Mechanisms of Hydrogen Sulfide against the Progression of Severe Alzheimer’s Disease in Transgenic Mice at Different Ages

Backgroud: Alzheimer disease is an age-related severe neurodegenerative pathology. The level of the third endogenous gas, hydrogen sulfide (H2S), is decreased in the brain of Alzheimer’s disease (AD) patients compared with the brain of the age-matched normal individuals; also, plasma H2S levels are negatively correlated with the severity of AD. Recently, we have demonstrated that systemic H2S injections are neuroprotective in an early phase of preclinical AD. Objectives: This study focuses on the possible neuroprotection of a chronic treatment with an H2S donor and sulfurous water (rich of H2S) in a severe transgenic 3×Tg-AD mice model. Method: 3×Tg-AD mice at 2 different ages (6 and 12 months) were daily treated intraperitoneally with an H2S donor and sulfurous water (rich of H2S) for 3 months consecutively. We investigated the cognitive ability, brain morphological alterations, amyloid/tau cascade, excitotoxic, inflammatory and apoptotic responses. Results: Three months of treatments with H2S significantly protected against impairment in learning and memory in a severe 3×Tg-AD mice model, at both ages studied, and reduced the size of Amyloid β plaques with preservation of the morphological picture. This neuroprotection appeared mainly in the cortex and hippocampus, associated with reduction in activity of c-jun N-terminal kinases, extracellular signal-regulated kinases and p38, which have an established role not only in the phosphorylation of tau protein but also in the inflammatory and excitotoxic response. Conclusion: Our findings indicate that appropriate treatments with various sources of H2S, might represent an innovative approach to counteract early and severe AD progression in humans.


Introduction
Alzheimer's disease (AD) is a severe, progressive and age-related neurodegenerative disorder that can be divided into a 3-stage model: early stage (characterized by mild AD: general forgetfulness) middle stage (moderate AD: forgetfulness, language and communication difficulty), and late stage (severe AD: inability to recognise familiar people and objects) [1]. Amyloid β (Aβ) accumulation in different areas of the brain, (as hippocampus and cortex, which are mainly involved in learning and memory) and tau hyperphosphorylation are the 2 main etiopathological factors of AD, leading to an excitotoxic and inflammatory response, with synaptic dysfunction, neurodegeneration, apoptotic death associated with neuronal loss and severe cognitive decline: this is the typical neurodegeneration process of AD [2][3][4][5][6][7][8]. The number of AD patients is constantly increasing. Indeed, according to estimates, 40 million people worldwide have AD, and this number is expected to redouble by 2050 [9]. So, to find new effective therapies is the urgent need of the hour.
Hydrogen sulfide (H 2 S) is best known as a highly toxic, colourless and flammable gas, but recently it has been demonstrated that it is also the third endogenous gas, after nitric oxide and carbon monoxide, and all these gases play important roles both in health and pathologic conditions [10]. The endogenous micromolar levels of H 2 S have been measured in the brain, blood and peripheral organs of rats, humans, and bovine and production differs considerably between the various mammalian species [11]; moreover, levels of H 2 S decrease with ageing [12]. Endogenous H 2 S is generated from cysteine metabolism through 2 enzymes: cystathionine-β-synthase and cystathionine-γ-lyase whose productions are mainly in the brain for the former and largely in the cardiovascular system for the latter [13,14]. Growing evidence shows that H 2 S is an important signalling molecule in various body systems, and it gives beneficial effects in animal models of inflammation, hypertension, and ischemia/reperfusion injury inducing anti-inflammatory, cytoprotective, immunomodulating and trophic effects [10,[13][14][15][16][17][18]. The relatively high endogenous concentrations of H 2 S in the brain suggest a neuromodulator role [19]; yet, to date, the detailed mechanisms involved have remained unknown. Furthermore, it has been reported that brain H 2 S synthesis is severely decreased in AD patients (up to 55%) [20], and plasma H 2 S levels are negatively correlated with the severity of AD [21]. Recently, it has been demonstrated that H 2 S donor sodium hydrosulfide reduces Aβ generation in cultured cells and subsequently, we have demonstrated this reduction also in vivo experiments with improvement of cognitive performance in rats and transgenic mice with mild (early stage) AD [22]. This study focuses on the possible ability of a chronic treatment (3 months) with an H 2 S donor and a sulfurous water, with high content in H 2 S [23], to counteract the progression also in a severe (late stage) AD, at different ages (6 and 12 months). In particular, in a severe 3×Tg animal model of AD, we investigated cognitive ability, brain morphological alterations, amyloid/tau cascade, excitotoxic, inflammatory and apoptotic responses.

Animals
For this study, male 6 and 12 month-old (at the start of the study) 3×Tg-AD mice and their wild-type littermates (The Jackson Laboratories, Bar Harbor, ME, USA) were used. These mice harbour human transgenes APP Swe , PS1 M146V and tau P301L overexpress mutant human APP, PS1 and tau protein respectively [22,24]. Animals were kept in air-conditioned colony rooms (temperature 21 ± 1 ° C, humidity 60%) on a natural light/dark cycle (lights on at 08: 00 am) with free access to food and water. Throughout the study, body weight was recorded. Animal sacrifice at the end of the study was performed under general anaesthesia with sodium pentobarbital intraperitoneally (i.p.; Sigma-Aldrich, Milan, Italy). All experiments were carried out in strict accordance with the European Community guidelines governing animal welfare and protection for scientific purposes (CEE Council 89/609; Italian D.L. No. 26,2014) and approved by the Committee on Animal Health and Care of Modena and Reggio Emilia University. All efforts were made to minimize animal suffering and to reduce the number of animals used in this study.

Drugs and Treatment Schedules
In this experimental model of severe AD, investigations with sodium hydrosulfide (NaHS), a H 2 S donor (Sigma-Aldrich, St. Louis, MO, USA) and sulfurous water (thermal-water with high H 2 S content; kindly provided by Salsomaggiore and Tabiano Thermae, Tabiano, Italy) were carried out. Sulfurous water originates from Tabiano's source Pergoli with the following characteristics: H 2 S level 125 mg/L (HS -43 mg/L and not ionized H 2 S 82 mg/L), pH 6.6, osmolarity 41 mOsm/L [23]. Sulfurous water (12 mL/kg i.p.) and sodium hydrosulfide (0.5 mg/kg, i.p.) were administered once daily for 12 weeks starting at 6 or 12 months of age; therefore, mice were sacrificed at 9 and 15 months. Control animals (3×Tg-AD mice and wild-type mice [WT]) received an equal volume of saline by the same route of administration. The doses of sodium hydrosulfide and sulfurous water, and time of animal sacrifice were chosen on the basis of our previous experiments [22]. The number of animals used for each group is indicated in figure legends.
Assessment of cognitive ability. As it is well known, Alzheimer disease is characterized by a progressive and severe cognitive decline. For this reason, in the present study, the Morris water maze test was used to assess spatial learning and memory, as previously described [6,22,[25][26][27]. The apparatus consisted of a circular white pool at a fixed position (100 cm in diameter and 55 cm in height), filled to a depth of 15 cm with tap water (26 ± 1 ° C) made opaque with milk. A white, circular-shaped refuge platform of 7 cm in diameter was submerged 1 cm below the water surface in one of the dials (target quadrant). The pool and the location of the platform were constant during all trials.
Assess spatial learning. The spatial acquisition test was conducted for 4 consecutive days and each day included one block of 4 trials. Each trial began when mice were randomly placed into the pool with face at the wall of pool from one of the 4 starting point of a quadrant, and latency to find the hidden platform was recorded (escape latency). Each animal had a period of 60 s to locate the hidden platform, and when they detected the platform, they could stay on for 15 s, to allow the formation of a spatial map. Those mice not reaching the platform within 60 s were gently guided to the platform and allowed to rest for 15 s on it.
Probe test: To examine spatial reference memory, a probe test was administered 24 h after ending spatial acquisition. During the test, the platform was removed from the pool and the mouse started the test from the opposite dial with the face facing the pool wall and swims freely for 1 min [28]. The time spent and the number of crossings, in the quadrant where the platform was first placed (target zone), were recorded as spatial memory retention.
Visible platform task. In order to evaluate the Visual-Motor coordination of mice 1 h after the probe test, a visual cue test was conducted [28]. For this test, the platform was set 1 cm above the water level and marked with black tape so that the mice could locate the platform for musing a local visual stimulus rather than relying on spatial orientation to extra-maze cues. The platform was placed in the opposite of the target quadrant and the mouse was tested once beginning from the opposite quadrant of the platform. The time spent to reach the platform was recorded.
After the last behavioural session, animals were sacrificed and then histological, biochemical and biomolecular studies were performed on their brain.

Histology
These studies were carried out in the mice experimental models. At the end of the last behaviuoral test, transcardial perfusion with ice-cold 4% paraformaldehyde (phosphate-buffered) was performed; then animal brains were removed and processed for histologic examination, as previously described [25][26][27]. Hippocampus and cortex morphology were studied in 7-µm thick paraffin-embedded sections, hematoxylin-eosin or Nissl method stained; the extent of Aβ plaques was assessed after ethanol-Congo red/Harris hematoxylin staining.

Isolation of Cytoplasmic Proteins and Western Blot Analysis
At the end of the last behaviuoral test, the whole hippocampus and cortex were dissected from the mouse brain and immediately frozen into liquid nitrogen and stored at -80 ° C. After the extraction of cytoplasmatic proteins (for analysis of phosphorylation state/level of APP, Tau, Aβ 1-42 , p-c-jun N-terminal kinases [JNK], p-extracellular signal-regulated kinases [ERK1/2] and p38) as previously described [6,22,[25][26][27], proteins (40 μg for each sample) were denatured, electrophoretically separated and transferred onto nitrocellulose membranes. The staining of the blots with Ponceau's solution showed that total protein amount was equal in each lane. The blots were then blocked and incubated overnight at 4 ° C with the following primary antibodies: p-APP, phospho (p)-tau, p-tau Ser396, Aβ (1-42 specific), JNK 54/46, ERK1/2 44/42 and p-p38 (Cell Signaling, Charlottesville, VA, USA). The day after, the membranes were incubated with a specific secondary antibody peroxidase-conjugated for 1 h at room temperature. To prove equal loading, the blots were analysed for β-tubulin expression (house-keeping gene) using an anti-β-tubulin antibody (Cell Signaling). The membranes were analysed by the enhanced chemiluminescence system according to the manufacturer's protocol (Millipore). Protein signals were quantified by scanning densitometry using a bio-image analysis system (Bio-Profil, Celbio, Italy) and expressed as relative integrated intensity in comparison with those of normal (naïve) mice. The level of all bands was expressed as relative integrated intensity normalized versus β-tubulin (= 100) [6,22,[25][26][27].

Statistical Analysis
All data were collected blinded to the treatment and are shown as mean ± SEM. Parametric data was analysed using SigmaPlot and Adobe Illustrator and SPSS17.0 software. The 2-way repeated measures ANOVA (behavioural data), or 1-way ANOVA (all other data), followed by the Student-Newman-Keuls' test were used to detect the significance from different groups. Aβ deposit data was analysed by means of the Kruskal-Wallis test followed by the Mann-Whitney U-test. A value of p < 0.05 was considered significant.

Sulfurous Water Improves Learning and Memory
Performance in a Severe Transgenic Mouse Model of AD WT, both 9 and 15 months, treated with saline, gave latencies to find the hidden platform continuously decreased during the 4 days of training (Fig. 1a, b).
3×Tg-AD mice, both 9 and 15 months, treated with saline (controls) showed impaired ability (as compared with WT) in platform finding during all 4 days of behavioural test (Fig. 1a, b). Conversely, the 2 groups (9 and 15 months old) of transgenic mice treated with sulfurous water (12 mL/kg i.p.) showed an improvement in spatial learning performance on the 4th day of training as compared with controls ( Fig. 1a, b). Treatment with H 2 S donor, sodium hydrosulfide (0.5 mg/kg i.p.) did not significantly modify Pharmacology 2019;103:50-60 DOI: 10.1159/000494113 the escape latency in 3×Tg-AD versus controls but showed only a trend to improve cognitive ability (Fig. 1a, b).
Twenty four hours after the Morris water test, animals were subjected to probe test. WT mice of both ages spent more time in quadrant where the platform was during the Morris test (original quadrant) respect to the 3×Tg-AD mice treated with saline (Fig. 1c, d), and also the number of crossings of this quadrant has been higher in WT mice respect to controls (Fig. 1e, f)   quadrant and a significant higher number of crossings with respect to control mice (Fig. 1d, f). Motor activity. All mice after 1 h of probe test were placed in the pool with the platform visible to evaluate they ability to swim. All animals showed a similar time of escape latency and identical swimming speed in the Morris water maze compared with control mice (data not shown).
Toxicity. No signs of toxicity were recorded throughout the 3 months of treatment with sodium hydrosulfide or sulfurous water (e.g., ruffled fur, diarrhea, lethargy, aggressiveness, hypothermia, alterations of spontaneous locomotor exploration and grooming), and body weight variations throughout the study were similar in all experimental groups (data not shown).

Sulfurous Water Preserves Hippocampus and Cortex Morphological Alterations in a Mice Model of AD at both Ages
To investigate the brain morphological features of AD in 9-and 15-months-old mice, we processed the hippocampus (data not shown) and cerebral cortex ( Fig. 2; 2 vulnerable regions of the AD brain) for histology. In both these areas and in both ages studied, in saline-treated 3×Tg-AD mice we found a number of neurons showing irreversible (pyknosis, nuclear dust) or reversible (swollen perikaryon, cellular shrinkage) alterations (Fig. 2c, i), and this led to a significant decrease of viable neurons (Fig. 2a). Saline-treated 3xTg-AD mice showed, in both ages and areas, a substantial amyloid deposit compared with WT mice (Fig. 2b-d, h-l).
On the contrary, treatments with sulfurous water significantly preserved morphological picture, with a higher number of viable neurons (Fig. 2a, g), and a significant reduction in amyloid deposit in cortex (Fig. 2b, e, f, h, m, n) compared with 3×Tg-AD saline-treated mice.
Treatments with sodium hydrosulfide did not significantly preserve hippocampus and cortex morphological alterations (not shown).

Sulfurous Water and/or Donor Sodium Hydrosulfide Reduce APP, Aβ. Tau and MAPK Expression/Activity Level in 3×Tg-AD Mice
In the hippocampus and cortex, we also investigated, by western blot analysis, the main proteins of the amyloid cascade and tau proteins. In both examined areas (hippocampus and cortex), at 9 and 15 months, 3×Tg-AD mice treated with saline showed a high phosphorylation level of APP activity (Fig. 3a, b) and high tau expression and tau activity phosphorylated at Ser396 (Fig. 3c, d), and treatment with sulfurous water and Na HS decreased significantly these markers (Fig. 3a-e).
In 3×Tg-AD mice treated with saline, the expression of Aβ1-42 (Fig. 4a-c) was significantly higher, compared with WT, both hippocampus and cortex. Treatments with sulfurous water or NaHS, significant decreased the expression of Aβ1-42 (Fig. 4a-c).
In neurodegenerative diseases, including AD, the MAPK play an important role in phosphorylation of tau protein [29]. It is well known that MAPK family members (JNK, p38 and ERK) are involved in the transcription regulation of several genes encoding for pro-inflammatory cytokines [29][30]. Our results showed, in the cortex and in the hippocampus of the saline-treated 3xTg-AD mice, at both age, an increase of activity of ERK (Fig. 5a, b), JNK (Fig. 5c, d) and P38 (Fig. 5f-g), as compared with WT mice. Treatment with sulfurous water or donor sodium hydrosulfide decreased significantly all these single MAPK ( Fig. 5a-g).

Discussion
Alzheimer's disease is a multi-factor pathology because, not only neurology but also cardiology, ophthalmology and other medical specialties are involved in the study of its etiopathology and therapeutic approach [31][32][33]. Indeed, in recent years, this broad spectrum of studies has led to demonstrate that endogenous H 2 S levels are decreased in the brains of patients and mice with AD [34,20]. This suggests that the restoration of H 2 S level could be a treatment for the Alzheimer's disease. Indeed, our previous data showed that sub-chronic and chronic treatments with H 2 S counteract the progression of early AD in rat and mouse models [22]. Herein, our present study demonstrates, for the first time, that sodium hydrosulfide (a donor of H 2 S) and sulfurous water (thermal-water rich of H 2 S content), administered for 3 months consecutively, preserve or slow down brain alterations and consequent cognitive deficit, in a severe 3×Tg-AD mouse model. In these triplex transgenic 6-month-old mice, senile plagues began to appear, and after 9 months of age, the plagues of beta-amyloid are prominent with notable learning and memory impairments, inflammation, oxidative stress and neuronal loss [22,24]. All together, these symptoms represent a severe level of AD progression in 3×Tg-AD mice. Our present results showed that the preservation or improvement in cognitive performance induced by H 2 S's treatments is associated with a trend of hippocampus and cortex reduction of Aβ deposits, low- ered phosphorylation of APP and tau protein, and diminished morphological damage including a decreased neuronal death in the CA1 subfield. It well known that the 2 main events in AD pathophysiology are Aβ deposits and tau hyperphosphorylation and it has been demonstrated that Aβ and tau exert toxicity both with separate or merge mechanisms [22,29,30]. Indeed, in vitro and in vivo studies suggest that Aβ may lead to tau pathology, tau may mediate Aβ toxicity and together Aβ and tau may synergistically target different component of the same system [2,29,34]. In addition to the Aβ and tau-dependent mechanisms, there are other several etiopathogenic mechanisms that could lead to AD, as apoptosis and inflammation [2,6,7,22,30]. Indeed, in this preclinical, transgenic AD study, we demonstrated, that, in a severe AD model, systemic chronic treatments with sulfurouswater rich in H 2 S or NaHS induced an anti-inflammatory response, thereby reducing the activity of the MAPK members JNK, p38 and ERK, in the hippocampus and cortex at both ages considered. It is well known that MAPK members play an important role not only in inflammation but also in phosphorylation of tau protein.
These biochemical and biomolecular results confirm important neuroprotective aspects of H 2 S, and demonstrate the ability to counteract the main established AD-related mechanisms of brain damage as inflammation. This antiinflammatory action of H 2 S on brain strengthens the well-known systemic anti-inflammatory response of H 2 S [35]. The identification of the sequence of events in the pathophysiology of AD is important for the novel identification of appropriate therapeutic approaches, leading to a series of potential therapies in AD because currently this sequence is still not well defined.
Several potential approaches for AD treatment such as cell and gene therapies are currently under investigation; however, a great number of compounds for AD failed to reach the goal [36].
One of the main aspects responsible for these failures of preclinical AD studies is that the compound under study affects only one of the pathophysiological responsible for neurodegeneration of AD, or shows toxic side effects [3,29,37,38]. For this reason AD remains a major cause of disability and mortality without an effective treatment.
Therefore, a winning strategy could be to identify substances able to affect simultaneously more pathophysiological mechanisms of AD. Our present data shows that an H 2 S donor and/or sulfurous water with high H 2 S content, in a severe model of AD, improve cognitive activity by reducing Aβ deposits, hyperphosphorylation of APP and tau isoform at Ser396, as well as inflammatory responses. Noteworthy, the chronic treatment of 3 months did not show any evident signs of toxicity (perhaps a sign of toxicity could be glimpsed in the cortex of 15-monthold animals treated with NaHS where beta-levels are higher than those of transgenic control animals). Again, our data are in agreement with a large number of evidence confirming that gaseous signalling molecule H 2 S exerts a cytoprotection in mammalian cells via multiple mechanisms (modulation of ion channels, enzymes, transcription factors and receptors) [10,15,16,[39][40][41]. For example, there is evidence demonstrating that H 2 S is a phosphodiesterases inhibitor: in fact, levels of second messengers cAMP and cGMP increase [41]. Phosphodiesterases inhibition could be an early step leading to neuroprotective mechanisms induced by H 2 S also in our experimental condition, and our next goal is to exam specifically this aspect.
A restraint of this work is that we administered the substances by i.p. injection to standardize doses; our next aim will be to study these substances administered by oral and inhaled route, which are routes commonly used in humans for sulfurous waters. Furthermore, levels of H 2 S in plasma and brain are under investigation in our laboratory, and will be the focus of our next paper.
In conclusion, our data supports the hypothesis that endogenous H 2 S might be physiologically involved in neuroprotection against AD and these positive results, obtained in a severe model of AD, should encourage further studies to better define the potential therapeutic value of H 2 S alone or sulfurous waters with high H 2 S content. immunoblots highlighting the activity of p-ERK, p-JNK, p-P38 and of house-keeping gene product β-tubulin.* p < 0.05, at least, versus the corresponding value of 3xTg mice treated with saline. # p < 0.05, at least, versus the corresponding value of wild type animals treated with saline. WT, wild-type mice; 3×Tg, 3×Tg-AD mice; S, saline; NaHS, donor of Sodium hydrosulfide; SW, sulfurous water.