. Kan MJ, Lee JE, Wilson JG, Everhart AL, Brown CM, Hoofnagle AN, Jansen M, Vitek MP, Gunn MD, Colton CA. Arginine deprivation and immune suppression in a mouse model of Alzheimer's disease. J Neurosci. 2015 Apr 15;35(15):5969-82. doi: 10.1523/JNEUROSCI.4668-14.2015. PMID: 25878270; PMCID: PMC4397598.

11. Polis B, Srikanth KD, Elliott E, Gil-Henn H, Samson AO. L-Norvaline Reverses Cognitive Decline and Synaptic Loss in a Murine Model of Alzheimer's Disease. Neurotherapeutics. 2018 Oct;15(4):1036-1054. doi: 10.1007/s13311-018-0669-5. PMID: 30288668; PMCID: PMC6277292.

Reviewer #2: Comments to Authors

In the present manuscript, “Multiple mechanisms of microdose lithium protect behavioral deficits and molecular mechanisms for memory formation in SAMP-8, a mouse model of accelerated aging”, authors examined the effects of microdose lithium on behavioral abnormalities, anxiety, plaque formation and neuroplasticity in SAMP-8 mice. The study is planned and executed well and findings are interesting and relevant to the current research in the area of Alzheimer’s disease. However, the following minor changes are required for the further improvement of the manuscript.

Title

1. The title is vague kindly rewrite it to make it clearer.

2. The word “mechanism” is used twice in the title, please avoid repetition.

Abstract

1. Alzheimer's disease (AD) is the most common affection.

Affection does not seem a suitable word.

Introduction

• Kindly state the objective/s of the study at the end of the introduction section.

Methods

Evaluation of neurons and astrocyte densities in the hippocampus: Page 10

1. Images of the hippocampal regions: GD, CA1, and CA3 were acquired using the Leica DMi8 177 inverted microscope.

What is meant by GD here? Is dentate gyrus (DG)?

Results

1. Figure 2A, B, and C: Provide the figure legend to represent the groups such as SAMR-1 and SAMP-8

2. Figure 2: Compare the results of SAMP-8 Li+ with SAMR-1 to further clarify the effects of lithium microdose.

3. Figure 4D: What could be the possible reason for the decrease of CaMK4 following low-dose lithium treatment? Please explain.

Reviewer #3: This paper addresses the effects of microdose lithium carbonate treatment (2-10 months of age) on Alzheimer-related brain phenotypes in the SAMP-8 mouse model of aging. Cognition was assessed using the Barnes maze longitudinally. Elevated plus maze was used to assess anxiety related behaviors. After treatment, brains were collected and stained for neurons, astrocytes, Thio-s and lysates were used to probe synaptic proteins and GSK-3Bser9 phosphorylation.

While the study is important and well written, there are major issues that call into question the reliability of the data presented.

1. One major issue is that it is impossible to know how much lithium each individual mouse received, as these may vary across the cage. 5 ug/day/animal. Is the entire mixture consumed daily? If ad libitum, this suggests the dose fluctuates.

2. More details on SAMR-1 and SAMP-8 mice are needed. What are these mice? How are they generated?

3. Line 89. There should be a SAMR-1 lithium treated group.

4. Line 152, 153, etc: typo in phosphorylated

5. Line 164: typo

6. Line 183: Concern that this method is not reliable. The detection range should not be altered between slices.

7. Line 184: relations should be relation

8. Line 192: typo

9. Line 204: Overall there are issues with the statistics. 2way ANOVA described in methods (with repeated measures). Looks like in some places only a t-test is used. The methods describe a 2way ANOVA with molecular assays, but there are not enough levels in the SAMR-1, because there was no lithium treated group, so it is unclear what the two-way ANOVA was comparing. P values of 0.05 are listed in the methods but not used if tests used were actually multiple t-tests.

10. Fig 2A, 2D: It is unclear why after day 2, latency to enter escape box and number of errors are worse than baseline for the SAMP-8 mice.

11. Figure 2C: If the SAMP-8 mice were divided to receive Li treatment, why do the SAMP-8 Li+ group already look better? Should have checked that the groups were balanced/equal before continuing with treatment.

12. Fig. 3: All individual data points should be shown.

13. Line 283: typo

14. Line 278: Full uncropped blots should be provided in supplement. Additionally, a single lane from one of each mouse is not sufficient.

15. Figure 4: It is unclear how these differences are tested. It should be one way ANOVA with appropriate post-hoc test. Appear to be multiple t-tests.

16. Line 326: abbreviation “a” appears as “b” in Fig 4H.

17. Line 328: n of 6 in 3C and F but legend says n of 7.

18. Overall, the use of WB for quantitation of synaptic proteins is antiquated because of its low sensitivity. Immunofluorescent staining and confocal imaging of serial sections with appropriate co-stains is more accurate.

19. It appears that the Actin band is re-used in Figs 4A,B,E,F and G. While it might be possible to probe multiple proteins of different molecular weights on the same blot, use secondaries with separate fluorescent channels, or even strip the plot to reprobe, the shape of the bands should be similar. In this case, Fig 4B is concerning because while the Actin bands are catching upward, the AMPA bands are catching downwards, suggesting they are from different gels/blots. The Actin and AMPA bands must be from the same blot. Showing the full blot would clear this up.

20. Fig. 5,6,7: Neuron number cannot be quantified by NeuN percent area. Use stereology.

21. Fig. 8: Should be labeled thio-s not AB. Thio-S can stain many other structures beyond just AB. Moreover, the Thio-s staining is not convincing.

22. Figure 8J: Individual data points from 5 mice should be shown.

23. Line 405: typo.

24. Line 406: this quote should be removed. This idea has since been dismissed. The increased prevalence for AD in women is equal to men at ages where the death rate is equal. Mice, which do not undergo menopause, do however show a lifeline decline in estrogen sensitivity in their tissues and this has been shown to relate to exacerbated pathology.

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Reviewer #1: Yes: Baruh Polis

Reviewer #2: Yes: Sanila Amber

Reviewer #3: No

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Attachment

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pone.0299534.s003.pdf^{(161.2KB, pdf)}

PLoS One. 2024 Apr 4;19(4):e0299534. doi: 10.1371/journal.pone.0299534.r003

Author response to Decision Letter 0

12 Jan 2024

Dear Editor and Reviewers,

We would like to express our sincere gratitude for your valuable feedback and thoughtful comments on our article titled " Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging". We truly appreciate the time and effort you have dedicated to reviewing our work and providing constructive suggestions for improvement.

First and foremost, we would like to address each of the points you raised individually:

Reviewer #1:

Arthur Antonio Ruiz Pereira et al., in their article "Multiple mechanisms of microdose lithium protect behavioral deficits and molecular mechanisms for memory formation in SAMP-8, a mouse model of accelerated aging.," apply chronic treatment with Lithium to show its effects on spatial memory, anxiety, and molecular mechanisms related to long- term memory formation during the aging process of a mouse model of accelerated aging (SAMP-8).

Critique:

I. Introduction.

1. The authors used long-term microdose lithium to treat SAMP-8 mice as a model of late-onset AD. The authors admit that the hypothesis is not novel, and Lithium was suggested as an anti-AD drug by several groups. The authors should reference the papers related to the topic.

a. Christopher Baethge (2020) stated that low-dose Lithium is efficient against dementia (1)

b. Robert Haussmann et al. (2021) suggested that Lithium is a therapeutic option in Alzheimer's disease (2).

c. Shanquan Chen et al. (2022) performed a retrospective cohort study to show the Association between lithium use and the incidence of dementia (3).

Answer: We thank the Reviewer for the suggestion. The authors cited the references (lines 53 and 54) together with the explanation of the hypothesis and experimental goals asked in item 2 below.

2. The novelty of the study is not presented clearly. The authors mention their clinical study with low-dose Lithium. Of note, short-term treatment with Lithium did not demonstrate promising results (4). However, long-term treatment protocol has been applied already. Orestes V. Forlenza et al. (2019) investigated the clinical and biological effects of long-term lithium treatment in older adults with amnestic mild cognitive impairment. They ran a long-term (24-month-long) randomised clinical trial to show the effects of Lithium (5).

Therefore, we know that micro-doses of Lithium (15-300 µg) are efficient against dementia and have little side-effects in long-term protocols.

Consequently, the authors must clearly state the hypothesis and experimental goals while considering animal welfare for long-term experiments.

Answer: We thank the Reviewer for the observation, and we are very happy that there is no doubt about the clinical effects of low-dose lithium. However, this low dose is not in current use by physicians to prevent Alzheimer’s disease or as an adjuvant strategy to MCI treatment. Our hypothesis for this fact is that there is not enough pre-clinical experimental data that supports the clinical use of it. In this way, our group is working on many projects to add information that can give this support. We add this comment to the text (lines 52 to 59).

II. Methods.

1. Treatments. The authors must justify the dose applied and present the calculations. Unfortunately, the reference they provide does not have it.

Answer: Lithium, in the form of lithium carbonate (Li2CO3), was dissolved in water and administered ad libitum. The dose used was the same as published by our research team before for mice treatment (Nunes et al., 2015; Malerba et al., 2021) and proportional to that used in a previous manuscript with Alzheimer’s disease patientes (Nunes et al., 2013). The dose used in mice corresponds to 1.5 mg of lithium carbonate/day or 0.006 mEq of lithium/Kg. The dose was adjusted according to the mice's pharmacokinetics profile (Rouaud et al., 1993; Wood et al., 1986). The final dose was 5ug/day/animal, corresponding to 0.25 mg/kg/day. The treatment lasted for a total of 8 months (from 2 to 10 months of age). The authors added this information to the text (lines 107-113).

2. Experimental design. The authors use only females in their study. Please clarify and justify it.

Answer: The rationale for exclusively utilizing female subjects in our study is explicated on lines 442 to 453. In this investigation, we opted to focus solely on female mice due to the well-documented higher prevalence of Alzheimer's disease in women compared to men. This observation has been supported by numerous studies, including those conducted by Niu et al. (2017), Beam et al. (2018), MacCarthy and Raval (2020), Meng et al. (2023). The prevailing hypothesis attributes this gender-specific discrepancy to the longer life expectancy of women compared to men. This argument and the references were added to the text (Lines 442-453).

3. Behavioral tests. Barnes maze. The authors apply a very unusual protocol for the Barnes maze, which generally assesses spatial memory acquisition and recall. And here. The authors repeated the task when animals were 5, 8, and 10 months old to assess memory recovery. So, please define the term "memory recovery" and present a reference showing the method.

Answer: We appreciate the reviewer's attention to the Barnes maze protocol employed in our study. While we understand the concern regarding the characterization of our protocol as "unusual," we would like to emphasize that our research group has previously utilized a similar Barnes maze protocol in other published works. For further details and reference, please consult the following articles, which are also cited in the text:

Nunes MA, Schöwe NM, Monteiro-Silva KC, Baraldi-Tornisielo T, Souza SI, Balthazar J, Albuquerque MS, Caetano AL, Viel TA, Buck HS. Chronic Microdose Lithium Treatment Prevented Memory Loss and Neurohistopathological Changes in a Transgenic Mouse Model of Alzheimer's Disease. PLoS One. 2015 Nov 25;10(11):e0142267. doi: 10.1371/journal.pone.0142267.

Malerba HN, Pereira AAR, Pierrobon MF, Abrao GS, Toricelli M, Akamine EH, Buck HS, Viel TA. Combined Neuroprotective Strategies Blocked Neurodegeneration and Improved Brain Function in Senescence-Accelerated Mice. Front Aging Neurosci. 2021 Aug 23;13:681498. doi: 10.3389/fnagi.2021.681498.

As for the term "memory recovery," in the context of our study, it pertains to the evaluation of spatial memory at different time points during the aging process (5, 8, and 10 months old). The repeated administration of the Barnes maze task allows us to assess the potential recovery or decline in spatial memory performance over time, providing insights into age-related changes in memory function.

Answer: We appreciate the Reviewer's diligence in examining our methodology for the evaluation of senile plaques with Thioflavin S. As correctly pointed out, the use of Thioflavin S to stain amyloid plaques is a well-established method commonly applied in transgenic AD animal models. While it is noted that aged SAMP-8 mice, in contrast to AD mice, were previously reported not to present amyloid-beta plaques detectable with Thioflavin S (Reference 7, Pouquet et al., 2015), we would like to draw attention to more recent findings. In a study conducted by our group and others, including Malerba et al. (2021) and Guo et al. (2022), Thioflavin S was able to stain senile plaques, as evidenced by the observed fluorescence in brain tissue from SAMP-8 animals. These newer investigations, including our own, suggest that Thioflavin S was capable of staining senile plaques in SAMP-8 mice. We have incorporated this latest reference into the manuscript (line 489-490) to reinforce the validity of our results and the chosen methodology.

III. Results.

1. Fig. 2. The authors applied repeated measures ANOVA to show the effects of treatment with low-dose Lithium on spatial memory, however, the presentation of the results is not correct. You cannot demonstrate statistical significance with repeated measures ANOVA by the way you did it (at the points of measurements) instead, you must analyze the entire plot and compare the curves.

Answer: We would like to clarify that there might be a misunderstanding regarding the presentation of results. Contrary to the perception that we analyzed the points of measurements individually, we indeed conducted a comprehensive analysis of the entire plot, comparing the curves across the treatment groups. Furthermore, to ensure the robustness of our findings, we employed the Bonferroni post-test to verify the differences among specific points. It's important to note that this approach aligns with our established and validated method for analyzing repeated measures ANOVA, which has been consistently applied in our previous works. For reference, we invite the reviewer to consult the following publications where similar methodologies were successfully employed:For references, please verify: Baraldi et al., Experimental Gerontology, 2013; Nunes et al., Plos One, 2015; Morzelle et al., Plos One, 2016; Malerba et al., Frontiers in Aging Neuroscience, 2021). We hope this clarification addresses the concerns raised.

2. Fig. 8. You are only displaying artifacts, no plaques. Look at reference #7 fig. 3.

Answer: We appreciate the Reviewer's attention to Figure 8 and the concern raised regarding the presence of amyloid plaques. We acknowledge the reference provided (#7, Fig. 3) and understand the importance of accurate representation in our figures. We would like to clarify that, in our study, we took meticulous steps to ensure the reliability of our findings. Specifically, we conducted a thorough analysis of the negative control to eliminate any potential artifacts. However, we understand that the Reviewer has highlighted the need for a clearer demonstration of amyloid plaques in Figure 8. In both this manuscript and a previously published work, as referenced earlier, we have consistently demonstrated that SAMP-8 animals exhibit a higher prevalence of amyloid plaques compared to SAMR-1 animals of the same age. The clarity of our images supports the indication of the presence of these plaques.

IV. Discussion. There is no molecular mechanism explaining the effects of low-dose Lithium.

I propose a theory that could be a good fit.

1. In 1986 Adlercreutz et al. showed that Lithium lowers renal, cardiac and splenic ornithine decarboxylase activity (ODC) in mice (8).

2. In 1992 Gad M. Gilad Gad Gilad et al. demonstrated that chronic lithium treatment suppresses ornithine decarboxylase (ODC) activity in the brain (9).

4. Polis et al. (2018) showed that arginase inhibition reverses cognitive decline and synaptic loss in a murine model of Alzheimer's Disease (11).

Answer: The proposed theory linking Lithium to the regulation of ODC activity and its implications in Alzheimer's Disease pathology is intriguing. The references provided, particularly Adlercreutz et al. (1986), Gilad et al. (1992), Kan et al. (2015), present compelling evidence on the connection between Lithium, ODC activity, and AD-like pathology. We acknowledge the importance of investigating these pathways further to enhance the depth of our understanding. While we find the proposed hypothesis to be of great interest and relevance, it's important to note that the primary focus of the present work was to examine the effects of low-dose Lithium on the density of proteins specifically related to memory formation and modulation. Regrettably, at this juncture, we do not have sufficient lysates to undertake additional experiments exploring the suggested molecular mechanisms. However, we genuinely appreciate the Reviewer's constructive suggestion and acknowledge the potential for future investigations in this direction. We will duly consider pursuing these avenues in subsequent projects where we can allocate the necessary resources to delve deeper into the molecular mechanisms involved. Your feedback is valuable, and we look forward to exploring these connections in future research endeavors.

V. It is necessary to improve the quality of scientific English language. This includes correcting any errors in spelling, grammar, and punctuation to make the text clearer and more precise.

Answer: Thank you for the suggestion. A review of the English writing was done.

Reviewer #2: Comments to Authors

In the present manuscript, "Multiple mechanisms of microdose lithium protect behavioral deficits and molecular mechanisms for memory formation in SAMP-8, a mouse model of accelerated aging", authors examined the effects of microdose lithium on behavioral abnormalities, anxiety, plaque formation and neuroplasticity in SAMP-8 mice. The study is planned and executed well and findings are interesting and relevant to the current research in the area of Alzheimer's disease. However, the following minor changes are required for the further improvement of the manuscript.

Title

1. The title is vague kindly rewrite it to make it clearer.

Answer: This title was suggested by other Reviewers. We can change it again to: "Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging."

2. The word "mechanism" is used twice in the title, please avoid repetition.

Answer: The authors thank you for the suggestion. The title was changed.

Abstract

1. Alzheimer's disease (AD) is the most common affection.

Affection does not seem a suitable word.

Answer: The authors changed the word “affection” to “neuronal disorder” (Line 2).

Introduction

• Kindly state the objective/s of the study at the end of the introduction section.

Answer: The objective of the work was stated in lines 57-59.

Methods

Evaluation of neurons and astrocyte densities in the hippocampus: Page 10

1. Images of the hippocampal regions: GD, CA1, and CA3 were acquired using the Leica DMi8 177 inverted microscope.

What is meant by GD here? Is dentate gyrus (DG)?

Answer: Yes. The complete information (granular layer of the dentate gyrus - GrDG) was added to the text (line 206).

Results

1. Figure 2A, B, and C: Provide the figure legend to represent the groups such as SAMR-1 and SAMP-8

Answer: As Figures A, B, and C have the groups’ representation in D, E, and F, respectively, we put the legend in these three graphs. The information was reinforced in the figure’s legend to become more clear (lines 284-286).

2. Figure 2: Compare the results of SAMP-8 Li+ with SAMR-1 to further clarify the effects of lithium microdose.

Answer: A comparison was done in the description of Figure 2 (lines 274-276).

3. Figure 4D: What could be the possible reason for the decrease of CaMK4 following low-dose lithium treatment? Please explain.

Answer: There is no decrease in CAMKIV following treatment with lithium. What is shown in Figure 4D is a decrease in CAMKIV in SAMP-8 animals that was not prevented with lithium treatment.

In the process of memory formation, Camk-IV plays an important role in activating the CREB transcription factor, promoting cell proliferation, differentiation, and protein synthesis (Benarroch, 2 Neurology 91(3): 125-132, 2018).

As stated, the difference shown in fig. 4D is between SAMR-1 animals and SAMP-8 animals without treatment. SAMR-1 animals do not show a cognitive decline. Therefore, the higher concentration of CAMK-IV in these animals may mean higher phosphorylation of CREB and greater maintenance of memory consolidation mechanisms, unlike what happens in SAMP-8 animals. In this way, the treatment with low-dose lithium did not influence the expression and density of this protein. This conclusion was added to the text (lines 335 – 336).

While the study is important and well written, there are major issues that call into question the reliability of the data presented.

1. One major issue is that it is impossible to know how much Lithium each individual mouse received, as these may vary across the cage. 5 ug/day/animal. Is the entire mixture consumed daily? If ad libitum, this suggests the dose fluctuates.

Answer: The authors agree with the Reviewer that there is no way to ensure the amount of lithium each animal receives. However, as the treatment was taken for 8 months uninterruptedly, we can assure that the blood concentration was stable and effective as per our experience in previous reports from our research team in both mice and human beings (Nunes et al., 2013; Nunes et al., 2015; Malerba et al, 2021). Unfortunately, we did not measure the blood concentration of lithium in this group, but we made the same protocol with male SAMP-8 mice, measured their lithemia, and found a stable concentration (these data were submitted recently for publication).

2. More details on SAMR-1 and SAMP-8 mice are needed. What are these mice? How are they generated?

Answer: The accelerated aging mouse line comes from AKR/J animals. In 1968, at Kyoto University, it was noticed, after brother-sister crossings, that some litters showed characteristics, such as decreased activity, periophthalmic lesions, and early death, but without any evidence of malformation. This new phenotype presented was inherited by other generations (TAKEDA, Neurobiology of aging 20(2): 105-110, 1999).

In 1975, the litters that presented the phenotypes described above were isolated as "senescence-prone" parents, and the animals that did not have an altered life course were "senescence-resistant" parents (TAKEDA, Neurobiology of aging 20(2): 105-110, 1999).

Today, it is known that there are several lineages of SAMP animals; the isolation of these lineages occurred due to the different characteristics presented between them. SAMP-8 animals present the hallmarks of accelerated aging, early death, memory and learning deficits, presence of amyloid plaques in the hippocampus, blood-brain barrier dysfunction, loss of neurons, and cortical atrophy (MIYAMOTO et al., Physiology & behavior 38(3): 399-406, 1986; YAGI et al., Journal of Neuropathology & Experimental Neurology 48(5): 577-590, 1989).

This information was added to the Methods (lines 74-85).

3. Line 89. There should be a SAMR-1 Lithium treated group.

Answer: The use of SAMR-1 animals in this study was to control SAMP-8 animals. The differences found between the two groups show that molecular mechanisms are altered in animals with accelerated aging. Our group understands that the focus of the study was to evaluate the effects of lithium microdoses in SAMP-8 animals, SAMR-1 animals only prove that SAMP-8 animals present spontaneous molecular and behavioral changes.

4. Line 152, 153, etc: typo in phosphorylated

Answer: Thank you for the observation. The corrections were made.

5. Line 164: typo

Answer: Thank you for the observation. The correction was made

6. Line 183: Concern that this method is not reliable. The detection range should not be altered between slices.

Answer: We thank the referee for the observation and we agree. There must be some mistake. The paragraph was rewritten (lines 213-214).

7. Line 184: relations should be relation

Answer: The sentence was dismissed.

8. Line 192: typo

Answer: Thank you for the observation. The correction was made.

Answer: There was a typo error. The test used in molecular data was one-way ANOVA.

10. Fig 2A, 2D: It is unclear why after day 2, latency to enter escape box and number of errors are worse than baseline for the SAMP-8 mice.

Answer: There is no statistical difference on day 2 for any group. This means that the behavior on day 2 is similar to day 1. Only from day 3 do animals begin to express their behavioral differences in the maze.

Answer: Animals were randomly assigned to the groups, and there were no statistical differences between the groups at that point.

12. Fig. 3: All individual data points should be shown.

Answer: Figures 5 to 8 were changed and now all individual points are shown.

13. Line 283: typo

Answer: Thank you for the observation. The correction was made

14. Line 278: Full uncropped blots should be provided in supplement. Additionally, a single lane from one of each mouse is not sufficient.

Answer: We appreciate the reviewer's request for full uncropped blots in the supplementary materials and understand the importance of providing comprehensive data. In our experimental approach, due to the optimization of experiments and the limited availability of cellular lysates, we employed a strategy where the same membrane was incubated with different proteins of varying molecular weights. To ensure the integrity and reliability of our results, we performed Ponceau staining on the membranes, confirming the successful transfer of all proteins and the efficacy of electrophoresis. This quality control step was essential to validate the accuracy of our Western blot data. In response to the Reviewer's suggestion, we have included in the supplementary materials the complete membranes stained with Ponceau, allowing for a comprehensive view of the entire lanes. Additionally, we have provided membranes with all the individual lanes for transparency and thoroughness. These supplementary data aim to enhance the transparency and reproducibility of our Western blot analyses.

15. Figure 4: It is unclear how these differences are tested. It should be one way ANOVA with appropriate post-hoc test. Appear to be multiple t-tests.

Answer: We thank the Reviewer for the observation. There was a mistake in the description of statiscal methods that was corrected (line 239). The test used was indeed one-way ANOVA.

16. Line 326: abbreviation "a" appears as "b" in Fig 4H.

Answer: The change was made.

17. Line 328: n of 6 in 3C and F but legend says n of 7.

Answer: We recognize these details and aim to offer clarification. In specific groups, the sample size is indeed 6 subjects, a decision made to exclude outliers during the analysis. This meticulous step was taken to enhance the robustness and accuracy of our results. Your attention to detail is invaluable, and we are grateful for the opportunity to address and rectify this discrepancy in our manuscript. If you have any further questions or concerns, please do not hesitate to reach out.

Answer: We appreciate the reviewer's observation and acknowledge the consideration of alternative techniques, such as immunofluorescent staining and confocal imaging, for the quantitation of synaptic proteins. While we recognize that WB may have lower sensitivity compared to the mentioned methods, it is a well-established technique for evaluating protein expression. Although it may not offer the same level of accuracy as immunofluorescent staining and confocal imaging of serial sections, WB provides a valuable insight into the expression profile of these proteins. For future research endeavors, we are grateful for the suggestion to explore alternative methodologies and will duly consider incorporating immunofluorescent staining and confocal imaging with appropriate co-stains. We understand the importance of employing the most precise and advanced techniques available to enhance the accuracy of our findings. Thank you for your insightful feedback, and we look forward to incorporating these considerations into our future work.

19. It appears that the Actin band is re-used in Figs 4A, B, E, F and G. While it might be possible to probe multiple proteins of different molecular weights on the same blot, use secondaries with separate fluorescent channels, or even strip the plot to reprobe, the shape of the bands should be similar. In this case, Fig 4B is concerning because while the Actin bands are catching upward, the AMPA bands are catching downwards, suggesting they are from different gels/blots. The Actin and AMPA bands must be from the same blot. Showing the full blot would clear this up.

Answer: As explained above, the original images were included in the supplemental material.

20. Fig. 5,6,7: Neuron number cannot be quantified by NeuN percent area. Use stereology.

Answer: Thank you for your suggestion. We agree that neuron quantification should be done by stereology. Our data show neuron semi-quantification based on the proportional labeled area of NeuN, which could be associated with changes in neuronal density.

21. Fig. 8: Should be labeled thio-s not AB. Thio-S can stain many other structures beyond just AB. Moreover, the Thio-s staining is not convincing.

Answer: We thank the Reviewer and we explained above the rationale of using Thio-S in this work to show senile plaques.

22. Figure 8J: Individual data points from 5 mice should be shown.

Answer: All graphs were changed and now it is possible to visualize the individual data points.

23. Line 405: typo.

Answer: Thank you. The correction was made.

Answer: The authors respectfully disagree with the Reviewer's suggestion to remove the quote from Lines 446-447, as recent studies continue to provide evidence regarding the differences in the prevalence of AD between men and women. Contrary to the notion that this idea has been dismissed, recent research, including the review by Subramaniapillai et al. (2021) and the article by Dubal (2020), supports the existence of disparities in AD prevalence based on gender. These studies emphasize the ongoing relevance of considering gender-specific factors in AD pathology. While it is acknowledged that mice do not undergo menopause, it's important to note that they exhibit a decline in estrogen sensitivity in their tissues, which has been demonstrated to relate to exacerbated pathology (Frick, 2009; Koeble et al., 2016). Therefore, the inclusion of the quote is intended to reflect the existing discourse on this topic and the evolving understanding of gender-related factors in AD. If there are additional studies or perspectives that the Reviewer recommends considering, we would be open to discussing them and potentially incorporating relevant information into the manuscript. Thank you for your thoughtful review, and we are committed to ensuring the accuracy and up-to-date nature of our literature references. A comment with references was included in the Discussion (lines 442 – 453).

Frick KM. Estrogens and age-related memory decline in rodents: what have we learned and where do we go from here? Horm Behav. 2009 Jan;55(1):2-23. Doi: 10.1016/j.yhbeh.2008.08.015. Epub 2008 Sep 16. PMID: 18835561; PMCID: PMC2664384.

Koebele SV, Bimonte-Nelson HA. Modeling menopause: The utility of rodents in translational behavioral endocrinology research. Maturitas. 2016 May;87:5-17. doi: 10.1016/j.maturitas.2016.01.015. Epub 2016 Feb 3. PMID: 27013283; PMCID: PMC4829404.

Dubal DB (2020) Sex difference in Alzheimer's disease: An updated, balanced and emerging perspective on differing vulnerabilities. Handbook of clinical neurology 175: 261-273.

Subramaniapillai S, Almey A, Rajah MN, Einstein G (2021) Sex and gender differences in cognitive and brain reserve: Implications for Alzheimer's disease in women. Frontiers in Neuroendocrinology 60: 100879.

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Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging

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Kyung-Wan Baek

22 Mar 2024

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PERMALINK

Microdose lithium improves behavioral deficits and modulates molecular mechanisms of memory formation in female SAMP-8, a mouse model of accelerated aging

Arthur Antonio Ruiz Pereira

Alessandra Macedo Pinto

Helena Nascimento Malerba

Mariana Toricelli

Hudson Sousa Buck

Tania Araujo Viel

Roles

Abstract

Introduction

Materials and methods

Animals

Treatment with microdose lithium

Experimental design

Fig 1. Timeline for the experimental design.

Behavioral tests

Evaluation of spatial memory with Barnes maze

Evaluation of anxiolytic-like effects with the elevated plus maze

Biochemical and histological analysis

Analysis of the density of proteins related to neuroplasticity and long-term memory

Evaluation of neurons and astrocyte densities in the hippocampus

Evaluation of the density of senile plaques

Statistical analysis

Results

Behavioral experiments

Chronic microdose lithium treatment preserved memory of SAMP-8

Fig 2. Effects of treatment with low dose lithium to spatial memory of SAMR-1 and SAMP-8.

Chronic microdose lithium treatment reduced the anxiety of SAMP-8

Fig 3. Effects of treatment with low dose lithium on anxiety-like behavior of mice.

Low-dose lithium preserved molecular markers related to memory and neuroplasticity

Fig 4. Effects of chronic treatment with low dose lithium for the density of proteins involved in long-term memory maintenance.

Effects of chronic microdose lithium treatment on neuronal, astrocytic and senile plaques density

Fig 5.

Fig 6.

Fig 7.

Fig 8. Senile plaques in the different animals’ hippocampus.

Discussion

Effects on molecular mechanisms involved in memory formation

Effects on neuronal and synapses survivor and senile plaques’ densities

Effects on α7 nicotinic acetylcholine and GABAA receptors

Inhibition of the GSK-3β

Fig 9. Global effects of treatment with microdose lithium for LTP.

Supporting information

Data Availability

Funding Statement

References

Author response to previous submission

Decision Letter 0

Kyung-Wan Baek

Roles

Author response to Decision Letter 0

Decision Letter 1

Kyung-Wan Baek

Roles

Acceptance letter

Kyung-Wan Baek

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Effects on α7 nicotinic acetylcholine and GABA_A receptors