Ex vivo investigation of betaine and boric acid function as preprotective agents on rat synaptosomes to be treated with Aβ (1–42)

Recent evidence suggests that ferroptosis, an iron‐dependent cell death process, may be involved in Alzheimer's disease (AD) pathology. The study evaluated the therapeutic potential of betaine and boric acid (BA) pretreatment administered to rats for 21 days in AD. Then, the rats were sacrificed, and morphological and biochemical analyses were performed in brain tissues. Next, an ex vivo AD model was created by applying amyloid‐β (Aβ1‐42) to synaptosomes isolated from the brain tissues. Synaptosomes were analyzed with micrograph images, and protein and mRNA levels of ferroptotic markers were determined. Betaine and BA pretreatments did not cause any morphological and biochemical differences in the brain tissue. However, Aβ (1–42) administration in synaptosomes increased the levels of acyl‐CoA synthetase long chain family member‐4 (ACSL4), transferrin receptor‐1 protein (TfR1), malondialdehyde (MDA), and 8‐hydroxydeoxyguanosine (8‐OHdG) and decreased the levels glutathione peroxidase‐4 (GPx4) and glutathione (GSH). Moreover, ACSL4, GPx4, and TfR1 mRNA and protein levels were similar to the ELISA results. In contrast, betaine and BA pretreatments decreased the levels of ACSL4, TfR1, MDA, and 8‐OHdG in synaptosomes incubated with Aβ1‐42, while promoting increased levels of GPx4 and GSH. In addition, betaine and BA pretreatments completely reversed ACSL4, GPx4, and TfR1 mRNA and protein levels. Therefore, betaine and BA pretreatments may contribute to the prevention of neurodegenerative damage by supporting antiferroptotic activities.


| INTRODUCTION
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that is characterized by the extracellular accumulation of amyloid-β (Aβ) plaques and the intracellular accumulation of neurofibrillary tangles (NFTs) in the brain. 1 AD is one of the leading causes of dementia in the elderly and is associated with cognitive impairment, memory loss, and other symptoms.The accumulation of Aβ plaques and NFTs is thought to cause neurotoxicity and synaptic loss, leading to the cognitive decline observed in AD.Aβ is a peptide that is produced by the cleavage of the amyloid precursor protein (APP), while NFTs are composed of hyper phosphorylated tau protein. 2 The exact mechanisms underlying the formation and accumulation of Aβ plaques and NFTs in AD are not fully understood.However, it is thought that several factors, including genetics, aging, and environmental factors, may contribute to their development.Several therapeutic approaches have been developed to target the accumulation of Aβ plaques and NFTs in AD, including immunotherapy, small molecule inhibitors, and gene therapy. 3However, despite significant efforts, there is currently no cure for AD, and treatment options remain limited.Research into the underlying mechanisms of AD and the development of novel therapeutic approaches remain an active area of investigation.Additionally, early detection and diagnosis of AD, as well as strategies to promote brain health and delay the onset of cognitive decline, are also important areas of focus in the field.
Synaptosomes as subcellular membranous structures or isolated nerve terminals that can be isolated from different parts of the brain. 4naptosomes are widely used as a model system to study various aspects of brain function, including the structure and molecular mechanisms of neurotransmission and neuroplasticity.Synaptosomes are composed of presynaptic nerve terminals and postsynaptic membranes, and they are capable of maintaining a variety of key cellular processes, including protein synthesis, plasma membrane potential, and ion homeostasis. 5These processes allow synaptosomes to remain metabolically and enzymatically active and to maintain their functionality as subcellular structures.One of the advantages of using synaptosomes as a model system is that they closely mimic the properties of intact neurons, allowing researchers to study the mechanisms underlying various aspects of synaptic transmission and plasticity in a controlled environment. 6In addition, synaptosomes can be easily isolated and manipulated, which makes them a valuable tool for studying the effects of drugs and other compounds on synaptic function. 7erall, synaptosomes are an important model system in neuroscience research, and their use has led to significant advances in our understanding of the structure and function of the brain, as well as the development of novel treatments for neurological disorders.
Brain iron accumulation has been increasingly implicated in the neurodegeneration observed in AD. 8 In AD, neuronal cells upregulate ferritin expression and downregulate ferroportin, resulting in increased iron uptake and reduced iron export. 9This leads to elevated levels of free Fe 2+ within cells and increased ferritin, ultimately leading to iron deposition in the brain.The deregulation of ferroptosis has been proposed as a mechanism linking brain iron burden in AD with neurodegeneration.Ferroptosis is initiated when iron-mediated peroxidation of membrane polyunsaturated fatty acids occurs, leading to the formation of lethal lipid hydroperoxides. 10Cell death ensues when the selenoprotein glutathione peroxidase 4 (GPx4), which acts as a checkpoint protector, is overwhelmed.Normally, peroxidation of membrane lipids occurs in cells but is controlled by GPx4, preventing cell disruption and paracrine propagation. 11GPx4 relies on reduced glutathione (GSH) for its activity, and depletion of GSH suppresses GPx4 function, allowing ferroptosis to progress.Considering that patients with AD exhibit iron deposits in their brain cells and excessive iron can exacerbate oxidative damage and cognitive deficits, the use of ferroptosis inhibitors holds promise as a potential therapeutic approach for treating AD.
Boron is an essential trace element that is naturally found in the environment, and it has low toxicity in mammals. 12Boron-containing compounds are ubiquitous in nature and can be found in a variety of foods, including fruits, vegetables, and nuts. 13Boric acid (BA) is one of the most common boron derivatives found in biological systems due to its water solubility.BA is a weak Lewis acid that can act as a proton acceptor, and it is often used as a preservative in food and cosmetic products due to its antimicrobial properties. 14In addition to its use in the food and cosmetic industries, boron and its derivatives have been investigated for their potential therapeutic applications.For example, boron-containing compounds such as boron neutron capture agents (BNCT) have shown promise in the treatment of certain types of cancer. 157][18] However, despite the potential therapeutic benefits of boron-containing compounds, it is important that more research must be conducted to determine safe boron levels.
Betaine is a naturally occurring compound that is nontoxic and has three additional methyl groups. 19It is commonly found in foods such as beets, spinach, and whole grains.A previous research has shown that betaine may have positive effects on several human diseases, including liver diseases, neurodegeneration, and cancer. 20In Alzheimer's disease, betaine has been shown to reduce levels of memory deficits. 21Betaine may also help protect against the development of neurodegenerative damages by regulating gene expression and reducing cell death pathways.
The objective of this study is to investigate comparatively the potential neuroprotective effects of BA and betaine pretreatment against neurodegenerative damage induced by amyloid-β (Aβ1-42) in synaptosomes obtained from rat cerebral cortex.In this study, rats were given appropriate concentrations of BA and betaine for 21 days.
Subsequently, to create an ex vivo AD model, Aβ (1-42) was applied to synaptosomes obtained from rat cerebral cortex.Next, several biochemical parameters were measured in brain tissue and synaptosomes to assess the neuroprotective effects of these compounds.These parameters include levels of acyl-CoA synthetase long chain family member 4 (ACSL4), GPx4, transferrin receptor 1 protein (TfR1), malondialdehyde (MDA), GSH, 8-hydroxydeoxyguanosine (8-OHdG), and total iron ions.Moreover, the histopathology of the brain tissue was examined by hematoxylin-eosin staining, and the morphological structures of the synaptosomes were visualized by transmission electron microscopy.

| Experimental protocol
The study used 21 Wistar albino male rats that were healthy, weighed between 250 ± 20 g, and were 2-3 months old.The rats were housed in rooms with controlled environmental conditions, including a 12-h light/dark cycle, automatic temperature adjustment of 24 ± 2 C, and humidity of 45% ± 5%.During the experiment, the rats were provided with standard pellet rat food and tap water.The cages used were made of polycarbonate material and were transparent, which allowed for easy monitoring of the rats.It is important to note that the use of healthy animals, as well as the provision of adequate housing and nutrition, are essential factors in ensuring the ethical treatment of animals in experimental studies.Kütahya Health Sciences University Animal Experiments Local Ethics Committee gave their approval for the experimental protocol under the decision number 2021.12.03.All animal experiments carried out the National Research Council's Guide for the Care and Use of Laboratory Animals.
The rats were randomly divided into three groups: (I) the control group (n = 7), which received 1 mL saline via gavage for 21 days, (II) the group (n = 7) that received 100 mg/kg/day of BA (prepared with saline) via gavage for 21 days, and (III) the group (n = 7) that received 250 mg/kg/day of betaine (prepared with saline) via gavage for 21 days.The doses and method of BA and betaine administration were determined based on earlier research conducted by Karimkhani et al. 22 and Ganesan et al. 23 All surgical procedures were conducted in a sterile environment using sterile surgical instruments.Twentyfour hours after the last BA and betaine treatment, intramuscular anesthesia of 10 mg/kg xylazine and 50 mg/kg ketamine was administered to the rats.After the animals were anesthetized, they were decapitated by cervical dislocation method.The brain tissues were then removed, and the cerebral cortex regions were dissected on ice.The brain tissue samples were stored at À80 C until the day of the experiment.

| Synaptosomes preparation and neurodegeneration model design
The modified method previously described was used to obtain synaptosomes from rat brains. 24A sucrose gradient was prepared by slowly adding 1.2, 0.8, and 0.3 M sucrose solutions to a centrifuge tube.The rat cortex regions were homogenized with 0.5 M HEPES and centrifuged, and the resulting supernatant was resuspended with solution A and then centrifuged again.The pellet was resuspended in solution B and added to the sucrose gradient tube before being centrifuged.The synaptosomal fraction was collected between the 0.8 and 1.2 M sucrose layers, resuspended with 1 mM NaHCO 3 , and stored at +4 C.
The synaptosome protein levels were determined using the method described by Lowry et al. 25 To create the ex vivo neurodegeneration model, we followed a specific protocol.Each of the synaptosomal fractions consisted of 100 mg of tissue in 3 mL of medium.The groups contained cerebral cortex regions from seven animals.To establish the ex vivo neurodegeneration model, the synaptosomes from the control, BA, and betaine groups were incubated with Aβ (1-42) as described: • Control group-synaptosomes were exposed to saline for 6 h at 37 C.

| Biochemical analyses in brain tissue and synaptosomal fractions
During the analysis, brain tissues were first weighed and then mixed in an ice-cold phosphate buffer solution using a homogenizer (Ultra-Turrax T-25).After the mixture was homogenized, it was centrifuged at 3500 Â g for 10 min at 4 C.The resulting supernatant was then used in biochemical analyses.

| Measurement of ferroptosis biomarkers
In order to investigate the role of BA and betaine in ferroptosis, we measured the levels of GSH, MDA, ACSL4, GPx4, and TfR1, which are important biomarkers of this process.These measurements were taken in both brain tissues and synaptosomal fractions, using commercially available kits (MBS9903690 for ACSL4, MBS934198 for GPx4, E-EL-R1009 for TfR1, E-EL-0026 for GSH, and E-EL-0060 for MDA).
The measurements were taken using a microplate reader (BioTek), following the manufacturer's instructions.

| DNA damage analysis
To determine DNA damage, 8-OHdG levels were analyzed using a commercial kit (E-EL-0028) according to the manufacturer's instructions.Shortly, 50 μL samples were added to each well, followed immediately by 50 μL biotinylated detection Ab working solution.
After incubating for 45 min at a temperature of 37 C, the plate was aspirated and washed three times.Next, 100 μL HRP conjugate working solution was added, followed by incubation for 30 min at 37 C.
After aspirating and washing the plate five times, 90 μL substrate reagent was added and incubated for 15 min at 37 C. Finally, 50 μL stop solution was added, and the plate was read at 450 nm.The concentration of 8-OHdG in samples was determined by comparing the optical density (OD) of the samples to that of the standard curve.
Results were reported as ng/mg protein.

| Total iron assay
The amount of total iron ions in brain tissues and synaptosomal fractions was measured using commercially available ELISA kit (MAK025).
Briefly, to measure levels of total iron in a sample, add 50 μL samples to sample wells in a 96-well plate and bring the volume to 100 μL per well using assay buffer.For ferrous iron measurement, add 5 μL of iron assay buffer to each sample, while for total iron measurement, add 5 μL of iron reducer to each sample well to convert Fe 3+ to Fe 2+ .Mix well and incubate for 30 min at 25 C, protecting the plate from light.After incubation, add 100 μL of iron probe to each well containing standard and test samples, mix well, and incubate for 60 min at 25 C while protecting from light.Finally, measure the absorbance at 593 nm.Results were expressed as nmol/mg protein.

| Quantitative polymerase chain reaction technique
The process of extracting total RNA from both brain tissues and synaptosomal fractions involved using TRIzol ® Reagent (Thermo Fisher Scientific) as per the manufacturer's instructions.In the reverse transcription reaction, 1 μg RNA from each group's samples was used with the Super-Script™ IV One-Step Real-Time polymerase chain reaction (PCR) System (Thermo Fisher Scientific).The amplification of complementary DNAs (cDNAs) was examined using the StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific), using SYBR Green Master Mix (Applied Biosystems).Amplification conditions were as described in our previous study (Hacioglu, 2022).Using the 2 ÀΔΔCt method, the relative expression levels of ACSL4, GPx4, and TfR1 were calculated, with β-actin serving as the internal standard.The results were presented as fold change from control.

| Western blot analysis
To determine the protein levels of ACSL4, GPx4, and TfR1 in brain tissues and synaptosomal fractions, a Western blot analysis was performed.The procedure followed the methodology described in our previous study. 26Briefly brain tissues and synaptosomal fractions lysed with RIPA buffer.The resulting lysate was loaded onto a 10% SDS-PAGE gel for electrophoresis, and the separated proteins were transferred to a nitrocellulose membrane.The membrane was blocked and incubated with primary antibodies against ACSL4, GPx4, and TfR1.
After washing to remove unbound antibodies, the membrane was incubated with secondary antibodies conjugated to horseradish peroxidase (HRP).The target protein bands were visualized, and the band intensities were analyzed using Image J software.The dilutions of the antibodies used were as follows: ACSL4 antibody at a dilution of 1:2000 (PA5-27137), GPx4 antibody at a dilution of 1:1000 (PA5-102521), TfR1 antibody at a dilution of 1:2000 (MA5-32500), and β-actin antibody at a dilution of 1:2000 (PA1-183; Invitrogen).HRP-conjugated anti-rabbit antibody (dilution 1:2000; Cell Signaling) and anti-mouse immunoglobulin G antibody (dilution 1:5000; Cell Signaling) were employed as secondary antibodies to detect primary antibodies.For protein band visualization, an enhanced chemiluminescence kit (34 579, Thermo Scientific) employing chemiluminescence was utilized.

| Imaging of synaptosomes by transmission electron microscopy
Transmission electron microscopy (TEM) analysis was used to compare the morphology of untreated (control) and treated (BA and betaine) synaptosomes.The synaptosomes were initially centrifuged and fixed the resulting pellet with glutaraldehyde 2.5% glutaraldehyde at room temperature for 2 h, and then centrifuged at 18 000 Â g for 10 min.The synaptosomes were then centrifuged again at 16 000 Â g for 10 min at +4 C, and the resulting pellets were washed twice with 0.1 M pH 7.4 phosphate buffer and fixed with 1% osmium tetroxide at room temperature for 2 h.The synaptosomes were then stained with uranyl acetate and lead citrate for 4 h, dehydrated with increasing concentrations of ethanol, and finally embedded in EPON resin.Sections were obtained from the embedded synaptosomes and were photographed using a JEOL JEM 1220 transmission electron microscope.

| Histological analysis of brain tissues
Hematoxylin-eosin (H&E) staining was performed to examine the histopathological effects of BA and betaine treatment in the brain tissue of rats.To prepare brain tissues for histological analysis, they were first fixed in 10% neutral formaldehyde.Next, they were immersed in a buffered neutral formaldehyde solution for 24 h to complete the chemical fixation process.The tissue was then dehydrated using increasing alcohol concentrations, made transparent using xylol, and blocked using paraffin.Sections (5 μm) were cut using a microtome and placed on poly-l-lysine coated slides after the paraffin was opened in a water bath.The sections were deparaffinized with xylol and stained with H&E after being kept overnight in a 37 C oven.The preparations were made permanent by sealing them with entellan.
The brain sections stained with H&E were examined at the light microscope level for histological analysis.All tissue sections were examined using the light microscope.

| Histopathology analysis in brain tissue
Figure 1 (A-F) displays light microscopy images of brain tissue sections from all of the experimental groups.Normal neurons and glial cells were present in the cortical area in the control, BA, and betaine groups.When the cerebral cortices were examined with H&E staining, it was observed that the tissues of the control group, BA group, and betaine group had normal histological structure.Neurons and glial cells appeared normal in all groups, and no pathological changes were observed in any animals (Figure 1).Thus, according to H&E staining, our results showed that BA and betaine treatments for 21 days did not cause any histopathological effect on the brain tissue.

| Biochemical markers in brain tissue
The study initially examined the levels of GSH, MDA, ACSL4, GPx4, and TfR1 in brain tissue by using ELISA measurement.The objective of the study was to investigate the effect of BA and betaine on the balance between oxidants and antioxidants in brain tissues.
The results from Figure 2A indicated that 100 mg/kg/day of BA and 250 mg/kg/day of betaine doses insignificantly decreased the MDA levels, which is a marker of lipid peroxidation ( p > .05).On the other hand, GSH levels displayed a meaningless increase in betaine group compared to the control group ( p > .05; Figure 2B).Furthermore, BA treatment provided a 19.5% increase in GSH levels in the brain tissue compared to the control group ( p = .0148).These results showed that the treatment of BA and betaine for 21 days could support antioxidant capacity while not affecting oxidative stress and lipid peroxidation in rat brain tissue.
The levels of ACSL4, GPx4, and TfR1 were then compared between untreated and treated rats.The results indicated that there was an insignificant reduction in the ACSL4 levels in brain tissue treated with BA, as shown in Figure 2C ( p > .05).In contrast, BA treatment caused a 12.8% increase in GPx4 levels compared to the control group ( p = .0209;Figure 2D).It is worth noting that there was no significant change in betaine groups ( p > .05).Moreover, treatment with BA and betaine in brain tissue did not cause any change in TfR1 levels compared to the control group ( p > .05; Figure 2E).
The study aimed to investigate if BA and betaine treatments caused DNA damage in brain tissue, by measuring 8-OHdG levels.
After 21 days of treatment, results showed that BA and betaine treatments had no significant effect on 8-OHdG levels in brain tissue (p > .05 vs. control; Figure 2F).
As for total iron ions levels, there was no change in the rats' brain tissue treated with BA and betaine compared to the control group (p > .05; Figure 2G).

| Analysis of mRNA and protein levels of target genes in the brain and synaptosomes
Consistent with the ELISA results, the mRNA levels of ACSL4, GPx4, and TfR1 showed variations depending on the BA and betaine treatments (Figure 3).In brain tissues, there was no difference in ACSL4 and TfR1 mRNA levels (p > .05 vs. control; Figure 3A,C), respectively, while there was a moderate nonstatistical increase in GPx4 mRNA levels in the BA group (p > .05 vs. control; Figure 3B).According to Western blot analysis, no statistically significant difference was detected in ACSL4, GPx4, and TfR1 protein levels ( p > .05 vs. control; Figure 3D).In contrast, synaptosomes treated with BA and betaine demonstrated an increase in GPx4 protein levels, suggesting a protective effect against Aβ (1-42) exposure.Additionally, the levels of ACSL4 and TfR1 proteins decreased in synaptosomes treated with BA and betaine, despite the presence of Aβ (1-42).These findings suggest that BA and betaine pre-treatments may counteract the effects of Aβ (1-42)   by regulating the expression of these proteins in synaptosomes.

| TEM images of synaptosomes
TEM analysis was performed to evaluate the morphological structures of synaptosomal fractions obtained using the sucrose gradient method.The results presented in Figure 5 showed that the isolated synaptosomes contained synaptic vesicles (SV) with uniform morphology.Additionally, synaptic connections (SJ) were prominently featured F I G U R E 2 Effects of boric acid and betaine treatment on rat brain tissues.A: MDA levels, B: GSH levels; C: ACSL4 levels, D: GPx4 levels; E: TfR1 levels; F: 8-OhdG levels; G: Total iron levels.*p < .05compared to the control group.8-OHdG: 8-hydroxydeoxyguanosine; ACSL4: acyl-CoA synthetase long chain family member 4; GPx4: glutathione peroxidase 4; GSH: reduced glutathione; MDA: malondialdehyde; and TfR1: transferrin receptor 1 protein.in all images, indicating that the isolated nerve terminals were functional.The TEM images of the control group synaptosomes showed smooth SV and SJ with preserved membrane integrity (Figure 5A).This preservation of synaptic vesicle structures allowed for the continued activity of the synaptosomes by ensuring the mitochondria (MT) remained within the vesicles.Treatment with BA and betaine did not result in any significant morphological changes in synaptosomes, and SVs, and SJs remained organized and intact, similar to the control group (Figure 5B,C).This suggested that BA and betaine treatment did not have any adverse effects on the structural integrity of synaptosomes.
Moreover, in the betaine group, there was a 32.5% increase in GSH levels compared to the Aβ (1-42) group ( p = .0277).
In Figure 6E, the synaptosomes showed a significant increase

| DISCUSSION
Synaptosomes, as subcellular preparations enriched in detached and resealed synaptic terminals, have been widely used as an ex vivo model system to investigate the pathophysiology of AD. 27 They provide a valuable tool for studying the effects of toxic intermediates, such as Aβ and phosphorylated tau, on synaptic function and their interactions with downstream effectors.Excessive synapse loss is a prominent feature of AD and is closely associated with cognitive decline.The loss of synapses occurs early in the progression of the disease and is considered a critical event in AD pathology. 28One of the primary contributors to synapto-toxicity in AD is the oligomeric  29 These soluble Aβ oligomers can disrupt synaptic function and impair neurotransmission, leading to synaptic dysfunction and ultimately synapse loss.Moreover, increasing evidence suggests a connection between dysregulated brain iron and the progression of AD, and this dysregulation has been implicated in the mechanism of neurodegeneration known as ferroptosis. 30,31Our results of the role of BA and betaine in synaptosomes treated with Aβ (1-42) has been expanded by biochemical analyses demonstrating that disruptions in synaptosomes function, either through lipid or DNA damages, make synaptosomes more susceptible to ferroptosis.For the first time in this study, we found that BA and betaine pre-treatment did not cause any morphological and biochemical changes in rat brain tissue.Moreover, we showed that BA and betaine pretreatments suppress ferroptosis by providing TfR1 regulation in Aβ (1-42) administered synaptosomes isolated in brain tissue.These findings open up new therapeutic strategies for interpreting the involvement of BA and betaine in AD and provide fresh insights into the potential mechanisms underlying the disease.
Iron is a crucial element for various biological processes in the brain.It is vital for fulfilling the high energy and metabolic requirements of neuronal tissue. 32However, disturbances in iron balance within the brain can lead to adverse effects.Iron dyshomeostasis can result in oxidative stress, which refers to an imbalance between the production of reactive oxygen species (ROS). 33Iron-related disturbances in the brain, such as iron overload or iron deficiency, can disrupt normal physiological processes and contribute to the development or exacerbation of neurological disorders. 34Indeed, accumulating evidence suggests a strong association between AD pathology and disturbances in iron homeostasis, as well as the involvement of ferroptosis. 35The transferrin (TF)/TfR pathway is a crucial mechanism for iron uptake into cells, including neurons, oligodendrocytes, and astrocytes. 36Iron in its inactive form (Fe 3+ ) is bound to transferrin (TF), a glycoprotein that transports iron in the blood.
The TF/Fe 3+ complex interacts with TfR1 on the cell surface, allowing the complex to be internalized into the cell via receptor-mediated endocytosis. 37Once inside the cell, the Fe 3+ is released from TF and is then available for cellular processes.TfR1 plays a dual role in iron metabolism and ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation.TfR1 not only facilitates the uptake of iron from the extracellular environment into the cell but also plays a role in intracellular iron redistribution. 38Inside the cell, TfR1 can promote the synthesis of ferritin, which is an iron storage protein.This contributes to maintaining the cellular iron pool and providing the necessary iron for cellular functions, including those involved in ferroptosis. 39Disruption of iron metabolism and dysregulation of the Tf/TfR pathway can lead to iron dyshomeostasis, which is associated with various pathological conditions, including neurodegenerative diseases.Regarding the TfR pathway, several studies have presented different findings regarding the alterations of TfR in AD.However, the data remain contradictory and lacks a definitive interpretation.As an example, Huang et al. 40 initially documented an increase in the expression of TfR1 in APP/PS1 transgenic mice, which was deemed crucial for the generation and accumulation of Aβ.Furthermore, the administration of a specific antibody targeting TfR1 in APP/PS1 transgenic mice led to a reduction in Aβ aggregation.On the contrary, contrary to earlier findings, there is a report indicating that the expression of TfR1 does not consistently increase throughout the progression of AD. 41 According to this report, TfR1 is upregulated during the early stages of AD, specifically in 3-month-old APP/PS1 Tg mice.However, the expression of TfR1 starts to decline when the mice reach 6 months of age.Furthermore, the researchers provided evidence suggesting that the nonaggregated form of Aβ may have significant involvement in both upregulating TfR expression and facilitating iron-induced production of Aβ during the initial stages of AD.In this study, we examined the levels of TfR1 levels in brain tissue and synaptosomes untreated/treated with Aβ (1-42).The rats after administering BA and betaine for 21 days, the levels of TfR1 mRNA and protein remained unchanged in brain tissues.In addition, BA and betaine pretreatment did not cause any difference in total iron levels.This inactivation results in the accumulation of lipid peroxidation, triggers the generation of additional ROS, and ultimately leads to ferroptosis. 43Recent research suggests that modulating ferroptosis could potentially offer benefits for neurodegenerative diseases.In particular, inhibiting ferroptosis through the action of GPx4 has been proposed as a protective mechanism against neurodegeneration.Studies have revealed a correlation between the low activity of GPx4 and the GSH system and the occurrence of ferroptosis in neurodegenerative processes. 44Notably, GPx4 has been found to exhibit reduced expression in AD.Suppression of GPx4 is considered a crucial factor contributing to the pathogenesis of ferroptosis in AD.In Gpx4BIKO mice, where GPx4 is knocked out specifically in the brain, various characteristics associated with AD pathology, including cognitive impairment and neurodegeneration in the hippocampus, have been observed. 45Moreover, these brain regions also exhibit increased lipid peroxidation, a hallmark closely linked to ferroptosis.Treatment with ferroptosis inhibitors has shown promising results in improving cognitive impairment and alleviating neurodegeneration in these mice.In APP NL-G-F knock-in AD mice, Majernikova and colleagues 46 demonstrated the upregulation of GPx4 and the downregulation of GSH and ACSL4 compared to WT mice.Although these findings indicate differential expression of these ferroptosis-related genes in APP NL-G-F knock-in AD mice, it is known that downregulation of GPx4 and upregulation of ACSL4 can promote the induction of ferroptosis.By inhibiting ACSL4, the availability of PUFAs for phospholipid synthesis is reduced, leading to a decreased susceptibility to lipid peroxidation and subsequent ferroptosis. 47Thus, suppressing ACSL4 activity effectively hampers the initiation of ferroptotic cell death.As for our study, we made several observations.Firstly, we found that the treatment of rats with BA and betaine for 21 days resulted in increased GPx4 and GSH levels in brain tissues, leading to the induction of antiferroptotic activity.Secondly, we found that Aβ (1-42) exposure of synaptosomes disrupted cellular iron homeostasis.This disruption subsequently led to an increase in MDA, ACSL4, TfR1, 8-OHdG, and total iron levels and a decrease in GPx4 and GSH levels.However, in the synaptosomes of rats pretreated with BA and betaine, we observed decreased levels of MDA, ACSL4, TfR1, 8-OHdG, and total iron levels and enhanced levels of GPx4 and GSH.Collectively, these findings suggest that the Aβ (1-42) exposure could lead to an increased iron burden within the synaptosomes and facilitate ferroptosis.Therefore, targeting TfR1 may represent a potential approach to suppress ferroptosis in AD.Overall, our study suggests that BA and betaine treatment might regulate cellular death pathway, including ferroptosis, in brain tissues by modulating the expression levels of TfR1 and ferroptotic markers.

| CONCLUSIONS
Synaptosomes are a type of ex vivo model system that closely mimic the in vivo conditions of synaptic function and are therefore suitable for studying neurodegenerative diseases such as AD.Understanding the complex interplay between iron metabolism, the TfR pathway and ferroptosis is crucial for unraveling the mechanisms underlying neurodegenerative diseases and developing potential therapeutic strategies to modulate iron homeostasis and prevent iron-induced cell damage.
According to this study, BA and betaine plays a significant role in regulating the susceptibility of synaptosomes to ferroptosis.It achieves this by inhibiting the expression of TfR1, which has multiple functions related to iron homeostasis.Activation or inhibition of TfR1 can have an impact on ferroptosis.Furthermore, TfR1 has been found to have a positive regulatory effect on ferroptosis.It is worth noting that there are currently no specific inhibitors available for targeting poly(rC)-binding protein 1 (PCBP1), which limits its potential for targeted therapy.The study demonstrated that regulation of TfR1 by BA and betaine pretreatment could modulate ferroptosis in synaptosomes exposed to Aβ (1-42).The results of this study may help to elucidate the mechanisms underlying the neuroprotective effects of BA and betaine and may provide new insights into the development of novel therapies for AD.However, further research is needed to fully understand the potential therapeutic applications of these compounds and their mechanisms of action in the context of neurodegenerative diseases.In addition, it is important to note that in vitro and ex vivo models have their own limitations as well.For example, these models do not fully capture the complexity of the brain and may not accurately reflect the in vivo conditions.Therefore, it is crucial to use a combination of in vitro, ex vivo, and in vivo models when studying neurodegenerative diseases.
However, TfR1 levels increased in synaptosomes after Aβ(1-42)   administration.Interestingly, in synaptosomes isolated in brain tissue treated with BA and betaine, TfR1 mRNA and protein levels showed a decrease compared Aβ(1-42)-treated group.To investigate the differences in iron metabolism induced by Aβ (1-42) exposure, we evaluated total iron levels in brain tissue and synaptosomes.Pretreatment with BA and betaine for 21 days caused no change in total iron levels in rat brains.Consistent with the TfR1 results, Aβ (1-42) administration increased total iron levels in synaptosomes, while BA and betaine pretreatments reversed this situation.Consequently, Aβ(1-42)induced synaptosomal toxicity was reversed in rats' brain synaptosomes treated with BA and betaine.These findings suggest that BA and betaine plays a crucial role in regulating intracellular iron levels by modulating the expression of TfR1 in response.Furthermore, dysregulation of TfR1 and alterations in iron metabolism have been implicated in the initiation and progression of ferroptosis, a form of regulated cell death that involves iron-dependent lipid peroxidation.Lipid peroxidation byproducts are very powerful triggers of ferroptosis.42This process can happen through enzymatic and nonenzymatic processes known as Fenton-type chemistry.GPx enzymes, specifically GPx4, are responsible for inhibiting lipid peroxidation by reducing phospholipid hydroperoxide (LOOH) to lipid alcohols (LOH) and preventing the formation of reactive lipid alkoxy (LO•) groups.Several steps are involved in the process of lipid peroxidation, starting with ACSL4 that catalyzes the esterification of arachidonoyl (AA) or adrenoyl acids (AdA) into phosphatidyl ethanolamine (PE).Finally, 15-LOX oxidizes AA-PE and AdA-PE into ferroptotic signals.During the process of ferroptosis, an important enzyme called GPx4 utilizes GSH as a substrate to catalyze the conversion of LOOH into LOH.As a part of this reaction, the sulfhydryl group in GSH is oxidized, leading to the formation of oxidized glutathione disulfide (GSSG).However, when the levels of reduced GSH decrease, GPx4 becomes inactivated.