Ghrelin regulates crosstalk between apoptosis, necroptosis and autophagy programmed cell death pathways in the hippocampal neurons of amyloid-β 1–42-induced rat model of Alzheimer’s disease

doi:10.21203/rs.3.rs-1726826/v1

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Ghrelin regulates crosstalk between apoptosis, necroptosis and autophagy programmed cell death pathways in the hippocampal neurons of amyloid-β 1–42-induced rat model of Alzheimer’s disease

https://doi.org/10.21203/rs.3.rs-1726826/v1

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Alzheimer's disease (AD) is a common progressive and irreversible neurodegenerative disorder. Neuronal loss in the brain is one of the important characteristic features of AD, which is along with memory and cognitive dysfunction. Activation of programmed cell deaths, especially apoptosis and necroptosis, and autophagy failure plays a critical role in the pathogenesis of AD. Ghrelin, an appetite-related hormone, exerts neuroprotective effects via the stimulation of growth hormone secretagogue receptor type 1a in the central nervous system. In this study, rats' cognitive impairment was induced by intra-hippocampal Cornu Ammonis 3 administration of amyloid-beta 1–42 (Aβ 1–42). Then animals were treated with ghrelin 80 µg/kg intraperitoneally for 10 consecutive days. The Morris water maze and passive avoidance learning test were used to evaluate of effect of ghrelin on learning and memory performance. After that, histopathological study on rat hippocampus was done, and the expression of pro-apoptotic protein Bax, anti-apoptotic protein Bcl-2, necroptotic proteins RIP1K and RIP3K and autophagic marker Beclin-1 were assessed using western blotting method. Our findings showed that ghrelin improves memory impairment in Aβ 1–42-induced rats and reduces Bax, RIP1K, and RIP3K expressions and also Bax/Bcl-2 ratio, as well as Beclin-1 expressions. In conclusion, our study suggests that ghrelin can provide neuroprotection via apoptosis and necroptosis inhibition and autophagy promotion and also crosstalk regulation between three cell death pathways in Aβ 1–42-induced model of Alzheimer's disease.

Alzheimer's disease

Amyloid-beta 1–42

Ghrelin

Apoptosis

Necroptosis

Autophagy

Alzheimer's disease (AD), generally known as senile dementia, is a neurodegenerative disorder characterized by progressive and irreversible cognitive decline, with oligomer-prone amyloid-beta (Aβ) peptides, phosphorylated tau, extensive synaptic and neuronal loss in the brain (Chavoshinezhad et al. 2019; Lin et al. 2019; Tian et al. 2019). Aβ peptides are an initial pathological signal in AD that gradually results in neurofibrillary tangles formation, neuronal cell death facilitation, and dementia (Leong et al. 2020). In AD patients, neural cell death, especially in the brain cortex and hippocampus, is attributed to declining learning and memory abilities (Yang et al. 2017). A newly emerging AD feature is the apparent activation of the apoptosis pathway in the neurons. This mechanism is the primary manifestation of neuronal loss and is responsible for extensive neuronal cell death (Rohn et al. 2001). Accumulations of Aβ peptides induce oxidative stress in the brain and leads to neuronal death by triggering the apoptotic signaling (Kim et al. 2020). Apoptosis is a form of programmed cell death characterized by dense chromatin, fragmented nuclear and wrinkled cell (Rohn et al. 2002). Bcl-2 family proteins, such as Bcl-2 and Bcl-xl as an anti-apoptotic subfamily, prevent apoptotic death in multiple cell types, including neurons and pro-apoptotic subfamily including Bax, Bak, Bad, Bim, are altered in the vulnerable neurons in AD. These family proteins are the central regulators of the evolutionally conserved pathway of apoptosis (Kudo et al. 2012).

On the other hand, neuroinflammation and overproduction of proinflammatory cytokines in the hippocampus of AD patients could be a possible cause underlying the enhanced necroptotic signaling via stimulation of the tumor necrosis factor (TNF) family death domain receptor (Motawi et al. 2020). The necroptotic pathway is a programmed cell death that has necrosis-like morphological characteristics. Necroptosis activation relies on the receptor-interacting protein (RIP) 1 and 3 and mixed lineage kinase domain-like protein (MLKL). Increasing evidence indicates that the necroptosis pathway may participate in neuronal death in several neurodegenerative disorders such as AD (Zhang et al. 2017). Therefore, it will be essential to study further whether apoptosis and necroptosis inhibition can provide neuroprotection in neurodegenerative models in vitro and in vivo.

Furthermore, multiple of evidence supports the hypothesis that autophagy failure plays an important role in AD pathophysiology. Autophagy is an intracellular degradation pathway that undesirable cytosolic contents are surrounded in autophagosomes, double-membrane vesicles, and then delivered to lysosomes for digestion (Di Meco et al. 2020). Beclin-1, a BH3-only domain protein family member, is a key molecule involved in autophagic flux initiation and autophagosome formation (Chen et al. 2018). Given the vital role of aggregate and misfolded proteins in AD and the established dysfunction of autophagic pathways in brain, now, activating autophagic flux to stimulate clearance of protein aggregates could be an ideal therapeutic candidate against AD (Cheng et al. 2018).

In recent years, AD has become a severe social and medical problem for the following three significant challenges: The continuously increasing of patients, problems in preclinical diagnosis, and insufficient methods for definitive treatment for AD (Jia et al. 2016). Therefore, research studies are critical to discovering the effective treatment for this disease.

Ghrelin is a 28-amino-acid peptide and is mainly secreted by the stomach cells, and first synthesis in the form of preproghrelin, which is cleaved to proghrelin. Proghrelin is further cleaved to produce unacylated ghrelin and acylated. Acyl ghrelin, representing 10% of the total circulating ghrelin, binds to growth hormone secretagogue receptor type 1a (GHSR1a) when the octanoylic chain is linked to serine by the enzyme ghrelin O-acyltransferase (GOAT) (Stoyanova 2014). GHSR1a, a G protein-coupled receptor, is expressed in peripheral tissues and extensively in the central nervous system, such as the brain cortex, pituitary gland, hypothalamus, thalamus, and hippocampus (Xu et al. 2009). Initially, it was thought that ghrelin is just an appetite-related hormone, but now we know that in addition to food intake, it plays an essential role in the function of multiple organs of the body. Furthermore, emerging evidence suggests that ghrelin acts in the central nervous system to control neuronal function and subsequently various brain functions, including memory, learning, neurogenesis, and neuroprotection (Li et al. 2013).

Therefore, according to neuroprotective effects of ghrelin, in this study, using the Aβ-induced model in male Wistar rats, we first examined the impact of intraperitoneal ghrelin injection on memory and learning functions in the Morris water maze and passive avoidance task, respectively. In the next step, after histopathological study, we assessed the effects of ghrelin treatment on the apoptosis, necroptosis, and autophagy cell death pathways via evaluation of Bcl-2, Bax, RIP1K, RIP3K, and Beclin-1 expression levels and crosstalk between 3 programmed cell death pathways in the extracted hippocampus.

Materials

Aβ-peptide (1-42) was provided by Sigma-Aldrich (Munich, Germany). Acylated ghrelin was purchased from GL Biochem Ltd (Shanghai, China). RIPA lysis buffer (sc-24948) supplied by Chem Cruz (Huizen, Netherlands). Western blot antibodies for Bax (sc-7480), RIP1K (sc-133102), RIP3K (sc-374639), and GAPDH (sc-47724) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Bcl-2 antibody (ab59348) was provided by Abcam (Cambridge, UK), and Beclin-1 antibody (D40C5) was supplied by Cell Signaling Technology (Danvers, MA, USA). Secondary HRP-conjugated antibodies, anti-rabbit IgG (A6154), and anti-mouse IgG (A8924) were obtained from Sigma-Aldrich (Munich, Germany). BCA protein assay kit (QG219577) was the product of Thermo Scientific (Rockford, USA). ECL Western blotting substrate kit (ab65623) was purchased from Abcam (Cambridge, UK). Other materials and reagents were obtained from Sigma-Aldrich (Munich, Germany).

Animals

24 adult male albino Wistar rats (Pasteur Institute, Tehran, Iran) weighing 250 ± 20 gr were housed in cages of three in a temperature-controlled holding room (23 ± 1ºC) and on a 12/12hr light/dark cycle (lights on at 8:00 am). Standard laboratory, ad libitum, and water were available. Animals were permitted to become habituated to the environment and were handled for a week before the tests. All experiments following the Guide for the Care and Use of Laboratory were accepted by the Research and Ethics Committee of Shahid Beheshti University of Medical Sciences (IR.SBMU.MSP.REC.1397.546) and were in accordance with National Institutes of Health guide for the care and use of Laboratory animals.

Experimental Design

The adult male rats were randomly sorted into four different groups, six rats in each: control, Aβ 1-42 (10 µg Aβ 1-42 / normal saline (2 mg/ml)), ghrelin (80 μg/kg ghrelin/PBS (1 mg/ml)) and Aβ 1-42 + ghrelin (10 µg Aβ 1-42 / normal saline (2 mg/ml) + 80 μg/kg ghrelin/PBS (1 mg/ml) groups. Fig.1 shows the study design schematically.

Drug administration

Microinjection of Aβ 1-42

The anesthetization was done with an i.p. injection of mixed ketamine (85 and 15 mg/kg, respectively). Adequate Persocaine-E was injected subcutaneously at the site of surgery to prevent bleeding and induces local anesthesia. Aβ 1-42 was dissolved in sterile PBS at a 2 mg/ml concentration and incubated under agitation Teflon-coated stirrer at 800 rpm at room temperature for 36 hours. After that, the Aβ 1-42 turbid solution was sedimented by centrifugation (10 min at 15000 g). Each rat was mounted on a stereotaxic apparatus. After the scalp was incised, the skull was cleaned, and 10 µg (2 mg/ml) aggregated Aβ 1-42 or 5 µl PBS as a vehicle was injected bilaterally into the Cornu Ammonis 3 (CA3) area of the hippocampus by using a 5 μl Hamilton syringe. The site of injections was identified on the skull as follows: AP: − 3.3 mm from Bregma; ML: ± 2.6 mm from the midline; DV: − 3.7 mm from the skull surface based on the Paxinos and Watson’s Atlas. Agents were injected slowly and two minutes after injection, the needle was slowly removed to avoid the backflow of the fluid.

Administration of ghrelin

Acylated ghrelin was freshly dissolved in the normal saline. One day after surgery, 80 μg/kg ghrelin (1 mg/ml) or 20μl normal saline as its vehicle was administrated i.p. for 10 consecutive days.

Behavioral assessment

Morris water maze (MWM) test

The MWM test was done to examine spatial cognitive performance. The experiment began on day 6 after surgery. The apparatus consisted of a circular metal tank placed in a soundproof room with several visual cues; also, it was assigned four quadrants of equal area and filled with water to a depth of 30 cm, which was maintained at 20 ± 1°C. A black platform was placed 1-2 cm below the midpoint of the target quadrant.

Before the training began, habituation was done by allowing rats to swim for 1 min without a platform. Animals received 4 training trials each day for 3 consecutive days (12 total training trials). The location of the platform was constant. However, the sequence of water-entry points was varied each day. The daily interval between trials was 30 s. The animal was allowed to stay on the platform for 20 s. If the rat failed to spot the platform within 60 s, it was guided to the platform and remained there for 20 s. On 3 days of the training phase, the escape latency to reach the platform was recorded for a maximum of 60 s in each trial. 24 hours after the last training day, rats were subjected to a probe trial test in which there was no platform in the tank. The animals were permitted to swim for 60 s to locate it. On the probe day, the number of platform crossing and the swimming pathway was recorded. At the end of the probe test, a visual assessment of rats was performed using a visible platform.

Passive Avoidance Learning (PAL) test

The passive avoidance test was performed 10 days after Aβ 1-42-injection. The apparatus was divided by a guillotine door into two light and dark compartments (20 × 20 × 40 cm). The evaluation of the learning and memory was performed on two consecutive days. Before the training trials, animals were habituated to the apparatus by placing them in the light room. After 5 s, the door was elevated. When the animal enters the dark compartment with all four feet, the door was closed, and the rat stayed there for 20 s. Then the animal was returned to a temporary cage. After 30 min, the rat was again positioned in the light compartment for 5 s. The door was opened to let the animal enter the dark chamber, and after the entrance, the door was shut, but this time animals received a foot shock (1.2 mA, 50 Hz, and 1.5 sec). 20 s after that, the rat was returned to the temporary cage. After 2 min, the same testing process was reiterated. The rats were again placed in the light room to see whether they stayed there for 2 min or not. It was considered successful learning when the animals did not enter the dark compartment during that period; otherwise, they would receive the same electric shock again until they refrain from entering the dark room. Indeed, during the learning session, rats learned not to enter the dark chamber for 120 s after giving electrical foot shock. After 24 hours, a retrieval test was done to assess memory retrieval. Each animal was positioned in the light compartment for 5 s, and then the door was elevated while no foot shock was applied in this trial. The time spent in the light compartment (TLC) and the number of crossing between the two compartments was recorded for 10 min.

Histopathological Study

A day after the conclusion of the behavioral assessment, three rats in each group was perfused transcardially with 4% paraformaldehyde in 0.1 M PBS after anesthetization. Then, all rats were decapitated, and whole brains were extracted and fixed overnight in fresh 4% paraformaldehyde at 4°C. After that, embedding in paraffin, coronal sections (8 μm) were cut by the slicer. For Nissl staining, sections were floated in 0.1% cresyl violet solution at 37°C for 20 min. After washing with distilled water, prepared sections were dehydrated in ethyl alcohol and xylene, fitted with coverslips, and examined with light microscope. Finally, shrunken and hyperchromic neurons with irregular shapes were considered dark cells in the CA1, CA3, and dentate gyrus (DG) regions.

Western blot

On day 11 of the experiment, after deep CO2 inhalation, animals were decapitated. The hippocampi were rapidly dissected on the ice and then transferred to the liquid nitrogen for 24 hours. Thereafter, all samples were stored at -80°C. For protein extraction, 400 μl ice-cold RIPA lysis buffer containing protease inhibitor cocktail was added to each sample and homogenized. Then samples were centrifuged for (15 min at 15000 g) at 4°C, and protein-containing supernatant was transferred to new microtubes. The BCA protein assay kit determined protein concentrations in supernatants. Next, samples were mixed with sample buffer and heated at 95 °C for 5 min. Then, in the SDS-PAGE stage, equal protein amounts of each sample were loaded in 15 % acrylamide gel and were run in the running buffer for 90 min with 90 V. Separated proteins in the gel were transferred to the nitrocellulose membrane for 120 min with 100 V. After that, the membranes were blocked in the 5% skimmed milk solution for 2 hours at 4 °C. Then, the membranes were subsequently incubated with different primary antibodies were diluted in the 0.1% TBST (1:1000) overnight at 4 °C. After three washes with 0.1 % TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or anti-mouse IgG as secondary antibodies (1:2000) for 120 min. After being washed another three times, membranes were smeared with ECL to evaluation their immunoreactivity, and then the images were captured with the Chemi Doc Imaging System (Bio-Rad, USA). Finally, the protein bands intensities were analyzed by Image J software and the density of each band was normalized compared with the GAPDH as internal control.

Statistical analysis

Statistical analysis was done through GraphPad Prism software, version 8.0. The results are expressed as the mean ± SEM. For data analysis, the one-way ANOVA was used. A pairwise comparison was made using Tukey’s post hoc test if a difference was found out to be significant. For MWM data, groups' differences in escape latency were analyzed using two-way ANOVA with repeated measures followed by Tukey’s multiple comparison tests with day and treatment as the sources of variation. The statistical significance was achieved when p<0.05.

The effect of ghrelin on spatial learning and memory

MWM test was used to evaluate the effects of ghrelin on spatial learning and memory. As shown in Fig.2A, in three consecutive days of training, according to Two-way ANOVA repeated measure analysis, the escape latency to reach the platform decreased in all groups on the second and third days during the training phase. While in the AD model group, latency time on the second and third days was close to the first day of training. Indeed, Aβ 1–42-injected rats had significantly longer escape latency on the second (p< 0.01) and third (p< 0.0001) days of testing compared to the control group. Furthermore, the Aβ 1-42 + ghrelin group tended to take less time to reach the platform on days 2 (p< 0.01) and 3 (p< 0.001) compared with the AD rats. Also, the decreased latency time on the third day in the ghrelin group was considerably different from the control group (p< 0.01).

In the next step, to assess the memory retention level of animals, a probe test was employed on day 4. As demonstrated in Fig.2B, One-way ANOVA analysis and post-hoc analysis by Tukey’s test of the mean number of platform crossing indicated a significant difference between AD and control groups (p< 0.01). The Aβ 1-42 + ghrelin rats showed a considerable increase in platform crossing compared to the AD group (p< 0.05). Furthermore, systematic injection to rats that received intra-hippocampal PBS could significantly increase this parameter compared to the control group (p< 0.05).

Animal’s performance in probe day is also shown in Fig.2C. The Aβ 1-42-injected rats swam randomly with a slight preference for the previous platform location than the control group. However, the Aβ 1-42 + ghrelin rats swam more in the last site platform than the model rats. Ghrelin and control rats swam nearly with a similar pattern in the previous platform area. In each image, the location of the platform is depicted by a blue circle.

The effects of ghrelin on passive avoidance test

As shown in Fig.3A, the AD model group had significantly reduced TLC when compared to the control group (p< 0.0001). Ghrelin administration in the Aβ 1-42 + ghrelin rats markedly increased the TLC compared to the Aβ 1–42 group (p< 0.0001). On the other hand, TLC in the ghrelin group showed a significant increase compared to the control group (p< 0.05).

According to Fig.3B, intra-hippocampal injection of Aβ 1-42 disrupts memory retrieval and increasing the number of crossing between chambers significantly compared with the control sham group (p< 0.01). Moreover, the Aβ 1-42 + ghrelin group showed a significant decrease in crossing compared with the model group (p< 0.01). The difference in the number of crossing in the ghrelin and control groups was significant (p< 0.05).

The histopathological findings in Nissl staining

In this study, the number of dark neurons per unit area in the CA1, CA3, and DG areas of rat hippocampus were counted and compared among the groups (Fig.4A.1, 4A.2, 4B.1, 4B.2, 4C.1, and 4C.2). Our results showed that in three areas, the number of dark cells in the Aβ 1-42 group had a significant incursion in comparison to the control group (p< 0.0001, p< 0.0001, and p< 0.001, respectively). Ghrelin treatment at the dose of 80μg/kg could significantly reduce dark cells in Aβ 1-42 + ghrelin group as compared to AD group (p< 0.01, p< 0.001 and p< 0.01) in the CA1, CA3 and DG regions respectively. These data indicate that ghrelin could protect the hippocampal neurons against Aβ 1-42 neurotoxicity.

The effects of ghrelin on apoptosis programmed cell death in the rat hippocampus

In this study, to evaluate the effects of ghrelin against Aβ 1-42-induced cell death, the amount of Bax was assessed by the western blotting method. The band for Bax protein was detected at 23 kDa. Accordingly, a significant difference was detected between Aβ 1-42 and control groups (p< 0.001). As expected, the post-verification analysis showed that Bax expression was significantly increased in the hippocampus of AD models. Furthermore, ghrelin (80μg/kg) could remarkably decrease Bax expression in Aβ 1-42 + ghrelin group compared to AD animals (p< 0.0001).

In this experiment, we found a significant decrease in the expression of Bcl-2 protein in the hippocampus of AD rats in comparison to normal rats (p< 0.001). Nevertheless, the hippocampal expression of Bcl-2 protein was significantly up-regulated in Aβ 1-42 + ghrelin group compared to Aβ 1-42 groups (p< 0.001). Bcl-2 antibody visualized a band at 26 kDa.

Also to determination of neuronal susceptibility to apoptosis, the Bax/Bcl-2 ratio was assessed. In the AD rats, the Bax/Bcl-2 ratio was increased compared with the control group (p< 0.001). Ghrelin treatment significantly reversed Bax/Bcl-2 ratio in Aβ 1-42 + ghrelin group, which exhibited decreased Bax/Bcl-2 ratio compared with the Aβ 1-42 group (p< 0.001) (Fig.5A).

The effects of ghrelin on the expression of rat hippocampal necroptosis pathway proteins

We found a significant increase between the expression levels of RIP1K protein in hippocampi of the AD model rats compared with the control group (p< 0.0001). On the other hand, repeated systematic ghrelin injection remarkably reduced RIP1K protein expression in the Aβ 1-42 + ghrelin group compared to the AD group (p< 0.0001). RIP1K band was showed in 76 kDa.

The expression of other necroptotic protein, RIP3K was increased significantly in the hippocampi of the Aβ 1-42 group compared with the control group (p< 0.0001). Also, ghrelin could significantly decrease levels of expression of RIP3K protein in the Aβ 1-42 + ghrelin group compared to the AD model group (p< 0.0001). RIP3K band was observed in 57 kDa (Fig.5B).

The effect of ghrelin on autophagy pathway in the rat hippocampus

To assess the ghrelin effects on the autophagy pathway, the protein expression of Beclin-1 was determined using western blotting in the hippocampus tissue. The band for Beclin-1 protein was detected at 60 kDa, as shown in Fig.5C. The amount of Beclin-1 protein significantly decreased in Aβ1–42 group compared with the control group (p< 0.0001). On the other hand, our finding indicated that Beclin-1 expression significantly increased in Aβ 1-42 + ghrelin group compared to Aβ 1–42-induced AD rats (p< 0.05).

In this research, we investigated the ghrelin effects on apoptosis, necroptosis, and autophagy programmed cell deaths and crosstalk regulation between these three pathways in the hippocampus of Aβ 1–42-induced rat model of AD. Our results demonstrated that repeated systemic administration of ghrelin could improve learning and spatial memory in AD rats. To assess ghrelin's effect on memory performance, we used MWM and PAL tests. Our data were consistent with previous studies that, ghrelin was injected intracerebroventricularly (i.c.v.) or via intrahippocampal route (Babri et al. 2013; Eslami et al. 2018; Goshadrou et al. 2013).

Moreover, the cognitive impairment found in AD can be partially explained by neuronal death, which is a mechanism that contributes to AD pathogenesis and progression (Yang et al. 2017). Neuronal death is a prominent feature of neurodegenerative diseases such as AD (Cotman and Anderson 1995). The present study showed that the Aβ 1–42 peptides administration to rat hippocampus induced apoptosis cell death via reduced anti-apoptotic protein Bcl-2, increased the apoptotic protein Bax expression and elevated Bax/Bcl-2 ratio in the AD rat hippocampus. Apoptosis is a predominant underlying mechanism of cell death resulting from AD, and the neuronal apoptosis hypothesis is a crucial aspect of AD pathology. Factors that induce apoptosis, such as Aβ accumulation, oxidative damage, and hypometabolism, are present in the brain of AD patients (Gu et al. 2016). In the specimens obtained from AD patients at postmortem, there were more TUNEL positive cells in the brain, massive loss of neurons, and shrinkage of the brain cortex, compared to normal-aging subjects. This data supports the notion that apoptosis is an important mechanism of neurodegeneration in AD (Olivares et al. 2012; Toiber et al. 2008). In the rat hippocampal neuronal cultures, exposure to Aβ 1–42 resulted in a significant reduction in Bcl-2 expression. A remarkable increase in Bax immunoreactivity in the mitochondrial fraction accelerates apoptosis by permeabilizing mitochondria (Justin Thenmozhi et al. 2017; Nilsen et al. 2006). Xinyi GU and et al.'s showed that the Bax/ Bcl-2 ratio increased in the cerebral cortex of transgenic mice models of AD (Gu et al. 2016).

Furthermore, our findings indicated that i.p. ghrelin treatment could inhibits apoptosis by increase Bcl-2 expression and decrease Bax expression and reverses the Bax/Bcl-2 ratio in the hippocampus of AD rats. These results were consistent with previous studies about ghrelin's effects on the apoptosis pathway. Immunohistochemical studies and TUNEL techniques on the brain tissue of the traumatic brain injury models revealed that ghrelin could reduce apoptosis and BBB damage in these animals (Lopez et al. 2012). On the other hand, hematoxylin and eosin (H & E) staining and TUNEL assay showed that ghrelin could increase the tolerance of hippocampal neurons to ischemic injury (Liu et al. 2006). Exposure of apoptotic fresh human neutrophils to ghrelin minimizes the ratio of Bax proapoptotic protein to anti-apoptotic protein Bcl-2 (Li et al. 2016). Also, ghrelin can inhibit apoptosis by increasing Bcl-2 expression and decreasing Bax expression in MC3T3-E1 osteoblast cells with serum deprivation in the environment (Liang et al. 2013). Therefore, due to the importance of the apoptosis pathway in AD pathogenesis, focus on mechanisms activating apoptosis and inhibiting them can be considered a practical approach in treating AD.

In addition to apoptosis, we examined the necroptosis in the Aβ 1-42-induced rat models. Our data revealed that RIP1K and RIP3K expressions increase in the AD rat hippocampus. Substantial evidence reveals that necroptosis is involved in AD pathogenesis. In this pathway, RIP1K binds to RIP3K, which in turn binds to MLKL to necrosome formation as an invariable marker of necroptosis activation. Then phosphorylated MLKL undergoes oligomerization and translocation to the plasma membrane, permeabilizing membrane, and ultimately neuronal cell death (Zhang et al. 2017). Numerous studies provide evidence for the presence of the three activated components of the necrosome machinery, i.e., RIP1K, RIP3K, and MLKL, in degenerating neurons in preclinical stages of AD pathology (Koper et al. 2020). These findings may open up a new phase of drug discovery for AD, focusing on identifying these proteins' small molecule inhibitors. A recent study demonstrated that the RIP1K levels have elevated microglia in postmortem cortical samples from AD patients and mouse models. Also, inhibiting the RIP1K kinase activity leads to increased clearance of Aβ in vitro by microglia, reduction of Aβ plaques in the CNS, and improvement in memory deficit in vivo (Ofengeim et al. 2017). In addition to Aβ, tau accumulation and pro-inflammatory cytokine elevation can be crucial for necroptosis activation in AD (Caccamo et al. 2017). So far, few studies have been the focused on the ghrelin effect on the necroptosis pathway. Therefore, we examined the impact of ghrelin injection on necroptosis cell death in a model of Aβ 1–42 -induced AD. Our finding indicated that systematic injection of ghrelin could inhibit necroptosis by reducing RIP1K and RIP3K expressions in the hippocampus of AD rats. This result was consistent with Felix N and et al.'s findings. They reported that, i.p., injection of unacylated ghrelin in rats with muscle damage could inhibits necroptosis by reducing RIP1K and RIP3K proteins (Ugwu et al. 2017).

In addition, this study evaluated the ghrelin effect on the autophagy pathway in the AD rat models via assessment of Beclin-1 expression. Our results demonstrated that Beclin-1 expression was reduced in the Aβ 1–42-induced rat model of AD, and ghrelin treatment could elevates Beclin-1 expression and prevent autophagy failure. Autophagy is a major elimination pathway for abnormal and misfolded proteins. The autophagy failure results in toxic aggregates and is associated with neurodegenerative diseases, such as AD (Cheng et al. 2018). Studies in postmortem human brains and mice models of AD indicate deficiencies in autophagy, contributing to the accumulation of Aβ containing autophagic vesicles within affected neurons in AD (Bolanda et al. 2008). In AD, the maturation of autophagolysosomes and their retrograde transport are impeded, which leads to a massive accumulation of autophagy intermediates and degenerating neuritis in large vesicles (Zhang and Zhao 2015). Beclin-1 is involved in autophagy initiation and autophagosome clearance. This autophagic marker deficiency alters amyloid precursor protein (APP) metabolism and shifts APP distribution to other subcellular compartments generating, such as Aβ peptides. Likewise, overexpression of Beclin-1 can decrease the Aβ deposits by promoting autophagy for neuroprotection and improve cognitive function (Jaeger et al. 2010; Pickford et al. 2008). Therefore, restoring Beclin-1 and autophagy pathways may be novel approaches to treat this disease. In line with our results, recent studies show that ghrelin can alter the expression of autophagy pathway proteins. This hunger hormone activates the autophagy pathway by increasing Beclin-1 expression (Wan et al. 2016). Also, ghrelin treatment of SH-SY5Y cells transfected with APP protein, an AD model, showed that the number of autophagosomes and the expression of LC3-II and Beclin-1 proteins increased compared to the control (Cecarini et al. 2016). On the other hand, stimulation of GHSR1a by ghrelin and its analogs prevents the accumulation of Aβ peptides and promotes destroyed autophagosomes containing Aβ (Jeon et al. 2019). Accordingly, autophagy dysfunction is a known mechanism in the pathogenesis of AD; activating the autophagy pathway can lead to the identification of new therapies approach for AD management.

Recent findings showed crosstalk between the apoptosis, necroptosis, and autophagy pathways. Figure 6 is an overview of the ghrelin effect on crosstalk regulation between apoptosis, necroptosis, and autophagy pathways. Autophagy and apoptosis can mutually inhibit each other. Under apoptotic conditions, the Beclin-1 triggers autophagy cleavage by Bax protein into Beclin-1N and Beclin-1C and causes autophagy failure. Then C-terminal fragment of Beclin-1 translocates to the mitochondria, which induces mitochondrial membrane permeability and apoptosis (El-Khattouti et al. 2013). On the other hand, the cleavage of Beclin-1 disrupts its interaction with Bcl-2 protein and leads to the bind the BH3 domain of Beclin-1 to other anti-apoptotic Bcl-2 family members. The competition of truncated Beclin-1 binding with anti-apoptotic Bcl-2 family members permits pro-apoptotic BH3-only molecules released from the Bcl-2/Bcl-xL complex to initiate intrinsic apoptotic (Wu et al. 2014). So, according to our results, ghrelin may regulate crosstalk between apoptosis and autophagy by increasing BCL-2 and downregulation of Bax and subsequently prevent Beclin-1 cleavage and inhibits apoptosis and activates autophagy in AD rats. In addition to mentioned proteins, caspases have an important role in directing autophagy-apoptosis crosstalk. Caspase-8 can cleave Beclin-1 and lead to autophagic flux inhibition. On the other hand, the autophagy-related proteins Atg4D and Atg5 are cleaved by caspase-3. Noteworthy, caspase-mediated cleavage of Atg5 and Beclin-1 switches autophagy to apoptosis, while truncated Atg4D results in increased autophagic activity.

In return, apoptosis inhibition by autophagy is mediated by the degradation of pro-apoptotic factors. Beclin-1 inhibits caspase-8 activation and subsequently blocks Bid to tBid cleavage, which triggers the reduction of mitochondrial membrane potential (Nikoletopoulou et al. 2013). So, according to our results, increasing in Beclin-1 expression and autophagy activation by ghrelin injection may inhibit apoptosis cascade via caspase-8 inactivation in AD rat models.

Furthermore, caspase-8 is an interfering factor between apoptosis and necroptosis, and the activity of caspase-8 determines apoptosis or necroptosis activation. Caspase-8, with its catalytic activity, can cleave RIP1K and RIP3K proteins, thereby blocking necroptosis (Chen et al. 2018). Therefore, autophagy activation and caspase-8, and apoptosis inhibition by ghrelin may be activating necroptosis by decreasing RIP1K and RIP3K cleavage. On the other hand, as a protective mechanism, autophagy can inhibit necroptosis. Beclin-1 can acts as a negative regulator of necroptosis by incorporation into the necrosome complex of phosphorylated RIP1K/RIP3K/MLKL. Beclin-1 interacts with MLKL and inhibits necroptosis by blocking MLKL oligomerization and the cell membrane perforation (Seo et al. 2020). Therefore, treating AD rats with ghrelin may inhibit the necroptosis pathway via elevation of Beclin-1 expression in the hippocampus. These early observations suggest a link between autophagy and necroptosis pathways. According to these different results, the molecular mechanism of crosstalk between autophagy and necroptosis is elusive and somewhat controversial.

In this study, we demonstrated that ghrelin could inhibit apoptosis and necroptosis pathways and activate autophagy flux in the Aβ 1–42-induced rat model of AD. Also, ghrelin can exert its neuroprotective effects by regulating the crosstalk between these three programmed cell death pathways.

None.

Acknowledgments

This article has been extracted from the Ph.D. thesis written by Mrs Faezeh Naseri in Faculty of Medicine, Shahid Beheshti University of Medical Sciences. This work was supported by the Shahid Beheshti University of Medical Sciences, Faculty of Paramedical Sciences, Tehran, Iran [grant number: 15750].

Author contributions

All authors contributed to the study conception and design. All authors read and approved the final manuscript. Faezeh Naseri contributed in methodology, data collection, data analysis and writing original draft. Majid Sirati-Sabet contributed in methodology, data collection, data analysis, review and editing. Fatemeh Sarlaki, Poneh Mokarram and Morvarid Siri contributed in methodology, data collection and data analysis. Mohammad Keimasi and Rasoul Ghasemi contributed in material preparation and methodology. Zahra Shahsavari and Fatemeh Goshadrou contributed in supervision, project administration, writing, review and editing.

Competing Interests

The authors declare that they have no conflict of interests.

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No competing interests reported.

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Ghrelin regulates crosstalk between apoptosis, necroptosis and autophagy programmed cell death pathways in the hippocampal neurons of amyloid-β 1–42-induced rat model of Alzheimer’s disease

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