Expression of SLC25A18 is positively correlated with Alzheimer's disease

doi:10.21203/rs.3.rs-1955488/v1

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Expression of SLC25A18 is positively correlated with Alzheimer's disease

https://doi.org/10.21203/rs.3.rs-1955488/v1

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Background: According to research, the neurotoxicity caused by the accumulation of the excitatory neurotransmitter glutamate is closely related to Alzheimer's disease. However, even after 50 years of research, little is known about the relationship between glutamate-related carriers and Alzheimer's disease, and it is unclear whether glutamate metabolism target genes are involved in the occurrence and development of AD.As a result, the goal of our investigations was to identify glutamate metabolism target genes involved in the onset and progression of AD, as well as to validate and mine the relationship between them.

Results: In this paper, we analysed the Gene Expression Omnibus (GEO) data further and discovered that the expression of glutamate carrier SLC25A18 is correlated with Alzheimer's disease by screening the differential genes in different regions of the brain of Alzheimer's disease patients. To verify the expression of SLC25A18 during Alzheimer's disease development, we created animal models (5×FAD transgenic AD animal model, chemically induced AD animal model, natural ageing animal model), and the results showed that the expression of SLC25A18 increased when Alzheimer's disease occurred in various animal models. After that, we checked the expression of SLC25A18 in Aβ1-42-induced Neuro 2A cells, and the results revealed that neurons had higher SLC25A18 expression after Aβ1-42 induction.

Conclusions: The results indicate that SLC25A18 is related to the onset and progression of Alzheimer's disease and that it could be a target for treatment.

Alzheimer's disease

SLC25A18

Glutamate

Mitochondria

Alzheimer's disease (AD) is a slowly progressing neurodegenerative disease characterized by the loss of neurons and synapses in the cerebral cortex, resulting in cognitive impairment and memory loss. The prevalence of Alzheimer's disease has risen steadily over the last 30 years, and by 2050, the global prevalence of the disease is expected to triple [1]. Scholars' efforts to combat Alzheimer's disease have never ceased in this dire situation. Scholars have proposed the amyloid cascade hypothesis and the Tau hypothesis in nearly 50 years of research, and believe that their accumulation is the primary factor leading to pathological changes [2]. Other potential AD initiating factors proposed by researchers include energy imbalance, metabolic dysregulation, infection, and toxins [3; 4]. However, no complete cure for AD has been discovered, so we will continue to work hard to discover new AD target genes.

Glutamate plays a role in almost every aspect of normal and abnormal brain activity. Glutamate is not only a neurotransmitter that excites neurons, but it is also a precursor and intermediate in metabolism. Olney's pioneering study provided the first evidence of the neurotoxicity of the excitatory neurotransmitter glutamate fifty years ago [5]. Multiple studies since then have confirmed that excessive glutamatergic signalling is a key feature of AD [6–8].

The glutamate in the synaptic cleft is primarily derived from the glucose metabolism pathway and the astrocyte glutamate uptake and recycling pathway. However, regardless of where glutamate comes from, its final oxidative metabolism necessitates the involvement of mitochondria. As a result, mitochondria play an important role in the theory of glutamate excitotoxicity and have emerged as the primary focus of AD drug development. However, no completely reliable drugs have been discovered among the many treatments targeting mitochondria (e.g., NMDAR inhibitors, antioxidants, mtPTP inhibitors, uncoupling agents, and so on). This demonstrates that the components involved in glutamate metabolism in mitochondria still require further investigation.

We discovered for the first time, using bioinformatics datasets, that the mitochondrial membrane glutamate transporter SLC25A18 is associated with AD. The use of AD animal and cell models confirmed that SLC25A18 expression was significantly increased, implying that SLC25A18 may be involved in the occurrence and progression of AD. This study introduces a novel concept for studying the pathogenesis of AD, as well as a new target for Alzheimer's prevention and treatment.

Bioinformatics investigation of SLC25A18

Enter the URL (https://www.ncbi.nlm.nih.gov/pubmed/) to download GSE5281 elderly AD gene expression data from the Gene Expression Omnibus (GEO) database of the National Center for Biotechnology Information (NCBI). The platform used was GPL570 [HG-U133 Plus 2] Affymetrix Human Genome U133 Plus 2.0 Array. There are 150 patients in total. The dataset is based on brain samples collected from three AD centres (Ariz. Alzheimer's Center, Duke University Alzheimer's Center, and Washington University Alzheimer's Center). The search keywords are 6 brain regions, they are: 1) entorhinal cortex (EC); 2) hippocampus (HIP); 3) temporal association cortex (TeA); 4) posterior cingulate (PC); 5) superior frontal gyrus (SFG); 6) primary visual cortex (PVC). Finally, 74 tissue samples in the control group and 87 tissue samples in the AD group were retrieved.

R software was used to analyze the gene expression data of the GSE5281 dataset respectively. After the t-test method, P < 0.05 and |log2FC| ≥ 1.5 were used as the screening conditions for differentially expressed genes (DEGs). DEGs were analyzed using Venny 2.1.0 (http://bioinfogp.cnb.csic.es/tools/venny/) to obtain co-expressed DEGs. Co-expressed DEGs were searched on the GeneCards website (http://www.genecards.org/) for their aliases and molecular functions.

Animal treatment

5×FAD mice (APP/PS1 transgenic mice were transfected with five familial inherited AD genes [APPK670N/ M671L (Swedish) + I716V (Florida) + V717I (London) + PS1 M146L + L286V]) were obtained from the Shanghai Model Organisms Limited Company (Shanghai, China). The wild type mice (C57BL/6J) were obtained from the Beijing HFK Bioscience Limited Company (Beijing, China). All experimental mice were male. The animals were housed in standard laboratory cages under a 12-h light/12-h dark cycle.

We divided the mice into three groups: 1)5×FAD transgenic AD (Tg-AD) animal model experimental group. 5×FAD mice were raised to 6 months of age, and wild type mice of the same age were chosen as the WT group. 2)Chemically induced AD animal model. For three months, 3-month-old wild type mice were treated orally by gavage with 20mg/kg/d AlCl₃ and intraperitoneally with 120 mg/kg/d D-galactose (D-gal) or the same volume of the corresponding vehicle (physiological saline). 3)Natural ageing animal model experimental group. The Young group consisted of 6-month-old wild type mice, while the Aged group consisted of 24-month-old wild type mice.

Morris water maze

The Morris water maze (MWM) test was performed to assess the learning and spatial memory of mice. The circular pool was filled with water containing dissolved milk powder and divided into four quadrants of equal area. A platform was hidden in the first quadrant at a position 1.2 cm beneath the surface of the water. Animals were individually started from one of the middles of the four quadrants facing the wall of the pool to train to find the platform for five consecutive days, four trials per day with a 30 s interval. A stationary array of cues outside the pool tub was used for mice to establish spatial orientation. For the training test, mice were allowed to find the platform within a period of 60 s and stayed on the platform for another 30 s. If the animal failed to find the platform within 60 s, we recorded the time as 60 s. Then, these mice were guided onto the platform and allowed to stay for 30 s. The probe trial was performed 24 h after the training test. The platform was removed and mice were individually placed into the quadrant as opposed to the location of the original platform. The time for the animal to arrive at the site of previous platform placement were automatically recorded by a video camera.

Sample preparation

Mice were sacrificed by exsanguination. A portion of each tissue sample was fixed with a 4% paraformaldehyde (Biosharp, USA) solution to allow the preparation of paraffin sections for immunofluorescent analyses. A portion of each tissue sample was immediately frozen in liquid nitrogen and stored at -80℃ until use.

Immunofluorescent staining and evaluation

Immunofluorescence staining was carried out on paraffin sections. Tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed with the Improved Citrate Antigen Retrieval Solution (Beyotime, China) for 5 min. Sections were blocked with 10% serum in TBS for 30 min at room temperature and stained with primary antibodies overnight at 4℃. Primary antibodies were as follows: anti-SLC25A18 (Proteintech, 17348-1-AP, 1:200), anti-beta Amyloid 1–42 (Abcam, ab201061, 1:1000). HRP-conjugated secondary antibodies (Alexa Fluor® 594) (Abcam, ab150080, 1:200) were applied for 1 hr at room temperature in dark. Stained slides were washed again in TBS before counterstaining with DAPI (Beyotime, China) and covered with cover glass. Images were taken on Zeiss Axio Imager Z2, using ImageJ software for image analyses.

RNA extraction, cDNA synthesis, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA from cells was extracted with Trizol reagent (Invitrogen, USA) or from a conditioned medium by RNA extraction kit (Qiagen, USA) according to the manufacturer's instructions. cDNA was synthesized from each 1 µg RNA sample by Reverse Transcription kit (TransGen Biotech, China), followed by RT-PCR reactions on a real-time detector (Bio-Rad, CFX96, USA) using an SYBR Green PCR kit (Roche, China). The detailed steps were carried out as follows: pre-denaturation at 95°C for 10 min followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 60 sec and elongation at 60°C for 60 sec. The primer sequences of SLC25A18 and GAPDH were: SLC25A18 forward, 5’-GCATCTCCGCAAAACTCATCAAT-3’; SLC25A18 reverse, 5’-TCCCCTGCTGGTTCTGTAGTC-3’; GAPDH forward, 5’-CCCCAATGTGTCCGTCGTG-3’ and GAPDH reverse, 5’-GCCTGCTTCACCACCTTCT-3’. The relative quantification of gene expression result was calculated by ΔCT-method and expressed as 2-ΔCT with setting GAPDH as the endogenous control.

Synthesis and Preparation of Aβ1–42

To a final concentration of 1 mmol/L, Aβ1–42 powder (GL Biochem, China) was added to pre-chilled 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) (Macklin, China). The mixture system was mixed and incubated at room temperature for 60 minutes. The Aβ-HFIP solution was kept on ice for an additional 10 minutes before being aliquoted in Eppendorf tubes under a fume hood to allow HFIP evaporation overnight. On day 2, the dried peptide film was completely dissolved in 5 mmol/L dimethyl sulfoxide (DMSO) (Solarbio, China). Without phenol red, dilute to 100 mol/L in DMEM/F12 medium (Procell, China). After 1 week of incubation at 4°C, centrifuge the supernatant at 14 000 g for 10 minutes in a refrigerated high-speed centrifuge.

Cell Culture

Neuro 2A cells (N2a) (ATTC, USA) were cultured in Dulbecco's Modified Eagle medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Clark, USA) and 1% antibiotics (penicillin and streptomycin) (Solarbio, USA). Cultures were maintained in a humidified incubator with 5% CO₂ at 37℃.

In vitro cytotoxicity of Aβ1–42 and L-Glutamine

N2a were seeded at a density of 0.5×10⁴ cells per well in 96-well plates and incubated overnight before being analyzed. L-Glutamine (L-Glu) (Solarbio, USA) or Aβ1–42 were used to treat the cells prepared as stock solutions in DMEM. The cells were incubated for 24 hours. Each well received 20 µl of 5 mg/ml MTT solution (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (Solarbio, China) to incubate for another 4 h at 37°C. The medium was then replaced with 150 µL DMSO. After completely dissolving the formazan, the absorbance at 490 nm was measured to calculate the cell proliferation profiles.

Cell treatment

Before performing various analyses, cells were seeded in 6-well plates at a density of 6.5×10⁴ cells per well and incubated overnight. Aβ1–42 treatment was carried out in DMEM (supplemented with 10% FBS and 0.01mmol/L L-Glu) at 10 mmol/L for 24 hours. The control cells were given DMEM supplemented with 0.01mmol/L L-Glu.

Protein extraction and Western Blot Analysis

For western blotting assays, cell lysates were arranged from cell lines with a RIPA lysis buffer kit (Beyotime Biotechnology, China), and the protein concentrations were quantified with a BCA protein quantification kit (Thermo, China). Equal amounts of protein were separated onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Solarbio, China) and transferred to a polyvinylidene difluoride (PVDF) (Merck Milliporemembrane, USA). The membrane was incubated overnight at 4℃ in primary antibodies in TBST buffer. Target proteins include SLC25A18 (Proteintech, 17348-1-AP, 1:200), and β-actin (Proteintech, 66009-1-Ig, 1:5000). The membrane was washed three times for 5 min each in TBST buffer to remove unbound primary antibodies. Incubation with HRP-conjugated Affinipure Goat Anti-Rabbit IgG or Anti-Mouse IgG (Proteintech, SA00001-1/SA00001-2, 1:2000) for 60 min at room temperature was followed by another three washes with TBST buffer. The membrane was incubated in Western ECL substrate (Thermo fisher or Proteintech, USA) and exposed to a Tanon imager, using ImageJ software for image analyses. The expressing amount of each protein was semi-quantitatively identified and the densities of blots for all proteins were normalized to β-actin.

Statistical Analysis

All experiments were performed at least three times. Data were expressed as mean ± SD and the statistical comparisons were performed using one-way analysis of variance (ANOVA) with subsequent Tukey's test. The data was considered to be statistically significant if p < 0.05.

Differentially expressed genes in AD.

We screened the GSE5281 dataset in the GEO database to look for unreported genes associated with AD. Differential expression analysis was performed on the data of gene expression profiles in six brain regions. A total of 2306 differentially expressed genes (DEGs) were identified in brain tissue samples from the AD and control groups, with 584 up-regulated genes and 1758 down-regulated genes (shown in Fig. 1a). There were no DEGs that were upregulated in the posterior cingulate or primary visual cortex.

The expression of SLC25A18 in multiple regions is positively correlated with AD.

The Venny 2.1.0 tool was used to intersect the up-regulated genes in EC, HIP, TeA, and SFG, yielding 67 up-regulated co-expression differential genes (shown in Fig. 1b). Then, one by one, we queried these genes on the GeneCards website to learn more about their molecular functions. SLC25A18, a multi-domain co-expressed DEG, was discovered to be an important glutamate transporter on the mitochondrial membrane. Mitochondrial dysfunction caused by disruptions in glutamate uptake, transport, and metabolism [9; 10] is important in neurodegenerative diseases such as AD [11]. SLC25A18 piqued our interest as a key glutamate translocator at the mitochondrial membrane.

The expression of SLC25A18 is elevated in the 5×FAD transgenic AD animal model.

Among the many theories about AD, the amyloid cascade hypothesis remains dominant, and Aβ accumulation is thought to be one of the primary factors leading to pathological changes [2]. We chose 5×FAD (familial Alzheimer's disease, FAD) transgenic mice as our model. A large amount of Aβ deposition began to appear in the brain of the transgenic mice when they were 4 to 5 months old, and the transgenic mice displayed spatial cognitive dysfunction [12]. As a result, 5×FAD transgenic mice can accurately mimic the pathological process in the AD brain.

We used RT-PCR to detect mRNA in brain tissue homogenates from 6-month-old WT mice and Tg-AD mice and discovered that SLC25A18 mRNA expression was increased in Tg-AD mice's brains (shown in Fig. 2a). We then stained mouse brain sections for Aβ and SLC25A18 with immunofluorescence and discovered Aβ deposition in the cortex and hippocampus of Tg-AD mice brains, as well as elevated expression of SLC25A18 in the same regions (shown in Fig. 2b). Based on the bioinformatics results, we looked at the EC, TeA, and HIP regions and discovered that SLC25A18 expression was higher in Tg-AD mice than in WT mice (shown in Fig. 2c). SLC25A18's elevation in the CA region was primarily in the CA3 region.

The expression of SLC25A18 is elevated in the chemically induced animal model.

AlCl₃ and D-gal can cause pathological changes in mice that mimic AD, such as cognitive impairment, oxidative stress, decreased immune response, and changes in gene transcription [13–15]. As a result, chemical induction has become one of the modelling methods for studying AD mechanisms and drug screening.

Behavioural testing was performed on AlCl₃ plus D-gal-induced AD model mice. The test results showed that the Model group mice spent significantly more time in the target position than the Control group mice (shown in Fig. 3a), indicating that the modelling was successful. Following that, immunofluorescence staining was performed on brain sections from both groups of mice. The expression of SLC25A18 was found to be higher in the EC, TeA, and CA3 regions of the brains of the Model group mice than in the Control group mice (shown in Fig. 3b).

The expression of SLC25A18 is elevated in the natural ageing animal model.

Ageing is one of the primary causes of AD, and the natural ageing animal model is a model that simulates age-related cognitive dysfunction and pathological changes in AD. In this study, wild-type mice were reared to the age of 24 months to create an animal model of ageing [16].

We stained brain sections of 6-month-old and 24-month-old WT mice with Aβ immunofluorescence and discovered that the 24-month-old mice had already developed Aβ pathological deposition (shown in Fig. 4a). This demonstrates that the model was successfully constructed. The brain sections of the two groups of mice were then immunofluorescently stained for SLC25A18. In aged mice, the expression of SLC25A18 was found to be increased in EC, TeA, and CA3 (shown in Fig. 4b).

The expression of SLC25A18 is elevated in the Aβ1-42-induced Neuro 2A cells model.

Increased extracellular glutamate levels and NMDA receptor activation have been linked to cognitive deficits and neuronal loss in the brains of AD patients [17; 18]. As a result, we used Aβ1–42 to induce neurons and a small amount of L-Glu to the culture medium to simulate excitatory neurotoxicity caused by NMDA receptor activation in postsynaptic neurons.

N2a cells were cultured in DMEM containing various glutamate concentrations, and it was discovered that L-Glu did not affect N2a cell viability at concentrations less than 2 mmol/L. (shown in Fig. 5a). Following that, we added various concentrations of Aβ1–42 to DMEM (containing very low concentrations of L-Glu). N2a cells were incubated in such media, and cell viability assays were performed to determine the degree of cytotoxicity of A1-42 to N2a cells (shown in Fig. 5b). We discovered a significant difference in cell viability when the concentration of Aβ1–42 reached 10 mmol/L. Finally, 10 mmol/L Aβ1–42 was used to induce N2a cells, and the induced cells were detected using Western Blot. SLC25A18 expression was found to be higher in the Model group shown in Fig. 5c).

Glutamate is the most abundant neurotransmitter in the mammalian central nervous system, where it exerts excitatory effects via ionotropic or metabotropic glutamate receptors. Excessive glutamatergic signalling is a hallmark of neurodegenerative disorders [6; 7]. Glutamate's role in the AD process is not limited to its role as a neurotransmitter that causes excitotoxicity, but it is also associated with a disruption in its metabolic balance [19]. However, little is known about the relationship between glutamate metabolism-related carriers and AD, and it is unknown whether glutamate metabolism target genes play a role in the onset and progression of AD. Our results suggest that SLC25A18 may be a key gene in the occurrence and development of AD, providing a new target for AD pathogenesis and therapeutic intervention.

SLC25A18 (Solute Carrier Family 25 Member 18, GC2) is a gene that codes for proteins. It is mostly found on the mitochondrial membrane, but it is also found in the inner mitochondrial membrane, the mitochondrial matrix, and the cytoplasm. As a result, it can connect the cytoplasm to the mitochondrial matrix and participate in glutamate transport in the inner mitochondrial membrane. SLC25A18 expression appears to correlate with inflammation level, as SLC25A18 levels are significantly reduced after spinal cord injury [20], whereas SLC25A18 expression is increased in macrophage cultures after treatment with IL4/IL13 [21]. The association of SLC25A18 with glutamate transport and inflammation suggests that SLC25A18 may play a role in neurological health and warrants further investigation in the context of clinical presentation. However, no research has been conducted to investigate the correlation between SLC25A18 and neurodegenerative diseases such as AD. Using bioinformatics techniques, we discovered that the expression of the mitochondrial glutamate carrier SLC25A18 is associated with AD. To validate this finding, we built several models to investigate the relationship between SLC25A18 and AD.

Behavioural studies have revealed that the hippocampus is essential for learning and memory [22]. TeA is an important part of the cortex that is primarily involved in the processing and integration of auditory information, as well as the transmission of information to areas such as the hippocampus [23]. The EC connects the hippocampus to the cortex [24]. We discovered Aβ aggregation in HIP, TeA, and EC immunofluorescence staining results in transgenic mice, which was consistent with clinical manifestations of cognitive and behavioural decline in AD [12]. We found elevated expression of SLC25A18 in these brain regions, indicating a link between SLC25A18 and AD clinicopathological features. Therefore, it can be speculated that the increase of SLC25A18 may play an important role in evaluating the clinical progress of AD.

Oxidative stress is another pathological feature of AD. AlCl₃ plus D-gal-induced animal model can better simulate AD oxidative damage than transgenic animal models [15; 25]. SLC25A18 expression was increased in the AlCl₃ plus D-gal-induced animal model in this experiment, indicating that SLC25A18 was involved in the process of oxidative stress damage in the development of AD pathology. Because SLC25A18 transports the raw material glutamate for mitochondrial energy metabolism, SLC25A18 elevation may be part of the process of mitochondrial energy metabolism imbalance.

The natural ageing animal model responds to the pathological changes associated with AD in a healthy state[16]. This model does not require any human intervention. In this model, we found that SLC25A18 expression increased with age. This suggests that SLC25A18 expression is involved in the natural ageing process. Because SLC25A18 is a glutamate carrier in mitochondria, its elevation must affect the energy metabolism balance. Interestingly, mitochondrial oxidative energy metabolism declines with age, resulting in a reduced brain's ability to cope with stress and pathological conditions [26]. As a result, we hypothesise that the compensatory expression of mitochondrial glutamate carriers increases in response to the progressive impairment of energy metabolism in the brain as we age.

During the development of AD, Aβ oligomers can directly activate glutamate NMDA receptors [27]. Aβ oligomers cause neurotoxicity in cortical and hippocampal neurons, which can be reduced with NMDA receptor antagonists [28]. As a result, we used Aβ1–42 to induce neurons and added a small amount of glutamate to the culture medium to simulate excitotoxicity caused by NMDA receptor activation in postsynaptic neurons, and found that SLC25A18 expression was increased. This suggests that SLC25A18 is involved in the occurrence and progression of excitotoxicity. The activation of neuronal NMDA receptors may affect mitochondrial glutamate metabolism.

In conclusion, we report for the first time that SLC25A18 expression has a positive correlation with AD, validate this finding using a variety of models, and investigate how SLC25A18 is linked to AD. SLC25A18 expression was found to be positively correlated with the clinicopathological features of AD, to be involved in the process of natural ageing, and to be closely related to the occurrence and development of oxidative stress injury and excitotoxicity in the pathological development of AD. This study lays the groundwork for future research into the correlation between glutamate metabolism-related carriers and AD. SLC25A18's ability to regulate AD will be studied further in the future.

AD: Alzheimer’s disease; GEO: Gene Expression Omnibus ; EC: entorhinal cortex; HIP: hippocampus; TeA: temporal association cortex; PC: posterior cingulate; SFG: superior frontal gyrus; PVC: primary visual cortex; DEGs: differentially expressed genes; Tg-AD: transgenic AD; D-gal: D-galactose; MWM: Morris water maze; WT: wild type; L-Glu: L-Glutamine; DMEM: Modified Eagle medium .

Ethics approval and consent to participate

All animal experiments followed the "Policies on the Use of Animals and Humans in Neuroscience Research" revised and approved by the Society for Neuroscience (USA) in 1995. This study protocol was reviewed and approved by the Ethics Committee of the Basic Medical of Jilin University, approval number 2021-028.

Consent for publication

Not applicable.

Availability of data and materials

The data used to support the findings of this study are available from the corresponding author upon request.

Competing interests

The authors declare that they have no competing interests.

Funding

This work has been supported by “Jilin Scientific and Technological Development Program” (grant number 20190304029YY) and “Jilin Province Development and Reform Commission”(grant number 2020C034-1).

Author Contributions

JS conceived the experiments. YJ contributed to the research data. JS drafted the manuscript. HH & XY & JZ participated in raising animals; MZ &JL & QZ &SW contributed to technical assistance. CW & LC & MZ revised the manuscript and supervised the analysis. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

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Expression of SLC25A18 is positively correlated with Alzheimer's disease

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Abstract

Figures

Introduction

Materials And Methods

Bioinformatics investigation of SLC25A18

Animal treatment

Morris water maze

Sample preparation

Immunofluorescent staining and evaluation

RNA extraction, cDNA synthesis, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Synthesis and Preparation of Aβ1–42

Cell Culture

In vitro cytotoxicity of Aβ1–42 and L-Glutamine

Cell treatment

Protein extraction and Western Blot Analysis

Statistical Analysis

Results

Discussion

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Author Contributions

Acknowledgements

References

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Version 1